key: cord- -n xvwkf authors: halstead, e. scott; umstead, todd m.; davies, michael l.; kawasawa, yuka imamura; silveyra, patricia; howyrlak, judie; yang, linlin; guo, weichao; hu, sanmei; hewage, eranda kurundu; chroneos, zissis c. title: gm-csf overexpression after influenza a virus infection prevents mortality and moderates m -like airway monocyte/macrophage polarization date: - - journal: respir res doi: . /s - - - sha: doc_id: cord_uid: n xvwkf background: influenza a viruses cause life-threatening pneumonia and lung injury in the lower respiratory tract. application of high gm-csf levels prior to infection has been shown to reduce morbidity and mortality from pathogenic influenza infection in mice, but the mechanisms of protection and treatment efficacy have not been established. methods: mice were infected intranasally with influenza a virus (pr strain). supra-physiologic levels of gm-csf were induced in the airways using the double transgenic gm-csf (dtgm) or littermate control mice starting on days post-infection (dpi). assessment of respiratory mechanical parameters was performed using the flexivent rodent ventilator. rna sequence analysis was performed on facs-sorted airway macrophage subsets at dpi. results: supra-physiologic levels of gm-csf conferred a survival benefit, arrested the deterioration of lung mechanics, and reduced the abundance of protein exudates in bronchoalveolar (bal) fluid to near baseline levels. transcriptome analysis, and subsequent validation elisa assays, revealed that excess gm-csf re-directs macrophages from an “m -like” to a more “m -like” activation state as revealed by alterations in the ratios of cxcl and ccl in bal fluid, respectively. ingenuity pathway analysis predicted that gm-csf surplus during iav infection elicits expression of anti-inflammatory mediators and moderates m macrophage pro-inflammatory signaling by type ii interferon (ifn-γ). conclusions: our data indicate that application of high levels of gm-csf in the lung after influenza a virus infection alters pathogenic “m -like” macrophage inflammation. these results indicate a possible therapeutic strategy for respiratory virus-associated pneumonia and acute lung injury. electronic supplementary material: the online version of this article ( . /s - - - ) contains supplementary material, which is available to authorized users. each year, influenza a virus (iav) affects a significant proportion of the population [ ] and causes pathologic changes both through direct cellular toxicity causing desquamation, de-ciliation, and cell death, and through indirect effects by stimulating an anti-viral immune response leading to collateral injury [ ] . this combination can lead to an ards-like syndrome characterized by increased capillary leak, oxygen diffusion difficulty and ventilation/perfusion mismatch [ ] . immune strategies that protect the host's lung function while still allowing for an adequate immune response to clear the viral load and resolve virus-induced pneumonia are needed. a number of pre-clinical studies have tested prophylactic gm-csf both as vaccine adjuvant and local supplementation against iav infection with encouraging results [ ] [ ] [ ] [ ] . the effect of local elevation of gm-csf on iav infection in the lung has been investigated in transgenic models with expression of gm-csf under the control of constitutive or doxycycline-inducible promoters in lungs of alveolar or small airway epithelial cells of gm-csf knockout (csf −/− ) mice [ , ] . differential effects on morbidity and mortality from iav infection in these studies was associated with increased alveolar macrophage (am) numbers in the constitutive gm-csf expression models [ , ] and am differentiation in the gm-csf-inducible model [ ] . differential results on morbidity and survival were also obtained after prolonged or brief administration of supra-physiological levels of gm-csf before or at the onset of iav infection [ ] . the question of whether therapeutic administration of gm-csf to the airways after establishment of the infection would confer protection has never been addressed. in this study we use a more clinically relevant model to examine whether supra-physiologic levels of gm-csf in the airways, induced after iav infection at the peak of virus replication, provided therapeutic benefit. using gm-csf-inducible mice on the wt c bl/ genetic background we show that airway gm-csf over-expression starting at days post infection (dpi) provides protection from mortality and prevents the degeneration of multiple lung mechanical properties. to examine the mechanism of protection conferred by therapeutic gm-csf levels, we measured respiratory and biochemical parameters of lower airway disease, and analyzed the transcriptome of facs-sorted ams and exudative macrophages (em) from iav-infected mice. our findings demonstrate that gm-csf restores proteostasis of exudate proteins and redirects responsiveness of ams and ems from an m -like to an m -like activation state, and prevents mortality from influenza-induced ards. the double transgenic gm-csf (dtgm) mice were bred as previously described [ ] , but this time on the wild-type c bl/ j background. littermate control (lm) mice were defined as being single transgenic littermates of dtgm mice that were only positive for the scgb a -rta and thereby did not have the cmv-gm-csf gene, which may potentially be virally induced in the absence of tetracycline (doxycycline) [ ] . dtgm mice and lm controls were exposed to mg/ml doxycycline in drinking water, and doxycycline-containing drinking water was replenished every - days. both male and female mice were used for all experiments; all mice were sex-and age-matched to control mice. the influenza strain a/puerto rico/ / (pr ) virus was a kind gift of dr. kevan hartshorn, and was grown in the chorio-allantoic fluid of ten ( ) day old specific pathogen free avian supplies (spafas) chicken eggs purchased from charles river laboratories (wilmington, ma) and purified on a discontinuous sucrose gradient as previously described [ ] . mice were anesthetized with ketamine/xylazine and intranasally (i.n.) infected with iav virus in μl of pbs. mice were infected in a bsl biosafety cabinet and housed within filter-top microisolator cages in the pulmonary immunology and physiology (pip) core, a bsl facility in the department of comparative medicine's animal facility at penn state university college of medicine. mice were observed at least twice daily during infections to assess morbidity and mortality. based on our experience at our facility in the last years and the variable clinical presentation of the influenza infection, we used other metrics to monitor morbidity in addition to mouse body weight curves [ ] . mice that exhibited immobility, ruffled hair, and labored breathing that had no chance of recovery, coinciding with approximately % of body weight loss, were euthanized by ketamine/xylazine overdose and cervical dislocation, and counted as dead. alternatively, mice that were sleeping but had normal breathing and body appearance, i.e., no ruffled hair or labored breathing, reached up to - % body weight loss and then began to recover normally. mice with favorable prognosis but with % body weight loss or greater were provided supportive care with food and hydrated gel packs at the bottom of the cage. we have not found a pattern of clinical disease specific to mouse genotype or gender in untreated mice. mice were anesthetized with ketamine/xylazine ( mg/kg and mg/kg, i.p., respectively). the trachea was cannulated via tracheostomy with a g blunt needle and the cannula was secured in place with a suture. mice were kept sedated using isoflurane inhalation (maintenance dosing, - % of inspired air) through the flexivent and were paralyzed with mg/kg vecuronium i.p. mice were ventilated using baseline settings of positive end expiratory pressure (peep) cm h o, tidal volumes (vt) ml/kg, respiratory rate (rr) breaths per minute (bpm) and an fraction of inspired oxygen (fio ) of . . oxygen saturations were measured using the mouseox plus pulse oximeter (starr life sciences, oakmont, pa, usa) via the thigh sensor. lung mechanic parameters were generated from the flexivent rodent ventilator using the forced oscillation technique as previously described [ ] . bronchoalveolar lavage (bal) protein measurements bal samples were collected as previously described, and after centrifugation at g for min, bal supernatants were removed and immediately frozen at − °c until batch analysis. proteins were measured with kits as detailed in additional file : table s . elisa plate absorbance was measured at nm with a spectramax m uv/vis/fluorescence - plate reader (molecular devices, sunnyvale, ca). cytokines were measured by elisa as described above or by procartaplex cytokine & chemokine -plex mouse panel a (thermo fisher scientific) via luminex magpix multiplex array (luminex). after iav infection, the entire lung was removed from each mouse and placed in ml of trizol (thermo fisher scientific, waltham ma), weighed, homogenized on ice using a polytron homogenizer for - s intervals, and frozen in aliquots at - °c until rna extraction. dna was then extracted using chloroform and rna was precipitated using isopropanol. quantitative rt-pcr for iav m copies per lung was performed as previously described [ ] using the following primers: influenza a/ /puerto rico/ m gene sense: '-aagaccaatcctgtcacctctga- ′ and antisense: ' caaagcgtct-acgctgcagtc - ′ primers, and the mediator probe sequence: ′-/ fam/ tttgtgttcacgctc-accgt/ -tamsp/ - ′. data are expressed as m viral copies per lung. single cell suspensions were prepared from bal and lung as described in supplemental methods. single cell suspensions from lung digests were placed at °c and then surface stained in hank's buffered saline solution (hbss) with % fbs with fluorochrome-conjugated monoclonal antibodies (additional file : table s ), and then stained with a fixable viability dye. for facs-sorting, bal cells were recovered and placed at °c and then surface stained in hank's buffered saline solution (hbss) with % fbs with fluorochrome-conjugated monoclonal antibodies (additional file : table s ) and -aminoactinomycin d ( -aad) was used to assess viability just prior to acquisition. all flow cytometric data were collected in the penn state hershey flow cytometry core facility using an lsr ii (becton dickinson, bd) instrument, and all facssorting was performed using a facsaria (bd) high speed cell sorter. cells were sorted directly into rna-bee to isolate rna for further rna-sequencing (rna-seq). all facs data analysis was performed using flowjo version . (treestar, mountain view, ca). rna was phase separated using chloroform and the aqueous phase containing rna was removed following centrifugation and precipitated overnight at − °c using ice cold isopropanol. rna was washed with % ethanol then solubilized in rnase-free water. optical density values of extracted rna were measured using nanodrop (thermo fisher scientific) to confirm an a :a ratio above . . rna integrity number (rin) was measured using bioanalyzer (agilent) rna pico kit to confirm rin above . the cdna libraries were prepared using the smarter® ultra® low input rna kit for sequencing -v (clontech) followed by nextera xt dna library prep kit (illumina) as per the manufacturer's instructions. the unique barcode sequences were incorporated in the adaptors for multiplexed high-throughput sequencing. the final product was assessed for its size distribution and concentration using bioanalyzer high sensitivity dna kit (agilent technologies) and kapa library quantification kit (kapa biosystems). the libraries were diluted to nm in eb buffer (qiagen) and then denatured using the illumina protocol. the denatured libraries were diluted to pm by pre-chilled hybridization buffer and loaded onto truseq sr v flow cells on an illumina hiseq (illumina) and run for cycles using a single-read recipe (truseq sbs kit v , illumina) according to the manufacturer's instructions. illumina casava pipeline (released version . , illumina) was used to obtain de-multiplexed sequencing reads (fastq files) passed the default purify filter. additional quality filtering used fastx-toolkit (http://hannonlab.cshl.edu/fastx_toolkit) to keep only reads that have at least % of bases with a quality score of or more (conducted by fastq_quality_filter function) and reads left with bases or longer after being endtrimmed with reads with a base quality score of b (conducted by fastq_quality_trimmer function). a bowtie index was built for the mouse reference genome (grcm ) using bowtie version . . . the rna-seq reads of each of the samples were mapped using tophat version . . [ ] supplied by ensembl annotation file; grcm . .gtf. gene expression values were computed using fragments per kilobase per million mapped reads (fpkm). differential gene expression was determined using cuffdiff tool which is available in cufflinks version . . [ ] supplied by grcm . .gtf. normalization was performed via the median of the geometric means of fragment counts across all libraries, as described in anders and huber [ ] . statistical significance was assessed using a false discovery rate threshold of . . we arbitrarily chose to further analyze the % most highly expressed gene transcripts in am or em cell populations from iav-infected dtgm or lm mice using ingenuity pathway analysis (ipa, www.qiagen.com/ingenuity) to identify upstream signaling pathways. significance was measured by fisher's exact test with a q < . cut-off. all statistical analysis was performed using jmp . . software (sas, cary, nc). normally distributed data were analyzed using student's t-test, and non-normally distributed data using wilcoxon signed-rank test. survival analysis was calculated by using the log-rank test. all data points are means ± standard error of the mean (sem) unless otherwise stated. graphs were created using prism for mac os x (graphpad, la jolla, ca). to characterize the pathogenicity of our h n pr iav preparation virus, wild-type c bl/ j mice (the jackson laboratory, ma) were purchased and we determined the lethal dose % (ld ) of our pr iav preparation. female wild-type mice were much more susceptible than males with an ld approximately fold lower than males ( vs. ffu, female and males respectively, additional file : figure s a -d). airway gm-csf levels were conditionally increased following iav infection using a doxycycline inducible promoter in the dtgm mouse model, formerly named the tet-gm +/+ , as previously described [ ] . in this conditional transgenic mouse model gm-csf is expressed and secreted by airway club cells via the club cell (cc , scgb a ) promoter after oral administration of doxycycline ( mg/ml in water ad libitum) (fig. a) . importantly, in the absence of infection, bal fluid levels of gm-csf in dtgm mice are near the limit of detection, similar to littermate controls (additional file : figure s a ), and their alveolar macrophages appear identical by multiparameter flow cytometry. once doxycycline is administered, bal levels of gm-csf peak after approximately h reaching levels of approximately pg/ml in . ml of recovered bal fluid and in preliminary experiments the dtgm mice were either administered or not administered doxycycline to create a condition of elevated vs. wild-type levels of airway gm-csf, respectively [ ] . however, these preliminary experiments demonstrated that low levels of gm-csf from the scgb a promoter in dtgm mice were endogenously induced by interferons during iav infection (additional file : figure s a ), a finding that has previously been reported [ ] . therefore, all subsequent experiments compared the dtgm to lm groups, both exposed to doxycycline, to examine the effect of elevated (dtgm) as opposed to wild-type (lm) levels of airway gm-csf, while also controlling for any off-targets effects of doxycycline. to address our research question of whether "treatment" with gm-csf during severe iav infection would improve survival, dtgm and lm mice were infected i.n. with approximately ld (differential dosing based on sex) of pr iav and were administered doxycycline in drinking water. gm-csf overexpression (dtgm) conferred a significant survival advantage as compared to wild-type levels (lm, fig. b , **p < . ). weight loss and recovery were similar in the two groups, however, because of survivor bias likely artificially elevating the average weights of surviving lm mice (fig. c) . of note, doxycycline treatment of lm mice had no effect on survival whereas doxycycline-untreated dtgm mice demonstrated a survival advantage over untreated lm mice, suggesting that even low levels of gm-csf can confer some survival benefit (additional file : figure s b ). lower respiratory tract iav infection can lead to impaired oxygenation due to v:q mismatch and decrease lung compliance due to the infiltration of inflammatory cells and an increase in lung water weight [ ] . given the ability of gm-csf to confer survival, we expected elevated gm-csf levels to improve arterial oxygen saturations (% spo ). however, gm-csf did not significantly increase median oxygen saturations (% spo ) levels as compared to lm mice at either or dpi (data not shown). to gain insight into whether gm-csf conferred any lung mechanical benefits, lung mechanics scans were performed by the forced oscillation technique and pv loop curves were generated (fig. a, b) . as expected, the pv curve flattens with iav infection due to decreased static compliance (cst), but we were surprised that compliance continued to fall from days to ( fig. a-c) . while gm-csf did not affect cst (fig. c) or total system resistance (rrs, fig. d ), dtgm mice demonstrated less tissue damping or peripheral airway resistance (g, cmh o/ml, fig. e) , and significant preservation of newtonian or central airway resistance (rn, cmh o*s/ml, fig. f ) and curvature of the deflation limb of the pv curve, a measure of maintenance of alveoli and small airway recruitment (k, /cmh o, fig. g ) at dpi. given that two of the lung mechanical parameters that are maintained by gm-csf, k and g, are correlated with dynamic processes at the small airway or alveolar level, namely alveolar size [ ] and changes in tissue physical properties of small airways [ ] respectively, and which can change with lung interstitial edema [ ] , we hypothesized that gm-csf may improve lung capillary barrier function and/or enhance alveolar fluid clearance. as surrogate of alveolar fluid content, we measured the concentration of total protein in bal fluid and found that gm-csf overexpression decreased bal fluid total protein levels at (peak inflammation) and dpi (early resolution phase) (fig. a) . to further investigate this difference in bal fluid protein content, we examined the concentration of various serum and lung-specific proteins including mouse serum albumin ( kda), as well as two larger proteins, alpha- -macroglobulin ( kda monomer, kda tetramer) and immunoglobulin m (igm, kda monomer, kda pentamer) as markers of capillary leak [ ] . gm-csf significantly decreased alpha- macroglobulin levels at dpi (fig. b) , but did not significantly decrease other markers of capillary leak including albumin or igm (additional file : figure s a-b) . we also directly assayed the lung epithelial barrier function with fitc-labeled dextran (mw , ), but surprisingly no differences in epithelial barrier function could be detected at dpi (data not shown). additionally, we also investigated whether gm-csf overexpression during iav increased bal levels of the epidermal growth factor family member, amphiregulin. gm-csf overexpression in uninfected mice elevated levels of amphiregulin, though iav infection also induced amphiregulin and gm-csf did not further increase these levels ( fig. c) . lastly, we assessed whether elevated gm-csf levels during active infection affected viral clearance. at dpi, the peak of virus levels in our model, we recovered - × m -copies total lung copies of iav pr matrix (m ) via rt-pcr and the viral copies decreased to . - × by dpi, though there was no statistically significant difference with gm-csf overexpression (fig. d) . alveolar macrophages have been shown to be necessary for protection from iav [ ] [ ] [ ] [ ] [ ] [ ] [ ] . gm-csf is known to mediate the proliferation and differentiation of monocytes and macrophages; studies using constitutive expression or gm-csf administration before iav infection models both documented an increase in am numbers [ , , ] . therefore we hypothesized that gm-csf would protect siglecf + ams from viral-induced depletion and would increase numbers of total airway (bal-recovered) macrophages. to investigate this immune cells were characterized and enumerated in single cell suspensions of bal and lung enzymatic digests by multi-parameter flow cytometry using a -color panel of macrophage and granulocyte surface markers (fig. a) . we specifically focused on the two predominant airway macrophage populations present during active iav infection: f / + , cd b neg/dim , siglecf + cells to discriminate alveolar macrophages (ams) and f / + , cd b + , siglecf neg/dim cells that have been termed exudative macrophages (ems) [ ] . our typical yield of ams recovered from bal fluid of an uninfected mouse is approximately , cells. at dpi, at the height of the inflammatory response to iav, the number of ams to simulate a therapeutic model of gm-csf administration doxycycline was administered to both dtgm and lm control mice starting days after i.n. infection with pr iav. doxycycline-containing water was protected from light and changed every three days (a). dtgm (n = , red circles/lines) and lm control (n = , black squares/lines) mice were administered approximately ld of iav pr virus i.n. and administered doxycycline in water starting on + dpi, and the effects on survival and body weight are shown. mice were euthanized if they lost > % body weight and were moribund. gm-csf over-expression (dtgm mice) conferred a significant survival benefit (b) but not a significant effect on weight loss/recovery (c) as compared to wild-type levels (lm mice). results shown represent three independent experiments (**p < . ) recovered was much lower, and gm-csf overexpression did not serve to increase this number (fig. b) . in contrast, ems become the predominant airway macrophage during iav infection at this time point, but again, gm-csf overexpression did not affect em cell numbers (fig. b) . while gm-csf overexpression did not change the number of macrophages, we hypothesized that it changed their phenotype. this is not a new concept as the primary function of gm-csf on ams is to induce differentiation and activation [ , , ] . despite attempting to discriminate the macrophage populations by multiple cell surface markers, we could not distinguish the iav-responding macrophages further than alveolar and exudative macrophages as described in fig. a . therefore, we sought to determine whether gm-csf affected the transcriptomes of the am and em populations independently by first facs-sorting the airway macrophages. facs-sorted airway macrophages from bal fluid were obtained at dpi, the time point where the survival curves of the dtgm and lm mice begin to diverge, and next generation rnasequencing was performed on the sorted am and em populations. using an unbiased approach, we identified the transcripts that were significantly affected by gm-csf over-expression during iav infection by comparing the mean value of each transcript from the dtgm and lm groups. for direct comparisons, transcripts that had a mean value of zero ( ) fkpm in one of the groups were not analyzed. of the , genes available in the reference genome, in the am population transcripts were significantly different between the groups with gm-csf over-expression leading to up-regulation of the chemokines ccl , cxcl , and ccl , and the down-regulation of cxcl , and arg , the prototypic marker of m macrophage polarization (fig. a) [ ] . in comparison to ams, in ems gm-csf induced more transcripts than it inhibited. only fig. flow cytometric discrimination of alveolar and exudative macrophages by surface marker expression. representative facs plots from an iav-infected lm mouse at dpi, which detail our -color flow cytometry gating strategy of single cell suspensions from bal and enzyme-digested lung (a). alveolar macrophages (am) were designated as f / + siglecf + cd b neg/dim , whereas exudative macrophages (em) were designated as f / + siglecf neg/dim cd b+. supra-physiologic gm-csf levels during iav infection had no effect on the absolute number of either airway (bal-recovered) am or em cell numbers at dpi (b) six transcripts were down regulated by gm-csf including lipg, cxcl and ccl , while gm-csf overexpression induced multiple transcripts in ems including dcstamp, retnla, irgc , mmp , and ccl (fig. b) . our unbiased analysis demonstrated that gm-csf overexpression during iav led to the up-regulation of some transcripts associated with m macrophages including matrix metalloprotease , mmp , and ccl , and the down-regulation of some m macrophage-associated transcripts such as cxcl and cxcl . therefore we examined the effect of gm-csf on multiple canonical and novel macrophage polarization markers [ ] . interestingly, while gm-csf tended to down-regulate m transcripts and up-regulate m transcripts, this effect was not absolute in either ams or ems (fig. c-f ). to validate these macrophage transcript differences we measured the chemokines ccl and cxcl , and the m -associated metalloprotease, mmp , in bal fluid by elisa. ccl was significantly induced by gm-csf (not only during iav infection, but also when gm-csf was induced in the absence of iav (fig. a) . in comparison, negligible amounts of cxcl were present in uninfected mice regardless of gm-csf induction, whereas with iav fig. characterization of the changes in transcriptome patterns of airway macrophages during iav infection. bal airway macrophages were sorted using the gating strategy described in fig. a and next generation rna-sequencing was used to profile the complete transcriptome data of ams (a, orange bars) and ems (b, blue bars) at dpi, the time point at which the survival curves diverge (n = mice per group). the effect of supraphysiologic gm-csf levels on each of the , sequenced macrophage genes was examined: differential gene expression was determined using with transcripts having a q-value < . being included. the relative expression of each transcript was calculated using the equation, log expression ratio (dtgm:lm) = log ðx transcript dtgm ) -log (x transcript lm ), and the differential expression of transcripts is shown. to investigate the impact of gm-csf on m /m macrophage polarization, the log expression ratios were plotted against known m and m macrophage-associated transcripts from ams (c, d) and ems (e, f) infection gm-csf overexpression there was a trend toward decreased expression (fig. b, p = . ). the concentration of mmp was approximately -fold higher in gm-csf overexpressing mice at dpi as compared to lms (fig. c, p < . ) . we also examined the ratio of the two chemokines (cxcl : ccl ) as an intrinsic property of the balf to probe macrophage polarization by chemokine expression and supra-physiologic gm-csf levels significantly decreased this ratio more than ten-fold in iav-infected mice (fig. d , p < . ). lastly, we attempted to determine which signaling pathways were affected by gm-csf overexpression during iav infection by analyzing our transcriptomes with ingenuity pathway analysis (ipa) software (qiagen). we analyzed the effect of gm-csf overexpression on the mean log expression ratios for the % most expressed genes in each of the macrophage type groups (am vs. em). the ipa software allows the construction of an upstream analysis that calculates the likelihood that an upstream regulator is involved given the gene set provided (p-value of overlap), as well as a composite score of activation depending on the state of downstream genes being increased or decreased in quantity (activation z-score). for both the upstream regulator analysis, and the subsequent canonical pathway analysis, we used a stringent p-value of overlap cutoff of e- . ipa predicted that gm-csf activates (table a ) several upstream regulators of signaling pathways in both ams and ems including il- receptor alpha (il ra), transcription factor tripartite motif-containing (trim ), and the atypical chemokine receptor (ackr ). conversely, ipa predicted that gm-csf over-expression inhibited multiple inflammatory signaling pathways in both ams and ems including interferon regulatory factor (irf ), irf , interferon gamma (ifng), interferon alpha/beta receptor (ifnar), tir domain-containing adapter molecule (ticam , or trif), signal transducer and activator of transcription (stat ), rapamycin-insensitive companion of mammalian target of rapamycin (rictor), toll-like receptor (tlr ), dexd/hbox helicase (ddx , or retinoic acid-inducible gene [rig- ]), and inhibitor of nuclear factor kappa-b kinase subunit beta (ikbkb). in terms of canonical pathway analysis one pathway, "eukaryotic initiation factor (eif ) signaling", was activated in both ams and ems, whereas "fc-γ receptormediated phagocytosis in macrophages and monocytes" was inhibited in both populations (table c, d) . "interferon signaling" was inhibited in ems (table d) , and trended towards significance in the am population [−log(p-value) . , z-score − . ], even though the levels of type i, ii and iii interferons were unchanged in fig. effect of gm-csf overexpression on airway levels of ccl , cxcl and mmp . mouse ccl (a), cxcl # (b), and mmp # (c) were measured by elisa in bal fluid from doxycycline-treated lm (black) and dtgm (red) uninfected and iav-infected ( dpi) mice. furthermore, the ratio of cxcl :ccl # in each bal sample was determined to examine the relative effect of supraphysiologic gm-csf levels on macrophage chemokine polarization (d). results from three independent experiments. ( # please note the log scale, *p < . , **p < . ) bal fluid from dtgm as compared to wt mice (additional file : figures s a-c ). in this study we examined the effect of elevated gm-csf levels during iav infection on clinical, lung physiologic and biochemical markers in a mouse model, and then used rna-sequencing to ascertain the differential effects of elevated gm-csf levels on the transcriptomes of the two predominant airway macrophages present during the peak of iav infection. our finding that elevation of airway gm-csf during active iav infection confers protection from mortality from iav is novel. multiple preclinical mouse studies have described the observations that the absence of gm-csf increases susceptibility to iav [ , , ] , while supra-physiologic levels of gm-csf achieved by constitutive overexpression or exogenous administration are beneficial [ , , ] . importantly, however, the publications that have demonstrated positive effects of supra-physiologic levels of gm-csf against iav infection have used either constitutive expression models [ ] [ ] [ ] or have administered gm-csf either before [ , ] or on the day of infection [ ] . to our knowledge this is the first description of the use of a therapeutic model of gm-csf wherein it is "administered" to the airways well after establishment of the infection (+ dpi) and still confers protection. table ingenuity pathway analysis predictions of the effects of supra-physiologic levels of gm-csf on airway macrophages during iav. bal airway macrophages were sorted and rna-sequencing was performed to compare the gene expression between iav-infected lm (n = mice) and dtgm (n = mice) treated with doxycycline at dpi. using the means of each group, the % ( genes) most expressed transcripts from each of the genotypes, dtgm and lm, were analyzed using qiagen's ingenuity pathway analysis (ipa) software. ipa was used to identify differential upstream regulators between ams (a) and ems (b) of dtgm and lm mice, and upstream regulators were included in the table if their p-value of overlap was < e- and the activation z-score was < − or > + . ipa was also used to identify differential effects of gm-csf on canonical pathways of ams (c) and ems (d). ingenuity canonical pathways were included in the table if their -log(p-value) was > and the z-score of pathway activation was < − or > + gm-csf over-expression led to an increase in macrophage expression and bal fluid levels of ccl and mmp , whereas a decrease in cxcl or monokine induced by gamma interferon (mig). these protein data, in addition to our macrophage transcriptome data, suggest that high levels of gm-csf push the typically classically activated m -like monocytes/macrophages in the lung during iav towards an m -like phenotype. interestingly, a recent investigation showed that the presence of m -like monocytes are a major determinant of iav pathogenicity in patients and strengthened this notion with a mouse model demonstrating that adoptive transfer of m as opposed to m macrophages results in better outcomes [ ] . the observation that gm-csf is pushing macrophages towards an m -phenotype is in stark contrast to a large body of in vitro literature that defines m monocytes/macrophages as being induced by gm-csf, whereas m monocytes/macrophages are differentiated by macrophage colony-stimulating factor (m-csf) [ ] [ ] [ ] . on the other hand, alveolar macrophages from gm-csf-deficient csf −/− mice exhibit a mixed m /m phenotype, not a strictly m phenotype as in vitro data would suggest [ ] . and our data also suggests that the polarization was not at all absolute: e.g., in ams, gm-csf led to lower transcript levels of the prototypic m macrophage marker, arg (fig. a) . thus, while the m /m macrophage polarization schema has been helpful [ , ] , perhaps a more nuanced view of macrophage polarization [ ] , where their intrinsic differentiation plasticity allows them to attend to specific needs of their local immune environment [ ] , could explain these results. ipa also predicted the activation of the il- receptor alpha-chain in both ams and ems. given that il- levels in bal fluid were not elevated in dtgm as compared wt mice (additional file : figure s d ), it is possible that gm-csf overexpressing during iav somehow potentiates il- signaling in the lung microenvironment. the role of interferons during iav infection is also nuanced. while it has been shown using ifnar −/− and ifngr −/− mice that interferon signaling is necessary for protection from iav [ ] , it is possible that this requirement only extends to epithelial cells. interferon-γ may not be necessary during iav infection and may in fact be detrimental, e.g., nitrogen oxide synthase deficient (nos −/−) mice are more protected from iav [ ] , and sun and metzger demonstrated that treatment with an anti-ifnγ mab clone xmg . had little effect on the course of the viral infection, but inactivation of ifn-γ protected against secondary bacterial pneumonia [ ] . recently, califano et al. showed that ifnγ −/− mice on both the balb/c and c bl/ backgrounds demonstrated improved survival to lethal iav infection [ ] . in their model, ifnγ serves to restrict protective innate lymphoid cell group (ilc ) function, whose production of il- and amphiregulin may improve lung barrier function. another group has also demonstrated that gm-csf can induce amphiregulin in a smoke model of copd followed by iav infection [ ] , however our data (fig. c) suggest that pretreatment with gm-csf is necessary for this effect on amphiregulin levels. furthermore, our gm-csf over-expression is started after iav infection, amphiregulin levels at dpi were not different in gm-csf over-expressing mice, and therefore amphiregulin is likely not an active player in our model. it is possible that our inducible gm-csf model may be replenishing gm-csf that otherwise would be produced by ilc s whose functions have been restricted by ifnγ [ ] . our data suggest that high levels of gm-csf inhibit interferon signaling in airway macrophages, though the mechanism is not clear. canonically, gm-csf signaling acts through jak /stat [ ] , though the beta-chain itself can activate nf-kb, and this activation is dependent on tnfr-associated factor (traf ) [ , ] , an e ubiquitin ligase with multiple immune functions [ ] . interestingly, our upstream analysis predicts that gm-csf activates trim , (aka tif α), a negative regulator of interferon signaling that acts by binding the retinoic acidresponsive element of the stat promoter [ ] , thus inactivating multiple interferon pathways. trim is also an e -ubiquitin ligase, and the tumor suppressor protein, p , serves as a ligand for both ligases: trim targets p for degradation [ ] while traf restricts p mitochondrial translocation [ ] . furthermore, a recent microarray study examining the relative pathogenicity of a mouse adapted strain of iav (ma-ca/ ) described negative inhibition of trim and early sustained interferon responses as important factors [ ] . however, we detected only very low levels of trim transcripts in our sorted airway macrophages, but gm-csf over-expression did lead to increased expression of another trim family member, trim , that also acts as a e ubiquitin ligase that can heterodimerize with other trims [ ] . future studies are needed to determine the exact cellular signaling pathways linking gm-csf and interferon. gm-csf enhanced exudative macrophage expression, and dpi bal fluid levels of mmp , or macrophage elastase, which is best known for its requirement for the development of smoke-induced emphysema in mice [ ] . however, it may also regulate acute inflammatory responses by proteolysis of chemokines [ ] , and through its divergent effects on ifn-α signaling depending on its intracellular (activating) vs. extracellular (inactivating) localization [ ] . a recent report demonstrated in two separate mouse models inflammation (peritonitis and arthritis) that macrophages resolve inflammation through multiple mechanisms via mmp including dampening neutrophil infiltration, clearing actin and fibrin from nets, terminating complement activation, and by activating prothrombin thus exhibiting procoagulant activity [ ] . cd + t cells and stat / , at least in a mouse model of pneumocystis pneumonia, are necessary for m macrophage mmp expression and relm-α and ccl production [ ] . while our data suggest that gm-csf may block m -like polarization in the lung during iav infection, it is not yet clear what in the lung microenvironment could promote m -like macrophage responses. recently it was shown that macrophage polarization may be pushed towards a il- dependent pathway in the lung and liver by the presence of surfactant protein a (sp-a) and complement component c q, respectively [ ] . the relationship between supraphysiologic gm-csf levels and sp-a during iav infection remains to be investigated and will be the subject of future studies. our current model of gm-csf induction on the wildtype background differs from our previous work using the inducible model generated on the gm-csf knockout (csf −/− ) genetic background [ ] . the present study is not confounded by prior immaturity of ams and defective surfactant catabolism, nor potential defects in migratory dendritic cell subsets, nk cells, and other myeloid cells outside the alveolar compartment in the lung and in other tissues of csf −/− mice [ ] [ ] [ ] [ ] , or disruption of gm-csf secretion by immune and non-immune cells that may elaborate gm-csf in response to infection. studies in csf −/− /spc-gm mice, in which t aecs express high levels of gm-csf constitutively, came to disparate conclusions as to the role of ams, dendritic cells and epithelial cells [ , ] in host resistance to iav infection. however, the life-long overexpression of gm-csf in the spc-gm +/+ model results in non-physiological proliferation of both t aec cells and ams [ ] that obscures assessment of temporal responses to iav. the spc-gm +/+ model also illustrates that prolonged lung exposure to supraphysiologic levels of gm-csf leads to desquamative interstitial pneumonia (dip) [ ] . we did not observe any similar findings of dip in our model, but this is not surprising given our model creates only a temporary doxycyclineinduced overxpression, and the overriding inflammatory effects of iav infection likely masks any differences. in our model, the ability of supra-physiologic levels of gm-csf to beneficially alter disease progression after iav infection delineates a time frame for possible future therapeutic intervention to arrest development of acute lung injury. in this regard, administration of gm-csf in humans has shown promise in the treatment of ards [ ] . concentration-dependent signaling via the gm-csf receptor affecting differentiation, proliferation, activation, and function of different effector cells has been studied extensively [ ] [ ] [ ] [ ] . lung function and organ dysfunctions in patients requiring mechanical ventilation during the influenza a (h n ) pandemic characterization of the human cd (+) t cell response following infection with pandemic influenza h n virus gm-csf in the lung protects against lethal influenza infection gm-csf modulates pulmonary resistance to influenza a infection alveolar epithelial cells orchestrate dc function in murine viral pneumonia delivery of gm-csf to protect against influenza pneumonia hsv- infected cell proteins influence tetracycline-regulated transgene expression effects of influenza a virus on human neutrophil calcium metabolism. in: pavlotsky and a i tauber information about subscribing to the journal of immunology is online at : metabolism pulse-oximetry accurately predicts lung pathology and the immune response during influenza infection comparative study of three flexivent system configurations using mechanical test loads sp-r (myo a) isoforms as intrinsic modulators of macrophage priming and activation how to map billions of short reads onto genomes transcript assembly and quantification by rna-seq reveals unannotated transcripts and isoform switching during cell differentiation differential expression analysis for sequence count data the tetracycline-responsive promoter contains functional interferon-inducible response elements influenza causes prolonged disruption of the alveolar-capillary barrier in mice unresponsive to mesenchymal stem cell therapy exponential analysis of the pressure-volume curve. correlation with mean linear intercept and emphysema in human lungs measuring the lung function in the mouse: the challenge of size changes in the mechanical properties of the respiratory system during the development of interstitial lung edema the relative balance of gm-csf and tgf-β regulates lung epithelial barrier function alveolar macrophages prevent lethal influenza pneumonia by inhibiting infection of type- alveolar epithelial cells lethal influenza infection: is a macrophage to 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mitochondrial translocation, apoptosis, and tumor suppression implication of inflammatory macrophages, nuclear receptors, and interferon regulatory factors in increased virulence of pandemic h n influenza a virus after host adaptation trim acts as an e ubiquitin ligase and can heterodimerize with other trim family members requirement for macrophage elastase for cigarette smoke -induced emphysema in mice macrophagespecific metalloelastase ( mmp- ) truncates and inactivates elr ϩ cxc chemokines and generates ccl , − , − , and − antagonists : potential role of the macrophage in terminating polymorphonuclear leukocyte influx a new transcriptional role for matrix metalloproteinase- in antiviral immunity macrophage matrix metalloproteinase- dampens inflammation and neutrophil influx in arthritis article macrophage matrix metalloproteinase- dampens inflammation and neutrophil influx in arthritis experimental pneumocystis lung infection promotes m a alveolar macrophage-derived mmp production 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molecular control and implications for immune homeostasis and therapy specific contributions of csf- and gm-csf to the dynamics of the mononuclear phagocyte system guide for the care and use of laboratory animals. national research council (us) committee for the update of the guide for the care and use of laboratory animals we would like to thank nate sheaffer and joseph bednarzyk from the penn state hershey flow cytometry core facility, as well as the institute for personalized medicine (ipm) at penn state hershey college of medicine, for assistance. we would also especially like to thank kevan hartshorn and mitchell white for providing the iav pr virus preparation used in all experiments. availability of data and materials all rna-seq data is available from the gene expression omnibus (geo) database, and the other datasets generated during and/or analyzed during the current study are available from the corresponding author on reasonable request. our data demonstrate that in vivo high airway levels of gm-csf profoundly rescue mice from lethal influenza pneumonia. while in vitro gm-csf is canonically described as an m -polarizing cytokine, our data demonstrates that in vivo, during iav infection, gm-csf instead temporizes the type ii interferon-induced m polarization of airway macrophages. the exact mechanism through which high levels of gm-csf block m macrophage polarization is still not known, and is the focus of our ongoing research. additional file : table s . all protein concentration measurements were made as described in the manuscript text using the reagents and kits listed. (tiff kb) additional file : table s . multi-parameter flow cytometry was utilized to characterize the alveolar and exudative macrophages as shown in fig the mouse influenza a virus infections and tissue harvesting were carried out by wg, md, tu, ly, sh and ekh. the rna sequence analyses were performed by yik, ps and jh. overall experimental design, analysis and interpretation were performed by esh with the mentorship of zcc. all authors read and approved the final manuscript.ethics approval and consent to participate all animal procedures were approved by the institutional animal care and use committee (iacuc) at pennsylvania state university college of medicine under protocols # and , , and were cared for as previously described [ ] . the regulation of the use of mice in research falls under the public health service policy on humane care and use of laboratory animals (phs policy), and is enforced by the office of laboratory animal welfare (olaw) under assurance number a - . in order to comply with the phs policy, our institution adheres to the us government principles for the utilization and care of vertebrate animals used in testing, research and training and the guide for the care and use of laboratory animals th edition [ ] . not applicable, the authors agree to pay the journal processing fee should the manuscript be accepted for publication. the authors declare that they have no competing interests.• we accept pre-submission inquiries • our selector tool helps you to find the most relevant journal submit your next manuscript to biomed central and we will help you at every step: key: cord- -t x w h authors: rowin, mark e.; xue, vivian; irazuzta, jose title: hypothermia attenuates β integrin expression on extravasated neutrophils in an animal model of meningitis date: journal: inflammation doi: . /a: sha: doc_id: cord_uid: t x w h brain injury in meningitis occurs in part as a consequence of leukocyte migration and activation. leukocyte integrins are pivotal in the inflammatory response by mediating adhesion to vascular endothelium and extracellular matrix proteins. we have demonstrated that moderate hypothermia early in the course of meningitis decreases leukocyte sequestration within the brain parenchyma. this study examines whether hypothermia alters neutrophil integrin expression in a rabbit model of bacterial meningitis. prior to the induction of meningitis, peripheral blood samples were obtained and the neutrophils isolated. sixteen hours after inducing group b streptococcal meningitis, animals were treated with antibiotics, iv fluids, and mechanically ventilated. animals were randomized to hypothermia ( – °c) or normothermia conditions. after hours of hypothermia or normothermia, neutrophils were isolated from the blood and cerebral spinal fluid (csf), stained for β and β integrins, and analyzed using flow cytometry. cerebral spinal fluid neutrophil β integrin expression was significantly decreased in hypothermic animals. beta- integrins can assume a higher affinity or "activated" state following inflammatory stimulation. expression of "activated" β integrins was also significantly decreased in hypothermic animals. beta csf neutrophil integrin expression was decreased in hypothermic animals, but failed to reach significance. these data suggest hypothermia may attenuate extravasated leukocyte expression of both total and "activated" β integrins. the process of inflammation is a complex interplay of agonists, cytokines, endothelial cells, and myeloid cells. polymorphonuclear leukocytes (pmns) are important in cellular defense of the host by releasing oxygen radicals and lysosomal enzymes necessary for the eradication of invading microorganisms. although pmns play a vital role in host defense, the accumulation of activated pmns at sites of tissue injury coupled with the excess release of bio-active products may increase tissue injury ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) . recently, specific therapies designed to attenuate excessive leukocyte activation have been examined. in inflammatory brain injury, blockade of neutrophil sequestration and activation decreases pmn-mediated tissue destruction ( ) ( ) ( ) . integrins are cell adhesion molecules that play a primary role in leukocyte sequestration. integrins are heterodimeric glycoproteins consisting of two non-covalently linked chains, the a and b subunits. integrins are commonly classified according to their b subunit. the major class of integrins present on pmns are b integrins (alb or lfa- ; amb or mac- ; and axb or p , ) ( ) . upon activation, b integrins mediate firm pmn adhesion to endothelium and subsequent transmigration of the leukocyte out of the vasculature. b integrins have also been described on pmns. they are a minority of the integrin population and their role is poorly understood. many researchers have speculated that the role of b integrins on human pmn is as receptors for extracellular matrix macromolecules. polymorphonuclear leukocytes express b integrins that recognize a domain on the extracellular matrix proteins fibronectin (a b ) and laminin (a b ) ( ) ( ) . recently, researchers have demonstrated that the b integrin plays a role in binding of pmns to tnfstimulated endothelial cells (a b ) ( ) , or expressed following transmigration through endothelial cells or a fibroblast layer (a , a and a b ) ( ) ( ) ( ) . integrins may also play a role in infectious or inflammatory disease processes. certain bacterial and viral infections are known to alter expression of b and b integrin subpopulations on both circulating granulocytes and lymphocytes ( ) ( ) ( ) . during inflammation, b integrins appear to play a critical role in directed lymphocyte migration. the a b integrin on t cells assists in lymphocyte recruitment into the subarachnoid space during viral meningitis ( ) . hiv infection causes increased expression of a b integrins on both acutely and chronically infected cd + t cells, with resultant increased adherence to extracellular matrix glycoproteins ( ) ( ) . multiple investigators have shown that hypothermia decreases pmn accumulation in animal models of brain injury ( ) ( ) ( ) ( ) . we have recently shown that hypothermia may be of benefit in the treatment of meningitis. the application of moderate hypothermia decreases brain myeloperoxidase levels, csf nitric oxide, and central nervous system (cns) excitatory amino acid levels ( ) ( ) . we have previously demonstrated that extravasated csf neutrophils in rabbits with group b streptococcal meningitis show a significant increase in expression of b , activated b , and b integrins ( ) . the mechanism(s) whereby hypothermia diminishes cns injury remains unknown. since the brain injury in meningitis occurs in part as a consequence of leukocyte activation and subsequent release of bioactive products, we speculate that hypothermia affects neutrophil function during the acute inflammatory response. to our knowledge, the effects of hypothermia on neutrophil integrin expression during meningitis have not been explored. thus, this article examines expression of b and b integrins on extravasated neutrophils in normothermic and hypothermic animals using bacterial meningitis as a model of cns inflammation. is a murine anti-human igg that binds to an activationdependent epitope expressed on the b subunit of b integrins ( ) . the anti-b igg monoclonal antibody (mab ) and the anti-b igg monoclonal antibody (mab ) were purchased through chemicon inc. (temecula, california). both of these antibodies react with the extracellular domains of their respective integrins. the hybridoma secreting the murine mab da . (igg ) directed against the m chain of human igm was obtained through american type tissue culture collection (rockville, maryland). monoclonalantibody da . was purified from ascites by octanoid acid precipitation and was provided by dr. john bohnsack (university of utah). the following reagents were purchased: hank's buffered saline (hbss) (gibco brl, gaithersburg, maryland), purified rat immunoglobulin g (calbiochem-novabiochem corporation, la jolla, california), phycoerythrin-conjugated f(ab′) goat anti-mouse igg and phycoerythrin-conjugated f(ab′) goat anti-rat igg (immunotech, miami, florida). animal model. male new zealand rabbits ( - kg) were used. standard animal husbandry and care practices were employed. a preliminary blood sample was obtained prior to induction of meningitis. to induce meningitis, a -gauge needle was introduced in the cisterna magnae and . ml of cerebral spinal fluid was withdrawn. after removal of csf, the animal's cisterna magnae was inoculated with . ml of a suspension containing × cfu / ml of group b streptococcus (gbs), serotype iii, diluted in sterile normal saline. sixteen hours post-inoculation the rabbits were treated with i.v. ceftriaxone ( mg / kg). the rabbits were then anesthetized and a tracheostomy was performed for continuous mechanical ventilation. the animals then underwent tracheotomy and continuous mechanically ventilated. animals were randomized to hypothermic (defined as - c) vs. normothermic (defined as - c) conditions. animals in the hypothermia group were rapidly cooled by covering their torso with a plastic bag containing ice. temperatures were maintained within set parameters with the use of warming / cooling blankets. intravenous fluids were administered over the next h. blood pressure, heart rate, and temperature were continuously monitored. pao and paco were monitored every h. a second cisternal tap was performed after h of hypothermia / normothermia and ml of csf was withdrawn. a concurrent blood sample was obtained at the time of the second cisterna magnae puncture. neutrophils were isolated as previously described ( ) . csf samples were centrifuged and the cell pellet was resuspended in buffered saline and evaluated for integrin expression. flow cytometry. neutrophils from peripheral blood and csf samples were isolated within min of collection and viability confirmed with trypan blue exclusion. neutrophils were harvested using dextran sedimentation and centrifugation through ficoll hypaque ( ) . peripheral and csf pmn samples were washed, centrifuged, and re-suspended in buffered saline solution to a concentration of × cells / ml. viability of all cells was confirmed by trypan blue exclusion and was consistently greater than %. histologic evaluation using direct hemocytometric observation was performed on all samples to confirm cells were predominantly pmns. cells were incubated with saturating concentrations of monoclonal antibodies directed towards the cd (b ) subunit, cd (b ) subunit, or an activation-dependent epitope located on the ligand recognition domain of the b subunit. pmns isolated from the csf were also stained with isotype matched controls to detect nonspecific binding. cells were then washed, counterstained with a phycoerythrin-conjugated igg antibody and finally fixed with % paraformaldehyde in pbs, ph . . labeled cells were analyzed on a facscaliber flow cytometer (becton dickinson inc., san jose, california). neutrophil populations were identified and gated by forward vs. right angle light scatter characteristics. cells, ( ) per gated determination were measured and mean channel fluorescence was analyzed. using available software, the mean fluorescence of the cell distribution was calculated (facscan  , becton dickinson, mountain view, california). relative fluorescence intensity was defined as the ratio of mean fluorescence of labeled csf pmns (after correction for nonspecific binding) compared to the fluorescence of circulating pmns. total neutrophil counts were performed on blood and csf samples by facscan  analysis. neutrophils were identified by size characteristics and nucleus to cytoplasm ratios, and expressed as number of cells per deciliter. statistical evaluation. all values are expressed as mean ± sem of n observations, where n represents the number of animals. significance is declared for p < . . for comparison of two groups, student's paired t-tests are used. when multiple variables were studied, all data was corrected for multiple comparisons. all statistics were performed using a commercially available statistical program (statview . ; abacus concepts, inc.) on a power g macintosh. the integrin family is strongly conserved across vertebrate species ( ) ( ) ( ) . multiple investigators have used anti-human integrin antibodies in rabbit models of various in vivo and in vitro experiments [ ] [ ] [ ] [ ] [ ] . we have previously shown the antibodies used in this experiment recognize rabbit integrin structures ( , ( ) ( ) . data were collected from animals with group b streptococcal meningitis ( in hypothermia group, in normothermia group). as part of our experiment, we initially evaluated whether hypothermia affects the amount of neutrophil extravasation into the subarachnoid space. animals exposed to hypothermia for h had a csf wbc count of cell / dl while normothermic animals had a csf wbc count of cell / dl. the increase in csf neutrophils did not reach significance between these two groups (p . ). similarly, there was no difference in the absolute neutrophil count in the peripheral circulation between normothermic and hypothermic animals (hypothermia . × cells / dl vs., normothermia . × cells / dl). flow cytometry data is expressed as relative fluorescence intensity (rfi) and is defined as the ratio of mean channel fluorescence of antibody stained-cells compared to fluorescence at baseline. baseline is defined as expression of the specific integrin on circulating neutrophils, prior to induction of meningitis. all samples were corrected for fluorescence secondary to nonspecific binding. we examined whether hypothermia affects total expression of b integrins on csf neutrophils. expression of total cell surface b integrins in normothermic animals increased from a baseline measurement of . to . (p < . ). as is shown in figure , hypothermia significantly decreased total b integrin expression on neutrophils when compared to normothermic animals, with the relative fluorescence intensity decreasing to near baseline levels (rfi . , p < . ). in addition to total surface number, b integrins are able to change their quartinary structure and assume a higher affinity or activated state. this change in affinity modulation can be detected by antibody recognition of a neoepitope expressed on the b integrin ligand recognition domain. as shown in figure , hypothermia sig- integrin expression on spinal fluid neutrophils in normothermic animals increased to . ± . . in animals exposed to h of hypothermia, b integrin expression significantly decreased to . ± . ( * p < . ). relative fluorescence intensity is the ratio of mean channel fluorescence of labeled neutrophils compared to pmns in circulation prior to induction of meningitis. mean channel fluorescence of extravasated pmns is corrected for nonspecific binding prior to analysis. the data represent the means ± sem of animals in each condition. normothermic animals. in animals exposed to hours of hypothermia, b integrin expression decreased to ± , but failed to reach significance (p . ). relative fluorescence intensity is the ratio of mean channel fluorescence of labeled neutrophils compared to pmns in circulation prior to induction of meningitis. mean channel fluorescence of extravasated pmns is corrected for nonspecific binding prior to analysis. the data represent the means ± sem of animals in each condition. nificantly decreased expression of the activation-dependent epitope located on the b integrin and recognized by mab / . relative fluorescence intensity decreased from . to . (p . ). these data suggest that hypothermia decreases total cell surface expression, as well as affinity modulation of the b integrin receptor. the largest percent of the neutrophil integrin population are the cd- (b integrin) receptors. figure compares expression of b integrins on extravasated csf neutrophils. a % decrease in relative fluorescence intensity was noted in extravasated pmns exposed to hypothermic conditions. however, this decrease failed to reach significance (p . ). we also evaluated whether hypothermic treatment of meningitis changes integrin expression on circulating neutrophils. b , activated b , and b integrin expression prior to and following hours of hypothermia did not significantly change on the circulating neutrophil population (table ) . regression analysis was performed on all three integrin groups studied and showed no relationship between changes in integrin expression on peripheral neutrophils and concurrently obtained extravasated neutrophils. integrin expression on circulating neutrophils did not significantly change as a consequence of hypothermia during meningitis. integrin expression on peripheral neutrophils was evaluated by flow cytometric analysis. numbers are expressed as relative fluorescence intensity (rfi) units. relative fluorescence intensity is the ratio of mean channel fluorescence of circulating neutrophils after h of hypothermia or normothermia during meningitis compared to baseline neutrophil fluorescence. baseline expression is defined as integrin expression prior to the induction of meningitis. the data represent the means ±sd of animals. hypothermia has been documented in numerous animal models to decrease pmn accumulation within injured or inflamed brain tissue ( ) ( ) ( ) ( ) . hypothermia decreased intracranial swelling and improved morbidity in severe head trauma ( ) ( ) ( ) . hypothermia also decreased pmn accumulation following ischemia and reperfusion injury ( ) . we have previously shown that hypothermia decreased pmn accumulation within the brain parenchyma during acute bacterial meningitis. in this rabbit model of bacterial meningitis, myeloperoxidase concentration within brain tissue decreased % in the hypothermic group ( ) . the temperature-dependent cellular signal responsible for decreasing pmn accumulation has not been defined. our data suggest that surface integrin expression may be one of the cellular pathways affected by hypothermia. both total and activated b integrin expression on exravasated neutrophils decreased to near baseline levels following hours of hypothermia during meningitis. integrin b expression on extravasated pmns also decreased, but failed to reach significance. in our model, hypothermia does not affect integrin expression on the circulating neutrophil population. the physiologic mechanism(s) that are responsible for the neuro-protective effect of hypothermia are poorly understood. originally, cns hypothermia was believed to preserve the high energy phosphate stores and to decrease cerebral metabolism ( ) . however, the beneficial effects from hypothermia appear to be secondary to suppression of the inflammatory cascade and not to a delay in the injury process ( ) . in our model of group b streptococcal meningitis, hypother-mia decreased the csf concentrations of glutamate and aspartate by - % and caused a significant decrease in the induction of -kda heat shock protein ( ) . in a traumatic brain injury model in rats. whalen et al. demonstrates hypothermia decreased neutrophil accumulation within the injured brain tissue ( ) . this effect was independent of endothelial e-selectin or icam- expression ( ) . our findings suggest one mechanism that may account for the decrease in neutrophil accumulation is the down-regulation of both total and activated b integrin expression on extravasated neutrophils. the beneficial effects induced by hypothermia were not secondary to modification of the degree of infection or bacterial load. there was no difference in the number of positive csf cultures between groups at the end of the observation period. one csf sample from each group grew gbs. the effects of hypothermia on circulating pmns are well described. in a rabbit model of hypothermia, circulating neutrophil counts significantly increased, but did not exhibit increased oxygen burst or phagocytic activity ( ) . in human studies, many of the studies evaluating the effects of hypothermia on granulocyte physiology and adhesion are reported in the cardiopulmonary bypass literature. hypothermia during cardiopulmonary bypass is associated with an initial decrease in circulating pmns, followed by a marked increase that persisted up to hours into the postoperative period ( ) . this effect appears to be independent of hypothermia-induced changes in endothelial-derived adhesion molecules ( ) . neutrophil activation, as measured by serum elastase release, decreases during hypothermic cardiopulmonary bypass ( ) . there are few studies in the literature with regards to the effects of hypothermia on integrin expression. finn et al. reported that in children undergoing cardiopulmonary bypass, a gradual rise in b expression on neutrophils was noted in a small segment of the granulocyte population ( ) . our studies showed no significant change in b integrin expression on circulating neutrophils as a consequence of hypothermia in meningitis. paugam et al. reported that b expression on neutrophils increased within min of cardiopulmonary bypass. however, this rapid increase in b expression was independent of both temperature and interleukin- production ( ). on neutrophils, b integrins facilitate firm binding to activated endothelium. while b integrins on neutrophils are known to function as an extracellular protein adhesion molecule, their role may be more complex. several clinical and laboratory observations suggest b integrin-independent mechanisms may contribute to neutrophil recruitment and activation in infectious states. pmns have been found post-mortem in the alveolar spaces of a patient with b integrin deficiency (leukocyte adhesion deficiency i) ( ) . hepatic sequestration of neutrophils in a rat model of endotoxemia has been shown to be b integrin-independent ( ) . migration of pmn into the alveolar space in both pneumonia and chemical lung injury models is not inhibited by anti-b monoclonal antibody ( , ) . similarly, anti-b integrin monoclonal antibody did not effect mortality nor total csf white blood cell count in a rat model of haemophilus meningitis ( ) . we have previously shown a marked increase in b (total and activated) and b integrin expression on extravasated neutrophils when compared to concurrent expression on circulating neutrophils in gbs meningitis. additionally, b integrin expression on extravasated neutrophils demonstrated a significant correlation with cns tissue pmn sequestration and blood brain barrier dysfunction ( ) . these studies suggest b integrins play an important role in the pathophysiologic consequences of pmn-induced tissue injury. this study was designed to evaluate the effect of short-term hypothermia on in vivo expression of b and b integrins on extravasated csf neutrophils. we have previously shown that b , activated b and b integrins on extravasated neutrophils significantly increase during group b streptococcal meningitis ( ) . we now demonstrate that hypothermia significantly decreases b integrin expression and activation on extravasated neutrophils. this effect is independent of total csf wbc count or alteration in integrin expression on the circulating neutrophil population. in our model of gbs meningitis, we note the absolute value of extravasated neutrophils in hypothermic animals was higher than in normothermic animals, but did not reach statistical significance. we speculate this finding may be secondary to the model design. meningitis was induced hours prior to condition randomization. animals randomized to hypothemic conditions demonstrated down-regulation of b integrin number and function. integrin b on neutrophils are known to bind to extracellular proteins, thereby allowing directed migration. thus, extravasated neutrophils in these animals may have impaired chemotaxis and sequester within csf. alteration in integrin expression on extravasted neutrophils during hypothermia is a descriptive finding. the exact cellular signaling mechanism(s) affected by hypothermia is uncertain. multiple pro-inflammatory chemokines are produced during bacterial meningitis. csf concentrations of tnfa, il- , groa, mcp- , mip- a and mip- b all increase early in the time course of meningitis ( ) ( ) . these cytokines activate leukocytes and cause an increase in adhesion molecule expression. additional studies to examine whether hypothermia affects the levels of these cytokines and whether they correlate with changes in expression of b integrins are ongoing. the relevance of integrin expression on neutrophils in specific infections is still under investigation. as pmns perform the majority of their phagocytic functions outside the circulation, they must adhere to extracellular matrix (ecm) macromolecules in a regulated, reversible fashion in order to crawl through the tissue to the site of inflammation or infection. better understanding the function of neutrophil integrin expression at sites of acute inflammation and the effects of hypothermia may suggest novel approaches to control the cascade of pmn-mediated pathophysiologic consequences associated with such processes as septic shock, ards, or the poor sequelae from meningitis. neutrophil activation and tissue neutrophil sequestration in a rat model of thermal injury alveolar epithelial barrier and acute lung injury a role for the beta integrin cd b in mediating experimental lung injury in mice neutrophils contribute to ischemia-reperfusion injury in rat liver in vivo inhibition of leukocyte rolling with polysaccharide fucoidin prevents pleocytosis in experimental meningitis in the rabbit bacterial meningitis: mechanisms of disease and therapy neutrophil mediated damage to isolated myocytes after anoxia and reoxygenation reactive oxygen intermediates contribute to necrotic and apototic neuronal injury in an infant rat model of bacterial meningitis due to group b streptococcus new antibodies as adjunctive therapies for gram positive bacterial meningitis a monoclonal antibody to the adherence-promoting leukocyte glycoprotein cd- reduces organ injury and improves survival from hemorrhagic shock and resuscitation in rabbits enhanced attenuation of meningeal inflammation and brain edema by concomitant administration of anti-cd monoclonal antibodies and dexamethasone in experimental haemophilus meningitis structure and function of the leukocyte adhesion molecules cd / cd divalent cation substitution reveals cd -and very late antigen-dependent pathways that mediate human pmn adherence to fibronectin human neutrophil adherence to laminin in vitro. evidence for a distinct neutrophil integrin receptor for laminin neutrophils can adhere via a b -integrin under flow conditions a novel beta -dependent adhesion pathway on neutrophils: a mechanism invoked by dihydrocytochalasin b or endothelial transmigration polymorphonuclear leukocyte migration through human dermal fibroblast monolayers is dependent on both beta- integrin (cd / cd ) and beta- integrin (cd ) mechanisms the b integrin, very late activation antigen- on human neutrophils can contribute to neutrophil migration through connective tissue fibroblast barriers measles virus-induced changes in leukocyte function antigen expression and leukocyte aggregation: possible role in measles virus pathogenesis the integrin vla- binds echovirus and extracellular matrix ligands by different mechanisms mycobacterium avium-m. intracellulare binds to the integrin receptor alpha v beta on human monocytes and monocyte-derived macrophages alpha integrin directs virus-activated cd + t cells to sites of infection hiv- infection of human t lymphocytes results in enhanced alpha beta integrin expression quantitative study of beta- integrin expression and fibronectin interaction profile of t lymphocytes in vitro infected with hiv the relationship between brain temperature and neutrophil accumulation after traumatic injury in rats the effect of brain temperature on acute inflammation after traumatic brain injury in rats importance of posttraumatic hypothermia and hyperthemia on the inflammatory response after fluid percussion brain injury: biochemical and immunocytochemical studies posttraumatic hypothermia reduces polymorphonuclear leukocyte accumulation following spinal cord injury in rats hypothermia in the treatment of severe bacterial meningitis hypothermia decreases excitatory neurotransmitter release in bacterial meningitis in rabbits integrin expression on neutrophils in a rabbit model of group b streptococcal meningitis alpha beta integrin-dependent cell adhesion is regulated by a low affinity receptor pool that is conformationally responsive to ligand integrins and other cell adhesion molecules antibodies to conserve cytoplasmic domain of the integrin beta- subunit react with proteins in vertebrates, invertebrates, and fungi molecular evolution of integrins: genes encoding integrin beta subunits from a coral and a sponge hu f g, an antibody recognizing the leukocyte cd / cd integrin, reduces injury in a rabbit model of transient focal cerebral ischemia monoclonal antibody against icam- and cd- attenuate cerebral vasospasm after experimental subarachnoid hemorrhage in rabbits a mab to the beta -leukocyte integrin mac- reduces intimal thickening after angioplasty or stent implantation in rabbits involvement of alpha beta integrin in matrix interactions and proliferation of chondrocytes role of endothelin in the pathophysiology of renal ischemia-reperfusion in normal rabbits role of beta integrins on csf neutrophils in a rabbit model of meningitis hypothermia attenuates b integrin expression on neutrophils in an animal model of meningitis marked protection by moderate hypothermia after experimental traumatic brain injury post-traumatic brain hypothermia reduces histopathologic damage following concussive brain injury in the rat a phase ii study of moderate hypothermia in severe brain injury mild hypothermia during reperfusion reduces injury following ischemia of the rabbit ear effect of body temperature change on cerebral oxygen consumption of the intact monkey treatment of traumatic brain injury with moderate hypothermia responses of neutrophils and lymphocytes in cold stress: effects of nonsteroid anti-inflammatory drugs changes in leukocyte count and soluble intercellular adhesion molecule- and e-selectin during cardiopulmonary bypass in children hypothermic versus normothermic cardiopulmonary bypass: influence on circulating adhesion molecules influence of temperature on neutrophil trafficking during clinical cardiopulmonary bypass changes in neutrophil cd b / cd and l-selectin expression and release of interleukin and elastase in paediatric cardiopulmonary bypass neutrophil expression of cd b / cd and il- secretion during northmothermic cardiopulmonary bypass leukocyte adhesion deficiency: clinical and postmortem observations sequestration of neutrophils in the hepatic vasculature during endotoxemia is independent of b integrins and intercellular adhesion molecule- cd -dependent and independent mechanisms of neutrophil emigration in the pulmonary and systemic microcirculation of rabbits disparate role of the b -integrin cd in the local accumulation of neutrophils in pulmonary and cutaneous inflammation in the rabbit anti-cd b monoclonal antibody in an infant rat model of haemophilus influenzae type b sepsis and meningitis tumor necrosis factor-alpha contributes to apoptosis in hippocampal neurons during experimental group b streptococcal meningitis chemokines and chemotaxis of leukocytes in infectious meningitis acknowledgments-this work was supported in part by a grant in aid from the children's medical center research foundation (#a ). key: cord- -le mc authors: liu, yong-juan; shao, li-hua; zhang, jian; fu, shan-ji; wang, gang; chen, feng-zhe; zheng, feng; ma, rui-ping; liu, hai-hong; dong, xiao-meng; ma, li-xian title: the combination of decoy receptor and soluble triggering receptor expressed on myeloid cells- for the diagnosis of nosocomial bacterial meningitis date: - - journal: ann clin microbiol antimicrob doi: . /s - - - sha: doc_id: cord_uid: le mc background: early diagnosis and appropriate antibiotic treatment can significantly reduce mortality of nosocomial bacterial meningitis. however, it is a challenge for clinicians to make an accurate and rapid diagnosis of bacterial meningitis. this study aimed at determining whether combined biomarkers can provide a useful tool for the diagnosis of bacterial meningitis. methods: a retrospective study was carried out. cerebrospinal fluid (csf) levels of decoy receptor (dcr ) and soluble triggering receptor expressed on myeloid cells- (strem- ) were detected by enzyme-linked immunosorbent assay (elisa). results: the patients with bacterial meningitis had significantly elevated levels of the above mentioned biomarkers. the two biomarkers were all risk factors with bacterial meningitis. the biomarkers were constructed into a “bioscore”. the discriminative performance of the bioscore was better than that of each biomarker, with an area under the receiver operating characteristic (roc) curve (auc) of . ( % confidence intervals (ci) . – . ; p< . ). conclusions: combined measurement of csf dcr and strem- concentrations improved the prediction of nosocomial bacterial meningitis. the combined strategy is of interest and the validation of that improvement needs further studies. nosocomial bacterial meningitis is a significant problem among hospitalized patients, which threatens patients' lives, extends their stay in hospital and increases the medical costs, sometimes even results in doctor-patient conflicts. despite emergence of antibiotics and improvement of clinical techniques, bacterial meningitis continues to be a significant cause of morbidity and mortality [ , ] . early effective antibiotic therapy is associated with better outcomes [ , ] and the choice of appropriate antibiotics is greatly influenced by the final identification of the specific pathogen. however, the diagnosis of this disease remains a challenge. firstly, the clinical signs of fever, headache, neck stiffness and mental status alteration are not specific [ , ] . secondly, laboratory tests are also not specific enough. cerebrospinal fluid (csf) culture is specific but lacks sensitivity, especially with previous use of antibiotics [ ] . in addition, the unnecessary use of antibiotics increases the chance of being infected by multidrug-resistant bacteria [ ] . based on the above reasons, the development of new indicators for the rapid diagnosis of bacterial meningitis is desirable. decoy receptor (dcr ), a soluble decoy receptor in the tnf receptor (tnfr) family, is capable of inhibiting apoptosis by neutralizing the activities of the following three members of the tnf superfamily: fas ligand (fasl) [ ] , light [receptor homologous to lymphotoxins exhibits inducible expression, competes with herpes simplex virus (hsv) glycoprotein d for hvem and is expressed by t lymphocytes] [ ] and tnf-like molecule a (tl a) [ ] . in addition, dcr can be a new potential biomarker for inflammatory disease, autoimmune diseases and cancer [ ] [ ] [ ] [ ] [ ] . our previous study has proved that levels of dcr are significantly elevated in patients with bacterial meningitis and it may act as a useful biomarker of bacterial meningitis [ ] . the triggering receptor expressed on myeloid cells- (trem- ) is a recently described receptor on the surfaces of monocytes and neutrophils [ ] . many previously published studies have indicated that the soluble form of trem- , called strem- , is a useful indicator for bacterial infection [ ] [ ] [ ] [ ] . indeed, several studies have suggested that strem- is valuable for the diagnosis of bacterial meningitis [ , ] before a positive result of csf culture. these data show the individual predictive value of these two biomarkers. combination of several biomarkers can improve diagnostic value in various diseases or conditions: such as sepsis [ ] , liver fibrosis [ ] , community-acquired pneumonia [ ] and severe malaria [ ] . however, the strategy of combined dcr and strem- has not yet been validated for bacterial meningitis. the aim of this study was to evaluate the combined diagnostic value of dcr and strem- . diagnosis of bacterial meningitis was based on a positive result of csf culture. nosocomial meningitis was defined as negative bacterial infection when the patient was admitted to the hospital, clinical evidence of an infection was found after hours on admission or within one month after discharge from the hospital where the patient had received an invasive neurosurgical procedure. otherwise, the patient was considered to have community-acquired meningitis [ , ] . external ventricular drain-related meningitis was diagnosed with bacterial infection being found within days of external ventricular drain removal [ ] . one hundred and twenty-three patients in this study were enrolled in qilu hospital of shandong university in china within november and october . all patients in this study were categorized into two groups. inclusion and exclusion criteria are described more extensively elsewhere. the mean age (mean ± sd) of patients with bacterial meningitis was . ± . years. the area under the receiver operating characteristic (roc) curve (auc) of dcr was . [ ] . the biomarkers associated with this study were csf dcr and strem- . committee of qilu hospital of shandong university (no. kyll- - ). written informed consents were taken from patients or clients before registration. the csf samples were centrifuged and supernatants were frozen at − °c until assay. levels of strem- and dcr in csf were determined in duplicate by elisa according to the kits instructions (cusabio, wuhan, china). the detection limits were . pg/ml for strem- and . ng/ml for dcr . descriptive results of strem- and bioscore were expressed as median ( th and th percentiles) and mann-whitney u-test was used to analyze the continuous data of strem- and bioscore. roc curve and auc were applied to evaluate the discriminatory power of each marker and combined csf markers. the youden index (j = max (sensitivity + specificity − )) was used to define the cut-off value. risk factors related to bacterial meningitis were explored by multivariate stepwise logistic regression analysis. hosmer-lemeshow goodness-of-fit test was used for the calibration of logistic regression model. all analyses were performed with spss, version . and a two-sided p < . was considered to be statistically significant. among the patients recruited in this study, patients were diagnosed with bacterial meningitis [ ] . in patients with bacterial meningitis, patients had external ventricular drain-related meningitis. the levels of csf dcr were significantly different between the groups of bacterial meningitis and non-bacterial meningitis. patients received antibiotic > h before csf sampling, among them patients with non-bacterial meningitis and patients with bacterial meningitis [ ] . concentrations of strem- in the patients with bacterial meningitis were significantly higher than those with non-bacterial meningitis ( . ( - . ) pg/ml vs. ( - ) pg/ml, p < . ). as shown in table , predisposing conditions were present in all patients, such as neurosurgery, pneumonia, csf leak, etc. all the patients with bacterial meningitis underwent neurosurgical operation (tables and ). the most frequently isolated microorganism was coagulasenegative staphylococci (table ). more than half of the patients (n= ) had intracranial tumor surgery. the conditions necessitating surgical interventions were shown in table . evaluation of strem- and dcr in discriminating bacterial meningitis from non-bacterial meningitis roc curve and auc were performed to determine the discriminative accuracy of csf strem- and dcr . combined with the published data, csf dcr yielded a higher discriminative value with an auc of . [ ] . the auc of strem- for predicting bacterial meningitis was . ( % confidence intervals (ci) . - . ; p < . , figure ) . a cut-off value of . pg/ml for strem- had a sensitivity of . % ( ci . % - . %), a specificity of . % ( % ci . % - . %), a positive likelihood ratio (plr) of . ( % ci . - . ), a negative likelihood ratio (nlr) of . ( % ci . - . ), a positive predictive value (ppv) of . % ( % ci . %- . %) and a negative predictive value (npv) of . % ( % ci . %- . %). as shown in table , csf leucocyte count yielded the best discriminative value, with an auc of . , a sensitivity of . % and a specificity of . %. multivariate stepwise logistic regression was applied to evaluate the independent predictors for bacterial meningitis. from univariate analysis, the following variables: csf leucocyte count, glucose, protein, lactate, dcr and strem- were associated with bacterial meningitis ( table ). all the six markers were taken into a multivariate stepwise logistic regression model. the result showed that only strem- and dcr were the independently risk factors with bacterial meningitis (odds ratio (or) = . , % ci = . - . , p = . for dcr ; or = . , % ci = . - . , p = . for strem- ; table ). in order to determine whether combined detection of the markers mentioned above could improve diagnostic accuracy, the biomarkers were combined into a "bioscore". for each biomarker, individual data were scored as or . if the data were higher than the critical value, the scores were recorded as ; and if the data were below the cutoff point, the scores were recorded as . the scores constituted the cumulative bioscore, which ranged between and . the biomarker score was significantly elevated for patients with bacterial meningitis ( ( - ) vs. ( - ), p < . ). roc curve was constructed as above for the bioscore. the auc for bioscore was . ( % ci . - . ; p< . ; figure ). when the bioscore was entered into the multivariate stepwise logistic regression model, the bioscore was also proved to be a significant factor for bacterial meningitis (or, . , % ci . - . ; p< . , table ). the possibility of being infected by bacteria grew with the increasing bioscore. the rate of bacterial meningitis ranged from . % for a bioscore of to . % for a bioscore of . according to the bioscore, the patients were divided into three groups. of patients ( . %) with a bioscore of or were diagnosed with bacterial meningitis (figure ). combination of several markers has been found to improve predictive value in various disorders [ ] [ ] [ ] [ ] . in this study, we combined the markers of dcr and strem- into a simple score, named as "bioscore", which was proved to be a useful predictor for the diagnosis of bacterial meningitis. furthermore, compared with the individual marker of dcr or strem- , the bioscore was found to be a better predictor of bacterial meningitis. except its neutralizing effects on fasl, light, and tl a, dcr also acts as a pleiotropic effector which can regulate cellular function via "non-decoy" activities [ ] . in addition, dcr may have pro-inflammatory functions [ , ] . in ards patients, higher plasma dcr levels are associated with multiple-organ dysfunction, longer duration of icu stay and ventilator dependence. serum dcr levels in non-survivors are higher than those in survivors regardless of the apache ii scores. therefore, dcr appears to possess the potential to serve as both a diagnostic and a prognostic marker of ards [ ] . moreover, levels of dcr are elevated in patients with figure receiver operating characteristic (roc) curves of dcr and strem- for the diagnosis of bacterial meningitis. areas under the roc curve (auc) are presented with % confidence intervals (ci). the auc of dcr has been published previously [ ] . in order to compare the auc of strem- with that of dcr , the figure of dcr is presented here. dcr : . ( % ci . - . ); strem- : . ( % ci . - . ). the performance of dcr and other csf markers has been published previously [ ] . in order to compare the diagnostic performance of the two biomarker with that of other csf markers, the data of dcr and csf markers are presented here. roc: receiver operating characteristic. sepsis and it may act as a clinically important biomarker of sepsis. dcr might be considered as a double-edged sword in sepsis and have the potential to be a novel target for the treatment of sepsis [ , ] . our previous study has showed that detection of csf dcr is useful for the diagnosis of bacterial meningitis. a cut-off point of . ng/ml is established, which has a sensitivity of . % and a specificity of . % [ ] . thus, similar to sepsis, dcr might turn out to be a novel target of bacterial meningitis. further studies are needed to determine the confirmatory role of abnormally high level of dcr in csf of patients with bacterial meningitis. as previously published studies [ , ] , strem- was also found significantly increased in patients with bacterial meningitis in present study. however, there are some discrepancies between our data and previously published data. csf strem- has a diagnostic auc of . , a sensitivity of % and a specificity of % [ ] . sensitivity of the study by bishara j and coworkers is . %, while the specificity is as high as % [ ] . however, the sensitivity of strem- in our study was poorer, only with a sensitivity of . %. several potential explanations for these discrepancies were as follows:the first explanation may be the difference of enrolled patients. the population enrolled in this study only included patients with nosocomial bacterial meningitis. the study by determann rm et al. has patients, among them patients being diagnosed with bacterial meningitis, with viral meningitis and healthy donors. only patients with community-acquired bacterial meningitis are recruited [ ] . another study by bishara j and coworkers has two groups and only has patients, including patients with a positive culture and patients with a negative culture, which is much less than that of this study. moreover, the study doesn't have the limit of wbc count [ ] . another important explanation might be technical. except for the elisa kits from different manufacturers, the sensitivity was different, which might lead to a lower cut-off value of strem- in present study. in addition to the individual diagnostic accuracy of each biomarker, the combination of dcr and strem- into a bioscore appeared to be an efficient and practical way to discriminate patients with bacterial meningitis from patients with non-bacterial meningitis. the or of biscore was . . the same as other disorders or diseases, such as sepsis and severe malaria [ , ] , the higher the score was recorded, the greater the possibility of being infected by bacteria was determined. bioscore could yield . % of patients with bacterial meningitis. with at least one of the two biomarkers higher than the critical point (bioscore ≥ ), about % of patients were found to be infected by bacteria. nonetheless, with a bioscore of , . % of patients could not be excluded. in addition, because serum of patients in this study were not collected, predictive value of combined bioscore in blood for bacterial meningitis was not determined. further studies are required to validate the potential value of combined bioscore in serum for the diagnosis of bacterial meningitis. currently, csf culture is still the golden standard for the diagnosis of bacterial meningitis. early diagnosis of bacterial meningitis is still a challenging problem for clinicians. in this study, although the negative predictive value of bioscore was not satisfactory, a strong positive predictive value was found if the bioscore was above . it seemed likely that the combined bioscore could strengthen the clinicians' decisions besides clinical work-up. our study had two major limitations. ( ) this retrospective study included unsatisfactorily sample size and firm conclusions were not concluded. the selection of non-consecutive samples might have resulted in a selection bias. predictive value of combined dcr and strem- needs validation in larger-scale prospective studies. ( ) only bacterial culture was used to determine the presence of bacterial meningitis. %- % of patients with bacterial meningitis are with negative results of bacterial culture [ ] . therefore, some patients with non-bacterial meningitis might be misclassified and limit the application of our findings. in conclusion, this retrospective study demonstrated that combination of dcr and strem- in csf could yielded a better diagnostic value for nosocomial bacterial meningitis than that of each biomarker. whether the bioscore be applied routinely in clinic needs further studies. clinical features and prognostic factors in adults with bacterial meningitis nosocomial bacterial meningitis inappropriately treated nosocomial meningitis is associated with higher bm: bacterial meningitis; non-bm: non-bacterial meningitis klebsiella pneumoniae (n= ), bacillus typhi suis (n= ), a mix infection of coagulase-negative staphylococci and acinetobacter baumannii (n= ) acinetobacter baumannii (n= ) and pseudomonas oryzihabitans (n= ). mortality and more neurologic sequellae than appropriately treated meningitis acute bacterial meningitis in children admitted to a rural kenyan hospital: increasing antibiotic resistance and outcome genomic amplification of a decoy receptor for fas ligand in lung and colon cancer a newly identified member of tumor necrosis factor receptor superfamily (tr ) suppresses lightmediated apoptosis tl a is a tnf-like ligand for dr and tr /dcr and functions as a t cell costimulator decoy receptor is highly expressed in patients with rheumatoid arthritis decoy receptor : a pleiotropic immunomodulator and biomarker for inflammatory diseases, autoimmune diseases and cancer serum decoy receptor , a potential new biomarker for sepsis decoy receptor , a novel inflammatory marker, and mortality in hemodialysis patients specific elevation of dcr in sera of sepsis patients and its potential role as a clinically important biomarker of sepsis predictive value of decoy receptor in postoperative nosocomial bacterial meningitis cutting edge: inflammatory responses can be triggered by trem- , a novel receptor expressed on neutrophils and monocytes soluble triggering receptor expressed on myeloid cells and the diagnosis of pneumonia soluble triggering receptor expressed on myeloid cells : a biomarker for bacterial meningitis soluble triggering receptor expressed on myeloid cells- for distinguishing bacterial from aseptic meningitis in adults diagnostic value of the soluble triggering receptor expressed on myeloid cells- in bacterial infection: a meta-analysis combination biomarkers to diagnose sepsis in the critically ill patient improving estimation of liver fibrosis using combination and newer noninvasive biomarker scoring systems in hepatitis c-infected haemophilia patients biomarkers improve mortality prediction by prognostic scales in community-acquired pneumonia combinations of host biomarkers predict mortality among ugandan children with severe malaria: a retrospective case-control study clinical characteristics and prognosis of acute bacterial meningitis in elderly patients over : a hospital-based study clinical characteristics and therapeutic outcomes of nosocomial super-infection in adult bacterial meningitis validation of an automated surveillance approach for drain-related meningitis: a multicenter study enhanced adhesion of monocytes via reverse signaling triggered by decoy receptor decoy receptor increases monocyte adhesion to endothelial cells via nf-kappa b-dependent up-regulation of intercellular adhesion molecule- , vcam- , and il- expression submit your next manuscript to biomed central and take full advantage of: • convenient online submission • thorough peer review • no space constraints or color figure charges • immediate publication on acceptance • inclusion in pubmed, cas, scopus and google scholar • research which is freely available for redistribution thanks are given to the patients enrolled in this study. the authors declare that they have no competing interests. key: cord- - px aq authors: griese, matthias; zarbock, ralf; costabel, ulrich; hildebrandt, jenna; theegarten, dirk; albert, michael; thiel, antonia; schams, andrea; lange, joanna; krenke, katazyrna; wesselak, traudl; schön, carola; kappler, matthias; blum, helmut; krebs, stefan; jung, andreas; kröner, carolin; klein, christoph; campo, ilaria; luisetti, maurizio; bonella, francesco title: gata deficiency in children and adults with severe pulmonary alveolar proteinosis and hematologic disorders date: - - journal: bmc pulm med doi: . /s - - - sha: doc_id: cord_uid: px aq background: the majority of cases with severe pulmonary alveolar proteinosis (pap) are caused by auto-antibodies against gm-csf. a multitude of genetic and exogenous causes are responsible for few other cases. goal of this study was to determine the prevalence of gata deficiency in children and adults with pap and hematologic disorders. methods: of patients with gm-csf-autoantibody negative pap, had no other organ involvement and had some form of hematologic disorder. the latter were sequenced for gata . results: age at start of pap ranged from . to years, patients were children. in half of the subjects gata -sequence variations were found, two of which were considered disease causing. those two patients had the typical phenotype of gata deficiency, one of whom additionally showed a previously undescribed feature – a cholesterol pneumonia. hematologic disorders included chronic myeloic leukemia, juvenile myelo-monocytic leukemia, lymphoblastic leukemia, sideroblastic anemia and two cases of myelodysplastic syndrome (mds). a year old child with mds and digeorge syndrome type was rescued with repetitive whole lung lavages and her pap was cured with heterologous stem cell transplant. conclusions: in children and adults with severe gm-csf negative pap a close cooperation between pneumologists and hemato-oncologists is needed to diagnose the underlying diseases, some of which are caused by mutations of transcription factor gata . treatment with whole lung lavages as well as stem cell transplant may be successful. pulmonary alveolar proteinosis (pap) is a rare disorder characterized by the progressive accumulation of surfactant in the alveoli of the lungs, leading to hypoxemic respiratory failure and, in severe cases, to death [ ]. pap is caused by (i) genetic diseases which result in dysfunction of surfactant or surfactant production (sftpc, sftpb, abca , ttf deficiency) mainly presenting during infancy, by (ii) disruption of gm-csf signaling from mutations in the receptor (gm-csfra, gm-csfrb) or from acquired autoantibodies against gm-csf, and by (iii) disorders that presumably impair surfactant clearance because of abnormal numbers or defective phagocytic functions of alveolar macrophages [ ] . the latter are caused by inhaled particles or by hematologic disorders affecting macrophage precursors. a broad spectrum of hematologic disorders have been associated with pap, most frequently myelodysplastic syndrome (mds), and more rarely acute (aml) and chronic myeloid leukaemia (cml), myelofibrosis, acute lymphoid leukaemia (all), adult t-cell leukaemia, aplastic anemia, lymphoma, multiple myeloma, plasmacytoma, essential thrombocytosis [ ] , congenital dyserythropoietic anemia [ ] , and status after unrelated stem cell transplant [ , ] . up to now only mutations in one specific gene -gata have been associated with this form of pap. gata is a zinc finger transcription factor essential for differentiation of immature hematopoietic cells [ ] . among many other functions gata regulates the phagocytosis of alveolar macrophages [ ] . alveolar macrophages treated with the sense gata- expression construct show an increase in their phagocytic activity by up to % compared to the antisense construct [ ] . gata deficiency is a recently described disorder of hematopoiesis, lymphatics, and immunity, caused by heterozygous mutations leading to haplo-insufficiency of the transcription factor gata . the disease presents with a complex array of diagnoses and symptoms of varying extent including mds, aml, chronic myelomonocytic leukemia (cmml), severe viral, disseminated mycobacterial and invasive fungal infections, pulmonary arterial hypertension, warts, panniculitis, human papillomavirus (hpv) positive tumors, epstein-barr virus (ebv) positive tumors, venous thrombosis, lymphedema, sensorineural hearing loss, miscarriage and hypothyroidism [ ] . pap has been reported in about % of all subjects with gata deficiency [ , ] . however, not much is known on the severity and clinical course of pap in these subjects, which were exclusively adults. pneumologists treating patients with severe pap not caused by autoantibodies against gm-csf frequently do not know the cause of these rare conditions [ ] . here, we investigate a cohort of children and adults with severe and chronic pap and hematologic disease for the presence of gata mutations. gata mutations were identified in a minority of patients only. one child with a gata variant was successfully treated initially by therapeutic whole lung lavages (wll) and eventually by stem cell transplant. all subjects with pap diagnosed based on the characteristic phenotype by lung biopsy or bronchoalveolar lavage (bal) and ct scan findings were identified from the kids-lung-register (http://www.kinderlungenregister.de/ index.php/en/) within the child-eu project (european register and biobank on childhood interstitial lung diseases, european commission, fp , ga ). in order to differentiate subgroups of pap, all patients with gm-csf-ra or rb mutations [ ] and a high gm-csf autoantibody level were excluded. subjects were identified of whom ( adults, children) had pulmonary involvement only. subjects suffered from pap and a hematologic disease -diagnosed by standard proceduresand were selected for further analysis (fig. ) . the gata gene was analysed by sanger sequencing. genomic dna was isolated from edta blood samples using the qiaamp dna blood mini kit (qiagen) and pcr amplified using hotstartaq polymerase (qiagen). pcr products were purified with the minelute uf pcr purification kit (qiagen). primers used for pcr and sequencing reactions are shown in table . sequencing was performed by gatc biotech ag (konstanz, germany). in subject ng of genomic dna extracted from formalin fixed tissue were fragmented to an average size of bp and desired fragment sizes were obtained with agencourt® ampure® xp beads (beckman coulter). sequencing libraries were prepared using accel-ngs- s library kit (swift biosciences, ann harbor, mi), sequenced on the hiseq (illumina) and aligned to the human genome assembly (hg ). the gata locus was covered by reads. follow-up data of the patients were collected until october . the study was approved by the institutional review board, the ethics commission of the medical faculty of the ludwig-maximilians university, munich, germany (ek - ). all parents or guardians gave their written informed consent, the children gave assent. age at onset of pap ranged from . to years. half of the patients were children (table ). beside chronic respiratory insufficiency due to pap, all patients suffered from recurrent respiratory tract infections or exacerbations. these were mainly common cold viral infections, in some cases non-tuberculous mycobacteria, herpes simplex virus (hsv), and cytomegalovirus (cmv) were identified. all patients were immunodeficient: in three patients primary immunodeficiency syndromes were identified: two with a monocyte deficiency not further specified (no. , ) and one with a di george type ii syndrome with combined table sequences of primers used for amplification of the gata gene exon forward reverse # ccc gca aag tga tgt cg caa acg gac caa gcg att c # acc tcg tgg tgg gac ttt g gat cct aca tcc ggg the hallmark of all patients of this study was severe pap with continuous need of oxygen (table ). all but two subjects (no. , ) were treated symptomatically with repetitive therapeutic wll to improve respiratory insufficiency. treatment was eventually not successful in patients who died from respiratory failure, complicated by infection or acute respiratory distress syndrome (ards). autoantibodies against gm-csf were determined in order to characterize pap further, they were negative in all patients (table ) . serum levels of gm-csf were not significantly increased in the patients with serum available. of the patients had sequence anomalies in gata ( table ) . two of these, a synonymous variant and a missense variant were predicted not to be damaging by polyphen- [ ] and sift [ ] . the other two were heterozygous point mutations predicted to be deleterious, both of these patients had a phenotype characteristic for gata deficiency. the courses of cases , , and are reported in more detail to illustrate characteristic presentations. a year old male patient presented with disseminated mycobacterium avium intracellulare infection, including the lungs, chronic labial herpes including a positive serum hsv pcr, and m. bowen of the skin. cellular immunodeficiency was suspected based on absent monocytes; the patient was hiv-negative. years later the patient developed respiratory insufficiency, the ct scan showed consolidations suspicious of cryptogenic organizing or atypical pneumonia. several courses of antibiotic therapy were followed by two pulses of cyclophosphamide; the patient deteriorated further, ct scan showed bilateral interstitial thickening and crazy-paving pattern. open lung biopsy revealed pap. bone marrow aspiration was normal. he was treated symptomatically with wll under ecmo (extracorporeal membrane oxygenation) support, developed anuric acute renal failure, and died from acute cardiac failure. the patient carried the mutation p.y d in the gata gene. a year old female patient presented with recurrent bronchitis and sinusitis, breathlessness during exercise, and interstitial markings on chest x-ray. an open lung biopsy was performed, histology revealed pap associated with cholesterol pneumonia. liver and bone marrow biopsies were normal. in the following months she developed progressive peripheral edema and cardiac insufficiency with mild pulmonary hypertension. she further developed a pseudomembranous glomerulonephritis with nephrotic syndrome and a pseudomonas urosepsis at years. at years cellular immunodeficiency was demonstrated based on the absence of monocytes. she developed condylomatous lesions of the vulva and hpvassociated (types and ) vulva intraepithelial neoplasia grade - (vin -vin ), which was resected. her course was determined by chronic respiratory failure (at rest lo /min; pao mmhg (normal > ), normal pac ). however, no wll were performed as the patient was lost on follow up at years. the patient carried the mutation p.r w in the gata gene. case no. a . year old girl with di george syndrome type ii from deletion of p (omim ) with severe immunodeficiency, single right kidney, inner ear deafness, [ ] , with good clinical and radiological response over a period of one year (fig. ) . a hla-identical ( / ) unrelated-stem cell donor was identified at the age of years and she received allogeneic stem cell transplant. within a few weeks after transplant, pap and respiratory distress resolved (fig. ) . a full-term boy with normocytic anemia (hb . g/dl) presented after a few weeks with paleness, fatigue, tachydyspnea, and failure to thrive. at the age of four month the hemoglobin level had decreased to . g/dl and a sideroblastic anemia was diagnosed. no genetic cause was identified; pearson syndrome and mutations in alas and slc a were excluded. his bone marrow showed massive erythroid hyperplasia with dyserythropoiesis and marked depression of myeloid cells. pulmonary infiltrates were initially interpreted as pneumonia. at the age of months a cmv infection was successfully treated with ganciclovir. at months he developed respiratory failure (pao mmhg, paco mmhg) with bilateral pulmonary infiltrates. the diagnosis of pap was made by analysis of bal fluid and open lung biopsy. from the age to months the boy underwent therapeutic wlls, improving his general condition and gas exchange. currently he maintains oxygen-saturation levels above % under oxygen support . - l of o /min). the anemia is stable with hb levels between . and . g/dl. the molecular cause of very rare sideroblastic anemia was not revealed, the associated depression of myeloid cells was likely the cause of pap. gata deficiency was not causal, as the patient showed no gata variant in our investigations; potential intronic variations were not investigated. this study shows that gata mutations in patients with hematologic diseases and severe pap occur at a relatively low frequency. gata analysis may help to diagnose the underlying hematologic condition. severe pap in children due to mds can be cured by stem cell transplant. severe pap is a rare but serious complication of hematologic disorders. here we differentiated two groups of diseases having in common a presentation with significant chronic respiratory insufficiency and recurrent pulmonary tract infections or exacerbations. these groups included (i) patients with gata deficiency, a protean disorder of hematopoiesis, lymphatics, and immunity [ ] , and (ii) patients with functionally or numerically reduced alveolar macrophages and/or their respective mononuclear precursors due to cml, jmml, call, mds or sideroblastic anemia in the absence of disease causing gata mutations. gata deficiency has a broad phenotype encompassing immunodeficiency, mds/aml, pulmonary disease, and vascular/lymphatic dysfunction. a precise history and knowledge of the disease course may help to select potential candidates with high confidence for specific genetic diagnostic. gata gene mutations were associated with pap in two cases; p.y d is a novel missense mutation demonstrated here to present with the classical mono-mac syndrome [ ] . p.r w has previously been described in six patients, of whom had a pap [ ] . here we add the histopathological feature of cholesterol pneumonia to this phenotype. of interest, further heterozygous variations in gata were identified in two other subjects; however these were predicted to be non-disease causing ( table ) . as not all subjects with a specific gata mutation causing gata deficiency develop pap [ ] , additional factors are involved which determine pap. patients with the same gata mutation need to be investigated further and experimental models will help to better understand the molecular defect(s) leading to pap. similarly, in non-gata deficient patients with pap and hematologic abnormalities, the mechanism of pap development is unknown. currently, their pap is presumed to be due to reduced monophagocytic function in the alveoli to clear surfactant [ ] . best proof for the critical role of bonemarrow derived alveolar macrophages for surfactant homeostasis is provided by the correction of pap by sct. this was shown here for the first time in a young child. shortly, i.e. within days after take of sct, there was clearing of pap, superseding rapidly the need for therapeutic wlls (fig. ) . here we also show that wlls are feasible also for children, even at very young age, with small sized airways and in the presence of severe respiratory distress, using techniques we have established previously [ ] . wll can be used to symptomatically treat all types of pap and to bridge until the underlying condition can be cured or alternative treatments have been implemented. due to relative immune deficiency state and irrespective of the cause, the impact of infections should be considered fig. long term course of a child suffering from digeorge syndrome type ii and pap due to mds with monosomy , trisomy , and a gata missense variant. successful treatment by therapeutic wlls, and definitive treatment of the pap by sct. (a) clinical course (b,c) ct at presentation (d,e) ct after first whole lung lavages (f) cxr before sct (g) cxr weeks after sct and (h) cxr year after sct carefully. it is likely that with often severe respiratory tract infections pap may be triggered to deteriorate. thus we recommend early diagnosis and proper antimicrobial treatment, in particular of organisms characteristic for these often lethal conditions, including mycobacteria, nocardia, herpes viruses, and fungi [ , , ] . in addition and concordant with the immune deficiency the extensive usage of systemic corticosteroids should be cautioned, as there is so far no evidence of their beneficial effect in pap. extensive usage might enhance immune deficiency and weaken antimicrobial defense further [ ] . after confirming the diagnosis of pap either by a combination of characteristic ct scan and bal findings and trans-bronchial or open lung biopsy, it is important to determine the etiology of pap. as more than % of pap in adulthood is caused by autoantibodies against gm-csf [ , , ] , this condition has to be initially excluded by analysis of serum [ , ] . none of our patients had increased levels of gm-csf autoantibodies. serum levels of gm-csf are low in autoimmune pap [ ] , whereas elevated values can be found in congenital gm-csfra or gm-csfrb defects and possibly in other forms of pap. thus, particularly in children these other entities need to be excluded. in our cohort of patients with severe pap and hematologic diseases, serum gm-csf level were low to intermediate, supporting some up regulation of gm-csf. next, genetic analysis of gata is helpful in order to diagnose the underlying hematologic entity more precisely, in particular in patients who meet the broad but characteristic phenotype of gata deficiency [ ] . this diagnostic algorithm for pap will allow differentiating important subgroups and may help to identify further genetic abnormalities, known or suspected to be associated with pap. when investigating a patient with severe pap, the pneumologist should be aware of the wide range of diseases which can cause secondary pap and consider interdisciplinary involvement of a hemato-oncologist. conversely, hemato-oncologists should include pap in the differential diagnosis of respiratory failure associated with pulmonary interstitial changes in hematologic diseases. atb-binding cassette sub-family a member ; all: acute lymphoid leukemia; aml: acute myeloid leukemia; ards: acute respiratory distress syndrome; bal: bronchoalveolar lavage; call: common acute lymphoblastic leukemia; cml: chronic myeloid leukemia; cmml: chronic myelomonocytic leukemia ecmo: extracorporeal membrane oxygenation; gm-csf: granulocyte macrophage colony-stimulating factor; hpv: human papilloma virus hsv: herpes simplex virus; jmml: juvenile myelomonocytic leukemia mds: myelodysplastic syndrome; pap: pulmonary alveolar proteinosis sftpb: gene encoding for surfactant protein b; sftbc: gene encoding for surfactant protein c; ttf : thyroid transcription factor ; wll: whole lung lavage. references . seymour jf, presneill jj. pulmonary alveolar proteinosis: progress in the first years pulmonary alveolar proteinosis: diagnostic and therapeutic challenges secondary pulmonary alveolar proteinosis complicating myelodysplastic syndrome results in worsening of prognosis: a retrospective cohort study in japan pulmonary alveolar proteinosis in association with congenital dyserythropoietic anemia: a case report. case rep pediatr secondary pulmonary alveolar proteinosis after unrelated cord blood hematopoietic cell transplantation gata deficiency: a protean disorder of hematopoiesis, lymphatics, and immunity master regulatory gata transcription factors: mechanistic principles and emerging links to hematologic malignancies downregulation of gata- transcription during pneumocystis carinii infection effect of transcription factor gata- on phagocytic activity of alveolar macrophages from pneumocystis carinii-infected hosts mutations in gata are associated with the autosomal dominant and sporadic monocytopenia and mycobacterial infection (monomac) syndrome secondary pulmonary alveolar proteinosis in hematologic malignancies characterization of csf ra mutation related juvenile pulmonary alveolar proteinosis a method and server for predicting damaging missense mutations predicting the effects of coding non-synonymous variants on protein function using the sift algorithm longterm follow-up and treatment of congenital alveolar proteinosis monomac syndrome in a patient with a gata mutation: case report and review of the literature the molecular basis of pulmonary alveolar proteinosis whole-lung lavage in infants and children with pulmonary alveolar proteinosis loss-of-function germline gata mutations in patients with mds/ aml or monomac syndrome and primary lymphedema reveal a key role for gata in the lymphatic vasculature gata haploinsufficiency caused by mutations in a conserved intronic element leads to monomac syndrome wash-out kinetics and efficacy of a modified lavage technique for alveolar proteinosis pulmonary alveolar proteinosis: new insights from a single-center cohort of patients anti-gm-csf antibodies in paediatric pulmonary alveolar proteinosis epidemiological and clinical features of idiopathic pulmonary alveolar proteinosis in japan granulocyte/macrophage-colony-stimulating factor autoantibodies and myeloid cell immune functions in healthy subjects the work of mg was kindly supported by else-kroener-fresenius stiftung (mg _a ), by the german federal ministry of education and research (eupapnet project inside erare, number gm a), european register and biobank on childhood interstitial lung diseases (european commission, fp , ga , child-eu). the authors declare that they have no competing interests.authors' contributions mg designed the study, collected the cases, analysed the data and wrote the draft of the manuscript. he is the guarantor of the entire manuscript. uc, amh, jl, kk, bf, ml, cs, ck and mg contributed and evaluated patients and performed chart review, zr, as, bh, sk, ckr, and ja performed laboratory analyses, cu, ckr and bf critically revised the manuscript, dt provided tissue samples and performed histopathological analyses, as, tw, uc and fb contributed to the long term collection of subjects, patient recruitment and evaluation. all contributors read and agreed to the final version of the manuscript.author's information this paper is devoted to prof. maurizio luisetti's outstanding contribution to the field of human alveolar proteinosis. key: cord- - ynhqmf authors: rauch, helene c.; montgomery, ilene nowicki; hinman, channing l.; harb, walid; benjamins, joyce a. title: chronic theiler's virus infection in mice: appearance of myelin basic protein in the cerebrospinal fluid and serum antibody directed against mbp date: - - journal: j neuroimmunol doi: . / - ( ) - sha: doc_id: cord_uid: ynhqmf myelin basic protein (mbp) appears frequently in the cerebrospinal fluid (csf) of mice with chronic demyelination following intracerebral infection with theiler's murine encephalomyelitis virus (tmev); antibody to mbp can frequently be found in the sera. the peaks of the immune responses to both mbp and tmev coincide with the time course of the clinical signs of disease. adsorption of mouse sera with tmev or mbp indicate the non-identity of the antigens and the specificity of the antisera as measured by elisa. immunoblot analysis of sera confirmed the elisa findings. the mechanism of induction of antibody directed against mbp and its role in tmev-associated demyelination remain to be determined. infection with the da isolate of theiler's murine encephalomyelitis virus (tmev, a picornavirus) produces an excellent model of delayed onset of chronic demyelination that appears to be an immunopathologic consequence of acute infection or complications of persistent infection (theiler ; lipton ; lipton and dal canto ; . although tmev is an enteric pathogen of mice, certain neurotropic strains when injected i.c. present a un;que biphasic disease (lipton ). the first phase, acute infection ( - weeks), is characterized by destruction of infected ventral horn neurons in the central nervous system (cns) gray matter. this phase depends upon dose and strain of virus and thus is not always clinically apparent in all infected mice. if present, the paralysis associated with lower motor neuron disease (lmnd) usually stabilizes. the second phase consists of chronic inflammatory demyelinating lesions which may persist for months (lipton ) . in this late-onset demyelinating disease that is associated with chronic tmev infection, the clinical signs observed are a gait abnormality and loss of righting reflex (rr). signs generally occur - weeks following infection, however, lipton and dal canto ( , ) have shown that demyelination is present as early as weeks post-infection, even while signs of progressive lmnd may be present. compromise of the immune system following treatment of hosts with antithymocyte sera or cyclophosphamide administration can forestall the demyelination process at an early stage following tmev infection (lipton and dal canto , ; roos et al. ) even though virus is present (rodriguez et al. ; lipton et al. ) . further evidence that the immune response may be significant in producing the demyelinating pathology derives from our finding of lymphoproliferative and antibody responses to tmev which correlate with the development of the clinical signs associated with demyelination . in this report, we present data showing that an immune response is induced to an autoantigen, myelin basic protein (mbp) in the form of serum antibody directed against mbp (ab [mbp] ). mbp appears in the cerebrospinal fluid (csf), presumably released as a consequence of myelin degradation following cns inflammation, as it is in experimental allergic encephalomyelitis (eae) ) and ms (cohen et al. ). specific ab [mbp] may thus be elicited if mbp enters the vascular compartment in an immunogenic form. we have eliminated tmev viral protein as a source of a common or cross-reacting epitope by both absorption and immunoblot analysis, but the mechanism of induction of ab[mbp] and its role, if any, in the clinical and pathologic development of tmev-associated demyelination remains to be determined. male sjl/j mice are infected i.c. upon arrival from jackson laboratories; they are between and weeks of age at the time of infection. each animal is given a fourth passage isolate of the da strain of tmev in . ml of a : dilution of a clarified homogenate of infected brain. the homogenate is obtained from i.c. infected swiss-webster suckling mice. the tmev stock used to establish the fourth-passage isolate is a third-passage virus stock supplied by dr. h. lipton, northwestern university. a small percentage (< %) of the i.c. infected animals develop an encephalitis and die within the first weeks when the stock is injected neat. however, we have established an infective dose of virus that does not produce encephalitis, does produce some lmnd coupled with a high incidence of the clinical signs associated with demyelination, particularly the loss of righting reflex. control mice are injected i.c. with a clarified homogenate of uninfected brain. infected mice are individually caged and are housed in a separate facility from the breeding colony and other experimental mice. mice are observed weekly. clinical signs of tmev-associated demyelination are noted such as gait abnormality and loss of righting reflex (rr). rr is scored on a scale of (+) to (+ + + ). the mouse's tail is held between the thumb and forefinger and gently twisted; if the animal can be turned on its back its rr is impaired. as soon as the animal is on its back, the tail is released. if the animal then rights itself immediately it is scored as (+). if it takes up to s to right itself it is scored as (++),greater than sis(+++). when animals are sacrificed, csf and cns tissues are removed. sections are prepared from tissue fixed in bouin's, paraffin embedded and stained with hematoxylin and eosin. all slides are scored without the examiner knowing the clinical signs exhibited by the animal. the pathologic signs are graded on three or more sagittal sections of the entire brain or cord based on a scale of to as follows: --meningitis; = meningitis and one or two vessels cuffed by infiltrating mononuclear cells per section; = several cuffed vessels per section; and = heavily cuffed vessels in every section and widespread inflammation. slides from a representative sampling of animals are screened; in general the histopathology reveals a relatively moderate inflammation resembling that of murine eae at the light microscope level. csf is obtained from the mice as demonstrated to us by d. griffin (johns hopkins university, baltimore, md). briefly, animals are deeply anesthetized by ether, the nuchal skin and muscles are laid open, and a specially drawn capillary tube is inserted through the meninges into the foramen magnum. usually - /~l of csf are collected prior to death. the volume of csf is immediately measured and the csf is transferred to a ml plastic microfuge tube previously coated with a protease inhibitor to prevent rapid enzymatic breakdown of any mbp present. the csf is stored at - °c until assay. only one assay is possible with each specimen. the assay is a modification of the inhibition-elisa of groome (groome ). mbp was isolated by the method of nakao et al. ( ) and hinman et al. ( ) . for coating plates, mouse mbp ( mg/ml ddh ) is diluted in . m carbonate coating buffer, ph . , with . % (w/v) sodium azide; final concentration of mbp is . /lg/ml. mbp is applied to the inner wells of polystyrene microtitration plates (immulon, dynatech, alexandria, va) and incubated overnight at °c. for the mbp standard curve, mbp is diluted in dpbs/t (dulbecco's phosphate-buffered saline, ph . , with . % tween ) containing mg/ml bsa such that mbp concentrations between ng/ml and . ng/ml are available for antibody binding in the standard curve; generally points are selected between and . ng/ml. /~ of each concentration is used per well, giving a sensitivity in the assay of from to . ng/well. diluted csf samples and standards are then incubated with anti-mbp antisera. the resulting preabsorbed standards are added in triplicate to the plate. each of the experimental mouse csf samples is also added to a well. after incubation, alkaline phosphatase-conjugated second antibody (sigma; goat anti-rabbit igg) is added to each well and again incubated. the enzyme substrate mg/ml of pnitrophenylphosphate (voller et al. ) is in % diethanolamine solution (ph . ) with . % (w/v) mgc and . % (w/v) nan . the enzyme-substrate reaction is allowed to proceed at rt until the od reading reaches . in the positive control well ( - min). enzyme activity is inhibited by the addition of /~ of m naoh to each well. substrate conversion is measured spectrophotometrically at nm against the reference blank, substrate-naoh, using a plate reader (biotek, burlington, vt, model el ). the standard curve is plotted using a least-squares cubic spline fit. for a standard curve to be acceptable, the standard deviation of the triplicate values of each of the curve points must be less than - % of the mean value of the triplicates. standard curves run on different days can be superimposed, indicating that the slope of the curve is relatively constant between assays. however, each plate has its own standard curve and positive control to allow for differing times of color development. positive control samples are normal csf pools which have tested negative in the assay and to which we add known amounts of mbp. the amount of mbp present in each sample is interpolated from the spline fitted curve, and allows for measurement of values as low as . ng/well. this program, written for an apple ii + or iie, is available upon request. mbp values are reported as ng//~l. in those samples which contain less than . ng of mbp, a value must be assigned to allow for calculation of sample popul.ation means. to determine the maximum possible value that could be present and not measured (i.e., below the sensitivity of the assay), the following calculation is made. . ng is divided by the sample volume and the sample is then assigned that value/#l for calculation purposes. therefore reported mean values may be slightly higher than the actual value. the range of mbp values in control animals was established using csf from uninjected normal sjl and other strains of mice. the mean value for uninjected mice was . _ . ng/btl. specimens are considered positive for mbp when they have more than . ng/~tl, the mean of the uninjected control mice plus sd. we monitored for the immediate release of mbp by assaying for mbp in the csf h after intracerebral injection. no significant levels of mbp were detected. to further assure that the presence of mbp in the csf was not due to non-specific inflammatory response to injury caused by intracerebral injection, we assayed csf from mice within weeks of injection. these animals were injected either with the virus stock or uninfected brain homogenate. thirteen did not show any mbp in their csf (< . ng/ml or . ng/well, the level of sensitivity of the assay). one sample obtained days after injection had > ng of mbp in the sample. we presume this was due to injury during injection. therefore as a further control for injury-related release, mbp values given in this paper are from animals sampled months or later after infection. the ab [mbp] in mouse sera is also determined by an elisa (groome ) . plates are coated with mouse mbp as previously described. the mouse sera are diluted for testing by adding # of sera to /~ of dpbs/t plus /~ of bsa ( mg/ml). the diluted sera are added to wells and incubated at °c for min. after incubation the plate is washed times with pbs/t, and a : dilution of goat anti-mouse igg alkaline phosphatase conjugate (sigma) is added. the plate is again incubated ( min, °c) and washed; the enzyme substrate is added and the procedure continued as described for the elisa determination of mbp. presence of ab[mbp] is determined with reference to positive control sera (from mice injected with mbp). levelsare reported as presence (+), absence (-) or equivocal (+) for ab [mbp] . positive sera are not further diluted to determine titer. antibody directed against tmev is determined using an elisa kit from litton bionetics (charleston, sc). in this procedure the tmev protein is coated on ferrous metallic beads. the beads are placed in the sera to be tested. they are removed with a magnet and transferred to another well containing the second antibody, a goat anti-mouse igg peroxidase conjugate. after incubation the bead is again transferred using the magnet, to a new well which contains the enzyme substrate. beads are thoroughly washed between exposures to reagents. the extent of substrate conversion is measured spectrophotometrically at nm with a plate reader and this reading is interpreted as presence (+), absence (-) of antibody or an equivocal reading (+) based on standards (+ and -) provided by the company. aliquots of protein are separated by sds gel electrophoresis, using a % acrylamide stacking gel and % running gel (greenfield at al. ) . proteins are transferred to nitrocellulose by electrophoresis at volts for h; a filter paper soaked in % sds is laid on the cathodal side of the gel (macklin et al. ) . after transfer, the blot is dried and proteins are stained using either (a) acid fast green to locate all proteins on the strip or (b) rabbit or mouse sera containing antibody directed against the proteins on the blot as described in the text. when the antisera are used, the blot is blocked with normal goat serum and % gelatin, then incubated with the antiserum, followed by goat anti-rabbit-pap or rabbit anti-mouse-pap; color is developed with -chloro-naphthol as substrate. protein standards are stained and compared with the results obtained with the experimental preparation to identify the component binding the immune serum. clinical appearance of tmev-associated demyelination coincides with the appearance of mbp in the csf, and serum antibody directed against both tmev and mbp (table , fig. ) antibodies directed against the virus (ab[tmev]) and against mbp (ab[mbp]) appear in the sera of infected mice (table ) between and weeks after i.c. injection of tmev. the incidence at weeks coincides with the mean onset of clinical signs of tmev-associated demyelination and the presence of mbp in the csf of these animals. of the samples assayed, / ( %) were considered positive for mbp ( . ng/~l, the mean of the controls plus sd). the average amount of mbp in the csf of all tmev-infected mice assayed was . + . . this value is higher than the mean for uninfected mice, . +_ . . this mean is for animals autopsied at least months post-infection. the mean value of mbp in csf of mice exhibiting severe clinical signs (rr ++, rr +++) at autopsy is . _+ . , with / ( %) of the specimens in the positive range (fig. , shaded squares) . the mean value for tmev-infected mice with minimal clinical signs (rr +) at autopsy is . +_ . , with / ( %) in the positive range. tmev-infected mice which were asymptomatic and autopsied months post-infection had a mean value of mbp in the csf of . _+ . , with / ( . %) of the specimens in the positive range. uninoculated mice held in the main animal quarters (tmev-infected mice are held in a separate facility) rarely possess ab[tmev]; when present it is due to the occurrence of wild-type tmev infection. sera pools are accumulated from uninfected animals (using groups of ten animals). these are often littermates and range in age from to weeks. no attempt is made to pool by age or sex; however, retired breeders, representing the oldest mice, are generally not used in these pools. pools of sera were screened in a random order until a positive pool was found. from this screening, and the quarterly viral screening of the colony (charles river biotechnology service), we estimate the occurrence of natural infection at - % of the mice in the colony. was not completely absorbed out. some antibodies present in the serum may be directed to an epitope on mouse mbp that is absent in gpmbp. b the increasing number of ( + ) reflects a relative increase or decrease in amount of antibody present compared to positive control sera. a change of one (+) is not always significant and is within the variation of the test. to insure that the ab[mbp] found in the infected animals did not develop spontaneously due to an aging phenomenon, we screened sjl breeders in our colony, ranging in age from months to months. of these , were negative, one had a score of (+) in our elisa assay and two had a score of (+). the sera of experimental mice subjected to an encephalitogenic cns tissue challenge (no viral inoculum) and which developed paralysis indicative of eae were used as the source of sera to be used as a positive control for ab [mbp] . ab [tmev] was not usually encountered in these eae mice; the frequency of its occurrence was as in the efitire colony. (table ) a question as to the specificity of these antibodies arose inasmuch as appearance of both ab [tmev] and ab[mbp] coincided in time. absorption of antibody by one antigen indicated specificity by a decrease in titer to only that absorbent antigen table a not every test could be done on every pool due to limited quantities. b the increasing number of (+) reflects a relative increase or decrease in amount of antibody present compared to positive control sera. a change of one (+) is not always significant and is within the variation of the test. abbreviations: bhk, baby hamster kidney cell lysate; tmev-bhk, lysate of tmev-infected bhk cells; rr, loss of righting reflex; ab[mbp], antibody to myelin basic protein; ab[tmev], antibody to theiler's virus; nd, not determined. and not to the other antigen. specificity was also indicated by specific absorption of the control sera (table ) . initially the viral protein preparation used for absorption was an infected bhk cell lysate containing virus and viral proteins (tmev-bhk) among other cell components (microbiological associates). a control of uninfected bhk cell lysate was subsequently tested as an absorbent when it was noted that some tmev-bhk preparations removed ab [mbp] . when the clarified lysate of tmev-infected bhk cells was used as the source of virus for absorption, the titer to both tmev and mbp was reduced, indicating cross-reaction between the bhk lysate and mbp. when uninfected bhk cell lysate was used as an absorbent, ab[mbp] titer was reduced but ab[tmev] remained relatively unchanged. absorption with purified mbp did not significantly reduce the ab[tmev]. inasmuch as uninfected bhk cell lysates absorbed ab[mbp], we repeated the absorptions with purified tmev (a gift of dr. h. lipton, northwestern university). the antisera were highly specific in that ab[tmev] titer was reduced following absorption with tmev protein but not with mbp. conversely the ab[mbp] titer was reduced following absorption with mbp and not following absorption with tmev. guinea pig mbp (gpmbp) was used to absorb ab [mbp] . since the induced antibody was presumably elicited against degraded mouse myelin, the difference between the absorbent antigen and the immunogen could account for the occasionally inadequate adsorptions of ab[mbp] by gpmbp. the bhk cell lysate may contain a protein wl~.ich has a partial analogy with mbp; this could account for the observed cross-reactivity. immunoblot discloses specific protein staining patterns with ab [tmev] and ab[mbp] (fig. ) when the tmev proteins were separated on sds gels, three bands were seen following silver staining of the gel. sera from tmev-inoculated mice (strips b-d) that contained ab [tmev] and ab[tmev] serum from a hyperimmunized rabbit (gift of dr. lipton) bound to the three bands following transfer of the proteins to nitrocellulose. these sera did not bind to any bands in the blots of either mouse spinal cord myelin extract or mouse spinal cord homogenate. strip d was incubated with a serum pool, from tmev-infected mice, that did not test positive for ab[tmev] using the commercial elisa kit. we noted faint bands on the immunoblot in the region of the tmev proteins. the hyperimmune rabbit ab[mbp] serum bound to the mouse mbp bands on a separate blot and to the corresponding bands of mouse spinal cord homogenate; this serum did not bind to tmev proteins (blot not shown). blots of the tmev-bhk lysate bound rabbit ab [tmev] and ab [mbp] as well as ab [tmev] from several of the mouse serum pools from infected animals, corresponding to the results obtained by elisa. as expected, the rabbit ab[tmev] sera bound to the tmev bands only. however, rabbit ab[mbp] sera bound to three bands in the - kda molecular weight region, which corroborated the data on the absorption of ab[mbp] by bhk lysate alone (blots not shown). we report here the appearance of mbp in the csf of mice weeks after cns infection with tmev. half of the infected animals had measurable levels of mbp, even those that were asymptomatic. this suggests that the quantity of mbp in the csf reflects the intensity of the chronic inflammatory response to the persisting infection rather than clinical signs. the ab [mbp] and ab[tmev] appear at the same time, particularly in animals with clinical signs, suggesting a relationship between immune responsiveness and these clinical signs. although the incidence of autoantibodies in general may be increased with age in animals, the appearance of ab [mbp] in uninoculated mice in our colony is a rare observation, and is not associated with age. ab[tmev] is seen at a frequency of - % in our uninfected mouse colony, presumably due to the occurrence of sporadic natural infection with wild-type virus. however, the experimental mice arrive from jackson laboratories at weeks of age, are immediately placed in isolation, and within h after arrival are inoculated i.c. with the da strain of the virus, thereby assuring that the observed ab[tmev] is due specifically to the i.c. infection of these animals. the ab [mbp] and ab[tmev] are specific and not cross-reactive as indicated by the cross-absorptions and immunoblot studies using purified mbp or tmev protein preparations. these studies would appear to rule out generation of ab[mbp] by tmev protein determinants. an interesting cross-reaction was noted, however, with the clarified freeze-thaw cell lysate from bhk cells. when the tmev-bhk and bhk cell lysate were both discovered to absorb ab[mbp], immunoblot analysis was undertaken using the tmev-bhk lysate. all ab[tmev] sera tested reacted with the three major tmev protein bands on the blot and not with bands in blots of mouse spinal cord homogenate or myelin extract. ab[mbp] sera from rabbits hyperimmunized with bovine or human mbp bound to three bands in the - kda area of the blot of tmev-bhk or control bhk lysate. it appears that the bhk cell lysate contains cross-reactive protein(s), the nature of which needs further clarification. hypotheses as to the induction of autoantibodies, as we observed in mice presenting signs of theiler's associated demyelination, often include a role for viruses. it is challenging to determine how such a virus infection in fact triggers an autoimmune response. in the target cell or tissue, infection may release or expose new antigens. this could be the case in the coronavirus infection or in the tmev infection as reported here. in other situations, extensive and chronic injury of a tissue or organ results in autoantibody production as in the case of severe chronic hepatitis (meliconi et al. ). this may be one explanation for the appearance of ab[mbp] following inflammatory demyelination in theiler's virus infection. myelin degradation may lead to immunogenic forms of mbp that could enter the vascular compartment to induce an immune response. in fact, when mbp is injected into the csf, it is transported through the blood-brain barrier and presented on the lumenal surface of endothelial cells of the cerebral and meningeal veins (vass et al. ). this indicates a means by which mbp from inflammatory demyelinating lesions may enter the vascular compartment and elicit an immune response. a virus infection could also result in an immunologically enhanced response due to presentation of normal brain constituents by antigen presenting cells, e.g. astrocytes. interferons (ifn) are produced following some viral infections by activated t cells shown to be present in certain inflammatory brain lesions. ordinarily brain cells express low levels of antigens encoded by the major histocompatibility complex (h- ), but in the presence of ifn, mhc gene expression was dramatically enhanced: most astrocytes, oligodendrocytes, microglia and some neurons expressed h- antigens. ifnv, also induced ia antigens on a subpopulation of astrocytes (nakamura et al. ; wong et al. ) . the capacity of ifn, produced as a result in viral infection, to induce h- or ia antigens may account for the findings of fontana et al. ( ) , who observed that astrocytes which displayed these antigens following contact with t cells could successfully present mbp to immunologically active encephalitogenic t cell lines. in fact there is precedent for finding an autoimmune response directed against mbp following a viral infection. watanabe et al. ( ) adoptively transferred eae-like lesions using lymphocytes obtained from lewis rats infected with murine coronavirus jhm and restimulated by mbp in culture. a virus infection could trigger an autoimmune response by not only infecting the target cell but could do so by infecting the immunologically active cell as well. epstein-barr virus infection appears to trigger b cells to express autoantibodies against an array of tissue antigens; the target cell is not infected (fong et al. (fong et al. , garzelli et al. ; robinson and stevens ) . infection of immunologically active cells outside the cns is a distinct possibility following i.c. inoculation of tmev (clatch et al. ; . a common epitope, i.e. molecular mimicry, shared by the cns and tmev has been ruled out thus far by our studies, but is still feasible in other virus infections as suggested by apparent sequence homology between some viral proteins and nervous system proteins (jahnke et al. ) . inasmuch as there are scattered reports of ab[mbp] among ms patients rabinowitz et al. ; richert et al. ; wajgt and gorny ; johnson et al. ) , these mechanisms warrant further consideration. characterization of theiler's murine encephalomyelitis virus (tmev) -specific delayed-type hypersensitivity responses in tmev-induced demyelinating disease: correlation with clinical signs radioimmunoassay of myelin basic protein in spinal fluid: an index of active demyelination age-associated changes in epstein-barr virus-induced human lymphocyte autoantibody responses selective induction of autoantibody secretion in human bone marrow by epstein-barr virus astrocytes present myelin basic protein to encephalitogenic t-cell lines epstein-barr virus-transformed lymphocytes produce monoclonal autoantibodies that react with antigens in multiple organs csf antibodies to myelin basic protein and oligodendrocytes in multiple sclerosis and other neurological diseases quaking mouse: isolation and characterization of myelin protein enzyme-linked immunoadsorbent assays for myelin basic protein and antibodies to myelin basic protein application of high performance liquid chromatography to the preparation of encephalitogenic myelin basic protein sequence homology between certain viral proteins and proteins related to encephalomyelitis and neuritis measles encephalomyelitis --clinical and immunological studies theiler's virus infection in mice. an unusual biphasic disease process leading to demyelination theiler's virus-induced demyelination: prevention by immunosuppression contrasting effects of immunosuppression on theiler's virus infection in mice the to strains of theiler's viruses cause 'slow virus-like' infections of mice theiler's virus antigen detected in mouse spinal cord ½ years after infection electroblot analysis of the myelin proteolipid protein hepatocyte membrane-bound igg and circulating liver-specific autoantibodies in chronic liver disease: relation to hepatitis b virus serum markers and liver histology effect of ifn-y on the immune response in vivo and on gene expression in vitro basic proteins from the acidic extract of bovine spinal cord. i. isolation and characterization endogenous myelin basic protein-serum factors (mbp-sfs) and anti-mbp antibodies in a patient with post-herpes simplex virus acute disseminated encephalomyelitis the role of the immune response in tmev infection and the development of late onset demyelination experimental allergic encephalomyelitis in mice: presence of myelin basic protein in cerebrospinal fluid expansion of antigenspecific t cells from cerebrospinal fluid of patients with multiple sclerosis production of autoantibodies to cellular antigens by human b cells transformed by epstein-barr virus persistent infection of oligodendrocytes in theiler's virus-induced encephalomyelitis the effect of short-term and chronic immunosuppression on theiler's virus demyelination spontaneous encephalomyelitis of mice, a new virus disease ultracytochemical distribution of myelin basic protein after injection into the cerebral spinal fluid the enzyme-linked immunosorbent assay csf antibodies to myelin basic protein and to myelin-associated glycoprotein in multiple sclerosis. evidence of the intrathecal production of antibodies adoptive transfer of eae-like lesions from rats with coronavirus-induced demyelinating encephalomyelitis inducible expression of h- and ia antigens on brain cells this work was supported in part by grant nincds ns- from the national institutes of health and a grant from the whittaker foundation.we would like to thank dr. howard lipton (northwestern university) for the original virus stock, purified viral protein and antibody directed against tmev. we would like to acknowledge the excellent technical assistance of sandra cola in performing both the assays for serum antibody and the assay for mbp in the csf and diane studzinski in performing the immunoblot assays. in addition we would like to acknowledge the work of jewell akins in caring for the mice, scoring them and collecting the csf. we would also like to acknowledge the assistance of keith kelly and cherine abdalla in collecting the csf. key: cord- -ndmf ekp authors: akins, paul taylor; belko, john; uyeki, timothy m.; axelrod, yekaterina; lee, kenneth k.; silverthorn, james title: h n encephalitis with malignant edema and review of neurologic complications from influenza date: - - journal: neurocrit care doi: . /s - - - sha: doc_id: cord_uid: ndmf ekp background: influenza virus infection of the respiratory tract is associated with a range of neurologic complications. the emergence of pandemic influenza a (h n ) virus has been linked to neurological complications, including encephalopathy and encephalitis. methods: case report and literature review. results: we reviewed case management of a -year old hispanic male who developed febrile upper respiratory tract signs and symptoms followed by a confusional state. he had rapid neurologic decline and his clinical course was complicated by refractory seizures and malignant brain edema. he was managed with oseltamavir and peramavir, corticosteroids, intravenous gamma globulin treatment, anticonvulsants, intracranial pressure management with external ventricular drain placement, hyperosmolar therapy, sedation, and mechanical ventilation. reverse transcriptase polymerase chain reaction analysis of nasal secretions confirmed h n virus infection; cerebrospinal fluid (csf) was negative for h n viral rna. follow-up imaging demonstrated improvement in brain edema but restricted diffusion in the basal ganglia. we provide a review of the clinical spectrum of neurologic complications of seasonal influenza and h n , and current approaches towards managing these complications. conclusions: h n -associated acute encephalitis and encephalopathy appear to be variable in severity, including a subset of patients with a malignant clinical course complicated by high morbidity and mortality. since the h n influenza virus has not been detected in the csf or brain tissue in patients with this diagnosis, the emerging view is that the host immune response plays a key role in pathogenesis. the current pandemic of influenza a (h n ) ( h n ) virus has presented challenges for clinicians the findings and conclusions in this report are those of the authors and do not necessarily represent the official position of the centers for disease control and prevention. worldwide. neurologic complications of seasonal influenza are likely under-recognized by neurologists and the frequency of acute or post-infectious neurologic complications with h n virus infection is unknown. it is worth noting the historical relationship between h n and neurology. following the - h n pandemic, an increase was observed in encephalitis lethargica cases [ ] . what have neurologists learned about complications of h n virus infections worldwide? we present a case report of h n -associated encephalopathy and review neurologic complications associated with seasonal influenza and h n virus infection. the kaiser permanente inpatient neurosurgery service maintains ongoing institutional review board approval for a prospective database registry for clinical research purposes. we identified a case of acute encephalopathy associated with h n virus infection of the upper respiratory tract referred from an outside kaiser community hospital for management. we conducted a detailed review of the electronic medical records. we also conducted a literature review using pubmed. mesh search terms included influenza, encephalitis, encephalopathy, h n , acute necrotizing encephalopathy, and meningitis. a previously healthy -year old male college student had days of non-productive cough, rhinorrhea, myalgias, and fever but no headaches or neck stiffness. on the th illness day, he presented to the emergency department of a community hospital with lethargy and confusion. he was electively intubated for airway protection. his chest x-ray (cxr) was normal. routine admission laboratory tests including hepatic transaminases were within normal range. a non-contrast head computed tomography (ct) did not reveal any abnormalities (fig. , top row) , and he underwent lumbar puncture. cerebrospinal fluid (csf) analysis showed wbc/ll with % lymphocytes, rbc/ll, protein mg/dl, and glucose mg/dl. he was diagnosed with meningoencephalitis and started on vancomycin, ceftriaxone, acyclovir, and oseltamivir ( mg twice daily per nasogastric tube). on the morning of the third-day of hospitalization, he experienced tonic-clonic seizures and remained comatose with extensor posturing afterwards. repeat head ct (fig. , bottom row) demonstrated diffuse brain edema and effaced basal cisterns. he received fosphenytoin, mannitol, and propofol. the treating physicians contacted the neuro-intensive care unit at kaiser sacramento for additional assistance. he was emergently transferred to the kaiser permanente sacramento neuro-intensive care facility (nicu). on arrival, his initial examination demonstrated a glasgow coma scale of (e v m ). his repeat cxr did not demonstrate on the bottom row, the small arrow points to effacement of basal cisterns (left) and subcortical brain edema (larger arrows, bottom row, left and right). this subcortical edema is confirmed on mr imaging (fig. ) any infiltrates or signs of acute respiratory distress syndrome (ards). an external ventricular drain was placed by the neurosurgeon at the bedside. he reported that the csf pressure noted at the time of initial catheter placement was elevated. the first recorded intracranial pressure (icp) was mm hg, and this reading was taken after the expected loss of csf during the procedure. on the second day of nicu hospitalization, his glasgow coma scale (gcs) score was (e v m ) and average icp was mm hg. throughout the remainder of the hospitalization, the recorded icp remained below mm hg. initial icp was maintained with external ventricular drainage at cm relative to the external auditory canal and a midazolam infusion ( mg/h). electroencephalogram (eeg) monitoring demonstrated diffuse, severe slowing in the delta range and no electrographic seizures. on hospital day , mri of the brain was obtained (see fig. ). he received days of dual neuraminidase inhibitor treatment (oseltamivir mg twice daily per nasogastric tube, peramavir mg iv daily); intravenous gamma globulin ( gm/ kg days); dexamethasone ( mg iv load, mg iv every h with taper over weeks); icp monitoring and management; ventilator support; and anticonvulsants (fosphenytoin, levetiracetam). his weekly glasgow scale scores showed delayed improvement ( , e v m , admission): (e v m , week ), (e v m , week ), (e v m , week ), (e v m , week ). the midazolam infusion was discontinued on hospital day , after clinical observation and eeg confirmation that he was not having electrographic seizures. thereafter he received intermittent doses of lorazepam as needed for sedation while on the ventilator. over weeks, neuroimaging demonstrated improvement in his brain edema with restoration of his basal cisterns, and the external ventricular drain was successfully weaned and removed. more rapid weaning of his external ventricular drain was not attempted due to severe neurologic impairments with gcs less than eight and radiographic appearance of diffuse brain edema and effaced basal cisterns. his nicu course was complicated by ventilatorassociated klebsiella pneumoniae and spontaneous pneumomediastinum on day of intensive care. chest ct demonstrated subcutaneous emphysema, mediastinal emphysema, bilateral lower lobe atelectasis, and no pulmonary interstitial emphysema, or pneumothorax. he did not develop adult respiratory distress syndrome or suffer periods of hypoxemia. rt-pcr of an admission nasopharyngeal swab was positive for h n virus at the california department of public health virology laboratory. rt-pcr analysis of csf samples was negative for influenza a and b viruses, herpes virus type , , and , varicella, enterovirus, and epstein barr virus. nasopharyngeal samples were negative for enterovirus and mycoplasma pcr. bacterial and viral cultures of csf were negative. test results from clinical specimens (blood, endotracheal aspirate, serum, and csf) sent to the california encephalitis project did not reveal an alternative cause. follow-up mri brain imaging (fig. b, d) was repeated at month. after weeks, he transitioned to acute rehabilitation, and month later returned home. because he had improved upper extremity use without recovery in his legs, the physiatry staff performed spine mr imaging and no specific cause was identified. at the time of this case report, the patient has returned home with his family. he is talking and interacting with his family normally. he has not returned to college. his gastromy tube has been removed. he has generalized rigidity without tremor or dyskinesia. he is ambulatory but requires a walker due to reduced endurance and leg weakness. fig. magnetic resonance imaging was done at the time of patient transfer a, c to the neuro-intensive care center and at month of treatment b, d with influenza-specific antiviral therapy, corticosteroids, and intravenous gamma globulin therapy. a coronal flair image shows diffuse brain edema with sulcal effacement and symmetric hyperintensities selectively affecting the white matter and sparing cortex and subcortical nuclei such as basal ganglia and thalami. b coronal flair image at month shows resolution of sulcal effacement, marked reduction in white matter hyperintensity, and relative brain atrophy ( year old patient). c diffusion-weighted imaging on admission showed some increased signal in the periventricular zones that were also bright on t and flair sequences consistent with t shine-through. d diffusionweighted imaging at month revealed hyperintensity in the caudate and putamen with corresponding decreased signal in adc map and lack of hyperintensities on t and flair sequences (see fig b) we present a case of a patient with acute encephalitis associated with febrile upper respiratory tract illness due to h n complicated by seizures and malignant cerebral edema. few adult cases of h n influenzaassociated acute encephalitis or encephalopathy have been reported to date. descriptions of h n -associated neurologic complications are limited to case reports and small case series, and have been more commonly reported among young children. given the current influenza pandemic, we provide an overview of neurologic complications associated with seasonal influenza and h n (fig. ) and review clinical management and rationale. influenza virus infections can cause human respiratory disease and have been associated with a variety of central nervous system disorders [ ] . influenza virus has been rarely detected in csf of patients that developed acute encephalitis/encephalopathy [ ] [ ] [ ] . the systemic inflammatory response syndrome (sirs) to influenza virus infection of the upper respiratory tract is hypothesized to play a prominent role in the more severe stages leading to cytokine dysregulation (''cytokine storm'') in influenzaassociated encephalopathy or encephalitis (iae) patients [ ] . elevated cytokines in serum and csf have been reported in patients with seasonal influenza-associated encephalopathy [ , [ ] [ ] [ ] [ ] . elevated csf to plasma ratios suggest activation of cytokine production within the cns may have occurred along with the respiratory tract and systemic cytokines [ , , ] . microglia and astrocytes are capable of producing cytokines in the cns [ , ] . it is known that influenza virus infects and replicates at the nasopharyngeal epithelium leading to extensive damage during infection. below the mucosa, the free nerve endings of the olfactory nerves may also become infected. as seen with herpes simplex viruses, some postulate that influenza virus could penetrate and replicate at the olfactory mucosa and the free nerve endings with resultant axonal transport of virions to the olfactory bulbs, to the olfactory tract, and finally to the brain [ ] . there is some literature to support this mechanism when one looks at h n , or avian influenza, where mice inoculated intranasally with h n developed cns lesions in the pons, medulla oblongata, and cerebellar nuclei. astrocytes and glial cells were positive for viral antigen but viral replication ceased before days [ , ] . further study is needed to elucidate the pathogenesis of cns disease complicating influenza a infection. neurologic symptoms associated with influenza can arise at different intervals after the initial influenza illness (fig. , table ). when assessing patients clinically, it is important to determine if the patient has active or recent symptoms (within days) of influenza or if the neurologic symptoms have appeared in a subacute manner. we will first discuss neurologic complications in the setting of recent influenza virus infection and then proceed to complications that present in a delayed manner the development of a confusional state in the setting of influenza illness symptoms and fever raises the possibility of influenza-associated encephalitis or encephalopathy. the degree of encephalopathy varies from a confusional state to obtundation. it is important to recognize that a small portion of cases can rapidly deteriorate to coma and subsequent brain death due to diffuse, malignant cerebral edema. focal and generalized seizures often occur and can be present with either mild or severe cases. the presence of fever and altered mental state should prompt clinicians to pursue csf analysis unless neuroimaging or laboratory studies reveal a contraindication. influenza illness may include upper respiratory symptoms, pneumonia, or diarrhea (more commonly in young children with seasonal influenza). a thorough medical assessment to exclude other causes such as sepsis, metabolic or toxic disorders, structural cns diseases, and other cns infections is warranted. we define encephalitis by the presence of inflammation in the csf or demonstration of viral infection in brain biopsy or autopsy specimens. we define encephalopathy when csf is acellular and brain biopsy or autopsy specimens have failed to demonstrate viral infection. in some cases, this distinction is arbitrary and the case has borderline csf pleocytosis or csf analysis was not performed due to malignant brain edema. a consistent observation is that patients with seasonal influenza-associated encephalopathy rarely ever have evidence of influenza viral rna in csf based on rt-pcr analysis of csf. furthermore, there is no evidence of seasonal influenza virus infection of brain specimens. in one case series, only one out of patients with acute seasonal influenza-associated encephalitis had influenza viral rna detected [ ] . terminology for post-infectious encephalitis can be confusing. for example, the international pediatric multiple chronic condition * sometimes classified as adem [ ] sclerosis study group [ ] listed ten terms that have been used to describe acute disseminated encephalomyelitis (adem). some terms focus on the triggering event, such as post-infectious encephalomyelitis; others on pathologic or pathophysiologic features such as acute demyelinating encephalomyelitis or hyperergic encephalomyelitis. these authors also classify acute hemorrhagic leukoencephalitis, acute necrotizing hemorrhagic leucoencephalitis (also known as acute necrotizing encephalitis, (ane)), and acute hemorrhagic encephalomyelitis as hyperacute forms of adem. these diagnostic terms are of great historical interest. they generally preceded modern neuroimaging and relied more on the clinical and pathologic details. the study group also lumps a diversity of neuroimaging findings under the diagnosis of adem including: ring-enhancing lesions; diffuse and multi-focal regions of t hyperintensity with and without associated hemorrhage; multi-focal lesions with associated mass effect (tumefactive lesions); and images with symmetric, bithalamic edema. while we prefer one term (adem) rather than ten terms to describe post-infectious encephalitis, we are concerned that the pathophysiology and outcome of a process leading to the formation of ring-enhancing lesions (demyelinating, for example, acute demyelinating encephalomyelitis) must be radically different than that causing bithalamic edema (necrotizing, for example, ane). in reality, iae presents along a spectrum ranging from milder cases with normal neuroimaging to more malignant cases with abnormal neuroimaging and less favorable outcomes. for the sake of discussion and literature review, we present a simplified classification scheme based on clinical and imaging findings. the iae benign variant can present with fever, confusional state, and seizures but neuroimaging with ct brain or mri brain does not demonstrate any acute abnormalities. csf analysis is within normal limits or has borderline findings. rt-pcr testing for h n influenza viral rna is positive in upper respiratory secretions but negative when csf is tested [ ] [ ] [ ] . these patients typically recover within week, and most cases have received oseltamavir and anticonvulsants. the initial reports of pediatric cases of h n encephalopathy in the us were not severe [ ] . similarly, other reported adult cases of h n iae without ards have not been severe with complete recovery [ , ] . a more recent pediatric case series of h n iae reported that / patients had imaging abnormalities and neurologic sequelae [ ] , so the treating physicians need to be aware that full recovery is not a certainty. the iae with splenial sign presents with acute febrile respiratory illness and additional neurologic symptoms with a characteristic mri abnormality. we found case reports associated with seasonal influenza but not with h n . it has been reported in children, but rarely in adults [ ] [ ] [ ] [ ] [ ] [ ] . encephalopathy is always present and can be severe. seizures are often present. mri imaging demonstrates increased t and flair signal and restricted diffusion in the splenium of the corpus callosum. this finding is reversible. the mri finding is not specific and has been reported with other infections, high-altitude brain edema, and certain metabolic states such as hypernatremia [ ] . csf analysis is unremarkable. these patients have been treated with oseltamavir and anticonvulsants, and typically recover within month. the iae with posterior reversible leucoencephalopathy syndrome (pres) presents as moderate to severe febrile encephalopathy. this subtype has been reported with seasonal influenza but not specifically with h n . the mri imaging appears radiographically identical to pres caused by more typical causes such as pregnancy or malignant hypertension [ , ] . vascular caliber changes have been observed in these cases; this is non-specific and can be related to infectious vasculitis or pres. given the diverse causes of pres including malignant hypertension, pregnancy, metabolic disorders, and certain medications such as chemotherapeutics and immunosuppressants; it is often difficult to distinguish the pathophysiology of iae in this clinical setting. therapy is focused upon antiviral treatment, corticosteroid administration, and supportive care. iae with malignant brain edema is one of the most challenging subtypes to diagnose and treat. both seasonal influenza and h n can be complicated by severe forms of acute encephalopathy and malignant brain edema [ ] [ ] [ ] [ ] . survival in some cases has been achieved with aggressive neuro-intensive case management with other therapies, including administration of antivirals, corticosteroids, immunoglobulin ( gm/kg in adult patients), hyperosmolar therapy, plasmapheresis, and hypothermia in some cases. one of the goals of treatment is to reduce viral expression with early antiviral treatment and thereby to reduce stimulation of the host inflammatory response. our case presentation illustrates the rapid time course for this complication (see fig. ) and neurocritical care treatment approaches. because of diffuse brain edema, a broad treatment approach using hyperosmolar therapy, intubation, fever control, and sedation were important. to the best of our knowledge, this is the only case description of iae in which an external ventricular drain was utilized, probably because it is difficult to place a catheter into the small, compressed ventricles of patients with diffuse brain edema associated with influenza. another adult case of h n encephalitis has been reported with radiographic findings similar to ours. fugate et al. [ ] described an adult with h n influenza-associated acute hemorrhagic leukoencephalitis. like our patient, their case also showed confluent areas of increased t signal in the periventricular white matter and centrum semiovale. because of the additional finding of microhemorrhages demonstrated on gradient echo mri sequences, they diagnosed acute hemorrhagic leukoencephalitis or hurst disease. their patient also had restricted diffusion in the basal ganglia (see fig. ). because their patient had severe adult respiratory distress syndrome (ards) with oxygen saturation readings in the range of - %, the authors attributed the basal ganglia findings to hypoxic brain injury. our patient did not have advanced pulmonary disease, hypoxia, or hypotension. care should be taken to distinguish iae with malignant edema from reyes' syndrome in which patients may present with lethargy, confusion, seizures, or coma accompanied by brain edema. reyes' syndrome most commonly occurs in children but has been reported in adults following influenza and aspirin ingestion [ ] . it can be distinguished based on the accompanying hepatic abnormalities, hyperammonemia, and hypoglycemia. caution should be taken with any neurosurgical procedures in reyes' syndrome due to increased risk of perioperative bleeding. one of the most devastating complications of seasonal and pandemic influenza is ane [ ] [ ] [ ] . patients develop rapid neurologic deterioration to coma. seizures are often present. initial brain ct may show decreased density in the thalami, and mri of brain demonstrates the characteristic bilateral thalami lesions. this finding may be initially mistaken for ischemic strokes (top-of-basilar syndrome) or venous infarction secondary to thrombosed internal cerebral veins, vein of galen, or straight sinus. it is interesting that there have been case reports for recurrent ane and also familial ane. this suggests that there may be a genetic susceptibility and a gene associated with familial seasonal influenza ane cases has been reported (nuclear pore gene, ranbp ; [ ] ). this condition is often fatal or accompanied by permanent neurologic sequelae in surviving cases. it is intriguing that the neuroanatomical changes found in the thalami, midbrain, and cerebellum on neuroimaging correlated with the clinical symptoms reported for encephalitis lethargica, specifically ''sleeping sickness'', ophthalmoparesis, quadriparesis, and delayed parkinsonism (see below). it is conceivable that survivors with less fulminant involvement could manifest a clinical syndrome with symptoms and signs that localize to brainstem structures. a pediatric case of h n -associated ane with bilateral thalamic imaging findings without associated malignant brain edema has been published [ ] , but detailed clinical follow-up was not reported. during the subacute period, additional classic neurologic syndromes associated with influenza have been described. post-influenzal cerebellitis is quite uncommon and has been reported rarely in adults [ ] [ ] [ ] . this syndrome was diagnosed in a -year old woman who developed ataxia, dysarthria, and truncal titubation month after influenza b virus infection, with neurologic symptoms that resolved gradually after an additional month. ct and mri brain imaging were unrevealing. csf studies detected evidence of the persistence of the np gene of influenza b virus in the csf from samples taken and weeks after the onset of initial influenza illness. a -year old woman gradually developed gait and speech problems after influenza a illness that was treated with oseltamavir. csf showed pleocytosis. the cerebellar cortex had increased t signal which resolved over an day period. she received pulse intravenous corticosteroid therapy. her symptoms resolved [ ] . plasmapheresis [ ] and ivig [ ] have also been used for this condition. some cases of cerebellitis following viral and mycoplasma illness have developed fulminant cerebellar swelling with secondary brainstem compression, obstructive hydrocephalus, with fatal outcome [ ] . interventions with posterior fossa decompression and external ventricular drain placement may lead to a favorable outcome in a child with this severe condition. antibodies to the glutamate receptor have been reported in patients with post-infectious influenza viral cerebellitis [ ] . guillain-barre syndrome (gbs) is a subacute, immunemediated disease predominantly affecting the peripheral nervous system. the diagnosis and treatment are wellknown to most neurologists and this condition has been extensively described and reviewed. gbs has been rarely reported in association with seasonal influenza virus infection [ ] , but it should be noted that influenza testing is rarely pursued in gbs cases and may be unrevealing. treatment for influenza-related gbs is identical to treatment for other gbs due to other associated causes. monitoring for respiratory compromise due to neuromuscular weakness with timely respiratory support if needed is critical. plasmapheresis or gammaglobulin treatments are also helpful. the precise pathophysiology is uncertain, but molecular mimicry of the infectious agent is presumed to stimulate autoimmune responses. this has been demonstrated to occur in campylobacter jejuni-associated gbs [ ] . influenza-associated myositis has been reported with seasonal influenza [ ] and h n variant [ ] . myalgias are a common symptom of influenza, but some patients develop frank weakness and have elevated serum levels of creatine phosphokinase (cpk). it is more common in children but has been seen in all age groups. the calf muscles are most suspectible, and patients may walk with a stiff gait or toe walk. onset is usually within the first week of infection and spontaneous improvement typically occurs within weeks in most cases. rarely, severe cases can result in myoglobinuria-associated renal failure and compartment syndromes requiring fasciotomies. influenza can also selectively attack specific muscle groups such as the heart (myocarditis). muscle biopsy shows necrosis, regenerating fibers, and occasionally inflammation. post-viral parkinsonism has been reported after an assortment of infections including influenza virus [ ] . an outbreak of these cases was temporally noted following the great influenza (h n ) pandemic of - [ ] . patients with this condition respond poorly to medical therapy, and it has an unfavorable prognosis. encephalitis lethargica is also known as von economo encephalitis and sleeping sickness [ ] . a wave of such cases was reported following the - influenza a (h n ) virus pandemic. the cardinal features of this condition are altered consciousness with prolonged somnolence and ophthalmoplegia. after intervals of months to years, survivors are at risk of developing parkinsonism. pathological findings include nerve cell destruction primarily in the midbrain, subthalamus, and hypothalamus [ , ] . using modern laboratory techniques, formalin-preserved autopsy brain specimens of encephalitis lethargica cases analysed for influenza viral rna were negative [ ] . scientists have proposed a ''hit-and-run'' model of early viral-mediated injury with late sequelae [ ] . the neurologist oliver sacks [ ] drew attention to this mysterious disorder and the discovery of l-dopa, in his book, awakenings later converted to a feature-length movie. the delayed appearance of restricted diffusion in the basal ganglia in our patient and others [ ] is concerning for this condition (fig. ) . we do not know if this indicates that our patient with h n is at risk of developing postviral parkinsonism, but long-term clinical follow-up will be important. a delayed diffusion neuroimaging abnormality was also reported in the dentate nucleus of a patient with seasonal influenza encephalopathy/splenial sign [ ] . we present a case of acute encephalitis associated with pandemic influenza a (h n ) virus infection, complicated by malignant brain edema. the emerging hypothesis about acute neurologic complications of seasonal influenza is that the immune response triggered by influenza virus infection of the respiratory tract plays a prominent role in the pathogenesis of neurological manifestations. this hypothesis regarding the development of acute encephalopathy and brain edema is analogous to current theories about the role of the immune system and cytokines in the development of ards with h n virus infection. we have also provided an overview of the spectrum of acute and post-infectious neurologic complications reported in association with seasonal and pandemic influenza virus infection of the upper respiratory tract. neurologists should be aware of the potential for a wide range of neurologic complications in association with the current h n pandemic and seasonal influenza. influenza, encephalitis lethargica, parkinsonism neuropathogenesis of influenza virus infection in mice pcr on cerebrospinal fluid to show influenza 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treatments elderly autopsy case of influenza-associated encephalopathy an adult autopsy case of acute encephalopathy associated with influenza a virus acute hemorrhagic leukoencephalitis and hypoxic brain injury associated with h n influenza influenza a virus and reye's syndrome in adults acute necrotizing encephalopathy in a child with h n influenza infection acute necrotizing encephalopathy in a child during the influenza a (h n ) pandemia: mr imaging in diagnosis and follow-up infection-triggered familial or recurrent cases of acute necrotizing encephalopathy caused by mutations in a component of the nuclear pore, ran-bp mr imaging in novel influenza a (h n )-associated meningoencephalitis an adult case of acute cerebellitis after influenza a infection with a cerebellar cortical lesion on mri probable post-influenza cerebellitis acute cerebellar ataxia and consecutive cerebellitis produced by glutamate receptor delta autoantibody plasmapheresis improves outcome in postinfectious cerebellitis induced by epstein-barr virus brain spect imaging and treatment with ivig in acute post-infectious cerebellar ataxia: case report acute near-fatal parainfectious cerebellar swelling with favourable outcome guillain barre syndrome and influenza virus infection carbohydrate mimicry between human ganglioside gm and campylobacter jejuni lipooligosaccharide causes guillain-barre syndrome benign acute childhood myositis: laboratory and clinical features melting muscles: novel h n influenza a associated rhabdomyolysis viral parkinsonism lack of detection of influenza genes in archived formalin-fixed, paraffin waxembedded brain samples of encephalitis lethargica patients from to new york: random house, inc key: cord- -gq omz u authors: griese, matthias; ripper, jan; sibbersen, anke; lohse, pia; lohse, peter; brasch, frank; schams, andrea; pamir, asli; schaub, bianca; muensterer, oliver j; schön, carola; glöckner-pagel, judith; nicolai, thomas; reiter, karl; hector, andreas title: long-term follow-up and treatment of congenital alveolar proteinosis date: - - journal: bmc pediatr doi: . / - - - sha: doc_id: cord_uid: gq omz u background: clinical presentation, diagnosis, management and outcome of molecularly defined congenital pulmonary alveolar proteinosis (pap) due to mutations in the gm-csf receptor are not well known. case presentation: a / years old girl was diagnosed as having alveolar proteinosis. whole lung lavages were performed with a new catheter balloon technique, feasible in small sized airways. because of some interstitial inflammation in the lung biopsy and to further improve the condition, empirical therapy with systemic steroids and azathioprin, and inhaled and subcutaneous gmcsf, were used. based on clinical measures, total protein and lipid recovered by whole lung lavages, all these treatments were without benefit. conversely, severe respiratory viral infections and an invasive aspergillosis with aspergilloma formation occurred. recently the novel homozygous stop mutation p.ser x of the gmcsf receptor alpha chain was identified in the patient. this mutation leads to a lack of functional gmcsf receptor and a reduced response to gmcsf stimulation of cd b expression of mononuclear cells of the patient. subsequently a very intense treatment with monthly lavages was initiated, resulting for the first time in complete resolution of partial respiratory insufficiency and a significant improvement of the overall somato-psychosocial condition of the child. conclusions: the long term management from early childhood into young adolescence of severe alveolar proteinosis due to gmcsf receptor deficiency requires a dedicated specialized team to perform technically demanding whole lung lavages and cope with complications. pulmonary alveolar proteinosis (pap) is characterized by a substantial and persistent increase in surfactant pool size [ , ] . there are several causes of this rare condition; mouse models with deletion of granulocyte-macrophagecolony stimulating factor (gm-csf) or the gm-csf receptor (gm-csfr) beta-chain showed the first evidence for involved molecularly mechanisms [ , ] . autoantibodies against gm-csf, blocking gm-csf signaling, are the cause for the most frequent form of pap, mainly found in adults and also called autoimmune pap [ ] . in the first two families with congenital pap and mutations in the alpha-chain of the receptor for gm-csf were described [ , ] and very recently another six families were reported [ ] . the patients presented with progressive dyspnea of insidious onset between the ages of . and years; some were asymptomatic. short term responsiveness to whole lung lavage (wll) treatment has been described, however not much information on the long term outcome of molecularly defined patients is yet available. other genetic conditions that lead to pap include a recently identified mutation in the beta chain of the gmcsf receptor [ ] , surfactant protein b or c deficiency [ , ] , niemann-pick type c disease [ ] and lysinuric protein intolerance [ ] . secondary pap develops in association with conditions involving functional impairment or reduced numbers of alveolar macrophages like inhalation of inorganic dusts, myeloic leukemia, myelodysplastic syndrome, immunosuppression related to organ transplantation, and some infections including pneumocystis [ ] . not much is known on the clinical spectrum, course and treatment options in patients with molecularly defined, congenital pap due to mutations in the gm-csf alpha chain. also the role of long term wll, which are be very demanding due to the small size of the airways, how to measure clinical response to lavage therapy and the relevance of glucocorticoid therapy have not been reported. here we present the successful management of a child with a severe congenital pap caused by the homozygous p.ser x mutation in exon of the csf ra gene and its follow up for more than a decade. these data may be helpful for future treatment of infants and children with this rare condition. the patient was the nd of living children; born at term in , with no immediate postnatal respiratory distress. the family history was unremarkable for pulmonary or other rare diseases; the parents were consanguineous and of turkish descent. at age / years during an acute respiratory tract infection with productive cough and fever with no response to antibiotics, intermittent cyanosis occurred and the child was referred to our centre because of chronic tachypnoea and weight loss. because of a typical chest computed tomography (ct) and chest x-ray ( figure a , b), bal macroscopic appearance ( figure g ) and microscopy ( figure c , h), and after exclusion of infectious or metabolic causes or malignancy, pap was suspected and confirmed by histology ( figure f , i). figure diagnosis of alveolar proteinosis was suspected based on typical radiological picture in chest x-ray and ct with groundglass attenuation and interstitial thickening, resulting in crazy paving pattern ( figure. a, b) . figure. e shows ct after whole lung lavage of the right lung. macroscopic appearance ( figure. g) and light microscopy of bal fluid stained with pas ( figure. c, magnification x ) showed extracellular positive proteinaceous material, lipid-laden macrophages ( figure. h, mgg stain, magnification x ), and after exclusion of infectious or metabolic causes or malignancy, pap was confirmed by histology (right lower lobe). he stained tissue demonstrated alveolar filling with eosinophilic material ( figure. at presentation the child had global respiratory insufficiency, combined with an elevated level of ldh and cea ( figure a ). based on the clinical diagnosis of alveolar proteinosis we initiated whole lung lavages and some additional treatments. therapeutic bronchoalveolar lavages of either the right or left lung were performed under general anaesthesia and paralysis. from age / to years we used a technique that isolated one lung with the help of a balloon catheter ( - fr, arrow, reading, usa) placed into one figure long term clinical course and treatments. lactate dehydrogenase (ldh), height, weight for height (wfh) expressed as the current weight of the child as a percentage of the normal weight for a given height, capillary co pressure, and carcino embryonic antigen, cea in serum ( figure. main stem bronchus through the cuffed endotracheal tube (id . - . ) and blocked there (see also figure a ). the position and fitting of the catheter was permanently monitored via an olympus bronchoscope (bfn , o.d. . mm), as described in detail [ ] . from age onward the size of the main bronchus was sufficient to allow double lumen tube and lavage of one lung, while ventilating the other. sterile . % nacl warmed to body temperature was used as lavage fluid. the lavage was done by manual injection and withdrawal of saline from a ml syringe, starting with smaller volumes of ml/kg body weight (used for diagnostic purposes) and increasing to about to ml/kg under continuous control of the correct position and tightness of the balloon. the recovered fluid was collected via a -way cock stop into ml bottles to allow judgement of turbidity and to follow the process of lavage [ ] . in , at age y, we tried to increase the yield of lipo-proteins recovered from the lungs with the help of perfluorocarbons (pfc) [ , ] . a total of ml of perfourodecalin (pharmpur, augsburg, germany) was instilled in aliquots after the th, th, or th ml wash during three consecutive wll sessions. this approach did neither improve the yield of phospholipids washed out ( figure a , b) nor that of total protein (not shown). empirically, we found that wll were the most efficient treatment. this was clearly shown for the short term; during instances investigated until the age of , the amount of nasal oxygen flow was reduced in after the lavages ( figure b ). this effect could be sustained for many years demonstrating long term efficacy. however the extreme value of wll was only very recently figure whole lung lavages were performed, due to the small sizes of the airways until the age of y, via a blocked endotracheal tube through which the child was ventilated ( figure. a, .) and through which also a pulmonary artery catheter ( figure. a, ., blue) was inserted and blocked in the left or right main stem bronchus (small syringe) and the lavage was done ( figure. a, ., large syringe). the tight fit of the blocked pulmonary artery catheter was continuously monitored via a . mm endoscope ( figure. a, ., black). figure. demonstrated unequivocally following implementation of our concept of very aggressive wll. up to the age of years, wll were done more or less to ameliorate partial respiratory insufficiency, i.e. to decrease the need for additional oxygen. from year onward, we performed one lavage per month, in order to try to completely clear the lung from its proteinosis load. this approach was very successful and resulted in complete resolution of partial respiratory insufficiency for the first time. the patient started puberty, growth and weight were sustained by oral nutrition without need of using the percutaneous tube and the dependency on supplemental oxygen up to that point in time, could be finished. this also led to increased self-confidence and better integration at school. also, the lung function improved very rapidly and chest radiograph cleared to almost normal ( figure d , e; figure c ). together somato-psychosocial condition substantially improved. a consecutive brief trial to increase the time lag between the lavages failed and an interval of about weeks was maintained. because at clinical diagnosis of the patient, both the exact cause of the pap and effective treatments in small children were unknown, empirical high dose glucocorticosteroids in pulses were used ( figure a ) and under the impression that they might be helpful, systemic corticosteroids were used for prolonged periods until the age of . years. during this time, azathioprine as a steroid saving agent was also used without any apparent benefit. however, we clearly observed that severe infectious complications were only observed during the time of increased immunosuppression by these agents (figure a, b) . at years of age an aspergillus fumigatus infection with formation of a cavity, leading to severe cardio-respiratory failure and resuscitation followed by resection of the left lower lobe ( figure b , figure a -d). at age . years she suffered a pulmonary para-influenza infection, leading to ards and necessitating mechanical ventilation. additionally, the child had many mild respiratory exacerbations, mostly believed to be induced by viral upper-and lower respiratory tract infections. figure at age y, during four consecutive whole lung lavage sessions perfluorocarbon (pfc) was instilled into the lungs after the th, th, or th of the ml wash in order to enhance the recovery of surfactant material. the concentration of phosphoplipids (and total protein (not shown)) washed out was not significantly altered compared to lavages done at that age without pfc ( figure. a, b) . from age to / years therapy with inhaled and subcutaneous injections of recombinant gm-csf was done (see also figure. b). although the intervals between consecutive therapeutic whole lung lavages were increased with gm-csf treatment the amount of protein removed was also increased, demonstrating no reduction of protein amount with gm-csf treatment ( figure. c, d) . note that not all lavages were available for total protein measurements. of interest, from age to / years, well before the molecular nature of the pap was determined, we used inhaled and subcutaneous recombinant gm-csf ( figure b) . a transient increase in peripheral blood eosinophils up to % of the neutrophils occurred [ ] (data not shown), but clearly no improvement of the alveolar proteinosis (figure a, b, figure c , d). due to the expectations of the treating physicians, the intervals between consecutive therapeutic wll were increased during gm-csf treatment: in parallel, this was associated with an increased load of protein, demonstrating a lack of an effect of gm-csf treatment ( figure c, d) . nutritional support was optimized with the help of a percutaneous gastrostomy (peg, figure b ) placed at the age of years, which was used regularly; the gastrostomy was changed to a jejunostoma at the age of / years, to completely exclude gastro-esophageal refluxes, although no such events had been demonstrated in phor impedance studies ( figure b ). at age years (in ) analysis of the patient's csf ra gene revealed the homozygous ser x stop-mutation in exon resulting in the almost complete absence of the gm-csf receptor alpha chain and causing the alveolar proteinosis we observed ( figure a ). the parents were heterozygous for the mutation (figure a, b) . mutations in sftpc, sftpb and abca were excluded. gm-csf level were increased in serum ( pg/ml, normal < pg/ml) of the child and normal in the parents. no anti-gm-csf autoantibodies were detected in serum [ ] . gm-csf-ra chain expression after stimulation with ng/ml gm-csf on peripheral mononuclear cells of the patient was markedly reduced and normal in both parents ( figure c ). in the absence of gm-csf stimulation, gm-csf-ra chain in the parents was only % of that of the controls ( figure a ), whereas gm-csf-rb chain and cd b were normal. after stimulation with gm-csf, gm-csf-ra and cd b remained low in the patient ( figure a, lower panel) , whereas the parents' levels were in the normal range level. gm-csf-rb chain without and with gm-csf stimulation was normal ( figure a ). cd b expression on the neutrophils was used to assess signal transduction after gm-csf stimulation. in the patient, similar as blocked by auto-antibodies from a subject with auto-immune pap, no dose-dependent stimulation of cd b was observed, demonstrating interruption of signalling ( figure b ). here we report a patient with molecularly defined severe congenital pap due to a previously undescribed autosomal recessive mutation in the alpha chain of the gm-csf receptor. this mutation leads to a stop of transcription and to a lack of functional protein. the gm-csf induced responses are mediated through activation of the transcription factor pu. and include increased surfactant catabolism and cd b expression [ ] . impairment of the latter was shown directly in mononuclear cells of the patient after stimulation with gm-csf. impaired gmcsf receptor activation of alveolar macrophages leads to decreased surfactant catabolism and accumulation of surfactant in the alveolar space, i.e. alveolar proteinosis. important messages from this study are related to the long-term management of this condition. first, persistent and aggressive removal of surfactant filling the alveolar space may eliminate gas exchange abnormalities and consecutive sequelae including developmental and growth failure, and restricted level of performance due to respiratory limitation. second, immune insufficiency, a figure. b) . flow cytometric analysis of peripheral blood cells demonstrates the absence of the alpha-chain ( figure. c) . isolated neutrophils were incubated with antibodies, washed with dulbecco's pbs twice and measured by means of flow cytometry. ten thousand cells were counted and analyzed by the facsdiva software. fc blocking and isotype controls were applied to exclude unspecific bindings. cd b (mouse monoclonal igg , pe conjugated), cd /gm-csf-r alpha (mouse monoclonal igg , pe conjugated), cd w/gm-csf-r beta (mouse monoclonal igg , purified) and secondary antibody for cd w (rat antimouse igg , pe-conjugated). problem also primarily resulting from abnormalities of the gm-csf signal transduction pathway [ ] , may be augmented by immunosuppressive therapy initiated to treat the condition empirically. therefore, molecular genetic definition of the basic defect in all children with pap is important. lastly, we describe the successful use of outcome measures of the efficacy of therapeutic wll, including oxygen demand, and amount of washed out protein and phospholipids. a major strength of this study is to demonstrate the feasibility of technically demanding repetitive wll in a very small child over extended periods of time. although therapeutic wll is generally accepted as the established treatment option for pap in adults, its optimal method, frequency of application and many other details are currently not known in infants or children. here we show that consecutive lavages via a small catheter located in a main stem bronchus ( figure a) can be used to efficiently remove accumulated surfactant from the alveolar space in a very small child. furthermore we show that it is helpful to monitor efficacy of the washing procedure by determination of proteins and lipids removed from the lungs [ ] . these measurements allowed us to demonstrate only a marginal, but not clinically significant increase in the removal of surfactant material from the lungs, by the use of pfc for lavage. in a case report on an infant with alveolar proteinosis due niemann pick disease the usage of pfc was recently shown not to be of benefit as well [ ] . although feasibility of the long term management of congenital pap with wll was demonstrated in this cd b. the purity of neutrophils was greater than % as assessed by differential cell counts of pappenheim cytospin preparations. cell viability was greater than % using trypan-blue exclusion method. neutrophils were treated with ng/ml gm-csf or the same volume of the vehicle buffer (aqua a.i.) for min at °c. in the absence of gm-csf stimulation, gm-csf-ra chain expression in the parents was only % of that of the controls ( figure. a) , whereas gm-csf-rb chain and cd b expression were normal. after stimulation with gm-csf, gm-csf-ra in the patient remained low, whereas the parents' level increased to almost control level. gm-csf-rb chain and cd b stimulation were normal ( figure. a) . cd b expression on the neutrophils was used to assess signal transduction after gm-csf stimulation. in the patient, similar as blocked by auto-antibodies from a subject with auto-immune pap, no dose-dependent stimulation of cd b was observed, demonstrating interruption of signalling ( figure. case of severe pap, molecular diagnosing pap as caused by a genetic deficiency of gm-csfra may have other important prophylactic and therapeutic implications. first, based on experiments in mice with pap bone marrow transplantation may cure the disease [ ] . currently we believe however that the risks of a bone marrow transplant (chronic graft versus host disease, among others) outweigh its benefits (elimination of need for wll). second, if diagnosed early in a family with an index case, the opportunity of early intervention by lavages at times of good clinical condition will help to reduce complications. subcutaneous injections or inhalations of gm-csf, which have been successfully utilized in adult patients with autoimmune pap [ , ] , were not helpful in our case to reduce alveolar filling as assessed by ct scanning (not shown) or improvement in gas exchange ( figure ). treatment with μg/kg of gm-csf per day subcutaneously was also shown to be ineffective for the child with congenital pap described by martinez-moczygemba et al. [ ] . immunosuppressive treatment was used empirically and because of the presence of neutrophils and some lymphocytes in the lavage specimens and in the interstitial space of the lung biopsy sample of the patient ( figure b ). unfortunately severe and prolonged infections occurred, including a cavity forming infection with aspergillus fumigatus which was treated by i.v. and inhaled amphotericin b and surgical resection of the cavity. sustained withdrawal of the systemic corticosteroids from age years onward did not alter the activity of the underlying pap, but reduced the rate of infectious respiratory complications considerably. our study exemplifies detailed long term management of severe molecularly defined alveolar proteinosis from childhood into young adolescence. it is of interest that a dedicated specialized team may be advantageous to maintain the appropriate expertise of complex procedures such as e.g. whole lung lavages in small children [ , , , ] . therefore a centralized approach, as it has been employed for rare lung diseases and pap in particular on a national basis in france [ ] , may be warranted. a web-based system to collect these rare cases, follow them and also to receive support is available at the kids lung register (http://www.kids-lung-register.eu). the novel whole lung lavages technique using an inflatable balloon catheter was feasible in very small sized airways. whereas empirical immunosuppressive therapy and inhaled and subcutaneous gmcsf were without significant benefit, a very intense treatment with wll resulted in complete resolution of respiratory insufficiency, and a normalisation of lung physiology and overall somato-psychosocial condition of the child. abbreviations pap: pulmonary alveolar proteinosis; gmcsf: granulocyte-macrophagecolony stimulating factor; gm-csfr: gm-csf receptor; wll: whole lung lavage; washing of a single right or left lung. pulmonary alveolar proteinosis pulmonary alveolar proteinosis: progress in the first years mice deficient for the il- /gm-csf/il- beta c receptor exhibit lung pathology and impaired immune response, while beta il receptordeficient mice are normal involvement of granulocyte-macrophage colony-stimulating factor in pulmonary homeostasis high-affinity autoantibodies specifically eliminate granulocytemacrophage colony-stimulating factor activity in the lungs of patients with idiopathic pulmonary alveolar proteinosis familial pulmonary alveolar proteinosis caused by mutations in csf ra pulmonary alveolar proteinosis caused by deletion of the gm-csfr alpha gene in the x chromosome pseudoautosomal region hereditary pulmonary alveolar proteinosis: pathogenesis, presentation, diagnosis, and therapy adultonset hereditary pulmonary alveolar proteinosis caused by a single-base deletion in csf rb mutations in the surfactant protein c gene associated with interstitial lung disease brief-report: deficiency of pulmonary surfactant protein b in congenital alveolar proteinosis respiratory disease in niemann-pick type c is caused by pulmonary alveolar proteinosis successful whole lung lavage in pulmonary alveolar proteinosis secondary to lysinuric protein intolerance: a case report whole-lung lavage in infants and children with pulmonary alveolar proteinosis therapeutic lung lavages in children and adults liquid ventilation in an infant with pulmonary alveolar proteinosis therapeutic lung lavage in the piglet model of meconium aspiration syndrome gcsf gene is expressed but not rearranged in a patient with isochromosome q positive acute nonlymphocytic leukemia anti-gm-csf antibodies in pediatric pulmonary alveolar proteinosis pulmonary alveolar proteinosis, a primary immunodeficiency of impaired gm-csf stimulation of macrophages bronchioalveolar lavage with perfluorochemical liquid during conventional ventilation an open-label trial of granulocyte macrophage colony stimulating factor therapy for moderate symptomatic pulmonary alveolar proteinosis inhaled granulocyte/macrophage-colony stimulating factor as therapy for pulmonary alveolar proteinosis pulmonary alveolar proteinosis in children partial and massive lung lavages in an infant with severe pulmonary alveolar proteinosis pre-publication history the pre-publication history for this paper can be accessed here long-term follow-up and treatment of congenital alveolar proteinosis the universities institutional review board approved the study (ek and . . ) and written informed consents of the patient, the parents of the index case and of the control subjects were obtained. written informed consent was obtained from the patient and the parents for publication of this case report and any accompanying images. a copy of the written consent is available for review by the editor-in-chief of this journal. supported by grants from e-rare and bmbf gold.net. authors' contributions mg designed the study, oversaw the biochemical analysis, participated in the calculation and presentation of the data, and wrote the draft of the manuscript. he is taking responsibility for the integrity of the work as a whole. jr has retrieved the data from the patients files, calculated and correlated the results, as, ap, asch and ah prepared the samples, performed cell sorting and in vitro work, as well as the biochemical analysis, pil and pel did extensive genetic analysis, fb did all pathology work. bs, om, cs, jg-p, tn, kr and mg were engaged in treating the patient, performing lavages, designing clinical interventions, discussing and putting the data together. all authors read and approved the final manuscript. the authors declare that they have no competing interests. key: cord- - rv p qh authors: zanella, m.-c.; lenggenhager, l.; schrenzel, j.; cordey, s.; kaiser, l. title: high-throughput sequencing for the aetiologic identification of viral encephalitis, meningoencephalitis, and meningitis. a narrative review and clinical appraisal date: - - journal: clin microbiol infect doi: . /j.cmi. . . sha: doc_id: cord_uid: rv p qh background: viral aetiologies are the most common cause of central nervous system (cns) infections. approximately one-half of cns infections remain of undetermined origin. high-throughput sequencing (hts) brought new perspectives to cns infection investigations, allowing investigation of viral aetiologies with an unbiased approach. hts use is still limited to specific clinical situations. objectives: the aim of this review was to evaluate the contribution and pitfalls of hts for the aetiologic identification of viral encephalitis, meningoencephalitis, and meningitis in cns patient samples. sources: pubmed was searched from january to august to retrieve available studies on the topic. additional publications were included from a review of full-text sources. content: among studies retrieved, used hts as a diagnostic technique. hts was performed in cerebrospinal fluid and brain biopsy samples of patients, including immunocompromised, immunocompetent paediatric, and adult cases. hts was performed retrospectively in studies and prospectively in . hts led to the identification of a potential causal virus in patients, with viruses known and ten not expected to cause cns infections. various hts protocols were used. implications: the additional value of hts is difficult to quantify because of various biases. nevertheless, hts led to the identification of a viral cause in % of encephalitis, meningoencephalitis, and meningitis cases in which various assays failed to identify the cause. hts should be considered early in clinical management as a complement to routine assays. standardized strategies and systematic studies are needed for the integration of hts in clinical management. meningitis, encephalitis, and meningoencephalitis are caused by various pathogens, but viral aetiologies are the most common cause [ e ] . among these, enterovirus (ev), herpes simplex type and (hsv- and hsv- ), and varicella zoster virus (vzv) are the most frequent viruses associated with encephalitis, meningoencephalitis, and meningitis in paediatric and adult populations [ , , e ] . the prevalence of other viruses varies according to the geographical location and immune status of the patient. during the last two decades, the implementation of molecular assays as a complement to serological assays, immunohistochemistry, and culture have improved the diagnosis of viral central nervous system (cns) infections. nevertheless, these assays have limitations because of their targeted approach. apart from technical limitations, the diagnosis of viral cns infections is subject to several issues, such as the type of sample (cerebrospinal fluid (csf) or brain biopsy), the timeline of sample collection, and the different pathogenic mechanisms of viruses. despite technical progress, approximately one-half of encephalitis, meningoencephalitis, and meningitis cases remain of unknown origin [ , , , ] . high-throughput sequencing (hts) has brought new perspectives to cns infection investigations. although hts has been recently integrated in encephalitis management guidelines [ ] , its use is still limited to specific clinical situations or research. the contribution of hts warrants a better appraisal for further implementation in cns infection management. this narrative review aims to evaluate the contribution and pitfalls of hts for the aetiologic identification of viral encephalitis, meningoencephalitis, and encephalitis in paediatric and adult patients. a comprehensive pubmed search was conducted from january to august to identify human studies using the following mesh and keywords research algorithm: '((central nervous system infection or cerebrospinal fluid or central nervous system) and sequencing) and virus'. additional publications were identified from a review of full-text sources. the title and abstract of each citation were screened by two reviewers and assessed for eligibility by detailed analysis. inclusion criteria were studies including patients with encephalitis, meningoencephalitis, or meningitis of unknown origin and reporting the use of hts for the aetiologic identification of a viral origin in cns samples. exclusion criteria were reviews, animal studies, other cns diseases, and studies addressing only technical aspects. a total of references were retrieved (fig. twenty-nine studies were selected for qualitative analysis ( case reports, ten case series) ( table ) . fourteen and studies concerned paediatric and adult cases, respectively, and two studies concerned both populations (table ) . hts was performed in cases ( adults; paediatric (< years); cases with no information on age). twenty-five studies reported patient age (median age, years; range, months to years). diagnostic criteria for encephalitis, meningoencephalitis, and meningitis were inconsistently reported. immune status was reported for patients and comprised immunocompromised (nine paediatric, adults) and immunocompetent patients ( paediatric, eight adults). studies came from a wide range of regions: europe (ten), north america (ten), asia (six), and oceania (three) ( table ) . when brain specimens were available, pathological examination provided proof of diagnosis of encephalitis. csf analysis results were reported in patients of studies, with the white blood cell count ranging from to cell/mm in encephalitis and meningoencephalitis cases and cell/mm in the only meningitis case [ ] ; three publications reported normal csf analysis without any description [ e ]. hts was performed on individual csf and brain specimens in and patients, respectively. csf samples of patients were pooled for hts analysis [ e ]. hts was performed on csf and brain specimens in five patients [ , e ] . positive results on both samples were obtained in a cache valley virus chronic meningoencephalitis case [ ] and positive results on brain biopsies only were reported in two human astrovirus (hastv)-va and tick-borne encephalitis (tbev) cases [ , ] . in one patient, hts analysis did not identify a viral cause, but balamuthia mandrillaris was identified in both csf and brain biopsy [ ] . despite limitations because of publication bias, the overall diagnostic yield for a viral aetiology according to sample type was estimated to be higher for brain specimens ( / ( . %) positive samples) than for csf samples ( / ( . %) positive samples). detailed microbiological investigations performed prior to hts varied according to local practice and were not reported in seven studies [ , e ] . in most studies, viruses identified with hts were not part of the microbiological work-up, except in three cases where hts identified a virus for which diagnostic assays were negative during routine investigation. these included a west nile virus (wnv) identified in the csf sample of a renal transplant recipient with meningoencephalitis and a negative serological assay [ ] . hsv- was identified in an encephalitis case [ ] . a vaccine strain of mumps virus was identified in a brain specimen of an immunosuppressed child with chronic encephalitis in whom (rt)-pcr for mumps on a csf sample was negative as the assay did not target vaccine strains [ ] . hts was performed retrospectively in studies and prospectively as part of the initial work-up in case reports with an impact on the clinical management of three immunocompromised patients: a child with encephalitis associated with hastv-va [ ] ; an adult with encephalitis associated with hastv-va [ ] ; and an adult with chronic meningoencephalitis associated with cache valley virus [ ] . turnaround times were reported in ten studies [ , , , , , , e ]. among publications in which hts was used prospectively, turnaround times ranged from hours to days [ , , , , , , ] . hts performed on pooled or individual samples and/or subsequent confirmatory assays allowed the identification of a potential causal virus in of patients ( . %), comprising paediatric cases (eight immunocompromised cases), adult cases (nine immunocompromised cases) and nine cases for whom age was not specified; median age was years (range months to years). fig. shows the distribution of viruses identified according to patient immune status and clinical manifestations. hts allowed the identification of viruses previously unknown or unexpected as a cause of cns infection (n ¼ ) and thus not screened during diagnostic investigations (table ) . these included parvovirus (two) [ ] , human coronavirus oc (one) [ ] , and novel hastv-mlb (one) [ ] identified in immunocompetent patients. a mumps virus vaccine strain (one) [ ] , hastv (undetermined specie; one) [ ] , hastv-va (four) [ , , , ] , and hastv-mlb (one) [ ] were identified in immunocompromised patients. a gemycircularvirus was also identified, but its causal role in encephalitis is under debate [ ] . three novel viral species or strains were identified in csf samples of patients with encephalitis (table ) : human csf-associated densovirus (hucsfdv ) [ ] ; cyclovirus viet-nam (cycv-vn) [ ] ; and lymphocytic choriomeningitis virus (lcmv)-related arenavirus [ ] . hts analysis also identified viruses known to be responsible for cns infections (n ¼ ) and not screened or detected by routine assays (hsv- , hsv- , vzv, epsteinebarr virus (ebv), tbev, wnv, cache valley, saint louis encephalitis, toscana, mumps, measles, and coxsackie a virus) [ , , , , , , , , ] (table ) . most studies performed nucleic acid extraction protocols dedicated to rna, or rna and dna. thirteen rna and seven dna viral species were identified (table ) . one study reported the identification of hsv- in a csf sample after rna extraction protocol [ ] . six studies where hts analysis was not restricted to the detection of viruses resulted in the identification of bacterial (brucella melitensis and leptospira santarosai) [ , ] , mycobacterial (mycobacterium tuberculosis) [ ] , and parasitic (balamuthia mandrillaris) [ , ] or fungal (candida tropicalis and fusarium solani and oxysporum) [ ] pathogens. the use of controls was not systematically reported. nine studies reported various negative control samples, such as brain specimens without encephalitis [ ] , csf samples from patients without infection [ , , , , , ] , serum samples, and water or elution buffer [ , , , ] . viral sequences of negative controls were not consistently described. positive controls, such as csf or serum samples positive for dna or rna viruses, were rarely reported or used [ , , , , ] . to address the specificity of hts results, other techniques were performed to confirm hts results in all studies, except one [ ] . (rt)-pcr assays were performed in samples from patients and were positive in at least one sample in patients. hts results were also confirmed with serological assays [ , , , ] , immunohistochemistry, and in situ hybridization [ , , , , , , ] . viral culture confirmed the presence of a replicative saint louis encephalitis virus in a csf sample [ ] , but was unsuccessful concerning a cache valley virus [ ] . hts offers the possibility of investigating viral aetiologies of cns infections by an unbiased approach when work-up according to guidelines fails to identify a causal pathogen. based on the studies retrieved, its diagnostic yield for a viral aetiology is difficult to estimate, particularly because of publication bias (high number of case reports), methodological heterogeneity, and a lack of systematic prospective studies. when focusing specifically on case series, the diagnostic yield for the identification of a viral cause was approximately %, but this result should be interpreted with caution in the light of the evolution of the technique from to . among the studies reviewed, the hts contribution is evident not only for the identification of a potential causal virus in cns infections of unknown origin, but also in the detection of novel or divergent viruses [ , , ] . similar to other techniques, the type of sample used for analysis is of particular importance. despite diverse hts protocols and publication bias, hts seemed to have a higher diagnostic yield in brain specimens than in csf samples. the diagnostic yield was particularly low in two studies where hts was performed on csf supernatant [ , ] . hts also shows its clinical value in situations where the viral pathogenic mechanisms and specific clinical situations impairs the results of conventional assays [ ] . among immunocompetent patients, hts led to the identification of viruses not previously associated with cns infections (parvovirus , cycv-vn, gemycircularvirus, and novel hastv-mlb ) [ , , , ] . hts clinical impact was mainly demonstrated among immunocompromised patients, with most studies dedicated to this population. it was performed prospectively in cases and led to a change in clinical management in three [ , , ] . the rapid decision to perform hts, short hts turnaround times, and the efficient interpretation of results were determinant for the management of these latter patients. among immunocompromised patients, hts contributed to the detection of viruses for which no assay was performed during conventional work-up: viruses known to cause cns infections (tbev, wnv, cache valley virus, saint-louis encephalitis virus, ebv) [ , , , , ] , viruses not known to be responsible for cns infections (novel hastv-va , hastv-mlb , human coronavirus oc , mumps virus vaccine strain) [ e , , , , , ] , and novel viruses (lcmv-related arenavirus) [ ] . focusing on novel hastv, hts brought new insights in our understanding of their association with cns infections [ e , , , ] . furthermore, all (rt)-pcr assays performed retrospectively confirmed hts results, thus highlighting the specificity of hts. in the absence of standardization, the methodological heterogeneity of studies is striking, not only concerning pre-analytic steps, but also hts per se, with the use of diverse hts platforms, single or paired protocols, as well as diverse bioinformatic pipelines and databases. the use of positive controls as quality controls was only reported in seven studies [ , e , , , ] . addressing the issue of contamination, only a few studies reported the use of negative controls [ , , , , , , , , ] . viral sequences assigned to viruses not considered as the cause of cns infection were not consistently performed: studies provided a description for one cns sample or more [ , , , , e , , , , ] . for most of these viral sequences, no interpretation of results was explicitly provided. among reads of viruses known to cause infections in humans, anelloviridae [ ] and herpesviridae were the most described in four and seven samples, respectively; human pegivirus reads were identified in one sample. the genome of the torque teno virus, a member of the anelloviridae family, and human pegivirus have been identified in cns samples, but without any association with a cns disease so far [ e ] . other viral sequences were mostly assigned to viruses infecting plants or non-vertebrates and were considered to be reagent contaminants. the minimal description of these hts 'background' results impairs the comprehension of the composition of the cns virome. furthermore, the integration of hts results in the clinical context is of particular importance and the absence of standardization of any reporting methods precludes an objective interpretation of these results. finally, hts-negative results could be interpreted in the context of several clinical and technical aspects that could impact on the sensitivity of the method. first, from a clinical point of view, differences in diagnostic yield from a biopsy compared with csf samples could be explained by several factors: patient selection (cases of encephalitis); the type of sample (e.g. multiple pooled post-mortem brain samples); and the timeline of sampling in the context of encephalitis (biopsy positivity could possibly be less affected by time than csf). from a technical point of view, it should be considered that this narrative review includes studies from to and thus takes into account the tremendous evolution of the hts technique over this last decade. several technical issues need to be considered for the interpretation of negative results: pre-analytic steps (e.g. the use of fresh, frozen, or paraffinembedded samples for analysis, extraction protocols, fragmentation methods, library preparation, paired-end versus single-end protocols); sequencing depth; sequencing platforms (table ) ; and the analysis of hts raw data (e.g. mapping software, viral databases, and pipeline precision). this review highlights that the use of hts in investigations concerning a viral cause of encephalitis, meningoencephalitis, and meningitis could extend not only to immunocompromised, but also to immunocompetent patients. considering the selection and publication bias of the literature reviewed here, the negative predictive value for the aetiologic identification of viral encephalitis, meningoencephalitis, and meningitis is difficult to quantify and further studies are needed. hts needs to be integrated in clinical management as a second-line technique or in parallel to first-line investigations when a standard work-up according to guidelines [ , ] and additional investigations considering local epidemiology and specific clinical situations fail to identify a causal agent. brain biopsy should also be considered. furthermore, hts is of particular interest for the screening of a large panel of viruses, particularly to avoid a restricted screening of low-volume clinical samples, such as in paediatric patients. hts brought new perspectives to the investigations of infectious diseases. notably, its unbiased approach is of particular interest in samples that would not usually be tested in specific syndromes. its use may not only be restricted to cns samples, but also extended to other clinical samples. this is illustrated by the positive results of (rt)-pcr assays performed on blood or plasma samples collected at the time of neurological manifestations, which allowed the identification of the same virus detected by hts in cns samples (parvovirus , lcmv-related arenavirus, novel hastv-va , novel hastv-mlb ) [ , , , ] . this could be of particular interest when a cerebral biopsy cannot be performed and a disseminated infection occurs or is suspected, particularly in immunocompromised patients. an early decision to perform hts, short hts turnaround times, and an efficient interpretation of results are major issues for allowing hts to contribute to clinical management. for prospective hts use in clinical routine, this timeframe should be as short as possible for clinical decision-making. among publications in which hts was used prospectively, reported turnaround times ranged from hours to days [ , , , , , , ] . hts use is still restricted to a limited number of diagnostic laboratories considering the cost of analysis and informatics infrastructures needed (e.g. costs of sequencing platforms, computing resources, data storage). despite the expanding use of hts in clinical microbiology, the surprisingly low number of studies retrieved for this review might be explained for several reasons, including financial limitations when considering the costs of the analysis, the need for shorter turnaround times, and the limitations cited above. addressing the question of the proof of causality, particularly in the context of pathogen discovery, lipkin proposed several criteria for pathogen causality with grading certainty according to confirmation with serological assays or culture for instance [ ] . most studies confirmed hts results with (rt)-pcr assays and a few with cell culture, serological assays, and immunohistochemistry. thus, hts should be implemented in clinical routine in association with other diagnostic tests. in most studies, the approach to establish causality was not explicitly described, but was reported as the temporal association of clinical manifestations and the identification of viral sequences of a specific virus using hts on cns samples at the time of manifestations. this process was only described in few studies. similar to other molecular tests such as (rt)-pcr, the detection of viral sequences or genome in a clinical sample should be interpreted with caution in the clinical context. in a near future, the process for the establishment of causality in hts analysis should be more transparent and should comprise multidisciplinary sessions involving infectious disease specialists and bioinformatics experts, not only for results concerning viruses unexpected to cause cns infections. finally, for hts implementation in clinical routine, the question of standardization has to be addressed concerning hts protocols, data analysis algorithms, reference databases and quality controls, and further prospective studies are needed [ ] . this review shows that hts contributed to the identification of potential viral aetiologies of encephalitis, meningoencephalitis, and meningitis of unknown origin in approximately % of cases and is of particular interest in immunocompromised patients. this unbiased or semi-unbiased approach led to the identification of novel viruses, viruses known or not expected to cause cns infections. standardized strategies are needed for the further implementation of hts in clinical management. in centres where available, the decision to perform hts should be considered early in the management of encephalitis, meningoencephalitis, and meningitis in as a second-line technique or in parallel to recommended 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next-generation sequencing tests for universal pathogen detection the authors would like to acknowledge rosemary sudan (geneva university hospitals, switzerland) for editorial assistance. key: cord- -y knt g authors: parkhomenko, taisiya a.; doronin, vasilii b.; castellazzi, massimiliano; padroni, marina; pastore, michela; buneva, valentina n.; granieri, enrico; nevinsky, georgy a. title: comparison of dna-hydrolyzing antibodies from the cerebrospinal fluid and serum of patients with multiple sclerosis date: - - journal: plos one doi: . /journal.pone. sha: doc_id: cord_uid: y knt g it was found that high-affinity anti-dna antibodies were one of the major components of the intrathecal igg response in multiple sclerosis (ms) patients [williamson et al., pnas, ]. recently we have shown that iggs from the sera of ms patients are active in the hydrolysis of dna. here we have shown, for the first time, that average concentration of total proteins ( -fold), total iggs ( -fold) and anti-dna antibodies ( -fold) in the sera is significantly higher than that in the cerebrospinal fluid (csf) of fifteen ms patients. the relative activities of total protein from sera and csfs varied remarkably from patient to patient. it was surprising that the specific dnase activity of the total protein of csf reparations were -fold higher than the serum ones. electrophoretically and immunologically homogeneous iggs were obtained by sequential affinity chromatography of the csf proteins on protein g-sepharose and fplc gel filtration. we present first evidence showing that iggs from csf not only bind but efficiently hydrolyze dna and that average specific dnase activity of homogeneous antibodies from csf is unpredictably ∼ -fold higher than that from the sera of the same ms patients. some possible reasons of these findings are discussed. we suggest that dnase iggs of csf may promote important neuropathologic mechanisms in this chronic inflammatory disorder and ms pathogenesis development. multiple sclerosis (ms) is a chronic demyelinating pathology of the central nervous system presenting a serious medical and social problem. its etiology remains unclear, and the most valid theory of its pathogenesis assigns the main role in the destruction of myelinproteolipid shell of axons to inflammation related with autoimmune reactions ( [ ] and refs therein). although the t-cell immune system plays a leading role in ms pathogenesis, normal functioning of the b-cell system is also important for the disease development. an enhanced synthesis of immunoglobulins (usually iggs), their free light chains, and of polyspecific dna binding antibodies (abs) interacting with phospholipids are observed in ms patients [ ] . new keys for understanding ms pathogenesis have appeared after cloning igg repertoire directly from active plaques and periplaque regions in ms brains and from b-cells recovered from the cerebrospinal fluid of a patient with ms with a subacute disease [ ] . it was found that high affinity anti-dna abs were a major component of the intrathecal igg response in ms patients. furthermore, dna-specific monoclonal abs derived from two ms individuals as well as a dna-specific ab derived from a systemic lupus erythematosus (sle) patient bound efficiently to the surface of neuronal cells and oligodendrocytes. for these abs, cell-surface recognition was dna-dependent. the findings indicate that anti-dna abs may promote important neuropathologic mechanisms in chronic inflammatory disorders, such as ms and sle [ ] . artificial abzymes (catalytic antibodies against transition state analogues of chemical reactions) and natural abzymes are novel biological catalysts that have attracted much interest in the last years (reviewed in [ ] [ ] [ ] [ ] [ ] ). artificial abzymes are abzymes against analogs of transition states of catalytic reactions [ ] [ ] [ ] [ ] [ ] or antiidiotypic abs induced by a primary antigen, which may show some of its features including the catalytic activity (for review also see [ ] [ ] [ ] [ ] [ ] ). during past two decades it has become clear that auto-antibodies (auto-abs) from the sera of patients with different autoimmune diseases can possess enzymatic activities and that their occurrence is a distinctive feature of autoimmune diseases (reviewed in [ ] [ ] [ ] [ ] [ ] ). it is thought that abzymes may play a significant role in forming specific pathogenic patterns and clinical settings in different autoimmune conditions through their broadened auto-ab properties [ ] [ ] [ ] [ ] [ ] . patients with autoimmune diseases produce abs to nucleoprotein complexes, to dna and to enzymes that participate in nucleic acid metabolism [ ] [ ] [ ] [ ] [ ] . in autoimmune diseases, there can be a spontaneous induction of anti-idiotypic antibodies, which are abs elicited by a primary antigen, including some with catalytic activity, or transition from polyreactive catalytic activity to autoantigen-directed activity. natural abzymes hydrolyzing dna, rna, polysaccharides, oligopeptides, and proteins are present in the serum of patients with several autoimmune and viral diseases (reviewed in [ ] [ ] [ ] [ ] [ ] ). healthy humans do not develop abzymes with detectable dnase and rnase activities, their levels being usually on the borderline of sensitivity of the detection methods [ ] [ ] [ ] [ ] [ ] . it has recently been shown that myelin basic protein (mbp)hydrolyzing activity is an intrinsic property of iggs, igms, and igas from the sera of ms patients [ ] [ ] [ ] [ ] [ ] [ ] . recognition and degradation of mbp peptides by serum auto-abs were confirmed as a novel biomarker for ms [ ] [ ] . the established ms drug copaxone appears to be a specific inhibitor of mbp-hydrolyzing abzyme activity [ ] . at the same time, as mentioned above, anti-dna abs were found to be a major component of the intrathecal igg response in ms patients [ ] . first dnase abzymes were found in the sera of sle patients [ ] . then, it was shown that iggs and igms from the sera of ms patients effectively hydrolyzed dna, rna, and oligosaccharides [ ] [ ] [ ] [ ] [ ] . whereas only and % of ms patients contained increased concentrations of abs to native and denatured dna, respectively, as compared with healthy donors, dnase abzymes were found in , - % of ms patients [ , , ] . since dnase abzymes of ms patients [ ] similarly to sle patients [ ] are cytotoxic, cause nuclear dna fragmentation and induce cell death by apoptosis, they can play an important role in sle and ms pathogenesis. taking these observations into account, analysis of relative concentrations of proteins, canonical enzymes and dnase abzymes in the cerebrospinal fluid (csf) of ms patients is of special interest. in the present study we have used different approaches to provide, for the first time, a very strong direct evidence that dnase activity is intrinsic to iggs from csf of ms patients and compared some other parameters characterizing the ms csf and sera. fifteen patients ( women and men) satisfying the criteria for clinically or laboratory-supported definite ms according to [ , ] were retrospectively selected for the study. of these, were relapsing-remitting (rr), and were primary progressive (pp) in agreement with the criteria of lublin and reingold [ ] . clinical course (rr and pp), clinical activity (relapse at time of sampling), and mri activity (the presence of gadolinium enhancing lesions at mri examination) were analyzed as described previously [ ] . the characteristics of the ms patients are summarized in table . it was interesting to compare some different indexes for csf and sera of ms patients. therefore, first we measured a relative concentration of total protein in csf and sera of ms patients ( table ). the relative concentrations of total protein of csfs (range . - . mg/ml) and sera ( - mg/ml) of fifteen ms patients varied in different ranges. the average concentration of the total protein in the serum ( . . mg/ml) was , -fold higher compared with csf ( . . mg/ml) and these values did not demonstrate good correlation (coefficient correlation (cc) = . ), table ). the relative concentrations of total iggs in the serum and csf were first measured using an immunoblotting test system. the relative concentration of total iggs in the serum (range . - . mg/ml; average value . . mg/ml) was -fold higher than that for the csf (range . - . mg/ ml; average value . . mg/ml) and there was not good correlation between these values, cc = + . . iggs from the serum and csf were purified by affinity chromatography on protein-g sepharose and igg-protein concentration was estimated in the peaks eluted from the sorbent by an acidic buffer (see below). the relative concentration of total iggs determined by two different methods was the same within the experimental error. interestingly, the concentration of total protein in csf was . -fold higher than total iggs, while this difference in the case of the serum ( . -fold) was by a factor of . lower. we estimated the relative concentration of anti-dna antibodies in csf and serum. the relative concentration of anti-dna antibodies in the csf of all patients was measured after dilution of csfs -fold; the values for all csfs were comparable (range . - . a units counting on a cerebrospinal fluid without dilution; average value . . a units), while in the case of the serum (dilution -fold) it was significantly varied (range - a units counting on serum without dilution; average value a units). since the relative concentration of anti-dna antibodies in csf was relatively low, we have removed all abs from csfs by passage through two columns: protein g-sepharose and protein a-sepharose. csfs passed through the columns and controls containing only a standard buffer demonstrated the same a values, which were # % in comparison with those for untreated csfs. thus, we have shown that the relatively low a values corresponding to untreated csfs are caused by anti-dna antibodies in these csfs. in addition, we have shown that dna-hydrolyzing activity is an intrinsic property of iggs from csf (see below). there was no good correlation between anti-dna antibodies in the sera and csfs, cc = + . (table ). in addition, the correlation was very low between the total concentration of iggs and anti-dna antibodies both in csf (cc = + . ) and the serum (cc = . ). at the same time, the average differences in the concentration of total iggs for the serum and the csf ( -fold) were . -fold lower than the difference for anti-dna antibodies of the serum and the csf ( -fold) ( table ) . it is known that human sera contain several major and especially minor proteins. the average concentration of the total protein in the serum was , -fold higher than in csf (table ) . it was interesting to compare major proteins of the sera and csfs using sds polyacrylamide gel electrophoresis (sds-page). fig. demonstrates major proteins in some samples of the sera and the csfs. one can see that the sera contain more major proteins, but in both cases iggs ( kda) and human serum albumin (, kda) are most major proteins. at the same time, in contrast to csfs, the sera contain several major proteins with mol. masses higher than kda as well as lower than kda, but higher than kda (fig. ) . it is interesting to see that in csfs there are several additional good visible proteins with electrophoretic mobility lower than kda in comparison with serum preparations. it was interesting to compare total dnase relative activity (ra) of all dna-hydrolyzing enzymes and abs in the sera and csf of ms patients. preparations of total protein were capable to hydrolyze supercoiled plasmid dna (scdna), forming single breaks in one strand of supercoiled dna (relaxed dna) and then multiple breaks yielding linear dna. finally, they hydrolyzed dna into short and medium-length oligonucleotides. however, after such a deep hydrolysis of dna, it was very difficult to estimate the relative activity of these preparations. the ras of total protein from sera and csf varied remarkably from patient to patient. fig. illustrates typical examples of cleavage of scdna by total protein from several ms patients after h of incubation. therefore, in order to estimate the dnase activity quantitatively, we found the concentration for each preparation and the time of incubation sufficient to convert scdna into the relaxed form ( - %; for example, lanes , , , , and of fig. c ) without further noticeable fragmentation after - h of incubation. since all measurements (initial rates) were taken within the linear regions of the time courses and protein concentration, the measured ras were normalized to standard conditions (pmole dna/ mg of protein/ h; standard units (su)) ( table ). the relative specific dnase activity of total protein of the serum preparations was varied in the range . - . pmole dna/ mg of protein/ h (average value . . su). it was surprising that the specific activity of the total protein of csf reparations were -fold higher (range - su, average value su) than the serum ones (table ) . at the same time, concentration of total protein in the case of csf was -fold lower than that of sera (table ) . therefore, in calculation on ml of liquid the average ra of total protein corresponding to the sera was only . -fold lower than that for csfs. recently, several strict criteria have been applied to show that the dnase activity is an intrinsic property of iggs from sera of ms patients but not from healthy donors [ ] [ ] [ ] . when searching abzymes in csf of ms patients, the igg fraction was purified by chromatography on protein-a sepharose in conditions to remove non-specifically bound proteins, followed by gel-filtration as in [ ] [ ] [ ] [ ] . the relative amount of csf preparations from only thirteen ms patients was enough for the purification of igg preparations. to analyze the ''average'' situation for ms iggs, we prepared a mixture of equal amounts of iggs from sera of thirteen patients (igg mix ). the homogeneity of the kda igg mix was confirmed by sds-page, which showed a single band before, and two bands corresponding to the h and l chains after ab reduction with dtt (silver staining) ( fig. a) . to prove that dnase activity of csf igg mix is its intrinsic property and is not due to copurifying enzymes, we applied some of well known rigid criteria [ ] [ ] [ ] [ ] [ ] ] ; a) electrophoretic homogeneity of igg mix ( fig. a) ; b) gel-filtration of igg mix in conditions of ''acidic shock'' (ph . ) did not lead to the disappearance of ab activity and the peak of activity tracked exactly with kda abs (fig. c) ; c) complete adsorption of the activity by sepharose bearing monoclonal mouse abs against human igg light chains and its elution from the adsorbent with buffer of low ph (fig. d ). in order to exclude possible artifacts due to any hypothetical traces of contaminating enzymes we analyzed additionally the dnase activity of igg mix using an in situ assay in sds-page gels containing dna. after incubation, in order to allow dnase renaturation and staining with ethidium bromide, a sharp dark band at the position of dna hydrolyzing proteins was revealed on a fluorescent background of dna-bound ethidium bromide (fig. b) . after the dissociation of the igg mix using dtt, dnase table . relative dnase activity of total proteins and iggs from csf and sera of patients with ms.*. serum ( ) csf ( ) serum ( ) csf ( ) serum ( activity was revealed only in the band of the separated light chains. similar data was obtained earlier for abzymes from serum of ms patients [ ] [ ] [ ] . since sds dissociates any protein complexes, and the electrophoretic mobility of hypothetical contaminating dnases cannot coincide at the same time with that of intact igg and its lchains, the detection of dnase activity in the gel region corresponding only to igg and its light chains, together with the absence of any other bands of the activity or protein, provides direct evidence that dnase activity is an intrinsic property of ms igg mix and is not due to copurifying enzymes. dnase activity was detected earlier in the case of iggs from sera of of ms patients (, %) but in none of healthy donors [ ] [ ] [ ] . there was not any possibility to get csf preparations from healthy donors. however, one can assume that csf, similarly to serum preparations from healthy donors, does not contain iggs with dnase activity. we compared the ras in the hydrolysis of dna of igg preparations from sera and csfs of thirteen ms patients. the ras of iggs from sera and csf varied markedly from patient to patient. fig. illustrates typical examples of cleavage of scdna by iggs ( . mg/ml) from the sera and csfs ( . mg/ml) of several patients after h of incubation at their fixed concentrations. to estimate the dnase activity quantitatively, we found the concentration for each igg preparation corresponding to the linear part of the rate dependences on ab concentration (the conditions of the reaction of the pseudo-first order), and the time of incubation sufficient to convert scdna into the relaxed form fig. a ). since all measurements (initial rates) were taken within the linear regions of the time courses and ab concentration curves, the measured ras for individual iggs were normalized to standard conditions similarly to those for the preparation of total protein (table ) . it was surprising, but csf iggs possess significantly higher specific dnase activity than serum ones. average specific dnase activity of serum iggs (average value . . pmole dna/ mg of ab/ h; range . - . units) is . -fold lower than that for csf abs (average value . . units; range . - . units) (table ). at the same time, there is no good correlation between catalytic activities of iggs from the sera and csf, cc = + . (table ) . interestingly, average specific ras of serum iggs are , -fold higher than those of serum total proteins, while this difference in the case of csf is significantly lower, only , . -fold (table ). there are no published data concerning csf abzymes with any catalytic activities. data reported in this paper provide strong evidence that dnase activity is an intrinsic property of iggs present in csf of ms patients: it is not due to copurifying enzymes. the presence of anti-dna abs and dnase iggs in csf of ms patients is in agreement with the detection of b-cells producing anti-dna abs directly in active plaques and periplaque regions in ms brain and cerebrospinal fluid of a patient with ms [ ] . since sera of ms patients contain greater number of different proteins than csfs (fig. a ) and the average concentration of total protein in the sera is , -fold higher than in csfs, it is not surprising that average specific ra of csf total protein is -fold higher than that of the serum one. the question is why specific ras of csf total iggs are significantly ( . -fold) more active than those from sera. in addition, relative concentration of abs (of a total pool) interacting with dna in sera is , -fold higher than in csf, while a difference in the concentration of total iggs is only -fold, a difference of . -fold (table ) . thus, a small specific fraction of anti-dna abs from the csf may be -fold more active (totally , -fold) than a similar small fraction of abs interacting with dna from the sera. in this context, some data from literature should be mentioned. overall, abzymes of ms patients may be significantly more active in the hydrolysis of dna than what we have found ( table ) . as it was previously shown, the fractions of abzymes with different catalytic activities, including nuclease ones, in the serum of autoimmune patients usually do not exceed - % of total immunoglobulins [ ] [ ] [ ] [ ] [ ] . since the specific activity was calculated using the total concentration of iggs, the specific dnase activities of the individual monoclonal subfractions in a polyclonal igg pool may be significantly higher than those of the nonfractionated iggs. in addition, the repertoire of polyclonal abs against different antigens in the case of sera from ms patients may be significantly wider than that of csfs. it may be one of the possible reasons of a lower specific activity of sera iggs. at the same time, an ever-growing number of observations suggest that autoimmune diseases originate from defects in hematopoietic stem cells [ ] . it was recently shown that the specific reorganization of immune system during the spontaneous development of a profound sle-like pathology in mrl-lpr/lpr mice is associated with changes in the differentiation profile and the level of proliferation of bone marrow hematopoietic stem cells and with the production of dnase, atpase, and amylase abzymes [ , ] . immunization of healthy mice with dna also leads to a production of abs with dnase activity; however, it is only associated with increased lymphocyte proliferation and suppression of apoptosis of lymphocytes in different organs (especially spleen), but not with a change in the differentiation of bone marrow cells [ , ] . thus, it is reasonable to suggest that b-cells of csf of ms patients can produce not only abs interacting with dna [ ] , but also specific anti-dna abzymes with higher dnase activity. abzymes produced by lymphocytes against dna in different organs of ms patients (and circulating in the blood system) may have a lower dnase activity in comparison with anti-dna abs of csf or may be different ratio of abzymes and anti-dna abs without catalytic activity in the csfs and sera of ms patients. we have estimated ccs between different characteristics of csf and sera. it was shown that there was no good correlation between several identical indexes characterizing csfs and sera of ms patients, ccs varying in the range of . to + . (tables and ). the correlation between the total protein concentration and: a) total concentration of iggs in csf (cc = + . ; columns and ) and sera (cc = + . ; columns and ); b) relative concentration of anti-dna abs in csf (cc = + . ; columns and ) or sera (cc = . ; columns and ) was low ( table ) . at the same time, low but still the best positive correlation was observed between the total protein concentration and relative dnase activity of total csf protein (cc = + . ; columns and ), whereas in the sera these values showed negative correlation (cc = . ; columns and ) (tables and ) . similar low ccs were observed for other estimated parameters. cc between the relative concentrations of iggs and anti-dna abs was relatively low in csf (cc = + . ; columns and ), and negative in the sera (cc = . ; columns and ) ( table ) . interestingly, cc between the concentration of iggs and the relative specific dnase activity of csf abs (cc = + . ; columns and ) was lower than that of the sera (cc = . ; columns and ) (tables and ). finally, the relative concentration of total anti-dna abs correlated with the relative specific igg dnase activity better in the sera (cc = + . ; columns and ) than in csf (cc = + . ; columns and ) (tables and ). an additional question is why there is no good correlation between various indexes, characterizing different ms patients. an analysis of correlation between titers of abs to dna as well as to mbp and different standard clinical parameters including poser criteria (indexes for evaluation of damage to functional systems: pyramidal functions; cerebellar functions; functions of brain stem; sensitive functions; functions of intestines and urinary bladder; visual functions; cerebral (psychical) functions and sum of these characteristics) in the case of patients with ms was carried out [ ] . for the whole group of ms patients, the absolute values of positive ccs between titers of anti-dna or anti-mbp abs and clinical poser indexes were very low (between . and . ), absent (, ), or even negative ( . to . ) and statistically non-significant. several ccs became higher and reached values up to . to . and . to . after the division of cohort into subgroups of patients with primary progressing, secondary progressing and remitting course of the disease [ ] . the groups of primary progressing remitting course and secondary progressing course of ms patients were not ''homogenous'' with respect to the patients' characteristics, and their further subdivision using cluster and factorial analysis revealed high statistically significant correlation coefficients [ ] . for example, for one sub-subgroup of the remitting course subgroup, a direct dependence between titers of anti-mbp and symptoms of lesions of the pyramidal tract was observed (cc = . ). in some cases, correlations of the opposite sign were observed for the same pairs of analyzed parameters for the three subgroups with different ms courses and their sub-subgroups obtained by cluster analysis from the subgroups. the absence of a definite dependence between titers of anti-dna and anti-mbp abs and these parameters with standard clinical indices may be due to several reasons. ms is an extremely multifactorial disease, in which similar pathomorphological and clinical indices manifested as ms may result from very different underlying processes and conditions [ , ] . for example, in each ms patient, the ''relative stability'' of different organs and their functions to the destructive effect of transient immune system errors can be significantly different depending on the genetic background and environmental stress factors, including geographic ones [ ] [ ] [ ] . some proteins of influenza, herpes, polyoma, epstein-barr and other viruses and of some bacteria have been reported to mimic human myelin proteins, and these infections can therefore lead to immunization with their proteins and stimulate the subsequent formation of abs to myelin and finally to the development of autoimmune reactions [ , [ ] [ ] [ ] [ ] . in individual ms patients, the development of autoimmune reactions can be stimulated by different viral or bacterial infections as well as various toxic chemicals. furthermore, it should also be taken into account that ms is pathology of at least two-phases [ ] . the cascade of reactions corresponding to the first inflammatory phase is very complicated and involves many proteins, enzymes, cytokines, and chemokines inducing macrophages and other cells producing no n radicals and osteopathin [ , ] . the complex and coordinated action of t-and b-cells, complement system, inflammation mediators, and auto-abs result in the formation of demyelinization nodi and interruption of axon conductivity. the neurodegenerative phase of ms that ensues later is directly connected with the neural tissue destruction in these patients [ , ] . therefore, any analysis of biochemical, immunological and clinical indices must take into account of the current stage of the disease. obviously, quite different characteristics of pathologic processes can be obtained in individual patients as the disease progresses against the background of the continually changing immunoregulation, including exhaustion of different compensatory and adaptive mechanisms and systemic metabolic changes. this makes the clinical course of ms hardly predictable in individual patients [ , ] . therefore, it is not surprising that we could not find a high statistically significant correlation of titers of abs to dna and ras of abzymes with all parameters measured, since each patient can be characterized by an individual combination of genetic, environmental, chronic, inflammatory, autoimmune, demyelinating, neurodegenerative and other factors. overall, all data obtained demonstrate that the dnase activity is an intrinsic property of iggs deriving from csf and sera of ms patients. these iggs are polyclonal and may consist of extremely different repertoires of dnase subfractions in the case of csf and sera. we have shown previously that the appearance of abzymes specifically hydrolyzing dna is among the earliest and clear signs of autoimmune reactions in a number of autoimmune diseases when titres of abs to dna or other auto-antigens have not yet increased significantly and correspond to their ranges for healthy donors [ , , [ ] [ ] [ ] . therefore, detection of dnase abs in the sera and csf of peoples can be considered as an additional index for early diagnostic of this pathology. most chemicals, proteins, protein g-sepharose, and the superdex hr / column were from sigma or ge healthcare. fifteen consecutive ms patients ( women and men; mean age = . years) satisfying the criteria for definite ms according to the classification of mcdonald [ ] and admitted to the multiple sclerosis center of the university of ferrara during the period from january to october were retrospectively selected for the study. disease severity was scored in all ms patients at the time of sample collection using kurtzke's expanded disability status scale (edss) [ ] (mean at entry = . . ; range from to . ). clinical course (rr and pp), clinical activity (relapse at time of sampling), and mri activity (the presence of gadolinium enhancing lesions at mri examination) were analyzed as described previously [ ] . at entry none of the patients had fever or other symptoms or signs of acute infections. moreover, at the time of sample collection none of the patients had received any potential disease-modifying therapies during the months before the study. the blood and csf sampling protocols confirmed the local committee for medical ethics in research (comitato etico della provincia di ferrara) that approved our study in accordance with helsinki ethics committee guidelines including written consent of patients confined to present of their blood and csf for diagnostics of a possible disease and scientific purposes. the protocol was approved at may and it was focused on the creation of a biological bank of csf and serum samples, and related clinical data of patients with ms and other neurological diseases including: a) a study of potential markers (especially proteins) for diagnostic and prognostic significance in diseases of the nervous system; b) specific antibodies directed against antigens potential exogenous and/or endogenous; c) presence of pathogens (mostly viruses or bacteria) for association studies and pathogenesis; d) neurotransmitters and their metabolites; e) a study of different properties of different markers. csf and serum samples were collected under sterile conditions and stored in aliquots at uc until assay. ''cell-free'' csf samples were obtained after centrifugation at room temperature of specimens taken by atraumatic lumbar puncture performed for purposes of diagnosis in the absence of contraindications. serum samples derived from centrifugation of blood specimens with-drawn by puncture of an anterocubital vein at the same time of csf extraction. paired csf and serum samples from ms patients were stored and measured under exactly the same conditions. informed consent was given by all patients before inclusion and the study design was approved by the regional committee for medical ethics in research. csf and serum igg levels were measured by immunochemical nephelometry with the beckman immage immunochemistry system (beckman instruments, inc. fullerton, ca. usa) according to the procedure of salden et al. [ ] . in all cases, protein concentration in the intact csf, sera of ms patients and final solutions of abs was measured using bradford assay with a bovine serum albumin standard. the concentration of iggs after their purification by affinity chromatography on protein g-sepharose (see below) was measured in the same way. relative concentrations of iggs in the intact csf and the sera of ms patients were analyzed using special quantitative isoelectrofocusing and immunoblotting test system in italy according to the standard manufacturer's protocol and equipment (igg ief, helena laboratories, gateshead, tyne and wear, uk). in addition, the relative concentrations of iggs in the intact csfs and the sera of ms patients were measured after abs purification by affinity chromatography on protein g-sepharose (see below). the titers of anti-dna abs were determined using standard assay plates with immobilized double-stranded dna, horseradish peroxidase-conjugated mouse abs against human igg, and tetraethyl benzidine as substrate according to the standard manufacturer's protocol (vector, russia). the preparations of human blood serum and csf were diluted respectively and times and ml of final solution was added to the strips. the reaction was stopped with sulphuric acid and optical density (a ) of the solutions was determined using a uniskan ii plate reader (mtx lab systems, usa). the relative concentration of anti-dna abs in the samples was expressed as the difference in the relative absorbance at nm (average of three measurements) between the experimental samples and the control samples containing no abs. as additional controls, we have used preparations complete devoid of abs after passage of csfs through protein g-sepharose and protein a-sepharose. there was no difference in the relative absorbance at nm of csf preparation containing no abs and controls containing a buffer only. finally, a values were recalculated on respective biological fluids without dilution. electrophoretically and immunologically homogeneous iggs were obtained by sequential affinity chromatography of the csf and serum proteins on protein g-sepharose and fplc gel filtration similarly to [ ] [ ] [ ] [ ] . in each case the protein corresponding to the central part of igg peaks was concentrated and used in further purification or analysis. iggs from csf were incubated in mm glycine-hcl (ph . ) containing . m nacl for min at uc. separation of the iggs under ''acid shock'' conditions was done by fplc gel filtration on a superdex hr / column equilibrated with mm glycine-hcl (ph . ) containing . m nacl as previously described [ ] [ ] [ ] [ ] . after - weeks of storage at uc in order to refold after the acid shock, the abs were used in the activity assays described below. in some cases, electrophoretically homogeneous iggs were chromatographed on sepharose bearing immobilized monoclonal mouse abs against light chains of human iggs as in [ ] [ ] [ ] [ ] . the protein was applied to the column ( ml) equilibrated with mm tris-hcl (ph . ) containing . m nacl and the column was washed with the same buffer containing . m nacl. abs were eluted in . m glycine-hcl (ph . ), neutralized, dialyzed and sterilized as described above. dna-hydrolyzing activity of total protein and igg preparations was analyzed using supercoiled (sc)dna as earlier described for the analysis of dnase i and human serum catalytic antibodies [ ] [ ] [ ] . the reaction mixture ( ml) contained mg/ml scdna pbluescript, mm mgcl , mm edta, mm tris-hcl (ph . ), and . - . mg/ml abs or initial preparations of the sera or csf (total protein) finally diluted respectively -and fold. reaction mixtures were incubated for . - h (standard time, h) at uc. the cleavage products were analyzed by electrophoresis in % agarose gel. the images of ethidium bromidestained gels were captured on a sony dsc-f camera and a relative amount of dna in different bands was analyzed using imagequant v . (molecular dynamics). the activities of igg preparations were determined as a decrease in the percentage of dna converted from the initial supercoiled form to the relaxed form, corrected for the distribution of dna between these bands in the control (incubation of pbluescript in the absence of abs). all measurements (initial rates) were taken within the linear regions of the time courses ( - % of dna hydrolysis) and then recalculated to the standard conditions (see tables) . sds-page analysis of abs for homogeneity and for the polypeptide spectrum of the sera and csf was performed in a - % gradient gel containing . % sds (laemmli system) as described in [ ] [ ] [ ] . iggs were used before and after their treatment with mm dithiothreitol. the polypeptides were visualized by silver and coomassie blue staining [ ] [ ] [ ] . in situ, experiments dnase activity of iggs after sds-page was analyzed in gel containing calf thymus dna ( mg/ml) under non-reducing conditions as in [ , ] . before the electrophoresis, igg samples were incubated at uc for - min in mm tris-hcl (ph . ) containing . % sds. to restore the enzymatic activity after sds-page, sds was removed by incubating the gel for h at uc in mm tris-hcl (ph . ) and washing the gel five times with the same buffer. to refold the protein after sds treatment and to assay it for dnase activity, longitudinal slices of the gel were incubated at uc for - h in the reaction buffer containing mm tris-hcl (ph . ), mm mgcl , and . mm cacl . to visualize the products of dna hydrolysis, the gel was stained with ethidium bromide. the same ethidium bromidestained or parallel longitudinal slices were used to detect the position of igg in the gel by coomassie blue staining. the results are reported as mean s.e. of at least three independent experiments for each sample analyzed. errors in the values were within - %. the correlation coefficients (cc) between sets of different samples were analyzed. the neuroimmunology of multiple sclerosis: possible roles of t and b lymphocytes in immunopathogenesis anti-dna antibodies are a major component of the intrathecal b cell response in multiple sclerosis antibodies as enzymes catalytic antibodies: evolution of protein function in real time from molecular diversity to catalysis: lessons from the immune system recent developments in catalytic antibodies opportunities at the interface of chemistry and biology abzyme generation using an anti-idiotypic antibody as the ''internal image'' of an enzyme active site antibody catalysis based on functional mimicry production and characterization of monoclonal anti-idiotypic antibody exhibiting a catalytic activity similar to carboxypeptidase a monoclonal anti-idiotypic antibodies as functional internal images of enzyme active sites: production of a catalytic antibody with a cholinesterase activity enzyme mimicry by the antiidiotypic antibody approach catalytic antibodies catalytic antibodies in healthy humans and patients with autoimmune and viral pathologies natural catalytic antibodies -abzymes. in: keinan e editor. catalytic antibodies natural catalytic antibodies in norm and in autoimmune diseases autoantibodies and natural catalytic antibodies in health, multiple sclerosis, and some other diseases hydrolysis of myelin basic protein by polyclonal catalytic iggs from the sera of patients with multiple sclerosis hydrolysis of myelin basic protein by igm and iga antibodies from the sera of patients with multiple sclerosis metal-dependent hydrolysis of myelin basic protein by iggs from the sera of patients with multiple sclerosis autoantibodies to myelin basic protein catalyze site-specific degradation of their antigen recognition and degradation of myelin basic protein peptides by serum autoantibodies: novel biomarker for multiple sclerosis catalytic heterogeneity of polyclonal dna-hydrolyzing antibodies from the sera of patients with multiple sclerosis immunoglobulins from blood of patients with multiple sclerosis like catalytic heterogeneous nucleases iggs containing light chains of the l and k type and of all subclasses (igg -igg ) from sera of patients with multiple sclerosis hydrolyze dna amylolytic activity of igm and igg antibodies from patients with multiple sclerosis novel functional activities of anti-dna autoantibodies from sera of patients with lymphoproliferative and autoimmune diseases recommended diagnostic criteria for multiple sclerosis: guidelines from the international panel on the diagnosis of multiple sclerosis rating neurological impairment in multiple sclerosis: an expanded disability scale (edss) defining the clinical course of multiple sclerosis: results of an international survey intrathecal production of chlamydia pneumoniae-specific high-affinity antibodies is significantly associated to a subset of multiple sclerosis patients with progressive forms catalytic hydrolysis of vasoactive intestinal peptide by human autoantibody organ-specific and systemic autoimmune diseases originate from defects in hematopoietic stem cells formation of different abzymes in autoimmune-prone mrl-lpr/lpr mice is associated with changes in colony formation of haematopoetic progenitors antibodies with amylase activity from the sera of autoimmune-prone mrl/mpj-lpr mice multiple sclerosis: molecular and cellular mechanisms oil and gas linkage disequilibrium between the mbp tetranucleotide repeat and multiple sclerosis is restricted to a geographically defined subpopulation in finland viral infections trigger multiple sclerosis relapses: a prospective seroepidemiological study concepts of viral pathogenesis of multiple sclerosis myelin basic protein and human coronavirus e cross-reactive t cells in multiple sclerosis affinity and catalytic heterogeneity of polyclonal myelin basic protein-hydrolyzing iggs from sera of patients with multiple sclerosis multiple sclerosis: a two-stage disease analytical performance of the three commercially available nephelometers compared quantifying protein in serum and cerebrospinal fluid this study was supported in part by grants from the presidium of the russian academy of sciences (molecular and cellular biology program, dnase antibodies cerebrospinal fluid plos one | www.plosone.org april | volume | issue | e . ; russian foundation for basic research ( - - , - - , and - - ), funds from the siberian division of the russian academy of sciences and funds from the regione emilia romagna, italy (ricerca sanitaria finalizzata). key: cord- -r z w authors: leinikki, pauli; shekarchi, isabel; iivanainen, matti; taskinen, eero; holmes, kathryn v.; madden, david; sever, john l. title: virus antibodies in the cerebrospinal fluid of multiple sclerosis patients detected with elisa tests() date: - - journal: j neurol sci doi: . / - x( ) - sha: doc_id: cord_uid: r z w the enzyme-linked immunosorbent assay (elisa) was used to determine levels of specific igg antibodies against measles, rubella, vaccinia, corona (oc ) and mumps viruses in cerebrospinal fluid (csf) and serum of patients with clinically definite multiple sclerosis (ms), patients with optic neuritis (on), patients with other neurological disease (ond), and control subjects without central nervous system disease. serum antibody levels were not significantly different between the four groups. differences in the frequency and levels of csf antibodies between the four groups were observed. control patients had serumcsf antibody ratios from . to . (log) with an average of . corresponding to a -fold difference between serum and csf antibody levels. ms patients had ratios from . to . with an average of . . the average was . for the on patients. the average for the ond patients was similar to the controls. the altered serumcsf ratios for several viruses within an individual patient was similar. these results suggest that nonspecific immunostimulation is responsible for the increased levels of csf virus antibodies. the enzyme-linked immunosorbent assay (elisa) was used to determine levels of specific igg antibodies against measles, rubella, vaccinia, corona (oc ) and mumps viruses in cerebrospinal fluid (csf) and serum of patients with clinically definite multiple sclerosis (ms), patients with optic neuritis (on), patients with other neurological disease (ond), and control subjects without central nervous system disease. serum antibody levels were not significantly different between the four groups. differences in the frequency and levels of csf antibodies between the four groups were observed. control patients had serum/csf antibody ratios from . to . (log) with an average of . corresponding to a -fold difference between serum and csf antibody levels. ms patients had ratios from . to . with an average of . . the average was . for the on patients. the average for the ond patients was similar to the controls. the altered serum/csf ratios for several viruses within an individual patient was similar. these results suggest that nonspecific immunostimulation is responsible for the increased levels of csf virus antibodies. introduction increased antibody titers against several viruses have been lbund in cerebrospinal fluid (csf) of multiple sclerosis (ms) patients. the most constant finding has been elevated levels of measles antibodies but elevated rubella, herpes simplex, vaccinia and mumps antibodies also have been found (haire ; norrby ) . the relative amount of csf antibodies compared to serum antibody levels is higher in many ms patients than in most controls and has been associated with an intrathecal synthesis of antibodies (frick and sheid-seydel ; tourtellotte and parker ; norrby ) . however, the low sensitivity of conventional serological techniques has limited the evaluation of csf antibody levels in normal controls due to the low amounts of antiviral antibodies present. we have recently developed a sensitive enzyme immunoassay for virus antibodies (leinikki and p~issil~i ; leinikki et al. ) . by using this technique, we studied the occurrence and levels of csf antibodies against unrelated viral antigens in ms and control groups. patients admitted to the outpatient department of neurology, university central hospital, helsinki, finland and in whom a lumbar puncture was performed at the admission were initially included. careful clinical and laboratory investigations subsequently established the diagnosis and placed the patient either to clinically definite ms, optic neuritis (on), other neurological disease (ond) or control group. the criteria for ms were those of schumacher et al. ( ) . the exclusion of neurological disease in the control group was based both on cfinical examination following admission and on a further clinical follow-up. altogether, ms patients, on, and ond patients were studied. the control group included subjects with symptoms such as headache, vertigo, pain in the neck, hyperventilation without evidence of central nervous system (cns) disease. csfs were coded before study in the laboratory. ( ) elisa for viral antibodies disposable polystyrene cuvettes and fp- photometer (labsystems, hetsinki, finland) were used as described earlier (leinikki and p/issil~i ) . measles (edmonston strain) and rubella (gilcrest strain) antigens (leinikki et al. ) were prepared by purifying virions in sucrose gradients. crude, commercially available vaccinia (microbiological associates, bethesda, md) and mumps (enders strain) were used (leinikki et al. ) . coronavirus antigen was prepared by growing strain oc in mouse brain and purified in sucrose gradient. heavy chain-specific, alkaline phosphatase-conjugated anti-human igg was prepared as described earlier (leinikki and p~issil~i ) . serum samples were diluted : and : while the csfs were diluted : . the amount of antibodies was calculated :from a logarithmic scale where the dilution of the sample was compared to the dilution fig. . distribution of mean serum/csf virus (measles, rubella, vaccinia, corona oc and mumps) antibody ratios (log) related to blood-brain barrier function in different patient groups. note that the ratios are highest in control subjects and lowest in ms patients without any relation to blood-brain barrier. © = intact blood-brain barrier; ~ = moderately impaired blood-brain barrier; • = severely impaired blood-brain barrier. associated with an impaired blood-brain barrier, but this was not the casein ms and on patients (fig. ) . the average serum/csf ratios of different viral antibodies in the study groups are presented in table . the control group showed relatively little variation from one virus antibody to another with an average for all antibodies of z . this corresponds to a -fold difference between serum and csf antibodies. in msthe average ratios varied from . for vaccinia to . for mumps virus antibodies with an overall average of . . the ratios in on were between those of ms patients and controls; only the vaccinia virus antibody ratio was less than . and this was derived from two patients. the average ratios in ond patients were slightly lower than in controls. the serum/csf ratios were analyzed statistically by a multiple regression analysis and compared with other data of serum and csf proteins including csf total protein, csf albumin and serum/csf albumin ratio. no significant correlation was found in ms or on groups indicating that the altered serum/csf antibody ratio was not due to altered blood-brain barrier. the serum/csf antibody ratio, in general, was very similar for different viruses in individual patients. this was equally true for control patients with normal ratios as well as for ms patients with abnormal ratios. a few examples are given in table . the sensitivity of the el sa test permits the detection of csf antibodies when the amount is too low to be detected by conventional serological methods. the serum/csf ratio for total igg is about ( . in log scale) in normal, healthy individuals. alteration of this ratio may be associated with in situ formation of antibody in the cns or impairment of the blood-brain barrier. the increased sensitivity of the present elisa technique made it possible to detect viral antibodies in csf in a majority of the control subjects. in fact, all individuals who had serum antibodies against measles and/or rubella had detectable levels in their csf. corona, vaccinia, and mumps virus antibodies when occurring in low concentrations in the serum could not be detected in the csf. the simultaneous elevation of multiple antibody levels in the csf of ms patients was clearly demonstrated in the present study. the role and evolution of viral antibodies in the csf of ms patients is not understood. the diversity of the viral agents that have been implicated in ms makes it improbable that any of these agents or some cross-reacting, unknown agent would cause the simultaneous multiple antibody stimulus observed in this study. the best explanation is that the viral antibodies produced within the cns of ms patients are related to the immunopathophysiology of the disease rather than a specific etiology. altered serum/csf virus antibody ratios have also been occasionally detected among patients with other neurological diseases (norrby ; iivanainen et al. ) . in diseases such as subacute sclerosing panencephalitis (sspe) the serum/csf antibody ratio is exclusively altered for measles virus antibodies (p. leinikki, d. mcfarlin, i. shekarchi and j. sever, unpublished observation) . it would be of interest to study multiple virus antibody ratios in acute inflammatory diseases such as mumps meningitis, uncomplicated measles, encephalitis, neurosyphilis and toxoplasmosis. untersuchungen mit j -markierten gamma-globulin zur frage der abstammung der liquoreiweissk rper significance of virus antibodies csf oligoclonal bands, immunoglobulins, and viral antibodies in progressive myoclonus epilepsy p~issil/i ( ) solid phase antibody assay by means of enzyme conjugated to antiimmunoglobulin, ". clin quantitative, semiautomated enzyme-linked immunosorbent assay for viral antibodies enzyme-linked immunosorbent assay determination of specific rubella antibody levels in micrograms of immunoglobulin g per milliliter of serum in clinical samples evaluation of enzyme-linked immunosorbent assay (elisa) for mumps virus antibodies immunoglobulin g and low molecular weight proteins in human cerebrospinal fluid viral antibodies in multiple sclerosis problems of experimental trials of therapy in multiple sclerosis --report of the panel on the evaluation of experimental trials of therapy in multiple sclerosis correlation between immunoglobulin g in cerebrospinal fluid and brain key: cord- -k xhbssu authors: norwood, jordan n.; gharpure, akshay p.; kumal, raju; turner, kevin l.; pistone, lauren ferrer; vander wal, randy; drew, patrick j. title: intranasal administration of functionalized soot particles disrupts olfactory sensory neuron progenitor cells in the neuroepithelium date: - - journal: biorxiv doi: . / . . . sha: doc_id: cord_uid: k xhbssu exposure to air pollution has been linked to the development of neurodegenerative diseases and anosmia, but the underlying mechanism is not known. additionally, the loss of olfactory function often precedes the onset of neurodegenerative diseases. chemical ablation of olfactory sensory neurons blocks the drainage of cerebrospinal fluid (csf) through the cribriform plate and alters normal csf production and/or circulation. damage to this drainage pathway could contribute to the development of neurodegenerative diseases and could link olfactory sensory neuron health and neurodegeneration. here, we investigated the impact of intranasal treatment of combustion products (laboratory-generated soots) and their oxygen functionalized derivatives on mouse olfactory sensory neurons, olfactory nerve cell progenitors, and the behavior of the mouse. we found that after a month of every-other-day intranasal treatment of soots, there was minimal effect on olfactory sensory neuron anatomy or exploratory behavior in the mouse. however, oxygen-functionalized soot caused a large decrease in globose basal cells, which are olfactory progenitor cells. these results suggest that exposure to air pollution damages the olfactory neuron progenitor cells, and could lead to decreases in the number of olfactory neurons, potentially disrupting csf drainage. air pollution, particularly small combustion particles (< . µm, pm . ), is a large contributor to global mortality (burnett et al., ) . these small particles are produced by combustion in internal combustion engines, jet aircraft engines, and during cooking. once generated, these particles can be oxidized over time (rattanavaraha et al., ; li et al., ; pourkhesalian et al., ) , generating surface functionalized oxygen groups which can increase their cellular toxicity (li et al., ; holder et al., ; li et al., ) . in addition to the many other adverse health effects of air pollution, there is a strong epidemiological link between exposure to air pollution, particularly pm . , to the development of neurodegenerative diseases (wang et al., ; forman and finch, ; peters et al., ) and to mental disorders (atanasova et al., ; hummel et al., ; buoli et al., ) . exposure to air pollution, particularly fine particulate matter (pm . ), also leads to reduced sense of smell and anosmia (ajmani et al., a; ajmani et al., b) and can damage nasal tissue (calderon-garciduenas et al., ) . interestingly, anosmia and a decline of sense of smell precede the onset of neurodegenerative disorders (doty, ; wilson et al., ; rahayel et al., ; growdon et al., ; ottaviano et al., ; roberts et al., ; murphy, ) and is also associated with depressive disorders (croy et al., ; kohli et al., ) . similar damage and sensory deficits have been implicated in covid- pathology (cooper et al., ) . the observed associations between particulate exposure, decreased olfactory function, and development of neurodegenerative and mental disorders suggests that some of the observed degeneration might originate from the damage to olfactory sensory neurons (osns) in the nasal epithelium. the movement of cerebrospinal fluid (csf) is thought to remove waste from the brain (iliff et al., ; nedergaard, ) , and disruption of normal csf turnover and circulation has been hypothesized to lead to the development of neurodegenerative diseases (albeck et al., ; stoquart-elsankari et al., ; simon and iliff, ; benveniste et al., ) . in addition to csf drainage pathways through meningeal lymphatics and arachnoid granulations (boulton et al., olfactory neuron axons, and chemical ablation of osns blocks this normal outflow, leading to decreased csf production and/or altered csf circulation (norwood et al., ) . thus, any damage to olfactory sensory neurons by air pollutants, in addition to impairing the sense of smell, might lead to disruption of normal csf circulation which can then contribute to the development of neurodegenerative diseases. olfactory sensory neuron cell bodies are located in the nasal epithelium and send their axons to the olfactory bulb through the holes (foramina) in the cribriform plate. because these neurons are exposed to the environment, they have a relatively short lifetime (several months (gogos et al., ) ), and are constantly replenished throughout the lifetime of the organism. olfactory sensory neurons are generated from a population of nearby stem cells (brann and firestein, ; liberia et al., ) , and the ongoing neurogenesis of olfactory sensory neurons continues throughout the life of the animal. there are two classes of stem cells in the nasal epithelia that give rise directly and indirectly to osns, horizontal basal cells (hbcs) and globose basal cells (gbcs) (child et al., ) . gbcs generate olfactory sensory neurons, while hbcs are usually quiescent and are involved in regenerating the nasal epithelial in response to injury. the capacity for regeneration has limits and is reduced with aging or repeated insults (child et al., ) . chronic nasal inflammation causes degeneration of olfactory neurons and their progenitor cells in both humans and animals (chen et al., ; hasegawa-ishii et al., ) . insults that kill olfactory sensory neurons and their progenitor cells will lead to shrinkage and loss of the nasal csf outflow pathways. insults that kill either gbcs or hbcs will decrease the population of stem cells, potentially resulting in a decrease in the number of osns later in life. to better understand the effects of air pollution on olfactory sensory neurons and their progenitor cells, we investigated the impact of intranasal treatment with surrogates for combustion generated 'soots' synthesized from carbon black precursors. carbon black is primarily composed of elemental carbon, but like combustion-produced soot, it is formed by the partial combustion or thermal decomposition of hydrocarbons (donnet, ) . the morphology consists of primary particles that are partially merged or appear "fused" into aggregates (fig. a) . such synthetic soots are also free of variable combustion-derived contaminants such as metals, ash, or condensed organics. we treated the mice with either non-functionalized soots, which resemble the combustion products immediately after their production, or functionalized soots that have been subject to oxygen functionalization, modifying their surface chemistry, mimicking the oxidation processes that would take place during atmospheric aging. we found that relative to vehicle controls, neither non-functionalized soots nor oxygen-functionalized soots had appreciable impact on olfactory sensory neurons. the effects of soot exposure on exploratory behavior was also minimal. however, oxygen-functionalized soots greatly decreased the levels of olfactory progenitor cells, suggesting that exposure to these particles can set up a long-term decrease in the number of osns. such a decrease could lead to anosmia and decreased csf movement. synthesis of soots: synthetic soot was produced by functionalizing commercial carbon black, (regal , cabot corp.) . carbon black was selected for its chemical purity and size similarity to diesel engine-produced soot. to introduce oxygen functional groups such as phenol, carboxyl and carboxylic, we used wet-chemical treatment based on acid etching (romanos et al., ) . in this preparation a gram of carbon black was treated with ml laboratory grade concentrated nitric acid (hno , > %) under reflux for a duration of hours at ˚c, just below the acid's boiling point of ˚c. the carbon-acid mixture was continuously stirred using a magnetic stirrer to ensure uniform exposure and functionalization. the mixture was maintained at a consistent simmer and was thereafter washed with distilled water, filtered, and dried to obtain functionalized carbon black as a synthetic, oxidized soot. any potential residual organic or aromatic compound present on the manufactured material as supplied would be oxidized and removed under these conditions. to visualize soot particles, we used transmission electron microscopy (fei talos f x instrument equipped with quad element eds detector capable of both transmission electron microscopy (tem) and scanning transmission electron microscopy (stem)). for imaging, a beam acceleration voltage of kev was used. beam current was kept less than na for which sample damage or alteration is negligible at these magnifications (< kx). image defocus was one or two steps before the eucentric position. images were captured using a ceta-cooled ccd. samples were dispersed and sonicated in methanol before being dropped onto mesh c/cu lacey tem grids. high angle dark field (haadf) images were obtained using an annular detector. eds for elemental analysis and mapping was performed in the tem. we also used the stem mode, which has a high spatial resolution on the order of the minimum probe size ( . Å). the instrument was fitted with a -quadrant sdd super-x eds detector for eds. the detection limit is typically < atomic percent (at. %) depending on collection parameters. typically, - regions of each material (nascent and functionalized forms) were sampled to gauge elemental representation. eds was performed in stem mode with a sample holder designed to provide low background signal for eds. xps experiments were performed using a physical electronics versaprobe ii instrument equipped with a monochromatic al kα x-ray source (hν = , . ev) and a concentric hemispherical analyzer. charge neutralization was performed using both low energy electrons (< ev) and argon ions. peaks were charge referenced to c-c band in the carbon s spectra at . ev. measurements were made at a takeoff angle of ° with respect to the sample surface plane. this resulted in a typical sampling depth of - nm ( % of the signal originated from this depth or shallower). quantification was done using instrumental relative sensitivity factors (rsfs) that account for the x-ray cross section and inelastic mean free path of the electrons. a thermogravimetric analyzer (ta , ta instruments) coupled to a discovery mass spectrometer (ms) was used to analyze mass loss and the composition of the evolved gases as a function of temperature. the temperature was ramped up at ˚c/min in an inert atmosphere. the tga features low volume, maximum temperature to ˚c and has an inert quartz liner. the ms is a quadrupole mass spectrometer with a heated capillary interface, offering a - amu range, unit m/z resolution. a horiba labram raman microscope was used to obtain raman spectra for the samples when exposed to a nm mw laser with a grooves/mm grating, providing a spectral resolution of cm - . xps was applied to dispersed powder to quantify both surface oxygen atom content (at. % basis) and distribution of oxygen functional groups (-c-oh, phenolic, -c=o, carbonyl, and -cooh, carboxylic), the nominal c s (energy loss) positions were , and . kev. casa was applied to deconvolve the high-resolution spectra, with group contributions ratioed to the total oxygen elemental content. as a baseline, nascent (untreated) carbon black was also subject to the same analytical procedure as a "blank" sample. wet acid reflux treatment of carbon black yielded ~ atomic % (near-surface) oxygen compared to the untreated carbon black, registering negligible surface content, (< at. %). by curve-fitting the c s spectral loss profile, the calculated distribution across function groups was determined as . % (phenolic, c-oh); . % (carbonyl, c=o); and . % (carboxylic, -cooh) (vander wal et al., ) . (the good agreement (± %) in the measured and calculated value of atomic oxygen indicates appropriate curve fitting for functional group identification.) the tga curve shows distinct regions of mass loss owing to functional groups leaving as temperature increases. the wt.% net mass loss corresponds to the gasification of the carbon by the chemisorbed oxygen groups. resolved by temperature, the tga spectrum supported xps identification of functional groups by successive mass loss stages for the oxygen group classes. temperature resolved mass loss curves reveal m/z peaks at amu (co ) arising predominantly from carboxylic groups and at amu (co) arising from carbonyl and phenol groups (kundu et al., ) . soot treatment protocol for mice: after the mouse had been rendered unconscious by a brief exposure to isoflurane, µl of soot (functionalized or non-functionalized, % in sterile h o) or vehicle control (sterile h o) was administered to the left nare dropwise using a pipette. the animal was then inverted to allow for excess fluid to exit the nasal cavity. this treatment was repeated every other day ( days a week) for one month. the animals were monitored and weighed daily after treatment. histology: mice were sacrificed via isoflurane overdose and perfused intracardially with confocal and images were processed using imagej (nih). cell quantification procedures: to quantify the mean fluorescence of pax and p antibody expression, images were first obtained on the olympus fluoview confocal. imaging settings were kept constant across samples to enable quantification of fluorescence. using imagej (nih), a rectangular roi was drawn ( µm in width and µm in height) along the apical side of the neuroepithelium. for every animal, the roi was drawn µm in the rostral direction from the cribriform plate within the neuroepithelium located on the dorsal side of the medial olfactory nerve. the mean fluorescence of the roi for each color channel (corresponding to each of the antibodies used) was obtained and averaged together for each treatment group. data was plotted and analyzed in graphpad prism , using one-way anova to test for significance. to measure any effects of intranasal soot treatment on behavior, mice were individually placed in a x x cm (l x w x h) plastic box one month after the start of the treatment. all experiments were performed between and zt. the acquisition and analysis were done with the experimenter blinded to the treatment, and the order of animals was randomized. mice were placed in the enclosure for minutes, and the behavior was quantified over this entire period. the enclosure was cleaned with % ethanol between mice. the amount of locomotion and rearing behavior were monitored using an intel® realsense™ depth camera d (hong et al., ) . this camera provides simultaneous visible light and depth information used to calculate the animal's distance from the camera. images were acquired at a nominal rate of frames/second using matlab (https://github.com/intelrealsense/librealsense). to track the distance the animal traveled, the distance between the centroid of the mouse was calculated between each successive frame. this distance between frames is then summed over the course of the minutes. rearing events were defined as when the mean of the highest % of pixels of the mouse exceeded cm from the bottom of the enclosure. a generalized linear mixed-effects model (matlab function fitglme) was used to evaluate the differences in rearing events, rearing duration, and distance traveled. each treatment (vehicle, non-functionalized and functionalized soot) was a fixed-effect, with the sex treated as a random effect. data availability: code for the acquisition, analysis and plotting of the behavioral data, as is the behavioral data plotted in figure , is available here: https://github.com/drewlab/norwood_gharpure_turner_ferrer-pistone_vanderwal_drew_manuscript transmission electron micrograph of a carbon black aggregate and primary particle is shown in fig. . the aggregate consists of pseudo-spherical primary particles, partially merged or fused together forming a fractal aggregate. a raman spectrum of the nascent carbon black is shown in fig. c . raman spectroscopy has been developed as a standard method for determining the planar coherence lengths (la) in graphitic carbon, which possesses limited long-range order (tuinstra and koenig, ) . the lower frequency "d" peak at ~ cm - arises from disorder-induced raman activity of zone-boundary a g phonons whereas the "g" peak at ~ cm - reflects the inplane stretching motion of the aromatic rings, designated as e g motions. their comparable intensity reveals considerable disordered carbon, characteristic of furnace blacks and representative of combustion-produced soot emissions (dennison et al., ; sadezky et al., ) . their intensity ratio is an accepted technique for determining la in disordered graphitic materials, given by the relation . (id/ig) - = la, calculated here as . nm, a value commensurate with the short lamellae viewed by hrtem (sadezky et al., ) . the asymmetry of the d-peak due to the extended low frequency (shift) tail is consistent with further disorder of the carbon lattice such as sp and sp carbon at the periphery of the crystallites, contributing vibrations of a g symmetry (sadezky et al., ; parent et al., ) . fluorescence from the oxygen groups and their auxochromic interactions with the electrons of the sp carbon network dwarfed the raman signature of the functionalized material, preventing its comparison to the nascent material. figure d shows a high angle dark field tem image of soot particles for reference and respective eds map displaying carbon (blue) and oxygen (red) for the nitric acid functionalized carbon black. while eds cannot point to a definitive volumetric vs. surface oxygen presence given its -d nature, nitric acid-treated carbon black shows oxygen appearing to be concentrated along the particle perimeter, reflecting a higher near-surface contribution near the particle edge along a) schematic showing the structure of a soot particle, which is an aggregate of smaller particles. b) tem image of a soot aggregate supported by the lacy mesh of the tem grid, illustrating the morphological structure of the particles. pseudo-spherical primary particles are coalesced, forming a branched aggregate whose d projection is shown in the image. c) raman spectrum of r- carbon black. the two peaks of similar intensity are indicative of unstructured (non-graphitic) carbon. d) left, a high angle, annular dark field (haadf) image of soot particles. formed by scattered (rather than transmitted) intensity, the uniformity illustrates the lack of crystallinity and absence of heavy elements such as metals. right, corresponding energy dispersive spectroscopy (eds) map. the elemental map reveals the spatial distribution of carbon and oxygen, integrated through the particle. the higher intensity at the particle perimeters shows that the oxygen is at the particle surfaces. the grid lacy mesh appears as arched support). e) photograph of suspended soot ( %) in water. the oxygen surface functionalization makes the particle hydrophilic, enabling stable dispersion in aqueous media. the beam path. in the -d image it must be noted that eds shows relative amounts of elemental carbon and oxygen and does not give information on what functional groups are present. an image of the % solution of soot in water that is applied intranasally is shown in figure e . soot accumulates in the nasal passageway and lungs, but does not change the structure of the olfactory nerve or osns we treated mice intranasally with vehicle, non-functionalized soot, or functionalized soot for one month. mice were briefly anaesthetized with isoflurane and an intranasal solution of soot (functionalized or non-functionalized, % in sterile water) or vehicle (sterile h o) was administered to the left nare. this treatment was repeated every other day (three times a week) for one month. we saw no appreciable differences between weight of mice of the different treatment groups (data not shown). after sacrifice, the skulls were rapidly decalcified (norwood et al., ) and sectioned. examples of thin sections of olfactory bulb/nasal cavity area are shown in fig a- c, and accumulation of functionalized soot in the olfactory epithelium, but not nonfunctionalized soot was observed. soot could be seen in the lungs of treated animals (fig d-f) . a total of . mg of soot particles was applied each day, though we conservatively estimate that < % remained in the nose after inverting the mouse. given that an average mouse respiratory volume over a day is ~ . l ( . ml tidal volume with breaths a minute this works out an effective dose equivalent to breathing air with a pm . level of ~ µg/m , comparable to the air quality in beijing (zíková et al., ) or new delhi (pant et al., ) . we also examined the status of olfactory sensory neurons to see if either type of soot had a detectable effect on their health. to assess any damage to the olfactory bulb or nerve caused by exposure to the soot particles, the area was examined histologically utilizing rapid decalcification and sectioning. olfactory sensory nerves enter the cranial compartment through the cribriform plate (bird et al., ) . we visualized the nerve in soot and vehicle-treated mice using an antibody against olfactory marker protein (omp) (fig. ) . we found no discernable difference in olfactory nerve labeling among the treatments, indicating that soot treatment does not have any obvious effect on olfactory sensory nerves for the treatment duration used. this is markedly different from intranasal treatment with zinc sulfate, a single treatment which causes the rapid and irreversible ablation of olfactory sensory neurons (norwood et al., ) . one important question is to what extent soot treatment impacts the behavior of the mice. we quantified locomotion and rearing behavior using an intel realsense d depth-sensing camera (hong et al., ) after intranasal soot exposure (fig. ) . treated mice from all three treatment groups were individually placed in a novel environment (white plastic container) after the one month of treatment and their movement and rearing behaviors were monitored for minutes. rearing behavior is a measure of anxiety (sturman et al., ) , and locomotion can be used to assay sickness and malaise (engeland et al., ) . no significant differences were observed in total rearing events or total rearing time for all treatment groups. however, a significant difference in total distance traveled was observed between the vehicle and both the functionalized and non-functionalized soot treatment groups. if the soot treatment causes pronounced health problems, we might expect large decreases in the amount of time rearing or locomotion behavior. as we did not observe pronounced changes in behaviors, this suggest the soot treatment does not cause any generalized decreases in health. comparisons of the effects of vehicle, non-functionalized, and functionalized soot on spontaneous rearing and locomotion behaviors over minutes. the data from each individual mouse is shown as a square (males) or circle (females). the mean of each group is shown as a diamond, standard deviation is denoted with error bars. a) plot of total number of rearing events for each treatment type. there was no significant difference in the number of rearing events between the control and either of the soot treatment groups (p < . non-functionalized, p < . functionalized). b) plot of total rearing time. there was no significant difference in the total rearing time between groups (p < . non-functionalized, p < . functionalized). c) probability distribution of individual rearing event durations for each of the treatment groups. (d) plot of total distance travelled by each mouse. treatment with non-functionalized soot (p < . ) and functionalized soot (p < . ) both significantly decreased the total distance traveled relative to the vehicle treated group. as we saw no obvious changes in olfactory sensory neurons and their axons, we then asked how soot treatment might affect other cell types in the nasal epithelium, particularly the progenitor cells that directly and indirectly give rise to olfactory sensory neurons. if these cells are damaged, then this could lead to a long-term decline in the number of osns as the animals age. we used immunofluorescence staining of the neuroepithelium to visualize changes in progenitor cells ( fig. a-b) . the expression of the anti-pax or anti-p primary antibody in the neuroepithelium was quantified to assess any disruptions in the number of globular basal cells . , p < . ) relative to non-functionalized soot (post-hoc unpaired t-test, t( ) = . , p < . ) and vehicle control (post-hoc unpaired t-test, t( ) = . , p < . ). g) no significant difference between group means of p fluorescence (one-way anova, f( , ) = . , p < . ). exposure. decreases in the number of gbcs could lead to decreases in the number of olfactory sensory neurons in the long term. in order to understand how air pollution might affect olfactory sensory neurons and their progenitor cells, we treated mice intranasally with surrogate soot-like particles that either had oxygen- functionalized surfaces or non-functionalized surfaces. we found that these compounds had minimal effects on behavior, the olfactory sensory nerve, or horizontal basal cells. however, oxygen functionalized soot greatly reduced the population of globular basal cells. our results are consistent with many other studies that have found that oxidized soots are more cytotoxic than un-oxidized soots (li et al., ; holder et al., ; pourkhesalian et al., ) . our results suggest a potential model of how long-term exposure to air pollutants could drive anosmia and decreased csf outflow into the nasal cavity (fig. ). exposure to oxidized soot particles reduces the number of gbcs. as olfactory sensory neurons senesce, the reduced population of gbcs leads to incomplete replacement of osns. the decrease in osns could then potentially lead to decreases in olfactory sensitivity seen with exposure to air pollution (ajmani et al., a; ajmani et al., b; hummel et al., ) . the decrease in osn axons could also reduce fluid outflow through the cribriform there are several limitations to our study. we do not know the mechanism by which oxygen functionalized soot preferentially damages gbcs. it could be that oxygen functionalized soot is more prone to accumulating in the nasal epithelium (fig. ) ( ) dna damage in nasal and brain tissues of canines exposed to air pollutants is associated with evidence of chronic brain inflammation and neurodegeneration. human and nonhuman primate meninges harbor lymphatic vessels that can be visualized noninvasively by mri effects of ambient air pollution exposure on olfaction: a review. lipopolysaccharide treatment in mice: a multivariate assessment of behavioral tolerance a critical review of assays for hazardous components of air pollution odor identification and alzheimer disease biomarkers in clinically normal elderly neuroplastic changes in the olfactory bulb associated with nasal inflammation in mice increased cytotoxicity of oxidized flame soot automated measurement of mouse social behaviors using depth sensing, video tracking, and machine learning position paper on olfactory dysfunction a paravascular pathway facilitates csf flow through the brain parenchyma and the clearance of interstitial solutes, including amyloid β the association between olfaction and depression: a systematic review functional groups on multiwalled carbon nanotube surfaces: a quantitative high-resolution xps and tpd/tpr study oxidant generation and toxicity enhancement of aged-diesel exhaust physicochemical characteristics and toxic effects of ozone-oxidized black carbon particles lymphatics in neurological disorders: a neuro-lympho-vascular component of multiple sclerosis and alzheimer's disease? outflow of cerebrospinal fluid is predominantly through lymphatic vessels and is reduced in aged mice rapid lymphatic efflux limits cerebrospinal fluid flow to the brain blocking cerebrospinal fluid absorption through the cribriform plate increases resting intracranial pressure olfactory and other sensory impairments in alzheimer disease quantification of cerebrospinal fluid transport across the cribriform plate into lymphatics in rats neuroscience. garbage truck of the brain anatomical basis and physiological role of cerebrospinal fluid transport through the murine cribriform plate olfaction deterioration in cognitive disorders in the elderly exposure in highly polluted cities: a case study air pollution and dementia: a systematic review effect of atmospheric aging on volatility and reactive oxygen species of biodiesel exhaust nano-particles the effect of alzheimer's disease and parkinson's disease on olfaction: a meta-analysis the reactive oxidant potential of different types of aged atmospheric particles: an outdoor chamber study association between olfactory dysfunction and amnestic mild cognitive impairment and alzheimer disease dementia silva amt, falaras p ( ) controlling and quantifying oxygen functionalities on hydrothermally and thermally treated single-wall carbon nanotubes raman microspectroscopy of soot and related carbonaceous materials: spectral analysis and structural information regulation of cerebrospinal fluid (csf) flow in neurodegenerative, neurovascular and neuroinflammatory disease aging effects on cerebral blood and cerebrospinal fluid flows exploratory rearing: a context-and stress-sensitive behavior recorded in the open-field test raman spectrum of graphite xps analysis of combustion aerosols for chemical composition, surface chemistry, and carbon chemical state toxicity of inhaled particulate matter on the central nervous system: neuroinflammation, neuropsychological effects and neurodegenerative disease olfactory impairment in presymptomatic alzheimer's disease longitudinal profiling of oligomeric abeta in human nasal discharge reflecting cognitive decline in probable alzheimer's disease cerebral oxygenation during locomotion is modulated by respiration on the source contribution to beijing pm . concentrations key: cord- -ewcgkx h authors: song, jong-am; han, kyung-yeon; park, jin-seung; seo, hyuk-seong; ahn, keum-young; lee, jeewon title: human g-csf synthesis using stress-responsive bacterial proteins date: - - journal: fems microbiol lett doi: . /j. - . . .x sha: doc_id: cord_uid: ewcgkx h we previously reported that under the stress condition caused by the addition of -hydroxyethyl disulfide, a thiol-specific oxidant, to growing cultures of escherichia coli bl (de ), a population of stress-responsive proteins [peptidyl-prolyl cis–trans isomerase b (ppib), bacterioferritin (bfr), putative hth-type transcriptional regulator yjdc (yjdc), dihydrofolate reductase (fola), chemotaxis protein chez (chez), and glutathione synthetase (gshb)] were significantly upregulated when compared with the nonstress condition. when those stress-responsive proteins were used as fusion partners for the expression of human granulocyte colony-stimulating factor (hg-csf), the solubility of hg-csf was dramatically enhanced in e. coli cytoplasm, whereas almost all of the directly expressed hg-csf were aggregated to inclusion bodies. in addition, the spectra of circular dichroism measured with the purified hg-csf were identical to that of standard hg-csf, implying that the synthesized hg-csf has native conformation. these results indicate that the bacterial stress-responsive proteins could be potent fusion expression partners for aggregation-prone heterologous proteins in e. coli cytoplasm. human granulocyte colony-stimulating factor (hg-csf) is a hematopoietic growth factor that plays an important role in hematopoietic cell proliferation, differentiation of hemopoietic precursor cells, and activation of mature neutrophilic granulocytes (metcalf, ; nomura et al., ) . in addition, hg-csf has been widely used for treating neutropenia caused by cancer chemotherapy. the mature hg-csf is an . -kda glycoprotein that predominantly consists of amino acids with two intramolecular disulfide bonds, although another minor form comprised of amino acids as a result of alternative splicing of mrna has also been described (nagata et al., ; lu et al., ; hill et al., ) . furthermore, native hg-csf has only one single o-glycosylation site at thr , which is not essential for biological activity (kubota et al., ) . therefore, nonglycosylated recombinant hg-csf has the same specific biological activity as natural glycosylated hg-csf (souza et al., ; oh-eda et al., ) . because hg-csf is synthesized in escherichia coli as inclusion bodies, solubilization and renaturation steps are necessary to obtain the native conformation (misawa & kumagai, ) , which means that highly complicated downstream processes with low-recovery yield are required. escherichia coli is one of the most widely used organisms for the commercial production of therapeutic and industrial recombinant proteins (baneyx & mujacic, ) for several reasons, including low manufacturing costs, rapid product accumulation, and well-established tools for genetic manipulation (eiteman & altman, ) . despite these advantages, overproduced heterologous proteins in e. coli often form nonproductive inclusion bodies, and fusion expression using solubility enhancer proteins as fusion partner has emerged as an efficient production strategy to overcome the inclusion body formation (baneyx & mujacic, ; sørensen & mortensen, ) . currently, shistosoma japonicum glutathione s-transferase (smith & johnson, ) , e. coli maltose-binding protein (bach et al., ), e. coli n utilization substance a (de marco et al., ) and e. coli thioredoxin (lavallie et al., are the most extensively examined fusion partners (davis et al., ; nallamsetty & waugh, ) , but they are not free from proprietary protection upon commercial application. moreover, the traditional solubility-enhancing fusion partner proteins are not equally effective in promoting the folding of fusion partners (dyson et al., ; park et al., ) . in this study, we report a novel and efficient strategy for the synthesis of soluble and correctly folded hg-csf using various e. coli stress-responsive proteins that we previously found through a proteome-wide analysis of e. coli proteins under -hydroxyethyl disulfide ( -heds)-induced stress condition . using the six stress-responsive proteins of e. coli, we successfully produced hg-csf with correct conformation as well as demonstrated using circular dichroism (cd). escherichia coli strain bl (de ) [f À ompt hsds b (rb À mb À )] was used for the hg-csf synthesis. after pcr amplification using appropriate primers, hg-csf gene and its various fusion mutants (yjdc<hg-csf, gshb<hg-csf, fo-la<hg-csf, bfr<hg-csf, chez<hg-csf, ppib<hg-csf) were inserted into the ndei-hindiii site of plasmid pet a (novagen) to construct the fusion expression vector (fig. ). after complete dna sequencing of all gel-purified plasmid vectors, the e. coli bl (de ) was transformed with the plasmid expression vectors, and kanamycin-resistant transformants were subsequently selected using luria-bertani (lb) agar plates supplemented with kanamycin ( mg l À ). for shake flask experiments, -ml erlenmeyer flasks containing ml lb media with mg l À kanamycin were incubated at c and r.p.m. when the culture turbidity (od nm ) reached . , gene expression was induced by the addition of isopropyl-b-d-thiogalactoside ( mm), and after an additional h of cultivation all of the recombinant cells were harvested by centrifugation (micro tr, hanil science industrial, korea); g for min. the cell pellets were then resuspended in ml lysis buffer ( mm tris-hcl, ph . , mm edta) and were disrupted using a branson sonifier (branson ultrasonics corp., danbury, ct). the cell-free supernatant and insoluble protein aggregates were then separated at g (micro tr, hanil science industrial) for min. the isolated inclusion bodies, if any, were washed twice with % triton x- . the cell-free supernatants and the washed inclusion bodies were then subjected to polyacrylamide gel electrophoresis (page) analysis, and coomassie-stained protein bands were scanned and analyzed by densitometry (duoscan t , bio-rad, hercules, ca). the purification of recombinant hg-scf was accomplished using metal affinity chromatography. briefly, polyhistidine-tagged hg-csf fusion mutants [(his) <fusion partner<(d k)<hg-csf] were loaded onto a probond resin (ni ) column. before sample loading, the resin was washed twice with column volumes of binding buffer ( mm potassium phosphate, mm kcl, mm imidazole, ph . ), which contained mm imidazole to minimize nonspecific binding of untagged protein contaminants. binding was then conducted in batch mode at c, after which the resin was washed twice with - ml tris-hcl ( mm tris, ph . ) before the enterokinase digestion step. enterokinase digestion was carried out in batch mode at c for h using u of enterokinase (invitrogen, ca). next, the proteolytic product was collected and centrifuged (micro tr, hanil science industrial); g for min, and the supernatant fraction was then analyzed by sodium dodecyl sulfate (sds)-page and western blotting. removal of the enterokinase was conducted following the manufacturer's protocol (ek-away tm resin, cat. no. r - , invitrogen, germany). hg-csf digests were resuspended in . m tris-hcl, ph . , % sds, % glycerol, . % bromophenol blue, % -mercaptoethanol, and then subjected to reducing % sds-page using tris-glycine gel. peptides were transferred onto a nitrocellulose membrane (schleicher & schuell bioscience gmbh, germany) for h at c in  transfer buffer (glycine . g l À , tris base . g l À , sds . g l À , meoh ml, distilled water ml). nonspecific antibody-binding sites on the membrane were blocked by incubating for h at room temperature with blocking buffer [ % nonfat skim milk in phosphate-buffered saline (pbs)]. after washing three times with pbs, the membrane was incubated for an additional h at room temperature with : diluted hg-csf primary antibody (mouse antihuman g-csf monoclonal antibody; clone d , santa cruz biotechnology inc., ca). the membrane was washed again as described above, after which it was incubated with : diluted goat anti-mouse immunoglobulin g-conjugated horseradish peroxidase (hrp) secondary antibody (santa cruz biotechnology inc.). after being washed, the membrane was developed using an hrp conjugate substrate kit (sigma-aldrich, mo). the cd spectra of commercial standard hg-csf [grasin prefilled-syringe (filgrastim), kirin, japan] and the purified hg-csf ( mg l À ) were measured using a jasco j- spectropolarimeter (korea basic science center, ochang, korea) at room temperature. direct expression of hg-csf gene in the cytoplasm of e. coli for the direct expression of mature hg-csf gene in e. coli cytoplasm, the plasmid vector, pt -gcsf was constructed by inserting the mature hg-csf gene into the ndei-hindiii site of the pt - vector. however, as previously reported by devlin et al. ( ) , the native hg-csf gene is hardly transcribed in e. coli because of the high (g c) contents at end of coding strand. therefore, the -end coding sequences of hg-csf gene were modified by changing the first five codons ( acc ccc ctg ggc cct ) to the high-frequency codon of e. coli ( act ccg tta ggt cca ) (jeong & lee, ) . as shown in fig. , the expression of the modified hg-csf gene resulted in the synthesis of a large amount ( % of total e. coli proteins) of recombinant hg-csf. however, most of the synthesized hg-csf was present in the form of insoluble inclusion bodies. strategy to enhance solubility of recombinant hg-csf using e. coli stress-responsive proteins the stress induced by overexpression of recombinant proteins resembles environmental stress conditions such as heat shock or amino acid starvation (sørensen & mortensen, ) . similarly, when proteins are exposed to oxidative stressors such as oxygen radicals and hydrogen peroxide (h o ), e. coli decreases the protein synthesis rate and enhances the protein degradation pathway (koo et al., ) . previously, vanbogelen et al. ( ) reported that the expression level of molecular chaperones such as dnak and groes increased in the presence of h o -derived oxidative stress. it has also been reported that addition of -heds, a thiol-specific oxidant, dramatically reduced the nadph/nadp ratio consistently in response to severe oxidative stress (halleck et al., ; ayene et al., ) . in our recent report , we investigated the e. coli proteome under -heds-induced stress conditions and found stress-responsive proteins that are significantly upregulated by the stressor through the comparative and quantitative analysis of e. coli proteomes under nonstress and stress conditions. because many proteins were aggregated by -heds-induced stress, it seems reasonable to assume that such stress-responsive proteins have an intrinsically higher folding efficiency than other proteins that are aggregated in response to the same stress conditions. among the stressresponsive proteins that we previously found , we were interested in the six stress-responsive proteins [peptidyl-prolyl cis-trans isomerase b (ppib, kda), bacterioferritin (bfr, kda), putative hth-type transcriptional regulator yjdc (yjdc, kda), dihydrofolate reductase (fola, kda), chemotaxis protein chez (chez, kda), and glutathione synthetase (gshb, kda)] that have relatively lower molecular mass compared with the other stressresponsive proteins. we found that these relatively small stress-responsive proteins were highly effective in enhancing the solubility of recombinant hg-csf when used as fusion partner, which will be discussed next. the six stress-responsive proteins (ppib, bfr, yjdc, fola, chez, and gshb) were used as n-terminus fusion expression partner to synthesize the hybrid proteins, i.e. nh -[fusion partner]<[d k]<[hg-csf]-cooh in e. coli cytoplasm. proteins with high molecular mass are usually not used as fusion partner because the big fusion partners may reduce the actual amount of target protein that is obtained after the fusion partner is removed. ppib ( kda), bfr ( kda), yjdc ( kda), fola ( kda), chez ( kda), and gshb ( kda) are relatively small proteins compared with the other proteins above and were also the most effective in enhancing the solubility of hg-csf. as shown in fig. a and b, the n-terminus fusion of six stressresponsive proteins led to a significant increase in both the expression level and solubility of hg-csf. inclusion body formation presumably occurs due to protein overexpression in a heterologous environment in which nascent polypeptide chains are continuously synthesized and then aggregate because of nonspecific hydrophobic interactions among the partially folded intermediates of the recombinant proteins, various host proteins, or a combination of the two within the cytoplasm. in many cases, broad exposure of hydrophobic surfaces on newly synthesized polypeptides results in intermolecular aggregation and misfolding. to prevent such occurrences, molecular chaperones (dnak/dnaj/grpe system, groel/groes, etc.) that recognize and interact with hydrophobic patches of nascent proteins can be used (maier et al., ) . hydrophobic amino acids are usually buried within the core of folded proteins but become exposed in the partially folded polypeptide intermediates (rüdiger et al., ; suh et al., ) . because of this, the solubilityenhancing function of stress-responsive proteins may be similar to that of cis-acting molecular chaperones in the context of fusion proteins (kapust & waugh, ) or as a 'chaperone magnet' (fox et al., ; ahn et al., ) that effectively recruits chaperone binding and prohibits undesired and nonspecific protein-protein interactions. purification and characterization of fusion partner-free hg-csf because the addition of a fusion partner to the native peptide can influence the biological activities and the physical properties of the protein (esposito & chatterjee, ) , the removal of the fusion tags is of crucial importance especially when the recombinant protein is a human therapeutic reagent. in order to purify the fusion partner-free hg-csf in e. (wülfing et al., ) . after loading the soluble fraction containing the fusion proteins onto the ni -nta column, we subsequently added enterokinase to the column to remove the fusion tags. because the sequence d k for enterokinase cleavage is downstream of the fusion tag, the cleaved fusion tags as well as any uncleaved fusion proteins still have the (his) tag and hence remains bound to the ni -nta column, while the released hg-csf is found in the flow-through from the ni -nta column. the flow-through containing the released hg-csf was centrifuged, and the insoluble pellet and supernatant were then analyzed by sds-page and western blot (fig. ) . figure clearly indicates that the recombinant hg-csf released from the fusion protein was still present in the form of soluble protein. with the purified hg-csf released from all the six fusion proteins, the spectra of cd were analyzed and compared with the cd spectrum of commercial standard hg-csf [grasin prefilled-syringe (filgrastim), kirin]. cd is very useful for the analysis of secondary structures of polypeptides and proteins (purdie et al., ) . recent studies also verified the proper folding of recombinant hg-csf using cd spectra (bae et al., (bae et al., , jeong & lee, ). in the other recent studies using cd spectrum, properly folded conformations of recombinant human and viral proteins were structurally analyzed (leopoldino et al., ; brucz et al., ; tolun et al., ; vamvakas et al., ) . the cd spectra of the purified fusion-free hg-csf (fig. a) ( fig. b) , thereby indicating that the recombinant hg-csf has a conformation with the correct secondary structure. it is also assumed that the recombinant hg-csf possesses biological activities similar to native hg-csf based on the proper conformation of recombinant hg-csf, even though the biological activity was not directly determined (bae et al., (bae et al., , jeong & lee, ) . we used the upregulated stress-responsive proteins as n-terminus fusion expression partners upon the synthesis of hg-csf, and then the hybrid proteins designated as nh -[fusion partner]<[d k]<[hg-csf]-cooh were synthesized in e. coli cytoplasm. the synthesized hg-csf was aggregated and formed inclusion bodies when directly expressed without the n-terminus fusion tag. however, the solubility of hg-csf in e. coli cytoplasm dramatically increased when the stress-responsive proteins were used as fusion partners, indicating that the fusion expression partners were highly effective solubility enhancers. in addition, even after the fusion partner was removed by enzymatic cleavage, the released, i.e. fusion-free recombinant hg-csf was present in the form of soluble protein. the cd spectra of the affinity-purified hg-csf was investigated, and the results showed that the recombinant hg-csf had a conformation with the correct secondary structure. consequently, the six stress-responsive e. coli proteins were highly effective cis-acting solubility and folding enhancers for the production of hg-csf and could be used for the bacterial synthesis of other aggregation-prone heterologous proteins in the form of soluble and active proteins. heterologous gene expression using self-assembled supramolecules with high affinity for hsp chaperone mutation in the glucose- -phosphate dehydrogenase gene leads to inactivation of ku dna end binding during oxidative stress escherichia coli maltosebinding protein as a molecular chaperone for recombinant intracellular cytoplasmic single-chain antibodies enhanced secretion of human granulocyte colony-stimulating factor directed by a novel hybrid fusion peptide from recombinant saccharomyces cerevisiae at high cell concentration improved process for production of recombinant yeast-derived monomeric human g-csf recombinant protein folding and misfolding in escherichia coli expression, purification and characterization of recombinant severe acute respiratory syndrome coronavirus non-structural protein new fusion protein systems designed to give soluble expression in escherichia coli the solubility and stability of recombinant proteins are increased by their fusion to nusa alteration of amino-terminal codons of human granulocyte-colony-stimulating factor increases expression levels and allows efficient processing by methionine aminopeptidase in escherichia coli production of soluble mammalian proteins in escherichia coli: identification of protein features that correlate with successful expression overcoming acetate in escherichia coli recombinant protein fermentations enhancement of soluble protein expression through the use of fusion tags single amino acid substitutions on the surface of escherichia coli maltose-binding protein can have a profound impact on the solubility of fusion proteins reduction of trans- , -dihydroxy- , -dithiane by cellular oxidoreductases activates gadd /chop and grp transcription and induces cellular tolerance in kidney epithelial cells multiple stressor-induced proteome responses of escherichia coli bl (de ) the structure of granulocyte-colony-stimulating factor and its relationship to other growth factors secretory production of human granulocyte colony-stimulating factor in escherichia coli escherichia coli maltose-binding protein is uncommonly effective at promoting the solubility of polypeptides to which it is fused a novel fermentation/ respiration switch protein regulated by enzyme iiaglc in escherichia coli structural characterization of natural and recombinant human granulocyte colony-stimulating factors a thioredoxin gene fusion expression system that circumvents inclusion body formation in the e. coli cytoplasm expression, purification, and circular dichroism analysis of human cdk folding and oxidation of recombinant human granulocyte colony stimulating factor produced in escherichia coli a cradle for new proteins: trigger factor at the ribosome the granulocyte-macrophage colonystimulating factors refolding of therapeutic proteins produced in escherichia coli as inclusion bodies the chromosomal gene structure and two mrnas for human granulocyte colony-stimulating factor solubility-enhancing proteins mbp and nusa play a passive role in the folding of their fusion partners purification and characterization of human granulocyte colony-stimulating factor (g-csf) o-linked sugar chain of human granulocyte colony-stimulating factor protects it against polymerization and denaturation allowing it to retain its biological activity solubility enhancement of aggregation-prone heterologous proteins by fusion expression using stress-responsive escherichia coli protein analytical applications of circular dichroism interaction of hsp chaperones with substrates single-step purification of polypeptides expressed in escherichia coli as fusions with glutathione s-transferase advanced genetic strategies for recombinant protein expression in escherichia coli recombinant human granulocyte colony-stimulating factor: effects on normal and leukemic myeloid cells structural features required for the interaction of the hsp molecular chaperone dnak with its cochaperone dnaj overexpression and purification of human calcitonin gene-related peptidereceptor component protein in escherichia coli expression, purification, and physicochemical characterization of the n-terminal active site of human angiotensin-i converting enzyme differential induction of heat shock, sos, and oxidation stress regulons and accumulation of nucleotides in escherichia coli an escherichia coli protein consisting of a domain homologous to fk -binding proteins (fkbp) and a new metal binding motif we thank professor hang chul shin at soongsil university for kindly providing the gene clones of hg-csf. key: cord- -ey h ry authors: eisenbeis, c. f.; winn, d.; poelman, s.; polsky, c. v.; rubenstein, j. h.; olopade, o. i. title: a case of pulmonary toxicity associated with g-csf and doxorubicin administration date: journal: ann hematol doi: . /s sha: doc_id: cord_uid: ey h ry the cytokine growth factor, g-csf (granulocyte colony-stimulating factor), is commonly used in oncologic practice and is generally believed to be a safe agent to administer. we describe here a case of pulmonary toxicity associated with the concurrent administration of g-csf and doxorubicin. we contend that g-csf contributed to the life-threatening lung injury in our patient, and discuss additional reports in the literature of pulmonary toxicity associated with the use of this agent. recombinant human granulocyte colony-stimulating factor (g-csf) has gained widespread popularity because of its use in diminishing the severity and duration of neutropenia associated with myelosuppressive chemotherapy and in mobilizing pluripotent bone marrow stem cells from healthy donors for harvesting for allogeneic stem cell transplants. in addition, it has been administered chronically without adverse effects as maintenance therapy for patients with severe congenital neutropenia and myelodysplastic syndrome. the overwhelming weight of evidence suggests that the administration of g-csf is safe, with the most common side-effects experienced being medullary bone pain, headache, fatigue, and nausea. nevertheless, there are numerous case reports suggesting that g-csf may, in rare cases, produce more severe adverse effects, including interstitial pneumonitis when given with cytotoxic chemotherapy and adult respiratory distress syndrome (ards). we report here a case of life-threatening respiratory dysfunction secondary to bronchocentric granulomatosis (bg) associated with the use of g-csf administered together with doxorubicin. a -year-old woman with stage iiia infiltrating ductal carcinoma of the right breast was treated by modified radical mastectomy. she subsequently received four cycles of adjuvant doxorubicin ( mg/m ) administered every weeks as per research protocol. because of the dose intensity of this regimen, g-csf support (filgrastim, amgen, thousand oaks, calif.; mcg sc daily) was given on days - of each -day cycle according to the protocol. on day of the third cycle, she developed low-grade fevers, which persisted without an obvious infectious source. on day of the fourth cycle, her temperature reached c, and she complained of mild dyspnea on exertion. a chest radiograph revealed a four-quadrant interstitial infiltrate in a reticulonodular pattern (fig. ) . on admission to the hospital, her temperature was . c. she was breathing comfortably and had a room air oxygen saturation of %. examination of the lungs revealed dry crackles in both lung bases. serum chemistries and complete blood count were normal except for mild anemia. blood and urine cultures were negative. serological tests for chlamydia psittaci and c. trachomatis were negative, as was a test for urine histoplasma antigen. bronchoscopy revealed no evidence of infection by bacteria, acid-fast bacillus, fungus, pneumocystis carinii, legionella, mycoplasma, herpes simplex virus, cytomegalovirus, epstein-barr virus, or parainfluenza. transbronchial biopsy showed no evidence of acid-fast bacillus, p. carinii or fungus. it did, however, reveal a peribronchial granulomatous inflammation with bronchial destruction consistent with bg, having features consistent with hypersensitivity or drug reactions (fig. ) . following bronchoscopy, the patient developed a pneumothorax and was placed on high-flow oxygen therapy. her pulmonary status rapidly worsened to respiratory failure, which ultimately necessitated mechanical ventilation. despite initial therapy for bacterial, fungal, and tuberculous infections, the pulmonary lesion worsened. once a diagnosis of bg was made, all antibiotics were dis- continued and methylprednisolone ( mg iv every -h) was started. the patient's respiratory function improved slowly, and she was successfully extubated after days on the ventilator. her slow improvement continued as her steroid dose was tapered, and she was discharged from the hospital after a -week stay. a chest radiograph obtained weeks after the initiation of steroid therapy revealed complete resolution of the pulmonary abnormalities. we have presented the case of a patient who developed bronchocentric granulomatosis while receiving doxorubicin concomitant with g-csf. the uncommon diagnosis of bg is a descriptive, pathologic one, and is not associated with a single disease entity. although it is seen as a manifestation of allergic bronchopulmonary aspergillosis (abpa) in asthmatic patients [ ] , a clear etiologic factor cannot be identified in many instances. an underlying hypersensitivity reaction to an unknown allergen residing within the bronchioles has been suggested as a possible pathogenetic mechanism. another hypothesis is that the inflammatory granulomas are a relatively non-specific pathologic response to a variety of airway injuries [ ] . in the present case, likely causes of pulmonary granulomatous disease were excluded. a carefully obtained history did not reveal environmental or occupational exposure. extensive studies of the bronchial lavage and lung biopsy samples revealed no evidence for infection, including that by uncommon organisms associated with granulomatous inflammation. lacking a clear explanation for the etiology of the lung injury in this case, we wondered whether it might have resulted from the administration of doxorubicin or g-csf, the only medications she had been receiving at the time. although some chemotherapeutic agents are known to cause toxic injury to lung tissue, doxorubicin has not been considered one of them. in addition, the granulomas in the biopsy specimen were more consistent with a hypersensitivity reaction than with a toxic drug reaction of the type associated with chemotherapy. although doxorubicin has been reported to cause a "flare" reaction of localized urticaria or erythema along the course of the vein into which it is being infused, generalized hypersensitivity and anaphylactoid reactions to this agent are rare [ ] , and delayed-type hypersensitivity has not been reported. recently, pneumonitis and pulmonary fibrosis have been observed in dogs inhaling doxorubicin for the treatment of lung tumors [ ] ; however, the radiologic and histologic features in these cases were suggestive of a direct toxic effect of the drug on lung tissue when administered using this novel approach. there is mounting evidence that g-csf administration may be associated with lung injury. initial attention to this issue was drawn by reports of a compelling increase in the pulmonary toxicity of combination chemotherapy regimens when g-csf was added to attenuate myelosuppression [ , , - , , ] . most patients were being treated for lymphoma, and a clear assessment of the contribution of g-csf to the toxicity of the chemotherapeutic regimens was difficult as they all contained agents (i.e., bleomycin, methotrexate, or cyclophosphamide) with known pulmonary toxicity. in contradistinction to these reports, others have shown, both retrospectively [ ] and in a randomized, placebocontrolled fashion [ ] , that the addition of g-csf did not increase the incidence or severity of pulmonary toxicity of a bleomycin-containing regimen. we were unable to find a single report in the literature of a case of pulmonary toxicity associated with the use of g-csf in combination with doxorubicin alone. evidence for a direct physiologic effect of g-csf on lung mechanics can be found in a report of a liver transplant recipient with ards who was given g-csf for antibiotic-induced neutropenia [ ] . administration of g-csf subcutaneously was reproducibly associated with oxygen arterial desaturation requiring % oxygen; the authors speculated that an increase in neutrophil activation secondary to g-csf administration resulted in worsening gas exchange. in addition, recent reports describe a fatal case of ards developing after the unnecessary administration of g-csf to a patient with anemia [ ] , and the development of ards in three patients being treated with g-csf for drug-induced neutropenia [ ] . it appears, therefore, that g-csf can exert an untoward effect on the lung even in the absence of known pneumotoxic drugs, possibly through enhanced neutrophil migration and activation with subsequent local production of superoxide radicals in the lung interstitium. the worsening of our patient's condition after supplemental oxygen therapy supports the involvement of superoxide radicals in this disease process. hypersensitivity reactions have been reported with g-csf administration [ , , , ] . in addition, both acute urticarial and delayed-type hypersensitivity reactions have been associated with polysorbate (tween ), a component of filgrastim [ ] . it remains a possibility that our patient's febrile illness and the granulomatous inflammatory response evident in her lung biopsy were the result of a delayed-type hypersensitivity reaction to the g-csf preparation. the time course of her fevers supports this hypothesis. it is becoming evident that the administration of g-csf is not without risk. additional investigation into the mechanism of g-csf-induced lung toxicity is needed, so that this negative effect can be minimized, thus allowing the continued safe use of this important therapeutic agent. anaphylactoid reaction in a normal donor given granulocyte colony-stimulating factor possible toxicity with the association of g-csf and bleomycin anaphylactic reaction after a first filgrastim (granulocyte colony-stimulating factor) injection ards during g-csf therapy for drug-induced agranulocytosis reply to letter entitled "subcutaneous granulocyte colony-stimulating factor and acute anaphylaxis bronchocentric granulomatosis: a review and thoughts on pathogenesis interstitial pneumonitis related to granulocyte colony-stimulating factor administration following chemotherapy for elderly patients with non-hodgkin's lymphoma inhalation chemotherapy for macroscopic primary or metastatic lung tumors: proof of principle using dogs with spontaneously occurring tumors as a model cytotoxic drug-induced pneumonia and possible augmentation by g-scf -clinical attention subcutaneous granulocyte colony-stimulating factor and acute anaphylaxis development of interstitial pneumonitis during treatment with granulocyte colony-stimulating factor serious pulmonary complications in patients receiving recombinant granulocyte colony-stimulating factor during bacop chemotherapy for aggressive non-hodgkin's lymphoma pulmonary toxicity of abvd chemotherapy and g-csf in hodgkin's disease: possible synergy bronchocentric granulomatosis. disease or diagnosis? interstitial pneumonia in patients receiving granulocyte colony-stimulating factor during chemotherapy: survey in japan - fatal g-csf-induced pulmonary toxicity pulmonary toxicity in patients with advanced-stage germ cell tumors receiving bleomycin with and without granulocyte colony stimulating factor impairment in gas exchange after granulocyte colony-stimulating factor (g-csf) in a patient with the adult respiratory distress syndrome polysorbate hypersensitivity hypersensitivity reactions to cancer chemotherapeutic agents pulmonary toxicity after granulocyte colony-stimulating factor-combined chemotherapy for non-hodgkin's lymphoma key: cord- -qht q l authors: takano, tomomi; azuma, natsuko; satoh, miyuki; toda, ayako; hashida, yoshikiyo; satoh, ryoichi; hohdatsu, tsutomu title: neutrophil survival factors (tnf-alpha, gm-csf, and g-csf) produced by macrophages in cats infected with feline infectious peritonitis virus contribute to the pathogenesis of granulomatous lesions date: - - journal: arch virol doi: . /s - - - sha: doc_id: cord_uid: qht q l feline infectious peritonitis (fip) is a feline coronavirus (fcov)-induced fatal disease of domestic and wild cats. the infiltration of neutrophils into granulomatous lesions is unusual for a viral disease, but it is a typical finding of fip. this study aimed to investigate the reason for the lesions containing neutrophils in cats with fip. neutrophils of cats with fip were cultured, and changes in the cell survival rate were assessed. in addition, the presence or absence of neutrophil survival factors was investigated in specimens collected from cats with fip. furthermore, it was investigated whether macrophages, one of the target cells of fipv infection, produce neutrophil survival factors (tnf-alpha, gm-csf, and g-csf). we showed that virus-infected macrophages overproduce neutrophil survival factors, and these factors act on neutrophils and up-regulate their survival. these observations suggest that sustained production of neutrophil survival factors by macrophages during fcov infection is sufficient for neutrophil survival and contributes to development of granulomatous lesions. feline coronavirus (fcov) belongs to group i of the family coronaviridae. fcov consists of three major proteins: nucleocapsid (n) protein, membrane (m) protein and peplomer spike (s) protein [ ] . fcov is classified into serotypes i and ii according to the amino acid sequence of its s protein [ , ] . both serotypes consist of two biotypes: feline infectious peritonitis (fip) virus (fipv) and feline enteric coronavirus (fecv). thus, there are types i and ii fecv and fipv in fcov. fecv is asymptomatic in cats, but fipv causes fip. it has been proposed that fipv arises from fecv by mutation [ , , ] , but the exact mutation and inducing factors have not yet been clarified. fip is a fatal disease, characterized by vasculitis associated with granulomatous inflammation containing b cells, neutrophils and macrophages. the infiltration of neutrophils into granulomatous lesions is unusual for a viral disease, but it is a typical finding of fip [ ] . macrophages/monocytes play an important role in the pathogenesis of fip. it has been reported that the difference in the proliferation of macrophages/monocytes is related to the difference in pathogenicity between fecv and fipv [ , ] . the possibility of feline vascular endothelial cell injury caused by metalloproteinase- and tnf-alpha produced by fipv-infected monocytes has also been reported [ ] . we reported that virus replication in macrophages induced tnf-alpha production, and the tnf-alpha produced was involved in aggravation of the fip pathology: tnf-alpha produced by fipv-infected macrophages was involved in lymphopenia and an increase in the level of the cellular receptor of type ii fipv, aminopeptidase n [ ] . tnf-alpha reportedly inhibits neutrophil apoptosis [ ] , suggesting its involvement in the infiltration of neutrophils into granulomatous lesions in cats with fip, but this has not yet been clarified. it is also unclear whether fipv-infected macrophages/monocytes produce factors other than tnfalpha that are related to the survival of neutrophils. in this study, we investigated the reason for the granulomatous lesions containing neutrophils in cats with fip. neutrophils of cats with fip were cultured, and changes in the survival rate were examined. the presence or absence of neutrophil survival factors in specimens from cats with fip was also investigated. furthermore, whether macrophages, one of the target cells of fipv, produce neutrophil survival factors was assessed. type ii fipv strain - ( tcid /ml) was administered orally to -to -month-old spf cats. nine cats that developed fip symptoms (fip cats), such as fever, weight loss, peritoneal or pleural effusion, dyspnea, ocular lesions, and neural symptoms, and nine -to -month-old spf cats administered a medium as mock infection controls were used in this study. fip diagnoses were confirmed upon postmortem examination, revealing peritoneal and pleural effusions and granulomatous lesions in major organs. all experiments were performed in accordance with the guidelines for animal experiments of kitasato university. felis catus whole fetus- (fcwf- ) cells were grown in eagle's minimum essential medium containing % l- medium, % fetal calf serum (fcs), u/ml penicillin, and lg/ml streptomycin. feline neutrophils and alveolar macrophages were maintained in rpmi growth medium supplemented with % fcs, u/ml penicillin, lg/ml streptomycin, and lm -mercaptoethanol. type ii fipv strain - was grown in fcwf- cells at °c. fipv strain - was supplied by dr. m. c. horzinek of state university utrecht, the netherlands. mab - - (igg a) used in the present study recognizes the s protein of type ii fipv, as demonstrated by immunoblotting. it has been reported that mab - - has virusneutralizing activity in assays carried out in fcwf- and crfk cells, but an enhancing activity in feline macrophage cultures, depending on the reaction conditions [ ] . blood collected from spf and fip cats using a heparinized syringe was centrifuged at , rpm for min, and the supernatant was used as a plasma sample. ascites fluid was collected from fip cats using a heparinized syringe and centrifuged at , rpm for min, and the supernatant was collected. heparinized blood ( ml) from spf and fip cats was diluted in twofold steps with phosphate-buffered saline (pbs) and subjected to ficoll-hypaque density gradient centrifugation at , rpm for min. after the removal of peripheral blood mononuclear cells and supernatant by aspiration from the top layer, the pellets were mixed with an equal volume of saline containing % dextran for granulocyte separation and allowed to stand for min at °c. the top clear layer was centrifuged at g for min, and the pellet was mixed with ml of . % nacl for min to eliminate contaminating erythrocytes and then mixed with ml of . % nacl. the cells were washed three times with pbs and resuspended with growth medium. cell purity was assessed to be more than % neutrophils by the examination of a smear stained with wright/giemsa solutions. to examine the effect of specimens from cats on the survival rate of neutrophils, feline neutrophils ( cells/ ll) were seeded into -well plates and cultured in the presence of fip-cat-derived ascites fluid (final concentration of : ), plasma (final concentration of : ), and spf-cat-derived plasma (final concentration of : ) for h. prior to and after incubation, ll of wst- solution (wst- cell proliferation assay kit; kishida chemical co., ltd, japan) was added. wst- is a tetrazolium salt that reacts with mitochondrial dehydrogenases, forming the formazan dye. expansion of viable cell numbers results in an increase in the activity of the mitochondrial dehydrogenases in the cells, corresponding to an increase in formazan dye metabolism. after the cells were returned to the incubator for h, the absorbance of the formazan produced was measured at nm with a -well spectrophotometric plate reader, as described by the manufacturer. the percent viability was calculated using the following formula: cell viability (%) = (after incubation od/prior to incubation od) . feline alveolar macrophages were obtained from spf and fip cats by broncho-alveolar lavage with hank's balanced salt solution (hbss) as described previously by hohdatsu et al. [ ] . rna isolation and cdna preparation rna isolation and cdna preparation were performed employing the method of takano et al. [ ] . determination of levels of feline gapdh mrna, tnf-alpha mrna, g-csf mrna, gm-csf mrna, and fcov n gene expression cdna was amplified by pcr using primers specific for feline gapdh mrna, tnf-alpha mrna, g-csf mrna, gm-csf mrna, and fcov n genes. the primer sequences are shown in table . pcr was performed using the method of takano et al. [ ] . the band density was quantified under appropriate uv exposure by video densitometry using scion image software (scion corporation, usa). tnf-alpha mrna, g-csf mrna, gm-csf mrna, and fcov n genes were quantitatively analyzed in terms of the relative density value compared to the mrna for the housekeeping gene gapdh. confluent fcwf- cell monolayers in -well multi-plates were inoculated with ll of the sample dilutions. after virus adsorption at °c, the cells were washed with hbss, and ml of growth medium containing . % carboxymethyl cellulose was added to each well. the cultures were incubated at °c for days, fixed in % buffered formalin, and stained with % crystal violet. inoculation of feline alveolar macrophages with fipv viral suspension (fipv strain - , tcid / . ml) and mab - - solution were mixed in an equal volume ratio and allowed to react at °c for h, and . ml of this reaction solution was used to inoculate feline alveolar macrophages ( cells) cultured in each well of -well multi-plates. as controls, medium alone and virus suspension alone were added to feline alveolar macrophages. after virus adsorption at °c for h, the cells were washed with hbss and ml of growth medium. the cells and culture supernatant were collected every h thereafter. the cells were used for measurement of the tnf-alpha mrna, g-csf mrna, gm-csf mrna, and fcov n genes. tnfalpha mrna, g-csf mrna, gm-csf mrna, and fcov n genes were quantitatively analyzed in terms of the relative density value compared to the mrna for the housekeeping gene gapdh. the culture supernatant was employed for determination of the virus titer. data were analyzed by student's t test. the data in fig. a , b were also analyzed using the mann-whitney test. p values \ . were considered to indicate a significant difference between compared groups. the neutrophil counts in peripheral blood of fip cats were examined and compared with those of uninfected spf cats. the count of neutrophils at the time of blood sampling is shown in fig. . the blood neutrophil counts in spf and fip cats were , ± , ll - (mean ± sd) and , ± , ll - , and median values were , and , ll - , respectively (p \ . ). to investigate the cause of increases in the neutrophil counts in fip cats, neutrophils were isolated from spf and fip cats, and the survival rates after -h culture were compared. the survival rate of neutrophils from fip cats was increased, but not significantly (p = . ), compared to that of spf cats (fig. ) . the survival rate of neutrophils in the presence of specimens from fip cats was significantly higher than those of neutrophils cultured with spf cat-derived plasma and medium (fig. ) . tnf-alpha, gm-csf, and g-csf mrna and fcov n gene expression levels in macrophages of spf and fip cats tnf-alpha, gm-csf, and g-csf mrna and fcov n gene expression levels were increased in alveolar macrophages derived from fip cats (fig. ) . the virus titer was significantly higher in the culture supernatant of macrophages infected with a mixture of fipv and mab - - than in that of macrophages cultured with medium and fipv alone. the neutrophil survival rates were significantly increased in the presence of the culture supernatant of macrophages infected with the mixture of fipv and mab - - compared to those in the presence of other supernatants (fig. ) . when spf-cat-derived alveolar macrophages were infected with a mixture of fipv and mab - - , the intracellular tnf-alpha, gm-csf, and g-csf mrna levels increased (fig. ). neutrophils are important for host defense against pathogens. viral infection generally reduces the number of neutrophils. the transfer of peripheral blood neutrophils to marginal tissues, destruction of neutrophils due to excess antibody production, and direct cell death caused by viral infection are considered to be the causes of neutropenia [ , , , ] . in human immunodeficiency virus and canine parvovirus infections, a reduced neutrophil count has been suggested to allow severe bacterial infection [ , ] . feline immunodeficiency virus and feline panleucopenia virus infections also affect stromal cells in the bone marrow, thus leading to a decreased production of neutrophils [ , ] . in contrast, severe acute respiratory syndrome (sars) coronavirus, which belongs to the same family as fipv, can also cause neutrophilia [ ] . neutrophilia has been also reported in cats with fip [ ] . paltrinieri [ ] reported that cytokines accelerate the delivery of neutrophils to the inflamed lesions, thereby prolonging the lifespan of circulating neutrophils. he also reported that the apoptosis of neutrophils is delayed by cytokines, thus increasing the life of neutrophils in lesions. it is likely that neutrophilia in cats with fip is associated with the infiltration of neutrophils into granulomatous lesions. however, there seems to be no established theory to explain the infiltration of neutrophils into granulomatous lesions in cats with fip. in humans, the lifespan of neutrophils is short. when neutrophils are isolated from peripheral blood and cultured in vitro, apoptosis is induced, and about half of the cells die within h [ ] . when neutrophils isolated from peripheral blood of spf cats were cultured for h, more than % died, suggesting that feline neutrophils also die due to apoptosis, similar to human neutrophils. furthermore, coculture with specimens from fip cats increased the survival rate of neutrophils, suggesting that the production of factors involved in the survival of neutrophils is enhanced in fip cats, and these factors act on neutrophils and prolong their lifespan. the tnf-alpha, gm-csf, and g-csf mrna levels were increased in macrophages of fip cats. these cytokine mrna levels were also elevated in macrophages infected with fipv and mab - - , clarifying the presence of neutrophil survival factors in the macrophage culture supernatant. it was suggested that: ( ) fipv-infected macrophages release tnf-alpha, gm-csf, and g-csf in response to virus replication, and ( ) these cytokines act on neutrophils and prolong their survival. we previously reported that fipv-infected macrophages produced tnfalpha and b-cell differentiation/survival factors, and these factors may have been involved in lymphopenia and hypergammaglobulinemia [ ] [ ] [ ] . kipar et al. [ ] suggested that fipv-infected monocytes are involved in feline vascular endothelial cell injury. cytokines and chemical mediators produced by fipv-infected macrophages/monocytes may play an important role in the pathogenesis of fip in cats. fig. the culture supernatant of fipv-infected macrophages promotes neutrophil survival. spf-cat-derived alveolar macrophages ( cells) were cultured with medium alone, fipv, or fipv and mab - - . the culture supernatant was collected after h. the virus titer in the culture supernatant was measured by the plaque assay method (left). the neutrophils of spf cats ( ml - ) were cultured at °c for h in the presence of each culture supernatant (final dilution of : ), and cell viability was assessed by the wst- assay (right). n = . nd not detected fig. relationship between tnf-alpha, gm-csf, and g-csf mrna expression and fipv replication in macrophages. spf-catderived alveolar macrophages ( cells) were cultured with medium alone, fipv, or fipv and mab - - . the cells were collected at h (as a control) and h. the intracellular tnf-alpha, gm-csf, and g-csf mrna expression levels were measured by rt-pcr. tnf-alpha, gm-csf, and g-csf mrna were quantitatively analyzed in terms of the relative density value to mrna for the housekeeping gene gapdh. n = neutrophil survival factors produced by macrophages in cats tnf-alpha, gm-csf, and g-csf are cytokines inhibiting the apoptosis of neutrophils [ , , , ] . these cytokines are secreted by macrophages and t cells. when these cytokines act on neutrophils, the intracellular apoptosis-inhibitory protein level increases, whereas the apoptosis-promoting protein level decreases, prolonging the survival of neutrophils [ , ] . in inflammatory diseases, such as cystic fibrosis and kawasaki disease, the pathological condition is aggravated as the survival time of neutrophils is prolonged [ , ] . moreover, the pathological aggravation of inflammatory diseases is associated with protease and reactive oxygen species produced by neutrophils [ , ] . these mediators may also be produced in excess and aggravate the granulomatous lesions in fip cats. we are now progressing with studies on the expression of apoptosis-inhibitory and -promoting proteins in neutrophils of fip cats. the enhancement of cathepsin b production in macrophages by elastase produced by neutrophils has been reported [ ] , while fipv reportedly requires cathepsin b to invade cells [ ] , suggesting that neutrophils are also involved in a viral replication enhancement mechanism, different from antibody-dependent enhancement (ade), in fipv infection. addie et al. [ ] reported that fcov re-infection of anti-fcov antibody-positive domestic cats might not result in the development of ade. the involvement of neutrophils in the enhancement of fipv production in macrophages and the influence of macrophages on neutrophil survival should be investigated further. virus production and tnf-alpha, gm-csf, and g-csf mrna expression were markedly enhanced in macrophages infected with fipv and mab - - compared to macrophages infected with the virus alone. we used immunofluorescence to detect fipv antigen: - % of cells were positive when spf cat-derived alveolar macrophages were infected with fipv and mab - - , whereas - % of cells were positive when alveolar macrophages were infected with fipv [ , ] . it seems that the increased number of virus-infected macrophages leads to the overexpression of neutrophil survival-factor-associated mrna in macrophages. in cats with fip, virus can be detected in macrophages of granulomatous lesions. to elucidate the mechanism of the infiltration of neutrophils into granulomatous lesions, we used alveolar macrophages, which can be readily collected in appropriate numbers. in addition, alveolar macrophages can be cultured without activation treatment, unlike monocytes. in this study, we showed that virus-infected macrophages overproduce neutrophil survival factors, and these factors act on neutrophils and up-regulate their survival. these observations suggest that sustained production of neutrophil survival factors by macrophages during fcov infection is sufficient for neutrophil survival and contributes to the development of granulomatous lesions. these findings may be important for elucidating the pathogenesis of fip. risk of feline infectious peritonitis in cats naturally infected with feline coronavirus gamma interferon/interleukin balance in tissue lymphocytes correlates with down modulation of mucosal feline immunodeficiency virus infection immune and idiopathic neutropenia replication of feline coronaviruses in peripheral blood monocytes cytokine-mediated bax deficiency and consequent delayed neutrophil apoptosis: a general mechanism to accumulate effector cells in inflammation polymorphonuclear leukocytemediated cell and tissue injury: oxygen metabolites and their relations to human disease neutrophil elastase up-regulates cathepsin b and matrix metalloprotease- expression the molecular genetics of feline coronaviruses: comparative sequence analysis of the orf a/ b transcription unit of different biotypes a study on the mechanism of antibody-dependent enhancement of feline infectious peritonitis virus infection in feline macrophages by monoclonal antibodies enhancement and neutralization of feline infectious peritonitis virus infection in feline macrophages by neutralizing monoclonal antibodies recognizing different epitopes the role of igg subclass of mouse monoclonal antibodies in antibody-dependent enhancement of feline infectious peritonitis virus infection of feline macrophages morphologic features and development of granulomatous vasculitis in feline infectious peritonitis spontaneous neutrophil apoptosis and regulation of cell survival by granulocyte macrophage-colony stimulating factor asymptomatic bacteriuria in puppies with canine parvovirus infection: a cohort study neutropenia, neutrophil dysfunction, and bacterial infection in patients with human immunodeficiency virus disease: the role of granulocyte colony-stimulating factor epstein-barr virus infects and induces apoptosis in human neutrophils marrow accessory cell infection and alterations in hematopoiesis accompany severe neutropenia during experimental acute infection with feline immunodeficiency virus virus infection of endothelial cells increases granulocyte adherence molecular cloning and sequence determination of the peplomer protein gene of feline infectious peritonitis virus type i comparison of the amino acid sequence and phylogenetic analysis of the peplomer, integral membrane and nucleocapsid proteins of feline, canine and porcine coronaviruses in vitro effect of recombinant human granulocyte colonystimulating factor on canine neutrophil apoptosis in vitro effect of recombinant human tumor necrosis factor-alpha on canine neutrophil apoptosis a review of feline infectious peritonitis virus: molecular biology, immunopathogenesis, clinical aspects, and vaccination the acute phase reaction some aspects of humoral and cellular immunity in naturally occurring feline infectious peritonitis pathogenesis of feline panleukopenia virus and canine parvovirus programmed cell death of the normal human neutrophil: an in vitro model of senescence feline infectious peritonitis and feline enteric coronavirus infections. . feline infectious peritonitis two related strains of feline infectious peritonitis virus isolated from immunocompromised cats infected with a feline enteric coronavirus differential role for low ph and cathepsin-mediated cleavage of the viral spike protein during entry of serotype ii feline coronaviruses acquisition of macrophage tropism during the pathogenesis of feline infectious peritonitis is determined by mutations in the feline coronavirus spike protein the role of eosinophils and neutrophils in inflammation a ''possible'' involvement of tnf-alpha in apoptosis induction in peripheral blood lymphocytes of cats with feline infectious peritonitis tnf-alpha, produced by feline infectious peritonitis virus (fipv)-induced macrophages, upregulates expression of type ii fipv receptor feline aminopeptidase n in feline macrophages b-cell activation in cats with feline infectious peritonitis (fip) by fip-virus-induced b-cell differentiation/survival factors delayed apoptosis of circulating neutrophils in kawasaki disease feline infectious peritonitis viruses arise by mutation from endemic feline enteric coronaviruses bcl-xl-and baxalpha-mediated regulation of apoptosis of human neutrophils via caspase- haematological manifestations in patients with severe acute respiratory syndrome: retrospective analysis neutrophil survival factors produced by macrophages in cats acknowledgments this work was supported by grant for encouragement of young scientists (no. e ) from the school of veterinary medicine, kitasato university. key: cord- - ihuqvei authors: thomas, william b. title: nonneoplastic disorders of the brain date: - - journal: clin tech small anim pract doi: . /s - ( ) - sha: doc_id: cord_uid: ihuqvei computed tomography (ct) and magnetic resonance imaging (mri) are helpful in the diagnosis of many nonneoplastic brain disorders in the dog and cat. the ability of ct and mri to depict normal and abnormal anatomy facilitates the identification of developmental anomalies, including hydrocephalus, chiari malformations, arachnoid cysts, and cerebellar hypoplasia. these imaging modalities also allow the detection of hemorrhage and infarction and are therefore useful in the evaluation of spontaneous cerebrovascular disorders and head trauma. finally, many inflammatory diseases, such as encephalitis, brain abscess, and parasite migration, cause abnormalities detectable by ct and mri. although more research on the imaging features of specific nonneoplastic brain disorders is needed, current information indicates that ct and mri are useful in the management of these disorders. imaging strategies for ct and mri are similar to those described for intracranial neoplasia. (refer to the article entitled "intracranial neoplasia" by kraft and gavin in the may issue.) throughout this chapter, the mri features of the various disorders are those depicted with spin echo imaging. readers should refer to the article entitled "advanced imaging concepts: a pictorial glossary of ct and mri technology" by tidwell and jones in the may issue for a description of other pertinent mri techniques such as gradient echo and inversion recovery pulse sequences, magnetic resonance angiography, and diffusion/perfusion imaging. disorders of brain development ecent advances in imaging techniques have greatly improved the ability to detect pathologic processes in the brain, localize lesions precisely, and predict the type of disease more accurately than ever before. although most reports of computed tomography (ct) and magnetic resonance imaging (mri) of brain disease in veterinary medicine have focused on the diagnosis of brain tumors, these imaging methods are also valuable in the evaluation of various nonneoplastic brain diseases. the purpose of this article is to provide an overview for imaging some of the more frequently encountered nonneoplastic diseases of the brain. clinical features of each disease are included because imaging can supplement but never replace a careful history and thorough physical and neurological examination. typical pathological changes are also mentioned, because ct and mri reflect gross and microscopic pathology and an understanding of the expected pathological changes can be very helpful in predicting imaging findings. ideally, a review article such as this would be based on systematic research and clinical data. unfortunately, the current literature on imaging of nonneoplastic brain disease in veterinary patients is fairly sparse. therefore, the information presented in this article is based on the limited data regarding veterinary patients supplemented by the much more complete information on comparable diseases in human patients, as well as my own experience. as more information becomes available, the conclusions in this article may have to be modified. hydrocephalus is a pathological condition in which there is accumulation of cerebrospinal fluid (csf) within the cranium. this usually occurs within the ventricular system (internal hydrocephalus) but can involve the subarachnoid space (external hydrocephalus)) hydrocephalus is not a specific disease, but rather a multifactorial disorder with a variety of pathophysiological mechanisms. csf is produced at a constant rate by the choroid plexuses of the lateral, third, and fourth ventricles; the ependymal lining of the ventricular system; and blood vessels in the subarachnoid space. the csf circulates through the ventricular system into the subarachnoid space, where it is absorbed by arachnoid villi. hydrocephalus can be caused by a blockage of the flow of cse called obstructive hydrocephalus, or secondary to decreased volume of brain parenchyma, termed hydrocephalus ex vacuo. (increased formation of csf is virtually never a cause of hydrocephalus.) a number of conditions, such as infarction and necrosis, can result in decreased volume of brain parenchyma and hydrocephalus ex vacuo. obstruction to csf flow can occur anywhere along the pathway from its formation to the site of absorption in the cranial and spinal arachnoid villi. obstruction within the ventricular system or at its outflow through the lateral apertures is called noncommunicating hydrocephalus because the ventricular system does not communicate with the subarachnoid space. obstruction within the subarachnoid space or at the level of absorption in the arachnoid villi is called communicating hydrocephalus. ~ hydrocephalus can be classified further as congenital or acquired. congenital hydrocephalus is most common in toybreed dogs. the causes are diverse and include genetic factors, developmental anomalies, and intrauterine or perinatal infection or bleeding in the brain. congenital hydrocephalus may be associated with a wide range of other nervous system anomalies, including meningomyelocele, chiari malformation, dandy-walker syndrome, and cerebellar hypoplasia. clinical signs of congenital hydrocephalus include an enlarged, domeshaped head with persistent fontanelles and open cranial sutures. neurological deficits include abnormal behavior, disturbed consciousness, ataxia, circhng, blindness, seizures, and vestibular dysfunction. in mature dogs, diseases such as tumors and inflammatory diseases can cause acquired hydrocephalus. neurological deficits are similar to those m puppms, but if hydrocephalus develops after the cranial sutures have closed, malformation of the skull does not develop. , ct and mri allow accurate assessment of ventricular size, extent of cortical atrophy, and the presence of any focal lesions that may account for the hydrocephalus. imaging is also useful in monitoring patients after surgical placement of ventriculoperitoneal shunts used for treatment. changes in ventricular size can be monitored, and the presence of complications such as subdural hematoma or hygroma can be evaluated. ventricular size is usually assessed subjectively, noting the progressively greater proportion of the intracranial volume occupied by the lateral, third, and/or fourth ventricles , ( fig ) . several investigators have provided quantitative measurements. when measured at the level of or caudal to the lnterthalamlc adhesion, hudson et al state that lateral ventricles are considered enlarged ff lateral ventncular height exceeds . cm or the ratio between the height of the lateral ventricle and the width of the cerebral hemisphere (ventriclehemisphere ratio) exceeds . . spaulding and sharp consider the lateral ventricles enlarged if the ratio between lateral ventricular height and the dorsoventral height of the cerebral hemisphere exceeds . . however, there is a poor correlation between clinical signs and ventricular size, and symmetric or asymmetric enlargement of the lateral ventricles is relatively common in normal adult dogs and puppies. q° therefore, diagnosis of hydrocephalus must be based on clinical features, not just ventricular size. hydrocephalus ex vacuo can be distinguished from obstructive hydrocephalus based on enlarged cortical sulci and subarachnoid space secondary to atrophy of brain parenchyma (fig ) . in obstructive hydrocephalus, the site of obstruction may be identified by dilatation of csf spaces proximal to the obstruction, and normal or collapsed spaces distally. for example, obstruction at the level of the third venmcle would be expected to result in enlarged lateral ventricles but no enlargement of the mesencephalic aqueduct and fourth ventricle (fig ) . dilatation of all the ventricles and the subarachnmd space implies an obstruction at or near the arachnoid vilh. unfortunately, this simplisuc approach has limited accuracy. for example, % to % of human patients with communicating hydrocephalus have little or no &latation of the fourth ventricle. obstructing masses, such as tumors, granulomas, and cysts, may be identified, especially on postcontrast images (fig ) . mri is more sensitive than ct in showing small focal lesions, especially those in the caudal fossa) periventricular edema may be identified in some patients with hydrocephalus. experimentally, acute obstructive hydrocephalus in dogs causes edema starting at the dorsolateral angles of the lateral ventricles and spreading into the adjacent white matter, n on ct, this is evident as blurring or loss of the normally sharp ventricular margins. the edema is best appreciated on t -welghted mri as increased intensity compared with normal white matter. ~ periventrlcular edema is most frequently associated with acute hydrocephalus and increased intraventricular pressure, rather than chronic, relatively compensated hydrocephalus with normal intraventricular pressure. imaging techniques make it possible to readily identify ventriculomegaly but may give little clue as to its clinical significance. it is therefore necessary to interpret the finding of ventriculomegaly in context with clinical features. chiarl described four unrelated types of malformations of the brainstem and cerebellum in human patients. the most common is chiari i malformation, which consists of caudal displacement of a portion of the cerebellum through the foramen magnum into the cervical portion of the vertebral canal. the cause is an underdeveloped occipital bone that induces overcrowding of the caudal fossa, which is accommodated by caudal displacement of the cerebellum. many human patients with this malformation develop hydromyelia (a dilatation of the central canal of the spinal cord that is lined by ependyma) and/or syringomyelia (an accumulation of fluid within the spinal cord that is not lined by ependyma). the term syringohydromyefia is often used because it can be &fficult or impossible to tell whether the cavity is lined with ependyma except at necropsy. syringohydromyelia in chiarl i malformation is caused by obstruction of csf flow at the foramen magnum. the brain expands as it fills with blood during systole, inducing a pressure wave in the csf that is accommodated in normal subjects by rapid movement of csf from within the skull to the vertebral canal. with obstruction to rapid movement of csf through the foramen magnum, the caudal portion of the cerebellum moves down- ward with each systolic pulse, acting as a piston on the surface of the spinal cord. this relentless compression of the spinal cord propels csf into and within the syrinx. some pauents also have hydrocephalus attributed to obstruction of csf flow in the crowded caudal fossa. clinical signs of chiari i malformation in human beings can develop in childhood or adulthood and include head and neck pain, myelopathy, or encephalopathy. , a similar malformation has been reported m adult dogs with neck pain and tetraparesls. ~a diagnosis is best made on sagittal mri of the craniocervmal junction (fig ) . the caudal portion of the cerebellum is displaced into the vertebral canal to a variable degree , the cerebellomedullary cistern is small or absent and the caudal fossa may appear subjectively overcrowded. hydrocephalus and syringohydromyelia may be evident. - syringohydromyelia often involves the caudal cervical or cranial thoracic segments, so this entire region should be imaged. all patients with syrmgohydromyelia should have imaging of the craniocervical region to rule out a chiari i malformation or other obstruction of csf flow. chiarl ii malformation, also known as arnold-chiari or cleland-chari malformation, involves caudal displacement of the brainstem and cerebellum through an enlarged foramen magnum. the fourth ventricle is elongated and extends caudal to the foramen magnum, and the brainstem may be kinked. chiari iii malformation is displacement of the cerebellum through a cervical spina bifida resulting m a cervmal encephalocele. chiari ii and iii malformations are not well described in small animals. chiari iv malformation is severe cerebellar hypoplasia and is discussed separately. in human patients, the dandy-walker complex is a group of malformations consisting of an enlarged caudal fossa, hypoplasia of the cerebellar vermis, and cystic dilatation of the fourth ventricle that nearly fills the caudal fossa. the cerebellar hemispheres are usually hypoplastic as well. other anomalies are also common, including hydrocephalus, hypoplasia of the corpus callosum, and syringohydromyelia. dandy-walker malformation is caused by outflow obstruction of the fourth ventricle in the developing embryo. this causes dilatation of the fourth ventricle, which enlarges upward between the developing cerebellar hemispheres, preventing their fusion. when the cerebellar hemispheres do not fuse, the vermis does not form. analogous malformations have been described in the dog and cat. ~< - in contrast to human patients, most dogs and cats with this syndrome do not have obvious enlargement of the caudal fossa, ls- neurological deficits typically occur at a young age and primarily reflect cerebellar dysfunction, including ataxia, hypermetria, intention tremor, and vestibular dysfunction, ls- on ct and mri, dandy-walker malformation is characterized by an enlarged caudal fossa filled by an enormous fourth ventricle and a small cerebellum. , , °, the cerebellar vermis may be absent, producing a cleft in the middle of the cerebellum. hydrocephalus may also be evident. . a variety of in utero msults and heritable defects may cause failure of normal development of the cerebellum, resulting in a hypoplastic or absent cerebellum. the most common cause in cats is in utero or permatal infection with the feline panleukopenia virus. cerebellar hypoplasia has also been reported in dogs, in which a heritable basis has been suspected in some cases, but in utero infections cannot be excludedy , single or multiple animals within a litter may be affected. there is nonprogressive ataxia, hypermetria, and intention tremor, first apparent at about the time the animal begins to walk. ct or mri shows a small or absent cerebellum. the remainder of the caudal fossa is filled with cse other anomahes, such as hydrocephalus, may also be apparent. = arachnoid cysts (also called intra-arachnold cysts) are accumulations of fluid surrounded by a membrane resembhng the arachnmd mater. they carl develop in the subarachnold space anywhere along the neuraxis. congenital arachnoid cysts are developmental anomalies originating from a splitting or duplication of the arachnoid mater. the cyst wall is formed by several layers of arachnoid cells. acquired arachnoid cysts may result from postinflammatory loculauon of csf caused by trauma, infection, or hemorrhage. in human beings, congenital intracranial arachnoid cysts may be asymptomatic or cause neurological deficits as the cyst progressively enlarges and compresses adjacent neural structures or interferes with csf circulation. enlargement of arachnoid cysts occurs because of fired production by the cyst wall or an anatomical communication functioning as a oneway valve between the cyst and the subarachnoid space. ~ intracranial arachnoid cysts have been reported in the dog and cat. z , the most common locatmn is the subarachnoid space between the cerebellum and the tentorium cerebelli. clinical signs may occur in immature or mature animals and include smzures, paresis, abnormal behavior, and cranial nerve deficits. in some patients, the cyst is an incidental finding on imaging. on ct, the cyst has well-defined margins and contains fired that is isodense wath csf (fig ) . there is mal, or intraventricular. epidural, subdural, and subarachnoid hemorrhages are often associated with trauma and are discussed later. intraparenchymal hemorrhage can be primary or secondary and can extend into the ventricular system. the cause of primary intraparenchymal hemorrhage is incompletely understood. people with this disorder often have systemic hypertension and fibrinoid degeneration of arteries in the b r a i n y although intracranial hemorrhage caused by hypertension is poorly documented in dogs and cats, primary hypertension and hypertension secondary to other disorders, such as renal disease, hyperadrenocorticism, and hyperthyroidism, are well recognized. these animals may be predisposed to cerebrovascular disease and intracranial hemorrhage. secondary causes of intracranial hemorrhage include trauma, infarction, congenital vascular malformations, intracranial tumor, vasculitis, and coagulopathies. [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] the clinical signs of intraparenchymal hemorrhage consist of a sudden onset of neurological deficits referable to a focal brain lesion. signs may progressively worsen as the hematoma expands, compressing adjacent neural structures and increasing intracranial pressure. - - computed tomography ct is exqmsitely sensitive to acute hemorrhage. because the attenuation of x-rays is primarily attributable to the globin portion of blood, there is a linear relationship between ct attenuation and hematocrit. ° the attenuation of whole blood with a hematocrit of % is approximately hounsfield units (hu). in comparison, normal gray matter has an attenuation of to hu, and normal white matter, to hu. ° acute hemorrhage in a patient with a normal hematocrit is therefore immediately evident as increased attenuation on ct ( fig ) . , hemorrhage in patients with severe anemia may be less obvious. an acute hematoma may he surrounded by a hypodense region corresponding to edema, and there is often an associated m a s s effect. ' the attenuation of extravasated blood increases for the first hours with clot formation and extrusion of low-density serum and subsequent increase in hemoglobin concentration. ° after that, the attenuation gradually decreases as the result of lysis and phagocytosls of erythrocytes, which starts at the periphery and progresses centrally. °~ the hematoma eventually becomes isodense at about month after onset. °- use of contrast agent is unnecessary in most acute hematomas and may obscure the inherent hyperdensity, confusing the diagnosis. however, use of contrast agent may be helpful if there is a small hemorrhage with mass effect out of proportion to the size of the hematoma. in this instance, the differential diagnosis includes bleeding into a tumor, and contrast may be necessary to visualize the tumor. ° in hematomas not associated with underlying neoplasia, enhancement first appears at the periphery of the hematoma at approximately to days because of neovascularization. . - a ring pattern of enhancement may persist for to weeks. ° although variations can occur, the mri features of most intracranial hematomas follow a predictable course over time. factors intrinsic to the lesion that affect images include the age of the hemorrhage, whether the bleeding is arterial or venous, the location of the bleeding, and the presence of any underlying lesions. operator-dependent factors that affect the images include the mare magnetic field strength, the exact pulse sequence parameters used, and the method of echo formation. hemorrhage can be thought of as an iron salvage pathway, in which iron from the extravasated erythrocytes is mobilized from hemoglobin and converted to short-term iron-containing proteins for transfer to the reticuloendothelial system, or to long-term storage proteins for local deposition. during this process, oxyhemoglobin is converted in a stepwise fashion to deoxyhemoglobin, methemoglobin, and finally ferritin and hemosiderin. these various forms of hemoglobin affect the appearance of hemorrhage in three ways: ( ) protein effects that are present with all forms of hemoglobin; ( ) paramagnetm relaxation caused by paramagnetic molecules, which is significant only with methemoglobin; and ( ) inhomogeneous susceptibility effects induced by paramagnetlc forms of hemoglobin (deoxyhemoglobin and methemoglobm) within erythrocytes. , protein effects. relaxation times are shortened in the presence of proteins in water solutions. this is caused by the changing magnetic fields of nearby nuclei as the molecules undergo brownian motion. protein effects are generally proportional to protein concentrations and result in shortening of t and t ; that is, increased intensity on tl-weighted images and decreased intensity on t -weighted images, compared with pure water. paramagnetic effects. biological substances containing unpaired electrons have magnetic properties dominated by the magnetic effects of these unpaired electrons. the magnetic fields of the unpaired electrons are normally oriented randomly such that there is no net magnetization. however, when an external magnetic field is applied, the unpaired electrons tend to align parallel to that field, resulting in enhancement (increased magmtude) of that applied field. substances that have no intrinsic magnetic field m the absence of an applied magnetic field, but align with and thus enhance that applied field, are termed paramagnetic. some forms of hemoglobin contain iron with paramagnetic properties, which greatly affects the mri appearance. if water molecules approach a paramagnetic center, there is an interaction between the nuclear magnetic dipoles of the water (protons) and the magnetic dipoles of the paramagnetic center (unpaired electrons). this proton-electron dipoledipole interaction shortens relaxation ume of the water protons. for proton relaxation enhancement to occur, however, the water proton must come within about . nm of the paramagnetic ions' unpaired electrons, because the magnitude of this interaction is inversely proportional to the sixth power of the distance between the dipoles. these paramagnetic effects are more prominent on tl-weighted images than on t -weighted images. they will therefore result in an increased signal on tl-weighted images, similar to the effects of gadolinium-based contrast agents. (paramagnetism of mri contrast medium is also discussed in the tidwell & jones article in the may issue.) inhomogeneous susceptibility effects. when paramagnetlsm is confined to a small region (such as within erythrocytes), there is an inhomogeneous distribution of paramagnetic susceptibility that creates microscopic field gradients. water molecules diffusing across the erythrocyte membrane experience a fluctuating magnetic field that results in dephasing and shortened t relaxation, a process called inhomogeneous susceptiblhty effect. in spin-echo sequences, inhomogeneous susceptibihty effects result in signal loss because of shortened t relaxation. this leads to decreased intensity on t -weighted images. hematoma evolution. these three relaxation mechanisms along with proton density contribute to image contrast during the various stages of hematoma formation (table ) . because numerous factors can effect the appearance of intracranial hemorrhage on mri, the following summary is necessarily oversimplified but generally applicable to spin-echo sequences at . to . t. (refer to the tidwell &jones article in the may issue for a discussion on gradient echo imaging of hemorrhage.) furthermore, the timing of various stages may vary significantly, and different stages often overlap. , the precise dating of specific hematomas is notoriously inaccurate, so the nomenclature of the temporal stages of hematomas is somewhat arbitrary. oxyhemoglobm. freshly extravasated blood from arterial bleeding is composed of intact erythrocytes containing primarily oxyhemoglobin. the iron in oxyhemoglobin is in the ferrous form (fe+ ). because there are no unpaired electrons in the iron (or other atoms), oxygenated blood is not paramagnetic. in the absence of paramagnetism, peracute hematomas appear as a high-proton-density region, with slightly shortened relaxation times, compared with water, mainly because of the protein content of blood. this peracute stage of a hematoma usually persists for fewer than hours. because of their transient nature, peracute hematomas are rarely seen in clinical practice. they are isointense to slightly hypointense on t weighted images and hyperintense on t -weighted images, and therefore appear similar to many other brain lesions. ,. thus, ct is the preferred imaging modality if peracute hemorrhage is suspected. deoxyhemoglobin. after a few hours, the oxygen tension within the hematoma is reduced, resulting in the formation of deoxyhemoglobin (acute hematoma). deoxygenation is usually complete by hours. removal of molecular oxygen changes the distribution of electrons in the ferrous ion, leaving four unpaired electrons in the outer shell. these unpaired electrons confer deoxyhemoglobin with paramagnetic properties. the paramagnetic iron of deoxyhemoglobm is contained within a hydrophobic crevice in the hemoglobin mdlecule. this prevents water from coming close enough to the paramagnetic ion to induce paramagnetic (dipole-dipole) interaction, thus, the hematoma is still isointense to slightly hypointense on t -weighted images. as long as the erythrocyte membrane is intact, the paramagneuc deoxyhemoglobln is localized within the cell. this results in inhomogeneous suscepub]lity effects# acute hematomas that contain intracellular deoxyhemoglobin consequently appear very hypointense on t -we]ghted images. at this stage, any surrounding edema appears as a hyperintense perimeter on t -weighted images) intracellular methemoglobin. as oxygen tension within the hematoma falls further, deoxyhemoglobin is oxidized to methemoglobin (subacute hematoma). in whole blood, methemoglobin begins to accumulate at the rate of about % per day, after the first days. s conversion to methemoglobin is maximal when the p is approximately mm hg. if the oxygen tension is higher or lower, conversion to methemoglobin is slowed, affecting mri contrast. the iron in methemoglobin is in the ferric (fe + ) state, which has five unpaired electrons in its outer shell and is highly paramagnetic. also, one of the coordination sites of the methemoglobln molecule is occupied by a water molecule that is rapidly exchanged with other water molecules in solution. as a result, water is positioned within . nm of the paramagnetic iron. this allows paramagnetic (dipole-dipole) interaction to shorten t relaxation, causing methemoglobin to be hyperintense on tl-weighted images. this hyperintensity begins at the periphery of an intraparenchyreal hematoma and progresses inward, probably because the outer rim of the hematoma has the optimal oxygen tension for the oxidation of hemoglobin. , at this stage the hematoma is still hypointense on t -welghted images other causes of t hyperintensity or t hypointenslty include fat, calcification, mucinous material, intratumoral melanin, flow effects, and enhancement with paramagnetic contrast agents extracellular methemoglobin. soon after methemoglobin formation begins, glucose reserves in the erythrocytes are depleted and hemolysis ensues. this eliminates the inhomogeneous susceptibility effect as the methemoglobin becomes uniformly distributed. this prolongs t , and the hematoma is once again hyperintense on t -weighted images (rebound hyperintensity). on the other hand, tl-weighted images are not affected and therefore continue to show hyperintensity. this paradoxical state of t and t hyperintensity may persist for month or more (fig ) . ferritin and hemosiderin. after to weeks, modified macrophages (gitter cells) infiltrate from surrounding brain and remove iron from the hematoma (chronic hematoma). this eventually eliminates the remaining protein and inhomogeneous susceptibility effects. the center of the hematoma eventually either collapses or is replaced by cse the iron is deposited at the periphery of the hematoma as hemosiderin and ferritin, which behave paramagnetically. the iron in these storage forms is not accessible to water, so paramagnetic (dipole-dipole) interaction does not occur, but the inhomogeneous susceptibility effect shortens t . a chronic hematoma has a rim that is hypointense on tl-weighted images and very hypointense on t -weighted images. the rim becomes thicker as the hematoma resolves. whenever intracramal hemorrhage is identified, it is important to determine if it is associated with an underlying tumor or is caused by a nonneoplastic lesion. the use of a contrast agent often allows identification of an underlying tumor. ° an important finding in a hemorrhagic tumor is persistent surrounding edema (decreased attenuanon on ct, hyperintensity on t -weighted images), whereas edema has usually resolved by the chronic stage of a nonneoplastic hematoma. in neoplastic hemorrhage, mri signal intensity patterns are more heterogeneous and the temporal evolution of intensity patterns is often delayed compared with nonneoplastic hematomas, probably because hypoxia within the tumor delays methemoglobin formation. °, neoplastic hemorrhage often lacks the well-defined hypointense rim characteristic of nonneoplastic hematomas in their subacute and chronic stage. °,~ this is because the persistently altered blood-brain barrier of tumors may allow more efficient removal of ferritm and hemosiderin. in situations in which the diagnosis is still uncertain, repeat scanning is often useful because this allows evaluation of the expected temporal changes. ° the most common is the arteriovenous malformation (avm). clinical signs are caused by spontaneous hemorrhage and are usually suggestive of a focal cerebral lesion. the onset of signs is usually acute and occurs in middle-aged to older animals. > the relatively late onset of signs associated with a congenital lesion can be explained by contmued hemodynamic stress and consequent attenuation of the abnormal vessels, which eventually leads to hemorrhage, z° when a cerebrovascular malformation has bled, early ct or mri usually shows intraparenchymal hemorrhage (fig ) . ct of capillary malformations is often normal, but may show an isodense to slightly hyperdense mass. contrast enhancement is usually minimal. mri is more sensitive, and shows a lace-like region of stippled contrast enhancement with no or subtle abnormality on unenhanced images. a a cavernous malformation appears on ct as a focal hyperdense region with variable calcification with mild contrast enhancement. on mri, a cavernous malformation is seen as a central region of mixed intensity, corresponding to methemoglobin, surrounded by circumferential rings of hypomtense hemosiderin and ferritin) whenever a spontaneous intraparenchymal hematoma is identified, it is important to look for any associated large vessels to suggest a vascular malformation. however, the failure to identify large vessels does not entirely exclude a vascular malformation, because compression or obliteration of vessels by adjacent hematoma, extremely slow flow, and thrombosis may obscure the abnormal vessels. , although ct and mri are useful in detecting hemorrhage associated with vascular malformations and depicting the vascular anatomy of some of these lesions, catheter angiography or magnetic resonance angiography (mra) is often necessary for complete assessment of cerebrovascular malformations m human patients. , ,~ (refer to the tidwell & jones article in the may issue for a discussion of mra.) obstruction of flow in the vessels of the brain can result in a sudden onset of neurological signs caused by infarction. arterial obstruction can be caused by thrombosis or embolism. a thrombus is a blood clot developing within a vessel that causes obstruction at the site of formation. embolism is occlusion of a vessel by a fragment of blood clot or other substance that has flowed to the site of obstruction from a distant location. because of abundant venous anastomoses, venous infarction is less common than arterial thromboembolism. in human patients, cerebrovascular thrombosis or embolism is often secondary to atherosclerosis. atherosclerosis also occurs in dogs, especially older dogs, dogs with hypothyroidism, and miniature schnauzers with idiopathic hyperlipoproteinemia. , , s atherosclerosis associated with these conditions can lead to cerebral thromboembohsm and neurological dysfunction. - other diseases associated with cerebral thromboembolism in dogs include sepsis, coagulopathy, neoplasia, and heartworm infection. > ° the hallmark of brain infarction is an acute onset of focal bram dysfunction. neurological deficits depend on the site of the lesion and are typically asymmetric. involvement of the forebrain, such as with thromboembolism of the middle cerebral artery, usually results in contralateral hemiparesis with decreased postural reactions. seizures are very common in dogs and cats with infarction affecting the forebrain. there may be contralateral bhndness with normal pupillary light reflexes, and the patient may circle or turn toward the side of the lesion. lesions in the cerebellum may cause ataxia, hypermetria, vestibular dysfunction, or opisthotonos. brainstem involvement is characterized by gait deficits ranging from ipsilateral hemlparesis to tetraplegia, cranial nerve deficits, and abnormal levels of consciousness, including coma. , , , , , , nonhemorrhagic infarction computed tomography. changes may be detected on ct as early as to hours after onset of signs and consist of a slight decrease in attenuation and subtle mass effect. these changes are related to edema and reach a maximum at to days (fig ) and resolve by to weeks after infarction. the location and shape of the lesion correspond with the distribunon of the involved vessel(s), most commonly the middle cerebral artery. abnormal contrast enhancement may be seen as early as hours but often does not become evident until about week after infarction. , enhancement is most apparent at the periphery of the lesion and reflects growth of new capillaries without a normal blood-brain barrier. enhancement of hypodense infarcts can result in isodense lesions, thereby masking their presence. for this reason, unenhanced as well as enhanced ct should be performed. in the chronic phase ( to weeks), the hypodense region becomes more sharply marginated as necrotic tissue is resorbed. ultimately there is a loss of parenchymal volume with attendant dilatation of adjacent sulci and ventricles. contrast enhancement does not usually occur in the chronic stage because the integrity of the blood-brain barrier is restored. magnetic resonance imaging. early changes on mri consist of a subtle increase in intensity on t -weighted and proton density-weighted images. these changes reflect edema and can be detected as early as hour after vascular occlusion. mri is thus more sensitive than ct in early infarction. also, mri is more sensitive in detecting small infarcts and those involving the brainstem. in acute infarction, tl-weighted mri is less sensitive than t -weighed and proton density-weighted images and may be normal. ~ parenchymal enhancement with gadolinium is uncommon within the first hours. , the use of functional mri techniques such as diffusion and perfusion studies for the detection of acute infarction is currently being investigated and is discussed in the arucle by tidwell &jones in the may issue. after the first hours, the hyperintensity on t -welghted and proton density-weighted images becomes more obvious (fig ) . ' ' during this time, tl-weighted images may show decreased signal. , gyral swelling and mass effect are more prominent, becoming maximal to days after onset of occlusion. parenchymal enhancement becomes evident within to days and may persist for to weeks. after several weeks, the signal changes seen on earlier scans become smaller and better defined. there is focal atrophy with dilatanon of nearby sulci and ventricles. there may be a collection of fluid with the s~gnal characteristics of cerebrospinal fluid. contrast enhancement does not usually occur. the above description applies to nonhemorrhagic or "bland" infarcts. however, many infarcts will have attendant bleeding caused by reperfusion of damaged blood vessels. this results in a hemorrhagic infarct. petechial hemorrhage into an infarct may result in an isodense lesion on unenhanced ct because of the combined effects of the brain edema, which decreases attenuation, and acute hemorrhage, which increases attenuation. mass effect caused by the edema usually provides a clue to the presence of a lesion. if hemorrhage is a large component of an acute infarct, unenhanced ct shows a hyperdense hematoma surrounded by hypodense edema. these changes are often confined to a vascular territory, a feature that is helpful in differentiating hemorrhagic infarctions from other causes of hemorrhage. ° in the subacute phase, the region around the infarct may enhance, increasing the possibility of hemorrhage within a tumor. mass effect from a hemorrhagic infarct is usually minimal or decreasing at the time of maximal contrast enhancement ( to weeks), which is useful in differentiating between hemorrhagic infarct and tumor. ~ the mri features of hemorrhagic infarction are similar to those of lntraparenchymal hemorrhage. ° compared with other causes of lntraparenchymal hemorrhage, hemorrhagic infarcts tend to have a higher p because of earlier revascularization and collateral perfusion. the higher p decreases the amount of deoxyhemoglobin, and therefore minimizes the acute t relaxation effect. uncontrolled seizures or status epileptmus may result in neuronal degeneration progressing to necrosis. the distribution of lesions varies somewhat among individual patients, but the hippocampus, basal nuclei, and frontal and pyriform lobes of the cerebrum are most severely affected. f° lesions are usually bilateral, but may affect one side more severely. f° these changes are thought to be the consequence of ischemia secondary to accumulation of cytotoxic agents and a mismatch between brain metabolism and blood flow during prolonged seizures. persistent or reversible mr lesions have been described in human patients after seizures, and similar abnormalities have there is also a smaller wedge-shaped region of hyperintensity in the vascular territory of the right middle cerebral artery. se, / , . t. been described in dogs, [ ] [ ] [ ] the most consistent finding is unilateral or bilateral lesions of the hippocampus, pyriform lobe, and frontal lobe. the lesions are hypointense on t weighted images and hyperintense on proton densityweighted and t -weighted images. there is minimal mass effect and no or moderate contrast enhancement tm (fig ) . these lesions may persist or disappear on subsequent scans. head trauma results from a variety of causes, including motor vehicle accidents, bites, kicks, and gunshot wounds. previously, imaging of head trauma was limited to skull radiography for the detection of fractures. plain radiography, however, cannot identify many traumatic brain lesions, such as hemorrhage, and therefore provides only limited diagnostic information. the development of ct provided a sensitive means for detecting and localizing intracranial hemorrhage, permitting expeditious surgical treatment, and improving outcome. mri has been shown to be similar in sensitivity for detection of hemorrhagic lesions, but is much more sensitive than ct for detecting nonhemorrhagic lesions, such as shearing injuries of white matter. neuroimaging should be considered early in the management of animals with head injury and marked impairment of consciousness or neurological deficits that progressively worsen despite initial medmal therapy. the most critical issue is to detect potential hematomas that may be treatable with sur-gery. in general, ct is the diagnostic study of choice for initial evaluation, because it can be completed quickly and is sensitive to acute hemorrhage, r ct also provides fine anatomic detail of bone when viewed at a wide window width allowing accurate characterization of any skull fractures (fig ) . although mri is slightly more sensitive than ct for detection of hematomas, those that are not seen on ct are usually small and typically managed conservatively. a disadvantage of mri is the longer examination ume and the difficulty in monitoring unstable patients in the mri environment. therefore, if ct is available, mr examination is usually delayed until the patient is stabilized. in patients with severe head trauma, mri should be considered in the first weeks after injury, because most parenchymal lesions are more easily detectable during this period. epidural hematomas accumulate in the potential space between the inner surface of the skull and the dura mater. they are usually found in the temporoparleteal region, and have been caused by laceration or tearing of the middle meningeal artery by skull fracture. the arterial force of the bleeding dissects the dura away from the bone, often resulting in a rapidly expanding mass. clinical signs may consist of rapidly progressing focal neurological deficits and deterioration in consciousness. however, patients with associated parenchymal brain injury may have severely impaired consciousness at the time of trauma. on ct, an acute epidural hematoma is typically a well-defined, biconvex lesion between the inner table of the skull and the underlying depressed dura and brain. tm the attenuation of the hematoma is initially greater than brain parenchyma and increases further during the first few hours with coagulation and clot retraction. with time, the attenuation decreases with clot retraction, erythrocyte lysis, and hemoglobin degradation. the hematoma becomes smaller and fades to isodense and then hypodense within or weeks. the inner surface of an epidural hematoma is dura mater, which normally enhances with intravenous contrast material, but this enhancing margin becomes even more prominent as a neovascular membrane develops over time. on mri, the intensity varies depending on the age of the hematoma, as described in the section on intracranlal hemorrhage and summarized in table . occasionally, an epidural hematoma may not have the classic lenticular shape or associated skull fracture, making it difficult to differentiate it from a subdural hematoma. mri can be helpful in this regard, because the medially &splaced dura mater is usually directly visualized as a thin line of low signal separating the hematoma from the underlying compressed brain parenchyma. mri is also superior in the detection of subacute and chronic hematomas, which may be isodense on ct subdural hematomas accumulate within the potential space between the pia-arachnmd and dura mater. they are usually caused by tearing of veins that traverse the subdural space. subdural hematomas appear to be less common m dogs and cats with head injury, compared with human patients. clinical signs may consist of progressive asymmetrical neurological deficits and decreased levels of consciousness. an acute subdural hematoma appears on ct as a hyperdense, crescentshaped collection conforming to the inner surface of the skull. tm mass effect is evident and may be compounded by contusion and edema of the underlying brain parenchyma. acute hyperdense hematomas may not be vislble on a narrow window width (soft tissue window), appearing only as an apparent thickening of the skull, since the bone and hematoma may have the same pixel brightness, r mass effect is usually present, however, providing a clue in their detectlon. widening the window width may be necessary to distinguish an acute subdural hematoma from the dense skull, rs as with other intracranial hematomas, the density of a subdural hematoma decreases over time, becoming isodense to gray matter by to weeks. accordingly, subacute subdural hematomas may be difficult to detect on ct but are readily detectable on mri. a chronic subdural hematoma ( to weeks old or older) is hypodense compared with normal brain on ct and is surrounded by a well-defined capsule (fig ) . this results in a more focal collection of blood with a straighter medial edge compared with crescent-shaped acute subdural hematomas. the capsule of a chronic subdural hematoma enhances with intravenous contrast material and may calcify. tm subarachnoid hemorrhage is bleeding into the csf-filled subarachnoid space. posttraumatic subarachnoid hemorrhage is relatively common and often associated with cortical contusions. acute subarachnoid hemorrhage is evident on ct as increased attenuation of the sulcl, fissures, or basal cisterns, with the degree of increased attenuation being related to the amount of blood in the subarachnoid space. with time the attenuation decreases and subarachnoid hemorrhage may not be detectable after the first week, unless rebleeding has occurred. acute subarachnoid hemorrhage is usually not detectable on mri, probably because the po of the subarachnoid csf is too high for the conversion of oxyhemoglobin to deoxyhemoglobin and methemoglobin. however, mri is excellent at detecting subarachnoid hemorrhage in the subacute or chronic stage. i brain contusions are common after head trauma and consist of heterogeneous regions of hemorrhage, edema, and necrosis, often located in the superficial gray matter. in human patients contusions tend to be multiple and bilateral and are much less likely to be associated with severe initial impairment of consciousness compared with diffuse axonal injur~ initial ct findings are often limited to faint, ill-defined hypodense areas mixed with tiny regions of hyperdense hemorrhage. contusions in which edema and necrosis predominate may not be visible imtially on ct but often become apparent several days later as regions of decreased attenuation and mass effect caused by edema. s mri, because of its greater sensitivity in detecting edema, is better at detecting early contusions, which appear hypointense on tl-weighted images and hyperintense on t -weighted images. head trauma that involves rapid angular acceleration may result in diffuse axonal injury. these shearing injuries result from differences in elastic and inertial properties between different but adjacent brain tissues/s, in human patients, these injuries are characterized pathologically by disruption of axons, especially at the junction of gray and white matter of the cerebrum, and at the corpus callosum, basal nuclei, and cranial aspect of the brainstem. there is subsequent axonal swelling and infiltration with macrophages. these patients present with severe impairment of consciousness starting from the moment of injury. " ,s mri is much more sensitive than ct in detecting diffuse axonal injury in human patients, although even mri findings usually underestimate the true extent of these injuries. ct is often normal, but may show scattered hemorrhages. the mri appearance reflects the prolonged t and t values of increased tissue fluid (edema). there are multiple, small elliptical lesions in the white matter. these lesions are hypomtense on tl-weighted images and hyperintense on t -welghted images. , infectious and inflammatory diseases inflammatory diseases are important diagnostic considerations for patients with brain disease. infectious agents, such as viruses, protozoa, and fungi, cause many inflammatory diseases, but for others the etiology is unknown. despite the numerous causes of mflammatory brain disease, the affected tissue can respond only in a limited number of ways. thus, many of these diseases appear similar on imaging studies and differentiating the potential etiologies based on imaging features alone may be impossible. furthermore, it may be difficult to &scriminate between inflammatory diseases and other categories of disease, such as neoplasia and vascular disorders. accordingly, results of imaging studies must be interpreted in context with clinical features and results of other laboratory tests, especially analysis of cse i all inflammatory brain diseases share a common pathological feature--an influx of leukocytes into the brain (cerebritis or encephalitis) or meninges (meningitis). because of the close anatomical association of these structures, more than one area of the nervous system can be involved in the inflammatory process. for example, inflammation of the meninges and brain is called meningoencephalitis, s the most common cause of meningitis in dogs is steroidresponsive meningitis-arteritis, a nonseptic suppurative meningitis of unknown etiology that responds to immunosuppressive dosages of corticosteroids, s infectious causes of memngitis are less common in small animals and include bacteria, viruses, fungi, and protozoa s pathologically. acute leptomeningitls results in congestion and hyperemia of the pia-arachnoid and distension of the subarachnoid space by an exudate containing leukocytes. clinically, affected patients show fever, spinal pain, cervmal rigidity, and stiff gait. several complications can occur m the ensuing days to weeks. there may be extension of the infection to the neural parenchyma, resulting in focal or diffuse encephalitis, myelitis, or abscess. inflammatory exudate may obstruct csf pathways, producing hydrocephalus. endogenous host inflammatory mediators can result in disruption of the blood-brain barrier, cerebral edema, and increased intracranial pressure. definitive &agnosis of meningitis is based on analysis of cse neuroimaging is useful in detectmg some of the complications associated with meningitis and when the differential diagnosis includes other diseases. neuroimaging features of experimental bacterial menmgitls in dogs is comparable with naturally occurring bacterial meningitis in human patients. r in human patients with uncomplicated early bacterial or viral meningitis, unenhanced ct and mri are often unremarkable or show mild dilatation of the ventricles or subarachnoid space (fig ) . with more severe involvement, there may be diffuse or patchy brain edema. postcontrast ct or mri may show abnormal enhancement of the leptomeninges. in experimental studies in dogs, tl-weighted mr images with gadolinium showed abnormal leptomeningeal enhancement better than ct. r mri also identifies complications such as encephahtis more effectively than ct. encephalitis generally refers to nonpurulent inflammation of the brain and is distinguished pathologically from suppurauve inflammation of the brain (cerebritis) assooated with bacterial infection. viral encephalitides of small animals include canine distemper and feline infectious peritonitis. other causes of encephalitis include rocky mountain fever, canine ehrlichiosis, toxoplasmosis, and neosporosls. finally, there are encephahtides of unknown etiology, such as pug dog encephalitis and granulomatous meningoencephalitis. distemper encephalitis. the two most common clinical forms of distemper encephalitis are acute encephalitis in young dogs and chronic encephalitis in mature dogs. immature dogs with distemper encephalitis typmafly suffer a rapid onset of systemic illness characterized by conjunctivitis, nasal discharge, cough, vomiting, and diarrhea. neurological dysfunction can occur during or after the systemic illness and includes seizures, abnormal behavior, blindness, and paresis. mature dogs are more likely to develop chromc, multifocal encephalitis with a predilection for the white matter of the brainstem. many of these dogs have an adequate vaccination history, and signs of systemm illness are often absent or transient. °- these patients often have slowly progressive gait deficits or vestibular dysfunction. °, ct of dogs with chromc distemper encephalitis may be normal or show focal or multifocal hypoattenuating lesions with a predilection for the white matter. these lesions may have uniform or ring-like enhancement (fig ) . some lesions may be associated wlth hypoattenuating edema and mass effect. lesions are typically hypointense or poorly defined on tl-weighted mri, hyperintense on t -weighted images, and enhance with contrast agent. feline infectious peritonitis. feline infectious peritonitis (fip) is a systemic disease of cats caused by an immune response to a corona virus. neurological signs are generally associated with the parenchymatous (dry) form of fie neurological deficits referable to brainstem involvement predominate and include ataxia, paresis, and vestibular dysfunction. [ ] [ ] [ ] imaging features of fip reflect the pathological changes, which consist of pyogranulomatous inflammation of the leptomeninges, choroid plexus, ependyma, and brain parenchyma. hydrocephalus is common and is probably secondary to obstruction by ependymltis. - the brainstem and fourth ventricle are consistently involved, but other regions of the central nervous system can be affected. the inflammatory process primarily affects the inner and outer surfaces of the brain with only secondary extension into the parenchyma. recognition of this surface-related pattern can be helpful in differentiating fip from other bram diseases in the cat. ct may be normal or show hydrocephalus. mri may show ependymitis, choroiditis, and memngitis. this is evident as hyperintensity of the ventricular lining, choroid plexus, and meninges, respectively, on t -weighted mri and abnormal enhancement with gadolinium-based contrast agent , (fig ) . fungal infections. many fungal agents can sporadically infect the nervous system, causing meningitis or granulomas. cryptococcus neoformans is the most common fungal infectmn to involve the nervous system of dogs and cats. in cats, this organism generally induces a mild, nonsuppurative meningitis or encephalitis, whereas affected dogs typically develop a granulomatous reaction in the brain and meninges. neurological deficits can be acute or chronic and include seizures, lethargy, ataxia, and vestibular dysfunction. °° on ct, mass lesions (cryptococcomas) appear as single or multiple isodense or hypodense masses with ring or solid enhancement and surrounding edema. , , i° leptomeningeal enhancement may also be apparent if the meninges are involved. ,i° hydrocephalus may occur secondary to meningitis or obstruction of csf pathways by the mass. , in human patients, mri is more sensitive than ct and may show clustered foci of signal abnormahties that are lsointense to csf on all sequences. these lesions represent small granulomas or dilated virchow-robin spaces filled with fungal organisms and mucoid. these are often bilaterally symmetrical, and are located in the basal nuclei and midbraln. these lesions do not enhance with gadolinium and are not associated with mass effect or edema. similar changes have been reported on mri of canine cryptococcosis.i° cryptococcomas appear as masses that are hypointense on tl-weighted images, hyperintense on t -welghted images, and enhance with gadolinium. < ° other fungal organisms sporadically infect the central nervous system, including blastomyces dermatldis, histoplasmosis capsulatum, aspergfllus spp, coccidiodes immitis, and phaeohyphomycosis (fig ) . l° qn necrotizing encephalitis (pug dog encephalitis). a necrotizing form of encephalitis has been recognized in pug dogs, maltese terriers, and yorkshire terriers between months and years of age. n -n signs include progressive seizures, abnormal behavior, blindness, ataxia, and walking in circles. pathological changes consist of multifocal necrosis and nonsuppuratlve inflammation, with a striking predilection for the white matter of the cerebrum. lesions are often bilateral but asymmetrical, n - ,n enlargement of the lateral ventricles secondary to shrinkage and cavitation of the cerebral hemispheres (hydrocephalus ex vacuo) is common n ,i n (fig ) . in yorkshire terriers, the brainstem may be preferentially revolved. the etiology is unknown. in acute forms of the disease, ct may show one or more focal hypodense lesions, most commonly affecting the cerebral hemisphere. the lesions may or may not enhance with contrast agent. mri shows the early edematous changes as increased signal intensity on proton density-weighted and t -weighted images and decreased signal intensity on tl-weighted images. in acute cases, there is often substantial mass effect and minimal if any abnormal enhancement with contrast medium (fig ) . differentials include neoplasia, other inflammatory lesions, and acute infarction. in more chronic cases, necrosis and cystic changes usually predominate. the centers of the lesions appears similar to csf; that is, hypodense on ct, very hypointense on tl-weighted images, and very hyperintense on proton density-weighted and t -weighted images - (fig ) . lesions are usually located in the white matter of the cerebral hemisphere, often in the area lateral to the ventricles. ii typically there is no mass effect or even a reverse mass effect (shift of surrounding tissue toward the lesion). lesions usually do not enhance, but may have a ring pattern of enhancement. , asymmetric enlargement of the lateral ventricles is common. the primary differential is chronic infarction. however, in necrotizing encephalitis the lesions are not confined to a specific vascular territory as is typical of infarction caused by thromboembolism. the onset of signs (sudden in infarction versus slow onset in chronic necrotizing encephalitis) ts also helpful. granulomatous meningoencephalomyelitis. granulomatous meningoencephalomyelitis (gme) is an inflammatory disease of the canine central nervous system characterized pathologically by an accumulation of mononuclear cells in the parenchyma and meninges of the brain and spinal cord. lesions may be disseminated or focal. in the disseminated form, lesions are distributed throughout the central nervous system, with a predilection for the white matter of the cerebrum, cerebellum, caudal aspect of the brainstem, and cervical spinal segments. the focal form is manifested as a single granulomatous mass, most commonly located in the cerebrum, with smaller disseminated lesions, ns-ln the cause is not known. adult dogs of any breed can be affected, although females and toy and terrier breeds are at increased risk. dogs with disseminated gme usually have rapidly progressive signs including neck pain, vestibular dysfunction, paralysis, and seizures. the focal form is manifested as chronic, gradually progressive signs, with seizures being the most common. iis- the clinical presentation of focal gme often mimics that of a tumor. on ct, disseminated gme is seen as multiple foci of ill-defined contrast enhancement involving the parenchyma and meninges. some lesions may be associated with hypoattenuating edema and mass effect. other inflammatory diseases are the primary differentials. focal gme appears on noncontrast ct as an isodense or hyperdense mass, most commonly located within the cerebrum or at the cerebellomedullary junction (fig ) . ' on mri, focal gme is usually isointense or slightly hypointense on tl-weighted images and hyperintense on proton density-weighted and t -weighted images (fig ) . enhancement is variable, including no enhancement, ring-pattern enhancement, or moderate homogenous enhancement. ,t ,i there may be edema in the white matter surrounding the mass. , , asymmetric enlargement of the lateral ventricles has also been reported. the primary differentials are neoplasia and other inflammatory lesions. biopsy is often necessary for definitive diagnosis. focal infection of the brain with pyogenic organisms is uncommon in small animals. bacteria may gain access to the brain through penetrating wounds; secondary to direct extension from infections in the eye, ear, nasal passages, or meninges; or via hematogenous spread from extracranial sources, i with hematogenous spread, lesions often arise at the graywhite matter junction of the cerebrum, s clinical signs reflect a progressively worsening focal brain lesion. i several reports detail the pathological and ct features of experimental cerebritis/abscess in dogs. , ° these imaging features are similar to those reported in human patients with spontaneous brain abscess. tm pathologically, serial changes occur over to weeks, starting as cerebritis and culminating in abscess. focal but poorly localized areas of scattered necrosis, edema, vascular congestion, and perivascular inflam- matory infiltrates characterize cerebritis. at this stage, unenhanced ct shows only an irregular, poorly circumscribed region of low attenuation, t - scans obtained immediately after contrast administration show ring-like enhancement. on delayed scans, contrast diffuses into the center of the lesion, starting peripherally, until the center of the lesion may be completely filled with contrast by minutes after admmistra-tlonj - t the inherent sensitivity of proton density-weighted and t -welghted mri to aheradons in tissue water enables earlier detection of cerebritis compared with ct. , these images show increased signal intensity indistinguishable from or slightly hypointense to surrounding edema, sg,j on t weighted images, cerebritis is isointense to slightly hypomtense to adjacent brain parenchyma, with associated mass effect. , over a period of to weeks, untreated cerebritis may progress to abscess formation when the central zone of necrosis becomes liquefied, better defined, and encircled by a collagen capsule, which is formed by fibroblast migration from the surrounding vessels. because of relatively poor vascularization of white matter, the medial aspect of the capsule may be somewhat thinner. this predisposes to expansion of the abscess into white matter, the formation of daughter abscesses medially, or rupture into the lateral ventricles, lz , °, the capsule is visible on unenhanced ct as an isodense rim that is visible because it is bordered medially by a hypodense liquid center and surrounded by hypodense edema (fig ) . the edema may be greater in volume than the abscess itself, causing much of the mass effect. on contrast-enhanced ct, the rim is usually smooth and brightly enhancing. there is no diffusion of contrast into the necrotic center on delayed scans as there is with cerebritis, n - on tl-weighted mri there is mild peripheral hypointensity representing edema and a more markedly hypointense liquid center. the capsule is a discrete rim that is isointense to slightly hyperintense. on t -weighted images the abscess center is isointense to mildly hyperintense to gray matter. the capsule is seen as a dramatic hypointense rim, possibly caused by paramagnetic free radicals within phagocytic macrophages. the ring pattern of enhancement parallels the enhancement seen on contrast-enhanced ct. the differential diagnosis for a ring-enhancing lesion includes primary brain tumor, metastasis, infarction, granuloma, and resolving hematoma. . helpful clues that may identify abscesses include the time course, location, temporal pattern of enhancement, and predisposition for the abscess rim to be thinner medially, s cuterebra. dogs and more commonly cats can suffer brain disease caused by aberrant migration of cuterebra larvae. [ ] [ ] [ ] fly larvae attach to the host and burrow into the subcutaneous tissue. the developing larva ( to mm long) may migrate under the skin and enter the brain, most likely through the nasal passages and cribiform plate. , pathological changes in the brain consist of multifocal meningoencephalitis with malacia and hemorrhage • - affected animals typically have access to outdoors and develop signs from june to october, coinciding with the larval migration portion of the cuterebra life cycle. a recent history of upper respiratory disease is common, likely reflecting migration of the larvae through nasal passages. there is an acute onset of neurological dysfunction, most commonly referable to a focal forebrain lesion. seizures, abnormal mentation, circhng, hemlparesis, and unilateral blindness are common. , clinical and pathological features in cats are similar to what has been previously reported as feline ischemic encephalopathy.i a mottled appearance to the brain has been reported on ct of an affected cat} based on my observations, ct may show focal or muhifocal regions of decreased attenuation with minimal mass effect. the may be one or more tract-like regions of contrast enhancement (fig ) . lesions may be hypointense on tl-weighted mri and hypermtense on t -weighted mri. edema is fairly minimal, and there may be small regions of hemorrhage. contrast enhancement is s~milar to that described for ct. an enhancing tract (arrows) extends from the cribiform plate through the right cerebral hemisphere. this cat, which lived outside, presented in october, days after suffering a sudden onset of sneezing followed by seizures, left hemiparesis, depression, and circling to the right. clinical signs gradually resolved over the next weeks, so the diagnosis was not confirmed by necropsy, but the clinical features are consistent with intracranial migration of a cuterebra larva. and does not enhance. i the intraparenchymal location is helpful in differentiating these lesions from arachnoid cysts, which are associated with the subarachnoid space. ,~- ct and mri are helpful in identifying and characterizing many nonneoplastic brain disorders in dogs and cats. the imaging features of some of these disorders are unique, permitting definitive diagnosis based on imaging results and clinical features. however, the imaging findings for other brain disorders are nonspecific. in these instances, ct and mri often allow detection and localization of abnormalities, but definitive diagnosis may require other laboratory tests or surgical biopsy. as the use of ct and mr] becomes more widespread in veterinary medicine, further research in the imaging findings associated with various nonneoplastic disorders will improve our ability to diagnose and manage these conditions. imaging the brain (first of two parts) canine hydrocephalus. comp contln ed pract vet computed tomography in evaluation of hydrocephalus diagnosis and management of an atypical case of hydrocephalus, using 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domestic cat magnetic resonance imaging cerebrovascular disease key: cord- -qp rrwr authors: martin, r.; martens, u.; sticht-groh, v.; dörries, r.; krüger, h. title: persistent intrathecal secretion of oligoclonal, borrelia burgdorferi-specific igg in chronic meningoradiculomyelitis date: journal: j neurol doi: . /bf sha: doc_id: cord_uid: qp rrwr in the cerebrospinal fluid igg of five patients with lymphomeningoradiculitis (bannwarth's syndrome) and radiculomyelitis studied by immunoblot technique an oligoclonal pattern was found. most of these oligoclonal bands were specific for borrelia burgdorferi. in patients suffering from chronic meningoradiculomyelitis, repeated csf examination by this technique showed persistent secretion of identical igg bands. thus, the specific humoral immune response and the disease activity could be documented over the course of the disease. lyme disease is a tick-borne spirochetal infection which, particularly in europe, often involves the central nervous system (cns). in most cases, the disease starts with a characteristic skin rash, erythema chronicum migrans (ecm), and symptoms of general illness such as fever, headache and arthralgia (stage ) [ , ] . weeks or months later, specific organs such as heart, joints and cns are involved (stage ) [ , ] . without appropriate treatment by antibiotics the disease may then progress into a subacute or chronic stage in which there may be acrodermatitis chronica atrophicans, chronic oligoarthritis or progresssive encephalomyelitis (stage ) [ , , ] . neurological manifestations of lyme disease include lymphomeningoradiculitis, radiculomyelitis or even progressive encephalomyelitis [ , , , , ] . characteristic cerebrospinal fluid (csf) findings are lymphocyte and monocyte pleocytosis, increased total protein and immunoglobulins. investigations of csf immunoglobulins during different stages consistently show the restricted heterogeneity of csf igg by the presence of oligoclonal bands [ , , ] . the diagnosis is confirmed by high titres of serum and csf antibodies, specific for borrelia burgdorferi, which has recently been identified as the aetiological agent of lyme disease and bannwarth's syndrome [ ] . the purpose of our study was to answer the questions whether the csf immunoglobulin g (igg) in lymphomeningoradiculitis is locally produced, whether its antigen specificity can be determined, and whether the persistence of a specific distribution pattern can be recorded over the course of the disease. the recently described immunoblot technique [ ] , which combines isoelectric focusing (ief) of unconcentrated or diluted csf with blotting to an antigen-loaded nitrocellulose filter, was used. the five patients studied were treated in our clinic in . their ages ranged between and years. three of the patients remembered a tick bite and/or ecm. the clinical diagnosis of meningoradiculitis or radiculomyelitis was confirmed by antibodies against b. burgdorferi [immunofluorescence test (ift): significant positive serum igg titre > : ; significant positive csf igg titre > : ; elisa: significant positive csf igg titre > : ] in the serum and the csf, elevated total protein and igg of csf and a lymphocytic csf pleocytosis. all five patients suffered from either meningitis with accompanying radiculoneuritis or from meningomyelitis (table ) and were treated with × units/day penicillin g for days either once or repeatedly, according to the persistence of clinical symptoms. according to the course of the disease and the persistence of pathological csf findings, we only used the term chronic meningoradiculitis or radiculomyelitis when both clinical and laboratory findings were present for longer than months without any improvement. relevant laboratory findings of the sera and csf, which were always collected on the same day, are shown in table . patient c had already been treated by corticosteroids and antibiotics when csf and serum were collected. the antibody titre was therefore comparatively low. in this patient, proliferative testing of peripheral blood lymphocytes with b. borgdorferi antigen further confirmed the diagnosis. all csf and serum samples were drawn with informed consent of the patients. as a positive control, monoclonal antibody h specific for b. burgdorferi (kind gift of dr.a.barbour, university of texas, san antonio, usa) was used. sera and csf samples of patients suffering from either meningeosis carcinomatosa or multiple sclerosis (both seronegative for b. burgdorferispecific antibodies) were taken (data not shown). determination of csf and serum protein concentrations. all protein and igg concentrations were determined by lasernephelometry (behring laser nephelometer, b ehring-werke, marburg, frg) and expressed in milligrams per decilitre (rag/ dl). antibodies to b. burgdorferi were tested by ift and elisa as described previously [ ] . the igg coefficient was calculated according to the method of delpech and lichtblau [ ] . b. burgdorferi antigen preparation. lyme disease spirochetes (b. burgdorferi strain m ; kind gift of dr. r.ackermann, department of neurology, university of cologne, frg) were prepared as described by pachner et al. [ ] . briefly, the , prepared as described above. the filters were incubated at room temperature overnight on a rocker platform and then rinsed in pbs for min. unoccupied protein binding sites were blocked by incubation in % bovine serum albumin (bsa; serva, munich, frg) in pbs (ph . ) for lh. after washing in pbs, . % np (sigma, taufkirchen. frg) in pbs and pbs ( min each cycle), the filters were ready for blotting. ief of csf and serum was carried out on the following agarose gel: % agarose (ief grade, pharmacia, freiburg, frg) containing % sorbitol (sigma, taufkirchen, frg), lml pharmalyte, ph - , . ml pharmalyte, ph - . (pharmacia, freiburg, frg). the dimensions of the gels were x x . mm (length, width, height). csf samples were adjusted to ~tg igg/ml by dilution with saline ( . %) and . gl aliquots were applied to the gel with application strips (serva, munich, frg; . x mm). ief was performed at w constant power in an lkb ultrophor electrofocusing unit (lkb, bromma, sweden) at °c for h. blotting of immunoglobulins to nitrocellulose filters was accomplished by afffinity-driven transfer. for this purpose, the moist filter was laid on top of the gel to avoid air bubbles. then it was covered with a pbs-moistened filter paper (lkb), three sheets of dry filter paper, a glass plate and x g weight. after lh, three washing cycles (as decribed above) followed. the filters were now incubated in ml pbs containing . mg/ml peroxidase-labelled rabbit anti-human igg (dakopatts, hamburg, frg) for h. after three washing cycles the filters were stained in a solution of mg -amino- -ethylcarbazole (sigma, taufkirchen, frg), , ml dimethylformamide (sigma), . ml mm/ sodium acetate solution (ph . ) and ~ hydrogen peroxide %. after staining for min a final washing in distilled water was performed. the five patients with typical clinical and laboratory findings of meningoradiculitis and radiculomyelitis showed raised total csf protein and elevated csf immunoglobulins with igg ranging up to mg/dl. as shown by ift (table ) , antibodies specific for b. burgdorferi were present both in the sera and the csf. although igg coefficients according to delpech and lichtblau were elevated in all cases, b. burgdorferi specific igg, determined by ift, was always lower in the csf ( table ). for the determination of a restricted pattern of intrathecally produced igg, immunoblotting of diluted csf and serum was performed using filters precoated with rabbit anti-human igg. thus, the oligoclonal nature of csf igg could be demonstrated. in fig. the igg distribution patterns within the sera and the csf are shown in parallel. the csf of each patient demonstrates an individual distribution of igg bands which cannot be compared with one another. in the sera, however, only homogeneous staining or single faint bands could be seen. it should be noted that sera and csf samples were adjusted to the same igg concentration of ~tg/ml. in a further step, we tried to identify the antigen specificity of the oligoclonal igg bands found. for this purpose, b. burgdorferi coated nitrocellulose filters were used for the immunoblotting procedure. figure the stable distribution pattern demonstrates the persistent secretion of specific antibodies by single b-cell clones. for the corresponding immunoblot for total igg, see fig. . pl, isoelectric point in addition, we studied the distribution pattern of intrathecally produced igg in one patient (e), who continuously suffered from radicular pain and paraparesis, over a period of months. laboratory values of five csf samples collected at approximately -month intervals are summarized in table . the igg concentration continuously dropped with time, whereas the ift titre for b. burgdorferi-specific antibodies remained the same. figure shows the configuration of oligoclonal total igg bands in all five csf samples of this patient. the corresponding immunoblot of b. burgdorferi-specific csf igg is demonstrated in fig. . both figures reveal that, during the -month period, no overt differences in the banding pattern could be detected, although the patient had three courses of treatment with penicillin g ( × units/day over days) in the meantime. in the present study, we used a rapid and sensitive immunoblotting technique [ ] to detect and characterize intrathecally produced igg in five patients suffering from chronic meningoradiculitis (bannwarth's syndrome) or radiculomyelitis. in earlier reports it was shown that the determination of b. burgdorferi-specific antibodies of either igg or igm type is a useful diagnostic criterion for lyme disease and bannwarth's syndrome [ ] . the patients studied in this report suffered from meningoradiculitis or radiculomyelitis in either a subacute or chronic stage. three patients (a, c, e) are still suffering from persistent clinical symptoms such as radicular pain, brisk tendon reflexes and positive babinski sign; for this reason the term "chronic" should only be used for these patients. after treatment by antibiotics patients b and d markedly improved within month and we would therefore prefer to consider these disease courses as subacute. all of them exhibited oligoclonal igg shown by both ief of unconcentrated csf and blotting to nitrocellulose filters coupled with rabbit antihuman igg. this observation is also well documented in other viral or bacterial cns infections such as tuberculous meningitis, neurosyphilis, subacute sclerosing panencephalitis and mumps meningitis [ , , , ] . in a further step the antigen specificity of csf igg for the aetiological agent b. burgdorferi was shown by using the same blotting technique, but coupling b. burgdorferi antigen to the nitrocellulose filters. thus, we found oligoclonal, b. burgdorferi-specific igg bands in the csf samples of all patients. the presence in three patients of minor bands in the sera which had been adjusted to the same igg content can be taken as an indicator of the intrathecal production of specific antibodies. this is confirmed by the high igg indices which had been calculated according to the method of delpech and lichtblau [ ] . the presence of weak bands seen in the serum track at the same position as the corrsponding csf bands may either be due to parallel formation of the antibodies in the csf and the peripheral blood or to passive diffusion of csf igg to the peripheral blood via the blood-csf barrier. apart from the demonstration of the antigen specificity of oligoclonal igg bands, this highly sensitive method can also be used to follow the persistent secretion of specific igg in single b cells. this issue was pursued by repeated immunoblotting of csf samples which had been collected over a longer period of time. owing to the restriction of space, the blots are only shown for patient e over a period of months. it is clearly demonstrated that the distribution pattern of both total igg and b. burgdorferi-specific igg remained exactly the same (figs. , ; tracks - ). this is an in-dication that a restricted number of b cells is continuously forming specific antibody. although unlikely, it cannot completely be excluded, however, that one single band contains two or more antibodies of different specificities but of the same electrophoretic mobility. as ift titres were comparatively low, it may be postulated that the persistence of b. burgdorferi-specific oligoclonal bands is a better indicator of disease activity than ift titres alone. in our patients, this notion is further supported by the co-existence of clinical symptoms. the presence of oligoclonal igg bands in the csf and not in the serum of patients suffering from meningoradiculitis or radiculomyelitis strongly favours the intrathecal production of these antibodies and was firstly demonstrated by kriiger et al. [ ] . murray et al. [ ] also investigated the question of antigen specificity of oligoclonal csf igg by staining ief bands with i-labelled b. burgdorferi antigen. in addition, they immunoprecipitated i-labelled b. burgdorferi antigen by either serum or csf and used this mixture for sds gel electrophoresis. applying such techniques, they detected b. burgdorferi-specific oligoclonal bands only in the csf of one out of nine patients. because few clinical data are given in their report, we cannot explain whether this difference resulted from a difference in disease severity or stage or whether their technique was less sensitive. henriksson et al. [ ] investigated igg-, igm-and iga-producing b-cells in the serum and csf, ig indices and also oligoclonal total igg bands over the course of lymphocytic meningoradiculitis. apart from a high proportion of igg-, igm-and iga-secreting b-cells and a prolonged igm response, they found oligoclonal igg bands in the csf of all patients. comparing our results with those reported in the literature, it seems that our method is more sensitive; it is rapidly performed and easily detects oligoclonal csf igg as well as characterizing its antigen specificity in patients suffering from subacute and chronic meningoradiculitis and radiculomyelitis. obvious advantages are the possibility of using unconcentrated or diluted csf and the rapid application with no need for radioactive substances. the technique can easily be used for the detection of intrathecally produced, b. burgdorferispecific igg because ief of csf is a standard procedure for the diagnosis of multiple sclerosis in most neurology clinics. the disadvantages of the immunoblot technique are the large amount of antigen necessary and the difficulty of identifying simultaneously the specificity of csf igg for subfractions of the b. burgdorferi antigen. for this purpose, western blot analysis, as described by wilske et al. [ ] , should be used. it should also be mentioned that the direct comparison of the patterns of oligoclonal igg bands with those specific for b. burgdorferi antigen may be difficult because the transfer conditions for total igg and b. burgdorferi specific igg are different, as already noted by d rries and ter meulen [ ] . in all patients, we were able to demonstrate prominent, oligoclonal igg bands in the csf but not in the serum. most of these bands were specific for b. burgdorferi, with only a few weak bands seen in the sera. the persistent secretion of specific igg is paralleled by the presence of clinical symptoms such as radicular pain, mono-and paraparesis over long periods of time. this is not a unique finding in meningoradiculitis, but can also be demonstrated after recovery from acute herpes virus encephalitis, mumps virus and varicella zoster meningoencephalitis [ ] [ ] [ ] and in relapsing subacute encephalomyelitis induced by corona virus in rats [ ] . in all these cases of persistent antibody secretion within the csf, the question arises whether live bacteria or viruses or only antigen fragments persist within the cns or whether the immune response against the invading pathogen cannot be suppressed in certain individuals. to elucidate further the cause of persistent csf antibody secretion, it will be necessary to isolate b. burgdorferi antigen from the csf and to characterize the cellular immune response within the cns, in patients suffering from chronic menigoradiculitis and radiculomyelitis, in more detail. progressive borrelien-enzephalomyelitis lyme disease, a tick-borne spirochetosis? lyme disease antibody response in lyme disease: evaluation of diagnostic tests l~tude quantitative des immunoglubulines get de l'albumine du liquide cephalorachidien detection and identification of virus-specific, oligoclonal igg in unconcentrated cerebrospinal fluid by immoblot technique analysis of the intrathecal humoral immune response in brown norway (bn) rats, infected with the murine corona-virus jhm immunoglobulin abnormalities in cerebrospinal fluid and blood over the course of lymphocytic meningoradiculitis (bannwarth's syndrome) characterization of antibody activity in oligoclonal immunoglobulin g synthesized within the central nervous system in a patient with tuberculous meningitis demonstration of oligoclonal immunglobulin g in guillain-barr syndrome and lymphocytic meningoradiculitis by isoelectric focusing viral antibodies in oligoclonal and polyclonal igg synthesized within the central nervous system over the course of mumps meningitis comparison of an indirect fluorescent-antibody test with an enzyme-linked immuno-sorbent assay for serological studies of lyme disease lyme disease specificity of csf antibodies against components of borrelia burgdorferi in patients with meningopolyneuritis garin-bujadoux-bannwarth demonstration of elec-trophoreticauy restricted virus-specific antibodies in serum and cerebrospinal fluid by imprint electroimmunofixation antigenspecific proliferation of csf lymphocytes in lyme disease neurologic abnormalities of lyme disease demyelinating encephalopathy in lyme disease mumps meningitis: specific and non-specific antibody responses in the central nervous system long-term persistence of intrathecal virus-specific antibody responses after herpes simplex encephalitis intrathecal synthesis of virus-specific oligoclonal igg, iga and igm antibodies in a case of varicella zoster meningoencephalitis neurosyphilis: intrathecal synthesis of antibodies to treponema pallidum chronic progressive neurological involvement in borrelia burgdorferi infection intrathecal production of specific antibodies against borrelia burgdorferi in patients with lymphocytic meningoradiculitis (bannwarth's syndrome) acknowledgements. the authors thank d. drenkard and n. gropp for excellent technical assistance. the study was supported by grant sti / - from the deutsche forschungsgemeinschaft. key: cord- -vdtpz u authors: dörries, r.; liebert, u.g.; ter meulen, v. title: comparative analysis of virus-specific antibodies and immunoglobulins in serum and cerebrospinal fluid of subacute measles virus-induced encephalomyelitis (same) in rats and subacute sclerosing panencephalitis (sspe) date: - - journal: j neuroimmunol doi: . / - ( ) - sha: doc_id: cord_uid: vdtpz u the intrathecal humoral immune response was analysed in patients with subacute sclerosing panencephalitis (sspe) and lewis rats with subacute measles virus (mv)-induced encephalomyelitis (same). sspe patients as well as same rats revealed oligoclonal, intrathecal antibody synthesis with mv specificity. same rats synthesized mv-specific antibodies intracerebrally to a higher extent than sspe patients. although a restricted isoelectric pattern of mv-specific antibodies was detected in the cerebrospinal fluid (csf) of sspe patients as well as of same rats, the heterogeneity within clusters of immunoglobulin bands was higher in the rat specimens. increase in the blood-brain barrier permeability for albumin was exclusively detected in same rats but not in sspe patients. these data suggest that the rat model offers excellent opportunities to study the initial humoral events in mv-induced encephalitides. measles virus (mv) is the agent responsible for the induction of subacute sclerosing panencephalitis (sspe), a chronic central nervous system (cns) disease process associated with a persistent mv infection in the cns. the infection is virologically characterized by restricted envelope gene expression, the absence of infectious virus particle formation and a pronounced intrathecal antiviral immune response, which fails to overcome the infection (ter meulen et al., ) . so far, the pathogenic mechanism which leads to mv persistence is unknown, but it has been suggested that viral antibodies play an important role in the establishment and maintenance of this infection (fujinami and oldstone, ) . in tissue culturc. extracellular mv antibodies can cause an antigenic modulation, which results in persistence and interferes with viral replication (barrett et al., ) . similar mechanisms may be of significance in sspe, but these cannot be evaluated in this disease since the early state of cns infection is clinically not detectable. therefore. these aspects can only be studied in an experimental model. recently, we have established a model in which a persistent mv infection in the brain of weanling lewis rats induces a subacute measles encephalomyelitis (same) after an incubation period of weeks to months (liebert and ter . like in sspe, infectious measles virus cannot be reisolated from diseased brain areas by conventional methods and the virus cell interaction reveals a restriction of mv gene expression. in infected brain cells only measles virus nucleocapsid (n) and phosphoprotein (p) can regularly be detected, whereas the levels of hemagglutinin (h), fusion (f) and membrane (m) proteins are either drastically reduced or not detectable. since the state of mv replication is similar to the one described in sspe, it was the aim of this study to characterize the intrathecal immune response in same diseased rats in comparison to sspe. the data obtained indicate that same in lewis rats represents an excellent model to evaluate the role of the intracerebral immune response in subacute cns diseases induced by mv. serum and csf samples from five sspe patients were obtained from the diagnostic laboratory of our institute. the permeability of the blood-brain barrier (bbb) was assessed by determination of csf/serum ratios for immunoglobulin g (igg) and albumin. to evaluate local antibody synthesis in the csf, measles virus-specific antibodies were titrated by an enzyme immunoassay (eia) and the clonal distribution of total as well as of mv-specific immunoglobulins was analyzed by affinity-mediated immunoblot (ami). a panel of lewis rats suffering from different stages of same after cerebral inoculation with measles virus strain cam/rbh into the left hemisphere ( /~ . × tissue culture (tc) ids ) were selected for this study. animals were sacrificed at different intervals after infection and serum and csf were taken to determine csf/serum ratios of immunoglobulin (ig), albumin and mv-specific antibody titers. isoelectric distribution of ig and mv-specific antibodies was visualized by affinity-mediated immunoblot. for histopathological evaluation brain and spinal cord were fixed in buffered paraformaldehyde, embedded in paraffin and stained with hematoxylin-eosin (he) and luxol fast blue for myelin. adjacent regions were snap frozen in liquid nitrogen and the distribution of mv proteins was assessed by indirect immunofluorescence on acetone-fixed cryostat sections (liebert et al., ) . for infection of animals mcasles virus cam/rbh was used as previously described (liebert and ter meulen, ) . viral antigen for use in eia and ami was prepared from vcro cells infected with the edmonston strain of measles virus. briefly, vero cells showing strong cytopathic effect (cpe) days post-inoculum (d.p.i.) were frozen at - °c, thawed and scdimented at × g for rain. the supernatant (s ) was kept for purification of virus particles. the sediment was homogenized in nte ( . m nac , . m tris, . m edta) by ten strokes in a glass homogenizer and centrifuged at × g for rain. the supernatant ($ ) was pooled with supcrnatant s and loaded on a % sucrosc cushion in nte buffer, ph . . after centrifugation ( min, × g, °c), the pellet was resuspended in nte, homogenized with ten strokes and clarified by low-speed centrifugation ( min, × g, °c). the supernatant was centrifuged through % sucrose onto a % sucrose cushion ( min, × g, °c) and the visible, virus containing band on top of the cushion was collected by a syringe. to remove the sucrose, the virus band was diluted in nte and viral antigens were pelleted for rain at × g. pellets were resuspended in phosphate-buffered saline (pbs), homogenized with strokes and kept at - °c until used as antigen in eia and ami. albumin concentrations were determined by rocket immunoelectrophoresis (laureli, ) . immunoglobulin concentrations in serum and csf from sspe patients were determined by radial immunodiffusion (mancini et al., ) and from rats by an eia as described previously (dorries et al., ) . the state of the blood-brain barrier was evaluated according to reiber ( ) . as a reference for the animal model csf/serum ratios for albumin (a) and immunoglobulin (i) were determined in healthy lewis rats and the normal range was defined by adding times the standard deviation to the mean values for a and i. for evaluation of the sspe cases the normal range determined by reiber ( ) was applied. a micro-enzyme immunoassay was used for determination of mv-specific titers in serum and csf specimens from sspe patients and diseased rats, mv antigen and vero cell control antigen ( /~ , /tg protein/ml) were coated onto the surface of round-bottom microtiter plates (immunoplates li, nunc, wiesbaden, f.r.g.) by overnight incubation at room temperature in coating buffer ( . m sodium carbonate buffer, ph . ). unbound antigens were removed by washing the wells times in /~ distilled water containing . % tween . serial dilutions of serum and csf from patients or rats in dilution buffer ( . m tris-hc , . m naci, % normal rabbit serum, % tween , ph . ) were incubated in /~ aliquots/well for min at °c. after a washing cycle as described mv-specific antibodies bound to the solid-phase antigen were detected by incubation with ~ per well of peroxidase-labeled rabbit anti-human immunoglobulin g (igg) or rabbit anti-rat immunoglobulin fraction (ig) antibodies (dako parts, hamburg, f.r.g.) for min at ° c. after a final washing cycle peroxidase-labeled secondary antibodies were visualized by incubation at /~ of . % o-phenylenediamine (opd) and . % hydrogen peroxide in . m citric acid-phosphate buffer, ph . per well for min at room temperature. after stopping the enzyme reaction by addition of btl of m sulfuric acid, the absorbance (abs) in each well was determined in a micro-colorimeter (bio-rad, munich, f.r.g.) at nm. the mv-specific titer was determined by the last dilution revealing a control antigen-corrected absorbance equal or higher than . abs. for determination of ab indices the procedure of ukkonen et al. ( ) was followed. csf/serum ratios of the reciprocal mv-specific antibody titers were divided by csf/serum ratios of total igg, intrathecal mv-specific antibody synthesis was assumed if the ab index was equal or higher than . . a modified procedure of the previously published ami (dorries et al., ) was applied to detect the distribution of total as well as mv-specific antibodies in serum and csf. prior to isoelectric focusing (ief) in an . mm agarose gel ( % ief agarose, . % pharmalyte ph . - . , % sorbitol, % np- ), serum specimens were diluted to contain the same amount of ig as the corresponding csf sample. focusing of /tl specimens, usually containing ng of ig, took place for volthours (vh) with a starting voltage of v. the established ph gradient was monitored with a marker protein solution (pharmacia, freiburg, f.r.g.). after focusing any visible moisture on the gel was removed by blotting with a cellulose acetate foil for a few seconds. subsequently the gel-fractionated antibodies were transferred to nitrocellulose strips coated either with measles virus antigen or the igg fraction of rabbit anti-rat ig or anti-human igg immunoglobulins. these strips were coated overnight on a rocker platform at room temperature, usually with a protein concentration of ~g/ml and ml/ cmz of nitrocellulose area in the case of the anti-immunoglobulin or with /,tg mv proteins/ml and ml/ cm of filter area in pbs. after coating the strips were blocked by % tween for h at room temperature, washed briefly in pbs and used for blotting. after transfer of the antibodies to nitrocellulose by an incubation of h at room temperature, the strips were removed from the gel, washed in pbs, pbs-tween ( . %) and pbs ( min each) and incubated in peroxidase-labeled rabbit anti-rat ig or anti-human lgg antibodies ( ml/ cm of filter area) diluted i : in pbs-tween ( . %) containing % of normal rabbit serum. to visualize transferred antibody clones, filters were washed as described and developed in -chloro-naphthol. the animals exhibited various degrees of clinical disease characterized by a weight loss of - %, unsteadiness, abnormal posturing and in some cases a mild paresis of one or more limbs. none of the animals reached a moribund stage. animals , , and survived a mild clinically apparent subacute disease by - days. in animals , , and no overt clinical signs were observed (table ) . histopathologically, all animals revealed a subacute measles encephalomyelitis (same) of varying degree characterized by perivascular lymphomonocytic infiltrations in the gray and white matter. demyelination was not observed. viral antigen was demonstrated using a human serum containing high titers of mv-specific antibodies specific for all viral structural proteins. animals , , and showed the presence of brightly fluorescing cells in the cortex and the basal ganglia, probably large neurons and probably some glia cells. in animals , and only few weakly staining cells were detected while in the remainder of animals no measles virus-specific antigen could be seen in the sections studied. however, the failure to localize mv antigen in some animals does not exclude the presence of mv antigen, since not the entire brain was available for this analysis. serum and csf specimens of lewis rats or sspe patients were tested for the presence of virus-specific antibodies by micro-enzyme immunoassay. as can be seen in table , out of rats developed mv-specific antibodies in the serum and in out of animals mv-specific antibodies were detectable in the csf. there was no correlation between the presence or titer of antibodies and the severity of the disease. all five of the sspe patients had mv-specific antibody titers in serum as well as in the csf and these were much higher than those of the rats (table ). in sspe it is known that most of the csf-derived mv-specific igg is produced intrathecaily (vandvik et al., ) . to enable direct comparison of the same diseased animals with the sspe cases, csf/serum ratios of mv-specific antibody titers (abmv) and immunoglobulin (i) were used to calculate mv-specific antibody indices (ab index) in both groups. as can be seen in figs. and , all animals with mv-specific antibody titers in the csf and all sspe patients synthesized these antibodies to a significant extent intrathecally. it is remarkable that in the animal model the ab indices are much higher than in the sspe cases, pointing to a stronger intrathecal mv-specific antibody synthesis in the animals compared to the patients. in order to correlate intrathecal synthesis of mv-specific antibodies and the state of the blood-brain barrier, a graphical analysis according to reiber ( ) was performed. disturbances of the bbb permeability for albumin could be observed frequently in the rat group (fig. ) . increased permeability was detectable with normal lg ratio (rats no. and ) or proportional increase of the ig ratio (rats no. and ), as well as in relation to disproportional increase of the lg ratio (rats no. and ). intrathecal synthesis of ig and increased permeability for albumin was found in five rats (nos. , , , and ) . together, almost % of the animals ( / ) revealed a dysregulated bbb. rats no. , , and synthesized lg intrathecally with a normal bbb permeability, whereas rats no. , , , and were within the normal range of csf/serum ratios for albumin and immunoglobulin. interestingly, two of these animals (nos. and ) produced mv-specific antibodies in the csf, a fact which did not result in an increase of the csf/serum ratio for lg. no animals were observed with an intrathecal lg synthesis in the absence of mv-specific antibody production in the csf. as expected, all of the sspe patients did show intrathecal increases in total igg and intact diffusion control by the bbb (fig. ) . one of the most prominent immunological markers of sspe is the marked oligoclonal distribution of mv-specific antibodies in the csf. to analyze this aspect an affinity-mediated immunoblot (d~srries et al., ) was applied. two representative patterns are shown in figs. and . distinct differences were noticeable between oligoclonal antibody patterns of serum and csf specimens obtained from same lewis rats (fig. ) and sspe patients (fig. ) . mv-specific antibody clones focus more cathodically and individual bands are more prominent in the sspe cases than in the same rat samples. however, clusters of antibody bands were observed in both diseases. moreover, fainter staining of antibody bands in the paired serum specimen as compared to the csf, verified intrathecal synthesis of mv-specific antibodies in same rats and sspe patients, also demonstrated above by calculation of ab indices (figs. and fig. . csf protein profile of five sspe patients. a=csf/serum ratios for albuminxl ; l= csf/serum ratio for immunoglobulin x ; area = disproportional increase of the bbb permeability for albumin; area ii = proportional increase of the albumin and immunoglobulin permeability; area iii = increased permeability for albumin either in conjunction with intrathecal ig synthesis or disproportional increase of the lg diffusion: area iv = increased permeability for albumin and intrathecal lg synthesis; area v = normal permeability for albumin and intrathecal lg production; • = intrathecal mv-specific antibody synthesis. ). the focused ig patterns detected in clinically severely diseased rats were not distinguishable from those with a mild disease or from clinically healthy, but histopathologicaily severely diseased animals. in this study, paired serum and csf samples were used to compare measles virus-induced subacute encephalomyelitis of lewis rats (same) and sspe. the state of the bbb was analyzed and mv-specific antibodies were characterized with respect to titers, site of synthesis and isoelectric distribution. mv-specific antibody titers in patients suffering from sspe were extraordinary high in serum as well as csf, a finding which is well known (ter meulen et al., ) . a similar phenomenon occurred in mv-infected rats. almost all same rats developed significant mv-specific titers in serum and csf, but not to the same high levels as observed in sspe samples. however, with respect to mv-specific ab indices, rats revealed much higher ratios than sspe patients, indicating a very strong intrathecal synthesis of mv-specific antibodies in the animals. this result suggests that same in rats represents probably the early events of a subacute mv-induced encephalitis, whereas sspe reflects the late consequences of a subacute encephalitis, characterized by the presence of a few cns-residing, antibody-secreting b cell clones, highly selected over the years by the persistent presence of a restricted pattern of mv-specific proteins in brain cells. correlation of these data to the graphic analysis of the state of the bbb revealed that in same rats as well as in sspe patients no increase of the csf/serum ratio for ig could be detected in the absence of an increased ratio for mv-specific antibody titers. for same rats this observation holds true whether the animals had an intact or disturbed bbb permeability. this observation seems to rule out the possibility that beside mv-specific antibodies, non-virus-specific antibodies have been produced in significant amounts in the cns. this is in contrast to coronavirus jhm-induced encephalomyelitis in lewis rats. in this model, jhm virus causes extensive primary demyelination in the cns which is sometimes accompanied by an intrathecal synthesis of non-virus-specific immunoglobulins of restricted heterogeneity in the absence of jhm-specific antibody clones (drrries et al., ) . whether these non-viral antibodies are directed against brain antigens as described in canine distemper virus-induced demyelinating encephalitis of dogs (krakowa et al., ; vandevelde et al., a, b) or theiler's virus encephalomyelitis in mice (rauch et al., ) is presently unknown. an intrathecal mv-specific antibody response of oligoclonal nature has also been reported in another animal model of mv-induced subacute encephalitis in ferrets (thormar et al., (thormar et al., , mehta and thormar, ) . however, the development of this subacute encephalitis could only be detected in animals which were pre-immunized with an mv vaccine. the necessity for pre-immunization in order to obtain a persistent mv infection of brain tissue is surprising, since sspe has never been observed in children after mv vaccination (ter meulen et al., ) . on the contrary, mv immunization seems to prevent this chronic human cns disease (halsey et al., ) . another interesting aspect is the observation that the intrathecal production of mv-specific antibodies in same rats is not always connected with an increase of the csf/serum ratio for total ig, a finding shared with mumps meningitis in man (link et al., ; ukkonen et al., ) and coronavirus-induced encephalomyelitis in lewis (di rries et al., ) and bn rats (d~srries et al., ) . obviously the amount of virus-specific antibody produced is not sufficient to result in an increase of the csf/serum ratio of total ig. therefore, an intact bbb and a normal csf/serum ratio for total ig does not exclude the possibility of intrathecal synthesis of virus-specific antibodies in persistent cns infections. analyzing the isoelectric distribution of ig and mv-specific antibodies in serum and csf samples, sspe-derived specimens revealed the typical oligoclonal banding of lgg in the alkaline region of the ph gradient. according to the classification of tourtellotte ( ) these bands would be assigned to area iii (ph . - . ) of the ph gradient. this is the area where oligoclonal igg bands from most inflammatory disorders of the cns in man are detected. to date, there is no satisfying explanation for this clustering of oligoclonal bands in the alkaline area of the ph gradient. as discussed by walsh et al. ( ) , the passage of proteins through the blood-brain barrier in man might be facilitated largely by a strong negative net charge. this could favor the entry of those b cell clones into the cns, which carry surface immunoglobulins with alkaline isoelectric points. most, if not all, of the oligoclonal bands could be characterized in sspe as mv-specific and parallel focusing of the same amount of serum and csf-derived igg verified that mv-specific ig clones were synthesized for the major part in the csf. although same rats showed similar results, subtle differences were detectable concerning the isoelectric distribution of mv-specific bands. as in sspe, clusters of focused bands were detectable, but the heterogeneity within these clusters seems to be higher in csf of same rats. this again supports the interpretation that same in rats is reflecting the early stages of a subacute encephalitis, where infiltrating b cell clones are not yet selected as in sspe. moreover, csf clones in rats were detected predominantly in area it (ph . - . ) of the gradient. since the same observation has been made in coronavirus-induced subacute encephalomyelitis in lewis and bn rats (d/ rries et al., , ) , and ig clones from serum usually do not focus at higher ph values, it seems that rat immunoglobulins are confined to a narrower ph band than human antibodies. in conclusion, same in lewis rats appears a promising model for the study of the intrathecal, humoral immune response during the course of an mv-induced subacute encephalomyelitis. in view of similarities between same and sspe with respect to the virus-specific cns-resident antibody response and the restriction of expression of mv-specific genes in the infected brain tissue, this animal model will provide the opportunity to obtain insight into the potential role of the mv-specific antibody response in the cns as a modulating factor in chronic persistent mv infection of the brain cells. effect of measles virus antibodies on a measles sspe virus persistently infected c rat glioma cell line detection and identification of virus-specific oligoclonal igg in unconcentrated cerebrospinal fluid by immunoblot technique murine coronavirus-induced encephalomyelitides in rats: analysis of immunoglobulins and virus-specific antibodies in serum and cerebrospinal fluid analysis of the intrathecal humoral immune response in brown norway (bn) rats, infected with the murine coronavirus j|tm antib~xly initiates virus persistence: immune modulation and measles infection subacute sclerosing panenccphalitis (sspe): an epidemiologic review myelin-specific autoantibodies associated with central nervous system demyelination in canine distemper virus infection quantitative estimation of proteins by electrophoresis in agarose gel containing antibodies virological aspects of measles virus-induced encephalomyelitis in l,ewis and bn rats restricted expression of measles virus proteins in brains from cases of subacute sclerosing panencephalitis viral antibodies in oligoclonal and polyclonal igg synthesized within the central nervous system over the course of mumps meningitis a single-radial diffusion method for the immunological quantitation of proteins measles antibcu..ties and oligoclonal lgg bands in subacutc sclerosing panencephalitis and subacute ferret encephalitis subacute sclerosing panencephalitis the discrimination between different blood-csf barrier dysfunction and inflammatory reactions of the cns by a recent evaluation graph for the protein profile of cerebrospinal fluid presence of oligoclonal immunoglobulin g bands and lack of matrix protein antibodies in cerebrospinal fluids and sera of ferrets with measles virus encephalitis measles virus encephalitis in ferrets as a model for subacute sclerosing panencephalitis do all clinical definite multiple sclerosis patients have evidence of intra-blood-brain-barrier lgg synthesis local production of mumps lgg and igm antibcxlies in the cerebrospinal fluid of meningitis patients immunological and pathological findings in demyelinating encephalitis associated with canine distemper virus infection demyelination in experimental canine distemper virus infection: immunological, pathologic and immunohistological studies oligoclonal measles virus-specific igg antibodies isolated from cerebrospinal fluids, brain extracts, and sera from patients with subacute sclerosing panencephalitis and multiple sclerosis lsoelectric point restriction of cerebrospinal fluid and serum igg antibodies to measles virus polypeptides in multiple sclerosis we thank andrea deutscher, susanne kotrla and marion sch/sller for expert technical assistance. dr. bert rima is gratefully acknowledged for critical reading of the manuscript. this work was supported by the deutsche forschungsgemeinschaft. key: cord- - v gor v authors: levine, gwendolyn j.; cook, jennifer r. title: cerebrospinal fluid and central nervous system cytology date: - - journal: cowell and tyler's diagnostic cytology and hematology of the dog and cat doi: . /b - - - - . - sha: doc_id: cord_uid: v gor v nan analysis of csf is an important adjunctive diagnostic tool in the workup of patients with cns disease and must be interpreted within the context of the patient's history, clinical signs, clinicopathological data, imaging studies, and other ancillary diagnostics. rarely is csf solely used to provide an etiological diagnosis (exceptions include cytological visualization of infectious agents or overtly neoplastic cells), but analysis may significantly narrow the field of pathophysiological differentials, guiding further diagnostic and therapeutic options. csf analysis is most sensitive in detecting inflammatory disease. positive findings in csf tend to be more diagnostically helpful compared with negative findings but are often nonspecific because many different diseases may cause a common csf pathology (e.g., neutrophilic pleocytosis). occasionally, the magnitude of change within the csf may be as instructive as the character of the change (e.g., a marked increase in protein concentration raising diagnostic concern for feline infectious peritonitis, marked neutrophilic pleocytosis raising diagnostic concern for steroid-responsive meningitis-arteritis in a young dog in pain). more frequently, however, specific disease etiologies will present with csf changes of variable character and magnitude. csf that falls within laboratory reference intervals should never be used to rule out a differential diagnosis because negative findings may represent early or mild disease, disease suppressed or masked by therapeutic intervention, or a disease process that does not present within the particular area of the extracellular space being sampled. csf analysis may or may not correlate with imaging studies; a retrospective study of cats receiving magnetic resonance imaging (mri) for spinal signs showed that abnormal csf was not a predictor for abnormal mri. in another study, approximately % of dogs with intracranial signs and inflammatory csf had normal brain mri results. the conventionally accepted theory of csf secretion and transport is based on the concept of active transport of ions within the ventricular ependymal cells and choroid plexi, subsequent passive flow of fluid, and circulation and drainage of csf into dural venous sinuses. these ideas have recently come under scrutiny as potentially simplistic and inconsistent with the past years of experimental evidence. analysis of past experiments, coupled with new data, supports a "global production" hypothesis-that instead of exclusive formation within the ventricles, csf is continually created and reabsorbed diffusely by cerebral capillaries that have slight variances in hydrostatic and osmotic pressure. canine studies have documented csf production within the ventricular system and the sas. a study of dogs with focal, noninflammatory disease showed that in cases of spinal lesions, csf was more likely to be abnormal if collected from the lumbar cistern, that is, caudal to the lesion. this observation may be explained by presupposing cranial to caudal flow of csf, but the traditionally held theory of csf flow has recently been contested. in canines, csf collected from the cerebromedullary cistern generally has lower microprotein concentrations compared with samples collected from the lumbar cistern. blood contamination may be more pronounced in lumbar collection, as the desired subarachnoid space is more difficult to enter and yields a smaller volume of fluid that tends to flow more slowly. , moreover, hemodilution may contribute to increased measured protein concentration. rare instances of csf contamination with hematopoietic precursors have only been reported from lumbar sites. a low, but potentially catastrophic, risk for puncturing the cervical spinal cord or caudal brainstem exists during cerebromedullary collection. because the spinal cord length is variable, spinal cord puncture is a possibility during lumbar collection, but it is associated with less severe adverse effects compared with injury following cisternal puncture. in a case series of four accidental cisternal parenchymal punctures (documented by using mri), three of the four patients suffered neurological decompensation and subsequently had to be euthanized. the following equipment should be assembled: anesthesia and monitoring equipment, clippers, aseptic preparation materials for the skin, sterile gloves, and a spinal needle with stylet. for cerebromedullary cistern collection in dogs weighing less than kg and for cats, a -gauge, . -inch spinal needle is usually adequate, and a -gauge, . -inch spinal needle is recommended for dogs weighing greater than kg. for lumbar puncture, a -gauge spinal needle up to inches long may be required for obese or extremely large patients. if available, fluoroscopic equipment may aid in the acquisition of cisternal or lumbar csf. at the authors' institution, fluoroscopy is often used before cisternal csf acquisition in toy-breed dogs to exclude the possibility of subclinical atlantoaxial subluxation. the anesthetized patient is placed in lateral recumbency (it is generally easier for a right-handed clinician to have the patient in right-lateral recumbency, and vice versa), with the neck and back flush to the edge of a sturdy table. for collection from the cerebromedullary cistern, the neck is flexed such that the dorsum of the muzzle is degrees to the long axis of the body (if needed, stabilizing the endotracheal tube to prevent kinking and deflating the cuff to prevent tracheal trauma), and the snout is propped up slightly, if necessary, to keep it parallel with the table and not angulated from the sagittal plane. a wide area ( - cm) around the atlanto-occipital joint (beyond atlas wings and axis spinous process and to the external occipital protuberance) is shaved and aseptically prepared, and landmarks are palpated with a gloved, nondominant hand. the needle is inserted at the intersection of two imaginary perpendicular lines that run ( ) along the dorsal midline (dividing the patient sagittally) from the occipital protuberance to the cranial spinous process of the axis (c ) and ( ) across the craniolateral aspects of the wings of the atlas (c ) (dividing the patient craniocaudally). for lumbar collection, the pelvic limbs are brought forward into full flexion, and the needle is inserted cranial and parallel to the dorsal spinous process of l for dogs and l for cats, advancing the needle until the ventral aspect of the vertebral canal is encountered; the needle is then retracted slightly and csf is collected from the ventral sas. , the pelvic limbs may be kicked or may twitch slightly during collection because of irritation of the cauda equina or spinal cord parenchyma. for either location, once landmarks are palpated, the needle is held stably with the dominant hand and very slowly advanced, stylet in place. the heel of the dominant hand may be supported against the table. for cisternal collection, it is important to advance the needle toward the point of the nose without angulation. the stylet is removed with the nondominant hand every to mm to check for fluid within the needle hub, waiting a few seconds. it is common to feel a decrease in resistance to forward needle movement once the thecal space is entered. if bone is hit or frank hemorrhage is observed from the needle, it should be withdrawn slowly and collection reattempted. if clear or slightly blood-tinged fluid is observed, advancement of the needle is stopped, and open tubes are placed directly under the needle hub to collect freely falling drops. csf is collected passively and should not be aspirated. there are no significant objective data regarding the maximal amount of csf that may be collected in dogs. several authors claim that it is safe to collect . milliliters (ml) of csf per kilogram of body weight ( ml/ kg); in other species much higher volumes of csf per body weight are acquired standardly. in general, . to ml of csf is adequate for routine diagnostic tests, including cell counts, protein concentration, and cytological analysis. larger volumes are necessary for additional diagnostics (cultures, titers, polymerase chain reaction [pcr], flow cytometry, protein electrophoresis, etc.). two sets of tubes should be readied and ideally handled by an assistant. an ethylenediaminetetraacetic acid (edta)-treated (purple-top) tube is used for cell counts, flow cytometry, and pcr testing for organisms, and plain (red-top) tubes are used for protein concentration, culture, or immunologic assays. some sources indicate that plain tubes are recommended, as edta could increase protein concentration. if csf analysis will occur rapidly (within hour), collection into a plain tube is adequate, whereas preservation of cells may be improved with collection into edta if analysis will be delayed. if low volume is present, priority is given to the edta tube. if csf appears red, then iatrogenic hemorrhage (puncture of a dural vessel) or actual cns hemorrhage has occurred. in this instance, the first few drops are allowed to collect into the first set of tubes, and the second set of tubes are reserved for the latter portion of the sample, as iatrogenic hemorrhage tends to clear over time. if the hemorrhage does clear, a decision may be made about discarding the first set of tubes or keeping them for ancillary testing not affected by the hemorrhage. after collection, the needle is withdrawn without the stylet, and the csf within the needle is allowed to drip into one of the tubes or is placed in an additional plain tube and saved for culture. as with other clinicopathological and cytological samples, evaluation of a fresh specimen is preferred to minimize cellular degradation, to which csf is particularly vulnerable because of its relatively low protein concentration. sample degradation will affect cell differential count to a greater extent than the total nucleated cell count or the protein concentration. a study of canine csf samples with pleocytosis concluded that delay of analysis up to hours was unlikely to alter interpretation, especially in samples with protein concentrations above milligrams per deciliter (mg/dl). preservative should be added to low protein samples unless analysis is to be completed within minutes (see next section), and a dilutional effect must then be factored into cell counts. samples to be shipped to a reference laboratory overnight should be kept at refrigeration temperature and shipped with ice packs for analysis within hours. , the reference laboratory should be prenotified to ensure prompt analysis. if analysis is likely to be delayed by more than hour and the csf sample has a protein concentration less than mg/dl, one of the following may be added as a protein source to maintain cellular integrity: ( ) hetastarch (add : volume), ( ) fetal calf serum ( . g/dl protein; add % by volume), or ( ) autologous plasma or serum (fresh or frozen; % by volume ≡ one drop from -gauge needle (approximately . ml) mixed into . ml csf). , , the sample should be labeled with the protein source and amount added to the sample. one study demonstrated better preservation of mononuclear cells in canine samples when fetal calf serum was used instead of hetastarch. all samples should be refrigerated at °c to minimize cellular degradation. a hemocytometer may be employed in practice to count nucleated cells and erythrocytes. both sides of the cover-slipped hemocytometer are loaded with unstained csf, which is then placed in a humidified container for to minutes to allow cells to settle on the glass. because the fluid is unstained, the microscope condenser is lowered to improve contrast. erythrocytes and nucleated cells are differentiated by size, refraction, granularity, and smoothness of plasma membrane. some laboratories stain csf samples with new methylene blue (nmb), as leukocytes will take up stain, whereas erythrocytes remain unstained, making differentiation of leukocytes (specifically small lymphocytes) and erythrocytes easier (fig. . ) . a small volume of csf is drawn into a capillary tube coated with nmb or a tube that has a small volume of nmb followed by an air pocket. the tube containing nmb and csf is gently rocked back and forth, allowing the cells to take up some stain without diluting the csf with a volume of nmb. the hemocytometer is then loaded, and each population is counted and totals are calculated, as follows: neubauer chamber: ( ) both areas of large nine squares are counted, and the average of the number of leukocytes and erythrocytes is found; ( ) the average is multiplied by to get the cells per microliter (cells/μl). the advia (siemens medical solution, fernwald, germany) hematology instrument has been validated for analyzing canine csf samples and shows excellent correlation with manual methods used in dogs with increased total cell counts (pleocytosis), but the instrument may overestimate the cell count in samples without pleocytoses and has not been validated for the identification of eosinophils. the automated differential count is also more accurate at higher cell numbers and thus should be compared with a traditional manual differential. the advia hematology analyzer displayed satisfactory agreement with the standard hemocytometer method. validation experiments using canine samples showed a sensitivity of % and specificity of % for accurately identifying samples with pleocytosis when manual counting was considered the gold standard (> cells/μl). the instrument tended to be less accurate at lower (within reference interval) nucleated cell counts. erythrocytes may be a source of interference, as a red blood cell (rbc) count of cells/μl was shown to elevate the nucleated cell count. with regard to differential cell count, the instrument performed better in the presence of pleocytosis, whereas monocytes were overcounted at lower nucleated cell counts. automated cell counts thus should not replace a manual differential but may be used as another level of quality control. automated instruments cannot recognize altered cell types, such as atypical neoplastic cells. measurement of csf specific gravity is not considered to be helpful because of low sensitivity for detecting abnormalities. csf microprotein may be semiquantitatively measured by using urine dipsticks that detect albumin. this assay has a lower detection limit of mg/ dl; therefore, it has low sensitivity for mild to moderate csf protein concentration elevations ( mg/dl to mg/dl). false-positive or false-negative reactions may occur if the dipstick reads at trace or +, but this method is useful if other techniques are not available. reference laboratories apply a similar but more sensitive methodology to measurement of csf microprotein as that of serum protein, using the trichloroacetic acid method, the ponceau s red dye-binding method, or the coomassie brilliant blue method. csf globulin production is typically screened for with the pandy reaction. in this test, a few drops of csf are added to ml of % carbolic acid solution, and the resulting turbidity is graded to +. any pandy score above zero is considered elevated. globulin concentration below mg/dl will be undetectable with either test. , protein electrophoresis and immunoelectrophoresis may be performed on csf and serum for maximum fractionation. the utility of protein electrophoresis or immunoelectrophoresis of csf lies in discriminating altered blood-brain barrier (bbb) permeability from increased localized production of immunoglobulin, which may be suggestive of (but not specific for) a disease entity for which an electrophoretic pattern has been established. cytological analysis is a critical component of csf evaluation because the differential count (percentages) of cells may be abnormal, even if the total nucleated count is within reference interval. cytology also enables examination for neoplastic cells, infectious agents, and evidence of prior hemorrhage. it may also serve as a quality control point, allowing for correlation between observed cellularity and the total count generated by a hemocytometer or an automated analyzer. because of its low cellularity, csf must be concentrated before cytological smear preparation. use of an in-house sedimentation chamber (sörnäs procedure) may be very useful and preserves cell-free fluid for ancillary testing. this technique will recover approximately % of total cells, which is sufficient for analysis. a syringe barrel (with the tip and needle aseptically removed with a scalpel blade) is turned upside down and the smooth, top side is placed in warm petroleum jelly and then onto a clean slide. once a seal has formed, fresh csf (at least . ml) is placed in the syringe and allowed to sit for minutes. , then, the supernatant is aspirated carefully with a pipette so as not to disturb the bottom layer contacting the slide. the syringe barrel is removed, and any excess csf is carefully absorbed with a small piece of filter paper or paper towel. the slide is completely and rapidly air-dried without heat (inadequate drying results in cellular distortion), excess petroleum jelly removed with a scalpel blade, and the slide is stained with routine romanowsky stains (e.g., diff-quik). if csf is sent to a reference laboratory, a cytological slide will likely be prepared using cytocentrifugation ( - revolutions per minute [rpm] for - minutes, either onto a slide coated with albumin or with the addition of . ml of % albumin for improved cell capture) for maximal concentration of nucleated cells onto one slide. cytocentrifuged cytology may show excellent cellular detail, but the preparation may enlarge cells slightly and create an artifactual foamy or vacuolated appearance. slides are air-dried and stained with conventional romanowsky stains. multiple cytospin preparations may be made to yield intact nucleated cells for classification. as it is rare for etiologic agents to localize only within the cns, all cases of suspected infection may be aided diagnostically by fine-needle aspiration (fna) cytology, biopsy with histopathology, culture of nonneural lesions, or all of these. bacterial culture and sensitivity testing of csf is recommended for most cases of neutrophilic pleocytosis, given the appropriate clinical index of suspicion for a septic lesion. even when organisms are visualized on csf cytology, speciation and susceptibility testing may help guide prognostic and treatment decisions. alternatively, bacterial or fungal culture may be negative regardless of cytological observation of organisms. , it must be remembered that bacterial cns infection is highly uncommon in dogs and cats compared with other domestic animal species. advanced techniques for neurological disease diagnosis are expanding rapidly. enzyme-linked immunosorbent assay (elisa)-based assays for antibody detection and pcr-based assays for nucleic acid detection of several medically important microbes have been developed for use on csf and may be instructive in the diagnosis of viral, rickettsial, protozoal, or fungal diseases. a large canine study that included a subset of dogs with neoplastic or inflammatory disease showed that csf titer provided diagnosis in % of cases. antibody assays should be interpreted cautiously because the presence of antibody may indicate prior exposure or vaccination rather than active infection. moreover, compromise to the bbb in states of inflammation may translate to the presence of antibodies within the csf without local production. occasionally cross-reactive antibodies may be present that do not represent presence of the disease agent under assessment. similarly, specimens for pcr should be submitted to a laboratory with strict quality control to minimize false-negative and false-positive results. poor collection technique may result in false-positive results, especially for bacterial species that are ubiquitous in the environment. as with other aspects of csf analysis, a negative pcr result does not definitively rule out the presence of a pathogen because of the sampling limitation of a small portion of the extracellular space. csf contains glucose, electrolytes, neurotransmitters, and enzymes, but these substances are not measured routinely, although this measurement represents a rapidly expanding area of research in the effort to give clinicians better tools for diagnosing patients and determining prognoses. csf enzymes originate from the bloodstream, the cns, or cells within csf. one study of cats with noninflammatory cns disease showed that measurement of csf activities of lactate dehydrogenase (ldh), aspartate aminotransferase (ast), and creatine kinase (ck) were not diagnostically sensitive but may be useful in detection of acute injury. multiple studies have correlated elevations in csf ck activity with poor prognosis in dogs with neurological disease or spinal cord injury. , immunoassays for vascular endothelial growth factor (vegf) and s- calcium-binding protein have shown elevations of both molecules in the csf of experimentally induced hypothyroid dogs, suggesting endothelial and glial contribution to increased bbb permeability in this population. myelin basic protein (mbp) has been found to be elevated in lumbar csf in dogs with degenerative myelopathy, supporting the conclusion that it is a demyelinating lesion. mbp concentration is elevated in the csf of dogs affected by intervertebral disk herniation (ivdh) and has been found to be an independent predictor of poor prognosis. beta- -microglobulin, a major histocompatibility complex i (mhc-i)-associated molecule, has been assayed by using elisa and found to be elevated in the csf of dogs with ivdh and inflammatory disease and also positively correlated with normal total nucleated cell count (tncc). the amino acids tryptophan and glutamine have been found to be elevated in the csf of dogs with portosystemic shunts because of abnormal ammonia metabolism. one study found increased oxytocin in the csf of dogs with spinal cord compression, where it is believed to have an analgesic effect. gamma-aminobutyric acid (gaba) and glutamate neurotransmitter concentrations have been measured in dogs with epilepsy. normal csf is clear and colorless, with few cellular elements and a protein concentration approximately to times less than that of plasma or serum. red or yellowish coloration indicates prior lesional hemorrhage or iatrogenic hemorrhage during collection. in the latter case, a pellet of rbcs will be present after centrifugation. true xanthochromia (yellowish color of hemoglobin breakdown products) that does not clear on centrifugation, cytological evidence of erythrophagia, or both indicate prior hemorrhage into the subarachnoid space. increased bilirubin leakage into the sas or high concentrations of csf protein (> - mg/dl) may cause xanthrochromia. increased turbidity of the sample may be caused by increased number of cells present (> rbcs/μl or > nucleated cells/μl) but is usually not affected by mild changes. , cell counts tncc is fewer than cells/μl in the dog and fewer than cells/μl in the cat, and elevation above this range is termed pleocytosis. grading of pleocytosis is somewhat subjective: in one reference, "mild" was defined as to cells/μl; "moderate" as to cells/μl; and "marked" as more than cells/μl. depending on laboratory-specific reference intervals, normal protein concentration is usually less than to mg/dl for cisternal csf and less than mg/dl for lumbar csf. , approximately % to % of csf protein is albumin, and % to % of csf total protein comprises gammaglobulins. eighty percent of csf protein is transferred from plasma, with the remainder produced within the cns. the latter population includes molecules also produced by other organs and proteins unique to the csf that may potentially be used as markers of cns tissue damage. experimental evidence and earlier literature support a gradient of increasing protein concentration from cranial to caudal within the subarachnoid space, which has been attributed to slower flow and greater blood-csf permeability caudally. normal csf is acellular or contains small numbers of small lymphocytes (figs. . and . ) and large mononuclear cells (macrophages, ependymal lining cells, meningothelial lining cells, choroid plexus cells) (figs. . and . ). large mononuclear cells may be vacuolated and contain phagocytized material ( fig. . ) . a low frequency of nondegenerate neutrophils (< %), which are usually indicative of blood contamination during collection, may be present. a study of samples of canine csf found a . % incidence of meningeal, choroid plexus, ependymal, endothelial cells, or all of these. no correlation existed between the presence of these cells and the presence of pleocytosis, elevated protein concentration, or the primary disease etiology. thus it is postulated that the presence of these cells is an artifact of collection and should not be overinterpreted. the authors recommended the term "surface epithelial cells" for the combined grouping (which cannot be distinguished cytologically), although not all of these cells (meningeal, endothelial) are of epithelial origin. occasionally, anucleate superficial squamous epithelial cells may be seen; these may be caused by contamination from the skin (fig. . ). occasionally, small amounts of granular, foamy extracellular material are present and are consistent with myelin or myelin-like material, which will stain positively with luxol fast blue stain. this material may consist of myelin fragments, which are generated from demyelination, or may consist of myelin figures (a nonspecific term for layered phospholipids exfoliated from damaged cells). the two cannot be distinguished with light microscopy. the significance of this material remains unclear because it may be observed in samples from patients with no discernible cause. a study of canine cerebromedullary and lumbar csf samples showed % incidence of myelin-like material, with a higher percentage in samples from the lumbar cistern or from small dogs (< kg). the presence of the material was not correlated with case outcome. similarly, in a study of cavalier king charles spaniels with chiari-like malformations, myelinlike material was observed in % of lumbar csf collections and % of cerebromedullary collections. thus myelin-like material may be a procedural artifact or may be consistent with a demyelinating (e.g., canine distemper virus, degenerative myelopathy) or potentially necrotizing disorder (e.g., ivdh, other spinal trauma, or a necrotic neoplasm). , normal csf should not contain erythrocytes, but hemodilution is a common occurrence. varying reports on the effect of blood contamination on tncc, leukocyte differential, and protein concentration have been published. [ ] [ ] [ ] [ ] deciding whether increased tncc or protein concentration is the result of hemodilution alone or a significant change concurrent with hemodilution necessarily remains, to an extent, a subjective assessment and must be critically evaluated in light of the magnitude of csf findings along with the other pertinent facts of the case. correction formulas for csf parameters in the face of hemodilution (e.g., adding nucleated cell/μl per or rbcs/μl) are unreliable. , in a recent study of canine csf samples without pleocytosis (tncc < /μl) but containing at least rbcs/μl, the mean percentage of neutrophils ( . % versus . %), percentage of samples with eosinophils present ( . % versus . %), and mean protein concentration ( mg/dl versus mg/dl) were found to be significantly increased in the samples with blood contamination when compared with controls. significant rbc contamination warrants repeat sampling, if possible. marked hemorrhage or evidence of prior hemorrhage (erythrophagocytosis, xanthochromia, hemosiderin-laden macrophages) may be useful in the diagnosis of cns trauma, which may be accompanied by neutrophilic to mixed cell pleocytosis and mild increase in protein concentration. elevated protein concentration in csf (> mg/dl) may occur with or without pleocytosis, and in the absence of pleocytosis is termed albuminocytological dissociation (acd). high protein concentration may be the result of leakage of plasma or cellular proteins across the bbb, localized production of immunoglobulin, localized tissue damage or necrosis, decreased clearance of protein into the venous sinuses, obstruction of csf circulation, or all of the above. as such, it is a nonspecific change that indicates cns damage or hyperproteinemic disease and is consistent with disease of any etiology (e.g., trauma, metabolic, infectious, inflammatory, degenerative, or neoplastic). caution should be exercised when diagnosing acd if the sample is hemodiluted (> rbcs/μl). as is true for pleocytosis, inflammation of the meninges and superficial regions of parenchyma will result in greater csf protein elevations than for lesions that are more remote from the sas. occasionally, an abnormal leukocyte differential (shifted from mononuclear predominance to neutrophil predominance) without pleocytosis occurs. this may only be detected if cytological analysis (after sedimentation or cytocentrifugation of csf) is performed. increased percentages of neutrophils may occur in early or mild inflammatory disease, noninflammatory cns disease, disease that is remote from the sas or sampling site, or in cases of hemodilution. an increased proportion of neutrophils is present when neutrophils comprise greater than % of all nucleated cells, and increased percentage of eosinophils occurs when eosinophils comprise greater than % of the differential. when present (with or without increased tncc), neutrophils should be evaluated for toxic change, degenerative change, and intracellular organisms or other inclusions ( fig. . ). increased percentage of neutrophils without pleocytosis has been associated with healthy dogs, blood contamination, degenerative disk disease, neoplasia, cerebrovascular accident, fracture, cns aspergillosis, and fibrocartilaginous embolism (fce). , , a study of cavalier king charles spaniels with chiari-like malformation documented that those with syringomyelia were more likely to have an increased percentage of neutrophils, but it was not reported whether this subpopulation also had a concurrent pleocytosis. in another study, cats with cns neoplasia had increased percentage of neutrophils or lymphocytes without a pleocytosis. although not a classic pattern, infectious or inflammatory disease should not be ruled out if increased neutrophils are visualized without pleocytosis. increased percentage of eosinophils has been reported in parasitic and protozoal diseases, such as neospora caninum infection. one cat with eosinophilic meningoencephalitis (eme) of unknown etiology had an increased percentage of eosinophils and lymphocytes without pleocytosis. the specific diseases mentioned in the next section on various categories of pleocytosis are a survey of the current literature and meant to be a helpful starting point in the generation of particular differential diagnoses. thus disease entities are listed in the section under which they are most commonly present, but it is important to note that for all disease entities, variability in the nature and the magnitude of pleocytosis may emerge in a particular patient at a particular point in time. wherever possible, other categories of pleocytosis that have been reported for a disease have been mentioned. generally, pleocytoses are defined by the cell type that comprises % or more of the nucleated cell population. if all cell types are % or less, the pleocytosis is classified as a mixed cell pleocytosis. and if, for example, lymphocytes are greater than % but less than %, some pathologists will classify the pleocytosis as mixed cell, lymphocyte predominant. a pleocytosis will be classified as eosinophilic if eosinophils compose at least % to % of the nucleated cell population. bacterial meningoencephalomyelitis. bacterial infections of the cns are unusual and represent a small portion of neutrophilic pleocytoses. typically, this pleocytosis is severe (could be over cells/μl), neutrophilic, and accompanied by significantly elevated protein concentration, but the cell population may change to mononuclear during the course of treatment. , , rare instances of brain abscessation secondary to sepsis (which may be a sequela of iatrogenic immunosuppression) may result in marked neutrophilic pleocytosis, markedly elevated protein concentration, visualization of bacterial organisms (see fig. . ) , and abnormal mri findings. staphylococcus intermedius was cultured from the csf of a dog presenting with a retrobulbar abscess and neurological signs. the csf showed a moderate neutrophilic pleocytosis ( cells/μl) and borderline elevation in protein concentration ( mg/dl). local extension of severe otitis interna resulting in meningoencephalitis and ventriculitis in a dog has been reported. this patient exhibited a severe neutrophilic pleocytosis ( cells/μl) and protein elevation (> mg/dl). pasteurella multocida meningoencephalomyelitis in a kitten was characterized by marked neutrophilic pleocytosis ( cells/μl) with mild protein elevation ( mg/dl) and rare extracellular and intracellular bacterial rods. bacterial culture and susceptibility testing are recommended but may yield false-negative results if organisms are not circulating in the extracellular space or if prior antibiotic therapy had been given. serology and csf-pcr (using organism-specific or universal bacterial [ub] pcr) are recommended. , cryptococcosis in dogs. cryptococcus spp. are a large genus of systemic dimorphic fungi with a predilection for cns tissue, which is infected hematogenously or via direct penetration of the cribriform plate. only two species at this time are medically important: ( ) cryptococcus neoformans (var. neoformans and var. grubii) and ( ) cryptococcus gattii. in a recent study of dogs with cryptococcosis, % had cns infection, with neurological signs being the most common reason for presentation. dogs and cats with cryptococcosis typically have pleocytoses and elevated protein concentrations, but pleocytoses may be variably neutrophilic, eosinophilic, mononuclear, or mixed. in a recent study of dogs with cns cryptococcosis, organisms were found in of csf samples (figs. . and . ). all affected dogs had pleocytoses that were mixed to mononuclear, whereas cats tended to have neutrophilic pleocytoses. of the samples, of also had increased protein concentrations (mean mg/dl), which were significantly higher than in cats in the same study (mean mg/ dl). capsular antigen latex agglutination testing on serum or csf is highly sensitive and specific and is recommended if cryptococcosis is suspected but organisms are not visualized cytologically. this test may yield negative results if disease is present but localized (i.e., within the respiratory tract), so appropriate clinical signs should guide testing. culture of csf may also be helpful and may distinguish c. neoformans from c. gattii with the use of selective media. the finding of inflammatory foci on mri may be supportive of the presence of fungal disease; cryptococcosis may result in mass lesions, meningitis, or pseudocyst formation. cryptococcosis is the most common systemic fungal disease of cats and is believed to infect the cns less frequently than in the dog. a recent study found that % of cats with cryptococcosis had cns infection, but respiratory signs were still a more common reason for presentation. mild to marked neutrophilic or mononuclear pleocytosis may occur, with variable and occasionally normal protein concentrations. a study of cats with cns cryptococcosis showed organisms in of of the csf samples, and a majority of cases ( of ) had neutrophilic pleocytosis and increased protein concentration ( of ). eosinophilic pleocytosis may also occur. capsular antigen latex agglutination testing on serum or csf is recommended for confirmation of cryptococcus spp. infection, with rare false-negative reactions if disease is highly localized. fungus that has been visualized in canine csf and may be extracellular or within leukocytes. ehrlichiosis. neutrophilic pleocytosis has been reported in cases of granulocytic ehrlichia spp. in dogs (fig. . ). neurological signs are uncommon in this disease, and affected dogs may display features ranging from ataxia to seizures. (fip) has been traditionally linked to marked csf changes, but the current literature paints a somewhat more varied picture. one study of natural fip infection showed neutrophilic pleocytosis (as defined by > % neutrophils) in the majority ( of ) of cases, with fewer cases of mononuclear ( of ; as defined by > % mononuclear cells) and mixed cell ( of ) pleocytosis, all of variable severity. most cases ( of ) also had differing degrees of elevated protein concentrations. diagnosis was confirmed by histopathology or suggested by elevated feline coronavirus antibody titers and reduced albumin-to-globulin ratios in both serum and body cavity effusions. a slightly older study of csf samples (natural and experimental infections) showed pleocytosis in of cases (neutrophilic and lymphocytic) and elevated protein concentration in of cases. in a larger study of cats with fip or non-fip disease, incidence of pleocytosis was highest in the neurological fip group, but % of these patients did not have a pleocytosis. additionally, protein concentrations were variably elevated and not statistically different in fip compared with non-fip neurological disease. another study of cats with cns fip showed of with unspecified pleocytosis and of with elevated protein concentration. in cats with cns disease, sensitivity of feline coronavirus (fecov) immunoglobulin g (igg) in csf for the diagnosis of fip was %, and specificity was %, with a positive predictive value of % and a negative predictive value of % (fip prevalence in this population was . %). definitive diagnosis of this disease remains challenging, with virus identification (pcr or immunohistochemistry) accompanied by pyogranulomatous inflammation in tissues being the gold standard. hypergammaglobulinemia, elevated serum α -acid glycoprotein (agp), mri abnormalities (typically involving the ventricular lining and meninges), and positive feline coronavirus igg titer or pcr from serum, tissue, or csf are supportive but not specifically diagnostic, and negative findings do not rule out disease. , , toxoplasmosis in cats. cats are the definitive hosts for toxoplasma gondii and may be subclinically infected; thus, diagnostics should only be performed on patients with appropriate clinical signs. cats typically present with mild neutrophilic or mononuclear pleocytosis and normal to mildly elevated protein concentration, but marked protein elevation may occur. mild lymphocytic pleocytosis is also reported. diagnosis may be confirmed by direct visualization of organisms in csf, aspirates of other inflammatory foci, histopathology of affected tissues, or fecal examination. serology must be interpreted cautiously because igg may remain elevated for up to years after exposure. therefore, paired serum igm-igg titers, indicating acute exposure, or documentation of rising serum igg titers are more useful, but the latter is difficult to document in the advanced state of disease. , spinal epidural empyema in dogs. epidural empyema is an uncommon disease in dogs, resulting from pyogenic infection in the epidural space. one study showed of dogs with neutrophilic pleocytosis of variable magnitude ( - cells/μl). no organisms were visualized on any of the samples. except for one case with a lumbar csf protein concentration of mg/dl, protein elevations were modest. three csf samples were cultured with no growth, and two dogs for which follow-up csf was obtained showed resolution of pleocytosis. these results are not surprising, as the dura likely provides a barrier to prevent infection extending from the epidural space to the subarachnoid space. reported in a young cat with a marked neutrophilic pleocytosis with intracellular and extracellular merozoites observed on csf cytology. diagnosis was confirmed with decreasing paired serologic titers, and speciation to the level of sarcocystis dasypi or sarcocystis neurona was conducted with pcr from blood. a case of systemic acanthamoeba spp. infection in a young boxer, diagnosed post mortem, had antemortem csf with marked neutrophilic pleocytosis ( cells/μl), marked increase in protein concentration ( mg/ dl), and subnormal csf iga concentration ( mg/dl; reference interval - mg/dl). postmortem pcr for the organism was positive on extraneural tissue but not on csf or spinal cord. the patient had been deliberately immunosuppressed on the basis of a preponderance of evidence of steroid-responsive meningitis arteritis at initial presentation and thus may have been infected either before or opportunistically after treatment. another case report of canine cerebellar balamuthia mandrillaris infection (diagnosed post mortem with immunohistochemistry) displayed a marked neutrophilic pleocytosis ( cells/μl), but other cases with lymphocytic pleocytosis have been reported. because of tissue encystment, it is suggested that extraneural tissue be used for immunohistochemistry or pcr for antemortem confirmation of amoebic infection; pcr of csf may be diagnostic but is not widely available. , two dogs with aberrant spinal migration of spirocirca lupi nematodes had moderate to marked neutrophilic to mixed or eosinophilic pleocytoses ( cells/μl with % neutrophils; cells/μl with % neutrophils, % eosinophils). steroid-responsive meningitis arteritis. steroid-responsive meningitis arteritis (srma) is presumptively an immune-mediated disease of mainly young, medium-and large-breed dogs: beagles, boxers, bernese mountain dogs, weimaraners, and nova scotia duck tolling retrievers are overrepresented. csf analysis is important in diagnosis and typically features a moderate to marked neutrophilic pleocytosis (a left shift may be present) and markedly elevated protein concentration. chronically, pleocytosis may change to a more mononuclear or mixed population (fig. . ) and may become mild or even fall into reference intervals. a study of affected dogs showed neutrophilic pleocytosis in of cases and mononuclear pleocytosis in of cases. concurrent elevations of serum and csf iga titers (elevated igg and igm fractions may be present), serum concentration of cross-reactive protein (crp), or serum α -macroglobulin is diagnostically supportive but not specific. , increases in iga have been linked to a t-helper (th )-dominated immune response driven by elevated interleukin- (il- ) and decreased il- and interferon-gamma (ifn-γ). serum amyloid a (saa), serum agp, and serum haptoglobin may also be elevated. another study of dogs with srma reported statistically significant elevations of csf and serum crp, but not serum α -macroglobulin, in dogs with srma compared with other neurological diseases. in a study of dogs, serum crp was positively correlated with csf tncc. additionally, serum haptoglobin and serum and csf iga remained increased throughout successful treatment, indicating that these parameters are more useful for diagnosis than for monitoring therapy. serum and csf concentrations of crp and saa have been documented to fall significantly during treatment, and repeat measurement of serum crp or saa may be used to guide therapy and predict relapse, which is less invasive and more sensitive than repeat csf sampling. , , rare cases have been documented in cats with marked mononuclear or mixed pleocytosis and mild to moderate protein concentration elevations. intervertebral disk herniation. csf from patients with ivdh may be extremely variable; data indicate that csf findings correlate with location of sampling, disk herniation location, chronicity of the lesion, and severity of spinal cord injury. bearing this in mind, it is no surprise that some reports in the literature state that neutrophilic, lymphocytic, mixed, and mononuclear pleocytoses are most common in dogs with ivdh. , , a study of cases of ivdh showed % with pleocytosis, of which % were neutrophilic, % were lymphocytic, % were mixed, and . % were mononuclear. of all cases, % had elevated protein concentrations. interestingly, a larger number of cases of lymphocytic pleocytosis were observed in the samples analyzed more than days after onset of clinical signs. the magnitude of pleocytosis, in general, was also shown to decrease with increasing time between clinical onset and sampling, and this observation has been corroborated by other studies. , prior treatment with corticosteroids was observed to reduce the number of observed lymphocytes in csf. the authors also found a higher incidence of pleocytosis in thoracolumbar disease ( %) compared with cervical disease ( %), but this may have been caused by exclusive sampling of lumbar csf closer to the lesion. ivdh is rare in cats and has been reported to feature mild mixed cell pleocytosis and elevated protein concentration. patients typically present with nonpainful, progressive, asymmetrical neurological signs. as only histopathology is confirmat ory, it is a multimodal diagnosis of exclusion. a study of dogs with presumptive fce, based on history, clinical signs, imaging, and outcome, showed % with normal csf, % with acd, and % with mild to moderate pleocytosis ( - cells/μl; median /μl). pleocytoses were neutrophilic or mixed. one study of confirmed cases in dogs showed that % had normal csf and the remainder displayed mild changes. another study looking at five dogs suggested that pleocytosis may be marked, up to cells/μl. fce is much less common in cats. in general, the disease process and clinical signs are similar to those in dogs, with the exception that the disease presents in cats in middle or older age, usually with cervical spinal cord signs. a case series of five cats showed csf ranging from normal to marked neutrophilic pleocytosis with moderately elevated protein concentration and variable correlation to clinical outcome. the case with the most severe csf changes had extensive myelomalacia at necropsy. it was suggested in this study that csf is more likely to be abnormal if collected closer to the lesion and that mri is helpful for localization and in supporting the diagnosis. , thiamine deficiency in cats. thiamine deficiency is a rare nutritional disorder of patients fed noncommercial, misformulated commercial, or irradiated diets. two case reports showed increased percentage of neutrophils or mild neutrophilic pleocytosis, presumptively from cerebrocortical necrosis. diagnosis is based on history, response to treatment, mri features compatible with the disease (cortical and brainstem hyperintensities), or histopathology. spaniels with chiari-like malformation showed that % of dogs with concurrent syringomyelia and cisternal csf sampling had mild (up to cells/μl) pleocytoses and increased percentages of neutrophils compared with the subpopulation without syringomyelia, but it was not specifically documented whether pleocytoses were, in fact, neutrophilic or mixed with an increased percentage of neutrophils. a positive correlation was also seen to exist between tncc and syrinx size. neoplasia. it is important to perform csf in neurology patients with suspected neoplasia, as definitive diagnosis may be achieved if neoplastic cells are directly observed via cytology. inflammatory pleocytoses or elevated protein concentrations are common in patients with cancer, tend to be mild to moderate in magnitude, and may represent paraneoplastic inflammation, compromise of the bbb, lesional necrosis, or all of these. normal csf is also a common finding in cases of neoplasia. moreover, in the absence of overtly neoplastic cells, no defined patterns connect specific tumors with specific types of inflammatory pleocytoses. neutrophilic pleocytosis of unspecified magnitude was found in the csf of of cats with spinal lymphoma and in of cats with nonlymphoma spinal neoplasia (astrocytoma or osteosarcoma). additionally, the remaining four cats with nonlymphoma spinal tumors (meningioma, peripheral nerve sheath tumor, plasma cell tumor) had either normal csf or acd of unspecified magnitude. metastatic tumors to the cns should also be considered in a patient with neurological signs. eosinophilic meningoencephalitis of dogs. eme is an idiopathic diagnosis of exclusion that is typically steroid responsive and is postulated to be triggered by an underlying hypersensitivity, allergy, or self-limiting infection. the disease may be overrepresented in rottweilers and golden retrievers. a study of dogs with eosinophilic pleocytosis (defined by > % eosinophils) showed cases of idiopathic eme, cases of infectious disease (c. neoformans, n. caninum, baylisascaris procyonis), and cases of ivdh. the magnitude of pleocytosis or the percentage of eosinophils could not be used to distinguish infectious versus eme cases, although ivdh cases tended to have milder pleocytoses (< cells/μl). in about half the eme cases, mri showed abnormal findings. peripheral eosinophilia may or may not be present. highly suggestive of protozoal (toxoplasmosis, neosporosis), fungal (cryptococcosis), parasitic (including cuterebra spp., dirofilariasis), and algal (protothecosis) infections and also rarely in cases of canine distemper and rabies viruses. , eosinophils have also been found in cases of granulomatous meningoencephalomyelitis (gme). eosinophilic pleocytosis has been documented in bacterial encephalitis as well. gondii infection tend to have neurological or neuromuscular signs. case reports are sporadic; documentation of mild acd ( mg/dl) and also a report of mild lymphocytic or eosinophilic pleocytosis ( cells/μl) with an elevated protein concentration of mg/dl exist in the literature. it is important to rule out other potential causes of the neurological signs because immunocompetent dogs tend to clear subclinical infections, and therefore paired serum igm-igg titers or sequential serum igg titers are preferable to a single serum igg titer. to the author's knowledge, no data on the life span of canine igg antibodies exist. reports in the literature are conflicting with regard to the cross-reactivity of t. gondii antibodies to other agents, such as n. caninum. , pcr testing for toxoplasma in serum, tissue, or csf is diagnostic. , rabies. pleocytoses may be lymphocytic and of varying severity. ancillary antemortem diagnostics include viral pcr on saliva or csf and the saliva antigen latex agglutination test. in a study of dogs under quarantine for suspected natural infection (subsequently confirmed positive), of were saliva-pcr positive, and of ( %) were csf-pcr positive. all animals with positive results on csf were also positive on saliva, and interestingly % correlation was seen between positive csf-pcr and the dull clinical presentation (all aggressive clinical presentations were csf-pcr negative). negative testing should never exclude diagnosis because viral load is highest within salivary glands and brain parenchyma. frequently made by exclusion when coupled with appropriate clinical signs. csf and mri findings are variable and may be normal in the acute stage of disease before inflammation has peaked. in a study of dogs with noninflammatory distemper, half ( of ) had normal csf. a study of eight dogs with natural infection (confirmed by cns tissue-pcr and histopathology) showed lymphocytic pleocytosis in all samples and normal protein concentrations. another case (confirmed by tissue-pcr and csf-pcr) in a -month-old dog displayed marked ( cells/μl) lymphocytic pleocytosis and a normal protein concentration. because this is a demyelinating disease, myelin-like material, which is amorphous, granular, pink, foamy, and stains positively with luxol fast blue, may be present. pcr testing of csf, serum, urine, epithelial or tonsillar tissue is available, and immunohistochemistry on biopsy specimens of nasal mucosa, haired skin, or footpad is % to % sensitive for detection of viral antigen. is present, it is likely to be lymphocytic. pleocytoses are typically mild to moderate, but severe lymphocytic pleocytoses have been reported. main differential diagnoses include other viral diseases, gme, or chronic bacterial infection. extranigral signs related to the gastrointestinal or the respiratory system, if present, may be helpful in distinguishing this disease from gme. in a study comparing four dogs with chronic cdv, six dogs with acute cdv, and controls, dogs with chronic cdv had markedly elevated csf igg concentration. the igg region was polyclonal, including a population of neutralizing antibodies for cdv. fungus acquired through inhalation, and most cases in the united states are observed in the southwestern region of the country. signs tend to involve respiratory or skeletal systems, and cns involvement is rare. one dog had a mild to moderate lymphocytic pleocytosis. complement fixation (detecting igg) or tube precipitation (detecting igm), or agar-gel immunodiffusion serological testing is recommended for confirmation. necrotizing meningoencephalitis. meningoencephalitis has been subcategorized as necrotizing meningoencephalitis (nme) and necrotizing leukoencephalitis (nle) on the basis of histopathological appearance. both nle and nme are believed to have an immune-mediated basis, and recent data support that in pugs with nme, canine leukocyte antigen gene aberrations exist. meningoencephalitis is rapidly progressive and affects a variety of generally young to middle-aged toy-breed dogs, including the pug, shih tzu, papillon, maltese, chihuahua, yorkshire terrier, french bulldog, pekingese, west highland white terrier, boston terrier, japanese spitz, and miniature pinscher breeds. , a study of csf from pugs with nme showed of with pleocytoses of varying severity (mean cells/μl). of these dogs, % had a lymphocytic pleocytosis, % had a mononuclear pleocytosis, and % had a mixed cell pleocytosis (fig. . ) ; of dogs had elevated protein concentrations (mean . mg/dl). another study of three dogs showed one with acd and two with moderate to marked ( - cells/μl) neutrophilic to lymphocytic pleocytosis. mri findings may help support a diagnosis, but only histopathological examination of lesions provides definitive proof. other. four cats with ischemic encephalopathy had mild (< cells/μl) lymphocytic pleocytosis. another study of feline ischemic encephalopathy reported one cat with normal csf and another with mononuclear to mixed pleocytosis ( cells/μl). a report of two cats with cerebrovascular disease (infarction or stroke) showed one with mononuclear pleocytosis and the other with acd. cerebrovascular disease was correlated in several other cases (without csf data) to hepatic lipidosis or fip. other. a case report of a pug with a mild mononuclear pleocytosis ( cells/μl), mild elevation in protein concentration ( mg/dl), evidence of hemorrhage, and direct visualization of angiostrongylus vasorum helminth larvae is found in the literature. eosinophilia was not observed within the csf or peripheral blood. the organism is endemic in europe and canada among foxes and canids, mainly causing respiratory signs or coagulopathy; neurological signs are typically caused by hemorrhage. two dogs with paraparesis and pyogranulomatous lumbar masses (one intradural, one extradural) had lumbar csf with mild mixed cell pleocytosis (lymphocytes and nondegnerate neutrophils) or lymphocytic pleocytosis. these patients were serologically pcr positive for bartonella vinsonii subsp. berkhoffi and presented with a nodular dermatosis. a dog with neurological signs and hepatozoon canis infection showed marked lymphocytic pleocytosis ( cell/μl) with mildly elevated protein concentration ( mg/dl). organisms were not visualized in csf but were found on cytology of peripheral blood, lymph node, bone marrow, and bony lesions. serology and positive pcr from a bone marrow sample were diagnostic. intrathecal contrast administration. contrast media or pharmacological agents, such as epidural anesthetics, may introduce preanalytical error into csf samples, artificially raising tncc and protein concentrations. in a study of healthy dogs given either iopamidol or metrizamide for emg, same-day csf sampling showed that of developed a mild to moderate mononuclear to mixed mononuclear or neutrophilic pleocytosis ( of were from iopamidol). in the same study, of (all metrizamide) developed mild protein elevation, but mean protein concentration for both groups stayed within the reference interval. in dogs given metrizamide, of had an increased pandy score after emg, which was considered a false-positive result because of the contrast agent. these data, plus histopathology from the same population, showed that the contrast agents caused low-grade leptomeningeal inflammation with no statistical difference between the two agents studied. another similar study over days showed that post-emg csf changes reversed after approximately days. granulomatous meningoencephalitis. gme is a progressive immune-mediated disease that is overrepresented in females, toybreed dogs, and terriers. it is a diagnosis of exclusion and has clinical presentations and mri findings that may be similar to various infectious and neoplastic diseases. csf may be unaffected or may display a mononuclear to mixed pleocytosis and protein concentration elevations, both of varying severity (figs. . and . ). in a study of csf samples from dogs with inflammatory neurological diseases, marked pleocytosis (> cells/μl) was found in cases of srma, bacterial encephalitis, or gme. pleocytosis may also be lymphocytic or neutrophilic. csf protein electrophoresis may be helpful, as several cases have been shown with increased β-globulin and gammaglobulin fractions. most of the diseases described previously in this chapter may manifest as mixed cell pleocytoses, depending on the time interval between disease onset and csf sampling, disease severity, and previous treatment administered. a mixed cell pleocytosis would be expected to occur during transition between different phases of the inflammatory response, where certain cells may predominate at specific times after injury. is typically rare and may involve chorioretinitis or focal cerebral granuloma in the cat. a study of two dogs with systemic blastomycosis and neurologic signs showed mild mixed cell pleocytosis ( cells/μl, mononuclear predominant; and cells/μl, lymphocytic predominant). using csf cytology or culture to diagnose the organism may be unrewarding. agar-gel immunodiffusion serologic testing has high sensitivity and specificity for canine antibodies and is recommended if appropriate clinical signs (respiratory signs or lymphadenopathy) are present. agar-gel immunodiffusion testing is less sensitive ( %- %) in the cat, as indicated by a limited number of reports. urine antigen enzyme immunoassay (eia) has good sensitivity for dogs and has been used successfully on at least one cat. eia may also be performed on csf. cytology of nasal, pulmonary, or dermal lesions is more likely to yield direct visualization of organisms. lymphoma. lymphocytic pleocytosis of inflammatory origin may be difficult to distinguish from lymphoma exfoliating into the csf (fig. . ). the size of lymphocytes and morphological atypia may be helpful, although these may be challenging to differentiate from artifactual morphological changes secondary to cytospin preparation. cats with neoplasia may have lymphocytic pleocytoses (suggestive of lymphoma), mild to moderate mononuclear to mixed cell pleocytoses (suggestive of nonlymphoma tumors), or normal csf. one study examined six cases of feline cns or multifocal lymphoma, which displayed pleocytoses of variable magnitude, absent to mildly elevated protein concentrations, and neoplastic cells visualized in of of the csf samples. in this study, eight cats with cns signs that were ultimately diagnosed with nonlymphoma tumors (e.g., meningioma, carcinoma, nerve sheath tumor) had mild csf protein elevations and either normal tncc ( of ) or mild to moderate mononuclear or mixed cell pleocytosis ( of ). another study of cats with spinal lymphoma showed neoplastic cells visualized in one case and hemodilution, acd, or neutrophilic pleocytosis in the remainder of cases. a case report of feline multiple myeloma involving lumbar vertebrae and associated soft tissues exhibited cisternal csf with an elevated protein concentration of mg/dl and mild pleocytosis ( cells/μl) consisting of a majority of neoplastic plasma cells. diagnosis was further confirmed by abnormal urine protein electrophoresis and bone marrow aspiration. histiocytic malignancies. malignant histiocytosis or histiocytic sarcoma tumor cells in canine csf have been documented in two recent case reports; csf cytology displayed marked mononuclear pleocytoses (> cells/μl) and mild to moderately elevated protein concentrations (< mg/dl). , tumor cells phenotypically resembled macrophages, displayed multiple criteria of malignancy, and reacted positively to cd c on immunocytochemistry, compatible with interstitial dendritic cell origin. , necropsy was confirmatory and found no evidence of neoplasia outside of the cns. , a case report of a gliomatosis cerebri (gc) neoplasm in a middle-aged poodle showed csf with a mild lymphocytic pleocytosis ( cells/μl) and protein concentration elevation. on histopathology, lymphocytelike perivascular cuffing and meningitis were noted. other case studies of canine gc have reported normal csf or mild acd. in a study of dogs with intracranial meningioma, in which csf analysis was performed, % had normal csf, % had acd, and % had pleocytosis ( of of these neutrophilic pleocytosis; of unspecified), with the overall incidence of neutrophilic pleocytosis at %. in this study, a positive correlation existed between elevated tncc and anatomical localization of the lesion to the caudal (versus middle or rostral) portion of the cranial fossa, and no association between pleocytosis and necrosis within the lesion was found. these findings contradict prior reports of a high percentage of abnormal csf findings in meningioma, and the authors reported that concurrent glucocorticoid therapy in some of the patients may have negatively biased the data. , a study of dogs with spinal meningioma showed no cases with exfoliating tumor cells, % with mild pleocytosis up to cells/μl (mean cells/μl), and normal or variably elevated protein concentrations up to mg/dl (mean mg/dl). both cisternal and lumbar csf samples were evaluated in this study and not found to be significantly different. interestingly, tumors of the lumbar region displayed higher mean tncc and protein concentrations compared with tumors of the cervical area ( versus cells/μl and versus mg/dl, respectively), which the authors postulated may be reflective of a higher number of lumbar csf samples with proximity to the lesion. other neoplasms. a case report of canine csf with cells/μl was characterized by atypical neoplastic round cells that were confirmed on immunocytochemistry and immunohistochemistry to be from a metastatic mammary carcinoma. inflammatory cells were of low numbers and were of a mixed population. a study of csf from dogs with choroid plexus tumors showed direct observation of tumor cells in % of the cases of carcinoma. mild to moderate mixed-cell pleocytosis was present in all cases of papilloma and in half of the carcinomas; when pleocytosis was present, no difference in magnitude existed between benign and malignant tumors. all cases had elevated protein concentrations, with median concentration for carcinoma being significantly higher ( mg/dl) than median concentration for papilloma ( mg/dl). a cutoff protein concentration of mg/dl yielded a sensitivity of % and a specificity of % for detection of choroid plexus carcinomas. another case report of canine choroid plexus carcinoma had a mononuclear pleocytosis of cells/μl, mildly elevated protein concentration of mg/dl, and numerous tumor cells visualized. a rise in the availability of stereotactic brain biopsy has facilitated increased cytological assessments of cns lesions. this technique offers several advantages, although significant equipment investment and time to perfect techniques is required. stereotactic biopsy often offers application accuracy for targeting lesions that approximate mm or less in all directions. in one study, diagnostic accuracy of stereotactic biopsy specimens submitted for histopathology (i.e., agreement with specimens obtained via open approaches) exceeded %. in experienced hands, stereotactic biopsy is believed to be a relatively low-morbidity procedure. a b um um cytological interpretation of brain biopsy specimens acquired via stereotaxy or open approaches may be challenging and does require a tumor that exfoliates well, a surgeon willing to provide multiple samples, and a cytologist with expertise in this area. a study of canine and feline cases of biopsy-or necropsy-confirmed cns lesions showed squash-prep smear cytology to have % sensitivity in accurately determining diagnosis, with an additional % of cases having partial correlation between cytology and histopathology. for the remaining % of cases, cytological interpretation did not correlate with final diagnosis. cytological interpretation of cns lesions may be very difficult, and biopsy with histopathological examination is recommended to confirm all diagnoses. it is important for cytological samples to be prepared in the same manner each time to avoid introducing additional cytological variations that the pathologist has to read through. some authors recommend wet-fixation of tissues followed by staining with hematoxylin and eosin (h&e). at the authors' institution, cns cytological samples are air-dried and stained with diff-quik or a modified wright stain. the reader is referred elsewhere for a complete discussion of normal cns cytology. clinical imaging findings, and signalment, should be considered carefully and may help the pathologist to formulate a list of potential differential diagnoses. it must be kept in mind that primary tumors may metastasize to the cns, and these should be included in the differential diagnoses, where appropriate. meningiomas are composed of neoplastic cells arising from the meningothelial cells of the leptomeninges of the cns. these tumors are the most common primary cns tumors of dogs and cats. histologically, these neoplasms are classified into at least nine subtypes based on appearance, and some tumors may be characterized by more than one pattern (fig. . ). cytologically, smears are often characterized by spindle-shaped cells draped around vessels and arranged in large whorling structures (fig. . ). some cells may contain nuclei that display intranuclear cytoplasmic pseudoinclusions, but this is not a feature reliably seen on a majority of tumors (fig. . ). as a whole, this group represents the second most common primary cns neoplasm seen in dogs and cats. glial tumors are more a b common than meningiomas in brachycephalic breeds. glial tumors arise from the supporting cells of the cns. astrocytomas are found most frequently in the cerebral hemispheres, although they have been reported to occur in various locations throughout the cns. astrocytomas arise from transformed astrocytes and are characterized cytologically by high cellularity, a high degree of nuclear pleomorphism, and fibrillar cytoplasmic processes. tumor cells will stain positively for glial fibrillary acid protein (gfap). oligodendrogliomas are derived from transformed oligodendrocytes and are found within the gray or white matter of the cns, with the highest incidence in the cerebral hemispheres. cytological preparations are characterized by large numbers of blood vessels surrounded by neoplastic cells (fig. . ) . neoplastic oligodendrocytes have small amounts of eosinophilic cytoplasm surrounding uniformly round nuclei. ependymomas are derived from the ependymal lining cells found on the surface of the ventricular system of the brain and central canal of the spinal cord. these tumors are rare and are found most often in the lateral ventricles. cytologically, smears are characterized by neoplastic cells palisading around branching vascular structures. cells are cuboidal to columnar in shape with high nuclear-to-cytoplasmic (n:c) ratios and eccentrically placed nuclei. choroid plexus tumors arise from the modified ependymal lining cells that contribute to the production of csf. they are more common in dogs than in cats. papillomas and carcinomas have a very similar cytological appearance and may only be reliably differentiated on the basis of histopathological examination. cytological preparations contain polygonal cells arranged in rafts, columns, or papillary projections around capillary structures (fig. . ). medulloblastoma arises within the cerebellum and is a type of primitive neuroectodermal tumor derived from a germinal neuroepithelial cell. cytologically, preparations are highly cellular, composed of individual round cells that are large in 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cytology-e-book: a color atlas and interpretation guide tumors in domestic animals what is your diagnosis? intracranial mass in a dog a true "triphasic" pattern: thoracolumbar spinal tumor in a young dog key: cord- -wqu t n authors: maideniuc, catalina; memon, anza b. title: acute necrotizing myelitis and acute motor axonal neuropathy in a covid- patient date: - - journal: j neurol doi: . /s - - - sha: doc_id: cord_uid: wqu t n a -year-old woman with covid infection developed acute necrotizing myelitis (anm) and acute motor axonal neuropathy (aman), a rare variant of guillain-barré syndrome (gbs) without systemic signs of infection. mri of the cervical spine demonstrated longitudinally extensive transverse myelitis, and emg was consistent with the diagnosis of aman. csf testing was negative for sars-cov- . high dose steroids followed by plasma exchange were administered, and the patient made a clinical recovery. immunotherapy has some role in fastening the improvement of immune-mediated neurological conditions associated with covid- . electronic supplementary material: the online version of this article ( . /s - - - ) contains supplementary material, which is available to authorized users. the variety of neurological disorders described in covid- could be attributed to one of several mechanisms of injury that have been described with other viral infections such as herpes simplex infections [ ] . there are several mechanisms involved for viral dissemination to the nervous system, including direct binding of the virus to the ace receptors expressed in the nasal epithelium or the olfactory bulb through a retrograde trans-synaptic mechanism, hematogenous, lymphatic and migration of infected immune cells. the critical test required for confirmation of the cns infection by sars-cov- is the detection of its rna by rt-pcr in the csf. however, the sensitivity of the csf sars-cov- rt-pcr is unknown. here we present a unique case of covid patients with acute necrotizing myelitis (anm) and acute motor axonal neuropathy (aman), a rare variant of guillain-barré syndrome (gbs) without systemic signs of infection. a -year-old right-handed woman with a history of hypertension, hyperlipidemia, hypothyroidism, and a remote history of nasopharyngeal and uterine cancer presented to the emergency department with progressive weakness. two weeks before her presentation, she had contact with a covid- positive coworker. a week before her presentation, she developed a runny nose and chills. she had no fever, cough or shortness of breath. three days before her admission, she started experiencing a tingling sensation in her fingers and toes. over the next day, symptoms progressed, and she lost feeling from the chest down and developed progressive weakness in her extremities, and lost her ability to walk. she has not had a bowel movement in a week and developed bladder retention. neurological examination showed increased tone in the lower extremities with weakness in the upper and lower extremities, worse in the lower extremities. reflexes were normal in the upper extremities but brisk in the lower extremities with upgoing toes bilaterally. the patient had a sensory level at c . nasopharyngeal sars-cov rt-pcr was positive. covid labs were all within the normal range (appendix). brain mri was normal. however, mri cervical spine showed patchy t hyperintensities within the central cord extending from below the foreman magnum, proximal electronic supplementary material the online version of this article (https ://doi.org/ . /s - - - ) contains supplementary material, which is available to authorized users. c -c , to cervicothoracic junction (fig. a--c) , with spinal cord slightly increased in overall caliber at c and c , and patchy enhancement obscured by the motion artifact (fig. e) . these changes were associated with a slight hypointense signal on the t images at the c -c level (fig. d) . mri thoracic and lumbar spine were normal. the patient had an extensive diagnostic work up, which was negative for nutritional deficiencies, infectious, autoimmune diseases (lupus, sjogren's vasculitis, syphilis, aquaporin- and myelin oligodendrocyte glycoprotein antibodies). aquaporin- antibody tested negative in both csf and serum. the patient had a spinal fluid analysis that showed a hemorrhagic tap (red blood cells /mm ) with normal white blood cells ( /mm ) elevated protein ( mg/ dl) and glucose ( mg/dl). csf igg index was normal ( . ), and no oligoclonal bands were present. csf gram stain and culture was negative. csf vdrl was negative. csf viral pcr for other microbes was recommended by the neurology team but was not collected. csf testing for sars-cov- was negative. csf paraneoplastic panel (mayo clinic, appendix) was also negative. the patient was treated with methylprednisolone g iv for days without improvements. the patient continued to progress and became quadriparetic. on neurological re-evaluation, weeks after her initial onset of symptoms, the patient was found to be areflexic in all extremities. she had a repeat spinal tap ( days after the first one), and an emg performed ( weeks after her initial presentation) to evaluate for gbs. repeat spinal fluid analysis demonstrated albuminocytological dissociation with elevated csf protein ( mg/dl) and normal white blood cell count ( / mm ), red blood cells ( mm ) , and glucose ( mg/dl). emg showed evidence of acute motor axonal neuropathy with normal sensory conductions (supplementary table) . the patient received five rounds of plasma exchange and was discharged to an inpatient rehabilitation facility. she started to make some clinical recovery - weeks after her clinical presentation. the patient started to stand up . spinal cord swelling is seen at c -c level with associated t hypointensity (d, white arrow). stir short inversion-time inversion recovery, flair fluid-attenuated inversion recovery, letm longitudinal extensive transverse myelitis with the assistance and was able to take few steps with the walker at the rehabilitation facility. acute necrotizing encephalitis, myelitis and variants of gbs such as axonal, demyelinating, and miller fisher syndrome have been reported with the covid [ ] [ ] [ ] [ ] . here we present the first case of covid patients who presented with gbs and anm at the same time without any systemic manifestation. in most of these cases, sars-cov- rt-pcr was positive in the nasopharyngeal swab but negative in the csf, including our case. all patients made a clinical recovery after immunotherapy. form these cases; we learn that the immunotherapy has some role in fastening the improvement of immune-mediated neurological conditions associated with covid- . funding not applicable. informed consent waiver for consent, consent is not needed for the case report per irb. availability of data and material available upon request. : . double-stranded dna-negative myeloperoxidase antibody-negative virus-induced neuronal dysfunction and degeneration covid- -associated acute necrotizing myelitis covid- -associated acute hemorrhagic necrotizing encephalopathy ct and mri features guillain-barré syndrome associated with sars-cov- miller fisher syndrome and polyneuritis cranialis in covid- key: cord- - lrmg z authors: ballinger, megan n.; standiford, theodore j. title: innate immune responses in ventilator-associated pneumonia date: - - journal: mucosal immunology of acute bacterial pneumonia doi: . / - - - - _ sha: doc_id: cord_uid: lrmg z ventilator-associated pneumonia (vap) is a common complication of mechanical ventilation, resulting in substantial morbidity, mortality, and health care cost. early upper airway colonization by pathogenic bacteria and microaspiration are the primary pathogenic events leading to vap. patients at risk for vap have defects in structural/mechanical defenses of the respiratory tract. in addition, critical illness, including sepsis, trauma, and postoperative states, is associated with profound defects in both innate and acquired antibacterial immunity, influencing antimicrobial effector functions of both leukocytes and structural/parenchymal cells. factors present within the lung microenvironment, including alveolar stretch, cyclical atelectasis, changes in oxygen tension, and respiratory tract microbiota, substantially impact antibacterial host responses. mechanisms accounting for dysregulated immune homeostasis are incompletely understood, but likely involve: ( ) alterations in the balance of pro- and anti-inflammatory cytokines; ( ) changes in pathogen recognition receptor and g-protein coupled receptor expression and downstream signaling cascades; and ( ) dysregulated cell death responses. antibiotics and preventive strategies are the mainstay of therapy in patients with vap. however, novel approaches are needed to reverse immunological reprogramming that occurs during critical illness and/or mechanical ventilation, and to identify patients who are most likely to benefit from immunomodulatory therapy. the most common cause of vap is by the gram-positive bacteria staphylococcus aureus , with methicillin resistant s. aureus (mrsa) representing over % of the s. aureus isolates in vap. other vap-causing pathogens include aerobic gramnegative bacilli such as pseudomonas aeruginosa , klebsiella pneumoniae , escherichia coli , enterobacter species, acinetobacter species, and stenotrophomonas maltiphila . legionella pneumophila is an obligate intracellular bacterial pathogen that is an etiologic agent in both community acquired pneumonia (cap), hap and vap. viruses and fungi are unusual causes of vap, although these organisms can modulate innate mucosal responses predisposing to the development of vap. while the bacterial pathogens that cause vap are similar to those that cause hap in nonintubated patients, vap is more frequently caused by pathogens with intrinsic resistance to multiple antimicrobial agents, including p. aeruginosa , acinetobacter species, s. maltiphila , and mrsa. mortality is considerably higher in patients with vap due to p. aeruginosa strains that express the type iii secretion system required for the secretion of pseudomonal exotoxins s, t, u, and y (roy-burman et al. ; sadikot et al. ) . a recent and disturbing trend is the increasing prevalence of community acquired stains of mrsa (ca-mrsa) as a cause of nosocomial infections, including vap (kashuk et al. ) . ca-mrsa, which is typically the usa strain, produce an array of exotoxins that promote extensive tissue necrosis and cavity formation. the intrinsic antibiotic resistance of these gram-positive and gram-negative bacterial strains contributes to increased mortality in patients with vap (fujitani et al. ) . however, these pathogens are generally less virulent and invasive than pathogens that cause pneumonia in otherwise healthy individuals in the community, and tend to be invasive in hosts with anatomic defects in the respiratory tract or substantial impairment in lung mucosal innate immunity. therefore, the presence of these bacterial species as pathogens identi fi es patients with profound anatomic defects or defects in lung innate immunity. the vast majority of vap cases develop as a result of microaspiration of bacteria colonizing the oropharynx (american thoracic society; infectious diseases society of america ) . oropharyngeal colonization occurs very rapidly in critically ill patients. for example, nearly % of patients with underlying lung disease and/or undergoing oropharyngeal intubation were found to be colonized by pathogenic bacteria within h of admission to the intensive care unit (garrouste-orgeas et al. ) . reservoirs contributing to oropharyngeal colonization include the nasopharynx, sinuses, and stomach. endotracheal tubes contribute to colonization by directly injuring mucosal surfaces of the upper respiratory tract, which facilitates bacterial adhesion. organisms that cause vap, including p. aeruginosa and s. aureus , promote bio fi lm formation with the endotracheal tube lumen, which can function as a nidus for direct inoculation of infected material into the distal airspaces. less common sources of bacterial inoculation include colonization of the ventilator circuit or direct inoculation via infected aerosols or instrumentation, particularly suction catheters or bronchoscopes. by comparison, hematogenous seeding of the lung as a cause of vap is considerably less common, accounting for < % of cases. notable exceptions are hematogenous seeding from an intravascular s. aureus infection or gut bacterial translocation that can occur in immunocompromised patients with neutropenia. microaspiration is a common event in both healthy and critical ill patients. these events rarely result in infection in healthy subjects, primarily due to highly effective means to eradicate infectious or toxic insults of the respiratory tract, which include ef fi cient mucocilliary clearance mechanisms and robust innate mucosal antimicrobial responses. in mechanically ventilated patients, impairments in both mucocilliary transport and innate cellular responses results in the establishment of pulmonary infection. a summary of factors contributing to the pathogenesis of vap is shown in fig. . . ciliated, pseudostrati fi ed columnar epithelial cells line the tracheobronchial tree. these ciliated cells are critical to effective mucocilliary transport and the cephalad movement of mucous, microbes, and acellular debris present within the conducting airways. damage to ciliated cells can occur as a direct result of endotracheal intubation the end result from a combination of host factors, medication, and instrumentation is the introduction of infectious material into the sterile lung environment. these factors along with the immune state of host, contribute to the development of vap or conditions that predispose the patient to respiratory failure (nicholls et al. ; piatti et al. ; pittet et al. ) . as discussed previously, denuding of columnar epithelial cells can result from the endotracheal tube or endotracheal tube cuff. moreover, lung conditions that can result in mechanical ventilation, such as copd, are associated with impaired mucocilliary transport (piatti et al. ) . moreover, certain forms of infectious lung injury, including severe acute respiratory syndrome (sars) is characterized by bronchial epithelial denudation and loss of cilia (nicholls et al. ) . similarly, in fl uenza infection predisposes to secondary bacterial infection, which is due not only to impairment in lung innate responses, but also disruption of mucocilliary transport mechanisms (pittet et al. ) . many forms of critical illness result in a profound state of immune suppression affecting both the cellular and acquired arms of host immunity. this syndrome of immune suppression has been best characterized and is perhaps most severe in sepsis, but has also been described in trauma patients, burn injury patients, and patients during the peri-operative period. sepsis is a complex clinical syndrome resulting from the interaction between microbe and host. clinically, it is de fi ned as the systemic in fl ammatory response syndrome (sirs) with evidence of infection (members of the american college of chest physicians/society of critical care medicine ) . changes in the population at risk for the development of sepsis, including an increase in the number of elderly and immunocompromised patients, has resulted in a steady rise in the incidence of severe sepsis (martin et al. ) . despite improvements in supportive care and immunomodulatory therapies, the mortality rate from severe sepsis remains unacceptably high (brun-buisson ) . host immune responses in critical illness, including sepsis can be conceptualized as occurring in distinct but overlapping phases. the initial response during critical illness, referred to as the systemic in fl ammatory response syndrome (sirs), is characterized by the release of a number of pro-in fl ammatory mediators, including early responses cytokines such as tumor necrosis factor-alpha (tnf-a ), interleukin- b (il- b ), interleukin- (il- ), interleukin (il- ) leukocyte-active chemokines, adhesion molecules, and in fl ammatory leukotrienes (dinarello ) . sirs is counter-regulated by the release of inhibitory molecules, including anti-in fl ammatory cytokines (e.g., interleukin (il- ), transforming growth factor-beta (tgf-b )), suppressors of pathogen recognition signaling cascades, immunomodulatory prostanoids and hormones. this counter-regulatory phase is referred to as the compensatory anti-in fl ammatory response syndrome (cars) (wesche et al. ; bone ) . molecules released during cars are believed to serve as a functional "brake" on systemic in fl ammation, and the expression of these mediators is induced by both microbial-derived and host-derived signals. sirs and cars overlap considerably, hence the overall immune status of the patient is dependent on which response predominates ( fig. . ) (van der poll and van deventer ) . recent evidence suggests a third response to an in fl ammatory insult, referred to as the mixed antagonist response syndrome (mars). this response is characterized by the secretion of both pro-and anti-in fl ammatory mediators (speci fi cally il- , il- , monocyte chemotactic protein (mcp)- , macrophage in fl ammatory protein (mip)- b , ifn-g , granulocyte-macrophage colony stimulating factor (gm-csf), and il- ) (tamayo et al. ) . consistent with this mixed systemic cytokine response, elevated levels of il- in circulation has been shown to predict the development of vap (ramirez et al. ) . whether the initial sirs response drives the expression of molecules that contribute to immune suppression or simply a marker of systemic in fl ammation remains to be determined. a summary of innate immune events in critical illness is shown in fig. . . the compensatory release of anti-in fl ammatory molecules in sepsis is believed to mediate immunosuppression during the peri-septic or post-injury period, during which time immune cell function is substantially impaired (historically referred to as critical illness-induced leukocyte "deactivation" or "immunoparalysis"). recently, since the altered leukocyte phenotype in critical illness involves selective regulation of some, but not all innate genes, this phenomenon is now more appropriated referred to as reprogramming. leukocyte reprogramming appears to be of considerable clinical signi fi cance, as higher rates of nosocomial infection and increased mortality are observed in postoperative, burn injury or septic patients who display evidence of monocyte deactivation, either in the form of decreased monocyte hla-dr expression, ex vivo cytokine production or impaired delayed-type hypersensitivity responses (appel et al. ; munoz et al. ) . septic patients are especially susceptible to nosocomial infections of the lung, particularly pneumonia from multidrug-resistant gram-positive and gram-negative organisms, including s. aureus and p. aeruginosa (richardson et al. ; mustard et al. ) . sepsis-induced immunosuppression is particularly prominent in patients with preexisting de fi ciencies in innate and acquired immunity, including the elderly and patients with chronic medical conditions (hotchkiss and karl ) . patients undergoing severe stress, including trauma, massive hemorrhage, burn injury, post-surgery, and sepsis exhibit signi fi cant defects in circulating and resident leukocyte populations. in addition, changes in the pulmonary microenvironment that occur as a result of mechanical ventilation substantially in fl uence lung innate responses. multiple leukocyte subtypes are affected and speci fi c defects are shown in fig. . . while sepsis and similar stress-associated events have been shown to in fl uence the effector activity of a variety of immune cells, the majority of studies have focused on peripheral blood monocytes (pbm), and to a lesser extent tissue macrophages. changes in monocyte/macrophage function in sepsis resemble but are not identical to those observed in endotoxin-tolerized macrophages. endotoxin tolerance describes the phenomena whereby upon initial exposure to lps, cells become refractory to a secondary stimulus with lps. pathogen-associated molecular patterns (pamps) other than lps can also induce a tolerance phenotype, and pamps of one type can induce cross tolerance to a different pamp. induction of tolerance results in suppression of multiple in fl ammatory genes, including both nf-k b and mitogen-activated protein kinase (mapk)-dependent genes (e.g., tnf-a , il- , inos). tolerance does not cause global suppression of all genes, as genes encoding certain antimicrobial and phagocytic proteins, including cathelicidin antimicrobial peptide, lipocalin, the scavenger receptor marco and the fmlp receptor, are indeed super-induced in response to sequential exposure to lps (foster et al. ) . it is also noteworthy that the induction of this phenotype is not restricted to myeloid cells, as structural cells, including alveolar epithelial cells, have been shown to develop a tolerance response upon repeated exposure to pamps. the lps or pamp tolerized phenotype is transient in nature and entirely reversible, and has been associated with remodeling of chromatin in the promoter region of several tolerizable genes (foster et al. ; chan et al. ) . critical illness, like endotoxin tolerance, leads to inhibition of a broad range of nf-k b-dependent in fl ammatory genes in monocytes. most notably, a signi fi cant reduction in the ex vivo production of in fl ammatory cytokines, including il- a , il- b , il- , and tnf-a has been observed in monocytes isolated from patients with sepsis (munoz et al. ) . this change in cytokine production may be a predictor of outcome, as peripheral monocytes isolated from those who survived sepsis regained their ability to produce cytokines in response to lps stimulation, and there are a variety of cellular, bacterial, and mechanical mediators which contribute to the impaired innate and acquired immune responses during critical illness. ( upward arrow ) represent effects that enhance expression/function ( downward arrow ) represents effects that reduce expression/function monocytes isolated from the nonsurvivors did not (munoz et al. ) . conversely, the production of certain anti-in fl ammatory proteins, including il- , il- receptor antagonist, and the tnf soluble receptor i and ii are enhanced in monocytes isolated from sepsis patients or patients with ventilator-induced lung injury (frank et al. ) . patients with sepsis or early trauma have reduced monocyte hla-dr expression (appel et al. ; adib-conquy et al. ) . this reduction in hla-dr expression has been reported to directly correlate with the magnitude of sepsis (volk et al. ) and may partially contribute to impaired cell-mediated immunity observed in patients with critical illness. similar critical illness-induced defects have been noted in macrophages residing in various tissues, which in some instances have been associated with evidence of enhanced macrophage apoptosis (ayala et al. ; gallinaro et al. ) . in particular, alveolar macrophage function has been shown to be impaired in the setting of sepsis. for example, alveolar macrophages recovered from mice with abdominal sepsis (cecal ligation and puncture) display reduced production of in fl ammatory cytokines, chemokines, eicosanoids, nitric oxide, and respiratory burst (reddy et al. ; goya et al. ) . importantly, these phenotypic alterations in alveolar macrophage effector function are associated with a markedly enhanced susceptibility to intrapulmonary challenge with both gram-positive and gram-negative bacterial pathogens (steinhauser et al. ; deng et al. ) . little is known about alveolar macrophage phenotype in critically ill patients at risk for the development of vap. however, we have performed affymetrix microarray analysis on adherence puri fi ed alveolar macrophages recovered from patients with sepsis-induced ali within days of onset of sepsis. relative to alveolar macrophages recovered from healthy subjects, lung macrophages from sepsis-induced ali patients displayed a hybrid tolerized/alternatively activated phenotype, as characterized by minimal change or suppression of nf-k b-dependent genes (e.g., tnf-a , il- b , il- , inos), induction of antimicrobial genes (antimicrobial peptides, chemoattractant, and phagocytosis genes), and expression of makers of alternative (m ) rather than classical (m ) activation (high arginase, ccr , il- r a , mmp expression; low inos, interferong , and ifn-inducible chemokine expression) (gordon and martinez ) . although this expression pattern may partially re fl ect the lung injury response, it is likely that the phenotype is shaped by systemic in fl ammation. alterations in neutrophils (pmn), resembling those described in monocyte/ macrophages, are present during the septic response and are predictive of adverse outcomes in these patients. systemic in fl ammation promotes cytoskeletal changes in pmn cell membrane rigidity and reduced cellular deformability, resulting in impaired recruitment to sites of infection and deleterious accumulation and activation of pmn in vascular beds of distant organs. directed migration is also impaired by nitric oxide-mediated inhibition of icam and vcam-dependent adhesion and transmigration of pmn, downregulation of the chemokine receptor cxcr , and inhibition of g-protein coupled receptor signaling (benjamim et al. ; cummings et al. ; czermak et al. ; huber-lang et al. ; swartz et al. ) . microarray analysis of pmn isolated from septic patients within h of onset reveals a global suppression of immune regulation and in fl ammatory response gene clusters, particularly genes regulated in an nf-k b-dependent fashion (tang et al. ) . conversely, the expression of selected suppressive genes was enhanced, including the nf-k b inhibitor nf k bia. the discovery of neutrophil extracellular traps (nets) has provided yet another role for neutrophils in the containment of infection. nets are complex structures composed of nuclear chromatin, histones, a variety of granular antimicrobial proteins and some cytoplasmic proteins (urban et al. ) . formation occurs in response to exposure of neutrophils to plasma from septic patients (clark et al. ) as well as direct contact with microbial pathogens (remijsen et al. ) . neutrophil elastase is released from azurophilic granules, assisting in the formation of nets via decondensation of nuclear chromatin, which along with other serine proteases confer antimicrobial responses (papayannopoulos et al. ) . netassociated myeloperoxidase directly contributes to bacterial killing of staphylococcus aureus in the presence of h o (parker et al. ) . nets are capable of physically ensnaring bacteria and facilitating the interactions between bacteria and antimicrobial effectors, ultimately leading to enhanced bacterial killing (mantovani et al. ) . despite their broad antimicrobial capacity, some bacteria express nucleases to degrade nets, thus avoiding capture and bacterial cell death (buchanan et al. ; berends et al. ; young et al. ) . in some cases, nets may exert detrimental effects to the host. increasing evidence links net formation to excessive in fl ammation and tissue damage in diseases such as sepsis (clark et al. ) . net formation has recently been demonstrated in the alveoli of mice with in fl uenza h n pneumonia, and these structures contribute to acute lung injury responses in these animals (narasaraju et al. ) . while the presence of nets has not been clearly established in experimental bacterial pneumonia or in patients with vap, it is tempting to speculate that these structures may contribute to lung injury that can occur in this setting. dendritic cells (dc) are the most ef fi cient professional antigen-presenting cells (apc) in the lung and have the unique ability to induce primary immune responses in naïve t cells. dc are prevalent centrally within the spleen, lymphatics, and at mucosal surfaces, most notably in gut and respiratory tract. systemic endotoxin administration in mice results in a brisk depletion in splenic dc by h post-lps. similarly, there is a prolonged loss of dc out to days post-induction of abdominal sepsis in both lung and spleen (wen et al. ) . in humans with lethal sepsis, follicular dc are substantially diminished early in the course of disease (hotchkiss et al. ) . similarly, reductions in blood myeloid dc and plasmacytoid dc ( and % of controls, respectively) have been observed in patients admitted to the hospital with pneumonia, and numbers of dc inversely correlated with procalcitonin levels, a marker of systemic in fl ammation (dreschler et al. ) . endotoxin-tolerized dc or dc isolated from animals or humans with sepsis produce low levels of il- and tnf-a , but high levels of il- (wen et al. ; wysocka et al. ) . this shift in cytokine pro fi les can persist for up to weeks post-abdominal sepsis (clp), and has been associated with posttranslational epigenetic modi fi cations of histones binding to the il- p and p promoters and increased susceptibility to pulmonary fungal challenge (wen et al. ) . regulatory dc, or "tolerogenic" dc, are a newly described dc population that can be induced by incubation of bone marrow-derived dc with il- , resulting in dc that preferentially secrete il- rather than il- , and induce t cell tolerance. a naturally occurring dc reg population has been identi fi ed in spleen (cd c low , cd rb high ), and adoptive transfer of this cell population to septic mice diminished in fl ammatory cytokine production and sepsis-induced lethality (fujita et al. ) . changes in the number, distribution, and function of these cells in lung, especially during critical illness, have not yet been explored. like other leukocyte populations, various lymphocyte populations are in fl uenced by and likely contribute to the immunosuppressive effects of critical illness. this effect can be directly due to changes in lymphocytes numbers or effector functions, or indirectly due to changes in apc function, most notably dc. studies consistently show that sepsis or other states of extreme stress (trauma, burn injury) generally result in anergy and a shift in t cell cytokine responses favoring a th -, rather than th -phenotype response. sepsis, trauma, and other critical states result in a substantial drop in the number of circulating lymphocytes. lymphopenia develops early after the insult, and the persistence and magnitude of lymphopenia correlates with risk of nosocomial infection and death (hotchkiss et al. ) . autopsy studies in septic patients revealed a profound loss of splenic b cells, cd + t cells, and follicular dendritic cells. no alterations in numbers of cd + t cells were observed. the loss of b and cd + t cells was mediated by caspase- -dependent apoptosis. similar changes, although not as uniform, could be observed in critically ill patients without sepsis (hotchkiss et al. ) . in addition to changes in the absolute number of lymphocytes, the septic response can induce considerable alterations in lymphocyte effector function. for instance, the memory/effector cd +/cd ro+ t lymphocyte subset in nonsurviving septic patients demonstrate signi fi cantly decreased ifn-g synthesis compared with survivors (zedler et al. ) . similarly, t cell proliferative responses and cytokine production (il- , tnf-a ) were signi fi cantly depressed in patients with abdominal sepsis, as compared to healthy controls, and the degree of il- and tnf-a suppression directly correlated with patient survival (heidecke et al. ) . the proportion of th t cells is increased in patients with sepsis, but not in non-septic critically ill control patients and healthy subjects (ferguson et al. ) . similar observations have been made in animal models of sepsis. splenocytes isolated from mice undergoing clp produced less il- , il- , and ifn-g , and more il- and il- than splenocytes isolated from healthy animals (ayala et al. ; o'sullivan et al. ) . given the importance of th phenotype responses in host defense against both intracellular and extracellular microbial pathogens, this shift away from an appropriate th -and towards a dysregulated th -phenotype response has obvious implications for antimicrobial host immunity. regulatory t cells (treg), are a limited but important population of cd +, cd + t cells that universally express the transcription factor forkhead box p (foxp ). treg inhibit cd + and cd + t cell effector functions, resulting in negative regulation of both innate and acquired immune responses. suppressive effects of treg are mediated by both direct cell-cell contact and through the release of soluble mediators, including but not limited to tgf-b and il- . an increase in the percentage (but not absolute number) of treg has been found in blood, lymphatics, or spleen in septic mice and humans with sepsis or trauma (venet et al. ; scumpia et al. ; wisnoski et al. ) . moreover, there is evidence of enhanced foxp expression and suppressive function of treg in mice with abdominal sepsis, and adoptive transfer of treg into septic mice reduced overzealous tnf-a production and improved mortality. however, the depletion of cd + cd + treg in mice with polymicrobial sepsis had little impact on sepsis-induced mortality (scumpia et al. ; wisnoski et al. ) . thus, the role of treg in controlling the systemic in fl ammatory response, or as a mediator of impaired innate and acquired immunity in critically ill patients at risk for vap, is uncertain and requires further study. a recently described b cell may play a critical role in innate responses during localized and systemic infection (rauch et al. ) . innate response activator b (ira-b) cells are a population of cd +, b + cells that produce large quantities of gm-csf during infection. this population expands in bone marrow and spleen in response to systemic lps administration or abdominal sepsis, and the genetic deletion of these cells resulted in marked reduction of systemic cytokine responses, gm-csf expression, and the ability to clear abdominal polymicrobial infection. microbes and microbial components that initiate the septic response are recognized by both cell surface and intracellular pathogen recognition receptors (prr), including toll-like receptors (tlrs) and nucleotide-binding oligomerization domain (nod)like receptors (nlr). toll-like receptors are a family of evolutionarily conserved type i transmembrane receptors that respond to pamps expressed by a diverse group of infectious microorganisms, resulting in activation of the host's immune system (aderem and ulevitch ; akira and hemmi ) . there exist distinct tlrs ( in humans and in mice) that have in common an extracellular domain with leucine rich repeats and an intracytoplasmic domain shared with the il- receptor (il- r). binding of ligands to tlrs initiates a signaling cascade involving myeloid differentiation marker (myd ), il- r-associated kinases (irak and ), and tnfr-associated factor (traf ), resulting in nf-k b translocation and mapk activation, culminating in expression of genes involved in antimicrobial host defense (aderem and ulevitch ; akira and hemmi ) . in addition, certain tlrs, such as tlr , tlr , and tlr can initiated a myd -independent signaling cascade that requires the adaptor proteins toll-il- receptor domain containing adaptor protein inducing interferon (trif) and trif-related adaptor molecule (tram), resulting in the expression of interferon responsive genes. the most relevant tlrs in lung antibacterial host defense include tlr , which recognizes speci fi c components of grampositive bacteria and fungi; tlr , which is the major receptor for lps; tlr , which recognizes and is activated by bacterial fl agellin; and tlr , which is activated by unmethylated cpg motifs present in microbial but not mammalian dna. in addition to pamps, tlrs can be activated by host-derived danger signals, referred to as damage-associated molecular patterns (damps) or alarmins, and include heat shock proteins and matrix components (ohashi et al. ) . also, high-mobility group box protein (hmgb ) is a molecule released during the septic response that has recently been shown to activate tlr and tlr (park et al. ) . this is of particular relevance in the setting of sepsis and acute lung injury. multiple tlrs participate in lung host immunity against gram-negative bacteria. for example, tlr recognizes the lipid a moiety of lps, and is the major tlr mediating early innate responses and clearance of non-fl agellated gram-negative organisms that cause vap, including k. pneumoniae , h. in fl uenza, and e. coli (schurr et al. ; bhan et al. ; wieland et al. ) . in addition, mice de fi cient in tlr display impaired dendritic cell-mediated responses during experimental klebsiella or legionella pneumonia, culminating in reduced lung bacterial clearance and decreased survival (bhan et al. (bhan et al. , . innate responses to the fl agellated extracellular bacteria p. aeruginosa are mediated by several myd dependent tlrs, predominantly tlr and tlr (hajjar et al. ; ramphal et al. ; skerrett et al. ) . interestingly, both bone marrow-derived and stromal cells contribute to myd -dependent innate responses to p. aeruginosa in the lung (hajjar et al. ) . toll-like receptors appear to play a lesser role in host defense against s. aureus . for example, while tlr has been shown to mediate in fl ammatory responses to the staphylococcal toxin panton-valentine leukocidin, neither tlr , tlr , nor myd is required for effective anti-staphylococcal host immunity during respiratory infection (skerrett et al. ; zivkovic et al. ) . the nucleotide-binding oligodimerization domain (nod)-like receptors (nlr) nod and nod , which recognize the peptidoglycan component muramyl dipeptide (mdp), have been shown to be important in in fl ammatory cytokine release and bacterial eradication in a murine s. aureus skin infection model (hruz et al. ; inohara et al. ) . more recently, mice de fi cient in rip , the shared nod / adaptor molecule, are considerably more susceptible to intrapulmonary challenge with s. aureus than wild-type mice, an effect which is dependent on downstream activation of in fl ammasomecaspase- -dependent il- b release (unpublished observations, j. deng). these later observations suggest that nlrs, rather than tlrs, may be the predominant contributors to anti-staphylococcal immunity in the lung signaling cascades some, but not all studies have identi fi ed changes in the cell surface expression of various tlrs during the septic response. in particular, either enhanced or reduced cell surface expression of tlr and tlr have been described in monocytes from sepsis patients and in tissue macrophages during experimental sepsis (deng et al. ; brunialti et al. ) . moreover, changes in monocyte cell surface expression of lps binding partners md , cd , and cd have also been observed in sepsis (brunialti et al. ; wolfs et al. ; williams et al. ) . disparate fi ndings are likely attributable to temporal differences in assessment of tlr expression and the heterogeneity of patient populations studied and animal models employed. the extracellular domains of certain tlrs can be shed from activated macrophages, and serve as sinks to bind extracellular pamps, and as a consequence dampen tlrmediated signal transduction. for instance, soluble tlr (stlr ) is released by human peripheral blood monocytes (pbm) and diminishes the cellular response to the tlr agonist pam cys without affecting cellular responses to lps (lebouder et al. ) . both naturally occurring and recombinant soluble tlr have been shown to diminish responses to lps (iwami et al. ; hyakushima et al. ) . the contribution of soluble tlr and tlr to impaired innate responses during critical illness remains to be determined. illuminating the importance of tlrs in lung innate immunity during critical illness, combined loss of function polymorphisms in both tlr and the tlr adaptor tirap/mal, or a homozygous tirap/mal polymorphism have been causally linked to reduced circulating in fl ammatory cytokine levels, reduced ex vivo monocyte cytokine expression, and increased risk for serious postoperative infections, including vap (ferwerda et al. ) . molecules have been identi fi ed that inhibit tlr signaling at multiple sites downstream of the receptor. interleukin- receptor-associated kinase (irak)- and - are key kinases necessary for both myd -dependent and il- receptor-mediated signal transduction. consequently, interruption of irak- and - phosphorylation or traf fi cking can have profound effects on the downstream expression of both nf-k b and mapk-dependent in fl ammatory or antimicrobial genes. interleukin- receptor-associated kinase-m (irak-m), also named irak- , is a member of the irak family. however, irak-m differs from irak- and irak- in that this protein lacks kinase activity and irak-m has been shown to be a negative regulator of tlr signaling by blocking the disassociation of irak- from the toll-il- signaling domain. bone marrow-derived or lung macrophages lacking irak-m display enhanced mapk kinase activation and in fl ammatory cytokine production in response to tlr agonists and live bacteria (wesche et al. ; kobayashi et al. ) . importantly, irak-m is induced by endotoxin, the nod- ligand muramyl dipeptide (mdp), and other pamps and is required for the development of tolerance to endotoxin and peptidoglycan (kobayashi et al. ; hedl et al. ; nakayama et al. ) . we have found that irak-m is upregulated in alveolar macrophages during experimental sepsis in a myd -dependent fashion, and mediates both the suppression of macrophage cytokine responses and impaired lung clearance of p. aeruginosa in septic mice (deng et al. ; lyn-kew et al. ) . irak-m has also been shown to suppress tlr-mediated responses in murine primary alveolar epithelial cells (seki et al. ) . emerging data suggests that irak-m may be a major mediator and perhaps a biomarker for severity of disease in sepsis. irak-m is substantially induced in monocytes from healthy subjects administered lps intravenously (van't veer et al. ) . in patients with gramnegative sepsis, blood monocytes demonstrate a more rapid and robust expression of irak-m when stimulated ex vivo with lps (escoll et al. ) . additionally, enhanced expression of irak-m mrna has been noted in pediatric patients with sepsis, and high irak-m mrna levels were associated with longer length of intensive care unit (icu) stay, need for mechanical ventilation and death (hall et al. ) . we have also observed high constitutive expression of irak-m mrna in alveolar macrophages and peripheral blood buffy coat cells isolated from patients with sepsis-induced ali, as compared to similar cell populations from healthy subjects (t. standiford, unpublished observations). in fact, irak-m was the only negative regulator of tlr signaling found to be signi fi cantly induced in this patient population. several other molecules have been causally linked with the development of endotoxin tolerance or hyperin fl ammatory responses to lps in genetically de fi cient mice. suppression of tumorigenicity (st ) is a transmembrane protein and soluble secreted protein that is expressed by a variety of cells, including t cells and macrophages. st inhibits myd -dependent signaling by interfering with the ability of mal/tirap and myd to interact with downstream signaling molecules. this protein appears to contribute to sepsis-induced impairment in lung antibacterial defense, at least in animal models (holub et al. ) . speci fi cally, clp-induced impairment in anti-pseudomonal lung host defense is reversed in mice de fi cient in st . interestingly, responsiveness of st −/− am was not altered, whereas the expression of ifn-g and tnf-a from cd + and cd + t cells was preserved in st −/− mice in the setting of abdominal sepsis, as compared to similarly treat wildtype animals. toll-like receptor signaling can also be modulated by both extracellular and intracellular decoys. single immunoglobulin il- r-related protein (sigirr) is a member of the il- receptor superfamily but is unable to signal. however, the extracellular domain of this molecule inhibits toll-il- signaling by interfering with binding of ligands to tlr , tlr , tlr , and il- receptor i, whereas the intracellular domain interferes with the complexing of irak- with traf- ( thomassen et al. ; wald et al. ; qin et al. ) . sigirr is expressed predominantly by epithelial cells, including alveolar epithelial cells, but also to a lesser degree in monocytic populations. mice de fi cient in sigirr have enhanced in fl ammatory responses to lps challenge. moreover, sigirr is upregulated in the pbm of septic patients, and is associated with the development of endotoxin tolerance in these cells (adib-conquy et al. ) . myd short (myd s) is an alternatively spliced variant of the parent molecule, myd . myd s functions as a dominant negative molecule by blocking recruitment of irak- to the toll-il- signaling domain, resulting in reduced phosphorylation of irak- (burns et al. ; rao et al. ) . the expression of myd s is induced in monocytes in response to lps and is constitutively expressed in blood monocytes isolated from patients with sepsis (adib-conquy et al. ) . tollip disrupts irak- and irak- interactions, whereas microrna (mirna ) post-transcriptionally inhibits irak- and traf expression (nahid et al. ) . the suppressors of cytokine signaling (socs) are a family of molecules that predominately inhibit jak-stat signaling, but also disrupt tlr signaling cascades through a yet unde fi ned mechanism. while these latter molecules could contribute to suppression of tlr-mediated responses during critical illness, there is no data to show enhanced expression and/or activity in blood monocytes or lung macrophages in patients at risk for the development of vap. initiation of mechanical ventilation (mv) is a vital therapeutic intervention in patients with respiratory failure. a consequence of mechanical ventilation is the inhomogeneous distribution of pressure and volumes to various regions of lung, resulting in excessive stretch in some alveolar units (referred to as volutrauma), and repeated alveolar collapse in other regions (referred to as atelectotrauma) (pugin et al. ) . excessive lung stretch results in activation of several transcriptional pathways, including the nf-kb and the mapk kinase pathway (fos, jun), which contributes to the release of various in fl ammatory mediators such as tnf-a , il- b , il- , and il- (gharib et al. ; halbertsma et al. ; jaecklin et al. ) . these cellular mediators not only trigger deleterious lung injury responses and possibly multiple organ dysfunction ) , but may also promote the reprogramming of leukocytes and structural cells that occurs in critical illness. importantly, mv at moderate to high lung volumes can also prime the lung for enhanced lung injury or systemic organ failure in response to an infectious challenge (e.g., second hit). for instance, as compared to spontaneously breathing animals, the intrapulmonary administration of s. aureus or e. coli to mechanically ventilated mice results in enhanced lung in fl ammation and lung injury, without changes in lung bacterial clearance . likewise, the i.p. administration of lps to mice undergoing high tidal volume mv substantially increased lung and systemic cytokine expression and extrapulmonary organ injury, as compared to non-mechanically ventilated controls . mechanisms accounting for synergistic interactions between lung stretch and infectious challenge have not been clearly de fi ned. however, previous work has shown that stretch of human alveolar epithelial cells increases the expression of tlr by sixfold (charles et al. ) . moreover, mechanical ventilation increased the relative expression of tlr and tlr in lung tissue and increased the generation of endogenous ligands for tlr in bronchoalveolar lavage fl uid (vaneker et al. ) . recent work has shown that mechanical ventilation also generates other tlr -independent and myd dependant endogenous tlr ligands (chun et al. ) . hyperin fl ation of the lung with high tidal volume not only promotes a signi fi cant increase in the expression of tlr , but also paradoxically induces the expression of irak-m, an important negative regulator of tlr signaling (villar et al. ) . a frequent consequence of mechanical ventilation and diseases that cause acute respiratory failure is alveolar collapse and atelectasis. alveolar collapse is due, in part, to reductions in surfactant that occur in patients receiving mechanical ventilation and in patients with vap (nakos et al. ) . atelectasis has been shown to promote bacterial overgrowth, and use of open ventilation strategies and administration of exogenous surfactant can reduce bacterial numbers in an animal model of vap (van kaam et al. ) . moreover, administration of positive end-expiratory pressure (peep) at - cmh o to non-hypoxemic mechanically ventilated patients can reduce the incidence of vap (manzano et al. ) . surfactant proteins a and d can agglutinate p. aeruginosa , and sp-d can serve as an opsonin to enhance phagocytosis of p. aeruginosa (mccormack ) . pseudomonal elastase has been shown to degrade sp-a and sp-d, and these proteins are decreased in the lungs of patients with cystic fi brosis (mariencheck et al. ) . however, changes in sp-a and sp-d levels during mechanical ventilation and/or vap have not been well characterized. administration of high concentrations of oxygen (fio > %) used during transient or prolonged mechanical ventilation is a common treatment for patients with respiratory failure (gore et al. ) . although therapeutically necessary, hyperoxia results in the generation of reactive oxygen species (ros), which promote the breakdown of critical barriers leading to systemic cellular and organ injury (lee and choi ) . in the lung, ros cause severe cellular damage and death, exposure of the basement membrane and disruption of the alveolar capillary membrane leading to increased pulmonary permeability, in fl ux of in fl ammatory cells, and impaired gas exchange (bhandari and elias ) . hyperoxic exposure can also exacerbate alveolar epithelial injury and apoptosis in response to infectious challenge with p. aeruginosa or l. pneumophila , resulting in increased bacterial dissemination (kikuchi et al. ) . moreover, high oxygen tensions inhibit the function of innate immune cells. for instance, macrophages exposed to elevated concentration of oxygen both in vitro and in vivo display reduced phagocytosis and killing of gramnegative bacteria which correlated with changes in cell morphology and actin polymerization (o'reilly et al. ) . in addition, in vivo hyperoxia exposure increased the susceptibility to k. pneumoniae lung infections, an effect that was partially attributed to reduced bal gm-csf levels and cell surface expression of tlr by am (baleeiro et al. ) . importantly, systemic treatment of these mice with gm-csf during hyperoxia preserved macrophage functionality and decreased the severity of lung infection (baleeiro et al. ) . taken together, hyperoxia is detrimental to the host by promoting greater alveolarcapillary injury, impairing local antibacterial responses, and increasing the risk of bacterial dissemination. emerging clinical and epidemiological data suggests a possible link between colonization with candida species and susceptibility to p. aeruginosa pulmonary infection. candida species is among the most common organisms recovered from endotracheal tube bio fi lm and tracheal secretions in patients with vap (adair et al. ) . historically, candida has been considered a commensal organisms rather than a true pathogen, and therefore believed to play no role in vap disease pathogenesis. however, an observational study found a statistical association between airway colonization with candida species and the development of p. aeruginosa vap (azoulay et al. ) . in a rat model of p. aeruginosa pneumonia, prior bronchial instillation of live but not heat-killed c. albicans resulted in increased susceptibility to subsequent bacterial challenge (roux et al. ) . mechanisms accounting for impaired in vivo clearance responses were not identi fi ed, but c. albicans was found to inhibit am respiratory burst ex vivo. while these intriguing fi ndings require con fi rmation in other experimental model systems, they do raise the possibility that candida and perhaps other commensal organisms may contribute meaningful to vap pathogenesis. antibiotics, prophylactic measures to reduce oropharyngeal colonization and microaspiration, and approaches to stimulate mucocilliary transport are the mainstay of therapy to prevent and treat vap. while these treatments are effective in some patients, adjuvant therapies are needed in others to bolster innate host responses, especially in the elderly and in patients with chronic immunosuppressive therapy. the recognition that critical illness can induce a profound state of immune dysregulation has prompted a reevaluation of potential immunologic approaches being used in the treatment of sepsis and other forms of critical illness (pockros et al. a ) . effective immunoadjuvant therapy must necessarily promote antimicrobial effects without exacerbating deleterious lung in fl ammatory responses. common features of both endotoxin tolerance and immune dysregulation of critical illness is impaired tlr signaling, nf-k b-dependent responses, reduced apc function, and a shift toward type rather than type immune responses. two cytokines that have been shown to partially reverse these changes in vitro and in vivo are ifn-g and gm-csf. in endotoxin-tolerized monocytes, treatment with ifn-g or gm-csf can reverse the tolerance phenotype, in part by facilitating interactions between irak and myd , resulting in enhanced downstream activation of nf-k b (adib-conquy and cavaillon ) . similarly, ex vivo treatment of blood monocytes from trauma patients with ifn-g or gm-csf, but not g-csf, enhanced lpsinduced cytokine production, and hla-dr expression (lendemans et al. ) . these preclinical studies served as the foundation for several small clinical trials in patients with sepsis. docke and colleagues administered ifn-g to patients with sepsis in an attempt to reverse the cytokine imbalance and restore monocyte function (docke et al. ) . in this uncontrolled study, nine patients with evidence of sepsis-induced immunosuppression (decreased blood monocyte hla-dr expression) were administered ifn-g at a dose of m g subcutaneously daily. treatment with ifn-g resulted in increased monocyte hla-dr expression in all patients, along with a restoration of monocyte tnf-a production to levels observed in monocytes isolated from healthy subjects. resolution of sepsis occurred in eight of the nine treated patients (docke et al. ) . in two small single center clinical trials, the i.v. administration of hrgm-csf to patients with sepsis resulted in improvements in ex vivo effector responses in pbms or neutrophils (nierhaus et al. ; presneill et al. ) . moreover, one of the studies revealed improvements in pao /fio ratios, as a measure of pulmonary gas exchange, suggesting reduced lung injury in the gm-csf treated group (presneill et al. ) . prevention of lung injury may be due, in part, to the fact that gm-csf is an alveolar epithelial cell mitogen and can protect the alveolar epithelium against hyperoxic and bleomycin-induced injury (baleeiro et al. ; moore et al. ) and in a murine model of in fl uenza pneumonia (sever-chroneos et al. ) . these preclinical and clinical fi ndings served as the basis for a multicenter randomized placebo controlled trial of subcutaneous gm-csf administration in patients with severe sepsis and evidence of monocyte deactivation (reduced hla-dr expression). as compared to the placebo group, gm-csf administration resulted in improved monocyte function (restored cell surface tlr / expression, tnf production, and hla-dr expression) and improved clinical outcomes, including reduced apache ii scores, shorter time of mechanical ventilation, and a trend toward decreased length of icu and hospital stay. these studies and others suggest that immunostimulatory therapy for treatment of critical illness-induced immune dysregulation or even end-organ injury appears to be a potentially viable therapeutic option that warrants larger controlled trials (luedke and cerami ) . an obvious concern of immunostimulatory therapy in patients with severe sepsis and/or pneumonia is the potential of exacerbating the "cytokine storm" of sirs. fortunately, neither ifn-g nor gm-csf has precipitated worsening of hemodynamic compromise or multiorgan failure, even in patient with severe sepsis or septic shock (docke et al. ; nierhaus et al. ; meisel et al. ) . additional consideration could be given to compartmentalized immunostimulatory therapy (e.g., aerosolized delivery) to prevent or treat vap. however, this approach may be limited substantially by ventilation-perfusion mismatching that occurs in patients with lung disease, and the concern that the leukocyte reprogramming that occurs during critical illness is not limited to the lung microenvironment but almost certainly occurs more broadly in leukocyte populations systemically. activation of the pi k/akt pathway in certain leukocyte populations can lessen nf-k b-mediated pro-in fl ammatory responses while stimulating pro-survival and antimicrobial responses (williams et al. ; wrann et al. ; zhang et al. ) . for example, the administration of selective activators of the pi k/akt signaling pathway (e.g., glucan, a -lipoic acid) to lps-challenged mice or mice undergoing clp reduced apoptosis, in fl ammatory cytokine release, and improved mortality (wrann et al. ; zhang et al. ) . interleukin is a pleurapotential cytokine that regulates dc, t, and nk cell activation, proliferation, and survival. the administration of il- to mice with abdominal sepsis (clp) has been shown to block sepsis-induced apoptosis of nk cells, dc, and cd t cells, and to restore nk cell production of ifn-g (inoue et al. ) . treatment with il- also mitigated sepsis-induced apoptosis of gut epithelium. importantly, il- not only reduced mortality in clp, but also in mice administered p. aeruginosa i.t. finally, caspase inhibitors have been shown to reduce lymphocyte apoptosis and increase survival in murine models of sepsis (hotchkiss et al. ) . a pan-caspase inhibitor (idn- ) have been employed in the treatment of liver disease in patients with hepatitis c (pockros et al. b ) . however, trials targeting caspases or other pro-apoptotic molecules or administration of pro-survival factors (e.g., akt activators, il- ) in patients with sepsis or nosocomial pneumonia have not yet been reported. in this review, we have de fi ned the clinical features of vap, and described the impact of critical illness and microenvironment factors introduced during mechanical ventilation on susceptibility to vap, with special attention to speci fi c molecules as potential mediators of immunosuppression and tissue injury. increases in microbial resistance, combined with a burgeoning population of patients at risk, are trends that clearly make vap a major clinical problem now and in the future. preventative strategies and optimal ventilator management have been paramount in reducing the incidence of vap. however, critical illness-induced reprogramming of leukocyte innate immune responses clearly contributes to susceptibility to vap and vapinduced tissue injury. given our past failures, a paradigm shift in how we approach patients with evidence of immune dysregulation is required. in order for novel therapies to proceed, better clinical markers are needed to distinguish a deleterious innate response (e.g. sirs) from a state of immunoparalysis (cars) or mixed antagonist response syndrome (mars) as the in fl ammatory response evolves (wesche et al. ) . differentiating these quite disparate but overlapping responses in a patient-speci fi c fashion will allow for better selection of patients in which immunoadjuvant therapy is more likely to be bene fi cial. implications of endotracheal tube bio fi lm for ventilator-associated pneumonia toll-like receptors in the induction of the innate immune response gamma interferon and granulocyte/monocyte colonystimulating factor prevent endotoxin tolerance in human monocytes by promoting interleukin- receptor-associated kinase expression and its association to myd and not by modulating tlr expression up-regulation of myd s and sigirr, molecules inhibiting toll-like receptor signaling, in monocytes from septic patients recognition of pathogen-associated molecular patterns by tlr family guidelines for the management of adults with hospital-acquired, ventilator-associated, and healthcare-associated pneumonia heme oxygenase- system, in fl ammation and ventilator-induced lung injury experimental and clinical signi fi cance of endotoxin-dependent hla-dr expression on monocytes polymicrobial sepsis selectively activates peritoneal but not alveolar macrophages to release in fl ammatory mediators (interleukins- and - and tumor necrosis factor) polymicrobial sepsis but not lowdose endotoxin infusion causes decreased splenocyte il- /ifn-[gamma] release while increasing il- /il- production candida colonization of the respiratory tract and subsequent pseudomonas ventilator-associated pneumonia sublethal hyperoxia impairs pulmonary innate immunity gm-csf and the impaired pulmonary innate immune response following hyperoxic stress role of nitric oxide in the failure of neutrophil migration in sepsis nuclease expression by staphylococcus aureus facilitates escape from neutrophil extracellular traps tlr is required for protective innate immunity in gram-negative bacterial pneumonia: role of dendritic cells toll-like receptor regulates the lung macrophage phenotype and host immunity in murine pneumonia caused by legionella pneumophila cooperative interactions between tlr and tlr regulate interleukin and production in a murine model of gram negative bacterial pneumonia cytokines in tolerance to hyperoxia-induced injury in the developing and adult lung sir isaac newton, sepsis, sirs, and cars the epidemiology of the systemic in fl ammatory response tlr , tlr , cd , cd b, and cd c expressions on monocytes surface and cytokine production in patients with sepsis, severe sepsis, and septic shock dnase expression allows the pathogen group a streptococcus to escape killing in neutrophil extracellular traps inhibition of interleukin receptor/toll-like receptor signaling through the alternatively spliced, short form of myd is due to its failure to recruit irak- endotoxin tolerance disrupts chromatin remodeling and nf-{kappa}b transactivation at the il- {beta} promoter mild-stretch mechanical ventilation upregulates toll-like receptor and sensitizes the lung to bacterial lipopeptide mechanical ventilation modulates toll-like receptor- -induced lung in fl ammation via a myd -dependent, tlr -independent pathway: a controlled animal study platelet tlr activates neutrophil extracellular traps to ensnare bacteria in septic blood expression and function of the chemokine receptors cxcr and cxcr in sepsis protective effects of c a blockade in sepsis sepsis-induced suppression of lung innate immunity is mediated by irak-m mechanical ventilation induces in fl ammation, lung injury, and extra-pulmonary organ dysfunction in experimental pneumonia proin fl ammatory cytokines monocyte deactivation in septic patients: restoration by ifn-gamma treatment altered phenotype of blood dendritic cells in patients with acute pneumonia rapid up-regulation of irak-m expression following a second endotoxin challenge in human monocytes and in monocytes isolated from septic patients t helper cell subset ratios in patients with severe sepsis functional and genetic evidence that the mal/tirap allele variant l has been selected by providing protection against septic shock gene-speci fi c control of in fl ammation by tlrinduced chromatin modi fi cations pathogenetic signi fi cance of biological markers of ventilator-associated lung injury in experimental and clinical studies regulatory dendritic cells act as regulators of acute lethal systemic in fl ammatory response pneumonia due to pseudomonas aeruginosa: part i: epidemiology, clinical diagnosis, and source alteration of mononuclear cell immune-associated antigen expression, interleukin- expression, and antigen presentation during intra-abdominal infection oropharyngeal or gastric colonization and nosocomial pneumonia in adult intensive care unit patients. a prospective study based on genomic dna analysis noninjurious mechanical ventilation activates a proin fl ammatory transcriptional program in the lung alternative activation of macrophages: mechanism and functions hyperoxia sensing: from molecular mechanisms to signi fi cance in disease characteristics of alveolar macrophages in experimental septic lung an essential role for non-bone marrow-derived cells in control of pseudomonas aeruginosa pneumonia cytokines and biotrauma in ventilator-induced lung injury: a critical review of the literature monocyte mrna phenotype and adverse outcomes from pediatric multiple organ dysfunction syndrome chronic stimulation of nod mediates tolerance to bacterial products selective defects of t lymphocyte function in patients with lethal intraabdominal infection lymphocyte subset numbers depend on the bacterial origin of sepsis the pathophysiology and treatment of sepsis caspase inhibitors improve survival in sepsis: a critical role of the lymphocyte sepsisinduced apoptosis causes progressive profound depletion of b and cd + t lymphocytes in humans depletion of dendritic cells, but not macrophages, in patients with sepsis nod contributes to cutaneous defense against staphylococcus aureus through alpha-toxin-dependent innate immune activation role of c a in multiorgan failure during sepsis interaction of soluble form of recombinant extracellular tlr domain with md- enables lipopolysaccharide binding and attenuates tlr -mediated signaling nod-lrr proteins: role in host-microbial interactions and in fl ammatory disease il- prevents apoptosis, reverses innate and adaptive immune dysfunction, and improves survival in sepsis cutting edge: naturally occurring soluble form of mouse toll-like receptor inhibits lipopolysaccharide signaling lungderived soluble mediators are pathogenic in ventilator-induced lung injury patterns of early and late ventilator-associated pneumonia due to methicillin-resistant staphylococcus aureus in a trauma population hyperoxia exaggerates bacterial dissemination and lethality in pseudomonas aeruginosa pneumonia irak-m is a negative regulator of toll-like receptor signaling soluble forms of toll-like receptor (tlr) capable of modulating tlr signaling are present in human plasma and breast milk pathways of cell signaling in hyperoxia differential immunostimulating effect of granulocyte-macrophage colony-stimulating factor (gm-csf), granulocyte colony-stimulating factor (g-csf) and interferon gamma (ifngamma) after severe trauma interferon-gamma overcomes glucocorticoid suppression of cachectin/ tumor necrosis factor biosynthesis by murine macrophages irak-m regulates chromatin remodeling in lung macrophages during experimental sepsis neutrophils in the activation and regulation of innate and adaptive immunity positive-end expiratory pressure reduces incidence of ventilator-associated pneumonia in nonhypoxemic patients pseudomonas aeruginosa elastase degrades surfactant proteins a and d the epidemiology of sepsis in the united states from through new concepts in collectin-mediated host defense at the air-liquid interface of the lung granulocytemacrophage colony-stimulating factor to reverse sepsis-associated immunosuppression: a double-blind, randomized, placebo-controlled multicenter trial american college of chest physicians/society of critical care medicine consensus conference: de fi nitions for sepsis and organ failure and guidelines for the use of innovative therapies in sepsis gm-csf regulates bleomycin-induced pulmonary fi brosis via a prostaglandin-dependent mechanism innate immune responses in ventilator-associated pneumonia dysregulation of in vitro cytokine production by monocytes during sepsis pneumonia complicating abdominal sepsis. an independent risk factor for mortality mechanistic role of microrna- a in endotoxin-induced differential cross-regulation of tlr signaling involvement of irak-m in peptidoglycan-induced tolerance in macrophages ventilator-associated pneumonia and atelectasis: evaluation through bronchoalveolar lavage fl uid analysis excessive neutrophils and neutrophil extracellular traps contribute to acute lung injury of in fl uenza pneumonitis lung pathology of fatal severe acute respiratory syndrome reversal of immunoparalysis by recombinant human granulocyte-macrophage colony-stimulating factor in patients with severe sepsis cutting edge: heat shock protein is a putative endogenous ligand of the toll-like receptor- complex mechanical ventilation interacts with endotoxemia to induce extrapulmonary organ dysfunction hyperoxia impairs antibacterial function of macrophages through effects on actin major injury leads to predominance of the t helper- lymphocyte phenotype and diminished interleukin- production associated with decreased resistance to infection neutrophil elastase and myeloperoxidase regulate the formation of neutrophil extracellular traps involvement of toll-like receptors and in cellular activation by high mobility group box protein myeloperoxidase associated with neutrophil extracellular traps is active and mediates bacterial killing in the presence of hydrogen peroxide effects of salmeterol on cilia and mucus in copd and pneumonia patients in fl uenza virus infection decreases tracheal mucociliary velocity and clearance of streptococcus pneumoniae oral idn- , an antiapoptotic caspase inhibitor, may lower aminotransferase activity in patients with chronic hepatitis c oral idn- , an antiapoptotic caspase inhibitor, may lower aminotransferase activity in patients with chronic hepatitis c a randomized phase ii trial of granulocyte-macrophage colony-stimulating factor therapy in severe sepsis with respiratory dysfunction activation of human macrophages by mechanical ventilation in vitro sigirr inhibits interleukin- receptor-and toll-like receptor -mediated signaling through different mechanisms systemic in fl ammatory response and increased risk for ventilator-associated pneumonia: a preliminary study control of pseudomonas aeruginosa in the lung requires the recognition of either lipopolysaccharide or fl agellin a novel splice variant of interleukin- receptor (il- r)-associated kinase plays a negative regulatory role in toll/il- r-induced in fl ammatory signaling innate response activator b cells protect against microbial sepsis alveolar macrophage deactivation in murine septic peritonitis: role of interleukin dying for a cause: netosis, mechanisms behind an antimicrobial cell death modality pulmonary infection complicating intra-abdominal sepsis candida albicans impairs macrophage function and facilitates pseudomonas aeruginosa pneumonia in rat type iii protein secretion is associated with death in lower respiratory and systemic pseudomonas aeruginosa infections pathogen-host interactions in pseudomonas aeruginosa pneumonia central role of toll-like receptor signaling and host defense in experimental pneumonia caused by gram-negative bacteria increased natural cd +cd + regulatory t cells and their suppressor activity do not contribute to mortality in murine polymicrobial sepsis critical role of il- receptor-associated kinase-m in regulating chemokine-dependent deleterious in fl ammation in murine in fl uenza pneumonia gm-csf modulates pulmonary resistance to in fl uenza a infection cutting edge: myeloid differentiation factor is essential for pulmonary host defense against pseudomonas aeruginosa but not staphylococcus aureus il- is a major mediator of sepsis-induced impairment in lung antibacterial host defense decreased systemic polymorphonuclear neutrophil (pmn) rolling without increased pmn adhesion in peritonitis at remote sites innate immune responses in ventilator-associated pneumonia pro-and antiin fl ammatory responses are regulated simultaneously from the fi rst moments of septic shock the use of gene-expression pro fi ling to identify candidate genes in human sepsis identi fi cation and characterization of sigirr, a molecule representing a novel subtype of the il- r superfamily neutrophil extracellular traps contain calprotectin, a cytosolic protein complex involved in host defense against candida albicans low-tidal-volume mechanical ventilation induces a toll-like receptor -dependent in fl ammatory response in healthy mice induction of irak-m is associated with lipopolysaccharide tolerance in a human endotoxemia model regulatory t cell populations in sepsis and trauma mechanical ventilation modulates tlr and irak- in a non-infectious, ventilator-induced lung injury model clinical aspects: from systemic in fl ammation to 'immunoparalysis' sigirr, a negative regulator of toll-like receptor-interleukin receptor signaling epigenetic regulation of dendritic cell-derived interleukin- facilitates immunosuppression after a severe innate immune response irak-m is a novel member of the pelle/interleukin- receptor-associated kinase (irak) family the myd -dependent, but not the myd -independent, pathway of tlr signaling is important in clearing nontypeable haemophilus in fl uenzae from the mouse lung granulocytemacrophage colony-stimulating factor induces activation and restores respiratory burst activity in monocytes from septic patients modulation of the phosphoinositide -kinase signaling pathway alters host response to sepsis, in fl ammation, and ischemia/reperfusion injury the contribution of cd + cd + t-regulatory-cells to immune suppression in sepsis increased release of smd- during human endotoxemia and sepsis: a role for endothelial cells the phosphatidylinositol -kinase signaling pathway exerts protective effects during sepsis by controlling c a-mediated activation of innate immune functions il- suppression during experimental endotoxin tolerance: dendritic cell loss and macrophage hyporesponsiveness neutrophil extracellular trap (net)-mediated killing of pseudomonas aeruginosa: evidence of acquired resistance within the cf airway, independent of cftr t-cell reactivity and its predictive role in immunosuppression after burns alpha-lipoic acid attenuates lps-induced in fl ammatory responses by activating the phosphoinositide -kinase/akt signaling pathway tlr and cd mediate innate immunity and lung in fl ammation to staphylococcal panton-valentine leukocidin in vivo key: cord- -fx gcd authors: pirko, istvan; noseworthy, john h. title: demyelinating disorders of the central nervous system date: - - journal: textbook of clinical neurology doi: . /b - - . - sha: doc_id: cord_uid: fx gcd nan demyelinating disorders of the central nervous system istvan pirko and john h. noseworthy multiple sclerosis (ms) is now known to be a common malady even though it was first recognized as a distinct clinicopathological entity less than years ago. the lack of clear medical reports before the early s is sometimes interpreted as evidence that ms is a relatively new disease. however, it is more likely that the evolution of medicine into science led to more precise observation and description of human diseases, including ms. saint lidwina of schiedam ( - ) developed a relapsing neurological disorder at the age of and may be the first case of clinically described ms. ollivier was the first to report a clinical case in the medical literature in . shortly thereafter, carswell illustrated a case of what is now clearly recognizable as ms in his atlas of anatomical pathology. cruveilhier published gross pathological and clinical descriptions of ms. vulpian first suggested the rubric of "sclerose en plaque" in . charcot was primarily responsible for establishing ms as a unique and recognizable syndrome. he also described the clinical spectrum and the histological appearance. pierre marie was the first to suggest an infectious cause of ms in , a hypothesis that is still debated. toxins were also considered to be responsible in the early s. a major advance toward the understanding of demyelinating diseases was the discovery of experimental allergic encephalomyelitis (eae) by rivers in . a variety of different demyelinating diseases have subsequently been described (table - ). myelin provides insulation for axons and is necessary for saltatory conduction. it is composed of tightly wrapped lipid bilayers with specialized protein constituents. peripheral nervous system (pns) myelin is formed by the extension of schwann cells, and central nervous system (cns) myelin is produced by oligodendrocytes. the myelin coating is interrupted at regular intervals (nodes of ranvier) where the axon membrane with its concentration of voltagegated sodium channels is exposed to the extracellular environment ( fig. - ) . the presence of myelin is essential to maintain conduction velocity; its loss or damage can lead to significantly slower conduction or conduction block. other factors affect conduction velocity including certain antibodies and chemicals like nitric oxide. in certain cases, blockade may be the initial event in the cascade of events leading to demyelination. cns and pns myelin differ in a number of important ways. schwann cells myelinate only one internodal segment from a single pns axon, whereas oligodendrocytes myelinate multiple cns axons. the proteins also differ. proteolipid protein (plp) accounts for approximately % of the cns myelin proteins. mutations in this highly conserved protein cause pelizaeus-merzbacher disease. protein zero is the major pns myelin protein and performs a function similar to plp in compacting the intraperiod line. myelin basic protein (mbp) makes up % of cns and % of pns myelin proteins. mbp is not an integral protein but binds to the cytoplasmic surface and is responsible for compaction at the major dense line. myelin associate glycoprotein accounts for about % of both peripheral and central myelin. myelin oligodendrocyte glycoprotein and cyclic nucleotide phosphodiesterase are minor constituents of cns myelin and are not found in the pns. peripheral myelin protein is a minor component of pns myelin. ms is an inflammatory relapsing or progressive disorder of cns white matter and is a major cause of disability in young adults. pathologically, it is characterized by multifocal areas of demyelination, loss of oligodendrocytes, and astrogliosis but with relative preservation of axons. while demyelination is the classic hallmark of ms, axonal and neuronal injury are important aspects of the disease and are gaining more recognition. although certain clinical features are characteristic of ms, investigative studies are often needed to confirm the clinical suspicion and exclude other possibilities. recently, there have been advances in understanding the etiology, mechanisms of myelin injury, and potential for repair, and several partially effective agents are now approved for use in relapsing-remitting and secondary progressive ms. the pathogenesis and pathophysiology of ms remains incompletely understood. several mechanisms may be important to ms plaque formation: autoimmunity, infection, bystander demyelination, and heredity. although convincing proof is lacking, dietary factors and toxin exposure have been hypothesized to contribute as well. these mechanisms are not mutually exclusive, and the true pathophysiology is likely to depend on more than one of them. autoimmunity. during ontogenesis, autoreactive lymphocytes normally undergo clonal depletion, but some escape and are merely suppressed, becoming tolerant to their antigens. low levels of autoreactive t and b cells persist even in normal individuals. autoimmune disorders occur when the tolerance of these cells toward their antigen is broken. the decreased suppressor activity of circulating lymphocytes from patients with ms and other presumed autoimmune diseases may reflect loss of tolerance. one potential mechanism that may break tolerance is molecular mimicry between self and foreign antigens. autoreactive t lymphocytes may become activated on exposure to structurally similar foreign antigens. some evidence suggests that molecular mimicry is relevant in ms. not only do several viral and bacterial peptides share structural similarities with mbp, but it has also been demonstrated that these antigens may activate mbp-specific t-cell clones derived from ms patients. blood-brain barrier leakage alone may break tolerance because it gives cnsreactive lymphocytes easy access to otherwise inaccessible antigens. alternatively, a primary event such as an infection or injury may release cns antigens into the periphery, where they may activate corresponding autoreactive cells. the major support for autoimmunity in the pathogenesis and pathophysiology of ms is by analogy to eae, the major animal model for ms. eae is, however, an artificial situation and there is no spontaneous autoimmune animal model of ms. while eae is the most commonly studied model of ms, several features of human ms can not be adequately captured by this model. , over a hundred different effective treatments have been described for eae; however, almost all of them are ineffective and some are harmful in human ms. a recent editorial discusses the merits and important limitations of eae as a model for ms. in eae, just like in classic human autoimmune diseases such as systemic lupus erythematosus (sle) or rheumatoid arthritis, the main target antigens are known. however, despite the discovery of several "weak" antigens in human ms, no dominant antigens have been identified to date. the only human demyelinating disease with an identified specific antigen is devic's disease (neuromyelitis optica), which appears to be a novel autoimmune chanellopathy with an antigen that is neither neuronal nor myelin related (see later discussion of devic's syndrome under neuromyelitis optica). infection. the role of viral infections in the initiation and maintenance of ms has been debated for some time. several viral infections are known to cause demyelination in animals, including visna virus of goats and sheep, canine distemper virus, and theiler's murine encephalomyelitis virus. viral infections in humans can also cause demyelination (progressive multifocal leukoencephalopathy [jc papillomavirus], subacute sclerosing panencephalitis [measles virus], and human t-cell lymphotropic or leukemia virus type [htlv- ]-associated myelopathy). the epidemiology of ms suggests that environmental factors may promote the disease state, possibly due to one or more viruses. a virus may be involved in the pathogenesis of ms in several ways: . transient or persistent infection outside the cns may activate autoreactive t cells by means of molecular mimicry or by other nonspecific means (as superantigens do). . transient cns infection may initiate a cascade of events that fosters autoimmunity (breach the blood-brain barrier, release cns antigens). . recurrent cns infections may precipitate repeated inflammation and demyelination. . persistent cns viral infection could either incite inflammatory reactions detrimental to oligodendrocytes or directly injure them. beyond speculation and epidemiological observations, there is insufficient evidence for a viral infection playing a causative role in ms. early serological studies are difficult to interpret because of nonspecific immune activation and resulting elevation of titers to many different viruses. many ms patients have elevated cerebrospinal fluid (csf) titers to measles and herpes simplex (hsv) viruses, but this finding appears nonspecific. virus has rarely been cultured from csf of ms patients, but a new strain of hsv (the ms strain) and a new virus (inoue-melnick virus) were first isolated from the csf of ms patients. , newer molecular techniques to search for a viral genome in csf and brain have rejected the claim that htlv- is associated with ms. the finding that human herpesvirus (hhv ), although present in % of brains from both control subjects and ms patients, is localized to the oligodendrocyte nuclei near plaques of ms patients and to oligodendrocyte cytoplasm in control subjects indicates that persistent cns viral infection is common. this raises the possibility that ms may depend on an aberrant host response to this normal condition or that a defective virus that lacks the ability to evade immune detection may be to blame. more recently, measles and canine distemper virus antibodies were found elevated in blood and csf samples of ms patients, although their relationship is not clear to the disease process. in a study from denmark, patients with serological markers for late-stage epstein-barr virus (ebv) infection had a threefold increase in the likelihood of developing ms. a follow-up study from sweden failed to reach this conclusion. in general, serum samples of ms patients may contain higher titers of antibodies to the following infectious organisms: adenovirus, canine distemper virus, hsv, hhv , and influenza, measles, mumps, parainfluenza, rubella, vaccinia, and varicella zoster virus (vzv). similarly, csf samples from ms patients may show higher titers of adenovirus; chlamydia pneumoniae; cytomegalovirus (cmv); ebv; hhv ; coronavirus; influenza viruses a and b; measles or mumps virus; mycoplasma pneumoniae; parainfluenza viruses , , and ; respiratory syncytial virus; rubella virus; vaccinia; and vzv. there has been an interest recently in a potential link between c. pneumoniae infection and the development of ms. no direct cause-and-effect relationship has been observed between any of these infections and ms. "bystander" demyelination. immune actions may mediate myelin injury in a nonspecific manner. many soluble products of the immune response other than immunoglobulins are known or suspected to be toxic to myelin and oligodendrocytes. activated complement is capable of lysing oligodendrocytes in an antibody-independent fashion. the proinflammatory cytokine tumor necrosis factor-a causes myelin disruption and oligodendrocyte apoptosis in vitro. arachidonic acid metabolites may also participate in myelinolysis, and reactive oxygen species released by macrophages cause lipid peroxidation that can damage myelin. other soluble substances that are potentially toxic to myelin include nitric oxide and vasoactive amines. histological subtypes of ms lesion development. through the groundbreaking work of lucchinetti and associates in the ms lesion project, it is postulated that the formation of ms lesions follows one of four patterns. patterns i and ii are related to immune-mediated damage to myelin sheaths. in pattern i, cellular mechanisms of injury seem to prevail (macrophages and t-lymphocytes) whereas in pattern ii humoral mechanisms of injury predominate (e.g., antibody and complement-mediated mechanisms). patterns iii and iv are related to oligondendrocye pathology: in pattern iii, a distal oligdendrogliopathy and apoptosis have been reported, whereas in pattern iv, primary oligodendrogliopathy and degeneration of oligodendrocytes have been described. currently these subtypes can be diagnosed only by biopsy; serum and magnetic resonance imaging (mri) markers are not yet known, although lesional t hypointense rims and response to plasma exchange may correlate well with pattern ii pathology. it is important to note that the patterns do not correlate with clinical subtypes of ms, with the exception of pattern iv, which has been identified only in primary progressive (ppms) patients. evidence to date suggests that the pattern of lesion formation remains the same within an individual patient; patients do not "switch" from one pattern to the other. also, the patterns do not seem to represent different chronological stages of lesion formation. gray matter involvement. it has been known since the late th century that ms affects both gray and white matter structures. the importance of gray matter involvement has received little attention until recently, largely due to the development of advanced mri techniques (see later) that indicate neuronal and axonal involvement even in the earliest stages of this disease. a classification system of gray matter plaques was proposed by peterson and associates they described three patterns of cortical demyelination: type i lesions are contiguous with subcortical white matter lesions; type ii lesions are confined to the cortex, and are often perivascular; type iii lesions extend from the pial surface to cortical layer or . besides cortical gray matter involvement, there is also evidence for prominent basal ganglia involvement, which can be seen in the early stages of ms, and may correlate better with motor outcome and cognitive measures than measures of white matter involvement. lucchinetti and associates demonstrated that biopsy samples from newly diagnosed demyelinating cases contain numerous infiltrating immune cells, and can be destructive. the pathological classification of cortical lesions as described by petersen can also be found in these early ms biopsy samples. approximately % of biopsy cases in which gray matter was also sampled had evidence of clear cortical demyelination. in , an extensive histological study by kutzelnigg and associates investigated the role of cortical demyelination in all clinical subtypes of ms. in this study, brains of ms patients (relapsing-remitting [rr], secondary progressive [sp], and ppms) and control subjects were studied using advanced quantitative morphological techniques. cortical demyelination and diffuse axonal injury in the normal appearing white matter (nawm) were reported as hallmarks of progressive forms of ms. cortical demyelination was mainly seen in the subpial layer of cortex, and was associated with significant inflammatory infiltrates in the surrounding meninges. diffuse inflammation was also found throughout the white matter of the progressive cases, associated with activation of microglia. no significant correlation was shown between focal white matter lesion load and cortical demyelination. this study defines three crucially important pathological hallmarks of ms-focal demyelinated white matter lesions, diffuse injury in the white matter, and cortical plaque formation-and concludes that white matter lesion formation predominates in active forms of ms, while cortical pathology and diffuse white matter injury characterizes the progressive forms. the authors of this landmark paper also established that these three processes are potentially independent of each other. heredity. epidemiological findings support a polygenic hereditary predisposition to ms. a number of candidate genes have been investigated, often with conflicting results. the only definitive genetic association in ms is with the serologically defined human leukocyte antigen (hla) dr , dq . this is one of the dr haplotypes, also known as dw in cellular terminology and drb * , dqa * , dqb * in molecular nomenclature. though its link to ms is well established, the risk conferred by this haplotype is small (relative risk of to ), and it is neither necessary nor sufficient for the development of ms. linkage to this locus has not been proved, indicating that it plays only a minor role in familial susceptibility. other susceptibility genes likely contribute, possibly the t-cell receptor variable b region and the igg heavy-chain variable region (especially the vh - gene). but their specific roles have not been established. other genes under study have been the mbp coding gene, the ctla- gene on chromosome q , and the interleukin- ra associated gene, in concurrence with the hla-dr haplotype. mitochondrial mutations are also under investigation, and an lhon-associated mtdna mutation may be an important cofactor in developing ms in some patients. the apoe gene, as in alzheimer's disease, has been associated with a higher incidence of ms. on the other hand, apoe is considered to have neurotropic, immunomodulatory, and antioxidant properties. these findings are yet to be confirmed by larger studies. twenty percent of ms patients have at least one affected relative. only about % of first-degree relatives of patients develop ms, but this represents a -to -fold increase in risk compared with the general population. unaffected family members sometimes have abnormal findings on cranial mri, implying that this risk is even higher. one study of ms rates in adopted relatives of ms patients verified that the familial distribution is due to genetic factors rather than shared environment. twin studies lend support to both genetic and environmental influences on ms development. genetically identical monozygotic twins are more often concordant for ms than dizygotic twins ( % and . %, respectively), indicating a genetic component; however, even after following monozygotic twins past age or using mri data, less than % are concordant, suggesting a role for environmental factors. epidemiology and risk factors. ms is not a rare disease. it affects millions worldwide and approximately , in the united states alone. symptoms usually begin during young adulthood, with the peak onset at age . approximately . % of ms cases are diagnosed before age . women are affected nearly twice as often as men. ms has a predilection for whites, especially those of northern european heritage. other races and ethnic populations are resistant to a variable extent. ms is virtually unknown among black africans but occurs in african-americans at half the rate of whites, possibly due to racial admixture or environmental factors. ms is rare in tropical areas, and the prevalence increases proportionally to the distance from the equator, excluding polar regions. the prevalence is less than cases per , in tropical areas; in high prevalence areas it can be higher than per , , reaching up to per , in selected areas. although usually interpreted as the effect of environmental factors, the prevalence gradient is at least partially due to racial susceptibility. perhaps the most incriminating evidence for the role of environmental factors in the development of ms is the changing risk with migration and the occurrence of ms clusters and epidemics. immigrant populations tend to acquire the ms risk inherent to their new place of residence. migration from high to low prevalence before the age of lowers the ms risk, whereas migration after this age does not affect risk. migration from low to high prevalence areas increases the risk of ms, but the effect of age is less clear. many clusters of ms have been reported. , the occurrences of ms epidemics in iceland and the faroe islands have been proposed to be the result of exposure to a pathogen brought by british troops during their occupation in world war ii. other environmental factors associated with the development of ms include cigarette smoking (odds ratio of . , ci: . - . ), animal fat intake, and deficiency of vitamin d. , epidemiological data support the view that ms is caused or triggered by an environmental factor in persons who are genetically susceptible. the familial frequency and distribution implies that several genes contribute to susceptibility, and this is consistent with the low relative risk conferred by the genetic loci studied so far. data from clusters, migration studies, and family studies reveal that there is a latent period of some years between exposure to the environmental factor and the development of clinical symptoms and that the age at exposure is around , the putative age at acquisition. the precise environmental events that lead to cns demyelination are uncertain. viral infection is the most plausible, but because of the nonspecific elevation of viral titers and long latent period, there is little direct evidence. minor respiratory infections precede % of relapses in patients with established ms. measles infection was found to have occurred at a later age in ms patients than control subjects, although the incidence of ms has not been reduced by immunization against measles. head injury and trauma have received attention as putative triggering events, but cohort studies have not verified any link. pregnancy does not alter the risk of developing ms, but it does seem to influence disease activity. the annualized relapse rate drops from approximately . to . by the third trimester, but this is offset by an increase to . in the first postpartum months. most studies have found no longterm effects of pregnancy on the prognosis for progression or disability, although one did report a favorable effect. a multitude of other environmental factors have been suspected to alter the risk for ms (cold climate, precipitation, amount of peat in the soil, exposure to dogs, and consumption of meat, processed meat, and dairy products), but none has been verified to be an independent risk factor. ms can cause a wide variety of clinical features. many signs and symptoms are characteristic, and a few are virtually pathognomonic for the disorder. conversely, some symptoms are atypical and some are so rare as to suggest a different diagnosis (table - ). the course of the illness is also variable, but it remains a critical consideration in the diagnosis of ms. sensory symptoms are the most common presenting manifestation in ms ( % to %) and ultimately develop in nearly all patients. loss of sensation (numbness), paresthesias (tingling), dysesthesias (burning), and hyperesthesias are common. these symptoms may occur in practically any distribution: one or more limbs, part of a limb, trunk, face, or combinations. the more distinctive sensory relapses of ms consist of the sensory cord syndrome and the sensory useless hand syndrome. a common scenario is that of numbness or tingling beginning in one foot, ascending first ipsilaterally and then contralaterally. the sensory symptoms may ascend to the trunk, producing a sensory level, or may involve the upper extremities. associated symptoms commonly include poor balance, weakness, urinary urgency, constipation, and lhermitte's sign (see later). brown-séquard syndrome may occur with sensory disassociation and hemiparesis. the sensory cord syndrome reflects an evolving demyelinating lesion that begins in the medial posterior column ipsilateral to the first symptoms. sensory cord syndromes are common in ms and suggest the diagnosis when they occur in young persons and remit spontaneously or in response to corticosteroids. patients with the sensory useless hand may note subjective numbness and lose discriminatory and proprioceptive function, resulting in difficulty writing, typing, buttoning clothes, and holding onto objects, especially when not looking at the hand. this problem can occur bilaterally even without lower extremity symptoms. the responsible lesion is in the lemniscal pathways either in the cervical spinal cord or in the brain stem. this syndrome usually remits over several months. the useless hand syndrome is a very specific symptom and is only rarely caused by other disorders. a large portion of ms patients have persistent sensory loss, usually consisting of diminished vibratory and position sensation in distal extremities (video , sensory ataxia). itching may occur in a dermatomal distribution with relapse or in paroxysms. pain is not a major manifestation of ms, but distressing lower extremity dysesthetic pain associated with spinal cord involvement, radicular pain from lesions at the root entry zone, paroxysms, and an uncomfortable sensation of pressure or tightness surrounding a leg or the trunk may be present. pyramidal tract dysfunction is common in ms and causes weakness, spasticity, loss of dexterity, and hyperreflexia (video , hyper-reflexia). motor deficits can occur acutely or in a chronic progression with weakness of one or more limbs and facial weakness, leg stiffness that impairs gait and balance, or extensor and flexor spasms (video , spastic gait). exercise or heat frequently worsens subtle deficits. muscle atrophy is usually due to disuse, but lesions of lower motor neuron fibers or of the anterior horn itself can cause a pseudoradiculopathy with segmental weakness, atrophy, and diminished reflexes. motor symptoms are presenting manifestations of ms in % to % of all cases; their prevalence is higher than % in long-standing ms. the initial symptom of ms is optic neuritis (on) in % to % of patients, and more than % experience a clinical episode of on during their lifetime. the most common manifestation is visual loss in one eye that evolves over a few days. periocular pain, especially with eye movement, usually accompanies and may precede the visual symptoms. bilateral simultaneous on is uncommon in adults, but formal visual field testing reveals unexpected defects in the clinically normal eye in a substantial number of patients. children and asian patients are more likely to have bilateral simultaneous on; it may also be seen in neuromyelitis optica (nmo) patients. examination shows an afferent pupillary defect, diminished visual acuity, subdued color perception, and often a central scotoma (video , afferent pupillary defect). funduscopic examination is usually normal but occasionally will reveal papillitis (more common in children) or venous sheathing. most patients begin to recover within weeks, and significant visual patients with frequent and severe on events in the first years were more likely to convert to nmo; they also had a higher likelihood for significant persistent vision loss. cerebellar pathways are frequently involved during the course of ms, but a predominately cerebellar syndrome is uncommon at onset. the manifestations include dysmetria, dysdiadochokinesia (video , dysdiadochokinesis), action tremor with terminal accentuation, dysrhythmia, breakdown of complex motor movements, and loss of balance (video , tremor with ataxia). patients with long-standing ms may develop a "jiggling" gait and an ataxic dysarthria with imprecise articulation, scanning speech, or varying inflection, giving it an explosive character. urinary urgency, frequency, and urge incontinence (due to detrusor hyper-reflexia or detrusor-sphincter dyssynergia) result from spinal cord lesions and are frequently encountered in ms patients. the combined incidence of bowel and bladder dysfunction in ms is thought to be higher than %. symptoms of bladder dysfunction may be transient and occur with an exacerbation but are commonly persistent. impaired vesicular sensation causes a high capacity bladder and may lead to bladder atonia with thinning and disruption of the detrusor muscle. incontinence results in constant dribbling of urine in this irreversible condition. interruption of brain stem micturition center input sometimes leads to cocontracture of the urinary sphincter and detrusor muscles (detrusor-sphincter dyssynergia). the resulting high pressure may lead to hydronephrosis and chronic renal failure if untreated. constipation is a common problem, occurring in % to % of ms patients, especially with limited activity and spinal cord involvement. fecal incontinence is a socially devastating symptom that is often associated with perineal sensory loss in ms patients. sexual dysfunction is seldom mentioned, even though it is a frequent problem in ms. nearly two thirds of patients report diminished libido. one third of men have some degree of erectile dysfunction, and a similar percentage of women have deficient vaginal lubrication. besides direct neurological impairment, sensory loss, physical limitations, depression, and fatigue additionally contribute to sexual difficulties in ms patients. in addition, the partner's attitude and psychological factors dealing with self-image, self-esteem, and fear of rejection may also lead to impotence or loss of libido. intense vertigo associated with nausea and emesis is an occasional manifestation of ms relapse. in the absence of a clear diagnosis of ms, these symptoms are often attributed to vestibular neuronitis. patients may also develop a persistent but mild vertigo that is precipitated by movement, or this may be a residual finding after an acute relapse. internuclear ophthalmoplegia, caused by a lesion in the medial longitudinal fasciculus, is the most common cause of diplopia in ms patients (video , afferent pupillary defect). when symptomatic, it produces horizontal diplopia on lateral gaze that usually remits. examination discloses incomplete or slow adduction of the eye ipsilateral to the lesion and nystagmus of the contralateral eye during abduction (see chapter ) . dissociated nystagmus may be the only finding of an old or subtle internuclear ophthalmoplegia (video , internuclear ophthalmoplegia). bilateral internuclear ophthalmoplegia is strongly suggestive of ms, although this rarely may occur with tumor, infarct, mitochondrial cytopathy, wernicke's encephalitis, and chiari malformation (video , wernicke's encephalopathy). vertical and diagonal diplopia usually results from skew deviation. nystagmus, slow saccadic movements, broken ocular pursuits, and ocular dysmetria are other eye findings produced by lesions of cerebellar and vestibular pathways (see chapters and ; video , saccadic dysmetria). abducens paresis occurs on occasion, but oculomotor and trochlear nerve impairment is rare. corticospinal, spinothalamic, lemniscal, vestibular, and cerebellar pathways can all be affected. cranial nerve impairment may be seen with lesions that affect brain stem nuclei or exiting and entering fibers. usually this occurs in association with other symptoms. because of the long spinal tract and nucleus, the trigeminal nerve is frequently involved (video , trigeminal neuralgia). facial nerve paresis does occasionally occur, but ms is an extremely rare cause of bell's palsy in patients without previous symptoms. acute unilateral hearing loss is an uncommon manifestation. dysphagia is often due to impairment of cranial nerves ix, x, and xii and generally appears late in the course of some patients. once thought uncommon, cognitive disorders are now known to be present in % to % of ms patients. age, duration of ms, and physical disability do not completely predict the presence of cognitive dysfunction, but classic mri measures like the total t -weighted lesion load does not seem to correlate well with the degree of cognitive decline. measures of cortical atrophy, venticular enlargement, and neuronal integrity seem to correlate better with the cognitive aspects of ms. the problems are often subtle and may not be detected on standard mental status evaluation. the pattern of cognitive decline is typified by decrease of episodic memory, processing speed, verbal fluency, and difficulty with abstract concepts and complex reasoning. to a lesser extent, executive functioning and visual perception, semantic memory, and attention span may also be also decreased. general intelligence is not typically affected. as expected, cortical symptoms such as aphasia, apraxia, and agnosia are unusual. homonymous hemianopia, which can be caused by cortical or subcortical lesions, is also uncommon. despite prominent cerebral white matter involvement, many of the disconnection syndromes such as alexia without agraphia, conduction aphasia, and pure word deafness have not been reported in ms patients. affective disorders are more frequent in ms patients than in the general population. these include both anxiety and depression. in long-term studies, the incidence of depression in ms patients is close to %. neither depression nor anxiety is related to physical or cognitive disability or mri lesion load. patients sometimes experience uncontrollable weeping or less commonly laughter incongruent with their mood. interruption of inhibitory corticobulbar fibers is responsible for these symptoms (pseudobulbar affect). fatigue is a pervasive symptom among ms patients that is not related to disability or depression. over % of ms patients experience fatigue during their disease course. a diurnal pattern is characteristic and follows the normal circadian pattern of body temperature fluctuations, with the worse symptoms occurring in afternoon hours (peak core body temperature) often giving way to improvement in the late evening. ms symptoms may fluctuate in a predictable fashion. transient worsening of symptoms frequently follows exercise or elevation of body temperature. one example is uhtoff's phenomenon, in which visual blurring occurs during strenuous activity or with passive exposure to heat. these episodes resolve when the body temperature cools to normal or after a period of rest. an intercurrent infection with fever can induce worsening of symptoms and may be confused with a relapse. heat sensitivity is presumably related to conduction block, as demyelinated axons are more prone to failed conduction than normal, myelinated fibers. paroxysmal symptoms are characteristic of ms and are believed to be due to the lateral spread of excitation (ephaptic transmission) between denuded axons in areas of demyelination. symptoms are typically brief (seconds to minutes) and recur frequently, occasionally dozens of times per day. they may be precipitated by hyperventilation, certain sensory input, or particular postures. tonic spasms (paroxysmal dystonia) most often affect the arm and leg on one side, but the face, one limb, or bilateral limbs are sometimes involved (video , tonic spasms). these spasms may result from lesions anywhere along the corticospinal tract. they often begin during the recovery phase after an acute relapse and remit after a few months. intense pain and ipsilateral or crossed sensory symptoms may accompany them. paroxysmal weakness occurs, but it is uncommon. a wide variety of paroxysmal sensory symptoms may occur with ms, including tingling, prickling, burning, or itching, and sharp neuralgic pain is common. trigeminal neuralgia may appear in patients with ms (video , trigeminal neuralgia). the occurrence of trigeminal neuralgia in a person younger than age is suggestive of ms. lhermitte's sign (transient sensory symptoms usually precipitated by neck flexion) is usually described as an electrical or tingling sensation that travels down the spine or into the extremities. although quite common in ms, lhermitte's sign can also occur with a wide variety of other disorders, such as vitamin b deficiency, spondylosis, chiari malformation, and tumors, and after cisplatin chemotherapy. several other paroxysmal symptoms are occasionally encountered, including paroxysmal dysarthria and ataxia, paroxysmal diplopia, and combinations of these symptoms. facial myokymia and hemifacial spasm are additional transient (lasting months) phenomena sometimes due to brain stem demyelination (video , facial myokymia; video , hemifacial spasm). trismus, kinesigenic dystonia, paroxysmal kinesigenic choreoathetosis, and segmental myoclonus have also been described in case reports of ms patients as rare and unusual examination findings. seizures occur in a larger proportion of ms cases compared to normal control subjects. a recently published review of case series of ms patients with epileptic seizures yielded a prevalence of . %. this represents an approximately three-to sixfold increase compared to the general adult population. cortical and juxtacortical lesions may be responsible for the increased incidence of seizures in ms patients. however, such plaques are common and seizures in ms are not, which suggests that other factors may also contribute to the relationship between epilepsy and ms. focal motor seizures, possibly with secondary generalization, are the most frequent. the occurrence of seizures usually follows one of two patterns. on occasion, focal onset seizures begin early in the course of ms and later remit. the start of seizures late in the course of ms more often poses a chronic problem and may be difficult to control. the eye is the only organ outside the nervous system that is sometimes involved in ms. uveitis and retinal periphlebitis each occur in at least % of ms patients. in a recent study, most patients with ms-associated uveitis were white females between and years of age. the diagnosis of ms preceded the onset of uveitis in %, followed it in %. in over % of the cases, the uveitis was bilateral. pars planitis was found to be the most frequent form of uveitis (over %), and concomitant anterior chamber inflammation was also common. usually ms-associated uveitis is benign from the standpoint of visual acuity. uveitis can involve the posterior, intermediate (pars planitis), or rarely anterior portion and resembles that seen in other inflammatory (e.g., sarcoid, reiter's syndrome, behçet's syndrome, inflammatory bowel disease, systemic lupus erythematosus) and infectious (e.g., syphilis, tuberculosis, lyme disease) conditions. periphlebitis is seen as venous sheathing on funduscopic examination and is histologically identical to the perivascular inflammation present in brain white matter. it is interesting that inflammation commonly occurs in the retina, which has a peripheral type of myelin produced by schwann cells. there are occasional reports of peripheral nerve or nerve root demyelination in ms patients as well as central demyelination in acute inflammatory demyelinating polyradiculoneuropathy and chronic inflammatory demyelinating polyradiculoneuropathy (see chapter ) . some of these cases may be due to the incidental occurrence of two unrelated disease processes. however, because the pns and cns share many antigens, including mbp, it is possible that an autoimmune reaction or a viral infection could involve both the cns and pns. persons with one autoimmune disorder generally have an increased risk of others. even though there are several reports of systemic and organ-specific autoimmune diseases in ms patients, population-based studies have not confirmed any increase in prevalence of these disorders among ms patients. in fact, there appears to be a negative association between ms and rheumatoid arthritis. multifocal cns involvement and acute relapses, remissions, and slow progression of neurological deficits typify ms. a single episode of neurological dysfunction can be suggestive of ms if it follows the typical time course of a relapse: progression over less than weeks (usually days), with or without a period of stabilization, and improvement or resolution (often over months). insidious progression of deficits localized to a single site in the cns can also be due to ms, but other causes must be excluded. the temporal course of ms can be described by one of four categories: relapsing-remitting (rr), secondary progressive (sp), primary progressive (pp), and progressive relapsing (pr). many physicians use the term relapsing progressive, which encompassed patients with spms, prms, and even those with rrms who have stepwise relapse-related worsening disability. this term has recently been abandoned. other terms that relate to the course of ms but have no consensus regarding their definition are sometimes encountered. benign ms generally refers to patients who have had ms for a long time but have little or no disability. malignant ms is sometimes used to describe patients with frequent relapses and incomplete recovery but is also used in reference to patients with acute fulminant demyelinating syndromes (see later). the term clinically isolated syndrome (cis) refers to patients presenting with their first episode of region-restricted episodes of cns inflammatory demyelination. this may remain an isolated syndrome (no recurrence), it may remain a forme fruste of acute disseminated encephalomyelitis (adem), or it may be the harbinger for one of the relapsing forms of ms. the probability of recurrent demyelinating episodes (e.g., clinically definite ms) has been the subject of several important investigations, and several clinical features and test results are of predictive value. optic nerve, spinal cord, and brain stem are the most common sites of these recurrent monosymptomatic events, and the time profile follows that of ms relapses. the pathogenesis, pathophysiology, epidemiology, clinical features, associated disorders, differential diagnosis, evaluation, and management are the same as in ms. the prognosis for visual recovery after each episode of on is good, and most patients regain normal visual acuity. profound visual loss, recurrent on, and age older than are associated with a higher risk for poor recovery. investigators have concluded that recurrent multifocal demyelinating episodes, fulfilling the diagnostic requirements of clinically definite ms, develop in % or more of patients after isolated on when follow-up is extended beyond years. most of this risk is incurred within the first few years, although significant risk may continue into the fourth decade after the event. children much more often develop simultaneous bilateral on and have a lower risk for subsequent ms than adults. factors that are associated with an increased risk of developing ms as a disseminated illness are the presence of venous sheathing, recurrent on, family history of ms, white race, previous vague or nonspecific neurological symptoms, and the presence of oligoclonal bands (ocbs), elevated igg index, or igg synthesis rate in csf. the severity of acute transverse myelitis is inversely related to the risk of acquiring further symptomatic demyelinating lesions. complete transverse myelitis with profound loss of motor, sensory, and sphincter function imparts a relatively low risk of to for the later diagnosis of ms. partial transverse myelitis with preservation of significant motor function at peak is associated with a much higher incidence of ms. although monosymptomatic brain stem demyelination is not as common as either on or acute transverse myelitis, similar conclusions have been reached. in the only study available, two thirds of these patients with cerebral white matter lesions detected on mri developed ms within years, compared with none of patients with normal head mri. , a recently published -year follow-up of the original queen square series continues to demonstrate the value of the baseline cranial mri study in determining risk of recurrence (ms risk). in this cohort study of cis patients, approximately two thirds had at least one asymptomatic lesion ( of , %) at baseline. after years of follow-up, slightly more than half with one to three asymptomatic baseline cerebral lesions had developed ms ( of ) compared with the majority of cases presenting with at least four baseline lesions ( of , %). after years of follow-up, the majority of patients with any asymptomatic cerebral lesions had developed definite ms ( of , %). the recently published -year follow-up data on this group of patients reveals that % of the initially mri positive patients developed ms versus % of the mri negative subgroup. this information is helpful for treating patients in the setting of cis. differential diagnosis. only a few diseases cause neurological deficits that regress spontaneously and relapse in different areas of the cns over the course of many years. however, because of the remarkable heterogeneity of ms, many disorders may resemble ms (table - ) , especially in the first years of active disease. other primary idiopathic inflammatory demyelinating cns disorders may be mistaken for ms. adem usually causes monophasic cns demyelination. although it frequently involves multifocal areas of white matter simultaneously, adem cannot be reliably differentiated from the initial clinical episode of ms. fulminant brain demyelination in persons without previous symptoms of ms is more likely due to adem or other conditions (schilder's myelinoclastic diffuse sclerosis, balo's concentric sclerosis, marburg's variant of ms). neuromyelitis optica differs from ms primarily in the topography and intensity of the lesions. several systemic or organ-specific inflammatory conditions can involve the cns white matter. on, myelitis, and other syndromes sometimes occur with systemic lupus erythematosus. whether this autoimmune disease increases the risk of developing ms or causes similar syndromes by a different pathological process is unknown. sarcoidosis can affect the nervous system in several ways, including multifocal, corticosteroid-responsive white matter lesions. sjögren's syndrome sometimes occurs with ms, but this may only represent a chance association. neuro-behçet's disease has a predilection for the brain stem. occasionally, isolated demyelinating syndromes are associated with inflammatory bowel disease. a wide variety of vasculitic syndromes (e.g., primary angiitis of the cns, periarteritis nodosa, wegener's granulomatous angiitis, vasculitis associated with rheumatoid arthritis, susac's syndrome, eales'disease) may mimic ms. however, these syndromes can usually be distinguished by involvement of the cortex, seizures, early dementia, personality changes, psychosis, infarcts involving large vessel territories on mri, and lack of improvement. findings characteristic to the particular vasculitis (uveitis and vitreal hemorrhage in eales' disease, retinal and cochlear involvement in susac's syndrome, upper and lower respiratory tract involvement in wegener's granulomatosis) also aid in the correct diagnosis. a few infections must also be considered in the differential diagnosis of ms. both lyme disease and syphilis may cause multifocal white matter lesions. htlv- causes a chronic progressive myelopathy (htlv- -associated myelopathy/tropical spastic paraparesis). acute or recurrent myelitis can be caused by vzv. progressive multifocal leukoencephalopathy and toxoplasma abscesses should be considered in immunocompromised patients with progressive neurological decline. bacterial endocarditis with brain abscess formation, subacute sclerosing panencephalitis, or chronic rubella encephalomyelitis may need to be considered in the appropriate circumstances. cerebrovascular disease is only rarely mistaken for ms. occasionally, an ms relapse has an abrupt onset that may mimic an infarct, especially in those not previously diagnosed with ms. the usual circumstance is that of a hemisensory or hemimotor deficit imitating a lacunar infarct. disorders with multiple cerebral infarcts (emboli, hypercoagulable states, sneddon's syndrome, cadasil, vasculitis) may produce an mri appearance and course resembling ms. vascular malformations may also produce symptoms similar to ms. additional neurological illnesses capable of producing multifocal lesions rarely mimic ms. metastatic tumors and multifocal gliomas are often cited examples, but rarely is this distinction difficult for an experienced clinician. lymphoma more commonly masquerades as ms because the lesions may involve the white matter, may be multifocal, and are corticosteroid responsive. in addition, demyelination sometimes presents as one (or a few) mass lesion(s). in this situation, biopsy may be needed for diagnosis. neoplasms can cause paraneoplastic syndromes that may be confused with ms. a high index of suspicion must be kept for older age at presentation, subacute ataxia, early dementia, and personality changes. a few metabolic disorders may resemble ms, such as vitamin b deficiency, vitamin e deficiency (seen in bassen-kornzweig syndrome, hypobetalipoproteinemia, and refsum's disease), and central pontine or extrapontine myelinolysis (video , pontine myelinolysis). leukodystrophies are usually not difficult to distinguish from ms. krabbe's disease (galactocerebroside-b-galactosidase deficiency), metachromatic leukodystrophy (mld; arylsulfatase a deficiency), and the usual adult form of adrenoleukodystrophy (ald) and adrenomyeloneuropathy (amn) exhibit both central and peripheral dysmyelination. blood leukocyte or fibroblast culture enzyme activity levels will confirm the diagnosis of krabbe's disease and mld, and elevated levels of very long chain fatty acids occur in ald/amn. mitochondrial disorders should also be given consideration because symptoms and mri appearance may be similar to ms. a relapsing remitting disorder identical to ms is sometimes seen in patients with the mutations responsible for leber's hereditary optic neuritis (lhon). this usually occurs in female patients, and there may not be a family history of visual loss. a number of rare biochemically defined illnesses and other genetic disorders may occasionally merit consideration (including cobalamin and folate dysmetabolism, adult polyglucosan body disease, hereditary spastic paraparesis, spinocerebellar degeneration, and hereditary cerebroretinal vasculopathy). several additional disorders must be excluded before diagnosing primary progressive ms (ppms). spinal cord compression from spondylosis or tumor may produce chronic progressive myelopathy. chiari malformations, syringomyelia, syringobulbia, other foramen magnum lesions, spinal arteriovenous malformations, and dural fistulas may also need consideration. careful imaging readily identifies these structural abnormalities. degenerative diseases such as olivopontocerebellar atrophy may mimic ppms. mri and csf examination will help distinguish between the two. conversion reactions and somatization disorders are commonly encountered in a busy referral practice and must be accurately diagnosed to afford optimal patient management. evaluation. the diagnosis of ms is based on the demonstration of white matter lesions disseminated in time and space in the absence of another identifiable explanation. ms remains a clinical diagnosis, although mri, evoked potentials, and csf examination can help clarify less certain cases. for research purposes, various categories of ms have been defined based on the certainty of the diagnosis. at least two attacks and evidence of two separate cns lesions (clinical or paraclinical) are required for the designation of clinically definite ms (cdms). two attacks and evidence of one cns lesion or one attack and evidence of two cns lesions (clinical or paraclinical) is considered clinically probable ms. cases that fulfill the criteria for clinically probable ms and have supportive csf findings are labeled as laboratory-supported definite ms. patients with a clear history of at least two attacks and supportive csf but a normal neurological examination and no paraclinical evidence of cns lesions are categorized as having laboratory-supported probable ms. suspected cases that do not fit any of these criteria may be regarded as possible ms. paraclinical evidence generally refers to abnormalities on evoked potential studies or imaging procedures. as a result of increasing availability of refined paraclinical diagnostic modalities (especially mri) and an overall better understanding of the disease process, new diagnostic criteria for ms were proposed by an international expert panel in . three out of four of the following findings should be present on mri: ( ) one gadolinium enhancing lesion, or nine t hyperintense lesions; ( ) at least one infratentorial lesion; ( ) at least one juxtacortical lesion; and ( ) at least three periventricular lesions. according to the clinical diagnostic criteria, if a patient had two or more attacks with objective evidence on examination of two or more anatomical areas involved, no additional data is required to make the definite diagnosis. however, if such diagnostic studies were done and are not supportive of a diagnosis of ms, then the diagnosis should be reconsidered. if a patient presents with a history of two or more attacks, but objective clinical evidence only suggests one lesion, the following additional data is needed to confirm the diagnosis: the disease process has to be disseminated in space as demonstrated by mri; alternatively, two or more mri-detected lesions consistent with ms plus positive csf would suffice to meet the newly defined criteria. the clinician also may elect to await a further attack implicating a different anatomical site. in case a patient had one attack, with objective clinical evidence of two or more lesions, dissemination in time as demonstrated by serial mris separated by at least months or a second clinical attack would clarify the diagnosis. if a patient has a clinically isolated syndrome, or "monosymptomatic" presentation, the following criteria should be met: dissemination in space as demonstrated by mri (again separated by at least months), or two or more mridetected lesions consistent with ms plus positive csf and dissemination in time on serial mri scans, or a second clinical attack. in case the patient presents with a progressive course, the presence of positive csf is required, and dissemination in space should be present, as suggested by nine or more t -weighted brain lesions, or two or more cord lesions, or four to eight brain lesions plus one cord lesion on mri. alternatively, abnormal visual evoked potentials (veps) with four to eight brain lesions, or fewer than four brain lesions plus one cord lesion, and dissemination in time on serial mri scans, or continued progression for a year would meet the diagnostic criteria. "positive csf" according to this set of criteria is defined by either the presence of oligoclonal bands detected by established methods (preferably isoelectric focusing on agarose gel followed by immunoblotting) different from any such bands in serum, or by a raised igg index. the presence of both enhancing and nonenhancing white matter lesions on a single mr image must not be used as evidence of dissemination in time as well as space, because these can also be seen in adem. oligoclonal bands (ocbs) and an elevated igg index provide supportive csf findings. ancillary tests are frequently required to confirm the diagnosis of ms and to exclude other possibilities in uncertain cases. laboratory tests on peripheral blood can help to exclude many of the infectious and other inflammatory disorders. a chest x-ray is generally needed to assess for sarcoid or paraneoplastic disorders if these are under consideration. an ophthalmological examination may be needed to search for alternative causes of visual loss. imaging studies, csf examination, and evoked potentials are often helpful because characteristic abnormalities are frequently present. mri of the head is the most sensitive imaging study for ms ( fig. - ). focal areas of increased t -weighted and decreased t -weighted signal reflect the increased water content associated with demyelinated plaques. the mri appearance of ms lesions, however, is not specific and similar abnormalities may be seen in normal aging, small penetrating vessel infarcts, lyme disease, tropical spastic paraparesis/htlv- -associated myelopathy, sarcoid, systemic lupus erythematosus, sjögren's syndrome, mitochondrial cytopathies, vasculitis, and adem. the specificity for ms can be increased by consideration of lesion number, size, location, and shape. this is especially important in persons older than age . mri characteristics, other than the ones suggested by the international criteria outlined previously, are size larger than mm, oval shape (often with the long axis directed perpendicular to lateral ventricles), and locations in the periventricular area, corpus callosum, and posterior fossa. longitudinal mri studies have shown the evolution of ms lesions. gadolinium enhancement, indicating blood-brain barrier disruption, sometimes precedes the development of t -weighted lesions and typically lasts for weeks in the brain (occasionally longer, especially in larger hemispheric lesions), and perhaps somewhat longer in the spinal cord. flair imaging is especially helpful for evaluating periventricular lesions that may go unnoticed on regular t -weighted scans. the disadvantage of the technique is its relative insensitivity to posterior fossa lesions. proton density weighted images are also part of the usual sets of images used in the mr diagnostics of ms. these images can be evaluated similarly to t -weighted images. technically, they are usually acquired together with the t -weighted datasets, as a first echo in conventional fast spin echo sequences, where the subsequent echoes can be used for generating the t -weighted images. new t weighted lesions have a fuzzy border and enlarge over a few weeks. after a period of stabilization, the t -weighted lesion regresses and becomes more sharply delineated from the surrounding white matter as edema resolves. most of the time, a residual abnormality with increased t weighting and decreased t -weighted signal remains, reflecting demyelination and gliosis. the low attenuation t signal, or "t black hole," is more often seen in secondary progressive ms and is thought to represent actual tissue loss. in several well-documented cases, hypointense lesions on t -weighted scans were described in subcortical gray matter structures in ms patients. on a molecular level these areas are thought to represent iron deposition; their significance in ms is not fully understood. the mri activity of disease, defined as either the number of new, recurrent, and enlarging lesions or the number of gadolinium-enhancing lesions, is usually higher than the clinical activity. this may be either because of the involvement of asymptomatic areas of the cns or because of a pathophysiological difference between symptomatic and nonsymptomatic lesions based on the presence or absence of axonal dysfunction. there is only poor correlation between disability and lesion load (volume of white matter abnormalities) determined by head mri. sometimes individuals have severe impairment and few mri abnormalities, and the converse may occur. this disparity is partially explained by variable spinal cord involvement, but a pathophysiological difference may account for some of the discrepancy. several mri markers of gray matter involvement correlate better than measures of white matter pathology with clinical functional outcome measures in ms. in a recent study edss showed the strongest correlation with gray matter volume loss and with t black hole volume increase (p < . ); both are considered to reflect neuronal and axonal pathology. ambulatory function, assessed as the -feet timed walk, also correlated well with gray matter volume loss and t black hole volume. on normal appearing gray matter magnetization transfer ratio (mtr) histograms, normalized peak heights inversely correlated with edss in rrms patients (r ¼ À . , p ¼. ). in a study evaluating a number of mri parameters (including brain t -hypointense and flair-hyperintense lesion volume, third ventricle width, brain parenchymal fraction and t hypointensities in the dentate nucleus), the best correlation with edss (and the only correlating parameter with -feet timed walk) was t hypointensity in the dentate nucleus. in ms patients, an mri study concluded that gray matter atrophy correlated with clinical status (edss, -feet timed walking and disease duration). a study of patients with ppms and rrms showed that neocortical volume as determined by mri correlated with edss scores across all the patients, but the strength of the correlation was stronger (p < . ) in the ppms (r ¼ À . , p < . ) than in the rrms group (r ¼ À . , p ¼ . ). mr spectroscopy is increasingly becoming an accepted diagnostic modality, where information can be obtained about the biochemical constituents of selected voxels of interest. with this technique, a cubic volume of interest is defined based on a regular mr image set. simultaneous acquisition of multiple volume units is possible. with long echo time (te) studies, naa (n-acetylaspartate), choline, creatinine, and lactate peaks can be identified on the mr spectrum. with short te studies, myoinositol, lipids, and some neurotransmitters may be identified. the resolution of the mr spectrum (the "number of lines" in the spectrum) is proportionate to the magnetic field strength used. naa is the second most abundant amino acid constituent in the brain after glutamate. it is localized almost exclusively in neurons and axons. creatinine is used as the "constant" peak in a mr spectrum, since it is the least likely to be altered by cns-specific processes. therefore, numeric mrs data are usually presented as ratios related to creatinine. the naa/creatinine ratio is decreased in areas of axonal or neuronal loss. it correlates well with disability. it can be decreased in normal appearing white matter, also in early stages of lesion formation, thus representing a challenge to the usual dogma of axonal loss being secondary to myelin damage. the decrease of the naa/creatinine ratio may return to normal following the resolution of the acute phase. this process may be related either to reversibility of neuronal injury or to disappearance of edema in the involved areas. in general, more reduced naa peaks are seen in progressive forms of ms with more profound tissue loss. if a relatively large hemispheric lesion shows decreased naa content, similar findings may be seen in the other hemisphere in a "mirror" location. the lactate peak can be elevated in a variety of acute processes, and as such, carries relatively low specificity. the short te spectrum is used less frequently; the "mobile lipid" peak (which is thought to represent macromolecular protein fragments) is increased in areas of acute demyelination. another newer mri technique used in ms research is magnetization transfer imaging. the principle behind this imaging modality is relatively simple. in complex macromolecular systems, there is a baseline magnetization exchange in equilibrium between macromolecular protons and mobile protons. if the macromolecular protons are saturated before each excitation (and subsequent data acquisition) with a prolonged off-resonance broadband pulse, then the signal intensity of the image will be reduced owing to magnetization transfer exchange between the saturated ("bound") and free ("mobile") protons. by obtaining duplicate sets of images (with and without magnetization transfer pulse), a magnetization transfer ratio can be calculated. the ratio reflects the integrity of the macromolecular environment. it is reduced by approximately % to % in areas of edema, but it is more significantly reduced in areas of demyelination or axonal loss. if the ratio "normalizes" in a lesion, no subsequent tissue loss is usually seen on other imaging modalities. despite these advantages, the magnetization transfer imaging is technically difficult because it produces variable findings depending on the technical environment and is not universally available. it has not become an accepted and standard technique for evaluation of ms patients. it may be very useful as a marker for remyelination and tissue repair in future neuroprotective or tissue restorative trials. diffusion-weighted imaging is well known from its widespread use in the diagnosis of ischemic stroke. this technique can show early stages of ms plaque formation. the increase in apparent diffusion coefficient correlates with acute plaques, and seems to best correspond with t -enhancing lesions; this technique may show the lesions at an even earlier stage. mri has become an important component of clinical trials in ms. because of the high sensitivity of mri for disease activity, it is reasoned that periodic mri may determine treatment efficacy more quickly than monitoring relapse rate or disability level. many studies have used mri as a secondary outcome, but clinical outcomes are still used as the primary outcome for definitive trials. additional mri techniques have also proved useful in the diagnosis of ms. mri of the spinal cord shows discrete lesions in about % of cdms patients. several semiautomatic methods exist to determine lesion volume, ventricle volume, or hemispheric volume. these are generally applied for research purposes only, and are not part of the usual workup or diagnostic follow-up of ms. csf evaluation remains a valuable diagnostic tool for ms. a lymphocytic pleocytosis occurs during acute exacerbations in about one third of patients, but this seldom exceeds cells. eighty percent of the lymphocytes are cd positive. the ratio of cd to cd cells is : . less than % of the cells are b cells. csf protein is normal in up to %; levels above mg/dl are unusual and may suggest a different disorder. the proportion of g globulin is high owing to the synthesis of immunoglobulins within the blood-brain barrier. the majority of csf immunoglobulin is igg, although igm and iga may also be elevated. measures of intrathecal igg production have been devised that are more useful than simple g-globulin levels. the igg index and synthesis rate are elevated in % to % of cdms patients and occasionally in other disorders. agarose gel electrophoresis, or the more sensitive isoelectric focusing of csf proteins, often reveals discrete bands of immunoglobulin, each a monoclonal antibody. it is pertinent to compare serum and csf banding patterns because peripheral monoclonal gammopathies may produce csf bands. to reduce false-positive results, only unique csf ocbs should be reported. between % and % of clinically definite ms patients have ocbs; however, early in the course they are not as prevalent. once present, ocbs persist and the pattern does not vary, although new bands occasionally appear. unlike subacute sclerosing panencephalitis, in which the majority of ocbs are antibodies specific for measles virus, the antigenic specificity of ocbs in ms is unknown; they are unlikely to be pathogen specific or autoantigen directed; there is some evidence that they may be genetically determined germline antibodies. five percent to % of noninflammatory cns samples and % of inflammatory samples are also positive for ocbs. more detailed recommendations about the inclusion of csf parameters to the diagnosis of ms were recently published suggesting that the cell count and differential should be completed within hours. the new and recommended method for the detection of ocbs includes immunoelectophorsesis on agarose gel followed by immunoblotting. the reported sensitivity of this technique is above %, with a specificity of % to %. in other inflammatory or infectious illnesses, ocbs are often transient features. their persistence is more suggestive of ms. the presence of myelin components, antimyelin antibodies, and kappa light chains in csf has also been used in the diagnosis of ms. however, the sensitivity and specificity of these products is less than that of ocbs. in late , a new set of recommendations were published based on the first years of using mcdonald's criteria in diagnosing ms. the original mcdonald criteria have been incorrectly interpreted by some as mainly relying on mri for making a diagnosis of ms. in reality, the mcdonald criteria cannot even be applied without careful clinical evaluation of the patient. neurological deficits must be evident to the examiner, and must be suggestive of ms. scans that "look like" ms (and meet the criteria of barkhof and tintore) but have never been accompanied by an obvious and documented neurological examination finding do not fulfill the mcdonald criteria. there was some sympathy among the international panel members revising the mcdonald criteria to allow selected symptoms that are clearly and specifically enunciated by the patient (e.g., lhermitte's symptom, trigeminal neuralgia, numbness ascending to the waist or higher) coupled with objective paraclinical (such as imaging and csf) findings to be sufficient as an indicator of a prior or current attack needed for an ms diagnosis. however, the panel was reluctant to endorse the diagnosis of ms in the absence of any objective clinical findings, even if objective paraclinical findings are in place, at least until such a scheme is tested in prospective settings. patients with imaging and csf findings suggestive of ms but not showing any objective evidence for neurological deficits commonly seen in ms require careful clinical and radiological monitoring. until objective evidence for neurological deficits are found, ms can not be diagnosed. ms may be the correct diagnosis with less stringent imaging criteria than originally proposed; however, the panel was uncomfortable making changes that would allow mri confirmation of dissemination in space based on lower stringency imaging criteria without appropriate prospective data. most studies performed to date have been inadequately designed to address this issue. advanced imaging technologies are constantly evolving and will likely one day be shown to aid in making the diagnosis of ms. visualization of intracortical lesions, use of higher field strength magnets, and analysis of "normal appearing brain tissue," may be conrnerstones of a future mri criteria for ms. preliminary evidence suggests that "occult" damage in normalappearing white and gray matter seen with magnetization transfer, diffusion tensor imaging, or spectroscopy is an early feature of ms, whereas it likely does not occur in other demyelinating conditions such as acute disseminated encephalomyelitis and nmo. important changes have been made to the original definition of "dissemination in time" by mri. in keeping with the definition that clinical relapses must be separated by at least month, it was agreed that new t lesions on mri should occur at least days after disease onset. this means that any new t lesion occurring at any time point after a so-called reference scan performed at least days after the onset of the initial clinical event is useful in meeting imaging diagnostic criteria for dissemination in time. it should be noted though that a new t lesion must be of sufficient size and location to exclude lesions that could have been missed previously for technical reasons of slice orientation, thickness or spacing, tissue contrast, patient motion, or other artifacts. this requires standardized scanning procedures with emphasis on careful repositioning, as well as input from qualified evaluators experienced in ms imaging. with the new revision, there are two ways to show dissemination in time using imaging: ( ) detection of gadolinium enhancement at least months after the onset of initial clinical event, if not at the site corresponding to the initial event; or ( ) detection of a new t lesion if this appears at any time compared with a reference scan done at least days after onset of the initial clinical event. spinal cord lesions can be important in differentiating ms from other white matter diseases; however, the original mcdonald criteria did not provide sufficient guidelines for the use of cord imaging in ms. spinal cord imaging that detects typical ms cord lesions (minimal or no swelling of the cord; clearly hyperintense on t -weighted imaging; at least mm in size, but less than two vertebral segments long; and occupying only part of the cord cross section) is particularly helpful if brain imaging does not detect dissemination in space in a patient suspected to have ms. for dissemination in space, a spinal cord lesion is equivalent to, and can substitute for, a brain infratentorial lesion, but not for a periventricular or juxtacortical lesion. an enhancing spinal cord lesion is equivalent to an enhancing brain lesion, and an enhancing spinal cord lesion can "count" doubly in fulfilling the criteria (e.g., a single enhancing spinal cord lesion can "count" for an enhancing lesion and an infratentorial lesion). individual cord lesions can contribute together with individual brain lesions to reach the required nine t lesions to satisfy the barkhof-tintore criteria (the mri criteria incorporated in the original mcdonald's criteria). the panel recognized that diffuse cord changes may occur in ms, especially in the progressive forms; however, these changes are not sufficiently reliable to allow for their incorporation into the diagnostic criteria at this time. repeat spinal cord imaging in patients without new symptoms of myelitis has a low yield in efforts to demonstrate dissemination of lesions in time. in other words, while it is common to see asymptomatic new brain lesions on repeated scans, new cord lesions generally do result in new neurological symptoms. therefore, repeat cord imaging is recommended only to support an ms diagnosis when there is a clinical reason to suspect a new cord lesion. important changes have been proposed in diagnosing primary progressive ms. these revised criteria stress clinical and imaging (brain or spinal cord) evidence for diagnosis and place less emphasis on csf findings. the new criteria for ppms is as follows: ( ) at least year of disease progression (retrospectively or prospectively determined) ( ) plus two of the following: (a) positive brain mri (nine t lesions or four or more t lesions with positive vep), (b) positive spinal cord mri (two focal t lesions), (c) positive csf (isoelectric focusing evidence of oligoclonal igg bands or increased igg index, or both). evoked potentials are summed cortical electrical responses to peripheral sensory stimulation that can be used to localize sites of disease and measure conduction velocity along sensory pathways. vep and somatosensory evoked potentials (sseps) may detect subclinical sites of demyelination, thus providing evidence of multifocality. brain stem auditory evoked potentials (baeps) are occasionally informative. more than % of persons with a history of on have an abnormal vep, and % of cdms patients have abnormalities on veps even when the history of on is absent. slowed conduction is present on ssep in nearly three fourths of patients with cdms. baes are the least sensitive, with abnormalities in less than %. mri has largely supplanted the use of evoked potentials in ms because of the greater sensitivity in the diagnosis and the detailed anatomical information it provides. in , the american academy of neurology released practice parameters regarding the usefulness of evoked potential studies in ms. according to these recommendations, veps are considered probably useful (class ii evidence) to identify patients at increased risk for developing clinically definite ms. sseps are possibly useful, whereas the evidence for baeps supporting the diagnosis of cdms is insufficient. management. there is no available prevention or cure for ms. treatments focus on three areas: treating acute exacerbations and hastening their recovery; altering the natural history of ms; and providing symptomatic relief of current symptoms by enhancing physical abilities and preventing or treating complications. a fourth management topic concerns special treatment issues related to pregnancy. acute exacerbations. corticosteroids are the most commonly used treatment for ms, although there have been few studies to address their efficacy. adrenocorticotropic hormone (acth) was shown to speed recovery from an exacerbation but had no effect on the ultimate degree of recovery. because of the unpredictable cortisone response to acth, oral prednisone and later intravenous methylprednisolone became the preferred treatments. the optic neuritis treatment trial verified that intravenous methylprednisolone but not prednisone increased the recovery rate and unexpectedly increased the time to the next relapse, thus delaying the diagnosis of cdms. moreover, the prednisone-treated group had twice as many recurrences. the finding was not replicated in a second study, but it has affected the practice of treating acute ms exacerbations. the current recommendation is to treat disabling attacks with to mg of intravenous methylprednisolone per day for to days with or without a short tapering dose of oral corticosteroids. according to the practice parameters for steroid treatment of acute on attacks released by the american academy of neurology in , oral prednisone in doses of mg/kg/day has no proven value. higher dose of oral or parenteral methylprednisolone may result in quicker and more thorough recovery of visual function. there is no evidence of long-term benefit for visual function. a study suggested that intravenous steroids may also have long-term effects on disease progression when given regularly. in this study, rrms patients randomized to receive regularly scheduled pulses of iv methylprednisolone (every months for years, then every months for years) demonstrated stability or improvement in disability measures, fewer "t black holes," and less brain atrophy than did control patients randomized to receive steroids only with relapses. these findings suggest a possible long-term benefit of pulsed iv methylprednisolone therapy on brain atrophy and disability. this as yet unconfirmed approach to long-term therapy might be considered a reasonable "control arm" in future phase iii trials of experimental therapies. up to one third of patients do not have an adequate recovery after a relapse despite the use of corticosteroids. plasma exchange (plex) alone was found beneficial in a substantial proportion of patients with severe inflammatory demyelinating episodes who had failed to improve following treatment with high-dose iv methylprednisolone. , a randomized, sham-controlled, double-blind trial in patients (seven exchanges over days) without concomitant use of immunosuppressants in acute demyelinating events confirmed these findings. moderate or greater clinical improvement was observed in over % of participants. a trial of seven plex treatments (alternate days) is a reasonable option for patients who fail to respond to conventional iv methylprednisolone therapy in acute, severe episodes of demyelinating diseases. the response to plex is stongly associated with the histological ms subtype. antibody and complement plays a crucial role in pattern ii lesion formation. in a study of biopsy-proven ms cases by keegan and associates, pattern ii cases showed good response to plex, whereas cases of pattern i or iii did not respond at all to plex. neuromyelitis optica, which is now considered an antibody-mediated condition with a known serological marker, also shows good response to plex: in a study by the mayo group, % of nmo patients showed moderate or marked improvement to plex, and an additional % showed mild improvement. alteration of the natural course. the primary goal of drug treatment is to alter the natural course of the disease (e.g., reducing the frequency and severity of relapses, preventing the chronic progressive phase, and slowing the progression of disability). the disease activity seen on mri is often used as a secondary outcome, although mri measures currently correlate imperfectly with clinical outcome. knowledge on altering the course of ms is largely restricted to three patient groups, those with clinically isolated syndromes, those with rrms, and those with secondary progressive ms. before we discuss the known data in each of these demyelinating disease categories, it may be worth while to review the use of the most important evidence-based medicine (ebm) statistics that are applied to measure the magnitude of treatment effect. relative risk reduction (rrr) is the metric most commonly cited in publications and promotional materials about clinical trials. the rrr is the degree that the treatment reduced the frequency of the outcome measure (experimental event rate, e.g., relapse, progression) compared with the control treatment (control event rate). the rrr is a ratio, not an absolute number, and is calculated as follows: rrr ¼ ðcontrol event rate À experimental event rateÞ= control event rate if the control event rate is low (making the denominator smaller), it will obviously inflate the rrr. an "impressive" % rrr may have a low biological significance if the outcome occurs infrequently. therefore, the absolute risk reduction (arr) should be calculated as this corrects for the frequency of the outcome. for most of the approved ms agents, the calculated arr is considerably less than rrr. this metric is usually not cited in reports of clinical trials of disease-modifying agents. to calculate risk reduction, one must have access to the data citing comparisons of proportions (ratios), and this is not always immediately available in publications. another useful measure of treatment effect is the "number needed to treat" (nnt). it is calculated as the inverse of the arr: overall, the nnts for the disease-modifying agents in ms are in the to range for treatment periods of to years. however, these nnts are for outcomes that have limited predictive value for long-term outcomes (e.g., relapse behavior does not precisely predict long-term disability) and the agents are expensive, inconvenient to use, and not without risk. we must also remember that clinical trials typically enroll patients with very restricted eligibility criteria (often a history of considerable recent disease activity or progression), and considerable efforts are in place in trials to optimize compliance with the treatment plan. as such, the nnt experienced in a practice setting (effectiveness) may considerably exceed what was reported in the trial setting. altering the course of a clinically isolated syndrome. when should treatment be initiated in patients with very early demyelinating disease? two recently published multicenter studies have addressed this issue in persons at high risk of developing ms. in the champs study, patients with their first episode of presumed demyelinating disease ("clinically isolated syndrome") in the setting of an abnormal, asymptomatic baseline cranial mri scan, were randomized to receive either weekly interferon-b a mg im or placebo after an initial course of steroid therapy. this study was terminated early when the primary outcome measure of conversion to "clinically definite ms" (cdms) status was reached in a greater number of placebo-treated patients. these findings were not unexpected given the known effect of interferons on reducing relapse rate but do provide some support for early treatment. the duration of follow-up in this study ( % year, % years, % years) is insufficient to determine long-term benefit from early intervention, however. it is also clear that the treatment is only partially effective, as % of interferon (ifn)-treated patients in the champs trial had clinical or mri evidence of recurrent disease within months of starting treatment. the analysis of treatment effect related to the champs trial reveal a rrr of %, an arr of . %, and an nnt of patients over years to prevent one conversion to "clinically definite ms." in a second placebo-controlled study of patients with either monosymptomatic ( %) or multifocal onset ( %) early demyelinating disease, early treatment with interferon-b a in an unusually low dose ( mg subcutaneously once weekly), reduced conversion to cdms ( % versus %) at years. again, there is no data on whether these treatments offer long-term benefit. the ebm calculations regarding this trial show an rrr of %, and arr of %, and an nnt of patients over years in order to prevent one conversion to "clinically definite ms." these two studies provide support for considering early treatment in patients presenting with first attack, in the presence of multiple asymptomatic mri lesions, but further studies are needed to determine whether this approach will provide a prolonged benefit on disease course. it is important to note that these studies do not provide guidance about clinically isolated syndromes that present with a brain mri that is not suggestive of ms (i.e., only one optic nerve lesion, or one brain stem or cord lesion explaining the cis symptoms). we do not recommend that cis patients with fewer than two asymptomatic mri lesions receive treatment with interferons. please see the discussions under "summary of recommendations for the treatment of rrms patients" for further advice on patient counseling and decision making about the use of the disease-modifying medications. altering the course of relapsing-remitting multiple sclerosis b-interferons. interferons are a class of peptides that have antiviral and immunoregulatory functions. both interferon-a and interferon-b are part of the anti-inflammatory t h response. interferon-b b (betaseron) was the first drug approved by the u.s. food and drug administration (fda) specifically for the treatment of ms. a large clinical study in rrms patients demonstrated a reduction in the frequency of relapses by about one third with subcutaneous injection every other day. the severity of relapses was also lessened. interferon-b b had a striking effect on mri measures of disease activity. the placebo-control group continued to accumulate white matter lesions, whereas patients in the high-dose arm ( million iu) had stabilization of their mri lesion load. no difference was found in the disability levels, however. side effects include injection site reactions, flu-like symptoms (low-grade fever, myalgias, headache; these lessen in frequency after treatment for a few months), mild liver enzyme elevation, and lymphopenia. depression and attempted suicide were more common in the treated groups. to illustrate the magnitude of treatment effect of the pivotal interferon-b b trial, the rrr was %, the arr was %, and the nnt analysis showed that patients are needed to be treated over years to increase the number of those who were relapse free by one. one particularly disturbing result was the production of neutralizing autoantibodies (nabs) in % of patients after years of treatment. not only do patients with these antibodies thereafter fail to respond to this drug, but there is also a concern that nabs may cross-react with natural interferon-b and interfere with its function. all positive sera for nabs seem to cross-react with both interferon-b a and b. switching from one preparation to the other does not change the pattern of antibody response. the long-term effects of nabs are unknown. recent studies seem to support that nab formation reduces clinical and mri effects although often nab formation subsides with time. there are no firm guidelines for monitoring nab formation. most physicians do not measure nabs but rather change therapies empirically when patients appear to be failing treatment. low titer nabs may be just transient phenomenon related to ifn treatment; persistent high titer nabs on two consecutive tests at least months apart is likely associated with poor treatment response to inf. interferon-b a (avonex) has the same amino acid sequence as natural interferon-b and differs from interferon-b b by one amino acid as well as by the presence of carbohydrate moieties. once-weekly intramuscular interferon-b a has been found to have effects similar to that of interferon-b b in reducing the frequency of ms relapses. in addition, a favorable effect on disability was also demonstrated and side effects were less common. in the original interferon-b -a intramuscular trial, the primary outcome measure was time to edss progression. the rrr was %, the arr was %, and the nnt was for years to prevent one patient from developing edss progression. the calculations for "proportion relapse free" show an rrr of %, and arr of %, and an nnt of over years ( patients need to be treated for years to increase the number of patients who were relapse free by one). nabs occurred half as often as with interferon-b b. interferon-b a has been approved by the fda for treatment of "relapsing ms." the "correct" dose of interferon continues to be debated. in a recent placebo-controlled trial, patients randomized to a high dose of interferon-b a ( mg three times per week) did better than those receiving half this weekly dose. both groups outperformed placebo and the high dose seemed to have more effect on relapse severity, hospitalizations, mri activity, and lesion volume accumulation, and possibly on delaying disability in the most severely disabled patients. at the end of the years of follow-up, placebo-treated patients were randomized to or mg subcutaneously three times weekly; patients on active treatment were continued on their original dose. the authors reported a benefit for the higher dose and for those treated for the full years, again suggesting that early treatment and perhaps higher doses of interferons may be beneficial. the primary outcome, however, was relapse count per patient per years and, as such, patients treated early had a significant advantage using this outcome measure. there were trends favoring the higher dose (relapse rate, mri volumes; not for time to first confirmed progression, however). the authors did not make statistical adjustments for multiple comparisons and there were many dropouts in the high-dose groups, making it difficult to draw firm conclusions. again, the answer to the question about the benefit of early treatment can best come from long-term (perhaps to years) studies using "hard outcomes" (e.g., time to progression, major milestones in disability). the ebm calculations based on the "proportion relapse free" data for the original interferon-b a (rebif) study show an rrr of %, and arr of %, and an nnt of over years to increase the number of relapse free by one. relative treatment advantages of interferon-b a and b have not been clearly established but are under study. , a pilot study in rrms patients suggests that interferon-a may also have a therapeutic effect. a study of interferon responders showed that younger patients with frequent relapses, and higher edss scores upon entry may be associated better response. laboratory monitoring of interferon products. it is important to note that even though the interferon products are generally safe to use, they can be associated with potentially harmful adverse reactions. we recommend that every newly starting patient should have a baseline complete blood count, liver function tests, and thyroid-stimulating hormone (tsh) test. the liver function tests and blood count studies should be repeated in week, month, and every months thereafter; the tsh should be repeated every to months. glatiramer acetate. glatiramer acetate (ga) is a synthetic mixture of polypeptides produced by the random combinations of four amino acids that are frequent in mbp. after a preliminary study suggested efficacy, a phase iii randomized, double-blind, placebo-controlled, multicenter trial showed a % reduction in relapse rate. the fda has approved this medication for use in rrms. even though this disease-modifying therapy requires daily subcutaneous administration, the side effects are relatively minor compared to the interferons, and patients do not need regular laboratory monitoring (table - lesions, lesion volumes, and the percentage of new lesions that will evolve into t "black holes," although the mri effect may be less pronounced compared to the interferon products, and is not apparent until the agent has been used for at least months. [ ] [ ] [ ] the ebm calculations for ga using the "proportion relapse free" data show an rrr of %, arr of %, and an nnt of over years to increase the number of relapse free by one. combined azathioprine and interferon-b b. a small trial at nih showed significant reduction in the number of contrast-enhancing lesions when azathioprine in an average maintenance dose of mg/kg/day was added to interferon-b b in a study of six rrms patients followed for a median period of months. the addition of azathioprine may be considered in "treatment failure" cases, but this study was hampered by the small number of patients, no control subjects, and no blinding. intravenous immunoglobulin. monthly treatment with low-dose ( . to . g/kg) intravenous immunoglobulin (ivig) in rrms patients resulted in fewer and less severe relapses in addition to slowing the accumulation of disability in a single randomized trial. the outcome was similar to that of injectable interferons. this therapy is less accepted in the united states. more studies with larger number of patients and extended follow-up are needed to confirm these limited observations. recent studies have failed to demonstrate that ivig administration reverses long-standing deficits from ms and on. [ ] [ ] [ ] ivig was also recently studied in acute on and failed to demonstrate benefit on any of the outcome measures. natalizumab. in late , natalizumab was approved for the treatment of rrms. natalizumab is a humanized a- integrin antibody that inhibits the migration of all leukocytes (except for neutrophils) to target organs. a phase study established that a -mg monthly dose reduced the number of gadolinium-enhancing lesions by % and the clinical relapse rate by over % compared to placebo. this study was followed by the affirm and sentinel phase iii studies. the affirm study enrolled over patients with rrms; none of them had been on other approved immunomodulators for longer than months. the annualized relapse rate at year was reduced from . in the placebo group to . in the treated group ( % relative reduction, p < . ). the proportion of relapse-free patients was % in the treated group, % in the placebo group. the number of enhancing lesions was reduced by %, and the number of new or newly enlarging t lesions was reduced by %. the proportion of patients without clinical and mri activity was % in the natalizumab group, and % in the placebo group. in the sentinel trial, the combination of intramuscular interferon-b a and natalizumab was studied against im interferon-b a and placebo in patients who had demonstrated an incomplete response (relapse suppression) to interferon therapy. the ebm calculations of the affirm data based on proportion with relapses suggests an rrr of %, an arr of %, and an nnt of over year to increase the proportion of relapse free by one. the senti-nel data shows an rrr of %, an arr of %, and an nnt of over year to increase the proportion relapse free. the original pilot trial data shows a rrr of % and an arr of %, with an nnt of over months to increase the proportion relapse free. based on these data, the fda granted expedited approval of natalizumab on november , . on february , , the medication was voluntarily withdrawn from the market by the sponsor (biogen-elan) after two cases of progressive multifocal leukoencephalopathy (pml) were reported in the sentinel study cohort. both patients were in the combined interferon and natalizumab arm. a third pml case was later identified from one of the phase iii inflammatory bowel trials of this agent. at the time of writing this manuscript, natalizumab is still off the market. the natalizumab story has received significant media attention. several consequences can be drawn from this failure. first, highly potent immunomodulators like natalizumab are best used by specialists in selected cases. a large number of prescriptions were written for natalizumab during its short months on the market, including prescriptions by general practitioners. widespread use of such medication in relatively stable cases of ms is not indicated. second, the combination of potent immunomodulators may result in unpredictable adverse outcomes. many ms experts anticipate that in the future ms therapies will need to be administered in combination to optimize therapeutic benefit. however, the exact effect of such combinations on the highly complex immune system is difficult if not impossible to predict. furthermore, our inability to treat ms more effectively does not stem from the fact that we can not provide powerful immunosuppression, as evidenced by the autologous bone marrow transplantation studies. ms is a complex disease with a prominent inflammatory component; however, increasing evidence suggests that the neurodegenerative component of this illness may be independent of the inflammatory component, and is just as important, if not more important, from the standpoint of long-term disability. third, in chronic diseases such as ms, a short -year trial, no matter how convincing the outcome may be, should not be considered sufficient to approve a medication, which will then be used in tens of thousands of patients on a "lifelong" basis. there clearly is a need for new and more effective medications for treating ms; however, clinical trials in chronic conditions are very difficult to sustain. to overcome this, many ms trials use primary mri outcome measures, since inflammation and new lesion formation-related mri markers respond more immediately to treatment; however, these markers do not correlate well with long term disability, as discussed earlier. summary of recommendations for the treatment of rrms patients. when making decisions about starting an ms patients on immunomodulators, several factors must be considered. one must realize that even though there are medications available for relapsing forms of ms, all the currently available therapies are only partially effective, the most reliable data is about short-term relapse rate reduction, and a relapse rate reduction does not necessarily translate into reduction of future disabilities. natural history data clearly suggest that a subset of ms patients will do very well without treatment (see discussion about the olmsted county cohort later); this information can be very useful when deciding about treatment in patients with a -or -year disease history and minimal disability (edss . ). in this patient group, a careful wait and see approach with appropriate monitoring is acceptable. counseling of newly diagnosed ms patients is of crucial importance, and it should usually include family members. most patients have easy access to an abundance of frequently misleading information on the internet, or from relatives and friends with ms. it is important to realize that every case is different; however, through the rational use of natural history data, clinical and mri features of the specific case, and the clear understanding of the available clinical trial data, the clinician should be able to provide customized and relevant advice to patients and families. considering that the treatments are only partially effective, the wishes of an educated patient constitute an important part in the decision-making process. ultimately, the treatment decisions should remain individualized between the patients and their treating physicians, and the physician's role as an information clearinghouse and educator cannot be overemphasized in this process. the three interferon products and ga represent the most commonly used ms immunomodulators in the united states; therefore, it is important to draw some practical conclusions about these agents. by now, several class i studies demonstrate that these agents are effective in reducing the relapse rate in rrms over a -to -year period; the reduction is roughly % with the high-dose interferons and ga. the above-mentioned nnt data are also very useful for the clinician and the well-informed patient when making treatment decisions. there is evidence for a dose-response relationship among the interferon products, mostly from the evidence and incomin studies. the double dose im interferon-b a study did not show a dose-response relationship; this may be related to the fact that the increased dose was given with the same frequency as the standard dose. the injectable immunomodulators have incomplete evidence for efficacy in disability-based outcome measures. many of the long-term extension studies suffer from several drawbacks, including open label unblinded design, significant dropout rate, and lack of control subjects; this is especially true for the ga extension data. the currently available few head-to-head comparison studies are also hampered with methodological issues; new comparative studies are under way. overall, these agents remain partially effective in relapsing forms of ms; their long-term effects on reducing the clinically most important feature of ms-disability-still remains unclear. altering the course of secondary progressive ms. within years of onset, almost % of rrms patients will enter the secondary progressive phase of the disease. treatment approaches aimed to affect the natural course of disease are available for these patients. interferons. interferon-b b may have a beneficial effect on the overall outcome of spms and may also alter mr lesions, , but this question remains incompletely answered. in the placebo-controlled european study of inteferon-b b in spms, the time to worsening was extended for treated patients. treated patients were less likely to be wheelchair-bound and had fewer hospitalizations. another analysis of this study confirmed the benefits, though the dropout rate in this study was relatively high. the patients who responded best to interferon therapy were those who experienced relapses during their disease course. mri monitoring suggested that the benefit on t lesion activity was seen early and persisted into the second half of the second year of treatment. t lesion load increased in placebo-but not interferon-treated patients in the first years of treatment. contrasting with these results, in another trial involving patients with spms, both high dose ( mg) and lower dose ( mg) interferon-b a failed to change the primary outcome of time to disability worsening. positive effects were seen on relapse rate and reduction of mri activity, but the effect on disability did not replicate the european interferon-b b report. a combined analysis of the american and european trials concluded that continued relapse activity and more rapid progression over the preceding year (by > on the edss scale) are the best predictors of response. ivig in secondary progressive ms. a recent european trial reported that ivig did not have a significant impact on clinical and disability related outcome measures. ivig did reduce the accumulation of brain atrophy in spms, but did not reduce the incidence of blood-brain barrier abnormalities. there was no statistically significant change on magnetization transfer mri measurements; however, a trend for conservation of normal-appearing brain tissue was found. overall recommendations for spms. in general, as the evidence that interferons alter long-term disability is limited and controversial, we generally do not newly start spms patients on interferon products. in a subset of patients still having disabling relapses, interferon therapy may be offered to specifically reduce relapse rate. the data by confavreux and associates, however, suggest that the edss in populations of spms patients continues to progress independent of relapses once a "fixed" baseline level of moderate disability has been reached. therefore, while interferons may reduce the relapse rate in spms, the rate of progression of disability may not be reduced by these treatments. more usually, spms patients are already on an injectable immunomodulator, and the question of whether it is worth continuing the therapy may come up, especially in patients who have a hard time tolerating these medications and feel that the side effects of the medications have a clear negative impact on their overall health. in these cases, we usually allow the patients to stop their medications. just like in the rrms cases, however, patient education about spms trials and realistic expectations about the treatment is a crucial element in the decision-making process. understanding the patient's needs and fears and clarifying potential misconceptions constitute a very important role of the treating neurologist. in those patients who continue their interferon therapy, we must continue to follow them for toxicity and disease activity. please also see the following discussion under mitoxantrone for recommendations on the potential use of that specific agent in spms. mitoxantrone. mitoxantrone (novantrone) is an anthracenedione chemotherapeutic agent licensed . . .for reducing neurologic disability and/or the frequency of clinical relapses in patients with secondary (chronic) progressive, progressive relapsing, or worsening relapsing-remitting multiple sclerosis (i.e. patients whose neurologic status is significantly abnormal between relapses). mitoxantrone is not indicated in treatment of patients with primary progressive multiple sclerosis. significant benefits were observed in a group of spms patients as in a european phase iii study of mitoxantrone. it has also been used in combination with methylprednisolone. several clinical and functional outcome measures were reported to stabilize or improve with every -month administration of this intravenous medication. secondary mri outcome measures, including enhancing lesion formation and overall t lesion load, were also better in the treated patients. the greatest concern regarding this medication is its cardiac toxicity: the cumulative lifetime maximum dose was established at mg/m . mitoxantrone can induce a seemingly dose-dependent cardiomyopathy, leading to potentially fatal congestive heart failure. we generally avoid exceeding a total lifetime dose of mg/m ( doses of mg/m ). patients receiving mitoxantrone should also be monitored every months with echocardiograms or muga (multiple gated acquisition) scans to determine the ejection fraction. reduction in the ejection fraction should prompt discontinuation of this therapy. besides the cardiac side effects, mitoxantrone may cause menstrual irregularities or overall ablation of the menstrual cycle, which may be permanent. in a review of the literature, ghalie estimated the risk of mitoxantrone therapyrelated acute leukemia in ms patients at . % to . %; in an international registry of ms patients taking mitoxantrone, the risk of leukemia seems somewhat higher. this therapy has been approved by the fda for treatment of spms, but no peer-reviewed full report of the mims study had been published until years after the initial report in an abstracts form. the study showed a treatment effect in rrms and spms patients with "recent rapid worsening." the study had a high dropout rate and a small sample size. most patients ( %) had relapses in the preceding years, suggesting this cohort mostly includes worsening rrms or prms patients, in whom a positive treatment effect is expected; however, it does not mean that for classic spms patients who no longer have relapses the study outcome is applicable, and it is especially not applicable to ppms cases. the primary outcome measure was a composite score comprised of five clinical measures: change in edss at years; change in ambulation index at years; change in the baseline standardized neurological status at years; number of relapses requiring corticosteroid treatment; and time to first relapse. seventy-seven percent completed months of follow-up. at months, benefit was reported in all five components of the composite measure for both active treatment arms, with the overall greatest benefit noted between placebo and the group receiving mitoxantrone at a dose of mg/m . the magnitude of the effect on edss was rather modest (mean edss change for high-dose mitoxantrone, - . [sd . ] versus þ . [sd . ] in the placebo group). the mri results of the mims trial were published in and are frankly disappointing. in a subset of patients (out of in the trial overall), the mg/m dose failed to reach a significant difference from placebo as measured by the primary mri outcome (total number of scans with gadoliniumenhancing lesions). the mg/m dose reduced the number of t -weighted lesions at month (p ¼ . ) and showed a trend at month (p ¼ . ). the number of active mr lesions showed a trend toward reduction in the mg/m group only at month (p ¼ . ). overall, the limited evidence to date supports the conclusion that mitoxantrone reduces relapse frequency and mri evidence for blood-brain barrier disruption in patients with very active ms. the benefit for patients with relapse-independent progression is uncertain at best. from the mims results, one would need to treat patients with secondary progressive multiple sclerosis for years to prevent one person from worsening by . edss point. this modest benefit must be carefully examined in light of the significant risk for toxicity. therapy of ppms. unfortunately, for classical ppms cases that present with insidious progression of usually myelopathy symptoms, none of the currently available treatments offer any clear benefit. the promise trial, in which over ppms patients were treated with ga, was terminated early owing to lack of effectiveness. the results of this trial have not yet been published. a small study with intramuscular interferon-b a was also negative. currently a large trial is under way with rituximab in csf ocb-positive ppms patients. until we clearly understand the pathophysiology of slow progression in ms, it is unlikely that we will find a treatment that has an important impact on this form of ms. symptomatic treatment modalities, including physical and occupational therapy, are very important, yet frequently overlooked in this patient population. other immunomodulator therapies. cyclophosphamide is an alkylating agent that has indiscriminate cytotoxic effects on rapidly dividing cells, including lymphocytes, making it a potent immunosuppressant. several studies have claimed a beneficial effect in both relapsing and progressive patients. because one of the major studies included acth, iv methylprednisolone is sometimes given with the cyclophosphamide. other trials have not found a favorable effect. because of the inconsistent results, high potential for serious side effects, and adverse reactions, including hemorrhagic cystitis and malignancy, cyclophosphamide is not widely used. some centers, however, use cyclophosphamide in patients with aggressive disease in whom more conventional treatments have failed. azathioprine, a purine analog antimetabolite, has marginal efficacy in the treatment of ms. a meta-analysis of all blinded, placebo-controlled studies confirmed a slight benefit of slowed progression and less frequent relapses. the toxicity of azathioprine and its slow onset of action have prevented its widespread use. besides the liver toxicity and hematological effects, the induction of malignancies has been a concern. one retrospective study did not find an increased incidence of cancer in ms patients treated with azathioprine, but this remains a potential risk. methotrexate is a folate antagonist that is effective in rheumatoid arthritis. weekly low-dose oral methotrexate was found to delay upper extremity dysfunction in spms patients, although it had no effect on the more traditional measures of disability, including the expanded disability status scale (edss). the use of cyclosporine in the treatment of ms has been evaluated in three clinical trials, none of which have demonstrated a convincing benefit. in addition, side effects such as hypertension and elevation of creatinine were common. numerous additional therapies have been tested, and many others are undergoing evaluation. the antiherpesvirus drug acyclovir has been shown to reduce relapse frequency in a small prospective trial. total lymphoid irradiation was found to slow the chronic progression of ms, but because this approach precludes the later initiation of immunosuppressant drugs and may be associated with a higher mortality rate, it is not widely used. cladribine is a nucleoside derivative that was found to decrease relapse rate and slow the progression in patients with spms in an initial investigation. the drug is better tolerated than other parenteral immunosuppressants, although bone marrow suppression is a risk. in a more extensive clinical trial, cladribine therapy did not change disability scores, but significant reduction in enhancing lesions and overall t lesion burden was observed with higher dose treatment. a study of a small number of patients treated with autologous stem cell transplantation suggested possible clinical stabilization or minor improvement over a -month period of follow-up in both secondary and primary progressive ms. the induction chemotherapy (beam regimen) resulted in one fatality in this trial; similar incidences are known in patients undergoing this procedure. the small number of patients and the different methods used (some patients received cd þ selected graft) makes the interpretation of this data very difficult. trials with higher number of patients under standardized circumstances are needed to verify the validity of these observations. symptomatic treatment of existing disabilities spasticity. spasticity is common even in patients with only minimal weakness (video , spastic gait). it is usually prudent to begin treatment of mild spasticity with a stretching program. a randomized controlled crossover trial of physical therapy ( -week blocks of therapy twice a week, for minutes per session) showed significant benefit on several outcome measures related to improved mobility. no apparent differences were observed between home-based or hospital-based therapy. the addition of an evening dose of benzodiazepine may help relieve extensor spasms and clonus that may interfere with sleep. as spasticity worsens, it becomes necessary to use baclofen. doses should be escalated slowly to prevent the occurrence of overt side effects, and up to mg/day may be required. although baclofen is well tolerated in most patients, limiting side effects such as sedation and increased muscle weakness may occur, and rarely a paradoxical increase in spasticity is noted. liver enzyme elevation and nonconvulsive status epilepticus presenting as encephalopathy have also been reported in association with baclofen. abrupt withdrawal of baclofen may result in hallucinations or seizures, making it necessary to taper doses. despite symptomatic improvement, antispasticity measures may not increase function or independence. in paraplegic patients with severe spasticity and intolerance to the required oral dose, intrathecal baclofen delivered by a subcutaneously implanted pump allows a much smaller dose and is often effective in alleviating intractable spasticity and may lessen urinary urgency. tizanidine seems to be as effective as baclofen, although it may be associated with more fatigue. dantrolene has been used for spasticity, although the therapeutic window is small. fatigue. for treating fatigue, medications are only partially effective. amantadine at mg twice a day is the standard initial treatment, although pemoline . mg daily is also superior to placebo. a recent, small pilot study by the mayo group suggested that high-dose aspirin ( mg/day) may sometimes be effective in the treatment of ms-related fatigue. this finding needs to be confirmed by a second, larger trial, however. the stimulating effects of the selective serotonin reuptake inhibitors may also be somewhat effective in combating ms-related fatigue. modafinil, a medication approved for the treatment of narcolepsy, has also been used with good success. often, however, patients need to limit activities and schedule rest periods. paroxysmal symptoms of ms. paroxysmal symptoms are highly responsive to medical treatment. a small dose of carbamazepine is often very effective. if not tolerated, several alternative medications may be tried, including phenytoin, acetazolamide, baclofen, and gabapentin. in addition, misoprostol has been claimed to be effective in ms-related trigeminal neuralgia. after about month of treatment, a periodic attempt at tapering off these medications is a reasonable approach because these symptoms usually remit. seizures in ms are treated no differently than in non-ms conditions. heat sensitivity. heat sensitivity may require avoidance of precipitating activities, but this depends on the nature of symptoms and the situation in which they occur. if the precipitating activity cannot be avoided, a cooling jacket may be an option. a potassium channel blocker, -aminopyridine, improves temperature sensitivity in some patients but occasionally causes seizures or disturbing paresthesias. action tremor. action tremor is a common disabling symptom (video , tremor with ataxia). unfortunately, it is often only marginally amenable to medical therapy. clonazepam may offer some relief, but tolerance frequently develops, necessitating increasing doses. isoniazid and carbamazepine have also been found marginally beneficial. one clinical trial showed ondansetron to reduce tremor-related disability. anecdotal reports suggest that gabapentin may be partially effective. improvements in stereotactic neurosurgery have made thalamotomy a legitimate option in those whose disability is mainly due to tremor and not ataxia. cognitive and memory problems. cognitive problems can also be seen in ms patients. these symptoms are generally not very severe; however, in some patients these may be one of their subjectively most bothersome complaints. it is important to make sure that such complaints are not depression related, as mood disorders are otherwise rather common in ms, and may explain the subjective cognitive impairment. while it is not fda approved for the treatment of ms related cognitive dysfunction, in a placebo controlled, randomized, -week long study of donepezil in patients, significant improvement was found on the selective reminding test (srt). this improvement was independent of ms subtype, gender, age, reading ability, and baseline srt results. the patients did not improve on other cognitive scales, but they were twice as likely to report cognitive improvement. dysesthetic pain. dysesthetic pains are difficult to control but sometimes respond to tricyclic antidepressants, carbamazepine, or baclofen. gabapentin, tramadol, and duloxetine may also be effective. standard analgesics are not often useful in ms-associated pain, and narcotics should be avoided in the treatment of chronic pain. emotional incontinence may be amenable to a low dose of a tricyclic antidepressant (video , dysarthria). symptoms of bladder dysfunction. symptoms of a hyper-reflexic bladder (urgency, frequency, and urge incontinence) are often manageable with anticholinergics such as oxybutynin, propantheline, or imipramine. a flaccid bladder can sometimes be aided by bethanechol, although intermittent self-catheterization is more often needed. symptoms that suggest urinary retention (a feeling of incomplete emptying, frequency, hesitancy, or a need to apply pressure to the lower abdomen to urinate) should prompt evaluation with urinalysis and a post-void residual urine measurement. residuals in excess of ml are abnormal; and if they are above ml, consideration should be given to urological consultation for more thorough investigation and to blood chemistries to determine urea and creatinine levels. detrusor-sphincter dyssynergia, diagnosed by cystometrography, is treated with anticholinergics, sometimes with the addition of an a- blocking agent (terazosin) or intermittent catheterization. it is important to reassess bladder function periodically, and residual urine volumes should be monitored if there are any persistent changes in function or symptoms. intermittent catheterization should be considered when post-void residuals reach ml. a chronic indwelling urinary catheter should be avoided if reasonably possible. it is usually not necessary to use antibiotics prophylactically in the prevention of urinary tract infections. urinary calculi may be prevented by acidification of the urine with cranberry juice. bowel dysfunction in ms. constipation can usually be managed with bulk laxatives and stool softeners. more severe cases may require osmotic agents, bowel stimulants, anal stimulation, suppositories, or enemas. bedridden patients may develop fecal impaction unresponsive to these measures and require manual disimpaction. fecal incontinence can be minimized by adherence to a schedule for bowel movements. fiber supplementation may be of some benefit even in these cases. sexual dysfunction in ms. the clinician should determine the precise nature of any sexual dysfunction in patients with ms. physical difficulty from spasticity may be alleviated by premedication with baclofen, and a fast-acting anticholinergic such as oxybutynin may calm urinary urgency. sexual dysfunction should not be automatically attributed to ms. it may be necessary to investigate hormonal levels and to obtain urological or gynecological consultation. manual lubrication with gel is a ready solution to vaginal dryness. erectile dysfunction responds very well to sildenafil. less frequently, vacuum devices, intracavernous injections of papaverine (or combinations of papaverine, prostaglandins, and epinephrine; triple agent), or penile implant are also used in the treatment of erectile dysfunction. thalamotomy or thalamic stimulation may provide some short-term clinical benefit to patients disabled by appendicular cerebellar tremor and ataxia. the benefits on disability and quality of life are much less clear, however, and the early benefits may wain within to years. , further studies are needed to clarify how best to select patients for these ablative and stimulation treatment interventions. general recommendations for ms patients. it is advisable for ms patients to attain good health habits, including proper diet and fitness. smoking, excess alcohol intake, and obesity should be avoided. exercise can help maximize function by increasing and maintaining joint mobility, strength, and stamina; may promote improved sleep hygiene; and may reduce the severity of fatigue. physical and occupational therapy can play an important role in regaining independence. canes, walkers, and wheelchairs or scooters may be needed to maintain safe mobility. hand controls can be installed in automobiles for patients with lower extremity dysfunction. in debilitated, immobilized patients, periodic shifts in posture to change weight-bearing regions and air or water mattresses prevent bedsores. passive range of motion exercises prevent contractures. when ventilatory dysfunction occurs, it should be evaluated and activity schedules should be appropriately modified. special considerations during pregnancy. before initiation of any drug in a woman of reproductive age, the potential for teratogenicity must be discussed. in general, immunomodulator therapy should be avoided if one is planning a pregnancy. the treatment of acute exacerbations is unchanged during pregnancy, although one might have a higher threshold for treatment. both corticosteroids and plasma exchange are relatively safe during pregnancy. none of the drugs used to alter the disease course, however, should be used during pregnancy. interferon-b drugs should be stopped to months before planning pregnancy. interferons are associated with an approximately % likelihood of abortions. this is close to eight times higher than the usually quoted approximately % spontaneous abortion rate in women. interferon-b use is also associated with lower birth weight. the cytotoxic immunosuppressants have teratogenic effects. the effects of many of the other drugs are unknown. it is best if these drugs are stopped several months before a planned pregnancy. prognosis and future perspectives. because most information on the prognosis of ms is reported in terms of the edss, it is important to have some understanding of this scale. the edss is a -point scale, with each increase representing worsening symptoms of function. the score is derived from severity scores in each of six systems as well as ambulation and work ability. a score of means no signs or symptoms; to represent mild disability with no or minimal impairment of ambulation; . to . refer to moderate disability and impairment of gait; the need for a cane to walk one-half block ( m) receives a score of ; an edss of refers to the need for a wheelchair and effective upper extremity function; an edss of refers to death related to ms. ms has a highly variable outcome, ranging from asymptomatic to fulminant with death ensuing in a matter of months. autopsy series have estimated that unsuspected ms may occur in as many as . % of the population. even when symptomatic, ms may cause only nuisance symptoms. benign ms, when defined as unrestricted ambulation or edss of or less years after onset, accounts for about one third of cases. however, many of these patients acquire more disability later. when considering all patients with ms, weinshenker found that years after onset, % had edss worse than , % had reached edss of or more, % were at edss , and % had died. the percentage of patients with initially rrms who develop spms increases steadily with disease duration. at years, % to % have continual deterioration; after years, approximately % have slow progression. in the most recent study published about the olmsted county ms prevalence cohort consisting of patients, the mean change in edss over years was point, and only % of patients had a change larger than . points. eighty-three percent of the patients with mild symptoms (edss < . ) were still ambulatory without cane years later. among the patients with an edss of to , % were using a cane; in the to edds range, % were wheelchair bound. strong predictors for the outcome were not identified in this study. population-based studies with complete ascertainment can effectively remove the bias of a referral practice, which is inherently biased towards the more active and more serious cases. these studies also provide some much needed balance to the "heavily skewed for recent disease activity" clinical trial experience. from the most recent extensions to the olmsted county ms cohort studies conducted by the mayo group, several conclusions can be drawn. the number of relapses in the first year of the disease do not predict long term outcome. the time to disability is not influenced by ongoing relapses once patients achieve an apparently permanent degree of moderate disability (edss . ). overall, % patients who are still classified as rrms into their second decade of disease continue to do well for to years with limited permanent disability. patients doing extremely well (edss . ) after years of ms generally do well in the next decade. however, very rarely patients doing well even for to decades may develop severe late disability. we advise that neurologists share these findings with patients who are in periods of prolonged remission during the discussions about the merits of beginning disease-modifying agents. natural history studies have identified several prognostic indicators that predict outcome to a limited extent. factors associated with a better prognosis (slower accumulation of disability, longer time before chronic progression) include young age at onset, female gender, rr course (as opposed to ppms), initial symptoms of sensory impairment or on, first manifestations affecting only one cns region, high degree of recovery from initial bout, longer interval between first and second relapses, low number of relapses in the first years, and less disability at years after onset (both edss and number of systems affected-sensory, motor, sphincter, brain stem, vision, cerebral). despite the indolent nature, a pp course is the worst prognostic factor, with the median time to reach edss of only years, compared with approximately years in rr patients. men and patients with an older age at onset are more likely to have ppms. the survival of ms patients is only slightly below expected. seventy-six percent of patients are alive years after onset, which is % of that seen in age-and sexmatched control subjects. ms is rarely the direct cause of death. complications of ms such as pneumonia, pulmonary emboli, aspiration, urosepsis, and decubiti are responsible for % of deaths. most of the other deaths are from heart disease, cancer, cerebrovascular disease, and trauma. suicide is the only cause of death that is overrepresented among these cases. the suicide rate among ms patients may be as high as two to seven times that of non-ms persons. neuromyelitis optica (nmo) is an uncommon neurological illness characterized by the occurrence of optic neuritis and myelitis. the names devic's syndrome, devic's disease, and nmo are often used interchangeably, although the first name encompasses all patients who fit the preceding definition and the second and third should only be used to refer to those patients presumed to have a distinct disorder. the term opticospinal ms is often used in the far east to denote patients with exclusive or predominant involvement of optic nerves and spinal cord, encompassing most patients with devic's syndrome. devic's disease (nmo) may be a monophasic illness, or may show a relapsingremitting course. it is the first inflammatory demyelinating disease with a known serum marker, the nmo-igg antibody. pathogenesis and pathophysiology. devic's syndrome may occur with adem, autoimmune disorders (e.g., systemic lupus erythematosus), ms, and possibly viral infections. also, patients with devic's disease may have other coexisting autoimmune conditions. classically, acute spinal cord lesions demonstrate diffuse swelling that extend over several levels or involve nearly the entire cross section of the cord. acutely, there is destruction with dense macrophage infiltration involving white and gray matter, loss of myelin and axons, and lymphocytic cuffing of vessels. in chronic lesions, the cord is atrophic and necrotic, occasionally with cystic degeneration and gliosis. in the absence of perivascular cuffing, these extensive lesions resemble infarctions. the prominent spinal cord swelling in the confines of the restrictive pia presumably may raise intramedullary pressure, leading to the collapse of small parenchymal vessels, further propagating tissue injury. proliferation of vessels with thickened and hyalinized walls similar to that seen after infarction or other extensive injury may occur. less fulminant lesions may coexist and are much more typical of inflammatory demyelination. the optic nerve lesions often involve the chiasm. even though nmo is usually restricted to the optic nerves and spinal cord, one may see classic ms like lesions in up to % of cases, and hypothalamic lesions have also been described in approximately %. the newly discovered serum marker, nmo-igg has a sensitivity of % and specificity of %. the discovery of this novel immune marker also clarified that most if not all cases of "opticospinal ms" reported in the japanese literature are also cases of nmo. to the surprise of the ms research community, the antigen is neither myelin nor neuron related: it is the aquaporin- water channel, a component of the dystroglycan protein complex located in astrocytic foot processes at the bloodbrain barrier. nmo thus may represent the first example of a novel class of autoimmune channelopathies. epidemiology and risk factors. devic's syndrome occurs in patients of varied ages (range, to years). the mean age at onset of monophasic devic's syndrome is , whereas relapsing nmo (see later) tends to occur in an older age group (mean age at onset of ). monophasic devic's syndrome affects males and females equally, whereas relapsing nmo affects females predominantly (f:m, . : ). one third of patients have a preceding infection within a few weeks of neurological symptom onset. most commonly this is a nonspecific upper respiratory tract infection, flu, or gastroenteritis. the most common specific infections preceding the development of devic's syndrome are chickenpox and pulmonary tuberculosis. devic's syndrome has also followed vaccination for swine flu and mumps. only a few instances of a possible familial occurrence of devic's syndrome have been reported, and in one of these families, a unique mitochondrial mutation was found. devic's syndrome is said to be more common in japan and east asia, although even there it is uncommon (less than per , ). three cases have been described in the literature with familial occurrence of devic's disease in the far east. in a genetic study, hla-dpb * was more frequently associated with "opticospinal ms," whereas hla-dpb * is the most strongly associated allele with conventional ms in the japanese. symptoms of on and myelitis develop over hours to days and are often preceded or accompanied by headache, nausea, somnolence, fever, or myalgias. continued progression of symptoms over weeks or months occasionally occurs. most patients (greater than %) develop bilateral optic neuritis. bitemporal or junctional field deficits, indicating chiasm involvement, are sometimes present early in the course of the on. visual loss is often accompanied by periocular pain, and myelitis onset is sometimes heralded by localized back or radicular pain. lhermitte's sign is common. severe degrees of neurological deficits are usual, and the degree of recovery is variable. approximately % of nmo patients have a monophasic illness, % develop relapses usually limited to the optic nerves and spinal cord (relapsing nmo or opticospinal ms), and rarely patients have a fulminantly progressive course without relapses or a course typical of ms. according to a study conducted at the mayo clinic, patients with a monophasic course usually presented with rapidly sequential events (median, days) with only moderate recovery. patients showing characteristics of the relapsing form of devic's had a median interval of days between index events, followed within years by clusters of severe relapses isolated to the optic nerves and spinal cord. most relapsing patients developed severe disability in a stepwise manner. approximately one third died from respiratory failure. predictors of a relapsing course in nmo include longer inter-attack intervals (relative risk [rr]: . per month increase), older age at onset (rr ¼ . per year increase), female sex (rr ¼ . ), and less severe motor impairment with sentinel myelitis event (rr ¼ . per severity scale point increase). autoimmune disease history (rr ¼ . ), higher attack frequency in first years (rr ¼ . per attack), and better recovery following index myelitis (rr ¼ . per point) are associated with increased mortality rate. features of nmo distinct from "typical" ms included normal initial brain mri, more than cells/ml in csf with polymorphonuclear predominance, and lesions extending over three or more vertebral segments on spinal cord mri. relapsing nmo is often associated with autoimmune disorders, most commonly systemic lupus erythematosus. these patients also frequently have an elevated erythrocyte sedimentation rate and nonspecific elevation of autoantibodies, including antinuclear antibodies, anti-ds-dna, and antiphospholipid antibodies. tonic spasms and neuropathic lower extremity pain are common sequelae to the spinal cord damage. symptoms referable to brain stem lesions (nystagmus, ophthalmoparesis, and vertigo) can occur in these patients as well. differential diagnosis. the differential diagnoses for devic's syndrome includes ms, adem, pulmonary tuberculosis, and viral infection (especially in the immunocompromised patient). in patients with an apparent affected family member, consideration should be given to mitochondrial disease. relapsing nmo should raise the suspicion for associated autoimmune disorders. because devic's syndrome can occur in persons older than age , when an unrelated ischemic optic neuropathy could occur, and because isolated or recurrent myelopathy may precede the on, additional consideration must be given to spinal cord compression, spinal cord tumor, and spinal arteriovenous malformation (avm) or dural fistula. evaluation. imaging is needed to exclude structural lesions and provide information on the pathological process. optic nerve or chiasm enlargement, t -weighted signal changes, and enhancement may be seen on head mri during the acute phase. increased t -weighted signal in the medulla is not uncommon and usually represents extension of high cervical lesions. spine mri characteristically shows cord swelling, signal changes, and enhancement extending over at least three levels ( fig. - ) . this appearance may resemble a spinal cord tumor, prompting consideration for biopsy. on magnetization transfer (mt) mri, no significant difference was found on normal-appearing white matter of devic's patients and control subjects, whereas ms patients had a significantly lower mt ratio peak and histogram average. t hypointense lesions in the cord and linear lesions that cross over more than two segments are more suggestive of devic's disease. an occasional patient may need prone and supine myelography to exclude a spinal dural-based avm. laboratory investigations reveal an elevated erythrocyte sedimentation rate in one third, positive antinuclear antibodies in nearly one half, and occasionally other autoantibodies (e.g., thyroperoxidase antibodies). it is reasonable to exclude syphilis, lyme disease, and human immunodeficiency virus by laboratory testing. in a few patients with the far east variety of devic's disease, hyperprolactinemia was described predominantly with optic nerve involvement. a chest x-ray helps to exclude pulmonary tuberculosis and sarcoidosis. csf examination is an essential part of the evaluation for devic's syndrome, and repeated studies are sometimes necessary to ensure that there is no infection in that the csf findings are sometimes atypical for inflammatory demyelination. a marked pleocytosis is often present, sometimes exceeding cells. moreover, neutrophils are commonly seen in csf and may predominate, a situation virtually unknown in ms. the protein concentration is often very high and in % exceeds mg/dl. anti-mog antibodies are the predominant autoantibody detected in csf; anti-mbp or anti-s b antibodies are less frequently seen. despite the intense inflammatory response, ocbs are conspicuously absent in the majority, being present in fewer than % of patients. csf serology for the herpesvirus family (hsv types and , vzv, ebv, and cmv) is important, and polymerase chain reaction testing should be done in cases suggestive of viral infection (immunocompromised patients). management. patients with acute or subacute devic's syndrome may respond to corticosteroids (e.g., intravenous methylprednisolone). they may respond to plasma exchange even when intravenous methylprednisolone does not produce significant improvement. attempts at preventing relapses and the subsequent disability are often disappointing even with the use of immunosuppressive agents. the classic injectable immunomodulators used in ms are insufficient to reduce the relapse rate in relapsing nmo. most commonly, a combination of azathioprine and prednisone is used for secondary prevention. other agents including mycophenolate mofetil, ivig, and mitoxantrone have been described to be effective in some cases. a small study of rituximab, a humanized anti-cd antibody showed a siginifcant reduction in the relapse rate of patients, making of relapse free. a large multicenter study of rituximab in nmo is in the planning stages. supportive care is important in the management of nmo. these patients are prone to many complications and require measures to prevent deep venous thrombosis and pulmonary embolism, urinary tract infection, decubiti, and contractures. mechanical ventilation may be needed either temporarily or permanently. patients with monophasic devic's syndrome generally have simultaneous or rapid onset of the on and myelitis (interval usually less than month). although some have significant residual disability, many recover remarkably and have little or no permanent deficits. a history of previous vague neurological symptoms or definite demyelinating events is predictive of future relapses, either typical of ms or relapsing nmo. those patients destined for recurrent myelitis and on have a longer interval between the onsets of myelitis and on. the vast majority of patients with relapsing nmo have very aggressive disease with frequent and severe exacerbations and a poor prognosis. adem is a monophasic inflammatory demyelinating disorder that characteristically begins within weeks of an antigenic challenge such as infection or immunization. it occurs more often in the young and causes the rapid development of multifocal or focal neurological deficits. perivenous inflammation, edema, and demyelination are the pathological hallmarks of adem, although these lesions commonly enlarge and coalesce, forming lesions pathologically indistinguishable from ms. moreover, perivascular changes typical of adem are common in patients with ms. there is considerable overlap in the epidemiological, clinical, csf, imaging, and pathological features between adem and ms, often making it difficult to distinguish between the two with reasonable confidence when encountering patients with a single demyelinating event. pathogenesis and pathophysiology. adem closely resembles the experimental allergic encephalomyelitis animal model of ms (eae) both clinically and pathologically, and is most likely due to a transient autoimmune response toward myelin. the occurrence of adem after vaccination with the rabbit spinal cord preparation of rabies virus led to the discovery of eae. infections and non-cnscontaining vaccinations may induce adem by molecular mimicry or by activating autoreactive t-cell clones in a nonspecific manner. lymphocyte reactivity toward mbp has been identified in blood and csf from patients with adem, but its absence in others indicates a role for other antigens. increased peripheral blood g interferon-producing t cells have been described in adem. epidemiology and risk factors. adem can occur at any age but perhaps because of the higher frequency of immunization and exposure to new antigens; it is most common during childhood. unlike ms, both sexes are affected with equal frequency. no association has been noted with pregnancy. adem has been reported to follow a number of different immunizations, usually within weeks, including those for pertussis, diphtheria, measles, mumps, rubella, influenza (postvaccination adem), tetanus, and yellow fever. in addition, there are case reports of adem following hepatitis b vaccination. however, the only epidemiologically and pathologically proved association is with rabies vaccination, which also causes demyelinating peripheral neuropathies. the original pasteur rabies vaccine, prepared in rabbit spinal cord, was associated with an incidence of adem of approximately per to per , vaccinations and is no longer in use. a later vaccine, made in duck embryo, which contains little neural tissue, carries a risk for adem of per , vaccines. the use of human diploid cell lines, which contain no nervous system tissue, for the production of rabies vaccine has virtually eliminated the risk of adem. the association of bee stings with adem has also been reported. parainfectious adem usually follows onset of the infectious illness, often during the recovery phase, but because of the latency between pathogen exposure and illness it may precede clinical symptoms of infection or the two may occur simultaneously. the most commonly reported associated illness is a nonspecific upper respiratory tract infection. there have been a vast number of specific infections associated with adem, such as virus infections (including rubella, mumps, vzv, ebv, cmv, influenza, coxsackievirus, and hepatitis c) and infection with mycoplasma, borrelia burgdorferi, and leptospira. measles carries the highest risk for adem of any infection, occurring in per to per cases. although adem has been reported in association with measles immunization, the risk is far lower than the risk of acquiring measles and its neurological complications. clinical features and associated disor-ders. a prodrome of headache, low-grade fever, myalgias, and malaise often precedes the onset of adem by a few days. in a german study of cases, the most frequent clinical signs were motor deficit ( %), followed by sensory deficits, brain stem signs, and cerebellar signs. csf findings were variable; normal results were present in up to % of patients. oligoclonal bands were positive in over %. almost all patients improved during the acute phase of the disease. of the patients with the final diagnosis of adem, had minor or no symptoms, died, the rest had moderate symptoms. compared to ms patients, the adem patients were older, and more often had a preceding infection, clinical signs of brain stem involvement, a higher csf albumin fraction, and infratentorial lesions. neurological symptoms develop rapidly in the acute phase and are commonly associated with encephalopathy, stupor, coma, meningismus, and seizures. peak severity occurs within several days, and recovery may begin soon afterward. occasionally, adem may evolve over a few months and there may be a second clinical deterioration or subacute progression for a time. in these unusual cases, the distinction from ms is difficult. three recent large retrospective series and an accompanying editorial have highlighted that there remain no clinical or laboratory features that accurately allow one to predict which adult or pediatric adem patients will develop. [ ] [ ] [ ] [ ] differential diagnosis. one of the primary concerns after a single demyelinating episode is whether other bouts can be expected (e.g., ms). several features may tip the balance toward one or the other, but the proper diagnosis becomes apparent only with time. classically, adem is characterized by the multifocal involvement at onset whereas ms often presents with monosymptomatic deficits such as on. however, adem may cause unifocal symptoms and ms may present with multifocal cns involvement, especially in children. the monosymptomatic deficits caused by adem are more commonly severe, such as bilateral on and complete transverse myelitis. although ocbs occur transiently in about one third of adem cases, their persistence implies a diagnosis of ms. the subsequent disappearance of ocbs, when performed by consistent techniques, is evidence against ms. the mri appearance of these two disorders is often identical, but the presence of basal ganglia or cortical lesions, or large globular white matter lesions, is more frequent in adem. the fulminant development of adem is distinctive but not pathognomonic, because a rare form of ms known as marburg's ms is also rapid in onset and often deadly. the appearance of brain stem, periventricular, and multiple, large cerebral white matter lesions and the presence of ocbs may distinguish marburg's variant from adem. on rare occasions, inflammatory demyelinating lesions may reach a large size and resemble tumors (especially lymphoma) on mri, necessitating biopsy for clarification. there is usually one dominant lesion, but smaller separate lesions may be identifiable. these have been referred to as both adem and ms in the literature. the prognosis for recovery is often quite good, although approximately one third suffer subsequent attacks. some develop typical ms, whereas others have recurring tumor-like lesions. the term multiphasic adem has been used when patients have large recurrences in the same location, and relapsing adem refers to recurrences at different sites. the relationship of these entities with ms is unclear. balo's concentric sclerosis refers to the pathological finding of alternating bands of demyelination and remyelination. these patients typically have large lesions and subacute deficits similar to those described earlier. typical demyelinating lesions commonly coexist, and rarely cdms patients are noted to have similar-appearing lesions. the reason for this peculiar alternating pattern is unknown. schilder's myelinoclastic diffuse sclerosis is another rare condition that may be confused with adem or other demyelinating conditions. this progressive demyelinating disorder usually begins in childhood. the features are often atypical and include dementia, aphasia, homonymous hemianopia, seizures, psychosis, elevated intracranial pressure, and the absence of ocbs. the most characteristic finding is the presence of two large, roughly symmetrical lesions on mri, one in each hemisphere. the diagnosis is made by excluding the known inherited leukodystrophies, especially adrenoleukodystrophy. management. treatment with intravenous methylprednisolone seems to halt progression and allow recovery to begin sooner, just as with ms. plasma exchange can be tried in those with severe deficits and little response to corticosteroids. ivig has also been used successfully according to case reports in the literature. one fulminant case responded to hypothermia only. genetics of multiple sclerosis utility of mri in suspected ms the use of mitoxantrone (novantrone) for the treatment of multiple sclerosis disease modifying therapies in multiple sclerosis natural history of multiple sclerosis a randomized study of two interferon-beta treatments in relapsing-remitting multiple sclerosis cortical lesions and brain atrophy in ms the pathology of multiple sclerosis management of multiple sclerosis: current trials and future options competing interests in multiple sclerosis 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allergic encephalomyelitis: a misleading model of multiple sclerosis igg marker of optic-spinal multiple sclerosis binds to the aquaporin- water channel isolation of herpes simplex virus type during first attack of multiple sclerosis isolation of virus from the spinal fluid of three patients with multiple sclerosis and one with amyotrophic lateral sclerosis plaque-associated expression of human herpesvirus in multiple sclerosis oligodendrocytes and oligodendrocyte/type- astrocyte progenitor cells of adult rats are specifically susceptible to the lytic effects of complement in absence of antibody experimental autoimmune encephalomyelitis: immunotherapy with anti-tumor necrosis factor antibodies and soluble tumor necrosis factor receptors evidence for pathogenic heterogeneity in multiple sclerosis transected neurites, apoptotic neurons, and reduced inflammation in cortical multiple sclerosis lesions cortical demyelination and diffuse white matter injury in multiple sclerosis a genetic basis for familial aggregation in multiple sclerosis. canadian collaborative study group genetics of multiple sclerosis multiple sclerosis: changing times epidemiology of multiple sclerosis in u.s. veterans: v. ancestry and the risk of multiple sclerosis unusual occurrence on a tropical island of multiple sclerosis when is an apparent excess of neurologic cases epidemiologically significant smoking is a risk factor for multiple sclerosis environmental risk factors in multiple sclerosis: causes, triggers, and patient autonomy pregnancy is associated with a lower risk of onset and a better prognosis in multiple sclerosis diagnosis of ms the natural history of recurrent optic neuritis the prevalence of cognitive impairment in a community survey of multiple sclerosis defining the clinical course of multiple sclerosis: results of an international survey. national multiple sclerosis society (usa) advisory committee on clinical trials of new agents in multiple sclerosis epilepsy and multiple sclerosis multiple sclerosis-associated uveitis a reappraisal of the epidemiology of multiple sclerosis in olmsted county optic neuritis: a population-based study in olmsted county the significance of brain magnetic resonance imaging abnormalities at presentation with clinically isolated syndromes suggestive of multiple sclerosis. a -year follow-up study interscanner variation in brain mri lesion load measurements in ms: implications for clinical trials the prognostic value of brain mri in clinically isolated syndromes of the cns. a -year follow-up a longitudinal study of abnormalities on mri and disability from multiple sclerosis occurrence of a multiple sclerosis-like illness in women who have a leber's hereditary optic neuropathy mitochondrial dna mutation genetic disorders that masquerade as multiple sclerosis new diagnostic criteria for multiple sclerosis: guidelines for research protocols recommended diagnostic criteria for multiple sclerosis: guidelines from the international panel on the diagnosis of multiple sclerosis assessment of mri criteria for a diagnosis of ms neuroimaging in multiple sclerosis the relationship between whole brain volume and disability in multiple sclerosis: a comparison of normalized gray vs. white matter with misclassification correction magnetization transfer ratio histogram analysis of normal-appearing gray matter and normalappearing white matter in multiple sclerosis mri t hypointensity of the dentate nucleus is related to ambulatory impairment in multiple sclerosis evidence of early cortical atrophy in ms: relevance to white matter changes and disability csf electrophoresis in one thousand patients early differential diagnosis of multiple sclerosis using a new oligoclonal band test diagnostic criteria for multiple sclerosis: revisions to the "mcdonald criteria the optic neuritis treatment trial: three-year follow-up results effects of iv methylprednisolone on brain atrophy in relapsing-remitting ms double-blind study of true vs. sham plasma exchange in patients treated with immunosuppression for acute attacks of multiple sclerosis plasmapheresis in acute episodes of fulminant cns inflammatory demyelination a randomized trial of plasma exchange in acute central nervous system inflammatory demyelinating disease relation between humoral pathological changes in multiple sclerosis and response to therapeutic plasma exchange plasma exchange for severe attacks of cns demyelination: predictors of response intramuscular interferon beta- a therapy initiated during a first demyelinating event in multiple sclerosis. champs study group interferon beta- a for early multiple sclerosis: champs trial subgroup analyses effect of early interferon treatment on conversion to definite multiple sclerosis: a randomised study interferon beta- b is effective in relapsing-remitting multiple sclerosis. i. clinical results of a multicenter, randomized, double-blind, placebo-controlled trial. the ifnb multiple sclerosis study group further study on the specificity and incidence of neutralizing antibodies to interferon (ifn) in relapsing remitting multiple sclerosis patients treated with ifn beta- a or ifn beta- b intramuscular interferon beta- a for disease progression in relapsing multiple sclerosis. the multiple sclerosis collaborative research group (mscrg) the long-term safety and tolerability of high-dose interferon beta- a in relapsing-remitting multiple sclerosis: -year data from the prisms study randomised double-blind placebo-controlled study of interferon beta- a in relapsing/remitting multiple sclerosis. prisms (prevention of relapses and disability by interferon beta- a subcutaneously in multiple sclerosis) study group magnetic resonance imaging results of the prisms trial: a randomized, double-blind, placebo-controlled study of interferon-beta a in relapsing-remitting multiple sclerosis. prevention of relapses and disability by interferon-beta a subcutaneously in multiple sclerosis ingested ifn-alpha: results of a pilot study in relapsing-remitting ms clinical characteristics of responders to interferon therapy for relapsing ms a pilot trial of cop in exacerbating-remitting multiple sclerosis copolymer reduces relapse rate and improves disability in relapsing-remitting multiple sclerosis: results of a phase iii multicenter, double-blind placebocontrolled trial. the copolymer multiple sclerosis study group glatiramer acetate reduces the proportion of new ms lesions evolving into "black holes european/canadian multicenter, double-blind, randomized, placebo-controlled study of the effects of glatiramer acetate on magnetic resonance imaging-measured disease activity and burden in patients with relapsing multiple sclerosis. european/canadian glatiramer acetate study group united states open-label glatiramer acetate extension trial for relapsing multiple sclerosis: mri and clinical correlates. multiple sclerosis study group and the mri analysis center longitudinal mri study: the effects of azathioprine in ms patients refractory to interferon beta- b the austrian immunoglobulin in ms (aims) study: final analysis a randomized trial of intravenous immunoglobulin in inflammatory demyelinating optic neuritis iv immunoglobulin does not reverse established weakness in ms placebo controlled pilot trial to study the remyelinating potential of intravenous immunoglobulins in multiple sclerosis a double-blind, randomized trial of iv immunoglobulin treatment in acute optic neuritis a controlled trial of natalizumab for relapsing multiple sclerosis progressive multifocal leukoencephalopathy complicating treatment with natalizumab and interferon beta- a for multiple sclerosis t( ) hypointense lesions in secondary progressive multiple sclerosis: effect of interferon beta- b treatment placebo-controlled multicentre randomised trial of interferon beta- b in treatment of secondary progressive multiple sclerosis. european study group on interferon beta- b in secondary progressive ms effect of interferon-beta- b on magnetic resonance imaging outcomes in secondary progressive multiple sclerosis: results of a european multicenter, randomized, double-blind, placebo-controlled trial. european study group on interferon-beta- b in secondary progressive multiple sclerosis final analysis of the european multicenter trial on ifnbeta- b in secondary-progressive ms the effect of interferon beta- b treatment on mri measures of cerebral atrophy in secondary progressive multiple sclerosis. european study group on interferon beta- b in secondary progressive multiple sclerosis randomized controlled trial of interferonbeta- a in secondary progressive ms: mri results interferon beta- b in secondary progressive ms: a combined analysis of the two trials european study on intravenous immunoglobulin in multiple sclerosis: results of magnetization transfer magnetic resonance imaging analysis relapses and progression of disability in multiple sclerosis mitoxantrone in progressive ms: a placebo controlled, randomized, observer blinded phase iii multi-center study: clinical results therapeutic effect of mitoxantrone combined with methylprednisolone in multiple sclerosis: a randomised multicentre study of active disease using mri and clinical criteria a study of therapy-related acute leukaemia after mitoxantrone therapy for multiple sclerosis mitoxantrone in progressive multiple sclerosis: a placebo-controlled, double-blind, randomised, multicentre trial effect of mitoxantrone on mri in progressive ms: results of the mims trial interferon beta- a in primary progressive multiple sclerosis overview of azathioprine treatment in multiple sclerosis lowdose ( . mg) oral methotrexate reduces the rate of progression in chronic progressive multiple sclerosis cladribine in treatment of chronic progressive multiple sclerosis cladribine and progressive ms: clinical and mri outcomes of a multicenter controlled trial. cladribine mri study group autologous stem cell transplantation in progressive multiple sclerosis-an interim analysis of efficacy controlled randomised crossover trial of the effects of physiotherapy on mobility in chronic multiple sclerosis a randomized controlled crossover trial of aspirin for fatigue in multiple sclerosis donepezil improved memory in multiple sclerosis in a randomized clinical trial stereotactic lesional surgery for the treatment of tremor in multiple sclerosis: a prospective case-controlled study a study of tremor in multiple sclerosis ventilatory dysfunction in multiple sclerosis the reproductive effects of beta interferon therapy in pregnancy: a longitudinal cohort the natural history of multiple sclerosis: a geographically based study. i. clinical course and disability change in ms-related disability in a population-based cohort: a -year follow-up study the clinical course of neuromyelitis optica (devic's syndrome) devic's neuromyelitis optica: a clinicopathological study of patients a serum autoantibody marker of neuromyelitis optica: distinction from multiple sclerosis neuromyelitis optica: clinical predictors of a relapsing course and survival mri and magnetization transfer imaging changes in the brain and cervical cord of patients with devic's neuromyelitis optica clinical, csf, and mri findings in devic's neuromyelitis optica neuromyelitis optica acute disseminated encephalomyelitis: a follow-up study of adult patients adem: distinct disease or part of the ms spectrum clinical and neuroradiologic features of acute disseminated encephalomyelitis in children acute disseminated encephalomyelitis. mri findings and the distinction from multiple sclerosis interactive study questions and referenced videos for this chapter are available on the dvd-rom key: cord- -cz y u authors: maziarz, eileen k.; perfect, john r. title: cryptococcosis date: - - journal: diagnosis and treatment of fungal infections doi: . / - - - - _ sha: doc_id: cord_uid: cz y u cryptococcosis is an infectious disease caused by the encapsulated fungi cryptococcus neoformans and cryptococcus gattii. once a relatively uncommon cause of human disease, cryptococcal infection can develop in apparently immunocompetent hosts and has emerged as an important opportunistic infection in humans over the past several decades as immunocompromised populations expand in the setting of hiv/aids, organ transplantation, malignancies, and treatment for other conditions. clinical manifestations are myriad but pulmonary and central nervous system (cns) infections are the most common. improvements in diagnostic testing and standardized approaches to antifungal therapy, when available, have made considerable impact in the management of this infection. while the widespread use of highly active antiretroviral therapy (haart) has improved the outcome of cryptococcosis in many hiv-infected patients, cryptococcosis remains an entity of considerable morbidity and mortality in many parts of the world, and restoration of host immunity can present management challenges that require individualized management. as immunocompromised populations continue to expand, it is likely that cryptococcosis will remain an important opportunistic fungal infection of humans requiring ongoing investigation. cryptococcosis is an infectious disease with a wide range of clinical presentations caused by pathogenic encapsulated yeasts in the genus cryptococcus. currently, there are two species of these fungi that commonly cause disease in humans: cryptococcus neoformans, which causes cryptococcosis in both immunocompetent and immunocompromised hosts, and cryptococcus gattii, which is primarily a pathogen in apparently immunocompetent patients but can also cause disease in the immunocompromised. c. neoformans was first identified as a human pathogen in by two german physicians, otto busse and abraham buschke, when they described a circular yeast-like microorganism in a lesion on the tibia of a woman; the microorganism was initially named saccharomyces hominis [ ] . the name c. neoformans has been consistently adopted in both the mycology and medical literature since [ ] . in the mid- s, when kwon-chung discovered two mating types of c. neoformans that produced fertile basidiospores, the organisms were subsequently separated into two varieties, var. neoformans (serotypes a and d) and var. gattii (serotypes b and c). these two varieties were recently separated into two species, c. neoformans and c. gattii, based on their genetic background and phylogenetic diversity, as proposed by kwon-chung in [ ] . it is possible, as more molecular information is gathered from genome sequencing, that c. neoformans var. neoformans (serotype d) and c. neoformans var. grubii (serotype a) will be divided into separate species as well as other cryptic species. the incidence of cryptococcosis began to rise in the late s. early case reports of cryptococcal infections were primarily associated with cancer, autoimmune diseases, organ transplantation, and receipt of corticosteroids as these immunocompromised populations expanded [ ] . a major surge in new cases of cryptococcosis occurred during the first two decades of the hiv pandemic, when cryptococcal infection was an important opportunistic infection (oi) in all parts of the world. furthermore, around , c. gattii strains (previously geographically restricted to tropical and subtropical regions) caused a localized outbreak of cryptococcosis in apparently immunocompetent individuals on vancouver island [ ] . this has increased recognition that these fungi can exploit new geographical environments and cause disease in both immunocompromised and apparently immunocompetent hosts. despite the development of highly active antiretroviral therapy (haart), which has decreased the rate of hiv-related cryptococcosis in developed countries, the burden of cryptococcal infection is still very high in developing countries and in those individuals without access to health care. it has been estimated that there are a million cases per year with more than , deaths due to cryptococcosis worldwide [ ] . the life cycle of c. neoformans and c. gattii involve asexual (yeast) and sexual (basidiospores/hyphae) forms. the asexual form is the encapsulated yeast that reproduces by narrow-based budding and is found most commonly in clinical specimens, whereas the sexual stage, which exists in one of two mating types, "alpha" or "a," is observed only under certain conditions, resulting in meiosis to form basidiospores. the vast majority of clinical infections and environmental isolates are caused by "alpha" mating-type locus strains. since the sexual stage of c. neoformans and c. gattii has been described, their teleomorphs were named filobasidiella neoformans and filobasidiella bacillospora, respectively. c. neoformans and c. gattii usually appear as white-tocream, opaque, and mucoid colonies that grow to several millimeters in diameter on the most routine agar within - h. with some strains, a few colonies occasionally develop sectors with different pigmentation or different morphologies (i.e., wrinkled, smooth, mucoid). both cryptococcal species will grow readily on most fungal culture media without cycloheximide at - °c in aerobic conditions. however, c. neoformans is generally more thermotolerant than c. gattii, and, within this species, serotype a is generally more tolerant than serotype d strains. in addition to their ability to grow at °c, the yeast produce a thick shedding polysaccharide capsule, melanin pigments, and the enzymes urease and phospholipase, which allow cryptococcus to be readily identified from other yeasts. these are also considered to be yeast virulence factors. cryptococcosis was considered an uncommon infection prior to the aids epidemic, associated with malignancies, organ transplantation, and certain immunosuppressive treatments. beginning in the early s, the incidence of cryptococcosis increased significantly and between and % of persons with aids developed cryptococcosis [ , ] . in fact, hiv/aids was found to be associated with % of cryptococcosis cases worldwide. cryptococcal infection is a major oi in hiv-infected patients as the cd + cell count falls below cells/µl. following widespread implementation of haart, the incidence of cryptococcosis among patients with hiv/aids has fallen significantly in most developed nations. the incidence of cryptococcal infection in persons not infected with hiv has remained stable during this time. moreover, in developing nations with limited access to haart, the prevalence of and morbidity and mortality associated with cryptococcosis remains unacceptably high, accounting for up to , deaths per year. besides hiv infection, other risk factors for acquiring cryptococcal infections include many conditions that result in an immunocompromised status (table . ). although both c. neoformans and c. gattii can cause cryptococcosis in apparently normal hosts, the percentage of c. gattii infections causing disease in such patients is significantly higher than for c. neoformans. c. neoformans is found throughout the world in association with excreta from certain birds such as pigeons and in tree hollows. c. gattii is commonly associated with several species of eucalyptus and other trees [ ] . while the link between the environmental source of infection and cryptococcosis cases is not precise, there is evidence to suggest an increased risk of cryptococcosis and asymptomatic cryptococcal antigenemia following intense bird exposures. recently, there has been a strong link between the c. gattii outbreak in humans on vancouver island and common environmental yeast exposures. although these fungi can be detected in endobronchial specimens from humans without disease (colonization), clinicians should be alert for subclinical disease or potential for disease when cryptococcus is isolated from any clinical specimen. approximately % of cryptococcal infections are caused by serotype a strains ( c. neoformans var. grubii) with the remaining - % of infections caused by serotype d ( c. neoformans var. neoformans) or serotype b and c strains ( c. gattii). whereas c. neoformans serotype a is found worldwide, serotypes b and c are found primarily in (table . ) [ ] . in australia and new zealand, serotypes b and c caused up to % of all cases of cryptococcosis cases in one study, but serotype a remains the predominant serotype even in these endemic areas [ ] . to date, only c. gattii strains have been reported to cause a widespread defined outbreak of disease [ ] . cryptococcosis occurs primarily by inhalation of the infectious propagules, either dehydrated (poorly encapsulated) yeasts or basidiospores, into pulmonary alveoli. direct inoculation into tissue due to trauma can be a portal of entry in occasional cases and, potentially, yeast may enter through the gastrointestinal tract. after the yeasts are inhaled into the lungs of a susceptible host, they encounter alveolar macrophages, and other inflammatory cells are recruited through release of cytokines and chemokines such as interleukin (il)- , il- , monocyte chemotactic protein (mcp)- , and macrophage inflammatory protein (mip)- α. cryptococcal infection primarily involves granulomatous inflammation, which is a result of a helper t cell (th ) response with cytokines including tumor necrosis factor, interferon-γ, and il- [ ] . in many circumstances, the yeasts remain dormant (yet viable) in hilar lymph nodes or pulmonary foci of an asymptomatic individual for years and then disseminate outside those complexes when local immunity is suppressed, similar to that which is observed in cases of reactivation tuberculosis or histoplasmosis [ ] . in a patient with severely compromised cellular immunity, the yeasts reactivate and proliferate at the site of infection and then disseminate to other sites causing progressive clinical symptoms. recent advances in the molecular biology of cryptococcus have confirmed several virulence factors. the three classical virulence factors of c. neoformans include: capsule formation, melanin pigment production, and the ability to grow well at °c [ , ] . the prominent antiphagocytic polysaccharide capsule, which is composed of glucuronoxylomannan (gxm), is unique to cryptococcus species and is considered an essential virulence factor that has multiple effects on host immunity. in addition, c. neoformans possesses an enzyme that catalyzes the conversion of diphenolic compounds to form melanin, which may have a biological role to protect the yeasts from host oxidative stresses and which may partially explain the organism's neurotropism. finally, its ability to grow at °c is a basic part of the virulence composite for most of the human pathogenic fungi including cryptococcus, as molecular studies have linked high-temperature growth with certain signaling pathways and enzymes that this yeast has acquired or adapted over time in order to enhance its pathogenicity. other virulence factors include phospholipase and urease production and multiple enzymes associated with protection against oxidative stresses. c. neoformans and c. gattii have a predilection for establishing clinical disease in the lungs and central nervous system (cns). other organs that may be involved in cryptococcosis include skin, prostate, eyes, bone, and blood [ , , , ] . in fact, this yeast may cause disease in any organ of the human body, and widely disseminated cryptococcal infection can affect multiple organs in severely immunosuppressed patients (table . ). the respiratory tract serves as the most important portal of entry for this yeast, and thus there are many clinical manifestations of pulmonary cryptococcosis, ranging from asymptomatic transient or chronic colonization of the airways or simply a pulmonary nodule on radiograph to life-threatening fungal pneumonia with acute respiratory distress syndrome (ards) [ , ] . in a normal host with cryptococcal infection, asymptomatic pulmonary cryptococcosis can occur in about one third of patients with pulmonary infection and patients may present to care with only an abnormal chest radiograph. the most common radiologic findings of cryptococcosis include well-defined single or multiple noncalcified nodules ( fig. . ) and pulmonary infiltrates ( fig. . ), but other less frequent radiographic findings include pleural effusions, hilar lymphadenopathy, and lung cavitation. patients with pulmonary cryptococcosis can present with symptoms of acute onset of fever, productive cough, respiratory distress, chest pain, and weight loss [ ] . the outbreak of c. gattii infections in vancouver island included several cases of severe symptomatic pulmonary cryptococcosis in apparently immunocompetent individuals. in an immunocompromised patient, especially those with hiv infection, cryptococcal pneumonia is usually symptomatic and can progress rapidly to ards, even in the absence of cns involvement. most immunocompromised patients with cryptococcal in-fection, however, present with cns rather than pulmonary symptoms. in fact, more than % of hiv/aids patients with cryptococcal infection already have cns cryptococcosis at the time of diagnosis, many of whom will have a paucity of respiratory complaints. the findings in chest radiographs of immunocompromised patients with pulmonary cryptococcosis are the same as those in immunocompetent patients, but alveolar and interstitial infiltrates tend to be more frequent and imaging can mimic pneumocystis pneumonia. accelerated presentations of cryptococcal pneumonia are more common among immunocompromised patients. in pulmonary cryptococcosis, if the infection is confined to the lung, serum cryptococcal polysaccharide antigen is usually negative. however, while a positive serum polysaccharide antigen may indicate the dissemination of the yeast from the lung, it does not confirm cns involvement. in immunocompromised individuals with pulmonary cryptococcosis, a lumbar puncture to rule out cns disease should be considered regardless of the patient's symptoms or serum polysaccharide antigen test results. the only setting in which screening a lumbar puncture may not necessarily need to be performed in a patient with cryptococcus isolated from the lung is in the asymptomatic, immunocompetent patient with disease that appears to be limited to the lungs. clinical manifestations of cns cryptococcosis include headache, fever, cranial neuropathy, alteration of consciousness, lethargy, memory loss, and signs of meningeal irritation [ , ] . these findings are usually present for several weeks and therefore cause a clinical syndrome of subacute meningitis or meningoencephalitis. however, on some occasions, patients can present more acutely or lack typical features including headache. in hiv-infected patients with cns cryptococcosis, the burden of fungal organisms in the cns is usually high. therefore, these patients may have a shorter onset of signs and symptoms, higher cerebrospinal fluid (csf) polysaccharide antigen titers and intracranial pressures (icps), and slower csf sterilization after starting antifungal treatment. different species may produce differences in clinical manifestations. for instance, one species may have a predilection to cause disease in brain parenchyma rather than the meninges. in certain areas of the world, c. gattii tends to cause cerebral cryptococcomas ( fig. . ) and/or hydrocephalus with or without large pulmonary mass lesions more frequently than c. neoformans. these patients with brain parenchymal involvement usually have high icp and cranial neuropathies, and respond poorly to antifungal therapy. cutaneous infections are the third most common clinical manifestations of cryptococcosis. patients can manifest several types of skin lesions. one common skin lesion is a papule or maculopapular rash with central ulceration that may be described as "molluscum contagiosum-like." these lesions are indistinguishable from those due to other fungal infections including histoplasma capsulatum, coccidioides immitis, and penicillium marneffei. other cutaneous lesions of cryptococcosis include acneiform lesions, purpura, vesicles, nodules, abscesses, ulcers ( fig. . ) , granulomas, pustules, plaques, draining sinus, and cellulitis. because there are many skin manifestations in cryptococcosis that mimic other infectious as well as malignant conditions, skin biopsy with culture and histopathology are essential for definitive diagnosis. skin lesions of cryptococcosis usually represent disseminated cryptococcal infection. primary cutaneous cryptococcosis is very rare and is usually associated with skin injury and direct inoculation of the yeasts. solid organ transplant (sot) recipients on tacrolimus seem to be more likely to develop skin, soft-tissue, and osteoarticular infections due to cryptococcus [ ] . tacrolimus has anti-cryptococcal activity at high temperatures, but loses this activity as environmental temperatures decrease; this may in part explain the increased frequency of cutaneous cryptococcosis in these patients. despite this series of patients, however, the most common site of disseminated infection in sot recipients still remains the cns, including patients receiving tacrolimus. prostatic cryptococcosis is usually asymptomatic, and the prostate gland is considered to be a sanctuary site for this yeast. the prostate may serve as an important reservoir for relapse of cryptococcosis in patients with a high fungal burden [ ] . latent c. neoformans infection has even been recognized to disseminate to the bloodstream during urological surgery on the prostate [ ] . cultures of urine or seminal fluid may still be positive for cryptococcus after initial antifungal treatment of cryptococcal meningitis in aids patients [ ] , strongly supporting the need for prolonged antifungal treatment to clear the prostate in these severely immunocompromised patients. in the early reports of cryptococcal meningitis before the aids epidemic, ocular signs and symptoms were noted in approximately % of cases [ ] . the most common manifestations were ocular palsies and papilledema. in the present hiv era, several other manifestations of ocular cryptococcosis have been identified, including the presence of extensive retinal lesions with or without vitritis, which can lead to irreversible blindness. furthermore, catastrophic loss of vision without evidence for endophthalmitis has also been reported [ ] . visual loss may be due to one of two pathogenic processes. the first is caused by infiltration of the optic nerve with the yeasts, producing rapid visual loss with few effective treatments. the second is due to increased icp and compression of the ophthalmic artery. in this setting, patients have slower visual loss and treatment with serial lumbar punctures or ventricular shunts can prevent or slow down visual loss. in addition to lung, cns, skin, prostate, and eye, c. neoformans can cause disease in many other organs (table . ). cryptococcemia can occur in severely immunosuppressed patients but rarely causes endocarditis. bone involvement of cryptococcosis typically presents as one or more circumscribed osteolytic lesions in any bone of the body, occasionally associated with "cold" soft-tissue abscesses, and has been associated with sarcoidosis. bone marrow infiltration can be observed in severely immunocompromised hosts. cryptococcal peritonitis [ ] and cryptococcuria are also reported in several case series. any organ of the human body can be a site of cryptococcal infections. there are several methods used for the diagnosis of cryptococcosis. these techniques include direct examination of the fungus in body fluids, histopathology of infected tissues, serological studies, and culture of body fluids or tissues. molecular methods, while available, are not currently used in routine clinical practice. the most rapid method for diagnosis of cryptococcal meningitis is direct microscopic examination for encapsulated yeasts by an india ink preparation of csf. cryptococcus can be visualized as a globular, encapsulated yeast cell with or without budding, ranging in size from to µm in diameter. it is easily distinguished in a colloidal medium of india ink when mixed with csf ( fig. . ). approximately - ml of specimen is recommended for use in the india ink preparation. india ink examination can detect encapsulated yeasts in a csf specimen with a threshold between and colony-forming units of yeasts/ml of fluid. the sensitivity of the india ink preparation technique is - % in non-aids-related cryptococcal meningitis and up to % in aids-related disease. some false-positive results can be found from intact lymphocytes, myelin globules, fat droplets, and other tissue cells. also, dead yeast cells can remain in the csf and be visualized by india ink preparation for varying periods of time during and after appropriate antifungal treatment. this is a limitation of direct microscopy of csf during the management of cryptococcal meningitis [ ] . cryptococcus can be identified by histological staining of tissues from lung, skin, bone marrow, brain, or other organs [ ] . histopathological staining of centrifuged csf sediment is more sensitive for rapid diagnosis of cryptococcal meningitis than the india ink method [ ] . peritoneal fluid from chronic ambulatory peritoneal dialysis, seminal fluid, bronchial wash, or bronchoalveolar lavage fluid can also be used for cytology preparations in the diagnosis of cryptococcal infections, whereas india ink preparations from these body fluids are difficult to interpret [ , ] . fine needle aspiration for cytology of peripheral lymph nodes, adrenal glands, or vitreous aspiration; percutaneous transthoracic biopsy under real-time ultrasound guidance; or video-assisted thoracoscopic lung biopsy on pulmonary nodules, masses, or infiltrative lesions can be used for obtaining tissues for cytology/histopathology [ ] . a variety of positive staining methods have been described to demonstrate the yeast cells in tissue or fluids, ranging from the nonspecific papanicolaou or hematoxylin and eosin stains to the more specific fungal stains such as calcofluor, which binds fungal chitin, or gomori methenamine silver (gms), which stains the fungal cell wall [ , ] ( fig. . ). several stains can identify the polysaccharide capsular material surrounding the yeasts. these stains can be especially useful in presumptively identifying cryptococcus when the organism does not grow or cultures are not obtained. they include mayer's mucicarmine, periodic acid-schiff (pas), and alcian blue stains [ ] . the fontana-masson stain appears to identify melanin in the yeast cell wall. the fungus is observed as a yeast that reproduces by formation of narrow-based budding with a prominent capsule. gram stain is not optimal for identification of this yeast, but may show diagnosis of cryptococcosis has improved significantly over the past several decades with the development of serological tests for cryptococcal polysaccharide antigen and/ or antibody. use of serum cryptococcal antibodies for diagnosis of cryptococcosis has not been adopted. in contrast, detection of cryptococcal capsular polysaccharide antigen in serum or body fluids by a latex agglutination (la) technique has been robust in its performance and is considered the gold standard diagnostic test for serological diagnosis of cryptococcosis. this test uses latex particles coated with polyclonal cryptococcal capsular antibodies or anti-gxm monoclonal antibodies and has overall sensitivities and specificities of - % and - %, respectively [ , ] . the false-positive rate of cryptococcal capsular polysaccharide antigen testing is - . % [ ] . the majority of false-positive results can be explained by technical error (improper boiling/treatment), presence of rheumatoid factor or interference proteins, and infections with trichosporon beigelii [ ] or some bacterial species [ ] . however, most of the false-positive results of la testing for cryptococcal polysaccharide antigen have initial reciprocal titers of or less [ ] . therefore, results of such low titers must be carefully interpreted within the clinical context. falsenegative results of the la test for cryptococcal polysaccharide antigen in cryptococcal meningitis are unusual but can be seen due to a prozone effect, and, therefore, high-risk negative specimens should be diluted and retested [ ] . low fungal burden, as in chronic low-grade cryptococcal meningitis or in the very early stages of cryptococcal infection, and improper storage of patient sera can also cause false-negative results in la cryptococcal polysaccharide antigen tests [ ] . enzyme immunoassays (eias) for detection and quantification of cryptococcal polysaccharide antigen of all four serotypes of c. neoformans in sera and csf have been developed to detect the major component of the polysaccharide capsule, gxm, with sensitivities and specificities of . - and %, respectively [ , ] . this methodology is automated and overcomes some of the practical limitations of la testing. previous studies have compared eia and la assays and revealed no significant difference between these testing methods. eia for cryptococcal polysaccharide antigen does not give discrepant results with rheumatoid factor or serum macroglobulins and is not affected by prozone reactions. both la and eia testing have been rigorously studied and are recommended for use in both serum and csf samples. recently, a lateral flow assay (lfa) was introduced in the diagnostic repertoire for cryptococcal infection and is food and drug administration (fda) approved for use in serum and csf. the semiquantitative lfa offers many advantages over other serological methods, including rapid turnaround (approximately minutes), minimal requirements for specialized laboratory infrastructure, stability at room temperature, and low cost [ ] . the lfa has been evaluated against both eia and culture, with sensitivities of - % for serum and plasma and - % for urine samples [ ] [ ] [ ] [ ] . this assay has good performance across a broad range of clinical settings, including resource-limited settings and among cohorts with low burden of hiv infection and high rates of c. gattii infection, for which some eia and la tests are known to be insensitive [ ] [ ] [ ] [ ] [ ] . the satisfactory performance of lfa combined with established cost-effectiveness and practical advantages of this approach support its use as a point-of-care testing (including preemptive screening of high-risk patients) in resource-limited settings [ , , ] . although the presence of cryptococcal polysaccharide antigen in serum is undoubtedly suggestive for dissemination of cryptococcal infection outside the lung, the precise value of cryptococcal polysaccharide antigen for diagnosis of nondisseminated pulmonary cryptococcosis remains less certain. generally speaking, detectable cryptococcal antigen in serum should make clinicians consider that infection is now also located outside the lung. in a high-risk patient with clinical symptoms suggestive of meningitis, identification of cryptococcal antigen in csf or serum is rapid, specific, noninvasive, and virtually diagnostic of meningoencephali- tis or disseminated cryptococcosis even when the india ink examination or culture is negative [ , ] . the la test for serum cryptococcal polysaccharide antigen is widely used for detecting cryptococcal infection in patients with aids, as an initial screening test for those with fever of unclear etiologies or neurological symptoms. in some patients, it may represent the only means of achieving an etiologic diagnosis of invasive cryptococcosis or early diagnosis prior to cns involvement. likely because of its sensitivity, the detection of cryptococcal polysaccharide antigen in the serum may precede clinically obvious disseminated cryptococcal disease ("isolated cryptococcal polysaccharidemia") in severely immunosuppressed patients [ ] [ ] [ ] . the management of these cases, in which there is a positive serum antigen and other nonspecific clinical findings in hiv-infected patients with negative fluid or tissue cultures, is uncertain. persons of high risk with isolated cryptococcal antigenemia probably do benefit from antifungal therapy to prevent or delay the development of overt cryptococcosis [ ] . generally, positive serum antigen tests at titers of : or more strongly suggest cryptococcal infections in these patients. baseline cryptococcal polysaccharide antigen titers in serum and csf may carry prognostic significance in patients with cryptococcal meningitis [ ] . a study in hiv-related acute cryptococcal meningitis indicated that a baseline titer of csf cryptococcal polysaccharide antigen of : or greater was a predictor of death during systemic antifungal treatment [ ] . after initiation of systemic antifungal therapy, patients may respond to treatment and titers of cryptococcal polysaccharide antigen fall. similarly, a rise in csf cryptococcal polysaccharide antigen titers during suppressive therapy has been associated with relapse [ ] . however, it is important to emphasize that the use of changing antigen titers to make therapeutic decision should be done with caution, as titers may not be equivalent across different serological modalities [ ] . the kinetics of polysaccharide elimination remains unclear and, despite the accuracy of commercial kits for general diagnosis, the accuracy of specific titers can vary from kit to kit even from the same clinical specimen. cryptococcus can be easily grown from biologic samples such as csf, sputum, and skin biopsy on routine fungal and bacterial culture media. colonies can usually be observed on solid agar plates after - h of incubation at - °c in aerobic conditions. antibacterial agents, preferably chloramphenicol, can be added to the media when bacterial contamination is considered. the yeast, however, do not grow in the presence of cycloheximide at the con-centration used in selective fungal isolation media ( µg/ ml). despite relatively rapid growth for most strains, cultures should be held for - weeks before discarding, particularly for patients already receiving antifungal treatment. conversely, cultures may be negative despite positive microscopic examinations (india ink) due to nonviable yeast cells, which may persist for a prolonged period of time at the site of infection. positive blood cultures are frequently reported in aids patients and may actually be the first positive test for cryptococcal infection in a febrile high-risk patient. c. neoformans colonies will appear on routine fungal media as opaque, white, creamy colonies that may turn orange-tan or brown after prolonged incubation. the mucoid appearance of the colony is related to the capsule size around the yeasts. cryptococcus does not routinely produce hyphae or pseudohyphae, or ferment sugars, but is able to assimilate inositol and hydrolyze urea [ ] . c. neoformans and c. gattii have the ability to use galactose, maltose, galactitol, and sucrose [ ] . there are special media such as canavanine-glycine-bromthymol blue (cgb) agar that can be used to differentiate c. gattii strains from c. neoformans strains [ ] . a number of molecular techniques have been developed for identification of cryptococcal species from biological specimens including single and multiplex polymerase chain reaction (pcr) fingerprinting, random amplified polymorphic dna (rapd), pcr restriction fragment length polymorphism (rflp) analysis, multi-locus sequence typing (mlst), and matrix-assisted laser desorption ionization time-of-flight (maldi-tof) mass spectrometry [ ] [ ] [ ] [ ] [ ] [ ] [ ] . these highly sensitive and specific methods have been evaluated with a variety of biologic samples [ ] and can rapidly identify to the species and subspecies/genotypic level, including identification of recognized and novel strains within geographical niches [ ] . while the expense and specialized techniques required of these methods preclude widespread use in clinical practice, their use in larger-scale investigations will continue to enhance our understanding of the epidemiology, pathogenesis, and nuances of antifungal management, as well as identify microevolution of different strains [ ] . the infectious diseases society of america clinical practice guidelines for the management of cryptococcal disease (summarized in tables . , . ), updated in , provide a suitable framework for therapeutic decision making [ ] . the updated guidelines provide detailed recommendations for specific "at-risk" populations and address different management strategies based on host, site of infection, and potential complications of cryptococcal infection. while subtle nuances exist based on host and site of infection, general principles for the management of cryptococcal infection can provide the cornerstone of a treatment plan in most cases. primary regimen ambd ( . - mg/kg/day) plus flucytosine ( -fc; mg/kg/day) weeks liposomal amb ( - mg/kg/day) or amb lipid complex (ablc; mg/kg/day) pls -fc( mg/kg/day) for patients predisposed to renal dysfunction weeks alternative regimens b - weeks ambd ( . - mg/kg/day) or liposomal amb c ( - mg/kg/day) or ablc ( mg/kg/day) if flucytosine-intolerant weeks ambd ( . - mg/kg/day) plus fluconazole ( mg/day) weeks fluconazole (> mg/day, preferably mg/day) plus -fc ( mg/kg/day) - months ablc amb lipid complex, amb amphotericin b, -fc flucytosine a initiate haart - weeks after beginning antifungal regimen. shorter duration (i.e., weeks) of induction therapy can be considered for certain low-risk patients b can be considered as alternative regimen in circumstances in which primary regimen not available but are not encouraged as equivalent substitutes c liposomal amphotericin can be safely administered in doses as high as mg/kg/day d after year of therapy, if successful response to arvs (cd count > and viral load low or undetectable for > months), discontinuation of antifungal therapy can be considered. consider reinstitution if cd count falls below e consider stepwise de-escalation of immunosuppressive regimen if allograft function permits f if csf culture remains positive at weeks of therapy or initial presentation with neurologic complications, longer therapy preferred g caution due to concomitant calcineurin inhibitor use amphotericin b deoxycholate (ambd) remains the foundation of treatment for disseminated cryptococcosis and severe cryptococcal infection. a standard induction dose of . - mg/kg/day is recommended. liposomal amphotericin b (ambisome) at - mg/kg/day has become a preferred alternative treatment with similar outcomes to that of ambd but with less nephrotoxicity and is specifically recommended for primary induction in organ transplant patients as well as patients at risk for renal dysfunction [ ] [ ] [ ] . higher doses of ambd have been shown to be more rapidly fungicidal [ , ] . flucytosine ( -fc) is primarily used in combination therapy with ambd for first-line therapy in cryptococcal meningitis or severe pulmonary cryptococcosis at a dosage of mg/kg/day in divided doses in patients with normal renal function [ , ] . the combination of ambd and -fc represents the most potent fungicidal regimen with more rapid sterilization of csf cultures at weeks as demonstrated by multiple studies [ , , ] . early studies from hiv infection demonstrated increased rates of csf sterilization and fewer relapses with the combination of ambd and -fc followed by itraconazole maintenance [ ] . this initial combination regimen has since been compared against multiple alternatives, with the superiority of its fungicidal activity consis-tently confirmed [ ] . similar results have been observed among the most severe cases of cryptococcal infection [ , ] . early mycological failure (as defined by persistently positive csf cultures at day ) has for many years been associated with late treatment failure and poor outcome [ ] , and lack of -fc has been independently associated with both early [ ] and late [ ] mycological failure. the improved fungicidal activity of combination therapy with ambd plus -fc has been shown to translate into a direct survival benefit compared with ambd monotherapy, with improved survival at weeks lasting up to months [ ] . -fc should be dose-adjusted for renal dysfunction, with therapeutic monitoring performed - days after initiation of therapy, to maintain -h post-dose levels under µg/ml (goal , to reduce its primary side effect of bone marrow suppression [ ] . though combination induction therapy with ambd and -fc remains the recommended standard of care for severe cryptococcosis including cryptococcal meningitis, limited availability of -fc in resource-limited settings presents significant challenges for managing patients in areas where the disease burden and mortality rates are highest. alternative combination therapies have been investigated, the most efficacious of which has been ambd ( . mg/kg/day) plus fluconazole ( mg/day), which has demonstrated improved rates of a composite end point of csf culture negativity, neurological improvement, and survival compared with ambd alone or in combination with lower doses of fluconazole [ ] . fluconazole (at doses of - mg/ day) in combination with ambd (standard dosing) has been shown to demonstrate similar rates of fungal clearance from csf as standard ambd plus -fc in a randomized study performed in hiv-infected patients in south africa [ ] and offers a potential viable option for effective initial therapy in settings where access to -fc is limited. whether the survival benefit observed with ambd plus -fc will be observed with this regimen remains uncertain. additional alternative regimens for primary therapy are available in the guidelines but their use is not encouraged based on limited data on the success of these regimens [ ] . use of fluconazole in the absence of a polyene is not recommended given the fungistatic nature of this drug, poor success, higher relapse rates, and increased resistance in relapse when used as monotherapy for induction [ , ] . however, in areas without access to ambd, high doses ( mg/day) of fluconazole should be commenced. csf sampling should be performed to rule out cns involvement b csf sampling can be considered but not required in the absence of neurological symptoms or high serum cryptococcal antigen c if successful response to arvs (cd count > and viral load low or undetectable for > months) and stable serum cryptococcal antigen, can consider discontinuation of antifungal therapy after months of therapy a three-stage regimen of induction/consolidation/maintenance is employed in the treatment of cryptococcal meningitis in all patients, irrespective of host risk factors [ , ] . in hiv-infected patients, the initial induction treatment usually begins with combination therapy with ambd plus -fc for at least weeks as above, followed by consolidation treatment with fluconazole - mg/day for weeks in patients who have demonstrated favorable response. following consolidation, a long-term suppressive/maintenance phase is commenced with oral fluconazole, - mg given once daily. this has been demonstrated to effectively reduce rates of relapse from ~ % to less than % in the pre-haart era [ ] . secondary prophylaxis can be discontinued after - years of antifungal therapy in patients who respond to haart with rise in cd + cell counts to greater than cells/µl and decline in viral load (hiv rna) to undetectable levels for at least months [ , , ] . itraconazole can be used as an alternative consolidation treatment for cryptococcosis, but first-line therapy is with fluconazole. despite its poor csf penetration and inconsistent oral bioavailability, itraconazole has been successfully used in the treatment of cryptococcal meningitis [ ] ; however, it has been shown to be inferior to fluconazole during the suppression phase [ ] and requires therapeutic drug monitoring due to its poor bioavailability. newer triazoles including posaconazole and voriconazole are not specifically incorporated into practice guidelines but are active against cryptococcal isolates in vitro and have been shown to demonstrate moderate efficacy in patients with refractory disease [ , ] . in patients with hiv-associated cryptococcal infection, haart has a major impact on long-term prognosis. however, given concerns regarding immune reconstitution inflammatory syndrome (iris), the optimum timing for haart initiation in the setting of ois has been a subject of much debate. early retrospective studies suggested an increased risk of iris among hiv-infected patients initiated on haart early after the diagnosis of an oi [ , ] . more contemporary studies have demonstrated conflicting results regarding outcomes of cryptococcal infection based on timing of haart initiation [ ] [ ] [ ] [ ] [ ] . the cryptococcal optimal art timing (coat) study provides the best evidence for current recommendations regarding timing of haart initiation in patients with cryptococcal meningitis [ ] . haart-naïve patients were randomized to receive immediate (within h) or deferred (greater than four weeks) haart following a minimum of days of antifungal therapy with ambd and high-dose fluconazole. this trial was stopped early after interim analyses suggested poorer early survival among patients receiving immediate haart ( % vs. %, p = . ), particularly among patients with altered mentation and low csf white blood cell count. although a trend toward increased rates of and earlier iris was observed in the immediate haart group, this was not statistically significant. the above data support recommendations to delay initiation of haart in patients with cryptococcal meningitis for a minimum of weeks after starting antifungal therapy (potentially longer if the primary regimen does not include ambd) and after demonstration of a sustained clinical response to antifungal therapy [ , ] . interruption of haart and/or corticosteroid treatment may be used to control symptoms if severe cryptococcal iris occurs. organ transplant recipients with cns cryptococcal infection are managed similar to hiv-infected patients, with the exception of preferential use of lipid formulations of amphotericin b to limit nephrotoxicity [ ] . the principles of induction, consolidation, and maintenance therapy remain the same. repeat csf sampling at weeks is recommended in this population and a longer course of induction therapy should be pursued if csf cultures remain positive at weeks, as this scenario is associated with increased -month mortality [ ] . unlike hiv-infected patients, relapse rates among organ transplant recipients are quite low (~ . %), such that a shorter course of maintenance therapy with fluconazole ( - months) can be pursued following standard consolidation [ , ] . drug interactions between fluconazole and immunosuppressive agents should be anticipated due to fluconazole-induced cyp a inhibition, and preemptive adjustment (reduction) in calcineurin inhibitors should be made. management of immunosuppression in the setting of cryptococcal infection requires recognition of the increased risk of iris associated with abrupt withdrawal or reduction of immunosuppression in organ transplant recipients with increased rates of allograft loss reported in some patients [ ] [ ] [ ] . stepwise reduction in immunosuppression is recommended, though the approach should be individualized for each patient. screening for hiv and cd lymphopenia is recommended among patients who present with cryptococcosis without apparent risk factors [ ] . very little prospective data are available on the management of cryptococcal infection among this heterogeneous group of "apparently immunocompetent" patients lacking classical risk factors for cryptococcosis. what is known is based on early studies that included a heterogeneous mix of patients and were performed prior to acceptance of the standard algorithm of induction, consolidation, and maintenance therapy and higher-dose polyene therapy [ ] . recommendations for longer induction therapy ( weeks or more) in this population are based on the recognition of poorer outcomes and higher mortality rates in this group of patients in both early [ , ] and contemporary [ ] studies. an additional weeks of therapy should be considered if -fc is not included in the induction regimen [ ] . recommendations for consolidation and maintenance parallel those for hiv-infected and transplant patients, and reflect early reports of relapse rates approaching % within the st year prior to introduction of consolidation and maintenance antifungal therapy [ , ] . criteria for stopping treatment in these patients include resolution of symptoms, generally following at least year of suppressive therapy. patients may have prolonged positive cryptococcal polysaccharide antigen tests and/or slightly abnormal csf findings for months during successful therapy, and if there are concerns about cure, follow-up csf culture should be considered. just as host factors influence management approaches for cryptococcal infection, site of infection also matters. airway colonization in a nonimmunosuppressed individual poses a low risk for invasive pulmonary infection (and dissemination) and treatment can be deferred. some experts would still favor treatment with fluconazole in this scenario, given the relative benign nature of this therapy. however, among immunocompromised patients with isolated pulmonary cryptococcosis, treatment is recommended to prevent dissemination [ ] . it should be emphasized that a thorough evaluation to rule out systemic disease/dissemination is warranted in this group of patients to provide optimal treatment. this includes blood and csf cultures as well as serum and csf cryptococcal antigen testing. if the results of the above evaluation are negative, symptoms are mild, and there is no evidence of diffuse pulmonary infiltrates or ards, oral fluconazole ( mg/day) is recommended for - months. however, in any patient in whom cryptococcemia is identified, symptoms are severe, ards is present, or csf examination reveals asymptomatic cns involvement, treatment for cryptococcal meningitis is recommended [ ] . cerebral cryptococcomas often can be managed with prolonged antifungal therapy without need for surgical removal unless mass effect or other evidence of obstruction is identified. at least weeks of induction therapy with ambd plus -fc, followed by - months of consolidation therapy with fluconazole ( - mg/day), is recommended for management. surgery should be considered for large lesions (> cm) or the presence of obstructive hydrocephalus [ ] . localized infection of extrapulmonary nonmeningeal sites can occasionally occur with direct inoculation, but more commonly represents disseminated infection. suspicion for the latter must be maintained when cryptococcus is identified from a sterile body site, as management strategies will differ if disseminated disease is present. consultation with ophthalmology is indicated in cases of cryptococcal eye disease [ ] . restoration of pathogen-specific immunity as a result of haart or following reduction of immunosuppression in sot recipients can result in a destructive inflammatory response known as the immune reconstitution inflammatory syndrome (iris). iris is best characterized in association with c. neoformans infection of the cns, particularly among hiv-infected patients, and is associated with significant morbidity and mortality [ , , , , [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] . proposed criteria for iris include onset of symptoms within months of haart initiation (with concomitant cd + recovery) [ ] . in addition, iris is estimated to occur in - % of sot recipients with cryptococcal infection and has been associated with an increased risk of allograft failure [ , [ ] [ ] [ ] [ ] [ ] ; cryptococcal iris may also be observed in non-hiv-infected, nontransplant patients [ ] . clinical features of cryptococcal iris are similar to cryptococcal infection, most commonly presenting as cns disease, although lymphadenitis, pneumonitis, multifocal disease, soft-tissue involvement, and mediastinitis have all been reported [ , ] . meningeal disease is the most frequent and most serious presentation [ ] ; aseptic meningitis with associated intracranial hypertension and csf pleocytosis is most commonly observed [ , , , , , ] . a hallmark histopathologic finding is suppurative or necrotic granulomatous inflammation with yeast seen in tissues despite negative tissue cultures [ , , , ] . the presence of a positive csf culture in cases of suspected cryptococcal iris should raise suspicion for direct antifungal failure or resistance, particularly in settings where fluconazole therapy is widely used as the standard of care [ ] . cryptococcal iris represents unchecked reversal of a th (anti-inflammatory) to th (pro-inflammatory) immune response in the setting of immune reconstitution [ ] . prospective cohort studies of haart-naïve individuals indicate that an ineffectual host immune response to initial infection is associated with a greater likelihood of future iris [ ] . a three-phase theory of cryptococcal immune reconstitution has been postulated, marked by: ( ) failure of antigen clearance due to inappropriate th response; ( ) lack of effector response despite inflammatory signaling; and, ultimately, ( ) vigorous pro-inflammatory responses (both th and th ) to residual antigen, recognized clinically as iris [ ] . there are no reliable diagnostic tests for iris, and establishing the diagnosis presents a considerable challenge [ , ] . the differential diagnosis includes progressive disease due to persistent immune deficiency, failure of antimicrobial therapy (due to resistance or nonadherence), coinfection with other ois, and drug toxicity. a high index of suspicion is necessary for recognizing atypical presentations or manifestations at distant sites. nevertheless, distinguishing between disease progression related to ongoing immune deficiency and clinical deterioration due to restoration of host immunity has important management implications. csf analyses and biomarkers may be useful in distinguishing between relapse and iris. prospective studies have demonstrated that csf opening pressure [ ] and wbc count [ , ] at the time of an iris event are significantly higher than baseline values for individual patients, and higher csf opening pressures can distinguish iris from relapsed infection [ ] . treatment options for cryptococcal iris are based largely on expert opinion [ ] . implicit in management is ensuring the efficacy of antifungal therapy, particularly in settings where access to ambd may be limited and fluconazole resistance may account for recurrent meningitis episodes [ , ] . in the absence of disease relapse or direct antifungal resistance, modification of antimicrobial therapy is not indicated [ ] . once the diagnosis of iris is suspected, consideration of disease severity is warranted. a significant proportion of minor cases will improve without specific treatment [ , , ] . corticosteroids have been shown to reduce the need for hospitalization and to improve short-term quality of life and functional status without increased risk of complications in paradoxical tuberculosis (tb)-associated iris [ ] ; the role of corticosteroids in cryptococcal iris, however, is not as well established and should be reserved for life-threatening cases, particularly in light of their association with increased mortality in one study [ ] . other antiinflammatory agents have been used in cryptococcal iris, but the number of patients treated with any of these agents is too small to draw substantive conclusions [ , , ] . other management strategies, including therapeutic lumbar drainage in the setting of intracranial hypertension [ , , ] and, at times, surgical drainage of suppurative lymph nodes [ , ] , are important adjunctive therapies that may be considered in severe disease. although no controlled studies have been performed, continuation of haart in the setting of iris is recommended and has been performed safely without adverse effects in several studies [ , , , , ] . similarly, withdrawal or reduction of immunosuppressive agents is standard practice in managing infectious complications in sot recipients [ ] . given the putative risk of iris with abrupt withdrawal or discontinuation of immunosuppressive agents in these patients, gradual de-escalation during the initiation of antifungal therapy is advised to reduce the risk of future iris [ , , ] . persistent and relapsed infection must be distinguished from iris, as management strategies will differ significantly. persistent disease can be defined as persistently positive csf cultures after month of antifungal therapy, whereas relapse requires new clinical signs and symptoms and repeat positive cultures (at same or distant sites) after initial improvement and fungal sterilization [ ] . surrogate markers, including biochemical parameters, india ink staining, and cryptococcal antigen titers, are insufficient to define relapse or alter antifungal therapy. general recommendations for management in these cases include resumption of induction therapy, often for a longer duration and at increased dosages, if tolerable, and pursuance of antifungal susceptibility testing [ ] . while routine in vitro susceptibility testing of cryptococcal isolates at the time of initial therapy is not recommended, there is a role for testing in cases of suspected relapse or persistent infection [ ] . it is generally recognized that primary antifungal resistance to most agents is rare, although reduced susceptibility to -fc has been observed in untreated patients [ ] and echinocandins have no reliable activity against this yeast. reduced susceptibility to fluconazole has been described in cases of culture-positive relapsed cryptococcal meningitis associated with prior fluconazole therapy [ , , ] (table . ). along with the optimization of antifungal therapy, management of increased icp is critically important. elevated icp is correlated with overall fungal burden, and is thought to be due to csf outflow obstruction by clumped yeast forms [ ] . an icp of mm h o or greater is considered elevated and is associated with increased morbidity and mortality [ ] . persistently elevated icp after weeks of treatment is associated with poorer clinical responses among patients with hiv-associated cryptococcal meningitis [ ] . intracranial imaging should be performed prior to lumbar puncture if impaired mentation or focal neurologic deficits are present. a baseline measurement of csf pressure should be obtained in all patients with suspected cryptococcal meningitis. aggressive attempts to control increased icp should occur, if elevated and if there are signs/symptoms to suggest increased icp (headache, mental status changes, and new focal neurological findings). treatment options for managing acutely elevated icp include repeated lumbar punctures (daily until pressure and symptoms are stable for > days), lumbar drain insertion, ventriculostomy, or ventriculoperitoneal (vp) shunt (table . ) [ ] . medical treatments such as corticosteroids (unless there is a component of iris), mannitol, and acetazolamide have been used in some cases, but are generally not recommended for use in management of increased icp in cryptococcal meningitis [ ] . some patients may develop symptoms of obstructive hydrocephalus necessitating the placement of a permanent vp shunt during the first - years of treatment, and occasionally at initial presentation. sterilization of csf is not required prior to placement of a vp shunt, which can be inserted once a patient is receiving the appropriate antifungal therapy [ ] . prevention of cryptococcal disease can be achieved by use of haart in hiv-infected patients. fluconazole prophylaxis has been shown to be effective for preventing cryptococcosis in aids patients with persistently low cd + cell counts (below cells/µl) [ , ] , but due to concerns regarding antifungal resistance, this approach is not currently recommended, and haart remains the best strategy for the prevention of cryptococcal disease in this population. routine screening for cryptococcal infection and/or prophylaxis are not recommended in sot recipients, even when immunosuppression is augmented in patients with previously (appropriately) treated infection [ ] . although cryptococcal gxm-tetanus toxoid conjugate vaccine and specific monoclonal antibodies to cryptococci have been developed, clinical trials have not been initiated to determine their usefulness 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cryptococcus neoformans var. neoformans, cryptococcus gattii, and hybrids identification by random amplification of polymorphic dna (rapd) of a common molecular type of c. neoformans var. neoformans in patients with aids or other immunosuppressive conditions simultaneous identification of molecular and mating types within the cryptococcus species complex by pcr-rflp analysis consensus multi-locus sequence typing scheme for cryptococcus neoformans and cryptococcus gattii maldi-tof ms enables the rapid identification of the major molecular types within the cryptococcus neoformans/c. gattii species complex matrixassisted laser desorption ionization-time of flight mass spectrometry-based method for discrimination between molecular types of cryptococcus neoformans and cryptococcus gattii detection of cryptococcus by conventional and molecular methods multilocus sequence typing reveals three genetically distinct subpopulations of cryptococcus neoformans var. grubii (serotype a), including a 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diseases mycoses study group and aids clinical trials group treatment of cryptococcal meningitis with combination amphotericin b and flucytosine for four as compared with six weeks combination antifungal therapies for hiv-associated cryptococcal meningitis: a randomised trial major role for amphotericin-flucytosine combination in severe cryptococcosis early mycological treatment failure in aids-associated cryptococcal meningitis determinants of disease presentation and outcome during cryptococcosis: the crypto a/d study antimicrobial therapy and vaccines a phase ii randomized trial of amphotericin b alone or combined with fluconazole in the treatment of hiv-associated cryptococcal meningitis comparison of the early fungicidal activity of high-dose fluconazole, voriconazole, and flucytosine as second-line drugs given in combination with amphotericin b for the treatment of hiv-associated cryptococcal meningitis combination flucytosine and high-dose fluconazole compared with fluconazole monotherapy for the treatment of cryptococcal meningitis: a randomized trial in malawi symptomatic relapse of hiv-associated cryptococcal meningitis after initial fluconazole monotherapy: the role of fluconazole resistance and immune reconstitution a placebo-controlled trial of maintenance therapy with fluconazole after treatment for cryptococcal meningitis in the acquired immunodeficiency syndrome discontinuation of secondary prophylaxis for cryptococcal meningitis in human immunodeficiency virus-infected patients treated with haart: a prospective, multicenter, randomized study discontinuation of maintenance therapy for cryptococcal meningitis in patients with aids treated with haart: an international observational study itraconazole therapy for cryptococcal meningitis and cryptococcosis a comparison of itraconazole versus fluconazole as maintenance therapy for aidsassociated cryptococcal meningitis. national institute of allergy and infectious diseases mycoses study group voriconazole treatment for less-common, emerging or refractory fungal infections activity of posaconazole in the treatment of central nervous system fungal infections incidence and risk factors for immune reconstitution inflammatory syndrome during haart incidence and risk factors of immune reconstitution inflammatory syndrome complicating hiv-associated cryptococcosis in france early haart reduces aids progression/death in individuals with acute opportunistic infections: a multicenter randomized strategy trial immune reconstitution inflammatory syndrome in hiv-associated cryptococcal meningitis: a prospective study cryptococcal immune reconstitution inflammatory syndrome after haart in aids patients with cryptococcal meningitis: a prospective multicenter study mbuagbaw l optimal timing for haart initiation in patients with hiv infection and concurrent cryptococcal meningitis early versus delayed initiation of haart for concurrent hiv infection and cryptococcal meningitis in sub-saharan africa haart initiation within the first weeks of cryptococcal meningitis is associated with higher mortality: a multisite randomized trial world health organization. rapid advice: diagnosis, prevention and management of cryptococcal disease in hiv-infected adults, adolescents and children. geneva: world health organization antifungal management practices and evolution of infection in organ transplant recipients with cryptococcus neoformans infection allograft loss in renal transplant recipients with cryptococcus neoformans associated immune reconstitution syndrome an immune reconstitution syndrome-like illness associated with cryptococcus neoformans infection in organ transplant recipients cellulitis revealing a cryptococcosis-related immune reconstitution inflammatory syndrome in a renal allograft recipient a comparison of amphotericin b alone and combined with flucytosine in the treatment of cryptococcal meningitis comparison of temporal trends of three groups with cryptococcosis: hivinfected, solid organ transplant and hiv-negative/non-tranpslant clinical features and serum biomarkers in hiv immune reconstitution inflammatory syndrome after cryptococcal meningitis: a prospective cohort study defining immune reconstitution inflammatory syndrome: evaluation of expert opinion versus case definitions in a south african cohort the role of immune reconstitution inflammatory syndrome in aids-related cryptococcus neoformans disease in the era of haart timing of cryptococcal immune reconstitution inflammatory syndrome after haart in patients with aids and cryptococcal meningitis outcomes of cryptococcal meningitis in uganda before and after the availability of haart paucity of initial cerebrospinal fluid inflammation in cryptococcal meningitis is associated with subsequent immune reconstitution inflammatory syndrome immune reconstitution inflammatory syndrome (iris) associated with cryptococcus neoformans infection in aids patients intramedullary abscess resulting from disseminated cryptococcosis despite immune restoration in a patient with aids cryptococcal immune reconstitution inflammatory syndrome: report of four cases in three patients and review of the literature cryptococcal immune reconstitution inflammatory syndrome in hiv- -infected individuals: proposed clinical case definitions th cells and il- receptor signaling are essential for mucosal host defense against oral candidiasis opportunistic infection-associated immune reconstitution syndrome in transplant recipients cellulitis revealing a cryptococcosis-related immune reconstitution inflammatory syndrome in a renal allograft recipient immune reconstitution syndrome after voriconazole treatment for cryptococcal meningitis in a liver transplant recipient novel immune regulatory pathways and their role in immune reconstitution syndrome in organ transplant recipients with invasive mycoses the poor prognosis of central nervous system cryptococcosis among nonimmunosuppressed patients: a call for better disease recognition and evaluation of adjuncts to antifungal therapy mediastinitis due to cryptococcal infection: a new clinical entity in the haart era hiv combination therapy: immune restitution causing cryptococcal lymphadenitis dramatically improved by anti-inflammatory therapy immunological profiles of immune restoration disease presenting as mycobacterial lymphadenitis and cryptococcal meningitis tuberculosis-associated immune reconstitution inflammatory syndrome: case definitions for use in resource-limited settings symptomatic relapse of hiv-associated cryptococcal meningitis after initial fluconazole monotherapy: the role of fluconazole resistance and immune reconstitution independent association between rate of clearance of infection and clinical outcome of hiv-associated cryptococcal meningitis: analysis of a combined cohort of patients randomized placebocontrolled trial of prednisone for paradoxical tuberculosis-associated immune reconstitution inflammatory syndrome diagnosis and management of increased intracranial pressure in patients with aids and cryptococcal meningitis. the niaid mycoses study group and aids cooperative treatment groups cryptococcal immune reconstitution syndrome during steroid withdrawal treated with hydroxychloroquine treatment of hiv-related inflammatory cerebral cryptococcoma with adalimumab paradoxical immune reconstitution inflammatory syndrome associated with previous cryptococcus neoformans infection in an hiv-positive patient requiring neurosurgical intervention aids: immune reconstitution inflammatory syndrome: a reappraisal flucytosine primary resistance in candida species and cryptococcus neoformans correlation of in vitro azole susceptibility testing with in vivo response in a murine model of cryptococcal meningitis correlation of fluconazole mics with clinical outcome in cryptococcal infection elevated cerebrospinal fluid pressures in patients with cryptococcal meningitis and acquired immunodeficiency syndrome a randomized, double-blind, placebo-controlled trial of acetazolamide for the treatment of elevated intracranial pressure in cryptococcal meningitis treatment of hydrocephalus secondary to cryptococcal meningitis by use of shunting primary prophylaxis with fluconazole against systemic fungal infections in hiv-positive patients a multicentre, randomized, double-blind, placebo-controlled trial of primary cryptococcal meningitis prophylaxis in hiv-infected patients with severe immune deficiency cryptococcosis in solid organ transplant recipients: current state of the science cryptococcus neoformans serotype a glucuronoxylomannan protein conjugate vaccines: synthesis, characterization, and immunogenicity therapeutic efficacy of monoclonal antibodies to cryptococcus neoformans glucuronoxylomannan alone and in combination with amphotericin b. antimicrob agents chemother suggested reading casadevall a, perfect jr. cryptococcus neoformans combination antifungal therapy for cryptococcal meningitis large-scale evaluation of the immuno-mycologics lateral flow and enzyme-linked immunoassays for detection of cryptococcal antigen in serum and cerebrospinal fluid a rare genotype of cryptococcus gattii caused the cryptococcosis outbreak on vancouver island (british columbia, canada) evaluation of a newly developed lateral flow immunoassay for the diagnosis of cryptococcus cryptococcosis in the era of aids- years after the discovery of cryptococcus neoformans discontinuation of maintenance therapy for cryptococcal meningitis in patients with aids treated with haart: an international observational study dolin r, editors. mandell, douglas, and bennett's principles and practice of infectious diseases clinical practice guidelines for the management of cryptococcal disease: update by the infectious disease society of america measurement of cryptococcal antigen in serum and cerebrospinal fluid: value in the management of aids-associated cryptococcal meningitis comparison of amphotericin b with fluconazole in the treatment of acute aids-associated cryptococcal meningitis. the niaid mycoses study group and the aids clinical trials group key: cord- - kfy hg authors: lye, patricia s.; densmore, emily m. title: fever date: - - journal: nelson pediatric symptom-based diagnosis doi: . /b - - - - . -x sha: doc_id: cord_uid: kfy hg nan temperature is controlled by the thermoregulatory center, located in the preoptic area of the anterior hypothalamus. the thermoregulatory center receives input from peripheral receptors and the temperature of the blood bathing the hypothalamus and acts on autonomic, endocrine, and behavioral mechanisms to maintain the body temperature at a particular set point. the hypothalamic set point normally maintains body temperature around °c, but there can be significant variation among individuals. normal temperatures range from - . °c, depending on the time of day, with the peak in the afternoon ( - p.m.) and the trough in the early morning ( - a.m.). although the circadian rhythm is not well established in infancy, it becomes more reliable by the nd year of life. the febrile response not only produces an elevation in body temperature but also causes physiologic changes that enhance the individual's ability to eliminate infection. production of acute-phase reactants and alterations in metabolism and endocrine function are examples of these changes. acute-phase reactants-proteins that are produced in response to infection or injury-include ceruloplasmin, c-reactive protein, haptoglobin, amyloid a, complement, and fibrinogen. hormones and cytokines, some of which are endogenous pyrogens, regulate the production of acute-phase proteins. exogenous pyrogens, such as bacteria or endotoxins, generate the production of endogenous pyrogens, which play a vital role in prostaglandin-related set point elevation and regulation of acute-phase responses. fever results when the thermoregulatory set point is elevated above the normal set point; the hypothalamus then generates physiologic changes involving endocrine, metabolic, autonomic, and behavioral processes. diversion of blood from peripheral vessels to central vessels causes coolness of the extremities but helps increase core temperature. shivering increases metabolic activity and heat production. the affected patient may feel cold and seek a warmer environment or add clothing to feel warmer and prevent heat loss. once these processes have resulted in increasing the core temperature to match the elevated set point, the thermoregulatory center works to maintain the temperature as it does during normothermia. the thermoregulatory point returns to normal once the infection is resolved. the hypothalamus then produces physiologic changes to decrease the core temperature; these include sweating, dilation of cutaneous blood vessels, and the sensation of feeling hot, which may lead to behaviors such as removing clothing or seeking a cooler environment. fever has both positive and negative effects. high body temperatures may impair the reproduction and survival of some invading microorganisms by decreasing required nutrients, such as free iron, or by increasing immunologic responses such as phagocytosis. however, at extremely high temperatures, immunologic responses may be impaired. fever increases the basal metabolic rate by - % for each degree celsius elevation of temperature. this increases oxygen consumption, carbon dioxide production, and fluid and caloric needs. fluid requirements increase ml/m /day for each °c rise in temperature above . °c. heat illness must be distinguished from fever as a cause for elevated body temperature. in heat illness, there is an unregulated rise in body temperature, despite the fact that the hypothalamic set point is normal. it can result from excessive heat production or inadequate heat dissipation. temperatures may reach extreme heights and can result in multiorgan dysfunction and death. restoration of normal body temperature in heat illness is mandatory (table . ). a child with fever of recent onset with no obvious historical or physical explanation for the fever is said to have fever without source (fws). bacterial pathogens account for a small but clinically significant number of cases. the risk of bacterial infection decreases with increasing age and is highest for infants less than months of age, compared to infants and toddlers - months of age, and even lower for children over the age of months. most of the patients in all age groups have a self-limited viral illness. the challenge is to identify which children have fever caused by bacterial pathogens, or other pathogens requiring treatment, in order to avoid the morbidity and mortality associated with delayed treatment, balanced against the risks of testing or treatment when neither is needed. bacterial infection must be considered in immunocompromised patients or those with central lines or shunts. studies in adults suggest that patients with high fever (> o f) and rigors have a higher risk of bacterial infection; exceptions to this include influenza and adenoviral infections. oral thermometry can be considered for cooperative patients who are older than - years of age. axillary temperatures are commonly done and tympanic membrane and temporal artery temperatures are newer modalities with some studies examining their reliability. axillary temperatures are less precise than rectal temperatures. there is a correlation between axillary and rectal temperature measurements; the axillary temperature is usually . - . °c lower. tympanic membrane thermometers are often inaccurate in children. temporal artery temperature measurement correlates well with rectal temperature in some studies, but has been shown to be inferior when patients are febrile. it can be considered in settings when children are not likely to be febrile and are over months of age. when detection of fever is critical for diagnosis and management, rectal temperatures should be used in the child years of age and younger. many children will have a source for fever identified on their history and/or physical examination. if no focus of infection is found on the physical examination, the clinician must rely on history and observation to determine the appropriate next steps. the child may appear ill or well. ill-appearing children are typically lethargic or irritable. they may show signs of shock, including weak peripheral pulses, tachycardia, poor perfusion, respiratory distress, mottling, cyanosis, or decreased mental status (table . ). after thorough clinical and laboratory evaluation, ill-appearing children should be admitted to the hospital, and will likely need empiric antibiotic treatment. infants and children with fever without source who do not appear ill create important decision processes in terms of evaluation and management. the physician's ability to make a hypothesis about the child's degree of illness, on the basis of observation, is critical in the evaluation. an objective scoring measure may be used in an effort to assess serious illness in young febrile children. the acute illness observation scale (aios) (table . ), also known as the yale observation score, is a -item predictive model graded on a scale of - . use of the aios in conjunction with the history and physical examination has a higher sensitivity for identifying serious illness than history and physical examination alone. the aios is most useful in patients younger than - months; it has not been shown to provide sufficient data to identify serious illness in -to -week-old infants, and has not been evaluated in infants less than weeks old. most children who present with fever without source (fws) are subsequently determined to have a self-limited benign viral infection. in study in - month old children who presented with fws, % had or more known pathogenic viruses found; % had adenovirus, human herpesvirus (hhv- : roseola), enterovirus, or parechovirus detected. other identifiable viruses include respiratory syncytial virus, parainfluenza viruses, influenza viruses, varicella (chickenpox), human metapneumovirus and parvovirus (fifth disease/erythema infectiosum). measles, mumps, and rubella are uncommon in developed countries but have been reported in epidemics following imported cases or in underimmunized communities. although rapid testing for viral pathogens is often readily available, a detailed investigation to identify a viral pathogen is not necessary unless the confirmation of a viral infection will change the acute diagnostic plan; treatment with antivirals is an option (hsv, influenza) if the fever is prolonged and evolves into fuo or if there is end-organ involvement, as in hepatitis, myocarditis, encephalitis, or meningitis. most viral infections do not have simultaneous co-infection with a bacterial pathogen. exceptions include croup due to parainfluenza history a detailed history may reveal a potential source for infection. a complete history addresses several important issues: ( ) onset and duration of fever; ( ) degree of temperature; ( ) by what method and in which anatomic site the temperature was taken; ( ) medications given, including antipyretics, antibiotics, or home remedies; ( ) environmental exposures; ( ) associated symptoms; ( ) ill contacts; ( ) recent immunizations, and ( ) recent travel. inquiry into the child's medical history may reveal important information such as recurrent febrile illnesses, primary or acquired immunodeficiency, or medications such as chemotherapy that alter host defenses. rectal temperature measurement is considered to be the gold standard for children years of age or younger. the most widely accepted definition of fever is rectal temperature of °c ( . °f) or higher. it is important to consider that infants, especially those younger than months of age, may have a blunted febrile (or hypothermic) response to infection. hence, lack of fever should not be used as a criterion for ruling out infection in infants. although rectal temperature measurement is the gold standard, it should be avoided in neutropenic immunocompromised patients, in whom rectal manipulation may seed the blood with bacteria. two of criteria, of which must be abnormal temperature or abnormal leukocyte count: . core temperature > . °c ( . °f) or < °c ( . °f) (rectal, bladder, oral, or central catheter) . tachycardia: mean heart rate > sd above normal for age in absence of external stimuli, chronic drugs or painful stimuli or unexplained persistent elevation over . - hr or in children < yr old, persistent bradycardia over . hr (mean heart rate < th percentile for age in absence of vagal stimuli, β-blocker drugs, or congenital heart disease) . respiratory rate > sd above normal for age or acute need for mechanical ventilation not related to neuromuscular disease or general anesthesia . leukocyte count elevated or depressed for age (not secondary to chemotherapy) or > % immature neutrophils sepsis plus of the following: . cardiovascular organ dysfunction, defined as: despite > ml/kg of isotonic intravenous fluid in hr: • hypotension < th percentile for age or systolic blood pressure < sd below normal for age or • need for vasoactive drug to maintain blood pressure or • of the following: • unexplained metabolic acidosis: base deficit > meq/l • increased arterial lactate: > times upper limit of normal • oliguria: urine output < . ml/kg/hr • prolonged capillary refill: > sec • core to peripheral temperature gap > °c ( . °f) . ards as defined by the presence of a pao /fio ratio ≤ mm hg, bilateral infiltrates on chest radiograph, and no evidence of left heart failure or sepsis plus or more organ dysfunctions (respiratory, renal, neurologic, hematologic, or hepatic) sepsis plus cardiovascular organ dysfunction as defined above mods presence of altered organ function such that homeostasis cannot be maintained without medical intervention virus, which may predispose to bacterial tracheitis and influenza, which may predispose to bacterial pneumonia. the sequence may be biphasic with viral symptoms followed by improvement, followed by worsening symptoms of the bacterial superinfection, or both phases may not be apparent as the child demonstrates no improvement or deterioration. respiratory syncytial virus (rsv) may predispose patients to otitis media. noninfectious conditions manifesting with fws are extremely rare. historical clues (recurrences, chronicity) or systemic signs usually indicate malignancy or rheumatic disorders. if the history and physical examination are not suggestive, these diagnoses need not be pursued. heat-related illness or drug ingestion may be considered if supported by the history. fever caused by immunizations may not be accompanied by other signs or symptoms, but the history should suggest immunization as the cause. utis are the most common serious bacterial infection in children less than months of age who present with fws. utis are almost always occult in children younger than months because the symptoms, except for fever, are nonspecific or nonexistent. uti occurs in % of febrile children younger than years. the prevalence of uti varies by height of the fever, duration of the fever, age, gender, race, and circumcision status. children with fever greater than °c are at a higher risk of utis. boys with fever for more than days and girls with fever for more than day are more likely to have a uti. higher rates of utis are found in girls, especially those younger than months of age. for febrile boys younger than months of age, . % of those who are uncircumcised have a uti; for circumcised boys the rate is . %. uti rates are higher among white infants than among black infants and among children with abnormal genitourinary tract anatomy or neurogenic bladder. urine specimens should be obtained from the following children with fws: those with a history of uti, those with a history of urinary tract anomalies or vesicoureteral reflux, all infants younger than months, girls younger than - months, uncircumcised boys younger than months, and circumcised boys younger than months. there is an age-associated risk of bacteremia with utis, particularly in infants. the incidence of bacteremia in patients younger than months with uti is %. the incidence of bacteremia in patients younger than months with uti ranges from - % depending on the setting. opinions regarding when to obtain blood cultures in infants with uti differ, but a reasonable approach would be to obtain blood cultures in children younger than - months with suspected uti, and in older infants with uti if they are ill-appearing (urosepsis). occult bacteremia is defined by the presence of a positive blood culture for pathogenic bacteria in a febrile patient who does not appear extremely ill and who has no focus of infection, excluding otitis media. following the introduction of the -valent pneumococcal vaccine in , invasive pneumococcal disease decreased dramatically. pneumococcal bacteremia decreased from % of the cases of bacteremia to %. most cases of bacteremia in children were not occult. bacteremic children were either ill or had a focus of infection, such as a uti. in study, the rate of occult bacteremia after was . %. after the -valent pneumococcal vaccine was introduced in , the incidence of invasive pneumococcal disease in children less than years old decreased again with state-based population study showing incidence rates dropping from / , to / , with the age group most involved being children - months of age. escherichia coli is the most common cause of bacteremia in children aged less than months, all due to utis. other less common causes of bacteremia in young as a marker for invasive disease caused by e. coli than by s. pneumoniae, thus its utility has declined with the reduction of the incidence of invasive pneumococcal disease. similarly, band counts are less commonly used, except in the - day old infant as part of identifying a low-risk cohort. a wbc count of , - , is generally considered normal for children over month of age. a wbc count less than , /mm or even leukopenia may be found in children with n. meningitidis bacteremia. a minority of children with occult nontyphoidal salmonella bacteremia have been found to have a wbc count exceeding , /mm . c-reactive protein (crp) and procalcitonin combined with a urine dipstick (the lab score) can be used to screen for bacterial infection. this combination of tests has been validated for children days to months of age. pcr is useful in identifying the cause of fever for common viruses such as respiratory syncytial virus, influenza viruses, parainfluenza viruses, enteroviruses, parechovirus, adenoviruses, or herpes simplex virus. additional methods available or in development that may be helpful to identify serious bacterial infections and distinguish bacterial from viral infections utilize molecular microbiology methods. gene expression profiles of the patient's peripheral blood leukocytes demonstrate different biosignatures of rna production that may differentiate bacterial from viral infections. this method does not identify the children are n. meningitidis, nontyphoidal salmonella, staphylococcus aureus, and group a streptococcus. neisseria meningitidis bacteremia is frequently associated with serious sequelae. children with n. meningitidis bacteremia are much more likely to progress to meningitis than are those with s. pneumoniae bacteremia. nontyphoidal salmonella bacteremia is often accompanied or preceded by enteritis. in some instances, particularly in young infants, the diarrhea is mild or even absent. the prevalence of salmonella bacteremia among patients with salmonella enteritis has been reported to be between % and %; fever is not always present. salmonella infection seldom causes serious complications in patients with normal host defenses and resolves spontaneously. infants younger than months, malnourished, and immunocompromised individuals are exceptions. evaluation is usually divided into different age ranges: younger than month, - months, - months, and older than months. testing for each individual age group is based on risks for diseases and prevalence of pathogens. most acute diarrhea and fever is caused by viral pathogens in developed countries. obtaining a stool culture is indicated if bacterial enteritis is indicated by the presence of risk factors in the history, such as blood in the stool or certain exposures (petting zoos) (see chapter ). febrile infants younger than months have a higher incidence of serious bacterial infections than older infants. the relatively high incidence of bacterial disease probably results from a combination of factors unique to this age group: decreased opsonin activity; decreased macrophage function; decreased neutrophil function; poor immunoglobulin g antibody response to encapsulated bacteria; and susceptibility to bacterial pathogens such as group b streptococci (gbs), gram-negative enteric organisms, and listeria monocytogenes. the incidence of early-onset group b streptococcal infections has decreased with routine screening and the intrapartum treatment of gbs-positive pregnant women; the incidence of late-onset gbs (> week) has not decreased. e. coli is the most common organism causing bacterial infections in neonates and young infants. in very young infants, clinical evaluation alone is inadequate for excluding serious bacterial infections. management of febrile infants younger than days includes a sepsis evaluation and hospitalization for parenteral antimicrobial therapy pending culture results. the reasoning for this conservative approach lies in the difficulty in evaluating the behavioral state of neonates, the rapid clinical deterioration of infants with bacterial infections, the immature neonatal immune system, and the possibility of life-threatening viral infections caused by herpes simplex viruses (hsv) or enteroviruses. sepsis evaluation should include culture of the csf, blood, and urine; a complete blood cell count with differential; examination of the csf for cells, protein, and glucose; and urinalysis. a chest radiograph should be considered if the patient has signs or symptoms of a respiratory infection. testing (blood and csf pcr for hsv) and treatment for possible hsv infection should be considered in ill-appearing infants, those with a seizure prior to presentation, and those with a vesicular rash consistent with hsv. a combination of clinical evaluation and laboratory studies can be used to define a specific population of infants aged - days who do not appear ill and are at low risk for bacterial infections. infants at low risk for bacterial infections are those who are previously healthy with no focus of bacterial infection on physical examination and who have negative laboratory screening results. a number of prospective studies have contributed to the development of specific low-risk screening criteria (table . ). the age groups included vary by study, ranging from - days to - days. because there are differences in study criteria used to define infants at low risk for bacterial infections the most conservative values have been used in the guidelines presented in this chapter. negative laboratory screening results consist of a wbc count of - , /mm ; fewer than bands/mm or a band-to-neutrophil ratio of less than . ; fewer than wbcs/hpf and no organisms on urinalysis; and fewer than wbcs/hpf and no organisms on csf gram stain. some experts also include a negative chest radiograph and, when diarrhea is present, a stool examination with fewer than wbcs/hpf. most experts suggest that febrile infants - days old who meet the low-risk criteria and have access to close follow-up can be managed as outpatients. blood, urine, and csf cultures should be obtained before empirical antibiotic treatment so that viral and bacterial causes specific pathogen. rapid multiplex pcr combined with standard blood culture methods may identify a specific pathogen much sooner (~ hours) than standard blood culture techniques. specific bacteria may be identified using s ribosomal rna bacterial gene detection. this method does not require bacterial growth. s rrna detection may be helpful when antibiotics were administered before the sample was obtained, and in patients with ventilator-associated pneumonia or bacteria that grow poorly or are present in effusions or tissues (heart valves). blood cultures are the gold standard for determination of bacteremia. although blood cultures do not provide immediate results, methods allow for continuous and more rapid detection of bacterial growth. blood cultures are easy to perform and provide essential information in the diagnosis and management of patients with possible bacteremia. preliminary blood culture results are typically available within hours, with positive identification of most organisms within hours. false-negative blood culture results may be due to prior treatment with antibiotics, missing an episode of bacteremia if it is intermittent, and inoculation of too little blood into the culture media. alternatively, too much blood inoculated into the blood culture bottle may yield a false-negative result because of ongoing killing of bacteria by neutrophils. three to ml of blood should be added to each blood culture bottle. false-positive results may be due to inadequate skin preparation, leading to contamination with skin flora. a positive urine culture was once considered the gold standard; current recommendations include a urinalysis that has pyuria (defined as > wbcs/high-power field [hpf] on the microscopic examination or a positive leukocyte esterase on dipstick) and a positive urine culture for a uropathogen in an appropriately collected specimen. fifty to , colonies of a single organism is considered positive (see chapter ). children should have a catheterized urine specimen obtained, unless they are toilet-trained and can supply a clean voided specimen. suprapubic aspiration is acceptable but requires technical expertise, and parents often perceive it as unsuitably invasive; it may be the only alternative for boys with severe phimosis. the use of plastic receptacles attached to the perineum should be discouraged because contamination from skin and fecal flora commonly occurs. lumbar puncture is indicated if the patient is younger than days or if a diagnosis of sepsis, meningitis, or encephalitis is considered, regardless of the child's age. normal cerebrospinal fluid (csf) findings, including chemistry, cell count with differential, gram stain, pcr, and culture, help exclude the diagnosis of meningitis. less than % of children with normal preliminary csf results have a positive culture; in most of these, the pathogen is n. meningitidis. thus, even in the presence of normal preliminary csf results, close follow-up is essential. chest radiographs are usually normal in children who have fws. respiratory signs or symptoms, such as tachypnea, retractions, crackles, wheezing, rhonchi, nasal flaring, grunting, cough, or hypoxia, may predict chest radiograph findings consistent with pneumonia. in practice, pneumonia can often be diagnosed solely on the basis of the clinical findings of fever, tachypnea, and crackles; chest radiographs are not always necessary. however, chest radiographs may be useful in evaluating for the presence of pleural effusion or other complications of pneumonia. screening urinalysis (ua) for uti should be considered in children with a history of uti, children with a history of urinary tract anomalies or vesicoureteral reflux, girls younger than - months, especially when the temperature is greater than . °c, uncircumcised boys younger than months, and circumcised boys younger than months. blood cultures are recommended for children with probable utis who are less than months of age. a febrile child with moderate leukocyte esterase on urine dipstick testing or pyuria on an appropriately collected specimen should be treated presumptively for a uti. urine cultures should be obtained for any patient with a suspected uti. the choice of antibiotics should be guided by knowledge of the common pathogens that cause utis and by patterns of antibiotic sensitivity in the community. hospitalization should be considered for the child who is vomiting, is dehydrated, or appears ill; for those in whom compliance is likely to be poor; and for any patient with underlying renal or urologic anomalies. examination and culture of the csf are the only tests to exclude the diagnosis of meningitis and encephalitis. they should be considered in any child in whom the diagnosis of sepsis, meningitis or encephalitis is suspected on the basis of the history, observation, and physical examination findings. outpatient management of children with fws is acceptable for those with a low probability of meningitis, good follow-up, and reliable caregivers. blood cultures should be obtained for all children in whom sepsis or meningitis is suspected. empiric treatment with antibiotics should be considered in those suspected of sepsis or meningitis after appropriate cultures are obtained. in summary, management of children aged - months with fever is based on clinical experience and numerous study results: • child who appears ill on initial evaluation or on follow-up: admit to the hospital for parenteral antibiotics after appropriate laboratory evaluation. • well-appearing children with fws should be screened for utis, based on their number of risk factors. risk factors for girls are: age < months, white race, temperature greater than °c, and fever for or more days. girls - months of age with or more of these risk factors have a greater than % probability of having a uti, and should be screened for a uti. for boys, the risk factors are uncircumcised status, nonblack race, temperature greater than °c, and fever for over hours. all uncircumcised boys less than months old, even if they don't have other risk factors, should be screened for a uti. for boys who are circumcised, or more of the other risk factors increases the risk to over % and they should be screened. • child with positive blood culture: reevaluation should occur in any child whose blood culture is presumptively positive. if the blood is found to contain n. meningitidis or haemophilus influenzae (which has been rare since the advent of h. influenzae b immunization), a csf sample and a repeat blood culture should be obtained, and the child should be admitted to the hospital for parenteral antibiotics, pending the results of the cultures. the child with occult pneumococcal bacteremia who appears well and is afebrile when returning for a follow-up may be managed as an outpatient with parenteral ceftriaxone followed by oral antibiotics according to the sensitivity of the organism. because of the concern of pneumococcal resistance to penicillin, a nd dose of intramuscular ceftriaxone may be given until sensitivity results are available. if the culture is positive for nontyphoidal salmonella organisms and the child is younger than months, full sepsis evaluation and intravenous antibiotics are recommended. oral antibiotics and close follow-up are recommended for older children with salmonella bacteremia. • child with positive urine culture: if the child is afebrile and appears well, treatment with oral antibiotics is recommended, according to the sensitivity of the organism. may be distinguished. an alternative strategy is to manage such infants as outpatients, without empirical antibiotic therapy, after blood, csf, and urine cultures are obtained. although most of the original studies on outpatient management of febrile infants included infants aged - months, many experts agree that infants aged - months can be managed safely according to the guidelines for infants and children aged - months (table . ). regardless of whether the clinician chooses to treat the patient with empiric antibiotics, all low-risk infants should be re-evaluated within hours. those who appear ill or who have positive culture results should be admitted for parenteral antibiotics. if a child appears well and all culture results are negative, close follow-up should be continued and a nd return visit made in hours. the risk of bacteremia for children with fws in this age group has decreased with the routine use of pneumococcal vaccines. the most common occult bacterial infection in this age group is uti. for children in this age group who appear ill, a full sepsis evaluation should be undertaken (table . ). infants are at low risk if they appear well, have a normal physical examination, and have a caretaker reachable by telephone, and if laboratory tests are as follows: • cbc: < , wbc/µl • urine: negative leukocyte esterase • csf: leukocyte count less than × /l infants are at low risk if they appear well and have a normal physical examination, and if laboratory tests are as follows: • cbc: < , wbc/µl; band: total neutrophil ratio < . • urine: < wbc/hpf; no bacteria on gram stain • csf: < wbc/µl; no bacteria on gram stain • chest radiograph: no infiltrate • stool: no rbc; few to no wbc infants are at low risk if they appear well and have a normal physical examination, and if laboratory tests are as follows: • cbc: , - , wbc/µl; peripheral absolute band count < , /µl • urine (enhanced urinalysis): wbc/µl and no bacteria on gram stain • csf: wbc/µl and negative gram stain; if bloody tap, then wbc:rbc ≤ : • chest radiograph: no infiltrate • stool: wbc/hpf with diarrhea infants are at low risk if they appear well and have a normal physical examination, and if laboratory findings are as follows: • cbc: , - , wbc/µl; absolute band count ≤ , /µl evaluation and management of ill-appearing children older than months with fever without source are similar to those of younger children. for children in this age group who do not appear ill, no screening diagnostic tests are indicated. close attention should be paid to environmental exposures and ill contacts because of the high likelihood of increased contacts in this school-aged cohort. bacterial meningitis is usually a disease of infants and young children. the attack rate is highest between the ages of and months; % of cases occur in children younger than years of age. bacterial meningitis is seen during all seasons; however, there may be a seasonal correlation between the presence of preceding respiratory pathogens in the upper respiratory tract and the subsequent development of bacterial meningitis. bacterial meningitis usually occurs sporadically. clusters of cases have been noted in day care centers, colleges, and other closed communities. bacterial meningitis occurs more frequently in children with traumatic fractures of the cribriform plate or paranasal sinuses or with a cochlear implant (pneumococci); in children who have undergone neurosurgical procedures such as ventricular shunts (s. aureus, s. epidermidis, corynebacterium species); in children with congenital or acquired immunodeficiencies (pneumococci, l. monocytogenes, meningococci); in children with anatomic or functional asplenia (pneumococci, meningococci); and in children with sickle hemoglobinopathies (pneumococci). there may be a genetic predisposition in some groups to the development of meningitis, inasmuch as there is an increased incidence of h. influenzae type b meningitis in navahos and eskimos. bacterial meningitis manifests in patterns. in the st, the symptoms develop slowly over several days, the initial symptoms being those of a nonspecific illness. the signs and symptoms of meningitis develop subsequently. in the nd pattern, the disease develops suddenly and quickly, the st indications of illness being the signs and symptoms of meningitis and/or sepsis. the manifestations of meningitis depend on the child's age. in infants, the findings are usually nonspecific and may be subtle; they include vomiting, diarrhea, irritability, lethargy, poor appetite, respiratory distress, seizures, hypothermia, and jaundice. only % of affected infants have fever; some present only with fever. it is uncommon for affected young infants to have a stiff neck; only % have a bulging fontanel. older children present with more specific meningeal signs. they complain of a headache that is described as being severe, generalized, deep-seated, and constant. they complain about neck stiffness, caused by inflammation of the cervical dura and reflex spasm of the extensor muscles of the neck. there is pain and limitation of motion on flexion of the short duration and inconsistent development of increased intracranial pressure, papilledema is usually not seen at presentation. when it is present, venous sinus thrombosis, subdural effusion, or an intracranial abscess must be considered. seizures occur before hospital admission in up to % of affected patients. children with meningitis may also present with cutaneous findings. although commonly associated with meningococcal disease, purpura, petechiae, or a diffuse nonspecific maculopapular rash may be present in meningitis caused by any of the common bacterial pathogens (see chapter ). septic arthritis may be seen simultaneously with bacterial meningitis. this has been assumed to be caused by simultaneous localizing infection after a primary bacteremia. reactive arthritis caused by immune complex deposition is also seen with bacterial meningitis. this arthritis affects large joint and appears - days after treatment for meningitis has started. in general, arthritis occurring acutely with meningitis should be assumed to be infectious (see chapter ). various eye disorders have also been described with acute bacterial meningitis, including transient cataracts, paralysis of the extraocular muscles, pupillary dysfunction, dendritic ulcers, endophthalmitis, cortical blindness, and conjunctivitis. recurrent episodes of bacterial meningitis rarely occur. potential etiologies include congenital csf fistulas (inner ear, dermal sinus, neuroenteric cysts, lumbosacral sinus tracts), traumatic or surgical csf fistula (skull fracture, postoperative nasal surgery, cochlear implant), immunodeficiency states and parameningeal infections (mastoiditis, sinusitis, craniofacial osteomyelitis). the definitive diagnosis of meningitis is based on examination of the cerebrospinal fluid (csf). the csf is usually obtained via a lumbar puncture (spinal tap). the lumbar puncture is performed by introducing a small-bore, short-beveled, spinal needle with a stylet into the subarachnoid space at the l -l or l -l level (figs. . to . ). a needle with a stylet is used to minimize the risk of introducing a nest of epidermal cells into the subarachnoid space that may later grow into a cord-compressing epidermoid tumor. approximately ml of fluid is removed for analysis. there are a few contraindications for the performance of a lumbar puncture. the st is cardiorespiratory compromise. performance of the lumbar puncture requires that the child be held in flexion to open the intervertebral spaces. in seriously ill children or children with significant underlying cardiac or pulmonary disease, this positioning may be enough to cause respiratory compromise. the lumbar puncture may need to be postponed, be performed cautiously with continuous oxygen saturation monitoring, or performed with the patient in the sitting position. second, children with increased intracranial pressure from a focal central nervous system (cns) lesion, such as brain abscess or tumor, or from illnesses associated with cerebral edema have a high risk of cerebral herniation after a lumbar puncture. if signs or symptoms of increased intracranial pressure are present, the lumbar puncture should be postponed until the increased pressure is lowered with appropriate treatment. if a lumbar puncture is delayed, appropriate antibiotic therapy should be initiated without further delay. third, a lumbar puncture should not be done if the spinal needle must pass through an area of infection on its way to the subarachnoid space. to do so might introduce pathogens into the cns that could cause meningitis. epidural hematomas causing lower limb paralysis may be a complication of lumbar punctures in children with bleeding disorders. therefore, in children with hemophilia, disseminated intravascular of the neck, but lateral movement of the neck may be normal and pain-free. they also complain of nausea, vomiting, anorexia, and photophobia. on examination, they demonstrate irritability, mental confusion or altered consciousness, nuchal rigidity, and, occasionally, hyperesthesia and ataxia. the clinician demonstrates nuchal rigidity by feeling resistance and observing a painful response while flexing the patient's neck. the stiffness may not be recognized until the end of flexion. the neck usually can be rotated without symptoms. in the child who is crying and tensing the muscles, nuchal rigidity may be demonstrated if the examiner places hand under the occiput of the supine patient and lifts the child. if the neck does not flex, it is stiff. alternatively, a sitting child may be observed following an object as it falls to the floor. the child who flexes the neck to look at the object does not have nuchal rigidity. in the presence of meningitis, flexion of the neck causes spontaneous flexion of the legs at the hips and knees, the brudzinski sign ( fig. . ) . the kernig sign is elicited when the patient lies supine and, with the knee flexed, the leg is flexed at the hip. the knee is then extended. a positive sign is present if this movement is limited by contraction of the hamstrings and causes pain. absence of nuchal rigidity is found in . % of older children with meningitis; it may be absent in children who have overwhelming infections, are deeply comatose, or who have focal or global neurologic impairment. as many as % of children with bacterial meningitis initially present in a comatose or semicomatose state (see chapter ). because a b specific agents) of pathogenic organisms. opening pressure measurements are obtained with the head of the bed flat and with the child relaxed and in the lateral decubitus position with the back no longer tightly flexed. the upper limit of normal value in children - years of age is less than cm of water. opening pressure is less than cm h o in premature infants and less than cm h o in normal newborns. opening pressure measurements are elevated if the lumbar puncture is performed with the patient in the sitting position and if the patient is combative or performing the valsalva maneuver. obstructive hydrocephalus, hyperventilation, or removal of fluid can all lead to lowering of the measurement. children with bacterial meningitis usually have a mean opening pressure of ± cm h o. normal csf is clear and colorless (table . ). blood in the csf indicates a traumatic lumbar puncture or a cns hemorrhage. obtaining a rbc count on tubes and may differentiate the conditions because the count is unchanged in cns hemorrhage but may decline in traumatic taps. centrifugation of the csf sample may also help differentiate between a traumatic tap and a cns hemorrhage. when blood has been present in the csf for several hours, the csf is xanthochromic after centrifugation. however, if the blood was recently mixed with csf, as in the case of a traumatic tap, the supernatant is clear. xanthochromic csf can also be caused by icterus or an elevated csf protein concentration. the normal values for wbcs in the csf are shown in table . . most children with bacterial meningitis have a wbc count of at least /mm in their csf, but, in general, more than /mm in children after the neonatal period is considered abnormal. normal values for neonates are - (mean: ) wbcs in the csf. an absolute neutrophil count exceeding /mm (neutrophils may be as high as %) is also considered abnormal and evidence of a coagulopathy, or thrombocytopenia, lumbar puncture should be postponed until the bleeding disorder is corrected, and extra care should be taken to avoid a traumatic lumbar puncture. such children should be monitored after the procedure for the development of neurologic deficits. empirical therapy may be started while the coagulopathy is corrected. other, rarer complications of lumbar puncture include cortical blindness from compression of the posterior cerebral artery against the tentorium cerebelli, causing ischemic infarction of the occipital lobes. cervical spinal cord infarction, with respiratory arrest and flaccid tetraplegia, may occur if intracranial hypertension causes herniation of the cerebellar tonsils through the foramen magnum with resulting compression of the anterior spinal artery or its penetrating branches. post-lumbar puncture headache may occur in up to % of older children and adults; it is presumably caused by persistent csf leakage at the lumbar puncture site. the csf is examined for red blood cells (rbcs), white blood cells (wbcs) and differential, glucose, protein, and the presence (by culture, by gram stain or other stain, or by antigen or dna-pcr testing for on occasion, the spinal needle is advanced too far and passes through the subarachnoid space and penetrates the richly vascularized ventral epidural space. blood is thereby introduced into the subarachnoid space, and the csf appears bloody. this occurrence is often called a traumatic tap. it is then difficult to know whether the wbcs seen on examination of the csf are caused by csf pleocytosis or are peripheral blood wbcs contaminating the csf. to aid in this determination, the ratio of wbcs to rbcs in the csf is compared with the ratio of wbcs to rbcs in the patient's peripheral blood. a higher ratio in the csf indicates the presence of csf pleocytosis. when the csf ratio is at least times higher than the blood ratio, bacterial meningitis is indicated, with a sensitivity of % and a specificity of %. conversely, the negative predictive value for the presence of bacterial meningitis of a less than -fold difference between the ratios is %. traumatic taps usually do not alter the csf glucose, gram stain, or culture findings, which are often abnormal with bacterial meningitis. when there is doubt about the validity of the cell count after a bloody tap, the lumbar puncture should be repeated after several hours by introducing the spinal needle intervertebral space above the original tap site. in normal csf, the glucose concentration is two-thirds that of serum glucose concentration. the csf glucose concentration is low in most infected infants and younger children and in % of school-aged children with bacterial meningitis. in children older than months of age, a csf/serum glucose ratio of less than . is % sensitive and % specific for the presence of bacterial meningitis. the presence of rbcs in a csf sample that is promptly analyzed does not affect the glucose concentration. the normal csf protein concentration is less than mg/dl in children older than months. the mean csf protein concentration is (range, - ) in full-term infants and (range, - ) in preterm infants. the csf protein concentration is elevated in more than % of younger children with bacterial meningitis but in only % of infected school-aged children. every rbcs in the csf (from a traumatic tap) increases the protein concentration by approximately mg/dl. the presence of bacterial pathogens in the csf should be investigated. microscopic examination of a gram-stained sample of the fluid is performed first. the sensitivity of this test is directly related to the number of organisms in the csf and is inversely related to the age of the patient. the gram stain identification of certain organisms, such as h. influenzae, may be problematic. a decision whether to treat a child for bacterial meningitis should not be based on the gram stain alone; the definitive diagnosis is based on the csf culture. rapid diagnostic tests for bacterial antigens in csf, including countercurrent immunoelectrophoresis and latex particle agglutination, suffer from variations in sensitivity and specificity that limit their value in clinical practice. some patients will have been treated with antibiotics before the lumbar puncture is performed. when the csf from such a child is examined, organisms may not be seen on gram stain or recovered on culture. however, abnormalities of csf cell count (including elevated leukocytes), protein concentration, and glucose concentration usually continue to suggest the diagnosis of bacterial meningitis. in this setting, presumptive treatment for bacterial meningitis is initiated. if an organism is identified by culture or antigen detection, definitive antibiotic treatment is administered. if no organism is identified, the decision to continue treatment depends on the clinical suspicion of bacterial meningitis and the exclusion of other causes of aseptic meningitis (tables . and . ). newer laboratory techniques that utilize pcr to detect bacterial pathogens are being developed and may be useful in the diagnosis of bacterial meningitis in patients who have been treated with antibiotics before lumbar puncture. routine computed tomography (ct) of the head is not indicated in children with suspected meningitis. even though children with bacterial meningitis have increased intracranial pressure, most ct scans are normal. in addition, most lumbar punctures do not result in cerebral herniation in patients with meningitis. ct should be reserved for children who show clinical signs of herniation or cerebral edema and for those who may have an intracranial mass causing signs and symptoms similar to meningitis. usually, the peripheral blood wbc and platelet counts are elevated with bacterial meningitis. a low wbc count and thrombocytopenia may also be seen; these are associated with overwhelming infection and a poor outcome. the sensitivity ( %), specificity ( %), and negative predictive value ( %) of the differential wbc count are too low to render the differential wbc examination useful in making the diagnosis of bacterial meningitis. blood cultures may be useful in identifying the bacterial pathogen of meningitis. however, a negative blood culture may be found in up to % of children with meningococcal meningitis, % of children with pneumococcal cases, and % of patients with h. influenzae type b meningitis. these numbers increase with prior antibiotic therapy. in addition, there is a negative correlation between the length of illness before diagnosis and the rate of positive blood cultures. a bacterial meningitis score has been developed to attempt to distinguish between bacterial and aseptic (nonbacterial) meningitis in patients with csf pleocytosis. the risk of bacterial meningitis is low if none of the following criteria are present: history of a seizure with the illness, blood neutrophil count ≥ × cells/l, positive csf gram stain, csf protein ≥ mg/dl, or csf neutrophil count ≥ × cells/l. this diagnostic tool is % sensitive and % specific for bacterial meningitis. it should only be applied to non-ill-appearing children older than months without petechiae, purpura, or other concerning findings on examination who have not been pretreated with antibiotics. aseptic meningitis is an inflammatory process of the meninges, most often characterized by acute signs and symptoms of meningeal irritation; csf pleocytosis, usually with a predominance of mononuclear cells; a normal or, less frequently, elevated csf protein concentration; normal or, less often, low csf glucose concentration; and no organisms demonstrable by gram stain or bacterial cultures. there are many causes of aseptic meningitis (see table . ). the most common cause is viral infection; up to % of cases are caused by enteroviruses and arbovirus. the definitive diagnosis is made by identifying the organism in the csf. however, this is not always possible, and other causes must be excluded by history, presence or absence of associated symptoms, and appropriate laboratory tests (tables . and . ). enteroviral meningitis occurs most often during the summer and early fall months. transmission is via the fecal-oral route, and young children exhibit increased transmission of the viruses and more severe disease in comparison with other age groups. initially, patients may have a respiratory tract infection, a nonspecific febrile illness, or vomiting and diarrhea. viral infection of the meninges occurs - days after initial exposure. the clinical course may be biphasic. virus from the oropharynx can be cultured only during the st - days of the illness but may be excreted in stool for - weeks. children with viral meningitis present with fever, nuchal rigidity, irritability, headache, and vomiting. less common symptoms are anorexia, drowsiness, photophobia, myalgia, and malaise. as in bacterial meningitis, affected young infants often lack meningeal signs. in addition, children may have an altered sensorium, but focal neurologic signs are rare. seizures are more common in infants. the number of wbcs in the csf varies from zero to several thousand (table . ). up to % of initial (early in the illness) csf specimens contain a predominance of polymorphonuclear cells. mononuclear cells predominate by days after the onset of symptoms. of children with enteroviral meningitis, % may have decreased csf glucose concentrations, whereas % may have elevated csf protein. treatment of enteroviral meningitis is supportive. admission to the hospital may be required while bacterial meningitis is being ruled out and for intravenous hydration. analgesics and antipyretics may also be indicated. the lumbar puncture performed to diagnose viral meningitis is often helpful in ameliorating the acute symptoms. the mechanism for this is not clear. the outcome is quite good for patients in whom common viral pathogens cause aseptic meningitis. sequelae in older children are rare. adverse outcomes are more common (but unusual) in children who have viral meningitis during the st year of life. speech and language development may be affected. treatment and outcome for the other types of aseptic meningitis depend on the underlying cause. tuberculous meningitis is an important treatable cause of aseptic meningitis. during the primary pulmonary tuberculous infection and enteroviruses and arboviruses cause most cases of infectious encephalitis in children. enterovirus encephalitis, uncommon without meningeal involvement, is suggested by epidemic occurrence and presence of typical prodrome or associated findings (table . ); prompt diagnosis is by pcr for enterovirus in csf, blood, throat, or stool specimens. a csf or blood specimen is preferred because pcr may identify enterovirus in throat and especially stool for weeks after the primary infection has resolved. arbovirus encephalitis is suggested by mosquito or tick exposure and epidemic occurrence and is diagnosed by findings of arbovirus immunoglobulin m in csf or blood or by paired serologic findings for immunoglobulin g. infections with herpes simplex virus (hsv) occur throughout the year. in neonates, hsv encephalitis usually occurs between and days of age; may produce focal or generalized cns disease; and may occur with or without conjunctivitis, oral mucosal involvement, vesicles on skin, or disseminated disease (hepatitis, pneumonia, septic appearance). after the neonatal period, hsv encephalitis is usually isolated to the cns and classically produces necrotizing encephalitis with a focus in the temporal lobe. symptoms in persons with hsv encephalitis range broadly from those suggesting mild aseptic meningitis to the presence of status epilepticus and coma and then death. in addition to neutrophils and monocytes, csf examination may show increased numbers of erythrocytes and elevated protein. ct, mri, and an electroencephalogram (eeg) may suggest a temporal lobe focus. specific diagnosis is by pcr of csf for herpes simplex dna. csf culture is usually negative. in the appropriate clinical setting, presumptive therapy with intravenous acyclovir, mg/kg/day given every hours, is indicated while the results of pcr of csf for hsv are awaited. autoimmune encephalitis. anti-d-methyl-d-aspartate receptor (anti-nmdar) encephalitis is a novel and relatively common form of encephalitis. data from the california encephalitis project showed that anti-nmdar encephalitis was the most common identifiable cause of encephalitis in their cohort, which included patients from months to years. most of the cases occurred in children and adolescents. patients present with similar features as viral encephalitis, but seizures, language dysfunction, psychosis, autonomic dysfunction, movement disorders, and eeg abnormalities are more common in these patients. in adults, fuo is defined as an illness lasting more than weeks, a fever higher than . °c ( °f) on several occasions, and uncertainty subsequent lymphohematogenous spread to extrapulmonary sites, tubercle bacilli produce local microscopic granulomas in the cns and meninges. if this primary cns infection is not contained by host defense mechanisms (t lymphocytes, monocytes), or if host defense mechanisms fail at a later period, tuberculous meningitis may result. meningitis occurs weeks to months after the primary pulmonary process. the symptoms of tuberculous meningitis are insidious and subacute (weeks to months). stage is a prodrome with nonspecific manifestations (apathy, poor school function, irritability, weight loss, fever, night sweats, nausea); stage is heralded by the onset of neurologic signs (headache, cranial neuropathy, nuchal rigidity, signs of increased intracranial pressure); and stage manifests with altered levels of consciousness (lethargy, stupor, coma). meningismus is not present in all patients. the diagnosis is supported by a history of contacts with adults with known active tuberculosis, a chronic cough, or human immunodeficiency virus (hiv) disease or by a history of immigration, poverty, or homelessness. in addition, the patient's chest radiograph is consistent with active or, more often, quiescent tuberculosis (parenchymal-hilar node calcifications, infiltrates, hilar adenopathy, and, in rare cases, endobronchial or cavitary lesions), and the patient's tuberculin skin test yields a positive result (see chapter ). cranial ct or magnetic resonance imaging (mri) may show the most intense meningeal inflammation around the base of the brain or inflammatory mass lesions (tuberculomas). the csf results (table . ) include profound hypoglycorrhachia, a high csf protein, lymphocyte-or monocytepredominant cells (usually cells/mm ), increased opening pressure, and, on occasion, tubercle organisms on acid-fast staining. pcr amplification of mycobacterium tuberculosis dna aids in making a more rapid diagnosis than does culture of csf, sputum, or gastric aspirates, which traditionally requires - weeks. the differential diagnosis depends on the stage of the illness. encephalitis is inflammation of the brain parenchyma, whereas meningoencephalitis is inflammation of the brain accompanied by inflammation of the meninges. meningoencephalitis is distinguished from aseptic meningitis by evidence of brain parenchymal involvement, including behavior or personality changes; altered level of consciousness (including agitation or coma); generalized seizures; focal neurologic signs, including focal seizures and focal motor defects (hemiparesis or ataxia); or movement disorders. bartonellosis in developed countries and brucellosis and typhoid in developing countries. often patients with an fuo have atypical manifestations of common childhood bacterial or viral diseases rather than unusual or uncommon disorders. the evaluation of a child with fuo centers on a detailed history and physical examination. taking the history should be repeated because parents often remember important details after the initial interview. the physical examination findings may also change during the course of the investigation revealing important clues (fig. . , table . ). the history should include the time of day of the fever, who measured the temperature, and the instrument that was used to measure the temperature. increased temperatures after exercise and in the afternoon often represent normal variations. the appearance of the of diagnosis after a -week study in the hospital (table . ). in pediatrics, the defined duration of fever is variable, from days to weeks (average, weeks). this may be dependent on the age of the patient, with shorter periods of fever in young infants and more traditional adult standards in adolescent patients. fuo is defined as a temperature higher than °c ( . °f) daily for at least - days and no diagnosis after an initial evaluation. the initial evaluation recommended varies but always includes a noncontributory history and physical examination, and nondiagnostic initial laboratory and radiologic tests. in accordance with this definition, the differential diagnosis for fuo in children is large ( child while febrile is also important. increased temperature without sweating might be seen in a child with ectodermal dysplasia or factitious fever. the pattern of fever should be noted (fig. . ). sustained fever, intermittent fever, and relapsing fever have been associated with different disease states. sustained or remittent fever remains elevated with little variation during the day and has been associated with enteric (typhoid) fever, tularemia, and rickettsial diseases such as typhus and rocky mountain spotted fever. intermittent fever normalizes at least once a day and is associated with tuberculosis, abscesses, lymphomas, juvenile idiopathic arthritis (jia), and some forms of malaria. children with relapsing fever have afebrile days between febrile episodes. relapsing fever has been associated with rat bite fever, borrelia species infection, malaria, brucellosis, subacute bacterial endocarditis, african trypanosomiasis, lymphomas, and lyme disease. saddle-back or double-hump fever lasts a few days, is followed by an afebrile day or , and then returns. it has been associated with some viruses and dengue fever. double quotidian fever ( fever spikes each day) occurs in kala-azar, malaria, and gonococcal endocarditis. periodic fevers occur as acute febrile episodes separated by prolonged afebrile, healthy periods. diseases to consider include cyclic neutropenia, familial mediterranean fever, and the syndrome of periodic fever, aphthous stomatitis, pharyngitis, and adenitis (pfapa). periodic fever syndromes have different prevalence patterns in different ethnic groups and different inheritance patterns. a detailed family history is particularly important when these diagnoses are considered (see chapter ). unfortunately, neither the fever pattern nor the duration is specific for a particular cause. fevers lasting for more than year are not usually infectious; factitious fever, rheumatic or granulomatous disorders, familial diseases, or malignancies need to be considered in these patients. a history of rash is important for diagnosing lyme disease, jia, and acute rheumatic fever (see chapter ). a history of pica is associated with visceral larva migrans and toxoplasmosis. exposure to domestic and wild animals should be identified to exclude zoonoses (see chapter ). the food history should be detailed and should include water sources, use of game meats, cooking practices, and consumption of unpasteurized, raw milk, or soft cheese. travel history is critically important in the establishment of a differential diagnosis. areas visited, accommodations, activities, prophylactic treatments, animal and insect exposures, and water and food sources should be reviewed. coccidioidomycosis, histoplasmosis, malaria, lyme disease, and rocky mountain spotted fever have regional distributions. children who have traveled to or have emigrated from developing countries are at increased risk for endemic diseases and m. tuberculosis (table . ) . previous medical records should be reviewed. weight loss is important for diagnosing many chronic diseases such as lymphoma, tuberculosis, and inflammatory bowel disease. poor weight gain and growth, with or without gastrointestinal symptoms, may be the only historical clue to inflammatory bowel disease (see chapter ). hiv risk factors in the parents and child should be reviewed. past and current medications should also be reviewed. the review of systems may reveal heat intolerance, palpitations, tremors, and declining quality of schoolwork in a child with hyperthyroidism. a history of severe head trauma may be associated with hypothalamic dysfunction and central fevers. whenever possible, the patient should be examined during a febrile episode. a high fever in the absence of an increased pulse may be present in a patient with factitious fever. to verify this diagnosis, the temperature of freshly voided urine may be recorded. tremor, tachycardia, palpitations, exophthalmos, lid lag, eyelid retraction, and smooth, flushed skin with diaphoresis are suggestive of hyperthyroidism. the ophthalmologic examination should include assessment of visual acuity, extraocular motion, visual field integrity, and gaze, as well as inspection of external structures and ophthalmoscopic examination (see chapter ). conjunctivitis, iritis-uveitis-scleritis, or both may be seen in a variety of infectious conditions, including epstein-barr virus (ebv) infection, leptospirosis, rickettsial infection, and cat-scratch disease. conjunctivitis, uveitis, or both occur with kawasaki disease, systemic lupus erythematosus (sle), polyarteritis nodosa, and jia. sarcoidosis may be associated with conjunctival and uveal tract nodules. a thorough ophthalmoscopic evaluation (and, if needed, slitlamp examination) should be performed. sarcoidosis may be accompanied by vascular occlusions, hemorrhages, vascular sheathing, and preretinal inflammatory exudates. cytomegalovirus (cmv) produces chorioretinitis associated with white infiltrates near vessels and confluent depigmented areas. histoplasmosis causes small atrophic spots and, in rare cases, focal granulomas of the retina and choroid. toxoplasma gondii is a common cause of recurrent retinochoroiditis. retinal changes also occur with bacterial endocarditis. tuberculosis can cause formation of choroidal tubercles and also ulcerative palpebral conjunctival lesions. slit-lamp examination may also reveal iridocyclitis in jia, behçet syndrome, and inflammatory bowel disease. the frontal and maxillary sinuses should be palpated for tenderness. the nares should be inspected for inflamed mucosa and purulent discharge. tympanic membranes should be viewed and insufflated (see chapter ). the mouth should be checked for lesions, inflammation, and tooth tenderness. behçet syndrome may manifest with oral aphthous lesions. inspection of teeth and gums may reveal a dental abscess. exudative and nonexudative pharyngitis is associated with ebv infection, tularemia, leptospirosis, and cmv. pfapa syndrome is characterized by periodic fever, aphthous stomatitis, pharyngitis, and cervical adenopathy. candida infection in the mouths of children older than years may result from immunodeficiency such as hiv or from the use of inhaled steroids. the neck should be examined for adenopathy or thyroid enlargement (see chapter ). the rest of the lymphatic system should be carefully examined. a single tender node may be seen with cat-scratch disease. generalized adenopathy can be seen in cmv infection, ebv infection, and systemic jia (see chapter ). the musculoskeletal examination should include assessments of strength and of active and passive range of motion and evaluation for warmth, tenderness, or swelling of joints. irritability and pain on palpation over a bone or disuse pseudoparalysis may be the st clue to osteomyelitis. bone pain may also result from neoplastic infiltration of the bone marrow or sickle cell anemia. unexplained fever, arthralgias, and arthritis may be present with acute rheumatic fever, jia, lyme disease, kawasaki disease, sle, polyarteritis nodosa, and behçet syndrome (see chapter ). myalgias occur commonly with viral diseases such as influenza, and may be present with rickettsial diseases, polyarteritis nodosa, takayasu arteritis, and dermatomyositis. careful auscultation of the heart and lungs is essential. a mitral or aortic regurgitant murmur may be the initial finding of endocarditis or of carditis in children with acute rheumatic fever. a pericardial friction rub may also suggest jia, sle, rheumatic fever, malignancy, or viral pericarditis. the abdomen must be carefully palpated for evidence of masses or hepatosplenomegaly (see chapters and ). abdominal tenderness may be present with abdominal abscesses, hepatosplenomegaly, and inflammatory bowel disease. a rectal examination should be performed, and stool should be tested for occult blood. sexually active girls should have a pelvic examination. pain on movement of the uterus during the pelvic examination may indicate pelvic inflammatory disease. specific serologic studies aid in the diagnosis of cmv, toxoplasmosis, brucellosis, tularemia, hepatitis a, b, and c, and leptospirosis. biopsies of lymph nodes, the skin, the liver, or bone marrow may be indicated. radiologic studies that may be of benefit if directed by the history, physical examination findings, and initial laboratory study results include sinus ct, abdominal imaging, or total body mri (to evaluate for occult osteomyelitis, malignancy, histiocytic disorders). the complete blood cell count with differential is neither specific nor diagnostic, except in rare circumstances, such as seeing lymphoblasts on the blood smear. approximately % of patients have abnormal white blood cell counts; % may have a left shift, lymphocytosis, atypical lymphocytes, or blasts. an elevated esr or crp indicates inflammation. the esr is usually ( - % of the time) high in children with fuo caused by infectious pathogens, malignancies, and rheumatic diseases. of patients with an esr less than mm/hr, % have a self-limited viral disease. urinalysis and urine culture identify occult infections, particularly in young girls. the urinalysis may also be abnormal in patients with endocarditis and rheumatic and other inflammatory disorders. unexpected consolidations, calcifications, interstitial changes, perihilar adenopathy, or cardiomegaly (heart failure, pericarditis) may be found on chest radiographs. chest films are abnormal in - % of patients with fuo. ct of the chest may reveal abnormalities not detected by a chest x-ray. specialized radiologic studies performed without specific diagnostic clues from the history, physical examination findings, or initial laboratory evaluation results have a low yield. whole body mri is another technique that may be useful in children with fuo. it is helpful in identifying abnormal areas in bones, such as with occult osteomyelitis. a wide variety of infections have been identified in children with fuo including subacute bacterial endocarditis, urinary tract infections (uti), sinusitis, abscesses, osteomyelitis, and rheumatic fever. bacterial endocarditis. bacterial endocarditis is rare in children; incidence increases with advancing age and history of preexisting heart disease (see chapter ). a new murmur or a change in the characteristic of an existing murmur may not be initially evident. vegetations also may not be visible initially by transthoracic echocardiography; a transesophageal approach is much more sensitive. serial blood cultures with anaerobic and aerobic media are necessary for definitive diagnosis. urinary tract infection. both upper and lower urinary tract infections may be asymptomatic, and leukocytes may not always be present in urine (see chapter ). sterile pyuria may be present with tuberculosis, nongonococcal urethritis, viral cystitis, kawasaki disease, reactive arthritis, interstitial nephritis, and other rheumatic diseases. renal ultrasonography may show areas of decreased echogenicity, enlarged echogenic kidneys, and renal or perinephric abscesses. kidneys may be enlarged with acute pyelonephritis. a ct scan with contrast may show infected parenchyma as a nonenhancing lucency. nuclear medicine renal scans also identify active areas of infection and old scars. sinusitis. factors that decrease the size and patency of the ostium, or impair the mucociliary transport system predispose a child to sinusitis. ethmoid and maxillary sinuses are present at birth. the frontal sinuses usually appear near or years of age but may be asymmetric or absent. sphenoid sinuses may be seen radiographically by years of age. prolonged nasal congestion, headache, purulent nasal discharge, sore throat, daytime cough, tender teeth, and halitosis may be present the skin must be inspected for evidence of rashes and other lesions (see chapter ). jia may manifest with an evanescent, salmon-colored macular rash over the trunk and joints that may appear and disappear rapidly and be evident only during febrile periods. dermatomyositis is characterized by a heliotropic rash of the upper eyelids and an erythematous eruption (vasculitis) over the extensor surfaces (gottron sign). sle may manifest with a butterfly rash over the nose and malar regions, signs of photosensitivity in sun-exposed areas, or vasculitis. the rash of kawasaki disease is erythematous and may manifest in many forms; it is most commonly a diffuse maculopapular rash. in rocky mountain spotted fever, there are macular erythematous spots on the wrists, ankles, or forearms that may become maculopapular and expand centripetally to the proximal extremities and torso; palms and soles may be involved and petechiae may develop later in the course of the illness. endocarditis may be associated with splinter hemorrhages or janeway lesions (painless, small, erythematous or hemorrhagic lesions on the palms and soles). lyme disease usually manifests with erythema migrans. this rash begins at the site of the tick bite and is erythematous with a pale center. the rash radiates out from the bite in a circular manner and persists for weeks; satellite secondary lesions may also appear. tularemia, salmonellosis, listeriosis, and ebv infections may feature generalized maculopapular rashes. laboratory evaluation should proceed in a stepwise, focused manner with emphasis on identifying serious illnesses with defined interventions (see fig. . ). initial studies should include a complete blood cell count with differential, erythrocyte sedimentation rate (esr) measurement, crp, blood cultures, urinalysis, urine culture, tuberculin skin tests with controls (anergy panel) or gamma interferon release assay, and chest radiograph. because ebv infection is common in childhood, viral-specific antibody titers may also be obtained at the initial evaluation (see chapter ). further studies should be directed from days to months. regional lymphadenopathy with or more nodes occurs proximal to the skin site - weeks after inoculation. the node or nodes become enlarged and tender and may have overlying erythema. the lymphadenopathy usually resolves after months but may last up to years. affected children may have adenopathy with fever, headache, malaise, anorexia, sore throat, and conjunctivitis (see chapter ). q fever. q fever is caused by coxiella burnetii, formerly classified as a rickettsia. it manifests with headache, fever, chills, malaise, and, on occasion, respiratory symptoms. hepatic, cardiac, and cns involvement may occur. rash is usually not seen. domestic farm animals, cats, rodents, and marsupials may be infected. pasteurization destroys the organism in milk. diagnosis is made by serologic testing. rat bite fever. rat bite fever is a relapsing fever caused by streptobacillus moniliformis or spirillum minus. s. moniliformis is a pleomorphic gram-negative bacillus transmitted by rat bite or by contaminated food or water. in - days after exposure, patients may exhibit fever, chills, malaise, and muscle aches. a rash may form on the extremities; arthralgias and arthritis may occur. complications include abscesses, pneumonia, endocarditis, myocarditis, and meningitis. diagnosis is made by blood culture or culture of other infected fluid, such as abscess aspiration. tularemia. francisella tularensis is the causative agent of tularemia. the disease is spread by contact with wild animals, such as rabbits and squirrels, and by insects that bite these animals, such as mosquitoes, ticks, and deer flies, as well as by contaminated water. a maculopapular nodule forms at the portal of entry and later becomes ulcerated. the child may present with fever, chills, and headache. lymphadenopathy, pharyngitis, conjunctivitis, hepatosplenomegaly, and pneumonia may also occur. diagnosis is made by serologic study. brucellosis. brucellosis is caused by gram-negative coccobacilli: brucella abortus, b. melitensis, b. suis, or b. canis. the microorganisms are found in sheep, goats, cattle, swine, and dogs. infection may occur by airborne spread or by ingestion of meat or milk. the child may present with fever, chills, malaise, headache, arthralgias, or myalgias. pneumonia, cardiac involvement, and cns involvement occur in rare cases. diagnosis is made by special culture techniques and serologic study. leptospirosis. leptospirosis is caused by members of the spirochete genus leptospira. infection is spread by contact with the urine of wild or domestic animals. in - weeks after exposure, patients experience the abrupt onset of fever, chills, nausea, vomiting, headache, and occasionally conjunctival suffusion and rash. liver, renal, and cns involvement may also occur. diagnosis is made by special culture techniques and serologic testing. blastomyces dermatitidis is a saprophytic fungus with both yeast and mycelial forms; it is found in the soil all over the world but is common in the americas. it is endemic in the southeast and midwest regions of north america. infections with this fungus may be disseminated or pulmonary. the diagnosis is made by visualization of single-budding yeast in clinical material, culture on sabouraud agar, or serologic tests. histoplasma capsulatum is a yeast found in soil in the ohio river valley and other locations in the united states that causes pulmonary and disseminated disease. diagnosis is made by the demonstration of the microorganism in biopsy specimens or by complement-fixing antibody. in children with sinusitis. ct studies may be helpful. rhinoscopy may show purulent material at the ostium of an infected sinus. infectious complications of sinusitis include dural space empyema or brain abscesses. abscesses. hepatic, renal, perinephric, pelvic, and subphrenic abscesses may present with fuo. internal jugular thrombophlebitis may manifest with prolonged fever and severe neck pain. liver abscess may manifest with right upper quadrant tenderness and hepatomegaly. blood cultures and liver function study results are often normal. the diagnosis may be made with mri, ct with contrast, or ultrasonography. the diagnosis of perinephric abscesses is made with ct with contrast or ultrasonography. ct or ultrasound guidance may be used to direct percutaneous drainage of many abscesses. pelvic abscesses should be suspected in children with fuo who have abdominal, rectal, or pelvic tenderness. osteomyelitis. osteomyelitis usually follows bacteremia, but it sometimes follows penetrating injury. tenderness to palpation over the infected site is common. abnormalities in plain films appear late ( weeks). mri is the imaging modality of choice. the blood or bone culture is often positive, and the esr is often elevated. suppurative myositis may mimic osteomyelitis and manifest as an fuo. rheumatic fever. acute rheumatic fever may cause fuo; the diagnosis is made by fulfillment of the jones criteria, updated in (see chapter ). initially, a child may present with polyarthralgia and an increased esr. elbows, wrists, knees, and ankles are frequently involved. the later migratory nature of the true arthritis differentiates rheumatic fever from jia. bacterial syndromes that cause fuo in children include agents of the following: • lyme disease • cat-scratch disease • q fever • rat bite fever • tularemia • brucellosis • leptospirosis lyme disease. lyme disease is caused by the spirochete borrelia burgdorferi and is transmitted by the ixodes dammini and i. pacificus ticks. the usual manifestation of early lyme disease is with erythema migrans, an erythematous, annular, expanding rash with central clearing. the rash resolves - days (usually weeks) after exposure. patients may exhibit fever, chills, fatigue, headaches, malaise, myalgias, arthralgias, and lymphadenopathy. early disseminated lyme disease follows - weeks after exposure; facial nerve palsy, peripheral neuropathy, cardiac conduction defects, myocarditis, and aseptic meningitis may occur. diagnosis is made clinically in early localized lyme disease because serology lacks sensitivity and specificity during early infection and because erythema migrans is so specific for lyme disease. diagnosis of early disseminated lyme disease requires a typical clinical illness, exposure to ticks known to carry b. burgdorferi and serologic evidence of infection with a -tier testing strategy. the initial test is an enzyme-linked immunosorbent assay (elisa) or immunofluorescent (ifa) test. if this result is equivocal or positive, a western immunoblot is performed. western blot should not be performed if the elisa is negative or has not been performed because it lacks specificity in this setting. cat-scratch disease. cat-scratch disease is a febrile illness associated with cats (usually kittens) and, more rarely, dogs. bartonella henselae, which may be transmitted by the cat flea and by cat saliva, is the etiologic agent. after a scratch or bite, a papule forms and may persist parasites fuo in children may be caused by parasitic infections, including ( ) babesiosis, ( ) toxoplasmosis, and ( ) toxocariasis. babesiosis is caused by babesia microtia and is a parasite of rodents transmitted to humans by tick bite. infection may result in fever, chills, nausea, vomiting, night sweats, myalgias, and arthralgias. identification of the organism in a thick smear of red blood cells is diagnostic. t. gondii is a protozoan parasite. children become infected from eating contaminated, undercooked meat or from exposure to the feces of domestic cats. most infections acquired postnatally are asymptomatic but children may develop a mononucleosis-like illness (see chapter ). toxocariasis (previously visceral larva migrans) results from ingestion of larvae of toxocara canis or from t. cati shed in dog and cat feces, respectively. infection results in fever, intense eosinophilia, hepatomegaly, and hypergammaglobulinemia. lung, heart, and cns involvement is rare. the eye may become infected. diagnosis is presumed with increased eosinophils and hypergammaglobulinemia, and elevated titers of isohemagglutinin to a and b blood group antigens. in a child who has traveled to or lives in a developing country, consideration must be given to the area, water sources, and activities. some causes of fuo to consider include malaria, hepatitis, typhoid fever, tuberculosis, and amebic liver abscess (table . ). malaria is transmitted by the bite of an infected mosquito carrying of the species of the plasmodium genus that cause disease in humans. the patient experiences chills, rigors, high fever, diaphoresis, and headaches. the incubation period varies among species, from week to several months. demonstration of the parasite on thick peripheral blood smear is diagnostic. risk for malaria can be checked for areas of the world on www.cdc.gov/malaria. hepatitis a may be contracted by ingestion of contaminated food or water. hepatitis b and c viruses are transmitted through blood products or sexual contact. diagnosis is by serologic testing. symptoms can include fever, malaise, jaundice, hepatomegaly, nausea, and anorexia. hepatitis b and c can become chronic (see chapter ). enteric fever is caused by infection with serovars of s. enterica, which includes s. typhi. after ingestion of contaminated water or food, incubation lasts from - weeks. persistent fever, headache, malaise, anorexia, and rose spots are clinical hallmarks of enteric fever. diagnosis is by blood culture. tuberculosis may manifest as fuo in children (see chapters and ). affected children may have pulmonary or extrapulmonary disease. the signs and symptoms of pulmonary disease may vary greatly from weight loss, tuberculin skin test conversion, and low-grade fever to mass effect from mediastinal lymphadenopathy and fulminant disseminated pulmonary involvement with miliary infiltrates or, in rare cases, cavitation. nonpulmonary tuberculosis more commonly manifests as fuo, inasmuch as positive chest radiograph findings and pulmonary signs may initiate an early work-up for tuberculosis. hematogenous spread may cause liver, heart, or renal involvement. ingested bacilli may result in gastrointestinal tuberculosis. the diagnosis requires demonstration of coccidioides immitis is found in soil in the southwestern united states. infections in humans are associated with a febrile pulmonary disease characterized by cough, rash, and chest pain. diagnosis is usually made serologically. cryptococcus neoformans is often found in pigeon droppings and can cause a variety of diseases. the diagnosis is made by culture or by identification of encapsulated yeast in collected specimens. psittacosis and lymphogranuloma venereum are chlamydial causes of fuo. chlamydia psittaci may be transmitted by infected birds and produces respiratory illness with fever. cardiac, liver, cns, and thyroid involvement are rare. diagnosis is made serologically. c. trachomatis is a sexually transmitted organism that causes urogenital infections, perihepatitis, invasive lymphadenopathy (lymphogranuloma venereum), neonatal conjunctivitis, and neonatal pneumonia. diagnosis is by cell culture and rapid antigen tests. rocky mountain spotted fever. rocky mountain spotted fever manifests with fever, headache, intense myalgias, and abdominal symptoms. a characteristic rash is usually present by the th day of illness. the rash covers the palms, wrists, soles, and ankles and progresses from macular to petechial. the disease can last up to weeks. many end organs, including the heart, kidneys, and cns, can be involved. transmission of the causative agent, rickettsia rickettsii, occurs by tick bite. diagnosis is made by pcr testing of blood. ehrlichiosis and anaplasmosis. these infections are caused by ehrlichia chaffeensis, anaplasma phagocytophilia, and e. ewingii and are transmitted by the lone star tick. anaplasmosis is caused by anaplasma phagocytophilia and is transmitted by the black legged deer tick. these illnesses are usually seen in the southeastern and upper midwestern united states, respectively, and have manifestations similar to that of rocky mountain spotted fever. the patient presents with headache, myalgias, fever, chills, nausea, vomiting, weight loss, thrombocytopenia, and leukopenia. rash is inconsistent but may be seen after week. pulmonary and renal complications can occur. mental status changes are less frequent. diagnosis is confirmed by pcr. cytomegalovirus infection. cmv may cause a mononucleosis-like syndrome in children. generalized or cervical adenopathy may be seen along with fatigue, malaise, fever, hepatosplenomegaly, and abdominal pain (see chapter ). a morbilliform rash may also be present. retinitis, hepatitis, colitis, and pneumonia may occur in children with impaired immune systems. the virus is transmitted by contact with secretions. infection is diagnosed by culture (nasopharyngeal, blood, urine) or by the detection of specific immunoglobulin g and immunoglobulin m antibodies. infectious mononucleosis. infectious mononucleosis is typically caused by ebv and may manifest with fever, exudative pharyngitis, malaise, and fatigue (see chapter ). the appearance of rash is sometimes preceded by amoxicillin therapy, but rash may occur without antibiotic administration. tender lymphadenopathy and hepatosplenomegaly may occur. the diagnosis may be made by nonspecific tests (heterophile antibody or monospot) in older patients, but these studies are unreliable for young children. specific antibody tests against viral capsid antigen, early antigen, and nuclear antigen are recommended in younger children. treatment is supportive. human immunodeficiency virus infection. infection with hiv or associated opportunistic infections or associated malignancies is another cause of fuo in children. neuroblastoma may manifest as abdominal, thoracic, or pelvic masses; spinal cord compression; bone pain; hypertension; hepatomegaly; diarrhea; and fever (see chapter ). diagnosis is aided by radiologic studies and urinary catecholamine measurements and is confirmed by biopsy. both acute lymphocytic leukemia and acute nonlymphocytic leukemia may manifest with lethargy, pallor, bleeding, fever, bone pain, lymphadenopathy, and arthralgias. diagnosis is made by blood smear and bone marrow biopsy. pheochromocytomas are rare catecholamine-secreting tumors; % occur in children. these tumors manifest with paroxysmal or sustained hypertension, headache, excessive sweating, fever, hyperglycemia, and palpitations. the tumors are usually in the adrenal medulla, but % of those occurring in children are multiple or extraadrenal. diagnosis is made by measuring urinary or plasma metanephrine or catecholamine levels. localization of tumor is by ct, mri, or iodine -metaiodobenzylguanidine scanning. familial mediterranean fever is an autosomal recessive trait seen in sephardic jews and people of middle eastern descent. the fever may be accompanied by joint, abdominal, and chest pain. anhidrotic ectodermal dysplasia is an x-linked recessive disorder associated with decreased ability to sweat, dental abnormalities, and sparse hair. eyebrows and eyelashes may be absent. fever may result from the inability of the body to cool itself. diagnosis is made by skin biopsy that shows an absence of eccrine glands. drug fever is a diagnosis of exclusion. some drugs are more likely than others to cause drug fever (α-methyldopa, quinidine, penicillins). there is no characteristic fever pattern. there is a highly variable lag time between the initiation of the drug and the onset of fever, and there is an infrequent association with rash or eosinophilia. some drugs may cause fever by virtue of physiologic side effects. anticholinergic drugs may decrease sweating and diminish the body's ability to cool itself. chronic salicylate intoxication can cause increased heat production by uncoupling oxidative phosphorylation. kawasaki disease may manifest with a variety of signs, including rash; lymphadenopathy; conjunctival hyperemia; strawberry tongue; erythematous lips; swelling of hands and feet; arthralgia; arthritis; myocarditis; late desquamation of hands, feet, and perineal area; and sterile pyuria. fever may be high and spiking. diagnosis is by fulfillment of clinical criteria (see chapter ). inflammatory bowel disease (ibd; ulcerative colitis, crohn disease) may manifest with fuo. ulcerative colitis may manifest with bloody diarrhea, fever, fecal urgency, and straining (see chapter ). pyoderma gangrenosum, arthritis, and erythema nodosum can also be seen. crohn disease (regional enteritis) may manifest with abdominal pain, fever, anorexia, and growth failure. diarrhea may develop later. arthritis, erythema nodosum, and finger clubbing may also occur. diagnosis of ibd is by endoscopy and histology. acid-fast bacilli from sputum, gastric aspirate, or the affected organ. skin testing may yield negative results even with positive controls. intestinal infection with entamoeba histolytica may produce invasion of the mucosal lining and spread to other organs such as the liver. amebic liver abscess may manifest with fever, weight loss, right upper quadrant pain, and anorexia. the patient may have painful hepatomegaly without splenomegaly. the abscess may be localized with abdominal ultrasonography or ct. diagnosis is by serologic study. rheumatic diseases as a cause of fuo are the nd most common identified cause of fuo after infections. in a systematic review, the most common causes were jia and sle (see chapter ). jia is a diagnosis that requires time to identify all of its manifestations and to exclude other entities. jia is defined by arthritis of unknown origin that begins in a child younger than years and persists for a minimum of weeks. jia is divided into subtypes: systemic, polyarticular, and oligoarticular. the systemic form often manifests with prolonged high fever. affected children often have a daily fever and may have a fine macular rash, arthralgias, arthritis, hepatosplenomegaly, or pericardial involvement. polyarticular jia may manifest with arthritis, low-grade fever, morning stiffness, anorexia, and weight loss. polyarteritis is a necrotizing vasculitis that may manifest with myalgia, arthralgia, fever, vasculitic skin lesions, and abdominal pain. cardiac, cns, and renal involvement may also occur. the esr usually is markedly elevated. biopsy and the presence of antibodies to proteinase and myeloperoxidase (antineutrophil cytoplasmic antibodies) are helpful. sle may manifest with fever, photosensitivity, mouth sores, weight loss, rash, myalgias, malaise, and hepatosplenomegaly. patients may also have serositis and renal involvement. laboratory tests that are helpful include lupus erythematosus cell preparation and those for antinuclear antibody, anti-smith antibody, anti-ribonuclear protein antibody, anti-ro (sjögren syndrome type a) antibody, and anti-la (sjögren syndrome type b) antibody. behçet syndrome is very rare in children but may manifest with fuo. patients may have aphthous stomatitis, arthritis, genital ulcers, uveitis, and erythema nodosum. hodgkin disease, lymphoma, neuroblastoma, and leukemia may all manifest as fuo. in young children, leukemia, neuroblastoma, and lymphoma should be suspected, whereas in adolescents, hodgkin disease and ewing sarcoma are more common as causes of fuo. hodgkin disease may manifest with firm, nontender adenopathy, fever, night sweats, and weight loss. diagnosis is made through biopsy. non-hodgkin lymphoma may manifest as painless adenopathy, cough or dyspnea from a mediastinal mass, abdominal mass, nerve compression, bone pain, fever, and weight loss. diagnosis is by biopsy. sweating, tachypnea, or tachycardia. if factitious fever is suspected, the temperature should be obtained while the patient is observed. the temperature of freshly voided urine can also be recorded. other patients may produce actual diseases that cause true fevers, such as by injecting infected pyogenic material subcutaneously or intravenously or by taking toxic levels of thyroid hormone. once the diagnosis is documented, psychiatric care is indicated. if no diagnosis is made, most patients are clinically well and asymptomatic on follow-up. some may be determined to be healthy from the start; most are in good health at follow-up, whereas few have symptoms at the end of evaluation. some may have relapses of fever for a few months. jia, inflammatory bowel disease, and pfapa syndrome may not be immediately diagnosed but usually manifest typical symptoms and signs within years of the onset of the fuo. hyperthyroid states may manifest with fuo. children usually have multiple symptoms, such as irritability, tremor, eyelid lag, and exophthalmos. diagnosis is made from thyroid function studies. factitious fever may be a form of factitious disorder imposed on self (formerly munchausen syndrome) or medical child abuse (formerly munchausen syndrome by proxy) (see chapter ). a variety of techniques have been used to falsely elevate a recorded temperature. a mercury thermometer may be rubbed between hands or placed near a light bulb. hot liquids may be placed in the mouth before an oral temperature is taken. hot rectal douches have also been reported to raise a rectally taken temperature. even with pathologic fevers, there is some circadian rhythm to the temperature curve; with factitious fever there is no rhythm. in addition, there is usually no vasoconstriction, many children with fever will have a source identified on their initial history and physical examination. red flags include patients with symptoms or signs of sepsis (tachycardia, hypotension) or meningitis or encephalitis (fever, headache, irritability, altered mental status and for the older child, meningismus). affected infants with meningitis are more likely than older children to have subtle and nonspecific symptoms. a child with fever of recent onset with no adequate historical or physical explanation for the fever is said to have fever without source (fws). because of the high volume of children with fws, it is important to have a reliable system for individual patient evaluation and management. although the majority of patients with fws have a self-limited viral illness, - % have an invasive bacterial infection, with young infants at highest risk. because of the potential for morbidity and mortality from the organisms that cause invasive disease, identification of patients at high risk is essential. although there is no single, timely series of tests that correctly categorizes all patients, the combination of careful clinical evaluation and appropriate laboratory screening criteria can help identify a level of risk in children of different ages. the reduction of bacteremia due to vaccine-serotype pneumococcus has led to a careful reduction in diagnostic testing, especially in the - month old child with fws. red flags include a history of immunodeficiency or other chronic medical illness, no prior immunizations, toxic appearance, signs of shock, petechiae or purpura, poor responsiveness, and other signs of altered mental status. some children, who are initially thought to have fws, develop into patients with fuo. definitions of fuo in children vary. a practical definition balancing different recommendations is fuo is a temperature higher than °c ( . °f) daily for at least - days and no diagnosis after an initial evaluation. work-up of the patient with fuo should proceed in a stepwise manner. it should be kept in mind that many patients with fuo have unusual, atypical, or complicated manifestations of common childhood illness, mainly infections. red flags include weight loss, night sweats, signs of organ system dysfunction or failure, or unstable vital signs suggestive of sepsis. only in this last category should a rapid diagnostic approach be performed and empirical antibiotic therapy initiated. temperature measurement in pediatrics: a comparison of the rectal method versus the temporal artery method randomized trial of rapid multiplex polymerase chain reaction - based blood culture identification and susceptibility testing comparison of temporal artery to rectal temperature measurements in children up to months effect of bundling and high environmental temperature on neonatal body temperature fever pathophysiology international pediatric sepsis consensus conference: definitions for sepsis and organ dysfunction in pediatrics the effects of bundling on infant temperature the future possibilities of diagnostic testing for the evaluation of febrile 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to identify serious infection in children in developed countries: a systematic review decline in invasive pneumococcal disease after the introduction of protein-polysaccharide conjugate vaccine comparison of the test characteristics of procalcitonin to c-reactive protein and leukocytosis for the detection of serious bacterial infections in children presenting with fever without source: a systematic review and meta-analysis central nervous system infection csf opening pressure in children with optic nerve head edema reference range for cerebrospinal fluid opening pressure in children normative cerebrospinal fluid profiles in febrile infants clinical features suggestive of meningitis in children: a systematic review of prospective data diagnosis and management of meningitis predictors of bacterial meningitis in the era after haemophilus influenzae the frequency of autoimmune n-methyl-d-aspartate receptor encephalitis surpasses that of individual viral etiologies in young individuals enrolled in the california encephalitis project lumbar puncture in pediatric bacterial meningitis: defining the time interval for recovery of cerebrospinal fluid pathogens after parenteral antibiotic pretreatment acute bacterial meningitis in infants and children natural history of neonatal herpes simplex virus infections in the acyclovir era safety and efficacy of high-dose intravenous acyclovir in the management of neonatal herpes simplex virus infections does this child have bacterial meningitis? a systematic review of clinical prediction rules for children with suspected bacterial meningitis cerebrospinal fluid opening pressure in children: experience in a controlled setting tuberculous meningitis meta-analysis of bacterial meningitis score validation studies central nervous system tuberculosis in children: a review of cases cerebrospinal fluid protein concentration in pediatric patients report of the committee on infectious diseases incidence of rash after amoxicillin treatment in children with infectious mononucleosis fever of unknown origin in children: a systematic review do penicillins really increase the frequency of a rash when given during epstein-barr virus primary infection? prolonged fever of unknown origin prolonged fever of unknown origin in children hemophagocytic syndrome in children: an important diagnostic consideration in fever of unknown origin prolonged fever in children: review of cases usefulness of scanning procedures for diagnosis of fever of unknown origin in children long-term follow-up of children with fever of unknown origin rocky mountain spotted fever in children key: cord- - tgecz authors: machado, gisele f.; melo, guilherme d.; souza, milena s.; machado, andressa a.; migliolo, daniela s.; moraes, olívia c.; nunes, cáris m.; ribeiro, Érica s. title: zymographic patterns of mmp- and mmp- in the csf and cerebellum of dogs with subacute distemper leukoencephalitis date: - - journal: veterinary immunology and immunopathology doi: . /j.vetimm. . . sha: doc_id: cord_uid: tgecz abstract distemper leukoencephalitis is a disease caused by the canine distemper virus (cdv) infection. it is a demyelinating disease affecting mainly the white matter of the cerebellum and areas adjacent to the fourth ventricle; the enzymes of the matrix metalloproteinases (mmps) group, especially mmp- and mmp- have a key role in the myelin basic protein fragmentation and in demyelination, as well as in leukocyte traffic into the nervous milieu. to evaluate the involvement of mmps during subacute distemper leukoencephalitis, we measured the levels of mmp- and mmp- by zymography in the cerebrospinal fluid (csf) and in the cerebellum of dogs naturally infected with cdv and uninfected dogs. the infected dogs presented high levels of pro-mmp- in the csf and elevated levels of pro-mmp- and pro-mmp- in the cerebellar tissue. active mmp- was detected in the csf of some infected dogs. as active mmp- and mmp- are required for cellular migration across the blood–brain barrier and any interference between mmps and their inhibitors may result in an amplification of demyelination, this study gives additional support to the involvement of mmps during subacute distemper leukoencephalitis and suggests that mmp- and mmp- may take part in the brain inflammatory changes of this disease. canine distemper is a viral, highly contagious and severe canine disease. the etiological agent belongs to the morbillivirus genus, paramyxoviridae family. the canine distemper virus (cdv) is a simple strand rna microorganism with negative polarity (beineke et al., ) . the virus initially infects the lymphoid tissue and reaches the central nervous system (cns) by means of meningeal macrophages and infected lymphocytes present in blood or in cerebrospinal fluid (csf), through the ependymachoroid plexus route (greene and appel, ; beineke et al., ) . due to the diversity of clinical manifestations, the brain lesions of dogs with distemper leukoencephalitis are classified as acute, subacute and chronic, depending on the amount of viral particles, the extension of the myelin loss and the composition and severity of the inflammatory infiltrate (alldinger et al., ; wünschmann et al., ; silva et al., ) . nevertheless, beineke et al. ( ) reported that lesions with different ages can be seen in the brain of the same dog. matrix metalloproteinases (mmps) are zinc-dependent enzymes involved in remodeling extracellular matrix and cell-matrix interactions. an imbalance in mmps and their inhibitors (timps) has been suggested as a cause of lesion initiation and progression during canine distemper virus infection (miao et al., ; gröters et al., ) . green et al. ( ) , working with human csf, demonstrated that the expression of mmps/timps in the csf during cns infections is determined by the etiologic agent, and that mmp- and mmp- are especially involved in inflammatory cell migration into the neuropil due to their ability to digest type-iv collagen, which is the major component of the basal membrane that involves the capillaries (rosemberg, ) . further, mmps and timps, together with other classical biomarkers, have been used to differentiate alzheimer's disease from vascular dementia (bjerke et al., ) , but biomarkers do not usually have relevance in clinical animal diagnosis and prognosis. therefore, with this study, we aimed to detect mmp- and mmp- in the csf and in cerebellar tissue by gelatin-zymography, as well as to identify these mmps and inflammatory cells in the cerebellum using immunohistochemistry. a total of dogs that died from canine distemper diagnosis were included as infected dogs. ten dogs that died with a negative diagnosis for canine distemper and without any brain alterations were included in the uninfected control group. the animals were provided from the veterinary teaching hospital of unesp -são paulo state university and from the zoonosis control center, both in the municipality of araç atuba, são paulo state, brazil. samples of csf and the whole brain were collected. the brain was sagitally sliced after removal; one hemisphere was fixed in % neutral buffered formalin and the other hemisphere was frozen at − • c. confirmative diagnosis was achieved by direct immunofluorescence test as per silva et al. ( ) , with a monoclonal anti-cdv antibody (vmrd, - -cdv) (fig. a) , and by rt-pcr with specific primers (sense: -gctcttgggttgcatgagtt- ; antisense: -gctgtttcacccatctgttg- ; genbank accession number af ) (fig. b) . the staging of the cdv infection as subacute (demyelination, astrogliosis and mild-to-moderate inflammatory infiltrate) was achieved by analyzing hematoxylin and eosin (he)-stained tissues by light microscopy ( fig. c and d) , according to the parameters described by beineke et al. ( ) and silva et al. ( ) . one of the hemispheres of each brain was frozen at − • c immediately after removal. fragments from the cerebellum and cerebellar peduncles were macerated at • c in the extraction buffer consisting of mm tris-hcl, mm nacl, mm cacl , . % (w/v) brij- , plus a protease inhibitor cocktail (complete, edta-free , roche). samples were then centrifuged at , × g for min at • c, and the supernatants were collected. for csf zymography, there was no pre-treatment of the samples, but centrifugation was performed. the total protein content was measured using the bicinchoninic acid (bca) method ( , pierce biotechnology). the gelatinolytic activity of these samples was detected by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (sds-page) gelatin zymography, according to the method previously described by machado et al. ( ) and by marangoni et al. ( ) . mmp identity and normalization between gels was achieved with a sample of canine mammary adenocarcinoma previously standardized with human recombinant mmp- (pf , calbiochem) and mmp- (pf , calbiochem). the gels were digitalized and the integrated density of the bands, expressed as arbitrary units, was calculated using the open-access software imagej . r (wayne rasband, national institutes of health, bethesda, md, usa; http://rsb.info.nih.gov/ij). for ihc, after dewaxing, endogenous peroxidase activity was blocked by incubating sections in % (v/v) hydrogen peroxide vol. diluted in % (v/v) methanol for min. pre-treatments for antigen retrieval were done according to the primary antibody specification (table ) . nonspecific binding was blocked with % (w/v) non-fat dry milk in pbs (phosphate-buffered saline) ph . for min. sections were incubated with the primary antibodies (table ) for - h at • c in a humidified chamber. slides were washed in pbs, incubated with a biotinylated secondary antibody and with streptavidin-hrp complex (lsab+ kit, dako, k ) according to the manufacturer's instructions. the reaction was developed with , -diaminobenzidine (dako, k ). the slides were then counterstained with harris's hematoxylin, dehydrated, cleared, and mounted with cover-slips. negative control sections were performed by replacing the primary antibody with pbs. lymph node tissue was used as a positive control for inflammatory cells. tissue samples were examined by light microscopy and the positive-stained area was measured by computerized image-analysis software (image-pro plus . , media cybernetics) as per melo and machado ( ) . the intensity of the positive staining was evaluated in the cerebellum and cerebellar peduncles, in a total area of , . m . the results are expressed as the percentage of the tissue's positive-stained area. all analyses were done "blind", without knowledge of the experimental groups. the differences between groups were determined by the mann-whitney test or by the wilcoxon signed-rank test, depending on the analyzed data. a value of p < . was considered statistically significant. data are expressed as the median (interquartile range). all statistical analyses were performed using prism software (graphpad). this study was approved by the institutional ethics and animal welfare committee (ceea -comissão de Ética e experimentaç ão animal, unesp, process number / ). representative zymograms from the csf and from the cerebellum/cerebellar peduncles of dogs are shown in fig. a and b. zymographic assays revealed gelatinolytic activity related to enzymes with molecular weights of kda (pro-mmp- ), kda (active mmp- ), kda (pro-mmp- ), and kda (active mmp- ). in the csf, the levels of pro-mmp- in the infected dogs were significantly higher than in the control dogs (p = . ). for active mmp- , even though there was no difference between table panel of antibodies used in this study to characterize astrocytes, t and b lymphocytes, macrophages, and matrix metalloproteinases and in the in the cerebellum of dogs with subacute distemper leukoencephalitis. polyclonal rabbit anti-human mmp- mmp- (active and pro-forms) ready-to-use tris-edta-tween ph . in steamer for min neomarkers, rb- -r groups (p = . ), the levels of this enzyme were elevated in some infected dogs. regarding pro-and active mmp- , these enzymes were not detected or detected in an inconsistent way in a few infected samples. pro-mmp- presented no difference between infected and control dogs (p = . ), and active mmp- , although not present in the control group, showed no difference between groups (p = . ) (fig. c ). in the cerebellum/cerebellar peduncles, the control group presented only faint kda bands corresponding to pro-mmp- . the infected group showed gelatinolytic bands with molecular weights consistent with pro-mmp- , active mmp- , and pro-mmp- . comparing the groups, the infected dogs presented higher levels of pro-mmp- (p = . ) and pro-mmp- (p = . ) than the control dogs. for active mmp- , even if there was detection in infected animals, its levels were statistically similar to those in the control animals (p = . ; fig. d ). this study, according to the authors' knowledge, is the first to use zymography to evaluate mmp- and mmp- during subacute distemper leukoencephalitis. zymographic analysis of mmp- and mmp- allowed the identification of these enzymes according to their molecular weights. in fact, this method was sensitive enough to allow the distinction between the active and the pro-forms of mmp- and mmp- (kleiner and stetler-stevenson, ) . in agreement with bergman et al. ( ) , only the pro-form of mmp- was detected in the csf of the clinically healthy dogs, whereas mmp- was not detectable in any form. during distemper leukoencephalitis, in the csf, increased activity of pro-mmp- was predominantly noticed; on the other hand, pro-mmp- and pro-mmp- were highly detected in the cerebellum/cerebellar peduncles, although active mmp- was also detected in some samples. mmps are involved in the pathogenesis of several viral infections in the cns and the imbalance between mmps and timps has been reported not only in humans with immunodeficiency virus (hiv) infections (conant et al., ) , but also in experimental murine infection with japanese encephalitis virus (shukla et al., ) , coronavirus (marten and zhou, ) and west nile virus (wang et al., ) . the detection of pro-mmp- and pro-mmp- in the cerebellum/cerebellar peduncles in this study is in agreement with miao et al. ( ) , that, by immunohistochemistry, detected an up-regulated expression of mmps- , - , - , - , - , - , - and - in the brain of dogs with distemper, in a phase-dependent manner. however, ihc detects both active and pro-forms of mmps together, whereas zymography is clearly able to differentially detect them. by means of ihc, it was possible to detect in which cell population the mmps were present. faint cytoplasmatic mmp- -staining was noticed in mononuclear cells, ependymal cells and in rod cells with microglial morphology (fig. e) . however, the main mmp- -positive cell population was composed of mononuclear cells in the white and gray matter and in the leptomeninges, with more intense staining in areas of malacia and demyelination (fig. f ). mmp- presented diffuse staining in areas of malacia (fig. g ) and cytoplasmatic staining in inflammatory cells inside blood vessels, in gitter cells within the demyelination plaques, and in some neurons, ependymal cells and perivascular cells (fig. h) . ihc also facilitated the identification and quantification of inflammatory cells trafficking through the cerebellum. an important component of the inflammatory infiltrate at the injured tissue was the cd + t lymphocytes, which were detected mainly in the perivascular cuffs and diffusely distributed along the demyelinization plaques. the perivascular infiltration was two to three cell layers thick (fig. a) . however, in some dogs, with the progression of demyelination, the vacuoles seemed to push the cells around them, and t lymphocytes were distributed in clusters in these cases. these cells comprised . % of the immunostained area in the infected group, while the control group presented only . %, representing a significant difference (p = . ; fig. c ). b lymphocytes were mainly observed in the leptomeninges and within the perivascular cuffs, also two to three cell layers thick (fig. b) , and even if in smaller number than t cells, cd ␣ + b lymphocytes were detected at higher levels in the infected group than the control group (p = . ; fig. c ). with regards to astrocytes, the main cerebellar alteration was astrogliosis. the presence of intranuclear inclusions (fig. d ) and gemistocytic cells was also a frequent finding. the astrocytes in the infected dogs presented an increase in the cytoplasm volume and in the length of the cytoplasmatic processes, and also a more intense cytoplasmic staining (fig. d) . the gfap immunoreactivity in the astrocytes of the infected dogs comprised an area of . %, while the area of the control dogs consisted of . %, characterizing a significant difference (p = . ; fig. f ). regarding macrophages/microglial cells, there was positive detection in only out of infected dogs, within and around the demyelination plaques (as gitter cells) (fig. e) . no positive staining was noticed in the control group. since the pathogenesis of distemper leukoencephalitis is phase-dependent, the source of mmps within the cns may vary according to the time of infection. mmp- , mmp- and timp- are expressed mostly in astrocytes and in macrophages/microglia in initial lesions, while infiltrating lymphocytes as well as activated macrophages/microglia are the main source of these components in advanced distemper leukoencephalitis (gröters et al., ) . the production of active mmp- and mmp- by t cells, monocytes and dendritic cells is required for their migration across the blood-brain barrier (bbb) and their subsequent invasion of the cns compartment (rosemberg, ; stamatovic et al., ) . by immunohistochemistry, we detected mmp- and mmp- preferentially in mononuclear cells. nevertheless, the role of mmps within the cns may start prior to leukocyte infiltration. in canine inflammatory brain lesions, microglial cells expressed a variety of mmps and timps mrnas, including mmp- and mmp- ; and some dogs also presented mmp- activity in brain-zymography (stein et al., ) . further, in theiler's murine encephalomyelitis (tme), a virus-induced model of multiple sclerosis, after stereotaxic injection of activated mmp- , - , and - into the caudal cerebellar peduncle of adult mice, the authors noticed a focally extensive primary demyelination, before the infiltration of inflammatory cells, as well as a reduction in the number of oligodendrocytes (hansmann et al., ) . in demyelination, mmps also act in myelin basic protein fragmentation (shiryaev et al., ) . due to the morphological similarities between canine distemper demyelinating leukoencephalitis and human demyelinating diseases, such as multiple sclerosis, canine distemper represents one of the few animal models for spontaneous demyelinating diseases to study the pathogenesis of myelin loss associated with immunomediated mechanisms . in multiple sclerosis, mmp- seems to play a critical role in early lesions (cossins et al., ) , whereas mmp- seems to have an ambiguous role in initial and advanced lesions (lindberg et al., ) . further, blood monocytes from patients with multiple sclerosis showed an elevated level of several mmps, including mmp- , - , - and - (kouwenhoven et al., ) . the characterization of separate roles for individual mmps is complicated by the wide variation of expression patterns, kinetics, substrate, and cell specificity in different inflammatory settings (savarin et al., ) . by zymography, there was no way of detecting a constant profile of mmps in the csf and the cerebellar tissue of the dogs and establishing a secure biomarker for the subacute inflammatory phase of the cdv infection; however, pro-mmp- was present in most of the csf samples and pro-mmp- and pro-mmp- were increased in the cerebellum. the mmps/timps imbalance may contribute to the complex mechanisms enrolled in distemper leukoencephalitis and therapies based on metalloproteinase inhibitors for dogs may be an additional aid to help in the recovery or stop the progression of the lesions in animals in the acute phase of disease. restricted expression of viral surface proteins in canine distemper encephalitis the pathogenesis of canine distemper virus induced demyelination-a biphasic process pathogenesis and immunopathology of systemic and nervous canine distemper characterization of matrix metalloproteinase- and - 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), as well as a.a. machado (grant number / - ) and d.s. migliolo (grant number / - ). none of the authors of this paper has a financial or personal relationship with other people or organizations that could inappropriately influence or bias the content of the paper. key: cord- -g q gpp authors: nan title: neurocritical care society th annual meeting date: - - journal: neurocrit care doi: . /s - - - sha: doc_id: cord_uid: g q gpp nan eighty-five patients were enrolled, underwent therapeutic hypothermia, and had a poor outcome. baseline characteristics did not differ between groups, except for the use of sedatives: % of hypothermia versus % of normothermia patients (p= . ). corneal reflex, motor response and neuron specific enolase (nse), performed sub optimally in both the hypothermia and normothermia groups. however, all predictors accurately predicted poor outcome (fpr %) in patients without sedation regardless of whether they received hypothermia *numbers indicate percentages ( % ci). ‡ data not available for all patients. sedation is a confounder in the prognostication of comatose survivors after cpr. patients treated with hypothermia are more likely to receive sedation in proximity of their -hour neurological examination. hypothermia did not affect the accuracy of predictors of poor neurological outcome in this limited data set. methods: continuous eegs performed in a pediatric icu were transformed into -channel cdsa and aeeg displays. neurophysiologists and eeg technologists were trained to identify seizures using cdsa and aeeg. participants were then presented with only the cdsa or aeeg displays and asked to mark events that they suspected to be seizures. their performance was compared to seizures previously identified using the conventional - channel eeg recording. the eeg recordings contained discrete seizures over hours. the sensitivity for seizure identification and false-positive rates across all recordings are shown below. values are median (range). false-positive rate (# / hour) sensitivity (%) false-positive rate (# / hour) . ( . - . ) . ( . - . ) . ( . - . ) . ( . - . ) for individual recordings, however, the median sensitivity for seizure identification varied from % to %, and the median false-positive rate varied from /hour to . /hour. factors reducing the sensitivity included focal and low amplitude seizures. factors increasing the false-positive rate included movement and electrode artifacts, and non-ictal eeg waveforms such as periodic epileptiform discharges and a burst-suppression pattern. cdsa and aeeg are equally sensitive and specific tools for seizure identification among critically ill children. their performance is likely even better in a clinical context, when reviewers have access to the underlying raw eeg. these findings support the use of cdsa and aeeg as screening tools, with the caveat that low amplitude and focal seizures are liable to be missed using these techniques. intermountain medical center, salt lake city, utah, united states, johns hopkins hospital, baltimore, md, united states recent studies suggest that glucose variability is an important predictor for mortality in a mixed critically ill population, however, the relationship of glucose variability with sich remains ill defined. we sought to evaluate the relationship between glucose variability on in-hospital mortality during the acute phase of sich. we performed a retrospective chart review of consecutively admitted patients with sich with a minimum of glucose readings during the first -icu days. data extracted included: patient characteristics, clinical features, glucose values/insulin use, and outcomes. blood glucose indices assessed over the days included: average glucose levels (glucavg), standard deviation of glucose (glucsd), coefficient of variance (gluccv), and peak glucose (glucmax). statistical assessment of the glucose indices was assessed in relationship to in-hospital mortality. there were a total of glucose readings in patients with an overall mortality of %. univariate analysis showed the only significant baseline patient characteristics were lower admission gcs and higher apache ii in the nonsurvival group. admission glucose ( ± vs. ± ; p= . ), glucavg ( ± vs. ± ; p= . ), glucsd ( ± vs. ± ; p< . ), gluccv ( ± vs. ± ; p= . ), and glucmax ( ± vs. ± ; p= . ) were significantly higher in the nonsurvival compared to the survival group (mg/dl, respectively). logistic regression analysis showed that significant predictors for in-hospital survival include admission gcs (or . ; % ci . - . ; p< . ), glucsd (or . ; % ci . - . ; p< . ), and gluccv (or . ; % ci . - . ; p= . ). findings from this study suggest that a large variability of glucose during the acute phase of sich is associated with higher in-hospital mortality. the variability appears to be a more important predictor for outcomes than admission glucose or average glucose levels. the proportion of deaths was higher among patients in the intensive arm but this was not statistically significant ( % vs. %, p= . ). when good versus poor outcome at months was dichotomized to mrs score - versus - , respectively, there was no difference in outcome between the two groups ( . % vs. % had a poor three month outcome, p = . ). there was also no difference in icu or hospital los or days on mechanical ventilation. hypoglycemia (< mg/dl) and severe hypoglycemia (< mg/dl) was more common in the intensive arm ( % vs. %, p= . ) and ( % vs %), respectively. there was no benefit to intensive insulin therapy in this small critically ill neurologic population. previous studies of glycemic control in non-neurologic icu patients have shown conflicting results. this is the first glycemic control study specifically examining critically ill neurologic patients and functional outcome. given these results and the increased resources required to implement intensive insulin therapy, it cannot be recommended over conventional control. to improve organ donation conversion rates, neurointensivists at a level ii trauma, community hospital and the local organ procurement organization (opo) decided to diminish decoupling. decoupling is when the explanation of brain death by the physician is separated from the request for organ donation by the family support coordinator (fsc). the fsc is not present for the final explanation of declaration of brain death and the physician familiar to the family, typically, is not present for the request. we were concerned that in the transition of physician to fsc, conversion was being lost. data was retrospectively reviewed from - when decoupling was still occurring and compared to data thus far from where the physician and fsc were present together for both the final explanation of brain death (by physician) and request for organ donation (by fsc). we also compared data from two similar local hospitals that have not changed decoupling. all first person consent donors were removed from the data. in - , our hospital's conversion rate of eligible to actual donors was % and % respectively. in the first six months of , where decoupling was diminished, the conversion rate was %. the conversion rates for two other local hospitals of similar potential and patient mix were % ( ), % ( ) , and % ( ) (community based hospital); and % ( ), % ( ) , and % ( ) (university based hospital). the presence of the physician familiar to the family during the organ donation request may enhance organ donation conversion in non-first consent potential donors. physicians should consider working with the opo and fsc to try this process especially if conversion rates for organ donation are low. previous studies suggest that dynamic autoregulation in the posterior cerebral artery (pca) is less efficient compared to the middle cerebral artery (mca). we examined the role of cerebral vasodilation due to metabolic activation (i.e. visual stimulus) on autoregulatory characteristics in the two vascular territories. blood flow velocity (bfv) in the pca and mca and mean arterial pressure (map) were measured continuously in healthy volunteers ( ± years) while seated with eyes open. additional subjects ( ± years) were examined with eyes closed and open. cerebrovascular resistance index (cvr i ) was calculated as the ratio of map to mean bfv in the pca and mca arterial territories. autoregulation was assessed using transfer function gains in both the pca and mca territories in the low ( . - . hz), high ( . - . hz) and cardiac (~ hz) frequency ranges. the effects of vascular territory (pca vs. mca) or visual activation (eyes-closed vs. eyes-open) on bfv, map, endtidal co , cvr i , and transfer function coherence, gains, and phases were assessed by using a repeated-measures two-way anova, respectively. with eyes open, gains were significantly higher in the pca compared to the mca in the low (pca: . ± . vs. mca: . ± . , p= . ) and high (pca: . ± . vs. mca: . ± . , p= . ) frequencies. opening eyes increased bfv and reduced cerebrovascular resistance index in the pca but not mca. this vasodilation in the pca was associated with increased gain in the low (autoregulatory) frequency while mca gain did not change (pca: . ± . vs. . ± . , mca: . ± . vs. . ± . , p= . ). dilation of the pca territory during visual cortex activation resulted in increased pca transfer function gain without changing mca gain. thus, impaired autoregulation in the pca reported in previous literature is likely the result of metabolic vasodilation and not an inherent difference in the autoregulatory characteristics of the posterior circulation. various scales have been devised for the prediction of vasospasm following aneurysmal rupture. all such tools require the measurement of sah burden by computed tomography to predict the likelihood of symptomatic vasospasm. especially prominent in these scales is their reliance upon a subjective assessment of clot thickness which allows for variability in grading across raters. the current study seeks to compare the inter-rater reliability of the fisher and newer frontera scales when a rigid definition of thick clot is used. cases of subarachnoid hemorrhage were randomly selected from our radiographic archives. initial head cts were independently reviewed by two raters and a score for both the fisher and frontera scale was assigned to each study. the following criteria were established to characterize thick clot: . hemorrhage in any major cistern appearing on two contiguous slices; . hemorrhage occupying > % of any major cistern on a single cut; . contiguous hemorrhage with a density approximating that of bone. hemorrhage was scored as "thick" if any two of the three criteria were met. the degree of agreement in scores between raters was then assessed by way of the spearman's rho and cohen's kappa for inter-rater reliability. for both the fisher and frontera scales, a high degree of inter-rater reliability was demonstrated with rho values of . (p=. ) and . (p. ) respectively. when cohen's kappa was employed, respective values of . and . were obtained. these kappa values, which reflect the use of a stringent definition for thick subarachnoid hemorrhage, were stronger than those previously reported by ah. kramer et.al. with the use of a stringent definition for thick subarachnoid hemorrhage, an assessment of subarachnoid clot burden can be made that shows a high degree of reliability across observers. although ventilator-associated pneumonia (vap) carries significant mortality there is scarce data on vap in the neurosurgical intensive care unit (nsicu). we sought to determine the clinical factors associated with vap in the nsicu. we analyzed all admissions to the nsicu requiring mechanical ventilation for at least hours to determine factors associated with vap. we collected demographics, medical history, admission diagnosis, admission glasgow and four coma scale, tracheostomy need, ventilator days, length of stay, and mortality. for statistical analysis we performed fishers exact test (categorical variables) and students t-test (continuous variables). we used the centers for disease control vap definition. we analyzed admissions to the nsicu over one year. the sample was comprised of males and females with a median age of ( - ). the median gcs was ( - ) and the median four scale was ( - ). the median length of stay was days . diagnosis included subarachnoid hemorrhage ( %), head trauma, ( %) intracranial hemorrhage ( %), subdural hematoma ( %), spinal cord injury ( %), neoplasms ( %), and others ( %). the incidence of vap was . %. mortality was significantly higher (p < . ) among patients with vap ( %) than in non-vap patients ( %). there was no difference in clinical risk factors, admission diagnosis, and need for tracheostomy. the mean age of vap patients was and that of non-vap patients was . age < was associated with vap (p< . ). the only clinical variables associated with vap were mechanical ventilation for > days and four score < (p = . and . respectively). in the nsicu vap is frequent and carries significant mortality. duration of mechanical ventilation and four coma score predict vap. poster blood pressure decreases due to general anesthesia for intra-arterial therapy for acute ischemic stroke are associated with decreased functional relative hypotension after large vessel stroke is associated with poor outcome. general anesthesia (ga) causes peripheral vasodilation and cardiac depression, leading to a decrease in systemic blood pressure. in initial analysis of the merci registry-a prospective, uncontrolled cohort of patients treated with the merci retriever-functional outcome after stroke was worse in those patients intubated for the procedure as compared with those in whom conscious sedation or deep sedation was performed. we hypothesized that the poor functional outcomes in intubated patients resulted from decreased systemic blood pressure. the study population consisted of all patients enrolled at our institution in the merci registry from october to july . during the study period, all ia stroke interventions were performed under ga. data regarding demographics, stroke severity (nihss on presentation), recanalization (tici grade), and functional outcome at days (modified rankin scale; mrs) were prospectively collected. we retrospectively reviewed the blood pressures on presentation, prior to intubation and after intubation. : patients were identified; of these, had adequate records of blood pressure before and after intubation as well as day follow-up assessments. the average age was and % were male. compared with pre-intubation baseline, significant reductions in sbp ( . vs . , p<. ) and map ( . vs . , p<. ) were observed following intubation. controlling for well established predictors of outcome (nihss, age, location of vessel occlusion, and recanalization), the first sbp and dbp recorded immediately after intubation were significantly correlated with mrs (p= . , p= . ) with lower measurements associated with poor functional outcomes. blood pressure declined significantly as a result of general anesthesia, and lower sbp and dbp following intubation were associated with worse functional outcomes. these findings suggest that blood pressure should be aggressively supported in acute stroke patients treated undergoing ia mechanical thrombectomy. posttraumatic vasospasm (ptv) is an under-recognized cause of ischemic damage following traumatic brain injury (tbi), but little is known about its pathogenesis and risk factors. although ptv significantly differs from aneurysmal vasospasm [ , ] , it shares certain characteristics [ ] [ ] [ ] [ ] that may provide insight into its pathogenesis. in particular, the risk of aneurysmal vasospasm is increased in patients with fever [ ] [ ] [ ] [ ] [ ] [ ] or leukocytosis [ - ], but these relationships have not been previously explored in ptv. a review of consecutive patients with tbi yielded patients with severe tbi that survived beyond hours. eight patients developed clinically significant posttraumatic vasospasm (csptv), defined as unexplained decline in neurological function or brain tissue oxygenation with ct angiogram evidence of arterial vasospasm. temperature and serum leukocyte counts were compared in severe tbi patients with and without ptv. admission temperature was significantly higher in patients that developed csptv ( . . ºc vs. . . ºc, p= . ), and fever on admission (t> ºc) was associated with significantly increased likelihood of vasospasm (or= . ). csptv did not occur in patients with hypothermia (t< ºc) on admission, while % of those with fever (t> ºc) developed csptv. admission leukocyte count was significantly higher in patients that developed csptv ( . . k/mm vs. . . k/mm , p= . ). . % of patients with leukocytosis on admission (wbc> k/mm ) developed csptv, compared to . % of patients without leukocytosis. hyperthermia on admission correlates with increased likelihood of developing clinically significant ptv. serum leukocyte count on admission is higher in patients who subsequently develop csptv, suggesting that activation of inflammatory pathways and/or early infection may be involved in the pathogenesis of vasospasm. the observation that csptv did not occur in patients with admission temperatures below ºc suggests a possible protective role for early hypothermia. subarachnoid hemorrhage (sah) frequently causes stunned myocardium (sm). the predictors of sm and its impact on clinical course and outcome are not fully defined. we evaluated consecutive sah patients enrolled in the sah outcomes project from february -june . patients were excluded due to history of cardiac disease, were excluded due to non-aneurysmal sah. sm was defined as wall motion abnormalities +/-elevated troponins. demographic, clinical, and outcome data was compared between those with and without sm. results: % (n= ) of patients were hunt & hess (hh) grade - . modified fisher score was >= in % (n= ). sm was diagnosed in % (n= ). on univariate analyses, sm was associated with loss of consciousness (loc) at onset, hh grade, hijdra score, posterior aneurysm location, female gender, tobacco non-use, bmi, ivh, systolic bp, heart rate (hr), glucose, and wbc count. in a logistic regression accounting for race and age, female gender (p= . ), loc at onset (p< . ), posterior aneurysm location (p= . ), hr (p= . ), systolic bp (p= . ), hh grade (p< . ) & tobacco non-history (p= . ) were independent predictors of sm. sm was associated with in-hospital development of fever, hyperglycemia, pneumonia, anemia, seizures, global cerebral edema (gce), sodium dysregulation, and arrhythmia. after accounting for age & gender, arrhythmia (p< . ), fever (p= . ), and gce (p< . ) were independently associated with sm. after adjusting for gender, age and known risk factors for poor outcome, sm was an independent predictor of mrs > and death at months. chronic brain atrophy is regionally specific and is regionally associated with reductions in oxidative brain metabolism but not ischemia. the temporal lobe exhibit the greatest extent of atrophy, which may be related to the extent of initial trauma. leão's spreading depression (sd) of electrocorticographic (ecog) activity describes a propagating wave of neuronal/astroglial depolarization in cerebral grey matter. sd occurring in normally perfused cortex may be benign, but similar peri-infarct depolarizations (pid) cause ischemic lesion growth. here we present results of a pilot study to determine the association of depolarizations with clinical outcome in traumatic brain injury (tbi). at five hospitals, subdural electrode strips were placed in patients who required craniotomy for surgical management of tbi. sd and pid events were identified by criteria of fabricius et al. (brain : - , ) in ecog recordings made during intensive care for a median duration of hr. six-month egos scores were dichotomized to good ( - ; n= ) and poor ( - ; n= ) outcomes. in / ( %) patients, depolarizations occurred. of these, were sd type, were pid, and were mixed. the proportion of poor outcomes was % ( / ) in patients with no depolarizations, compared to % ( / ) in patients with sd and % ( / ) for patients with pid. the occurrence of pid and either type of depolarization were both significantly associated with worse outcomes (fisher exact test, p= . and , p= . , resp.), while sd alone was not ( , p= . ). there was no association of pupil reactivity ( , p= . ), gcs motor score ( , p= . ), pre-hospital hypotension ( , p= . ), or subarachnoid hemorrhage ( , p= . ) with outcome. ages of patients with good ( ± s.d.) vs. poor ( ± ) outcome did not significantly differ (p= . ). these data suggest that depolarization activity is significantly associated with poor outcome, with predictive power at least as great as established outcome predictors. prevention of depolarizations by pharmacologic or physiologic therapy may represent a novel strategy to improve tbi outcomes. an increased sample size is required for improved statistical power and to determine the independence of depolarizations from co-variates. introduction: vasospasm (vs) represents a substantial source of morbidity and mortality in patients with subarachnoid hemorrhage (sah). transcranial ultrasound (tcus) velocities indicating vs in the anterior cerebral artery (aca) are not well established. the purpose of this study is to identify aca velocities that correlate to ipsilateral aca infarction. the aca mean velocities of consecutive sah patients undergoing routine twice daily tcus were prospectively collected. the maximum (max), minimum (min), and first (fir) mean velocity value for each vessel was determined, as were the ratios for max/min and max/fir. this process was performed for the entire group, and then for only patients having at least days of readings. determination of aca territory infarction was made by evaluation of serial head ct scans performed up to day following the ictus. velocity comparisons were made between patients having and those not having aca territory infarctions on ct. for the entire group, data was available for vessels, of which had associated infarction. max velocity was somewhat greater in patients with aca infarctions ( cms/s vs cms/s, p=. ), and min velocities were substantially greater ( cms/s vs cms/s, p=. ). the group having at least days of tcus constituted vessels, of which had associated infarction. max velocity was again somewhat greater ( cms/s vs cms/s, p=. ) and min velocity was again significantly greater ( cms/s vs cms/s, p=. ). no correlation was observed for fir, max/min, or max/fir. patients ultimately developing aca infarctions have greater min velocities and tend to have greater max velocities. since only the use of max velocities is practical, our findings suggest that velocities between - cms/s may identify those vessels at risk for infarction, which is consistent with the available literature. cerebral infarction following subarachnoid hemorrhage (sah) contributes to morbidity and mortality. vasospasm (vs) has traditionally been considered the main cause, yet recent literature suggests other potential etiologies. anterior cerebral artery (aca) infarctions may result in permanent deficits of intellect and behavior. the purpose of this study is to document the prevalence of aca infarctions and to characterize the etiology of these infarctions in patients with aneurysmal sah. consecutive sah patients underwent review of cerebral ct scans as close to weeks after the ictus as possible so as to identify sah related aca infarctions. earlier scans were reviewed in patients found to have aca infarctions to determine the timing of the infarction. vs related infarctions were defined as those beginning at least days after the ictus. infarcts occurring less than days after the ictus were considered to be non-vs related. imaging was available for patients ( aca territories). overall, . % of patients developed aca infarctions in . % of aca territories. of these, only . % of patients and . % of territorial infarcts were deemed likely due to vasospasm. most aca infarct patients ( %) had aca/acomm aneurysm ruptures. of patients with aca infarction and aca/acomm aneurysms, % had infarcts within the first days (p=. ). all bilateral aca infarctions with aca/acomm aneurysms had infarctions within days of the ictus. aca infarctions are not rare in patients with sah. patients with aca/acomm aneurysms were more likely to have aca infarcts in the acute phase, prior to the usual onset of vs. the etiology of these infarctions remains to be determined, but may be related to vessel thrombosis at the time of hemorrhage, procedural/operative complications, or early vs. telemedicine holds promise as a technology-intensive method of providing rapid acute neurology expertise to local hospitals with available ct scanning, and has been proposed as a way to increase access to limited specialty expertise in a cost-effective manner. we here report the experience of a multi-state telemedicine company, working in joint effort with academic hospitals, providing acute neurological consultations to community-based hospitals. specialists on call (soc) is a california-based telemedicine company providing / specialist physicians consultations to urban, suburban and critical access hospitals via videoconferencing technology. neurological consultations are conducted by board-certified neurologists. consults requests are responded within minutes. initially, the specialists discusses the case by telephone and in a second step, the video-conference is started. teleneurological exam is conducted following established and validated guidelines, especially for nihss. recommendations and further steps are discussed with patients, family members and consulting physician. between january and may , a total of teleneurology consults were performed, among community hospitals in states. only hospitals had over beds ( and ), the rest ranged between and beds. stroke was the diagnosis in cases ( . %), of which ( %) were acute ischemic events (aie) (stroke or tia) and ( %), intracranial hemorrhages. ( . % of aie) received thrombolytic therapy with intravenous tpa. seizure was the diagnosis in patients ( . % of the total) and other diagnosis (including headache, dizziness, vertigo and chronic pain) in ( . %) patients. telestroke consultation can be useful in increasing the use of intravenous tpa at community hospitals without access to adequate on-site stroke expertise. besides thrombolytic decisions, teleconsultation can improve the care of other neurocritical conditions, including seizures, or intracerebral hemorrhage and triage to centers with neurocritical-care capability. increased intracranial pressure (icp) is associated with poor outcome in acute brain injury. in this study we examined how episodes of increased icp (> mmhg; > minutes) affected brain metabolism. twenty-one patients (mean age . + . years) with severe brain injury (gcs< ) were studied prospectively. lactate, pyruvate, and glucose were measured each hour using cerebral microdialysis (cma). brain oxygen (pbto ), mean arterial pressure (map), icp and cerebral perfusion pressure (cpp) were recorded continuously. linear mixed effects models were used to examine the relationship between episodes of increased icp and the lactate:pyruvate ratio (lpr). there were episodes of increased icp, episodes of compromised pbto (< mmhg) and episodes of brain hypoxia (pbto < mmhg). median icp ( % - % iqr) was greater during brain hypoxia ( . [ . ] vs. . [ . ]; p < . ). gee models indicated that icp > mmhg was associated with more than double the odds of brain hypoxia (or= . ; % ci: . , . , p= . ) or compromised pbto (or= . ; % ci: . , . , p= . ). however the frequency of increased icp (> mmhg) was similar among patients with compromised pbto (p= . ) or brain hypoxia (p= . ) compared to normal pbto . only half the patients with brain hypoxia had increased icp. elevated lpr (> ) was rare (n= [ . %] of icp episodes). median lpr ( % - % iqr) was greater during episodes of brain hypoxia than normal pbto ( . ( . ) vs. . ( . ), p< . ) and only slightly greater in episodes with compromised pbto compared to corresponding episodes with normal pbto ( . ( . ) vs. . ( . ), p= . ). lpr did not increase when icp was > mmhg. evidence for brain energy dysfunction is very rare when icp is > mmhg and any icp effect on lpr may be indirect and depend on pbto . rafael badenes, pablo gonzalez, laura alcover, armando maruenda, javier belda hospital clinico universitario, valencia, spain this was a pilot study to compare the cerebral neurochemical changes in patients with traumatic brain injury (tbi) who underwent conventional blood glucose level (bgl) control and intensive bgl control with continuous titrated insulin. this prospective, randomized study was conducted in traumatic brain injury patients in a surgical and trauma intensive care unit. patients admitted over an -month period with tbi were prospectively divided into two groups according to the method used for bgl control: the 'intensive' group consisted of patients who underwent continuous titrated insulin infusion to maintain a lower normoglycemic level of - mmol/l, and the 'conventional' group consisted of patients whose bgl was maintained at between . and . mmol/l using conventional 'sliding scale' bolus subcutaneous insulin administration. data on cerebral haemodynamics, interstitial brain oxygenation (ptio( )) and neurochemical monitoring were collected via microcatheters inserted in the penumbral region. we analyzed cerebral microdialysis samples. in patients treated with intensive insulin therapy, there was a reduction in microdialysis glucose by % of baseline concentration compared with a % reduction in patients treated with a conventional blood glucose level control. intensive insulin therapy was associated with increased incidence of microdialysis markers of cellular distress, elevated glutamate ( +/- % vs. +/- %, p<. ), elevated lactate/pyruvate ratio ( +/- % vs. +/- %, p<. ) and low glucose ( +/- % vs. +/- %, p<. ), and increased global oxygen extraction fraction. cerebral microdialysis glucose was lower in nonsurvivors than in survivors ( . +/- . vs. . +/- . mmol/l, p < . ). intensive glycaemic control using insulin induced a decrease of cerebral glucose and an increase in microdialysis markers of cellular distress. in patients with severe brain injury, tight systemic glucose control is associated with increased mortality. brain tissue oxygen (bto ) monitoring is used in severe traumatic brain injury (tbi) patients. how cerebral hypoxia should be treated and its response to treatment is not clearly defined. we examined which medical therapies restore normal bto in tbi patients. severe tbi (gcs less than ) patients were enrolled in a prospective observational cohort study. intracranial pressure (icp), cerebral perfusion pressure (cpp) and bto were monitored. episodes of cerebral hypoxia (bto less than mmhg) and medical interventions and therapies that improved bto were identified. three hundred seventy nine episodes of cerebral hypoxia were recorded and treated in forty nine patients (mean age +/- years). medical management successfully reversed % of the cerebral hypoxia episodes. ventilator manipulation, cpp augmentation, and sedation were the most frequent interventions. increasing fio restored bto % of the time. cpp augmentation and sedation were effective in % and % cerebral hypoxia episodes, respectively. icp reduction using mannitol was effective in % of treated episodes. phenylephrine was the most frequent vasopressor administered and improved bto % of the time. other interventions including head repositioning, airway suctioning, and blood transfusions, were effective in %, %, and % treated episodes, respectively. successful medical treatment of cerebral hypoxia was associated with improved outcome. survivors had a % rate of response to treatment (n= ) and nonsurvivors had a % rate of response (n= ; p= . ). cerebral hypoxia occurs in tbi patients despite traditional practices to maintain cpp. medical interventions other than those to treat icp and cpp can improve bto , increasing the number of therapies for severe tbi in the icu. poster intravenous dantrolene for the treatment of cerebral vasospasm after subarachnoid hemorrhage -final results of a prospective phase i cerebral vasospasm (cvsp) after subarachnoid hemorrhage (sah) is the major cause of disability and death. treatment options are limited. dantrolene blocks ryanodine receptor-mediated intracellular calcium release from the sarco-endoplasmic reticulum. it attenuates cerebral vasoconstriction, potentates the action of nimodipine on cerebral vessels and is neuroprotective in animal models. we performed a prospective phase i study examining the safety and effects of a single-dose of dantrolene on cvsp after sah. in an irb approved, prospective, open-label single-blinded phase i study, sah patients with elevated transcranial doppler (tcd) velocities and lindegaard indices suggesting cvsp were enrolled. after baseline tcds by a single, trained operator, patients receive a one-time infusion of dantrolene over minutes with dose escalation (first five patients . mg/kg, the following five patients . mg/kg). infusions, ventilator and ventriculostomy settings were kept unaltered, so that physiological data could be followed. hr, bp, icp, cpp, cvp and body temperature were recorded at infusion start (time ), every min during the infusion and with every tcd thereafter. serum abg, chem and osmolarity were measured at time and min, and lfts at time and hrs. tcds were repeated at , and min after time . statistical analysis was performed with repeated measures anova for the physiological values and change ( ) in systolic, mean and diastolic tcd in the vessel in cvsp from time , followed by post-test bonferroni's multiple comparison test with bonferroni p-value adjustment for significant findings. laboratory values were analyzed by wilcoxon matched pairs test. ten patients (n= each group with . mg/kg and . mg/kg dantrolene) were enrolled. over the entire study period, hr, map, dbp, icp, cpp and body temperature remained stable, except for sbp which decreased (p= . ). posttest bonferroni's multiple comparison test with p-value adjustment (p= . ) showed a trend towards a difference between time points and min (mean - . mmhg), and and min (mean - . mmhg), although this was not significant. significantly different laboratory changes were na (mean - . meq/l, p= . ), cl (mean - . meq/l, p= . ) and alkaline phosphatase (mean - . mg/dl, p= . ); the degree of change, however, was considered clinically insignificant. none of the other laboratory values changed. systolic and mean tcd velocities decreased significantly over time compared to time (systolic p= . ; diastolic p= . ; mean p= . ). post-anova linear trend testing indicates the magnitude of change: systolic (slope - . , p= . ); diastolic (slope - . , p= . ) and mean tcd (slope - . , p= . ). a one-time infusion of dantrolene appears safe, although the mild changes in na, cl and alkaline phosphatase warrant monitoring. most importantly, transaminases did not change. dantrolene decreases tcd velocities over time, presumably due to inhibition of cerebral vasoconstriction. we have insufficient data to comment on the duration of this effect. our results warrant further study with repeated or continuous dantrolene dosing for treatment or prevention of cvsp after sah or other vasoconstriction syndromes. status epilepticus carries a mortality rate up to %. newer, intravenously (iv) applicable antiepileptic agents might be powerful adjunctive therapies. we report our experiences with iv levetiracetam in a prospective patient cohort with status epilepticus. we treated patients with convulsive status epilepticus with an institutional protocol consisting of iv lorazepam followed by iv phenytoin, iv levetiracetam as third line agent or second line agent if there were contraindications for iv phenytoin, and iv propofol and/or midazolam as fourth line agents. primary outcome was treatment success of iv levetiracetam. secondary outcome measures were time to treatment success, modified rankin scale (mrs) at and months, and complications. of the patients had cerebral structural abnormalities, an infection, and hyponatremia as underlying etiology for status epilepticus. median age was (range - ) years, patients were male. baseline gcs was ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) . levetiracetam successfully treated status epilepticus in patients. median time to treatment success was ( - ) minutes in all patients. at months patients had died, support had been actively withdrawn in patients. the mrs was in patients, in patients, in patient, in patients, in patients and in patients. at months, patient with a mrs of had progressed to . the most common complications included hyperglycemia ( %), hypotension ( %), acute renal failure ( %), anemia ( %), thrombocytopenia ( %), urinary tract infection ( %), and seizure recurrence ( %). levetiracetam offers a feasible alternative strategy to break status epilepticus as adjunctive third line therapy in a paucity of patients before administration of sedatives with a broad spectrum of adverse effects and needs to be studied in a standardized trial. stroke is the third leading cause of death in industrialized nations.the treatment pathway for ischemia is often determined by assessing the extent of permanent damage aided by imaging modalities such as mri.diffusion weighted imaging (dwi) is an accepted mri technique that is sensitive to water diffusion.the restriction of water in damaged cells is used as a surrogate measure of cell death.although cells may have taken on water,this surrogate measure may be inaccurate,frequently leading to overestimation of the severity of ischemia.measures of sodium are reported to be more accurate indicators of cell death and can also be used to determine stroke onset time because of a linear relationship between ischemia and sodium concentration.we have developed a multinuclear coil and radiofrequency current source that permit simultaneous high-speed proton and sodium imaging so that treatable ischemia can be better ascertained. an -channel,broadband,radiofrequency,phased array and current source were built enabling targeted,accelerated,and simultaneous spectroscopic imaging of water,helium,and sodium on a . t ge mr scanner.it was tested on phantoms of known sodium and water concentrations and in rats with known inhaled helium concentrations. high quality imaging of protons and sodium in phantom models of known sodium concentration were obtained.additionally, high quality helium imaging in rats demonstrated the ability of the system to image other nuclei and proved the quantitative and qualitative imaging capabilities of the array in vivo. mars imaging provides a new, simultaneous multinuclear approach to determine the extent of ischemia quickly and quantitatively.high quality images with this system can be obtained in humans with the same hardware and can be used with standard . t mr scanners. septic shock is often associated with relative vasopressin (avp) deficiency that may be related to impaired avp synthesis and release by the neurohypophyseal system, which includes the neurohypophysis and magnollecular neurons of the paraventricular and supraoptic nuclei. neurohypophyseal system has never been assessed in human septic shock and only partially in experimental sepsis. we investigated avp synthesis and release by the neurohypophyseal system in septic rats and in human septic shock. design: ex vivo human and animal study. setting: university research laboratory in the human study, post-mortem examination of the neurohypophyseal system was performed in patients who died from septic shock (n= ) or other causes (n= ). in the experimental study, sepsis was induced by fecal peritonitis in conscious, fluid-resuscitated male adult wistar rats. rats either early died spontaneously from septic shock in average at hours (septic early death, n= ) or were sacrificed in average hours after induction of sepsis (septic, n= ). post-mortem examination was performed in both groups. comparisons were made against sham operation controls (n= ). avp protein and mrna were assessed by immunohistochemistry and in-situ hybridization. in both septic shock patients and septic rats with early death, the avp content in the neurohypophyseal and supraoptic magnocellular neurones was decreased while it was increased in the paraventricular magnocellular neurones. no significant change was observed in avp mrna expression in either paraventricular or supraoptic magnocellular cells. in septic shock, avp post-transcriptional synthesis and transport are altered in the supraoptic and paraventricular magnocellular neurones, respectively. this suggests that supraoptic and paraventricular nuclei are liable to distinct pathogenic mechanisms, which may account for relative avp deficiency. introduction: dual brain death (dbd) examination has been historically followed to determine irreversible brain damage. a policy was introduced in our hospital to utilize single brain death examination (sbd) including an apnoea test and a confirmatory test for cerebral blood flow in patients with catastrophic neurological injuries to determine brain death. we investigated if organ procurement would be affected by sbd. the database of gift of life (the designated organ recovery organization for michigan), was screened for our institution patients meeting brain death criteria between jan and july . for each patient, age, sex, primary cause of mortality, number of brain death examinations performed, type of confirmatory tests used to declare brain death, medical exclusions for organ donation and number of organs procured was obtained. continuous variables were analyzed using the student t-test and categorical variables using fischer's exact test with p values set at . . seventy patients met brain death criteria between january and july and were excluded due to incomplete records. there was no difference between the age and sex composition between the two groups. twenty seven patients were diagnosed with brain death using sbd while were diagnosed using dbd. twenty four patients with sbd and with dbd were eligible for organ donation (p= . ). each of eligible sbd and dbd patients donated organs (p= . ); organs were procured from each group (p= . ). single brain death examination did not preclude the rate of organ donation in our patient cohort. dual brain death examination can be substituted by a single brain death examination along with a confirmatory test for cerebral blood flow in patients with catastrophic neurological injuries without affecting the rate of organ donation. this may result in a less time delay before declaring death and minimize physician workload. etiologies for spontaneous intracerebral hemorrhage (ich) or intraventricular hemorrhage (ivh) vary. mri can often identify underlying vascular lesions, but conventional catheter angiography remains the gold standard. guidelines for the use of catheter angiography are non-specific. we aimed to determine the diagnostic yield of catheter angiography in addition to mri in patients with ich or ivh who met pre-defined criteria. consecutive patients with spontaneous ich or ivh were enrolled. in addition to non-contrast brain ct and laboratory testing, all patients underwent gadolinium enhanced mri/mra. catheter angiography was pursued if the following criteria were met: . lobar ich or isolated ivh and age years or . deep ich and no history of hypertension and age years or . any other indication based on the opinion of the treating neurointensivist. of prospectively enrolled patients, ( %) met criteria for catheter angiography. seven were excluded from angiography because a definitive ich cause was established by mri and because of a coagulopathy explaining the ich. forty-four ( %) patients underwent catheter angiography, which identified the ich etiology in ( %). in of these, the diagnosis was already suspected based on mri, but in cases catheter angiography increased the diagnostic confidence. in one patient a small avm was diagnosed by angiography alone. thirteen patients ( %) had both contrast angiography and pathology. of these, had a vascular abnormality as the cause of the ich. five of these were diagnosed by angiography. in one patient the pathology showed an avm while the angiogram and the mri were negative. two patients with cavernous malformations were diagnosed by mri alone. the diagnostic yield of catheter angiography in spontaneous ich or ivh is limited if patients also undergo gadolinium enhanced mri/mra. patients with anticoagulation-related intracerebral hemorrhage (ich) commonly are treated with fresh frozen plasma (ffp) for anticoagulation reversal. one risk of ffp is thought to be related to volume overload and pulmonary edema (ped). however, this has neither been validated, nor quantified compared with the natural risk following ich. we hypothesized that patients with anticoagulation-related ich are at higher risk of ped in-hospital, and that this increased risk would be related to dose of ffp used. retrospective review of a prospectively collected cohort of consecutive patients with primary ich presenting to a single center between august -may . of included patients, % were male, and mean age was +/- years. % were on warfarin at presentation, with a median inr of . (iqr . - . ). of these patients, % received ffp, at a median dose of (iqr - ) units. overall, patients ( . %) developed ped, at a median time of (iqr - ) days after presentation, and these patients showed a longer hospital length of stay [median (iqr - ) days vs. (iqr - ) days, p= . ]. anticoagulated patients were at higher risk of developing ped during hospitalization ( % vs. %, p= . ). patients receiving ffp were also at higher risk than those who did not ( % vs. %, p< . ). in multivariable analysis with a cox proportional hazards model, use of ffp was an independent predictor of developing ped (hr . per unit given, % ci . - . ), and the effect of warfarin fell out of the analysis when ffp was included. patients with anticoagulation-related ich are at increased risk of ped during hospitalization, accounted for by ffp use. each additional unit of ffp confers an approximately % increased risk of this complication. ventilator associated pneumonia (vap) is an infrequently studied morbidity in neurointensive care, despite high historical rates ranging from . to . infections per device days. in comparison, mean rates in medical icu's have been substantially lower: . - . . in our neurocritical care unit (nccu), vap incidence was initially near the national nosocomial infection surveillance (nnis) th percentile for neurosurgical icus ( . infections per ventilator days) necessitating an evidence-based performance improvement initiative to reduce vap rates. prospective surveillance study of vap incidence in a -bed nccu over a month period. vap rates were defined by national healthcare safety network (nhsn) criteria. interventions included an aggressive hand hygiene campaign, use of the ventilator bundle, oral care every hours, and introduction of the hi/lo endotracheal tube. ventilator bundle compliance was assessed. : patient days with a total of ventilator days were monitored. in september , concomitant to an acinetobacter outbreak, vap rates were infections per ventilator days. by january , hand hygiene compliance had increased from to % and rates were . following institution of frequent oral care in combination with the emphasized vap bundle, by may , rates had decreased to . at this point, all new intubations performed in the unit employed the hi/lo ett. by june , vap incidence decreased to . infections per ventilator days (< th nnis percentile). compliance with components of the ventilator bundle ranged from % to %. despite caring for patients at high risk of vap, concerted efforts with multiple evidence-based performance measures and interventions can significantly reduce the incidence of infection. feedback with compliance may be essential to maintain low vap rates. the use of hmg-coa reductase inhibitors (statins) has increased among subarachnoid hemorrhage (sah) patients for cerebral vasospasm prophylaxis. statins increase risk of myopathy, but additional factors may also be causative. myopathy rates in this group of critically ill patients are not well characterized. sah patients were prospectively entered into an institutional database; those with myopathy were retrospectively identified. serum creatine kinase (ck), aldolase (at time of suspected myopathy diagnosis), and catecholamines (at admission), muscle biopsy results, and medication administration records were evaluated. four ( . %) of aneurysmal sah patients treated between jan , through july , were newly diagnosed with myopathy. two were hunt/hess , and each: grade and . one was fisher , others were fisher . all had symptomatic hydrocephalus, were treated endovascularly with paralysis paraprocedurally, given simvastatin mg daily within hours of sah (none had prior statin use). all were insulin resistant, requiring high dose sliding scale insulin. two received steroids. all had elevated catecholamines. maximum ck levels (u/l) were , , , and , respectively. aldolase was checked and elevated in patients. the forth underwent muscle biopsy revealing necrotic fibers; this patient had the lowest maximum ck. the myopathy rate in this cohort is times higher than that reported in healthy patients treated with statins. since all sah patients did not receive statins, this rate is underestimated and much higher than reported in other trials evaluating statins in sah, assuming risk is solely attributable to statins. the contribution of other etiologic variables (critical illness, paralytic, steroid) is not clear, and potentially are additive. aldolase may be an additional means of identifying subclinical cases. these findings need to be confirmed and pathogenic factors better elucidated. neurocritical care is a relatively new discipline and its practitioners come from a variety of backgrounds. salaries are likely to differ based on primary appointment, geographic location, practice setting, and time spent on clinical effort. it is not known how many neurointensivists practice full-time critical care, and it is likely that many also have responsibilities as consultants, researchers and administrators. a survey will be emailed to all members of the neurocritical care society. information that will be collected regarding salary, icu directorship, primary appointment, practice setting, hospital type, geographic location, percent effort on clinical responsibilities, sources of income including salary incentives, patient population, board certification and subspecialty training, etc. the results will be reported at the meeting and compared to the previous survey done by the authors. the information gathered in this survey enhances the understanding of the current practice of neurocritical care throughout the united states. this data may be valuable to neurointensivists during contract negotiations, to hospital administrators trying to assess the feasibility of hiring a neurointensivist, and to neurologists-in-training as a way of generating interest in neurocritical care as a career choice. therapeutic hypothermia (th) is being implemented with an increase for multiple indications in the neuro-icu. the risk of developing renal dysfunction with th is thought to be low, but is not clearly defined in neurologic patients. retrospective chart review of prospectively identified patients. per institutional th protocol, patient's goal temperature was c. baseline serum creatinine (cr) level and creatinine clearance (crcl) were obtained and followed at least daily during th and rewarming. data was evaluated for changes in cr and crcl during and following th induction and any impact these changes had on treatment. thirty five patients received th (for post-cardiac arrest and intracranial pressure control related to subarachnoid haemorrhage, intracerebral haemorrhage or traumatic brain injury). maintenance of goal temperature varied from to hours. nine ( %) had an increase in cr and crcl within normal limits; of these, occurred following induction, occurred during th maintenance, and during rewarming. three ( %) patients had an elevation in cr above normal limits; all of these elevations arose after beginning rewarming, and none led to chronic renal failure. there was a direct relationship in the change in cr and crcl (p< . ). of the patients who had an abnormal change in cr, had an abnormal change in crcl. overall, % of the patients demonstrated some form of elevation in cr and decrease in crcl. no patient experienced clinically significant changes in renal function requiring changes in therapy. this cohort experienced changes in renal function that were not associated with clinical relevance. the majority of changes occurred during th or rewarming and were not chronic. any contribution of th induced muscle injury to cr changes would need to be assessed with future study. initial hematoma size, coagulopathy, and hypertension are recognized predictors of hematoma progression in intracerebral hemorrhage (ich). we aimed in our study to assess if the absolute number of wbc and/or increase in the wbc number within hours of progression can predict hematoma progression. data of consecutive patients with primary, supratentorial ich, admitted within h of onset were reviewed, identifying patients with progression (wp) and no progression (np). hematoma progression was defined as % increase of hematoma size, subsequent intraventricular bleeding or increase of the preexistent amount of intraventricular blood. we compared the two groups for demographic data, risk factors, admission neurological status, neurological deterioration occurence, and wbc, coagulation profile, and blood pressure (bp) at admission or within hours of hematoma progression, using univariate and multivariate analysis. we identified cases (np) and (wp). baseline variables were similar, except for the systolic bp that was higher in wp than in np group ( + mmhg versus + mmhg, p= . ). neither wbc at admission ( . + . x /mm versus . + . x /mm , p= . ) nor the variation of the wbc admission -within h of progression ( . + . x /mm versus . + . x /mm , p < . ) was significantly different between the np and wp groups. neurological deterioration and mortality were more frequent in the wp than np group ( % versus %, p< . ; % versus %, p= . respectively). logistic regression showed that the change in wbc from admission to within hours of progression and systolic bp were associated with hematoma progression (wald statistic . , p< . ; wald statistic . , p < . ). the variation of wbc within hours of progression and systolic bp seem to be independent predictors of hematoma progression. heparin-induced thrombocytopenia (hit) is a dreaded complication of heparin related products. we analyzed the risk factors and outcomes of subarachnoid hemorrhage (sah) patients in whom hit was suspected and either confirmed as present or absent by platelet factor (pf ) antibody test. all patients with presumed aneurysmal, non-traumatic sah and a pf test were identified through the massachusetts general hospital's research patient database. charts, laboratory values and images were analyzed retrospectively. we identified patients with sah who were tested for hit. of these patients, ( %) had a positive antibody test. there was no difference between mean platelet nadirs of hit+ and hit-patients, vs. th/mm , respectively. univariate analysis identified gender, magnesium prophylaxis, fisher group , clipping vs. coiling, presence of angiographic spasm, number of vasospasm treatments and day of hit testing as potential risk factors associated with hit. a multivariate analysis showed that female gender (or . , %ci . - . ), greater number of vasospasm treatments (or . , %ci . - . ), later day of hit testing (or . , % . - . ) increased the risk of hit and coiling reduced the risk compared to clipping (or . , %ci . - . ). those patients in whom hit was present had more infarcts on ct, longer icu and hospital stays and worse modified rankin scores on discharge. the presence of hit in sah has adverse consequences and is more likely in female patients, who have undergone aneurysm clipping and require more than one endovascular vasospasm treatment. coagulopathy-associated intracerebral hemorrhage (cich) leads to over % mortality and is associated with secondary thromboembolic (te) complications. rapid coagulopathy reversal improves cich outcome. activated factor viia (fviia) rapidly reverses coagulopathy and causes local hemostasis, but is associated increased te. we examine a large case series of cich patients treated with fviia to determine te rates in this population all cich patients are treated with standardized protocol with emergent intravenous vitamin k, fresh frozen plasma (ffp), and are eligible for fviia mcg/kg. we identified consecutive fviia-treated cich patients from database from - and identified patients. were excluded for no identifiable coagulopathy or fviia use for severe trauma. were analyzed. we collect data on diagnosis, coagulopathy etiology, history of ischemic heart disease (cad) and te. we examined the incidence of troponin elevation, ekg changes, symptomatic coronary ischemia, venous thrombosis, and stroke following fviia use. subjects had average age . years. over % had abnormal ekg on presentation. % fviia-treated cich patients had history of venous thrombosis (dvt) or pulmonary embolism (pe), % had cad, % had atrial fibrillation, and % stroke. troponin elevation above . ng/ml developed in % patients. only / patients developed clinically symptomatic cardiac ischemia. % developed dvt/pe, and / ( . %) developed ischemic stroke. there is a trend towards correlation of cad history with degree of troponin elevation (p= . ). coagulopathy-associated ich patients have high burden of prior ischemic heart disease and venous thromboembolism. though low level troponin elevations occur, incidence of fviia-related symptomatic cardiac ischemia, stroke, or venous thrombosis is low in fviia-treated cich patients. this low incidence justifies a prospective controlled study to evaluate risk versus benefit of fviia use for emergent coagulopathy reversal in cich. assessing neurological function is important in critical illness, but in sedated patients neurological examination is considered to be non interpretable. this prospective multicentre observational study assessed neurological responses in critically ill patients who required to be sedated with midazolam (± subfentanyl). their relationship with -day mortality and altered mental status (delirium or coma within three days after sedation discontinuation) was also assessed. daily neurological examination included the glasgow coma scale, the assessment to intensive care environment score (atice), eye position and movement, pupil size and response to light, corneal reflex, oculocephalic response, grimace to noxious stimuli and cough reflex. at awakening, mental status was assessed with using atice or confusion assessment method for the icu ( neurological examination is interpretable and may be useful for prediction of outcome of critically ill sedated patients. daniel evans , gail tudor , deborah cushing , jeffrey florman , david seder maine medical center, portland, me, united states, husson college, bangor, me, united states, we evaluated complication rates, outcomes, and the cost of care of patients with good-grade (hunt and hess grades i-iii) aneurismal subarachnoid hemorrhage (ggsah) admitted directly to an intermediate care unit (imc). retrospective chart review of all ggsah admitted to a tertiary referral center from to . we recorded demographics, vital signs, and pertinent aspects of the hospital course. a multivariate logistic regression model including hunt and hess grade was employed to evaluate for association between admission location and radiographic or clinical vasospasm or infarction. among ggsah admissions to imc or the intensive care unit (icu), mortality was . %. thirty-three grade i patients ( %), grade ii patients ( %), and grade iii patients ( %) were admitted directly to imc. none of these patients died, and ( %) suffered cerebral infarction. factors associated with imc admission were lower hh grade (p< . ), gcs of (p< . ), and no ventricular drain placement (p=. ). age, medical comorbidities, and clipping vs. coiling were not associated with admission location. eight patients ( %) admitted to imc were subsequently transferred to icu. patients admitted to icu were more likely to die ( % vs. %, p=. ), to suffer respiratory failure ( % vs %, p=. ), and fever ( % vs %, p=. ). in multivariate logistic regression, imc admission was unrelated to vasospasm or infarction. admission to icu was associated with higher median patient charges ($ , . vs. $ , . , p< . ). we found no evidence that imc admission (primarily among hunt and hess i and ii patients) was associated with increased morbidity, and the in-hospital mortality rate of imc admissions over years was zero. given the higher cost of care among patients admitted to icu, it may be appropriate to consider imc admission for selected patients. wei xiong, matthew koenig, xiaoxu kang, xiaofeng jia, adrian puttgen, nitish thakor, romeryko geocadin johns hopkins university, baltimore, md, united states neurologic injury from cardiac arrest (ca) continues to be a significant problem, in part due to the lack of real-time monitoring of brain injury and recovery. somatosensory evoked potentials (sep) are a reliable marker of poor outcome because they are relatively resistant to physiologic and therapeutic perturbations. we tested the hypothesis that early recovery of cortical sep would be associated with better outcome after resuscitation from ca. sixteen adult male wistar rats were subjected to asphyxial cardiac arrest. half underwent mins of asphyxia (group ca ) and half underwent mins (group ca ). continuous seps from median nerve stimulation were recorded from these rats for hours immediately following ca. additional serial seps were recorded at , , and hours after ca. clinical recovery was evaluated using the neurologic deficit scale ( - , normal = ), which was performed at , , and hours after ca (primary outcome measure). all rats in group ca survived to hours, while only rats in group ca survived to that time. mean nds values in the ca group at , , and hours after ca were . , . , and . ; while in group ca , they were . (pvalue . ), . (p-value . ), and . (p-value . ), respectively. the n (first negative peak at approximately ms) amplitude differed significantly between the two groups within hour after ca. rats that suffered longer ca durations showed later recovery of n . the n latency was similar between the two groups. although early recovery of n showed a trend towards better -hour nds scores, this was not significant. a smaller n peak was consistently observed to recover earlier in all rats, which may represent the thalamic component of sep. the delayed recovery of n is associated with longer ca times in rats. early recovery of n shows a trend towards better outcomes. n , which may represent thalamic activity, reappears much earlier than cortical responses (n ), suggesting thalamocortical desynchrony in early recovery. sep after ca is a dynamic and promising tool to monitor early neurologic recovery after ca. evidence suggests a role for inflammation in vasospasm after subarachnoid hemorrhage (sah). recent studies suggest that systemic inflammation may lead to vasospasm. to test the hypothesis that systemic inflammation worsens vasospasm we evaluated the effect of lps on vasospasm. c bl/ j mice received either ug/animal lps i.p., or saline. hours later, animals had either sah induction or sham surgery. in a separate group, neutrophils were depleted prior to lps administration. to test whether neutrophils in the csf from the sah are required for vasospasm, we injected blood from lps-sensitized, neutrophil-depleted mice to the csf of lps-sensitized, non-depleted mice and the converse (adoption studies). at hours post injection, animals were perfused with saline, formalin and india ink, and the brains were removed for quantitative evaluation of basal cerebral vasculature for vasospasm. the mean differences in diameter of mca segments at mm distal to bifurcation were compared. a separate set of animals were perfused with saline and formalin for immunohistochemical staining of neutrophils and microglia. in saline-injected animals with sah, the mean vessel diameter was significantly smaller compared to the salineinjected sham group. there was no difference in the means of vessel diameter between saline-or lps-injected sham groups. lps injection in the animals with sah exacerbated the vasospasm. neutrophil depletion prior to lps ameliorated vasospasm. neutrophil extravasation into the brain and microglial activation was increased in the lps group compared to controls but was reversed by neutrophil depletion. in the adoption studies, depletion of neutrophils in the csf blood ameliorates vasospasm but neutrophil depletion in the systemic circulation did not. systemic inflammation induced by lps exacerbates vasospasm. the effect is reversed by neutrophil depletion in the csf. this suggests that inflammation in the brain is a more important contributor than systemic inflammation in vasospasm. malnutrition in the intensive care setting is associated with increase mortality presumably secondary to increased infections. acute ischemic and hemorrhagic stroke patients in the icu often experience a delay of enteral nutrition due to delays in swallowing evaluation and diagnostic procedures that require a period of food abstinence. serum albumin levels are often used as markers for malnutrition. this study was retrospective analysis of patients admitted from january/december with the diagnosis of ischemic or hemorrhagic stroke. the goal of the study was to determine the association between albumin levels less than . mg/l during the first hours of icu admission and mortality. t-tests were used to identify significant difference between means. chi-square tests were used to examine the distribution of categorical variables across discharge statuses. after identifying variables that were significantly different, a logistic model was built to determine if admission day albumin levels are independently associated with mortality. there was no difference in mean serum albumin levels between non-survivors ( . mg/l) or survivors ( . mg/l (p= . )). there was no difference between non-survivors and survivors in day albumin levels or in the change from day to day . a logistic model controlling for age and dyslipidemia (factors significantly or marginally significantly elevated in non-survivors) showed that admission day albumin was not an independent predictor of outcome. in our study, there was no correlation between serum albumin levels and mortality. we did not analyze the incidence of infections in this study population. this study validates the may critical care medicine guidelines on nutrition support in the icu. they concluded that albumin was not a valid nutrition assessment tool in the icu. future studies should examine the relationship between hypoalbuminemia, prealbumin levels and the incidence of infections in the stroke patient population. tnf-is an inflammatory cytokine that plays a central role in promoting the cascade of events leading to an inflammatory response. recent studies have suggested that tnf-may play a key role in the formation and rupture of cerebral aneurysms, and that the underlying cerebral inflammatory response is a major determinate of outcome following subrarachnoid hemorrhage (sah). we studied comatose sah patients who underwent multimodality neuromonitoring with intracranial pressure (icp), cerebral microdialysis, and brain tissue oxygen (pbto ) as part of their clinical care. continuous physiological variables were time-locked every hours and recorded at the same point that brain interstitial fluid tnf-was measured in brain microdialysis samples. significant associations were determined using generalized estimation equations. each patient had a mean of brain tissue tnf-measurements obtained over an average of hours of monitoring. tnf-levels rose progressively over time. predictors of elevated brain interstitial tnf-included higher brain interstitial fluid glucose levels ( = . , p< . ), intraventricular hemorrhage ( = . , p< . ), and aneurysm size > mm ( = . , p< . ). there was no relationship between tnf-levels and the burden of cisternal sah; concurrent measurements of serum glucose, or lactate-pyruvate ratio. brain interstitial tnf-levels are elevated after sah, and are associated with large aneurysm size, the burden of intraventricular blood, and elevation brain interstitial glucose levels. experimental studies have demonstrated that tumor necrosis factor-(tnf-) plays a crucial role in the onset of hemolysis-induced vascular injury and cerebral vasoconstriction [ ] . we hypothesized that tnf-measured from brain interstitial fluid would correlate with the severity of vasospasm following aneurysmal subarachnoid hemorrhage (asah). from a consecutive series of asah patients who underwent cerebral microdialysis (md) and evaluation of vasospasm by computed tomographic angiogram (cta) or digital subtraction angiography (dsa), tnf-levels from md were measured at hour intervals from sah days - using enzyme-linked immunosorbent assay (elisa). a blinded attending neuroradiologist independently evaluated each cta and dsa and assigned a vasospasm index (vi). five patients had vi< and patients had a vi> , where the median vi was (range - ). median log tnf-area under the curve (auc) was . (pg/ml)*day (interquartile range . - . ) for the vi< group, and . (pg/ml)*day (interquartile range . - . ) for the >= group (p< . ). in this small series of poor-grade asah patients, the area under the curve of tnf-levels from sah days - correlates with severity of radiographic vasospasm. further analysis in a larger population is warranted based on our preliminary findings. mild therapeutic hypothermia (th, - ºc) reduces mortality and improves neurologic outcomes after ventricular fibrillation cardiac arrest (ca). the relationship between time to achieve th and outcomes remains undefined. we hypothesized that a shorter interval from ca to achieve th would be associated with improved neurologic outcome. we retrospectively reviewed all subjects with in-or out-of-hospital ca treated with th between november and april at our institution. the time to target temperature was defined as the interval between witnessed ca and first measurement of hypothermia ( ºc) and further categorized as early (< hours) or delayed (> hours). outcomes were assessed at the time of death or discharge by the cerebral performance category score (cpc). good neurologic outcome was defined as cpc or . fisher's exact test was used to assess the univariate relationship between time to target temperature and neurological outcome. patients were treated with th after in-hospital ( %) and out-of-hospital ( %) ca. subjects that did not reach target temperature or with unwitnessed ca were excluded. of the remaining patients, % ( / ) survived to discharge and % ( / ) achieved a good neurologic outcome. five patients ( / ) reached early target temperature; % ( / ) of those had a good neurological outcome. % ( / ) of subjects with delayed target temperature achieved a good neurological outcome. the univariate relationship between time to target temperature and neurological outcome was statistically significant (p= . ). attaining th within hours of in-or out-of-hospital ca is associated with a greater likelihood of a good neurological outcome at discharge. time from ca to achieved th should be included as a clinically important covariate in future studies of predictors of outcome after ca. cerebral autoregulation tests have gained importance for the assessment of patients with a variety of brain disorders. in critically ill patients, testing of dynamic autoregulation is safe and practical, but the ability to respond to steady state change in blood pressure is probably more clinically relevant. the purpose of this study was to compare static autoregulation testing with two different dynamic autoregulation tests (cuff deflation and carotid compression tests) in patients with severe traumatic brain injury. twenty-two studies were performed in tbi patients. changes in middle cerebral artery flow velocity (mcafv) were observed by transcranial doppler. static autoregulatory index (sari) was determined from the steady-state response of mcafv to phenylephrine-induced rise in blood pressure. dynamic autoregulatory index (dari) was determined by the cuff deflation method as described by aaslid ( ) and the transient hyperemic response ratio (thrr) was calculated as described by smielewski ( ) . these dynamic tests were performed in triplicate at baseline prior to inducing hypertension with phenylephrine, and the values were averaged to give a single index value. since the anatomy of the brain injury varied from patient to patient, the autoregulatory indices were summarized for the worst and best sides of the brain based on the appearance of the initial ct scan. the sari averaged . + . on the side of the brain that was more injured, and . + . on the less injured side. thrr was closely correlated with the sari, both on the side that was more injured (r=. , p=. ) and on the less injured side (r=. , p=. ). the dari was significantly correlated with sari only on the side that was more injured (r=. , p=. ). these data suggest the ability of dynamic autoregulation to predict static autoregulation may vary with the type of test chosen. published guidelines from the american society for gastrointestinal endoscopy considers peg tube placement a high-risk procedure for bleeding and recommends discontinuation of clopidogrel, to days before peg placement. unfortunately the perioperative time period is associated with increased ischemic events, length of stay (los) in the hospital and resource consumption. this is a retrospective review of prospectively collected data that sought to examine the safety of peg tube placement in patients while on clopidogrel alone or in combination with aspirin. patients admitted into our neuro-icu who met the set criteria during the period january to july were included in the study. mean duration on antiplatelet therapy prior to peg placement was days. one patient had a new stroke during hospitalization, unrelated to the procedure. no post-operative complications, bleeding or neurologic changes were noted in any of the patients. relevant blood indices remained largely unchanged. although peg tube placement is considered a high-risk procedure for bleeding, in the absence of pre-existing bleeding disorder it may be safe to perform this procedure in patients taking clopidogrel. there may be no need to consider reversion to aspirin alone, in those on combination therapy. timely placement of peg tubes in this subgroup of patients may reduce their los and decrease the risk of new ischemic events. introduction: hunt and hess grade sah is accompanied by high rates of mortality and severe disability. mortality in these patients is driven by withdrawal or limitation of care. some patients do enjoy good functional outcomes. we seek to describe the frequency and predictive factors for good outcome following grade sah. we will also describe risk factors for limitation of care following grade sah. we identified consecutive patients with sah and worst hunt and hess grade of within hours of admission (mean age years; % female) in a prospectively collected registry of aneurysmal sah patients. the frequency of good outcome (modified rankin through ) at months was calculated. we performed univariate analysis of pre-admission and admission characteristics to identify associations with good outcome. independent risk factors were identified through multiple logistic regression analyses. we performed a multivariate analysis of risk factors for limitation of care. good functional outcome occurred in ( %) of patients. white ethnicity (or . %ci . - . ), employment at the time of sah (or . %ci . - . ), lack of limitation of care (or . %ci . - . ), and normal papillary reactivity on admission (or . %ci . - . ) were independently associated with good outcome. in a subgroup of patients in whom care was not limited, ( %) had good outcomes; white ethnicity(or . %ci . - . ), employment status(or %ci . - . ), and absence of fever(or . %ci . - . ) independently predicted good outcome. among patient characteristics analyzed, admission gcs(p< . ) predicted limitation in care. a substantial proportion of patients with grade sah who receive full medical support enjoy a good recovery at months. age, pre-morbid co-morbidity, and clinical and radiographic measures of hemorrhage severity do not predict good outcome in our study, but socioeconomic factors may. intracerebral hemorrhage (ich) is the most lethal type of stroke. hypertensive ich (hich) is the most frequent ich subtype. we aimed to evaluate predictors of -day mortality after hich. retrospective cohort. this study was approved by our irb. we found patients with hich amongst patients admitted to our hospital from july to june . mortality was % (n = ). thirty-two patients ( %) were male, and ( %) were black. mean age was ± years ( patients were years). initial pulse pressure ± mm hg, and mean gcs score was ± . mean ich volume was ml (range, . to ml) measured on first head ct scan with the use of the abc/ method, and patients ( %) had intraventricular hemorrhage (ivh). fifty-five patients ( %) had supra-and patients had infra-tentorial ich. the mean ich score (hemphill, et al. stroke. ; : - ) was . points (range, to points). one of patients with ich score of , and patients with scores of or died. in univariable logistic regression modeling, all independent predictors used to develop the ich score, except age years (p = . ), were associated with day mortality: initial gcs (p = . ), ich volume (p = . ), ivh (p = . ), and infra-tentorial ich (p = . ), and the ich score (p = . ) accurately predicted mortality at days. in multivariable logistic regression analysis, only gcs alone was predictive of -day mortality. the roc/auc analysis demonstrated that gcs was a powerful predictor of mortality with an auc = . . gcs was the most powerful predictor of mortality. our study suggests that gcs is a powerful predictor of -day mortality after hich. further research is warranted. pneumocephalus is found in % of postcraniotomy computed tomographies of the head (ctoh) and is considered a benign complication of surgery. occasionally, however, it may lead to lethargy, headache and, if under tension, signs of elevated intracranial pressure or brain herniation. high percentage supplemental oxygen is frequently used as a treatment, but data regarding its effectiveness are very limited. postcraniotomy patients admitted to the neuro-intensive care unit with pneumocephalus received % fio on-off every hours for at least hours (treatment subgroup). during the off period, this subgroup, as well as the controls remained on room air (or % fio if mechanically ventilated). the assignment to each subgroup was based on the neurosurgeon's preference. the intracranial air volume on the ctoh was measured before and after the intervention via an image j analysis package. twenty-two treated patients and controls (mean age and years, and % women, respectively) were identified. the most common diagnoses were subdural hematoma (in % vs % for the treatment and control subgroups, respectively) and tumor ( % in both). there was no difference in the number of ventilated patients or in those with external ventricular drainages, in the lapsed period between the initial and final ctoh and the initial and final volume of air between the two subgroups. the percentage of air volume change (after adjustment for the lapsed time) and the rate of air absorption were significantly higher in the treated group ( % vs %, p = . and . . %/hour vs . . %/hour, p = . , respectively). this pilot study suggests that intermittent oxygen administration in patients with craniotomy decreases the pneumocephalus volume and increases the rate of intracranial air absorption. in a recent publication (wijdicks et al. neurology. oct ; ( ): - ), the safety of apnea testing in the declaration of brain death was evaluated at a single tertiary care center. one major conclusion was that apnea testing was safe in hemodynamically compromised patients in most circumstances and rarely aborted. determinants of apnea test completion failure are unknown. we calculated the alveolar-arterial oxygenation gradients (a-a gradient) in the previously studied cohort. arterial blood gas values were obtained prior to the initiation of apnea testing. patients that completed the procedure during the declaration of brain death were compared to those whose studies were aborted. statistical analysis was performed using nonparametric wilcoxon rank-sum test. of the original patients studied, a-a gradients were calculated for patients. seven of these patients had aborted apnea testing because of hypoxemia and/or hypotension. seventy-nine percent of patients that completed apnea testing had gradients larger than mm hg compared to % in those whose study was aborted, % versus % with gradients greater than mm hg, and % versus % with gradients greater than mm hg. the a-a gradient median values for completed and aborted apnea tests were mm hg (range: - - ) and mm hg (range: - ), respectively (p value= . ). the apnea test can be performed safely in most hemodynamically compromised individuals with large a-a gradients undergoing brain death evaluation. a larger percentage of patients that failed completion of apnea testing had significantly greater a-a gradients. predicting apnea test failure with this respiratory parameter warrants further validation in a larger population. cerebral glucose metabolism and energy production are affected by serum glucose levels. the objective of this study was to assess whether serum glucose variability and the ratio of cerebral-to-serum glucose are associated with cerebral metabolic distress and outcome after severe brain injury retrospective cohort study conducted in a neurological intensive care unit of a university hospital. we studied consecutive comatose patients with subarachnoid or intracerebral hemorrhage, traumatic brain injury, or cardiac arrest who underwent cerebral microdialysis and intracranial pressure monitoring.continuous insulin infusion was used to maintain target serum glucose levels of - mg/dl. general linear models of logistic function utilizing generalized estimating equations were used to relate these predictor variables to cerebral metabolic distress (defined as a lactate/pyruvate ratio [lpr] ) and mortality. the ratio of brain-to-serum glucose was calculated every to hours. daily serum glucose variability was expressed as the standard deviation (sd), mean amplitude glycemic excursion (mage), and glycemic lability index (gli) of all serum glucose measurements. a total of neuromonitoring hours and days were analyzed. after adjustment for glasgow coma scale scores, cerebral perfusion pressure, and serum glucose levels, brain/serum glucose ratios below the median ( . ) were independently associated with increased risk of metabolic distress (adjusted or= . [ . - . ], p< . ). increased serum glucose variability was also independently associated with higher risk of cerebral metabolic distress (adjusted or= . [ . - . ], p< . for sd and adjusted or= . [ . - . ], p= . for mage). low brain/serum glucose ratios and all three measures of increased serum glucose variability were also independently associated with in hospital mortality after adjusting for age and apache-ii scores (all p . ) reduced brain/serum glucose ratios and increased serum glucose variability are associated with cerebral metabolic distress and increased hospital mortality after severe brain injury. in critically-ill neurological patients, cerebral perfusion may be optimized by manipulating cerebral perfusion pressure and cardiac output. the objective of this study was to investigate the relationship between cardiac output (co) response to a fluid challenge and changes in brain tissue oxygen pressure (pbto ) in patients with severe brain injury prospective observational study conducted in a neurological intensive care unit of a university hospital. normal saline ( ml) or albumin % ( ml) boluses were given according to a standardized fluid management protocol. the relationship between co and pbto was analyzed using generalized estimating equations with an exchangeable correlation structure we studied fluid challenges administered to consecutive comatose patients that underwent multimodality monitoring with co, intracranial pressure (icp), and pbto . diagnoses included subarachnoid hemorrhage (n= ), intracerebral hemorrhage (n= ), cardiac arrest (n= ), traumatic brain injury and status epilepticus (n= each). of the fluid boluses analyzed, ( %) resulted in a % increase in co. median absolute (+ . vs+ . mmhg) and percent ( % vs %) changes in pbto were greater in co responders than in non-responders. in a multivariable model, a co response was independently associated with pbto response (adjusted or . , %ci . - . , p= . ) after adjusting for mean arterial pressure, icp and end-tidal co . stroke volume variation showed a good ability to predict co response with an area under the roc curve of . and a best cutoff value of %. bolus fluid resuscitation resulting in augmentation of co can improve cerebral oxygenation after severe brain injury little current data exists regarding outcome, cost and length of stay after subdural hemorrhage (sdh). we sought to examine predictors of discharge disposition, icu and hospital length of stay (los) and direct, indirect, icu, surgical and imaging charges for sdh. a retrospective review was conducted of acute, chronic and subacute sdh patients, aged > years admitted to our hospital between - . disposition was characterized as dead or poor (discharged to a nursing home, hospice, subacute or chronic care facility). multivariable logistic regression analysis was performed to identify predictors of each outcome variable. of sdh patients, the median age was . ( - ), and the median admission glasgow coma scale (gcs) was ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) . the sdh was characterized as acute in ( %), subacute in ( %), chronic in ( %) and acute, subacute and chronic in ( %). craniotomy was performed in ( %) of patients, burrhole drainage in ( %) of patients and both in ( %) of patients. death occurred in ( %) of patients and poor outcome in ( %). significant predictors of death or poor outcome included age, admission gcs and hospital los (all p< . ). surgery was protective against poor outcome (odds ratio [or] . , % confidence interval [ci] . - . , p= . ). median hospital los was ( - ) days and median icu los was ( - ) days. both were associated with gcs (all p< . ). median total direct charges for hospitalization were $ , ($ -$ , ). icu and hospital los were significant predictors of direct charges, overhead, imaging and surgical charges (all p< . ). herniation, sdh thickness, type of sdh, type of surgery and gender did not predict discharge disposition, cost or los. despite good admission neurological status, death or poor discharge outcome is common after sdh. though surgery mitigates against poor discharge disposition, los and charges remain high. sathees thayapararajah, bryan young, irene gulka, ahmed al-amri, sujit das lhsc, university of western ontario, london, on, canada introduction: acute fulminant hepatic failure (afhf) is common in tertiary care centers with transplant facilities. cerebral edema frequently threatens the lives of such patients. we reviewed cases of afhf in the neuroicu, noting the incidence of cerebral edema with ct scans and factors associated with mortality. patients were captured through hmri classification of acute liver/hepatic failure. chart review included tabulation of: demographics, inr; serum bilirubin, creatinine, albumin; in-hospital mortality. ct scans were re-read with blinding to clinical information and catalogued for changes in sulcal markings, ventricular size and gray-white differentiation (gwd). inclusion criteria: age greater than years, encephalopathy, hepatic failure within weeks of onset of liver disease, ct scans of head performed. acetaminophen toxicity was the most common etiology ( cases). twelve patients had cerebral edema on ct, including of the with acetaminophen toxicity. decreases in sulcal markings and ventricular size preceded conspicuous alterations in gwd. fourteen died, including all with cerebral edema. none of the hematological or biochemical variables correlated significantly with mortality. acetaminophen toxicity is a common cause of afhf; this combination has a strong association with cerebral edema. early development of cerebral edema occurs in almost all the afhf cases with acetaminophen overdose and can be detected in its early stages. this facilitates management for prevention of fatal brain herniation. afhf patients develop the changes on brain parenchyma within hours of onset of symptoms, during grade i-ii encephalopathy, most strikingly with aod. levetiracetam is increasingly being considered for seizure prophylaxis following tbi. although its acquisition cost is higher than phenytoin, the complete cost of therapy remains unknown as levetiracetam does not require therapeutic drug monitoring and has less pharmacokinetic variability. we developed a cost-minimization model to compare total costs associated with phenytoin and levetiracetam when used for seizure prophylaxis following tbi. five scenarios were tested based on drug, initial method of administration (iv vs. po) and whether or not po conversion occurred. factors considered in the analysis were drug costs, monitoring costs and likelihood of achieving a therapeutic concentration. treatment duration consisted of days. for arms that included po transition, po therapy began after day . decision trees were developed and a single-payoff method was used to identify the least costly scenario. hospital acquisition costs using us dollars were used to assess all costs. the scenario associated with the lowest cost was iv phenytoin followed by po levetiracetam ($ /patient). this was followed by iv levetiracetam transitioned to po levetiracetam ($ ), iv phenytoin transitioned to po phenytoin ($ ), iv phenytoin ($ ) and iv levetiracetam ($ ). the factor associated with the most variability in the model was timing of po transition. a two-way sensitivity analysis which altered timing of po transition revealed iv phenytoin followed by po levetiracetam as the least costly scenario except when iv to po transition occurred after day . in this scenario, iv levetiracetam followed by po levetiracetam was preferred. iv phenytoin followed by po levetiracetam will result in the lowest overall cost when used for a total -day course in patients with tbi. this illustrates the importance of considering all costs associated with a therapy when evaluating the total cost of medication therapy. the joint commission accreditation standards require hospitals to develop policies which address donation after cardiac death (dcd). optn/unos published the "model elements" for dcd protocols that describe suitable dcd candidates to primarily have "non-recoverable and irreversible neurological injury" resulting in ventilator dependency; a description we suspected to be highly inaccurate. we sought to more accurately clinically characterize those considered dcd eligible to facilitate constructive improvement of relevant policies and processes. local opo quarterly audits over months identified patients who were considered eligible for dcd and died within the requisite minutes of treatment withdrawal (dcdep). all cases were reviewed to determine the frequency/nature of any neurological abnormalities and whether they were "non-recoverable and irreversible." we also characterized the mechanism of respiratory failure and death. ( %) of dcdep had an identified neurological injury. only / ( %) had "non-recoverable and irreversible neurological injuries. / ( %) of dcdep were seen by a neuro-specialist, and / ( %) had brain imaging. / ( %) had a neurological injury that could compromise ventilatory drive, and at least / ( %) died of airway compromise with variable approaches to palliative care regarding sedation and oral airway usage. . % of dcdep had no neurologic injury. . only patients ( %) had neurologic injuries that could be correctly characterized as "non recoverable and irreversible" leading to "ventilatory dependency." . airway compromise is an important cause of death in dcdep and demands better uniformity of palliative care to assure equivalent treatment of dying patients independent of "donor status." . the published "model elements" for dcd protocols do not accurately represent the patient population. ventilator-associated pneumonia (vap) is the most common nosocomial infection among medical intensive care unit (icu) patients and associated with increased mortality and length of stay (los). neurologic disease is a risk factor for vap development, but the relationship between vap and outcomes in neurologic patients remains largely unknown. all mechanically-ventilated patients over a two-year period with neurovascular disease were included. data collected included patient demographics, dates of admission and discharge, los, and ventilator hours. vap was defined using standard published criteria. comparisons between neurologic patients who did and did not develop vap were made using univariate and multivariate analysis. of intubated neurovascular patients, ( . %) developed vap. compared with those who did not develop vap, those with vap were younger ( . ± . versus . ± . , p= . ), had increased los ( . ± . days versus . ± . , p< . ), and more ventilator hours ( ± versus . ± , p< . ). there was no difference in mortality between patients with and without vap ( . % versus . %, p= . ). vap was not an independent predictor of mortality in a multivariate model (or . , p= . ). subsequent case-control analysis of patients with and without vap demonstrated an increase in transports for cross-sectional head imaging ( . transports versus . , p= . ). vap in neurocritical care patients is associated with increased los and ventilator hours, but does not lead to increased mortality, contrary to prior studies in medical icu patients. the significance and frequency of vap in neurologic patients is different from patients in other icus because reasons for intubation vary. neurologic patients with vap have more imaging-related transports compared to controls, suggesting an association with ventilator disconnections. introduction: ich causes the highest mortality of all strokes. admission to a neuro-icu has been associated with reduced mortality following ich. this is leading to several hospitals routinely transferring ich patients to hospitals with neuro-icus. however, delays in optimizing management prior to and during transfer often leads to deleterious consequences. our objective was to compare functional outcomes in ich patients admitted to our neuro-icu directly from our ed with inter-hospital transfer admissions. records of consecutive spontaneous supratentorial ich patients admitted to our neuro-icu were reviewed. patients with ich related to trauma or underlying lesions (brain tumors, aneurysms, avm) were excluded. we compared outcomes at discharge in patients admitted directly from our ed and inter-hospital transfers (iht) using dichotomized modified rankin scale. other factors potentially impacting outcomes such as age, ich volume, ivh volume and admission gcs were included in the multiple logistic regression analysis. patients were included in the analysis (ed . %; iht . %). there were no significant differences between the groups in mean age (ed . +/- . ; iht . +/- . , p . ), ich volume (ed . +/- . ; iht . +/- . , p . ), ivh volume (ed . +/- . ; iht . +/- . , p . ) and gcs (ed . +/- . , iht . +/- . ; p . ). . % ed patients had good outcomes at discharge compared to . % iht. this difference was statistically significant following univariate (p= . , % ci= . - . ) and multivariate analysis (p= . , % ci= . - . ). odds (adjusted) of ed admissions having good outcomes was times higher than inter-hospital transfers. ich patients brought to the neuro-icu directly from our ed had significantly better outcomes than inter-hospital transfers. although this could possibly be caused by delays in optimizing ich management, other equally plausible hypotheses need to be prospectively tested. isis duran, storm liebling, michael moore, andrew naidech northwestern university, chicago, united states hospital acquired pneumonia (hap) is a significant cause of morbidity and mortality. hap increases costs, impacts quality metrics and will soon be designated as a medicare "never event". the us centers for disease control have published standard guidelines for the diagnosis of pneumonia, but few confirmatory data exist. we sought to determine the inter-rater reliability of diagnosing pneumonia by cdc criteria in patients admitted for brain hemorrhage. patients with intraparenchymal or subarachnoid hemorrhage admitted to our neuro/spine icu in were included in this irb-approved study. utilizing cdc criteria, pneumonia was diagnosed prospectively by a neurointensivist and institutional infection control (ic) personnel. following a thorough review of the electronic medical records, chest radiographs, and microbiology results, a neurocritical care fellow and a pulmonary critical care attending physician made an independent retrospective assessment of the diagnosis. analysis of the inter-rater reliability of the diagnosis of pneumonia was performed using kappa statistics. one hundred three patients were identified. the male:female ratio was : . pneumonia was diagnosed in patients by ic personnel, by the neurointensivist, by the fellow, and by the pulmonologist. overall inter-rater reliability was poor, with a median kappa value of . [ . - . ]. the highest inter-rater agreement was between the fellow and the pulmonologist (kappa= . ), while the lowest was between the pulmonologist and ic personnel (kappa= . ). the diagnosis of hap by cdc criteria, despite highly trained reviewers and clear diagnostic criteria, had poor interrater reliability in a sample of high risk patients. the diagnosis of hap should not be a measure of quality of care, nor should it be used as a determinant of payment unless the inter-rater reliability can be markedly improved. recent studies have reported excess hospitalization costs for aneurysm coiling compared to clipping after subarachnoid hemorrhage (sah). we aimed to compare categories of charges, length of stay (los), and discharge disposition in patients who underwent surgical versus endovascular aneurysm repair. a retrospective review was conducted of spontaneous sah patients between / - / . charges captured in the hospital database and were categorized as direct, overhead, icu, surgical and radiographic/angiographic. analysis was adjusted for age, hunt-hess grade, aneurysm size, aneurysm location and los. discharge disposition and los were compared between clipped and coiled patients using logistic regression or mann whitney u-nonparametric test. of sah patients, ( %) were clipped and ( %) were coiled. coiled patients were significantly older ( versus years; p= . ), and had larger aneurysms ( versus mm; p= . ). there were no differences in hunt-hess grade, aneurysm location, or modified fisher score. compared to coiled patients, median radiographic/angiographic charges were lower in the clipped group ($ versus $ , , adjusted or [aor] . , % ci . - . , p< . ), but median surgical charges were higher ($ , versus $ , aor , % ci - , , p< . ). total median direct charges were similar ($ , for clipped versus $ , for coiled patients, p= . ), as were icu direct charges ($ , versus $ , , p= . ) and overhead ($ , versus $ , , p= . ). median icu los ( days for each group) and hospital los ( days for each group) were similar as were discharge dispositions after adjusting for age, hunt-hess grade and aneurysm size: % of clipped patients died versus % of coiled patients (p= . ) and % versus % had a poor discharge disposition (p= . ). though surgical and radiographic/angiographic charges differed between sah patients who had surgical versus endovascular repair, icu charges, overhead and total direct charges were similar as were icu and hospital los and discharge disposition. osmotic diuretics and hypertonic saline (hs) are commonly used to treat traumatic brain injury (tbi). the untoward effects of mannitol, including hypotension, rebound intracranial hypertension, decreased potency and effect duration have lead to research of alternative treatments. hypertonic saline has been increasingly used to treat cerebral edema, however, efficacy and safety of repeated boluses has not been established. this preliminary prospective trial assesses the ability of single ml . % saline bolus to lower icp without losing potency while maintaining hemodynamic stability. thirty-five individual boluses of . % saline were given in tbi patients (aged - ) during a -month period. included tbi patients sustained icp elevation (> mmhg x minutes) despite full sedation, paralytics, temperature control and minimal stimulation. starting at bolus initiation, icp, cerebral perfusion pressure (cpp), heart rate (hr), systolic blood pressure (sbp), sodium level (na), and serum osmolality (sosm) were recorded regularly for hours. if repeated boluses were given in the same patient (icp re-elevated > mmhg x minutes), recording of parameters was restarted at a new zero time-point to assess the effect of each bolus individually. statistical analysis included power analysis, normalization testing, anova (analysis of variance) and scheffe test. within minutes of administration a statistically significant decrease in icp was sustained up to hours (power > %, p< . ). mean icp at initiation declined from mmhg to < mmhg by minutes (> % reduction, p< . ). the mean cpp before treatment increased from mmhg to mmhg by minutes ( % rise, p< . ). mean hr and sbp remained constant. sodium levels ranged from to and sosm from to . small volume . % saline boluses can be used repeatedly in patients with tbi to significantly lower icp and improve cerebral perfusion. repeated boluses resulted in a sustained magnitude and duration of icp reduction up to hours. introduction: intraventricular hemorrhage (ivh) can result from different etiologies all are intracranial in location. in this unique case we describe a case of ivh secondary to an extracranial vascular source. retrospective chart analysis for a patient that was taken care of at our institution's neurocritical care unit with cerebellar hemorrhage. patient was a year old male who presented with severe headache and imbalance. ct scan revealed a cerebellar hemorrhage and minimal intraventricular hemorrhage. patient underwent suboccipital craniectomy with evacuation of the hematoma. patient had an external ventricular drain (evd) placed in the or through the occipital horn. patient recovered over the following few days with minimal neurological deficits. suddenly patient suffered from a profuse bleeding from the scalp site of the evd. bleeding was controlled by pressure and suture. next day patient suffered from a similar episode. during the control of the bleeding the patient deteriorated neurologically and had to be intubated. patient ct scan showed massive intraventricular hemorrhage. angiography revealed an occipital artery pseudoaneurysm that was the cause of the bleeding and probably resulted from the evd insertion. the aneurysm was coiled without complication and patient was discharged later to long term care facility. pseudoaneurysms of the external carotid artery branches could result from trauma induced during evd insertion. in the presence of evd tract the hemorrhage that occurs from these pseudoaneurysms could track along under pressure to cause intracranial hemorrhage. this is an unusual and unfortunate experience that we wanted to raise awareness about. veena yashaswi, ravi patel, adham kamel, jonathan naysan, juliuse gene latorre, tara ramachandran, ziad el-zammar, yahia lodi upstate medical university, syracuse, ny, united states surgical treatment of fusiform intracranial aneurysm is extremely difficult and associated with poor outcome. endovascular stent-assisted treatment of fusiform intracranial aneurysm without sacrificing the parent artery has been introduced into clinical practice recently as an alternative option. objective: the objective of our study is report our experience of stent-assisted treatment of fusiform intracranial aneurysm. consecutive patients who underwent stent-assisted treatment for fusiform intracranial aneurysm were enrolled from to . patient's demographics including the hunt & hess grade, fished scale, location and size of aneurysm including the rate of radiographic evidence of aneurysm occlusion were collected. additionally a days outcome measurement was obtained using glasgow outcome scale (gos). five female patients, median age years (ranges to ) with five unruptured symptomatic intracranial fusiform aneurysms were treated with neuroform stent. four of which required staged coiling in addition to stenting and one required stenting only. three aneurysms were located at the internal carotid artery (two at the carotid bifurcation, one at the origin of ophthalmic artery) one at the middle cerebral artery and one at the vertebral artery. there was no intraoperative or post operative complication related to the stent-assisted treatment. immediate near complete occlusion was observed in one and subtotal occlusion in cases. in months follow-up angiography, complete occlusion of aneurysm was observed in patients (vertebral artery and carotid bifurcation ), near complete occlusion in two (carotid ophthalmic one and carotid bifurcation one) and subtotal in one (middle cerebral artery). good outcome was observed in all cases (gos ). endovascular stent-assisted repair not only provides a safe alternative option for the treatment of intracranial fusiform aneurysm, but also improve progressive occlusion of aneurysm with good outcome. wilson cueva, obi iwuchukwu, fernando goldenberg, agnieszka ardelt, jeffrey frank university of chicago medical center, chicago, il, united states hyperammonemia is a well recognized precipitant of cerebral edema (ce) and an important cause of death in acute liver failure. however, isolated hyperammonemia can occur in patients with enzymatic deficiencies important for ureagenesis. extreme hyperammonemia from newly diagnosed ureadysgenesis in adults has been reported, most often leading to disabled outcome or death from ce. we present the clinical, therapeutic and outcome details of two patients with newly symptomatic ureadysgenesis-induced hyperammonemia who developed profound ce and intracranial hypertension (ich). both are the only survivors ever reported with their degree of extreme hyperammonemia (peak and mcg/dl) with normal neurological outcome. case- : a healthy year-old male developed seizures followed by profound encephalopathy associated with ammonia level of mcg/dl without liver failure. case- : after a successful lung transplant, a year-old man developed severe encephalopathy associated with ammonia level of mcg/dl without liver failure. ornithine transcarbamylase deficiency was discovered in case and acquired glutamine synthetase deficiency was suspected in case . steroids provoked symptomatology in both cases leading to severe ce and ich. both required intracranial pressure monitoring, cerebral perfusion pressures directed therapy, promotion of ammonia clearance (cvvhd, lactulose), catabolism limiting treatments (hypothermia, insulin administration, infection control, nourishment), protein restriction, and the use of alternative pathway therapy. both patients fully recovered. -hyperammonemia should be suspected in patients presenting with unexplained ce even in the absence of liver failure. -multidimensional contemporary neurocritical care strategies can optimize survival and improve functional outcome from this historically disabling and deadly condition. -extreme hyperammonemia should not deter aggressive proactive management in these patients now that we report normal neurological outcome in these unique survivors. we is a known neurological complication of thiamine deficiency. although it usually manifests among alcoholics, na patients with either malabsorption, poor dietary intake, severe vomiting or increased metabolic demands are prone to develop we. we present three clinical cases in whom typical brain mri and pathology findings led to the diagnosis of na-we. case- : year-old na female became comatose after a two months history of severe vomiting secondary to a gastrointestinal disease. case- : year-old na female with breast cancer became comatose in the setting of weeks history of severe vomiting after chemotherapy. case- : year-old na female became comatose status-post cardiac arrest of very short duration, not enough to explain the severity of the encephalopathy. severe alteration of the level of consciousness without focal deficits was the prominent clinical finding in all patients; nystagmus was present in case . brain mri showed t /flair signal abnormalities in bilateral mamillary bodies, thalamus and periaqueductal area in cases and and an autopsy in case revealed findings consistent with we. despite intravenous thiamine supplementation, cases and did not improve clinically and eventually expired. case had complete neurological recovery within the first hours of treatment with intravenous thiamine. -we should be considered in all patients with unexplained confusion or deteriorating mental status even in the absence of a prior history of alcohol abuse. -in the presence of atypical or incomplete clinical picture of we, appropriate brain mri findings can help establishing the diagnosis. -early diagnosis of we is critical given that the success of the treatment depends on the urgent thiamine supplementation. -failure to recognize and treat we may result in devastating neurological outcome. arterial venous carbon dioxide removal (avco r) is a technique that uses a pumpless extracorpeal circuit for carbon dioxide removal. avco r has been used in adult and pediatric patients with severe hypercapnea. the system is placed at bedside using the seldinger technique to cannulate the femoral vessels. normally this system requires anticoagulation but can be performed without anticoagulation. case reports of avco r used to control ph and pco after neurotrauma. a male suffered extensive head injury after mvc progressing to brain death. organ harvest was planned. because of extensive lung injury complicated by hypercapnea and academia, donor viability was in jepody with paco rising to and ph of . . avco r was placed for ph and carbon dioxide control. the paco and ph promptly corrected to and . . after a brain death exanimation he remained on avco r until organ harvest. a male presented with a cervical injury at the c- level. imagining showed cervical fractures, disc herniation and cord contusion involving c- to t- .the patient developed respiratory distress. chest x-ray revealed ards. in aprv the ph was . and pco of . the patient had a subsequent cardiopulmonary arrest. after successful resuscitation avco r was started. within hours the ph and pco were . and without manipulation of the ventilator settings. he was on aco r for days and eventually weaned off mechanical ventilation after surgical decompression and fusion and discharged to rehabilitation. neither case required anticoagulation. . avco r is a simple extracorpeal technique that can be used to manage life threatening hypercapnea in patients with critical neurologic illness or injury . the technique can be inserted at bedside and used without anticoagulation continuous renal replacement therapy (crrt) is preferred over intermittent hemodialysis (ihd) in patients with acute brain injury (abi) due to increased intracranial pressure (icp) seen during ihd [ , ] . despite the preference for crrt in this patient population limited data is available on icp changes during therapy. there is some support for the early stability of icp for patients with fulminant hepatic failure that underwent continuous arteriovenous hemofiltration [ , ] . retrospective observational study (over a year period) of patients with abi and icp monitoring whom also underwent crrt. icp and fluid volumes were analyzed for the hours before and after initiation of crrt. four patients met criteria. table- describes the sample population. three patients had developed refractory intracranial hypertension (rih) prior to initiation of crrt ( in pharmacologic coma) and patient developed intracranial hypertension on ihd that resolved with crrt. no changes in medications were made in the hours prior to starting crrt except pentobarbital coma was initiated one patient six hours prior to crrt without lowering of icp. no attempts were made to lower icp in the hours following crrt. a decline in icp was seen at , , and hours following initiation of crrt in rih patients ( given the decrease in icp at one hour and relatively small percentage of total fluid balance removed, it seems unlikely that fluid removal or improved systemic oxygenation decreased icp. early improvement in icp may be due to removal of cytokines and myocardinal depressants seen with ultrafiltration and membrane absorption which is maximal during the first hour of filter use due to filter charge [ ] . given the mortality and morbidity associated with rih, further research is warranted. isoflurane, an inhalational anesthetic, is an alternative treatment for refractory status epilepticus (rse). it is effective, has rapid onset of action, and is easily titrated to produce burst-suppression patterns on the electroencephalogram. little is known regarding potential human toxicities caused by isoflurane. we present two cases of prolonged rse treated with prolonged high dose isoflurane who developed abnormal t hyperintensity lesions on magnetic resonance imaging (mri), which improved after taper or discontinuation of isoflurane. we report two patients with prolonged refractory status epilepticus who were treated with prolonged high-dose isoflurane (defined as an average end tidal concentration > . % for seven or more days) and developed new changes on brain mri. we collected demographic information, daily dosing of all antiepileptic medications and anesthetics received. we reviewed and analyzed the results of serial mri scans. patient one had prolonged rse for days and was treated with isoflurane for days with . % concentrationhours. patient two, currently hospitalized, has had rse for at least days and was treated with isoflurane for days with . % concentration-hours. in both patients, serial brain mris showed progressive t signal hyperintensity involving bilateral thalami, cerebellar hemispheres, and cerebellar vermis after treatment with - weeks of high dose isoflurane. these findings improved following taper and/or discontinuation of isoflurane. these cases raise the possibility that isoflurane is neurotoxic when used in high doses for prolonged time periods. though we cannot be certain of the exact cause of brain lesions, the timing of their appearance after isoflurane initiation and subsequent improvement after taper or discontinuation suggest a possible association with isoflurane. further studies are needed to clarify the safety of prolonged isoflurane use in rse cerebral edema is common in severe brain injury and can lead to harmful elevations in intracranial pressure (icp). hyponatremia, typically associated with excess levels of vasopressin (adh), frequently complicates acute brain injury and can worsen edema and icp. conivaptan, a vasopressin-receptor antagonist, has been shown to correct hyponatremia in these high-risk patients by inducing loss of free water (aquaresis). it is unknown whether raising sodium with a bolus of conivaptan can also acutely reduce icp. we prospectively assessed the change in serum sodium (na + ), icp, and cerebral perfusion pressure (cpp) after a bolus of conivaptan was given for the treatment of hyponatremia in a patient with cerebral edema associated with traumatic brain injury (tbi). a -year old suffered severe tbi with left carotid dissection, complicated by hemispheric infarcts and worsening edema. conivaptan mg iv was given as a bolus when na + rapidly dropped to meq/l. its aquaretic effect peaked between and hours after the dose, with hourly urine outputs of ml/hour. eight hours postadministration, na + had risen to meq/l. icp had been stable at - mm hg for several hours prior and remained in this range for the first hours after conivaptan, but then fell to mm hg at hours, remaining mm hg after hours. cpp, initially stable at - mm hg, rose to mm hg after hours. a single bolus of conivaptan not only resulted in rapid correction of hyponatremia but also a significant fall in icp temporally associated with peak aquaresis. confirmation of this novel osmotic effect is required, as is further delineation of the role of such agents in the management of brain edema. financial support: the authors have received speaking honoraria from astellas pharma. seizures are a known complication of aneurysmal subarachnoid hemorrhage (sah). they can increase cerebral metabolic demand and lead to cardiopulmonary compromise. this could be detrimental in the setting of delayed cerebral ischemia (dci), when brain tissue is vulnerable to further reductions in oxygen delivery or increases in demand. an association between seizures and worsening ischemia could influence the decision to use antiepileptic drug (aed) prophylaxis in patients with vasospasm. case report of a patient who developed irreversible neurological deficits and cerebral infarction immediately after a seizure in the setting of initially stable vasospasm with dci. a year-old woman developed confusion, aphasia, and right hemiparesis on day after sah. angiography confirmed severe anterior circulation vasospasm. the patient responded to hypertensive therapy with almost complete resolution of her ischemic neurological deficits. on day , however, she had a single generalized seizure and required intubation after brief oxygen desaturation. she had a concurrent drop in blood pressure, necessitating an increase in previously stable dose of vasopressors post-ictally she developed recurrent aphasia and worsening hemiparesis which did not resolve despite further hemodynamic augmentation. subsequent head cts revealed new infarcts in the left anterior and middle cerebral artery territories. she had received prophylactic phenytoin for only the first days of her icu stay per our sah protocol. aed prophylaxis is typically used early after sah when risk is high and a seizure may precipitate aneurysmal rebleeding. this case illustrates how a seizure occurring later, in the setting of vasospasm, can lead to decompensation of dci with potential for irreversible infarction. therefore, patients with vasospasm may benefit from extended duration of prophylaxis to prevent such complications. dural sinus thrombosis is a rare cause of stroke. anticoagulation is the preferred treatment; however, some patients experience rapidly progressive neurological deficits and poor outcomes despite adequate anticoagulation. mechanical thrombectomy via a trans-femoral approach is an effective alternative treatment, but technical limitations can make this approach impossible in some patients. we report two cases in which angiojet® (medrad-interventional/possis) thrombectomy was performed via a transjugular approach. in the first patient, trans-jugular access was preferred due to the presence of bilateral deep vein thromboses in the femoral and iliac veins and an inferior vena cava filter. in the second patient, the trans-femoral approach was impossible due to the length of the catheter ( cm xmi), which was insufficient to access to the anterior two thirds of the superior sagittal sinus via a trans-femoral approach. in both patients, the trans-jugular access was obtained utilizing direct ultrasound and fluoroscopic guidance. a stabilizing guidewire was placed to deliver the angiojet® catheter to the superior sagittal sinus. the first patient was a year-old woman with heparin-induced thrombocytopenia, a large intracranial hemorrhage and refractory increased intracranial pressure. the second patient was a year-old man who presented with dehydration and a rapidly declining neurological exam. in both patients, antegrade blood flow was restored within the dural sinuses after mechanical thrombectomy via the trans-jugular approach. computed tomography scans after thrombectomy did not show evidence of increased hemorrhage and there were no complications from the procedure. the first patient died despite successful thrombectomy from other complications of her underlying disease. the second patient made a full recovery. mechanical thrombectomy has been shown to be a successful treatment for dural sinus thrombosis for patients with progressive symptoms despite adequate anticoagulation. the trans-jugular approach is a valuable alternative variation of mechanical thrombectomy in patients who have contraindications to the trans-femoral approach. we retrospectively reviewed data from patients with stbi admitted to the neuroscience icu (nsicu) of an urban tertiary care level trauma center who had a cerebral oxygen monitor (licox ) in place and were administered inhaled nitric oxide (ino) per institutional protocol. data were collected from bedside flow sheets. two patients met inclusion criteria. patient # was admitted after a motor vehicle collision with stbi and pulmonary contusions. she developed adult respiratory distress syndrome (ards) on hospital day # requiring ino at ppm. prior to ino therapy, pbto was . mm hg, pao = mm hg and icp = cm h o; within hours of ino initiation, pbto climbed to mm hg (+ %), pao rose to (+ %), and icp remained cm h o (+ %). patient # sustained a stbi and pulmonary contusions after a motorcycle collision. he developed ards on hospital day # requiring ino at ppm. prior to ino therapy pbto was . mm hg, pao = mm hg and icp = cm h ; within hours of ino initiation, pbto rose to mm hg (+ %), pao rose to mm hg (+ %) and icp climbed to cm h o (+ %). patients admitted to the nsicu after stbi may develop complex physiologic derangements, including ards. the use of ino may benefit both cerebral and pulmonary dysfunction, and may warrant further investigation. amar dhand, kazuma nakagawa, wade smith, tarik tihan university of california, san francisco, san francisco, ca, united states introduction: ventricular free wall rupture is a fatal complication of myocardial infarction (mi). although described in mi patients who receive thrombolytic therapy, this complication is not well known in ischemic stroke patients who receive intravenous (iv) t-pa. here, we report a patient who had cardiac rupture and hemopericardium immediately following iv t-pa administration. case report. a -year-old woman with history of coronary artery disease presented with acute onset of left hemiparesis and right gaze preference (nihss ). ct angiography showed right middle cerebral artery (mca) occlusion at the bifurication, a filling defect in the left atrial appendage suggestive of left atrial thrombus, and right segmental pulmonary embolism. an electrocardiogram showed st elevations in the v -v distribution with initial troponin i level of . ug/l. iv t-pa was administered hour from symptom onset. one hour after completing t-pa infusion, the patient suddenly became unresponsive, bradycardiac, and rapidly demonstrated an asystolic arrest. given the established dnr/dni status, she was not resuscitated. autopsy study showed subacute myocardial infarction ( - days old), rupture of the anterolateral wall of the left ventricle, and cc of hemopericardium. pathological study of the brain showed an old hemorrhagic infarction in the left occipital lobe, evidence of remote hypoxic/ischemic injury and % occlusion of the basilar artery, but no evidence of intracranial hemorrhage. this case report illustrates a fatal cardiac complication of iv thrombolytic therapy that was used for acute ischemic stroke treatment in the setting of subacute myocardial infarction. the speculated mechanism of this phenomenon is alteration of collagen metabolism by thrombolytic therapy. although mi is not an absolute contraindication for iv t-pa administration, clinicians should be aware that cardiac rupture may occur when iv t-pa is given to patients with concomitant stroke and mi. anti-nmda-receptor encephalitis (nmdare) is a rare autoimmune encephalitis associated with antibodies that antagonize nmda receptors. although nmdare is an uncommon disorder, we present confirmed cases treated in our neuro-icu over months. we report three cases of nmdare including the first reported case during pregnancy. all patients were women between and years of age. all had a prodrome of psychiatric symptoms and had orofacial and limb dyskinesias at presentation. each developed progressive unresponsiveness, required mechanical ventilation secondary to hypoventilation and had autonomic instability. one required a transvenous pacer for symptomatic bradycardia. all were evaluated for ovarian teratoma. all underwent oopherectomy. two were found to have ovarian teratomas by pathology. patients were treated with a combination of ivig, steroids and plasma exchange. all patients improved and were discharged from the hospital to inpatient rehabilitation. one patient was weeks pregnant at admission. she delivered via cesearean section at weeks gestation. the neonate had mildly increased tone but appeared otherwise healthy and was discharged home at days. the csf of the neonate was negative for nmda antibodies. nmdare is a reportedly rare cause of encephalitis which may be more common than reported. it has characteristic features that should not go unrecognized. high suspicion for ovarian teratoma is appropriate. imaging may not accurately differentiate between benign cysts and teratoma. oopherectomy for any ovarian abnormality may be reasonable, given poor correlation of pathologically confirmed teratoma with radiographic imaging. this is the first reported case of nmdare during pregnancy. nmda receptors play an important role during fetal development and the long-term sequelae for children exposed to nmdar antibodies in utero are unknown. csf hypovolemia is typically diagnosed in patients presenting with positional headaches. however, severe intracranial hypotension and brain sagging may cause orthostatic coma. we present a case that illustrates this uncommon presentation. a yr old male who presented with acute onset of headache, nausea, vomiting and disequilibrium. extensive diagnostic work-up, including head ct, mri/mra and lp, was initially unremarkable. his headaches became progressively worse with a prominent postural component. a csf leak was suspected at the lumbar level diagnosed with ct myelogram and treated with a blood patch at the outside hospital with temporary resolution of his symptoms. a repeat mri/mra revealed bilateral subdural hematomas without mass effect and diffuse dural enhancement, consistent with decreased csf pressure. due to worsening level of consciousness, the patient underwent urgent evacuation of the left subdural without any change in his mental status subsequent imaging showed reaccumulation of the hematoma. over the next few days, the patient became increasingly stuporous and had an acute respiratory decline requiring intubation. the patient was subsequently transferred to our institution. head mri revealed dramatic sagging of the brain showing the pontomedullary junction at the opening of the foramen magnum. there was reproducible improvement in his cognitive status and cheyne stokes breathing with trendelenburg positioning. he underwent a repeat ct myelogram which showed a csf leak at t and possibly at c -c . the csf leaks were repaired with localized blood patches with significant improvement in his neurological exam. csf hypovolemia may cause coma from distortion and downward displacement of the thalamus-brainstem structures. evacuation of subdural fluid collections-typically without mass effect-may be detrimental in these patients contributing to further reduction of csf volume. instead, identification and treatment of the responsible csf leak is curative. infection with human immunodeficiency virus (hiv) has been associated with the development of intracranial aneurysms. although the pathogenesis of aneurysm formation in hiv infected patients is unclear, one purported mechanism is direct invasion of cerebral vessels by the virus itself. here, we report a unique case of an hiv infected patient whose intracranial aneurysm rapidly enlarged during a period of anti-retroviral therapy non-adherence. case report. a year-old hiv infected female (cd count cells/ml) was admitted with a subarachnoid hemorrhage. cerebral angiography revealed a dilating vasculopathy of multiple large intracranial vessels along with fusiform aneurysms of the right and left proximal anterior cerebral arteries (aca). a saccular aneurysm measuring x . mm arose from the right fusiform aca aneurysm and was successfully treated with endovascular coiling. four weeks later, the patient was re-admitted with a decreased level of consciousness. head ct revealed recurrent subarachnoid hemorrhage. the patient had not adhered to her anti-retroviral therapy and her cd count upon re-admission was cells/ml (hiv load copies/ml). cerebral angiography revealed enlargement of the previously coiled aneurysm which now measured x x . mm. infectious vasculitides were excluded with serum and cerebrospinal fluid (csf) testing, including negative blood and csf cultures, negative serum and csf antibodies for syphilis, and negative serum antibodies and csf polymerase chain reaction for varicella zoster virus. the co-occurrence of rapid aneurysmal enlargement with non-adherence to antiretroviral therapy suggests an elevated hiv burden may accelerate vasculopathy. rapid enlargement and re-rupture of intracranial aneurysms may be seen in hiv infected patients with an elevated viral burden. identifying the unique clinical and radiological features of hiv vasculopathy may lead to earlier recognition and novel therapeutic approaches. patients with de novo refractory status epilepticus are often referred to as having norse. the clinical course is often prolonged (range: - days), and morbidity and mortality is high ( %). cjd is a rare cause of refractory convulsive and non-convulsive status epilepticus. we describe here a patient with norse who had probable sporadic cjd. year old, kg, caucasian female with a past medical history of systemic hypertension, pulmonary hypertension, and prior pulmonary mai complex presented to the er with delirium and accelerated hypertension. initial examination revealed encephalopathy. patient had generalized tonic clonic seizures, with rapid progression to status epilepticus which was refractory to dilantin ( mg tid), keppra ( mg bid), phenobarbitone ( mg tid), midazolam ( mcg/kg/min), and propofol ( mcg/kg/min) infusion. she was then put in pentobarbital coma ( m/kg/hr) for hours, and was found to be refractory to withdrawal of pentobarbital. on day of her status epilepticus the patient had an episode of massive pulmonary hemorrhage and went into pea, from which she couldn't be revived. workup for norse including mri brain, ct chest, abdomen and pelvis, failed to reveal any evidence of stroke, press, neoplasm, meningo-encephalitis. paraneoplastic antibody panel was negative. toxicology, metabolic, haematological, vascular, and immunological workup was negative. csf analysis revealed a wbc of , protein and glucose and protein - - was positive. to our knowledge this is the first case report of norse complicating cjd. norse complicating cjd is associated with high mortality. our case is also unique for its acute onset, absence of myoclonus, and absence of extra pyramidal features commonly seen in cjd. in patients with refractory status epilepticus with no obvious cause, cjd should be considered in the differential diagnoses. predicting recovery after cardiac arrest continues to challenge neurointensivists. updated aan practice parameters add two new evidence-based elements to traditional clinical examination criteria ) absence of bilateral n response on sseps and ) neuron-specific enolase (nse) > µg/dl as measured within days. concurrently moderate hypothermia has emerged as an efficacious therapy, with the possibility of modifying the predictive power of criteria established independent of such intervention. case: a year old woman undergoing breast lumpectomy was resuscitated following interoperative asystole. rosc was secured by mins; but she arrived to nsicu comatose with only minimal pupillary and corneal reflexes. moderate hypothermia with target temperatures of - o c was achieved by hours and maintained for hours before slow re-warming. at hours, n s were bilaterally present, but nse was µg/dl. brain mri at day was normal. over weeks she remained comatose with absent motor response requiring aggressive therapy for bouts of refractory non-convulsive status epilepticus. she continued to have intermittent transient myoclonic movements months after cardiac arrest. eye opening without awareness of surroundings began at icu week with gradual return to consciousness. subsequently she has made slow steady improvement, conversing appropriately with memory of family names and past experiences. now months post-arrest on the inpatient rehabilitation unit she moves all extremities with - / muscle strength. her most recent fim score is . prognostication after cardiac arrest remains complex. application of hypothermia may alter the validity of predictors established previously. confounders and convergent evidence must be considered over any single data point. as in the past, time remains the final arbiter of certainty. near infrared spectroscopy is a non-invasive method of monitoring cerebral oxygenation. by employing time and spatial resolution of several light wavelengths, cortical blood flow, volume and oxygenation can be quantified (cerebral oximetry). we present a case utilizing cerebral oximetry in a patient with cerebral vasospasm after subarachnoid hemorrhage (sah) with concurrent use of brain tissue partial pressure of oxygen (pbto ) monitoring. a year old woman developed severe diffuse vasospasm following sah. we monitored intracranial pressure (icp) as well as tissue oximetry (pbto ) via licox (integra) catheter placed in the distribution of the left mca. over a -hour period during the third day of vasospasm, cerox (ornim) monitoring was applied over the left fronto-temporal area to evaluate the relationship between pbto and non-invasive cerebral oximetry. there were episodes of pbto desaturation (< mmhg for > mins) over the period of dual monitoring. over % of these pbto desaturations were preceded by > % decline in cerox values from baseline. there were episodes of cerebral oximetry desaturation (< % for > mins). less than % of cerox desaturations were temporally related to a decline in pbto to less than mmhg. hemoglobin was stable at mg/dl and icp was well controlled (< mmhg) during the entire hours. in this subject, desaturations of pbto appeared to be related to desaturations by non-invasive cerebral oximetry; the converse was not the case. perhaps cortical oxygen desaturations (cerox) occur with increased frequency compared to subcortical oxygen desaturations (pbto ) in diffuse vasospasm after sah. cerox monitoring may provide an enhanced understanding of oxygen delivery and utilization during periods of ongoing cerebral ischemia. further studies are required to substantiate these findings. status epilepticus refractory to conventional anti-epileptic drugs typically carries a poor prognosis, but patients may recover well if seizures can be stopped. case reports suggest that electroconvulsive therapy (ect) may stop seizures in patients with refractory status epilepticus, and we sought to examine its effectiveness in a series of patients. three consecutive patients with refractory status epilepticus at our institution were treated with ect after other therapies had failed. all patients were women, with age ranging from to years, and none had a significant medical history. extensive diagnostic testing was unrevealing, and all patients were empirically treated for infectious and autoimmune encephalitis. ect was begun because of ongoing seizures despite potent combinations of conventional anti-epileptic drugs, multiple trials of complete eeg suppression with anesthetic agents, and trials of more infrequently used therapies such as inhaled anesthetic agents and ketamine. ect stopped seizures in of patients. one patient recovered completely, and in outpatient follow-up had a normal neurological examination and a score of on the mini mental state examination. the second patient was left with mild cognitive impairment and epilepsy, but returned to independent living. in the third patient, seizures continued despite ect, and care was withdrawn at the family's request. autopsy revealed evidence of active meningoencephalitis despite treatment with antiviral therapy and high-dose steroids. ect stopped seizures in of patients with refractory status epilepticus. our results and those of prior case reports suggest that ect may be an effective therapy for refractory status epilepticus, and warrants further study for this indication. wendy wright, bill asbury, susan samuel, jane gilmore, owen samuels emory university hospital, atlanta, ga, united states conivaptan, an avp-receptor antagonist, has been used in neurocritical care patients to treat euvolemic hyponatremia. therefore, it would stand to reason that the aquaretic effect of conivaptan could also be used to induce a state of therapeutic hypernatremia. therapeutic hypernatremia is one of the standard modalities for the treatment of cerebral edema. conivaptan bolus +/-continuous infusion was administered to three patients with cerebral edema in the neurocritical care unit. all patients were initially treated with conventional measures to induce therapeutic hypernatremia, yet were not meeting the desired serum na goal. conivaptan was used in these patients to augment the effects of hypertonic saline. one patient received a single mg bolus of conivaptan in addition to . % nacl and his [na] increased an average of meq/l. one patient received a single mg bolus in addition to . % nacl + % nacl + nacl tablets + fludrocortisone and his [na] increased an average of meq/l. a third patient received conivaptan boluses + infusion in addition to % nacl and his [na] increased an average of meq/l. fluid balances were not adversely affected in any of these patients. conivaptan added to hypertonic saline therapy appears to be a rational strategy for achieving therapeutic hypernatremia in patients with cerebral edema without adversely affecting fluid balance. further study is needed to assess the effects of conivaptan on intracranial pressure, cerebral perfusion pressure and intravascular volume. financial support: dr. wright has served as a consultant for astellas pharma us delayed cerebral ischemia from vasospasm is an under-recognized, yet potentially treatable cause of morbidity and mortality in meningitis. while cerebral vasospasm has been documented via transcranial doppler sonography in patients with meningitis, few reports document vasospasm by cerebral angiography in this population. we report two patients who suffered neurological decline resulting from angiographically documented vasospasm during treatment for meningitis. the first patient was a -year-old woman who developed acute aphasia and hemiplegia during treatment for meningitis. formal cerebral angiography demonstrated left anterior circulation vasospasm. she was treated with verapamil into the left internal carotid artery and aggressive hypervolemia and hypertension. within hours, she was neurologically normal. the second patient was a postpartum woman with meningitis who presented with aphasia and hemiplegia. magnetic resonance imaging showed areas of diffusion restriction consistent with her examination. although she initially made clinical improvement with antibiotic therapy and was discharged, she re-presented days later with severe left anterior circulation vasospasm and massive left hemisphere stroke and later died. in our cases, as well as those described by the tcd literature, neurological decline and vasospasm occurred within days from the diagnosis of meningitis. this suggests that the "window" for vasospasm secondary to meningitis may be similar to that of vasospasm from sah. the development of focal neurologic symptoms in patients with meningitis should prompt radiographic evaluation for vasospasm. current treatment algorithms do not include the routine use of cerebrovascular imaging during treatment for meningitis, and thus this potentially treatable complication may be under diagnosed. prospective studies evaluating cerebrovascular complications in acute meningitis using neuroimaging coupled with directed hypervolemichypertensive therapies should be undertaken and may lead to a reduction in the persistently high morbidity and mortality associated with this common disease. opsoclonus-myoclonus syndrome (oms) is typically associated with a paraneoplastic syndrome or viral encephalitis. various locations, including the crerebellum, have been proposed as anatomic correlations to this syndrome. oms as a result of posterior reversible encephalopathy syndrome (pres) has not been previously described. a yo man with past medical history of poorly control hypertension, hyperlipidemia, and peripheral neuropathy presented with confusion and visual difficulties. initial exam demonstrated a fever of , bp= / (max / ), and lethargy. laboratory studies revealed acute renal insufficiency (ari) (cr= . ) and rhabdomyolysis (cpk= ). mri showed faint hyperintensities in the cerebellum and paieto-occipital subcortical areas (image ). the patient subsequently developed agitation with diffuse multifocal myoclonus and pronounced opsoclonus. repeat mri (image ) showed extensive hyperintensities in the subcortical hemispheres bilaterally and in the cerebellum, consistent with pres (image ). eeg showed diffuse slowing, and lp showed elevated protein ( mg/dl). csf cultures, vdrl, lyme antibody, listeria antibodies, west nile virus pcr, hsv pcr, and jc virus pcr were all negative. ct scan with contrast of the chest, abdomen and pelvis, revealed no neoplasms. results of serum paraneoplastic antibodies are pending. elevations in spep and upep were determined to be due to monoclonal gammopathy of unknown significance. we present a unique case of pres presenting as oms. involvement of the cerebellum may have been causative in this case. the most likely explanation for the development of pres was hypertension with ari. the acute onset, negative viral studies and body ct scan, resolution of symptoms with control of hypertension and reversal of ari, and the characteristic mri findings all supported pres as the cause of oms. we describe the clinical, imaging, and follow-up details of two patients who developed bsevad in the peripartum period to enhance the early recognition of this uncommon but important and potentially disabling complication. both patients were initially misdiagnosed with post-dural puncture headaches (pdph). both patients were in their thirties (ages and ), had epidural anesthesia, and developed their symptoms within days of delivery. patient # developed postural headache within hours of delivery, and patient # developed severe neck pain and bioccipital headache days after delivery. both received epidural blood patch for presumptive diagnosis of pdph without any significant relief. patient # developed nausea, vomiting, and ataxia days postpartum with a follow-up mri revealing acute bilateral cerebellar infarcts and a unilateral pontine infarct. patient # performed unusual physical positions during her pushing phase of labor (drawing provided). initial neuroimaging with ct, mri brain in both were reported as normal. however, -vessel cerebral angiography in both patients revealed bsevad. both patients did well with medical therapy without sequelae. the risk factors, diagnostic clues, and therapeutic considerations are discussed. . bsevad is a rare peripartum complication. . bsevad presents with clinical features that resemble pdph but have some distinguishing features to facilitate differentiation. . bsevad can lead to stroke similar to non peripartum cerebral arterial dissections. . early recognition of this rare complication can potentially lead to protecting patients from devastating posterior circulation strokes. the late-onset form of pompe's disease presents generally with limb girdle weakness. respiratory failure develops later and is the most frequently reported cause of death. we describe a case of late onset pompe's disease emphasizing the need to incorporate this rare entity into the differential diagnosis of patients with ventilatory failure a year old man presented with progressive weakness and shortness of breath. - years previously he noticed gradual lower extremity weakness. he began having difficulty breathing while lying flat. he was admitted to the neuroicu due to progressive respiratory failure. neuromuscular junction disease, neuropathy and typical myopathies were excluded. emg: normal nerve conduction studies. needle exam showed complex repetitive discharges, myotonic discharges and fibrillation potentials consistent with a myopathic process. muscle biopsy: myopathic changes associated with features of a vacuolar myopathy with abnormal glycogen accumulation and markedly increased acid phosphatase reactivity consistent with acid maltase deficiency. dried blood spot serum assay for acid -glucosidase was undetectable. pompe's disease is a rare condition that is now recognized as a treatable entity. therefore, it should be included in the differential diagnosis of adult patients with gradually progressive myopathy and respiratory muscle weakness. its treatment, enzyme replacement with recombinant human alpha-glucosidase (rhgaa), although not yet fda-approved for patients over years of age, has shown significant clinical benefit when started early in the course of the disease. owing to the success of therapeutic hypothermia (th) post cardiac arrest, additional indications are now being explored. this case report documents successful application of th for treatment of refractory intracranial hypertension due to poor grade subarachnoid hemorrhage (sah) without the need for decompressive hemicraniectomy. a -year-old male (da) became unconscious after complaining of a bad headache. in the ed, da was listed as unresponsive to all stimuli. ct revealed a hunt/hess grade sah. an aneurismal clipping was performed the following day. on day , he experienced severe vasospasm not amendable to angioplasty and refractory to osmotherapy, cerebrospinal fluid drainage, and mild hyperventilation. at one point the intracranial pressures exceeded mmhg and his left pupil became fixed and dilated. a ct showed extensive edema and a worsening midline shift. it was then decided to initiate th in anticipation for worsening vasospasm. once the target temperature of degrees c was achieved, the icp stabilized. attempts to re-warm on days and led to increases in icp therefore aborted. finally, on day (th day ), da was re-warmed successfully. ct results that originally showed a large area of edema and midline shift was resolved. da was extubated day , a vp shunt was placed on day and discharged to home after a rehabilitation stay with a good neurological recovery on day . considering the mortality of a high grade sah can exceed %, we believe th contributed significantly to a good neurological outcome. a recent study described the need for a decompressive hemicraniectomy prior to attempting mild hypothermia. our case report documents successful application without invasive surgery and may be an option for others. amaurosis is an uncommon complication of pregnancy encountered by neurologists. two common causes of blindness in during the peripartum period are: ( ) reversible posterior leukoencephalopathy syndrome (rpls), and ( ) preeclampsia. case report: a -year old woman delivered twins at -weeks because of severe preeclampsia. in the early postpartum period, she developed altered mental status, and by postpartum day # , she was responsive only to first name, unable to follow commands, and increasingly combative. blood pressures were elevated up to mmhg systolic, and mmhg diastolic. she had limited vision by absence of blink response to confrontation bilaterally. head ct and eeg were unremarkable. based on findings on cerebral magnetic resonance imaging (mri), a diagnosis of hypertensive encephalopathy was made. however, the occipital lobes are clearly spared. her condition substantially improved, and by postpartum day she was able to cooperate with visual acuity and funduscopic examinations. visual acuity was / bilaterally. bilateral fundi showed discrete patches of retinal whitening located between the arterioles and venules, few retinal hemorrhages, and normal optic discs .by discharge, her blood pressure and mental status were at baseline, her vision improved to / , and repeat mri showed resolution of the earlier findings. cause of blindness in this patient was related to purtscher's retinopathy. we believe that this is the first documented case of a patient with a hypertensive encephalopathy and purtscher's retinopathy. this observation indicates that transient visual loss in the setting of elevated systemic blood pressures does not have to be cortical in nature. continuous eeg monitoring of neurocritical care patients is becoming more common. over % of critically ill patients with altered sensorium are diagnosed with non-convulsive seizures or status epilepticus. as continuous eeg is increasingly used with critically ill patients, it is important for practitioners to recognize artifacts that may mimic clinically relevant pathologic discharges. we describe a newly discovered artifact from an invasive hemodynamic monitoring device. seven patients who had a ref/ox (edwards lifesciences) continuous cardiac output pulmonary artery catheter (cco) placed for hemodynamic monitoring and were simultaneously monitored with continuous eeg were retrospectively identified. all patients were cared for in a bed neuroscience icu in an urban level trauma center. all eegs were interpreted by a board certified epileptologist. all patients' eegs demonstrated a distinctive artifact believed to be associated with the cco catheter. this artifact has not been previously described with other pulmonary artery catheters. the eeg was characterized by an intermittent high amplitude, narrow complex, spike-like artifact followed by a high amplitude slow wave. it is hypothesized that this signal results from current flow to the thermal element of the catheter. neurocritical care patients frequently undergo multi-modality monitoring. this newly identified eeg artifact with cco monitoring has an appearance that may be confused with epileptic spike/wave discharges or burst suppression. the impact of this potential artifact generated by the use of cco devices requires further characterization. as neurocritical care patients increase in complexity and are subjected to more invasive monitoring, the identification of new eeg artifacts may become more common. the diagnosis of gullian-barre syndrome (gbs) is based on a combination of clinical and laboratory features. gbs typically presents as a monophasic, subacute, symmetrically, predominantly motor neuropathy. in rare cases, gbs can present with acute quadriparesis and cranial nerve involvement. we report two cases of patients who presented in a state mimicking brain death with complete dysfunction of efferent nerves which turned out to be fulminant gbs. two cases with rapidly progressive weakness presented to our institution with very rapid deterioration requiring mechanical ventilation. over a very short course of time, both patients became paralyzed with complete absence of brainstem reflexes. brainstem function tests were performed as part of full neurological examination which revealed that both patients had non-reactive mydriasis with complete internal and external ophthalmoplegia. rest of the neurological exam including deep tendon reflexes showed no reponse. due to lack of identifiable cause of patient s condition, further diagnostic test were carried out. both patient s underwent csf analysis which revealed evidence of albuminocytological dissociation. a diagnosis of severe guill i a n-barre syndrome with involvement of peripheral and cranial nerves was suspected. electrophysiological studies were performed that showed this was suggestive of severe, axonal, sensorimotor peripheral polyneuropathy with profuse ongoing denervation in bulbar, cervical and lumbosacral innervated muscles. extensive laboratory evaluation including gq b antibody were carried out. after prolonged course, both patients made some functional recovery. both these cases proved that in rare cases, gbs can present with signs of coma and absent brainstem reflexes. brain-death protocols require that before the declaration of brain-death, an etiology needs to identified that could explain the clinical picture and all reversible causes are excluded. these cases illustrate the importance of electrophysiologic, laboratory and imaging studies in patients with suspected brain death where a cause is not clearly determined. hyperperfusion syndrome is a serious complication after carotid revascularization procedure associated with poor outcome, developing between day to days after procedure. in addition to increased cerebral blood flow, clinical manifestation include headache, seizure and intracerebral hemorrhage. equally rare is stent thrombosis especially in patients who were adequately treated with antiplatelets prior to procedure. distinction between the two condition requires prompt diagnosis to achieve good outcome. case report and medical record review m presented with acute l hemiparesis and was treated with iv tpa. nihss was on admission and after hours. ct brain did not show any acute infarction. cerebral angiogram showed % r ica stenosis.the patient underwent r carotid stenting on day . postprocedure map was maintained between - mmhg using oral and iv antihypertensive agents. hours post procedure the patient developed l hemiparesis, dysarthria, right gaze preference and l hemianopia. blood pressure was augmented to map - mmhg while en route to ct/mri and angiography for suspected acute stent occlusion. patient's hemiparesis worsened. ct brain showed unilateral subtle r hemispheric edema but no hemorrhage. mri showed patchy dwi along r hemisphere with subtle cortical and meningeal enhancement. emergent angiography showed patent stent. blood pressure was immediately controlled to map - mmhg and patient improved. within hours, patient was ambulatory. repeat ct brain did not show any acute infarction. months after discharge, he was asymptomatic. hyperperfusion syndrome can develop even with relatively controlled blood pressure post-carotid revascularization. emergent vascular imaging is necessary to differentiate acute stent occlusion from hyperperfusion syndrome so that appropriate measures may be done. when aggressively managed, symptoms associated with hyperperfusion syndrome are fully reversible if not associated with hemorrhage. fulminant hepatic failure (fhf) or diffuse anoxic injury can lead to the development of cerebral edema and increased intracranial pressure. hypothermia has been utilized in both clinical scenarios in attempt to prevent the development cerebral edema and manage elevated intracranial pressure. in this case, we sought to determine a correlation between brain and core temperatures in specifically in intravascular therapeutic hypothermia (iht) this observation was conducted in a year-old woman with grade iii hepatic encephalopathy (he) due to fhf. iht using coolgard® icy catheter (zoll medical) was started immediately as the patient progressed to grade iii he. esophageal and foley temperature probes were utilized for recording core body temperature. monitoring of brain temperature and intracranial pressure was conducted via licox® system (integra). goal temperature range for iht was between - ° c. brain and core temperatures were recorded hourly during iht which was a period of hours. data was collected and plotted to show correlation between the three temperatures over time. measurements were obtained over the course of hours to log temperatures. the results showed: brain temperature: y=- . x + . . r = . . bladder temperature: y = - . x + . . r = . . esophageal temperature: y = - . x + . . the results show a direct linear correlation between brain, esophageal, and bladder temperatures accurate correlation between brain and core temperatures was demonstrated during ihs. further investigation using larger number of subjects is needed to confirm this. the cerebral circulation is normally pulsatile except for short periods of time in patients subjected to extracorporeal circulation, commonly used during cardiac surgery. a new generation of left ventricular assist devices (lvad) generates continuous, non-pulsatile blood flow. in patients with implantable continuous flow lvad (cflvad), peripheral arterial pulsatility will exist as long as the native heart is capable of maintaining enough contractility to generate some stroke volume during systole. we explored the intracranial circulation with transcranial doppler (tcd) in patients that had a cflvad implanted and were neurologically intact. doppler insonation was performed through the routine temporal and occipital bone windows and proximal intracranial vessels were surveyed. transthoracic echocardiogram was performed in all patients to assess the lv function. / patients ( %) that had some preservation of the native heart function exhibited an intracranial flow pattern consisting of: high end diastolic flow velocity, very low pulsatility index and sometimes a sinusoidal wave appearance coincident with the native heart's systolic contraction. / patients ( %) that had extremely poor heart contractility exhibited a distinctive pattern of continuous flow where it was impossible to distinguish between systolic and diastolic flow. with the advent of new mechanical cardiac support for patients with end-stage heart failure, new peripheral and cerebral blood flow patterns develop and clinicians need to be aware of these distinctive and novel findings. this scenario opens an enormous opportunity to understand and better characterize a new physiological situation. it could also limit the usefulness of bedside tcd as a complementary method for the diagnosis of cerebral circulatory arrest given the lack of pulsation, known generator of the isolated systolic spikes or the "to and fro" pattern considered pathognomonic findings of the absence of intracranial circulation. edgar samaniego , gregory kapinos the advanced cardiovascular life support (acls) and advanced trauma life support (atls) provider courses are excellent resuscitation tools directed towards respiratory and hemodynamic stabilization, nevertheless, survival rates with good neurological outcome are dismal. the neurological content of acls and atls training manuals was reviewed. an advanced neurological life support (anls) course is proposed based on the deficiencies of acls and atls (table) . the neurological content of acls is % and covers only ischemic stroke, with no mention of hemorrhagic stroke or other neurological emergencies. the neurological content of atls is %, with a brief description of intracranial hemorrhages, increased intracranial pressure and spinal cord injuries management. both courses overlooked frequent devastating neurological emergencies like status epilepticus, anoxic encephalopathy, acute paralysis and meningitis. many basic concepts of neurological critical care management are missing in advanced resuscitation courses. we advocate the creation of an anls provider course to improve neurological outcomes of patients who undergo resuscitation. animal studies have shown that even a temperature elevation of one degree celsius can worsen neuronal injury after brain ischemia. since the skull acts as a thermal insulator, we hypothesized that decompressive hemicraniectomy lowers brain temperature by facilitating the heat convection from the brain to its surrounding air. fifty patients with severe brain injury (tbi= , ich= ) requiring continuous brain temperature monitoring (licox, integra lifesciences, plainsboro, nj) from january to march were retrospectively studied and grouped into "hemicraniectomy" (n= ) or "no hemicraniectomy" group (n= ). the core body (t core ) and brain (t br ) temperature measurements were recorded at -min intervals over ± icu days. as a surrogate marker for the degree of external heat loss from the brain, t br-core was calculated as the difference between t br and t core with each recording. t-tests were used to initially assess the difference between groups. then, in order to account for clustering of observations in individual patients, generalized estimating equations (gee) were used to assess association of hemicraniectomy with t br-core , adjusting for core body temperature and diagnosis. ). however, after adjusting for intraindividual variability using gee, only higher core body temperature, but not hemicraniectomy, was associated with difference in t br-core (p= . for t core ; p= . for hemicraniectomy). this suggests that the t br-core temperature difference is larger at higher body temperature. substantial variability exists in the brain-to-body temperature gradient across patients and core body temperatures. however, this difference is not due to the presence of a hemicraniectomy. the assumption is often made that young people would want decompressive hemicraniectomy after a large stroke as a life-saving measure. however, this assumption favoring aggressive life-saving treatment, and the perception of quality of life after neurological disability, have not been adequately studied. we conducted a cross-sectional questionnaire-based survey that consisted of demographic information (age, sex, race, marital and family status, religion, income, education level, access to healthcare), and attitude towards neurological disability (based on the highest acceptable modified rankin scale (mrs) that they would be "willing to live with"). young adults in the los angeles county were surveyed and grouped by whether or not they would want hemicraniectomy after a large stroke despite a high likelihood of disability. findings from the two groups were compared using student's t-test and chi-square test. logistic regression analysis was used to determine the factors predicting willingness to accept decompressive hemicraniectomy. in this pilot study, young adults (mean age: ± ) were surveyed. the highest acceptable mrs ( - ) participants felt "willing to live with" were: . % ( ), . % ( ), . % ( ), . % ( ), . % ( ), . % ( ) . despite a high likelihood of severe disability, of ( %) reported they would undergo hemicraniectomy after a severe stroke. neither the demographic factors nor the highest acceptable mrs were associated with the willingness to seek aggressive treatment and hemicraniectomy. the results from our preliminary study support the commonly held assumption that young adults are generally willing to accept decompressive hemicranietomy as a life-saving measure. however, a substantial subset (~ %) were not willing to accept this aggressive measure, which emphasizes the importance of discussing the individual's previously stated wishes, even in the young population. further study in larger populations is needed to better characterize the factors impacting young adults' decisions regarding aggressive care. . ) , but a trend was noted for ne levels (p=. ), and a significant correlation was seen for dhpg (p=. ). our study supports the theory of a cns mediated adrenergic mechanism for nc, based on the presence of increased csf levels of ne and its dhpg metabolite. recent studies have suggested that recurrent stroke during aspirin treatment might have been caused by biochemical aspirin resistance (bar). we hypothesized that patients with bar would develop early recurrent ischemic lesions (erils) on diffusion-weighted imaging (dwi) more than those without bar. we included consecutive patients who: ) were admitted to our center within hours of stroke onset; ) had a final diagnosis of acute ischemic stroke, confirmed by dwi, or tia; ) underwent follow-up dwi within seven days after initial dwi; ) received aspirin therapy; and ) underwent tests for bar. aspirin was administered to patients soon after initial imaging study. bar was measured using the veryfynow rapid platelet function assay-aspirin (accumetrics inc., san diego, ca). an aru was defined as bar. erils were defined as new lesions on followup dwi with decreased apparent diffusion coefficient, which were not detected on initial dwi scans. the bar is associated with the development of erils during the first week after development of ischemic stroke. this suggests that increased thrombogenicity is one of important mechanisms of erils and that aggressive antiplatelet therapy is warranted during the acute phase. in this study we examined the effects of mannitol % on brain metabolism and brain tissue oxygenation (pbto ) in severely brain-injured patients with intracranial hypertension. twenty-two episodes of raised intracranial pressure (> mm hg) resistant to standard therapy that required infusions of mannitol were prospectively studied in comatose patients with multimodality monitoring of intracranial pressure (icp), pbto , and microdialysis. we compared mean arterial blood pressure (map), icp, cerebral perfusion pressure (cpp), pbto , and brain lactate, pyruvate, and glucose using cerebral microdialysis, for hours preceding and hours after hyperosmolar therapy. time series data were analyzed using a multivariable general linear model (glm) utilizing generalized estimating equations (gee) for model estimation to account for within-subjects and betweensubjects variations over time. g/kg of % mannitol solution led to a maximal reduction of icp at minutes (from ± to ± mm hg, p < . ). cpp increased at a peak of minutes (from ± to ± mm hg, p = . ) after mannitol infusion was started, whereas map and pbto did not change significantly. compared to lactate-pyruvate ratio (lpr) at the time of osmotherapy ( ± ,) , mannitol resulted in a an % decrease over hours (to ± , p = . ). brain glucose levels remained unaffected. mannitol effectively reduces icp and augments cpp, and appeared to benefit oxidative metabolism as measured by the lpr. twenty-eight comatose sah patients that underwent multimodality monitoring with intracranial pressure and microdialysis were studied. mc was defined as lactate/pyruvate ratio (lpr) and brain glucose < . mmol/l. time series data were analyzed using a multivariable general linear model with a logistic link function for dichotomized outcomes. multimodality monitoring included hours of observation (mean ± hours per patient). in exploratory analysis, serum glucose significantly decreased from . mmol/l ( mg/dl) hours before to . mmol/l ( mg/dl) at the onset of mc (p< . ). reductions in serum glucose of % or more were associated with new onset mc (adjusted odds ratio [or] . , % confidence interval [ci] . - . ). this association was independent of the absolute serum glucose level. in a second model we chose an elevation of the lpr by % or more as the outcome variable. again, reductions in serum glucose of % or more were independently associated with an lrp rise (adjusted or . , % ci . - . ). all analyses were adjusted for significant covariates including glasgow coma scale and cerebral perfusion pressure. acute reductions in serum glucose, even to levels within the normal range, may trigger brain energy metabolic crisis and lpr elevation in poor-grade sah patients. hyponatremia develops in up to one-third of patients after subarachnoid hemorrhage (sah), and is usually attributed to cerebral salt wasting or siadh. our goal was to identify risk factors for hyponatremia after sah, and to determine its impact on outcome. we analyzed consecutive sah patients enrolled in the columbia university sah outcomes project between july and june . hyponatremia was defined as sodium level meq/l occurring at any point during hospitalization. multivariate analysis was performed to identify risk factors for hyponatremia. functional disability was evaluated at discharge and months with the modified rankin scale (mrs, score - ) and barthel index (bi, score < ) the frequency of hyponatremia in or cohort was % ( / ). hyponatremia developed on median post bleed day with most cases occurring between days and . logistic regression adjusted for gender and initial hunt- hyponatremia occurs in % of sah patients, is predicted by older age, fever, renal failure and hydrocephalus, and is associated with reversible functional disability at discharge. failure to correct hyponatremia my potentially interfere with rehabilitation and recovery after sah. previous studies have reported that younger patients have a higher incidence of clinical deterioration from vasospasm after subarachnoid hemorrhage. we sought to determine the relationship between age, with the incidence of vasospasm defined by angiographic, tcd, or clinical criteria. we analyzed consecutive sah patients enrolled in the columbia university sah outcomes project between july and june . vasospasm was assessed using angiography and/or a mean flow velocity greater than cm/s in any vessel. symptomatic vasospasm was defined as clinical deterioration (i.e. a new focal deficit, decrease in level of consciousness, or both) and asymptotic vasospasm included a new infarct on ct that was not visible on the admission or immediate postoperative scan. a tcd velocity greater than cm/s was observed in % of patients and of the patients that had follow-up angiography performed ( %), % of those patients had vessel narrowing consistent with angiographic vasospasm. in contrast symptomatic vasospasm was observed in only % of all patients with % of patients suffering infarction attributed to vasospasm. in total % of patients had either symptomatic vasospasm or asymptomatic infarction from vasospasm. multivariable logistic regression revealed that after accounting for disease severity (hunt & hess) , modified fisher score, gender, and history of smoking, younger age was significantly related to the occurrence of angiographic (or: . , % ci: . - . ) and tcd> cm/s (or: . , % ci: . - . ) spasm, but was not significantly associated with symptomatic vasospasm (p=. ) or delayed infarction from vasospasm (p=. ). our data support the findings that younger patients are more likely to experience vasospasm defined by tcd and angiography than older patients, but in our cohort we did not observe a higher incidence of clinical vasospasm or infarction. aggressive treatment of tcd-based and angiographic vasospasm with intra-arterial vasodilators or balloon angioplasty may mitigate the effect of age. post-traumatic vasospasm (ptv) occurs in - % of patients with severe traumatic brain injury (tbi), and is an independent predictor of neurological outcome. although ptv incidence has been associated with injury severity, there are conflicting reports regarding patterns of intracranial hemorrhage that may correspond with development of posttraumatic vasospasm. some authors report that subarachnoid hemorrhage or subdural hematoma is necessary to develop ptv, while others have reported significant ptv in the absence of these lesions. we performed a review of prospectively collected ct scan data from consecutive head injured patients treated at a tertiary level i trauma center. rotterdam ct score data was reviewed from all patients in the tbi registry, and admission head ct scans from patients with severe tbi (gcs ) with (n= ) and without (n= ) clinically significant ptv (csptv) were re-evaluated by a 'blinded' investigator. csptv was defined as demonstrated neurological decline with ct angiographic evidence of arterial vasospasm. rotterdam ct score significantly correlated with the development of csptv(p= . ). the components of this score were further investigated. we found no correlation between epidural hematoma, subdural hematoma, midline shift, or cisternal compression and the development of csptv. the presence of intraparenchymal hemorrhage (p= . ) and cisternal subarachnoid hemorrhage (p= . ), however, significantly correlated with risk of csptv. all cases of csptv were diffuse in anatomic distribution, and, therefore, did not correlate with side of maximal injury. rotterdam ct score, intraparenchymal and cisternal subarachnoid hemorrhage on admission ct are significantly correlated with the incidence of csptv. this suggests that risk of cerebral vasospasm following traumatic brain injury is increased not only in subarachnoid hemorrhage, but also intraparenchymal hemorrhage, and that rotterdam ct score may be a useful metric for assessing risk of csptv in severe tbi patients. we reviewed patients from a tertiary level trauma center tbi registry and identified patients with clinically significant ptv (csptv), defined as demonstrated neurological decline with ct angiographic evidence of arterial vasospasm. patient charts were reviewed to characterize the natural history, treatment and efficacy of treatment in csptv. treatment strategies for patients with csptv included observation, "triple-h" therapy, oral statins, intra-arterial verapamil infusion and tba. the decision to pursue intra-arterial therapy was based on severity of spasm and clinical exam. observation alone was used in patients with mild, diffuse spasm on cta and rapid clinical improvement (n= ), whereas those with persistent signs of spasm all underwent medical therapy (n= ). intra-arterial verapamil infusion was used in patients with moderate to severe spasm (n= ). tba was performed in patients who had severe and diffuse spasm (n= ). in all cases, therapy was effective in reducing (n= ) or reversing (n= ) ptv. three month functional outcome data revealed no significant differences between patients with and without csptv. treatment of ptv is effective in reducing or reversing arterial vasospasm. a variety of therapies exist, which should be chosen based on clinical exam and the degree and distribution of spasm. although it is unknown if treatment improves outcome, our data suggest that patients with csptv have similar outcomes to those without csptv when they are adequately treated. a clinical pathway is presented to aid in the screening, diagnosis, and treatment of ptv. tracheostomy and gastrostomy are common procedures in patients suffering neurologic insults. we report our current data of these procedures performed simultaneously at bedside by a neurointensivist using percutaneous techniques. database of all tracheotomies, gastrostomies and combined procedures performed by the neuro-critical care team was retrospectively analyzed. also, satisfaction surveys by nursing and house staff were employed to reassess and refine the service. all procedures were completed at bedside in the neurointensive care or other intensive care units utilizing two critical care fellows, an intensive care nurse and a respiratory therapist under the direction of the neurocritical care attending. the team followed each patient daily and reported any complication until discharge. complications were categorized as major (requiring additional surgical intervention) or minor (no additional surgical intervention). to date the team has performed over combined percutaneous tracheostomy and gastrostomiesin patients with primary neurologic pathology. there were two major complications and five minor complications reported. the neurologic pathology was mixed as was the,age and weight ranges combined tracheostomy and peg tube placement can be performed safely by a neurointensivist complications rates are low and no catastrophic events reported. attendings, house staff and nursing supports the continuation of this programs the neuro-critical care service now performs the majority of these procedures in our institution based on the success of the service, non-neurologic related services are consulting the neuro-critical care team to perform these procedures. introduction: traumatic brain injury (tbi) is a complex disease state that includes disruption of the blood-brain barrier (bbb) and inflammatory changes. angiopoietins are a family of growth factors integral in maintaining endothelial integrity and controlling inflammation. angiopoietin i (ang ) induces phosphorylation of the tie ligand enhancing endothelial integrity. angiopoietin ii (ang ) inhibits this action. in animal models, ang is up regulated in tbi while ang appears unchanged. injury models in other tissues suggest that the ratio of ang to ang may be significant. little is known about their role in humans with tbi. we collected csf from patients with tbi ( patients) and compared it controls ( patients). individual levels and ratios were compared. each non-tbi csf had < cells/um, negative gram stain and cultures and normal protein and glucose levels. csf samples were collected from the tbi group within hours of drainage placement.. ang and ang were analyzed using an elisa method and reported in pg/ml. the levels of ang in the control group and tbi group were not significantly different (p value of . ). there was significant increase in ang- in the tbi group (p = . ). comparing the ratios of ang and ang , ang was times higher ( : ) in the control group than in the in the tbi group ( . : ). this data correlates with animal data that shows an increase in ang after tbi. this data further demonstrates a significant change in the ratio of ang to ang after tbi. what happens over time and how this relates to severity and prognosis is yet to be investigated. restoring an ang- to ang- ratio to normal may be a therapeutic strategy worthy of investigation. external ventricular drains (evd) and intracranial pressure monitoring equipment are used frequently in neuroscience intensive care units. because of the potential for the development of nosocomial infection prevention is important. current practice varies from using no antibiotics to continuous antibiotics while devices are in place. there is inadequate foundation to support a particular practice. to define practice patterns, a survey was sent to > , neurosurgeons, critical care, neurocritical care, and infectious diseases specialists. the same survey was also submitted to members of the neurocritical care society but filtered to exclude redundancy. ten percent of practitioners solicited responded to the survey. eighty seven percent of respondents were from north america, followed by asia, europe, and south america. twothirds practiced in academic centers and had > years experience. seventy seven percent of respondents were neurosurgeons, followed by neurocritical care, infectious diseases, and critical care. o % peri-operative abx o % use none. one third of respondents use antibiotic coated evd's there are differing practices among the specialties surveyed. a majority of the respondents use abx for the duration the devices are in place. there are differences in practice among respondents based on specialty, geography, years of practice and type of practice. eighty percent of respondents think a randomized trial comparing abx strategies is needed. retrospective chart review of prospectively identified patients. per institutional protocol, patients were cooled to a nadir of c. baseline prothrombin time (pt), partial thromboplastin time (ptt) and platelet count were obtained and followed at least daily during th and rewarming. data was evaluated for the development of new abnormalities following th induction. thirty six patients received th for various clinical indications, including cardiac arrest and intracranial pressure control (related to subarachnoid hemorrhage, intracerebral hemorrhage, ischemic stroke, and traumatic brain injury). duration of goal temperature maintenance varied from hours to hours. after induction of th, / ( . %) showed abnormal pt, / ( . %) had abnormal ptt, and / ( . %) patients developed thrombocytopenia (platelet count < , /µl). in those developing abnormalities, normalization was not seen for any parameter within hrs of rewarming. overall, % of the patients demonstrated some form of new abnormality following th, none of which had clinically significant bleeding episodes. overall, -day mortality was %; no mortality was attributable to th. we concur with the previously reported findings that th is associated with coagulation abnormalities. a high proportion of patients were found to demonstrate such abnormalities, which persisted following rewarming; the exact clinical significance of these findings is not clear. in addition to this standard laboratory testing, changes in radiographic imaging may serve as a more sensitive adjunctive measure to evaluate the significance of th related coagulopathy. transtentorial herniation (tth) is a clinical syndrome consisting of pupillary dilatation with loss of pupillary light reflex and decreased level of consciousness in the setting of a large intracranial mass lesion. reversal of tth is defined as return of pupillary light reflex with or without immediate improvement in level of consciousness. the role of renal function in the mechanism of hypertonic therapy remains unclear. we evaluated the efficacy and safety of . % saline in tth in patients with end-stage renal disease (esrd) on hemodialysis. patients with clinically defined tth and esrd on hemodialysis treated with . % saline ( to ml) were included in the analysis of a retrospective cohort. of subjects over years, we identified patients with esrd that had tth events. lesions were related to stroke (n= ), intracerebral hemorrhage (n= ), and subdural hemorrhage (n= ). all patients received a . % saline bolus, along with mannitol ( % of events), hypertonic saline maintenance fluids ( %), ventriculostomy (n= ), and hemicraniectomy (n= ). clinical reversal of tth occurred in / events ( %); of patients survived to discharge. in patients, icp recording of tth events showed a reduction from icp of . mmhg (mean sem) with tth to . . mmhg (p= . ) one hour after the . % saline bolus. serum sodium increased from . mmol/l to . mmol/l hours after . % saline bolus (p= . ). no patients were undergoing hemodialysis at the time of the tth event, and the post-infusion serum creatinine did not change. treatment with . % saline was associated with rapid clinical reversal of tth and reduction in icp in this small cohort of patients with esrd. this finding supports that hypertonic saline may be effective in cases of esrd. introduction: intubated patients with subarachnoid hemorrhage (sah) may spontaneously hyperventilate despite minimal ventilatory support. the impact of this is unclear, although hypocapnea may be harmful in traumatic brain injury. we set out to determine the incidence of spontaneous hyperventilation in patients with sah and its association with clinical outcomes. we identified consecutive, intubated patients with spontaneous sah from clinical databases ( ) ( ) ( ) . demographics, clinical and ventilation data (for the first days post-bleed) were collected. hypocapnea was defined as an arterial pco <= mmhg. primary outcomes were ( ) the presence of symptomatic vasospasm (defined by both angiographic vasospasm and clinical symptoms); ( ) death in the intensive care unit. associations between hypocapnea and outcomes were explored with multivariate analysis. we identified patients with sah and a median duration of ventilation of days [iqr - ]. hypocapnea was observed on at least one day in patients ( %), and patients ( %) had at least pco < mmhg. all hypocapnea was associated with alkalemia. ventilatory support was minimal (cpap or ps cm h o) in % of hypocanea measurements. sedation normalized pco in % of cases, and use of neuromuscular blockade was rare. median duration of hypocapnea (at least one pco <= mmhg each day) was days [iqr - ]. duration of hypocapnea was associated with increased odds of symptomatic vasospasm (or . for each day with hypocapnea; p= . ) after adjusting for fisher ct grade. duration of hypocapea was not associated with icu mortality after adjustment for apache ii and wfns grade (p= . ). the incidence of spontaneous hyperventilation is high in intubated patients with sah, despite minimal ventilator support. duration of hypocapnea was independently and statistically significantly associated with symptomatic vasospasm. few studies have evaluated physician-family interactions and decision-making in the neurocritical care unit (nccu). we sought to determine if the icu team's use of a structured checklist for family conferences (fc) would improve family satisfaction. we conducted a prospective pilot pre-and post-intervention study. we designed an -item checklist of key content for fc conducted with the intent of making significant patient management decisions. phase i was observational, with a nurse covertly documenting the key content covered during the fc. phase ii was interventional. we asked the icu team to use the checklist during fc to cover all key content. a family member and the icu team member completed an immediate post-fc written survey, and the fs-icu , a family satisfaction survey, was mailed to the family months after nccu discharge. families enrolled ( phase i; phase ii), with patient age ± years, apache iii score ± and nccu los ± days. patients died ( -pi; -pii). median key content covered was items in phase i and items in phase ii (p= . ). in phase ii, icu team member self-report of key content was higher than documented content ( vs. items; p= . ). post-fc survey scores increased from . (phase i) to (phase ii) (p= . ). the fs-icu decision-making subscale median score was . in phase i and in phase ii (p= . ). use of a fc checklist in the nccu marginally improved coverage of key elements in family conferences, however post fc family satisfaction was improved. further evaluation of the influence of checklists on patient outcomes and family satisfaction for family conferences in the nccu is warranted. h. adrian püttgen , jai madhok , xiaofeng jia , anil maybhate johns hopkins university medical institutions, baltimore, md, united states, johns hopkins university school of medicine, baltimore, md, united states, johns hopkins university, baltimore, md, united states sep's represent the brain's response to sensory electrical stimulus. current clinical methods require averaging a large number of sep waveforms for meaningful prognostication. automated sep monitoring could be used as a noninvasive bedside tool for conditions that severely affect somatosensory conduction due to elevating intracranial pressure (icp) such as cerebral oedema or intracerebral haemorrhage. adult wistar rats were used in this pilot study. to model intracranial hypertension, a latex micro-balloon ( µl maximum volume) was surgically inserted into the epidural space via a burr hole on the left hemisphere ( mm off the sagittal suture). using a micro-pump, the balloon was slowly inflated with water at µl/min for two min periods with a min pause. seps were recorded after electrically stimulating the hind limb at . hz. icp was recorded using a transducing catheter inserted in the subdural space over the right hemisphere. balloon inflation was accompanied by a steady increase in the icp. the increase in icp beyond a certain level was accompanied by the sudden disappearance of sep's within a few seconds ( to sweeps). in our pilot experiments, the peak to peak amplitude of the sep dropped steeply from about + µv to + µv before a complete and sudden disappearance when the balloon volume reached approx. + µl. this pilot study demonstrates the effect of an intracranial mass on the integrity of the somatosensory pathway. the finding of a threshold of lesion magnitude after which further expansion causes a dramatic disappearance of sep points to the possibility of using continuous sep for monitoring rapidly evolving mass lesions such as cerebral oedema or intracerebral haemorrhage. we studied the feasibility of intracortical electroencephalography (ice) including quantitative eeg (qeeg) analysis for the detection of vasospasm in a series of poor-grade sah patients. from a consecutive series of sah patients who underwent ice placement, the alpha/delta ratio ( - hz/ - hz; adr) was calculated at twenty second intervals from the ice and scalp eeg recordings. percent changes between averaged values over - hours of the baseline eeg and the eeg prior to angiography were calculated. the entire continuous qeeg recording for each patient was then reviewed to determine optimal automated alarm criteria. ice recordings revealed an improved signal-to-noise ratio when compared to surface eeg recordings. the adr calculated from the ice decreased between baseline eeg and follow-up eeg on average by % (mean adr decrease . ± . to . ± . ) for those with vasospasm (n= ) compared to % ( . ± . to . ± . ) for those without vasospasm (n= ). a sustained decrease in the adr by at least % from baseline for a minimum of hours occurred in patients with vasospasm - days before angiographic confirmation of vasospasm. this was not seen in patients without angiographic vasospasm. eeg recordings from ice are promising to reliably detect vasospasm in severely brain injured sah patients. absence of artifact allows for automated qeeg analysis of ice recordings. raising the head-of-bed (hob) js a very important step in taking care of critically ill patients, particularly in the neurocritical care, as it influences abdominal pressure, decreases incidence of pneumonia associated with aspiration secondary to the decrease in gastroesophageal reflux and reduces intracranial pressure, improving cerebral perfusion pressure. nevertheless, this relatively simple maneuver is still not widely applied. after explaining the goals of raising the hob individually, each health-care worker (hcw) attending in our -bed neurocritical care service was requested to position the hob between and degrees. as our beds are able to measure hob angulation, it was later conferred. after this simple procedure, hcw were again explained and a folder was distributed. it contained a questionnaire and pictures of hob at , , and degrees. later on, they were again requested to position hob at the adequate position. the first poll revealed that out of participating nurses did not or only partially knew the reasons hob should be positioned between and degrees and almost % of the attempts resulted in failures. among physicians, . % only partially knew the reasons. they could rightly position hob in % of the attempts. after the questionnaire, every and each one of the hcw could name the reasons of hob positioning and almost % of the attempts resulted in right results. hcw should be constantly reminded the importance of simple tasks in the care of neurocritical patients. hob elevation should not be regulated by random trials. automatic beds are very important devices, particularly when some features are present, such as hob angulation and, even though it could seem an expensive device, it will finally allow cheaper and less risky expenditures. posterior reversible encephalopathy syndrome (pres) can be caused by hypertensive crisis and is often associated with rapid fluctuations in blood pressure (bp). the role of these bp changes in the pathogenesis of pres has not been formally studied. we sought to analyze the relationship between blood pressure (bp) fluctuations and the occurrence of pres. consecutive hospitalized patients who developed pres were compared with randomly selected controls matched for age, gender, and history of hypertension. systolic bp (sbp) and diastolic bp (dbp) were collected every hours for hours before developing pres symptoms. sbp, dbp, mean arterial pressure (map), and pulse pressure (pp) changes over a -hour window was summarized for each individual by calculating an m value as described by service et al ( ) . m values were compared using wilcoxon signed rank test. tests were two sided and p values less than . were considered statistically significant. we analyzed the bp profiles in cases of pres and controls. median age of pres patients was years (range - ). of them ( %) had pre-existing hypertension. hypertensive encephalopathy was considered the etiology of pres in patients ( %). at symptomatic onset of pres, mean sbp was ± mmhg (range - ), dbp ± mmhg (range - ), map ± (range - ), and pp ± (range - ). while bp was higher in pres cases, hypertension severity was variable and bp fluctuations were not significantly more common than in controls (p values for sbp, dbp, map, pp were . , . , . , . , respectively). bp fluctuations do not appear to be more common in hospitalized patients who develop pres compared with matched controls. other predisposing factors must therefore contribute to the development of pres. technologies allowing emergent detection of focal cerebral hypoxia would be of great utility in the treatment of ischemic stroke by facilitating diagnosis, tracking reperfusion, and identifying re-thrombosis. non-invasive brain oxymetry using near-infrared reflectance spectroscopy (nirs) technology incorporated into the invos device (somanetics, troy, mi), provides direct measurement of regional oxyhemoglobin saturation (rso ) within the cerebral cortex. this study utilized the invos device to determine the predictive value of cortical rso monitoring in the assessment of ischemia in patients presenting with large hemispheric strokes. patients exhibiting acute ischemic strokes involving proximal mca or ica occlusions on ct angiography were enrolled prospectively. the invos device was applied according to the manufacturer's recommendations. rso data was recorded at s intervals for at least hr in each patient. concomitant vital signs, hgb, oxygen saturation, pao , and paco were collected. three out of patients underwent emergent cerebral angiography. a neuroradiologist, blinded to the invos results, evaluated all ct, ct perfusion, and cerebral angiography studies. ct perfusion imaging confirmed large hemispheric strokes in all patients. mean time from symptom onset to start of rso monitoring was hours (range = - ). data analysis consistently demonstrated mean ro saturation levels on the ischemic hemisphere to be either the same or higher than that of the non-ischemic hemisphere. rso levels were independent of bp, hgb, oxygen saturation, pao or paco levels. cerebral angiography demonstrated significant collateral flow over the affected hemisphere despite deep large vessel occlusions. these findings suggest that nirs technology has limited utility in the assessment of patients with acute ischemic stroke. patency of cortical collaterals and increased tissue oxygen extraction during ischemia, among other factors, may offset a decrease of cortical rso within the affected hemisphere. venous thromboembolism is a common problem in critically ill patients. neurosurgical patients even though at higher risk; often do not receive timely pharmacological thromboprophylaxis for fear of bleeding risks. recent literature points towards the safety and efficacy of early prophylaxis (scd's + heparin/lovenox); however this has not been tested extensively in a randomized controlled trial. a retrospective chart review of patients with a primary diagnosis of subarachnoid hemorrhage (sah), intracerebral hemorrhage (ich), and subdural hematoma (sdh) admitted from january to june was conducted for icd- codes of dvt and or pe, and for presence of associated risk factors. all patients received intermittent compression devices (scd's) on all patients from time of admission to time of discharge, surveillance doppler ultrasound evaluation of both lower extremities once every week, and doppler screening of symptomatic upper extremities. overall incidence of dvt was . % (n= ). the incidence of dvt was . % in sah, . % in ich, and . % in sdh. the incidence of pe was . %.the presence of intraventricular hemorrhage was seen in . % of patients with sah who had dvt. this study shows almost double the incidence of dvt than reported in the recent literature. picc line and central lines were associated with higher incidence of dvt.the timing of the diagnosis of dvt falls in a time window where intracranial bleeding risks from anti-coagulation are far less than in the acute stage. this study will provide us a unique cohort of patients whom we can compare in a prospective manner to patients who will receive subcutaneous heparin along with scd's in the future, since we are changing our policy to implement heparin thromboprophylaxis. the presence of intraventricular hemorrhage (ivh) is predictive of worse outcomes following aneurysmal subarachnoid hemorrhage (sah) [ ] . however, the amount of ivh can vary considerably. no previous studies have assessed the association between actual hematoma volume (in ml) and subsequent complications or outcomes. we performed a cohort study involving consecutive patients with concomitant sah and ivh. with investigators blinded to subsequent events, ct scans were graded using two systems. first, to determine the volume of ivh, we used the ivh score, recently shown to correlate exceptionally well with computerized volumetric assessment [ ] . second, to examine the relative amount of subarachnoid blood, we applied the sah component of the hijdra score [ ] . using logistic regression to adjust for sah score and other potential confounders, we assessed the association between ivh volume and poor neurological outcomes (glasgow outcome scale - ), as well as symptomatic vasospasm and delayed infarction. compared with patients who had a favourable outcome, those with poor outcomes had significantly larger baseline ivh volumes (mean . ml vs. . ml, p= . ). in the multivariable analysis, ivh volume remained an independent predictor of poor neurological outcome (or per ml: . , . - . , p= . ). patients in the highest quartile for ivh volume were far more likely to progress to poor outcomes compared with those in the lowest quartile (or . , . - . , p= . ). in contrast, ivh volume was not associated with either vasospasm or delayed infarction. interobserver agreement in the determination of ivh score was good. volume of ivh is a strong, independent predictor of death and poor neurological recovery, even when one adjusts for the amount of concomitant subarachnoid blood. future studies should assess whether measures aimed at accelerating the clearance of ivh (e.g. intraventricular thrombolysis) can modify this association. andrew naidech, kimberly levasseur, storm leibling, rajeev garg, michael shapiro, michael ault, sherif afifi, hunt batjer northwestern university, chcago, il, united states while many icus have implemented protocols for tight glucose control, there are few data on relative hypoglycemia and neurologic outcomes. we addressed the hypothesis that lower glucose leads to worse neurologic outcomes after subarachnoid hemorrhage (sah). one hundred seventy-two ( ) consecutive patients were treated with a protocol designed to achieve serum glucose - mg/dl. we prospectively ascertained patients on admission and recorded medical history and clinical events. glucose measurements from the hospital laboratory were electronically retrieved. (a separate analysis of bedside glucose results found similar results.) cerebral infarction was prospectively documented with neuroimaging. outcomes were assessed with the modified rankin scale (mrs) at days, days and months. worse neurologic injury at admission (p< . ) and a history of diabetes (p= . ) were associated with increased glucose variance. patients with radiographic cerebral infarction ( ± vs. ± mg/dl, p= . ), symptomatic vasospasm ( ± vs. ± mg/dl, p= . ) and angiographic vasospasm ( ± vs. ± mg/dl, p= . ) had lower nadir glucose, but maximum and mean glucose were not different. glucose < mg/dl was earlier and more frequent in patients with worse functional outcome (p< . ). progressive reductions in nadir glucose were associated with increasing functional disability at months (p= . ) after accounting for neurologic grade and mean glucose. severe hypoglycemia (< mg/dl) occurred in one patient. in patients with sah, nadir glucose below the < mg/dl is associated with cerebral infarction, vasospasm, and worse functional outcomes in multivariate models. protocols for target glucose - mg/dl effectively control hyperglycemia, but may place patients with sah at risk for vasospasm, cerebral infarction and poor outcome even when severe hypoglycemia does not occur. andrew naidech, rajeev garg, storm liebling, kimberly levasseur, micheal macken, stephan schuele, hunt batjer northwestern university, chicago, il, united states there are few data on the effectiveness and side effects of anti-epileptic drug (aed) therapy after intracerebral hemorrhage (ich). we tested the hypothesis that aed use is associated with more complications and worse outcome after ich. we prospectively enrolled patients with ich and recorded aed use as either prophylactic or therapeutic along with clinical characteristics. aed administration and free phenytoin (pht) serum levels were retrieved from the electronic medical record. patients with depressed mental status underwent continuous eeg monitoring. outcomes were measured with the nih stroke scale and modified rankin scale (mrs) at days or discharge, and the mrs at days and months. we constructed logistic regression models for poor outcome at months with a forwardconditional model. seven ( %) patients had a clinical seizure, five on the day of ich. pht was associated with more fever (p= . ), worse nih stroke scale at days ( [ - ] vs. [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] , p= . ) and worse mrs at days, days and months. in a forward-conditional logistic regression model pht prophylaxis was associated with an increased risk of poor outcome, or . ( . - . ) p= . , entering after admission nih stroke scale and age. excluding patients with a seizure did not change the results. levetiracetam was not associated with demographics, seizures, complications, or outcomes. pht was associated with more fever and worse outcomes after ich. posterior reversible leukoencephalopathy syndrome (pres) is characterized by seizures, headache, encephalopathy and visual disturbances associated with reversible vasogenic edema on brain imaging. status epilepticus (se) has been infrequently described as an initial manifestation of pres. the clinical and radiological features of patients with pres and se have not been well described. patients with se were identified from a mostly prospectively collected database of patients (n= ) with pres. we collected data on general demographics, clinical presentation, history of epilepsy, peak systolic and diastolic blood pressures, and predisposing conditions. brain mris were analyzed independently by two neuroradiologists for lesion location and distribution, severity, presence of hemorrhage and presence of restricted diffusion. of patients with pres, ( %) presented with se. only had a prior history of epilepsy. mean peak sbp was mm hg ( - ) and mean dbp was mm hg ( - ). etiologies of pres included hypertension (n= ), cytotoxic medications (n= ), sepsis (n- ) and other (n= ). renal failure was present in ( %) cases and ( %) had pre-existing chronic kidney disease. twelve patients ( %) had a history of autoimmunity. among patients with brain mri, ( %) demonstrated mild edema and ( %) had moderate-severe edema. the cortex was involved in only patients ( %). almost all had edema in the parietal-occipital region (n= , %). when compared with the rest of our pres cohort, we did not identify any significant clinical or radiological predictors of se. se is not an infrequent presentation of pres. its occurrence is not correlated with the severity of radiologic edema and the great majority of patients actually lack cortical involvement. recognition that pres may present with se is important because, besides anticonvulsants, appropriate treatment requires identifying and treating the underlying cause of pres. hypervolemia is known to lead to peripheral and pulmonary edema however the effect on intracranial pressure (icp) following traumatic brain injury (tbi) is unclear. there is no direct evidence in humans linking hypervolemia independently to elevated icp. compelling evidence suggests that fluid restriction should be avoided and limited evidence suggest significant hypervolemia may be associated with worse outcome following tbi [ ] . the use of fluids and vasopressors to elevate cerebral perfusion pressure (cpp) > mmhg has been to shown to increase the risk of pulmonary complications (but not clearly effect icp) following tbi and is not recommended by guidelines [ , , ] . despite this, some ancillary monitoring protocols recommend elevating cpp to treat episodes of cerebral hypoxia. retrospective observational cohort study of severe tbi patients admitted over a -year period to a neuro-trauma unit. data extracted: characteristics; fluid balance; development of refractory intracranial hypertension (rih); pulmonary complications; use of vasopressors; ancillary monitoring. patients with unsurvivable injuries, early withdrawal of care or the development of refractory intracranial hypertension (rih) within hours were excluded. forty-one patients with mean age . ; % male; % automobile accidents; % polytrauma; average best gcs of . (a subgroup presented with higher gcs with declined secondary to neurological injury). rih was associated with lower fluid balance but not hypervolemia (overall q = %, iq = %, q = %). an early low fluid balance and hypervolemia both are associated with more pulmonary complications. the use of vasopressors, and to a lesser extent licox monitoring is associated with a higher incidence of pulmonary complications and possibly rih. [tables - ] % ( / ) % ( / ) % ( / ) q = first quartile, iq = interquartile, q = fourth quartile; rih = failure of first tier therapy by brain trauma foundation; pulmonary complications = ards or pulmonary edema with p/f ratio < ; use of vasopressors = for > hours and > hours to maintain cpp > mmhg following severe tbi hypovolemia should be avoided as it's associated with increased icp and pulmonary complications [ ] . extreme hypervolemia should be avoided, if possible, to minimize pulmonary complications. ancillary monitoring protocols should be used with caution, as the components that may improve outcome versus those that may harm are incompletely defined. without correction for patient demographics, severity of illness, and head ct findings further conclusions cannot be made. invasive mechanical ventilation is required in one third of patients with guillain-barré syndrome (gbs). there are few early indicators of subsequent progression to respiratory failure. adrenal function has rarely been studied in patients with gbs. we assessed the relationship between plasma cortisol level and gbs related complications, notably respiratory failure. plasma cortisol levels were measured before (t ) and minutes (t ) after corticotrophin test in gbs patients at admission, ( %) of which were ventilated within hours from admission, ( %)ventilated after the th hour and ( %) never ventilated. the volume of subarachnoid hemorrhage (including intraventricular blood) following aneurysmal rupture is associated with the development of vasospasm. intraventricular catheters (ivc) facilitate cerebral spinal fluid (csf) drainage and may reduce the incidence or severity of vasospasm but little evidence exists from which clinicians may determine the best practice. the purpose of this study was to provide the foundation for designing a trial that will explore how different methods of csf drainage may impact outcomes in these patients. this observational pilot study enrolled adult sah patients. data was collected through chart abstraction. attending neurosurgeons determined whether each patient's ivc was primarily left open to drain csf resulting in intermittent icp monitoring (drain-first group), versus an ivc that was primarily set to monitor icp resulting in intermittent csf drainage at a set pressure threshold (monitor-first group). subjects were primarily female ( %), mean years old. subjects in the drain-first group (n= ) and the monitor-first group (n= ) had similar hunt/hess (p= . ) and fisher scores (p= . ). although there are no statistically significant differences between groups, this pilot study was not designed to test a hypothesized difference. the monitor-first group had lower mean csf output ( vs ml/day), lower rates of vasospasm ( % vs %), lower incidence of complication ( % vs %), shorter length of stay ( vs days), and lower modified rankin scores at discharge ( . vs . ). this observational study suggests that the method of ivc management may impact clinical outcomes. although the monitor-first group method appears to be favourable, it is difficult to attribute differences in a non-randomized trial. a larger randomized controlled clinical trial is now in progress. introduction: subarachnoid hemorrhage (sah) patients whose initial angiogram does not locate a bleeding source are often classified as having perimesencephalic hemorrhages. however, many patients do not fit into this benign picture and are non-perimesencephalic, angiogram-negative sah (npan-sah). though the conventional angiogram remains the gold standard for diagnosis, multiple non-invasive imaging tests, beyond a second angiogram, are often performed in the acute evaluation of npan-sah. with irb approval, we retrospectively reviewed non-traumatic sah patients admitted to our institution from january , to june , . hunt-hess and fisher scores, in-hospital complications, and imaging data were abstracted from medical charts. non-perimesencephalic angiogram-negative sah has a worse prognosis compared to perimesencephalic sah. additional non-invasive neuroimaging provided no diagnostic yield in either patient population. guidelines suggest an ideal time from injury to surgical decompression of less than four hours in patients with acute traumatic subdural or extradural haematoma. previous audits at our centre showed this standard was not consistently achieved. we looked for a relationship between the length of this time interval and adverse neurological outcome at six months. we retrospectively reviewed all patients with acute traumatic subdural (asdh) or extradural (aedh) haematoma transferred to our neurosurgical centre over a three year period (december -november ) for emergency surgical decompression. we identified the time elapsed from presentation at the emergency department to commencement of surgical decompression. we then assessed neurological function at six months post surgery using a glasgow outcome score. we were able to include patients in our study ( asdh, aedh). the mean time from presentation to surgery was : hours. at six months . % of patients had a good neurological outcome (gos - ), . % had a poor outcome . of those presenting with gcs < , % had a good outcome compared to % of those with an admission gcs of or above. achieving definitive surgery within four hours of presentation, let alone injury remains elusive. we were unable to associate prolonged length of transfer time with worse neurological outcome at six months. our study was retrospective and the numbers were small. our unit accepts a significant number of patients from outside its normal referral area, meaning there may already be a significant delay in many cases. in most cases there was no single identifiable reason for delay and a few cases showed that transfer could be achieved very rapidly. sah patients who had hunt-hess grades - , a ventriculostomy, and tcus performed for at least days were included in this study. csf ml was collected from each patient during the first hours and assayed by hplc for levels of epinephrine (epi), norepinephrine (ne), and dihydroxyphenylglycol (dhpg). mca vs was defined as a mean velocity (mv) > cms/s with a mca/ica ratio of > at any time. analyses were calculated on a per-case and per-vessel basis. of the initial patients included, were excluded due to incomplete data as a result of early mortality or absent bone windows. from the remaining patients, had only ipsilateral bone windows, resulting in a total of vessels amenable to insonation. mean age was yo, and % were female. ct scores (frontera et al.) were = %, = %, = %, & = %. on a per-case basis, patients with mca vs were younger ( yo vs yo, p=. ), but no correlation was observed between mca vs and adrenergic levels. on a per-vessel basis, hh grade tended to correlate with mca vs (p=. ), but again no association was observed between mca vs and adrenergic levels. no connection was seen between csf adrenergic levels and mca vs. our study is limited by small numbers, but our findings are consistent with the available literature whereby the association between the sympathetic nervous system and vs remains uncertain. marek mirski hospital of the university of pennsylvania, philadelphia, pa., united states, johns hopkins medical institutions, baltimore, md., united states dexmedetomidine is an alpha- adrenoreceptor agonist with sedative, analgesic and anxiolytic properties approved for the intubated adult patient in the icu setting. it possesses well described attributes for the neurological population; a rapid ability to sedate and awaken the patient allowing continuing neurologic assessment and no relevant respiratory depression. properties including neuroprotection, cardioprotection and renoprotection have been proposed and investigated in various settings. demonstrated clinical benefits in the icu neuroscience setting are just emerging. this synopsis reviews the literature in regards to clinical studies conducted to evaluate dexmedetomidine in the neurosciences. characteristics of the studies were categorized by study design, setting, patient population, and comparisons to other agents. human clinical studies were identified through a search of pubmed from - . key words include dexmedetomidine, nicu, neurocritical care and cea. study designs include randomized, observational, retrospective and case series. twenty-seven studies were included in the final analysis. the majority are case-studies or anecdotal and the literature consists of mostly surgical patients vs. the icu population. the leading hypothesis is that dexmedetomidine is safe and efficacious in the neurosurgical population and may provide neuroprotection. consistent findings are the attenuation of hemodynamic and endocrine response, smoother extubation and facilitation of neurological assessment. dexmedetomidine has gained popularity in applications beyond its labeled indication and dosage, in various icu's, and in special populations. the literature points to gained acceptance and favorable conditions for sedation without toxicity on cns parameters and a rapidity of onset and offset. there is brevity of literature which demonstrates positive outcomes in the neuroscience setting and the primary data does not represent level or evidence. more studies should be done to validate this drug for common use as there appears to be great advantages in the neuroscience population. the hijdra scale was developed to quantify the volume of blood following aneurysmal subarachnoid hemorrhage (asah). we investigated the relationship of hemorrhage quantity utilizing the hijdra scale and aneurysm size among patients with asah. we prospectively followed up a cohort of sah patients annually to document outcome events after obtaining informed consent. we abstracted demographic, clinical, and past medical history data by chart review on a subset of patients with documented asah after excluding those with cryptogenic, traumatic, and non-aneurysmal sah. the primary outcome of interest, hemorrhage quantity, was analyzed as both an ordinal measure (small, moderate, large) using tertiles and a dichotomous measure using the median. proportional odds logistical models for ordinal response measures and simple logistical regression for dichotomous responses were constructed to investigate the relationship between hemorrhage quantity and aneurysm size. from / to / a total of patients were enrolled in the rush university sah database. of these, we identified patients with documented asah; % were female; % white, % black, % hispanic; and the mean age was ± years. the mean ruptured aneurysm size was . mm and the median hijdra score was (range - ). we found no relationship between ordinal (p= . ) and dichotomous (p= . ) hemorrhage quantity and aneurysm size. no relationships were found between hemorrhage quantity and age, sex, race, apache ii score, and history of anti-platelet use. there was a trend for significance among patients with a past medical history of hypertension and having large hemorrhage quantity (or . , . - . ; p= . ). we found no relationship between aneurysm size and quantity of hemorrhage among patients with asah. future studies should focus on clinical variables such as hypertension and their role in hemorrhage quantity. stroke is the third leading cause of death in the united states. among the stroke subtypes intracerebral hemorrhage (ich), subarachnoid hemorrhage (sah), and ischemic stroke (is), ich and sah are associated with the highest mortality, followed by is. most deaths due to stroke occur within the first days, though it is unclear if any specific stroke subtype carries a significantly higher risk of early mortality (within the first hours from presentation) when compared to the other subtypes. with irb approval, we retrospectively reviewed stroke patients transferred to our institution between november and april who died during hospitalization. we collected data including primary diagnosis, confirmed by ct or mri, and time from presentation to our institution to death from any cause. among the in-hospital stroke deaths, ich was the diagnosis in ( %), sah in ( %), and is in ( % amongst in-hospital stroke deaths, ich was the stroke subtype associated with the highest likelihood of early mortality. this may indicate the severity of the disease process and a lack of effective early therapeutic measures available for ich. cerebral vasospasm is a common complication of sah and remains a major cause of death and disability after aneurysm rupture. the enos promoter (- t>c) cc genotype has been associated with a three-fold increased risk of angiographic cerebral vasospasm, however, its effect on adverse neurologic outcomes after sah has yet to be determined. we hypothesize that enos genotype would predict worse outcome, likely through its effect on risk of vasospasm. subjects included patients with confirmed aneurysmal sah enrolled in a longitudinal cohort study. we analyzed data from subjects for whom we had genotype information as well as -month follow-up assessment. patients who died prior to follow-up were excluded. univariate analyses used chi-square, wilcoxon ranksum or students t-test for the individual predictor variables. modified rankin scale score (mrs) was our primary outcome. logistic regression analysis for poor outcomes (mrs > ) included genotype and adjusted for age, sex, race/ethnicity, and hunt-hess grade. of the subjects, % were dependent or severely disabled (mrs > ) at months. older age, higher hunt hess grade and presence of vasospasm were associated with poor outcomes. the enos (- t>c) cc genotype was associated with mrs> with an adjusted or of . ( %ci . - . ). our results support a trend between enos (- t>c) cc genotype and -month poor functional outcome (mrs> ). although these results are not as robust as the association with angiographic cerebral vasospasm, it demonstrates the ability to integrate genetic information with clinical outcomes. limitations are primarily the small sample size and ability to adequately adjust for all clinical factors that could influence outcome. whether the effect on outcome of enos genotype is related to cerebral vasospasm risk will require further study. therapeutic hypothermia has been utilized in various brain injury models, including aneurysmal subarachnoid hemorrhage (sah). hypothermia has been used to treat refractory cerebral edema or severe vasospasm in this setting. however, there is very little data on hypothermia as a prophylactic measure before potential complications of sah have occurred. we evaluated the safety and feasibility of prophylactic hypothermia in patients with aneurysmal sah. we conducted a retrospective chart review of patients admitted with aneurysmal sah at a tertiary stroke center from july , to june , , who were also treated with induced hypothermia. only patients who had hypothermia initiated prior to symptomatic vasospasm onset were included. a total of out of patients were treated prophylactically with mild hypothermia ( - degrees celsius). three patients presented with hunt and hess grade i-iii, and seven patients with grade iv-v sah. average time at initiation of hypothermia was on sah day (range day - ). six ( %) patients underwent treatment of aneurysm by endovascular coiling or surgical clipping within hours of symptom onset. the average duration of hypothermia was days (range - days). nine ( %) patients developed evidence of vasospasm on computed tomography angiography or transcranial doppler. five ( %) patients survived to discharge. causes of death included irreversible global hypoxia from cardiac arrest ( ), severe refractory cerebral vasospasm ( ), and malignant mca infarction ( ) . all four patients with grade v sah died. when these patients were excluded, of the remaining patients, ( %) survived to discharge, and ( %) died. prophylactic hypothermia may be effective and safe in selected patients with aneurysmal sah. additional studies are needed to further define timing and parameters for therapeutic hypothermia in this setting. melatonin and pinoline are indolamines which have shown an antioxidative direct and indirect protection effect in vitro and in vivo models. fourteen -week-old male, c b mice underwent reversible middle cerebral artery occlusion ischemia ( . hours) followed by hr of reperfusion. the animals received pinoline ( mg/kg i.p.; n= ), melatonin ( mg/kg i.p.; n= ) or vehicle (n = ) at ischemia, immediately upon reperfusion, and at and . hr post-ischemia. another three animals in each group received the same doses but were sacrificed at . hours and used for protein oxidation quantification by western blot. rectal temperature, surgical time, time to ischemia and time to reperfusion were recorded continuously. initial neurological damage by modified stroke score was grossly assessed at ischemia, reperfusion, and at hr. infarction volume was quantified using , , -triphenyltetrazolium chloride (ttc) staining, digital photography, and imaging analysis software. means (± sd) were calculated and compared using student's t-test or anova. p . was set as statistically significant. total hemispheric infarction volume was reduced in the pinoline and melatonin-treated mice compared with the nontreated group ( ± % vs. ± %; p < . ) and ( ± % vs. ± %; p < . ) respectively. pinoline score was . vs. . in the control group at h. no statistical difference was observed in the melatonin group. optical net intensity ratio was statistically significance at cortical level on the kd band in the melatonin and kd on the pinoline groups. pinoline and melatonin treatment appeared to confer neuroprotection on a cerebral ischemia in vivo model. although its anti-ischemic mechanism needs to be elucidated, both molecules are potent free radical scavenging properties may offer a potential therapy. manisha gupte, jay joshi, sayona john, shyam prabhakaran, vivien lee rush university medical center, chicago, il, united states the "weekend effect" phenomenon suggests that admission day of the week is an independent predictor of mortality. we evaluated the effect of weekend admission on sah in-hospital mortality at a single academic center. with irb approval, we retrospectively reviewed consecutive sah patients admitted to our institution from august , to june , . weekend was defined as saturday or sunday. data was collected on day of the week admission, in-hospital mortality, aneurysm type and treatment. ct images were reviewed by the study neurologist and scored for fisher grade. of sah patients admitted to our institution, ( %) were female. the mean age was . years (range, to ). ct brain fisher score was as follows: fisher ( %), fisher ( %) and fisher ( %). the cerebral aneurysm distribution was acom ( %), pcom ( %), mca ( %), multiple ( %), and angiogram negative ( %). surgical clipping was performed in ( %) and endovascular treatment was performed in ( %). the overall sah in-hospital mortality rate was %. weekday admission accounted for %, and weekend admission occurred in %. age, fisher grade, and treatment modality were not significantly different between weekday versus weekend admission. the mean time from admission to treatment of aneurysm was . days and did not differ significantly by weekend versus weekday admission (p . ). mortality rate was % for sah patients admitted on a weekend versus % for sah patients admitted on a weekday (p . ). the weekend effect does not appear to be a significant factor in mortality outcomes of sah patients. the time to definitive aneurysm treatment does not appear to be impacted by weekend admission. the ich score is a simple clinical-radiographic scale in patients with intracerebral hemorrhage (ich) that helps estimate -day mortality. we hypothesize that the ich score can be applied to patients with warfarin ich (w-ich) to help estimate thirty-day mortality. anemia is a highly prevalent condition among hospitalized patients. we hypothesize that patients with acute cerebrovascular disease and anemia on admission have poor prognosis in terms of death, length of stay and disposition. a retrospective analysis of patients admitted to our institution with acute stroke (ischemic, hemorrhagic, subarachnoid hemorrhage) between october and march was performed. they were dichotomized based on hematocrit levels of < , >/= for women and < and >/= for men using the who definition of anemia. covariates used include diagnosis, demographic information and past medical history. the best admission hematocrit cutoff points for distinguishing between those with increased risk of death, disposition to snf (skilled nursing facility), increased los (length of stay) were identified. of the patients, ( . %) were female with a mean age of . years. of these patients ( . %) were anemic. ten patients died and nine were dispositioned to snf. while the relationship between disposition and anemic status was not significant (p= . ), there was evidence that those who died were more likely to be anemic (p= . ). los did not differ statistically between anemic and those without anemia. none of the variables were statistically significant on univariable analysis for mortality. anemia on admission did not predict death, disposition to snf or los, but there was a tendency that patients who died were more likely to be anemic. the admission hematocrit cutoff point for distinguishing risk of death, disposition to snf was slightly lower and increased los was higher than the who definition of anemia. abciximab, a glycoprotein iib/iiia receptor inhibitor (gpiib/iiia), is used during neuroendovascular procedures both to prevent and treat ischemic sequelae. experience with abciximab in this setting is limited and major bleeding complications, including fatal intracranial hemorrhage (ich), are of particular concern. we report our multicenter experience with ich following administration of abciximab during neuroendovascular procedures. we identified neuroendovascular procedures in which abciximab was used at three academic institutions from november through april . cases of periprocedural ich were identified and pertinent demographic, historical, procedural, laboratory, and radiographic data were collected. clinical outcome was measured by the glasgow outcome scale (gos) either at death or discharge. abciximab was used in neuroendovascular procedures; ich cases ( . %) were identified. procedures performed and indications for abciximab use varied. route of abciximab administration included iv bolus only (n= ), ia bolus and iv infusion (n= ), iv bolus and iv infusion (n= ), and iv infusion without preceding bolus (n= ). all patients but one received periprocedural antiplatelet, anticoagulant, or thrombolytic agents. all ich were detected within hours of abciximab administration, (except patient ; hours); were detected within hours. ich patterns varied and included subarachnoid hemorrhage (sah) with intraventricular hemorrhage (ivh) (n= ); intraparenchymal hemorrhage (iph) with ivh (n= ); sah, ivh, and iph (n= ); and a combination of sah, ivh, iph, and subdural hemorrhage (n= ). four patients died following ich (i.e. gos score of ); gos scores at discharge for the remaining cases were (n= ), and (n= ). ich was common ( . %) after neuroendovascular procedures using abciximab and was associated with a % mortality. future management strategies should focus on earlier recognition of gpiib/iiia-related-ich; development of direct gpiib/iiia antidotes; comparisons with shorter-half-life gpiib/iiia drugs; and identification of optimal abciximab dose and route. cerox is a novel noninvasive brain and tissue oxygen saturation monitor based on nirs and ultrasound technology. the purpose of this prospective observational study of patients with both traumatic and non-traumatic brain injuries is to determine if the cerox correlates with existing measures of cerebral oxygen metabolism which are currently used as part of regular care in the management of patients with severe brain injury. we enrolled patients with severe brain injury (tbi = , ich = ) who had at least one invasive cerebral oxygen monitor in addition to an intra-cranial pressure monitor. cerox adhesive patches were placed bilaterally over the frontal regions of the scalp and optical probes were attached to the patch clips. monitoring with cerox continued for up to days. high density physiological data, e.g., map, brain tissue oxygen, jugular venous saturation, icp, were collected at q minute intervals into our neurocritical care database. physiological data were then merged with cerox measurements. ten patients requiring invasive neuromonitoring were enrolled during this -month study period. the duration of noninvasive recording was - days (mean= days) with maximum length of uninterrupted recording being -hours. cerox measurements ranged from - . % (mean = %) on the left and - % (mean = . %) on the right. in this group of patients, the brain tissue oxygen tension ranged from . - . mm hg, the jugular venous saturation was . - % and the cerebral blood flow varied from . - ml/ gm/min. continuous monitoring with cerox is safe and feasible in neurocritical care setting. it has the potential of providing information about cerebral metabolism needed for close monitoring and management of patients with severe brain injury deep vein thrombosis (dvt) is a common complication of intracerebral hemorrhage (ich) and has been associated with immobility in the lower extremities. [ ] atherosclerotic risk factors (hypertension, diabetes mellitus (dm) and hypercholesterolemia) are associated with arterial thrombosis and have been postulated play a role in venous thrombosis. [ ] we hypothesized that a history of atherosclerotic risk factors increases the risk of dvt in ich patients. retrospective analysis of patients diagnosed with spontaneous ich at our institution between january and december was performed. demographics, history of hypertension, dm or hypercholesterolemia; systolic blood pressure at presentation; presence of immobility or hemiparesis and diagnosis of dvt were collected. logistic regression analysis was used to predict the risk of dvt. of patients with spontaneous ich were immobile and were selected for analysis. all patients had sequential compression devices applied on admission. the overall incidence of dvt diagnosed by lower extremity doppler was % and pulmonary embolism was . %. mean time to diagnosis of dvt was . (sd . ) days. after stepwise logistic regression analyses, significant predictors of dvt in immobilized ich patients were, history of hypercholesterolemia (or . p= . ) and sbp on admission > (or . p= . ). immobilized ich patients with a history of hypercholesterolemia were three times more likely to develop dvt. a sbp > on admission was five times more likely to predict dvt. thus atherosclerotic risk factors may play a role in the pathophysiology of dvt in immobilized ich patients, suggesting a possible etiopathologic link between arterial and venous thrombosis. acute ischemic stroke due to the occlusion of the internal carotid artery (ica) is associated with malignant stroke and poor outcome. without revascularization of ica perfusion to the middle cerebral artery (mca) and anterior cerebral artery (aca) is not possible. objective: objective of our study is to evaluate the technical feasibility of emergent carotid artery revascularization using stent and to evaluate the impact of stenting in distal cerebral perfusion. from an established stroke database consecutive patients with acute ischemic stroke who underwent emergent carotid stenting and thrombolysis/clot retrieval of the mca and aca from july to december were enrolled. patients' demographics including presenting national institute of health stroke scale (nihss), degree of revascularization, hemorrhagic conversion and days outcome data using glasgow outcome scale (gos) were collected. successful ica stenting was possible in / ( %) patients. the average age of patients was years (ranges - ) and average nihss was (ranges - ). carotid stenting facilitated successful revascularization of the mca and aca using tpa and merci clot retriever device in patients ( . %), ( %) of which has achieved complete recanalization in the mca and aca. in complete recanalization group a point or higher nihss improvement was observed in / ( . %) patients. symptomatic intracranial hemorrhage was observed in . % patients. seven of patients who achieved complete recanalization had a good outcome. five of patients who did not achieve complete recanalazation of the mca and aca died and had nihss . nihss was associated with incomplete recanalization of the mca and aca with poor outcome. emergent carotid revascularization is not only technically feasible in patients with acute ischemic stroke due to the carotid occlusion, but it also facilitates successful renalization of the mca and aca. further study is necessary. stent-assisted coiling of wide neck intracranial aneurysm requires therapeutic dose of antiplatelets to prevent stent thrombosis. stent-assisted coiling of the ruptured intracranial aneurysms also requires a loading of both loading dose of aspirin and plavix. objective: to report any potential complication associated with the use of both aspirin and plavix in stent-assisted coiling of ruptured wide neck intracranial aneurysm. consecutive patients who underwent stent-assisted coiling for ruptured wide neck intracranial aneurysm were enrolled from to . patient's demographics including the hunt & hess grade, fished scale, use of ventriculostomy catheter, location and size of aneurysm were collected. any complication such rupture of aneurysm, ventriculostomy associated hemorrhage or systemic bleeding was recorded. additionally a days outcome measurement was obtained using glasgow outcome scale (gos). results: patients with mean age of ± underwent stent-assisted coiling. a loading dose of plavix ( mg to mg) and aspirin mg were given prior to stent placement. patients received ventriculostomy catheter, cases before and cases after the procedure. there was no intraoperative ruptured of aneurysm or hemorrhage related to ventriculostomy or systemic hemorrhagic event. there were two episodes of stent thrombosis; one was an asymptomatic which developed during stent-assisted coiling procedure and resolved spontaneously, the other was symptomatic required intra-arterial administration of thrombolytic. there was no mortality and good outcome was observed in % of patient. stent-assisted coiling of the ruptured wide neck intracranial aneurysm using therapeutic dose of aspirin and plavix is not associated with increased bleeding complication such as rupture of aneurysm or intracranial hemorrhage related to ventriculostomy. however, the thromboembolic events remain the main challenge in stent-assisted coiling of ruptured intracranial wide neck aneurysm. therefore, antiplatelets should not be withheld prior to a stent-assisted coiling of ruptured wide neck aneurysm. we have used mild therapeutic hypothermia in patients with severe traumatic brain injury. in this study we investigated the effects of hypothermia on brain tissue oxygenation. brain tissue oxygen tension (pbto ) in addition to intracranial pressure (icp), cerebral perfusion pressure (cpp), and jugular venous saturation (sjo ) were monitored in consecutive patients with a glasgow coma scale score of to (ages to years). patients were cooled to a target temperature of . o c. patients with good recovery and moderate disability on the glasgow outcome scale were regarded as having favorable outcomes. a retrospective review of a six-month period in a university nccu was performed where patients were treated according to the above hypothesis. anticoagulation was usually started with heparin units sq q hr within the first hours and increased to units sq q hr after hours. anticoagulation was increased in many cases to enoxaparin mg sq q hr after another hours. of the patients who received care during the six-month period, patients ( . %) were diagnosed with lower extremity dvt that were asymptomatic in % of the cases. ivcf's were placed in patients ( % of those with dvt). two patients were diagnosed with pulmonary emboli (. %). there were no fatal pulmonary emboli. there were no significant bleeding complications or ivcf complications. surveillance lower extremity venous dopplers every - days, scd's, cs's, and escalating doses of anticoagulation as is tolerated and safe lower the risk of dvt in this high risk population and identify early asymptomatic dvt. fatal pe can be prevented with ivcf placement and more aggressive anticoagulation as permitted by the diminishing risk of bleeding as time passes from the acute injury. heparin induced thrombocytopenia (hit) is a common yet under-recognized condition in the neuro icu. it is caused by an autoimmune reaction to heparin-platelet factor (pf ) complexes which causes activation of platelets and leads to thrombosis. patients with aneurysmal subarachnoid hemorrhage treated by endovascular means are exposed to large doses of unfractionated heparin and therefore may be at high risk for hit. the medical records of consecutive patients with aneurysmal sah were reviewed. diagnosis of hit was made by clinical determination. clinically diagnosed hit is common in the sah population. patients with hit are at higher risk for cerebral infarction, in-hospital mortality and disability. a high suspicion for hit is appropriate in patients with aneurysmal sah treated by endovascular means. mild hypothermia ( - °c) has been investigated in a variety of neurologic diseases and disorders. since the s research has shown that hypothermia provides vital neuroprotection after sustaining brain/spine injury from a trauma, stroke, or cardiac arrest. hypothermia reduces increased icp and improves neurologic outcomes. [ ] [ ] [ ] [ ] translation of the research to clinical practice poses many challenges such as determination of the most effective method of cooling, maintaining hypothermia, and slowly re-warming back to normothermia. a neuro hypothermia protocol was instituted in march . patients underwent mild hypothermia using a hydrogel-pad cooling system. this retrospective study analyzed the data related to induction start times and associated variables (bmi and bsa) and sought to determine whether any correlation existed between the variables and degree/hour induction to goal temperature - + . °c. additionally, data was collected related to hours at °c and assessment of device control of ascent rate to °c. using the pearson correlation coefficient and the bonferroni standard correction method, patient charts were reviewed and data assessed to determine the statistical relevance of several variables: gender-males, age , bmi . , bsa . , induction start temperature . , target temperature . , hour to target temperature . , and temperature descent of . degrees/hour. it was determined that there was a significant statistical association between temperature changes (degrees/hour) and bmi/bsa values. the p-values for the bmi was determined to be . and the bsa . . target temperatures were maintained at °c with minimal variances. the ascent to °c was controlled at . °c/hour for the brain injured patients and . °c/hour for the spinal cord injured patients. the final analysis of the data revealed that an individual's bmi and bsa does directly affect both the induction of hypothermia and the controlled re-warming back to the targeted normothermic goal. mechanical ventilation is associated with worse outcome after intracerebral (ich) and subarachnoid hemorrhage (sah). we sought to examine the predictors of duration of mechanical ventilation. we prospectively identified patients with spontaneous ich and sah who required invasive mechanical ventilation. ventilator settings and measurements were recorded daily from the initiation of ventilation. complications were prospectively recorded. data are presented as mean +/-sd or n(%) when appropriate. variables for multiple linear regression were chosen with a stepwise algorithm (in order of decreasing significance). patients with aneurysmal sah of all clinical grades were prospectively studied. regional anterior alpha power was quantitatively analysed. we assessed alpha power and variability using the product of standard deviation and mean power over a -hour duration, repeated along a window sliding by minutes and graphically displayed. an independent clinician predicted the status of patients as improvement, deterioration or no change from the previous day. this was first done using only clinical data. ceeg trends prior to that day were then presented and another prediction made. results were compared with the true clinical states that were determined independently. clinical evolution in patients who were treated for vasospasm was correlated with daily mean alpha power. coiling followed by clot evacuation is associated with a faster time to aneurysm protection and similar outcome, los, and cost as clipping and evacuation. this may be a viable alternative treatment strategy. using cdsa, icu nurses were the most sensitive at identifying seizures, however they also demonstrated the highest false-positive rate. neurophysiologists and eeg technologists demonstrated slightly lower sensitivity, and much lower false-positive rates. however, on individual eeg recordings performance varied greatly, with group median sensitivities ranging from at % to %. neurophysiologists, eeg technologists and bedside nurses demonstrated comparable performance in seizure identification using cdsa. the observed differences in sensitivity and false-positive rates between different groups of reviewers are smaller than the variability in their performance on individual eeg recordings. coagulation disorders are common after traumatic brain injury (tbi), and may contribute to morbidity and mortality ( ) . these disorders are complex and dynamic over time, making clinical evaluation of coagulation status of the patients difficult ( ) . thromboelastography (teg) has been suggested as a tool for rapid assessment of such states. teg is a test of clot formation and lysis, providing a holistic assessment of clot formation time and strength. it is an easy to perform, point of care test that enables clinicians to differentiate hypo or hypercoagulability, and the factors contributing to each, and evolution over time. the aim of this work is to show the contribution of teg to the evaluation of coagulopathies in patients with isolated tbi. we have retrospectively inspected teg records and routine coagulation studies from patients with isolated tbi, and checked for signs of either bleeding tendency or signs of hypercoaguilation ten patients with isolated traumatic brain injury were evaluated using teg. reasons for tests included workup of suspected bleeding, assessment of hypercoagulable states, or planned invasive procedures. three of these patients showed increased ly , indicating thrombolysis, and two patient showed prolongation of the r value, indicating prolonged clotting time. two patients showed increased maximal amplitude (ma), indicating a hypercoagulable state. thromboelastography is a useful adjunct tool in the assessment of coagulation status in isolated tbi patients, and may help in clinical decision making in such patients. further work, relating thromboelastography results, prognosis and management are warranted. christine hartney, kathryn keim, diane sowa, richard temes rush university medical center, chicago, il, united states the objective of this study was to compare differences in resting energy expenditure (ree) results of critically ill neurology patients based on gender, body mass index (bmi) class and race. this study was a retrospective chart review of patients admitted to the neurosciences intensive care unit at an urban medical center who were started on enteral nutrition support. the research methods received approval from the institutional review board for human studies. the differences between gender, bmi class and race may not have been detected as a limitation of the sample size. research is needed to further explore the relationship among gender, bmi class and race and use of established predictive equations for the critically ill neurology patient. many critically ill neurologic and neurosurgical patients undergo a significant change in functional status or require end-of-life care. therefore, palliative care is an integral part of care provided by neurocritical care physicians and midlevel providers. at times, the needs of the patients and families can overwhelm these clinicians, whose focus is often on curative measure, so there may be a benefit to integrating a formal palliative care consultation service into the neuroicu. an anonymous survey was conducted among the four neuro icu physicians and nine nurse practitioners regarding the integration of a palliative care consult service into the neurocritical care service. the survey consisted of seven "yes" or "no" questions and a write-in section for comments. all providers thought that it was helpful to have the palliative care consult team in the icu, and that they provided added support not just for families, but to the physicians and nurse practitioners. / respondents stated that palliative care was only appropriate for families that wanted to decelerate care. only / responded that they were aware of the existence of formal criteria that were designed to trigger the consultation of the palliative care team. feelings were mixed regarding nurse-driven consults, with only respondents feeling that this was appropriate. in general, the formal palliative care consult service was felt to be a welcome addition. clearly, the existence of formal criteria to trigger a palliative care consult had not been emphasized enough to physician and midlevel providers, and the concept of nurse-driven consults was not accepted by the majority of number of providers. additional comments obtained will be used to improve the process by which palliative care services are obtained. most clinical trials in traumatic brain injury (tbi) have failed to demonstrate a therapeutic benefit. one factor implicated in these failures is an inadequate estimate of the smallest clinically meaningful beneficial effect -the minimal important difference (mid). in this study we surveyed the neurocritical care society (ncs) membership to determine an mid for tbi clinical trials. a survey approved by the ncs research committee was developed to assess the mid that would lead physicians to recommend a new therapy for tbi patients. the survey was distributed online to all ncs members with a -week response period. there were responses ( . %) from ncs members. respondents included neuro-intensivists ( . %), neurologists ( . %), and neurosurgeons ( . %); % were in academic practice on average . years. two-thirds ( . %) cared for to severe tbi patients monthly and . % had participated in tbi clinical trials. one third believed that % of patients would consent to minimal risk trials. the preferred primary outcome measures were mortality, glasgow outcome score (gos), and gos extended, while the sf- , neuropsychological measures and sliding dichotomy were the least preferred. the preferred secondary outcome measures were intracranial pressure (icp) control, therapeutic intensity level (for icp) and repeat imaging. organ dysfunction scores and biomarkers were least preferred. a reduction in unfavorable outcome of % (iqr - %) was reported as the mid needed to introduce a new therapy. mid rather than "number needed to treat" was the preferred method to describe trial efficacy. in this ncs survey, the preferred primary outcome measure for tbi trials was mortality or gos. a % reduction in unfavorable outcome is considered the mid. this information can be used to help define sample size for future tbi clinical trials. compare the quality and sensitivity of electroencephalography signals (eeg) obtained with a disposable template system to eeg obtained by certified eeg technicians. prospectively acquired eeg data were obtained in hour blocks (matched pairs) from leads placed by a certified eeg technician vs. those placed using a disposable template system (brainet®). quality measures included start and end recording of impedance, elapsed time from physician's order to first recorded eeg, and a blinded subjective evaluation of data quality. all segments of data were de-identified and will be read by a blinded reviewer highly experienced in eeg interpretation. analysis from the first subjects of a subject trial is presented here. average impedances in the brainet® group were within recommended guidelines, but were slightly higher than technician applied leads. groups had similar impedance variance, lead failure rates, and maximal difference in impedance at the beginning and at the end of the hour blocks. the difference in mean time to first eeg for the brainet® group ( mins) vs. technician applied leads ( mins) was statistically significant (p< . ). evaluation of sensitivity is pending collection of the remaining data sets. preliminary analysis indicates the use of a disposable template system that allows a non-technician healthcare provider to place eeg leads is feasible and safe. no significant differences in eeg quality during hours of recording were found, and use of brainnet® leads was associated with a significant reduction in the time from order to the first page of eeg data. this preliminary assessment does not allow for conclusions about the overall quality and sensitivity of disposable template leads; the complete set of eeg segments needs to be collected and undergo blinded review. outcome following aneurysmal subarachnoid hemorrhage (sah) is related to various demographic and clinical factors. biomarkers are an increasingly employed means for determining outcome in neurologically injured patients. the purpose of this study is to correlate cerebrospinal fluid (csf) adrenergic compound and metabolite levels to clinical and outcome measures. consecutive sah patients with ventriculostomy had csf collected ml within d of onset. csf was assayed for epinephrine (epi), norepinephrine (ne), and dihydroxyphenylglycol (dhpg) by hplc. levels were compared to various demographnic, clinical, and radiological measures, and to mortality at days. mean age was yo and % were female. hh grade was in %, in %, and in %. no correlation was found for age, but women had greater dhpg levels ( pg/dl vs pg/dl, p=. ). dichotomized hh score demonstrated greater epi levels in g / patients compared to g patients ( pg/dl vs pg/dl, p=. ). patients who died had also greater epi levels ( pg/dl vs pg/dl, p=. ) yet lower dhpg levels ( pg/dl vs pg/dl, p=. ), but regression analysis incorporating hh grade eliminated these associations. in sah, women demonstrate greater elevations in the ne metabolite dhpg, and greater elevations in epi are present in hh grade / patients. patients who die have greater csf epi levels which appears related to the severity of the disease. gail pyne-geithman, opeolu adeoye, jordan bonomo, carolyn koenig, jed hartings, lori shutter university of cincinnati, cincinnati, oh , united states as in clinical neurocritical care (ncc) practice, effective ncc basic science research requires organized interdisciplinary collaboration. the purpose of this abstract is to share our experience in building a basic science collaborative to facilitate the efforts of others and foster discussion regarding engagement of basic scientists in the neurocritical care society (ncs) and ncc research efforts. our institution is active in interdisciplinary ncc clinical practice and fellowship training, in addition to conducting various clinical trials relevant to ncc. collaborating with the clinicians is a core of basic scientists who are working to integrate their funded research into the fabric of care in the neurosciences icu. the composition of the team is truly interdisciplinary, spanning multiple clinical and basic science departments and colleges within our institution. frequent meetings among ncc physicians, surgeons, research nurses in the division of clinical trials and basic scientists have resulted in fruitful collaborations in teaching, research and funding. clinical responsibilities of fellows and residents limits time for bench research, so joining an existing project allows time and resources to be used productively. ncc fellows are teamed with a basic science mentor, and these collaborations often continue beyond the tenure of the fellowship. the basic scientists benefit, as current basic research needs to have translational potential to the clinical setting. basic scientists attend clinical rounds, reinforcing the benefits of truly translational research. gaining a reputation for quality research that enables consistent funding and earns respect from the ncc community requires engagement and input of basic scientists. individual institutions can solicit interested basic scientists to join in their research planning and execution and augment the training of residents and fellows, thus preparing the next generation of research-trained clinicians. the methodist hospital, houston, texas, united states the purpose of this study was to evaluate the association between tight glucose control and the incidence of ventriculitis in neuro intensive care unit with evds the hospital's computer system was used to identify patients admitted between january , and december , to the neuro icu with documented evd placement. patients' years of age or older and deemed to require insulin therapy by the admitting physician were included in the study. we excluded patients if they had evd placement or documented csf infections before admission to the unit, were treated with antibiotics a week prior to admission, and length of icu hospitalization less than seven days. the primary outcome measure was evd related infection. the secondary outcome measures were in hospital and icu length of stay and in hospital death the association between glucose control and positive csf cultures was described using the morning blood glucose for seven consecutive days stratified based on the number of blood glucose readings that fell between - mg/dl. the binary logistic regression model showed that patients with a higher percent of readings in tight blood glucose range were more likely to have cns infection (odds ratio . ; p <. ). the secondary outcomes could not be measured because we did not have enough readings to stratify our data into categories contrary to our hypothesis, the results from our study suggest a possible association between tight blood glucose and an increase in evd related infections. at this time we are unable to make recommendations based on these results given the inherent limitations of our study ; i.e., small sample size, retrospective design, single centered and single morning blood glucose reading to assess glucose control following aneurismal subarachnoid hemorrhage (sah) half the patients' die and only one third of survivors make a full recovery. the optimal hemoglobin (hgb) after sah, however, is uncertain. higher-goal hgb and more red blood cell transfusion (rbct) lead to worse outcome in general critical care. clinical series suggest that rbct may increase vasospasm risk and exacerbate outcome after sah. however other studies suggest that a higher hgb may be associated with better outcome and less cerebral infarction after sah. we now will examine the hypothesis that patient outcome after sah is better when an hgb level of . g/dl rather than an hgb of g/dl triggers transfusion. we propose a multi-center, prospective, phase iii randomized, clinical trial involving adults admitted to university based nicus within hours of sah.. eligible sah patients will be randomly assigned to one of two treatment groups, ) restrictive (hgb of . g/dl) or ) liberal (hgb of . g/dl) transfusion triggers stratified by center and sah severity. the primary objective is to determine if sah subjects who have a restrictive transfusion trigger during the first days of care, are more likely to have a favorable -month outcome than subjects transfused with a liberal trigger. the trial is designed to detect an overall absolute difference of % in the proportion of favorable outcomes (glasgow outcome score of good or moderate disability). secondary objectives include: ) determine if a restrictive policy is associated with less vasospasm. ) examine the relationship between hgb and month outcome and cerebral infarction. the number of randomized subjects is expected to be . the number of randomized subjects is expected to be . to date centers have agreed to participate. this group will receive administrative, statistical and data coordinating support from the university of pennsylvania center for clinical epidemiology and biostatistics. the proposal is under review at the nih. background: brain death (bd) is diagnosed clinically by documentation of coma, absence of brainstem reflexes, and apnea unresponsive to hypercarbia. in argentina (like other countries) other confirmatory tests are required as a part of the diagnostic criteria. the utility of cta with cerebral perfusion was reported by qureshi in and then evaluated by combes et al and they found % false negative rate for the test; moreover, greer et al reported one case of false positive. in spain, otero has reported a sensitivity of % in a series of patients who had cta and ct perfusion. accuracy of cta and ctp must be assessed. we propose that cta and ctp is a reliable confirmatory test for bd, with particular interest in cases where barbiturates or other cns depressant drugs difficult to diagnose clinically or by electrophysiological studies. prospective multicenter study to determine the accuracy diagnostic of brain death with cta & ctp in patients with suspicion of bd according clinical criteria (cc) defined by neurological criteria, apnea test; compared with electrophysiological methods and tcd evaluation. all adults of at least years of age who meet the cc of bd. intensive care unit, emergency department, neurocritical care unit, stroke unit in hospitals with availability of tcd, eeg and multi-row ct hours. in patients with cc of bd, we will be performing ctp and then cta. all the case will be made an eeg and tcd evaluation main outcome measure: evidence of cerebral circulatory arrest. absence of cerebral perfusion sensitivity and specificity, ppv and npv for cta & ctp compared with cc, eeg & tcd. accuracy of cta & ctp in patients with recent utilisation of cns depressor or nm blockers to confirm bd before eeg and tcd. complications rate associated with the use of contrast. renal failure post contrast use. we need at least patients to to achieve a sample size that allows the analysis of sensitivity, specificity and construction of roc. not started. funding is needed to support the project. quantitative diffusion-weighted imaging mri (dwi) in comatose post-cardiac arrest survivors holds promise as a prognostic tool. between and days more than % of brain volume with an adc value < x - mm /sec identifies poor outcome patients with % specificity and % sensitivity (ann neurol ; : - ). this threshold needs validation in an external dataset. we hypothesize that the capacity for recovery of consciousness in comatose cardiac arrest survivors can be predicted with quantitative dwi. multicenter observational study of dwi mris in comatose cardiac arrest survivors obtained between and days after the arrest. patient data will be recorded using a web-based data entry form including baseline characteristics, neurological examinations, results of neurophysiological testing, cause of death, and -day outcome. patients may be entered retrospectively and prospectively. brain dwi scans will be blindly analyzed centrally and an outcome measure (survival versus death or vegetative at days) will be assigned. patients who remain comatose after cardiac arrest and who have undergone dwi between and days after the arrest. main outcome measure: the specificity of the predefined dwi threshold (adc of x - mm /sec) for prediction of poor outcome (defined as death or failure to recover consciousness at days). the sensitivity of the predefined dwi threshold in comparison with the -hour neurological examination and, if available, sseps and peak serum levels of neuron specific enolase. assuming a % survival rate, and % specificity of dwi for poor outcome, patients are needed to achieve a % false positive rate for poor outcome with a % confidence interval of to %. centers are invited to participate. several investigators have expressed interest. background: there have been several randomised controlled clinical trials of weaning from mechanical ventilation which has shown quicker weaning and shorter ventilation time in abrupt discontinuation of mechanical support as opposed to gradual step-wise withdrawal. however, there have been no substantial trials of ventilatory weaning in acute brain injury patients or those with neuromuscular diseases. the neurocritically ill patient on mechanical ventilation will require slower step-wise weaning from mechanical ventilation. multicenter, randomized, non-blinded phase trial feasibility and safety trial. all patients in the neurocritical care unit expected to be on mechanical ventilation for more than days. routine scheduled post-operative patients and patients transferred from outside hospitals already on mechanical ventilation for more than hours will be excluded. neurocritical care unit. patients will be randomized to slow step-wise simv wean versus pressure support wean on cpap. length of time on mechanical ventialtion pneumonia, urgent re-intubation, hypoxia from previous studies in the medical intensice care units, patients, half with acute brain injury, half with neuromuscular diseases. despite the use of appropriate antimicrobial therapies, the morbidity and mortality associated with bacterial meningitis remains high. cerebrovascular complications from meningitis, including vasospasm, have been shown to contribute to this poor outcome. several series have reported tcd velocity elevation correlates with clinical decline and occurs in up to % of patients with bacterial meningitis. to date no systematic large trial has been completed to detect or treat this complication. . phase ii: clinically significant vasospasm in bacterial meningitis results in higher mortality compared with those with normal tcd velocities. . phase iii goal directed therapy: triple h, intra-arterial verapamil and angioplasty will increase survival in patients with bacterial meningitis at high risk for vasospasm. patients admitted to the icu with the diagnosis of bacterial meningitis. phaseii: bacterial meningitis enrolled within hours of diagnosis. subjects receive baseline cta of head/neck, daily tcds, angiography when mean velocities > , daily nihss, mortality rate at one month, and mrs at months. significant vasospasm will be defined as angiographic vasospasm with corresponding increased nihss of at least points. phaseiii: target population from phase ii. all subjects will undergo testing and data collection as outlined in phase ii. subjects randomized to aggressive vasospasm treatment vs. standard-care. in the treatment group, vasospasm will be treated with goal-directed therapy. one month mortality. independent predictors of the presence of vasospasm. rankin score at months. phase ii and iii: inference of portions (alpha . , beta . , delta . , n= subjects) current status: big idea. the outcome of devastating neurologic disease like massive ischemic stroke, intracranial hemorrhage, status epilepticus, and subarachnoid hemorrhage is presumed to be poor. mortality in studies may be influenced by premature withdrawal of care, and not by natural history or chronic complications. if these patients are given maximal supportive care chronically, their outcome will be better than expected based upon commonly accepted morbidity/mortality. randomized, controlled non-blinded clinical trial. patients with devastating neurologic conditions as listed above who require mechanical ventilation (mv) and feeding tube placement (tf). neurocritical care unit of major tertiary care centers as part of a multi-center trial. families are offered usual standard of care-either withdrawal of care or full supportive care. for those not certain about which course to take, enrolment is offered. the trial would necessarily require initial full supportive care such as tracheostomy and feeding tube placement. the patients are randomized to one of two treatment regimens: ) aggressive, long-term supportive care involving treatment of intercurrent medical complications and full resuscitation; ) basic supportive care including mv and tf but not involving these aggressive measures. modified rankin scale at one, two, and five years. continued need for mv/tf, barthel index, correlation with initial hospital care in a specialized neurocritical care unit. assuming a % event rate (severe disability or mortality) in the control group and expecting a % relative risk reduction (about % absolute risk reduction), the estimated sample size with an % power and an alpha of . is patients total ( in each arm). proposal status. how to structure randomization in concert with ethical principles and which supportive measures can be ethically restricted must be determined. intervention: continuous veno-venous hemofiltration via femoral access with total effluent rate of ml kg - h - , blood flow rate between - ml/min, high permeability glycerine free polyethersulfone membrane, filter change every hours, pre-filter replacement fluids: prismasate bgk / / . (osmo ) and sodium-citrate anticoagulant. fluid management per attending physician. minimum duration of hours with termination after - hours of icp control or at hours. secondary outcome measures: change in icp at hours and every hours thereafter; neurological outcome (glasgow outcome scale score dichotomized unfavorable ( , , ) / favorable ( , )); cytokine removal and complications. power analysis: a sample of size will be obtained to attain a power of . at % level of significance current status: idea phase renal replacement therapy in the patient with acute brain injury renal replacement therapy for the patient with acute traumatic brain injury and severe acute kidney injury early changes in intracranial pressure during haemofiltration treatment in patients with grade hepatic encephalopathy and acute oliguric renal failure continuous arteriovenous hemofiltration in patients with hepatic encephalopathy and renal failure cytokine removal during continuous hemofiltration in septic patients fluid thresholds and outcome from severe brain injury adult respiratory distress syndrome: a complication of induced hypertension after severe head injury prevention of secondary ischemic insults after severe head injury combination therapy with hypothermia for treatment of cerebral ischemia clinical study of mild hypothermia treatment for severe traumatic brain injury management of pitfalls for the successful clinical use of hypothermia treatment multicenter trial of early hypothermia in severe brain injury guidelines for the management of severe traumatic brain injury. rd edition none s neurocrit care early decompressive craniectomy for patients with severe traumatic brain injury and refractory intracranial hypertension--a pilot randomized trial renal replacement therapy for the patient with acute traumatic brain injury and severe acute kidney injury continuous renal replacement therapy for refractory intracranial hypertension introduction: many authors suggest using pentobarbital when elevated intracranial pressure or seizures are refractory to other agents. due to the lack of outcome data after the use of this agent, we investigated the outcomes of patients treated with pentobarbital over the past five years. patients were identified using a pharmacy database that tracks inpatient medication dispensing at our tertiary referral center. all patients, older than , cared for in adult icus, who received pentobarbital between and were included. inpatient mortality was compared between patients older and younger than as well as those who required vasopressors using fisher's exact test. twenty-two patients received pentobarbital. the mean patient age was (sd= ). just over half ( %) were men. pentobarbital was used in % of the patients to treat intracranial hypertension; the remainder were treated for refractory status epilepticus. the most frequent underlying disorders were toxic-metabolic disease processes.fourteen patients ( %) died in the hospital. care was withdrawn in %. of the patients ( %) who were alive at the time of discharge: ( %) were discharged to acute rehabilitation, ( %) to an extended care facility, ( %) to sub-acute rehabilitation, ( %) to hospice, and ( %) to home.hypotension, renal failure, and pneumonia were common ( - %) in patient receiving pentobarbital. there was no significant association between inpatient mortality and reason for pentobarbital use. age and need for vasopressors were not significantly associated with in hospital mortality. pentobarbital use was associated with significant morbidity and mortality (greater than %), but % of patients were discharged home or to acute rehabilitation facilities. further study is needed to better clarify the risks and benefits of pentobarbital to treat refractory intracranial hypertension and status epilepticus. the neurologic mechanism leading to unresponsiveness after acute traumatic brain injury is not well understood. posturing reflex examination in evaluating comatose patients is ubiquitous. the reliability of this practice has not been systematically evaluated. from the trauma service registry at a level trauma center, all admissions between from / / to / / where the patient had a head component of the abbreviated injury score > were identified. from this group of , patients, the records of the patients with a glasgow coma scale (gcs) on presentation and a brain ct scan performed in the ed were evaluated. ct scans were scored for injury by location and the motor component of the gcs (gcs m ) was noted from ed documentation. the study population was young (mean age . ) and predominantly male ( %). the gcs m was (indicating extensor posturing or no response) for patients and > (indicating flexor response or better) for patients. on univariate analysis, intra-axial injury above the thalamus did not correlate with the gcs m ( with gcs m , with gcs m > , p= . ). a second analysis of intra-axial injury above the midbrain again showed no reliable correlation with gcs m ( with gcs m , with gcs m > , p= . ). patients with extensor or worse exams were less likely to have a glasgow outcome score > ( ( %) with gcs m , ( %) with gcs m > , p= . ). gcs m responses of flexor or worse did not reliably correlate with injuries at the level of the thalamus or below. however, lower gcs m was still associated with poor gos. this study points to a need for reinvestigation into the neuroanaotmic basis for posturing and unresponsiveness to enhance the understanding and improve acute management of these patients. jeff chen, sandy cecil, patrick chen, susan rowland, sarah callaway, david adler legacy emanuel medical center, portland, oregon, united states since the cma cerebral microdialysis analyzer received fda approval for clinical use in in the united states, cerebral microdialysis has gained increasing acceptance as an adjunct in the multimodality monitoring of the brain after traumatic brain injury, subarachnoid hemorrhage, and stroke. we describe a single institutional four year experience with cma and recent iscus flex . the cma and iscus flex analyzers, cma pump, and cma microdialysis catheters were obtained from cma microdialysis (solna, sweden). perfusion fluid cns (artificial csf) was perfused at . ul/min, and samples collected hourly. lactate, pyruvate, glucose, glycerol, and glutamate levels were entered into the icu pilot program along with neurophysiologic parameters to analyze relationships/trends. all cerebral microdialysis catheters were implanted by board certified attending neurosurgeons at a single community-based hospital. catheters were implanted directly into the brain via a mm diameter corticectomy at the time of craniotomy. catheters were placed via twist drill hole/bolt when craniotomies were not performed. status epilepticus (se) affects , americans yearly. - % of cases fail initial therapy. refractory cases requiring midazolam, pentobarbital, or propofol fail in - % of patients. outcome is independent of the anaesthetic agent or extent of eeg suppression. hypothermia (ht) in rodent models of se abates epileptic discharges and neuronal death. case reports demonstrate ht as an effective adjunctive or primary treatment for refractory se (rse). ht effectively treats rse. multicenter, randomized, non-blinded phase iia trial of ht in rse evaluating target temperature and duration. inclusion criteria: rse patients failing initial benzodiazepines and phenytoin treatment, > yo, and > o c upon admission. exclusion criteria: immunosuppression, active infected, unstable cardiac rhythm, coagulopathy, se secondary to cardiac arrest/anoxia, active chf, pregnancy, in-place ivc filter, or dnr/dni status. eeg monitored patients with rse will be randomized. controls will be managed at the intensivist's discretion. remaining patients will be varied by ht duration ( hr v hr) and target temperature ( - o c v - o c). initially, seizure will be treated with midazolam while endovascular cooling catheters are placed, and a cold saline bolus is given. at goal temperature, midazolam will be weaned to off or the lowest dose necessary for absence of seizure activity. anaesthetic medication requirements and seizure burden (i.e. seizure frequency, duration, and number), from achieving goal temperature until icu discharge, as compared with control. total iv anaesthetic icu duration vasopressor/inotrope requirements modified rankin score at discharge infectious, device, and coagulopathic complications power analysis: > patients (> each arm) would be % powered to detect a % difference in ht vs control (alpha= . ), and a % difference of each arm vs control (alpha= . ), in the primary outcome measures of anaesthetic and seizure burden. a phase i study is underway at washington university and henry ford hospital. this study remains unfunded. traditional dogma has mandated that brain injured patients with a glasgow coma score of < need to remain intubated for airway protection. recent prospective studies have suggested that in brain injured patients with intact airway reflexes, prolonged intubation leads to an increase in nosocomial pneumonias and worse outcomes. a recent randomized trial suggested randomization of brain injured patients into early and delayed extubation is safe and feasible. brain injured patients with a gcs < and intact airway reflexes will not have worse outcomes if extubated early compared to a similar extubation group with delayed extubation until their gcs becomes > . multi-center non blinded randomized phase trial. a non inferiority trial of immediate versus delayed extubation accounting for a . change in modified rankin scores would require patients in each treatment arm for % power. immediate extubation in stable brain injured patients with intact airway reflexes as evaluated by an airway care score. hospital discharge modified rankin score. hospital and icu length of stay, nosocomial pneumonias, reintubations. all intubated adult patients with severe brain injury defined as a gcs< are potentially eligible. exclusion criteria includes: patients < , lack of surrogate informed consent, intubation for therapeutic interventions, anticipated medical or neurological worsening, intubation for airway edema, or prolonged intubation > weeks. a feasibility trial has been completed and published. the study is in search of centers and funding. outcome of patients with ich is dismal. the majority of patients succumb in the first hours to the effect of the hemorrhage causing tissue shifts and herniation. furthermore, many are comatose, ventilator-dependent because of alteration of consciousness brought upon by pressure on midline diencephalic structures. both early hemostatic therapy and surgical evacuation failed to improve outcome simple yet large decompression with durotomy in patients with large, unilateral hypertensive capsulo-ganglionic ich preceded by administration of rfviia for stabilization of clot and thereby prevention of hematoma growth upon decompression should result in less hematoma expansion (safe), reduce pressure on midline structure, and improve mentation and overall outcome study design: randomized, controlled but not blinded, feasibility and safety multi-center trial patients with spontaneous, hypertensive large ( ml) ich located in the putamen and internal capsule with evidence of mass effect on midline structure ( mm septum pellucidum shift), who are not moribund (decerebrate posturing, absent pupillary light and oculocephalic reflexes), and who present within hours from onset of bleed are eligible to be enrolled. patients should have no contraindication to receiving rfviia. patients are screened upon arrival to the er or nicu if transferred directly there intervention: mcg/kg rfviia is given prior to patients undergoing a large, fronto-temporo-parietal decompressive craniotomy with durotomy without clot evacuation. further care like blood pressure control and osmotic therapy will not be standardized. hematoma expansion, improvement in midline shift by % deterioration by points on nihss within first hours following decompression, good outcome at months (mrs ), icu and hospital los, days on mechanical ventilation with a beta level of . and alpha . , an assumed hematoma expansion rate of % in the non intervention group (fast trial placebo group hematoma growth rate) and % in the intervention group, patients are needed to be randomized not initiated yet cerebral vasospasm (cv) after aneurysmal subarachnoid hemorrhage (asah) remains a significant cause of morbidity. intravenous nicardipine has been previously studied clinically as a neuroprotectant, and shown to decrease the incidence of angiographic and symptomatic vasospasm in asah, and has the potential to avoid rescue intraarterial rescue therapy and the resultant complications. hypertensive hypervolemic vasodilatory hhvd for cv will result in a reduction in duration cv, fewer delayed ischemic neurologic deficits (dinds) and better functional outcome. randomized placebo controlled trial of hhvd versus hh therapy. patients with asah, ages - , without a history of coronary artery disease (cad), ischemic cardiomyopathy, neurodegenerative disorder, or chronic kidney disease not on hemodialysis. intervention: hhvd therapy using norepinephrine and continuous nicardipine infusion at mg/hr initiated at the onset of cerebral vasospasm, for a duration of days. intracranial hypertension (ih) is the most powerful predictor of poor outcome in severe tbi. indomethacyn (im) is a cox inhibitor with a potent vasoconstrictive effect in cerebral arterioles that has been used in tbi, avm's and intracranial neoplasm. there is a little of evidence that supports its utility in the treatment of ih with special emphasis in type a waves in patients with an impaired cerebral vasoreactivity, improving both cerebral perfusion and response to other second-tier therapeutic tools. however, large, prospective, randomised and controlled studies have not yet been performed to confirm its benefit in patients with tbi the im could be effective to treat refractory ih in severe tbi with impaired cerebral autoregulation and poor response to other therapeutic strategies. im can improve indices of cerebral haemodinamics and cerebral oxygenation decreasing neuronal ischemic damage. this therapeutic approach can reduce til to control intracranial pressure, also the length-of-stay in icu can be reduced too. im can improve long term functional outcome in severe tbi. multicenter, randomized controlled trial to evaluate the efficacy and safety of im in patients with severe tbi that presents intracranial hypertension which have reached a therapeutic intensity level that includes second-tier therapies (ie,deccompresive craniectomy, controlled hypothermia, etc) all patients (older than years old) that presents with glasgow coma scale or <, with icp above torr and with any evidence of hyperaemia (tcd, sjvo , avdo etc) or increased cbv (ct perfusion, etc) despite standard therapy (includes mechanical ventilation, evacuation of intracranial mass, profound sedation, osmotherapy, etc). intensive care unit or neurocritical care unit or neurotrauma unit at a hospital that has multimodal monitoring modality (icp, cpp, etco or pco , sjvo or ptio , tcd etc) and neuroimaging with evaluation of cbv (ct perfusion, etc) indomethacyn . - . mg/kg at loading dose, followed by continous infusion . - . mg/kg/hr or placebo in patients who develop high icp despite standard therapy for icp control. icp control (reduction of icp below torr or torr in dc), normalization of cerebral oxygenation (avdo , sjvo or ptio ). improvement of cerebral perfusion measured by cuantitative or cualitative methods neurological outcome (egos) at discharge, , , and days. overall mortality at month , and qol at month , and evidence of long-term ischemic damage we need a sample of at least patients to find statistically significative difference between intervention and placebo group. not started. financial supports is needed. hyperglycemia is very common in acute brain injury (abi) from ischemic stroke, hemorrhage or trauma and is associated with poor outcome. tight glucose control is effective in improving outcome in medical/surgical icu but its role in abi is uncertain. studies investigating brain metabolism using microdialysis showed increased brain metabolic crisis with tight glycemic control. currently the optimal glucose control for patients with acute brain injury is unclear. aggressive hyperglycemia management will result in improved outcome in abi compared with standard glucose management. multicenter, randomized, single-blinded phase ii feasibility and safety trial. inclusion criteria: patients admitted to icu for management of abi with high likelihood of requiring at least hours of intensive care. absence of health care proxy to sign consent. patient with do not resuscitate and do not intubate orders on admission history of allergy or known contraindication to insulin moderate to severe baseline disability (pre-abi modified rankin scale or greater) severe terminal concurrent medical illness with expected survival of less than three months. neurocritical care unit. treatment arm will receive continuous insulin infusion targeting blood sugar level - mg/dl. control group will receive subcutaneous insulin injection and/or insulin infusion targeting blood sugar level - mg/dl. modified rankin scale at months rate of medical complications, including infection, new neurologic abnormality, hypoglycaemia and in-hospital mortality.length of icu, hospital stay. based on our retrospective study with expected good clinical outcome of % and % relative difference in outcome between groups, the study will need patients in each arm with % power and % two-sided alpha level. protocol complete. for submission for funding. rebleeding on the first day following asah is as high as - %, and approximately half of these occur within hrs of onset. unanticipated delays in asah diagnosis and result in failure to secure aneurysms during the period of maximal rebleeding. a novel approach of acute antifibrinolysis (< h duration) has demonstrated safety, however, there have been no trials powered to demonstrate a difference in long-term outcome. therefore, a clinical trial evaluating the impact of -aminocaproic acid (eaca) on outcome following asah is warranted. acute treatment with intravenous eaca will improve twelve-month outcome in patients with asah. neurological emergency treatment trials (nett)-based multi-center, randomized, double-blind, placebo-controlled phase-iii trial. all adult asah patients presenting to nett facilities will be screened for enrollment. patients must receive study drug within h of asah onset. those with aneurysm-negative sah, anticipated treatment within h, or recent thromboembolic disorder will be excluded. subjects will be enrolled and treatment initiated in the emergency department and continued during transfer and the referral-center intensive care unit. patients will receive an intravenous placebo or eaca. a g loading dose, will be followed by infusion of g/hr, to a maximum h. favorable -month modified rankin score( - ) the barthel and lawton scales(disability scales), sip(quality-of-life scale), and a psychometric battery(cognitive/intellectual domains) will be assessed as secondary outcomes. known sequelae of asah and antifibrinolytic therapy including rebleeding, vasospasm, hydrocephalus, and thrombotic complications will be tracked. based on a analysis with = . and a power of %, subjects will be randomized. this calculation is based on conservative estimates from past studies that demonstrate % increase in favorable outcome for patients receiving acute eaca. based on a analysis with = . and a power of %, subjects will be randomized. this calculation is based on conservative estimates from past studies that demonstrate % increase in favorable outcome for patients receiving acute eaca. additionally, there are also associated nonlinear hospital system factors, hi, probably accounting for positive preclinical and single center studies, followed by multicenter failure. the severity regression equation can now be described asthis leads to the notion that the current widely accepted methods of evaluating single facet therapy to attenuate such multifaceted complex problems is generally a fruitless waste of public resources which has produced innovation paralysis.preclinical studies have demonstrated the potential for dramatic breakthrough level neuroprotection with a multifaceted approach but a rational systematic method for introduction of multifaceted therapeutic bundles is needed. multifaceted neuroprotective bundles can be used to demonstrate robust neuroprotection. can the plan-do-study-act (pdsa) qi method be used to incrementally add and evaluate individual facets of neuroprotective therapeutic bundles? single center pdsa therapeutic bundle development followed by multicenter randomized trial of a therapeutic bundle. patients with acute tbi or brain ischemia syndromes setting: ed, or, and icu multi mechanism multifaceted therapy incrementally and sequentially implemented during active post insult secondary pathophysiologic processes. surrogates for functional outcome with sequentially added facets in a therapeutic bundle ongoing evaluation of functional neurologic outcome. none yet background: refractory intracranial hypertension (rih) is associated with death or poor neurological outcome in - % of patients and clinical equipoise often exists among management [ ] . for patients with cerebral edema or intracranial hypertension who require renal replacement therapy, continuous (crrt) modes are preferred due to limited data showing improved intracranial stability over traditional intermittent hemodialysis (ihd). this is attributed to better cardiovascular stability, less rapid fluid shifts, bicarbonate and osmolality changes as well as more biocompatible, and highly permeable membranes [ ] . anecdotal reports have suggested improvement in intracranial pressure (icp) [ and verbal] during crrt and we have observed this in patients (table , figure - patients with persistent disorders of consciousness, defined as the absence of response to simple orders, days after the event unexplained by sedation. signed informed consent. inclusion of tbi and non tbi comatose patients (ischemia, sah, hematoma and cerebral anoxia). neuroicu. mri under mechanical ventilation. inclusions during years. follow up at months and one year by phone interview. electronic crf. multimodal mri with mrs (pons and csi) and dti under mechanical ventilation. controls per center. predictability of dichotomized gos at year using a composite index combining clinical data and quantified indicators from mrs (naa/cr in specific brain regions) and dti. design of specific algorithms according to the etiology of coma. with subjects, % of power to detect a variable with an or by standard deviation of . ( = %, bilateral test, proportion of patients with poor outcome = %, nquery advisor® . ). % of lost to follow-up within year and % of drop-out. total tbi to be included = . same reasoning for non tbi patients. founded in france €. european actively including patients following a similar protocol. patients already included (tbi patients, anoxia , intracerebral hematoma , sah , arterial ischemia ). mortality rate at one year %.financial support: none key: cord- -evnfvc l authors: nemunaitis, john; stanbery, laura; senzer, neil title: severe acute respiratory syndrome coronavirus- (sars-cov- ) infection: let the virus be its own demise date: - - journal: nan doi: . /fvl- - sha: doc_id: cord_uid: evnfvc l there has been a collaborative global effort to construct novel therapeutic and prophylactic approaches to sars-cov- management. although vaccine development is crucial, acute management of newly infected patients, especially those with severe acute respiratory distress syndrome, is a priority. herein we describe the rationale and potential of repurposing a dual plasmid, vigil (pbi-shrna(furin)-gm-csf), now in phase iii cancer trials, for the treatment of and, in certain circumstances, enhancement of the immune response to sars-cov- . sars-cov- dual therapeutic review cleavage at viral entry as compared with sars-cov [ , ] . following the attachment of the receptor-binding domain (s ) to the ace -binding cellular site, the affinity of which is -to -fold higher than sars-cov [ ] , the s subunit is shed resulting in a stable and accessible fusion domain (s ) subunit [ ] . sars-cov- utilizes the plasma membrane fusion pathway rather than the more immunogenic endosomal membrane fusion pathway, which is used by sars-cov. amino acid sequence differences in the sars-cov- hr region enhances binding affinity between heptad repeat- (hr ) and hr thereby accelerating viral membrane fusion [ ] . the presence of a unique furin cleavage site (rrar) at the s /s boundary and the furin-like s site located between fusion peptide (fp) and internal fusion peptide (ifp) sites on the s subunit may provide a gain-of-function allowing cleavage during viral egress thereby directly or indirectly contributing to increased replication rate, transmission and disease severity [ ] . note that proteolytic cleavage of the s glycoprotein can determine whether the virus can cross species, e.g. from bat. while structurally similar to sars-cov- , the ratg / virus lacks a unique peptide prra insertion region at the s /s boundary [ ] . further, the s glycoprotein from a mers-like coronavirus isolated from ugandan bats can bind to human cells but cannot mediate virus entry unless incubated with trypsin prior to transduction allowing s glycoprotein cleavage and virus entry [ ] . these observations suggest that cleavage of the s glycoprotein may be a prerequisite to coronavirus cross-species transmission. a recent publication from nankai university (tianjin, china) on sars-cov- reported that genome sequence analysis revealed a section of genes that was not present in sars-cov that had a cleavage site similar to hiv and ebola which carry viral proteins necessary for fusogenic activity of viral species to the human cell membrane. to be activated, the viral fusogenic surface glycoprotein has to be cleaved by furin [ ] . as mentioned, viruses contain surface glycoproteins which when cleaved by furin or other proprotein convertases (pc) are activated and viral propagation is achieved (i.e., avian influenza, hiv, ebola, marburg and measles viruses) [ ] [ ] [ ] . another pc necessary for viral entry is the transmembrane serine protease tmprss , which is known to contribute to efficient sars-cov cell entry and hoffman et al., has produced in vitro data showing that sars-cov- also uses tmprss priming [ ] . however, further assessment of furin cleavage in vivo is appropriate given fusion-mediated cell entry of sars-cov- rather than sars-cov via endocytosis, the presence of a unique furin cleavage site (rrar) at the s /s boundary and the furin-like s site in sars-cov- and the combination of cell membrane entry fusion and differences in the sars-cov- hr domain, which may contribute to the typical syncytium growth pattern in infected cells rarely reported in sars-cov [ ] . inhibition of furin may be a therapeutic approach that has efficacy in sars-cov- and other viruses that contain a furin cleavage domain. another immunotherapeutic intervention would be to increase the pulmonary expression of gm-csf, which, in vivo, redirects macrophages from an m state of activation to an m activation state and enhances expression of anti-inflammatory mediators and perhaps allow more time for patients to mount an effective immune response against sars-cov- [ ] . in addition to interfering in viral dynamics, a therapy-targeting host proteases rather than a viral epitope could also reduce the development of vaccine resistance due to mutation of nonessential viral-targeted antigens. for both reasons, furin is an attractive therapeutic target. it is highly conserved and genomically unrelated to viral replicative functions and antigenic drift [ , ] . we do not know how effective vaccination will be with sars-cov- given the low titers of na in patients with covid- and antigenic drift characteristic of human host rna viruses. vaccination for influenza virus is only effective in % of individuals due to rapid antigenic evolution. furin, was first described in and is the product of the fur gene [ ] . it is an evolutionarily conserved family member of the proprotein convertases which contain a subtilisin-like protease domain and was the first proprotein convertase (pc) to be identified in humans [ ] [ ] [ ] . furin is a type i transmembrane protein that is ubiquitously expressed in vertebrates and invertebrates [ ] . it is localized to the golgi and trans-golgi network where it cleaves multiple proteins and is also located on the outer membrane where pathogens utilize it to cleave glycoproteins, a step essential for entry into host cells [ , ] . it can be secreted as a soluble, truncated active enzyme [ , , ] . the correct folding of furins catalytic domain relies on the inhibitory function of the n-terminal -amino acid propeptide [ ] . to gain its enzymatic activity, the inhibitory propeptide is removed during transport from the endoplasmic reticulum to the trans-golgi network [ ] . in order to be released into the extracellular space, the membrane localization is cleaved at the c-terminus [ ] . due to furin's ubiquitous expression and localization it is able to process a large amount and variety of proteins including growth factors, cytokines, hormones, adhesion proteins, collagens, membrane proteins, receptors as well as other classes [ ] . furin cleavage can also inactivate other proteins [ , ] . its cleavage consensus sequence is arg-xaa-(lys/arg)-arg↓-xaa [ , ] . many viral pathogens including, coronavirus, flavivirus, pneumovirus, avian influenza, influenza a and hiv, utilize furin-mediated membrane glycoprotein cleavage facilitate viral entry and, for certain viruses, egress from target host cells [ ] . hiv- utilizes furin to cleave the viral membrane protein (env) gp into gp and gp- prior to mature virion assembly. conversely, flavivirus rely on furin cleavage after formation of packaged virions. sars-cov- , as noted above, is cleaved at two sites, s /s furin cleavage site (prrar↓sv) and a furin-like s cleavage site (kr↓sf) [ ] . rna viruses such a sars-cov- have several critical functions dependent upon protease activity. consequently, modulation of protease activity may provide therapeutic function in sars-cov- in a variety of other rna viruses. furin is a particularly promising opportunity for therapeutic intervention. as previously described it cleaves and activates numerous mammalian, viral and bacterial substrates [ ] . becker et al. optimized preclinical therapeutic performance of several peptidomimetic furin inhibitors and demonstrated 'in vitro' significant inhibition of highly pathogenic h n influenza virus propagation [ ] . although mechanisms have evolved enabling rna viruses to invade host cells, host defense mechanisms have also evolved. innate and adaptive immune responses have been shown to target viral antigens. additionally, targets critical to viral entry, protein assembly and egress are also of high therapeutic value. these are 'virus dependency factors'. various host proteins such as ifi and samhd have been shown to inhibit both rna and dna viral gene expression and replication, respectively [ , ] . furin is critical for viral membrane fusion, protein assembly and propagation, particularly as related to sars-cov- . multiple furin inhibitors have been developed and tested in vitro and in animal models. initial targets were peptide and protein inhibitors which target active sites and competitively inhibit binding sites. as example, two ifnχ-inducible gtpases, guanylate-binding proteins and (gbp and gbp ), with inhibitory furin activity have demonstrated cleavage inhibition of the hiv env precursor gp and reduced hiv virion infectivity [ ] . control of furin expression with protease activated receptor (par ), impacts downstream furin function and processing of human metapneumovirus f protein in hiv [ ] . associated neurocognitive disorders also provides evidence of resistance mechanisms that can occur while inhibiting spread of hiv- [ ] . another example, α- antitrypsin portland (α -pdx) inhibits both pc k and furin. α -pdx has been shown to inhibit processing of hiv- env and measles virus f [ , ] . moreover, peptides involving the cleavage site of influenza a virus hemagglutinin compete for furin activity [ , ] . activation of mmp is also inhibited by autoinhibitory propeptide of furin [ , ] . these data support therapeutic development involving furin inhibition against sars-cov- . interestingly, corneal damage in mice related to pseudomonas aeruginosa has been shown to be reduced by non-d-arginine (d r) and other furin inhibitors [ ] . nonpeptidic furin inhibitors have also demonstrated antifurin activity in the nanomolar dose range [ ] . , -dideoxystreptamine shows unusual furin inhibiting activity whereby a complex is formed with furin involving two molecules with separate functions, which interfere with the catalytic triad conformation and binding to an adjacent peptide stretch to inhibit furin activity [ ] . toxic effects related to furin inhibitors have not been observed outside of embryonic models. a study of furindeficient mice demonstrated a critical role of furin during embryogenesis in which knock-out of the fur gene led to death by day due to the failure of ventral closure and embryonic turning [ ] . therefore, furin inhibition should be limited to the non-pregnant population. liver-specific interferon-inducible furin knock-out mice have not demonstrated adverse effects outside of embryogenesis implying that other proprotein convertases may compensate for furin deficiency given overlapping activity [ , ] . targeting furin, a host enzyme, also avoids the emergence of resistance due to viral antigenic drift as described earlier as furin genome is highly conserved and maintains a stable genomic structure, while sars-cov- target sites undergo mutational changes throughout the viral life span and pandemic period [ ] . furin inhibitors also function as mentioned previously via knockdown at the rna level [i.e., regnase- (zc h a), roquin (rc h )] [ ] . a concern, however, with modulation of regnase- and roquin is that both agents will most likely result in off-target effects as these products both degrade off target mrna. the results outlined and safety profile support potential role of furin inhibitors within a pandemic and possibly even within the anti-terrorist government protection 'tool box'. similar to sars-cov- , alveolar epithelial cells are the primary target of influenza virus (iv) and are the first site of entry and support for viral propagation and replication. proinflammatory immune response is rapidly initiated toward viral cytopathogenic effect which leads to alveolar epithelial cell (aec) apoptosis [ ] . however, when infection persists and viral propagation continues leading to intensified inflammatory response, capillary and alveolar leakage occurs, followed by severe hypoxemia and eventually ards which requires hospitalized management, oxygen support and often ventilation assistance [ , ] . clearance of the viral pathogens from the lung by immune effector cells and the initiation of epithelial repair processes including expansion of local epithelial progenitor cells to begin resealing of the epithelial layer are critical for medical recovery and prevention of hospitalization, oxygen and ventilation support in iv-induced lung injury. the majority of mortality in relation to sars-cov- infection has been related to ards leading to hospitalization and ventilation support which is testing our medical capacity [ , ] . however, the inflammatory immune response against the virus needs to be balanced between the elimination of virus and toxic effect of immune-mediated pulmonary injury in order to limit damage to the respiratory tract and alveolar cells which prevent ards [ ] . mononuclear effector cells (macrophages, dendritic cells, cd + , neutrophils and lymphocytes) carry the bulk of the load in iv clearance and 'balanced' immune response against iv [ ] . similar activity demonstrated with iv is important for clearance of sars-cov- . gm-csf has been shown to promote proliferation, differentiation and immune activation of monocytes, granulocytes, macrophages [ , ] . gm-csf in the lungs is mainly expressed by aec type ii cells [ ] and is a first cytokine responder in protection of the lung environment, aec survival and function, and is a positive prognostic factor in clearance of iv infection. expressed gm-csf in pulmonary secretions can potentially be used as an indication in bronchial lavage samples of early response and resistance thereby affecting medial need involving o support. other cell types produce gm-csf, but aecs have been shown to upregulate gm-csf in the distal lung parenchyma upon iv infection, and then produce high levels of gm-csf in the alveolar surrounding secretions [ ] . aec gm-csf secretion with iv infection resolution appears to be further mediated via hgf/c-met and tgf-α/egfr signaling [ ] . relationship of gm-csf to immune response activation against cancer and viral infection is well described [ ] [ ] [ ] [ ] [ ] . gm-csf also regulates the differentiation, proliferation and activation of alveolar macrophages [ , ] . in vitro studies indicate that gm-csf causes rapid proliferation of alveolar type ii epithelial cells thereby serving in repair and barrier protection of the respiratory epithelium at early stages of acute inflammation [ ] . it is also known that gm-csf expression from alveolar type ii epithelial cells facilitates surfactant homeostasis further enhancing protection of viral induced pathology [ , , ] . gm-csf also enhances the antiviral responses of alveolar macrophages. indeed, elevated levels of gm-csf may elicit a biphasic m ↔m response pattern [ ] . although a number of studies show that gm-csf and type i interferon act together to modulate macrophage polarization toward the m state of activation, recent in vivo studies conclude the opposite [ ] [ ] [ ] . gm-csf enhances viral clearance through expression of scavenger receptors, sr-a and marco [ ] [ ] [ ] [ ] . these two receptors aid in viral clearance through activation of receptors tlr- , tlr- , nod- and nalp- [ ] [ ] [ ] . gm-csf enhances mucosal immune responses and the effectiveness of dna vaccines [ , ] . recombinant human gm-csf has been delivered to the lung and conferred resistance to iv infection [ , ] . transgenic mice that constitutively expressed human gm-csf exposed to iv, were able to mount and effective antiviral response that resulted in increased numbers of human alveolar macrophages [ ] . halstead et al., also showed how gm-csf overexpression after iv virus infection in a gm-csf transgene mouse model prevents mortality [ ] . protective effects of gm-csf against iv-a pneumonia have been seen in mice with constitutive and inducible gm-csf expression models in alveolar type ii epithelial cell transgenic mice with gm-/-and gm +/+ pulmonary specific promoters (sftpc, scgb a ), respectively [ ] . this model was able to show gm-csf enhancement of alveolar cell activity as indicated by increased expression of sp-r and cd c expressive mononuclear cells. in mice lacking sr-a and marco, two receptors regulated by gm-csf, marco was shown to increase expression of sp-r on alveolar macrophages and decrease resistance to iv. however, although continuous sp-c-gm+/+ transgenic mice resisted early mortality from iv, concern was raised to continuous high gm-csf exposure over prolonged time. late assessment of lung tissue sections revealed the histological features of degenerative desquamative interstitial pneumonia at day . degeneration of alveolar structure and large spaces containing desquamated cells characterized the lungs of high gm-csf exposed mice. the results indicate that excessively high levels of gm-csf impair appropriate tissue healing resulting in development of interstitial lung disease secondary to iv pneumonia and provide guidance for early large animal assessment and phase i monitoring of patient safety. however, results support that the conditional gm-csf expressive mice do well and have long-term survival advantage to iv infection and have significant advantage over untreated controls. expression of gm-csf either through transgenic or pulmonary delivery conferred survival advantage to influenza virus compared with wt mice that did not survive infection. when alveolar phagocytes were depleted, the protective effect also diminished suggesting that these cells are necessary to induce the innate immune response [ ] . as described, infection with sars-cov- can progress to rapid induction of viral pneumonia and ards resulting in fatal outcome [ ] . aecs play a critical role in orchestrating the pulmonary antiviral host response [ ] . however, with early iv and sars-cov- infection aec's release gm-csf. gm-csf heightens immune function of alveolar cells which leads to improved epithelial repair processes. during iv infection, aec-derived gm-csf also enhances a lung-protective mechanism. similar results are seen with local rhgm-csf application. this early use and/or enhancement of immune function and alveolar protection with elevated gm-csf expression appears to overwhelmingly benefit clinical response. however, gm-csf expression late in the inflammatory lung response is less well characterized. although correspondence by herold s et al. in using recombinant gm-csf (leukine, bayer healthcare pharmaceuticals, wa, usa) and an aeroneb solo nebulizer to administer leukine ( μg/dose) demonstrated significant clinical benefit in four of six patients with ards related to infectious pneumonia (including two with h n virus) [ ] . immune function enhancement was also shown in the leukine treated patients compared with untreated ards patients in analysis of pulmonary immune response, which is similar to preclinical evidence (in vitro and animal models) [ , , , ] . gm-csf treated patients demonstrated alveolar cell protection, enhanced alveolar cell activity toward viral and other infectious clearance and shift to m response as assessed by increased alveolar cd + cells and cd drop. these results support enhancement of gm-csf expression even late in pulmonary inflammatory response to viral infection may be of benefit which suggest therapy benefit in late stage ards patients. safe administration of gm-csf via inhalation therapy in patients with autoimmune pulmonary alveolar proteinosis was also demonstrated to show benefit by ohashi et al. [ ] . elevated il- in bronchial alveolar lavage fluid was shown as a gm-csf induced cytokine and may serve as a biomarker associated with benefit. sever-chroneos et al. [ ] , found worsening iv infection and response in gm-csf deficient mice was due to impaired iv clearance by macrophages. this work is supported by berclaz et al. who demonstrated that the fcγ receptor (fcγr)-mediated opsonophagocytosis of invaded pathogens by alveolar macrophages is related to gm-csf [ ] . t-cell-produced interferon γ (ifn γ) also effects alveolar macrophage fcγr expression which in turn stimulate production of ifn γ and other cytokines such as il- and il- supporting involvement of both innate and adaptive immunity turn on. elevated alveolar gm-csf level in transgenic mice also improves resistance of alveolar cells in association with iv infection [ ] . gm-csf has also been shown to be an important stimulator of cd + t lymphocytes and further enhances their role to activate dc priming in lymphoid tissue, thereby providing a positive feedback in further stimulation of cd + t cell expansion [ ] . greter et al. [ ] , also showed gm-csf to be critically important for induction of cd + t-cell immunity. chen et al. [ ] also found gm-csf to promote b-cell maturation and production of iv specific antibodies. during iv pneumonia, extensive additional in vivo data support the role of gm-csf as a lung barrier-protectant and positive immune response factor [ , , , ] . aec-expressed gm-csf directly benefits the injured epithelium and is important in enhancing epithelial proliferation in the setting of hypoxic lung injury via repair of barrier function, reduction of capillary leak and return of tissue to homeostasis [ , ] . the data discussed above regarding targeting furin and increasing gm-csf expression warrants further investigation to target sars-cov- infection. vigil, which combines bifunctional shrna targeting furin and incorporating a gm-csf dna sequence in a plasmid delivery vehicle (pbi-shrna furin -gm-csf) has been described as the most advanced anti-furin technology in clinical testing [ ] . vigil is an autologous tumor cell vaccine, with dual function that knocks down furin expression as seen by decreased expression of downstream proteins tgfβ / and expresses gm-csf [ ] . it has demonstrated clinical success in several cancer populations but especially ewing's sarcoma and ovarian cancer [ ] [ ] [ ] [ ] . it has a demonstrated safety profile with no evidence of grade product related toxicity effect following doses in cancer patients. the potential efficacy and use of vigil for covid- is an example of the rational repurposing of drugs from indicated to nonprimary target disease alternatives. such an approach could accelerate the clinical development process particularly urgent given the current covid- pandemic. vp constructed by gradalis, inc. (tx, usa), consists of two stem-loop structures with a mir- a backbone [ ] . the bi-shrna furin dna as shown in figure a uses a single targeted site to induce both mrna cleavage and sequestration in p-bodies (translational silencing) and/or gw-bodies (repositories) [ ] . by the use of this [ ] . (b) adapted with permission from [ ] . proprietary process, the encoding bi-shrna can accommodate mature shrna loaded onto more than one type of rna induced silencing complex (risc) [ ] . also, with the bi-shrna furin molecular design focusing on a single site potential toxic effects are reduced. targeting of multiple sites increases chance for a 'seed sequence' being induced and leading to off-target effect that could result in increased clinical toxicity. synthetic complementing and interconnecting oligonucleotides via dna ligation were used to assemble the two stem-loop double stranded dna sequences [ ] . the base pair dna constructed with bam hi sites at both ends was inserted into the bam hi site of a prior clinically validated plasmid called tag [ ] in which we removed a tgfβ antisense dna sequence and placed the bi-shrna furin -gmcsf dna sequence. orientation of the inserted dna was validated by the appropriate pcr primer pairs designed to screen for the shrna insert and orientation. safety profile defined with the prior tag clinical therapeutic was used to support clinical advancement of vp in experimental cancer management testing under fda guidance.vigil is designed with the mammalian promoter cytomegalovirus [cmv] that drives the cassette. in between the gm-csf gene (with a stop codon) and furin bi-shrna there is a a ribosomal skip peptide followed by a rabbit poly-a tail. the picornaviral a sequence allows the production of two proteins from one open reading frame, by causing ribosomes to skip formation of a peptide bond at the junction of the a and downstream sequences [ ] . since we previously demonstrated the a linker to be effective for generating similar expression levels of gm-csf and anti-tgfβ transcripts with the tag vaccine and we observed robust activity in product release testing of therapeutic effector components expressed with this plasmid design along with clinical benefit and safety, we maintained the same design for vp. transient expression of bi-shrnafurin-gm-csf plasmid and diluted expressive cell numbers in patients would not be expected to approach continuous toxic effect of transgenic models. gradalis has been clinically testing this plasmid since [ ] . aldevron (nd, usa) and waisman (wi, usa) have participated in lot manufacturing. this plasmid, which consists of a bi-shrna furin dna sequence and a gm-csf dna sequence (figure b) , has been validated for fda registration trial based on significant cancer patient benefit for use of transfection (via electroporation) into an autologous tumor for vaccine (vigil) construction in ewing's sarcoma [ , [ ] [ ] [ ] [ ] [ ] . additional benefit has also been suggested in a variety of other cancer types in phase i and ii testing, most notably ovarian cancer [ , , [ ] [ ] [ ] . the current active lot of vigil plasmid was manufactured in by waisman. the plasmid concentration per vial is . mg/ml which provides μg of plasmid. yearly stability testing has passed all measures of evaluation under us fda review. recent double blind randomized control trial involving nationally acclaimed sites unblinded and revealed os advantage (hr: . , p = . ) and rfs advantage (hr: . , p = . ; stratified cox's proportional hazard model) [ ] . viral pneumonia, particularly in elderly or immune compromised patients, can be associated with devastating medical consequence [ ] . pulmonary delivery via aerosolized systems are simple, nonexpressive, noninvasive and allow for pain-free access of therapeutic and minimization of possible systemic side effects [ , ] . aerosols have been shown to deliver plasmid dna droplets with size ranging from and μm, which are able to disperse to the bronchial and alveolar epithelial cells. this enables pdna entry and maximizes subsequent gene expression [ ] . rajapaksa et al. successfully demonstrated the use of a saw liquid nebulization device for the generation of aerosolized pdna with suitable size and stability characteristics to facilitate effective pulmonary delivery particularly for iv vaccination [ ] . in vivo studies have shown successful pdna delivery in both small and large animals. saw nebulization used to deliver a plasmid vaccine demonstrated expression of protective anti-hemagglutinin (ha) antibodies. anti-ha antibody titers detected were comparable to vaccination outcomes of other similar pdna influenza vaccines not using a nebulizer [ ] . these results support use of naked pdna for effective delivery via pulmonary distribution while also demonstrating product stability and function. following pdna vaccination in rats, revealed higher serum hemagglutination inhibition (hai) titers which were identified as protective according to who standards [ ] . however, at this time, the saw nebulizer approach has not demonstrated scale up capability for use in a pandemic event. aerosolized ribavirin however has demonstrated large volume capacity and adequate aerosolized delivery and clinical benefit including use in morbid condition patients. ribavirin is indicated therapy for severe rsv infection in children. the conventional continuous treatment of mg of ribavirin/ml for h was found to be effective. aerosolized ribavirin (administered mg/ml for h three times daily) has also been effective in cancer patients with rsv infection [ ] . ribavirin inhalation method at intermittent high doses ( mg/ml) over the same schedule in immune suppressed children with rsf infection was also well tolerated. moreover, results demonstrated similar improved clinical response compared with standard therapy. there was also less adverse exposure to healthcare workers [ , ] . parainfluenza virus is associated with potentially serious complications in high morbidity patients (i.e., heart-lung transplant, allograft rejection, bronchiolitis obliterans [ ] . inhaled ribavirin in this population was associated with clinical improvement [ ] . aerosolized ribavirin ( mg/ml) was also effective against iv-a and b infections in mice [ ] . recently, aerosolized ribavirin ( mg/ml) was shown to be effective in mice infected with lethal iv-a h n virus, and resulted in > % survival when given early (within - h) after infection [ ] . aerosolized ribavirin treatment has been used with success against metapneumovirus pneumonia [ ] . moreover, in treatment of pneumotropic human adenovirus, aerosolized ribavirin demonstrated greater benefit over intravenous ribavirin likely related to the more robust drug concentration achieved in the alveoli with aerosolized product compared with intravenous ribavirin therapy. additionally, the aerosolized delivery did not appear to lead to cytotoxic effect [ ] . s-flu immunization provides a broad cell-mediated immune response to conserved viral antigens. data reveal that immunization with s-flu-expressing h ha (h s-flu) dna reduces the viral load in lungs after aerosolized challenge with the closely matched pdmh n virus strain [ ] . the reduction of viral load was shown to be optimal using aerosol administration when compared with intravenous s-flu. however, viral neutralizing ab was not observed in s-flu-immunized pigs, and the reduction of viral load in the h are group correlated with the presence of ifng-producing cd or cd /cd double-positive cells in the bronchoalveolar lavage suggest adequate product delivery. these data provide proof of principle that s-flu dna can be efficiently delivered by aerosol to a large animal, supporting possible use of a nebulizer device as a method of immunizing patients. aerosolized delivery may be further optimized with use of lipid-dna complexes [ ] . others have also shown successful aerosol delivery of measles vaccine in humans and/or exosome/viral delivery [ , ] . the challenge to this approach is how to introduce plasmid dna into the lungs without loss or damage to the plasmid. plasmid dna is highly prone to shearing, therefore methods with low shear forces are necessary for effective delivery of the supercoiled dna. both nebulizers and dry powder inhalers use low amounts of shear forces. nebulizers however use aerosol droplets to deliver particles into the lungs, which may not be an effective method to deliver plasmid dna, as dna degrades while in solution if not stored appropriately. additionally, nebulizers limit the concentration of product that can be delivered due to solubility. dry powder inhalers are not limited by solubility and plasmid dna would not need to be stored in solution [ ] [ ] [ ] . this method also reduces shear stress and thermal degradation which results in a high concentration of quality plasmid delivered directly into the lungs. accumulating knowledge of intracellular viral processing, molecular biology, viral dynamics, host immune mechanisms and immunokinetics will allow for the development of tools and methods to protect lung function, delay or prevent ards, enhance anti-viral resistance and institute prophylactic measures. the unique role of furin and the demonstration of robust viral clearance in all patients who survive sars-cov- infection supply the rationale and support for repurposing vigil for treatment of patients with covid- . knockdown of furin with vigil would target multiple steps of viral propagation, including viral, entry, protein assembly and egress. expression of gm-csf would provide further therapeutic benefit, enhancing the immune response and aec protection. the data, limited as it is, showing no obvious correlation between seroconversion and viral clearance, gives additional support to a multifunctional therapeutic approach to covid- , in other words, combining inhibition of a protease critical to viral entry and cell to cell transmission with an immune response modulator. vigil is already involved in fda characterization with a known product safety profile. further testing will be necessary, including in vitro activity assessment against sars-cov- and large animal safety. future perspective sars-cov- presents unique challenges for clinical management and infection containment. while vaccination is important to foster immunity and protect at-risk populations, finding relevant and effective therapeutics that could work across multiple viral pathogens will remain important. logical drug repurposing and compound testing will be critical for rapid response to not only this pandemic, but future pandemics as well. • sars-cov- relies on furin cleavage for multiple steps of the viral replication process, including viral entry, protein synthesis and viral egress. • gm-csf provides a lung protective effect, which may help to prevent ards and therefore allow natural immune clearance and antibody generation. • vigil plasmid is an example of logical drug repurposing and targets multiple viral propagation steps. clinical course and risk factors for mortality of adult inpatients with covid- in wuhan, china: a retrospective cohort study analysis of the first patients diagnosed with covid- early transmission 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stage ovarian cancer randomized double-blind placebo controlled trial of primary maintenance vigil immunotherapy (vital study) in stage iii/iv ovarian cancer: efficacy assessment in brca / -wt patients (late breaking oral presentation) fang vaccine: autologous tumor cell vaccine genetically modified to express gm-csf and block production of furin gw-bodies and p-bodies constitute two separate pools of sequestered non-translating rnas characterization of endogenous human argonautes and their mirna partners in rna silencing self-splicing of a group i intron reveals partitioning of native and misfolded rna populations in yeast tag vaccine: autologous tumor vaccine genetically modified to express gm-csf and block production of tgfb expression of heterologous genes in oncolytic adenoviruses using picornaviral a sequences that trigger ribosome skipping ewing's sarcoma: development of rna interference-based therapy for advanced disease pilot trial of fang immunotherapy in ewing's sarcoma preclinical justification of pbi-shrna ews/fli lipoplex (lpx) treatment for ewing's sarcoma innovative exploratory clinical approaches for relapsed and/or refractory metastatic ewing's sarcoma case report: immune-mediated complete response in a patient with recurrent advanced ewing sarcoma (ews) after vigil immunotherapy follow-up of bi-shrna furin/gm-csf engineered autologous tumor cell (eatc) immunotherapy vigil r in patients with advanced melanoma summary of bi-shrna/gm-csf augmented autologous tumor cell immunotherapy (fang) in advanced cancer of the liver case report: vigil therapy in pathology defined high-risk differentiated thyroid cancer compounded by post ablation high-risk factors randomized double-blind placebo controlled trial of primary maintenance vigil immunotherapy (vital study) in stage iii/iv ovarian cancer: efficacy assessment in brca / -wt patients (late breaking oral presentation) inhaled therapeutics for prevention and treatment of pneumonia rapid-response vaccines-does dna offer a solution? pulmonary delivery of nucleic acids pulmonary vaccine delivery effective pulmonary delivery of an aerosolized plasmid dna vaccine via surface acoustic wave nebulization enhanced lung gene expression after aerosol delivery of concentrated pdna/pei complexes progress and challenges for malaria vaccines strategies to enhance immune function in hematopoietic transplantation recipients who have fungal infections difficulties with fungal infections in acute myelogenous leukemia patients: immune enhancement strategies recombinant interferon gamma b immune enhancement in patients with hematologic malignancies and systemic opportunistic infections treated with donor granulocyte transfusions invasive aspergillosis in the setting of cardiac transplantation epidemiology of invasive mold infections in allogeneic stem cell transplant recipients: biological risk factors for infection according to time after transplantation aerosol amphotericin b is effective for prophylaxis and therapy in a rat model of pulmonary aspergillosis efficacy of prophylactic aerosol amphotericin b lipid complex in a rat model of pulmonary aspergillosis infectious complications among consecutive heart transplant patients at stanford university medical center safety of aerosolized amphotericin b lipid complex in lung transplant recipients aerosol delivery of a candidate universal influenza vaccine reduces viral load in pigs challenged with pandemic h n virus aerosolization of cationic lipid-dna complexes: lipoplex characterization and optimization of aerosol delivery conditions measles aerosol vaccine project a randomized, controlled trial of an aerosolized vaccine against measles in vitro and in vivo performance of dry powder inhalation formulations: comparison of particles prepared by thin film freezing and micronization dry powder insufflation of crystalline and amorphous voriconazole formulations produced by thin film freezing to mice amorphous powders for inhalation drug delivery the authors would like to acknowledge b marr for her competent and knowledgeable assistance in the preparation of the manuscript. the authors have no relevant affiliations or financial involvement with any organization or entity with a financial interest in or financial conflict with the subject matter or materials discussed in the manuscript. this includes employment, consultancies, honoraria, stock ownership or options, expert testimony, grants or patents received or pending, or royalties.no writing assistance was utilized in the production of this manuscript. key: cord- - stsx je authors: singh, m.; foster, d.j.; child, g.; lamb, w.a. title: inflammatory cerebrospinal fluid analysis in cats: clinical diagnosis and outcome date: - - journal: j feline med surg doi: . /j.jfms. . . sha: doc_id: cord_uid: stsx je the medical records of cats with clinical signs of central nervous system disease and accompanying inflammatory cerebrospinal fluid (csf) analysis were examined retrospectively to determine if signalment, clinical signs, csf analysis and ancillary testing could accurately predict the type of central nervous system disease that was present. an inflammatory csf was defined as one in which a total nucleated cell count was greater than cells/μl or one in which the total nucleated cell count was normal but the nucleated cell differential count was abnormal. sex, degree of csf inflammation, neuroanatomical location and systemic signs provided little contributory information to the final diagnosis. in % of the cases a presumptive diagnosis could be made based on a combination of clinical signs, clinicopathological data and ancillary diagnostic tests. csf analysis alone was useful only in the diagnosis of cats with feline infectious peritonitis, cryptococcus species infection, lymphoma and trauma. overall, despite extensive diagnostic evaluation, a specific diagnosis could not be made in % of cats. the prognosis for cats with inflammatory csf was poor with % of cats surviving less than year. the medical records of cats with clinical signs of central nervous system disease and accompanying inflammatory cerebrospinal fluid (csf) analysis were examined retrospectively to determine if signalment, clinical signs, csf analysis and ancillary testing could accurately predict the type of central nervous system disease that was present. an inflammatory csf was defined as one in which a total nucleated cell count was greater than cells/ml or one in which the total nucleated cell count was normal but the nucleated cell differential count was abnormal. sex, degree of csf inflammation, neuroanatomical location and systemic signs provided little contributory information to the final diagnosis. in % of the cases a presumptive diagnosis could be made based on a combination of clinical signs, clinicopathological data and ancillary diagnostic tests. csf analysis alone was useful only in the diagnosis of cats with feline infectious peritonitis, cryptococcus species infection, lymphoma and trauma. overall, despite extensive diagnostic evaluation, a specific diagnosis could not be made in % of cats. the prognosis for cats with inflammatory csf was poor with % of cats surviving less than year. i nflammation of the central nervous system (cns) is common in cats of all ages. despite this, few attempts have been made to evaluate how well cerebrospinal fluid (csf) analysis, clinical signs and other clinicopathological data correlate with the disease occurring in the cns. causes of cns inflammation in cats include viral, protozoal, fungal, parasitic and bacterial infection, 'immune mediated' and 'idiopathic' disorders of uncertain aetiology (eg, non-suppurative and eosinophilic meningoencephalitis) (munana ) . in addition to primary inflammatory cns disorders, there are diseases that can induce cns inflammation secondary to tissue damage and necrosis. such diseases include neoplasia, trauma, intervertebral disc disease, cerebrovascular disease and nutritional disorders (eg, thiamine deficiency). the purpose of this study was to determine if signalment, clinical signs, csf analysis, additional clinicopathological data and diagnostic imaging could be used to determine the specific aetiology of the cns disease in cats with inflammatory csf. prognosis for the various disease classifications was also evaluated. records were searched at three referral centres in sydney, nsw from january to december for cats that had csf analysis. csf collection was performed in cats. of these, cats could be classified as having inflammatory cns disease. csf was collected by percutaneous puncture from either the cerebello-medullary cistern or via lumbar puncture from either the l el or l el intervertebral space. csf analysis included total white cell count and red cell count by haemocytometer, cytomorphology by stained sediment (idexx laboratories pty ltd) or stained cytocentrifuge smears (the university of sydney department of clinical pathology) and total protein by evaluation of microprotein on a commercial biochemistry autoanalyser (olympus au , idexx laboratories; cobas mira, the university of sydney department of clinical pathology). the reference ranges used by our laboratories for feline csf were: white cell count % cells/ml, red cell count cells/ml and total protein ! . g/l (canfield and martin , raskin and myer ). an inflammatory csf analysis was defined as one in which a total nucleated cell count was greater than cells/ml or one in which the total nucleated cell count was normal but the nucleated cell differential count was abnormal (o % neutrophils, o % eosinophils) (canfield and martin , raskin and myer ) . csf nucleated cell counts were classified as: , normal: % cells/ml; , mildly elevated: e cells/ml; , moderately elevated: e cells/ml and , markedly elevated: o cells/ml. the nucleated cell population was classified as: , suppurative: o % neutrophils; , non-suppurative: o % mononuclear cells; , eosinophilic: o % eosinophils and , mixed: no predominance of any one cell type (canfield and martin , raskin and myer ) . csf total protein was classified as , normal: % . g/l; , mildly elevated: . e g/ l; , moderately elevated: e g/l and markedly elevated: o g/l. csf erythrocyte counts were classified as: , normal: cells/ml; , mildly elevated: e cells/ml; , moderately elevated e cells/ml and , markedly elevated: o cells/ml (table ). the contribution of csf red cell contamination to the total csf white cell count was corrected for by subtracting nucleated cell for every red cells (rand et al ) . cats with csf nucleated cell count that was less than or equal to that predicted from blood contamination were excluded from the study. haematology was performed on standard automated counters (celldyn, idexx laboratories; sysmex k- , university of sydney clinical pathology). differential cell counts were performed via stained (diff quick) blood smear examination. biochemistry was performed using standard automated chemistry analysers described previously. cryptococcal antigen titres were measured by latex agglutination (negative titre z ), toxoplasma gondii igg antibody titres were measured by indirect haemagglutination (negative z ! : ), t gondii igm titres were measured by indirect fluorescent antibody (negative ! : ), feline leukaemia virus (felv) antigen was measured by elisa, feline immunodeficiency virus (fiv) specific antibody was measured by elisa and feline coronavirus (fecov) antibody was measured by indirect fluorescent antibody (negative titre z ! : ). owners and referring veterinarians were contacted to determine the outcome after discharge from hospital. the evaluation period ranged from . months to years. the following parameters were evaluated for each cat: , signalment; , duration of clinical signs; , neurological findings and localisation of lesion; , systemic signs; , cerebrospinal fluid testing; , ancillary testing including results of culture, diagnostic imaging, blood evaluation and , outcome. sixty-two cases had sufficient data and met the criteria of inflammatory changes within the cns. there was no age or sex distribution pattern. the majority of cats were domestic shorthair ( %), while there were four burmese, four persians and three ragdolls. the remainder represented many different breeds including himalayan, chinchilla, birman, burmilla, siamese, cornish rex, devon rex, russian blue, oriental shorthair, abyssinian and domestic longhair. there were no obvious breed risks associated with the inflammatory changes within the cns. the age range was months to years, with a median age of years. thirteen cats ( %) presented with acute clinical signs of less than weeks' duration. forty-nine cats ( %) presented with clinical signs longer than weeks duration. fifty-two cats ( . %) presented with gait abnormalities that included ataxia, hypermetria, paresis/paralysis or unilateral lameness. fifteen cats ( . %) presented with seizures, partial or generalised. twelve cats ( . %) exhibited nystagmus, cats ( . %) had a head tilt, cats ( . %) had other cranial nerve deficits (facial nerve or trigeminal nerve paralysis), eight cats ( %) had reduced mentation and eight ( %) had abnormal postural reactions. other signs encountered included circling, spinal pain, anisocoria and behavioural abnormalities. in cats ( %) the presentation suggested multifocal cns disease while in cats ( %) the lesions appeared focal (cerebral cortex in nine cats, cerebellum in two cats, central vestibular in five cryptococcus toxoplasma other meningoencephalitis thiamine deficiency lymphoma other had a total protein of . e g/l, two cats had a total protein of e g/l and three cats had a total protein of o g/l. eleven cats ( . %) did not have a total protein measured because of insufficient sample. twelve cats had more than red cells/ml. three cats had csf characteristics indicative of haemorrhage characterised by activated macrophages and large numbers of haemosiderophages with erythrophagocytosis. in the remaining cases the increased csf erythrocyte count was consistent with blood contamination during collection. the most common ancillary testing procedures were haematology in cats ( %), biochemistry in cats ( %) and cryptococcal antigen titres in cats ( %). fifteen cats ( . %) had a myelogram performed, cats ( . %) had t gondii igg antibody titres measured and cats ( . %) had felv antigen tested. fiv antibody, thoracic radiographs, skull radiographs and abdominal ultrasound were performed in nine cats each. surgical biopsy was performed in five cats. fecov antibody titre measurement and computed tomography (ct) scan were performed in four cats each. urinalysis and fine needle aspirate biopsy were performed in four cats each. blood pressure, t gondii igm antibody titre, magnetic resonance imaging (mri), thyroid hormone concentration and cholinesterase concentration were performed in two cats each. ammonia tolerance testing, blood lead concentrations and bone marrow aspiration were performed in one cat each. the most frequent abnormalities seen were increased serum globulins in cats ( %), anaemia in nine cats ( . %), an abnormal myelogram in cats ( %) and a peripheral neutrophilia in five cats ( %). thirty-five cats ( . %) survived less than month after presentation. three died or were euthanased immediately after cerebrospinal fluid collection. of these, one died due to uncontrollable seizures and two were euthanased because of persistent apnoea. ten cats ( . %) survived e months, three cats ( . %) survived e months and cats ( . %) survived greater than months and were still alive at the time of writing. of the cats that died or were euthanased, ( %) had a post mortem performed, cats ( %) did not. two cats were lost to follow-up. based on the results, clinical information, clinical pathology and ancillary testing procedures the following classifications of disease could be made: , feline infectious peritonitis (fip); , cryptococcus species infection; , toxoplasma species infection; , other meningoencephalitis; , thiamine deficiency; , lymphoma; , other neoplasia; , trauma; , intervertebral disc disease; , spinal cord granuloma and , undiagnosed (table ) . eleven cats ( %) were diagnosed with fip. a presumptive diagnosis of fip was made based on age, a suppurative or mixed inflammatory csf, poor response to treatment, elevated serum or body cavity effusion fecov antibody titre or a reduced albumin:globulin ratio (! . ) of serum or body cavity effusions. the diagnosis was confirmed at necropsy in eight cats and on surgical biopsy in one other ( % of the cases). the remaining two cats had a diagnosis of fip made based on ancillary diagnostic tests (serum and body cavity effusion albumin:globulin ratio ! . , and elevated fecov antibody titre in serum and body cavity effusions). the ages ranged from months to years of age with a median of months. breed distribution did not differ from the total population of cats studied. the most common neurological signs were gait abnormalities in seven cats and reduced mentation in three cats. systemic signs included pyrexia in six cats, lethargy in three cats, ocular abnormalities in six cats and weight loss in four cats. four cats had an abdominal effusion and one cat a pleural effusion. ten cats had clinical signs consistent with multifocal lesions of the central nervous system. these were referrable to a combination of cerebral cortex and brainstem (often central vestibular) signs. one cat had cerebellar signs only. csf analysis was characterised as suppurative in seven cats, mixed in one and mononuclear in three. five cats had a marked elevation in the csf white cell count (o cells/ml), three cats had moderate elevations ( e cells/ml), two cats had mild elevations ( e cells/ml) and one cat had insufficient sample for a white cell count. seven cats had increased csf protein concentrations. four cats had a mild elevation ( . e g/l), one cat had a moderate elevation ( e g/l) and two cats had marked elevations (o g/l). two cats had normal csf protein concentrations and two cats did not have csf protein concentrations measured because of insufficient sample. all cats had haematology and serum biochemistry preformed. six cats had a mild, non-regenerative anaemia and three cats had a mild-moderate mature neutrophilia. no cat was lymphopenic. ten cats had elevated serum globulin concentrations, two had elevated alanine aminotransferase (alt) and one cat had an elevated bilirubin concentration. of the two cats without a histopathological diagnosis, one had a serum fecov antibody titre of : and an fecov antibody titre in ascitic fluid of : . this cat had an albumin:globulin ratio of . in the ascitic fluid and a csf albumin:globulin ratio of . . the second cat had a serum fecov antibody titre of : and serum albumin:globulin ratio of . . no cat survived longer than days after presentation. one cat has a seizure and died immediately after csf collection. four cats were diagnosed with central nervous system cryptococcosis based on a positive serum antigen titre and/or the presence of cryptococcal organisms in the csf. all were spayed female. the age range was e years with median age of years. three cats were domestic shorthair and one was a burmese. all four cats had gait abnormalities. other clinical signs observed were reduced mentation, spinal pain, seizures, circling, reduced postural reactions and nystagmus. systemic signs observed included bradycardia, inappetence and ocular abnormalities. the location of the neurological signs was variable, encompassing all central regions except the cerebellum. two cats had suppurative csf and two had mononuclear csf. three cats had large numbers of cryptococcal organisms in the csf. one had no organisms detected in the csf and was initially diagnosed with cns toxoplasmosis (based on a moderate mixed inflammatory csf, t gondii igg of : and igm of o : ). at necropsy months later, a large cryptococcal granuloma was detected in the cerebrum. this cat had a premortem cryptococcal antigen titre of : . csf nucleated cell numbers ranged from to cells/ml. the protein concentration was normal in two cats, markedly elevated in one cat ( g/l) and not measured in the other. three cats had positive serum cryptococcal antigen titres, although in one cat it was very low ( : ). an antigen titre was not measured in one case. two cats had elevated serum globulins and one cat had a moderate mature neutrophilia. two cats recovered and survived more than years after diagnosis. one cat was euthanased days after presentation and did not undergo a post mortem examination. two cats were diagnosed with cns toxoplasmosis. a presumptive diagnosis was made based on a mild suppurative-mixed csf inflammation, normal-mildly increased csf protein levels and positive igg antibody titre. both cats had the clinical diagnosis confirmed at necropsy (t gondii tachyzoites observed in the cns). one of the cats was diagnosed with suppurative meningitis of unknown cause. it was euthanased months after presentation and necropsy revealed cns toxoplasmosis. both cats presented with gait abnormalities, head tilt and anisocoria. one cat had mild neutrophilic csf inflammation with a mildly elevated protein ( . g/l) and did not have t gondii antibody titres measured. the other cat had mild mononuclear csf inflammation. the csf protein levels were not measured in this cat because of insufficient sample. one had a positive t gondii igg antibody titre ( : ). both cats presented with a chronic course of disease and multifocal neurological signs. both cats had elevated serum globulins and abnormal thoracic radiographs (a single nodular opacity present in both). survival times were and weeks. five cats were diagnosed with meningoencephalitis of unknown cause. they were subclassified, according to response to treatment, into nonsuppurative meningoencephalitis and steroid responsive meningoencephalitis. three cats were diagnosed with non-suppurative meningoencephalitis on the basis of a mononuclear pleocytosis in the csf or histopathology indicating a perivascular mononuclear infiltrate. one cat recovered from the disease over days and was clinically normal years later. the other two cats were euthanased within weeks because of persistent neurological signs. both cats received a post mortem examination that revealed a mononuclear perivascular cellular infiltrate in the cerebral cortex of one cat and in the cerebral cortex and brainstem of the other cat. the ages of these cats ranged from to years with a median age of years. the main neurological signs were seizures in two, and one each of nystagmus, gait abnormalities, behavioural changes and reduced mentation. extra-neural signs included one each of weight loss, inappetence, pyrexia and ocular abnormalities. all three cats presented with chronic signs of illness. two cats had multifocal signs and one had focal cerebral signs. all three cats had mononuclear csf inflammation with mild-moderate elevations in the csf leukocyte counts. csf protein concentrations were normal in two cats and not measured in one because of insufficient sample. no cat had serological testing for infectious diseases. two cats had a marked inflammatory pleocytosis on csf analysis but made a complete recovery with corticosteroid therapy and were diagnosed as a steroid responsive meningoencephalitis. one cat was a -year-old female spayed ragdoll, the other a -year-old male castrate russian blue. the ragdoll presented with acute signs of seizures, circling, proprioceptive deficits and ataxia. the csf nucleated cell count was markedly elevated ( cells/ml) with a mixed inflammatory pattern and moderate blood contamination ( cells/ml). the csf protein concentration was mildly elevated ( . g/l). fip was the initial clinical diagnosis and prednisolone (macrolone; mavab) was prescribed. the cat made a dramatic improvement within days of prednisolone therapy and was gradually weaned off prednisolone over months. twelve months later the cat was clinically normal. the russian blue presented with a chronic history of hindlimb paresis with localisation of signs to the thoracolumbar spinal cord. the nucleated cell count was moderately elevated ( . cells/ml) with moderate blood contamination ( cells/ml). the csf protein concentration was moderately elevated ( . g/l). the inflammation was mononuclear. complete blood count, biochemical profile and spinal radiographs did not reveal any abnormalities. the cat was treated with prednisolone (macrolone; mavlab) and gradually improved over the following days. this cat was also gradually weaned off prednisolone over months and was clinically normal years later. two cats were diagnosed with thiamine deficiency on the basis of a clinical history of a thiamine deficient diet, response to thiamine supplementation and a mild suppurative inflammatory csf. a -year-old female spayed himalayan had an acute onset of ataxia, behavioural abnormalities, absent menace and absence of physiological nystagmus. there was a mildly elevated csf nucleated cell count ( . cells/ml) that was suppurative. the cat had been fed a diet of sulphur dioxide preserved kangaroo meat exclusively for weeks. treatment included thiamine (thiamine hydrochloride; natural health products), mg po q h and a diet change to a balanced cat food. the cat showed a dramatic improvement within h of therapy and was clinically normal at long term follow-up years later. the other was a -year-old male castrated domestic shorthair that also had with an acute onset of ataxia, depression, nystagmus, postural reaction deficits and weight loss. the csf leukocyte count was normal ( cells/ml) but consisted entirely of non-degenerate neutrophils. the cat was treated with clindamycin (antirobe; pharmacia and upjohn) as blood tests revealed elevated bilirubin, alanine aminotransferase (alt), alkaline phosphatase (alp), and a toxoplasma species igg of o . the cat was euthanased days later because of marked deterioration. necropsy revealed changes consistent with thiamine deficiency and hepatic lipidosis. fourteen cats ( . %) with inflammatory cerebrospinal fluid were diagnosed with neoplasia. of these cats, six ( %) had a confirmed diagnosis of lymphoma based on a mononuclear inflammatory csf with abnormal lymphocytes, or when needle aspiration/biopsy of extracranial sites confirmed malignant lymphoid cells. eight ( %) were diagnosed with other neoplasms on the basis of chronic progressive clinical signs, mild mixed inflammatory csf and ancillary laboratory tests (radiographs, myelogram, mri, ct, surgery and biopsy, table ). only three of these cases had a confirmed histopathological diagnosis of the type of neoplasia. six cats were diagnosed with cns lymphoma. the ages ranged from to years with a median of . years. the main clinical signs were gait abnormalities in six cats, reduced conscious proprioception in three cats and a head tilt in two cats. one cat had multiple cranial nerve deficits (nystagmus, anisocoria and facial nerve paralysis). systemic signs included anorexia in one cat and ocular abnormalities in one cat. five cats had a chronic course of disease while one was presented with acute clinical signs. in three cats the clinical signs localised the disease to the spinal cord. two cats had multifocal disease and one cat focal cerebral disease. all six cats had mononuclear inflammation and in five cats, large atypical lymphoid cells were observed in the csf. in the sixth cat, lymphoma was diagnosed via fine needle aspirate biopsy of the kidneys. four cats had mildly elevated csf leukocyte counts ( e cells/ml). the remaining two cats had moderate ( cells/ml) and marked ( cells/ml) cns inflammation. one cat had a normal csf protein level and four had mild elevations ( . e g/l). in one cat the protein was not measured due to insufficient sample. the most common abnormal ancillary tests were non-regenerative anaemia in three and leukocyte abnormalities (varying combinations of eosinophilia, neutrophilia, monocytosis and abnormal peripheral lymphocytes) in three. one cat had abnormal thoracic and abdominal radiographs (sternal lymphadenopathy and hepatomegaly), one had an abnormal myelogram (spinal cord compression), one had lymphoma revealed by fine needle aspirate biopsy of the kidneys and one had a computed tomography scan which revealed a mass in the frontal lobe. the cat with hepatomegaly had lymphoma confirmed by transabdominal hepatic fine needle aspirate biopsy. the cat with the abnormal myelogram had lymphoma of the spinal cord confirmed by surgical fine needle aspirate biopsy. three cats survived less than month, two survived e months and one cat was lost to follow-up. no cat hct z haematocrit, tpp z total protein, bun z blood urea nitrogen, fna z fine needle aspirate, lcat z latex cryptococcal antigen titre, ct z computed tomography, mri z magnetic resonance imaging, other me z other meningoencephalitis (non-suppurative meningoencephalitis and steroid responsive meningoencephalitis), ivdd z intervertebral disc disease, def z deficiency, e z post mortem not undertaken. was recorded to have survived more than months after presentation. no cat had a necropsy performed. of these eight cats, one cat was still alive at the time of the study ( months after presentation), six were euthanased and one died. the ages ranged from to years with a median age of . years. the main clinical signs were gait abnormalities in seven, reduced proprioception in three and other cranial nerve deficits (facial nerve paresis, reduced pupillary light response, absent gag reflex) in three. a variety of systemic signs were observed (upper respiratory tract infection, heart murmur, pale mucous membranes). seven cats had a chronic course of disease while one cat presented acutely. the clinical signs localised the disease to the spinal cord in four cats, brainstem in two cats, and central vestibular system in one cat. in one cat the lesions appeared multifocal (vestibular and spinal). in six cats the inflammation in the csf was mixed and in two it was mononuclear. in one cat the csf nucleated cell count was normal ( cells/ml), in six cats it was mildly elevated ( e cells/ml) and in one cat it was moderately elevated ( cells/ml). six cats had mildly elevated csf protein concentrations ( . e g/l). in two cats the csf protein was not measured because of insufficient sample. five cats had abnormalities on myelogram (widening of spinal cord, intradural mass lesions). one cat had abnormalities on skull radiographs (bilateral radio density in tympanic bullae). one cat had abnormal ct scan (soft tissue density in ethmoid turbinates invading cribiform plate and rostral brain). one cat had an abnormal magnetic resonance image (mass in cerebello-pontine angle, invading petrous temporal bone). other abnormalities seen were one each of leukocytosis, elevated globulins, elevated liver enzymes, elevated creatinine kinase and an abnormal biopsy (nasal carcinoma). four cats survived less than month, one survived months and two cats survived e months. one cat was still alive at the time of the study ( months later). this cat was a -year-old female, spayed chinchilla with neurological signs of a thoracolumbar spinal lesion. myelogram revealed an extramedullary-intradural lesion. surgical removal was performed which revealed an osteosarcoma of the articular facets. the cat was well but mildly paretic on its hindlimbs months after surgery. two cats had a post mortem examination that revealed a cerebral meningioma in one cat and an undifferentiated nerve root tumour in the other. three cats were diagnosed with cns trauma based on history, myelogram and a marked haemorrhagic csf. all had acute onset of disease ( e h). one cat was years of age and two were years of age. the main clinical signs were gait abnormalities in two cats, and one each of increased reflexes, spinal pain, reduced conscious proprioception, urinary and faecal incontinence. no systemic clinical signs were seen. in all three cats the neuroanatomical location was the spinal cord (thoracolumbar in two cats and lumbosacral in one cat). the csf inflammation was mixed in two cats and suppurative in one cat. all cats had a mild elevation in csf protein concentration. myelography was performed in all three cats. this revealed intramedullary swelling in one, loss of spinal cord opacity in another and under opacification with compression of the spinal cord in the third cat. csf haemorrhage was marked in all three cats with evidence of erythrophagia and xanthochromia, however the total nucleated cell counts were higher than could be accounted for with blood contamination alone. all three cats made a full recovery. one cat was diagnosed with intervertebral disc disease. it was a -year-old male castrate domestic shorthair with chronic signs of hindlimb paresis localised to the thoracolumbar spinal cord. the cat had a mild mixed inflammatory csf ( . cells/ml) with a mildly elevated total protein ( . g/l). myelogram revealed spinal cord compression. hemilaminectomy was performed and the cat recovered. two cats were diagnosed with a spinal cord granuloma of unknown cause. both cats had a chronic history of hindlimb paresis and the lesions were localised to the spinal cord in both cats. one cat had a mild mixed inflammatory csf ( cells/ml). the other cat had a moderate suppurative inflammatory csf ( cells/ml). the first cat did not have csf protein measured while the second had a mild elevation in csf protein ( . g/l). myelography revealed extradural spinal cord compression in both cats. surgery and biopsy in both cats revealed granulomatous spinal cord lesions but no underlying cause was found. the first cat was euthanased months later because of a recurrence of clinical signs. a post mortem examination was not performed. the second cat recovered and was still alive years later. eighteen cats ( %) remained without a diagnosis often despite extensive clinicopathological testing (table ) . of these, one cat made a complete recovery and was euthanased years later for renal failure. one cat was lost to follow-up. the remaining cats died or were euthanased. the age range was monthse years with a median age of years. the predominant neurological signs were gait abnormalities ( cats), seizures (nine cats), reduced proprioception (six cats), head tilt (four cats), postural reaction deficits (three cats) and other cranial nerve deficits (facial nerve, trigeminal nerve paralysis) (three cats). the main systemic signs were inappetence, weight loss and pyrexia in two cats each. sixteen cats had a chronic course of disease and two cats had acute clinical signs. the neuroanatomical location was multifocal in eight cats, cerebral in four cats, brainstem in five cats, central vestibular in two cats and spinal cord in three cats. the type of inflammation was mixed in six cats, suppurative in eight, mononuclear in three and eosinophilic in one cat. three cats had a normal csf white cell count, had mild elevations in the csf nucleated cell counts ( e cells/ml) while three cats had moderate elevations ( e cells/ml). the csf protein concentration was normal in four cats and mildly elevated in cats ( . e g/l). in two cats the csf protein concentration was not measured due to insufficient sample. three cats had blood leukocyte abnormalities (neutropenia, lymphopenia and monocytosis). three cats had elevated globulins, two had azotaemia, one cat had elevated t and one had elevated liver enzymes. eleven cats survived less than month, five cats e months, one cat was lost to follow-up and one cat recovered. two cats underwent a post mortem examination, in which a cause for chronic partial seizures, behavioural changes and hemiparesis in one cat and seizures with cranial nerve deficits in the other cat was not found. the purpose of this study was to establish if an accurate diagnosis in cats with inflammatory csf could be established by clinical signs, csf analysis and ancillary diagnostic tests. a previous study (rand et al b) failed to demonstrate any significant difference in csf parameters between cats diagnosed with primary inflammatory, degenerative, or neoplastic disease of the cns. this may reflect the presence of secondary inflammation and degeneration associated with many cns disorders. categorising csf as 'inflammatory' depends on a number of factors such as the method of collection (particularly peripheral blood contamination), laboratory reference ranges, loss of cells in csf due to delays between sample collection and analysis and experience of the technician interpreting the sample. to control these variables, all the reference ranges chosen were those used by the laboratories involved in processing the sample. all samples were evaluated by an experienced veterinary pathologist within h of collection. peripheral blood contamination is a common confounding problem in csf collection despite good technique. normal csf should not contain any erythrocytes, however up to red blood cells (rbcs/ml) is considered 'normal' contamination as it does not have a significant effect on the csf white cell count (rand et al ) . correcting for peripheral blood contamination using the ratio of red cells to white cells in the peripheral blood may not be an accurate indicator of the effects of blood contamination on csf fluid (wilson and stevens ) . rand et al ( ) suggested a formula for the correction of csf white blood cell counts based on the csf red blood cell number. this study also showed that the formula was likely to overestimate the degree that blood contamination elevated csf white cell counts in approximately % of cases. the maximum expected increase in white cells is calculated based on the assumption that there is one additional white cell/ml of csf per red cells/ml of csf. in this study, cases were excluded if blood contamination was significant enough to explain the elevated white cell count. thus, all cats with true cns inflammation would be included but is possible that some cats with true inflammation were excluded. location of csf collection may alter protein concentration and cell counts. lumbar csf white cell counts can be less and protein concentrations higher than that of cisternally collected csf in dogs. the reason for the reduced white cell counts in the lumbar csf is not known. possible causes include cell lysis, less cells entering the lumbar csf or migration of white cells from lumbar csf to blood (bailey and higgins ) . increased protein concentration in the lumbar csf may be caused by sluggish spinal csf circulation resulting in protein accumulation. similar differences have been observed in feline csf protein levels (hochwold et al ) . changes in white blood cell counts between lumbar and cisternal punctures have not been compared in cats. csf was collected from both sites as this study encompassed cases from three different referral hospitals and the method of collection was chosen according to the preference of the veterinarian involved. often, the area of collection was not specified in the data. differences may have occurred but this could not be accounted for in the present study. fip was the most common cause of inflammatory csf and accounted for % of the cases. this is similar to a previous study (rand et al a (rand et al , b where % of cats with primary inflammatory and primary non-inflammatory cns disease were diagnosed with fip. fip is a difficult disease to diagnosis antemortem. the 'gold standard' for diagnosis is histopathology which reveals a perivascular proliferation of macrophages, lymphocytes plasma cells and neutrophils with a central area of necrosis (hartmann et al ) . it has been shown that evaluation of albumin:globulin ratio (! . ) and anti-coronavirus antibody in body cavity effusions (any titre) had a good positive predictive value for the diagnosis of fip. in addition, the highest serum anti-coronavirus antibody titre ( : ) also had a good positive predictive value however, any serum antibody titre less than this was not valuable (hartmann et al ) . while nine cases of fip in this study were diagnosed via histopathology, two cases were diagnosed via the clinical and serological criteria. the next most common disease category in our study was neoplasia, with both confirmed lymphoma and undetermined forms accounting for . % of the cases. in contrast, rand et al ( a rand et al ( , b found that % of cases (combined primary inflammatory and non-inflamma-tory cns disease) were diagnosed with nonsuppurative meningoencephalitis of suspected viral origin. quesnel et al ( ) found % of cats presented for seizure disorders to have nonsuppurative meningoencephalitis of possible viral cause. our study found that only three cats had findings that were supportive of non-fip viral infection. the diagnosis is suggested by mononuclear meningitis without visceral lesions of fip and perivascular cuffing as the only histologic lesion (lundgren ) . it has been postulated that certain viruses, eg, parvovirus, feline herpesvirus, calicivirus, fiv and felv could all cause these histopathological lesions. the arboviruses have received special interest in canada. there are six strains of arbovirus known to cause encephalitis in humans in canada (artsob and spencer ) . some of these are also known to be endemic in the wild animal population and experimental infections in cats (powassan virus) have caused non-suppurative meningoencephalitis (keane et al , rand et al a . there are more than strains of arbovirus in australia, only two of which are known to cause encephalitis in humans (murray river and kunjiin virus) (boughton ) . the cause of feline non-suppurative meningoencephalitis is not known but the apparent variation in the diagnosis of this condition may reflect differences in causative agent(s) seen in the two geographical locations. the median age for cats in this study was years and was reflected in all disease categories except fip, which was markedly lower (median age year). overall, the most common systemic clinical signs were weight loss ( %) and ocular abnormalities ( %), apart from fip where pyrexia was the most common clinical finding ( %). in all groups, the majority of cats had gait abnormalities as the major neurological sign ( %). neuroanatomical location did not help in differentiating between disease categories as many cats were diagnosed with multifocal/ diffuse disease ( %). exceptions to this were cats with lymphoma, other neoplasia, spinal cord granuloma and trauma in which the spinal cord was the most frequent neuroanatomical location. csf protein concentration may provide some help in categorising disease groups. the only cats with moderate or marked csf protein elevations (o g/l) were cats with fip ( %), one cat with cryptococcus ( . g/l) and one case of other meningoencephalitis (steroid responsive meningitis) ( . g/l). cats with fip had, by far, the highest csf protein levels ( and g/l). hence, markedly elevated protein concentrations should increase the index of suspicion for fip. the lack of protein measurements in % of the cases as a consequence of insufficient sample, may have skewed the interpretation. the degree of cns inflammation was inconsistent within groups. severe inflammatory csf was observed more commonly in fip cases ( %). other categories had variable degrees of inflammation. the type of inflammatory pattern was helpful only in cats with lymphoma as all had a mononuclear inflammation. mixed inflammation of the csf was the most common type of inflammatory pattern observed ( . %) and when observed did little in aiding the diagnosis. interestingly, both cats with thiamine deficiency had a mild suppurative inflammation. to the author's knowledge, there are no reports in veterinary or human literature on the csf findings with thiamine deficiency. as csf collection is a diagnostic test, it is not required if the diagnosis is known. a diagnosis of thiamine deficiency can often be suspected from dietary history and clinical signs. thiamine deficiency results in cerebrocortical necrosis (read and harrington , steel ) that could result in a mild suppurative csf infiltrate. seventy-seven percent of cats survived less than year. three cats died or were euthanased immediately after csf collection. this represents almost % of cases and serves to illustrate that cats with central nervous system disease can be poor anaesthetic risks and that csf collection can be associated with significant morbidity and mortality. cats diagnosed with cryptococcus had a better prognosis ( % still alive more than years after presentation), as did those diagnosed with trauma and steroid responsive meningitis ( % still alive more than months after presentation). survival times were variable in cats with toxoplasmosis, non-suppurative meningitis and thiamine deficiency (table ). this study revealed that csf analysis alone was helpful only in cns fip, cryptococcus, lymphoma and trauma. in other conditions a presumptive diagnosis could be made using a combination of antemortem findings (age, csf cellular and protein levels, serum and effusion antibody titres, biopsy and outcome). neuroanatomical location provided some indication of the diagnosis in cats with lymphoma, other neoplasia, spinal cord granuloma or trauma (spinal cord). the degree of csf inflammation, csf protein concentration and systemic signs provided little contributory information to the final diagnosis other than in cats with fip. despite often extensive diagnostic evaluation, % of cats was left without a premortem aetiological diagnosis. future prospective studies with thorough post mortem examination are warranted as post mortem evaluation was only performed in this retrospective study in of the cases. the increased use of advanced imaging (ct/mri), csf antibody, molecular biological methods, and cns biopsy may aid in the diagnosis of cns disease in the living patient. arctic and tropical arboviruses comparison of total white blood cell count and total protein content of lumbar and cisternal cerebrospinal fluid of healthy dogs arboviruses and disease in australia veterinary cytology: a bench manual for the canine and feline practitioner comparison of different tests to diagnose feline infectious peritonitis california serogroup: powassan virus infection of cats feline non-suppurative meningoencephalomyelitis. a clinical and pathological study consultations in feline internal medicine diagnostic evaluation of cats with seizure disorders: cases ( e ) reference intervals for feline cerebrospinal fluid: cell counts and cytological features clinical, cerebrospinal fluid, and histological data from twenty-seven cats with primary inflammatory disease of the central nervous system clinical, cerebrospinal fluid, and histological data from thirty-four cats with primary non-inflammatory disease of the central nervous system experimentally induced thiamine deficiency in beagle dogs: pathologic changes of the central nervous system thiamine deficiency in a cat associated with the preservation of 'pet meat' with sulphur dioxide effects of blood contamination on cerebrospinal fluid analysis the authors would like to thank dr jody braddock, dr carolyn o'brien, the reception staff at the university veterinary centre sydney and dr elizabeth dill-macky, the animal referral hospital, sydney for their assistance in providing cases for this study. we would like to thank the staff of central sydney imaging, royal prince alfred hospital medical centre for providing computed tomography, magnetic resonance imaging equipment and assistance in interpretation. we would also like to thank dr colin dunlop, advanced anaesthesia specialists for anaesthesia of the above cases, idexx laboratories and the clinical pathology department of the university of sydney for laboratory testing. key: cord- -suiqh gv authors: lafolie, jérémy; labbé, andré; l'honneur, anne sophie; madhi, fouad; pereira, bruno; decobert, marion; adam, marie noelle; gouraud, françois; faibis, frédéric; arditty, francois; marque-juillet, stéphanie; guitteny, marie aline; lagathu, gisele; verdan, matthieu; rozenberg, flore; mirand, audrey; peigue-lafeuille, hélène; henquell, cécile; bailly, jean-luc; archimbaud, christine title: assessment of blood enterovirus pcr testing in paediatric populations with fever without source, sepsis-like disease, or suspected meningitis: a prospective, multicentre, observational cohort study date: - - journal: lancet infect dis doi: . /s - ( ) - sha: doc_id: cord_uid: suiqh gv background: enteroviruses are the most frequent cause of acute meningitis and are seen increasingly in sepsis-like disease and fever without source in the paediatric population. detection of enterovirus in cerebrospinal fluid (csf) specimens by pcr is the gold standard diagnostic test. our aim was to assess a method of detecting enterovirus in blood specimens by pcr. methods: we did a prospective, multicentre, observational study at french paediatric and emergency departments in hospitals. we recruited newborn babies (aged ≤ days) and infants (aged > days to ≤ years) with fever without source, sepsis-like disease, or suspected meningitis, and children (aged > years to ≤ years) with suspected meningitis, who were admitted to a participating hospital. we used a standardised form to obtain demographic, clinical, and laboratory data, which were anonymised. enterovirus pcr testing was done in blood and csf specimens. findings: between june , , and oct , , and between june , , and oct , , we enrolled patients, of whom had enterovirus pcr testing done in blood and csf specimens. enterovirus was detected in ( %) patients in either blood or csf, or both ( newborn babies, infants, and children). detection of enterovirus was more frequent in blood samples than in csf specimens of newborn babies ( [ %] of vs [ %] of ; p= · ) and infants ( [ %] of vs [ %] of ; p= · ), and was less frequent in blood samples than in csf specimens of children ( [ %] of vs [ %] of ; p< · ). detection of enterovirus was more frequent in blood samples than in csf specimens of infants aged years or younger with fever without source ( [ %] of vs [ %] of ; p= · ) or with sepsis-like disease ( [ %] of vs nine [ %] of ; p= · ). detection of enterovirus was less frequent in blood than in csf of patients with suspected meningitis ( [ %] of vs [ %] of ; p< · ). interpretation: testing for enterovirus in blood by pcr should be an integral part of clinical practice guidelines for infants aged years or younger. this testing could decrease the length of hospital stay and reduce exposure to antibiotics for low-risk patients admitted to the emergency department with febrile illness. funding: university hospital clermont-ferrand. enteroviruses are the most frequent cause of paediatric aseptic meningitis and are attributed to more than % of viral meningitis cases in which a microorganism is identified. , detection of enterovirus by rt-pcr from cerebrospinal fluid (csf) specimens is recom mended for diagnosis of meningitis caused by entero virus. [ ] [ ] [ ] paediatricians are also confronted frequently with young infants with fever without source or sepsis-like diseases. these febrile illnesses account for · - · % of cases seen in emergency departments. symptoms can result either from severe bacterial infection requiring admission to hospital and empirical antibiotic treatments or, most typically, from benign and spontaneously resolving viral infection; therefore, diagnosis is a challenge. additional molecular tests are needed to speed up diagnosis of conditions asso ciated with enterovirus infections. several studies have evaluated testing blood specimens, [ ] [ ] [ ] [ ] [ ] [ ] but as yet no assessment has been done in a large cohort of paediatric patients. the aim of our multicentre study was to assess detection of enterovirus by pcr in blood specimens of newborn babies, infants, and children with fever without source, sepsis-like disease, or suspected meningitis. we did a prospective, multicentre, observational study at paediatric and emergency departments in french hospitals. we restricted enrolment of patients to the seasonal period of increased enterovirus circulation in countries with a temperate climate. we enrolled newborn babies (aged ≤ days) and infants (aged > days to ≤ years) with fever without source, sepsis-like disease, or suspected meningitis, and children (aged > years to ≤ years) with suspected meningitis. all participants were admitted to one of the participating hospitals. we required an edta blood sample (plasma) obtained by venepuncture for participation. we also did lumbar puncture when clinically indicated. we defined fever without source as a body temperature of °c or higher for less than days in a child whose medical history and physical examination did not reveal the cause of the fever. we defined sepsis as suspected or proven infection and at least two of another four criteria, one of which had to be abnormal temperature (> · °c or < °c) or abnormal white-blood-cell count (elevated [> × ⁹ per l] or depressed [< × ⁹ per l] for age), and the other criterion could be either tachycardia or bradycardia, or tachypnoea. further criteria for sepsis were a platelet count lower than × ⁹ per l and c-reactive protein greater than mg/l. we defined meningitis as either the presence of age-specific pleocytosis or the presence of at least two of these neurological signs or symptoms: headache, nuchal rigidity, photophobia, bulging fontanelle, irritability, lethargy, nausea, vomiting, or positive kernig's or brudzinsky's signs. we defined pleocytosis as a white-blood-cell count in the csf of more than per µl for newborn babies (aged ≤ days), ten per µl or more for infants (aged - days), and five per µl or more for older children (aged > days). exclusion criteria were refusal of consent from parents, absence of or insufficient blood samples, and bacterial or other viral infections in blood or csf specimens. we also excluded patients (at a later stage) who were diagnosed evidence before this study we searched pubmed up to feb , , for papers reporting paediatric enterovirus diseases and enterovirus pcr testing or molecular detection of viruses in cerebrospinal fluid (csf) or blood specimens of patients with aseptic meningitis, sepsis and sepsis-like disease, or fever without source. we used the search terms "enterovirus", "nonpolio enterovirus", "meningitis", "viral meningitis", "aseptic meningitis", "enterovirus meningitis", "acute meningitis", "sepsis", "sepsis-like disease", "fever", "fever without source", "genome detection", "enterovirus detection", "enterovirus rt-pcr", "molecular detection", "viremia", "viremic", "virus load", "blood", "plasma", and "cerebrospinal fluid". we also reviewed references from relevant articles not identified in the original search. our search identified studies in which enterovirus detection was reported in blood and csf. most studies were retrospective, the number of patients recruited varied between and , and blood samples were not obtained in all patients whose csf was tested. two studies of and patients aged days or younger with enterovirus infection were referenced to discuss our enterovirus detection frequency in the blood and csf of febrile infants. in a study of patients aged years or younger with aseptic meningitis, % had enterovirus detected in blood samples by pcr. however, in that study, age groups were not analysed separately. in all these studies, symptom duration before lumbar puncture or venepuncture, and time between csf and blood collection, were not recorded. our study of patients with laboratory findings of enterovirus infection is, as far as we are aware, the largest prospective, multicentre, observational study to assess pcr testing for enterovirus in both blood and csf samples from newborn babies (aged ≤ days) and infants (aged > days to ≤ years) with fever without source, sepsis-like disease, or suspected meningitis, and children (aged > years to ≤ years) with suspected meningitis. to our knowledge, our study is the first to show that sensitivity of enterovirus detection in blood samples is related to patients' age and clinical presentation. detection of enterovirus was more frequent in blood samples than in csf specimens of newborn babies and infants with fever without source or sepsis-like diseases, and it was less frequent in blood samples of patients with suspected meningitis. furthermore, our study showed that enterovirus positivity in blood was related inversely to patient's age with meningitis. at present, blood samples from febrile patients in the paediatric emergency department are not sent routinely for enterovirus pcr testing, and only csf samples are sent for infants younger than days or for patients with suspected meningitis. guidelines for biological management of patients aged years or younger with febrile illness in the emergency department need to be reconsidered. when blood is sampled for a complete blood count, an additional blood tube should be obtained for enterovirus pcr testing, which can now be done sufficiently rapidly to have a real effect on infection management. pcr testing of blood samples done in routine practice could result in a more accurate assessment of the actual number of positive cases in patients with suspected meningitis, sepsis-like diseases, and fever without source. a positive enterovirus diagnosis could affect beneficially decisions about a patient's management, by reducing antibiotic or antiviral therapy, avoiding ancillary tests, lowering hospital-related costs, and allowing earlier discharge. with infections in other biological specimens (eg, urine, nasopharyngeal aspirate, or stool). the study was approved by the french ethics committee (au ), under the institutional review board number . we obtained verbal consent for use of clinical samples for research from parents or guardians of children aged years or younger and from children older than years. within h of admission, a doctor completed a standardised questionnaire for every patient, with details of the nature and duration of preadmission symptoms and signs and the results of a physical examination done at the time of admission. laboratory findings comprised the date and time at which biological specimens were taken, csf and full blood count characteristics, c-reactive protein assay, and the results of other bacteriological and virological testing of samples recorded by biologists. symptom duration was the interval between onset of symptoms and venepuncture (and lumbar puncture, if indicated). we estimated the onset of symptoms as either am, pm, pm, or am when symptoms were recorded in the morning, afternoon, evening, and night, respectively. csf protein concentration was classified as normal if it was · g/l or lower for newborn babies (aged ≤ days) and · g/l or lower for older children (aged > days). investigation for urinary-tract infection in febrile patients was done with urine dipsticks and confirmed by culture of specimens obtained from urethral catheters. a urinary-tract infection was diagnosed as leucocytosis (≥ ⁴ cells per ml) and clinically significant bacteria (≥ ⁵ colony-forming units per ml) in urine culture. all samples were submitted for routine bacteriological and virological investigations at every participating hospital, according to local practice. a senior paediatrician and the study team reviewed the final diagnosis at discharge. we did pcr testing for enterovirus in blood and csf specimens at microbiology laboratories of five participating hospitals: centre hospitalier universitaire (chu) de clermont-ferrand (clermont-ferrand), cochin hospital (assistance publique-hôpitaux de paris, paris), grand hôpital de l'est francilien, site de meaux (meaux), centre hospitalier de versailles andré mignot (versailles), and chu de rennes (rennes). we used commercial (xpert ev, cepheid, sunnyvale, ca, usa [only used with csf samples]; enterovirus r-gene, biomérieux, marcy l'etoile, france; and o-diaent, diagenode, seraing, belgium) or in-house rt-pcr assays. a diagnosis of enterovirus was established with positive pcr findings in either plasma or csf, or both. for specimens negative for enterovirus on pcr testing, and if the volume of sample remaining was sufficient, we did a specific rt-pcr parecho virus assay in blood and csf (parechovirus r-gene, biomérieux). we typed enterovirus-positive specimens at the national reference centre for enteroviruses and parechoviruses (clermont-ferrand, france) by amplification and sequencing of the vp capsid protein. we did statistical analyses with stata . statistical tests were two-sided with a type i error set at an α of · . we presented continuous data as median (iqr) for every age group (newborn babies, infants, and children). we estimated κ coefficients and sensitivity, specificity, and predictive values (with % cis) for blood enterovirus pcr testing and compared these with values in csf to ascertain validity. we judged κ values according to recommendations: less than · (negligible), · - · (low or weak consistency), · - · (moderate agreement), · - · (substantial or good agreement), and greater than · (excellent agreement). for comparisons between groups, we used χ² or fisher's exact tests for categorical variables, then (when appropriate) we did marascuilo's procedure. for quantitative parameters, we used student's t test or the mann-whitney test when assumptions of the t test were not met. we did a regression model for newborn babies and infants to identify factors that were associated independently with viraemia, using a stepwise (backward and forward) approach. we ascertained covariates according to univariate results (entry at p= · ) and relevant biological and clinical variables-eg, csf whiteblood-cell count, duration of symptoms, tachycardia, hypotonia, irritability, and seizure (adjustment factors). we paid attention to multicollinearity. we expressed results as odds ratios (ors) and % cis, and we represented findings using forest plots. we did a sensitivity analysis for multivariate analysis, which we applied to all groups. we also did a sensitivity analysis to assess the effect of any inaccurate dates or times of symptom onset recorded by parents. the funder had no role in study design, data collection, data analysis, data interpretation, or writing of the report. ca and al had full access to all data in the study and had final responsibility for the decision to submit for publication. csf=cerebrospinal fluid. *parents did not agree to participation of their child after reading the information leaflet. †pcr inhibitors were present either in blood (n= ) or in csf (n= ) samples. of those with pcr inhibitors in blood, two had negative csf (one infant and one child) and one had positive csf (newborn baby). of those with pcr inhibitors in csf, one had negative blood (infant) and two had positive blood (newborn baby and infant). three patients had enterovirus infection. all six patients were excluded from the analysis. data are n (%) or median (iqr), unless otherwise indicated. csf=cerebrospinal fluid. +=enterovirus pcr positive. -=enterovirus pcr negative. *time between onset of symptoms and venepuncture or lumbar puncture was significant between children and infants and between children and newborn babies (p< · ). †time between onset of symptoms and lumbar puncture was significant between infants and newborn babies (p= · ). a multivariate analysis that adjusted for age, duration of symptoms, hypotonia, irritability, and all factors judged significant in univariate analysis confirmed that patient's age, tachycardia, and exposure to a sick contact were associated significantly with viraemia in patients aged years or younger (figure a). in a further multivariate analysis, including patients in all three age groups, the time between onset of symptoms and venepuncture (< h, or · , % ci · - · ; p= · ; h to < h, · , · - · ; p= · ) and pleocytosis ( · , · - · ; p< · ) were also associated with viraemia ( figure b) . data for patients in each age group with and without enterovirus infection, who had samples available for pcr testing, were compared for differences in symptoms and laboratory characteristics ( (p= · ), hypotonia (p= · ), and nausea or vomiting (p< · ) than were those without infection. patients of all ages infected with enterovirus were more likely to have been exposed to a sick contact than were those without infection (p≤ · ). newborn babies infected with enterovirus were more likely to show symptoms of irritability (p= · ) and hypotonia (p= · ) compared with those testing negative for enterovirus. tachycardia was more frequent in infants infected with enterovirus than in those not infected with enterovirus (p= · ). all patients recovered from the enterovirus infection. pleocytosis was recorded in ( %) of patients infected with enterovirus compared with ( %) of who were not infected (p= · ). pleocytosis increased with patient's age, with ( %) of newborn babies, ( %) of infants, and ( %) of children having pleocytosis. amounts of protein in csf were similar in patients infected and not with enterovirus. blood lymphocyte counts were lower in patients of all ages infected with enterovirus than in those not infected (p≤ · ). prospective enterovirus typing was done for ( %) of patients whose csf or blood specimens, or both, were positive for enterovirus (appendix p ). viral strains were assigned to different types, eight within the entero virus a species ( patients) and within the enterovirus b species ( patients). the enterovirus genotypes e , e , e , and cvb were more frequent in newborn babies than in infants and children; patients with suspected sepsis were more likely to be infected with e genotype than were patients with other clinical presentations; and those with suspected meningitis were more frequently infected with e , e , or cvb genotypes. our study shows that, in newborn babies and infants, the sensitivity of enterovirus pcr testing is higher in blood samples than in csf specimens. this finding substantiates those of previous single-centre studies [ ] [ ] [ ] and lends support to use of blood enterovirus testing as a diagnostic adjunct to rapidly identify newborn babies and infants admitted with fever without source, sepsislike disease, or suspected meningitis whose antibiotic treatment can be discontinued and who are eligible for discharge. our study also shows that blood enterovirus pcr testing in children with suspected meningitis has no additional benefit compared with pcr testing in csf. seizures tachycardia exposure to a sick contact hypotonia irritability time between onset of symptoms and blood sample ≥ h time between onset of symptoms and blood sample - h time between onset of symptoms and blood sample < h white blood cell count pleocytosis c-reactive protein level (> mg/l) this lower sensitivity, compared with that recorded in newborn babies and infants, was attributable mainly to the long period between onset of symptoms and venepuncture. to our knowledge, we report here the largest, prospective, multicentre, observational study to show that positive detection of enterovirus in blood is associated with patient's age and clinical presentation. detection of enterovirus was significantly higher in blood samples than in csf specimens from newborn babies and infants and varied by clinical presentation, with detection higher in patients admitted with fever without source or sepsis-like diseases than in those with suspected meningitis. a positive result for enterovirus in blood samples from patients with suspected meningitis was related inversely to age, with higher detection in newborn babies ( %), then infants ( %), then children ( %). compared with previous reports, enterovirus was detected in blood samples from newborn babies and infants more frequently in our study. in a study of infants (aged ≤ days) with fever who were infected with enterovirus, pcr yielded equally positive results in blood samples and csf specimens ( % and %, respectively). in another study of infants with suspected sepsis, detection of enterovirus was similar in blood and csf samples ( % vs %). our study followed guidelines from the uk national institute for health and care excellence (nice) for clinical management in emergency departments of newborn babies, infants, and children with febrile illness, . these data suggest that viraemia occurs early and is of short duration, a finding rarely noted in other reports and only with small patient populations. , in some biological diagnostic practices, csf pcr testing is done only in patients with pleocytosis. in our study, we did lumbar puncture in patients, of whom did not have pleocytosis (data not shown). in these patients, however, enterovirus was detected in csf (n= ) and blood (n= ). without detection of enterovirus in blood or csf samples, these patients without pleocytosis would have been discharged without aetiological diagnosis. thus, the diagnostic practice to do enterovirus testing solely in patients with pleocytosis can lead to enterovirus infections being missed. moreover, patients had enterovirus pcr testing done only in blood samples, of whom ( %) had enterovirus infection-mostly newborn babies or infants with fever without source. ahmad and colleagues detected enterovirus in blood samples from ( %) of neonates with sepsis-like illness. the diversity of prevailing enterovirus genotypes during the two seasons of the present study and differences in the distribution of genotypes among age white-blood-cell count (× ⁹ per l) harvala and colleagues reported higher or similar viral loads in csf compared with plasma in children younger than years with cns disease. they found low or undetectable csf viral loads and high plasma viral loads in children with sepsis. a previous study by our group showed that among patients with acute meningitis, enterovirus viral loads in csf were higher in newborn babies than in infants and adults and that genotypes were associated with different viral loads. the analysis of clinical and biological characteristics in patients aged years or younger showed that viraemia was detected more frequently in younger infants (median age days [iqr - ]) with acute-stage disease, as suggested in earlier retrospective studies of smaller patient populations. , clinically, tachycardia was present in ( %) of young patients with viraemia and fever without source, sepsis, or suspected meningitis. other reports have cited fever, irritability, lethargy, and poor feeding in patients with enterovirus viraemia. , in our study, patients with enterovirus viraemia were also more likely to have been exposed to a sick contact. all patients with viraemia in our study were febrile. amounts of c-reactive protein in serum greater than mg/l were detected more frequently in patients without viraemia. pleocytosis was noted in ( %) of patients with enterovirus viraemia, mainly those with suspected meningitis (data not shown). dagan and colleagues reported an inverse relation between viraemia and pleocytosis. the number of lymphocytes and monocytes was significantly lower in patients with viraemia than in those without viraemia. this finding and earlier data , suggest a detrimental effect of enterovirus infection on populations of mononuclear leucocytes during viraemia. this effect can be caused by virus replication in mononuclear leucocytes, because some enterovirus genotypes can replicate in vitro in human peripheral blood mononuclear cells. , accordingly, the concentrations of circulating mono nuclear cells at different ages and the ability of entero virus genotypes to replicate in these cells can affect the sensitivity of blood enterovirus detection. concomitant bacterial infections, mostly urinary-tract infections, occurred in a small proportion ( [ %] of ) of patients with fever and enterovirus infection. amounts of c-reactive protein in serum greater than mg/l were noted in five ( %) of these patients (data not shown). the frequency of concomitant enterovirus and bacterial infections was consistent with that in other studies. , , no enterovirus and bacterial coinfections were identified in csf of patients with meningitis and in blood samples of patients with sepsislike disease. our study has three limitations. first, the date and time of symptom onset were recorded less reliably by parents of children than by those of newborn babies and infants. a sensitivity analysis (data not shown) excluding the inaccurate dates and times of symptom onset for ( %) of patients did not affect our results. second, virology management (including detection of viruses other than enterovirus) and bacteriology management varied between the hospitals taking part. in ( %) of patients, no diagnosis was established, a figure similar to that reported by ahmad and colleagues ( %). third, four different rt-pcr assays were used in our multicentre study. it is unlikely that this variability affected the overall results, because methods used are assessed annually by an external quality assess ment programme, and all yielded correct results. in conclusion, detection of enterovirus in blood improved diagnostic yield in newborn babies and infants admitted for fever without source, sepsis-like disease, or suspected meningitis, compared with detection in csf. the high frequency of detection of enterovirus in blood samples from very young patients with fever without source and sepsis-like disease suggests that enterovirus febrile illnesses are underdiagnosed. it is important to reconsider the guidelines for biological management of patients aged years or younger with febrile illness in emergency departments, to obtain-at the time of blood sampling-an additional tube for enterovirus pcr testing, which can be done sufficiently rapidly to have a real effect on management. a positive enterovirus blood diagnosis could beneficially affect patient management decisions by reducing antibiotic or antiviral therapy, avoiding ancillary tests, lowering hospital-related costs, and allowing earlier discharge. blood enterovirus genome can also be used as an alternative biomarker in case of contraindications for csf sampling and failure of lumbar puncture. service urgences pédiatriques inserm, cic service de microbiologie clinique des hupssd service de microbiologie clinique des hupssd service pédiatrie générale et urgences pédiatriques service de microbiologie et hygiène, hôpitaux universitaires service de pediatrie-néonatologie laboratoire de biologie médicale viral infections of the central nervous system in spain: a prospective study hospital admissions for viral meningitis in children in england over five decades: a population-based observational study impact of rapid enterovirus molecular diagnosis on the management of infants, children, and adults with aseptic meningitis improvement of the management of infants, children and adults with a molecular diagnosis of enterovirus meningitis during two observational study periods recommendations for enterovirus diagnostics and characterisation within and beyond europe epidemiology of a pediatric emergency medicine research network: the pecarn core data project diagnosis and outcomes of enterovirus infections in young infants a polymerase chain reaction-based epidemiologic investigation of the incidence of nonpolio enteroviral infections in febrile and afebrile infants days and younger prospective comparison of the detection rates of human enterovirus and parechovirus rt-qpcr and viral culture in different pediatric specimens epidemiology of sepsis-like illness in young infants: major role of enterovirus and human parechovirus frequency of enterovirus detection in blood samples of neonates admitted to hospital with sepsis-like illness in kuwait enterovirus and parechovirus viraemia in young children presenting to the emergency room: unrecognised and frequent surveillance of enteroviruses in france management of fever without source in infants and children report on the expert meeting on neonatal and paediatric sepsis defining cerebrospinal fluid white blood cell count reference values in neonates and young infants rapid routine detection of enterovirus rna in cerebrospinal fluid by a one-step real-time rt-pcr assay prospective identification of enteroviruses involved in meningitis in through direct genotyping in cerebrospinal fluid quality criteria were proposed for measurement properties of health status questionnaires fever in under s: assessment and initial management-clinical guideline (cg ) viremia in hospitalized children with enterovirus infections the correlation between the presence of viremia and clinical severity in patients with enterovirus infection: a multi-center cohort study point sur les infections à entérovirus au décembre distinct systemic and central nervous system disease patterns in enterovirus and parechovirus infected children variations in cerebrospinal fluid viral loads among enterovirus genotypes in patients hospitalized with laboratory-confirmed meningitis due to enterovirus characteristics of young infants in whom human parechovirus, enterovirus or neither were detected in cerebrospinal fluid during sepsis evaluations replication of enteroviruses in human mononuclear cells enterovirus receptors and virus replication in human leukocytes enterovirus neurological disease and bacterial coinfection in very young infants with fever this study was supported by a grant from the university hospital of clermont-ferrand aoi (appel d'offre interne) . we gratefully acknowledge the blood enterovirus diagnosis infection (bledi) in paediatric population study team, who contributed to data collection from french participating hospitals. we thank emilie leroy and nathalie rodde for assistance with virus genotyping; and jeffrey watts for help preparing the article (funded with a grant from the university hospital of clermont-ferrand aoi ). key: cord- - ny j ny authors: cuddon, paul a title: the weak and ataxic or paralyzed cat date: - - journal: problem-based feline medicine doi: . /b - - - - . - sha: doc_id: cord_uid: ny j ny nan ataxia and paresis occur when there is physical or functional disruption to both the motor and sensory pathways of the nervous system. paresis is a deficit of voluntary movement, leading to weakness in one limb (monoparesis), both pelvic limbs (paraparesis), the limbs on one side of the body (hemiparesis), or all four limbs (tetraparesis). it is caused by disruption of the voluntary motor pathways anywhere from the cerebral cortex through the brainstem and spinal cord to the spinal cord segments and the peripheral nerves supplying muscles. neurological testing to determine whether a cat is paretic includes gait analysis and postural reaction testing, which includes hopping, wheel-barrowing, hemi-standing/hemi-walking, and the extensor postural thrust. the term paralysis is reserved for the patient who has a complete loss of any voluntary movements. ataxia is a lack of coordination of the limbs or trunk produced by disruption of the sensory proprioceptive pathways of the spinal cord and brainstem. lesions of the cerebellum and the vestibular system also can produce ataxia. sensory ataxia, the disruption of sensory proprioceptive pathways, can be assessed via such tests as proprioception and tactile placing. most cats with spinal cord disease have a combination of both ataxia and paresis, since most myelopathies cause disruption of both the motor and sensory systems. in many circumstances, therefore, separation of ataxia and paresis becomes almost impossible on routine neurological examination. cats presenting solely with ataxia and paresis/paralysis most commonly have spinal cord disease. weakness and ataxia can also occur with cerebral and brainstem disease. • however, cats with cerebral disease will usually show seizures, behavior change, aimless wandering, and pacing along with the ataxia and contralateral hemiparesis or tetraparesis. • cats with brainstem disease will demonstrate cranial nerve abnormalities as well as an ipsilateral hemiparesis or tetraparesis. most polyneuropathies manifest as generalized weakness without ataxia. in many cases of peripheral nerve disease, sensory tests such as proprioception and tactile placing will be normal despite obvious muscle weakness. localization of myelopathies is dependent on determining whether the thoracic and pelvic limbs have upper (umn) or lower motor neuron (lmn) signs. cats with umn paresis will demonstrate normal to increased segmental spinal reflexes (myotatic or tendon reflexes, muscle tone and withdrawal reflexes) whereas cats with lmn paresis will show decreased to absent segmental spinal reflexes. an umn bladder, likewise, will have increased tone and be difficult to express due to external (+/− internal) urethral sphincter hypertonia, whereas a lmn bladder will be flaccid (or atonic) and easily expressed due to external urethral sphincter hypo-to atonia. the classic spinal cord divisions when localizing a myelopathy are as follows: • cervical spinal cord (c -c ) -umn tetraparesis to tetraplegia. • cervical intumescence (c -t ) -lmn signs to thoracic limbs and umn signs to pelvic limbs. the most common causes of spinal cord ataxia and paresis in cats are infectious (including feline infectious peritonitis virus (coronavirus)), neoplasia (lymphosarcoma) and trauma. primary spinal neoplasia (meningioma), inflammation (feline polioencephalomyelitis), and ischemia (aortic thromboembolism and fibrocartilaginous embolism) are less common. intervertebral disc disease is very rare. spinal lymphosarcoma*** • chronic progressive ataxia. • asymmetric paraparesis. • focal areas of spinal pain. lymphosarcoma is the most common feline neoplasia. - % of cats with lymphoreticular malignancies develop neurologic involvement. % of cats with cns involvement show thoracic and lumbar myelopathy, although the brain may also be affected. epidural lymph channels and extramedullary hematopoietic tissue are possible sites for development of primary spinal lymphosarcoma. tumor growth commonly occurs longitudinally along the spinal canal (the epidural space is a low-resistance channel). % of cats with spinal lymphosarcoma have solitary epidural lesions. multifocal lesions are also possible, as is tumor involvement of multiple nerve roots. spinal lymphosarcoma is most commonly seen in young cats (≤ years). males are more commonly affected. most cats present with an initial insidious course of neurologic dysfunction followed by acute deterioration. tumors occur predominantly between t -l . involvement of the brachial intumescence and plexus also can occur. cervical spinal cord involvement is rare. the most common clinical signs include progressive ataxia, asymmetric paraparesis and focal spinal pain. if there is involvement of the brachial or lumbosacral intumescences, lmn signs will occur. nerve root involvement may result in lameness and limb pain. acute exacerbation of signs is commonly associated with hemorrhage. weight loss is the most common extraneural sign. the most common extraneural tumor site associated with spinal lymphosarcoma is the kidney. complete blood count, biochemistry parameters and urinalysis are often normal, although non-regenerative anemia or leukemia may occasionally be seen. many cats are seropositive for felv or positive on indirect fluorescent antibody testing of bone marrow. a monomorphous population of neoplastic lymphocytes will be seen in some cases. survey spinal radiographs are usually normal. mri scan or myelography shows single or multiple asymmetric extradural spinal cord compression(s). infectious diseases, such as feline infectious peritonitis, toxoplasmosis and cryptococcosis also may produce signs of progressive spinal cord dysfunction. however, unlike spinal lymphosarcoma, most infectious conditions usually result in multifocal neurologic dysfunction, with involvement of the cerebrum, brainstem and/or cerebellum. spinal lymphosarcoma is radiosensitive. • rapid reduction in tumor volume can occur within hours of delivering a single large palliative dose of radiation ( - gy). chemotherapy is important for systemic control of lymphosarcoma although most chemotherapeutic agents do not cross the blood-brain barrier. cytosine arabinoside may also help control spinal lymphosarcoma. • this anti-metabolite drug crosses the blood-brain barrier and reaches appropriate csf levels. • the recommended dosage is mg/m /day as a constant rate infusion intravenously for days. • an alternative regimen is mg/m subcutaneously twice daily for days. • potential toxicity includes myelosuppression (at - days); vomiting; and anorexia. • tumor cells rapidly become resistant to this drug's action. surgical decompression of the spinal cord and/or nerve roots, and tumor resection or debulking is a potential treatment option, especially if the tumor is localized. follow-up radiation and chemotherapy is recommended. prognosis is poor for long-term survival, especially when most cats are infected with the felv virus. the only effective means of prevention is test and removal programs directed at controlling the spread of felv. feline infectious peritonitis (fip) may produce a nonsuppurative meningoencephalitis in % of infected cats. the cns signs are usually associated with the noneffusive ("dry") form of fip (cell-mediated immunity is defective but not absent) the neurological form of fip has no breed or sex predilection, although most cases are < - years of age. in most cases, the neurological signs are multifocal with a predominance of caudal fossa signs (brainstem, vestibular nuclei and cerebellum). • signs can include nystagmus (positional, nonconstant, rotatory, horizontal or vertical in nature), head tilt, body lean or rolling, facial paralysis, an ipsilateral hemiparesis or tetraparesis, intention tremor, and hypermetria or dysmetria. the spinal cord and the cerebrum also can be involved. • signs of cerebral disease can include seizures, behavior change, decreased mentation, decreased menace response, compulsive walking, ipsiversive wide circling and head pressing. • spinal cord involvement will manifest as umn and/or lmn tetraparesis or paraparesis with ataxia. tetraplegia or paraplegia are the most severe consequences of spinal cord involvement. myelopathy is seen either as the sole abnormality or as part of a multifocal distribution in % of cases. the onset of fip can be acute, although most cats have a chronic progressive course over week to months. extraneural signs may include fever, cachexia, poor body condition, dehydration, lethargy, muscle atrophy, chorioretinitis, dyspnea and gastrointestinal/hepatic signs. many cats present with the neurological form of fip without extraneural signs. the diagnosis is best established via csf analysismarked elevation in protein (usually greater than the csf fip viral titer is usually positive if cns signs are present. it is probable that csf anti-fip viral immunoglobulin g is produced within the cns. serum biochemical changes often consist of an elevation in total protein and hypergammaglobulinemia. serum fip antibody titers are not reliable, and are elevated in only % of cats with the neurological form of fip and are even negative in some cats. numerous other infectious and immunological diseases can produce meningitis and encephalomyelitis in cats. felv-associated cns lymphosarcoma can usually be differentiated by csf analysis, and a positive felv serum titer. cryptococcal organisms are usually visible in csf with confirmation made by a latex agglutination assay or culture. cns toxoplasmosis is rare in the cat, usually producing signs of intracranial disease rather than myelopathy. recent or active infection is diagnosed by demonstrating a positive igm titer, a four-fold increase or decrease in igg serum titer, a csf igm antibody titer or a csf igg titer present at a higher csf:serum ratio than another antibody present in serum that is not produced in csf, e.g. calicivirus. feline polioencephalomyelitis usually produces only a mild to moderate increase in protein (mean of . g/l [ mg/dl] with a range of . - . g/l [ - mg/dl]) and a mild mononuclear pleocytosis (mean of cells/mm [ cells/μl] with a range of - cells/mm [ - cells/μl]) on csf. this is not typical of fip. no effective therapy is known against fip. immunosuppressive corticosteroid therapy may slow the disease course ( - mg/kg prednisone or prednisolone orally twice daily). all cats with the neurological form of fip will eventually die from their disease. • acute variably located spinal cord dysfunction (umn or lmn signs). • focal spinal hyperesthesia. • spinal crepitus. • external abrasions and bruising. external spinal trauma can result from automobile accidents, gunshot injuries, falls from heights and blunt trauma. sacrocaudal fractures most often occur when the cat's tail is forcefully pulled away from the body. • this most commonly occurs when the cat's tail is trapped under the tire of an automobile. the rotating tire luxates the tail at the sacrococcygeal junction or between two coccygeal vertebrae, resulting in damage to the coccygeal, sacral and lumbar nerve roots (cauda equina). most spinal fractures occur at the junction of mobile and immobile segments of the spine, such as the thoracolumbar, lumbosacral and sacrocaudal junctions. traction on the cauda equina in sacrocaudal luxations can also damage the nerve cell bodies in the caudal lumbar and sacral spinal cord due to cranial transmission of the forces through the dura mater and filum terminale of the cord. the degree of spinal cord compression; the length of time of compression; and the velocity of the initial impact injury are important contributors to the resultant spinal cord injury. there are two forms of spinal cord injury -mechanical (primary) injury, consisting of physical disruption of vessels and axons and ischemic (secondary) injury. the latter leads to energy compromise within the spinal cord, cellular accumulation of calcium and resultant intracellular stimulation of enzymes. this leads to protein and lipid breakdown (phospholipid hydrolysis), release of arachidonic acid, free radical and eicosanoid formation, and cell death. gray matter hemorrhages and white matter edema occur within minutes following acute spinal cord injury, progressing rapidly over the - -hour posttraumatic period. signs depend on the injury level and the degree of physical or functional disruption of the cord. signs in cats with sacrocaudal injuries range from hyperesthesia over the tail base to flaccid analgesic tails with varying degrees of urinary reflex dyssynergia or lmn urinary/fecal incontinence. • reflex dyssynergia involves a failure of urethral relaxation when the cat attempts to urinate and the bladder undergoes contraction. this leads to the production of only very small quantities of urine. the bladder will still be large and the cat will often dribble urine in between attempts to urinate. many cats with sacrococcygeal trauma also show signs of lmn paraparesis (sciatic nerve injury), consisting of dragging of the hind paws on their dorsum and a failure to flex the pelvic limb(s) when walking or when the withdrawal reflex is performed. there will also be a hypo-to areflexia of the cranial tibial and gastrocnemius reflexes. palpation of the sacrocaudal area of the spine will result in crepitus and pain. the tail will often be malaligned with the sacrum and the rest of the vertebral column. cats with spinal trauma commonly have concurrent thoracic, abdominal or pelvic trauma. neurological and physical examination determine the localization and severity of the cord injury. non-contrast spinal radiographs (lateral and across the table ventrodorsal views of the entire spine) establish the type of vertebral disruption (fracture vs. subluxation) and the location(s) of the vertebral trauma. myelography, mri or ct scans will determine the degree of spinal cord compression. any other cause of acute myelopathy must be considered as a differential diagnosis in cats with spinal trauma. most other causes can be eliminated based on history and physical examination -external abrasions, bruising, splintering of claws and spinal pain are seen with trauma. sacrocaudal fractures must be differentiated from aortic thromboembolism, which also can produce an acute onset of lmn paraparesis to paraplegia, with pain. • however, cats with this disease do not have dysfunction associated with the perineum, bladder, rectum and tail. • the lack of femoral pulses and the presence of cold, cyanotic pelvic limbs would strongly support aortic thromboembolism. pathologic vertebral fracture secondary to neoplasia will present as an acute, painful myelopathy, and may appear superficially similar to a traumatic fracture on non-contrast spinal radiographs. however, closer examination should reveal areas of lysis within the involved vertebral body. ischemic myelopathy secondary to fibrocartilaginous embolism most commonly involves the lumbosacral intumescence as is the case with sacrococcygeal trauma. • a differentiating feature of ischemic myelopathy is the absence of spinal pain. initial medical emergency treatment should include intravenous methylprednisolone sodium succinate ( mg/kg), administered within hours (and preferably within hours) of the spinal trauma. continued treatment consists of a constant rate intravenous infusion of methylprednisolone at . mg/kg/hour or, if not possible, a second bolus intravenous injection ( mg/kg) at hours after the initial treatment, followed by mg/kg at hours and then times daily for - hours. most cases of spinal trauma will require emergency surgical intervention (spinal cord decompression [laminectomy and/or hemilaminectomy] and spinal fixation). the type of decompression and stabilization technique is determined by non-contrast and contrast spinal radiographs or ct/mri scans. surgical spinal stabilization is rarely necessary in sacrocaudal fractures/luxations and, due to the oftenmarked separation of the involved vertebrae, is difficult to achieve. surgical decompression of the cauda equina in sacrocaudal trauma is also unnecessary since it is a traction, not compressive, injury. if the cat is incontinent, attention should be paid to cleanliness, bladder and bowel management, and prevention of decubital ulcers, urine scalding, and cystitis. if there is umn or lmn urinary incontinence, manual expression or indwelling catheterization of the bladder is required. pharmacological management of umn urinary incontinence or reflex dyssynergia, related to sacrocaudal trauma, consists of phenoxybenzamine, an alpha-adrenergic receptor antagonist, to relax the internal sphincter ( mg orally three times daily) together with diazepam ( . - mg/cat orally two to three times daily) to relax the external sphincter. bladder detrusor dysfunction can then be treated with bethanechol, a parasympathomimetic agent ( - mg orally two to three times daily). bethanechol may help lmn urinary incontinence (encourages detrusor muscle contraction). fecal retention can be treated by increasing the bulk of the feces with bran, psyllium or canned mashed pumpkin. in cats with sacrocaudal injury with only tail paresis, conservative medical management will often result in return of tail function. • if this does not occur after - months, amputation of the tail is recommended. prognosis is based on the severity of the spinal cord injury. the milder the neurological deficits, the better the prognosis for recovery. cats with severe myelopathy or cauda equina injury with analgesia have a very poor to hopeless prognosis since they commonly have physical or functional spinal cord or cauda equina transection. • only % or less of cats with flaccid tail, perineal analgesia and lmn urinary and fecal incontinence from a sacrocaudal injury will regain neurologic function. • % of cats with only tail flaccidity can regain tail function. prognosis should never be determined based on the degree of radiographic displacement of the involved vertebra(e). the only preventative measure is for cats to avoid situations where trauma is a distinct possibility. • chronic, progressive umn or lmn tetraparesis or paraparesis dependent on tumor location. • focal areas of spinal pain. although most meningiomas are found intracranially, they may also develop along the spinal cord with an intradural, extramedullary predilection site. meningiomas arise from any cell of the meningesblood vessels, fibroblasts or arachnoid cells. meningiomas produce signs by compressing the adjacent spinal cord causing vasogenic edema occasionally, other tumors such as an astrocytoma occur. most cats are > years of age. the nature of the ataxia and paresis (umn versus lmn signs) depends on the tumor's location. a focal area of spinal pain or more diffuse spinal discomfort may occur weeks prior to the development of neurological dysfunction. signs are slowly progressive. non-contrast spinal radiographs may reveal thinning or deformation of the vertebral lamina secondary to pressure necrosis from the expanding tumor. lumbar csf most commonly reveals increased protein (> . g/l [ mg/dl]) without an accompanying pleocytosis. tumor cells are rarely seen. any cause of chronic, progressive myelopathy should be considered. the focal nature of the myelopathy with meningioma eliminates most infectious and inflammatory myelopathies, with the exception of felv-associated spinal lymphosarcoma. ischemia and trauma produce an acute myelopathy and can usually be eliminated. age of disease onset for meningiomas would eliminate congenital and inherited myelopathies. differentiation from nerve root tumors often requires histopathology following surgical removal. surgical removal of the tumor is the only definitive treatment. post-operative radiation therapy is recommended. corticosteroids only transiently improve neurological function. the long-term prognosis is guarded to fair if surgical removal of the tumor appears complete. follow-up radiation probably further improves this prognosis. without surgery, the long-term prognosis is poor. there is no known prevention of spinal meningiomas in cats. • acute lmn paraplegia. • severe pain associated with the pelvic limb musculature. • vocalization and anxiety. • weak or absent femoral pulses. • cyanotic and cold nail beds and foot pads. aortic thromboembolism results from a thrombus that is dislodged from within the left heart or aorta, leading to obstruction of the aortic trifurcation and severe ischemia to pelvic limb muscles and nerves. in cats it is most commonly associated with hypertrophic, dilated and restrictive cardiomyopathy, with thrombus formation in the left atrium. it also occurs with thyrotoxic cardiac disease. thrombus formation requires either damage to the endocardium as occurs in cardiomyopathy, especially the dilated left atrium; blood stasis which also occurs in the dilated left atrium; or altered blood coagulability. disseminated intravascular coagulation (dic) was found in % of cats with cardiomyopathy and was associated with consumptive or liver-mediated coagulopathy or thromboembolism. embolism occurs when the thrombus lodges in a blood vessel. the thrombus most often lodges in the distal aorta at the trifurcation, leading to a saddle thrombus and occlusion of blood supply to the hindlimbs. ischemic neuromyopathy occurs and is most severe distal to the stifle. signs are usually asymmetrical. occasionally the right brachial artery is occluded, resulting in lameness or paresis of the right forelimb. occasionally cats with thromboembolic disease do not have underlying heart disease. thrombosis may also occur with infectious or neoplastic disease. there is no breed or age predilection (average years, range - years) although males are twice as likely to be affected. aortic thromboembolism usually produces an acute onset of lmn paraplegia and severe pain. the hindlimbs drag behind the cat, as the hocks cannot be flexed. hip extension and flexion is present. less severe ischemia leads to mild to moderate paraparesis or pelvic limb lameness. the gastrocnemius muscles usually are firm, but soften - days after embolization. femoral pulses are weak or absent and the nail beds and footpads are cyanotic and cold. the distal limbs are often swollen. extraneural signs include vocalization, tachypnea/ dyspnea, a cardiac murmur and arrhythmias. heart failure is present in about % of cats with aortic thromboembolism. dehydration and hypothermia are often present. tail movement, perineal reflexes and urinary function remain intact. increases in serum creatine kinase (often - iu/l [normal: - iu/l]) occur secondary to severe muscle damage. creatinine and bun are increased in more than % of cats and may be prerenal or renal. in some cats, concurrent embolization of the renal artery occurs. thoracic radiography reveals cardiomegaly ( - % of cats) and pulmonary edema and/or pleural effusion ( %). electrocardiography most commonly reveals sinus tachycardia. atrial fibrillation, and supra-/ventricular arrhythmias can also be seen. doppler (color flow) will often demonstrate the decrease in blood flow through the site of thromboembolism. mri angiography will reveal a complete or partial blood flow obstruction at the aortic trifurcation. a major feature that differentiates aortic thromboembolism from traumatic, infectious, neoplastic and ischemic myelopathies is the presence of paraparesis to paraplegia without involvement of the perineum, tail and bladder. cold, cyanotic pelvic limbs without femoral pulses and firm gastrocnemius muscles also are unique. with current treatment modalities, the results of pallative therapy are as good as aggressive thrombolytic therapies or physical removal of the thrombus, and considerably less expensive. • physical removal is via surgery or balloon embolectomy. • thrombolytic agents include tissue plasminogen activator, urokinase and streptokinase. -current information should be consulted on administration and monitoring of thrombolytic and intensive anti-thrombotic therapies if they are to be used. • reperfusion effects are a major problem with aggressive thrombolytic therapies and physical removal. rapid onset of severe hyperkalemia associated with reperfusion is common and often fatal. renal hemorrhage may also occur during thrombolysis, adversely affecting survival. clinical hemorrhage may require a blood transfusion to control. pallative therapy consists of relieving pain, heparin to help prevent another clot forming in the left atrium, fluid, electrolyte and nutritional support, warmth, physiotherapy (passive massage and flexing/extending legs) and treatment of underlying heart disease. potassium concentrations should be monitored carefully. injectable analgesics and a fentenyl patch should be used to provide adequate levels of pain relief. excessive licking or chewing of the affected limb may occur resulting in self-mutilation. loose bandaging of the limb or an elizabethan collar are usually effective. dehydration and electrolyte imbalances, especially hyper-and hypokalaemia, need correcting. nutritional needs should be met in anorexic cats by the placement of a nasoesophageal tube for feeding. acutely, heparin ( u/kg subcutaneously followed by u/kg subcutaneously three times daily) can be given to prevent further activation of the coagulation cascade, although it will not have any effect on the formed thrombus. continue palliative therapy for - days and look for rewarming of the toes and returning pulses as an initial sign of improvement. doppler is useful for detecting blood flow. if there is no sign of reperfusion after days, the prognosis is hopeless and the cat should be euthanized. various other treatments have been advocated but there are no studies showing increased survival. these include aspirin, periactin, acepromazine and vasodilator agents. aspirin may be beneficial during and after an episode of thromboembolism due to its antiplatelet effects and decreased production of the vasoconstrictor thromboxane a , as well as analgesic effects. the dosage is / × mg adult aspirin ( mg) every second to third day. blockade of prostacyclin production by the endothelium with aspirin is of concern, and some advocate lower doses of aspirin more frequently ( - mg/cat q h). prostacyclin inhibits platelet aggregation and vasoconstriction. acepromazine ( . - . mg subcutaneously three times daily) can be used for sedation and vasodilation. vasodilation with alpha-blockers is advocated by some but unproven. long-term warfarin therapy may decrease the frequency of rethrombosis, but needs careful monitoring using the international normalization ratio (inr) for pt to achieve a inr between and . doses of . - . mg/kg po q h have been advocated. a lower dose of . mg/cat po q h has also been used. only - % of cats survive the initial episodes and go home. most will re-embolize -long-term prognosis is poor (average survival is - months with therapy) with less than % surviving year. • % of cats have rethrombosis even when treated with warfarin. most cats that survive an initial episode will show varying degrees of neurological recovery to their pelvic limbs, although this often takes weeks to months. rarely does the neuromuscular function fully recover. hypothermia and azotemia prior to therapy and hyperkalemia during thrombolysis are negatively associated with survival. echocardiographic evidence of another thrombus in the left atrium also decreases the long-term prognosis. little can be done to prevent aortic thromboembolism if the underlying cardiac disease has not been previously recognized. if cardiac disease is diagnosed, the risk of thromboembolism may be reduced by appropriate treatment of the cardiomyopathy (see chapter ). efficacy of longterm aspirin (low dose - mg/cat q h or a regular dose mg/cat, q - h po) or warfarin therapy in preventing thromboembolism has not been demonstrated, and most cats rethrombose. • stiff staggering gait. • inability to jump. • chronic pelvic limb ataxia and paraparesis. • inability to retract their claws. • thoracolumbar hyperesthesia. • thoracic limb paresis and ataxia. • decreased mentation and seizures. • fever (in % of cases). feline polioencephalomyelitis is a chronic, progressive disease affecting the spinal cord and brain of cats. the cause is unknown, although neuropathology is suggestive of a neurotrophic virus. recently, specific antibodies to the borna disease virus have been found in % of cats with feline polioencephalomyelitis in sweden. feline polioencephalomyelitis is a sporadic worldwide disease. affected cats range from months to years of age. both male and female cats are affected. all domestic cat breeds as well as large non-domesticated cat species can contract the disease. neurologic signs usually develop over - months with a subacute to chronic progressive course. the most striking neurologic signs include a stiff staggering gait, inability to jump, pelvic limb ataxia and paraparesis. • some cats have decreased spinal reflexes and some are unable to retract their claws. other signs may include thoracic limb ataxia and paresis, hyperesthesia over the thoracolumbar and lumbosacral regions, decreased mentation, behavior change, decreased pupillary light reflexes, hypersensitivity to external stimuli, impaired vision, seizures, increased salivation, intention tremors and pruritis. with disease progression, paraplegia eventually occurs. a fever is present in approximately % of cats. serologic testing for fiv and felv antibodies is negative. definitive diagnosis can only be made on necropsy. histopathology reveals a disseminated meningoencephalomyelitis with lymphocytic perivascular cuffs that is most severe in the spinal cord (gray matter) and brainstem. infectious diseases (fip and toxoplasmosis) also may produce signs of progressive spinal cord dysfunction, making differentiation based on neurological signs difficult. uveitis, chorioretinitis and optic neuritis are often present with cns infections, but not with polioencephalomyelitis. cats with spinal cns lymphosarcoma commonly are felv positive and may have other systemic signs of tumor involvement. csf analysis may help differentiate between the infectious diseases, lymphosarcoma and feline polioencephalomyelitis (see sections on lymphosarcoma and fip), although the range of values overlap between the diseases. immunosuppressive steroid therapy (prednisone - mg/kg bid) is the recommended treatment against this disease, although data are lacking. this may be combined with other immunosuppressive agents such as cytosine arabinoside. aggressive and persistent therapy may result in remission and possible cure after several months. prognosis is guarded. the disease tends to be progressive if untreated. since this disease is of unknown etiology and very sporadic, there are no recommendations for prevention. • umn or lmn paraparesis or tetraparesis dependent on the anomaly and its location. • scoliosis (lateral curvature of the spine), lordosis (downward arching spine), or kyphosis (upward arching spine) resulting from a hemivertebra. • cervical pain and umn tetraparesis to paralysis (atlantoaxial subluxation). • lmn paraparesis to paraplegia (spina bifida and sacrococcygeal dysgenesis). • lmn urinary and fecal incontinence (spina bifida and sacrococcygeal dysgenesis). spinal cord and vertebral anomalies are classified into abnormalities originating in the tissues of mesodermal origin (vertebral body and intervertebral disc) and those arising from tissues of ectodermal origin (spinal cord and meninges). • block vertebrae. -caused by improper segmentation of somites, resulting in stable vertebral fusion. • butterfly vertebrae. -abnormal persistence of the notochord (the embryological precursor of intervertebral discs), producing a midline cranial to caudal cleft in the vertebral body (when viewing the vertebra from the dorsoventral direction). • hemivertebrae. -produced by fusion of one lateral somite to one on the contralateral side that is not directly opposed or by a lack of vascularization, leading to a failure of ossification in part of the vertebral body. this produces a wedge-shaped vertebra, and scoliosis, lordosis or kyphosis. • transitional vertebrae. -sacralization of the last lumbar vertebra is the most common in cats. in this condition, the last lumbar vertebra has characteristics of both lumbar vertebrae and the sacrum. clinical signs are dependent on the type of congenital malformation and its location. block vertebrae are usually stable. • occasionally they can be stenotic or angulated, causing extradural spinal cord compression. • disc extrusion can occur at disc spaces immediately cranial and caudal to the block vertebrae. butterfly vertebrae are generally incidental findings. hemivertebrae commonly lead to scoliosis, lordosis or kyphosis. • signs are due to spinal cord compression or repeated trauma associated with vertebral instability. instability produces osseous changes that secondarily compress the spinal cord. • signs may be acute, chronic, progressive or intermittent, but are usually first noted within the first - years of life. • conformation may be visibly or palpably abnormal. breed association (manx cat). signs are most commonly observed in immature animals (< months of age). the cat's conformation may be visibly or palpably abnormal (scoliosis, skin dimple, etc.). • block vertebra -joining of two adjacent vertebral bodies. • butterfly vertebra -sagittal vertebral body cleft. • hemivertebra -abnormal vertebral wedging and shortening. • atlantoaxial subluxation -abnormally shaped or absent dens. • spina bifida -vertebral arch defect. mri scan or ct myelography reveals soft tissue abnormalities associated with a spinal cord that is tethered (fixed) by either meningocutaneous attachments (spina bifida) or by a failure of the terminal cord-like attachment from the spinal cord to the dura to stretch with growth. they will also reveal cord compression. major differentials for hemivertebra include traumatic or pathologic fracture with secondary collapse of the vertebral body. • hemivertebrae, however, have smooth end plates and well-formed adjacent disc spaces. • difficulty in differentiation arises when vertebral osteophytes are present. differential diagnoses for block vertebrae include vertebral fusion secondary to discospondylitis, vertebral fracture/luxation, collapsed disc space secondary to disc extrusion, or previous disc surgery. however, all have associated reactive bone, which is not present with block vertebrae. atlanto-axial subluxation and spina bifida have few rule-outs, except trauma. spinal cord compression from a hemivertebra can be treated via surgical decompression and stabilization (if necessary). treatment for atlantoaxial subluxation involves stabilization of the atlantoaxial joint via ventral crosspinning or screws (preferred) or dorsal wiring. specific treatment for spina bifida and sacrococcygeal dysgenesis is rarely attempted. • if signs are associated with spinal cord tethering or there is a meningocutaneous fistula without primary spinal cord anomalies, reconstructive surgery may be possible. prognosis depends on the type of congenital anomaly and the degree of dysfunction. cats with mild signs due to a surgically treatable hemivertebra or block vertebra, have a guarded to good prognosis for improvement. morbidity and mortality is high with surgical stabilization of atlantoaxial subluxation. • with successful stabilization, however, cats with initially mild to moderate neurological dysfunction have a fair to good prognosis. prognosis for cats with spina bifida or sacrococcygeal dysgenesis with anomalies associated with the spinal cord or cauda equina, is grave. these cats should be euthanized if the neurological dysfunction is incompatible with a good quality of life. cats with spinal cord tethering (spina bifida) have a potentially fair to good prognosis with surgery. prevention of further congenital malformations is best achieved by a spay/neuter program. • cervical and thoracic limb hyperesthesia and rigidity. • thoracic limb lameness. • chronic progressive ataxia. • reluctance to move. • chronic tetraparesis to paralysis. hypervitaminosis a is a skeletal disease, secondary to excessive intake of vitamin a (liver or vitamin a supplementation (cod liver oil) (> iu/ml). affected cats are usually - years of age. signs include cervical and thoracic limb hyperesthesia and rigidity due to extensive confluent exostosis in the cervical and thoracic spine, thoracic limb lameness, ataxia, reluctance to move, and tetraparesis to paralysis. other signs include lethargy, anorexia, constipation, weight loss and an unkempt haircoat due to an inability to groom. exostoses develop insidiously with the above signs occurring only after the disease is advanced. non-contrast spinal radiographs show new bone formation involving the cervical vertebrae. • the sternum, costal cartilages, and long bone metaphyses also show new bone formation. • joints may show arthrodesis. analgesics may symptomatically treat the clinical signs. • aspirin - mg/kg po q h. • narcotic analgesics such as morphine ( mg/ml) - . mg/kg po three times to four times daily. • non-steroidal anti-inflammatory drugs. • fentanyl patch. • acute, non-progressive, non-painful, asymmetrical lmn paraparesis to paraplegia. • lmn urinary and fecal incontinence. spinal cord infarction secondary to fibrocartilaginous embolism is uncommon. the embolus is histochemically identical to the fibrocartilage of the nucleus pulposus. it is unknown how the embolus reaches the spinal vasculature from its origin. embolization of arteries, veins or a combination of the two may occur. sudden increases in intra-abdominal pressure (hard exercise) may facilitate retrograde passage of disc material through the venous sinuses and spinal veins. embolism results in segmental hemorrhagic necrosis and malacia of the spinal cord. clinical signs reflect the location of the lesion along the spinal cord. in cats, the lumbosacral intumescence is the most common site of myelopathy, resulting in an acute lmn paraparesis to paraplegia, and lmn urinary and fecal incontinence. other findings include a lack of spinal pain, lack of disease progression and marked asymmetry of neurological dysfunction. diagnosis is based on exclusion of other etiologies. history and neurological exam are important. survey radiographs are normal. csf analysis is variable, ranging from normal, hemorrhagic, or showing a mild mixed pleocytosis ( - cells/mm [ - cells/μl]). myelography will either be normal or occasionally show cord edema. mri diffusion studies may reveal the ischemic area of spinal cord, if the affected area is large enough. definitive diagnosis can only be made at necropsy. differential diagnosis includes any cause of acute paraparesis to paraplegia. the unique clinical signs and usual absence of abnormalities on work-up separate fibrocartilaginous embolism from other acute myelopathies. immediate treatment consists of methylprednisolone sodium succinate (see spinal trauma). embolic myelopathy is non-surgical. long-term corticosteroid therapy is not recommended. prognosis depends on density of neurological dysfunction and degree of irreversible cord damage. cats with severe lmn paraplegia, absent pain sensation and lmn urinary incontinence associated with involvement of the lumbosacral intumescence have a poor to hopeless prognosis. cats with less severe signs have a guarded to favorable prognosis for partial to full recovery. there are no preventive measures that can be undertaken. a review of the clinical diagnosis of feline infectious peritonitis viral meningoencephalomyelitis jr (ed) consultations in feline internal medicine ed) textbook of veterinary internal medicine congenital abnormalities of the spinal cord and vertebrae, chapter jr (ed) consultations in feline internal medicine feline non-suppurative meningoencephalomyelitis. a clinical and pathological study inflammatory disorders of the central nervous system encephalitis and meningitis. in: bagley r (ed) veterinary clinics of north america small animal practice -intracranial disease feline thrombotic disease veterinary information network vascular disorders, chapter based on the clinical and neurological examinations, other differentials include infectious meningitis and myelitis, vertebral neoplasia, mucopolysaccharidosis and discospondylitis (rare).non-contrast spinal radiographs eliminate all differentials with the possible exception of mucopolysaccharidosis vi, a rare autosomal recessive storage disease produced by deficiency of the lysosomal enzyme, arylsulfatase b. • mucopolysaccharidosis vi occurs in - -monthold siamese cats, producing spinal cord compression secondary to fusion of the cervical and thoracolumbar vertebrae with bony proliferation. • these cats also have a flat, broad face, widely spaced eyes, corneal clouding and enlarged feet. remove excess vitamin a from the diet to prevent further development of exostoses.use a balanced commercial cat diet.skeletal improvement is monitored by radiography and neurological examinations. key: cord- - rukc y authors: bergman, robert l. title: miscellaneous spinal cord diseases date: - - journal: consultations in feline internal medicine doi: . /b - - - / - sha: doc_id: cord_uid: rukc y nan spinal cord diseases in cats vary in the severity and in progression of neurological dysfunction. diagnosis of spinal cord disease in cats can be a challenge. clinical signs often are vague and insidious. advanced imaging techniques have improved diagnostic capabilities and recognition of new disorders. infectious inflammatory disease is the most common categorical differential diagnosis in cats with spinal cord dysfunction. other common disease categories include neoplasms, trauma, and degenerative disorders. veterinarians must think beyond the more common differential diagnoses to consider unusual diseases and different diagnostic approaches. this chapter emphasizes newly recognized spinal cord diseases and provides a review of the current literature. signs of neurological dysfunction dictate the neuroanatomical localization of a lesion within the spinal cord. spinal reflexes and paraspinal hyperesthesia assist with lesion localization. localization is specified to the spinal cord regions c -c , c -t , t -l , and l -s , based on upper motor neuron or lower motor neuron signs of limb weakness. cats with spinal cord compressive disease or meningomyelitis often exhibit paraspinal hyperesthesia. pathology of the spinal cord tissue itself usually does not have hyperesthesia as a clinical sign. orthopedic, polyneuropathic, myopathic, and neuromuscular junction disorders can mimic signs of spinal cord dysfunction. careful interpretation of the neurological examination differentiates among these disorders (see chapter ) . signalment and history aid in formulation of a list of probable differential diagnoses. young cats are more likely to be diagnosed with feline infectious peritonitis (fip), lymphosarcoma, or a congenital anomaly. middle-age and older cats may be diagnosed with nonlymphoid neoplasia or intervertebral disc disease. history is important for determining temporal onset (acute, insidious, or episodic) and progression (rapid, gradual, or static). trauma, vascular insults, and some inflammatory and neoplastic diseases present acute in onset. cats with spinal cord dysfunction require thorough physical examination and routine laboratory diagnostic testing. other disorders that cause paresis can mimic spinal cord disease; for example, neuropathy, myopathy, junctionopathy, polyarthropathy, and cardiovascular disease. routine laboratory testing consists of a complete blood count (cbc), serum chemistry profile to include creatine phosphokinase (ck) enzyme activity, and urinalysis. evaluation of ck activity aids in identification of a myopathy, which can mimic spinal cord dysfunction. patient infection with feline immunodeficiency virus (fiv) or feline leukemia virus (felv) must be identified. additional serology for infectious disease is dependent on suspicion of other diseases. survey radiography of the spine is recommended for cats with spinal cord dysfunction. sedation or general anesthesia often is necessary to allow for proper patient positioning and relaxation of the spine. some findings are nonspecific, but discospondylitis, vertebral tumors, or spinal trauma usually have more obvious radiographic abnormalities. orthogonal or multiple views are recommended strongly, because a single view may not always provide complete information about the extent of the lesion (figure - ). myelography consists of injection of a nonionic contrast medium ( . to . ml/kg of iohexol [ mg/ml] or iopamidol [ mg/ml]) into the subarachnoid space of the low lumbar spine (l -l or l -l ) or the cerebellomedullary cistern. myelography is an imaging technique used commonly to identify the location and extent of spinal cord compression. additional information may be used when combining the myelographic findings with computed tomography (ct). magnetic resonance imaging also is a more sensitive technique for evaluation of the spinal cord tissue. cerebrospinal fluid (csf) analysis is useful for detection of evidence of spinal cord disease, particularly when an inflammatory disorder is suspected. however, in most cases, a definitive diagnosis is not provided by csf analysis alone, even in cats with overt cns inflammatory disease. exceptions include finding the inciting organism in the csf (e.g., cryptococcus neoformans) or identifying neoplastic cells (e.g., lymphosarcoma). collection of fluid from the lumbar region may be preferable, because csf flows in a caudal direction. additional diagnostic procedures include electrophysiology, csf protein electrophoresis, serology, and exploratory surgery. infectious inflammatory diseases account for per cent of all feline spinal cord diseases. common infectious inflammatory spinal cord diseases include fip, cryptococcosis, felv infection, and toxoplasmosis. approximately per cent of cases of meningomyelitis in cats are bacterial or suspected to be bacterial in origin. bacterial infections may occur secondary to hematogenous spread or, more likely, as a result of direct extension of a local wound (e.g., cat bite abscess). pasteurella spp. and staphylococcus spp. are common pathogens. discospondylitis often is caused by a bacterial infection and involves the intervertebral disc and associated vertebral endplates. discospondylitis has been reported infrequently in cats and, if not treated appropriately, may progress to severe neurological dysfunction. , polioencephalomyelitis, an inflammatory disease of unknown cause, is associated with per cent of cases of feline spinal cord disease and may present with clinical signs of paraparesis. although not well described in the literature, eosinophilic/histiocytic meningomyelitis accounted for per cent of inflammatory spinal cord diseases in cats. feline immunodeficiency virus. fiv has been reported to cause a degenerative myelopathy that can be detected histologically with changes including myelin sheath splitting and intramyelinic vacuoles. clinical signs of spinal cord dysfunction are not evident in experimentally infected cats or in cats with naturally occurring fiv infection. presenting signs and pathogenesis. fip accounts for more than half of the infectious inflammatory causes of myelitis in cats, and per cent of all spinal cord diseases reported in cats. clinical signs of fip result from the immune response of susceptible cats when infected by a mutant form of the feline enteric coronavirus (fecv), which reproduces within macrophages. , the dry, or noneffusive, form of the disease is associated most commonly with cns signs as opposed to the "wet" or effusive form, which involves the visceral organs and causes abdominal effusion. pyogranulomatous inflammatory lesions involve the meninges, choroid plexus, and ventricular system. the immune response associated with fip causes a vasculitis and an ependymitis that subsequently may obstruct flow of csf. signs of systemic illness occur in approximately per cent of cats with fip. typical signs include weight loss, anorexia, intermittent fever, and ocular changes (anterior uveitis or chorioretinitis). about one third of cats with fip have presenting clinical signs of neurological dysfunction. young, purebred, sexually intact male cats are at a higher risk for developing fip. a genetic susceptibility of about per cent exists for development of fip. all cats in one study of patients with neurological signs of fip were from multiple-cat households. although younger cats are most susceptible to fip infection, cats of any age can develop the disease. in a case series of cats with spinal cord-related signs, more than per cent were younger than years of age. most cats with fip have intracranial signs but often manifest signs of spinal cord dysfunction: pelvic limb ataxia, generalized ataxia, and paraspinal hyperesthesia. in a small case series of cats with confirmed fip, four of had paresis or paralysis as the presenting clinical sign. paresis was evident in two of seven of these cats with diffuse fip. in another study, of cats with fip had histological lesions that predominated in the cervical spinal cord and brain. diagnosis. antemortem diagnosis of fip is difficult. diagnosis is suspected based on assimilation of history, signalment, hematology, and other supportive diagnostic tests that include serology, csf analysis, findings on imaging, and tissue biopsies. a typical history includes acquisition of the cat from a cattery or shelter, and a fever that waxes and wanes and does not improve with antibiotic therapy. common hematological and biochemistry abnormalities include neutrophilia or lym- phopenia, low albumin with increased globulins, or a high serum fibrinogen. serological testing and polymerase chain reaction studies to assess for viral load can be beneficial. serology only confirms exposure to feline coronavirus. some cats with fip may have high antibody titers, but this is not absolute. a recent study of histopathologically confirmed cases of fip found that serological testing provided further support for tissue biopsy procedures. high antibody titers ( : ) provide a per cent probability of active fip infection. a titer that was positive but below : suggested only a per cent probability that cats had fip. the titers in per cent of cats with fip were negative, which suggests a compromised immune system. definitive diagnosis of fip is made by histopathology of abdominal organs obtained by tissue biopsy. immunofluorescent assay/immunohistochemistry techniques can detect presence of coronavirus antigens within macrophages. diagnostic evaluation of the cns aids in an indirect diagnosis of fip. results of csf analysis often reveal a marked increase in protein concentration and a neutrophilic pleocytosis. , comparisons of antibodies in serum and csf may provide additional information, but false negatives and false positives are possible. presence of antibodies in csf must be interpreted in light of blood-brain barrier breakdown. adjunctive comparison of other infectious disease antibody titers in serum and csf can assist with determination of intrathecal production of antibodies. common abnormalities on mri and ct include presence of hydrocephalus and periventricular contrast enhancement. overall the most consistent diagnostic findings in cats with the cns form of fip include a positive coronavirus igg titer in csf, a high serum total protein concentration, and abnormalities in brain imaging. treatment. no treatment has been proven effective for fip, and the long-term prognosis is poor. overall mortality rate is per cent. supportive therapies consist of antiinflammatory doses of prednisone ( mg/kg/day po) and immunomodulation with cyclophosphamide or interferon. a recent report found use of recombinant feline interferon combined with corticosteroids more effective in cats with the effusive form than with the noneffusive form (n = ) of fip. other therapeutic recommendations include a diet with high nutritional value and stress reduction. presenting signs and pathogenesis. cryptococcus neoformans is a saprophytic fungal organism that can cause systemic or focal disease. transmission occurs through inhalation of the organism that lives in the soil or bird excrements. cns signs are reflective of meningitis or focal granuloma formation within the brain parenchyma. fungal masses within the extradural space cause secondary compression ( figure - ). cryptococcal infection can cause focal spinal cord disease in some cats. the mean age for cats infected with cryptococcus is years; however, the age range can vary. , approximately per cent of cats diagnosed with cryptococcus spp. were considered primarily outdoor cats. systemic signs are variable and commonly include depression/lethargy, fever, poor body condition, or anorexia. in one case series, approximately per cent of the cats with cryptococcosis had cns signs, per cent had ocular signs, and per cent had respiratory signs. cutaneous lesions also can occur. another case series reported that only per cent of cats showed signs of neurological dysfunction. in this series, nasal signs were more common. clinical signs of spinal cord dysfunction, including paraspinal hyperesthesia and paresis, have been reported in at least one case series. c. neoformans accounted for per cent of infectious causes of spinal cord disease in cats. diagnosis. csf analysis is one of the most useful diagnostic tests in cats with cns cryptococcosis. neutrophilic and eosinophilic pleocytosis often are present. in some cases, the organism is identified. diagnosis also is based on detection of capsular antigen using a latex agglutination test in serum and csf. cats with focal granulomas in the cns may have negative antigen titers. latex agglutination tests can have falsenegative results (or interference), which makes definitive diagnosis difficult. in these cases, cultures, cytology, or histopathology of other tissues, such as skin, may be necessary. also important is documentation of the felv and fiv status of cats with cryptococcosis, because concurrent infection may be common. additionally, cats with other concurrent viral infections tend to have a higher incidence of treatment failure. treatment. treatment of cns cryptococcosis consists of long-term administration of systemic antifungal agents. itraconazole and fluconazole are considered the drugs of choice. fluconazole ( to mg/kg po q h) is recommended, because it crosses the blood-brain barrier readily and has high lipid solubility. duration of treatment ranges from to months; however, a longer duration may be required to prevent relapse. , antiinflammatory doses of corticosteroids help to decrease the inflammation and edema that can worsen neurological signs during treatment. therapeutic monitoring is based on clinical response and serial serum antigen titers. antigen titers often remain positive for a considerable period of time after clinical signs have resolved. cats that have a reduction in antigen titer during the course of treatment have a better prognosis. surgical removal of a fungal granuloma may be considered in conjunction with antifungal therapy. presenting signs and pathogenesis. toxoplasma gondii is an infrequent cause of spinal cord disease in cats; it was reported as the cause for only per cent of all infectious diseases resulting in spinal cord dysfunction. t. gondii is a protozoal coccidian parasite for which cats are the definitive host. transmission occurs congenitally through the placenta from an infected queen or more commonly by ingesting the organism. healthy cats may be positive on serology but rarely develop clinical disease. predisposing factors for clinical disease include immunosuppression as a result of fiv and/or felv infection, administration of corticosteroids or chemotherapy, and diabetes mellitus. toxoplasmosis causes a nonsuppurative meningoencephalomyelitis. the organism also may infect muscle and peripheral nerves. systemic signs include anorexia, weight loss, fever, and pneumonia. diagnosis. definitive diagnosis of toxoplasmosis is difficult. a suspected diagnosis is based on clinical signs, the exclusion of other cns diseases, serology, and response to treatment. the amount of csf pleocytosis is variable, and the cellular differential count usually consists of mononuclear cells. albuminocytologic dissociation may be the only abnormality. t. gondii-specific igg and igm can be assayed in serum and csf. paired titer evaluations may detect an increase in serum igg; however, the disease course still may be static. an igm titer greater than : may indicate an active or recent infection. antibodies in csf are compared with serum antibody titers for accurate interpretation of blood contamination and intrathecal antibody production. a definitive diagnosis is made by detecting the organism in a tissue biopsy. clindamycin ( . mg/kg po q h for weeks) is recommended for treatment of cns toxoplasmosis. an alternative drug therapy is trimethoprim-sulfonamide ( mg/kg po q h). one author reported a fair to good outcome in three cats treated for toxoplasma-induced myelitis. clinical signs can be residual and response to therapy may be slow. clinical presentation and pathogenesis. feline leukemia virus, an oncogenic retrovirus, can cause spinal cord dysfunction. felv can cause myelopathy by indirect and direct pathogenic mechanisms. felv can predispose to the spinal form of lymphoma indirectly or cause a degenerative myelopathy directly. felv-associated myelopathy reflects primary pathology within the spinal cord. light microscopic examination revealed swollen axons and myelin sheaths in the brain stem and spinal cord of affected cats. immunohistochemical staining revealed felv antigens in neural tissue. a previously reported case of degenerative myelopathy in a felv-positive cat may have been felv-associated myelopathy. this disease is associated with chronic infection with felv. cns signs develop on average years after the first positive felv test. mean age of affected cats is years. signs of felv-associated myelopathy include progressive ataxia and hyperesthesia, and paralysis develops within year after onset of paraparesis. urinary incontinence occurs in a small percentage of cats. diagnosis and treatment. a suspected antemortem diagnosis is based on ruling out other diseases. positive felv tests should heighten suspicion for this disease. csf analysis usually is not helpful. advance imaging studies have not been evaluated in cats with felv-associated myelopathy. myelography is normal. no treatment options have been described. neoplasia is a common cause of spinal cord dysfunction in cats. with regard to relative incidence in one case series, neoplasia affected per cent of cats diagnosed with spinal cord dys-function. lymphosarcoma made up per cent of neoplasiarelated spinal cord cases; however, this disease is becoming less common with the reduction in incidence of felv infection. , , lymphosarcoma presenting clinical signs and pathogenesis. spinal lymphoma historically has been the most common cause of spinal cord neoplasms in cats. cns lymphoma accounted for . per cent of all cases of lymphoma and, of these cases, per cent had spinal cord involvement. the disease is especially common in young felv-infected cats, with a mean age reported between . and years. , cats younger than years of age make up approximately per cent of the cases. clinical signs associated with spinal lymphoma may be associated with a focal myelopathy that can occur in any region of the spinal cord. paresis has been reported in approximately per cent of cats with spinal lymphoma. , evidence of spinal hyperesthesia may be focal or multifocal with more extensive distribution. the disease course can be rapidly progressive, with some cats showing signs for a week or less. , neurological signs are related to the location of the lymphoma. although lymphosarcoma generally is a multicentric disease, more than per cent of cats with cns involvement lack systemic signs or hematological changes. renal lymphoma is likely to metastasize to the cns. diagnosis. evidence for systemic disease on physical examination includes enlargement of lymph nodes and abdominal organs. a cbc may show anemia, leukopenia, and thrombocytopenia. circulating lymphoblasts may be present on a differential white blood cell count. a positive correlation between serological testing and spinal lymphoma has been reported. , the safest and most reliable method of obtaining a diagnosis of cns lymphoma is confirmation of the presence of lymphoma in other visceral organs. bone marrow aspiration may be diagnostic for neoplasia in up to per cent of cases with this disease. csf analysis is not always diagnostic for lymphosarcoma because of its extradural location. one case series reported that of cats had neoplastic lymphocytes in the csf. myelography can determine lesion extent and detect presence of extradural, intradural-extramedullary, or intramedullary involvement. an extradural lesion is the most common myelographic finding. fluoroscopic aspiration and cytology may allow definitive diagnosis of the spinal lesion. mri may detect intramedullary lesions. treatment. positive felv status in cats has been shown to be a negative prognostic indicator for spinal lymphoma. the prognosis for cats with paresis or paraplegia is considered poor. treatment options for spinal lymphoma consist of chemotherapy, surgical resection, and focal irradiation. no superior treatment strategy for chemotherapy has been documented. currently, multidrug protocols are advocated. , a laminectomy procedure facilitates diagnosis and decompression until other therapies can take effect. clinical presentation and pathogenesis. nonlymphoid tumors involving the spinal cord are less common in cats. tumors may be categorized based on expected locations: intramedullary, extramedullary/intradural, and extradural. intramedullary tumors are considered uncommon and make up per cent of all reported spinal cord neoplasms in cats. documented tumors include astrocytoma and ependymoma. , intradural/extramedullary tumors make up per cent of spinal neoplasia cases and include meningiomas, meningeal sarcomas, and malignant nerve sheath tumors. feline meningiomas usually occur rostral to the foramen magnum; only per cent of meningiomas found in cats affected the spinal cord. levy, mauldin, kapatkin, et al reported five cases of spinal meningiomas in cats: one in the cervical region, three in the thoracic spine, and one in the lumbar spine. another case report described a meningioma affecting the spinal cord at the c -c spinal cord segment. extradural spinal cord compression can result from spinal canal masses or tumors of the surrounding bone and vertebrae. these make up about per cent of spinal cord-associated neoplasms in cats, with vertebral and bone tumors accounting specifically for per cent of all spinal tumors. reported tumor types include chondrosarcoma, lipoma, osteosarcoma, and multiple myeloma. nonlymphoid spinal neoplasia typically occurs in older cats, with a median age of years in one case series. these tumors are not associated with felv or fiv infection. clinical signs of myelopathy are dependent on tumor location. focal pain and paresis are most typical. intramedullary neoplasms usually do not cause spinal hyperesthesia until later in the disease course. the clinical course of spinal neoplasms also varies. chronic progressive spinal dysfunction may be expected; however, peracute signs (e.g., pathological vertebral fracture) may present (figure - ) . diagnosis. the process of diagnosis of nonlymphoid tumors begins with survey spinal radiographs. evidence of bony lesions can be evident in osteosarcoma and multiple myeloma. myelography determines extent and location of spinal involvement. advanced imaging (ct and mri) may assist further with determination of lesion extent. findings on csf analysis often are nonspecific. fluoroscopic aspiration or surgical biopsy may yield a definitive diagnosis. treatment. specific treatment regimens are based on histopathological diagnosis of the tumor. treatment often consists of palliative corticosteroids (i.e., prednisone . to mg/kg/day po) to control edema, and pain management. surgical removal/debulking of various tumor types has been described and may improve survival times. a reasonable survival time can be expected for cats with meningiomas after surgical resection. osteosarcomas may be associated with long survival times and appear to be less aggressive than the canine form of this disease. a treatment regimen reported for multiple myeloma in a maine coon cat years of age consisted of a combination of chemotherapy and irradiation. clinical presentation and pathophysiology. spinal trauma is an important cause of spinal cord dysfunction in cats. cats have been the subject of multiple laboratory studies of spinal cord injury. [ ] [ ] [ ] [ ] [ ] [ ] [ ] information learned from this research must be interpreted from the standpoint that the mechanism of spinal cord trauma is controlled in the laboratory environment. naturally occurring spinal trauma in cats is not a well-described phenomenon because cats often do not survive the inciting incident. spinal injury occurs more frequently in younger cats. cats (n = ) in a retrospective study of spinal injury were years of age or younger; per cent were younger than year of age, and per cent were between and years of age. the mean age of another study of cats was . years (range months to years). common causes of spinal trauma are height-related and vehicular injuries. in a large report of high-rise syndrome in cats, per cent sustained spinal fractures or luxations. other sources of trauma include dog attacks and gunshot wounds. , compression-type fractures occur in more than two thirds of the cases with spinal fractures. twenty per cent of cats with spinal injuries also have acute disc extrusions secondary to the trauma. spinal cord contusion injury without evidence of fractures also may occur after a fall. other injuries that occur in conjunction with spinal trauma include pneumothorax, pulmonary contusions, abdominal organ trauma, and head trauma. although all segments of the spine are susceptible to trauma, the cervical and sacral/caudal regions are much less likely to sustain injury. the thoracic and lumbar regions make up per cent and per cent of spinal column injuries, respectively. traditionally, the most likely location for spinal column injury is at a site characterized by a transition from rigid stability to less stability, such as t -l or l -s . this was challenged recently in a larger study consisting of cats, in which per cent of the spinal trauma cases occurred between t and t . the segments between l -l and l -l also were significant sites in per cent of the cases. the t through t vertebrae have been reported to be affected in per cent of spinal injuries in cats. a case series reported by voss showed that per cent of spinal fractures were located between the l and l vertebrae. diagnosis. in cases of suspected spinal injury, the neurological examination is performed with caution to minimize movement of the cat with suspected spinal instability. evaluation of deep pain perception is most important with regard to determining prognosis. spinal radiography using orthogonal views should localize the lesion. radiography documents spinal alignment during that time without knowledge of the amount of displacement at the time of the injury. the entire spine should be radiographed, because multiple spinal fractures are common. advanced imaging of the spine has been recommended because plain film radiography and myelography may underestimate the degree of fractures or luxations present. myelography or mri defines the extent of spinal cord compression more accurately. treatment. goals of therapy are to prevent further mechanical damage to the spinal cord and reduce secondary injury processes. treatment recommendations often are adapted from laboratory studies that involve species other than cats. drawing firm conclusions for optimal treatment of spinal trauma in cats is difficult. management of a cat with spinal trauma should focus first on systemic stabilization. management consists of following the abcs of trauma. airway is assessed for patency and adequate ventilation. appropriate fluid therapy helps to maintain cardiovascular function. aggressive fluid therapy is important to maintain spinal cord perfusion. hypotension is one factor shown to worsen outcome in human beings with spinal cord injury. isotonic crystalloids (lactated ringer's solution or . per cent sodium chloride) at shock doses, initially ( ml/kg/hr iv in cats) are given to effect until heart rate, capillary refill time, and pulse quality improve. hetastarch ( per cent) is a large molecular weight colloid that consists of a branched polysaccharide, amylopectin. its molecular properties provide a long intravascular half-life. the dose is to ml/kg given to effect up to ml/kg/hr. hetastarch is administered intravenously to cats to effect up to to ml/kg; the dose is increased in ml/kg increments every to minutes to avoid nausea and vomiting. hypertonic saline ( per cent) also may be used to expand blood volume quickly. the dose ( to ml/kg) is administered as an intravenous bolus over to minutes. the disadvantage associated with the use of hypertonic saline is that it remains in the vascular space for only to minutes. blood products also are used to expand volume and provide increased oxygen delivery. whole blood is administered intravenously at a dose of to ml/kg/hr, over to hours in stable patients and faster in unstable patients. the goal is to restore the hematocrit to to per cent and albumin to more than g/dl. use of high-dose methylprednisolone sodium succinate (mpss) is becoming more controversial but is still considered standard of care in human medicine. experimental spinal cord injury studies in cats have shown that increased lactate levels in spinal cord immediately after injury most likely were attributed to decreased spinal cord perfusion. high doses of mpss minutes after induced trauma attenuated the secondary injury process dramatically. the intent of high-dose mpss is to provide adequate tissue concentrations of steroid at the site of injury. the original dose for this regimen was mg/kg iv initially, then a dose of mg/kg given and hours later, followed immediately by a . mg/kg/hr infusion, which is continued for hours. the total dose of mpss administered is mg/kg. recent prospective clinical studies that have used modifications of this protocol have come under intense scrutiny for various reasons, including statistical manipulation, lack of proven efficacy, and increased rates of complications in human beings and dogs. [ ] [ ] [ ] [ ] [ ] administration of mpss is time dependent and has shown efficacy if administered within minutes of the injury. use of high-dose mpss is not recommended for administration if the time has been longer than hours after sustaining the injury. supportive medical management alone is useful if spinal instability is not detected or when there is financial constraint. , cats may not tolerate body splints. strict cage confinement is relied upon for to weeks after the injury to initiate the healing process. surgical management. surgical management of spinal trauma is recommended in cases of instability or severe spinal cord compression. the timing of surgery relative to the injury is somewhat controversial; however, adequate medical stabilization before surgery is essential. early decompression has been supported in the laboratory setting in cats, but the optimal time to perform surgery still is unknown. immediate surgical decompression of the affected site is a controversial subject in human spinal trauma, and some studies have not shown a benefit to early surgery. , the technique of surgical stabilization depends somewhat on the fracture type. decompression alone may be sufficient in some cases in which instability is not present. decompression was needed in cats that sustained displacement of the intervertebral disc or endplate into the spinal canal. a dorsal laminectomy, preserving the articular processes, suffices for adequate decompression. common techniques of internal spinal fixation/stabilization include the use of pins with polymethylmethacrylate and spinal stapling. spinal stapling involves the use of rigid intramedullary pins that are secured to the spine after reduction of the fracture/luxation. intramedullary pins are secured to the lamina at the base of the spinous process (figure - ) . spinal stapling is considered technically easier to perform than other described forms of internal stabilization. limited information is available for the long-term outcome. problems associated with this type of surgery include fragility of the spinous processes and migration or breakdown of implants. a recent case series involving cats with thoracolumbar trauma described using a figure- tension band technique as a modification of spinal stapling. complications from this technique were not observed. successful use of pins and polymethylmethacrylate to stabilize a lumbar fracture in a cat has been described. this form of treatment provides significant stability, particularly for rotational forces. optimal placement of pins within the vertebral body may be difficult because of the small size of typical feline vertebral bodies. complications of this procedure include pin migration, pin breakage, pneumothorax, and additional trauma to soft tissue structures. outcome for cats with spinal trauma is guarded. survival rate in one study was only per cent. cats that did not survive were euthanized or died within days of the injury. cats with spinal fractures and absence of deep pain perception almost always have a hopeless prognosis. the return of motor function does not equate necessarily with return of voluntary urination. spinal walking: from laboratory to clinics. prognosis for return of voluntary motor function in cases of absent deep pain perception generally is considered grave. however, the cat has been the subject of extensive experimental work studying the return of ambulation after spinalization, or complete transection of the spinal cord. "spinal walking" is a clinical term used for return of ambulation in an animal with no deep pain perception in the pelvic limbs. in the laboratory setting, this phenomenon is known as spinal locomotion. pelvic limbs are under no voluntary control, and the thoracic limbs move asynchronously with the pelvic limbs when on a treadmill. spinal locomotion may be evident within a few days of the injury. the spinal cord generates this pattern of limb movement, which allows for the placement each foot, weight-bearing, and alterations of speed with change in treadmill velocity. the animal also is capable of stepping over objects placed in its way. cats have been trained to develop spinal locomotion after complete experimental spinal cord transection at t . the underlying mechanism may be the result of a spinal locomotor generator. spinal locomotion is dependent on the development and preparation of a spinal locomotor pattern generator, stimulation of cutaneous receptors, alterations of intraspinal neurochemistry, and input from the midlumbar spinal cord. plasticity occurs within the spinal cord as a result of training. lesion location within the spinal cord also can affect the ability to walk; for example, a lesion at the l -l spinal segment is not conducive to development of spinal locomotion. spinal walking in a cat with a complete spinal cord injury is much less likely to occur without training. animals with complete spinal cord transections and no training can begin to take steps within weeks of the injury. cats with naturally occurring spinal trauma had a low success rate in development of spinal locomotion after injury. reasons for the low success were attributed to less controlled spinal injury and inadequate physical therapy/training. training for minutes daily days a week provides an per cent success rate of weight-bearing in the pelvic limbs. without appropriate rehabilitation the rate drops to per cent. variability exists among cats as to when walking movements begin to occur. repeated training of a cat by placing the thoracic limbs on a nonmoving platform and the pelvic limbs on a treadmill resulted in better walking and weight-bearing ability in the pelvic limbs. this process involves the use of a treadmill, tail support, and various forms of stimulation. cutaneous stimulation is important for afferent sensory input. younger animals tend to have a better recovery rate for walking. training activities resulted in almost all cats regaining the ability to walk. early intensive training allowed for better walking. spinal locomotion is maintained only for a finite period after discontinuation of training activities and begins to show decline after weeks. long-term management of a deep pain-negative cat. much has been learned in cats after experimental spinal cord injury regarding optimal medical management of deep pain-negative cats. bladder expression is required at a minimum of twice daily. some cats urinate without expression, but the bladder is not emptied completely. stimulation of the perineum initiates a mass reflex and partial emptying of the bladder. researchers report that treadmill training also stimulates urination and defecation in cats with complete spinal cord injuries. inadequate emptying of the bladder predisposes to chronic urinary tract infections (see chapter ) . suggestions for care to prevent this problem include adequate bladder expression and water intake. chronic bladder infections weaken the muscular wall, further complicating manual emptying of the bladder. fecal elimination usually can occur without assistance and is aided by perineal stimulation. diarrhea and constipation still can occur as complications. clinical presentation and pathogenesis. intervertebral disc disease (ivdd) is recognized commonly in cats with approximately published cases. several case series have been published in recent years. [ ] [ ] [ ] the incidence of ivdd as a significant clinical problem compared to other diseases that affect cats has been reported to be . per cent. earlier clinical reports of disc disease in cats were postmortem studies that described cervical and, to a lesser extent, lumbar disc disease in older cats. [ ] [ ] [ ] [ ] [ ] these discs were mostly hansen type ii, with bulging of the annulus fibrosus into the spinal canal, and were described as incidental findings. characteristics of a degenerated intervertebral disc suggest some degree of chondroid degeneration of the discs. more recent literature describes hansen type i, with extrusion of nucleus pulposus into the spinal canal, and recognizes this type to be the most common form of disc-related spinal cord compression in cats. ivdd also can occur spontaneously in cats having no history of trauma. ivdd occurs more frequently in middle to older aged cats. mean age for all reported cases is years. the age range varies somewhat in different reports, between and years, and and years (mean age of . ). no gender or breed predilections exist for ivdd in cats. clinical signs of disc disease vary on lesion location and in severity and can consist of back pain and paresis/plegia. lesion involvement in the thoracolumbar region of the spinal cord is common. cervical disc disease is uncommon in cats, with two reported cases confirmed by necropsy, and one presumed case diagnosed with mri. [ ] [ ] [ ] disc spaces between the t and l vertebrae are affected in per cent of cats with clinical signs of ivdd. the l -l disc interspace also is a common site in per cent of the reported cats with ivdd. ivdd at l -s disc was described in a cat with lower motor neuron signs, flaccid tail, and urinary and fecal incontinence. diagnosis. survey spinal radiographs may reveal typical evidence of disc disease: narrowed disc spaces and evidence of mineralized material in the intervertebral foramen. collection of csf is performed to eliminate other potential inflammatory diseases. findings on csf analysis in cats with ivdd are not specific and may show a mild neutrophilic pleocytosis and increased protein concentration. myelography is used to localize the site of the disc extrusion/herniation more precisely. computed tomography can detect hyperdense material within the spinal canal at the affected disc space. findings on mri suggestive of ivdd include evidence of dehydration of the disc with loss of signal intensity on t -weighted sequence. treatment. conservative medical management has been used successfully in cats with ivdd; however, in severe spinal cord compression, this form of treatment should not replace surgery. based on a limited number of case reports, medical management alone may result in a poor outcome. conservative management still may be a better option in cases with a small amount of extruded disc material in the canal. medical management usually consists of pain control with use of a combination of narcotics and corticosteroids. corticosteroid therapy (prednisone . to mg/kg/day po) is used short-term in combination with strict cage confinement for to weeks. physiotherapy also may aid the long-term outcome of neurological function. surgical decompression for removal of extruded disc material may be accomplished with use of either a hemilaminectomy or dorsal laminectomy procedure. , surgery offers a higher rate of success and more rapid and complete neurological recovery when compared with conservative treatment. , many cats still have residual neurological deficits that include paresis and urinary and/or fecal incontinence. , clinical presentation and pathogenesis. fibrocartilaginous embolism (fce), or embolic myelopathy, has been described in many species including cats (table - ) . [ ] [ ] [ ] this is a rare disease in cats, with about per cent of spinal cord diseases attributed to vascular causes. in this disease, a small portion of fibrocartilaginous tissue, which is presumed to be intervertebral disc material, occludes the vascular supply to the spinal cord, resulting in an acute onset of asymmetrical spinal cord dysfunction. typically, fce is nonprogressive and not painful. lesions in the cervical and lumbar spinal cord regions have been reported. the mean age from the limited case reports available is . years, with a range between and years of age. diagnosis. diagnosis of fce is based on elimination of other causes of myelopathy. csf analysis may reveal a neutrophilic pleocytosis and an increased protein concentration. , similar abnormalities also have been reported with intervertebral disc disease and may simply indicate necrosis. , case reports have lacked definitive imaging results, except in one case in which myelography showed evidence of intramedullary swelling. mr images can be expected to show an increased signal intensity relative to surrounding tissues of the spinal cord parenchyma on a t -weighted sequence. treatment. treatment strategies have been extrapolated from treatment options recommended in other species with fce. this consists of high doses of mpss (if the drug can be administered within hours of the onset of clinical signs), adequate fluid therapy, bladder management, and supportive care. once the cat is stabilized, physical therapy may aid in recovery. prognosis in these cases is difficult to predict because the literature in this area shows some bias as definitive diagnosis requires necropsy. prognosis is presumed guarded to fair in cats that have deep pain perception intact. syringomyelia is an abnormal fluid-filled cavity within the parenchyma of the spinal cord. hydromyelia often occurs with syringomyelia and is defined as dilation of the central canal. pathophysiology of syringohydromyelia is associated with alterations in flow of csf often secondary to a congenital anomaly, infectious disease process, or trauma. syringohydromyelia has been reported in cats but is not well described. clinical signs include paraspinal hyperesthesia and paresis. the syrinx can be detected using mri. treatment usually is directed toward the underlying cause. an antiinflammatory dose of prednisone ( . to mg/kg/day) may reduce edema and inflammatory response. clinical presentation and pathogenesis. diverticulum within the subarachnoid space results in accumulation of csf and compression of the spinal cord, which causes neurological dysfunction. these diverticula are not true cysts but rather leptomeningeal cavitations that are filled with csf. spinal arachnoidal cysts have been reported to cause paresis in cats. [ ] [ ] [ ] [ ] another case report documented an intradural epithelial-lined cyst found at the vertebral body of c in a / year-old female burmese cat. location of these cyst formations is variable in cats and can occur in the cervical, thoracic, and lumbar spine. the cause of arachnoidal cysts is unknown, but may be related to factors that include previous trauma, inflammation, and developmental or congenital malformations. an arachnoidal cyst in a -year-old spayed female domestic shorthair cat with paraparesis was associated with a lordotic malalignment of the caudal thoracic spine. affected cats usually are young to middle-age with a range between and years of age. clinical signs usually are chronic and progressive and reflect the location of the cyst. duration of clinical signs is chronic and progressive in onset. a cat in one report showed signs for only a few weeks. diagnosis. diagnosis of spinal arachnoidal cysts is made using myelography, ct-myelography, or mri. the diverticulum is identified with myelography as a teardrop shape within the subarachnoid space ( figure - ) . magnetic resonance imaging can document a spinal arachnoidal cyst on a t weighted sequence as an area of hyperintensity. treatment. reports of treatment protocols for spinal arachnoidal cysts in cats have been limited. surgical fenestration has been reported. , , a hemilaminectomy or dorsal laminectomy is used to expose the cystic structure for dural fenestration and possible excision of the cyst. outcome in these cases has been excellent with complete recovery. residual neurological deficits may occur. histopathology of the cyst wall is recommended to rule out other lesions. palliative medical management consists of antiinflammatory doses of corticosteroids. prevalence of diseases of the spinal cord of cats intervertebral disc disease and myelography inflammatory cerebrospinal fluid analysis in cats: clinical diagnosis and outcome. australian college of veterinary scientists science week analysis of cerebrospinal fluid from the cerebellomedullary and lumbar cisterns of dogs with focal neurologic disease: cases ( - ) spinal cord diseases in cats bacterial discospondylitis in a cat lumbar diskospondylitis and meningomyelitis caused by escherichia coli in a cat neuropathology in cats experimentally infected with feline immunodeficiency virus: a morphological, immunocytochemical and morphometric study diagnostic features of clinical neurologic feline infectious peritonitis recommendations from workshops of the second international feline coronavirus/feline infectious peritonitis symposium spinal cord disorders an axial ct image after a myelogram of the lumbar spine from a -year-old male castrated domestic shorthair cat with a history of progressive upper motor neuron paraparesis. the cat also had a spinal malformation inflammation and changes in cytokine levels in neurological feline infectious peritonitis epidemiology of feline infectious peritonitis among cats examined at veterinary medical teaching hospitals comparison of different tests to diagnose feline infectious peritonitis back to the cat": feline spinal cord disease use of recombinant feline interferon and glucocorticoid in the treatment of feline infectious peritonitis a cryptococcal granuloma in the brain of a cat causing focal signs feline cryptococcosis: a retrospective evaluation clinical and serologic evaluation of cats with cryptococcosis cryptococcal infection in cats: factors influencing treatment outcome, and results of sequential serum antigen titers in cats cryptococcosis in cats: clinical and mycological assessment of cases and evaluation of treatment using orally administered fluconazole infectious diseases of the dog and cat toxoplasmosis and neosporosis feline leukemia virusassociated myelopathy in cats degenerative myelopathy in a cat feline lymphoma and leukemia therapeutic choices for the medical management of feline lymphoma. waltham feline medicine symposium feline spinal lymphosarcoma: a retrospective evaluation of cats spinal lymphoma in cats: cases feline lymphoma ( cases): proliferation indices, cluster differentiation immunoreactivity, and their association with prognosis in cats principles of treatment for feline lymphoma spinal cord astrocytoma in a cat a glioma in the spinal cord of a cat meningiomas in animals use of magnetic resonance imaging for diagnosis of a spinal tumor in a cat nonlymphoid vertebral canal tumors in cats: cases ( - ) multiple myeloma in cats: variable presentation with different immunoglobulin isotypes in two cats effects of a single large dose of methylprednisolone sodium succinate on experimental posttraumatic spinal cord ischemia. dose-response and time-action analysis the neuroprotective pharmacology of methylprednisolone correlation of methylprednisolone levels in cat spinal cord with its effects on (na + k + )-atpase, lipid peroxidation, and alpha motor neuron function lactate and pyruvate metabolism in injured cat spinal cord before and after a single large intravenous dose of methylprednisolone effects of multi-dose methylprednisolone sodium succinate administration on injured cat spinal cord neurofilament degradation and energy metabolism evaluation of an intensive methylprednisolone sodium succinate dosing regimen in experimental spinal cord injury pretreatment with alpha tocopherol enhances neurologic recovery after experimental spinal cord compression injury management of spinal trauma in cats survival rates and outcomes in cats with thoracic and lumbar spinal cord injuries due to external trauma high-rise syndrome in cats: cases tension band stabilization of fractures and luxations of the thoracolumbar vertebrae in dogs and cats: cases combined medical and surgical treatment after acute spinal cord injury: results of a prospective pilot study to assess the merits of aggressive medical resuscitation and blood pressure management methylprednisolone for acute spinal cord injury: an inappropriate standard of care is the role of steroids in acute spinal cord injury now resolved? high dose methylprednisolone in the management of acute spinal cord injury -a systematic review from a clinical perspective gastric hemorrhage in dogs given high doses of methylprednisolone sodium succinate complications of methylprednisolone sodium succinate therapy in dachshunds with surgically treated intervertebral disc disease evaluation of time-dependent spread of tissue damage in experimental spinal cord injury by killedend evoked potential: effect of high-dose methylprednisolone nonsurgical management of thoracic and lumbar spinal fractures and fractures/luxations in the dog and cat: a review of cases management of vertebral column fractures in dogs and cats: cases ( - ) spinal fracture or luxation reversible spinal cord trauma in cats. additive effects of direct pressure and ischemia an evidence-based review of decompressive surgery in acute spinal cord injury: rationale, indications, and timing based on experimental and clinical studies current use and timing of spinal surgery for management of acute spinal cord injury in north america: results of a retrospective multicenter study principles of vertebral fracture management use of pins and methylmethacrylate in stabilization of spinal fractures and luxations the rotational stabilizing effect of spinal fixation techniques in an unstable vertebral model recovery of locomotion in the cat following spinal cord lesions determinants of locomotor recovery after spinal injury in the cat locomotor capacity attributable to step training versus spontaneous recovery after spinalization in adult cats effects of training on the recovery of full weight bearing stepping in the adult spinal cat return of weight supported locomotion in adult spinal cats chronic spinal cord-injured cats: surgical procedures and management retention of hindlimb stepping ability in adult spinal cats after the cessation of step training feline intervertebral disc disease: a review of the literature intervertebral disc extrusion in six cats intervertebral disk disease in cats spontaneous lumbar intervertebral disc protrusion in cats: literature review and case presentations disc protrusions in the cat: distribution of dorsal protrusions along the vertebral column disc protrusions in the cat: age incidence of dorsal protrusions disc protrusions in the cat: ventral protrusions and radial splits degeneration of the intervertebral disc in the cat protrusion of the intervertebral disc in the cat intervertebral disc syndrome in the cat intervertebral disc protrusion in a cat acute intervertebral disc extrusion in a cat: clinical and mri findings lumbosacral disc disease in a cat radiographic diagnosis: intervertebral disc extrusion in a cat fibrocartilaginous embolic myelopathy in a cat tetraparesis in a cat with fibrocartilaginous emboli fibro-cartilaginous embolism in a cat syringomyelia and hydromyelia in dogs and cats subarachnoid cyst in a cat correlative imaging findings in seven dogs and one cat with spinal arachnoid cysts spinal subarachnoid cyst in a cat intradural epithelial cyst in a cat key: cord- -wj xmp h authors: karlin, l; darmon, m; thiéry, g; ciroldi, m; de miranda, s; lefebvre, a; schlemmer, b; azoulay, É title: respiratory status deterioration during g-csf-induced neutropenia recovery date: - - journal: bone marrow transplant doi: . /sj.bmt. sha: doc_id: cord_uid: wj xmp h exacerbation of prior pulmonary involvement may occur during neutropenia recovery. granulocyte colony-stimulating factor (g-csf)-related pulmonary toxicity has been documented in cancer patients, and experimental models suggest a role for g-csf in acute lung injury during neutropenia recovery. we reviewed cases of noncardiac acute respiratory failure during g-csf-induced neutropenia recovery. half the patients had received hematopoietic stem cell transplants. all patients experienced pulmonary infiltrates during neutropenia followed by respiratory status deterioration coinciding with neutropenia recovery. neutropenia duration was ( – ) days, and time between respiratory symptoms and the first day with more than leukocytes/mm( ) was (− . to ) day. of the patients, received invasive or noninvasive mechanical ventilation, including patients with acute respiratory distress syndrome (ards). five patients died, with refractory ards. in patients with pulmonary infiltrates during neutropenia, g-csf-induced neutropenia recovery carries a risk of respiratory status deterioration with acute lung injury or ards. clinicians must maintain a high index of suspicion for this diagnosis, which requires eliminating another cause of acute respiratory failure, g-csf discontinuation and icu transfer for early supportive management including diagnostic confirmation and noninvasive mechanical ventilation. exacerbation of prior pulmonary involvement may occur during neutropenia recovery. granulocyte colony-stimulating factor (g-csf)-related pulmonary toxicity has been documented in cancer patients, and experimental models suggest a role for g-csf in acute lung injury during neutropenia recovery. we reviewed cases of noncardiac acute respiratory failure during g-csfinduced neutropenia recovery. half the patients had received hematopoietic stem cell transplants. all patients experienced pulmonary infiltrates during neutropenia followed by respiratory status deterioration coinciding with neutropenia recovery. neutropenia duration was ( - ) days, and time between respiratory symptoms and the first day with more than leukocytes/mm was (À . to ) day. of the patients, received invasive or noninvasive mechanical ventilation, including patients with acute respiratory distress syndrome (ards). five patients died, with refractory ards. in patients with pulmonary infiltrates during neutropenia, g-csf-induced neutropenia recovery carries a risk of respiratory status deterioration with acute lung injury or ards. clinicians must maintain a high index of suspicion for this diagnosis, which requires eliminating another cause of acute respiratory failure, g-csf discontinuation and icu transfer for early supportive management including diagnostic confirmation and noninvasive mechanical ventilation. prognosis; cancer; hematological malignancies acute respiratory failure is a major cause of morbidity in cancer patients. about % of patients with solid tumors or hematological malignancies present with a respiratory complication, and pulmonary disease occurs in about half the patients with neutropenia or bone marrow transplantation. acute respiratory failure is the main reason for icu admission in cancer patients; - the mortality rate ranges from to %, and a need for mechanical ventilation is the main predictor of death. however, recent advances including strategies for early diagnosis, noninvasive mechanical ventilation, and better knowledge of clinical patterns such as leukemic pulmonary infiltration have translated into improved outcomes. [ ] [ ] [ ] [ ] in cancer patients, neutropenia recovery may be associated with a deterioration in oxygenation and exacerbation of pre-existing pulmonary disease. in a previous study, we found that the main risk factor for acute respiratory distress syndrome (ards) during neutropenia recovery was the occurrence of pneumonia during the neutropenic period. human granulocyte colony-stimulating factor (g-csf) is the most important regulatory cytokine capable of stimulating neutrophil production from committed hematopoietic progenitor cells, both in vitro and in vivo. , g-csf is widely used in cancer patients to reduce the duration of chemotherapy-induced neutropenia, most notably after peripheral blood stem cell transplantation and lymphoma treatments. in this last case, g-csf allows closer spacing of chemotherapy courses, thereby substantially improving the prognosis. , although g-csf is generally safe and well tolerated, there have been several reports of acute respiratory failure during g-csf-induced neutropenia recovery. , g-csf is believed to enhance cytokine production and to activate the oxidative burst within circulating or resident alveolar neutrophils and macrophages. , because early diagnosis and treatment are key determinants of survival in cancer patients with acute respiratory failure related to neutropenia recovery, a careful and close clinical monitoring of respiratory symptoms (respiratory rate, heart rate, and oxygen saturation) must be proposed in this condition. to help clinicians bear in mind the increased risk of acute respiratory failure during g-csf-induced neutropenia recovery in cancer patients, we describe cases seen in our intensive care unit (icu) over a -month period. to the medical icu of the saint-louis teaching hospital, paris, france, between june , and april , , were prospectively included. for each patient, the following were recorded: (a) demographics; characteristics of the cancer; (b) reason for icu admission; (c) simplified acute physiology score (saps ii) and logistic organ dysfunction score (lod); , (d) characteristics of respiratory involvement, including chest radiograph changes, type of respiratory support needed, daily values of pao /fio , and presence of ards; (e) duration of neutropenia, of g-csf administration, time from neutropenia recovery to respiratory symptoms; and icu-mortality. all patients underwent a thorough evaluation to rule out pulmonary infection. when the clinical status allowed, bronchoscopy was performed, as previously described. bronchoalveolar lavage (bal) specimens were sent to laboratories for microbiological studies (bacteria, mycobacteria, viruses, parasites, and fungi) and cytological studies. microbiologically documented pneumonia was defined as a protected distal sample culture showing colony-forming units/ml or a bal fluid culture showing colony-forming units/ml for bacterial pneumonia, or a positive nonquantitative screen for aspergillus fumigatus, pneumocystis carinii, respiratory syncytial virus, and other respiratory viruses. also, cultures of blood, intravascular catheters, urine, protected distal samples, peritoneal fluid, pleural fluid, and cerebrospinal fluid were performed as clinically indicated. results are reported as medians and quartiles ( th- th percentiles) or numbers (%). patient characteristics were compared using the w test or fisher's exact test, as appropriate, for categorical variables and the nonparametric wilcoxon test or the kruskal-wallis test for continuous variables. vital status at hospital discharge was available for all patients. in all, patients ( men and nine women, median age ( - ) years) fulfilled our inclusion criteria. all patients experienced acute respiratory failure during g-csf-induced neutropenia recovery. patient characteristics are reported in table . the diagnosis was lymphoma in patients ( %), leukemia in six ( %), myeloma in two ( %), and solid tumor in two ( %). all patients received cancer chemotherapy (table ) and seven also underwent radiation therapy (including five patients given total body irradiation). none of the patients had a diagnosis of chemotherapy-induced pulmonary toxicity. before icu admission, patients ( %) received peripheral blood stem cell transplantation after high-dose chemotherapy and two ( %) received allogeneic hematopoietic stem cells. icu admission occurred ( - ) days after the last chemotherapy course. median neutropenia duration was ( - ) days and time from neutropenia recovery to respiratory status deterioration was (À . to ) day. leukocyte counts were ( - )/mm on the day of icu admission, ( - )/mm on the day of neutropenia recovery, and ( - , )/mm on the last day of g-csf. median duration of g-csf was ( - ) days. g-csf was discontinued in all patients, within the period ranging from days before to day after neutropenia recovery. all patients had acute respiratory failure at icu admission. in addition, shock was present in six ( %) patients, acute renal failure in two ( %), and coma in one ( %). median saps ii score was ( - ) and median lod score was six, - indicating a % risk of death. during neutropenia, prior to icu admission, pulmonary infiltrates developed in all patients and was documented by microbiological tests in patients (table ). the organisms were aspergillus fumigatus (n ¼ ), staphylococcus aureus (n ¼ ), escherichia coli, pseudomonas aeruginosa, enterococcus faecalis, klebsiella pneumoniae, pneumocystis carinii, and respiratory syncytial virus. in all, patients had pre-existing heart disease, but in none of them was the acute respiratory failure ascribable to cardiac pulmonary edema. radiographic changes were present in all patients at acute respiratory failure onset and consisted in alveolar infiltrates in patients, interstitial pneumonia in two, mixed alveolar and interstitial changes in three, and nodules in two. in patients, hypoxia was considered too severe to allow bronchoscopy and bal. of the patients who did undergo these investigations, five had tests showing a pathogen; alveolar cells consisted only in alveolar macrophages. ards occurred in ( %) patients. figure shows the day-to-day changes in pao /fio ratio and leukocyte count within the -day period centered on neutropenia recovery. pao /fio was lowest on the day of neutropenia recovery. among ards patients, received conventional mechanical ventilation (mv) only, two received noninvasive mv followed by invasive mv, and one required only noninvasive mv. among the six patients without ards, four were treated with a high-concentration oxygen mask, one with noninvasive mv, and one with mv. duration of ventilatory support was six ( . - . ) days. patients who required noninvasive mechanical ventilation only were ventilated for five ( - ) days, and patients who had received invasive mechanical ventilation were ventilated for ( - ) days. the overall icu mortality rate was %; the five patients who died were among the with ards. neither neutropenia duration nor g-csf treatment duration predicted ards. neutropenia duration, g-csf duration, and maximum leukocyte count, were not significantly different in patients with and without ards. respiratory status deterioration and abnormal lung microvascular permeability during neutropenia recovery without g-csf is a well-known entity. , the possibility that g-csf may induce pulmonary toxicity has been investigated, particularly during neutropenia recovery. , we describe critically ill cancer patients who experienced noncardiac acute respiratory failure during g-csf-induced neutropenia recovery. in all patients, pulmonary infiltrates developed during the neutropenic period and worsened at neutropenia recovery. these cases support published evidence that g-csf-induced neutropenia recovery carries all patients received cancer chemotherapy agents known to cause pulmonary toxicity. however, none was considered to have chemotherapy-related pulmonary toxicity, and most of the survivors received further cancer chemotherapy without experiencing recurrent pulmonary disorders. however, as previously suggested, g-csfrelated pulmonary toxicity occurs more frequently in patients who received previously at least three courses of cancer chemotherapy, suggesting that g-csf enhances alveolar inflammation after repeated endothelial damage by chemotherapy agents. , , the first pulmonary event in our patients was pulmonary infiltrates during neutropenia without a need for icu admission. acute respiratory failure requiring icu admission occurred later, during neutropenia recovery. although pathogens were recovered in only half our patients, previous data suggest that g-csf and neutropenia recovery merely exacerbate previous lung injury, possibly via accumulation of neutrophils in the lungs. , however, as previously reported in this entity, macrophages, but not neutrophils, were recovered from bal fluid, suggesting that acute lung injury during neutropenia recovery may be nosologically similar to ards during neutropenia. , in a lamb model of experimental lung injury, g-csf-enhanced alveolar neutrophil functions, increasing both cytokine production and oxidative burst. , g-csf upregulates the production of cytokines that increase alveolar permeability or neutrophil influx, such as tumor necrosis factor (tnf) a, interleukin (il) b, and il- . , in vitro studies also found enhanced secretion of proinflammatory cytokines by isolated alveolar macrophages obtained during neutropenia recovery from rats that received g-csf, compared with rats that did not receive g-csf, providing a possible explanation for lung injury exacerbation during g-csf-induced neutropenia recovery. our study has some limitations, however. first, we only report cases of critically ill patients with acute lung injury or ards during g-csf-induced neutropenia recovery, but we do not report the magnitude of the risk. a prospective observation of the incidence and risk factors for respiratory symptoms (whatever the severity) during g-csf-induced neutropenia recovery is timely. second, further study should also seek to evaluate biological markers reflecting epithelial and endothelial injury that characterizes the disease, so as to help clinicians easily diagnose ards during g-csf-induced neutropenia recovery. third, g-csf has been investigated in large randomized trials in cancer and noncancer patients without any report of pulmonary toxicity. however, these studies were not powered for detecting such pulmonary complications. moreover, we believe that noncancer patients receiving g-csf are not expecting to present with pulmonary toxicity since they do not receive cancer chemotherapy. indeed, without neutropenia recovery, nor chemotherapy-related repeated endothelial injuries, the clinical condition we describe should not occur. last, we did not demonstrate that g-csf withdrawal resulted in respiratory improvement. however, g-csf discontinua- tion is in order after neutropenia recovery, and since g-csf-related pulmonary toxicity has been reported, all nonmandatory drugs potentially harmful for the lung should be stopped in case of severe respiratory failure. survival of cancer patients admitted to the icu for acute respiratory failure has improved in recent years. in the present study, the % mortality rate was far lower than expected in a population of critically ill cancer patients with severe disease, and a high rate of mechanical ventilation. this encouraging survival rate may be related mainly to early g-csf discontinuation (at acute respiratory failure onset), early diagnosis, and effective early supportive care. this study, together with previous data from animals and humans, strongly suggests the harmful effect of g-csf in some cancer patients who experience pulmonary infection during neutropenia. g-csf is widely used in cancer patients and has substantially improved the safety of cancer chemotherapy and the prognosis of many malignancies. this agent is generally safe. nevertheless, it may exacerbate respiratory disorders related to chemotherapyassociated pulmonary toxicity. the exacerbation occurs during neutropenia recovery. physicians should be aware of this risk and should exercise great caution when using g-csf in neutropenic patients with pulmonary involvement. in summary, in patients with pulmonary infiltrates during neutropenia, g-csf-induced neutropenia recovery carries a risk of respiratory deterioration due to acute lung injury or ards. it must be emphasized that g-csf is useful in many patients to reduce the duration of neutropenia and to allow intensive chemotherapy protocols. however, clinicians must maintain a high index of suspicion for g-csf-related acute respiratory failure so that they can discontinue g-csf therapy early and immediately transfer the patient to the icu for early noninvasive mechanical ventilation, adequate supportive care, and appropriate diagnostic investigations. outcomes of 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physiology score (saps ii) based on a european/north american multicenter study the logistic organ dysfunction system, a new way to assess organ dysfunction in the intensive care unit the american-european consensus conference on ards: de´finitions, mechanisms, relevant outcomes, and clinical trial coordination delayed pulmonary toxicity syndrome following high-dose chemotherapy and bone marrow transplantation for breast cancer the uncertain role of the neutrophil increased permeability pulmonary edema neutrophils and the adult respiratory distress syndrome occurence of the adult respiratory distress syndrome in neutropenic patients adult respiratory distress syndrome in patients with severe neutropenia effect of granulocyte colony-stimulating factor on acute lung injury in the rat effect of granulocyte colony-stimulating factor on bleomycin-induced acute lung injury and pulmonary fibrosis g-csf and il- but not gm-csf correlate with severity of pulmonary neutrophilia in acute respiratory distress syndrome association of endogenous g-csf with anti-inflammatory mediators in patients with acute respiratory distress syndrome we thank a wolfe md for helping with this manuscript. key: cord- -j nn g authors: fridholm, helena; Østergaard sørensen, line; rosenstierne, maiken w.; nielsen, henrik; sellebjerg, finn; bengård andersen, Åse; fomsgaard, anders title: human pegivirus detected in a patient with severe encephalitis using a metagenomic pan-virus array date: - - journal: j clin virol doi: . /j.jcv. . . sha: doc_id: cord_uid: j nn g we have used a metagenomic microarray to detect genomic rna from human pegivirus in serum and cerebrospinal fluid from a patient suffering from severe encephalitis. no other pathogen was detected. hpgv in cerebrospinal fluid during encephalitis has never been reported before and its prevalence in cerebrospinal fluid needs further investigation. metagenomic methodologies are excellent complement in cases where a diagnosis is difficult to establish with conventional laboratory tests and its usage is ever increasing. metagenomic approaches reveal presence of both pathogenic and commensals in patient samples where the focus is on identifying an underlying ethological agent for a specific disease condition. we report a case of severe encephalitis where the only microbe detected in the cns was human pegivirus (hpgv), hitherto only known to cause asymptomatic infections in humans. one previous report describes the detection of hpgv in brain tissue and csf [ ] . in both cases it is uncertain if hpgv is pathogenic but it is noteworthy to detect a virus at a high viral load in the cns. in other cases, hpgv infections have been associated with beneficial outcomes in patients dually infected with hpgv and hiv or ebola [ ] [ ] [ ] [ ] . a -year old danish female was admitted to the hospital for abdominal pain, vomiting, dizziness and lower extremity pain. she was working as a bartender on a cruise ship, was sexually active but had no travel history outside scandinavia or exposure to blood transfusions, intravenous drugs or close contact to animals albeit recently received a tattoo. her past medical history included radiofrequency catheter ablation for wolf-parkinson-white syndrome and she awaited elective cholecystectomy due to prior gallstone attacks. upon admission she was alert and circulatory stable with a fever of . • c, glasgow coma score of and a bmi of . routine blood test showed haemoglobin of . mmol/l, white blood cell count of . × /l with lymphocytopenia, normal platelet count, c-reactive protein (crp) of mg/l, alanine aminotransferase u/l, lactate dehydrogenase u/l, alkaline phosphatase u/l and bilirubin of mol/l. an acute laparoscopic cholecystectomy was performed but no pathology of the gall bladder was found. immediately following the surgery she developed increasing abdominal pain and fever and she underwent an explorative laparoscopy, again with normal findings. post-operatively the patient complained of headache and diplopia, which both disappeared within h and she was discharged from the hospital. however, she was readmitted the following day with fever, headache, sudden behavioral change, photosensitivity and ataxia. she presented with somnolence and neck stiffness on physical examination (glasgow coma score ). vital signs were within normal limits but she had a fever of . • c. peripheral blood showed crp of mg/l, white blood count . pleocytosis of cells/mm ( % lymphocytes) and increased protein concentration of . g/l, indicative of a viral infection. an mri of the brain revealed leptomeningeal enhancement over the right hemisphere together with parenchymal changes, consistent with meningoencephalitis. she was treated with aciclovir for suspected viral encephalitis and with meropenem for possible bacterial infection. over the following days the patient worsened with mental status deterioration and progressed into coma and was transferred to an intensive care unit for mechanical ventilation. repeated lumbar puncture on day disclosed an increase of mononuclear cells to leukocytes/mm ( % lymphocytes) with a protein concentration of . g/l. she received dexamethasone, methylprednisolone and later prednisolone. serum and csf were tested for relevant pathogens, all returned negative ( table ) . because of the lack of a specific diagnosis serum and csf were sent to statens serum institut, copenhagen, where the specimens were run on a lawrence livermore pan-microbial array. this array contains probes against all sequenced bacteria and viruses present in the ncbi database as of [ , ] . the only positive signal was for human pegivirus (hpgv) (acc. nr. gse ), and two separate diagnostic laboratories subsequently confirmed hpgv rna in both serum and csf [ , ] . the ct value for hpgv during the acute phase in serum and spinal fluid was . and . , respectively. an rnaseq library was prepared from serum and sequenced on an illumina platform to obtain type information. the reads mapped to the entire reference genome (acc. nr. nc ) with a mean sequence depth of , a pariwise identity of . % (nt) and , % (aa), respectively. the assembled sequence (acc. nr. kp ) clustered within genotype [ ] . after eight days with severe neurological symptoms the patient gradually recovered and was discharged from the hospital four weeks later for rehabilitation. five weeks after discharge she was still viremic for hpgv in serum but viral load had decreased times (ct . ). hpgv is a flavivirus, characterized by enveloped virions with a single-stranded, positive-sense rna genome and is closely related to hepatitis c virus [ ] . upon discovery hpgv was initially thought to cause acute hepatitis but this has later been disproven and an hpgv infection has not been linked to any clinical disease in humans [ , ] . on the contrary, an infection has been associated with a beneficial outcome in hiv patients and also recently in ebola infected individuals [ , ] . in radkowski et al. screened csf samples from patients suffering from aseptic meningitis or encephalitis by rt-pcr but could not detect hpgv in any of the samples [ ] . more recently, both positive and negative rna-strands of hpgv was detected in post mortem brain tissue from a multiple sclerosis (ms) patient, implying that the virus was replicating in the brain tissue [ ] . the authors also detected hpgv in the csf of the same patient, but screening of csf from an additional ms patients were all negative. in developed countries - % of healthy blood donors are viremic for hpgv [ ] [ ] [ ] [ ] [ ] [ ] ( . % in denmark) [ ] whereas in developing countries and for people with blood-borne or sexually transmitted diseases the prevalence reaches - % [ ] . clearance of hpgv coincides with the appearance of hpgv e antibodies [ ] [ ] [ ] and healthy individuals normally clears the infection within two years. between - % of the population are seropositive against the glycoprotein e reflecting a previous infection [ , ] . hpgv is not routinely tested for during illnesses of the cns and it was surprising to detect it in csf of our patient as the only positive finding. we analyzed an additional csf samples from encephalitis patients and from patients with a relapse of ms for hpgv by qpcr. for of the encephalitis samples we tested serum collected concurrently with the csf sample. all csf samples where negative for hpgv but one encephalitis patient was positive in serum (ct . ). interestingly, this patient also had csf pleocytosis, as our case patient. as hpgv is lymphotropic [ ] the presence of hpgv in the csf could be attributed to passive transport of the virus as the cells are recruited to csf. it is uncertain if the hpgv infection and its presence in csf in any way influenced the clinical presentation but this possibility cannot be excluded. a few weeks after discharge she was still viremic for hpgv but the viral load in serum had decreased from ct . to . . it seems that on rare occasions hpgv can enter the cns in high viral titers but it is unknown for how long it persists. clearance of virus from the csf is not a well understood process. herpesvirus (cmv, ebv, vzv and hsv / ) and flavivirus (tbev) can persist in individuals after encephalitis and may cause recurrent encephalitis. we have also excluded the possibility that the encephalitis is a result of a reactivation event due to the surgical procedure as the patient was negative for hsv and the symptoms coincided with the time of surgery. several viruses, bacteria and antibodies are known to be able to induce encephalitis but in many clinical cases the etiology remains unknown [ , ] . metagenomic methodologies target a multitude of microbes simultaneously thereby reducing the number of tests and the total cost. due to their unbiased presentation of the nucleic acids present in a sample they have an unprecedented potential as a diagnostic tool for differential diagnosis. ngs has been used to resolve the presence of leptospira santarosai [ ] and an astrovirus [ ] in csf in cases of encephalitis, where comprehensive microbiological testing was inconclusive. more precise interpretations need to be developed as both pathogenic and non-pathogenic microbes will yield a signal and perhaps disclose known virus with unexpected pathogenetic potential. whether presence of hpgv in csf affects disease progression remains to be established but these findings needs to be reported as they add to our knowledge of the microbial flora and aids in pinpointing which microbes warrants further attention and study. deep sequencing for the detection of virus-like sequences in the brains of patients with multiple sclerosis: detection of gbv-c in human brain gb virus c: the good boy virus? diagnostic value of anti-gbv-c antibodies in hiv-infected patients gb virus c coinfections in west african ebola patients acquisition of gb virus type c and lower mortality in patients with advanced hiv disease the microbial detection array for detection of emerging viruses in clinical samples-a useful panmicrobial diagnostic tool a microbial detection array (mda) for viral and bacterial detection multiplex real-time pcr for the detection and quantification of latent and persistent viral genomes in cellular or plasma blood fractions hepatitis c virus and gb virus c/hepatitis g virus viremia in swedish blood donors with different alanine aminotransferase levels evidence for extensive genotypic diversity and recombination of gb virus c (gbv-c) in germany the gb viruses: a review and proposed classification of gbv-a, gbv-c (hgv), and gbv-d in genus pegivirus within the family flaviviridae g-pers creepers, where'd you get those papers? a reassessment of the literature on the hepatitis g virus gb virus-c-a virus without a disease: we cannot give it chronic fatigue syndrome lack of gb virus c/hepatitis g virus sequences in cerebrospinal fluid in patients with central nervous system infections a case-control study of transmission routes for gb virus c/hepatitis g virus in swedish blood donors lacking markers for hepatitis c virus infection gb virus c/hepatitis g virus infection in patients investigated for chronic liver disease and in the general population in southern sweden prevalence of gb virus c (also called hepatitis g virus) markers in norwegian blood donors seroprevalence of gb virus c and persistence of rna and antibody a novel t cell evasion mechanism in persistent rna virus infection humoral immune response to the e protein of hepatitis g virus is associated with long-term recovery from infection and reveals a high frequency of hepatitis g virus exposure among healthy blood donors gb virus c epidemiology in denmark: different routes of transmission in children and low-and high-risk adults detection of antibodies to a putative hepatitis g virus envelope protein antibody to gbv-c second envelope glycoprotein (anti-gbv-c e ): is it a marker for immunity? prevalence of gbv-c/hepatitis g virus rna and e antibody among subjects infected with human immunodeficiency virus type after parenteral or sexual exposure evidence that the gbv-c/hepatitis g virus is primarily a lymphotropic virus causes of encephalitis and differences in their clinical presentations in england: a multicentre, population-based prospective study etiology of encephalitis in australia actionable diagnosis of neuroleptospirosis by next-generation sequencing diagnosis of neuroinvasive astrovirus infection in an immunocompromised adult with encephalitis by unbiased next-generation sequencing the authors declare no conflicts of interests. this study was partially funded by familiefonden egetrae. key: cord- -azzy t a authors: rahman, asma; niloofa, roshan; de zoysa, ishan m; cooray, akila d; kariyawasam, jayani; seneviratne, suranjith l title: neurological manifestations in covid- : a narrative review date: - - journal: sage open med doi: . / sha: doc_id: cord_uid: azzy t a covid- , a respiratory viral infection, has affected more than million individuals worldwide. common symptoms include fever, dry cough, fatigue and shortness of breath. some patients show neurological manifestations such as headache, dizziness, cerebrovascular disease, peripheral nerve and muscle symptoms and smell and taste impairment. in previous studies, sars-cov- and mers-cov were found to affect the nervous system. given the high similarity between sars-cov- and sars-cov- , effects on the nervous system by sars-cov- are a possibility. we have outlined the common neurological manifestations in covid- (information are up-to-date as of june ) and discussed the possible pathogenetic mechanisms and management options. two large cohort-based studies on neurological manifestations of covid- have been reported so far. in a study done in wuhan, china, mao et al. noted neurological manifestations in . % of covid- patients and these were significantly more common in patients with severe disease. central nervous system (cns) and peripheral nervous system manifestations were seen in . % and . %, respectively. in a study conducted in france, helms et al. found % of patients admitted to the intensive care unit because of acute respiratory distress syndrome due to covid- to have neurological signs. the differences in percentage between the two studies may be because the second study focused on more severely affected covid- patients. table summarizes the common neurological manifestations in covid- . the main cns manifestations observed are headache, dizziness, cerebrovascular disease (cvd), encephalopathy, delirium and other related manifestations as discussed below. headache and dizziness are the most common neurological manifestations recorded in several studies (table ) . , it is uncertain whether these two manifestations were caused by a direct effect of the infection on the nervous system or due to other factors such as stress, fear or anxiety. li et al., who analyzed the cohort of patients described by mao et al. and seven more, found % to have acute ischemic stroke. stroke was also reported in five patients younger than years from new york. based on brain magnetic resonance imaging (mri) findings from of their patients, helms et al. reported two to have acute cerebral ischemic stroke and one to have subacute cerebral ischemic stroke. in a retrospective cohort-based study from new york, . % had imaging-proven acute ischemic stroke and most ( %) strokes were cryptogenic, possibly related to an acquired hypercoagulability. a recent systematic review showed the incidence of acute ischemic stroke in covid- to be . % to . % with a mortality rate of %. in addition, several case reports of stroke in patients with covid- have been published ( table ). the cvd in covid- may be due to high levels of inflammation and/or a hypercoagulable state. raised serum interleukin and c-reactive protein concentration have been reported, and coagulation abnormalities are increasingly noted with raised d-dimer concentration pointing to a poorer prognosis. awareness by clinicians of the possibility of cvd in covid- patients may lead to more timely management decisions and thus a reduction in both morbidity and mortality. dixon et al. acute necrotizing encephalopathy a -year-old female patient with aplastic anemia reported seizures and reduced consciousness days after fever. ct and mri showed swelling of the brain. patient did not respond to steroid treatment and died on the th day after hospitalization. filatov et al. encephalopathy a -year-old female patient presented with encephalopathy and tested positive for covid- . csf tested negative for virus. pilotto et al. encephalopathy a -year-old female with covid- who progressively developed severe encephalopathy. improved with high doses of steroid treatment. poyiadji et al. acute necrotizing encephalopathy a female patient in her late s presented with acute necrotizing encephalopathy and later tested positive for covid- . the csf was not tested for sars-cov- virus. zayet et al. encephalopathy two cases having symptoms of encephalopathy followed by being positive for covid- . csf was tested negative for sars-cov- , and mri showed no signs of inflammation. avula et al. acute stroke a series of four patients presenting with radiographic evidence of acute stroke. beyrouti et al. acute ischemic stroke a series of six covid- patients with acute ischemic stroke. co et al. stroke a -year-old female patient with a past history of stroke presented typical symptoms of covid- initially. developed right upper and lower extremity weakness and severe dysarthria. fara et al. stroke a series of three patients presenting with stroke-like symptoms. the patients were found to have subocclusive severe stenosis of the common carotid artery. none had severe respiratory symptoms. gunasekaran et al. stroke a -year-old female patient presenting with typical symptoms initially, developed stroke during hospitalization immovilli et al. stroke a series of patients having stroke; two cases of hemorrhagic stroke and cases of ischemic stroke. reports an association between stroke and pneumonia severity in covid- patients. morassi et al. acute ischemic and hemorrhagic stroke a series of six patients who developed stroke. mainly presented severe pneumonia and multiorgan failure. oxley et al. large vessel stroke a series of five patients younger than years of age presented with large vessel stroke. al saleigh et al. one patient showing hunt and hess (h&h) grade aneurysmal subarachnoid hemorrhage, and the second patient having had an ischemic stroke with massive hemorrhagic conversion. csf tested negative for sars-cov- virus. valderrama et al. ischemic stroke a -year-old male patient with hypertension who tested positive for covid- . the patient presented to the hospital with symptoms of a stroke days later. zhai et al. acute ischemic stroke a -year-old male patient showing symptoms of a stroke and diagnosed with covid- . other cns manifestations bernard-valnet et al. acute meningoencephalitis both patients developed meningoencephalitis few days after diagnosis of covid- having mild respiratory and general symptoms. csf was tested negative for sars-cov- virus. chaumont et al. meningoencephalitis a -year-old patient developed meningoencephalitis week after infection. csf was tested negative for sars-cov- virus but the virus was detected in bronchoalveolar lavage. first munz et al. acute transverse myelitis the patient had typical respiratory symptoms of covid- and was discharged from the hospital. admitted back and diagnosed with multifocal myelitis. csf was negative for sars-cov- virus. al-olama et al. meningoencephalitis a -year-old male patient initially with typical covid- symptoms developed meningoencephalitis with intracerebral subdural hematomas. fluid from chronic subdural hematoma tested positive for sars-cov- rna. sharifi-razavi et al. intracerebral hemorrhage a -year-old with a history of fever and cough who tested positive for covid- . the patient presented with intracerebral hemorrhage after few days. reichard et al. acute disseminated encephalitis like pathology a patient was admitted due to coronary heart disease and underwent surgery. subsequently developed covid- . the patient died after weeks in hospital and autopsy revealed neuropathological lesions. sarma and bilello acute transverse myelitis a -year-old female patient with sars-cov- presenting lower back pain, bilateral symmetric upper, and lower extremity numbness. diagnosed with acute transverse myelitis. valiuddin et al. acute transverse myelitis the patient first presented with generalized weakness, following bilateral lower and upper extremity weakness. the csf tested negative for sars-cov- virus. vollono et al. focal status epilepticus a -year-old female patient whose primary presentation was focal status epilepticus. csf analysis was not carried out. wong et al. rhombencephalitis a -year-old male patient who initially had fever developed with acute brainstem dysfunction. mri showed changes in inflammation of the brainstem and upper cervical cord. csf was not tested for the virus due to low sample quantity. ye et al. encephalitis patient first presented with typical symptoms of covid- , followed by deteriorated consciousness. csf tested negative for virus. the condition gradually improved with the clearance of the virus and treatment. zanin et al. seizure/brain and spine demyelinating lesions a -year-old female patient who was found unconscious at home tested positive for covid- . the mri revealed demyelinating lesion in the brain and spine. csf tested negative for sars-cov- . zhang et al. adem first described case of adem with covid- . the mri showed patchy areas of abnormal signals in certain areas of the brain. zhao et al. there are reports of encephalopathy in covid- (table ) , and healthcare workers need to consider testing for sars-cov- in such patients. , delirium has been reported to occur in covid- , especially among older persons. beach et al. presented a case series, where three of the four covid- patients had delirium, without the presence of significant respiratory symptoms. at present, in most reported studies, csf had not been tested for sars-cov- . in the patient described by filatov et al., csf was tested and found to be negative. encephalopathy and delirium may be due to direct invasion of the cns by sars-cov- , inflammation secondary to a cytokine storm or as a result of septic encephalopathy. there are reports of encephalitis and meningitis in covid- . for instance, the sars-cov- virus has been detected in the csf of two patients with encephalitis, , raising the possibility of direct cerebral effects of the virus. mao et al. reported seizures and hemiplegia in one and two patients, respectively, prior to the onset of respiratory symptoms. however, in a retrospective study, lu et al. did not find an increased risk of symptomatic seizures in covid- patients. at present, it is uncertain whether the seizures are coincidental or due to sars-cov- viral effects or the drugs used in treatment. in the study by mao et al., . % of patients had peripheral nervous system manifestations. the common manifestations include guillain-barré syndrome (gbs) and other related variants and loss of the sense of taste and smell. currently, a total of reports on gbs and its variants in covid- have been reported. pathogen-associated antibodies that attack peripheral nerves due to molecular mimicry have been previously put forward as a disease mechanism in gbs. covid- -related gbs is mainly seen in the elderly while typical gbs can occur in all age groups. none of the patients with post-covid- gbs tested positive for sars-cov- in the csf, points to an immune mechanism such as inflammation secondary to a cytokine storm as a possible cause. some variants of gbs such as miller fisher syndrome and polyneuritis cranialis have been reported in two covid- patients from italy; both recovered fully within weeks. twenty cohort studies have reported on loss of smell (anosmia) and taste (ageusia) as early symptoms of covid- (table ) . these symptoms may appear early in the course of the disease or in otherwise asymptomatic individuals. a european study of covid- patients, conducted across four counties, found . % and % to have impairment of the sense of smell and taste, respectively. at present, there have been only a few studies on this aspect from the asia-pacific region. for instance, mao et al. found . % and . % of their cohort to have taste and smell impairment, respectively. compared with the european studies, the frequency of smell and taste impairment in the chinese study was low, which may be because the latter study was not specifically designed to assess this aspect. smell and taste impairment may also vary across different populations; individuals with a strong preference for spicy foods may have a reduced taste sensitivity than those with a lower preference. the variations may also be attributed to the method of testing, as most of the studies were questionnaire based. for instance, in a study by lechien et al., of the . % of patients self-reporting olfactory disorders, . % were subsequently found to be normal on objective testing. a standard quantifiable test needs to be developed to validate the variations of smell and taste impairment. increased awareness that olfactory and gustatory dysfunction is common and early symptoms in covid- would allow earlier diagnosis and thus effective self-isolation. currently, although complete recovery has been reported in the majority of patients, it may be too early to comment on the longer-term implications. it is still uncertain whether the taste and smell alterations are due to inflammation of the nasal tract or damage to the sensory neurons in the olfactory bulb. a large number of cells in the nasal epithelium express the angiotensin-converting enzyme (ace ) receptor which is the cell entry receptor for sars-cov- . however, brann et al. noted an absence of ace receptors in the olfactory sensory neurons and suggested inflammation may be the primary cause for small impairment. a previous mouse study found sars-cov- to be able to enter the brain through the olfactory bulb. ace receptors are found to be expressed in olfactory sustentacular cells and other non-neuronal cells in the olfactory epithelium. these cells maintain the integrity of the sensory neurons and damage to these may lead to alterations in smell and taste. skeletal muscle injury was recorded in . % of the covid- patients studied by mao et al. creatine kinase (ck), d-dimer, c-reactive protein and lactate dehydrogenase levels were found to be elevated in patients with skeletal muscle injury. in another report, myalgia was noted in . % of the studied covid- patients. clinicians should be aware of the range of neurological manifestations in covid- , as this would facilitate early recognition and appropriate management. further studies from different regions of the world, using appropriate brain imaging, electroencephalography (eeg) and csf analysis, could provide evidence for the neuro-invasive potential of sars-cov- . such studies would also shed further light on why many neurological manifestations are more common in the elderly with severe covid- . neurological involvement in covid- may be due to direct sars-cov- viral damage to the nervous system or through indirect means. ace receptors are highly concentrated in the substantia nigra and ventricles of the brain. it is also found in many neurons, astrocytes, oligodendrocytes, middle temporal gyrus and posterior cingulate cortex. a mouse cellculture study found ace receptor expression on astrocytes. ace receptors are also expressed on endothelial and arterial smooth muscle cells of blood vessels in the brain. these studies suggest that major cns manifestations are possible if the virus invades the brain. a recent autopsy study found sars-cov- viral particles, on electron microscopic examination, in the frontal lobe of the brain. virus-like particles were observed budding out of endothelial cells in the blood vessels of the frontal lobe, thus pointing to a hematogenous pathway of spread through the blood-brain barrier. in a study by ding et al., sars-cov- virus was detected exclusively in the neurons of the brain. the sars-cov- virus has also been found in the csf. a transgenic-mouse study found sars-cov- entry into the brain via the olfactory bulb, and a similar pathway has been postulated in humans. the entry of sars-cov- to the olfactory bulb through the cribriform plate might explain smell impairment in covid- . li et al. suggest the sars-cov- virus may spread to the medullary cardiorespiratory center in the brainstem via chemo and mechanoreceptors in the lung, as has been observed with some other respiratory viruses. this raises the possibility of a neurological mechanism for respiratory failure in some covid- patients. while ace- receptors are found in the alveolar epithelium of the lung, the mechanism of viral movement from the lungs to the nervous system remains unclear. the detection of the sars-cov- in csf or brain biopsies would further clarify this potential pathway. no previous studies have been published of such a mechanism in either sars-cov- or mers-cov infections. in addition to inflammatory effects in the brain, neurological manifestations may also be caused by hypoxia-related injuries, as alveolar and interstitial lung inflammation may lead to cns hypoxia. this in turn may cause cerebral vasodilation and cerebral edema. questionnaire boscolo-rizzo (italy) telephone surveys giacomelli (italy) questionnaire . hornuss (germany) the possibility that medications used to treat covid- may cause neurological manifestations should be remembered. for instance, other neurological infections that may occur due to the immunosuppressive effect of the medications or seizure thresholds may be reduced. headache is a common side effect of the monoclonal antibody tocilizumab and chloroquine. cases of tocilizumab-associated multifocal cerebral thrombotic microangiopathy and tocilizumabrelated demyelinating disorders have been reported. , chloroquine and hydroxychloroquine are also known to have certain neurological side effects such as seizure, balance disorder, peripheral neuropathy, parasthesia and hypaesthesia. considering the high transmission rate of the sars-cov- virus, carrying out autopsy studies are challenging. however, the findings from such studies would contribute to and shed light on the potential neurological mechanisms and prognosis in covid- and direct more evidence-based treatment plans. individuals with ms and neuromuscular disorders may be prescribed medications which suppress the immune system and thus are at a higher risk of developing severe covid- . however, guidelines specifically recommend having discussions with neurologists prior to modifying any courses of medication. patients who suffer with cvd have a . -fold higher risk of getting severe covid- . covid- seems to have a worsening effect on patients with parkinson's disease too. hainque and grabli report two patients with parkinson's disease where early diagnosis of covid- was challenging and thus associated with poorer outcomes. currently, there is no evidence that individuals with epilepsy are at a higher risk of developing covid- . limitations of this review include the small number of studies reporting on certain neurological manifestations, thus making it difficult to provide more definitive conclusions on these aspects. it is possible that subtle neurological findings were not documented (and thus underestimated) due to the high workload during the early part of the pandemic. further well-conducted studies from different regions of the world in the coming months would help expand this evidence base and thus provide better answers to the many questions at hand. ours is a broad overview on the main reported neurological manifestations in covid- and a more comprehensive clinical picture would emerge in the coming months. during the covid- pandemic, if a patient has neurological symptoms such as loss of the sense of smell and taste or delirium, testing for sars-cov- should be considered irrespective of them not having the other typical symptoms. at present, the long-term effects of neurological manifestations are still uncertain but should become better defined as more studies using brain imaging, eeg and csf findings become available. detailed and systematically conducted histopathology and autopsy studies should shed light on aspects of pathogenesis and pathology that are still undefined and uncertain. neurological manifestations have been reported in some covid- patients. the detection of sars-cov- in the csf of two patients and in endothelial cells of blood vessels of the frontal lobe of another provides evidence for a neurotropic potential of this virus. the nervous system may also be affected via indirect methods such as hypoxia, inflammation or an immune-mediated damage. future studies using brain imaging, eegs, csf analysis and histopathology would provide a clearer understanding of the effect of sars-cov- on the nervous system. the author(s) declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article. the author(s) received no financial support for the research, authorship, and/or publication of this article. suranjith l seneviratne https://orcid.org/ - - - neuroinvasion by human respiratory coronaviruses acute and persistent infection of human neural cell lines by human coronavirus oc neurological manifestations in severe acute respiratory syndrome organ distribution of severe acute respiratory syndrome (sars) associated coronavirus (sars-cov) in sars patients: implications for 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cerebrovascular disease is associated with an increased disease severity in patients with coronavirus disease (covid- ): a pooled analysis of published literature rapid worsening in parkinson's disease may hide covid- infection key: cord- -i bfuvq authors: macdonald-laurs, emma; koirala, archana; britton, philip n.; rawlinson, william; hiew, chee chung; mcrae, jocelynne; dale, russell c.; jones, cheryl; macartney, kristine; mcmullan, brendan; pillai, sekhar title: csf neopterin, a useful biomarker in children presenting with influenza associated encephalopathy? date: - - journal: eur j paediatr neurol doi: . /j.ejpn. . . sha: doc_id: cord_uid: i bfuvq purpose: neurological complications of influenza cause significant disease in children. central nervous system inflammation, the presumed mechanism of influenza-associated encephalopathy, is difficult to detect. characteristics of children presenting with severe neurological complications of influenza, and potential biomarkers of influenza-associated encephalopathy are described. methods: a multi-center, retrospective case-series of children with influenza and neurological complications during was performed. enrolled cases met criteria for influenza-associated encephalopathy or had status epilepticus. functional outcome at discharge was compared between groups using the modified rankin scale (mrs). results: there were children with influenza studied of whom / had encephalopathy and / had status epilepticus. only one child had a documented influenza immunization. the biomarker csf neopterin was tested in / children with encephalopathy and was elevated in / . mri was performed in all children with encephalopathy and was abnormal in ( %). treatment of children with encephalopathy was with corticosteroids or intravenous immunoglobulin in / ( %). in all cases oseltamivir use was low ( %) while admission to the intensive care unit was frequent ( / , %). clinical outcome at discharge was moderate to severe disability (mrs score > ) in the majority of children with encephalopathy ( / , %), including one child who died. children with status epilepticus recovered to near-baseline function in all cases. conclusion: raised csf neopterin was present in most cases of encephalopathy, and along with diffusion restriction on mri, is a useful diagnostic biomarker. lack of seasonal influenza vaccination represents a missed opportunity to prevent illness in children, including severe neurological disease. severe neurological complications from seasonal influenza, including influenza-associated encephalopathy/encephalitis (iae), cause considerable morbidity and mortality in healthy children, and those with pre-existing neurological disease. e recent estimates indicate the annual incidence of iae in australia is . per , , in children under years, with around % of hospitalized influenza cases associated with iae. other populations show similar or higher incidence, with japan's annual incidence of iae recorded as e / , , . , neurological complications attributed to influenza range from a mildly altered mental state, vertigo and brief febrile seizures to life threatening complications such as status epilepticus, meningitis, stroke, and demyelinating disease. antiviral agents, predominantly neuraminidase inhibitors, and immunomodulatory treatments (corticosteroids, intravenous immunoglobulin), are used to treat patients with influenza-associated neurological disease but there is limited evidence on their efficacy. while it is thought that more extensive changes on mri correlate with disease severity there are no other available biomarkers that predict outcome. the australian influenza season typically occurs between july and october. the season saw the highest levels of influenza reported since the pandemic year. the authors of this report noted an apparent increase in iae and other severe neurological complications during . here, we describe the clinical presentation, laboratory testing, neuroimaging, treatment and short-term outcome of these cases. in addition, we observed elevated cerebrospinal fluid (csf) neopterin e a marker of central nervous system (cns) inflammation e amongst children with iae that has not previously described. we compared the frequency of iae during the influenza season with previously published incidence estimates. materials and methods we identified children aged e years, with evidence of influenza and associated severe neurological disease including status epilepticus or moderate to severe encephalopathy, admitted to two paediatric hospitals which comprise the sydney children's hospital network, the largest paediatric network in australia. cases were ascertained between april st and october st, . at the children's hospital at westmead, cases were identified from those recruited under pre-existing surveillance studies: the australian childhood encephalitis study (ace), and the influenza complications network (flucan) surveillance study. , at sydney children's hospital, children were identified from neurology consultation databases. children were included if they required hospital admission and consultation from a paediatric neurologist for a neurological complication or worsening of a pre-existing neurological condition due to proven influenza. all children included either presented with status epilepticus (for min or longer) or reached level diagnostic certainty on the brighton encephalopathy score. children were excluded if: influenza was not confirmed, neurological symptoms were mild, hospital admission was not required, and when an alternative diagnosis could better explain the presentation. data were retrospectively collected from electronic medical records. we collected demographic data, presenting clinical characteristics, intensive care unit (icu) admission, and length of stay, laboratory results including csf testing, and influenza testing. csf analysis included cell count, protein, glucose, microscopy, lactate, oligoclonal bands, neopterin and influenza pcr. an elevated csf neopterin result was defined as > nmol/l. electroencephalogram reports and brain magnetic resonance imaging (mri) (t weighted, flair and diffusion weighted imaging) were assessed by a neurologist (s.p.) and neuroradiologist (c.c.h.). the neuroradiologist was blinded to diagnosis during review of the mri. influenza was most commonly acutely diagnosed through the detection of influenza rna in respiratory samples. both hospitals used multiplex pcr assays (seegene, south korea) which detected up to respiratory viruses. the assay has targets for both influenza a and b and was performed daily with a turnaround time of e days. at sydney children's hospital, the assay also had targets for subtypes of influenza a: h strains and the pandemic strain h n / . when influenza serology was requested to diagnose a recent influenza illness, this was performed using a complement fixation assay (virion, germany). treatment given including oseltamivir, empiric aciclovir, or rd generation cephalosporins, ivig, corticosteroids, and e u r o p e a n j o u r n a l o f p a e d i a t r i c n e u r o l o g y ( ) e plasmapheresis were recorded. time from admission to commencement of oseltamivir was recorded. each case was assigned a modified rankin scale (mrs) based on the examination at presentation and discharge from hospital. the mrs ranges from (no symptoms) to (death). a poor outcome was an mrs score of > which indicates at least moderate disability requiring assistance. the relation between categorical variables was investigated using the two tailed fisher exact test. the mannewhitney u test was used to determine the relation between continuous variables. ethics approval was granted by the sydney children's hospitals network ethics committee (lnr/ /schn/ ). twenty two children were included in this case series; % ( / ) were female. the median age at presentation was five years (range: . e years). eight children ( %) had preexisting epilepsy and/or developmental delay. one child had an immunodeficiency (hypogammaglobulinemia) and receives monthly ivig. this child, who presented with status epilepticus, was the only child recorded as having received the seasonal influenza vaccination. eleven children ( %) met level brighton criteria for encephalitis and were designated as influenza associated encephalopathy/encephalitis (iae). two children with iae had previous episodes of acute disseminated encephalomyelitis (adem) with complete clinical and radiological recovery ( table cases & ). one child had mosaic tetrasomy x, while another had epilepsy and developmental delay. the remaining children in our series had status epilepticus. in contrast to the children with iae, over half of this group (n ¼ ) had preexisting neurological disease including two children with refractory genetic epilepsies (dravet syndrome and cdkl ). the majority of children had a fever ( %) and two-thirds had respiratory symptoms. half presented with neurological symptoms within two days of onset of their influenza illness. sixteen children ( %) presented with an altered level of consciousness. seizures occurred in children ( %) at any stage of illness and status epilepticus was frequent (n ¼ , %). other neurological findings at presentation were weakness (n ¼ , %), pyramidal signs (n ¼ , %), movement disorder (n ¼ , %) and ataxia (n ¼ , %). hallucinations, meningism, cranial neuropathy and pupillary changes were infrequent (< %). the majority of children (n ¼ , %) had influenza a. of those sub-typed (n ¼ ) half were h ( ) and half were h . four children had other respiratory pathogens co-identified on npa (rhinovirus, coronavirus, mycoplasma pneumoniae). enterovirus was detected in the npa of one child but was absent in csf. one blood culture was positive for staphylococcus epidermidis, and this was assessed to be a contaminant. lumbar puncture was performed in children where it was considered clinically indicated ( table ) . of those who did not have a lumbar puncture performed most were children who presented with status epilepticus alone, usually with known pre-existing epilepsy. one case, with acute necrotising encephalopathy (ane), who was deemed to be too unwell to undergo a second lumbar puncture for measurement of neopterin. where sampled, csf showed pleocytosis and elevated protein in only a third (each n ¼ ). influenza pcr on csf was positive in of children tested, in an immunocompetent previously well year old. the csf neopterin was elevated in of children tested; in seven children it was considerably elevated ranging from to nmol/l (normal < nmol/l), one had a borderline result ( nmol/l). csf neopterin was measured in one child presenting with status epilepticus and was . nmol/l (borderline result). while most children with iae had a raised csf neopterin ( / , %), only three had csf pleocytosis ( , , cells/mm ) and two had an elevated csf protein ( . g/l and . g/l). oligoclonal bands were measured on serum and/or csf in / children with iae and were not present in any. some children with iae had anti-neuronal antibodies testing performed on serum and/or csf, usually nmda and vgkc (table ). these were negative apart from two cases with mildly elevated anti-thyroid antibodies. other routine laboratory data were normal or only mildly abnormal in most children (table ) . mri brain was performed in children and showed new abnormalities in eight ( %), all with iae. the common acute mri abnormalities were the presence of t -flair hyperintensities, diffusion restriction (each, n ¼ ), and gadolinium enhancement (n ¼ ). the spectrum of radiological features are shown in fig. aei . diverse clinico-radiological syndromes were diagnosed including: ane (n ¼ ), acute encephalopathy with biphasic seizures (aesd) (n ¼ ), posterior reversible encephalopathy (pres) (n ¼ ), hemiconvulsion hemiplegia syndrome (hhs) (n ¼ ), and cerebellitis. genetic testing of ran-binding protein (ranbp ) was performed in the child with ane (table case ) and the child who died from an ane-like illness ( table , case ). both were negative. one child who met criteria for iae was subsequently found to have a mutation in the polymerase gamma (polg) gene (table , case ). fourteen ( %) children were admitted to the icu and nine ( %) required mechanical ventilation. thirteen ( %) children received oseltamivir. median time to commencement of oseltamivir from presentation was day (mean . days, iqr e days), but > days in five cases. two icu ventilated children were commenced on oseltamivir nine days after admission. in contrast, nineteen ( %) children were treated with a rd generation cephalosporin, while ( %) received aciclovir. nineteen ( %) received anticonvulsants and ( %) continued these on discharge. first-line e u r o p e a n j o u r n a l o f p a e d i a t r i c n e u r o l o g y ( ) e immunomodulatory treatment (corticosteroids and/or ivig, plasmapheresis) was given to nine children with iae (corticosteroids (n ¼ ), ivig (n ¼ ) and plasmapheresis (n ¼ )) but none of those with status epilepticus alone. the median length of icu and hospital stay was four days (range e ) and days (range e days) respectively. children with iae were more likely to have both longer hospital (mean . days vs . days; p ¼ . ) and picu admissions (mean . days vs days; p ¼ . ) compared to children with status epilepticus. one child with iae died following an ane-like illness, although mri findings were atypical (table , case ). her post-mortem was inconclusive: showing generalised cerebral oedema and some features of acute hemorrhagic leucoencephalitis. ten out of children with iae had an mrs score of (normal) at baseline. at discharge from hospital / ( %) of children with iae had a higher in mrs score (mrs > ; moderate disability) compared to those with status epilepticus. the change in mean mrs was significant between the two groups: children with iae had a change in mrs of . points while those in the status epilepticus group had a mean increase of mrs of . points (p-value: . ). nearly all children ( / , %) with mri diffusion restriction had a poor outcome. the child with mri diffusion restriction and a good outcome had posterior reversible encephalopathy syndrome (pres). among the children with iae and a considerably elevated neopterin, four had an mrs > while three had mild deficits (mrs , and ). there was a mean increase in the mrs of . points the seven children with highly elevated csf neopterin ( e nmol/l). in this case series we observed two groups of children who presented with severe influenza related neurological disease. one group of children fulfilled criteria for iae, while the other group, most often with pre-existing neurological disease, presented with status epilepticus but otherwise did not fulfill criteria for iae. amongst cases of iae, elevated csf neopterin appeared to correlate with the presence of diffusion restriction on mri brain and adverse outcome. use of oseltamivir was infrequent among all cases, although use of antibiotics occurred in the majority. only one child had documented influenza vaccine, even amongst those with pre-existing neurological co-morbidity, despite the fact that this is recommended in australia. influenza associated neurological complications are thought to occur due to an inflammatory or immunemediated response to influenza infection rather than direct viral invasion. , among those with iae we observed, similar to previous authors , , that csf pleocytosis and detection of influenza in the csf occurred in a minority (n ¼ ; %, and n ¼ / ; %). , , , however csf neopterin, a biomarker of inflammation, was elevated in most children with iae (n ¼ , %). neopterin, a catabolic product of guanosine triphosphate (gtp), is synthesized by human macrophages upon stimulation from interferon gamma and can be measured in urine, serum and csf. while serum neopterin levels are useful in the diagnosis and monitoring of systemic infectious or inflammatory diseases, such as hiv, csf neopterin, reflecting intrathecal production by microglial cells, more accurately detects cns inflammatory diseases (infectious or immune mediated). , a recent review assessing biomarkers of csf inflammation found, among clinically available tests, that csf neopterin performed better than the presence of oligoclonal bands or csf pleocytosis in detection of cns inflammation. there is limited data regarding prognostically significant biomarkers in iae. pro-inflammatory cytokines may impair the blood brain barrier and induce apoptosis of neurons. elevated cytokines such as interleukin- (il- ) and tumour necrosis factor alpha have been demonstrated in children with iae and correlates with poorer outcome. , , testing csf il- outside the research setting is currently unavailable. clasmatodendrosis, abnormal morphological changes in astrocytes, occurring presumably due to the effect of proinflammatory cytokines, has recently been suggested to be a pathological feature of iae on autopsy. clasmatodendrosis was found in the cerebral white matter, thalamus, corpus callosum, cerebellum, thalamus and hippocampus of children with iae and may correlate mri changes commonly seen. previous authors have associated abnormalities on mri brain with poorer outcome. in our cohort mri brain abnormalities were diverse and common, particularly diffusion restriction in the subcortical white matter. diffusion restriction correlated with a poor outcome, apart from in the child who had pres, and was associated with an elevated csf neopterin in most cases. further studies of iae are required to evaluate whether significant elevations of csf neopterin, particularly in combination with diffusion restriction and other mri changes, could predict short and long-term outcome. oseltamivir, a neuramidase inhibitor which prevents release of influenza virus from infected cells has been shown to reduce influenza symptoms in otherwise healthy children by h ( % ci e h,p ¼ . ). only % ( children) were treated with oseltamivir and there was a significant delay in commencement in cases (> days in hospital). in contrast, empirical rd generation cephalosporin ( %) and aciclovir use ( %) was more frequent. this may be related to the perception among practitioners that antiinfluenza therapy has little benefit. we suggest in accordance with local guidelines, that children with encephalitis should be empirically treated with oseltamivir during the influenza season (may to october). the evidence for use of immunotherapy (ivig, corticosteroids) in iae, is limited , however, in our case series, most children with iae were treated with first-line immunotherapy with uncertain benefit. no serious side effects were reported. in , the burden of influenza in australia (particularly the eastern states) was the highest seen since the pandemic. based on iae incidence estimates published by britton et al. from the e influenza seasons in australia and the population coverage of our hospitals, we calculated that we would expect . ( . e . ) cases of iae in children (< years) per year. the iae case frequency observed in our cohort was twice the expected point estimate based on these previous incidence estimates but within the % confidence interval, and so contribute to validating the estimates from britton et al. the short-term outcome of our cohort, particularly those with iae, was alarming with % having a poor outcome. while there was a significant rate of icu admission among the group of children with status epilepticus ( %) this was not as high as children with iae ( %) and, most often, non-iae children did not experience a significant change in their mrs. this supports previous observations that survivors of iae during the h n pandemic, and in more recent nonpandemic influenza seasons in australia, experienced significant ongoing disability. , we have previously shown in a large retrospective encephalitis cohort study that icu admission, mri diffusion restriction and status epilepticus and were risk factors for a long-term abnormal outcome. these risk factors were common ( %, %, %) in children with iae from our cohort. the medium and long-term outcome in our cohort should be assessed including formal neuropsychological testing. further research is required to understand and modulate the cns inflammatory cascade present in iae in order to modulate long term neurodisability. the overall influenza immunisation rate during in australia was low at %, however a recently observed rate of vaccine receipt among children was even lower at . %. four age-specific quadrivalent influenza vaccines containing two strains of influenza a (h n [michigan] and h n [hong kong]) and two strains of influenza b (brisbane and phuket) were available in . children older than six months were eligible to be vaccinated and the vaccine was provided free to children with neurological disease. in our cohort just one child had a documented influenza vaccination, although a third of children were eligible for free immunisation and the remainder could have received an immunisation at the cost of around $ e aud. we emphasise that the severe syndromes and adverse outcomes observed here should be considered preventable. following high rates of influenza related morbidity in (including these cases), new south wales and other australian states have introduced universal funded seasonal influenza immunisation to all children aged months to years. our series has limitations. we describe children with severe influenza-associated neurological complications but did not include children with mild neurological complications. children with pre-existing epilepsy may not have always been tested for influenza and may be under-represented. the collection of clinical data was retrospective, and some electronic data were incomplete. seasonal influenza immunisation status was not always clearly recorded, although we reviewed the australian immunisation register to verify vaccination status where possible. influenza sub-typing from npa samples and csf influenza pcr testing was not routinely performed. in the se group, csf studies, including csf neopterin were performed infrequently and mri brain infrequently requested. due to this we were unable to use this group as a direct control for the finding of elevated csf neopterin the iae group. serial csf neopterin to assess treatment and clinical progress were not performed. this is the first series to demonstrate that elevation of csf neopterin, a marker of cns inflammation, occurs commonly in children with iae. csf neopterin may be a useful diagnostic marker for iae while its role as a prognostic marker e u r o p e a n j o u r n a l o f p a e d i a t r i c n e u r o l o g y ( ) e requires further evaluation. mri diffusion restriction was associated with a poor outcome in iae. short-term outcomes of children with neurological complications of influenza, especially within the iae group, were alarming, with nearly two-thirds of children having a poor outcome despite receipt of icu support, anticonvulsants, first-line immunotherapy and, in some, anti-viral treatment. given the severity of influenza associated neurological complications, we recommend a "treat and test" approach to the use of oseltamivir in children presenting with acute encephalopathy/encephalitis during the influenza season. finally, seasonal influenza vaccination should be universally provided to children and those at risk of severe influenza, with better education and awareness to increase uptake in the paediatric population. this research did not receive any specific grant from funding agencies in the public, commercial, or not-for-profit sectors. neurological manifestations of influenza infection in children and adults: results of a national british surveillance study the spectrum and burden of influenza-associated neurological disease in children: combined encephalitis and influenza sentinel site surveillance from australia neuroinfluenza: evaluation of 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results from the paeds-flucan collaboration influenza vaccine effectiveness against pediatric deaths seasonal influenza vaccination the authors have stated that they had no interests, which might be perceived as posing a conflict or bias. this manuscript has been contributed to, seen, and approved by all the authors. all the authors fulfill the authorship credit requirements. no honorarium grant or other form of payment was received for the preparation of this manuscript. supplementary data to this article can be found online at https://doi.org/ . /j.ejpn. . . . r e f e r e n c e s key: cord- -xwclh ih authors: kim, faith; reichman, victoria; hooven, thomas a title: human herpesvirus- meningitis in a premature infant with fevers: a case and literature review date: - - journal: clin med insights case rep doi: . / sha: doc_id: cord_uid: xwclh ih human herpesvirus- (hhv- ) is a common virus that can cause nearly universal infection in infancy and early childhood. it typically manifests as an acute febrile illness. we describe a case of a premature infant with congenital hydrocephalus secondary to aqueductal stenosis with a ventriculoperitoneal shunt in place who developed intermittent fevers while she was admitted to the neonatal intensive care unit. she was ultimately diagnosed with acute hhv- meningitis. in addition to this report, we present a literature review regarding this virus’s potential modes of transmission and forms of clinical presentation in the neonatal period. human herpesvirus- (hhv- ) is a ubiquitous dna virus that causes near-universal infection in childhood. similar to other herpesviruses, it can establish latency in monocytes and macrophages and is capable of reactivation. there are expressed variants including hhv- a and hhv- b, the latter more prevalent and known to cause roseola infantum or sixth disease, which is associated with % to % of acute febrile seizures in young children. infants and children between months and years of age acquire primary hhv- infection following the loss of protective maternal antibodies with seroprevalence of hhv- reaching more than % in children greater than years of age; most have evidence of infection by months of age. , there is a wide clinical spectrum of hhv- infection ranging from asymptomatic disease to more serious disease including neonatal hepatitis, infectious mononucleosis-like syndrome, hemophagocytic syndrome, or viral myocarditis, particularly in the immunocompromised population. , although infrequent, primary hhv- infection is associated with neurological disease including meningitis and encephalitis that can lead to longterm neurologic sequelae and poor outcomes. congenital hhv- infection, which is defined as presence of hhv- dna in an infant at birth, has been previously reported in approximately % of cord-blood samples by hall et al. however, congenital infection could originate from multiple sources-one of which is by hereditary transmission of chromosomally integrated hhv- (cihhv- ), which has recently become recognized as an inherited condition with a unique mode of transmission. , although the majority of hhv- + cases are due to latent cihhv- , a significant subset of hhv- + neonates acquire active infection transplacentally, usually from mothers with cihhv- that has activated and produced replicating virions during pregnancy. , the significance of this virus along with its potential neurologic predilection and reactivation capabilities remain unclear in the neonatal period. we report a case of a premature infant with congenital hydrocephalus and a ventriculoperitoneal (vp) shunt in place who presented with intermittent fevers ultimately found to have acute hhv- meningitis. a baby girl was born at weeks' gestation via tertiary repeat cesarean section. she was born to a -year-old woman who presented unregistered to our institution in active preterm labor with vaginal bleeding concerning for abruption versus premature rupture of membranes. prenatally, the fetus was noted to have severe congenital hydrocephalus and bilateral cleft lip and palate diagnosed at weeks' gestation at a previous institution. she had a fetal brain magnetic resonance imaging (mri) suggestive of aqueductal stenosis. the mother had scant prenatal care with her last visit at weeks' gestation. her initial labs included a negative urine toxicology screen on admission; her routine prenatal serology tests ultimately returned negative. the baby was born with apgar scores of and at and minutes, respectively. her birth weight was . kg with a head circumference of . cm (> th percentile). her exam was notable for macrocephaly with bulging fontanelles, large bilateral cleft lip/ palate, nasal encephalocele, and bilateral microphthalmia. her neurologic exam was notable for diffuse hypotonia but spontaneous movement of her extremities. during her admission to the neonatal intensive care unit (nicu), she underwent a vp shunt placement on day of life and a gastrostomy tube placement on day of life given her inability to feed by mouth with clinical medicine insights: case reports her severe craniofacial anomalies. she was extubated to room air following each surgery with no difficulties and was tolerating full enteral feeds awaiting placement in a long-term care facility, formal adoption, and staged cleft repair at months' corrected age. her medications included vitamin d and iron supplementation. in terms of a genetic work-up for her underlying anomalies, she had a negative microarray and fluorescence in situ hybridization (fish) analysis with no further genetic testing performed. on day of life , she had an axillary temperature of °c, pulse of per minute and blood pressure of / . her exam at the time of the first fever was notable for tachycardia, cool and clammy distal extremities, and overall fussiness but an otherwise unchanged neurological exam. her gastrostomy tube and vp shunt sites were not suggestive of a skin infection, and her respiratory status was stable. blood and urine cultures were drawn, a respiratory viral panel was negative, which tests for common respiratory viruses and bacteria via molecular identification at our institution (table ) . her initial complete blood count (cbc) demonstrated a mild leukopenia with white blood cell (wbc) count . × ul − , hemoglobin . g/dl, mean corpuscular volume . fl, and platelets of × ul − . manual differentiation showed % segmented neutrophils, % bands, and % lymphocytes. a repeat cbc on her fifth day of fever demonstrated leukocytosis with increased wbc count . × ul − and elevated platelet count × ul − with % segmented neutrophils, % bands, and % lymphocytes on a manual differential. there were no atypical lymphocytes noted. her initial c-reactive protein (crp) was . mg/l, which peaked at . mg/l on her second day of fever. her basic metabolic panel was within normal limits. she had a chest radiograph that did not show a focal infiltrate. she was initiated on vancomycin and ceftazidime at meningitis treatment doses. she continued to have intermittent fevers as high as °c multiple times per day for the next days and would defervesce each time with acetaminophen. with her fevers she would become tachycardic and diaphoretic, but in between fevers, her exam was at her baseline. after hours of negative blood and urine cultures, her antibiotics were stopped. she had increased nasal congestion and secretions, so a repeat respiratory viral panel was performed on her second day of fever but returned negative. on her fifth day of fever, her exam was notable for increasing irritability and more persistent tachycardia with elevated inflammatory markers, so a repeat blood culture was drawn and a lumbar puncture was performed. she was also given her first transfusion of packed red blood cells. her lumbar puncture demonstrated ul − total nucleated cells, ul − red blood cells with manual differentiation of % lymphocytes, % neutrophils, and % monocytes. gram stain was negative and culture showed no growth. her cerebrospinal fluid (csf) glucose was mg/dl, while serum glucose was mg/dl and the csf protein concentration was mg/dl. a biofire filmarray meningitis/encephalitis csf multiplex polymerase chain reaction (pcr) panel returned positive for hhv- ( table ) . antibiotics were not re-initiated. after days, her fevers resolved, and she was presumed to have hhv- meningitis but was clinically improving thus antiviral therapy was not started. she was monitored closely for seizures given her intracranial abnormalities. she had a routine electroencephalogram (eeg) weeks earlier that did not demonstrate any definite seizures. she remained at her neurological baseline once she recovered from her viral illness. she was noted to have a diffuse maculopapular rash on her face, neck, and chest about hours after her fevers resolved. she was transferred on day of life to a long-term rehabilitation facility. we describe a case of a premature infant with multiple congenital anomalies including congenital hydrocephalus status post placement of a vp shunt, bilateral cleft lip and palate, bilateral microphthalmia, and nasal encephalocele who was found to have acute hhv- meningitis in the setting of intermittent fevers during her nicu course. the diagnosis of hhv- central nervous system (cns) infection was supported by her clinical presentation and detection of viral dna in her csf from a lumbar puncture. in terms of risk factors for both a bacterial and viral infection, she had a vp shunt and a newly placed gastrostomy tube and had been admitted to an nicu for more than weeks in the midst of the winter season. in the setting of her fevers, which are defined as a temperature ⩾ °c in the neonatal population, etiologies considered included bacterial pneumonia, viral upper or lower respiratory tract infection, urinary tract infection, bacteremia, cellulitis in the setting of indwelling devices, and meningitis given her intracranial anomalies with an indwelling device in place. she had laboratory evidence of inflammation with leukopenia initially and elevated inflammatory markers with negative blood cultures, urine culture, respiratory viral panels, and csf culture that was drawn hours after cessation of broad-spectrum antibiotics. her csf demonstrated elevated nucleated cells with a lymphocytic predominance, a low glucose, and elevated protein concentration outside the range of normal in a neonate; however, these numbers are difficult to interpret given her elevated red blood cell count. because she had a shunt, we presumed she was at higher risk for acquiring bacterial meningitis; however, a viral etiology fit more with her clinical picture of a self-limiting illness with an improved fever curve and resolution of symptoms without reinitiating antibiotic therapy. in the literature, there are only a few cases of hhv- meningitis or encephalitis reported in infants, and to our knowledge, this is the first reported case of cns infection in a premature infant. huang et al reported cases of hhv- meningitis in a -month-old boy and -month-old girl who had typical courses of roseola infantum with high fever for a few days followed by a skin rash eruption with evidence of csf pleocytosis, serum lymphocytosis, and serum serological evidence of igm anti-hhv- . it is well-established that passively transferred maternal antibodies gradually decrease until the lowest level is reached around to months after birth, so these infants were in the expected time period to manifest a primary infection. in addition, sugimoto et al reported neonatal cases of exanthema subitum caused by hhv- in a -day-old full-term boy and a -day-old full-term boy who each presented with high fevers followed by a classic skin rash. they both had igm antibodies in the acute phase and pcr detection of hhv- dna in the serum at high copy numbers suggestive of a primary infection despite presence of preexisting maternal antibodies, which the authors isolated from both mothers. although classic descriptions of primary hhv- infection typically occur after months of age, authors have speculated that the level of passive maternal antibodies may not be uniformly protective as in these previous cases. in a prospective study evaluating for incidence of primary hhv- infection as the cause of acute febrile illnesses, infants who acquired their infection in the first few months of life had lower mean antibody titers than infants who did not have a primary infection. providers have speculated that the level of viremia targeted to blood mononuclear cells likely causes symptomatic infection once the level of passive maternal antibodies has declined or if the viral load is particularly high. multiple modes of transmission of hhv- have been described in the literature including both vertical and horizontal transmissions, as well as a unique mechanism secondary to chromosomal integration of hhv- (cihhv- ), which can remain latent or undergo transplacental passage in utero. the virus can become activated in immunosuppressed individuals as well as during pregnancy, although the exact mechanism behind reactivation remains unknown. both in vivo and in vitro studies have shown that certain drugs including steroids like progesterone, anti-epileptics, antibiotics, and even nonsteroidal anti-inflammatory drugs can activate the virus. , viral dna has also been detectable in vaginal swabs with an incubation period of about days suggesting that horizontal transmission from mothers to babies is possible. maternal-fetal infection with hhv- has also been described and may be linked with a higher rate of fetal loss. in one study, hhv- antibodies were assayed in mothers with spontaneous abortions in the first trimester, and the authors found that % of the cohort were positive for hhv- igm antibody, while the hhv- antigen was detected in the majority of abortive villous tissue suggesting that viral infection could predispose mothers to fetal loss. similarly, a group from the united kingdom investigated occurrence of viral infection in fetal death and detected viral dna in % of tissue samples including detection of hhv- and hhv- in samples. another group analyzed over a thousand samples from multiple sites including tissue biopsies for detection of hhv- by pcr and identified a case of primary hhv- a seroconversion occurring in a young pregnant woman with subsequent transmission to the fetus and unfortunately a spontaneous abortion at weeks. in general, hhv- b is transmitted through contact with infected oral secretions with previous detection of this strain in the oropharynx of asymptomatic adults thus representing a major source of transmission to young children; however, there is no known information about how hhv- a is spread. moreover, congenital infections detected as hhv- dna in cord blood have been identified as another source of transmission similar to congenital cytomegalovirus (cmv) infections. but in contrast to cmv, the majority of congenital hhv- infections are thought to be secondary to chromosomal integration of the virus into different human chromosomes within the whole genome, which is a proven phenomenon. in a prospective study that examined the frequency and characteristics of cihhv- , % of infants with congenital infections were primarily from cihhv- , while the remaining % were secondary to transplacental infections who did not inherit cihhv- . infants with congenital infection due to cihhv had evidence of high viral loads in the cord blood and detection of hhv- dna in hair follicles in both the infants and at least one parent. the transplacental transmission was primarily from cihhv- mothers, while only a small proportion of congenital hhv- infections resulted from the activation of hhv- from a mother with inherited cihhv- . in other words, they found no evidence of transplacental infections except from mothers with cihhv- who suffered a reactivation of their integrated virus during pregnancy. , importantly, the identification of cihhv- in an infant does not rule out active transplacental infection because the chances of cihhv- + infants acquiring a transplacental infection should be the same as in infants without evidence of cihhv- . studies have attempted to investigate the congenital transmission of active hhv- virus prior to the discovery of cihhv- . one study analyzed cord-blood specimens for evidence of congenital hhv- infection in random samples that were originally collected to assess for congenital cmv infection. only samples were repeatedly positive for hhv- -specific igm antibody identified by enzyme-linked immunosorbent assay (elisa) method but were negative for hhv- genomic dna by pcr testing representing active infection at approximately two-thirds the rate of congenital cmv infection in this same group. the authors speculated that congenital transmission of hhv- similar to cmv is plausible yet rare, but given the retrospective nature of this analysis there were no information regarding the clinical presentations of both infants. however, if both these infants were asymptomatic, then the presence of hhv- igm in their blood would be indicative of intrauterine transmission of either reactivation of hhv- and/ or activation of hhv- in mothers with cihhv- resulting in transmission of replication virions. because breast milk is another mode of transmission for cmv, another study was done to assess for hhv- in breastmilk. the authors evaluated randomly selected human breast milk samples and tested them for hhv- dna by pcr. however, none of the specimens had evidence of hhv- , suggesting no transmission through breastmilk consumption. although there have been cases of neonatal hhv- infection characterized by fever and classic rash despite evidence of maternal antibodies, our patient who was otherwise considered immunocompetent developed a presumed cns infection due to hhv- at an early age. as previously mentioned, congenital infections are mostly asymptomatic, but in our patient's case, the question of mode of transmission was challenging to decipher. however, studies have demonstrated that % of active infections investigated were acquired from mothers with cihhv- that had activated during pregnancy suggesting that transmission of activated maternal cihhv- is the primary cause of non-inherited congenital infection. , thus, there are possibilities of congenital hhv- infection in an infant like our patient: . she had cihhv- but no active infection; however, these patients would be asymptomatic with evidence of hhv- dna but no igm antibodies. . she had cihhv- with active infection from a cihhv- + mother who reactivated the virus during pregnancy and subsequently transmitted the active virus, but it is impossible to differentiate active infection in a cihhv- patient using pcr methods alone. . she did not have cihhv- but had evidence of active infection from a cihhv- + mother who reactivated and transmitted the replicating hhv- virus transplacentally. . she acquired it transplacentally from a mother reinfected with hhv- or whose latent hhv- reactivated with no evidence of maternal cihhv- . . she acquired it postnatally from another person in the nicu about weeks prior to the onset of symptoms. we do not know the viral state of the maternal hhv- dna to determine whether reactivation or transcription of viral genes from an integrated genome were a possibility or if this was an acquired primary hhv- meningitis with a high enough viral load that overwhelmed her passive maternal antibodies. we hypothesize similarly to previous authors that she may have been exposed to a significant amount of viral replication leading to viremia with clinical symptoms, or potentially her maternal antibodies were low to begin with given her manifestation of cns disease. the role of hhv- cns disease continues to be an area of ongoing investigation particularly given its ranges of manifestations from febrile seizures to meningitis, encephalitis, and demyelinating disorders as reported previously in the literature. currently, the route of hhv- entry into the cns is unknown in the literature despite its association with a wide kim et al variety of neurologic disease from meningoencephalitis to multiple sclerosis in adults. using autopsy specimens, harberts et al demonstrated that hhv- potentially infects the cns via the olfactory pathway with highest prevalence of the virus in olfactory issues and the nasal cavity. we speculate that our patient may have been more susceptible to transmission of the virus to her cns given her significant craniofacial anomalies and nasal encephalocele. in one prospective study that assessed complications of primary hhv- infection, seizures were the principle complication accounting for approximately one-third of first-time febrile seizures among children less than years old. although investigators have been unable to successfully culture hhv- from csf, it is often detected in csf and other bodily fluids by pcr, as in our patient. there have been conflicting results in the literature regarding the frequency of hhv- pcr identification in csf with one study reporting positivity in up to % to % of children who had neurologic symptoms during their primary hhv- infection. , another study examined csf samples from pediatric patients who underwent evaluation for possible sepsis or neurologic symptoms and tested them for hhv- dna by pcr and found evidence of it in patients who were all less than months of age. in of the patients who were full term and less than month old, they presented with fevers and had evidence of csf pleocytosis defined in this study as > leukocytes × l − . both were diagnosed with aseptic meningitis at discharge and found to have hhv- dna in their csf samples when tested retrospectively. the last patient was a former -week baby boy who developed positive blood cultures for candida on day of life but had no evidence of csf pleocytosis with negative bacterial, viral, and fungal cultures and also tested positive for hhv- dna, which was thought to be an incidental finding with no clinical implications. the authors concluded that the clinical features of the first patients and the absence of other identified infectious agents were secondary to hhv- as a cause of aseptic meningitis. however, similar to our case in which the patient's csf results may have been from contamination by blood, the presence and magnitude of a pleocytosis are difficult to interpret with the number of red blood cells. the authors also commented that it was difficult to say with certainty that the hhv- dna isolated from the csf was not due to contamination from peripheral blood. in addition, the assay used to detect hhv- dna pcr is an important consideration. our institution routinely uses the biofire diagnostics filmarray meningitis/encephalitis pcr (mep) panel, which provides a rapid pcr-based detection in the csf of various bacteria and viruses compared to conventional diagnostic methods of culture and pathogen-specific pcr testing. one retrospective study compared the real-world performance of mep panel compared to conventional testing in csf samples and reported an overall agreement of % and a mean time to hour diagnosis of hours compared to hours, respectively; however, the authors did comment that the mep panel can potentially detect persistent, reactivated, or cihhv- that may not be causing active infection and must be interpreted with caution in the right clinical context. finally, congenital hhv- may be compared to congenital cmv in the literature as they are closely related, and while it is true that most cihhv- congenital infections are asymptomatic, at least % of congenital infections are the same as congenital cmv with active virus transmitted to the fetus during pregnancy. , cytomegalovirus is an abundant virus that is known to cause neurodevelopmental disability including sensorineural hearing loss and other developmental disabilities. congenital cmv infection results from primary infection, reinfection or reactivation of latent virus in the mother compared to congenital hhv- infection, which does not appear to be commonly due to reactivation or reinfection in the mother. caserta et al performed a prospective double-blind controlled study comparing infants with congenital hhv- infection versus those without infection using a set of neurocognitive assessments to determine whether hhv- has an impact on early neurodevelopmental outcomes similar to cmv. infants with congenital hhv- infection were defined as having hhv- dna present in their cord-blood mononuclear cells, and chromosomal integration was confirmed by detecting dna in hair follicle specimens. major findings included significantly lower scores at months of age on bayley-mental development index scores in the congenital infection group even after controlling for covariates potentially linking hhv- infection and neurologic disease; however, there were no specific clinical manifestations identified at birth such as hearing loss. although there are limited longitudinal studies regarding the potential impact on neurodevelopmental impairment in this cohort that has yet to be confirmed, hall study brings to light this important question particularly if there are progressive, detrimental effects over time. consequently, the question remains regarding the clinical significance of this finding in the neonatal population given this virus's potential pathogenic role in the cns. hhv- is very common in young infants accounting for % of visits among -to -month-old infants presenting to the emergency department for febrile illnesses, but the frequency of mild or asymptomatic primary infection as well as its longterm impact remains undefined. in hall et al's study, of the patients diagnosed with a primary infection, % were under months of age with a mean duration illness of days and a higher rate of hospitalization compared to age-matched healthy infants. thus, it is important for clinicians to maintain a high index of clinical suspicion for viral infections in this population as well as its complications prenatally and postnatally. in one cohort, pityrasis rosea, which is a rash often associated with both hhv- and hhv- , was noted in % of pregnant women who ultimately miscarried with evidence of hhv- dna in fetal tissue in out of stillborns, suggestive of an association between hhv- and potential fetal death. it is still unclear about the role congenital hhv- infections play in the perinatal and neonatal period particularly when it clinical medicine insights: case reports comes to neurodevelopmental outcomes; however, there does not appear to be a severe congenital hhv- syndrome unlike congenital cmv, which often occurs in women who acquire primary infection early in pregnancy. hhv- infection occurs in essentially all children within the first few years, therefore preexisting immunity in the mother may be protective against such severe disease in most infants. increasing use of pcrbased pathogen detection panels in the neonatal population may lead to more frequent diagnoses of hhv- and improved understanding of its epidemiology and natural history. in summary, we present a case of a premature infant with multiple anomalies who acquired acute hhv- viral meningitis in the setting of intermittent high fevers, elevated inflammatory markers, and diagnostic testing from her csf that confirmed the diagnosis. given her hydrocephalus and vp shunt, neurology and neurosurgical subspecialists will continue to follow her as an outpatient. with her intracranial abnormalities, she remains high risk for seizures. she also failed her hearing tests multiple times throughout her course likely as a complication of her intracranial and craniofacial anomalies; her prematurity and comorbidities place her at high risk for significant neurodevelopmental impairment now possibly increased following her hhv- cns infection. it is important to consider hhv- infection as an etiology of fevers even during the neonatal period as timely diagnosis may prevent further unnecessary treatment with empiric antimicrobials and diagnostic evaluations. there are several limitations of this case report. it is based on a single case instead of a case series given the low prevalence of this infection in the premature population. the patient also has multiple underlying craniofacial abnormalities with a potentially underlying genetic diagnosis that has not yet been uncovered making it difficult to generalize her case to other infants, particularly if she is predisposed compared to others. finally, there was a lack of confirmation in hhv- detection in other bodily fluids or serum due to unavailable routine testing at our institution by either culture, serology, or pcr that could have strengthened this finding of an active infection based on her clinical presentation. laboratory and clinical aspects of human herpesvirus infections update on human herpesvirus biology, clinical features, and therapy human herpesviruses and and central nervous system infection in children clinical impact of primary infection with roseoloviruses review part : human herpesvirus- in multiple non-neurological diseases congenital infections with human herpesvirus (hhv ) and human herpesvirus (hhv ) transplacental human herpesvirus (hhv- ) congenital infection caused by maternal chromosomally integrated virus sequence analysis of transplacentally acquired human herpesvirus dna is consistent with transmission of a chromosomally integrated reactivated virus meningitis caused by human herpesvirus- human herpesvirus- infection in neonates: not protected by only humoral immunity human herpesvirus- infection in children: a prospective study of complications and reactivation chromosomal integration by human herpesviruses a and b possible progesterone-induced gestational activation of chromosomally integrated human herpesvirus b and transplacental transmission of activated human herpesvirus b hhv and hhv : persistence and vertical transmission hhv- infection during pregnancy and spontaneous abortion viral infection in hydrops fetalis, spontaneous abortion and unexplained fetal death in utero detection of hhv- in over a thousand samples: new types of infection revealed by an analysis of positive results chromosomal integration of human herpesvirus is the major mode of congenital human herpesvirus infection serological evidence for congenital transmission of human herpesvirus examination of human breast milk for evidence of human herpesvirus by polymerase chain reaction human herpesvirus and neuroinflammation human herpesvirus- entry into the central nervous system through the olfactory pathway association of human herpesvirus infection of the central nervous system with recurrence of febrile convulsions human herpesvirus dna in cerebrospinal fluid specimens from allogeneic bone marrow transplant patients: does it have clinical significance? potential clinical impact of the film array meningitis encephalitis panel in children with suspected central nervous system infections early developmental outcomes of children with congenital hhv- infection evidence of human herpesvirus- and - reactivation in miscarrying women with pityriasis rosea we are grateful to the family of our patient, the clinical microbiology laboratory at morgan stanley children's hospital of new york-presbyterian, and members of the neurosurgery team who consulted on this case. written informed consent from the current legal guardian was obtained and is maintained by the primary author. faith kim https://orcid.org/ - - - key: cord- -vwgai k authors: nan title: publication only date: - - journal: bone marrow transplant doi: . /bmt. . sha: doc_id: cord_uid: vwgai k nan introduction & objectives: literature states that human postnatal dental pulp stem cells (hdpscs) have the ability to differentiate to osteoblastic cells. the purpose of this paper is to present the results obtained in the differentiation of hdpscs with three different media and to compare their osteogenic ability. materials & methods: human dental pulp was extracted from teeth of healthy adult subjects aged to years. the pulp was gently removed and immersed in a digestive solution for h at cº. after digestion, cells were cultured and adherent cells were isolated. after the second pass the cells were placed in three different fl asks with three classes of differentiation media. medium : osteodiff (miltenyi®); medium : alpha-mem supplemented with % fetal bovine serum (fbs), u/ml penicillin, . mg/ml streptomycn, and . mg/ml amphotericin b; medium : alpha-mem medium, supplemented with % fbs, mm p-ascorbic acid, mm l-glutamine, u/ml penicillin, . mg/ml streptomycin, and . mg/ml amphotericin b. flasks were incubated at ºc in a % co and the medium changed twice a week for days. to quantify the different amount of mineralized nodules the absorbance rate was used. results & discussion: hdpscs were obtained at a good rate and differentiated with any of the three media into osteoblastic cells that developed mineralization nodules (clusters), as revealed by alizarin red staining. this staining was signifi cantly more intense with medium than medium and medium (absorbance values . , . and . respectively). conclusions: this study demonstrates the ability of hdpscs to differentiate into osteoblasts. the medium (osteodiff medium, miltenyi®) , was the best to differentiate these cells to the osteogenic lineage. long-term haematopoietic reconstitution and clinical evaluation of autologous peripheral blood stem cell transplantation after cryopreservation of cells at - °c in a mechanical freezer for longer than months l. calvet, a. cabrespine-faugeras, n. boiret-dupre , e. merlin, c. paillard, m. berger, j.-o. bay, o. tournilhac, p. halle chu (clermont-ferrand, fr) controlled-rate freezing in or % of dmso and storage in the nitrogen is the standard technique for cryopreservation of hematopoietic progenitor cells (phs). the main inconveniences are its high cost and dmso toxicity. many teams try to reduce dmso infused by phs concentration before cryopreservation or wash before infusion. however, labor intensive increases the cost and not free of cell loss. we developed an easier and cheaper technique, the cryopreservation of the phs at - °c, an uncontrolled rate freezing with . % hes, % albumin and only . % of dmso allowing infusion without wash. this technique preserves the functional capacities of phs, can produce successful engraftment and reduces toxicity during infusion. does the cryopreservation of the phs at - °c allow a long-term hematopoietic reconstitution and clinical course even if storage is greater than months? patients who had undergone autografts ( adults, children) were studied. the median storage time of the phs cryopreserved was . months [ . with . % ( / ) preserved more than months (median , [ - ] ). the median recovery of nucleated cells and cd + cells were similar, for the preserved phs <or > months ( % versus % , p= . ) and ( % versus % , p= . ), respectively. only mild infusion-related toxicity was observed in . % (nauseas/vomiting . %, shivers . %). median time to reach . x /l granulocytes (pn), and x /l platelets (pl) were , and days respectively. delay to reach hematopoietic reconstitution was similar between phs preserved < or > months except for pl > x /l. this delay was signifi cantly longer for phs kept > months versus [ - ] (n= . ) with a correlation between cd + cells dose and the number of days need to reach x /l pl. in order to assess long term hematopoietic reconstitution, only patients without other treatment (n= ) were studied at , and months. median values were , and x /l for the platelets and , , , and , x /l for the pn at , and months respectively. mortality at post-autograft days was of . %. median overall survival was months and years survival rate was of %. the long term hematopoietic reconstitution was satisfactory. this easier and cheaper cryopreservation method leads to successful engraftment even if phs had been cryopreserved more than months. improve mobilization in these patients have been described. another exciting option for these patients is the new cytokine, amd . this agent is an inhibitor of sdf binding to cxcr and appears to promote mobilization of cd + cells into the circulation. the use of this amd in combination with g-csf in patients unable to collect adequate cd + cells with g-csf alone was recently reported in patients with lymphoma and multiple myeloma (mm) . in this study g-csf was given at a dose of mcg/kg per day and amd was started at mcg/kg on day of mobilization. in contrast, clinical studies showed that aml, cll and pcl cells may also be mobilized by amd via cxcr inhibition. due to these concerns, aml, cll and pcl patients are excluded from amd trials. we here report patients ( female/ male) with non hodgkins lymphoma (n= ), mm (n= ) and germ cell cancer (n= ) who failed stem cell mobilization after chemotherapy and g-csf administration (patient characteristics table ). patients received x µg/ kg daily of g-csf for days followed by µg/kg of amd given subcutaneously - hrs before collection on day . our aim was to assess the effect of amd on the mobilization of cd + cells. administration of g-csf and amd were continued daily until end of collection cycle. adequate collection of cd + cells ( . and . x cd + cells/kg) were achieved in patients. in patients additional bone marrow collection were performed, patient failed mobilization with amd . until now patients underwent autologous transplantation with . , . , . and . x cd + cells/kg respectively and achieved sustained leukocyte and platelet engraftment. in conclusion, amd in combination with g-csf was generally safe and offers a new treatment to collect cd + cells for autologous transplant from poor mobilizers. due to the reported mobilization of leukemic cells, amd should be restricted to patients with lymphomas, mm and solid tumors. evaluating the effect of substance p on expansion of human umbilical cord blood cd + haematopoietic stem cells in a serum-free media s. shahrokhi ( ) , m. ebtekar ( ) , k. alimoghaddam ( ) , m. kheirandish ( ) , a. pourfathollah ( ) , a.r. ardjmand ( ), a. ghavamzadeh ( ) ( )tarbiat modares university (tehran, ir); ( ) hematology, oncology and bone marrow transplantation research center (tehran, ir) ex vivo expansion of cord blood hematopoietic stem cells has been progressively interested as alternative sources for stem cell transplantation. using different combination of growth factors especially cytokines has been investigated in most reports, but there are little evidence about regulatory roles of other factors including neuropeptides in this way, then we choose substance p (sp) to evaluate its effect on expansion. material and methods: cd + purifi ed from umbilical cord blood by macs, were cultured in a serum-free liquid culture system. different concentration of sp used in combination with cytokine cocktail of scf, fl, tpo, il and il . phenotypic and functional analysis of the cells produced in culture, was performed by fl owcytometry. count and percentage of cd + cells were compared in different groups of treated cells. results: ex vivo expansion cultures of cd + cells of ucb were signifi cantly increased, in cells cultivated in "sp + cytokine cocktails" group compared cytokine groups alone. conclusion: consideration of the role of other growth factor such as sp along with cytokines, may enable us to overcome the diffi culties before us in ex vivo expansion of cord blood cells. our studies indicate that sp could act as a superior supplement for expansion of ucb-hsc cytokine cocktails. additional studies are needed to establish the functional activity of expanded ucb-hsc as well as the effects of substance p. standard protocols for cryopreservation of peripheral blood progenitor cells (pbpc) use rate-controlled freezing and storage in liquid nitrogen, which are both time-consuming and expensive. in the last years we used a simplifi ed method (galmes et al ) consisting of storage in a mechanical freezer at - ºc, with dmso as the sole cryoprotectant. this study evaluates the safety of this approach, in terms of infusion-related toxicity and hematopoietic reconstitution, in consecutive autologous transplantations performed from / to / in patients (median age ; underlying disease: lymphoma in , myeloma in , acute leukaemia in , breast cancer in ). after mobilization with g-csf ± chemotherapy (usually cyclophosphamide . g/m²) pbpc were collected in a cs + separator (fenwall), mixed in autologous plasma and dmso (to a fi nal concentration of %) and frozen in plastic bags (cryocyte, fenwall) at - ºc. median cd + count was . x /kg and median storage duration was days ( - ). infusion-related toxicity was frequent ( %) and generally mild (transient hypoxemia, broncospasm, hypertension or arrhythmia, and abdominal pain, nausea or diarrhea) but there were cases of acute congestive heart failure and anaphylactic shock (probably related to dmso). engraftment to neutrophils and , platelets/ul occurred on days + and + (median). bacteremia occured in % transplantations, and grade or toxicity in %. median hospitalisation duration was days. mortality at day + and + was . and . % respectively. an engraftment delay beyond d+ was seen in cases. there were no secondary graft failures. with a median follow up of months, % patients are alive. these results confi rm the feasibility and safety of this simpler and cheaper cryopreservation methodology. belarus y. isaikina, n. minakovskaya, o. aleinikova belarusian center for ped oncohematology (minsk, by) introduction: recent studies suggest that cotransplantation of mesenchymal stem cells (mscs) can improve the engraftment of allogeneic hematopoietic stem cells and prevent graft-versus-host disease (gvhd) due to their immunomodulatory properties. we analyzed the clinical effect of msc infusion on day + after hsct for prophylaxis of gvhd and applying of mscs for treatment of severe steroid-resistant gvhd. patients and methods: eight pts after allogeneic hematopoetic stem cell transplantation (hsct) underwent mscs infusions (median age of pts was years, male/female: / ) between and . diagnoses included:all- , aml- , aa- , mds- .gvhd prophylaxis for pts with all, mds consist of csa and mtx mg/m² (n= ); for pts with aa -csa+mmf; for pts with aml -csa and mtx mg/m² (n= ). for the treatment of gvhd all pts received metylprednisolon - mg/kg. mscs were prepared applying technique of expansion in vitro from bone marrow of hla-identical siblings, haplo-identical and haplo-nonidentical family donors and unrelated donors. four pts received mscs once and four -twice. for three pts mscs was used for prophylaxis of gvhd on day + after hsct and the median dose was , ( , - , )x /kg and fi ve pts received mscs for treatment of steroid-resistant gvhd with medium time of mscs infusion after hsct ( - ) days and the dose was , ( , - , )x /kg. results: there was no evidence of early and late side effect of msc infusion. one patient died from pulmonary gvhd month after cotransplantation mscs and seven pts-alive. all pts (n= ), who received mscs on day + for prophylaxis gvhd developed grades ii-iv gvhd and needed the secondary mscs infusion and the median time between mscs infusions were ( - ) days. four pts out of fi ve with steroid-resistant gvhd showed signifi cant improvement of clinical sign of gvhd that allowed reducing immunosuppressive therapy and stopping the steroids. conclusion: our experience demonstrates the absence of positive gvhd prophylactic effi cacy when infusion of mscs was done on day + . however, we observed decreasing of gvhd grades from iii-iv to -ii, when mscs were used as treatment of steroid-resistant gvhd. clinical characteristics of early-onset acute graft-versushost disease after allogeneic haematopoietic stem cell transplantation t. yamashita, y. najima, t. kikuchi, h. muto, c. sakurai, w. munakata, m. yamamoto, k. ohashi, h. sakamaki, h. akiyama tokyo metropolitan komagome hospital (tokyo, jp) acute graft-versus-host disease (gvhd) is one of the major factors that have infl uence on the outcomes of allogeneic hematopoietic stem cell transplantation (hsct). traditionally, acute gvhd has been defi ned as a syndrome after neutrophil engraftment within the fi rst days following hsct. but in our practice, we sometimes encounter acute gvhd that may occur s both early, even before engraftment, and late, beyond day . the latter has been defi ned as "late-onset acute gvhd", but the former may not be clearly identifi ed yet. in this retrospective study, we evaluated the incidence, clinical manifestations and outcomes of "early-onset acute gvhd", defi ned as that occurring before engraftment after transplantation, among consecutive myeloablative allogeneic hscts at our hospital. of patients, the median age was years. ninety-three percent of patients received allogeneic hsct for hematologic malignancies. thirty-eight percent of patients received an hlamatched related donor transplant, % received hla-matched unrelated donor grafts and % received hla-mismatched unrelated donor grafts. the stem cell source was bone marrow in % of patients and peripheral blood in %. the conditioning regimen was tbi-based for % of patients and % received busulfan-based conditioning. forty-three percent (n= ) of the cases developed grade ii-iv acute gvhd. of these, ( %) cases were described as early-onset acute gvhd (group e). other cases of acute gvhd occurred after engraftment (group c). the median onset date of acute gvhd is day in group e and day in group c. grade iii-iv acute gvhd was seen in % of group e and in % of group c (p= . ). the frequency and severity of each involvement site were comparable in both groups. major primary therapy for acute gvhd was mpsl - . mg/kg/day, but % cases in group e were refractory for this primary therapy and % in group c (p= . ). three-years overall survival (oas) was % in group e and % in group c (p= . ). in group c, oas of cases without gi symptoms was %, whereas oas of cases with gi involvement was % (p= . ). in group c, oas was not affected by with or without gi-gvhd (p= . ). in conclusion, early-onset acute gvhd accounts for a substantial proportion of acute gvhd after allogeneic hsct. patients with early-onset acute gvhd tend to be refractory to steroid therapy and will have poor prognosis if gi involvement exists. contrast enhanced ultrasound sonography in intestinal acute graft-versus-host disease e. benedetti ( ) a year old female with high risk acute b cell leukemia received a fully ablative peripheral blood stem cell transplant from a allele (at the b locus) mismatched unrelated donor. conditioning consisted of cy/tbi and gvhd prophylaxis of cyclosporine (csa) and short course mtx. on day + she developed steroid refractory (biopsy proven) acute skin gvhd. photopheresis was started with major skin improvement. on day + she developed nausea, vomiting and profuse diarrhea. standard endoscopy with gastric biopsies showed gvhd. infections were ruled out. a trans-abdominal sonography (ta-us) revealed mucosal oedema and thickening of the terminal ileum ( . mm) and the ascending colon. moreover, pillcam capsule endoscopy showed mucosal oedema, erosions and lymphagectasies. infl iximab at mg/kg was added and, after doses, despite a major clinical improvement, her terminal ileum was still thickened. to investigate if this thickening was associated with residual active gvhd she underwent a contrast enhanced ultrasound sonography (ceus) using a linear phased-array . -mhz transducer. a sulphur hexafl uoridebased with a phospholipid shell microbubble contrast agent (sonovue®, bracco) was injected i.v. as a bolus ( . ml) followed by ml saline fl ush. sonovue® is a blood pool second generation contrast agent. ceus showed an intense and sustained enhancement in the arterial phase involving the whole ileum wall with a late phase wash out. such enhancement pattern has been previously described in active crohn disease. given the clinical improvement, infl iximab was discontinued to reduce the risk of infections. however, as ceus revealed active gvhd she continued on budesonide, beclometasone, csa and prednisone. forty days later her abdominal symptoms had completely resolved and a ta-us showed a normal terminal ileum. four months later her intestinal gvhd (confi rmed by colon biopsies) fl ared. ceus was performed on descending colon (most involved intestinal tract by standard ultrasonography) and showed intense arterial phase enhancement with late phase wash out. rituxan and mmf were added with slow resolution of symptoms and normalisation of us features. in conclusion ceus showed residual gvhd activity despite the improved clinical symptoms. moreover, good concordance with clinical symptoms and standard colonoscopy when gvhd fl ared was also shown. further prospective studies are needed to evaluate its usefulness in monitoring intestinal gvhd. extensive chronic graft-versus-host disease is a frequent complication after peripheral blood stem cell transplantation -results of long-term follow-up d. stamatovic, l. tukic, b. balint, o. tarabar, m. elez, g. ostojic, b. todoric zivanovic, z. tatomirovic, o. tasic, b. cikota, m. malesevic, s. marjanovic military medical academy (belgrade, rs) introduction: many studies have compared effi cacy of allogeneic stem cell transplantation (sct) from peripheral blood (pb) with bone marrow (bm), but fi nal conclusion concerning this treatment modality is still not well defi ned. aim: to compare effi cacy of pbsct with bmt in the treatment of hematological malignancies with respect to engraftment, transfusion need, frequency and severity of acute and late complications and overall survival (os). methods: we have analyzed patients (pts), median age years ( - ), m/f / , with various hematological diseases (saa- , cml- , aml- , all- , mds- , mm- , mh- , granulocytic sarcoma- ) in whom we perfomed allogeneic sct from till . in pts we perfomed secondary allogeneic sct in due to graft rejection ( ) or relapses ( pts). pts were divided into two groups concerning sc origin- pts in bm group and pts in pb group. all pts had hla-dr sibling transplant ( singeneic, fully matched, mismatched and haploidentical). sc were collected from bm up to standard method and from pb with one apheresis after fi ve days aplication of granulocytic growth factor. all pts have received unmanipulated suspension of sc. conditioning were adjusted to primary diseases and gvhd prophylaxis was mostly combination of cyclospirine a and metothrexate. prevention of infections were standard. results: pts with sc originate from pb have received signifi cantly more mononuclear cells ( , ± , vs , ± , , p< , ) in comparisson with bm. engraftment was more rapid (p< , ) in the pb group approximately for days. transfusion requirements were much higher in bm group (p< , ). those pts had more frequent oropharingeal mukositis grade - ( , % vs , %, p< , ). there were no difference in the incidence of acute ( , % vs , %, ns) or chronic gvhd ( , % vs , %, ns). pts with pbsct had signifi cantly more frequent extensive cgvhd ( , % vs , %, p< , ). there were no difference considering trm ( , % vs , %, ns) or relapses ( , % vs , %, ns). pts with bmt had better overall survival but with no statistical signifi cancy. conclusion: results of this analysis mostly corresponds with other studies showing that pbsct have rapid engraftment and less acute complications. pbsct is connected with more frequent extensive chronic gvhd that is potentialy fatal, making results of this particular treatment option less better. future will bring defi nite estimation of pbsct effi cacy. a preliminary study of human natural killer t-cell recovery post allogeneic stem cell transplantation b. rees ( ) , r. morse ( ) , s. robinson ( ) , j. hows ( ) , c. donaldson ( ) ( )centre for research in biomedicine, university of the west of england (bristol, uk); ( )university hospitals bristol nhs foundation trust (bristol, uk) natural killer t cells (nkt), defi ned by their cell surface immunophenotype cd +, v alpha +, v beta + and their specifi c activation pathway by the glycolipid alpha-galactosyl ceramide are a unique and small ( . - . %) subset of lymphocytes. these cells may play a key role in the cure of leukaemia after stem cell transplantation (sct) through activation of the graft versus leukaemia (gvl) effect. they have the ability to stimulate both innate and adaptive immune responses through cytokine production and the activation of 'classical' t, b and natural killer (nk) cells. campath, a complement fi xing monoclonal antibody targets the cd antigen expressed by t, b and nk cells and may be used in vitro and/or in vivo for donor lymphocyte depletion during stem cell transplantation. our previous work has shown that cd +, v alpha +, v beta + nkt cells also express the cd antigen and so are also susceptible to damage by campath. twelve patients (median age . years, range - ) on the bmt unit, university hospitals bristol were recruited. diagnoses were aml ( ), all ( ), anll ( ), cml ( ), mds ( ), nhl ( ) , and hd ( ) . seven received reduced intensity conditioning, tbi and / received campath. all patients received adult stem cells, from matched siblings, from unrelated donors. nine survived more than year, including the patient with hd who relapsed months post autologous sct and is alive months post matched unrelated sct. the normal range for nkt cell numbers in adult blood was established, mean . x /l (sd . ) (n= ). cells stained with cd -pecy , v alpha -fitc and v beta -pe were analysed using the becton dickinson facs vantage se cell sorter with cell quest software. recovery of nkt cells was studied up to - months post transplant, with mean levels of . ± . x /l. all individual values were below those in the normal adult population. recovery of other lymphocyte subsets was comparable with those reported in previous studies. nk cells recovered to within their normal range to months post sct, cd t cells numbers were within the normal range by approximately months and cd t cells only attained values in their normal reference range by months. the slow recovery of nkt-cells has not been previously reported and this may contribute to a reduced gvl effect. n. nakano, a. kubota, m. tokunaga, y. takatsuka, s. takeuchi, t. itoyama, a. utsunomiya imamura bun-in hospital (kagoshima, jp) background: adult t-cell leukemia/lymphoma (atll) has a poor prognosis because of its chemo-resistance. many chemotherapeutic regimens have been created but none of them have shown suffi cient results. we proposed allogeneic stem cell transplantation (allo-sct) for atll patients and showed an improved survival rate. however, relapse or progression of atll is one of the major limiting factors of survival in post sct patients. objectives: in order to establish a better treatment strategy for poor responders after sct for atll, we analyzed the outcome of relapse or progression cases after allo-sct. we paid special attention to the graft versus atll (gvatll) effect. methods: there were atll patients in which allo-sct was performed in imamura bun-in hospital (ibh) from june to november . twenty seven cases survived over days after sct. sixteen of the patients relapsed. using data in medical records of ibh, we analyzed transplant characteristics and the outcome of these patients retrospectively. results: disease status at sct was cr in pts, pr, sd, and pd. eight patients received conventional stem cell transplantation (cst) and the other eight patients received reducedintensity stem cell transplantation (rist). fourteen patients in obtained remission ( cr and pr), but the remaining did not ( sd and pd) after sct. the sites of relapse or progression in were skin in patients, lymph node, peripheral blood, central nervous system, and bone. all patients discontinued immunosuppressants after relapse or progression. eleven patients obtained remission. especially, in out of patients, remission was obtained only by discontinuation of immunosuppressants, and the time to remission after discontinuation of immunosuppressants was between to days. twelve patients were complicated with acute gvhd (grade i-iv). twelve patients died after sct. the causes of death were disease progression of atll in patients, acute gvhd, infectious complications, and interstitial pneumonia. four patients who were complicated with acute gvhd survived over months. conclusions: a certain number of patients obtained remission only by the discontinuation of immunosuppressants. four patients survived more than years with their complication of acute gvhd. these results suggest that the gvatll effect after sct exists and plays an important role in longer survival for poor responders of post allo-sct in atll patients. adoptive immune transfer in paediatric and young adult patients with refractory malignancies p. sovinz, w. schwinger, h. lackner, m. benesch, a. moser, c. urban medical university graz (graz, at) background: patients with metastatic malignancies refractory to or relapsing after conventional ± high-dose chemotherapy have a poor prognosis. graft-versus-tumor (gvt) effects have been reported in small numbers of patients for various solid tumors. patients and methods: eight pediatric and young adult patients (male: female = : ; age . to years) underwent allogeneic hematopoietic stem cell transplantations (allohsct). diagnoses were relapsed/ refractory neuroblastoma (n= ), second relapse of hodgkin's disease, refractory mediastinal large-b-cell-lymphoma, metastatic ewing sarcoma/ osteosarcoma /wilms tumor, respectively. five patients had received high-dose chemotherapy with autologous stem cell rescue. conditioning regimens consisted of fl udarabine (n= ) combined with melphalan ±atg (n= ) or melphalan/thiotepa/okt (n= ) or treosulfan/thiotepa/okt (n= ); and treosulfan/melphalan (n= ). haploidentical donors (parents, n= ) underwent aphereses: one product was cd / depleted, the other cd selected; grafts from matched donors (siblings:n= , unrelated: n= ) were not manipulated. median cd -number was . x /kg; median cd -number in haploidentical grafts was . x /kg. in the absence of graft-versus-host disease (gvh) immunosuppression was stopped median on day + . to date, a median of donor lymphocyte infusions (dli; - ; dose range: . x to x ) were given to / patients, starting on median day + . results: neutrophil engraftment (> . x /l) was achieved median on day + . acute gvh of the skin (i-ii) developed in patients, of skin+liver (iii) in one; chronic gvh occurred in patients (skin:n= , gut:n= ) there was no transplant-related mortality; / patients survive for a median of days (range: - ) in complete (cr; n= ) or partial remission (pr; n= ) with ongoing regression (disease status not yet evaluated: n= ). two patients who were transplanted in disease progression showed partial response after allohsct but eventually died of progressive disease on day + (mediastinal large-b-cell-lymphoma) and + (neuroblastoma, after the second allohsct). conclusions: eight heavily pretreated pediatric and young adult patients with poor-prognosis metastatic malignancies tolerated the conditioning regimens well. all patients showed at least transient partial response to allohsct ±dli; six patients in partial remission or better before allohsct survive in cr or pr with evidence of further tumor regression. cmv infection in seropositive patients with haematologic malignancies after allogeneic peripheral blood stem cell transplantation t.-d. tan koo foundation sun yat-sen cancer center (taipei, tw) objective: to investigate the incidence and outcomes of cmv infection in our seropositive population patients after allotransplant as compared with other western patients. we also investigate the impact of post-transplant occurrence of acute graft-vs-host disease and the use of anti-thymocyte globulin upon the outcome of our patients. methods: cmv seropositive patients of various hematologic malignancies underwent allogeneic peripheral blood stem cell transplantation at our institute between march and november . we used weekly cmv pcr to monitor cmv infection following neutrophil engraftment until day + or when any infectious complication occurred. when two consecutive pcrs were positive with > copies present or cmv was found histopathologically, we treated patients with intravenous ganciclovir mg/kg q h for to days. results: patients (median age . , ~ ) of various hematologic malignancies including aml (n= ), cml (n= ), all (n= ), nhl (n= ), hl (n= ), myeloma (n= ), myelodysplastic syndrome (n= ), underwent myeloablative or non-myeloablative allotransplant ( vs ). the source of stem cells includes related ( patients), unrelated ( patients), and umbilical cord blood stem cell ( patients). cmv infection or reactivation rate was . % ( in ) with median date of occurrence ranges + to + days with the median of + days and the immediate cmv-related mortality rate was . % ( in ). the incidence of cmv infection in patients with grade ~i vs ii~iv acute gvhd are . % vs . %, respectively, with risk ratio (p= . ). the occurrence of cmv infection in patients with or without the use of anti-thymocyte globulin use was . % vs . %, respectively, with risk ratio . (p= . ). the -year event-free survival and overall survival of our patients with or without cmv infection are . % vs . %(p= . ), and . % vs . %(p= . ), respectively. conclusions: our cmv seropositive patients do not have higher incidence of cmv infection or reactivation than other lower seropositive patients reported in the western world. there is an increased incidence of cmv infection in the patients who suffer from grade ii~iv acute gvhd, and there are signifi cant differences in efs and os between patients with or without cmv infection. on the contrary, the impact of atg use in our patients is not clear. objectives: patients after hematopoietic stem cells transplantation (sct) have markedly increased susceptibility to moulds infections. according to recent data, the moulds of fusarium spp are emerging as human pathogens associated with significant morbidity and mortality in immunocompromised patients. in current report we are describing disseminated invasive fungal infections caused by fusarium incarnatum in three recipients of allogeneic hematopoietic stem cells, a pathogen not earlier reported for such patients. methods: blood samples were analyzed using automatic bact/ alert system. the culture and identifi cation were performed according to conventional microbiological procedures. the sabouraud agar was used for strain's isolation and the samples were incubated in °c for days. the cream to nut-brown mould's colonies were suggestive for fusarium incarnatum. also the microscopic analysis of direct samples revealed microand macroconidias typical for fusarium genus. results: the -years-old male and a -years-old female patients, with relapsed and refractory acute myelogenous leukemia (aml) have been treated by allogeneic sct from matched unrelated donors after myeloablative conditioning. the third patient, a -years-old woman with hodgkin's lymphoma relapsed after autologous sct was transplanted from hla-matched sibling donor after reduced intensity conditioning. all patients suffered from neutropenic fever which did not respond to broad-spectrum antibiotics and fl uconasole. the appearance of nodular, painful skin lesions with characteristic dark red colour and central necrotic area in later stadium suggested skin microembolism caused by infectious microorganism. the mycological analysis confi rmed fusarium incarnatum as a pathogen. i.v. voriconazole in standard doses was started as soon as invasive fungal infection was suspected. the two female patients responded well to voriconazole with gradual resolution of fever and skin lesions. this corresponded with neutrophil engraftment. the male patient with aml died of disseminated fusariosis (autopsy confi rmed) before achieving engraftment. conclusions: we identifi ed fusarium incarnatum as a new mould pathogen which can cause disseminated fatal infections in immunocompromised patients and sct recipients. although the voriconazole was proven to be an effective agent to treat these patients, the hematological recovery seems to be a prerequisite factor needed to survive the disseminated fusariosis. background: infections are the most common complications of stem cells transplantation and chemotherapy induced neutropenia. bacterial infections predominate during the early stage after transplantation. during this phase deep neutropenia and central venous catheter are the most important risk factors. because of high rate of mortality due to gram-negative bacteria, prophylaxis against this microorganisms is mandatory, but this strategy offer gram-positive predomination in all sites of isolation. despite low rate of mortality due to gram-positive bacteria, infections caused by streptococcus today became a real problem. material and methods: during a years period we have performed stem cells transplantation in patients with different hematological malignancies(aml: ; all: ; cml: ; cll: , nhl: ; hodgkin diseases: ; multiple myelomas: ; aplastic anaemia: ;myelofi brosis: ewing sarcoma: ; male: female . median age: years ( - ). in order to monitoring local micro-fl ora we perform in all patient two times a week: blood-culture, sputum, urine-culture, and simples from central venous catheters. cultures were performed using standard microbiological tools. patients were treated in sterile room conditioned with hepa fi lters, gram-negative prophylaxis with ciprofl oxacine , gr. per day, low bacterial diet. results: gram-positive cocci were predominantly isolated microorganisms ( %), then gram-negative bacteria ( %) and fungi ( %). the most frequent isolated bacteria was staphylococcus coagulaza negative, from central venous catheter, while streptococcus pneumonia was the most common bacteria isolated after day + , predominantly from sputum. meticillin resistant staphylococcus aureus (mrsa) was isolated in % from all gram positive bacteria. we have no vancomicyn-resistant enterococcus isolation. conclusion: the epidemiological pattern of bacterial infection continues to evolve globally and locally at the institutional level, as do patterns of susceptibility and resistance. these trends are often associated with local treatment practices and have a signifi cant effect on the nature of empirical antibiotic prophylaxis and therapy. in our center gram positive bacteria were isolated predominantly. gram-positive prophylaxis is doctrinary used in some centers, but there is a problem with gram-positive resistance. heptavalent pneumococcal vaccination may be reasonable choice. background: invasive fungal infections (ifi) are an important life-threatening complication after allogeneic hematopoietic stem-cell transplant (ahsct). risk factors that further increase the risk of ifi in these patients include prolonged neutropenia, graft failure, immunosupression and graft-versus-host-disease (gvhd). aim: to evaluate the effi cacy and safety profi le of posaconazole as prophylaxis of invasive fungal infection after ahsct. material and methods: in patients at high risk who received posaconazole for prophylaxis we analyzed the incidence of ifi during the treatment period. demographic, clinical, laboratorial and radiologic variables of all patients were studied including age, gender, underlying disease and it´s status at allogeneic transplantation, presence of gvhd, treatment with steroids, adverse events, galactomannan antigen in plasma and high resolution computed tomography (ct-scan). adverse events were also analyzed. results: from a total of patients received posaconazol patients were included in the study, among them received ahsct. during the treatment period there were no proven ifi reported. probable ifi were reported in patient. no serious adverse events related to treatment were reported. during the observational period the overall mortality was % ( patients) and none of them died due to ifi. patients ( , %) were receiving steroids during the treatment period and none of them developed ifi. the incidence of global gvhd was %. acute gvhd incidence was %. patients had galactomannan positive and ct-scan were performed in all of them without found ifi in any case. conclusions: posaconazole prophylaxis is a useful and safe approach in order to prevent ifi avoiding systemic antifungal treatment in patients who had undergone ahsct. mucormycosis are an emerging form of invasive fungal infections (ifi) with high mortality rate ( %). early treatment contributes to improve prognosis. posaconazole is a broad spectrum azole that prevents ifi in patients with aml and in patients receiving an immunosuppressive treatment for gvhd. we describe two cases of mucormycosis (cunninghamella bertholletiae) in patients receiving posaconazole prophylaxis. the fi rst received allogeneic haematopoietic stem cell transplantation with reduced-intensity conditioning for myeloma in relapse. because of grade ii cutaneous gvhd, corticosteroids s were added to ciclosporine months later associated with posaconazole prophylaxis. however, the patient developed a digestive gvhd. at this date, cunninghammella bertholletiae was found in bronchioalveolar lavage cultures. amphotericin b was added. the patient died with disseminated infection. autopsy confi rmed multiple pulmonary lesions of mucormycosis. the second patient was hospitalised with aml for induction therapy. posaconazole was introduced on the fi rst day. ten days after, a febrile episode occurred without documentation. liposomial amphotericin b was substituted. five days later, mucormycosis was identifi ed in skin biopsy. despite anti-fungal treatment associating amphotericin b and posaconazole, he died months later with disseminated infection. residual concentrations of posaconazole were assessed retrospectively by hplc, using sera conserved at a temperature of °c (therapeutic residual plasma concentration: . and mg/l). for the fi rst patient, the serum concentration was below detection threshold (< . mg/l). for the second patient, two sera were collected at prophylaxis and curative treatments ( . and . mg/l, respectively). in both cases, the pathogens were susceptible to posaconazole (in vitro minimal inhibitory concentrations values). our second patient had probably been imunocompromised for several months (long-lasting neutropenia preceding the onset of aml, and history of diabetes). our fi rst patient had an intestinal gvhd with major diarrhoea, which was likely responsible for the very low (undetectable) levels measured when mucormycosis was diagnosed. in conclusion, our report stresses out the necessity to closely evaluate the use of broad spectrum prophylactic antifungal therapy. the prophylaxis in patients with gvhd and/or diarrhea must be used with caution. we recommend to systematically monitor posaconazole levels at least in these cases. inhalation of mold spores can lead in immunocompromised patients to an invasive disease and pneumonia. invasive fungal infection (ifi) has still a high mortality rate. mold-dna can be detected by a polymerase chain reaction (pcr) based method. using it for the bronchio-alveolar lavage (bal) can help to detect an ifi in an early stage. the pcr can discriminate between different mold species and directs the treatment. in our study on patients, a mold pcr from bal was conducted in addition to routine diagnostics. the pcr with primers specifi c for mitochondrial aspergillus-dna and ribosomal s dna for zygomycetes. our results show that mold pcr is more sensitive than standard fungal diagnostics. based on these pcr results, an intensifi ed therapy was undertaken successfully. hence, mold pcr from bal is a useful additon of the microbiological investigations. the mould pcr allows the proof of a zygomycosis at an early stage and thereby ensures successful treatment. further investigations are to show if computer-tomography of the lung combined with mold pcr are suffi cient to diagnose for sure a pulmonal mold infection. introduction: cartilage hair hypoplasia (chh)is a rare autosomal recessive disorder caused by mutations in the ribonuclease rna-processing rmrp complex. hsct has resulted in immune restoration, yet fails to correct the chondrodysplasia. we describe a patient with chh and combined immune deficiency who developed granulomatous infl ammation. treatment with anti-tnf-alpha monoclonal antibodies (moab) caused reactivation of jc virus with ensuing progressive multifocal leukoencephalopathy (pml). case report: at age y a female chh patient ( c>t and a-g mutation in rmrp) with combined immune defi ciency developed painful non-caseating granulomas. no infectious agent was identifi ed and antibiotic therapies failed. finally at age y anti-tnf-á moab(infl iximab) was started with partial response. after the rd administration she developed a debilitating intentional tremor of the right hand. mri t and flair showed demyelination in the right cerebellum. jc virus pcr was (+)in blood and in cerebrospinal fl uid (csf) and (pml) was diagnosed. weekly administrations of cidofovir, followed by two-weekly administrations for month resulted in a partial response. cidofovir was continued two-weekly. months after diagnosis of pml, hsct with a / unrelated donor was performed with reduced intensity conditioning according to ebmt-esid guidelines. there was neutrophil engraftment at d+ and stable donor chimerism of > % at d+ . at d+ , the patient complained of dizziness, with evidence of a cerebellar syndrome. mri and csf polyoma virus copies were stable. at d+ , she presented with hypertensive encephalopathy including convulsions reminiscent of posterior reversible encephalopathy. discontinuation of ciclosporine led to resolution of the encephalopathy. however, pml progressed despite restoration of t cell function, with increasing cerebellar and brain stem symptoms including ataxia, dysarthria, aphasia, n. facialis and n. glossopharyngeus paralysis with corresponding mri imaging and increase in jc virus pcr copies in the csf. despite intensifi cation of cidofovir treatment, trials of steroids, fl uoroquinolones, mirtazapine, lefl unomide as well as high dose ivig and cytarabine iv, the neurodegeneration was progressive and the patient died of respiratory failure at d+ . conclusion: we describe the fatal course of pml due to jc virus reactivation in a patient with chh, despite successful hsct in terms of myeloid engraftment and restoration of t cell function. a. tomaszewska ( ), b. nasilowska-adamska ( ), t. dzieciatkowski ( ), b. marianska ( ) ( introduction: viral infections still are a serious diagnostic and therapeutic problem in patients undergoing alternative donor transplants. betaherpesviruses (hhv , hhv , hhv ) are recognized pathogens in this group of patients. we report a case of hhv encephalitis complicated by guillain-barré syndrome (gbs) in a hematopoietic stem cell transplant (hsct) recipient with preceding reactivation of cmv infection. methods: a year-old-man with a history of chronic myeloid leukemia underwent hsct from a matched unrelated female donor in october . sero-status for cmv was igg positive in the recipient and igg negative in his donor. on the day + patient developed acute graft-versus-host disease successfully treated with iv methylprednisolone. in march he was admitted to our unit due to cmv infection reactivation. he started pre-emptive therapy with iv gancyclovir. after weeks of treatment he revealed high fever, uroschesis, paraparesis, impaired consciousness and generalized epileptic seizure. computed tomography of his brain was normal. a lumbar puncture revealed pleocytosis ( /µl) and elevated level of protein ( . mg/dl). investigation of cerebrospinal fl uid (csf) by pcr for infective causes of patient's neurological decline including hsv t. / , vzv, adenovirus, cmv and dna candida and aspergillus were negative as well as csf culture, real-time pcr revealed in his csf presence of hhv dna. according to these fi ndings and neurological status of our patient we made a diagnosis of an hhv encephalitis complicated by gbs. the therapy with foscarnet (all symptoms revealed during pre-emptive therapy with gancyclovir) and ivig was started. due to gbs diagnosis we performed procedures of plasmapheresis. we observed gradual improvement in neurological status. after discharging home the therapy was continued with cidofovir given once a week during four weeks. at present, . year after this episode, the patient remains in a good condition without cmv and hhv reactivation, with slight neurological defi ciency. conclusions: betaherpesviruses are emerging pathogens in the hsct setting and may cause central nervous system disease. gbs is a very rare complication among stem cell transplant recipients and usually has been attributed to infection. our successfully diagnosed and treated case of hhv neuroinfection complicated by gbs suggests that hsct recipients with cns signs and symptoms should have their csf investigated for hhv as well as other pathogens. zygomycosis is a rapidly growing systemic fungal infection, commonly fatal, despite intensive antifungal treatment. it almost always occurs among patients with an immunosuppressive background, diabetes mellitus, prolonged neutropenia, recent chemotherapy and an excessive iron overload. iron is essential for the growth, development and virulence of many fungi, and particularly of the zygomycetes, which are incapable to grow under iron-deprived conditions. we report on a -year old male patient, who at the age of was diagnosed with cd + b-cell acute lymphoblastic leukemia and achieved a cr following chemotherapy of hyper-cvad type. the patient remained relapse-free for almost years, but when he relapsed, he was treated with the g-mall protocol and a second cr was obtained after cycles of treatment. at that point a fully matched related pbsc allograft, obtained from his -year old sister was offered. he engrafted on day+ , and the post-transplant period was complicated by cmv reactivation and mild chronic gvhd. the patient relapsed on day+ and he was treated with high dose cytosine arabinoside days - and -h infusional mitoxandrone on days + and + . during the aplastic phase he was complicated by histologically proven, extensive left rhinocerebral and pulmonary zygomycosis, with left facial nerve paresis. at that time point he had a transferring saturation of % and ferritin ng/ml. the patient was refractory to initial treatment was surgical debridement and a combination of liposomal amphotericin-b and posaconazole. since no signifi cant improvement was obtained despite a second surgical intervention, deferasirox mg/kg of body weight was added to his antifungal regimen. following weeks of treatment with the triple combination fever was rapidly subsided, as did both, nasal and facial symptoms and lesions. the pulmonary lesions were clearly improved. transferrin saturation decreased to % and ferritin to ng/ml. unfortunately, chemotherapy produced a minor response and months later leukemia reappeared. the patient fi nally succumbed from pulmonary hemorrhage, following salvage treatment with clofarabine and cyclophosphamide, without any sign or symptom of recurrence of his previous zygomycosis. introduction: despite the relatively high transplant-related mortality (trm), the management of the end-life care is poorly understood issue and the problems of providing palliative care to patients submitted to stem cell transplantation (sct) may be underestimated. in this regard, the use of palliative sedation therapy (pst) in the sct setting remains a major concern. patients (pts) and methods: in order to address this issue, a retrospective study on the use of pst in our tertiary sct unit was performed. search criteria were: death and previous sct. data regarding symptoms, symptoms control and use of pst were collected. we identifi ed dead pts. last line of therapy before death was sct and a salvage treatment given for a post sct relapse in and patients respectively. near the death, / patients experienced a total of refractory symptoms and in cases more than one of them was present. intractable symptoms were: excruciating dyspnoea in ( %), agitated delirium in ( %), severe pain in ( %) and massive bleeding in ( %). results: pst was started in all patients, at a median of ( - ) days before death. the most used sedative drug was midazolam, that was administered to / pts as single agent and in cases in association with promazine; pt received the latter agent alone. at the start of pst, pts with pain were receiving parenteral morphine. symptoms control was adequate in cases (complete and partial symptoms control in and respectively) and not adequate in . conclusion: pst is a controversial issue in palliative medicine, although it has been clearly claimed that when it has the intent to provide symptom relief, pst should be considered a proportionate intervention. sct failure represents a so strongly discouraging event to determine diffi culties to recognize end life status. as a consequence, the risk of an inadequate symptoms assessment and of an inappropriate palliation should be considered. in our experience, in a patient closed to the death, when other treatments failed to relieve the intolerable suffering from refractory and otherwise intractable symptoms, pst represented a valid palliative care option by a reduction in patient consciousness, using appropriate drugs carefully titrated to the patient's comfort. adequate symtom control was obtained in more than % ( / ) of pts. an internal operative protocol is under construction to improve those results. donor lymphocyte infusion as therapy for persistent pure red cell aplasia following major abo-incompatible stem cell transplantation a. lübking, i. winqvist, s. lenhoff lund university (lund, se) pure red cell aplasia (prca) after abo-mismatched allogeneic stem cell transplantation (sct) is not uncommon. however, spontaneous remissions within months are frequent. we here report a case of long-lasting prca refractory to multiple therapies that eventually responded to donor lymphocyte infusion (dli). a year old woman received peripheral blood cells from an unrelated hla-identical donor following myeloablative conditioning six months after diagnosis of aml. there was a major abo incompatibility between recipient ( +) and donor (b+). engraftment of granulocytes (> , x /l) and platelets (> x /l) was noted on day and respectively. due to the absence of reticulocytes, bone marrow analysis was performed on day showing the total absence of erythroid precursors. initial treatment with steroids, erythropoetin and withdrawal of immunosuppressive therapy was not successful. four doses of rituximab were given from day without any effect. starting on day immunoabsorbtion on three consecutive days was performed followed by methylprednisolone, cyclophosphamide and immunoglobulin infusions. although the igg and igm antidonor isoagglutinins were reduced from : to : and : respectively, the prca persisted. from day she received doses of dli within months in escalating doses ( , , and million cd /kg). three months after last dli she developed signs of a mucosal gvhd accompanied by moderate eosinophilia. concomitantly, stable reticulocytosis occurred from day and she became transfusion independent. since residual recipient b-and plasma cells are presumed to be responsible for production of anti-donor-isoagglutinins causing prca, inducing gvhd by withdrawal of immunosuppressive therapy or dli might be a reasonable option. there are previously published cases of successful dli treatment for prca, but in many cases dli was given relatively shortly after transplantation, i.e. when spontaneous remission still was possible and the time between dli and reappearance of reticulocytes varied. in our case stable reappearance of reticulocytes occurred concomitant with signs of gvhd. we therefore fi nd our case highly suggestive of that inducing gvhd with dli can overcome post-sct prca refractory to almost all other therapy options. cystatin c level as a marker of renal function in haematopoietic stem cell transplantation h. muto, k. ohashi, m. ando, r. hanajiri, t. kikuchi, w. munakata, c. sakurai, m. yamamoto, t. kobayashi, t. yamashita, h. akiyama, h. sakamaki tokyo metropolitan komagome hospital (tokyo, jp) hematopoietic stem cell transplantation (hsct) recipients have an increased risk of acute kidney injury (aki) or chronic kidney disease (ckd). however, serum creatinine level may underestimate the prevalence of these renal complications because of decreased lean body mass or concurrent liver disease, which was frequently observed in a hsct setting. cystatin c measurement may be more sensitive for detecting impaired kidney function. we retrospectively reviewed the medical records of hsct ( allogeneic and autologous) recipients who had at least one chance to monitor serum cystatin c level during last years in our institution, and evaluated cystatin c as a possible new marker which can predict subsequent renal dysfunction. the occurrence of aki was defi ned by the rifle classifi cation and ckd staging was based on kdoqi criteria. of transplant recipients, patients developed aki after median days (range - days) after hsct, while worsening ckd stage was observed in patients during observational periods. cystatin c level was not infl uenced by autologous transplant (p= . ), but signifi cantly elevated after allogeneic transplantation (p< . ). pretransplant advanced disease status also had an infl uence on cystatin c level before transplantation (p= . ) multivariate analysis disclosed that the use of calcineurin inhibitor was a major cause of cystatin c elevation (odds ratio . , p= . ). there was also a strong inverse correlation between cystatin c and estimated gfr (r=- . , p< . ). proportional hazard modeling analysis revealed that the episode of aki after transplantation were a great risk for substantially worsening ckd stage (hazard ratio . , p< . ). cystatin c measurement could be a useful clinical tool to identify hsct recipient at increased risk for ckd. control of severe bleeding from acute gvhd by treatment with tranexamic acid j. hasenkamp ( ) acute graft-versus-host disease (agvhd) is a major cause of morbidity and mortality after allogeneic hematopoietic stem cell transplantation. % of the cases with intestinal agvhd are refractory to standard treatment regimen. these patients suffer frequently from severe agvhd grades to including massive gastro-intestinal bleedings. we report from clinical courses of two cases treated with tranexamic acid for diffuse, life-threatening gastro-intestinal bleedings caused by steroid-refractory agvhd. the agvhd was confi rmed by biopsy and histopathology. immunosuppression consisted of tacrolimus, mycophenolate mofetil, prednisolone and second line treatment with alemtuzumab. one patient received additionally extra-corporal photopheresis and mesenchymal stem cells. global coagulation and factor xiii plasma levels were kept in normal ranges by substitution. thrombocytopenias were compensated by adequate transfusion of cell separated thrombocytes concentrates. bloody stool volumes of and kg in h lead to dropped hemoglobin levels despite massive transfusion of erythrocyte concentrates. because of this persistent, diffuse gastro-intestinal bleedings, both patients were treated additionally with mg tranexamic acid i.v. every h. after three infusions of tranexamic acid the bleedings in both patients stopped. treatment with tranexamic acid was discontinued without reoccurrence of the bleedings. there were no adverse events of tranexamic acid observed. local hyperfi brinolysis in the gastro-intestinum may contribute to bleedings from tissue damage caused by agvhd. tranexamic acid is indicated for prophylaxis and treatment of bleedings by systemic and local hyperfi brinolysis after e.g. surgery or plasminogen activator treatment. abortion of hyperfi brinolysis can contribute to stabilization of coagulation. prophylaxis or control of severe agvhd is preferred for prevention of hemorrhage. however, tranexamic acid is a treatment option in otherwise unmanageable gastro-intestinal bleeding caused by agvhd. further studies are desired to charge the signifi cance of tranexamic acid in this indication. a low or high body mass index is not predictive for outcome following allogeneic haematopoietic stem cell transplantation j. auberger, j. clausen, b. kircher, g. gastl, d. nachbaur innsbruck medical university (innsbruck, at) objectives: recently it was hypothesized that a low (< ) bodymass index (bmi) is signifi cantly correlated with an increased transplant-related mortality, decreased survival and relapsefree survival after allogeneic sct (k le blanc, haematologica ; : ). patients: patients receiving a fi rst allogeneic transplant were studied. underlying diagnoses were acute myeloid leukemia (aml) (n= ), acute lymphoblastic leukemia (all) (n= ), lymphoma (n= ), and other diseases (n= ). median patient age at time of transplant was (range, - ) years. patients were grafted from an hla-identical sibling donor and patients received grafts from volunteer unrelated donors. conditioning was myeloablative in and of reduced intensity in the remaining patients. results: overall survival for the entire cohort was % ( %- %, % confi dence interval, ci). there was a trend for a poorer outcome in patients with < % and > % percentile bmi (i.e. bmi ≤ and ≥ ) (os % vs %, p= . log rank test) due to a higher non-relapse mortality in this patient cohort ( % vs. %). these differences were observed in both, the myeloablative as well as reduced intensity transplant cohorts. the bmi had no infl uence on relapse incidence in either patient cohort. conclusion: by deviding patients into percentiles bmi had no signifi cant impact on outcome and non relapse mortality neither following myeloablative nor following reduced intensity allogeneic stem cell transplantation. autoimmune thyroiditis after haplo-identical stem cell transplantation for severe combined immunodefi ciency f. dogu ( ) introduction: thyroid dysfunction is a well known complication in survivors of hematopoietic stem cell transplantation, and is reported after tbi as well as radiation-free conditioning. the most common disorders after radiation free conditioning are euthyroid sick syndrome(ets) and compansated hypothyroidism. autoimmune thyroiditis is rarely reported after hsct in children and it has never been described after hsct for scid. here we report an autoimmune thyroiditis developed months after the third haploidentical stem cell transplantation for scid. case: a -months-old girl was referred to clinic with the diagnosis of t-b-nk+ scid. as she didn't have a fully matched sibling donor and her clinical condition was unstable she received peripheral blood stem cell transplantation(pbsct) from his haploidentical father after cd + cell selection without conditioning. engraftment wasn't achieved on day + and she received second haploidentical cd + selected pbsct from her mother. third transplantation was performed months after the second one, due to graft failure and this time she received bu/cyclo for conditioning and csa for gvhd prophylaxis. myeloid and platelet engraftments were achieved on day+ and + respectively. grade i acute gvhd developed on + and treated with corticosteroid for ten days. she was discharged on day+ with full donor chimerism. thyroid hormone levels which were normal before hsct revealed compansated hypothyroidism at posttransplant months in a routine follow-up visit. elevated antithyroid proxidase ( iu/ml) and anti-thyroglobulin ( iu/ml) titers were all consistent with the diagnosis of autoimmune thyroiditis(hashimoto). levothyroxin treatment was started. since the thyroid hormone levels were normal and antithyroid antibodies were negative in her mother, the transfer of autoimmune disorder was excluded. conclusion: regular screening of thyroid functions is important and necessary to detect and treat thyroid illness, especially in young children following hsct. once-daily intravenous busulfan as myeloablative reduced-toxicity conditioning regimen in haematopoietic stem cell transplantation s. santarone, e. di bartolomeo, p. bavaro, p. di carlo, p. olioso, g. papalinetti, p. di bartolomeo bmt center (pescara, it) postulating favorable antileukemic effect with reduced toxicity and improved safety, we used i.v. busulfan (bu) associated with either cyclophosphamide (cy) or fl udarabine (flu) as conditioning therapy for hematopoietic stem cell transplantation (hsct) in patients affected by aml (n= ), mds (n= ), all (n= ) and thalassemia major (n= ) between may and june . patient age was - (median ) years. five of them were older than years. nine patients received flu at a dose of mg/m /day for days (from - to - ) immediately followed by bu given in single i.v. administration over hours at a dose of , mg/kg day for days (total dose , mg/kg). five patients received the same dosage of bu from day - to day - followed by cy mg/kg/day from - to - . donors were hlaidentical (n= ) or antigen mismatched siblings (n= ) and were matched unrelated (mud). the graft-versus-host disease (gvhd) prophylaxis included cyclosporine and short course methotrexate for all patients with the addition of antithymocyte globulin for the mud transplants. eleven patients received bone marrow cells (median dose of nucleated cells , x / kg, range , - , ) and were given peripheral blood stem cells (median dose , x /kg cd + cells, range , - , ). all patients achieved primary engraftment. the median time to . x /l neutrophils and x /l platelets was (range, - ) and days (range, - ) respectively. chimerism studies revealed that of were complete chimeras ( % donor) at year post-hsct. acute gvhd was observed in patients (grade i in , grade ii in , grade iii in ). two patients had mild to moderate chronic gvhd. there was no death due to the transplant procedure. the transplant-related complications were limited. grade iii who hepatic toxicity occurred in patients, hemorrhagic cystitis in , moderate oral mucositis in and a single episode of seizures in . six patients developed cmv reactivation between day and post-transplant (median, day ). two patients relapsed and died. as of december , patients ( %) are alive and disease-free after a median follow-up of days (range, - ) . although the small number of patients does not permit any fi nal conclusion, our hsct protocol treatment confi rms that i.v. bu, associated either with flu or cy, is a well tolerated reduced-toxicity myeloablative conditioning regimen and deserves further study with more patients and longer follow-up. background: down's syndrome (ds) is associated with higher incidence of both haematological and non haematological neoplastic diseases, if compared with general population. reduced susceptibility to chemo-and radiotherapy and the frequent comorbidities limit the use of high dose treatments, especially required in adult patients. case report: a year old male with ds developed an acute myelogenous leukaemia, fab m , aml /eto rearranged, in september . he received standard induction treatment obtaining complete remission (cr), consolidation therapy and, in february , autologuos transplantation with bu-mel conditioning regimen using mobilized peripheral blood stem cells. patient relapsed in february , at the age of , was treated with mec schedule, obtaining a second cr. hla typing showed the presence of an identical sibling. a full clinical evaluation revealed mild reduction of the ejection fraction due to corrected congenital fallot tetralogy (ef= %) and a pulmonary hypertension. a reduced intensity conditioning regimen was proposed, consisting of thiotepa ( mg/kg iv /d x dd) and fludarabine ( mg/kg iv /d x dd) followed by allogeneic stem cell reinfusion in june . standard gvdh prophylaxis was given; engraftment was achieved at day + for anc > , x /l and + for plt > x /l; grade who was recorded for liver toxicity and grade for mucosal toxicity. full donor chimerism was documented at day + . the patient developed stage agvhd; however, months after transplantation relapse was diagnosed with immunological features of all. immunosoppression was suspended, although blast percentage increased rapidly to % and a salvage therapy with all active drugs was started. discussion and conclusion: few data are reported on allogeneic stem cell transplantation in adult patients with ds. this is the fi rst case of ric allosct in a ds adult. those sporadic data do not allow conclusions about outcome on ds adult. a retrospective analysis on large database and a prospective study would be useful to address this issue, helping physicians on treating adult ds pts, when immunogenic effect of allosct play a crucial role to prevent relapse. severe immune hemolysis and pure red cell aplasia after haploidentical non-myeloablative allogeneic stem cell transplantation g. nair, a. mischo, g. stüssi, u. schanz university hospital (zurich, ch) haploidentical stem cell transplantation (sct) offers potential cure to patients without hla-identical donor. recently nonmyeloablative conditioning regimens with in-vivo t-cell depletion have been introduced. severe immune hemolysis rarely occurs after hla-identical sct, but little is known about the occurrence after haploidentical sct. here, we describe patients receiving haploidentical sct for high-risk or relapsed aml ( ), cml after blast crises ( ), and as a rescue therapy in a patient with all after primary graft failure following hla-identical mud sct. all patients were in morphological remission at the time of sct. conditioning regimen included fl udarabine ( mg/qm x days), cyclophosphamide ( mg/qm x days), and alemtuzumab ( mg x days) for in-vivo t-cell depletion. gvhd prophylaxis comprised mycophenolate mofetil (day - ) and cyclosporine (day - - ) and all patients received prophylactic antibiotic treatment. g-csf was administered until hematologic recovery. peripheral blood sct was performed over - days with a median number of . x ( . - . ) cd positive cells. the early posttransplant course was uneventful, the median time of aplasia was ( - ) days. acute gvhd occurred in / patients (i: ; ii: ; iv: ). four patients experienced posttransplant infectious complications ( cmv, bk, fungal infections, one pulmonary infection). two patients experienced severe immune hemolytic anemia and concomitant pure red cell aplasia in the bone marrow and months after sct. in both patients relapse was diagnosed shortly before or after the onset of hemolysis. the direct antiglobulin test was positive for igg and c d. the serum of both patients reacted with all cells in a cell antibody search panel without evidence for cold-reacting antibodies and no antibody specifi city could be evaluated. one patient was treated with steroids, ivig, rituximab, and high-dose cyclophosphamide, but eventually died due to fatal hemolysis. the second patient is currently being treated with steroids, ivig and high dose cyclophospamide with a marked reduction of hemolytic activity. the remaining three patients are currently in complete remission without evidence of hemolysis. in conclusion, nonmyeloablative conditioning regimens in haploidentical sct offer new possibilities for patients without a hla-identical donor. however, physicians should be aware of the potentially fatal complication of severe immune hemolysis. one of the major side effects poorly tolerated, especially in children, is represented by emesis post-chemotherapy. the use of antiemetic during chemotherapy (three to four doses for day) is necessary to reduce this complication. in this work was evaluated using a single dose of palonosetron intravenous for the prevention of nausea and vomiting secondary to chemotherapies. methods: since we have used the palonosetron in pediatric patients of which males and females, undergoing bone marrow transplantation, allogeneic (both sibling that mud) and autologous. the median age is years (range - ) and the median weight is kg (range - kg). the diseases in young patients are reported in table , the conditioning transplantation are listed in table . the dosage used, including scientifi c literature data, was mcg /kg body weight. the palonosetron was considered effective when the emesis was not more than episodes in hours and nausea no more than nd grade. results: it was encouraging, having achieved a good control of nausea and/or vomiting induced by chemotherapy, in fact, only seven patients ( %) was necessary to resort to a second dose of antiemetic, in four of seven ( % of total) was repeated the success with palonosetron a distance of four days after the fi rst dose using the same dosage. in patients ( %) has not been no emetic episode while in the remaining group ( %) episodes were occasional and not have needed any treatment. in all patients was not noted any adverse event or side effect. conclusions: our experience, although on a small sample, it suggests that palonosetron can be considered an effective drug in preventing the nausea induced by chemotherapy, is also a drug that not have adverse events, so well-tolerated and easily manageable, it is necessary a single dose within hours before the start of chemotherapy, not least the assessment of the reduction in costs compared to conventional antiemetic. allogeneic or autologous haematopoietic stem cell transplantation (hsct) is an established mode of treatment of different diseases. loss of protective immunity to pathogens has been consistently demonstrated in patients referred to hsct. impairment of humoral and cell-mediated immunity is commonly seen after transplantation. the degree of immunodefi ciency is determined by many factors, particularly by the type of disease and transplant, the presence of graft-versus-host disease (gvhd) or ongoing immunosuppressive treatment. the aim of the study was to evaluate ) immunogenicity of a revaccination schedule in pediatric hsct recipients ) quality of recipient immune reconstitution and protection against ordinary pathogens. patients and methods: twenty one patients (pts) . - (average . ) years old, boys and girls after autologous ( , %) and allogeneic ( , %) hsct were included in revaccination program. indications to hsct were: solid tumors - , hematological malignancies - , immunodefi cency states - and aplastic anemia pts. time interval between hsct and begining of vaccination protocol was . - (av. . ) years. vaccines used in protocol were as follows: diphtheria and tetanus toxoids, pertussis (for patients < years old), hbv, vzv, haemophilus infl uenzae type b conjugate, -valent pneumococcal polysaccharide, inactivated infl uenza, inactivated polio and attenuated measles-mumps-rubella vaccines. plasma samples to determine specifi c antibodies by elisa tests were collected before and after vaccinations. results: with the exception of one patients presented with repeated fevers, lymph nodes enlargement, muscles and joints pain, no important side effects of vaccinations were observed. a meningococcial meningitis developed in one patient who refused vaccinations. plasma antibody concentrations before and after vaccinations were as follows: antidiphteria ( - , mean . ; - , mean ), antitetanous ( - , mean ; - , mean ) and antihbv ( - , mean ; - , mean ) iu/ml. conclusions: ) systemic immunization is necessary at appropriate time intervals following transplantation to re-establish immunity. ) a signifi cant increase of antibodies titer after hbv, diphtheria and tetanus toxoids was detected. ) vaccinations in patients after hsct are effi cient and well tolerated. ) a delay in begining of vaccination can result in life threatening complications. ministry of science rp, grant number /g/ . according to the world bank data, released in the report, romania has an upper-middle-income economy. the hematopoietic stem cell transplantation (hsct) program started in romania in and more than transplants (auto and allo) were performed. we analyzed the outcome for patients who underwent an allogeneic hematopoietic stem cell transplantation from matched related donor for acute leukemia ( patients) and aplastic anemia ( patients). for of the patients the procedure was performed in romania and for patients abroad. for both categories the follow-up after transplant was done in hematology units in romania. the overall survival was . months, with the longest survival of months and respectively shortest outcome for less than one month. on the st november , there were patients alive, between and months from the procedure, with a median survival of months. sixteen patients died, the median survival being months after transplant. four out of patients died during the fi rst month after transplant, and a total of patients died during the fi rst months after transplant. the transplant related mortality was . %, . % died due to relapsed disease and . % died of graft failure. for these results, there could be incriminated the irregular and inadequate drugs and reagents supplies in the romanian health system, an ineffi cient follow-up system and registry and home-care facilities defi ciencies in romania. in conclusion, the gross national income (gni) per capita and the human development index (hdi) are very important factors for the outcome of recipients of hematopoietic stem cell. background: umbilical cord blood stem cell transplantation has many advantages over bone marrow transplantation or peripheral blood stem cell transplantation. but, there are some problems to be solved in order to be applied to adults. the main problem is limitation of volume, which can be collected from one placenta was only between ml and ml. to overcome this problem, the ex vivo expansion of cryopreserved umbilical cord blood stem cells is needed. the object of this study was to evaluate the effect of cryopreservation on ex vivo expansion potential and viability of umbilical cord blood stem cells. methods: after normal delivery, cord blood was drawn from umbilical cord vein and was used to evaluate the mononuclear cell count, the cell viability and clonogenic capacity of cord blood stem cells before and after cryopreservation. results: before cryopreservation, the mononuclear cell count of umbilical cord blood was . ± . x /ml, cell viability was ± . %, total colony count was . ± . and percentages of cfu-gm, cfu-gemm, bfu-e were . ± . %, . ± . % . ± . %, respectively. the mononuclear cell count of umbilical cord blood cryopreserved for days was . ± . x /ml and cell viability was ± . %. total colony count of umbilical cord blood cryopreserved for days was . ± . and percentages of cfu-gm, cfu-gemm, bfu-e were . ± . %, . ± . %, . ± . %. but, there were few colony count which could be observed after cryopreserving for days. conclusion: there was no difference of clonogenic capacity of umbilical cord blood stem cells before and after cryopreservation. the cell viability of umbilical cord blood stem cells was decreased after cryopreservation but there was no difference between umbilical cord blood cryopreserved for days and days. therefore, it is possible that suffi cient umbilical cord blood stem cells could be obtained by ex vivo expansion of cryopreserved umbilical cord blood in order to be used for adult patient. objective and methods: combined hematopoietic stem cell transplants (hsct) plus solid organ transplants (sot) have been rarely reported. the majority of patients with a previous history of liver transplants were children that underwent hsct for aplastic anemia after viral hepatitis. here we report an adult patient who received a cord blood hsct after a preceding liver transplantation. results: in a year old man required orthotopic liver transplantation for cirrhosis after b viral hepatitis. in april acute myeloid leukaemia m citotype , normal karyotype, flt-itd positive was diagnosed and a fi rst complete remission was reached after induction and consolidation cycles. at that time the patient was not considered eligible for a transplant program due the previous history of sot. in february the patient relapsed and came to our centre: he was treated with high-dose cytosine-arabinoside chemotherapy, that was complicated by a pulmonary aspergillosis , but reached a second complete remission. we decided to start a cord blood donor search, since siblings were not available and he could not wait for an unrelated donor search. a cord blood with hla locus a allelic mismatch and locus c antigenic mismatch was identifi ed. patient's comorbidity index according sorror at transplant was . in may a preparative regimen containing treosulfan, fludarabine and atg fresenius was administered and , x /kg cd + cells were reinfused. grade i mucositis and grade ii hepatoxicity were observed. a bacterial pneumonia and cmv reactivation occurred at day and at day respectively and both rapidly resolved. a neutrophil count > x /l was reached at day and platelet counts > and > x /l platelet count were reached at day and day respectively. no acute and chronic gvhd were observed. a % donor chimerism has been reached in whole peripheral blood and in cd + cells since days onwards. no minimal residual disease has been detected by marrow immunophenotyping and by wt- gene expression until last follow-up, at day . conclusion: to our knowledge this is the fi rst report of a successful cord blood allogeneic hsct in an adult patient with a history of liver transplantation. this case might encourage physicians to propose allogeneic hsct by any stem cell source to patients with high-risk haematological diseases, who had previous liver or other sot's. double unit cord blood transplantation(cbt) has been established as an alternative source of donor cells for allogeneic haematopoietic stem cell transplantation (hsct). we reported here an interesting case of long-term mixed full donor chimerism during the regular follow-ups of one year after hsct. a year-old woman with acute lymphoblastic leukaemia in second complete remission received two units of cord blood after a myelo-ablative conditioning regimen. the cord blood units were hla / identical with the recipient ( b mismatches and a+ b mismatches). total nuclear cell doses infused were respectively . x and x , whereas the cd +cells number was identical in the cbus and the cd +cells number was higher (x ) in the fi rst one (table ). neutrophil recovery was observed at day and platelets engraftment at day after cbt. only one event of acute graft versus host disease (gvhd)grade i was reported at day . currently the patient does not have chronic gvhd and is disease free. analysis of chimerism was performed by str-pcr or rq-pcr on whole blood and specifi c lineage cells (cd +, cd + and cd +). follow-up was done at , , and months post transplant. full donor chimerism (fd) was achieved on day . each of the two units contributed at different levels to the donor chimerism in specifi c lineage cells: whole blood and cd were about % cbu /cbu , cd cells were preferentially from cbu origin ( %), and cd cells were preferentially from cbu origin ( %). this mixed origin of donor cells was detected early and was constant during regular follow-ups. usually, recipients of double unit cbt were engrafted predominantly with one of the units after months. the mechanism of a long-term mixed full donor chimerism is still unknown for our patient. kir ligand analysis showed an absence of mismatch in gvh direction between recipient (c -c ) and each cb unit (c -c and c -c ) while there was a mismatch between the units. in this case, a state of full tolerance settled down between the various lineages, either immune mediated interaction between host/graft or between graft/graft could explain s this chimerism pattern, but it will have to be clarifi ed: a specifi c study of treg cells is in progress. optimising cd yields in pbsch: a comparative analysis of mobilisation regimens c. black, t. elston, m. streetly, m. kazmi guy's hospital (london, uk) cyclo/g-csf (cyclophosphamide/ granulocyte-colony stimulating factor) has been the mobilisation regime of choice when collecting peripheral blood stem cells (pbscs) for transplantation yet pbsc harvests post chemotherapy produce effi cacious yields. this data seeks to compare and inform current mobilisation strategies in this centre. dhap (p= . , and ara-c (p= . , t-test) therapy yielded signifi cantly better cd results compared to cyclophosphamide. mm patients mean cd for cyclo mobilisation (n= ) were . x /kg (range: . - . ), g-csf only (n= ) . x /kg (range: . - . ), and ara-c ( n= ) . x /kg ( . - . ). myeloma patients post ara-c yielded signifi cantly more cd + cells (p= . ) compared to cyclo than those mobilised with g-csf only. nhl patients mean cd harvest results for cyclo mobilisation (n= ) were . x /kg (range: . - . ), g-csf only (n= ) . x /kg (range: . - . ), and dhap (n= ) . x /kg (range: . - . ). cd yield of nhl patients mobilised with cyclo compared with those harvested post dhap, a signifi cantly higher harvest result was noted (p= . ) than those mobilised with g-csf only (p= . ). paired mm data (n= ) compared patients fi rst mobilised with cyclo-gsf and post ara-c, (p= . , paired t-test). this study suggests that harvesting of patients post ara-c, or post dhap is valuable, giving greater cd yields than traditional agents and should be considered. paired data also indicates that ara-c could be used for effective second mobilisation. can type of delivery infl uence cord blood units' quality? g. pucci, a. pontari , d. marcuccio, i. bova, r. monteleone, d. princi, g. gallo, a. dattola, e. spiniello, c. garreffa, t. moscato, p. iacopino ao bianchi melacrino morelli (reggio calabria, it) the cord blood banks use the total nucleated cell (tnc) number as principle to proceed or not to cryopreservation of the cord blood (cb) units. we know that tnc and cd -positive cells infused on unrelated umbilical cord blood transplantation in haematological disease are fundamental for the engraftment of haematopoietic stem cells (hsc) background: immunomagnetic cd + selection is a procedure used both for autologous grafts to perform cellular purging and for allogenic transplant. in aploidentic transplant the purpose of cd + is to reduce the quantity of cd + and cd + cells so as to reduce the incidence rate of the graft versus the host disease (gvhd). aims: in this study we have valued the purity and the cellular recovery after immunomagnetic selection performed with clini-macs automatic system (miltenyi biotec, germany); a group of concentrates has been selected after incubation manually performed, while another group has been submitted to incubation and to the subsequent washings using an automated system (cytomate (baxter oncology, chicago il). methods: in our study we subjected peripheral blood stem cells (pbsc) concentrates taken from donors with microcythemia to immunomagnetic cd + selection, in order to perform aploidentic grafts on children affected by beta-thalassemia major. concentrates have been submitted to washings pre and postincubation and to incubation using the automatic system cytomate, while concentrates have manually been worked. cell count of nucleated cells (nc) was performed using an electronic cell counter while cd +, cd +, and cd + were quantifi ed using fl ow cytometry. results: the following table shows the results. conclusion: immunomagnetic selection in microcythemic donors determines according to our experience, a less recovery in comparison to the data reported in literature, nevertheless in our study results evident, even though casuistry is not very ample, that the use of an automatic system for the washing and the incubation of the cellular concentrates has determined a greater recovery and a greater purity in comparison to the procedures manually performed. allogeneic transplantation from hla identical family donor is a common therapeutic approach in patients with intermediate risk aml in fi rst cr. we present an unusual onset of acute leukemia in a female patient that was a healthy donor of pbsc (previously mobilized with g-csf mcg/kg) for her hla identical sister with diagnosis aml (fab-m ). the transplantation was preformed in january with myeloablative bu-cy conditioning and conventional cy+mtx gvhd prophylaxis. at day + acute gvhd gr i/ii was observed and resolved with addition of median dose of corticosteroids. in a period of years after this occasion, acute leukemia (aml-m no cytogenetic abnormalities) was diagnosed in the donor and treatment with chemotherapy was started. the induction chemotherapy was provided with dae regimen. with consecutive cycles cr was achieved. the patient followed consolidation treatment with hd-arac and anthracikline. further treatment with allogeneic transplantation was on schedule and the source of stem cells would be taken under consideration. can a person with aml and years surviving in a complete remission become a donor of its own donor (hla dna identical) with the same diagnosis is a question that has to be resolved in a higher number of patients. clinical outcome and characteristics of donor graft failure in patients with haematopoietic disease given donor cell boost, second allogeneic transplantation or no treatment r. ahmed nacer, m. benakli, f. mehdid, r. belhadj, n. rahmoune, m. baazizi, a. talbi, f. kaci, r.m. hamladji pierre and marie curie center (algiers, dz) introduction: donor graft failure (gf) is a life-threatening complication of allogeneic hematopoietc stem cell transplantation(hsct), determined when anc had not reached , /l by day (primary gf) or when anc decreased irreversibly after engrafment (secondary gf). frequency of gf is variable in function to hematopoietic disease. material and methods: from may to december , patients (pts) underwent allogeneic hsct from hla-identical sibling donor. pts are appraisable for this study and gf was diagnosed in pts ( , %) : primary gf: pts, secondary gf: pts with median time engrafment to ; median age at transplant years ( - ); sex ratio (m/f) , ; hematologic non malignant disease (hnmd; n: ): aplastic anemia: / ( , %), major athalassemia: / ( %) and hematologic malignant disease (hmd): / ( , %); pts had received more than transfusions before allograft; abo incompatibility between donor/recipient was seen in d/r pair; median interval from diagnosis to transplant months ; pts was multiparous; pts received myeloablative conditioning regimen (mcr ) and one pt reduced intensity conditioning (ric); pts received peripheral blood stem cell (pbsc) and pts bone marrow transplant (bmt). the chimerism testing was performed in cases: predominant host population (host population: - %) in pts and mixed (host population < %) in pts. pts were given donor cell boost with no additional conditioning with median time gf to treatment days ( - ) and fi ve pts a second hsct within one a third hsct (mcr: ; ric: ) with median time gf to treatment days ( - ). pts had not been treated. at november maximal follow up is months and minimal months. results: pts are alive ( %) within pts (donor cell boost: pts; second hsct: pt) with success engraftment(donor population: %) after median follow up months ( - ) and pts (second hsct: pt; no treatment: pts) with autologous reconstitution (donor population: %). pts died ( %) within pts after given donor cell boost, pts after second hsct and pts before given donor cell boost. os at years is %. conclusion: gf is rare but serious and concern hnmd more than hmd. better outcome can be obtained after chimerism testing study to choice treatment : predominant donor population will have donor cell boost and predominant host population second hsct with another donor. mixed haematopoietic chimerism: how the initial dynamics of mixed chimerism correlate with later chimerism status k assing ( ), c. heilmann ( ) ( )herlev university hospital (herlev, dk); ( )university hospital, rigshospitalet (copenhagen, dk) background: the natural history of mixed (hematopoietic) chimerism (mhc) has been extensively studied in hematopoietic stem cell recipients, in order to fi nd determinants for relapse or graft rejection. methods enabling quantitative prediction of later mhc status have not been devised. methods: recipients, receiving hematopoietic stem cells due to non-clonal disorders and displaying at least % donor chimerism at minimum one time point, were serially tested for whole blood chimerism over a median period of . years (range: . - . years). relative changes in the host fraction (termed alfa ) between the median time points: . and . weeks post-transplantation were correlated with later mhc status. the predictivity of alfa values for later mhc outcome was assessed in a linear regression model. findings: all recipients engrafted. subsequently, . % became mixed chimera and . % achieved complete donor chimerism. weekly chimerism fl uctuations prior to six months posttransplantation ( . % points; . - . % points) exceeded those after six months time ( . % points; . - . % points, p< . ). at seventeen weeks, alfa values correlated with endpoint mhc levels at . years (r = . , p< . ). negative alfa values predicted ( % confi dence intervals) the presence of less than % host cells, while alfa values between: . - . , were predictive of mhc with ≤ % host cells. the only recipient experiencing rejection ( . %) displayed the largest alfa value and had a predicted mhc outcome of . % host cells ( % ci: . - . %). interpretation: we have devised a simple mathematical method enabling us, early post-transplantation, to predict later mhc status and thus determine at an early time point, where intervention is needed in order to prevent rejection or poor graft function. feasibility of out-patient autologous stem cell transplantation for malignant haematologic disorders a. ghavamzadeh, a. allahyari, k. alimoghaddam, a. karimi, r. aboulhasani, a. manookian, m. asadi, a.r. shamshiri hematology-oncology and sct research center (tehran, ir) introduction: high-dose chemotherapy with autologous stem cell support is utilized for the treatment of a variety of malignancies including non-hodgkin's lymphoma, hodgkin's lymphoma, and acute leukemias. the aim of this study was to explore the feasibility and safety of performing autologous stem cell transplantation (asct) on an out-patient basis. material and methods: total of patients affected by malignant hematologic disorders( cases of hl, cases of nhl, cases of aml) with median age of y (range : - y) and in complete remission and without medical problem were selected. they received conditioning regimen (ceam for nhl and hl, busulfan and etoposide for aml) and stem cell infusion in hospital. the day after sct, patients were discharged and followed by outpatient sct team; include a general physician, staff nurse and care giver during their neutropenic period, and to be rehospitalized in the case of febrile neutropenia, after sepsis workup and performing chest x-ray, they were received the fi rst dose of antibiotic in hospital and treatment continued in their home. results: median time for wbc recovery was days (range: - days), median time for plt recovery was days (range: - days), median number of transfused single donor plt was . units (range: - unit). mucusitis grade was seen in patients, median duration of neutropenic fever was days (range: - days), patients was rehospitalized because of the neutropenic fever, median duration of rehospitalization in these patients was days, median follow up of patients was days (range: - days), all patients were alive and in complete remission. conclusion: results show that out-patient autolgous sct in malignant hematologic disorders (hl, nhl, and aml) is feasible and its complication is manageable. haplo-identical sct as a salvage therapy in haematological malignancies: a single-centre experience o. paina, y. stankevich, i. kazantsev, n. stancheva, a. golovacheva, e. babenko, a. alyanskiy, n. ivanova, e. semenova, p. krugliakov, d. polyntzev, l. zoubarovskaya, b. afanasyev spb state i. pavlov medical university (st. petersburg, ru) background: allogeneic hematopoietic stem cell transplantation (allo-hsct) is the one of curative option for patients (pts) with acute leukemias, though its usage is often limited by lack of matched related donor or the time required for search of unrelated one. usage of haploidentical donors allows to avoid these problems and to perform allo-hsct in time. patients and methods: very high risk pts underwent haploidentical sct: all - ( %) pts, aml ( %) pts, jmml - pt, cml- pt, and resistant neuroblastoma- pt. the total number of resistant/in progression pts was ( %), ( %) pts were in remission. ( %) pts were children (age - ), ( %) were adults (age - ). in all cases reduced intensity conditioning regi-mens (ric) were used: fl udarabine and atg with addition of different alkylating agents (busulphan, melphalan or thiotepa). sources of hsc -peripheral blood stem cells (pbsc) and bone marrow. for pbsc cd + positive selection clinimacs was used. the mean cd + count was , x /kg ( , ) . in pts agvhd prophylaxis consisted of csa and short course of mtx with or without mmf. in pts tacrolimus and mmf were used. in pts at d- used mesenchymal stem cells (msc) from third -party donors were used prevention of agvhd, in pts, msc were used for treatment of acute gvhd. results: the incidence and severity of agvhd weren't higher, than in other types of allohsct: ( %) pts had grade iii-iv agvhd with skin and gut involvement, one pt died. when mcs using in conditioning regimen agvhd, i stage was observed. treatment of agvhd with msc was successful: in pts in cr. the toxicity of the conditioning regimen was acceptable, ( %) developed grade ii-iii organ toxicity. ( %) pts had invasive aspergillosis and ( %) pts of them had cmv reactivation. the -year os is %, with mediam observation terms of , months ( to months). pts died in relapse and in cr (infection - pt, another failed to engraft and acute gvhd of the gut). conclusions: haploidentical hsct with ric is characterized by acceptable toxicity and agvhd control, stable engraftment. it proved to be a good option for the group of pts with poor prognosis. randomized clinical trials are necessary for estimation of therapeutic effect of mscs in haploidentical hsct pts. results: there were donation requests involving donors ( females, males). one donor was contacted but declined to donate dli. one donor donated twice for the same recipient. for / donors no details of donation are available. the median age at time of donation was . years (range - years). there were no failed collection procedures. / donors experienced mild citrate toxicity. / donors had a vasovagal episode, but both recovered rapidly and collection was able to be completed. donor required central access for dl collection: she had also previously required central access for pbsc donation. a median . donor blood volumes was processed (range . - . ). no late donor complications were reported. in total, donors had dl collected. among the prospective recipients ( female; male), indications for dl were: mixed chimerism(n= ); residual disease(n= ); molecular relapse(n= ); clinical relapse(n= ); ebv reactivation(n= ); pancytopenia of uncertain cause(n= ); no data(n= ). of patients for whom data were available ( %) actually received dl infusions. for the remaining , reasons for not proceeding were: spontaneous improvement in blood counts; death from ebv; death from relapsed disease; development of gvhd prior to dli; and spontaneous resolution of mixed chimerism. an escalating-dose regimen was used at -monthly intervals depending on response: the median number of doses reinfused was (range - ). patients ( %) developed gvhd s following dli. the dli was successful in treating the stated indication in patients ( %). there were recipient deaths: relapsed disease(n= ), infection(n= ), gvhd(n= ) and progressive ebv(n= ). only the two gvhd deaths were considered dl-related. conclusions: our single-centre experience confi rms that dl are frequently an effective treatment for mixed chimerism or early relapse post-hsc transplant, and that donor experiences are generally good. although requirement for dl is itself an adverse prognostic factor following hsc transplant, % of recipients had a successful outcome. nowadays, haematopoietic stem cell transplantation (hsct) remains the single curative approach to the treatment patients (pts) with the resistant primary and secondary aml. these pts have extremely poor prognosis with the level of relapse at least % and the risk of trm - %. as known, high level of blasts to the moment of transplantation infl uences on dfs and os. patients and methods: at the russian children research hospital between october and june hsct were made in refractory aml pts ( m/ f). the median age was ( - ) years. fab-type: m - pts, m - pts, m - pt, m - pts, m - pts, m - pts, mx - pts. primary refractory aml was diagnosed in pts and secondary refractory -in pts. kids were transplanted from msd, pts -from mud ( mmud) and pts -from mmfd ( haplo-pbsc) with the usage of cd /cd -depletion ( pts) or cd -selection ( pt) of the graft. the median level of blasts in bone marrow prior to hsct was % ( % - %). the myeloablative conditioning regimens were used in hsct and non-myeloablative regimen -in second hsct. pts received double-phase conditioning regimens. a median dose of cd ±cells was ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) x /kg. pts received dli on median day ( - ); pts received prophylactic dlis and pts received treated dlis due to increasing of mrd level or the mixed chimerism. results: the engraftment level was % with a median time to neutrophil recovery days ( - ) and to platelets recovery - days ( - ). % pts achieved cr to day , pts had the progression of disease, one pt died before engraftment, and the rejection was documented in pt. acute gvhd developed in pts ( %), chronic gvhd -in ( %) from pts who were alive to day after hsct. pts had the liver toxicity grade - and two pts had the pulmonary toxicity grade - (who-classifi cation). trm level was %. relapse was diagnosed in pts (dfs %). at this time pts are alive in cr, pts died ( -from relapse, -from gvhd and sepsis and one pt -from dag). os for all pts was % with a median time months ( - ) . but after double-phase conditioning regimen os and dfs were % both and trm was %. overall rfs depended on the presence of gvhd, the type of donor, the using of dli. conclusion: our results show that even for very high risk aml pts hsct may be performed successfully without the significant increasing of trm particularly with prophylactic dlis. a.a. hamidieh, a. ghavamzadeh, m. jahani, k. alimoghaddam, a. mosavi, m. iravani, b. bahar, a. khodabandeh, m. jalili hematology-oncology and sct research center (tehran, ir) objective: hematopoietic stem cell transplantation (hsct) has been extensively used in the treatment of pediatric leukemia. this is follow-up report of pediatric patients (< years) with acute myeloid leukemia (aml) and acute lymphoblastic leukemia (all) whom transplanted in hematology-oncology and stem cell transplantation research center, tehran, iran. methods: pediatric patients boys and girls (median age= years) with acute leukemia ( patients with aml and patients with all) received hsct between and . the most common conditioning regimen was cyclophosphamide + busulfan. they have received allogeneic ( aml/ all) or autologous ( aml/ all) hsct from bone marrow ( aml/ all), peripheral blood ( aml/ all) or cord blood ( aml). donor type for allogeneic transplantation in patients( aml/ all) were stem cell from hla matched siblings, patients ( aml/ all) received from other related (hla-matched confi rms with high resolution method) and patients(aml) from other related with one or more than one antigen mismatch. prophylaxis regimen for graft-versushost disease (gvhd) was cyclosporine a and methotrexate or cyclosporine a alone. results: ( . %) patients are alive, ( . %) patients died ( aml/ all). the most common cause of death was relapse of disease in . %. among patients who received allogeneic transplantation acute gvhd occurred in % and chronic gvhd in . %. two years overall survival and disease free survival of aml patients were % and % respectively. two years overall survival and disease free survival of all patients were % and % respectively. no statistical difference between aml group and all. conclusions: our results of overall survival and disease free survival are compatible with literatures. autologous haematopoietic stem cell transplantation with busulfan and etoposide as conditioning regimen for acute myelogenous leukaemia patients s. mousavi, k. alimoghaddam, f. khatami, m. jahani, m. iravani, b. bahar, a. khodabandeh, a. jalali, a. ghavamzadeh hematology-oncology and stem cell transplantation research center (tehran, ir) introduction: acute myelogenous leukemia(aml) is a potentially lethal disease. hematopoietic stem cell transplantation(hsct) has increased disease free survival (dfs) and overall survival (os) of patients more than conventional treatment. the result of autologous hsct in patients without suitable donor is near to allogeneic transplantation. we performed autologous transplantation with busulfan and etoposide as conditioning regimen for patients who didn't have suitable donor. methods: since january until oct , patients received autologous transplantation. we included all children and adult with aml in fi rst or second complete remission without suitable donor and end organ failure who can tolerate high dose chemotherapy. mobilization regimen was cyclophosphamide g/m² for one day and g-csf µg/kg for days. we have done stem cell harvesting when patient's white blood cell (wbc) count raised to /µl then the patients received oral busulfan mg/kg(from - to - ) and etoposide mg/kgiv (from - to - ) as conditioning regimen. after that patients have transplanted with their peripheral blood stem cells. results: median age at time of transplantation was . years (age range: - ), male/female: / . patients were in fi rst complete remission and patients were in second complete remission. median infused mononuclear cells was . * /kg despite of the progress in the treatment of acute myeloid leukemia allogeneic hematopoietic stem cell transplantation (hsct) remains the single curative approach to the treatment of resistant aml. these patients (pts) have extremely poor prognosis with the level of relapse - % and the risk of trm - %. high level blasts to the moment of transplantation infl uences on dfs and os. the usage double-phase conditioning regimens with the aim to reduce the level of blasts maximally to the transplantation date can improve dfs and os without the elevation of trm. patients and methods: we reviewed the records of refractory aml pts ( m/ f) who underwent hsct at the russian children research hospital between october and june . the median age was ( - ) years. fab-type: m - pts, m - pts, m - pts, m - pts, mx - pt and secondary aml - pt. primary refractory aml was diagnosed in pts and secondary refractory -in pts. kids were transplanted from msd, pts -from mud, pt -from mmud and pts -from mmfd with cd /cd -depletion of graft. the median level of blasts in bone marrow prior to hsct was % ( - ). first phase included flam ( pts), flag/go ( pts), ham ( pts), flae ( pt), go ( pt) or dacogen ( pt). mylotarg doses were - mg/m². the interval between -st and -nd phase of conditioning protracted - days. second phase of conditioning was treosulfan-( pts) or bu-based ( pts) . a median dose of cd ±cells was , x /kg. seven patients received dli on median day ( - ). results: engraftment was documented in all patients with a median time to neutrophil recovery days ( - ) and to platelets recovery - days . patients achieved cr to day and one patients had pr. acute gvhd developed in patients, chronic gvhd -in from patients who were alive to day . patients had the liver toxicity grade - , and one patient had the pulmonary toxicity grade - . trm level was , %. relapses were diagnosed in patients (dfs %). nowadays pts are alive in cr, pt has bm-relapse, pts died ( -of the relapse, pt -of viral infection, pt -of vod). os amounted % with a median time months. rfs depended on the presence of gvhd and type of donor. conclusion: our results show that the usage of double-phase conditioning regimen generally doesn't increase the level of toxicity and trm and allows to achieve the long-term survival in pts with very high risk aml. allogeneic haematopoietic stem cell transplantation for acute myelogenous leukaemia in other than fi rst complete remission status k. alimoghaddam, f. khatami, a. jalali, m. jahani, s. mousavi, m. iravani, a. khodabandeh, b. bahar, a. alimohammadi, n. bahar, a. ghavamzadeh hematology-oncology and stem cell transplantation research center (tehran, ir) introduction: for patients with aml in whom an initial remission cannot be achieved or for those who have a relapse after chemotherapy, stem cell transplantation from an hla-identical sibling offers the best chance for cure. this study reports the result of years allogeneic hsct for aml in other than fi rst complete remission status. patients and methods: from march until november , aml patients, male and female with median age of years old (range: - yrs) received allogeneic hsct. the status of them before transplantation was other than cr (including second cr, third or more cr, relapse and primary induction failure). source of hematopoietic stem cells was peripheral blood, bone marrow, and cord blood. result: median time to absolute neutrophil count ≥ . * l was days post hsct and median time to platelet count ≥ * l was days post hsct. forty-two recipients developed acute graft versus host disease (gvhd) and twenty-four developed chronic gvhd. at present ( %) patients are alive. the most common cause of death was relapse. median follow up period was month (range: - month). six month disease free survival (dfs) and overall survival (os) were % (se= %) and % (se= %), respectively. year dfs and os were % & % (se= %). conclusion: allogeneic hsct for aml in other than fi rst complete remission could be advice and can improve the result of treatment in these high-risk patients. outcome of haematopoietic stem cell transplantation for patients with acquired aplastic anaemia at a cancer center, amman, jordan: experience of a young hsct program in a developing country f. abdel-rahman, i. al-sadi, a. badeeb, h. el taani, a. ahmed, r. rihani, a. al zaben, m. sarhan king hussein cancer center (amman, jo) purpose: to evaluate the outcome of hsct in patients with acquired aplastic anemia at khcc. patients and methods: between ( / - / ) , patients had allogenic hsct for aplastic anemia. there were adults ( %), and children ( %), with a median age of years (range: - years). there were patients ( %) with severe aplastic anemia and patients ( %) with very severe disease. the source of stem cells was bone marrow in patients ( %), and peripheral blood in patients ( %). the median time from diagnosis to transplantation was days. among the group, patients had a full hla matched-related donor, one had / matched related donor and had / donor. the conditioning regimens were cyclophosphamide +antithymocyte globulin (atg) in patients, and different conditioning in the other patients. results: the main end points of the study are overall survival for the whole group, and overall survival according to the age, severity of the disease, occurrence of graft versus host disease, and degree of hla match. the median duration of follow up was . months ( . - . months) . the median time for the wbc engraftment was engrafted the wbc or the platelets, and patients never engrafted the platelets. the median survival for the whole group was . months. from the patient, patients are still alive. from the deaths, patients died from sepsis and one from massive gi-bleeding secondary to gut gvhd. from the adult patients are alive ( %), while from the pediatric patients are alive ( %) from the patients with severe aplastic anemia are alive while from the patients with very severe disease are alive. from the patients who had transplant from / hla matched related donor, are alive ( %). the three other patients who received mismatched graft died. acute gvhd was associated with increased mortality. six of nine patients who develop gvhd died while only out of patients who did not develop gvhd died. four patients had second transplant, two of them are still alive. conclusion: the important predictors of the outcome are: -degree of hla match: survival % in / hla match, versus % for the mismatch transplant. -occurrence of gvhd: survival is % in patients without gvhd, and % in patients with gvhd. therefore, the most important factor for predicting survival is the degree of hla match. our plan is not to transplant aa from mismatched donors except according to an international study protocol. ( ) diabetes type is caused by immune destruction of insulinproducing b cells of pancreas. it has recently been shown that immunoablation combined with transplantation of autologous hematopoietic stem cells may alter the course of the disease and alleviate exogenous insulin requirement [voltarelli et al. jama, ; : - . we report a year old patient with an early diabetes type (typical clinical course, presence anti-gad antibodies, diagnosis weeks prior to study inclusion) with sustained presence of c-peptide in the blood, in good clinical condition without other serious comorbidities who has been chosen for treatment after signing informed consent for study protocol earlier accepted by local bioethics committee. treatment consisted of plasmapheresis followed by mobilization with cyclophospamide ( g/m²) and granulocyte colony stimulating factor (g-csf) analogues at µg/kg from day + . three x cd + cells/ kg were obtained by leukapheresis and were later used for transplantation without further selection. conditioning regiment consisted of cyclophosphamide ( mg/kg for days - to - each) with atg (thymoglobuline , g /kg over days - to - ). and was followed by transfusion of collected peripheral blood cells on day . results: the transplantation was performed on the . . . patient engrafted on day + . during the cytopenic period no major complications were observed. the patient insulin requirement was: , iu/kg -before moblization, , iu/kg on the transplantation day, , iu/kg on engraftment. insulin was discontinued shortly after the regeneration (+ ). glucose monitoring showed normal glucose levels without the need for insulin injections from that day on. hba c levels at diagnosis were , %, , % after months from transplantation and , % months after the transplantation. continuous glucose monitoring was performed around months after the transplant and showed normal values (glucose - mg/dl) -fi gure . intravenous glucose tolerance test showed normal values of glucose levels after minutes, however the st phase of insulin secretion was not present . conclusion: this case support the notion that immunoablation followed by autologous stem cell transplantation in patients with early diabetes type may at least temporary alleviate insulin requirement with excellent control of glycemia. introduction: the allogeneic stem cell transplantation (hsct) represent an effective curative treatment in cml but treatmentrelated morbidity and mortality can be substantial. with the era of bcr-abl kinase inhibitor the place of sct is in discuss for children. we report the results of myeloablative allogeneic hsct underwent in patients (pts) under years of age. material and methods: from december to april ( months period) pts under years of age with cml (chronic phase: , accelerated phase: ) underwent myeloablative allogeneic hsct from hla-identical sibling donors; median age at transplant years ( - ); sex ratio m/f , ; median interval from diagnosis to transplant months ( to ). all patients received chemotherapy based conditioning regimen: tutshka (n: ), tutshka with additional vp (n : ) and santos (n: ). pts received peripheral blood stem cell with median cd + cell , /kg body weight (bw), pts bone marrow transplant with median nuclear cell ( nc ) , /kg bw, one pt blood cord transplant with , nc/ kg bw. graft-versus-host disease (gvhd) prophylaxis consisted of association ciclosporin and methotrexate. the molecular bcr-abl transcripts diagnosis concerned pts (m(b a ): , m(b a ): and double transcripts m (b a ; b a ): ). molecular monitoring of disease using real-time quantitative polymerase chain reaction (rq-pcr) concerned pts. at july maximal follow-up is months and minimal months. results: the median time of aplasia was days ( - ). eighteen pts ( %) are alive in complete hematological remission (complete molecular remission: ; major molecular remission: ; no evaluated: ) after median follow-up time months . acute gvhd occurred in pts ( %) with grade ii-iv, and chronic gvhd in pts ( , %) with extensive. disease relapse occurred in pts ( %) within are in complete remission with imatinib. seven pts died ( %): acute gvhd grade iv (n : , trm: %) ; relapse (n: ; %). the os and event free survival (efs) at years are respectively % and %. conclusion: our results confi rm that trm is low in young pts and the mayor problem is still relapse disease. the relapse after graft can be treated with bcr-abl kinase inhibitor ( pts in our study). the question about using allogeneic hsct or bcr-abl kinase inhibitor in children with cml is still open. reduced-intensity conditioning allogeneic stem cell transplantation in advanced chronic lymphocytic leukaemia. the impact of conditioning regimen on the non-relapse mortality. a single-centre experience j. el-cheikh, c. oudin, l. wang, c. faucher, s. furst, d. blaise institut paoli calmettes (marseille, fr) purpose: a unicentric retrospective study to determine the transplant related toxicity in patients with advanced chronic lymphocytic leukemia (cll) after reduced intensity conditioning hematopoietic stem-cell transplantation (hsct) including or not antithymoglobuline (atg). patients and methods: we studied patients with progressive or relapsing chronic lymphocytic leukemia (cll) treated with hematopoietic stem cell transplantation (hsct) in our cancer centre of marseille. males and females, (median age: years). all patients received a reduced intensity conditioning regimen. we compared patients ( %) receiving a non myeloabaltive conditioning including atg with fl udarabine, busulfan (atg group) to patients ( %) receiving fl udarabine, total body irradiation (tbi gy) and anti cd without atg (non atg group). patients ( %) had a matched related and patients ( %) a matched unrelated donors. graft-versus-host disease (gvhd) prophylaxis consisted of cyclosporine alone in the atg group or a combination with mycophenolate mofetil (mmf) in the non atg group. results: after a median follow-up of months, patients ( %) still alive and in complete remission (to date). mrd was monitored in those patients with cr; all patients achieved a molecular cr. patients had acute and/or chronic gvhd, ( % in atg group vs % in non-atg group). at the last follow up patients died ( %), and the cause of death in all of them was the treatment related complications (infections and/ or gvhd); the trm at days was %; % at one year and % at three years of transplantation. ( % in atg group vs % in the non atg group); overall survival (os) at three years was % in the atg group vs % in the non atg group. the os at one and three years was % and % respectively. fig . in conclusion: despite the small effective, we can conclude that hsct after reduced conditioning is effective and has the capacity to induce a long term complete remissions, the real impact of atg should be revaluated on further large multicentric studies. dasatinib: optimal bridge to stem cell transplant in chronic myeloid leukaemia blast crisis a. gozzini, s. guidi, c. nozzoli, b. bartolozzi, r. saccardi, b. scappini, a. bosi bmt unit (florence, it) pts presenting cml-bc have a survival of - months and scarce response to imatinib. dasatinib (bms- ) is an oral, multi-targeted kinase inhibitor, currently being used in pts with imatinib-resistant advanced cml or relapsed/refractory ph+ all. most of these pts will be evaluated for sct, even though for them this curative therapy showed higher incidence of gvhd, vod and trm. we report here fi ve pts affected from cml-lb who received dasatinib prior to allosct. donors were matched siblings ( ), matched unrelated ( ) or blood cord unit ( ) . were male and female with a median age of , ( - ) years. first line therapies included chemotherapy (vcr) plus high dose imatinib. all pts after - months from diagnosis received dasatinib mg bid. t i mutation occurred in patients, y and e k in patients, and a non codifi ed mutation in patient. dasatinib induced complete hematological response (chr) in pts, and complete (n= ) and partial cytogenetic response (pcyr) (n= ) prior to sct. patients did not achieved a complete haematological response presenting % marrow blasts and % respectively prior to sct. all pts were conditioned with myeloablative protocol. gvhd prophylaxis consisted of csa and mtx (n= ) or micofenolate association until + (n= ). pts received a mobilized peripheral blood stem cell graft with , - , x cd + cells/kg (n= ) and cord blood unit with , x cd + cells/kg (n= ). dasatinib was stopped days before transplant procedure. / pts successfully engrafted reaching anc > . x /l on day + ( - ) and plt > x /l on day + ( - ). dasatinib was introduced again in patients days after sct. one of them stopped therapy because of haematological toxicity after weeks. / patients presented chimerism was - %. transplant related toxicities were grade i/ii. no pts developed hyperbilirubinemia or vod. hyperacute extensive gvhd (gr iii) was observed in only pts at + . five patients are alive, all of them in complete molecular response with a median follow-up of . ( - ) months, died of agvhd. we may conclude that in pts undergoing sct following dasatinib there is no evidence of adverse effect on sct outcome, organ toxicities. larger studies and longer follow-up are obviously indicated to confi rm our preliminary results. both t i positive patients are alive in chr. dasatinib represents an effi cient bridge to transplant to improve the outcome of this subset of patients. inmatinib combined myeloablative allogenetic haematopoietic stem cell transplantation for advanced chronic myeloid leukaemia y. luo, y. tan, j. shi, x. han, g. zheng, x. zhu, x. lai, h. huang zhejiang uninversity school of medicine (hangzhou, cn) improved strategies are needed to treat patients with advanced chronic myeloid leukemia (cml) in order to reduce the need for lifelong therapy. we treated patients with advanced cml ( in ap, in bc) with myeloablative allogeneic stem cell transplantation (allo-sct) combined with pre-transplantation imatinib. the donors included hla-matched and -locus mismatched unrelated volunteers (n= ), and hla-matched siblings (n= ). graft-versus-host disease (gvhd) prophylaxis consisted of cyclosporine, mycophenolate mofetil and short-term methotrexate. out of ( . %) evaluable patientsdeveloped ii-iv agvhd, ( . %) patients suffered from agvhd grade iii-iv. two patients suffered from intensive chronic gvhd. after a median follow-up of months (range ¨c months), the overall survival was ( . %) / . the ten patients were all in molecular remission. imatinib combined with allo sct could provide a safe, well-tolerated therapeutic option for patients with advanced cml. this conclusion needs to be tested in prospective randomized clinical trials. p-beam group. both groups did not differ in terms of time of hospital stay, days of iv antibiotics, mucositis and infections. with the median follow-up of ( - ) years, the probability of overall survival at years equaled % for p-beam and % for chopp-cbv group (p= . ). the probability of progressionfree survival was % and %, respectively (p= . ). conclusions: p-beam and chopp-cbv protocols followed by autopbsct are effective and well-tolerated salvage therapies for patients with advanced hl. prolonged administration of the therapy seems to be appropriate for this group of patients. towards safer autotransplants in patients with non-hodgkin's lymphoma: cardiac pre-evaluation, angiotensin-converting enzyme inhibition in patients with decreased left ventricular function, antimicrobial prophylaxis and vigilant supportive care e. jantunen, s. hämäläinen, t. kuittinen, k. penttilä, m. pyörälä, a. juutilainen, i. koivula, t. nousiainen kuopio university hospital (kuopio, fi) autologous stem cell transplantation (asct) for nhl is associated with an early non-relapse mortality rate of - % most commonly due to sepsis. during - nhl patients received asct at our department. seventeen patients ( %) experienced severe sepsis and nine ( . %) died due to septic shock. severe sepsis was caused by gram-negative bacteria including pseudomonas in a signifi cant proportion of the patients (hämäläinen et al. scand j infect dis ). subclinical anthracycline cardiomyopathy may be important in regard to the development of severe sepsis in some nhl patients. since january we have applied prospectively cardiac pre-evaluation (radiocardiography), angiotensin converting enzyme inhibition in patients with decreased left ventricular ejection fraction (lvef) (< %), ciprofl oxacin prophylaxis and start with ceftazidime plus tobramycin in patients with neutropenic fever in nhl patients undergoing asct. febrile patients are observed closely with measurements of pro-brain type natriuretic peptide (bnp) and c-reactive protein (crp) for three days. also blood pressure, blood oxygen saturation, hydration and diuresis are monitored. until nov , altogether patients with nhl ( m, f) with a median age of years (range - ) have received beam followed by pb infusion according to this protocol. lvef was < % in six patients ( %) pre-transplant and they received enalapril during the peritransplant period. neutropenic fever was observed in patients ( %). no cases with gram-negative bactereamia or severe sepsis have been observed. the median peak crp value was mg/l ( - ) and was reached in a median of two days after rise of fever. serum bnp values were above normal limit in / patients with fever on day . elevated bnp values were observed in / patients on day , in / patients on day , and in / patients on day , respectively. whether severe sepsis or early deaths could be prevented with this approach remains to be seen in upcoming years with larger number of patients. outcome of refractory/relapsed patients affected by hodgkin's lymphoma treated with or without peripheral blood stem cells autografting: a single-centre experience f. angrilli, s. falorio, f. fioritoni, s. santarone civic hospital (pescara, it) introduction: despite a high curability rate, to % of patients (pts) affected by hodgkin lymphoma (hl) fail to respond or relapse after front-line treatment with polychemotherapy alone or combined with radiotherapy. the treatment of choice for refractory or early relapsed pts is high-dose chemotherapy (hdc) followed by peripheral blood stem cells autografting (pbsca), while late relapsed pts may be treated with either conventional therapy or hdc plus pbsca. methods: from to december , untreated pts with hl have been admitted in our institution. after front-line therapy, ( %) pts obtained a complete remission (cr) and pts ( %) were refractory to standard treatment. overall, pts relapsed within months after diagnosis of hl, while pts experienced late relapse. the aim of this retrospective study is to evaluate the outcome of the our refractory/relapsed pts according to the type of salvage treatment. twenty-six pts received as salvage treatment - courses of igev (iphosphamide, gemcytabine, vinorelbine), patient courses of coppebvcad (cyclophosphamide, carmustine, melphalan, epirubicin, vinvristine, vinblastine, prednisone) and patient courses of abvd (doxorubicin, bleomycin, vincristine, dacarbazine). today, pts completed salvage chemotherapy. of them, ( with refractory hl and with relapsed disease) have been submitted to pbsca. conditioning regimen consisted of beam in all cases. results: pts were male and female (m/f ratio , ). median age was years (range - ). overall, pts obtained a cr ( %) and pts had progressive disease ( %). in particular the cr were ( %) in the group of the pts receiving pbsca and ( %) in the other pts (p < . ). one patient died in cr of beam toxicity prior pbsca and pts died of progressive hl. after a medium follow-up of months, overall survival was % for the pts who received pbsca and % for those who received conventional treatment (p= . ). conclusions: our data confi rm the benefi t of hdc plus pbsca both in relapsed and in refractory patients with hl. nevertheless, a portion of refractory or early relapsed pts fail to respond to pbsca and died of hl. for these pts tandem pbsca or allogeneic stem cell transplantation should be proposed, especially if they are not in cr prior to pbsca. t cell lymphoma is a heterogeneous group of aggressive lymphomas associated with poor prognosis with standard chemotherapy and autologous hematopoietic progenitor cells transplantation (hpct) is offered as consolidation in fi rst remission or at relapse. in this study we conducted a retrospective analisis of patients underwent hpct from december to august . seven patients had diagnosis of peripheral t-cell lymphoma, four patients of systemic anaplastic large cell, and fi ve patients of linfoblastic lymphoma. five patients were transplanted in fi rst complete or partial response, ten patients in second or beyond complete or partial response and one patients in second refractory disease. median age was , years; seventy-fi ve percent preesented advanced (iii-iv) ann arbor stage, % had b symptoms, % had high lactate dehydrogenase. with a median follow-up of months from diagnosis and , months from transplantation, the -year progression-free survival (pfs) and overall survival (os) were , % and , % respectively. based on these preliminary results the hpct as consolidation therapy may offer a durable survival benefi t. the chimeric anti-cd monoclonal antibody rituximab offers new therapeutic options in the treatment of b-cell nhl (non-hodgkin's lymphoma). the addition of rituximab to chop (cyclophosphamide, doxorubicin, vincristine, and prednisolone) or cvp (cyclophosphamide, vincristine, and prednisolone) regimen was found to signifi cantly improve the response rates and survival in patients with untreated diffuse large b-cell lymphoma (dlbcl) and is now considered as the standard therapy option. rituximab also has been shown to improve response rates when combined with salvage chemotherapy. there are few studies regarding the effects of rituximab on mobilization. we compared the effi cacy of rituximab plus eshap (etoposide, metil prednisolone, cytosine arabinoside, cisplatin) with eshap alone as mobilization regimen in ( %) hodgkin's and ( %) non-hodgkin's lymphoma patients. ( %) patients were dlbcl. ( %) relapsed and ( %) refractory patients were involved. ( %) patients were treated with r-eshap and ( %) patients with eshap regimen. aphaeresis were evaluated. median number of aphaeresis was . days for r-eshap patients and . days for eshap patients. median number of mononuclear cell aphaeresis was . * per kg (kilogram) and . * per kg respectively. total number of cd + cells was . * per kg in the r-eshap group and . * per kg in the eshap group. toxicities were similar in both groups. there were no engraftment delays in the r-eshap group. so we conclude that r-eshap is effective and feasible as eshap regimen for mobilization. total number of cd + cell aphaeresis was slightly lower in the r-eshap group but did not have an effect on engraftment. prospective randomized studies are needed to evaluate whether rituximab really decreases mobilization adequacy or not. no benefi t of autologous stem cell transplantation as consolidation for high and high-intermediate risk diffuse large b-cell lymphoma in .cr after r-chop therapy -a single-centre experience m. karas, k. steinerová, p. jindra, d. lysák, s. vokurka, v. vozobulová, m. schützová, l. mohammadová, v. koza charles university hospital pilsen (pilsen, cz) objectives: the role of high-dose therapy (hdt) and autologous stem cell transplantation (asct) for patients (pts) with high and high-intermediate (h/hi) risk diffuse large b-cell lymphoma (dlbcl) in .cr was not clearly defi ned especially after addition of rituximab (r) to fi rst line chemotherapy (cht) and the use of rituximab also as maintenance therapy. therefore, we retrospectively analysed outcome of pts treated in our transplant centre with hdt and asct for h/hi risk dlbcl in .cr after - cycles of r-chop- chemotherapy and we compared their outcome with a control group of pts with h/hi risk dlbcl in .cr treated only with chemoimmunotherapy. patients and methods: between and (median follow-up months, range - months) consecutive pts with median of age years (range - years) with h/hi risk dlbcl in .cr after - cycles of r-chop- underwent hdt (beam) and asct. the median of time from diagnosis to asct was months (range - months). source of stem cells was peripheral blood and median of infused cd + cells was , x /kg (range , - , x /kg). the control group consisted of consecutive pts with h/hi risk dlbcl in .cr treated only with chemoimmunotherapy ( - cycles of r-chop- , % maintenance therapy with rituximab). the control group except for the older age did not differ in any prognostic parameters. results: in the transplanted group pts ( %) are alive in cr. pts ( %) relapsed and died. no patient died due to transplant-related mortality (trm). the estimated probabilities of -years disease-free survival (dfs) and overall survival (os) were % and %. in the chemoimmunotherapy treated group pts ( %) are alive in cr. patient ( %) relapsed and died. the estimated probabilies of -years dfs and os were % and %. we did not observe between both groups any significant difference in cumulative relapse incidence (p= , ), dfs (p= , ) and os (p= , ). conclusion: our data suggest that hdt with asct in pts with h/hi risk dlbcl in .cr after r-chop chemotherapy was well-tolerated with no trm death but in comparison with pts treated only with chemoimmunotherapy we did not observe any improvement of outcome among transplanted pts. of course relatively lower number of evaluated pts and retrospective type of analysis could infl uence our results and only prospective randomized studies can fi nally defi ne the role of frontline hdt with asct for h/hi risk dlbcl in .cr after chemoimmunotherapy. kyrcz-krzemien medical university of silesia (katowice, pl) background and aims: autologous peripheral blood stem cell transplantation (autopbsct) is widely used for the treatment of poor-risk patients with hodgkin's lymphoma (hl), however, the optimal preparative regimen has not been established. we assumed that patients with advanced hl may benefi t from receiving intensive pre-transplant therapy with prolonged administration of cytostatics and the addition of oral drugs, such as procarbazine or chlorambucil. therefore, we modifi ed the commonly used beam and cbv protocols by incorporating oral agents and prolonging the distribution of the total doses to and days, respectively. the goal of this pilot study was to evaluate safety and effi cacy of those regimens. patients and methods: patients ( males and females, median age years, range - ) with relapsed hl were included in this study. previous therapy consisted of median ( - ) lines of treatment and ( - ) chemotherapy cycles results: / patients died due to septic complications in chopp-cbv group, whereas no procedure related mortality was observed among patients treated with p-beam. all remaining patients engrafted. time to achievement anc > . g/l was signifi cantly shorter in p-beam vs. chopp-cbv group: ( - ) days vs zevalin-beam conditioning in transformed follicular lymphoma; acceptable toxicity and possible therapeutic benefi t a hdt-sct) using beam conditioning has become standard therapy for relapsed fl, however recurrent disease especially in transformed follicular lymphoma (t-fl) remains the commonest cause of death. the addition of zevalin (ibritumomab tiuxetan), a cd targeted radiolabelled antibody to beam is safe and may improve the effi cacy of hdt we analysed patients aged to with advanced stage t-fl who had received a median of (range - ) lines of therapy prior to zevalin-beam sct. the median time from diagnosis to hdt-sct was months (range - . ) and all patients had chemosensitive disease in partial remission bcnu mg/m , etoposide mg/m , cytarabine mg/m , melphalan mg/m ) from day - to - . the median stem cell dose was the remaining patients remain stable at a median of months (range - ) post-sct conclusion: the zevalin-beam protocol is as well tolerated as standard beam conditioning. the disease free survival in this small cohort of high risk patients with t-fl is encouraging but needs longer follow-up rituximab or not? a historical comparison of eshap and r-eshap as mobilisation regimen in patients non-myeloablative allogeneic stem cell transplantation in patients with high-risk lymphoma: a multicentre experience g. console ( ), g. irrera ( ), m. martino ( ) a. meliadò ( ), c. rigolino ( ), t. del vecchio ( ), o. iacopino ( ), m.c. cannatà ( ), p. scaramozzino ( ), i. bova ( ), d. marcuccio ( ), c. stelitano ( ), s. molica ( ), r. cantaffa ( ), l. nocilli ( ), a. mele ( ) pugliese-ciaccio" (catanzaro, it); ( )osp patients (pts) ( females and males), median age , years (range - ) underwent nst for high risk hodgkin disease (hd, cases) and non hodgkin lymphoma (nhl, cases) conditioning regimens consisted of fludarabine, thiotepa and cyclophosfamide in cases, tli and atg in cases, fludarabine and cyclophosfamide in cases, fludarabine and thiotepa in case, fludarabine, melphalan, thiotepa and atg in case, campath- , fludarabine, melphalan and tbi were employed in cases. in case tbi and fludarabine. cyclosporine-a (cya) and methotrexate (mtx) were used as gvhd prophilaxys in cases, in campath- and moftil micofenolate (mmf) were combined and in cases cya and mmf were used. a mean number of . x /kg cd + cells (range , - , ) were infused. pts received a mean of . (range - ) packed red blood cells after a median follow up of , months, (range - ), pts are still alive ( in cr, pr, in relapse), experienced cgvhd ( cutaneos w.h.o. grade - , pneumonial w.h.o. grade , liver w.h.o ). pts died for liver agvhd, patient died for cerebral vasculitis at months to the transplant, patient died for ards at months from transplant. pts for acute bacterial pneumonia. pts for mof respectively at and months from transplant, for aptt, at months from transplant. patient for interstitial pnemoniae at months to the transplant, pts died for disease recurrence at , and months post-transplant respectively here we report on a single centre, retrospective analysis evaluating the outcome of patients (pts.) with dlcl treated with high-dose chemotherapy and autologous (auto) or autologous-allogeneic (auto-allo) hsct. patients and methods: in total, ( , %) male pts. and ( , %) female pts. with dlcl underwent auto ( pts.) or autoallo hsct ( pts) between . . and . . . pts. received auto hsct as part of fi rst-line therapy (group ). in pts auto ( pts, group ) or auto-allo ( pts., group ) hsct was performed as second-line therapy. the median patient age was years : / / , group : / / , group : / / . patients who received auto hsct as fi rst-line therapy (group ) tended to have a better median os ( vs. d, p= . ), rfs ( vs. d, p= . ) and -ysr-os ( % vs. %, p= . ) compared to pts of group . furthermore patients of group had a signifi cant better os and -yrs-os in the auto-allo group of pts died ( pts died from severe infection with multiorgan failure and patient from relapse of disease). in contrast, none patient died from trm after second auto hsct, but died from progressive disease and pt from relapse. conclusion: the survival of patients with relapse of dlcl could not be improved by using the therapeutic approach of auto-allo hsct compared to an auto hsct based regime, due to the high trm in the auto-allo group. however, for interpretation of these results some facts have to taken into account since year , patients with primary myelofi brosis ( females and males age - y median . ) received allo hsct ( sib and unrelated donors matched at allele level). according to the dupriez prognostic system: , , patients were in high, intermediate an low risk of the disease. the diagnosis was proved by trephine biopsy, which revealed that all patients were at advanced stage of fi brosis, all patients had splenomegaly and abnormal blood smear with the presence of erythroblasts. the length of the disease duration was from to months (median ). six patients were transfusion dependent because of anaemia and thrombocytopenia, three patients were on steroids and six on hydroxycarbamide. splenectomy prior to transplantation was performed in two patients. two patients received myeloablative conditioning (busulfan mg/ kg cyclophosphamide mg/kg) and eight reduced intensity conditioning (busulfan mg/kg, fludarabine - mg/m² or melphalan - mg/m² and low dose atg). all patients were transplanted with pbpc with cd dose from . to . x /kg (median . x /kg). two patients died due to transplant toxicity (one with additional ebv reactivation and sepsis and one with vod symptomatology). in other patients toxicity was mild and there was no agvh exceeding grade i. two patients transplanted with major blood group incompatibility developed prca. plasmapheresis and erythropetin were successfully employed in those patients. finally all surviving patients reconstituted haematologically. a trephine biopsy performed months post transplant documented the process of bone marrow remodeling with a normal picture six months post transplant. all patients except one had full chimerism. eight out of ten patients are alive and with normal hematopoesis during observation period from to months (median ). the post transplantation course was similar in patients having and lacking jak mutation. in conclusion haematopoetic stem cell transplantation in primary osteomyelofi brosis is associated with rather low risk and results in sustained haematological recovery. nutritional assessment of children undergoing haematopoietic stem cell transplantation for primary immunodefi ciency or severe autoimmune disease m. slatter, c. ferguson, e. rogerson, a. yurasova, p. askew, t. flood, m. abinun, a. cant, s. bunn, j a major challenge post hsct is adequate nutrition, as poor nutritional status adversely affects outcome. patients undergoing hsct for pid often fail to thrive pre-hsct due to underlying disease. we aimed to document nutritional intake of pid children undergoing hsct at our centre. children who underwent hsct for pid or severe autoimmune (ai) disease from april -january were evaluated. the following prospective data was collected: diagnosis, age, donor, conditioning, presence of infection and growth. nutritional intake, biochemical indices, use of antiemetics and complications were documented on admission, after weeks, then monthly until day , + , + , + , + , + then monthly until discharge home. patient characteristics: patients had scid, had other pid. had severe ai disease. age at transplant ranged from months to . years (median months). were ≤ yr. had unrelated ( cords), matched family, matched sibling and haploidentical donor. all had chemotherapy conditioning - bu/cy, flu/melph, treo/flu, treo/cy. results: all received supplementary feeding via nasogastric ( ) or percutaneous jejunal tube ( ) . only required total parenteral nutrition, with severe ai disease and with persistent norovirus enteritis. all received at least anti-emetic. had viral enteritis - norovirus, adenovirus. in patients for whom adequate data was available, all had a reduction in calorie and protein intake in the - weeks following hsct, because of fl uid restriction. had grade ii skin gvhd, none developed gut gvhd. had mucositis requiring morphine. only patient lost weight overall from time of admission to discharge, one had static weight, but gained weight, by time of discharge. further evaluation of nutritional indices is required. the time around hsct is the most challenging to support adequate nutrition. careful nutritional assessment of patients undergoing hsct is critical and should direct nutritional support. patients should be optimised nutritionally prior to hsct, as the high metabolic demands around the time of hsct are unlikely to be met over the immediate transplant period. thalidomide+dexamethasone and partly b, b+dexamethasone, b+adriamycin+ dexamethasone (pad). the b group had a post-tx maintenance therapy with b weeks: , mg iv doses weekly + dexamethasone mg days. results: length of survival times (os) without and with b were signifi cantly different. further analysis of the curves in complete remission indicated % survival probability and % disease free survival (dfs) in patients in a -month period. in the very good partial remission (vgpr) group ( pts) the os was %, however, the dfs was only %. the survival curves were signifi cantly worse when tx was made in partial remission (os: %, dfs: % by pts). conclusions: . the author›s data support the fi nding that lasting survival can be expected when tx is performed in cr or vgpr. . in the interest of this, in cases of a more aggressive disease, the fi rst line pad protocol before tx is the best therapy. after the tx a consolidation therapy with b+dexamethasone is very useful. . in a slightly less aggressive disease or with accompanying diseases a thalidomide+dexamethasone fi rst therapy may also be possible. . tx performed in partial remission maybe dangerous. at this time needed put in "the therapy arsenal". acute renal failure in myeloma patients during mobilisation procedures for autologous transplantation a. pivkova ( ) during the last years blood cell separation, generally referred to aphaeresis, has established a central role in both blood donor programmes and therapeutics. the technological advances in aphaeresis equipment have made procedures safer, faster and more effective. we present cases ( males and female) with multiple myeloma treated at our department during until . initial chemotherapy treatment was provided with thalidomide based regimens (c-thal dex cycles or thaldex in cycles) or cycles of vad in one patient. all patients before diagnosis and during initial treatment had normal and stable renal function. after completing remission in all, mobilisation of pbsc was preformed with g-csf mcg/kg in duration of days. the number of wbc count prior collection was median x /l ( - ) with median lymphomonocyte percent , ( - ). aphaeresis was preformed at day with cobe spectra cell separator and large volume aphaeresis. in all patients after fi nishing the fi rst procedure we registered increase of renal degradation products in the serum during the fi rst hours post aphaeresis and complete anuria which revealed in acute renal failure (renal type) treated with haemodialysis in several consecutive occasions. one month after resolving the renal impairment the patients continued with second mobilisation procedure with the same regimen and obtained a minimal mnc count of , x /kg. autologous transplantation followed by melphalan reduced dose conditioning mg/m². engraftment was registered for ne> , x /l and plt > x /l on median day + ( to ). the patients had no need for blood transfusions. all are in cr med mths ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) after transplant. in one patient months after, a double transplant was preformed. concerning the small group of patients, we can evaluate the possible impact of large volume aphaeresis in the renal impairment in these patients or the infl uence of cytokine mobilised cells on renal tubules. key: cord- -t ryky f authors: kamal, yasmine mohamed; abdelmajid, yasmin; al madani, abubaker abdul rahman title: cerebrospinal fluid confirmed covid- -associated encephalitis treated successfully date: - - journal: bmj case rep doi: . /bcr- - sha: doc_id: cord_uid: t ryky f the covid- pandemic that attracted global attention in december is well known for its clinical picture that is consistent with respiratory symptoms. currently, the available medical literature describing the neurological complications of covid- is gradually emerging. we hereby describe a case of a -year-old covid- -positive patient who was admitted on emergency basis. his clinical presentation was primarily neurological, rather than the covid- ’s classical respiratory manifestations. he presented with acute behavioural changes, severe confusion and drowsiness. the cerebrospinal fluid analysis was consistent with covid- encephalitis, as well as the brain imaging. this experience confirms that neurological manifestations might be expected in covid- infections, despite the absence of significant respiratory symptoms. whenever certain red flags are raised, physicians who are involved in the management of covid- should promptly consider the possibility of encephalitis. early recognition of covid- encephalitis and timely management may lead to a better outcome. the covid- pandemic that attracted global attention in december is well known for its clinical picture that is consistent with respiratory symptoms. currently, the available medical literature describing the neurological complications of covid- is gradually emerging. we hereby describe a case of a -yearold covid- -positive patient who was admitted on emergency basis. his clinical presentation was primarily neurological, rather than the covid- 's classical respiratory manifestations. he presented with acute behavioural changes, severe confusion and drowsiness. the cerebrospinal fluid analysis was consistent with covid- encephalitis, as well as the brain imaging. this experience confirms that neurological manifestations might be expected in covid- infections, despite the absence of significant respiratory symptoms. whenever certain red flags are raised, physicians who are involved in the management of covid- should promptly consider the possibility of encephalitis. early recognition of covid- encephalitis and timely management may lead to a better outcome. the covid- virus, classified as sars-cov- , emerged in wuhan, china, and was initially identified as the new coronavirus disease. the who eventually named it as covid- on february . later on june , the who officially announced that covid- has infected individuals and claimed more than lives worldwide. covid- is not the first coronavirus to infect humans. other human coronaviruses (hcov) include six other members designated as sars-cov, middle east respiratory syndrome-cov, hcov-hku , hcov-nl , hcov-oc and hcov- e. as described in the literature, covid- possesses neuroinvasive potentials, which makes the central nervous system (cns) an important target. there are multiple proposed mechanisms of cns involvement, including retrograde movement from the olfactory nerve, entry into cns via circulating lymphocytes or entry via permeable bloodbrain barrier. there are several neurological manifestations that have been described in patients with severe respiratory distress. but this case is unique due to the fact that the patient's symptoms were mainly neurological in nature, that was preceded with a mild, self-limiting cough. what also enhances the uniqueness of this case is the presence of a very few reported cases of established encephalitis alongside an objective evidence of the virus itself in cns. on may , a -year-old previously healthy man, who happened to live in a particular area with uncontrolled covid- spread in dubai, started experiencing some mild, self-limiting cough symptoms without any episode of fever. this was not brought to medical attention and resolved spontaneously within days. on may , he started to become physically and verbally aggressive, as stated by his acquaintances. on may , he presented to the emergency department in rashid hospital with an altered mental state and abnormal behaviour. the patient's acquaintances clearly stated that the patient does not suffer from unusual presentation of more common disease/injury any comorbidities and denied any history of alcohol intake or substance abuse. the patient was afebrile. his heart rate was /min, blood pressure was / mm hg, respiratory rate was /min and oxygen saturation on room air was %. neurological examination revealed acute confusion state associated with severe agitation and fluctuations in the level of consciousness. cranial nerves examination was unremarkable. the motor examination including tone, power in upper and lower limbs, and deep tendon reflexes was normal as well. coordination was difficult to assess at this point. no neck stiffness or other meningeal signs were evident. chest examination, including inspection, auscultation, percussion and palpation, revealed no abnormalities. abdominal examination revealed a soft abdomen and present bowel sounds, without any evidence of tenderness or organomegaly. ► brain ct without contrast revealed multiple hypodensities in the external capsules bilaterally, the insular cortex and the deep periventricular white matter of the frontal lobes bilaterally (figure ). another brain ct was performed hours after the initial one, but did not reveal any significant interval changes (figure ). ► chest x-ray was unremarkable. ► pulmonary ct showed normal attenuation in both lungs without any appreciable air space consolidations, pneumothorax or pleural effusion. no evidence of ground glass opacities. ► pulmonary ct angiogram shows good flow of contrast of the main pulmonary trunk, right and left main pulmonary arteries, as well as the lobar, segmental and subsegmental branches without any appreciable filling defects. no evidence of pulmonary embolism. ► abdominal ct was normal ► brain mri with contrast, performed after weeks to comply with our hospital's protocol that only allows covid- -negative patient to get in contact with the mri machine, revealed abnormal signal intensity in the temporal lobe cortex bilaterally in a rather symmetrical fashion. in addition, the involvement of the parasagittal frontal lobes bilaterally was evident as well, displaying bright signals on t -fluid attenuated inversion recovery and t -weighted images with corresponding diffusion restriction. these findings are suggestive of encephalitis (figures [ ] [ ] [ ] . electrophysiological studies ► electroencephalogram did not display any significant epileptic discharges. that could possibly be due to the masking effect of lorazepam that was given to the patient to manage his agitation. living in an area where there is a higher infection rate of covid- is a red flag by itself. given the presenting symptoms, covid- encephalitis should be considered, as well as acute metabolic disorders, such as renal and hepatic encephalopathies. our patient had an initially elevated bilirubin level; however, the alanine aminotransferase, aspartate aminotransferase and gamma-glutamyl transferase were within normal limits (table ) . hepatitis c antibodies and hepatitis b surface antigen were negative, and abdominal ct scan was normal too. the elevated bilirubin normalised within weeks, indicating that this elevation was non-specific. acute cerebrovascular accident or toxic insults should also be wisely ruled out. nevertheless, viral, bacterial, parasitic, mycobacterial and fungal encephalitis should be excluded. covid- encephalitis should be also considered in the differential diagnoses, particularly nowadays. such life-threatening conditions need proper screening for all the above mentioned to avoid uninvited complications. the patient was admitted in an isolated high dependency care unit. primary care was initiated, including nasogastric tube and foley's catheter insertion, oxygen supplementation by nasal cannula, as well as intravenous fluids for the purpose of hydration. the following treatment plan was decided on and was immediately started, and it included chloroquine mg two times per day for weeks, along with two tablets of lopinavir-ritonavir two times per day for weeks. seven hundred and fifty milligrams of intravenous acyclovir sodium, three times per day, was started empirically before the cerebrospinal fluid (csf) results were obtained, addressing the possibility of herpes simplex virus (hsv) i and ii encephalitis. the decision to continue the acyclovir for a further duration of weeks was made, despite of the absence of evidence of hsv in the csf, based on the fact that the patient was gradually improving, and there might be possibility of a false negative herpes simplex pcr csf test. levetiracetam g two times per day was started empirically, tackling the suspicion of non-convulsive seizure as a possible cause for the altered level of consciousness. in addition, mg of intravenous lorazepam and . mg of intramuscular haloperidol two times per day were given as required, whenever needed. enoxaparin mg subcutaneously once a day and pantoprazole mg daily were prescribed for deep venous thrombosis prophylaxis and gastrointestinal prophylaxis, respectively. for supplementation, ml of calcium-magnesium-d -zinc (osteocare) mg- mg- unit- mg/ ml syrup was given as well. after week of his admission, the patient's level of consciousness improved dramatically, despite his fluctuating confusion and agitation. the same management plan was resumed, except an increment in the enoxaparin dose to mg subcutaneously two times per day was made, as a result of the patient's elevated d-dimer levels of . mg/dl (table ) . the patient eventually became fully conscious and well coherent, with a complete resolution of his psychosis and agitation. after weeks, he was successfully able to resume his normal life routine. fifteen days after admission, covid- rna pcr test was performed again on samples from both the nasopharynx and the csf (table ) . both results turned out to be negative. in addition, the bilirubin level improved as well. the patient was safely discharged from the hospital on june , retaining his normal baseline condition. on discharge, he was only prescribed vitamin c and zinc supplements. he did not require further anticoagulation as his d-dimer fell back to its normal limits and his pulmonary angiogram was unremarkable. a telephonic follow-up consultation was held with the patient, where he confirmed that he remains unquestionably in a good condition. sars-cov- is acknowledged to affect the nervous system and induce polyneuropathy, encephalitis and acute ischaemic strokes. the mechanism by which coronavirus affects the cns is not yet fully understood. it is sensible to agree that the mechanism of neuroinvasion could be either the traditional viral entry into cns via circulating lymphocytes, or its entry via a permeable blood-brain barrier. one would debate that the acuteness of our patient's neurological symptoms, as displayed in the symptomatology, brain imaging, as well as the elevated d-dimer, might suggest a viral influence on the vascular network of the cns. nevertheless, the possible mechanism of injury of the brain's vascular endothelium could be some disruption in the vascular structures, eventually leading to clotting and infarction. [ ] [ ] [ ] this, however, was not suggested in the presented case. a detailed look at the mri of the brain (figures - ) study reveals an abnormal distribution that is symmetrical bilaterally, affecting mainly the frontal and temporal lobes. this picture highly suggests a viral pathology rather than a vascular insult. as explained earlier, the behavioural changes, acute psychosis, acute confusional state and drowsiness were the initial and main presenting symptoms in a patient with covid- without major respiratory symptoms, except for the self-limiting episode of mild cough that resolved spontaneously, prior to his presentation, without medical interference. the early suspicion of covid- encephalitis and performing the appropriate csf studies was the key to establishing the correct diagnosis and timely management. despite the absence of csf pleocytosis, the suspicion of cns encephalitis should still be considered. upadhyayula suggested that viral meningoencephalitis may occur frequently in the lack of csf pleocytosis. in addition, erdem et al also suggested that the suspicion of cns infections should not be underestimated despite the lack of csf pleocytosis. there are also several published case series that described patients without csf pleocytosis in relation to bacterial meningitis, herpes simplex encephalitis and enteroviral meningitis. [ ] [ ] [ ] patients with meningoencephalitis associated with covid- may not present with the commonly known flu-like illness, as with the presented patient. we conclude that early establishment of the diagnosis and the immediate commencement of a management plan may contribute to a better outcome. ► a red flag of the possibility of covid- encephalitis should be raised whenever patients present with abnormal behaviour, acute psychosis, confusion state or drowsiness. ► prompt and specific investigations to diagnose this condition should not be hindered in absence of the more common respiratory covid- symptoms such as anosmia, dysgeusia, flu-like symptoms, headache, and sensory or motor deficits. ► early diagnosis and management of such cases is important to avoid further undesired complications. who. coronavirus disease situation report epidemiology and clinical presentations of the four human coronaviruses e, hku , nl , and oc detected over years using a novel multiplex real-time pcr method neuroinfection may contribute to pathophysiology and clinical manifestations of covid- steroid-responsive encephalitis in coronavirus disease a first case of meningitis/encephalitis associated with sars-coronavirus- encephalitis as a clinical manifestation of covid- neurological manifestations in severe acute respiratory syndrome large artery ischaemic stroke in severe acute respiratory syndrome (sars) coronavirus disease (covid- ) and cardiovascular disease coagulopathy and antiphospholipid antibodies in patients with covid- ace -ang-( - )-mas axis in brain: a potential target for prevention and treatment of ischemic stroke . incidence of meningoencephalitis in the absence of csf pleocytosis central nervous system infections in the absence of cerebrospinal fluid pleocytosis cerebrospinal fluid white cell count: discriminatory or otherwise for enteroviral meningitis in infants and young children? analysis of clinical outcomes in pediatric bacterial meningitis focusing on patients without cerebrospinal fluid pleocytosis normocellular csf in herpes simplex encephalitis covid- -associated meningoencephalitis complicated with intracranial hemorrhage: a case report we would like to thank dr raheel ahmed for his assistance in direct patient care, as well as dr maria khan, for the final review of the article. the contributors of this work include ymk for direct medical care and writing the structure of the article; ya for direct patient care, editing the article and the submission process; and aaaam for direct patient care, as well as being the most responsible physician. we would also like to express our gratitude for the infectious disease team, the psychiatry team and the nursing team in rashid hospital for helping us provide the complete medical care that the patient needs. last but not least, we are thankful to the patient for consenting to publish his case.funding the authors have not declared a specific grant for this research from any funding agency in the public, commercial or not-for-profit sectors. provenance and peer review not commissioned; externally peer reviewed.this article is made freely available for use in accordance with bmj's website terms and conditions for the duration of the covid- pandemic or until otherwise determined by bmj. you may use, download and print the article for any lawful, non-commercial purpose (including text and data mining) provided that all copyright notices and trade marks are retained. yasmin abdelmajid http:// orcid. org/ - - - key: cord- -awhnjwyc authors: hoon‐hanks, l.l.; mcgrath, s.; tyler, k.l.; owen, c.; stenglein, m.d. title: metagenomic investigation of idiopathic meningoencephalomyelitis in dogs date: - - journal: j vet intern med doi: . /jvim. sha: doc_id: cord_uid: awhnjwyc background: meningoencephalomyelitis of unknown origin (muo) is a common and life‐threatening neuroinflammatory disease in dogs. features of the disease are suggestive of an underlying immune‐mediated process, but the association of this disease with a pathogen is still unknown. hypothesis/objectives: to search for candidate etiologic agent associated with cases if muo using next generation metagenomic sequencing. animals: twenty‐two dogs diagnosed with either muo ( / ; csf and brain), or noninflammatory cns diseases inconsistent with muo ( / ; csf and brain) that served as negative controls. methods: a case control study was performed by identifying muo and non‐muo cases. samples were blindly processed and then unblinded for comparative analyses. inclusion criteria for muo cases included consistent mri lesions and inflammatory csf with a negative pcr panel for infectious agents or histopathologic diagnosis. dogs with glucocorticoid therapy within weeks of sample collection were excluded. fresh‐frozen cerebrospinal fluid (csf; ) and brain ( ) samples were collected and rna and dna were extracted separately for shotgun metagenomic sequencing. known positive samples were used as controls to validate our sequencing and analysis pipelines and to establish limits of detection. sequencing results were analyzed at a nucleotide and protein level for broad comparison to known infectious organisms. results: no candidate etiologic agents were identified in dogs with muo. conclusions and clinical importance: these results support but do not prove the hypothesis that muo is not associated with infectious agents and might be an autoimmune disease. muo is thought to account for up to % of cases of inflammatory cns disease in dogs. the prognosis of untreated muo is poor, but treatment with immunosuppressant drugs such as corticosteroids can alleviate clinical signs and delay progression of disease. this suggests that muo is an immune-mediated disease. however, a study targeting the inflammatory components of gme found a predominance of mhc class ii and cd + t cells, which might be the result of a delayed hypersensitivity reaction. therefore, whether the immune response is targeting an infection is a critical open question that this study sought to answer. currently, the etiology of muo remains unknown. studies searching for an infectious etiology have failed to reveal a consistent infectious agent. , [ ] [ ] [ ] prior studies have utilized polymerase chain reaction (pcr), serology, culture, immunohistochemistry, or a combination of these tests to investigate viruses commonly implicated in cns disease, including herpesviruses, adenoviruses, parvoviruses, canine parainfluenza virus, encephalomyocarditis virus, bunyaviruses, coronaviruses, enteroviruses, flaviviruses, paramyxoviruses, and parechoviruses. [ ] [ ] [ ] although the overwhelming majority of these studies have been negative or inconclusive, they have been limited by targeted testing for specific agents as opposed to utilizing less biased methodology to search for pathogens. this limitation has impacted our understanding of human neurologic disease as well: in large analyses of human encephalitis cases, targeted methods failed to detect an infectious agent in up to % of suspected cases. [ ] [ ] [ ] in this study, we utilized a pathogen discovery technique that bypasses many of the limitations of specific diagnostics: next-generation metagenomic sequencing. this technology has emerged in human and veterinary medicine as an invaluable tool for pathogen discovery in neurologic and other diseases of unknown etiology. [ ] [ ] [ ] [ ] in metagenomic sequencing, total nucleic acids from a clinical or environmental sample are randomly sequenced and are taxonomically categorized by comparison to known sequences in public databases. in this study, the cerebrospinal fluid (csf), brain, or both of dogs with or without muo were sampled by antemortem and postmortem techniques. in previous investigations of muo in dogs, only brain samples were tested for infectious agents; however, csf is a common sample utilized in the clinical evaluation of neurologic disease for the detection of infectious agent nucleic acids, especially by pcr. [ ] [ ] [ ] [ ] [ ] [ ] csf is more readily available as an antemortem sample and therefore more clinically relevant for the antemortem diagnosis of muo. additionally, csf has been successfully utilized in previous metagenomics sequencing studies to detect infectious organisms in encephalitis. therefore, based on sample availability and clinical relevance, this study utilized csf and brain to attempt to identify a candidate etiologic agent of muo. to be included in the diseased group for this study, dogs had to be greater than six months of age with a neurologic examination consistent with focal or multifocal neurological dysfunction. additional inclusion criteria included negative pcr tests on cerebrospinal fluid, whole blood, or both for the infectious agents caused by members of the species or genera toxoplasma gondii, neospora caninum, ehrlichia canis, ehrlichia ewingii, anaplasma, neorickettsia, bartonella, and rickettsia; the presence of multifocal t -weighted hyperintense lesions on mri; and csf pleocytosis with a nucleated cell count of greater than cells/ll with greater than % mononuclear cells and a red blood cell count of less than , cells/ll. for cases in which a necropsy was performed, histopathologic confirmation of disease was accepted in the absence of infectious disease testing, mri, and csf. because of the inflammatory nature of muo, any potential subject to whom glucocorticoids were administered within two weeks of csf or antemortem brain collection were excluded from this study; however, this criteria was not used for postmortem brain samples. three animals in the diseased group and one animal in the control group received antimicrobials within several days of sample collection, which could have altered the results of the infectious disease testing. animals in the control (non-muo) group were subject to the same age criteria as the diseased group (muo). the control cases had a low index of suspicion of inflammatory disease based on a noninflammatory csf analysis, inconsistent mri findings, histologically confirmed non-muo disease process, or a combination of these findings. samples of brain tissue were obtained using aseptic surgical technique and sharp dissection. in the diseased group, the samples were taken from the regions of the brain showing abnormalities on mri or from areas of the brain associated with the clinical signs when mri was not available (ie multiple areas of the cerebellum were sampled in dogs with cerebellar signs). in animals from the control group, samples were taken from the cerebral cortex. portions of the tissue samples were then placed aseptically into sterile containers and stored at À °c. the remainder of the sample was placed in % neutral buffered formalin and processed using standard paraffin-embedding and histologic techniques for microscopic evaluation of lm hematoxylin and eosin-stained tissue sections. brain samples collected at the time of necropsy were processed identically. csf was collected under general anesthesia via a cerebellomedullary cisternal or lumbosacral centesis using aseptic technique. for csf collection, the area was surgically prepped using % chlorhexidine gluconate scrub. a . inch or . inch or gauge spinal needle was used to collect csf in a sterile tube. the csf was stored at À °c. all animals in both the diseased (muo) and control (non-muo) groups received a physical and neurologic examination. in the muo group, of animals had a complete blood count; of animals had a serum blood chemistry; of had csf analysis; of had infectious disease testing of whole blood or csf; of had an mri; of animals were necropsied with histopathology and one of these animals also had an antemortem brain biopsy collected. of the animals in the inflammatory group, of were euthanized, of died as a result of their disease, of is currently stable, and of have been lost to follow-up (table ). in the control group, of animals had csf analysis; of animals had a cbc and serum blood chemistry; of had an mri; of were tested for infectious diseases by whole blood or csf; of animals were necropsied with histopathology, and of of these animals also had an antemortem brain biopsy collected (one necropsied). all of the animals in the control group had diagnoses inconsistent with muo (see table for listing). of the animals in this group, of were euthanized, of is currently stable, and of were lost to follow-up (table ) . total rna was extracted from fresh-frozen csf and brain samples from dogs (canis familiaris) that fit the inclusion or control criteria described above. these samples were blinded as to their case or control origin before processing. additionally, rna was extracted from postmortem brain samples from a mule deer (odocoileus hemionus), green tree python (morelia viridis), american crow (corvus brachyrhynchos), and american robin (turdus migratorius), all of which had previously been tested by pcr, metagenomic sequencing, or both, and were found to be infected with specific known infectious agents. these were used as positive controls. , rna was extracted using a combination of trizol (tissue; ambion life technologies) or trizol ls (body fluid; ambion life technologies) with rna clean and concentrator columns (cc- ; zymo research). approximately, mg of brain tissue was added to ml of trizol, and ll of body fluid (csf, serum, or blood) was added to ll of trizol ls and incubated at room temperature (rt) for minutes. tissue samples were macerated using a single sterile metal bb shaken in a tis-suelyzer (qiagen) at hz for minutes. then, ll of chloroform (sigma-aldrich) was added, shaken for seconds by hand, and incubated at rt for minutes. samples were spun at , rpm for minutes at rt. the aqueous phase was removed (approximately ll) and was added to a mixture of ll of rna-binding buffer (cc- ; zymo research) and ll of % ethanol (etoh). this was added to an rna clean and concentrator column (cc- ; zymo research). the interphase and organic phase were set aside for dna extraction (see below). the rna column was washed with ll rna wash buffer and then incubated with u dnase enzyme (neb), dnase buffer (neb), and rna wash buffer for minutes. the column was spun to remove dnase mixture and then washed with ll rna prep buffer. additional washes with and ll rna wash buffer were performed, the column was dried with a minute high-speed spin, and then rna samples were eluted in ll of rnase-free water. all csf samples had undetectable concentrations of rna by fluorometric quantification. these samples, along with a no template control, were reverse transcribed, the second dna strand synthesized, and total dna amplified using the ovation rna amplification system v (nugen) according to the manufacturer's protocol. for extracted rna of brain samples, approximately nanograms of rna was added to pmol of a random hexamer oligonucleotide ( -nnnnnn; mds- ) and incubated for minutes at °c; a separate no template control was also used for these samples. reverse transcription reaction mixture containing superscript iii fs reaction buffer (invitrogen), mm dithiothreitol (invitrogen), mm each deoxynucleoside triphosphates (dntps), and u superscript iii reverse transcriptase enzyme (invitrogen) was added to the rna-oligomer mix ( ll total reaction volume) and incubated for minutes at °c, then minutes at °c, then minutes at °c. then, u rnase h (neb) diluted in ll superscript iii fs reaction buffer and pmol mds- was added to the reaction mixtures, which were incubated at °c for minutes followed by °c for minutes. then, single-stranded cdna was converted to doublestranded dna by adding . u klenow dna polymerase ( to exo-neb) in ll superscript iii fs reaction buffer and diseased cases ( of ) represent animals diagnosed with muo based on clinical presentation and antemortem diagnostics, with or without postmortem assessment. antemortem diagnosis could not be further classified into the muo subtypes. postmortem diagnosis was made in of cases, two of which were diagnosed as either nme or gme and two of which had meningoencephalitis but lesions were not specific for any subset of muo (see discussion). control cases ( of ) are animals with noninflammatory csf and either a definitive non-muo diagnosis or additional clinical findings inconsistent with muo. for "diagnostics" and "sample used," if a fraction is not specified, then it applies to all in the group. muo, meningoencephalomyelitis of unknown origin; me, meningoencephalitis; nme, necrotizing meningoencephalitis; gme, granulomatous meningoencephalomyelitis; mm each dntps and incubated at °c for minutes. dna was purified using sera-mag speed beads at a . : bead/dna volume ratio according to the manufacturer's protocol. dna was eluted in ll molecular grade water (sigma-aldrich). the interphase and organic phase from the trizol extraction described above were used for dna extraction according to the manufacturer's protocol (invitrogen) with minor alterations. briefly, ll of % etoh per ml trizol was added to the interphase and organic phase, gently mixed, and incubated for minutes at rt. samples were centrifuged for minutes at rt, and the supernatant was removed and discarded. the dna pellet was washed twice in ml of . m sodium citrate in % etoh ph . (per ml trizol), with a minute rt incubation, minute centrifugation, and removal of the supernatant. the dna pellet was then resuspended in % etoh, gently mixed, and incubated for minutes at rt. the samples were then centrifuged for minutes, the supernatant discarded, and the pellet air-dried for minutes. the dna pellet was then resuspended in ll molecular grade water (sigma-aldrich), heated to °c for minutes, and then centrifuged for minutes at °c. the supernatant containing dna was then transferred to a . ml conical new tube and purified using sera-mag speed beads as previously described. all csf samples were amplified to generate detectable levels of dna for fluorometric quantification. this was performed using phi isothermal strand displacement amplification. five ll of template, including a no template control, was added to lm of random hexamer primer and incubated at °c for minutes and then placed directly on ice. template and primers were then added to a mixture containing phi buffer (neb), bovine serum albumin (neb), . mm each dntps, mm dithiothreitol (invitrogen), and u phi dna polymerase (neb). samples were incubated at °c for hours then °c for minutes. the dna concentration from each sample (both rna and dna derived samples) was measured fluorometrically, and ng was used as a template in . ll of tagment dna buffer and . ll tagment dna enzyme (illumina). the mixture was incubated at °c for minutes and then placed directly on ice. tagmented dna was cleaned with sera-mag speed beads as previously described and used as a template ( . ll) in the addition of fulllength adaptors with unique bar-code combinations by pcr. the ll pcr reaction contained kapa real-time library amplification master mix (kapa biosystems), . lm (each) mds- and mds- primers ( caagcagaagacggcatacg and aatgatacggcgaccaccga , respectively), and . lm each of adapter and bar-coded primers. thermocycling conditions in consecutive order were °c for minutes, °c for seconds, and cycles of °c for seconds, °c for seconds, and °c for minutes. relative concentrations of libraries were measured in quantitative pcr (qpcr) reactions containing home-made qpcr master mix ( mm tris-hcl ph . , mm kcl, . mm mgcl , . mm of each dntp, % glycerol, . % np- , . % tween- , sybr green (life technologies) and . u taq polymerase) and . lm mds- and mds- primers. equivalent amounts of dna from each sample were pooled and then cleaned using sera-mag speed beads as previously described. the pooled libraries were run on a % agarose gel and size selected ( - nucleotides) by gel extraction with a gel dna recovery kit (zymo) according to the manufacturer's protocol. size-selected pooled libraries were amplified once more in a pcr mixture containing kapa real-time library amplification mix, pmol of mds- and - each, and ll of library template in a ll total reaction volume. this pcr also included single reactions of separate fluorometric standards (kapa). thermocycler conditions were °c for seconds and cycles of °c for seconds, °c for seconds, and °c for minutes, which was when the sample curve passed standard . dna was purified using sera-mag speed beads as previously described. library quantification was performed with the illumina library quantification kit (kapa biosystems) according to the manufacturer's protocol. sequencing was performed on an illumina nextseq instrument with a nextseq / high output kit v ( cycles). sequences were trimmed using cutadapt (version . . ) to trim adaptor sequences and low-quality bases, and remove trimmed sequences that were less than nt long. quality base was set to (default) and quality cutoff was set to for the and ends. the first base of each sequence was also trimmed. the cd-hit-dup sequence clustering tool was then used to collapse reads with % global pairwise identity, leaving only unique reads remaining. host-derived sequences were then filtered using the bowtie alignment tool (version . . ). first, a bowtie index was generated from the host genomic sequence (assembly canfam . for dogs, assembly python_molurus_bivittatus- . . for the green tree python, bos_taurus_umd_ . . for mule deer, assembly asm v for american crow, and all available assemblies in the ncbi assembly database in the order passeriformes for the robin [asm v , gwvir . , gwplu . , passer_domesticus- . , taeniopygia_guttata- . . , ficalb . , geofor_ . , pse-hum . , zonotrichia_albicollis- . . , sca , asm v , asm v , asm v , hooded_crow_genome, sturnus_vulgaris- . , parus_major . . , lepidothrix_coronata- . ]) and then sequences aligning with a local mode alignment score greater than were removed. spades genome assembler (version . . ) was used to generate contiguous sequences (contigs). then, to taxonomically categorize sequences, the ncbi nt database was queried with all contigs greater than nt using the blastn alignment tool (version . . +). , any hit with an expect value less than À was assigned taxonomically according to the sequence with the highest alignment score. , additionally, to attempt to categorize contigs that were too divergent to produce a high scoring nt-nt alignment, the ncbi nr database was queried in a rap-search (version . ) with a minimum length of amino acids and an expect value of . . the same process was performed using all the reads that did not form contiguous sequences from spades genomic assembly, except gsnap alignment tool (version - - ) was used instead of blastn. raw sequence data was deposited in the ncbi short read archive database (accession srp ). we then looked for taxa that were specifically associated with cases and not controls. samples were unblinded, and datasets were identified as either muo or non-muo (nm). all taxonomic identifications (taxids) present within muo samples that were also present in nm samples were removed from further analysis. next, remaining taxids were compared between muo samples. a fraction was generated for each taxid to determine the number of muo samples that had alignments to the specific taxid over the total number of samples evaluated. if a taxid occurred in two muo samples or more, the sequences associated with the taxid were manually inspected by again querying using ncbi blastn and blastx to corroborate initial taxonomic assessment. , this was performed four times for each sample using the different sequencing outputs: spades generated contiguous sequences queried to ( ) blastn and ( ) rapsearch and individual reads queried to ( ) gsnap and ( ) rapsearch . eleven cases of muo were collected for this study (table ) . seven cases were diagnosed based on an inflammatory csf that was collected prior to immunosuppressive therapy, a negative infectious disease panel, and mri findings. four of these cases were diagnosed with meningoencephalitis by postmortem histopathology, and two of these cases were definitively diagnosed with subtypes of muo: nme and gme. the gme case included full diagnostics with an mri, imageguided brain biopsy, and postmortem histopathology showing classical histologic features of gme. diagnosis in this case was ultimately made by postmortem microscopy of necropsy brain tissue as histopathology of the brain biopsy obtained antemortem was considered "nondiagnostic." neither of these two cases received immunosuppressive therapy prior to postmortem evaluation of the brain. the other two histologically examined cases were diagnosed as meningoencephalitis (me). these cases had small numbers of macrophages, lymphocytes, and plasma cells within the meninges and around cerebral blood vessels in the gray and white matter, as well as variable regions of vacuolization within the neuropil, axonal degeneration, and encephalomalacia, consistent with me. however, these cases did not exhibit classical histologic patterns associated with any specific subtype of muo. in contrast to the histologically diagnosed gme and nme cases, both of these animals had received doses of glucocorticoids (either prednisone or dexamethasone) and chemotherapy (cytarabine) prior to postmortem evaluation; one for six months and the other for three days prior to euthanasia or death. the remaining cases (control group) were diagnosed with diseases inconsistent with muo, including neoplastic, degenerative, traumatic, and idiopathic epilepsy (table ) . these served as negative control cases. rna and dna were extracted from csf and brain samples from muo dogs and non-muo dogs as well as multiple positive controls samples. nucleic acids were then converted into sequencing libraries and sequenced on an illumina nextseq instrument. the datasets contained on average . , -nucleotide sequences per sample. a stepwise data analysis pipeline was used to remove adaptor sequences and low-quality reads, collapse sequences to unique reads, and filter out dog-derived sequences. approximately, % of sequences remained in each sample after filtering (table s ). remaining sequences were assembled into longer contiguous sequences (contigs), which were queried against databases of nucleotide and protein sequences to identify possible pathogen-derived sequences. sequences from no single organism were found in more than muo samples (of ), and organisms were inconsistent between dna and rna from the same tissue as well as brain and csf collected from the same animal. a majority of sequences lacked specificity to any single organism based on nucleotide and protein sequence analysis. this was because of either poor quality of the read, or sequences that were low complexity or highly conserved, and thus taxonomically ambiguous. this was the case for all eukaryotic organisms detected. a number of bacterial-aligning reads were also detected; however, because of the range of bacterial species and the inconsistency of any given organism among samples, these were deemed environmental contaminants. the most common bacteria detected were pseudomonas, streptococcus, and staphylococcus species. a low number of viral species were detected, but all that were present solely within muo samples were bacteriophages, and therefore unlikely to be associated with disease. overall, a consistent and specific candidate etiological candidate was not detected. we sequenced and analyzed in parallel a number of known positive samples to validate our approach and to establish limits of detection. these included ( ) brain from a captive green tree python positive for python nidovirus ; ( ) brain from a wild mule deer positive for caprine herpesvirus ; ( ) brain from a wild-caught american robin experimentally infected with west nile virus (wnv) ; and ( ) brain from a wild-caught american crow experimentally infected with wnv. these samples had previously tested positive by metagenomic (green tree python and mule deer) or targeted next-generation sequencing (crow and robin). we used an identical analysis pipeline for positive control samples, except we used different, appropriate genome assemblies for filtering host sequences. as expected, we detected python nidovirus, caprine herpesvirus , and wnv in the green tree python, mule deer, and crow, respectively, using our metagenomic sequencing approach (table s ) . we did not detect wnv in the experimentally infected robin brain, but confirmed that the sample was positive for wnv rna by qrt-pcr. we quantified the wnv copy number in the bird brain samples at genome copies/ll rna in the robin and . genome copies/ll rna in the crow. muo is an idiopathic inflammatory neurologic disease, including gme and the necrotizing encephalitides (nme and nle). the pathogenic mechanisms underlying muo remain unknown. similar to previous targeted diagnostic studies, , [ ] [ ] [ ] [ ] our study using a less biased approach failed to detect any infectious agents that were consistently associated with canine muo cases. there are several possible biological and technical explanations for our study's inability to identify a candidate etiologic agent for muo, including the underlying pathogenesis of the disease, sample type and collection methods, case inclusion criteria, sensitivity of diagnostics, and database limitations. first, it is possible that the inflammation observed in muo does not have an infectious etiology. second, it might be that muo has an infectious cause, but that we are sampling at a point in the natural history of the disease when the initiating pathogen is no longer present in detectable amounts. this possibility could be investigated by the development of a comprehensive serological panel of known canine pathogens that would enable retrospective sampling of dogs with and without muo. third, cns lesions could be secondary to a primary infection elsewhere in the body, resulting in a systemic response that manifests as meningoencephalitis. or the lesions could be a disproportional response to a very low-level cns infection. the evaluation of multiple tissue types in dogs diagnosed with muo, beyond cns samples, could help assess this possibility. fourth, it might be that we sampled the wrong regions of the cns. muo, like many other neurologic diseases, can be focally or multifocally distributed. this limitation is likely to apply more to biopsy/postmortem samples than to pathogen detection in csf. however, low or inconsistent shedding of organisms into the csf could reduce the likelihood of detection. future studies could benefit from more consistent use of antemortem image-guided biopsies (only of of our muo cases) and sampling of multiple sections of the cns postmortem (only of muo cases), as well as multiple time-separated csf sample collections. furthermore, although four of the diseased cases were histologically confirmed as having inflammatory brain disease, seven cases were presumptively diagnosed with muo. strict inclusion criteria were used for antemortem diagnosis in this study. however, the lack of histopathology does not definitively rule out other disease processes, such as lymphoma. therefore, it is possible that not all of the presumptively diagnosed muo cases were gme, nme or nle. additionally, only two of the four cases evaluated by histopathology yielded a definitive diagnosis of gme or nme, whereas the other two were diagnosed as meningoencephalitis of undetermined subtype. the use of a greater number of cases with histologic confirmation could have strengthened the diagnostic certainty of each case and allowed for a more specific investigation of muo based on histologic type. there are also several possible technical reasons that could have prevented us from identifying an infectious agent underlying muo. first, it might be that we lacked the necessary sensitivity. although metagenomic sequencing can detect any pathogen, it is generally less sensitive than targeted methods such as pcr. the sensitivity of pcr is typically defined in absolute units (eg genome copies in a quantitative pcr reaction), but the sensitivity of metagenomic sequencing is limited by read depth and the relative pathogen concentration. for example, if a metagenomic dataset contains million unique sequences and if a pathogen's nucleic acid is present at a concentration lower than part per million host nucleic acid molecules, then it is unlikely to be detected. the development and use of methods to deplete mammalian nucleic acids could have improved the sensitivity of our study by eliminating dog sequences and enriching for microorganismal nucleic acids. our analysis of bird brain samples with high and low wnv copy numbers illustrates this sensitivity threshold. we detected wnv by sequencing in the crow brain, which had . viral rna copies per microliter of rna but did not detect wnv in the robin brain, which had . genome copies per microliter of rna. it can, therefore, be deduced that our limit of detection lies somewhere between these values. this range is large, and the use of wnv-positive samples with intermediate copy numbers could have allowed us to narrow this empirically determined limit of detection. additionally, csf has inherently low nucleic acid content because of the low number of nucleated cells present when compared to tissue. therefore, dna and rna extraction generally have a low yield and further amplification is required for library preparation in these samples. amplification can introduce base-composition bias and increases the number of nonunique reads, contributing to reduced sequencing quality and read depth. finally, it is also possible that the cause of muo is an infectious agent so divergent from known pathogens that its sequence was unrecognizable. this is not likely, however. eukaryotic and bacterial pathogens typically have characteristic conserved sequences that are easily recognizable (eg ribosomal rna sequences), and viruses can typically be recognized by viral polymerase sequences, especially when compared at the protein level, as we did. in summary, we applied the best available molecular methods to continue the search for an muo etiology, and did not find a candidate agent. there are several technical and biological reasons that could have prevented us from doing so. however, the thoroughness of our approach, our inclusion of internal positive controls, similar negative results from previous studies, and the clinical responsiveness to immunosuppressant therapy all provide support for the hypothesis that muo is a primary autoimmune disease. perspectives on meningoencephalomyelitis of unknown origin idiopathic granulomatous and necrotising inflammatory disorders of the canine central nervous system: a review and future perspectives a necrotizing meningoencephalitis of pug dogs necrotizing encephalitis in yorkshire terriers necrotizing meningoencephalitis of maltese dogs necrotising encephalitis in a french bulldog necrotizing meningoencephalitis associated with cortical hippocampal hamartia in a pekingese dog canine necrotizing encephalitis associated with anti-glomerular basement membrane glomerulonephritis diagnosis of inflammatory and infectious diseases of the central nervous system in dogs: a retrospective study immunohistochemical characterization of inflammatory cells in brains of dogs with granulomatous meningoencephalitis polymerase chain reaction screening for dna viruses in paraffin-embedded brains from dogs with necrotizing meningoencephalitis, necrotizing leukoencephalitis, and granulomatous meningoencephalitis broadly reactive polymerase chain reaction for pathogen detection in canine granulomatous meningoencephalomyelitis and necrotizing meningoencephalitis non-suppurative meningoencephalitis of unknown origin in cats and dogs: an immunohistochemical study viral pathogen discovery isolation and molecular identification of sunshine virus, a novel paramyxovirus found in australian snakes experimental evolution of an rna virus in wild birds: evidence for hostdependent impacts on population structure and competitive fitness cutadapt removes adapter sequences from highthroughput sequencing reads cd-hit: a fast program for clustering and comparing large sets of protein or nucleotide sequences basic local alignment search tool rapsearch : a fast and memoryefficient protein similarity search tool for next-generation sequencing data fast and snp-tolerant detection of complex variants and splicing in short reads xenosurveillance: a novel mosquito-based approach for examining the human-pathogen landscape the authors acknowledge the colorado state university center for companion animal studies for its work testing and processing the samples collected for this study, justin lee and the csu ngs facility for assistance with sequencing, and the colorado state university college of veterinary medicine and biomedical sciences college research council for funding.conflict of interest declaration: authors declare no conflict of interest.off-label antimicrobial declaration: authors declare no off-label use of antimicrobials. additional supporting information may be found online in the supporting information tab for this article: table s . average reads per sample and sequencing analysis summary in dog datasets. table s . positive control sequencing summary. key: cord- - fq wxz authors: kent, marc title: the cat with neurological manifestations of systemic disease. key conditions impacting on the cns date: - - journal: journal of feline medicine & surgery doi: . /j.jfms. . . sha: doc_id: cord_uid: fq wxz practical relevance a number of systemic diseases are associated with neurological deficits. most systemic diseases that impact on the nervous system result in multifocal neurological signs; however, isolated deficits can also be observed. this article reviews the clinical signs, pathophysiology, diagnosis, treatment and prognosis of four important systemic diseases with neurological consequences: feline infectious peritonitis, toxoplasmosis, hypertension and hepatic encephalopathy. clinical challenges early recognition of systemic signs of illness in conjunction with neurological deficits will allow for prompt diagnosis and treatment. while neurological examination of the feline patient can undoubtedly be challenging, hopefully the accompanying articles in this special issue will enable the clinician to approach these cases with more confidence. evidence base the veterinary literature contains numerous reports detailing the impact of systemic disease on the nervous system. unfortunately, very few references provide detailed descriptions of large cohorts of affected cats. this review summarises the literature underpinning the four key diseases under discussion. knowledge of the epidemiology of fcov infection is important in order to understand the pathogenesis of fipv. feline coronavirus infection is virtually endemic, with studies revealing that: ✜ approximately % of cats in the united states and europe have antibodies against fcov; ✜ in australia, the seroprevalence in owned cats is approximately %; ✜ in the uk, % of show cats, % of breeding cats and % of single-cat homes have anti-fcov antibodies; ✜ in italy, the seroprevalence is % in cats from breeding colonies, shelters and homes; ✜ in switzerland, similarly % of breeding cats, and % of free-roaming cats, test positive for anti-fcov antibodies. the significance of this worldwide distribution relates to the fact that fipv is the result of spontaneous mutation of fecv, which means that cats worldwide are susceptible to developing fip. despite this, approximately % of cats in multicat homes and a smaller percentage of cats in single-cat homes develop fip. , , notably, it is young cats and immunosuppressed cats that are most susceptible to developing fip. in addition, certain purebreed cats, specifically the birman, ragdoll, bengal, rex, abyssinian and himalayan breeds, have a greater risk of developing fip, which suggests a genetic influence on susceptibility. transmission of fcov is through infected fecal material via the orofecal route, leading to enteric infection. enteric fcov infection typically results in inappetence and/or mild gastrointestinal signs such as vomiting and diarrhea. infected cats shed fecv for up to months post infection; thereafter infected cats shed virus intermittently or continuously, serving as chronic carriers and thereby perpetuating reinfection of other individuals. as with all coronaviruses, fecv undergoes a high rate of mutation. the degree of mutation, and therefore the development of a mutation leading to fip, appear greater in susceptible individuals as well as in individuals with a high viral load. , in part, this may explain the fact that more than % of cats with fip are under a year of age. the initial step in the pathogenesis of fip is the mutation of fcov, in the process of which the virus gains the ability to replicate within macrophages. once this occurs, the virus can be disseminated throughout the body. ultimately, fip is an immune complex disease that is a consequence of virus or viral antigens complexed with antiviral antibodies. after distribution by macrophages, the virus may enter tissue and replicate, resulting in attrac-consisting primarily of lymphocytes, macrophages, and varying numbers of plasma cells forming perivascular cuffs. subependymal periventricular inflammatory infiltrate may obstruct the mesencephalic aqueduct leading to obstructive hydrocephalus. similarly, obstruction of the central canal of the spinal cord may lead to hydromyelia. occasionally, infiltrate extends into the superficial neuropil and cranial nerve roots. hematology in affected cats usually reveals a normocytic, normochromic, nonregenerative anemia, leukocytosis consisting of a neutrophilia, and lymphopenia. approximately % of cats with the effusive form and % of cats with the dry form have increased serum proteins, primarily comprising a hyperglobulinemia. protein electrophoresis discloses a polyclonal gammopathy, mainly involving the γ-globulins. other biochemical changes may be observed depending on the severity of involvement of other organ systems including abnormal liver enzyme, bilirubin, blood urea nitrogen and creatinine levels. although common clinicopathologic abnormalities in affected cats have been defined, changes in routine hematology and biochemical evaluations are often non-specific. consequently, establishing a definitive ante mortem diagnosis of fip is extremely challenging. definitive diagnosis can only be achieved through histopathological identification of pyogranulomatous inflammation within tissue coupled with identification of the virus. viral identification in tissue samples can be performed using immunohistochemistry or through pcr testing. however, there are several tests that may help support a presumptive ante mortem diagnosis. importantly, interpretation of results from such tests should be evaluated in conjunction with clinical signs and results of other diagnostics. taken outside the context of signs and other clinicopathologic data, most tests are unable to provide a definitive diagnosis. when present, effusions should be analyzed. typically, effusion from an affected cat should be consistent with a modified transudate. cats with neurological signs should undergo magnetic resonance imaging (mri) of the brain. this may disclose hydrocephalus. additionally, t -weighted and t weighted flair images may reveal periventricular hyperintensities consistent with periventriculitis. although these findings are not pathognomonic, mri of the brain should be pursued in order to eliminate the potential that clinical signs may be a consequence of a disease process other than fipv. analysis of cerebrospinal fluid (csf) often reveals increased protein content ( - mg/dl) with a pleocytosis consisting of neutrophils, lymphocytes and macrophages. , , the evaluation of fcov antibody titers (often erroneously referred to as an 'fip titer') in blood and other fluids has been extensively studied. however, despite years of investigation, caution should be exercised when interpreting fcov antibody titers in blood and effusions as high titers can be observed in healthy cats and low to absent titers in affected cats. ✜ fipv is fatal and treatment is mainly palliative. since fip is an immune-mediated disease process, therapy has been directed at immunosuppression and/or immunomodulation with the goal of providing symptomatic care. immunosuppressive therapy using corticosteroids (prednisone at - mg/kg/day) may allow mildly affected cats to maintain an acceptable quality of life for weeks to months. in addition to corticosteroids, a wide array of drugs including chemotherapy agents (cyclophosphamide and melphalan), an antiviral (ribavrin), a thromboxane synthetase inihibitor (ozagrel hydrochloride) and a variety of immunomodulating drugs (promodulin, human interferon-α, propionibacterium acnes, and feline interferon-ω) have been investigated. the interpretation of results from most studies has been hindered by a lack of control groups and the difficulty in establishing a definitive diagnosis of fip in treated cats. still, most therapeutic trials have failed and, disappointingly, an effective treatment regime remains elusive. presence of neurological fipv. cerebrospinal fluid fcov antibody titers correlate with serum fcov antibody titers but, most importantly, elevated csf fcov antibody titers may also be observed in cats affected by neurological diseases other than fip. while pcr assays can be performed on blood and effusions in affected cats, they are unable to distinguish between the mutated fcov causing fip, and the non-pathogenic fcov. in addition, healthy cats can be viremic with fcov. therefore, pcr identification of virus in blood or effusions does not provide a definitive diagnosis. while its application in csf has not been studied, pcr identification of virus in csf may allow a definitive diagnosis of fip. measurement of serum α -acid glypoprotein (agp), an acute phase protein that increases during inflammation, has been used in the diagnosis of fip. , in cats with signs and clinicopathologic data highly suggestive of fip, elevation in serum agp provides strong supportive evidence of fipv infection. however, agp may also increase in other conditions associated with inflammation, such as feline immunodeficiency virus (fiv) infection, or in cats with a high viral load of fecv, which may limit its potential as a diagnostic tool for fip. , prognosis unfortunately, the prognosis for cats with fip is grave as all affected cats succumb to the disease. to date, no therapy has been shown to alter the eventuality of humane euthanasia or death of affected cats. toxoplasmosis is caused by an obligate intracellular protozoan parasite, toxoplasma gondii. the definitive hosts are the domestic cat and other felidae. many mammals can become infected with t gondii and serve as intermediate hosts; however, fecal shedding of infective oocysts occurs only in cats. systemic and ocular toxoplasmosis have been well described in cats. the emphasis in the following discussion is on central nervous system (cns) toxoplasmosis. there are three methods of transmission of t gondii: fecal-oral, ingestion of tissue cysts, and congenital. reproduction of the organism can involve both a sexual and asexual phase. the asexual phase occurs in many mammals and birds, which serve as intermediate hosts. as the definitive host, the sexual phase of the life cycle can only occur in cats and it does so within the intestinal tract. as a result, unsporulated oocysts, which are non-infectious, are passed in the feces. these oocysts require - days for sporulation to occur, at which point they become infectious. ingestion of sporulated oocysts by another cat begins another cycle. toxoplasma gondii also displays an extraintestinal life cycle. after infectious oocysts have been ingested, the organism is capable of penetrating the wall of the intestinal tract and disseminating to multiple organs. within these other organs, asexual reproduction occurs, giving rise to tissue cysts -bradyzoites (so named given their slow replication) and tachyzoites (in which replication is rapid). ingestion of bradyzoites in tissue is probably responsible for the majority of infections. in cats it can result in intestinal replication, while ingestion of bradyzoites by other animals can only lead to extraintestinal infection. ingestion of infectious organism during gestation can also lead to congenital infection. cysts that form as a result of extraintestinal infection are likely to persist for life. , encysted bradyzoites are the most probable source of continual release of antigen and reactivation of infection. reactivation of infection is thought to occur secondarily to immunosuppression. cats infected with fiv appear to be predisposed to the development of acute toxoplasmosis. there appears to be a high seroprevalence of t gondii infection in cats co-infected with fiv. , the significance of this relationship is unknown as the seroprevalence of t gondii infection in fiv-infected cats is similar to that in the general population. , immunosuppression as a result of ciclosporin therapy has also led to acute toxoplasmosis. , with the availability of renal transplantation in cats, the role of immunosuppression in reactivation of infection and the development of clinical disease has gained importance. [ ] [ ] [ ] clinical signs clinical infection with t gondii is not common in cats. infections can be considered acute or chronic. acute toxoplasmosis typically affects younger cats. the most common clinical signs are anorexia, lethargy, fever, dyspnea and sudden death. [ ] [ ] [ ] [ ] chronic toxoplasmosis typically affects older cats and manifests over weeks to months. signs are similar to acute infection and may include vomiting, diarrhea, anorexia, weight loss, fever and icterus. , [ ] [ ] [ ] with the exception of finding t gondii in tissue, no single test provides a definitive diagnosis. serology for the detection of igg and igm anti-t gondii antibodies is widely used. after experimental inoculation, an igm response is detected in - weeks and an igg response in - weeks. immunoglobulin m responses peak within weeks and persist for - weeks. in cats co-infected with fiv, there is a delayed conversion from an igm to an igg response. unfortunately, an igm response does not necessarily correlate with active disease as occasionally igm responses can be detected in clinically normal cats with chronic infection. likewise, a single high igg response does not predict active disease, as igg responses can last up to years. a rising titer is strongly suggestive of active disease, however, and maximal titers are reached within - weeks. in practice, given the insidious nature of the disease many cats have reached maximal immune responses by the time they are examined by a veterinarian, making documentation of a rising titer difficult. theoretically, identification of an immune response in the cns, an immunoprivileged site, would suggest infection. however, immunoglobulins may extravasate from the blood into the csf in other inflammatory diseases that disrupt the blood-brain barrier. defining a ratio between serum and csf igg responses may help eliminate the possibility of passive cross over of antibodies secondary to another disease that compromises the integrity of the blood-brain barrier. a serum:csf igg response > suggests local cns production of immunoglobulin. in experimental oral inoculation, cats remain clinically normal yet develop a detectable igg response in the csf - weeks post inoculation and again - weeks after secondary exposure. importantly, an igg response in the csf can occur after exposure to killed tachyzoites in previously infected cats. therefore, observation of an igg response in csf does not necessarily document infection. experimental inoculation does not result in an igm response in the csf. potentially, therefore, detection of an igm response in the csf may be indicative of active disease, but this is yet to be confirmed. clinical signs reflecting organ involvement include lymphadenopathy, myocardial disease, pancreatitis, hepatitis, anterior uveitis and chorioretinitis. , , [ ] [ ] [ ] , the diagnosis of clinical toxoplasmosis can be challenging. hematologic findings are nonspecific, often consisting of non-regenerative anemia, neutrophilic leukocytosis, lymphocytosis and monocytosis. , , biochemical abnormalities generally reflect organ involvement and include azotemia, elevation in liver enzymes, hyperbilirubinemia and hyperproteinemia. , , thoracic radiographs may show a diffuse interstitial to bronchial pattern in which infiltrate may coalesce into areas of patchy alveolar patterns (fig ) . [ ] [ ] [ ] neurological signs ✜ central nervous system involvement occurs in almost all clinically affected cats. neurological signs typically reflect a multifocal distribution and include hypothermia, behavioral changes, seizures, ataxia, blindness, anisocoria, torticollis, vestibular disease, muscle hyperesthesia, and paresis/paralysis. cerebrospinal fluid analysis typically reveals a mild lymphocytic pleocytosis predominantly, although other cell types may be observed; protein may be elevated to up to mg/dl. neutrophilic pleocytosis has also been reported. with the exception of identifying t gondii in tissue, no single test provides a definitive diagnosis. a presumptive diagnosis is based on a combination of clinical signs, evidence of recent or active infection (gained via serology for immunoglobulins or immune complexes, or pcr), exclusion of other disease processes, and response to therapy. hypertension has been defined as a sustained increase in systolic blood pressure ≥ - mmhg. although the prevalence of hypertension in cats has not been accurately established, one study documented hypertension in % of healthy cats. in cats referred for evaluation of disease associated with hypertension, or animals with clinical signs compatible with hypertension, a % prevalence was found. hypertension can be divided into three categories: white coat, secondary and idiopathic. ✜ white coat hypertension is an artefactual increase in blood pressure that develops secondarily to excitement or anxiety, and is likely to be the result of activation of the sympathetic nervous system. it is observed in cats, and results in a median increase in systolic blood pressure of . mmhg ± . mmhg. although pcr assays have not been performed on csf for the detection of t gondii, pcr assays have been utilized in the aqueous humor, another immunoprivileged site. , toxoplasma gondii can be identified in the aqueous humor of cats with uveitis using pcr; however, the organism can also be detected in the aqueous humor of clinically normal cats that have naturally been exposed to t gondii. consequently, pcr detection of t gondii in aqueous humor does not provide definitive proof of active disease. a similar interpretation of pcr analysis of csf is likely. unfortunately, the prognosis is poor for cats displaying neurological signs or severe respiratory disease as most will succumb to the disease. , [ ] [ ] [ ] , despite this, cats with focal cns toxoplasmosis may achieve long term remission. since the initial description of systemic hypertension in cats, the impact of hypertension systemically and on the nervous system has become increasingly recognized. a testament to this is a recent consensus statement from the american college of veterinary internal medicine that has established guidelines for identification, evaluation and management of hypertension in dogs and cats. in healthy cats, normal systemic blood pressure, which is often reported as a systolic measurement, is - mmhg. [ ] [ ] [ ] the wide range in the reported normal values is likely to reflect a lack of standardization in technique and equipment used to measure blood pressure. many factors affect blood pressure measurement including recording device, cuff size and operator skill, as well as patient factors such as size and demeanor of the cat. although increasing age was found to be associated with increased blood pressure in one study, other reports have found no effect of age on blood pressure. [ ] [ ] [ ] the treatment of choice for cats with clinical toxoplasmosis is clindamycin hydrochloride at - mg/kg divided per day. , clindamycin is almost completely absorbed after oral administration and achieves high concentrations in most tissues, including the lung. concentrations in csf are low; , however, the concentration in the brain may be higher given the lipophilic nature of the drug. clindamycin is well tolerated, with only minimal side effects (eg, vomiting and diarrhea) reported at dosages two and a half to four times the recommended dosage. parenteral formulations can be used in animals unable to receive oral medication or those experiencing gastrointestinal toxicity. reports of successful treatment are rare, which may reflect the difficulty of establishing a definitive diagnosis. systemic clinical signs typically show improvement within - h of initiation of treatment. cats with systemic or ocular disease treated with antibiotic therapy may achieve clinical remission; however, recurrence of signs is likely as antibiotic therapy is unlikely to eliminate the organism entirely. chronic systemic hypertension has a variety of pathological consequences that collectively are referred to as end-organ or target organ damage. important target organ damage is observed in the kidneys, eyes, heart and nervous system. in the kidney, this leads to an accelerated decline in renal function, proteinuria and death. hypertension can exist in animals at any stage of renal disease, and may be seen in nonazotemic animals. in the eye, hypertension leads to hypertensive retinopathy and choroidopathy (fig ) . exudative retinal detachment, retinal hemorrhage, multifocal retinal edema and tortuosity of the retinal vessels may be observed, and commonly result in blindness. , , , , [ ] [ ] [ ] in the heart, hypertension may result in cardiomegaly and left ventricular hypertrophy. , , , a systolic murmur, gallop rhythm and congestive heart failure may be observed. , in the nervous system, hypertension may result in a hypertensive encephalopathy. , , , , two studies have variously documented neurological signs in % and % of cats with hypertension. , clinical signs since hypertension in most cats can be cat egorized as secondary, clinical signs typically reflect the underlying disease process. consequently, affected cats often demonstrate signs relating to renal disease or hyperthyroidism, given the high prevalence of hypertension with these disorders. although the pathophysiology underlying hypertensive encephalopathy remains unclear, it is thought to involve the development of vasogenic edema, which predominantly affects the white matter. , with acute hypertension, the autoregulatory capacity of the brain vasculature may be exceeded, leading to hyperperfusion, breakdown of the blood-brain barrier, and cerebral edema. , in experimental acute hypertension in cats, gross findings include coning of the vermis of the cerebellum, cerebel-lar herniation into the foramen magnum (fig ) , rostral displacement of the colliculi, and widening and flattening of the cerebral gyri, all of which reflect raised intracranial pressure. microscopically, the consequences of edema are observed such as marked pallor of the cerebral white matter, accentuation of the separation between axons and myelin sheaths, and widening of the perivascular space. in chronic hypertension, brain vasculature may be chronically vasoconstricted leading to hypertrophy and hyperplasia of the smooth muscle. as a result, fibrous changes develop, allowing leakage of plasma which ultimately causes degeneration of the vasculature predisposing to ruptures and microhemorrhages. multifocal cerebral arteriosclerosis with hemorrhages has been observed in cats with spontaneous hypertension. a presumptive diagnosis of hypertensive encephalopathy is relatively straightforward and requires the documentation of hypertension (systolic blood pressure ≥ - mmhg) with contemporaneous neurological signs. the gold standard for blood pressure measurement is invasive intra-arterial monitoring. however, this is often not feasible in clinical practice. consequently, indirect blood pressure monitoring is used most commonly. accurate and reliable indirect blood pressure measurements can be performed using doppler flow ultrasonography and oscillometry. minimal, mild, moderate and severe risk categories for target organ damage have been defined based on blood pressure recordings (see below). identification of hypertension should prompt investigation for an underlying disease process. a complete blood count, biochemistry profile and urinalysis should be performed in all hypertensive cats. in cats older than years of age, serum thyroxine level should also be measured. when indicated, endocrinological testing for cushing's disease or diabetes mellitus should be performed. in cats with suspected or confirmed renal disease, quantification of a proteinuria should be performed. thoracic radiographs should be obtained to assess cardiovascular structures, and abdominal ultrasonography should be performed to assess renal structure and identify any concurrent disease. echocardi ography is warranted in cats with a murmur, gallop rhythm or other signs consistent with cardiac disease. in all cats with hypertension, echocardiography allows assessment of any secondary cardiac changes. in cats with severe neurological dysfunction mri may be warranted. in addition to assessing cns pathology related to hypertension, mri allows exclusion of other disease processes that can produce similar neurological signs. given the potential for raised intracranial pressure and brain herniation in hypertensive encephalopathy, caution should be exercised prior to advanced imaging; the requirement for general anesthesia can lead to deterioration or death in animals with severe raised intracranial pressure. in humans with hypertensive encephalopathy, mri of the jfms clinical practice brain discloses hyperintensities in the white matter of the parietal and occipital lobes of the cerebrum on t -weighted images. less frequently, similar findings may be observed in the brainstem. magnetic resonance imaging in hypertensive cats has not been studied; however, given the gross and microscopic changes observed in affected cats, similar findings would be expected. unfortunately, control of hypertension does not appear to have a significant effect on survival time. , , , however, amlodipine does seem to reduce the degree of proteinuria in cats with renal disease, and a reduction in proteinuria appears to have a positive effect on survival time. , unless animals are showing evidence of target organ damage, or are at severe risk of developing target organ damage (see box on page ), there is no requirement for immediate therapeutic intervention. instead, repeated blood measurements over a period of time, combined with identification and treatment of any potential underlying disease process leading to hypertension, may be all that is needed to control blood pressure. in cats that remain hypertensive despite control of an underlying disease process, or those with idiopathic hypertension in the mild to moderate risk category for target organ damage, the decision to pursue hypertensive therapy requires a dedicated owner as treatment is generally lifelong and involves frequent re-evaluations. in animals displaying signs consistent with hypertensive encephalopathy, prompt intervention should be pursued. the treatment of choice for hypertension in cats is amlodipine besylate, a calcium channel blocker. , , - a dose of . - . mg/cat orally once to twice daily reliably reduces blood pressure with minimal risk of causing hypotension. , - furthermore, treatment with amlodipine is not associated with increases in blood urea nitrogen and creatinine in cats with chronic renal failure. , in cats in which amlodipine is ineffective at controlling hypertension, adjunctive therapy with the β selective β-blocker, atenolol, may be instituted at . - . mg/cat po q - h. alternatively, the angiotensin-converting enzyme inhibitor, benazepril, at . - . mg/kg po q - h can be used. however, benazepril therapy is associated with only a small but significant reduction in blood pressure in cats with chronic renal failure. in acute hypertension, subcutaneous hydralazine ( . - . mg/cat) has been effective at reducing blood pressure without significant risk of hypotension. while parenteral hypotensive medications may be preferable in the setting of severe hypertensive encephalopathy, the use of such medications requires continuous, direct arterial blood pressure measurement and is associated with a significant risk of hypotension. in cats with severe neurological dysfunction that do not respond to a reduction in blood pressure, treatment for raised intracranial pressure due to brain edema may be warranted. this entails diuretic therapy with mannitol ( . to g/kg iv over - mins often combined with furosemide . mg/kg iv), or other hypertonic agents. note, however, that diuretic therapy should not be used until blood pressure has normalized, as these agents can transiently increase blood pressure. tre a t m e n t o f h y p e r t e n s i o n hepatic encephalopathy is the clinical syndrome of abnormal neurological function caused by portosystemic shunting, with or without intrinsic liver disease. as a result, he can develop in cats with acquired or congenital liver disorders. by far the most common cause of he in cats is portosystemic shunting of blood secondary to a congenital vascular anomaly. regardless of the cause of the underlying liver disease, the clinical signs of he are similar and can be divided into systemic and neurological signs. affected cats often display intermittent clinical signs that may be associated with eating. cats with portosystemic shunts are generally small in stature, fail to thrive and grow, and lose weight. [ ] [ ] [ ] [ ] pytalism is a common clinical sign, occurring in approximately % of cats. , [ ] [ ] [ ] [ ] [ ] other, less common clinical signs include gastrointestinal signs such as decreased appetite, anorexia, pica, vomiting, diarrhea or constipation. , , cats may demonstrate polydipsia, and polyuria, pollakiuria and stranguria may occur as a consequence of cystic calculi. , , affected cats may have copper-coloured irises. although a complete understanding of the mechanisms underlying he remains elusive, it is clear that the pathophysiology involved is multifactorial. despite numerous potential factors, ammonia remains key in the development of he. ammonia is produced by bacteria in the gastrointestinal tract, primarily the colon, as a consequence of protein metabolism. it is also produced by the gastrointestinal cells as a result of metabolism of glutamine, the main cellular energy source for the epithelium. a further source of ammonia is the kidneys, during states of hypokalemia or alkalosis. normally, the liver efficiently removes ammonia from the portal vasculature, ultimately converting it to urea. in animals with hepatic failure or portosystemic shunts, hyperammonemia may develop. however, the severity of the neurological signs does not always correlate with the degree of hyperammonemia. in fact, blood ammonia levels may be normal in cats with he. this relates to a greater rate of uptake of ammonia in the cns in he, leading potentially to a high cns ammonia level in the face of a normal blood ammonia level. although he is a syndrome of neuronal dysfunction, the neuropathological consequences of increased cns ammonia are played out in the astrocyte. normally, cns ammonia undergoes energy-dependent metabolism to glutamine by the astrocytes. with increased cns ammonia, astrocytes become overwhelmed, leading to energy depletion. additionally, the increased concentration of glutamine in astrocytes may act as an osmotic stress, leading to cell swelling. increased numbers of swollen astrocytes -referred to as alzeheimer type ii astrocytes -is the only structural change observed microscopically in the brain in he. as a consequence of cellular edema, neurotransmitter processing in the astrocytes is affected, resulting in upregulation of neuronal benzodiazepine receptors and the production of neurosteroids which increase γ-aminobutyric acid (gaba) neurotransmission and thereby ultimately affect neuronal function. ammonia may also have a direct toxic effect on neurons. while ammonia remains a focal point in the pathogenesis of he, other factors are also involved. mercaptans are formed during the degradation of sulfur-containing amino acids. these substances exert a neurotoxic effect through inhibition of atpase activity, thereby potentiating the effect of ammonia. short and medium chain fatty acids are derived from bacterial metabolism of carbohydrates or from incomplete β-oxidation of long chain fatty acids in the liver. like mercaptans, these molecules may inhibit energy metabolism as well as inhibiting urea cycle enzymes in the liver. a putative role for mercaptans, short and long chain fatty acids is unknown, but they may act synergistically with ammonia. hepatic encephalopathy may develop through an imbalance of inhibitory (gaba) and excitatory (glutamate) neuro transmitters. there is evidence to implicate exces- sive gabaergic tone in he, which would result in global inhibition of neurological function. in humans with he, there are increased gaba concentrations in the cns leading to excessive gabaergic tone. in addition, there may also be increased concentrations of endogenous benzodiazepines in the cns. , benzodiazepines also bind to the gaba receptor, potentiating the effect of gaba. false neurotransmitters may also play a role in he. in liver disease, the production of branched chain amino acids is reduced. branched and aromatic amino acids compete for the same transporter into the cns. as a consequence of reduced branched chain amino acids, there may be a relative increase in the aromatic amino acids; these so-called false neurotransmitters may act like gaba and other inhibitory neurotransmitters. ultimately, the clinical signs of he may be a result of global inhibition of neurotransmission. a complete blood count, biochemistry and urinalysis should be performed in all animals with clinical signs suggestive of he. on hema tol ogy, microcytosis may be present. biochemical abnormalities may include low blood urea nitrogen, increased liver enzymes, decreased total protein and albumin concentrations, and hypocholesterolemia. urinalysis may disclose hyposthenuria and ammonium urate crystals. a presumptive diagnosis can be made by documenting altered liver function in the setting of clinical signs consistent with he. an elevated fasting blood ammonia level helps confirm the clinical suspicion. in order to provide accurate results, blood samples should be treatment should be directed at the underlying cause of he as well as controlling the clinical signs of he. one of the primary aims is to reduce blood ammonia levels. for animals with mild to moderately severe clinical signs, which are capable of taking oral medications, lactulose should be administered at a starting dose of ml po q - h. the dosage is adjusted based on stool consistency and response. in more severely affected cats, lactulose can also be administered per rectum. prior to rectal administration, warm water enemas should be performed to remove fecal material. lactulose is a non-aborbable disaccharide that undergoes extensive metabolism by colonic bacteria, first to constituent monosaccharides and then to volatile fatty acids. ultimately, lactulose decreases the production/absorption of ammonia. this is accomplished in several ways: namely, by decreasing the colonic luminal ph, leading to conversion of ammonia (nh ) to ammonium (nh + ), trapping it intraluminally; decreasing transit time through the osmotic cathartic effect of lactulose; and interfering with intestinal absorption of glutamine, thereby decreasing the production of ammonia. antibiotic therapy is often combined with lactulose administration. antibiotics with activity against ureaseproducing bacteria are effective at reducing ammonia production. neomycin is an oral aminoglycoside antibiotic that undergoes minimal systemic absorption. it is administered at mg/kg po q h. despite the limited systemic absorption, systemic concentrations capable of causing side effects are possible. metronidazole is also effective at reducing urease-producing microbes, and is administered at mg/kg po q h. reduced hepatic metabolism in animals with liver disease may result in an increased incidence of neurotoxicity. alternatively, ampicillin or amoxicillin-clavulanate can be administered. in severely affected animals, antibiotics (ampicillin, amoxicillin or metronidazole) should be administered parenterally. there are several precipitating factors that can lead to he, and these should be identified and corrected in the individual animal. correction of dehydration, hypoglycemia and hypokalemia is imperative. animals with clinical signs suggestive of gastrointestinal bleeding, such as melena, anorexia and vomiting, should be treated with h -blockers. in moderately to severely affected animals, food should be withheld until therapy results in significant improvements. once a clinically significant improvement is obtained, affected animals should be fed a diet restricted in protein, with limited aromatic amino acids and short chain fatty acids. key points: hepatic encephalopathy placed in a heparinized tube and transferred to the laboratory on ice for immediate testing. red blood cells contain large amounts of ammonia; hence hemolysis may result in falsely elevated blood ammonia levels. alternatively, a presumptive diagnosis of he can be made by demonstrating altered liver function through fasting bile acid stimulation testing and on the basis of response to therapy. a suspected or confirmed diagnosis of he should prompt investigations for congenital or acquired portosystemic shunting. portosystemic shunts are most commonly diagnosed by ultrasonography or per rectal portal scintigraphy using m technetium pertechnetate. [ ] [ ] [ ] [ ] positive contrast portography also can be performed; however, this necessitates general anesthesia and laparotomy. the prognosis for animals with he is dependent on the underlying liver disease. with treatment, most animals experience an improvement in the clinical signs related to he. severely affected animals may not respond to therapy, however. animals with increased blood ammonia tend to respond better to treatment than those with normal blood ammonia. feline infectious peritonitis, part feline infectious peritonitis and feline enteric coronavirus infections. i. feline enteric coronaviruses clinical, cerebrospinal fluid, and histological data from twenty-seven cats with primary inflammatory disease of the central nervous system inflammation and changes in cytokine levels in neurological feline infectious peritonitis use of anti-coronavirus antibody testing of cerebrospinal fluid for diagnosis of feline infectious peritonitis involving the central nervous system in cats detection of feline coronavirus rna in feces, tissues, and body fluids of naturally infected cats by reverse transcriptase pcr detection of feline coronaviruses by culture and reverse transcriptase-polymerase chain reaction of blood samples from healthy cats and cats with clinical feline infectious peritonitis development of a nested pcr assay for detection of feline infectious peritonitis virus in clinical specimens critical assessment of the diagnostic value of feline alpha -acid glycoprotein for feline infectious peritonitis using the likelihood ratios approach value of agr -acid glycoprotein in the diagnosis of feline infectious peritonitis serum [alpha] -acid glycoprotein (agp) concentration in non-symptomatic cats with feline coronavirus (fcov) infection treatment of cats with feline infectious peritonitis diagnosis of induced toxoplasmosis in neonatal cats toxoplasmosis in cats infectious diseases of the dog and cat feline immunodeficiency virus predisposes cats to acute generalized toxoplasmosis clinical and epidemiological aspects of feline immunodeficiency virus and toxoplasma gondii coinfections in cats clinical feline toxoplasmosis: serological diagnosis and therapeutic management of cases prevalence of toxoplasma gondii infection in cats in georgia using enzyme-linked immunosorbent assays for igm, igg, and antigens antemortem diagnosis and treatment of toxoplasmosis in two cats on cyclosporin therapy a case of fatal systemic toxoplasmosis in a cat being treated with cyclosporin a for feline atopy bsava manual of canine and feline neurology neurological history and examination. in: handbook of veterinary neurology feline infectious peritonitis some important disorders of cats feline infectious peritonitis: isolation of a coronavirus feline infectious peritonitis viruses arise by mutation from endemic feline enteric coronaviruses seroprevalence study of feline coronavirus in owned and feral cats in sydney feline coronavirus antibodies in uk cats comparison of serologic techniques for the detection of antibodies against feline coronaviruses feline coronavirus serotypes and : seroprevalence and association with disease in switzerland feline coronavirus antibodies in cats serologic studies of naturally occurring feline infectious peritonitis prevalence of feline infectious peritonitis in specific cat breeds patterns of feline coronavirus infection and fecal shedding from cats in multiple-cat environments risk factors for feline infectious peritonitis among cats in multiple-cat environments with endemic feline enteric coronavirus an overview of feline enteric coronavirus and infectious peritonitis virus infections topics in feline neurology diagnostic features of clinical neurologic feline infectious peritonitis a review of coronavirus infection in the central nervous system of cats and mice feline infectious peritonitis with neurologic involvement: clinical and pathological findings in cats results of magnetic resonance imaging in cats with meningoencephalitis laboratory profiles in cats with different pathological and immunohistochemical findings due to feline infectious peritonitis (fip) an appraisal of the value of laboratory tests in the diagnosis of feline infectious peritonitis feline infectious peritonitis in a closed breeding colony acute toxoplasmosis following renal transplantation in three cats and a dog diagnostic predictors of complications and survival after renal transplantation in cats evaluation of the prevalence of infections in cats after renal transplantation: cases histologically confirmed clinical toxoplasmosis in cats: cases ( - 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) the treatment of hepatic encephalopathy new mode of action for lactulose medical management of animals with portosystemic shunts available online at www.sciencedirect.com key: cord- -sxd t tz authors: nan title: poster presentations date: - - journal: dev med child neurol doi: . /dmcn. sha: doc_id: cord_uid: sxd t tz nan what are the perspectives and understanding of healthcare professionals including occupational therapists on treatment and care of babies with infantile spasms and early-onset epilepsy? a qualitative design dm middleton university of roehampton-online, birmingham, uk objective: to explore the perspectives and understanding of allied healthcare professionals (occupational therapists, physiotherapists, speech & language therapists) that work with children and epilepsy in order to guide and advocate for this population group. methods: a qualitative study design using interpretive thematic analysis with the data from participants in semi-structured interviews. results: the professionals had worked across acute and community settings and had previous experiences of working with children with epilepsy with some awareness of these needs. there were themes that emerged: ( ) housing and social needs, ( ) epilepsy, psycho-social and mental health needs, ( ) therapy approaches, ( ) training for allied healthcare professionals, and ( ) adolescents, young girls, women and epilepsy. conclusions: there are gaps in service provision for certain areas and will be shared within the presentation. epilepsy requires additional considerations for safety that other conditions may not require. it is crucial in the interests of public health for children and families with epilepsy to be able to advocate for resources and their specific needs. poster no. time to onset of cannabidiol (cbd) treatment effect and resolution of adverse events in patients with dravet syndrome: pooled analysis of two randomised controlled trials solution) at mg/kg/day (cbd ; gwpcare ) or mg/ kg/day (cbd ; both trials) or placebo for weeks. cbd treatment started at . mg/kg/day and reached mg/kg/day on day and mg/kg/day on day . percent reduction in cumulative convulsive seizure frequency for each treatment day (including previous treatment days) and timing of adverse events (aes) were assessed. results: overall, patients were randomised to cbd and to placebo. cbd led to significantly greater percent reductions in convulsive seizure frequency than placebo in gwpcare (cbd % vs placebo %, p= . ) and gwpcare (cbd %, cbd % vs placebo %, p= . and p= . ). in the pooled data, treatment differences in seizure reduction emerged during titration and were maintained throughout the study, with nominal significance (p< . ) achieved by day for cbd and day for cbd . onset of the first reported ae occurred during titration in % of patients with aes. aes resolved within weeks of onset in % of patients and by the end of the study in %. increases in alt/ast (> upper limit of normal) occurred in ( %) patients for cbd , ( %) for cbd , and ( %) for placebo; all were on concomitant valproate. all elevations resolved, either spontaneously while continuing cbd, after discontinuing cbd, or after reducing cbd, valproate, and/or clobazam dose. conclusions: cbd treatment effect (seizure reductions and aes) may occur early, during titration. the majority of aes resolved during the study. poster no. low dose fenfluramine hydrochloride oral solution provides long-term, clinically meaningful (≥ %) reduction in seizure frequency in dravet syndrome: interim analysis of a long-term openlabel extension study objective: to characterize long-term safety and durability of effect for adjunctive fenfluramine (ffa) in treating dravet syndrome (ds). methods: patients ( - y) with ds entered a long-term openlabel extension (ole) ( ) after completing one of two phase studies: study ( wks; placebo or ffa . or . mg/kg/d [max, mg/d] or study ( wks; placebo or ffa . mg/kg/d [max, mg/d]) . stiripentol was excluded in study but mandatory in study . in , patients received ffa . mg/kg/d for month ; dosing was titrated to effect thereafter. effectiveness and tolerability were assessed at months , , and , then at -month intervals. results: at interim analysis ( -mar- ) , / patients continued into ole; % completed months of ffa (mean dose, . mg/d; median duration, d [range, - d] ). during the entire ole, median percentage change in monthly convulsive seizure frequency (mcsf) for ffa vs pretreatment phase study baseline was - . % (p< . ); clinically meaningful (≥ %) and profound (≥ %) mcsf reduction from baseline were % and %. at month , median and mean longest interval between convulsive seizures were and days (range, - d); % of caregivers and % of investigators rated patients 'much improved/very much improved'. the most common adverse events included appetite decrease, pyrexia, nasopharyngitis, and diarrhea. no valvular heart disease or pulmonary hypertension was observed in any patient. conclusions: treatment with ffa resulted in robust, sustained reductions in mcsf and was generally well tolerated. no valvular heart disease or pulmonary arterial hypertension was observed in any patient at any time. ffa may be an important, novel antiepileptic drug for long-term ds treatment. poster no. zx (low dose fenfluramine hydrochloride oral solution) provides long-term, clinically meaningful reduction of convulsive seizure frequency in young (< years old) dravet syndrome participants: analysis from a long-term open-label study results: a total of of ( . %) participants who enrolled in the ole were < years old upon entry into the phase studies. the median baseline monthly convulsive seizure frequency (mcsf) before double-blind treatment was . seizures/month (range, . - . ) in this patient subgroup (< y). at the time of the ole interim analysis, the median decrease in mcsf in the < years subgroup over the entire observation period compared to baseline was - % (p< . ) compared with - % in the overall study population ( - y). the most frequently reported adverse events included pyrexia, upper respiratory tract infections, decreased appetite, and diarrhea. no valvular heart disease or pulmonary arterial hypertension was observed. conclusions: treatment with zx provided sustained, clinically meaningful reduction in mcsf in ds participants < years old. importantly, effective control of seizures in this young age group might be expected to mitigate the negative neurodevelopmental outcomes reported to be associated with treatment-refractory seizures. the improving provision of epilepsy care for children in england and wales methods: all relevant health boards and trusts (hb/t) were invited to register to participate and identify a hb/t lead. a snapshot survey was completed via a bespoke online platform by the hb/t lead describing local provision as of april . data was analysed by the rcpch including regional and national aggregates and longitudinal comparison to previous , reports. results: hb/t with a paediatric epilepsy service across england and wales registered to participate and submitted data. . % ( / ) of hb/t employed a consultant paediatrician with expertise in epilepsy; . % ( / ) had some epilepsy specialist nurse (esn) provision; . % ( / ) had a defined epilepsy clinic seeing patients at secondary level. . % ( / ) of hb/t had agreed referral pathways to tertiary paediatric neurology services. satellite paediatric neurology clinics were hosted in . % ( / ) of hb/t. conclusions: there are improvements in the overall numbers of epilepsy nurse specialists, paediatricians with expertise and specific clinics for children and young people with epilepsies. the findings led to comprehensive recommendations to hb/ t and commissioners, informed updates to the epilepsy best practice tariff and themes within the nhs long term plan. poster no. diagnosing and managing seizures on picu: an explanatory sequential mixed methods approach tonic clonic seizures. awake and sleep eeg showed temporal focal slowing. she was labelled as non lesional focal epilepsy after a normal mri scan and was discharged on keppra. she had multiple admissions with cluster of brief seizures at the age of , , , , , and months associated sometimes with febrile illness with poor response to intravenous aed's. she was diagnosed with autism at months. nd child: months old younger sibling had seizure onset at months. seizures were tonic in nature, brief, multiple and in clusters over a period of to days. eeg's showed non-specific slowing during seizures. array cgh revealed chromosome p . microdeletion. keppra was commenced and increased but recurrent cluster of seizures at the age of , and months required admission with poor response to iv aed's. family history revealed that half-sister (biological father's daughter who had epilepsy and global developmental delay) was diagnosed with pcdh epilepsy. gene tests were requested on both siblings and both were heterozygous for pcdh mutation. she had delayed social and communication skills from years with a diagnosis of autism at months. rd child: year old half sibling (father's th child from rd relationship) has tested positive for pcdh . her development is normal and so far there have been no seizures. conclusions: pcdh epilepsy is increasingly recognised as one of the early onset infantile encephalopathies. gene testing is likely to yield a diagnosis with a family history or with a typical phenotype. poster no. seizure, developmental and cognitive outcomes in children post hemispherotomy tt tay , dr reed , vj josan , sr rust , jt tan university of manchester, manchester, uk; neuropsychology team, paediatric psychosocial service, royal manchester children's hospital, manchester, uk; neurosurgery, salford royal nhs foundation, manchester, uk; paediatric neuropsychology, royal manchester children's hospital, manchester, uk; paediatric neurology, royal manchester children's hospital, manchester, uk introduction: patients with focal refractory epilepsy secondary to structural hemispheric changes have been shown in retrospective studies to have significantly improved seizure outcomes following hemispheric disconnection. the aim of this study was to report the seizure and cognitive outcomes in our cohort and investigate prognostic factors for seizure outcomes. methods: this was a single-centre retrospective study on children and adolescents who had hemispherotomy for refractory epilepsy in the royal manchester children's hospital between and . results: twenty-two patients were included with median (range) age of seizure onset and of surgery of ( - ) and ( - ) months respectively. median (range) time from seizure onset to surgery was . ( - ) months. the most common aetiologies were antenatal/perinatal middle cerebral artery infarct (n= ) and malformations of cortical development (n= ). at year after surgery and at last follow-up (median [range] [ - ] months), % ( / ) and % ( / ) achieved complete seizure freedom. the number of anti-epileptic medications decreased for ( %) at last follow-up. lateralisation of ictal and interictal eeg (p= . , p= . ), aetiology (p= . ), age of first seizure (p= . ) were not associated with seizure recurrence. five who had formal neuropsychological testing using the wechsler intelligence scale for children (wisc) showed improvement in cognitive abilities across all subsets post-surgery. ten children showed reduction in median vineland adaptive behaviour score, from to . , indicating a failure to progress rather than regression of skills. nine ( %) had newly reported behavioural or psychiatric issues including sleeping problems, challenging behaviours, autistic spectrum disorder. sixteen ( %) were reported by parents/carers to show improved verbal abilities postoperatively while the rest had unchanged verbal abilities. conclusions: we present a cohort of children with early onset seizures who had hemispherotomy at a relatively early age. our cohort showed good seizure outcomes and cognitive improvements. there were no prognostic factors for seizure outcome identified in this small group. the mri phenotype of atp a -related disease contrast, all ahc patients with mri abnormalities ( %) had a hypoplastic corpus callosum. the only patient with normal mri was the patient carrying mutation p.g r, associated with a mild clinical phenotype. of the patients with clinical ataxia (n= ), ( %) had cerebellar atrophy on mri; patients with cerebellar atrophy were not ataxic. two ( %) of the patients with severe intellectual disability had cerebral atrophy. conclusions: atp a mutations have subtle radiological findings, clustering around callosal dysmorphisms, as well as pontine and cerebellar abnormalities that seem to form distinctive mri phenotypes for ahc and capos. study of larger cohorts is required to more accurately define mutation-specific phenotypes and allow for quantitative analysis. poster no. long-term safety and efficacy of adjunctive perampanel in paediatric patients (aged to < y) with partial-onset seizures (pos) or primary generalised tonic-clonic seizures (pgtcs) in study r flamini , a patten , ly ngo pediatric and adolescent neurodevelopmental associates, atlanta, ga, usa; eisai ltd., hatfield, hertfordshire, uk; eisai inc., woodcliff lake, nj, usa objective: study (nct ) was a multicentre, openlabel, single-arm study of perampanel oral suspension ( . mg/ ml) in paediatric patients (aged to < y) with pos (with/ without secondarily generalised seizures [sgs] ) or pgtcs. here, we report long-term ( y) safety and efficacy data of adjunctive perampanel in paediatric patients from study . methods: this analysis included cumulative data from all enrolled patients in the core study ( wks of treatment) and extension phase a ( wks of treatment). assessments included monitoring of treatment-emergent adverse events (teaes), median percent change in seizure frequency per days from baseline, and % responder and seizure-freedom rates. results: of patients enrolled in the core study (pos, n= ; sgs, n= ; pgtcs, n= ), patients entered extension a. of these, patients discontinued extension a; most common primary reasons for discontinuation were adverse events ( . %) and inadequate therapeutic effect ( . %). for all patients, mean (standard deviation [sd] ) time since diagnosis was . ( . ) years and mean (sd) duration of exposure was . ( . ) weeks. during baseline, . % of patients received two concomitant anti-seizure medications. teaes were reported in ( . %) patients; somnolence was the most commonly reported ( . %). median percent reductions in pos, sgs and pgtcs frequencies at weeks - were . %, . % and . %, respectively; these were maintained at weeks - and were . %, . % and . %, respectively. seizure-freedom rates for pos, sgs and pgtcs at weeks - were . %, . % and . %, respectively. conclusions: long-term ( y) adjunctive perampanel is generally safe, well tolerated and efficacious in paediatric patients aged to < with pos (with/without sgs) or pgtcs. poster no. long-term adjunctive perampanel and healthrelated quality of life (hrqol) in paediatric patients (aged to < y) with partial-onset seizures (pos) or primary generalised tonicclonic seizures (pgtcs): study ea portillo , a patten , g meier , m malhotra , ly ngo paediatric neurology unit, department of paediatrics, hospital universitario virgen del roc ıo, sevilla, spain; eisai ltd., hatfield, hertfordshire, uk; eisai inc., woodcliff lake, nj, usa objective: study (nct ) was a multicentre, openlabel study of adjunctive perampanel oral suspension in paediatric patients (aged to < y) with pos (with/without secondarily generalised seizures [sgs] ) or pgtcs. we report long-term ( y) hrqol data using the euroqol dimensions-youth (eq- d-y) scale from study . methods: this analysis included cumulative data from all enrolled patients in the core study and extension phase a ( and wks of treatment, respectively). eq- d-y was assessed at baseline, week and week , and included five domains (mobility, self-care, doing usual activities, pain/discomfort, feeling worried/sad/unhappy). the eq- d-y visual analogue scale (vas) was also assessed; increases in vas correspond with improvements. data are for observed cases. results: all enrolled patients were included in the eq- d-y analyses. the proportion of patients reporting 'a lot of problems' was similar during baseline vs week : mobility, / ( . %) vs / ( . ); self-care, / ( . %) vs / ( . %); doing usual activities, / ( . %) vs / ( . %); pain/discomfort, / ( . %) vs / ( . %); feeling worried/sad/unhappy, / ( . %) vs / ( . %). outcomes were also similar for 'no problems' during baseline vs week : mobility, / ( . %) vs / ( . %); self-care, / ( . %) vs / ( . %); doing usual activities, / ( . %) vs / ( . %); pain/discomfort, / ( . %) vs / ( . %); feeling worried/sad/unhappy, / ( . %) vs / ( . %). mean (standard deviation) change in eq- d-y vas from baseline at week was . ( . ). conclusions: long-term adjunctive perampanel treatment (up to y) does not negatively affect hrqol (based on all eq- d-y domains) in patients aged to < years with pos (with/without sgs) or pgtcs. poster no. efficacy and safety of adjunctive perampanel for partial-onset seizures (pos) in adult, adolescent and paediatric populations (studies , , , ) . in study , patients received perampanel ≤ mg/day (without enzyme-inducing anti-seizure medications [eiasms] ) or ≤ mg/day (with eiasms) ( -week treatment period). efficacy assessments included median percent change in seizure frequency/ days from baseline, % responder rate and seizure-free rate. safety assessments included the incidence of treatment-emergent adverse events (teaes). results: the median percent reduction in seizure frequency/ days was greater with perampanel at ( . %), ( . %) and mg/day ( . %) vs placebo ( . %; p< . ) in adolescent/adult patients and was . % in paediatric patients. the % responder rate during the maintenance period was greater with perampanel at ( . %), ( . %) and mg/ day ( . %) vs placebo ( . %; p< . ) in adolescent/adult patients and was . % in paediatric patients. seizure-freedom rates were greater with perampanel at ( . %), ( . %) and mg/day ( . %) vs placebo ( . %; p< . ) in adolescent/ adult patients and was . % in paediatric patients. teaes occurred in . %- . % of adolescent/adult patients with perampanel - mg/day (vs . % in placebo patients), and in . % of paediatric patients. teaes observed in pediatric patients were similar to those reported in adolescents and adults. conclusions: these studies suggest perampanel is efficacious and generally safe in paediatric, adolescent and adult patients with pos (with/without sgs). methods: patients who completed either of the rcts could enter this ole trial (gwpcare /nct ). patients received gw pharmaceuticals' plant-derived highly purified cbd medicine ( mg/ml oral solution). the primary endpoint was safety. the secondary efficacy endpoints were median percentage change from baseline in drop and total seizure frequency. results: overall, % ( / ) of eligible patients with lgs entered the ole. median follow-up was weeks ( d to wks); patients ( %) withdrew. mean age: years; % ≥ years; % male. baseline median seizure frequency/ days: drop seizures; total seizures. during the extended follow-up, the incidence of adverse events (ae) was %; serious aes %; aes leading to discontinuation %. most common aes (≥ %): diarrhoea, convulsion, pyrexia, somnolence, vomiting, upper respiratory tract infection, and decreased appetite. aes of alanine aminotransferase increased occurred in % of patients. there were deaths; none deemed treatment-related by the investigator(s). median percentage reductions in seizure frequency ( -wk windows over wks) was - % for drop seizures and - % for total seizures. conclusions: long-term treatment with add-on cbd in patients with lgs produced sustained seizure reductions, with no new safety concerns. poster no. management of status epilepticus in children with dravet syndrome jaa holland, u rajalingam paediatrics, hinchingbrooke hospital, huntingdon, uk objective: status epilepticus is reported to be the second greatest cause of mortality in children with dravet syndrome. we aimed to review the evidence on convulsive status management in children with dravet syndrome to guide local practice. methods: literature review. results: pubmed search using search terms 'dravet' or 'scn a' and 'status epilepticus' returned results, of which were relevant. only one of these articles presented specific data on reported effectiveness of medications used in acute seizure management; this was based upon retrospective questionnaire data and defined status epilepticus as seizures lasting minutes or longer. here, the most efficacious agents reported to terminate such seizures within minutes were intravenous barbiturates ( of patients) and benzodiazepines ( of patients). rectal benzodiazepines, chloral hydrate and intravenous phenytoin or lidocaine were reported as less effective. the remaining articles presented expert and consensus opinion, all advising early administration (some at seizure onset) of buccal or intravenous benzodiazepines. provision of rescue medication for home use, with individualised plans, is recommended. one author advocated giving three doses of benzodiazepines sequentially. an article summarising a consensus panel described sodium valproate as a preferred second line option where benzodiazepines are ineffective, but there was no overall agreement on other possible medications. several articles advised caution in using phenytoin in acute seizure management. one source discusses possible harm from high dose barbiturates. conclusions: status epilepticus management for children with dravet syndrome should feature early, rapidly acting benzodiazepine administration. for second line treatment, phenytoin and barbiturates are commonly used in 'standard' status epilepticus management protocols, but there are potentially concerns around their use in this patient group. these concerns, however, appear largely theoretical. in the absence of evidence favouring a specific management protocol, individualised care plans should be designed with involvement of patients and their carers. poster no. zx (low dose fenfluramine hydrochloride oral solution) significantly reduces frequency of generalized tonic-clonic seizures in dravet syndrome: pooled analysis from two phase clinical trials jh cross , a gil-nagel , b gunning , d battaglia , k riney , g farfel , , a mistry , , b galer , , g morrison , , a gammaitoni , , k pagano , great ormond street hospital, london, uk; servicio de neurologia, hospital ruber internacional, madrid, spain; stichting epilepsie instellingen, zwolle, the netherlands; gemelli hospital, rome, italy; mater children's hospital, brisbane, qld, australia; zogenix, inc and int, emeryville, ca, usa; zogenix, inc and int, maidenhead, uk objective: zx (low dose fenfluramine hcl oral solution) significantly reduced the frequency of convulsive seizures in patients with dravet syndrome (ds) in two phase clinical trials. we conducted a pooled analysis of these trials to analyze the effect of zx on the frequency of tonic-clonic seizures (tcs), recently identified as a major risk factor for sudden unexpected death in epilepsy. methods: the frequency of generalized tcs and focal-to-bilateral tcs in patients with ds enrolled in one of two phase clinical trials of zx added to current antiepileptic drug regimens were analyzed. results: patients ( % male, mean age y) were enrolled and randomized to placebo (n= ), or zx . (n= ), . (n= ), or . (n= ) mg/kg/day. the median baseline monthly frequency of generalized tcs ranged from . to . /month in the four dose groups, and decreased during treatment by %, %, and % in the zx . , . , and . mg/kg/day groups, respectively, and by % in the placebo group. focal-to-bilateral tcs were experienced by fewer patients and had a median baseline frequency of . to . / month. during treatment, median percentage reductions in focal-to-bilateral tc frequency were %, %, and % in the zx . , . , and . mg/kg/day groups, respectively, and % in the placebo group. most common adverse events included decreased appetite, diarrhea, and fatigue. no valvular heart disease or pulmonary arterial hypertension was seen in any participant at any time. conclusions: zx substantially reduced the frequency of tcs. zx may be an important, effective new treatment option for ds patients. objective: mutations affecting tbc d have been associated with an expanding spectrum of phenotypes including developmental delay, hearing impairment, doors syndrome and a range of epilepsies. a number of different movement disorders, including ataxia, spasticity and episodic paroxysmal dystonia have also been described. here we report two unrelated patients with biallelic tbc d variants, in whom exerciseinduced dystonia was a major disease feature. methods: both patients were diagnosed through whole-exome sequencing. clinical information was obtained by a review of the medical notes, clinical correspondence and available video footage. results: both patients were found to have compound heterozygous mutations in tbc d , associated with an episodic dystonic/dyskinetic movement disorder reliably triggered by exertion. in the case of patient , exertion of specific body parts induced specific localised symptoms: for example, singing would precipitate orolingual dyskinesia. both girls experienced truncal dystoniaspecifically, lateral flexion of the trunkbrought on by prolonged walking. both girls also had epilepsy; of note, the exercise-induced movements and postures were captured on eeg and had no ictal correlate. conclusions: although tbc d mutations are an established genetic cause of epilepsy, our study further confirms that not all paroxysmal events in people with tbc d mutations are epileptic in nature. tbc d should be included in the genetic differential diagnosis of patients with complex neurological syndromes associated with paroxysmal exercise-induced dyskinesia. objectives: heterozygous de novo rhobtb mutations have recently been reported in developmental and epileptic encephalopathy, but the associated movement phenotypes are not fully delineated. in order to better define the expanding phenotype and movement disorder in rhobtb -related disease, we report a series of unrelated patients presenting with complex movement disorders as well as epilepsy and developmental impairment. methods: cases were identified both in the uk (through the neurogenetic services at great ormond street hospital and the national hospital for neurology and neurosurgery, london), and from international collaborating centres. data were collected retrospectively by the patients' clinicians, using a standardised proforma. results: nine individuals were identified, aged from to years. / had epilepsy. of these, / had achieved seizure freedom at their last review. the commonest seizure types were focal onset with impaired awareness and/or focal to bilateral tonic-clonic seizures. / also had a paroxysmal movement disorder, which included hemiplegic or asymmetrical episodic weakness in / , generalised dyskinesia in / , episodic focal dystonia in / and episodic ataxia in / . all individuals affected by a movement disorder had at least two different types of episodes. movement disorders improved significantly after treatment with carbamazepine in three children. cognitive ability varied from average to severe intellectual disability and in all but one case, developmental delay predated the onset of epilepsy. conclusions: rhobtb mutations cause a complex neurological phenotype associated with both epileptic and non-epileptic paroxysms. paroxysmal events occurring in people with known rhobtb mutation should therefore not be assumed always to be epileptic in nature. our study confirms that a wide variety of movement disorders are reported, including some which fall within the spectrum of alternating hemiplegia of childhood (ahc). rhobtb should thus be considered as a potential gene for ahc, other complex movement disorder phenotypes and epilepsy-dyskinesia syndromes. poster no. evaluating seizure recognition and the use of electroencephalography in the paediatric intensive care unit objective: in the paediatric intensive care unit (picu), seizures are challenging to detect given patient complexity, comorbidity and sedation. this has led to both over-and under-treatment of seizures. there is growing literature on the use of continuous electroencephalography in picu, considered gold standard but not universally available, but little on standard electroencephalography (eeg). this study aims to investigate the indications for eeg requests, their efficacy and the use of antiepileptic drugs (aed) in picu, hypothesising a difficulty in clinically differentiating between epileptic and non-epileptic events and suboptimal use of aeds. methods: this retrospective study examined eeg reports over years at a tertiary picu. data was collected on participant characteristics, eeg indications and findings and aed use. results: eeg reports from participants were included. median age was months (iqr mo- y mo). indications for eeg (often multiple per eeg) included suspected clinical seizures ( %), suspected subclinical seizures ( %), prognostication ( %) and suspected encephalopathy ( %). % of participants with suspected seizures were sedated and % of all participants were encephalopathic. clinical episodes suspected to be seizures were captured in / eegs. only % of these were eeg-confirmed seizures. captured movements shown not to be seizures are qualitatively described. % of patients with suspected seizures had electrographic seizures with no clinical correlate. most confirmed seizures were in participants without pre-existing epilepsy. antiepileptic(s) were changed prior to / captured events. seizures were present in % of these cases, while % had neither clinical nor electrographic seizure activity. / participants with confirmed clinical seizures had aeds changed. conclusion: it is challenging for clinicians to differentiate between seizure and non-seizure movements in picu. moreover, there are issues of over-medication and low event-capture rate with eeg. we propose a multidisciplinary education strategy and investment in ceeg to address these issues. introduction: glucose transporter deficiency syndrome (glut -ds) is a rare neurometabolic disorder causing impaired glucose transport into the brain. in the majority of patients, it is caused by an autosomal dominant heterozygous mutation in the scl a gene. ketone bodies generated by a ketogenic diet (kd) provide the brain with an alternative energy source and is gold standard therapy. we report our experience for our cohort of patients at royal manchester children's hospital. methods: retrospective case note review of patients with glut -ds at royal manchester children's hospital from to . results: patients - male, female. age range to years. average age at diagnosis was years months (range mo- y). there was a history of seizures in of patients with average seizure onset of years months. seizures types were absences ( / ), generalised tonic-clonic ( / ), myoclonic ( / ), myoclonic astatic ( / ), tonic ( / ) and focal to bilateral tonic-clonic ( / ). ketogenic diet was used in all patients for a range of months to years months. no significant adverse effects occurred that required discontinuation. six patients complying with kd are seizure free and not taking antiepileptic drugs (aeds). one of these patients had occasional tonic-clonic seizures with illness and loss of ketosis but has been seizure free for > months. five patients are non-compliant with kdtwo have good seizure control with aeds, potentially limiting motivation, and two (siblings) have a parent with glut -ds and learning difficulties. learning difficulties were reported in patients. other symptoms included ataxia ( / ), dysarthria ( / ), tremor ( / ) and dystonia ( / ). one patient presented with episodic hemiplegia. conclusions: patients with glut -ds are a heterogeneous group leading to challenges in diagnosis, management and prognosis. ketogenic diet has been effective in managing this cohort but compliance was a limiting factor. objective: to conduct a survey regarding the management of relapse in children epilepsy in following weaning off aeds. methods: we conducted an online survey in the east of england (eoe) via the eastern paediatric epilepsy network (epen) regarding the management of relapse in children epilepsy after weaning aeds. epen is a network of paediatricians and nurse specialist with eoe who manage lead in management children with epilepsy within all the dgh's in the region. the questions in the survey asked about various aspects of management of patients after relapse, including the choice of anti-epileptic medication restarted, if started, any further investigations undertaken, and finally, the length of aed treatment before a second attempt at weaning might be considered. results: we received responses from paediatricians in dgh's across eoe. there was a large degree of variation in the responses to all of the questions in the survey. the frequency and semiology of seizures on relapse seemed to play a key role in decision making, as did the thoughts and views of the family and patient themselves. it was interesting to note there was a variation in response to whether any further investigations would be undertaken and if these were deemed necessary. most clinicians responded that they would continue aeds for another years before attempting weaning again. conclusions: there is variability in the management of epilepsy relapse in the eoe and we suspect that this may also be the case nationally. to investigate this further, we would envisage extending the survey nationally, via open ukwhich is an organisation that links the various regional paediatric epilepsy networks across uk. this would enable establishing a standardized guideline for management of epilepsy relapse in the future. objective: de novo dominant mutations in dhdds were recently identified as a cause of developmental and epileptic encephalopathy. dhdds encodes dehydrodolichyl diphosphate synthase, which is essential for dolichol monophosphate synthesis and protein glycosylation. we report two half-siblings with a new pathogenic, maternally inherited dhdds missense variant, c. g>a(p.arg gln), identified through whole-exome sequencing. method: case note and literature review. results: sibling , aged years, presented at months with global developmental delay, hypotonia and frequent absences with eyelid myoclonia. from age , she developed atonic drop attacks, myoclonic seizures, tremor, ataxia and facial dyskinesia. dyskinesia and mobility deteriorated from age and she is now largely non-ambulant. severe learning disability with possible cognitive deterioration and insatiable appetite are also features. sibling , aged years, developed blank spells associated with eyelid flickering at months and atonic drop attacks aged . development delay is present, but progress is greater than her sibling. dyskinesia, tremor, ataxia and deterioration in mobility are features. neither is dysmorphic. eegs on both showed bursts of irregular generalised spike wave associated with head nods and eyelid flutter. photosensitivity was not shown but both were treated with anti-epileptic medication. mri scans are normal. clobazam and zonisamide improved seizure control in both. mother has mild learning difficulties, tremor and dyspraxia. she had generalised tonic clonic seizures, from age to years, well controlled with lamotrigine. compared to the six known cases in the literature, our report confirms atonic seizures and dyskinesia as important features of this disorder, in addition to common characteristics of myoclonic component to seizures, hypotonia and tremor. learning disability is of variable severity. conclusions: this is the first report of familial inheritance of dhdds related developmental and epileptic encephalopathy and describes variable severity of the phenotype within family members. the features described are consistent with those previously observed. objectives: to evaluate whether the duration of treatment has an effect on the relapse rate in children with cae attending a paediatric neurology centre in cyprus, and whether the eeg can be used as a prognostic tool. methods: electronic patient database review of patients with cae, who have discontinued treatment attending the paediatric neurology clinic between years - . results: fourteen patients with cae, off treatment were identified ( male). age at presentation ranged from to years (median . y). all patients underwent an eeg to confirm diagnosis and those who presented with seizures other than absences were excluded. twelve patients were treated with valproic acid (depakine) and with ethosuximide (zarontin). in , absences resolved on first line monotherapy, whilst were refractory requiring combination therapy. positive family history was present in (non-identical twins), attention deficit in , and learning difficulties in patient. all initial eegs were consistent with cae, patients also underwent an eeg post seizure control to confirm resolution. mean time to seizure cessation was . months, mean duration of treatment . years; patients discontinued treatment after year of seizure freedom. prior to withdrawing treatment all patients had an eeg (normal , mildly abnormal with brief generalised discharges , photosensitivity , brief electrographic absence ). relapse occurred in patients who required re-instigation of treatment. mild abnormalities on eeg prior to coming off treatment did not correlate with a higher relapse rate. there was no difference in relapse rate in patients on treatment for , or more years. patients were followed up for a mean of . years. conclusions: treating patients with cae for less than years does not affect relapse rate provided patients are seizure free, also confirmed by eeg normalisation, which may be used as an additional predictor. background: mutations in unc , encoding part of the unc -unc -nalcn channel complex, causes autosomal-recessive severe infantile encephalopathy, this is a rare case of profound global developmental delay with psychomotor retardation. only individuals have been reported to date. unc deficiency is characterized by hypotonia, strabismus, oral motor dysfunction, postnatal growth deficiency, and developmental delay. the majority of individuals do not learn to walk. all individuals lack expressive language. additional features can include nystagmus, extremity hypertonia, a highpitched cry, repetitive and self-stimulatory behaviours, constipation, clubfeet, joint contractures, and scoliosis. there is no loss of skills suggestive of neurodegeneration. case presentation: -year-old, with a recent confirmed diagnosis of unc gene mutation and microcephaly. she had profound global developmental delay, learning disability, bilateral squint with cortical blindness, seizure disorder, sleep apnoea, head drops, movement disorders, feeding difficulty, scoliosis and constipation. term baby with normal anti-natal history. induction of labour for iugr. good apgar, at birth but developed respiratory distress with cardiac problems. birth weight g. neurologically: floppy with reduced muscle tone and microcephaly. had a short neonatal admission and discharged with cardiac, endocrine, neonatal and neurodevelopmental delay follow-up. she had no spoken words and communicated by crying. she was only able to sit transiently and never walked. she was wheelchair bound with gmfcs, macs, cfcs and edacs level each, needing full -hour support from patents and carers. ddd study confirmed unc gene mutation, her cousin was also noted to have same gene mutation via exome sequencing and with similar clinical picture. conclusion: early diagnosis is key for genetic counselling for further children and ensuring global support as reported individuals span ages from birth to year. the diagnosis is established in a pro-band with developmental delay and hypotonia by identification of bi-allelic pathogenic variants in unc on molecular genetic testing. poster no. cognition and disease burden in scn a positive dravet syndromea -year follow-up study development, disease burden and sleep profile of patients with dravet syndrome. methods: this is a follow-up to a study previously involving ds patients with detailed developmental and clinical information available. participants completed a structured postal questionnaire on epilepsy severity and disease burden, the adaptive behavioural assessment system (abas- ), the sleep disturbances scale for children, pediatric quality of life inventory (pedsql) and the strength and difficulties questionnaire. results: / from the original cohort were contactable and ( %) of carers completed the outcome measures. the developmental quotient at follow-up was significantly lower compared to the earlier study (p= . ), and % of affected individuals had a severe or profound learning disability. we observed the steepest decline in cognitive functioning in those that were youngest (age - y) at original study onset (p= . ). poorer developmental quotients correlated with early onset of initial developmental concerns (rs= . ; p= . ), later mobility problems (rs= . ; p= . ), higher levels of behaviour problems (rs= . ; p= . ) and worse pedsql scores (rs= . ; p= . ). carers health and wellbeing was negatively affected in % of cases and in %, at least one of the two carers quit their job due to their child's illness. sleep problems as measured by total sleep scale score were reported in % of patients, whilst % had at least one abnormal sleep scale category. only % of individuals with abnormal sleep scores received treatment. rs=spearman rho correlation coefficient. conclusions: this study highlights the ongoing cognitive decline in ds, particularly affecting younger patients, alongside often untreated sleep problems and a significant disease burden on primary carers. with new therapeutic opportunities on the horizon, early interventions appear crucial to avert the observed severe cognitive decline. poster no. forced normalisation as a factor in behaviour deterioration on the ketogenic diet e hassan , vj whiteley , , hj tan department of neurology, royal manchester children's hospital, manchester, uk; therapy and dietetics, royal manchester children's hospital, manchester, uk; school of health and society, university of salford, salford, uk introduction: there have been a number of reports that demonstrate a correlation between improved seizure management and deterioration of behaviour with psychosis in adults and children. forced normalisation is a concept where there is deterioration in behaviour when better seizure control is achieved with antiepileptic drugs (aeds) or epilepsy surgery. the ketogenic diet (kd) is a treatment option for children with refractory epilepsy with approximately to % showing at least % reduction of seizures and % of those patients reaching seizure freedom months after treatment. although forced normalisation has been discussed in literature following aeds and neurosurgical interventions, it has not been reported following the use of kd. cases: of the children that have commenced on kd over the last years at royal manchester children's hospital, . % responded to the diet (at least % improvement in seizures) and . % were non-compliant. we present patients under the care of the kd service, whose behaviour deteriorated on kd when seizure reduction was > %. the behaviour changes described by parents included poor sleep, unsettled, agitation, head-banging and shouting. five out of the six patients stopped kd treatment, with subsequent improvement in behaviour. conclusions: there are reports that patients on the ketogenic diet with seizure freedom show improvement in their behaviour, unlike our small cohort whose behaviour deteriorated. forced normalisation has been explored in paediatric patients as a cause for behaviour deterioration following surgical and medical management for intractable seizures. the associated factors of deteriorating behaviour have not yet been explored in depth with the ketogenic diet. objective: we wanted to determine how lacosamide was being used in children locally, and what their outcomes were at a year. methods: we undertook a registered, retrospective, clinical audit using hospital electronic records. we ascertained every patient aged < years who had been dispensed lacosamide january -january . the electronic health records were reviewed, and data collected using a standard proforma, including: patient demographics, age of seizure onset, seizure type, ecg and mri findings, baseline seizure frequency, seizure frequency , , and months on lacosamide, maximum dose prescribed (mg/kg/day), and adverse effects at , , and months. results: / ( %) patients were male, the median age was years (range mo to y), with a mean age of onset of years (range mo to y). / ( %) had epileptiform activity on eeg and / ( %) had an abnormal mri. / ( %) had focal seizures. / ( %) had a minimum of one seizure a week. / ( %) had previously tried or more antiepileptic drugs (aeds), and / ( %) had drug resistant epilepsy prior to starting lacosamide (already failed previous aeds). all patients had lacosamide alongside another aed. the mean daily dose of lacosamide was . mg/kg/day (range . - . ). at months, / ( %) of patients reported a > % reduction in seizure frequency. / ( %) remained on lacosamide year after starting, and / ( %) experienced an adverse side effect. conclusions: in this local audit, lacosamide was mostly prescribed for drug resistant epilepsies and was used in polytherapy. a third of patients saw a significant reduction in seizure frequency on lacosamide, although some were also started on other treatments during this period. most patients remained on lacosamide after months, and about in experienced one or more adverse side effect. cacna a is a large gene which encodes for the alpha subunit of a neuronal ion channel and it is expressed widely throughout the central nervous system (cns). pathogenic variants in this gene have been associated with many phenotypes. most commonly episodic ataxia type (ea ) and spinocerebellar ataxia type (sca ). rarer phenotypes include, familial hemiplegic migraine, paroxysmal tonic upgaze, epilepsy, and intellectual disability with autism. here we present the case of an month old girl who presented with new onset paroxysmal abnormal eye movements during an intercurrent illness. the referring clinicians felt these episodes may be epileptic. however, an electroencephalogram (eeg) captured these movements which were non-epileptic downbeat nystagmus. all other initial investigations including cerebrospinal fluid glucose and neurotransmitters were normal as was her neuroimaging. over the next year her nystagmus became constant. she had otherwise normal development. at months her gait was noted to be abnormally unsteady and broad based (even accounting for age). the nystagmus and ataxia changed in severity from day to day, she could have to more severe days followed by to better days, they never completely resolved. she had no family history of abnormal eye movements or ataxia. subsequent genetic testing revealed a cacna a c. g>a p(glu lys) missense variant described only twice previously in ea , both with very different phenotypes to our patient. neither of her parents carried the same genetic variant. this case is the first reported case of this cacna a variant presenting as downbeat nystagmus followed by ataxia. both her age of presentation and her initial presenting features are very different to the typical phenotypes associated with this gene. it broadens the phenotype of cacna a, and also broadens the differential diagnoses associated with abnormal eye movements in infancy. the importance of following up as yet undiagnosed patients who may go on to develop new and revealing symptoms is highlighted. objective: to undertake a questionnaire-based survey retrospectively exploring parents'/carers' recall of, and views on, the safety and risk advice given at the time of their child's epilepsy diagnosis. methods: questionnaires were distributed throughout scotland via scottish paediatric epilepsy network (spen). parents'/ carers' of to -year-old children were asked to complete the questionnaire prior to their seizure clinic appointment. results: questionnaires were suitable for inclusion. seizure burden was evenly distributed: % < seizure/month, % > seizure/month, % > seizure/week and % had absences only. respondents could recall post-diagnosis information being provided on: water safety ( %), taking medication regularly ( %), sports/activities ( %), seizures in sleep ( %), first aid ( %), prolonged seizures ( %) and/or sudep ( %). there was no statistically significant difference in the duration of epilepsy diagnosis between those who could recall information being given (m= . y, sd . ) and those who could not (m= . y, sd . ; t test p= . ). the majority of information was given via clinic discussions ( %). % received written information, % directed to websites and/or independent search ( %). most information was 'just right' ( % water safety, % on taking aed regularly, % on sports/activities, % on seizures in sleep, % on first-aid for seizures). approximately % of respondents want more information on seizures in sleep, water safety and sports/activities. % of respondents felt worried following information about seizures in sleep, % about prolonged seizures and % regarding sudep. conclusions: a substantial proportion of parents'/carers' do not recall receiving safety information on epilepsy despite this being standard practice through spen. this appears to be unrelated to the duration of their child's epilepsy. repeated timely reinforcement may be of benefit. a high proportion of parents'/carers' felt concerned following information provided on nocturnal seizures, prolonged seizures and sudep. this should be recognised with support in place for further discussions. poster no. seizure outcome in responsive vagus nerve stimulation therapy in children and young people v rasiah, n barnes, s carter, k das, r robinson, z tahir, s varadkar great ormond street hospital for children nhs foundation trust and ucl gosh institute of child health, london, uk aim: vagus nerve stimulation (vns) therapy is an established treatment for pharmacoresistant epilepsy. newer responsive-vns (rvns) systems use ictal-tachycardia detection as a biomarker of seizure onset and automatically deliver additional stimulation on detection to abort the seizure. we reviewed the seizure outcomes in children and young people (cyp) implanted with rvns at great ormond street between to . methods: data were collected prospectively on patients who had an aspiresr â rvns inserted during time period of to . reduction in seizure frequency and severity, wean of medications and treatment complications or side-effects were assessed at time-points of year, months, years and years post-implantation. results: cyp (mean age . y) had rvns inserted. at year, % ( / ) had a positive response graded as > % reduction in seizure frequency or severity (i.e., duration), % ( ) had benefit though < % benefit, and % ( ) of cyp were non-responders. this increased at months to % ( / ) of children showing response > %, a further % showed response % and only % ( / ) non-responders. response was over-all sustained, with response lessening in only children between months and years. reduction of medication burden was achieved in % ( / ) (not attempted in all cyp). no patients achieved seizure freedom. replacement of vns from an older model to rvns showed further benefit. complications were infrequent: % ( / ). device removal for infection was required in one child of small body size; successful replacement was possible within the year. conclusions: vns is a useful treatment option for cyp with pharmacoresistant epilepsy. seizure outcomes with rvns in cyp are better than with standard vns. response is sustained. benefit may not be seen by year; therapy should be continued until at least months. in our patients who responded later than -year, further optimisations of duty cycle and current were made. replacement of older vns. devices with rvns led to additional benefit. these findings are consistent with reported outcomes from adult series, though seizure freedom is not seen in cyp. in our centre, cyp who seemed to benefit most were those whose epilepsy was of structural aetiology and those with focal seizures, although our numbers were not large enough to assess the significance of this. poster no. atp a mutation in twins presenting with apnoeic episodes, suspected seizures and possible dystonic events objective: we report the case of monochorionic diamniotic twins presenting at the age of months with infantile seizures and apnoeic episodes. the eiee gene panel revealed a mutation in the gene atp a , supporting the clinical diagnosis of alternating hemiplegia of childhood (ahc). methods: case report. results: twin r presented with staring episodes, eye deviation and tonic posturing of limbs. episodes occurring mainly in clusters, affecting either side and requiring rescue medications for termination. profound apnoeic episodes needing resuscitation were also noted. twin s presented few weeks later with a very similar presentation. developmentally making satisfactory progress with twin r showing only very mild delay. the array cgh from twin r was normal. several investigations were performed including two normal standard eegs, a normal sleep eeg, a normal ecg, an echo showing a small pfo and an mri scan demonstrating a left sided mesial temporal lobe sclerosis. similar mri findings were reported in twin s. investigations such as urine organic acids and amino acids, plasma amino acids, carnitine, acylcarnitine, transferrin glycoforms, mucopolysaccharidosis screen, ammonia and lactate were all normal. pyridoxine was tried with no improvement, levetiracetam was added and afterwards changed to carbamazepine. sodium valproate was commenced eventually after an episode of prolonged clinical seizure. the eiee gene panel revealed a de novo atp a mutation in both twins. flunarizine was commenced following this result. a video telemetry managed to capture both epileptic and non-epileptic episodes in twin r. the epileptic episode was characterised solely as apnoeic episode due to a left temporal seizure activity spreading onto the opposite hemisphere which is concordant with the imaging finding of left mesial temporal sclerosis. conclusions: knowledge of the atp a mutation allowed clinical correlation of a diagnosis of ahc, matching the wide clinical spectrum of ahc including paroxysmal dystonia and epilepsy. poster no. epilepsy in a child development centre population pc kenyon, njv cordeiro, gl duffy rainbow house child development centre, irvine, ayrshire, uk objective: to assess all the cases in a child development centre (cdc) population with epilepsy, to enable characterisation of the caseload. methods: all case notes of children with an epilepsy diagnosis coded on the cdc database were retrospectively reviewed for demographic, investigation and treatment data. results: children were identified. % were diagnosed before a year of age, and over half before their third birthday. % of patients had an eeg, and of these, % had an abnormal eeg. % had genetic testing performed, and of these, % had a genetic cause of their epilepsy identified. % had an mri scan, and of these, % had a structural cause for their epilepsy identified. % had global developmental delay, and % had a diagnosis of learning disability. one third have a diagnosis of cerebral palsy, % have autism spectrum disorder, and % have a hemiplegia. % are seizure free, the majority of whom have their epilepsy controlled with one medication. % had adherence concerns identified. conclusions: compared to a general paediatric epilepsy clinic, this group of children were diagnosed earlier in life, had higher rates of genetic or structural causes identified, and were less likely to be seizure free. results: case . a -year-old with autism spectrum disorder (asd) developed hand waving in front of her face in bright light from years. multiple myoclonic seizures occurred with screen use and her family live in complete darkness. eeg demonstrated photosensitivity, generalised spike-slow wave after hand waving and - . hz spike-wave with myoclonia. clonazepam has been commenced. case . a -year-old with learning difficulties and a family history of generalised seizures presented aged years with forehead rubbing leading to loss of part of her eyebrow. eeg showed photosensitivity and the generalised spike-wave of absences and eyelid myoclonia (em). sodium valproate was used but replaced with lamotrigine due to weight gain. case . a -year-old with a family history of generalised seizures presented aged years with hand waving in front of her face, requiring her nursery to provide a dimly-lit setting. eeg demonstrated generalised polyspikewave, -hz spike-wave and myoclonia with photic stimulation. lamotrigine was ineffective and replaced with sodium valproate. case . an -year-old with likely asd developed hand waving in front of her face in bright light aged years, triggering generalised tonic convulsions (gtc). she had a non-induced gtc in dappled sunlight. there was on-going anxiety and thoughts of self-harm. eeg showed photosensitivity and bursts of spikes-polyspikes. lamotrigine was ineffective; seizures stopped with sodium valproate. conclusions: the self-induced seizures of sunflower syndrome are difficult to treat and are associated with physical, psychological and social impairments. sodium valproate is the most effective medication which may be problematic in this predominantly female patient group. what are the information needs of parents whose child is diagnosed with glutaric aciduria type to help preserve neuro-developmental outcome? objectives: to assess the information needs and support of parents at the time of diagnosis of ga in their child, and how to support them in preventing metabolic decompensation and preserving neuro-developmental function. methods: a focus group with five parents was conducted using a topic guide to direct the discussion, which was recorded and fully transcribed. data were analysed using thematic analysis. two researchers were involved in initial coding of data and key analytic decisions. results: two main themes were identified. 'understanding the condition' explored parent's needs to understand the scientific complexity of ga and to be aware of the 'worst case scenario' associated with loss of metabolic control, and brain injury. parents reported clinicians did not give then enough information on the ga , and were forced to use other information sources, sometimes seeking out scientific papers. information on managing crises was insufficient, with parents not understanding what the doctor meant about commencing the emergency regime when their child was 'sick'. parents reported living in terror of their child experiencing metabolic decompensation and permanent brain injury. 'managing the condition' explained how parents coordinated and controlled the involvement of other carers and outlined parents' need to be active partners in medical management to feel in control. parents wanted to know the results of regular biochemical tests for reassurance, but found they were not easily accessible. parents could not leave their child in the care of another adult because they did not have sufficient knowledgeable about ga or were known to 'cheat' by offering the child food they should not have. the transition into school was a particular challenge. conclusions: the study highlights the importance of addressing parents' initial and ongoing informational needs so they can fulfil their role and protect their child from metabolic decompensation and permanent brain injury. poster no. normal transferrin isoelectric focusing in a child with cog related congenital disorder of glycosylation objectives: congenital disorders of glycosylation (cdg) are a large group of rare multisystem diseases caused by defective linkage of oligosaccharides to newly synthesised proteins or lipids. several cdg subtypes are the result of mutations in subunits of the conserved oligomeric golgi (cog) complex. this includes cog -cdg, an autosomal recessive disorder caused by pathogenic variants in the cog gene. in the two cases previously reported transferrin isoforms were abnormal, consistent with defective n-glycosylation. methods: we describe a -year-old female born to non-consanguineous parents. she presented with severe global developmental delay, dysmorphic features, postnatal progressive microcephaly, complex epilepsy, rhizomelia, spastic quadriplegia, and feeding problems from early infancy. results: mri brain showed global cerebral atrophy, predominantly supratentorial, with relative cerebellar sparing. trio exome sequencing and analysis identified compound heterozygous cog variants in the proband, a maternally inherited pathogenic splice site variant c. + g>a and a paternally inherited likely pathogenic splice site variant c. + g>a. messenger rna analysis showed that the c. + g>a variant caused aberrant splicing, with skipping of exon and the introduction of a premature stop codon in exon , likely to result in nonsense mediated decay. analysis of transferrin isoforms was normal (by both isoelectric focussing and mass spectrometry). since cog -cdg also affects o-glycosylation apolipoprotein ciii (apociii) isoelectric focussing was undertaken, however this too was normal. conclusions: transferrin and apociii isoelectric focusing are screening tests for n-and o-glycosylation defects. however, both have their limitations and some cases of cdg have normal transferrin or apociii glycoforms escaping those screening tests. this is the first case of a patient with cog -cdg with normal biochemical markers to be described. this case also demonstrates the diagnostic power of next generation sequencing for rare metabolic disorders, where the biochemical screening may be inconclusive. poster no. developmental delay in a young infant with nonclassical combined malonic and methyl malonic aciduria (cmamma) caused by homozygous missense mutation in acsf gene kd dayasiri , eg goh , sk kodagali , jb baruteau , ga anand oxford university hospitals nhs foundation trust, oxford, uk; great ormond street hospital, london, uk introduction: acylcoa synthetase family member (acsf ) activates malonylcoa and methymalonylcoa into their respective thioesters. acsf deficiency causes non-classical cmamma, a rare inborn error of metabolism characterised by presence of methyl malonic acid in higher concentrations than malonic acid in urine. the reversal is seen with classic cmamma caused by malonylcoa decarboxylase deficiency (mcd). case report: -and-a-half-month old male infant second born to consanguineous south asian parents presented with severe failure to thrive and recurrent vomiting. his older sibling who had failure to thrive and neuro-developmental delay died at months without a genetic diagnosis. initial blood tests revealed metabolic acidosis, pancytopenia and coagulopathy. neuroimaging was unremarkable. subsequent evaluation revealed normal levels of methionine, homocysteine and red cell folate. significant methylmalonic aciduria with mild malonic aciduria without evidence of other abnormal metabolites (propionyl-coa metabolites: hydroxypropionate, and methylcitrate or tiglylglycine) in urine suggested the diagnosis of non-classic cmamma, confirmed by homozygous missense variants in acsf gene revealed by trio-exome sequencing. neurodevelopmental assessment at months revealed global developmental delay with general hypotonia; gross motor ( - mo); fine motor ( - mo); speech ( - mo) and social (under mo), and without any regression. carnitine was supplemented to avoid secondary depletion caused by the excretion of mma. parents were advised to avoid prolonged fasting and to provide emergency regimen (powdered carbohydrate drink mix) in the event of acute deterioration. conclusions: this report describes an unusual paediatric presentation of non-classic cmamma. urine organic acids allows identification of increased ma and mma excretion and highly suggestive of the diagnosis, thus avoiding additional investigations determination of urinary mma/ma ratio can help differentiating between classical and non-classical forms. methods: we conducted retrospective case note analysis of the five paediatric cases with confirmed diagnosis of the late onset pompe disease, referred to the highly specialised metabolic service. results: three of five patients (current age - y) presented with delayed motor milestones in early childhood (mean age: . y). one patient initially presented with episodes of thigh pain and high ck. in addition, when years he re-presented with recurrent abdominal pain with high ck. the remaining one presented with muscle pain upon exercise with high ck. all apart from one had muscle weakness affecting limb girdle muscles and axial muscles. the remaining one presented with proximal muscle weakness by the age of years. all patients remained ambulant, one developed scoliosis and two were on non-invasive ventilation. cardiac involvement as ventricular dysfunction requiring targeted treatment was observed in one. pathology showed vacuolated deposits in three patients and non-specific myopathic changes in one. four are on enzyme replacement therapy (ert) and tolerated well. conclusions: late onset pompe disease is a multisystem disease and should be considered in cases of isolated respiratory problems, lower back pain, rigid spine, and myopathy or exercise intolerance with elevated serum ck if these symptoms cannot be attributed to another disorder. poster no. delay in diagnosis and misdiagnosis of ataxiatelangiectasia: a systematic review pubmed, scopus). the cochrane library was also searched. the search protocol is available. the inclusion criteria were: all dates, all languages, all ages, human participants and clinical relevance. the exclusion criteria were: no reference to ataxia-telangiectasia within the article, not an original article, animal studies, article not clinically relevant. results: search returned articles; titles and abstracts were reviewed after removing duplicates. full text review includes articles of which case series and case reports were identified ( , exclusions; , articles not found or not accessible). mean age of first sign or symptom of a-t in cases reviewed to date was . months (range - d to mo). the mean age of diagnosis in cases in which it has been reported was . months (range - d to mo). there was a mean time of . months from presentation to clinician, to diagnosis of a-t (range - to mo, median mo) in the cases in which this was reported. / ( . %) cases had a documented alternative diagnosis prior to the diagnosis of a-t. / ( %) of these children were incorrectly diagnosed with cerebral palsy and / ( %) with hyper-igm syndrome. the mean delay from incorrect diagnosis to a diagnosis of a-t was months with the longest delay months. conclusions: this study is the first comprehensive systematic review of scientific literature on ataxia-telangiectasia. we aim to describe the natural history of the condition and, along with results from the natural history of a-t (n-hat) study, systematically define, where possible, the conditions presentation, course, and prognosis. ( %) people with a-t presented with gait ataxia or disturbance, and / ( . %) with truncal ataxia. the most common presenting feature in cases without ataxia were developmental delay, or regression, and choreoathetoid movements. the second most common neurological presenting sign was dysarthria in / ( . %) cases, and at least of these had no associated ataxia. dystonia was a presenting sign in / ( . %) cases, including / ( . %) with no associated ataxia. / ( . %) initially presented with no neurological signs or symptoms. conclusions: this study is the first comprehensive systematic review of scientific literature on ataxia-telangiectasia. these results show that . % of people with a diagnosis of a-t presented initially with at least one neurological sign or symptoms. this completed review will lead into the natural history of a-t (n-hat) study, a longitudinal, retrospective and cross-sectional study. the role of serum oxysterol in the diagnosis of niemann pick c m alcheikh, g connolly, n cluskey, s osullivan royal hospitals belfast health and social care trust, belfast, uk introduction: neimann pick c is a neurovisceral disease that is caused by cellular cholesterol trafficking disruption. historically, the diagnosis of niemann pick c was made using filipin staining and skin fibroblast cultures. recently genetic testing of npc an npc genes are available. mutations of either gene also affect cellular trafficking of cholesterol and detecting oxidative cholesterol metabolities can also be diagnostic of nieman pick c. serum oxysterol can be used as a first line test with subsequent genetic confirmation and has a positive predictive value of > %. methods: we present a case of an -year-old boy who was referred to genetics initially with absence of up gaze, severe restricted downward gaze, developmental delay, regression of skills and frequent falls. in the last year his parents and school observed progressive deterioration of his symptoms with gelastic cataplexy, markedly decreased tone, increasing difficulty with memory loss and slurred speech. these symptoms are strongly suggestive of niemann pick c disease and oxysterols were requested which showed elevated oxysterol level of . ng/ml (normal range . - ). he was started on miglustat. genetics confirmed the diagnosis. results: overall the child's parents report that since commencing the miglustat he is more confident and they have recently seen him hop and skip which they haven't seen in quite a while. conclusions: oxysterol is suitable biomarker for neimann pick c disease and can be used as first line with the genetic confirmation of gene npc and npc at later stage. as modifying treatment with miglustat is available it is important to attempt diagnosing the condition as early as possible and oxysterol level can be used as screening test for neimann pick c when clinically suspected. introduction: biotinidase deficiency is a rare autosomal recessive inborn error of biotin metabolism. biotinidase catalyses biocytin to biotin, a deficiency of which can present with neurological symptoms including hypotonia, seizures, feeding difficulties, lethargy, optic atrophy, and sensorineural deafness. case: a -week-old female presented with a -week history of seizures and developmental delay. examination revealed generalised and axial hypotonia and delayed smile. she continued to have seizures despite initial treatments including levetiracetam and carbamazepine. mri brain was normal and initial interictal eeg on day post admission revealed no significant abnormalities. ambulatory eeg on day showed a focal onset epileptic seizure with sharp and slow wave activity originating predominantly from the left occipito-parietal region. normal investigations included paired plasma and csf glucose, lactate and culture, csf neurotransmitters, microarray and epilepsy gene panel. on day post admission her biotinidase result was reported showing no activity. biotin was commenced at mg once daily and her seizures abruptly stopped. she was discharged home on day and weaned off levetiracetam and carbamazepine. her development was normal at -month follow-up. genetic testing was declined by the family. discussion: biotinidase deficiency can present from the neonatal period up to years of age with a mean age of . months. in recent years our understanding of pathogenic changes in the biotinidase gene has increased through sequencing for novel mutation. this has important implications for families and consideration should be given to offering affected families genetic counselling. treatment is available with oral biotin that rapidly improves symptoms, with seizures usually resolving within days and other symptoms showing improvement within weeks. objective: we present a previously well -year-old boy with a known m. t>c mutation experiencing weeks of vomiting, lethargy and exercise intolerance. method: he was mildly dehydrated, fully consciousness but tachycardic and hypotensive. his ph was . with be - . , hco . and lactate of . . electrolytes, fbc and inflammatory markers were normal. he was admitted to picu for fluid management and sodium bicarbonate. tachycardia persisted during the first hours though he remained stable. he had a good urine output, was on non-invasive monitoring and an echocardiogram was normal. his gas lactate ranged from . to . . the following day he deteriorated with kussmaul respirations, tachypnoea, increased tachycardia and hypotension. venous blood revealed a ph . , pco . hco . be - and lactate . . he became unresponsive with refractory hypotension and multiorgan failure. arterial blood showed a ph . , pco . and a lactate > with indeterminable be or hco . for over hours he had a blood lactate level of > . rhabdomyolysis and acute kidney injury occurred with a ck of > requiring haemofiltration. encephalopathy, with multiple white matter microhaemorrhages on mri brain, and acute liver failure, with thrombocytopenia and coagulopathy, ensued. multiple inotropes were required. the prognosis was very guarded. off sedation he was unresponsive, apnoeic and areflexic however an eeg showed an alpha rhythm which prompted on-going heroic efforts. he required prolonged haemofiltration, ventilation and inotropes with days intensive care. results: the patient made an astounding recovery. he required month of neurorehabilitation and returned to his cognitive baseline, achieving a grades at gcse months later. liver and renal dysfunction resolved. conclusions: this case demonstrates that mitochondrial metabolic crises in melas can be severe and result in profound acid-base derangements. our patient was expected to not survive but uniquely did so without significant neurodisability. neuronal ceroid lipofuscinosis in children from central africa gl fisher, n shah, p watts, ja te water naude noah's ark children's hospital, university hospital wales, cardiff, uk objective: the effective and rapid diagnosis of neuronal ceroid lipofuscinoses (ncls) has become more relevant with the advent of disease-modifying treatments. some ncls have a more stereotyped clinical presentation: we describe two cases in a non-consanguinous family, originally from the democratic republic of the congo, with variant ncl. methods: retrospective case series. results: the index case was initially diagnosed with a focal epilepsy: on review this child had myoclonic seizures in the context of a slow developmental decline, with mild spasticity. ophthalmology was not diagnostically helpful, and a putative diagnosis of ncl was suggested by lymphocyte inclusions. neither enzyme nor dna analysis was available at that stage, although dna was retained. a brother presented with nystagmus and visual inattention in : examination showed myoclonic jerks with a bull's eye maculopathy and an abnormal peripheral retinal vascular leak. mri imaging showed some element of cerebellar atrophy, which on review was also the case with his brother's scans. some lymphocytes ( %) contained fingerprint bodies, suggestive of variant ncl. this was confirmed by dna sequencing which was consistent with a diagnosis of mfsd /cln -related ncl. the identical dna alteration was also found in the index case. conclusions: ncl is not described in children from central africa: the presentation, investigations and laboratory findings and evolution are consistent with that for other children with variant ncl. obesity screening of patients affected by duchenne muscular dystrophy (dmd) in a tertiary paediatric neuromuscular centre and the effectiveness of metformin use in weight control in those with confirmed insulin resistance m neocleous , s spinty , r madhu , p dharmaraj , c degoede , k cooke , c greaves alder hey hospital, liverpool, uk; royal preston hospital, preston, uk objective: to assess whether patients affected by duchenne muscular dystrophy (dmd) who are currently followed up in alder hey hospital, uk are receiving obesity screening when clinically appropriate. to assess whether metformin use in those who are insulin-resistant, has been effective in controlling weight. methods: using the neurology department records, a list of patients with dmd currently under our services and those transitioned to adult services in the last years, was generated. the patient record system, meditech, was used to collect patients' demographics, latest weight/height value and bmi. for patients classified as overweight/obese, completion of obesity screening was assessed as well as initiation of appropriate treatment (metformin). for patients on metformin, the following parameters were collected: weight pre and post-steroids, age of start of excessive weight gain, confounding variables and medications, weight/height/bmi at initiation of metformin and at -monthly intervals, side-effects, cessation of medication and reasoning. results: sixteen out of patients were found to be above the th weight centile. in patients, weight was at or more centiles above height. using the non-dmd standardised bmi classification, patients were identified as being overweight/ obese. patients received obesity screening; were found to be insulin resistant. of those were started on metformin. patients overall were started on metformin. of those exhibited overall weight loss. patients were found to have gained weight and patient showed weight increase up to months post-metformin initiation with subsequent weight loss. conclusions: there is a need for a validated and agreed bmi classification in dmd. screening for insulin resistance in this patient group should be considered for implementation as standard practice, especially if patient is classified as overweight/obese. a larger-scale study would be required to assess the effectiveness of metformin in this patient population. objective: niemann-pick disease (npd) is an autosomal recessive metabolic disorder with a prevalence of . to . / worldwide, marked by varying degrees of lipid storage and foam cell infiltration in tissues, associated with hepatosplenomegaly, pulmonary insufficiency or central nervous system involvement. npd type a and b are allelic disorders caused by mutations in the sphingomyelin phosphodiesterase- gene, smpd ( p . - . ), characterized by a primary deficiency of acid sphingomyelinase activity, resulting in an accumulation of sphingomyelin. in contrast to npd-a, npd-b is the milder, lateronset form, with no neurological involvement. in this paper, we report on three paediatric cases with npd-b who present an atypical phenotype marked by neurological involvement. methods: the three patients were diagnosed at the age of with hepatosplenomegaly. the first is a girl who presented psychomotor regression at the age of and epileptic seizures at the age of . she died at the age of . the second is an years old girl who presented growth retardation, kyphosis and neurodevelopment regression since the age of . the third is a years old boy with a mild phenotype marked by developmental delay and an aggressive behaviour. splenectomy was performed at the age of . results: genetic testing was performed, and all patients presented mutations of the smpd gene, confirming the diagnosis of niemann-pick type b. the c. t>g(p.trp gly) mutation was common in all cases. in addition, heterozygous mutations c. delt(p.ser alafs* ) and del c. _ del were found in the first and second case, respectively. conclusions: all cases present a complex phenotype, marked by psychomotor regression which is atypical for npd type b. the severity of the disease seems to be correlated to the genetic mutation-the most severe phenotype was associated with c. delt. further work is necessary to more clearly delineate genotype-phenotype relationship in npd. objective: acute encephalitis syndrome (aes) is a group of symptoms and signs, which help diagnose encephalitis. since there is no definite treatment for most, role of fluids seems crucial. therefore, the objective of our study was to describe the association of low admission weight and weight loss in the hospital (as clinical marker of dehydration) with outcome of patients of acute encephalitis syndrome. to describe the association between changes in weight and blood lactate levels (at admission and discharge as indicators of hydration and acid base status) and outcome in children with acute encephalitis syndrome. methods: all children aged month to years with fever and altered sensorium and/or new onset seizures from september to september attending kanti children's hospital, kathmandu, nepal were recruited. weight-for-age (wfa) using z score and serum lactate were assessed at admission and discharge. total fluid input and output was monitored daily. results: of the patients, % had low admission wfa or lost weight-after-admission (lwaa) (group a) and % no low wfa or didn't lwaa (group b). there was times risk of death and times risk of bad outcome (death or sequelae) in group a compared to b. bad outcome was significantly associated with less admission wfa, more fluid deficit, and trend for higher admission serum lactate. death was significantly more in those with low wfa, more lwaa, longer illness, more % dextrose and . normal saline, higher sodium and higher urea at admission. methods: we conducted a prospective cross-sectional study recruiting children aged between month to years attending kanti children's hospital, kathmandu, nepal with altered sensorium and two of the following: fever, seizure, focal neurological deficit, csf pleocytosis, electroencephalogram and computer tomography suggestive of encephalitis, over year. in these patients, ve was if csf cell count was < cells/ mm (lymphocyte predominance) and absence of non-viral pathogens in the csf or blood. bm was csf cell count > cells/mm (polymorph predominance) and csf protein > . g/l and csf/plasma glucose < %, and/or positive gram stain and/or bacterial culture. je was ve with ≥ units of anti je-igm in the csf and/or serum. all cns infections were defined as, suspected cases by treating clinician with or without fever with lp showing csf cells > /mm . results: out of , bm was found in %, je % and other causes in %. although who definition of aes was not significantly associated with all cns infections (p= . ), it was significantly associated with ve (p< . , sensitivity %, specificity %, ppv %, npv %) and bm (p< . , sensitivity %, specificity %, ppv %, npv %). conclusion: we validate who aes definition of bm and ve as a significantly useful screening tool for children with these diseases specially in resource poor settings, endemic areas and where confirmatory tests were not easily available. objective: the aim of this case series is to raise awareness of this autosomal recessive encephalopathic syndrome that presents after birth in the multi-ethnic population in england. although aicardi goutieres syndrome (ags) is rare, its importance lies in the fact that that its presentation may be mistaken for other neurological conditions associated with congenital infections. results: all of the patients were seen in our paediatric outpatient neurology clinic. the age of presentation ranged from the neonatal period to the first weeks of life. all patients were of pakistani origin and were from consanguineous marriages. they all had an uneventful antenatal period with normal birth weight and head circumference. the initial presentation seems to be of poor feeding and irritability. further observations include truncal hypotonia, limb spasticity intermittent dystonic posturing coinciding with the onset of poor head growth and chilblains. of our patients had abnormal movements with diffuse slow wave electroencephalogram activity. nystagmus with visual inattention and poor visual acuity were a typical finding in all of them by the age of months. the ct scans showed cerebral calcification in all of them and mri suggested brain atrophy. the most striking abnormality was a raised level of csf interferon-alpha (infa) in an absence of other infection or metabolic disorder. csf inf a is a reliable diagnostic marker and can thus be used to differentiate patients with ags from other conditions. three of our patients had the same gene mutation, rnaseh c. conclusions: ags is a rare disorder, however in patients from consanguineous marriages that depicts microcephaly, poor tone and global developmental delay, diagnosis of ags should be considered. as ags is a progressive neurological condition, early support and prognosis can be provided for affected families. t thomas , ht yeo , sv barron , paediatric neurology, kk hospital, kampong java, singapore; university of newcastle, newcastle, uk objective: we report the association of mild encephalopathy with a reversible splenial lesion (mers) with a primary dengue virus infection. this case implies the existence of a wider spectrum of neurological involvement in dengue virus infections. case description: a -year-old girl presented with acute confusion, dysarthria and bilateral limb weakness following a -day history of fever. symptoms resolved after hours; neurological examination was completely normal. she later experienced a second episode of slurred speech, dysphasia and right arm weakness which lasted an hour. a contiguous lesion involving the genu, body and splenium of the corpus callosum and bilateral posterior periventricular white matter was evident on the mri brain scan, with restricted diffusion and t -hyperintensity. cerebrospinal fluid analysis showed no inflammation and polymerase chain reaction assay for respiratory viruses was negative. her clinical and radiological features were consistent with mild encephalopathy with a reversible splenial lesion (mers). on day of admission, she developed a generalised maculopapular rash with leukopenia (white blood cell count . /l) and thrombocytopenia (platelets /l). serology (igm/igg) for dengue virus was negative and a positive dengue ns antigen was thus indicative of a primary dengue virus infection. she was given fluid rehydration and advised bedrest. at discharge (day admission) she was well with no sequelae. conclusions: mers is a mild form of virus associated encephalopathy (vae), which are a spectrum of clinico-radiological syndromes associated with common childhood viral infections. the clinical and neurological symptoms in our patient occurred early in the course of illness (typical to vae) as opposed to after or late in the illness as is typical for postinfectious encephalopathy syndromes associated with dengue virus infections (e.g., acute disseminated encephalomyelitis). shouldering the burden of sepsis norfolk and norwich university hospital nhs trust, norwich, uk; sheffield children's hospital, sheffield, uk aim: we report a case of a -year-old boy who presented with right brachial plexus neuritis secondary to meningococcal group b sepsis. brachial plexus neuritis or neuralgic amyotrophy (also known as parsonage -turner syndrome) is a rare disorder affecting the brachial plexus. it can be caused by various infectious agents and is characterized by acute onset of intense pain in the shoulder and arm followed by weakness, sensory loss and atrophy. methods: a -year-old boy, previously fit and well presented to the emergency department with an acute onset of excruciating pain in his right shoulder, radiating down his arm and hand with associated paresthesia. few hours later, he developed an evolving non-blanching purpuric rash to the chest, back, shoulder and right arm. he gradually developed weakness in the right arm and sensory loss over the ulnar aspect of the right hand. he then began to complain of headache, photophobia with subsequent vomiting. he was treated for meningococcal sepsis with intravenous ceftriaxone and received three fluid boluses for hypotensive shock with vitamin k correction for his associated coagulopathy. he received analgesia for right shoulder pain. results: blood cultures and blood pcr confirmed neisseria meningitidis group b type, nt subtype. mri of the shoulder showed inflammation consistent with brachial plexus neuritis with motor impairment affecting the right side c to t myotomes and sensory impairment involving the right c dermatome. the patient was treated with oral prednisone and gabapentin whilst receiving neurorehabilitation from physiotherapy and occupational therapy. he made a very pleasing recovery after few months and currently has no motor or sensory deficit of his right shoulder and arm. conclusion: brachial plexus neuritis should be considered in the differential when a child presents with sudden onset pain and weakness of the shoulder and arm. in review of literature, brachial plexus neuritis associated with meningococcal infection has not been described previously. to the best of our knowledge, this is the first reported case of its kind. introduction: previous cohort studies on paediatric multiple sclerosis (ms) have reported very low frequencies for a primary progressive ms course (ppms) ranging from to %. an age-dependent increase in the rate of primary-progressive courses has been well described in the adult ms population. objectives and methods: we describe five patients presenting prior to the age of years and fulfilling the mcdonald criteria for ppms. patients were identified from the national hospital for neurology and neurosurgery (nhnn) and the uk childhood inflammatory demyelination (uk-cid) network. results: patients presented at a median age of years (range: - y), with at least -year history of progressive deterioration of their balance (n= ) or progressive worsening of lower limb function (n= ). over time, all patients developed lower limb spasticity, three patients developed cognitive difficulties, three had visual problems, three had bladder involvement. median edss at years was (range: to ). cerebrospinal fluid (csf) oligoclonal bands were detected in all patients tested. dissemination in space on first mri was seen in all patients with peri-ventricular (n= ), cortical juxtacoritcal (n= ), infratentorial and spinal cord (n= ) lesions. all patients showed new lesions on repeat mri imaging. contrast enhancement was present in out of ( %) during the disease course. three patients had genetic investigations to exclude other mimics. a trial of iv methylprednisolone was unsuccessful in patients. all patients were on symptomatic treatment for spasticity and pain, including oral/intra-thecal baclofen, gabapentin and sativex. conclusions: given the rarity of primary progressive course in paediatric ms, presentation with progressive neurological symptoms and signs in young people should prompt evaluation for genetic causes. nevertheless, our five patients presented with clinical, mri and immunological features consistent with a diagnosis of primary progressive multiple sclerosis. objective: clinical course in nmdar-antibody encephalitis is variable and difficult to predict. we aimed to identify clinical features in the presenting disease episode associated with worse functional outcome and/or relapsing disease course. methods: systematic review of the literature was conducted to identify published cases with individually reported data. clinical and treatment characteristics at first episode, outcome at ≥ months, and monophasic vs. relapsing disease course were recorded. results: cases were identified from articles ( % female; % ≤ years old at onset). % received immunotherapy at first episode: corticosteroids in %, ivig in % and therapeutic apheresis in %. second-line immunotherapies were used in % at first episode, most frequently rituximab ( %), cyclophosphamide ( . %), or both ( . %); emerging second-line treatments (intravenous/intrathecal methotrexate, subcutaneous/intravenous bortezomib, intravenous tocilizumab) were used in . %. life-threatening adverse events or death related to immunotherapy occurred in . %. in a univariate analysis of cases with ≥ months follow-up data, poor final outcome (defined as modified rankin scale [mrs] score - ) occurred in % and was associated with very young or elderly age at onset, movement disorder, decreased consciousness, autonomic dysfunction, mechanical ventilation, higher mrs score in the acute phase, longer hospital stay, extreme delta brush on eeg, abnormal mri, csf pleocytosis and elevated csf protein (all p< . ). a subset of cases followed up for ≥ months were analysed to identify associations with relapsing course, which occurred in %. in univariate analysis, factors protective against relapse were < days delay in first-line immune therapy, therapeutic apheresis, ivig, rituximab and other second-line treatments at first episode (all p< . ). conclusions: worse functional outcome of nmdar-antibody encephalitis is associated with very young or elderly age at onset and worse disease severity in the acute phase. relapsing disease course is associated with delayed or insufficient early immunotherapy. objective: we describe three children with familial hemophagocytic lymphohistiocytosis (fhlh), who presented with an atypical chronic demyelinating illness. an initial working diagnosis of 'clippers' (chronic lymphocytic inflammation with pontine perivascular enhancement responsive to steroids) was made in two cases. methods: retrospective case series. results: case : an -year-old girl presented with diplopia and squint with evolving ataxia. mri showed multiple enhancing white matter lesions in the pons, medulla and cerebellum raising the possibility of 'clippers'. her symptoms and neuroimaging responded only partially to treatment with ivig and steroids. genetic testing revealed a compound heterozygote mutation in the rab a gene consistent with griscelli syndrome type with fhlh. case : a -year-old boy had nocturnal headaches with a squint evolving over time. mri showed demyelination and swelling predominantly in the cerebellum. significant radiological resolution with steroids was followed by recurrence of demyelination on weaning steroids. a brain biopsy lesion was consistent with 'clippers'. genetic testing revealed a heterozygote variant in the stxbp consistent with fhlh v. case : a -year-old girl had a -month of intermittent fevers, deranged lfts and recurrent bilateral optic neuritis responsive to steroids/ivig. mri brain showed multiple areas of demyelination largely in the subcortical white matter. oligoclonal bands were positive in the csf. she developed a pleural effusion, high ferritin, deranged coagulation, lymphadenopathy and a rash found to be a cutaneous t-cell lymphoma. genetic testing revealed a homozygous mutation in the unc gene consistent with fhlh . all three children are awaiting stem cell transplantation. conclusions: fhlh can present with an isolated atypical demyelinating illness or with neuroimaging suggestive of 'clippers'. there may be no signs of systemic inflammation. we propose that all children with atypical recurrent cns inflammation and presentations consistent with 'clip-pers' undergo genetic panel testing for fhlh and natural killer cell functional testing. objective: the most common paediatric presentation of mog-ab disease is with acute disseminated encephalomyelitis (adem), or optic neuritis (on). with increasing recognition of the association of mog-abs with seizures in children, we present a case series of affected children. methods: retrospective anonymised case note review of affected children presenting between - , from uk paediatric neurology centres. patients were followed up for median of years (range . - ). results: cases ( female) of mog-ab-positive epilepsy patients were identified; median age at first presentation was (range - y). the most common preceding mog-ab disease was multiphasic adem (mdem ; adem ; adem-on ; adem and transverse myelitis ; nmosd ; on ). median time to recurrent seizure onset was months (range - ). focal epilepsy/seizures were most common ( / ). eeg abnormalities were found in / patients, all demonstrated slowing/encephalopathy which was generalised or focal; epileptiform discharges were reported in patients. brain mri was abnormal in all patients ( with multifocal hazy/ poorly marginated lesions involving grey and white matter; leukodystrophy-like pattern, cortical encephalitis and reported with subtle changes in brainstem). patients received immunotherapy, all required at least anti-epileptic drug (aed) and children continue to have on-going breakthrough seizures. median mrs (modified rankin scale) at last follow-up was (range - ), indicating no significant disabilities despite symptoms in clinical examination, age appropriate behaviour and development. conclusions: focal epilepsy is more common and more likely to follow an mdem presentation in children with mog-abs. with the relapsing nature of mog-ab disease there is a high risk of long-term cognitive impairment. further preclinical studies are urgently required to determine whether this epilepsy is due to ongoing inflammation or as a result of the mri changes commonly seen. this will help inform future management decisions regarding immunotherapy. poster no. maternal mid-gestation cytokine dysregulation in mothers of children with autism spectrum disorder autism spectrum disorder (asd) is a developmental disorder characterised by a spectrum of deficits in social interactions/ communication combined with stereotypical, repetitive behaviours. recent evidence suggests maternal immune activation (mia) during pregnancy may predispose offspring to asd. the aim of this study was to examine the mid-gestation cytokine profile in mothers of children with a subsequent asd diagnosis. maternal-child dyads were recruited to a prospective population-based pregnancy study; the scope study, new zealand. children with confirmed diagnosis of asd at years were enrolled in the nested cohort, along with matched neurotypical controls. cytokine concentrations (pg/ ml; mean [sem]) were examined in maternal serum samples taken at and weeks gestation using mesoscale discovery proinflammatory, cytokine and chemokine assays. of mothers recruited to the scope-nz study, children completed follow-up and had reported asd at years. these were analysed alongside neurotypical matched controls. downregulation of il- a occurred at weeks gestation in cases when compared to controls (mean [sem]). -way anova revealed a relationship between il- a concentration and weeks' gestation f( , )= . ; p= . , and also il- a concentration and asd status f , )= . ; p= . . posthoc uncorrected fisher's lsd revealed a significant difference between cases (- . [ . ] ) and controls (- . [ . ]) at weeks gestation p= . . ifnc is also downregulated at weeks gestation in cases when compared to controls. -way anova revealed a relationship between ifny concentration and weeks gestation f( , ) = . ; p= . . posthoc uncorrected fisher's lsd revealed a significant difference between cases ( . [ . ] ) and controls ( . [ . ]) at weeks gestation p= . . we have shown altered cytokine expression at weeks gestation in mothers of children who progress to develop asd. this adds to the growing body of evidence that maternal immune regulation may play a role in foetal neurodevelopment. objective: acute disseminated encephalomyelitis (adem) is an immune mediated inflammatory cns disorder, predominantly affecting white matter, with a wide differential ( ). here we describe a rare mimic of adem that is essential to consider in order to avoid a catastrophic outcome. case history: a -year old girl presented with a day history of confusion, dysarthria, ataxia and left-sided squint, preceded by weeks of general malaise and headache. examination confirmed encephalopathy and a left third cranial nerve palsy. mri brain was suggestive of adem and mri spine was normal. a recommended work up for adem was performed. rapid resolution of her symptoms occurred following intravenous methylprednisolone for days. years later she presented with acute left lower limb ischaemia and underwent emergency embolectomy of a popliteal arterial obstruction, with myxomatous material identified. preoperative echocardiogram confirmed a large atrial mass which was surgically removed. pathology confirmed an atrial myxoma (am). retrospective review of her initial mri images concluded that embolic phenomena from the am was the most likely explanation of her first presentation. conclusions: am is a very rare primary cardiac tumour and left sided am can embolise to the cerebrovascular system. early identification of am is important as, untreated, it can cause multiple embolic events and sudden cardiac death. % of adults with am present with neurological symptoms and this can mimic multiple sclerosis. am presenting with acute neurological symptoms masquerading as adem in paediatrics has not been previously reported. careful follow up is essential as late neurological complications (including cerebral arterial aneurysms) are recognised. this case highlights that adem is a diagnosis of exclusion and that mimics for acute focal neurology with encephalopathy and t hyperintensities on mri require careful consideration, including embolic phenomena. clinical examination alone does not exclude am and consideration of echocardiography is recommended. we describe an year old boy with a rare demyelinating disorder: balo's concentric sclerosis. a routine optician review raised concerns about papilloedema in an asymptomatic child. a ct brain identified a well-demarcated high-density lesion in the left posterior frontal lobe with surrounding oedema. mri brain with contrast identified the lesion had central rim enhancement and slight diffusion restriction. alternating layers of high and low signal intensity gave it an 'onion bulb' appearance. the differential included balo's concentric sclerosis, a single demyelinating lesion, or a tumour. routine blood tests were unremarkable. csf oligoclonal bands were normal as were other csf indices. markers of immunology, including ana, anca, anti-mog, anticardiolipin, and aquaporin antibodies were all negative, with normal complement levels. esr was mildly elevated when taken during an episode of acute tonsillitis. imaging months later demonstrated an increase in the size of the lesion. a biopsy revealed inflammatory demyelinating pathology mediated by a perivascular and parenchymal t-and b-lymphocyte infiltrate and macrophage activity with associated demyelination lacking a perivenular distribution. there was no evidence of neoplasia, vasculitis, granulomas, or viral infection (including negative pcr testing). no treatment with corticosteroids was given, and the child remained asymptomatic. imaging, at months from presentation, revealed the lesion had substantially reduced in size. there was still a rim of enhancement. as with all single demyelinating lesions, it is difficult to predict the clinical course. we opted to adopt a 'wait and see policy' and offer surveillance imaging and clinical review. balo's concentric sclerosis is a rare demyelinating disease, characterized by concentric lamella of alternating demyelinated and partially myelinated tissues. mri shows one or more concentrically mulitlayered ring-like lesions, usually in the cerebral white matter. most case reports describe cases predominantly in young adults, with few reports of cases in children. objective: cognitive and acquired neurodevelopmental deficits have been reported in children with opsoclonus-myoclonus syndrome (oms) and are associated with more severe and relapsing disease course. however, there is a paucity of data regarding cognitive dysfunction in children with stable neurological disease. we report on serial cognitive assessments of children with oms demonstrating evolving cognitive dysfunction with milder disease course. methods: retrospective analysis of clinical features at presentation, investigations, treatments, clinical course including relapses and neuropsychological testing. results: four children (m:f : ) diagnosed with oms between and months were followed up for to years. neuroblastoma was identified in one child. oms severity scores ranged between to / at presentation. patients underwent immunotherapy in accordance with european oms protocol. all patients were in remission by months (range - mo), with treatment maintained for year. one child remained relapse free whilst other children had one clinical relapse which was immunotherapy responsive again. in all cases, progressive cognitive dysfunction was reported despite being in remission and stable off treatment for months (range of - mo; oms score / and one / ). sequential neuropsychological testing scores showed mean declines in fsiq of ( - ) , viq of ( - ) and piq of (- to ) between time of oms remission/stable disease and longer term follow-up time point ( - y). conclusions: our cases demonstrate progressive cognitive dysfunction occurring in children with oms who have a milder disease and long after completion of treatment. children continued to develop but with a widening gap in comparison with peers. damage to cerebellar-cortical circuits at onset of the disease that becomes more apparent with time or indeed persistent ongoing low grade inflammation may explain this deterioration. the past decade has seen increasing cases of acute flaccid paralysis (afp) associated with enterovirus d (evd ) infection. it presents in clusters approximately every years. we report three cases of afp due to evd that presented in november-december . cases: two patients developed afp following a viral upper respiratory tract infection and one developed lower limb hypotonia and weakness during an inpatient admission with refractory epilepsy. two required admission to the paediatric intensive care unit for respiratory compromise and required tracheostomy ventilation. mri showed acute flaccid myelitis (afm) consistent with evd in / cases, consisting of central cord t hyperintensity in the cervical region over multiple segments with subsequent enhancement of the thalamus and cauda equina on follow-up imaging in one patient; and ventral surface enhancement of the conus and cauda equina in another. mri in the third patient was normal. electromyography and nerve conduction studies were normal in two patients but revealed a severe generalised motor axonal poly-neuronopathy in the third. two patients received intravenous immunoglobulin, corticosteroids or plasma exchange therapy and showed slow motor improvement in a distal to proximal pattern. the one patient without mri changes had received long-term oral corticosteroids but received no additional treatment and returned to baseline neurological function within weeks. discussion: our cases demonstrate the range and clinical course of peripheral neurological presentations secondary to evd infection. we highlight the importance of sending repeat samples from multiple sites when the diagnosis is suspected, given that initial samples tested negative, and of sending samples to a national surveillance laboratory for confirmatory testing. ongoing national and multinational surveillance studies will hopefully continue to advance our understanding and treatment of this disease. poster no. nmda receptor encephalitis a potential complication of biologic therapy for juvenile idiopathic arthritis? n abbassi , t rossor , r close , a skippen , k armon , p bale , g ambegaonkar paediatric neurology, addenbrookes hospital, cambridge, uk; paediatric rheumatology, addenbrookes hospital, cambridge, uk a -year-old girl presented with headaches, confusion and agitation, followed by seizures. she had presented at months with polyarticular arthritis and was managed over subsequent years on methotrexate alone ( y) with added etanercept (anti-tnf alpha, y), tocilizumab (anti il- , y) and abatacept (cd / t-cell modifier, y prior to presentation). flares of disease following a period of control necessitated the changes in monoclonal antibody therapy. at this presentation she was managed on methotrexate, abatacept and mg prednisolone for polyarticular rheumatoid factor negative juvenile idiopathic arthritis (jia). at presentation the patient was agitated, non-verbal and had one generalised seizure. examination demonstrated acute confusion, with increased tone, brisk reflexes and bilateral clonus in her lower limbs. iv ceftriaxone, acyclovir and clarithromycin were commenced. full blood count, liver function tests and inflammatory markers were unremarkable. infective serology was non-contributory. csf was unreactive with normal protein and jc virus pcr negative. mri brain days after presentation showed increased t and flair signal intensity in the white matter of the right parietal lobe, in-keeping with an inflammatory process. eeg showed diffuse slowing with delta brush. she commenced iv methylprednisolone, followed by prednisolone. she continued to deteriorate and underwent plasmapheresis for treatment of presumed nmda receptor encephalitis, subsequently confirmed by anti-nmda receptor antibodies in serum and csf. she was commenced on rituximab (b-cell depletion, anti-cd ), and continued to undergo plasmapheresis over the course of weeks. she gradually improved and was discharged home after a -week inpatient stay. several adult and two paediatric cases of nmda receptor encephalitis are reported in patients on biologic therapy for autoimmune disease. autoimmune diseases are more common in those already affected by one autoimmune condition. it is unclear what contribution an autoimmune history or immunomodulation made on the development of this condition. introduction: the incidence of acute flaccid myelitis (afm) associated with enterovirus infection occurring in biennial clusters since has been reported in the us (bmj , ; :k ) and recently in europe. previously, cases with transverse myelitis (tm) with anterior-horn cell or peripheral nerve involvement have been collectively termed tm-plus (neurology ; ;s -s ). we aimed to identify cases of tm-plus from a retrospective cohort of children to identify potential cases of 'undiagnosed' afm, and to evaluate the clinical and radiological features alongside long-term outcome. methods: consecutive cases of children (< y of age) who presented to a large paediatric neurosciences centre from to fulfilling the transverse myelitis consortium working group criteria modified for the paediatric population (neurology ; : - ) were retrospectively evaluated for additional features of anterior horn-cell involvement (fulfilling criteria for afm (current treat options neurol ( ) : )) or peripheral nervous involvement; and were collectively evaluated with the contemporary tm-plus cohort ( ) ( ) ( ) . results: cases of tm were identified, of which were excluded from further analysis; ms (n= ), adem (n= ). cases of tm-plus were identified, before and after, all associated with a viral prodrome. flaccidity (n= ) and asymmetry (n= ) was noted at presentation, with corresponding nerve conduction studies revealing a motor axonopathy with sensory sparing (n= ) and anterior horn cell involvement confirmed in cases. all cases with anterior horn cell involvement had poor outcomes while both cases with good outcomes had peripheral involvement and normal mri brains. conclusions: tm-plus was detected in our cohort from to with biennial clusters noted in and . the clinical presentation, investigations and long-term outcomes appear consistent in both groups. acute necrotising encephalopathy is more severe when associated with influenza background and objective: acute necrotising encephalopathy (ane) is a rare but potentially life-threatening condition associated with viral infections. a familial and recurrent form (ane ) has been identified by mutations in the nuclear pore ran binding protein (ranbp ). we report the morbidity and mortality when associated with influenza infection. methods: we performed a review of paediatric ane cases from to evaluating the clinical, biochemical, microbiological and neuroimaging appearances as well as outcomes. results: the cohort comprised children ( boys), age ranging from months to years months (< y n= ), of which had a confirmed genetic diagnosis and were ranbp negative. there were episodes of encephalopathy, with recurrences in cases ( ane ). of these episodes had infectious aetiology identified: coronavirus n= , parainfluenza n= , adenovirus n= , h influenzae n= , influenza (h n n= , h n n= ). clinical features of fever and encephalopathy were consistent ( %), and seizures and sixth nerve palsies prominent ( % each). csf revealed absent pleocytosis, normal-elevated protein and negative virology. symmetric involvement of the thalami bilaterally was present in all cases, and all ane cases were associated with haemorrhage and external capsule/claustrum involvement ( % specific and sensitive). the outcome following influenza infection was striking, death n= , vegetative n= , limb motor and movement disorder n= . of these cases had previous episodes of encephalopathy with noninfluenza infection and did have recovery, albeit with moderate to severe disability. all cases were never immunised against influenza infection and suffered grave outcomes. conclusions: influenza infection in ane has the poorest outcome therefore vaccination should be a mandatory consideration for the known cases of ane. introduction: acute flaccid paralysis (afp) is characterized by a rapid onset of limb weakness. we describe six cases of afp in children aged months to years of age who presented to a tertiary paediatric neurology service in the east of england over a -year period from november -november . retrospective analysis of the six cases was performed, reviewing their clinical course and management, as well as their radiological, electrophysiology and laboratory results. results: case , a -month-old boy, presented in november with a viral urti requiring escalation of care to picu due to a significant respiratory compromise. only on subsequent recovery was the child found to have a unilateral upper limb flaccid paralysis. this child was positive for enterovirus serotype d . cases and in our series presented in with acute weakness of the lower limbs with an mri brain and spine showing enhancement of the lumbar spinal roots. both have made a good recovery. enterovirus was not detected in either case. the final three children in our series presented in autumn with weakness of a unilateral upper limb following a viral urti, with all three being positive for enterovirus. unfortunately, they have shown minimal recovery of motor function of the affected upper limb. one child, a year-old girl, showed a more severe clinical course involving a prolonged period of intensive care and a tracheostomy for long-term ventilation. she has undergone neurorehabilitation and an upper right arm nerve transfer. conclusions: in our case series, four patients presented with an acute viral urti associated with an upper extremity weakness, and subsequently all four were positive for enterovirus. clusters of acute limb weakness in paediatric patients have been linked to outbreaks of non-polio enteroviruses, termed acute flaccid myelitis (afm). objective: to present an interesting case of recurrent anti-mog demyelinating disease and provoke discussion regarding possible immunomodulatory therapy. methods: a year old girl presented, initially at years of age, with headache and vomiting. she was initially treated as atypical tuberculosis meningitis based on csf cell counts, but later developed a th nerve palsy and was diagnosed with optic neuritis. anti-mog antibodies were positive and they were commenced on iv methylprednisolone. she clinically improved and was discharged on weaning oral prednisolone, antibodies were negative following treatment. results: a few months later she had her first relapse, with an acute decline in visual acuity. an mri showed new lesions in her optic chiasm and both optic nerves with associated bilaterally reduced visual evoked potentials. she was again treated with iv methylprednisolone, with rapid improvement, followed by a switch to weaning oral prednisolone. anti-mog antibodies were negative following treatment. she was symptom free for years until her second relapse, when she presented with facial palsy, swallowing difficulties and slurred speech. mri showed brainstem and periventricular white matter demyelination, with positive anti-mog antibodies. she was again treated with iv methylprednisolone, followed by oral prednisolone, but was maintained on low dose prednisolone as her anti-mog antibodies remained weakly positive despite being symptom free. these were stopped at patient request due to low mood and abdominal pain in february , with no recurrence of symptoms as yet. repeat mog antibodies have been sent and are awaited. conclusions: this case shows an interesting relapsing/remitting pattern of anti-mog demyelinating disease, which appears to be very steroid responsive, however on her second relapse her anti-mog antibodies remained weakly positive despite steroid therapy. discussion is welcomed on whether prophylactic immune modulation therapy should be considered with this child, such as azathioprine, mycophenolate mofetil or rituximab. objective: to highlight the utility of early mr imaging in children presenting with acute severe encephalopathy and to consider whether there might be a subgroup of children with myelin oligodendrocyte glycoprotein antibody (mog-ab)associated demyelination who might be candidates for early intense immunomodulation. methods: we report two cases with mog-ab-associated acute demyelination who relapsed with new neurological symptoms after initial steroid therapy had been discontinued. results: two toddlers were initially admitted to intensive care with acute encephalopathy and acute symptomatic seizures. both had an initial ct head during picu admission; one was reported normal; the other was suggestive of diffuse cerebral oedema. both children improved with supportive care only and were discharged home within a week. both children presented again over the following to weeks with new neurological symptoms but without encephalopathy. mr imaging demonstrated demyelination and they were treated with steroids. both children relapsed as steroids were being weaned and/or stopped. repeat mr imaging at this stage demonstrated new enhancing lesions. it was subsequently found that both children were mog-ab positive. conclusions: reliance on cranial ct imaging in the context of a young child with acute encephalopathy and seizures can be misleading. prediction of the severity of mog-ab-associated demyelinating syndrome at onset is challenging. mr imaging in the acute phase with early follow up imaging may identify this subgroup. case: hb is a year old boy who presented with sudden onset diplopia and painful ophthalmoplegia, following a day history of frontal headache. on examination, he had a left vith nerve palsy, partial left iiird nerve palsy and normal right eye movements. the remainder of his neurological and general systems examination was normal. his initial ct head scan and pre-contrast mri of the brain were normal. he was discharged from the local hospital following normal blood tests and imaging. clinical course: nine days later, his symptoms worsened including severe vomiting, worsening frontal headache and photophobia. he was treated for a possible underlying infective cause with ceftriaxone and acyclovir. given the broad underlying differential diagnoses, hb had extensive infectious and immunological blood workup which was unremarkable. a repeat mri brain scan with contrast revealed a lesion in the left cavernous sinus, possibly of vascular origin. differentials included cerebrovascular venous sinus thrombosis, tumour and inflammatory causes such as tb, sarcoid and zoonoses. hb continued to be conservatively managed and completed his course of ceftriaxone and acyclovir. repeat mri imaging months later showed some resolution of the lesion and a diagnosis of tolosa-hunt syndrome was provisionally made. hb's symptoms continued to improve and further repeat mri scan months later showed ongoing resolution of the lesion. discussion: according to the ichd- beta classification of tolosa-hunt syndrome, hb fulfilled the diagnostic criteria given his presentation of unilateral headache, granulomatous inflammation of the cavernous sinus on mri, palsies of ipsilateral iiird, ivth and vith cranial nerves. steroid use has been reported to be beneficial although more evidence is required in the paediatric population, refractory cases may respond to azathioprine and methotrexate. objective: we present a case of internuclear ophthalmoplegia unresponsive to steroid treatment which clinically improved with folinic acid supplementation. method: a retrospective chart review. results: our patient presented with acute internuclear ophthalmoplegia, ataxia and bilateral ptosis. she had a background of hypoplasia of the corpus callosum and optic atrophy with visual impairment, learning difficulties and asd. mri brain demonstrated symmetrical high signal intensity in the region of the medial longitudinal fasciculus and periaqueductal grey matter. she was investigated to exclude an inflammatory cause and was treated with high dose steroids. follow up mri did not show any improvement post steroids and there was no clinical improvement. subsequent csf investigations showed a low level of -methyltetrahydrofolate of nmol/l (normal range - nmol/l). she was commenced on folinic acid mg once daily and her symptoms improved. on follow up her eye movements had significantly improved as had her ptosis. follow up mri brain showed partial resolution of the areas of abnormal signal in the periaqueductal grey matter. conclusion: internuclear ophthalmoplegia is a sign usually associated with an inflammatory or demyelinating cause. our case did not respond to steroid treatment but has associated low levels of -methyl tetrahydrofolate and has responded to treatment with folinic acid. this is sometimes associated with underlying mitochondrial disorders but muscle biopsy in this case did not show any evidence of mitochondrial disease. mri brain has shown partial resolution of the abnormal signal in the periaqueductal grey matter. introduction: paediatric intracranial aneurysms are rare. the pattern of disease is different to that in adults and there is far less literature available. i provide a case as an example of the presentation and progress of a child with a dissecting vertebrobasilar artery aneurysm. presentation: -year-old boy presented to his local hospital with sudden-onset headache, photophobia and vomiting. bloods and observations were normaldischarged. symptoms recurred more severely the following day. managed as meningitis. lumbar punctures were 'bloody' and considered failed. mri brain days post-admission demonstrated a vertebrobasilar aneurysm. transferred to the regional neurosurgical centre. transfer to neurosurgical/neurovascular centre: cerebral angiography revealed dissecting vertebrobasilar aneurysm ( mm). fusiform component extending beyond aici/ pica origins. wide-necked saccular component. procedure and progress: loaded with aspirin and clopidogrel. underwent endovascular procedure the following daycoil embolization, flow-diverting stent to the aneurysm. ongoing low dose dual anti-platelet therapy. made an excellent recovery with no neurological deficits. further imaging -x-ray cervical spine for possible arcuate foramen or atlantoaxial instability, normal. ultrasound liver/spleen, normal. discharged home days post-transfer. patient background: past medical historyunder the gp for months of headaches. traumasignificant fall from bicycle years before with forced lateral flexion of the neck. posited that this may have been a contributing factor in aneurysm development. no significant family history. lifestylean active boy, enjoys weightlifting and motocross. weightlifting also posited as a contributing factor. conclusions: i provide a case which i hope will raise awareness of paediatric intracranial aneurysms and stimulate discussion concerning their management and aetiology. poster no. cavernoma in children: cddft experience of two cases d jayachandran, s chandraiah darlington memorial hospital, cddft, darlington, uk aim: to report two cases of congenital cavernoma diagnosed in children presenting with neurological symptoms. cases: case : -year-old female presented with multiple left facial focal seizures in the form of twitches with full awareness. there was no family history of epilepsy. a typical event was captured lasting seconds during the awake eeg and was reported as focal seizure arising from right hemisphere. an mri brain scan showed popcorn balls lesions within the frontal/fronto parietal lobes, one on each side (right>left) with evidence of bleeding. she remains seizure free on carbamazepine. ccm gene results are awaited and neurosurgeon opinion was not for intervention and for local follow up. case : -year-old male had presented with confusion at years of age. he also had transient loss of vision, vomiting, headaches with a few minutes unresponsive episode during admission. with a diagnosis of migraine, he had an mri brain scan as op that showed multiple cavernous haemangiomas in the cerebrum and cerebellum with the largest demonstrating a fluid level in the left parieto-occipital region. family history revealed that father had seizures secondary to brain cavernomas. he was positive for krit (ccm ) mutation. neurosurgeons advised active monitoring and he presented again at years with focal onset seizures with impaired awareness. eeg was normal but mri showed a new cavernoma in the left temporal horn with bleeding. he remains well on carbamazepine with a plan for yearly mri scans. conclusions: congenital cavernomas of brain can be sporadic or familial, can be multiple and in any location including brain stem and can result in physical disability secondary to bleeding. in the majority of cases bleeding is spontaneous and diagnosed on mri scans after a neurological event. objective: foxp -related intellectual disability syndrome is characterised by developmental delay, variable physical features and autism. diagnosis has increased with better access to broad genetic testing. we present a case report of a child with foxp mutation whose presentation was notable for significant cerebral venous ectasia. case: child s presented aged year with gross motor delay (not sitting or weightbearing) along with relative macrocephaly ( st- th centile), strabismus and prominent superficial forehead veins. intracranial imaging was arranged in which ct angiogram raised a possibility of an arteriovenous fistula. subsequent catheter angiography excluded this but demonstrated extensive tortuous cerebral venous ectasia. the venous ectasia was not felt to explain her developmental difficulties. initial genetic testing including microarray and pik ca and pten analysis was normal. s made some developmental progress but remained globally delayed compared with her peers. reanalysis of her dna against a panel of genes associated with intellectual disability identified a de novo heterozygous pathogenic variant in the foxp gene, c. c>t, p.(arg ter) (east anglian medical genetics service). discussion: foxp acts as a transcription factor and is likely to be involved in the development of many different tissue types. a wide range of genetic aberrations affecting foxp , including point mutations and large structural anomalies, lead to overlapping clinical phenotypes. this patient demonstrates many relevant clinical features including developmental delay with autistic traits, relative macrocephaly with a prominent forehead, strabismus and early gross motor developmental delay. however, descriptions of associated neurovascular anomalies in foxp syndrome are scarce, with a single case report recording a venous angioma and none of cerebral venous ectasia to our knowledge. foxp follow up studies and whole exome or genome sequencing may help determine whether there is an additional genetic cause for this child's cerebral venous ectasia or whether it is foxp related. objective: a rcpch guideline was published to increase awareness of stroke in children and standardize best practice. the need for urgent (within h) ct angiography in children presenting with suspected stroke and criteria for thrombolysis were set out. we aimed to review the acute management and investigation of pais in children ( mo to y) since the introduction of the guideline with an emphasis on identifying candidates for thrombolysis. methods: retrospective notes review in a single regional neurology centre over a -month timeframe. results: eighteen cases were identified ( f: m) with two mortalities. age range months- years (mean . y). / presented in peripheral hospitals and / in the regional centre. no cases had pednihss score documented on presentation. / had dedicated vascular imaging (cta/ mra) on initial imaging. / presented with a hemiparesis, / with seizures, / dysphasia, / headache, / ataxia. / cases had a stroke post cardiac surgery, / idiopathic stroke, / post varicella angiitis, / arterial dissection, / cardiomyopathy and / embolic stroke as a complication of central line insertion. / presented with an acute hemiparesis with a clear time of onset. no cases received thrombolysis. / was imaged within hour of presentation. / had vascular imaging (mra or cta) on presentation. / cases was discussed acutely with paediatric neurology; this case was not suitable for thrombolysis due to cardiomyopathy. in retrospect the other / cases were suitable candidates for thrombolysis. conclusions: the results highlight the continued need for enhanced awareness of paediatric stroke as a medical emergency. most acute strokes will present to peripheral hospitals. therefore, there is a need for regional multidisciplinary integrated pathways amongst emergency department physicians, paediatricians and radiologists to ensure prompt vascular neuroimaging and discussion with a paediatric neurologist about the possibility of thrombolysis in suspected pais. poster no. 'a nudge, a fall and a weakness'a common but missed cause of paediatric stroke r shyam , r bahri , , a kumar , v jain department of paediatric neurology, santokba durlabhji hospital and medical research institute, jaipur, india; norwich medical school, university of east anglia, norwich, uk objective: this study retrospectively analysed paediatric strokes with further evaluations and outcomes of strokes related to minor injury, to isolate characteristic features and outcomes in these patients. methods: paediatric patients ( mo to y age), presenting with acute stroke between january to january were retrospectively recruited from a tertiary care hospital in north india. from this cohort, strokes following minor injury were analysed for clinical profile, investigations and outcomes (measured by international paediatric stroke study scoring system, ipss). results: of the total cases, ( . %) were post-minor injury (m: f . : ; mean age . ae . mo). of the remaining (n= ; mean age ae . y) most common aetiologies were moya-moya disease (n= , %) and transient unilateral arteriopathy (n= , %). the post-minor injury group revealed a median time of minutes from trauma to stroke onset. more than / rd ( %; n= ) had transient episodic hemi-dystonia on the hemi-paretic side after a median of days of symptoms onset. % ( / ) of children where results were available had anaemia. ct head in all (n= ) showed calcification of the lenticulo-striate vessels. subsequent brain mri (n= ) confirmed ct findings of basal ganglia ischaemia. mr angiogram and thrombophilia screen (n= ) done in the first few patients were normal and hence not pursued subsequently. follow ups of / ( - mo; median= mo) showed good recovery in the majority ( %; n= / ). the median ipss score for these children was . . conclusions: trivial injury leading to basal ganglia stroke was the most common cause of paediatric stroke, occurring exclusively in less than year-olds. ct head was diagnostic (calcification in lenticulo-striate blood vessels and ischemia) with no further information revealed from vascular imaging or thrombophilia work-up. children were commonly anaemic, a potential causative association. often transient episodic hemidystonia of the hemiparetic side after a few days was witnessed. neurological outcomes in most children with this entity is good. introduction: rcpch launched guidance on paediatric stroke in march . we present a series of paediatric patients who presented with stroke following this publication. we audited this group of patients against the key standards of the rcpch guidance. methods: we retrospectively analysed cases between april and april . we identified cases from a neurology database and audited acute management using rcpch guidance as standard. results: we identified children with stroke in the last years. ages ranged from months to years with a mean age of years at presentation. children had haemorrhagic stroke and had ischaemic stroke. the majority involved the anterior cerebral circulation ( / ). underlying aetiology was identified in patients, of whom had haemorrhagic stroke. of the patients died. only out of patients had brain imaging within an hour of presentation. only one patient was eligible for thrombolysis, however due to contraindication she underwent thrombectomy. discussion: stroke pathways are well developed in adult services. due to the rarity of stroke in childhood and challenges with recognising symptoms, treatment is often delayed. symptoms in children need a high index of suspicion. the findings of this audit support the development of an all wales paediatric stroke pathway. we aim to facilitate activation of the stroke pathway when children present with the fast symptoms. we also hope to increase awareness of stroke in childhood. withdrawn. objective: stroke is a common childhood neurological disorder, affecting at least children in the uk each year. the majority of children have residual sequelae across a wide domain of functions, with significant personal and societal consequences. recent rcpch guidelines have proposed criteria for hyperacute treatments; this would require rapid recognition of the potential diagnosis by clinicians. here we describe the acute care pathway of a group of children with confirmed arterial ischaemic stroke (ais). methods: parents of children aged > days and < years referred to gosh (ais) ( - ) were approached and sent a questionnaire exploring their experience of navigating through the healthcare system on initial presentation. responses were tabulated where possible and reported as frequencies; qualitive results were thematically coded and categorised for analysis. results: / eligible parents responded. and the gp were the first port of call for the majority (n= for each). ten parents stated they had 'no idea' what initial symptoms might represent. when directly asked if they had suspected a stroke, nearly / stated 'no'. f.a.s.t features (f: face a: arm s: speech t: time) were noted in a third of patients and only patients were given a diagnosis of stroke at first presentation. on initial discharge, a correct diagnosis of stroke was provided to patients. notably, the need for improved education of paediatric stroke, for healthcare providers was raised by nearly % of parents surveyed. conclusions: the study demonstrates the need for further education to be delivered in pre-hospital, primary and secondary healthcare settings for recognising acute stroke in children. this will be an essential step in the delivery of hyperacute treatments. background: hereditary spastic paraparesis refers to a heterogeneous group of inherited neurodegenerative disorders characterized by progressive lower limb spasticity and weakness. there is marked genetic heterogeneity in hsp with all modes of inheritance described for the different loci and causative genes implicated up to now. we present a case of a child with a complex diagnosis of hsp with a homozygous missense mutation in nt c underlying hereditary spastic paraplegia spg . case presentation: this child had initially been diagnosed as having bilateral cerebral palsy with diplegic pattern and gmfcs ii. he had gross motor and speech and language delay. there was a family history of consanguinity. mri scans had initially been described as showing evidence of periventricular leukomalacia and he had been referred for consideration of sdr. following tertiary assessment there were clinical concerns that the overall diagnosis may need to be reexplored. review of his mri scans demonstrated similarities to a case series of hsp with a rarer form of hsp (spg ). he subsequently tested positive for spg nt c . a second case with similar clinical findings has now been identified in our region. mri scan findings will be presented. summary: spg is a rare but important cause of a cerebral palsy phenotype and testing for nt c should be considered in the differential diagnosis and investigation of patients presenting with cp. recommendations on patient suitability for sdr has focused on children functioning at gmfcs level ii or iii. sdr has been considered for gmfcs levels iv and v however the decision to progress can be challenging. the goals in these children are significantly different with the focus being around comfort and pain relief rather than mobility. we describe the case of a boy with a mixed pattern movement disorder involving limbs which had initially been managed with oral medications baclofen, trihexiphenidyl and intramuscular botulinum toxin. due to the child having a vp shunt the family didn't wish to consider itb pump. despite optimisation of oral medications and intramuscular botulinum toxin spasticity was a significant issue adversely impacting on quality of life. escalation of baclofen was unsuccessful due to loss of head control and duration of action of botulinum toxin injections appeared to be limited to to weeks before tone increased again. this resulted in significant difficulties with moving and handling. in view of this the option of selective dorsal rhizotomy was explored. the goal was to improve ease of personal care and sleep. post-operative outcomes greatly exceeded the family expectations. along with a reduction in tone in the lower limbs they were delighted to report several unexpected improvements. these included: improved functional ability with the left upper limb which facilitated switch access; improved truncal control making moving and handling easier; a reduction in extension posturing which had been particularly problematic pre-operatively; significant improvement in sleep; improved mood. this resulted in the local team exploring eye gaze technology with him. these functional benefits have significantly improved the quality of life for not only the patient but his family. further research into the benefit of sdr in this group of patients should encompass care and comfort outcomes, sleep assessment and measurement of pain. introduction: clonidine has become increasingly repurposed for the management of childhood dystonia. one potential advantage of clonidine is the availability of patches for transdermal delivery. we aimed to review the use of transdermal clonidine patches in our institution. methods: a retrospective notes review of children and young people (cayp) with dystonia issued transdermal clonidine patches as identified from pharmacy records. results: a total of cayp were identified, median age at initiation of clonidine of years months (range mo to y and mo). prior to initiation of patches / cayp were already receiving clonidine, including acutely receiving iv clonidine infusions. one child with difficult iv access experiencing an acute deterioration of dystonia was lost to follow up following transfer to local services. the commonest indications for transdermal clonidine were concerns about 'on/off' effect of enteral doses (n= ) and concerns about enteral absorption (n= ). transdermal clonidine was discontinued by / cayp ( as patches wouldn't adhere, receiving patches as a temporary bridge from iv to enteral clonidine and due to severe local cutaneous reaction). one additional clonidine naive child experienced significant hypotension with a . µg/kg/hour transdermal dose but tolerated a reduced dose of . µg/kg/hour. follow up for the remaining / children ranged from months to . years (median y). the median transdermal dose was . µg/kg/hour (range . - . µg/kg/h). additional enteral clonidine was used by / cayp. efficacy of transdermal clonidine was difficult to determine, but / cayp retrospectively scored on the clinical global improvement-scale, suggesting significant improvement. conclusions: in cayp receiving enteral clonidine switching to transdermal clonidine patches appears to be well tolerated, with . % of cayp continuing with longer term use. further prospective work is required to determine the efficacy and safety profile of transdermal clonidine. objective: to review the indications for and outcomes following itb for children and young people (cayp) at our centre. methods: patients were identified undergoing itb pump insertion from to . had reports available. a retrospective note review was performed, with data extracted using a standardised data collection proforma. results: median age at itb pump insertion was (range to ). hypertonia was described as dystonia, spasticity and/or dyskinesia. median length of follow up was years (range: mo to y). choice of outcome measure was dependent upon the goals identified for surgery. care provider child health index living with disability (cpchild) data was available for cayp at baseline and cayp at year. cpchild improved from a median score of . to . at year (p= . , wilcoxon signed rank test). burke-fahn-marsden dystonia rating scale (bfmdrs) data was available for cayp at baseline (median: . for motor, for disability), cayp at year (median . for motor, median for disability), all with a clinical picture of dystonia. gross motor function measure (gmfm) was available for cayp at baseline (median= ), cayp at year (median ) and cayp beyond year (median= . ). gmfm and bfmdrs were not statistically significant. pain was measured with paediatric pain profile, available for cayp at baseline, cayp in year and cayp beyond year. median of most troublesome pain improved from at baseline to in year. conclusions: for a heterogeneous cohort of cayp with motor disorders, itb appeared to improve ease of care and comfort, indicated by change in cpchild. multiple measures are required to fully capture benefits seen in this cohort, which should be focused on their individual needs for intervention. objective: to evaluate neurosurgical interventions and outcomes in the management of hemidystonia. methods: the pubmed database was searched including terms 'hemidystonia', hemi-dystonia', 'unilateral dystonia', 'dystonia and (pallidotomy or thalamotomy)', 'dystonia and (dbs or 'deep brain stimulation')' and 'dystonia and (itb or 'intrathecal baclofen')', up to may . papers were included if written in english and presenting outcome data for human participants undergoing a neurosurgical intervention for management of hemidystonia. reference lists of included papers were also reviewed. individualised patient data was extracted. to facilitate comparison across patients with and without validated dystonia scale scores individual patient outcomes were categorised on a -point scale ranging from 'worsened compared to baseline' to 'very marked improvement'. results: we identified reports meeting inclusion criteria, describing unique patients ( < y of age). ablative methods ( / cases) most commonly targeted the thalamus, and dbs ( / cases) the gpi. in recent years dbs is reported far more commonly then ablative surgery. reported follow up ranged from months to years. one patient underwent itb but no further individual data was available. out of the cases had individual outcome data. objective measures were available in dbs and ablation cases, most commonly the bfmdrs. reported outcomes in dbs patients were / worsening compared to baseline, / no change, / slight improvement, / moderate improvement, / marked improvement and / very marked improvement. for the ablative cases / worsened, / no change, / slight improvement, / moderate improvement, / marked improvement and very marked improvement. complications were reported in dbs cases ( shielded battery syndrome, one infection) and ablative cases ( depression and transient hemiplegia). conclusions: available evidence for the neurosurgical treatment of hemidystonia is of low quality, but suggests generally positive results, with few complications reported. introduction: acute flaccid myelitis (afm) is a recently characterised condition causing multiple muscle paralysis and life-changing disability in children. no medical treatment is effective. however, recovery of denervated muscle function is possible via nerve transfer surgery. such treatment is complex, specific to the individual and should be carried out by specialist teams. objective: to describe the clinical features, management and outcomes of nerve transfer surgery following the afm outbreak. methods: retrospective analysis of patients with afm treated with nerve transfer surgery in . surgical criteria: persistent motor deficits (paralysis) months post onset with neurophysiologic signs of denervation and donor nerve availability. results: eight patients (m=f, aged - months; mean ) were referred between march and july . at initial onset/ infection: / had involvement of all four limbs and trunk and / had involvement of the phrenic nerve. mean date of initial assessment within specialist centre was . months post onset (range - ). at this time had upper limb paralysis ( right, left) and had bilateral lower limb paralysis. following consultation, declined surgical intervention and are awaiting surgery. / patients have proceeded to surgery: / cases presented with three-or-more nerve root involvement. nerve-transfers have been performed (median per patient). no surgical complications were encountered. early clinical functional outcomes from this cohort following surgery are currently being collated and evaluated and will be presented in full at conference. conclusion: this study supports international experience that nerve transfer surgery can improve functional outcomes in afm. the delivery of care in the nhs requires coordination and referral to specialist centres. experiences with this cohort will inform decision making and improve patient outcomes and family expectations during the next outbreak of afm. poster no. a catastrophic case of acute flaccid myelitis t chakrabarty , c lundy , g doherty , d bhattacharya , p moriarty , d peake royal belfast hospital for sick children, belfast, uk; royal victoria hospital, belfast, uk objective: acute flaccid myelitis (afm) is a rare but serious neurological condition characterised by acute onset of flaccid weakness in one or more limbs with distinct abnormalities of the spinal cord grey matter on magnetic resonance imaging (mri) and without any features suggesting an upper motor neuron disorder. in recent years, there has been a global increase in the incidence of afm associated in some cases with a non-polio enterovirus, ev-d . the long-term prognosis in most cases remain poor. we present a severe case of afm in a -year-old boy with catastrophic consequences. method: retrospective review of clinical course, neuroimaging, treatment and neurorehabilitation. results: a -year-old boy presented with clinical encephalitis. he deteriorated over hours and developed an encephalopathy, multiple cranial nerve deficits, and complete flaccid paresis requiring picu support. csf weeks apart showed a resolving lymphocytic pleocytosis with increased protein ( . to . g/dl). serial mri brain and spinal cord showed extensive signal abnormality involving thalamus, dorsal pons, medulla, cervical cord, conus along with cauda equina. nerve conduction studies were consistent with a severe acute motor axonal neuropathy. eeg showed a posterior dominant encephalopathy. infective (including edv- ), metabolic, immunological (aqp and mog abs) and genetic (ranbp ) tests were negative. immunomodulation therapies (methylprednisolone, ivig, and plasma exchange) resulted in no clinical improvement. resolution of signal changes in the thalami, brainstem and spinal cord along with mild generalised cerebral atrophy was noted in repeat mri after months. months following presentation he remains fully ventilated with no significant motor improvement despite intensive rehabilitation including use of passive range of movement devices and functional electrical stimulation. conclusion: this is one of the most severe cases of afm, representing the wider spectrum of afm involving encephalopathy, dominant bulbar signs and quadriparesis. mirror movement disorder (mmd) is a rare movement disorder with a prevalence of less than in a million in which involuntary symmetrical movement is observed in the limb contralateral to the voluntary limb movement. we report children with an mmd. case : a -year-old girl following an uneventful pregnancy and normal delivery, born to nonconsanguineous parents presented with difficulties with fine motor activities like writing. parents noticed from age of eight that her left hand would make similar movements to her right hand in activities like writing, combing etc. maternal grandfather has mmd. case : a -year-old girl following an eventful pregnancy and normal delivery to non-consanguineous parents presented with an mmd at age of years first noticed whilst writing and this continues affect her activities of daily living. there is no family history of mmd. both children are neurodevelopmentally normal and have normal mri brain and spine. mmd have been described congenitally due to prenatal insult before weeks gestation, kallman syndrome and klippel-feil syndrome or as an acquired due to hemiplegic stroke and parkinson's disease in adults. pathogenesis is thought to be due to lesions in supplementary motor area, corpus callosum or cervical lesions. mutations in dcc and rad gene are present in % of the cases. the multimodal mri and neurophysiological studies have revealed that the motor system is completely disorganised with abnormal crossing at brain stem level and abnormal communication between both brain hemispheres in these children. there is a positive family history in some cases. the upper limbs are commonly involved. diagnosis is usually clinical and treatment is symptomatic with support at school and limiting repeated complex and sustained movements involving both hands. poster no. acute spinal cord infarction in children: a review of the presentation, aetiological investigations and outcomes in children m shehata, ar hart, cd rittey, e davies department of paediatric neurology, sheffield children's hospital nhs foundation trust, sheffield, uk aims: acute spinal cord infarction is poorly understood in the paediatric population. we reviewed cases presenting to a single paediatric neurology centre in uk between and to explore common themes of presentation, aetiology and outcome. material and methods: cases of spinal cord infarction presenting to a single centre were identified from our spinal database and medical records were reviewed to determine clinical presentation, aetiological investigations, management and outcomes. results: six children/young people were identified, male, female, age range to years. participants presented with symptoms after seemingly trivial movements or trauma, including: being kicked in the back whilst playing football, bending forwards to tie hair up, getting up to walk in the garden, and performing a crab gymnastic movement. had no obvious precipitants. initial presentation was neck or back pain in all patients, progressing to bilateral lower limbs weakness, sensory deficit, lost reflexes, and urinary/bowel involvement. mri imaging failed to reveal the diagnosis when performed early in participant. the level of lesions for each participant were: t - ; t - ; t -t ; c -t ; c -t ; and isolated to the conus. aetiological investigations, including thrombophilia screens, failed to reveal a cause in any participant. initially received steroids because the differential diagnosis included an inflammatory disorder. patients received aspirin, / gained motor improvement but none returned to normal. all had residual bladder problems, had bowel sequelae. conclusions: spinal cord infarction may be related to minor trauma or movements. the association with a chiari malformation is previously described in adults. outcome is poor. although motor improvement can be seen, children do not return to normal functionally. aetiological investigations and treatments vary within a single centre. national recommendations are required to standardise practice. cost of care for long-term ventilation patients r belderbos, v kumar, l alkhalidi east lancashire nhs trust, lancashire, uk background: advances in neonatal and paediatric intensive care have increased the survival of children with life threatening or life limiting conditions. there has been a significant rise in children on long-term home invasive ventilation. high profile cases have been in the media recently with debate on whether such interventions should be implemented focussing on ethics but without evidence of cost benefit analysis. children on long-term invasive ventilation are a high cost group with complex and varying underlying medical conditions requiring input from multiple teams, including hour carers, medical and multidisciplinary team input as well as recurrent hospital and picu admissions. in addition, the cost of equipment and drugs makes this a costly intervention. in any limited healthcare system rationing decisions have to be made: drug and other therapies are subject to health economic analyses. this study aims to assess cost per annum for ltv and a cost benefit analysis. objective: identify patients on ltv including comorbidities. assess cost of ltv to quantify cost-benefit analysis. measure outcomes: death/admissions/recovery. methods: review of patients requiring home long-term invasive ventilation july -july . analysis of costs: clinic visits, hospital admissions, costs of equipment; cost of medication. outcomes and quality of life: mortality, admissions and length of stay; decannulation, ability to communicate and mobility analysis; ability of parents to work. results: patients: died (aged , and y), decannulated, ongoing ltv (aged mo, and y), night package £ pa, accessories £ pa, replaceables/service £ , average cost home ltv around £ pa. conclusions: ltv ventilation is an expensive treatment: its use should be analysed on a cost benefit analysis in a similar way to other available treatments. objective: to assess the feasibility of recruiting to a study, performing and interpreting aeeg in preterm infants, and to assess family and staff members' views. methods: a prospective feasibility study. preterm neonates between and weeks postmenstrual age were recruited for continuous aeeg monitoring using adhesive electrodes whilst receiving nicu care. we studied optimal methods of attaching leads, impedance data, number of electrode changes, and preliminary aeeg findings. staff and parents were asked for feedback on the process and their involvement. results: we recruited . % of eligible babies. nuprep and sorbaderm were the most effective combination for skin preparation. the aeeg recording was good quality if staff were engaged and knew when electrodes needed to be changed. four of the seven ( . %) babies showed seizure activity on aeeg, none of which were diagnosed clinically. babies with seizures were born earlier, had lower birthweights, and had more complications than babies without seizures. feedback showed parents and staff were positive, although staff reported caring for the baby was harder. . % of parents and . % of staff would 'definitely' recommend the study to parents with a premature baby. conclusions: the use of continuous aeeg in preterm neonates in feasible, with similar recruitment rates to other studies in the department, and a positive experience for parents and staff. a high rate of electrical seizures was detected. background: the 'magnetic resonance imaging (mri) to enhance the diagnosis of fetal developmental brain abnormalities in utero' (meridian) study showed improved diagnostic accuracy and confidence for detecting fetal neurological abnormalities compared to ultrasound. the additional information provided by in utero mri altered prognosis in % of women. the meridian study did not report whether the neuro-developmental prognoses given to women varied between clinicians or were accurate. objectives: to assess the variation in prognosis given to pregnant women by clinicians in feto-maternal units for different fetal brain abnormalities. methods: we contacted one clinician at each of the meridian feto-maternal units and asked what percentage chance of normal neuro-developmental outcome they would give pregnant women for fetal neurological abnormalities: isolated ventriculomegaly to mm; unilateral hypoplasia of the cerebellar hemisphere, isolated hypoplasia of the cerebellar vermis, isolated cisterna magna, and isolated blake's pouch cyst. respondents were asked to give a percentage chance of normal outcome, although some used free text to answer. results: responses were received from senior obstetricians in feto-maternal units. there was general agreement for isolated mild ventriculomegaly with respondents replying that to % would have normal developmental outcome. wider variation was seen for posterior fossa abnormalities, with the suggested chance of normal outcome for one condition ranging from to %. conclusion: estimating long-term neuro-developmental outcome based on antenatally detected neurological abnormalities is challenging due to limited high-quality data. our data highlights there is high variation in outcomes offered by different clinicians for the same abnormality. further work is needed to determine what advice is given by obstetricians on the potential developmental outcomes of a wide range of fetal brain abnormalities in current practice, how well these agree with published evidence, and whether the involvement of paediatricians with experience in neuro-developmental disorders improves prognostication. background: in-utero mri (iumri) detects fetal brain abnormalities more accurately than ultrasonography (uss) and provides additional clinical information in around half of pregnancies. there is little published data on whether postnatal neuroimaging beyond months of age changes the diagnostic accuracy of iumri nor its ability to predict developmental outcome. methods: families enrolled in the meridian study whose child survived to years of age were invited to have a case note review and assessment of developmental outcome with either the bayley scales of infant and toddler development, the ages and stages questionnaire or both. a paediatric neuroradiologist, blinded to the iumri results, reviewed the postnatal neuroimaging if the clinical report differed from iumri findings. diagnostic accuracy was recalculated. a paediatric neurologist and neonatologist categorised participants' development as normal, at risk, or abnormal, and the ability of iumri and uss to predict developmental outcome were assessed. results: participants had case note review, of whom ( . %) had mri after months of age. the diagnostic accuracy of iumri remained higher than uss (absolute differ-ence= %, % ci % to %, p< . ). developmental outcome data was analysed in participants: ( %) were normal, of whom ( %) had a normal or favourable prognosis on uss and ( %) on iumr (difference in speci-ficity= %, % ci % to %, p= . ). no statistically significant difference was seen in infants with abnormal outcome (difference in sensitivity= %, % ci - % to %, p= . ). conclusions: iumri remains the optimal tool to identify fetal brain abnormalities. it is less accurate at predicting developmental outcome, although iumri is better at identifying children with normal outcome than uss. further work is needed to determine how the prognostic abilities of iumri can be improved. poster no. introducing hypothermia or not decision algorithm (honda) guideline in the assessment of neonates following hypoxic insult at birth in a district general hospital a sproule, j courtney, m mcgowan ulster hospital, belfast, uk introduction: hypoxic-ischaemic encephalopathy (hie) accounts for up to % of cases of cerebral palsy. hie can be caused by multiple events and occurs in / births. hypoxic insult at the time of birth can result in an encephalopathic state characterised by: need for resuscitation at birth, neurological depression, seizures and electroencephalographic abnormalities. the toby study demonstrated that induction of moderate hypothermia within hours of birth for hours duration in infants who had perinatal asphyxia resulted in improved neurologic outcomes in survivors. therapeutic hypothermia is the only proven neuroprotective treatment for hie. an assessment tool was required as there was no standard proforma for neurological assessment for babies with a low cord ph (< . ) in our district general hospital (dgh). this was to ensure that all infants who met the toby criteria received the appropriate treatment within the recommended timeframe. methods: the honda assessment tool was developed for use in the tertiary neonatal intensive care unit. this assessment tool was adapted to use in a dgh as a guideline. the honda included the criteria from the toby study with a user-friendly flow chart. a comprehensive neurological examination is outlined with text and images to ensure reliable and repeatable findings by different clinicians over time. results: the honda tool ensured a standard algorithm was used to assess those infants who had a hypoxic insult at birth. it has standardised record keeping and repeated neurological examination of at-risk infants. conclusions: the honda is a comprehensive and userfriendly algorithm to ensure those infants who meet requirement for therapeutic hypothermia are being appropriately identified and treated. poster no. foetal exposure to misoprostol and mobius syndrome s tilib-shamoun, a siddiqui, v ramesh king's college hospital, london, uk background: mobius syndrome is a rare condition comprising a collection of specific congenital anomalies, usually congenital lower motor neuron th and th cranial nerve palsies. hydrocephalus, cerebellar hypoplasia, orofacial and limbs deformities have been reported in some. the literature links mobius syndrome to early foetal exposure to misoprostol, a synthetic prostaglandin e analogue widely used for medical termination of pregnancy. for abortions it is used by itself or with the anti-progestogen mifepristone; the combination is % effective during the first trimester of pregnancy. the mechanism by which misoprostol disrupts brainstem development resulting in hypoplasia or absence of central brain nuclei is not elucidated as yet. suggested mechanisms include selective vulnerability to hypo-perfusion and ischaemic injury of the foetal brain stem due to direct disruption of the foetal vasculature or to global foetal hypoxia because of uterine contractions and placental ischaemia. clinical case: we report a case of an infant with known exposure to misoprostol from failed medical termination of pregnancy (top) at weeks gestation, who presented with an abnormally increased head circumference, multiple lower motor neuron cranial nerve palsies ( , and th cranial nerves). his mri scan showed hydrocephalus due to cerebral aqueduct stenosis, inferior vermian hypoplasia and loss of bulk of the right facial collculus of the pons. conclusions: it is vitally important to counsel expectant mothers following exposure to misoprostol and failed top of possible congenital anomalies if the woman elects to continue with the pregnancy. poster no. neurological examination in unwell neonates: health care professionals' perspectives a fadilah, ar hart department of paediatric neurology, sheffield children's hospital, sheffield, uk objective: to explore health care professionals' opinions of neurological examination in unwell neonates. methods: a questionnaire designed to assess views on examining unwell neonates neurologically was distributed to all uk neonatal and paediatric neurology units. questions included likert scales, with scores ranging from to . scores of to were taken to be positive, to negative or equivocal. answers were compared between consultants and other staff members using chi-squared testing, with p< . assumed statistically significant. results: responses were received, although not every question was answered. / ( . %) responders were based in general paediatrics, / ( . %) in tertiary neonatal units, and / ( . %) in paediatric neurology. / ( . %) were consultants. / ( . %) performed a neurological examination in all unwell neonates, / ( . %) in most. . % of consultants felt confident performing a neurological examination, compared to . % of other health care professionals (p< . ). consultants were also more confident at interpreting the results and using them to formulate management and prognosis (all p< . ). / ( . %) did not find a high-quality neurological examination documented routinely in medical notes of half or more unwell neonates. / ( . %) reported using the classical neurology examination adapted for neonates, ( . %) used the hammersmith neonatal neurological examination or an adapted version. the most difficult aspects were fundus and cranial nerve examination. the most frequently cited challenges were: effect of medication; difficulties in interpretation; equipment and lines; experience; time limitations; and risks of handling unwell neonates. / ( . %) wanted a new standardised neurological examination for unwell neonates; ( . %) did not. conclusions: non-consultant grade health care professionals feel less confident performing a neurological examination in unwell neonates. all responders highlighted a number of challenges to performing and interpreting the results. around three-quarters of responders want a new, standardised neurological examination for unwell neonates, which could address these challenges. use of re-standardised griffiths scales of child development ( rd edition) in a healthy cohort at to months of age objective: the griffiths scales of child development (gscd) is an established tool for the developmental assessment of children from birth to years. in , the gscd underwent significant revisions, and was re-standardised using contemporary cohorts. to date, no studies have reported on its use in healthy children post-marketing. our aim is to examine the use of the gscd-iii in a healthy population of infants aged to months and to provide the first published data on the use of the revised griffiths-iii. methods: in a prospective observational study of healthy, fullterm infants, participants were recruited into a randomized controlled trial of infant massage. griffiths iii assessments were performed by aricd-trained practitioners across subscales and a general development quotient (gd) at to months. results: children were considered in the analysis, male: female ratio / . mean (sd) birth weight was . ( . ) kg and mean birth gestational age was . weeks (sd . ). mean (sd) age at assessment was . ( . ) months, with ( . %) children being assessed according to / rounded norms, and ( . %) to / norms. no difference was found in either arm of the study in any subscale. scores were considerably greater than average (dq - ) in all subscales but particularly subscales b, d and gd. mean (sd) developmental quotients (dq) in a= . ( . ); b= . ( . ); c= . ( . ); d= . ( . ); e= . ( . ) and in gd= . ( . ). using the published cutoffs, we found that . % (n= ) of our cohort scored 'above average' or greater in gd. conclusions: we have provided the first data on the use of griffiths-iii in a healthy cohort. scores were higher than expected across all sub-scales. this may be due to the characteristics of our cohort but raises concern that griffiths-iii may overestimate ability in young infants. objective: metabolic investigations are important in the investigation of children with disordered development. the aim of this audit was to determine if paediatric metabolic investigations were ordered as per current best practice evidence at tallaght university hospital, dublin, republic of ireland. methods: we used recommendations from seven publications to guide this audit and identified indications for performing metabolic investigations. we reviewed metabolic investigations sent on paediatric patients at tallaght university hospital from january to december . we identified the clinical indication for investigating patients by reviewing dictated clinic letters available on the hospital intranet and confirmed investigation results by reviewing scanned copies available on the hospital intranet. we compared the indications for metabolic investigations with published expert guidelines. results: metabolic investigations were performed on patients from january to december . six patients had inconclusive results and were referred to the metabolic team at temple street children's university hospital dublin for further assessment. there have been no metabolic diagnoses made to date as per tallaght university hospital dictated letters. of the patients, had a diagnosis of autism spectrum disorder (asd). of those with asd, had a confirmed or suspected intellectual disability. patients ( %) met best practice recommendations for metabolic investigations. of the patients who did not fulfil recommendations, ( %) were for children with asd. conclusions: we identified two areas that could improve patient care by optimising diagnostic yield and improving resource utilisation at the hospital. first, we recommend clinicians send targeted investigations and avoid blanket investigations for children with disordered development, including asd. second, we recommend clinicians include relevant clinical details on request forms to improve diagnostic yield. finally, we question the value of metabolic investigations for intellectual disability in the absence of other clinical risk factors or comorbidities and suggest this requires further study. the early developmental course of babies with sturge-weber syndrome n thapa , t fosi , , v siyani , j sloneem , h richardson , s aylett , university college london medical school, london, uk; ucl great ormond street institute of child health, london, uk; neurodisability, great ormond street hospital, london, uk background: sturge-weber syndrome (sws) is a rare neurocutaneous condition which arises from a mutation in g protein subunit alpha q. the hallmark is leptomeningeal angiomas often associated with a facial port-wine birthmark. seizures, stroke-like episodes and hemiplegia are common clinical presentations. objective: to describe clinical features of infants with sws under years and their developmental trajectory in relation to seizure onset. methods: a retrospective case note review was conducted on children aged below years with sws under clinical review at our centre. the medical history and standardised developmental test results (language, cognition, motor and visuospatial skills) contained in patients' assessment reports were analysed. results: common clinical features of children with sws aged under years were: seizures in patients ( %), hemiplegia in patients ( . %) and glaucoma in patients ( . %). their developmental trajectory was a decrease in the mean percentiles (for language, cognition and motor skills) and mean developmental quotients (for visuospatial skills) over the first months. infants with unilateral brain involvement had significantly higher cognitive percentiles than those with bilateral brain involvement (p< . ), but both groups showed the aforementioned pattern. children with epilepsy had worse language (p= . ) and cognitive outcomes (p= . ) than children without seizure onset. there was seizure onset in the first year in infants ( . %). in these patients, earlier seizure onset was associated with a higher language percentile (p= . ) at age months or at the time of seizure onset. conclusions: following treatment of early seizures in sws language recovery appears to occur over time relative to cognition. the functional plasticity of language might account for these observations. it is proposed that seizure prevention and optimal seizure control in the crucial first year of life will benefit cognitive and language development in patients with sws. objective: rett syndrome (rtt) is a rare neurodevelopmental disorder primarily affecting females, characterized by loss of speech, stereotypies, abnormal hand movements, motor and cognitive impairment. diagnosing rtt before regression occurs remains a challenge and there is an increasing interest in early diagnosis, due to the ongoing gene therapy clinical trial in rtt. methods: retrospective case notes review. the patient was born at term after induction of labour for reduced movements, with meconium-stained liquor, but was well. poor crying and suck noted at birth with gradual deterioration of feeding, with frequent chest infections, necessitating peg-feeding at months. peripheral/axial muscle weakness and hypotonia were noted at this time. mri brain showed mild underopercularisation of sylvian-fissures; thin corpus-callosum. mrs, mri spine, echocardiogram, and eeg were normal. vitamin b deficiency was found, treated with hydroxycobalamin. sleep study showed hypoventilation with frequent apnoeas and low respiratory rate, leading nocturne bipap. emg was myopathic. muscle biopsy showed marginal loss of complex-i activity in the respiratory-chain-enzymes analysis. results: videofluoroscopy showed delayed swallow and disorganised pharyngeal stage leading to peg feeding. over the following months, no regression noted but only minimal motor progression seen; she was interactive and smiled. at months, regression in her motor abilities was notedshe stopped fixing, following and smiling with progressive microcephaly and hand writhing movements. eeg showed epileptic encephalopathy with tonic/myoclonic jerks. whole-exomesequencing showed a de-novo pathogenic mutation in the mecp gene (nm_ . :c. _ del,p.[leu fs] ) and the diagnosis of rtt was confirmed. after months, she restarted smiling and fixing/following and making motor progress but continues to have seizures. conclusions: this case illustrates early-onset features in atypical rtt with central breathing abnormalities, bulbar insufficiency, generalized hypotonia before regression. evidence of mitochondrial dysfunction is in keeping with recent reports suggesting neuronal redox imbalance in rtt as one of the disease pathogenic contributors. objective: neurodegeneration with brain iron accumulation (nbia) comprises a group of rare genetic disorders characterized by progressive extrapyramidal and other neurological symptoms due to focal iron accumulation in the basal ganglia. b-propeller protein-associated neurodegeneration (bpan) is the most recently identified subtype of nbia caused by heterozygous variants in wdr at xp . . we report a new subtype of bpan caused by a de novo wdr variant in a -year-old girl. methods: case report on a new subtype of b-propeller proteinassociated neurodegeneration (bpan) caused by a de novo wdr deletion in a -year-old girl and review of the literature. results: we report a year old girl with bpan due to a large ( bp) de novo chrx:g. , , _ , , del (hg ) deletion in wdr , presenting with early-onset global developmental delay, hypotonia, seizures, and speech apraxia. the patient presented at the age of months with hypotonia and motor developmental delay, following a normal birth history, and at months developed complex partial seizures and later on steroid-responsive electrical status epilepticus of slow-wave sleep (eses). she has made minimal developmental progress and has remained profoundly globally developmentally delayed and cognitively impaired, and has still not achieved independent ambulation. conclusion: we have described the clinical, neurophysiological and neuro-imaging findings in a -year-old girl, the unique combination of which may assist in the diagnosis of further similar cases. bpan is an exceedingly rare, severe and debilitating disorder with a broad spectrum of clinical heterogeneity and variable age at presentation with early-onset symptoms. early detection and diagnosis are very important in order to offer proper genetic counselling to affected families and provide symptomatic treatment to patients. next-generation broad-spectrum genetic analyses will enable early detection of bpan in the paediatric age group in order for patients to be diagnosed prior to reaching adulthood. introduction: how to measure the effectiveness of an early intervention program in low resource setting. can assessments lead to interventions? and with improvements leads to new interventions, can new assessment lead to new interventions? can this system be measured for its effectiveness and improved based on feedbacks and results? an attempt to set up child development centres in low resource countries using software, apps and e-learning. method: years of data in early interventions was analysed in lucknow, india. in phase , children with non-progressive neurological problems were given best available local interventions. only % compliances and improvement were seen. based on the feedback algorithms were written to create individual profile of children based on their skills (uk curriculum of excellence), disability score, information processing preference, educational and behaviour problems. based on the score each child generates an individualised profile and an intervention plan delivered by app for parents (p-bac-drv) and app (t-bac-drv) at child development centres. the assessment is repeated every months and new individualised profile is generated with new set of intervention. in total more than interventions are developed, and the algorithm helps in deciding which main areas to target at one time. result: the current system in low resource settings have either no service or results are close to % prevent disability in non-progressive neurological problems. our system has shown to prevent disability in about % of children. supported by government start up initiative the program has won in top data innovations in india. conclusion: use of technology to provide training, exams and support professionals in the low resources areas is the solution to provide effective services. pattern recognition is the key delivered by software and auto audits has been placed to measure and improve the system. introduction: medical advances in the treatment of cns tumours has enhanced survival but also impacted on levels of residual morbidity and participation. service provision has not increased alongside the improvement in survival, with many patients not being able to return to their previous level of activity and participation following their oncology treatment. nice cancer services for children and young people state: 'children and young people who have had a central nervous system malignancy should receive a specialist neuro-rehabilitation care package'. robbie's rehab, a charity funded physiotherapy service embedded within the southampton children's hospital therapy service, launched june , provides supplementary physiotherapy for children diagnosed with brain and spinal tumours under the care of southampton children's hospital. objective: to accurately identify and quantify the need for this service. method: prospective data was collated and reviewed june -may . results: year : patients; year : patients. new episodes of physiotherapy (average . per month), direct clinical contacts. reasons for accessing the service: need for enhanced intensity of rehabilitation on discharge (n= ), enhanced inpatient rehabilitation (n= ), bridge the gap whilst awaiting community services (n= ), change in symptoms (n= ), pre-op assessment (n= ), support for palliative care planning (n= ), support for complex social and emotional needs (n= ), disease progression (n= ), higher level rehabilitation not fulfilling community criteria (n= ), facilitate access to local exercise facilities (n= ), within oncology clinic for assessment/one-off treatment (n= ), post-op assessment (n= ), individualised goal orientated participation (n= ). patients had an estimated weeks reduction in acute bed days. conclusions: robbie's rehab referrals are for a variety of multifactorial reasons with rereferral often needed within their pathway. it has enabled earlier discharge, improved transition to community services and opportunities for therapy access previously not available. results: our proband presented at weeks with marked stridor and bulbar weakness after a normal pregnancy. he subsequently developed respiratory failure requiring nocturnal bipap and was found to have a type i laryngeal cleft. initially he met developmental milestones but at months developed features of axial weakness with further regression at months with limb weakness and loss of deep tendon reflexes. emg confirmed denervation in genioglossus, as well as proximal and distal limb muscles without evidence of neuropathy. genetics for smn gene and smard were negative. inherited peripheral neuropathy gene panel testing identified a heterozygous missense variant c. t>c, p.(cys arg) in exon . the variant is predicted to alter a highly conserved amino acid, has not been reported before and has not been identified in control databases. in silico prediction tools supports the pathogenicity of the variant. mutagenesis of the equivalent amino acid in mice produces impaired motor control and denervation. conclusions: the gars gene encodes an ubiquitously expressed glycyl trna synthetase which has an integral role in protein synthesis in all eukaryotic cells. missense gars mutations can lead to distal hereditary motor neuropathy as well as a sensorimotor neuropathy phenotype (cmt d) typically with adolescent or early adulthood onset. objective: to discuss sma, which is one of the differentials in a hypotonic child and bring to light the diagnosis is not always cerebral palsy (cp). method: descriptive case report. results: case : -month-old girl admitted to icu with severe pneumonia. case : -month-old boy, both admitted to the intensive care unit with severe pneumonia. case : -month-old girl, presented to outpatient with progressive 'floppy' limb weakness and swallowing and breathing difficulties. case : attended opd with worsening respiratory distress, difficulty feeding, difficulty managing secretions. all had perinatal histories of uncomplicated deliveries but subsequent early respiratory distress and oxygen requirement for the first few days of life. all had been 'floppy' since birth, with severe gross motor delay, feeding difficulties, poor weight gain and recurrent chest infections. cases , and had been diagnosed with cp despite having normal neuroimaging. examination of all children was similar and consistent with clinical diagnosis of sma. findings included an alert, interactive child; frog-legged posture; -limb hypotonia and weakness with legs more affected than arms; absent deep tendon reflexes; bell-shaped chest; and tongue fasciculation. genetic testing for all confirmed homozygous deletion of exon of the smn gene. in all the cases creatinine kinase levels were normal, ruling out myopathy. conclusions: the incidence of sma is / livebirths. it can be diagnosed clinically from pathognomic features when genetic testing is unavailable and should be considered in any hypotonic child, irrespective of perinatal history. a wide clinical spectrum that ranges from early death to near-normal adult life exists. families must be counselled regarding implications of this genetic diagnosis. correct early diagnosis and multidisciplinary intervention can vastly improve outcomes. poster no. syros studylong-term reduction in rate of respiratory function decline in patients with duchenne muscular dystrophy (dmd) treated with idebenone l servais , c lawrence , oh mayer , cm mcdonald , u schara , t voit , e mercuri , gm buyse respiratory function decline in dmd is caused by the underlying weakness and degeneration of the respiratory muscles leading to impaired inspiratory and expiratory effort and associated complications. idebenone reduced the rate of respiratory function decline over weeks in the phase iii delos trial. syros is a long-term study in former delos patients who transitioned to idebenone under expanded access programs following a variable untreated period. here, we aimed to characterize the long-term effects of idebenone on respiratory function. patients were managed according to routine clinical practice. respiratory function was assessed by calculating the annualized decline in forced vital capacity (fvc) and peak expiratory flow (pef), expressed as percent predicted (%p). comparisons were made between treated and untreated periods and to matched external controls. data on bronchopulmonary adverse events (baes) and hospitalizations were collected. data from / former delos patients were available. at delos baseline, mean (sd) age and fvc%p were . ( . ) years and . % ( . %); all patients were glucocorticoid non-users and . % were non-ambulatory. patients were treated for an average (min-max) of . ( . - . ) years compared to an average untreated period of . ( . - . ) years. the annual rate of fvc%p decline was almost halved ( . % vs . %) when comparing these periods. for the external comparisons, declines remained lower across all treatment years (up to y) compared to the matched group of untreated patients. comparable results were seen for pef%p. the risk of baes was reduced by % during longterm idebenone treatment versus untreated periods, leading to fewer hospitalizations due to respiratory causes ( . vs . events per year). long-term treatment with idebenone results in a consistent and sustained reduction in the rate of respiratory function decline for up to years. two placebo-controlled trials of -week duration (phase ii delphi, phase iii delos) showed that idebenone consistently reduced respiratory function decline rate in patients with advanced dmd. long-term data from the delphi-extension (delphi-e) study and syros (delos patients who transitioned to idebenone under an expanded access program) are now presented. the aim was to assess the consistency of the long-term effect of idebenone on respiratory function across both placebo-controlled trials and their respective long-term data collections. delphi-e and syros patients with abnormal (< %) forced vital capacity (as percent predicted, fvc%p) were treated with idebenone for an average of . and . years respectively. annualized fvc%p decline rates were compared to untreated patients from syros or matched external controls (matched for baseline fvc%p) from the cinrg duchenne natural history study (cinrg-dnhs). mean (sd) baseline age was . ( . ) and . ( . ) years in delphi (n= ) and delos (n= ), respectively, and fvc%p was . % ( . %) and . % ( . %). for the first -year period, where data were available for both studies, the average annual decline rate was comparable in treated patients ( . % and . % in delphi-e and syros) and lower than in untreated syros patients and external controls ( . % untreated and . % in cinrg-dnhs). during years to , the annual decline rate was consistently lower than for matched controls. treatment with idebenone resulted in a sustained reduction in the rate of decline in respiratory function across both placebo-controlled week studies and across both long-term data collections, with follow-up time of up to years. the consistency observed across independent datasets adds to the robustness of the treatment effect of idebenone and its potential to modify the course of respiratory function decline in dmd. poster no. a rare mutation in dync h causing a mixed clinical phenotype of spinal muscular atrophy with lower extremity predominance and hereditary spastic paraplegia: a case series in a family el goh , , s jayawant , g anand objective: to describe the identification of a rare mutation in the dync h gene as a cause of a mixed clinical phenotype of spinal muscular atrophy with lower extremity predominance (sma-led) and hereditary spastic paraplegia (hsp). methods: case series of three family members (father and two sons) across two generations. results: there was a history of early childhood-onset, progressive lower limb muscle weakness and atrophy. no relevant family history of neuromuscular disorders was reported on both the paternal and maternal sides of the family. examination revealed markedly diminished tone and power in the lower limbs, with wasting and a positive crossed adductor reflex. there were no abnormalities detected in the upper limbs and sensation was preserved throughout. neurophysiological testing showed moderate to severe chronic denervation of the lower limb muscles with sparing of the peripheral sensory nerves. hsp panel was negative but charcot-marie-tooth (cmt) panel demonstrated a heterozygous sequence change in the dync h gene: c. a>t p.(glu val), which was present in all affected family members. discussion and conclusions: mutations in the dynein gene are typically associated with sma-led or cmt. a mixed sma-led and hsp phenotype has previously been shown to be caused by mutations in bicd . bicd encodes a golgin, which is a component of dynein-based transport, and plays a key role in mrna transport during oogenesis and embryogenesis. we present the first case series of a mixed clinical phenotype of sma-led and hsp occurring due to a mutation in dync h . this was the first observation of the c. a>t p.(glu val) variant at our laboratory and was not listed in the genome aggregation database, suggesting an extremely rare variant. opening the lid on unilateral ptosis in paediatric nf e hassan , e witter , z mughal , l robinson , d weisburg , sa roberts , e hupton , j eelloo , e burkitt wright , , s garg , l lewis , dg evans , , sm stivaros , , , g vassallo , , introduction: neurofibromatosis type (nf ) is a genetic disorder with a birth incidence of in - individuals and prevalence of around in worldwide. ptosis is a welldocumented feature in this condition and is known to be associated with plexiform neurofibromas or in the noonan phenotype, with bilateral ptosis. unilateral ptosis in the absence of a plexiform neurofibroma is not a common feature in nf . we describe a number of patients with nf who demonstrated unilateral ptosis. methods: a retrospective cohort study was carried out using the patient database within the nf service based in st mary's hospital, manchester, uk. children and young adults aged to years with nf were identified via the patient database and patients with a presentation of ptosis were identified. results: six children with unilateral ptosis were identified, four females and two males (ages - , mean= . y). five had unilateral ptosis affecting the right and one the left, with no differences observed between sporadic or familial disease. five patients had complex disease; however, none had any other associated complication to account for the unilateral ptosis apart from nf . they did not meet the diagnostic criteria for noonan syndrome, and none had plexiform neurofibromas in the orbital or peri orbital area. discussion: it is unclear why there is an increased incidence of unilateral ptosis in our cohort of nf patients, in the absence of plexiform neurofibromas and noonan's syndrome. ptosis in nf has been associated with a noonan syndrome phenotype in nf patients. the general hypotonia and myopathy observed in these patients could also factor into the causes for ptosis. further research is necessary to investigate the aetiology of increased unilateral ptosis in nf patients. objective: sma is a severe neuromuscular disorder characterised by progressive muscle atrophy and weakness. scoliosis is a highly prevalent complication and surgery is almost invariably required in 'sitters'. data on secondary outcomes are limited, and this study investigates post-surgical respiratory (fvc%) and motor function, weight gain, pain and satisfaction. methods: we retrospectively reviewed the notes of patients who never walked or lost ambulation (sma type ii/iii) who successfully underwent scoliosis surgery at great ormond street hospital: spinal fusion ( ), magnetic ( ) or traditional ( ) growth rods. we performed phone interviews and run a focus group for families on pre and post-surgical satisfaction. results: median follow-up before and after surgery was . ( . - . ) and . ( . - . ) years respectively. mean annual rate of fvc% decline improved post-surgery in sma ii: - . versus - . (p< . ), with similar trajectories in sma iii. mean annual rate of hammersmith functional motor scale's scores decline did not change significantly (- . vs - . , p< . ), while the revised upper limb module's scores showed a less progressive deterioration (- . vs - . , p= . ). a negative deviation from previous weight curve after surgery was observed in / requiring food supplements ( ); one/ with significant weight loss (> % of total weight) needed gastrostomy. pain was frequently documented, especially hip pain ( / ) requiring painkillers ( ), intra-articular steroids ( ) and surgery ( ). nine/ families participating in the phone interview reported major improvements in posture, physical appearance, self-image; all rated the procedure as very successful. however, / did not report significant improvements in quality of life due to reduced mobility and increased unmet care needs. five families attended the focus group reporting on both positive and negative aspects of their experiences. conclusions: this study provides relevant data and suggestions to improve the current multidisciplinary approach of scoliosis surgery in children with sma. poster no. sunfish part : -month safety and exploratory outcomes of risdiplam (rg ) treatment in patients with type or spinal muscular atrophy (sma) objective: spinal muscular atrophy (sma) is a severe, progressive neuromuscular disease caused by reduced levels of survival of motor neuron (smn) protein due to deletions and/or mutations of the smn gene. while smn produces fulllength smn protein, a second gene, smn , produces only low levels of functional smn protein. risdiplam (rg / ro ) is an investigational, orally administered, centrally and peripherally distributed small molecule that modulates smn pre-mrna splicing to increase smn protein levels. sunfish (nct ) is an ongoing, multicentre, double-blind, placebo-controlled study (randomised : , risdiplam:placebo) in patients aged - years, with type or sma. methods: sunfish part (n= ) is a dose-finding study assessing the safety, tolerability and pk/pd of risdiplam; pivotal part (n= ) assesses the safety and efficacy of the risdiplam dose level that was selected based on results from part . sunfish part included patients of broad age ranges and clinical characteristics (functional level, scoliosis and contractures). an interim analysis of part (data cut-off, july ) showed a sustained, > -fold increase in median smn protein versus baseline after year of treatment. adverse events were mostly mild, resolved despite ongoing treatment and reflective of the underlying disease. despite not being designed to detect efficacy, risdiplam improved motor function measures over months versus natural history. results: safety, tolerability and pk/pd will be reported from all patients in part who have received treatment with risdiplam for a minimum of months. updated part exploratory efficacy data, including motor outcome measures, will also be presented. the clinical benefit of risdiplam is being assessed in part , which is ongoing worldwide. objective: ryr encodes the skeletal muscle ryanodine receptor, an intracellular calcium-release channel that is crucial to excitation-contraction coupling in muscle. gene variants can cause heterogeneous myopathies, including dominantly inherited central core disease. both autosomal dominant (ad) and autosomal recessive (ar) pattern of inheritance have been reported. methods: retrospective case notes review. results: sibling : female, presented during the first year of life with motor developmental delay. at years of age she is able to sit unsupported and crawl but not stand or walk. she has facial weakness, but no feeding difficulties or ophthlamplegia. she has axial and proximal weakness with antigravity power in neck flexors and hip flexors (mrc / ) and sub-gravity power in hip abductors/extensors and knee flexors/extensors (mrc / ). there are severe hip, knee and ankle fixed contractures. power and joint range is normal in upper limbs. muscle biopsy showed type- fibre predominance and core-like structures. sibling : female, presented at birth with feeding difficulties. at months of age she is fully nasogastric fed. there is no facial weakness or ophthlamplegia. she has good head control with active head lift in prone, and antigravity power in hips, knees and ankles. she has mild hip and knee contractures and shoulder girdle weakness. both siblings have been confirmed to be heterozygous for a ryr pathogenic frameshift variant (c. _ dupca p.(met fs)) inherited from father and a likely pathogenic missense variant (c. g>c p.(lys asn)) inherited from mother. both parents are asymptomatic. conclusions: the clinical and pathological features of ad ryr -related myopathy are well recognized but much less is known about ryr -related disorders secondary to an ar pattern of inheritance. we report two siblings with ar ryr related myopathy with similar genotypes but different phenotypic features demonstrating intra-familial variability and expanding current knowledge on this disorder. results: our probands were the second and third children of consanguineous irish parents who were fourth cousins. antenatally, reduced fetal movements and amniotic fluid was noted with both probands. at birth, both had arthrogryposis and the second sibling required prolonged intubation at birth. both had significant developmental delay; a more severe phenotype in the younger. on examination, both had myopathic facies, inability to bury eyelashes, full eye movements, high arches palates, drooling, a weak cry, micrognathia but a preserved suck. they both had long thin fingers, with thumbs held adducted and dimpling of elbows and hands. peripheral reflexes were absent but there were good anti-gravity movements of the lower limbs. both were noted to have pectus excavatum and progressive scoliosis. muscle biopsies showed dystrophic features of fibrosis, hypertrophy and atrophy of fibres and variation in fibre size with increased fibrous connective tissue. occasional central cords and multiple mini cords were also seen in the second proband. whole exome sequencing identified the compound heterozygote mutation (ttn c. delc in combination with ttn c c>tp. (ser leu) and ttn c. g >a p. (asp asn)). conclusions: mutations in the titin gene (ttn) have been implicated in several skeletal and or cardiac phenotypes to date. each individual variant of the compound heterozygote has not been reported as pathogenic mutations and have been detected in the general population at . % frequency. however, the presence of the triple count may certainly account for the severe phenotype of our probands. poster no. gene-replacement therapy (grt) in spinal muscular atrophy type (sma ): long-term follow-up from the onasemnogene abeparvovec phase / a clinical trial objective: sma is a rapidly progressing neurologic disease caused by biallelic survival motor neuron gene (smn ) deletion/mutation. the smn grt onasemnogene abeparvovec (formerly avxs- ; approved in us) treats the genetic root cause of sma and is designed for immediate, sustained smn protein expression. in a phase / a trial (start [cl- ]; nct ), sma patients received a one-time onasemnogene abeparvovec infusion at low dose (cohort , n= ) or high dose (cohort , n= ), and demonstrated improved outcomes versus natural history. no patients in start received nusinersen during the -month follow-up after dosing. sma patients in start could rollover into a long-term follow-up study (study lt- ; nct ). primary objective: long-term safety. methods: sma patients have annual visits ( y), then phone contact (additional y). patient record transfers are requested. safety assessments include medical history and record review, physical examination, clinical laboratory evaluation, and pulmonary assessments. efficacy assessments include developmental milestone evaluation to determine maintenance of the highest achieved milestone in the parent study. results: as of march , patients (cohort , n= ; cohort , n= ) had enrolled in study lt- and had a baseline visit. for patients in cohort , the mean (range) age and time since dosing were . ( . - . ) years and . ( . - . ) years, respectively. all patients in cohort ( / ) were alive. no developmental milestones achieved in start were lost, and new milestones have been achieved, supporting the durability of onasemnogene abeparvovec. updated data will be presented. conclusions: one-time onasemnogene abeparvovec administration at the high dose continues to provide prolonged and durable efficacy with milestone development in lt- . poster no. micu -myopathy: a mitochondrial disorder that mimics a congenital muscular dystrophyreport of siblings with variable phenotypes m fernandez, m sa, v gowda evelina london children's hospital, london, uk objective: micu encodes a selective calcium-channel subunit within mitochondrial inner membrane whose function is essential for buffering cytosolic ca + transients and activating atp production. mutations in micu have been reported in different families with muscle weakness, fatigability and developmental delay, with normal lactate despite being a mitochondrial disorder and persistently elevated creatine kinase (ck) usually in the range of congenital muscular dystrophies (cmd). the phenotypic spectrum is highly variable and keeps expandingother features include progressive extrapyramidal signs, learning disabilities, nystagmus and cataracts. we report the clinical features of siblings from a consanguineous family with the homozygous c. - g>c splicing mutation in micu . methods: retrospective case notes review. results: sibling : boy, older sibling, presented aged years. he had short stature which was investigated when found to have ck of iu/l. he complained of occasional cramps. muscle biopsy showed mild dystrophic changes with reduced alpha-dystroglycan that indicated a possible congenital disorder of glycosylation. his mri brain was normal. he was diagnosed with autism. sibling : girl, diagnosed antenatally with cerebellar hypoplasia, confirmed postnatally as inferior-vermis hypoplasia. presented at years with occasional cramps, mild tightness of tendo-achilles, and ck of iu/ l. her height and weight were on nd centile. muscle mri showed a small area of high signal in the left adductor magnus related to a group of normal vascular structures. neurophysiology studies were normal. no other systemic involvement was seen in either of them. next generation sequencing revealed the micu mutation described. conclusions: our work expands the phenotypical spectrum of micu deficiency and highlights the variability in patients within the same family. targeted analysis of the micu gene in patients with high ck levels resembling a cmd picture may be warranted, even in the absence of prominent muscle features. the role of dystrophin brain isoforms on early motor development and motor outcomes in young children with duchenne muscular dystrophy half of patients have central nervous system (cns) manifestations. two dystrophin isoforms, dp and dp , play an important role in cns function. those lacking dp have a more severe cns phenotype, most marked in those lacking dp and dp . our objective is to determine whether lack of dp and dp also has an adverse impact on early motor development. methods: the northstar ambulatory assessment (nsaa) is a scale of motor function. clinical information for dmd participants was classified by dmd mutation location and effects on isoform expression as follows: dp +_dp + (dp absent, dp /dp present), dp -_dp + (dp /dp absent, dp present) and dp -_dp -(dp /dp /dp absentall isoforms affected). results: amongst to year olds, median total nsaa scores were lower in the dp -_dp + (p= . ) and dp -_dp -groups (p= . ) than the dp +_dp + group, most markedly in the dp -_dp -group. for example, for -year olds, median total nsaa scores were (dp +_dp +), (dp -_dp +) and (dp -_dp -). amongst to year olds, a lower percentage of participants achieved a full score of (normal, achieves goal without assistance) for the nsaa sub-items in the dp -_dp + (p< . ) and dp -_dp -(p< . ) groups than in the dp +_dp + group, most markedly in the dp -_dp -group. for example, amongst -year-olds, percentage of visits for which a full score was recorded for jump were as follows: % (dp +_dp +), % (dp -_dp +) and % (dp -_dp -). conclusions: in addition to the known cns phenotype, young dmd patients lacking dp also exhibit lower median total nsaa scores and greater early motor delay, most markedly seen in those lacking both dp and dp (lacking all dystrophin brain isoforms). this has important implications for patient prognostication and clinical trial design. background: most commonly known as a rare subtype of guillain-barr e, miller fisher syndrome (mfs) has evolved since it was first described in . the syndrome is characterised by a triad of ophthalmoplegia, ataxia and areflexia but clinical variations do occur. it occurs more often in men than woman (ratio : ) with the average age of onset . years. mfs is associated with positive anti gq b antibodies, which is concentrated in cranial nerves iii, iv and viexplaining the link with ophthalmoplegia. clinical case: we present an unusual case of a -year-old boy with background of macrocephaly and pre-existing developmental delay with a previous mri which showed mild signal change in periventricular white matter bilaterally. he was admitted with a subacute history of proximal muscle weakness and fatiguability. he had no obvious focal neurological signs apart from intermittent lid hopping and ptosis. differential diagnosis included myasthenia, demyelinating disorders or an underlying pre-existing luecodystrophy. anti gq b antibodies were checked along with extensive metabolic investigations, lumbar puncture, muscle biopsy, anti-cholinesterase and antimusk antibodies along with repeat mri. all investigations were negative including mri which showed no significant change from previous. the only findings were strongly positive anti gq b antibodies. in the interim, the patient was started on trial with pyridostigmine with significant clinical improvement. conclusion: atypical variants of mfs should be a differential in children with subtle eye signs without ophthalmoplegia. lid hopping and fatiguability should raise the suspicion of mfs and anti gq b antibodies should be tested. pyridostigmine has been reported to be effective in mfs. potential utility of muscle mri in congenital myasthenia syndrome secondary to agrn mutation found on whole exome sequencing (wes) congenital myasthenia syndromes (cms) are caused by genetic defects affecting neuromuscular transmission, resulting in muscle weakness and fatigability. agrin, an extracellular matrix molecule released by the nerve is essential at the neuromuscular junction. the large coding gene agrn, has a number of exons and with increasing variants found on wes, it is time consuming and complex to undertake functional studies to define pathogencitiy. previous reports of agrn mutations have a phenotype with prominent distal leg weakness and changes in the soleus on mri. we describe a differing presentation and striking changes on mri, especially in the posterior compartment of the thigh. a -year-old presented with deterioration in his gait and difficulty climbing the stairs. he was born at term, via a normal vaginal delivery. his parents were consanguineous, and he had three well siblings. he was reported to walk by years. on examination he had a waddling gait and was unable to run or hop. he had proximal weakness with a positive gowers sign, together with weak eye closure. muscle biopsy showed non-specific myopathic features, however an mri of the lower leg found widespread fatty muscle atrophy of the thigh and calf with relative preservation of the adductor longus, rectus femoris and semitendonosis. wes revealed an agrn mutation (c. _ del) and a homozygous mutation in the nebulin gene (not felt to be clinically relevant). single fibre emg confirmed electrodecrement on repetitive nerve stimulation. the patient has been commenced on treatment with salbutamol. our patient had very distinctive changes on mri and nonspecific muscle biopsy changes. muscle mri changes prompted further genetic testing when symptoms fitted a clinical diagnosis of a congenital myasthenic syndrome. with increasing variants found of unknown significance in these patients, collation of mri imaging to try and elucidate patterns of changes will be important. mcardle disease (glycogen storage disease type v) is an autosomal recessive condition caused by pathogenic mutations in both copies of the muscle glycogen phosphorylase (pygm) gene encoding the muscle-specific isoform of glycogen phosphorylase, 'myophosphorylase' exclusively affecting skeletal muscle. it is the commonest form of glycogenosis. mcardle disease shows significant clinical variability, with symptoms ranging from mild discomfort during exercise to marked muscle weakness and rhabdomyolysis with myoglobinuria. the second wind phenomenon is unique to mcardle disease and consists of improved exercise tolerance with a decrease in heart rate after a rest. despite the majority of patients recalling symptoms during the first years of life, mcardle is infrequently diagnosed in children, % of patients being diagnosed after years of age according to a recent review. here we report two patients diagnosed with mcardle disease at the age of and years respectively. case one presented with fatigue and inability to increase pace of walking from the age of . hills lead to earlier fatigue. she was able to participate in gymnastics and dancing. presentation was with fluctuating ck levels ( to ). she had no second wind phenomenon or myoglobinuria. case two presented at years with a history from months of reduced exercise tolerance and myalgia after low intensity physical activity with no evidence of myoglobinuria or second wind. on formal assessment there was no evidence of muscle weakness or functional impairment. ck was persistently raised ranging from to . in the light of symptoms and ck levels a rhabdomyolysis panel was requested in both cases leading to diagnosis. objectives: to explore characteristics of anxiety experienced by young males with duchenne muscular dystrophy (dmd) using: . qualitative analysis of focus group discussions with dmd boys and their parents. . parent-report scales of anxiety/emotional problems. methods: eight boys aged to years with dmd and dmd parents participated in separate child and parent focus groups. perspectives on anxiety were elicited using semi-structured discussions, and framework analysis was applied to identify themes. scores on five parent-report scales were determined and scales were compared for content and sensitivity. results: from group discussions, six characteristics of anxiety were recurrently reported: catastrophic conclusions; rigidly held anxieties; extreme distress; unexpected/unfamiliar; social anxieties; physical changes and needs. many features echo the anxiety phenotype in autism spectrum disorder (asd). four further themes described relevant contextual factors: individual, family, social and environmental responses. from parent-report scales, younger dmd boys ( - y; n= ) had significantly higher total, general and social anxiety scores compared to population means on at least two scales (p< . ; p< . ; p< . ). the older dmd group ( - y; n= ) trended towards higher scores in total, general and separation anxiety (p= . ; p= . ; p= . ) compared to population norms. different scales varied in their diagnostic sensitivity and item content, which may influence their utility in dmd. conclusions: anxiety can be a pervasive and impactful issue in dmd. it appears to have some shared traits with anxiety in asd and may be influenced by situational factors, such as living with a disabling, life-limiting condition. screening with standard anxiety scales may not accurately capture the full spectrum of the phenotype in dmd, therefore further evaluation to determine optimal screening instruments in dmd is warranted. however, multi-modal assessments tailored to dmd are key to identifying those in need of support to optimise the mental well-being of young people with dmd. objective: as part of the clinical psychology service in paediatric neurology we developed a tic management group to support young people and their parents to develop positive coping in relation to their tics. the group combined psychoeducational, emotional regulation and habit reversal therapy (hrt) components. this evaluation aimed to establish the effectiveness of these groups in reducing tics and associated distress. method: twenty-eight children, aged to years and their parents attended one of seven tic management groups facilitated between february and november . these children had been referred to the clinical psychology for support with tics. each group consisted of weekly, -hour sessions with a review session weeks later. a parent group was held in parallel. both the young persons and parent groups were facilitated by the clinical psychology team. homework tasks were provided to support hrt skill practice and consolidation of learning of the group content between sessions. the following pre and post group measures were completed by the young people and their parents: the paediatric index of emotional distress, the yale global tic severity scale, the parent tic questionnaire and session rating scales. measures were collated and descriptive data reviewed. results: % of children found the group was helpful in the management of tics. % of children were 'less bothered by their tics'. % of children felt more confident in controlling their tics. parents reported a greater understanding of tics and a reduction in the severity of their child's tics. conclusions: results indicate the tic management group is effective in building young peoples' understanding of tics, confidence in tic management whilst providing peer support. the findings also indicate that parents found the groups informative and valued the opportunity to share experiences with others. background: patients with epilepsy often have deficits in cognitive, physical, psychological and social functioning, and treatment should aim to alleviate these deficits. epilepsy surgery is considered for medication refractory epilepsy with aims to improve patient quality of life. a recent study highlighted the importance of a multidisciplinary workup prior to epileptic surgery, including a neuropsychiatric assessment. part of this assessment should identify patient expectations of epilepsy surgery, so that these can be addressed peri-operatively. at king's college hospital (kch), london, these assessments are routinely performed by the paediatric liaison service as part of the children's epilepsy surgery service (cess). aim: to analyse retrospective data of pre-operative patient and carer expectations between october -september at kch. methods: a record of patient and carer expectations is routinely recorded as part of kch cess neuropsychiatric assessments. the responses were compiled and analysed using qualitative content analysis. results: a preliminary survey of cases with an average age of (range - ) identified responses that were grouped into broader classifications (cognitive, seizure experience, social process, school experience, mental state and general improvements). simple analysis showed carers most often expected surgery to reduce the need for medications ( %), ablate seizures ( %), increase school performance ( %), independence ( %) and overall quality of life ( %). this compared to child responses, where the most common expectations were a reduction in lifestyle restrictions ( %), a cure for epilepsy ( %), decrease in medications ( %) and increased independence ( %). conclusion: consideration of both child and carer expectations during pre-epilepsy surgery neuropsychiatric assessments is important in order for services to manage each individual's expectations. unmanaged unrealistic expectations may lead to a negative psychological outcome for either child or carer. expectations should be weighed up against an individual's clinical profile. poster no. neural correlates of conversion hemianaesthesia in an adolescent: a novel fmri case study m ray, a zaman, t alam leeds teaching hospital nhs trust, leeds, uk aim: to highlight the novel functional magnetic resonance imaging (fmri) findings in an adolescent with rare conversion hemianaesthesia. methods: we hereby report a right-handed y old boy who presented with inability to perceive sensations on the right half of body without any motor weakness causing him to have frequent injuries on his right leg as well as burns on his right hand without realizing. when he wore a jacket, he felt warm on one side of the body more than the other. his birth and developmental history were non-contributory. neurological examination was unremarkable except for right hemisensory disturbance. the mri of brain and spine, peripheral nerve conduction studies and somatosensory evoked potentials did not show any evidence of dysfunction were normal. he underwent fmri on a t philips achieva. the paradigm consisted of stimulating both the right and left hands and feet with three dissimilar stimuli (cold, brushing, pin-prick-pain) . the order of the stimuli was pseudorandomised and after each stimuli delivery, feedback was obtained. results: both the hand and foot sensory motor cortices were successfully stimulated. irrespective of which hand was being stimulated, there was left hemisphere sensory motor cortex dominance (with the brushing and cold stimuli), however selfreport from the participant confirmed detection of stimuli on the left-side only. there was more sensory-motor activation when the stimuli were delivered to the right hand. pain stimuli successfully activated parts of the 'pain matrix', furthermore enhanced attention effects (frontal pole activations) were observed with right-sided stimulation (supports lack of stimuli detection ability). the pain stimuli were more effective on the hands than foot, reflected by increasing activation and also self-report from the participant. conclusions: the fmri findings are unique and support the evidence of neuroplasticity and the current study paves the way for future studies investigating conversion hemianaesthesia. poster no. chronic paroxysmal hemicrania presenting as facial pain in a child with autism and bipolar disorder: diagnostic challenges case presentation: a boy diagnosed with disintegrative psychosis aged , revised to autism with bipolar disorder, had been on carbamazepine with risperidone for poor mood control. withdrawal of risperidone produced tardive oromotor diskinesia responsive to clonidine. aged , when mood improved on aripiprazole, carbamazepine was withdrawn. he then presented with episodes of distress preceded by withdrawal, unilateral but not side-locked facial flushing, with additional flushing of neck, back and wrists. behaviours included hitting wrists off walls, chewing of hard objects and requesting pressure to his head. episodes occurred - times/ day, lasting - minutes. he showed rhinorrhea and tearing, attributed to crying, during events. the attacks self-terminated. results: mri and electroencephalogram were normal. failed pharmacological trials included paracetamol, amitriptyline, gabapentin and oxcarbazepine. diclofenac provided mild pain relief and morphine reduced the incidence of attacks. reintroduction of carbamazepine resulted in improvement at mg/kg/day but did not eliminate pain. sequencing of scn a was normal. a plan to wean morphine alongside a trial of indometacin, initially at mg twice-daily was successful at mg twice-daily. episodes ceased, including all autonomic features. exacerbation at weeks occurred in context of an intercurrent illness and was managed with an additional dose of indometacin. conclusions: cph is underreported in the paediatric age group. in our case, the patient's inability to describe events, and an additional psychiatric diagnosis added complexity. the possibility of pain as a cause for early psychotic breakdown in a developmentally vulnerable child cannot be excluded. criteria emphasising side-locked headache and autonomic features, and not recognising associated symptoms elsewhere may also delay recognition in children. objectives: piih can be a challenging condition to diagnose and manage with risks of misdiagnosis, permanent sight loss and frequent comorbidities. we aimed to review our practice to identify areas of uncertainty to help formulate important questions to address within a clinical guideline. methods: a single centre retrospective case notes review of all cases referred to neurology with suspected piih (papilloedema confirmed by a consultant ophthalmologist in all cases) during an -month period. results: ( f: m) cases were identified. age range to years. mean years. / had a bmi > th centile. one case was referred to an obesity service. / had a comorbid headache disorder and / had anxiety/depression. all cases had neuroimaging ( mri, ct) with / having dedicated venography. in / cases lumbar punctures (lp) were conducted under general anaesthesia (ga). in / cases lp was not done; due to presence of a chiari malformation and due to procedural failure related to body habitus. intracranial pressure (icp) monitoring was done in one of these four cases. all children were treated with acetazolamide as first line therapy. frusemide, zonisamide and topiramate were also used in single cases. / children had repeat lps due to failure of resolution of symptoms. / cases had sight threatening piih with permanent visual loss in one case. / cases were discussed with neurosurgery. one child with evolving visual failure had an emergency ventriculo-peritoneal shunt. conclusions: important questions raised were: should all obese children with piih have access to a specialised obesity service? should all children have dedicated venographic imaging? how reliable is measuring csf opening pressure under ga? where lp is not possible should icp monitoring always be done? should repeat lps be done for persistent symptoms? should csf diversion surgery be restricted to cases of sight threatening piih only? objective: to describe a case of revesz syndrome due to a de novo missense variant in tinf . case report: a male infant was born at weeks gestation by emergency lscs due to maternal hypertension and reduced amniotic fluid. from week's gestation, reduced fetal growth was identified. the proband was born by at weeks. birth weight was . kg ( . th- nd centile) and occipitofrontal circumference (ofc) was . cm ( nd- th centile). he spent days in the scbu. he developed thrombocytopenia (nadir: /l), which resolved pre-discharge. periventricular calcifications on cranial ultrasound prompted torch screen and ophthalmology review. a right pre-retinal haemorrhage with overlying organised vitreous haemorrhage was identified, which remained stable on subsequent reviews. aged weeks, he was smiling, fixing and following with good head control. aged months, he developed new wobbly eye movements and was no longer fixing or following. bilateral retinal detachments were identified. ct and subsequent mri showed diffuse calcification within the thalami, posterior limb of the internal capsule, deep white matter, cerebellar atrophy and thin corpus callosum. findings on examination included ofc of . th centile, rotatory nystagmus and central hypotonia. whole-exome sequencing identified a pathogenic de novo variant in tinf (c. g>a, p.arg his). he subsequently developed thrombocytopenia and anaemia and is transfusion dependent. discussion: trf interacting nuclear factor- (tinf ), protein regulates telomerase and prevents telomere shortening. revesz syndrome is a severe form of dyskeratosis congentia, with multi-system involvement and early onset in-utero. revesz syndrome is characterised by intrauterine growth retardation (iugr), microcephaly, cerebellar hypoplasia, bilateral exudative retinopathy, intracranial calcifications and progressive bone marrow failure. revesz syndrome is distinguished from hoyeraal-hreidarsson syndrome by the presence of retinopathy. telomere disorders should be considered in infants with a background of iugr, thrombocytopenia, retinopathy and intra-cranial calcifications with a negative torch screen, as early features mimic congenital infection. objective: currently the most commonly reported neurological complication of sca is overt stroke. reversible cerebral vasoconstriction syndrome may be more frequent in patients with sickle cell anaemia than reported at present. the scarcity of prevalence studies however makes it difficult to improve diagnostic accuracy in these patients. methods: a -year-old ghanaian female was rushed to the paediatric emergency room with first episode of sudden severe global headaches initially started hours prior to arrival. the headache was so excruciating that she described it as her heart was beating in her head. there was associated neck pain, back pain, dizziness, and vomiting. there was no fever or dark urine. she was first diagnosed with sickle cell anaemia (genotype ss) at years of age after she was treated for dactilitis. she had since then been in her usual state of health with no history of blood transfusions or surgeries or admissions. she was compliant with her medications (folic acid mg daily). a physical examination and all investigations were also normal. on day six of admission patient had a generalized tonic clonic seizure with some degree of left sided weakness after having her bath. this was aborted with intravenous diazepam and a magnetic resonance imaging (mri) of the brain was requested. the mri of the brain revealed diffuse narrowing of the cerebral arteries with no areas of bleeding or oedema. reversible cerebral vasoconstriction syndrome was therefore suspected. results: the headache rapidly improved after starting nimodipine and repeat angiography at months showed no vasoconstriction, confirming the diagnosis. on follow up she is doing well academically with no neurological deficits. conclusions: the true incidence of rcvs in patients with sickle cell is uncertain, thus sensitizing medical practitioners is important. introduction: status dystonicus (sd) is a life-threatening disorder of generalised, painful dystonic movements and muscular spasm in patients with severe neurodisability. while rare, it may be complicated by rhabdomyolysis, multi-organ dysfunction, and death. infection, pain, gord, and medication failure are common triggers, but in approximately one-third of cases, sd is idiopathic. mordekar et al. ( ) identified a series of patients in whom sd occurred secondary to gi dysfunction. assisted feeding (e.g., via gastrostomy), and aberrant bowel peristalsis may trigger the onset of sd. this was a retrospective analysis which aimed to estimate an incidence rate for feed-induced sd (fisd). methods: patients presenting to sheffield children's hospital over a -year period with sd were identified. episodes were studied to assess for the nature of the onset of sd and as to the likelihood that the trigger was feed related. incidence of fisd as a proportion of total sd was calculated and or calculation performed to explore relative risk of sd between individual trigger factors. results: twenty-four individual episodes of sd were identified. ( %) arose from non-feed-related sources (nfisd), and were felt to be fisd ( %). additional patients were entered into a feed-induced dystonia (fid) group, whom showed clinical evidence of dystonia in relation to gi sources, but not sd. with the exception of infection, the relative risk of sd secondary to gi dysfunction was significantly higher than pain/gord and medication failure combined (or . ( % ci . - . ) and . ( % ci . - . ) respectively). conclusion: gi dysfunction coupled with severe neurodisability could serve as a trigger in a number of previously idiopathic sd cases through disruption of the neuro-enteric axis. however, overlap between triggers for fisd and nfisd, and significant variation between groups is evident, in addition to a lack of statistical study power. large, prospective studies are needed in the future to corroborate with these findings. poster no. dystonia can twist the patient, physician and the scans: hypermanganesemia, a rare cause of dystonia in children r kumar, s ali liaqat national hospital, karachi, pakistan introduction: manganese (mn) is a chemical element with symbol mn and atomic number . mn in the environment can cause toxicity with dystonia and other movement disorders. waterborne mn has a greater bioavailability than dietary manganese. according to results from a study, higher levels of exposure to mn in drinking water are associated with increased intellectual impairment and reduced intelligence quotients in school-age children. we have recently reported a suspected autosomal recessively inherited syndrome of hepatic cirrhosis, dystonia, polycythemia, and hypermanganesemia in cases without environmental mn exposure. the rarity of the disease can become a challenge for the physicians to recognize this as a cause of dystonia in children. it also has a characteristic finding on mri with t hyperintensity in basal ganglia rather than on t . case report: we present a case of a -year-old girl with dystonia who was previously healthy. she has been suffering from this for the last months and currently one of her years old sister started showing similar symptoms. physical examination revealed marked dystonia (score of on baryalbright dystonia scale) and polycythemia (haematocrit ). magnetic resonance imaging (mri) brain showed basal ganglia hyperintensity on t weighted images. hypermanganesemia was suspected and samples send for serum level which came out to be high. water samples were tested, which came out to be normal. chelation was done and the dystonia improved. conclusions: dystonia in children should be thoroughly investigated and rare, treatable causes should not be ignored. objectives: sodium valproate is used primarily for the treatment of epilepsy in children. it is a well-established teratogen, with in babies at risk of developmental disorders and in babies at risk of birth defects. this risk has been known since the s and yet it is estimated that since then children in the uk have been left with disabilities as a result. in , the medicines and healthcare products regulatory agency released guidance for its use, which included a risk acknowledgement form. patient safety alerts were issued in asking all organisations to identify females taking this medication. we aimed to identify all girls taking sodium valproate in the south eastern trust under paediatrics requiring annual risk assessment; patients under the additional care of a neurologist; patients receiving an annual review. methods: patients were identified through paediatric epilepsy nurse records and data collected through the electronic care record and medical notes from august to december . results: % (n= ) of girls with epilepsy currently taking sodium valproate, % under the age of years, % profound learning difficulties/disability and considered to be at low risk of pregnancy, % (n= ) potentially currently at risk, % were under the additional care of a neurologist, % reviewed in the past year. conclusions: sodium valproate must not be considered first line treatment in girls with epilepsy and > % of girls in our trust are not receiving it. of those receiving it, the majority are felt to be low risk due to young age and/or profound disability. we identified two patients at risk and steps were taken to ameliorate this. we have demonstrated good awareness; however lifelong education of families is crucial to reducing the burden of fetal valproate syndrome. rett syndrome (rtt) is neurodevelopmental disorder affecting approximately in - live female births, most commonly associated with mutations in the mecp gene. hand stereotypies and gait disturbance, as well as spasticity and dystonia, have been noted in rtt since the first descriptions of the syndrome. objective: this review aimed to explore the prevalence of reported movement disorders in rtt. data sources and extraction: pubmed and embase databases for papers describing features of movement disorders in rett syndrome. papers were selected for inclusion to be reviewed if they included description of case report, cohort or case-series of patients with rtt which included a description of clinical features of their movement disorder. selected papers were divided into epochs: (i) pre- , (ii) to , and (iii) onwards. results: studies ( in the first epoch, in the second epoch and in the third epoch) reported on movement disorders including stereotypies in rtt patients. hand stereotypies were almost universal in reported cases, diminishing but not disappearing over time. gait disturbance and ataxia/tremor were also very common (> % cases). elements of hypertonia were also common, increasing with age. in earlier descriptions spasticity was commonly described, with more frequent reference to dystonia/rigidity in more recent reports. myoclonus and choreoathetosis are uncommonly reported in rtt. conclusions: movement disorders beyond hand stereotypies are common in rtt, most notably tremor. hypertonia is a common feature seen in rtt, increasing in prevalence with age, and with an apparent change in nomenclature over time, (i.e., early epoch spasticity, late epoch dystonia). dystonia was specifically reported in / cases. further work is required to explore the relative contribution of dystonia and rigidity to hypertonia in rtt, as well as the impact of these impairments when present. introduction: headache is the common complaint in children, and the source of it, a great deal of worry for general practitioners and parents. one of the commonest causes of headache in paediatrics is migraine. methods: a prospective study was conducted to evaluate the demographic data, clinical spectrum and grading the child with migraine by using the paediatrics migraine disability assessment (ped-midas) questionnaire and to start prophylactic treatment for those with the higher grades in the department of paediatrics in tertiary care hospital. all children with migraine from age to years were included while all other types of headache cases were excluded. results: total children with migraine were studied. approximately % children complained of bilateral frontotemporal headache in which . % presented with throbbing type. other associated features were photophobia, phonophobia, nausea and vomiting. % had skipped meal, followed by altered sleep and exam stress as aggravating factors. . % required medication for headache relief. headache duration and frequency was approx. days and days/month. . % cases were diagnosed migraine without aura and . % cases were diagnosed as migraine with aura. loss of full school days due to headache was approx. days for period of months. based on ped-midas score, % of children with migraine had grade i disability while . % and . % cases had grade ii and grade iii disability respectively. correlation of ped-midas score with frequency and severity were significant (p< . ) while with duration of headache was insignificant (p< . ). conclusions: all patients with higher ped-midas grade are warranted prophylactic treatment. both ped-midas scores and grading can be successfully used for assessing the migraine disability and its easier, less time consuming, bedside diagnostic tool, can be used widely in routine clinical evaluation and management. objective: to review the cases referred to this uk-wide study of children with possible variant creutzfeldt-jakob disease (vcjd) and report the differential diagnosis in children presenting age years or older. methods: children meeting the case definition for progressive intellectual and neurological deterioration (pind) were identified via the british paediatric surveillance unit. details were obtained by standard questionnaire. results: between april and august , children had been notified to the study. were found not to meet the pind case definition. had an underlying diagnosis to explain their deterioration, with over different disorders including vcjd cases (the last identified in ). there were children who presented to clinicians when aged years or over, including all the vcjd cases. of the other disorders in this age group the commonest were: mitochondrial cytopathy , adrenoleukodystrophy , lafora body disease , huntington's disease , neuronal-ceroid lipofuscinoses , niemann-pick type c , metachromatic leukodystrophy , sspe , wilson's disease . when reviewed in there was no underlying diagnosis in pind cases; of them had diedonly underwent autopsy. the recent identification of the first patient with vcjd who was mv heterozygous at prnp codon reinforces the need for continued vcjd surveillance, particularly as a study of archived appendix samples from uk hospitals published in indicated that approximately in of the uk population is carrying abnormal prion protein in the gastrointestinal tract. in the absence of a validated vcjd screening test the pind study remains the only means of performing systematic surveillance of the neurodegenerative diseases that make up the differential diagnosis of vcjd. objective: transient lesions in the splenium of corpus callosum (scc) are rare findings in mri brain in paediatrics. in literature, it has been described as reversible splenial lesions syndrome (resles) and mild encephalitis/encephalopathy with reversible splenial lesions (mers). the condition has diverse aetiology and widely variable neurological presentation but the prognosis is usually favourable. we present two cases of resles with predominantly expressive dysphasia but varying causal associations. method: retrospective review of resles case series exploring clinical course, investigations, neuroimaging, treatment and recovery. result: case : a year-old-girl presented with confusion, fever and low oxygen saturation. she had alagille syndrome and partially corrected tetralogy of fallot. her neurological manifestations were expressive dysphasia, dysarthria and difficulties with spatial awareness. interestingly she was able to use occasional words that were abusive in nature. mri showed prominent focus of abnormal signal and restricted diffusion in scc. her blood culture grew staphylococcus aureus and echo revealed infected shunt. treatment involved shunt replacement and prolonged iv antibiotics. repeat mri showed resolution of splenial lesions. she continued to improve neurologically. case : a year-old-girl presented with paroxysmal episodes of head turning, head drop and staring for a few seconds. she refused to feed. she showed emotional lability with expressive dysphasia but preservation of expletives. neurologically she was intact. mri brain showed high signal with restricted diffusion in scc. her blood and csf investigations including mog, aquaporin, nmdar, lyme antibodies were negative except asot was . her eeg was normal. she received a course of ivig and azithromycin. her repeat mri showed resolution of the lesion in splenium. she made complete recovery over next few months. conclusion: splenial lesions are rare but clinically significant but not 'non-specific'. expressive dysphasia is a prominent symptom. awareness of resles/mers will avoid unnecessary investigations and assist in the prognostication. background: the evidence-base on managing paediatric-headaches is sparse resulting in wide variation in practice with nice guidelines commencing over years of age. this study aims to evaluate outpatient management of paediatric headaches. objective: to investigate paediatric headache referrals to a tertiary hospital over a -year period, exploring patient demographics, headache type, role of neuroimaging, management and outcome. methods: this prospective study reviewed headache referrals for the year - . the data was collected following weekly emails to relevant clinicians. the patient demographics, headache classification, imaging, management and outcome were collated on a proforma from the electronic patient-records. results: there were patients. the median age of patients at first outpatient appointment was years (range - y); . % were female. incidence of headaches increased with age. female preponderance of headaches existed in all age groups and was most substantial post-puberty with a . : female-to-male ratio in patients aged to years. migraine was the most common diagnosis, affecting . % of patients. % of referred patients underwent a brain mri scan, all of whom had a normal neurological examination. no mri scans found pathology contributing to headache presentation. % of patients were discharged from neurology clinic after first or second neurology appointment. non-pharmacological management was the most common intervention and consisted of: headache diary, lifestyle advice, education, relaxation techniques. the most common medications prescribed bar simple analgesia were sumatriptan ( %), propranolol ( %) and pizotifen ( %). conclusions: a multidisciplinary and biopsychosocial approach to managing paediatric headaches, consisting of non-pharmacological and pharmacological methods resulted in a positive outcome, with majority discharged from tertiary care after first appointment. prescription of sumatriptan and propranolol first line for acute and prophylactic management respectively, was in accordance with current clinical recommendations. the role of mri scanning for paediatric headaches requires further exploration and perhaps more stringent guidelines. objective: the head-up tilt test (hutt) is the gold standard autonomic function test for identifying disorders of blood pressure (bp) and heart rate (hr) regulation, specifically with excessive falls in bp and or hr, as well as excessive postural tachycardia (pt). the minute active standing test (ast) is quicker and easier to apply, e.g., in an outpatient clinic, and may be more sensitive in demonstrating pt. we aimed to compare the yield of these abnormalities when using ast vs hutt. methods: this was a retrospective, clinical notes review, and registered clinical audit of unselected consecutive children and young people undergoing hutt immediately preceded by an ast. results: data was available on children and young people, ( %) female, aged to years (median ). / ( %) with complete data sets for the first minutes of hutt and the ast had abnormally large drops in bp and or hr on hutt. only / positive on hutt was also positive on ast. however, an additional / ( %) were positive on ast but not on hutt, giving / ( %) positive in total. / ( %) with hr data sets for minute hutt and ast had abnormally large rises in hr on hutt. only / positive on hutt were also positive on ast. however, an additional / ( %) were positive on ast but not on hutt, giving / ( %) positive in total. while hutt yielded more cases with significant falls in bp and or hr than ast ( % vs %), combining the tests gave the highest yield ( %). while ast yielded more cases with significant rises in hr than hutt ( % vs %), combining the tests gave the highest yield ( %). conclusions: we recommend routinely undertaking a minute ast prior to the minute °hutt, in children and young people. objective: the aim of our work is to describe the respiratory function trajectories and their correlation with motor function in a cohort of spinal muscular atrophy type and non-ambulant sma paediatric patients. methods: this is a retrospective -year study in patients recruited in the ismac natural history study (uk, italy, us). the following respiratory data were collected: lung function data (forced vital capacity absolute (fvc) and fvc% predicted, non-invasive ventilation (niv) requirement. recumbent or ulnar length were used as surrogate for height in fvc%pred. calculation. comorbidities affecting lung function such as aspiration were collected. anthropometrics and motor function scores as hammersmith functional motor scale (hfms), revised performance of upper limb (rulm) were noted. we excluded patients in interventional clinical trials and nusinersen therapy. results: data were available for patients: sma , sma . mean age at first visit was . (ae . ) and . (ae ) years for sma and . / ( %) sma and / of significant lesions. a review of practice of asking routine or non-urgent mri requests should be considered in view of an unlikely significant result. retrospective review of brain magnetic resonance imaging referrals in children less than years (r[ ] ) is an ultrarare disease characterised by drug-refractory epilepsy, cognitive impairment and behavioural problems. non-pharmacological treatments should be considered alongside antiepileptic drugs (aeds) early after diagnosis to benefit prompt seizure control and preserve cognitive function. we aimed to understand the use and experience of ketogenic diet therapy (kdt) in r( ) by patients families carers (pfcs) and healthcare professionals (hcps), assessing its efficacy and safety, and contrasting nhs kdt service provision with patient demand. methods: literature searches were conducted on use of kdt in r( ) and similar complex epilepsies. two surveys were developed to gather demographic, diagnostic and clinical care information. surveys were qualitative and descriptive with patient and expert collaborators assessing content accuracy and readability. responses were discussed at a patient and expert workshop. results: the number of responses ( pfcs, hcps) was considered significant given the ultra-rare status of r( ). % of pfcs had tried kdt. seizure activity, behaviour and cognitive outcomes were ranked equally important by hcps and pfcs. significant improvement in seizure activity, cognition and alertness were reported; side-effects were typically mild but with one report of increased seizure frequency. the high rate of comorbidities, older age at presentation, behavioural problems and cognitive impairment can make implementing kdt in r( ) challenging. pfcs report quality of life would be most improved with reduced aed side-effects; hcps report they would consider reducing or withdrawing aeds where kdt is successful. conclusions: kdt may not be suitable for every r( ) patient, but there is a strong consensus that it should be considered as an early intervention. in the uk, nhs kdt services are predominantly available for paediatric patients, with very limited adult access. a detailed health economic analysis illustrating reduced acute care costs and improved quality of life may encourage more widespread kdt implementation. objectives: whole exome sequencing (wes) with a -week result turnaround time has become available on the nhs for children in an intensive care setting. we aimed to determine the diagnostic utility and impact on clinical care of wes in a regional paediatric neurology centre. methods: retrospective case notes review. results: six cases ( m, f) were identified. three patients were dependent on long-term respiratory support. a pathogenic mutation was detected on wes in / cases ( %). one case required 'reverse phenotyping' with an abnormal transferrin glycoform electrophoresis confirming that two heterozygote variants of the rtf gene were consistent with a congenital disorder of glycosylation (cdg). no other variants of unknown significance were found. three children presented with neonatal onset epileptic encephalopathy (two cases had scn a, one case wwox), one child with intractable epilepsy from months of age (rft mutation associated with cdg) and one child with hypotonia and ventilator dependence after a respiratory infection at months of age (ighmbp mutation associated with spinal muscular atrophy with respiratory distress). wes found no pathogenic mutation in a -year-old with intracranial calcification, microcephaly, epileptic encephalopathy and severe developmental regression. in / cases other single genes/panels had been sent prior to initiation of wes with multiple single genes/panels sent in / cases. in / cases wes detected the gene thought most likely based on clinical phenotyping on the request form. conclusions: wes has a high diagnostic yield in this cohort of patients. reaching a prompt diagnosis facilitated withdrawal of care in one case (ighmbp ) and helped to exclude an epilepsy surgery hypothesis in four cases as well as guide prognosis in all cases. wes should be considered as a cost-effective alternative when multiple single genes and/or genetic panels are being sent off in parallel due to clinical urgency. objective: the care provided in the time surrounding the death of a child shapes long-term memories and has potential to impact on the grieving process. there are no specific guidelines for picu staff in relation to what good care looks like at this time. we sought insight into practice across the uk to build an evidence base, improve care provided and share good practice. conclusions: from the survey feedback, we found this was an area that all units believe can be improved. in relation to acps, we hope this will be more widely introduced. we know that % of patients admitted to picu are life limited. these difficult conversations with family help guide management, understand wishes, and formal documentation ensures all staff are aware. several units with higher uptake of hospice/home care found early conversations with families beneficial. units with a dedicated palliative nurse stated this allowed more time with families. we believe this should become a standard of care. staff training is limited in most units. for something so difficult and frequently encountered, it is vital we equip staff better. prioritising children with epilepsy in the first seizure clinic cp white, s brown, ka hapgood, s tuohy children's epilepsy service, morriston hospital, swansea, uk objective: rising demand for limited first seizure clinic appointments was leading to increasing waiting times and the feeling that children with epilepsy were waiting too long for their first assessment. concern was also expressed that families were not receiving our first seizure leaflet or given instructions about capturing any further episodes either on video or by making a written record when initially seen. methods: from april all families were sent a letter acknowledging the referral and asking them to contact the specialist epilepsy nurse if they had any concerns prior to the appointment. later modifications included a seizure information leaflet and a seizure record document. we have analysed the results of the first year of using this system. patients were identified from the clinic database and further information was obtained by reviewing clinic letters. results: our initial concern that the specialist nurse would be inundated with phone calls from worried parents were not realised as only % ( / ) of parents contacted the service before their appointment. these were invariably parents whose children had had a second episode ( / ). had had further generalised tonic clonic seizures. children had eegs performed before their first appointment. this included all the children given a diagnosis of epilepsy. % of these children ( / ) were given a diagnosis of epilepsy made compared to . % of other referrals (p< . ). although a higher percentage of families who were reminded about videoing any further episodes did so the difference between the two groups was not statistically significant. unfortunately, overall waiting times were not affected. conclusions: a simple change to the way in which the service is delivered has led to earlier identification of those children with epilepsy. we are looking at other ways of improving the accuracy and timeliness of the appointments. introduction: paediatric idiopathic intracranial hypertension (iih) is an uncommon disorder and presentation is varied with children presenting to paediatricians, paediatric neurologist, and ophthalmologists. there are many areas of diagnosis and management where evidence is limited. a national adult evidence and consensus-based guideline was published in . no national paediatric guideline exists to aid the further investigations and management of the cases. the iih meeting at the bpna conference in and january set the scene for collaborative work on this topic. aim: to develop a national paediatric iih guideline based on the available literature such as modified dandy criteria, friedman classification and icdh- classification and consensus amongst various members of bpna chan group, members of rcpch, ophthalmology, neurosurgery and radiology, and patients. methods: a core children's headache iih guideline development group was established and has met at four national special interest group meetings between november and september . topics discussed include incidence of papillioedmea, csf dynamics in iih, bpnsu iih data, ophthalmology good practice, regional iih pathways in uk and setting up of the delphi process. the paediatric iih study day in september with invited patient/parent representatives highlighted the impact on families with the disorder with need for better communication about the disorder, clear guidelines and sharing of good practice amongst clinicians. an email list of bpna chan group, rcpch members, ophthalmologists interested in the guideline was created. results: a set of statements were drafted for delphi consensus work. these are currently being reviewed by the core guideline development group prior to being circulated to the wider working group. conclusions: goal setting for the next process with the delphi process to work with the core committee and a wider working group will be presented at the bpna conference . objective: nhs england's marginal rate emergency threshold (mret) and readmission fund funded the chameleon project (twitter account: @chameleonproje ), to improve children's end of life care. this funded a lead disability paediatrician with expertise in paediatric palliative care ( h/wk), a children's palliative care nurse ( d/wk) a network administrator ( d/wk), and additional hours for paediatricians in the critical care, oncology, and neonatal units, and in each of the local district general hospitals (total h/wk). methods: tools were developed to aid identification of children in the last year of life and to support anticipatory care planning. the team attended ward rounds and provided teaching sessions, advice and support. children who died an expected death in the months of the project were ascertained from the child death review teams. non-elective admissions, bed days, and costs were tabulated. we also evaluated the documentation of care plans and post bereavement family feedback questionnaires. results: children died an expected death. the same number died during the previous months. the median number of non-elective admissions reduced from to per child, specialist ward bed days reduced from to ( % reduction). for children admitted to picu in the last months of life, the total picu bed days reduced from to ( % reduction), the median length of stay reduced from days to days, and the maximum length of stay reduced from days to days. the percentage of children who died an expected death who had documented anticipatory care plans rose from % to %. conclusions: the network of clinicians with expertise in paediatric palliative care working together across a region improved anticipatory care planning and reduced admissions and bed days for children in their last year of life: better care with reduced costs. child a, a boy with speech and language delay, presented at years of age with self-resolving episodes of floppiness, ataxia, and disorientation. there was associated muscle weakness and drooling. these unusual episodes occurred to times per week and were often triggered by excitement, especially physical and emotional overstimulation. they lasted from a few minutes to an hour, with no residual deficit. episodes could also be triggered by having late meals (fasting episodes). investigations including an mri/mra brain, eeg, sleepdeprived eeg and video telemetry did not reveal any significant abnormalities. metabolic and endocrine tests were normal. as his presenting symptoms were consistent with episodic ataxia, possibly a periodic paralysis spectrum, a trial of acetazolamide was given, which showed some improvement in the number and severity of the episodes. at years of age, genetic sequencing results revealed child a has a recessive kb deletion within the long arm of chromosome , band q . . this is a homozygous intragenic deletion within the tango gene. tango is a 'transport and golgi organization ' homolog. the function of tango is unknown; however, in previous studies, depletion in drosophila s tissue culture cells was observed to cause fusion of the golgi with the er. a recent study of individuals with tango , illustrated that child a has a clinical phenotype which is consistent with those previously reported in the literature. although seizures are present in % of individuals with tango , child a has not had any seizures to date. although no effective treatments for this rare condition are known, early diagnosis is important so that individuals and their families are aware of the potential encephalomyopathic crises and arrhythmias which occur. further research in elucidating the structure and function of the tango protein may lead to effective therapies in the future. objective: hie affects around . / live births. prognostication relies on clinical progress, neurophysiology, neurological examination, and magnetic resonance imaging (mri). there is limited information on the relationships between mrs brain results and visual appearances of the brain on mri and clinical features. this work studied the use of mrs in this cohort. methods: mrs is used routinely in all neonates with hie in our unit, so approval for this service evaluation was obtained from our clinical governance department. we identified neonates with hie between jan and march who had mri and mrs in the first days of life. medical notes were reviewed, and mri results categorised as normal or abnormal. mrs results and clinical features were compared between mri groups using parametric or non-parametric testing. correlation and regression analyses studied relationships between clinical features and mrs results. p-values of < . were assumed to be significant. results: participants were identified, were excluded because they did not meet our inclusion criteria. data from a total number of neonates were analysed using r studio. babies with abnormal mri scans had significantly lower birth weight (p= . ), gestational age (p= . ), and higher scores in the sarnat staging scale (p= . ). the analysis of the mrs data also revealed that these babies had lower levels of n-acetylaspartate (naa) in their parieto-occipital region (p= . ), as well as higher levels of lactate and lactate to choline both in the parietooccipital region (p= . and p= . respectively). finally, these significant mrs variables were significantly correlated with time to normalisation of lactate in single linear regression. background: more children and young people are surviving with an acquired central nervous system injury (traumatic or non-traumatic). the first nhs england (nhse) specialist specification for paediatric neurorehabilitation services was written in . evidence for benefits of early neurorehabilitaion after adult stroke are compellingevidence for early neurorehabilitation in cyp is emerging. methods: service information was collected from all england and wales pnr units in . results: / units contributed. activity is increasing ( ( / ); ( / ); ( / ). / ( %) are major trauma units ( %) have dedicated coordinators. several units cannot offer daily therapy. most units discharge cyp home. conclusions: considerable neurorehabilitation in-patient activity is taking place but there remains an absence of secure funding, adequate staff, dedicated beds, key members of the mdt, protected time for pro-active patient specific discharge planning. neurorehabilitation is an integral part of the neuroscience clinical pathway and our children deserve a fully resourced service as described in the service specification. tuberculosis/sarcoidosis ( ). out of the sub-categories, the group of refractory seizures/status epilepticus were most likely to have repeated imaging with either ct or mri within years ( %), followed by the group of ventriculo-peritoneal shunt blockage ( %), space occupying lesion ( %) then head injury ( %). out of patients with refractory seizures/ status epilepticus, were already known to have epilepsy. also, most repeated imaging included a subsequent head mri. conclusions: most common indications for ct head were head injury and shunt blockage (as this was a neurosurgical centre). the groups most likely to have repeated imaging were refractory seizures/status epilepticus and shunt blockage. with children presenting with known epileptic seizures in the emergency department, it is important to consider clinical data and seek to devolve decision to image. poster no. isolated radial nerve palsy, a rare presentation of congenital wrist drop c duggan, n mcsweeney cork university hospital, cork, ireland isolated congenital radial nerve palsies are a rare phenomenon and typically spontaneously recover within months. the true incidence is not known, but in a recent study . % of infants presenting to a brachial plexus injury clinic had an isolated congenital radial nerve palsy. patient a is a -week-old male who presented at birth with a left sided wrist drop following a non-traumatic elective caesarean section at + / . his birthweight was . kg ( st centile). movement of the wrist and digits were impaired to absent with preservation of function at the shoulder and elbow. there was a nodule noted in the left upper limb, anatomically superficial to the radial nerve. it was a normal pregnancy with no antenatal or postnatal issues. he attended physiotherapy and occupational therapy who provided a splint. on examination at weeks, there was weakness of the extensors of the left wrist. the rd, th and th digits remained fully flexed at rest and could be extended passively but not actively. extension of the thumb and index finger had recovered at -week review. function at the shoulder and elbow joints were preserved with normal flexion of the wrist and digits. a scar was noted superficial to the radial nerve at the same location as the lesion described at birth. the remaining systemic and neurological examinations were normal with typical development and appropriate growth. the working diagnosis at present is an isolated radial nerve palsy likely caused by in-utero compression. the nodule and scar noted above are consistent with lesions described in a previous case series. these were hypothesised to be areas of fat necrosis secondary to compression; resulting in the palsy. patient a's lack of further neurology such as a generalised brachial plexus palsy makes a birth injury less likely. further investigations and follow-up are awaited. background: valproate is an effective antiepileptic medication. if a woman becomes pregnant while taking valproate, her baby is at risk of congenital malformations ( in ) and developmental disorders ( in ). furthermore, it is associated with an increased risk of autism spectrum disorder and adhd. in , the nhs/hse recommended new restrictions on the use of valproate, including a national pregnancy prevention program (prevent) and avoidance in prescribing to female patients of childbearing potential unless other treatments are ineffective or not tolerated. objective: to review the use of valproate in a well-defined population of at risk females with moderate to profound intellectual disability (id). identify the patients at risk and imbed the guideline into our practice. methods: a retrospective chart review was carried out of all girls aged between and years, attending the daughters of charity disability service (doc) in dublin, ireland. data such as diagnosis, valproate use, degree of id/gross motor function classification system (gmfcs), documentation of menarche and discussion regarding risk of valproate use were recorded. results: in total females aged between and years where identified as currently using valproate out of charts reviewed ( %). of the patients identified, / had moderate id (gmfcs iii) and / had severe to profound id (gmfcs iv-v). / had menarche documented. / had the risk of valproate discussed. conclusions: in our cohort, a significant number of girls remain on valproate. % complied with new guidelines regarding discussions around the risks of valproate; highlighting the % of patients in need of counselling. an annual risk acknowledgement form was placed in their charts to prompt discussion next visit. in children with intellectual disability, conversations regarding contraception are difficult but essential. if valproate is used, then the risks must be fully understood by parents and carers. evaluation of the management of children up to age of years with cerebral palsy in southend university hospital large district university general hospital against nice guidelines (nice guidelines ng ). methods: clinic notes of all registered children with cerebral palsy (cp) up to years of age as of june were included in the study. this was because there was no early data on children above years age. results: patients were age to years and patients age to years. were male and females. had hemiplegic, quadriplegic, diplegic and dystonic cp. only ( %) had gmfcs levels recorded. ( %) were < weeks, ( %) were to weeks and ( %) were term. mri head findings: white matter changes including pvlin ( %), ( %) hie changes, ( %) basal ganglia changes, ( %) congenital brain malformation, ( %) infarction. migrated to area with no mri report. all the children received multidisciplinary team (mdt) input including physiotherapy. comorbidities werechildren on medications for gastro-esophageal reflux - (with peginsertions). for epilepsy - , for dystonia/spasticity - , for constipation - , for poor salivary control - . behavioral issues noted in and was on adhd medications. had botulinum toxin injections and had selective dorsal rhizotomy for spasticity. documented discussion of diagnosis with family was in ( %) patients and none in patients ( %). only % patients had vitamin d levels checked. conclusions: management was in line with nice guidelines. they all had mdt input. there is a need to improve documentation of -evidence of discussion with parents, gmfcs level by age ½ years plus, hip surveillance from age years for gmfcs level iii to v and annual vitamin d levels especially for gmfcs level iii to v, peg fed and children on multiple anti-epileptic medications. poster no. intracranial hypertension in children: an updated systematic review l di genova , n desai , s esposito , p prabhakar pediatric clinic, department of surgical and biomedical sciences, universit a degli studi di perugia, perugia, italy; department of paediatric neurology, great ormond street hospital nhs foundation trust, london, uk; neurosciences, great ormond street hospital nhs foundation trust, london, uk objective: our goal is to provide an overview on paediatric intracranial hypertension. methods: given that the last update of the diagnostic criteria of idiopathic intracranial hypertension was published in , a thorough medline search of all english articles was conducted between and . results: intracranial hypertension may be primary, with a paediatric annual incidence ranging between . and . per children or arise from a secondary cause. misdiagnosis or delayed intervention can lead to poor quality of life and morbidity. in , this condition was reconsidered, due to new accepted values for opening pressure and advances in neuroimaging; the importance to develop effective therapeutic strategies in order to prevent blindness was thus highlighted. to date, the main strategies described involved both medical and surgical approaches; nevertheless, there have been no paediatric intervention studies. disease monitoring plays a key role in the definition of the best timing and modality of treatment. recently, a risk stratification has been proposed with the aim to facilitate an adequate evaluation and proper care of children with intracranial hypertension: visual monitoring could represent an objective tool to manage these patients. in recent years, important evidence for the efficacy of acetazolamide emerged in the idiopathic intracranial hypertension treatment trial. surgical treatment is the modality of choice in children with worsening vision impairment, intractable headaches despite maximal medical management or in case of intolerance to medical therapy. conclusions: there are poor evidences about paediatric intracranial hypertension's outcomes. unfortunately, children's quality of life is heavily influenced by pain and permanent vision loss. standardized therapeutic strategies remains uncertain, highlighting the need for longitudinal studies to identify the best treatment in childhood. in order to alleviate symptoms and prevent permanent chronic sequelae, careful clinical evaluation and ophthalmological monitoring could be a useful guide to better manage this medical condition. objective: cerebral sinovenous thrombosis in childhood is a life-threatening neurological entity with uncertain epidemiology, potentially complicated by secondary intracranial hypertension. in the literature, there is a lack of evidence supporting the main strategies to approach both these medical conditions. our objective is to highlight the value of a prompt diagnosis aiming to define a tailored management approach based on children monitoring. methods: we review the main findings regarding cerebral sinovenous thrombosis and intracranial hypertension in children through illustration of a case with otogenic sinus thrombosis and secondary intracranial hypertension. results: a -year-old boy developed a local venous sinus thrombosis because of the spreading of a primary infective process from his middle ear into the sigmoid sinus complex, facilitated by anaemia and dehydration. the venous outflow disturbances led to secondary intracranial hypertension. the management aimed to treat cerebral thrombosis with anticoagulants and intracranial hypertension through medical and surgical strategies. the insertion of the lumbar-peritoneal shunt was necessary when medical approached failed and visual function deterioration was evident. careful clinical evaluation and ophthalmological monitoring helped us in the tailoring of the best treatment with the aim to alleviate symptoms and prevent sequelae of increased intracranial pressure. in the literature, no paediatric intervention studies regarding the main strategies to reduce intracranial pressure have been published. moreover, there is a lack of evidence supporting the safety of anticoagulation therapy, reducing the possibilities to safely manage cerebral thrombosis in childhood. conclusions: in children, a multidisciplinary approach is essential to manage both cerebral thrombosis and intracranial hypertension and ensure an optimal follow-up, aiming to prevent visual and therapy-related complications, possible relapses and their early diagnosis. from our perspective, monitoring our patient with clinical manifestations and visual status helped us to plan the best timing and modality of treatment and intervention. yearly rate of progression of fvc% predicted (available in n= ) was . % in sma and . % in sma . in sma , fvc% predicted declined steeply from to years of age, followed by a levelling. conversely, in sma patients fvc% predicted declined slower but steadily from years of age. / ( %) sma and / ( %) required non-invasive ventilation due to respiratory infections or hypoventilation conclusions: the results of this ongoing collaborative work suggests that in sma and lung function declines from age and respectively. lung and motor function correlate well in both sma and . this data will help the assessment of the long-term efficacy of new treatments for sma this review aims to study the appropriateness of mri brain referrals following implementation of local changes to improve compliance to the nice cg standard. methods: following an earlier survey (es; / / - / / ) of mri brain referrals for headaches in children over years, key recommendations included adding pop-ups in the neuroimaging request system (ice) of nice cg and headsmart clinical guideline v as well as verbal consent obtained from senior paediatrician before request was made. following these implementations, requests for mri brain were analysed during / / - / / in the same district general hospital. referral was deemed compliant if the nice guideline cg standard were met. results: children were referred for mri brain scan (mean / month vs /month in es). ( %) referrals were compliant (vs % compliance in es). ( %) referrals were 'urgent' (vs % urgent es) and ( %) 'routine' or non-urgent (vs % routine es). ( %) of urgent referrals (vs % es) and ( %) of routine referrals (vs % es) were compliant mri brain guidelines for neuroimaging in less than years exist (headsmart clinical guideline v and nice epilepsy qs ) but are limited. this study aimed to assess current practice of mri brain referrals in children under years. methods: retrospective review of mri brain referrals in children under years performed between mri brain scans were done (m:f . : ) ( %) referrals under headsmart and ( %) of these were urgent requests; significant brain abnormality was seen in ( %) in urgent and ( . %) in nonurgent cases. ( %) referrals under epilepsy qs and ( %) were urgent requests; significant brain abnormality in ( %) in urgent and ( %) in non-urgent cases. ( %) were miscellaneous requests and ( %) were urgent; significant brain abnormality in ( %) in urgent and ( %) in non-urgent cases. overall mri brain showed significant abnormality in urgent requests ( %) compared to non-urgent requests ( %) poster no. investigating factors that influence unplanned admissions and a&e attendances in those with pre-existing neurological conditions in childhood s dowsell , k kananaviciute , r parslow , am childs university of leeds, leeds, uk; paediatric neurology, leeds general infirmary, leeds, uk objective: previous papers have shown increasing demands and costs to the nhs in relation to the inpatient care of children with neurological conditions. unplanned admissions may reflect a lack of effective care and have been shown to correlate with high outpatient clinic did not attend (dna) rates . the aim of this study was to determine factors underlying unplanned admissions and accident & emergency (a&e) attendances in a cohort of patients under the care of the leeds regional paediatric neurology service over a -year period. methods: all children < years who had paediatric neurology outpatient appointments in were identified using hospital databases. clinical and demographic data was extracted from electronic case notes. those without a definitive neurological diagnosis or who had moved to adult services during the study period were excluded. the cohort was cross referenced to a&e databases and admission records from to . poisson regression was used to identify any correlation between specific predetermined factors to assess their influence on a&e attendance and admission rates. results: a cohort of patients was established and had a total of unplanned admissions during the study period. patients had a&e attendances with a total of attendances. higher dna rates, younger age and certain diagnostic categories correlated with increased rates of unplanned admissions. the role of emergency care plans in preventing admission was unclear as only / patients with epilepsy had care plans in place. conclusions: this study confirms the association between increased rates of a&e attendances and unplanned admissions in children with specific neurological disorders and high dna rates. this is relevant for service planning as it highlights the need to target scarce resources towards 'higher' risk patients with more complex diagnoses where more integrated care and support may prevent or reduce unplanned hospital attendances.poster no. audit comparing great ormond street hospital headache clinic diagnoses and management of patients aged to years to nice clinical guidelines a ward , , p prabhakar neurology, great ormond street hospital, london, uk; university of glasgow, glasgow, uk introduction: between / / and / / the gosh headache clinic saw new patients aged to years. the nice clinical guideline (cg ) on the diagnosis and management of headaches in over s covers tension-type headache, migraine, cluster headache and medication overuse headache. this audit aims to compare gosh diagnosis and management to those of the cg . methods: using the patient list from the headache clinic data was gathered by accessing outgoing clinic letters via epic. raw data was collected on; age, gender, description of headache (pain location, quality, intensity, duration and frequency) and associated symptoms, triggers, previous imaging, previous and current treatments. the management data collected include: diagnosis and treatments offered, as well as whether gosh offered lifestyle advice, psychology, occipital nerve block or riboflavin. this data was then compared to cg . results: ( . %) of diagnoses made by gosh matched the cg diagnosis. ( . %) diagnoses differed, with of these due to discrepancy between chronic/episodic and/or presence of aura and due to the vague diagnoses of migraine-type, new daily persistent, migrainous etc. fitting the cg definition of chronic migraine. all but one patient was managed in line with the guidelines. . % of patients had brain imaging prior to attending the clinic, with . % of these reporting positive findings. discussion: despite patients' diagnosis differing between gosh and cg , all but one patient was managed in line with the guidelines. this is likely due to nice recommended management being the same for any type of migraine. improvements could be made in documentation of frequency and duration of headache and aura, as well as more routinely offered lifestyle advice, psychology and riboflavin recorded in outgoing clinic letters. objective: to review the purpose of ct head requests from emergency department of a busy tertiary hospital as part of quality improvement. due to increasing evidence of ct scan radiation predisposing to leukaemia and brain tumours, it is best to keep ct scans to the minimum if clinically indicated. this project reviewed the indications for ct head and also looked at patients who had repeated ct or mri head scans within years. methods: data was collected retrospectively looking at a snapshot period of months between september-november . patients were < years of age and they had a ct head from emergency department at king's college hospital, london. trauma patients were excluded. data was collated with aid of the neuro ct department 'cris' system. results: out of patients, reasons for ct included: head injury ( ), ventriculo-peritoneal shunt blockage ( ), refractory seizures/status epilepticus ( ), space occupying lesion ( ), orbital cellulitis ( ), intracranial haemorrhage ( ) objectives: mutations in kif a are associated with a wide range of neurological disorders, ranging from hereditary spastic paraparesis (hsp) to sensory neuropathies to a severe infantile neurodegenerative disorder. collectively, they are extremely uncommon but likely to be under-recognised. we aim to report the spectrum of kif a-related disorders from a single tertiary neurology centre, with a view to improving understanding and awareness of these rare conditions. methods: affected individuals known to great ormond street hospital were identified through liaison with consultants involved in the care of children and young people with movement disorders. clinical information was collected through a retrospective review of case notes. results: twelve individuals in families were identified. all had heterozygous kif a mutations including three previously unreported variants. severity ranged from a fatal neonatalonset disorder with contractures, absence of visual development, and agenesis of the corpus callosum on mri to hsp with preservation of ambulation into the second or third decade of life and entirely normal mri. upper motor neuron signs were found in / children and a primarily sensory neuropathy was present in / children assessed. / children also had extrapyramidal signs (dystonia). some degree of learning difficulties and/or disorders of mood or behaviour were present in all children. optic atrophy, mr brain white matter changes and epilepsy were also common, especially in those children who were more severely affected overall. conclusions: kif a related disorders are so diverse that it is arguably misleading to consider them as a single disease entity. features common to the majority of affected patients include upper motor neuron involvement, and neuropathy (even in the absence of an obvious sensory deficit), with high risk of other neurological and neurobehavioural comorbidities. objective: ataxia with oculomotor apraxia type (aoa ) is a slowly progressive, autosomal recessive disease characterised by the triad of ataxia, oculomotor apraxia, and sensorimotor neuropathy that results from mutations in the gene encoding senataxin (setx), a dna/rna repair protein essential for genomic stability. we investigated a -year old male with a history of unsteady gate for genetic and molecular changes associated with aoa . in this report we describe a case of aoa with two clear pathogenic setx mutations, one of which is novel, as well as two further setx changes likely to be in cis polymorphisms that have previously been reported as pathogenic. methods: two independent lymphoblastoid cell lines obtained from the patient were used for western blotting of senataxin and protein markers of other autosomal recessive cerebellar ataxias. the setx gene was sequenced to identify possible disease-causing mutations. results: western blotting showed reduced levels of senataxin. serum afp level was elevated at lg/l (normal . - . lg/l). genetic sequencing revealed two clear pathogenic setx mutations. one of these was a novel mutation, c. delg; p.(cys phefster ), a deletion causing a reading frameshift resulting in truncation and loss of expression of senataxin protein from this allele. the other, c. c>t; p.(pro leu) was a missense mutation within the helicase domain which has previously only been reported in the homozygous state in a japanese aoa patient. two further sequence changes, c. a>g; p.(asn asp) and c. c>a; p.(gln lys), were also identified in our patient. conclusions: the reduced senataxin expression and elevated afp levels support a diagnosis of aoa in our patient. genetic analysis found a novel pathogenic mutation and documented the first case of another pathogenic mutation in the helicase domain outside of japan. the case contributes to the growing diversity of setx mutations known to be responsible for aoa . key: cord- -e ic pnc authors: yang, jiancheng; carey, patrick; ren, fan; lobo, brian c.; gebhard, michael; leon, marino e.; lin, jenshan; pearton, s.j. title: nanosensor networks for health-care applications date: - - journal: nanosensors for smart cities doi: . /b - - - - . - sha: doc_id: cord_uid: e ic pnc functionalized transistors provide effective sensors for a variety of viruses (zika, severe acute respiratory syndrome), toxins (botulinum), cancers (breast and prostate), and disease or injury biomarkers (troponin, cerebrospinal fluid). a hallmark of this approach is high specificity, rapid response (< minutes), and ability to be integrated with wireless data transmission capabilities. the ultimate goal is hand-held point-of-care detection that can streamline patient diagnosis. there is a need for continued development of selective, cost-effective hand-held biosensors with rapid response and detection sensitivities compared to existing lab assay methods. these could play a significant role in speeding patient diagnosis and, in some cases, reducing emergency room overcrowding. for biological and medical sensing applications, disease diagnosis by detecting specific biomarkers (functional or structural abnormal enzymes, low molecular weight proteins, or antigen) in blood, urine, saliva, or tissue samples has been established using a number of approaches, such as enzyme-linked immunosorbent assay (elisa), particle-based flow cytometric assays, electrochemical techniques based on impedance and capacitance, electrical measurement of micro-cantilever resonant frequency change, and conductance measurement of semiconductor nanostructures [ À ] . for some of these techniques, here are some drawbacks related to assay time and throughput. elisa allows only one analyte measurement at a time. particle-based assays allow for multiple detections by using multiple beads, but the whole detection process is generally longer than hours, which is not practical for in-office or bedside detection. electrochemical devices are low cost, but improvements in sensitivities are still needed for clinical samples. microcantilevers are capable of detecting concentrations as low as pg/ml but suffer from an undesirable resonant frequency change due to the viscosity of the medium and cantilever damping in the solution environment. nanomaterial devices have provided an excellent option toward fast, label-free, sensitive, selective, and multiple detections for both preclinical and clinical applications. in this chapter, we describe the use of semiconductor transistor-based systems in which specific functional layers are placed directly on the gate region of the transistor or connected to it from disposable glass or plastic slides to provide a sensor capable of fast response and excellent detection sensitivity. examples are given of detection of various viruses, cancers, or disease biomarkers using this approach, as well as the integration with wireless data transmission capability. networked systems like this are attractive in health-care applications. severe acute respiratory syndrome (sars) outbreaks are capable of causing a large number of fatalities and severe economic disruption [ ] . the sars coronavirus is the cause of the condition. the virus replication occurs through a coronavirus protein, nucleocapsid protein (n protein), and encapsulating the coronavirus genomic rna. sars-n protein is a nucleic acid-binding agent capable of interacting with rna and dna [ À ]. functionalized algan/gan high electron mobility transistors (hemts) have been utilized as an assay for sars and to examine the binding between double-stranded dna and the sars coronavirus nucleocapsid protein [ , ] . these hemts have been widely used as biosensors, as summarized in table . . by functionalizing the gate region with appropriate layers, highly specific sensing of antigens or other biomarkers can be achieved. in the case of sars, the binding was detected through a change in current in the hemt and allowed extraction of the dissociation constants of the nucleotideÀprotein interaction. this is a good example of the use of hemts not only as sensors but also for determining the binding affinity of ligandÀreceptor complexes [ ] . investigating the nucleotideÀsars-n protein interaction can assist in constructing a genome packaging model. in the past, electrophoretic mobility shift assay and filter-binding assays have been used to study proteinÀnucleic acid interactions [ À ]. these methods require labeling of fluorescent probes or isotope elements on nucleic acids to provide detection. the assay cost is high and the labeling may alter the binding affinity of molecules. the number of binding sites and the dissociation constants of a receptor are related to the ratio of the number of ligand-bound receptors to the total number of receptors on the sensor and can be obtained in either one-binding or multiple-binding site models [ , ] . the use of hemts allows rapid determination of these parameters. similar studies have been performed for the binding affinity of nonnucleoside reverse transcriptase inhibitors (nnrtis) to the reverse transcriptase (rt) of hiv- for determining the efficiency of the drug performance. the hiv- rt immobilized hemts were used to find the dissociation constant of nnrtis [ ] . the zika virus (zikv) is a flavivirus, primarily transmitted via the aedes mosquito [ À ] and leads to abnormal brain development in fetuses [ À ]. zikv can be detected using rna in human urine, serum, and saliva using the reverse transcription polymerase chain reaction [ À ] . however, the degradation of rna in saliva may occur during the saliva collection, storage, and processing [ ] . reverse transcription loop-mediated isothermal amplification was also used to detect zikv rna in unprocessed biological samples, such as urine, plasma, and zika-infected mosquito carcasses, with a detection limit of . pfu [ ] . these methods are time consuming and require a well-trained technician to perform the tests. we have shown that functionalized hemts may be used for zikv detection. fig. . shows the schematic of the sensor, consisting of an antibody-functionalized cover glass and an algan/ gan hemt. pulsed biasing of the electrode fabricated on the cover glass and functionalized with zika antibody was used for detection [ ] . the target zika antigen (recombinant zikv ns ) solutions were diluted with bovine serum albumin (bsa) in pf . pbs solution with . , , , or ng/ml concentration. the reversible antigen and antibody binding through active sites on these two protein molecules and the hemt drain current changes could fit with the langmuir extension model [ ] . in addition, the hookean spring model was employed to simulate the relaxation portion of the time-dependent drain current. since the drain current is proportional to the gate voltage of the hemt or the stretched distance of the antibody and antigen molecules, the solution for the stretched distance is directly proportional to the drain current (i d ) as where c* is the ratio of antibody bound with antigen to the total available antibody on the functionalized contact window, τ and τ are the relaxation time constants of antibody and antigen molecules, respectively, a, b, and e are constants. fig. . shows the modeled drain current for pbs solution without antigen and pbs solutions with different antigen concentrations. the ratio of antibody bound with antigen to the total available antibody on the functionalized contact window increased from . at . ng/ml to . at ng/ml, with the ratio scaling faster than concentration due to increased interaction probability. a wide range of zika antigen . À ng/ml was detected. clostridium botulinum neurotoxins are deadly toxins, with a lethal dose in unvaccinated humans of only ng/kg [ À ]. conventional methods of detection involve the use of high-performance liquid chromatography, mass spectrometry, and colorimetric elisa assays; but these methods must be carried out at centralized locations and are too slow to be used in the field. antibody-immobilized algan/gan hemts have been used to detect botulinum toxin type-a with a limit of detection (lod) below ng/ml [ , ] . the antibody was anchored to the gate area through immobilized thioglycolic acid and the toxin detected through bonding to the botulinum antibody and the signal detected by changes in the hemt drain current [ , ] . the sensor saturated above ng/ml of the toxin, with a lod below ng/ml in pbs buffer solution. the sourceÀdrain current change was nonlinearly proportional to botulinum toxin concentration. the long-term stability was tested by storing the sensor stored in pbs at c and periodically testing over months at room temperature. the sensitivity losses were %, %, and % after , , and months, respectively [ ] . the toxicity of heavy metal ions, including mercury(ii) hg , lead(ii) pb , copper(ii) cu , and zinc(ii) zn , is a chronic environmental problem [ À ]. mercury is released into the environment through the combustion of fossil fuels, mining, volcanic emissions, and solid waste incineration. mercury and lead impact on wildlife ecology and human health. some types of bacteria convert inorganic hg ions into neurotoxic organic mercury compounds, which bioaccumulate through plants and the food chain. bare au-gated and thioglycolic acid-functionalized au-gated algan/gan hemts can detect mercury(ii) and copper(ii) ions [ À ]. fast detection of less than seconds was achieved for thioglycolic acid-functionalized sensors. the thioglycolic acid-functionalization increased the sensitivity for detection of mercury by . over the bare au-gated surface. the limit of mercury(ii) ion detection was m and s selectivity of more than for detecting hg over na or mg ions. the sensors could be recycled using a de-ionized (di) water rinse, as shown in fig. . . mortality in breast cancer patients can be reduced by increasing the screening frequency [ À ]. most patients are screened by mammography, which is invasive (radiation) and limits the frequency of screening. a % survival rate is predicted to be achievable if patients could be screened every months, but this would require low cost, point-of-care technologies that can screen more frequently and noninvasively [ , ] . salivary testing for markers of breast cancer may be used in conjunction with mammography [ À ]. saliva-based diagnostics for the protein c-erbb- , a prognostic breast marker assayed in tissue biopsies of women diagnosed with malignant tumors, shows potential. soluble fragments of the c-erbb- oncogene and the cancer antigen À were found to be significantly higher in the saliva of women who had breast cancer than in those patients with benign tumors. to fully realize the potential of salivary biomarkers, technologies are needed that will enable facile, sensitive, and specific detection of breast cancer at home with concomitant wireless data transmission into the clinic. if cheap technologies that can wirelessly detect breast cancer are developed, early diagnosis will significantly lower mortality. antibody-functionalized, au-gated algan/gan hemts were used to detect c-erbb- , a breast cancer marker [ ] . the antibody was anchored to the gate area through immobilized thioglycolic acid. the sensor showed a response of less than seconds when target c-erbb- antigen in a buffer at clinically relevant concentrations from . to . μg/ml was added to the antibody-immobilized surface. this electronic detection of biomolecules is a significant step toward a compact sensor chip, which can be integrated with a commercially available hand-held wireless transmitter to realize a portable, fast response, and high sensitivity breast cancer detector. prostate cancer is the second most common cause of cancer death among men in the united states and the most common form of cancer among men, other than skin cancer [ À ]. the most commonly used serum marker for diagnosis is prostate-specific antigen (psa). one in six men will be diagnosed with prostate cancer during their lifetime [ À ]. the american cancer society recommends health-care professionals offer the psa blood test and digital rectal exam yearly for men above the age of . psa is made by cells in the prostate gland and is found in semen and in the blood. when prostate cancer develops, the psa level usually goes up above ng/ml. about % men with a psa below will have prostate cancer on biopsy. if the patient's psa level is between and , their chance of having prostate cancer is b %. if the patient's psa level is above , there is more than % chance of prostate cancer. psa testing approaches are costly, time consuming, and need sample transportation. antibody-functionalized au-gated algan/gan hemts were used to detect psa in saline solutions [ ] . the psa antibody was anchored to the gate area through the formation of carboxylate succinimdyl ester bonds with immobilized thioglycolic acid. the hemt drainÀsource current showed a response time of less than seconds when target psa in a buffer at clinical concentrations was added to the antibody-immobilized surface. the devices could detect a range of concentrations from μg/ml to pg/ml, two orders of magnitude lower than the cutoff value of psa measurements for clinical detection of prostate cancer. fig. . shows the real-time psa detection in pbs buffer solution using the source and drain current change with constant bias of . v. no current change can be seen with the addition of buffer solution or nonspecific bsa, but there was a rapid change when ng/ml psa was added to the surface. the ultimate detection limit appears to be a few pg/ml [ ] . cerebrospinal fluid (csf) is a physiologically critical extracellular liquid secreted from the choroid plexus in the cerebral ventricles [ À ]. csf covers the brain and spinal cord, held in the central nervous system by the meninges. in addition to acting as a physiological buffer solution, providing nutritional and waste transport, it also helps to maintain intracranial pressure and acts as a physical shock absorber, cushioning the brain in the case of sudden movement or force. csf is constantly replenished. in a normal human adult, there is À ml of csf at one time, which is replenished every hours, so approximately À ml of csf is produced daily. leakage of csf leak is a serious complication that can result traumatically, iatrogenically, or spontaneously. while imaging studies can often elucidate the site of a leak, the standard for detection of csf is an assay for beta transferrin (b ) in nasal secretions or other drainage [ À ]. the primary methods of detection for b are immunofixation electrophoresis (ife) and elisa. consistent ife results down to μg/ml can be obtained in patient samples but require a . hour testing period, which is not expedient for real-time feedback during ent surgeries. to achieve good sensitivity and handle the inherently low concentration of β transferrin in csf, laboratory procedures have required samples to be concentrated by as much as -fold or the sample to be run in duplicate to ensure accurate detection. these tests are performed only at limited sites throughout the country due to cost and expertise, resulting in real-life return times on the order of days to a week. to alleviate the slow turn-around times of hospital laboratories and limited lower lods, there has been interest in electronic detection methods using biologically functionalized transistors, which provide an electronic readout and are readily integrated with wireless transmission of data. we developed a disposable testing slide externally integrated with a transistor to detect b at concentrations, from . ng/ml to μg/ml. a disposable testing slide was externally integrated with a si mosfet to detect csf from . ng/ml to μg/ml. a si mosfet pcb was designed to simplify the testing setup. human pooled csf was diluted in ph . pbs and wt% bsa with . ng/ml to μg/ml. we recognize that the concentration of csf is not the concentration of β t, it is actually much lower since it is only a minor constituent of csf. to test the csf sensor, diluted csf solution was applied to the sensing electrode and allowed to bind for minutes before measurement. time-dependent detection of csf dilutions from . to ng/ml is shown in fig. . (left) , while the response as a function of concentrations is shown in fig. . (right). cardiac troponin i (ctni) and the complex involving ctni, cardiac troponin t, and cardiac troponin c in the cardiac muscle tissue are standard clinical biomarkers for acute myocardial infarction (ami) and diseases that produce cardiac muscle damage [ , ] . their concentrations in the blood rise quickly following the onset of ami as they are released from myocardial cells following cell death. elevated troponin concentrations can be detected in the blood within a few hours up to several days following the onset of angina (where myocardial cells suffer reversible damage) to ami where myocardial cells die [ , ] . the time dependence of concentration of these species is commonly detected by radioimmunoassay, elisa fluorimetric, luminometric, colorimetric, and electrochemical methods, many of which are time consuming and require trained personnel to perform tests [ À ]. the measurement of blood troponin concentrations can decide whether ami has occurred or that chest pain and other symptoms are due to other causes. inexpensive techniques to provide rapid, accurate blood troponin concentrations would be welcome in managing treatment of emergency room patients. sarangadharan et al. [ , ] reported an electrical double-layer gated high-field algan/gan hemt biosensor in which the gating mechanism overcomes charge screening effects that are prevalent in traditional field effect transistor (fet)-based biosensors, allowing detection of target proteins in physiological solutions. they were able to detect troponin i in blood samples in the range . À ng/ml, using antibody or aptamer functionalization [ , ] . the cover glass approach leads to an increase in the pulsed current, as shown in fig. . [ ] . in this configuration, the receptor immobilization produces a decrease in total capacitance of the solution plus dielectric capacitance and thus a decrease in effective gate voltage and an increase in current. the electronic double-layer hemt designs enhance the current gain of the sensor in high ionic strength solutions, resulting in increased sensitivity and specificity in detection of troponin i. the ability to use a simple, functionalized glass slide as the active sensing area opens up the possibility of inexpensive cartridge sensors. radio frequency (rf) communication circuits can be integrated with sensors, enabling robust wireless sensors which transmit their data to a central location [ ] . a simple wireless sensor network is therefore needed to manage the collection and process of data from multiple sensors. the rf transceiver should be simple to reduce size and power consumption. radio frequency identification (rfid) tags can be monolithically integrated on a chip. the device consumes little power and can operate with a small battery or without a battery. in the latter case, the tag device is powered by the external interrogation signal from the base station. its low data bandwidth and low power operation make it suitable to integrate with sensors for wireless sensing, since the data bandwidth and operating power of sensor are also very low. once the detected sensor data are digitized, it can be merged with the id code and transmitted together. the rfid part of the integrated wireless sensor functions as a simple rf transceiver which receives base station signal to turn on the transmitter and sends back the sensor data. this type of wireless sensor is suitable for short-range operation similar to personal area network. the network consists of multiple sensors of the same function or different functions. each sensor is connected to an rfid tag with a unique id code that identifies its sensing function and location. in active operation mode, it sends out data to a central monitor station whenever the sensor detection is positive and triggers the rfid tag transmitter [ ] . in passive operation mode, the sensor is activated by the central monitor interrogation signal and responds with the data. fig. . shows the block diagram of the integrated wireless sensor with rfid. the sensor can be powered by the external interrogation rf energy from base station or an integrated battery. the sensor data are merged with rfid code, which modulates the antenna load and the reflected signal from base station. wireless sensor array using rfid is similar to the earlier except that multiple wireless sensors are integrated together on one substrate. for applications where size and power consumption are not critical, but operating distance is a major concern, the wireless sensor architecture in fig. . can be used. the device consists of multiple sensors, a memory stores its id code, a data processor that collects sensor data and merges with id code, and transmitter with power amplifier and antenna array for long-range transmission. the beam-forming antenna array can be preprogrammed to point the antenna beam to the central station [ À ] . for wireless transmission of data, an instrumentation amplifier can be used for the detection circuit to sense the change of current due to antigen detection. the current variation, embodied as a change in the output voltage of the detection circuit, will be fed into a microcontroller. the microcontroller will calculate the corresponding current change and control a zigbee transceiver which will transmit data to a wireless network server. the block diagram of the sensor module and wireless network server is shown in fig. . . the transceiver module is completely turned off for most of the time and is turned on to transmit data in extremely short intervals. when the sensor module is turned on, it is programmed to power up for the first seconds. following the initialization process, the detection circuit is periodically powered down for seconds and powered up again for another second, achieving a . % duty cycle. the zigbee transceiver is enabled for . ms to transmit the data only at the end of every cycle. this gives an rf duty cycle of only . %. data acquisition and transmission, if continuous, consumes most of the power in a wireless sensing system. since the sensor only needs to acquire and transmit data for a few seconds in every minutes during normal operation, the average power consumption can be significantly reduced by using low duty cycle operation. fig. . illustrates the package sensors mounted on a circuit board containing the detection circuit and microcontroller and the wireless transmitter for data collection. the sensor module is fully integrated on an fr pc board and is packaged with battery. the dimension of the sensor module package is . v . v v. the maximum line of sight range between the sensor module and the base station is m. the base station of the wireless sensor network server is also integrated in a single module ( . v . v . v) and is ready to be connected to a laptop by a usb cable. the base station draws its power from the laptop's usb interface and thus does not require any battery or wall ac transformer, which reduces its form factor. in detection of medical biomarkers, many different methods have been employed. there is also a need for small, handheld sensors with wireless connectivity, which have fast response. for medical sensing applications, disease diagnosis by detecting specific biomarkers (functional or structural abnormal enzymes, low molecular weight proteins, or antigen) in blood, urine, saliva, or tissue samples has been established. most of the techniques mentioned earlier, such as elisa, possess a major limitation in that only one analyte is measured at a time. particle-based assays allow for multiple detection by using multiple beads but the whole detection process is generally longer than hours, which is not practical for in-office or bedside detection. semiconductor-based sensors can be fabricated using mature techniques from the si chip industry and/or novel nanotechnology approaches. the goal is to realize real-time, portable, and inexpensive biological sensors and to use these as hand-held exhaled breath, saliva, urine, or blood monitors with wireless capability. frequent screening can catch the early development of diseases, reduce the suffering of the patients due to late diagnoses, and lower medical costs. there are still some critical issues. the sensitivity for some antigens needs to be improved to allow sensing in body fluids other than blood (urine, saliva). second, a sandwich assay allowing the detection of the same antigen using two different antibodies needs to be tested. third, integrating multiple sensors on a single chip with automated fluid handling and algorithms to analyze multiple detection signals, and fourth, a package that will result in a cheap final product is 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biosensors and the sensor array the work at uf was partially supported by a grant from the nsf i/ucrc of the multi-functional integrated system technology (mist center) iip- . the authors acknowledge the use of the nanoscale research facility (nrf) in the nanoscience institute for medical and engineering technology at the university of florida. key: cord- -f pbd authors: bosteels, cedric; maes, bastiaan; van damme, karel; de leeuw, elisabeth; declercq, jozefien; delporte, anja; demeyere, bénédicte; vermeersch, stéfanie; vuylsteke, marnik; willaert, joren; bollé, laura; vanbiervliet, yuri; decuypere, jana; libeer, frederick; vandecasteele, stefaan; peene, isabelle; lambrecht, bart title: sargramostim to treat patients with acute hypoxic respiratory failure due to covid- (sarpac): a structured summary of a study protocol for a randomised controlled trial date: - - journal: trials doi: . /s - - - sha: doc_id: cord_uid: f pbd objectives: the hypothesis of the proposed intervention is that granulocyte-macrophage colony-stimulating factor (gm-csf) has profound effects on antiviral immunity, and can provide the stimulus to restore immune homeostasis in the lung with acute lung injury post covid- , and can promote lung repair mechanisms, that lead to a % improvement in lung oxygenation parameters. sargramostim is a man-made form of the naturally-occurring protein gm-csf. trial design: a phase academic, prospective, arm ( : ratio), randomized, open-label, controlled trial. participants: patients aged - years admitted to specialized covid- wards in belgian hospitals with recent (< weeks prior to randomization) confirmed covid- infection and acute respiratory failure defined as a pao /fio below mmhg or spo below % on minimal l/min supplemental oxygen. patients were excluded from the trial in case of ( ) known serious allergic reactions to yeast-derived products, ( ) lithium carbonate therapy, ( ) mechanical ventilation prior to randomization, ( ) peripheral white blood cell count above . /μl and/or active myeloid malignancy, ( ) high dose systemic steroid therapy (> mg methylprednisolone or equivalent), ( ) enrolment in another investigational study, ( ) pregnant or breastfeeding or ( ) ferritin levels > μg/ml. intervention and comparator: inhaled sargramostim μg twice daily for days in addition to standard care. upon progression of disease requiring mechanical ventilation or to acute respiratory distress syndrome (ards) and initiation of mechanical ventilator support within the day period, inhaled sargramostim will be replaced by intravenous sargramostim μg/m( ) body surface area once daily until the day period is reached. from day onwards, progressive patients in the active group will have the option to receive an additional days of iv sargramostim, based on the treating physician's assessment. intervention will be compared to standard of care. subjects progressing to ards and requiring invasive mechanical ventilatory support, from day onwards in the standard of care group will have the option (clinician's decision) to initiate iv sargramostim m μg/m( ) body surface area once daily for days. main outcomes: the primary endpoint of this intervention is measuring oxygenation after days of inhaled (and intravenous) treatment through assessment of a change in pretreatment and post-treatment ratio of pao /fio and through measurement of the p(a-a)o gradient (pao = partial alveolar pressure of oxygen, pao =partial arterial pressure of oxygen; fio = fraction of inspired oxygen). randomisation: patients will be randomized in a : ratio. randomization will be done using redcap (electronic iwrs system). blinding (masking): in this open-label trial neither participants, caregivers, nor those assessing the outcomes will be blinded to group assignment. numbers to be randomised (sample size): a total of patients with confirmed covid- and acute hypoxic respiratory failure will be enrolled, in the active and in the control group. trial status: sarpac protocol version . (april ). participant recruitment is ongoing in belgian hospitals (i.e. university hospital ghent, az sint-jan bruges, az delta roeselare, university hospital brussels and zna middelheim antwerp). participant recruitment started on march (th) . given the current decline of the covid- pandemic in belgium, it is difficult to anticipate the rate of participant recruitment. trial registration: the trial was registered on clinical trials.gov on march (th), (clinicaltrials.gov identifier: nct ) - retrospectively registered; https://clinicaltrials.gov/ct /show/nct ?term=sarpac&recrs=ab&draw= &rank= and on eudract on march th, (identifier: - - ). full protocol: the full protocol is attached as an additional file, accessible from the trials website (additional file ). in the interest in expediting dissemination of this material, the familiar formatting has been eliminated; this letter serves as a summary of the key elements of the full protocol. the primary objective is to investigate whether the administration of inhaled sargramostim (leukine®) at a dose of mcg daily during days improves oxygenation in covid- patients with acute hypoxic respiratory failure. the secondary objectives are: -to study if early intervention with sargramostim is safe (number of aes/saes) -to study if early intervention with inhaled sargramostim affects clinical outcome defined by duration of hospital stay, -point ordinal scale, clinical sign score, sofa score, news score -to study if early intervention with sargramostim affects the rate of nosocomial infection -to study if early intervention with inhaled sargramostim affects progression to mechanical ventilation and/or ards -to study if treatment with sargramostim affects all-cause mortality rate at and weeks post inclusion -to study if treatment with sargramostim affects features of secondary haemophagocytic lymphohistiocytosis, defined by hs score -to study if treatment with sargramostim has a favourable effect on long term - week follow up . . subjects . . . number of subjects a total of patients with confirmed covid- and acute hypoxic respiratory failure will be enrolled, in the active and in the control group. confirmed covid- patients with acute hypoxic respiratory failure admitted to the covid- isolation ward. inclusion and exclusion criteria the following patients will be enrolled: -recent (≤ weeks prior to randomization) confident diagnosis of covid- confirmed by antigen detection and/or pcr, and/or seroconversion or any other emerging and validated diagnostic test. -in some patients, it may be impossible to get a confident laboratory confirmation of covid- diagnosis after h of hospital admission because viral load is low and/or problems with diagnostic sensitivity. in those cases, in absence of an alternative diagnosis, and with highly suspect bilateral ground glass opacities on recent (< h) chest-ct scan (confirmed by a radiologist and pulmonary physician as probable covid- ), a patient can be enrolled as probable covid- infected. in all cases, this needs confirmation by later seroconversion. -presence of acute hypoxic respiratory failure defined as (either or both)  saturation below % on minimal l/min o  pao /fio below -admitted to specialized covid- ward -age - -male or female -willing to provide informed consent exclusion criteria -patients with known history of serious allergic reactions, including anaphylaxis, to human granulocyte-macrophage colony stimulating factor such as sargramostim, yeast-derived products, or any component of the product. -mechanical ventilation before start of study -patients with peripheral white blood cell count above . per microliter and/or active myeloid malignancy -patients on high dose systemic steroids (> mg methylprednisolone or equivalent) -patients on lithium carbonate therapy -patients enrolled in another investigational drug study -pregnant or breastfeeding females (all female subjects regardless of childbearing potential status must have negative pregnancy test at screening) -patients with serum ferritin > mcg/ml (which will exclude ongoing hlh) . . study interventions confirmed or highly suspect covid- patients with acute hypoxic respiratory failure (saturation below % on minimal l/min o or pao /fio < ) will be randomized to receive sargramostim mcg twice daily for days as a nebulized inhalation on top of standard of care (active group), or to receive standard of care treatment (control group). upon progression of disease requiring initiation of mechanical ventilatory support within the day period, in patients in the active group, inhaled sargramostim will be replaced by intravenous sargramostim mcg/m body surface area once daily until the day period is reached. from day onwards, progressive patients in the active group will have the option to receive an additional days of iv sargramostim, based on the treating physician's assessment. in the control group with progressive disease requiring mechanical ventilatory support, from day onwards, the treating physician will have the option to initiate iv sargramostim mcg/m body surface area once daily for days. safety data, including blood leukocyte counts, will be collected in all patients. efficacy data will also be collected and will include arterial blood gases, oxygenation parameters, need for ventilation, lung compliance, organ function, radiographic changes, ferritin levels, triglyceride levels, etc. as well as occurrence of secondary bacterial infections. patients will stop the investigational drug if there is unacceptable toxicity according to investigator's judgement. / . . . imps and dosage leukine® (sargramostim) prepared and administered for inhalation using nebulizer leukine for injection is a sterile, preservative-free lyophilized powder that requires reconstitution with ml normal saline solution. once reconstituted, leukine can be inhaled as an aqueous aerosol using either a vibrating mesh nebulizer (philips innospirego) or jet nebulizer, per manufacturer instructions. (nebulizers studied include: akita apixneb, pari lc-plus set, pulmoaide, pan lc, aeroneb solo device). use reconstituted leukine® solution for inhalation within hours following reconstitution and/or dilution. nebulizing is preferably done in an isolation negative pressure chamber, and if not, personnel should use an ffp mask. patient should self-administer the medication and where possible, the room should not be entered within one hour after administration. for patients that are on a mechanical ventilator and cannot be treated with leukine® inhalation:  the recommended dose is mcg/m /day administered intravenously over a -hour period once daily for up to days.  for intravenous injection: administer leukine injection in . % sodium chloride injection, usp. dilute leukine for intravenous infusion in . % sodium chloride injection, usp. if the final concentration of leukine is below mcg/ml, add albumin (human) at a final concentration of . % to the saline prior to addition of leukine to prevent adsorption to the components of the drug delivery system. to obtain a final concentration of . % albumin (human), add mg albumin (human) per ml . % sodium chloride injection, usp (e.g., use ml % albumin [human] in ml . % sodium chloride injection, usp). . . . schematic overview of the data collection & interventions . . study duration the total treatment duration of the study is days, and the entire study duration is - weeks to final follow up visit. sargramostim (leukine®) is a yeast-derived recombinant humanized granulocyte-macrophage colony stimulating factor (rhugm-csf, sargramostim) and the only fda approved gm-csf (leucine package insert). gm-csf, a pleiotropic cytokine, is an important leukocyte growth factor known to play a key role in haematopoiesis, effecting the growth and maturation of multiple cell lineages as well as the functional activities of these cells in antigen presentation and cell mediated immunity ( ). since its initial fda approval in , over , patients have received leukine®, providing extensive clinical and post-marketing data in a broad range of treated individuals -from preterm neonates to the elderly and including males and females -representing a well-characterized safety profile for leukine®. leukine® administered as a subcutaneous or intravenous injection is approved for six indications including use as a medical countermeasure for radiation exposure. the us government currently holds leukine® in the strategic national stockpile. leukine® may benefit patients with beginning signs of acute respiratory distress syndrome (ards) due to covid- infection. gm-csf is a critical cytokine for the health of lungs. the alveolar macrophages are dependent on gm-csf for differentiation and normal functioning. in addition, gm-csf is an immunomodulator that plays a critical role in host defense by promoting differentiation of dendritic cells, and stimulating antiviral immunity ( - ). as described in detail below, it is being studied as an adjuvant therapy in the management of lifethreatening infections to boost the hosts innate immune response to fight infection, reduce the risk of secondary infection, and in varied conditions to prevent infection during critical illness ( ) ( ) ( ) ( ) . in addition, it has been studied in pulmonary conditions that affect alveolar macrophages, such as autoimmune pulmonary alveolar proteinosis ("apap"), with beneficial outcomes ( , ) . we propose based on preclinical and clinical data and the safety data from more than , adult and pediatric patients treated with leukine®, that patients with beginning signs of acute lung injury and/or ards due to covid- infection be given leukine®. ards due to covid- carries a high mortality rate ( ) and leukine® may confer benefit by both active management of this complication as well as in prevention of secondary infections. in animal models of postviral ards and mortality, gm-csf has demonstrated immunomodulatory effects that improve the clinical response and symptoms associated with influenza and other viral respiratory infections ( ) ( ) ( ) , and represents a promising candidate for the prevention of ards in patients with covid- . the proposed development plan was guided by three specific considerations: the biology and effects of gm-csf on the lung, specifically alveolar macrophages and epithelial cells, as well its immunomodulatory activities in stimulating antiviral immunity make gm-csf a critical cytokine for healthy pulmonary function and defence. detailed studies have shown that gm-csf is necessary for the maturation of alveolar macrophages from fetal monocytes and the maintenance of these cells in adulthood ( ). gm-csf has a wide array of effects on myeloid cells. gm-csf has been shown to be a myelopoietic growth factor that has pleiotropic effects not only in promoting the differentiation of immature precursors into polymorphonuclear neutrophils, monocytes/ macrophages and dendritic cells, but also in controlling the function of fully mature myeloid cells ( ) . gm-csf is also known to reverse immunoparalysis seen in sepsis by immune activation, resulting in beneficial outcomes ( ). there is a large body of evidence generated with gm-csf in animal studies suggesting the potential use in ards and infections ( ) . for the purpose of brevity, we will point to the data that reflects the potential value in viral lung infections and preventing secondary bacterial infections and progression to ards: halstead and colleagues demonstrated that in vivo high airway levels of gm-csf profoundly rescue mice from lethal influenza pneumonia. while in vitro gm-csf is canonically described as an m polarizing cytokine, their data demonstrated that in vivo, during influenza a virus infection, gm-csf instead temporizes the type ii interferon-induced m polarization of airway macrophages and reduces inflammation induced damage ( , ) . unkel and colleagues demonstrated gm-csf-dependent cross-talk between influenza virus infected alveolar epithelial cells and cd + dendritic cells is crucial for effective viral clearance and recovery from injury and thus pointing to the potential use of gm-csf treatment in severe influenza virus pneumonia ( ) . investigations have shown that gm-csf conferred resistance to influenza in mice via alveolar phagocytes and through alveolar macrophages which became more resistant to influenza-induced apoptosis. delivery of intranasal gm-csf to wild-type mice also conferred resistance to influenza ( ) . there is evidence that inhaled gm-csf prevents bacteremia in post influenza bacterial pneumonia primarily through locally-mediated improved lung antibacterial resistance to systemic bacteremia during influenza a viral infection ( ) . conclusions: gm-csf confers resistance to influenza by enhancing innate immune mechanisms that depend on alveolar macrophages, which are dependent on gm-csf for their health and normal functioning. pulmonary delivery of this cytokine has the potential to reduce morbidity and mortality due to viral pneumonia. this is summarized in the diagram below: / . experience: use of leukine® has beneficial effect in the treatment of conditions that are similar to ards seen with covid- . a small ( patient) double blind randomized placebo controlled clinical trial of low-dose ( mcg/kg daily for days) intravenous gm-csf treatment in adult patients with severe sepsis and respiratory dysfunction, led to the conclusion that gm-csf treatment was associated with improved gas exchange and might play a homeostatic role ( ) . in a phase ii study, patients with severe sepsis with respiratory dysfunction were randomized to gm-csf ( mcg/m intravenously daily for days) or placebo. the results showed an improvement in day mortality on gm-csf; this did not reach statistical significance due to the small sample size ( ). herold and colleagues used leukine® by inhalation route on a compassionate basis in six patients with moderate to severe community-acquired pneumonia or ventilator-associated pneumonia ards who were not improving despite all measures and at least days of mechanical ventilation ( ) . mcg of leukine® were applied by aeroneb solo device (covidien, neustadt, germany) at an interval of hours. compared to historical controls, the authors observed significant improvement in oxygenation and lung compliance with gm-csf therapy. this resulted in improved morbidity using standard scoring systems and of the six patients recovered and were discharged from the hospital. there is an ongoing study of inhaled gm-csf across multiple centers in germany (gi hope; nct ) recruiting patients with diagnosis of pneumonia associated ards. there is a large body of evidence of inhaled leukine® in autoimmune pulmonary alveolar proteinosis (apap), which results in accumulation of surfactant in alveolar sacs with resultant hypoxia. tazawa and colleagues conducted a phase ii study of inhaled leukine® at pulmonary centers throughout japan in patients with unremitting or progressive apap with hypoxia and symptoms ( ) . patients received mcg daily by inhalation, using an lc-plus nebulizer with a manual interrupter valve connected to a pari turbo boy compressor, for days and this cycle was repeated every other week for six cycles (total weeks). the treatment was well tolerated with no serious adverse events. adverse events were reported in just of the patients oxygenation, radiological changes as well as symptoms. following these results, a larger randomized phase study (page study) was conducted by the japanese investigators in centers. patients with mild to moderate apap with hypoxia were randomized to receive placebo or leukine® ( patients) at a dose of mcg twice a day for days followed by a week of no treatment. this two-week cycle was repeated times over a period of weeks. the treatment was again well tolerated with no significant differences in adverse events between the two groups. the gm-csf treated patients had significantly improved hypoxia parameters and radiographic changes ( ) . this clinical experience of use of leukine® in viral pneumonia suggests salutary effects. in addition, these studies establish the safety of inhaled leukine® and provide evidence for activity of inhaled leukine®. . expediency: toxicology, pharmacologic and safety data supports the immediate clinical use of leukine® in hypoxic respiratory failure with acute lung injury leading to ards due to covid- . investigator brochure is available and contains detailed information on toxicity. risk/benefit assessment covid- poses a very significant risk of mortality of - % and this percentage rises to mortality of % in patients with co-morbidity ( , ) . of all infected patients, some - % develop severe respiratory symptoms necessitating hospital admission. around % of infected patients will require invasive mechanical ventilation, and many of those ( - % will die). the current world-wide pandemic of covid- is putting unforeseen stress on the entire primary, secondary and tertiary medical system, leading to unseen triage of patients that potentially benefit or not from admission to icu units when they develop respiratory failure. gm-csf (sargramostim, leukine®) has been given systemically to almost . patients in the past. it is therefore a well characterized product. inhalation of gm-csf has also been used to treat patients with interstitial lung disease and reduced oxygen saturation (i.e. partial acute hypoxic respiratory failure) with few significant side effects above the placebo arm. the protocol is set up to give twice daily inhalation with gm-csf, followed by intravenous administration if the patient would move to the icu unit on mechanical ventilation. although gm-csf has been given systemically and via inhalation to patients with pneumoniaassociated ards, there are no current data on the safety profile of this drug in patients with covid- . given the severity of the clinical syndrome caused by covid- , and the prior triage of patients before hospital admission to the covid- ward, this trial will be performed in a hospital setting on a covid- ward with close monitoring of vital parameters (continuous ecg, oxygen saturation, temperature, vital clinical signs), which will allow intermediate intervention should serious side effects occur. once on the icu unit, patients will be intensively monitored for all vital parameters, as part of the routine icu monitoring. there are currently no treatments directed at improving lung repair and local immunity in covid- patients, and no treatment that attempt to halt the progression from manageable acute hypoxic respiratory failure to ards. preventing such progression to ards could have a huge impact on the foreseeable overflow of the icu units. we therefore believe the benefits of administering inhaled gm-csf treatment in early stage covid- acute hypoxic respiratory failure outweighs the risks associated with a phase imp administration via a different route and unknown indication. there is a large number of covid- infected patients that are currently being hospitalized across the globe. in just days time, our covid- ward at ghent university hospital has admitted confirmed cases, of which a significant portion ( %) already fulfill eligibility criteria for the current proposed protocol. we therefore believe that given the current ascending part of the epidemiology curve, with numbers of patients rising sharply, there will be no shortage of patients that are eligible. partner therapeutics has offered to give (free of charge) enough gm-csf to treat patients for a day period and an additional controls for days (should deterioration occur). there are large / amounts of gm-csf in the united states strategic national stockpile, so should this therapy work, there might be immediate worldwide application of a gm-csf inhalation therapy. primary objectives this is phase academic, prospective, randomized, open-label, interventional study designed to investigate the efficacy of sargramostim (leukine®) in improving oxygenation and short-and long-term outcome of covid- patients with acute hypoxic respiratory failure. there are currently no treatments directed at improving lung repair and local immunity in covid- patients, and no treatment that attempt to halt the progression from manageable acute hypoxic respiratory failure to ards in patients with covid- infection. justification for our objective is that preventing progression from early acute hypoxic respiratory failure to ards could have a huge impact on the foreseeable overflow of the icu units, that is already happening in some countries and is bound to happen on a global scale. the outcome of our study could thus have large impact from a medical, ethical and economic perspective. the hypothesis of the proposed intervention is that gm-csf has profound effects on antiviral immunity, can provide the stimulus to restore immune homeostasis in the lung with acute lung injury post covid- , and can promote lung repair mechanisms, that lead to a % improvement in lung oxygenation parameters. this hypothesis is based on experiments performed in mice showing that gm-csf treatment can prevent mortality and prevent ards in mice with post-viral acute lung injury. to address our hypothesis, we will randomize patients with confirmed covid- with acute hypoxic respiratory failure (saturation below % on minimal l/min o or pao /fio < ) to receive sargramostim mcg twice daily for days as a nebulized inhalation on top of standard of care (active group), or to receive standard of care treatment (control group). upon progression of disease requiring initiation of non-invasive or invasive mechanical ventilatory support within the day period, in patients in the active group, inhaled sargramostim will be replaced by intravenous sargramostim mcg/m body surface area once daily until the day period is reached. to measure the effectiveness of sargramostim on restoring lung homeostasis, the primary endpoint of this intervention is measuring oxygenation after days of inhaled (and intravenous) treatment through assessment of pretreatment and post-treatment ratio of pao /fio and through measurement of the p(a-a)o gradient, which can easily be performed in the setting of clinical observation of patients admitted to the covid - ward or icu covid- unit. during the day treatment period, we will perform daily measurements of oxygen saturation (pulse oximetry) in relation to fio , and the slope of alterations in these parameters could also be an indicator that our hypothesis is correct. comparison will be between active group a receiving sargramostim on top of standard of care and control group b receiving standard of care. data from the wuhan covid- epidemic show that patients that deteriorate are facing a prolonged period of mechanical ventilation. therefore, from day onwards, progressive patients in the active group will have the option to receive an additional days of iv sargramostim, based on the treating physician's assessment. this group will be called group c. in the control group, for patients with / progressive disease requiring (non)-invasive mechanical ventilatory support, from day onwards, the treating physician will have the option to initiate iv sargramostim mcg/m body surface area once daily for days. this group will be called group d. comparisons of group a (early day intervention with sargramostim) with group d (late day intervention with sargramostim) will also be very informative. secondary objectives -to study if early intervention with sargramostim is safe (number of aes/saes) -to study if early intervention with inhaled sargramostim affects clinical outcome defined by duration of hospital stay, mean change in -point ordinal scale between day and day mean change in clinical sign score between day and day time to clinical sign score < maintained for h mean change of sofa score between day and day or between day and day . mean change news score score between day and day or between day and day . tine to news score less than for at least h to measure the effectiveness of sargramostim on restoring lung homeostasis, the primary endpoint of this intervention is measuring oxygenation after days of inhaled (and intravenous) treatment through assessment of pretreatment (day ) and post-treatment (day ) ratio of pao /fio and through measurement of the p(a-a)o gradient, which can easily be performed in the setting of clinical observation of patients admitted to the covid - ward or icu covid- unit. preferentially, this measurement should be done in the upright position, while breathing room air for a minimum of minutes.. if this is impossible due to need for supplemental oxygen, fio and oxygen supplementation method should be recorded in patient record, so that a-a gradient can be normalized for age expected normal a-a gradient while on supplemental oxygen use. during the day treatment period, we will perform daily measurements of oxygen saturation (pulse oximetry) in relation to fio , and the slope of alterations in this parameters could also be an indicator that our hypothesis is correct. if the patient leaves hospital prior to the day analysis point, oxygenation at day of discharge will be used as value for measuring primary endpoint. -to study if early intervention with sargramostim is safe (number of aes/saes) although sargramostim has been given previously by inhalation to patients with ards and interstitial lung disease, data on safety in patients with covid- infection are currently lacking. since we are randomizing against days of no sargramostim treatment, comparison of aes and saes between group a and group b will be very informative. -to study if early intervention with inhaled sargramostim affects clinical outcome defined by length of hospital stay mean change in -point ordinal scale change between day , day and -to study if early intervention with sargramostim affects the rate of nosocomial infection patients with viral respiratory infection are at risk of secondary bacterial infections. as part of routine clinical care, sputum samples will be collected in patients suspected of secondary bacterial pneumonia, and checked for the presence of bacteria. -to study if early intervention with inhaled sargramostim affects progression to mechanical ventilation and/or ards decreasing oxygenation often leads to the need for non-invasive or invasive mechanical ventilation, and if severe enough to a diagnosis of ards. we will therefore as a secondary endpoint also study if early intervention with inhaled sargramostim prevents progression to criteria-defined ards (according to the american-european consensus conference (aecc) diagnostic criteria for ards: acute onset; ratio of partial pressure of arterial oxygen to fraction of inspired oxygen (pao /fio ) of or less, regardless of positive end-expiratory pressure; bilateral infiltrates seen on frontal chest radiograph; and pulmonary artery wedge pressure of mm hg or less when measured, or no clinical evidence of left atrial hypertension), requiring high-flow oxygen devices, non-invasive mechanical ventilation, mechanical ventilation, by measuring the day from admission when this diagnosis is made or therapies are initiated. -to study if treatment with sargramostim affects all-cause mortality rate at and weeks post inclusion. -to study if treatment with sargramostim affects features of secondary haemophagocytic lymphohistiocytosis. a large subset of patients with severe covid- developing respiratory failure might have a cytokine storm syndrome, designated as secondary haemophagocytic lymphohistiocytosis (shlh). shlh is an under-recognised, hyperinflammatory syndrome characterised by a fulminant and fatal hypercytokinemia with multi-organ failure. cardinal features of shlh include unremitting fever, cytopenias, and hyperferritinaemia; hypertriglyceridemia, pulmonary involvement can present as ards. a cytokine profile resembling shlh is associated with covid- disease severity, characterised by increased interleukin (il)- , il- , granulocyte-colony stimulating factor, interferon-γ inducible protein , monocyte chemoattractant protein , macrophage inflammatory protein -α, and tumour necrosis factor-α. predictors of fatality from a recent retrospective, multicentre study of confirmed covid- cases in wuhan, china, included elevated ferritin (mean · ng/ml in non-survivors vs · ng/ml in survivors; p< · ) and il- (p< · ), suggesting that mortality might be due to virally driven hyperinflammation. to address the effect of sargramostim treatment on shlh, we will measure levels of ferritin, these chemokines and cytokines at the beginning of the trial day and after the initial day treatment. pbo including leukocytes and lymphocytes are performed on a routine clinical basis in these patients. -to study if treatment with sargramostim has a favourable effect on long term - week follow up at - weeks after discharge from hospital, patients will be seen on routine check-up by pulmonologist, who will perform a clinical exam, pulmonary function tests (including fvc, tlc and diffusion capacity), and a laboratory (ferritin, lymphocytes, leukocytes). this is phase academic, prospective, randomized, open-label, interventional study designed to investigate the efficacy of sargramostim (leukine®) in improving oxygenation and short-and long-term outcome of covid- patients with acute hypoxic respiratory failure. there are currently no treatments directed at improving lung repair and local immunity in covid- patients, and no treatment that attempt to halt the progression from manageable acute hypoxic respiratory failure to ards in patients with covid- infection. justification for our objective is that preventing progression from early acute hypoxic respiratory failure to ards could have a huge impact on the foreseeable overflow of the icu units, that is already happening in some countries and is bound to happen on a global scale. the hypothesis of the proposed intervention is that gm-csf has profound effects on antiviral immunity, can provide the stimulus to restore immune homeostasis in the lung with acute lung injury post covid- , and can promote lung repair mechanisms, that lead to a % improvement in lung oxygenation parameters. this hypothesis is based on experiments performed in mice showing that gm-csf treatment can prevent mortality and prevent ards in mice with post-viral acute lung injury. we will randomize patients with confirmed covid with acute hypoxic respiratory failure (saturation below % on minimal l/min o or pao /fio < ) to receive sargramostim mcg twice daily for days as a nebulized inhalation on top of standard of care (active group), or to receive standard of care treatment (control group). upon progression of disease to requiring invasive mechanical ventilatory support within the day period, in patients in the active group, inhaled sargramostim will be replaced by intravenous sargramostim mcg/m body surface area until the day period is reached. dosing of inhaled and systemic sargramostim are based on prior experience of this drug in patients with pulmonary alveolar proteinosis (inhaled) and with pneumonia associated ards (inhaled and intravenous). the inhaled route is preferred first, because high local concentrations of gm-csf have a favourable effect on lung immunity, lung homeostasis and lung repair. the switch to intravenous treatment with deterioration requiring initiation of mechanical ventilation is necessitated by the fact that patients with covid- poorly tolerate ventilation in the absence of high level positive end expiratory pressure (peep), especially when they develop ards. for giving the sargramostim via inhalator in a ventilated patient, this would involve peep-free ventilation for at least - minutes, which will not be tolerated in covid- associated severe hypoxic respiratory failure and/or ards according to expert opinion (prof. dr. pieter depuydt, intensive care unit, uz ghent). to measure the effectiveness of sargramostim on restoring lung homeostasis, the primary endpoint of this intervention is measuring oxygenation after days of inhaled (and intravenous) treatment through assessment of pretreatment and post-treatment ratio of pao /fio and through measurement of the p(a-a)o gradient, which can easily be performed in the setting of clinical observation of patients admitted to the covid - ward or icu covid- unit. supplemental oxygen use will be recorded, and if needed a-a gradient will be normalized against expected age-and supplemental oxygen dependent a-a gradient. during the day treatment period, we will perform daily measurements of oxygen saturation (pulse oximetry) in relation to fio , and the slope of alterations in this parameters could also be an indicator that our hypothesis is correct. if the patient leaves hospital prior to the day analysis point, oxygenation at day of discharge will be used as value for measuring primary endpoint. comparison will be between active group a receiving sargramostim on top of standard of care and control group b receiving standard of care. data from the wuhan covid- epidemic show that patients that deteriorate are facing a prolonged period of mechanical ventilation. therefore, from day onwards, progressive patients in the active group will have the option to receive an additional days of iv sargramostim, based on the treating physician's assessment. this group will be called group c. in the control group with progressive disease requiring invasive mechanical ventilatory support or developing ards, from day onwards, the treating physician will have the option to initiate iv sargramostim mcg/m body surface area once daily for days. this group will be called group d. comparisons of group a (early day intervention with sargramostim) with group d (late day intervention with sargramostim) will also be very informative. the subject has completed the study if he or she has completed all phases of the study, including the last visit (week - clinical follow up visit) or the last scheduled procedures, as described in this protocol (see section " . study specific procedures"). overall, the end of the study is reached when the last study procedure for the last subject has occurred: last subject, last visit (lslv). as soon as the whole study has ended (cfr. the definition above), the co-ordinating investigator shall notify the hiruz clinical trial unit, so that the competent authority and the ethics committee can be informed in a timely manner according to the regulatory requirements (within days after end of the study, or if the study had to be terminated early, this period must be reduced to days and the reasons should clearly explained). there is a large number of covid- infected patients that are currently being hospitalized across the globe. in just days time, our covid- ward at ghent university hospital has admitted confirmed cases, of which a significant portion ( %) already fulfill eligibility criteria for the current proposed protocol. we therefore believe that given the current ascending part of the epidemiology curve, with numbers of patients rising sharply, there will be no shortage of patients that are eligible. we estimate the study to terminate in weeks, including last clinical follow up visits. the following patients will be enrolled recent (≤ weeks prior to randomization) -confident covid- diagnosis confirmed by antigen detection test and/or pcr and/or positive serology, or any emerging and validated diagnostic laboratory test for covid- within this period. -in some patients, it may be impossible to get a confident laboratory confirmation of covid- diagnosis after h of hospital admission because viral load is low and/or problems with diagnostic sensitivity. in those cases, in absence of an alternative diagnosis, and with highly suspect bilateral ground glass opacities on recent (< h) chest-ct scan (confirmed by a radiologist and pulmonary physician as probable covid- ), a patient can be enrolled as probable covid- infected. in all cases, this needs confirmation by later seroconversion. -presence of acute hypoxic respiratory failure defined as (either or both) saturation below % on minimal l/min o pao /fio below -admitted to specialized covid- ward -age - -male or female -willing to provide informed consent -patients with known history of serious allergic reactions, including anaphylaxis, to human granulocytemacrophage colony stimulating factor such as sargramostim, yeast-derived products, or any component of the product. -mechanical ventilation before start of study -patients enrolled in another investigational drug study -pregnant or breastfeeding females (all female subjects regardless of childbearing potential status must have negative pregnancy test at screening) -patients with peripheral white blood cell count above . per microliter and/or active myeloid malignancy -patients on high dose systemic steroids (> mg methylprednisolone or equivalent) -patients on lithium carbonate therapy -patients with serum ferritin > mcg/ml (which will exclude ongoing hlh) / . . . screen failures screen failures are defined as subjects who consent to participate in the clinical study but are not subsequently randomly assigned to the study intervention or entered in the study. a minimal set of screen failure information will be kept to ensure transparent reporting of screen failure subjects. there is a large number of covid- infected patients that are currently being hospitalized across the globe. in just days time, our covid- ward at ghent university hospital has admitted confirmed cases, of which a significant portion ( %) already fulfill eligibility criteria for the current proposed protocol. similar numbers of patients are currently being seen in all centers.. we therefore believe that given the current ascending part of the epidemiology curve, with numbers of patients rising sharply, there will be no shortage of patients that are eligible. the number of subjects that will be included in this study is: . these are divided into following sub-groups: group a : active sargramostim treatment group, treatment for initial days, no deterioration after days number of patients : group b : control group : no treatment with sargramostim in first days number of patients : group c and d : data from the wuhan covid- epidemic show that patients that deteriorate are facing a prolonged period of mechanical ventilation. therefore, from day onwards, progressive patients in the active group will have the option to receive an additional days of iv sargramostim sargramostim mcg/m body surface area once daily, based on the treating physician's assessment. this group will be called group c. it is estimated that some % of patients might deteriorate and require noninvasive or invasive mechanical ventilation, giving potentially rise to patients that progress from group a to group c, if the clinician decides to move forward with the drug. in the control group progressing to requiring invasive or non-invasive mechanical ventilatory support, from day onwards, the treating physician will have the option to initiate iv sargramostim mcg/m body surface area once daily for days. this group will be called group d. it is estimated that some % of patients might deteriorate to mechanical ventilation or ards, giving potentially rise to patients that progress from group a to group c, if the clinician decides to move forward with the drug comparisons between group a (early sargramostim) versus group b (no sargramostim) at day will be important for reaching primary endpoint, and for key secondary endpoints. comparisons of group a (early day intervention with sargramostim) with group d (late day intervention with sargramostim) will also be very informative for secondary endpoint analysis. subjects are free to withdraw from participation in the study at any time upon request. an investigator may discontinue or withdraw a subject from the study for the following reasons: • allergic reactions (anaphylactic shock) to sargramostim • pregnancy  progression to non-invasive or invasive mechanical ventilation and/or ards between screening and randomization • significant study intervention non-compliance • if any clinical adverse event (ae), laboratory abnormality, or other medical condition or situation occurs such that continued participation in the study would not be in the best interest of the subject • if the subject meets an exclusion criterion (either newly developed or not previously recognized) that precludes further study participation in all cases, the reason why subjects are withdrawn must be recorded in detail in the ecrf and in the subject's medical records. if a patient decides to leave hospital before day of the study, for example because of clinical improvement, the oxygenation parameters at day of discharge will be used to calculate the primary endpoint measurement. the following actions must be taken if a subject fails to return to the clinic for a required study visit (visit at - weeks post end of study) : • the site will attempt to contact the subject and reschedule the missed visit within weeks and counsel the subject on the importance of maintaining the assigned visit schedule and ascertain if the subject wishes to and/or should continue in the study. • before a subject is deemed lost to follow-up, the investigator or designee will make every effort to regain contact with the subject (where possible, telephone calls and, if necessary, a certified letter to the subject's last known mailing address or local equivalent methods). these contact attempts should be documented in the subject's medical record or study file. • should the subject continue to be unreachable, he or she will be considered to have withdrawn from the study with a primary reason of lost to follow-up. subjects will be recruited at the covid- hospitalization ward at the participating centers. the study will be proposed by the treating physician to all subjects with pcr-confirmed covid- infection and a presence of acute hypoxic respiratory failure. there will be no compensation for study participation. partner therapeutics inc. is providing sargramostim to the study subjects, free of charge. since this is a hospital based trial, taking place over a minimum of five days in which patients are severely ill, we suspect the retention in the trial to be high. patients will be informed about the study by the treating physician. after receiving full explanation, having received sufficient time to considerer the trial, asking questions and receiving satisfying responses to all questions, patients will be asked to sign icf. a serum pregnancy test will be done (female patients only). medical history will be checked for review of exclusion criteria and relevant subject information. patients will be continuously monitored on the covid- ward. exams (standard of care) include, but are not limited to: -ecg -chest x-ray, and ct-scan -laboratory tests for leukocyte formula, kidney and liver function, ferritin levels -vital signs -pulse oximetry, arterial blood gas, capnography as soon as all in-and exclusion criteria are checked and patient is considered eligible, patient can be randomized. there is no minimal window to randomize the patient. the producer and distributor of leukine® is partner therapeutics inc, an integrated commercial-stage biotech company focused on the development and commercialization of therapeutics that improve health outcomes in the treatment of cancer. the distribution of imp will be done by tanner pharma. for inhalation: leukine® is a sterile, preservative-free lyophilized powder that requires reconstitution with ml normal saline solution, to reach a concentration of , mcg/ml. once reconstituted, leukine® can be inhaled as an aqueous aerosol using either a vibrating mesh nebulizer or jet nebulizer, aerosolizing ml twice daily. reconstituted leukine® solution for inhalation should be used within hours following reconstitution and/or dilution. dosage for inhalation: mcg twice daily via nebulizer. nebulizing is preferably done in an isolation negative pressure chamber, and if not, personnel should use an ffp mask. patient should self-administer the medication and where possible, the room should not be entered within one hour after administration. for intravenous injection: leukine® injection in . % sodium chloride injection, usp. dilute leukine® for intravenous infusion in . % sodium chloride injection, usp. if the final concentration of leukine® is below mcg/ml, add albumin (human) at a final concentration of . % to the saline prior to addition of leukine to prevent adsorption to the components of the drug delivery system. to obtain a final concentration of . % albumin (human), add mg albumin (human) per ml . % sodium chloride injection, usp (e.g., use ml % albumin [human] in ml . % sodium chloride injection, usp). once diluted for infusion, leukine® is stable for h. dosage for intravenous injection: mcg/m /day over a -hour period for up to days. no dose adjustments and interruptions are permitted during this trial. in case of anaphylaxis or severe ae, the drug will be immediately interrupted. leukine® will be administered for days, with a possible day extension to a maximum of days in case of progression of disease and need for mechanical ventilation. . . . packaging and labeling of the imp leukine® (sargramostim) for injection is a sterile, preservative-free, white lyophilized powder supplied in a carton containing five mcg single-dose vials. leukine® (sargramostim) injection is a sterile, clear, colorless solution preserved with . % benzyl alcohol supplied in a carton containing one mcg/ml multiple-dose vial and a carton containing five mcg/ml multiple-dose vials (ndc - - ). storage and handling : leukine should be stored at °c. drug will be labeled by pharmacy uz ghent (for uz ghent enrolment) for inhaled or intravenous use. store leukine® vials refrigerated at °c- °c ( °f- °f) in the original carton to protect from light. do not freeze or shake. do not use beyond the expiration date printed on the vial. leukine® is to be shipped refrigerated at °c- °c ( °f- °f). the medication will be delivered to the pharmacy of the participating centers. temperature during shipment and storage is to be monitored continuously. whenever a temperature deviation occurs, partner therapeutics inc. should be contacted. partner therapeutics inc. might allow further use of the medication vials depending on the duration and intensity of the temperature excursion. the co-ordinating investigator should be informed of this deviation as well. to date, there have been no new safety signals associated with leukine® (sargramostim). observed side effects with aerosolized leukine® at mcg dose and in at least one evaluation have included: bronchospasm, cough, dyspnea, a decrease in vital capacity and/or forced expiratory volume associated with bilateral infiltrates, pleural effusions, increased phlegm, throat irritation, and back pain. there are no restrictions regarding concomitant/rescue medication. patients will be informed about the study by the treating physician. after receiving full explanation, having received sufficient time to considerer the trial, asking questions and receiving satisfying responses to all questions, patients will be asked to sign icf. the icf process will be performed before any other study related procedure. in this open label trial patients will be randomized in a : ratio. randomization in belgium will be done using redcap (electronic ivrs system). this is a hospital based intervention trial, in which patients with covid- will be treated at least for days with sargramostim. patients with covid- infection and respiratory failure are severely ill, and will require multiple daily clinical exams, blood sampling, vital parameter measurements, blood oxygenation measurements, and chest x-rays. these are all part of the clinical management plan of the patients, and data stored in the electronic patient file will be used as part of the assessment of efficacy and safety profile of sargramostim. on screening, blood sample will be taken, preferentially during routine blood sampling, to determine exclusion criteria (pregnancy, high ferritin level). on day , prior to sargramostim treatment in group a, and during the day in group b control patients, a tube of blood serum ( ml) and an edta tube ( ml) will be collected for measuring blood cytokine and chemokine levels, and activation of immune cells in selected centers. also in each center, an arterial blood gas determination via arterial puncture will be taken. this sample should be taken in an upright position, while breathing room air for a minimum of minutes.. if this is impossible due to dependency on supplemental oxygen, fio , oxygen flow rate, and method of oxygen delivery should be noted in the patient file. if arterial blood gas is taken within h before first dose administration, as described in point° the arterial blood gas of screening can be used as d value. on day or on day of discharge before day , a tube of blood serum ( ml) and an edta tube ( ml) will be collected for measuring blood cytokine and chemokine levels, and activation of immune cells in selected centers. also in each center, an arterial blood gas determination via arterial puncture will be taken. on days - , patients in group a will inhale sargramostim mcg twice daily for days as a nebulized inhalation using a philips innospire go portable mesh nebulizer on top of standard of care. this device is a handheld mesh nebulizer that can be fitted with a facial mask. patients will be instructed prior to receiving the first dose on how to use this simple device, by a physician. this procedure is finished in - minutes, and will be performed twice daily, in the morning (between a.m. and a.m.) and evening (between p.m. and p.m.). upon progression of disease requiring initiation of invasive mechanical ventilatory support within the day period, in patients in the active group, inhaled sargramostim will be replaced by intravenous sargramostim mcg/m body surface area once daily until the day period is reached. this administration will occur via a centrally placed catheter or peripheral catheter, that will be in place as part of routine medical care at the icu. on a final clinical visit between week - an additional serum tube ( ml) and an edta tube ( ml) will be taken in selected centers. . patient demographics age, sex, ethnicity, day of admission . day of covid- pcr positivity, and conversion to negative . patient biometry weight, length, bmi, body surface area . clinical and laboratory parameters on screening day and during trial -first day of illness, potential source of infection -clinical examination findings (cyanosis, crepitation's and rales, heart murmurs, peripheral edema) -vital signs (temperature, blood pressure, heart rate, breathing rate) -pulse oximetry data (sao ) -clinical blood gas sampling (pao , paco , hco ) -clinical chemistry sampling (ferritin, leukocyte formula, platelets, kidney and liver function, fibrinogen, triglycerides) -chest x-ray and/or ct characteristics and radiology clinical report -in case of admission to icu : invasive monitoring data (arterial blood pressure, pcwp, continuous o saturation, continuous ecg, ventilatory parameters (tidal volume, fio , peep pressure, peak pressure, minute ventilation) . all standard care drugs used during the trial and on day of enrolment of the trial, including oxygen flow rate. . basic clinical data on prior medical history (prior lung diseases, smoking history, prior lung function measurements (preferentially within preceding years), prior gas exchange measurements) and medication use will be collected from electronic medical record. . study specific measurements . sampling in selected centers only: d : serum blood sample ml, edta blood sample, ml d or discharge before day : serum blood sample ml, edta blood sample, ml w - follow-up visit : serum blood sample ml, edta blood sample, ml these samples are to be taken on d and d (or discharge if before day ) and on final follow up visit between week and . there's no time window allowed. in selected centers samples will be taken during hospitalization together with the blood draw for standard of care. after clotting for - minutes the samples will be processed at rpm or g during minutes at room temperature. aliquots will be filled and frozen at - °c until further analysis. centrifugation and storage will be done by qualified personal. edta blood samples will be processed to purify peripheral bloodmonocytes and stained for flow cytometric analysis of number of monocytes, hla-dr expression on monocytes and dendritic cells, and lymphocyte activation. multiple cytokines and chemokines will be measured by multiplex bead based elisa assay. flow cytometry will be performed on paraformaldehyde fixed samples. development of anti-drug antibodies (ada) will be measured using protocol developed by partner therapeutics on serum samples taken at day and follow up visit. serum samples and frozen pbmcs will be stored at temperature monitored facilities of the participating research centres. . storage conditions: - °c. initially samples will be stored for the use as described within this protocol. if at a later time point samples will be stored for future use, they will be stored in fagg certified biobank the outcome(s) on which the sample size calculation is based upon, is the primary endpoint measurement of oxygenation, defined as ratio of pa /fio and p(a-a)o . sample calculation and power analysis have been performed using genstat. the target difference is the difference measured at the primary endpoint (at day ) between the control and the treated group. given a sample size of patients each, a minimal improvement of % in the treated group relative to the control group will be detected as significant at a significance level of , with a power of . . the error variance was set at units, corresponding with a standard deviation of units. the statistical test to be used will be an f-test. a two-sample t-test is expected to give similar results. the statistical analysis will be performed by gnomixx, a biostatistics consultancy company based in ghent (dr marnik vuylsteke). http://www.gnomixx.com/ subjects that are included in the study , will be assigned a unique study number upon their registration in redcap.. on all documents submitted to the coordinating center, sponsor or ci, patients will only be identified by their study number. the subject identification list will be safeguarded by the site. the name and any other directly identifying details will not be included in the study database. an electronic data capture (edc) system, i.e. redcap, will be used for data collection. data reported on each ecrf should be consistent with the source data. if information is not known, this must be clearly indicated on the ecrf. all missing and ambiguous data will be clarified. only the data required by the protocol are captured in the ecrf. the ecrfs and the database will be developed, based on the protocol. the final ecrf design will be approved by the co-ordinating investigator. all data entries and corrections will only be performed by study site staff, authorized by the investigator. data will be checked by trained personnel (monitor, data manager) and any errors or inconsistencies will be clarified. the investigator must verify that all data entries in the ecrf are accurate and correct. redcap is provided and maintained by vanderbilt university; a license for use was granted to the health, innovation and research institute (hiruz). redcap is a web-based system. the study site staff is responsible for data entry in redcap. the data is accessed through a web browser directly on the secure redcap server. the server is hosted within the uz ghent campus and meets hospital level security and back-up requirements. privacy and data integrity between the user's browser and the server is provided by mandatory use of transport layer security (tls), and a server certificate issued by terena (trans-european research and education networking association). all study sites will have access to redcap. site access is controlled with ip restriction. the investigator and sponsor specific essential documents will be retained for at least years. at that moment, it will be judged whether it is necessary to retain them for a longer period, according to applicable regulatory or other requirement(s). direct access will be granted to authorised representatives from the sponsor, host institution and the regulatory authorities to permit study-related monitoring, audits and inspections. login in redcap is password controlled. each user will receive a personal login name and password and will have a specific role which has predefined restrictions on what is allowed in redcap. furthermore, users will only be able to see data of subjects of their own site. any activity in the software is traced and transparent via the audit trail and log files. term definition adverse event (ae) any untoward medical occurrence in a subject to whom a medicinal product has been administered, including occurrences which are not necessarily caused by or related to that product. an adverse event, the nature or severity of which is not consistent with the applicable product information (e.g., investigator's brochure for an unapproved investigational product or package insert/summary of product characteristics for an approved product). adverse reaction (ar) an untoward and unintended response in a subject to an investigational medicinal product which is related to any dose administered to that subject. the phrase "response to an investigational medicinal product" means that a causal relationship between a study medication and an ae is at least a reasonable possibility, i.e. the relationship cannot be ruled out. all cases judged by either the reporting medically qualified professional or the sponsor as having a reasonable suspected causal relationship to the study medication qualify as adverse reactions. a serious adverse event is any untoward medical occurrence that:  results in death  is life-threatening  requires inpatient hospitalisation or prolongation of existing hospitalisation  results in persistent or significant disability/incapacity  consists of a congenital anomaly or birth defect other 'important medical events' may also be considered serious if they jeopardise the subject or require an intervention to prevent one of the above consequences. note: the term "life-threatening" in the definition of "serious" refers to an event in which the subject was at risk of death at the time of the event; it does not refer to an event which hypothetically might have caused death if it were more severe. an adverse event that is both serious and, in the opinion of the reporting investigator, believed with reasonable probability to be due to one of the study treatments, based on the information provided. a serious adverse reaction, the nature and severity of which is not consistent with the information about the medicinal product in question set out:  in the case of a product with a marketing authorisation, in the summary of product characteristics (smpc) for that product  in the case of any other investigational medicinal product, in the investigator's brochure (ib) relating to the study in question attribution definitions an adverse event is considered associated with the use of the drug if the attribution is possible, probable or definitive. an adverse event which is not related to the use of the drug. an adverse event for which an alternative explanation is more likely -e.g. concomitant drug(s), concomitant disease(s), and/or the relationship in time suggests that a causal relationship is unlikely. an adverse event which might be due to the use of the drug. an alternative explanation -e.g. concomitant drug(s), concomitant disease(s), -is inconclusive. the relationship in time is reasonable; therefore the causal relationship cannot be excluded. an adverse event which might be due to the use of the drug. the relationship in time is suggestive (e.g. confirmed by dechallenge). an alternative explanation is less likely -e.g. concomitant drug(s), concomitant disease(s). an adverse event which is listed as a possible adverse reaction and cannot be reasonably explained by an alternative explanation -e.g. concomitant drug(s), concomitant disease(s). the relationship in time is very suggestive (e.g. it is confirmed by dechallenge and rechallenge). ae's will be recorded from the first drug administration until the end of the study, as defined in section . . special attention will be given to those subjects who have discontinued the study for an ae, or who experienced a severe or a serious ae. all ae's should be recorded in the patient's file and in the crf. sae's occurring during the entire study period will be reported as below. all serious adverse events (initial and follow up information) and pregnancies occurring during this study must be reported by the local principal investigator within hours after becoming aware of the sae to: -the local ethics committee (it is the responsibility of the local pi to report the local sae's to the local ec) -hiruz ctu of the university hospital ghent -the national coordinating investigator (in case of multicenter studies) the company partner therapeutics that provides the imp this reporting is done by using the appropriate sae form. for the contact details, see below.  study team informs company that provides the imp susar notify to hiruz ctu within hours after becoming aware of the susar  hiruz ctu will submit to the central ec.  hiruz ctu will submit to the famhp  study team informs company that provides the imp in case the (su)sar occurs at a local participating site, the local pi or study team should also contact: -the local ethics committee -the co-ordinating investigator . . events, excluded from reporting covid- infection is a very recent syndrome, on which few data are available. normal symptoms and natural disease course symptoms that will not be reported as adverse events are dyspnea, coughing, malaise, fever, drop in oxygen saturation, progression to respiratory failure, progression to ards, severe drop in blood pressure in the icu,progression to multi-organ failure. all study medication is registered and used in current practice. despite the known safety profile of the study medications and study design, a dsmb is foreseen. the coordinating investigator will provide dsurs once a year throughout the clinical study, or on request, to the competent authority (famhp in belgium), ethics committee and sponsor. this dsur will include all sae's (who were not categorized as sar's and were not immediately reported to the ec). the report will be submitted within days after the start of the study, and will subsequently be submitted each year until the study is declared ended. hiruz ctu can provide a template that can be used to complete this dsur. monitoring/auditing/inspection . . monitoring monitoring of the study will be performed in compliance with gcp e (r ) and the applicable regulatory requirements. the study team will be trained in an initiation visit by the monitor. a training and delegation log will be held. a detailed description of the monitoring tasks can be found in the latest version of the (study-specific) 'monitoring plan'. monitoring services will be provided by hiruz ctu. all relevant contact details (e.g. primary contact person, can be found in the 'monitoring plan'. monitoring services will consist of the following (non-exhaustive list): -review of informed consents and the followed process -check on recruitment status -checking for protocol deviations/violations -checking gcp compatibility -check on safety reporting compliance -imp handling and storage -review of study data … this study can be inspected at any time by regulatory agencies during or after completion of the study. therefore access to all study records, including source documents, must be accessible to the inspection representatives. subject privacy must be respected at all times, in accordance to gdpr, gcp and all other applicable local regulations. the investigator/study team should immediately notify the sponsor if he or she has been contacted by a regulatory agency concerning an upcoming inspection. sponsor and all investigators agree to take any reasonable actions to correct protocol deviations/violations noted during monitoring/inspection, in consultation with the monitoring team. all deviations must be documented on a protocol deviation log by the study team that is kept available at any time for monitoring/inspection purposes. under emergency circumstances, deviations from the protocol to protect the rights, safety or well-being of human subjects may proceed without prior approval of the sponsor and the ec. ethical and legal aspects . the study will be conducted cfr the latest version of the ich e (r ) gcp guidelines, creating a standard for the design, conduct, performance, monitoring, auditing, recording, analyses and reporting of clinical studies that provides assurance that the data and reported results are accurate and that the rights, integrity and confidentiality of study subjects are protected. eligible subjects may only be included in the study after providing written (witnessed, if needed) ethics committee-approved informed consent, or, if incapable of doing so, after such consent has been provided by a legally acceptable representative(s) of the subject. informed consent must be obtained before conducting any study-specific procedures (as described in this protocol). prior to entry in the study, the investigator must explain to potential subjects or their legal representatives the study and the implication of participation. subjects will be informed that their participation is voluntary and that they may withdraw consent to participate at any time. participating subjects will be told that their records may be accessed by competent authorities and by authorized persons without violating the confidentiality of the subject, to the extent permitted by the applicable law(s) and/or regulations. by signing the informed consent form (icf), the subjects or legally acceptable representatives are authorizing such access. after this explanation and before entry to the study, written, dated and signed informed consent should be obtained from the subject or legally acceptable representative. the icf should be provided in a language sufficiently understood by the subject. subjects must be given the opportunity to ask questions. the subject or legally acceptable representative will be given sufficient time to read the icf and to ask additional questions. after this explanation and before entry to the study, consent should be appropriately recorded by means of either the subject's or his/her legal representative's dated signature or the signature of an independent witness who certifies the subject's consent in writing. after having obtained the consent, a copy of the icf must be given to the subject. in case the subject or legally acceptable representative is unable to read, an impartial witness must attest the informed consent. subjects who are unable to comprehend the information provided or pediatric subjects can only be enrolled after consent of a legally acceptable representative. any significant change or addition to the protocol can only be made in a written protocol amendment that must be approved by the central ethics committee (and the famhp if applicable). only amendments that are intended to eliminate an apparent immediate safety threat to patients may be implemented immediately. notwithstanding the need for approval of formal protocol amendments, the investigators are expected to take any immediate action, required for the safety of any subject included in this study, even if this action represents a deviation from the protocol. these actions should always be notified to the sponsor. all study data will be handled in accordance with the law on general data protection regulation (gdpr) and institutional rules [belgian law dated on july and aug. ]. the collection and processing of personal data from subjects enrolled in this study will be limited to those data that are necessary to fulfill the objectives of the study. these data must be collected and processed with adequate precautions to ensure confidentiality and compliance with applicable data privacy protection laws and regulations. appropriate technical and organizational measures to protect the personal data against unauthorized disclosures or access, accidental or unlawful destruction, or accidental loss or alteration must be put in place. sponsor and site personnel whose responsibilities require access to personal data agree to keep the identity of subjects confidential. the informed consent obtained from the subject includes explicit consent for the processing of personal data and for the investigator/institution to allow direct access to his or her original medical records (source data/documents) for study-related monitoring, audit, ethics committee review and regulatory inspection. this consent also addresses the transfer of the data to other entities, if applicable. privacy and confidentiality of data generated in the future on stored samples will be protected by the same standards applicable to all other clinical data. stored samples will be pseudonymized throughout the sample storage and analysis process and will not be labeled with personal identifiers. the sponsor has taken a no fault insurance for this study, in accordance with the relevant legislation (article , belgian law of may , critical issues that significantly affect patient safety, data integrity and/or study conduct should be clearly documented and will be communicated with the coordinating investigator, hiruz ctu and possibly both the applicable ethics committee(s) and competent authority. (please contact hiruz ctu asap in case of a serious breach: hiruz early determination of the study (in a specific center or overall) may be necessary in case of major noncompliance. . . end of study notification if all subjects have completed the study, a notification of the end of the study should be submitted to the (central) ethics committee and famhp. this notification should be made within days of the end of the clinical study the cloning of gm-csf alveolar macrophages develop from fetal monocytes that differentiate into long-lived cells in the first week of life via gm-csf gm-csf treatment prevents respiratory syncytial virus-induced pulmonary exacerbation responses in postallergic mice by stimulating alveolar macrophage maturation yolk sac macrophages, fetal liver, and adult monocytes can colonize an empty niche and develop into functional tissue-resident macrophages a review of gm-csf therapy in sepsis a randomized phase ii trial of granulocyte-macrophage colony-stimulating factor therapy in severe sepsis with respiratory dysfunction a randomized trial of recombinant human granulocyte-macrophage colony stimulating factor for patients with acute lung injury inhaled granulocyte/macrophage colony-stimulating factor as treatment of pneumonia-associated acute respiratory distress syndrome inhaled granulocyte/macrophage-colony stimulating factor as therapy for pulmonary alveolar proteinosis inhaled gm-csf for pulmonary alveolar proteinosis pathological findings of covid- associated with acute respiratory distress syndrome gm-csf overexpression after influenza a virus infection prevents mortality and moderates m -like airway monocyte/macrophage polarization lower respiratory tract delivery, airway clearance, and preclinical efficacy of inhaled gm-csf in a postinfluenza pneumococcal pneumonia model lung epithelial gm-csf improves host defense function and epithelial repair in influenza virus pneumonia-a new therapeutic strategy? the pleiotropic effects of the gm-csf rheostat on myeloid cell differentiation and function: more than a numbers game frontline science: coincidental null mutation of csf ralpha in a colony of pi kgamma-/-mice causes alveolar macrophage deficiency and fatal respiratory viral infection alveolar epithelial cells orchestrate dc function in murine viral pneumonia gm-csf in the lung protects against lethal influenza infection pulmonary pathological features in coronavirus associated severe acute respiratory syndrome (sars) screening follow-up ( - weeks after d ) d d d (or discharge before day ) informed consent x inclusion/exclusion criteria check serum pregnancy test x vital signs (incl. height and weight)* ° this sample should be taken in an upright position, while breathing room air for a minimum of minutes.. if this is impossible due to dependency on supplemental oxygen, fio , oxygen flow rate, and method of oxygen delivery should be noted in the patient file. § if arterial blood gas is taken within h before first dose administration, as described in point° the arterial blood gas of screening can be used as d value ∞ patients randomized in the treatment group will receive inhaled sargramostim from d untill d . in case of progression requiring mechanical ventilation within the first days, iv sargramostim can be initiated until the day period is reached. from day onwards, progressive patients in the active group will have the option to receive an additional days of iv sargramostim, based on the treating physician's assessment. patients in the control group will have the option to receive days of iv sargramostim in case of progression requiringmechanical ventilation, based on the treating physician's assessment.order of assessments: imp should always be administered after other assessments, where possible. there are no subject restrictions during this trial.in case the coordinating investigator, in consultation with hiruz ctu, decides the sae is a susar (suspected unexpected serious adverse reaction), hiruz ctu will report the susar to the central ec and the famhp within the timelines as defined in national legislation. the coordinating investigator reports the susar to all local pi's. in case of a life-threatening and fatal susar the entire reporting process must be completed within calendar days. in case of a non life-threatening susar the reporting process must be completed within calendar days. notify to hiruz ctu within hours after becoming aware of the sae  hiruz ctu will submit to the central ec this study will be registered at clinicalstudies.gov, and results information from this study will be submitted to clinicalstudies.gov. in addition, every attempt will be made to publish results in peerreviewed journals. appendices appendix : uspi (us package insert) key: cord- -thywse g authors: hwang, yoon jung; myung, heejoon title: engineered bacteriophage t as a potent anticancer agent in vivo date: - - journal: front microbiol doi: . /fmicb. . sha: doc_id: cord_uid: thywse g oncolytic viruses (ovs) induce antitumor effect by both direct lysis of target cells and eliciting immunogenic response to the virus and ultimately to the target cells. these viruses are usually natural human pathogens. bacteriophages are natural pathogens of bacteria that do not infect human and have greater advantages in safety, manipulation, and production over human viruses. we constructed an engineered bacteriophage t displaying a peptide, which targets murine melanoma cells and harbors a mammalian expression cassette of the cytokine granulocyte macrophage-colony stimulating factor (gm-csf) in viral genomic dna. the engineered phage was successfully transduced to b f melanoma cells both in vitro and in vivo. gm-csf was expressed from the transduced phage dna. all mice treated with the phage intravenously survived for days until the end of experiment, while only % of those not treated survived. during the days of phage treatment, phage t displaying homing peptide and expressing gm-csf inhibited tumor growth by % compared to the untreated control. serum cytokine levels of il- α, tnf-α, and gm-csf were seen to increase during the treatment. immunohistochemical analysis of tumor tissue revealed infiltration by macrophages, dendritic cells (dcs), and cd (+) t cells. migration of murine macrophages to bacteriophages was also observed in in vitro transwell assays in both time- and dose-dependent manners. taken together, the recombinant bacteriophage t efficiently inhibited tumor growth by changing the tumor microenvironment and recruiting anti-tumor immune cells. oncolytic viruses (ov) are viruses, which are known to attack and lyse cancer cells (raja et al., ; reale et al., ) . they destroy tumor mass by infecting and multiplying within the mass. in addition to viral lysis, the tumor mass, due to the presence of immunogenic viruses, is subject to attack by the immune system. partial remission of tumor mass when patients contracted viral diseases was observed by physicians as early as the beginning of twentieth century (kelly and russell, ). an hsv- based oncolytic virus (t-vec) has already been approved by the fda and is currently in clinical use (puzanov et al., ) . attenuated human pathogens, which have been tested as potential ovs, include the adenovirus (shaw and suzuki, ) , the vaccinia virus (haddad, ) , the measles virus (aref et al., ) , the mumps virus (ammayappan et al., ) , and the influenza virus (pizzuto et al., ) . other viruses known to be poor human pathogens, which have been tested as ovs, include the newcastle disease virus (tayeb et al., ) and the vesicular stomatitis virus (bishnoi et al., ) . potential ovs are often not powerful enough for solid tumors and safety has not always been matched with efficacy, as ovs are known to exhibit a certain range of toxic effects (raja et al., ; reale et al., ) . in addition, live viruses have been shown to be transferable from the primary treatment patient to healthcare workers and people inhabiting the same household (robilotti et al., ) . bacteriophages are viruses naturally infecting bacteria. since their discovery independently by frederick twort and felix d'herelle in early twentieth century, phages have mainly served as antibacterial agents and for the exploration of the basic mechanisms of life at the molecular level. in , accumulation of phages in cancer tissue and the inhibition of tumor growth were observed, and in , the binding of phages to cancer cells was seen both in vitro and in vivo (budynek et al., ) . phage t and its substrain hap were shown to bind melanoma cells and inhibit lung metastasis in a murine model (dabrowska et al., ) . interaction between lysine-glycine-aspartic acid (kgd) motif on phage capsid protein and the β integrin receptor on cell surfaces was hypothesized as being responsible for the activity. the same group of researchers showed that dendritic cells (dcs) primed with phage t and tumor antigens initiated differentiation accompanied by exhibiting an enhanced ability to prime t cells (pajtasz-piasecka et al., ) . application peritumorally of primed dcs retarded tumor growth in a mouse colon cancer model. eriksson et al. reported that peritumoral injection of m phages displaying peptide receptors or fab fragments in mouse melanoma cells led to delayed tumor growth and increased survival of tumor bearing mice (eriksson et al., ) . later, by investigating the tumor microenvironment, the same group of researchers showed that tumor destruction was caused by the activation of tumorassociated macrophages (eriksson et al., ) . hybrid adenoassociated virus/phage (aavp), a filamentous phage capsid packaging the cis-acting aav genomic element, was developed to facilitate targeted gene delivery without natural cell tropism (hajitou et al., ) . arginine-glycine-aspartic acid (rgd) peptide-displaying aavp expressing tnf-α with radiation therapy inhibited melanoma by modifying the tumor microenvironment in a mice model (quinn et al., ) . in this study, we developed phage t as an oncolytic phage (op). it displayed peptides targeting mouse melanoma cells and at the same time harbored a mammalian expression cassette for the cytokine granulocyte macrophage-colony stimulating factor (gm-csf). we tested its anti-tumor efficacy in vivo. the homing peptide used was pep (ctvalpggyvrvc) targeting grp on cancer cells (kim et al., ) . both strands of oligonucleotides encoding the peptide were synthesized (bioneer, korea) and annealed and ligated between ecori and hindiii sites in multiple cloning sites of t select - cloning kits (novagen, canada). the resulting t select vector was used for electroporation into escherichia coli blt (novagen, canada) to produce peptide-displaying phages. additionally, a cassette expressing gm-csf under cmv promoter was synthesized (bioneer, korea) and used to clone into the above described t genomic dna (genbank accession number v . ), which was cut with the restriction enzyme paci at , ~ , basepair. the synthesized cassette consisted of cmv promoter, kozak sequence, the orf encoding murine gm-csf (gene id ), and bgh polya signal. the recombinant phage was used to infect freshly cultured e. coli blt in a ml culture media at the multiplicity of infection (moi) of . . the mixture was incubated at room temperature for phage adsorption for h followed by shaking incubation at °c for h. chloroform was added to the culture at a final concentration of % (v/v) for complete lysis of bacteria and the culture was then incubated with shaking for more min. nacl was subsequently added at a final concentration of % (weight/volume) and the culture was incubated at °c for h. to remove any remaining bacterial cells or debris, the mixture was subjected to centrifugation at , × g for min. the supernatant was recovered and peg was added at a final concentration of % (weight/volume). the mixture was again subjected to centrifugation at , × g for min. supernatant was discarded and the pellet was resuspended in ml of sm buffer ( mm tris-hcl ph . , mm nacl, and mm mgso ). one milliliter of chloroform was added and the mixture was rigorously vortexed and subjected to a centrifugation at , × g for min. the upper phase was recovered and subjected to an ultracentrifugation. three milliliter of % glycerol was poured into an empty tube followed by the slow addition of % glycerol. the upper phase containing phages was then added to the tube and the remaining space was filled with sm buffer. the tube was centrifuged at , × g for hour. supernatant was discarded and the pellet containing phages was recovered by resuspension in ml of sm buffer. the method was described previously (branston et al., ) . triton x- was added to the phage sample at a final concentration of % (v/v) and the mixture was rigorously vortexed. after incubation on ice for min, the mixture was rigorously vortexed and subjected to centrifugation at , × g, °c for min. the upper phase was recovered and used for phage experiments in vitro and in vivo. the b f mouse melanoma cell line (kclb ) was obtained from the korean cell line bank at seoul national university. cells were grown in dulbecco's modified eagle's medium (dmem, thermo fisher scientific, usa) supplemented with fetal bovine serum (fbs, cellect, usa) at a final concentration of % (v/v) and penicillin/streptomycin (sigma aldrich, usa) at a final concentration of % (v/v). for staining bacteriophage t after transduction, × b f cells were seeded in a six-well plate with coverslip and grown in a co incubator. media was discarded after h and × pfu of phages in sm buffer were added to each well and incubated for min. unbound phages were washed out and the cells were fixed with cold acetone. blocking solution ( % bovine serum albumin in pbs) was added and the mixture was incubated for min. cells were then treated with : diluted anti-t tag antibody (ab , abcam, usa) for h followed by washing with pbs three times. secondary antibody ( : diluted anti-goat antibody, ab abcam, usa) was added and the mixture was incubated for h followed by washing with pbs three times. the nucleus was stained with ', -diamidino- -phenylindole (dapi) for min. a laser confocal microscope (lsm , carl zeiss, germany) was used for observations. for masking the receptor grp prior to phage transduction, : diluted anti-grp antibody (ab , abcam) was added to the cell culture and incubated for h followed by washing with pbs. visualization was performed by adding an alexa -labeled secondary antibody (anti-grp rabbit antibody, ab , abcam). for the labeling of phage dna, brdu (thermo fisher scientific, usa) was added at a final concentration of μm at the time of phage infection to the bacterial culture and the resulting progeny phages were collected. a total of × b f cells were seeded in a -well plate and incubated overnight. bacteriophages were added to the well at multiplicities of infection (moi) of or and the mixture was incubated for h followed by -( , -dimethylthiazol- -yl)- , -diphenyltetrazolium bromide (mtt) assay (cell viability assay kit, dong-in ls, korea) in accordance with the manufacturer's instructions. a total of × b f cells were seeded in a six-well plate and incubated overnight. engineered bacteriophage t displaying homing peptide (pep ) and expressing gm-csf was added to the culture at an moi of and cells were incubated for days. cells were harvested and lysed with cell extraction buffer ( mm tris-hcl, ph . , mm nacl, . % triton x- , . % sodium dodecyl sulfate, mm sodium orthovanadate, and mm naf) and the lysate was subjected to an sds-page analysis. expression was confirmed using the anti-gm-csf antibody (ab , abcam, usa) and the anti-rabbit secondary antibody (ab , abcam, usa) in a western blot analysis. the same lysate was used for the extraction of total rna using the trleasy total rna ultrapurification kit (rbc, taiwan). fifty milliliter of extracted rna was treated with unit of dnase i (solgent, korea) at room temperature for min for complete removal of dna as described (rio et al., ) . the mixture was cleaned up through a column included in the above rna isolation kit. five microgram of total rna was mixed with oligo (dt) primer and , u of reverse transcriptase (dynebio, korea). the mixture was annealed at °c for min followed by incubation at °c for min to allow for enzyme reaction. the reaction was then stopped by incubation at °c for min. real-time pcr (rt-pcr) was performed with the resulting cdna, primer, and sybr green qpcr x premix (dynebio, korea). the primer sequence used was forward: 'ggccttggaagcatgtagag ' and reverse: 'ccgtagaccctgctcgaata '. as a control, the extracted rna with dnase i treatment was subjected to a pcr reaction to detect any remaining dna. all animal studies were approved by, and complied with, the regulations and guidelines of the ethical committee for animal experiments of hankuk university of foreign studies (approval number hufs- - ). six-week-old female balb/c mice were obtained for the experiments (young bio, korea). for tumor size measurement, mice were divided into six groups. in vitro cultured × b f cells were subcutaneously injected into the right flank of each mouse. tumor mass was allowed to grow for week until its diameter reached ca. mm. treatment started week post melanoma cell graft. group contained control mice with sm buffer treatment. group mice were treated with × pfu of wild type bacteriophage t every day for days. group mice were treated with × pfu of pep -displaying bacteriophage t every day for days. group mice were treated with × pfu of pep -displaying bacteriophage t harboring expression cassette of gm-csf every day for days. group mice were treated with × pfu of pep -displaying bacteriophage t and ng of gm-csf (catalogue number z , genscript, usa) every day for days (sun et al., ) . group mice were treated with ng of gm-csf every day for days. all treatments were injected intravenously (iv) in the tail vein. tumor volume was measured during the treatment period. after days of treatment, the mice were sacrificed and tumor mass was removed for immunohistological analysis. for serum cytokine analysis, μl of orbital blood collection was performed for each mouse. for survival observations, mice were divided into six groups as above and survival was monitored for days. when tumor mass exceeded % of total body weight, the mouse was euthanized. the survival graph was plotted in accordance with kaplan-meier plot and drawn with prism, graphpad software. in vivo imaging was described previously (kelly et al., ) . briefly, × pfu of pep -displaying bacteriophage t or wild type bacteriophage t was fluorescently labeled with . mg/ ml of fluorochrome-hydroxyl-succinate ester (cy . ) in a dark room at room temperature for h. cy . -labeled phages were injected in tail veins of balb/c mice bearing b f grafted tumor mass and in vivo live imaging was performed using an fmt -lx imager (institute pasteur korea) after h. cytokine elisa serum was obtained from mouse blood by centrifugation at × g for min. mouse cytokines il- α, tnf-α, and gm-csf were measured using multi-analite elisarray kits (qiagen, germany) in accordance with the manufacturer's instruction. the assay was performed in triplicate. tumor-bearing mice treated with various phages and/or cytokine were sacrificed and tumor masses were removed. these were then fixed in % formalin and hematoxylin-eosin (he) staining and immunohistochemistry (ihc) were performed (logone bio convergence research foundation, seoul, korea). all experiments were performed in triplicate and statistical significance was obtained using one way anova followed by tukey's test (prism, graphpad software). p < . was considered as statistically significant. kaplan-meier analyses were used and the log-rank mantel-cox test was employed to determine any difference between the survival curves of the groups. p < . was considered as statistically significant. twelve millimeter transwell with . μm pore (corning transwell polyester membrane cell culture inserts, cls ) was used. a total of × b f cells were seeded in the lower chamber cell viability assay of b f cells after exposure to bacteriophages. two different concentrations (multiplicity of infection of either or ) of native t or engineered t were added to the culture and incubated for h before mtt assay was performed. control was treated with sm buffer. (c) homing and internalization of phage particle, and nuclear localization of phage dna. first and second row: wild type phage t (wt t ) or t displaying the homing peptide (t -pep ) was added to in vitro cultured b f melanoma cells and binding was observed under a fluorescent laser scanning confocal microscope. the nucleus was stained with dapi (blue) and the phage particle was stained with anti-t antibody (green). third row: b f cells were first treated with anti-grp antibody (red) to mask the receptor for pep . then t -pep (green) was added and binding was observed. fourth row: t -pep was produced in the presence of brdu (green) to label the genomic dna and added to cultured b f cells. internalized phage dna to dapi (blue) stained nucleus is shown. (d) real-time pcr (rt-pcr) analysis of mrna encoding gm-csf from t -pep or t -pep _g transduced b f cells. relative amounts of mrna encoding gm-csf from cells treated with t -pep or t -pep _g are shown in black or white bars, respectively. control was treated with sm buffer. t -pep , phage t displaying pep and t -pep _g, phage t displaying pep and expressing gm-csf. (e) western blot analysis of gm-csf from t -pep _g transduced b f cells (lane ) and empty cell (lane ). gapdh was used as an internal control. for statistical analysis, one way anova was performed and then tukey's test was conducted. *above each vertical bar indicates statistical significance of each test to the control. *above each horizontal bar indicates statistical significance of each test between corresponding pairs. and incubated at °c for h. a total of × pfu of phage t displaying pep harboring expression cassette of gm-csf was added to the confluently grown cells and incubated for h. then, × raw . cells were loaded in the upper chamber and incubated at °c for , , or h. media were discarded and migrated cells on membrane surfaces were fixed with ml of % ethanol at room temperature for min followed by drying for min. fixed cells were stained with . % crystal violet at room temperature for min followed by washing with distilled water three times. cells were then observed under a light microscope. the engineered bacteriophage t displaying homing peptide (pep ) and harboring a mammalian expression cassette of murine gm-csf was produced and the phage solution was nearly completely cleared ( %) of endotoxins ( figure a) . we first tested if this preparation exerted any toxicity in vitro. an mtt assay was performed with the results indicating that neither wild type t nor its engineered version had a significant effect on the viability of murine melanoma cells ( figure b) . next, we verified whether the engineered phage homed into b f cells in vitro. both wild type t and its engineered version were added to cultures of murine melanoma cells and stained with anti-t antibody followed by observation under laser scanning confocal fluorescent microscope ( figure c) . as anticipated, wild type t was washed out while engineered t displaying pep remained attached to melanoma cells. grp is known to be the receptor for pep (kim et al., ) . when the melanoma cells were treated with anti-grp antibody prior to the addition of phages, phage t displaying pep could not bind to the cells. we then grew phage t in the presence of brdu to label the phage genomic dna and used the phage for transduction. we observed localization of brdu labeled dna to the nucleus, suggesting that phage genomic dna enters the nuclei of melanoma cells. since the phage genomic dna harboring the expression cassette of gm-csf was localized to nuclei, we expected production of cytokine gm-csf from the transduced culture of melanoma cells. we could observe the transcription of cassette in a time-dependent manner using rt-pcr analysis ( figure d) . expression of gm-csf was also observed by western blot ( figure e ). as homing of the engineered phage was seen in vitro, we next investigated whether it homed into targets in vivo. after grafting of in vitro grown b f cells in the mice and after their tumor masses had built up, either wild type phage t or the engineered version was given to the mice intravenously and in vivo live imaging was performed (figure ) . fluorescently labeled phage t displaying pep localized to tumor masses four times more than wild type t . the location of wild type t looked peritumoral, rather than intratumoral, while the location of the majority of the engineered t looked intratumoral. since the engineered phage t was seen to home in tumor mass in vivo and to express gm-csf in vitro, we next explored whether this phage could inhibit tumor growth in vivo. in vitro cultured b f murine melanoma cells were implanted into mice and allowed to grow for days before the start of phage treatment. phages were intravenously injected into mice once every day for days and the survival, and changes in, tumor masses were observed (figures a,b) . tumor mass was measured , , , and days post implantation with various treatments (figures c-e) . phage t displaying homing peptide and expressing gm-csf (t -pep _g) inhibited tumor growth by % compared to the untreated control. treatment with phage t displaying pep (t -pep ) or in combination with externally added protein gm-csf led to a decrease in tumor volume by %. no additional effects were observed at the given concentration of gm-csf. the amount of total gm-csf available in the body could be one determining factor for the efficacy of this treatment (see figure ) . alternatively, the availability of gm-csf in the tumors' microenvironment could be another determinant. isolated tumor mass after sacrifice is shown for each treatment (figure c ). forty percent of untreated mice survived, while % of mice treated with phage t displaying the homing peptide and expressing gm-csf (t -pep _g) survived at the end of the experiment (figure f) . untreated mice began to die from day and % died by day . sixty percent of mice treated with wild type t displaying homing peptide (t -pep ) or with protein gm-csf survived, but protein gm-csf-treated mice had earlier deaths. eighty percent of wild type t or t displaying pep plus added gm-csf survived, but wild type t -treated mice had earlier deaths. one can note the following, in figure , the live imaging was performed at days post graft and hours after iv injection of phages. thus, the tumor sizes were identical at that particular time point. in figure , tumor mass was measured from to days post graft, where effects of injected phages were observed. thus the initial tumor sizes between the two experiments were different. animal cells are not natural hosts for bacteriophages and we cannot expect cell lysis from phage multiplication. one possibility for the lysis of tumor cells is immunological attack. since the recruitment of immune cells to the tumor could be mediated by various cytokines, we next checked the increase in serum cytokine after the administration of the phages (figure ) . three inflammatory cytokines, il- α, tnf-α, and gm-csf, were measured in mice serum after each treatment. eight-or three-fold increases in serum il- α were observed when the mice were administered with phage t displaying pep and expressing gm-csf (t -pep _g) or phage t displaying pep plus the externally added protein gm-csf (t -pep + g), respectively. neither the phage alone nor the protein gm-csf alone lead to any increases ( figure a) . however, homing phage t displaying pep (t -pep ) alone could strongly increase serum tnf-α ( figure b) . the latter effect decreased in the presence of gm-csf. t -pep alone induced tnf-α, but not other cytokines. in case of viral infection, inflammatory cytokines are induced at different levels. for example, epstein-barr virus infection in b or t cells induced tnf-α, but not il- α (lay et al., ) . in another example, sars-cov infection led to a preferential production of tnf-α, resulting in loss of germinal center cells (kaneko et al., ) . phage t may preferentially induce tnf-α rather than other cytokines. phage t displaying pep and expressing gm-csf (t -pep _g) induced the highest level of the cytokines (figure c) . t displaying the homing peptide (t -pep ) alone did not induce the production of gm-csf. taken together, for statistical analysis, one way anova was performed and tukey's test was conducted; * or **above each vertical bar indicates statistical significance of each test to the control; * or **above each horizontal bar indicates statistical significance of each test between corresponding pairs. (f) survival of mice bearing tumor mass was monitored for each treatment for days post implantation (n = /group). log rank test was used to test the significance. both t displaying the homing peptide and gm-csf were the determinants for the induction of il -α, while the presence of t displaying the homing peptide is the major determinant for the induction of tnf-α. t displaying pep (t -pep ) was not an inducer of gm-csf in these mice. as the expression of cytokines could lead to the activation and recruitment of immune cells to tumor mass, mice bearing tumor mass were treated with phages and/or cytokine gm-csf, and immunohistochemical observation was performed (figure ) . massive necrotic or damaged tumor cells were seen after treatment with the recombinant phage ( figure a) . tumor destruction and the shrinkage of cells were most prominent in mice treated with phage t displaying pep and gm-csf, which was either expressed from the phage or externally added. wild type t , t displaying pep , or externally added protein gm-csf alone, induced a limited destruction of tumor mass and shrinkage of cells. the highest degree of macrophage infiltration was observed when both t displaying pep and gm-csf were present ( figure b ). lesser infiltration was seen when t displaying pep or gm-csf alone was treated. for dc or cytotoxic t cells, t displaying pep and expressing gm-csf strongly recruited the immune cells (figures c,d) . considering the amount of total gm-csf detected (figure c) , gm-csf seems to play a significant role in recruiting the immune cells. for natural killer (nk) cells, little recruitment was seen with any treatment (figure e ). multiple sections of tumor tissue were stained immunohistochemically and observed for each immune cell. to observe phage-induced macrophage infiltration quantitatively in vitro, we performed a transwell migration assay. various doses of either wild type t or t displaying pep and expressing gm-csf (t -pep _g) were added and macrophage migration was detected at various points in time (figure ) . it was seen that wild type t hardly induced any migration of macrophages, while t expressing gm-csf induced a massive recruitment of macrophages in both dose-and time-dependent manners. in this experiment, a very efficient homing of phage t displaying pep , targeting cell surface receptor grp , was verified both in vitro and in vivo. in addition, in vivo imaging showed that accumulation of wild type t (without tropism for b f cells) occurred at the tumor, although at a much lesser degree. earlier literature reported the accumulation of phages in cancer tissue and the inhibition of tumor growth in (budynek et al., ) . this observed accumulation was both intratumoral and peritumoral. we speculate that the peritumoral accumulation of wild type phages in the latter experiment was in the tumor vasculature (forbes et al., ) . for example, bacteria were also shown to accumulate in the chaotic vasculature of tumors. bacteria are better at infiltrating inside tumors thanks to their motility, which phages do not have (forbes, ) . nonetheless, a small portion of phages were seen inside tumor masses in vivo in this experiment. an earlier experiment using the continued) bacteriophage lambda also reported that unmodified phage particles were capable of transducing mammalian cells in vivo, although much less efficiently than the surface modified phage targeting mammalian cells (lankes et al., ) . once attached to the cell surface, phage t particle was efficiently internalized and phage genomic dna was delivered to nuclei followed by the expression of gene encoding gm-csf from the cassette. it is coherent with previously reported gene deliveries using native phage lambda (lankes et al., ) , phage lambda displaying cyclizable rgd peptide to cos- cells (dunn, ) , phage m displaying epidermal growth factor (egf) to cos- cells (kassner et al., ) , m targeting her- receptor (urbanelli et al., ) , or aavp displaying rgd peptide (hajitou et al., ; trepel et al., ; kia et al., ; stoneham et al., ; smith et al., ; przystal et al., ) . trafficking of internalized phage particles was shown to be mediated by clathrin-mediated endocytosis followed by endo-lysosomal delivery (stoneham et al., ) . the latter is a common pathway for internalized materials and escaping from the endo-lysosome seems to be critical for more efficient expression of phage transgenes. increases in serum cytokines may be a factor leading to tumor regression in this experiment. recently approved oncolytic virus t-vec harbors two copies of gene encoding gm-csf (conry et al., ) . gm-csf promotes dc accumulation at sites of inflammation and enhances antigen presenting cell functions (kaufman et al., ) . since gm-csf was expressed from transduced tumor cells, the local concentration is expected to be higher than in total serum. two other proinflammatory cytokines, il- α and tnf-α, were also increased in the presence of t displaying homing peptide and harboring expression a b figure | transwell migration assay of macrophages. cultured b f cells in the lower chamber were treated with three different titers (moi , , or ) of wild type t (wt t ) or t displaying the homing peptide and harboring an mammalian expression cassette of gm-csf (t -pep _g). macrophages (raw . ) in the upper chamber were allowed to migrate for three different time periods. (a) staining and visualization of membrane after migration. white pores are seen from the membrane and macrophages are stained with crystal violet. (b) three random fields were chosen and the migrated cells were quantitated. for statistical analysis tukey's test was performed. *above each vertical bar indicates statistical significance of each test to the control. *above each horizontal bar indicates statistical significance of each test between corresponding pairs. cassette of gm-csf. in addition, tnf-α increased in the presence of t displaying the homing peptide without expression of gm-csf, suggesting that locally accumulated, high titer of the phages themselves could stimulate cytokine production. interestingly, less increases of tnf-α were observed, where gm-csf was expressed. therefore, there may be interference between the productions of the two cytokines in this experiment. changes in the tumor microenvironment, including locally accumulated phages and increases in proinflammatory cytokines accompanied by the recruitment of various immune cells, seems responsible for tumor regression. in fact, macrophages were found in tumor tissue when various phages and/or gm-csf were administered. on the other hand, dc and cd + t cells were predominantly found in tumor tissue when the t displaying the homing peptide and harboring expression cassette of gm-csf was administered. local expression of gm-csf might lead to enhanced t-cell priming by dc. antigen priming by dcs and the direct killing of tumor cells by t cells are the most powerful way of bringing about immune-mediated tumor regression. we could observe the same set of immune cells clearly infiltrating tumor tissue, where homing phages accumulated and gm-csf was produced by the tumor cells themselves, which could lead to locally increased concentrations. in case of nk cells, we could not observe any strong recruitment. one study reported a dual role of gm-csf in recruiting nk cells (nandagopal et al., ) . at high concentrations, gm-csf strongly induced migration of nk cells, while at low concentrations, it induced hyper-polarization, immediate arrest of nk cells, and little/or no nk-cell migrations. the observation that macrophage, one of the immune cells recruited in vivo, also migrated to tumor cells treated with phage t displaying the homing peptide and harboring expression cassette of gm-csf in time-and dose-dependent manners in an in vitro transwell assay is coherent with in vivo results. the immuno-modulatory nature present in the mucosal immunity of bacteriophages was recently reported (gogokhia et al., ) . the authors described that bacteriophages or phage dna could stimulate ifn-γ production via tlr- sensing, resulting in exacerbated colitis. a greater and greater body of evidence suggests relationships between phages and mammalian immunity. there have been several reports describing tumor regression in the presence of phages. in this experiment, we have demonstrated advancements in the utilizing of phages for this purpose. first, by displaying that the homing peptide (pep ) is more highly selective for target cells when compared to kgd or rgd peptides previously described (dabrowska et al., ; quinn et al., ) . although a report had already described the peritumoral injection of phage m displaying a homing peptide (eriksson et al., ) , we utilized intravenous injections and obtained greater tumor regression, suggesting better targeting methods and the increased stability of our system. t has much more packaging capacity when compared to m , and the double stranded nature of genomic dna has a better expression and prolonged stability inside mammalian cells. unlike aavp (hajitou et al., ) , t has no dna of animal virus origin, leading to less possible side effects. taken together, the combination of tumor-targeting bacteriophages and intratumoral expression of gm-csf from transduced phage dna can efficiently stimulate the immune system, leading to tumor regression. the datasets generated for this study are available on request to the corresponding author. the animal study was reviewed and approved by 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immunotherapy in the war on cancer: infection control considerations recent advances in oncolytic adenovirus therapies for cancer an aavpbased solid-phase transducing matrix for transgene delivery: potential for translational applications clathrin-mediated endocytosis and subsequent endo-lysosomal trafficking of adeno-associated virus/phage gm-csf quantity has a selective effect on granulocytic versus monocytic myeloid development and function therapeutic potential of oncolytic newcastle disease virus: a critical review. oncolytic virother a heterotypic bystander effect for tumor cell killing after adeno-associated virus/phage-mediated, vascular-targeted suicide gene transfer targeted gene transduction of mammalian cells expressing the her / neu receptor by filamentous phage hm conceived and designed the experiments. yh performed the experiments and generated the data. hm wrote the manuscript. all authors contributed to the article and approved the submitted version. key: cord- -eq zhtd authors: nan title: abstracts of oral presentations and posters date: journal: ann hematol doi: . /bf sha: doc_id: cord_uid: eq zhtd nan since the blood cells are primarily concerned with host defense, the introduction of the csfs as therapeutic agents offers the opportunity to develop unique therapeutic strategies designed to enhance overall host defense, particularly with relevance to cancer and aids. administration of csfs is associated with profound changes in cellular function, and treatment strategies will need to consider the potential deleterious effects of heightened host cell activity and potential effects on nonhematopoietic cells. memorial sloan-kettering cancer center, new york, ny , usa a cytokine the majority of hemopoietic stem cells in the steady state marrow are dormant in the cell cycle. using serial observation (mapping) of blast cell colony formation from bone marrow cells of mice that have been treated with a high dose -fluorouracil ( -fu), we have identified a number of cytokines that appear to control the cell cycling of the primitive hemopoietic progenitors. the early-acting cytokines may be divided into three groups. the first group would consist of il- , gm-csf, and il- . the second group consists of il- , g-csf, il- , il- , and lif/dia. the third group consists of steel factor (sf). according to our studies in culture, cytokines in each group can interact with those in other groups to initiate cell division in the cell cycle dormant primitive progenitors. while studies using retrovirally-labeled murine stem cells demonstrated unequivocally the presence of lymphohemopoietic progenitors that are capable of producing both lymphoid and myeloid progenies, it has not been possible to identify and quantitate these progenitors in culture. recently, we have developed a two-step methylcellulose culture method to quantitate murine lymphohemopoietic progenitors that are capable of producing myeloid cells and and pre-b-cells. after establishing the primary culture system initially with medium conditioned by pokeweed mitogen stimulated spleen cells, we characterized combinations of cytokines that are able to maintain the b-lymphoid potentials of the primary colonies. we observed that two-factor combinations including sf such as sf plus il- , sf plus g-csf, sf plus il- , sf plus il- were effective in maintaining the proliferation of b-cell progenitors. somewhat less effectively il- -based combinations such as il- plus il- and il- plus il- also supported the b-cell potentials of the the primary colonies. interestingly, il- -based combinations were unable to maintain the b-cell potentials of the primary colonies even though the cells in myeloid lineages proliferated strongly. we also found that addition of il- to an effective two-factor combination such as sf plus il- inhibit the b-cell potentials of the primary colonies. our cell culture for the murine lymphohemopoietic progenitors may provide an important tool for studying the mechanisms regulating the early process of lymphopoiesis. the survival and proliferation of haemopoietic stem cells, induced by growth factors, occurs concomitantly with differentiation and developmeat of the stem cells and their progeny, into mature blood cells. are the growth factors simply permissive for these processes? or do they have an inductive role to play in lineage commitment of stem cells and subsequent maturation into phenotypically mature cells? from various experiments, it is clear that the outcome of the response (that is the types of mature cells produced) is a reflection of the range of growth factors to which the cells are exposed-suggesting that combinations of growth factors may well influence the choice of lineage options taken by multipotent cells. the problem, of course, is that in all systems studied to date, no detailed examination has been made of cell death, and that even in growth factor combinations where, for example, no erythropoiesis is found, it is possible that erythroid progenitors are being produced as a consequence of differentiation of the multipoteni cells but that these cells are then dying due to lack of availability of the appropriate survival (growth factor) stimulus. we have recently been able to circumvent some of these problems and have shown that, orovided the cells receive a survival stimulus, differentiation can occur hi the absence of added growth factors and also that proliferation it not a prerequisite for acquisition of a mature cell phenotype. in other words, the growth factors may act primarily as survival and mitogenic stimuli and not as "inducers" of differentiation. expression of p bcr/abl by transfection converts interleukin- (il- )-dependent cell lines to factor independence and transforms immature hematopoietic cells in vitro. we tested the hypothesis that p bcr/abl may induce factor-independence by constitutively activating signal transduction pathways which are normally regulated by il- /gm-csf. in both the il- -dependent murine myeloid cell line, dcl , and the il- /gm-csf-dependent human line, mo e, p bcr/abl induces rapid factor-independence despite continuous growth in il- -containing medium. one-and two-dimensional antiphosphotyrosine immunoblotting showed that most proteins tyrosine phosphorylated by p bcr/abl are different that those phosphorylated in response to il- . several signaling molecules have been found to be activated or phosphorylated by both il- /gm-csf and p bcr/abl, including raf- , map kinase, shc, vav, and probably pi k. other signal transducing proteins were found to be phosphorylated only by pz bcr/abl (p rasgap, two rasgap associated proteins, and c-fes), or only by . in order to better define the biochemical activities of p bcr/abl which lead to mitogenesis, a series of cell lines were constructed in which the functional expression of pz bcr/abl was inducible. the uninduced cell lines had a wild-type phenotype while the induced cell lines displayed markedly reduced apoptosis in the absence of growth factor, and some were hyper responsive to growth factors. the phenotypes of these cell lines have been stable in culture, and the lines should be useful to define biochemical activities of p obcr/abl which are important for mitogenesis. r.e. donahue, m.r. kirby, p.d. lawman, s.e. sellers, s.w. kessler, and m.j.p. lawman a novel factor has been purified to homogeneity from a cell line derived from a human mixed germ cell tumor. by western blot analysis, using a polyclonal rabbit antibody raised to the purified native protein, scpf was found to be expressed both as a kd secreted and as a kd membrane-bound protein. to further evaluate scpfs' ability to support cd + cell growth in culture, scpf was used in short-and long-term cultures using immunoselected cd + cells. for short-term culture studies, cd + cel~s were evaluated prior to and subsequent to a six day exposure to either media alone or media supplemented with il- , scpf, or il- +scpf. the greatest expansion of cd + cells was in those expressing od . compared to pre-culture, cultures maintained in scpf, il- , or il- +scpf had, respectively, a . , . , or . -fold increase in cd +cd + numbers. there was also a consistent increase in the ratio of large cd + + cells to small cd +cd + cells. presumptively, this change represents an increase in the number of cells in either the g /m or s phase of the cell cycle. of the cytokine combinations evaluated, only the combination of il- + scpf led to a t . -fold expansion of cd + cd -cells above baseline values. by themselves, scpf and il- led to a . and . -fold reduction in cd +cd -numbers. when compared, however, to the number of cd + cd -cells present in media alone after the days in culture, scpf, il- , and il- + scpf hat respectively a . , . , and . -fold greater number of cd +cd -cells. in long-term culture assays in the absence of scpf, the cultures deteriorated rapidly and were test by day . in the presence of scpf, cell numbers were maintained over the initial - days in culture, with proliferation becoming evident days post-culture. at day , some of the cells were removed from culture media containing scpf and replatad in culture media alone. after an additional days in culture the cells that were no longer exposed to the scpf had differentiated. interestingly, the cells that were cultured in scpf continued to proliferate. after days in culture, these cells were predominantly cd +, cd +, cd +, cd +, cd +, and hi.a-dr +, and failed to express thy t, cd , cd , cd , od , or cd . karyotypic analysis demonstrated that these cells hat multiple chromosomal aberrations, civin, a. gewirtz, p. rockwell, l. witte we have cloned the cdna for stk- (stem cell tyrosine kinase ), a human growth factor receptor tyrosine kinase, and investigated its expression in bone marrow, leukemias, and leukemic derived cell lines. stk- expression is restricted to the cd + fraction of normal human bone marrow, the fraction containing all of the hematopoietic stem cell activity of marrow. experiments in which cd + cells grown on irradiated bone marrow stromal feeder layers were exposed to stk- antisense oligonucleotides resulted in inhibition of colony formation. stk- is also expressed in most cases of aml, b lineage all, and t cell all. a number of hematopoietic tissue culture cell lines which express stk- have been identified, including kmt , kgla, kg , ml- , hl- , nalm- , and reh. ml- cells stop growing and differentiate after exposure to phorbol esters and other agents. stk- expression is completely shut off by this treatment. antipeptide antibody generated against several regions of stk- identifies a doublet of proteins of kd and kd, probably corresponding to different degrees of glycosylation, in several of the cell lines. these data imply a possible role for the stk- receptor in the normal proliferation of hematopoietic stem cells and the abnormal proliferation of leukemic cells. further antisense experiments and the isolation of the growth factor for this receptor will be necessary to fully understand its role in hematopoiesis and leukemia. murine long term repopulation and double transplantation assays have clearly demonstrated irreversible damage to early stem cells by repeated doses of alkylating agents or anti-metabolites. the ability to protect from the short term effects of chemotherapy-induced myelosuppression by administration of hematopoietic growth factors has obscured the potential problem of long term stem cell insufficiency. indeed in murine models involving repeated cyclophosphamide administration, csf administration has been reported to protentiate stem cell damage. the development of techniques for isolation of human hematopoietic stem cells in a cd + lin -re fraction of marrow and blood, together with long term culture-initiating assays on marrow stroma or long term ex vivo expansion with cytokine combinations, permits quantitative analysis of human stem cell proliferation potential. it is becoming apparent that extensive chemotherapy treatment gravely compromises the population of primitive hematopoietic stem cells as reflected in their impaired capacity to peripheralize and to be represented in the blood cd + population following csf treatment with or without cytoxan. five strategies are currently under evaluation: ) upfront harvesting of marrow and/or elicited peripheral blood prior to onset of chemotherapy with subsequent "rescue" following chemotherapy; )fine tuning of cytokine and chemotherapy administration to take advantage of "rebound quiescence" of stem cells; )administration of negative regulators to suppress stem cell proliferation. transforming growth factor , macrophage inflammatory protein e and tumor necrosis factor have all proved protective in preclinical models. ) utilize cytokines, eg il- , that protect stem cells by increasing drug enzymatic inactivation, decreasing drug influx and/or increasing drug efflux, and inducing dna repair or decreasing dna damage. ) ut ze gene therapy to introduce into hematopoietic stem cells drug resistance genes such as mutated dihidrofolate reductase that confers methotrexate resistance or enhance the expression of the multi-drug resisting gene (mdr) expression. interleukin- : a novel hematopoietic cytokine possessing multiple biological activities in vitro and in vivo. j.p. leonard and s.j. goldman, genetics institute, cambridge, ma. interleukin- is a multifunctional hematopoietic cytokine which was originally identified in the conditioned medium from an il- stimulated primate stromal cell line. the human cdna was subsequently cloned from a fetal fibroblast cell line enabling the expression and purification of the human protein. the in vitro biological activities of rhll- result predominantly from synergistic interactions with other growth factors. in combination with other cytokines, rht[,- has been shown to support the formation of primitive human blast ceil coionies from bone marrow, promote erythroid burst formation and stimulate both early and late stages of megakaryocyte proliferation and differentiation. in addition, rhll- alone directly increased the size and ploidy of enriched megakaryocytes. although rhll- has no inherent cell growth factor activity, rhll- has been shown to stimulate immunoglobulin producing b cells both in vitro and in vivo. rhll-i is biologically active in mice, rats, dogs and primates when administered as a single agent in vivo. the predominant effect of rhll- in naive animals was on cells of the megakaryocyte lineage, increasing the number of bone marrow megakaryocyte progenitors, stimulating megakaryocyte endoreplication and increasing peripheral platelet counts in a dose dependent fashion. in a variety of murine rtiodels of myelosuppression, the effects of rhtl- were multitineage, stimulating the recovery of megakaryocyte, erythroid, and granulocyte and macrophage progenitors in the bone marrow, rhll-i administration reduced the platelet and hematocrit nadirs and the overall duration and severity of thrombocytopenia and anemia in these models. in a murine bone marrow transplant model, rhll- also accelerated neutrophil recovery. the results from ongoing preclinical studies continue to confirm the broad spectrum of biological activities possessed by rhil- in vitro and suggest this cytokine may be an effective agent in the treatment of myelosuppression associated with cancer chemotherapy and bone marrow transplantation. preclinical biology of human il- t.l. nagabhushan, s.k. narula and m.i. siegel human il- (hull- ) is a amino acid polypeptide synthesized by a number of different cell types. it is a pleiotropic factor with both immunosuppressive and immunostimulatory activity. recombinant hull- expressed in cho cells is not glycosylated, and when expressed in e. coil the protein retains the biological activity of the cho-derived product. in the presence of antigen presenting cells, hull- inhibits cytokine synthesis in t cells. hull- downregulates ,f-ifn and il- induced mhc class ii antigen expression on monocytesmacrophages. in contrast, it has no effect on class ii expression on purified tonsillar and peripheral b cells. hull- also inhibits the synthesis of il- % il- /l il- , il- , tnf-m gm-csf and g-csf at the protein and rna levels in monocytes activated by lps or lps and .r the y-ifn or gm-csf induced phagocytosis of opsonized yeast particles by human peripheral blood-derived macrophages and granulocytes is downregulated by hull- . il- induced ige synthesis by pbmnc is inhibited by hull- . the protein also potentiates a strong lak activity in human pbmnc. these results will be discussed along with some early in vivo biology in rodents. a monomeric pentapeptide (peedck) inhibits murine hematopoiesis in vitro and in vivo while its dimer counterpart (peedck): formed through a disulphide bridge has a stimulatory effect in the same systems. stable peptides of the rimer have been made by substitution of the disulphide bridge with a dimethylene bridge. in vitro the dimer seems to have no direct effect on gm-cfc or on purified lin-scal + cells in the hpp-cfc assay ( cells per culture, - % pe). the monomer does not affect gm-cfcs, but inhibits approximately % of the purified hpp-cfcs. the dimer stimulates human or mouse stromal cells to produce m-csf and possibly other cytokines which augment colony formation in vitro and also activate human pbl as measured by an increased expression of cd lb. in ex vivo experiments it was found that the dimer increases and the monomer decreases cell cycle rate of cfu-gm and cfu-s in the bone marrow. intraperitoneal injection of the dimer ( to ng/kg) into mice, led to increased progenitor cell number in the bone marrow and also increased survival of mice given a lethal dose of cyclophosphamide ( mg/kg). at present receptors of the peptides have not been identified. these studies have shown that the dimer has an indirect effect on hematopoiesis as a stimulator of cytokine production, while the monomer seems to act both as an antagonist of dimer action and is also able to directly inhibit early myeloid progenitor cells. the possibility that these two compounds have therapeutic efficacy in diseases involving a myelosuppressed bone marrow is indicated. there is evidence that basic fibroblast growth factor (bfgf) plays a role in the regulation of normal blood cell proliferation and differentiation. basic fgf is produced by and is a potent mitogen for human slromal cells. it is found in megakaryocytes and cells of the granulocyte lineage, in vivo, and it enhances megakaryopoiesis and myelopoiesis in human long term bone marrow cultures. it stimulates progenitor cell growth and augments the proliferation of progenitor cells when added in conjunction with other hematopoietic growth factors. in addition, it counteracts the suppressive effects of transforming growth factor beta. basic fgf also synergizes with stem cell growth factor to augment granulocyte macrophage colony stimulating factor stimulated progenitor cell growth. based on the observations in normal hematopoiesis, the role of bfgf in malignant bematopoiesis is presently an area of active investigation. scf is one of the earliest-acting hematopoietic growth factors. pre-clinical studies have demonstrated its multi-lineage hematopoietic effects. we have conducted a phase i trial of scf and report now on the first patients (pts) with stage iiib (n= ) or iv (n= ) breast cancer. the study was designed to evaluate the toierabllity and biologic effects of scf. pts received scf prechemo (cycle o) and following cycles - of c/a chemotherapy. cohorts of lots were randomized in a : ratio to receive either scf (n= ) by subcutaneous injection at dosages of i , , and so/~g/kg/day for days or no scf as a parallel c/a control group (n= ). various physiologic, biochemical, pharmacokinetic, and hematologic parameters were studied. bone marrow (bm) and peripheral blood (pb) progenitors were assayed. scf administration was associated with pb progenitor mobilization in cycle at all dose levels. absolute neutrophil counts demonstrated modest doserelated increases over baseline ranging from median values of % ( pg/kg/d) to % (so/jg/kg/d). no reproducible effects were seen on red blood cells or platelets. the principal adverse events were derrnatologic in nature. they included mild to moderate reactions at the injection site in all pts, and moderate to severe reactions distant to the site in i pts (primarily at higher doses). at pg/kg/d, / pts experienced dose-limiting respiratory symptoms including cough, hoarseness, and laryngospasrn. because of scfs known mast cell effects, prophylaxis with h /h blockers and bronchodilators is being evaluated. no pts developed antibodies to scf. evaluation of effects following chemotherapy is ongoing. we conclude that scf is an active hematopoietin capable of stimulating production of bm and pb progenitor cells as well as peripheral neutrophils. g. trinchieri nksf/il- is a heterodimeric cytokine produced by monocytemacrophages, b cells and other accessory cells in response to various stimuli including bacteria and bacterial products. nksf/il- acts on t cells and nk cells inducing cytokine production, proliferation, and enhancement of cytotoxic activity. nksf/il- is a particularly efficient inducer of ifn- production, acting alone or in synergy with other ifn- inducers such as il- , antigens, anti-cd or anti-cd antibodies, mitogens, and phorbol diesters. nksf/il- appears to play a major role in regulation of natural resistance: when produced by monocyte-macrophages, it directly activates the cytotoxic activity of nk cells and induces both nk and t cells to produce ifn- and other cytokines with important effects on activation of phagocytic cells. the important role of nksf/il- in response to bacterial products is clearly demonstrated by the ability of anti-nksf/il- antibodies to inhibit in vivo in mice the ifn-y production induced by lps ii~ a toxic shock model. nksf/il- is also an important factor in the regulation of adaptive immune response, by inducing the differentiation and growth of t helper cells type (th- ) and by preventing the differentiation of il- producing th- cells. a possibly obligatory role of nksf/il- for th- cell differentiation can be demonstrated in vitro in the human lymphocyte response to allergens or bacteria-derived antigens and both in vitro and in vivo in the murine system, e.g. in the immune response to leishmania infection. production of nksf/il- by accessory ceils is stimulated by ifn- , a product of th- cells and suppressed by il- or il- , products of th- cells. these results suggest that nksf/il- represents an important link between natural resistance and adaptive immunity and is at the center of a cytokine network that regulates the equilibrium between cellular and humoral immunity. work with alpha-interferon in cml started at mdacc in and has evolved over the years to allow us interpretation of response rate in a large number of patients with relatively long follow up. newly diagnosed patients (<i year from diagnosis) were treated with a starting dose of mu/m of interferon. of such patients followed in different studies, we noted a % complete hematologic remission; % cytogenetic response, and % complete and partial cytogenetic response (< % phi). in long-term analysis of patients, achieved cytogenetic response ( %) of which ( %) were complete; ( %) were partial, and ( %). were minor ( %) of the cytogenetic responses are durable. of major interest is the fact that % of the complete cytogenetic remissions are durable ( of ) but only a minority of the partial and minor cytogenetic responses are durable ( % and %, respectively). this clearly set our goal on improving the incidence of complete eytogenetic remission. median survival ranges between - months. however, of particular interest is the excellent survival rate of the complete cytogenetic responders ( of alive at time of follow-up). current studies are focusing on idealizing combination therapies and will be discussed. cml is a paradigm of tumor progression; therefore, we were looking for characteristics associated with disease progression and identified evidence of autocrine growth factor loop formation with disease progression and the overproduction of il-ib. finally, we are also focusing on the study of minimal residual disease in patients with complete cytogenetic remissions and have identified residual ph i progenitor cells in the majority of these patients, including patients with unmaintained remission. the nature of such a remission will be discussed as well. the mxa-protein: a new and excellent marker for endogenously produced type-i-ifn d.jakschies , k.u.nolte , r.zachoval , h.deicher , and p.v.wussow the human mxa-protein is a recently identified human ifn-induced protein, which is synthezised by peripheral blood cells specifically and dose-dependently upon stimulation with type-i-interferons both in vitro and in vivo. mononuclear cells (mnc) both from healthy persons and cancer patients (n= ) are usually (in the absence of a virus infection) mx-negative. patients starting an ifn-a-therapy become mxa-positive (n= ) within hours and remain positive throughout their therapy. furthermore, two groups of patients are known to have frequently measurable serum ifn-titer: sle-patients and aids-patients (n= ) studied expressed significant mxa-levels in their mnc. while all patients with measurable ifn-titer had mxa-levels above u/ . mnc, also the vast majority of serum ifn-negative patients possessed detectable mxa-levels indicating that also these patients produce endogenous ifn, although due to its rapid clearence from the circulation it may not be measurable in serum. in addition, patients with acute hepatitis were investigated for the mxa-protein. surprhingly large difference in the endogenous tfnproduction were observed. only in the first two weeks after the onset of clinical symptoms hepatitis a patients (n= ) had both high mxaprotein and - -a-synthetases levels, but not thereafter. the levels of both ifn-induced activities reached concentrations found in cancer patients treated with - -mill. iu rifn-c~ per dose. in acute hepatitis b and c (n= ;n= ) only marginally elevated mxa and - -as levels were found. in summary, the mxa-protein is a new and excellent marker for endogenously produced type-i-ifns. m.e. rybak interleukin (il- ) is a cytokine with pleiotropic immunomodulatory activities. these actions include b cell stimulation and co-stimulation, enhancement of il- mediated generation of cytotoxic t cells, increase in the expression of hl-a class ii antigens by monocytes and the inhibition of the production of a number of monokines including il- , il- and tnf~. human il- has been cloned and expressed in e. coli. the mature protein has amino acids, mw , d. studies of rhuil- in malignancy were prompted by i__nn vitro data demonstrating the ability of il- to inhibit the proliferation of chronic myelomonocytic cells, non-hodgkin's lymphoma and myeloma cells in culture, and to inhibit the il- induced proliferation of chronic lymphocytic leukemia. inn vivo models utilizing murine il- demonstrate an immune response to tumors (e.g lung, sarcoma and melanoma) following systemic administration of il- or the administration of tumor cells transfected with the il- gene. initial phase i trials of rhuil- have been completed in patients. forty-seven patients received daily subcutaneous (s.c.) rhuil- , . - . ~g/kg. side effects seen in ~ % of patients included headache, fatigue, fevers (< ~ myalgias and reversible elevation of sgot, sgpt and alkaline phosphatase. nausea, pedal and periorbital edema, and rhinitis were also seen. the mtd for rhuil- was approximately ~g/kg/d s.c. activity has been seen in hodgkin's disease, non-small cell lung cancer, chronic lymphocytic leukemia, and multiple myeloma. current studies include phase ii investigations in advanced non-small cell lung cancer, refractory hodgkin's disease, and relapsed multiple myeloma. an update on these studies will be presented. this lecture will review the evidence concerning the possibility that sustained overstimulation by growth factors might provoke or accelerate myeloid leukemia development. the regulatory factors likely to be relevant are the four colony stimulating factors although a possible role for scl and il- probably also needs consideration. in general, prolonged overstimulation by growth factors in the mouse either using transgenlc mice or engraftment of hemopoietic cells engineered to produce growth factors leads to hyperplasia but not leukemia. in contrast insertion of csf genes into immortalized, but non-leukemic cell lines leads to prompt leukemic transformation. if fdc-pi cells are engrafted in preirradiated animals, these can undergo delayed leukemic transformation often by activation of csf genes by inserted ~ particles. normal cells can be transformed to leukemic cells by retroviral insertion of a combination of a csf gene with fox . , the apparent function of the hox . being to dysregulate the process of self-generation in early hemopoietic precursor cells. thus two distinct changes appear to be necessary for leukemic transformation -abnormal self-generation and an acquired capacity for autocrine growth factor production. all the above studies involve autocrine growth factor production but we have recently completed a study indicating that sustained stimulation by exogenous growth factors can also accelerate leukemic transformation. this study involved fdc-pi cells engrafted in gm-csf transgenic mice. again the actual transformation process often involved rearrangement of csf genes with autocrine csf production. the latter experiments raise the potential clinical hazard of prolonged csf treatment in myelodysplastic patients but this possibly should not be overinterpreted. since granulocyte (g-csf) and granulocyte macrophage (gm-csf) colony stimulating factors were first introduced in to clinical trials in , there have been a vast number of clinical trials demonstrating the utility of these agents in reducing nentropenia, and therefore infection in patient groups ranging from congenital nentropenia to bone marrow transplantation. the discovery that a large number of progenitors are released into the peripheral blood by these agents either alone or after chemotherapy was not one of the major indications predicted in . the rapid reconstitution of both nentrophils and platelet counts when these peripheral blood progenitors (pbpc) are used as autologous rescue after myeloblative treatment, is encouraging many centres to use this type of rescue instead of abmt. interesting studies are at present indicating that these pbpcs can be expanded in vitro prior to clinical use. attention is now moving to the stimulation of platelet productions with il- , il- , and l- . there is increasing evidence from experimental haematologn/ that combinations of hgfs give optimal stimulation often of more primitive cells than previously thought. clinical studies are now being performed, but the logistic problem of testing all the possible comb'mations is daunting. as the use of haemopoietic growth factors successfully protecta the patients against haematological problems, other organs such as the gi tract, become dose limiting, often keeping the patient in hospital after haemopoietic recovery. repeated high dose treatments also deplete the haemopoietic stem cells. great interest is now being found in protecting the normal haemopoietic and other organ stem cells from the effects of radiochemotherapy. this may be possible using molecules such as mip alpha or tgf beta. other workers have used the timing of the use of stimniatory hgfs to induce quiescence of the haemopoietic stem cells. the optimum use of hgfs would probably be to in sequence, prime the haemopoietic progenitors, then protect them from radiochemotherapy, followed by rapid expansion under the influence of probably a mixture of hgfs. crc deft of medical ontology, christie hospital, wilmslow road, withington, manchester, m bx. il- is the first cytokine available for human use which was predicted to have both a activity on early hematopoietic progenitor cells, as well as specific action leading an increase in the number of circulating platelets. phase i/ii clinical trials have confirmed these predictions and have demonstrated, in addition, moderate activity on the late myeloid cell lineage leading to increased numbers of circulating neulrophils and eosinophils, events predicted from monkey studies, but not from in vitro and rodent studies. phase i studies provided a clear definition of the maximum tolerated dose ( #g/kg daily iv or s.c.), since at /lg/kg an unacceptable frequency of severe headache, flushing and tissue swelling were observed. at doses between . - #g/kg, depending on the bone marrow reserve, cytotoxic chemotherapy and malignant disease, il- prevented or reduced the severity of neulropenia and thrombocytopenia, reduced the number of platelet transfusions needed and allowed adherence to myelotoxic chemotherapy. in combination with other cytokines, such as gm-csf or g-csf, enhanced myeloid responses were seen including increased numbers of peripheral blood progenitor cells. these observations have led to now ongoing phase iii clinical trials using il- in conjunction with cytoxic chemotherapy in several malignancies and in conjunction with autologous bone marrow transplantation. there appears little doubt at present that il- will be an important contribution to management of the patients with oncologic or hematologic diseases. extensive phase ii studies have shown a reduction in the severity and duration of neutropania when filgrastim (r-methug-csf) is administered soon after chemotherapy. two phase iii studies demonstrated that filgrastim, given to small cell lung cancer patients from the day after chemotherapy, reduced the incidence of febrile neutropenic episodes by approximately % (crawford et al, ; trillet-lenoir et al, ) . these studies administered filgrastim from the day after chemotherapy, and they suggest that filgrastim is highly effective when used as an adjunct to chemotherapy to prevent fever and neutropenia. in a recent study (maher el al, in press), pilgrastim administration was delayed until after the onset of fever and antibiotic therapy. patients enrolled in lhis study were a median of days after their last dose of chemotherapy. it was found that this later commencement of filgrastim could significantly accelerate neutrophil recovery, produce a shorter duration of febrile neutropenia, and decrease the risk of prolonged hospitalization; however, maximal benefit appears to be derived from adjunctive use of filgrastim prior to the onset of fever and neutropenia. from in vitro and animal studies it is known that il- is a stimulator of thrombopoiesis, a stimulatory factor for t and b cells, that it stimulates hepatocytus and can have antitumor activity. an ongoing phase i-ii study with rhll- before and after chemotherapy (ct) is performed. patients (pts) with breast cancer or non-small cell lung cancer receive e. coil derived rhll- (sandoz, basle) in doses of . , , . , and #g/kg/d, with > pts per dose step. before ct rhll- is given dl iv and d - sc. ct (mitoxantrone mg/m and thiotepa mg/m , iv dl) starts d , q wks. after ct rhll- is given d - se in all cycles (c). before ct pts were treated. rml~ related side effects were: fever who gri-ii ( %), headache ( %), myalgia ( %), local erythema ( %) and starting at . #g nausea ( %), increase of liver enzymes who gri-ii ( %) and anemia ( %). symptoms were controllable with acetaminophen (n= ) and antiemetics (n= ). upto #g them was a dose response related platelet increase compared to dl (r= . , p< . ), with highest count after rml- cessation. at . /~g max. increase was . + . (se), at ~g . + . %. on d a leukocyte increase compared to dl was observed starting at . #g upto /~g (dl . + . , d . + . , p< . ) mainly due to neutrophil increase (p< . ). at d increase of lymphocytes occurred compared to dl (dl . + . , d . + . , p< . ). there was a reversible hemoglobin reduction especially at and i ,gg, starting d with a max. decrease at dg ( /zg . + . g/l, #g . + . g/l), at d initial values were almost reached without intervention. a dose related acute phase response was observed, with max. value of crp (mg/l) • at . /zg and • at /tg (r= . , p< . ), and max. value of serum amyloid a (rag/l) at . /zg of • and at #g + (r= . , p< . ). after ct c (no c= ) were evaluable upto dose #g, in these e rhll- related side effects were: fever ( %), headache ( %), myalgia ( %), local erythema ( %), nausea ( %) and increase in liver enzymes ( %). uptu c there was no difference in platelet nadir, moment and duration of platelets < x /l between . - /~g and . - #g doses. the same was observed for leukocytes. in conclusion, rml- upto #g/kg/d has a acceptable toxicity profile and results, before ct, in dose dependent platelet and acute phase proteins increase. after ct, evaluable now for the first cycle, there is a faster platelet recovery at higher dose steps. university hospital groningen, division of medical oncology, department of internal medicine, oostersingel , ez gronlngen, the netherlands. g-csf in acute lymphoblastic leukemia. d.hoelzer, o.ottmann rhg-csf given in acute lymphoblastic leukemia (all) after induction/consolidation therapy or allogeneic/autologous bmt can shorten the recovery time of neutrophils by - days, with reductions of the infection rate in some studies. granulocytopenia is the major dose limiting toxicity not only after but also during chemotherapy. for example, severe neutropenia occurs in ~ of patients during the intensive wk induction regimen of the german all trials. therefore, the effect of parallel administration of g-csf (filgrastim) and chemotherapy was explored, rhg-csf ( ~g/kg) was given concurrently with cyclophosphamide, arac, -mercaptopurine, prednisone and prophylactic cranial irradiation, rhg-csf was continued until neutrophil counts exceeded /~i, but for at least seven days following the last dose of chemotherapy. in a pilot study of pts, the median duration of granulocytopenia < /~i was days compared to days in a historical control group. when rhg-csf was applied throughout the entire wk induction treatment (scherrer et al) in pts, the cr rate was %, the tim~ of nm,trophil reeoverv to > ~o~l~l after the end of chemotherapy was reduced from , days to , days, and the infection rate was lower in the g-csf treated patients. from these studies it was concluded that the concomitant administration of rhg-csf and intensive chemotherapy in adult all is leasable, well tolerated and without major side effects; in particular, there was no evidence of prolonged neutropenia due to stimulation of hemopoietic stem cells by g-csf and their subsequent elimination by chemotherapy. the clinical effects of rhg-csf given concomitantly to chemotherapy require confirmation in a randomized trial. in an ongoing randomized multicenter trial~ an interim analysis of pts revealed a highly significant reduction of the total duration of severe neutropenia (< /~l) in the patients receiving g-csf. further analysis of the potential clinical benefits of this treatment modality is in progress. we performed a phase h trial to assess the ability of g-csf -mobilized pbpc to rapidly and completely restore hemopeiesis after high dose chemotherapy in the absence of bone marrow infusions, with selection for pbpc-only infusions based on yield of granulocyte -macrophage colony -forming cells (gm-cfc) after g-csf treatment. twenty-nine adults with acute lymphoblastic leukemia, non-hodgkin's lymphoma, or hodgkin's disease who were eligible for autologons bone marrow transplantation were treated. g-csf was given at or ixg/kg/d for - d and mononuclear cells collected by apheresis on days , , or , , . yield of pbpc was assessed by assay for granulocyte-macrophage colony forming cells (gm-cfc). high yield was defined as total gm-cfc collected > x /kg. high dose bnsulfan ( mg/kg/d x d) an& cyclophosphamide ( mg/kgtd x (: ) were administered and hemopoiedc cells infused• and recovery of hemopoiesis mouitored. progenitor cell yield was high in of patients. patients given infusions of g-csf-mobilized pbpc, but without bone marrow infusion, experienced recovery of hemopoiesis in all cases. no patient given pbpc alone required bone marrow infusion in up to months of follow up. kinetics of recovery of both the platelet and neutrophil counts were more rapid in the high yield (pbpc-alone) group than in the low yield group (pbpc pins bone marrow). pintelels recovered to > x / at a median of days and neutrophils to > . x / at days in the high yield group, compared with days and days in the low yield group (p= . and . respectively). fewer platelet transfusions were required in the high yield group (median packs per patient vs. , p--- . ). we conclude that in patients with a high yield of pbpc after g-csf therapy, infusion of g-csf-mobilized pbpc results in rapid and sustained restoration of hemopoiesis. use of g-csf mobilised pbpc to support multiple cycles of hdc for treatment of high-risk stage ii and iii breast carcinoma is now being examined in a phase i study pursued by our group. our data on pbpc moblisation without chemotherapy allows consideration of g-csf-mobilised pbpc for haemopoietie ceil allotmnsplantation and for gene therapy trials. our approach for high-dose (hd) chemotherapy is to first treat patients eligible for dose intensification with a standard dose chemotherapy (vip: vp = etoposide: mg/m , ifosfamide: g/m , cis-platinum: mg/m ) followed by the application of colony stimulating factors (g-csf, gm-csf or il- + gm-csf) in order to combine a regimen with broad anti-tumor activity with the recruitment of peripheral blood progenitor cells (pbpcs). pbpcs are reinfused into the patients after high dose intensification chemotherapy (according to underlying disorder: hd-vip, vic-e or beam). highest numbers of pbpc were recruited by the sequential administration of _- + gm-csf (median of cd + cells/iji and . total progenitors/ml, including multilineage as well as megakaryocytic progenitors). csf-mobilized pbpcs include lineage negative cd + cells, -hc resistant precursors as well as long term culture initiating cells (ltc-ic). so far, a total of patients were supported with pbpos and csfs following hd-chemotherapy. the period of severe neutropenia (< //ji) as well as thrombocytopenia (< . o//ji) was reduced to a median of or days respectively. in order to decrease the number of potentially contaminating tumor cells in the leukapheresis preparations, we have started to positively select cd + cells by an avidin-biotin immunoadsorption column (provided by r. berenson, cellpro, usa). in all these patients, recovery data were comparable to the patients who received unseparated cells. to provide sufficient numbers of pbpcs for repetitive use or in patients with low progenitor cell yield as well as to possibly avoid leukapheresis, we investigated the ability of hematopoietic growth factor combinations to expand pbpcs ex vivo. chemotherapy + g-csf recruited cd + cells from patients were enriched by immunoadsorption (purity: . -+ . ) and cultured in suspension. a combination of stem cell factor, erythropoietin, interleukin- , il- , and il- was identified as the optimal combination for the expansion of clonogenic progenitors. proliferation peaked at day with a mean -fold increase (range - ) of clonogenic cells. interferon-gamma synergized with the -factor combination, whereas the addition of gm-csf or g-csf decreased the number of clonogenic cells. large-scale expansion of cd + cells in auto-iogous plasma supplemented with the -factor combination resulted in an equivalent expansion. our data indicate the feasibility of large-scale pro+genitor cell expapsion in cancer patients, starting from small numbers of cd ceils. given the pressures on health care resources new health technologies need to be assessed to establish whether their benefits justify their costs. g-csf is an important new technology that has profound impacts on cancer chemotherapy and patients outcome. as clinical studies show, the main economic benefits of g-csf come from * a significant reduction of hospitalisation during chemotherapy, * reduced expenditure on inpatient and outpatient i.v. and oral antibiotic treatment, * and reduced platelet and rbc transfusions. in addition, it is expected that a therapy with g-csf during cancer chemotherapy shows intangible and non-monetary benefits: * increase in the rate of treatment as intended (time, number of cycles) * increase of quality of life of patients * reduction of drop-outs * ability to intensify dosage recently coducted economic cost-cost studies show that the cost of managing neutropenia and infection and the cost of chemotherapy are lower for patients receiving g-csf than those who do not. future studies have to focus mainly on the quality-of-life cost-effectiveness of g-csf. the aim of this study is to evaluate the effects of recombinant human erythropoietin (rhu epo) on hematopoietic regeneration after allogeneic or autologous bone marrow transplantation. patients were randomized to receive either u rhu epo/kg body weight or placebo as continuous intravenous infusion from day after bmt until independence from erythrocyte transfusions for days or until day . the randomization was performed per each center and stratified according allogeneic or autologous bmt and major abo-blood group incompatibility. at the time of the planned interim analysis with patients treated, the time to erythrocyte transfusion independence after allogeneic bmt was shorter in group a (n = ) than in group b (n = ). after autologous bmt no difference between group a (n = ) and b (n = ) could be detected so far concerning time to transfusion independence or the number of transfusions applied, considering either erythrocyte or thrombocyte transfusions. there were no major differences in side effects between groups a and b. as of october , the study was finished with a total of patients. since rcc patients have been screened for entry into clinical trials and entered onto surveillance as first management policy until symptoms or serial radiology indicated tumour progression. + ( %) have demonstrated unexplained "spontaneous" cr+pr(median duration mths), and ( %) "stable" disease(median mths). none of not nephrectomised regressed, while cr+pr was % of nephrectomised at diagnosis of metastasis, % of developing metastases - mths after nephrectomy and % of developing metastases more than mths after nephrectomy. it was % of with lung only verses % of with other sites. possible evidence for relief of potentially immunosupressive influences have been demonstrated in of patients demonstrating unexplained "spontanous regression. study of hla class i and ii antigen abnormally on these tumours and their influence on tumour infiltrating lymphocytes will be reviewed. patients seen to progress on surveillance were entered into treatment trials, the remainder being too old, sick or having bone or brain metastases needing radiotherapy. + ( %) achieved cr+pr. + of ( %) of patients with lung only verses + of ( . %) of those with other sites of metastasis. although these results suggest that no harm has come from a period of preliminary surveillance, the fact that the therapeutic benefits including durable complete remission from therapy are confined almost entirely to the good risk small volume asymptomatic patients makes it difficult to justify a policy of surveillance for such patients. auto bmt, as alternative of allo bmt, lack{ immunotherapical effect delivered by allo graft. however immune reconstiturion after auto bmt shows features recalling the post allo setting. futhermore in vitro and in vivo in rll is able to stimulate the proliferation and the lyric functions of nk and t cells, major effectors of this gvl effect. for these reasons from , we have so far treated patients with acute leukemia (aml- ;all: ) in cr with auto bmt followed with rll . all conditioning regimen included tbi. median rime between diagnosis and bmt was r~. mths ( - mths).arll was started as soon as platelets reach x ~/ and anl . x ~/ . rll was given in cycles of days (c ) and days (c to c ), starting on d ,d ,d ,d ,d ~. rll was given as a continuous infusion at a median of m iu/m~/d ( m- m). no patient died of rll related toxicity. platelet toxicity was obvious during the starting cycle but did not impair the long term hematological recovery. immune stimulation was major and intense for both nk and t cells and both lak and nk activity (all p < . ). long term infusion conducts to privilegiate nk stimulation. with a median follow-up of mths, relapse and survival probabilities are respectively % and % fo all and % and % for aml comparying favorably with historical control ; these data invited us to set up a randomised study in cr aml and all after auto bmt. started in in france, italy and england, pts have been so far included. we have shown that local, inhalative il- application in combination with low dose systemic il- and ifna is highly effective and has low toxicity (j urol , , ) . here we report about longterm follow-up from to month of an outpatient schedule in patients with pulmonary metastasis of renal cell carcinoma. treatment protocol based on time daily inhalation of il- ( x . i j) combined with low dose i.v. il- ( x u/ days every weeks) in patients or low dose il- s.c. injection of . u per day in patients. all patients received x s.c. ifna three times weekly. median treatment duration was month (range - month). toxicity of il- inhalation was low, no fever, no vasculary leakage. even after up to month of continuous inhalative treatment no evidence for irreversible pulmonary damage due to il- induced immunomodulation occured in patients. in pulmonary metastases complete response ( month), partial response ( , , , , + , , , , ) and stable disease ( , , , + , , , +) were achieved. of patients responded with nonpulmonary metastasis. overall tumor response considering both, pulmonary and nonpulmonary metastases was complete response, partial responses stable diseases, mixed responses and progressive disease. while il- per inhalation had to be stopped because of grade ii toxicity in patient only, ifna systemically had to be stopped in of and il- i.v. cycles in of patients. patients are alive with median actual survival of all patients of . month which seems to be prolonged compared to risk factors. inhalafive treatment with il- combined with low dose systemic cytokines represents a highly valuable model for the low toxicity of lccal il- application, resulting in an effective long term treatment schedule with long term responses. recent clinical trials for the biological therapy of solid tumors have used recombinant human cytokines in combination with conventional chemotherapy. in patients with metastatic renal cell carcinoma, we established a -drug combination of subcutaneous recombinant human interferon-u (ifn-u), interleukin- (il- ) and -fluorouracil ( -fu) as outpatient regimen. treatment consisted of eight weeks each of ifn-~ ( million u/m' x per week sq) combined sequentially with il- ( - million iu/m' x per week sq for four weeks) and -fu ( mg/m iv weekly for four weeks). among consecutive patients treated, there were complete ( . %) and ( . %) partial responders, with an overall objective response rate of . % ( % confidence interval, - %). median response duration was calculated at + months, and no relapse has occurred among complete responders. systemic toxicity was mild to moderate, with no severe -fu related mucositis or diarrhea. there were no dose limiting adverse effects of sq il- and no toxic deaths. in summary, this outpatient biochemotherapy was as effective as the most aggressive inpatient iv il- regimen; it appeared to significantly improve the therapeutic index in patients with metastatic renal cell carcinoma. medizinische hochschule, d- hannover, germany metastatic melanoma: immunotherapy with ifna and il- , dtic/ifna as second line treatment, ifne(/il- + dtic or cddp. u keilholz, c scheibenbogen, w tilgen, d maclachlan, p brossart, th m hler, w hunstein. this abstract summarises our -year experience in the treatment of metastatic melanoma with sequential or combined chemo-immunotherapy. patients with progressing metastatic melanoma have been treated with ifna and il- . mill u/sqm ifn(~ were given s.c. on days - , and a new decrescendo regimen of il- was used: lmg/sqm/ hours, followed by mg/sqm/ hours, and mg/sqm/ hours, and . mg/sqrrv hours x . the current response rate is % ( cr, pr, mr/sd, pd). patients not responding to ifnedll- (sd and pd) were eligible for subsequent chemotherapy with dtic, mg/sqm day , followed by ifna, mill u/day - . the response rate for this second line regimen is % ( cr, pr, sd, pd, n= ). using this sequential approach, the overall response rate in this cohort is %, and the median survival is months. in preparation of a randomized trial comparing chemo-immunotherapy and immunotherapy a pilot study was performed. patients not responding to the standard ifnedll- regimen received a single dose of dtic, mg/sqm (n= ) or cddp, mg/sqm (n= ) on day one, followed by ifn~il- according to the identical protocol as previously without chemotherapy. in the case of cddp, grade nephrotoxicity was observed in / patients. pharmacokinetics of il- were not influenced by previous chemotherapy, except in the patients with cddp-associated nephrotoxicity. induction of secondary mediators (tnfa, ifn'~, neopterin, scd ) by il- was not diminished by previous chemotherapy. patients unresponsive to immunotherapy alone showed tumor shrinkage upon chemo-immunotherapy. conclusion~: with initial immunotherapy followed in nonresponders by chemotherapy a response rate above % is achieved. combined chemoimmunotherapy is feasible, and the immunologic response to il- is not diminished by previous chemotherapy. a randomised trial is necessary to determine, whether combined chemo-immunotherapy is superior to immunotherapy alone. inflammation is characterized by an accumulation of leucocytes at the site of injury. it hab been well established that the disease associated lesions are caused by a plethora of soluble mediators released by both the infiltrated leukocytes as well by tissue cells, including many degrading enzymes, lipid mediators, reactive oxygen species, or cytokines. among those, cytokines play a crucial role in the inflammatory process as their release appears to represent the initial response which mediates the secretion of subsequent effector molecules responsible for the pathophysiological effects (e.g. changes of vascular resistance) in an autocrine or paracrine fashion. this holds true for the acute inflammation during which circulating granulocytes or monocytes are activated directly by a noxious agent; an example being the pivotal function of tumor necrosis factor (tnf) in septic shock. chronic inflammatory diseases are initiated and perpetuated by immune reactions. cytokines represent an important link between immune reactions and the recruitment and activation of blood borne infiltrating inflammatory ceils. thus interferon r and tumor necrosis factor , secreted by activated t lymphocytes, are potent stimulators of mononuclear phagocytes. the activated macrophages themselves secrete cytokines such as interleukin-t (il-t), tnf a or interleukin- (il- ) which have been termed collectively as inflammatory cytokines, il- and tnf again in an autocrine or paracrine way upreguiate the synthesis of the ultimate pathogenic mediators in macrophages-e.g. the secretion of degrading enzymes as in rheumatoid arthritis-. equally important they induce functional changes in vascular and autochthonous tissue cells which are thus recruited to contribute to the inflammatory lesions. il- , and similarly the lymphocyte derived rantes, chemotactically attracts to the site of lesion and activates granulocytes, which then participate in the inflammatory reaction. while this rapidly recruits circulating phagocytic cells, the secretion of colony stimulating factors by activated t lymphocytes and macrophages also increases the pool of granulocytes and monocytes by stimulating their synthesis in the bone marrow. in turn il- an tnf a synthesized by monocytes (and also some tissue cells) are coactivators of t lymphocytes, and thus contribute to al local immune reaction. besides this general role of cytokines in inflammation their involvement in specific situations becomes increasingly apparent, an important example being the allergic inflammation. cytokines synthesized by t lymphocytes such as interleukin- or interleukin- not only regulate immunogtobulin synthesis of b lymphocytes, including ige, but equally important modulate the functions of mast cells or eosinophilic leukocytes to become effective effector cells of allergic reactions. the detailed functions of cytokines in different acute or chronic inflammatory diseases will be discussed in the subsequent contributions. clinical results e holier, r hintermeier-knabe, b ertl, hj kolb, m kaul, u behrends, s thierfelder, w wilmanns experimental as well as clinical studies suggest dual involvement of proinflammatory cytokines as tnfalpha and ill in complications and gvhd following allogeneic bmt: nonspecific activation of recpient tissues during pretransp~ant conditioning results in early release of tnfa which strongly enhances donor t cell activation; in the effector phase, t cell stimulation is amplified by ifngamma and lps mediated release of tnf and ill. the role of these mediators as critical effectors of gvhd related tissue damage could be confirmed by application of cytokine antagonists early after murine bmt: both, anti-tnfa and ill-receptor-antagonist (illra, as shown by j ferrara, boston) resulted in a > % reduction of gvhd related mortality in mice, while pentoxifylline proved to be ineffective. in human bmt, phase l/ll studies analyzing anti-tnfa and illra in refractory gvhd report substantial improvement in - % of patients, though reoccurrence of symptoms after cessation of treatment in most patients indicates a late effector function of cytokines at least in advanced gvhd. contribution of tnfa to recipient related induction of gvhd is further suggested by a recent phase / study performed in our institution as prophylactic application of anti-tnfa during pretransplant conditioning suppressed occurrence of early gvhd in high risk patients as compared to historical controls. though these data indicate a future role of a variety of cytokine antagonists in management of clinical complications of bmt their impact on long term survival has to be evaluated and compared to classical strategies (e.g.the use of corticosteroids and t-cell-manipulation) in randomized studies. the cytokines il- and tnf play central roles in inflammatory responses which can lead to tissue injury, naturally occurring antagonists such as soluble cytokine receptors or receptor antagonists are also produced during inflammatory responses. these soluble receptors and antagonists may act as a buffer system to reduce and limit tissue injury induced by cytokines. however, in some disease states this naturally occurring buffer system may not be sufficient to inhibit the detrimental actions of an inflammatory response. responses to il- are mediated via the type receptor (il- r, type i). the type ii receptor (il- r, type ii) has never been shown to signal, but instead appears to be shed as an il- antagonist. both recombinant soluble il- r, type i an il- r, type ii are capable of binding il- and inhibiting responses in vitro. in a phase i clinical study evaluating soluble l- r (type ) in modifying cutaneous allergic responses, il- r was a potent inhibitor of allergen induced latephase inflammation in the skin, with a high safety profile. responses to tnf are mediated via the ps or pt tnf receptor. soluble forms of both the p and tnfr occur naturally and are increased in many inflammatory states. dimeric constructs of the p tnf have been engineered to form a soluble fc fusion protein with two monomeric tnfr extraceuular portions contained on a truncated ig heavy chain (tnfr:fc). the tnfr:fc possesses higher affinity than a monomeric soluble receptor and the ig-like fc structure imparts a longer half life. animal studies have shown that soluble il- r, type i and tnfr:fc are effective antagonists of inflammation. in animal models for arthritis and pulmonary inflammation, il-ir and tnfr:fc reduced inflammatory celt infiltration and tissue damage. both molecules are currently in clinical trials and hold promise for treatment of autoimmune and allergic diseases such as rheumatoid arthritis and asthma. recently, evidence was raised that pentoxifylline (pof} is able to suppress the synthesis of tumor necrosis factor-alpha [tnf} in cell cultures, in vivo, and to protect experimental animals against endotoxin shock: it was found that pof selectively inhibits the formation of tnf but not interleukin- (il- ]. we could confirm these pharmacological effects of pof in humans under controlled conditions of endotoxlnemia. ten healthy volunteers recieved a bolus injection of endotoxin [ ng of lps of s.a.e.], which caused a transient increase of circulating tnf and il- . weeks later the voluntecrs were again injected with endotoxin and pof was also infused. due to pof administration, there was no rise in circulating tnf, whereas il- levels rose in parauel with body temperature, comparable to those seen in the first part of the study. treatment of allograft transplant recipients with the murine anti-cd monoclonal antibody okt leads to an acute cytokine release syndrome. especially tnf seems to play a pivotal role in the pathophysiology of the okt first-dose reaction. pretreatment of patients with pof prior to okt administration was able to reduce the endogetlous tnf formation significantly as compared to controls and, thus, prevents severe clinical side effects, whereas il- formation and febrile response were not affected. severe pulmonary tuberculosis is associated with a chronic cytokine release syndrome [elevated levels of circulating tnf and il- ]. in patients pof treatment inhibited chronic tnf release selectively, and, thus, reduced tnf-dependent caehexia without affecting chronic il- formation and related symptoms, such as fever and night sweat. in conclusion, we suggest that pof may improve therapeutic strategies in cases of acute and chronic cytoklne release syndromes. tumor necrosis factor (tnf) plays a central role in the maintenance of the inflammatory events in rheumatoid arthritis. we evaluated the expression of p and p tnf receptors (tnfr) and of tnf-a and tnf-i~ on the surface of synovial fluid mononuclear cells in patients with rheumatoid arthritis (ra) (n = ), spondylarthropathy (spa) (n = ), and traumatic effusions (n= ). synovial t-lymphocytes from ra patients express in all cases the p tnfr on the cell-membrane, in / cases also a weak expression of the p tnfr is detectable, both mrnas can be detected by polymerase chain reaction (pcr). synovial macrophages also express the p tnfr and low amounts of the p tnfr. patients with active ra also have circulating p tnfr positive t-lymphocytes in their blood. high concentrations of soluble tnfr (stnfr) are found in the joint effusions of ra patients: up to ng/ml of p stnfr and ~jp to ng/ml p stnfr. significantly lower stnfr levels are found in spa effusions. both receptors are also more elevated in the serum of ra patients ( . _+ . ng/ml p stnfr and . + . ng/ml p stnfr) as compared to spa patients ( . _+ . ng/ml p stnfr and . + . ng/ml p stnfr, p < . ). tnf-a could be detected in the synovial fluid of ra patients (up to pg/ml), but not in the serum. the soluble tnfr are biologically active and neutralize the effects of tnf-a in a cytotoxicity assay. the high levels of soluble tnfr in the inflammatory effusions may reflect tnf neutralizing activity in an environment where enhanced tnf synthesis has occured. we have generated several anti-sense-tnf-a oligonucleotides (as-tnf), in order to down-regulate tnf biosynthesis at the mrna level. with as-tnf- we could achieve more than % inhibition of tnf secretion in pha-stimulated peripheral blood or synovial fluid lymphocytes. the effects of as-tnf- on the expression of tnfr and on the synthesis of other cytokines are currently being investigated. to investigate the effects of g-csf (r-methug-csf, aragon, thousand oaks, ca) on neutrophil production, blood distribution, survival and function, daily subcutaneous injections of g-csf were administered to healthy, young (y) ( - years) and healthy elderly (o) ( - years) subjects for days at three dose levels, mcg (n= ), mcg (n= ), and mcg (n = ). daily cbcs and serial measurements of neutrophil oxidative activity by chemiluminescence were made. in addition, blood neutrophil kinetics were determined (day ), transit time of the marrow neutrophil post mitotic pool (ntt) (days to ), neutrophil migration to skin chambers (days and ), and blood colony forming cells (cfu-gm) (day and ), as well as routine marrow morphology studies (days , and ) were performed. baseline neutrophil counts were similar in the y and o subjects and counts increased similarly for the two age groups, with peak counts of . --+- . for the mcg dose. g-csf significantly shortened the ntt from . - . days (control) to . - . days ( mcg) and . - . days ( mcg) (p< . ). a concomitant of the shortened ntt was a dose dependent increase in the chemiluminescence responses, reflecting higher myeloperoxidase activity. the shortened ntt was also reflected by a decreased proportion of marrow cells in the post mitotic pool (metas, bands and pmns) and apparently lengthened blood half life of the circulating pmns. neutrophil migration to cutaneous inflammatory sites was also decreased in a dose dependent fashion. comparison of the age groups showed the only significant difference to be in the mobilization of blood colony forming cells with blood cfu-gm increasing fold in the young versus fold in the elderly over days ( mcg). no significant toxicities were observed in these normal subjects. these studies demonstrate the dose dependent stimulation of neutrophil production with g-csf administration, which is not affected by the age of the subjects. the neutrophils released into the peripheral blood have enhanced oxidative responses but somewhat reduced migratory capacities, probably reflecting the accelerated production and early release of the developing neutrophils. these changes are remarkably similar to changes in neutrophil production and function observed with bacterial infections in hematological normal individuals. the systemic regulation of the host response during acute inflammatory states remains poorly understood. among the regulatory systems that are likely to play a role in controlling host responses to bacterial infection is the neuroendocrine axis. the pituitary for example, is ideally situated to integrate central and peripheral stimuli and among other effects initiates the systemic increase in glucocorticoid production that accompanies host stress responses. we studied the secretory response of the murine pituitary cell line att- in vitro and whole pituitaries in vivo after endotoxin (lps) stimulation. we identified macrophage migration inhibitory factor (mif), a previously described t-cell cytokine, as a major secreted protein of att- cells that were stimulated by sub-nanogram amounts of lps. to study the expression of pituitary-derived mif in vivo, balb/c mice were injected ip with sub-lethal amounts of lps. pituitaries and serum were collected at intervals and pituitary mif mrna and pituitary and serum mif protein were measured. pituitary mif mrna specifically increased with time and reached a plateau - hr after lps challenge. mif protein, which is present constitutively in the pituitary, decreased within hr. determination of serum mif in normal, athymic and hypophysectomized balb/c mice indicated that the pituitary is an important source of serum mif that appears in the post-acute phase ( - hr), whereas t cell mif contributes primarily to the hyper-acute rise in serum mif ( hr). these data suggest that mif plays a central role in the systemic response to endotoxin and that pituitary mif is likely to reflect the interplay of diverse neurohumoral stimuli that regulate acute and chronic inflammation. pretreatment of mice or rats with granulocyte colony-stimulating factor (g-csf) protected against endotoxin-induced lethal shock or against endotoxin-induced liver injury in galactosamine-sensitized animals. in the animals protected by such pretreatment, the systemic tumor necrosis factor r (tnf) bioactivity was significantly suppressed. various macrophage populations taken ex vivo from g-csf-pretreated animals showed an attenuated tnf release following lps stimulation. however, g-csf had no such effects on macrophages when directly added in vitro to the cells. these findings show that g-csf protects against septic shock via tnf suppression in a way requiring the participation of additional circulating cells or factors. pretreatment of rodents with granulocyte macrophage colony stimulating factor (gm-csf) greatly enhanced the susceptibility of the animals to endotoxin and led to a tremendous increase of the systemic tnf found following an lps challenge. an anti-gm-csf antibody significantly protected animals against septic organ failure. considerable amounts of endogenous gm-csf are released following endotoxin chaitenge with a maximum at h. the enhancement of lps-inducible tnf release from rnacrophages takes also place in vitro. it is concluded that gm-csf is a pivotal mediator of sepsis as well as a directly acting enhancer of lpsinducible tnf release. we have previously demonstrated that protein production and mrna expression of gm-csf, g-csf, and il- were decreased in activated mnc from umbilical cord (c) compared with adult (a) peripheral blood (cairo, et al, pediatr res : , , and : , ) . rhg-csf + recombinant rat stem cell factor (rrscf) administration in newborn rats has, however, resulted in a significant induction of neutrophilia, an increase in bone marrow post-mitotic pool, and is synergistic with antibiotics during experimental group b streptococcal sepsis (cairo, et al, blood : , , and : , . to assess the toxicity and efficacy of rhg-csf in newborns with presumed sepsis, nb < days ( - wks) with a diagnosis of presumed sepsis were randomized to either placebo (p), . , . , or . pg/kg q hrs, . or . /lg/kg q hrs of iv rhg-csf x days. cbc, differential, and platelet counts were obtained at time , , , , , and hrs. tibial bone marrow aspirates were performed at hrs and bone marrow nsp, npp, cfu-gm, and cfu-gemm were determined. serum for g-csf levels was obtained before and , , , , , , and hrs after rhg-csf dosing and measured by a sandwich elisa assay. rhg-csf induced significant neutrophilia at hrs vs. placebo ( + %) following both . ( + %, p< . ) and . ijg/kg/d ( -+ %, p< . ). significant neutrophilia continued at and hrs at both . (p< . and < . ) and h'g/kg/d (p< . s and < . ) respectively. rhg-csf also significantly induced a dosedependent increase in the bm post-mitotic pool (p vs. ijg/kg/d) ( +- . vs. + . %) (p< . ). t /z of g-csf in the nb was . _+ . hrs (r= - . ) with peak g-csf levels occurring within hrs. to date there has been no evidence of acute or chronic toxicity secondary to rhg-csf. future studies are required to determine the clinical implications of the biological efficacy of rhg-csf in newborns with presumed sepsis. children's hospital of orange county, orange, california usa depenbrock, t. block, h. vogelsang, ch. fellbaum, j. rastetter, a.-r. hanauske tgf-po is known to function both as inhibitory and stimulatory factor depending on the type of cell line investigated. the purpose of our study was to determine the effects of tgf-~ and tgf- (concentrations: . - - ng/ml) on soft agar colony formation of freshly explanted i~uman tumors in vitro. of specimens, had to be excluded from further analyses ( confirmed benign, bacterial/fungal contamination). of the remaining tumors showed evaluable growth in control capillaries ( %). at ng/ml, tgf-po inhibited colony formation (_< . x control) in specimens ( %): / renal, / non hodgkin's lymphoma, / breast, / ovarian, / melanoma (median: . x control, range: . - . ). at ng/ml, tgf-e,= showed inhibition of colony formation in specimens ( %) with a similar spectrum of activity (median: . x control, range . - . ). stimulation (colony formation _> . x control) was observed in only / specimens. combination of tgf-po with epidermal growth factor (egf) reversed the inhibitory activity in / specimens ( %). combination of tgf-p~ with platelet derived growth factor (pdgf) reversed the inhibitory activity in / specimens ( %). similar results were observed for tgf z. we conclude that tgf-i~ and tgf-i~ inhibit a subgroup of freshly explanted clonogenic tumor cells. their activity, however, appears to be modulated by other growth factors. a korfel. z yon marschall. d ol~erbera. e thiel. and we berdel mip-i~ is a member of a family of proinflammatory cytokines produced by activated macrophages which has been shown to be a negative regulator of early hematopoietic stem cell progenitors. our group investigates the interactions between hematopoietic cytokines and non-hematopoietic malignant cells. here, we describe results testing rhmip-lc~ (rh stem cell inhibitor, sci; kindly provided by dr. wolpe, genetics institute, cambridge, ma, usa) on the clonal growth of different human non-hematopoietic tumor cell lines in vitro. cell lines tested included the following histologies: gtioblastoma x, neuroblastoma lx, head and neck carcinoma x, lung carcinoma lx, colorectal carcinoma x, gastric carcinoma lx, pancreatic carcinoma l x, breast carcinoma l x, bladder carcinoma lx, prostate carcinoma lx, choriocarcinoma lx, ovary carcinoma lx, osteosarcoma lx, melanoma x. mip- ( , , , ng/ml) was tested in human tumor cloning assays (htca) in mixtures of methylcellulose and agar. htca has previously been shown to detect positive and negative growth control by cytokines. plating efficacy of the cells in the controls was > % (median %, range . - . %) in this series of experiments: tumor cells were continuously exposed to the cytokine for the complete assay period. clonal growth of none of the celt tines was significantly and reproducibly stimulated or inhibited by mip-lm since mip-lc~ may enter clinical trials for indications such as protecting hematopoietic stem cells from damage caused by cytotoxic chemotherapy in tumor patients, further experiments should be performed in vitro and in vivo. dfg be / - klinikum steglitz, freie universitaet berlin, berlin, germany clinical studies have demonstrated the activity of single hematopoietic growth factors (hgf) in restoring bone marrow function after chemotherapy. these studies have prompted clinical trials using multiple growth factors to promote the maturation of precursor cells at various stages of differentiation. however, hgf also have the capability to stimulate growth of non-hematopoietic tumor ceils at least in long-term cell cultures. we have assessed the growth-modulating activity of combinations of various hgfs on freshly explanted human tumor colony forming units in vitro. a total of tumor specimens were exposed for - days to il- , gm-csf, g-csf, scf (a~l at final concentrations of ng/ml) and il- (final concentration: ng/ml) or combinations of these hgfs using a capillary soft agar cloning system. specimens ( %) showed evaluable growth in control capillaries. stimulation of colony growth was observed in / tests ( . %) with / ( %) specimens expressing sensitivity to individual hgfs, / ( %) to combinations of two hgf, and / ( %) to combinations of more than two hgfs. inhibition of colony growth was observed in a total of / tests ( . %) with / ( %) specimens expressing marginal sensitivity to individual hgfs, / ( %) to combinations of two hgf, and / ( %) to combinations of more than two hgfs. for inhibitory effects, median colony survival for combinations > hgfs was . x control (range . - . ). for stimulatory effects, median colony survival for combinations > hgfs was . x control (range: . - . ). our data indicate that combinations of hgf will not substantially alter the pattern of clonal proliferation of the majority of freshly explanted tumor cells in vitro. however, growth modulation may occur in a minority of tumors. klinikum rechts der isar der technischen universit&t m nchen, ismaninger str. , m nchen supported by grants w / /ha- - from the deutsche krebshilfe and . . from the withe~m-sander stiftung is granulocyte colony-stimulating factor an angiogenic factor in human glioblastoma? s. corbacioglu, k. welte and t. pietsch granulocyte-colony stimulating factor (g-csf) belongs to a family of glycoproteins winch regulate growth, differentiation and function of hematopoietic ceils of the myelomonocytic lineage. in addition, g-csf induces migration and proliferation of endothelial cells in vitro. to investigate the effects of g-csf on vascularization of glioblastoma in vivo we transplanted the human glioblastoma cell line u- mg which is capable of producing g-csf in high amounts. after s.c. inocculation of these cells into the backs of athymic mice, they developped highly vascularized solid tumors. in order to block the effect of g-csf directly or to inhibit the production of g-csf by tumor cells neutrolizing monoclonal antibody (mab) a against g-csf or dexamethasone were injected intravenously. at -day intervals the tumor volumes were measured. after seven days the mice were sacrificed and the tumors were explanted. blood was collected for differential blood counts and serum was tested for g-csf. fresh frozen sections of the tumors were specifically stained for capillaries using bandeiraea (griffoina) simplicifolia lectin i isolectin b (bsl b ). morphometric studies of the stained sections were performed in order to quantitate the vascularization of the tumors. the differential blood counts showed significantly increased neutropinls due to the effect of human g-csf produced by the glioblastoma cells. tins effect was inhibited by anti-g-csf mabs or dexamethasone. however neither g-csf mabs nor dexamethasone could inhibit tumm growth compared untreated tumor bearing mice. dexamethasone significantly decreased the tumor vascularization whereas anfi-g-csf mabs did not have any effects on tumor vascularization. these findings suggest that g-csf is not an essential angiogenic factor in vivo. pediatric hematology/oncology, medical school hannover, konstanty-gutschow-str. , harmover , germany michael martin, torsten spencker, karen welch*, and andreas strasser* the spgm cell line was established from a transplantable mouse progranulocytic/promacrophage tumor (d hrsen u et al. , leukemia : - ) . extensive phenotyping of this mouse progenitor line revealed the properties of a typical cd positive pre-b cell, spgm being positive for pb , b (cd r), and the pre-b cell immunoglobulin receptor complex, comprising p-heavy chains, xs-and vpree-surrogate light chains and the igma and igl~ co-receptor molecules. southern blot analysis revealed clonal rearrangement of the p-heavy chain locus and germline light chain loci. interestingly, spgm readily formed blast cell, macrophage and occasional granulocytic colonies in soft agar in the presence of interleukin (il- ). in suspension cultures il- also induced macrophage differentiation, the cells becoming larger, adherent and independent of -mercaptoethanol in the culture medium. il- induced an initial burst of proliferation during differentiation, accompanied by loss of self renewal capacity, subsequently followed by a decrease and cessation of proliferation. the earliest changes were detectable at hours by northern blot analysis. il- treatment increased mac mrna, induced cfms mrna (m-csf receptor) and down regulated mrna for p, x , vpree, and mbl. after to days the cells morphologically, phenotypically and functionally resembled macrophages, expressing strongly mac and f / , and phagocytosing latex beads (martin met al. j.immunol. in press). thus, il- induced the cd positive pre- cell line spgm to switch its differentiation program in a coordinate fashion from a pre-b cell to a macrophage. igf is known to be mitogenic for a variety of cell lines in vitro. we have studied the effects of insulin-like-growth-factor i [igf-i] and insulin-like-growth-factor ii [igf-ii] on freshly explanted human tumors using a capillary soft agar cloning system. specimens had to be excluded from further analyses ( confirmed benign, bacterial/fungal contamination). / specimens ( %) showed adequate growth in controls ( renal, breast, lymphoma, colorectal, miscellaneous) with a median colony formation of . colonies/capillary (range: . - . ). at final concentrations between lr m and . m, igf-i significantly stimulated colony formation (_> . x control) in / evaluable specimens ( %) with a median of . x control (range: . - . ) and inhibited colony formation ( _< . x control) in / specimens ( %, median: . x control, range: . - . ). igf-ii stimulated / specimens ( %, median: . x control, range: . - . ) and inhibited / specimens ( %, median: . x control, range: . - . ). the optimal concentration for stimulation was found to be -~ m for igf-i and igf-ii. of specimens not significantly stimulated by either igf-i ( g m) or epidermal growth factor (egf, ~ m), ( %) were significantly stimulated by the combination of the two factors. ( %) specimen was stimulated by a combination of igf-ii ( -~ m) and egf. we conclude that igf modulates the clonogenic growth of a subgroup of freshly explanted human cancer cells in vitro. the myeloid growth factors g-csf and gm-csf are increasingly introduced in therapy trials in neutropenic disorders and in mds. in a series of therapy trials . % up to . % of patients treated with gm-csf displayed a stimulation of blast cells (antin , estey , ganser , vadhan-raj . recently, we could demonstrate a growth advantage for monosomy -cells in gm-csf containing bone marrow cultures (haase, ) which is supported by results from others (andreeff ). now, further data are available corroborating an association of monosomy and leukemic blast stimulation by myeloid growth factors. in a cytogenetic follow-up study of patients with mds under gm-csf (in preparation) we could observe a cytogenetic, clinical, and cytologic progression in a patient with monosomy within days. in a patient with kostman's syndrome, receiving g-csf, we performed sequential cytogenetic analyses. the patient's disease progressed to mds and finally to aml. he initially had a mosaic karyotypeof normal and monosomy cells but displayed a rapid karyotype evolution with supersession of normal cells and gain of additional abnormalities. a recent publication from a japanese group adds further information to a possible association of monosomy with stimulation of leukemia in % of neutropenic children treated with g-csf (kojima, ) . besides the need for further cytogenetic follow-up studies in growth factor therapy trials, data are accumulating that monosomy is a risk factor in gm-csf and g-csf therapy. ii- is a cytokine with multilineage activity and stimulates proliferation of immature hemopoietic progenitors (yang, ) . recently, il- has been introduced in clinical trials in mds (dunbar, ; ganser, ) . as in gm-csf therapy one major concern attributes to an il- mediated stimulation of leukemic cells, since a rise in blast counts has been observed in two of patients reported (ganser, ) . however, as yet it is not known whether the blasts stimulated belong to the normal or leukemic population. we performed cytogenetic analyses of bone marrow cultures with and without addition of i ng/ml il- (behringwerke, marburg). the influence of this cytokine on the clonal compostion in patients ( anll, mds) with mosaic karyotypes was examined. in all patients independent from diagnosis and chromosome abnormality the normal cell population was promoted by il- with different intensity. individual data are outlined on the the effect of stem cell factor (scf) on peripheral blood b-cll-celis were studied in vitro by bromodeoxyuridine/propidiumiodide (brdu/pi) cell-cycle-analysis. peripheral blood cells from patients with b-cll were examined with cd-markcrs and prepared as follows: after ficoll centrifugation and lysis of monocytes by leucine-methyl-ester (lme) t-lymphocytes were depleted with a cd monoclonal antibody and magnetic cell sorting. the cells were grown in serum free culture medium (cg-medium) containing #mol/l brdu, and . ng/ml up to ng/ml scf. controls were grown without cytokines. samples were drawn repeatedly at , , , , and hours. cell-cycle-analysis was performed after double dna staining with propidiumiodide and anti-brdu-antibodies and determinated by flow cytometry. minimal alterations were observed with t-cell depletion, the b-cll cells from patients were stimulated by scf during the first hours reaching a maximum of - . % compared with the controls after hours in cultures containing ng/ml scf. on the other hand, cultures without t-cell depletion showed no effect for scf. we conclude that scf has only a minimal stimulatory, early effect in inducing the proliferation of b-cll cells. stem cell factor (scf) is known to promote proliferation of hematopoieticprogenitors and mast cells.however, the spectrum ot its biological effects is not completely understood. since scf may be able to accelerate hemopoietic recovery after chemotherapy or in other situations where severe immunosuppression is present, we were interested in its effects on t cells. thus, we have studied the influence of human recombinant scf on t cell proliferation invitro. when cultured for days in serum-f~ee medium, h-thymidine incorporation of unstimulated peripheral blood mononuclear ee ells s (pmnc) resulted in -+ epm without growth factors, +_ clam with .- ( ng/ml), +_ cpm with scf (lon~/ml), and _+ c.m with both . and scf (n=fi~ addition of scf to one way" mixed lymphocyte cultures (mlc) increased thymidine incorporation by % ( - %); addition of scf plus id increased thymidine incorporation by % ( - %; id alone %, n= ). after depletion of monocytes and the majority of b cells from pmnc, the proliferation of the remaining eeu fraction, which consisted mainly of t cells, was not enhanced by id , scf or il- + scf. we conclude that scf can promote proliferation of unstimtdated or allostimulated t cells in the presence of id . since this effect requires monoeytes or other accessory ceils, a direct influence of scf on t cells does not become evident from our data. it is, however, still unclear whether ltb acts in this regard directly or indirectly by stimulating the release of chemotactic and inflammatory cytokines. here we report that ltb induces synthesis of interleukin (il)- by human blood monocytee through transcriptional activation of the il- gene. we furthermore demonstrate that this process involves activation of the transcription factor nf-rb and, to a lesser extent, of nf-il , white the activity of the transcriptior~ factor ap- , shown to otherwise confer il- inducibility, appeared to be unaffected by ltb . involvement of nf-~:b and nf-il in induction of il- transcripts by monocytes was demonstrated using deleted forms of the il- promoter. activation of the il- promoter by ltb was not only associated with accumulation of the respective transcripts but resulted in synthesis of functional il- protein as well. in addition, ltb mediated transcactivation of a heterologous promoter construct containing the nf-~:b or the nf-il enhancer, but not the ap- enhancer. the signalling events mediating this effect appeared to involve the release of h , since ltb failed to induce nf-rb or nf-il in the presence of the scavenger of h , n-acetyi-l-cysteine. both interleukin- (il- ) and granulocyte-macrophage colony-stimulating factor (gm-csf) have been previously identified to induce rapid phosphorylation of the map-kinase (blood : (blood : , . however, little is known about signalling events initiated by il- /gm-csf which occur downstream of the map-kinase. map-kinase has been shown to phosphorylate the ap- transcription factor and to activate two kinases designated insulin-stimulated protein kinase- (ispk- ) and mapkapkinase . we here report that il- and gm-csf induce mapkap-kinase activity in the human megakaryoblastic leukemia cell line mo e and phos-phorylate the human small heat shock protein hsp on serine residues. in contrast, neutrophils failed to phosphorylate hsp upon il- , while gm-csf induced hsp phosphorylation in a similar range as observed in mo e cells suggesting that mapkap-kinase mediated hsp activation occurs independently of proliferation. hsp phosphorylation is dose-dependent and occurs as early as minutes following exposure to il- or gm-csf. moreover, hsp phosphorylation is inhibited by tyrosine kinase inhibitors such as genistein or herbimycin a. in addition, we show that protein tyrosine phosphatase and protein phosphatase a (ppa ) interfere with the ability of il- or gm-csf to induce serine phosphorylation of hsp . taken together, our findings indicate that tyrosine phosphorylation of map-kinase is a prerequisite for serine phosphorylation of hsp mediated by mapkap-kinase . hsp is localized in the nucleus and has been linked to the cellular stress response. its precise function, however, is largely unknown. our data identify hsp as a target of il- /gm-csf stimulation via map-kinase and mapkap-kinase . further-more, our results indicate that hsp may also exert phosphorylation-activation functions involved in growth signalling pathways in unstressed cells. in recent years it has been shown that the mechanism of betalactam antibiotic-induced agranulocytosis involves a direct inhibition of the replicative dna polymerases alpha, delta, and epsilon. in this report we examine, as a representative of these antibiotics, the effect of freshly prepared ceftazidime (cef) and degradation products of cef on myelopoiesis. we investigated freshly dissolved cef and cef incubated for hours at + (cefd) on the production of myeloid cells in the supernatant (sn cells) and colony stimulating activity (csa) by the stroma. one week after drug treatment of the mltbmc a significan~ dosedependent inhibition of the myeloid cell production (xl ~ and csa (assessed by cfu-gm assay) occured, as summarized in the following we here report that human lung fibroblasts respond to x-ray treatment (xrt) with release of interleukin (il)- . synthesis of il- upon ionizing radiation is preceded by an increase of il- transcript levels resulting from transcriptional activation of the il- gene. analysis of deleted fragments of the il- promoter indicated that transcriptional activation of the il- promoter was due to enhanced binding activity of the transcription factor nuclear factor (nf)-~b. although activation protein (ap)-i did not participate in the rapid induction of the il- promoter, its binding activity was also enhanced by xrt. in contrast to binding kinetics observed with nf-~:b, ap- binding following xrt was biphasic with the second peak being dependent on de novo protein synthesis. in contrast, however, nf-il- activity was not enhanced by xrt in fibroblasts. the introduction of both the nf-~b-and the ap- recognition sequence, conferad inducibility by xrt to a heterolgous promoter, with reporter gene activity being maximal hours or hours following xrt, respectively. sequential acitvation of two distinct transcription factors might thus contribute to synchronize transcriptional activation of different genes participating in the x-ray (xr) response. on the basis of study of functional and morphological characteristic of bone marrow stromal tissue of human fetuses, - week gestation, and of adults aged - years, and in experiments on animals, the role of the sinusoidal endothelium, reticular, fat and endosteal cells in hemopoiesis regulation has been concretely defined. the endothelium of sinuses forms the histo-hematic barrier "bone marrow-blood', ensures the wanscellular migration of stem cells and mature blood cells, releases hemopoiesis-regulating factors and is involved in the erythroid cell maturation. bone marrow reticular cells participate in the formation of intramedallar supporting frame-work, regulate the transvasal and intramedullar cell migration, form the extracellular matrix, produce humoral regulators of hemopoiesis, effect the cell differentiation by way ot their intercellular contacts with hemopoietic precursors and give origin to adipocytes, lntramedullar adipocytes present an energetic depot of hemopoietic and stromal tissues and in the stage of preadipocytes they effect, by means of contacts, the granuloeyte development. the endosteal cells of bone marrow are the source of intramedullar stromal tissue, they participate in the anchorage of the hemopoietic stem cells and form the microenvironment of the latter, regulate the endosteal ca ion levels and might be a possible source of hemopoietic tissue (population of cells of the residual embryonal mesenchyma). the thesis on the mechanism causative of the impaired regulation of precursor proliferation and differentiation in hematologic diseases is proposed. the self-renewal capacity of cfu-s (spleen colony-forming unit) following the treatment of (cbaxc b )f female mice with recombinant human granutocyte colony-sffmulating factor (rhg-csf) was investigated. the possibility of synergism between erythropoietin (epo) and rhg-csf in blood cfu-s mobilization was also studied. during the investigation the following observations were made: and mkg/kg/day injection of rhg-csf expended the number of cfu-s- in circulation -and -fold, accordingly. the self-maintenance potential of peripheral blood cfu-s-i did not change significantly. the treatment of mice with mkg/kg/day of rhg=csf resulted in two fold increase of spleen cellularity and fold augmentation of cfu-s- number, without noticeable changes in their self-renewal capacity. moderate changes in cfu-s- number were observed after epo administration in spleen and peripheral blood, however no significant synergistic effect of epo with both doses of rhg-csf was detected. the multifold increase of cfu-s- number in peripheral blood following rhg-csf administration with no reduction in their self-maintenance potential suggests that mobilized with rhg-csf blood stem cells provide an appropriate source for reconstitution of the hematopoietic system. we used the brdu-incorporation method to show the effects of l- ( , , u/ml),il- ( . , , ng/ml) and il- ( u/nil) plus il- ( ng/ml) on b-cll-ceiis. after ficollseparation, lysis of the erythrocytes (nh ci) and lysis of monocytes ( - eucin-methyl-ester), cells were divided into two groups. group was cultured in a serum free medium (+brdu +cytokine) without any t-cell depletion. group was cultured in a serum free medium (+brdu +cytokine) after t-cell (cd +) elimination by the macs (magnetic activated cell sorting). samples were taken after h, h, h, l h and h. after staining with anti-brdu fitc and propidiumiodide (pi) proliferation was measured by flow cytomemy (facs scan). . p=proliferation n=no effect i=inhibition conclusions: i[,- shows a proliferative effect on b-cll-cells independent of t-cells. il- shows heterogeneous effects. by itself it has most often no effect on proliferation, but sometimes it inhibits or increases the prolifoation. this effect does not seem to depend on t-cells. it could depend on the dosage or some unknown patients' characteristics. further on il- inhibits ,- induced proliferation in nearly all cases independent of t-cells. ii-i and tnf are inflammatory cytokines with overlapping biological activities. human vascular endothelial cells express ii-i and tnf receptors and respond to ii-i and tnf stimulation by elaboration of colony stimulating factors such as g-csf and gm-csf.however quantitative data are required in order to evaluate the contribution of ii-i and tnf to the activation of inflammatory hemopoietic cells such as granulocytes and macrophages by csfs. therefore we quantified the production of g-csf and gm-csf in endothelial cell cultures subsequent to treatment with il-l~ ( .l- u/ml) and tnf( - u/ml)or ll- in combination with tnf.a dosedependent stimulation of g-csf and gm-csf secretion was found following ll-i and tnf treatment of endothelial cells.ll-i was a more potent inducer of g-csf secretion than was tnf using approximately equipotent doses of il-i ( u/ml) and tnf ( u/ml) regarding the induction of gm-csf.in addition significant snperadditive stimulation of g-csf and gm-csf production was found with a low dose of il- ( iu/ml) and a saturating dose of tnf( oou/ml) in combination.however the costimulatory effect of ii-l(iu/ml) was significantly more pronounced with g-csf than with respect to gm-csf.in summary the differential modulation of g-csf and gm-csf production ~n endothelial cells by ii- and tnf may indicate 'a disparate role of ii-i and tnf in vascular inflammatory processes and atherosclerosis regarding recruitment and activation of inflammatory leukocytes. cytokines are known to be involved in the pathophysiology of graft versus host disease (ghvd) and to effect lymphohematopoetic progenitor cell growth after bone marrow transplantation. the use of t-cell depleted marrow in human transplantation is associated with suppression of gvhd but decreased rates of engraftment. we have shown in rodent models that uvb irradiation (uvb) of donor inoculum inhibits gvhd while preserving engraftment. to determine the effects of uvb on eytokine production by cells associated with gvhd, human marrow mononuclear cells isolated by ficoll density gradient were uvb-irradiated at doses of - j/m and then stimulated with pha/lps or allogeneic cells. elisa assays were used to measure the production of gm-csf, il- , lif, il-lbeta, il- , il- , tnf-alpha, and lfn-gamma by stimulated cells . two week methylcellulose cultures were used to determine viability of cfu-gm, bfu-e and cfu-gemm progenitor cells after uvb. all results were compared to non-uvb-irradiated marrow and to marrow depleted by soybean agglutination and sheep erythrocyte rosetting. progressively increasing doses of uvb produced progressively decreasing levels of all cytokines except il- , which remained unchanged. t-cell depleted marrow produced decreased levels of all cytokines except il- . uvb at j/m resulted in higher levels of gm-csf and il- as compared to t-cell depleted marrow. this same dose of uvb essentially preserved cfu-gm, bfu-e, and cfu-gemm colonies. we conclude that uvb may inhibit the cytokine cascade active in gvhd while preserving progenitor cell growth at uvb jim . uvb may serve as gvhd prophylaxis in clinical marrow transplantation and in vivo studies on non-human primates are in preparation. the ability to migrate is fundamental for the acquisition of invasive properties by tumor cells. a tumor derived cytokine was identified by its ability to induce direct and random migration via a receptor mediating signal pathway, i.e. autocrine motility factor (amf). we identified the receptor for amf (hamf-r) and found ~at hamf-r plays a role in invasiveness and metastasis in human bladder carcinomas. we investigated the expression pattern of hamf-r in fresh frozen bladder cancer specimens by immunofluorescence technique on the translation level and found a strong correlation (p< . ) to tumor stage and grade. furthermore we have shown that patients who were found to be hamf-r positive have an increased risk for early tumor progression (p< . ). a large number of substances from the working place and in the general environment such as quartz and coal mine dusts are causing silicosis and leading to lung fibrosis. alveolar macrophages are the primary target for the noxious effect of quartz dust. quartz dust incubated human macrophages release in vitro a cytokine, which stimulates cell proliferation of human lung fibroblasts (wistar ). in recent studies we found that beside fibroblasts also epithelial cells of the alveolar unit, such as pneumoeyte type ii cells (a- ) respond to the cytokine with strong proliferation. supernatants of quartz dust exposed macrophages were concentrated by ultrafiltration and thereafter fractionated by gelfiltration (sephadex g , pharmacia). biological activity of the factor eluted within a molecular range of - kd. furthermore, the factor was purified by anion exchange chromatography (q-sepharose). fractions revealing biological activity were further analysed by sds-gel eleetrophoresis (page) and showed two protein bands with apparently molecular masses of and kd,respectively. after addition of the supernatant initially spindle shaped pneumocytes were detected, followed by epithelial-like cells when cell proliferation progressed. induction of spindle-shaped pneumocyte type ii cells could also be seen after addition of pure tumor necrosis factora. however, in this case no cell proliferation was observed. we assume that beside the cytokine, which is responsible for the induction of cell proliferation tnf~ is present in supernatant. differences in a variety of immunologic parameters. in peripheral blood however differences to healthy persons have hardly been described. in this investigation we compared serum levels and concentrations of interferon- (fin-y) and il-l--ct in stimulated samples of whole blood. ixl whole blood of healthy controls (ref) and patients with sareoidosis, without treatment (pot) and under corticoid medication (put), was incubated in medium at ~ and % co . con a was used for stimulation of ifn-y and lps for il- --m concentrations were determined with an elisa. results: without stimulation no measatalg, e amounts of ifn--y could be found. after stimulation ref showed median coneentrations of ng/ml, pob ng/ml and put ng/ml. the difference between ref and put respectively pot was statistically significant. il-l-m without lps the differences were not significant. under stimulation pot had si~ificantly higher values ( pg/ml) compared to ref ( pg/ml) and also to put ( pg/ml). in conclusion we were able to demonstrate that in conlrast to serum levels stimulation of peripheral whole blood reveals significant differences in concentrations of ifn--y and il- --ct between patients with sarcoidosis and healthy references. we established a modified polymerase chain reaction protocol for the detection and semiquantitative assessment of mrna-transcdpt levels for il- , il- , il- , il- , ifn- , tnf-cl, gm-csf, tgf- and il- -receptor-c{ (il- r). the method was shown to distinguish -fold differences in template concentration after rounds each of amplification in the range from to cycles; the lower threshold of sensitivity was at copies per pcr-reaction. reproducibility was > % for a positive result after rounds of amplification; it decreased to % after rounds. this corresponded to the detection of , and template copies, respectively, per pcr-reaction. human peripheral blood mononuclear cells (pbmc) and tumor cell lines were evaluated for mrna-expression with or without stimulation and these results were compared to secretion of the corresponding cytokine. for pbmc, constitutive mrna-expression was found positive for tnf-o~, ifn- , il- , il- , tgf- and il- r, whereas detectable expression of il- , il- and gm-csf was induced only after stimulation. using ~-actin as an intemal standard, the pcr could demonstrate relative differences in cytokine transcripts after stimulation with (a) ]u/ml il- , (b) % lymphocyte-culture conditioned media (ccm) and (c) pma ( ng/ml) plus a ( ng/ml). for il- transcripts no detectable expression was found without stimulus or after addition of il- , whereas ccm resulted in a , -and pmna in a , -fold increase. other mrnatranscripts increased t -fold (tnf-(~) up to , -fold (gm-csf) with or without differences between the stimulating agents. the cell lines caki- (renal cell carcinoma) and daudi (burkitt lymphoma) also expressed comparable levels for cytokine transcripts, with a strong induction after stimulation with pmna . the relative ifn~ mrnacontent in caki- increased from to , , for gm-csf from to , . the influence of different cytostatics on il- production by peripheral blood mononuclear cells (pbmc) was studied. pbmc were pre-incubated with or without phytohemagglutinin p (pha-p), then pulse exposed during hr to different cytostatics in their therapeutical concentrations, washed three times and incubated additionally during - hrs. then the supernatants were collected and il- biological activity was measured in mttmodified b -cells biological assay. though significant individual variations of the il- production were found, all the studied drugs can be separated on severa) groups: ) adriamycine and methotrexate induced late increase of il- production ( - hrs ); ) cytarabine strongly increased the early as well as the late il- production ; ) the pretreatment with etoposide and rubomycine suppressed subsequent production of il- by pbmc during - hrs, the late production was increased; ) in contrast, the cyclophosphamide pretreatment stimulated the early production and strongly suppressed the late one. the changes of il- production by pbmc was not due to the cellular death. the pha-p stimulated pbmc produced usually more il- that unstimulated cells did. these data suggest that the cytostatics possess the different effects on il- production by pbmc that could be important in the therapy of malignancies. recent studies implicating a deficiency of interleukins in several diseases have underlined the importance of measuring in vitro the dna synthesis and the cytokine production (il- , il- , il- , tnfalpha) in the same cell system. previously had found that normal peripheral blood mononuclear cells (mnc) of patients suffering from high-malignant non-hodgkin lymphomas showed a decreased capability to proliferate after mitogenic stimulation (pha, con a, pwm). here we have studied the dna synthesis and the production of different cytokines (il- , il- , il- , tgf-i~ and tnf-alpha) by pokeweed mitogen (pwm) stimulated mnc from healthy control subjects and from patients with nhl. the il- production of pwm-stimulated mnc of patients with nhl was found to be significantly decreased, wheras the il- , il- and tnf-alpha release were not changed significantly. these data showed a good correlation with the reduced capability of mnc from patients with nhl to proliferate after mitogenic stimulation. the multifunctional cytokine transforming growth factor-ii (tgf-i ) is known to inhibit the dna synthesis, as well as the il- production of mitogenstimulated mnc. however, tgf-i~ release was not significantly changed in call culture supernatants from patients with nhl in comparison to healthy controls. we conclude that the suppressed dna synthesis and _- production of mnc from patients with nhl is not the consequence of a deceased tgf- level secreted by these cells. is a co~on problem after chemotherapy and requires supportive care until normal hemopoiesis has recovered. to study the importance of endogenously produced cytokines for regeneration of bone marrow progenitors we measured serum levels of g-csf and ii- . blood samples were obtained before and hours after chemotherapy, during the stage of leukopenia (< /ul) and recovery (> /ul)of leucocyte counts. in / patients we found a more than -fold increase in serum g-csf levels at the stage of leukopenia. highest amounts were observed in two patients with lethal outcome. there was no correlation between thrombocytopenia and levels of g-csf or il- . serum g-csf (pg/ml, mean, range) before (leuko> ) after (< /ul) chemotherapy aml dav ( -i ,n= ) ( - ,n= ) all 'h~izer" ( - ,n= ) ( we measured different hematopoietic cytokines as g-csf, gm-csf and il- in amniotic fluid and cord blood to ctearify their physiological and palhophysiological role in fetal and neonatal development amniotic fluid was available from the th to the st week of gestation (n= ), cord blood from the th to the nd week of gestation (n- ). activity levels of cytokines were determined by stimulation of the ceii lines nfs- , d , and tf-i. which are responding specifically to g-csf, il- , and gm-csf. specificity has been proved by neutralizing antibodies. calculation of cytokine levels was done by standards of recombinant growth factors. sensivity for g-csf and il- : pg/ml for gm-csf: oo pg/ml. in amniotic fluid g-csf tanged from to i .ooo pg/m[ and il- from to . pg/ml, whereas gm-csf was not detectable. in cord blood g-csf was between and . pg/ml and [l- between and pg/mh in most of the samples (qo%~ gm-csf was beyond the sensivity limit. in % ( of cases) g-csf levels were elevated over pg/ml and associated with amnion infection syndrome, while green amnioflc fluid alone during delivery did not stimulate the production of g-csf, the levels of the hematopoietic cytokines showed no influence by the gastational age. identical twins without maternal infection showed the same g-csf levels. inflammation of the amniotic membrane and maternal sepsis is associated with elevated g-csf levels in cord blood. gm-csf can normally not be detected in cord btcod and amniotic fluid. detectable levels of gm-csf in cord blood and amniotio fluid maybe give a hint for a pathological situation during pregnancy. total number of children with all, boys and girls, aged from to years were included to the study. tnf production was studied acc. the method based on growth inhibition of sensitive to tnf l mice fibroblasts, ill- production ace. method based on inhibition of autologous rosette formation by thymoeyteg of cba mouse and l- production ace. to conventional el sa genzyme-test. twenty five healthy children served as the control group. it was found that in children with all during the whole period of therapy the il- and il- production, was significantly lower than that observed in the control group of healthy children (p . ). the tnf production in all children before therapy was lower in comparison with the control group values. during cytostafic therapy was higher and grew up above the normal limits after cessation of the therapy. il- production grew up after the end of the therapy but never reached the value of the control group. efs at ruth in all children with il- production < /~ before therapy was higher than that in children with il- < /~ (efs % v %). the il- production seems to be a good prognostic marker. (pts) with active autoimmune disorders as well as with malignant lymphomas. in addition, cd and its soluble form (scd ) is thought to be involved in the regulation of b-cell proliferation. therefore, we examined scd , scd , and scd in pts with b-cell chronic lymphocytic leukemia (b-cll) in order to assess their role as indicators of disease activity. fifty-five pts with b-cll were studied so far. staging was performed according to the classification systems of rat and binet, respectively. serum samples were freshly stored in liquid nitrogen until further processing. levels of scd , scd , and scd were measured by a sandwich elisa technique using commercially available assays (biermann, germany). advancing rat stages were associated with a progressive increase of all the three serum factors (scds: rat + u/mt vs rat iv + u/ml, scd : rat - - u/ml vs rat iv __. u/ml; scd : rat + u/ml vs rat iv +_ u/ml). this progression was also evident when binet's classification was applied. occurrence of b-symptoms was associated with high levels of scd (p< . ), whereas scd and scd were found also to be increased but without statistical significance. high levels of all the three factors strongly correlated with a lymphocyte doubling time < months, a lymphocyte count > . /tzl, and with the presence of hepato-and splenomegaly. interestingly, occurrence of bulky lymph nodes (i.e. at least one nodule of > cm in diameter) was linked with high levels of scd only (p< . ). in summary, ( ) progressive serum levels of scd , scd , and scd correlate with advancing stages of disease in b-cll. ( ) b-symptoms were associated with high levels of scd . ( ) we found scd to be the more sensitive marker of the total tumor load than scd and scd . thus, scd may be useful in monitoring pts with b-cll. cytosine arabinoside (ara-c) is one of the most active single agenls in the treatment of acute myeloid leukemia (aml). its cytotoxic activity mainly depends on its phosphorylation to ara-ctp and on its incorporation into the dna. based on recent in vitro studies showing that hematopoietic growth factors like gm-csf and il- enhance the cytotoxicity of ara-c on clonogenic leukemic cells, the gm-csf priming concept is currently explored in clinical phase ii and iii studies. in an ongoing study at the universities of muenster and goettingen gm-csf ( /~g/m /d) is started hrs before induction chemotherapy (tad /ham) until recovery of blood ceil counts. this study provided a means to assess the effect of gm-csf on the intraceunlar ara-c metabolism in vivo in pts with aml. enzyme activities of deoxycytidine kinase (dck), thymidine kinase (tk), dcoxycytidine deaminase (dcd), dna polymerase (pol) and dna polymerase alpha (pola) were determined before therapy, hrs after the administration of gm-csf and hrs after the administration of ara-c. in addition, ara-c incorporation into the dna was measured after hrs ara-c administration. enhancement of enzyme activity was observed in / , / , / , / and / cases for pola, pol, tk, dck and dcd, respectively. increases ranged from - % for pola (median %), - % for pol (median %), - % for tk (median %), - % for dck (median %) and - % ( %) for dcd. inadequate blast cell reduction after tad (> % blast cells on day or ) was associated with significantly higher dcd blast cell activities compared to the dcd activity values obtained for pts with adequate blast cell clearance (median values: . vs . nmol/min x mg, p< . ). cases with dcd activities < nmol/min x mg showed significantly higher ara-c incorporation into the dna compared to pts with dcd activities > nmol/min x mg ( . vs . ng/ cells). furthermore, inadequate blast cell clearance was associated with lower ara-c incorporation into the dna (median . vs . ng/ cells) and lower pola activities (median . vs . pmol/min x mg). in pts we investigated simultaneously the effect of gm-csf pretreatment on ara-c metabolism in vitro. enzyme activities of pola, tk and pol correlated significantly in vivo and in vitro (rp~ rtk=o. , rp~ p< . ). these data demonstrate that gm-csf enl/ances dna synthesizing enzyme activities in vivo and in vitro. furthermore, these data suggest that gm-csf might improve the therapeutic response to induction chemotherapy by increasing dna polymerase alpha activity and thereby increasing the ara-c incorporation into the dna. the effects of interferon-alpha (ifn-alpha), interferon-beta / lnterleukin- (il- ) and interferon-gamma (ifn-gamma) in inducing megakaryocytic differentiation of blast cells from acute megakaryoblastic leukaemia (amegl) patient determined by the increase in cd and cim b expressions using monoelonal antibodies in apaap technique were investigated in liquid suspension culture. after six days of culture, the percentage of cd and cd b positive cells increased in control cultures from , % and , % on day to , i , % and , • , %, respectively. the addition of ifn-alpha significantly increased the number of cimi and cim b positive cells by about two to three fold compared to control cultures, p < , and by about four to six fold compared to day , p < , . similarly, ifn-beta /il- induced a significant increase in cd and dc b positive cells. on the other hand, ifn-gamma failed to increase the number of cim and cd b positive cells in comparison to control cultures on day and instead induced a significant increase in the number of monocytes/macrophages from only , _+ , % in control cultures to , + , %, , + , %, , • , % and , • , % in , , and units/nil ifn-gamma-treated cultures, respectively, p < , . the present results suggest that megakaryocytic differentiation of blast cells in amegl could be induced by ifn-alpha and il- and support a clinical role for il- in the treatment of amegl patients. also, the present results showed that monocytic differentiation of blast cells in amegl could be induced by ifn-gamma~ supporting the multipotent stem or progenitor cell origin of the amegl subtype of acute myeloid leukaemia. a monoclonal antibody-based elisa and bioassay were used to measure leukemia inhibitory factor (lif) protein levels, activity and the functional role of lif in superuatants of cultured stromal cells derived from patients with acute and chronic myelogenous leukemia, myelodysplastic syndrome, and hairy cell leukemia and from normal controls. we demonstrate that biologically active lif protein is constitutively produced and secreted by coltured bone marrow stromal cells from all subjects studied. in addition, adherent-layer conditioned-media lif protein levels were significantly elevated in samples from patients with all hematologic malignancies studied as compared to samples from normal controls. confluent adherent layers exposed for hours to interleukin (il) or tumor necrosis factor- (tnf-a) showed a significant increase in lif protein levels, whereas exposure to il- (sterling drug inc., great valley, pa) resulted in a dose-dependent decrease in lif levels. . (i. - . ) . ( . - . ) . interestingly, neutralizing antibody against lif caused a % reduction in normal progenitor proliferation derived from the superuatant but not from the adherent layers, and this effect was reversible by the addition of recombinant lif protein. we conclude that (i) biologically active lif protein is constitutively produced by adherent layers from normal donors, (ii) tnf-ct and il- increase and il- decreases adherent layers lif protein levels, (iii) the steady state levels of lif protein produced by adherent layers from leukemic patients is significantly elevated, and (iv) lif may participate in the interaction between adherent layers and hematopoietic progenitors to maintain normal hematopoietic colony growth. it is well known that bone marrow stromal cells have crucial impact on haemopoietic cell proliferation. little is known about stromal humoral factors leukemic cell proliferation. the aim of this study is to evaluate the effect of stromal cell conditioned media (sccm) on the h-thymidine uptake by normal and leukemic target cells. patients with aml were studied treated with " + " based regimens. long term bone marrow cultures were established in non-leukemic and leukemic patients (before and during treatment). target cells for sccm were normal haemopoietic cells and leukemic blasts. the results are the comparison of the effects of leukemic and non-leukemic stromal cells. a part of the patients revealed high stimulative activity upon non-leukemic cells (+ + %, p< . ) and inhibited proliferation of leukemic cells (- • %, p< . ) this group entered complete remission. sccm of another group of patients inhibited proliferation of nonleukemic cells (- _+ %, p< . ) and stimulated blast cell proliferation (+ + %, p< . ). the magnitude of the figures was even more profound later: during treatment, bone marrow aplasia, recovery. this group of patients failed to achieve remission. it seems that stromal cells has significant on impact on restoration of normal or leukemic hemopoiesis after chemotherapy. peripheral b-cll-cells from patients were investigated upon the proliferating effect of ifn~, tnf~ or combination of both in serum free culture medium (cg-medium). blood cells were drawn from patients and lymphocytes separated by ficoll hypaque and monocyte-lysis (leucinemethyl-ester incubation). t-cells were depleted using a, cd mab and macs (magnetic activated cell separat r, miitenyi biotec). at each step heterogeniety of the population was controlled by facs analysis with different mab (cd , , , o, , , , ) . the homogeneous population (contamination less than %) was co-incubated with both cytokines ( . - ng/ml) and bromodeoxyuridine (brdu) in cgmedium. after , , , and t hours cells were harvested an analysed for brdu-incorporation into the genome. ifnu and tnfc~ measurings ( in total) were almost similar: patients were non-responder and showed no stimulatory effect on cells; patients showed an inhibitory effect; cells from patients were responding upon cytokine cultivation. the combination of both ifn~ and tnf~ produced in these cells an additive effect ( out of ). best results could be observed when the control population (without cytokine) was minimal proliferating compared to no proliferation. a high correlation was observed between cytokine response and pre-treatment: without glucocorticoid treatment of patients prior to measurements the influence of cytokines on resting b-cll-cells was significantly higher (with methyl-prednisolone %, without we-treatment % were responders). functional defects of the monocyte/macrophage system probably contribute to the increased rate of severe infections in patients with myelodysplastic syndromes (mds). therapeutic trials with hematopoietic growth factors (hpgf) have resulted in substantial improvement of cytopenia, especially neutropenia. however, little is known about the alterations of the monocyte/macrophage system during these therapeutic interventions. it therefore was the aim of the present study to analyze the capacity of monocytes/macrophages from mds patients prior to and after hpgf therapy to secrete il- ~, tnfe, il- , and il- upon in vitro stimulation with lipopolysacharide (lps). sixteen patients were studied: t had a refractory anemia, had a refractory anemia with excess of blast cells. prior to therapy, the capacity of adherent monocytes/macrophages to secrete il- ~, tnfc~, il- , and il was significantly reduced by - percent as compared to normal controls. on the other hand, oxygen radical release was normal in mds patients tested. treatment with gm-csf ( - /~g/m /d sq x - ; n= ), il- ( - /~g/m /d sq x - ; n= ), and g-csf ( - fg/kg/d sq x in combination with all-trans retinoic acid; n= ) normalized the potenital of monocytes/macrophages to secrete il-ti~, tnf~, and il- . il- secretion was only improved by il- or gm-csf dosages _> /~g/mz. oxygen radical release was significantly stimulated by both gm-csf and il- . these results indicate that treatment of mds patients with gm-csf, il- , and g-csf (the latter in combination with all-trans retinoic acid) can restore deficient monocyte/macrophage secretory function to normal. depletion of cd positive t ceils has been used in human patients for prevention of gvhd. we studied depletion of cd + cells from canine marrow for induction of gvh-tolerance across a complete dla-haplotype difference. prompt engraftment and fatal gvhd occurs in a littermate combination of dla-homozygous donors and dla-heterozygous recipients when undepleted marrow is given. aiiogeneic marrow depleted with a crossreactive antibody to human cd and immunomagnetic beads was given to dogs. one dog died with haemorrhage on day due to thrombocytopenia, dogs showed complete hematopoietic recovery. dogs became tolerant chimeras and one dog died with gvhd due incomplete depletion. chimerism was mixed early after transplantation, became complete later and is still complete in / dogs after - years. dogs received cd depleted marrow grafts and loijg/kg/d s.c. r-canine g-csf starting on day after transplantation. although all dogs had fast recovery of granulocytes, dogs receiving . x mnc/kg died of marrow aplasia on days and without recovery of thrombopoiesis. two dogs receiving x mnc/kg had sustained engraftment with delayed recovery of thrombocytes compared to dogs without g-csf. facs analysis of depleted marrow showed complete depletion of cd + cells but about % of cd + cells; cfu-c growth and nk-activity was retained after depletion. cd depleted marrow inhibited the generation of cytotoxic cells. these experiments indicate that qualitative t-cell depletion is effective, since cd recognizes only subpopulations of t-cells. the application of r-canine g-csf enhanced the recovery of granulocytes but led to graft failure in dogs receiving a low number of marrow cells. gsf-inst. fdr immunologic, marchioninistr. , mqnchen supported by the wilhelm-sander foundation we wished to analyse the factors which may affect the yield of pbpc (cfu-gm) to be collected by leukapheresis following high-dose cyclophosphamide (hd-cyc: g/m ). we retropectively studied the following criteria in patients with high-risk mm of which received gm-csf (sandoz sa/ schering-plough) after hd-cyc: time from diagnosis to hd-cyc, number of chemotherapy cycles (ctc) (fermand, ) , b microglobulin, bone marrow plasma cell count before hd-cyc, administration of gm-csf after hd-cyc, "slow" or "fast" rate of platelet and wbc recovery (jagarmath, schwartzenberg, ), "poor" or "good" mobilization ofpbpc (jagannath, ), differential wbc count between "day x ~ and "day x-l" during haematopoietic recovery. each variable was studied as continuous (regression analysis) and discontinuous (t-or chisquare tests). when the differential wbc count was < wbc/ial, % of the leukapheresis procedures performed on day x yielded more than x cfu-gm vs % when it was _> /tjl (p< . ). the infusion of gm-csf was asaocaated with a higher yield of cfu-gm (bidt, ). the patients with "good ~ pbpc mobilization (> x cfu-gm in >_ leukapheresis) could all be transplanted with pbpc alone (vs % of those with ~poor" mobilization). they had a shorter duration of aplasia after transplantation than the other patients (p< . ). the "fast" wbc recoverers had a higher yield of cfu-gm than the other patients (p< . ). when only the patients who did not receive gm-csf after hd-cyc were considered, a higher yield of cfu-gm was achieved in patients who underwent < crc as compared to those who underwent > ctc before hd-cyc (p<o, ). no other criteria had a significant prognosis value. these criteria will help to define which patients may need the collection of bm or the infusion of gm-csf (i) to collect pbpc and (ii) after transplantation. haemopoietic cells released into the circulation by g-csf (lenograstim) alone or in combination with intense chemotherapy: assessment of progenitors and stem cells. i.baumann*, ng testa*, mehm van hoef ~, c.lange*, e.de wynter*, a.howell $, tm dexter*. we report results of a phase i/ii recombinant human granulocyte colony stimulating factor trial for dose intensification in advanced breast cancer. sixteen patients were studied. seven patients underwent the phase i g-csf trial before entering the phase ii dose intensification study plus chemotherapy. nine patients were entered into the phase ii study with four cycles of dose escalating chemotherapy. the release of progenitor cells into the circulation was determined by clonogenic assays. two stage long term bone marrow cultures were used to assess the repopulating capacity and therefore the stem cell quality of the mobilised cells. normal bone marrow from allogeneic transplant donors (with informed consent) were used as controls. we found equivalent total numbers of progenitors in both cycle and cycle of g-csf plus chemotherapy peaking at day ia- , respectively. in contrast, we found significant differences between the progenitor cells of cycle and generated from circulating haemopoietic cells when seeded onto preestablished irradiated bone marrow stroma. this indicates that peripheral blood stem collection should be performed in an early stage of chemotherapy due to an increased damage of the stem cell pool with increased levels of chemotherapy. the capacity of haemopoietic cells released by g-csf to repopulate bone marrow stroma was equivalent to normal bone marrow as recently reported. in autologous bone marrow transplantation (abmt) acute phase responses usually occur between day and day following abmt, and increases in bilirubin levels are observed in early endothelial complications such as endothelial leakage syndrome and veno-occlusive disease (vod). il- is the central cytokine modulating acute phase responses. therefore we performed a prospective study analyzing il- , tnf-u, sil- r and g-csf levels in serum samples of patients undergoing abmt ( all, aml, hodgkins disease, non hodgkins lymphoma, ewing sarcoma). patients received g-csf post abmt, patients were transplanted without g-csf. during leukopenia < /gl g-csf levels of _+ pg/ml ( - pg/ml) were measured in patients not receiving g-csf. during regeneration, when leukocyte counts exceeded . /tal, the endogenous g-csf levels declined to the normal range (< pg/ml). severe transplantation associated complications were defined as vod or interstitial pneumonia. il- levels increased from +_ pg/ml before therapy to • pg/ml during febrile leukopenia (p<. ). patients with complicated abmt had significantly higher il- levels ( -+ pg/ml n= vs • pg/ml n= ) (p<. ). sil- r-levels increased from + u/ml before abmt to _+ u/ml during leukopenia. in patients receiving g-csf sil- r-levels further increased to _+ u/ml during regeneration, while in patients not receiving g-csf the sil- r-levels were • pg/ml (p<. ). tnf-a levels remained unchanged during the course of abmt; there was no correlation with febrile neutropenia or severe complications. these data suggest a direct feedback regulation of endogenous g-csf release by increases in peripheral granulocyte counts. monitoring of il- serum levels during abmt may give insights into pathophysiology of abmt associated complications resulting from activation of monocytes and endothelial cells. we have previously reported that the rate of hematopoietic recovery following autologons blood stem cell transplantation (absct) could be influenced either by the type of conditioning regimen or by the underlying disease. we also reported that peripheral blood stem cell (pbsc) growth was sensitive to the stimulation of allogeneic irradiated stmmal layers. thus we undertook a study to examine the quality of the bone marrow (bm) raiemenvironment of patients who had undergone absct for either malignant lymphoma (ml) or multiple m~,eloma (mm) after either myeloablative chemotherapy or tbi conditmning regimens. thirteen patients with ml and eight patients with mm underwent absct using cells collected during recovery phase after intensive chemotherapy. we used the long term culture system (ltc) to investigate the quahty of the bm microenvimnment from patients (pts) with ml and patients with mm. all bm were collected after absct. twenty-two ltc established with bm from patients with ml were investigated; pts received myeloablative chemotherapy (cbv) as conditmning regimen and out of ioof these pts developed a complete confluent stmmal layer. three of the ml pts received tbi and / of these pts developed a complete confluent stromal layer. eight ltc were established with bm from patients with mm, who had att received" till" prior to absct. three ~f the mm pts de~ a complete confluent stromal layer. we evaluated the cfu-gm production by the stmmal layer from all patients. the type of disease (ml vs mm at d p= , and di p= , ) , the conditioning regime used prior to absct o'b! vs cbv at d p= , and di p= , ) , the presence or absence of a complete, confluent stromal layer (at d p= , and d p= , ) did not influence the production of cfu-gm by bm stromal layers from autografted patients. nor was there a correlation between the development of a confluent stromal layer and the duration between ltc and absct or the date of the cytapheresis as compared with the date of diagnosis or the number of naeleated cells and cfu-gm used to engraft the patients. interestingly, when we examined the ability of confluent and non confluent stmmal layer to support hematopoiesis, we observed no difference in the total cumulative production of nucleated cells or cfu-gm, suggesting that the quality of the bm stromal miemenvironment after absct cannot explain the differences seen in hematopoetic reconstitution. the present study was designed to estimate the immunologic and hematopoietic reconstitution after several consecutive chemotherapy courses (ctx). patients selection: all (n = / ctx-cycles), aml (n = / ), nhl (n = / ), m. hodgkin (n = / ), and non-seminomatous germ cell tumor pts. (nsgct, n = / ). ctx was performed according to the current treatment protocols used at our institution. the nhl and nsgct pts. received g-csf before ctx for mobilization of stem cells for apheresis. g-csf application ( "pg/kg) after ctx was used in all nhl, all and nsgct and aml pts. the collected pbsc were reinfused after each following ctx cycle to all nhl and nsgct pts. the cd , cd , cd , cd , cd , cd , cd and hla-dr positive cells in peripheral blood were analyzed, using dual color fluorescence and flow cytometry. cfu-gm from peripheral blood mnc's were used as control for the clonogenic capacity of cd * cells. samples were obtained before, immediately after ctx and several times after the wbc's reached > lxlog/l. a positive correlation of the rising and decreasing subpopulation counts within the mnc's were noticed (r=. -. ), however the cd * were in inversed ratio to the cd § cells (r =-. ). the percentages of cd * and especially of the cd + cells showed an increment immediately after ctx, where the proportions of cd " and cd § cells tended to fall. there was also a correlation between cd " and cd § cells (r=. , p<. ) and between cd § ceils and cfu-gm growth (r= . , p<. ). an increased clonogenity was associated with low numbers of cd * cells: cd ~: cfu-gm : before ctx; : immediately after ctx; : during the regeneration phase. the same phenomenon could be seen by intensively pretreated compared to less intensively pretreated patients. the hematopoietic reconstitution parameters (median) were as follows in the pbsc-rescued vs non-rescued pts: platelet transfusions - vs (p<. ), rbc transfusions - vs , days with platelets < // - vs (p<. ), duration of neutropenia with wbc< //ji - vs , days with fever - vs . . the augmentation of cd § cells correlated with rising numbers of mnc's and especially of cd § but not with cd *. the correlation between cd " cells and cfu-gm in peripheral blood was convincing. dose-escalated cytotoxic therapy with stem cell support may be considered for patients with stage ii multiple myeloma, because of the poor median survival of only . years with conventional treatment. a threshold quantity of x cd + eells/kg bw is necessary for a rapid and sustained engr~ent following myeloablative conditioning therapy. since june six patients (median age: years, range - ) with mm received pg g-csf/kg bw (neupogan r, amgen) so. daily at the time of best response with conventional treatment. the content of cd + cells in the peripheral blood was monitored by facs each day. leukapheresis were started when a detectable population of cd + cells appeared. in of steady-state leukaphereses, more than , xl ~ cd +cells were harvested. after the therapy with high dose eyclophosphamide ( pts g/m , pt g/m ) plus g-csf more than , xl cd + cells were collected in of leukapheresis. to date two pts have undergone myeloablative conditoning therapy with hyperfractioned total body irradiation ( , gy) and melphalan ( mg/mz). one patient received mg/sqm melphalan as eonditoning therapy. after the reinfusion of the g-csf-mobilized pbsc, a rapid engraftment was achieved with median time of days ( range - ) to reach , x / neutrophils and days (range - ) for . x / platelets. no hematopoietic growth factors were given post-transplantation. in this pilot study, high dose eyclophosphamide and g-csf is an effective method for harvesting pbsc. evaluation of the mobilization of hematopoietic stem cells during steady-state hematopoiesis using higher doses of g-csf is planned. were treated with mtx, ifo, ara-c, prednisolone and escalating doses of vp . pts. (n = ) with relapsed or advanced non-seminomatous germ cell tumors were treated with cisplatinum, escalating doses of vp and ifo. the protocol design was similar: g-csf before ctx ( x pg/kg/die s.c.) with pbsc-aphereses at days to (nhl) or days , (nsgct} followed by to ctx-courses. cytaphereses were also performed after ctx when the total wbc's recovered above lxl ~ the ctx-cycles were followed by reinfusion of the previously collected pbsc (n = ) and application of g-csf ( pg/kg/d.; n = ) up to the last day of the subsequent stem cell collection. the cd § cells, the clonogenic peripheral blood progenitor cells (cfu-gm & bfu-e) and light density cell (ldc) counts were determined in cytapheresis samples. the ctx/g-csf courses contributed to substantially higher progenitor cell amounts than g-csf alone (n= / ; p< , ), without a difference in the collected ldc ( . • vs . • ldc; . • vs . • ~ cd § cells; . + . vs . • . x cfu-gm; . • . vs . • . x bfu-e/kg/apheresis; n = / ; mean + sem), but with approximately two times lower clonogenity. two or three leukaphereses were enough to rescue ctx-courses with a minimal dose of x cd + cells/kg/patient. the optimal time to initiate pbsc-collection after ctx in the studied patient group was proved to be at the l't to ~h day after reaching leukocyte > pl. the original total leukocyte, light density cell (ldc) and platelet counts in the peripheral blood at start of leukapheresis played an essential role for the efficiency of the procedure (eff), as shown by regression analysis: eff to total leukocyte count correlation was r =- . (p< .ot); eff to ldc-count r =- . (p<o. ); and eff to platelet count r =- . (p= . ). the results of pbsc retransfusion on hematopoietic recovery were as follows: days to reach leukocytes> pl and days -platelets> ooo pl; duration of neutropenia was days; and days to become platelet transfusion-independent (median). total number of children, girls and boys, with neoplasia aged from - years were treated with gm-csf-leucomax sandoz and g-csf filgrastim hoffmann-la roche, during severe myelosupression occuring after intensive polychemotherapy. in children gm-csf was applied twice after consequent courses of chemotherapy. one child received gm-csf four times after chemotherapy courses. twenty children with malignancies served as historical control group. gm-csf was given at dose #g/kg, g-csf - #g/kg daily s.c. duration of therapy ranged from - days with median days. after cytokines therapy increase of mean and median numbers of total wbs, neutrophils, monocytes and eosinophils was observed. the median time to hematopoietic recovery was shorter in the group of children treated with cytokines when compared with the control group. ( v days). in of children signs of infection disappeared even before granulocyte count increase. also shorter median time of febrile days, v days, in comparison with the control group was observed. no serious side effects during cytokines therapy were noticed. only in one patient local erythema in injection place was observed. in two children transient retrosternal pain was seen. our results showed that gm-csf and g-csf administered in children with neoplasia after chemotherapy shortens the period of neutropenia and infection duration. for both hodgkin's disease and non-hodgkin's lymphoma the outcome of chemotherapy has been shown to correlate closely with the dose intensity of treatment. however, dose intensification is limited most often by severe myelosuppression with the subsequent risk of fever and infections. we performed a clinical trial in patients with hodgkin's disease or non-hodgkin's lymphoma to evaluate whether r-methug-csf could facilitate the safe and timely administration of an intensive chemotherapy regimen. patients who developed neutropenia _< . x /l for more than two days and / or fever _> . ~ and / or signs of infection after a cycle of chemotherapy (ceboppnim protocol administered at intervals of days), as well as patients in which chemotherapy had to be delayed due to an anc _< . x /l on day , were eligible for treatment with r-methug-csf. in the subsequent cycles r-methug-csf was given subcutaneously at a dose of #g/kg/d from day to . of patients were evaluable, one patient had received only day of r-methug-csf treatment. of the evaluable patients experienced neutropenia with an anc of less than . x /l during the chemotherapy course preceeding r-methug-csf treatment, whereas only patients had ancs _< . x , l after the subsequent therapy with r-methug-csf (p< . ).overall analysis showed that the duration of anc nadirs < . x /l was on average . days in cycles without r-methug-csf compared to . days in cycles with r-methug-csf treatment. the administration of chemotherapy had to be delayed only for . days (mean value) during cycles with r-methug-csf. side effects probably related to r-methug-csf, were moderate muscle and joint pain in patients and chills in one patient. in general, r-methug-csf was well tolerated. under this treatment regimen patients reached complete remission, patients reached partial remission and one patient had stable disease. one patient was treated adjuvantly after gastrectomy. in conclusion, r-methug-csf allowed the safe and timely administration of this intensive chemotherapy regimen and reduced myelosuppression for patients with hodgkin's disease and non-hodgkin's lymphoma. based on previous studies (cancer res. : , , blood, : , we know that many patients (pts) cannot receive ct consisting of carboplatin (cbdca) mg/m and cyclophosphamide (cyclo) mg/m for oc every wks without hematopoietic growth factor support. the desirable dose of rhil- based on a phase i/ii study in this setting was or gg/kg/day. a study was designed to determine whether rhil- would allow ct administration every wks with pts treated to date. cyclo was administered mg/m and cbdca was dose adjusted to creatinine clearance: - ml/min: mg/m , - : mg/m , - : mg/m , > : mg/m . a total of cycles (c) was administered. rhil- ( or /zg/kg/d) was given sc d - in each c. at and #g doses are ( c) and ( c) pts evaluable for toxicity and i ( c) and ( c) pts for efficacy. side effects were fever and headache controllable with acetaminophen. at #g rhll- in three c ( pts) and at #g in six c ( pts) urticaria occurred. in episodes dyspnea and/or oedema was observed. this reaction only occurred during c ~ and was controlled with antihistamine and prednisolone. ct could be administered every wks in / c ( / c at /~g, and in / c at /zg (ns)), every wks in / c and > wks in / c. no platelet transfusions were required. thus, in % of c it was possible to give a ct dose intensification of %. if full dose ct were to be given every wks it would have been possible to administer in % of c in time. conclusion: with rhil- ct dose intensification of % is possible by reducing ct intervals, while no platelet transfusions were required with rhil- . dept. of medical oncology. university hospital groningeu. oostersingel , ez groningen. the netherlands. k. mempel, a. reiter, e. yakisan, e. odenwald, m. pfetsch, g. schwab, h. riehm, k. welte. in the multicenter trial all-bfm , we have initiated a phase iii study of rhg-csf in children with high risk all. high risk (hr) patients are characterized by at least one of three criteria: . prednisone poor response ( /mm absolute blasts number in the blood at day after days' exposure to prednisone), . failure to achieve complete remission at day of induction therapy, and . t( ; ). the primary objective was to test whether rhg-csf reduces the incidence of febrile neutropenic episodes. the second objective was to examine whether rhg-csf administration allows closer adherence to planned dosing schedule and to determine the overall response to chemotherapy. hr-all pts are randomized to receive either cycles of chemotherapy (hrg i) or cycles of chemotherapy (day - ) followed by rhg-csf (day - ; hrg li). up to date, pts have been treated according to this protocol (hrg i: pts, hrg i : pts). in hrg ii, rhg-csf is well tolerated without g-csf related adverse events. in each arm, one pt relapsed. the incidence of neutropenia was % in hrg i and % in hrg i . more importantly the incidence of febrile neutropenia was % in hrg i and % in hrg ii. these data demonstrate that rhg-csf allows for reduction of the incidence of febrile neutropenia in hr-all-patients. the patient has experienced complete resolution of stomatitis, fever and malaise. the administration of g-csf in patient with idiopathic neutropenia significantly increased the absolute ueutrophil counts (p < . ). g-csf was effective in reducing the severity of neutropenia and infectious complications in our patient. hansen f., stenbygaard l. and skovsgaard t. twenty patients with recurrent metastatic breast cancer treated with high-dose myelosuppressive antjr eoplastic drugs (cyclophosphamide , g/m or epirubicin mg/m both q . weeks) as first or second line chemotherapy were randomized in a prostective study to gm-csf (n= ) microg/ kg/dag for ten days after cessation of chemotherapy or control (n= ). compared to the control-group highly significant reduction in granulocyte nadir duration (two days ( - ) with gm-csf vs. seven ( - ) days) and severity (wbc . x /i with gm-csf vs. . x i /i) was found. no difference in frequency of neutropen fever or antibiotic use could be observed. even though the patients treated with gm-csf at random were more heavily pretreated with chemotherapy, there was a surprisingly higher responserate in these patients as compared to the control-group, namely % vs. %, resp. no severe side effects were seen, but presumably due to gm-csf one patient developed an allergic type i reaction and one patient developed a possible pericardia[ exudation. both were fully reversible after cessation of gm-csf treatment. keywords: gm-scf, chemotherapy, breast cancer. twelve adult patients with chronic neutropenia, including patients with idiopathic sporadic neutropenia, with idiopathic familial neutropenia and with cyclic neatropenia have been treated with rhu-met-g-csf (amgen, thousand oaks, usa). treatment has been started in all patients with #g/kg/d sc once daily. doses have been modified according to wbc. all patients had a rapid increase of absolute neutrophil counts. data are shown for idiopathic neutropenia (base line, after , and weeks). doses required ranged from . to #g/kg/d. treatment has been continuously given up to three years in patients with severe preceeding infections. the clinical efficacy of the treatment was excellent with abrogation of significant infections. one patient with idiopathic i sporadic neutropenia recovered after days of treatment with an anc of "~ ~o o > /# after stop of g-csf. in a patient with familial cyclic neutropenia cycle length shortened from to days. in another patient with acquired idiopathic cyclic neutropenia the cycle length of days remained constant but re t w w w the nadir of anc rose from to /# . this patient was taken off therapy because of urticaria related to g-csf on day . there were no further significant adverse events. no loss of effectivity was observed during long-term treatment. we conclude that g-csf is safe for long-term treatment of idiopathic neutropenie with severe preceeding infections. as response to treatment is quic, it may also be an effective interventional treatment in acute infections in these patients. cytokines and growth factors are widely used to promote growth and proliferation ofbematologic cell populations. improvement or wonnu healing by stimulation with g-csf has been relxn'ted in patients suffering from kostmann syndrome, felty syndrome or from neutro-~ nia due to chemotherapy. e report on two patients with mds/ra (ha, female, age ; sh, male age ); duration of disease was months and years, respectively. ha was admitted for neutropenia (neuu'ophils: . - . g/l), epistaxis and a growing ulcerous wound in the pubic area (diameter mm) already pretreated with antibiotics for days. surgery was not possible due to poor heart condition and thrombocytopenia refractory to donor platelets. mu g-csf were administered subcutaneously daily for days resulting in neutrophil counts of . g/ and effective wound granulation and epithelialisation. the patient died of cardiac failure on day . sh was adnutted for infected hematoma of the left thigh. subcutaneous infection progressed due to severe neutropenia (neula-ophils < . g/l). incision and resection (ulcus diameter ram with deep invasion into the fascia) was performed. days later the defect measured x mm, reaching the knee joint, and the patient underwent a second surgical interventaon. enterococcus, staphylococcus epidermidis and bacterium xerosis could be cultured from direct swabs. therapy with g-csf at a dose of mu s.c. was started on day . neutrophils reached . g/i and g-csf was reduced to every other day. complete wound healing without any further surgical intervention was achieved by day and sh was dismissed. after discontinuation of g-csf the patient is well and has normal differential blood counts. we conclude that g-csf is successful in promotion of wound healing in mds patients due to enhancement of neutrophil production. leukemic conversion of mds during g-csf was not observed. we report on a year old male patient presenting in / with moderate thrombocytopenia~ transfusion dependent macrocytic anemia and normal., wbc. trephine biopsy showed hyperceuular marrow without fibrosis wzm trilineage dysplasia (mds/ra). cytogenetic analysis was ,xy. in / vasculilas ~as diagnosed. from / to / three cycles of gm-csf ( #g/m s.c. - days) were administered, resulting in both transient leukocytosis and increased platelet counts_ bone marrow aspirations showed dysplasia but no blast proliferation. in / vasculitis progressed, splenomegaly and hemolytic anemia developed requiring prednisolon. in / high dose erythropoietin was started ( iu/kg i.v. twice weekly) and continued till / . there was no change clinically, bone marrow smears and cytogenetics. in / the patient complained of pain in the lumbosacral region and neuralgia in both legs developed; a ct scan was negative. both pain and neurological symptoms (paraplegia and sensibility disorders) progressed. act scan and an mrt showed an intraspinal tumor (d -d ). "although severe thrombocytopenia refractory to high dose i.v. immnnoglobulin and platelet support (hla class i & lymphocytotoxic antibodies) developed, therapeutic laminectomy was performed in / , but only a part of the tumor could be resected. histologically the tumor consisted only of erythropoiesis with dysplasia without excess of blasts. wound healing was without complicatmns. after surgery gamma irradiation and therapy with ifnc~ ( #g s.c. times/week) were performed. the patient recovered totally from neurological disorders and is still alive but iransfusion dependent because of severe cytopenia. we conclude: intraspinal extramedullary hematopeiesis is a rare symptom in mds. althoughthis infiltration was diagnosed months after gm-csf a_ad hd-epo therapy, it could be induced by cytokine therapy. ( monoclonal antlbodles elther' with or without gam crosslinking. in addition, we added the cytokines. the phenotypic change of expression of fcy receptors was measured. ~o production and calcium flux using the dihydrorhodamine (dhr) and fluo- am methods, resepectively. there were no changes in expression of fcy receptors, but a significant enhancement of pmn activation via fc receptors by all three cytokines. we observed an increase of h production . fold by g-csf, fold by gm-csf and . fold by il- . a fcyriii-b specific monoclonal antibody fgr pmn (id ), which was alone unable to mobilize ca i+, together with all three cytokines was a potent stimulator. the effects of gm-csf and g-csf were calcium independent, in contrast, il- also enhanced calcium mobilization significantly. in summary, all three cytokines potentiate the fcy receptor activation of pmns and therefore play a significant role in inflammatory granulocyte activation as in leukocytoclastic vasculitis. g-csf is a hematopoetic growth factor required for proliferation and differentiation of hematopoeitic progenitor cells. it is now being successfully used to overcome neutropenias of various etiologies. recently, we demonstrated that rhg-csf induced neutrophils from patients with severe congenital neutropenia showed altered surface marker expression (upregulation of fcyri (cd } and cd and downregulation of fcvriilicd )) as well as decreased chemotaxis towards a variety of chemoattractants including fmlp. to separate the effects of the underlying disease from those of the rhg-csf therapy, we investigated neutrophils from patients receiving cytotoxic chemotherapy (n = ) and healthy adults (n = ) after application of rhg-csf. results: neutrophils from patients receiving daily application of rhg-csf (neupogen ~ #g sc.) were studied ex vivo one day before, three times during and - days after cessation of rhg-csf treatment. expression of fcvri, cd and cd (measured by flow cytometry) increased during therapy reaching a maximum at - days after initiation of rhg-csf therapy, whereas expression of fcvrll! decreased to a minimum after - days. chemotaxis of neutrophils under agarose towards fmlp was also reduced during therapy. investigation of surface marker expression and chemotaxis - days after cessation of rhg-csf revealed return to levels before therapy. to exclude the possibility that the observed alterations were caused by the underlying disease or chemotherapy, five healthy adults were treated with a single dose of rhg-csf (neupogen', pg, sc.). a continuous upregulation of fcvri and cd starting h after application with a maximum after hours (fc~l) and hours (cd ) and a downmodulation of fcrriii reaching a minimum at hours was observed. chemotaxis towards fmlp decreased to h after application and returned to normal after h, whereas expression of fcrri, cd and f%riii showed baseline values after hours. conclusions: the results obtained from the healthy test subjects clearly demonstrate that neither the malignant disease nor chemotherapy, but rhg-csf induced the profound alterations of fcr receptor and cd expression and chemotaxis in neutrophils in vivo. the continuous character of the surface marker alterations without appearance of subpopulations and the increase in cd expression suggests that preactivation rather than immaturity of rhg-csf induced neutrophils alone might be responsible for the observed phenomena. fraunhofer institute ita, nikolai-fuchs-sral e , w- hannover severe congenital neutropenia (scn) is a disorder of myelopoiesis characterized by a maturation arrest on the level of promyelocytes with absolute neutrophil counts below /pt in the peril~heral blood. in this study we investigated the expression of receptors for the granulocyte colony-stimulating factor (g-csf) on neutrophils from patients with scn during g-csf therapy. the normal g-csf receptor expression on neutrophils is in the range of - receptors per cell. neutrophils from scn patients express increased numbers of receptors in the range of - receptors per cell. the dissociation constant of the binding of g-csf to the g-csf receptor is not altered as compared to healthy donors. in contrast neutrophils from patients suffering from cyclic neutropenia express normal g-csf receptor numbers ( - receptors per ceil). in addition, we have compared the g-csf receptor cdna of neutrophils from healthy donors and scn patients using the polymerase-chain-reaction technique. we could not detect any major alterations in the g-csf receptor cdna in scn patients. preliminary cdna sequencing data also did not reveal any point mutation. from this data we conclude that there is no defect in g-csf receptor expression and no alteration in the sequence of the g-csf receptor mrna in scn. pediatric hematology and konstanty-gutschow-str. , d- hannover oncology, medical school hannover, severe congenital neutropenia is a disorder of myelopoiesis characterized by severe neutropenia secondary to either a maturational arrest of myelopoiesis at the level of promyelocytes (kostmenn's-syndrome; scn) or regular cyclic fluctuations in the number of blood neutrophils with a median anc below / ~ (cyclic neutropenia). we have treated patients with scn and patients with cyclic neutropenia. thirty of patients with scn and all patients with cyclic neutropenia responded to rhg-csf treatment with an increase of the median anc to above /ixl. the doses needed to achieve and maintain the response varied between . and i~g/kg/d. long-term treatment did not exhaust the myelopoiesis: the mean anc remained stable up to years of treatment. the increase in anc was associated with dramatic clinical responses: significant reduction of severe bacterial infections, reduction of intravenous antibiotic treatment episodes, and reduction of hospitalizations. no severe bacterial infections occured in any of the rhg-csf responders during long-term treatment. severe adverse events, most likely associated with the underlying disease, included the development of mds/leukemia in two patients, and osteopenia/osteoporosis in patients. these results demonstrate the benefical effects of rhg-csf treatment in severe congenital neutropenia patients. fifty-two patients (pts) (median age - years) with philadelphia chromosome positive (ph +) chronic myeloid leukemia (cml) have been treated with ifn ( x units/m ) within six months of diagnosis (median . months (mths), range - mths). we divided the pts into three groups according to sokars classification: low risk group (n = ), intermediate risk group (n = ) and high risk group (n = ). forty-three pts acheived a complete hematological response (chr) as defined by the houston criteria. the cytological response was evaluable in pts: pts ( . %) demonstrated a partial or major eytogenetical response (more than % ph i negative metaphases). the hematological and cytogenetical responses were influenced bsr the risk factors, os the percentage of chr and cytogenetical responses was higher for the pts from the low or intermediate risk groups ( % and % respectively) than for the high risk group ( % and %). transformation occurred in four pts who did not demonstrate a cytogenetical response. the estimated chance of surviving at three years was % for the overall population. toxicity was mild but ifn had to be internpted in four pts for cardiac (n = ), liver (n = t) or neurological (n = ) tocxic effects. these results confirm that ifn is a very effective treatment for cml. the effect of rhg-csf on platelets was studied in healthy volunteers with the thrombometer, a specially developed device which is described in detail. additionally, conventional aggregation tests were performed low doses of rhg-csf enhance functional platelet activity, as shown by significant acceleration of the occlusion of the thrombometer channel. similar results were found in conventional aggregation tests using collagen for induction. with g-csf concentrations of , and , ng/ml the time of response was significantly accelerated and the maximum response was observed in a higher proportion ofplatelets. however, the second phase of aggregation induced by epinephrine was significantly inhibited by , ng/ml g-csf. the expression of cd , cd and cd on platelets' surface was determined in ten patients before and niter administration of g-csf (facscan flow cytometer).quality controls were done by calculating the events positive for cd and cd , which were expressed in nearly t % of the platelets without being changed by the cytokine. the expression of cd in the platelets' surface however was significantly enhanced indicating a depletion of the c~-granules. no platelet aggregation was observed. cd expressed on thrombocytes' or damaged endothel cells' membrane is a receptor for macrophages. this property facilitates rapid adhesion of leucocytes to endothelium at regions of tissue injury as well as platetetleucocyte interactions at areas of inflammation and hemorrhage.-in contrast cd can also have an antiinflammatory function because exposure of tnfa-activated neutrophils to plasmatic cd inhibits their cdl -dependent adhesion to resting endothelium and superoxide production. ifn a has a unique activity in cml leading to complete and partial remissions in - % of the patients. to improve these results, we are currently treating patients with ph'+ cml with a combination of cytosine-arabiuoside at a maximum dose of mg/m z sc on days per week and ifn c~- b. ifn u- b is started at a dose of muim sc dally and escalated to the maximum tolerated dose. patients ( male, female, median age years) have been entered into the trial. patients have been pretreated with other regimen for a median time of months. patients are without pretreatment. the treatment has been well tolerated. besides the ifn a related side-effects some patients experienced gastrointestinal toxicity with nausea and vomiting after prolonged ara-c application. the median observation time in the study is now months and patients are still entered. up to now complete hematologic remissions have been achieved in patients, and partial ones in patients. the rate of complete hematologic remissions was higher in patients without pretreatment ( %) compared to patients who have been pretreated ( %). five partial eytogenetic remissions have been observed and minor reductions in the ph'+ cell done. all cytogenetic responses have been found in patients without pretreatment. we conclude that a combination of cytosine-arabinoside and ifn u- b is well tolerated in patients with cml. early results are encouraging. longer follow up times are necessary to evaluate whether combination therpy will give superior results compared to a txeatment with ifn a alone. during rlfn-~ therapy a minority of patients develops high-titered antibodies neutralizing the injected rlfn-a . the rlfn-c~-andbodies from six of such patients, who lost their clinical response to rlfn-c~ and showed a relapse of their leukemia ( cml, hcl) despite continuous rlfn-c~ a-therapy, as well as ifn-c~-specific antibodies from two patients with systemic lupus erythematusus (sle) were characterized. the anti-ifn activity was purified by sequential protein g -and rifn-c~ affinity chromatography and was found to consist only of igg-antibodies. these antibodies were further tested for their capacity to neutralize the antiviral and antiproliferative activity of various rifns-c~-subtypes. all six sera tested showed a common pattern of neutralization (mdbk-vsv bioassay) distinct from the sleantibodies. all six neutralized rifn-c~a and rifn-ak consistently with a higher titer against aa. three of the six sera neutralized aa, ak, ~c, ~c/j and m, but not m, m and some other subtypes. therefore, from the structure of the c/jl-hybrid, it seems that one epitope recognized by these three sera is at the nh -terminal half of the molecule. in contrast, the sle associated antibed]es neutralized the antiproliferative and antiviral activity of every subtype tested. these data indicate that the therapy-induced antibodies against rifn-c~ recognize very selected epitopes on the rifn-cd-molecule suggesting that only a part of the rifn-~ molecule is immunogenic. in vitro experiments indicate higly synergistic effects of combining ifn with cytostatic drugs such as anthracylines (a). in a phase i/ii-study patients (pts) with pro$ressive inoperable hcc were treated with e mg/m z weekly x and ifn mio iu/m s.c. x weekly for weeks, followed by one week off treatment. in case of at least no change (nc) and tolerable toxicity the therapy was continued. escalation of e in steps of mg/m z per cycle was attempted. pts characteristics: median age years ( - ); male , female pts. pretreated with a pts. toxicity and treatment: total number of cycles ; median ( - ) per pt. worst toxicity per pt (who): wbc ~ %, ~ %; platelets ~ %, ~ %; diarrhoea ~ %, ~ ~%; n~isea/vumitinq ~ %; ~ %; ifn related fever (maximal ~ in %. ifn-related wasting syndrome pts, no severe organ toxicity. divisione di ematologia -ospedale san camillo -roma from november to october six patients with acute leukaemia, who achieved their first complete remission with standard chemotherapy followed by autologous bone marrow transplantation (abmt), were consecutively treated with r.interferon alfa- a (ra-ifn). patients ( all and anll) were from ii to years old, of them ( anll and all) were reinfused with autologous bone marrow purged with asta-z i mcg/ml/ x cells and in the remaining all patients immunomagnetic purging was employed. conditioning regimens were bucy in patients and cy-tbi in the others, ra-ifn started at median time of months ( - ) after abmt when complete consolitated hemopoietic recovery occurred. the ra-ifn dose was . iu/sqm times a week for years. none of the patients presented significant toxicity and only short suspensions occurred for fever or alt level increase. the incidence of infectious complications were particularly low compared with other autotransplanted groups of patients who received similar antinfectious prophilaxis. one case six months after abmt and days after acyclovir prophilaxis interruption presented a mild herpes-zoster complication which required new acyclovir therapy and resolved i days later. the amount of these patients is extremely low because the study was early interrupted to start a new protocol including il- ; but the long duration of the good continous complete remission ( / years after abmt) in all these unselected and consecutively treated patients is very interesting. surgical procedures may be associated with an inceased risk of tumor spreading due to surgical mobilisation of the tumor and transient postoperative immunosuppression. recurrences may result either from early growth of micrometastases already present at the time of surgery or from the seeding of malignant cells shed during operative manipulation of the tumor. imrnunomodulators have been proposed to correct the immunological impairement induced by surgical procedures. from / to / , patients with advanced stage cancer underwent surgical resection with peri-operative interferon-alpha administration. patients received interferon alpha- a (roferon-a), by daily subcutaneous injection for fourteen days, starting on three days before surgery. incremental doses were , , , , and x iu for , , , , and patients respectively. peripheral blood lymphocyte (pbl) subset numbers were assessed using flow cytometric analysis the day before injection (d- ), before surgery (d-i), at the day and . absolute numbers of total t cell (cd +) and nk cell (cd +, cd -) were determined, as well as auxiliary t cell (cd +), activated t cell (cd +, dr+), and b cell (cd +) counts. short-term cytotoxicity of pbmc against k and daudi target celts in a -hour standard chromium release assay were determined. no w.h.o. grade iv toxicity were observed. a significant post-operative fall of the total pbl count, of cd +; cd +; cd +; cd - + occured from d- to d . the decrease were not significant for cd +dr+. values were not significantly different, between d- and d , only for cd dr+ and cd - +. cytotoxicity against k and daudi target ceils increased significantly from d- to d-l, and from d-i to d , with a significant fall of cytotoxicity against daudi from d- to d . peri-operative interferon alpha administration is well tolerated even at the . iu doses. in spite of treatment and increasing cytotoxicity activity, we observed a post-operative fail for the majority of the imunological parameters. further studies are necessary to compared with a control group, with more patients treated with . tiiu daily. patients: six patients have been treated (med. age yrs.; - yrs.) four patients had acute myeloid leukemia (aml m : , m : , m eo: ). two patients had acute lymphocytic leukemia (both t-all). all patients had manifest disease with more than % blasts in the bone marrow. patients were selected not to have rapidly progressive diesease allowing the application of the cytokine. treatment and toxicities: ifn a was given for a median of ( - ) days at a dose of - mu sc daily. the following toxicities _>grade who were observed: fever , gpt , pulmonal , infection ( pneumonia), pains . the patients with aml ware transsusion dependent for platelets and erythrocytes. no significant additional bleeding was observed. results: one aml patients had stable disease and three had disease progression. of the all patients one had disease stabilization and one progression. conclusions: ifn a is well tolerated in patients with refractory aml and all if they ere in a relatively stable condition. the effectivity of ifn a- b as a single agent is poor in patients with refractory aml or all. freund et al. eds, springer-verlag, ) have confn'med the wide range of clinical usefulness of ifn alphabased therapy in cancer patients (pts). we present in this paper a retrospective analyses of cases with advanced neoplasias treated between - by a sequential administration of ifn alpha and standard chemotherapy. there were w, m, aged - y, with solid cancers pats and lymphomas pts. the treatment consisted of a sequential association of ifn alpha (also with ra all-trans) and cht plus tamoxifen (for appropriate cancers). the ifn schedule was of monthly series, one series consisting of million iu/d for consecutive days. cht, appropriate for each primary cancer also administered in monthly series. the results are grouped according to the status of the disease (subsets of pts) at the onset of ifn-based therapy, and refer especially to long term survival ( - y +). for minimal residual disease (mrd) from cases there were cr ( / breast ca, / cr melanomas, each sts and rcc / rc, / gastric and head and neck cancers together, /t lymphoma. for progressive disease, pre-lfn-based therapy there were pts, and post ifn-cht treatment there were sd and pd. in of failure pts association of ifn alpha and bropirimine (ifn-inducer) appeared an unusual good response. conclusions: ) ifn alpha-based therapy is a very useful one especially in mrd. ) the therapy must be individualized for selected subsets of pts and for each patient day to day. authors have used inf alpha b in cases of haematological malignancies for three years. the number of cases not too high, and non fo cml cases was so called "early" cml. our cases are: cml, non-hodgkin malignant lymphoma, myeloma multiplex, essential thrombocythemia. the tnf alpha b was used as monotherapy in the cases of low grade non-hodgkin malignant lymphoma, and essential thrombocythemia, and was combined with chemotherapy in the other cases. on the basis of our initial results, we recommend the inf alpha b in the treatment of haematological malignancies in suitably selected cases. department of haematology and oncology, hospital of ministry of interior, vftrosligeti fasor - the influence of low oral doses of human leukocyte derived interferon alpha on the immune system of chronically hbv infected patients with depressed immunological response. low oral doses &the interferon were given to a group &seven children with limphoblastic acute leucaemia in the state of remission, chronically hbv infected. interferon alpha was produced by hayashibara biochemical laboratories inc. okayama, japan, in tablets of iu and iu respectively. immunological response was checked by measuring population and subpopulations of limphocytes, level ofimmunoglobulin and complement c fraction. a distinct stimulation of cellular immune response was observed: the fraction of activated limphocytes t increased significantly, index cd /cd became normal and population of limphocytes b increased gradually. there was no influence of interferon treatment on immunoglobulin and complement c fraction serum level. the interferon treatment improved the patients' general condition and shortened the period of intoxication after cytostatic treatment. no side effects were observed. none of the children eliminated the hb virus during the six months treatment. cytokines play an important role in activating the immune system against malignant cells. one of these cytokines, il- has entered clinical phase i trials because of its immunoregulatory potency. in the present study we report that rhll- has direct antiproliferative effects on some human lung cancer cell lines in vitro as measured by a human tumor cloning assay (htca). this activity could be abolished by neutralizing antibody against rhll- . the biological response of the tumor cells to the cytokine is correlated with expression of receptors for bll- on both the mrna level and the protein level. the most responsive cell line ccl secretes il- after being incubated with rhll- . on the other hand, neutralizing antibodies against il- showed no influence on the growth modulatory efficacy of rhil- in this cell line. furthermore, ccl does not show detectable production ot il- , tnf-~ or ifn-y alter incubation with rhll- . thus, the response to rhll- is not mediated through autoeriee production of these cytokines triggered by rhll- . in a next series of experiments the ceillines were xenotransplanted to balb/c nu/nu mice. subsequently, the mice were treated for > days with twice , mg/m rhll- (rhll- was a kind gift from dr. urdal, immunex, seattle, usa) or control vehicle subcutaneously per day. treatment with rhll- yielded a significant inhibition of tumor growth versus control in the responsive cell lines ccl and htb , but no therapeutic effect in the non-responsive cell lines. plasma levels of rhtl- were sufficient for in vitro growth-inhibition in the responsive lines. histology of the tumors in both groups showed no marked infiltration of the tumors with murine hematopoietic and lymphocytic cells consistent with the species specificity of il- . we conclude that rhll- has direct antiproliferative effects on the growth of some human lung tumor cell lines in vitro and in vivo which together with its regulatory effects on various effector cell populations makes this cytokine an interesting candidate for further investigation in experimental cancer treatment. the combination of systemic chemotherapy and immunotherapy comprising interleukin- and alpha-interferon leads to significant tumor regressions in patients with advanced malignant melanoma. in contrast to chemotherapy by itself, the combination produces a significantly extended remission duration in the majority of treatment responders. we conducted phase ii studies.to assess the potentially additive or synergistic effects of chemotherapy and immunotherapy in metastatic malignant melanoma patients: the first study comprised two cycles of carboplatin ( mg/m ) and dacarbazine ( mg/m~); the sesond study included up to four cycles of cisplatin ( mg/m x days), dacarbazine ( mg/m x days), bcnu ( mg/m , cycle i+ ) and tamoxifen ( mg daily). chemotherapy was followed by up to cycles of a -week immunotherapy comprising interleukin- ( - million iu/m ~ sc x weekly) and alpha-interferon ( - million u/m sc x weekly). among evaluable patients in study i, there were ( %cr, %pr) objective responders; median remission duration was + months for complete, and + months for partial responders. chemotherapy intensification in study ii lead to an increased response rate of % ( out of patients). in both studies, the progression free interval was significantly extended when compared to patients who received chemotherapy, only (historic controls). the role of immunotherapy as maintenance in patients with advanced metastatic malignant melanoma is currently being evaluated in a prospective randomized trial. integrin receptors play a crucial role in cell-cell and cell-matrix adhesive function, and thus are supposed to influence invasion and metastasis. very little is known about the impact of interleukins on integrin regulation in tumor cell lines. therefore, we investigated the expression of of and i integrin subunits on well (ht ) and poorly differentiated (sw ) human colon cancer cell lines using a panel of specific monoclonal antibodies and cdna probes. ht and sw expressed similarly high levels of ~ , a , ~ gt, and f~ subunits on the cell surface. no a , ~ , and ~ was detected on either cell line. while a was not expressed on ht , sw showed higher levels of the laminin receptor ~ . the poorly differentiated cell line sw was resistant to il- , whereas ht was sensitive. treatment with il- induced a decrease in ~ , a , '~ , ~v, l~l, and integrin expression. however, ~ subunit was markedly upregulated. in contrast to il- , there was no evidence that il-lfi could modulate integrin expression on these celt lines. the function of integrin receptors was assessed by measuring adhesion to collagen, laminin, vitronectin, and fibronectin. il- significantly increased the adhesion of ht to fibronectin, while attachment to collagen, laminin, and vitronectin remained unchanged. these results suggest differential integrin expression pattern on well and poorly differentiated tumor cell lines. we provide evidence that integrin expression may be selectively regulated by il- , but not by il-ib. furthermore, il- can alter adhesive behavior of tumor cells. since il- is currently studied in clinical trials, the metastatic potential of malignant tumors should be monitored thoroughly. immunotherapy with ifncz and il- is an active regimen in malignant melanoma and has shown response rates of - %. in previous studies no prognostic parameters for response could be identified. patients with progressive metastatic melanoma have been enrolled in various immunotherapy trials including ifno~ and high dose il- since with an overall response rate of %. patients with mm treated in our phase ii trials could be analysed to identify possible prognostic parameters for response. patients were divided into three groups: responder ( cr/ pr), stable disease ( sd/ mr), and nonresponder ( pd). all patients had measurable tumor, a karnofsky index of > %, no cns metastasis, and no severe cardiorespiratory or renal disease. we examined the following pretreatment parameters for prognostic relevance of response: age, sex, performance status, time from diagnosis to onset of first metastases/ to begin of immunotherapy, tumor toad, number of metastatic sites, organ sites of metastases, ldh, ap, esr, and hla-type. of these several variables were found to significantly correlate with response: tumor load (p= . ), number of metastatic sites (p= . ), serum ldh (p= . ) and ap (p= . ). tumor load, ldh and ap are no independent parameters. while time from diagnosis to onset of first metastasis is of no prognostic significance for response, the time between first diagnosis and begin of immunotherapy, usually reflecting metastatic disease necessetating systemic treatment, significantly correlates with probability of response (p= . ). since several hla class i alleles have been shown to function as restriction elements for recognition of melanoma cells by specififc t cells in vitro, namely a , a , b , and cw , we compared the frequency of these hla antigens between responder and non-responder. we found a , b and cw to be increased in responder vs. non-responder. our results indicate that in patients with mm tumor load, number of metastatic sites, ldh, and time from diagnosis to begin of immunotherapy are prognostic parameters for response to immunotherapy. these parameters may be useful to determine patients with good and poor risk for response to immunotherapy and are of relevance for stratification in randomized clinical trials. dept of medicine, university of heidelberg, hospitalstr. , heidelberg, germany surgery of metastatic melanoma following successful il- based immunotherapy. u keilholz , e stoelben , c scheibenbogen , hd saeger , k neumann , w hunstein surgery of advanced metastatic melanoma is of limited value and usually not recommended. immunotherapy using high dose il- is effective in a substantial proportion of patients, however, the duration of responses is limited, and benefits in survival are not yet proven. this evaluation was done to determine the value ot resection of residual tumor lesions following successful immunotherapy. patients with progressive metastatic melanoma have been enrolled in various immunotherapy trials including ifna and high dose il- since . patients showed evidence of antitumor response ( cr, pr, mr/sd). in patients responding to immunotherapy, residual lesions were resected, whenever technically possible and patients agreed to surgery ( patients). of the responding patients without surgery relapsed, the median time to progression was months (range - ), almost all initial relapses occured locally, patients died so far. of patients who underwent surgery ( pr, mr/sd) were converted into cr by surgery. of the patients disease-free after surgery relapsed, locally ( , , and months after surgery), and one cns months after surgery. patients are still free of recurrence ( +, +, +, +, +, +, + months after surgery) and of are still alive. in the patient with cns relapse complete resection of this lesion was again possible, and there was no evidence of recurrence for months after this second surgery. histology revealed vital tumor cells in almost all resected specimens, however in of patients profound necrosis of the tumor tissue was observed. of special importance is the observation that patients with minor response or sd according to imaging procedures were found to have an almost complete response histologically. interestingly, almost all metastases resected after immunotherapy had developed a fibrous capsule. surgical reevaluation and resection of residual lesions should be considered in patients with partial response after immunotherapy, and in selected cases also with stable disease. this approach offers the chance for extended disease free survival, and may be curative in certain patients. t-cell-receptor (tcr) vcz~ usage of tumorinfiitrating t-cells in primary, regressing and progressing melanoma metastases following [mmunotherapy with ifn~ and il- : evidence for a specific t-cell response. mshler, t., willhauck, m., scheibenbogen, c., pawlita, m.#, bludau, h.#, brossart. p.. keilholz. u. the identification and characterization of immunological effector cells mediating tumor regression in immunotherapy with il is of great interest for understanding and further development of this therapeutic approach. tumor infiltrating lymphocytes specific for autologous tumor cells can be expanded from certain melanoma tissues. t cells recognizing the same antigen use a limited tcr repertoire with a certain vc~ and [ variable region, determining the specifity of their receptor. we therefore analyzed t-cell receptor v-regio,q distribution in tumor tissue from melanoma patients prior to and following immunotherapy with il- . we used a highly sensitive rna-pcr method. after rna-extraction from tissue and subsequent cdna-synthesis semiquantitative pcr with different primers for all known vc~-and vii-t-cell receptor gene families ( vc~ and v~) was pedormed. tumor tissue samples were analysed including samples of primary malignant melanoma and tumor samples of three patients after immunotherapy. the results were compared to control tissues (peripheral blood, unatfected skin, and liver tissue). the analysis of primary malignant melanoma tissue showed a weak overexpression of different v[ -families. preferential usage of different tcr-v~,genes was more obvious in tumor tissue of patients alter immunotherapy. of special interest is a patient with a mixed response to immunotherapy with progressing and regressing skin metastases, in the regressing lesion we could demonstrate a predominant usage of tcr-v[~ -gene almost lacking in the progressing lesion. this suggests a role of v[ -expressing t-cells in mediating tumor regression in this patient. cloning and sequenzing analysis are currently performed to assess wether this represents a true clonal t-cell proliferation. recently a highly sensitive assay combining reverse transcription and polymerase chain reaction (rt/pcr) to assess for melanoma cells in peripheral blood has been developed. the detection of tyrosinase mrna, a tissue spezific enzyme in melanocytes and melanoma cells in peripheral blood indicates the presence of melanoma cells, we used rt/pcr assay to determine malignant melanoma ceils in peripheral blood of patients with malignant melanoma in different stages of disease. in none of patients with stage i (localised tumor) but in of patients in stage ii (regional lymph node metastases) tyrosinase transcripts were detected. tyrosinase mrna was found in all patients with distant metastases (stage iii). this method may be helpful to define a group of patients at high risk for development of hematogenous metastases, that would be a possible target group to explore adjuvant treatment strategies. we then examined blood samples and bone marrow aspirates of patients with metastatic malignant melanoma for presence of melanoma ceils prior to and after therapy with ifn-a and il- . patients showed antitumor response to immunotherapy: complete remissions (cr) and ? partial remissions (pr r. hilse, m.meffert, j.grosse, h.kirchner, h.poliwoda, and j.atzpodien we investigated the use of pcr for a semiquantitative estimation of cytokine expression patterns in pbmc before and after administration of il- to patients with advanced renal cell carcinoma or malignant melanoma, mrna of cytokines was measured using a modified polymerase chain reaction protocol, which could detect -fold differences in mrna-contents of stimulated pbmc in vitro. weekly rna-samples of patients receiving a total of treatment cycles were examined for long term changes, in patients frequent samples were taken immediately after tl- -administration for transcript-kinetics, mrnaexpression for il- , il- , il- , ifn-)', tnf-c~, gm-csf, tgf-~ and il- receptor-c~ was clearly detectable in most of the samples, including four healthy donors. however, our method could not detect significant changes in transcript-levels of pbmc during days following injection of (a) mio.lu or (b) x mioju dl- daily. this was in marked contrast to cytokine secretion assayed by elisa. thus, serum il- peaked - hours after administration followed by secondary cytokines with a peak - hours later. increases for tnf-m ifn- , il- and il- r serum levels were significant (p< , ) with the highest response found for il- , increasing -(a) and -fold (b) at day , or -/ -fold at day . comparing normal individuals to patients, only small differences in constitutive cytokine expression were seen (< -fold) with no distinct pattern. during therapy, changes could be seen for all cytokines except for il- and tgf-i . in one patient, a -fold increase for il- , tnf-c~ and ifn- transcripts was observed during week of the second treatmentcycle, other changes were approximately -fold. abt. h&matologie und onkologie, medizinische hochschule hannover, d- hannover , germany regional immunotherapy: perfusion of liver metastases with lak cells u. keilholz, c. scheibenbogen, m. brado, w. tilgen, and w. hunstein a regional approach of adoptive immunotberapy with interleukin- and lymphokine activated killer cells for the treatment of liver metastases is reported. the treatment consists of continuous infusion of interleukin- i.v. or into the splenic artery, and transfer of ex vivo generated lymplaokine activated killer cells into the portal vein or the hepatic artery. patients with malign ant melanoma, with renal cell carcinoma, and with thyroid carcinoma have been treated. all had progressive liver metastases. trafficking studies using indium-oxine labelled cells revealed that > % of the lak cells remained in the liver after regional adoptive transfer. in patients with liver metastases of cutaneous melanoma, cr ( and + months), pr ( + months, converted to cr by surgery), sd ( and months), ad pd were observed. the lesion in the patient with pr was resectable after two cycles of treatment, and histology revealed almost completely necrotic tumor tissue surrounded by a dense fibrous capsule. no responses were observed in patients with liver metastases of ocular melanoma, suggesting an immunologic difference between these two melanoma subtypes. pr ( months) and sd ( months) were achieved in patients with renal cell carcinoma, and sd ( months) in the patient with thyroid carcinoma. evidence for the crucial role regional cell transfer is provided by the observation in a patient with an anatomic variation of hepatic blood supply in whom we achieved complete and durable tumor regression. in this case anti-tumor responses were only observed in anatomic areas of the liver which were perfused with lak cells. depts. of internal medicine, diagnostic radiology, and dermatology, hospitalstrage , heidelberg, germany in a randomized phase ii study we evaluated the response and side effects of a combined administration of interleukin- (il- ) and interferon-alpha b (ifn-alpha b) versus interferon-gamma (ifn-gamma) in patients with metastatic renal cell cancer. patients in group a received subcutaneous (sc.) meg ifn-gamma once a week. in group b patients were treated with a (sc.) combination therapy of ill- ( x iu/m in week and , x' iu/m in week , , and , twice a day for days) and ifn-alpha b ( x u/m ) over weeks once a day times a week. up to now patients were treated, patients in each group. toxicity of ifn-gamma treatment was absent. the therapy with il- and ifn-alpha b led to sideeffects grade (who): fever, chivering, fatigue and weight-loss. treatment were withheld in %, follow up after months ( - months) showed stable disease in patients and progression inl patients in group a in group b there were complete remissions, partial remission and patients with progressive disease. although the combination therapy showed % objective response (p< . , fisher-test) no significant improvement on survival was seen (p = . logrank test). patients with locally advanced renal carcinoma are at high risk of relapse after initial radical surgery. we initiated a clinical phase ii trial using autologous tumor vaccines for the surgical adjuvant therapy of renal cancer patients. seventy-two patients (pts) ( female, male; median age, yrs; range, - yrs) with locally advanced renal carcinoma (pt b- pn or ptxni- m ) received autologous newcastle disease virus modified and lethally irradiated tumor vaccines in combination with . million iu of il- and . million u of ifn-~ , once weekly over consecutive weeks. toxicity was very mild with transient flu-like symptoms. among evaluable patients, there were relapses ( pts, pt ani- ; pts, pt bn ); the median relapse-free survival was + months with a range from to + months; survival probability in this vaccine treated cohort was significantly better than in all historic controls. using western blot analyses, we could demonstrate a vaccine specific in-vivo b-cell response in all patients receiving ndv tumor vaccine. a subset of peripheral blood natural killer (nk) cells has been found to exhibit high density surface expression of the nk associated cd antigen; it has been suggested that these nk cells respond to lower concentrations of il- when compared to the majority of nk cells expressing cell surface cd at low density. we evaluated density of the cd antigen on circulating nk cells of patients with advanced renal cell carcinoma by flow cytometry. patients received a combination of low-dose subcutaneous recombinant interleukin- (ril- ) at million iu/m /day on days and , followed by . million iu/m /day, days per week, over consecutive weeks, in combination with recombinant ~-interferon (rifn-~) at million iu/m , three times weekly. antigen density of cd before therapy was found . -fold higher (p< . ) in patients who subsequently achieved a complete or partial tumor remission (n= ) when compared with patients who presented with progressive disease on therapy (n=ll). after a -week treatment cycle, nk cells of treatment responders expressed significantly ( .l-fold; p< . ) more cd antigens than nk cells in nonresponding patients. these results suggested a potential role of both pre-and posttreatment nk antigen density levels as a biologic correlate to treatment response in tumor patients receiving low-dose ril- and rifn-a. intravesical immunotherapy against superficial bladder tumor recurrences and carcinoma in situ is a recognized and highly effective regimen in urology. to further clarify the mode of action of this approach, the local immune response of patients was investigated: the cytokines il- , il- , and tnf were determined in the urine before and after intravesical instillation by elisas and biological assays. furthermore, bladder biopsies taken before and after the treatment course were analysed by means of immunohistology for the presence of mononuclear cell subsets. the results show a significant increase of urinary cytokines with a maximum - hours after the instillation of bcg which returned to baseline values within hours. this intense locai immune activation was further reflected by the accumulation of activated mononuclear cells, predominated by t cells as demonstrated with bladder biopsies. the local t-helper/t-suppressor cell ratio shifted towards the t-helper subset. these changes persisted for more than year after the initial treatment course. in conclusion, this local immune response may be associated with the therapeutic success of bcg. further analyses will dissect the role of each factor with regard to antitumor cytotoxicity against bladder carcinoma. enhancement of therapeutic effect of intarleukin- (il- ) by association with cyclephcsphamide (cy) was studied on el-/+ lymphoma maintained in ascitic form in syngeneic c bl/ (h- b) mice and lymphoreticulosarcoma (spontaneous origin) maintained in solid form in syngeneic cba (h- k) mice. immunotherapy with il- (obtained by in vitro stimulation of el- lymphoma cells with phorbol myristate-acetate) was applied hours after transplantation of el- lymphcma and i days after transplantation of lym phoreticulosarcoma be administration i.p. (intratumorel) in el- bearing mice and s.c.(peritumoral) in lymphoreticulosarcoma bearing mice for three consecutive days. cyclophosphamide was administered at a dose of mg/kg i.p. six hours before the immune treatment with il- . the results obtained demonstrated that the prolongation of the survival rate expressed by the median survival time (mst) and the percentage of increasing llfe span (ils) of the groups treated with il- associated with cy was significantly higher than that of the groups which receveid a single treatment with il- or cy. enhancement of therapeutic effects of il- in association with cy on lymphoreticulosarcema was revealed by inhibition of tumor growth with a marked regression in the vol~tme of the established tumors and even resorption in some cases. we conclude that the antitumoral effect of il- treatment was enhancemented by association with c a well known cytoreductive drug which selectively removed t-suppressor l~mphocytes from the tumor bearers. this could be conside red as an alternative to immune-or chemotherapy in cancer. to investigate the toxicity and clinical efficacy of aerosolized nil- (biotest) patients presenting with advanced malignancy were entered into a phase i trial. patients suffered from metastasizing renal cell carcinoma, patients from advanced bronchial carcinoma. at start the patients received either , or u nil applied as a single dose. if no adverse events were observed, treatment was continued with the same dose times daily for six weeks. in addition to standard invetigations detailed evaluation of the respiratory function was performed once weekly. soluble interleukin receptor serum levels and the effect on numbers and/or phenotype of lymphocytes in the bronchoalveolar lavage fluid were measured for assessment of biological response to inhalative nil- treatment. treatment with aerosolized nil- was well tolerated. most prominent toxicity appeared to be resistant cough in all patients treated with x u/d. no febrile reactions or other constitutional side effects were observed. a dose-dependent increase of the numbers of memory t lymphocytes, macrophages and eosinophil granulocytes could be demonstrated in bal fluid. in addition, the treatment resulted an increased expression of adhesion molecules on lymphocytes. patient suffering from renal cell carcinoma achieved a partial remission after weeks of treatment with x u/d. we conclude that treatment with aerosolized nil- is biologically active and well tolerated and should be further tested in clinical phase ii trials. divisions of hematology and pulmology of the iiird department of internal medicine, medical center of the johannes gutenberg university, w- mainz, department of urology, univ. hospital eppendorf, hamburg. soluble interleukin- receptors (sil- r) exert a potential role in immunoregulation. we investigated the ex vivo effects of sil- r on several interleukin- (il- )-dependent activation events. proliferation of the il- -dependent mouse cell line ctll- and isolated human pbmc stimulated with recombinant il- (ril- ) was suppressed by sil- r added to the culture medium in a dose-dependent way. preincubation of sil- r with ril- did not enhance this suppression. cytotoxicity of ril- -stimulated human pbmc against the human cell lines k and daudi was correlated inversely to the concentration of sil- r in the culture medium during ril- stimulation. sil- r concentrations higher than . pm produced a significant decrease in cytotoxicity (p< . ). light microscopy of il- -stimulated pbmc revealed no signs of cellular activation when high dosages of sil- r had been added. the effect of different sil- r concentrations added to cultured human pbmc on secondary il- and sil- r production was tested by elisa. initial supply with high sil- r dosages yielded weak increase and subsequent slow reduction of il- levels. in contrast, strong secondary il- production followed by rapid clearance was observed when low sil- r concentrations had been added. endogenous shedding of sil- r in response to ril- was abrogated by the initial exogenous addition of high amounts of sil- r whereas low exogenous addition of sil- r was followed by a continuing endogenous production of sil- r after five days of culture. our studies may lead to a better understanding of il- -related immunoregulation in the preclinical and clinical settings. we investigated the effect of interferons (ifn) on expression of il- and other cytokines regulating inflammatory responses in various cellular models in vitro and in vivo. in peripheral blood mononuclear cells (pbmnc) of healthy individuals il- gene expression which was upregulated in vitro was significantly reduced in presence of ifn-o~. in dose titration experiments a reduction of the il- protein was detected at ifn concentrations as low as u/ml. in cml patients with constitutive expression of il- a reduction of il- mrna expression was seen after therapeutic administration of ifn-a. by contrast, in lps stimulated granulocytes ifn failed to inhibit il- expression in vitro. we investigated the mechanism of il- inhibition in the thp- cell line more in detail. nuclear run on assays and rna decay analysis in presence of acinomycin d suggested that the effect was regulated predominant/y at a posttranscriptional level. de novo protein synthesis was not required since the inhibitory effect was also detected in presence of cycioheximide. in addition to il- expression we studied the effect of ifn-o~ on the synthesis of il- , tnf, il- and il- ra in pbmnc and bone marrow stromal cell cultures. these experiments revealed an antagonistic effect of il- action by ifn x at two levels which was most striking in bone marrow stromal cells. expression of il- mrna was downregulated whereas the production of il- ra was enhanced by ifn-c~. in contrast expression of il- and tnf was enhanced by ifn. we conclude that ifn-o~ differentially regulates proinflammatory cytokines. the inhibition of il- and il- action suggest an antiinflammatory role of type i ifns. in a phase i clinical trial of recombinant human interleukin- (il- ), patients were entered to receive daily subcutaneous injections of il- over days followed by a two week observation period and another weeks of daily il- injections. doses varied between . and pg/kg body weight. patients were evaluable for studying immune functions. at all dose levels il- administration led to a marked increase in serum levels of c reactive protein and complement factor c . natural killer (nk) cell activity was reduced at doses exceeding pg/kg. similarly lymphokine activated killer (lak) cell activity induced by in vitro culture over days in the presence of u/ml interleukin- (il- ) was suppressed at and p.g/kg, as was the proliferative response to il- in vitro. however no changes were observed in the proliferation induced by phytohaemagglutinin, pokeweed mitogen or fixed staphylococcus aureus. there were no changes in peripheral blood lymphocyte subpopulations as measured by cd and cd , nor in the expression of hla dr. serum levels of immunoglobulins iga, igm and igg remained unaffected by il- treatment. in contrast we found consistent elevations in levels of ige all over the dose range. we conclude that il- inhibits nk and lak activity in vivo which may be of interest in future studies with cytokine combinations and that the role of il- in ige related diseases might be more important than previously thought. one of the most potent stimulatory agents for the induction of cytokines in myeloid cells is the bacterial ceuwallproduct lps (liopopolysaccharide or endotoxin). in the bloodstream it forms a complex with lbp (lps binding protein) and is recognized by effector cells via the cdi receptor. here we report on studies performed with human peritoneal macrophages that were stimulated in vitro with lps and a synthetic lps homologue in the presence and absence of serum. as revealed by elisa-based analysis of the cellsupernatants, strong, serum-dependent resonses were seen for tnf-, il- , il- and g-csf production, while unstimulated cells produced basically only il- . repeated stimulation of the cells with lps resulted in adaptation that was different for certain groups of cytokines. the "adapted" ceils produced much less tnf and il- while il-i and g-csf was superinduced. stimulation of the cells with the lipid a anolog mrl showed a similar picture, given that mrl had to be used in higher concentration. "adapatation" of the cells with mrl also resulted in an "adapted" response to a challenge with a high dose of lps so that it might be useful as a therapeutic agent for preventing the septic shock syndrome, e.g. northern blot analysis showed that the differentiated response of the cells towards a low dose lps stimulation regarding cytokine production after an lps challenge occured on transcriptional level, as mrna levels were regulated accordingly. these results give evidence that two different pathways for lps dependent stimulation of myeloid cells for cytokineproduction exist and experiments to further elucidate this phenomenon and possibly discover cdi independent and dependent pathways are underway. the neutrophil-activating peptide (nap- ), a member of the "intercrine"-family of chemotactic and reparative host defense cytokines, represents one of several n-terminally truncated cleavage products that originate from platelet-derived -thromboglobulin through proteolytic processing. here we present evidence that there exists also a naturally occurring c-terminally truncated form of nap- that is about four times more potent in eliciting neutrophil degranulation than the original cytokine. the novel molecule was detected in concentrates of culture supernatants from peripheral blood mononuclear ceils and could be separated from authentic nap- by several steps of column chromatography. according to amino acid sequence analysis it had a n-terminus identical to nap- , whereas electrophoretic analyses indicated a lower molecular weight as well as a higher isoelectric point. immunochemical analyses performed with epitope-characterized antibodies raised against nap- c-terminal synthetic peptides identified limited truncation at the c-terminus of the variant molecule. comparison of reactivity patterns of these antibodies in western blots as well as in a nap- biologic assay (pmn degranulation assay) confirmed that the variant nap- was truncated by at least one and by maximally three residues. thus, there is for the first time evidence that proteolytic processing at the n-terminus is not necessarily the only mechanism regulating the formation of neutrophilactivating peptides, but that modification at the c-terminus may assist in the fine-adjustment of biological activity. cytokines like interleukin- -beta (ill), interleukin- (il ), interleukin- (il ) and tumor-necrosis-factor-alpha (tnf) are involved in the pathogenesis of fever and infection. however, intra-and inter-individual values differ considerably and there is only limited data on early.cytokine serum levels and their evolution in febrile neutropenic patients. therefore. we measured cytokine levels in adults with chemotherapy-induced aplasia and fever. concentrations of ill. il and tnf were determined by irma. il by elisa in specimens per patient. ill and tnf were elevated in of patients with peak values of pg/ml and pg/ml, respectively. il and il were elevated in all patients with a maximum of . pg/ml (median pg/ml, range - pg/ml) for il and . oo pg/ml (median pg/ml, range - . ) for il . both cytokines showed a high correlation (r= . ). the individual il concentration-time curve closely paralleled the temperature curve. in of patients il was elevated before onset of fever and peaked at or one hour before the temperature maximum in seven cases. so also in the cytopenie patient lacking a main source of cytokine producing cells and cytokine target cells consistently high il and il serum levels can be detected very early in the course of fever and infection. the biological and clinical significance of this cytokine response and its regulation mechanisms remain to be determined. interleukin (il- ) and the neutrophil-activating peptide (nap- ) are two closely related members of the "intercrine" family of host defense cytokines. the expression of at least two different receptor classes for il- on human neutrophils (pmn) exhibiting similar affinities has been demonstrated recently. using iodinated ligands we could directly demonstrate that si-nap- specifically bound to pmn with two different affinities, characterized by kd-values of about . nm and . nm, respectively. cold -residue il- competed with iodinated nap- for binding to the high affinity site(s) with practically equivalent efficacy, while it was significantly more effective in displacing %nap- from its low affinity site(s) than was cold nap- itself. as new findings, unlabeled il- could completely displace ~ -nap- and vice versa, indicating that there are no distinct binding sites for either cytokine on pmn. in contrast to il- , nap- did not induce pmn degranulation at concentrations ( nm), engaging solely its high affinity site. however, short-term priming of pmn with the same amount of nap- dramatically down-regulated degranulation inducible by higher concentrations of nap- as a secondary stimulus. the il- -induced secondary response was also diminished, but to lower extents. these phenomena correlated with the rapid downregulation and internalization of nap- high affinity binding sites from the cell surface. thus, our data provide direct evidence for a regulatory function of nap- at very low concentrations, obviously occurring through the modulation of nap- and il- receptor expression on pmn. determination of cytokine plasma levels possesses many promising features concerning monitoring and studying of an array of different diseases including febrile reactions. detailed analysis of the role of these factors is of crucial importance for the understanding of the complex cytokine network. investigation of cytokine blood levels however is complicated by their short half-fife in circulation, the presence of soluble inhibitors and the ill-defined beginning of fever. looking for a suitable in-vivo-model allowing a sequential and well-defined analysis of cytokine plasma levels we chose the acute toxicity after intravenous amphotericin b (am b) application consisting of fever, chills and hypotension. these side-effects were reported to be mediated by release of pro-inflammatory cytokines such as tnf a and interleuldn (il- ). in order to compare mutual interations and different temporal patterns of liberation we determined a panel of cytokines including tnf a, s-tnfreceptor (s-tnf-r), interleukin- , interleukin- -receptor-antagonist (il- -ra), intefleukin- and interleukin- from patients suffering from acute leukemia and fungai infections. serial edta-plasma samples were obtained before and up to hours after start of am b infusion. samples were immediately centrifuged and stored at - ~ c until analysis by elisa (medgenix and r&d systems). patients experiencing adverse reactions showed tnf c~ peak plasma levels - minutes after starting am b infusion reaching maximum concentrations of pg/ml. concentrations declined m base levels within the following - hours. il- as welt as il- concentrations showed similar, but delayed changes of plasma concentration. compared to tnf a s-tnf-rlevels peaked about minutes later with a prolonged decrease to basal concentrations. in contrast to tnf c~ no circulating il-i- could be detected, while il- -ra demonstrated up to -fold increases in concentration. we conclude that this model of a drug induced acute-phase-reaction offers wide possibilities for studying the behaviour of inflammatory cytokines and their inhibitors by means of plasma level determination. inflammatory processes following severe trauma were found to be associated with an abnormal high secretion of inflammatory cytokines. these cytokines are discussed to be involved in neutrophil activation associated with the release of high amounts of destructive lysosomal proteases into the extracellular space. the task of our investigations was to evaluate the possible regulation of the degranulation of neutrophils by the immunostimulatory cytokine il- and the immunosuppressive factor tgf-i.~. we analysed the concentration of the cqantltrypsin-complex of the lysosomal protease elastase as markers for the degranulation of neutrophils as well as the levels of il- and tgf-i~ in the plasma of patients with multiple trauma or after severe surgeries. the time courses of the plasma levels of il- and the elastase-inhibitor-complex were found to be highly correlated, suggesting a possible regulatory role of this cytokine on the neutrophil degranulation. however the plasma concentrations of tgf-~, were not significantly altered in comparsion to the control group. in additional experiments, the effect of both cytokines on the degranulation of healthy donors was investigated in vitro. pathological high concentrations of rh~l- up to ulml (as detected in several probes from the surgical area) were found to be capable to induce a significant degranulation of the azurophilic granules ( , _+ , % of the total cellular enzyme content) under serum free conditions as detected by measurement of elastase release by elisa technique and enzymatic methods. in contrast to this, the degranulation of neutrophils was found to be uneffected by tgf-i~. in conclusion, these data suggest that the inflammatory cytokine il- may contribute to the activation of neutrophil granulocytes in acute inflammatory processes following severe irauma, whereas the immunosupresive factor tgf-fil seems to have no direct regulatory effects beside the described chemotactic effects on neutrophils. we determined serum concentrations of soluble tumor necrosis factor receptor (stnf-rs) in hiv infected individuals. eighty-five percent of these had increased serum concentrations of stnf-r type i (p ) (stnf-r ) and % had increased stnf-r type ii (p ) (stnf-r ). the extent of the increase of stnf-r was greater in more advanced hiv infection (p= . ) as it was measured by dividing the individuals into two groups according to the median of the cd + t cell count, stnf-r- did not differ between these two groups. a strong correlation was found between stnf-r and the soluble immune activation markers b -microglobulin (rs= . , p< . ) and urinary neopterin rs= . , p< . ), and a less strong correlation with interferon gamma (rs= . , p= . ). the correlations observed for stnf-r were also significant but were always weaker than that for stnf-r . a weak inverse correlation was found between the number of cd + t cells and stnf-r (rs=- . , p= . ), no such correlation was observed with stnf-r . our findings suggest that increased concentrations of serum stnf-rs in hiv infection are linked to immune activation where synergistic action of interferongamma and the tnf-alpha system are likely to play an important role. to answer the question whether il- is of hematogenous origin, immunohistochemistry and in situ hybridization with il- specific s labeled rna probes was established to study the frequencies of il- expressing peripheral blood mononuclear cells. a two color immunofluorescence assay with antibodies to cd and il- was utilized to correlate il- mrna expression and il- protein production in monocytes of patients and control individuals. - % of circulating monocytes spontaneously expressed il- mrna compared to % in normal individuals. a strong correlation of il- protein production and il- mrna was found in monocytes of patients and controls. semiquantification of il- mrna and il- i mrna by pcr demonstrated that about to fold higher amounts of mrna were found in untreated patients compared to normal individuals. in contrast, we did not find evidence for excessive synthesis of tnfa mrna. the overproduction of il- and il- in the absence of detectable tnfn mrna establishes a specific cytokine pattern in circulating monocytes of pmr and gca patients. we analyzed the il- mrna expression in biopsy specimens from gca patients applying in situ hybridization. autoradiographs were analyzed by visual examination and by using an image-analysis system. tissue infiltrating macrophages expressed il- mrna, however, the proportion of il- mrna + cd t was lower than in the peripheral blood. also, immunohistochemistry with an il- specific antibody demonstrated that only a small fraction of macrophages produced this cytokine. these data suggest that in pmr and gca, circulating monocytes are activated and produce il- and il- . tissue infiltration of macrophages is not accompanied by a local activation, in contrast, cytokine production is downregulated. our objective was to evaluate the prognostic value of p protein levels in patients with advanced hivinfection during cytokine therapy (ifn or ifn/combination). methods: p serum levels were measured every two months by elisa in hiv-infected patients during a period of to months (median , ) and compared with clinical stage (wr), disease progression and response to therapy. patients suffered from an infection or an inflammation disease during therapy and from kaposi sarcoma (ks). results: in patients with stable disease constant p levels (• ng/ml) were observed. changes of disease correlated with distinct increasing levels of p (in patients with acute infection and/or inflammation disease, in patients with progress of ks). after start of azt-therapy, chemotherapy and antibiotic/ antimycotic therapy p level decreased. the highest amount of p (up to ng/ml) was found in phases with opportunistic infections. the lowest amount of p ( ng/ml) was observed in a clinically stable patient without an evident progress of disease. conclusions: here, we detected increased levels of p in hiv-i infected patients under ifn-therapy during acute opportunistic infection. protein p may be a useful marker for the activation of the immunesystem and a prognostic marker during the course of ifn-therapy. previous studies have shown that the diacetylated synthetic pentapeptide splenopentin (dac-sp- ) accelerates hemopoietic recovery following sublethal irradiation and nutologons bone marrow transplantation in mice, but the effects of the peptide on human bone marrow cells were still unknown. in this study, human granulocyte-macrophage (rhgm-csf), macrophage (rhm-csf), and granulocyte colonystimulating factor (rhg-csf) as well as interleukin-l~ (rhil-l~) and interleukin- (rhll- ) were compared for their stimulatory activity on human granulocytemacrophnge colony-forming cells (cfc-gm) alone and in combination with dac-sp- . after depletion of accessory cells from bone marrow mononuclear cells (bmmnc) in semi-liquid cultures the combination of rhgm-csf plus splenopentin stimulated the growth of cfc-gm/-m in dose-depended manner. furthermore, similar effects were seen in combination of dac-sp- plus rhil-la and and rhil- , but not in rhg-csf and rhm-csf plus splenopentin. in unseparated as well as in bmmnc enriched for cd + cells comparable stimulatory effects of sp- alone were missed. additionally, in bmmnc enriched for cd +/cd + population, the preculture with dac-sp- for l days enhanced the ability of rhgm-csf, rhil-lct and rhll- to induce colony formation from this cell source. however, in all these combinations, mainly differentiation to macrophage lineage has been observed. pheuotypie analysis of precultured bmmnc with splenopentin leads to the suggestion that this compound may recruit a cell population being more sensitive to gm-csf, il-la and il- , because the percentage of cd + cells decreased rapidly, whereas the expression of hla-dr + was enhanced. therefore, this potentially interresting molecule might be a candidate as a therapeutic adjuvant with hemopoietic growth factors. although, the examination of gm-csf, il-lcc and . the role of t-lymphocyte subsets in the development of aplastie anaemia (aa) remains poorly understood. therefore we analysed by cytophomotry the contribution and proportion of lymphocyte subpopulationa in the peripheral blood (pb) and bone maxrow (ibm of patients with aa before, after weeks of treatment and additionally after weeks of therapy with anti-lymphocyte globulin (alg), methylprednisolon, and eyelosporine a (csa). for double labeling immtmofluorescence studies mouoelonal antibodies directed against the following specifities were used: tcrct~-, tcr~ , tcs , cd , cd , cd , cds, cd , cd , cd , and hla-dr. hi patients with aa a sigmficant decrease of cd positive t-cells was observed after weeks of therapy (pb , . , ; bm , • as compared with normal controls (pb , • bm , + , ). before therapy, the cd /cd ratio in pi and lqm did not differ ~om the ratio in the control population; however a reversed ratio (< ) was present in pb and bm after and weeks of therapy. the number of activated t-cells defined by the antigens cd , hla-dr and cd were low or in the normal range,and did not fu~er decrease during therapy in contrast to the non-activated t-cells. ? -t-cells were significantly decreased betbre and after weeks of therapy as compared with healthy controls. however, the proportion of the ~/~-subpopulation ~tcsi was markedly increased betbre (pb +_ , ; bm +_ , i) after weeks (pb , + , ; bm , +_ , ) and esspeciauy after weeks of therapy (pb ,.t~l . ) as compared with that in normal subjects (pb _+ , ; bm , _+_ , ). these data indicate that tcs -t-celis are a t-cell population not affected by treatment with alg, methylpredrtisolone and csa . ftwther studies have to show whether tiffs subpopulafion has an effect on the hematopoiesis of patients with aa. il- j , il- , tnf-i , g-csf and other cytokines are characterised to have proline in the second position of the n-terminal peptide sequence. from this they could be potential substrates of the dipeptidyl peptidase iv (dp iv). using the method of capillary electrophoresis, here we show that purified soluble dp iv is capable of hydrolysing oligopeptides with sequences analogous to the n-terminal part of human il-lb, il- , tnf-b and mouse il- up to a length of amino acids. furthermore, it could be demonstrated that hydrolysis rates are negatively correlated the with chain length of the oligopeptides. glycosylation of threonine in the third position of the il- hexapeptide sequence has no effect on the hydrolysis rate of this peptide by the dp iv. in contrast to these results, no degradation was found in the case of rll-lp, rll- , natural il- , and rg-csf, using up to -fold higher dpiv concentrations than in the experiments with oligopeptides. after incubation with both, dp iv and aminopeptidase n, also no cytokine degradation was found. possible explanations for this results will be discussed. hemopd~'etic growth factors are now being tested in several institutions, in an effort to reduce the duration of neutropenia after bone marrow trasnplantation (bmt). in the past years, we have conducted or participated to several double blind multicentric studies using gm-csf and g-csf (schering-plough/sandoz, behring/hoechst and roche lab.) in patients (pts) with nhl. we wish to summarize these studies and also report on our own observations. -gm-csf post-abmt: double blind international studies and the french national trial that we conducted (blood (blood , , (blood - have clearly demonstrated the gm-csf infusion post-abmt significantly accelerates neutrophil recovery by to days, both in pts receiving unpurged marrow or marrow purged by mafosfamide. the duration of hospitalization is reduced by days with a possible cost benefit. -in pts with documented cmv infection requiring dhpg treatment, myelosuppression was not seen in those who received gm-csf and dhpg concomitantly while in contrast a severe neutropenia developped in those who received dhpg after administration and discontinuation of gm-csf (lancet, , . - pts. in our institution received gm-csf in a compassionate use for delayed enraftment or engraftment failure after autologous (abmt : ) or allogeneic bone marrow transplantation (bmt : ). the pretransplant regiment included total body irradiation (tbi) in and consisted of high dose polychemotherapy only in . interestingly pts were transplanted for acute myelocytic leukemia (aml), a disease in which the presence of receptors to gm-csf has been detected in vitro in about % of cases, pts hat nhl, and all. of the abmt, pts received marrow heavily treated in vitro by mafosfamide and marrow purged by long term marrow culture (ltc). in (aml , all , nhl ), granulopoietic recovery occured within days ( - ). this included a pt with refractory all who received ltc marrow, developped cytomegalovirus (cmv) infection and had not engraftment by day : in this pt the absolute nucleated count (anc) peaked to . /i, days later. an additional pt with aml had not engrafted by day when gm-csf was administered for days with no efficacy. infusion of back up marrow (which has never been effective in our past experience) combined with gm-csf and cyclosporine a was follwoed by sustained engraftment occuring days later. dt (aml) had a minor and transient response and failed. additional pts in the context of entgraftment failure have since received in various sequences gm-csf and cyclosporine a + back up marrow after no response to gm-csf. have recovered sufficient hemopdfesis strongly suggesting a role of cycloporine a in engraftment failure (manuscript in prepration). we conclude that gm-csf is now the first line treatment for poor engraftment and/or engraftment failure. we do not freeze any longer back up marrow in pts autografted after pretransplant regimens not containing tbi. in pts with engraftment failure post tbi, who do not respond to first line gm-csf, we suggest that the combination of back up marrow and cyclosporine a on top of gm-csf should be further evaluated. -we administered gm-csf at a dose of #g/m /day to pts with resistant nhl and bone marrow involvement after the beam myeloablative regimen; reconstitution occurred within the same delays than observed after abmt in . we propose that gm-csf may replace abmt in highly selected cases of non-hodgkin's lymphoma with progressive disease and bone marrow involvement (lancet , , ) . -we have transplanted since september nhl pts with cd purified stem cells followed by infusion of gm-csf. successful engraftment was obtained in all with recovery to . x pmn/i by day ( - ) and to x plts/l by day ( - ). the expansion of cd + cells in short term liquid culture with cytokines (including gm and g-csf) is another approach toward transplantation with total abrogation of neutropenia. overall the introduction of hemopoietic growth factors in transplant units has considerably changed the situation in several aspects. further studies will combine gm-csf and g-csf to other cytokines. dept. of hematology, bone marrow transplant unit -h pital st-antoine, paris, france alter a pre-phase of vcr . rag/@ (maximum rag) i.v. days , and pradnisolone rng/n~ p.o. days - the high-dose chemotherapy consisted of prednisolone rag/n? p.o. days - , ifosfamide days - , methotrexate , mg/m day as a hour infusion, cytosine-arabinoside , mg/m i.v. days + , and etopeside i.v. days + . etoposide has been escalated from mg/rn to rng/ nf at the present time. the dose of itostamide is currently escalated from , rag/ rlf to , mg/rtf and finally , mg/rn as a continuous hour infusion. the high-dose chemotherapy is repeated for a maximum of four times. during the prephase pg/kg fiigrastim (recombinant g-csf) are given twice daily. apheresis of apbsc is done on days - . apbsc are reinfused after the high-dose chemotherapy and fiigrastim is given at a dose of p.g/kg. with wbc rising to > , /p.i aphereses are performed repeatedly. the pre-phase for induction of pbsc was excellently tolerated and a median of we report here the effects of long-term g-csf subcutaneous administration in patients (congenital n= , cyclic n= , idiopathic n= ) treated for - years. a sustained anc response was seen in / congenital patients, in / cyclic patients, and in / idiopathic patients. the g-csf doses needed to maintain these responses ranged between and #g/kg/d. the anc responses were associated with a significant decrease in the incidence of severe infections and the need for intravenous antibiotics. g-csf has been well tolerated in the majority of patients and resulted in a dramatic improvement in the quality of life. the adverse events noted included: osteopenia (n= ), vasculitis (n= ), mesangioproliferative glomerulonephritis (n= ), and the development of mds/leukemia (n= ). these adverse events are most likely associated with the underlying disease and not caused by g-csf treatment we have evaluated recombinant human granulocyte-macrophage colonystimulating factor (rhgm-csf) (sandoz-schering/plough) as an adjunct for cop-blam in the primary treatment of high grade malignant non-hodgkin's lymphomas (nhl). patients (n = , stage ii-lv, age - years), were randomized to rhgm-csf ( #g) or placebo for days s.c. following chemotherapy. efficacy was analyzed for patients receiving at least % of study medication (n = ). the frequency of clinical relevant infection was reduced by rhgm-csf ( vs infections, vs patients, p = . ) with a cumulative probability of remaining infection free in % vs % (p = . , log rank test at days). periods of neutropenia (p _< . in / courses), days with fever ( . vs . , p = . ) and days of hospitalization for infection ( . vs . days, p = . ) were significantly reduced. complete response (cr) rates, assessed by prognostic risk patients (pts) entered an outpatient protocol designed to test the tolerance, and the clinical and biological effects of extended administration of sc low-dose il- following high dose chemotherapy with autologous bone marrow rescue (bmt). two il- regimens were used. protocol a consisted of a once daily dose administered in -day cycles of millions iu/m z sc ril- (ru kindly provided by roussel-uclaf) every other week. the pts were treated at home and the treatment was scheduled for months (i.e. cycles for months) in the absence of major disease progression or sideeffects. protocol b consisted of a once daily dose of millions iu/m sc il in days/cycles for consecutive weeks (i.e. cycles for weeks). il- was started to days following bmt. blood lymphocyte subsets and nk/lak cytotoxic activity were determined monthly. pts received regimen a and pts were given regimen b. in both regimens, inflammatory skin reactions were the main side effect, leading to interruption of treatment in case. no capillary leak was observed. flue like syndrom was occasionny observed in protocol b. hematological parameters were not adversely affected by il- . at the lowest dose levels (regimen a), long term administration of il- did not produce any changes of blood lymphocyte subsets. on the other hand, the administration of millions iu/m il- resulted in a significant increase of cd +, cd + cells and cd -cd + ceils, and nk/lak cytotoxic activity of fresh pbls. this study confirms the feasability of long-term administration of sc lowdose il following autologous abmt. a dose of millions iu/m /d resulted in detectable activation of circulating lymphocytes. further studies are needed to assess the clinical impact of prolonged low-dose il- in this clinical setting. aim: to study the incidence of infectious episodes (ie) in a single centre series of autologous bmt patients who received il- in an attempt to prevent relapse. patients. methods: il- was given as a oontinuous i.v. infusion through a hickman catheter with a portable pump: . iu/m /d for the first week and . iu/m /day thereafter during weeks. il- was started when full neutrophil engraftment was achieved and platelet counts remained stable over . platelets/mm (median: day + . range: - ).median age was years ( - ). were mate. bmt was performed for acute lymphoblastic leukemia ( ), lymphoma ( ), and myeloma ( ). results: il- planned therapy was completed in patients ( early relapse, refusal) in a median of days ( - ). median intensity dose of il- was . x iu/m /day ( . x - . x ). infectious episodes who precised admission to the hospital were observed in patients. ol these infections were microbiologically documented: % of isolations corresponded to gram negative and % to gram positive. first ie was detected at a median of days ( - ) after the starting of il- . median cumulative tl- dose until first infectious episode was x iu/m ( . x ~' - x loe). catheter were removed in of them. in our experience, low dose continuous i.v. administration ol il- is associated with a high incidence of infectious episodes. sr (sanofi recherche) is a cho-derived r-il , glycosylated on the threonine in position . its specific activity is identical to native il . dose escalation studies were performed using sr either as iv bolus or iv continuous infusion in advanced-stage cancer patients. treatment schedule consisted in three -day cycles with -day rest in between. pts received iv bolus q - hrs at dose levels ( ; ; ; millions iu/m-'/bolus) and pts received iv continuous infusion at dose levels ( . ; . ; ; ; millions iu/m /day). mtd were found to be millions iu/m q hrs/day and millions iu/m /day for bolus and continuous iv infusion respectively. toxieities were similar in nature to those described under treatment with non-glycosylated rll- . dlt were mainly related to capillary leak. pr (nhl) was obtained. a significant rise of t-cell and nk cell subsets as well as nk/lak cytotoxicity in blood was observed at day . stimulation was already maximum for the lowest dose levels. at . millions iu/mz/day, the mean number of cd + cd + cells increased from to /mm -~ on day . similarly, cd + cells increased from to /mm , and cytotoxicity of fresh pbl against k increased from % to % (e/t ratio = : ). in vivo, sr appears to exhibit effects similar to those observed with higher doses (x - fold) of non glycosylated rll . although intravesical therapy with bacillus calmette-gutrin (bcg) against superficial bladder cancer recurrences and carcinoma in situ is highly effective, its mode of action is still unclear. the bladder tumor cell lines bt-a, bt-b (grade transitional cell carcinoma), sbc , and sbc (grade transitional cell carcinoma) are nearly resistant to natural killer cell activity in vitro. we could demonstrate that these cell lines are susceptible to lymphokine-activated killer cells generated by interleukin (il)- or interferon (ifn)-% we have now investigated whether bcg itself is able to activate a lak cell-like reaction against bladder tumor cells. our results show that activation of pbmc with viable bcg is a potent way of generating cytotoxic effector cells against bladder tumor cells. in contrast, killing of the nk and lak cell-sensitive k cell line was not enhanced by bcg-induced pbmc. because of their different target cell pattern and their, compared to lak ceils, distinct way of activation these cytotoxic effector cells were termed "bcg-activated killer (bak) cells". antibodies neutralizing ifn-t activity blocked the induction of bak cell cytotoxicity, indicating that this cytokine is playing a crucial role during this process. with respect to the phenotype of bak cells, our data show that the effector cells belong to the cd +/cd § lymphocyte subset. depletion of either cd + or cd + cells from bcg-induced pbmc led to a decrease of cytotoxicity against bladder tumor cells. furthermore, positively selected cd + cells could maintain the level of cytotoxicity exerted by bcg-induced pbmc whereas the depletion of cd § cells from this population also eliminated the cytotoxic effect. a direct involvement of cd + cells or macrophages in the killing of bladder tumor cells was not observed. in patients with superficial bladder cancer lak and bak cells might play an important role in the maintenance of the relapse free state. in this report we summarize our experience with high-dose therapy and peripheral blood stem cell (pbsc) autografting in advanced malignant lymphoma. since may , patients ( male / female) were included into this study. the median age was years (range - ). patients had hodgkin's disease and non-hodgkjn's lymphoma ( low-grade / highgrade nhl). four patients received recombinant g-csf (filgrastim) ( i~g/m / day s.c.) during steady-state hematopoiesis, while in the remaining patients recombinant g-csf (filgrastim) was started hours after conventional chemotherapy. for all patients, a target quantity of . x /kg total nucleated cells (tnc) was reached. a wide interindividual range with respect to the level of circulating hematopoietic progenitor cells ( -fold for cfu-gm/ml and fold for cd + cells/id) was observed. with a median number of leukaphereses (range - ), a median of . x cfu-gm/kg bw and . x cd + celts/kg bw could be harvested, respectively. high-dose therapy consisted of either tbi ( . gy, hyper-fractionated)/cyclophosphamide ( mg/kg) or the beam-protocol. with the exception of patient who died of a respiratory distress syndrome days following autografting, transplant-related toxicity was moderate. the low toxicity is reflected by the kinetics of hematological reconstitution: (median) days for . x / pmn and days for x ~/i platelets. platelet reconstitution was closely related to the number of cd + cells reinfused. twenty-six patients are evaluable for response: patients ( %) relapsed after a median of months (range - ) posttransplant. the remaining patients are alive in remission with a median follow-up of months (range - ). in summary, recombinant g-csf (filgrastim) is highly efficient in mobilizing pbsc capable of restoring hematopoiesis after myeloablative conditioning therapy. the quantity of cd + cells harvested was inversely related to the amount of previous cytotoxic chemotherapy. therefore, high-dose therapy with stem cell support should be considered as an upfront treatment modality in poor prognosis nhl patients.department of internal medicine, university of heidelberg, heidelberg, germany. many of the in vivo effects of the haemopoietic cell growth factors may well have been anticipated based on their known ability to stimulate proliferation and development of multipotent and lineage-restricted progenitor ceils in vitro. what was not predicted from these in vitro studies, however, was the observed ability of at least some of these growth factors to mobillse large numbers of haemopoietic progenitor cells from the bone marrow into the peripheral blood. this was an added "oonus" effect of growth factors and one that is now being widely exploited in a variety of clinical situations.initially these mobilised pbpc were used, in combination with bone marrow cells, to facilitate the more rapid recovery of myeloid cells following transfer into patients receiving high dose cytotoxic therapy. but when experimental studies showed that the mobilised pbpc also contained primitive (repopulating) stem cells -this encouraged the use of pbpc alone as a source of cells for transplantation. in most of these studies however, collection of the pbpc was performed over several cycles of apheresis -and this was somewhat limiting to their widespread use.because of this, our approach has been to define the optimal growth factor regimen for mobillsation of pbpc such that apheresis is not required, i.e. to use whole blood and then to identify groups of patients who may benefit from receiving these cells either for autologous or for allogeneic marrow recenstitution. using a sensitive immunohistochemical technique, . -> . logs of breast cancer cell depletion was documented in the positively-selected fractions of marrow ( patients) and -> logs from the pbpc fractions ( patients), in whom tumor was initially detected. to date no tumor has been detected in the cd + pbpc fractions. the engraftment rates for cohorts and were significantly faster than those of the other cohorts. the preliminary data suggest that since cd + pbpcs alone are capable of restoring hematopoiesis following high-dose therapy, a marrow fraction may no longer be needed for this purpose. longer follow-up will be required to assess the ultimate therapeutic effect of the entire treatment program. the principal morbidity and mortality of high dose chemotherapy with autologous bone marrow support (abmt) relates to the infectious complications which occurs during - week aplasia until the marrow autografi recovers. progenitor cells can be mobilized into the peripheral blood compartment by hematopoietic growth factors, alone or used after chemotherapy. we describe four trials using cytokine-mobilized peripheral blood progenitor cells (pbpc). in the first trial, pbpc collected after gm-csf administration are used to augment marrow. reconstitution of tririneage marrow function occurred quickly, resulting in short hospital stays and fewer platelet transfusions. in a second study, gm-csf/chemotherapy-mobilized pbpc were used as the sole hematopoietic support during high dose chemotherapy. granulocyte and platelet reconstitution was rapid. time to hematopoietie recovery, transfusion requirements and duration of hospital stay were all significantly improved for the patients receiving pbpc compared with similar patients receiving marrow alone. however, some patients had poor platelet engraftment. two recent trials were designed to explore multiple high-dose therapy. in the third trial, pbpc with and without marrow made it feasible to deliver two sequential cycles of high dose therapy. the fourth trial utilizes pbpc in addition to cytokines to deriver four cycles of dose-intensive therapy utilizing doses of chemotherapy that could not be given with cytokine support alone. pbpc appears to make multiple course combination high dose therapy feasible, is particularly useful to support platelets, and may enhance the safety, tolerance and cost of high dose therapy. severe neutropenia and functjonal defects of neutrophils are an increasing problem in treating advanced hiv infected p. neutropenia may result from bone marrow failure due to hiv by itself, or maybe.secondary to drug toxicity (mainly zidovudine, ganciclovir, chemotherapy) or bone marrow infiltration (mycobacterial infection, neoplasms). p. with severe neutropenia are at a higher risk of developing bacterial infections. since both gm-csf and g-csf have been-successfully used in cancer to optimalize dose intensity and to prevent neutropenia and secondary infections in p. receiving myelotoxic agents, clinical trials have recently been conducted in arc and aids p. with neutropenia to determine their potential benefits and toxicity profile. our previous experience in treating p. with non hodgkin's lymphoma (nhl) with a -cycle third generation regimen (promnce-cytabom), with gm-csf given at alternate cycles, clearly demonstrated that doses of chemotherapy following the adjunction of gm-csf were significantly higher than doses given without prior gm-csf administration. gm-csf was largely well tolerated in those p., with no sign of active viral replication as measured by hiv- p antigenemia. we have also performed an open clinical trial in p. with severe neuttopenia to assess the efficacy and tolerance of gm-csf with a three weekly subcutaneous administration in p. who initially responded to a daily dose. out the enrolled p. (median duration : days) showed a rise of the absolute neutrophil count (anc) above the target value of /mm after a median period of day at mcg/kg/d. only / p. were given antiretrovirals while other concomitant drugs included mainly ganciclovir ( ) and pyrimethamine ( ). of the p. who entered the maintenance phase at weekly doses, ( %) showed a sustained increase of the anc > , while in the remaining p, a new episode of neutropenia was observed.in contrast to p. with nhl, adverse reactions were commonly observed, mostly fever ( %) and flu-like symptoms ( %). furthermore among the p. without antiretroviral therapy who were p ag negative at baseline, a detectable p antigenemia was found in p. these preliminary data on this original schedule of gm-csl = administration was effective in preserving anc levels at > in % of the responders. such an approach could enhance p.'s compliance and have a direct impact on the cost/benefit evaluation of such therapy. in addition, antiretrovirals should always be associated with gm-csf to reduce the increased risk of viral replication. attempts to further improving the curative treatment for aml have to overcome the problem of residual disease represented by leukemic cells surviving in a therapy resistant state. a novel approach aims at recruiting those cells into a sensitive state by the use of gm-csf before and together (priming) with chemotherapy. in vitro, the recruitment of leukemic blasts to colony forming cells in presence of gm-csf (blood : , ) is documented by numerous reports. given to patients with newly diagnosed aml gm-csf priming decreased the proportion of leukemic cells in go and increased cells in sensitive cycle phases within - h (blood : , ) . a similar priming with preinfusion of gm-csf for a variable period of - days before chemotherapy started resulted in significantly inferior outcome and more persistent leukemias than in historical controls suggesting protective effects of gm-csf (blood : , . in a first study we showed that gm-csf following chemotherapy in high risk aml effectively reduced both neutrophii recovery and early mortality and had no adverse impact on leukemic regrowth and remission duration (blood : (blood : , . in current randomized study in patients with newly diagnosed aml we give gm-csf from h before chemotherapy and then on to neutrophil recovery which is repeated in each of the initial treatment courses and is compared to chemotherapy alone. / years from study start and after entering patients (median age , range - years) this update in the gm-csf group and controls shows % and % cr, and persistent leukemias and a clearance of day b.m. blasts to < % in % and in %, and remission duration shows a trend in favour of the gm-csf group. in addition to recruitment, other effects of gm-csf like enhancement of ara-c cytotoxicity (leukemia : , ; ch. router et al. leukemia in press) may contribute to this strategy. thus, gm-csf appears not to antagonize antileukemic chemotherapy. whether gm-csf priming and longterm administration ultimately improves the cure rate in aml should be shown some later from the multiple course strategy used in this study.medizinische universit~itsldinika (h~imatologie/onkologie), albert-schweitzer-str. , d- mfinster in this multicenter trial was evaluated, whether rhu gm-csf given concomitant and after chemotherapy (ct) can improve the outcome of aml patients by increasing the cytotoxic effect of ct and by reducing the rate of infectious complications. induction and early consolidation therapy included cytarabine (ara-c, mg/m , day - civi), daunorubicin ( mg/m , iv, day - ,) and etoposide ( mg/m , day - , h iv infusion) with reduced dosages in the second induction and early consolidation course. late consolidation included one cycle with high-dose ara-c ( g/m , doses) and daunorubicin ( mg/m , day - ) for patients aged years and younger, whereas patients over years received a reduced dose of ara-c ( . g/m , doses). patients were randomized after the first induction course to receive either rhu gm-csf (e. coil, pg/m /day, s.c.) or placebo starting hours prior to the second induction and the subsequent courses and given throughout chemotherapy until absolute neutrophil count had recovered > /pl. eighty out of patients (median age = years) included into the study could be randomized to receive either gm-csf (n = , median age = years) or placebo (n = , median age = years). out of patients ( %) achieved a complete remission (cr). patients ( . %) were treatment failures. two patients died and in one patient the response to therapy was not evaluable. there was no statistically significant difference in cr rate between patients of the gm-csf ( %) and placebo group ( %) nor between patients aged years or less and those over years old. the proportion of relapse free survival at a median follow up of months is % in the gm-csf versus % in the placebo group (p = . ). the proportion of survival of the gm-csf group at months is % versus % in the placebo group (p = . ). gm-csf did not significantly shorten the period of critical neutropenia and prolonged the period of critical thrombocytopenia especially in patients aged over years. the overall incidence of infectious complications as well as the non-hematological toxicity was similiar in boths groups. thus gm-csf therapy is feasible in aml therapy while its influence on remission quality and survival remains still open.dept. int. med. iii, university of uim, uim/d, frg.al key: cord- -g lajsp authors: han, jae-ik; chang, dong-woo; na, ki-jeong title: a multiplex quantitative real-time polymerase chain reaction panel for detecting neurologic pathogens in dogs with meningoencephalitis date: - - journal: j vet sci doi: . /jvs. . . . sha: doc_id: cord_uid: g lajsp meningoencephalitis (me) is a common inflammatory disorder of the central nervous system in dogs. clinically, me has both infectious and non-infectious causes. in the present study, a multiplex quantitative real-time polymerase chain reaction (mqpcr) panel was optimized for the detection of eight canine neurologic pathogens (blastomyces dermatitidis, cryptococcus spp., neospora caninum, borrelia burgdorferi, bartonella spp., toxoplasma gondii, ehrlichia canis, and canine distemper virus [cdv]). the mqpcr panel was subsequently applied to cerebrospinal fluid (csf) samples collected from dogs with me. the analytic sensitivity (i.e., limit of detection, expressed as molecules per µl of recombinant vector) was . for cdv, . for ehrlichia canis, . for bartonella spp., . for borrelia burgdorferi, . for blastomyces dermatitidis, . for cryptococcus spp., for neospora caninum, and . for toxoplasma gondii. among the tested csf samples, seven ( %) were positive for the following pathogens in decreasing order of frequency: cryptococcus spp. ( / ), blastomyces dermatitidis ( / ), and borrelia burgdorferi ( / ). in summary, use of an mqpcr panel with high analytic sensitivity as an initial screen for infectious agents in dogs with me could facilitate the selection of early treatment strategies and improve outcomes. meningoencephalitis (me) results from concurrent inflammation of the meninges (meningitis) and brain (encephalitis) [ ] . me can be classified as granulomatous meningoencephalomyelitis, necrotizing meningoencephalitis, or necrotizing leukoencephalitis based on histopathologic features [ ] . however, in veterinary practice, this classification is challenging because it requires biopsy and histopathologic examination. thus, a simple and rapid strategy for identifying the causative agent of me would be useful for selecting an appropriate therapeutic regimen. multiple infectious agents can cause me in dogs. [ , , , , , , ] . the pathogenesis of me can also involve immune-mediated or idiopathic etiologies that provoke aseptic inflammatory responses in the central nervous system (cns). rapid and reliable detection of the causative pathogens of me is essential for implementation of timely and effective therapeutic strategies. routine techniques in veterinary diagnostic laboratories include bacterial and fungal cultures, immunohistochemistry, antigen-capturing enzyme-linked immunosorbent assay (elisa), and polymerase chain reaction (pcr) [ , , , , , , ] . these methods are laborious, expensive, and time-consuming, and their sensitivity depends on specimen quality, timing of sampling, and prior antimicrobial therapy [ ] . furthermore, different assays for detecting the various pathogens have variable sensitivities and specificities, leading to biased outcomes [ ] . recent studies have used multiplex quantitative real-time pcr (mqpcr) as a diagnostic tool for multifactorial diseases because this approach can simultaneously detect multiple pathogens with good diagnostic performance and is faster than conventional methods [ , ] . the present study was conducted to develop an mqpcr-based diagnostic approach for me (hereafter, me mqpcr) that simultaneously detects eight infectious agents (b. dermatitidis, c. neoformans, n. caninum, b. burgdorferi, bartonella spp., t. gondii, e. canis, and cdv) and to assess the prevalence of these pathogens among cases of canine me in south korea. type strains of c. neoformans (atcc ), staphylococcus aureus (atcc , , baa- , baa- , and baa- ), escherichia coli (atcc ), and malassezia pachydermatis (atcc ) were purchased from the american type culture collection (atcc; usa). field isolates of b. burgdorferi (n = ), t. gondii (n = ), e. canis (n = ), e. ewingii (n = ), anaplasma phagocytophilum (n = ), candidatus mycoplasma haematoparvum (n = ), s. pseudintermedius (n = ), candida spp. (n = ), malassezia spp. (n = ), microsporum canis (n = ), microsporum gypseum (n = ), aspergillus spp. (n = ), canine parvovirus (n = ), canine coronavirus (n = ), and cdv (n = ) were identified at chungbuk national university veterinary diagnostic laboratory. recombinant plasmid vectors for b. dermatitidis, c. neoformans, n. caninum, b. burgdorferi, bartonella spp., t. gondii, e. canis, and cdv were used to assess the analytical specificity and as positive controls. nucleic acids (dna and rna) extracted from whole blood in two dogs were used as negative controls. all field isolates were identified by culture, conventional pcr, or antigen-capturing elisa and verified by dna sequencing. the vectors were constructed by amplifying synthetic oligonucleotides based on sequences obtained from the genbank database (national center for biotechnology information, usa) using the primers listed in table and cloned into a t-blunt pcr cloning kit (solgent, korea) according to the manufacturer's instructions. correct insertion into the vector was verified by sequencing. each recombinant vector was prepared in diethylpyrocarbonatetreated distilled, deionized water as a series of -fold serial dilutions and then used to assess the analytic sensitivity of the test. total nucleic acid was extracted from the samples using the magmax total nucleic acid isolation kit (applied biosystems, usa) according to the manufacturer's instructions. briefly, l of each sample were added to a bead tube containing zirconia beads, followed by l of lysis/binding solution. the bead tube was then placed on an ambion vortex adapter and beaten with a vortex-genie (scientific industries, usa) at maximum speed for min. after beating, the tubes were centrifuged at , × g for min, and the supernatant was then carefully transferred into clean microcentrifuge tubes. following another centrifugation step at , × g for min, l of the supernatant was transferred to a -well, deep-well microplate containing l of paramagnetic beads and l of % isopropanol. the plate was shaken for min on an orbital multi-well-plate shaker at maximum speed without spilling the sample, after which it was placed on a magnetic stand for min and the supernatant was carefully discarded. the bead pellet was then washed twice each with wash solution i and ii ( l). next, the plate was vigorously shaken for min, after which the resultant pellet was resuspended in l of elution buffer. finally, the extracted total nucleic acid in elution buffer was stored at − o c until use in pcr. primer and probe sequences used for me mqpcr were based on published reports [ , , , , , , , ] and are listed in table . all primers and probes were synthesized and purchased from bioneer (korea), except probes for minor groove binder (applied biosystems). a bp oligonucleotide was used as an internal control (ic) to monitor false negatives resulting from failure of pcr due to the presence of inhibitory substances. the ic contained a non-specific s ribosomal rna gene sequence flanked by the uvrc gene sequence of mycobacterium (m.) bovis to minimize cross-reactivity with m. bovis. the ic ( . m) was spiked into each reaction, which resulted in a positive signal with threshold cycle (c t ) values between and . primers and probes were categorized as mqpcr a, b, c, and d (table ) . each mqpcr reaction included two primer/probe sets for the target pathogen, one primer/probe set for the ic, and a reference dye. to minimize cross-interference of the dyes for each probe, a combination of fam and hex was used for the target probes. cy was used for the ic, and rox was used as the reference dye. pcr amplification was conducted using an eco real- time to compare and optimize the pcr conditions, analytical sensitivity was assessed for each pathogen in triplicate as a singleplex assay and the multiplex panel by using the corresponding recombinant vector. the singleplex pcr assay was performed using the same regents and pcr conditions as the multiplex panel. the reaction for e. canis was performed using two mqpcr kits (path-id multiplex one-step real time-pcr kit; applied biosystems; quantitect multiplex pcr; qiagen) with the recombinant vector as a template to determine whether the two kits had comparable sensitivity. a total of csf samples collected from dogs with me or submitted to chungbuk national university between january and august and six csf samples collected from healthy dogs were included in this study. all dogs with me presented with non-specific neurological symptoms, such as seizure, incoordination, or altered consciousness, and had no laboratory abnormalities consistent with a metabolic disorder or radiographic abnormalities (i.e., complete blood count, serum biochemistry profile, urinalysis, x-ray and abdominal ultrasonography) that would suggest non-cns disease as the cause of symptoms. the criteria for case selection included: ( ) evidence of focal or multifocal brain lesions during neurological examination without signs of spinal cord lesions or lesions in the peripheral nervous system; ( ) abnormal result of csf examination indicating inflammation and tissue necrosis (reference interval: ＜ white blood cells/l, total protein ＜ . g/l); and ( ) evidence of focal or multifocal intracranial lesions upon computed tomography (ct) or magnetic resonance imaging (mri) [ , , , , ] . after diagnosis, the residual csf samples were used for the mqpcr assay. all mqpcr assays detected only the target pathogen and did not produce any non-specific amplification products or products of other pathogens in repeated experiments. in addition, there were no false positives due to cross-talk between dye signals within each reaction. standard curves for all multiplex assays generated from -fold serial dilutions of each template showed correlation coefficients (r ) ranging from . to . and slopes of . - . (fig. ) , indicating good linearity of pcr amplification. next, the performance of the mqpcr panel and singleplex pcr for each of the eight pathogens were compared directly to identify any negative effects of multiplexing on pcr detection. the analytical sensitivity (i.e., limit of detection) of the pcr was estimated using serially diluted recombinant vectors with known copy numbers (molecules per l). the estimated mqpcr detection limits of the mqpcr panel were . for cdv, . for e. canis, . for bartonella spp., . for b. burgdorferi, . for b. dermatitidis, . for c. neoformans, for n. caninum, and . for t. gondii ( table ). the results of singleplex and multiplex pcr were identical except for cdv, and ct differences between pcr reactions were not significant, demonstrating that multiplexing did not have a significant negative effect on sensitivity of the pcr. the ct value obtained for e. canis using the path-id multiplex one-step rt-pcr kit (applied biosystems) was slightly lower than that obtained with the quantitect multiplex pcr kit (qiagen); however, the difference in ct was less than , suggesting that the kits had comparable performance. of the csf samples obtained from dogs diagnosed with me, seven ( %) were positive for the target pathogen. the most common pathogens were c. neoformans ( / ), b. dermatitidis ( / ), and b. burgdorferi ( / ). in contrast, conventional csf analysis and culture was negative for all pathogens. none of the samples were positive for more than one pathogen. for the seven samples with discrepant results between the mqpcr panel and routine test methods, agarose gel electrophoresis, sequencing, and comparison with sequences available in the genbank database confirmed the presence of the identified pathogen. among the six csf samples from healthy dogs, all were negative for the eight pathogens of the mqpcr panel, suggesting that mqpcr did not falsely detect normal csf constituents as target materials. in the present study, a panel of four mqpcr assays (one qrt-pcr and three qpcrs) that can simultaneously detect eight pathogens (b. dermatitidis, c. neoformans, n. caninum, b. burgdorferi, bartonella spp., t. gondii, e. canis, and cdv) was optimized and applied to csf samples. the analytic sensitivity of the panel was as good as each singleplex assay, and there was no interference among primer/probe sets, suggesting that the panel could be substituted for the singleplex assay. the panel optimized in this study was also designed for use with one-step extraction and pcr procedures in a -well format. accordingly, simultaneous testing of a large number of samples can be achieved at a lower cost, with less time and effort than routine diagnostic techniques, such as individual pcr, cultures, microscopic examination, and elisa. me is a common inflammatory cns disease in dogs [ ] . histopathology can be used to classify me based on the tissue manifestations of the disease. however, histopathological diagnosis has little relevance for clinical outcome because treatments are not specific for the me subtype. a previous study employed pcr to examine the presence of viral agents that cause human me in a small number of canine me cases and found that none were positive for human me viruses [ ] . in contrast, the present study focused on eight known neurologic pathogens that have caused outbreaks in korea and confirmed their relatively high prevalence among dogs with me ( / ). therefore, it is possible that the prevalence of neurologic infections causing me in dogs has been underestimated. although the cases of me that tested positive for pathogens were not confirmed by histopathology, one case of infection with cryptococcus spp. exhibited dramatic clinical improvement after a two-month treatment with oral fluconazole. moreover, after observation for one year, there was no relapse of neurological signs, suggesting the importance of early pathogen detection for the selection of an effective treatment strategy. in the present study, five of the seven cases of infection were with a fungal agent that could not be eradicated by conventional antibiotics or anti-inflammatory agents, demonstrating the importance of proper diagnosis. although uncommon, fungal infections in dogs pose a diagnostic and therapeutic challenge [ ] . symptoms of fungal infections vary depending on the location and distribution of the lesion, making it difficult to distinguish them from noninfectious inflammatory or neoplastic disorders based on clinical manifestations [ ] . for example, cryptococcus spp., the most common fungal pathogen in this study, tends to infect the cerebellum, resulting in non-specific neurological symptoms, such as seizures and obtundation [ , ] . b. dermatitidis, which is endemic to north america, is seldom associated with cns infection [ ] and usually presents with lymphadenopathy or pulmonary infiltration [ ] . b. dermatitidis infection is rare among humans in korea [ ] ; however, it was found to be the second most common pathogen in the present study. interestingly, these cases did not exhibit typical clinical signs, indicating that appropriate laboratory tests are critical for achieving a definitive diagnosis. this is the first report to implicate b. dermatitidis in canine me in korea. lyme disease is a zoonotic tick-borne disease caused by b. burgdorferi that affects various organs in dogs, including the skin, lymph nodes, aorta, and kidney [ , ] . although the bacterium also infects the meninges and choroid plexus, cns involvement is not pathognomonic of lyme disease [ ] . in the present study, two cases positive for b. burgdorferi had a history of ixodid tick infestation. in both cases, the locations of cns lesions included the choroid plexus, which can be associated with neurologic signs in dogs afflicted by neuroborreliosis. however, it was not possible to confirm whether the lesions were caused by b. burgdorferi because the owners declined additional tests and therapeutic trials. in conclusion, an mqpcr-based test panel for eight canine neurologic pathogens was developed and applied to csf samples in this study. the panel with high analytic sensitivity confirmed that seven ( %) samples were positive to the target pathogen, suggesting the necessity for screening of infectious agents in dogs with me. because treatment strategies for different histopathologic subtypes of me are not specific, initial screening for infectious agents using the developed panel followed by ct or mri would be useful for selecting an appropriate treatment in a timely manner. blastomycosis in dogs: cases ( - ) cryptococcosis of the nervous system in dogs. part : epidemiologic, clinical, and neuropathologic features use and evaluation of molecular diagnostics for pneumonia etiology studies experimental induction of chronic borreliosis in adult dogs exposed to borrelia burgdorferi-infected ticks and treated with dexamethasone characteristics of magnetic resonance imags of granulomatous meningoencephalomyelitis in dogs comparison of magnetic resonance imaging sequences in dogs with multi-focal intracranial disease case-control study of microbiological etiology associated with calf diarrhea development of a panel of multiplex real-time polymerase chain reaction assays for simultaneous detection of major agents causing calf diarrhea in feces detection of borrelia burgdorferi dna in tissues from dogs with presumptive lyme borreliosis multiplex real-time pcr for detection of anaplasma phagocytophilum and borrelia burgdorferi development of a novel genus-specific real-time pcr assay for detection and differentiation of bartonella species and genotypes detection of medically important ehrlichia by quantitative multicolor taqman real-time polymerase chain reaction of the dsb gene detection of canine distemper virus in dogs by real-time rt-pcr magnetic resonance imaging findings in histologically confirmed pug dog encephalitis fatal meningitis and encephalitis due to bartonella henselae bacteria detection of neospora caninum in dog organs using real time pcr systems update on canine and feline fungal diseases molecular and pathological investigations of the central nervous system in borrelia burgdorferi-infected dogs fungal infections of the central nervous system in the dog and cat real-time pcr for quantitative detection of toxoplasma gondii meningoencephalitis of unknown origin: investigation of prognostic factors and outcome using a standard treatment protocol treatment of dogs with meningoencephalomyelitis of unknown origin with a combination of prednisolone and cytosine arabinoside ocular histopathology of ehrlichial infections in the dog laboratory experience in phenotypic and molecular identification of blastomyces dermatitidis first isolated in korea acute canine distemper encephalitis is associated with rapid neuronal loss and local immune activation polymerase chain reaction screening for dna viruses in paraffin-embedded brains from dogs with necrotizing meningoencephalitis, necrotizing leukoencephalitis, and granulomatous meningoencephalitis real-time pcr assay for identification of blastomyces dermatitidis in culture and in tissue idiopathic granulomatous and necrotising inflammatory disorders of the canine central nervous system: a review and future perspectives one-step multiplex real time rt-pcr for the detection of bovine respiratory syncytial virus, bovine herpesvirus and bovine parainfluenza virus real-time polymerase chain reaction detection of cryptococcus neoformans and cryptococcus gattii in human samples review of idiopathic eosinophilic meningitis in dogs and cats, with a detailed description of two recent cases in dogs the authors thank the staff of nowon n and helix animal medical centers for assistance collecting samples and interpreting the mri results. this study was supported by the wildlife center of chungbuk and anidap laboratories in korea. there is no conflict of interest. key: cord- - s ffoy authors: keyhanian, kiandokht; umeton, raffaella pizzolato; mohit, babak; davoudi, vahid; hajighasemi, fatemeh; ghasemi, mehdi title: sars-cov- and nervous system: from pathogenesis to clinical manifestation date: - - journal: j neuroimmunol doi: . /j.jneuroim. . sha: doc_id: cord_uid: s ffoy since the coronavirus disease (covid- ) pandemic, caused by severe acute respiratory syndrome coronavirus (sars-cov- ), a growing body of evidence indicates that besides common covid- symptoms, patients may develop various neurological manifestations affecting both the central and peripheral nervous systems as well as skeletal muscles. these manifestations can occur prior, during and even after the onset of covid- general symptoms. in this review, we discuss the possible neuroimmunological mechanisms underlying the nervous system and skeletal muscle involvement, and viral triggered neuroimmunological conditions associated with sars-cov- , as well as therapeutic approaches that have been considered for these specific complications worldwide. the first reports of an atypical pneumonia epidemic emerged out of wuhan, china in december , and by early january the world health organization (who) started reporting on the issue (world health organization (who), b). cases were associated with a novel strain of coronavirus, retrieved from lower respiratory tract samples of cases on january , which is from the same family of viruses that are associated with severe acute respiratory syndrome (sars) and middle east respiratory syndrome (mers) . subsequently the virus was named as severe acute respiratory syndrome coronavirus (sars-cov- ) (gorbalenya et al. , ) , and the disease was classified as coronavirus disease (covid- among other cranial nerves, trigeminal nerve and vagal nerve could be more plausible way of transmission. while sars-cov- involves lung and gastrointestinal tract very commonly, neuro-invasion through retrograde neuronal transport within vagal nerve afferents (li, bai, , toljan, has been postulated. local peripheral nerves located in the enteric nervous system, may also get infected by other cells in the gastrointestinal tract (bostancıklıoğlu, , lima et al. , , wong et al. , b . experimental studies have demonstrated this retrograde route for the influenza virus (matsuda et al. , ) and hemagglutinating encephalomyelitis virus (hev) (andries and pensaert, b) . moreover, trigeminal nerve, which usually supplies nociceptive cells in nasal cavity as well as sensory fibers in conjunctiva, might be a potential source of cns involvement. accordingly, sars-cov- rna has been found in patients with conjunctivitis (lima, siokas, , sun and guan, ) . hematogenous spread, through the destruction of the blood-brain barrier (bbb), has been proposed as yet another pathway of viral invasion to the brain, as found in influenza and other coronaviruses (desforges, le coupanec, , koyuncu, hogue, , wang et al. , . this can be through the direct invasion of the cns by sars-cov- or infected leukocytes entering the cns (bostancıklıoğlu, ) . additionally, sars-cov- can attack angiotensin-converting (mhv) models (bleau et al. , , cabirac et al. , , cowley and weiss, . mhv studies demonstrated that coronaviruses might be capable of disrupting tight junctions of brain microvascular endothelial cells, leading to increase in permeability. mhv can cause myelitis, encephalitis and cns demyelinating disease. interestingly, mhv infected mice can be used as an experimental mouse model mimicking multiple sclerosis (mecha et al. , ) and causing demyelination in both brain and spinal cord. viral-like particles of sars-cov- were also found in post mortem brain endothelial capillary pericytes, supporting hematogenous cns infection in covid- patients (paniz-mondolfi et al. , b) . moreover, the presence of sars-cov has been confirmed in the cerebrum of patients with sars (ding et al. , ) . the first and foremost important indication of viral infectivity is receptor recognition and presenting a combination of amino acids for the strongest binding of virus-host receptor. if a new virus makes a stronger bond than a prior one, it would be chosen by natural selection. rna viruses generally adapt to their hosts more rapidly, due to high mutation rates. their high adaptation capacity favors them for transmitting between animals to humans (wu et al. , ) . coronavirus family are all spherical or oval and have spike (s) glycoproteins throughout their envelope, which gives them the shape of a crown under electron microscopy. hence, they are named as corona (crown) viridae (schoeman and fielding, , wu, xu, b) . the trimeric s proteins and their receptor biding domains have a similar d structure and homology in both sars-cov and sars-cov- . they both have strong affinity toward the human ace- receptor , ziegler et al. , . a series of mutations led to a different j o u r n a l p r e -p r o o f affinity of sars-cov receptor domains toward human ace- receptor. for example, there is a salt bridge between lys and glu under hydrophobic environment in human ace- hot spot (yin et al. , ) . first in the civet sars-cov receptor binding domain, the residues that counter reacts with hot spot in ace- was also a lysine (yin, feng, ) . lysine in both sides causes steric and electrostatic interference with civet-sars-cov and ace- salt bridge counterpart. later as result of a point mutation (k n) at lysine residue was substituted by asparagine, which guaranteed a stronger interaction between sars-cov and ace- and facilitated transmission of sars-cov to human. as such mutations happened through coronaviruses evolution, some of them were more advantageous for human-sars-cov- host interaction while still some are in favor of human-sars-cov. however, generally speaking, all the selected mutations in human-cov enhance their interaction with human ace- comparing to civet-cov (yin, feng, ) . ace- receptor is expressed in multiple tissues within human body including the cns and skeletal muscle (hamming, timens, ) . it is mainly detected over glial cells and neurons (baig, khaleeq, , palasca et al. , . thus, if the virus reaches out to cns or pns, neurons and glial cells would be potential targets. within the brain, ace- receptors are particularly present in the brainstem and medulla as part of reticular activating system function involved in regulation of cardiovascular system (xia and lazartigues, ) . interestingly though, expressing virus receptors, like ace for sars-cov or dipeptidyl peptidase (dpp ) for mers-cov, did not seem to be the only mechanism for the host cell infection with coronavirus family. it was first postulated that the level of receptor expression in the cell is the determinant factor for its infectability but marked infection of liver by coronavirus despite very low to undetectable level of ace receptor suggested other mechanisms than ace theory j o u r n a l p r e -p r o o f (prabakaran et al. , , to and lo, ) . another proposed pathway is through synaptic routes of nerve cells. hev, which is also a member of β-coronaviridae seems to infect cns retrogradely via peripheral sensory nerves (andries and pensaert, a, hara et al. , ) . after infecting nerve cells hev particles bud from endoplasmic reticulum-golgi intermediate compartments. afterward they form into virion vesicles through golgi apparatus and finally are secreted into the surrounding matrix. virions would then be up taken by adjacent nerve cells (figure ) (hara, hasebe, , steardo et al. , . viral transport through olfactory nerve seems to be a feasible channel for introducing the virus from endothelium to olfactory nerves and bulb and finally passing to brain. olfactory epithelial cells also express ace- . this pathway also explains the anosmia caused by sars-cov- infection. but whether interacting with ace- receptor is the primary mechanism for anosmia commonly found in sars-cov infection or disruption of ciliary nasal epithelium similar to hcov- e is key, and is yet to be determined (koyuncu, hogue, , lechien, chiesa-estomba, , troyer et al. , , wu, xu, b . notably, when transgenic mice expressing human ace- were infected intranasally by sars-cov, the viruses were found to enter the brain by day post infection primarily via the olfactory bulb resulting in a rapid, transneuronal spread to the connected areas of the brain (netland, meyerholz, ) . another coronavirus, hcov-oc , has demonstrated a similar behavior (dube et al. , ) . in this case, the virus spreads to the piriformis cortex, brain stem, and spinal cord by day post infection. interestingly, administration of zinc sulfate, that causes degeneration of the olfactory sensory neurons, almost completely stopped the virus to gain entry to the cns (dube, le coupanec, ) . moreover, when transgenic mice expressing human dpp were infected intranasally by mers-cov, brain disease was observed, with the greatest involvement noted in the thalamus and brain stem (li et al. , ) . the temporal course j o u r n a l p r e -p r o o f of brain tissue infection suggested retrograde virus spread from olfactory neurons. altogether, these data support the critical role of the olfactory pathway and ace- in the neuroinvasion process. to date, the full pathway for nerve and glial cells infections is not convincingly explained. however, in prior case reports and autopsies, sars-cov and mers-cov particles were found within neurons and glial cells as well as the cerebrospinal fluid (csf) proving that cells in the nervous system can be infected (he et al. , , lau et al. , , li, wohlford-lenane, , xu et al. , . after cell infection with coronavirus, the cell ultimate endpoint is death whether it would happen through autophagy, apoptosis, pyroptosis, elimination through innate immune cells, or other pathways (varga et al. , , yang and . viral antigens were detected in respiratory brain stem centers like nucleus of the solitary tract and nucleus ambiguous. damage to these centers may be a contributor to cardiac or respiratory arrest (li, bai, , steardo, steardo jr, , xia and lazartigues, . additional effect of sars-cov- on cns is through systemic and local inflammatory response causing cytokines storming and immune cells reactivation (shi et al. , , steardo, steardo jr, . earlier epidemiologic studies showed that about % of cases might advance to severe disease and the rate is higher in people older than years of age (guan et al. , ) . later some european countries like italy showed higher case fatalities from what china and most of other countries witnessed (onder et al. , ) . the determinant factors for progressing to severe stages are not completely understood; and is the most striking question. there is always a tug-of-war between viruses and host immune response. through years the host immune system either succeeds in clearing the pathogen or adapts in a way causing chronic viral infections. when a virus surpasses a species after years of co-evolution, the new host would respond to it with a more severe immune reaction that can even damage host tissues. accordingly, possible severe immune response to sars-cov- is expected (fung et al. , , rahman et al. , , wagstaff et al. , . there are several pathways proposed to be involved in human immune response towards sars-cov- , and all include two general phases, innate and adaptive immune responses (figure ). innate immune response includes activation of neutrophils, macrophages and natural killer cells and adaptive response involves cytotoxic cd + cells, cd + t helper cells and b cells (steardo, steardo jr, ) . what has been observed so far in severe and fatal covid- infection is a reduction in the absolute number of t cells as well as monocytes, eosinophils, and basophils. at the same time neutrophilic response is enhanced, leading to increased neutrophil-lymphocyte ratio. despite absolute reduction in total number of t cells, including both cd + and cd + cells, the main reduction is among memory t helpers and regulatory cells, while naïve t cells and pro-inflammatory t helper cells were even boosted in number (karakike and giamarellos-bourboulis, , lagunas-rangel, , qin et al. , . because of this pro-inflammatory cell shift, immune cells hyper-react by producing excess levels of inflammatory cytokines. whether the cytokine storm is a part of the "cytokine release syndrome (crs)", or the "secondary haemophagocytic lymphohistiocytosis (shlh)" also called "macrophage activation like syndrome (mal)", the outcome is a robust increase in the highly inflammatory cytokines such as interleukin (il)- , il- , il- , granulocytecolony stimulating factor (gmcsf), and tumor necrosis factor-α (tnf-α) (mehta et al. , . j o u r n a l p r e -p r o o f crs is commonly seen after car t cell therapy and sepsis following organ transplantation and is also reported after viral infections. clinical features of crs include headache, fever, encephalopathy, hypotension, coagulopathy, cytopenia and multiorgan failure , zhang, wu, a . most of these features are shared with mal, which also could occur secondary to infections and hematological malignancies (karakike and giamarellos-bourboulis, ). one of the main outcomes commonly found in both crs and mal is an upsurge in il- , which is reported to be also augmented in moderate to severe cases of sars-cov- infection (wan et al. , ) . inflammatory conditions leading to increased il- and tnf-α also might facilitate disruption of bbb (ichiyama et al. , , linker et al. , which in turn might be responsible for encephalitis, acute necrotizing encephalopathy and demyelination in the cns and even guillain-barré syndrome (gbs) in the pns. there are several case reports of such complications due to sars-cov- infection as we discuss in the next sections (alberti et al. , , mcabee et al. , , moriguchi et al. , , poyiadji et al. , , zanin et al. , . interestingly, tocilizumab (a recombinant, humanized monoclonal antibody against the il- receptor), which is an fda approved medication for treatment of t cell induced crs, also showed some benefit over severe cases of covid- infection (le, li, , xu et al. , a . however, as of yet, there is not sufficient evidence to clarify the exact role of systemic inflammation versus local inflammation due to the direct viral infection or hypoxia, which is a common complication of sars-cov- infection. the brain and the lungs have a close inter-relation. a disease process in one would potentially cause complications of the other (abdennour et al. , ) . brainstem centers for respiratory and cardiovascular systems are potential targets of sars-cov- and neural cell death in these centers might be responsible for a central cause of respiratory/cardiovascular arrest (li, bai, , steardo, steardo jr, , xia and lazartigues, . sars-cov- lung infection has been reported to cause an atypical form of ards, while patients usually show relatively wellpreserved lung mechanics not matching the severity of hypoxemia. this may be due to the dysregulation of lung perfusion and hypoxic vasoconstriction, which may have a central cause as well (gattinoni et al. , ) . on the other hand, through a process called "infectious toxic encephalopathy" usually seen in toxic metabolic disorders or acute infections, alveolar gas exchange problem might lead to anaerobic metabolism in brain cells, and cause cns hypoxia. the hypoxemia and increased acidity within the brain causes cell swelling, interstitial edema, obstructive hydrocephalus, and increased intracranial hypertension leading to an altered mental status and even coma (abdennour, zeghal, , wu, xu, b . hypoxic injury to the brain also may cause cerebrovascular accidents like stroke or seizures, and again would activate the loop of both local microglial activation and systemic inflammation mccullough, , wu, xu, b) . another clinical and scientific significance of sars-cov- infection is widespread observation of hypercoagulable state indicated by elevated d-dimer level, prolongation of prothrombin time (pt), activated partial thromboplastin time (aptt), and thrombocytopenia (violi et al. , b) . coagulopathy was previously observed in infection with other coronoviridae viruses including sars and mers (giannis et al. , , merad and martin, ) . although there are some prospective studies currently looking at incidence of thrombotic events, early studies have already confirmed increased frequency of intravascular thrombosis leading to pulmonary embolism, myocardial infarction, ischemic strokes, and even cerebral venous sinus thrombosis. j o u r n a l p r e -p r o o f a thrombotic event was sometimes reported as the first presentation of covid- infection (hughes et al. , , klok et al. , . in a retrospective study on covid- patients, about % presented with acute cerebrovascular events, mainly ischemic strokes (cantador et al. , ) . a small number of stroke patients with covid- infection presented with cerebral hemorrhage (cantador, nunez, ) . recent investigation has also found that covid- patients have increased serum nox overactivation, which is an important trigger for vascular dysfunction through excess production of reactive oxygen species (violi et al. , a) . interestingly, it was more up-regulated in more severe covid- cases and also those with thrombotic complications (violi, oliva, a) . one possible underlying mechanism is the reduced expression and function of ace- in sars-cov- infected cells. ace- regulates the cerebral blood flow and its altered signaling can lead to subsequent hypertension and predisposition to developing hemorrhagic stroke from arterial wall rupture (sharifi-razavi et al. , ) . another possible mechanisms is the underlying coagulopathy induced by the infection with thrombocytopenia . since the early phases of the global pandemic, numerous studies have demonstrated clinical symptoms and signs of covid- which mainly include fever, cough, sore throat, dyspnea, diarrhea, nausea, vomiting, anorexia, and fatigue ( j o u r n a l p r e -p r o o f consciousness ( . %), ageusia ( . %), anosmia ( . %), stroke ( . %), nerve pain ( . %), visual impairment ( . %), seizure ( . %), and ataxia ( . %) (mao et al. , ). an important finding was the significantly higher rate of neurological manifestations (in general) and impaired consciousness, stroke and skeletal muscle injury (in particular) in patients with severe covid- infection than those with non-severe infection (mao, jin, ) . another study demonstrated that the major factor associated with neurologic complications was age over , which was also a strong risk factor for mortality (xiong et al. , ) . when patients with covid- infection were compared at the same level of severity, new-onset of neurologic critical events (e.g. impaired consciousness and stroke) was later found to increase the risk of death by six-fold (xiong, mu, ) . overall, the most common neurological symptoms described in patients are fatigue/malaise, myalgia, headache, impaired consciousness, dizziness, ageusia, and anosmia; and less common reported symptoms include visual impairment, nerve pain, occipital neuralgia, ataxia, tremor, and tic. growing number of case reports and/or series indicate that a variety of neurological conditions and post-viral triggered autoimmune complications, as we discuss below, occur in association with sars-cov- infection which mainly include guillain-barré syndromes (gbss) (table ), myopathy and rhabdomyolysis (table ) , encephalopathy, meningoencephalitis, encephalomyelitis, and myelitis (table ) . gbss characteristically manifest with acute (< weeks) ascending muscle weakness accompanied by decreased/absent deep tendon reflexes, mild-moderate sensory loss, occasionally cranial nerve involvement, and radicular or muscle pain. although gbss are commonly demyelinating (i.e. acute inflammatory demyelinating polyneuropathy [aidp]), j o u r n a l p r e -p r o o f primary axonal injury may occur, known as acute motor and sensory axonal neuropathy (amsan) or acute motor axonal neuropathy (aman). miller-fisher syndrome (mfs) is another gbs variant which is characterized by the triad of ophthalmoplegia, gait ataxia, and areflexia (rocha cabrero and morrison, ). gbs is considered as an autoimmune neurologic disease that can be triggered by a variety of viruses. about % of cases may have a viral illness - weeks prior to neurologic symptoms (wakerley and yuki, ) . gbs outbreak has been observed with viral epidemics including those with coronaviruses (i.e. mers-cov and sars-cov) (kim et al. , , wakerley and yuki, ) . the first case of sars-cov- related gbs was reported from the major covid- hotspot, wuhan, china . the patient was a years old woman who, one week after her trip from wuhan, developed rapidly progressive ascending limb weakness over days accompanied by areflexia and later distal sensory changes. the csf study (day ) revealed albuminocytologic dissociation and electromyography/nerve conduction study (emg/ncs) on day showed a demyelinating neuropathy at early stage. laboratory results on admission were notable for lymphocytopenia and thrombocytopenia. she was treated with intravenous immunoglobulin (ivig). notably, she developed fever, cough, and pneumonia eight days after the onset of neurological symptoms. the sars-cov- rrt-pcr was positive in oropharyngeal swabs at that time. the patient had a full recovery after weeks and the repeat test for covid- was negative (zhao, shen, a) . overall cases of gbs variants have been reported worldwide in patients with confirmed covid- since the pandemic (table ), with mostly typical manifestation of rapidly progressive flaccid limbs weakness and areflexia, with and without facial muscle weakness, and distal paresthesia or numbness. cranial nerve involvements have been observed in ( %) cases (bigaut et al. , , gutiérrez-ortiz et al. , , reyes-bueno et al. , j o u r n a l p r e -p r o o f al. , ). either unilateral or bifacial weakness was present in ( %) cases. in patients, covid- symptoms were variably present between and days prior to the onset of gbs symptoms, including cough ( %), fever ( %), gastrointestinal symptoms (e.g. diarrhea, nausea and vomiting, %), dyspnea ( %), myalgia ( %), ageusia ( %), anosmia ( %), fatigue/malaise ( %). ageusia and anosmia were also present on admission in % and % of these cases, respectively. three cases (all from spain) had mfs (gutiérrez-ortiz, méndez, , reyes-bueno, garcía-trujillo, ). among patients that underwent csf study, elevated protein levels ( to mg/dl) with normal leukocyte counts (i.e. albuminocytologic dissociation) were found in ( %) patients. positive serum anti-gd b igg antibody was reported in one patient with mfs (gutiérrez-ortiz, méndez, ). however, serum and csf anti-ganglioside antibodies (including anti-gm , gq b, and gd b antibodies) checked in and cases, respectively, were negative (table ). variable degrees of leukocytopenia (mainly lymphocytopenia; %) and elevated acute phase reactants (e.g. erythrocyte sedimentation rate [esr] , c-reactive protein [crp], ferritin, or fibrinogen; %) were also reported among these cases. except for five patients (two with mfs) that did not undergo emg/ncs, cases (including one with mfs) ( %) had demyelinating features (i.e. aidp), ( %) had amsan, and one ( %) had aman in emg/ncs. this implies that different gbs variants could occur in association with sars-cov- . except for patients (including one mfs and one aidp case who received plasma exchange therapy), all other patients received a -day course of ivig ( . mg/kg/day, one patient received only one dose) with an additional cycle for patients. overall, the outcome was favorable with partial to complete recovery in ( %) cases. two patients passed away due to severe, progressive respiratory failure within hours after initiation of ivig (alberti, beretta, , marta-enguita et al. , ). an important finding in these reported cases is that covid- rrt-pcr was negative in all those checked csf samples, indicating no active intrathecal sars-cov- replication or root infection. this finding combined with relatively favorable outcome post-ivig therapy and positive anti-gd b antibody in one case may suggest an underlying autoimmune process triggered by post-sars-cov- viral infection in these cases. there is evidence that the sars-cov- s protein can bind to sialic acid-containing glycoprotein and gangliosides on cell surfaces (fantini et al. , ) , increasing its viral transmissibility. therefore, it is possible that crossreactivity between epitopes within the sars-cov- s protein-bearing gangliosides and surface peripheral nerve glycolipids may occur, serving as an underlying mechanism in sars-cov- triggered autoimmune gbs. accordingly, most gbs variants (aidp, aman, amsan, and mfs) have been reported in sars-cov- patients (table ). checking anti-ganglioside antibodies in future cases may provide more detailed information about this hypothesis. it is also noteworthy that some of the reported gbs cases received hydroxychloroquine in addition to ivig or plasma exchange therapy. chloroquine is shown to bind to sialic acids and gm gangliosides preventing binding to sars-cov- s protein, thereby inhibiting virus entry to the cells. therefore, adjunctive therapy with chloroquine in sars-cov- -associated gbs could be an interesting consideration in future studies. and shabarek, ), association of sars-cov- with either viral or necrotizing autoimmune myositis is still elusive. two reported cases may indirectly suggest a sars-cov- triggered necrotizing autoimmune myositis tong, , suwanwongse and shabarek, ) . the first case is a man, aged years, from new york, presenting with acute, painful bilateral proximal lower limb weakness and hyperckemia ( u/l) (suwanwongse and shabarek, ) who was found covid positive and started on hydroxychloroquine, and his weakness and ck levels improved one week later. the second case is a man, aged years, from wuhan, with -day covid-positive pneumonia and fever who days later, despite improvement in his clinical condition, developed painful proximal muscle weakness with hyperckemia ( u/l) and elevated crp, and benefited from ivig therapy (jin and tong, ) . a more recent study also reported six intensive care unit (icu)-admitted cases (age between and years old) with covid- who had acute flaccid quadriplegia (madia et al. , ) . emg/ncs showed myopathic features in all of these patients and ck levels were normal to mildly elevated (highest level of u/l), suggesting the presence of critically illness myopathy (madia, merico, ) . overall, these observations may necessitate pursuing more investigations such as muscle biopsy and antibody screening in some covid- patients with signs of skeletal muscle injury, as treatment with ivig may potentially improve functional outcomes in these patients. notably, ace- is shown to be expressed in skeletal muscles (cabello-verrugio et al. , ) ; thus, evaluation of direct sars-cov- infection of skeletal muscle fibers would be a highly interesting topic for future studies. human coronavirus infections have been associated with encephalopathies in the past and are known to have human neurotropic and neuroinvasive potentials, mainly through the olfactory bulb or hematogenous route, causing inflammation, and demyelination (desforges, le coupanec, ) . the underlying pathophysiology is still not well understood, and includes abnormal host immune responses with autoimmunity and/or direct cns damage due to viral replication and infiltration (desforges, le coupanec, ) . more in details, ace- and transmembrane protease, serine (tmprss ) are documented co-receptors for sars-cov- entry and they are expressed in the oligodendrocytes, suggesting a direct involvement of white matter in case of encephalitis related to covid- infection (needham et al. , , sellner et al. , . a retrospective study from turkey (kandemirli et al. , ) included patients in icu, of which ( %) developed neurological symptoms. the authors collected mri data from / patients to show neurotropism related to covid- . the findings included cortical flair (fluid attenuated inversion recovery) abnormalities ( %) with cortical diffusion restriction, leptomeningeal enhancement, or cortical blooming artifact in a non-specific pattern. less frequently subcortical and deep white matter flair lesions were reported. unfortunately, the findings were not correlated with the patients' symptomatology. csf data were also obtained in half of the patients with cortical flair abnormalities, showing elevated proteins, normal cell count, glucose level, igg index, oligoclonal bands and albumin as well as negative rrt-pcr for sars-cov- (kandemirli, dogan, ) . another report from uk described a patient with fever and respiratory symptoms who developed progressive unsteady gait, diplopia, limb ataxia, altered sensation in the right arm, hiccups, and dribbling when eating, found to have a rhomboencephalitis in the mri with involvement of the right inferior cerebellar peduncle (table ) . csf showed normal protein, normal white blood cell (wbc) counts and negative bacterial j o u r n a l p r e -p r o o f culture (wong et al. , a) . unfortunately, the sars-cov- pcr test was not administered, and there were no results for the myelin oligodendrocyte glycoprotein and aquaporin antibodies sent as part of the workup. the presence of brain inflammatory changes related to covid- was also confirmed by neuropathology findings of foci of perivascular lymphocytes, focal leptomeningeal inflammation in brain specimens of encephalopathic patients, although these findings were reported as rare and did not support an underlying diagnosis of encephalitis. immunohistochemical analyses to detect sars-cov- by rrt-pcr performed in the tissues were negative, and the virus was detected at low levels in only patients, possibly as a result of viral direct infiltration in the brain or viral rna coming from blood (solomon et al. , ). increasing evidence indicates that encephalopathy is one of the several presenting symptoms or complications of covid- . encephalitis represents an inflammatory process of the brain and surrounding tissues and its symptomatology can include altered mental status, headache, behavioral changes, psychiatric disturbances in association with focal neurological signs (e.g. paresthesia, weakness, etc.). on the other end, meningitis is an inflammatory process of the meninges and spinal cord and gives typical symptoms such as fever, headache, photophobia, phonophobia and neck stiffness. seizures could also be part of the encephalitis and meningitis presentation (asadi-pooya, , sohal and mossammat, ). the severity of the above symptoms can vary and sometimes it is difficult to make a proper diagnosis particularly in patients with mild symptoms. to add complexity in the diagnosis and management of encephalopathies, there is the inability to distinguish the underlying process (infectious or toxicmetabolic) only based on the symptoms. indeed, many patients with severe covid- infection j o u r n a l p r e -p r o o f may present with altered mental status from the toxic metabolic processes due to hypoxia, electrolyte derangements, metabolic disturbances, and multiorgan failure, without necessarily presenting involvement of the cns. moreover, two cases of acute necrotizing encephalopathy (ane) in patients with covid- positivity from nasopharyngeal and oropharyngeal swab, but without csf pcr for sars-cov- data, were reported in the literature (poyiadji, shahin, , radmanesh et al. , . ane is characterized by neuroinflammation secondary to cytokine storm with multifocal symmetric lesions in the gray and white matter without direct viral damage. in addition, there is a report of possible demyelinating lesion in the white matter and globus pallidus in a -year-old woman admitted initially for respiratory distress due to covid- infection with only history of mild elevated blood pressure under treatment. her glasgow coma scale score was with altered sensorium but her neurological exam was non-focal on admission, then rapidly deteriorated and required endotracheal intubation and received hydroxychloroquine in addition to azithromycin and amoxicillin/clavulanic acid. sedation was discontinued two days later but the patient remained obtunded for a long period afterwards, and this prompt neuroimaging and further investigations. brain mri eventually revealed bilateral asymmetric restricted diffusion lesions without hemorrhage or enhancement in the supratentorial periventricular white matter and globus pallidus, without involvement of the thalamus, striatum and posterior fossa. subsequent mri obtained days later showed homogeneous contrast enhancement of the lesions, brain vascular images were negative. csf studies, performed twice (on admission and days after), were reportedly unremarkable, including rrt-pcr for sars-cov- . the patient was treated with steroid for suspected demyelination twelve days later after her hospitalization upon negative nasopharyngeal pcr. the patient reported residual right-side hemiplegia and there are no data about her response to steroids, which could be supportive of the j o u r n a l p r e -p r o o f diagnosis of demyelination (brun et al. , ) . despite that, the images were suspicious of demyelination, which has been described associated with coronavirus, both in murine animal models (wu et al. , ) , and in a pediatric patient with acute disseminated encephalomyelitis (adem) (yeh et al. , ) . however, other diagnoses could not be completely excluded in the case described above. in a retrospective observational case series from wuhan (mao, jin, ) that collected data from patients with laboratory-confirmed diagnosis, . % had neurological manifestations. more in detail, cns manifestation was present in . % of the patients, and in particular . % had encephalopathy. the authors noticed that cns manifestation were significantly more common in patients with severe infection compared to non-severe infection, with encephalopathy present in . % of cases versus . % (p < . ), respectively. the patients with severe infection were older, with higher blood pressure and with less typical symptoms such as fever or cough on admission. furthermore, those patients were more prone to develop neurological symptoms few days after the admission, with associated higher mortality rate. they also had a marked inflammatory response with higher levels of wbc counts, neutrophil counts, blood urea nitrogen, d-dimer and crp, and reduced lymphocyte and platelet counts than in those with less severe infection, pointing to a multiorgan involvement and immunosuppression as underlying pathogenic mechanism for the neurological manifestations. the study did not further investigate the etiology of the encephalopathy, either toxic-metabolic or infective. other case reports described encephalopathic patients who first presented to the hospital with new onset of seizure as manifestation of underlying meningoencephalitis in the setting of covid- infection (asadi-pooya, , moriguchi, harii, , sohal and mossammat, . as of now, there are no csf or serologic biomarkers available worldwide to help diagnosing cases of covid- with cns involvement (baig, , kandemirli, dogan, ; therefore, proper neurological evaluation of encephalopathic covid- patients can help early diagnosis, better tailor the treatment, and possibly improve the outcome. the workup for encephalopathic patients should not only include detailed documentation of the neurological symptoms but also electrophysiological studies (i.e. electroencephalography or eeg), csf analysis, and perhaps brain imaging (asadi-pooya and simani, , liu et al. , b , oxley et al. , . moreover, seizures should be suspected in case of altered mental status in patients with covid- since cases with clinical or subclinical seizures or status epilepticus have been reported, either as a direct consequence of the brain damage from the virus or secondary toxic-metabolic derangements (asadi-pooya, ). anti-epileptic drugs (aeds) should be administered in patients with seizure as initial presentation, to prevent further episodes, for a period of weeks and then taper and discontinue the aed in - weeks (asadi-pooya, ). since these covid- patients are usually critically ill, intravenous formulations and aeds with less side effect on respiratory and cardiac status are recommended, such as levetiracetam and brivaracetam. moreover, since some patients may require extracorporeal membrane oxygenation which affects the pharmacokinetic of highly protein-bound aeds, phenytoin and valproic acid should be avoided (asadi-pooya, ). the diagnosis of covid- meningoencephalitis is based on clinical and laboratory studies such as csf characteristics and possibly detection of the virus in the csf. the first case of covid- with associated laboratory-confirmed viral encephalitis was reported in beijing in a patient j o u r n a l p r e -p r o o f with altered mental status, seizures, persistent hiccups, hyperreflexia, meningeal irritation and slow pupillary response (table ) . notably, csf studies showed normal range wbc, glucose and protein, but an increased opening pressure and positive pcr for sars-cov- (oxley, mocco, , sun and guan, ) . this case report was published in chinese and it seems to lack further clinical and laboratory data to corroborate the diagnosis. a recent paper discussed about a woman with encephalitis and no respiratory symptoms with sars-cov- positivity in both nasopharyngeal swab and csf . another case of meningoencephalitis from covid- in japan (moriguchi, harii, ) described a young patient who presented with headache, fatigue, fever and few day later was found unconscious with an episode of generalized tonic seizure while transported to the hospital. he had clear meningeal signs, pleocytosis in the blood, negative ct scan of the head, with sars-cov- detected only in the csf but not in the nasopharyngeal swab. interestingly csf showed elevated opening pressure and wbc mainly mononuclear; mri showed dwi (diffusion-weighted imaging) positivity along the wall of the inferior horn of the right ventricle and flair abnormalities in the right mesial temporal lobe and hippocampus. these are the only three encephalitis cases based on our knowledge that were associated with csf viral detection. it is noteworthy that false positivity of the pcr has been reported given the risk of sample contamination from shed airborne virus with this diagnostic technique (needham, chou, ). on the contrary, there is also a case report of a patient who presented with fever, cough and typical multiple ground-glass opacities on ct of the lungs, who later developed focal neurological symptoms and altered mental status suggestive of meningoencephalitis (yin, feng, ) . notably, the throat swab was positive for covid- but the csf pcr was negative (yin, feng, ) . other csf results suggestive of viral infection were the elevated opening pressure j o u r n a l p r e -p r o o f and proteins (yin, feng, ) . the patient was monitored, treated with antivirals and supportive care. over time the lung infections improved, he started requiring less oxygen supplementation and concomitantly his neurological exam improved consistently. at that time two further throat swab were done and resulted negative. other case reports of patients with suspected viral meningoencephalitis with positive nasopharyngeal swab but negative csf pcr for sars-cov- have been reported in the literature. in the majority of the reports, csf showed increased cells, mainly lymphocytes, and elevated protein levels , dogan et al. , . more in details, a case series of icu patients reported subjects intubated for severe ards and no improvement of their mental status or agitated delirium after extubation with subsequent neurological workup (dogan, kaya, ) . neurological involvement was diagnosed in of the intubated patients ( . %). these patients had increased levels of acute-phase reactants such as ferritin, crp, il- , fibrinogen. mri findings showed white matter and cortical abnormalities and contrast enhancement compatible with meningoencephalitis in / patients. csf data revealed elevated proteins without pleocytosis in all cases with negative pcr for viruses including sars-cov- . an underlying autoimmune etiology was suspected for both mri positive and negative patients, and they underwent treatment with plasmapheresis. improvements of the clinical status were observed in / patients, and mri findings were reversible in all patients with positive mri. another case from italy (pilotto et al. , ) described a -year old man with mild respiratory symptoms that developed akinetic mutism and nuchal rigidity. mri and eeg were negative for any abnormalities. csf showed elevated protein level and lymphocytic pleocytosis, as well as increased il- , il- and tnf-α but negative for sars-cov- and for other neurotropic viruses. covid- infection was established by nasopharyngeal swab. the patient was started on antibiotic and antiviral coverage j o u r n a l p r e -p r o o f initially, as well as hydroxychloroquine. an improvement was seen after high dose of steroids were initiated. patient was discharged after days of iv steroids with oral prednisone taper with normal neurological examination. although the exact pathogenetic mechanism of autoimmune encephalitis in the setting of covid- is unclear, it may be related to cytokine storm with direct damage to the bbb and increased leukocyte migration to the brain (sohal and mossammat, ), as well as dysregulation of viral immunity mediated by molecular mimicry (pilotto, odolini, ) . a trial with immunomodulatory therapies can be crucial to diagnose autoimmune encephalitis. further case reports of patients with suspected viral or autoimmune meningoencephalitis and detailed description of their presentation and workup are also needed. the cases reported above showed that csf pcr may be not reliable for the diagnosis since sars-cov- dissemination in the brain can be transient and its csf titer may be extremely low (ye et al. , a) . furthermore, the test is not widely available. a proper neurological examination, eeg, csf studies, and brain imaging are for now the only tools that can guide to the diagnosis of covid- -associated meningoencephalitis, and appropriate treatment should be initiated promptly. a retrospective study of patients with covid- reported altered mental status on hospital admission in % of patients who expired and % among those who recovered from the infection. this hints towards a possible negative prognostic factor related to encephalopathy as initial presentation. headache without associated neurological symptoms or signs was reported in % of the deceased patients, versus % of the recovered patients. metabolic derangements were more common in deceased patients than in recovered patients, and j o u r n a l p r e -p r o o f % of the deceased patients suffered from what was classified as hypoxic encephalopathy related to pulmonary inflammation. in this study neurological symptoms other than headache, neurological signs, and seizures were not reported or considered as possible manifestation of the disease. furthermore, no laboratory studies were carried to rule out a viral encephalitis/meningitis, underlying seizures, and non-convulsive status epilepticus. it is possible that some of the cases described to have toxic encephalopathy were indeed suffering from a viral encephalitis. one of the first case reports published at the beginning of the pandemic (filatov et al. , ) reported the case of a -year old man with chronic obstructive disease (copd), atrial fibrillation and prior cardioembolic stroke in the left posterior cerebral artery. he presented to the emergency department with fever and cough, initial negative workup for pneumonia, and initially discharged with possible copd exacerbation. he was readmitted to the emergency department after hour with severe altered mental status and headache. neurology was consulted at that time and a full work up included a ct scan of the head, eeg and csf studies were conducted. the head ct scan showed the old stroke, related encephalomalacia; and csf was not suggestive for infection, although sars-cov- pcr was not performed. eeg showed diffuse slowing and focal sharply contoured waves in the left temporal region which raised the suspicion for subclinical seizures. he eventually tested positive for covid- . he was treated with aeds as well as hydroxychloroquine, lopinavir, and ritonavir. unfortunately, there is no follow-up report about his response to the treatments. this case report is an example of covid- related toxic metabolic encephalopathy and epileptic activity from lowered seizure threshold secondary to severe underlying metabolic process (delanty et al. , ) . another study from france (helms et al. , ) showed that % of patients with ards and covid- presented with neurological signs such as agitation ( %), corticospinal tract signs ( %) and dysexecutive syndrome with inattention, disorientation, or poorly organized movements in response to command ( %). mri of the brain was performed in of the patients reported in the study. notably, these patients did not have any focal signs suggestive for stroke, although % had an underlying ischemic stroke, % had leptomeningeal enhancement, and all the patients who underwent perfusion imaging ( ; %) had bilateral frontotemporal hypoperfusion. a small proportion of the patients ( out of ) had an eeg that showed nonspecific changes or bilateral frontal slowing; and a smaller proportion ( ) acute myelitis is a known complication of viral infections, mainly attributed as an autoimmune response, although it could also be an early manifestation of other neuroimmunological disorders such as multiple sclerosis and neuromyelitis optica spectrum disorders. little is known about association between sars-cov- and acute myelitis. up to date, cases of acute myelitis, alone ( cases) or combined with brain involvement ( cases), have been reported in relation to covid- (alketbi et al. , , munz et al. , , novi et al. , , sarma and bilello, , valiuddin et al. , , wong, craik, a j o u r n a l p r e -p r o o f zanin, saraceno, ) . six patients ( - years age range, . % female) variably had symptoms of covid- (fever, dyspnea, malaise, chills, and rhinorrhea) to days prior the onset of neurologic symptoms of myelitis (table ) . sars-cov- rrt-pcr were checked in the csf of patients and all were negative. overall, the functional outcome was favorable in ( . %) patients after treatment with either steroids or plasmapheresis. additionally, a recent case report from spain (sotoca and rodríguez-Álvarez, ) has presented a years old woman who developed a -day radicular neck pain, right facial numbness, left hand numbness and weakness, gait instability, and general hyperreflexia. she had fever and cough days prior these symptoms. the cervical spine demonstrated a t- hyperintensity extending from the medulla oblongata to c , suggestive of acute transverse myelitis. she had negative blood testing for anti-antiaquaporin- (aq ), -myelin oligodendrocyte glycoprotein (mog), and antiphospholipid antibodies, but had elevated protein level and pleocytosis in the csf study with no oligoclonal band, normal igg index, no anti-neuronal surface antibody, and negative sars-cov- pcr. however, nasopharyngeal swab for sars-cov- rrt-pcr was positive. she was treated with -day methylprednisolone ( gr/day). few days later her symptoms worsened, and she developed bladder/bowel incontinence, bilateral hands weakness and paresthesia, and paraparesis. the repeated spinal mri showed a new area of central necrosis at the t level with peripheral enhancement. this case was indeed the first case of acute necrotizing myelitis in association with covid- . she was additionally treated with plasmapheresis and -day methylprednisolone ( gr/day) followed by slow tapering oral prednisone with favorable outcome after weeks. the exact underlying mechanism in acute necrotizing myelitis is still elusive; however, a post-viral triggered autoimmune cytokine storm has been suggested j o u r n a l p r e -p r o o f (kansagra and gallentine, ) . thus, this may imply in the case of sars-cov- , especially with observed clinical improvement after steroid and plasmapheresis therapy. there is growing evidence about psychiatric manifestations as potential complication of sars-cov- infection. accordingly, a worldwide exacerbation of mental health disorders during the pandemic has been reported, which includes but not limited to delirium, cognitive impairment, mood alterations, psychosis and suicide (orsini et al. , ) . more in details, delirium has been noticed more in % of covid- patients whose conditions require icu level of care versus - % rates documented in the past (kotfis et al. , ) . cognitive dysfunctions have been reported to be possibly a direct consequence of covid- infection to the cns, and in particular to the hippocampus, which appears to be vulnerable to coronaviruses infections, with possible acceleration of hippocampal degeneration as occurs in alzheimer's disease (ritchie et al. , ) . cognitive impairment can be also a consequence of acute respiratory syndrome and relative hypoxia which have been associated to cerebral atrophy and ventricular enlargement (hopkins et al. , ) and worsening attention, executive functions and verbal memory (hopkins et al. , ) . anxiety, depression, post-traumatic stress disorder, insomnia and obsessive-compulsive symptomatology appear to be very common in covid- survivors, particularly in females, and with worsened scores on psychopathological measures in those with previous psychiatric comorbidities (mazza et al. , ) . a recent study reports an incidence of psychosis in infected patients between . % and % versus a median value of . ( . - . ) per , previously described (mcgrath et al. , ) . increased j o u r n a l p r e -p r o o f rates of suicide have been also reported, with possible contributing factors found in the social isolation/distancing, economic recession and social discrimination (thakur and jain, ). it is unclear whether the above psychiatric symptoms are a direct consequence of the cns viral infection (i.e. viral meningoencephalitis), cerebrovascular accidents, hypoxia, and the immunological and inflammatory responses, which may play important roles in major depressive disorder (ghasemi et al. , , wohleb et al. , and psychosis (ferrando et al. , ) , or whether they are related to increased psychosocial stress of this severe and potentially fatal disease and difficulties accessing to health care related to the pandemic infection (zhou and yao, ). this has been posing increased challenges in the treatment of infected patients, especially those in the icu, requiring accurate multidisciplinary approaches and early interventions to decrease overall morbidity and mortality (ojeahere et al. , ). as described in above sections, several treatment approaches have been used to treat manifestations or consequences of the sars-cov- −related nervous system injury, such as ivig for gbs and skeletal muscle injury, iv/oral steroids and plasmapheresis for autoimmune encephalitis and acute myelitis, and aeds for seizures. with regards to medications aimed to modulate the immune response to viral infection and to induce viral clearance, antimalarial drugs (e.g. hydroxychloroquine), dexamethasone, rna-dependent rna polymerase inhibitors (e.g. remdesivir), hiv- protease inhibitors (lopinavir/ritonavir), and biological agents like tocilizumab, interferons and convalescent plasma have shown some beneficial effects (chibber et al. , ) . among these, the fda granted emergency use authorization for remdesivir as an emergency medication for severely ill hospitalized adult and pediatric patients with proved or j o u r n a l p r e -p r o o f suspected sars-cov- infection (lamb, ) . this drug, which has a broad-spectrum antiviral activity against several rna viruses, can inhibit sars-cov- replication, alleviate symptoms, fasten the recovery rate, and reduce mortality rate (frediansyah et al. , ) . more in details, the final report of the adaptive covid- treatment trial (actt- ), a double-blind randomized placebo-controlled trial of intravenous remdesivir in affected adults with evidence of lower respiratory tract infection, showed a median recovery time of days ( % confidence interval [ci], to ) versus days ( % ci, to ) in those in the placebo group. mortality estimates by day were . % with remdesivir versus . % with placebo and those by day were . % with remdesivir and . % with placebo (paladugu and donato, ) . given the lack of head to head comparison, it is unclear if remdesivir offers a superior benefit over dexamethasone, which is widely available and less expensive (mccreary and angus, ). the randomized evaluation of covid- therapy (recovery) trial has shown that dexamethasone resulted in lower -day mortality in covid- patients receiving either invasive mechanical ventilation or oxygen alone at randomization but not in those receiving no respiratory support (horby et al. , ) . the authors of the actt- trial also adjusted the data for glucocorticoid use suggesting that the benefit of dexamethasone may be additive to that of remdesivir (beigel et al. , ) . however, it is still unclear whether remdesivir or dexamethasone have beneficial effects on the neurological manifestation of covid- . with regards to possible therapeutic strategies aimed to ameliorate the neuronal damage mediated by covid- , high doses of melatonin seem promising in immunomodulation and reducing neuroinflammation, with no direct effect on viral replication or transcription. melatonin seems to act via an anti-inflammatory, anti-oxidative and immune-enhancing mechanism with ability to restore the bbb hemostasis (romero et al. , ). there are ongoing worldwide clinical trials for the development of a vaccine to prevent covid- , which is not currently available and that poses some challenges due to safety, efficacy, and long-lasting effects without further risks of re-infection, particularly in the elderly population (jamwal et al. , ) . the covid- pandemic with its variety of manifestations, not only pulmonary or neurological, is an international public health emergency that requires efforts from all countries to develop effective drugs and vaccines as early as possible. evolving data indicates that patients with covid- may variably develop neurologic manifestations prior, during and even after the onset of common covid- symptoms. the commonly reported neurological symptoms and signs include dizziness, headache, myalgia, fatigue, impaired consciousness and confusion, ageusia, anosmia, neuropathic or radicular pain, occipital neuralgia, visual impairment, seizure, and ataxia. based on a growing number of case reports and series, both the cns, pns and skeletal muscles can be involved in covid- presenting with a variety of neuroimmunological conditions including gbss, myopathy and rhabdomyolysis, encephalopathy, meningoencephalitis, encephalomyelitis, and acute myelitis. the exact etiology of these complications remains to be fully elucidated. however, suggested mechanisms are direct sars-cov- infection to the nervous system, neuroinflammation, postviral triggered autoimmune response, hypercoagulability, and metabolic or hypoxic injury. in general, therapeutic strategies for covid- are based on three main directions: (i) targeting sars-cov- with antivirals, neutralizing antibodies or convalescent plasma therapy, (ii) targeting inflammatory storm using immunomodulatory medications and cytokine inhibitors, and (iii) developing vaccines to prevent the disease manifestation (for comprehensive review see j o u r n a l p r e -p r o o f journal pre-proof (vabret et al. , ) ). however, it is still too early to find out whether even with successful treatment of the active infection, post-viral triggered autoimmune neurological complications of covid- (e.g. gbs, myositis, cns demyelination, and myelitis) will be also lowered in frequency and/or severity. additional studies are clearly needed to address this issue. no conflict of interest exists in relation to the submitted manuscript. this research did not receive any specific grant from funding agencies in the public, commercial, or not-for-profit sectors. ( ) myalgia ( ), headache ( ), dizziness ( ), impaired consciousness ( ) fever ( ), cough ( ), fatigue ( ), anorexia ( ), dyspnea ( ), chest tightness ( ), pharyngalgia ( ), hemoptysis ( ), nausea ( ), vomiting ( ), abdominal pain ( ) leukocytosis ( ) ( ) myalgia ( ), headache ( ), dizziness ( ), impaired consciousness ( ) fever ( ), cough ( ), fatigue ( ), anorexia ( ), dyspnea ( ), chest tightness ( ), pharyngalgia ( ), hemoptysis ( ), nausea ( ), vomiting ( ), abdominal pain ( ) leukocytosis ( ), lymphocytopenia ( ),  albumin ( ) & k + ( ),  ast ( ), alt ( ), k + ( ), na + ( ), ddimer ( ), ldh ( ), crp ( ), il- β ( ), il- r ( ), il- ( ), il- ( ) ( ) leukopenia ( ) headache ( ), myalgia ( . ), malaise ( ) fever ( ), cough ( ), dyspnea ( . ), rhinorrhea ( ), arthralgia ( ), chest pain ( ), vomiting ( ), ards ( ) aki ( ), cardiac injury ( ) ( ), rhinorrhea ( ), chest pain ( ), diarrhoea ( ), nausea/vomiting ( ), ards leukocytosis ( ), lymphocytopenia ( ) , thrombocytosis ( ), thrombocytopenia ( ), anemia ( ),  albumin ( ) j o u r n a l p r e -p r o o f adc, apparent diffusion coefficient; aki, acute kidney injury, alt, alanine aminotransferase; ards, acute respiratory distress syndrome; ast, aspartate aminotransferase; bnp, b-type natriuretic peptide; bun, blood urea nitrogen; ck, creatine kinase; cns, central nervous system; crp, c-reactive protein; dic, disseminated intravascular coagulation; eeg, electroencephalography; esr, erythrocyte sedimentation rate; f, female; fgf, fibroblast growth factors; gcsf, granulocyte colony-stimulating factor; gmcsf, granulocyte-macrophage colonystimulating factor; icu, intensive care unit; ifn-γ, interferon-γ; il, interleukin; ip, induced protein; iqr, interquartile range; ldh, lactate dehydrogenase; mcp, monocyte chemoattractant protein; mip, macrophage inflammatory protein; pdgf, platelet derived growth factor; pro-bnp, pro-brain natriuretic peptide; pt, prothrombin time; rantes, regulated on activation and normally 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(covid- ):a multi-center study in wenzhou city clinical course and outcomes of critically ill patients with sars-cov- pneumonia in wuhan, china: a single-centered, retrospective, observational study encephalitis as a clinical manifestation of covid- . brain, behavior, and immunity the pathogenesis and treatment of the `cytokine storm' in covid- detection of coronavirus in the central nervous system of a child with acute disseminated encephalomyelitis concomitant neurological symptoms observed in a patient diagnosed with coronavirus disease sars-cov- can induce brain and spine demyelinating lesions the cytokine release syndrome (crs) of severe covid- and interleukin- receptor (il- r) antagonist tocilizumab may be the key to reduce the mortality clinical features and short-term outcomes of patients with covid- in wuhan clinical characteristics of patients infected with sars-cov- in wuhan covid- -associated acute disseminated encephalomyelitis: a case report. medrxiv epidemiological, clinical characteristics of cases of sars-cov- infection with abnormal imaging findings guillain-barré syndrome associated with sars-cov- infection: causality or coincidence? the lancet neurology acute myelitis after sars-cov- infection: a case report social support and acute stress symptoms (asss) during the covid- outbreak: deciphering the roles of psychological needs and sense of control a novel coronavirus from patients with pneumonia in china sars-cov- receptor ace is an interferon-stimulated gene in human airway epithelial cells and is detected in specific cell subsets across tissues leukopenia ( . ),  neutrophils ( ), neutropenia ( . ), lymphocytopenia ( . ), thrombocytosis ( . ), thrombocytopenia ( . ),  albumin ( . ),  d-dimer ( . fever ( . ), cough ( . ), anorexia ( . ), dyspnea ( ), diarrhea aidp, acute inflammatory demyelinating polyneuropathy; aki, acute kidney injury, alt, alanine aminotransferase; aman, acute motor axonal neuropathy; amsan, acute motor and sensory axonal neuropathy; ards, acute respiratory distress syndrome; ast, aspartate aminotransferase; cbc dif , complete blood counts with differential; ck, creatine kinase; cmap, compound motor action potential; crp, c-reactive protein; covid- , coronavirus disease ; csf, cerebrospinal fluid; emg/ncs, electromyography/nerve conduction study; esr, erythrocyte sedimentation rate; f, female; il, interleukin; ino, internuclear ophthalmoparesis; inr, international normalised ratio; ivig, intravenous immunoglobulin; ldh, lactate dehydrogenase; lft, liver function test; m, male; nr, not reported; pt, prothrombin time; rft, renal function test; rrt-pcr, real-time reverse transcription polymerase chain reaction; sars-cov- , severe acute respiratory syndrome coronavirus ; siadh, syndrome of inappropriate antidiuretic hormone secretion; snap, sensory nerve action potential; wbc, white blood cell. key: cord- -xmqs ax authors: romoli, michele; jelcic, ilijas; bernard‐valnet, raphaël; garcía azorín, david; mancinelli, luca; akhvlediani, tamar; monaco, salvatore; taba, pille; sellner, johann title: a systematic review of neurological manifestations of sars‐cov‐ infection: the devil is hidden in the details date: - - journal: eur j neurol doi: . /ene. sha: doc_id: cord_uid: xmqs ax background: we systematically reviewed available evidence for reports of neurological signs and symptoms in coronavirus disease (covid)‐ patients to identify cases with severe acute respiratory syndrome coronavirus‐ (sars‐cov‐ ) infection or immune‐mediated reaction in the nervous system. methods: we followed prisma guidelines and used the medline, embase, google scholar, medrxiv and chinaxiv databases to search for papers on covid‐ and nervous system involvement which were published from january (st) to april (th) . data on design, sample size, neurologic assessment and related work‐up were extracted. biases were assessed with the newcastle‐ottawa scale. results: we analysed publications on potential neuroinvasive or parainfectious neurological complications of covid‐ . the reports focused on smell and taste (n= ) and evaluation of neurological symptoms and signs in cohorts (n= ). there were cases of guillain‐barré syndrome/miller‐fisher syndrome/cranial neuropathy ( cases), meningitis/encephalitis ( cases) and various other conditions ( cases). patients with cerebrospinal fluid (csf) examination and in particular sars‐cov‐ pcr was negligible. amongst, two had a positive sars‐cov‐ pcr exam of csf specimen. the study of potential parenchymal involvement with magnetic resonance imaging was rare. only reports received a rating for the highest quality standards. conclusion: this systematic review failed to establish comprehensive insights to nervous system manifestations of covid‐ beyond immune‐mediated complications as aftermath of respiratory symptoms. the authors therefore provide guidance for more careful clinical, diagnostic and epidemiological studies to characterize the manifestations and burden of neurological disease caused by sars‐cov‐ on behalf of the infectious disease panel of the european academy of neurology. the clinical spectrum of sars-cov- infection is wide, and encompasses asymptomatic infection, mild upper respiratory tract illness, and severe viral pneumonia with respiratory failure and sometimes death. from a neurobiological and translational viewpoint, neurological manifestations can be expected in covid- . this is substantiated on the one hand by a few cases with neurologic signs and symptoms and detectable virus load in cerebrospinal fluid (csf) during the sars-cov- epidemic in [ ] . sars-cov- and sars-cov- share genetic sequences but sars-cov- has a - times higher binding affinity to ace [ ] . on the other hand, angiotensin converting enzyme- (ace- ), the functional receptor utilized by sars-cov- and - for cell entry, is not only expressed in the lungs but also in the central nervous system (cns) [ , ] . expression of ace- is found in neurons and non-neuronal cells, the latter include astrocytes, oligodendrocytes and olfactory support cells [ , ] . moreover, infection of neurons with sars-cov- has been proven in transgenic mice and several presumed routes of cns entry were described in preclinical models [ ] . in a report from wuhan, china more than a third of the hospitalized covid- patients had some sort of nervous system-related clinical signs or symptoms [ ] . these included on the one hand more specific conditions such as loss of sense of smell or taste, myopathy, and stroke. there were non-specific symptoms such as headache, impaired level of consciousness, dizziness, or seizure on the other hand. from a neuroinfectiologic viewpoint, the relevance of these findings is limited; these conditions can be coincidental, secondary to systemic complications or a side-effect of therapy. only further diagnostic details such as focused neuroimaging, evaluation cardiovascular risk factor and comorbidities, assessment of prothrombotic or systemic hyperinflammatory states, presence of intrathecal inflammation and systematic exclusion of differentials would enable a placement within the spectrum of complications. this study therefore aimed to identify clinical cases of confirmed nervous system invasion or postinfectious neurological disease in the available covid- literature on the basis of a systematic review. hereupon, members of the infectious disease panel of the european academy of neurology (ean) compiled guidance for the diagnostic approach, which emphasizes the need for precise case definitions and standards for reporting. a systematic review was carried out to study all cases reporting nervous system involvement in patients with proven sars-cov infection. the protocol followed the prisma guidance for reporting of systematic reviews. medline, embase, google scholar, medrxiv and chinaxiv database were searched for papers accepted article published from st january to april th regarding the nervous system and covid- . the search strings for pubmed were as follows: (("covid"[all fields] or "coronavirus"[all fields] or "sars-cov- "[all fields]) and (("neurology"[mesh terms] or "neurolog*"[all fields]) or ("brain" [mesh terms] or "brain"[all fields]) or ("neuro"[all fields]) or ("nervous system"[mesh terms] or "nervous system"[all fields])) and (" / / "[pdat] : " / / "[pdat])). we also hand-searched reference lists of all articles identified in the electronic search using common search engines (e.g. google, bing). the search selected studies reporting neurological features of patients with sars-cov- infection. studies were identified after search and data were extracted regarding: study design, sample size, neurological assessment and diagnostic workup including brain imaging and csf analysis. biases were assessed with newcastle-ottawa scale [ ] . the systematic search yielded papers, of which were eligible for full-text assessment ( figure for prisma flow-chart). four were excluded; these were commentaries, response letters and review articles proposing on sars-cov- nervous system invasion but did not comprise clinical findings. twenty-six publications reporting neurological disturbances in patients with sars-cov- infection were evaluated, the major readouts are shown in table . bias assessment revealed low to fair quality more than half of the studies ( / , %), as shown in figure . this was mainly due to selection and reporting bias, as well as on the basis of uncertain exposure and lack of testing for sars-cov- with pcr in csf. only reports reached a rating for high quality; one study evaluated neurological diagnoses of deceased patients with covid- [ ] , another studied neurological signs and symptoms in a cohort of hospitalized covid- patients [ ] , and a case series of peripheral nervous system dysfunction with in-depth phenotyping and diagnostic workup [ ] . the fourth study described the clinical characteristics, laboratory features, treatment, and outcomes of cerebrovascular disease complicating sars-cov- infection [ ] . among the consecutively admitted patients with covid- , ( %) had acute ischemic stroke, ( . %) cerebral venous sinus thrombosis (cvst), and ( . %) cerebral hemorrhage. five observational studies evaluated smell and taste dysfunction. the first study used an internet-based platform in adults who underwent testing for covid- and found a higher rate of smell and taste impairment in sars-cov- positive patients [ ] . the second report was a european multicenter study of mild-to-moderate covid- patients which used standardized questionnaires [ ] . they found olfactory accepted article and gustatory dysfunction in . % and . %, respectively. the third study used an online checklist inquired for self-reported anosmia/hyposmia [ ] . in that cohort, % had hyposmia or anosmia and the onset was reported as sudden in %. the fourth report was a case-control study of smell and taste disorders among patients positive for sars-cov- on nasopharyngeal swab examination and also included sars-cov- negative patients as controls [ ] . they found that covid- patients were significantly younger ( %) and a high rate of smell ( %) and taste disorders ( .%). the fifth study smell dysfunction in % of the sars-cov- nasopharyngeal swab pcr positive patients and reported that this was evident for all odorants studied [ ] . the study also had a age-and sex-matched control group. none of these five studies provided data csf analysis or brain imaging. five studies examined neurological disorders in cohorts. in the first study, hypoxic encephalopathy was the cause of death in % of patient who died from covid- [ ] . the second report assessed neurologic manifestations in a cohort hospitalized at three dedicated covid- inpatient centers [ ] . they found that . % had various neurologic manifestations that involved the cns, peripheral nervous system (pns), and skeletal muscles. brain imaging, csf analysis and further workup were reported in neither of the studies. the third study reported neurologic features in . % of consecutive patients admitted because of acute respiratory distress syndrome (ards) due to covid- and treated at the icu [ ] . confusion, agitation, pyramidal signs and dysexecutive syndrome were the most common clinical manifestations. cerebral magnetic resonance imaging (mri) was performed in / ( %), there was evidence for leptomeningeal enhancement in % and ischemic stroke in %. eeg ( / , %) and csf examinations were performed in some patients ( / , %). none of the patients had a pleocytosis in csf. a multicentre retrospective study evaluated the occurrence of seizures in covid- patients [ ] . there was not a single case of symptomatic seizures or status epilepsy among this cohort in which patients with epilepsy were excluded a priori. the fifth study has been already covered above and concerned the rate of acute cerebrovascular events ( %) in a cohort of covid- patients [ ] . the average of time from symptoms of sars-cov- infection to clinical manifestation of cerebrovascular disease was days (interquartile range - ). the patients with cerebrovascular disease were significantly older, more likely to suffer from severe respiratory disease and more likely to have cardiovascular risk factors and medical history of cerebrovascular disease. they were also more likely to have an increased inflammatory response and hypercoagulable state. four publications reported eight cases of guillain-barre syndrome (gbs) in patients with confirmed sars-cov- infection. nerve conduction studies disclosed both demyelinating and axonal neuropathies (n= and n= , respectively). all but one case occurred with a time lag from the respiratory symptoms, the accepted article range was to days. in the remaining case the clinical manifestation of gbs preceded covid- symptoms by days [ ] . in the case series of five gbs patients, three patients had high protein levels and all tested negative for sars-cov- in csf, as well as for anti-ganglioside antibodies [ ] . the other reports did not perform pcr for sars-cov- or testing for immunoglobulin levels, and did not investigate antiganglioside antibodies in csf or serum. in addition to the gbs cases, a case of miller-fisher syndrome with positive gd b-igg and a case of multiple cranial neuropathies, both with negative sars-cov- pcr in csf, were found [ ] . nine cases of encephalitis/meningitis and presumed association with covid- were reported in publications. amongst was a case of encephalitis in a patient with negative sars-cov- testing in both nasopharyngeal swab and csf (normal cell count), no mri was performed [ ] . another patient with presumed encephalitis had normal cell count and negative sars-cov- pcr in csf, an mri was not performed [ ] . a similar constellation was reported for another case [ ] . there is a patient with a diagnosis of covid- related encephalitis for which data could only be retrieved from the hospital report [ ] . in that case, the neurological symptoms included seizures and hiccups and sars-cov- pcr of csf was positive. for the case of acute necrotizing encephalitis, a sars-cov- pcr was not performed in csf [ ] . a pathogenesis triggered by a covid- -related cytokine storm was subsequently assumed. a positive sars-cov- pcr in csf was present in a patient with right temporal lobe encephalitis and ventriculitis [ ] . two patients were classified as meningo-encephalitis in association with covid- [ ] . both had encephalitic symptoms, including non-convulsive status epilepticus and mental changes, with normal mri and negative sars-cov- pcr in csf. a case of meningoencephalitis was described, the patient had meningism, headache, fever and seizures, and was pcr was negative for sars-cov- in csf [ ] . there were further case reports, which were related to various aspects. a patient in whom the authors assumed myelitis as final diagnosis [ ] . in detail, the patient had a myelopathic syndrome days after the onset of respiratory symptoms but was not evaluated with mri nor lumbar puncture. there is a case of presumed acute disseminated encephalomyelitis (adem) with only minimal contrast-enhancement on brain mri [ ] . csf examination was normal (cell count, protein, glucose), sars-cov- pcr was only performed for nasopharyngeal swab, which was positive. furthermore, there is a patient with pre-existing epilepsy related to herpes-simplex virus encephalitis who presented with non-convulsive status epilepticus in the context of covid- infection [ ] . the authors discuss fever as the cause of lowering the threshold for seizures in a brain with structural damage. the case of intracerebral hemorrhage which occurred days after fever and respiratory symptoms did not have obvious coagulation disturbances [ ] . this article is protected by copyright. all rights reserved vascular imaging and csf diagnostic were not performed. a patient with headache, altered mental status, fever, and cough was classified as acute encephalopathy [ ] . eeg ruled out status epilepticus, csf showed normal results and sars-cov- pcr in csf was not done. overall, there were patients positive for sars-cov- pcr of csf among the examined patients [ , ] . our systematic search yielded only a limited number of studies and a significant reporting bias. this does not enable an in-depth characterization of neuroinfectious diseases associated with covid- . indeed, quality, design and sample size of the available studies detains us from conclusion on possible direct neuroinvasive disease caused by sars-cov- . the available literature does, however, provide evidence for unspecific symptoms commonly seen in viral infections including smell and taste disturbances, and the chance of immune-mediated peripheral nerve involvement. our analysis also suggests that there is an overdiagnosis of neurological disorders due to the inappropriate use of case definitions and restricted exclusion of potential mimics. nervous system involvement has been reported during previous coronavirus epidemics. interestingly, the analysis of the sars-cov- and mers epidemic identified only a few anecdotal case reports and could not provide comprehensive insights to the clinical and radiological picture of neurological disease [ ] . moreover, there are preclinical studies reporting the neuroinvasive potential of coronaviruses and their immunogenicity [ ] . the gbs we identified in our analysis are more consistent with a parainfectious disorder, i.e. a syndrome occurring during or soon after the viral syndrome, rather than a postinfectious syndrome. the limited literature for the covid- outbreak could be seen in the restricted documentation due to the tying up of resources posed by the medical challenges. indeed, it is conceivable that most emphasis was placed on the management of severe respiratory symptoms and restricted icu capacity. it is obvious that neurologists are required for the care of covid- patients [ , ] . their active involvement is not only mandatory for the work-up of presumed infectious and immune-mediated conditions but also for patients with reduced consciousness and nervous system complications of cardiac, pulmonary and coagulation disturbances related to sars-cov- [ ] . moreover, hypoxic brain injury may be the reason for clinical deterioration in a subgroup of patients. the potential association of sars-cov- with cerebrovascular diseases needs to be assessed in more detail; prospective trials with systematic use of ancillary investigations to confirm direct and indirect mechanism of action are mandatory in order to accepted article gain further insights. from a neuroinfectiologic viewpoint, the major limitation of the available reports were that precise case definitions were not used, csf testing was performed only in a subgroup of patients and exclusion of potential other diagnoses were reported only on occasion. there were just cases with positive sars-cov- pcr in csf among patients with potential neurologic symptoms and proven covid- . however, to date nothing is known about the sensitivity of this detection method for the examination and csf. indeed, csf examination for tick-borne encephalitis virus with pcr is not a standard due to a low sensitivity of the method and probably also transient presence of the virus in csf. the best diagnostic approach to diagnose cns infection with sars-cov- or parainfectious immune reaction associated with sars-cov- remains to be elucidated. until now, no reports about intrathecal sars-cov- -specific igg synthesis in these cases is available but could be key for diagnosis. in addition, a better understanding of the reported non-specific symptoms including olfactory and gustatory disturbances, impaired consciousness and encephalopathy is needed. the systemic inflammatory response is a relevant feature of severe covid- and could explain some of these scenarios. the current analysis tells us that we do need more careful clinical, diagnostic and epidemiological studies to define the manifestations and burden of neurological disease caused by sars-cov- . in this regard, we see a clear need for the use of precise case definitions and focused diagnostic work-up to distinguish nonspecific complications of severe disease and focused reporting of neurological involvement in association with sars-cov- infection. moreover, appropriate investigations are required to rule out other established causes of brain infections and parainfectious disease before attributing a condition to sars-cov- . it also needs to kept in mind that sars-cov- causes a large number of asymptomatic or mildly symptomatic infections. a coincidental infection may exacerbate a so far asymptomatic or known neurological disease of other causes. here, we provide guidance for assembling key clinical and paraclinical data which are required to establish insights to true spectrum of direct and indirect effects of sars-cov- infection on the nervous system ( table ) :: . the timing and results from nasopharyngeal swab pcr needs to be reported. most important is the relation to the development of respiratory and neurological signs/symptoms. as soon as the antibody testing gets more widely available, this also pertains to this method. both igm and igg need to be reported. for all detection methods, the testing kit and ideally the exact values need to be mentioned. . potential differentials need to be ruled out: frequent mimics in a report from spain of patients evaluated for sars-cov- infection included hypercapnia, renal or liver failure and side-effects of accepted article drug therapy [ ] . comorbidities are frequent in certain patients and risk factors for neurological complications need to be identified. . if neuroinvasion or immune-mediated disease of the nervous system is suspected, it is mandatory to perform pcr testing for sars-cov- in csf and anti-sars-cov- igm/igg testing in serum and csf to check for intrathecal humoral immune reaction. it will be of major importance to determine whether pcr of csf specimen is sensitive enough and define the time window of potential sars-cov- detection in relation to respiratory and neurological symptoms. the list of differential diagnoses for meningitis, encephalitis and myelitis is extensive [ , ] . a guidance is shown in table . we recently summarized potential indications for csf examination [ ] . briefly, a permissive strategy for csf testing should be exerted  on suspicion of encephalitis  new focal neurological deficit of no plausible differential etiology/no better explanation  delirious condition of no plausible differential etiology or no better explanation  acute cerebrovascular disorders  convulsive or non-convulsive seizures of no plausible differential etiology or no better explanation and  icu patients with disorders of consciousness of no plausible differential etiology or no better explanation. the distinction of encephalopathy and encephalitis needs to be done according to standard criteria [ ] . brain mri is critical and contrast-enhanced sequences are mandatory [ ] . coagulation disorders are relatively frequently encountered among covid- patients and need to be considered in the workup of cerebrovascular disorders [ , ] . in cases of peripheral nervous system involvement nerve conduction studies and electromyographic findings need to be reported, and antibodies to specific for immune-mediated conditions to provide differentials with critical illness neuropathy, acute non-inflammatory neuropathies and myopathy [ ] . . reporting of timing and type of treatment. there is currently no study evidence for efficacy of a specific treatment for sars-cov- [ ] . guideline for the management of respiratory symptoms and systemic complications are outlined elsewhere, many be regionally distinct and are likely to be updated on a regular basis. with regard to neuroinfectious manifestations, one should be adhere to guidance on the management of viral meningitis and encephalomyelitis [ ] . the management of immune-mediated conditions including gbs and miller-fisher syndrome should follow standard guidelines, with intravenous immunoglobulin (ivig) or plasma exchange as first- this article is protected by copyright. all rights reserved line options [ ] . coagulation disorders and other systemic complications of sars-cov- are likely to be of relevance for neurological care and complications. while an increased risk of seizures has not been reported so far, the potential interaction of antiepileptics and antiviral/-microbial therapy need to be kept in mind. . we do need information on critical care illness, prognostic factors and outcome. the authors confirm that the data supporting the findings of this study are available within the article. this article is protected by copyright. all rights reserved what did we learn from the previous coronavirus epidemics and what cen we do better: a 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reserved key: cord- -y gjh authors: wilson, m.r. title: meningitis, viral date: - - journal: encyclopedia of the neurological sciences doi: . /b - - - - . - sha: doc_id: cord_uid: y gjh this article provides an overview of the pathogenesis, epidemiology, causes, clinical presentation, laboratory diagnosis, and treatment of the most common causes of viral meningitis in the united states. it also summarizes other infectious and noninfectious causes of lymphocytic or aseptic meningitis. the concept that agents other than bacteria can invade the central nervous system (cns) began with the emergence of poliomyelitis as an epidemic infection and, subsequently, with the realization that similar meningeal inflammation and cerebrospinal fluid (csf) pleocytosis occurred in as many as % of patients with mumps parotitis. that meningitis could be caused by other 'filterable agents' (i.e., viruses) was demonstrated by rivers and scot, who in recovered lymphocytic choriomeningitis virus (lcmv) from the csf of an affected patient. it is now known that a wide and constantly changing variety of viruses may invade the cns to produce meningitis or encephalitis. viral meningitis is important in three respects. first, viral meningitis must be differentiated from the much more dangerous condition, bacterial meningitis: until this is accomplished, patients presenting with signs and symptoms of meningitis must be considered medical emergencies, and antibiotic treatment for presumptive bacterial meningitis must be instituted. second, viral meningitis, although rarely fatal, may produce clinical impairment that can persist for weeks to months, especially in the immunosuppressed patient. finally, 'lymphocytic' or 'aseptic' meningitis may be caused by agents other than viruses, and the possibility of other, more readily treated (and sometimes more dangerous) conditions must be kept in mind when diagnosing a patient with presumed viral meningitis. before meningitis can occur, the causative agent must first penetrate the body from the external environment and then gain entry to the cns across the blood-brain barrier (bbb). entry of the virus into the host may occur by gastrointestinal inoculation (as is the case with the enteroviruses), cutaneous inoculation (as occurs with the arthropod-borne agents), the respiratory route (as is the case with mumps), or transmucosal penetration or intravenous inoculation (as occurs with human immunodeficiency virus (hiv)). early workers in the field of viral cns infections believed that invasion of the nervous system occurred by the spread of viruses along neurons, as is the case with rabies virus. currently, however, it is known that the majority of viral meningitis results from hematogenous dissemination of virus following symptomatic or clinically inapparent systemic infection. penetration across the bbb may occur at the choroid plexus or through meningeal capillaries. exceptions to this include meningitis following genital herpes simplex infection, meningitis associated with herpes zoster, and mollaret's meningitis, in which reactivated infection of herpes simplex virus type (hsv- ) within the dorsal root ganglia leads to repeated episodes of meningitis. viral replication in the meninges, superficial brain or spinal cord parenchyma, and the ventricular system elicits an inflammatory response, which is predominantly lymphocytic. this results in an alteration of the bbb so that protein levels increase within the csf. unlike bacteria or fungi, however, viral replication does not lower glucose within the csf, nor does it usually results in altered transport of glucose across the bbb. thus, in contrast to bacterial, mycobacterial, or fungal infections, csf glucose concentrations during viral meningitis are usually normal. meningitis is the most common phenotype of a neuroviral infection, and it is a far more common condition than bacterial or fungal meningitis. approximately cases of lymphocytic or presumed viral meningitis occur each year in the united states although numbers are difficult to ascertain because it is not a reportable illness. csf pleocytosis has also been reported in individuals infected with measles, mumps, and hiv without signs and symptoms of meningeal irritation. similar asymptomatic cns involvement probably occurs with other viruses as well. although viral meningitis affects all age groups, it is predominantly a disease of childhood. the agents causing viral meningitis can be divided into three broad groups: ( ) common agents of viral meningitis, including the enteroviruses, arthropod-borne agents, and hsv- ; ( ) less common agents, including hiv, mumps, lcmv, human herpesvirus (hhv- ), and parvovirus b ; and ( ) agents known to cause lymphocytic meningitis only in rare cases. outside the united states, new viruses like toscana virus and chikungunya virus are emerging causes of viral meningitis. in addition, a number of nonviral, and occasionally noninfectious, conditions may cause a clinical syndrome indistinguishable from viral meningitis. enteroviruses account for approximately % of cases in which the causative virus is identified ( table ) . enteroviruses are small, nonenveloped single-stranded positive-sense ribonucleic acid (rna) viruses within the family picornaviridae. although more than serotypes of enteroviruses have been identified, coxsackievirus a and echoviruses e , e , e , e , and e account for % of all cultured isolates of csf. poliovirus, although no longer found in developed countries due to mass vaccination efforts, still causes aseptic meningitis and paralytic disease in countries like pakistan, afghanistan, and nigeria where cultural taboos, lack of development, and war have stymied vaccination efforts. enteroviruses are disseminated by fecal-oral spread, and cases in developed countries cluster during summer months when sanitation tends to be most relaxed. recent studies employing polymerase chain reaction (pcr) methods, however, confirm older observations that enteroviral cns infections occur throughout the year, and many previously undiagnosed cases of viral meningitis occurring during winter months are also caused by these viruses. coxsackieviruses, echoviruses, and enteroviruses may cause encephalitis and, rarely, paralytic disease, especially in neonates and the immunosuppressed. this was vividly illustrated in with the enterovirus outbreak in cambodia that killed more than children. arthropod-borne viruses, or arboviruses, include agents from several different families whose hosts are typically small mammals or birds. they spread to humans through the bite of an arthropod vector ( table ) . although these agents are most commonly considered to cause encephalitis, all of them, with the exception of eastern equine encephalitis, more frequently cause meningitis. the most common domestic arthropodborne agents associated with viral meningitis include st. louis encephalitis virus, the california/lacrosse group of viruses, colorado tick fever, and west nile virus, which caused an explosive outbreak when it first arrived in the united states in and more recently in . these agents, like enteroviruses, have a peak incidence in summer and early fall. the exception to this rule is colorado tick fever, which is more frequently transmitted in the spring and early summer. herpesviridae are enveloped, double-stranded dna viruses. hsv- and hsv- , varicella-zoster virus (vzv), epstein-barr virus (ebv), cytomegalovirus (cmv), and hhv- all cause viral meningitis. of these, however, only hsv- has been associated with a significant number of cases. older data suggest that hsv- accounts for - % of viral meningitis cases. recent work employing pcr suggests that it may be the most common cause of viral meningitis in adult women. hsv- most often causes meningitis following primary genital infection. occasional cases may also follow primary genital infection with hsv- . patients with recurrent (mollaret's) meningitis often have recurrent infection due to hsv- . csf pleocytosis occurs during both chicken pox and herpes zoster with or without skin lesions; this pleocytosis is usually asymptomatic but may occasionally be associated with meningitic symptoms. hsv- is also the most common cause of nonepidemic viral encephalitis, commonly producing an asymmetric lesion in the anterior and medial temporal lobes. in the immunosuppressed and elderly, vzv can cause a multitude of other cns syndromes including stroke due to vasculitis, herpes zoster ophthalmicus, myelitis, encephalitis, and cranial neuropathies. cmv can cause ventriculitis and radiculitis in patients with hiv. lastly, hhv- is now appreciated to produce a bilateral medial temporal lobe encephalitis in bone marrow transplant patients. hiv has been associated with both acute and persistent lymphocytic meningitis. onset is most commonly at the time of seroconversion, and for the astute clinician, this represents an opportunity to make an early diagnosis of hiv during a period of time when the patient's viral load is very high, and thus, is at high risk for transmission. the course may be uniphasic, chronic, or, occasionally, recurrent. in recurrent cases that happen despite the patient achieving systemic virologic control with antiretroviral medications, the differential diagnosis should include the possibility of cns hiv escape either due to a resistant strain of hiv having evolved in the cns compartment or due to inadequate cns penetration of antiretroviral medications. hiv rna can be identified in csf using reverse transcriptase-polymerase chain reaction (rt-pcr) methods, which also allow an assessment of viral burden and the resistance mutation profile. although hiv itself typically causes a csf pleocytosis of less than cells/mm with a as discussed previously, hsv- , hhv- , vzv, cmv, and ebv occasionally cause meningitis. mumps virus, like measles virus, is a paramyxovirus, containing a single-stranded negative-sense rna genome. before the advent of the mumps vaccine, mumps was the most common cause of viral meningitis, accounting for more than % of isolates. currently, mumps virus meningitis is rare in developed countries. the virus is still a common cause of cns infection in underdeveloped countries and countries like japan in which vaccination programs have been suspended due to vaccine-induced cases of lymphocytic meningitis. in these countries, mumps virus is an important cause of sensorineural deafness. in experimental animals infected in utero, mumps can cause aqueductal stenosis, and there are approximately human cases of this in the literature. lcmv is an arenavirus containing a single-stranded ambisense rna genome. wild and laboratory mice are the natural hosts, and there is recent evidence that snakes harbor these viruses as well. lcmv is associated with human cases of meningoencephalitis as a consequence of exposure to laboratory or wild mice, and in rare epidemics it is associated with pet hamsters. cases are more common in impoverished areas with poor hygiene. important outbreaks of fatal lcmv meningoencephalitis have also occurred in clusters of solid organ transplant patients infected by organs from asymptomatic donors. lcmv meningitis typically occurs during autumn and early winter, and it has been suggested that this reflects more extensive mouse-human contact as mice move inside to escape winter weather. in studies before , the virus was thought to account for - % of cases of viral meningitis. in recent years, reports of meningitis due to lcmv have been rare. however, congenital lcmv infection is a significant, often unrecognized cause of chorioretinitis, hydrocephalus, microcephaly or macrocephaly, and mental retardation. acquired lcmv infection likewise may be an underappreciated illness. the meningitis caused by lcmv may be extremely persistent and has been associated with symptoms and csf abnormalities lasting for months. acquired lcmv infection may also be associated with encephalitis, transverse myelitis, a guillain-barré-type syndrome, and both transient and permanent hydrocephalus. parvovirus b most commonly causes an acute febrile illness, accompanied by erythema infectiosum. the virus can also produce meningitis and meningoencephalitis in both immunocompetent and immunocompromised patients. the combination of rash and signs of meningeal irritation may mimic acute meningococcal infection. other items to always consider in the differential of rash and meningitis include west nile virus, syphilis, rickettsial infection (i.e., rocky mountain spotted fever), enteroviruses, lyme disease, human granulocytic anaplasmosis, and human monocytic ehrlichiosis, many of which are treatable. csf findings are typical of viral infection. occasionally, csf may be normal. rare causes of viral meningitis include influenza a and b viruses, parainfluenza viruses, rotaviruses, coronaviruses, measles virus, and adenoviruses. lastly, although jc virus is typically associated with progressive multifocal leukoencephalopathy, it can also cause viral meningitis, especially in the immunosuppressed. onset of viral meningitis may occur following a symptomatic, systemic illness, or as an isolated event following inapparent systemic infection. patients can present with fever ( %), headache ( %), photophobia, neck stiffness ( %) and/or back pain. significant alteration of consciousness is far less common than in bacterial meningitis. immunocompromised patients may have even more subtle exam findings and histories, and there should be a low threshold for further investigation with lumbar puncture in these cases. seizures or focal neurological signs are unusual although cranial neuropathies are seen with certain viruses like vzv, hsv, west nile virus, and hiv. focal signs should raise concerns about a concomitant viral encephalitis or a focus of infection, such as a brain abscess. patients are usually uncomfortable but do not appear severely ill. physical examination may reveal evidence of systemic illness, including rash, lymphadenopathy, pharyngitis, or splenomegaly, depending on the infectious agent. neurological examination commonly reveals nuchal rigidity with the patient unable to touch chin to chest. resistance to passive neck flexion and kernig's and/or brudzinski's signs may be present but are inconsistent. both signs may be absent in milder cases. a useful test of nuchal rigidity is to ask the patient to touch forehead to knee; this will often be positive when all other tests of meningeal irritation are questionable or absent. papilledema is rare. routine blood studies may reveal a lymphocytic leukocytosis. liver function tests may be elevated if there is hepatic involvement. the most important diagnostic test in viral meningitis is the lumbar puncture. this should be preceded by head magnetic resonance imaging (mri) or, less optimally, computed tomography if focal signs are present, there is significant altered mental status, or if there is any suspicion of increased intracranial pressure. spinal fluid will usually show mildly elevated opening pressure, lymphocytic pleocytosis, elevated protein, and normal glucose ( table ) . the cell count is usually less than cells/mm . protein is usually in the range of - mg/dl. exceptions exist to this csf formula, however. cell count may be as high as cells/mm . during the first - h of infection, csf may contain a mixture of polymorphonuclear leukocytes and lymphocytes with even a polymorphonuclear predominance early in infections with west nile virus, hsv, and vzv. glucose concentrations, although usually % of blood values, may be significantly depressed: this has been reported with meningitis due to herpes zoster, mumps, and lcmv. return of csf parameters to normal may be extremely prolonged following viral meningitis, and isolated reports have described persistent csf pleocytosis and elevated protein over periods of weeks to months. before the advent of pcr, diagnosis of viral meningitis was difficult and often an exercise in futility: viruses may take considerable time to grow in culture, and many viral agents cannot be readily grown from csf. viral serologies comparing acute and convalescent sera have been used for retrospective diagnosis, and serological diagnosis can be accelerated by comparing serum and csf antibody titers to identify the synthesis of specific antiviral antibodies within the cns; however, serological tests only rarely allow rapid enough diagnosis to direct therapy. the advent of pcr methods has revolutionized the diagnosis of both meningitis and encephalitis. pcr for enteroviruses, hsv- , hsv- , vzv, and cmv are readily available in many laboratories, and pcr diagnosis of other agents is often available through larger commercial laboratories or the centers for disease control. although pcr is highly specific, it has limited sensitivity in certain circumstances. it is relatively insensitive in the first days of infection with hsv as well as after day of infection. the overall sensitivity of pcr for vzv is %; if vzv is suspected, immunoglobulin g (igg) and immunoglobulin m (igm) antibodies should always be sent from the csf as well. in the case of hiv, rt-pcr methods are available not only for diagnosis but also for determining viral load and resistance mutations. even with the use of pcr, the causative agents in many cases of viral meningitis remain undiagnosed. viral meningitis should be considered in the differential diagnosis of any patient presenting with headache, photophobia, and neck stiffness. however, the presence of these findings also makes it mandatory to exclude bacterial infection. although patients with viral meningitis are less severely ill, bacterial meningitis may also appear mild in its early stages. conversely, patients with viral meningitis may deteriorate. many other infectious conditions can also cause lymphocytic meningitis. these include secondary syphilis, leptospirosis, brucellosis, infections by mycobacterium tuberculosis, lyme disease caused by borrelia burgdorferi, infections due to ehrlichiae or, rarely, other rickettsial agents, mycoplasma pneumoniae, and fungi (particularly cryptococcus neoformans, histoplasma, blastomyces coccidioides, and candida). tuberculous and fungal meningitis are often, but not always, accompanied by a significant decrease in csf glucose. lyme meningitis may produce csf findings identical to those seen in viral meningitis. however, erythema migrans, multiple cranial neuropathies and polyradiculopathies are common features of lyme meningitis. similarly, patients with lyme meningitis tend to have fewer white blood cells (mean, vs. /mm ) and a significantly greater percentage of mononuclear cells than patients with viral meningitis. both m. tuberculosis and m. pneumoniae are difficult to culture but are readily detectable by pcr; pcr tests for ehrlichiae are in limited use. noninfectious etiologies can also cause a lymphocytic or aseptic meningitis including side effects of a number of medications like nonsteroidal anti-inflammatory drugs, serum immunoglobulins, carbamazepine, lamotrigine, and trimethoprim sulfamethoxazole. recurrent meningitis can occur in patients with periodic leakages from dermoid or epidermoid cysts abutting the meninges. in such patients, the diagnosis can be made by a detailed mri examination of the brain and the spinal cord. lastly, autoimmune disease can manifest as a lymphocytic meningitis, sometimes representing the initial presentation of systemic lupus erythematosus and sarcoidosis. it can also be seen associated with sjö gren's syndrome and rheumatoid arthritis, particularly in patients who take nonsteroidal anti-inflammatory drugs. most cases of viral meningitis are self-limited, and antiviral therapy is usually not indicated. controlled studies of antiviral csf during the first h of viral meningitis may contain a mixture of lymphocytes and polymorphonuclear leukocytes. in these cases, in contrast to bacterial meningitis, csf glucose is usually normal and follow-up lumbar puncture h later will often but not always show lymphocytes only. c positive gram stain requires approximately colony-forming units (cfu)/ml of csf. approximately % of gram stains will be positive if csf contains cfu/m. prior antibiotic treatment will reduce this amount by %. abbreviations: afb, acid-fast bacillus; csf, cerebrospinal fluid; pcr, polymerase chain reaction. agents in viral meningitis have not been reported in detail. recent data from controlled studies presented in abstract form, however, suggest that virological and clinical improvements are better in patients with severe enteroviral meningoencephalitis treated with the antiviral agent pleconaril than with placebo. similarly, depending on the severity of illness, consideration should be given to the therapy for hsv meningitis with acyclovir or similar agents. use of antiviral agents in the treatment of viral meningitis is still essentially experimental and must be balanced against the severity of disease and complications of the therapy. an exception to this is hiv meningitis, in which diagnosis of hiv infection is in itself an indication for highly active antiretroviral therapy. viral meningitis is almost always a self-limiting disease. recovery may occur within days. however, symptomatic illness is not infrequently prolonged, and patients may require weeks or months to return to full health. permanent neurological deficits or intellectual impairment are rare and, if present, should prompt a more thorough workup for nonviral etiologies of meningitis. see also: encephalitis, viral. herpesviruses, human. meningitis, bacterial. meningitis, fungal aseptic meningitis, a disease of diverse etiology: clinical and etiologic studies on cases the rational clinical examination. does this patient have acute meningitis lymphocytic choriomeningitis virus: reemerging central nervous system pathogen cerebrospinal fluid in central nervous system infections prospective analysis of cases of enteroviral meningitis: interest of systematic genome detection in cerebrospinal fluid irrespective of cytologic examination results lymphocytic choriomeningitis virus: a neglected pathogen of man viral infections of the nervous system diagnosis and surveillance of herpes simplex virus infection of the central nervous system the outbreak of west nile virus infection in the new york city area in cerebrospinal fluid findings in aseptic versus bacterial meningitis laboratory diagnosis of common viral infections of the central nervous system by using a single multiplex pcr screening assay viral meningitis clinical significance of enteroviruses in serious summer febrile illnesses of children treatment of potentially life-threatening enterovirus infections with pleconaril prolonged fever caused by parvovirus b -induced meningitis: case report and review key: cord- -t hyfum authors: rifino, nicola; censori, bruno; agazzi, emanuela; alimonti, dario; bonito, virginio; camera, giorgia; conti, marta zaffira; foresti, camillo; frigeni, barbara; gerevini, simonetta; grimoldi, maria; la gioia, sara; partziguian, tania; quadri, stefano; riva, riccardo; servalli, maria cristina; sgarzi, manlio; storti, benedetta; vedovello, marcella; venturelli, elisabetta; viganò, martina; callegaro, annapaola; arosio, marco; sessa, maria title: neurologic manifestations in covid- patients admitted to papa giovanni xxiii hospital, bergamo, italy date: - - journal: j neurol doi: . /s - - - sha: doc_id: cord_uid: t hyfum objectives: evidences from either small series or spontaneous reporting are accumulating that sars-cov- involves the nervous systems. the aim of this study is to provide an extensive overview on the major neurological complications in a large cohort of covid- patients. methods: retrospective, observational analysis on all covid- patients admitted from february rd to april th, to asst papa giovanni xxiii, bergamo, italy for whom a neurological consultation/neurophysiological assessment/neuroradiologic investigation was requested. each identified neurologic complication was then classified into main neurologic categories. results: of covid- patients, presented neurologic manifestations that manifested after covid- symptoms in pts and was the presenting symptom in . neurological manifestations were classified as: (a) cerebrovascular disease [ pts ( . %)] including ischemic and haemorrhagic strokes, transient ischemic attacks, cerebral venous thrombosis; (b) peripheral nervous system diseases [ ( . %)] including guillain–barrè syndromes; (c) altered mental status [ ( . %)] including one necrotizing encephalitis and cases with rt-pcr detection of sars-cov- rna in csf; (d) miscellaneous disorders, among whom patients with myelopathy associated with ab anti-sars-cov- in csf. patients with peripheral nervous system involvement had more frequently severe ards compared to patients with cerebrovascular disease ( . % vs %; difference = . % % ci . – . ; χ( )= . ; p < . ) and with altered mental status ( . % vs . %; difference = . % % ci . – . %; χ( )= . ; p < . ). conclusion: this study confirms that involvement of nervous system is common in sars-cov- infection and offers clinicians useful information for prevention and prompt identification in order to set the adequate therapeutic strategies. in december , an outbreak caused by a novel coronavirus ( -ncov), now named severe acute respiratory syndrome coronavirus (sars-cov- ), occurred in china and has rapidly spread all over the world causing a pandemic. the disease caused by sars-cov- was named covid- [ ] . in europe, the first case was reported in the lombardy region. although soon after all italian regions reported patients with covid- , the highest number of cases was in eastern lombardy [ ] , specifically in the bergamo's province with , confirmed covid- patients up to april th [ ] . the typical spectrum of disease severity of sars-cov- infection is highly variable, ranging from asymptomatic carriers to severe acute respiratory distress syndrome (ards) leading to death. with the increasing number of confirmed cases and the accumulating clinical data, it is now well established that, in addition to the predominant respiratory symptoms, a significant proportion of patients has neurological manifestations [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] . despite available studies have drawn attention to the neurological component of sars-cov- infection and provide detailed description of the neurological complications, they still have limitations. data were collected either from limited or selected cohorts, and in the only nation-based study, case notification was on voluntary basis; diagnosis was mainly based on patient's description as cerebrospinal fluid analysis and instrumental examinations were avoided in order to reduce the risk of cross infection [ ] . moreover, scarce information regarding the impact of the neurologic involvement on clinical outcomes are available. here, we describe the neurologic manifestations observed in patients among patients with laboratory or radiology-confirmed diagnosis of covid- , admitted to the asst papa giovanni xxiii in bergamo, in the epicentre of italian pandemic, between february rd and april th, . we provide an extensive overview on the different neurological complications involving both central (cns) and peripheral (pns) nervous system, and on their impact on outcome. the present study is a retrospective, observational analysis conducted at asst papa giovanni xxiii on all adult patients of both sexes, with confirmed covid- disease, admitted from february rd to april th, . our hospital was designated to treat patients with sars-cov- infection. within a few days from the beginning of the outbreak, % of beds were reconverted to covid- patients and % staff doctors, regardless of their specialty, were redeployed to covid- units. we evaluated all neurological consultations/neurophysiological assessments/ neuroradiologic investigations requested for covid- patients during hospitalization. we included in our analysis the following main diagnosis/syndromes: (a) cerebrovascular diseases (cvd) including ischemic and haemorrhagic stroke, transient ischemic attack, and cerebral venous thrombosis, (b) altered mental status including encephalitis/ meningitis/meningo-encephalitis, (c) peripheral nervous system disorders including guillain-barrè syndromes (gbs), critical illness myopathy and neuropathy (crimyne), plexopathies and peripheral polyneuropathies. a few additional patients presenting symptoms/signs that did not fit these categories were classified as miscellaneous. wherever possible, informed consent was collected verbally. however, in most cases, due to the inability to provide informed consent by the patient or the inability to collect it in compliance with the contagion prevention measures, the principle of secondary use of data was used in accordance with art. , paragraph , letter b) of the november th, law, n. , included in the legislative decree / of art. -bis. the institutional review board at asst papa giovanni xxiii provided approval for the study ( / , / / ). demographic (age, sex), comorbidities (hypertension, diabetes, dyslipidemia, atrial fibrillation, peripheral artery disease, cardiac disease or cvd, malignancy, dementia and lung disease), onset of neurological symptoms (before, concomitant, after covid- onset), severity of ards according to the horowitz index, clinical, laboratory and imaging data were extracted from medical records using a standardized anonymized data collection form by study physicians (nr, ms, sg). covid- diagnosis was confirmed: ( ) by real-time reverse-transcriptase polymerase-chain-reaction (rt-pcr) on nasopharyngeal specimens [ ] ; or ( ) by rt-pcr on bronchoalveolar lavage (bal) obtained by bronchoscopy in case of high clinical suspicion of sars-cov- infection and negative test results on at least two nasopharyngeal swabs performed at least h apart; or ( ) in the presence of characteristic radiological interstitial pneumonia associated with typical symptoms (fever, dry cough, dyspnea), even with negative rt-pcr, with no other possible aetiologic explanation. the neurology department was reconverted to neuro-covid with neurologists specifically dedicated and supported by a team of infectious disease specialists, pneumologists and intensivists. in addition, other staff neurologists were dedicated to the emergency department and consultations in all the other covid wards. patients complaining of neurological symptoms were evaluated by means of standard neurological examinations, neurophysiological exams including electroencephalography (eeg), evoked potentials (ep), electroneurographic and electromyographic recordings (eng-emg), and brain computerized tomography (ct) and/or magnetic resonance (mr) according to standard care. cerebrospinal fluid (csf) examination, when performed, included protein and glucose levels, cell count, cytological evaluation, research for bacteria and common neurotropic viral agents (herpes simplex - , varicella zoster, cytomegalovirus, epstein barr and human herpes virus ) and for sars-cov- genome by real-time pcr, cobas ® sars-cov- test (roche) and by genefinder covid- (elitech group). ab anti-sars-cov- on serum and csf were tested by vivadiagtm covid- igm/igg immunochromatographic assay from vivachektm biotech (china), performed according to manufacturer's instructions. frequencies were compared by means of the χ test with yates' correction. continuous variables were compared by means of the wilcoxon rank sum test. because of multiple comparisons, the significance level was set at . . from february rd to april th, a total of covid- patients were admitted to asst papa giovanni xxiii and either discharged ( ) or still hospitalized ( ). among them, ( . %) developed symptoms/signs of cns/pns involvement. table shows the observed neurological complications. the more represented neurological manifestations fell into the following three categories: cvd ( patients; . %), pns diseases ( patients; . %), altered mental status ( patients; . %). their average age was . ± . (range - years); % of the patients were female. the most frequent comorbidity was hypertension (in pts; . %); diabetes was present in pts ( . %), dyslipidemia in ( . %), cardiovascular disease in ( . %), malignancy in ( . %), pulmonary disease in ( . %), and previous neurological disease in ( . %). the average number of comorbidities was . . in patients ( . %), diagnosis of sars-cov- infection was made by rt-pcr from pharyngeal swab; in the remaining ( . %) patients with a negative swab, diagnosis was formulated in the presence of characteristic radiological interstitial pneumonia associated with typical symptoms, in some cases confirmed by positive bal. pao /fio ratio was available at admission in / pts. sixty-nine patients ( . %) had a moderate or severe ards (pao /fio ratio lower than ) according to the horowitz index. fifty-five patients ( . %) required admission to intensive care unit (icu). icu admission was significantly more frequent in patients with a moderate/ severe lung injury compared to those with a non-severe respiratory distress ( . % vs . %; diff. . %- % ci . - . ; χ = . ; p < . ). the neurological complication was the presenting symptom in patients, of whom never manifested fever and there was a trend versus higher mortality in patients with cvd when compared to patients with pns involvement ( . % vs . %; χ = . ; p < . ), but not when compared to patients with altered mental status ( . % vs . %; χ = . ; ns). however, in the multivariable analysis that included age as a covariate, mortality was not significantly associated with any neurological group (data not shown). a -year-old woman with a history of hypertension, ischemic cardiomyopathy, mechanical aortic valve replacement in anticoagulant therapy, and recent hospitalization for sars-cov- infection was readmitted to the emergency department (ed) for acute onset of left sensorimotor hemiparesis and dysarthria (nihss ). pharyngeal swab for sars-cov- was negative. chest radiography showed evidence of previous bilateral interstitial pneumonia. brain ct scan was normal; ct-angiography revealed a thrombus in her basilar apex. intravenous alteplase was administered followed by mechanical thrombectomy with excellent angiographic (tici ) and clinical outcome (nihss ). the following day, for the acute onset of left hemiplegia, hypoesthesia and dysarthria, a brain and angio-ct was repeated showing occlusion of the m segment of right mca. thrombectomy was successfully performed. an additional pharyngeal swab for sars-cov was positive. cardiological investigations were unremarkable. the patient was discharged home at day fully recovered. forty-nine patients manifested disturbances of vigilance and/or consciousness. all performed either ct scan or brain mri. twenty-one pts underwent csf analysis, in all of whom sars-cov- rt-pcr was performed. based on clinical characteristics, csf data and neuroimaging, we concluded with the diagnosis of encephalitis in patients, among whom one hsv -related, one necrotizing encephalitis, and two patients with detection of sars-cov- by rt-pcr in csf. a otherwise healthy -year-old man presented to ed because of headache and confusion. he left the hospital from the waiting room, and was later found wandering and confused. on readmission, he was afebrile and eupneic, with prominent agitation followed by a generalized tonic-clonic seizure. chest radiography was normal. blood tests revealed elevated levels of d-dimer, fibrinogen, ldh, il- , and c-reactive protein; blood cell counts were normal. electroencephalogram and brain ct scan were unremarkable. csf analysis showed normal protein level and cell count; culture was sterile; rt-pcr was negative for common neurotropic virus. rt-pcr for sars-cov- resulted positive both on pharyngeal swab and csf. hydroxychloroquine ( mg twice daily) was started together with aripiprazole at a daily dose of mg. the patient was discharged home at day fully recovered. patients ( . %) manifested pns involvement: gbs, critical illness myopathy and neuropathy (crimyne), brachial plexopathies, and peripheral polyneuropathies (pnp) ( table ) . all patients performed emg-eng studies. for the diagnosis of gbs, hadden criteria were applied [ ] . csf analysis was generally avoided as the majority of the patients were treated with low molecular weight heparin at high doses in primary prevention of sars-cov- induced thrombophilia. it was performed in patients ( with gbs, with crimyne, and with pnp). moderate/severe ards was significantly more frequent in patients with pns involvement compared to patients with cvd ( . % vs %; diff. . %- % ci . - . ; χ = . ; p < . ) and to patients with altered mental status ( . % vs . %; diff. . %- % ci . - . ; χ = . ; p < . ). twenty-eight were admitted to icu. the emg-eng study confirmed the diagnosis of crimyne in nine and of gbs in sixteen patients. albumino-cytological dissociation was present in / patients with gbs. csf rt-pcr for sars-cov- was performed and negative in / . in one pt the diagnosis of gbs was formulated on clinical ground and csf results. thirteen gbs patients underwent a blink reflex test, which showed a demyelinating pattern in either the facial and/or the trigeminal nerve in all cases, suggesting a frequent cranial nerve involvement. mean length of stay was significantly longer in pts with pns involvement compared with patients with cvd (z = . ; p < . ). in addition, we observed patients ( . %) with myelitis, patients ( . %) complaining of headache, patients ( . %) with seizures, and patients ( . %) with movement disorders. patient : a otherwise healthy -year-old man presented to ed complaining of back-pain irradiating to lower extremities, sensory changes, weakness, and constipation. neurological examination showed bilateral leg motor weakness (grade / mrc scale) and diminished sensation below the t sensory dermatome. the tendon reflexes were normal bilaterally, with normal cutaneous plantar reflex. sars-cov- nasopharyngeal swab and thoracic ct scan were negative. serology testing revealed the presence of igg antibodies to covid- ( au/ml). brain mri was normal; spine mri showed diffuse degenerative changes. patient : a -year-old man had been in his usual health state until days before admission, when fever, anosmia, and ageusia developed and lasted for days. no further investigations had been performed and no treatment started. two weeks later, he complained of walking disturbances and numbness involving both lower extremities. on admission, he had bilateral leg weakness (grade / mrc scale), with reduced sensation to touch and acroparesthesia. deep tendon reflexes were brisk with bilateral distal clonus and normal cutaneous plantar reflex. a spastic paraparetic gait was observed. the nasopharyngeal swab for sars-cov- and the chest radiograph were negative. however, serology testing revealed the presence of igg antibodies to covid- ( . au/ml) and chest ct scan demonstrated small ground-glass opacities, suggestive of previous pneumonia. brain mri was normal; spine mri showed diffuse degenerative changes, with a conglutinated appearance of the roots of the cauda, which present a slight hyperintense signal in t sequences. in both patients, csf analysis revealed normal cell count and slightly increased protein level; rt-pcr for bacteria, common neurotropic virus, and sars-cov- were negative; igg anti-sars-cov- were positive. in both, emg-eng showed a reduction of maximal voluntary activity; sep and mep recorded from lower limbs showed a bilateral medullar conduction block of the long motor and sensitive pathways. therapy and outcome: pat. -the patient received methylprednisolone gr intravenously for days, followed by immunoglobulins ( . g/kg die). because of clinical worsening, immunoglobulins were stopped after days and plasma exchange started. oral steroid treatment was tapered gradually. pat -the patient received days of methylprednisolone g intravenously, followed by rounds of plasma exchange. oral steroid treatment was tapered gradually. at discharge to rehabilitation unit, both patients were able to ambulate without support, although with a paretic gait, with residual mild sensory deficits. although respiratory distress is the most distinctive clinical picture of sars-cov- infection, neurologic manifestations have been described. however, available evidence derives mainly from scanty series [ ] , selected populations [ ] , or single case descriptions [ ] [ ] [ ] [ ] [ ] . a larger study, launched at the beginning of april, has been recently published [ ] , which reports cases prospectively notified through online portals developed across the principal uk neuroscience bodies. the national and interdisciplinary structure of the study permitted to capture both neurological and psychiatric diagnoses nation-wide. however, although physicians were permitted to notify retrospectively recent cases, the majority of neurological complications that occurred in march are likely to be missed. in addition, because of the clinical demands of the pandemic, awareness of the study was likely to be scarce and voluntary-based case notification underreported. clinical, laboratory, and radiological findings of cases of covid- neurological disease from queen square hospital have been recently published [ ] . the spectrum of neurological syndromes was similar to what encountered in our population, apart from inflammatory syndromes. contrary to paterson's cohort, we did not observe any case of acute disseminated encephalomyelitis, despite the majority of patients with altered mental status performed brain mr (data not shown). paterson's cases were discussed in the context of multidisciplinary team meeting, thus representing a bias towards more complex and severe cases. here we report unselected neurologic manifestations on the largest covid- population ever described within a single hospital. among patients with laboratory or radiology-confirmed diagnosis of covid- disease, consecutively admitted to the asst pg in bergamo from february rd to april th , patients ( . %) developed a neurologic complication. the incidence of neurologic events observed in our population is much lower than the incidence reported by mao et al. [ ] . however, in mao series, data regarding neurologic complications are based on subjective descriptions derived from medical records, and include aspecific symptoms such as dizziness or nerve pain. on the opposite, we have included only neurologic complications documented by neurologic consultancy. in addition, we have extensively evaluated patients with neurologic involvement by appropriate instrumental tests. on the contrary, in available literature, instrumental evaluations were not systematically performed either to reduce the risk of cross infection, as in the chinese report, or likely due to the well-known difficulties in transferring patients admitted in icu for instrumental tests, as in helmes et al's report [ ] . the mean age of our patients was . years, higher than in wuhan series. the mean age observed in our series is in line with that reported from a large cohort of covid- patients admitted to icus in lombardy [ ] , and likely reflects the higher median age of the italian population. on the opposite, it is lower than the mean age of uk cases where, according to authors, an older population could be overrepresented [ ] . it is worth noting that more than % of our patients were below years of age. this observation should alert physicians that neurologic involvement is not a exclusive prerogative of the older population. in our cohort, neurologic event was the presenting manifestation in pts, % of whom never developed other covid- symptoms during hospitalization. this observation emphasizes that sars-cov- infection can manifest with predominant neurological symptoms and prompts the adoption of all specific measures to prevent contamination among patients and health professionals. as in varatharaj's study [ ] , the three principal groups of neurologic manifestations observed in our population were cvd, impaired consciousness and confusional states, and pns involvement. considering the entire covid- population analysed ( pts in bergamo cohort and pts in wuhan cohort), the prevalence of acute cvd was similar ( % vs . %). on the opposite, the prevalence of impaired consciousness and confusional states, as well as pns involvement was inferior in our cohort when compared to that in wuhan cohort ( . % vs . % and . % vs . %). the observed differences are likely due to the fact that we considered only neurologic manifestations classified by means of appropriate laboratory and/or instrumental tests into specific nosographic categories. it would be useful to compare the numbers of admissions for strokes, confusional states, and acute peripheral neuropathies in the same period of the previous year. regarding stroke, the comparison is biased by the fact that, during pandemic, lombardy region redesigned regional networks for acute conditions, centralizing stroke management in a few hubs. diagnosis of confusional states and consciousness impairment are poorly traced in electronic records, making comparison impossible. on the contrary, it is worthwhile to note that we observed a dramatic increase in gbs diagnosis, when comparing with the equivalent period of the previous year ( vs ). mean lag time from covid- symptom onset to neurologic manifestations in our population was . days, with a median of days. this observation is consistent with previous studies describing neurologic events during infections by other coronaviruses, such as sars and mers [ ] [ ] [ ] . on the contrary, mao et al. [ ] report that most neurologic manifestations occurred early at a median time of - days, but exclude from the analysis cvd and impaired consciousness, which are the main complications observed in our and varatharaj's [ ] cohorts. it is of note that, even if almost half of the complications occurs within the first weeks, still a relevant percentage can manifest late in the course of the disease, . % between and weeks and . % beyond weeks from covid- symptom onset. it has to be underlined that, among patients with detection of neurological signs beyond weeks, patients ( %) were admitted to icu, and neurologic findings were recorded when sedation and neuromuscular blockade were withheld; in eight of them a diagnosis of gbs was confirmed by emg-eng analysis. considering that response to immunoglobulin treatment shortens recovery and improves disability score when administered within the early stage of the disease [ ] , our observation is particularly relevant as it points out the need to monitor icu covid- patients either by means of sedation windows or instrumentally by neurophysiologic tests for the appearance of pns disorders. in our cohort, we describe two patients with altered mental status and two cases of myelitis with detection in csf of sars-cov- rna and igg anti-sars-cov- , respectively. direct demonstration of the sars-cov- genome has been reported only in two patients presenting with acute encephalopathy from japan [ ] and china [ ] respectively. only one case of acute necrotizing myelitis in a covid- patient has been reported so far. however, sars-cov- pcr in csf was negative and csf antibodies to covid- were not tested [ ] . an additional case presenting with acute flaccid paralysis of bilateral lower limbs and sphincter incontinence has been described. however, no csf analysis nor spinal mri were performed, making diagnosis of myelitis presumptive [ ] . our additional cases further support the neuroinvasive potential of sars-cov- and strongly suggest the need to systematically perform csf examination with pcr for sars-cov- genome and search for ab anti-sars-cov- in patients with symptoms related to cns involvement, even in the absence of respiratory syndrome at clinical and instrumental evaluations. to the best of our knowledge, this is the first study with the aim of intercepting all neurological complications of sars-cov- infections occurred in the biggest covid- population ever reported. nevertheless, it has several limitations. first of all, it is a retrospective study. however, thanks to the reorganization of the hospital, the redeployment of the neurologists in staff to covid units, and the awareness of healthcare personnel on the involvement of ns during sars-cov- infection, we are pretty confident that we could intercept the majority of relevant neurologic complications. moreover, we immediately designed a database where collecting demographic, clinical, and instrumental data of patients within an observational study approved by our institutional review board. as we did not include patients discharged by the ed, we might have lost some patients with mild neurological symptoms. however, we can speculate that patients with evolving neurological problems would have been readmitted to the ed and therefore captured later. conversely, we might have lost patients who died in the ed for severe ards and concomitant neurologic involvement. it has to be said that we evaluated all brain ct scan requested for covid- patients and therefore we should not have missed evident acute brain lesions. because of the overwhelming access of severely ill covid- patients, especially in the first weeks of the pandemic, we did not trace records of ageusia and anosmia at onset of covid- symptoms. this is a relevant limit as these symptoms may testify the involvement of the olfactory bulbs, considered one of the possible access route of the virus to cns. to circumvent this limit, we are planning to include this item in the medical history record to be administered to the patients during follow-up visits, fully aware that we will miss the data in deceased patients. even if we have data on short-term outcome, we are lacking data on the medium term. to accomplish this aim, we have planned a follow-up out-patient service for clinical and instrumental evaluations of all covid- patients discharged from asst pg , which includes neurologic evaluation at and months, together with neuropsychologic, neurophysiologic and neuroradiologic exams, when appropriate. this study confirms that involvement of ns is common in sars-cov- infection and offers clinicians useful information either for prevention or to early intercept neurologic manifestations and set the adequate therapeutic strategies. during the current pandemic caused by a novel virus, the importance of sharing worldwide data from large populations is crucial to prepare the national health systems facing all the potential complications and saving the highest number of lives. in addition, a better knowledge on the epidemiology and relevance of neurologic involvement may shed lights on the pathophysiology of the disease, help in understanding the impact on the severity of respiratory distress as well as on long-term outcome, and contribute to the development of preventive strategies against viral entry into the cns. covid- : the first documented coronavirus pandemic in history coronavirus disease (covid- ) in italy neurologic manifestations of hospitalized patients with coronavirus disease in wuhan, china neurologic features in severe sars-cov- infection guillain-barré syndrome associated with sars-cov- guillain-barré syndrome associated with sars-cov- infection: causality or coincidence? guillain barré syndrome associated with sars-cov- a first case of meningitis/encephalitis associated with sars-coronavirus- first case of novel coronavirus disease with encephalitis neurological and neuropsychiatric complications of covid- in patients: a uk-wide surveillance study the emerging spectrum of covid- neurology: clinical, radiological and laboratory findings diagnostic testing for severe acute respiratory syndrome-related coronavirus electrophysiological classification of guillain-barré syndrome: clinical associations and outcome. plasma exchange/sandoglobulin guillain-barré syndrome trial group baseline characteristics and outcomes of patients infected with sars-cov- admitted to icus of the lombardy region large artery ischaemic stroke in severe acute respiratory syndrome (sars) severe neurologic syndrome associated with middle east respiratory syndrome corona virus (mers-cov) neurological complications of middle east respiratory syndrome coronavirus: a report of two cases and review of the literature intravenous immunoglobulin for guillain-barré syndrome covid- -associated acute necrotizing myelitis acute myelitis after sars-cov- infection: a case report conflicts of interest the authors declare that they have no competing interest.ethical standard statement wherever possible, informed consent was collected verbally. however, in most cases, due to the inability to provide informed consent by the patient or the inability to collect it in compliance with the contagion prevention measures, the principle of secondary use of data was used in accordance with art. , paragraph , letter b) of the november th, law, n. , included in the legislative decree / of art. -bis. the institutional review board at asst papa giovanni xxiii provided approval for the study ( / , / / ). all procedures followed were in accordance with the ethical standards of the responsible committee on human experimentation (institutional and national) and with the helsinki declaration of , as revised in . key: cord- -a x lws authors: nan title: eosinophils in human disease date: - - journal: eosinophils in health and disease doi: . /b - - - - . - sha: doc_id: cord_uid: a x lws nan in healthy individuals, eosinophils represent a minor leukocyte subpopulation, accounting for less than % of total circulating white blood cells. tissue compartments with abundant resident populations of eosinophils include bone marrow, primary, and secondary lymphoid tissues, the uterus, and most of the gastrointestinal tract (with the exception of the esophagus under homeostatic conditions). these tissues share features of substantial cellular turnover and regenerative capacity. to a large extent, eosinophils serve as effector cells, capable of inducing significant tissue damage as a result of their release of preformed cytotoxic mediators, including the granule proteins, major basic protein, and eosinophilic cationic protein. these mediators lead to the production of reactive oxygen species and generate an array of lipid mediators. the role of eosinophils was previously considered to be defensive in the setting of parasitic infections or offensive in the development of an allergic response to an environmental allergen. this binary expression of the role or function of eosinophils, especially in the context of human disease, has recently undergone considerable evolution. eosinophilia and eosinophil products are now centrally positioned in ongoing immune responses through production of pivotal cytokines and chemokines, expression of features of antigen-presenting cells, ligation of toll-like receptors, and the elicitation of thelper (t h ) immune responses. in these activities, eosinophils have been shown on the one hand to enhance local inflammatory responses, while on the other to dampen such responses. with this extensive array of activities, it is not surprising that a role for eosinophils has been demonstrated in normal tissue homeostasis and in many disease states. this chapter details the unique positions eosinophils play in a wide range of disease states, in both pathological and protective roles. in chapter . , per venge begins by addressing the proteome of human eosinophils and the differences in molecular forms of many of the proteins in healthy and allergic subjects. identification of the spectrum of proteins produced and the genetic polymorphisms of the major secretory molecules is fundamental to our understanding of the role of eosinophils in health and disease and provides the potential for targeted regulation of specific functions. eosinophilia is a hallmark of allergic disorders characterized by the activation of selective hematopoietic processes during the onset and maintenance of allergic inflammation. the appearance of eosinophils in the circulation and in tissue involves processes in the bone marrow that lead to accumulation, differentiation, and proliferation of eosinophil lineage-committed hematopoietic progenitors at tissue sites. in chapter . , gavreau and denburg summarize mechanisms that lead to the accumulation of eosinophils and their progenitors in the airways of allergic asthmatics. the role of eosinophils in asthma is further developed by thomas and busse. recognizing the controversial relationship between airway eosinophilia and asthma severity, they focus on the dynamic contribution of eosinophils to asthma. what emerges are two major roles, one in which the eosinophil serves as an effector cell in airway remodeling, the other as a biomarker for asthma exacerbations. the link between eosinophils and asthma is strengthened with the recognition that viruses are a primary cause of asthma exacerbations. in chapter . , bivins-smith and jacoby explore the association of eosinophils with virus-induced asthma, especially eosinophil contact with airway nerves, which become activated and release mediators that cause dysfunction. in releasing excess acetylcholine, the altered airway nerves modulate airway smooth muscle responses and induce the development of bronchoconstrictive responses. moving from the airways to the skin, in chapter . simon and simon discuss eosinophil infiltration of the skin in a wide variety of disorders, both allergic and nonallergic. however, it remains somewhat unclear what mechanisms are responsible for eosinophil recruitment and activation in the skin, especially as conditions and pathogenic roles vary from disorder to disorder. a similar scenario is observed in the various primary eosinophilic gastrointestinal disorders. in chapter . , davis and rothenberg identify common and uncommon features of these disorders and the surprising and somewhat frightening increases in prevalence of these conditions, especially eosinophilic esophagitis. beyond the association of eosinophilic infiltration of the airways, skin, or gastrointestinal tract, the entity hypereosinophilic syndrome has emerged, in which there is introduction almost years ago, the eosinophil granule major basic protein (mbp) was purified by gleich and colleagues from guinea pig cells , and the eosinophil cationic protein (ecp/ rnase ) purified from human leukemia cells by our group. , these achievements were the starting points for the study of the proteome of the human eosinophil, with the subsequent purification of mbp from human eosinophils and the identification and purification of the two other major eosinophil granule proteins, eosinophil protein x/eosinophilderived neurotoxin (epx/edn) , and eosinophil peroxidase (epo). , remarkably, these four highly basic proteins make up about % of the proteins contained in the secretory granules of the human eosinophil. the eosinophil is regarded to be a secretory cell and it was consequently assumed that the biological activities of the human eosinophil are governed to a large extent by the activities of these proteins. thus, in attempts to understand the role of the human eosinophil in health and disease, a detailed study of the proteins and their genetics in relation to human disease therefore seemed logical. in this context, it should be emphasized that the granule content of human eosinophils is unique and shared only with other primates, since the duplicated gene products ecp and epx/edn are rapidly evolving and highly divergent orthologues are present in nonprimate mammalian species. such findings indicate that the interpretation of activities of eosinophils of other species should be extrapolated to humans with care. in this subchapter, i will describe some of the key activities of the four major granule proteins and the experience of assaying these proteins in various biological materials in human disease. i will also describe recent attempts to map the protein content further using modern proteomics techniques. at the end of the subchapter, i will summarize the genetic findings of the proteins and the associations of single nucleotide polymorphisms (snps) of the genes encoding these proteins with disease. more details of the biological activities of the four proteins are found throughout this volume. the four major granule proteins of human eosinophils will be considered in turn (table . . ). ecp is a single chain, highly basic protein [isoelectric point (pi) ranging from . to ] with apparent molecular masses ranging from . kda to kda. the heterogeneity is largely due to glycosylation of the protein. , the gene encoding ecp comprises two exons and one intron and is located on the q arm of chromosome ( q). exon is the coding dna sequence for ecp. ecp is located in the secretory granules of human eosinophils and is unique to humans and primates. minute amounts of ecp may be produced by monocytes and neutrophils under certain conditions. e however, most of the ecp located in neutrophils probably derives from the active uptake of ecp from the environment. ecp belongs to the large family of rnases and is also named rnase . in addition to being an rnase, ecp is a true multifunctional protein with both cytotoxic and noncytotoxic activities. the cytotoxic activities are determined by post-translational glycosylations and the majority, if not all, of ecp stored in the granules is richly glycosylated and noncytotoxic. , , upon release from the eosinophil, the molecule is deglycosylated and acquires cytotoxic capabilities. several snps have been found in the dna sequence of ecp; however, only two are in the coding part of exon . , the most commonly found snp is located at position , in which guanine (g) is replaced by cytosine (c). in scandinavian populations g is most commonly found, whereas in african populations c is the most common. thus, in scandinavia about % of the population carry the gg genotype and about % the cc genotype, whereas the reverse is the case in a ugandan population. the g>c snp results in an amino acid shift from arginine at position to threonine and a fundamental change in biological activity, since the cytotoxic activity is lost. whether the loss in cytotoxic activity is due to the amino acid shift per se affecting the cytotoxic site of the molecule or due to the fact that the replacement of arginine with threonine potentially creates a new glycosylation site that might disguise another cytotoxic site is at present not entirely clear. attempts to identify the bactericidal active sites were made by engineering recombinant protein and peptides. these experiments suggest a location for the activity at the n-terminal portion of ecp. the presence in the ecp molecule of several active sites, possibly with different targets, seems likely. the snp c>t in the coding region of the ecp gene is much less common and gives rise to a replacement of arginine at position with cysteine. the possible functional consequences of this amino acid shift are unknown, but it is predicted to have a great impact on the molecular structure. other biological activities of ecp, such as the rnase activity and the ability of ecp to activate fibroblasts, are not affected by the amino acid shift from arginine to threonine, which shows that these capabilities of ecp are dissociated from each other. other snps in the ecp gene are associated with protein expression. thus, the g>c snp in the utr region was found to closely correlate with the cellular content of ecp. the affected sequence is a binding site for the transcription factor, retinoic acid receptor (rxr), which in turn acts as a cofactor to the transcription factor, sp . sp was shown to affect ecp synthesis by binding to the promoter region of the gene. also, the intronic snp c.- a>c has been shown to relate to the ecp content of eosinophils. thus, several parts of the ecp gene seem to affect ecp production. in a japanese population, a promoter polymorphism - c>t is common, but is not found at all in the scandinavian population. this mutation is closely related to serum levels of ecp, thus suggesting an impact on ecp production. the activities of ecp are counteracted by heparin, but also by a protease-modified a -macroglobulin. eosinophil protein x/eosinophil-derived neurotoxin epx/edn is a single chain protein of . kda that shares % homology with ecp, since the formation of the two proteins is the result of a gene duplication e million years ago. , the ancestral gene was an rnase and this property has been conserved by the gene product epx/edn, but almost completely lost in the gene product ecp, which has instead acquired cytotoxic properties. the protein was independently described, purified, and named (epx) by our group and the group of gleich (named edn) and both names are used in the literature. for the sake of clarity the name eosinophil derived neurotoxin (edn) will be used throughout this volume, since this is the more commonly used name and also because the gene of eosinophil peroxidase (epo) recently has been renamed epx. the edn gene is located close to the ecp gene at chromosome . chromosome is also the location of the genes of the rnase a superfamily to which ecp and edn belong; hence, the alternative name of rnase . edn is also a highly basic protein (pi of about ), although less so than ecp. edn is produced in small amounts by macrophages and neutrophils, but also by liver cells. edn is stored in the eosinophil within the secretory granules together with ecp, but is also stored in a separate compartment of easily mobilized secretory vesicles. the biological activities of edn are related to its rnase activity and involve antiviral properties. however, recent studies indicate several other activities of great interest. thus, edn has been added to the growing list of alarmins, which are proteins that attract and enhance the activities of antigen-presenting cells, such as dendritic cells. activation of cells through toll-like receptor (tlr ) further links the activity of edn to components of innate immunity. the neurotoxic activity, which is the basis for the name edn, suggests cytotoxic properties for edn, although the cytotoxic activity of the molecule against any other cell is modest and mostly absent. in our previous studies, we could show some alterations in purkinje cells in the cerebellum of rabbits following injection of edn, thus resembling the gordon phenomenon. however, the injection of times lower amounts of ecp had much more detrimental consequences, with the disappearance of purkinje cells and the rapid development of ataxia and other neurological disturbances. the neurotoxic activities of the eosinophil proteins and the development of the gordon phenomenon may therefore be the combined actions of the potent rnase edn and the cytotoxic ecp. four snps were identified in the edn gene in a scandinavian population, none of which gives rise to an amino acid shift. one snp, g>c, is located in the intron and is closely related to the cellular content of edn. this locus is the binding site for several different transcription factors that may be involved in the expression of edn. epo is a two chain heme-binding protein with one heavy chain of about kda and one light chain of about kda. the gene is located on chromosome q and consists of exons and introns. the amino acid sequence shows an almost % homology with that of myeloperoxidase and also considerable homology with other members of the peroxidase family of proteins. epo is a highly basic protein with a pi of > . it is located in the matrix of the secretory granules and is probably specific to eosinophil granulocytes, since no other locations have been identified in mature cells. epo is difficult to extract from mixed blood leukocytes, since it has a high affinity for neutrophil membrane structures. the biological activities of epo are partly related to its peroxidase activity and partly to other properties of the molecule. the peroxidase catalyzes halidation reactions leading to the formation of long-acting hypohalides, such as hypobromous acid, oxidation of thiocyanate, and nitration of tyrosine. , such radicals may act on cellular membranes and take part in defense reactions against a variety of microbes. numerous mutations and polymorphisms have been found in the epo gene, five of which result in amino acid shifts. the possible consequence to functional activities of these amino acid shifts is unknown. eosinophil mbp was named from findings in guinea pig eosinophils, since it appeared to make up the majority of the proteins contained in the secretory granules. , e in human eosinophils the content of mbp is in the range of the other three major proteins, i.e., e mg/ eosinophils. the mass of mbp is . kda and its pi is . . the mbp gene is located on chromosome q and consists of six exons and five introns. mbp is apparently produced as a much larger preproprotein, and an acidic portion of prombp is cleaved off upon storage in the eosinophil granules. this acidic portion of prombp may serve to protect cellular structures from its cytotoxic activities during synthesis and packaging. the larger prombp, however, has been identified in immature bone marrow cells. prombp has also been found in placental cells in complex with the metalloproteinase pappalysin- , or pregnancy associated protein a (papp-a), and shown to inhibit the activities of papp-a. the mbp molecule makes up the typical crystals seen in the specific granules of human eosinophils. an mbp homologue was identified, characterized, and named mbp . this protein was purified from human eosinophils and has a molecular mass of about . kda and a much lower isoelectric point of . . the gene encoding hmbp is located in close proximity to the gene of mbp at chromosome q and has five exons. mbp is expressed in several cell types other than human eosinophils, such as basophils and placental cells, whereas hmbp seems to be located only in eosinophils. the biological activities of mbp are predominantly related to its cytotoxic capabilities, but numerous noncytotoxic activities have also been identified, many of which will be described throughout this volume. in the mbp genes, several mutations and polymorphisms have been identified, five of which may result in an amino acid shift. consequences to the activities of mbp resulting from these amino acid shifts have not been described. proteins in addition to the major proteins described above. these include large numbers of adhesion molecules, chemokines, cytokines, and others. in an attempt to gain further insight into the biology of human eosinophils, modern proteomics techniques may be applied to map the major protein content of normal and diseased eosinophils. in this regard, several different approaches may be applied. one is the description of as many proteins as possible and another is the selected description of proteins based on criteria such as extraction procedures or detection methods, e.g., based on the identification of phosphorylated proteins only. one study incubated eosinophils with sonicates of mast cells and the cytokines granulocyte-macrophage colony-stimulating factor (gm-csf) and tumor necrosis factor (tnf-a) and used [ s]methionine to monitor protein synthesis. extracts of eosinophils were run on two-dimensional ( -d) gels and the number of protein spots increased dramatically following these stimuli compared to control cells. in addition, the position of the spots differed depending on the stimuli used, which suggests that eosinophils respond differently to these stimuli. unfortunately, no attempts were made to identify the proteins in these spots. another study showed differences in spots between healthy subjects and those affected by atopic dermatitis. one such difference was downregulation of the grb adaptor protein in cells from patients, which may relate to eosinophilia of the patients and antiapoptotic features of these cells. overall, spots were identified in healthy subjects and spots in the eosinophils of atopic dermatitis patients, which emphasizes that circulating eosinophils of such patients are exposed to various stimuli that induce protein synthesis. one upregulated spot of particular interest in atopic dermatitis relates to increased expression of the low-affinity receptor for immunoglobulin e (ige). a different approach involved the study of phosphoproteins in an acute myelogenous leukemia (aml) eosinophil cell line after exposure to dexamethasone or il- . fourteen phosphoproteins showed significant changes, i.e., were either phosphorylated or dephosphorylated, after il- and after dexamethasone. phosphorylation of the translation initiation factor elf- subunit was increased by il- and was also found to be increased in patients with atopic dermatitis. interestingly, phospho-apolipoprotein e (p-apoe) was induced in eosinophils by dexamethasone but was decreased by il- treatment. p-apoe levels could therefore be used as an indicator of proliferation or apoptosis of eosinophils. a -d gel of a survey of proteins in whole eosinophil extracts and extracts of membrane fractions of eosinophils of healthy subjects and of eosinophils obtained from allergic subjects during a pollen season is shown (fig. . . ). altogether more than spots were identified by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (maldi-tof ms), representing different proteins. among the proteins identified were the four major granule proteins described above and a number of other proteins hitherto not associated with human eosinophils. as expected, the proteins represent, to a large part, cytoskeletonrelated proteins such as actin, but more than % of all proteins are of granular origin ( fig. . . ). the study also showed large differences in the expected pis of several proteins. thus, the cytoskeleton-related proteins cofilin- , profilin- , adenylyl cyclase-associated protein (cap ), and coronin- a were all found to be significantly acidified, whereas edn and mbp were much more basic than expected. the actual biological significance of alterations to cytoskeleton-related proteins is uncertain, but may relate to the well-documented migrating capacity of eosinophils. in eosinophils obtained from allergic subjects exposed to pollen, several intriguing changes were observed (table . . ). one such was a change of more than three units in the pi of two protein spots identified as the heavy chain of epo due to heavy chain glycosylations. we speculate that such heavy glycosylation may interfere with the enzymatic activity of epo. in support of such speculations are our previous findings that the peroxidase-dependent luminol-enhanced chemiluminescence reaction of blood eosinophils purified from allergic subjects during the pollen season is significantly reduced. altogether spots were significantly changed in eosinophils from pollen-exposed allergic subjects, five of which were identified by maldi-tof ms. the other three identified spots were heat shock cognate protein (hsc ) and the a and b subunits of cap . as indicated above, cap subunits are involved in cell motility. the upregulation of these proteins, therefore, indicates that eosinophils from allergic subjects have an increased potential to respond to chemoattractants, a capacity that is well documented, since eosinophils harvested from the blood of allergic subjects show increased migration toward several chemoattractants. the upregulation of hsc has a number of biological implications of interest, such as changes in protein folding, intracellular protein transportation, and antigen presentation. the latter may lend further support to the eosinophil being actively involved in antigen presentation. the eosinophil marker that has become most widely used in the everyday clinical routine of the allergist is ecp, although several reports have shown that the measurement of edn, epo, or mbp may also be useful. the measurement of any of these eosinophil proteins may indicate the activity and turnover of the eosinophil granulocyte. currently, ecp is measured in serum/plasma, but measurements in nasal lavage fluid, sputum, and possibly saliva are interesting alternatives, since ecp in these biological fluids more accurately reflects the local process. the advantages and disadvantages of measuring ecp in various biological fluids will be discussed below, and the current evidence that ecp may be a useful complement to the diagnostic armamentarium for monitoring and characterizing disease activity in the allergic patient. the emerging evidence of the clinical usefulness of urine measurement of edn as alternative to serum ecp measurement to reflect eosinophil turnover and activity will also be considered. instrument for characterizing and monitoring inflammatory processes in the airways. , this has been particularly shown in patients with asthma, chronic obstructive respiratory disease, and cystic fibrosis. in most cases, sputum has to be induced by hypertonic saline and cells in the sputum need to be separated from the supernatant in order to analyze mediators released from inflammatory cells. the relatively time consuming and complicated procedures required to achieve this are probably the main obstacles for a more widespread use of sputum measurement as a clinical tool. an alternative and much simpler procedure is the measurement of specific markers of various cells in whole sputum extracts. the numbers of eosinophil granulocytes in sputum have been estimated using ecp and several publications show that the numbers of eosinophils measured in this way correlate well with disease activity in asthma and are reduced as a consequence of corticosteroid treatment. an interesting alternative to sputum is saliva, since we showed recently that asthmatics have significantly raised levels of ecp in saliva that are reduced by corticosteroid treatment. still, however, we do not know what the ecp levels in saliva actually reflect, as they may be indicative of either systemic or local eosinophil activity. in addition, the measurements of specific cell markers in nasal lavage fluids or ear secretions have been widely used, and the usefulness of such measurements in the understanding of cellular involvement has been clearly indicated. however, their clinical application is still not established. ecp may be measured in both serum and plasma. if plasma is chosen, the blood should be anticoagulated with ethylenediaminetetraacetic acid (edta) or citrate in order to prevent spontaneous extracellular release of ecp and subsequent interaction with heparin. the levels of ecp in serum are consistently higher than in plasma, due to the fact that eosinophils in the test tube continue their extracellular release of ecp ex vivo. this is an active process that is both time and temperature dependent, which means that higher extracellular levels are achieved with increasing time and ambient temperature, and vice versa. thus, if ecp is measured in serum, strict standardization of the blood sampling procedure and handling of the blood sample are necessary in order to avoid unacceptable variations in ecp levels. our recommendation is that blood should be taken in tubes with a gel separator and that coagulation is allowed for h at room temperature ( c) before centrifugation and separation of serum. both plastic and glass tubes may used. however, differences in the material and the inclusion of coagulation activators in the tubes may affect measurements. this means that normal ranges of ecp have to be determined in each laboratory that does not follow the recommendations of the manufacturer. the levels of ecp in edtaeplasma probably correctly reflect the circulating levels of ecp at the time of blood sampling. these levels are the consequences of production and elimination of ecp, i.e., local or systemic release of ecp to the circulation as well as variations in the turnover rate of ecp. normally, turnover is quite rapid, with a half-life (t ½ ) of about min. for several reasons, we can assume that the turnover is more rapid in subjects with ongoing inflammation. this means that an increased release of ecp to the circulation in certain diseases does not always lead to the anticipated increase in plasma levels, since the increased release may be partly or fully counteracted by an increased elimination rate. the dynamics of such counteracting principles may be the main explanation of the fact that in most cases clinical information obtained by edtaeplasma measurements of ecp is less clear and less useful than the information obtained by serum measurements. in addition to the circulating levels, serum levels of ecp also reflect the secretory activity of the eosinophil population in the blood and, since the levels in serum are often e times those in plasma, we may draw the conclusion that it is above all the secretory activity of the eosinophils that determines ecp levels. the propensity of blood eosinophils to release ecp is increased in subjects exposed to allergen. my own interpretation of serum ecp levels is that they reflect the propensity of the eosinophil population to release ecp in the local process, e.g., in the lung of asthmatics. the higher this propensity is, the more damage is inflicted on the patient. in order to eliminate the influence of eosinophil counts on the serum levels of ecp and thereby obtain a purer reflection of eosinophil activity, serum levels may be divided by the eosinophil count, thus forming an ecp/eosinophil ratio. in some studies, such a ratio was found to be more closely related to disease severity in asthma. more than a thousand papers have been published dealing with the relation between ecp levels and allergic or other inflammatory diseases. , , , the majority of these publications indicate that ecp provides novel information about the process and that the information may be used in treatment stratification and monitoring of the disease, since ecp levels are closely related to exacerbation propensity and severity in diseases such as asthma and atopic dermatitis. recent data also indicate that serum levels of ecp and epo predict the further development of allergic disease. thus, serum levels of the two proteins were significantly elevated in a group of patients with allergic rhinitis who developed asthma-like symptoms years later. the prediction was not seen for blood eosinophil counts or nasal lavage findings. several publications, though, have questioned and sometimes rejected ecp as a clinically useful marker. one reason for this may be the simplified view that asthma is one disease and that the disease is caused by one cell, the eosinophil, and that one marker such as ecp will solve all clinical problems. this is obviously not true, since we know today that the involvement of eosinophils in the asthmatic process is very variable between individuals. another cause of variations in the results is probably the lack of awareness of the importance of correct sample handling. still another possibility may be related to the recent discovery of several genetic variants of ecp and the fact that these variants are related to the expression of allergic symptoms and serum levels of ecp. the levels of ecp in blood or any other biological fluid are not disease specific, but provide us with information about the extent to which eosinophils are involved in the particular disease process. in a search for noninvasive means to monitor eosinophil involvement in inflammatory diseases, urine measurement of edn has emerged as a promising candidate, particularly in children. e in order to minimize the influence of differences in water dilution of the urine, edn levels have to be adjusted to creatinine concentrations unless -hour samplings are carried out. it is also useful to bear in mind that all eosinophil markers, including the excretion of edn, show circadian rhythms with the highest levels occurring at night. thus, for the sake of standardization of blood or urine measurements of eosinophil markers, sampling should always be carried out at the same time of the day. several studies have shown that edn in urine is elevated in asthma and atopic dermatitis, is related to disease activity, and reduced by anti-inflammatory treatment, i.e., corticosteroid treatment. elevated levels of edn in urine in wheezy children also seem to predict the development of asthma. as mentioned above, two major granule proteins of human eosinophils are unique to humans and primates. thus, knowledge of the role of these proteins in human disease cannot be extrapolated from mouse gene knockout experiments. the alternative is therefore to search for human counterparts to such knockouts, i.e., snps or mutations that have an impact on the biological activities of these proteins either with regard to their functional alterations or altered production. as shown above, only one snp is known to lead to a functional alteration in any of these proteins: the ecp g>c gene polymorphism, in which the replacement of g with c in the dna sequence results in the production of a noncytotoxic protein with the amino acid threonine at position instead of arginine. in the first study examining the possible association of the ecp g>c snp with human disease, we found strong associations with the development of allergic symptoms, both in a group of medical students and in a group of subjects with allergic or nonallergic asthma. in the group of medical students, the diagnosis of allergy was based on a self-assessment questionnaire and in the asthma group the distinction was based on a clinical diagnosis. we found that the genotype ecp gg, giving rise to the production of the cytotoxic species, was more common in those experiencing allergic symptoms than in nonallergic subjects or those with nonallergic asthma. however, most notable was the absence of any allergic manifestations in the two cohorts carrying the ecp cc genotype. the ecp cc genotype therefore seemed to exclude the development of allergic symptoms and provided strong support for a key role for eosinophils in allergy. in a larger community-based study on randomly selected subjects in estonia and sweden [the european community respiratory health survey (ecrhs)], symptoms and signs of allergy were based on a structured interview. the results of this study were much less clear, although the ecp g>c genotypes (ecp gg, ecp gc, and ecp cc) showed significant associations with various expressions of allergic symptoms. however, it also became clear that ethnicity, gender, and smoking habits are important confounders. one intriguing finding of the ecrhs study was the associations of the ecp g>c genotypes with lung function with reduced lung functions found in both women and men carrying the ecp g>c g-allele compared to those carrying the c-allele. if confirmed, these findings suggest a detrimental effect of the cytotoxic ecp on lung tissues. in a norwegianedutch study, no associations between allergy and the ecp g>c genotype were found. in contrast, this study showed an association with nonallergic asthma, which was also the case in our ecrhs study. an association with allergic rhinitis of the ecp g>c genotype was also negated in a korean study. the association between asthma/allergy and the ecp g>c genotype is at present confusing and the seeming differences in findings not easily explained. ethnicity and gender differences may have an impact, but the definition of the phenotypes studied is probably more important. it is important to clarify these relationships, since the cytotoxic activities of ecp could be targets for therapeutic interventions if such associations are confirmed and established. ecp has the capacity to kill schistosoma mansoni larvae. knowing that the cytotoxic capacity is lost by an amino acid shift from arginine to threonine at position , we conducted a study on subjects living in uganda in an endemic area of s. mansoni infections. the ecp g>c genotype distribution in this population was dominated by subjects carrying the ecp cc genotype, i.e., the opposite of the distribution found in non-african populations. thus, the majority of people living in these endemic areas have a genotype that gives rise to the production of a noncytotoxic ecp and possibly a poorer defense against s. mansoni infection. we examined parasite egg excretion in feces, to reflect the level of defense against the infection, and indeed found higher numbers in subjects carrying the c-allele, thus suggesting the involvement of ecp in this defense reaction. we also found that subjects carrying the g-allele are much more prone to develop liver fibrosis, one of the serious consequences of the infection that affects about % of those infected by s. mansoni. thus, it seems that the capacity to produce the cytotoxic ecp has some effect on defense against s. mansoni, but that the host's reaction to the infection is the major threat to the infected subject. in this regard, cytotoxic ecp may play a key role. as mentioned above, the ecp g>c genotype is closely related to the cellular content of ecp, with the lowest levels found in those carrying the ecp cc genotype. we found few, if any, associations between this genotype and the expression of allergic symptoms or asthma, but close relations to gender, with a higher prevalence of the g-allele in women, and relations to smoking habits. similar findings were seen for the g>c genotypes and may relate to the fact that these two genotypes are in strong disequilibrium. in the korean study, the ecp g>c callele was found to be more prevalent in allergic rhinitis. in the norwegianedutch study, no apparent relations to allergy, asthma, and ecp levels were found, whether evaluated alone or as part of a haplotype. the intronic snp ecp c.- a>c is located in a part of the gene that may be involved in regulating ecp production. the norwegianedutch study showed a higher proportion of elevated serum levels of ecp and ige, as well as higher proportions of subjects with asthma and bronchial hyperreactivity, in those carrying the adenine (a)-allele. most of these associations, though, were only seen in the dutch population. in a japanese study, no association between the intronic snp and serum ecp levels were found. in our ecrhs study, we found an intriguing association between the ecp c.- a>c genotypes and atopy. among males, but not among women, atopy was associated with the ecp c.- (a>c) genotypes, with a significantly higher frequency of the cc genotype. in a logistic regression analysis, the ecp c.- cc genotype was independently associated with an increased risk of atopy with an odds ratio of . and confidence interval (ci) of . e . when adjusted for gender, ethnicity, and smoking habits. the ecp - c>t snp is located in the promoter region of the ecp gene. this snp has only been described in the japanese population. interestingly, the - c>t genotypes are related to serum levels of ecp, with undetectable levels in subjects carrying the tt genotype. eosinophil protein x/eosinophil-derived neurotoxin, eosinophil peroxidase, and major basic protein genotypes and clinical findings one study examined a large number of edn and ecp snps in a south indian population with microfilaria infection and tropical pulmonary eosinophilia. no associations between either of these conditions and the snps examined were seen. however, the south indian population seemed to have unique snps and haplotypes of the edn and ecp genotypes compared to asian and scandinavian populations. in two japanese reports, snps in the coding parts of the epo gene were found to be associated with cedar pollinosis. , in particular, an snp in exon , resulting in the amino acid shift from proline to leucine and which might affect the activity of the protein, showed a strong association. in the second study, an association with a silent snp in exon was shown in addition to the exon snp. in a recent czech study on allergic rhinitis, the exon snp was also found to be associated, whereas the exon snp was not present in this population. these reports suggest the involvement of epo in the allergic process, although whether it involves the peroxidase activity of epo or reflects other mechanisms is unknown. one report from germany studied patients with atopic dermatitis and possible associations between nine different snps located in the four major eosinophil granule protein genes. however, no associations with atopic dermatitis were found for any of the snps studied, despite the fact that eosinophils are regarded to be important effector cells in this tissue eosinophilia with or without associated blood eosinophilia is observed in a number of skin disorders, including allergic diseases, autoimmune diseases, bacterial or viral infections, hematologic diseases, parasitic infestations, as well as in association with tumors. , the presence or absence of eosinophils in skin specimens is often used for differential diagnoses by dermatopathologists. for instance, eosinophils in the upper dermis might be a clue for the diagnosis of early lesions of bullous pemphigoid (bp), even in the absence of blisters. in addition, the detection of eosinophils might indicate the differential diagnosis of drug reactions, which often cannot be distinguished from other inflammatory skin diseases. in hematoxylin and eosin (h&e) stained skin specimens, eosinophils are seen as round shaped cells stuffed with coarse eosinophil granules ( fig. . . a) . disrupted oval-shaped eosinophils may also be found, e.g., in subacute and chronic eczematous lesions ( fig. . . a, b). depending on the disease, eosinophils are located among other inflammatory cells in the perivascular areas (e.g., in eczema), between collagen bundles in the dermis (e.g., in eosinophilic cellulitis), or in the epidermis (e.g., in pemphigus foliaceus; fig. . . bed). moreover, in eosinophilic pustular folliculitis, eosinophils enter the hair follicle. extracellular granular proteins can be detected in varying amounts either as separate deposits or as a thin coating on collagen bundles. the latter are called flame figures and are typically seen in eosinophilic cellulitis. recently, deposition of eosinophil granule proteins in association with extracellular dna traps was reported in several allergic, autoimmune, and infectious skin diseases. immunofluorescence staining using antibodies directed against eosinophil cationic protein (ecp) or major basic protein (mbp) allows a more sensitive detection of eosinophils and extracellular granular protein depositions than h&e staining. under physiological conditions, eosinophils are usually not detectable in the skin. therefore, primary causes (intrinsic) or stimuli (extrinsic) are required for initiating the increased production, recruitment, and/or survival of eosinophils under pathological conditions. myeloproliferative forms of hypereosinophilic syndromes (hes) represent intrinsic eosinophilic disorders, in which mutations of multipotent or pluripotent hematopoietic stem cells occur, with subsequent increased eosinophil proliferation, often affect the skin (table . . ). cutaneous manifestations vary from multiple erythematous papules, plaques, and nodules, to generalized erythematous maculopapular eruptions, often associated with pruritus. , clonal eosinophilia can occur as a consequence of gene rearrangements that result in increased tyrosine kinase activity. as a consequence, patients with hypereosinophilia due to fusion of the platelet-derived growth factor receptor a (pdgfra) and fip like (fip l ) genes respond to imatinib therapy. extrinsic eosinophilic disorders due to cytokine release by either t cells or tumor cells are more common than intrinsic hes forms due to genetic abnormalities within hematopoietic stem cells (table . . ). cytokines involved in the development of skin eosinophilia are interleukin- (il- ), il- , and granulocyte/macrophage colony-stimulating factor (gm-csf). the expression of il- in association with eosinophilic skin disorders has been reported for atopic dermatitis (ad), bp, cutaneous t cell lymphoma, episodic angioedema with eosinophilia, eosinophilic cellulitis, eosinophilic fasciitis, eosinophilic folliculitis, exanthematous drug reactions, hypereosinophilic syndrome with skin involvement, and urticaria. il- expression has been detected in blister fluids of bp and blood leukocytes of hes patients. e in langerhans cell histiocytosis, as well as in ad, atopy patch test reactions, and cutaneous late phase reactions, the expression of both gm-csf and il- has been shown. the chemokine eotaxin/c-c motif chemokine (ccl ) is important for tissue recruitment and activation of eosinophils. eotaxin expression has been observed in ad, autoimmune-blistering diseases like dermatitis herpetiformis and bp, drug reactions, eosinophilic folliculitis, and parasitic dermatoses, and also in lymphomas, e.g., cutaneous t-cell lymphoma and hodgkin disease. the primary function of eosinophils was originally thought to be related to the protection against helminth parasites. recently, a novel mechanism of eosinophil function in innate immunity has been reported. by releasing mitochondrial dna and granule proteins, eosinophils form extracellular structures that can bind and kill bacteria invading the gastrointestinal tract. such extracellular dna structures generated by eosinophils have recently also been reported in inflammatory skin diseases. furthermore, eosinophils are presumed to cause tissue damage. , in addition, eosinophils play an important role in repair and remodeling processes, as well as in immunomodulation. , the role of eosinophils under pathological conditions has mostly been studied in parasitic infections and bronchial asthma. in contrast, the role of eosinophils has not been explored in substantial depth in skin diseases. with regard to skin diseases, it can be assumed that eosinophils directly contribute to or amplify pruritus (itch) in the skin, by releasing neurotrophins (nerve growth factor and brain-derived neurotrophic factor) or indirectly by acting on mast cells. pruritus is associated with most eosinophilic skin diseases, in particular with ad, bp, cutaneous t-cell lymphoma, and parasitic infections. in the following sections, we summarize the current knowledge on the activation and function of eosinophils in selected eosinophilic skin disorders. tissue eosinophilia is a typical feature of eczema, in particular of ad. the numbers of eosinophils in the skin are usually modest ( . cells/mm ; range e . ) and correlate with disease severity, as well as with the degree of spongiosis in acute exacerbations and marked epidermal hyperplasia in chronic stages. besides eosinophils, eosinophil-derived products, such as ecp, eosinophilderived neurotoxin (edn), and mbp, are present in increased amounts in the blood and the skin of ad patients. immunostaining with antibodies to ecp and mbp, as well as electron-microscopic evaluation revealed eosinophil granule proteins inside eosinophils, but also in the extracellular spaces, and the near absence of intact eosinophils, suggesting eosinophil degranulation and degeneration. , in ad, eosinophil production, differentiation, recruitment, survival, and activation are under tight control of cytokines, particularly gm-csf, il- , and il- , and chemokines, including eotaxin and rantes (c-c motif chemokine ; ccl ), as well as adhesion molecules, complement factors and leukotrienes. however, the pathogenic role(s) of eosinophils in ad have not yet been defined. the release of granule proteins suggests a role in host defense and/or tissue damage. furthermore, eosinophils release a broad spectrum of mediators such as cytokines [gm-csf, il- , il- , il- , il- , il- , il- , il- , and transforming growth factor (tnf)] and leukotrienes (in particular, the cysteinyl leukotrienes ltc , ltd , and lte ) and thus they can regulate immune responses or initiate tissue repair processes. improvement of ad upon both systemic and topical therapy is usually associated with a decrease in eosinophils and other inflammatory cells in the skin. however, the administration of an anti-il- antibody showed only moderate effects on clinical symptoms, although blood eosinophils were almost completely depleted. currently, it remains unclear whether anti-il- antibody treatment reduces eosinophil tissue infiltration in lesional ad skin. although the development of pruritic wheals in urticaria is attributed to the release of histamine by mast cells and basophils, other cell types, including eosinophils, neutrophils, and macrophages, and t cells, are also present in extracellular deposits of mbp have been observed as granular deposits and coating tissue fibers in the dermis, as well as in small blood vessel walls. ecp may stimulate histamine release by mast cells and basophils. by generating eicosanoid mediators and secreting neuropeptides, such as substance p, eosinophils may contribute to vasodilation. vascular endothelial growth factor, which is elevated in the plasma of chronic urticaria patients and correlates with disease severity, has been reported to be predominantly expressed by eosinophils. recently, an involvement of the coagulation cascade in the pathogenesis of chronic urticaria has been suggested. eosinophils that were shown to express tissue factor in urticarial lesions may activate the tissue factor pathway of coagulation, resulting in the generation of thrombin, which stimulates mast cells for histamine release. eosinophilic cellulitis is characterized by an intense infiltration of eosinophils, extracellular granule deposition, and flame figures in the dermis. recently, high numbers of eosinophils generating extracellular dna traps in association with ecp have also been observed in this eosinophilic disorder. patients present with recurrent episodes of acute pruritic dermatitis and occasionally with blisters, painful edematous swellings, or persistent urticarial eruptions. the cause is unknown, but some patients develop eosinophilic cellulitis in association with hematological disorders, infections, or anti-tnf-a therapy. corticosteroids are usually helpful in this disease. in % of patients with hes, the skin is affected. skin manifestations of hes include blisters, eosinophilic cellulitis, erythematous macules, lichenoid eruptions or urticarial lesions, necrosis, papules or nodules, pruritus, purpura, and ulcerations. cutaneous symptoms are usually present in a subgroup of patients with hes, in which il- -producing t cells exhibiting an abnormal immunophenotype have been identified. anti-il- antibody therapy has been shown to improve skin symptoms in hes patients. eosinophilic pustular folliculitis (epf) presents with recurring clusters of sterile follicular papules and pustules, predominantly on the face and trunk. epf may affect immunocompetent subjects (ofuji disease), but is most commonly seen together with immunosuppression. epf has been reported in association with infections, in particular acquired immunodeficiency syndrome (aids), autoimmune diseases, and medications, as well as autologous peripheral blood stem cell and allogeneic bone marrow transplantation. the histology shows a dense follicular and perifollicular infiltrate of eosinophils and scattered lymphocytes, and sometimes follicle destruction. a pathogenic role for eosinophils in response to fungi (malassezia), demodex mites, and bacteria has been suggested. the histopathology of bp reveals eosinophil infiltration in and below blisters and along the basement membrane, as well as in nonblistering, urticarial, or eczematous lesions of bp. patients with active bp exhibit increased eotaxin and il- levels, as well as eosinophil numbers in the blood compared with patients in clinical remission and healthy controls, e associated with significant eosinophil infiltration in the skin, as well as disease intensity. , whether bp and/or bp autoantibodies of immunoglobulin g (igg) or ige types can activate eosinophils with or without preceding priming has not been investigated so far. eosinophils exhibit cd , the fc-g receptor, and degranulate upon stimulation with igg immune complexes. furthermore, il- -primed eosinophils from these patients release granule proteins upon stimulation with complement c a. it has been hypothesized that in the presence of complement, eosinophils release enzymes and reactive oxygen onto the basement membrane, causing tissue destruction and blister formation in bp. eosinophil granule protein depositions have been observed in both blistering and evolving lesions. recently, extracellular dna traps generated by eosinophils were also described in bp. mmp has been reported to be expressed by eosinophils in lesional skin, as well as in blister fluids of bp. moreover, mmp cleaves the extracellular, collagenous domain of bp autoantigen in vitro. eosinophilic spongiosis can be observed in early pemphigus including pemphigus foliaceus. the presence of eosinophils may be due to il- as part of the mixed thelper / (t h )/t h cytokine profile that has been found in pemphigus vulgaris. complement-fixing antibodies were shown to induce eosinophil infiltration in pemphigus. charcoteleyden crystals have been observed in pemphigus vegetans. eosinophils can be seen in the papillary dermis in dermatitis herpetiformis, although neutrophils and leukocytoclasis are more characteristic in this disorder. furthermore, both neutrophils and eosinophils are the predominant infiltrating cells in linear iga bullous dermatosis. epidermolysis bullosa acquisita following gm-csf therapy has been related to eosinophil infiltration and deposition of eosinophil peroxidase (epo) and mbp at the dermaleepidermal junction. in drug reactions, the presence of eosinophils in the skin is quite a striking finding, despite various clinical and histopathological presentations [e.g., acute generalized exanthematous pustulosis (agep), erythema multiforme, maculopapular rashes, and pseudolymphomatous and granulomatous drug reactions]. drug reaction with eosinophilia and systemic symptoms (dress) is a drug reaction with both blood and tissue eosinophilia and systemic symptoms. it presents with an acute, severe skin eruptions that may develop from a maculopapular rash into erythroderma, as well as with blood eosinophilia, fever, hepatitis, lymphadenopathy, and other organ involvement. eosinophils accompanied by other inflammatory cells are found in the skin and lymph nodes. severe hepatitis, in which eosinophilic infiltration or granulomas as well as hepatocyte necrosis and cholestasis are striking features, may result in liver failure, accounting for the high mortality rate of %. the treatment is based on high-dose corticosteroids. drugs known to cause dress are anticonvulsants, anticancer drugs, antidiabetics, antimicrobial agents, nonsteroidal anti-inflammatory drugs, and sulfa drugs. in langerhans cell histiocytosis (lch), among the infiltrate of langerhans cells (lc), scattered or clusters of eosinophils can be found in the papillary and deeper dermis, respectively. since activated lc generate a broad spectrum of proinflammatory cytokines, e.g., chemokines and gm-csf, they are able to recruit and activate eosinophils directly or via stimulation of other cell types. a predominant t h type cytokine production by cutaneous t-cell lymphoma (ctcl) results in eosinophilia, extracellular granule protein depositions, as well as increased il- levels in the skin and/or peripheral blood. in angiolymphoid hyperplasia with eosinophilia, clonal t cell populations have been identified. currently, it is not known whether the eosinophils inhibit or modulate proliferation of the malignant cells. eosinophilia is a common histological finding in a number of diseases characterized by marked tissue fibrosis, such as cutaneous forms of systemic sclerosis, drug induced scleroderma-like illness (e.g., bleomycin), eosinophiliae myalgia syndrome, eosinophilic fasciitis, localized scleroderma, and toxic oil syndrome. elevated eosinophil granule protein levels have been detected in the serum of patients with diffuse cutaneous forms of systemic sclerosis, suggesting eosinophil activation and degranulation. as they produce fibrogenic cytokines, e.g., transforming growth factor b (tgf-b), eosinophils are thought to contribute to skin fibrosis. together, these examples show that most observations on eosinophils in skin diseases are rather descriptive. the exact mechanisms whereby eosinophils are recruited and activated, as well as their pathogenic role(s), are not yet fully understood. depending on the skin disease, a role in host defense, immunoregulation and/or remodeling, and fibrosis can be assumed. further research is therefore required in order to understand the function of eosinophils in skin diseases and to develop new therapeutic strategies. asthma is characterized by variable airflow obstruction, bronchial hyperresponsiveness, and chronic airway inflammation, all of which are likely to be intertwined and interdependent. the immune processes involved in the development of these characteristics in asthma are complex, redundant, and interactive, making it difficult to specifically define which factor, or factors, are the principal contributors to these processes and the eventual pathophysiology of asthma. as has also become apparent, asthma is represented by multiple phenotypes in which the clinical profiles and patterns of inflammation have distinct, though overlapping, characteristics. to appreciate the mechanisms of disease in asthma and the role of eosinophils, it is helpful to explore the contribution of individual cells to the pathophysiology of asthma. as early as the turn of the th century, the eosinophil was identified as a prominent cell associated with asthma. , for example, postmortem examinations of the lungs of patients who died from status asthmaticus showed, in many cases, sheets of eosinophils infiltrating the airways. these telling findings led to the long-held belief that eosinophils are an inherent characteristic and possibly an essential component of asthma, particularly in severe exacerbation of disease. as the role of airway inflammation became more fully defined, the assumption that eosinophils are a primary, if not the principal, contributor to asthma was integrated into concepts of asthma, at least until the last decade. this positioning into asthma was further supported by animal models, which strengthened the hypothesis that eosinophils are a principal contributor to inflammation, airflow obstruction, and airway hyperresponsiveness (ahr). from animal models, it was possible to separate individual components that contribute to inflammation, and the cytokine, interleukin- (il- ), was found to be responsible not only for terminal differentiation of eosinophils, but by ablating il- with a specific monoclonal antibody, many of asthma-like airway responses to inhaled antigen were inhibited. based upon these findings, eosinophils and il- became a major pathway in allergic inflammation and a target to regulate and more mechanistically control asthma. these theories would subsequently be tested with the administration of anti-il- to patients with asthma. as expected, there was a significant reduction in circulating and sputum eosinophils but, surprisingly, little to no impact on features of asthmadairflow obstruction or ahrdand only a % reduction in bronchial mucosa eosinophils. based upon these data, the role of the eosinophil in and contribution to asthma appeared less apparent. what has evolved since these dramatic shifts from an early appreciation of the eosinophil to asthma has been a more accurate and informed picture of this cell's involvement and contribution to asthma, which is the objective of our subchapter. to most fully appreciate current views of eosinophils in asthma, we feel that it is helpful to trace the key observations that have attempted to define this cell's role in asthma. retracing these discoveries has led to a more well-defined elucidation of the eosinophil's role in asthma. clinical evidence to suggest that eosinophils are key players in asthma first emerged from examinations of peripheral blood samples, largely because of the ease and safety of such studies. peripheral eosinophilia had been recognized as a feature of asthma for decades, with clinical correlations arising between airflow obstruction and the magnitude of peripheral blood eosinophilia. in , a pivotal study by horn et al. found that the total peripheral blood eosinophil count in a cohort of asthmatic patients was greater in those individuals with a more severe disease. this was an important early observation and provided a clue to what the eosinophil may contribute to asthma as an association arose between the degree of airflow obstruction and peripheral blood eosinophil counts. these and subsequent observations suggested that circulating eosinophils may be a key clinical feature of asthma and, because of these associations, contribute to airflow obstruction and hence disease severity. in a subsequent study of patients with chronic asthma, bousquet et al. assessed clinical symptoms of patients in relationship to peripheral blood eosinophil counts. these investigators found a positive correlation between the eosinophil count and disease severity. to extend the value of peripheral blood eosinophils to features of asthma, taylor et al. was also able to find correlations between peripheral blood eosinophilia and another key characteristic of asthma, bronchial hyperresponsiveness. collectively, these studies were helpful in gaining an understanding of the role of eosinophils in asthma as they showed that the level of circulating eosinophils may serve as a biomarker not only for the presence of disease but also to indicate the degree of severity. how did the eosinophil's biology further link this cell type to asthma? the eosinophil was named for the ability of eosin to stain its basic granules and thus impart the cell's well-recognized red color. these eosin-staining granules, however, were found to be more than a marker for this cell type. eosinophil-derived granule products have been found to have multiple actions, some of which can produce pathological and physiological features associated with asthma, including injury to respiratory epithelium (desquamation) and ahr. the eosinophil granules consist of four major cytotoxic cationic proteins: major basic protein (mbp), eosinophil peroxidase (epo), eosinophil-derived neurotoxin (edn) or nonsecretory ribonuclease, and eosinophil cationic protein (ecp). mbp accounts for % of the eosinophil granule content, is toxic to parasites, and has similar cytotoxic effects on the respiratory epithelial cells of both animals and humans (table . . ). , in vitro studies have shown that the application of mbp to respiratory tissue, at concentrations consistent with values found in sputum and bronchoalveolar lavage (bal) fluid of asthmatic patients ( e mg/ml), leads to almost complete erosion of tracheal epithelium and parallels the observed desquamation of respiratory epithelium in the bronchial mucosa of subjects with asthma. these effects of mbp suggest a potential mechanism by which eosinophils may contribute to airway injury, initiate repair (i.e., remodeling), and subsequently induce hyperresponsiveness. to extend these observations, flavahan et al. incubated guinea pig tracheal rings with mbp and found bronchial smooth muscle tension to acetylcholine was significantly enhanced compared to tissues not exposed to eosinophils. in a dog model, the intraepithelial administration of mbp also increased the bronchoconstriction response. ecp also causes cytotoxic effects on respiratory tract cells. by adding small amounts of ecp to the respiratory tract, dahl et al. found damage and denudation of bronchial and tracheal epithelial cells, which paralleled observations in asthma. elevated ecp values are found in bal fluid of asthmatic subjects during the late-phase response (lpr) to an allergen challenge, as well as in lavage fluid of patients with chronic, persistent asthma. in guinea pig models, the application of increasing concentrations of epo to tracheal mucosa caused epithelial cell exfoliation, ciliostasis, and bleb formation. serum edn levels are elevated in asthma and return to normal levels following treatment with prednisolone and the achievement of disease control. finally, when compared to normal subjects, edn concentrations in urine are greater in asthmatic children. eosinophils also produce other inflammatory products such as cysteinyl leukotrienes, platelet activating factor (paf), reactive oxygen species (ros), and substance p. in in vitro studies designed to assess eosinophil biology, paf stimulated the release of granule products and led to superoxide anion generation. paf was also chemotactic for the eosinophil, as reflected by the induction of an eosinophilic infiltrate of the airway after local or systemic delivery of paf in guinea pigs. , these early findings in animals suggested that the release of paf by eosinophils could potentially create a self-perpetuating cycle to further the development of eosinophilic inflammation and hence airway injury. paf also has direct proinflammatory properties. when administered to humans, paf causes rapid bronchoconstriction, which can last up to h. similar to changes that follow an airway allergen challenge, paf causes a prolonged increase in bronchial responsiveness. although a role for paf in human asthma has not been established, the biology of this eosinophil product is associated with airway changes reflective of asthma and also implies an important role for eosinophils in this process. eosinophils also generate the cysteinyl leukotrienes, ltc and ltd , which can cause both an increase in vascular permeability and bronchoconstriction. ltc is secreted by eosinophils and is increased in asthmatic children. activated eosinophils can also release substance p, a neuropeptide, to increase vascular permeability and promote plasma extravasation. eosinophils, like other phagocytes, generate ros, which are produced in greater concentrations in asthma. in asthma, ros can cause mucus secretion, which leads to increased vascular permeability and airway obstruction. when damaged by ros, respiratory epithelium produces fewer bronchodilatory substances, such as prostaglandin e (pge ) and nitric oxide, with the net result of increased airflow obstruction ( fig. . . ). although t cell-derived cytokines may be the major source of factors that influence eosinophil development and function, eosinophils themselves can also produce several cytokines to perpetuate their own inflammatory biology and that associated with asthma. transforming growth factor b (tgf-b) is a profibrotic cytokine produced by eosinophils and found in increased concentrations in asthma. tgf-b stimulation of fibroblasts leads to a thickening of the reticular lamina of the airways, and thus provides a conceptual link between eosinophilic inflammation and structural remodeling of the airway in asthma. eosinophils also produce several other mediators/cytokines, such as il- , matrix metalloproteinase- (mmp- ), tissue inhibitor of metalloproteinases (timp- ; or metalloproteinase inhibitor ), and vascular endothelial growth factor (vegf), which have also been implicated in matrix remodeling in asthma. il- , il- , il- , il- , il- , il- , il- , il- , and il- are all produced by eosinophils in varying concentrations. il- , initially observed in mice and later found in hypereosinophilic patients, is associated with hla-dr expression and is thought to contribute to the eosinophil's function as an antigen-presenting cell. il- and granulocyte macrophage colony stimulating factor (gm-csf), are also produced by eosinophils, and can function in an autocrine fashion to prolong the cell's survival. il- , which upregulates vascular cell adhesion molecule (vcam) receptors on endothelial cells and is a cofactor in immunoglobulin e (ige) isotype switching, is produced gm-csf, granulocyte-macrophage colony-stimulating factor; il, interleukin; paf, platelet-activating factor. by eosinophils from patients with atopic asthma. il- , which regulates terminal differentiation and survival of eosinophils, is found in eosinophils obtained from bal fluid of asthmatic patients. il- release also follows eosinophil stimulation by iga, igg, or ige immune complexes. the presence of eosinophils in sputum has been a characteristic finding of asthma since the early th century. bousquet et al. increased sputum and peripheral blood eosinophils in asthma patients. sputum eosinophilia is also a feature of nonasthmatic eosinophilic bronchitis, a relatively nonspecific term. in contrast, sputum eosinophilia is not a feature of chronic obstructive pulmonary disease (copd). in addition, previous studies have suggested a relationship between bal fluid eosinophilia and the level of bronchial responsiveness. the concentrations of ecp and mbp in bal fluid were also found to directly relate to the percentage of eosinophils, implying that eosinophils in asthmatic airways are activated and have undergone degranulation. to extend these observations, uchida et al. evaluated the effect of mbp on airway responsiveness in an animal model. within h of direct instillation of mbp onto the trachea of rats, significant increases in airway responsiveness to methacholine occurred. finally, subjects with asthma were identified, half of whom were treated with inhaled corticosteroids (ics) and the other half given only bronchodilators, to be used as needed. ics use led to a fall in serum and bal ecp levels but, interestingly, the eosinophil count was unchanged in both the corticosteroid or bronchodilator treated groups. collectively, eosinophil-derived products can have a significant influence on the function and pathohistology of the airway and mirror many features of asthma. in the s, a further relationship of asthma to eosinophils was supported by the finding of a marked infiltration of eosinophils in the lung tissue of patients who died suddenly of status asthmaticus. in cases of death from acute, severe asthma, eosinophilic infiltrates were found in the lung parenchyma, bronchial lumen, and entire thickness of the bronchial wall. bronchial biopsies obtained by bronchoscopy from patients with mild disease also showed eosinophils as a prominent cellular infiltrate. in addition to the presence of eosinophils and their granule products in airways of asthmatic patients, the airway histology showed epithelial desquamation, impaired ciliary function, basement membrane thickening and mucous plugs. , expanding upon these findings, ohashi et al. found the eosinophilic infiltration of mucosal tissue was associated with opening of tight junctions of bronchial epithelial cells. these histological analyses showed eosinophil infiltration of the airways was associated with epithelial damage and possibly linked to subsequent airway hyper-responsiveness. when subjects with allergic asthma inhale allergens to which they are sensitized, there is an immediate, or early, response, characterized by an acute fall in forced expiratory volume in one second (fev ). approximately % of subjects will go on to develop an lpr, which occurs e h after allergen exposure. in lpr, eosinophils are the prominent airway cellular infiltrate both in animal models and human subjects. to define the kinetics of eosinophil recruitment and eotaxin/c-c motif chemokine (ccl ) generation, humbles et al. found the chemokine eotaxin was increased e h postallergen challenge in sensitized guinea pigs. by e h postallergen challenge, there was a significant increase in eosinophil numbers in the bal fluid, but no further rise in eotaxin. these findings in the guinea pig suggest that eosinophil recruitment to the airway following an inhalation of allergen is, in part, regulated by eotaxins. findings of an early recruitment of eosinophils and the later development of a late-phase airflow obstruction to allergen suggested that these events reflect processes in asthma that could provide insight into how eosinophils may contribute to asthma. histopathological findings of the airway in lpr are also similar to events found in patients with chronic asthma and persistent airway obstruction. in guinea pigs sensitized to ovalbumin, h after an inhalation challenge with ovalbumin the cellular composition in bal fluid is predominantly neutrophils. , by h post antigen exposure, % of bal cells are eosinophils. in addition, the subsequent eosinophilic infiltration of the peribronchial smooth muscle and epithelium persists for up to days. eosinophils in the bal of immunized guinea pigs with an lpr were found to be activated, suggesting that these cells are primed during recruitment to the airway. similarly in humans, de monchy et al. found significant eosinophilia only in patients who developed lpr to inhaled antigen. moreover, the eosinophils in the bal fluid had undergone degranulation, as evidenced by an elevated ecp:albumin ratio. there was also evidence that peripheral blood eosinophils and ecp concentrations are increased prior to antigen challenge in those subjects who eventually develop an lpr. subsequently, cockcroft et al. evaluated the lpr in a population of asthma patients who had been given a single dose of inhaled beclomethasone dipropionate, inhaled salbutamol or inhaled cromoglycate in a randomized, double-blind, placebo-controlled crossover trial. while beclomethasone had no effect on the allergeninduced early pulmonary obstructive response, there was a significant inhibition of the lpr at h and h later. collectively, these data suggest that allergen provocation of allergic inflammation is likely to be regulated by eosinophils and can be controlled by corticosteroids, which block both the late-phase rise in recruited eosinophils and the subsequent reduction in lung function. thus, a logical conclusion from work at this time was that a direct association between eosinophil recruitment and altered lung function existed in asthma, a finding substantiated by kidney et al. in , gauvreau et al. extended these observations when she evaluated the development of the lpr and recruitment of eosinophils in asthma patients who were initially treated with inhaled budesonide for week and then underwent an inhaled allergen challenge. budesonide administration reduced the intensity of the lpr and also inhibited eosinophil recruitment to the airway, reflected by sputum eosinophils and the parallel increase in peripheral blood eosinophils h after challenge. from these data, it was assumed that allergic activation of the airway and the subsequent development of the lpr were caused by eosinophils recruited to the lung. to extend observations from whole-lung allergen challenges, a number of groups developed the technique of using a bronchoscope to deliver antigen into single segments of the airway. to perform these studies, a bronchoscope is introduced into the lung and then wedged into an isolated airway segment, where a dose of allergen is introduced and a lavage performed immediately; the cells and lavage fluid obtained at this time represent events associated with the acute or early response. bronchoscopy is repeated e h later, the challenged segment of the airway identified, and lavage performed: the airway events analyzed at this time model the lpr. this approach, while not allowing for measures of pulmonary function, provides a direct measure of cells, mediators, and retrieval of cells for ex vivo study to compare early-and late-phase allergic reactions. when antigen is introduced to airway segments in allergic patients, there is a strong eosinophilic response e h postchallenge. the analysis of bal is characterized by large concentrations of granule proteins, ecp, edn, epo, and mbp, as well as ltc , suggesting that the recruited eosinophils are activated when they appear in the airway. additionally, when eosinophils are retrieved from bal fluid, they are phenotypically distinct from circulating cells and have greater superoxide anion release, collagen adherence and cell surface adherence receptors compared to peripheral blood eosinophils. these findings suggested that lpr-associated eosinophils, which are recruited to the lung during the late phase and are terminally differentiated, have a greater capacity to generate inflammation. bal levels of il- were also increased and correlated with the eosinophils, suggesting a key role for this cytokine in these processes. from these studies, a more expanded picture of the allergic inflammatory response was made, along with the identification of key mediators and evidence for an enhancement of the eosinophil's inflammatory potential. calhoun et al. used segmental antigen challenges in subjects with allergic rhinitis who had been inoculated with rhinovirus to further evaluate the interactions between viral upper respiratory infections and allergic reactions, and gain insight into mechanisms of asthma exacerbations. during an acute rhinovirus infection, bal fluid obtained h after antigen challenge contained increased numbers of eosinophils. in some subjects, the increase in eosinophils persisted for up to month following the acute viral infection and initial antigen challenge. this augmented antigen-induced eosinophil recruitment was thought to be a possible mechanism for an intensified inflammatory airway response during viral infections and to account for greater asthma symptoms at that time. alternatively, virus-induced epithelial damage, with a subsequent increase in mucosal permeability, was hypothesized to increase allergen contact with immune cells, thus creating a greater inflammatory response. the presence of eosinophils is regulated by apoptosis and modulated by cytokines, growth factors, and lipid mediators, which are released during allergic inflammation but suppressed by glucocorticoids. consequently, the persistence of airway eosinophilia in some patients with asthma was attributed to cells that had developed resistance to corticosteroids. the reduction in airway eosinophils with corticosteroids and improved asthma control further supported a central role for eosinophils in asthma. under both circumstances, either a reduction or persistence of eosinophils, and their correlation with symptoms of asthma, supported a direct link of eosinophils to the pathobiology of asthma. in , evans et al. measured eosinophil numbers in asthmatic subjects days following the initiation of inhaled budesonide treatment. airway responsiveness to methacholine was improved and was associated with a fall in peripheral eosinophils. these findings also suggested that eosinophil production, maturation, and differentiation may take place in the lung, as well as the bone marrow. as corticosteroid treatment reduced serum, sputum, and tissue eosinophils, and these reductions were associated with improved asthma symptoms, the eosinophil's place as a primary contributor to asthma appeared further substantiated. however, not all patients with asthma have eosinophilia, nor are all asthma patients responsive to corticosteroids. these observations, in the face of data already discussed, raised questions as to how essential the eosinophil is to all of the clinical features of asthma. while many studies of asthma confirm the presence of elevated eosinophils, circulating and bal fluid eosinophilia are not always present in asthma. , theories as to these differences included the possibility that eosinophils may be present only during an exacerbation and their absence may result from the actions of medication, particularly ics. , these observations also led to an emerging theory that at least two asthma phenotypes exist, based on the presence or absence of tissue eosinophils. persistent eosinophilic inflammation, despite treatment, was found to be more common in adult onset asthma and less common in classic allergic asthma in patients receiving ics. while up to % of cases of severe asthma appear to start later in life, the presence of eosinophils in patients with late-onset asthma is also more variable. interestingly and importantly, patients with asthma and existing eosinophilia had greater airway remodeling and more exacerbations, despite treatment. to begin to dissect and clarify these relationships, woodruff et al. used a number of innovative approaches for study. firstly, to determine the molecular basis for asthma heterogeneity and the involvement of eosinophils, the effect of an underlying t-helper type (t h )-mediated profile of inflammation was evaluated in a cohort of patients with mild to moderate asthma and healthy control subjects. the recruited subjects were stratified based on high or low expression of il- -inducible genes in samples of their airway epithelium. using the response of their epithelium to stimulation with il- , investigators were able to classify the reaction as th- -high vs. th- -low. the t h -high asthma group was indistinguishable from the t h -low asthma group in relationship to demographics, lung function, and response to bronchodilators. however, the t h -high group had significantly greater ahr, total ige levels, and bal and peripheral blood eosinophils. the presence of airway remodeling was also evaluated in this study population, and both the reticular basement membrane thickness and epithelial mucin stores were increased in subjects with a t h -high profile. following this classification, subjects were randomized and treated with either inhaled fluticasone or placebo to determine if there was a difference in clinical response to ics based upon their t h profiles. in the t h -high group, ics improved the fev , whereas no change occurred in the low t h group. this study further supported the concept of heterogeneity in the pathogenesis and pathophysiology of asthma that can be characterized by the presence of t h -driven inflammation with eosinophilia and responsiveness to corticosteroids as markers of this profile. these findings also indicated that other phenotypes of asthma exist but their features were poorly understood. t h cell-derived il- has been identified as the major cytokine involved in terminal differentiation of eosinophils, figure . . interleukin- . interleukin- (il- ) is a signaling molecule that stimulates eosinophil proliferation, maturation, and activation. an antigen-stimulated immune response in tissues leads to the secretion of il- by cells such as eosinophils, mast cells, and t-helper type (t h ) cells. il- then acts on the bone marrow to mobilize existing eosinophils and induce further eosinophil production. these maturing eosinophils become responsive to eotaxin/ c-c motif chemokine (ccl ), produced by the endothelium, allowing for their exit from the bone marrow to tissue. ccr , c-c chemokine receptor type ; il- r, interleukin- receptor. activation of mature eosinophils, and prolongation of eosinophil cell survival ( fig. . . ). il- enhances eosinophil degranulation, chemotaxis, antibody-dependent cytotoxicity, and adhesion to endothelium. van der veen et al. identified patients with mild to moderate, dust mite-sensitive allergic asthma and found a significant correlation between the magnitude of the lpr, the allergenspecific proliferative response of peripheral t lymphocytes, and an increase in il- in vivo following inhaled antigen. extending beyond observational studies of an elevation of il- in relationship to lpr, inhaled il- was found to cause, or significantly contribute to, ahr. shi et al. also demonstrated that inhaled il- in asthma acted as an eosinophil chemoattractant and activator of the recruited eosinophils to the airway. in a subsequent, blinded, placebocontrolled crossover study of eight patients with allergic bronchial asthma, shi et al. found a significantly enhanced methacholine pc (the dose of the inhaled antagonist that provokes a % drop in fev ) responsiveness h and h after an inhalation of il- , as well as a significant increase in sputum eosinophils and ecp. using a mouse model with il- knocked out, inhaled allergen did not lead to eosinophilia or an increase in ahr. il- was also decreased during treatment with corticosteroids that improved asthma control. from these and other data, il- emerged as the dominant cytokine regulating the eosinophil's involvement in allergic airway disease. the anti-il- monoclonal antibody is an igg antibody that binds with high affinity to free il- , thus preventing its binding to the il- receptor on the surface of eosinophils and their progenitors. van oosterhouet et al. used ovalbumin to challenge sensitized guinea pigs and induce airway eosinophilia, neutrophilia, and tracheal hyperreactivity. when the sensitized guinea pigs were treated with anti-il- and then challenged with ovalbumin, airway eosinophilia, not neutrophil recruitment, was suppressed as well as the allergen challenge-induced increase in ahr. when sensitized guinea pigs were treated with the anti-il- monoclonal antibody by akutsu et al., there was a decrease in ahr, tracheal wall eosinophil infiltration, and the lpr. animal studies in which anti-il- decreased airway eosinophilia, ahr to allergen, and lpr provided further support for the hypothesis that eosinophils are central to many important aspects of the pathogenesis of asthma, and suggested that if eosinophil migration to the airway could be inhibited, signs and symptoms of asthma could be controlled or prevented. in , leckie et al. conducted a randomized, doubleblind, placebo-controlled trial in which a single infusion of either of two doses of anti-il- humanized monoclonal antibody or placebo was administered to men with mild allergic asthma. the study goal was to evaluate the effect of anti-il- on the lpr to inhaled antigen, on the premise that this treatment would ablate eosinophil recruitment and hence the airway responses to allergen, including the development of the lpr and an associated increase in ahr. the investigators found anti-il- to decrease blood and sputum eosinophils following inhaled allergen challenge. surprisingly, anti-il- treatment had no effect on either the lpr or postallergen increase in airway responsiveness to histamine. thus, in striking contrast to animal studies, there was no significant effect of anti-il- on the development of an lpr, despite the absence of eosinophils in the blood or the airways following antigen exposure. this study, though conducted in a small numbers of patients, prompted a total reevaluation of the eosinophil's role in asthma, including whether it had one at all. extending this study, flood-page et al. evaluated anti-il- monoclonal antibody treatment in asthma patients by examining its multidose effect on blood and sputum, as well as bone marrow and airway tissue eosinophils. in a randomized, double-blind, placebo-controlled study, patients with mild asthma received three intravenous doses of mepolizumab (i.e., il- monoclonal antibody) or placebo for weeks. within weeks of the first anti-il- dose, there was a significant decrease in peripheral blood eosinophils. at weeks and of the study, there was nearly a % reduction of eosinophils in blood and sputum samples following anti-il- treatment. in contrast, eosinophils were reduced by only % in bone marrow aspirates. similarly, bronchial mucosa eosinophils were reduced by % from baseline. despite this reduction of intact eosinophils, staining of the bronchial biopsy for intracellular mbp was unchanged by mepolizumab. despite these reductions in eosinophils, there was no change in ahr, exacerbations, fev values, peak flow measurements, or symptoms between the anti-il- and placebo-treated groups. while a reduction in blood and bal fluid/sputum eosinophils occurred with anti-il- , there was only a % reduction in bone marrow and bronchial eosinophils. this finding confirmed that, despite anti-il- therapy, residual airway eosinophils persisted and the total amount of mbp present in airway tissues was unchanged. from these observations, it was also hypothesized that the residual eosinophil population in the airway continued to exist and release, or retain, granule proteins, despite anti-il- treatment. furthermore, it was proposed that these persistent effects may be responsible for the lack of improved asthma control, fev , and ahr. these findings with anti-il- contrasted sharply with effects noted with oral corticosteroids, which caused an % decrease of bronchial mucosal eosinophilia and led to significant clinical improvements of asthma symptoms. given the possibility that, despite anti-il- treatment, residual airway eosinophils may be sufficient to continue to exert their influence on clinical outcomes, including lung function and symptoms, the eosinophil should not have been excluded as a target for asthma therapy, although the outcome may not be symptoms or airflow obstruction. in , rothenberg et al. reported the results of a randomized, double-blind, placebo-controlled trial of anti-il- in prednisone-dependent ( e mg/d) patients with hypereosinophilic syndrome. in addition to a significant lowering of peripheral blood eosinophilia, % of the mepolizumab-treated subjects were able to reduce their oral prednisone dose to mg/d compared to only % in the placebo group. in addition, mepolizumab significantly reduced the likelihood of an exacerbation of their hypereosinophilic disease. while the effect of therapy on hypereosinophilic syndrome patients may not be extrapolated to asthma, this study did show that mepolizumab has the ability to decrease eosinophils, the prednisone requirement to maintain disease control, and exacerbations from the hypereosinophilic syndrome. expanding on this theme, green et al. examined a strategy designed to determine the effects of treatment directed toward reducing sputum eosinophil counts rather than administering a dose of ics based on symptoms alone, i.e., a guidelines approach. seventy-four patients with moderate to severe asthma were identified and randomly assigned to either a management strategy based upon british thoracic society guidelines or one using a dose of ics that reduced sputum eosinophils to < %. the sputum management strategy group had an average eosinophil count that was % lower than that of the guidelinemanaged group during the study. as an apparent consequence of this reduction in eosinophils, patients in the sputum management group had significantly fewer asthma exacerbations and were admitted less frequently to the hospital for asthma ( fig. . . ). however, total asthma quality of life scores, mean peak flow measurements, postbronchodilator fev , and the use of rescue bronchodilators were not different in the two management groups. while this study did not show a relationship between sputum eosinophils and variable airflow obstruction and other parameters, such as daily symptoms, it did support the notion that eosinophils play a central role in asthma by either serving as a marker for exacerbation risks or being possibly linked to an increased susceptibility for exacerbations. it can be argued that previous studies of anti-il- had examined patients with only mild asthma and with outcome measures that were not specifically related to ongoing eosinophilic inflammation. to extend upon this hypothesis, haldar et al. and nair et al. published results of studies designed to evaluate mepolizumab treatment in patients with severe asthma, persistent eosinophilia in sputum, and frequent exacerbations. in an approach similar to that of green et al., haldar et al. evaluated the effect of a reduction in sputum eosinophils with anti-il- . in this randomized, double-blind, placebo-controlled trial, patients were enrolled with asthma and sputum eosinophilia that was refractory to treatment and a history of recurrent exacerbations. patients were given anti-il- monoclonal antibody or placebo monthly for year. the mepolizumab group had exacerbations requiring prednisone (a mean of . exacerbations per year per subject) compared to exacerbations ( . exacerbations per subject per year) in the placebo group ( fig. . . ) . the mepolizumab treatment group also had a greater improvement in scores of the asthma quality of life questionnaire, with a mean improvement of . compared to . in the placebo group. similar to findings of flood-page et al., , the anti-il- treatment group had significantly lower eosinophil counts in bal, blood, and bronchial wash, but less of a decrease in bronchial mucosa eosinophils. as noted in previous studies, there were no significant changes from baseline in ahr, bronchodilator use, or fev in the mepolizumab group. interestingly, when prednisolone was given after the mepolizumab treatment, there was an improvement in exhaled nitric oxide or lung function, suggesting these symptoms and pathways may be dissociated from eosinophilic inflammation, and are possibly mediated through other mechanisms. reticular basement membrane thickening in asthma is associated with the presence of bronchial mucosal eosinophils. using allergen-sensitized mice, humbles et al. found eosinophil-deficient mice were protected from the development of peribronchiolar collagen deposition and increased airway smooth muscle mass following allergen challenge. however, increases in ahr and mucus secretion occurred in the eosinophilic deficient mice, just like wildtype mice. the authors concluded that eosinophils contribute substantially to airway remodeling, but are not obligatory for allergen-induced lung dysfunction. as noted earlier by flood-page et al., residual bronchial wall eosinophils persists despite treatment with anti-il- , even when blood and sputum eosinophils are nearly eliminated. in addition, anti-il- has little effect on the mbp found in the airways, airflow obstruction, and ahr. these observations raise the possibility that bronchial mucosal eosinophils are a privileged cell or are in a privileged location when lodged in tissues, and their levels are not responsive to previously used methods of eosinophil depletion. furthermore, these findings suggest that another important contribution of eosinophils to the pathophysiology of asthma is airway remodeling, which is supported by other studies. in a -year study, sont et al. evaluated an asthma treatment strategy aimed at reducing ahr compared to treatment that followed international guidelines and was based primarily on symptom and lung function assessments. in this randomized, prospective, parallel trial of adults with mild to moderate asthma, patients were placed in the reference strategy group, with a treatment based on current guidelines, while patients were placed in the ahr group with a treatment strategy based on guidelines as well as a reduction in ahr. in addition to measuring bronchodilator use, fev , peak expiratory flow (pef) and symptoms, ahr was also quantified following methacholine challenges. patients in the ahr strategy group received higher doses of ics to reduce ahr. this use of higher doses of ics and reduction in ahr was associated with a lower incidence of exacerbations and a greater improvement in lung function. of the patients in the study, also underwent bronchial biopsies before and after their individual treatment approaches. in ahr strategy group subjects, who received an additional mg/d of ics, there was a significantly greater decrease in subepithelial reticular layer airway thickness and bronchial mucosa eosinophils compared to the reference group ( fig. . . ). the study also showed that the decrease in mucosal eosinophils related to an accompanying improvement in ahr. when the investigators evaluated the relationship between improvements in ahr and changes in the histology of the airway biopsies, they found a correlation with the reduction in tissue eosinophils ( fig. . . ). these findings further support for a role of eosinophils in airway remodeling. the haldar et al. study also evaluated the effects of anti-il- on features of airway remodeling. airway wall thickness and airway wall area were evaluated by chest x-ray computed tomography (ct). changes in these assessments of airway structure were made following a year of treatment in both the mepolizumab and placebo groups. the investigators found a significant reduction in airway wall thickness and total wall area (ta and wa) in the mepolizumab-treated group ( fig. . . ) . flood-page et al. also hypothesized that a reduction in bronchial wall eosinophils with anti-il- would reduce markers of airway remodeling. twenty-four mild atopic asthma patients received three monthly infusions of mepolizumab and had bronchial biopsies taken before and after each infusion. in an attempt to determine if bronchial mucosa eosinophilic inflammation was associated with an increased deposition of extracellular matrix (ecm) proteins, researchers found a positive correlation between the thickness, density and expression of the ecm protein tenascin, and bronchial mucosal eosinophil numbers. as noted previously, mepolizumab caused a significant, though incomplete elimination of bronchial mucosal eosinophils. associated with these changes in eosinophils was a decrease in the thickness and density of tenascin, as well as density of the ecm proteins lumican and procollagen iii, in the reticular basement membrane. in addition, there was a decrease in expression of both tenascin and lumican. bal fluid in the mepolizumab-treated group also had a significant decrease in tgf-b. not only did this study confirm that the expression of ecm proteins is greater in asthma, it also showed that a reduction in eosinophil numbers is associated with a decrease in ecm protein deposition in the airway. figure . . relationship between changes in eg þ eosinophils and changes in methacholine pc (the dose of the inhaled antagonist that provokes a % drop in fev , the forced expiratory volume in one second) during years of treatment according to the reference and airway hyperresponsiveness (ahr) strategies. the greater the decrease in number of eg þ eosinophils, the greater the improvement in ahr to inhaled methacholine. eg , antibody marker for activated esinophils. (reproduced with permission from sont jk, et al. ) figure . . individual changes in reticular layer thickness beneath the epithelium in bronchial biopsy specimens before and after treatment for years according to the reference and ahr strategies. bars indicate mean values at the visits for both strategies. there is a significant decrease in reticular layer thickness within the ahr strategy group, which is significantly greater than in the reference strategy group. (reproduced with permission from sont jk, et al. ) thus, the above studies provide convincing evidence for a relationship between the presence of eosinophils and features of airway remodeling. firstly, an increased ics dose leads to a decrease in bronchial mucosal eosinophils and airway thickness, as well as a reduction in bronchial hyperresponsiveness. anti-il- studies built on such observations by showing that a decrease in bronchial mucosal eosinophils is associated with a reduction in airway thickness, as assessed by ct scans of the chest. finally, evidence for a decrease in tgfb and ecm proteins following the use of anti-il- provides further support of the link between bronchial mucosa eosinophilic inflammation and airway remodeling in asthma. since the discovery of the eosinophil in the late s, this cell has been considered to be a characteristic feature and possibly an essential and etiological component of the pathophysiology of asthma. these assumptions were supported by demonstrating tissue eosinophilia in postmortem analyses of the lungs of patients who died in status asthmaticus, as well as by finding parallel relationships between blood eosinophilia and asthma severity. the belief in the central role of eosinophil in asthma was further strengthened by animal models that convincingly found anti-il- strategies to reduce ahr, blood and lung eosinophils, and the lpr to inhaled antigen. these studies identified eosinophils as a central contributor to asthma. surprisingly, when anti-il- was evaluated in patients with asthma, airway and peripheral eosinophils dramatically diminished, but there were no significant improvements in daily symptoms of asthma or airflow obstruction. what has emerged in the wake of anti-il- studies is perhaps a more informed opinion on two major roles for eosinophils in asthma: as effector cells of airway remodeling and exacerbations. the role of the eosinophils as key players in the pathophysiology of asthma has been debated, despite evidence that the cells are present and activated in the airway lumen and tissue of patients with current asthma; are increased in number when asthma is uncontrolled or severe and decreased when asthma is controlled ; and treatment strategies that aim to control airway eosinophilia are significantly more effective and less expensive in improving asthma control , and decreasing asthma exacerbations compared to guideline-based clinical strategies. cynicism was fueled by observations that in murine models of allergic sensitization, airway hyperresponsiveness could be induced without eosinophils. skepticism grew stronger when therapy using monoclonal antibodies against interleukin- (il- ), which has no known clinically relevant biologic activity other than targeting eosinophils, failed to demonstrate improvement in asthma outcomes despite decreasing airway and blood eosinophil numbers. the molecule did not reduce allergen-induced airway constriction or hyperresponsiveness, airflow limitation, exacerbations, or symptoms. the likely explanations for this apparent paradox are inappropriate methodology, inadequate sample size, or an inadequate reduction in bronchial mucosal eosinophil numbers. this subchapter will describe the clinical studies that demonstrated an improvement in asthma control using treatment strategies that aimed to normalize sputum eosinophil count using corticosteroids; to evaluate critically the clinical trials that failed to demonstrate an improvement in asthma using monoclonal antibodies directed against il- ; and to present evidence from a prospective audit of clinical outcomes of patients managed by normalizing sputum cell counts. airway eosinophilia can be reliably and relatively noninvasively assessed in sputum. in clinical practice, approximately % of patients with asthma attending a tertiary clinic have eosinophilic bronchitis. more severe asthma and more severe airflow limitation are associated with more intense sputum eosinophilia. two studies in adults and one study in children have evaluated the outcomes of titrating anti-inflammatory treatment with the intention of normalizing eosinophils in sputum. the first single center, -year trial that examined the effect of treating asthma to reduce eosinophils to % resulted in a significant reduction of severe exacerbations compared with a control group treated without sputum eosinophil counts. the large number of exacerbations and their severity was probably a result of the policy at the time to reduce corticosteroid use further if control was maintained for months. the second trial was a multicenter trial conducted over years, and differed in that the minimum dose of corticosteroid to maintain sputum eosinophils at % was determined first and then maintained for the duration of the study. exacerbations were few and mild compared with the first study and were reduced by about % compared with the group treated with the same bestguideline approach to treatment without sputum cell counts. the active treatment reduced eosinophilic exacerbations but had no effect on neutrophilic exacerbations, which were regarded to be probably of viral cause. the benefits in both studies were achieved without any increase in corticosteroid dose over that required by the control group. in contrast, a similar study in children showed a nonstatistically significant effect on reducing exacerbations using a sputum strategy that aimed to keep eosinophil levels to below . %. the modest benefit was most likely due to the inadequate control of eosinophils in the treatment arm that was probably related to the inadequate dose of inhaled corticosteroids allowed in the study. the effectiveness of using sputum eosinophils as a marker to decrease exacerbations in adults and children with moderate to severe asthma was recently confirmed in a systematic review and meta-analysis. a critical review of the recently published clinical trial literature reveals that the reduction in exacerbation reported in all the most recent large clinical trials for either asthma or chronic obstructive pulmonary disease (copd) for any new medication compared to placebo is significantly less than those reported for strategies employing the judicious use of currently available medications guided by cell counts in sputum (table . . ). in addition to a significant reduction of exacerbations at a reduced cost, the adverse consequences of new therapies and suboptimal treatment may also be avoided. the beneficial effects of corticosteroids are not limited to decreasing eosinophil numbers in the airways. they can also reduce the numbers of other cells, such as lymphocytes and mast cells, and decrease some markers of remodeling. thus, it is not possible to conclude definitively the pathobiological role of eosinophils in asthma from those studies. definitive proof would be obtained by reducing eosinophil numbers in the airway using treatments that directly target the eosinophils. recently, the availability of monoclonal antibodies directed against il- has provided us with the opportunity to examine this question. in two recently published randomized controlled trials (rcts) on the effect of mepolizumab , and a clinical trial on the effect of reslizumab, these drugs reduced sputum eosinophils numbers to almost zero. this reduction was associated with a reduction of exacerbations compared with the placebo group in the first mepolizumab study and a prednisonesparing effect and improvement in clinical outcomes in a small sample number in the second. in the larger reslizumab clinical trial, the reduction in sputum eosinophils was associated with an improvement in the forced expiratory volume in one second (fev ) and in asthma controls over a -month period in patients with moderate to severe asthma. the results of these three studies contrasted with the negative results of five other trials, in which the effect of the antieosinophil drug was not examined in patients with asthma and current sputum eosinophilia. in two of the five studies that measured sputum eosinophils and in the three rcts, the greater the certainty that an increase in eosinophils was persistent, the greater the success of the treatment (table . . ). clinical outcomes are significantly improved when patients who require daily prednisone are monitored using sputum cell counts. sixty-three patients with asthma ( men; mean age, years; mean bmi, . ) were followed for a median period of years (range, . e year). thirty-seven patients had associated chronic airflow limitation (postbronchodilator fev /vital capacity < %). twenty had never smoked. forty-two percent were nonatopic. significant comorbidities included gastroesophageal reflux disease ( %), nasal polyps and sinusitis ( %), and sensitivity to nonsteroidal anti-inflammatory drugs ( %). ethmoid and sphenoid sinusitis were the most important predictors of persistent airway eosinophilia. at the time of their initial assessment, the majority of the patients were not on daily prednisone (median daily dose, mg; sputum eosinophils: mean, . %; median, . %; minimum, %; maximum, %). monitoring with the aim of keeping sputum eosinophils at < % resulted in higher doses of corticosteroids (median daily dose of prednisone was mg and for inhaled corticosteroids was mg of fluticasone equivalent), and this was associated with predictable significant adverse effects. over the period of follow-up, despite decreasing the eosinophilic exacerbations to . years/patient, there were noneosinophilic neutrophilic exacerbations. overall, there was no significant loss of lung function over the period of follow-up (mean decrease in fev , ml/year). corticosteroids are very effective in reducing eosinophil numbers and activation in the airways of most patients with asthma. therefore novel treatment strategies such as anti-il should probably be reserved for patients who require high doses of inhaled or regular ingested corticosteroids to control their airway eosinophilia and asthma. although a larger number of antisense molecules, monoclonal antibodies, and small molecules are currently being evaluated to target a number of relevant cytokines or chemoattractants involved in eosinophil recruitment into the airway, such as eotaxin/ c-c motif chemokine (ccl ), il- , and il- , none of them have yet been demonstrated to be effective in suppressing an airway eosinophilia that persists despite being treated with prednisone. these are currently being investigated. eosinophils, a prominent feature of asthma, are found in increased numbers in the circulation and airways in relation to the severity of asthma. , inhaled allergen challenge in asthmatic subjects results in the appearance and accumulation of mature and immature eosinophils in the bone marrow, blood, and airways. e the kinetics of eosinophilia are compartment-specific ( fig. . . ), , , and the number of eosinophils correlates with the severity of the late asthmatic reaction. this will be covered in detail elsewhere in this book. eosinophils are terminally differentiated myeloid leukocytes that migrate to tissues as effector cells in a number of inflammatory processes, including allergic diseases and helminth infections. the migration and accumulation of eosinophils is highly regulated via signaling of cytokines and chemokines through cell-surface receptors, and by induction of adhesion molecule expression. since allergic asthma is primarily a t-helper type (t h )-mediated disease, it is not surprising that cytokines driving eosinophilia are t h cell products: specifically, granulocyte-macrophage colony-stimulating factor (gm-csf), interleukin- (il- ), and interleukin- (il- ), which signal through specific high-affinity cell-surface receptors linked to a common b-chaindall of which can act as eosinophil growth factors that promote formation of eosinophil/basophil (eo/b) colony-forming units (cfu) in functional assays. the common b-chain is especially important for eo/b proliferation, production of cytokines from eosinophils, and eosinophil migration to effector sites. of the three eo/b differentiation-inducing cytokines, il- is necessary for mobilization of eosinophil progenitors from the bone marrow and their terminal differentiation. other t h cytokines, such as il- and il- , are known to regulate transmigration of eosinophils from the vascular bed into the tissue compartments by augmenting expression of adhesion molecules on the endothelium and inducing expression of potent eosinophil chemokines, such as eotaxin/c-c motif chemokine (ccl ) and rantes/ccl in the airways. , il- has also been shown to play a supportive role in both mast cell and eo/b differentiation. , once in the inflamed tissue, eosinophils contribute to the manifestation of symptoms through release of granule proteins and proinflammatory mediators. hemopoietic progenitors, which are found in the circulation under steady-state and disease conditions, exist in the bone marrow at various stages of lineage commitment; in the latter compartment, these progenitors can be allowed or selected to differentiate in specific directions in response to various ambient stimuli, such as hemopoietic cytokines engaging specific cell-surface receptors. this hemopoietic activity occurs in the bone marrow proper under the influence of stromal cells and microvascular endothelial cells, and in tissues, also in response to epithelial cells. each of these resident cell populations can respond to inflammatory stimuli by increasing transcription and translation of hemopoietic growth factors and/or cytokines. the bone marrow environment itself, as well as primed tissues, can each support and accelerate production of eosinophils for either release into the circulation or amplification of tissue eosinophilic inflammation, respectively. , the process of tissue amplification of eosinophilic inflammation through progenitor differentiation at the site has been termed in situ hemopoiesis (figure . . ). , hemopoietic progenitors express the cell stage-specific antigen, cd , which is present at the highest levels on early hemopoietic myeloid progenitors and is progressively lost on terminally differentiating cells. these cd þ hemopoietic progenitors contribute to the ongoing recruitment of eosinophils and basophils to sites of allergen challenge in allergic diseases, including asthma. indeed, the number of cd þ cells, as well as both mature and immature eosinophil cell numbers, are higher in blood and bone marrow of atopic subjects compared with nonatopic controls; , mild asthmatic subjects have fewer circulating cd þ cells than severe asthmatic subjects, after airway allergen inhalation challenge in atopic asthmatic subjects, cd þ cells from bone marrow synthesize mrna and protein for membrane-bound il- receptor subunit a (il- ra), permitting them to further respond to the eosinophilopoietic cytokine, il- . progenitor cells from the bone marrow of atopic subjects show increased responsiveness to il- , probably due to increased levels of bone marrow cd þ il- ra þ cells, a unique feature of atopic disease. the phenotype of bone marrow cd þ cells from mild asthmatic subjects has been carefully examined by flow cytometry, demonstrating a higher proportion of cd þ il- ra þ after allergen challenge in those subjects who developed airway eosinophilia and increased methacholine airway responsiveness. , taken together with in vitro experiments demonstrating il- -induced expression of il- ra on human progenitor cells, , these data demonstrate eosinophil lineage skewing of cd þ cells in response to an allergic stimulus. thus, increased production of eo/b lineage-committed progenitors within the bone marrow, the subsequent development of blood and tissue eosinophilia, and the maintenance of an allergic inflammatory response , have been linked. reports of antigeninduced increases in cd þ il- ra þ cell numbers in murine bone marrow coincident with enhancement of il- -dependent eosinophilopoiesis provide further evidence that eosinophil production occurs as a result of expansion of the relevant eosinophil progenitor population rather than exclusively from demargination or release of sequestered mature eosinophils into the circulation. e the rapid increase in expression of il- ra on cd þ cells from the bone marrow of atopic asthmatics , clearly demonstrates that progenitor cells in these subjects are primed to respond to il- ; indeed, cd þ il- ra þ cell numbers circulate at higher numbers in subjects with asthma compared to controls, and correlate with asthma severity. although controversial, chemokines such as eotaxin may also play a role not only in the migration but also in the differentiation of progenitor cells. since cd þ progenitor cells are found to express c-c chemokine receptor (ccr ), which is upregulated in a t h environment, it is possible that signaling through this receptor also causes progenitor cells to differentiate into eosinophils independently of gm-csf, il- , and il- . colony assays are functional measures for the quantification of hemopoietic differentiation potential. eo/b cfu are clusters of immature, nascent eosinophils and basophils derived from single progenitors e present in nonadherent mononuclear or purified cd þ cell populations seeded into a semisolid medium in the presence of hemopoietic growth factors. there are significantly greater numbers of eo/b cfu in the peripheral blood of subjects with various allergic airway disorders, including asthma, nasal polyposis, and rhinitis. , greater numbers of cd þ cells are detected in blood and bone marrow of atopic than nonatopic subjects, correlating positively with higher numbers of eo/b cfu grown from the blood and marrow of these subjects. the higher levels of cd þ cells and il- -responsive eo/b cfu in atopic subjects indicate a role for eo/b progenitors in allergic diseases, such as asthma. the growth of eo/b cfu is also influenced by exposure to specific antigen, which initiates a cascade of events with a stimulatory effect on the bone marrow to produce and release newly formed inflammatory cells. importantly, allergic individuals can exhibit fluctuations in the numbers of circulating progenitors and tissue eosinophils during an allergen pollen season. , an initial increase in circulating eo/b cfu at the beginning of seasonal allergen exposure is followed by a significant decline at the peak of the season, coincident with nasal symptoms and inflammation, probably reflecting the migration and differentiation in situ of progenitor cells from the blood to the inflamed tissue. e this increase in eo/b cfu has also been demonstrated in allergic asthma exacerbation. , supporting the latter in vivo observation, higher numbers of bone marrow and circulating eo/b cfu can be measured h after allergen inhalation. studies conducted in animal models of allergic asthma that carefully investigate the trafficking of cells from bone marrow confirm that allergen-induced airway inflammation is associated with increased numbers of newly formed cells in the blood and airways. , in mouse models of allergic rhinitis and/or asthma, changes in bone marrow eosinophilopoiesis, accompanied by appropriate peaks of hemopoietic cytokines and chemokines, such as eotaxin, gm-csf and il- , and changes in cell surface receptors for these factors, were shown to occur as early as h after allergen challenge, , , e indicating that progenitor cell fluctuations and hemopoietic processes contributing to eosinophilic airways inflammation can occur quite rapidly in response to allergen challenge. studies to assess changes in cytokines levels within the bone marrow of sensitized mice or atopic asthmatics following allergen exposure have detected increases in levels of il- consistent with the kinetics of eosinophil lineage commitment. e , , , using colony-forming assays, bone marrow progenitor cells from asthmatic subjects developing late-phase responses and airway eosinophilia postallergen challenge were shown to be more responsive to il- than cells from subjects without these allergen-induced responses. a subsequent investigation demonstrated that allergen inhalation by allergic asthmatic subjects induces a time-dependent change in the levels of growth factors and cytokines in the bone marrow: subjects with elevated levels of airway and circulating eosinophils postchallenge had increased numbers of il- -responsive progenitors at h and h postallergen ( fig. . . ), coincident with increased il- protein levels in the bone marrow. as such, allergeninduced activation of an eosinophilopoietic process highlights the relationship between increased bone marrow il- levels and the regulation of eosinophil production from bone marrow progenitor cells. given the observed delayed interferon g (inf-g) increase in the marrow of these subjects, it could be postulated that activated t cells migrate from the airways to the bone marrow and release cytokines such as il- and il- that may locally orchestrate activation of hemopoietic events during an allergic inflammatory response. e to this end, investigations of the cell-associated cytokine production within the bone marrow have shown that cd þ cells , and t cells e , produce il- during the course of the allergic inflammatory response. nascent eosinophils and basophils picked from eo/b cfu have also been shown to constitute autocrine sources of gm-csf and il- , , e , , which could further amplify the process of differentiation and proliferation. more recently, it has been shown that cd þ cells from blood and bone marrow release more il- following stimulation with calcium ionophore and phorbol- -myristate- -acetate than do the equivalent number of cd þ t-lymphocytes. traditionally, the focal point of the differentiation and maturation process involving hemopoietic progenitors has been the bone marrow, under the regulation of its proper microenvironment. however, there is now an abundance of evidence demonstrating that eosinophil progenitors can traffic as fully or partly undifferentiated cells to allergic inflammatory tissues in the upper and lower airways, where they can and do differentiate into mature eosinophils under the control of local stimuli. indeed, eo/b cd þ cells are found in tissues such as the bronchial mucosa, nasal polyps, and even in atopic dermatitis lesions. despite the importance of systemic and local il- production for the differentiation of cd þ cells in the bone marrow, it is apparent that eo/b can differentiate similarly at sites of tissue inflammation. this has been demonstrated functionally in several studies. firstly, inflamed tissue from subjects with allergic rhinitis and nasal polyposis has been shown to produce hemopoietic cytokines that promote the differentiation and maturation of eo/b cfu. , e next, mononuclear cells extracted from nasal polyp tissue have the potential to produce eo/b cfu in response to growth factors such as il- . , , this supports the concept of in situ hemopoiesis, in which locally elaborated growth factors can drive the differentiation and maturation of hemopoietic progenitors into mature eosinophils ( fig. . . ) . finally, phenotypic evaluations have identified cd þ cells in various airway compartments, including the mucosa of the upper and lower airways of subjects with allergic rhinitis and asthma, respectively. , , the already elevated levels of cd þ cells in induced sputum samples collected from mild asthmatic subjects are further increased after allergen inhalation challenges compared to healthy controls. with respect specifically to in situ eosinophilopoiesis, increased numbers of cells double positive for cd and il ra mrna are found in nasal biopsies and lung biopsies of allergic asthmatic subjects compared to normal controls, , suggesting that il- -responsive cd þ cells committed to the eo/b lineage are present within the inflamed tissue. furthermore, cd þ cells appearing in sputum of allergic asthmatic subjects stain positive for il- and il- intracellularly, suggesting that they themselves may act as proinflammatory effector cells in the microenvironment of the inflamed tissue. autocrine production of growth factors in eo/b cfu grown from the blood of allergic asthmatic subjects has likewise been documented, which suggests that cytokine expression by differentiating progenitors may provide an additional stimulus to enhance differentiation in situ. , a similar autocrine upregulation of both eosinophilopoietic factors and their receptors has recently been demonstrated for tlr-ligated cd þ cells in cord blood. , in situ il- -dependent differentiation of eosinophil progenitors can be triggered in the nasal mucosa of allergic rhinitic donors: ex vivo nasal mucosa cultures stimulated with allergen were shown to contain reduced numbers of cd þ il ra mrna þ cells and increased numbers of major basic protein (mbp) immunoreactive cells, thus shifting cells locally from an immature to a mature phenotype. in a mouse model of allergen-induced airway inflammation, newly produced cd þ ccr þ cells, isolated from lung and stimulated with eotaxin- /ccl or il- , differentiated into significant numbers of cfu; cell cycle analysis showed significant increases in the number of cd þ ccr þ proliferating cells in allergenexposed animals. these data support the notion of local eo/b differentiation within tissues, suggesting that the ccr /eotaxin pathway is also involved in the regulation of this allergen-driven in situ hemopoiesis, at least in mice. recently, it has been shown that human airway smooth muscle of allergic asthmatics can also stimulate increased eo/b differentiation in vitro, adding to the complexity of systemic and local hemopoietic processes in the generation of eosinophilic tissue inflammation. the evidence described above for in situ hemopoiesis is also in keeping with a recent observation that il- /thymic stromal lymphopoietin (tslp) is sufficient to induce the differentiation of cd þ progenitors into eosinophils, in addition to activating t h and mast cells. a major recent discovery is that tslp promotes t h pathways (via il- and il- ) that induce the development of a c-kit int sca- þ multipotent progenitor (mmp; also known as 'nuocytes') population in gut-associated lymphoid tissue; , this provides further support for in situ hemopoiesis, and promotes the idea that tslp provides a critical link between adaptive and innate immunity in the process of hemopoietic cd þ cell differentiation within tissues ( fig. . . ). as mentioned above, recent data showing that cd þ il- þ and cd þ il- þ double-positive cells can be detected in sputum of asthmatic patients, with increases after allergen challenge, are consistent with the concept of allergen activation, via tslp, of multipotent as well as lineage-committed progenitors, some of which can thus potentially function as inflammatory effector cells without further differentiation. preliminary data show that recombinant tslp, with or without il- as a coligand, can also upregulate il- ra on cd þ cells, rendering them more responsive to the effects of il- , with concomitant functional eo/b cfu differentiation. moreover, stimulation of cd -enriched human cord blood cells with the toll-like receptor (tlr) agonists, lipopolysaccharide (lps) or cpg-oligodeoxynucleotides (cpg odn), induces increased expression of tlr- , tlr- , and tlr- on cd þ cells, as well as increases in gm-csfra, il- ra, and il- ra. , these observations demonstrate additional mechanisms through which innate immunity can regulate the responsiveness of eo/b progenitors. cd þ cells stimulated with a combination of tlr agonists and hemopoietic cytokines have been shown to give rise to more eo/b cfu responsive to il- and il- than through hemopoietic cytokine stimulation alone. , collectively, these data point to tslpetlretlr ligand interactions that can result in autocrine upregulation of t h cytokines (e.g., il- , il- ) in cd þ cells, , , and further support the idea that tslp provides a critical link between adaptive and innate immunity in the process of hemopoietic cd þ cell differentiation within tissues (fig. . . ). il- is central for upregulation of myeloid progenitors in the bone marrow after airway allergen challenge, , and for trafficking from the marrow to the airways in several animal models of either upper or lower airways inflammation. , , airway, blood, and nasal eosinophilia are completely inhibited by either neutralizing the biologic effects of il- or through deletion of the gene encoding il- . although the asthmatic lung can release abundant amounts of il- , studies in animal models have led to debate regarding the distribution and sources of il- required to drive airway eosinophilia. studies in mice have shown that circulating, rather than local, il- in the lung is critical for the development of allergic airways eosinophilia. this was investigated through systemic il- gene transfer to il- -knockout mice, which effectively supported ovalbumin (ova)-induced eosinophilia in the blood, bone marrow, and lung. this contrasts with il- gene transfer into the airways, which did not induce eosinophilia following ova challenge. thus, systemic il- seems to be necessary for the development of eosinophilia in the mouse. however, this is more controversial in humans. further studies have demonstrated that inhalation of il- by allergic asthmatic subjects leads to the development of peripheral blood and sputum eosinophilia, and inhaled il- induces airway eosinophilia accompanied by increased airway hyperresponsiveness (ahr). in contrast, three subsequent studies reported no effect of il- inhalation on eosinophil levels in blood, airways, or ahr in allergic asthmatics. e by inhalation, il- was found to significantly decrease cd þ il ra mrna þ cells within the bronchial mucosa and decrease the percentage of cd þ ccr þ cells in the bone marrow of atopic asthmatics. it has been hypothesized that lung-derived il- provides a signal to a population of cells within the bronchial mucosa that traffic to the bone marrow, where they locally induce the efflux of cd þ ccr þ cells. , elegant experiments involving perfusion of the femoral bone marrow of guinea pigs confirmed that whereas il- induces chemokinesis of bone marrow eosinophils, mobilization of mature eosinophils by il- occurs synergistically with, and requires, the presence of eotaxin. eotaxin is a potent and eosinophil-specific chemoattractant. e eotaxin- is thought to be more important than eotaxin- for inducing mobilization of eosinophils and their progenitors from the bone marrow into the blood circulation. , a reduction of eosinophil numbers was observed in the lungs of mice treated with eotaxin- blocking antibodies , and in strain-specific eotaxin- knockout mice. , eotaxin- is released from lung structural cells, and is released at increased levels following allergen challenge. human endothelial progenitor cells also express eotaxin- , and have been shown to be rapidly mobilized to the lung after allergen challenge in sensitized mice and atopic asthmatic subjects, where they also can contribute to the development of lung eosinophilia through the expression and secretion of eotaxin- . more recently, both eotaxin- and eotaxin- have been shown to induce migration of murine bone marrow and blood cd þ ccr þ cells using an in vitro transmigration assay. these data suggest that the ccr /eotaxin pathway is involved in the regulation of allergen-driven accumulation/mobilization of eosinophil lineage-committed progenitor cells in the lung. the receptor for eotaxin, ccr , is upregulated on cd þ cells after allergen challenge, thereby facilitating eotaxin-mediated progenitor cell mobilization from the bone marrow to the peripheral circulation. , other migration signals studies in sensitized mice have indicated that t cells are the major gatekeepers regulating eotaxin and il- levels, and thus eosinophilia. reductions in airway, bone marrow, and peripheral blood eosinophil levels in cd À/À and cd À/À mice suggest that these t cell populations are critical regulators of allergen-induced eosinophilia. furthermore, reduced serum il- and bronchoalveolar lavage eotaxin- levels in cd À/À mice suggest that cd þ t cells are obligatory for the development of allergen-induced airway eosinophilia. in addition to eotaxin and il- , many other mediators have been shown to induce responses in both mature eosinophils and eo/b progenitors. cysteinyl leukotrienes, which are released in the airways following perturbation by allergen, are chemoattractants for mature eosinophils, but have also been shown to aid differentiation and induce chemotaxis and in vitro transendothelial migration of eo/b progenitors. preliminary work has shown that both il- and il- can prime hemopoietic progenitor cells in a transmigration assay, and studies are under way to investigate whether progenitors are also responsive to the eosinophil chemoattractants prostaglandin d receptor (crth ; reviewed in ) and peroxisome proliferatoractivated receptor g (pparg). , in the allergen challenge model, there is attenuation of cxcr (sdf- a receptor) expression on bone marrow cd þ cells from mild asthmatic subjects, as well as a reduction in sdf- a levels in the bone marrow. this demonstrates a mechanism whereby retention of progenitors in the bone marrow regulates their egress into the blood. the discoveries that il- is a specific eosinophil growth factor in humans and that eotaxins can selectively induce eosinophil recruitment, as described above, were instrumental for the development of drugs targeting the eosinophil. this is reviewed in detail elsewhere. anti-il- treatment in mild atopic asthmatic subjects has been shown to induce a reduction of airway eosinophils, arrest bone marrow eosinophil maturation, and decrease eosinophil progenitors in the bronchial mucosa. , though initial clinical trials of il- blockade in patients with asthma were unsuccessful in demonstrating clinical efficacy, , e a number of issues may have contributed to the failure of these studies, including lack of depletion of tissue eosinophils and their granule products; patient selection; and methodological problems. subsequent studies, conducted in a subgroup of asthmatic patients selected on the basis of having persistent eosinophilic asthma, have demonstrated that blocking il- with the humanized il- antibody, mepolizumab, has a steroidsparing effect, reduces exacerbations, and improves quality of life for asthma patients. , the anti-il- approach is troubled by the inability to completely abolish eosinophilia, consistent with murine observations (reviewed in matthaei et al. ), since it is hypothesized that if eosinophilia were solely controlled by il- then a more complete suppression of eosinophils would ensue from therapeutic anti-il- interventions. a propos this possibility, however, medi- is a humanized anti-il- ra monoclonal antibody that binds il- ra with high affinity and mediates cell lysis via antibody-directed cell-mediated cytotoxicity. as such, medi- kills all cells bearing il- ra, including eosinophil progenitors, and has been shown to eliminate eosinophils from the circulation of subjects with mild asthma. , this antibody is currently under investigation in other clinical models of asthma. that eosinophils can remain in the lung tissue of asthmatic subjects despite il- blockade suggests that additional signals promote eosinophil survival. indeed in il- -deficient mice, responses to gm-csf and il- are normal, despite absence of eosinophilia in the nasal mucosa and the bone marrow, significantly lower numbers of il- -responsive eo/b cfu and maturing cfu eosinophils, and reduced expression of il- ra on bone marrowderived cd þ cd þ progenitor cells. these results indicate that redundant cytokine mechanisms can compensate for il- deficiency and highlight the multifactorial nature of allergic inflammation, indicating that combined, as opposed to single-line, therapies may be more effective in the treatment of diseases such as asthma. one such therapy is currently being tested in clinical trials of allergic asthma. tpi-asm is a combination of two antisense oligonucleotides, one blocking translation of the il- /il- /gm-csf receptor common b chain, and the other blocking translation of ccr . as such, tpi-asm prevents expression of receptors for gm-csf, il- , il- , eotaxin- , and eotaxin- , and will thus hypothetically inhibit many of the signals shown to be crucial for eosinophilopoiesis, as well as eosinophil migration, activation and survival. following allergen challenge in mild atopic asthmatic subjects, inhaled tpi-asm inhibited accumulation of mature eosinophils and cd þ il- ra þ cells in the sputum, in addition to inhibiting the late asthmatic response. , despite advances made in the development of eosinophilspecific therapies, corticosteroids remain the gold standard for the treatment of allergic inflammatory diseases like asthma. e local delivery using inhalers is intended for topical treatment of the affected tissue; however, the antiinflammatory actions of corticosteroids have been shown to extend beyond the environment of the airways, probably due to a small amount of systemic availability. these systemic effects are beneficial for regulating hemopoietic mechanisms that originate in the bone marrow. stepwise withdrawal of inhaled corticosteroids results in a rapid and substantial increase in eo/b progenitors assayed in peripheral blood, which returns to baseline when treatment is reinstated. only week of treatment with inhaled corticosteroid is sufficient to significantly attenuate allergen-induced levels of circulating eo/b cfu and reduce baseline numbers of bone marrow eo/b cfu in mild allergic asthmatic subjects, further demonstrating the efficacy of corticosteroids on progenitors in peripheral blood. however, the inhaled steroid has no effect on the allergen-induced increase in the number of bone marrow cd þ cells, the increase in il- ra expression on these cells, or the number of eo/b cfu. these findings suggest that topical corticosteroids may exert indirect suppressive effects on the differentiation of eosinophil progenitors. eosinophil progenitors are now emerging as effector cells that can migrate to inflamed tissue where they rapidly proliferate in response to allergic stimuli. understanding communication between eosinophil progenitors and the innate immune system will require further exploration. asthma is a chronic pulmonary disease characterized by airway remodeling, airway inflammation, and bronchoconstriction. patients with asthma cycle between periods of exacerbations, which result in significant morbidity, and recovery. virus infections are the leading cause of asthma exacerbations in children and adults, yet specific treatment and prevention strategies for virus-induced asthma are limited. the tissue damage caused during virus-induced asthma is divided into two categories: damage caused by the virus itself and damage from the host immune response. eosinophils play a significant role in both virus clearance and immunity-mediated tissue damage. as many as % of childhood cases and % of adult cases of asthma attacks have an identifiable underlying virus infection. , diagnosis of virus infection in acute asthma is often presumptive and based on patient history and physical examination, but other diagnostic laboratory techniques include serology, virus culture, and reversetranscription polymerase chain reaction. respiratory rna viruses are the main types of virus that induce asthma attacks, with rhinovirus (the common cold virus) accounting for approximately two-thirds of viruses identified. coronavirus, influenza, parainfluenza, and respiratory syncytial virus (rsv) comprise the remainder of respiratory rna viruses that induce asthma attacks. the immune response to viral infections is t-helper type (t h )-driven and involves the production of proinflammatory cytokines such as interleukin- (il- ) and interferon g (ifn-g). studies show that t h responses in patients with asthma are impaired, which results in decreased t h cytokine production during viral infection. asthma also skews the immune system away from a t h response toward a t h immune environment through increased production of t h cytokines, such as il- , il- , and il- . il- is the central cytokine involved in eosinophil proliferation, maturation, and survival. when the immune environment of a virus-infected patient with asthma shifts from t h to a t h response, these patients experience more severe symptoms of infection and delayed pathogen clearance, which likely contribute to increased asthma exacerbations and hospital admissions. , the association of eosinophils with virus-induced asthma is well documented. eosinophil products are present in the sputum of patients with virus-induced asthma, and histology studies show that in patients who have died from severe asthma, eosinophils are clustered around airway nerves (fig. . . ) . in the following sections, we will present the mechanisms of eosinophil pathophysiology in virus-induced asthma, focusing on virus-induced airway inflammation, viral activation of eosinophils, virus-induced eosinophil-mediated neural changes, and t celleeosinophil interactions during virus infection. we then discuss the beneficial role of eosinophils in virus-induced asthma and potential targets for prevention and treatment of the disease. respiratory virus infection induces airway inflammation in all individuals, but the inflammatory response is different in patients with asthma from in nonasthmatic individuals. experimental rhinovirus infection of human subjects increases eosinophil numbers in the bronchial epithelium in both nonasthmatic and asthmatic volunteers. eosinophils remained in airway tissues of virus-infected patients with asthma weeks longer than in infected, nonasthmatic controls. these data suggest that virus-induced eosinophil influx into the airways occurs in individuals both with and without asthma and that the eosinophil response to viral infection is accentuated in patients with asthma. the airway epithelium is the primary site of respiratory virus infection. virus-infected epithelial cells release a wide array of proinflammatory cytokines that recruit inflammatory cells into the airways. among these proinflammatory cytokines are eotaxin/c-c motif chemokine (ccl ), granulocyte-macrophage colony-stimulating factor (gm-csf), mip- a/ccl , and rantes/ccl , which are chemoattractants and activators of eosinophils. e virus infection increased eosinophil inflammation in the airways of antigen-sensitized and antigen-challenged mice compared to sensitized-challenged, mock-infected animals. in addition, virus infection of epithelial macrophages induced the release of eotaxin, and the blockade of eotaxin bioactivity with a neutralizing antibody inhibited airway eosinophilia in sensitized-challenged, virus-infected but not sensitized-challenged, mock-infected mice. these data suggest that virus infection recruits eosinophils to the lungs of sensitized-challenged animals and that this is dependent on eotaxin. virus infections of allergic individuals may lead to the development of eosinophilic inflammation as well as features of asthma. calhoun et al. showed that patients with allergic rhinitis who were experimentally infected with rhinovirus had increased eosinophil influx into the airways following allergen challenge. these patients also had increased histamine release and edema from fluid leakage, which are consistent with asthma. these data suggest that virus infection may induce asthma in individuals with preexisting allergic respiratory diseases. the t h immune environment that allergic rhinitis elicits closely resembles the cytokine profiles of asthma, so a subsequent viral infection may tip airway physiology toward an asthma phenotype. eosinophils are activated during viral infection. studies of patients with rsv bronchiolitis showed the presence of eosinophil cationic protein (ecp) and eosinophil-derived neurotoxin (edn) in their lower airway secretions. , other studies have shown that cell surface expression of cd b, a marker of cellular activation, is upregulated in eosinophils of rsv-infected patients compared to uninfected volunteers. these data suggest that eosinophils are activated and degranulate following viral infection. toll-like receptors (tlrs) are pattern recognition receptors that recognize pathogen-associated molecular patterns (pamps). many groups have shown that tlrs are present and functional in eosinophils. tlrs mediate both the innate and adaptive immune responses, and some believe they are involved in controlling sensitization. studies suggest that polymorphisms in tlr , tlr , tlr , and tlr are associated with an increased risk of developing asthma. e human eosinophils express tlr , tlr , tlr , tlr , and tlr mrna. tlr recognizes single-stranded rna (ssrna) found in respiratory rna viruses. nagase et al. showed that r (a synthetic ligand for tlr and tlr ) increases cd b expression on human eosinophils, induces superoxide generation, and promotes cell survival. phipps et al. showed that mouse eosinophils express tlr on their cell surface and tlr , tlr , and tlr in endosomes. furthermore, ssrna treatment of mouse eosinophils increases eosinophil peroxidase (epo) release, increases cd b expression, and induces degranulation. eosinophil tlr responses to stimulation may differ between atopic and nonatopic individuals. mansson et al. showed that eosinophils treated with il- and then stimulated with polyi:c (a tlr agonist) had increased il- release compared to il- treatment alone. in addition, il- pretreatment potentiates r (a tlr -specific synthetic ligand)-induced il- release. moreover, eosinophils collected from atopic patients and stimulated with r release increased il- compared to r -stimulated eosinophils from nonallergic volunteers. these data suggest that atopy affects eosinophil tlr responses and that aberrant virus-induced eosinophil activation in patients with asthma may exacerbate their airway disease. eosinophils can be infected and activated by respiratory viruses. eosinophils can be activated directly by virus binding to receptors on either the cell surface or in endosomes, depending on the virus's mechanism of entry. rsv mediates entry into target cells by binding tlr . although it has not been demonstrated that rsv binds tlr on eosinophils, in vitro culture of rsv with human eosinophils prolongs cell survival. transmission electron microscopy studies showed that rsv is internalized by human eosinophils and identified virions in phagocytic vacuoles near the cell surface. in addition, infected eosinophils underwent piecemeal degranulation, suggesting that these cells were activated upon infection. in some cases, virus-induced eosinophil activation requires priming prior to infection and, in other cases, viruses prime eosinophils to respond to treatment with additional stimuli. handzel et al. showed that rhinovirus binds intracellular adhesion molecule- (icam- ) on human eosinophils primed with gm-csf and that gm-csf treatment upregulates icam- on eosinophils. additional studies showed that rsv increased superoxide generation and cd b in eosinophils that were first treated with platelet activating factor. rsv infection of eosinophils potentiates phorbol- -myristate- -acetate (pma)-induced superoxide generation and leukotriene c release. these data suggest that virus-induced eosinophil activation in asthma may lead to an exaggerated host immune response, resulting in airway inflammation and constriction. during virus-induced asthma, eosinophils cause dysfunction of parasympathetic nerve signaling. airway parasympathetic nerves provide the dominant control over airway smooth muscle. these neurons release acetylcholine (ach), which binds m muscarinic receptors (m rs) on smooth muscle to cause bronchoconstriction. ach release is normally limited via negative feedback inhibition of ach on neuronal m muscarinic receptors (m rs). when neuronal m rs are dysfunctional, ach release by neurons is not inhibited, and increased levels of ach binding to m rs on airway smooth muscle cause airway hyperresponsiveness (ahr). in antigen-sensitized, antigen-challenged guinea pigs, eosinophils mediate ahr. parasympathetic nerves recruit eosinophils to the nerves via eotaxin signaling. cholinergic nerves express icam- and vascular cell adhesion protein (vcam), which bind to eosinophils. eosinophils bind to nerves and release major basic protein (mbp), which blocks m rs on nerves. this causes loss of m r function, resulting in increased ach release and bronchoconstriction. , viral infection of nonsensitized guinea pigs causes the loss of m r function and ahr, but this loss is not eosinophil-mediated (not prevented by antibody to il or antibody to mbp). in nonsensitized guinea pigs, the virus-induced loss of m r function appears to be on the level of reduced m r gene expression. this appears to be the result of production of tnf-a, which decreases m r mrna stability. in contrast, in sensitized guinea pigs, virusinduced loss of m r function and ahr is mediated by eosinophils. adamko et al. showed that depletion of eosinophils with an anti-il- antibody prevents virus-induced ahr in sensitized guinea pigs, but not in nonsensitized, virus-infected animals. additional studies showed that blockade of mbp bioactivity inhibits virus-induced ahr and m r dysfunction in sensitized guinea pigs. collectively, these data suggest that in the context of asthma, eosinophils are recruited to airway nerves, virus infection activates the eosinophils to release mbp, and mbp antagonizes m rs, resulting in bronchoconstriction (fig. . . ). in addition to eosinophilenerve interactions, eosinophils communicate with t cells in virus-induced asthma. as mentioned above, eosinophils mediate virus-induced ahr and m r dysfunction in sensitized guinea pigs but not in nonsensitized animals. adamko et al. demonstrated that depletion of cd þ t cells prevents virus-induced ahr and m r dysfunction in sensitized but not in nonsensitized guinea pigs. sensitization increases the number of eosinophils in the airways, and virus infection of both nonsensitized and sensitized guinea pigs reduces the total number of eosinophils in the airways and eosinophils associated with nerves, suggesting that virus infection induces eosinophil degranulation. cd þ t cell depletion inhibits virus-induced eosinophil cytolysis, suggesting that cd þ t cells promote virus-induced eosinophil degranulation. these data suggest that cd þ t cells interact with eosinophils in the airways of virus-infected, sensitized animals to promote ahr and m r dysfunction. schwarze et al. showed that cd þ t cells are also necessary for ahr and lung eosinophilia during rsv infection of mice. in vitro studies have shown that eosinophils directly interact with t cells to induce eosinophil degranulation and promote antiviral, t cell-mediated immunity. when human eosinophils are incubated with parainfluenza virus in the presence of t cells and antigen-presenting cells (apcs; macrophages or dendritic cells), they release epo. uvinactivated virus also induces epo release from eosinophils, suggesting that this process occurs in the absence of viral replication. additional studies showed that rsv is also able to induce epo release from eosinophils in the presence of t cells and apcs. handzel et al. showed that eosinophils present rhinovirus antigens to cd þ t cells, inducing their clonal expansion and the release of ifn-g. these data suggest that eosinophils directly interact with cd þ and cd þ t cells, and that their interactions result in eosinophil degranulation, which may augment symptoms of asthma as well as initiate antiviral adaptive immunity. despite the detrimental role eosinophils play in the pathophysiology of asthma, the antiviral effect of eosinophils may also be beneficial in the context of virus infection and asthma. the antibacterial and antiparasitic effects of eosinophils are well documented, yet their antiviral properties have been studied only recently. while eosinophils have historically been acknowledged for their pathophysiological role in asthma, recent studies suggest that these cells also play a beneficial antiviral role in virus-induced asthma. adamko et al. showed that guinea pigs sensitized to ovalbumin had reduced parainfluenza titers in their lungs days following infection compared to nonsensitized, virus-infected animals. furthermore, depletion of eosinophils with an anti-il- antibody increased viral levels in sensitized virus-infected animals to above those observed in nonsensitized infected animals. these data suggest that eosinophils serve an antiviral role in the context of virus infection and allergy. additional studies have investigated the mechanisms by which eosinophils promote virus clearance in vivo. phipps et al. showed that il- transgenic mice, which have increased levels of eosinophils, have improved rsv clearance compared to wild-type rsv-infected mice. myd is the adaptor molecule for many tlr signaling pathways, and studies show that adoptive transfer of wildtype eosinophils, but not eosinophils deficient in myd , into the lungs of infected wild-type mice improves virus clearance and inhibits ahr. blockade of nitric oxide synthase (nos- ; inducible nitric oxide synthase) activity inhibits rsv clearance in vivo. these data suggest that eosinophils contribute to rsv clearance in both a myd -dependent and nos-dependent manner. furthermore, these data suggest that eosinophils may aid the prevention of rsv-induced ahr. as mentioned above, eosinophils can be activated by viruses to degranulate. upon degranulation, eosinophils release granule proteins and reactive oxygen species, which can cause damage to pathogen moieties. several groups have shown that eosinophils increase superoxide generation in response to virus exposure. , klebanoff et al. showed that human eosinophils stimulated with pma are virucidal against human immunodeficiency virus (hiv) type in vitro and that purified epo, when incubated with hydrogen peroxide and a halide (its substrate), is also virucidal against hiv. others have demonstrated an antiviral effect of edn against hiv in vitro. , collectively, these data suggest that eosinophils inactivate rna viruses by a number of different mechanisms involving granule proteins and reactive oxygen species. figure . . viral infection activates airway eosinophils in a cd d t cell-dependent process. activated eosinophils release major basic protein, which binds to inhibitory m muscarinic receptors on parasympathetic nerves. blocking these receptors increases acetylcholine (ach) release, causing bronchoconstriction. m , m muscarinic receptor. while some studies have shown that eosinophils destroy virus, other groups have shown that eosinophils are productively infected by viruses. as mentioned previously, eosinophils can bind and internalize viruses. kimpen et al. showed that rsv can bind to the eosinophil membrane and observed virus in the phagocytic vacuoles of eosinophils. , dyer et al. showed that rsv and mouse pneumovirus productively infect human and mouse eosinophils, respectively, resulting in the release of infectious virions and proinflammatory cytokines in vitro. in contrast, our laboratory has presented evidence suggesting that parainfluenza virus abortively infects human eosinophils. parainfluenza virus can enter eosinophils and replicate the viral rna genome, but infectious virus particles are not released. a productive infection of eosinophils could potentially serve to induce cytokine release and attract other immune cells, while an abortive infection would remove extracellular virus from the local environment and/or promote the presentation of virus antigens to t cells. collectively these data suggest that interactions between eosinophils and different types of viruses are varied and complex. standard treatments for patients suffering from virusinduced asthma exacerbations consist of b-agonists in combination with anticholinergics and steroids. b-agonists relax smooth muscle by stimulating adenylate cyclase activity and closing calcium channels, while anticholinergics block the effects of the neurotransmitter ach on airway smooth muscle constriction. viruses and interferon downregulate inhibitory m rs on parasympathetic nerves, thereby increasing ach release, and steroids can reverse these effects. steroids can also reduce eosinophil influx into the lungs, icam- expression on nerves, and neuronal m r dysfunction. , while eosinophil in sensitized animals can prevent virus-induced hyperreactivity, the effects of eosinophil depletion in human virus-induced asthma attacks are less clear. while eosinophil depletion with anti-il has limited effects on the response to antigen challenge in human subjects and a clinical study of anti-il had unimpressive effects in unselected asthmatics, when asthmatics with sputum eosinophilia were treated with anti-il , steroids were withdrawn without exacerbation in the majority of cases. whether eosinophil depletion can prevent virusinduced asthma exacerbation awaits further studies. another potential treatment for virus-induced asthma is the correction of deregulated cytokine production. as mentioned previously, the immune environment of patients with asthma is skewed toward a t h phenotype, with decreased interferon production. treatments that promote a t h phenotype, the immune response typically observed in viral infection, may prove clinically valuable. the most direct method of treatment or prevention of virus-induced asthma is targeting the viral infection itself. however, such treatments are at present limited. treatment and prevention of rhinovirus, the cause of twothirds of virus-induced asthma exacerbations, presents a significant challenge because over serotypes of the virus exist. the varying antigenicity among serotypes further hinders vaccine design. as mentioned previously, rhinovirus uses icam- binding for entry into cells, thus the blockade of cellular infection by blocking virus binding to target cells presents one promising mechanism for drug design. while ribavirin, which is believed to interfere with viral rna synthesis, has activity against a range of viruses, including rhinovirus and rsv, its clinical efficacy is limited and it is not widely used in the treatment of rhinovirus. rsv vaccine development has been problematic, with paradoxical worsening of the clinical response being an issue. palivizumab is a monoclonal antibody that prevents rsv infection by interfering with the rsv fusion protein and, thus, viral entry into cells. in addition, studies show that type iv phosphodiesterase inhibitors inhibit rsvinduced ahr and eosinophilia in the lungs. effective vaccines for influenza are recommended for patients with airway disease, including asthma. although it is difficult to prove that influenza-induced asthma attacks are reduced by vaccination, this may be due to the relatively small number of asthma attacks caused by influenza. treatment for influenza viral infections consists of neuraminidase inhibitors. again, the role of neuraminidase inhibitors in preventing asthma attacks is difficult to demonstrate. currently no treatments or vaccines exist for parainfluenza virus and coronavirus. improved immunological understanding of these viruses and of virus interactions with the host, and eosinophils specifically, will aid in the advancement of future vaccines and treatments. respiratory virus infections are the primary cause of asthma exacerbations in children and adults. in asthma, eosinophils are recruited to the airways as a part of the t h immune response, where they contribute to asthma pathophysiology. however, eosinophils may play a dual role in virus-induced asthma; on the one hand recognizing viruses, releasing virucidal mediators, and presenting antigens, and on the other hand becoming activated and increasing airway reactivity. current treatments for virus-induced asthma are not specific to virus infection and instead focus on smooth muscle relaxation and reduction of airway inflammation. further investigation of the interactions between eosinophils and viruses is warranted and will likely lead to more targeted treatments and prevention. primary eosinophilic gastrointestinal disorders (egid) are diseases with eosinophilia in the absence of known causes (e.g., drug reactions, malignancy, and parasitic infections). these disorders include eosinophilic colitis, eosinophilic enteritis, eosinophilic esophagitis (eoe), eosinophilic gastritis, and eosinophilic gastroenteritis, a term used when more than one gastrointestinal (gi) segment is involved. , the symptoms of egid include abdominal pain, diarrhea, dysphagia and food impaction, failure to thrive, gastric dysmotility, irritability, and vomiting. both genetic and environmental factors have a role in egid. approximately % of patients with egid have a first-degree relative that also has egid. it appears that allergy may play a role. in fact, approximately % of egid patients have atopy, allergen-free diets have been shown to be therapeutic, and tissue specimens show mast cell degranulation. animal studies of egid also provide evidence of allergic etiology. although food-specific immunoglobulin e (ige) is common in egid patients, food-induced anaphylactic responses occur in only a minority of patients. thus, egid appears to fall between pure ige-mediated food allergy and cellularmediated hypersensitivity disorders ( fig. . . ). in fact, a recent study shows a higher incidence of eoe in patients with celiac disease, supporting a cell-mediated hypersensitivity or t-helper type (t h )-mediated mechanism, although another recent study demonstrates a lack of celiac disease-associated alleles in eoe. the chief differential diagnoses include gastroesophageal reflux disease (gerd), inflammatory bowel disease (ibd), and specific infections including parasites and helicobacter pylori. , the prevalence of egid has not been rigorously calculated, but they appear to be widespread and not uncommon. for example, eoe has been reported in australia, brazil, england, italy, japan, spain, and switzerland. in one study approximately % of pediatric patients with gerd-like symptoms who were unresponsive to acid blockade had eoe. another study reported that % of children with chronic esophagitis have eoe. prevalence estimates vary from : , adults in australia to : children in cincinnati, usa. the increased prevalence of the disease is primarily due to increased recognition, as the disease accounted for approximately % of refractory chronic esophagitis in the s and s; however, a bona fide increase in disease incidence is also occurring. it appears that egid may be even more prevalent than pediatric ibd. the blood eosinophil level in eoe patients is typically not dramatically elevated, although it averages twice the normal value. the relative normal value of blood eosinophilia compared with esophageal eosinophilia highlights the tissue-specific pathogenesis of the disease. however, some egid patients have peripheral eosinophilia high enough to meet criteria for hypereosinophilic syndrome (hes), defined by sustained peripheral blood eosinophilia (> cells/mm ) and end-organ involvement in the absence of known causes of eosinophilia. , while hes often involves the gi tract, the other organs typically affected in hes (heart and skin) are rarely involved in egid. in most tissues, eosinophils are present in minute amounts. organs with substantial eosinophils include gi tract, lymph nodes, spleen, and thymus. interestingly, in a large study of healthy patients, on autopsy eosinophil degranulation was only seen in the gi tract. eosinophils are normally present in the lamina propria of the colon, small intestine, and stomach, but are not normally present in the epithelium or peyer patches, although they infiltrate these regions in egid. eosinophil homing to the gi tract appears to occur independent of gut flora, as evidenced by prenatal, adult, and germ-free mice having eosinophils in similar locations and concentrations. additionally, mice deficient in innate signaling responses (i.e., myd deficient) have normal numbers of gi eosinophils. together these data indicate that eosinophils respond to signals distinct from those of most other intestinal inflammatory cells that typically require gut flora-induced signaling. in fact, eotaxin/c-c motif chemokine (ccl ) is the unique signal that induces localization of eosinophils to the gi tract. in vitro, the granule components of eosinophils are toxic to many tissues, including intestinal epithelium. the eosinophil cationic proteins major basic protein (mbp), eosinophil peroxidase (epo), and eosinophil cationic protein (ecp) are cytotoxic to epithelium at concentrations similar to those found in biological fluids from patients with eosinophilia. ecp can render cell membranes porous. mbp increases smooth muscle reactivity via vagal muscarinic m receptors and can trigger mast cell and basophil degranulation. in patients with eosinophilic gastroenteritis, mbp and ecp are deposited extracellularly in the small bowel, and ultrastructural changes in eosinophil secondary granules (indicating degranulation and mediator release) are found in duodenal biopsies. furthermore, charcoteleyden crystals are commonly found in feces, and disease severity correlates with mucosal eosinophil numbers. eosinophils can secrete a number of different cytokines, suggesting that they may modulate multiple aspects of the immune response, as well as epithelial growth, fibrosis, and tissue remodeling. in addition, tissue eosinophils have distinct cytokine expression patterns under inflammatory versus noninflammatory conditions. for example, esophageal eosinophils from eoe patients express high levels of t h cytokines and transforming growth factor b (tgf-b). other molecules secreted by eosinophils include halide acids, hydrogen peroxide, and leukotrienes, which increase vascular permeability and mucus secretion and stimulate smooth muscle contraction. in ibd, eosinophils form only a small percentage of the infiltrating leukocytes, but their level has been proposed to be a negative prognostic indicator. observations of eosinophilia in ibd suggest that eosinophils mediate axonal necrosis. recently, it has been shown that eotaxin is upregulated in intestinal macrophages and epithelial cells in pediatric ulcerative colitis and thus may be a future target for therapy. common symptoms of egid patients include abdominal pain, diarrhea, dysphagia, failure to thrive, gastric dysmotility, hypoproteinemia, irritability, microcytic anemia, and vomiting. patients with these refractory problems, especially individuals with a strong history of allergic diseases, peripheral blood eosinophilia, and/or a family history of egid, should be evaluated for egid. evaluation for egid starts with a comprehensive history and physical examination. symptoms vary depending on the intestinal segment involved (e.g., abdominal pain and dysphagia are most common in eosinophilic gastroenteritis and eoe, respectively). in most egid patients, peripheral eosinophilia is not present. total ige levels may help classify patients with atopy or those with occult parasite infection. in atopic egid patients, food allergy is common and can be verified by skin prick and skin patch testing. there are no pathognomonic symptoms or blood tests for diagnosing egid. therefore, the diagnosis of egid is based on endoscopic biopsy procurement and appropriate clinical information. the diagnosis of egid is dependent on histological evaluation of biopsy samples, contingent upon the quantity, location, and characteristics of the eosinophilic inflammation. egid patients often have a variety of endoscopic findings, but it is not uncommon for the lumen to look normal endoscopically; thus, a microscopic evaluation of biopsy samples is required. egid often has patchy involvement, requiring analysis of biopsies from multiple intestinal segments. while the normal esophagus is devoid of eosinophils, the rest of the gi tract contains readily detectable eosinophils. with the lack of diagnostic criteria, diagnosis of egid depends on clinical and histopathological expertise. diagnosis of egid depends on several factors including the following: ( ) eosinophil quantification, ( ) the histological location of eosinophils, ( ) associated histopathological abnormalities, and ( ) the absence of pathological features suggestive of other primary disorders. recently, gene expression profiles (transcriptomes) have been proposed to be helpful for the diagnosis of eoe; elevated levels of esophageal eotaxin- /ccl in a single biopsy specimen has approximately % sensitivity. other disease processes, such as drug hypersensitivity, collagen vascular disease, malignancy, or infection, should be ruled out (table . . ). evaluation for intestinal parasites via stool samples, colonoscopy aspirates, or antibody titers should be performed, especially if patients have high-risk exposure (e.g., drinking well water, living on a farm, or travel to endemic area). in one study of eosinophilic enteritis, the dog hookworm ancylostoma caninum was identified as the cause in % of cases. infection with strongyloides stercoralis should be excluded before treating for egid, as immunosuppressants can be life threatening with this infection. the esophagus is normally devoid of eosinophils. , disorders associated with esophageal eosinophil infiltration include allergic vasculitis, bullous pemphigoid, carcinomatosis, drug injury, eoe, eosinophilic gastroenteritis, esophageal leiomyomatosis, gerd, hes, ibd, myeloproliferative disorders, parasitic and fungal infections, periarteritis, pemphigus vegetans, recurrent vomiting, and scleroderma. , eosinophil-associated esophageal disorders are classified as primary or secondary (i.e., due to another known disease process) (table . . ). the primary subtype includes the atopic, nonatopic, and familial variants, and the secondary subtype is subdivided into those with and without systemic eosinophilia. eoe is familial in about % of patients. the etiology of eoe is poorly understood, but food allergy has been implicated. most eoe patients have specific ige to foods and aeroallergens, but only a few have experienced anaphylaxis. esophageal eosinophilia may also be linked to allergic airway disease. several independent studies have linked eoe to allergic etiology. for example, repeated delivery of allergens or interleukin- (il- ) to murine lungs (via direct delivery or transgenic overexpression) induces experimental eoe; patients with allergic rhinitis have increased esophageal eosinophils; intranasal delivery of indoor insect allergen induces eoe in mice; patients with eoe commonly report seasonal variations in symptoms; and there is a seasonal variation in eoe diagnosis. , in addition to eosinophils, t cell and mast cell numbers are increased in esophageal biopsies, suggesting a chronic t h -associated inflammation (fig. . . ) . mast cells have been shown to be elevated in the esophagus and to degranulate in eoe, and they appear to be stimulated by the kit ligand. carboxypeptidase a and tryptase appear to be good surrogate markers for mast cell involvement. is overproduced in the esophagus of eoe patients. in addition, in experimental systems, such as il- lung transgenic systems, il- induces eosinophilic esophagitis and tissue remodeling with features both dependent and independent of eosinophils. consistent with t h activation, il- overexpression induces eoe, and il- neutralization completely blocks allergen-or il- -induced eoe in mice. however, anti-il- therapy in humans has not yet been shown to be effective at ameliorating clinical aspects of the disease, although esophageal eosinophilia improves. , interestingly, a cytokine with more of a t h skew, il- , appears to mediate cd þ t cells and to be involved in the pathogenesis of eoe. in a recent genome-wide microarray expression profile analysis of esophageal tissue from patients with eoe and normal controls, an eoe transcriptome was found to contain changed expression of approximately % of the entire human genome. interestingly, eotaxin- was the most prominently overexpressed gene in eoe patients, and levels correlated with disease severity. furthermore, a single nucleotide polymorphism (snp) in eotaxin- was overrepresented in eoe patients. conversely, mice lacking the eotaxin receptor (c-c chemokine receptor type ; ccr ) were protected from developing experimental eoe. notably, eotaxin- is induced by il- . two recent studies have implicated genetic susceptibility for eoe with genetic variants of the tslp gene; tslp encodes thymic stromal lymphopoietin, an epithelial gene product that targets dendritic cells and promotes their t h polarizing ability. these studies included a genome-wide analysis study of common variants, as well as a largescale candidate snp approach. notably, sex-associated tslp receptor polymorphisms on xp . /yp . may explain male predisposition to eoe. symptoms of eoe include dysphagia, epigastric or chest pain, gi obstructive problems, and vomiting. patients are predominantly male and have robust esophageal eosinophilia, extensive epithelial hyperplasia, and are commonly atopic compared to gerd patients. in adults with eoe, dysphagia and food impaction are common complaints. one study presented a symptom scoring tool that can help identify patients with eoe and correlates with tissue inflammation. however, another study demonstrated dissociation between symptom scores and histology in treated eoe patients. at present, it is important to develop validated disease instruments so that these important parameters can be tracked, especially in clinical trials. eoe is associated with esophageal dysmotility/ dysphagia, which may be related to motor dysfunction of the esophagus rather than to physical narrowing. esophageal ultrasound shows dysfunctional muscularis mucosa in eoe. radiographic and endoscopic studies demonstrate furrowing, mucosal rings, polyps, strictures, ulcerations, and whitish papules. it appears that a small diameter on barium esophagography can help diagnosis of eoe, although many patients have normal barium assessments and need further evaluation by endoscopy. recently, it has been demonstrated that esophageal distensibility, defined as cross-sectional area following intraluminal pressure, is significantly reduced in eoe patients. the number and location of eosinophils helps distinguish eoe from gerd. greater than eosinophils per high-power field (hpf) and a lack of response to protonpump inhibitors suggest eoe. proximal and distal esophageal eosinophilia suggests eoe, whereas eosinophilia confined to the distal esophagus suggests gerd. histopathological changes in eoe include esophageal mucosa thickening with basal layer hyperplasia and papillary lengthening. tissue eosinophil counts may underestimate eosinophil involvement, particularly with marked degranulation. eosinophil-derived neurotoxin (edn) staining of biopsy specimens may be useful for diagnosis and management. clinical assessment of eoe includes analysis of food and aeroallergen sensitization and exclusion of gerd as well as other causes of eosinophils in the esophagus. however, eoe and gerd are not mutually exclusive and may coexist in the same patient ( the inflammatory response in eoe. food antigens trigger t-helper type (t h ) cells to release interleukin- (il- ) and il- , which stimulate eosinophils and esophageal epithelial cells, respectively. il- induces epithelial cells to produce eotaxin- /c-c motif chemokine (ccl ) and downregulate filaggrin. reduced production of filaggrin might inhibit esophageal barrier function, which could perpetuate the inflammation by maintaining local food antigen uptake. il- and eotaxin- activate eosinophils to release major basic protein (mbp) and eosinophil-derived neurotoxin (edn), which activate mast cells and dendritic cells, respectively. eosinophils and mast cells also produce tgf-b, which activates fibroblasts and muscle cells and contributes to dysmotility, fibrosis, and hyperplasia. , ecp, eosinophil cationic protein; epo, eosinophil peroxidase; tgf-b, transforming growth factor b. specific food antigen and aeroallergen avoidance, identified by skin testing or history, is indicated for patients with atopic eoe. if feasible, an elemental diet is recommended, as it improves symptoms and reduces the number of eosinophils in the esophageal biopsies of patients with eoe (allergic or nonallergic subtypes). elemental diet therapy frequently requires placement of a gastrostomy tube to achieve adequate caloric intake. glucocorticoids are also effective. systemic steroids are used for acute exacerbations, and topical steroids provide day-to-day control. for topical steroid delivery, the patient is instructed to swallow the dose to promote deposition on the esophageal mucosa. topical fluticasone lowers the level of eosinophils, cd þ cells, and cd þ cells in the proximal and distal esophagus and improves symptoms. side effects of inhaled glucocorticoids are less likely with swallowed fluticasone, as this drug undergoes extensive first-pass metabolism in the liver. however, local esophageal candidiasis may occur. in addition to inhaled steroids, an oral suspension of budesonide can be used. viscous budesonide improves symptoms and endoscopic/histological appearance and also appears to reach the distal esophagus. a recent study shows fk binding protein (fkbp ) transcript levels increase with glucocorticoid exposure, thus helping to distinguish responders from nonresponders and treated from untreated. this also provides the best evidence that swallowed steroids induce local effects in the esophagus. although food antigen avoidance and glucocorticoids are the mainstay of treatment, additional therapies may be beneficial. in eoe patients for whom impaction has become an issue, esophageal dilation appears to be safe and effective. , even if gerd is not present, neutralizing gastric acidity (with proton-pump inhibitors) may improve symptoms and esophageal pathology. on the horizon, an anti-il- antibody is a promising future therapy. in an animal model of il- -induced esophageal eosinophilia, an anti-il- antibody was effective, and clinical studies are now under way. additionally, anti-il- antibodies prevent experimental eoe in mice , and appear to decrease eosinophilia of the human esophagus in earlystage clinical trials. human clinical trials are currently under way. although the natural history of eoe is not fully known, it is not uncommon for children with eoe to have a parent with a history of chronic esophageal strictures. esophageal biopsies in some of these parents have revealed eoe. the following symptoms occur in order of increasing age: feeding problems, vomiting, abdominal pain, dysphagia, and food impaction. thus, if left untreated, eoe is likely to progress to esophageal scarring and dysfunction. recent data show that pediatric eoe patients, diagnosed by retrospective biopsy analysis, are at increased risk of developing persistent disease characterized by dysphagia, food impaction, a need for esophageal dilation, and food allergy. , despite this, the development of barrett esophagus in eoe has not been studied. however, it appears that eoe requires chronic treatment. having eoe increases the risk of developing other forms of egid. thus, routine surveillance guided by symptoms of the entire gi tract by endoscopy is recommended. at baseline, the stomach and intestine have readily detectable eosinophils. therefore, diagnosis of enteritis, eosinophilic gastritis, and gastroenteritis is more complex than diagnosis of eoe. these diseases are characterized by selective infiltration of eosinophils in the stomach and/or small intestine with variable involvement of the esophagus and/or large intestine. eosinophilic enteritis, gastritis, and gastroenteritis can be divided into primary or secondary. the primary disorders have also been called idiopathic or allergic gastroenteropathy. primary eosinophilic gastroenteritis is subdivided on the basis of the level of histological involvement into mucosal, muscularis, and serosal forms. endoscopic biopsy can be normal in the latter two subtypes. importantly, many other disorders feature eosinophil infiltration in the stomach, including allergic vasculitis, drug hypersensitivity, drug injury, hes, ibd, myeloproliferative disorders, parasitic and bacterial infections (e.g., h. pylori), periarteritis, and scleroderma. because total ige is elevated and food-specific iges are detectable in most egid patients, an allergic mechanism is suspected. however, even though most patients have positive skin tests to a variety of food antigens, they do not have typical anaphylactic reactions, suggesting a delayed form of food hypersensitivity syndrome. in support of an allergic mechanism, mast cells are increased in egid. also, eosinophilic gastroenteritis can be induced by feeding enteric-coated allergen beads to sensitized mice. these mice develop eosinophil-associated gi dysfunction, including delayed food transit, gastromegaly, and weight loss. in addition, duodenal lamina propria t cells in egid preferentially secrete t h cytokines (especially il- ) when stimulated with milk proteins. furthermore, elevated secretion of il- and il- by peripheral blood t cells has been observed in eosinophilic gastroenteritis. iga deficiency can also be associated with eosinophilic gastroenteritis and may be related to the increased rate of atopy or to occult gi infection in these patients. in general, these disorders present with symptoms related to the degree and area of the gi tract affected. the mucosal form of eosinophilic gastroenteritis (the most common variant) is characterized by abdominal pain, bloody stools, diarrhea, failure to thrive, iron-deficiency anemia, malabsorption, protein-losing enteropathy, and vomiting. in the muscularis form, thickening of the bowel wall may result in gi obstructive symptoms. the serosal form is characterized by exudative ascites. there are no standards for the diagnosis of eosinophilic gastritis or gastroenteritis, but the presence of increased eosinophils in biopsy specimens, infiltration of eosinophils within intestinal crypts and gastric glands, lack of involvement of other organs, and exclusion of secondary causes of eosinophilia support a diagnosis of eosinophilic gastroenteritis. patients with eosinophilic gastritis may have micronodules (and/or polyposis), and these lesions often contain aggregates of lymphocytes and eosinophils. food allergy and peripheral eosinophilia may be present but are not required for diagnosis. eliminating foods implicated by skin prick or radioallergosorbent (rast) testing has variable results, whereas complete resolution is generally achieved with amino acidbased elemental diets. once remission has been achieved by dietary modification, specific food groups are reintroduced (at approximately -week intervals for each food group), and endoscopy is reperformed to identify sustained remission or disease flares. systemic or topical steroids are the main therapy when diet restriction has failed or is not feasible. for systemic steroid therapy, a course of e weeks of therapy with relatively low doses seems to work better than a -day course of glucocorticoid bursts. various topical glucocorticoid preparations are designed to deliver drugs to specific segments of the gi tract [e.g., budesonide tablets (entocort ec) targeted to the ileum and proximal colon]. as with asthma, topical steroids have a better riskebenefit ratio than systemic steroids. in cases refractory to or dependent on glucocorticoid therapy, parenteral nutrition or antimetabolite therapy (azathioprine or -mercaptopurine) may help. drugs such as cromoglycate, ketotifen, mycophenolate mofetil (an inosine monophosphate dehydrogenase inhibitor), suplatast tosilate, and various alternative therapies are not generally very useful, although successful long-term remission of eosinophilic gastroenteritis has been reported following montelukast treatment. use of proton-pump inhibitors can improve symptoms and the degree of esophageal and gastric pathology, even if gerd is not present. the natural history of eosinophilic gastritis, enteritis, and gastroenteritis is not well documented; however, they are often chronic, relapsing/remitting diseases. when the disease presents in infancy and specific food sensitization can be identified, remission is likely by late childhood. in food antigen-induced disease, abnormal levels of circulating ige and eosinophils often serve as markers for tissue involvement/relapse. as noted earlier, due to concern for hes, routine surveillance of the cardiopulmonary system is recommended. colonic eosinophilia occurs in a variety of disorders, including allergic colitis of infancy, drug reactions, eosinophilic gastroenteritis, infections (e.g., helminths), ibd, and vasculitis (e.g., churgestrauss syndrome). allergic colitis in infancy (or dietary protein-induced proctocolitis of infancy syndrome) is the most common cause of bloody stools in the first year of life. similar to other egid, these disorders are classified into primary and secondary. in contrast to other egid, eosinophilic colitis is usually not ige associated. some studies point to a t cell-mediated process, though the exact mechanism is unclear. allergic colitis of infancy may be an early expression of proteininduced enteropathy. cow's milk and soy proteins are the foods most frequently associated, but other food proteins can also induce this disorder. interestingly, this condition may more commonly occur in infants who are exclusively breastfed and can occur in infants fed with protein hydrolysate formulas. an association between allergic colitis and the later development of ibd has been reported but remains controversial. similar to eosinophilic gastroenteritis, the symptoms of eosinophilic colitis vary depending on the degree and location of tissue involvement. although diarrhea is a classic symptom, symptoms that can occur independent of diarrhea commonly include abdominal pain, anorexia, and weight loss. in infants, bloody diarrhea precedes diagnosis by several weeks, and anemia due to blood loss is not uncommon. most infants affected do not have constitutional symptoms and are otherwise healthy. there is a bimodal age distribution, with the infantile form presenting with a mean age at diagnosis of approximately days, and the second group presenting during adolescence and early adulthood. there is no single gold standard diagnostic test, but peripheral blood eosinophilia or eosinophils in the stool are suggestive of eosinophilic colitis. on endoscopic examination, patchy erythema, loss of vascularity and lymphonodular hyperplasia are seen; findings are mostly localized to the rectum but can affect the entire colon. histological examination often reveals preservation of mucosal architecture, with focal aggregates of eosinophils in the crypt epithelium, lamina propria, and muscularis mucosa and occasionally, multinucleated giant cells in the submucosa. treatment of eosinophilic colitis varies according to the disease subtype. clinical symptoms of eosinophilic colitis of infancy resolve within hours of withdrawal of the offending protein. treatment of eosinophilic colitis in older patients, for which ige-associated triggers are rarely identified, usually requires medical management. antiinflammatory drugs, including aminosalicylates and systemic or topical steroids, are commonly used and appear to be efficacious, but clinical trials have not been conducted. several forms of rectally delivered glucocorticoids treat the distal colon, though eosinophilic colitis typically also involves the proximal colon. in cases refractory or dependent on systemic glucocorticoid therapy, parenteral nutrition or antimetabolite therapy (azathioprine or -mercaptopurine) are alternatives. eosinophilic colitis presenting in the first year of life has a very good prognosis with the vast majority of patients able to tolerate the culprit food(s) by e years of age. the prognosis for eosinophilic colitis developing in adolescence or adulthood is less favorable. as with eosinophilic gastroenteritis, the natural history has not been studied, and this disease is considered to be a chronic relapsing/remitting disorder. because eosinophilic colitis can often be a manifestation of other disease processes, ruling out autoimmune disease and other secondary causes of eosinophilia is recommended. the term hes describes patients with systemic symptoms due to marked eosinophilia. diagnostic criteria for hes include persistent eosinophilia of at least cells/mm for a sustained duration; lack of known causes of eosinophilia; and symptoms and signs of organ system involvement. patients with egid and blood eosinophil counts greater than /mm meet these diagnostic criteria but generally do not have the high risk of life-threatening complications associated with classic hes (i.e., cardiomyopathy or central nervous system involvement). as is true in any patient with prolonged and marked eosinophilia, hes patients are prone to develop eosinophilic endomyocardial disease with embolization. thus, routine echocardiograms are warranted in patients with egid and peripheral blood eosinophilia. additionally, the diagnosis of hes should be considered in patients with egid who develop extra-gi manifestations. additional testing may include bone marrow analysis (searching for myelodysplasia), serum tryptase and vitamin b levels (both moderately elevated in classic hes), and genetic analysis for the fip -like /platelet-derived growth factor receptor-a (fip l epdgfra) fusion event. a major advance in our understanding of hes has come about through treatment of hes with imatinib mesylate, a tyrosine kinase inhibitor. in many hes patients, treatment with imatinib mesylate dramatically reduces peripheral blood and bone marrow eosinophils, suggesting that certain hes patients express a kinase that is sensitive to imatinib mesylate. a fusion gene, the result of an -kb deletion in chromosome , yields a novel activated kinase (fip l epdgfr). fip l epdgfr is inhibited by imatinib in vitro, explaining the sensitivity of hes patients to imatinib. those responsive to imatinib are typically e -year-old males who present with marked peripheral eosinophilia (i.e., classic hes). these patients have been shown to meet minor criteria for systemic mastocytosis, having elevated levels of serum mast cell tryptase and high numbers of dysplastic mast cells in the bone marrow. egid are now being recognized more frequently. they have strong genetic and allergic components and share clinical and immunopathogenic features with asthma. egid are associated with a variety of nonspecific common gi symptoms and laboratory findings, making their diagnosis dependent on microscopic examination biopsies and ruling out of other eosinophil-associated disease. eosinophils have potent proinflammatory effects mediated by their cytotoxic granule constituents and various lipid mediators and cytokines. in t h -associated gi inflammatory conditions, eosinophilia in the lamina propria is dependent on il- and eotaxin. esophageal eosinophilia can be induced experimentally by pulmonary deposition of aeroallergens or the t h cytokine, il- . many new therapeutic approaches are now being developed for egid, including eotaxin- receptor/ccr antagonists, eotaxin- blockers, humanized anti-il- , and il- /il- inhibitors. however, although much progress has been made concerning gi eosinophils and egid, there is still a paucity of knowledge compared with other cell types and gi diseases that may be even less common (e.g., ibd). a better understanding of the pathogenesis and treatment of egid will emerge by combining comprehensive clinical and research approaches involving experts in the fields of allergy, gastroenterology, nutrition, and pathology. although case reports describing eosinophilic infiltration of tissues accompanied by pronounced peripheral eosinophilia have existed since the s, the concept of a hypereosinophilic syndrome (hes) was not introduced until in a landmark paper by chusid and colleagues. the original description of hes was based largely on clinical findings highlighting the heterogeneous presentations of hes and proposed the following diagnostic criteria: blood eosinophilia > /mm for at least months (or death before months with signs and symptoms of eosinophilic disease); lack of evidence for allergic, infectious, parasitic, or other known causes of eosinophilia; and presumptive signs of organ involvement. since that time, the evolution of diagnostic testing has led to the identification of several hes variants with defined etiologies. furthermore, the availability of novel targeted therapies precludes waiting months for a diagnosis. as a result, several new conflicting classification schemes have been proposed. the world health organization (who) classification specifically distinguishes between hes and eosinophilic disorders with proven clonal populations of myeloid or lymphoid origin. although correct from a diagnostic standpoint, there are many issues that are not addressed, namely the clinical overlap between patients with a detectable clonal population and those with similar disease in whom the mutation is not detectable with current techniques, and the heterogeneity of patients with nonclonal hes. in contrast, an hes classification scheme proposed at a nih-sponsored workshop, and subsequently refined by the same authors, groups clinically similar disorders with known and unknown etiologies into hes variants. although useful from the standpoint of treatment approaches, this approach creates confusion with respect to diagnostic labels, since a patient with proven clonal eosinophilia is classified as having both hes and chronic eosinophilic leukemia (cel). despite these limitations, for the purposes of this subchapter, hes will be defined as ( ) blood eosinophilia of > /mm on at least two separate occasions or evidence of prominent tissue eosinophilia associated with symptoms and marked tissue eosinophilia, and ( ) exclusion of secondary causes of eosinophilia, such as drug hypersensitivity, hypoadrenalism, and parasitic infection. the approach to classification, diagnosis, pathophysiology, and treatment of the various forms of hes and other rare manifestations of eosinophilic disease will be discussed. hypereosinophilic syndromes are rare diseases, and correct estimates of incidence and prevalence are unavailable. by extrapolation from the surveillance, epidemiology and end results (seer) database of cancer statistics in the united states, the incident rate of hes has been estimated to be between . and . per , person-years in the period from to . the calculated prevalence was estimated at . e . per , . these estimates are approximations and rely on coding of eosinophilia by individual physicians. in addition, the lack of specific codes for hes variants precludes a more specific characterization of incidence. unified comprehensive patient databases would be useful in this regard. hes characteristically develops between the ages of and years; however, young children and the elderly can also be affected. a male predominance has been described, although this is due primarily to overrepresentation of males with the fip -like /platelet-derived growth factor receptor-a (fip l / pdgfra) mutation in most series. hypereosinophilia may be attributable to secondary causes requiring specific treatment. therefore, a careful history and physical examination are of paramount importance for making the correct diagnosis (fig. . . ) . the history should include the degree and duration of eosinophilia, with documentation if available. specific symptoms related to individual organ systems should be elicited, as patients can present with a multitude of findings ranging from the most common complaints of skin involvement to more rare presentations of connective tissue or ocular involvement. pertinent medication, occupational, and travel histories should also be obtained to exclude drug allergy, hypersensitivity reactions, and helminth infections, respectively. a family history of eosinophilia should also be investigated. the initial physical exam should be complete, with a focus on examination for organ involvement commonly seen in eosinophilic syndromes. notably, careful examination of the skin, abdomen (to assess organomegaly), cardiovascular system (to assess evidence of heart failure and valvular disease), lungs, neurological system (to exclude neuropathy) should be performed. finally, laboratory and diagnostic testing for eosinophilia should be directed by the history and prior physical findings, but should include basic testing to assess endorgan involvement (fig. . . ). initial laboratory evaluation should include, at a minimum, a complete blood count (cbc) with a differential, chemistry panel to assess creatine kinase, creatinine, electrolytes, erythrocyte sedimentation rate (esr), liver enzymes, quantitative immunoglobulin e (ige), levels serum b , and serum tryptase. assessment of cardiac status should include echocardiogram, electrocardiogram (ekg), and measurement of serum troponin. spirometry with assessment of lung volumes and diffusion capacity should be performed to assess occult pulmonary involvement. often the initial evaluation will determine the need for further testing. for example, a patient with asthma and sinusitis should be screened for churg-strauss syndrome (css) with serum anti-neutrophil cytoplasmic antibody (anca). electrolyte abnormalities with eosinophilia might necessitate a workup for hypoadrenalism. findings of anemia, neutropenia, or thrombocytopenia would initiate a search for hematological disorders, including cel. a pertinent drug history should prompt discontinuation of potentially offending agents to see if the eosinophilia resolves. exceedingly uncommon in secondary eosinophilia, an elevated serum b or tryptase level should prompt evaluation for cel and systemic mastocytosis (sm). if no secondary or reactive cause is identified, one can reasonably proceed to further evaluation for hes and other rare eosinophilic disorders. additional screening tests at this point should include computed tomography (ct) of the abdomen, chest, and pelvis, and bone marrow examination to assess cellularity, dysplastic changes of either eosinophils or mast cells, eosinophil precursors, mast cell numbers, and myelofibrosis. cytogenetic analysis of dividing cells should be performed, as well as specific testing by real-time polymerase chain reaction (rt-pcr) or fluorescence in situ hybridization (fish) for the most common genetic abnormalities associated with eosinophilia, including pdgfr-associated cels. d v kit analysis should also be performed to exclude sm in patients with elevated serum tryptase and/or suggestive findings on bone marrow biopsy. t-cell clonality should be assessed by rt-pcr for t-cell receptor rearrangement and/ or flow cytometry. phenotypic assessment should also be performed to identify aberrant populations of t cells that may or may not be clonal and should include cd , cd , cd , and if possible cd and cd . aberrant t-cell populations often exhibit elevated intracellular interleukin- (il- ), il- , and il- levels; however, intracellular cytokine analysis is not routinely available except at specialized academic centers. various biomarkers have been clinically correlated with particular variants of hes, including serum b and tryptase elevation in myeloproliferative forms of hes and elevated serum ige and thymus and activation-regulated chemokine (tarc)/c-c motif chemokine (ccl ) levels in lymphocytic variant hes and are discussed further in the next section. it is important to recognize that the diagnosis of hes is an iterative process and may be revisited if new clinical findings develop and as new biomarkers and diagnostic tests become available. as stated in the introduction, the current classification of hes variants is in evolution as specific etiologies become better defined. despite the molecular uncertainties and current unknowns, it remains useful to subdivide patients into hes variants based on clinical characteristics and a combination of molecular and biologic data (fig. . . ) , since this has important implications regarding prognosis, monitoring, and treatment. unfortunately, the vast majority ( e %) of patients with hes remain classified as idiopathic. in the following section, we discuss hes variants in lymphocytic variant hes (l-hes) is defined by the expansion of an aberrant and/or clonal t-lymphocyte population with increased production of eosinophilopoietic cytokines in a patient who meets criteria for hes. historically, the evidence for t-cell-mediated pathogenesis came to light when peripheral t-cell clones generated from a patient with hes were cultured with bone marrow progenitors, leading to outgrowth of eosinophilic colonies in culture. further studies demonstrated that il- was overproduced; however, other cytokines such as il- , interferon g (ifn-g), and additional t-helper type (t h ) cytokines, il- and il- , may also be increased in individual clones. l-hes affects males and females in equal proportions. patients frequently have elevated serum ige levels and skin involvement, ranging from urticaria and eczematous rashes to subcutaneous involvement with angioedema. rare patients present with cyclic eosinophilia and angioedema in the setting of an abnormal lymphocyte population (gleich's syndrome). lymphadenopathy and splenomegaly are uncommon except in the case of occult lymphoma, and bone marrow examination is generally normocellular with increased eosinophils. in contrast to the myeloproliferative variant, life-threatening end-organ involvement, including endomyocardial fibrosis, neurologic complications, and hypercoagulability are extremely rare in l-hes. furthermore, l-hes is usually glucocorticoid-responsive, although moderate to high doses may be necessary to control symptoms. blood counts reflect elevated eosinophil levels; there may be lymphocytosis, but this is uncommon. there is often a polyclonal expansion of igg or igm, and markers of inflammation, such as esr and c-reactive protein (crp), and t-cell activation, such as ccl , may be elevated. thrombocytosis may also be present. the majority of aberrant t-cell populations in l-hes are detectable by flow cytometry using standard panels. although cd À cd þ is the most common phenotype, cd þ cd À cd À and cd þ cd dim/À have also been described in some patients. it is important to include appropriate markers to distinguish these cell populations from monocytes, especially if cd expression is decreased, and to exclude tcell lymphomas that may present with eosinophilia, including cutaneous t-cell lymphoma and angioimmunoblastic t-cell lymphoma. clonal populations of il- producing cells with a normal surface phenotype have also been described in l-hes. consequently, t-cell receptor (tcr) rearrangement analysis and/or assessment of clonality by flow cytometry using tcr-vb staining should be performed. demonstration of increased t h cytokine production by the aberrant and/or clonal t-cell population by intracellular flow cytometry is also diagnostic, but is impractical at most centers. the decision to treat l-hes patients depends on the nature and extent of disease. patients with clinical manifestations attributable to the eosinophilia should be treated initially with corticosteroids ( e mg prednisone/d depending on the severity of the signs and symptoms), reducing slowly to the lowest dose that suppresses symptoms and eosinophilia. steroid-sparing agents should be considered for patients with elevated eosinophil counts and symptoms requiring moderate to high dose (> e mg prednisone equivalent daily) corticosteroids. aims of therapy should be to reduce disease manifestations and prevent organ dysfunction. a number of steroid-sparing agents targeting t cells have been tried with variable success. among currently available agents, ifn-a (at a dose range of e mu daily) has shown the greatest efficacy. due to in vitro data demonstrating decreased apoptosis of the clonal population in the presence of ifn-a, concomitant low dose corticosteroid therapy is recommended in patients with l-hes. more recently, anti-il- therapy with mepolizumab (available only for compassionate use through glaxosmithkline) has been shown to be safe and effective as a steroid-sparing agent in this subgroup of patients. it is important to monitor patients with a clonal t-cell population for progression to t-cell lymphoma throughout the course of their disease. this may be preceded by lymphadenopathy, expansion of the clone, and/or the development of cytogenetic abnormalities. yearly bone marrow examination with karyotyping has been recommended, although the impact of this approach on prognosis is unknown. the most common cause of marked eosinophilia with myeloproliferative features is an interstitial deletion in chromosome q between fip l and the tyrosine kinase, pdgfra, that results in the fusion gene product fip l /pdgfra. other pdgfra fusion partners have been identified, but are less common. colony forming assays using an fip l /pdgfra reporter showed that expression of the fusion gene in human hematopoietic progenitors induces differentiation of erythrocytes and neutrophils in addition to eosinophils, and multiple lineages, including b-and t-lymphocytes, monocytes, mast cells, and neutrophils, can express the fusion gene in affected patients. current data suggest, however, that the detrimental clinical effects are mediated primarily by eosinophils. in fact, the clinical presentation of fip l/ pdgfra-positive cel is indistinguishable from that of a subset of idiopathic hes patients with myeloproliferative features. a number of other myeloproliferative disorders (mpds) can present with marked blood and bone marrow eosinophilia and have been defined at the molecular level. these include pdgfrb-associated chronic myelomonocytic leukemia (cmml), janus kinase (jak )-associated mpds, q-syndrome and d v kitassociated sm. although sm shares bone marrow features with fip l /pdgfra-positive cel, the clinical presentation is often quite different from hes and the eosinophils are not usually implicated in disease pathogenesis. myeloproliferative variant hes (mhes) is the most aggressive form of hes, with fatality rates of up to % at years prior to the availability of imatinib therapy. patients are predominantly male with a near % male predominance in pdgfra-associated disease. mhes typically presents between the ages of and years, although children and the elderly can be affected. the clinical presentation ranges from fatigue and malaise to endomyocardial fibrosis and stroke. splenomegaly is common and characteristic laboratory findings include anemia and thrombocytopenia, occasional neutrophilia, and elevation of serum b and tryptase levels. bone marrow examination reveals a hypercellular marrow with increased and dysplastic eosinophils and eosinophil precursors, fibrosis, and, in most cases, a concomitant increase in atypical mast cells. eosinophilia is unresponsive to corticosteroids in most patients with mhes. although the existence of a myeloproliferative (leukemic) variant of hes had long been recognized, the availability of molecular testing has revolutionized diagnosis of this syndrome. the fip l /pdgfra fusion gene can be detected either by rt-pcr or fish in peripheral blood. additional testing, including cytogenetics, fish, and/or quantitative pcr, should be performed, as indicated, to identify other mpds associated with peripheral eosinophilia. the discovery that the tyrosine kinase inhibitor, imatinib, can decrease eosinophilia and improve symptoms in patients with hes facilitated the discovery of the fip l / pdgfra fusion gene and has dramatically improved prognosis in mhes. some patients with fip l epdg-fra-negative mhes also respond to imatinib. therefore, a trial of imatinib in such patients is reasonable if they fail low dose corticosteroids, particularly in the setting of myeloproliferative features. most fip l /pdgfrapositive patients achieve clinical and molecular remission within e weeks of beginning imatinib therapy and can be maintained on low-dose therapy ( mg daily) for many years without disease progression. although fip l /pdgfra-negative patients may require longer to respond, a trial of imatinib mg daily for weeks is sufficient to assess response. side effects of therapy are comparable to that seen in the treatment of chronic myeloid leukemia (cml), but rarely lead to discontinuation of therapy, and the development of resistance appears to be extremely rare. unfortunately, imatinib is not curative in mhes. since the clinical symptoms may be rapidly progressive, any new diagnosis of hes with myeloproliferative features should be evaluated without delay so that treatment can be initiated rapidly. ekg, troponin, and echocardiogram should be performed prior to initiating therapy. if cardiac dysfunction is present or serum troponin is elevated, corticosteroids should be initiated concurrent with imatinib therapy, due to reports of necrotizing myocarditis after initiation of therapy in patients with preexisting cardiac involvement. initial monitoring on imatinib therapy should include weekly complete blood counts, liver function tests, and serum troponin levels. bone marrow examination should be repeated at e weeks, even in the setting of hematologic response, to exclude occult leukemia or lymphoma that may have been masked by the marked eosinophilia. long-term monitoring should include echocardiograms every e months to assess progression of disease, as well as to monitor for possible treatment-related cardiomyopathy. for patients with fip l /pdgfra-positive mhes who fail or are intolerant to imatinib therapy, a trial of one of the newer tyrosine kinase inhibitors with activity against the most common resistance mutations is indicated. for fip l /pdgfra-negative mhes patients who fail imatinib, a step-wise approach of commonly used medications for reduction of eosinophils should be employed, balancing toxicities of the drugs and the individual patient's comorbidities. drugs that have been used successfully include hydroxyurea, ifn-a, anti-il- therapy, and vincristine. patients with rapidly progressive or aggressive disease unresponsive to standard therapies should be considered for nonmyeloablative allogeneic bone marrow transplant, a strategy that has proven curative in a number of cases. by definition, the mechanism of pathogenesis in idiopathic hes is unknown. similar to other forms of hes, pathogenesis is due to tissue infiltration by eosinophils, with deposition of granule proteins and release of inflammatory mediators. patients with idiopathic hes may have relatively few or no symptoms (benign eosinophilia) or a wide variety of manifestations attributable to eosinophilic infiltration of target organs. the most commonly affected organs are the gastrointestinal tract, lungs, and skin ( fig. . . ). nonspecific symptoms, including arthralgias, fatigue, malaise, and myalgias, are also common. cardiac manifestations, including eosinophilic myocarditis and endomyocardial fibrosis, and neurological involvement each occur in e % of patients with idiopathic hes and are major causes of morbidity and mortality in this patient group. two areas of confusion with regards to secondary forms of eosinophilia may delay treatment for true hes. the first involves appropriate testing to rule out parasitic disease. prompt referral to an infectious disease specialist can target helminth infection testing based on the patient's travel history and likelihood of exposure. whether to empirically treat for strongyloides according to centers for disease control and prevention (cdc) guidelines is an area of controversy. recommendations of the cdc are that all patients who are 'at risk of disseminated strongyloidiasis should be treated.' the drug of choice for treatment of uncomplicated strongyloidiasis is ivermectin. the second area of confusion is that of medications causing hypersensitivity syndromes such as drug reaction with eosinophilia and systemic symptoms (dress). many drugs can cause eosinophilia starting from days to years after initiation. simplification of drug regimens can vastly improve the ability to make the diagnosis of hes. once the diagnosis of hes is made, the decision to treat is tailored to the likely etiology. since patients with idiopathic hes are heterogeneous in their presentation, virtually all treatment must be individualized based on the presence of signs and symptoms and the likelihood of disease progression with end-organ involvement. although the discovery of imatinib has dramatically altered prognosis in myeloproliferative hes, and in particular for patients with fip l /pdgfrapositive cel, patients with idiopathic hes are much less likely to respond. corticosteroids remain first-line therapy for the majority of patients with hes, including l-hes, although many patients require moderate to high doses or develop significant steroid-related toxicity. patients who fail to respond to corticosteroids or have significant side effects from prolonged high dose therapy should be considered for second-line therapy. a short ( e week) trial of imatinib, the only united states food and drug administration (fda)-approved drug for the treatment of hes, may be warranted in steroid-resistant patients. hydroxyurea is the most commonly used of the cytotoxic medications and has been used alone or in combination with ifn-a at doses ranging from mg to g daily. it can be associated with cytopenias or other adverse effects at high doses, thus limiting its use as a solitary agent. furthermore, hydroxyurea may be associated with an increased risk of secondary malignancy with prolonged use. of the immunomodulatory medications, ifn-a is most frequently used and at doses of e mu daily is effective in up to % of patients. prolonged remission of clinical symptoms and eosinophilia has rarely been reported after prolonged ifna use or when inf-a is used in combination with cytotoxic medications. other cytotoxic and immunomodulatory agents have been used with varied success and include cytosine arabinoside (ara-c), vincristine, alemtuzumab, , intravenous immunoglobulin, cyclosporine, cyclophosphamide, azathioprine, and methotrexate. however, bone marrow transplantation remains the only curative therapy. familial hypereosinophilia is an autosomal dominant disorder discovered in one kindred family. eosinophilia is present at birth in affected individuals and remains remarkably stable over time. although the index case and his sister presented with eosinophilic end-organ involvement (endomyocardial fibrosis and peripheral neuropathy) that progressed despite therapy, most affected family members have followed a benign course despite eosinophil counts ranging from e /mm over many years without treatment. there have been isolated reports of additional families with eosinophilia, but no clear genetic inheritance pattern has been found. common environmental exposures, including helminth infection, must be excluded prior to attributing the eosinophilia to a familial origin. the genetic defect in familial hypereosinophilic syndrome is not known. the gene has been mapped to an area on chromosome q harboring the cytokine gene cluster; however, sequencing of a number of candidate genes in this region, including gm-csf, il- , and il- , has failed to identify polymorphisms that could account for the affected phenotype. additional sequencing is currently under way. eosinophils from affected family members appear to be less activated than those from patients with hes, coincident with the general lack of eosinophil-mediated pathology. no further family members have developed organ involvement, thus precluding further studies of disease pathogenesis in this disorder. single organ eosinophilic tissue infiltration has been described for nearly all organ systems, although isolated involvement of the skin, lung or gastrointestinal tract is most common. the mechanism whereby certain tissues are targeted preferentially over others is not well understood. some organ-restricted disorders, such as eosinophilic esophagitis, have distinct clinical presentations and rarely progress to multiorgan involvement even in the presence of marked peripheral eosinophilia. other disorders, such as chronic eosinophilic pneumonia (cep), may overlap considerably in presentation with, or be the initial manifestation of, systemic hes. treatment varies depending on the specific clinical manifestations, but local or systemic corticosteroids are often effective. it is beyond the scope of this subchapter to discuss all organ-restricted eosinophilic syndromes, several of which are covered in other subchapters. consequently, only a few examples are provided to illustrate the diversity of syndromes seen. eosinophilia restricted to the lung, with or without peripheral eosinophilia, is associated with a wide variety of allergic, infectious, inflammatory, and neoplastic disorders, including asthma, allergic bronchopulmonary aspergillosis, drug hypersensitivity reactions, fungal pneumonia, helminth infection, and sarcoidosis. pulmonary eosinophilia can also be the first indicator of systemic eosinophilic diseases, including css. when no trigger is identified and disease remains restricted to the lung, pulmonary eosinophilia can be classified into two distinct syndromes: acute eosinophilic pneumonia (aep) and cep. aep typically occurs in healthy men between the ages of and years without a history of asthma. symptoms include the abrupt onset of dyspnea, fever, malaise, night sweats, nonproductive cough, and pleuritic chest pain, and respiratory failure requiring mechanical ventilation is common. physical examination may reveal bibasilar rales or rhonchi. radiological findings include diffuse alveolar infiltrates or reticular opacities in the early stages and bronchoscopy reveals increased absolute eosinophil counts as well as an increased percentage of eosinophils (> %) relative to the total inflammatory cell content. whether an environmental exposure plays a role in the pathophysiology remains controversial. patients typically respond well to high-dose corticosteroid therapy despite respiratory failure at presentation, and long-term sequelae are extremely uncommon. cep presents as a subacute illness, often mistaken for asthma in the early stages. it progresses to involve similar symptoms of cough and dyspnea, and is occasionally associated with constitutional symptoms of fever, sweats, and weight loss. physical exam findings are similar to aep, with wheeze and or crackles being present in most cases. radiological findings typically show peripheral infiltrates. bronchoalveolar lavage fluid (balf) shows an eosinophil predominance. while patients may respond initially to corticosteroids, many relapse and may require long-term corticosteroid treatment. peripheral eosinophilia of > /mm is common, but not universal. eosinophilia of the liver may occur in primary liver diseases, such as primary biliary cirrhosis or autoimmune hepatitis, in the setting of hepatobiliary involvement by helminth infections, including clonorchiasis and schistosomiasis, or secondary to a wide variety of prescription and nonprescription drugs. primary eosinophilic hepatitis with or without peripheral eosinophilia is, however, relatively rare. early studies using immunohistochemistry implicated eosinophils in the pathogenesis of progression to chronic hepatitis by demonstrating major basic protein and activated, degranulated eosinophils in close proximity to affected hepatocytes. hepatic cholangiopathy, fibrosis and liver failure have been reported, although most patients in the literature have been responsive to corticosteroids or hydroxyurea. eosinophilic cystitis is a rare disorder, primarily seen in children, that presents with dysuria, gross hematuria, or suprapubic pain. urinalysis may reveal microscopic hematuria or pyuria. cystoscopy often reveals bladder wall erythema, edema, or nodules, and the diagnosis is made histologically from biopsies of the bladder. infrequently, necrosis or fibrosis can be present with delayed diagnosis. it is important to exclude bladder cancer, since eosinophilic cystitis may be present in the setting of bladder cancer. treatment with antihistamines, nonsteroidal antiinflammatory medications, fulguration and steroids were reported to be successful in one series. overlap syndromes with hypereosinophilic syndrome a number of systemic disorders have overlapping clinical presentations with hes. of these, the two most common are sm and css. sm is a myeloproliferative disorder, most commonly associated with a d v mutation in kit. patients typically present with symptoms related to mast cell tissue infiltration and mediator release, including anaphylaxis, diarrhea, flushing, and urticaria. diagnosis is based on the presence of major and minor criteria according to the who classification. d v-positive sm with eosinophilia (sm-eo) is clinically distinct from fip l -pdgfra-positive cel and can be distinguished using molecular and clinical findings. css is a distinct multisystem disorder that is characterized by the presence of eosinophilic vasculitis in the setting of asthma, pulmonary infiltrates, sinusitis with polyps, and marked peripheral eosinophilia. anca may be present and appears to be associated with a more severe course, often with renal involvement. definitive diagnosis can be made by tissue biopsy demonstrating granulomata and necrotizing eosinophilic vasculitis. significant overlap in clinical presentation with idiopathic hes can cause diagnostic confusion, particularly since corticosteroids must often be initiated to prevent serious end-organ damage before appropriate biopsies can be obtained. marked eosinophilia can be seen in association with immunodysregulation of varied etiologies, including primary immunodeficiencies, secondary immunodeficiencies [e.g., human immunodeficiency virus (hiv) infection], and autoimmune disorders (table . . ). in general, the peripheral eosinophilia seen in these disorders is not associated with characteristic end-organ manifestations. exceptions include omenn syndrome and dock deficiency, which may present with eosinophilic tissue infiltration involving the skin or other organs. , in some disorders, including autoimmune lymphoproliferative syndrome (alps) and hiv, peripheral eosinophilia reflects more profound immunodysregulation and in some cases may portend a worse prognosis. , novel therapies for hypereosinophilic syndrome improved understanding of the pathogenesis of eosinophilic disorders, including eosinophilic asthma, has led to the development of a number of novel agents, including agents targeting il- and its receptor, although none are currently fda approved. these are discussed in chapter . additional agents, including alemtuzumab, a monoclonal antibody that targets cd on aberrant t cells, and tyrosine kinase inhibitors with activity against imatinibresistant fip l /pdgfra-positive mutations, have also been used with success in small numbers of patients with l-hes and cel, respectively. the complexity of diagnosing and treating hes arises from the difficulty in excluding secondary forms of eosinophilia and knowing when to treat and with what medications. an understanding of underlying pathogenesis has improved genotypeephenotype classification of some eosinophilic disorders and reinforced the perception of hes as a heterogeneous group of rare disorders with differing pathogenesis, prognosis, and treatment options. advancing knowledge of molecular pathogenesis and new genetic discoveries will provide the foundation for development of novel targeted therapies, as well as a promising outlook for the care of patients with hes. advanced epithelial tumors typically undergo necrosis with subsequent release of damage-associated molecular pattern molecules (damps). tumor cells are dependent on the host-created microenvironment, including endothelial cells, inflammatory cells, and stromal cells. this makes it difficult to cultivate more than a minority of tumor cells in vitro but, as is increasingly being understood, provides unique opportunities for cancer therapy. thus, when evaluating a tumor, it is important to assess three elements within the microenvironment: . factors released by tumor cells and their surrounding cells, consisting of epithelial and endothelial cells, fibroblasts, specialized local mesenchymal cells, and infiltrating leukocytes; . the quantity and quality of tumor-associated cells, specifically leukocytes; . their state of activation. the mammalian immune system reciprocally interacts within dynamic networks of tissue-associated nonimmune cells, enabling metabolic homeostasis, orderly scheduled cellular maturation and replacement, the timely eradication of effete cells, the repair of damage, and protection against pathogens. the simultaneous tolerance to self-antigens and reciprocal reactivity to new or occult antigens often occurs in settings of tissue damage and wound healing. when tissue homeostasis is perturbed, mast cells, granulocytes, and macrophages are recruited and rapidly release mediators such as cytokines, chemokines, matrix remodeling proteases and reactive oxygen species (ros), and bioactive mediators such as histamine. these in turn induce mobilization and infiltration of additional leukocytes into damaged tissue (causing inflammation). subsequently, the process of wound healing begins, characterized by phagocytosis of cellular debris and apoptotic cells, immune suppression, reepithelialization, and synthesis of extracellular matrix (ecm). thus, inflammation is resolved, thereby restoring tissue homeostasis. tumor cells, paradoxically growing in the setting of substantial necrosis and (chronic) inflammation, harness the collaborative capabilities (see below) of immune cells and local nonmutated but injured tissues to promote cell survival and proliferation, partly by releasing factors such as transforming growth factor b (tgf-b) and interleukin- (il- ), leading to restoration of barrier function in epithelial tissues. the host therefore enables tumor cells to escape from eradication and to release tissue-healing factors, thereby providing neovascularization and subsequent nutritional supply to tumor cells. wound healing and tumor stroma formation share many important properties. nevertheless, wound healing is itself a self-limiting process, whereas tumors addicted to death release damps, thereby sustaining tissue proliferation, angiogenesis and continuous leukocyte recruitment. prolonged (chronic) inflammation is often associated with carcinogenesis. ros generated largely intracellularly or at the cell membrane by nadph oxidases can also promote mutagenic changes in cells when aerobic denaturation of extracellular damps is ineffective. tumor necrosis factor a (tnf-a) and matrix metalloproteinases promote recruitment of inflammatory cells and tissue remodeling, but also facilitate tumor metastasis. a state of metabolic symbiosis e between the tumor and the surrounding stroma, or within central hypoxic tumor cells and those at the growing rim of the tumor, allows regional variations in oxidative phosphorylation and autophagy that depend on nutrient supply and availability of molecular oxygen. compartments with abundant resident populations of eosinophils include tissues with substantial cellular turnover and regenerative capacity, such as the bone marrow, primary and secondary lymphoid tissues (e.g., lymph nodes, spleen, and thymus), the uterus, and almost the entire gastrointestinal tract (with the exception of the esophagus, except under abnormal states). , this link with cell turnover and tissue repair may also explain the presence of eosinophils at sites of wound repair and the commonality of an eosinophil infiltrate among solid tumors. eosinophil localization to the lamina propria of the gastrointestinal tract is critically regulated by eotaxin/ c-c motif chemokine (ccl ), a chemokine constitutively expressed throughout the gastrointestinal tract. nevertheless, eotaxin expression within the gastrointestinal tract (e.g., the esophagus) is by itself insufficient to induce eosinophil accumulation, because the esophagus is normally devoid of these granulocytes. this suggests the potential involvement of other eosinophil chemoattractants and activating factors that contribute to tissue-specific accumulation and degranulation. in particular, the correlation of eosinophil recruitment/activation with tissue damage and cell death associated with these inflammatory responses suggests that damps may represent previously overlooked signaling molecules that elicit eosinophil agonist activities (fig. . . ) . consistently, high mobility group box (hmgb ; a prototypic damp molecule) serves as a chemoattractant and survival factor for eosinophilic granulocytes. eosinophilic granulocytes are found within necrotic tissues and the pseudocapsule surrounding tumors. these immune cells contain, and can release, several cationic proteins that, in addition to their toxic tissue damaging character, are also potentially important for tissue remodeling and clearing cellular debris. eosinophils are thought to be, in part, responsible for the cell death and tissue damage commonly observed in disease states that are associated with increased eosinophil numbers and tissuespecific eosinophil recruitment. since the mid- s, eosinophils have been known to mediate their effects via at least three independent mechanisms in addition to the release of cytotoxic granule proteins, which enable them to modulate the intensity of inflammation, as well as to elicit cell death leading to the loss of tissue integrity: in addition, they initiate an acute inflammatory response to recruit, activate, and polarize b cells, cytotoxic lymphocytes, dendritic cells (dcs), and monocytes. chronic exposure to active damps promotes angiogenesis and immunosuppression by recruiting and activating endothelial cells, fibroblasts, and leukocytes such as t-regulatory (t reg ) cells, and myeloid-derived suppressor (mds) cells, which are important for promoting wound healing. atp, adenosine -triphosphate; hmgb , high mobility group box ; hsp, heat shock protein; s , protein s t h , t-helper type . a growing body of literature suggests that both immunoregulation and tissue repair/remodeling may represent important nonoverlapping eosinophil effector functions. a quantitative assessment of eosinophil recruitment/accumulation in solid tumors showed that the tissue eosinophilia is apparently mediated by one or more factors released directly from necrotic tissues within the tumor. studies linking eosinophil recruitment and activations with cell death and necrosis abound. , , thus, damps released from damaged/dying epithelial cells may represent a previously underappreciated signaling event capable of mediating both eosinophil recruitment and the execution of effector functions leading to (and/or promoting) tissue repair and remodeling. in addition to their capacity to synthesize and release a variety of immunoregulatory molecules, some studies have suggested that eosinophils may function as antigen presenting cells (apcs) or enhance dc maturation. eosinophils may also affect local t cell responses by modulating the balance of t-helper type (t h ) and t h immune responses [e.g., through eosinophil-derived indoleamine , -dioxygenase (ido) production of kynurenine ]. importantly, ido appears to be essential for the induction of tolerance by tissue recruited t cells. thus, similar to other eosinophil-mediated immunosupressive activities (e.g., the potential induction of t-regulatory cells through tgfb production), eosinophil-derived ido may also play a crucial role in immunosuppression and potentially facilitate tissue repair and, as a by-product, tumor growth. the eosinophil plays a somewhat passive role in the tumor. eosinophils are frequently observed following immunotherapy with il- , il- , , gm-csf, repeated vaccination, and antibodies to ctla- , but the significance of their appearance remains largely unknown. in particular, the antitumor effects of successful cytokine therapy of cancer with il- has been associated with the identification of degranulating eosinophils within the tumor, suggesting that eosinophil effector functions (e.g., direct or antibodydependent tumor cell lysis or the immunoregulatory capacity of eosinophils modulating the local tumor microenvironment) may play a role in the anticancer activities mediated by systemic il- administration. , however, despite the promise of these potential eosinophilmediated antitumor activities, the presence of eosinophils is not an important prognostic indicator in high-dose il- treated patients. mouse studies suggesting a link between eosinophils and the therapeutic value of antitumor responses associated with il- administration have led to clinical trials to evaluate these responses in cancer patients. in a phase i clinical trial of il- administered to cancer patients, sosman and colleagues showed that il- therapy induced systemic eosinophil degranulation associated with increased serum and urine major basic protein (mbp) levels. moreover, the increase in serum mbp was il- dose dependent. unfortunately, the link of antitumor activities with eosinophil numbers in these patients is only correlative and similar to observations made in patients following il- administration. no definitive conclusions can be made as to whether and how eosinophils modulate tumor growth. efforts to demonstrate experimentally a role for eosinophils in tumor immunity have also been fraught with complicating factors yielding qualified interpretations. most notably, considerable excitement was generated by data from the elegant studies of tepper and colleagues , , that demonstrated in athymic nude mice that malignant cell lines transfected for constitutive expression of interleukin (il- ) elicited a tumor associated macrophage and eosinophil infiltrate that led to the attenuation of tumor growth. this provoked a series of tumor xenograft studies that attempted to define the cellular and molecular mechanisms of the apparent il- -mediated antitumor effect. although these studies have shown that spontaneous tumors showed evidence of tumor regression, associated with tumor-infiltrating eosinophils, none of these studies has resolved the role(s) of eosinophils in tumor rejection reactions. recently, colon tumor eradication by eosinophils in murine models suggested a more conventional cytolytic response. in this study, coculture of eosinophils with colorectal tumor cells led to the release of eosinophil cationic protein and eosinophil-derived neurotoxin, as well as tnf-a secretion. interestingly, this may be related to the ability of eosinophils to both lyse and promote clearance of stressed or damaged cells. eosinophils accumulate at unique sites in response to cell turnover, thus regulating tissue homeostasis, and are regulators of local immunity and/or remodeling/repair according to the liar hypothesis, suggesting a more nuanced and interesting role for these cells in damaged tissues and tumors. within several tumor types, including gastrointestinal tumors, tumor-associated tissue eosinophilia (tate) is associated with a significantly better prognosis. the converse is true in other tumor types, such as differentiated oral squamous cell carcinoma or hodgkin lymphoma, , where eosinophils may be involved in promoting cancer cell growth. the mechanism by which eosinophils, in particular, are recruited into tumor tissues is largely unknown. we could characterize damps, including the nuclear protein hmgb , as candidate factors eliciting eosinophil chemotaxis into tumor tissue. thus, eosinophil activities are likely to have multiple roles, dictated by specific circumstances, which were originally adapted to maintain tissue homeostasis. eosinophils are not only able to destroy tissue but are also attracted and activated by stressed and damaged cells, as we and others have demonstrated. , it is likely that stressed cells attract and activate eosinophils by expressing molecules such as nkg d ligand (letal), major histocompatibility complex class i chain related a (mica), and micb, as well as other nkg d ligands or ul -binding proteins (ulbps). these stress-associated molecules all serve as ligands for nkg d, first described on natural killer (nk) cells and subsequently on eotaxin-activated eosinophils and t cells. thus, tumorassociated eosinophils appear to have at least two dominant nonoverlapping activities: . destructive effector functions that may limit tumor growth and induce recruitment and activation of other leukocytes; . immunoregulatory and remodeling activities that suppress immune response and release cytokines, thus promoting wound healing. consistent with the hypothesis that damps initiate innate immune cell activation when encountering microbes or parasites, eosinophils are often first responders to tissue damage and likely mediate some aspects of tissue remodeling and repair. the presence of damps such as hmgb in the necrotic areas of tumors may, in part, elicit both eosinophil tissue recruitment and localized execution of effector functions such as degranulation. the available data, however, suggest that while all threatened epithelial cells, including cancer cells, release damps, not all eosinophil tissue infiltration is associated with tumor eradication. , this conclusion again suggests that the relationship of eosinophils with the modulation of tumor onset/growth is complex and that expression of damps, the emergent role of autophagy, and redox chemistry e is likely to be only one of several inflammatory mechanisms capable of eliciting eosinophil effector functions. interestingly, of all tested biologic activities, eosinophils respond most sensitively to the presence of damps, including hmgb , with generation of peroxide leading to oxidative degradation and thus inactivation of damps. these damps with or without oxidation, play differential roles in the recruitment of mesenchymal stem cells as well as t regulatory cells which may confound and limit the antitumor response. e in summary, while the role of eosinophils in tumor onset and growth is unresolved, recent studies suggest that eosinophils are a common and robust tumor infiltrate and that much interesting biology remains to be explored. specifically, do eosinophil activities limit tumor growth through destructive effector functions or do eosinophilderived immunoregulation and tissue repair/remodeling promote tumor growth and metastasis? how does the critical role of redox inform eosinophil function in relation to damps? the resolution of these questions could inform the initiation of eosinophil-based modalities and, in turn, novel therapeutic approaches to treat cancer patients. eosinophils have been associated with numerous diseases affecting multiple organ systems and, as this volume attests, their activities have been implicated as a cause of tissue pathology in a wide range of these conditions. eosinophils are strongly implicated in the pathogenesis of allergic diseases, including allergic rhinitis, asthma, atopic dermatitis, food allergy, and others. although chronic rhinosinusitis (crs) is not strictly an allergic disease, it is often associated with allergy and the appearance of intense eosinophilia in tissues from the upper airways and sinuses of patients suffering from this condition has been known for the better part of a century. the purpose of this subchapter is to review the evidence showing an association between the eosinophil and crs; the mechanisms of recruitment of eosinophils into sinonasal tissue in crs patients; the roles that eosinophils play in the pathogenesis of this disease; and the potential for treatment modalities based on targeting the eosinophil. crs is a disease of the upper airways and paranasal sinuses that affects e % of the general population. it is typically characterized by one or more of the following symptoms: copious secretion of mucus, facial pressure and headache, fatigue, loss of smell, and nasal obstruction. the physical exam may be unremarkable, although in some cases nasal endoscopy may reveal the presence of purulent drainage and/or nasal polyps. historically, crs has been subdivided according to many of its pathophysiological features, including appearance of fungal mucins, comorbidity with asthma, formation of polyps, hyperplasia of connective tissue, and sensitivity to aspirin. more recently, partly for the sake of simplifying the study of this condition, it has generally been divided into crs with nasal polyps (crswnp) and crs without nasal polyps (crssnp), a terminology that we will use here. the vast majority of crs cases are idiopathic with an unpredictable clinical course; however, a minority have a more characteristic clinical picture. specifically, crs with fungal mucins together with fungal atopy is often viewed as a distinct condition known as afs (allergic fungal sinusitis). patients with aspirin allergy, asthma, and nasal polyps have a syndrome known as samter's triad, characterized by a severe form of recurrent polyposis. lastly, crs occurs in nearly all patients with cystic fibrosis, commonly with nasal polyposis, and this is frequently categorized as a discrete entity as well. in the united states, there are in excess of , surgeries performed annually to relieve the suffering of patients with crs, and the discarded tissue from these procedures has provided access to tissue for those interested in studying the pathogenesis of the disease. the formation of nasal polyps occurs in roughly e % of patients with crs and the inflammatory process within the polyp is particular intense and may differ from inflammation that occurs in crssnp. the level of eosinophilia is greater in crswnp and this form of the disease has been the most extensively studied. consequently, we primarily focus the discussion in this review on the role of the eosinophil in crs with nasal polyps. the appearance of eosinophils in pathological conditions in the airways was noticed shortly after paul ehrlich developed the acidic dyes that continue to be used to detect these cells today. crs is one of the most intensely eosinophilic diseases, rivaling hypereosinophilic syndromes, eosinophilic esophagitis and eosinophilic gastroenteritis, in terms of tissue density of eosinophils (fig. . . ). , bachert and his colleagues have shown that average eosinophil numbers are elevated in both crssnp and crswnp, but they are higher in crswnp and highest within nasal polyps themselves. in our own studies, eosinophilia is the highest in patients with samter's triad (see belowdunpublished observations). although some controversy exists as to whether there are relative differences in the eosinophilia of nasal polyps in populations of african, caucasian, and chinese descent, bachert has reported low levels of eosinophilia in chinese patients with crs. e such studies are often complicated by the fact that the degree of eosinophilia in sinonasal tissue of patients with crs can be dramatically altered by therapies that patients receive prior to surgery, especially oral glucocorticoids, which can diminish the eosinophil number by an order of magnitude (see below). it has been suggested that some intranasal chinese herbal remedies may also alter eosinophil numbers. along with elevated levels of eosinophils, nasal polyps also demonstrate elevated levels of b cells, dendritic cells, neutrophils, macrophages, mast cells, and t cells, and thus represent a tissue undergoing a robust immune and inflammatory response. although mast cells are not the subject of this volume, it must be acknowledged that they are likely to play an important role in the pathogenesis of crs, especially in the tissue swelling and formation of nasal polyps that occur. , detection of eosinophils and eosinophilia in crs has employed assays for eosinophil granule proteins, such as eosinophil cationic protein (ecp), as well as specific antibodies against eosinophil granule proteins, notably eg , which is considered to specifically stain activated eosinophils. , studies using these approaches have confirmed the presence of eosinophilia in crs and provided evidence for activation of the tissueresident eosinophils. e persson and colleagues have emphasized the importance of cytolytic eosinophil degranulation and release of clusters of free granules. evidence has been provided to demonstrate that eosinophils in sinonasal tissue of crs patients undergo both piecemeal degranulation and cytolytic degranulation. , recently, a method has been developed to purify eosinophils from human tissues, including nasal polyps, that should allow further studies into the activation state of eosinophils and gene expression in crs. crs is often comorbid with asthma, and one of the most severe forms of the disease, nasal polyps with asthma and aspirin sensitivity (referred to often as samter's triad), manifests both severe polyposis and severe, often glucocorticoid-resistant, asthma. crs with nasal polyps is also comorbid with churg-strauss syndrome, a systemic vasculitic disorder in which eosinophils feature prominently. several groups have compared the level of eosinophils in sinonasal tissue with asthma severity, levels of eosinophils in bronchial lavage, or sputum samples from the lower airways. in one study, levels of eosinophils in lavage from the middle meatus correlated with fev (forced expiratory volume in one second) in asthmatics with crs. in another study, the presence of eosinophils in sinonasal tissue was higher in crs patients undergoing sinus surgery who had comorbid asthma than in those who did not have asthma. mehta et al. found that the extent of crs disease as measured by sinus computed tomography (ct) scores correlated with eosinophils in blood and sputum, suggesting that systemic elevation and activation of eosinophils may be a feature of crs. patients with crswnp were found to have increased asthma prevalence, as well as increased exhaled nitric oxide. the etiology of crs has yet to be definitively ascribed to infection with any single pathogen or class of pathogens. it is clear that patients with crs experience frequent acute, presumably infectious, exacerbations of disease, and crs patients are generally treated frequently with antibiotics, suggesting that treating physicians suspect the presence of bacteria. along these lines, it has been suggested that patients with crs may have a defect in the innate immune barrier, making them more susceptible to infection or colonization with microorganisms in general. , another line of investigation implicates fungi, or at least allergy to fungi, in crs, based on the presence of activation of lymphocytes to express cytokines in response to fungal extracts. , while atopy to aeroallergens is frequently seen in patients with crs, nearly half of crs patients are nonatopic according to standard tests, and based on this present state of knowledge, crs should not be viewed as a classical allergic disease. yet another line of investigation suggests that staphylococcus aureus and the toxins it produces are important inciting factors in crs. e according to the superantigen hypothesis of crs, staphylococcus-derived superantigens drive a t-helper type (t h )-skewed inflammatory response that is responsible for the eosinophilia observed in crs (the mechanism of eosinophilia in crs is discussed below). superantigens activate large numbers of t cells by cross-linking class ii mhc on antigen-presenting cells with specific vb regions on the t-cell receptor, leading to profound release of cytokines, in some cases skewed toward t h . reports on the extent of staphylococcal colonization in crs have been variable but most have demonstrated normal or supranormal frequencies. bachert et al. and desrosiers et al. have reported that e % of crs patients with nasal polyps have colonization with staphylococcus. , staphylococcal superantigens can also act as allergens, and several reports have detected staphylococcal superantigens in the airways of crs patients and/or demonstrated the presence of functional immunoglobulin e (ige) antibodies directed against the staphylococcal superantigens. e a recent study by bachert et al. has implicated the presence of interleukin- (il- ) and ige directed to staphylococcal enterotoxins in crs comorbid with asthma. a longitudinal study demonstrated that high numbers of eosinophils in the nasal mucosa or in mucus collected from crs patients are strong risk factors for recurrence of disease and the need for subsequent surgery in a -year follow-up study. in summary, eosinophil numbers are increased in sinonasal tissue of patients with crs and increased the most in those with nasal polyps, those with asthma, and especially those with all three conditions. the correlation of eosinophils with disease severity implicates them as either a biomarker of the pathogenic process or a mediating cell responsible for disease. the molecular mechanisms by which eosinophils are recruited to tissues have been reviewed and are the topic of another subchapter in this volume. we restrict our comments therefore to the available evidence on recruitment of eosinophils in crs. in general, several important processes occur. one is the priming and survival-promoting effects of cytokines on eosinophils, especially including granulocyte-macrophage colony-stimulating factor (gm-csf) and il- , both of which also play a role in the generation of eosinophils. another is the local expression of eosinophil-attracting chemokines by epithelium and other tissue cells. the expression of adhesion molecules by endothelium, especially vascular cell adhesion protein (vcam- ), is equally important in the rolling, arrest and transmigration of these cells into the affected tissue. an extensive literature that shows the elevation of these factors in sinonasal tissue of patients with crs will be summarized next. the primary receptor driving chemokine-mediated recruitment of eosinophils is c-c chemokine receptor (ccr ), which has a number of ligands, notably rantes (or c-c motif chemokine ; ccl ), eotaxin (ccl ), eotaxin- (ccl ), eotaxin- (ccl ), and mcp- (ccl ), with less activity for mcp- , mcp- , and mcp- . , mice lacking ccr have severe, but not totally diminished, eosinophil infiltration in allergic inflammation, and ccr antagonists are under investigation in clinical trials. beck et al. found elevated levels of rantes mrna and tissue staining in nasal polyp tissue but not in normal turbinate tissue. rantes expression is primarily found in epithelial cells, an observation leading to speculation that localization of eosinophils to the epithelium and lamina propria may be related to epithelial chemokine expression. e similar findings have subsequently been made with other ccr ligands, including eotaxins e and mcp- . e a careful study by jahnsen et al. found that levels of mrna for eotaxin- , eotaxin- , and mcp- are elevated in nasal polyp tissue and that levels of eotaxin- and mcp- in turbinates from crswnp patients are higher than in normal turbinates. taken together, it is clear that both surrounding tissue (i.e., turbinate) and affected tissue (nasal polyps, which commonly emerge from the ethmoid sinuses and other deeper tissues) in patients with crs have elevated levels of ccr ligands that are likely to play a role in eosinophil recruitment to both of these regions in crs. regulation of chemokine expression by epithelium is complex, but both nf-kb and signal transducer and activator of transcription (stat ) play important roles in the response, and t h cytokines including il- and il- are important inducers. , other stimuli may also be important in activating ccr ligand expression in crs, and chitin has recently been shown to induce eotaxin- in human sinonasal epithelial cells in vitro. eosinophils not only respond to ccr agonists but also release eotaxin, eotaxin- , eotaxin- , and rantes. , the release of ccr agonists from eosinophils may be involved in the local eosinophil accumulation in nasal polyps. recently, our group has found that mpif- (ccl ) is elevated in eosinophilic nasal polyps and that eg þ eosinophils are major mpif- -producing cells in nasal polyps. a mpif- is a ligand for ccr and is known to recruit monocytes, macrophages, and dendritic cells. , these findings indicate that activation of eosinophils may further enhance local inflammation via secondary recruitment of additional cells in nasal polyps. exposure of eosinophils to gm-csf or il- leads to several notable phenotypic changes that are likely to be relevant to their accumulation in crs, including increased expression and function of adhesion molecules, increased transendothelial migration, and increased survival. the earliest studies on crs were by denburg and colleagues, and showed elevated levels of gm-csf in eosinophils in nasal polyp tissue, , and that gm-csf is prominently expressed by epithelial cells, in agreement with earlier in vitro studies showing that epithelial cells are a rich source of this cytokine. several studies have shown that conditioned medium from nasal polyps stimulated with antigen or from cultured nasal polyp epithelial cells can prolong eosinophil survival or activate eosinophils in vitro and that the activity can be significantly blocked by anti-gm-csf antibodies. e additional studies have shown that il- is also an important eosinophil priming and survival factor in nasal polyp tissue. , e this finding is supported by success in recent clinical trials by bachert et al. using anti-il- antibodies (see below). the relative importance of il- and gm-csf as priming cytokines in sinonasal tissue is unknown. liu and busse found that lung-migrating eosinophils have reduced expression of the il- receptor in asthmatics and postulated that the main effects of il- (and therefore probably anti-il- ) may be in the bone marrow and circulation. whether the same is true in the upper airways and sinuses in patients with crs requires further investigation. in vitro, vcam- is known to be an important and relatively selective endothelial adhesion molecule that mediates eosinophil rolling, firm adhesion, and transendothelial migration. several groups have demonstrated increased endothelial expression of vcam- in nasal polyps and shown that vcam- levels correlate with the presence of eosinophils, leading to the hypothesis that recruitment of eosinophils in crs is partially mediated by vcam- . , e using the stamperewoodruff assay, symon, wardlaw, and collaborators have demonstrated that eosinophils bind to nasal polyp tissue in vitro via interactions with p-selectin and on this basis suggested a role for p-selectin as well. in general, the t cell cytokine milieu that most drives eosinophilic inflammation is a t h pattern, including the vcam- activators il- and il- as well as the eosinophil priming cytokine il- . although most crs is probably t h driven, it has been suggested that in some cases of crs, including nonallergic crs, other t-cell cytokines may be involved. the discussion above firmly establishes that eosinophils are present in high numbers in the sinonasal tissue of patients with crs, especially the polypoid form, and that these tissue eosinophils are activated. together, these two lines of investigation provide circumstantial evidence that eosinophils may play a role in alterations to nasal physiology, tissue structure, and clinical phenotype. this section discusses some features of crs that may be mediated by activated eosinophils. in most cases, these discussions should be viewed as hypothetical or speculative, as there is no validated animal model of crs and in most cases it is not possible to definitively assess mechanisms in human subjects (other than some recent studies with antibodies against il- or ige, discussed in the next section). in our view, it is clear that crssnp and crswnp are distinct disease entities that are primarily related by their frequent resistance to treatment, need for surgery (probably greater in crswnp), and eosinophilia (probably greater in crswnp). however, they are distinct in many of the clinical and inflammatory processes that we and others have been investigating at the molecular level. e as to the roles of eosinophils in pathogenesis, by far the most data are available for polypoid crs, and we will restrict our comments here to crswnp, even though they may in many cases apply to crssnp. although eosinophils are elevated in crs and express the activation marker eg (see above), eg has not proven to be a reliable marker of degranulation; eosinophils in nasal polyps are undoubtedly activated, as studies of eosinophil morphology by erjefält and persson have demonstrated extensive piecemeal degranulation and cytolytic degranulation in eosinophils in nasal polyp tissue. , the most obvious and prominent feature of crswnp is the formation of the polyp itself, although details of the mechanism of this metamorphosis of sinonasal tissue are not well understood. perhaps the best studies have been those by bachert et al., who studied early-phase and established nasal polyps. they found formation of a subepithelial eosinophilic cap at the upper surface of the tissue outgrowth and implicated fibronectin and deposition of albumin and extracellular matrix proteins in the early and late phase, respectively. the presence of il- and eotaxin- correlated with the process, thus leading them to propose an important role for eosinophils. based on the deposition of albumin, we can presume that vascular leakage is occurring in crs, although the stimuli for the leak are not well understood. studies by steinke, borish, and others have shown that the -lipoxygenase ( -lo) pathway is activated in crs, especially in aspirin-sensitive disease, and that eosinophils express -lo and ltc synthase within the polyp tissue. , increased eicosanoid metabolism has been reported in crs and correlates with ecp and il- . although eosinophil cyclooxygenase and lipoxygenase metabolites might be important in vascular leakage driving polyp formation, in general the clinical experience with inhibitors of both of these pathways has been disappointing and they are unlikely to be the primary mediators driving vascular leak in a forming polyp. the relative importance of mast cell and eosinophil mediators in polyp formation is unknown. di lorenzo et al. measured ecp, histamine, and tryptase in nasal lavage and found only tryptase and ecp to correlate with symptom scores. since mast cells are highly elevated in nasal polyps and are capable of releasing numerous mediators of vascular leakage, their potential role in polyp formation must be seriously considered. considerable evidence has accumulated to demonstrate elevations of both ige and iga in nasal polyp tissue, and it is not unreasonable to speculate that these antibodies play a role in activating mast cells and eosinophils, respectively, in crswnp. with respect to eosinophils, activation by ige, if it occurs at all, most likely occurs indirectly as a result of the action of mediators released from basophils, mast cells, and other cells that clearly express a functional ige receptor (e.g., inflammatory macrophage-like cells). kita and colleagues have shown that igg and iga, but not ige, can prolong eosinophil survival, and induce cytokine expression and effector function. , however, convincing data demonstrate that eosinophils are activated to degranulate by cross-linking receptors for iga. , more uncertain is the nature of the antigen systems that might drive iga-mediated degranulation in nasal polyp eosinophils. as discussed above, only half of patients with crswnp are atopic, and it is difficult to implicate typical aeroallergens as anything more than exacerbating factors. recent studies by tan and colleagues have demonstrated the presence of autoantibodies of both igg and iga isotypes in patients with recalcitrant crs requiring revision surgery. suh et al. found correlations between both total igg and total ige with the number of eg þ cells, but not iga or secretory iga. clearly, further work is required to establish the importance of immunoglobulins in eosinophil activation in crs. in both asthma and crs there is evidence for epithelial damage mediated by eosinophils. gleich and colleagues demonstrated that the presence of eosinophils and extracellular levels of the toxic eosinophil granule protein major basic protein (mbp) correspond to regions of epithelial injury in patients with crs. this occurs in asthma as well, and there is ample evidence for a similar pathological process occurring in the two diseases. chanez, bousquet, and colleagues and gaga et al. demonstrated a strong correlation between eosinophils in the nose and lung in asthmatics. e bresciani et al. found that % of asthmatics have crs, as determined using clinical and ct scores, and observed a correlation of clinical scores to blood eosinophils in those with mild and moderate asthma. eosinophil-derived granule mediators are well established to be toxic to epithelium, to activate mast cells and basophils, and to drive inflammation. an ultrastructural investigation of epithelial damage in asthmatic and nonasthmatic nasal polyps revealed reduced length of desmosomes in allergic crswnp and in asthmatics. several studies suggest that the epithelium in both asthma and crs presents a poor barrier. , , whether damage from eosinophils occurs as a result of the cationic granular proteins, the respiratory burst and peroxidase activation, protein nitration, or another mechanism is worthy of further investigation. , recent studies indicate reduced spink in epithelial tissue from crs patients. spink is a protease inhibitor that can regulate barrier function in the skin via preventing activation of par and subsequent induction of thymic stromal lymphopoietin (tslp). briot et al. found that high levels of tslp in the skin drive a highly eosinophil and mast cell rich inflammation even in the absence of t cells. kamekura et al. presented evidence that tslp itself can induce claudins and occludins and enhance tight junction function in nasal epithelial cells in vitro, suggesting that its influences may be complex. tslp is elevated in crs, and whether tslp regulates barrier function in crs and/or contributes to eosinophilia needs further clarification. e studies in the lower airways indicate that eosinophilic inflammation is often associated with fibrotic changes, including the laying down of extracellular matrix proteins or repair proteins (e.g., lumican, procollagen, and tenascindsee chapter ). , some nasal polyps are characterized by the deposition of collagen and other matrix proteins, and it is possible that eosinophils may play a role in this process. studies by ohno et al. have demonstrated that transforming growth factors a and b (tgf-a and tgf-b ), as well as platelet-derived growth factor receptor (pdgf), are expressed by eosinophils in nasal polyps and suggest that eosinophil-derived growth factors may alter the structure of the affected nasal mucosa. , eosinophil production of tgf in nasal polyps was confirmed by elovic et al. ultrastructural studies in nasal polyposis using anti-il- antibodies, such as the ones used by flood-page, kay et al. in asthma, will be required to assess the role of eosinophils in remodeling of the sinonasal tissue in crs. at present, there are no specific approved therapies for the treatment of crs. in general, a significant number of patients that present with the diagnosis have failed treatment with antibiotics and/or intranasal glucocorticoids. since nasal polyps commonly grow out of the sinuses, it is a challenge for intranasal glucocorticoid sprays to penetrate to the area of relevant inflammation. treatment with oral steroids is often effective, especially in crswnp, and is frequently used to treat crswnp, although most patients and physicians prefer to avoid chronic treatment with oral glucocorticoids. glucocorticoids continue to be the most effective antiinflammatory drugs available for a wide variety of autoimmune and allergic chronic inflammatory illnesses. their mechanisms are pleiotropic and represent actions exerted upon numerous cell types known to participate in crs, including epithelial, t h , and t h cells. other important cells, such as endothelial cells, mast cells, neutrophils, and t h cells, are relatively unresponsive to glucocorticoid treatment and are unlikely to be important targets in crs. among the steroid responsive cells are eosinophils, and the actions of glucocorticoids on eosinophils have been reviewed. , in , patiar and reece performed a cochrane review of the literature and identified only one randomized-controlled study comparing oral steroids with no intervention or placebo. , as found in earlier uncontrolled trials, oral treatment with prednisone or a similar systemic steroid shrank polyps, reduced symptoms, and improved olfaction in this study. alobid et al. also found that continued treatment with intranasal steroid maintained the benefits of the -week treatment with oral steroids for nearly year. subsequently, hissaria et al. performed a controlled trial testing a short course of prednisolone in crswnp, and found improved symptom scores and a reduction in polyp size, as determined both subjectively and objectively by mri. recently, van zele, bachert, and collaborators performed a double-blind placebo-controlled multicenter trial of oral methylprednisolone in crswnp. in addition to shrinking polyps, improving olfaction and reducing symptoms, the oral steroids improved congestion, nasal peak flow rates, postnasal drip, and rhinorrhea. importantly, in this study methylprednisolone reduced ecp, ige, and il- in nasal secretions and decreased blood eosinophils, ecp, and soluble il- ra in the serum. in vitro studies have identified numerous effects of glucocorticoids on eosinophils (for a summary, see ) . glucocorticoids diminish eosinophil survival in vitro by promoting apoptosis and this effect is blocked by survivalpromoting cytokines such as gm-csf or il- . glucocorticoids also diminish production of these specific cytokines by many cell types, including epithelial cells and t lymphocytes. steroids have a similar suppressive effect in vitro on the expression of cytokines that activate endothelial adhesion molecule expression [e.g., il- , il- , il- , and tumor necrosis factor (tnf)] and of chemokines known to cause eosinophil migration (e.g., ccr agonists). thus, theoretically, glucocorticoids should diminish eosinophil recruitment to the sinuses and nasal cavity and hasten the disappearance of the eosinophils once they arrive in the sinonasal cavity. numerous in vivo studies have addressed these theoretical effects of glucocorticoids by administering intranasal or oral glucocorticoids to patients with crs prior to surgery or biopsy. a general feature of these studies is that glucocorticoids uniformly reduce the number of eosinophils found in nasal polyps. kanai et al. also found treatment with budesonide to reduce the proportion of activated (eg þ ) eosinophils, along with reducing levels of total eosinophils, t cells, the icam- adhesion molecule, and hla-dr, a marker of adaptive immune activation. similar findings were made by hamilos and coworkers, who additionally found reduced p-selectin (but not il- b, tnf, or vcam- ) in polyps from patients treated with intranasal fluticasone propionate. they also found reduced numbers of cells expressing t h cytokines (il- and il- ). delbrouck et al. reported that budesonide decreases levels of both icam- and vcam- , and to a lesser extent p-selectin. as mentioned above, denburg, dolovich, and collaborators identified gm-csf as an important eosinophil-priming and survival-promoting cytokine in the nose of patients with allergic rhinitis and crs. nonaka et al. from this group found that budesonide reduces survival of peripheral blood eosinophils in vitro but not eosinophils extracted from nasal polyps, suggesting that the polyp eosinophils are primed and rendered glucocorticoid resistant as a result of the gm-csf exposure within the polyp. it is unclear whether gm-csf or il- is the most important eosinophil survival cytokine in crswnp, and this is not a question that can be easily addressed, since both of these cytokines have effects on eosinophils outside of the sinonasal cavity. nonetheless, bolard et al. found a correlation between levels of il- and eosinophils in the nasal secretions of patients with nasal polyps and that intranasal steroids reduce both of these parameters. whether glucocorticoid treatment directly induces apoptosis in tissue eosinophils or indirectly induces eosinophil death as a result of suppressing survival-promoting cytokines is still an open question. as to the expression of chemokines that attract eosinophils, jahnsen et al. found that nasal polyp tissue expresses highly elevated levels of eotaxin, eotaxin- , mcp- and rantes, and that after treatment with oral glucocorticoid these factors are all profoundly reduced along with a reduction in eosinophil numbers in the tissue. importantly, in association with the reduction of eosinophils and of the cytokines that induce their accumulation, treatment of crs with intranasal glucocorticoids also leads to a reduction in damage to the epithelium that is thought to be mediated by eosinophils and is accompanied by the restoration of epithelium integrity. perhaps the most valuable data implicating eosinophils in the pathogenesis of crswnp is from the group of gevaert, bachert, and collaborators. e these researchers noted increased levels of il- and soluble il- ra in patients with nasal polyposis and found that treatment of crswnp patients with an antibody against il- led to a reduction in nasal polyp size, particularly in patients with elevated il- . the same patients had a corresponding reduction in the level of eosinophilia, as measured by total eosinophils in the blood and levels of ecp in both the serum and in nasal secretions. e studies using anti-il- antibodies to reduce eosinophilic inflammation are still ongoing and this approach continues to show promise for the treatment of crs. as kinase inhibitors that block the development of eosinophils are developed, it is possible that some will emerge that have some specificity for the eosinophilic lineage of cells and will thus have utility in a variety of diseases including crs. treatment of mice with antibodies against fas caused widespread apoptosis of eosinophils but lacked specificity and thus killed many other important cell types. treatments based on siglec- activation may induce apoptosis more selectively in eosinophils, and in the related allergic cells mast cells and basophils, and thereby provide some benefit without undue safety concerns (see also chapter . ). in addition, recent studies from our group demonstrate that b cells and immunoglobulins, especially iga which is a potent eosinophil activator, are highly elevated in crs. , therapies targeting b cells, such as antibodies against b cell activating factor belonging to the tnf family (baff) or b cells (e.g., anticd ), may prove to diminish the activation of eosinophils in crs if iga and secretory iga are important in activating eosinophils in crs. the ability of organisms to repair themselves is an indispensable requirement for their survival. most members of the animal kingdom inevitably encounter external or internal environmental pressures that can threaten their existence. examples of these are, respectively, injury inflicted by a traumatic event, such as a stab wound, and parasites that inappropriately reside in various tissues. a complex series of reactions to these survival challenges has evolved among vertebrates to correct tissue damage traumatically imposed by a foreign object (i.e., a knife) or to completely remove an injurious agent (i.e., an infection). these shared mechanisms of self-preservation are collectively referred to as healing. in many individuals, healing events in the coronary vasculature occur and can be characterized by both types of reactions, including correction of vessel damage inflicted by circulating substances (i.e., coronary artery disease) and subsequent introduction of a foreign agent (i.e., a stent), as well as to mount an immune response (i.e., hypersensitivity) aimed at eliminating such a foreign body. the most common type of healing occurrence in blood vessels is, paradoxically, also responsible for the most common type of coronary artery disease. indeed, atherosclerosis is likely the result of a response to injury that occurs predominantly in medium-sized muscular vessels, including the left anterior descending, left circumflex, and right coronary arteries. the injury is thought to be primarily inflicted on endothelial cells (ecs), which, from their position as a lining of the vessel lumen, normally provide a nonthrombogenic, nonadhesive surface despite their direct contact with flowing blood. agents suspected of damaging ecs include elevated blood cholesterol levels and oxidized low-density lipoprotein. the outcome of vessel healing is often manifest as an eccentrically oriented lesion consisting of, in its most mature form, lipid-laden macrophages and smooth muscle cells (or so-called foam cells), and lymphocytes enclosed by a fibrous cap comprised of collagen, elastin, and proteoglycans. consequently, these lesions, or plaques, in their stable form can compromise critical oxygen supply to the energetic myocardium by significantly impeding blood flow and, in their unstable or vulnerable forms, can cause myocardial infarction and death by providing sites for platelet adherence and activation of clotting factors to promote formation of a fully occlusive thrombus. although lifestyle changes and medications, including cholesterol synthesis inhibitors (or statins), effectively reduce the risk factors for developing atherosclerosis, its place as a predominant cause of morbidity and mortality in industrialized nations has not wavered. indeed, its recurrent detection is afforded by sophisticated angiographic evidence through catheterization of vessels suspected of harboring lesions. once detected, a patient's physician has several choices for revascularizing occluded coronary vessels. since the late s, a variety of minimally invasive, catheter-based procedures have evolved to displace occlusive atherosclerotic plaques in coronary vessels. andreas gruentzig and colleagues performed the first such procedure in by using a catheter to guide a balloon to the site of atherosclerosis. once positioned, the balloon was expanded to crush the plaque against the vessel wall. this procedure, referred to as percutaneous transluminal coronary angioplasty (ptca), or simply angioplasty, was at least acutely successful for providing a less-invasive treatment, compared to traditionally used coronary artery bypass grafting (cabg), to restore adequate blood flow to the myocardium. approximately months following the procedure, however, approximately % of patients experienced a renarrowing (restenosis) of the blood vessel at the original atheromatous site. since then, two major modifications to ptca have been made in an effort to reduce the incidence of restenosis, including balloon-mediated deployment of bare metal stents (bms) and drug and polymer addition to such stents to form a device collectively referred to as a drug-eluting stent (des). the former amendment to ptca originated with the recognition that restenosis was at least partially attributable to elastic recoil of the vessel as an immediate reaction to balloon-induced vessel expansion, while the addition of drug came with an appreciation for the significant contributions of vascular smooth muscle cell (vsmc) proliferation and migration to restenosis following bms insertion into coronary arteries. the cell proliferative component is similar to that which promotes benign tumor formation and, therefore, was as a good candidate for being disrupted by antiproliferative agents such as those on first-generation des that emerged from johnson and johnson in (cypher des; cordis, miami lakes, fl) and boston scientific corporation in (taxus express , maple grove, mn), and which included sirolimus and paclitaxel, respectively. vascular responses to stent implantation in coronary arteries occur in sequence to culminate in what is collectively referred to as healing. in the traditional sense, vascular wound healing is orchestrated by platelet-and clotting factor-mediated hemostasis, cytokine-/chemokineand leukocyte-mediated inflammation, and vsmc-and fibroblast-mediated tissue remodeling. although healing naturally connotes a process that is favorable, it can be characterized by an exaggeration of the processes outlined above, leading to unfavorable consequences, such as reocclusion, or restenosis of the vessel and/or thrombosis. restenosis can occur in conjunction with stent use. moreover, thrombosis rates associated with des implantation are higher than those related to bms deployment after year, possibly owing to insufficient ec coverage of the stent itself and of the local vascular area in which a des resides. the fate of the vessel wall localized to the area of stent implantation is determined, for the most part, by a balance between the interactions of cells and the soluble factors that they secrete. these events, in turn, may be dictated by the circumstances of des deployment itself, such as the extent of mechanical injury imposed by stent insertion. additionally, des composition may play a significant role in determining the prognosis of des use as a therapy to alleviate occlusive coronary vascular disease. along with their differential abilities to prevent restenosis and association with thrombosis, des and bms platforms may be distinguished by inflammatory cell infiltrates, based on the extent of eosinophil presence that follows deployment of each. clinically used variations of des currently feature controlled drug release from polymers that coat the metal stent skeleton. twenty-eight days following their deployment, overlapping taxus and cypher des each had more eosinophils associated with them compared to their respective overlapping bms controls in rabbit iliac artery. these des are composed of different drugs and nonerodible polymers, but the same metals ( l stainless steel), suggesting that drugs and/or polymers may ultimately attract eosinophils to stent insertion sites. john and colleagues found that a critical amount of polymer may selectively incite eosinophil recruitment in rabbit iliac artery. cypher was associated with significantly more luminal eosinophils than a polymer-free sirolimuseluting stent, a polymer-free sirolimuseestradiol-eluting stent, or a bms, but only when each stent platform was overlapped upon itself (i.e., one stent on top of another of the same kind) in the vessel. eosinophil recruitment following des implantation in rabbits appears not to be specific to this species, since eosinophil accumulation was observed around cypher stent struts, followed by less infiltration near both taxus and bm stents, in porcine coronary arteries. table . . provides a summary of studies citing the occurrence of eosinophil infiltration in response to a variety of catheter-based revascularization interventions, including purely balloon-mediated, or plain old balloon angioplasty (poba), and bms or des implantation. such reactions to foreign-body stent substances suggest an eosinophil-mediated hypersensitivity, which has been implicated in thrombosis and restenosis following stent insertion into blood vessels, as described below. several instances of thrombosis occurring after bms or des implantation and their correlation with histological evidence of eosinophil infiltration have been recorded. zavolloni and colleagues reported that inflammatory infiltrate observed in thrombectomy material retrieved from right coronary artery previously implanted ( years) with a bms for myocardial infarction contained a prevalence of eosinophils. data such as these contrast with previous reports that associated stent thrombosis and eosinophil recruitment particularly with des rather than bms. virmani and colleagues provided histological evidence of thrombus formation in left circumflex coronary artery of a -year-old male who had received overlapping cypher stents in that vessel months earlier. aneurysm formation and inflammatory prominence consisting of eosinophils, giant cells, lymphocytes, macrophages, and plasma cells within the vessel area localized to stent placements were evident. the authors referred to these phenomena as a 'hypersensitivity vasculitis,' which has since been described by other investigators following des implantation in conjunction with repeated thromboses associated with implantation of cypher stents into the left circumflex and also in autopsy specimens. the latter study is especially informative, given that it compared eosinophil presence among thrombotic events according to classification, and in the context of acute myocardial infarction or not, and time-frame, including early ( days) after bms or des [cypher, taxus, and endeavor (medtronic, inc. minneapolis, mn); drug is zotarolimus] deployment, late ( e days) after bms deployment, and very late (> year) after des deployment. here, eosinophil accumulation and the fraction of leukocytes accounted for by eosinophils around cypher stents was substantially greater than that measured in association with taxus or endeavor stents. furthermore, eosinophils were most evident in thrombi that occurred very late following des (predominantly of cypher) deployment. interestingly, vessel remodeling was exclusively related to very late stent thrombosis that occurred after des, with its extent varying according to the number of eosinophils found in thrombi. the authors speculated that vessel remodeling likely caused stent malapposition and subsequent thrombosis. taken together, these studies indicate eosinophilic reactions to bms or des deployment and suggest a role for eosinophils in stent thrombosis. at least two questions may be drawn from these conclusions: . what factors related to coronary stents and/or their deployment attract eosinophils to stented vessel segments? . how might eosinophils contribute to stent-related thrombosis? eosinophilic inflammation occurs following both poba and stent insertion. however, eosinophilic recruitment is more robust following stent placement, e suggesting that balloon-mediated vessel expansion, which is common to both poba and stent placement, is not completely responsible for enhanced eosinophilic recruitment associated with stent placement. indeed, stent components and prophylactic dual antiplatelet (medicinal) therapies (dapt) that are prescribed for use after stent implantation, including the platelet adenosine diphosphate (adp) receptor antagonist, clopidogrel (plavix; bristol-myers squibb, new york, ny, and sanofieaventis, paris, france) and acetylsalicylic acid (aspirin), an inhibitor of prostaglandin g/h synthase (inhibits thromboxane production), can elicit hypersensitivity reactions. the well-recognized role of eosinophils in allergic reactions and their suggested role in a localized hypersensitivity reaction to stent placement are in agreement with positive correlations between allergic sensitivity to metal stent components, including nickel , and molybdenum, and eosinophil recruitment to stented vessel segments. , however, restenosis incidence may or may not positively correlate with allergic reactions to nickel and molybdenum. in addition, drug-eluting stent-related hypersensitivity reactions appear not to guarantee subsequent thrombosis, as revealed by a study showing that a minority (approximately . %) of total des-specific hypersensitivity reactions (based on hypersensitivity cases reported) were accompanied by thrombosis. these hypersensitive, thrombotic cases, however, were characterized by eosinophilic inflammation and incomplete stent coverage, or so-called delayed healing, % of the time. overall, the majority of des-specific hypersensitivity cases were associated with cypher implantation (approximately %) and likely attributable to the poly n-butyl-methacrylate and polyethylene-vinyl acetate cypher copolymer allergens, but not to sirolimus, since the latter can reduce eosinophil infiltration. consistent with this, finn and colleagues reported that / cases of late stent thrombosis were associated with hypersensitivity reactions, with four occurring after cypher and one after taxus implantation. in these episodes, too, eosinophilic inflammation was always present. taken together, these studies suggest that the risk of hypersensitivity to stent components is likely to be small among patients that have or will receive des, with the onset of thrombosis occurring in such hypersensitivity cases also to be small. however, histological evidence shows that the incidence of such rare cases is consistently associated with eosinophil accumulation at stent sites and seems to be especially common with cypher implantation. , at least one study found the combination of hypersensitivity and delayed healing (i.e., endothelialization) of stent struts to be a risk factor for developing late-stent thrombosis. identification of patients prone to each of these circumstances is not performed and may, in fact, be unpredictable considering that hypersensitivity reactions to stents appear not to be unequivocally associated with thrombosis and, further, the incidence of eosinophil involvement in such reactions, which correlates with hypersensitivity-related thrombosis, , is also unclear. thus, without objective, basic study-and clinical trial-based reasoning for differential postprocedural prescription to des recipients, it is essential that all patients that receive des comply with using dapt medicines for their full recommended terms. currently, the exact duration of dapt therapy following stent deployment is questionable, but would appear to be optimized by an awareness of when stent struts are completely healed (or endothelialized) such that the risk of platelet adhesion and activation is dramatically diminished. unfortunately, the time required for adequate, antithrombotic endothelialization of stent struts and the local vascular wall likely varies among patients, because endothelialization rates among des patients afflicted with complicating clinical backgrounds, such as diabetes, may vary, especially compared to des patients without complicating ancillary medical conditions. the concern, then, pertaining to eosinophil involvement in thrombus formation associated with stent deployment is that dapt may be suspended before stent struts, whether covered by polymer or not, are masked enough by healing processes so as not to be a potentially chronic stimulus for eosinophil recruitment and activation. indeed, the nonspecific nature of antiproliferative drugs currently included on clinically used des inhibit not only vsmc proliferation and migration, two major contributors to restenosis, , but also attenuate the same properties of ecs. , therefore, the risk of eosinophil-related thrombosis following des deployment may be assessed by ( ) whether the stent is bms or des and ( ) evidence of allergies to stent components that may predispose the patient to a hypersensitivity reaction that involves eosinophils. fortunately, research efforts have provided suggestive evidence concerning the mechanism of eosinophil recruitment to stented vessel segments and the mechanism through which eosinophils contribute to thrombus formation. these pieces of information may be used to direct future therapies intended to mitigate the purported role of eosinophils in promoting thrombosis following stent deployment. descriptions of two hypersensitivity reactions (i and iv) have been suggested to provide hypothetical explanations for eosinophilic involvement in inflammatory responses to stent insertion. as suggested by virmani and coworkers, eosinophil recruitment to segments stented with cypher and their association with thrombosis in this context may be due to a type iv hypersensitivity reaction, in which t-helper lymphocyte liberation of t-helper type (t h ) cytokines and interleukins and attract eosinophils. another hypothesis includes a two-phase cascade of cell-and soluble factor-mediated reactions being initially orchestrated by ige-activated mast cells and the mediators that these cells elaborate (approximately e h post-stent deployment) to promote secondary infiltration of basophils, eosinophils, macrophages, neutrophils, and t lymphocytes (approximately e h post-stent deployment) that can remain chronically situated around stent struts. thus, occurrence of the first phase of hypersensitivity would coincide with onset of acute stent thrombosis, while the latter time frame would fit the onset of both late and very late stent thromboses. evidence of a role for eosinophils in thrombogenesis includes observations made in hypereosinophilic patients. furthermore, endothelial cells are likely targets for the highly basic-charged major basic and eosinophil cationic proteins (mbp and ecp) of eosinophils, considering that ecs express a negatively charged glycocalyx on their luminal surface. once bound to ecs, these proteins may inflict damage or even kill these cells, as suggested by their cytotoxic capabilities. alternatively, direct activation of platelets by mbp and/or eosinophil peroxidase (epo), or disruption of thrombomodulin function by mbp, may explain the contribution made by eosinophils to stent thrombosis. eosinophil presence in the context of stent-related thrombosis cases that may be secondarily related to tissue remodeling and consequent stent malapposition raises the possibility that eosinophils directly or indirectly possess tissue-remodeling properties. both may be true, since eosinophils express matrix metalloproteinase- (mmp- ), which is a collagenase capable of degrading type iv collagen, a major component of subendothelial layer basement membranes. furthermore, eosinophils secrete interleukin- , a chemokine that has been shown to induce release of mmp- (a collagenase) and mmp- from ecs. eosinophils also express vascular endothelial growth factor (vegf) and heparanase. these factors are likely to partially mediate the ability of eosinophils to promote angiogenesis, by inducing ec growth (vegf) and degradation of perlecan (heparanase), a heparan sulfate proteoglycan component of basement membranes. the relevance of this is that angiogenesis can occur in the context of the granulation stage of vascular healing following stent insertion. the cumulative occurrence of any thrombi that may form as a result of angiogenic events may culminate as a thrombus of significant size, with the ability to dramatically or completely block blood flow in the main stented vessel. table . . summarizes investigations that have linked stent-related thrombosis with eosinophilic inflammation. kawano and coworkers reported that a patient who received a bms to relieve total occlusion of the left coronary artery experienced repeated episodes of restenosis after stent implantation. examination of the restenotic lesion revealed granulation tissue with eosinophil infiltration. the patient displayed positive reactions to allergic patch tests for nickel and molybdenum, both of which are components of the l stainless steel bms that the patient received. thus, through their direct association with granulation tissue formation, eosinophils may contribute to both thrombotic and restenotic mechanisms that pertain to stent deployment. details concerning the former are outlined above, while the latter may be due to activation of platelets by eosinophilic proteins and subsequent platelet degranulation to release promitogenic factors, such as plateletderived growth factor and fibroblast growth factor, capable of stimulating vsmc migration and proliferation. restenosis of revascularization attempts by bms or des implantation are associated with eosinophil infiltration. this was documented nearly years ago, when the transition from poba to poba with bms deployment was being tested for clinical use. karas and colleagues compared histological patterns of restenosis between poba and insertion of a bm tantalum stent into swine coronary arteries. inflammation accompanied by greater vsmc proliferation was observed in association with in-stent restenosis. the inflammatory infiltrate consisted of eosinophils, macrophage-like histiocytes, and t lymphocytes surrounding stent struts. consistent with this, a more recent report found vsmc proliferation to be primarily responsible for restenosis following stenting, but not following poba, in swine. macrophages were selectively found in stented lesions and were accompanied by neutrophils and eosinophils. in another investigation, t lymphocytes were found in restenotic lesions of both poba and bms; however, significantly more vsmcs and eosinophils were associated with in-stent restenosis. these studies suggest a positive correlation between inflammation characterized by eosinophil presence and vsmc proliferation and, similar to observations made relating eosinophils to stent thrombosis, a hypersensitivity reaction to a stent component that ultimately attracts eosinophils to the stented vessel segment. such a relationship between the vsmc proliferative component of restenosis and eosinophil infiltration is significant, given the importance of vsmc growth in restenosis and the fact that des are loaded specifically with antiproliferative compounds primarily intended to block vsmc mitogenesis. interestingly, other studies have shown the selective association of eosinophils, among other circulating cells including inflammatory and bone marrowderived progenitor cells, with in-stent restenosis. gabbasov and colleagues found that the number of osteonectinpositive progenitor cells, but not granulocytes, in blood were higher in patients afflicted with ischemic heart disease (ihd; n ¼ ) than in healthy individuals (n ¼ ). in contrast, only elevations in eosinophils were detected in ihd patients that subsequently received cypher des and experienced restenosis (n ¼ ). blood eosinophil levels were not increased in patients that did not undergo restenosis (n ¼ ). together, histological and blood analyses have established a link between eosinophil presence and restenosis that is particularly associated with stent deployment, compared to poba. these study observations suggest that eosinophils are equipped to contribute to the mechanism of restenosis. however, the question remains: are eosinophilic contents biomarkers of restenosis and thrombosis or do they actively contribute to these processes? niccoli and colleagues showed that serum ecp levels prior to cypher or taxus implantation predicted whether patients would experience a major adverse cardiac event (mace), including cardiac death, recurrent myocardial infarction, or target lesion revascularization (tlr), which was defined as being necessary if > % stenosis occurred within mm upstream or downstream of the stent. the majority ( %) of mace onset occurred days after stent insertion, while % of such cases happened more than year following deployment. clopidogrel was prescribed for months and aspirin for a lifetime after stent insertion. some patients had allergies, none of which were confirmed to stent components, such as metals or polymers. furthermore, ecp levels were nearly equivalent between allergic and nonallergic patients that did not experience mace. what factor(s) could preelevate ecp levels in individuals who had not yet been exposed to potential allergens contained in des? elevated ecp levels may be explained by prior observations indicating that eosinophil count positively correlated with ihd development and that eotaxin/c-c motif chemokine (ccl ) levels may play a role in atherosclerosis. taken together, these studies suggest that ecp may play a causative role in mace, particularly tlr, which represented the majority of mace cases in this study, since it was elevated before stent implantation. related to this issue, experimental evidence implicates eosinophilic contents as having the potential to promote prorestenotic events during the healing process post-stent deployment. for example, as discussed earlier, activation of platelets by both mbp and epo may liberate growth factors from platelets that are capable of inducing migration and proliferation of vsmcs. by secreting eotaxin and transforming growth factor b (tbf-b), eosinophils may directly stimulate vsmc migration and extracellular matrix production by fibroblasts and vsmcs, respectively. each of these manifestations of eosinophilic secretory products would contribute to restenotic lesion development. masu and colleagues described that a heat-labile, unidentified constituent of < kda in eosinophilic lysates can promote proliferation of airway smooth muscle cells (smcs), suggesting the ability of eosinophils to also stimulate vascular smc growth. table . . provides a summary of studies that have reported an association between eosinophilic inflammation and restenosis. further studies involving genetic deficiency or mrna silencing may delineate which specific eosinophil component is responsible for this and other prorestenotic activities. in summary, healing in coronary vessels can be a deleterious phenomenon in two instances, including forming atherosclerotic lesions following ec injury and forming thrombi and restenotic lesions following stent implantation. ironically, treatments to alleviate atherosclerotic burden have evolved to include use of a minimally invasive therapeutic mode, namely deployment of bms and des, that potentially incites yet further, exaggerated, healing responses that can manifest as clinical concerns. this subchapter explains that healing events associated with stent deployment may be two-fold, including standard hemostatic, inflammatory, and tissue-remodeling phenomena that are likely common to all occurrences of stent implantation and, in a minority of individuals, superimposition of such standard healing responses by hypersensitivity reactions to the stent itself. to date, data suggest that both bm and polymer components of des are candidates to stimulate involvement of factors and cells that mediate such hypersensitivity reactions, including eosinophils. basic science studies have revealed the possibility that eosinophils contribute to thrombosis and restenosis associated with stent implantation, by virtue of the potentially prothrombotic effects of their granule proteins, such as ecp, epo, and mbp, on platelets and thrombomodulin, and on smc growth. as the incidence of hypersensitivity reactions as sequelae to stent implantation becomes more evident, routine prophylactic measures may be warranted, in addition to postprocedural dapt prescription, in candidate stent recipients, to include prescreening individuals for allergies to stent components. of course, the integrity of such tests is encumbered by the caveat that positive allergic patch tests may not predict the occurrence of stent-related thrombotic or restenotic events. alternatively, the use of next-generation bioabsorbable stents, which have relatively limited residency times in vessels compared to nonbioabsorbable stents such as cypher and taxus, would theoretically eliminate the stimulus for eosinophilic responses relatively quickly and serve as a reasonable and useful way to reduce complications due to hypersensitivity in vulnerable patients. clearly, observations made of eosinophils in the vicinity of stent struts in association with thrombosis and restenosis are highly suggestive of a role for these cells in such adverse events. further work, perhaps involving eosinophil-deficient animals or cultured eosinophils deficient in granule proteins, is needed to more precisely define the role of eosinophils and the extent of medical attention deemed necessary to mitigate their presumed involvement in stentassociated thrombosis and in restenosis. eosinophils are a minor leukocyte subset in healthy subjects, representing less than % of circulating white blood cells, and present in discrete locations, specifically the bone marrow, digestive tract, mammary glands, thymus, and uterus. , eosinophils belong to the myeloid lineage, and their differentiation and proliferation in the marrow is controlled successively by specific transcription factors [including gata binding protein (gata ) and transcription factor pu. ] and growth factors [granulocytemacrophage colony-stimulating factor (gm-csf), interleukin- (il- ), and il- ]. among the latter, only il- is specific for the eosinophil lineage in humans, as eosinophil precursors express the ligand-binding il- ra on their surface. increased production of il- in vivo by cd þ t cells or transformed cells (e.g., carcinomas or reed sternberg cells ) results in increased eosinophilopoiesis and peripheral eosinophilia in blood and/or tissues. various factors contribute to preferential eosinophil transendothelial migration and homing in tissues, including adhesion molecules (vascular cell adhesion protein ; vcam- ), cytokines (il- ), chemokines [specifically, eotaxins , and , as well as rantes (c-c motif chemokine ) and monocyte chemoattractant protein (mcp)], and arachidonic acid metabolites [leukotriene b (ltb ), cysteinyl-leukotrienes, and prostaglandin d (pgd )], in addition to the more general signals generated under conditions of cell stress and death. eosinophils were long considered as exclusively effector cells, able to induce significant tissue damage and dysfunction by releasing preformed, highly cytotoxic mediators, including the granule proteins, major basic protein and eosinophil cationic protein, producing reactive oxygen species, and generating arachidonic acid metabolites such as platelet activating factor (paf) and ltc (reviewed in ). these effector functions were considered potentially beneficial in the setting of parasitic infections, and harmful in the setting of allergic responses to environmental antigens. recent studies have shattered this paradigm. it is now well established that eosinophils are active participants in ongoing immune responses through the production of cytokines and chemokines, and through previously unrecognized functions of their granule proteins [e.g., the ability of eosinophil-derived neurotoxin, a natural toll-like receptor (tlr ) ligand, to induce dendritic cell maturation and to promote antigen-specific t-helper type (t h )-biased immune responses ] , and that they possess many characteristics of antigen-presenting cells, enabling them to elicit antigen-specific cd þ t cell responses. indeed, eosinophils express major histocompatibility molecule (mhc) class ii and co-stimulatory molecules (cd , cd , and cd ), and were shown to be able to process antigen (ovalbumin; ova) and present it to naive cd þ t cells in lymph nodes, in a murine model of allergic pulmonary inflammation. further upstream in allergic pulmonary inflammation, eosinophils have been shown to be required for the recruitment of antigen-specific effector cd þ t cells to the lungs and the development of typical histopathological changes after allergen challenge in mice. their ability to induce production of the t h chemokines thymus and activation-regulated chemokine (tarc) and macrophage-derived chemokine (mdc) in the lung is critical in this process. in parallel with these proimmune functions, eosinophils also have the potential to modulate local inflammatory responses, for example dampening t h -dominated inflammation through release of il- and il- . production of galectin- (also known as charcoteleyden crystal protein), il- , indoleamine , -dioxygenase, and transforming growth factor b (tgf-b) may confer eosinophils with a regulatory role on effector t-cell responses. e eosinophils also contribute to processes of remodeling and repair. although eosinophils produce several potentially relevant factors, including fibroblast growth factor (fgf- ), matrix metalloproteinase- (mmp- ), and vascular endothelial growth factor (vegf), mechanistic studies establishing a causal role in remodeling are lacking; in contrast, several studies strongly suggest that eosinophilderived tgf-b contributes to airway remodeling in allergic asthma. , a series of observations on eosinophil behavior in health and disease unaccounted for by our current understanding of eosinophil biology, together with the paucity of experimental data supporting a causal relationship between eosinophil cytotoxicity and tissue damage and/or disease, have generated the local immunity and/or remodeling/ repair (liar) hypothesis, which has recently been presented for scientific scrutiny. the authors propose a central role for eosinophils in modulating liar, with recruitment of these cells to sites of cell death and turnover where stem cell activities are operative, in order to maintain tissue homeostasis. the accumulation and functions of eosinophils are dependent on various factors in the local microenvironment, including the presence of other specific immune effector cells, and of soluble growth factors liable to sustain eosinophil survival and activation. at physiological sites of high cell turnover, such as the endometrial lining and the gut, it is assumed that eosinophils dampen immune responses that could be triggered by such active metabolic activity. similarly, eosinophils may inhibit the immune response elicited by tissue-infiltrating helminth parasites, favoring cohabitation between host and pathogen. in contrast, the local production of eosinophil growth-promoting and activating cytokines by other immune cells in allergic inflammation may favor positive feedback loops that sustain and amplify the immune response. , , finally, in addition to the increasing complexity of eosinophil contributions to adaptive immunity, a role for eosinophils in innate immunity was recently suggested by a study showing that eosinophils express variable levels of cd and g/d t-cell receptors, which are involved in antimycobacterial and antitumor immune responses. the inflammation that develops in solid organ transplants in the setting of an alloimmune response (i.e., transplant rejection) may contain, and in some instances be dominated by, eosinophilic infiltrates. whether eosinophils are directly involved in the damage to foreign tissue and thus actively contribute to rejection through release of their cytotoxic mediators, or are engaged in liar activities, is currently unknown. the local release of small molecule mediators, such as damage-associated molecular pattern molecules, by dying cells within the transplant may contribute to very early eosinophil recruitment. eosinophils could theoretically contribute to initiation of the allogeneic response by cross-presentation of foreign mhc antigens; they may also favor local recruitment of allospecific effector t cells, as is seen in allergic inflammation. in mouse models of acute graft rejection in the setting of deficient cd þ t-cell effector functions, namely mhc class ii-incompatible skin grafts and fully histoincompatible cardiac transplants in cd -deficient recipients, marked eosinophilic infiltrates emerge that are dependent on il- produced by antidonor cd t cells. however, il- neutralization or silencing, and associated eosinophil depletion, fails to prevent rejection in these stringent alloreactive models, indicating at most a partial contribution of eosinophils to rejection. this is not surprising, since allograft rejections are mediated by multiple redundant pathways. nevertheless, turning off t-cell cytotoxicity revealed a role for il- and eosinophils in a model of acute and chronic rejection of mhc class ii-incompatible skin grafts. , , in the chronic rejection model, the t h component of alloreactivity and t-cell cytotoxicity were dampened by repeated injections of anti-cd antibody. similarly, treatment with anti-cd and a depleting cd monoclonal antibody ab resulted in eosinophilic infiltration in a model of transplant arteriosclerosis. , in other experiments, the adoptive transfer of alloreactive noncytotoxic t h clones into t cell-deficient mice induced the rejection of skin or cardiac allografts characterized by a dense eosinophil infiltrate. , finally, il- or il- neutralization, as well as eosinophil depletion through repeated injections of anti-ccr (c-c chemokine receptor type ) antibody, prevented the rejection of weakly immunogenic skin grafts bearing a single minor antigen disparity in mhc class i-deficient recipients. the role of eosinophils in renal transplantation has aroused little attention. although publications described the presence of eosinophils during renal allograft rejection in the early s, the first systematic reviews on the subject only appeared in the mid- s. , this may be due to technical reasons. indeed, conventional staining techniques, like hematoxylineeosin (h&e), underestimate the presence of tissue eosinophils and do not usually detect their degranulation. motivated by a case report of marked hypereosinophilia and eosinophilic infiltration in a rejected renal allograft, in weir and coworkers decided to investigate retrospectively a cohort of renal transplant recipients ( biopsies) with episodes of acute rejection. they concluded that increased eosinophils in the blood or renal biopsy represented an adverse prognostic factor for renal outcome. this was followed by a prospective study by kormendi and coworkers analyzing cellular infiltrates using fine-needle aspiration in a cohort of renal allograft recipients during the first month posttransplantation. tissue eosinophilia exceeding % was considered a useful cutoff with a predictive accuracy for serious or irreversible rejections of % (sensitivity: %; specificity: %, with a prevalence of acute rejections of . %). in contrast, blood eosinophil counts were found to be less reliable. in another study, ten and coworkers showed eosinophils in the kidney interstitium and in tubular casts. of note, eosinophil degranulation was evaluated by the extracellular localization of the eosinophil granule major basic protein (mbp) as revealed by immunofluorescence. in this small cohort of patients, eosinophils and extracellular mbp were more frequently observed in acute rejection ( % and %, respectively) than in cyclosporine toxicity ( patients; % and %), whereas both features were absent in controls (normal kidney donors). similarly, urinary levels of mbp were also elevated in acute rejections and acute interstitial nephritis while they remained normal in cyclosporine nephrotoxicity. in another study, eosinophils and extracellular eosinophil cationic protein (ecp) were prominent features of acute vascular rejection rather than interstitial rejection, and eosinophil density increased in areas bordering necrotic tissue and in arteries with necrotic lesions. a correlation between eosinophil infiltrates and rejection severity was also observed by meleg and coworkers, who reviewed allograft nephrectomies. they concluded that a significant interstitial graft eosinophil infiltrate called sige was statistically associated with vascular rejections but not iatrogenic interstitial nephritis. this was already reported by hongwei and coworkers, who also found a correlation between the density of the eosinophil infiltrate and the rate of graft loss by rejection. all together, these observations reinforce the possibility of a nonincidental, causative association between eosinophils and acute allograft rejection (table . . ). eosinophils are also linked to chronic allograft rejection characterized by interstitial fibrosis, obliterative arteriopathy, and tubular atrophy. indeed, nolan and coworkers reported the presence of eosinophils in % of renal allografts undergoing chronic rejection. they were located in the intimal and adventitial space of the thickened arteries, as well as in the interstitium. interestingly, in vitro experiments revealed that eosinophil by-products (see above) enhanced fibroblast and vascular smooth muscle cell proliferation in murine and human experiments, perhaps reflecting a pathogenic mechanism involved in obliterative arteriopathy. although these data strongly suggest a pathogenic role for eosinophils and by-products in kidney allografts, their presence may be related to other functions, as evoked in the liar hypothesis. although eosinophils are described in chronic and acute lung allograft rejection, their role in these processes remains unclear. e acute lung rejection classically occurs during the first year after transplantation and its diagnosis is essentially based on transbronchial biopsies (tbb). the current international guidelines, published in by the international heart and lung transplantation society (ishlt), establish acute lung rejection as the presence of, firstly, perivascular and interstitial mononuclear cell infiltrates (grade a) and, second, small-airway inflammation, namely lymphocytic bronchiolitis (grade b). grading is scaled depending on the composition, extension, and intensity of the infiltrate. eosinophils are not a feature of grade a (minimal rejection), but are found in grade a (mild vascular rejection) and, importantly, are considered to be a common finding in severe vascular rejection (grade a ). regarding airway inflammation, eosinophils are considered to be occasional in low-grade (b r) and common in high-grade (b r) inflammation. chronic lung allograft rejection, synonymous with bronchiolitis obliterans syndrome (bos), occurs in up to % of recipients. bos is characterized by a persistent decrease in expiratory flow and is potentially life threatening, requiring retransplantation. although eosinophils are not taken into consideration in the ishlt working formulation for this condition, a recent prospective cohort study reported that recurrent tissue eosinophilia (with higher concentrations of il- and il- ) is significantly associated with an increased risk of developing bos. increased eosinophilia in lung transplant recipients should be interpreted with caution for two reasons. firstly, there are numerous nonrejection-related causes of graft eosinophilia. among these, infectious diseases are dominant, including fungi (e.g., aspergillus and coccidioidomycosis), bacteria (e.g., tuberculosis), helminths (e.g., toxocara canis and ascaris lumbricoides), and even viruses (e.g., cocksackies). high-dose steroid treatment could be detrimental under these conditions. drug reactions are also a common cause of pulmonary eosinophilia (e.g., antibiotics, diuretics, or methotrexate). the second reason is that blood eosinophilia does not always reflect bronchoalveolar lavage fluid or lung tissue eosinophilia. lung biopsies are therefore crucial for assessing the role of eosinophils after lung transplantation. there are many similarities regarding eosinophilia in liver transplantation compared with other transplanted organs already discussed. il- and eosinophils were rapidly identified during liver allograft rejection. e there was a consensus for considering eosinophils and il- as mediators of a nonclassical pathway of rejection (i.e., non-t h -mediated rejection). ; indeed, liver allografts with evidence of rejection showed concomitant intragraft il- mrna and activated eosinophils releasing mbp. in pediatric recipients, elevated biliary and serum il- correlate with rejection. along the same lines, another study reported that blood eosinophilia and serum ecp are early indicators of acute liver allograft rejection and precede alterations of conventional liver function tests by several days. however, the use of increased serum ecp as a rejection marker is limited by its association with infections. these pioneering findings were confirmed and refined by more recent studies that identified graft and blood eosinophilia as an independent highly specific marker of acute liver allograft rejection. , , , , in addition, an elevated blood eosinophil count may predict the severity of rejection, just as in the case of lung and kidney transplantation. however, the use of this potential marker of rejection is limited in patients with hepatitis c infection, and those treated with corticosteroids, as both these circumstances decrease eosinophil levels. inflammation plays a pivotal role in the complex pathogenesis of acute and chronic graft-versus-host disease (gvhd). conventionally, acute gvhd (agvhd) is described as a t h disease associated with the release of interferon g (ifn-g) , il- , il- , and tnf-a. e the gastrointestinal tract, liver, and skin are the most common targets of gvhd, and diarrhea, jaundice, and skin rash are its most common manifestations. chronic gvhd has features resembling autoimmune and other immunological disorders, such as bronchiolitis obliterans (see above), chronic immunodeficiency, sjögren syndrome, and systemic sclerosis. although the pathophysiology has not been fully elucidated, chronic gvhd appears to be mediated by the overproduction of t h -type cytokines, namely il- and il- . , the role of eosinophils in acute and chronic gvhd is a matter for speculation. thirty years ago, shulman and coworkers showed for the first time that eosinophilia after allogeneic hematopoietic stem cell transplantation (hsct) was often present at the time of diagnosis of chronic gvhd. afterwards, it was shown that eosinophilia could precede, sometimes by several months, the onset of chronic gvhd symptoms, and seemed to have a strong predictive value for the subsequent development of this condition. , should this be confirmed by additional prospective trials, this observation may have significant clinical impact. more recently, eosinophilia was also observed among patients who developed agvhd and, similar to chronic gvhd, was seen before the beginning of symptoms in some cases. , the pathophysiology behind eosinophilia in the setting of gvhd remains unclear. t h cytokine production may be involved, as suggested by the finding that serum il- concentrations are elevated in patients with symptoms of agvhd. , however, no correlation with blood eosinophilia has been observed. studies focusing on the prognostic importance of eosinophilia have produced conflicting data. in chronic gvhd, some retrospective data suggest a better outcome for hsct recipients with eosinophilia, while others found no correlation, suggesting that eosinophilia may just be a bystander of cgvhd rather than a prognostic biomarker. , , among young patients with malignant diseases treated by hsct, those with eosinophilia showed increased event-free survival and a lower relapse rate than those without eosinophilia, suggesting that eosinophils could be involved in the graft versus leukemia effect. in agvhd, observational studies showed that patients with eosinophilia after allogeneic hsct have a milder disease than patients without eosinophilia. one could hypothesize that improved prognosis of agvhd when eosinophils are present is related to the fact based on the histological examination of muscle biopsy sections. infiltration of skeletal muscle tissue by eosinophils is an unusual event, observed especially during parasite infections (including taenia solium, trichinella spiralis, and sarcocystosis) or more rarely bacterial infections (borreliosis). several immune disorders, such as sarcoidosis or rheumatoid arthritis, may also be accompanied by eosinophilic infiltration of the skeletal muscle tissue. these forms must be identified as they may benefit from specific therapeutic management. additionally, some toxic causes have been implicated in the formation of eosinophilic infiltrates in muscle tissue. those include, in particular, the ingestion of certain plant oils (which caused spanish toxic oil syndrome in ) and the eosinophiliamyalgia syndrome, caused by the ingestion of l-tryptophan and presenting with a histological aspect resembling eosinophilic fasciitis, but associated with multisystemic manifestations. e the exclusion of the aforementioned different etiologies determines the diagnosis of idiopathic eosinophilic myositis. depending on the localization of the eosinophilic infiltrate, idiopathic eosinophilic myositis can be classified into three subgroups (reviewed in e ): focal eosinophilic myositis, eosinophilic polymyositis, and eosinophilic perimyositis. focal eosinophilic myositis includes eosinophilic infiltration of muscle tissue with invasion of muscle fibers, and is associated with necrotic fibers. clinically, myopathy preferentially affects the lower limbs, without involvement of skin or fascia. eosinophilia is usually observed, and is associated with elevated serum creatine phosphokinase (cpk) levels. spontaneous or corticosteroid-induced recovery may be observed, but with frequent relapses. at the histological level, eosinophilic polymyositis combines diffuse eosinophilic infiltration of the muscle tissue and at perivenular locations, associated with necrotic fibers. , unlike focal eosinophilic myositis, infiltration is instead located at the perimysium, without muscle fiber invasion. clinical presentation associates myositis with severe systemic symptoms, including possible cardiac and skin involvement. myopathy is preferentially proximal and high serum cpk levels are observed, reflecting extensive muscle damage. corticosteroid treatment can allow recovery, but with possible relapses if not continued long term. in eosinophilic perimyositis, infiltrates predominate at the superficial fascia and perimysium. , there is usually no damage to muscle fibers and, in particular, no necrosis. clinically, a prodromal phase (abdominal pain, arthralgia, and fever) precedes muscle damage, which involves preferential impairment of lower limbs with localized induration. eosinophilia is rare, and serum cpk levels are usually normal. the evolution can be spontaneously favorable. shulman's syndrome, or eosinophilic fasciitis, is a distinct entity characterized by eosinophilic infiltration of the deep fascia, without systemic manifestations, and about half the cases are responsive to corticosteroid treatment. the identification of capn mutations as the first genetic cause involved in eosinophilic myositis indicates that mutations in other genes may also be causal, or act as modifiers, of this pathophysiology. in this regard, a case of dystrophinopathy in which a muscle biopsy shows the appearance of eosinophilic myositis has been described, and baumeister and coworkers recently reported a case of idiopathic eosinophilic myositis caused by a homozygous mutation in the g-sarcoglycan gene, which is implicated in another form of lgmd. interestingly, these 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and chronic rhinosinusitis: detection of staphylococcal exotoxins in nasal polyps specific immunoglobulin e for staphylococcal enterotoxins in nasal polyps from patients with aspirin-intolerant asthma presence of il- protein and ige antibodies to staphylococcal enterotoxins in nasal polyps is associated with comorbid asthma chronic rhinosinusitis: risk factors for the recurrence of chronic rhinosinusitis based on -year follow-up after endoscopic sinus surgery cloning and functional expression of cc ckr , a human monocyte cc chemokine receptor selective for mip- a, mip- b, and rantes cloning, expression, and characterization of the human eosinophil eotaxin receptor detection of the chemokine rantes and endothelial adhesion molecules in nasal polyps characterization of the eosinophil chemokine rantes in nasal polyps detection of the chemokine rantes and endothelial adhesion molecules in nasal polyps the role of rantes in nasal polyposis increased eotaxin-mrna expression in non-atopic and atopic nasal polyps: comparison to rantes and mcp- expression glucocorticosteroids inhibit mrna expression for eotaxin, eotaxin- , and mcp- in airway inflammation with eosinophilia localization and quantitation of eotaxin mrna in human nasal polyps eotaxin- , - , and - immunoreactivity and protein concentration in the nasal polyps of eosinophilic chronic rhinosinusitis patients activation of eotaxin gene transcription by nf-kb and stat in human airway epithelial cells interleukin- , interleukin- , signal transducer and activator of transcription factor , and allergic asthma chitin stimulates expression of acidic mammalian chitinase and eotaxin- by human sinonasal epithelial cells in vitro human eosinophils express messenger rna encoding rantes and store and release biologically active rantes protein increased expression of the chemokine cck in eosinophilic chronic rhinosinusitiswith nasal polyps characterization of the signal transduction pathway activated in human monocytes and dendritic cells by mpif- , a specific ligand for cc chemokine receptor molecular and functional characterization of two novel human c-c chemokines as inhibitors of two distinct classes of myeloid progenitors growth and colony-stimulating factors mediate eosinophil fibroblast interactions in chronic airway inflammation human nasal polyp epithelial basophil/mast cell and eosinophil colony-stimulating activity production of granulocyte-macrophage colony-stimulating factor by cultured human tracheal epithelial cells chronic hyperplastic sinusitis: association of tissue eosinophilia with mrna expression of granulocyte-macrophage colony-stimulating factor and interleukin- allergen-induced release of gm-csf and il- in vitro by nasal polyp tissue from atopic subjects prolongs eosinophil survival the effect of nasal polyp epithelial cells on eosinophil activation comparison of the role of nasal polyp and normal nasal mucosal epithelial cells on in vitro eosinohpil survival. medication by gm-csf and inhibition by dexamethasone il- and rantes immunoreactivity and mrna expression in chronic hyperplastic sinusitis with nasal polyposis (np) bronchial interleukin- and eotaxin expression in nasal polyposis. relationship with (a)symptomatic bronchial hyperresponsiveness direct demonstration of delayed eosinophil apoptosis as a mechanism causing tissue eosinophilia decreased expression of membrane il- receptor alpha on human eosinophils: ii. il- down-modulates its receptor via a proteinasemediated process detection of the chemokine rantes and activation of vascular endothelium in nasal polyps protein biochip array of adhesion molecule expression in peripheral blood of patients with nasal polyposis vcam- and eosinophilia in diffuse sino-nasal polyps eosinophil infiltration in nonallergic chronic hyperplastic sinusitis with nasal polyposis (chs/np) is associated with endothelial vcam- upregulation and expression of tnfalpha eosinophil infiltration is related to increased expression of vascular cell adhesion molecule- in nasal polyps eosinophil adhesion to nasal polyp endothelium is p-selectin-dependent mediators in nasal polyposis evidence for distinct cytokine expression in allergic versus nonallergic chronic sinusitis t-cell regulation in chronic paranasal sinus disease evidence for altered activity of the il- pathway in chronic rhinosinusitis with nasal polyps role of interleukin- a in the eosinophil accumulation and mucosal remodeling in chronic rhinosinusitis with nasal polyps associated with asthma genetic evidence for a role of il in nasal polyposis association of stem cell factor expression in nasal polyp epithelial cells with aspirin sensitivity and asthma expression of eicosanoid receptors subtypes and eosinophilic inflammation: implication on chronic rhinosinusitis protease-dependent activation of nasal polyp epithelial cells by airborne fungi leads to migration of eosinophils and neutrophils local complement activation in nasal polyposis innate immunity of the sinonasal cavity: expression of messenger rna for complement cascade components and toll-like receptors clinical factors influencing the eosinophil infiltration of nasal polyps distinct features of chronic rhinosinusitis with and without nasal polyps pathogenesis of chronic rhinosinusitis epithelium, inflammation, and immunity in the upper airways of humans: studies in chronic rhinosinusitis degranulation patterns of eosinophil granulocytes as determinants of eosinophil driven disease nasal polyposis: from cytokines to growth expression of -lipoxygenase and cyclooxygenase pathway enzymes in nasal polyps of patients with aspirin-intolerant asthma cysteinyl leukotriene expression in chronic hyperplastic sinusitis-nasal polyposis: importance to eosinophilia and asthma measurement of inflammatory mediators of mast cells and eosinophils in native nasal lavage fluid in nasal polyposis secretory iga induces antigen-independent eosinophil survival and cytokine production without inducing effector functions antigen-specific igg and iga, but not ige, activate the effector functions of eosinophils in the presence of antigen iga-induced eosinophil degranulation iga is a more potent inducer of nadph oxidase activation and degranulation in blood eosinophils than ige evidence for intranasal antinuclear autoantibodies with patients with chronic rhinosinusitis with nasal polyps role of igg, iga, and ige antibodies in nasal polyp tissue: their relationships with eosinophilic infiltration and degranulation a clinical and pathologic study of chronic sinusitis: the role of the eosinophil comparison between nasal and bronchial inflammation in asthmatic and control subjects eosinophils are a feature of upper and lower airway pathology in non-atopic asthma, irrespective of the presence of rhinitis rhinosinusitis in severe asthma ultrastructural investigation of epithelial damage in asthmatic and nonasthmatic nasal polyps epithelium dysfunction in asthma mechanisms of eosinophil-associated inflammation protein nitration in chronic sinusitis and nasal polyposis: role of eosinophils epithelial genes in chronic rhinosinusitis with and without nasal polyps kallikrein induces atopic dermatitis-like lesions through par -mediated thymic stromal lymphopoietin expression in netherton syndrome thymic stromal lymphopoietin is released by human epithelial cells in response to microbes, trauma, or inflammation and potently activates mast cells tlr -and th cytokine-dependent production of thymic stromal lymphopoietin in human airway epithelial cells role of thymic stromal lymphopoietin in the pathogenesis of nasal polyposis anti-il- treatment reduces deposition of ecm proteins in the bronchial subepithelial basement membrane of mild atopic asthmatics structural and cellular changes in asthma eosinophils as a potential source of platelet-derived growth factor b-chain (pdgf-b) in nasal polyposis and bronchial asthma transforming growth factor b (tgfb ) gene expression by eosinophils in asthmatic airway inflammation expression of transforming growth factors-alpha and beta messenger rna and product by eosinophils in nasal polyps pharmacology of glucocorticoids in allergic disease glucocorticoid effects on human eosinophils the effects of glucocorticoids on human eosinophils oral steroids for nasal polyps. cochrane database syst rev:cd severe nasal polyposis and its impact on quality of life. the effect of a short course of oral steroids followed by long-term intranasal steroid treatment short course of systemic corticosteroids in sinonasal polyposis: a double-blind, randomized, placebo-controlled trial with evaluation of outcome measures oral steroids and doxycycline: two different approaches to treat nasal polyps nasal polyp inflammation. effect of topical nasal steroid effect of intranasal fluticasone on cellular infiltration, endothelial adhesion molecule expression, and proinflammatory cytokine mrna in nasal polyp disease award lecture atherosclerosis: a defense mechanism gone awry lipid lowering drugs in atherosclerosisdthe hmg-coa reductase inhibitors nonoperative dilatation of coronary-artery stenosis: percutaneous transluminal coronary angioplasty restenosis after percutaneous transluminal coronary angioplasty (ptca): a report from the ptca registry of the national heart, lung, and blood institute neointimal tissue response at sites of coronary stenting in humans. macroscopic, histological and immunohistochemical analysis migration of smooth muscle and endothelial cells: critical events in restenosis angiographic findings of the multicenter randomized study with the sirolimus-eluting bx velocity balloon-expandable stent (ravel): sirolimus-eluting stents inhibit restenosis irrespective of the vessel size taxus ii study group. randomized study to assess the effectiveness of slow-and moderate release polymer-based paclitaxel-eluting stents for coronary artery lesions restenosis following implantation of bare metal coronary stents: pathophysiology and pathways involved in the vascular response to injury long-term outcomes following coronary drug-eluting-and baremetal-stent implantation safety and efficacy of sirolimus-and paclitaxeleluting coronary stents vascular responses to drug eluting stents. importance of delayed healing restenosis and the proportional neointimal response to coronary artery injury: results in a porcine model differential response of delayed healing and persistent inflammation at sites of overlapping sirolimus-or paclitaxel-eluting stents differential healing response in polymer-and nonpolymer-based sirolimus-eluting stents comparison of inflammatory response after implantation of sirolimus-and paclitaxel-eluting stents in porcine coronary arteries eosinophils: biological properties and role in health and disease inflammatory substrate with eosinophils may be present in bare-metal stent thrombosis localized hypersensitivity and late coronary thrombosis secondary to a sirolimus-eluting stent. should we be cautious? repeated stent thrombosis after des implantation and localized hypersensitivity to a stent implanted in the distal portion of a coronary aneurysm thought to be sequela of kawasaki disease: autopsy report correlation of intravascular ultrasound findings with histopathological analysis of thrombus aspirates in patients with very late drug-eluting stent thrombosis coronary intimal proliferation after balloon injury and stenting in swine: and animal model of restenosis mechanisms of restenosis after coronary intervention. difference between plain old balloon angioplasty and stenting eosinophilic infiltration in restenotic tissue following coronary stent implantation drugeluting stent thrombosis. the kounis hypersensitivity-associated acute coronary syndrome revisited granulation tissue with eosinophil infiltration in the restenotic lesion after coronary stent implantationda case report nickel and molybdenum contact allergies in patients with coronary in-stent restenosis evaluation of metal allergies in patients with coronary stents hypersensitivity cases associated with drug-eluting coronary stents. a review of available cases from the research on adverse drug events and reports (radar) project rapamycin inhibits airway leukocyte infiltration and hyperreactivity in guinea pigs pathology of drug-eluting stents in humans. delayed healing and late thrombotic risk the effects of high glucose on human endothelial cell growth and gene expression are not mediated by transforming growth factor-beta drug-eluting stents: sirolimus and paclitaxel differentially affect cultured cells and injured arteries comparative characterization of cellular and molecular antirestenotic profiles of paclitaxel and sirolimus requirement for il- independently of il- in experimental asthma cardiovascular manifestations of hypereosinophilic syndromes activation of platelets by eosinophil granule proteins major basic protein binding to thrombomodulin potentially contributes to the thrombosis in patients with eosinophilia eosinophils as a source of matrix metalloproteinase- in asthmatic airway inflammation il- directly enhanced endothelial cell survival, proliferation, and matrix metalloproteinases production and regulated angiogenesis expression of vascular endothelial growth factor by human eosinophils: upregulation by granulocyte macrophage colony-stimulating factor and interleukin- eosinophil major basic protein: first identified natural heparanase-inhibiting protein human peripheral blood eosinophils induce angiogenesis biology of the eosinophil the eosinophil human th and th subsets: doubt no more non-small-cell lung cancer associated with excessive eosinophilia and secretion of interleukin- as a paraneoplastic syndrome detection of interleukin- messenger rna in reed-sternberg cells of hodgkin's disease with eosinophilia eosinophil-derived neurotoxin acts as an alarmin to activate the tlr -myd signal pathway in dendritic cells and enhances th immune responses airway eosinophils: allergic inflammation recruited professional antigen-presenting cells allergic pulmonary inflammation in mice is dependent on eosinophil-induced recruitment of effector t cells human eosinophils constitutively express multiple th , th , and immunoregulatory cytokines that are secreted rapidly and differentially eosinophils: singularly destructive effector cells or purveyors of immunoregulation? cutting edge: human eosinophils regulate t cell subset selection through indoleamine , -dioxygenase human cd þcd þ regulatory t cells: proteome analysis identifies galectin- as a novel marker essential for their anergy and suppressive function a role for eosinophils in airway remodelling in asthma intravenous anti-il- monoclonal antibody reduces eosinophils and tenascin deposition in allergen-challenged human atopic skin eosinophils in health and disease: the liar hypothesis eosinophils promote allergic disease of the lung by regulating cd (þ) th lymphocyte function schistosoma mansoni egg-induced early il- production is dependent upon il- and eosinophils a functional gammadeltatcr/cd complex distinct from gammadeltat cells is expressed by human eosinophils a role for eosinophils in transplant rejection il- mediates eosinophilic rejection of mhc class ii-disparate skin allografts in mice il- and eosinophils mediate the rejection of fully histoincompatible vascularized cardiac allografts: regulatory role of alloreactive cd (þ) t lymphocytes and ifn-gamma multiple pathways to allograft rejection critical roles for il- , il- , and eosinophils in chronic skin allograft rejection intragraft interleukin- mrna expression after short-term cd blockade may trigger delayed development of transplant arteriosclerosis in the absence of cd þ t cells critical role for il- in the development of transplant arteriosclerosis in the absence of cd -cd costimulation type immune deviation has differential effects on alloreactive cd þ and cd þ t cells heterogeneity of t cell clones specific for a single indirect alloantigenic epitope (i-ab/h- kd e ) that mediate transplant rejection il- deficiency prevents eosinophilic rejection and uncovers a role for neutrophils in the rejection of mhc class ii disparate skin grafts eosinophilia associated with acute allograft kidney rejection the prognostic value of the eosinophil in acute renal allograft rejection the importance of eosinophil cells in kidney allograft rejection role of the eosinophil in chronic vascular rejection of renal allografts eosinophil granule major basic protein in acute renal allograft rejection activated eosinophil infiltration and deposits of eosinophil cationic protein in renal allograft rejection abundance of interstitial eosinophils in renal allografts is associated with vascular rejection eosinophils in acute renal allograft rejection eosinophilic granulocytes and interleukin- level in bronchoalveolar lavage fluid are associated with the development of obliterative bronchiolitis after lung transplantation eosinophilic infiltrates in a pulmonary allograft: a case and review of the literature graft eosinophilia in lung transplantation revision of the working formulation for the standardization of nomenclature in the diagnosis of lung rejection grading of cellular rejection after orthotopic liver transplantation the eosinophil as an effector cell of the immune response during hepatic allograft rejection morphometric inflammatory cell analysis of human liver allograft biopsies eosinophil cationic protein's role in human hepatic allograft rejection blood and graft eosinophilia as predictors of rejection in human liver transplantation evidence for a nonclassical pathway of graft rejection involving interleukin and eosinophils elevated biliary interleukin as an indicator of liver allograft rejection monitoring eosinophil activation and liver function after liver transplantation applications and limitations of blood eosinophilia for the diagnosis of acute cellular rejection in liver transplantation peripheral eosinophil count both before and after liver transplantation predicts acute cellular rejection new perspectives on the biology of acute gvhd pathophysiologic mechanisms of acute graft-vs.-host disease th cytokines (il- , il- and il- ) and il- mrna expression by concanavalin a-stimulated peripheral blood mononuclear cells during chronic graft-versus-host disease biology of chronic graft-versus-host disease: implications for a future therapeutic approach chronic graft-versus-host syndrome in man. a long-term clinicopathologic study of seattle patients eosinophilia after allogeneic bone marrow transplantation using the busulfan and cyclophosphamide preparative regimen peripheral blood eosinophilia has a favorable prognostic impact on transplant outcomes after allogeneic peripheral blood stem cell transplantation eosinophilia predicts better overall survival after acute graft-versus-host-disease hypereosinophilia as a presenting sign of acute graft-versushost disease after allogeneic bone marrow transplantation serum cytokine concentrations and acute graft-versus-host disease after allogeneic peripheral blood stem cell transplantation: concurrent measurement of ten cytokines and their respective ratios using cytometric bead array significance of eosinophilia after stem cell transplantation as a possible prognostic marker for favorable outcome kinetics of serum cytokines after allogeneic bone marrow transplantation: interleukin- as a potential marker of acute graftversus-host disease blood eosinophilia as a marker of favorable outcome after allogeneic stem cell transplantation donor cd -enriched cells of th cytokine phenotype regulate graftversus-host disease without impairing allogeneic engraftment in sublethally irradiated mice pretreatment of donor mice with granulocyte colony-stimulating factor polarizes donor t lymphocytes toward type- cytokine production and reduces severity of experimental graft-versus-host disease ex vivo rapamycin generates donor th cells that potently inhibit graft-versus-host disease and graft-versus-tumor effects via an il- -dependent mechanism combined th cytokine deficiency in donor t cells aggravates experimental acute graft-vs-host disease production of il- by alloreactive sibling donor cells and its influence on the development of acute gvhd a possible association between the presence of interleukin- -secreting cells and a reduction in the risk of acute graft-versus-host disease th and th mediate acute graft-versus-host disease, each with distinct endorgan targets reciprocal differentiation and tissue-specific pathogenesis of th , th , and th cells in graft-versus-host disease eosinophilia indicates the evolution to acute graft-versus-host disease activated eosinophils in upper gastrointestinal tract of patients with graft-versus-host disease immunophenotypic profile of peripheral blood eosinophils in acute graft-vs.-host disease acute flare-up of conjunctival graft-versus-host disease with eosinophil infiltration in a patient with chronic graft-versus-host disease sparing effect by montelukast treatment for chronic graft versus host disease: a pilot study eosinophils and severe forms of graft-versus-host disease mast cells are essential intermediaries in regulatory t-cell tolerance defining a link with asthma in mice congenitally deficient in eosinophils deletion of a high-affinity gata-binding site in the gata- promoter leads to selective loss of the eosinophil lineage in vivo musculoskeletal syndromes in parasitic diseases clinical epidemiology of toxic-oil syndrome. manifestations of a new illness association of the eosinophilia-myalgia syndrome with the ingestion of tryptophan eosinophilia-myalgia syndrome associated with ingestion of l-tryptophan: muscle biopsy findings in patients idiopathic eosinophilic myositis eosinophilic myopathic syndromes relapsing eosinophilic perimyositis diffuse fasciitis with eosinophilia: a new syndrome eosinophilic fasciitis. a pathologic study of twenty cases capn mutations in patients with idiopathic eosinophilic myositis the calpain system sequence comparison among musclespecific calpain, p , and calpain subunits calpain : a key regulator of the sarcomere? insertion sequence of muscle-specific calpain, p , acts as an internal propeptide suppressed disassembly of autolyzing p /capn by n a connectin/titin in a genetic reporter system calpain is activated through autolysis within the active site and lyses sarcomeric and sarcolemmal components mutations in the proteolytic enzyme calpain cause limb-girdle muscular dystrophy type a adults with eosinophilic myositis and calpain- mutations eosinophilic myositis in calpainopathy: could immunosuppression of the eosinophilic myositis alter the early natural course of the dystrophic disease? calpainopathy and eosinophilic myositis eosinophilic major basic protein and interleukin- in eosinophilic myositis characterization of a new p -like calpain form in human lymphocytes eosinophilia of dystrophin-deficient muscle is promoted by perforin-mediated cytotoxicity by t cell effectors human muscle protein degradation in vitro by eosinophil cationic protein (ecp) becker muscular dystrophy presenting as eosinophilic inflammatory myopathy in an infant eosinophilic myositis as presenting symptom in g-sarcoglycanopathy our research is supported by the stanley thomas johnson foundation and swiss national science foundation, bern, switzerland. inflammometry. this chapter is dedicated to the fond memory of professor freddy hargreave who tirelessly argued for the implementation of measurement of bronchitis by sputum examination in clinical practice. the authors would like to thank drs guy delespesse and zoulfia allakhverdi, laboratory on allergy, chum research center, notre dame hospital, montreal, canada, for providing input from their research into tslp effects on progenitor cells (fig. . . ) . thanks also to lynne larocque for her expert assistance with the preparation of the manuscript. funding for this research was provided by the canadian institutes of health research (cihr), and the allergy, genes and environment network of centres of excellence (allergen nce inc.). that this reflects t h , rather than t h (classically considered as the effectors of agvhd cell activation). in agreement, murine studies have shown that several cytokines that are known to favor t h polarization of donor t cells, such as gm-csf, il- , or rapamycin, can reduce agvhd. e in humans, it has been shown that an increased number of il- -and il- -producing cells are associated with reduced severity or absence of agvhd, , also suggesting a possible protective role for t h cytokines in agvhd. nevertheless, reports indicate that t h subsets may actually cause agvhd by targeting other organs than those targeted by t h subsets. , this argues against a rigid paradigm according to which agvhd is a t h process and chronic gvhd a t h process. in support of this hypothesis, prospective data have shown that bone marrow eosinophilia after hsct may be a predictive marker of severe agvhd. similarly, the presence of eosinophils in duodenal biopsy specimens taken during acute flares correlates with intestinal gvhd severity. there is also evidence that eosinophils show signs of activation in both blood and target organs of patients during agvhd flares. , the bad reputation of eosinophils in gvhd generated by these observations has led some investigators to test the efficacy of montelukast (an orally active leukotriene antagonist that inhibits eosinophils) as a supplement to standard therapy for patients with chronic gvhd, with promising preliminary results. for the time being, the role of eosinophils in target organ damage still needs to be assessed in the setting of well-conducted experimental studies. in conclusion, compelling evidence links activated eosinophils with allograft rejection and graft-versus-host disease. in contrast to mast cells, they are not (yet) linked to transplantation tolerance. their presence seems to be correlated with the gravity of tissue damage, which may reflect effector functions contributing to rejection, or eosinophil accumulation in response to tissue damage, in agreement with the liar hypothesis. to date, the roles played by il- and eosinophils in the redundant pathways of allograft rejection and the potential graft healing processes remain cryptic. the recent availability of genetically engineered mice lacking eosinophils (phil and ddblgata) provides a unique opportunity to clarify their contribution to these processes. , chapter . eosinophils and calpain- mutation: a genetic cause implicated in idiopathic eosinophilic myositis eosinophilic myositis is a rare histopathological entity characterized by infiltration of skeletal muscle tissue by eosinophils, possibly in association with peripheral blood and/or bone marrow hypereosinophilia. these characteristics distinguish eosinophilic myositis from other idiopathic inflammatory myopathies, such as polymyositis and dermatomyositis. the diagnosis of eosinophilic myositis is at the current state of knowledge, eosinophilic myopathies constitute a heterogeneous group of rare diseases without a causal factor being identified in most cases.in , our group reported an unexpected clinical observation: a -year-old boy was diagnosed with idiopathic eosinophilic myositis and muscle biopsy analysis revealed a calpain- protein defect. calpain- is a muscle-specific protein, belonging to the family of calpains, nonlysosomal calcium-dependent cysteine proteases. the most well studied of the calpains are the ubiquitous heterodimeric calpains (m-calpain and mcalpain). the human calpain- gene is located on chromosome q . eq . . the predominant product of this gene is encoded by exons corresponding to a bp mrna expressed mainly in adult skeletal muscles. the translation of the main calpain- gene (capn ) product leads to the formation of a kda protein comprising amino acids and consisting of a short n-terminal region (domain i), a papain-type proteolytic domain (domains iia and iib), a c -like domain (domain iii), and a calcium-binding domain composed of five ef-hand motifs (domain iv). in addition, calpain- possesses three unique sequences not found in other calpains: the ns (n-terminal sequence), and the is and is (inserted sequences and ) sequences. is is a polypeptide of about amino acids encoded by an alternative exon . it is composed of an a-helix flanked by loops that close the catalytic cleft, thus blocking access to substrates and inhibitors. it also contains autolytic sites involved in the initiation of calpain- proteolysis by opening the catalytic cleft. immunolocalization studies carried out in human and mouse demonstrated that calpain- located in the n a, m-line, and z-line regions of the sarcomere. in addition to these localizations, calpain- has also been found at costameres and near the triad of the t-tubule. calpain- functions to promote proteolysis of several substrates located in the costameres, sarcolemma, and sarcomere and seems to be most important in fully differentiated fibers (for review see ) . in adult fibers, calpain- participates in sarcomere adaptation by cleaving cytoskeletal proteins during muscular adjustment, in accordance with the distribution of known substrates. mutations in the capn gene cause the most prevalent form of autosomal recessive limb-girdle muscular dystrophy (lgmd), type a (lgmd a), also referred to as calpainopathy. based on the calpain- protein defect identified in the boy with idiopathic eosinophilic myositis mentioned above, the capn gene was analyzed, revealing mutations and thus a genetic cause associated with eosinophilic infiltration in this case. nonspecific inflammatory features may be associated with a variety of muscle dystrophies, possibly leading to misdiagnosis of polymyositis in some cases (e.g., congenital muscle dystrophy, fascioscapulohumeral muscular dystrophy, and dysferlinopathies). however, eosinophilic infiltration had not been previously characterized as a component of inflammatory features in muscular dystrophies.we subsequently identified five additional children diagnosed with idiopathic eosinophilic myositis (exemplified in one patient in fig. . . ) and identified capn disease-causing mutations in all cases, either in a homozygous or compound heterozygous state. following our publication of these pediatric cases, two adult cases of idiopathic eosinophilic myositis and capn mutations were reported by amato. in , oflazer and colleagues reported another pediatric case of idiopathic eosinophilic myositis associated with capn mutations, in which a positive effect of immunosuppressive therapy was observed. since our initial report, we have characterized five additional unrelated patients with idiopathic eosinophilic myositis and capn mutations (one adult and four children, unpublished data). importantly, except for the initially identified boy presenting with a calpain- defect, inclusion criteria for capn mutation screening of the other cases were based only on the particular histopathological presentation, without any identified etiological factors.noteworthy, capn mutations identified in patients with idiopathic eosinophilic myositis do not appear to constitute any particular mutational spectrum as compared to typical lgmd a. our findings demonstrate that at least a subset of idiopathic eosinophilic myositis has a genetic origin, caused by mutations in the capn gene and with an autosomal recessive mode of inheritance. on the other hand, as eosinophilic infiltration is not known to be a typical feature of lgmd a, it is possible that eosinophilia may be a transient feature in the natural course of this disease.the explanation for eosinophilic infiltration correlating with defective calpain- needs to be further evaluated. t lymphocytes may be a key component in this process as: . together with macrophages, they are the main components of inflammatory lesions in the vicinity of damaged muscle fibers. . they play a central role in the chemoattraction of eosinophils by secreting interleukin- , which induces local eosinophil accumulation and they express calpain- . regarding the latter, no relevant function for calpain- in t lymphocytes has been identified to date. the presence of eosinophils has been previously reported to be a component of muscular dystrophy in mdx mice, promoted by perforindependent cytotoxicity of effector t cells. in addition, eosinophils play a specific role in muscle fiber degradation, due to degradation of myofibrillar and membrane-associated proteins by eosinophil cationic protein. key: cord- -gx c mk authors: nan title: cell and tissue reactions date: journal: forensic neuropathology and associated neurology doi: . / - - -x_ sha: doc_id: cord_uid: gx c mk similar types of tissue reaction result as a final common pathway from a wide array of different internal brain pathophysiological states and external insults. since these cellular and tissue reactions are largely independent of the specific type of insults, they are, therefore, non-specific. the tissue reactions are to be differentiated according to their specific pathogenetic mechanisms, though these mechanisms as well as the phenomena are overlapping as demonstrated in fig. . ; brain ischemia as a type of metabolic disturbance, edema, intracranial pressure, necrosis, herniation and inflammation are influencing themselves and are dependent on each other. some will be mentioned again in later chapters as viewed from different forensic aspects; therefore, a certain redundancy is unavoidable. immediately following, we offer a survey of the individual types of reaction and their fundamental pathophysiological principles and morphology. similar types of tissue reaction result as a final common pathway from a wide array of different internal brain pathophysiological states and external insults. since these cellular and tissue reactions are largely independent of the specific type of insults, they are, therefore, non-specific. the tissue reactions are to be differentiated according to their specific pathogenetic mechanisms, though these mechanisms as well as the phenomena are overlapping as demonstrated in fig. . ; brain ischemia as a type of metabolic disturbance, edema, intracranial pressure, necrosis, herniation and inflammation are influencing themselves and are dependent on each other. some will be mentioned again in later chapters as viewed from different forensic aspects; therefore, a certain redundancy is unavoidable. immediately following, we offer a survey of the individual types of reaction and their fundamental pathophysiological principles and morphology. if brain volume increases, both blood and csf are displaced until the intracranial pressure (icp) increases. the consequent pression of the brain against the inelastic dura mater (fig. . ) and the skull can lead to a lethal series of complications in clinical neurology. the following remarks are based largely on ironside and pickard ( ) . brain volume depends on the following factors: . water content (cerebral hydration). the brain has a normal water content of about %. a disturbance of the blood−brain barrier (bbb) can lead to an increase in the fluid content, with a consequent increase in brain volume. . intracranial blood volume (hirai et al. ). this can be driven upward by a number of factors: arterial hypertension (marshall et al. ); enhanced cerebral blood flow secondary to elevated cerebral perfusion pressure (artru et al. ); a decline in the cerebrovascular resistance of arterioles, capillaries, and postcapillary vessels (langfitt et al. ) due to hypercapnia, hypoxemia associated with severe elevation of arterial pressure (marmarou et al. ) , or due to obstruction of the venous outflow of the brain. elevated cerebral blood volume, also known as "brain swelling," is a congestive process. . cerebrospinal fluid (csf) pressure. the central nervous system (cns) of the average adult contains a csf volume of approximately − ml. among the causes of a rise in csf pressure is acute obstructive high pressure hydrocephalus (see pp. f). a number of additional factors may also influence brain volume. the congested brain expands particularly rapidly under high arterial pressure (leech and miller ) . nawashiro et al. ( ) used experimental closed brain injury in rats to demonstrate a rapid and widespread increase in regional cerebral blood flow and impaired cerebral autoregulation. in humans a variety of factors act in concert after the incidence of severe brain injury. cerebral computed tomography (cct) and magnetic resonance imaging (mri) studies have shown that brain edema is the major fluid component of brain swelling (marmarou et al. (marmarou et al. , . a reactive hyperemia is an additional factor and may be the mechanism underlying mechanical/ischemic brain injury (seida et al. ). moreover, regional cerebral ischemia additionally is attributed to a compromised, leaky microvasculature rather than to vasospasm of larger vessels (schröder et al. ). the conclusion that brain swelling is due primarily to edema and not congestion of blood appears to be valid also for children (see chap. , pp. f) . cerebral blood flow in children with severe head injuries is not substantially increased over that in uninjured children (zwienenberg and muizelaar ) . this has also been demonstrated following experimental generation of brain trauma in newborn and juvenile pigs (armstead and kurth ) . the experimental findings of biagas and coworkers ( ) , in contrast, demonstrated a delayed rise in cerebral blood flow following experimental contusion in young and adult, but not in elderly, rats. normal adult icp is less than kpa ( kpa= . mm hg= . torr), mild elevations in pressure range from kpa to kpa, moderate elevations attain kpa, while major intracranial hypertension exceeds kpa (miller and ironside ) . normal csf pressure in adults is − . kpa, with an upper limit of kpa. while a short-term rise in icp pressure of up to kpa may be tolerated so long as it does not cause distortion of the brain (johnston and paterson ) , a mbi-induced rise of more than kpa with distortion of the brain can result in herniation and brain death syndrome. the classic symptoms associated with elevated icp are vomiting, headache, papilledema, and coma. schematic demonstration of overlapping pathogenetic mechanisms that are associated with different types of tissue reactions by a causal link: metabolic disturbance, i.e., edema, increasing cerebral blood volume, perfusion pressure and herniation, i.e., brain swelling and cortical necrosis distortion or pressure on the floor of the fourth ventricle are most likely responsible for the vomiting, while stretching and distortion of the dura mater and major intracranial blood vessels, all sensitive to pain, probably account for the headache. the papilledema is not a direct result of the rise in water content, but the consequence of an accumulation of axoplasm in the optic papilla secondary to the blockade of axoplasmic flow from ganglion cells along the optic nerve. papilledema is a common symptom of chronic intracranial hypertension. it is not, however, a common feature of mbi (selhorst et al. ) , and its absence does not necessarily mean that icp is normal. a further increase of icp leads to a loss of consciousness and coma. elevated icp affects other organ systems as well. it often induces arterial hypertension and systolic blood pressure may climb to kpa or more. the arterial hypertension is caused by an increase in sympathetic activity. cases of raised icp with myocardial involvement often exhibit pathological alterations consisting of subendocardial hemorrhage and widespread focal myocardial necrosis as well as electrocardiographic changes such as t-wave inver- sion and elevation of the st−segment, which point to myocardial ischemia (connor ) . respiratory disturbances associated with elevated icp often precede apnea. central neurogenic hyperventilation is observed in connection with the midbrain lesion. in patients with raised icp, neurogenic pulmonary edema can complicate the clinical course. the mucosa of the digestive and urogenital tracts can become hemorrhagic, eroded, and ulcerated, gastric erosions being particularly common in comatose patients with elevated icp. a fundamental distinction must be made between global and focal cerebral edema. the former follows acute systemic hypoxic events, e.g., transitory cardiac arrest or chronic hypoxia in respiratory diseases. global cerebral edema may also be associated with metabolic diseases, intoxications, and inflammation. focal cerebral edema results from focal tissue destruction or alteration of brain tissue that has undergone membrane failure in cells and vessels due to infarction or traumatic hemorrhage or tumor. the tissue surrounding the central lesion has passed only the upper threshold of electrical silence and thus retains the capacity to recover if perfusion is restored in time (harding and copp ) . this zone resembles the penumbra surrounding the moon in full eclipse (astrup et al. ) . because these tissue changes are partly reversible, they are of considerable therapeutic interest (see pp. f). information on the incidence of intracranial hypertension has been gained mainly from survivors of mbi. miller and his associates ( ) reported that icp levels exceeded . kpa for min or longer in % of patients in one series and in % of another series of patients (miller et al. ) . raised icp was found in more than % of cases in a more detailed prospective study of elevated icp in victims with severe head injury by marmarou and colleagues ( ) . jones and colleagues ( ) found elevated icp in more than % of brain injury patients ( severe, moderate and minor) undergoing artificial ventilation with icp monitoring. these findings indicate that intracranial hypertension is a common event, especially in comatose patients. certain features detected by cct are consistently associated with elevated icp. loss of the images of the third ventricle and perimesencephalic csf cisterns, and dilatation of the lateral ventricle contralateral to a mass lesion, as well as the absence of these features are no guarantee that icp will remain normal (teasdale et al. ; o'sullivan et al. ) . elevated icp is also common in patients who are comatose from causes other than brain injury (chan-dler and kindt ) . among the possible causes are liver failure (hepatic coma), intracranial hemorrhage (subarachnoid and intracerebral), post-hypoxemic states (cardiorespiratory arrest, near drowning), infection (meningitis, abscess, and encephalitis), as well as various other types of inflammation and intoxication. adams and graham ( ) published neuropathological criteria for determining at autopsy whether icp in victims of brain injury was elevated during life. the same team (graham et al. ) compared the nature of the brain damage in patients with and without elevated icp after suffering a non-missile brain injury who had survived long enough to receive treatment in a neurosurgical unit. pressure necrosis of the parahippocampal gyrus, an indicator of high supratentorial icp and tentorial herniation, was present in twothirds of the patients in their most recent study. it was closely associated with skull fracture, brain swelling, diffuse axonal injury, hypoxic brain damage, and extensive supratentorial hematoma. the brain stem was damaged in % of victims with pressure necrosis, the anterior lobe of pituitary was necrotic in %, and there was hemorrhagic infarction in the distribution of the posterior cerebral artery in %. klatzo ( ) distinguishes two types of edema: vasogenic edema and cytotoxic edema (table . ). this distinction continues to be of both theoretical and practical value (cf. kimelberg a, b; mendelow et al. ) . blood vessels in the cns are uniquely restricted in their permeability. many substances are exchanged between the blood and brain parenchyma at slower rates and the concentrations in cns at steady state are lower than in other organs (lee ) . dyes as well as proteins, drugs, and microorganisms introduced into the csf enter the brain freely, while those introduced into the blood stream do not. this limited permeability is attributed to the bbb, a specialized feature of the cns that restricts the entry of viruses and bacteria, emigration of immune cells, and diffusion in the cns of drugs and soluble molecules from the systemic compartment. intravenously applied evans blue will bind to serum albumin and permeate the bbb only under pathological conditions. this phenomenon is demonstrated after experimentally induced ischemia of one hemisphere by means of macroscopic observation ( fig. . a) and by means of fluorescence micros- copy ( fig. . b). the demonstration of albumin in human brain using immunohistochemistry will only succeed during the very early postmortem interval ( fig. . d) − before a general diffusion of blood serum occurs within the brain parenchyma − as a morphological marker of the diffuse postmortem bbb disturbance. under experimental conditions an accumulation of plasma proteins in purkinje cells gencic a, ikegaya et al. ) takes place. anatomically the bbb consists of a capillary endothelium containing intercellular tight junctions and specialized enzymes, such as transpeptidases, dehydrogenases, decarboxylases, and monoamine oxidases (reese and karnowsky ; brightman and reese ; lee ) . the intercellular junctions are most conspicuous near the luminal surface where the cell membranes fuse. a basement membrane in contact with astrocytic foot processes surrounds the endothelial cells. pericytes are enclosed by an envelope of the perivascular basement membrane, which splits to enclose the pericyte. brain capillaries are almost totally invested by astrocytic processes. astrocytes exert inductive actions during development and are thereby largely responsible for the special attributes exhibited by endothelial cells, such as the presence of tight junctions between the cells (abbott et al. ) . astrocytes and microglia both contribute to the formation of the bbb (prat et al. ) . there is an inverse hemodynamic relation between icp and cerebral blood flow (cbf): the higher the icp, the lower the cbf. if cerebral circulation and circulatory autoregulation are normal, a drop in icp will induce only a slight increase in cbf until a threshold level of about kpa is attained. cbf is regulated by mechanisms such as compensatory dilatation of small arteries and arterioles. patients suffering from acute mbi, intracranial hemorrhage, or hypoxic brain injury need a mean arterial blood pressure above kpa to maintain perfusion. the brain damage in such circumstances is associated with a rise in cerebrovascular resistance due to the vessels' spastic reactivity. baseline icp levels may even need to be higher in order to drive sufficient blood through the brain tissue (chan et al. ) . should cbf drop below (ml/min)/ g, potassium levels in the intercellular spaces rise, while intracellular sodium and calcium increase. cellular edema causes the cells to swell and a calcium influx triggers a series of autodestructive processes. the bbb can be compromised by the following three mechanisms (see miller and ironside ) : . enhanced vesicular transport and creation of transendothelial channels by perturbation of endothelial plasmalemma, increased pinocytic activity, the activity of free oxygen radicals, or an increase in superoxides. subarachnoid application of hemoglobin and hemoglobin degradation are known to cause brain edema (huang et al. ) . . disconnection of the interendothelial tight junctions, e.g., by substances of very high osmolarity. . structural or biochemical modification of the endothelial membrane that intensifies its permeability. it has also been known for a long time that the ability of certain substances to pass through the bbb depends on their specific properties: ▬ their nature regarding the capacity of the bbb for active transport (broman and steinwall ) ▬ their affinity for carrier molecules (lajtha ). ▬ their molecular radius (thompson ) . ▬ their lipid solubility (oldendorf ) . as shown in detail later, the permeability of the bbb also depends on age. a good example of this is bilirubin encephalopathy, which is caused by bilirubin crossing the bbb of certain nuclei during the perinatal period − a feat it is incapable of in later life − and inflicting damage on nerve cells and, to a lesser vasogenic perifocal edema in a case of a traffic accident associated with liver failure and elevated bilirubin level. a massive intracerebral hemorrhages and green-colored edema; b focal hemorrhage; perifocal as well as contralateral greencolored edema degree, on astrocytes (for further information see pp. ff). thus for bilirubin at least the bbb appears to be less efficient at birth than in children or adults (haymaker et al. ) . in mbi with intracerebral hemorrhages and associated elevation of the bilirubin level the perifocal edema can be marked by a green color demonstrating extravascular bilirubin as demonstrated in fig. . . in senile and mentally disturbed patients, the bbb has been found to have a lower rate of transport, a reduced uptake of glucose and other nutrients, plus a diminished outflow of metabolic wastes (quadbeck (quadbeck , . the movement of water from the vascular compartment to intracellular or interstitial spaces is not regulated biochemically by the bbb since hydrostatic and more powerful osmotic gradients enable free water to diffuse passively across capillary membranes. the passage of ions and molecules of various sizes is controlled by lipid-soluble substances in the endothelial wall and by ionic channels and active pumps. the white matter of the brain is % water, the gray matter % (adachi and feigin ) . a rise in brain water content entails an increase in brain volume, i.e., brain edema. as a result of energy failure (deprivation of oxygen and glucose), which disables the sodium-potassium membrane atpase pump system, water accumulation within the cells follows an osmotically obliged response to an increase in intracellular sodium and loss of potassium ( fig. . c) . the influx of osmotically drawn water causes swelling of the cell. the energy failure is accompanied by an influx of sodium and chloride and an efflux of hydrogen ions, potassium, and bicarbonate. there is a parallel disturbance of the voltage-and ligand-gated mechanisms that regulate the entry of calcium into the cell that initiates a calcium-mediated destructive sequence. cytotoxic edema is the result of the action of various cytotoxic agents, e.g., of cyanide or triethyl tin (also see chaps. , ). brain cells can also swell without a concomitant increase in brain volume if fluid shifts from an extracellular to an intracellular space. although this does not produce an immediate rise in icp, cellular edema ultimately draws water from the vasculature into the brain, increasing brain volume and precipitating a rise in icp. ischemic edema is a cytotoxic edema whose clinical effects depend on how much and for how long cerebral blood flow is reduced (bell et al. ) . klatzo ( ) showed that the initial cytotoxic edema following permanent occlusion of a major blood vessel causes irreversible ischemic cell damage, resulting in a secondary vasogenic edema when endothelial cells are damaged. even temporary ischemia with subsequent reperfusion will induce reactive hyperemia and secondary endothelial damage that produces a secondary vasogenic edema (greenwood ) . as a whole, the brain is resistant to pure hypoxia (diminished oxygen supply) (pp. ff, see also miyamoto and auer ) , which causes no or only partial breakdown of the bbb and which may be reversible after recovery (bakay and lee ; auer ) . anoxia (complete lack of oxygen), by contrast, results in a rapid rise in bbb permeability that becomes irreversible after just a few minutes (pp. ff). if anoxia acts in combination with complete ischemia secondary to ligation of both common carotid and subclavian arteries, the bbb can retain its impermeability for as long as h (broman ; blank and kirshner ) . incomplete ischemia, however, will rapidly and completely break down the bbb (bakay and lee ) . kimelberg and colleagues ( ) could demonstrate the potential toxic mechanisms of this type of edema on neurons. they describe a primary cytotoxic effect on astrocytes that induces astrocytic swelling. this swelling in turn leads to the release of excitatory amino acids such as glutamate, whose levels increase in extracellular spaces following the incidence of mbi (kanthan and shuaib ) . a rise in extracellular glutamate levels causes cell death due to an influx of ca + via the neurons' activated ionotropic glutamate receptors (choi and rothman ) . other authors have offered a somewhat different explanation for the irreversible injury: it may be induced by simultaneously generated free radicals or extravasated plasma components that stimulate the activation of nitric oxide synthase (nos) in reactive cells. nitric oxide thus generated may contribute to diffuse degeneration of the white matter (gotoh et al. ). the accumulation of plasma proteins within neurons and microglia in combination with cytochrome-c release by astrocytes can lead to dna fragmentation and cell death (matz et al. ). klatzo's ( ) classification of brain edema has proved to be a useful aid in distinguishing between various pathogenetic mechanisms and their sequelae. in experimental and clinical practice however it must be assumed that brain swelling is caused by a combination and/or a temporal overlapping of a number of processes, as described by kimelberg et al. ( ) . non-invasive diffusion-weighted (dw) mri is able of calculating changes in the apparent diffusion coefficient (adc) of water protons in the brain (garcia et al. ; chu et al. ) . a decline in adc has been attributed mainly to a reduction of the extracellular space and a rise in intracellular volume, although other contributing factors are possible (pierpaoli et al. ) . in this manner cellular (cytotoxic) edema can be differentiated from extracellular (vasogenic) edema and a correlation made with the severity of injury and consequent deficit. because dw-mri (see also mendelow et al. ) enables edema types to be determined intra vitam under clinical conditions, current classifications of edema types could be revised in light of new findings. however, we should remember that all edema ultimately arises from the blood. it is the size of the leak in the brain vasculature that gives rise to the artificial distinction between cytotoxic and vasogenic edema, cytotoxic edema being mainly water and vasogenic edema including proteins also derived from the blood. brain swelling caused by edema, congestion or a rise in csf pressure can obliterate the subarachnoid space, flatten the gyri, reduce ventricular size (squier , fig. . ), and cause herniation (see pp. ff). at autopsy the white matter seems softer in consistency and paler than normal. the normal, sharp demarcation between gray and white matter is lost, often with thinning of the cortex overlying the zone of white matter edema. the arcuate fibers are spared. in rare cases of vasogenic edema associated with liver insufficiency combined with elevated bilirubin levels in serum the spread of edema may be characterized by a greenish-yellow color ( fig. . ) . under normal conditions bilirubin is not able to permeate the bbb, with one exception: the newborn (see pp. ff). cytotoxic edema, which predominates in gray matter, is characterized by astrocytic swelling and the enlargement of perineuronal and perivascular spaces indicative of the swelling of astrocytic foot processes around neurons, capillaries, and arterioles ( fig. . ). the hallmarks of vasogenic edema include swelling of pericapillary astrocytic processes and of oligodendrocyte cytoplasm, plus the spread of exudate in the extracellular space of white matter ( fig. . ). macroscopically vasogenic edema induces a slight green discoloration of the white matter. histologically, edema, vasogenic edema in particular, features extensive cytoplasmic vacuolation in the white matter with status spongiosus where a clear space surrounds small vessels and nuclei ( fig. . ) . ultrastructurally, few visible changes are evident in the cerebral capillaries. the brain parenchyma, in contrast, exhibits swelling of glial processes or dendrites, splits in the myelin laminae and, less often, enlargement of the extracellular space. vacuolation may be especially prominent in myelinated fiber bundles and constitute the earliest and most consistent elementary edema-induced change. following immersion fixation, however, these phenomena can often be difficult to distinguish from (postmortem) artifacts. these phenomena may be associated in the beginning with a leukocyte emigration ( fig. . a) and in the last stage with astrocytic hyperplasia and hypertrophy ( fig. . c, d). astrocytes and macrophages also ingest extravasated plasma proteins. myelin sheaths undergo increasing fragmentation and macrophages phagocytose lipid breakdown (figs. . f, . b, . ) . oligodendrocytes are much less likely to partake in the alterations of edematous brain tissue. most cases of brain edema exhibit a combination of cytotoxic and vasogenic edema. inhibition of ion pumping or secondary retrograde reaction can cause swelling of neurons. the usual reaction is neuronal shrinkage, commonly combined with swelling of neighboring glial cells, especially of astrocytic processes. irreversible changes in the myelin sheath are unequivocally manifested by the apposition of mac-rophage reaction in the form of compound granular cells. involvement of the white matter by edema of this severe degree coincides with a so-called edematous necrosis (jacob ) . the final phase of terminal edema can be marked by cystic alteration or glial scaring. brain swelling is one of a wide variety of neurological conditions, among them tumors, hemorrhages, and ischemia/hypoxia, that can induce an increase in icp. a rise in icp leads not only to compression of the brain, but to diminished csf volume, shifting, and herniation, as well as to secondary complications such as ischemia and hemorrhage. if not treated, icp can rapidly progress to death due to brain stem compression secondary to cerebellar or uncal herniation (meyer ) . focal expanding mass lesions must be distinguished from diffuse space-occupying processes. ▬ diffuse brain lesions such as inflammation, bilateral intracranial hemorrhage, total brain ischemia (cardiac arrest) or intoxication are macroscopically characterized ( fig. . ) by a tension of the dura, gyral flattening, and by narrow ventricles that are symmetrically compressed. no lateral shift of midline structures is seen, but rather central herniation of the diencephalon (centrencephalic herniation) and by cerebellar tonsillar herniation and compression of the medulla oblongata. bilateral herniation may occur and various types of herniation result from caudal displacement of the brain parenchyma (for types of herniation, see below). ▬ focal intracranial processes such as abscess, tumor, infarction or subdural hemorrhage ( fig. . a, b) are also capable of inducing a life-threatening homolateral rise in icp. because they allow time for intrinsic compensatory mechanisms to operate, particularly reduced csf volume, slowly expanding focal lesions are less likely to cause an early increase in icp and brain shift. however, the distortion and herniation of the brain in such cases can be considerable. rapidly expanding focal lesions, by contrast, are more likely to produce an immediately elevated icp. brain death often supervenes in such cases before much distortion or herniation can occur. distortion of the brain results from compressive forces exerted by adjacent structures, which leads to overall expansion of the hemispheres. the dura mater may become so tense as to compress the terminal branches of the cerebral arteries, with consequent ischemic or hemorrhagic necrosis of cortical structures (lindenberg ) or impairment of perfusion (janzer and friede ) accompanied by perisulcal infarcts. continued expansion of the mass may provoke contralateral displacement of the midline structures (see chap. and fig. . a). if the contralateral foramen of monro is obliterated, the contralateral lateral ventricle may become enlarged, triggering a further rise in icp. a lesion that expands in the frontal lobe may displace the free margin of the anterior part of the falx cerebri ( fig. . a, d). if a lesion expands in the temporal lobe, a disproportionately pronounced shift of the third ventricle will occur ( fig. . a), with upward displacement of the sylvian fissure and neighboring branches of the middle cerebral artery. the ultimate result of the space-occupying process is development of lateral and then downward herniation, visible at several loci: . falx cerebri (cingulate, or subfalcine herniation). . tentorium cerebelli (lateral, or uncal herniation). . thalamus/hypothalamus (central, or diencephalic herniation) which may result in downward displacement and hemorrhage in the midbrain and pontine tegmentum. . foramen magnum (tonsillar herniation). a bilateral expanding supratentorial mass can cause herniation-induced notches as well as hemorrhages of the uncal area. this in turn exerts downward pressure on the medial part of the parahippocampal gyrus toward and through the tentorial incisura (figs. . b, . ). the clinical and morphological se- d a hemorrhage in the upper frontal lobe may lead to a notch, a hemorrhage or a softening of the corpus callosum; e a rare tentorial displacement caused by an infratentorial space-occupying process is demonstrated by notches on the upper cerebellar surface (arrows) quelae of uncal herniation depend on the magnitude of the supratentorial pressure and on anatomical variations in the size of the tentorial notch, position of the brain stem within the notch, position of the oculomotor or third nerve, and the inter-oculomotor nerve angle. they also depend on the structure and course of the posterior cerebral artery, known to play a role in herniation syndromes (adler and milhorat ) . herniation of the parahippocampal gyrus ( fig. . ) creates narrowing of the midbrain along its transverse axis and compression of the aqueduct. this pushes − in the case of a unilateral expanding mass − the contralateral cerebral peduncle against the opposite free tentorial edge ( fig. . b), pinning the ipsilateral oculomotor nerve between the petroclinoid ligament or free edge of the tentorium and the posterior cerebral artery. the lesion of the ipsilateral oculomotor nerve is associated clinically with ptosis and dilatation of the ipsilateral pupil, which becomes unresponsive to light. the elevated icp produces a wedge of hemorrhagic necrosis along the parahippocampal gyrus groove (so-called pressure necrosis, to be differentiated from "herniation contusion" − see figs. . b, . b) . pressure necrosis can result from an icp exceeding . kpa (see adams and graham ) . it is analogous to necrosis due to brain retractor pressure in neurosurgery. clinically, uncal herniation is accompanied by an abrupt worsening of the patient's neurological status, with loss of consciousness and onset of decerebrate rigidity, both due to midbrain impairment caused by pressure coming from above. compression of arteries can also cause secondary necrosis: if the anterior choroidal artery is occluded, the result can be infarction of the medial part of the globus pallidus; posterior cerebral artery occlusion can cause hemorrhagic infarction of the thalamus, of the medial and inferior surfaces of the cortex of the occipital lobe (figs. . , . ) , and of the temporal lobe including the hippocampus. herniation of the ipsilateral cingulate gyrus under the free edge of the falx results from the unilateral growth of a mass in the frontal or parietal lobe and causes selective displacement of the pericallosal arteries away from the midline (fig. . a, d) . should this compromise circulation through the pericallosal arteries, the parietal parasagittal cortex can become infarcted, which is expressed clinically as weakness or sensory loss in one or both legs. a frontal or parietal mass lesion can induce downward axial displacement of the diencephalon (fig. . a) and rostral brain stem (figs. . c, . a) . the consequent symmetrical herniation of both parahippocampal gyri (fig. . a , b) may be manifested clinically by bilateral ptosis and failure of upward gaze. the final clinical result is loss of consciousness, decerebrate rigidity, and bilateral dilatation of the pupils with loss of the pupillary light reflex. the blood pressure rises due to increased sympathetic activity. hemorrhage or necrosis of the midbrain and/ or pons are the possible sequelae of supratentorial space-occupying processes located adjacent to the midline (figs. . a, . a, . ). these lesions are caused by caudal displacement and anterior-posterior elongation of the rostral brain stem and by sideto-side compression by the tentorial hernia, coupled with relative immobility of the basilar artery. progressive displacement stretches and narrows the central perforating branches of the basilar artery which supply the rostral brain stem, causing spasm, infarction or hemorrhage. an early complication of expanding masses in the posterior cranial fossa is displacement of the cerebellar tonsils through the foramen magnum (figs. . d, . b, c) . this may also be caused, however, by lesions occupying the supratentorial space. morphologically, the tips of the tonsils display hemorrhagic necrosis and grooving of the ventral surface of the medulla where it impinges on the anterior border of the foramen magnum. clinically these changes give rise to apnea, which can occur while the victim is still conscious. among the other common neurological deficits are decerebrate rigidity and impairment of brain stem reflexes. upward tentorial displacement (fig. . e) is produced by enlargement of an infratentorial mass in the posterior cranial fossa. both the fourth ventricle and aqueduct become compressed and displaced contralaterally. there is upward herniation of the superior surface of the cerebellum, which is distinguished clinically by the abrupt manifestation of bilateral extensor rigidity and loss of pupillary light response. a number of clinical complications associated with brain swelling, brain edema, and bbb can arise during diagnostic and therapeutic interventions in the cns carried out by physicians or nursing personnel. since the sequelae are foreseeable − and in most cases avoidable − these complications will be dealt with briefly in the following. ▬ in patients with elevated icp, a lumbar puncture of the csf can give rise to herniation. papilledema, though not always associated with icp, must be excluded before every csf puncture. should other clinical signs of high icp be present, a ct examination must be carried out prior to puncture. ▬ pharmacotherapy must not be performed without knowing whether the agent can permeate the bbb and affect the cns. this is especially true of substances such as antibiotics or cytostatics that are intended to reach and act upon the cns. other substances are not intended to reach the cns because they are toxic there; thus, the contraindication for intrathecal application of vincristine (see p. ). ▬ if cns edema already exists, the bbb may be (pathologically) permeable to substances not intended to reach the cns, some of which can then have a toxic effect. a mbi-induced perifocal edema, for example that arises in the context of polytrauma-induced shock, can produce greenish discoloration of the perifocal edema as a consequence of an accompanying hepatic insufficiency, which can have an additional neurotoxicologic effect on the neurons. ▬ the status of the bbb may well be age dependent, its postnatal status differing from that of adults. blood group incompatibility between mother and child during pregnancy or after birth can cause bilirubin encephalopathy (see pp. ff). the bbb appears to be less permeable in the elderly. ▬ a cytotoxic effect can be mediated by alcohol in mbis with consequent loss of neurons. alcohol lowers cerebral perfusion pressure (cbf) and depresses ventilation. it diminishes respiratory drive in response to elevated paco levels. ethanol-induced respiratory depression and hypotension can increase the morbidity and mortality associated with brain injury. the theoretical considerations of kimelberg et al. ( ) appear to contradict these empirical findings, arguing rather that alcohol inhibits both the excitotoxin receptor function of neurons (simson et al. ) and the influx of ca + via nmda receptor ion channels. hydrocephalus is characterized by abnormal accumulation of fluid within csf spaces, i.e., within the cerebral ventricles and subarachnoid space. by this time, there is atrophy of the brain parenchyma and additional ventricular enlargement. csf is formed by the choroid plexus at a rate that remains unchanged within a wide range of icp values: − ml/h will avert a long-lasting imbalance between its formation and absorption. elevated csf pressure is associated only with acute or obstructive hydrocephalus (see below). the subarachnoid space and ventricular system are connected via the foramina of luschka and magendie in the basal cisterns. csf is absorbed by the arachnoid villi, which do not fully develop until adolescence or young adulthood (grassman and potts ) . in fetuses and infants, csf is absorbed mainly through nerve roots and periventricular and arachnoid veins. external hydrocephalus ex vacuo involves diffuse loss of gray matter that gives rise to external atrophy, with dilatation of the subarachnoid space ( fig. . a , b). diffuse loss of white matter can cause expansion of the ventricular system, the so-called internal hydrocephalus ex vacuo (fig. . c ). the causes of the gray and white tissue damage may vary, but csf kinetics and anatomic pathways are important considerations (see also pp. f). a distinction is made between normal pressure hydrocephalus of still unknown etiology (adams et al. ; hurley et al. ) , low pressure hydrocephalus, and high pressure hydrocephalus. the latter is caused by accumulation of fluid secondary to elevated csf production (hypersecretory hydrocephalus) or insufficient resorption (malabsorptive hydrocephalus or communicating hydrocephalus). disten-sion of the ventricles results from pressure-induced fluid build up in the cerebral ventricles (reversible) or from pressure-induced (irreversible) atrophy as a result of parenchymal loss. fluid may also enter the periventricular tissue (trans-ependymal resorption of csf seen on neuroimaging). normal pressure hydrocephalus can feature repeated brief episodes of elevated icp, possibly in the form of an intermittent pressure or occult hydrocephalus. normal pressure hydrocephalus can also result from a hydrocephalus ex vacuo, which is associated with primary ventricular system enlargement and white matter destruction. it is often found in victims of severe brain injury, in alcoholics, and vascular disease patients with multi-infarct dementia or other types of progressive degenerative brain disorders, especially age-dependent dementia (miller and ironside ) . hydrocephalus ex vacuo can also be caused by long-lasting generalized brain edema with high icp. the causes of obstructive hydrocephalus are mbi, subarachnoid hemorrhage, meningitis and arachnoid fibrosis, arnold-chiari malformation, aqueductal stenosis (for complete list see table . , for review see garton and piatt ) . the latter can be either congenital due to atresia or acquired due to inflammation, compression or reactive gliosis, hemorrhages or tumor. the responsible congenital abnormalities consist in most cases of replacement of the aqueduct lumen by numerous random, narrow channels or ependymal nests. external hydrocephalus, whatever the causes are, exhibits dilation of the subarachnoid space, with no increase in collagenous fibers or cellular elements, but an increase in csf. the causative encephaloclastic disease may be diagnosed on the basis of other phenomena, such as liver cirrhosis in alcoholics, or loss of neurons in the presence of plaques and tangles in senile dementia or alzheimer's disease. typical macroscopic features of internal hydrocephalus include an enlarged ventricular system ( fig. . a, b) (weller and shulman ) , interstitial edema, disruption of the ependymal cells lining the ventricle, and axonal and myelin destruction in the periventricular white matter (del bigio ) . secondary changes in neurons reflect compensation to the stress or ultimately the disconnection. proliferating astrocytes and/or gliosis (fig. . c) replace in part the interrupted ependymal cell line. these glial nodules appear granular or like small tumors (fig. . c ) upon macroscopic inspection of the inner surface of the ventricles (fig. . b) . there is also a partial reestablishment of flattened ependymal cells, and a decline in the number of axons with parallel proliferation of glial fibers in the periventricular white matter. in chronic hydrocephalus with high pressure hydrocephalus, a flattening of the gyral crests is seen (fig. . a ); in addition a small reactive glial zone around the ventricular system (fig. . b ) may develop which can be separated from the intact white matter at autopsy (fig. . c) . in adults, the clinical symptoms of hydrocephalus are non-specific; in infants and children they may be specific (see chap. − pp. f) and depend on the causes and the time course of the hydrocephalus. the salient symptoms comprise psychopathological alterations such as dementia, memory disturbances, and loss of orientation, culminating in the most severe cases in loss of consciousness. the first step in diagnosing hydrocephalus involves the use of imaging techniques, mri and cct, to establish its presence. the second step seeks to determine the underlying cause, and again employs as its methods of choice mri and cct, in combination with clinico-chemical (fishman ) and cytological analysis of the csf itself (oehmichen a ). "necrosis" (for review see lindenberg ) is commonly used to designate the death of tissue components, including that of cells and their processes in a defined area of blood and oxygen supply. after a severe episode of ischemia, mbi or epilepsy, it is typical to find necrotic cell death within the injury core. in addition, a substantial number of neurons in regions surrounding the injury core have been observed to die via the programmed cell death pathways due to secondary effects derived from the various types of insults (liou et al. ) . "apoptosis" (for review see vermes et al. ) is applied to the selective (programmed) death of one or more individual cells. apoptosis is the more active and physiological form of cell death. in necrotic cell death, a stimulus such as ischemia, hemorrhage, mechanical or chemical damage alters cell homeostasis thus causing cell death, whereas in apoptosis an internal death stimulus triggers the innate cellular suicide program, the latter (not the stimulus itself) mediating the cellular demise (beal ) . in the following, the various pathogeneses and mechanisms of these types of cell death will be described, along with their different morphologies and underlying molecular factors. the most common cause of necrosis is ischemia. other causes include mechanical injury (contusion fig. . a−c. internal hydrocephalus. a expanding ventricular system associated with an atrophy of white matter; b the ventricular surface is commonly marked by a granular surface structure, which (c) microscopically is characterized by multiple glial nodules which replace lost segments of the ependymal layer (h&e, magnification × ) necrosis), toxic agents (formic acid in methyl alcohol), heat (thermocoagulation), freezing (cryosurgery), infections (poliovirus), and overexposure to ultrasound. each case, however, involves the action of additional factors independent of the type of primary traumatic event. chief among these factors are free radicals and nitric oxide (no). reaction products of no and o , including potent oxidizing molecules such as peroxynitrite and nitrogen dioxide, can be more toxic than no itself. the type of brain necrosis depends in large part on the duration of local circulatory arrest: . transient ischemia only destroys neurons and oligodendrocytes, inducing incomplete necrosis or selective neuronal necrosis (scholz ). . prolonged ischemia, termed "infarction," gives rise to complete necrosis of all tissue components. morphologically cell necrosis, especially neuronal necrosis, features irreversible changes of the cytoplasm (condensation, hydropic swelling, intense eosinophilia, loss of structure, homogenization) ( fig. . ) and of the nucleus (pyknosis, karyolysis, karyorrhexis) (majno and joris ) . time course. the data vary on how soon after the onset of ischemia the first microscopic neuronal changes become evident. some authors report an interval of min (jacob and pyrkosch ) , others / h (müller ) . the data may differ in part due to prolongation of the necrotic process for as long after death as brain temperature remains favorable (lindenberg ). prolonged ischemia-induced tissue necrosis is termed "infarction" or liquefactive necrosis. the infarcted area displays macroscopically evident pallor on h&e, nissl, and myelin preparations within − h as an indication of acidosis. a narrow halo of even greater pallor surrounds the necrotic area ( fig. . a) . around the necrotic area, vessels are distended and release fluid into the infarcted tissue and surrounding tissue by way of a vascular network (perifocal edema). neurons become thorny and severely shrunken within − h, with darkly staining incrustation of their pericellular structures. a survival time of h leads to homogenization of the cytoplasm and nuclear and cytoplasmic pallor. between h and h, the neurons disappear except for the nuclei (see fig. . c) . within − h the necrotic tissue is characterized by an emigration of neutrophil leukocytes (fig. . a, b) . within h the necrotic area exhibits proliferation and extensive activation of microglial cells (fig. . c−f). along the infarct margin, macrophage numbers increase. hypertrophic astrocytes appear along the border zone within the brain parenchyma after − days ( fig. . ) . the infarct liquefies at its center and macrophages phagocytose the debris. the final stage of cortical liquefactive necrosis is termed "laminar necrosis" (fig. . a−c) associated with intense gliosis during the final phase (fig. . d) . the final stage of ischemic involvement of the basal ganglia and the thalamic nuclei is a cystic necrosis (fig. . ) . ischemic damage of the hippocampal area is characterized by a segmental loss of neurons in the hippocampal cortex ( fig. . a, b) associated with compensatory (early) microglial activation (fig. . c ) and secondary gliosis (fig. . d) . another type of brain tissue necrosis is the rare phenomenon "persistent coagulative necrosis" first described by spielmeyer ( ) (fig. . ) , which affects cell tissue elements. within gray matter the outlines of dead neurons are recognizable, their cytoplasm is intensely eosinophilic and usually contains numerous, often large, vacuoles, and the nucleus stains poorly with hematoxylin. this picture, which is recognizable within the first − h, is followed by decreasing stainability of nucleus and cytoplasm until a barely recognizable ghost cell is all that re-mains. acute and enduring deprivation of blood supply causes necrosis of cells and tissue components, the lesion remaining in a state of "coagulation" for a prolonged period. the cells appear only as shadows and the necrotic tissue persists within the brain as a foreign body and sometimes becomes encapsulated in mesenchymal tissue. the pathogenesis of this rare type of necrosis is still unknown (escolá and hager ; cervos-navarro and ferszt ) . apoptosis can be differentiated from necrosis based on differences in their pathogenesis, cell reactions, and morphologic features. apoptosis is the programmed death of a cell as regulated by specific death genes (for review see clarke ). it initiates a delayed secondary death of neurons in response to environmental changes, deficient metabolic and trophic supply, and changed gene transcription. during apoptosis, the integrity of mitochondria is compromised and various pro-apoptotic proteins are released into the cytoplasm. this results in activation of caspases, proteases that orchestrate the death of the cell (waterhouse ). apoptosis requires active protein synthesis (mcintosh et al. ; raghupathi et al. ) . a single cell can undergo a switch between the two types of cell death based on several pathways (mcconkey ; fiskum et al. ; see also leist et al. ) (for further informations, see p. ). the characteristic morphology of apoptosis exhibits cleavage of the internucleosomal chromatin that can be identified in situ using the terminal deoxynucleotidyl transferase-mediated dutp nick end-labeling (tunel) method (gavrieli et al. ) . apoptosis causes pyknosis of the nucleus and condensation and shrinkage of the cell body. as it progresses, budding and karyorrhexis occur, and ultimately a breakup into clusters of apoptotic bodies (majno and joris ) . the time course of apoptosis following a traumatic event is as follows: about h after a traumatic event, apoptosis begins, and remains demonstrable for about days (yakovlev and faden ) . three major factors are known to participate in the apoptotic cascade of "delayed" neuronal death (for review kermer et al. ; see also huppertz et al. ): . immediate early gene transcription factors (cjun, jun-b, jun-d, c-fos, ap- , atf, nf-κb) . proteases (calpains, caspases) . glutamate-mediated toxicity (free radicals, protein-kinases, ca + homeostasis, second messenger systems) the death-inducing activity of the bax, bad, bid, bcl-x s family members is thought by raghupathi et al. ( ) to be in dynamic equilibrium with their survival-promoting cognates bcl- , bcl-x l . shifts in the protective intracellular bcl- -family-protein levels can tilt the balance toward cell death by activating the death-inducing cysteine proteases, caspases (thornberry and lazebnik ) . the death of single cells releases insufficient quantities of chemoattractants to allow effective concentrations of molecular species to reach the vascular endothelium. for this reason a genuine cell reaction does not occur. neighboring cells that are not professional phagocytes cannibalize the cell debris in a process specific to apoptosis (majno and joris ) . in inflammatory diseases, an essential factor in the resolution of the inflammatory attack is the clearance of apoptotic leukocytes by tissue-specific phagocytes (platt et al. ). this process has been termed the "safe, phagocytic clearance of dying, yet intact leukocytes undergoing apoptosis" involving rapid recognition, uptake, and degradation. microglial cells were recently shown to be capable of protecting neurons, cerebellar granule neurons in particular, from apoptosis (polazzi et al. ): molecules are released by apoptotic neurons that enable the anti-apoptotic activity of microglia. in vitro, normal microglia release molecules capable of rescuing neurons from apoptotic death. microglia, astrocytes, and oligodendroglia may participate in apoptotic or necrotic processes. the reaction of neurons highly sensitive to injuries such as ischemia, hypoglycemia, infection, and mechanical trauma are described above and classified systematically in table . . a review by rosenblum ( ) includes a comparison of various hypotheses regarding the underlying causes of "delayed" neuronal death, among them excitotoxicity, calcium, and apoptosis. rubin ( ) and abe ( ) review the phenomenon of neuronal apoptosis as it appears in various neurological diseases. astrup et al. ( ) first defined the ischemic penumbra as brain tissue perfused at a level within the thresholds of functional impairment and morphologic integrity, which has the capacity to recover if perfusion is improved. because tolerance of tissue to ischemic damage is dependent on residual flow and duration of flow disturbance (heiss and rosner ) , the ischemic penumbra is a dynamic process; it exists for a short period even in the center of ischemia, from which the conversion into irreversible necrosis propagates over time to the neighboring tissue. focal ischemia results in necrosis at the infarct core and activation of complex signal pathways for cell death and cell survival in the penumbra. increased expression of caspase- , - , - , and - , and of cleaved caspase- has been observed in the penumbra (for details, see ferrer and planas ) . but for a limited time interval, penumbral tissue has the potential for recovery and, therefore, is the target for interventional therapy in acute ischemic stroke (time window, heiss ). relatively little is known about the effects of injury of the dendritic processes of neurons. axotomy of a motor neuron induces loss of some presynaptic terminals and the retraction of processes (blinzinger and kreutzberg ; summer and watson ) . abnormalities in dendritic spines have been report- fig. . a−f. dendritic and axonal injury. a dendritic processes and neuronal perikarya are reactive with a map antibody which (b) lose their reactivity within a short time after an ischemic period as well as in cases of (c) mbi-induced hemorrhage; d axonal injury is demonstrable by axonal bulbs or balls using h&e stain as well as (e, f) by their reactivity to β-app antibody (magnification a × ; b × , c × ; d−f × , ) ed during the perinatal period in cases of developmental retardation (purpura (purpura , . li et al. ( ) showed that expression of microtubule-associated protein (map ) in perikaryons and dendrites is a sensitive marker of dendritic lesions in spinal cord trauma (fig. . b ; see also chap. − pp. f). as early as h after moderate or severe compression of the spinal cord, loss of map immunoreactivity in dendrites and nerve cell bodies became evident in the injured segment. this phenomenon continued for the duration of the -day experimental period. how much map immunoreactivity is lost depends on how hard the cord is impacted (li et al. ) . the loss of immunoreactivity may result from an (impact-induced) influx of calcium, activating calcium-dependent proteolytic enzymes capable of degrading map (inuzuka et al. ). the same phenomena may be observed as a result of hypoxic lesions in the hippocampal area ( fig. . a, b) . axonal injury is characterized by an interruption of axonal fibers (fig. . b ) as demonstrable − for example − as a result of a gunshot: the axons will be fragmented (figs. . b, . ) . moreover, axonal injury induces an anterograde (wallerian) and retrograde degeneration of the injured axons. the terms "anterograde" and "retrograde" refer to the directions of conduction of the nerve impulse along the axon, i.e., the degeneration following focal damage proceeds in a centrifugal or centripetal direction (for review see brodal ). an interrupted anterograde flow of proteins along the axon can cause the phenomenon of axonal injury. this phenomenon was once demonstrated by h&e stain and by silver staining techniques within − h after a traumatic event (strich ; adams et al. ) . but since the injured axon is selectively characterized by expression of β-amyloid precursor protein (β-app) (fig. . d−f; cf. gentleman et al. ; sherriff et al. ) , it is now routinely confirmed in − min (blumbergs et al. ; oehmichen et al. ) , in adult brains as well as in infant brains (reichard et al. ) . β-app expression is seen even if the axonal injury is moderate or the axotomy delayed or incomplete (povlishock ) . although axonal injury was long thought to be a morphological correlate of mbi, it is now known to be a non-specific phenomenon also associated with acute intoxication (nies et al. ) or hypoxia/ischemia (oehmichen et al. ) . in a recent study graham et al. ( ) evaluated the pattern of β-app immunoreactivity. the authors identified three different types: . a diffuse − multifocal type − in mbi, co poisoning and hypoglycemia; . a type corresponding to the outlines of an infarct or hematoma with evidence of raised intracranical pressure; . a mixture of ( ) and ( ) which was seen in serve mbi. it is still unclear which events trigger axonal degeneration within the cns and pns. kapoor et al. ( ) suggest a link between nitric oxide (no) and subsequent molecular events that previously have been indicated as contributors to reversible and irrevers- ible axonal injury. waxman ( ) demonstrates the axonal death cascade induced by hypoxia, ischemia, mechanical trauma, inflammation or no. the molecular events involving sodium channels and the na + /ca + exchanger lead to an increase in intracellular calcium, which can provoke axonal degeneration. axonal injury of myelinated axons is always accompanied by a demyelinating process, i.e., a loss of myelin, which will be eliminated by mononuclear phagocytes (see: fig. . b) . the sequelae will be a glial scar lacking myelin as demonstrable by luxol fast blue stain (fig. . a) or by immunohistochemistry using an antibody against myelin basic protein ( fig. . b, c) . interrupted axons or injured neurons exhibit disintegration of the distal stump. breakdown of the distal axonal stump is known as "wallerian degeneration" and begins several days after a traumatic event. its salient morphological features are lysis of axons and myelin, schwann cell proliferation, and phagocytosis by invading macrophages. axonal and myelin degeneration follow a time course described in detail by brodal ( ) (see also p. ). axotomy induces a characteristic centripetally directed morphological change of the neuron termed retrograde axonal degeneration. this retrograde alteration is characterized by rounding of the neuronal contours, swelling of the neuronal cytoplasm, and a decline in the number of nissl bodies at the center of the cytoplasm (central chromatolysis − see fig. . c). the nucleus becomes slightly deformed and is displaced to the periphery of the perikaryon. these changes correspond to the changed or increased neuronal gene expression after axotomy (graeber et al. ) . axotomy is followed in hours by rapid upregulation of the immediate early genes, such as cfos, c-jun, jun-b (haas et al. ) , in association with the upregulation of heat shock proteins (kalmar et al. ) . the one constant change associated with acute retrograde changes may be chromatolysis. these changes are sometimes accompanied by local proliferation of satellite glial cells whose time course has also been described by brodal ( ) (see also pp. and f). it is accepted as common knowledge that plasticityassociated molecular and structural events occur in the injured brain. these are at least partly responsible for functional recovery. increases in dendritic arborization, spine density, and synaptogenesis in both peri-injury and intact cortical areas are the potential morphological strategies that enable the brain to reorganize its neuronal circuits (keyvani and schallert ) . on the other hand we have to consider that the scarring process is an ineffective regenerative process which is associated with cell proliferation. the cell proliferation will be − especially within the first h after the traumatic event − non cell-specific, as immunostaining with markers for immature and mature astrocytes, activated microglia, neural precursors, and mature neurons will be negative (chirumamilla et al. ) . nevertheless, it is also accepted that both retrograde degeneration and the axonal injury-induced bulbs and swelling of the proximal axonal stump are markers of a regeneration process. although the cns has little innate capacity for repair, this capacity does exist for the peripheral nervous system. it is not known why axons in the adult cns are not capable of better regeneration; it is known that they do in fact regenerate, as was recently reconfirmed by von euler et al. ( ) . the observations of schwab and caroni ( ; schwab ) are of major importance. they show that proteins released by oligodendrocytes inhibit axonal elongation. this inhibitory protein has since been identified and cloned (chen et al. ; grand-pré et al. ; prinjha et al. ) . these authors also identified the protein (nogo) of a previously unknown gene that encodes the inhibitory myelin protein in rats and humans. the myelin-derived axon outgrowth inhibitor nogo protein binds to an axonal nogo- receptor and at least accounts for the lack of cns repair (li and strittmatter ; mcgee and strittmatter ) . it remains unclear, however, what effect other factors may have, whether negative (e.g., the release by neurons of large quantities of glutamate following the incidence of spinal cord injury), or positive [e.g., release of tissue growth factors (ramer et al. ) and cytokines, or induction of the macrophage scavenger function (see also schwab ) ]. it is now thought that neurons are renewed throughout life from endogenous stem cells and added to the dentate gyrus. adult neurogenesis could be demonstrated in the subgranular and subventricular zones of the hippocampus (kempermann et al. ; cameron and mckay ) and the olfactory bulb (craig et al. ). these are the only zones where differentiation of neural stem cells into neurons is known to take place in the intact adult cns (reilly ) . because this process does not occur in the adult spinal cord, svendsen ( ) postulates that the growth of neurites can only be stimulated by newly regenerated astrocytes. additional findings suggest that adult neurogenesis is actively regulated in large part by astrocytes (reilly ; song et al. ) . gage ( ) and colleagues (kempermann et al. ) were able to show that exposure to an environmental challenge evokes much greater neurogenesis in old than in young animals. more recent data suggest that the old brain reacts quickly with a neurogenic response to functional challenges, a type of cellular plasticity associated with the continued sensory stimulation and activity of the aging brain (kempermann et al. ; mckhan ) . the cns was once described as an "immunologically privileged site" (medawar ) . this hypothesis was based mainly on the existence of the bbb (pachter et al. ) , which restricts diffusion of soluble molecules and the migration of immune cells out of the systemic circulation into the cns (oehmichen ) . pericytes, endothelial cells, microglia and astrocytes, by contributing to bbb function (see above), participate in cns immune regulation (prat et al. ) . meanwhile a number of basic processes are known (bauer et al. ) that explain how the cns responds to inflammatory attack by regulating local antigen presentation, by the different cell types, and by its special cytokine environment. it also eliminates inflammation via emigrating macrophages (oehmichen et al. − for review oehmichen cserr and knopf ) and destruction of apoptotic t-cells. in vitro and in vivo studies have elucidated the mechanisms underlying immune-mediated (via cytokines, macrophage/microglial toxins, and antibodies) tissue damage, featuring many different potential pathways. the normal cns has limited expression of major histocompatibility complex (mhc) class i antigens, primarily on the endothelium and glial cells, and no expression of mhc-ii and the various immunoactive adhesion molecules. fontana et al. ( ) were the first to point out that cytokines such as interferonγ (ifn-γ) are able to stimulate astrocytes to express mhc class ii to secrete cytokines (il- , il- , tnf-α), and to present antigens such as myelin basic protein (mbp) to specific t-cell lines (fontana et al. (fontana et al. , massa et al. ) . frei et al. ( ) focused on microglia and found that they too could respond to ifn-γ by upregulating mhc-ii. if ifn-γ or ifn-γ and tnf-α are introduced directly into the cns, there is independent, progressive upregulation of both mhc classes and of adhesion molecules, such as the intercellular adhesion molecule- (icam- ), which are expressed first by perivascular macrophages (hickey and kimura ) , subsequently by microglia and macrophages throughout the cns, and finally by astrocytes (massa et al. ). experimental induction of autoimmune encephalitis (eae) revealed that perivascular macrophages are the chief presenters of cns antigens to circulating t-cells (hickey and kimura ) . the role of the endothelium in antigen presentation within the cns is ambiguous. astrocytes too are thought (waksman ) to play an ambiguous role. when presenting antigen to specific t-cells in vitro, they are lysed. it is not known whether t-cells are induced to proliferate or to release inflammatory cytokines, or possibly both, or whether they shut down for lack of suitable co-stimulatory signals (so-called clonal anergy). the duality of the inflammatory response is crucial to host repair and defense. it may also however cause loss or impairment of function, i.e., although otherwise beneficial, inflammation may impair neuronal function (perry et al. ). instead of the bbb being a limiting factor of the cns immune response as once believed, today it is thought that the brain itself is an immune system organ (fabry et al. ; chao et al. ). this conclusion is based on the numerous cytological and immunological findings of the past two decades showing that the targets to be protected are neurons, axons, and myelin. in the absence of protection these can become necrotic or succumb to apoptosis, be phagocytosed and disappear. ultimately all signs of degenerative alteration can be demonstrated. there is no direct correlation however between leukocyte emigration and parenchymal cell death in vivo (schmid-schönbein et al. ) . glial cells outnumber neurons in the cortex, where there are eight glial cells for every neuron. among glial cells in the cortex, astrocytes comprise %, microglia %, and oligodendroglia % (chao et al. ). these cells possess many immunological features marking them as important immunoregulatory cells. hallmarks of cns inflammation in particular are activated microglia and astrocytes. inflammation undoubtedly serves primarily as a host defense mechanism in peripheral tissue, facilitating essential repair processes by altering local blood flow in the injured tissue, with accumulation of fluid and specialized cells. the brain possesses cellular host defense mechanisms since activated t-cells are capable of crossing the bbb (hickey et al. ) . cellular mediators of cns inflammation in the brain have been shown to differ with regard to type and number from those of the periphery (see below). these differences are mainly due to the brain's tight regulatory environment and the balance between inflammation-induced tissue damage and tissue repair (parsons and hunter ) . the specificity of the immune response appears to be controlled largely by cns antigen presentation (sedgwick and hickey ) , though the precise nature of the control remains unresolved. cns antigen presentation according to hart and fabry ( ) occurs outside and inside the cns, with the bbb playing a major role in the regulation of cns immune function. parsons and hunter ( ) showed that the early events leading to t-cell activation by antigenpresenting cells result from mhc binding. co-stimulatory molecules then bind to the antigen, presenting cell−t-cell complexes for further development of the cascade. mhc is expressed not only on astrocytes and microglia, but under certain conditions also on neurons and oligodendrocytes (sedgwick and hickey ) . mhc ii antigens are also expressed under normal conditions in a population of macrophages inhibiting the perivascular space, subarachnoid space, and choroid plexus. this expression may indicate that these cells perform a modulatory function at the blood/csf interface (matyszak et al. ) . under highly specific circumstances, physiological response mechanisms resembling those at the periphery also appear to take place in the brain. under pathological conditions, hematogenous cells that are absent or extremely rare under normal circumstances appear to accumulate in the brain, i.e., platelets (fig. . a) , neutrophilic leukocytes ( fig. . b,c) , lymphocytes (fig. . d) , and macrophages. the number and type of inflammatory cells in the cns vary widely depending on the attracting stimulus or on their inherent ability to attack a cns antigen. the intravascular cells, especially the leukocytes, interact with vessel walls as determined by integrins (hynes ) , selectins (bevilaqua ) , and immunoglobulins of the supergene family (springer ). among the heterogeneous supergene family are mhc molecules, t-cell receptors, and icam- . during emigration, i.e., extravasation, leukocytes initially come into loose contact with the walls of vessels of the microcirculation via selectin molecules or lectins, producing a rolling motion along the vessel wall (mcever ) . they are then expressed on the endothelium. the selectin molecules are e-selectin (elam- ), l-selectin (lam- , leccam- ), and p-selectin (cd ). p-selectin plays a role in recruitment of neutrophils to the brain parenchyma (bernardes-silva et al. ) , while e-selectin is thought to participate in both neutrophil and cd + t-cell adhesion (harlan and liu ) . endothelial selectins can be rapidly upregulated following wounding, p-selectin within minutes (< h − zoppo ) , and e-selectin within a few hours (granger and kubes ; mcever ) . among the stimuli for expression of endothelial cell adhesion receptors are tnf-α and il- . adhesion of leukocytes to endothelial cells is mediated by adhesion molecules such as mac- , the intracellular adhesion molecules (icams), lymphocyte function-associated antigen- (lfa- ), and vascular cell adhesion molecule- (vcam- ), all of which, as already mentioned, are upregulated in endothelial cells. in the cns, icam- and vcam- ( baron et al. ) are constitutively expressed on perivascular cells and some astrocytes. in areas of cns inflammation they are readily upregulated on endothelial cells and astrocytes (sobel et al. ) , which stimulates recruitment of neutrophils to the site of injury. such neutrophil emigration can be experimentally induced by extravasal injection of cytokines such as il- β (bernardes-silva et al. ) or tnf-α (schmid-schönbein et al. . neutrophils are the first circulating leukocytes to reach the site of injury. increase in vascular permeability is caused by the release of free radicals and lysosomal enzymes, giving rise to edema (weiss ) . despite mechanisms evolved to restrict entry of neutrophils into the brain parenchyma, neutrophil recruitment is clearly a feature of acute brain injury, such as that caused by stroke or mechanical trauma. numerous studies employing a transient or permanent model of focal ischemia in rats and mice have demonstrated that cerebral tissue injury is lessened by neutrophil depletion (jean et al. ) . a correlation between the development of cerebral edema and neutrophil recruitment has also been shown in models of mbi (schoettle et al. ). this deleterious effect of neutrophil recruitment contrasts with their beneficial function and phagocytic ability as scavenger cells in cases of bacterial inflammation and of particular importance in cases of sterile inflammation. the aforementioned recruitment of t-lymphocytes to the site of injury clearly depends on an accumulation of adhesion molecules, especially of vcam- , in the endothelial wall. when activated, t-cells express lfa- and can bind icam- on endothelium (van kooyk et al. ) , thus facilitating entry of t-cells into the cns (baron et al. ) . few data have been published on the migratory requirements of b-cells. it is thought, however, that b-cells in their fully mature form as plasma cells have no or only limited migratory potential. immunization of rats with a foreign, non-pathogenic antigen behind the bbb was found to result in the presence of b-cells and plasma cells specific for that antigen in the cns (knopf et al. ) . this finding appears to indicate that, after entering, b-cells remain in the cns at least in part because they have found their specific antigen . after entering the cns, the function of t-and bcells is to recognize their antigen. among the potential antigen-presenting cells of the cns are endothelial cells and astrocytes. under inflammatory conditions, microglial and perivascular cells (members of the mononuclear phagocyte family residing in the cns) constitute the chief antigen-presenting cells. as already mentioned (pp. f), a distinction must be made between activated and resident microglia, and leptomeningeal, perivascular, and choroidal macrophages. all of these cells represent different functional stages of blood monocytes (oehmichen and huber ; oehmichen a oehmichen , b, oehmichen , cf. also perry and gordon ) . microglia residing in the white matter do not express the mhc i antigen, and only a few express mhc class ii (hart and fabre ) . leptomeningeal, perivascular, and choroidal macrophages, in contrast, do express mhc class ii. the resident microglia also feature a downregulation of other antigens, the leukocyte common antigen (lca), and ed- or cd . monocytes emigrate under pathological conditions into the brain parenchyma, where their morphology and antigenic characterization both change. they now participate in the immunological process as macrophages and express mhc class ii antigens. they also scavenge cell debris and myelin fragments left over from damaged tissue. astrocytes are among the first local cells to respond to cns injury. the main responses are reactive gliosis and swelling of reactive (hypertrophic) astrocytes upregulating gfap. reactive astrocytes express acute phase reactive protein (koo et al. ) , mhc class ia (frank et al. ) and mhc class ii antigens (fierz et al. ) , il- (griffin et al. ) , plus multiple other factors (for review see norenberg ) . astrocytes are known to act in conjunction with cells of the immune system and to be involved in immune/ inflammatory processes. they are immunocompetent cells capable of augmenting, amplifying, and sometimes even of regulating an immune response. they produce many immune mediators and can in return be affected by them. by helping to eliminate infectious or foreign agents, astrocytes may contribute to a beneficial response. given their direct contact with leukocytes in the blood, endothelial cells constitute the ideal site for antigen recognition in the cns (sedgwick and hickey ) . the scarcity of t-cells in the cns may be an indication that endothelial cells are the sole site capable of adequate t-cell antigen−mhc interaction. the finding that activated t-cells can enter the cns through an intact bbb (hickey et al. ) , however, is a clear sign that antigen recognition at the endothelial cell surface need not occur. endothelial cells are thus regarded as major players in the inflammatory and immune response (sternberger et al. ) and simultaneously guarantee the bbb. they also enable alterations in the form of receptor-mediated events (dietrich ) , i.e., an inflammatory response (for details, especially on the expression of adhesion receptors, see above). endothelial cells proliferate at the site of brain wounds (fig. . a) . therefore, the number of capillaries increases and − in a final phase − decreases near hemorrhages or infarcts (fig. . b, c) in association with an increase in collagen fibers, especially collagen type iv (fig. . c ). this process leads to a network of collagen fibers and glial fibers, which is the last stage in the formation of a brain scar. among the inflammatory mediators are cytokines and their subgroups, chemokines, in the sense of adhesion molecules. other mediators of inflammation include effector molecules such as no, nos, reactive oxygen species (ros), and free radicals. cytokines mediate the initiation, propagation, regulation, and suppression of immune and inflammatory responses (benveniste ) . they are proteins with low molecular weight and are synthesized during effector phase immunity. they are secreted by cells and are also expressed on their surfaces. many different cell types are capable of producing the individual cytokines, which for their part can have a variety of effects on different cell types. usually acting locally, cytokines begin to act on target cells by binding to specific cell-surface receptors, which generally have a high affinity for their ligands. it takes only minute amounts of a cytokine to evoke a biological response. in multiple sclerosis or experimental allergic encephalitis, ifn-γ and il- are known to be products of activated t-cells. tnf-α and il- derive from activated astrocytes and macrophages. astrocytes can be activated by ifn-γ and/or tnf-α, produce il- and il- , tnf-α, transforming growth factor β (tgf-β), granulocyte-macrophage colony-stimulating factor (gm-csf), and other types of molecules such as prostaglandin e (pge ) (for review see waksman ) . in human brains, tnf-α, il- , and il- in particular can be induced by mbi or cerebral ischemia. brain ischemia triggers rapid production of tnf-α mrna, which peaks − h after ischemia and subsides − days later. it remains above baseline, however, for up to days (barone ) . neuronal cells in and around the ischemic tissue acutely express tnf-α in a so-called penumbra, but it also turns up several days later in macrophages in the infarcted tissue. tnf-α triggers adhesion molecule expression on activated glial cells and the endothelium, in this manner regulating gliosis, tissue remolding, and scar formation. il- levels rise before and during glial activation and neuronal damage (rothwell et al. ). chemokines are chemoattractant cytokines. during inflammation they mediate leukocyte entry into the cns. among their known functions is the interaction of leukocytes with the endothelial surface, a multistep and sequential process mediated by selectin molecules by which the leukocyte rolls on the endothelium. the end result is firm adhesion. the entire process is mediated by interaction of icam- and vcam- expressed by endothelial cells and their leukocyte-associated ligands. moderate levels of icam- and very low levels of vcam- , two molecules responsible for the adhesive properties of granulocytes and of t-cells, are expressed by brain endothelial cells. chemokines constitute a subgroup of small cytokines ( − kda) that attract certain inflammatory cell populations, among them lymphocytes, neutrophils and monocytes, to the target tissue (meeusen et al. ; bonecchi et al. ). the number of known chemokines and chemokine receptors continues to expand rapidly (for review see prat et al. ) . three classes of chemokines are known, as defined by the arrangement in the mature protein of conserved cysteine (c) residues, cxc or α-chemokines, cc or βchemokines, and of cc or γ-chemokines. astrocytes, endothelial cells (zach et al. ; weiss et al. ), perivascular cells, and macrophages (simpson et al. ) produce and release chemokines. ros and no are generated in astrocytes and activated macrophages (hartung et al. ). ros, no, and other free radicals are effector molecules that contribute to the inflammatory cascade and tissue damage. what role complement plays in cns damage is not clear (morgan ) . the enzyme no synthase (nos) synthesizes no. inducible nos (inos) is an isoform of nos that is induced transcriptionally by immunological stimuli. inos, which synthesizes large quantities of no, participates in inflammation-induced cytotoxicity. in the brain, inos message, proteins, and enzymatic activity are induced de novo by cerebral ischemia (iadecola ) . inos is expressed by neutrophils in permanent ischemia and in vascular cells in transient ischemia. high levels of no are synthesized by human astrocytes upon stimulation with ifn-γ, tnf-α, il- and potentiate il- . dalkara et al. ( ) showed that no plays a detrimental role in experimentally induced cerebral infarction in neuronal nos knockout mice. the nos knockout infarcts h after permanent vessel occlusion were % smaller than those of wild type. these finding seem to indicate that expression of inos is a factor contributing to ischemic brain damage. an apoptotic pathway mediates no-induced neuronal cell death and an nmda receptor antagonist blocks no-mediated neurotoxicity. neuronal cell death was shown by chao et al. ( ) to be initiated by the release of il- by the microglial cell, this in turn inducing the generation of astrocytes. neurons are destroyed by no via nmda receptor-linked apoptosis. cerebral trauma, ischemia, and reperfusion are known to generate hydrogen peroxide and superoxide radicals, which then produce ros and hydroxyl radicals (chan ) . under normal conditions and following reperfusion injury, mitochondrial respiration creates ros. microglia and astrocytes that have been activated by cytokines produce vast quantities of neurotoxic free radicals. an intramitochondrial antioxidant enzyme, manganese superoxide dismutase (mnsod), scavenges superoxide radicals and thus constitutes the first line of antioxidant defense. as already pointed out, inflammatory responses are based on an inflammatory cascade, whose details have become increasingly clear in recent years. three basic types of inflammatory response are known: sterile inflammation, cell-mediated inflammation, and antibody-mediated inflammation. these types of response are mutually exclusive, but characterized by occasional overlapping. in cases of mechanical violence, spontaneous intracerebral hemorrhage or stroke, sterile inflammation features an initial phase of infiltrating neutrophils and a second phase of infiltrating mononuclear phagocytes (bone marrow-derived monocytes, i.e., activated microglia and macrophages). macrophages and neutrophils produce cytotoxic cytokines such as tnf-α, proteolytic enzymes (anthony et al. ) , reactive oxygen intermediates (cross et al. ) , cell death-inducing surface molecules such as fasligands (d'souza et al. ) , or even excitotoxins (lipton ) . the macrophages in particular take up the scavenger function and eliminate tissue and cellular debris. they also release mediators that promote the scarring process, i.e., induce fibroblasts to produce collagenous fibers and stimulate astrocytes to proliferation and produce fibrils, aiding healing by the production of a fibrillary glial-collagenous scar. multiple sclerosis (ms) is the classic model of t-cellmediated inflammation whose inflammatory infil-trates are chiefly comprised of t-lymphocytes, fewer b-lymphocytes, as well as activated microglial cells and macrophages (brück et al. ; gay et al. ) . ms features local expression and/or upregulation of markers of t-lymphocyte and macrophage activation (brück et al. ) , of class i and class ii mhc antigens (traugott ) , of chemokines and adhesion molecules in addition to their receptors (lassmann ) , and of co-stimulatory molecules (windhagen et al. ) . diseases with an autoimmune background such as ms or virus-induced inflammatory diseases exhibit a uniform cellular and mediator profile. unlike ms, lesions associated with viral inflammation of the brain display considerable differences in topography and in their patterns of structural damage. they also vary with regard to the nature of the immune response and its associated cellular tropism. in lesions of virus encephalitis cd + -lymphocytes abound; in ms lesions both active and inactive cd + -cells usually outnumber cd + -cells (gay et al. ) . virus-infected cells generally evoke a cell-mediated immune response, although humoral mechanisms play a role as well (for review see esiri and kennedy ) . phagocytosis of infected cells by macrophages can also be promoted by antibodies. antibody-dependent cell-mediated cytotoxicity (adcc) is a process in which lymphocytes bearing fc receptors for igg lyse virus-infected cells bearing relatively small amounts of surface-bound antibody. the immunological specificity of the reaction derives from the antibody not the lymphocytes, which are not specific and have been designated killer cells. an important cell-mediated specific mechanism for killing infected target cells is provided by virus-specific cytotoxic t-cells. the cytotoxic effect is seen even if an antibody is lacking and is restricted by mhc class i antigens. viral antigens on the surface of infected host t-cells are recognized by virus-induced cytotoxic t-cells in association with class i mhc antigens. the infected target t-cells are then killed by these t-cells only if they share the same mhc antigens, i.e., the t-cell killing is restricted by mhc (zinkernagel and doherty ) . viral infection induces secretion of cytokines within the cns, either by lymphomononuclear cell infiltrates or infected brain cells. cytokines play an important role in the induction of mhc molecules. they stimulate humoral and cell-mediated immune responses by acting on immune system cells and neighboring brain cells, evoking the expression of surface recognition molecules such as mhc antigens and antiviral proteins such as mx (campbell ) . viral infections can provoke or amplify mhc class ii expression on the surface of astrocytes and microglial cells, which is important in light of the significance of these antigen-presenting cells in the cns. this process can occur as a direct effect of the infection even in the absence of ifn-γ, as shown for measles virus-infected astrocytes and the murine coronavirus j. howard mueller virus (massa et al. ) . in viral and bacterial inflammation, both the cell-mediated immune response and the humoral response are highly important, the latter also usually dominating. blood-borne dissemination of virus from the primary infection site to other organs is restricted by circulating antibodies, igg or igm. antibodies in tissue spaces can stop the spread of infection from one cell to another by neutralizing extracellular viruses. however, viruses able to fuse cell membranes, such as measles or herpes viruses, can elude this mechanism without ever being exposed to antibody. viruses can be inactivated by antibodies in a variety of ways. antibodies can assist phagocytosis by coating the surface of the virion, or they may thwart attachment of the virus to specific receptors on vulnerable cells, or in the case of enveloped viruses they may promote viral lysis via attachment and activation of complement. in the absence of antibody, direct viral lysis can also be produced by complement alone. edema can produce an increase in tissue pressure that disturbs the microcirculation of portions of the cns that are anatomically prevented from swelling by bone or tight meningeal constraints. it is thought that this mechanism contributes to the formation of necrotic lesions in transverse myelitis. the inflammatory process often has a vasculitic component (gray ) that causes occlusion of small veins and venules and is commonly associated with massive tissue damage. ischemia can contribute to the development of structural damage and functional deficits in inflammatory cns lesions. involvement of arteries and veins is rare in bacterial inflammation but the exudate is frequently accompanied by strands of fibrin. the edematous cortex exhibits large artificial perineuronal spaces and a spongiform neuropil. the cytoplasm of neurons often reveals ischemic cell necrosis and is acidophilic. if the course is subacute, fibrinoid necrosis and thrombosis may appear in a few blood vessels in the exudate, resulting in small foci of cortical necrosis. in the field of neuropathology both gross and microscopic changes can be misinterpreted. only a few aspects will be discussed here, each involving routine immersion fixation with % buffered formalin, dehydration and embedding in paraffin (see above). a detailed overview of the problems associated with postmortem changes, artifacts, and misinterpretation has been provided by lindenberg ( ) . autolysis, a process involving self-digestion of tissue, can cause slides of the adult brain to be discolored or poorly staining. the morphology of the changes associated with autolysis are identical with those of respirator brain (brain death). they arise if fixation is done too late, i.e., if the interval between intracranial circulatory arrest and autopsy or brain fixation is too long. depending on the ambient conditions, the brain will liquefy after a certain postmortem interval without fixation. the brain emits a foul odor if it has undergone microorganism-induced putrefaction, and the central parts of brain slides display a faint pink coloration. variably large bullous cavities ( fig. . a, b) give an appearance of swiss cheese to large sections of the brain. such cavities are created by the activation of gas-producing microorganisms due to poor quality formalin. macroscopic necrosis of the cerebellar granular layer was interpreted by ikuta et al. ( ) as a postmortem phenomenon, whereas lindenberg ( ) thought that the necrotic process precedes or accompanies the onset of sublethal hypoxemia, i.e., shortly before death. neurons in the substantia nigra and locus coeruleus of the brains of infants and children up to years of age normally possess no melanin pigment. this finding is neither an artifact nor pathological. the spinal cord is especially sensitive to postmortem mechanical injury in an unfixed state. in this manner the so-called toothpaste-artifact can occur (hughes ) . if the tissue in a segment of the spinal cord is artificially constricted, proximal portions of the cord are squeezed upward, distal portions downward, thus appearing in histological sections at the wrong level. any tissue processing produces artifacts that have to be interpreted: fixation procedures as well as staining techniques. the artifacts are dependent on the time periods between death and fixation, the duration of the fixation process, the temperatures, etc. for example the freezing process of (brain) tissue leads to crystalline vacuoles within the tissue sections ( fig. . a, b) . additionally, the forensic neuropathologist fundamentally needs to be able to reliably distinguish between vital and postmortem changes (cf. oehmichen ) . this requires among other things the testing and use of novel histochemical and immunohistochemical staining techniques that can help to establish the length of the postmortem interval. so-called dark neurons (fig. . c) can appear among otherwise normal neurons. dark neurons are irregularly contoured, shrunken neurons that are created by excessive pressure on unfixed postmortem tissue (scharrer ; cammermeyer cammermeyer , . apical dendrites also have a dark coloration, sometimes in combination with a corkscrew-like appearance. there is a danger of confusing dark neu-rons with ischemic cell necrosis. in our own investigations (oehmichen and gencic a) we could observe that most, but not all dark neurons have a potent albumin uptake, an indication that they represent lesions of neuronal metabolism (and/or membrane) . animal experiments demonstrate that the following three types of altered neurons appear at different postmortem intervals in rats (oehmichen and gencic b) : . shrunken, hyperchromatic neurons, whose number declines as the postmortem interval proceeds. . swollen and autolytic neurons, with a pale perikaryon and nucleoplasm, and an absence of nissl bodies. the nucleus can no longer be differentiated from the vacuolized and autolysing cytoplasm. the cells themselves have lost their contours and appear swollen and spherical. the swelling in particular represents the most fundamental postmortem change (whose differential diagnosis is retrograde degeneration). . pericellular spaces surrounding neurons may be caused by postmortem autolytic processes that mimic edema, especially in the gray matter. children two years old and younger almost invariably exhibit periventricular and perivenous accumulation of cytoplasm-poor, lymphocyte-like mononuclear cells which suggest an encephalitis (so-called pseudoencephalitis). those cell aggregations consist of neuroblasts as an indication of development, not of an inflammatory process. this age group also regularly shows a superficial granule layer of the cerebellar cortex composed of germinal cells (matrix cells). these usually disappear some time between the second and fourth years of life. in cases of sudden death with brief agony or if tissues have been poorly fixed (hirano ) , extensive acute swelling of oligodendroglia is common. in addition, a generalized swelling and clasmatodendrosis of astrocytes is often seen. these changes are rather slight compared to the neuronal changes. they must also be regarded as non-specific and do not constitute markers of edema. lindenberg ( ) points out that the state of the brain before circulatory arrest also plays a role. if the agony was brief (healthy 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ced-related genes modulating neuronal apoptosis expression of a chemotactic cytokine (mcp- ) in cerebral capillary endothelial cells in vitro immunological surveillance against altered self components by sensitized t-lymphocytes in lymphocytic choriomeningitis selectins, icams, and integrins in cns injury severe pediatric head injury: the role of hyperemia revisited key: cord- -u g authors: lang, frederick m.; lee, kevin m.-c.; teijaro, john r.; becher, burkhard; hamilton, john a. title: gm-csf-based treatments in covid- : reconciling opposing therapeutic approaches date: - - journal: nat rev immunol doi: . /s - - - sha: doc_id: cord_uid: u g therapeutics against coronavirus disease (covid- ) are urgently needed. granulocyte–macrophage colony-stimulating factor (gm-csf), a myelopoietic growth factor and pro-inflammatory cytokine, plays a critical role in alveolar macrophage homeostasis, lung inflammation and immunological disease. both administration and inhibition of gm-csf are currently being therapeutically tested in covid- clinical trials. this perspective discusses the pleiotropic biology of gm-csf and the scientific merits behind these contrasting approaches. mutations that ablate gm-csfr function) causes pulmonary alveolar proteinosis (pap), a life-threatening interstitial lung disease in which dysfunctional alveolar macrophages cannot clear surfactant . patients with pap have increased susceptibility to opportunistic infections due to defective antimicrobial function of alveolar macrophages and basal circulating neutrophils, caused by impaired gm-csf signalling , . in mice, gm-csf has been reported to be required for the steady-state maintenance of non-lymphoid tissue-resident cd + dendritic cells across multiple tissues, and this population of cells was shown to be critically important for the initiation of cd + t cell responses in the lung , . gm-csf thus serves a crucial role in normal lung health and can be important for host defence. during inflammation, gm-csf can be secreted by several different cell types, including epithelial cells and leukocytes, and is a critically important cytokine that can drive both innate and adaptive immune responses ( fig. ). gm-csfr is mainly expressed on myeloid cells, generally restricting the direct-acting function of gm-csf to cells of this lineage. gm-csf broadly serves two important roles during the immune response: it polarizes mature myeloid cells into a pro-inflammatory phenotype (paracrine/autocrine function), and it governs 'emergency myelopoiesis' , expanding and mobilizing progenitor myeloid cells to sites of inflammation (endocrine function) . gm-csf-activated myeloid cells can secrete reactive oxygen species and express elevated levels of pro-inflammatory cytokines (such as il- , il- and tumour necrosis factor (tnf)) and a variety of chemokines (such as ccl , il- and ccl , which can attract monocytes, neutrophils and lymphocytes, respectively) . gm-csf can also enhance the ability of dendritic cells to prime t cells during antigen-specific immune responses , . a distinct subset of cd + t helper cells (t h cells) that produce primarily gm-csf has been identified [ ] [ ] [ ] [ ] . these t cells can heighten the immune response by activating pro-inflammatory myeloid cells and recruiting them to sites of inflammation [ ] [ ] [ ] . thus, it has this perspective provides a brief overview of the pleiotropic biology of gm-csf and examines the preclinical and clinical studies supporting the use of both sargramostim and gm-csf-targeting mabs in covid- . macrophage colony-stimulating factor (m-csf), granulocyte colony-stimulating factor (g-csf) and gm-csf are implicated in myelopoiesis, the production of monocytes, macrophages, dendritic cells and granulocytes (neutrophils, eosinophils and basophils) from progenitor cells. m-csf and g-csf appear to be involved in steady-state myelopoiesis, given that null mutants of the encoding genes in mice cause severe phenotypes (for example, skeletal and sensory defects and neutropenia) [ ] [ ] [ ] . by contrast, gm-csf is barely detectable in the blood of healthy individuals and is thought to serve less of a role in homeostatic myelopoiesis, as evidenced by the fact that gm-csf-deficient mice have a virtually normal lifespan and have less dramatic alterations in the basal myeloid system , . importantly, however, gm-csf is known to be a critical homeostatic factor in lung alveoli, where it is produced at low levels for the development and long-term maintenance of alveolar macrophages , . severe deficiency of gm-csf (for example, due to autoantibodies to gm-csf or been proposed that gm-csf serves as a primary communication conduit between inflammatory lymphoid and myeloid cells . role in disease. the aberrant expression of gm-csf is known to drive excessive inflammation, pain, chemotaxis and tissue damage and to enhance the production of other pathogenic cytokines , . given the proposed role of gm-csf at the interface of lymphoid and myeloid cells, it has been postulated that a 'gm-csf network' promotes disease by driving inflammatory responses to become persistent or hyperactive . this network is defined as a positive-feedback loop involving the interdependent secretion of gm-csf and pro-inflammatory cytokines/chemokines across monocytes/macrophages, t h cells and neighbouring cell populations. the cytokines most prominently implicated in this network are il- , il- and tnf , which have been targeted successfully in various inflammatory diseases and have now been suggested as potential targets in covid- (refs , ). gm-csf has been shown to be upregulated either systemically and/or in the diseased tissues of patients with autoimmune conditions (such as rheumatoid arthritis) , as well as in conditions that show similarities to late-stage covid- , including severe acute respiratory syndrome (sars) , acute respiratory distress syndrome (ards) , cytokine release syndrome (crs) , haemophagocytic lymphohistiocytosis (hlh) , hyperinflammation associated with graft-versus-host disease (gvhd) and other inflammatory diseases of the lung , heart - and nervous system , , , . gm-csf-producing t h cells have been identified as being involved in the pathogenesis of various immunological disorders (for example, rheumatoid arthritis , multiple sclerosis , and sepsis ), reminiscent of the pathogenic t h pathway known to drive disease pathology in multiple autoimmune contexts (for example, psoriasis) . gm-csf inhibition via neutralizing antibodies has shown beneficial effects in a diverse range of preclinical models, including those of many of the aforementioned diseases . in humans, treatment with gm-csf-targeting mabs has demonstrated efficacy across multiple phase ii clinical trials for rheumatoid arthritis, with some potential advantages (for example, fewer off-target effects and decreased infection susceptibility) over standard-of-care therapeutics, such as disease-modifying antirheumatic drugs, tnf-targeting agents and janus kinase inhibitors , (table ) . during this response, gm-csf can act locally in inflamed tissue to induce the survival, proliferation and/or differentiation of myeloid cells, such as monocytes/macrophages and neutrophils. more specifically, gm-csf can potentially do the following: activate mature myeloid cells to a pro-inflammatory phenotype with enhanced cytokine (for example, il- , il- and tumour necrosis factor (tnf)) and chemokine (for example, ccl , il- and ccl ) secretory capacity; recruit immature myeloid cells from the circulation and aid in their terminal differentiation; and develop/stimulate dendritic cells to prime the adaptive immune response. activated lymphocytes (for example, gm-csf-producing t helper cells) can migrate into diseased tissue and the circulation, serving as a source of gm-csf, thereby aiding in the recruitment and activation of new myeloid cells. gm-csf levels can also be elevated systemically to induce 'emergency myelopoiesis', expanding and mobilizing immature myeloid/progenitor haematopoietic cells in the circulation and bone marrow. these gm-csf-dependent responses thus heighten the inflammatory response in inflamed or diseased tissue. the broad range of immunological activities of gm-csf can form part of positive-feedback loops/networks that can initiate and maintain disease-causing hyperactive or chronic immune responses. gm-csf has also been shown to enhance antimicrobial host defence and lung barrier repair (not shown). blue arrows mean 'secretes', black arrows mean 'acts on', dotted arrows indicate movement or differentiation and 'host cell' refers to various haematopoietic and non-hematopoietic cell types. sars-cov- , severe acute respiratory syndrome coronavirus . www.nature.com/nri epidemics , a three-phase clinical staging model has been proposed for covid- : ( ) fever, cough and other relatively mild symptoms accompanying an increase in viral load; ( ) severe pneumonia that persists, despite a decline in viral load, due to a hyperactive immune response; and ( ) continuation of significant immune dysregulation resulting in pulmonary destruction, cardiac instability, multiorgan failure and death . it has become increasingly well appreciated that the characteristic hyperactive immune response driving covid- progression consists of a 'cytokine storm' , overwhelming infiltration of inflammatory myeloid cells into the lungs (particularly monocytes, macrophages and neutrophils), and even a disease phenotype resembling secondary hlh (often referred to as 'macrophage activation syndrome') , [ ] [ ] [ ] [ ] [ ] . a subset of patients also experience acute myocardial injury and/or neuropsychiatric symptoms, which are associated with poor outcomes and may be caused by systemic inflammation , . therapies aimed at increasing viral clearance, strengthening lung tissue and/or reducing the excessive host immune response may be able to reduce the morbidity and mortality associated with covid- . recombinant human gm-csf (sargramostim) is fda-approved for multiple indications, and its administration may provide several benefits to patients with covid- . as mentioned already, gm-csf is required to maintain pulmonary function and lung sentinel cell-mediated immunity , . overexpression of gm-csf in mice prevented hyperoxia-induced lung injury by strengthening the resistance of alveolar wall cells to apoptosis and protecting against secondary bacterial infection , . early elevated expression of gm-csf in bronchoalveolar lavage fluid (balf) of patients with acute lung injury and ards correlated with increased survival, potentially owing to the enhanced survival of alveolar macrophages . on the basis of these data, a randomized controlled clinical trial was conducted to study the use of intravenously administered recombinant human gm-csf in patients with acute lung injury or ards . this trial failed to demonstrate reduction of ventilator-free days or mortality over the -day observation period. however, the study was underpowered owing to a slow recruitment pace (n = of planned participants) , and it has been hypothesized that local delivery of high nature reviews | immunology levels of gm-csf directly to the lungs may be required for a therapeutic effect , . across many preclinical models of viral and bacterial pneumonia, gm-csf expression in the lung has been shown to serve a beneficial role by enhancing repair of injured lung tissue and by activating innate and adaptive immune responses to clear pathogens , , [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] . in this context, gm-csf is thought to act mainly on alveolar macrophages and tissue-resident cd + dendritic cells, and there is even evidence that gm-csf directly modulates alveolar epithelial cells , , . pretreatment with intranasally administered gm-csf protected mice from lethal influenza-induced lung injury , , and lung-specific overexpression of gm-csf after influenza viral infection in an inducible transgenic mouse model significantly increased survival . inhaled gm-csf also protected against secondary bacterial infection in a postinfluenza pneumococcal pneumonia mouse model . conversely, gm-csf-deficient mice showed no survival hours after intratracheal inoculation with gram-negative bacteria (compared with % survival in controls) due to impaired alveolar macrophage bactericidal function . during infection resolution, gm-csf was shown to mediate macrophage-epithelial cell crosstalk, stimulating alveolar epithelial cell proliferation and barrier repair , . in six patients with pneumonia-associated ards, compared with an external control group, increased oxygenation and lung compliance were observed following treatment with inhaled gm-csf on a compassionate use basis; however, these results will need to be verified in controlled studies . currently, a randomized, open-label, investigator-initiated trial is ongoing to assess inhaled and/or intravenously administered sargramostim in patients with acute hypoxic respiratory failure due to covid- (n = , nct ) . a randomized, double-blind, placebo-controlled study has also been initiated to assess intravenously administered sargramostim in patients with respiratory failure due to covid- (nct ) (table ). this strategy may prove useful for stabilizing alveolar macrophage and epithelial cell function, increasing sars-cov- clearance, protecting against secondary infection and contributing to lung repair mechanisms. sargramostim is typically used clinically to expand bone marrow progenitor cells and promote myeloid reconstitution, for example, in cases of neutropenia following chemotherapy or autologous bone marrow transplantation. in apparent contradiction to the data presented in the previous subsection, the sargramostim fda label cautions against treating patients with hypoxia because its administration has been shown to induce respiratory symptoms due to excessive granulocyte mobilization into the lungs from the circulation . it was demonstrated in healthy volunteers that neutrophils primed ex vivo with gm-csf are sequestered in the pulmonary vasculature, whereas little to no retention was observed with non-primed cells . considering that neutrophil accumulation in the lung is a hallmark of ards , it is noteworthy that acute lung injury has been reported as a potential complication of sargramostim use in rare cases . gm-csf has been shown to boost neutrophil survival in ards, and thus inhibition of the gm-csf pathway has been proposed as a potential ards therapeutic approach , , , contrary to recommendations in many of the aforementioned reports. given the aforementioned literature, careful monitoring will be needed with sargramostim use in the covid- setting, particularly as late stages of covid- are thought to be driven by host overactive immunity rather than high viral load . gm-csf administration can induce flu-like symptoms, leukocytosis and capillary leak syndrome , therefore posing the potential risk of exacerbating the sars-cov- -induced hyperinflammatory response. balf analyses of patients with covid- have shown that alveolar macrophages are depleted in patients with severe covid- (refs ), indicating perhaps that gm-csf administration may be more beneficial in patients with earlier-stage covid- . indeed, the covid- trials assessing gm-csf administration exclude patients with ferritin levels greater than , µg ml − (consistent with ongoing hlh) and thus may treat patients before they progress to an overt hyperinflammatory phenotype. anti-inflammatory therapies have attracted great interest in covid- , and an immunomodulatory agent that is able to prevent or reduce the disease-driving hyperactive immune response could be a beneficial therapy for late-stage covid- (ref. ). in covid- and other coronavirus-mediated diseases, pathogenic myeloid cell overactivation is thought to be an important mediator of tissue damage, hypercoagulation and the cytokine storm , . balf analyses from patients with mild or severe covid- showed that patients with severe covid- experienced significant lung infiltration by circulating inflammatory monocyte-derived macrophages . due to its role as a myeloid cell growth factor and pro-inflammatory cytokine, gm-csf may be a key driver of the immunopathological sequelae of covid- . although virtually undetectable in the circulation of healthy individuals , gm-csf was recently noted as being upregulated in the serum of a subset of patients with covid- (ref. ). it was reported that the percentages of gm-csf-expressing cd + t cells, cd + t cells, natural killer cells and b cells were significantly higher in the blood of patients with covid- who were admitted to an intensive care unit (icu) than in healthy controls . this pan-cellular observation was not seen with il- and tnf expression in the respective populations. furthermore, a gm-csf + ifnγ + cd + t cell signature, which is associated with gvhd and autoimmune arthritis , encephalomyelitis and diabetes , was found in the peripheral blood of the patients in the icu. these t cell responses were accompanied by a significant increase in the numbers of cd + cd + inflammatory monocytes, and a high percentage of monocytes secreted gm-csf and il- (ref. ). the reported immunological changes appeared to be more pronounced in patients admitted to an icu than in those who did not require icu care and thus appear to correlate with clinical severity. similarly, a study in patients with sepsis demonstrated that an increased percentage of circulating gm-csf-producing t h cells is predictive of poor outcome and is correlated with il- and il- expression; these cells exhibited a memory phenotype and were reported to be mediators of dysfunctional neutrophil activity . however, given the role of gm-csf in pathogen clearance and lung repair, it is important to consider that gm-csf levels may be elevated as a compensatory mechanism or as a background consequence of increased covid- severity. further studies are therefore needed to determine whether increased production of gm-csf in patients with covid- represents a physiological response to infection or a pathogenic driver of disease. we, along with others , , suggest that in patients with covid- , dysregulated gm-csf expression could induce overactivation of myeloid cells that secrete pro-inflammatory mediators and destructively infiltrate tissue, such as www.nature.com/nri the lungs and potentially even the heart and the nervous system. this suggestion is consistent with the disease-driving mechanism of action of gm-csf proposed in many preclinical models with pathologies similar to that of late stages of covid- , including models of chimeric antigen receptor (car) t cell-related crs and neurotoxicity , gvhd-associated crs , septic shock , , neuroinflammatory disease , inflammatory lung conditions , - and acute cardiovascular diseases (myocarditis , myocardial infarction and vasculitis ). therapeutic inhibition of gm-csf has shown benefit (including survival advantages) in all of these preclinical models by decreasing the production of multiple pro-inflammatory cytokines/chemokines and reducing tissue infiltration by inflammatory immune cells , . of note, a recent report described an outbreak of 'kawasaki-like' disease in sars-cov- -infected children , and gm-csf neutralization via mab in a mouse model of kawasaki disease led to significant reductions in disease incidence and severity . with respect to the lung, systemic monoclonal anti-gm-csf administration after intranasal lipopolysaccharide challenge in mice reduced the lung accumulation of myeloid cells in a dose-dependent manner . a similar benefit was achieved with the use of gm-csf neutralization to treat inflamed lungs in multiple other mouse studies [ ] [ ] [ ] [ ] . a phase ii trial of monoclonal anti-gm-csf administration in patients with asthma demonstrated no benefit in the overall population but statistically significant improvement versus placebo on the primary outcome measure in prespecified subgroups . in mouse models of sars-cov infection, gm-csf was proposed as a mediator of the lethal sars-cov-induced infiltration of inflammatory monocytes/macrophages into the lungs . gm-csf was upregulated before all other cytokines (il- , tnf and ifnβ) and chemokines (ccl , ccl and ccl ) that were measured, indicating that gm-csf might be involved in the initiation of this immunopathological process. in these studies, genetically modified mice (ifnar −/− mice, which cannot respond to type i interferon) did not experience the early upregulation of gm-csf and were protected from the cellular infiltration and death . experimental depletion of inflammatory monocytes and macrophages resulted in significantly reduced morbidity and mortality ( % survival out to ~ weeks versus ~ - % in controls) and increased numbers of virus-specific t cells in the lungs, demonstrating the therapeutic potential of downregulating inflammatory myeloid cells in coronavirus infections . together, these data suggest that the use of mabs to gm-csf or gm-csfr might be a promising therapeutic strategy for curbing the hyperactive host immune response observed in covid- . a number of large clinical trials in patients with covid- are currently assessing similar immunomodulatory strategies. these include il- targeting via sarilumab or tocilizumab, the latter of which is fda-approved for car t cell-related crs , and il- blockade with anakinra or canakinumab , . recently, a data monitoring committee analysis of an ongoing phase ii/iii randomized controlled trial of sarilumab showed a large reduction in c-reactive protein levels and an increase versus placebo on ventilator-free survival in 'critical' patients with covid- (requiring high-flow oxygenation, mechanical ventilation or icu care at study entry) (n = receiving placebo, n = receiving high-dose sarilumab therapy, no p values reported) . the data monitoring committee recommended stopping the assessment of low-dose treatment, as well as discontinuing the enrolment of patients with "severe" disease (requiring supplemental oxygen without mechanical or high-flow oxygenation) and patients exhibiting multiorgan system dysfunction, demonstrating the importance of timing and dose strength for the use of immunomodulatory biologics in covid- (ref. ). the careful assessment of the designs and results of these types of cytokine-targeting mab clinical trial will be important for setting expectations and implementing amendments during the ongoing gm-csf-targeted mab clinical trials in patients with covid- . because gm-csf can stimulate the expression of il- , il- , tnf and other pro-inflammatory cytokines and chemokines, a gm-csf-targeting strategy might have broader effects than other immunomodulatory approaches when one is seeking to therapeutically dampen overactive immune responses. this hypothesis is supported by data from clinical trials in which gm-csf-targeted therapy was shown to be efficacious in patients with rheumatoid arthritis who were unresponsive to tnf-targeted therapy , . in a head-to-head study comparing gm-csf blockade with monoclonal anti-tnf therapy in patients with rheumatoid arthritis, gm-csf blockade induced a sustained reduction in the levels of markers of inflammation, such as c-reactive protein and il- , whereas monoclonal anti-tnf therapy did not in the particular population under study . even given the benefits of tocilizumab in crs, it has been speculated that patients can become refractory owing to early and sustained upregulation of gm-csf , , , and clinical trials are ongoing or planned to assess the benefit of gm-csf-targeting mabs in car t cell-related crs and in crs associated with gvhd - . in summary, these data suggest that gm-csf can have a master regulatory effect on cytokine expression and myeloid cell-mediated hyperinflammation, including in the lung. many of the preclinical and clinical data from the gm-csf-targeting mab therapeutic class come from inflammatory disorders not caused by a viral pathogen, making extrapolation to covid- difficult. however, as mentioned earlier, late stages of covid- appear to be driven not by active viral replication and cell lysis but instead by host immunopathology -particularly myeloid cell immunopathology -that is similar to many aspects of these disorders , . thus, the putative pathogenic role of gm-csf in immune overactivation across many studies provides a rationale for the initiation of the ongoing randomized controlled trials using gm-csf-targeting mabs for the treatment of patients with covid- (table ) . given the homeostatic role of gm-csf in the lung, blocking gm-csf action in patients with covid- comes with the potential risks of compromising alveolar macrophage function and hindering pathogen clearance. as with any anti-inflammatory approach under investigation in covid- , close monitoring for evidence of viral exacerbation will be needed. importantly, mabs to gm-csf and gm-csfr have demonstrated a strong safety profile to date across more than , patients treated in multiple phase ii trials , including a long-term safety study where patients were receiving the therapy for a median of . years . although secondary infections could have been expected (as can be observed in patients receiving tnf-or il- -targeted therapy), no increase in tuberculosis and other serious infections has so far been noted . while pap is of theoretical concern, no patient has developed this disease in any monoclonal anti-gm-csf or monoclonal anti-gm-csfr trial to date. it has been hypothesized that primary pap can develop only from dramatic and sustained gm-csf neutralization by polyclonal antibodies (for example, autoantibodies) . in the covid- setting, therapeutic intervention will occur over a short time frame (likely weeks or less), lessening the risk of lung toxicity. furthermore, the timing of mab administration may be very important. although gm-csf could be beneficial for maintaining alveolar macrophage function during the viral assault in the early disease phase, neutralizing gm-csf may be able to reduce the primary pathology of the cytokine storm and myeloid cell-induced lung destruction in later disease stages. a number of clinical trials of systemically administered mabs to gm-csf or gm-csfr have been completed or are ongoing for inflammatory/ autoimmune conditions; recently, six companies initiated clinical studies assessing these mabs for the treatment of covid- (table ) . encouraging data were obtained from an open-label cohort study of patients with covid- treated with the gm-csfr mab mavrilimumab (n = ), compared with a matched contemporaneous untreated control group (n = ) . benefits in the mavrilimumab-treated group were reported across multiple clinically relevant end points, including time to hospital discharge and mortality; mavrilimumab was observed to be well tolerated in all patients, with no infusion reactions . however, these findings need to be confirmed in larger studies that are placebo controlled. as of may , six randomized, double-blind, placebo-controlled trials were ongoing for gm-csf-targeting mabs in covid- (table ). the lenzilumab trial (n = , nct ) excludes patients with ards, and the mavrilimumab trials (n = , nct ; n = , nct ) exclude patients receiving mechanical ventilation at the time of randomization. by contrast, the otilimab (n = , nct ), gimsilumab (n = , nct ), and tmj (n = , nct ) trials allow inclusion of these patients. the differing target patient populations in these studies should indicate whether targeting gm-csf may be effective at early and/or late stages of covid- . of note, there is expected to be little difference between targeting the gm-csf ligand versus the receptor because both strategies block the same interaction. indeed, preclinical and clinical trial data in rheumatoid arthritis have shown similar benefits for these two approaches . we have provided the rationale and risks for both therapeutically administering and inhibiting gm-csf in covid- . given the pleiotropic roles of gm-csf in lung health, host defence and inflammation, care should be taken with respect to dose, route and timing of administration for each therapeutic approach. gm-csf administration in patients with covid- may improve lung function by strengthening the alveolar wall and enhancing viral clearance, and this approach may thus provide particular benefit in early stages of covid- . by contrast, gm-csf or gm-csfr blockade could be a beneficial treatment for the cytokine storm and inflammatory myeloid cell tissue infiltration associated with moderate-to-severe covid- . the gm-csf blockade strategy may have broad immunomodulatory effects given that it could affect the secretion of multiple pro-inflammatory cytokines and chemokines by myeloid cells. in our view, the gm-csf-based therapies are worthwhile investigational approaches during the urgent global search for effective covid- therapeutics. pathogenic t cells and inflammatory monocytes incite inflammatory storm in severe covid- patients gm-csf in inflammation anti-colonystimulating factor therapies for inflammatory and autoimmune diseases fda approves initiation of humanigen's phase iii study of lenzilumab in covid- patients i-mab announces ind clearance from fda for tjm to treat cytokine release syndrome (crs) associated with severe coronavirus disease (covid- ). i-mab biopharma kiniksa reports data for mavrilimumab in covid- pneumonia and hyperinflammation and for vixarelimab in diseases characterized by chronic pruritus initiation of two-centre compassionate use study involving namilumab in the treatment of individual patients with rapidly worsening covid- infection in italy roivant doses first patient in pivotal breathe clinical trial evaluating gimsilumab in covid- patients for the prevention and treatment of acute respiratory distress syndrome gsk taps experimental arthritis antibody to calm the cytokine storm hitting covid- patients total absence of colony-stimulating factor in the macrophage-deficient osteopetrotic (op/op) mouse mice lacking granulocyte colony-stimulating factor have chronic neutropenia, granulocyte and macrophage progenitor cell deficiency, and impaired neutrophil mobilization targeted disruption of the mouse colony-stimulating factor receptor gene results in osteopetrosis, mononuclear phagocyte deficiency, increased primitive progenitor cell frequencies, and reproductive defects granulocyte/macrophage colonystimulating factor-deficient mice show no major perturbation of hematopoiesis but develop a characteristic pulmonary pathology from growth factor to central mediator of tissue inflammation alveolar macrophages develop from fetal monocytes that differentiate into long-lived cells in the first week of life via gm-csf pulmonary alveolar proteinosis gm-csf autoantibodies and neutrophil dysfunction in pulmonary alveolar proteinosis gm-csf controls nonlymphoid tissue dendritic cell homeostasis but is dispensable for the differentiation of inflammatory dendritic cells alveolar epithelial cells orchestrate dc function in murine viral pneumonia a novel subset of helper t cells promotes immune responses by secreting gm-csf gm-csf in neuroinflammation: licensing myeloid cells for tissue damage fate-mapping of gm-csf expression identifies a discrete subset of inflammation-driving t helper cells regulated by cytokines il- and il- β functional inflammatory profiles distinguish myelin-reactive t cells from patients with multiple sclerosis trials of anti-tumour necrosis factor therapy for covid- are urgently needed covid- : consider cytokine storm syndromes and immunosuppression synovial cd + t-cell-derived gm-csf supports the differentiation of an inflammatory dendritic cell population in rheumatoid arthritis expression profile of immune response genes in patients with severe acute respiratory syndrome neutrophil apoptosis in the acute respiratory distress syndrome car-t-cell neurotoxicity: hope is on the horizon primary and secondary hemophagocytic lymphohistiocytosis: clinical features, pathogenesis and therapy graft-versus-host disease, but not graft-versus-leukemia immunity, is mediated by gm-csf-licensed myeloid cells phase , randomised placebocontrolled trial to evaluate the efficacy and safety of an anti-gm-csf antibody (kb ) in patients with inadequately controlled asthma the infarcted myocardium solicits gm-csf for the detrimental oversupply of inflammatory leukocytes sca- + cardiac fibroblasts promote development of heart failure gm-csf pathway signature identified in temporal artery biopsies of patients with giant cell arteritis rorγt drives production of the cytokine gm-csf in helper t cells, which is essential for the effector phase of autoimmune neuroinflammation dysregulation of the cytokine gm-csf induces spontaneous phagocyte invasion and immunopathology in the central nervous system high levels of circulating gm-csf + cd + t cells are predictive of poor outcomes in sepsis patients: a prospective cohort study the th axis in psoriatic disease: pathogenetic and therapeutic implications pharmacodynamic biomarkers and differential effects of tnf-and gm-csf-targeting biologics in rheumatoid arthritis characteristics of and important lessons from the coronavirus disease (covid- ) outbreak in china: summary of a report of cases from the chinese center for disease control and prevention pathogenic human coronavirus infections: causes and consequences of cytokine storm and immunopathology covid- illness in native and immunosuppressed states: a clinicaltherapeutic staging proposal targeting potential drivers of covid- : neutrophil extracellular traps complex immune dysregulation in covid- patients with severe respiratory failure single-cell landscape of bronchoalveolar immune cells in patients with covid- cytokine release syndrome in severe covid- cardiovascular implications of fatal outcomes of patients with coronavirus disease (covid- ) are we facing a crashing wave of neuropsychiatric sequelae of covid- ? neuropsychiatric symptoms and potential immunologic mechanisms lung epithelial gm-csf improves host defense function and epithelial repair in influenza virus pneumonia-a new therapeutic strategy? transgenic overexpression of granulocyte macrophage-colony stimulating factor in the lung prevents hyperoxic lung injury gm-csf and the impaired pulmonary innate immune response following hyperoxic stress modulation of neutrophil apoptosis by granulocyte colony-stimulating factor and granulocyte/macrophage colony-stimulating factor during the course of acute respiratory distress syndrome a randomized trial of recombinant human gm-csf for patients with acute lung injury inhaled granulocyte/macrophage colony-stimulating factor as treatment of pneumoniaassociated acute respiratory distress syndrome delivery of gm-csf to protect against influenza pneumonia gm-csf modulates pulmonary resistance to influenza a infection local delivery of gm-csf protects mice from lethal pneumococcal pneumonia tlr -dependent gm-csf protects against lung injury in gram-negative bacterial pneumonia protective effects of recombinant human granulocyte macrophage colony stimulating factor on h n influenza virusinduced pneumonia in mice gm-csf overexpression after influenza a virus infection prevents mortality and moderates m -like airway monocyte/macrophage polarization lower respiratory tract delivery, airway clearance, and preclinical efficacy of inhaled gm-csf in a postinfluenza pneumococcal pneumonia model role of granulocyte macrophage colony-stimulating factor during gram-negative lung infection with pseudomonas aeruginosa macrophage tumor necrosis factor-alpha induces epithelial expression of granulocyte-macrophage colony-stimulating factor: impact on alveolar epithelial repair gm-csf enhances lung growth and causes alveolar type ii epithelial cell hyperplasia in transgenic mice partner therapeutics announces initiation of clinical trial to evaluate leukine® in respiratory illness in patients with covid- at singapore general hospital leukine® (sargramostim) package insert pulmonary retention of primed neutrophils: a novel protective host response, which is impaired in the acute respiratory distress syndrome neutrophils in the initiation and resolution of acute pulmonary inflammation: understanding biological function and therapeutic potential acute lung injury following the use of granulocytemacrophage colony-stimulating factor neutrophil gm-csf receptor dynamics in acute lung injury pathological inflammation in patients with covid- : a key role for monocytes and macrophages clinical features of patients with novel coronavirus in wuhan foxo transcription factor drives pathogenic t helper differentiation by inducing the expression of eomes foxp -independent mechanism by which tgf-β controls peripheral t cell tolerance gm-csf inhibition reduces cytokine release syndrome and neuroinflammation but enhances car-t cell function in xenografts gm-csf signalling boosts dramatically il- production intravenous delivery of granulocytemacrophage colony stimulating factor impairs survival in lipopolysaccharide-induced sepsis lipopolysaccharide-induced lung inflammation is inhibited by neutralization of gm-csf granulocyte/macrophage-colonystimulating factor (gm-csf) regulates lung innate immunity to lipopolysaccharide through akt/erk activation of nfkappa b and ap- in vivo interleukin- α controls allergic sensitization to inhaled house dust mite via the epithelial release of gm-csf and il- gm-csf but not il- is critical for the development of severe interstitial lung disease in skg mice gm-csf produced by the airway epithelium is required for sensitization to cockroach allergen il- a + gm-csf + neutrophils are the major infiltrating cells in interstitial lung disease in an autoimmune arthritis model gm-csf intrinsically controls eosinophil accumulation in the setting of allergic airway inflammation gm-csf mediates autoimmunity by enhancing il- -dependent th cell development and survival gm-csf is a pro-inflammatory cytokine in experimental vasculitis of medium and large arteries an outbreak of severe kawasaki-like disease at the italian epicentre of the sars-cov- epidemic: an observational cohort study gm-csf primes cardiac inflammation in a mouse model of kawasaki disease dysregulated type i interferon and inflammatory monocyte-macrophage responses cause lethal pneumonia in sars-cov-infected mice interleukin- blockade with high-dose anakinra in patients with covid- , acute respiratory distress syndrome, and hyperinflammation: a retrospective cohort study regeneron and sanofi provide update on u.s. phase / adaptivedesigned trial of kevzara® (sarilumab) in hospitalized covid- patients a randomized phase iib study of mavrilimumab and golimumab in rheumatoid arthritis efficacy and safety of namilumab, a human monoclonal antibody against granulocytemacrophage colony-stimulating factor (gm-csf) ligand in patients with rheumatoid arthritis (ra) with either an inadequate response to background methotrexate therapy or an inadequate response or intolerance to an anti-tnf (tumour necrosis factor) biologic therapy: a randomized, controlled trial tocilizumab-refractory cytokine release syndrome (crs) triggered by chimeric antigen receptor (car)-transduced t cells may have distinct cytokine profiles compared to typical crs mechanisms of acute toxicity in nkg d chimeric antigen receptor t cell-treated mice mavrilimumab, a fully human granulocyte-macrophage colony-stimulating factor receptor α monoclonal antibody: long-term safety and efficacy in patients with rheumatoid arthritis neutralization and clearance of gm-csf by autoantibodies in pulmonary alveolar proteinosis the authors sincerely thank s. lowry for his guidance during the ideation for and writing of the manuscript. the authors also thank b. dorner, a. berri and t. lin for their support during the preparation of the manuscript. all authors contributed significantly to all aspects of the article. of zurich, holds a patent on the use of neutralizing gm-csf in acute gvhd following stem cell transplantation and has a license agreement with humanigen inc., which is manufacturing such a gm-csf-neutralizing mab. nature reviews immunology thanks m. dougan and the other, anonymous, reviewer(s) for their contribution to the peer review of this work. key: cord- -hcbs a authors: ifergan, igal; miller, stephen d. title: potential for targeting myeloid cells in controlling cns inflammation date: - - journal: front immunol doi: . /fimmu. . sha: doc_id: cord_uid: hcbs a multiple sclerosis (ms) is characterized by immune cell infiltration to the central nervous system (cns) as well as loss of myelin. characterization of the cells in lesions of ms patients revealed an important accumulation of myeloid cells such as macrophages and dendritic cells (dcs). data from the experimental autoimmune encephalomyelitis (eae) model of ms supports the importance of peripheral myeloid cells in the disease pathology. however, the majority of ms therapies focus on lymphocytes. as we will discuss in this review, multiple strategies are now in place to target myeloid cells in clinical trials. these strategies have emerged from data in both human and mouse studies. we discuss strategies targeting myeloid cell migration, growth factors and cytokines, biological functions (with a focus on mirnas), and immunological activities (with a focus on nanoparticles). myeloid cells play critical roles in the health and diseases of the central nervous system (cns). for example, myeloid cells constitute a significant proportion of the cells found within perivascular infiltrates in cns lesions of multiple sclerosis (ms) and its animal model, experimental autoimmune encephalomyelitis (eae) ( ) ( ) ( ) . myeloid cells are also critically involved in the secondary damage in spinal cord injury (sci) and traumatic brain injury (tbi) ( ) ( ) ( ) . these myeloid cells have the ability to attract other immune cells, release neurotoxic factors, phagocytose proteins and debris and promote the expansion, and polarization of antigen-specific t cells in the cns. in addition to their capacity to induce and sustain inflammation, myeloid cells are also critically involved in communication with glial cells and neurons, as well as in promoting and maintaining peripheral tolerance ( - ). ms is an inflammatory autoimmune disease wherein cells of the immune system initiate an attack against myelin in the cns that supports axonal conduction. the immune response in ms is thought to be mediated by autoreactive t lymphocytes that recognize myelin peptides. typically, demyelination is associated with an accumulation of t lymphocytes (lymphoid component of infiltrates) and monocytes/ macrophages/ dendritic cells (myeloid cells component of infiltrates) that arise from the migration of peripheral blood immune cells across the cns microvascular endothelium ( - ).as they infiltrate the cns, encephalitogenic t lymphocytes require the presence of these blood-derived antigen-presenting cells (apcs) to further sustain lymphocyte proliferation and cytokine polarization in the cns compartment ( - ). the role of these peripherally-derived myeloid cells in cns inflammation will be the focus of the present review. experimental autoimmune encephalomyelitis is a commonly utilized mouse model of ms that recapitulates many aspects of the human disease such as the cns inflammation, encephalitogenic t cell infiltration, and attack of oligodendrocytes resulting in demyelination. although not perfect, eae has allowed uncovering some of the molecular pathways governing the pathogenesis of ms such as elucidating the pathogenic role of t h lymphocytes. in addition, eae models were critical in identifying and testing new therapeutic agents such as glatiramer acetate (ga) and natalizumab ( ). although myeloid apcs play a prominent role in the pathogenesis of ms, there has been little consideration given to targeting these cells as an ms therapy. some of the current ms disease-modifying therapies may act on myeloid cells even if these cells were not the original intended targets ( ). however, interfering directly with myeloid cell has proven to be efficacious in other diseases including psoriasis with multiple drugs targeting il- (guselkumab, risankizumab, and tildrakizumab) or il- and il- (ustekinumab) ( ), crohn's disease and ulcerative colitis targeting il- and il- (ustekinumab) ( ), rheumatoid arthritis targeting il- (anakinra) ( ), systemic juvenile idiopathic arthritis targeting il- β (canakinumab) ( ), and many others. there are ongoing clinical trials in rheumatoid arthritis, stroke, atherosclerosis, and cancer using agents that target myeloid cells and their products. biber et al. have provided a recent comprehensive review of drugs in clinical trials targeting myeloid cells in cns diseases such as alzheimer's disease, brain tumors, and inflammatory pain, as well as for other cns diseases ( ). as we will discuss in this review, multiple tools have been developed in the eae models of ms demonstrating significant regulation of disease progression by various approaches blocking myeloid cell activation and effector function, but to date, these approaches have not been tested for therapeutic efficacy in ms patients. the first strategy we will discuss is interference with peripheral myeloid cell migration to the cns. the blood-brain barrier (bbb), composed of tightly bound endothelial cells (ecs), regulates the entry of blood-borne molecules and immune cells into the cns. under physiological conditions, a limited number of peripheral blood immune cells gain access to the cns, a process called immune surveillance ( ). during an inflammatory process, meningeal, and bbb-ecs amplify the migration of immune cells into the cns parenchyma, in a multistep process that involves selectins, chemokines and cell adhesion molecules ( ). bbb-ecs express cell adhesion molecules such as intercellular adhesion molecule (icam)- , vascular cell adhesion molecule (vcam)- , activated leucocyte cell adhesion molecule (alcam), and melanoma cell adhesion molecule (mcam) which mediate at least in part, the adhesion process and the transmigration of leucocytes to the cns through their interaction with integrins αlβ [leucocyte function-associated antigen (lfa)- ], α β [very late antigen (vla)- ], cd , and mcam respectively ( - ). interfering with immune cell trafficking across the bbb by targeting adhesion molecules has proven to be beneficial in reducing clinical disease activity and pathological indices in ms ( ). indeed, natalizumab, which blocks vla- , the ligand of vcam- , is reported to reduce migration of most leukocyte subtypes, including myeloid cells, into the brain. more recently, a new adhesion molecule expressed by bbb-ecs called nerve injury-induced protein (ninjurin)- was described ( ). ninjurin- is a membrane protein known to interact in a homophilic manner through an extracellular residue-binding motif ( ). on immune cells, ninjurin- was weakly expressed by lymphocytes, but highly expressed by peripheral myeloid apcs including monocytes, macrophages and dendritic cells (dcs), in humans and mice. interestingly, ninjurin- was also found to be expressed in ms lesions. ninjurin- neutralization specifically abrogated the adhesion and migration of human monocytes across a monolayer of bbb endothelial cells, without affecting lymphocyte recruitment. moreover, ninjurin- blockade during the course of eae reduced infiltration of peripheral myeloid cells and reduced clinical disease activity and histopathological indices of eae ( ). another adhesion molecule involved in the migration of peripheral myeloid cells is junctional adhesion molecule (jam)-like (jaml). jams are type i transmembrane proteins differentially expressed at the junctions of ecs, epithelial cells, and on various leukocytes ( ). similarly to ninjurin- , jaml can interact in a homophilic manner ( ). it was observed that jaml is expressed by bbb-ecs, and has an increase expression in ms lesions compared to normal appearing white matter ( ). in addition, human monocytes and cd + t cells were found to express jaml, and its level was significantly increased on rrms patients when compared control subjects: vs. % for monocytes, and . vs. . % and for cd + t cells. these data reveals that jaml might be a more important adhesion molecule for monocytes than for cd + t cells. however, migratory capacity of both cell types was significantly compromised when jaml was blocked. chemotactic cytokines (chemokines) are secreted proteins that regulate the migration of leukocytes. chemokine receptor signaling plays a central role in cell migration during inflammatory responses in autoimmune and infectious diseases as well as in cancer. there are ∼ chemokines and receptors known at this time. blockade of ccr and ccr have been the two majors targets in a half dozen ms clinical trials ( ). the chemokines ccl (macrophage inflammatory protein- α-mip- α) and ccl (regulated on activation, normal t cell expressed and secreted-rantes) bind to ccr , while ccl (monocyte chemoattractant protein -mcp- ) binds to ccr . both lymphoid and myeloid cells express ccr and ccr , with monocytes/macrophages/dcs the cells where these chemokine receptors are most abundant ( - ). in animal models of ms, it was shown that ccr -deficient animals developed a less severe disease ( ), while ccr -deficient mice were completely resistant to disease induction ( , ), highlighting the importance of signaling through these chemokine receptors for disease initiation. in addition, it has been shown that ccr + ly- c hi monocytes are rapidly recruited to the inflamed cns in eae and are crucial for the effector phase of disease. selective depletion of this specific monocyte subpopulation through engagement of ccr significantly reduced disease severity ( ). ccr + and ccr + macrophages were both found in active ms lesions ( , ). the role of chemokines and their receptors are now well-characterized in ms and other inflammatory diseases. the potential for a therapy targeting this signaling pathway is well-recognized. however, none of the chemokine-directed ms clinical trials has shown robust clinical efficacy. similar lack of clinical responses have also been reported in therapeutic trials targeting chemokines in other diseases such as rheumatoid arthritis, psoriasis, asthma, and many others ( ). the issue may lie in the redundancy of chemokine/chemokine receptor action, in which case, it may be beneficial to develop strategies employing multiple antagonists simultaneously. as innate cells, myeloid cells express pattern recognition receptors (prrs). prrs include toll-like receptors (tlrs), rig-i-like receptors, nod-like receptors, and c-type lectin receptors (clrs) ( ). selectins, which are part of the c-type lectins family, are known to play a crucial role in the control of leukocyte trafficking and homing to sites of inflammation ( ). selectins are particularly important for the rolling of cells on endothelial cells, an important component of migration of myeloid cells into tissue sites of inflammation ( ). more recently, it was uncovered that clec a, a clr, was involved in facilitating binding and transmigration of dcs across the bbb in response to ccl chemotaxis ( ). in eae, clec a −/− mice displayed delayed disease onset and significantly reduced disease severity. additionally, in a chronic model of eae, anti-clec a antibody treatment initiated at disease initiation also delayed onset and lessened disease severity. anti-clec a antibody administration to mice undergoing relapsing-remitting eae after disease onset, resulted in less severe disease relapse ( ). although the ligand of clec a is currently unknown, it was suggested that the ligand is present on bbb endothelial cells ( ). abundance of immune cells as well as cytokines, chemokines and immunoglobulins in ms plaques and their accumulation in the cerebrospinal fluid (csf) of ms patients, support the notion that ms is an inflammatory disorder. these observations lend support to the idea that immune cell products, especially cytokines, have an important role in both the induction and progression of ms. targeting cytokines has been a successful strategy used in therapy of other inflammatory diseases. for example, blockade of tumor necrosis factor (tnf) has shown positive results in rheumatoid arthritis and crohn's disease ( , ) . as of december , tnf inhibitors were the world's leading drug class, with sales of more than us $ billion and used in more than seven million patients ( ) . in ms, the first treatment approved for rrms was interferon (ifn)-β, thus showing that cytokines manipulation is potentially a good strategy. current data suggests that ms, and its animal model, eae, are driven by both t h lymphocytes, producing ifn-γ, interleukin (il)- and tnf, and t h lymphocytes, producing il- , il- , il- , and granulocyte-macrophage colony-stimulating factor (gm-csf also known as csf- ). surprisingly, ifn-γ, il- , il- a, il- f, il- , and il- have all been shown to be dispensable for the development of eae [reviewed in ( ) ; discussed here ( ) ]. however, in , the cns pathogenicity of t h cells was reported to be primarily associated with their production of gm-csf ( , ) . gm-csf production by t cells has been correlated with pathogenesis in several autoimmune diseases, including ms, rheumatoid arthritis, and myocarditis. it was reported that il- β-and il- -induced production of gm-csf by cns-infiltrating cd + t cells is essential for the induction of eae ( , ) . gm-csf is a hematopoietic growth factor produced by a number of hematopoietic and non-hematopoietic cell types including activated cd + t cells, monocytes/macrophages, b cells, nk cells, endothelial cells and epithelial cells. gm-csf has a wide array of functions, notably the survival and activation of myeloid cells, the ability to induce differentiation of dendritic cells (dcs), the polarization of macrophages toward a pro-inflammatory m phenotype, enhanced antigen presentation, the induction of complement-and antibody-mediated phagocytosis, and the mobilization of monocytes and other myeloid populations from bone marrow to blood ( ) ( ) ( ) . the gm-csf receptor (gm-csf rc) is a heterodimer comprised of a specific low-affinity α chain (cd ; gm-csf rα) and a common β chain (cd ; gm-csf rβ) that is shared by il- and il- ( ) . the gm-csf rc is expressed in multipotent myeloid progenitor cells and continues to be expressed throughout myeloid development on monocytes, dcs, macrophages and neutrophils ( ) ( ) ( ) . it is not expressed by t and b lymphocytes ( ) . thus, most of the suspected direct effects of the gm-csf in diseases are focused on myeloid cells (peripheral and cns resident). findings related to the function of gm-csf signaling in eae pathology have been recently reviewed ( ) . in brief, in eae, gm-csf is necessary for disease as gm-csf kos were found to be resistant to disease induction ( ) . disease can be rescued by the administration of recombinant gm-csf. adoptive transfer using cytokine-deficient mice showed that wild-type, il- a −/− , and ifnγ −/− t cells induced eae with similar kinetics. by contrast, gm-csf −/− t cells were incapable of inducing eae and invading the cns ( ) . due to the variety of cells gm-csf can stimulate, it became important to determine the cell population in which signaling was necessary for disease. a bone marrow chimera study determined that peripheral myeloid cells, but not microglia, are key responders ( ) . this corresponds with earlier observations that gm-csf administration stimulated cd b + ly c hi inflammatory monocytes into the circulation ( ) . circulating ly c hi monocytes traffic across the blood-brain barrier, upregulate pro-inflammatory molecules, and differentiate into central nervous system dcs and macrophages ( ) . these data were confirmed recently using conditional gene targeting in which the β chain of the gm-csf receptor (csf rb) was deleted in specific subpopulations throughout the myeloid lineages ( ) . it was found that deletion of csf rb in ccr + ly c hi monocytes phenocopied the eae resistance seen in complete csf rb-deficient mice. in humans, gm-csf levels in the csf are higher in patients with active ms than in patients in remission ( ) . also, untreated ms patients had significantly greater numbers of cd + gm-csf + t cells and cd + gm-csf + t cells in peripheral blood compared with healthy controls and with ifn-β-treated ms patients ( ) . in addition, ifn-β significantly suppressed gm-csf production by t cells in vitro. more recently, the canadian b cells in ms team uncovered a subset of memory b cells producing gm-csf ( ) . in vitro, gm-csf-expressing b cells efficiently activated myeloid cells in a gm-csf-dependent manner, and in vivo, b cell depletion therapy resulted in a gm-csf-dependent decrease in pro-inflammatory myeloid responses of ms patients. in light of the critical role of gm-csf in the pathogenesis of ms and other inflammatory diseases, multiple tools have been developed targeting either the cytokine or the receptor. first tested in eae, it has been shown that blocking antibodies against gm-csf in chronic (c)-eae ( ) or antibodies against gm-csf rα in c-eae and relapsing-remitting (rr)-eae ( ) were able to prevent disease if given at the time of eae induction (day ). mice treated with anti-gm-csf after disease onset completely recovered within days of treatment in a model of c-eae ( ) . therapeutic treatment with anti-gm-csf rα ameliorated progression of c-eae and resulted in a significant reduction of the relapse severity of rr-eae ( ) . blockade of the gm-csf rα led to a reduction of activated mdcs, and reduced pro-inflammatory cytokine production by cd b + ly c + inflammatory monocytes. additionally, anti-gm-csf rα altered the expression of chemokine receptors, leading to the possibility that antibody treatment may impede cell migration ( ) . logically, the next step is to test the therapeutic potential of gm-csf targeting in humans. a review of tools developed for clinical trials can be found here ( ) . at this time, gm-csf blocking antibodies have been tested in rheumatoid arthritis and have shown promising results. as for ms, only one drug has been tested in clinical trials: mor- (also known as gsk or otilimab), a human antibody to gm-csf. the results of a phase ib clinical trial employing mor- in patients with relapsing-remitting or secondary-progressive ms have shown the drug to be safe and well-tolerated, although with modest efficacy ( ) . at this moment, there are no ongoing clinical trials targeting gm-csf or gm-csf receptor in ms. another important growth factor regulating myeloid cell function is macrophage colony-stimulating factor (m-csf also known as csf- ). m-csf is ubiquitously produced in the steady state by a variety of cells, including endothelial cells, fibroblasts, osteoblasts, smooth muscle, and macrophages, and can be detected in plasma at ∼ ng/ml ( ) ( ) ( ) . the levels of circulating m-csf are upregulated in pregnancy ( ) as well as in many different pathologies including cancer, autoimmune diseases and chronic inflammation ( ) ( ) ( ) ( ) ( ) . m-csf stimulates progenitor cells from bone marrow and plays an important regulatory role in the survival, proliferation (in mice), differentiation, phagocytosis, and chemotaxis of myeloid cells, including monocytes, macrophages, dcs, and microglia ( ) ( ) ( ) . the effects of m-csf are mediated by signaling through the type iii tyrosine kinase transmembrane receptor csf- r (cd ), which is encoded by the c-fms proto-oncogene ( ) . il- is also able to bind csf- r with similar outcomes as to m-csf binding ( ) . however, m-csf and il- present differences in their spatiotemporal expression patterns, and thus seem to play complementary roles in their biological activities on target cells ( , , ) . csf- r is expressed by myeloid cells such as monocytes, macrophages, dcs, and microglia, as well as by trophoblasts, neural progenitor cells and epithelial cells ( , ) . there is ongoing debate about whether m-csf is a pro-inflammatory or pro-repair cytokine. m-csf seems to be essential for the survival and renewal of tissue-resident macrophages, but not for circulating myeloid cells. indeed, in the osteopetrotic csf op /csf op mouse, which harbor an inactivating mutation in the coding region of the csf- gene and are m-csf deficient, the functions and numbers of several tissue macrophage populations are altered while there is no difference in monocyte populations in the blood ( ) . these findings were later confirmed in mice deficient for a specific enhancer for csf- r gene, the fms-intronic regulatory element (fire) ( ) . csf r fire/ fire mice present a deficit in tissue resident macrophages in the brain (microglia), skin, kidney, peritoneal, and heart without significant differences in blood monocytes. during inflammation, the presence of monocytes in inflamed tissue is critical for proper immune responses, notably due to their capacity to traffic to draining lymph nodes and their ability to present antigens to t cells ( , ( ) ( ) ( ) ( ) ( ) ( ) ( ) . while tissue resident macrophages also participate in inflammatory processes, their role in promoting tissue repair and regeneration is critical ( , ) . for example, m-csf favors kidney and liver repair after acute injury ( ) ( ) ( ) . moreover, m-csf is used to drive human and in mouse macrophage differentiation in vitro into an anti-inflammatory (m ) phenotype ( ) ( ) ( ) . in eae, it was shown that peritoneal apcs treated with m-csf and pulsed with mog − , the disease initiating peptide, were able to suppress ongoing eae when injected at the time of disease initiation or significantly reduce the severity of the disease when injected at day post-immunization ( ) . these m-csf activated apcs were demonstrated to induce a treg profile from cd + t cells (cd + foxp + ) with increased secretion of il- and decreased secretion of il- , ifn-γ, and tnf ( ) . however, as mentioned earlier, elevated levels of m-csf are also observed in different pathologies. there are multiple publications linking m-csf/il- and csf- r signaling in models of arthritis ( ) ( ) ( ) ( ) , diabetes ( ) , systemic lupus erythematosus ( , ) , cancer ( ) ( ) ( ) , amyotrophic lateral sclerosis ( ), parkinson's disease ( ) , and alzheimer's disease ( ) ( ) ( ) . in an effort to determine the role of m-csf/il- and csf- r signaling in ms, different groups used potent cfms tyrosine kinase inhibitors, which block m-csf signaling. ki ( ) , imatinib ( ) , gw ( , ) , sorafenid ( ) , and plx ( ) are all tyrosine kinase inhibitors that have shown to effectively treat c-eae. gw has the greatest apparent specificity for csf- r vs. the other kinase inhibitors ( ) . amelioration of eae using ki was associated with the suppression of myeloid cell expansion in the spleen and reduction in mog-specific t-cell proliferation ( ) . gw and sorafenib suppressed tnf-α production by macrophages whereas imatinib and sorafenib both abrogated pdgf-induced proliferation of astrocytes ( ) . plx effect was associated with microglia and macrophage ablation from the white matter ( ) . however, in the cuprizone model of cns demyelination, which allows study of the remyelination process with little involvement of the peripheral immune cells ( ), injection of m-csf reduced demyelination by boosting microglia activity ( ) . tamoxifen-induced conditional deletion of the csf- r in microglia from cuprizone-fed mice caused aberrant myelin debris accumulation and reduced microglial phagocytic responses ( , ) . these data indicate that m-csf plays an important role in ability of microglia to clear myelin debris and to support proper remyelination, and suggest m-csf functions as a critical factor in tissue repair. these divergent results exemplify the various functions of m-csf/il- and csf- r signaling on cells. the possible contribution of m-csf signaling to both inflammatory and repair processes suggest that targeting m-csf in ms may be problematic. however, although there is an increase of myeloid cells in ms lesions, the expression of csf- r is lower in ms lesions when compared to normal appearing white matter ( ) . it is thus possible to hypothesize that a therapeutic treatment targeting m-csf in ms would primarily target peripheral myeloid cells rather than those in the cns. there are now multiple tools targeting m-csf signaling approved for human therapy, especially for cancer. imatinib was the first tyrosine kinase inhibitor approved for the treatment of chronic myelogenous leukemia ( ) . imitanib is also now in clinical trials for the treatment of different pathologies, such as rheumatoid arthritis, type i diabetes and asthma, for which positive results of a phase clinical trial were recently published ( ) . sorafenib is approved for the treatment of primary kidney cancer and advanced primary liver cancer ( ) . although there are side effects related to these inhibitors, an important advantage of tyrosine kinase inhibitors is the fact they can be administered orally to the patients. in september , a phase clinical trial for rrms was started testing the efficacy of evobrutinib, a bruton's tyrosine kinase inhibitor. although this is not a csf- r inhibitor, it shows: ( ) the desire to develop oral treatments in ms, and ( ) the possibility of targeting tyrosine kinases in ms. bruton's tyrosine kinase are critical for b cell receptor signaling and is also involved in tlr signaling as well as inflammasome activation in myeloid cells ( ) cytokines as mentioned earlier, blockade of myeloid specific cytokines il- β, il- , and il- have proven to be efficient therapies in multiple diseases such as crohn's disease, ulcerative colitis, rheumatoid arthritis, psoriasis, and systemic juvenile idiopathic arthritis. these cytokines are all involved in cd + t lymphocytes differentiation. while il- is critical for t h induction ( ), il- β and il- are both involved in t h differentiation and promote the encephalitogenic capacity of these cells by inducing gm-csf expression ( , , ) . in eae, mice lacking il- β, or the receptor, il- r, developed a milder disease than wt animals ( , ( ) ( ) ( ) ( ) . moreover, specific ablation of il- r on cd + t cells resulted in significantly reduced disease severity ( ) , confirming the importance of il- β signaling on t cells to induce a full eae. in addition, rats treated with an il- receptor antagonist (il- ra), which blocks the biological activity of il- β, developed milder signs of eae compared to control animals ( ) . as il- β secretion is the result of inflammasome activation, mice treated with a blocking agent for the inflammasome component nlrp exhibited decreased eae severity ( ) . in ms, it was shown that il- r expression is significantly higher in cd + t cells from rrms patients than from healthy controls ( ) . il- β expression was also found to be significantly increased in ms lesions when compared to tissue from other neurological diseases ( ) . interestingly, multiple treatments used in ms [e.g., ifn-β, glatiramer acetate, and natalizumab] have shown to increase il- ra expression and/or to decrease il- β production ( , ) ]. multiple tools have been developed to block il- β activity: the recombinant il- ra anakinra used for rheumatoid arthritis, the neutralizing il- β antibody canakinumab used for systemic juvenile idiopathic arthritis as well as cryopyrin-associated periodic syndrome, and the soluble decoy il- receptor (rilonacept) also use for cryopyrin-associated periodic syndromes ( ) . at this time, anakinra is the only il- β-targeting drug in clinical testing for ms. this phase i/ii clinical trial just started a few months ago, and at this time, it is still in the recruitment phase (nct ). il- and il- are heterodimeric cytokines that share a common subunit il- p . the other subunit needed to form il- is il- p , while the other subunit to form il- is il- p . il- signals through the il- receptor (il- r) composed of the il- rβ and il- rβ subunits, while il- signals through il- r and il- rβ ( ) . thus, il- rβ is required for biological response to both il- and il- . when specific gene ablation was tested for the different receptor chains of il- and il- , it was found that il- rβ −/− mice were completely resistant to eae ( ) . however, il- rβ −/− mice developed severe eae, extensive inflammation and demyelination, and higher production of pro-inflammatory cytokines than wt animals ( ) . finally, similar to il- rβ −/− mice, il- r −/− mice were completely resistant to eae induction ( ) . as for the cytokines, mice deficient for the subunits il- p or il- p were resistant to eae. by contrast, mice in which the subunit il- p was deleted were highly susceptible to eae ( ) . in addition, treatment with anti-il- p antibodies inhibited both murine and primate models of eae ( ) ( ) ( ) . treatment with anti-il- p antibodies reduced the clinical severity and prevented relapsing eae by inhibiting epitope spreading ( ) . these results led to the conclusion that il- was a more critical factor than il- in the inflammatory response observed in eae. nevertheless, there are multiple studies linking both cytokines to ms pathology. it was demonstrated that peripheral blood monocytes from progressive ms patients produced increased amounts of il- compared to controls and that il- production correlated with disease activity ( ) . another study showed an augmented level of il- mrna-expressing cells in the peripheral blood and the csf of ms patients when compared to controls ( ) . there was also elevated levels of il- p detected in plasma from ms patients compared to healthy individuals. a more recent report showed that both rrms and secondary progressive ms patients had increased levels of il- p mrna compared with controls during the development of active lesions ( ) . il- p and il- p have also been detected in human ms lesions ( , ) . based on this and other data, there was hope that ustekinumab, an il- p neutralizing antibody, would be efficacious for treatment of ms. however, disappointingly no clinical improvement in the treatment group compared to the placebo was found ( ) . possible reasons for the failure of ustekinumab are the broad range of ms patients in the trial, many having very severe symptoms and long-standing disease. also, there may be weak bioavailability of the drug as ustekinumab may be inefficient in crossing the bbb ( ) . at this time there are no ongoing trials targeting il- /il- in ms despite the impressive results in the various animal models of the disease. micrornas (mirnas) are small non-coding rnas of - nucleotides that regulate gene expression by inducing mrna degradation or by interfering with translational machinery of mrnas ( ) . it is predicted that more than % of protein-coding genes are regulated by mirnas ( ) . they are key regulators of various biological processes including immune cell lineage commitment, differentiation, maturation, and maintenance of immune homeostasis and normal function [reviewed in ( ) ]. extensive evidence demonstrates that mirnas play crucial roles in the development, differentiation, and function of different immune cells, such as b and t lymphocytes, dcs and macrophages ( ) ( ) ( ) ( ) . in the last few years, mirnas have drawn a lot of interest due to their involvement in the pathogenesis of cancer, inflammatory and autoimmune diseases [reviewed in ( ) ]. in ms patients, expression studies using whole blood ( ), pbmcs ( ) , as well as brain sections ( ) identified multiple deregulated mirnas. of these mirnas, three were consistently upregulated across multiple studies and directly affecting myeloid cell functions: mir- , mir- and mir- a. mir- is induced by the myeloid transcription factors pu. and ccaat/enhancer-binding protein-β (c/ebpβ) ( ) . mir- expression is mainly confined to myeloid cells and is induced during the lineage differentiation of myeloid progenitor cells. it was shown to negatively regulate both the proliferation and activation of neutrophils ( ) . moreover, mir- −/− macrophages exhibited enhanced pro-inflammatory m , but decreased regulatory m responses ( ) . it was later described that mir- is required for efficient m -associated phenotype and function ( ) . moreover, a low functional level of the mir- is essential for monocyte differentiation. in ms patients, mir- was found significantly increased in blood, pbmcs and active ms lesions compared with control subjects ( , ) . during eae development, the expression level of mir- is dramatically increased in myeloid cell populations, but not in other cell types, and was maintained at comparable levels between disease onset and peak of disease ( ) . surprisingly, although mir- expression is associated with m macrophages and microglia ( ) , it was shown that mir- ko mice present a milder course of eae than wt mice ( ) ( ) ( ) . reduced disease severity was also observed in adoptive transfer eae induced by transfer wt t lymphocytes into mir- ko recipient mice compared to transfer into wt recipient mice, demonstrating the importance of mir- on the apcs side rather than on the t cells side ( ) . our group demonstrated that while m -like macrophages were upregulated in ko mice, dcs showed a reduced inflammatory profile characterized by increased pd-l expression and decreased expression of il- β, il- , and il- , all cytokines involved in differentiating and sustaining a t h profile ( ) . significantly, apcs from mir- ko mice have a comparable ability to drive t h cells, but possess a reduced capacity to drive t h cells ( ) . moreover, it was shown that monocytic-myeloid-derived suppressor cells (mo-mdscs) isolated from mir- −/− suppressed t cell proliferation and cytokine production in vitro and regulated eae more efficiently than mo-mdscs derived from wt animals ( ) . the enhanced suppressive function of mir- −/− mo-mdscs was associated with higher expression of arg and stat , which are mir- target genes ( ) . interestingly, stat controls the expression of pd-l on apcs ( ), consistent with the previous observation of pd-l upregulation on dcs in mir- −/− animals. although these results point to mir- as a potential therapeutic target in ms, it is important to note that in a model of lysolecithin-induced demyelination, the absence of mir- was demonstrated to lead to impaired cns remyelination and myelin debris clearance ( ) . the impaired capacity of m polarization by macrophages and microglia is likely a significant factor contributing to the decreased remyelination capacity in mir- ko mice. in particular, microglia adopting an m profile are critical for proper remyelination ( , ) . thus, when targeting mir- in ms, it is important to keep in mind the different implications of such therapy. mir- has drawn a lot of attention for its possible role in ms as detailed in a recent review ( ) . mir- has been shown to be upregulated in active ms lesions ( ) as well as in cd + monocytes isolated from the blood of rr-ms patients compared to control donors ( ) . while mir- expression is limited to myeloid cells, multiple immune cell populations express mir- such as b cells, t cells, macrophages and dcs ( ) . mir- is found at low levels in both myeloid and lymphoid cells, but its expression is upregulated following cellular activation via antigen, toll-like receptor (tlr) ligands, and inflammatory cytokines. an important target of mir- is src homology (sh )-domain containing inositol- ′phosphatase (ship- ) ( ) . ship- is an enzyme that inhibits phosphoinositide -kinase (pi k) activity, which governs cellular responses to multiple stimuli, cell proliferation and cell survival ( ) . thus, it is believed that mir- dysregulation would have critical consequences. indeed, forced expression of mir- in hematopoietic stem cells by a retroviral vector leads to severe splenomegaly as well as increased myeloid cell populations in the bone marrow and in circulation ( ) . in addition, it has been reported that in absence of mir- , mice displayed altered immune responses to infectious agents, due to defective functions of b cells, t cells, and dcs ( ) . focusing on myeloid populations, it was shown that dcs lacking mir- are less competent at inducing antigen-specific t cell activation ( ) . more recently, it was demonstrated that overexpression of mir- in dcs is a critical event that is alone sufficient to break selftolerance in an animal model of diabetes, and promote a cd mediated autoimmune response in vivo ( ) . human cd + monocytes and macrophages overexpressing mir- exhibit increased production of pro-inflammatory cytokines, including il- β, il- , and tnf, and decreased production of the antiinflammatory cytokine il- ( ) . mir- -deficient mice display a delayed course and reduced severity of clinical symptoms of eae ( , ) . decreased disease severity in mir- −/− mice was associated with reduced t h and t h responses. in addition to the direct effect on t cells, it was also shown that the decreased ability of mir- ko mice to mount inflammatory t cell responses was linked to dcs secreting less cytokines critical for driving t h and t h responses, mainly il- β, il- , il- , il- , and tnf ( ) . mir- is induced in macrophages and dcs after exposure to a variety of inflammatory cytokines such as ifnβ, ifn-γ, and tnf-α ( , ) . it is thus possible to speculate that following the first wave of inflammation, these myeloid apcs upregulate mir- leading to an accentuation of the inflammatory response. in addition, more recently, it has been demonstrated that mir- plays an essential role in driving the inflammatory phenotype of m macrophages ( ) , which would also impact the severity of the disease. lastly, treatment with a mir- inhibitor after eae onset reduced the clinical disease severity ( ) . considering the important role of mir- in driving inflammatory responses in general, and specifically in myeloid apcs, fine tuning the expression of this mirna in ms would most certainly prompt beneficial results in terms of slowing the inflammatory loop. it is noteworthy that mir- is the most consistent mirna found to be upregulated in ms being reported in eight independent studies ( ) . a third mirna that has been shown to regulate myeloid cell activation is mir- a. like mir- , mir- a is upregulated following cell stimulation and its induction is nf-κb dependent ( ) . however, contrary to mir- and mir- , mir- a represses inflammatory responses by targeting two adapter proteins, tnf receptor-associated factor (traf ) and il- receptor-associated kinase (irak ), that are crucial for pro-inflammatory signaling ( ) . mir- a ko mice develop a spontaneous autoimmune disorder, characterized by splenomegaly, lymphadenopathy, and multiorgan inflammation ( , ) . in addition, mir- a ko mice display excessive production of myeloid cells and develop flank tumors in their secondary lymphoid organs. consistent with the repression of inflammation, mir- a expression promotes m -macrophage polarization by targeting notch- ( ) . multiple studies have indicated that mir- a plays pivotal roles in the pathogenesis of several autoimmune diseases, such as systemic lupus erythematosus, rheumatoid arthritis, and sjögren's syndrome ( ) . in ms, mir- a is upregulated in active lesions ( ) , as well as in pbmcs of rrms patients ( , ) . expression of mir- a is reported to be significantly downregulated in glatiramer acetate treated rrms patients ( ) . logically, upregulation of this mirna would seem to be beneficial in reducing the ongoing inflammation observed in ms patients leading to the possibility that the upregulation observed in ms patients is the result of the ongoing inflammation rather than a pathological expression. however, when studied in animal models of ms, there was no consensus on the suppressive effects of mir- a. one study using the cuprizoneinduced demyelination model found that mir- a-deficient mice displayed reduced inflammatory responses, demyelination, axonal loss, and numbers of infiltrating macrophages compared to wt controls ( ) . however, a second study found that mir- a-deficient mice developed more severe eae characterized by exaggerated t h responses ( ) , going along the possible beneficial effect of upregulation of this mirna in ms. more recently, it was shown that mir- a mimic treatment of mice with rr-eae at day improved neurological function, increased the number of newly generated oligodendrocytes, which may facilitate remyelination in the cns ( ) . in addition, the treatment increased the number of regulatory m macrophages while reducing the number of pro-inflammatory m macrophages ( ) . currently, targeting mirnas is a challenge since they control a myriad of immune and non-immune related functions. however, there is a strong interest in pursuing this approach, not only in ms, but also in many different diseases. identification of technology to target mirnas in a cell specific manner would appear to be the desired way to safely and effectively employ this targeting strategy. in the meantime, an abundance of researchers are also exploring the use of mirnas as biomarkers of diseases pathogenesis and therapy. the final strategy we will discuss is the use of nanoparticles to target myeloid cells for disease therapy which has been pioneered in our laboratory. in the recent past, many studies have focused on characterizing the ability of nanoparticles to modulate immune responses and ultimately to be used as potential therapeutics for immune-related diseases. here we will focus on the "carboxylated" poly(lactic-co-glycolic acid) (plga) nanoparticles, which are particles without any protein or peptide attached to the surface or encapsulated inside. phagocytic cells have the extraordinary ability to engulf dead cells, invading microbes and other particles, and this property of phagocyte cells led to the idea of using carriers such as apoptotic cells ( ) ( ) ( ) , liposomes ( ), extracellular vesicles ( ), or nanoparticles ( ) ( ) ( ) ( ) ( ) to deliver molecules to modify the immune response. we will restrict our discussion to the use carboxylated plga nanoparticles for the modulation of inflammatory monocytes for treatment of cns inflammation for multiple reasons. firstly, they can be easily manufactured under gmp conditions. secondly, they more specifically target inflammatory monocytes by their affinity of binding via the macrophage receptor of collagenous structure (marco) ( ) as compared to liposomes and extracellular vesicles. thirdly, they directly carry out immune-modulatory effects on monocytes without the need for add-on agents such as would be required with liposomes. lastly, they have been proven to be safe and efficacious for use in celiac disease patients treated via intravenous infusion of gliadin encapsulating plga nanoparticles for induction of immune tolerance in a phase / a clinical trial ( ) . nanoparticles have diameters between and , nm. smaller particles (< nm) are able to cross tissue barriers and traffic directly to the lymph nodes. larger particles (> nm) require uptake by phagocytic cells ( ) . nanoparticles administered subcutaneously or intradermally may be taken up by tissue resident apcs or their precursor cells and are ultimately transported to the draining lymph nodes. systemic administration of nanoparticles favors accumulation in the organs such as the spleen and liver ( ) . also, the shape of nanoparticles dictates efficiency of uptake by phagocyte cells. for example, phagocyte cells internalize spherical-shaped nanoparticles more easily than stretched-shaped structures ( ) . and although positively charged particles are taken up more avidly, negatively charged particles have been shown to exhibit lower toxicity ( ) ( ) ( ) ( ) . nanoparticles can be made from different materials, metallic (e.g., silver, gold, and copper), magnetic (e.g., iron) (useful for imaging), ceramic, carbon-based, silica, lipid-based, or polymeric such as poly(amino acids), polysaccharides and poly(alpha-hydroxy acids). our group was one of the first to test the ability of nm non-biodegradable carboxylated polystyrene (ps) particles to modulate immune responses in inflammatory settings in vivo. intravenous infusion of ps nanoparticles led to a reduction in trafficking of ly c hi inflammatory monocyte into the cns and increased survival in a mouse model of west nile virus (wnv) encephalitis ( ) . it was discovered that these inflammatory monocytes were redirected to the spleen of treated animals and resulted in a dramatic reduction of mortality in wnv-infected mice by preventing the release of a pro-inflammatory "cytokine storm" in the cns. robust antiinflammatory effects induced by infusion of ps nanoparticles were also observed in other inflammatory diseases such as peritoneal inflammation and inflammatory bowel disease. to enhance the clinical relevance of the nanoparticle targeting approach, we next investigated the potential of biodegradable carboxylated plga nanoparticles for regulation of myeloid cell-dependent inflammation. plga is one of the best characterized and most used biodegradable polymers. the hydrolysis of plga leads to metabolite monomers, lactic acid and glycolic acid. the two monomers are endogenous and easily metabolized by the body via the krebs cycle. there is minimal systemic toxicity associated with the use of plga ( , ) . because plga is a safe material, it has been approved by the united states food and drug administration (fda) and european medicine agency (ema) in various drug delivery systems in humans. indeed, plga can be engineered to deliver, alone or in any combination with small-molecule drugs, proteins, peptides, dna, mirnas, and even clustered regularly interspaced short palindromic repeat (crispr) ( ) . we have shown that administration of negatively charged nm plga nanoparticles resulted in reduced inflammatory monocytes accumulation and overall robust beneficial effects in disease severity in multiple mouse models of inflammatory disease such as eae ( ) , sci ( ), tbi ( ), myocardial infarction ( ) , and herpes simplex virus infection of the cornea ( ) . the exact mechanisms behind immunomodulatory effects of plga therapy are still under investigation. however, in all these models, it has been shown plga particles are selectively recognized and bound by inflammatory monocytes. these monocytes undergo sequestration and eventual apoptosis in the spleen, culminating in reduced immune pathology at sites of inflammation. phenotypic changes were also observed on dcs and macrophages in the inflammatory sites, showing decreased expression of activation markers such as mhc ii and cd . in the sci study, plga nanoparticle administration led to reduced m macrophage polarization. while our group has also shown that antigen (ag)-coupled or encapsulated plga nanoparticles can have important immunomodulatory effects ( , , , ) , other strategies using plga nanoparticles have also been shown to regulate eae ( ) . for example, cappellano et al. showed that simultaneous subcutaneous injection of plga nanoparticles loaded with either mog − or il- ameliorated the course of eae ( ) . tgf-β, another immunoregulatory molecule, coupled to the surface of plga nanaopartlces containing plp − peptide were shown to improve the tolerogenic effect of ag-plga nanoparticles ( ) . another example is maldonaldo et al. using plga nanoparticles loaded plp − together with rapamycin, an inhibitor of the mtor pathway, and demonstrating that a single dose of these particles injected at the peak of disease were able to protect from relapses ( ) . also, pei et al. aimed to develop plga nanoparticles which function as a direct modulator of t cells, without the involvement of apcs ( ) . for that purpose, tgf-β encapsulated nanoparticles were coupled with target antigens for cd and cd t cells (mog − /h- d b -ig dimer and mog − /i-a b multimer), regulatory molecules (anti-fas and pd-l -fc) and a "self-marker" cd -fc ( ) . these particles were injected in eae mice on day , , , and after immunization with mog − , and induced a significant reduction in eae symptoms that lasted for more than days. moreover, the authors observed a decrease of t h and t h mog − -specific cells as well as t c and t c mog − specific cells, an increase of regulatory t cells, inhibition of t cell proliferation and augmentation of t cell apoptosis in the spleen ( ) . in addition to regulating the immune response, plga nanoparticles have also been used as a transporter to help in the remyelination process. indeed, rittchen et al. encapsulated leukemia inhibitory factor (lif), which is a cytokine known to promote oligodendrocyte maturation thus favoring remyelination ( ) . to specifically target oligodendrocytes, the lif-plga nanoparticles were coupled with anti-ng antibodies. the authors showed that intra-lesion delivery of lif-plga nanoparticles improved cns remyelination increasing the percentage of remyelinated axons and their thickness ( ) . in conclusion, the mechanism(s) of action of plga nanoparticles are still incompletely understood, but studies in multiple models have shown their capacity to limit inflammatory events by targeting inflammatory monocytes. plga nanoparticles can also be used as delivery vectors, like liposomes and extracellular vesicles. however, a critical advantage of carboxylated plga nanoparticles, as compared to liposomes and extracellular vesicles, is their ability to act directly to modulate the function and trafficking of inflammatory monocytes based on their ability to engage the marco scavenger receptor. because of the safety record of plga nanoparticles, they can be easily translated into clinical use. in fact, cour pharmaceuticals successfully completed a phase iia clinical trial for celiac disease showing the safety and efficacy of systemic infusion of plga nanoparticles encapsulating gliadin for inducing gluten-specific immune tolerance in celiac disease patients undergoing oral gluten challenge. takeda pharmaceuticals has acquired the exclusive license for future development of this therapy for celiac and other gi diseases. cour pharmaceuticals is currently developing antigen encapsulating plga nanoparticle-based tolerance clinical programs for treatment of ms and peanut allergy and clinical programs using carboxylated "naked" plga nanoparticles targeting inflammatory monocytes for treatment of acute respiratory distress in covid- infection and treatment of tbi. the importance of peripheral myeloid cells in ms pathology is profound. there is an extensive presence of these cells and their products in ms lesions as well as in the csf of ms patients. studies in animal models of ms have clearly demonstrated the beneficial effects in targeting peripheral myeloid cells for the different forms of the disease. multiple tools have now been developed targeting these cells including blockade of their migration to the cns, their activation and cytokine production, their biological functions and their immunological activity (figure ) . however, contrary to other inflammatory disorders, no drug is currently approved targeting specifically these cells in ms. multiple pro-inflammatory cytokines including gm-csf, il- β, il- , il- , m-csf all represent potential ms therapeutic targets. treatments targeting these cytokines have been shown to be well-tolerated and safe in patients for different diseases. additionally, non-specific blockade of leukocyte entry to cns using natalizumab is beneficial in ms, however this carries the risk of severe side-effects from infections. however, specifically impeding the migration of myeloid cells would limit such adverse effects. clec a, ccr , ccr , jam-l, and ninjurin- represent interesting options to inhibit cns migration of peripheral myeloid cells. altering the biological functions of myeloid cells via through mirna modulation is an appealing strategy for treating ms and other chronic inflammatory diseases. mir- a, mir- , and mir- are all upregulated on myeloid cells from ms patients. lastly, nanoparticles represent one of the most exciting new tools for regulating myeloid cell functions. the biodegradable plga particles are particularly interesting due to their approval by the fda and ema for use in humans, as well as 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(lif) co and also a member of the scientific advisory board and consultant for cour pharmaceutical development co as well as a shareholder.the remaining author declares that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.copyright © ifergan and miller. this is an open-access article distributed under the terms of the creative commons attribution license (cc by). the use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. no use, distribution or reproduction is permitted which does not comply with these terms. key: cord- - nrfipz authors: jay, taylor r.; von saucken, victoria e.; landreth, gary e. title: trem in neurodegenerative diseases date: - - journal: mol neurodegener doi: . /s - - - sha: doc_id: cord_uid: nrfipz trem variants have been identified as risk factors for alzheimer’s disease (ad) and other neurodegenerative diseases (ndds). because trem encodes a receptor exclusively expressed on immune cells, identification of these variants conclusively demonstrates that the immune response can play an active role in the pathogenesis of ndds. these trem variants also confer the highest risk for developing alzheimer’s disease of any risk factor identified in nearly two decades, suggesting that understanding more about trem function could provide key insights into ndd pathology and provide avenues for novel immune-related ndd biomarkers and therapeutics. the expression, signaling and function of trem in ndds have been extensively investigated in an effort to understand the role of immune function in disease pathogenesis and progression. we provide a comprehensive review of our current understanding of trem biology, including new insights into the regulation of trem expression, and trem signaling and function across ndds. while many open questions remain, the current body of literature provides clarity on several issues. while it is still often cited that trem expression is decreased by pro-inflammatory stimuli, it is now clear that this is true in vitro, but inflammatory stimuli in vivo almost universally increase trem expression. likewise, while trem function is classically described as promoting an anti-inflammatory phenotype, more than half of published studies demonstrate a pro-inflammatory role for trem , suggesting that its role in inflammation is much more complex. finally, these components of trem biology are applied to a discussion of how trem impacts ndd pathologies and the latest assessment of how these findings might be applied to immune-directed clinical biomarkers and therapeutics. human genetic studies have provided crucial insight into neurodegenerative disease (ndd) pathogenesis. alzheimer's disease (ad) is a prime example of how advances in genetic technology have facilitated the evolution of our understanding of the etiology of ndds. early studies using genetic linkage approaches identified familial mutations in proteins related to amyloid beta production, amyloid precursor protein (app), presenilin (psen ) and psen , as well as the late onset ad (load) risk variant apolipoprotein e (apoe ) [ ] . these studies provided important insight into amyloid as a critical factor in ad pathogenesis and prompted application of molecular approaches and animal models to understand the disease. since [ ] , case-control genome wide association studies (gwas) have identified many novel ad-associated genetic variants [ ] . though many of these individually confer only modestly elevated risk for developing ad, collectively these studies provide broad insight into the pathways and processes involved in load. many identified genetic linkages are implicated in modulating immune function [ ] , demonstrating an important role for the immune response in ad. more recently, next generation sequencing technologies have made possible the identification of rare variants, some of which may confer higher disease risk and therefore can provide important insight into genes with strong biological roles in disease [ ] . the application of whole exome sequencing [ ] and gwas with imputation based on predicted genetic associations [ ] to ad led to the identification of relatively rare variants in the gene triggering receptor expressed on myeloid cells (trem ) that are associated with a high risk for developing ad. heterozygous trem variants confer similar risk for ad as one copy of apoe . significantly, the adassociated trem variants are largely coding variants, in contrast to most of the single nucleotide polymorphisms (snps) identified in gwas [ ] , making it more straightforward to translate into in vitro and in vivo models and perhaps also into therapeutics [ ] . trem variants have now also been linked to other ndds, suggesting that trem is critically involved in shared disease mechanisms. the excitement in the field following identification of these ad-associated trem variants was also driven by its implications, providing a clear link between the innate immune system and ndd pathogenesis. while it has long been known that immune cell function is dysregulated in ad and other ndds, it was not clear whether this actively contributed to disease pathogenesis and progression or was just a secondary response to ad-related pathology. however, this debate was largely settled in favor of the former when trem variants were found to be significantly associated with risk for ad and other ndds, and to form a genetic basis of polycystic lipomembraneous osteodysplasia with sclerosing leukoencephalopathy (plosl, also known as nasu-hakola disease). because trem is exclusively expressed on immune cells, these genetic associations were hailed as providing conclusive evidence that immune dysregulation can be a primary, causal contributor to ndd pathogenesis [ , ] . thus, ndd-associated trem variants provide a new avenue to investigate the important roles that the immune system plays in neurodegeneration [ ] . in the years since trem variants associated with ad risk were identified, many groups have developed research programs aimed at understanding trem genetics, expression, structure, signaling, function, and its relationship to ndd pathologies and applied these findings to clinical biomarkers and therapeutics. progress in these areas has clarified our understanding of the biology of the trem receptor. while it was previously thought that trem expression was decreased by proinflammatory stimuli and mediated anti-inflammatory effects, it is now clear that its roles are more complex. in vitro, inflammatory stimuli decrease trem expression but in vivo trem expression is increased in inflammatory contexts. more than half of studies report that trem has a pro-inflammatory effect, suggesting that there must be cell type-and context-dependent functions of the receptor. recent studies have also illuminated new aspects of trem biology which necessitate a reevaluation and reinterpretation of previous literature. one example is the finding that soluble trem is produced in ad in a disease progression-dependent manner [ ] and that this soluble form of the receptor may have distinct biological effects [ , ] . other fundamental aspects of trem biology are also under intense investigation, including epigenetic and posttranslational modification of trem that affect expression and function, the ontogeny of trem expressing cells in the brain, and how noncanonical signaling pathways may contribute to trem function. this review offers a comprehensive synthesis of these studies alongside previous trem literature to identify areas of consensus and emerging questions in the field. this understanding will be crucial to support informed design and interpretation of studies of trem and the immune response in ndds moving forward. diverse trem variants are associated with ndd risk there is great diversity in the trem variants that have been associated with ndds, including single amino acid substitutions, frameshift and nonsense mutations, and changes in splice sites predicted to alter the inclusion or exclusion of particular exons [ ] . and, while most of the trem variants identified are present in the coding sequence, there have also been disease-associated variants found in the 'utr [ ] , and upstream of the transcription start site [ ] . the first ndd-associated trem variants identified were w x and w x, which result in premature truncation of the protein, a variant at the consensus splice site which results in exclusion of exon , and the k n mutation, which disrupts association of trem with its obligate intracellular signaling adaptor, dap [ ] . these all likely result in a loss of trem function, and a patient with the e x nonsense variant of trem had no detectable trem transcript levels [ ] . subsequently, a host of diverse trem variants have been identified. despite their structural diversity, all of the ndd-associated trem variants identified have been suggested to confer loss of function through different mechanisms. however, whether loss of function truly unifies all of these variants is still very much an open question. trem variants are the genetic basis of plosl and some familial ftd cases trem was first identified as a genetic cause of plosl, also commonly known as nasu-hakola disease [ , ] , which is characterized clinically by bone cysts and fractures, neuropsychiatric symptoms and dementia [ ] . neuropathologically, plosl patients have axonal degeneration and white matter loss, as well as cortical atrophy [ , ] . this is accompanied by an inflammatory response consisting of increased microglial density and activation and astrocytosis [ ] . these neurological manifestations can also occur in the absence of fractures [ ] or bone cysts [ ] . paloneva and colleagues [ ] were the first to link trem variants with plosl, and since then, many studies have identified homozygous trem variants that form the genetic basis of plosl [ ] [ ] [ ] [ ] [ ] [ ] . studies in families with frontotemporal dementia (ftd) [ ] or frontotemporal lobar dementia (ftld) found that plosl-associated trem variants t m [ , ] , w x [ ] , q x and y c [ ] in either homozygosity or heterozygosity could also cause ftd [ ] [ ] [ ] (fig. ) . case-control studies were then performed to assess whether trem variants might increase risk for ftd in the general population. initial studies suggested that there was a positive association between trem variants as a whole and risk of ftd [ , ] with a significant association found between ftd risk and individual trem variants including t k and l p [ ] and r h [ ] . others failed to replicate this association with trem variants and ftd or ftld [ , , , ] . however, an association between trem variants and specific endophenotypes of ftd, including reduced white matter volume, seizures and motor symptoms has been reported [ ] . together, it is not clear whether trem variants increase risk for ftd outside of specific familial cases, but they may influence specific clinical manifestations of the disease. fig. diverse trem variants are associated with ndds. genetic variants in the trem gene (shown above) result in diverse changes in the protein structure (shown below). these variants occur in almost every exon (black boxes) and impact known protein motifs (sequences highlighted in blue) and flank many sites of known protein modifications (amino acid number and type of modification detailed inside black boxes). trem variants have been found to be significantly associated with many ndds, including ad (variants shown in yellow), ftd or ftld (pink), pd (purple) and plosl (red). the table shows genetic variants that have been found to be significantly associated with disease risk, with supporting references shown in dark green and references that provide strong counterevidence shown in red. references shown in light green did find a significant association between the trem variant and disease risk, but only in one or multiple populations they examined or only after inclusion of previously published literature into metastudy analyses. while these variants have been significantly associated with disease risk, many more studies find suggestive but not significant associations between additional trem variants and ndd risk which are not represented here [ , , , , , , , , - , - , - , - , , , , , , , , , , , , , , - ] trem variants are associated with risk for ad it was investigated whether trem variants could also confer risk for alzheimer's disease. while it was first suggested that plosl-associated genes might confer risk for ad in [ ] , a small, case-control study in failed to demonstrate a significant association with ad risk [ ] . however, larger studies in found that heterozygous expression of the trem r h [ , ] and d n variants [ ] were significantly associated with ad risk. the association of trem variants with ad has been extensively replicated [ , , [ ] [ ] [ ] [ ] and the r h variant [ , , , , [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] validated in neuropathologically-confirmed cases [ ] . other variants have also been consistently shown to confer ad risk, including d n [ , ] , r h [ , ] , l p and t k, and h y [ , ] (fig. ). while rare, individuals with ad homozygous for the r h [ , , ] and l p [ ] variants have also been identified. studies examining the association of trem variants with particular ad endophenotypes yielded somewhat inconsistent results, likely due to the small sample size of patients with these trem variants. some found that r h trem variant carriers had a decreased [ ] or a trend toward decrease [ ] in the age of ad onset, though others found no significant association [ , ] . additionally, some found that disease progression was accelerated in r h carriers [ , ] , though this was not observed in all studies [ ] . other variants were also found to increase [ ] or decrease [ ] the rate of ad progression. despite possible differences in ad onset or progression, the clinical presentation of ad in r h carriers is similar to non-carriers [ , ] , though there may be a higher incidence of some endophenotypes including seizures and motor symptoms [ ] . trem variants have also been assessed as potential risk factors for other neurodegenerative diseases, though the findings in these other disease contexts are less definitive. in amyotrophic lateral sclerosis (als), one study found a significant association of the r h variant and als risk, as well as an inverse correlation between trem levels in the spinal cord and survival in als patients [ ] , though this was not replicated by others [ ] . the trem r h variant has also been reported to be associated with increased risk of parkinson's disease [ ] by some [ , , ] but not all studies [ , , ] . in order to understand these differences, lill and colleagues [ ] divided their groups by ethnicity and found that the odds ratio of the r h variant was significantly higher in their northern european population compared to non-northern europeans. others identified another parkinson's disease (pd)-associated snp kb upstream of trem [ ] , though its effect on trem expression is not known. the trem r h variant has also been investigated as a risk factor for posterior cortical atrophy [ ] , multiple system atrophy [ ] , essential tremor [ ] , multiple sclerosis [ ] and creutzfeldt-jakob disease (cjd) [ ] , though these studies were not conclusive. one family has been identified in which a mutation in trem is thought to result in progressive non-fluent aphasia [ ] , though other cases will be necessary to confirm this association. so far, evidence suggests that trem variants are not significantly associated with dementia with lewy bodies [ ] , ischemic stroke [ ] or progressive supranuclear palsy [ ] . because many of these diseases share overlapping clinical features with ad and ftd, it will be important to validate any associations of trem with other ndds in neuropathologically confirmed cases. overall, the association of trem variants with these other ndds is less clear, and future studies with large sample sizes in diverse but well-matched populations will be required to definitively establish whether trem variants confer risk for ndds other than plosl, ftd and ad. importantly, the association of trem variants with multiple ndds suggests it may underlie common disease mechanisms. trem dysfunction may provide insight into mechanistic links among these diseases. epidemiologically, the prevalence of trem variants differs greatly among individuals from different genetic backgrounds [ ] . in caucasian populations, the minor allele frequency (maf) of the r h trem variant ranges from . - . % in the united states, to up to % in some specific british populations [ , , ] . while the r h variant is virtually absent in east asian individuals, [ , , [ ] [ ] [ ] [ ] [ ] [ ] [ ] nine other trem variants were present in east asian populations and collectively associated with ndd risk [ ] . similarly, the maf of the r h and r h variants are much lower in african americans compared to european american populations [ , ] . however, exonic sequencing of trem revealed variants that had much higher mafs in the african american population compared to european americans, and some of these variants were significantly associated with ad risk in that population [ ] . because of the small mafs, it is not clear whether the effect sizes of these variants differ among different ethnic groups as well. the low mafs and diversity in the frequency of trem variants across populations necessitates that studies have large study populations and be well-matched for ethnicity. it will also be important to take advantage of the identification of these trem variants across diverse populations to gain a full understanding of how trem variants confer ad risk and how they might interact with other genetic differences among individuals from distinct genetic backgrounds. several groups have examined the relationship between trem and other ndd risk factors. variants in siglec- (cd ) are significantly associated with ad risk and cd levels on human blood monocytes were found to inversely correlate with surface trem levels [ ] . the ad-associated cd allele increased its surface expression, effectively decreasing trem signaling. another ad-associated gene, membrane-spanning -domain family a (ms a), was found to be co-enriched with trem in the human brain and is substantially upregulated in monocytes derived from patients with ploslassociated trem variants [ ] . an ad-associated snp in ms a correlated with altered soluble trem (strem ) levels in cerebrospinal fluid (csf), suggesting that ms a may also regulate trem expression or processing [ ] . trem levels are also increased in ad mouse models lacking pgrn expression, which models lower pgrn expression observed with an ad and ftld-associated pgrn genetic variant [ ] . components of the trem signaling pathway have also been associated with ndds, including its intracellular signaling adaptor dnax activation protein of kda (dap , also termed tyrobp). dap and trem variants produce virtually indistinguishable phenotypes in plosl patients [ , ] . however, satoh and colleagues [ ] found that dendritic cells derived from monocytes of plosl patients with trem or dap mutations had very different patterns of gene expression, suggesting that they may produce the same phenotype through different molecular mechanisms. a rare dap variant at the site of interaction with trem also confers risk for developing early onset ad [ ] and dap was found to play a central role in ad-related molecular networks [ ] . as its relationship to ndds might predict, dap deficient mice also have synaptic degeneration and reduced myelination [ ] , though recent evidence suggests that dap deficiency may be neuroprotective in an ad mouse model [ ] . the precise mechanisms underlying these changes are not yet understood. variants in additional proteins associated with the trem signaling pathway, ship and colony stimulating factor receptor (csf r), have been associated with ad risk and leukoencephalopathy with spheroids [ , ] . in addition, apoe, a putative trem ligand [ ] [ ] [ ] , is clearly established as an ad risk factor [ ] . finally, environmental risk factors for ad including traumatic brain injury [ , ] , diabetes [ ] and age [ ] , all alter trem expression in the brain. together, the identification of variants in genes involved in these common immune pathways suggest that trem , along with its interaction partners, together play an important role in modifying ndd pathology. co-regulation of trem and other members of the trem family trem is located on human chromosome in a gene locus containing several trem and trem-like genes, such as trem , triggering receptor expressed on myeloid cells like transcript (treml ) and treml , which likely originated from duplication events but now have relatively diverse sequences [ ] [ ] [ ] . many of these genes, including trem , are highly conserved between humans and mice, while others are present only in mice (trem and treml ) or humans (treml and nkp ). there may be some shared mechanisms of gene regulation across the locus. for example, there is a retinoid x receptor (rxr) binding site upstream of the entire locus that is thought to result in coordinate regulation of these genes [ ] . however, in some cases, opposing regulation of these different genes has been shown, such as between trem and trem [ ] , and between trem and treml [ , ] . it is not known what factors contribute to these inverse correlations in expression. snps within this locus can also result in changes in expression of multiple trem genes. variants in the treml [ ] and treml [ ] gene have been shown to increase brain trem and treml expression levels. chan and colleagues [ ] found that ad-associated variants in trem result in reduced trem expression on human monocytes and increased trem expression. moreover, an intronic variant in trem which decreases its expression leads to increased amyloid accumulation and cognitive decline in ad patients [ ] . however, non-ad associated variants decreased both trem and trem expression, leading the authors to suggest that the ratio of trem and trem expression rather than the absolute changes in expression may be important for disease. additionally, variants in treml increase pd risk [ ] , and other disease-related snps that alter treml levels [ ] associate with ad in gwas analyses [ ] . another variant of treml was found to be protective against developing ad [ ] . an intergenic variant associated with ad risk was shown to alter rna levels of treml [ ] . these data highlight the importance of characterizing the expression patterns of all trem members in an effort to pinpoint co-regulatory mechanisms among these genes physiologically and in the context of disease. trem expression is highly cell-type and context specific. however, the molecular mechanisms governing this highly specific regulation of trem expression are just beginning to be understood. while there are several predicted transcription factor binding sites in the utr's and promoter region of trem , only a few have been functionally validated. pu. , a master regulator of myeloid cell fate specification, is present in the trem promoter [ ] . the rxr agonist bexarotene was found to enhance rxr occupancy of known binding sites [ ] upstream of trem in mice, though this did not correlate with increased trem rna levels [ ] . however, in ad mouse models, bexarotene did enhance gene expression of trem [ ] , suggesting that rxr may effectively induce trem transcription when cells are already primed in a particular context. nkκb [ , ] , protein e [ ] , rankl, and nfat [ ] have all been shown to regulate trem expression in different cell types, but it is not clear whether these factors directly regulate expression. one group has suggested that nfκb may instead regulate trem expression by increasing levels of microrna a which, in reporter assays, decreased trem expression [ , [ ] [ ] [ ] [ ] [ ] . there are also epigenetic changes that have been shown to influence trem expression. in humans, hippocampal enrichment of -hydroxymethylcytosine ( hmc), a marker of active demethylation, at the trem transcription start site and in exon were found to positively correlate with trem mrna levels [ ] . methylation upstream of the trem transcription start site was also increased in ad, a context in which trem levels are also increased [ ] . additionally, methylation at the cpg sites in intron negatively correlated with trem mrna levels in human leukocytes, and methylation at these sites was reduced in ad patients [ ] . others have found that h kme and h kme , histone modifications which typically are associated with active gene transcription, are increased at the trem locus in db/ db mouse adipose tissue [ ] and in cultured dendritic cells and macrophages during differentiation [ ] , contexts in which trem mrna levels are elevated. finally, there are likely post-transcriptional mechanisms of regulation of trem expression which contribute to differential expression in different contexts. trem mrna stability can be dynamically regulated. the half-life of trem mrna in cultured human peripheral blood mononuclear cells went from . h to h after toll-like receptor (tlr) ligation [ ] . furthermore, hu and colleagues [ ] found that trem mrna expression strongly correlated with surface levels of trem protein on monocytes, but only weakly correlated with protein expression on granulocytes in the plasma. this suggests that, in addition to differential regulation of trem transcription through transcription factors and epigenetic markers, trem expression can be further differentially controlled at the mrna and protein level in distinct cell types through mechanisms which are not resolved. how trem variants affect trem expression is a topic currently under investigation. as discussed above, some trem variants are known to reduce trem expression. the nonsense mutations e x [ ] and q x [ ] were both found to eliminate trem protein expression. trem rna levels were reduced in patients harboring the variant t m [ ] and a splice donor mutation in intron [ ] . heterozygous expression of a variant that affects trem splicing in intron , which is associated with early onset dementia, also affected the expression of the common variant allele of trem , reducing it by more than half [ ] . it is unclear how these variants result in reduced trem transcript levels, though it has been suggested that epigenetic changes may partially account for these effects. in contrast, the r h variant either did not change [ ] or trended toward increasing trem transcript levels [ ] in individuals with ad. although there were no changes found in gene methylation upstream of trem 's transcription start site in r h carriers compared to controls [ ] , variants tend to cluster around exon where trem expression was found to be correlated with hmc enrichment [ ] . whether variants affect regulation of trem rna expression at the level of epigenetics, transcription factor binding, rna stability or altering the cell phenotype in a manner that indirectly drives alterations in trem transcription is not yet clear. at the protein level, in transfected cell lines t m and y c trem variant proteins were found to be degraded by the proteasome, leading to decreased protein expression [ ] . in ad patients with r h variants, there was a trend toward decreased trem protein levels in one study [ ] , but a trend toward an increase in another [ ] . it will be crucial to continue collecting data from trem variant carriers to gain a clearer picture of how trem expression is altered by different disease-associated variants. in plosl patients with dap mutations, there was a variable effect on dap expression levels, in some cases increasing and in others decreasing expression [ ] . it may be that trem variants also alter trem expression in distinct ways. trem is expressed on many cells of the myeloid lineage, as its name suggests, including dendritic cells [ , , , [ ] [ ] [ ] , granulocytes [ ] , bone marrow and monocyte derived macrophages [ , , , , ] , and tissue macrophages like splenocytes [ ] , kuppfer cells [ ] , alveolar macrophages [ , ] , and osteoclasts [ , , ] (fig. ) . trem is not reported to be expressed on lymphocytes [ ] . its expression on circulating monocytes remains controversial. initially it was thought that trem was only expressed after differentiation of monocytes into macrophages [ ] , and others have provided further data to support a lack of trem expression or expression on only a small subset of monocytes in humans [ , , , ] and mice [ ] . however, others have detected trem expression in whole blood [ , [ ] [ ] [ ] and specifically on human [ , ] and mouse [ ] monocytes. these disparate findings may be due to differences in sensitivity of detection or other technical factors, but it will be important to resolve moving forward. it is clear that trem is expressed in the brain and most evidence documents that trem is expressed exclusively within the brain by microglia. all published studies to date find that primary cultured microglia express trem [ , [ ] [ ] [ ] [ ] . in vivo, most studies detect trem expression in mouse microglia [ , , [ ] [ ] [ ] [ ] [ ] [ ] , though some find that it is expressed by only a subset of these cells [ , [ ] [ ] [ ] [ ] and others could detect trem rna but not protein expression in mouse microglia [ ] . furthermore, studies demonstrated that when microglia are acutely depleted from the brain through csf r antagonist treatment in vivo, [ ] through cd b-hsvtk depletion in brain slices [ ] , or chronically in pu. knockout mice [ ] , trem is no longer detectable in the cns. in humans, experimental outcomes have been more variable, with some detecting high levels of trem expression across all microglia [ ] , others finding lower levels of trem expression [ ] and others not detecting microglial expression of trem [ ] . with a few exceptions, most evidence is in strong agreement that trem is expressed, at least under normal physiological conditions, specifically in microglia within the cns. can be present as a full-length protein (shown at top) or as a soluble product. this can occur through proteolytic cleavage by adam followed by γ-secretase to produce soluble trem (strem ), a c-terminal fragment (ctf) and an intracellular domain (icd). trem can also be alternatively spliced to produce soluble isoforms. one alternative transcript has been validated in mice, while two have been validated in humans and four others are predicted to occur [ , , , , , , , ] trem expression changes throughout neurodevelopment and varies across brain regions trem expression in the cns is also regulated throughout development and its expression pattern varies across different brain regions. trem is first detectable in the mouse cns at e and continues to be expressed through adulthood [ ] . however, at p , all brain myeloid cells express trem rna, but not all of these cells still express detectable levels by p , which has also been supported in vitro [ ] . this early elevated trem expression also occurs in other organs following macrophage infiltration during organogenesis [ ] . trem expression is also regulated differentially across brain regions in humans and mice. trem was found to be highly expressed in white matter [ , ] , hippocampus [ , , ] and spinal cord [ , ] , among other regions. while this could suggest that particular microenvironmental niches induce trem expression locally within distinct microglial populations, it may also simply reflect the high density of microglia in these regions [ ] . trem expression has been shown to be dramatically altered in the contexts of inflammation, injury and disease. in vitro, application of classically pro-inflammatory molecules (tnfα [ , ] , il β [ , , ] , ros [ , ] , ifnγ [ ] , tlr agonists, including lipopolysaccharide trem is expressed in many immune cells, and is localized to microglia in the cns. trem expression has been assessed in a variety of human and mouse cell types. these data represent trem expression in these cells under homeostatic conditions, though, as discussed in the next section, trem expression can change in the contexts of inflammation or pathology. references shown in green are supportive of trem expression in the cell type listed while those in red did not detect trem expression using the listed method of detection. references in yellow provide evidence of expression, but at low levels or in a small percentage of cells assayed. the graph represents the cell types in which trem expression has been examined at a size relative to the number of studies and methods used to detect trem expression in that cell type. they are graphed along the y-axis according to the percentage of these findings which support trem expression on these cells [ , , , , , , , , , , - , - , , - , , , , , , ] (lps) [ , , , , [ ] [ ] [ ] [ ] , cpgs [ ] and other tlr ligands [ , , , ] , mitochondrial lysates [ ] and bacteria [ ] ) decreased trem expression, while anti-inflammatory molecules (vasoactive intestinal peptide [ ] and il [ ] ) increased trem expression. in contrast, in vivo, inflammation and different disease states almost universally increase trem expression. stimuli that induce an inflammatory response in the lung increase trem expression in alveolar macrophages [ , , [ ] [ ] [ ] [ ] [ ] [ ] . high fat diet [ , ] increased inflammation and trem expression in adipose tissue, liver and brain. trem is also upregulated in numerous other inflammation-related contexts, including sepsis [ ] , rheumatoid arthritis [ ] , corneal infection [ ] , macular degeneration [ ] , glioma [ ] , oral [ ] , esophaegeal [ ] , and liver [ ] cancers, following prosthetic joint implants [ ] , osteoporosis [ ] , colonic mucosal injury [ ] , colitis [ ] , gastrointestinal mucositis [ ] and muscular sarcoidosis [ ] . in the cns, trem expression is increased in the context of traumatic brain injury [ , ] , stroke [ , ] , spinal nerve transection [ ] , als [ ] , pd [ ] , prion disease [ , ] , models of demyelination [ , , [ ] [ ] [ ] and following beta-amyloid (aβ) vaccination [ ] . only one study found reduced trem expression in in vivo inflammatory contexts, following lps injection and middle cerebral artery occlusion in mice [ ] . trem has also been shown in almost all cases to positively correlate with aging, both in mouse models [ , ] and in humans [ ] . soluble trem , a product of full-length protein cleavage or alternative splicing, is detectable in csf and its levels were also positively correlated with age [ , , , ] , though this was not reflected in blood [ ] . taken together, these studies largely demonstrate that, in vitro, inflammatory stimuli decrease trem expression, while in vivo, inflammatory stimuli predominantly increase trem expression, clearly suggesting that the dogma based on early studies that trem expression is universally reduced in inflammatory contexts is not applicable to in vivo contexts. why this occurs is not clear, but may reflect differences in cell recruitment, acute versus chronic signaling, non-cell autonomous signaling pathways, or phenotypic changes in myeloid cells that occur when they leave their native environment. the most comprehensive assessment of changes in trem expression has been performed in the context of alzheimer's disease. studies in ad patient brain tissue almost exclusively show increased trem expression [ , , , , [ ] [ ] [ ] [ ] , and some [ , , ] but not all [ ] found this was also reflected in increased trem levels in monocytes from ad patients. trem levels in the brains of ad mouse models are also increased. one study reports a reduction in trem rna levels before the onset of pathology in tg mice [ ] , though after the onset of pathology, all amyloid models of ad examined have increased trem rna and protein levels [ , , [ ] [ ] [ ] [ ] [ ] . this upregulation of trem expression occurs shortly after the onset of pathology and largely seems to correlate with amyloid burden [ , , ] and the association of myeloid cells with amyloid plaques [ , ] . tau models of ad also show increased trem levels [ , ] , however, trem is only increased long after neurofibrillary tangle development in these models [ ] , consistent with trem upregulation at late stages of disease progression in postmortem ad brain tissue [ ] . several studies have sought to determine what aspect of ad pathology drives trem expression. trem is upregulated in myeloid cells associated with plaques [ , , , , , ] , and specifically, trem is highly expressed on myeloid cell processes in contact with plaques [ ] . in support of a plaque-driven upregulation in trem expression, varvel and colleagues [ ] depleted microglia from the brains of an ad mouse model and allowed the brain to repopulate with new myeloid cells. these new cells that repopulated the brain initially failed to associate with plaques, but eventually became plaque-associated, and coordinately upregulated trem expression. furthermore, stereotactic injection of beta-amyloid (aβ ) into the cortex and hippocampus of wild-type mice was sufficient to induce an upregulation in trem transcripts within h [ ] . together, these findings suggest that amyloid can increase trem expression in myeloid cells. to determine whether this effect was cell-autonomous, cultured microglia were treated with aβ, though these studies have so far produced inconsistent results with respect to trem expression [ , ] . significantly, melchior and colleagues [ ] found that there was no effect on cultured microglia treated with beta-amyloid (aβ ) on trem levels, but if they added aβ to mixed glial cultures, this did result in upregulation of trem on microglia by flow cytometry. this suggests that, at least this aβ species may drive trem expression through feedback from other cell types, though the signals that mediate aβ-induced upregulation of trem on myeloid cells are not yet known. not all myeloid cells associated with plaques express trem [ , ] . investigation of which subset of myeloid cells upregulated trem in the ad brain has yielded conflicting results. some findings suggest trem + cells may be peripherally derived macrophages rather than brain resident microglia. jay and colleagues [ ] found that trem was expressed on cd hi myeloid cells which expressed the monocyte marker ly c and not the microglial-specific marker p ry . following toxoplasma gondii infection in xfad mice, mohle and colleagues [ ] found that trem was expressed most highly by c-c chemokine receptor type (ccr ) + ly c lo f / + cells in the brain, a marker signature of peripherally derived macrophages. trem expression in other disease models, including basal cell carcinoma [ ] , sciatic nerve transection [ ] and colonic mucosal injury [ ] , was found to be upregulated coincident with the peak of macrophage infiltration into the tissue. in contrast, others have detected trem expression on "dark" microglial cells in the brain, which are d + and c − , a marker signature consistent with resident microglia [ ] . wang and colleagues [ ] also performed experiments in which age-matched cd . wt donors were parabiosed with cd . ad mouse models and were not able to detect cd . cells in the brains of the ad mice. indeed, the contribution of peripherally derived macrophages to ndd pathologies has a long and controversial history. however, if trem is expressed on these cells, it suggests they may play a key role in modulating pathology, and thus this issue should continue to be explored. the structure of full-length trem trem is a single pass transmembrane protein whose ligand binding domain includes an extracellular ig-like domain [ ] with n-linked glycosylation sites [ , , , ] , phosphorylation sites and disulfide bonds which are thought to perform important structural roles. the transmembrane domain anchors trem to the membrane and contains the intramembraneous lysine residue necessary for association with its intracellular membrane adaptor, dap . this is followed by a short cytoplasmic tail with no established function. diseaseassociated variants of trem alter many of these structural elements (fig. ). many variants are found in exon and may change the structure of its ligand-binding domain, impacting the affinity of trem for different ligands. for example, the y c variant associated with plosl and ftd is predicted to alter an important flanking sequence of the cysteine residues which form trem 's disulfide bonds [ , ] . likewise, glycosylation is affected in cells transfected with y c and t m trem variants [ ] , though not in several other variants examined [ ] . glycosylation of the r h variant was also found to be reduced [ ] , though not to the same extent as the other trem variants in vitro. in humans expressing the r h variant there were no significant differences in the level of glycosylation [ ] but there were differences in the pattern of glycosylation [ , ] . trem variants can also affect other important structural motifs of the trem protein such as those required for dap association, and overall protein folding and stability. t m and y c variants [ ] along with v g are predicted to be important for protein packing. consistent with impaired protein folding, t m and y c variants exhibit enhanced proteasomal degradation [ ] . the r h variant has been predicted to impair protein stability [ ] , but transfected r h-trem constructs actually have an increased half-life relative to wt trem and are resistant to proteasomal degradation in the endoplasmic reticulum (er) [ ] . ndd-associated trem variants located on the surface of the protein (r h, t k, d n and r h), are not predicted to substantially alter trem structure [ , ] but instead affect ligand binding [ ] . as the structure of trem and disease-associated variants continue to be resolved, we will gain better insight into the organization of structural features essential for trem function. the structure and production of soluble trem trem can also be produced as a soluble protein (fig. ) . soluble trem (strem ) has been detected in the supernatants of mouse [ ] and human cells in culture [ , ] . it has been proposed that strem could be produced by both alternative splicing and proteolytic cleavage. insertions [ ] or frameshifts [ ] preceding exon terminate the transmembrane domain and are predicted to yield a soluble product. in human brain tissue, at least three trem isoforms have been detected [ ] , with isoform , encoding the full-length protein, being the most highly expressed [ ] . meanwhile, the transcript encoding a -residue splice isoform is expressed to a lesser extent than isoform in the hippocampus of ad patients [ ] , whereas the degree of expression of the -residue splice isoform has yet to be resolved. based on rna sequencing data in ad mice, - % of transcripts were predicted to be alternatively spliced [ ] . notably, these alternative transcripts have been identified in human monocytes [ ] , and in ad brain tissue [ , ] . evidence of elevations in expression of trem exons and in advanced ad cases by microarray-based gene expression analysis [ ] is suggestive of trem alternative splicing in ad [ , , ] . the expression of full length and splice isoforms of trem are strongly correlated in ad tissue, suggesting all trem isoforms may be coordinately regulated [ ] . dna methylation within the body of the gene has been shown to impact alternative splicing [ ] , and as trem can be methylated within exon [ ] , it is possible that context-dependent changes in methylation may also result in altered splicing. however, direct evidence that alternatively spliced mrnas are translated is lacking. the sequential proteolytic processing of trem has definitively been shown to generate strem [ , , ] . in vitro inhibitor studies revealed full-length trem is proteolytically cleaved by a disintegrin and metalloproteinase domain-containing protein (adam ) resulting in shedding of the ectodomain [ , , ] . detection of trem c-terminal fragments (ctfs) within cell lines [ , , ] and human brain extracts [ ] is suggestive of a two-step proteolytic cleavage event of trem . following ectodomain shedding by adam , the remaining membrane-associated trem c-terminus undergoes intramembrane proteolysis by γ-secretase to release its intracellular domain [ ] . γ-secretase cleavage of trem results in accumulation of trem ctfs, without a corresponding increase in full-length trem at the plasma membrane upon γ-secretase inhibition [ , , ] . it is not known how this process is regulated, but it is known that trem 's adaptor protein dap is required for strem production, at least in some contexts [ ] . whether the production of strem in ad occurs by alternative splicing or ectodomain shedding is unclear. it may be cell-type and context dependent as different cell types showed either an up-(dendritic cells) or downregulation (microglia, monocytes) of the trem splice transcript levels when stimulated by lps [ , ] . il and il were also shown to enhance strem production in bone marrow derived macrophages [ ] . this is consistent with studies examining other members of the trem family as trem [ ] [ ] [ ] and tremlike transcript- [ ] which also produce cell-specific protein products. trem variants linked to ad and other neurodegenerative diseases can alter soluble trem generation. trem variants can drive novel trem splicing [ , ] such as the plosl-associated trem variant c. + t > c in which conversion of a single nucleotide at a splice-donor consensus site within intron results in the deletion of exon in addition to exon and/or exon [ ] . this is proposed to produce soluble protein products [ ] lacking either the transmembrane (tm) domain or both the tm and ectodomain. while most trem variants are expressed in all isoforms of trem , there are diseaseassociated variants expressed only in the alternatively spliced isoforms of trem [ , , , , ] . several reports have also demonstrated that trem variants t m and y c yield significant changes in strem release by cultured cells [ , ] . this was also evident in individuals homozygous [ , ] and heterozygous [ ] for the t m variant, exhibiting a loss or reduction in csf levels of strem , respectively. other case reports showed disease-associated trem variants lowered csf strem levels compared to a non-carrier group [ ] . while some have reported decreased levels of strem and trem intracellular domain (icd) production in vitro with the r h variant [ ] , the trem r h variant acted similarly to wild-type in strem production in other in vitro assays [ ] and ad patients carrying the r h variant had elevated levels of csf strem [ ] . there was a trend for higher transcript levels of a -residue trem splice variant in cortices of late-stage ad cases with the r h risk allele compared to significant elevation in this splice isoform in non-carrier ad cases [ ] . these data altogether suggest that different trem variants have distinct effects on strem levels. in addition to altering proteolytic cleavage, the localization of trem within the cell can greatly impact trem signaling, and its trafficking appears to be a highly dynamic process. in homeostatic conditions, trem seems to be primarily found intracellularly [ ] , associating with the trans-golgi network [ , , ] and in a population of exocytic vesicles [ ] . these vesicles appear to be continuously shuttled to the membrane, a process which can be rapidly induced by increases in ca + in response to ionomycin [ ] . it is not clear what other specific stimuli or disease contexts result in changes in trem localization within the cell, but this will be critical to understand trem 's functional role in these contexts. trem is recycled from the membrane in clatherin-coated vesicles in a beclin- [ ] and vps dependent manner [ , ] . vps mediates recycling of trem from the membrane via retromer complexes [ ] . when this process is blocked, trem increases its association with the lysosome and is degraded [ ] . trem variants can impact trem localization within the cell. in cells in which trem variants were transfected, t m and y c [ , , , , ] as well as other variants [ ] significantly reduced trem surface expression. these variants increased the localization of trem with the er [ , ] , which may indicate impaired protein folding. the r h variant was found to either not alter surface expression [ , ] or reduce surface expression of trem to a lesser extent [ ] . unlike the other variants, r h trem was mostly localized to the trans-golgi network rather than the er, comparable to the wt receptor [ , ] . however, yin and colleagues [ ] did find that the r h variant had reduced association with vps , resulting in increased lysosomal degradation following recycling of the receptor from the surface. overall, this suggests that variants may, in part, impact trem function by altering the localization of trem within the cell. while trem has been proposed to play a functional signaling role exclusively on the cell surface, this could also impact possible functional roles of trem in other cellular compartments. despite substantial efforts, the identity of the biological ligands of trem remains controversial (fig. ) . trem is known to modulate myeloid cell activity in response to microbial products [ ] , which led several groups to test bacteria as a possible source of trem ligands. using a trem -fc fusion protein that consists of the extracellular ectodomain of trem attached to the fc-portion of human igg, trem was found to bind to some bacteria [ , ] , including gram-positive (s. aureus) and gram-negative species (e. coli, f. tularensis), but not s. cerevisiae [ , ] , salmonella or typinmurin [ ] . trem was specifically found to bind highly anionic bacterial products [ ] and pertussis [ ] and cholera [ ] toxins. in addition to microbial products, trem was also reported to bind to high molecularweight nucleic acids [ ] and heat-shock protein [ ] , that were further shown to initiate trem signaling in reporter cell lines. while some of these interactions were found to be of relatively low affinity [ ] , recognition of many molecules like carbohydrates and glycans induce only minimal biological signaling at low densities, but at high densities when receptors are forced into closer contact, can create strong biological effects [ ] . indeed, glycosaminoglycans and specifically heparin sulfate were found to modulate trem binding [ ] and it has been suggested that this may result in clustering of trem on the membrane, thus potentially modulating trem binding to other ligands [ ] . trem signaling and function. trem has been proposed to bind to a variety of different ligands, categorized here by lipids, lipoproteins and ligands associated with damage-or pathogen-related molecular patterns. these ligands bind to the trem receptor. following ligand binding, trem can associate with dap homodimers or dap /dap heterodimers to mediate downstream signaling. this signaling requires phosphorylation of the adaptor, following which activating (shown on left in green) or inhibitory (shown on right in red) signaling components can bind. these activating components have been shown to initiate different downstream pathways that lead to cell proliferation and differentiation, survival, phagocytosis, chemotaxis and inflammation. while many other signaling components are thought to play a role downstream of trem activation, only those validated as part of the signaling pathway responsible for the listed functions have been included here. association with inhibitory components is thought to prevent activation of these downstream pathways. lipids: pe [ , ] , ps [ , , , , ] , pa [ , , ] , pg [ , , ] , pc [ , , , , ] , pi [ , , ] , cl [ , , ] , sm [ , , , ] , la [ , ] , sa [ , , , ] , apo-/lipoproteins: apoe [ , , ] , apoj [ ] , apoa- [ , ] , apoa-ii [ , ] , ldl, hdl [ , , ] , pamp/damps: [ , ] , (−) bacterial products (lps, lta) [ ] , nucleic acids [ ] , heat shock protien [ ] , apoptotic cells [ , , , , ] , macropahages [ ] , astrocytoma cells [ ] unidentified trem ligands were also detected by trem -fc binding to the cell surface of macrophages [ ] , human astrocytoma cells [ ] , dendritic cells [ ] , n a cells [ , ] , thp- cells [ ] and apoptotic cells [ ] . in support of these findings, trem deficiency partially impairs microglial recognition of apoptotic cells [ , , ] . recent studies have demonstrated that trem binding to these cells is sensitive to proteinase k, suggesting the receptor binds to protein [ ] , perhaps complexed with proteoglycans. it had previously been shown that members of the trem family recognized lipid ligands [ ] , and lipids may also help mediate the interaction of trem with ligands on the cell surface. polar lipids found on the cell surface were assessed as possible trem ligands [ , , ] , using lipid arrays [ ] and reporter assays [ , ] . while anionic lipids seemed to produce among the highest levels of response, additional factors that influence the particular lipids that trem recognizes requires further study. this lipid binding may allow trem to sense changes in the local environment as exemplified by trem -positive cells binding to externalized phosphatidylserine on apoptotic cells [ ] and myelin debris [ ] . some studies suggest that trem may bind lipids in cis with other protein-based interactions [ ] . trem has also been reported to bind to lipoproteins, including apolipoprotein a (apoa-i), clusterin (clu), and low density lipoprotein (ldl), though apoe has been most widely demonstrated to bind to soluble trem -fc. this binding seems to occur independent of apoe isoform [ ] [ ] [ ] ] and be dependent on residues - [ ] . because trem binds to lipids, the lipidation status of apoe and other apolipoproteins could dictate their binding affinity to trem . several studies [ , , ] demonstrate that trem -apoe binding is not dependent on lipid loading. however, others have found that lipidation was necessary to drive trem binding [ ] . lipid association is reported to be necessary for trem binding to apoe, apoa-i and apoa-ii from cynomolgus macaque csf and serum [ ] . apoe binding to trem was found to induce trem signaling in nfat reporter cell lines [ ] , though how its binding to trem would alter signaling in vivo remains to be determined. because apoe can bind to apoptotic cells [ ] and amyloid plaques [ , ] , it has been proposed that an interaction between trem and apoe may indirectly allow it to mediate recognition and phagocytosis of these substrates. this may be important in the recognition of ad-related stimuli by trem , because trem was found not to bind to plate-bound aβ [ ] , but did bind to areas around amyloid plaques in an ad mouse model [ ] . however, the possibility of trem binding directly to aβ, in addition to these indirect interactions, has not been fully excluded. together, these data indicate that, in addition to proteinproteoglycan complexes, trem may also bind to lipids and protein-lipid complexes. ad-associated trem variants including r h, r h, d n and t k, are found on the surface of the protein, and impact ligand binding [ ] . studies employing a trem r h-fc chimeric protein revealed the r h mutation significantly reduces trem binding to cells [ ] , apoe [ ] including all three isoforms [ ] and its lipidated form [ ] , other lipids [ , ] , apolipoproteins [ , ] and lipoproteins [ ] . trem variants at either the same residue (r a, r e and r a) [ ] or with a similar r-to-h substitution (r h) [ , ] as r h similarly disrupted trem recognition of apolipoproteins [ ] or cells [ ] . however, r h and r h variants demonstrated relatively comparable lipid detection to wt trem [ ] . other variants residing within the trem ectodomain (y c, t m, k m) effectively abolished trem binding to proposed ligands [ ] , while those located on the ectodomain proximal to the stalk region of trem (d n, t k) exhibited enhanced interactions with some ligands [ , ] , while decreasing association with others [ ] . trem variants within the stalk region (h y, e k, r w) or intracellular domain (l p) had no significant impact on ligand binding. the differences among these diseaseconferring trem variants' recognition of cells, lipids, lipoproteins and apolipoproteins may alter how they impact downstream signaling. however, it remains unclear whether the ligands identified thus far are the relevant binding partners of trem in vivo and mediate the receptor's ability to respond to damage or infection in the cns. future work establishing the full array of physiological trem ligands, and how variants impact these interactions will be instrumental in elucidating the role of trem on myeloid cells in response to different pathologic stimuli. because the major isoform of trem has only a short cytoplasmic tail, it requires the intracellular adaptor dap [ , ] to mediate several of its signaling functions [ , ] . along with trem , dap is required for signaling of other trem family members [ ] , mdl- , and siglecs and can be used as an adaptor for other receptors critical for regulating myeloid cell function including csf r and toll like receptors [ ] . in some contexts, cross-talk among these receptors has been shown to occur at the level of dap availability. in order to associate with the membrane, dap requires the presence of its receptors [ ] . indeed, dap is clustered at the same area of the membrane where trem is highly upregulated on myeloid cell processes in contact with plaques in an ad mouse model [ ] . at the membrane, dap can associate with other dap molecules to form homodimers through cysteine residues in its short extracellular domain [ ] . in some contexts, dap can also heterodimerize with dnax activation protein of kda (dap ) [ ] which can modify downstream signaling cascades. it is not known whether dap complexes constitutively associate with trem or whether this is induced upon ligand binding in vivo but recent data using a split luciferase assay found that trem -dap association in transfected hek cells was primarily driven upon trem stimulation [ ] . interestingly, the t m trem variant but not r h or s c variants enhanced the constitutive association with dap in this system [ ] . regardless, activation of trem and other dap -associated receptors results in tyrosine phosphorylation of dap within its immunoreceptor tyrosine-based activation motifs (itam) by src family kinases casein kinase ii at residues - and at residues - by pkc [ ] . this phosphorylation occurs only when dap is receptorassociated [ ] and serine / threonine phosphorylation of the itam motifs are required for signaling [ ] . these phosphotyrosine residues on the dap itam serve as docking sites for a number of molecules that initiate signaling cascades that activate an immune response. there are several immune stimulating molecules that associate with dap in response to trem activation (fig. ) . crosslinking trem , commonly used to mimic trem activation, can result in recruitment of dap , pi k or lab to the trem -dap complex. in turn, these molecules are activated through tyrosine phosphorylation, principally by itam-associated syk and go on to activate downstream signaling components, including akt, rac, vav and mapks, including erk [ , , , , , [ ] [ ] [ ] [ ] . these initiate changes in gene expression and cytoskeletal rearrangement which mediate many downstream cellular functions associated with immune cell activation [ ] . while itam domains are typically activating, they can be inhibitory in certain contexts [ , ] . when the itam motifs of dap are partially phosphorylated [ ] , inhibitory phosphatases ship, shp and the adaptor downstream of kinase (dok ) are recruited to the trem -dap signaling complex. these molecules inhibit immune activation [ , [ ] [ ] [ ] , possibly through blocking dap , pi k, and syk association with the trem -dap complex and preventing activation of erk, vav and calcium mobilization [ ] . whether signaling through dap results in activation or inhibition of the immune response seems to be receptorand stimulus-dependent. activation of trem and myeloid dap -associating lectin (mdl ) but not sirpβ enhanced association of the inhibitory ship with dap [ ] . similarly, macrophage colony stimulating factor (mcsf) alone but not mcsf and rankl induced localization of ship to dap [ ] . even different levels of the same stimulus can induce association of trem -dap with different downstream signaling components. for example, peng and colleagues [ ] found that a low dose, but not a high dose of lps resulted in association of dap with dok . this served to dampen the cellular response to lps, as dok deficiency increased downstream signaling components, cytokine production and death of mice administered an otherwise sub-lethal dose of lps. however, at high doses of lps treatment, dok did not associate with dap . ship was also shown to moderate the response of trem -induced proliferation. when ship deficient preosteoclasts were exposed to an activating trem antibody, osteoclast formation was upregulated an additional -fold [ ] . whether dap serves to activate or inhibit the immune response depends on the receptor it is associated with, the stimulus used to activate that receptor and the strength of that stimulus. it may also depend on availability of different downstream signaling components locally at the membrane, their relative expression in the cell [ ] or other environmental factors [ ] . much of the signaling data thus far was performed in cultured osteoclasts, and future studies may find that other cell types use distinct signaling mechanisms. whether trem signaling is activating or inhibitory in the context of disease is also not known, and a greater understanding of the different trem pathways that are relevant in disease will be instructive. interestingly, ship variants also confer risk for ad, and it is thought that these variants result in a change in the transcriptional start site of ship resulting in a protein that lacks its sh domain which is necessary for association with itams and immunoreceptor tyrosine-based inhibitory motifs (itims) [ ] . this suggests that these inhibitory components that associate with trem deserve attention moving forward in understanding immune-related pathways that are important in ad. there is a general consensus that trem likely acts as a homodimer or homomultimer to induce downstream signaling. this is a common mechanism of activation of other receptors with similar structures to trem in which ligand binding induces complex formation and initiates downstream signaling cascades. almost all studies examining trem signaling have provoked trem dimerization using antibody-mediated crosslinking to induce signaling [ ] . however, there is also evidence that trem can associate with other receptors, including plexina [ , ] . other studies are suggestive that trem could also bind to treml [ ] through co-ip experiments or csf r due to their close linkage in network analyses [ ] and the strong commonalities in their downstream pathways [ , ] . while these last two interactions remain to be validated, it is certainly possible that trem also acts through these alternative heteromeric complexes. whether this is regulated by the cell type or context, and whether this is an important role of trem endogenously in the context of ndds, remain to be determined. the biological roles of strem have been controversial. initially strem was postulated to act as a decoy receptor opposing full-length trem function. a soluble version of a closely related trem family member, strem , modeled by a trem -fc fusion protein, competes with its membrane-bound form to block trem signaling [ ] and produces opposing effects on inflammation and survival following lps injection in mice [ ] . soluble trem may similarly act as a decoy receptor to negatively modulate trem signaling [ , ] . in support of this, in vitro a chimeric trem -fc protein used to model soluble trem inhibited osteoclastogenesis, a process that requires trem -dap signaling [ ] . recent studies suggest that strem may have its own biological function. exogenously applied strem was internalized by cultured bone marrow derived macrophages, and promoted survival in cells lacking trem expression [ ] . however, strem failed to rescue phagocytosis in trem deficient bone marrow macrophages in culture [ ] . recent data also demonstrate that treatment of microglial cell lines with trem -fc or a hek-cell produced strem peptide increases survival, in line with full-length trem function [ ] . this study also found that strem strongly induced an inflammatory response in culture models of microglia. these data suggest that there are important biological roles of strem other than acting as a decoy for the full-length trem receptor. the mechanisms underlying strem function are not yet well understood, but interestingly, does not require the presence of full-length trem or its intracellular adaptor dap [ ] . the signaling role of trem ctfs is also starting to be explored. in the absence of γ-secretase activity, membrane-associated trem ctfs have been proposed to either promote trem anti-inflammatory signaling in response to lps [ ] , or impair trem signaling by sequestering dap from interacting with full-length receptors [ ] , reducing dap phosphorylation and downstream plcγ activation [ , ] along with trem -mediated phagocytosis [ ] . production or stabilization of ctfs on the membrane may also provide a point of cross-talk through which trem could modulate the signaling of other dap -associated receptors. it is clear that further study will be necessary to gain insight into how these soluble trem products impact signaling both of trem and other pathways critical to innate immunity. while trem expression and signaling are contextdependent, there are some commonalities in trem function that have been found across the diverse cell types and environments in which it has been studied (fig. ) , one of which is regulating myeloid cell number. the impact of trem on myeloid cell number outside of the context of disease or stimulus is not completely clear. while knocking down trem in primary microglia lead to reduced cell number [ ] , it had no effect on osteoclasts derived from peripheral blood mononuclear cells (pbmc's) from plosl trem e x carriers lacking trem expression [ ] and microglial numbers were the same up to year of age in mouse models lacking trem expression. however, crosslinking trem did promote an increase in osteoclast number in culture [ ] . what is clear is that trem has an effect on increasing myeloid cell number in response to inflammation or disease. trem deficiency was shown to prevent increases in the brain myeloid cell populations in response to traumatic brain injury [ ] , ischemia [ , ] , aging [ ] , and in the initial response to demyelination [ ] , though it did increase the number of cells in a model of sepsis [ ] . trem deficiency also prevented local increases in myeloid cells around plaques in ad. amyloid plaques are typically surrounded by a rapidly recruited [ , ] cluster of activated myeloid cells in ad human brain tissue [ , ] and in ad mouse models [ ] . recent evidence demonstrates reduced myeloid cell accumulation around amyloid plaques in trem hemizygous [ , , ] , and trem - [ , , , , ] and dap -deficient [ ] ad mouse models, as well as in postmortem ad human brain tissue from individuals harboring the trem r h variant [ ] . these data illustrate that trem , and its adaptor protein, dap , are required for myeloid cell accumulation around amyloid plaques. while wang and colleagues [ , ] found that trem deficient ad mice had a decrease in total brain myeloid cells, others found that this was primarily driven by the specific loss of plaque-associated myeloid cells [ , ] . together, this suggests that trem is important for myeloid cell expansion in response to disease. evidence suggests that in various contexts, trem is important for myeloid cell survival, proliferation and chemotaxis, all of which could lead to disease-associated increases in myeloid cell number. trem has been shown in multiple contexts to be important for cell survival. osteoclasts [ ] and bone marrow derived macrophages [ ] from trem deficient mice, and liver cancer [ ] and glioma cell lines [ ] in which trem was knocked down had increased levels of caspase , bcl- -associated x protein (bax), annexin v and tunel positivity, all suggesting that trem deficiency enhanced apoptosis. similarly, primary microglia and the bv microglial cell line with reduced trem expression had decreased survival, along with decreased levels of elements of the survivalrelated wnt/β-catenin pathway [ ] . conversely, trem activation through receptor crosslinking increased survival of monocyte-derived dendritic cells [ ] and osteoclasts [ ] . in culture, microglia derived from trem deficient mice did not show more cell death at baseline, but when levels of csf , an important factor for the maintenance of microglial survival, were reduced, trem deficient microglia were more likely to undergo apoptosis [ ] . this was also found to be the case in bone marrow derived macrophages [ ] . in an ad mouse model, trem deficiency also increased the number of plaque-associated myeloid cells which were tunel+ [ ] . taken together, these studies suggest that trem is protective against apoptosis, especially under stressful cellular conditions. trem may also increase cell number through promoting myeloid cell proliferation. in glioma cell lines [ ] , liver cancer cell lines [ ] , and primary microglia [ ] , reduced levels of trem led to cell cycle arrest. the number of proliferating myeloid cells were also decreased in vivo in response to demyelination [ ] , colonic mucosal injury [ ] and in ad mouse models [ , ] lacking trem expression. while the mechanisms of this regulation of proliferation are not clear, trem deficiency in cultured osteoclast precursors prevented csf -mediated proliferation [ ] , a process also critical for proliferation of many macrophage populations, including brain myeloid cells. it has been suggested that trem may interact with the csf- receptor to mediate these effects. in dendritic cells derived from plosl patient pbmcs expressing q x and v g trem variants, gene expression profiling identified "negative regulation of proliferation" as a genetic pathway which was significantly increased in variant carriers compared to controls [ ] . in addition to being important for directing proliferation of the cells in which it's expressed, trem might also promote a myeloid cell phenotype that directs proliferation of other cells in the surrounding microenvironment. trem is highly upregulated during organogenesis when macrophages release factors to promote proliferation of surrounding cells [ ] , in tumor associated macrophages where analogous macrophagedriven trophic support occurs [ ] , and following cns trauma where myeloid cells serve as an important source of neurotrophic support during tissue repair [ ] . interestingly, trem is strongly upregulated by neural stem cells [ ] and esc-derived oligodendroglial precursors [ ] . a relationship between trem expression and neurogenesis has not yet been explored, but given the influence of trem of proliferation on other cell types, this may warrant further examination. trem may also influence cell differentiation. differentiation was impaired in osteoclasts derived from plosl patients expressing trem variants [ ] and in raw macrophages deficient for trem through a plexina dependent pathway [ ] . however, otero and colleagues [ ] demonstrated that mouse-derived trem deficient preosteoclasts differentiated into osteoclasts faster. though the role of trem in cell differentiation is not completely clear, this step in cell phenotype determination may also contribute to the changes in cell numbers and population observed in the context of trem deficiency. another potential contributor to trem 's role in expanding the myeloid cell population in the context of disease or inflammation is by modulating chemotaxis or migration of these cells. in culture, knocking down trem reduced chemotaxis of glioma cells in a boydon chamber assay in response to serum [ ] , and in the bv microglial cell line in a scratch assay [ ] . microglia from trem deficient brain slices exhibited reduced chemotaxis into co-cultured brain tissue from old or ad mouse models [ ] . in addition, trem deficient mice had fewer microglia migrate to the site of apoptotic neuron injection in the brain and had slower process extension toward a brain laser lesion as measured using two photon microscopy [ ] . conversely, trem crosslinking increased ccr -dependent chemotaxis [ , , ] . trem was also found to be co-enriched with genes involved in purinergic signaling, a key pathway directing microglial chemotaxis in network analyses [ ] , though whether trem regulates p r-receptor mediated chemotaxis has not been examined experimentally. however, others did not see a deficit in chemotaxis in plosl patient-derived osteoclasts [ ] . kiialainen and colleagues [ ] found that pbmc's cultured from patients with plosl-associated trem variants had both up-and down-regulated components of the chemotactic response. it may be that different components of the chemotactic pathway and therefore different types of chemotaxis are differentially regulated by trem . studies have examined the effect of trem on specific chemotactic pathways that are involved in tissue infiltration by myeloid cells. wang and colleagues [ ] found that trem deficient glioma cell lines downregulated cxcl , cxcr , mmp and mmp which are all important in tissue invasion. in network analyses, trem was significantly co-enriched with dock and dock which are involved in tissue transmigration [ ] and mice deficient for trem had reduced leukocyte infiltration following experimental induction of colitis [ ] . there was also decreased neutrophil recruitment to mouse lungs in response to bacterial infection in trem deficient mice [ ] . in vitro, cells lacking trem expression had reduced chemotaxis toward ccl [ ] . mice lacking dap were found to have significantly reduced recruitment of peripheral macrophages in vivo in response to cigarette smoke or intranasal c-c motif chemokine ligand (ccl ) administration, and these dap -chemotactic deficits were found to be rescued by reintroducing a trem -dap fusion construct [ ] . trem deficient mice also had reduced levels of ccl [ ] and fewer peripheral immune cells in their brain following middle cerebral artery occlusion (mcao) [ ] . there was a trend toward a correlation between ccl and strem levels in the csf of human ad patients, which may suggest that trem plays a role in mediating ccl -mediated chemotaxis of cells in the context of ad as well. however, other studies have found no link between trem and monocyte trafficking into inflammatory tissues [ ] . future studies will be necessary to assess which chemotactic pathways are influenced by trem and whether that includes pathways related to peripheral immune cell infiltration into the cns in ndds. a well-characterized function of trem is to enhance phagocytosis. trem is expressed in a subset of myeloid cells within the cns that have high phagocytic capacity [ ] . across numerous in vitro studies, loss of trem results in reduced phagocytosis of a variety of substrates, including apoptotic neurons or neuronal cell lines [ , , , ] , bone [ , ] , bacteria and bacterial products [ , , , ] and lipids [ , ] . conversely, trem activation or overexpression enhanced uptake of these substrates [ , , ] . trem expression correlated with aβ uptake in bv cells in which trem was knocked down or overexpressed [ ] . aβ uptake was also reduced in trem deficient primary microglia [ , ] and in the n microglial cell line expressing a non-functional trem when plated onto brain slices from ad mouse models [ ] . in agreement with these findings, in vivo, trem deficient mice have reduced localization of aβ within cd + phagosomes in ad mouse models [ ] and reduced uptake of deposited aβ three hours after injection into the brain [ ] . together, these findings suggest that trem is important for aβ uptake by brain myeloid cells. however, in culture, trem expression was no longer found to correlate with aβ uptake after pretreatment of cells with lps [ ] . a similar effect was observed in a mouse model of sepsis where injection of myeloid cells overexpressing trem enhanced bacterial phagocytosis and survival, but not if the mice were pretreated with lps [ ] . these findings suggest that the mechanisms of trem -dependent phagocytosis can be modified by other signals in the microenvironment. interestingly, the other modulatory components present in the brain microenvironment change throughout the course of ndds, which could explain some of the differences in trem function at different stages of disease progression. outside of ad, trem is important for clearance of myelin in experimental autoimmune encephalomyelitis (eae) [ ] and peri-infarct tissue in mice following mcao [ ] . however, it does appear that the effect of trem on phagocytosis can be cell type specific. sharif and colleagues [ ] found that bone marrow macrophages derived from trem deficient mice had reduced phagocytosis, but trem deficient alveolar macrophages had increased uptake of bacteria in vitro and in vivo. r h [ ] and r h trem variants had impaired phagocytosis [ ] . this was also true in hek cells transfected with trem -dap fusion constructs expressing r h, t m and y c variants [ ] . interestingly, while all of these variants impaired uptake of polystyrene beads, t m and y c but not r h impaired uptake of e. coli particles [ ] , suggesting that different trem variants could affect recognition of specific phagocytic substrates as well as induce changes in basal phagocytic activity reflected in the fluid phase uptake of beads. more studies will be required to parse out the role of trem in basal phagocytosis and cargo-driven phagocytosis of specific substrates. the mechanism underlying trem -dependent uptake of various substrates is not clear. while transfection of a trem -dap construct into cho cells was shown to be sufficient for uptake of neuro a cells [ ] , it may be that trem does not have to directly bind to its phagocytic substrates, as trem binding to hsp was sufficient to increase phagocytosis of bacteria [ ] . if trem does not directly bind to these substrates, then it must interact with other phagocytic pathways. it is possible that trem impacts fluid-phase phagocytosis rather than cargo driven phagocytosis. trem may also interact with other phagocytic receptors. for example, mertk is essential for the phagocytosis of apoptotic cells [ ] and is upregulated on the same cell population as trem in ad [ ] . network analyses have also shown that trem is co-enriched with genes involved in fcγr and complement-mediated phagocytosis [ ] . in support of an association between trem and fc-dependent phagocytic pathways, stimulating cells lacking trem function with an antibody against the desired phagocytic substrate did increase internalization of the substrate, but did not rescue it back to wt levels [ ] . trem deficient alveolar macrophages were found to increase phagocytosis and this was found to be dependent on the upregulation of first component of complement q (c q) in these cells [ ] , which acts to opsonize phagocytic substrates. it may also be that strem plays a role in binding and directing phagocytosis of substrates by these other pathways, as adam inhibitors reduced strem production and decreased phagocytosis of e. coli [ ] . some have also suggested that these findings may reflect changes in degradation of phagocytic substrates rather than their uptake. forabosco and colleagues [ ] found that genes associated with lysosome activity were co-enriched with trem across the brain and in monocyte-derived macrophages. in plosl patients, there is an accumulation of large cd + myeloid cells, suggesting that phagocytic uptake by these cells may be intact [ ] . in a cuprizone model of demyelination, trem deficiency was found not to impair uptake of myelin debris, but that this debris remained in cells longer than in controls, suggesting that degradation was specifically impaired [ ] . this was also found to be true in trem deficient macrophages which were able to take up bacteria at comparable levels to cells expressing wt trem but were unable to kill and degrade them [ ] . however, jiang and colleagues [ ] found that in primary microglia in which trem was knocked down, aβ degradation was unaffected. together, the exact role of trem in phagocytosis and other means of cellular uptake and degradation of substrates from the microenvironment remain unclear, though it clearly does play an important role in these processes. trem interacts with many other inflammation-related pathways. while trem has been touted as being antiinflammatory, it seems that the interaction between trem and other inflammation related pathways is actually more complex. depending on the precise stimuli, the strength [ ] and duration [ ] over which they are presented, the cell type and the context, trem can play different roles in the inflammatory response. in support of this, network analyses found that trem was co-enriched with both classically pro-and antiinflammatory gene clusters in the brain [ ] . likewise, a microarray analysis of macrophages derived from a plosl patient pbmc's showed components of the inflammatory response and innate immune response were both up-and down-regulated, respectively, relative to controls [ ] . outside of the context of injury or disease, the transcriptional profiles of trem deficient [ ] or overexpressing [ ] myeloid cells compared to controls was fairly similar. it is in the context of disease where trem seems to heavily influence changes in inflammation-related pathways. trem has been classically described as being antiinflammatory and several in vitro and in vivo studies are supportive of an anti-inflammatory role for trem in certain contexts. knocking down trem in cell lines increases levels of proinflammatory mediators such as inos, tnfα, il β and il [ ] in response to apoptotic neuronal membrane components [ ] , tlr ligands [ ] , including lps [ , , , ] and aβ [ ] . a transient knock down of trem in the p s tau model and in the samp model of accelerated aging also increased inflammatory cytokine production [ , ] . trem deficiency also resulted in increased levels of ifnγ, tnfα and inos [ ] following colonic mucosal injury and trem knockdown or antibody-mediated inhibition increased expression of many inflammationrelated cytokines following corneal infection [ ] . moreover, overexpressing trem in cell lines, amyloid [ ] and tau models of ad [ ] reduced levels of these pro-inflammatory transcripts. together, these studies suggest that in some contexts, trem can attenuate inflammatory responses. however, many other studies also support that trem can mediate or amplify inflammatory responses. for instance, trem knockdown impaired ros production [ , ] . trem deficient microglia are more ramified in culture, a morphological signature of reduced activation [ ] . trem deficient ad mouse models have reduced levels of inflammation-related transcripts in both unbiased rna sequencing approaches [ ] and in the genes il β and il in targeted analyses [ , ] . plaque-associated cells in ad mouse models deficient [ ] or haploinsufficient [ ] for trem also had decreased cell soma size, surface area and increased process length, indicative of reduced activation [ ] . recent work using single cell sequencing approaches indicates that trem is required specifically for a second phase of the myeloid cell response in ad which allows cells to fully adopt a neurodegeneration-associated phenotype [ ] . this may be true in diverse disease contexts as pro-inflammatory cytokine levels were also reduced in trem deficient mice following traumatic brain injury [ ] , ischemia [ ] , lung infection [ , ] and demyelination [ ] , where trem deficient brain myeloid cells exhibited a less activated morphology [ ] . conversely, activation of trem in a macrophage cell line increased no release [ ] , agonizing trem following spinal nerve transection increased tnfα and il β [ ] and overexpression of trem increased expression of il , tnfα and mcp in mouse adipose tissue [ ] . because these studies examined gene expression in whole tissue, it is not clear whether these changes are due to changes in immune cell phenotype or alteration in cell number in the affected tissues. however, taken together, these findings clearly indicate that trem can also promote inflammatory responses in certain contexts. this body of data strongly opposes the often-cited descriptor of trem as an anti-inflammatory receptor. future studies will be required to delineate the molecular and environmental determinants that govern how trem contributes to the inflammatory response in different contexts. while the number of studies have been limited, trem variants associated with ndds also seem to have mixed effects on inflammatory responses. the r h variant impaired inflammatory responses in bv cells [ ] , yet r h carriers with ad had increased expression of genes related to inflammatory pathways compared to non-carriers [ ] . a variant within intron of trem , which is prevalent in african american individuals, was found to be significantly associated with levels of c-reactive protein (crp), a systemic marker of inflammation, whose expression is primarily driven by il and il β [ ] . however, it is not clear whether this represents a direct relationship between this trem variant and systemic inflammation. in addition to impacting the inflammatory responses of myeloid cells, trem also seems to be able to indirectly feedback onto the inflammatory response in other cells within the microenvironment, including astrocytes. astrocytosis, measured by glial fibrillary acid protein (gfap) levels, was reduced across all stages of pathology examined in trem deficient ad mouse models [ , ] , in areas of active demyelination [ ] and trended toward a reduction in gfap area in mice following ischemia [ ] . however, gfap levels were unchanged in trem deficient mice at acute and chronic time points following traumatic brain injury [ ] , suggesting that trem must work in tandem with context-dependent signals to alter astrocyte activation. one of the characteristic features of plosl is astrocytosis [ ] and in a plosl patient with a trem variant, gfap levels were significantly increased in frontal lobe tissue [ ] . this suggests that trem can play multiple roles in regulating astrocyte activation depending on the precise context. while regulating cell number, phagocytosis and inflammation are the best studied roles for trem , other studies have suggested additional roles for the receptor, such as regulation of synaptic pruning and monitoring of synaptic function [ ] . because of the cross-talk between trem and complement pathways and a clear role of trem and complement in phagocytosis in disease [ ] , it would be of interest to assess whether trem influences synaptic function by modulating synaptic pruning, either normally during development or aberrantly in the context of ndds. others have suggested that, due to the close apposition of trem + cells to oligodendrocyte precursors during development, they may support their function [ ] . trem has also been shown to be important for angiogenesis following stroke [ ] . because of trem 's proposed lipid-related ligands, and the strong links between lipid metabolism and ndds, it would not be surprising if trem also played roles in this pathway. in support of this, lipid metabolism was the most strongly altered pathway in trem deficient mouse brains following cuprizone-induced demyelination [ ] . how trem affects these normal functions within the brain has not been studied, but may represent important future areas of investigation. outside of the brain, studies have proposed additional roles for the trem receptor. trem has been proposed to play a role in adaptive immunity. myeloid cells expressing higher levels of trem were able to increase t cell proliferation better than those expressing lower trem levels [ ] . trem was also found to be coenriched with genes related to adaptive immunity in gene network analyses [ ] . however, others have found that activating trem through crosslinking did not upregulate molecules involved in antigen presentation [ , ] , suggesting that trem -mediated stimulation of adaptive immune responses may be indirect or require additional environmental factors. trem also seems to be important for cell maturation [ ] and in particular multinucleation of osteoclasts [ , , ] . it is not yet known how trem might mediate these additional functions. the observed changes in trem expression, signaling and function with disease-associated genetic variants ultimately translate to changes in ndd pathology. many studies have focused on how trem and diseaseassociated variants impact ad-related pathologies. studies examining loss of trem function in amyloid mouse models initially appeared to support contradictory conclusions. some groups found that trem deficiency reduced [ , ] while others found that it increased [ ] amyloid pathology. however, recent evidence has harmonized these results by demonstrating that trem deficiency has a changing role throughout ad progression, reducing amyloid pathology early but increasing it at later stages of disease [ ] . studies overexpressing trem in ad mouse models also found a temporal effect of this overexpression, which reduced pathology early in disease progression [ ] but no effect at a later time point [ ] . this is supported by korvatska and colleagues [ ] who demonstrate accelerated disease progression in r h carriers compared to non-carriers with ad. this may also explain discrepancies in human studies of r h carriers who found no association between r h carriers and non-carriers in amyloid deposition [ ] and others who found that r h carriers had significantly more plaques compared with noncarriers [ ] . this changing role for trem throughout progression of amyloid pathology may also reflect a dynamic role for myeloid cells themselves. as hickman and el khoury [ ] posit, these brain myeloid cells may be protective early through clearance of aβ, but detrimental later in disease progression when they enhance the inflammatory response without being effective phagocytes. alternatively, it could reflect how trem impacts the phenotype and abundance of distinct myeloid cell subsets, or perhaps other microenvironmental cues which change trem downstream signaling to favor alternative pathways. while mouse models so far have recapitulated several aspects of trem localization and function observed in human brain tissue, it is worth noting that there is a caveat to studying trem in mouse models of ad with psen mutations since γ-secretase is also important for trem ctf cleavage [ ] . it is not clear whether changes in γ-secretase activity would be likely to greatly alter trem function in vivo, but this should be a consideration when interpreting these studies. interestingly, there also appears to be a difference in the mechanism by which trem affects pathology early and late in disease progression. early, trem deficiency decreases the number of plaques, while later in disease progression, increase in pathology is instead driven by increased plaque size [ ] . while it's not clear exactly how trem could modulate aβ proteostasis early in disease progression to impact plaque number. trem was shown to impact app processing in a genome-wide sirna screen [ ] , though since it is not neuronally expressed, this would likely occur through indirectly altering neuronal phenotype. conversely, later in disease, the association of myeloid cells with plaques has been proposed to limit plaque growth by forming an insulated microenvironment or barrier [ , ] . trem deficiency appears to impede the formation of this barrier, and in doing so, cause a shift from compact to diffuse plaques [ ] . trem has also been studied in ad in the context of modifying neuritic dystrophy. several studies have found increased neuritic dystrophy around plaques in trem deficient mice [ , ] and in human r h carriers with ad [ ] . trem overexpression in -month but not -month-old app/ps mice had increased levels of synaptophysin, suggesting that enhanced trem expression may protect against aβ-driven synapse loss [ , ] . one possible mechanism for this lies in the larger, more diffuse plaques with high soluble aβ affinity [ ] observed in trem deficient mice late in disease progression [ ] . the relative toxicity of soluble aβ is well documented, including its roles in blocking longterm potentiation [ ] and inducing tau hyperphosphorylation and aggregation [ , ] . together, these data suggest that functional trem is necessary for microglial clustering around amyloid plaques and may thereby form a barrier around plaques which limits neuritic dystrophy. however, not all data support a protective role of plaque-associated myeloid cells on ad pathology. microglia can serve as synaptotoxic agents in ad through complement-mediated synaptic pruning [ ] . in this way, the loss of plaque-associated myeloid cells due to trem deficiency could be beneficial. others suggest that it may not be that more dystrophic neurites are formed around plaques in trem deficient mice and r h carriers, but that trem deficient myeloid cells are not as effective at clearing them [ ] . further evidence will be required to assess the formation and clearance of dystrophic neurites across stages of ad in the context of trem deficiency or trem variants to assess these possible mechanisms. the impact of trem on tau pathology in ad has also been examined. the effect of trem on phosphorylated tau (p-tau) accumulation in dystrophic neurites in ad is not clear, with some studies showing an increase [ , ] and others showing a decrease [ ] in hyperphosphorylated tau markers surrounding plaques in trem deficient amyloid mouse models of ad. these different outcomes are likely related to disease progression dependent effects on the amyloid pathology driving this accumulation. less work has been done in tau models of ad, but overexpressing trem under the cd b promoter in the p s tau model of ad resulted in reduced hyperphosphorylated tau levels, coordinate with a decrease in activation of two of the known tau kinases, cyclin dependent kinase (cdk ) and gsk β [ ] . opposite effects were observed in p s mice in which trem was knocked down [ ] . in humans, trem protein levels in the temporal cortex of ad patients correlated with tangle score and paired helical filament (phf) levels [ ] and strem levels in csf are correlated with csf tau levels early in clinical ad progression [ ] , suggesting an important relationship between trem and tau pathology in humans. r h patients had higher levels of csf p-tau [ , ] , and a variant located upstream of trem was associated with increased tau pathology in the brain [ ] . together, these findings suggest that trem variants may also have an impact on tau-related pathologies in ad, though the mechanisms governing this association are less clear. studies have also examined how trem and its variants impact neuronal and tissue loss and cognition in ad. trem protein levels in the temporal cortex of ad patients were positively correlated with cleaved caspase and negatively correlated with the presynaptic marker snap [ ] , suggestive that loss of trem could impact synapse pathology. there was also a significant reduction in neurons in layer v of the cortex in trem deficient amyloid models of ad [ ] and a substantial rescue of neuronal loss when trem was overexpressed in amyloid [ ] and tau [ ] ad mouse models. these changes in neuron number also correlated with a rescue in behavioral deficits [ , ] . interestingly, however, trem expression levels on peripheral monocytes correlated with lower mmse [ ] and moca scores as well as reduced gray matter volume [ ] . r h variant carriers with ad also had reduced gray matter volume [ ] in the temporal cortex and hippocampus [ ] . there was also a trend toward a reduction in hippocampal volume [ ] and significant decreases in other brain regions [ ] in r h carriers even in the absence of clinical ad. though no changes in cognitive function were reported in middle-aged r h carriers [ ] , the variant did correlate with cognitive deficits in older adults with r h variants [ ] . this was also true in healthy individuals heterozygous for nhd variants [ ] . these findings suggest that trem variants may have direct effects on neuronal loss, even in the absence of ad pathology. although not as extensively studied as in ad, the impact of trem on other ndd-related pathologies has also been assessed. plosl patients have severe white matter dystrophy [ ] and oligodendrocytes that survive in plosl patient white matter express markers of cell stress [ ] suggesting a role for trem deficiency in white matter degeneration. in cuprizone-mediated demyelination models, trem deficiency impaired recovery and increased levels of axonal degeneration markers [ , ] . in addition, injecting mice with trem transduced myeloid cell precursors prevented eae-induced demyelination and ameliorated motor phenotypes [ ] . in addition to affecting oligodendrocyte survival and recovery following demyelination, trem variants in plosl have also elucidated other roles of trem in ndds. plosl patients often experience seizures [ , ] , resulting in excitotoxicity. one patient with a predicted loss-of-function trem plosl mutation had a reduction in many synaptic components, including nine gaba receptor subunits, which could play a role in mediating this enhanced excitability [ ] . however, the mechanism underlying this phenomenon is not well understood and requires further study. the effect of trem on inflammation-related pathologies trem has also been shown to modify tissue loss and behavior in several neuroinflammatory contexts. trem deficiency reduced hippocampal volume loss and improved some behavioral outcomes at chronic time points following traumatic brain injury [ ] . in contrast, trem deficient mice had increased infarct volume in one mcao model [ ] , though no change in another study [ ] . treatment with a trem agonist induced pain behavior in mice in a dap -dependent manner even in the absence of nerve injury [ ] . outside of the brain, trem deficiency results in increased body weight and glucose and insulin intolerance in mice fed a high fat diet [ ] . trem variant carriers may also have increased risk of systemic infection [ ] and trem deficiency is detrimental in the context of bacterial infection [ , , , ] . overall, in the brain and the periphery, despite great advances in assessing how trem alters pathology, there is still no clear picture of how trem mediates these diverse functional impacts across inflammatory and disease contexts. this will require a greater understanding of trem expression, signaling and function and how these features change in the context of pathology. while these studies do point toward some common mechanisms by which trem might modify aspects of pathology relevant to multiple ndds, it is clear that the role of trem in ndds is not simple. since the identification of ndd-associated trem variants, and the detection of strem in the csf and plasma of ad patients, there has been much excitement about how this may translate into immune-related ndd biomarkers and therapeutics. elevated levels of strem were first detected in the csf of patients with multiple sclerosis (ms) and other inflammatory neurologic diseases [ ] and were found to be significantly elevated in ms patients [ ] . these findings served as an impetus to examine whether csf strem might also be changed in ad patients. groups have reported elevations [ ] , reductions [ ] or non-significant changes [ ] in csf strem in ad cohorts not stratified by disease stage (table ) . however, as shown in table , studies that did divide subjects by stage of disease progression, csf strem levels were found to be significantly higher in patients with ad-related mild cognitive impairment [ , , ] and mild dementia [ , ] compared to controls and ad cases [ ] . cross-sectional studies in patients with dominantly inherited ad confirmed a significant increase in csf strem starting years before expected onset of clinical dementia, but found no significant differences between ad patients and controls beyond years after symptom onset [ ] . together, these studies suggest a specific elevation in csf strem levels in the early symptomatic stages of ad. interestingly, strem was also found to be significantly elevated specifically in early stages of als pathology before returning to baseline at late stages of disease [ ] . while it's not clear whether these disease-stage dependent effects are due to a common mechanism, it will be interesting to see whether this pattern continues to be consistent across ndds. the detection and reported changes with disease progression in csf levels of strem have raised questions of its origin and biological meaning in health and disease. in ad patients, csf strem was not associated with changes in csf aβ [ , , , ] but positively correlated with csf biomarkers total tau [ , ] and phosphorylated tau [ , , ] , including in cross-sectional cohorts with dominantly inherited ad [ ] . several groups have proposed that csf strem levels may signify microglial activation in response to ad-related pathology [ , , , , ] . evidence of csf strem positively correlating with glial protein ykl- in csf [ , ] , another proposed ad immune biomarker, in addition to immunosuppressive agents causing a reduction in csf strem levels [ ] are consistent with this theory. changes in strem appear to be independent of apoe status [ ] [ ] [ ] . further work assessing how strem generation changes with microglial phenotype will be needed to definitively validate strem as an indicator of microglial activation. a recent clinical study proposes a neuroprotective role for strem , reporting higher gray matter volume in areas susceptible to ad pathology for mild cognitive impairment (mci) and ad patients with high csf strem levels [ ] though this correlation was not found in all studies. higher levels of csf strem at late stages of als also correlated with longer survival [ ] . these findings, in combination with the positive correlation between csf tau and strem , could indicate that elevated strem production occurs as a protective response to neurodegeneration, though there is no definitive consensus yet as to the biological significance of strem . the current data on strem illustrate limitations for its use as an ndd biomarker. several ndds including ms, ad and ftd, are associated with elevated strem in csf [ , ] , suggestive of a common innate immune mechanism in these distinct pathologies. it has also been suggested as a biomarker for welding fume exposure [ ] and, as discussed above, can be regulated in many other inflammation-related contexts. this lack of disease and context specificity in strem changes raises concerns for its utility as a diagnostic tool for ad. recent data show csf strem levels are altered differentially throughout ad progression. these observations raise questions about its utility as a diagnostic readout for ad disease status in diverse neurologic cohorts, though does suggest that strem levels could be helpful in identifying stage of ad in coordination with other biomarkers [ ] . moreover, heslegrave and colleagues [ ] acknowledged that strem levels in ad patients and controls, while significantly different, substantially overlap, thereby further limiting its diagnostic utility. transcriptome-based studies found dysregulation of several innate immune genes in blood from ad patients [ ] , which led others to assess whether trem expression might also be changed in blood. while studies did find a correlation between trem expression on blood monocytes and an ad diagnosis [ ] , strem in plasma was shown to not correlate with csf strem levels [ ] and plasma samples yielded non-significant differences in strem of ad and ftd patients compared to healthy controls [ , ] . while strem alone does have clear limitations as a biomarker, it may have a potential application as part of a biomarker panel to assess the immune response in ad and other neurodegenerative diseases. in addition to its potential as a biomarker, many have suggested that trem -directed therapeutics may prove to be a novel target for ndds. there are several factors to consider in developing trem therapeutics. first, while trem variants confer as strong a risk for developing ad as one copy of the apoe allele, the minor allele frequency of trem variants are substantially lower, with less than % for trem to approximately % for apoe [ , ] . thus, though some have suggested that therapeutics might want to restore wt trem function in these variant carriers as a potential therapeutic, correcting trem variants are not likely to be a broadly applicable therapeutic approach. rather, studying trem variants that confer risk for ndds will illuminate components of the immune response centrally important in immune modulation of pathology, and serve as a prerequisite to developing targeted immune-directed therapeutics. so far, the field has identified potential changing roles for immune cell function throughout progression of ad, and possibly identified a key role for peripherally derived immune cells in ad pathology, which would greatly aid in therapeutically targeting the immune cells relevant to ad pathology. common functions of trem have been identified across multiple ndds which suggest therapeutic targets could be relevant to multiple disease contexts. however, we have not found a simple explanation for what trem does across cell types and contexts. based on its disease progression dependent effects, it does not appear that simply activating or inhibiting trem would be beneficial even in the context of ad. there may also be sex-dependent effects of trem , as some [ ] but not all [ ] have shown differences in strem levels in csf between male and female subjects. likewise, a trem variant was associated with markers of systemic inflammation specifically in women not men, and was hormone-independent [ ] . with the lack of strong biomarkers to stage ndds and the variability in clinical progression among patients, it is not likely that increasing or decreasing trem will be the universal solution to ndd pathologies. rather, understanding when, where and how trem is working is more likely to provide insights into immune function that can be modulated throughout disease progression. however, the emphasis on understanding trem in ndds began just years ago and we still have a long way to go to understand trem 's expression, signaling, function and effects on these various pathologies. it will also be essential to start to dissect how the diverse array of trem variants result in ndd risk. while this understanding of trem variants may not directly translate into trem -directed biomarkers or therapeutics at this time, the insight into how the immune system actively participates in ndd pathology promises to provide many avenues for a new class of immune-directed therapeutic targets for ndds. genetics of alzheimer's disease evidence for novel susceptibility genes for late-onset alzheimer's disease from a genome-wide association study of putative functional variants alzheimer's disease risk genes and mechanisms of disease pathogenesis targeting microglia for the treatment of alzheimer's disease trem variants in alzheimer's disease variant of trem associated with the risk of alzheimer's disease genetic variations underlying alzheimer's disease: evidence from genome-wide association studies and beyond variant trem as risk factor for alzheimer's disease essential role of the microglial triggering receptor expressed on myeloid cells- (trem ) for central nervous tissue immune homeostasis how neuroinflammation contributes to neurodegeneration trem and the neuroimmunology of alzheimer's disease dominantly inherited alzheimer n. early changes in csf strem in dominantly inherited alzheimer's disease occur after amyloid deposition and neuronal injury trem promotes microglial survival by activating wnt/β-catenin pathway soluble trem induces inflammatory responses and enhances microglial survival mutation analysis of the ms a and trem gene clusters in a case-control alzheimer's disease data set investigating the role of rare heterozygous trem variants in alzheimer's disease and frontotemporal dementia genetic determinants of disease progression in alzheimer's disease mutations in two genes encoding different subunits of a receptor signaling complex result in an identical disease phenotype dap /trem deficiency results in impaired osteoclast differentiation and osteoporotic features a lipid metabolic disease-"membranous lipodystrophy"-an autopsy case demonstrating numerous peculiar membrane-structures composed of compound lipid in bone and bone marrow and various adipose tissues neuropsychiatric and genetic aspects of a new hereditary disease characterized by progressive dementia and lipomembranous polycystic osteodysplasia nasu-hakola disease: the first case reported by nasu and review variable expression of microglial dap and trem genes in nasu-hakola disease nasu-hakola disease (polycystic lipomembranous osteodysplasia with sclerosing leukoencephalopathy -plosl): a dementia associated with bone cystic lesions. from clinical to genetic and molecular aspects immunohistochemical characterization of microglia in nasu-hakola disease brains polycystic lipomembranous osteodysplasia with sclerosing leukoencephalopathy (plosl): a new report of an italian woman and review of the literature a case of nasu-hakola disease without fractures or consanguinity diagnosed using exome sequencing and treated with sodium valproate nasu-hakola disease with a splicing mutation of trem in a japanese family absence of trem polymorphisms in patients with alzheimer's disease and frontotemporal lobar degeneration the genetic causes of basal ganglia calcification, dementia, and bone cysts dap and trem an italian family affected by nasu-hakola disease with a novel genetic mutation in the trem gene targeted sequencing approach to identify genetic mutations in nasu-hakola disease a novel mutation in trem gene causing nasu-hakola disease and review of the literature french res network ff-a. trem mutations are rare in a french cohort of patients with frontotemporal dementia homozygous trem mutation in a family with atypical frontotemporal dementia using exome sequencing to reveal mutations in trem presenting as a frontotemporal dementia-like syndrome without bone involvement variants in triggering receptor expressed on myeloid cells are associated with both behavioral variant frontotemporal lobar degeneration and alzheimer's disease heterozygous trem mutations in frontotemporal dementia investigation of the role of rare trem variants in frontotemporal dementia subtypes trem in neurodegeneration: evidence for association of the p.r h variant with frontotemporal dementia and parkinson's disease r h trem variant increases risk of typical early-onset alzheimer's disease but not of prion or frontotemporal dementia the role of trem r h as a risk factor for alzheimer's disease, frontotemporal lobar degeneration, amyotrophic lateral sclerosis, and parkinson's disease movement disorders and genetics in frontotemporal dementia lipomembranous polycystic osteodysplasia (brain, bone, and fat disease): a genetic cause of presenile dementia alzheimer's dis neuroimaging i. gene-based rare allele analysis identified a risk gene of alzheimer's disease the rare trem r h variant exerts only a modest effect on alzheimer disease risk trem and neurodegenerative disease rare trem variants associated with alzheimer's disease display reduced cell surface expression more evidence for association of a rare trem mutation (r h) with alzheimer's disease risk trem analysis and increased risk of alzheimer's disease coding variants in trem increase risk for alzheimer's disease trem is associated with the risk of alzheimer's disease in spanish population missense variant in treml protects against alzheimer's disease assessing the role of the trem p.r h variant as a risk factor for alzheimer's disease and frontotemporal dementia assessment of trem rs association with alzheimer's disease in a population-based sample: the cache county study triggering receptor expressed on myeloid cells variant is rare in late-onset alzheimer's disease in han chinese individuals the triggering receptor expressed on myeloid cells (trem ) is associated with enhanced inflammation, neuropathological lesions and increased risk for alzheimer's dementia a rare coding variant in trem increases risk for alzheimer's disease in han chinese association study of the trem gene and identification of a novel variant in exon in iranian patients with late-onset alzheimer's disease r h variant of trem associated with alzheimer disease in a large late-onset family clinical, genetic, and neuropathological study trem and neurodegenerative disease a trem variant alters the accumulation of alzheimer-related amyloid pathology frontobasal gray matter loss is associated with the trem p.r h variant trem variant p.r h as a risk factor for sporadic amyotrophic lateral sclerosis convergent genetic and expression datasets highlight trem in parkinson's disease susceptibility trem rare variant p.r h is not associated with parkinson's disease evaluating pathogenic dementia variants in posterior cortical atrophy assessment of trem rs association with parkinson's disease and multiple system atrophy in a chinese population trem r h variant and risk of essential tremor: a cross-sectional international multicenter study no evidence for shared etiology in two demyelinative disorders, ms and plosl ariffin r: a case of trem mutation presenting with features of progressive non-fluent aphasia and without bone involvement trem p.r h substitution is not associated with dementia with lewy bodies trems in alzheimer's disease: genetic and clinical investigations genetic analysis of trem variants in chinese han patients with sporadic parkinson's disease association study of trem polymorphism rs with leucoaraiosis or parkinson's disease in the han chinese population assessment of trem rs association with amyotrophic lateral sclerosis in a chinese population triggering receptor expressed on myeloid cells variants are rare in parkinson's disease in a han chinese cohort association study of trem polymorphism rs with late-onset alzheimer's disease in chinese han population investigation of trem , pld , and unc c variants in patients with alzheimer's disease from mainland china lack of genetic association between trem and late-onset alzheimer's disease in a japanese population lack of genetic association between trem and alzheimer's disease in east asian population: a systematic review and meta-analysis trem is associated with increased risk for alzheimer's disease in african americans trem : a new risk factor for alzheimer's disease cd modulates trem : convergence of alzheimer loci cerebrospinal fluid soluble trem is higher in alzheimer disease and associated with mutation status opposing effects of progranulin deficiency on amyloid and tau pathologies via microglial tyrobp network tyrobp genetic variants in earlyonset alzheimer's disease integrated systems approach identifies genetic nodes and networks in late-onset alzheimer's disease osteopetrosis and thalamic hypomyelinosis with synaptic degeneration in dap -deficient mice deficiency of tyrobp, an adaptor protein for trem and c receptors, is neuroprotective in a mouse model of early alzheimer's pathology mutations in the colony stimulating factor receptor (csf r) gene cause hereditary diffuse leukoencephalopathy with spheroids meta-analysis of , individuals identifies new susceptibility loci for alzheimer's disease trem binds to apolipoproteins, including apoe and clu/apoj, and thereby facilitates uptake of amyloid-beta by microglia apolipoprotein e is a ligand for triggering receptor expressed on myeloid cells (trem ) the triggering receptor expressed on myeloid cells binds apolipoprotein e the role of apolipoprotein e in alzheimer's disease triggering receptor expressed on myeloid cells deficiency alters acute macrophage distribution and improves recovery after traumatic brain injury differential detection of impact site versus rotational site injury by magnetic resonance imaging and microglial morphology in an unrestrained mild closed head injury model chronic consumption of a western diet induces robust glial activation in aging mice and in a mouse model of alzheimer's disease insights into trem biology by network analysis of human brain gene expression data the human trem gene cluster at p . encodes both activating and inhibitory single igv domain receptors and includes nkp trem and trem-like receptors in inflammation and disease trems in the immune system and beyond the active enhancer network operated by liganded rxr supports angiogenic activity in macrophages opposing roles of the triggering receptor expressed on myeloid cells and triggering receptor expressed on myeloid cells-like transcript in microglia activation missense variant in treml protects against alzheimer's disease a candidate regulatory variant at the trem gene cluster associates with decreased alzheimer's disease risk and increased treml and trem brain gene expression no association of trem rs and trem rs with alzheimer disease reply genome-wide association and population genetic analysis of c-reactive protein in african american and hispanic american women a comprehensive profile of chip-seq-based pu. /spi target genes in microglia rnasequencing reveals transcriptional up-regulation of trem in response to bexarotene treatment expression of the phagocytosis-essential protein trem is down-regulated by an aluminum-induced mirna- a in a murine microglial cell line divergent neuroinflammatory regulation of microglial trem expression and involvement of nf-κb e proteins regulate osteoclast maturation and survival regulation of itam adaptor molecules and their receptors by inhibition of calcineurin-nfat signalling during late stage osteoclast differentiation microrna- a-mediated down-regulation of the microglial-enriched triggering receptor and phagocytosis-sensor trem in age-related macular degeneration deficits in the mirna- a-regulated endogenous trem phagocytosis sensor-receptor in alzheimer's disease (ad); an update regulation of trem expression by an nf-kappa b-sensitive mirna- a aluminum-induced amyloidogenesis and impairment in the clearance of amyloid peptides from the central nervous system in alzheimer's disease over-expressed pathogenic mirnas in alzheimer's disease (ad) and prion disease (prd) drive deficits in trem -mediated a beta peptide clearance trem upregulation correlates with -hydroxymethycytosine enrichment in alzheimer's disease hippocampus increased dna methylation near trem is consistently seen in the superior temporal gyrus in alzheimer's disease brain dna methylation changes at trem intron and trem mrna expression in patients with alzheimer's disease in vivo evidence of enhanced dimethylation of histone h k on upregulated genes in adipose tissue of diabetic db/db mice genome-wide promoter analysis of histone modifications in human monocyte-derived antigen presenting cells inhibition of rank expression and osteoclastogenesis by tlrs and ifngamma in human osteoclast precursors increased expression of trem in peripheral blood of alzheimer's disease patients triggering receptor expressed on myeloid cells (trem ) promotes adipogenesis and diet-induced obesity impaired differentiation of osteoclasts in trem- -deficient individuals mutations in trem lead to pure early-onset dementia without bone cysts expression and processing analyses of wild type and p.r h trem variant in alzheimer's disease brains disease-associated mutations of trem alter the processing of n-linked oligosaccharides in the golgi apparatus molecular characterization of antigen-peptide pulsed dendritic cells: immature dendritic cells develop a distinct molecular profile when pulsed with antigen peptide transcript profiles of dendritic cells of plosl patients link demyelinating cns disorders with abnormalities in pathways of actin bundling and immune response a dap -mediated pathway regulates expression of cc chemokine receptor and maturation of human dendritic cells characterisation and trophic functions of murine embryonic macrophages based upon the use of a csf r-egfp transgene reporter empty liposomes induce antitumoral effects associated with macrophage responses distinct from those of the tlr / agonist pam( )csk( ) (blp) clearance of apoptotic neurons without inflammation by microglial triggering receptor expressed on myeloid cells- trem governs kupffer cell activation and explains belr genetic resistance to malaria liver stage infection dap is required for macrophage recruitment to the lung in response to cigarette smoke and chemotaxis toward ccl inflammatory response in rat lungs with recurrent exposure to welding fumes: a transcriptomic approach trem , a dap -associated receptor, regulates osteoclast differentiation and function alzheimer's disease-associated trem variants exhibit either decreased or increased ligand-dependent activation trem -mediated early microglial response limits diffusion and toxicity of amyloid plaques trem mrna expression in leukocytes is increased in alzheimer's disease and schizophrenia monocytes following knockdown of dap , a causative gene for nasu-hakola disease higher peripheral trem mrna expression levels are related to cognitive deficits and alzheimer's disease and amnestic mci trem- promotes macrophage survival and lung disease after respiratory viral infection different mechanisms of apolipoprotein e isoform-dependent modulation of prostaglandin e- production and triggering receptor expressed on myeloid cells (trem ) expression after innate immune activation of microglia analysis of the host transcriptome from demyelinating spinal cord of murine coronavirus-infected mice dap and trem , molecules involved in innate immunity and neurodegeneration, are coexpressed in the cns a role for trem ligands in the phagocytosis of apoptotic neuronal cells by microglia developmental regulation of trem and dap expression in the murine cns: implications for nasu-hakola disease triggering receptor expressed on myeloid cells- is involved in prion-induced microglial activation but does not contribute to prion pathogenesis in mouse brains distribution and signaling of trem /dap , the receptor system mutated in human polycystic lipomembraneous osteodysplasia with sclerosing leukoencephalopathy dementia upregulation of trem ameliorates neuropathology and rescues spatial cognitive impairment in a transgenic mouse model of alzheimer's disease dual induction of trem and tolerancerelated transcript, tmem b, in amyloid transgenic mice: implications for vaccine-based therapies for alzheimer's disease trem -transduced myeloid precursors mediate nervous tissue debris clearance and facilitate recovery in an animal model of multiple sclerosis triggering receptor expressed on myeloid cells (trem ) deficiency attenuates phagocytic activities of microglia and exacerbates ischemic damage in experimental stroke heterogeneous expression of the triggering receptor expressed on myeloid cells- on adult murine microglia trem deficiency eliminates trem (+) inflammatory macrophages and ameliorates pathology in alzheimer's disease mouse models colony-stimulating factor receptor signaling is necessary for microglia viability, unmasking a microglia progenitor cell in the adult brain trem protein expression changes correlate with alzheimer's disease neurodegenerative pathologies in post-mortem temporal cortices differential modulation of trem protein during postnatal brain development in mice microrna- a-mediated down-regulation of the microglial-enriched triggering receptor and phagocytosis-sensor trem in age-related macular degeneration dj- deficiency triggers microglia sensitivity dopamine toward a pro-inflammatory phenotype that is attenuated by rasagiline cutting edge: trem- attenuates macrophage activation silencing of triggering receptor expressed on myeloid cells- enhances the inflammatory responses of alveolar macrophages to lipopolysaccharide triggering receptor expressed on myeloid cells- fine-tunes inflammatory responses in murine gram-negative sepsis enhanced inhibitory effects of a novel cpg motif on osteoclast differentiation via trem- down-regulation mitochondrial lysates induce inflammation and alzheimer's disease-relevant changes in microglial and neuronal cells the triggering receptor expressed on myeloid cells inhibits complement component q effector mechanisms and exerts detrimental effects during pneumococcal pneumonia vasoactive intestinal peptide re-balances trem- /trem- ratio in acute lung injury intratracheal instillation of single-wall carbon nanotubes in the rat lung induces time-dependent changes in gene expression microarray-based analysis of the lung recovery process after stainless-steel welding fume exposure in sprague-dawley rats impact of cigarette smoke on the human and mouse lungs: a gene-expression comparison study differential regulation of dap and molecules associated with dap during host responses to mycobacterial infection adipose tissue transcriptome changes during obesity development in female dogs triggering receptor expressed on myeloid cells- protects against polymicrobial sepsis by enhancing bacterial clearance the immunoreceptor tyrosine-based activation motif (itam) -related factors are increased in synovial tissue and vasculature of rheumatoid arthritic joints trem- promotes host resistance against pseudomonas aeruginosa infection by suppressing corneal inflammation via a pi k/akt signaling pathway overexpression of trem enhances glioma cell proliferation and invasion: a therapeutic target in human glioma whole genome expression profiling in chewing-tobacco-associated oral cancers: a pilot study diagnostic marker signature for esophageal cancer from transcriptome analysis trem sirna inhibits cell proliferation of human liver cancer cell lines polyethylene particles stimulate expression of itamrelated molecules in peri-implant tissues and when stimulating osteoclastogenesis in vitro gene expression profile of the bone microenvironment in human fragility fracture bone efficient colonic mucosal wound repair requires trem signaling bacterial sensor triggering receptor expressed on myeloid cells- regulates the mucosal inflammatory response molecular characteristics of high-dose melphalan associated oral mucositis in patients with multiple myeloma: a gene expression study on human mucosa th -m immunity in lesions of muscular sarcoidosis and macrophagic myofasciitis attenuated inflammatory response in triggering receptor expressed on myeloid cells (trem ) knock-out mice following stroke trem /dap signal elicits proinflammatory response in microglia and exacerbates neuropathic pain systemic inflammation modulates fc receptor expression on microglia during chronic neurodegeneration trem sustains microglial expansion during aging and response to demyelination astrocyte-targeted production of interleukin- reduces astroglial and microglial activation in the cuprizone demyelination model: implications for myelin clearance and oligodendrocyte maturation identification of soluble trem- in the cerebrospinal fluid and its association with multiple sclerosis and cns inflammation t cells specifically targeted to amyloid plaques enhance plaque clearance in a mouse model of alzheimer's disease neuroprotective effect of trem- in aging and alzheimer's disease model triggering receptor expressed on myeloid cells knockdown exacerbates aging-related neuroinflammation and cognitive deficiency in senescence-accelerated mouse prone mice strem cerebrospinal fluid levels are a potential biomarker for microglia activity in early-stage alzheimer's disease and associate with neuronal injury markers cerebrospinal fluid soluble trem in aging and alzheimer's disease elevated trem mrna expression in leukocytes in schizophrenia but not major depressive disorder changes in the expression of genes related to neuroinflammation over the course of sporadic alzheimer's disease progression: cx cl , trem , and ppar gamma transcriptomics and mechanistic elucidation of alzheimer's disease risk genes in the brain and in vitro models neocortical and hippocampal trem protein levels during the progression of alzheimer's disease chf (csp- ) induces microglia alternative activation in plaque-free tg mice and primary glial cultures exposed to beta-amyloid viral gene transfer of apps alpha rescues synaptic failure in an alzheimer's disease mouse model a genome-wide gene-expression analysis and database in transgenic mice during development of amyloid or tau pathology trem is upregulated in amyloid plaque-associated microglia in aged app transgenic mice nuclear receptors license phagocytosis by trem (+) myeloid cells in mouse models of alzheimer's disease upregulation of trem ameliorates neuropathology and rescues spatial cognitive impairment in a transgenic mouse model of alzheimer's disease dark microglia: a new phenotype predominantly associated with pathological states silencing of trem exacerbates tau pathology, neurodegenerative changes, and spatial learning deficits in p s tau transgenic mice replacement of brain-resident myeloid cells does not alter cerebral amyloid-beta deposition in mouse models of alzheimer's disease immune hyperreactivity of aβ plaque-associated microglia in alzheimer's disease trem haplodeficiency in mice and humans impairs the microglia barrier function leading to decreased amyloid compaction and severe axonal dystrophy chronic toxoplasma gondii infection enhances beta-amyloid phagocytosis and clearance by recruited monocytes acute injury in the peripheral nervous system triggers an alternative macrophage response trem lipid sensing sustains the microglial response in an alzheimer's disease model neurodegenerative disease mutations in trem reveal a functional surface and distinct loss-of-function mechanisms the alzheimer's disease-associated r hvariant of trem has an altered glycosylation pattern and protein stability trem mutations implicated in neurodegeneration impair cell surface transport and phagocytosis evidence of trem variant associated with triple risk of alzheimer's disease preparation, crystallization, and preliminary crystallographic analysis of wildtype and mutant human trem- ectodomains linked to neurodegenerative and inflammatory diseases mycobacterium bovis bcg cell wall-specific differentially expressed genes identified by differential display and cdna subtraction in human macrophages transcriptomics and mechanistic elucidation of alzheimer's disease risk genes in the brain and in vitro models sequential proteolytic processing of the triggering receptor expressed on myeloid cells- (trem ) protein by ectodomain shedding and gamma-secretase-dependent intramembranous cleavage expression and processing analyses of wild type and p.r h trem variant in alzheimer's disease brains functional involvement of gamma-secretase in signaling of the triggering receptor expressed on myeloid cells- (trem ) metalloproteinases shed trem- ectodomain from lipopolysaccharide-stimulated human monocytes a soluble form of the triggering receptor expressed on myeloid cells- modulates the inflammatory response in murine sepsis production of soluble triggering receptor expressed on myeloid cells by lipopolysaccharide-stimulated human neutrophils involves de novo protein synthesis tlt- s, alternative transcripts of triggering receptor expressed on myeloid cell-like transcript- (tlt- ), inhibits the triggering receptor expressed on myeloid cell- (trem- )-mediated signaling pathway during osteoclastogenesis a split-luciferase complementation, real-time reporting assay enables monitoring of the disease-associated transmembrane protein trem in live cells triggering receptor expressed in myeloid cells (trem ) trafficking in microglial cells: continuous shuttling to and from the plasma membrane regulated by cell stimulation microglial beclin regulates retromer trafficking and phagocytosis and is impaired in alzheimer's disease vps -dependent recycling of trem regulates microglial function the trem receptor family and signal integration campylobacter jejuni targets immunoglobulin-like receptor lmir -o-sulfo-beta-d-galactose moiety of endogenous sulfoglycolipids is a potential ligand for immunoglobulin-like receptor lmir pattern recognition by trem- : binding of anionic ligands trem- (triggering receptor expressed on myeloid cells ) is a phagocytic receptor for bacteria the innate immune response to salmonella enterica serovar typhimurium by macrophages is dependent on trem -dap pertussis toxin targets the innate immunity through dap , fcrgamma, and myd adaptor proteins evidence for tlr and fcr gamma-card activation by cholera toxin b subunit and its direct bindings to trem and lmir receptors the surface-exposed chaperone, hsp , is an agonist of the microglial trem receptor lectins as pattern recognition molecules: the effects of epitope density in innate immunity trem -ligand interactions in health and disease cutting edge: inhibition of tlr and fcr responses in macrophages by triggering receptor expressed on myeloid cells (trem)- and dap trem- , triggering receptor expressed on myeloid cell- , negatively regulates tlr responses in dendritic cells specific lipid recognition is a general feature of cd and trem molecules the alzheimer's disease risk factors apolipoprotein e and trem are linked in a receptor signaling pathway cloning and characterization of a novel mouse myeloid dap -associated receptor family the expanding roles of itam adapters fcrgamma and dap in myeloid cells trem haplodeficiency in mice and humans impairs the microglia barrier function leading to decreased immunoreceptor dap bearing a tyrosine-based activation motif is involved in activating nk cells trem -and dap -dependent activation of pi k requires dap and is inhibited by ship trem and beta-catenin regulate bone homeostasis by controlling the rate of osteoclastogenesis the linker for activation of b cells (lab)/non-t cell activation linker (ntal) regulates triggering receptor expressed on myeloid cells (trem)- signaling and macrophage inflammatory responses independently of the linker for activation of t cells trem- promotes macrophagemediated eradication of pseudomonas aeruginosa via a pi k/akt pathway inflammation, microglia and alzheimer's disease microglial activatory (immunoreceptor tyrosinebased activation motif)-and inhibitory (immunoreceptor tyrosine-based inhibition motif)-signaling receptors for recognition of the neuronal glycocalyx you say itam and i say itim, let's call the whole thing off: the ambiguity of immunoreceptor signalling monophosphorylation of cd a and cd b itam motifs initiates a ship- phosphatase-mediated inhibitory signaling cascade required for b cell anergy the src homology domaincontaining inositol -phosphatase negatively regulates fcgamma receptormediated phagocytosis through immunoreceptor tyrosine-based activation motif-bearing phagocytic receptors the sh -containing ′-inositol phosphatase (ship) is tyrosine phosphorylated after fcγ receptor clustering in monocytes a physical interaction between the adaptor proteins dok and dap is required to inhibit lipopolysaccharide signaling in macrophages environmental factors determine dap deficiency to either enhance or suppress immunopathogenic processes genetics ignite focus on microglial inflammation in alzheimer's disease plexin-a and its interaction with dap in immune responses and bone homeostasis ddr (discoidin domain receptor ) suppresses osteoclastogenesis and is a potential therapeutic target in osteoporosis sporadic adult-onset leucodystrophy with axonal spheroids and pigmented glia with no mutations in the known targeted genes trem- ligand expression on platelets enhances neutrophil activation trem- amplifies inflammation and is a crucial mediator of septic shock contribution of nuclear factor of activated t cells c to the transcriptional control of immunoreceptor osteoclast-associated receptor but not triggering receptor expressed by myeloid cells- during osteoclastogenesis trem -deficiency reduces the efficacy of immunotherapeutic amyloid clearance dap stabilizes the c-terminal fragment of the triggering receptor expressed on myeloid cells- (trem ) and protects against lps-induced pro-inflammatory response dynamics of the microglial/amyloid interaction indicate a role in plaque maintenance rapid appearance and local toxicity of amyloid-beta plaques in a mouse model of alzheimer's disease relationship of microglia and astrocytes to amyloid deposits of alzheimer disease the complex of microglial cells and amyloid star in three-dimensional reconstruction microglial response to amyloid plaques in appsw transgenic mice altered microglial response to a beta plaques in appps - mice heterozygous for trem disease progression-dependent effects of trem deficiency in a mouse model of alzheimer's disease trem regulates microglial cell activation in response to demyelination in vivo innate immunity: the missing link in neuroprotection and neurodegeneration? microglial activation mediates host neuronal survival induced by neural stem cells immunomodulation by transplanted human embryonic stem cell-derived oligodendroglial progenitors in experimental autoimmune encephalomyelitis trem deficiency impairs chemotaxis and microglial responses to neuronal injury the signaling adapter protein dap regulates multinucleation during osteoclast development biology of the tam receptors trem modifies microglial phenotype and provides neuroprotection in p s tau transgenic mice a unique microglia type associated with restricting development of alzheimer's disease do glia drive synaptic and cognitive impairment in disease? complement and microglia mediate early synapse loss in alzheimer mouse models clearance of tissue debris by trem -transduced myeloid cells promotes recovery of experimental autoimmune encephalomyelitis trem overexpression has no improvement on neuropathology and cognitive impairment in aging appswe/ps de mice pathway-based analysis of genome-wide sirna screens reveals the regulatory landscape of app processing microglia constitute a barrier that prevents neurotoxic protofibrillar abeta hotspots around plaques naturally secreted oligomers of amyloid protein potently inhibit hippocampal long-term potentiation in vivo molecular mechanisms of amyloid oligomers toxicity tau pathogenesis is promoted by abeta - but not abeta - the effect of increased genetic risk for alzheimer's disease on hippocampal and amygdala volume investigation of triggering receptor expressed on myeloid cells variant in the wisconsin registry for alzheimer's prevention neuropsychological tests and functional nuclear neuroimaging provide evidence of subclinical impairment in nasu-hakola disease heterozygotes lc , an autophagosome marker, is expressed on oligodendrocytes in nasu-hakola disease brains a japanese case with nasu-hakola disease of dap gene mutation exhibiting precuneus hypoperfusion the genetic causes of basal ganglia calcification, dementia, and bone cysts: dap and trem trem regulates microglia activation in response to cns demyelination soluble trem- in cerebrospinal fluid from patients with multiple sclerosis treated with natalizumab or mitoxantrone increased cerebrospinal fluid soluble trem concentration in alzheimer's disease in vivo differential brain clearance and catabolism of monomeric and oligomeric alzheimer's aβ protein cerebrospinal fluid strem levels are associated with gray matter volume increases and reduced diffusivity in early alzheimer's disease a data-driven approach links microglia to pathology and prognosis in amyotrophic lateral sclerosis the apoe ε genotype modulates csf ykl- levels and their structural brain correlates in the continuum of alzheimer's disease but not those of strem csf strem : marking the tipping point between preclinical ad and dementia? perturbation of the transcriptome: implications of the innate immune system in alzheimer's disease csf biomarkers and incipient alzheimer disease in patients with mild cognitive impairment a metalloproteinase disintegrin that releases tumour-necrosis factor-alpha from cells trem variants and risk of alzheimer's disease: a metaanalysis a novel compound heterozygous mutation in trem found in a turkish frontotemporal dementia-like family we thank erin g reed-geaghan for providing her insight and critical feedback. her contributions substantially improved the conceptual organization and clarity of the manuscript. this work was supported by the alzheimer's association (bfg- - , to gel), nia grant rf ag (to gel), nia grant r ag (to gel), nia national research service award f ag (to trj). • we accept pre-submission inquiries • our selector tool helps you to find the most relevant journal submit your next manuscript to biomed central and we will help you at every step: key: cord- -p y rrpb authors: kipar, anja; meli, marina l.; failing, klaus; euler, tatjana; gomes-keller, maria a.; schwartz, dirk; lutz, hans; reinacher, manfred title: natural feline coronavirus infection: differences in cytokine patterns in association with the outcome of infection date: - - journal: vet immunol immunopathol doi: . /j.vetimm. . . sha: doc_id: cord_uid: p y rrpb natural and experimental feline coronavirus (fcov) infection leads to systemic viral spread via monocyte-associated viraemia and induces systemic proliferation of monocytes/macrophages. in the majority of naturally infected animals, fcov infection remains subclinical and is associated with generalised b and t cell hyperplasia, but no other pathological findings. a minority of cats, however, develop feline infectious peritonitis (fip), a fatal systemic granulomatous disease. this is generally accompanied by b and t cell depletion. the obvious functional differences of lymphatic tissues in fcov-infected cats with and without fip suggest that they contribute to the outcome of fcov infection. this study attempted to evaluate the functional changes in haemolymphatic tissues after natural fcov infection, with special emphasis on the magnitude, phenotype and function of the monocyte/macrophage population. the spleen, mesenteric lymph nodes and bone marrow from naturally fcov-infected cats with and without fip and specific pathogen-free (spf) control cats were examined for the quantity and activation state of monocytes/macrophages both by immunohistology and by quantitative real time pcr for the transcription of interleukin (il)- β, il- , il- , il- p , tumour necrosis factor (tnf), granulocyte colony stimulating factor (g-csf), macrophage-csf (m-csf) and gm-csf. compared to cats with fip, fcov-infected cats without fip exhibited significantly higher il- levels in the spleen and significantly lower levels of il- , g- and m-csf in mesenteric lymph nodes. in cats with fip, however, il- p levels were significantly lower in lymphatic tissues in comparison to both spf cats and fcov-infected cats without fip. in comparison to spf cats, fip cats had significantly higher il- β levels and lower tnf levels in mesenteric lymph nodes and lower m-csf levels in the spleen. findings indicate that fcov-infected cats which do not develop fip are able to mount an effective fcov-specific immune response and can avoid excessive macrophage activation and fip, possibly by upregulation of il- production. development of fip, however, might be due to a lack of il- which inhibits an effective cellular immune response and allows for monocyte/macrophage activation and the development of fip. feline infectious peritonitis (fip) is a well-known and widely distributed coronavirus (fcov)-induced systemic disease in cats, characterised by fibrinousgranulomatous serositis with protein-rich effusions into body cavities, granulomatous-necrotising phlebitis and periphlebitis and granulomatous inflammatory lesions in several organs (hayashi et al., ; weiss and scott, ; kipar et al., kipar et al., , . fcov is transmitted via the faecal-oral route and primarily infects enterocytes (pedersen, ) , but subsequently spreads systemically via monocyteassociated viraemia (gunn- moore et al., ; kipar et al., ; meli et al., ) . however, fcovinfected circulating monocytes are not only responsible for viral dissemination, but also, in an activated state, for the development of vasculitis (weiss and scott, ; jacobse-geels et al., ; pedersen, ; kipar et al., ) . despite the generally high prevalence of fcov infection in the cat population, fip morbidity is low and rarely surpasses % (addie et al., ; pedersen, ; gunn-moore et al., ) . this is due to the fact that virulent fcov are predominantly generated within the individual infected host (vennema et al., ) . in virulent fcov, deletions in genes encoding non-structural proteins of yet unknown function, which develop during replication, have been observed (vennema et al., ; kennedy et al., ) . previous studies revealed major differences in the composition and functional state of lymphatic tissues of fcov-infected cats with and without fip (kipar et al., (kipar et al., , a . in cats with fip, t and b cell depletion is consistently observed (weiss and scott, ; kipar et al., a) . in experimentally infected animals with fip, enhanced lymphocyte apoptosis in b and t cell zones of the spleen and mesenteric lymph nodes was also described (haagmans et al., ; dean et al., ) . prior to lymphocyte depletion, however, most animals appear to develop a specific, systemic b cell response with formation of secondary follicles (kipar et al., a) . in addition, lymphatic tissues contain increased numbers of monocytes/ macrophages, some of which are proliferating (kipar et al., (kipar et al., , a . in contrast, fcov-infected cats without fip exhibit generalised b and t cell hyperplasia with a high rate of lymphocyte proliferation (kipar et al., (kipar et al., , a . regardless of the development of fip, fcov infection induces a specific systemic immune response with fcov antibodies, circulating fcov-specific immune complexes and the presence of plasma cells positive for fcov-specific antibodies in lymphatic tissues (osterhaus et al., ; kipar et al., ; meli et al., ) . in fip lesions, b cells are the dominant lymphocyte subtype; they gradually replace the macrophages and seem to develop into plasma cells positive for fcov-specific antibodies (kipar et al., (kipar et al., , . these findings suggest that animals susceptible to fip fail to avoid the potential detrimental sequelae of fcov infection and therefore develop both fip and lymphatic depletion (kipar et al., a) . based on morphological and limited functional studies, the pathogenesis of fip lesions is now relatively well understood (hasegawa and hasegawa, ; foley et al., ; berg et al., ; kipar et al., ) . however, data on the immunological processes associated with the development of the disease are limited and mainly restricted to experimentally infected animals (haagmans et al., ; dean et al., ) . in the present study, an attempt was made to evaluate the potential contribution of haemolymphatic tissues to the outcome of natural fcov infection in the individual cat. examinations focused on the assessment of the functional state of the spleen, mesenteric lymph nodes and bone marrow in cats with fip, in comparison to fcov-infected cats without fip and uninfected specific pathogen-free (spf) cats, with special emphasis on the magnitude, phenotype and function of the monocyte/macrophage population. it included quantitative real time pcr for feline interleukin (il)- b, il- , il- , il- p , tumour necrosis factor (tnf), granulocyte colony stimulating factor (g-csf), macrophage-csf (m-csf) and gm-csf. the study was performed on necropsied cats which had died or been euthanased with lesions of fip (group ). animals ranged from months to years of age (table ) . group consisted of apparently healthy spf cats, aged year, which had been housed for weeks with cats developing fip (kipar et al., ) . all group cats had shown positive results in tests for cov antibodies, circulating fcov-specific immune complexes and monocyte-associated fcov viraemia (kipar et al., ) . the heamolymphatic tissues from group and group cats had also tested positive for fcov rna by rt-pcr (kipar et al., ) . nine spf cats aged . months and five spf cats aged months served as uninfected controls (group ). all animals were necropsied at times ranging from a few minutes to h after death, and samples from the spleen, mesenteric lymph nodes and bone marrow were collected and frozen at À c for rna extraction. the tissue samples from the spleen and mesenteric lymph nodes were taken from areas without macroscopic fip lesions. additional haemolymphatic tissue and organ samples were fixed in % buffered formalin and routinely embedded in paraffin. sections ( mm) were stained with haematoxylin and eosin (he) or by immunohistology (ih). in haemolymphatic tissues monocytes, recently blood-derived macrophages and myelomonocytic cells were demonstrated by ih, using a cross-reacting mouse monoclonal antibody against the human myeloid/histiocyte antigen (clone mac , dakocytomation gmbh, ely, uk; kipar et al., kipar et al., , a kipar et al., ,b, . expression of the cd component of b -integrin adhesion molecules was shown by a mouse monoclonal antibody against feline cd (clone fe . f , leukocyte antigen biology lab., university of califonia, davis, usa; kipar et al., ) . both haemolymphatic tissues and organ specimens with histologically confirmed fip lesions were examined for the presence of fcov antigen (clone fcv - , custom monoclonals int., west sacramento, usa; kipar et al., kipar et al., , . the percentage of cells labelled for myeloid/ histiocyte antigen and cd in relation to the total cell population in the different compartments (e.g. the splenic red pulp) was blindly evaluated semiquantitatively (i.e. by counting the immunolabelled cells and the total number of nucleated cells in several high power fields) by light microscopy. the scoring was made in % steps. using a previously described evaluation protocol on he-stained sections (kipar et al., a) , activity and composition of haemolymphatic tissues in spf cats was considered as normal (controls). in spf cats, splenic white pulp was represented by small primary and/or secondary follicles and few, cell-rich periarter- ial lymphocyte layers in t cell zones; splenic red pulp exhibited moderate numbers of mononuclear cells. mesenteric lymph nodes generally exhibited cell-rich secondary follicles, broad, cell-rich t cell zones and mild histiocytosis of the medullary sinuses. lymphocyte depletion and hyperplasia, both in follicles and t cell zones were graded as mild (+), moderate (++) or severe (+++), based on the degree of reduction or increase in cellularity of the tissue compartments (kipar et al., a) . grading as +/++ or ++/+++ was used when variability between different areas of the organ was observed. bone marrow activity was graded as low (+), moderate (++), moderate to high (++/+++) or high (+++), based on the cellularity of a cross section of the femoral bone marrow cylinder. in spf cats, bone marrow activity was mostly high and occasionally moderate to high. for rna extraction, approximately mg of tissue was homogenised with a tissue homogeniser (pcr tissue homogenizing kit, süd-laborbedarf gmbh, gauting, germany) and lysed with ml lysis buffer. total rna was extracted with a commercially available kit (rneasy mini kit; qia-gen, hilden, germany) according to the manufacturer's protocol (kipar et al., b) . rna was dissolved in ml rnase-free water and digested with rnase-free dnase i (promega gmbh, mannheim, germany) and subjected to the synthesis of cdna using avian myoblastosis virus (amv) reverse transcriptase (promega) according to published protocols (leutenegger et al., ; kipar et al., b) . taqman-pcr for feline il- b, il- , il- , il- p , tnf, g-csf, m-csf and gm-csf quantitative real time taqman pcr, using an automated fluorometer (abi prism sequence detection system) was used for the relative quantification of feline (f) il- b, fil- , fil- , fil- p , ftnf, fg-csf, fm-csf and fgm-csf transcription (leutenegger et al., ; kipar et al., b) . feline gapdh levels served as internal controls. for each target gene, two primers and an internal oligonucleotide as a probe (fam ( -carboxyfluores-cein)-labelled at the end (reporter dye), tamra ( carboxytetramethylrhodamine)-labelled at the end (quencher dye), phosphate-blocked at the end to prevent extension by amplitaq gold dna polymerase) were used. primer (f, r) and probe (p) sequences were as previously published for the following systems: il- b ( bp pcr product; kipar et al., b) , il- ( bp pcr product; kipar et al., a,b) , il- ( bp pcr product; leutenegger et al., ) , il- p ( bp pcr product; leutenegger et al., ) , tnf ( bp pcr product; kipar et al., b) and gapdh ( bp pcr product; leutenegger et al., ) . primer and probe sequences for the csfs were as follows: g-csf (ncbi-accession no. y ; bp pcr product): fgcsf. f from every cdna sample, parallel reactions were performed in duplicate in separate tubes for the detection of fgapdh and all cytokines. amplification conditions and assay compositions were identical for all reactions and followed previously published protocols (kipar et al., b) . primer and probe concentrations were nm and nm, respectively, for fgapdh, fil- b, fil- , fil- , fil- p and ftnf, nm and nm for fgm-csf primers and probe, and nm and nm for fg-csf and fm-csf primers and probes. the specificity of the taqman pcr systems for feline gapdh, il- b, il- , il- , il- p and tnf was demonstrated previously (leutenegger et al., ; kipar et al., b) . taqman pcr products for feline g-csf, m-csf and gm-csf were run on a % agarose gel. fragments were cloned, using a topo ta cloning pcr . -topo kit (invitrogen bv, groningen, the netherlands), propagated in e. coli (top f'one shot e. coli) vector, and sequenced with a fluorescence-based automated sequencing system (abi dna sequencer; seqlab, sequence laboratories göttingen gmbh, göttingen, germany) to confirm the specificity. relative quantification of cytokine signals was done by the comparative c t method and was reported as relative transcription or the n-fold differences relative to the calibrator cdna (fgapdh) (leutenegger et al., ; kipar et al., b) . for each sample, differences between the target and internal control c t were calculated and served to normalize for differences in the amount of total nucleic acid added to each reaction and the efficiency of the reverse transcriptase step as previously described (kipar et al., b) . statistical analysis was performed, using the statistical programme package bmdp (dixon, ) . the kruskal-wallis test, followed by the nemenyi comparison, was applied to compare cytokine transcription levels in organs of all three groups of cats. additionally, the wilcoxon mann whitney test was used to compare cytokine transcription levels in organs of cats with fip and spf cats. lesions typical for fip were represented by a fibrinous to granulomatous peritonitis and pleuritis, often associated with effusion, and/or by granulomatous lesions in various organs as well as lymph nodes and the central nervous system (table ). brain and spinal cord involvement was characterised by a granulomatous leptomeningitis, often with granulomatous phlebitis and periphlebitis. the latter was also observed occasionally in eyes, renal cortices and lungs. immunohistologically, fcov antigen was demonstrated within macrophages in the lesions. cats with fip exhibited mild to severe follicular depletion in spleen ( / ) and mesenteric lymph nodes (mes lnn, / ). primary and/or secondary follicles were observed in both organs. t cell zones were mildly to moderately depleted in both the spleen ( / ) and mes lnn ( / ). in seven cases, the splenic red pulp was very cell-rich and predominantly composed of macrophage-like mononuclear cells. in cases, mes lnn exhibited mild to severe histiocytosis, mainly of marginal sinuses, which was often associated with marked dilation of the sinuses. fcov-infected cats without fip generally exhibited secondary follicles both in the spleen and mes lnn. in the spleen, these were normal ( / ) or hyperplastic ( / ) and in only two cases were mildly depleted. in the mes lnn, secondary follicles were normal ( / ) or hyperplastic ( / ). t cell zones were mainly normal ( / ) in the spleen and often hyperplastic ( / ) in the mes lnn. myeloid/histiocyte antigen (m/h ag)-positive monocytes/macrophages represented less than % of the cells in the splenic red pulp of spf cats; they were more numerous in some fcov-infected cats without fip and often represented the dominant cell population in cats with fip ( figs. a and a and b) . in spf cats, up to % of cells in the red pulp were cd -positive; in fcov-infected cats without fip, the number of cd -positive cells was often higher and generally surpassed % in cats with fip (figs. b and c and d). in the mes lnn, the m/h ag was only expressed by scattered sinus histiocytes in all groups of cats. in spf cats and fcov-infected cats without fip, cd expression was also restricted to few, faintly positive sinus histiocytes. whereas in cats with fip, sinus histiocytes generally exhibited a strong cytoplasmic and peripheral cd reaction (fig. ) . bone marrow activity was moderate to high in all groups of cats. in the majority of spf cats, m/h ag expression was seen in up to % of nucleated cells. the number of positive cells was often higher in fcov-infected cats without fip, whereas m/h agpositive cells generally represented the dominant cell population in cats with fip (fig. c) . in spf cats, the amount of cd -positive cells was rarely above %, whereas it ranged between % and % in fcovinfected cats without fip. in cats with fip, more than % of nucleated cells were cd -positive in the majority of cases (figs. d and a and b) . the demonstration of fcov antigen by ih yielded negative results in haemolymphatic tissues of all cats, except for the granulomatous lesions of cats with fip (see above). in general, cytokine transcription levels were very variable (fig. ) . in spf cats, all cytokines were constitutively transcribed. gm-csf mrna was only detected in % ( / ), but all other cytokines were detected in more than % of the samples. il- b, il- , il- p , tnf and particularly m-csf were transcribed at relatively high levels (fig. a) . cats with fip and fcov-infected cats without fip also exhibited constitutive transcription of all cytokines, with lowest rates of detection for gm-csf ( / ( %) fcov-infected cats without fip; / ( %) cats with fip). comparing the three groups of cats, average il- b, il- and tnf transcription levels were highest in fcov-infected cats without fip (fig. a) . the increase in transcription was significant in the case of il- when comparing the fcov-infected cats without fip with the fip cats (table ). in contrast, cats with fip exhibited the lowest il- p and m-csf transcription levels (fig. a) . the reduction in transcription of these cytokines was significant in cats with fip compared to spf cats (table ) . in spf cats, all cytokines were constitutively transcribed, confirmed by demonstration of mrna in at least % of the samples. highest average transcription levels were observed for tnf, followed by m-csf (fig. b) . cats with fip and fcov-infected cats without fip also exhibited constitutive transcription of all cytokines, with at least % of samples positive for the respective mrna. comparing the three groups of cats, average il- , g-csf and m-csf transcription levels were lowest in fcov-infected cats without fip (fig. b) . the reduction in transcription of these cytokines was significant in fcov-infected cats without fip compared to fip cats (table ). in cats with fip, there was evidence for increased transcription of il- b and m-csf and decreased transcription of il- p and tnf (fig. b) . the reduction in il- p transcription was significant in comparison to both other groups of cats. the increase in il- b transcription and the decrease in tnf transcription were significant in comparison to spf cats (table ) . in the red pulp, the vast majority of nucleated cells express the myeloid/histiocyte antigen. peroxidase anti-peroxidase method. papanicolaou's haematoxylin counterstain. bar = mm. (c) spf cat. in the red pulp, a few cells express cd . in the follicle, cd expression is restricted to a few cells in the center (follicular dendritic cells). avidinbiotin peroxidase complex method. papanicolaou's haematoxylin counterstain. bar = mm. (d) cat with fip. in the red pulp, the vast majority of nucleated cells express cd . in the follicle, cd expression is restricted to a few cells in the center (follicular dendritic cells). avidin-biotin peroxidase complex method. papanicolaou's haematoxylin counterstain. bar = mm. (for interpretation of the references to colour in this figure legend, the reader is referred to the web version of the article.) in spf cats, constitutive transcription was mainly seen for il- b, il- , tnf and m-csf, as mrna from all other cytokines was often not detected (fig. c) . in general, transcription levels for all cytokines were lower than in the spleen and mes lnn, and il- , g-csf and gm-csf were transcribed in the lowest amounts. the overall cytokine transcription pattern was similar in both other groups of cats (fig. c) . in fcovinfected cats without fip, il- and gm-csf transcription was not detected at all. average il- and m-csf transcription levels were highest in cats with fip (fig. c ). there was no evidence of statistically significant differences in the transcription of cytokines in the bone marrow of the three groups of cats. there was no evidence to suggest a relationship between cytokine transcription patterns and type and distribution of fip lesions or the presence or absence of fip lesions in the mes lnn and splenic serosa, respectively. also, there was no distinct evidence of higher transcription levels of any cytokines examined either in the spleen or the mes lnn, of those cats where a cell-rich red pulp or a moderate to intense sinus histiocytosis in the mes lnn were observed. we have evaluated the activity and composition of haemolymphatic tissues in naturally fcov-infected cats with particular emphasis on the macrophage population and the transcription levels of cytokines that are produced by, and/or act on, monocytes/ macrophages (namely il- b, il- , il- , il- p , tnf, g-csf, m-csf and gm-csf). this is the first study to evaluate the constitutive transcription of cytokines in feline haemolymphatic tissues. the spleen, mes lnn and (although at a generally far lower level) bone marrow constitutively transcribe all cytokines examined, which indicates their role in the homeostatic balance of healthy cats. although the study was performed on a relatively homogeneous group of young adult spf cats, high variability in individual transcription levels was observed. a similarly high variability has previously been described in isolated feline monocytes (kipar et al., b (kipar et al., , . such an obviously wide biological range of cytokine levels renders comparative studies, in particular on natural diseases, difficult. not unexpectedly, therefore, equally variable cytokine transcription levels were seen in both fcov-infected cats without fip (again a relatively homogeneous group of cats with regard to age and previous spf status) and cats with fip (a group heterogeneous both in age and background). the haemolymphatic tissues of both fcovinfected cats without fip and cats with fip exhibited evidence of proliferation and activation of monocytes/ macrophages and their precursors. bone marrow of these two groups contained increased numbers of m/h ag-positive, myelomonocytic cells (horny et al., ) . in particular in cats with fip, m/h ag-positive cells were also very numerous in the splenic red pulp, where they represent monocytes or recently bloodderived macrophages (rugtveit et al., ; kipar et al., ) . in both organs, the same cell types table statistical analysis of differences in the relative quantity of cytokine transcription levels in the spleen, mesenteric lymph nodes and bone marrow exhibited b -integrin upregulation, as shown by increased cd expression. in the bone marrow, where increased cd expression was most pronounced in cats with fip, it is a feature of myelomonocytic cell maturation (kim and feldman, ) and can be induced by cytokines, such as gm-csf and m-csf (aglietta et al., ; kamps et al., ) . in cats with fip, the mes lnn exhibited pronounced sinus histiocytosis, again with b -integrin upregulation, represented by intense cd expression in particular on the cell surface. like in the splenic red pulp, this indicates activation of the macrophages (freyer et al., ; fernandez-segura et al., ) . in contrast to the splenic macrophages, the majority of sinus macrophages in the mes lnn were m/h ag-negative, which suggests they originated from local macrophages and thus proliferated locally, instead of being monocytes recruited into the lymph nodes (rugtveit et al., ) . interestingly, cytokines known to be produced by macrophages and to stimulate macrophages to proliferate and increase in activity, such as g-, m-and gm-csf, il- and/or tnf (aglietta et al., ; orikasa et al., ; springer, ; kamps et al., ) were not upregulated in either group of cats despite the obvious abundance of activated macrophages in the haemolymphatic tissues. accordingly, a systemic effect seems likely, possibly via cytokines released from circulating monocytes, as they (a) trigger fcov viraemia, (b) are activated in the course of fip (gunn- moore et al., ; kipar et al., kipar et al., , meli et al., ) and (c) produce the respective effector cytokines (clark and kamen, ; brandt et al., ; sallerfors and olofsson, ; aglietta et al., ; kerst et al., ; springer, ; kipar et al., ) . with regards to the lymphocyte populations and the overall cytokine transcription patterns, however, major differences were seen between the groups of cats. healthy fcov-infected cats without fip exhibited the generalised b and t cell hyperplasia already described in a previous study (kipar et al., (kipar et al., , a and displayed overall significantly higher il- levels in the spleen as well as significantly lower il- , g-csf and m-csf levels in the mes lnn when compared to cats with fip. reduced il- and csf transcription could be an effect of il- which is known to downregulate these cytokines in humans (de waal malefyt et al., a; hannen et al., ) . il- also inhibits release of other cytokines, such as il- , tnf and il- b (de waal malefyt et al., a; koch et al., ) . this might explain why in fcov-infected cats without fip, transcription levels of these cytokines were not significantly higher than in spf cats despite the abundance of activated macrophages in the tissues (de waal malefyt et al., a; trinchieri, ) . in a fip dna vaccination study where il- was co-expressed with viral antigens, enhanced susceptibility of cats to fip was observed in the presence of il- (glansbeek et al., ) . these findings suggest that prevention of il- release is essential to avoid disease in fcov-infected cats. il- reduces the antigen-specific proliferation of t cells by downregulating mhc ii expression in monocytes and macrophages (de waal malefyt et al., b ) which might in fcov infection limit the specific immune response despite the persistent antigen challenge in the course of prolonged viraemia (gunn- moore et al., ; kipar et al., ; meli et al., ) . il- also stimulates nk cells and upregulates both fcg receptor i expression and antibody-dependent cellular cytotoxicity in monocytes (te velde et al., ; moore et al., ) . thus, it might contribute to viral clearance and account for the generally lower viral load observed in healthy fcov-infected cats in comparison to cats with fip (kipar et al., ) . il- may also contribute to the b cell hyperplasia present in healthy fcov-infected animals (kipar et al., a) , as it induces both the proliferation of antigen receptor-activated b cells and ifn-g secretion (rousset et al., ; lauw et al., ) . on the other hand, il- downregulates the b integrin expression in monocytes (petit-bertron et al., ) which may reduce their capacity to adhere to endothelial cells and thereby to initiate the fip- fig. . box and whisker plots, demonstrating levels of cytokine transcription in spf cats, fcov-infected cats without fip and cats with fip. the amount of target was calculated by Àdct , resulting in evaluation of the experimental samples as an n-fold difference relative to the calibrator fgapdh (kipar et al., b) . the plots display the range of values, the minimum and maximum, low (first) and high (third) quartiles and the median. (a) cytokine transcription levels in the spleen; group: ( ) spf cats, ( ) fcov-infected cats without fip, ( ) cats with fip; (b) cytokine transcription levels in mesenteric lymph nodes; group: ( ) spf cats, ( ) fcov-infected cats without fip, ( ) cats with fip; (c) cytokine transcription levels in bone marrow; group: ( ) spf cats, ( ) fcov-infected cats without fip, ( ) cats with fip. specific vasculitis (kipar et al., ) . taken together, our results indicate that il- is a key cytokine in fcov infection, ensuring an effective specific immune response, but avoiding the inflammatory processes associated with the development of fip (kipar et al., ) , by inhibiting the virus-induced macrophage activation. in lymphatic tissues, il- is produced by monocytes/macrophages and b and t cells (benjamin et al., ; moore et al., ; dean et al., ) . it remains to be clarified which cells in the spleen are responsible for the il- production in fcov-infected cats without fip, whether il- upregulation represents a direct viral effect on these cells and which factors determine whether il- is produced or not. in cats with fip, lymphatic tissues exhibit generalised t and b cell depletion, albeit with evidence of previous activation (kipar et al., a) , and significantly lower il- p transcription levels than both other groups of cats. il- p , the inducible monomer of il- is secreted predominantly by activated mononuclear phagocytes and dendritic cells (trinchieri, ) . the reduced production of il- observed in lymphatic tissues in this and a previous study (dean et al., ) , and in brains with fip lesions (foley et al., ) in the presence of abundant activated macrophages in haemolymphatic tissues and granulomatous infiltrates in cats with fip indicates impairment of the immune response, in particular of the th response (trinchieri, ; o'garra, ) . monocytes/macrophages produce il- when in contact with activated t cells and under the influence of ifn-g and gm-csf in particular (shu et al., ; kennedy et al., ) . interestingly, neither ifn-g (data not shown) nor gm-csf were transcribed at lower levels in haemolymphatic tissues of cats with fip despite the obvious t cell depletion in these animals. also, il- is obviously not responsible for the reduced il- transcription as overall il- levels were not elevated in cats with fip (koch et al., ) . results of this investigation are difficult to compare with those of a previous study which examined viruspositive and virus-negative lymphatic tissues of cats with fip (dean et al., ) ; there, the presence of virus was associated with increased il- and decreased il- transcription, but potential differences between fcov-infected cats with and without disease were not assessed. still, the general t cell depletion (haagmans et al., ; kipar et al., kipar et al., , a de groot-mijnes et al., ) together with the obvious lack of significant ifn-g production in organs or lymphatic tissues with fip lesions (dean et al., ; foley et al., ) indicate impairment of the t cell-mediated macrophage stimulation in fip. as previous studies did not identify a direct effect of fcov on t cells (haagmans et al., ; dean et al., ; de groot-mijnes et al., ) , a lack of t cell activation by macrophages has to be considered, possibly due to fcov-induced impairment of antigen presentation and/or inhibition of il- production by monocytes/macrophages. the latter seems likely due to the fact that fcov induces high antibody titres (osterhaus et al., ; kipar et al., ; meli et al., ) and that fcg ligation can lead to suppression of il- transcription in macrophages (grazia cappiello et al., ) and indicates direct inhibition of il- production by fcov. we also demonstrated significantly reduced tnf transcription, but increased il- b transcription in the mes lnn of cats with fip in comparison to spf cats. considering that both these cytokines are generally released under similar circumstances and have similar functions (abbas and lichtman, ) , a direct, selective effect of fcov on the function of sinus histiocytes appears likely. increased il- b production may contribute to both the remittent fever observed in the course of fip and the systemic changes in cats with fip, such as the generalised activation of endothelial cells (dinarello, ; kipar et al., ) . the reduced tnf production, however, cannot readily be explained, in particular as a previous study provided evidence of increased tnf production in viruspositive, depleted lymphatic tissue of cats with fip (dean et al., ) . in summary, our findings suggest that, regardless of the development of fip, fcov induces proliferation and activation of monocytes/macrophages which is likely mediated by fcov-infected circulating monocytes. thereby, a constant supply of cells for viral replication and spread is ensured. in parallel, fcov provokes a specific systemic immune response. healthy, long-term fcov-infected cats mount an effective t and b cell response with general t and b cell hyperplasia; here, the immune response might be limited by upregulation of il- . the presence of fip lesions, however, is associated with t and b cell depletion and reduced il- production in lymphatic tissues. a lack of il- might be responsible for the obviously impaired cellular immune response and a failure to reduce/limit the viral load in fip (kipar et al., ) . despite this fip is associated with general activation of monocytes/macrophages, as reflected by their upregulation of adhesion molecules and cytokine production (dean et al., ; kipar et al., ) . this indicates that the development of granulomatous inflammatory processes and fatal fip are the consequence of exaggerated activation of monocytes/macrophages. the cells that are directly affected by fcov and the mechanisms that elicit these effects need to be investigated further. it also needs to be clarified whether the virus or the immunophenotype of the individual cat are responsible for the variable effect of fcov infection on the host's lymphatic tissue. fcov virulence is associated with deletions in non-structural genes of yet unknown function and increased viral replication (vennema et al., ; kennedy et al., ; kipar et al., ) . further studies have to clarify whether viral gene products interact with effector molecules, such as cd , cd ligand or mhc ii, or have a direct effect on gene transcription in infected cells. cellular and molecular immunology risk of feline infectious peritonitis in cats naturally infected with feline coronavirus granulocyte-macrophage colony stimulating factor and interleukin : target cells and kinetics of response in vivo human b-cell interleukin- : b-cell lines derived from patients with acquired immunodeficiency syndrome and burkitt's lymphoma constitutively secrete large quantities of interleukin- cellular composition and interferon-gamma expression of the local inflammatory response in feline infectious peritonitis (fip) effect of recombinant human granulocyte-macrophage colony-stimulating factor on hematopoietic reconstitution after high-dose chemotherapy and autologous bone marrow transplantation the human hematopoietic colonystimulating factors in vivo cytokine response to experimental feline infectious peritonitis virus infection natural history of a recurrent feline coronavirus infection and the role of cellular immunity in survival and disease interleukin (il- ) inhibits cytokine synthesis by human monocytes: an autoregulatory role of il- produced by monocytes interleukin (il- ) and viral il- strongly reduce antigen-specific human t cell proliferation by diminishing the antigen-presenting capacity of monocytes via downregulation of class ii major histocompatibility complex expression the biological properties of interleukin- topographic distribution of cd integrin on human neutrophils as related to shape changes and movement induced by chemotactic peptide and phorbol esters inflammation and changes in cytokine levels in neurological feline 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induced feline infectious peritonitis role of macrophage colony-stimulating factor in the differentiation and expansion of monocytes and dendritic cells from cd + progenitor cells cd /cd ligand interactions are required for t celldependent production of interleukin- by mouse macrophages deletions in the a orf of feline coronavirus associated with an epidemic of feline infectious peritonitis interleukin- is a survival factor for committed myeloid progenitor cells activated fes protein tyrosine kinase induces terminal macrophage differentiation of myeloid progenitors (u cells) and activation of the transcription factor pu. cellular composition, coronavirus antigen expression and production of specific antibodies in lesions in feline infectious peritonitis histopathological alterations of lymphatic tissues in cats without feline infectious peritonitis after long-term exposure to fip virus evaluation of lymphatic tissue activity in cats with spontaneous feline infectious peritonitis cytokine mrna levels in isolated feline monocytes morphological features and development of granulomatous vasculitis in feline infectious peritonitis natural fcov infection: cats with fip exhibit significantly higher viral loads than healthy infected cats high level il- production by murine dendritic cells: upregulation via mhc class ii and cd molecules and downregulation by il- and il- proinflammatory effects of il- during human endotoxemia quantitative real-time pcr for the measurement of feline cytokine mrna high viral loads despite absence of clinical and pathological findings in cats experimentally infected with feline coronavirus (fcov) type i and in naturally fcov-infected cats interleukin- interleukin- and the interleukin- receptor cytokines induce the development of functionally heterogeneous t helper cell subsets induction of macrophagic and granulocytic differentiation of murine bone marrow progenitor cells by , -dihydroxyvitamin d seroepidemiology of feline infectious peritonitis using transmissible gastroenteritis virus antigen an overview of feline enteric coronavirus and infectious peritonitis virus infections adherence influences monocyte responsiveness to interleukin- interleukin is a potent growth and differentiation factor for activated human b lymphocytes expression of the l antigen (calprotectin) by tissue macrophages reflects recent recruitment from peripheral blood rather than upregulation of local synthesis: implications for rejection diagnosis in formalin-fixed kidney specimens granulocyte-macrophage colonystimulating factor (gm-csf) and granulocyte colony-stimulating factor (g-csf) secretion by adherent monocytes measured by quantitative immunoassays activated t cells induce interleukin- production by monocytes via cd -cd ligand interaction traffic signals for lymphocyte recirculation and leukocyte emigration: the multistep paradigm il- stimulates monocyte fc gamma r surface expression and cytotoxic activity. distinct regulation of antibody-dependent cellular cytotoxicity by ifn-gamma, il- , and il- interleukin- and its role in the generation of th cells feline infectious peritonitis viruses arise by mutation from endemic feline enteric coronaviruses pathogenesis of feline infectious peritonitis: pathologic changes and immunofluorescence we wish to thank dr. m. linenberger, division of hematology, department of medicine, university of washington, usa, for kindly providing the sequence of the feline m-csf gene to design primers and probes. we are grateful to dr. c. leutenegger key: cord- - exenodj authors: alkan, ali; uncu, asaf; taşkıran, irmak; tanrıverdi, Özgür title: double-edged sword: granulocyte colony stimulating factors in cancer patients during the covid- era date: - - journal: clinics (sao paulo) doi: . /clinics/ /e sha: doc_id: cord_uid: exenodj nan to the editor, the whole world is struggling because of coronavirus disease , and it has already triggered a series of crises. patients with comorbidities are highly susceptible to infection and thus have a poor prognosis. cancer patients have been heavily impacted by the onset of this pandemic. a few recent studies on cancer patients have shown that they are at an increased risk for severe acute respiratory syndrome-coronavirus- (sars-cov- ) infection. furthermore, infected patients were found to have an increased risk of admission to the intensive care unit, requiring invasive ventilation, or death ( ). oncologists are providing optimal cancer care and, simultaneously, are trying to protect these fragile patients from sars-cov- infection and its consequences. cytopenia, especially neutropenia and lymphopenia, is the most frustrating complication of cancer treatment and can increase the susceptibility to infection. both the european society for medical oncology (esmo) and the national comprehensive cancer network (nccn) have recommended expanding the indications for use of granulocyte colonystimulating factors (g-csfs) in cancer patients to decrease the risk of neutropenic fever and prevent them from having to visit a hospital ( , ) . the efficacy of g-csfs in preventing febrile neutropenia and treatment-related hospitalizations has been well documented in earlier studies. moreover, a -day course of g-csf prophylaxis in most chemotherapy regimens has been reported to be effective ( ); however, limited data are available on its efficacy in covid- patients. g-csfs are growth factors that stimulate the survival, proliferation, differentiation, and function of neutrophil precursors and mature neutrophils via signal transduction pathways. studies on healthy volunteers revealed that the administration of g-csfs resulted in increased the levels of interleukin (il)- , soluble tumor necrosis factor (tnf) receptors (stnf-rs), il- , il- , and il- and reduced the levels of tnf-a, interferon (ifn)-g, and granulocyte-macrophage colony-stimulating factor (gm-csf) ( ). data from a limited number of covid- patients have shown a cytokine storm in critically ill patients. besides, the levels of il- , il- , il- , and ifn-g were found to be higher in severe cases of covid- than in mild cases, and a strong inflammatory response during its clinical course was reported to be associated with high morbidity and mortality ( ) . clinical outcomes are also unpredictable in covid- patients with high levels of inflammatory cytokines undergoing g-csf prophylaxis. furthermore, there are chances of acute respiratory failure during g-csf-induced neutropenia recovery. g-csfs have been reported to be associated with activation of the oxidative burst mechanism within circulating or resident alveolar neutrophils and macrophages, further complicating the situation ( , ) . therefore, devastating results can potentially be seen in covid- -positive patients administered g-cfss or in those exposed to infection while undergoing g-csf therapy. however, very limited data on the outcomes of chemotherapy in cancer patients in the covid- era are currently available, with no specific subgroup analysis in patients treated with g-csfs. although the described scenarios are theoretical and speculative, both nccn and esmo have concluded that the benefits of g-csf administration outweigh its risks and have, therefore, recommended its use with caution. until more data are available, clinicians should be more cautious with the use of g-csfs in cancer patients in the covid- era. ' references cancer patients in sars-cov- infection: a nationwide analysis in china supportive care strategies during the covid- pandemic hematopoietic growth factors. short-term recommendations specific to issues with covid- (sars-cov- ) a multicentre, randomised trial comparing schedules of g-csf (filgrastim) administration for primary prophylaxis of chemotherapy-induced febrile neutropenia in early stage breast cancer effect of granulocyte colony-stimulating factor treatment on ex vivo blood cytokine response in human volunteers longitudinal characteristics of lymphocyte responses and cytokine profiles in the peripheral blood of sars-cov- infected patients. version deterioration of previous acute lung injury during neutropenia recovery respiratory status deterioration during g-csf-induced neutropenia recovery key: cord- - xoubkxb authors: samannodi, mohammed; hansen, michael; allana, ambreen; hasbun, rodrigo title: compliance with international guidelines in adults with encephalitis date: - - journal: j clin virol doi: . /j.jcv. . sha: doc_id: cord_uid: xoubkxb background: encephalitis is associated with significant neurological disability and mortality. many guidelines are published for encephalitis management but compliance with them is unknown. objectives: to evaluate the appropriate management and compliance to the current guidelines in adults with encephalitis. study design: a retrospective multicenter study at hospitals in the greater houston area from august , through september , . all cases met the definition for possible or probable encephalitis as per the international encephalitis consortium guidelines. results: a total of adults (age > years) with encephalitis were enrolled. the most common etiologies were unknown ( . %), viral ( . %) and autoimmune ( . %). an adverse clinical outcome was seen in % with . % in hospital mortality. a high compliance with guidelines (> %) was only seen in obtaining a brain computerized tomography (ct) scan, blood cultures and cerebrospinal fluid (csf) gram stain and culture. a csf herpes virus simplex (hsv) polymerase chain reaction (pcr) was done in % and only repeated in . % of patients with an initial negative result. furthermore, only two-thirds of patients were started empirically on intravenous acyclovir and antibiotics. evaluation for other etiologies were not uniformly performed: arboviral serologies ( . %), csf anti-n-methyl-d-aspartate receptor (nmda) receptor antibody ( . %), and csf varicella zoster virus (vzv) pcr ( %). the highest yield for the tests were arboviral serologies ( %), anti-nmda antibodies ( . %) and vzv pcr ( . %). conclusion: the management of encephalitis as per current guidelines is suboptimal leading to underutilization of currently available diagnostic tests and empirical therapy. encephalitis is an inflammatory process of the brain associated with mortality ranging between - % of patients depending on the etiological agent, country of the study, immunosuppression, and inclusion of adults or children or both [ , ] . furthermore, up to two-thirds of survivors from viral encephalitis (herpes simplex virus, japanese encephalitis, west nile virus) have neurological and/or neurocognitive sequelae [ ] [ ] [ ] . the incidence of encephalitis varies by country between . - cases per , persons [ ] . infections and autoimmune etiologies are considered to be the most important causes of encephalitis representing approximately % and %, respectively when systematically evaluated [ ] . however, between - % of encephalitis cases remain with unknown etiologies [ , [ ] [ ] [ ] . in the past, brain biopsy was the gold standard for diagnostic method for encephalitis but currently it is infrequently done as less invasive and highly sensitive diagnostic tools such as polymerase chain reaction (pcr) for viruses in the cerebrospinal fluid (csf) was introduced [ ] . the variability in the proportion of unknown etiologies between studies is most likely driven by the availability and use of molecular diagnostic techniques, and testing for arboviral serologies and autoimmune etiologies [ ] . in this study, we are evaluating the work up, management and outcome of adults with encephalitis based on the majority of current guidelines recommendations in literature [ ] [ ] [ ] [ ] . to our knowledge, this is the first study evaluating the compliance to the current guidelines in adults with encephalitis https://doi.org/ . /j.jcv. . received february ; received in revised form april ; accepted april . methods the study was approved by the ut health committee for the protection of human subjects, by the memorial hermann hospital research review committee and the harris health research committee. we conducted a retrospective study of adults with a diagnosis of encephalitis. the study was collected from hospitals from the memorial hermann health (mhh) system and harris health system (hhs) in the greater houston area between august of and september of . primary outcome of the study was assessing encephalitis management and compliance to current guidelines in our population. as summarized in (supplemental digital content - table ), all guidelines of encephalitis management have major parts in evaluating and managing patients with encephalitis; exposure evaluation, appropriate utilization of diagnostic and neurodiagnostic studies, and proportion and timing of empirical antibiotic and antiviral therapy [ ] [ ] [ ] [ ] [ ] . in our study, we followed these major parts and compared them with our population. as per iec guidelines, an encephalitis case was determined by the presence of the major criterion (presentation of altered mental status without alternative cause lasting more than h) and at least minor criteria: a. fever h before or after presentation, b. new onset seizures, c. new onset focal neurological findings, d. white blood cell count > mm in the cerebrospinal fluid (csf), e. new neuroimaging findings, f. abnormal electroencephalogram consistent with encephalitis. case outcomes were defined using the glasgow outcome scale (gos), where, is considered a good recovery, being consistent with moderate disability, with severe disability, with a vegetative state, and with death. anything that was scored as a or less was defined as an adverse clinical outcome (aco) [ ] . herpes viral infections (hsv, vzv, cmv and ebv) were identified by csf polymerase chain reaction (pcr). arboviral diseases were diagnosed via a panel of testing that included immunoglobulin m (igm) for st. louis encephalitis virus, eastern equine encephalitis virus, venezuelan equine encephalitis virus, la crosse virus, and west nile virus. bacterial diseases were identified either through csf culture or via relevant serological testing (igm) for rickettsia, brucella and mycoplasma. fungal infections were identified by csf culture or csf antigen. parasitic infections were identified by microscopic examination of brain tissues or csf antigen. patients were considered positive for an autoimmune encephalitis if the csf antibody test was present in an abnormal range in the absence of any other identified cause of encephalitis and presence of current autoimmune disease such as hashimoto thyroiditis. also positive csf antibodies for n-methyl-d-aspartate (nmda) or voltage -gated potassium channel (vgkc) defined as autoimmune encephalitis. medical records were reviewed independently by two physicians and all cases met the definition for possible or probable encephalitis as per the international encephalitis consortium (iec) guidelines [ ] . data extraction from the medical record was done utilizing a standardized form. extracted data included information on demographics, clinical presentation, diagnostic testing, and treatment. all patients were assigned as infectious, autoimmune or idiopathic. a total of , patients were identified with an international classification of disease (icd)- discharge diagnosis code of encephalitis. after review of the medical records, we excluded the following patients: did not have a central nervous system infection (n = ), healthcare-associated ventriculitis or meningitis (n = ), community-acquired bacterial meningitis (n = ), aseptic meningitis (n = ), fungal meningitis (n = ), tuberculous meningitis (n = ), parasitic infection (n = ), and incomplete medical records (n = ). only ( %) met the definition of encephalitis. statistical analysis was performed with spps version (ibm, austin, tx, usa). univariate analysis was performed using fisher's exact test, chi square, and student's t-test as appropriate. the demographic and clinical characteristics are shown in (table ) . a total of adults with encephalitis were enrolled. the median age was years of age with the majority of patients being male ( %), non-caucasian ( . %), and with private insurance ( . %). during our study period, average houston population was about , , and the majority of houston population were hispanic (∼ %) followed by caucasian (∼ %) then african american (∼ %) as reported by texas department of state health services. however, our study showed that african americans ( . %) were disproportionally affected. a total of patients ( %) were immunocompromised with ∼ % of them being human immunodeficiency virus (hiv) positive (n = ), recent chemotherapy due to malignancy (n = ), chronic immunosuppressive therapy due to autoimmune diseases (n = ) and solid organ transplantation (n = ).the median duration of symptoms prior to admission was days. the most common presenting symptoms included fever, headache, photophobia, respiratory symptoms and nausea/vomiting. a high opening pressure (> cm h o) was documented in % of the patients with the most common clinical signs being focal neurological deficits ( . %), seizures ( . %), and obtundation ( %). coma (gcs < ) and status epilepticus was seen in % and %, respectively. laboratory findings and etiologies are summarized in (table ). the csf profile consisted of a mild pleocytosis (median csf serum wbc was cells/mm ) with a lymphocytic predominance, a normal glucose and mildly elevated protein. the serum wbc count was , cells/ml with leukopenia and thrombocytopenia being documented in . % and . %, respectively. in terms of etiology, the most common cause of encephalitis was idiopathic ( . %) followed by viral ( . %) and autoimmune ( . %). the most common causes of viral encephalitis were west nile virus (wnv) and herpes simplex virus (hsv) were ( %, % respectively) followed by varicella zoster virus (vzv) ( . %). the most common cause of autoimmune encephalitis was due to anti-n-methyl-d-aspartate receptor ( . %). regarding bacterial etiologies, streptococci (group a streptococcus, s. pneumoniae) and staphylococci (s. aureus) were the most common etiologies. mycobacterium tuberculosis was seen in patients. toxoplasma gondii was the only parasite isolated in our population. fungal etiology was not common in our study with total of three cases. the infectious disease society of america (idsa), british, australian, international consortium, and french guidelines recommend that clinicians evaluate for potential exposures and risk factors and to perform appropriate utilization of diagnostic studies in patients with suspected encephalitis. as shown in (table ) , we found poor documentations of exposure risk factors in patients' medical charts. for example, high-risk sexual activity, recent travel history and occupational history was documented in about %, %, and . % of patients, respectively. all other exposure risk factors (e.g., insect, animal or, ill contact, fresh water exposure or recent vaccination history) were infrequently documented. although exposure and risk factors were poorly documented, most of them were had high yield results. as shown in (table ), the utilization of diagnostic studies was variable. most of patients had chest x-ray and blood culture ( %, . % respectively). other diagnostic tests were variable such as testing for hiv ( . %), arbovirus ( . %), syphilis ( . %), and for mycobacterial (sputum afb culture) or fungal etiologies (histoplasma urinary antigen, serum cryptococcal antigens) (< %). testing for mycoplasma and rickettsial antibodies (igm) was done in a minority of patients. we found that when done many of the diagnostic studies have high yield results. the highest yield and the most significant test was serum arboviral antibodies ( %) neurodiagnostic studies are summarized in (table ). brain ct and mri were performed in . % and . % respectively. electroencephalography was underutilized in our study with a compliance rate of %. csf gram stain and bacterial culture were done in the majority of patients. csf hsv pcr was obtained in about % of patients reflecting that clinicians understand the importance of hsv in encephalitis. csf enterovirus pcr was obtained in about half of patients only. the tests with the highest yield were csf anti-nmda antibodies ( . %) and csf vzv pcr ( . %). csf infectious process was identified in % of all immunocompromised patient (n = ). all reviewed guidelines of encephalitis management recommend starting empiric antibiotics and acyclovir as soon as possible, pending results of diagnostic studies [ ] [ ] [ ] [ ] . as demonstrated in (table ) , the compliance to empiric intravenous antibiotics and acyclovir was not favorable. additionally, there was significant delay in initiation of empiric intravenous antibiotics and acyclovir. about % of our population developed acute kidney injury, which can be attributed to antimicrobials in addition to other causes. also, most of the guidelines recommends to repeat csf hsv pcr in - days in undiagnosed cases of encephalitis in which patients have clinical features or neuroimaging findings of hsv encephalitis [ ] [ ] [ ] [ ] . we found undiagnosed cases with suspected features of hsv encephalitis, only . % had repeat csf hsv pcr and all were negative. in terms of outcome, approximately half of the patients had an adverse clinical outcome with a all reviewed guidelines recommend a thorough exposure evaluation to patients with encephalitis [ ] [ ] [ ] [ ] [ ] . as shown in (table ) , the compliance to exposure evaluation was the lowest. however, most of exposures and risk factors yielded important information in a substantial proportion of patients highlighting the importance in documenting them. the most common cause of encephalitis was idiopathic which is consistent with most studies [ , , , ] . the most common infectious cause of encephalitis in our study was wnv. in the encephalitis study done in england, hsv was the most common cause of infectious encephalitis with no cases of wnv reported [ ] . in contrast, wnv is endemic in the us. furthermore, patients ( . %) with wnv encephalitis were diagnosed during the endemic season (between june and october) [ ] . regarding diagnostic tests, most guidelines recommend obtaining hiv test and blood culture in all patients with encephalitis and other diagnostic tests can be done based on clinical clues and epidemiological risk factors [ ] [ ] [ ] [ ] [ ] . the compliance rate was favorable in ordering blood culture ( . %) but suboptimal in hiv ( . %). in terms of neurodiagnostic evaluations, it is recommended by all guidelines to obtain csf analysis, csf gram stain and culture, csf hsv pcr, csf vzv pcr, csf enterovirus pcr, brain imaging which mri is preferable, and electroencephalography (eeg). further neurodiagnostic testing will be based on clinical necessity [ ] [ ] [ ] [ ] [ ] . most of our patients had csf analysis, csf gram stain and culture, and brain imaging. eeg was underutilized in our study and was only performed in two third of patients. hsv is the most common treatable cause of encephalitis and the guidelines recommend that all patients should be tested. in our study, a csf hsv pcr was done in . %. additionally, the guidelines advocate to repeat a csf hsv pcr in undiagnosed cases of encephalitis in which patients have clinical features or radiological findings of hsv encephalitis [ ] [ ] [ ] [ ] [ ] . in our study, a repeat csf hsv pcr was only done in . % of undiagnosed patients with suspected features of hsv encephalitis. this raises the concern of underdiagnosing the most common treatable etiology. a csf vzv pcr were not uniformly done but had the highest yield in our study. additionally, [ ] . although anti-nmda antibodies were checked only in about % of our population, anti-nmda antibodies was the most common cause of autoimmune encephalitis in our study. all guidelines in agreement that empiric antibiotics and acyclovir should be started as soon as possible in patients with suspected encephalitis. in our study, empiric intravenous antibiotics and acyclovir were not started in about one third of the patients; this is worrisome as delays in therapy are associated with an increase adverse outcomes in both bacterial meningitis and herpetic encephalitis [ , , [ ] [ ] [ ] . furthermore, the median time to initiate intravenous acyclovir was h from arriving to the emergency room. a recent study of patients with herpetic encephalitis documented unfavorable outcomes in . % of patients with age and glasgow coma scales as independent prognostic factors [ ] . the authors were also able to correlate a delay in the initiation of acyclovir with a linear increase in adverse clinical outcomes and recommended to start antiviral therapy urgently in all patient with suspected encephalitis. the mortality rate in our study was . %; similar to the - % rates reported in the literature [ , , ] . overall, the management of encephalitis in our population was suboptimal. the reason behind this fact was not fully clear. a possible reason could be due to the lack of awareness of the encephalitis diagnosis or guidelines and/or due to the relative lack of experience on the treating clinicians in this relative rare disease. our study had several strengths. it is the first study to evaluate the compliance with multiple encephalitis guidelines. second, it was a multicenter study done in hospitals that included a wide variety of community hospitals and academic medical centers. third, we only included cases that met the iec definition of encephalitis and provided a comprehensive review of all the diagnostic and treatment modalities as recommended by guidelines. despite the strengths, our study had some limitations. first, due to the retrospective nature of the study, some missing data could limit the conclusion of the study. furthermore, the study was conducted in houston and the findings needs validation in other centers. finally, diagnosis of encephalitis still challenging and sometimes we proceed with invasive diagnostic measures such as brain biopsy to find the causative factor [ ] . despite our currently available diagnostic tools, a large proportion of patients still have unknown etiologies. metagenomic sequencing of csf has recently been shown to aid in the further identifying infectious etiologies in patients with meningitis and encephalitis and could be a promising clinical tool in the near future [ ] . adults with encephalitis continue to have high rates of unknown etiologies and adverse clinical outcomes. encephalitis management and compliance with the guidelines were suboptimal accounting for the underutilization of currently available diagnostic tests and empirical therapy in a significant proportion of patients. we hope that this study will increase awareness of encephalitis guidelines among clinicians with goals to improve the care and outcomes of this devastating disease. ms contributed to conception and design of the study, acquisition and analysis of data, and drafting a significant portion of the manuscript. mh contributed to conception and design of the study, and acquisition and analysis of data. aa contributed to acquisition and analysis of data. rh contributed to conception and design of the study, acquisition and analysis of data, and drafting a significant portion of the manuscript. this study was supported by the grant a starr foundation. dr. hasbun is a speaker and has research support from biofire®. all other authors do not have any conflicts of interest. acute encephalitis in immunocompetent adults epidemiology of infectious encephalitis causes in the epidemiology, clinical features, and long-term prognosis of japanese encephalitis in central sarawak results of a multinational study suggest the need for rapid diagnosis and early antiviral treatment at the onset of herpetic meningoencephalitis the neurocognitive and mri outcomes of west nile virus infection: preliminary analysis using an external control group in search of encephalitis etiologies: diagnostic challenges in the california encephalitis project causes of encephalitis and differences in their clinical presentation in england: a multicenter, population-based prospective study challenge of the unknown. a systematic review of acute encephalitis in non-outbreak situations herpes simplex virus encephalitis use of testing for west nile virus and other arboviruses the management of encephalitis: clinical practice guidelines by the infectious diseases society of america management of suspected viral encephalitis in adults -association of british neurologists and british infection association national guidelines consensus guidelines for the investigation and management of encephalitis case definitions, diagnostic algorithms, and priorities in encephalitis: consensus statement of the international encephalitis. consortium guidelines on the management of infectious encephalitis in adults meningitis with a negative cerebrospinal fluid gram stain in adults: risk classification for an adverse clinical outcome focal encephalitis following varicellazoster virus reactivation without rash in a healthy immunized young adult anti-nmda receptor encephalitis: report of ten cases and comparison with viral encephalitis adult herpes simplex encephalitis: fifteen years' experience outcome of and prognostic factors for herpes simplex encephalitis in adult patients: results of a multicenter study atypical manifestations and poor outcome of herpes simplex encephalitis in the immunocompromised predictors of outcome in acute encephalitis clinical metagenomic sequencing for diagnosis of meningitis and encephalitis we want to thank mr. and mrs. starr for their continued support of our research. key: cord- -isdsestc authors: hosseini, akram a.; shetty, ashit k.; sprigg, nikola; auer, dorothee p.; constantinescu, cris s. title: delirium as a presenting feature in covid- : neuroinvasive infection or autoimmune encephalopathy? date: - - journal: brain behav immun doi: . /j.bbi. . . sha: doc_id: cord_uid: isdsestc nan the most common symptoms of covid- are related to systemic and respiratory involvement. we report two cases of severe acute respiratory syndrome coronavirus- (sars-cov ) infection with acute onset of altered mental status and delirium with normal respiration and metabolic balance in the first hours. informed consent for publication was obtained from both patients. a -year-old male presented with two days history of headache followed by acute hypoactive delirium, disinhibition, confusion, but no fever, cough, or systemic manifestations. he had no previous medical or psychiatric illness, smoking, alcohol or illicit drug use. he was apyrexic but encephalopathic, nonverbal, and unable to follow commands. there was no weakness, but he had ankle clonus, brisk reflexes and babinski's sign. baseline brain ct and blood tests were unremarkable (table ) . nasopharyngeal swab real-time reverse-transcriptase-polymerase-chain-reaction (rrt-pcr) test was positive for sars-cov . after days, he developed fever and status epilepticus requiring sedation, neuromuscular blockade and intubation. the fraction of inspired oxygen (fio ) remained at % and no additional respiratory support was needed. chest x-ray only showed patchy consolidation without significant progression on subsequent imaging. cerebrospinal fluid (csf) showed mildly raised protein and oligoclonal bands, and was negative for sars-cov . neuromuscular blockade was withdrawn on day , when the new symptom of cough emerged. despite normal brain ct at hours, mri on day showed three hyperintense foci on diffusion-weighted images, but no overt restriction, consistent with t -shine-through suggesting cellular infiltration/inflammation or small infarcts ( figure ). the presistent diffusion-weighted hyperintensity on subsequent mri supported neuroinflammation. cranial arteries were normal on ct angiogram. empirical treatment included ceftriaxone, aciclovir and sodium valproate. . he noted hyposmia and dysgeusia during the convolescence. despite improvement after a week, he had impaired verbal fluecy, linguistic abstraction, phrase repetition, and delayed recall memory. he scored / on montreal cognitive assessment on day . a -year-old female was admitted to hospital following a seizure resulting in injuries to her face. according to her relatives, the symptoms started a few hours earlier with confusion and verbal communication difficulties. there was no preceding illness. she had no fever, cough, or respiratory symptoms at presentation. vital signs were normal. there were no sensory or motor deficits. she had dysphasia and impaired orientation, attention and memory. the first brain mri only showed chronic small vessel ischemic changes. chest ct scan only demonstrated bilateral pleural effusion. after hours, she had two generalized seizures, followed by fever and low oxygen saturation. neurological examination confirmed poor mental state, with glasgow coma scale scores between and . repeat blood tests showed hyponatremia. the second nasopharyngeal sample rrt-pcr was positive for sars-cov . csf revealed normal constituents (table ) . ct scan and repeat brain mri showed new changes in the limbic system with partial diffusion restriction, suggestive of limbic encephalitis ( figure ). the patient's persistent delirium improved over days with mild respiratory symptoms, hyposmia and dysgeusia. on day , she scored / on montreal cognitive assessment with impaired verbal fluency, repetition, abstraction, and delayed recall memory. there is emerging evidence that sars-cov can present with neurological features and concomitant encephalopathy. , however, there is currently no report of limbic encephalitis associated with covid- that presented with delirium in the absence of respiratory, metabolic or systemic features, while patients may be hidden sources of spreading the virus in busy clinical settings. the detection of sars-cov in the csf in a patient with meningo-encephalitis supports neurotropic and neuroinvasive potential of the virus presumably through the blood vesselrich meninges once the blood brain barrier is damaged. the evidence of sars-cov viral particles in brain capillary endothelial cells of an infected patient suggests hematogenous cns entry. the olfactory neuronal pathway could explain hyposmia. angiotensinconverting enzyme (ace ) receptors of vascular endothelium in respiratory system and meningeal capillaries can be the binding target for sars-cov proteins. in the absence of detectable virus in the csf, it is plausible that sars-cov causes an immunologic response that results in parenchymal inflammatory injury, cerebral edema and clinical manifestations of encephalopathy. the presence of oligoclonal bands in one of our patient's csf and serum supports immune-mediated response that is not restricted to intrathecal production of immunoglobulins. increased interleukin- in severe sars-cov disease highlights the occurrence of immunologic response and indicates intracranial cytokine storms. post-infection or non-infectious autoimmune encephalitis are known to be associated with antibodies against neuronal cell-surface or synaptic proteins. the clinical phenotype similarly includes neurological and psychiatric presentations without csf pleocytosis, and early immunotherapy improves outcome. in patients with recent episodes of psychosis, higher prevalence of antibodies against four coronaviruses strains are reported , which supports the possibility of sars-cov encephalopathy and psychosis. it is unknown if immunotherapy is required to improve neurocognitive outcome in patients with sars- neurologic manifestations of hospitalized patients with coronavirus disease a first case of meningitis/encephalitis associated with sars-coronavirus- evidence of the covid- virus targeting the cns: tissue distribution, host-virus interaction, and proposed neurotropic mechanisms central nervous system involvement by severe acute respiratory syndrome coronavirus - (sars-cov- ) human coronaviruses and other respiratory viruses: underestimated opportunistic pathogens of the central nervous system? viruses tissue distribution of ace protein, the functional receptor for sars coronavirus. a first step in understanding sars pathogenesis nervous system involvement after infection with covid- and other coronaviruses covid- : consider cytokine storm syndromes and immunosuppression we acknowledge the help from dr rob dineen, dr yasser falah, dr key: cord- - u f authors: costela-ruiz, víctor j.; illescas-montes, rebeca; puerta-puerta, jose m.; ruiz, concepción; melguizo-rodríguez, lucia title: sars-cov- infection: the role of cytokines in covid- disease date: - - journal: cytokine growth factor rev doi: . /j.cytogfr. . . sha: doc_id: cord_uid: u f covid- disease, caused by infection with sars-cov- , is related to a series of physiopathological mechanisms that mobilize a wide variety of biomolecules, mainly immunological in nature. in the most severe cases, the prognosis can be markedly worsened by the hyperproduction of mainly proinflammatory cytokines, such as il- , il- , il- , ifn-γ, and tnf-α, preferentially targeting lung tissue. this study reviews published data on alterations in the expression of different cytokines in patients with covid- who require admission to an intensive care unit. data on the implication of cytokines in this disease and their effect on outcomes will support the design of more effective approaches to the management of covid- . cardiac damage, and evidence of pulmonary ground-glass opacities. in some patients, ground-glass opacities were detected in subpleural regions in both lungs, which may lead to both systemic and localized immune responses, exacerbating inflammation. in some cases, treatment with inhalers and interferon not only had no positive effects but also worsened the clinical symptoms, with the progression of pulmonary opacities [ , ] . patients of advanced age and those with underlying conditions (e.g., hypertension, chronic obstructive pulmonary disease, diabetes, and/or cardiovascular disease, etc.) are at higher risk of a rapid progression to acute respiratory distress syndrome, septic shock, metabolic acidosis, coagulation dysfunction, arrhythmia, kidney damage, heart failure, liver dysfunction, and/or secondary infection, often resulting in death [ , ] . treatment options against this new disease are mainly limited to the mitigation of clinical symptoms, especially those affecting the respiratory system, including the application of oxygen therapy, with the provision of extracorporeal membrane oxygenation for patients with refractory hypoxemia. treatments with hyperimmune plasma and immunoglobulin g have also been received by some critically ill patients [ , ] , while the administration of antivirals and corticosteroids is contraindicated [ , ] . remdesivir, an adenosine nucleotide analog, has proven effective in in vitro and animal studies and in one patient in the usa [ , ] . antiretrovirals used against the human immunodeficiency virus (hiv), such as lopinavir/ritonavir, have also been found to reduce the viral load of sars-cov- [ ] . chloroquine has also been proposed as a candidate drug for the treatment of covid- based on its immunomodulatory properties and capacity to suppress tumor necrosis factor  (tnf-) and interleukin- (il- ) and to inhibit autophagy [ , ] . in general, and especially in patients suffering from a cytokine storm, clinicians should focus on the reduction of uncontrolled inflammation by blocking il- and tnf- or eliminating cytokines via hemoperfusion [ ] (table ) . cytokine hyperproduction is often followed by edema, gas exchange dysfunction, acute respiratory syndrome, acute cardiac damage, and secondary infection [ ] . with regard to the pathophysiological mechanism of the virus, it enters the cell via angiotensin-converting enzyme- (ace- ), mainly through the toll-like receptor- the majority of patients infected with covid- have normal or reduced white cell counts and lymphocytopenia, and those with severe disease have shown significantly elevated levels of neutrophils, dimer-d, and urea in blood, with a continuing decrease in lymphocytes. increases in certain cytokines and chemokines (e.g., il- , il- , and tnf-) have also been observed in these patients. thus, patients admitted to intensive care units (icus) have been found to have elevated serum levels of il- , il- , il- , macrophage colony-stimulating factor (m-csf), granulocyte colony-stimulating factor (g-csf), granulocyte-macrophage colony stimulating factor (gm-csf), kd interferon-gamma-induced protein (ip- ), monocyte chemoattractant protein- (mcp- ), macrophage inflammatory protein - (mip -), and tnf- [ , , ] (fig. ) . it is essential to analyze the factors underlying the physiopathology of this pandemic disease, and certain cytokines appear to play a key role. the objective of this study was to review data on the cytokines that influence the progression of covid- in order to support efforts to manage this highly virulent disease. the immediate immune response to infection by viruses, bacteria, or other microorganisms involves the mobilization of cells and molecules and draws on energetic, enzymatic, and biosynthetic resources; i.e., metabolic resources [ ] [ ] [ ] . metabolic dysfunctions caused by viral infection requires a reprograming of the host metabolism to generate effective antiviral defense responses. data published on interferences between the actions of viruses and cytokines reveal the molecular mechanisms underlying the innate immune response against viral infection [ ] [ ] [ ] . cytokines are a group of polypeptide signaling molecules responsible for regulating a large number of biological processes via cell surface receptors [ ] . key cytokines include those involved in adaptive immunity (e.g., il- and il- ), proinflammatory cytokines and interleukins (ils) (e.g., interferon (ifn)-i, -ii, and -iii; il- , il- , and il- ; and tnf-); and anti-inflammatory cytokines (e.g., il- ). in response to stress-generating internal processes (e.g., cancer or microbial infection), host cells secrete cytokines with a highly important role in cell metabolism reprogramming as a defensive response [ , , ] . concerning covid- disease, blanco-mello et al. described a distinctive and unsuitable inflammatory response related to sars-cov- infection. these authors revealed that an "inappropriate and weak immune response" appears more frequently in patients with comorbidities. thus, this could favor virus replication and enhance complications related to severe cases of the disease [ ] . in the short time since the emergence of covid- , numerous studies have described abnormal levels of the following cytokines and chemokines in the patients: il- , il- , il- , il- , il- , il- , il- , il- , il- , m-csf, g-csf, gm-csf, ip- , ifn-, mcp- , mip -, hepatocyte growth factor (hgf), tnf-, and vascular endothelial growth factor (vegf) [ , , , , ] (table ). the key point in sars-cov- infection could be the depletion of antiviral defenses related to innate immune response as well as an elevated production of inflammatory cytokines [ ] . il- actively participates in the inflammatory response to infection [ ] , and its main sources are activated monocytes and macrophages [ ] . sars-cov- appears to act on the activation and maturation of il- , which in turn activates other proinflammatory cytokines, such as il- and tnf- [ ] [ ] [ ] . hence, il-  forms part of the cytokine storm produced by coronavirus infections [ ] [ ] [ ] [ ] [ ] [ ] [ ] . yang et al. detected elevated levels of the antagonistic receptor of il- (il- ra) in severe cases of covid- , and this marker has been associated with increased viral load, loss of pulmonary function, lung damage, and mortality risk [ ] . liu et al. also found elevated il-  levels in patients with severe covid- , and these were strongly associated with lung injury [ ] . il- levels are related to the virulence of the process, and significantly higher serum levels have been observed in sars-cov- cases with severe symptoms than in mild cases or in those infected with the sars-cov or mers coronavirus [ ] . most covid- patients with severe symptoms have elevated levels of il- , which has been associated with sars, hypercoagulation, and disseminated intravascular coagulation [ ] . for this reason, some therapeutic strategies have used the inhibition of il- in an attempt to avoid the cytokine storm [ , ] . in this way, mesenchymal stem cells (mscs) have been used to inhibit proinflammatory cytokines such as il-  and tnf- [ ] . il- plays a key role in the proliferation of t cells and in the generation of effector and memory t cells [ ] . it is involved in adaptive immunity and increases glucose metabolism to promote the proliferation and activation of t, b, and nk cells [ ] . hence, il- participates in the prevention of autoimmune diseases and is essential to control immune responses and maintain self-tolerance [ ] . the absence of this interleukin has been associated with a poor control of effector cells and the consequent development of autoimmunity [ ] . huang et al. detected elevated levels of il- or its receptor il- r in patients with covid- , and it has been reported that these increases are directly proportional to the severity of the disease [ , , , [ ] [ ] [ ] , , , ] . elevation of this interleukin and its association with disease severity have also been reported in patients with other types of coronavirus [ ] [ ] [ ] . il- is also involved in adaptive immunity, playing a crucial role in the immune regulation governed by activated t helper (th) cells. it preferentially acts through activation, proliferation, and differentiation of b lymphocytes and promotion of the immunoglobulin e isotype. it therefore decisively intervenes in the induction of humoral immunity-regulating th cells [ , ] . it has been proposed that il- has an anti-inflammatory function that is specific to the tissue in which it is present, reflecting the metabolic plasticity of different tissues [ ] . with respect to viral infections that target the respiratory system, bot et al. observed that its expression during infection with an influenza virus had negative effects on cd + memory t cells [ ] .various studies of covid- patients have detected elevated il- levels as part of the cytokine storm associated with severe respiratory symptoms [ , , , ] . j o u r n a l p r e -p r o o f il- is involved in inflammation, the immune response, and hematopoiesis [ ] . this pleiotropic biomolecule is secreted by multiple cell types and regulates a wide variety of physiological processes [ ] . during initial stages of inflammation, secreted il- travels to the liver and induces a large number of acute-phase proteins, including creactive protein (crp), serum amyloid a (saa), fibrinogen, haptoglobin, and  antitrypsin. in addition, il- has been found to diminish the production of fibronectin, albumin, and transferrin [ ] . it has been reported that it has regenerative and antiinflammatory effects mediated by the conventional signaling process, although it also exerts proinflammatory effects mediated by trans-signaling, as in the case of viral infections [ ] . in relation to the different types of coronavirus, elevated il- levels have been observed in sars cases and related to the severity of symptoms [ , , ] and in sars-cov, being implicated in possible t-cell dysfunctionality. it has been observed that sars-cov-induced cytokines may damage the capacity of t cells in relation to dendritic cells, compromising the viability of these cells and macrophages to eliminate the pathogen [ , ] . similar observations have been made in relation to mers [ , ] . proportional association between elevated il- levels and t cell counts [ ] , and another study of patients with severe symptoms described elevated serum levels of il- and crp [ ] . a study of patients infected with sars-cov- also reported that the elevation of il- levels was more marked with more severe symptoms [ ] . these levels have been higher than those observed in patients with sars-cov or mers [ , ] . il- levels were also found to be markedly higher in patients who died from covid- than in those who recovered [ ] . as noted above, activation of il-  by sars-cov- in turn activates il- and tnf- [ ] [ ] [ ] . it has also been demonstrated that a high expression of il- in patients with covid- can accelerate the inflammatory process, contributing to the cytokine storm and worsening the prognosis [ ] . the cytokine storm, including elevated levels of il- , has also been associated with cardiac damage in these patients [ ] . with regard to the important role of il- in the sars-cov- -induced cytokine storm and its evasion, it has been reported that the monoclonal antibody tocilizumab acts by blocking the receptor of il- and has been reported to reverse cytokine hyperproduction [ , [ ] [ ] [ ] , inflammation, and pulmonary fibrosis [ , ] . in fact, a multicenter clinical trial is under way in china on the usefulness of tocilizumab to treat patients with covid- (nct & nct ), and it has already been included in clinical practice guidelines in china and italy [ , ] . the polyol myoinositol was also described by bizzarri et al. as a potential candidate drug to reduce il- levels and the risk of cytokine storm [ ] . in addition, the capacity of the macrolide azithromycin in combination with hydroxychloroquine, to reduce nasopharyngeal levels of sars-cov- has been attributed to its capacity to block il- and tnf- [ , ] . chloroquine also has the capacity to inhibit il- and tnf-, and is being tested against covid- [ , ] . finally, blood purification therapy has been proposed to eliminate pathological antibodies and il- , among other cytokines, given its successful application against other diseases [ ] . il- plays an important role in lymphocyte differentiation, participating in the development of t cells and peripheral homeostasis [ ] . all of the main cd t cell subgroups (cd + immature, memory, and th cells) depend on this cytokine for peripheral homeostasis [ ] [ ] [ ] [ ] . il- activates t cells, increases the production of proinflammatory cytokines, and negatively regulates transforming growth factor beta (tgf-). the role of this biomolecule depends on il- [ ] , and it has been suggested that il- secretion can be induced by viral infections [ ] . as in the case of other cytokines, it has been reported that il- levels are elevated in patients with covid- and directly related to disease severity [ , , ] . il- is a type cytokine that inhibits the production of proinflammatory cytokines (e.g., ifn, tnf, il- , and il- ) in various cell types and prevents dendritic cell maturation by blocking il- . it hampers expression of the major histocompatibility j o u r n a l p r e -p r o o f complex and costimulatory molecules, which have an important role in cell immunity. however, il- can have immunostimulatory effects, including the stimulation of ifn production by cd + t cells. it is also a powerful factor for the growth and differentiation of b cells, mast cells, and thymocytes [ ] [ ] [ ] [ ] [ ] [ ] [ ] . numerous authors have described viruses (epstein barr virus, cytomegalovirus and herpesvirus) that contain il- homologs, which may contribute to the presentation of different binding profiles to receptors and biological activities that can increase viral resistance [ , , [ ] [ ] [ ] [ ] [ ] [ ] . however, il- regulation is decreased in infection with some viruses, such as hiv, contributing to t-cell depletion [ ] [ ] [ ] . animal studies have shown that the antibody-mediated inhibition of il- signaling improves the t-cell response and contributes to eliminating viral resistance. hence, the effective blocking of il- signaling may be useful against resistant viral infections [ , ] . various authors have detected this interleukin in patients with covid- and related its levels to disease severity and progression, as in the case of other cytokines [ , , , , , , , , ] and it has been reported to have possible prognostic value [ ] . in fact, some authors indicate that il- may be hyper-expressed in anti-sars-cov- immunity, being higher in patients of advanced age with respect to a "hyperinflammatory response", possibly related to the reduction of t-cell receptors in the elderly [ ] . as with other cytokines, il- levels were found to be higher in patients with covid- than in those with sars-cov or mers [ ] . il- is one of a group of heterodimeric biomolecules with distinctive characteristics, including pairing versality (also observed for il- , il- , and il- ), which are involved in molecular processes and functions with a crucial role in positive and negative feedback [ , ] . il is a proinflammatory/prostimulatory cytokine produced in response to microbial agents by dendritic cells, b cells, monocytes, and macrophages, among others. it has key functions in the development of th and th cells [ ] [ ] [ ] [ ] . il- also induces the production of ifn- by t and nk cells in a positive feedback mechanism [ ] . the action of this cytokine in viral infections is based on its direct chemotactic effects on the infiltration of nk cells, increasing their binding to vascular endothelial j o u r n a l p r e -p r o o f cells. nk cells secrete ifn-, which participates in positive feedback by increasing the production of il- [ , ] . viral infections rapidly induce the gene expression of il- , which also acts after viral replication [ ] [ ] [ ] . for instance, il- is endogenously induced during the pneumonia produced by influenza and activates nk cells, which secrete ifn- and thereby inhibit viral replication. il- has been found to produce some improvement in the response of cd + t cells [ ] . elevated serum il- levels have been observed in patients infected with sars-cov- , [ , , ] and in those infected with other coronaviruses such as sars-cov [ ] . mscs inhibit the secretion of il- , as well as ifn- and tnf-, and have been proposed as an effective therapy against covid- [ , ] . il- is secreted by activated th cells, constituting a counter-regulatory system for the th -type immune response. it is considered an important regulator of immune responses mediated by th -type cytokines [ ] . il- has varied functions and has been implicated in the development of bronchial asthma by inducing the production of tgf-, eotaxin- , and mucin [ ] [ ] [ ] [ ] . it also participates in the activation of mast cells (de vries je, ). both il- and il- are involved in allergic processes, asthma, and the regulation of th lymphocytes [ ] . il- mediates tissue responses to infections, including the mobilization of eosinophils and the expulsion of parasites [ ] . few data are available on the presence of il- in patients with covid- . huang et al. found no difference in serum il- levels between those requiring icu admission and those who did not [ ] . however, liu et al. observed a directly proportional association between il- levels and the viral load of sars-cov- [ ] . il- is synthetized by th lymphocytes and is elevated in inflammatory processes and autoimmune diseases [ ] [ ] [ ] [ ] . it is also produced by cd + cells and by various sets of immature lymphocytes, including gamma-delta t cells, nk cells, and group innate lymphoid cells [ ] . il- , alongside il- and tnf-, also induces the j o u r n a l p r e -p r o o f production of antimicrobial peptides [ ] . hence, il- is a proinflammatory cytokine that plays a role in tissue damage, physiological stress, and infection. these functions vary according to the tissue in which il- is expressed, being particularly important in the gastrointestinal tract and skin [ , ] . elevated il- levels have been reported in patients with sars-cov- as part of the cytokine storm [ ] , and they have been associated with the viral load and disease severity [ ] . in contrast, wan et al. found that il- levels were normal in patients with covid- , with no significant differences between patients with severe versus mild symptoms [ ] . elevated il- levels were previously described in patients with sars-cov or mers [ , ] . the fact that th cells can produce il- , among others, has led to proposals for a therapeutic approach to covid- focused on janus kinase (jak ) inhibitor named fedratinib. this jak inhibitor decreases il- expression by th cells in murine models [ ] . m-csf, also known as colony-stimulating factor- , is a primary growth factor that belongs to the family of colony-stimulating factors [ ] . it regulates the growth, and even higher in those requiring icu admission [ ] . this could lead the multiorgan failure related to severe cases [ , ] . wu d and yang xo ( ) associated g-csf levels with the response of th lymphocytes in patients with sars-cov- , finding that the il- produced by these cells can induce the production of g-csf, among others. they also reported that th contributes to the cytokine storm triggered by sars-cov- [ ] . liu et al. directly related g-csf levels to the viral load of sars-cov- and the associated lung damage [ ] . gm-csf is a heterodimeric complex that consists of a specific alpha chain of gm-csf and a signal transduction subunit shared with il- and il- receptors. it is a single-membrane protein that homodimerizes after binding to g-csf [ ] [ ] [ ] . the main sources of this biomolecule are fibroblasts and endothelial, epithelial, stromal, and hematopoietic cells [ ] , and it is secreted by epithelial cells in the lung [ , ] . unlike m-csf and g-csf, gm-csf is virtually undetectable in the blood, although it has been found to be locally produced and activated in tissues affected by inflammatory processes. serum gm-csf levels can also be elevated in response to endotoxins. hence, gm-csf plays an important role in the inflammatory process [ ] [ ] [ ] , stimulating the proliferation and activation of macrophages, eosinophils, neutrophils, monocytes, dendritic cells, and microglial cells. besides its role as a hematopoietic growth factor, this cytokine increases the production of proinflammatory cytokines, favors antigenic presentation and phagocytosis, and promotes chemotaxis and leukocyte adhesion. it is of major importance in maintaining immune homeostasis in lung and gut [ ] . elevated serum gm-csf levels have been detected during the acute phase of infection by sars-cov- in comparison to healthy individuals, both in those who required icu admission and those who did not [ ] . th cells produce gm-csf, and high th counts have been associated with elevated gm-csf levels [ ] . it has also been observed that the activation of th cells by pathogens generate gm-csf, among other cytokines [ ] . ip- was initially identified as a chemokine whose secretion is induced by ifn-. ip- is secreted by neutrophils, endothelial cells, keratinocytes, fibroblasts, dendritic cells, astrocytes, and hepatocytes. through its binding to chemokine receptor (cxcr ), it regulates immune system responses by activating and recruiting leukocytes, including t cells, monocytes, and nk cells. therefore, ip- and cxcr play a key role in recruiting leukocytes to inflamed tissues and in perpetuating inflammation, thereby making a major contribution to tissue damage [ ] . elevated ip- concentrations have been found in numerous infections, mainly viral infections [ ] . serum ip- levels were found to be elevated in patients with covid- and even higher in those who required icu admission, suggesting their relationship with lung damage and disease severity [ ] . high levels of this biomolecule were previously found in patients with sars-cov [ ] , and the levels are even higher in those with sars-cov- [ ] . thus, the hyperproduction of ip- , among others, is considered to contribute to disease progression [ ] . liu monocytes, macrophages, dendritic cells, and neutrophil granulocytes can also produce this cytokine. although numerous cells can be the source of ifn-, it is mainly produced by t and nk cells. mscs can also secrete low ifn- levels to regulate hematopoiesis [ ] . ifn- participates in numerous immune and adaptive immunological functions and in inflammatory processes. it promotes macrophage activation and antigen presentation and is highly involved in anti-bacteria and anti-virus immunity and in signal transduction. it is difficult to classify ifn- as a pro-or anti-inflammatory cytokine, given its complex and varied roles [ ] . ( ) observed that elevated ifn- levels were associated with greater viral load and lung damage [ ] . sun et al. found that ifn-, il- , and il- levels were higher in patients with infection by sars-cov- but did not differ between patients who required icu admission and those who did not [ ] . in fact, these authors found that levels of this cytokine were lower in cd + t-cells from patients with severe versus mild symptoms and suggested that the infection may initially affect cd + and cd + t-cells, reducing the production of ifn- [ ] . this protein belongs the c-c chemokine family and is a powerful monocyte chemotactic factor that is constitutively produced or induced by oxidative stress, cytokines, or growth factors. it can be expressed by endothelial cells, fibroblasts, epithelial cells, smooth muscle cells, mesangial cells, astrocytes, monocytes, and microglial cells, which play an important role in the antiviral response in the peripheral circulation and tissues. monocytes and macrophages are the main source of mcp- , which regulates the migration and infiltration of monocytes, memory t cells, and nk cells [ ] . huang et al. found that mcp- levels were higher in patients with covid- and even higher among those admitted to icu [ ] . it has been reported that mcp- increases rapidly in the early acute phase of infection and then progressively decreases with the advance of the disease [ ] . xiong et al. detected elevated levels of mcp- and other cytokines in the bronchoalveolar lavage fluid of patients with covid- and associated the pathogeny of the virus with these cytokines [ ] . elevated levels of this protein have also been detected in the lung tissue of patients infected with sars-cov- [ ] . tnf- is produced by various cell types, such as monocytes, macrophages, and t cells, among others. this cytokine has been related to proinflammatory responses mediated by il-  and il- . alongside other cytokines, tnf- is involved in the regulation of inflammatory processes, infectious diseases, and malignant tumors [ ] . it has been observed that serum tnf- levels are elevated in patients with covid- and are higher with more severe disease [ , , ] . diao et al. reported similar results in a sample of patients with covid- and found an inverse relationship between tnf- levels and t-cell counts [ ] . in contrast, wan et al. described normal tnf- levels in patients with covid- [ ] . tnf- was one of the cytokines whose overproduction was related to a poor prognosis in patients with sars-cov and mers [ ] [ ] [ ] ] . zhang et al. proposed that the administration of certolizumab, an anti-tnf- antibody, might have beneficial effects on patients with covid- [ ] . another possible therapeutic approach is to use mscs to inhibit tnf- and il- , among other cytokines [ ] . other biomolecules besides cytokines have shown increased expression in patients with covid- , including growth factors. j o u r n a l p r e -p r o o f vegf vegf is essential for vascular endothelial homeostasis and is present in numerous cells and tissues. it has also been found to participate in the pathogenesis of tumor growth and metastasis. vegf plays an essential role in endothelial cell activation by binding to cell surface receptors (vegfr). the integrity of the endothelial barrier in lung tissue is crucial for alveolar immune regulation. severe inflammation and the associated immune responses induce the apoptosis of epithelial and endothelial cells, increasing the production of vegf and exacerbating edema and the extravasation of immune cells [ ] . the pathogenic effects of this factor are related to its action on vascular permeability and neoangiogenesis [ , ] . vegf hyper-regulation is observed in various viral infections, and attempts have been made to stimulate this factor by using vegf homologs present in some viruses or by activating inflammatory mediators that trigger the overexpression of vegf [ ] . serum vegf levels were found to be higher in patients with sars-cov- , although they did not differ between those who were admitted to icu and those who were not [ ] . as noted above, the therapeutic application of mscs is under consideration due to their regenerative and immunomodulatory potential. besides vegf, mscs can also secrete vegf, among others, which would be useful in the approach to respiratory distress syndrome and in the regeneration of lung tissue and treatment of lung fibrosis induced by infections [ ] . hepatocyte growth factor (hgf) is synthesized by fibroblasts and by endothelial and hepatic cells, among other cell populations. the active form is produced by the action of a serine protease enzyme released by damaged tissues. in the lungs, hgf is sequested by pulmonary fibroblasts, and its secretion is increased when the tissue is damaged [ ] . serum hgf and mip - levels were found to be elevated in patients with covid- , and even higher mip - levels were found in those requiring icu admission [ ] . the immunological reaction triggered by infection with sars-cov- mobilizes numerous cytokines, mainly of proinflammatory character. changes in their levels are associated with the presence of the disease and a more severe prognosis. this study summarizes findings on the role of these cytokines in the onset and outcomes of sars-cov- infection and on possible therapeutic approaches involving the inhibition of their activity. the authors declare no competing or financial interests. chen et al. [ ] retrospective study to describe the epidemiological and clinical features of sars-cov- pneumonia in patients from wuhan jinyintan hospital approximately % of the subjects who developed the disease had been exposed to the huanan seafood market. it affects men more often, with an average age of . approximately half of the subjects had other comorbidities. the main symptomatology is characterized by fever, cough, shortness of breath, muscle pain among others. ct scan revealed that % of patients developed bilateral pneumonia and some cases evolved into acute respiratory distress syndrome (ards) or died from multiple organ failure. huang et al. [ ] descriptive study to describe the epidemiological, clinical, laboratory and radiological characteristics, treatment and outcomes of patients infected with covid- in wuhan and make a comparison between patients in the intensive care unit (icu) and those who are not infection by sars-cov- caused clusters of severe respiratory disease and was associated with icu admission and high mortality. most of the infected patients were male, with different comorbidities and an average age of years. the disease is mainly manifested by fever, cough, and myalgia or fatigue. dyspnea occurred in more than % of the patients. lymphopenia was present in % of the cases. patients admitted to the icu were found to have higher plasma levels of il , il , il , gscf, ip , mcp , mip a, and tnfα. liu et al. [ ] descriptive study to describe the epidemiology, clinical features, possible treatments and prognosis of patients infected with sars-cov- in hubei the initial manifestations of sars-cov- were fever, cough and muscle pain or fatigue. most patients had a normal or decreased white blood cell count, and . % had lymphocytopenia. lung involvement was observed in all cases. treatment was based on symptom control and respiratory support. immunoglobulin g was administered to some critically ill patients while systemic corticosteroids showed no significant benefit. the risk of death was related to age, comorbidities and the period between initial symptoms and dyspnea. wang et al. [ ] retrospective study to describe the cytokine release syndrome-like (crsl) that occurs in patients affected by covid- pneumonia and to identify risk factors and possible treatments. average age: years old. gender: men mostly. extensive pulmonary inflammation and ards: , %. symptoms: fever, hypoxia and shock ( , %). laboratory findings: decrease of cd , cd , cd , nk cells. increase of il- , cd /cd ratio. crsl: , %, manifested by: pulmonary inflammation, decrease of cd , cd and nk, increase of il- and dysfunction of non-pulmonary organs. management: ventilation, anti-inflammatory therapy, mechanical-ventilation chen et al. [ ] literature review to review the literature on the relationship between covid- and the cytokine storm as well as the possible immutherapic treatments available the effectiveness of some therapies was demonstrated such as the use of stem cells that inhibit the activation of t-lymphocytes, macrophages and induce their differentiation into regulatory subpopulations of t-cells and anti-inflammatory macrophages, but also inhibit the secretion of il- α, tnf-α, il- , il- , and γinterferon, controlling the cytokine storm. conti et al. [ ] literature review to clarify the relationship between il- and il- pro-inflammatory cytokines and lung inflammation. anti-inflammatory strategies infection with covid- causes a release of il- β and il- which will lead to lung inflammation, fever and fibrosis. the therapeutic potential of several emerging coronaviruses: genome structure, replication, and pathogenesis china novel coronavirus investigating and research team, a novel coronavirus from patients with pneumonia in china the origin, transmission and clinical therapies on coronavirus disease (covid- ) outbreak -an update on the status understanding of covid- based on current evidence the reproductive number of covid- is higher compared to sars coronavirus pattern of early human-to-human transmission of wuhan novel coronavirus ( -ncov) clinical characteristics of coronavirus disease in china the covid- epidemic updated understanding of the outbreak of novel coronavirus ( -ncov) in wuhan novel wuhan ( -ncov) coronavirus epidemiological and clinical characteristics of cases of novel coronavirus pneumonia in wuhan, china: a descriptive study clinical features of patients infected with novel coronavirus in identification of a novel coronavirus causing severe pneumonia in human: a descriptive study hospitalized patients with novel coronavirus-infected pneumonia in wuhan ct imaging of the novel coronavirus ( -ncov) pneumonia novel coronavirus ( -ncov) situation reports convalescent plasma as a potential therapy for covid- zhonghua jie he he hu xi za zhi coronavirus susceptibility to the antiviral remdesivir (gs- ) is mediated by the viral polymerase and the proofreading exoribonuclease washington state -ncov case investigation team, first case of novel coronavirus in the united states case of the index patient who caused tertiary transmission of covid- infection in korea: the application of lopinavir/ritonavir for the treatment of covid- infected pneumonia monitored by quantitative rt-pcr chloroquine is a potent inhibitor of sars coronavirus infection and spread quinoline-based antimalarial drugs: a novel class of autophagy inhibitors why the immune system fails to mount an adaptive immune response to a covid - infection clinical characteristics of novel j o u r n a l p r e -p r o o f coronavirus cases in tertiary hospitals in hubei province the definition and risks of cytokine release syndrome-like in covid- -infected pneumonia critically ill patients: disease characteristics and retrospective analysis, intensive care and critical care medicine how low cholesterol is good for anti-viral immunity interferons induce changes in core metabolism that are critical for immune function electrophilic properties of itaconate and derivatives regulate the iκbζ-atf inflammatory axis ordered recruitment of chromatin modifying and general transcription factors to the ifn-beta promoter the interplay between central metabolism and innate immune responses viral activation of cellular metabolism cytokine synergy: an underappreciated contributor to innate anti-viral immunity cytokines and chemokines: at the crossroads of cell signalling and inflammatory disease immunology of covid- : current state of the science ncov) infections trigger an exaggerated cytokine response aggravating lung injury advances in the research of cytokine storm mechanism induced by corona virus disease and the corresponding immunotherapies chloroquine and hydroxychloroquine equally affect tumor necrosis factor-alpha, interleukin , and interferon-gamma production by peripheral blood mononuclear cells inhibition of nf-κb-mediated inflammation in severe acute respiratory syndrome coronavirusinfected mice increases survival severe acute respiratory syndrome coronavirus envelope protein ion channel activity promotes virus fitness and pathogenesis severe acute respiratory syndrome coronavirus orf a protein activates the nlrp inflammasome by promoting traf -dependent ubiquitination of asc cytokine responses in severe acute respiratory syndrome coronavirus-infected macrophages in vitro: possible relevance to pathogenesis chemokine up-regulation in sars-coronavirusinfected, monocyte-derived human dendritic cells middle east respiratory syndrome coronavirus efficiently infects human primary t lymphocytes and activates the extrinsic and intrinsic apoptosis pathways severe acute respiratory syndrome: historical induction of pro-inflammatory cytokines (il- and il- ) and lung inflammation by coronavirus- (covi- or sars-cov- ): anti-inflammatory strategies hlh across speciality collaboration, uk, covid- : consider cytokine storm syndromes and immunosuppression characteristics of lymphocyte subsets and cytokines in peripheral blood of hospitalized patients with novel coronavirus pneumonia (ncp) exuberant elevation of ip- , mcp- and il- ra during sars-cov- infection is associated with disease severity and fatal outcome, infectious diseases (except hiv/aids) -novel coronavirus ( -ncov) infections trigger an exaggerated cytokine response aggravating lung injury dysregulation of immune response in patients with covid- in the use of anti-inflammatory drugs in the treatment of people with severe coronavirus disease (covid- ): the perspectives of clinical immunologists from china immunotherapeutic implications of il- blockade for cytokine storm mast cells contribute to coronavirus-induced inflammation: new anti-inflammatory strategy il- : the first effective immunotherapy for human cancer the importance of regulatory t-cell heterogeneity in maintaining self-tolerance revisiting il- : biology and therapeutic prospects clinical and immunologic features in severe and moderate coronavirus disease zhonghua jie he he hu xi za zhi. ( ) e an interferon-gamma-related cytokine storm in sars patients t cell responses to whole sars coronavirus in humans virus-specific memory cd t cells provide substantial protection from lethal severe acute respiratory syndrome coronavirus infection association of tgf-beta , il- and il- gene polymerphisms with asthma in a chinese population, asian pac interleukin- regulates lipid metabolism by inhibiting adipogenesis and promoting lipolysis local il- expression in the lung reduces pulmonary influenza-virus-specific secondary cytotoxic t cell responses the ebv il- homologue is a selective agonist with impaired binding to the il- receptor il- in inflammation, immunity, and disease role of il- in asthma and other inflammatory pulmonary diseases interleukin- and the acute phase response il- trans-signaling via the soluble il- receptor: importance for the pro-inflammatory activities of il- plasma inflammatory cytokines and chemokines in severe acute respiratory syndrome cytokine regulation in sars coronavirus infection compared to other respiratory virus infections severe acute respiratory syndrome (sars) coronavirus-induced lung epithelial cytokines exacerbate sars pathogenesis by modulating intrinsic functions of monocyte-derived macrophages and dendritic cells comparative and kinetic analysis of viral shedding and immunological responses in mers patients representing a broad spectrum of disease severity comparative replication and immune activation profiles of sars-cov- and sars-cov in human lungs: an ex vivo study with implications for the pathogenesis of covid- reduction and functional exhaustion of t j o u r n a l p r e -p r o o f cells in patients with coronavirus disease possible vertical transmission of sars-cov- from an infected mother to her newborn tocilizumab treatment in covid- : a single center experience potential effect of blood purification therapy in reducing cytokine storm as a late complication of severe covid- sars-cov- : a storm is raging clinical predictors of mortality due to covid- based on an analysis of data of patients from wuhan, china clinical features of severe pediatric patients with coronavirus disease in wuhan: a single center's observational study clinical features of cases with coronavirus disease risk factors associated with acute respiratory distress syndrome and death in patients with coronavirus disease detectable serum sars-cov- viral load (rnaaemia) is closely associated with drastically elevated interleukin (il- ) level in critically ill covid- patients, infectious diseases (except hiv/aids) clinical course and risk factors for mortality of adult inpatients with covid- in wuhan, china: a retrospective cohort study aberrant pathogenic gm-csf+ t cells and inflammatory cd +cd + monocytes in severe pulmonary syndrome patients of a new coronavirus covid- and the heart effective treatment of severe covid- patients with tocilizumab mechanisms and management of chimeric antigen receptor t-cell therapy-related toxicities the cytokine release syndrome (crs) of severe covid- and interleukin- receptor tocilizumab may be the key to reduce the mortality biomarkers of cytokine release syndrome and neurotoxicity related to car-t cell therapy elevation of il- and il- levels in serum associated with lung fibrosis and skeletal muscle wasting in a bleomycin-induced lung injury mouse model stanford medicine covid- pharmacotherapy information vademecum per la cura delle persone con malattia da covi- inositol and pulmonary j o u r n a l p r e -p r o o f function. could myo-inositol treatment downregulate inflammation and cytokine release syndrome in sars-cov- ? macrolide activities beyond their antimicrobial effects: macrolides in diffuse panbronchiolitis and cystic fibrosis hydroxychloroquine and azithromycin as a treatment of covid- : results of an open-label non-randomized clinical trial remdesivir and chloroquine effectively inhibit the recently emerged novel coronavirus ( -ncov) in vitro il- r expression and il- signaling confer a distinct phenotype on developing human b-lineage cells interleukin- mediates the homeostasis of naïve and memory cd t cells in vivo il- is critical for homeostatic proliferation and survival of naive t cells interleukin and t cell receptor signals regulate homeostasis of cd memory cells the many faces of il- : from lymphopoiesis to peripheral t cell maintenance il- engages multiple mechanisms to overcome chronic viral infection and limit organ pathology hepatic interleukin- expression regulates t cell responses interleukin : a novel stimulatory factor for mast cells and their progenitors interleukin is a potent growth and differentiation factor for activated human b lymphocytes interleukin- increases th cytokine production and cytotoxic potential in human papillomavirus-specific cd (+) cytotoxic t lymphocytes interleukin- and the interleukin- receptor human cytomegalovirus-encoded interleukin- homolog inhibits maturation of dendritic cells and alters their functionality the regulation of il- production by immune cells il- elicits ifnγ-dependent tumor immune surveillance homology of cytokine synthesis inhibitory factor (il- ) to the epstein-barr virus gene bcrfi a single amino acid determines the immunostimulatory activity of interleukin differential il- r expression plays a critical role in il- -mediated immune regulation human cytomegalovirus harbors its own unique il- homolog (cmvil- ) shaping phenotype, function, and survival of dendritic cells by cytomegalovirusencoded il- same structure, different function crystal structure of the epstein-barr virus il- bound to the soluble il- r chain il- , t cell exhaustion and viral persistence the role of il- in regulating immunity to persistent viral infections recent insights into microbial triggers of interleukin- production in the host and the impact on infectious disease pathogenesis interleukin- determines viral clearance or persistence in vivo resolution of a chronic viral infection after interleukin- receptor j o u r n a l p r e -p r o o f blockade immune-epidemiological parameters of the novel coronavirus -a perspective interleukin- : odd one out or part of the family? molecular interactions within the il- /il- cytokine/receptor superfamily regulation of interleukin- production in antigenpresenting cells regulation of t(h) differentiation--controlling the controllers il- and il- : master regulators of innate and adaptive immunity new il- -family members: il- and il- , cytokines with divergent functions il- family cytokines: immunological playmakers interleukin- is chemotactic for natural killer cells and stimulates their interaction with vascular endothelium il- and viral infections interleukin- gene expression after viral infection in the mouse activation of type iii nitric oxide synthase in astrocytes following a neurotropic viral infection herpes simplex virus type -mediated up-regulation of il- (p ) mrna expression. implications in immunopathogenesis and protection mesenchymal stem cells tune the development of monocyte-derived dendritic cells toward a myeloid-derived suppressive phenotype through growthregulated oncogene chemokines transplantation of ace -mesenchymal stem cells improves the outcome of patients with covid- pneumonia transcriptional regulation of th /th polarization hematopoietic cell phosphatase associates with the interleukin- (il- ) receptor beta chain and down-regulates il- -induced tyrosine phosphorylation and mitogenesis specific recruitment of sh-ptp to the erythropoietin receptor causes inactivation of jak and termination of proliferative signals regulation of colony-stimulating factor receptor signaling by the sh domain-containing tyrosine phosphatase shptp signalling by the kit receptor tyrosine kinase is negatively regulated in vivo by the protein tyrosine phosphatase shp how are th -type immune responses initiated and j o u r n a l p r e -p r o o f amplified? divergent expression patterns of il- and il- define unique functions in allergic immunity interleukin -producing cd + effector t cells develop via a lineage distinct from the t helper type and lineages il- drives a pathogenic t cell population that induces autoimmune inflammation a genome-wide association study identifies il r as an inflammatory bowel disease gene secukinumab sustains improvement in signs and symptoms of psoriatic arthritis: year results from the phase future study innate il- -producing cells: the sentinels of the immune system il- : there is a gap in our knowledge interleukin- and innate immunity in infections and chronic inflammation the il- family of cytokines in health and disease cell host response to infection with novel human coronavirus emc predicts potential antivirals and important differences with sars coronavirus distinct immune response in two mers-cov-infected patients: can we go from bench to bedside? th responses in cytokine storm of covid- : an emerging target of jak inhibitor fedratinib from growth factor to central mediator of tissue inflammation macrophage colony-stimulating factor and cancer: a review regulation of myelopoiesis by proinflammatory cytokines in infectious diseases role of granulocyte colony-stimulating factor in infection and inflammation neutropenia, neutrophil dysfunction, and bacterial infection in patients with human immunodeficiency virus disease: the role of granulocyte colonystimulating factor novel coronavirus-important information for clinicians reconstitution of functional receptors for human granulocyte/macrophage colonystimulating factor (gm-csf): evidence that the protein encoded by the aic b cdna is j o u r n a l p r e -p r o o f a subunit of the murine gm-csf receptor a human high affinity interleukin- receptor (il r) is composed of an il -specific alpha chain and a beta chain shared with the receptor for gm-csf molecular analysis of the granulocyte colony-stimulating factor receptor alveolar macrophages develop from fetal monocytes that differentiate into long-lived cells in the first week of life via gm-csf induction of the nuclear receptor ppar-γ by the cytokine gm-csf is critical for the differentiation of fetal monocytes into alveolar macrophages studies on the bone marrow colony stimulating factor (csf): relation of tissue csf to serum csf il- , il- , and granulocyte/macrophage colony-stimulating factor, in allergen-induced late-phase cutaneous reactions in atopic subjects pivotal roles of gm-csf in autoimmunity and inflammation ip- in autoimmune thyroiditis trail and ip- as biomarkers of viral infections in the emergency j o u r n a l p r e -p r o o f department interferon gamma in autoimmunity: a complicated player on a complex stage mers-cov infection in humans is associated with a pro-inflammatory th and th cytokine profile monocyte chemoattractant protein- (mcp- ): an overview hypothesis for potential pathogenesis of sars-cov- infection-a review of immune changes in patients with viral pneumonia transcriptomic characteristics of bronchoalveolar lavage fluid and peripheral blood mononuclear cells in covid- patients targeting tnf and tnf receptor pathway in hiv- infection: from immune activation to viral reservoirs covid- : melatonin as a potential adjuvant treatment vegf upregulation in viral infections and its possible therapeutic implications vegf in signaling and disease: beyond discovery and development this study was supported by research group bio (junta de andalucía) and the department of nursing of the university of granada. cytokines, such as il- and , capable of inhibiting molecules such as those previously mentioned, has been demonstrated mehta et al. [ ] correspondenceto define the cytokine storm syndrome that occurs in severe covid- states and to identify possible treatments cytokine storm syndrome is characterized by increased interleukin (il)- , il- , il- granulocyte-colony stimulating factor, interferon-γ inducible protein , monocyte chemo-attractant protein , macrophage inflammatory protein -α, and tumour necrosisfactor-α. immunosuppressors such as tozulimab or anakinra appear to work in states of hyperinflammation as described in severe covid- stateswan et al. [ ] observational study to characterize the state of the immune system and the implications of different cytokines in sars-cov- patients and to study their relationship with the severity of the process lower levels of cd +t and cd +t and higher levels of il- and il- were observed in the more severe ncps. these markers may help predict the worsening of mild patients.yang et al. [ ] observational study to study the different cytokine profiles in patients infected with sars-cov- and their relationship to the severity of the disease.a total of cytokines were shown to be elevated in patients with covid- , with particularly high levels of ip- , mcp- , and il- ra in severe patients. these markers were predictors of the evolution of the disease towards more severe and even fatal states.liu et al. [ ] retrospective study to characterize the cytokine storm that occurs in patients with covid- patients with covid have hypercitokinaemia that manifests itself with an elevation of of the cytokines measured. patients with lung lesions were observed to have an upregulation of m-csf, il- , ifn- , il- , il- , ip- , il- , il- ra, g-csf, il- , ifn-γ, il- , il- , hgf, and pdgf-bb. these biomarkers may be useful as predictors of the severity of the pathology.qin et al. [ ] retrospective study to analyze the expression of different biomarkers, inflammatory cytokines and lymphocyte subsets in patients infected with covid- , and to determine their relationship with the severity of the process.an increase in neutrophil-lymphocyte-ratio and t lymphocytopenia (especially cd + t cells) was observed, which was more pronounced in severe patients. elevated serum levels of tnf-α, il- and il- and il- were also found in severe cases.zhang et al. [ ] literature review to study the use of anti-inflammatory drugs in the therapeutic approach of patients infected with covid- treatment with anti-inflammatory drugs may be useful in managing the cytokine storm that develops in critically ill patients. however, possible immunological alterations of the host should be considered before starting therapy and their individual characteristics should also be considered. kritas et al. [ ] literature review to determine the cytokine potential of various cytokines of the il- family as a new strategy in the management of inflammation il- , which inhibits il- , may be considered in the treatment of patients with covid- because of its anti-inflammatory activity that would help control fever and inflammation.chen et al. [ ] retrospective study to establish and compare cytokine profiles between moderate and severe stages of covid- in patients from tongji hospital.severe patients more frequently presented dyspnea, lymphopenia and hypoalbuminemia, with higher levels of alanine aminotransferase, lactate dehydrogenase, c-reactive protein, ferritin and d-dimer, as well as significantly higher levels of il- r, il- , il- and tnf-α. the numbers of t-lymphocytes, j o u r n a l p r e -p r o o f chen et al. [ ] retrospective study to establish the clinical features of covid- and to study the relationship between the cytokine storm detected in serum and the severity of the process in patients from tongji hospital.the main symptoms of -ncov pneumonia were fever with or without respiratory symptoms and other systemic symptoms. serum white blood cell counts were normal or decreased, lymphocyte count decreased, hs-crp increased, procalcitonin normal, ldh increased and albumin decreased. high resolution ct showed single or multiple frosted glass shadows accompanied by septal thickening.significantly higher levels of interleukin- receptor (il- r) and il- were found in the most severe patients. no differences in serum tnf-α, il- , il- , il- , hs-crp, lymphocyte count and ldh levels were found between the groups chu et al. [ ] ex vivo study to study replication, cell tropism and the route of immune activation by sars-cov- in human lung tissues sars-cov- infected and replicated in human lung tissues more efficiently than sars-cov. in lung tissue, sars-cov- infected type i and ii pneumocytes and alveolar macrophages but did not stimulate production of type i, ii, or iii interferons and only increased expression of il , mcp , cxcl , cxcl , and ip diao et al. [ ] retrospective study to analyze figures and markers of t-cell exhaustion in patients with covid- a marked reduction in the overall t-cell count was observed in patients with covid- . total t-cell, cd +t-cell, or cd +t-cell counts below /μl, /μl, or /μl are negatively correlated with patient survival and serum levels of il- , il- , and tnf-α. in addition, it was observed that pd- (a marker of tcell exhaustion) is higher in patients with covid and that its expression is related to the severity of the process.dong et al. [ ] retrospective study study the possible vertical transmission of the sars-cov- a newborn presented igm antibody to sars-cov- as well as cytokine alterations although he was negative for rt-pcrspedersen & ho [ ] literature review to describe the cytokine storm that occurs in severe sars-cov- infected patients increased levels of il- , il- and tnf-α, lymphopenia (in cd + and cd + t cells) and decreased expression of ifn-γ in cd + t cells in severe patients were evidenced.ruan et al. [ ] retrospective study to establish predictors of mortality in patients infected with covid- predictors of mortality in patients infected with sars-cov- include age, presence of comorbidities, presence of secondary infection, and elevated inflammatory markers in the blood such as lymphocytes, platelets, albumin, total bilirubin, blood urea nitrogen, blood creatinine, myoglobin, cardiac troponin, c-reactive protein (crp), and interleukin- sun et al. [ ] retrospective study to describe epidemiological and clinical features, imaging and laboratory data, clinical treatments and outcomes of severely or critically ill pediatric patients infected with covid- in wuhan.most of the subjects were males between the ages of months and years. the main manifestations included polypnea, fever and cough. patch-like shadows and ground glass opacity were observed in most patients on chest ct scans. the analyses showed an increase in c-reactive protein, procalcitonin and lactate dehydrogenase, cd , cd , cd , il- , il- and ifn-γ and a decrease in cd + j o u r n a l p r e -p r o o f cd and th/ts. treatment was based on symptom control and respiratory support. two subjects required invasive mechanical ventilation. wu et al. [ ] retrospective cohort studyto describe the clinical features and outcomes in patients with ards or who died from covid- pneumonia [ ] retrospective study to analyze the relationships between the incidence of rnaaemia and the cytokine storm as well as the severity of the disease there seems to be a relationship between rnaaemia and the severity of the patient's condition, as well as with il- levels, which is particularly high in this group of subjects and may act as a therapeutic target for the management of critically ill patients.zhou et al. [ ] retrospective multicenter study zhou et al. [ ] retrospective study to analyze blood samples from patients with sars-cov- severe pneumonia in order to identify their immune characteristics.covid- disease leads the activation of cd + t cells and generate gm-csf, among others. the infection generates the secretion of several cytokines that induce inflammatory cd + and cd + monocytes with the consequent increase of il- expression and the acceleration of inflammatory process. akhmerov & marban [ ] literature [ ] literature review to clarify the pathogenesis of sars-cov- and compare it with other infections caused by coronavirusthe virus enters the body through the nasal and pharyngeal mucous membranes and reaches the lung parenchyma where it can be incorporated into the circulation causing viremia. once in the bloodstream it can affect organs that express ace . the period from the first symptoms to ards is approximately days. at this point a second, much more aggressive stage of the infection begins. in infected patients, lymphopenia and an increase in pro-inflammatory cytokines are detected.xiong et al. [ ] retrospective study to study transcriptional changes in bronchoalveolar lavage fluid (balf) and peripheral blood mononuclear cells (pbmc) specimens from covid- patientsan association was observed between the pathogenesis of covid- and the excessive release of cytokines such as ccl /mcp- , cxcl /ip- , ccl /mip- a and ccl /mip b. in addition, it was postulated that the ability of the virus to activate apoptosis and the p signaling pathway in lymphocytes may be the cause of lymphopenia in patients. j o u r n a l p r e -p r o o f key: cord- - f niif authors: tadavarthy, silpa n.; finnegan, kerriann; bernatowicz, gretchen; lowe, elisha; coffin, susan e; manning, marylou title: developing and implementing an infection prevention and control program for a covid- alternative care site in philadelphia, pa date: - - journal: am j infect control doi: . /j.ajic. . . sha: doc_id: cord_uid: f niif background: on march , , the city of philadelphia was given permission by temple university to convert the liacouras center gymnasium to an alternate care site (acs) to treat low-acuity covid- patients. acs's, especially those created to specifically care for infectious patients, require a robust infection prevention and control (ipc) program. methods: the ipc program was led by a physician and nurse partnership, both of whom had substantial experience developing ipc programs in u.s. and low-resource settings. the ipc program was framed on a previously described conceptual model commonly referred to as the “ s's”: space, staff, stuff, and systems. results: the gymnasium was transformed into red, yellow and green infection hazard zones. the ipc team trained staff in critical ipc practices and personal protective equipment (ppe) standards. systems to detect staff illness were created and over staff health screening surveys completed. discussion: use of existing guidance and comprehensive facility and patient management assessments guided the development of the ipc program. program priorities were to keep staff and patients safe and implement procedures to judiciously use limited resources that affect infection transmission. conclusion: planning, executing and evaluating ipc standards and requirements of an acs during a pandemic requires creative and nimble strategies to adapt, substitute, conserve, reuse, and reallocate ipc space, staff, stuff and systems. on january , , the first case of laboratory-confirmed infection due to the novel virus severe acute respiratory syndrome coronavirus (sars-cov- ) in the united states was identified in seattle, washington. six weeks later, in early march, the first person in pennsylvania was diagnosed with coronavirus disease- (covid- ) disease. the subsequent rapid growth in covid- cases in the philadelphia region led most acute care hospitals to suspend non-urgent procedures and hospitalizations by mid-march. very quickly, hospitals were required to assess their surge capacity in preparation for a possible largescale, public health emergency. despite individual facilities' efforts to accommodate a surge in patients with moderate-to-severe covid- , multiple acute care hospitals in philadelphia began to experience a surge in demand just three weeks after the first confirmed case was identified. on march , temple university granted the city of philadelphia permission to use the liacouras center as an overflow medical facility for low-acuity covid- patients. setting: philadelphia is the sixth largest city in the u.s. with a population of over . million people. it is also the poorest large city in the country. most hospital beds are in facilities that are members of extensive healthcare networks. according to data, philadelphia county has approximately , adult staffed medical-surgical beds and intensive care unit (icu) beds, although pandemic planning included identifying additional hospital beds in each facility in the event of a surge of demand. the temple university liacouras center is known as a premiere basketball facility and provides a unique and flexible space, which is also used for concerts, banquets and trade shows. it is one of the largest indoor, public assembly venues in philadelphia. the initial material assets of the csf-l were provided by fema. key materials included: cots, commodes, walkers, bathing equipment, medical monitoring equipment, portable non-plumbed sinks, sharps containers, infectious waste receptacles, alcohol-based hand rub, and personal protective equipment (ppe). method: we utilized a previously described conceptual model to assess disaster responses and surge capacity, commonly referred to as the - s's‖: space, staff, stuff, and systems. [ ] [ ] [ ] this framework guided our development of a novel ipc program for this surge facility. in this article, we describe the - s's‖ of our program developed for the csf-l and the related challenges at a covid- alternative care site. the rapid creation and unusual configuration of this facility, together with the challenges of new clinical teams unfamiliar with one another, and working together in uncomfortable ppe to provide high-quality patient care, necessitated some basic approaches to the development of our ipc program. these included: . use of existing guidelines and other resources from expert groups whenever available , . adapt existing guidance to apply to the unique conditions of the surge field hospital . standardize ipc processes to ensure the safety of patients and staff because the liacouras center was neither designed nor engineered to care for patients, a comprehensive environmental and occupational risk assessment was undertaken prior to facility opening. environmental health and safety experts, together with leaders of the ipc team, conducted an -all-hazard‖ risk assessment of the site for actual or potential risks to patients or staff; this team produced a comprehensive health and safety plan for the csf-l. the plan identified the need for engineering controls (e.g. specifications for heating, ventilation, and air conditioning systems) and specified occupational ipc health and safety requirements, including ppe standards, daily monitoring of staff for acute illness, sanitation standards for both hand hygiene and equipment sanitation, as well as laundry and waste management recommendations. the identified ipc hazards and risk reduction plans, priorities and progress were reported and addressed by the csf-l team before the facility moved forward in development. a facility map was created that designated -red, yellow, and green zones,‖ each with a different level of infection risk and expectation for ipc practices and ppe use. colored tape was placed on the floor to provide visual cues. separate entrance and exit paths were designated for both staff and patients. the patient care and decontamination areas were designated as -red zone‖, requiring the highest level of ppe and to which physical access was strictly controlled. the -green zone‖ included the facility entrance and hallways leading to the staff locker room; only surgical masks were required while in these areas. the -yellow zone‖ was the interface between -red zone‖ and -green zone‖ where staff donned and doffed ppe. nearby liacouras offices were converted into ppe storage and distribution rooms. the ipc team was led by a member of the pdph's healthcare-associated infections and antimicrobial resistance team (sec) and a highly experienced nurse certified in ipc (mlm). collectively, these coleaders have over years of experience serving as local, national and international consultants and trainers for ipc programs. additional critical ipc team members included temple university medical students (st and kf), a nurse practitioner experienced in family medicine and college health and a registered nurse experienced in ipc (gb and el). our team worked in concert with the pdph and oem staff onsite as well as the csf-l leadership team. given the unique setting, heterogenous background of staff and challenges preventing nosocomial transmission of the sars-cov- virus and other potential healthcare associated infections, infection preventionist (ip) coverage of the -red zone‖ on all shifts was considered an integral component of the ipc plan. a call for volunteers from the local chapter of the association for professionals in infection control and epidemiology (apic) was released via the chapter listserv. interested and available ips were instructed to register through the philadelphia mrc website. however, recruiting these ips was a lofty goal given the intense increase of ip workload in their own facilities, so we began to seek ip designees, such as nurses or public health experts with advanced ipc knowledge. monitor. it was important to have a core group of individuals assigned to these roles as their responsibilities included being familiar with policies and providing focused coaching to ensure staff adherence to essential infection prevention practices. the fema medical station cache provided resources for a -bed facility. the included ipc resources included , n respirators of various sizes and models, fit test kits, surgical masks, disposable isolation gowns, face shields, and over boxes of non-sterile examination gloves of various sizes. the cache also included portable, non-plumbed sinks and alcohol-based hand rub. additional ppe resources were continuously being sought and obtained through vendors as well as private and public donations. the availability and maintenance of the ppe inventory was critical for csf-l operation. prior to opening, a baseline inventory of every item was established and the ppe distribution room was organized to maximize space and to improve the efficiency of distribution. it was staffed hours per day by a consistent group of registered nurses and two members of the dod to standardize the process. all staff entering the patient care area (-red zone‖) received an isolation gown, a face shield, and a fit-tested n respirator from the ppe distributor. their name and the items they received were recorded by hand in the ppe distribution log. this process was repeated each time the staff member entered the patient care area at the start of their shift and after each scheduled break. staff received a new or reprocessed n respirator each time they entered or re-entered the -red zone.‖ a running count of all items distributed was recorded every six hours on the daily ppe inventory tracking form. stock delivered to the ppe distribution room and items returned to stock after reprocessing were also recorded here. the numbers from the previous hours were reconciled at the start of each day and entered by hand into the master inventory spreadsheet. key process indicator reports outlining the number of days on hand of each item were generated daily and shared with the leadership team. all ppe, except gloves and surgical masks, was reprocessed. face shields, safety glasses and goggles were disinfected on site by the decontamination staff in a designated, well-ventilated area away from patient care and all other activities, with a hospital-grade disinfectant. n respirators were reprocessed using a bioquell hydrogen peroxide vapor decontamination facility developed by a local hospital to maintain their own ppe supply. the used n respirators were prepared and packaged for transport by the waste management staff and were transported to and from the reprocessing facility every other day. isolation gowns were reprocessed daily by a medical laundry service. all reprocessed items were then returned to the ppe distribution room and logged into the inventory tracking form. one point of entry into csf-l was established for all staff to ensure security and facilitate health screening. this area was staffed hours per day by security personnel and a staff entrance surveillance monitor. staff entering the building were required to wear a personal face mask and remain six feet apart from other personnel at all times; if someone did not have a mask, a surgical mask was provided. surveillance was intended to identify individuals with clinical signs or symptoms suggestive of covid- or other acute illness, or recent exposure to sars-cov- . the daily entrance survey was accessed and completed by volunteers and staff using a qr (quick response) code on their smartphone or if they had no smartphone, on a paper survey. staff monitors verified that the survey was complete, asked about any positive answers and took each volunteer's temperature using a no-contact infrared thermometer. the names of all individuals who reported symptoms of an acute illness or a temperature > . °f were recorded for investigation; ill staff were instructed to return home and given instructions for self-monitoring and when to seek care. staff who cleared the screening process signed in, performed hand hygiene using an alcohol-based hand rub, and proceeded into the facility. staff entrance screening began on april and responses were monitored daily through may . during that time period approximately , surveys were completed. no staff were noted to have a fever upon temperature check or a positive symptom screen at facility entry. staff were recruited from the philadelphia mrc (a group who serve the city during public health emergencies and large-scale events), contracted staffing agencies and vendors, and the dod. this meant clinical staff came with varying experiences and approaches to infection control and nonclinical/support staff had little to no experience with ipc measures. we operated under the assumption that all staff needed training in csf-l-specific ipc standards and measures. thus, we developed orientation materials and training procedures in order to ensure that staff would be adequately protected and trained. we created an -infection prevention and control orientation‖ presentation that described proper protocols for entering the csf-l with the screening survey, hand hygiene, ppe standards and processes, mask use and reuse, cleaning and disinfection, sharps safety and occupational exposures, including needlesticks. this presentation also included videos from the cdc demonstrating proper donning and doffing technique. the ipc presentation and live ppe demonstration took approximately minutes and was included with other ori-entation presentations on the facility and its mission, safety measures and a tour of the patient care area. after completion of the orientation, clinical staff were fit tested using osha respirator fit testing protocol by environmental health and safety consultants for the available n respirators. they were required to don and doff the ppe that would be available at csf-l with trained ipc team members assisting and observing the techniques. eleven orientation sessions were held between april and april and were attended by staff. given the unique clinical environment, rapidity of development of ipc standards, and challenges with equipment procurement, we used a process of rapid cycle tests of change to adapt the ppe process, while remaining aligned with current cdc guidelines. during the duration of the csf-l development and use, every person on site was required to wear a face covering (either a cloth face covering or surgical mask). plastic full-face shields were the standard eye and face protection for every person working in the patient care area. safety glasses and goggles were provided as an alternate strategy for eye protection. following the ppe standards obtained from the emergency field hospital opened at the jacob k. javits convention center in new york city and the most current cdc recommendations, the ipc team initially recommended that only providers of hands-on patient care would wear n respirators, while non-patient care staff, such as environmental services, would wear a surgical mask. after further consideration of the open patient care environment, uncertainty of the infectivity of the patients, and goal of providing as much assurance of safety as possible to staff, we established a standard that all staff present in the patient care area (-red zone‖) would wear an n respirator and eye protection. because the number of disposable isolation gowns was limited, the ipc team, with support from a vendor, was able to obtain reusable, fluid resistant isolation gowns for use by all staff while in the patient care area. hand hygiene with alcohol-based hand rub was required before donning gown and gloves and after doffing gloves and gown, face shield and n respirator. clean, intact gloves were required to be worn by all volunteers present in the patient care area. hand hygiene with alcohol-based hand rub and glove change was required between each patient contact and when moving from dirty to clean activities. although portable, non-plumbed sinks were available, they were ultimately not used in the patient care area because they had only a fivegallon reservoir of water and therefore posed more challenges than benefits including needing to be refilled and cleaned often. thus, wall mounted alcohol hand rub dispensers were placed on the headwall of each bed space and table top dispensers were available at nursing stations and other staff work areas. safety and inventory were two guiding principles used in creating quality improvement measures at the csf-l. when we experienced a % loss of n respirators during the first round of reprocessing, primarily due to makeup use, we added a strongly worded request to the orientation that all staff refrain from make-up use while in the facility. we also provided makeup removal wipes and posted reminders to not wear makeup along with our respirator loss rate in the locker room and staff lounge restrooms. after implementing these interventions, our respirator loss rate significantly decreased to < %. due to some variability in ppe donning/doffing training received by staff during different orientation sessions (as a result of rapidly and continuously evolving cdc guidelines and best practice standards) a ppe and hand hygiene quality improvement donning/doffing evaluation tool was developed. the purpose was to assess proper donning and doffing procedures use by each staff member entering and leaving the -red zone‖ as well as correcting staff when needed. this was completed by the donning/doffing assistant and included ) assessment of an n respirator seal check, ) proper hand hygiene use during donning, ) use of the appropriate ppe doffing sequence, ) hand hygiene at appropriate moments during doffing sequence, and ) verification that no ppe other than a personal mask was worn in the -yellow zone‖ and -green zone‖. when it was realized that there was confusion and concern around proper hand hygiene in the -red zone,‖ we developed a hand hygiene quality improvement evaluation tool to be completed by the red zone infection preventionist. this tool assessed the proper doffing of gloves, use of hand hygiene (alcoholbased hand rub for seconds), and donning of new gloves between patients by providers. although we designed these measures with the intention to implement all of them, we were unable to do so due to the lack of further need for and closure of the csf-l. in this report, we describe the development, implementation and management of an ipc program for a covid- acs. key lessons learned included the need to: develop strategies to cope with real and potential shortages of critical supplies; adapt existing guidance for unique sites of care; standardize and continually assess staff use of ppe and fundamental ipc practices; and the importance of communication of ipc principles and concerns throughout the planning and management of this covid acs. a critical component of preparedness plans is surge capacity or the ability to adequately care for a significant influx of patients and be prepared for demands on supplies, personnel and physical space. although much of disaster and surge capacity planning focuses on hospital-based care, the covid- pandemic required various buildings and structures of opportunity across the country be converted to temporary field hospitals with the goal of increasing healthcare capacity and capability as needed. the liacouras center in philadelphia was such a structure and rapidly converted to function as an acs to assist regional health care facilities by providing non-acute care for adults with mildly to moderately symptomatic covid- . the csf-l ipc team, reporting to the chief nursing officer, was quickly established. the team leaders had previously worked together, were well-versed on cdc ipc guidelines, and had extensive experience in establishing ipc programs in non-traditional and resource-limited settings nationally and internationally. this worked to the team's advantage as we quickly identified program aims and delineated priorities. the team relied on real-time, action-oriented learning using the plan-do-study-act (pdsa) cycle for testing our initiatives -by planning it, trying it, observing the results, and acting on what is learned. this approach led to quick, early successes. for instance, we quickly realized that although fema provided resources for a -bed facility, only beds could be set-up in order to maintain at least six feet of distance between patients. another example, one of our first tasks was to establish the staff wellness check-in/surveillance procedure. working closely with our facility operations and security colleagues, a single point of building entry was identified. the ipc team explored several options for collecting volunteer screening data. based on convenience and ease of use we selected the free online qr code generator to create a code for the survey, while concurrently configuring the physical space to accommodate the related activities. we conducted multiple pdsa cycles to improve the original concept, resulting in an efficient, effective, standardized process. a similar approach was used to standardize ipc staff orientation and ppe donning and doffing competency check-offs. pdsa cycles were also used to navigate the ipc implications of the proposed system for facility access and flow of patients and for the support services of pharmacy, respiratory therapy, laboratory, patient linen and laundry, patient and staff food delivery, and waste (including medical waste and sharps) and garbage removal. predictably, the greatest challenge centered on managing ppe standards and clinical staff expectations. due to the critical shortages of ppe and alcohol-based hand rub across the country, the cdc revised its recommendations for the safe and appropriate use of ppe several times during our planning stages. this dynamic combined with the initial uncertainty of the resources available to the csf-l, made it difficult to develop ipc policies and procedures specific to this setting at the outset. there were also significant clinical staff concerns and anxieties surrounding ppe use. staff from throughout the us, varied practice settings (e.g., intensive care units, emergency departments, medical-surgical units) and without prior experience working together had to adapt to the csf-l ipc policies and procedures. having an ip or ip designee present hours a day, seven days a week in the -red zone‖ was invaluable in managing staff ipc expectations. they provided real-time staff ipc adherence monitoring, education, coaching, support and csf-l updates. in addition, a frequently asked question sheet with answers and rationale to many commonly asked questions was created. it included questions such as -why are we not double gloving?‖, -why are we not using hand sanitizer on top of gloves?‖, -why are we not wearing a surgical mask over the n respirator?‖ two factors underscored the importance of standardizing ipc practices in the csf-l. first, the risk of exposure to covid- in the csf-l environment was possibly increased as com-pared to other practice settings given the open ward structure and minimal engineering controls available. additionally, it was critical to establish a shared model of safe practice given the diversity of staff knowledge and experience with general and covid- specific ipc practices. less expected, was the complexity of ppe inventory management. there was no computer access in the ppe distribution area, so inventory management was a labor-intensive, manual process prone to error. this risk was mitigated by assigning designated staff to the ppe distribution room. had the csf-l remained opened, tools such as the the national institute for occupational safety and health (niosh) ppe tracker mobile app could be used. however, future acs's should utilize computerized inventory management systems, staffed by skilled personnel, to track all inventory. one of the most important aspects of disaster and emergency response is ensuring effective, frequent and timely information exchange. information exchange and management should be based on a system of collaboration, partnership, and sharing. while collaboration and partnership were a part of preparing the csf-l for patients, real-time information sharing to increase the ipc team's situational awareness of csf-l's capabilities and resource needs, was at times challenging, given the plethora of agencies, personnel, and teams working independently, yet simultaneously in an effort to prepare for occupancy. all acs, particularly those developed in response to an emerging infectious threat such as sars-cov- , will benefit from close partnerships between leaders, front-line and support staff, and ipc experts. finally, we believe our approach may have utility beyond the pandemic. use of the - s's‖ framework, coupled with actionoriented learning using pdsa cycles, could be used in other surge situations. the ipc team worked quickly and efficiently to manage the constantly evolving circumstances and the time constraints that accompanied the opening of a covid- pandemic acs. despite the growing scarcity of ppe, the csf-l goals of ensuring an adequate supply of ppe and provid-ing the safest environment for both patients and staff were achieved. the ability to leverage our collective ipc knowledge, skills, abilities and energies to this situation has been extremely rewarding. in the spirit of volunteerism, we had the opportunity to work with an extraordinary group of people dedicated to a common goal. agency for healthcare research and quality: surge capacity-education and training for a qualified workforce city provides update on covid- for friday the pennsylvania department of health &the hospital and healthsystem association of pennsylvania ( ) exploring the concept of surge capacity. ojin:the online journal of issues in nursing factors associated with preparedness of the us healthcare system to respond to a pediatric surge during an infectious disease pandemic: is our nation prepared? hospital surge capacity: the importance of better hospital pre-planning to cope with patient surge during dengue epidemics -a systematic review considerations for alternate care sites infection control in healthcare personnel: infrastructure and routine practices for occupational infection prevention and control services hospital infectious disease emergency preparedness: a survey of infection control professionals pan american health organization. information management and communication in emergencies and disasters: manual for disaster response teams critical care surge response strategies for the covid- outbreak in the united states key: cord- -zynor b authors: eisenhut, michael title: neopterin in diagnosis and monitoring of infectious diseases date: - - journal: j biomark doi: . / / sha: doc_id: cord_uid: zynor b neopterin is produced by activated monocytes, macrophages, and dendritic cells upon stimulation by interferon gamma produced by t-lymphocytes. quantification of neopterin in body fluids has been achieved by standard high-performance liquid chromatography, radioimmunoassays, and enzyme-linked immunosorbent assays. neopterin levels predict hiv-related mortality more efficiently than clinical manifestations. successful highly active antiretroviral therapy is associated with a decrease in neopterin levels. elevated neopterin levels were associated with hepatitis by hepatitis a, b, and c viruses. serum neopterin levels were found to be a predictor of response to treatment of chronic hcv infection with pegylated interferon combined with ribavirin. neopterin levels of patients with pulmonary tuberculosis were found to be higher in patients with more extensive radiological changes. elimination of blood donors with elevated neopterin levels to reduce risk of transmission of infections with known and unknown viral pathogens has been undertaken. neopterin measurement is hereby more cost effective but less sensitive than screening using polymerase chain reaction based assays. in conclusion neopterin is a nonspecific marker of activated t-helper cell dominated immune response. it may be a useful marker for monitoring of infectious disease activity during treatment and for more accurate estimation of extent of disease and prognosis. neopterin was first isolated from larvae of bees, in worker bees and in royal jelly in , and subsequently from human urine by sakurai and goto in [ ] . neopterin or -amino- -hydroxy- -(d-erythro- , , trihydroxypropyl)-pteridine is produced from guanosine triphosphate via guanosine triphosphate cyclohydrolase i (gtpch i) by activated monocytes, macrophages, dendritic cells, and endothelial cells and to a lesser extent in renal epithelial cells, fibroblasts, and vascular smooth muscle cells upon stimulation mainly by interferon gamma and to a lesser extent by interferon alpha and beta with its release being enhanced by tumor necrosis factor [ , ] . gtpch i mrna expression is synergistically and independently induced by interferon gamma through the jak /stat pathway of nuclear transcription regulation and through tnf by the nf-kappab pathway (see figure ) [ ] . release in response to cytokines released by t-lymphocytes and natural killer cells make neopterin an indicator of activation of cell mediated immunity including release by infections associated with activation of t-lymphocytes and natural killer cells, malignancies, autoimmune diseases, rejection of transplanted organs, and atherosclerosis. at its first isolation in the s neopterin was detected in the pupae of bees by anion exchange chromatography followed by paper chromatography [ ] . in the seventies gas chromatographic-massfragmentographic methods were described allowing measurement in urine. subsequently detection and quantification of neopterin succeeded in serum, urine, and other body fluids using standard high pressure and by reverse-phase high-performance liquid chromatography with fluorescence detection. later simpler radioimmunoassays and more recently enzymelinked immunosorbent assays have been developed which are suitable for large numbers of samples [ ] . semiquantitative measurement with a dipstick system using polyclonal antineopterin antibodies has been validated and may be suitable for bedside testing and in the setting of developing countries [ ] . levels have been observed, which correlate with the activity of disease. this was first described in [ ] and subsequently neopterin elevations were noted in infections with hepatitis viruses, epstein-barr, cytomegalo, measles, mumps, varicella zoster, rubella, and influenza viruses [ , [ ] [ ] [ ] [ ] [ ] . elevated neopterin levels in body fluids were found at the end of the incubation period before the onset of clinical symptoms. the highest neopterin levels occur just before specific antibodies against the virus become detectable, which is about two to four weeks after onset of increased neopterin production. in acute varicella zoster virus infection peak neopterin levels were observed at the end of the appearance of the rash and in measles virus infection one to three days after appearance of the rash [ , ] . immunisation with live viruses, for example, measles, mumps and rubella and virus vaccine, resulted in a significant increase of neopterin independent of presence of any symptoms. in measles vaccination neopterin levels were observed to rise at a median of days after vaccination about days before the appearance of antibodies [ ] . these investigations point to a future application of measurements neopterin as a correlate of a successful vaccination. neopterin should be investigated as a marker to evaluate protective efficacy of vaccines stimulating cell mediated immunity against mycobacterial, parasitic, or viral diseases. the magnitude of the elicited neopterin levels could be put into relationship to incidence of the disease immunised against the population of immunised children. serum neopterin levels were also found to be significantly elevated in symptomatic dengue virus infections with levels higher than in measles and influenza virus disease [ ] . levels correlated with duration of fever and severity of disease [ , ] . investigations into the physiological functions of neopterin in viral infections revealed that it is able to delay the development of the cytopathic effect of coxsackie b virus in hep- cells [ ] . a proposed mechanism is the stimulation of inducible nitric oxide synthase expression leading to an increase in nitric oxide production. other mechanisms include the induction of the translocation of the nuclear factor-kappa b to the nucleus. infection. testing of samples of hiv infected individuals found that / ( . %) of samples, which were hiv- rna and p antigen positive had elevated neopterin levels (> nmol/l). / ( . %) samples, which were both hiv- and p antigen negative had elevated neopterin levels [ ] . neopterin levels were also found to be significantly elevated in hiv- infection compared to controls [ ] . studies investigated markers of immune activation for their usefulness as prognostic markers in hiv infection and showed an increase of neopterin levels in people with hiv infection compared to patients without hiv infection [ ] [ ] [ ] [ ] . neopterin levels hereby were found to increase early in the course of hiv infection preceeding cd +-t-cell decline and clinical manifestations of aids [ , ] . plasma neopterin levels were found to correlate with plasma hiv viral load [ ] . neopterin levels were found to predict hiv related mortality [ , ] . a retrospective study compared microglobulin, immunoglobulin a, g, and m, adenosine deaminase, and neopterin levels above normal range as predictors of clinical or immunological deterioration in patients with hiv infection. changes in microglobulin levels showed the greatest sensitivity to detect worsening ( %) with neopterin slightly less sensitive ( . %) followed by immunoglobulin levels ( . - . %) and adenosine deaminase levels with . % having the lowest sensitivity [ ] . marker for viral load to monitor response to antiretroviral treatment. in a land mark study the effects of dual reverse transcriptase inhibitor (rt) therapy and highly active antiretroviral therapy (haart) on neopterin levels in patients with hiv infection were compared to hiv uninfected controls, hiv infected patients not on treatment, and patients who had stopped treatment [ ] . rt inhibitor treatment decreased circulating levels of neopterin (mean of . for treated versus a mean of . ng/ml for untreated hiv patients, < . ). haart decreased neopterin levels significantly further. this confirmed results of a previous study on the effects of haart on neopterin levels [ ] . neopterin levels in patients who discontinued haart became similar to untreated hiv patients. neopterin may be a particularly useful surrogate marker for monitoring of control of hiv replication in settings in developing countries where hiv rna viral load measurement is not available and may be a cheaper alternative particularly if semiquantitative dip stick tests are used for urine samples [ ] . longitudinal serial measurements in the same individual could overcome difficulties with interpretation in settings where chronic parasitic (malaria) or bacterial schistosoma mansoni praziquantel blood serum levels normalized on treatment [ , ] (tuberculosis) infections may elevate the baseline neopterin level and could allow monitoring of response to antiretroviral treatment in the absence of resistance testing and provide means to monitor compliance in the outpatient setting (see table for list of diseases in which neopterin levels have been used to monitor treatment response). hepatitis. the first study investigating the role of neopterin in specific forms of viral hepatitis tested urinary levels in patients with hepatitis a, hepatitis b, and non-a, non-b hepatitis virus infection [ ] . the authors noted that in patients with acute viral hepatitis patients had elevated urinary neopterin levels with the highest levels found in patients with acute hepatitis a. while all patients with active hepatitis b had elevated neopterin levels, / hbsag carriers ( %) had normal urinary neopterin levels. the authors noted that neopterin levels were not a reflection of hepatocellular damage as patients with alcoholic hepatitis had normal urinary neopterin levels. in order to address the question whether neopterin is a useful marker for early detection of viral infection in donated blood products before seroconversion, one study investigated neopterin levels in anti-hcv-negative specimens, which were hcv rna and hcv core antigen positive. the investigators found that / ( . %) had elevated neopterin levels (> nmol/l). / ( . %) specimens positive for hbv dna had elevated neopterin levels [ ] . in patients with thalassemia major receiving multiple blood transfusion significantly more patients with histologically proven chronic hepatitis ( / were anti hcv antibody positive) had elevated blood neopterin levels compared to patients with siderosis of the liver [ ] . alanine aminotransferase levels in hcv infected persons correlated significantly with neopterin levels. serum neopterin levels were found to be a useful predictor of response to treatment of chronic hcv infection with pegylated interferon combined with ribavirin. neopterin concentrations were evaluated in hcv patients treated by pegylated interferon combined with ribavirin. mean and median pretreatment neopterin concentrations were lower in patients with sustained virological response than in nonresponders. the rate of response was twofold higher among patients with pretreatment neopterin levels < nmol/l than in patients with neopterin levels ≥ nmol/l, even after controlling for hcv genotype status [ ] . a recent study investigated specifically whether serum neopterin levels can discriminate between patients with replicative ( = ) and nonreplicative ( = ) hbv carriage [ ] . replicative hbv carriage was defined as hbv dna > pg/ml by hybrid capture system. neopterin levels had a mean of . nmol/l in replicative versus . nmol/l in nonreplicative hbv carriers ( < . ). this result was not reproducible in another study, which found that in patients with replicative hbv infection ( = ) mean serum neopterin level was . nmol/l and in nonreplicative hbv ( = ) . nmol/l a difference, which was not statistically significant [ ] . this may have been due to large standard deviations and small numbers in groups. a more recent investigation found that in chronic hepatitis the mean ± sd serum neopterin levels were . ± . nmol/l, . ± . nmol/l in patients with liver cirrhosis and . ± . nmol/l in the control group. serum neopterin levels were significantly higher in patients with chronic hepatitis ( = . ) and cirrhosis patients ( = . ) than in control subjects. cirrhotic patients had significantly higher serum neopterin levels than patients with chronic hepatitis ( = . ). there was a positive correlation between serum neopterin levels and alanine aminotransferase levels in patients with chronic hepatitis ( = . , = . ) and cirrhotic patients ( = . , = . ). positive correlations were detected between serum neopterin levels and inflammatory score in patients with chronic hepatitis ( = . , = . ) and cirrhotic patients ( = . , = . ) [ ] . infections. neopterin has been investigated as a marker to distinguish viral from bacterial lower respiratory tract infections. the investigators found that serum neopterin levels were elevated (> nmol/l) in % of patients with viral lrti. the median serum neopterin concentration was almost -fold higher in the viral lrti group than bacterial lrti patients ( . versus . nmol/l) and -fold higher than those in healthy controls. the specificity for correct identification of viral lrti was . % for a cut-off of > nmol/l [ ] . serial monitoring of serum neopterin levels in patients with severe acute respiratory syndrome (sars) associated virus revealed that all ( = ) investigated patients had elevated neopterin levels by day [ ] . duration of pyrexia in sars patients correlated positively with neopterin levels. patients on steroid therapy had significantly lower neopterin levels. measurement of neopterin in isolation and in relationship to other inflammatory markers like procalcitonin and c-reactive protein were investigated for discriminatory power between viral and bacterial lower respiratory tract infections. investigators used the crp/neopterin ratio (c/n ratio) to discriminate viral and bacterial etiology of respiratory tract infections. in a study conducted in hong kong sera obtained on the day of hospitalization for lrti from patients with confirmed bacterial etiology and patients with viral etiology were examined. a further sera from healthy chinese subjects with no infection were included as controls. the area under the receiver operating characteristic (roc) curve (area under curve [auc]) for distinguishing bacterial from viral infections was . for crp and . for pct. the auc for distinguishing viral from bacterial infections was . for neopterin. when the markers were used in combination, auc of roc of the c/n ratio was . , whereas (crp × pct)/neopterin was . [ ] . in a subsequently reported study the median of the c/n ratio was times higher in patients with bacterial aetiology than with viral aetiology ( . versus . mg/nmol; < . ) and times higher than those in healthy subjects ( . versus . mg/nmol; < . ). the area under the receiver operator characteristic curve for the c/n ratio was . . a cut-off value of "c/n ratio > " for ruling in/out bacterial/viral infection yielded optimal sensitivity and specificity of . % and . %, respectively [ ] . early studies showed elevated neopterin levels in cerebrospinal fluid (csf) of patients with aseptic meningitis and herpes simplex and measles virus encephalomyelitis [ ] [ ] [ ] . csf levels of neopterin seem to reflect intrathecal production by microglia as pterins have a low permeability across the blood brain barrier with a serum-to-csf distribution at a quotient of / [ ] . it has recently been established that normal csf neopterin is brain-derived. the interindividual variation of csf neopterin in healthy adults was found not to depend on serum neopterin concentration variation (coefficient of variation, cv-csf = . % < cv-serum = . %). additionally individual normal csf neopterin concentrations were found to be invariant to the variation of the albumin quotient, qalb; that is, csf neopterin does not derive from leptomeninges [ ] . patients with viral meningitis had elevated csf neopterin levels compared to healthy controls but normal serum levels [ ] . csf neopterin levels correlated hereby with csf monocytic cell count. patients with various forms of encephalitis including those caused by herpes simplex virus, varicella zoster virus, and tick borne encephalitis virus had significantly elevated csf neopterin levels compared to controls and higher levels than in patients with viral meningitis without overlap of levels in the two conditions. in hiv infection there was a clear relationship between the severity of aids-related dementia and csf neopterin levels [ , [ ] [ ] [ ] . higher csf hiv viral loads were associated with higher csf neopterin levels [ ] . after commencement of combination antiretroviral therapy (art), csf neopterin decreased markedly but remained slightly above normal levels in a substantial number of patients despite several years of receiving art [ , [ ] [ ] [ ] . even patients with systemic virological failure exhibit a substantial reduction of csf neopterin concentrations, though above that of virologically suppressed patients [ ] . in patients on combination art, the lowest csf neopterin levels have been found in patients with the lowest csf viral loads (< . copies/ml) [ ] . no significant difference in csf neopterin concentrations was found between those treated with protease inhibitor-and nonnucleoside reverse transcriptase based regimens in combination with nucleoside analogues [ ] . this would support the idea that viral replication within or close to the csf, at least to some extent, is partly driving the inflammatory response. it has also been suggested that an inflammatory response, once triggered, may lead to a self-sustaining state of cellular activation as has been seen in patients with herpes simplex virus type- encephalitis [ ] . findings in this study are consistent with these reports. hiv rna levels measured in csf or plasma were not significantly associated with csf neopterin trajectories. in addition, all study participants had experienced virologic control to the limit of standard detection as a result of their treatment and csf neopterin levels were the only factor strongly associated with subsequent decay rates and the ultimate set-point levels [ ] . patients with bacterial infections with species other than mycobacteria showed significantly lower urinary neopterin levels compared to patients with viral infections in one study [ ] but no statistically significant difference in a more recent study [ ] . within the group of bacterial infections it was shown that patients with symptoms for at least days had significantly higher neopterin concentrations than patients with acute illness. this applied particularly to bacterial pneumonia. patients with urinary tract infections were found to have similar levels to patients with viral infections with data on urinary neopterin concentrations but not serum concentrations. thus it remains unclear whether local production of neopterin takes place in urinary tract infections and serum neopterin would stay low. there was no significant difference in neopterin levels between patients with febrile neutropenia and underlying haematological and oncological conditions and gram-negative versus gram-positive infections [ ] . in patients on an intensive care unit with sepsis journal of biomarkers and septic shock urinary neopterin/creatinine ratios were found to be significantly higher compared to patients with other forms of systemic inflammatory responses syndromes [ ] and serum neopterin levels were higher in nonsurvivors compared to survivors of sepsis and multiorgan failure scores correlated with neopterin levels [ ] [ ] [ ] [ ] . in this context it was however noted that neopterin levels correlated negatively with reduced renal function reflecting renal failure causing a reduced excretion of neopterin. future studies could correct for reduced excretion due to reduced renal function by calculation of the serum neopterin/creatinine ratio. investigations on critically ill patients on intensive care units evaluated neopterin levels as tool to discriminate patients with systemic inflammatory response syndrome with and without infectious etiology. neopterin levels were found to have a specificity of % for discriminating infectious and noninfectious etiology of critical illness [ ] . bacterial meningitis was associated with both elevated serum and csf neopterin levels compared to controls [ ] . in lyme neuroborreliosis-a late complication of infection by the tick-born spirochete borrelia burgdorferi-high neopterin concentrations were found in csf of patients, whereas serum neopterin levels were not markedly increased, confirming intrathecal neopterin production [ ] . infection with treponema pallidum subsp. pallidum (syphilis) was not associated with elevated neopterin levels [ ] . in melioidosis by pseudomonas pseudomallei neopterin concentrations were found to be significantly higher than controls [ ] . in brucellosis neopterin levels were with a mean . mmol/ml significantly higher than healthy controls and patients with tuberculosis [ ] . in leprosy caused by mycobacterium leprae % of patients with tuberculoid and lepromatous leprosy presented with elevated urinary neopterin excretion [ ] . on the basis of in vitro and in vivo data showing that macrophages release neopterin in response to stimulation by t-lymphocytes [ , ] fuchs et al. [ ] investigated urinary neopterin levels by hplc in patients with culture confirmed pulmonary tuberculosis and compared them with normal controls. % of patients had levels above the upper tolerance limit (containing with % probability . % of healthy controls). neopterin levels were higher than age and gender matched controls for every extent of pulmonary disease and correlated with its extent. the correlation with extent of pulmonary tuberculosis was also demonstrated for serum levels and levels found in bronchioalveolar lavage fluid [ ] . subsequent studies showed higher levels of neopterin in serum and pleural effusions of patients with pulmonary tuberculosis compared to controls [ , ] . peripheral blood mononuclear cells (pbmnc) from tuberculosis patients showed a significantly higher spontaneous production of neopterin. stimulation with phytohaemagglutinin or purified protein derivative did not yield higher neopterin production in pbmnc of patients with tuberculosis showing that it is not the production capacity for neopterin which is different [ ] . elevated serum neopterin levels were also found in hiv infected patients with tuberculosis and decreased significantly on antituberculous treatment [ ] . a relapse of tuberculosis was in cases characterized by increase in neopterin levels. further studies compared neopterin levels in urine, serum, and bronchoalveolar lavage fluid and found that they correlate significantly in patients with tuberculosis [ , ] . the elevation of neopterin in patients with tuberculosis was more pronounced in urine than in serum or bronchoalveolar lavage [ ] . diseases. patients with pulmonary tuberculosis had significantly higher urinary neopterin levels compared to patients with lung cancer or pneumonia with more than twice the concentration reported in adults [ ] . pleural fluid neopterin levels were investigated for its ability to differentiate between tuberculous and malignant pleural effusion and were found to be significantly higher in patients with pleural tuberculosis but performance characteristics including receiver-operating characteristics curve analysis was inferior to adenosine deaminase [ ] . waiting for the results of susceptibility tests to select an effective antituberculosis drug regimen often causes a delay in effective treatment which can be disastrous, especially in children. because the x-ray changes tend to resolve very slowly and may get even worse after starting therapy because of a paradoxical reaction due to immune-reconstitution even in otherwise immune-competent patients, other than clinical status there is no reliable parameter to reflect success or failure of the drug regimen [ ] . urinary neopterin levels declined on twice weekly measurements in all monitored patients with pulmonary tuberculosis on treatment and fell to below tolerance limits within weeks of treatment in / patients [ ] . measurement of serum neopterin levels in patients on treatment for microbiologically confirmed pulmonary tuberculosis confirmed this observation and showed a significant decline of levels to near normal levels within months of treatment [ ] . in the context of emerging multiple drug resistance and difficulties in monitoring compliance and drug absorption neopterin needs to be explored as a tool for monitoring of success in treatment of mycobacterium tuberculosis infection. it may also help to distinguish active from latent disease. people with hiv-m. tuberculosis coinfection with active tuberculosis responded with a reduction of plasma neopterin to antituberculotic treatment but neopterin levels remained above the baseline levels of hiv negative tuberculosis patients and levels were higher in patients with lower cd count [ ] . the first study of neopterin levels in parasitic infections included measurements of urinary neopterin by hplc in patients with plasmodium falciparum and vivax infections including patients with low grade parasitemia [ ] . all patients had elevated urinary neopterin levels compared to uninfected controls to a level of to micromol neopterin/mol creatinine. levels in patients treated with quinine sulphate and levels in untreated patients were not significantly different. a subsequent detailed interventional study provided data on urinary neopterin levels in volunteers experimentally infected with plasmodium falciparum [ ] . serial monitoring revealed that urinary neopterin levels were not elevated until peripheral blood parasite densities had increased through to cycles of intraerythrocytic schizogony. a sharp rise in urinary neopterin was detectable at the beginning of day after infection. there was an increase one day after onset of fever. in one patient a urinary neopterin increase was noted without the occurrence of fever. neopterin production in falciparum malaria seems to be a direct effect of plasmodial antigens on monocytes/macrophages. in vitro studies showed that the monocytic cell line u could be stimulated to produce neopterin with lysates of plasmodium falciparum parasitized human erythrocytes and recombinant p. falciparum proteins [ ] . at a cut-off point of . ng/ml, neopterin had a positive and negative predictive value of . and . for detection of severe falciparum malaria [ ] . chloroquine treatment was followed by a reduction of urinary neopterin levels. when clinical disease resolved within - days of treatment, neopterin levels normalized rapidly [ ] . neopterin levels in nonimmune patients and young children were higher than were those of semiimmune individuals. csf (csf) neopterin levels were investigated for ability to discriminate between different stages of cerebral trypanosoma (t.) brucei (b.) gambiense infection. in an investigation of t. b. gambiense patients originating from angola, chad, and the democratic republic of the congo csf igm and neopterin were the best in discriminating between the two stages (s and s ) of disease with . % and . % specificity, respectively, at % sensitivity. when a validation cohort ( patients) was tested, neopterin ( . nmol/l) correctly classified % of s and s patients, confirming its high staging power [ ] . serum neopterin was also assessed as a disease marker in human schistosoma mansoni infection and levels were found to reflect the extent of hepatic involvement with higher levels found in patients with hepatomegaly. treatment with praziquantel led to a normalisation of serum neopterin levels as a result of a reduction of egg induced immunopathology [ , , ] . the detection of new blood borne viruses including hiv and non-a non-b hepatitis viruses led to investigations into new ways of excluding transmission of blood borne viruses by transfusion of blood products. the government of tirol in austria introduced routine measurement of neopterin levels in all donated blood in . a cut-off of nmol/l was used and led to the exclusion of . % of donors (total number of donors = ). the most common cause of elevated levels was in ( %) of cases a viral respiratory tract infection. donors with elevated neopterin had an acute toxoplasmosis and had hiv infection or non-a-non b-hepatitis [ ] . in another study . % of donations with increased neopterin levels were positive for cmv igm indicating acute infection. . % of patients with low neopterin levels had cmv igm. seroconversion was detected in patients with initially elevated neopterin levels on a second serum sample indicating that neopterin may precede the appearance of cmv antibodies by - weeks [ ] . a further study of austrian blood donors showed that neopterin levels were significantly higher in early compared to late infection or carrier state. all early infections (seroconversions) had elevated neopterin levels while only % of late and carrier states [ ] . a recent study found that using a neopterin elisa % of cmv dna-positive samples had elevated neopterin levels [ ] . with regard to other viruses . % of donors with above normal neopterin had epstein-barr virus igm generating an almost threefold greater chance of acute ebv infection in donors with increased neopterin (odds ratio: . ( % confidence interval, . - . ). with regard to parvovirus b infection / ( . %) donors were found to be seropositive for parvovirus b igm [ ] . a later study by the same group found no hpv dna positive results amongst patients with normal neopterin levels [ ] . an investigation of the association of neopterin levels with chronic hepatitis c virus infection revealed that significantly more patients with elevated neopterin levels and hcv antibodies were hcv pcr positive for hcv rna (odds ratio: . = . ) [ ] . neopterin is a nonspecific marker of activated cell mediated immunity involving release of interferon gamma. neopterin may be a useful marker for more accurate estimation of extent of disease and hence prognosis. knowledge of all potential causes of its elevation can overcome problems with reduced specificity in a patient known to have a specific infectious disease. longitudinal serial measurements in the same individual could overcome difficulties with interpretation in settings where chronic parasitic (malaria) or bacterial (tuberculosis) infections may elevate the baseline neopterin level and could allow monitoring of response to antiretroviral, antituberculous, and antiparasitic treatment in the absence of resistance testing and provide means to monitor compliance in the outpatient setting (see table ). this is particularly important in the current context of emerging multiple drug resistance of hiv and mycobacterium tuberculosis. neopterin for which high quality elisa systems to measure urine and blood levels are commercially available is an underused marker in clinical practice and is suitable for introduction into the routine clinical laboratory practice. the author declares that there is no conflict of interests regarding the publication of this paper. neopterin measurement in clinical diagnosis potential role of immune system activation-associated production of neopterin derivatives in humans cytokine-stimulated gtp cyclohydrolase i expression in 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and parvovirus b in blood donations with above-normal neopterin concentration human parvovirus b detection in asymptomatic blood donors: association with increased neopterin concentrations association between chronic hepatitis c virus infection and increased neopterin concentrations in blood donations key: cord- -rp kmd authors: ayatollahi, parisa; tarazi, apameh; wennberg, richard title: possible autoimmune encephalitis with claustrum sign in case of acute sars-cov- infection date: - - journal: the canadian journal of neurological sciences. le journal canadien des sciences neurologiques doi: . /cjn. . sha: doc_id: cord_uid: rp kmd nan predominating over the frontocentrotemporal regions bilaterally. cerebrospinal fluid (csf) analysis showed normal opening pressure ( mm h o), increased white blood cells ( cells/ul, % lymphocytes), no red blood cells, normal protein ( mg/dl), glucose ( mg/dl), and ldh ( iu/l) levels. csf gram stain and culture negative; csf pcr for herpes simplex virus negative, csf pcr for sars-cov- negative. abdominal/pelvic ultrasonography and ct showed mild free fluid in the pelvic cavity and fluid in the endocervical cavity; lung spiral ct revealed no abnormalities. the patient was empirically treated with intravenous acyclovir on admission and administered intravenous valproate sodium. the next day, she developed myoclonic jerks involving primarily the right side of the face and right leg, occurring in brief runs lasting a few seconds; the jerks decreased and did not involve the leg after increases in valproate sodium dosage. on the third day of admission, new behavioral changes appeared including elated mood, inappropriate laughing, anxiety, and insomnia, leading to treatment with clonazepam, risperidone, and sertraline, in addition to sodium divalproex. she began to complain of chest pain and dyspnea; electrocardiography (ecg) showed inverted t-waves in leads v - with normal echocardiography and negative troponin. by the fourth day of admission, she was experiencing severe generalized pruritis, for which hydroxyzine was started. she thereafter stabilized, albeit with persistent memory deficits and occasional facial jerks, and was discharged from hospital after days. six days later, however, she was re-admitted after experiencing two generalized tonic-clonic seizures. on this second admission, frequent episodes of right-sided or occasionally bilateral lower facial myoclonic or clonic jerks were noted, lasting from a few seconds up to a minute. eeg again showed moderate bilateral nonepileptiform abnormalities. levetiracetam was prescribed as additional antiseizure treatment. the patient continued to complain of pruritis, anxiety, insomnia, and chest pain. repeat ecg was unchanged and troponin negative; d-dimer level was elevated (> mcg/ml). repeat brain mri showed signal hyperintensities on fluidattenuated inversion recovery (flair) and t -weighted sequences in the claustrum bilaterally, which were not present on the initial scan weeks earlier. the hyperintensities extended slightly to the external/extreme capsules, with questionable involvement of some adjacent areas of anterior insular cortex; the mesial temporal structures were uninvolved (figure ). serological testing (tissue indirect immunofluorescence) was negative for the following neural antibodies (n-methyl-d-aspartate receptor, α-amino- -hydroxy- -methyl- -isoxazolepropionic acid receptors, γ-aminobutyric acid b / receptors, dipeptidyl peptidase-like protein , contactin-associated protein-like , and leucine-rich glioma inactivated ). onconeural antibodies were not screened and csf was not tested for neural antibodies. with a diagnostic presumption of "antibody-negative" autoimmune encephalitis, , intravenous methylprednisolone pulse therapy was administered ( g/day for days). on the third day of steroid treatment, the myoclonic jerks stopped, along with *received august , . final revisions submitted september , . date of acceptance september , improvement of the pruritis and insomnia. she was discharged on clonazepam, sodium divalproex, levetiracetam, and sertraline, with weekly outpatient pulse steroid treatments. follow-up mri month after the abnormal scan showed near-complete resolution of the claustrum hyperintensities ( figure ). seizures have not recurred ( -week follow-up); recent memory deficits persist. claustrum flair/t hyperintensities have been proposed as a marker for autoimmune encephalitis/epilepsy, often appearing after status epilepticus, , the encephalitis typically heralded by fever and unassociated with the currently screened autoimmune encephalitis antibodies. [ ] [ ] [ ] the mri abnormality ("the claustrum sign") may extend to external/extreme capsules and insular cortices and typically resolves in weeks or months. , concomitant mesial temporal hyperintensities are present in %- % of patients. , the claustrum is one of the most densely connected areas in the brain: the mri hyperintensities may represent a focused concentration of hyperactivity in epileptic networks connected to the claustrum, perhaps especially cortical and subcortical areas around the frontal-temporal-opercular region. autoimmune encephalitis has rarely been reported as a presentation of covid- , and the possibility of a coincidental association in this and other described cases cannot be excluded. [ ] [ ] [ ] [ ] the patient described here had only mild non-neurologic symptoms and signs attributable to covid- (fever, malaise, macular rash, urinary retention, and endocervical inflammation); the pruritis was presumed to be of central origin. csf pcr for sars-cov- has been nearly invariably negative not only in presumptive covid- encephalitis patients but also in patients with severe systemic covid- illness and neurological/ neuroimaging abnormalities, suggesting neurological abnormalities may not be due to glioneuronal infection with sars-cov- . , instead, negative csf pcr for sars-cov- may reflect that the underlying neuropathogenesis is autoimmune, which would align with steroid responsiveness. the finding of the claustrum sign on brain mri, not previously reported in a covid- patient, [ ] [ ] [ ] [ ] [ ] provides further support for the idea that acute sars-cov- infection may present as an autoimmune encephalitis. claustrum hyperintensities: a potential clue to autoimmune epilepsy a clinical approach to diagnosis of autoimmune encephalitis new-onset refractory status epilepticus with claustrum damage: definition of the clinical and neuroimaging features a first case of meningitis/ encephalitis associated with sars-coronavirus- a case of limbic encephalitis associated with asymptomatic covid- infection neurological manifestations in covid- infection: a systematic review and meta-analysis neurological and neuropsychiatric impacts of covid- pandemic neurologic and neuroimaging findings in covid- patients: a retrospective multicenter study follow-up brain mri month after scan shown in figure . (a) claustrum hyperintensities no longer evident on axial flair or t -weighted (far right panel) images. (b) small areas of residual hyperintensity at anterior extent of claustrum and external/extreme capsules (left panel, arrows) on coronal t -weighted images the authors have no conflicts of interest to declare. all authors contributed equally and approved the final version of this manuscript. key: cord- -nfu rdvh authors: muccioli, lorenzo; rondelli, francesca; ferri, lorenzo; rossini, giada; cortelli, pietro; guarino, maria title: subcortical myoclonus in covid‐ : comprehensive evaluation of a patient date: - - journal: mov disord clin pract doi: . /mdc . sha: doc_id: cord_uid: nfu rdvh nan myoclonus has been reported as a possible manifestation of coronavirus disease (covid- ), yet its neurophysiology and pathogenesis were poorly investigated. [ ] [ ] [ ] [ ] we describe a middle-aged man with covid- who underwent extensive examinations for his disabling myoclonus. a -year-old hypertensive man with a one-week history of fever and cough presented to the emergency department with dyspnea. a nasopharyngeal swab tested positive for sars-cov- . the patient was admitted to the icu after one week and placed on invasive mechanical ventilation due to respiratory distress. he was treated with hydroxychloroquine, tocilizumab and remdesivir. respiratory status quickly improved, thus he was extubated after five days and oxygen therapy was progressively weaned off. two days after icu discharge, he became markedly agitated. his mental status normalized in hours, however at this point he developed multifocal myoclonus elicited by action and tactile stimuli, predominant in the right proximal inferior limb muscles, preventing his ability to stand [video ]. cognitive deficits were not observed. electrolytes, renal and liver function tests were unremarkable. cerebrospinal fluid (csf) analysis, performed eight days after myoclonus onset, demonstrated leukocytes/μl, elevated protein levels ( mg/dl) and csf/serum albumin ratio ( . ), and negative sars-cov- rt-pcr. cytokine analyses revealed il- at . pg/ml in csf ( . pg/ml in serum, reference< . ) and il- at pg/ml in csf ( pg/ml in serum, reference< ). a serologic panel of autoantibodies against neuronal intracellular and cell surface antigens was negative. brain mri showed cerebral small vessel disease of moderate severity. eeg was unremarkable. polymyography confirmed the presence of multifocal positive myoclonus with a burst duration of - ms. back-averaging analysis did not show eeg time-locked discharges [ figure ]. the patient was treated with clonazepam and levetiracetam, resulting in marked amelioration of the myoclonus within five days. this case report confirms that myoclonus can occur in the context of diffuse inflammation related to covid- . in previously published reports, myoclonus has been described as spontaneous or action-induced, multifocal or generalized, with a non-specific distribution. [ ] [ ] [ ] [ ] this article is protected by copyright. all rights reserved. in our patient, the prominent involvement of axial and proximal limb muscles, myoclonus stimulussensitivity, the absence of cortical discharges at eeg jerk-locked back-averaging and the long duration myoclonic bursts, are consistent with subcortical myoclonus, possibly secondary to brainstem involvement. defining the underlying pathophysiology is challenging. myoclonus may present in the context of other viral infections, with concomitant encephalopathy/encephalitis or as an isolated postinfectious phenomenon. - sars-cov- may theoretically access subcortical structures involved in myoclonus generation via invasion of the olfactory bulb. in our patient, however, clinical course (including the absence of hyposmia), mri and csf findings argue against a direct pathogenic role of cns viral invasion. even though myoclonus appeared after a period of intubation, our patient did not suffer anoxic brain injury, thus excluding lance-adams syndrome. myoclonus onset timing and clinical course were also not consistent with an adverse drug reaction, a mechanism suggested in the form of serotonin syndrome in two patients treated with lopinavir/ritonavir. , agitation and myoclonus were preceded by severe cytokine release syndrome, a distinctive feature of covid- . csf analysis showed blood-brain barrier disruption, slightly elevated csf il- levels and elevated il- csf/blood ratio. these abnormalities may have been more pronounced if assessed at myoclonus onset. interestingly, cytokine-mediated neuroinflammation induced by sars-cov- has been implicated in steroid-responsive covid- -associated encephalopathy. in addition to marked agitation in our patient, previous reports also had clinical/instrumental findings suggestive of encephalopathy, including dysexecutive syndrome, delirium, somnolence, eeg slowing, elevated inflammatory markers, variable responses to immunotherapies and a benign clinical course, - further suggesting an immune-mediated/inflammatory pathogenesis. in conclusion, subcortical myoclonus should be considered among the neurological manifestations associated with covid- . the pathogenic role of cytokine-mediated neuroinflammation should be addressed in future studies. this article is protected by copyright. all rights reserved. generalized myoclonus in covid- serotonin syndrome in two covid- patients treated with lopinavir/ritonavir this article is protected by copyright. all rights reserved mixed central and peripheral nervous system disorders in severe sars-cov- infection delirium in covid- : a case series and exploration of potential mechanisms for central nervous system hyperkinetic movement disorders associated with hiv and other viral infections isolated" postinfectious myoclonus cytokine release syndrome in severe covid- steroid-responsive encephalitis in covid- disease we thank prof. paolo tinuper for helpful discussion on the case, the neurophysiology technologists soraia garrossi and rita signorelli for their quality work during this pandemic, and olivia j. henry for english language editing. the authors confirm that approval of an institutional review board was not required for this work.informed written consent for publication was obtained from the patient. we confirm that we have read the journal's position on issues involved in ethical publication and affirm that this work is consistent with those guidelines. key: cord- -hel h h authors: brown, julianne r.; bharucha, tehmina; breuer, judith title: encephalitis diagnosis using metagenomics: application of next generation sequencing for undiagnosed cases date: - - journal: j infect doi: . /j.jinf. . . sha: doc_id: cord_uid: hel h h background: current estimates suggest that even in the most resourced settings, the aetiology of encephalitis is identified in less than half of clinical cases. it is acknowledged that filling this gap needs a combination of rigorous sampling and improved diagnostic technologies. next generation sequencing (ngs) methods are powerful tools with the potential for comprehensive and unbiased detection of pathogens in clinical samples. we reviewed the use of this new technology for the diagnosis of suspected infectious encephalitis, and discuss the feasibility for introduction of ngs methods as a frontline diagnostic test. methods: a systematic literature review was performed, using mesh and text word searches for variants of “sequencing” and “encephalitis” in medline and embase, and searching bibliographies and citations using the web of science database. two authors independently reviewed, extracted and summarised data. findings: the review identified articles reporting case reports of patients with suspected encephalitis for whom ngs was used as a diagnostic tool. we present the data and highlight themes arising from these cases. there are no randomly controlled trials to assess the utility of ngs as a diagnostic tool. interpretation: there is increasing evidence of a role for ngs in the work-up of undiagnosed encephalitis. lower costs and increasing accessibility of these technologies will facilitate larger studies of these patients. we recommend ngs should be considered as a front-line diagnostic test in chronic and recurring presentations and, given current sample-to-result turn-around times, as second-line in acute cases of encephalitis. encephalitis is defined as inflammation of brain parenchyma associated with neurological dysfunction. , it is strictly a pathological diagnosis. recent epidemiological studies suggest that the global burden of encephalitis has been grossly underestimated, with current incidence suggested to be over annual cases in the uk, and , worldwide. , the syndrome encapsulates a myriad of diverse diseases, with distinct global distributions, presenting features and clinical courses. , infections represent the most frequently identified aetiology, with data suggesting this accounts for - % of cases. , , hundreds of pathogens have been associated with encephalitis, with the most frequently identified including herpes simplex virus (hsv), varicella zoster virus (vzv), enteroviruses, measles morbillivirus, mumps virus, japanese encephalitis virus (jev), influenza viruses, adenoviruses and mycoplasma pneumoniae. hsv, jev and rabies are the chief causes in europe, asia and africa respectively. the main alternative aetiology to infection is immune mediated, for which management includes immune suppression. it is critical to differentiate between autoimmune and infectious causes of encephalitis; immune suppression in cases where the cause is an undiscovered pathogen could be devastating. strikingly, more than one third of cases of encephalitis remain unidentified, even in the best-equipped medical centres. , , there are well-recognised challenges and inadequacies in current diagnostics and treatment, and these correspond with the poor reported outcomes. [ ] [ ] [ ] [ ] overall mortality is estimated at %, but is highly variable and dependent on the aetiology and access to supportive care. a high proportion is left with complex disability. the introduction of management guidelines for cases of acute encephalitis syndrome over the last decade has aimed to improve outcomes. , [ ] [ ] [ ] [ ] [ ] [ ] [ ] notably, there are many differences between guidelines in the approach to diagnostic evaluation, such as standard and extended diagnostic testing, or whether to administer empirical aciclovir. these variations may reflect geographically contrasting aetiologies, timing of publication and the rapid acceleration of technologies, as well as available resources. further, there is a lack of a systematic approach to access to pathogen discovery methods, discussed below. it is only the newest french guidelines in which ngs is mentioned at all, and the authors reasonably state that the clinical role is still to be evaluated. current diagnostic techniques for suspected infectious cases rely on prior knowledge of the likely causative agent. informed by clinical presentation, epidemiological data, guidelines and local resources, a laboratory will perform targeted tests for a disease. these are largely confined to specific polymerase chain reaction (pcr) or serological assays. this approach has fundamental limitations, and contributes to the relatively high proportion of encephalitis cases that remain undiagnosed. aside from the difficulties of testing for the myriad of rare pathogens that might be expected to cause encephalitis, this approach does not permit the identification of new or unexpected pathogens. undeniably, methods for novel pathogen discovery such as electron microscopy and cell culture have existed for many years, however they are cumbersome, time-consuming, lack sensitivity and specificity and are often no longer routinely available. furthermore, there are groups of patients, such as immunosuppressed patients, who frequently present with subtle or non-specific symptoms and signs, are high-risk for encephalitis, infection with unexpected or unusual pathogens, and are seen to have more severe outcomes. there is a need for improved diagnostic methods for encephalitis. a method which has recently been applied to pathogen detection in cases of encephalitis is metagenomic analysis using next generation sequencing (ngs). proof of concept for its use in the diagnosis of encephalitis has been demonstrated in the literature, however its suitability for routine diagnosis has not been assessed and is the subject of this review. ngs, also known as deep sequencing, generates a single sequence from each fragment of dna, or cdna, present in a specimen. downstream analysis allows differentiation between the origin of sequence fragments, for instance human, a specific bacterial species or a particular virus. this means mixed specimens, that contain host and microbial sequences, can be resolved (fig. ). sequencing the total dna or rna (known as metagenomics) from a biopsy or body fluid allows the identification of genetic material from any microorganism present in the specimen, and thus potentially causing encephalitis. this approach overcomes the limitations of targeted diagnostic methods such as pcr as it requires no prior knowledge or assumptions about the type of pathogen causing infection therefore enabling detection of novel and unexpected pathogens. the majority of readily available sequencing methods to date are dna based, however sequencing only total dna would exclude detection of viruses with rna genomes. consequently, an alternative approach is to synthesise metagenomics for diagnosis of encephalitis complementary dna (cdna) from total rna, which will enable detection of viruses with rna genomes but also the rna transcripts of organisms with dna genomes. once sequences are generated, complex downstream bioinformatic analysis is required to identify the presence of any pathogen sequences. in brief, any reads mapping to the human genome are removed, after which all remaining nonhuman sequences are compared to a database of known sequences to identify the provenance of the unknown sequences (fig. ) . the possibility of incorporating unbiased pathogen discovery technology into routine diagnostics for encephalitis would represent a paradigm shift in diagnostic algorithms. we and others are validating the use of metagenomics for clinical use and prospective studies are already underway to examine whether application of ngs at the outset of management pathways improves patient outcome and costs, namely the precision diagnosis of acute infectious disease (pdaid) study. , nonetheless, there is a paucity of evidence in this field which is largely limited to case reports. we aim to perform a rigorous summary and critical review of existing evidence, to assess the utility of ngs in diagnosis of encephalitis. only articles reporting application of ngs in csf or brain biopsies in suspected encephalitis and published in english between january and april were included. the web of science database was also used to search bibliographies and citations of relevant article. two authors independently reviewed, extracted and summarised included literature. data was extracted in the first instance by the first reviewer into a custom data extraction excel sheet with pre-defined data headings (supplementary file ), designed for the purpose of this review; additional miscellaneous data or observations were also noted where relevant. the extracted data and each included study were independently reviewed by the second author, with a focus on technical and scientific aspects of each study. consensus extracted data was used in analyses; both review authors were in full agreement on the extracted consensus data. relevant manuscripts that were not identified through the initial search, but were identified in the reference list of included literature, were also included. the funding bodies had no role in the decision to write, the analysis, manuscript preparation or the decision to submit for publication. twenty-five articles were identified from the search (fig. ) . all the included articles were case reports, or case series of - patients. altogether cases were reported in which ngs provided a diagnosis in otherwise undiagnosed cases of encephalitis (table ). an exponential temporal increase in cases has been observed over the last decade (fig. ) . country of origin of cases included australia, china, france, germany, india, ireland, japan, poland, sri lanka, uk, usa, and vietnam. samples were analysed in laboratories largely in the usa and europe (uk, france, germany, poland), but also in china, japan and vietnam. among the cases for which age was documented, the median age was years (interquartile range - ). of the cases that reported immune status of the patient, % ( / ) were immunocompromised. there was uniformly poor reporting of encephalitis or meningoencephalitis case definitions, and limited explanation of diagnostic assays performed and algorithms used for testing. none of the studies reported adherence to published or unpublished clinical guidelines. in of the known cases, well-established causes of encephalitis were detected which could have been identified by rapid and specific primary screening methods such as pcr. these organisms included hsv, coxsackievirus a , measles virus, vzv, mumps virus, epstein-barr virus, jc virus and mycobacterium tuberculosis. however, in the remaining cases novel ( / ), rare ( / ) or unexpected ( / ) organisms were detected which could not (in the case of novel organisms) or are unlikely (in the cases of rare and unexpected pathogens) to have been detected using specific pcr assays. the five unexpected cases were known human pathogens but novel causes of encephalitis. although diagnostic pcr assays may exist for some of these viruses, they are unlikely to have been considered in the differential diagnosis and therefore would not be routinely tested. this included two cases of human parvovirus (parv ), first described in when it was associated with a viraemic patient in whom an acute viral infection was suspected ; one case of human coronavirus oc- , typically a human respiratory pathogen never previously described in a human case of encephalitis but known to cause encephalitis in mice ; one case of human astrovirus mlb , of mumps vaccine virus in a child who was vaccinated prior to a primary immunodeficiency diagnosis. the five cases in which rare causes of encephalitis were identified were brucella melitensis, candida tropicalis, leptospira santarosai and two cases of balamuthia mandrillaris. eighteen cases were considered to be novel pathogens. three ( / ) of the identified organisms were arenaviruses, three cases were a variegated squirrel bornavirus, four were a novel astrovirus (astrovirus va /hmo-c), three were cycloviruses, three were gemycircuarlviruses and one was a densovirus. the three arenavirus cases occurred in three solid organ transplant recipients who all received organs from the same donor, who was later shown to be antiarenavirus igm and igg seropositive. the three cases of variegated squirrel bornavirus occurred in three breeders of variegated squirrel and was retrospectively detected in one of the breeder's squirrels. the novel astrovirus va /hmo-c was initially detected in an adolescent with primary immunodeficiency; it has since been shown, through four further case reports identified by ngs, , , , as an emerging under recognised cause of encephalitis in immunosuppressed patients that should be included in the differential diagnosis of encephalitis in this patient group. the clinical significance of the densovirus, cyclovirus and gemycircularviruses is doubtful, and discussed in detail further on in this review. an additional advantage of using ngs for the diagnosis of encephalitis is that, aside from pathogen identification, in instances where virus titre and read depth is high enough it is possible to generate partial or full genome sequences for the pathogen. pathogen sequences can be used for phylogenetic analysis to elucidate the strain , or possible source of the organism, as was the case for morfopoulou et al., who demonstrated . % homology between the mumps virus found in the brain of an encephalitic child with primary immunodeficiency and the vaccine batch used to immunise the child. ngs is a powerful tool for pathogen detection, allowing us to detect organisms that may not previously have been described or associated with the disease in question. however, as with all molecular tools, detection of a microorganism does not prove causality. to provide further evidence for an aetiological role in encephalitis of the identified pathogen, a challenge reviewed in detail elsewhere, some reports make use of additional clinical and laboratory indicators to exclude the possibility that the detected organism is an incidental finding. seroconversion to the pathogen in question is highly suggestive of etiological significance of an organism however preinfection or follow-up serum samples are rarely available; of the cases identified in this review seroconversion was demonstrated in only two. , a further eight were able to demonstrate the presence of specific antibodies, but without knowledge of the sero-status prior to onset of symptoms. although detection of pathogen-specific intrathecal antibodies is also highly suggestive of a causal relationship and recommended by uk guidelines, none of the cases identified in this review reported intrathecal antibody testing. in cases where encephalitis is caused by reactivation of a dormant pathogen, rather than primary infection, or where the pathogen does not cause a strong systemic antibody response, serology may not be useful. in the case of a novel or emerging cause of encephalitis for which the clinical significance may be unclear, proving causality is particularly important. in this instance organismspecific immunostaining or in situ hybridisation in affected tissues will provide additional evidence of the cellular distribution of infection and exclude the possibility of reagent or tissue contamination; of the reviewed cases report confirmatory immunostaining or in situ hybridisation. in this context brain biopsies are a more useful specimen than cerebrospinal fluid (csf) since it allows immunostaining of the affected tissue. moreover, in encephalitis caused by mutated pathogens such as in subacute sclerosing panencephalitis (sspe) caused by chronic measles infection or cases of mumps vaccine encephalitis the pathogen may not be detected in csf but only in brain parenchyma. an alternative molecular method, such as pcr, can be used to confirm the presence of the detected organism and exclude the possibility that the identified organism is an artefact of the bioinformatics analysis. in this review / cases confirmed the presence of the organisms by pcr. pcr supports the identity of the organism sequenced by ngs, however does not contribute to proving causality. six cases identified novel small circular ssdna viruses in the csf of patients with encephalitis of unknown aetiology; three cycloviruses , and three gemycircularviruses. however, the clinical significance of these is doubtful. phan et al. confirmed, via repeat dna extraction using an alternative method, that the source of the cyclovirus is not reagent contamination. nevertheless, whilst screening csf samples for cyclovirus by pcr, tan et al. detected cyclovirus in the csf of patients in whom the aetiology of their central nervous system (cns) disease had already been confirmed as japanese encephalitis, dengue virus or bacterial meningitis. the only identified cellular host for gemycircularviruses is fungi. in the absence of other evidence of pathogenicity, such as seroconversion or demonstration of the pathogen within cells in the brain, the probability remains that detection of cyclovirus or gemycircularvirus in csf is an incidental finding. similarly the detection of densovirus, a small linear ssdna virus, in csf may be incidental since it was detected in a case with confirmed n-methyl d-aspartate (nmda)-receptor encephalitis. autoimmune antibodies have previously been co-detected with herpesvirus dna in csf from cases of encephalitis, however the host range of densoviruses is to date exclusively invertebrates; the authors suggest a possible explanation for detection in csf is the passive transfer of virus from an insect bite or csf contamination from skin flora or an environmental source. in these cases, further evidence is required before assigning a pathogenic role. as with other molecular tests, including pcr which has become the gold standard of virological diagnostics, results from metagenomics applied to cases of encephalitis should be interpreted in the context of other clinical and laboratory findings, particularly when a novel or unexpected organism is detected. the majority of reports concerning the use of metagenomics for diagnosis of encephalitis are comprised of single case reports, therefore it is difficult to assess the diagnostic yield (number of positive results/number of cases tested), and thus utility, of metagenomics for encephalitis. five reports included testing multiple cases of encephalitis; in these the diagnostic yield was % ( / ), . % ( / ), % ( / ), % ( / ) and % ( / ). the first three studies with low diagnostic yield of - % tested only csf supernatant which is cell-free, therefore only cell free viruses or cell-free microbial nucleic acid can be detected; moreover csf often contains a lower pathogen load then brain biopsies and so pathogen detection is more challenging. the aforementioned studies also included only samples for which primary routine diagnostic testing using standard methods was negative therefore the utility of ngs as a first-line test cannot be assessed. a higher diagnostic yield was reported where specimens were tested using metagenomics as a firstline screening tool; % using whole csf and % using brain biopsies. the use of brain tissue rather than csf may increase diagnostic yield. in three cases a pathogen was detected in brain biopsy but not in csf , , while the opposite was not observed, although in one instance the proportion of pathogen reads was greater in csf than brain biopsy. in our hands, the diagnostic yield for metagenomics in encephalitis is, to date, % ( / ) all of which were brain biopsies. the eight identified pathogens were coronavirus oc- , two cases of vaccine derived mumps virus, , toxoplasma gondii (unpublished) and four cases of astrovirus va /hmo-c , (two cases unpublished). all of our positive results were in immunocompromised patients and in the majority of cases there was a high index of suspicion of infection, suggesting metagenomics may be best applied to a targeted population in whom it will be most rewarding. quality assurance prior to providing a new diagnostic test, extensive validation must be undertaken to ensure the service is fit for purpose, such as determining the specificity and sensitivity of an assay, the purpose of which is to ensure a robust, accurate and reproducible result all of which is part of quality assurance. the regulatory requirements that should be fulfilled for the validation of metagenomics for pathogen detection is discussed in detail elsewhere, however one aspect that must continue beyond the validation stage is the use of positive and negative controls. in the context of metagenomics for pathogen detection a positive control is a specimen (real or constructed) that is known to be positive for one or multiple organisms; a negative control is one that is known to be negative for any pathogens. these should be included in every sequencing run; if the positive control fails (i.e. the known pathogen/s is not detected) this invalidates the results of all clinical specimens processed in parallel for which no pathogen was identified. conversely if the negative control fails (i.e. an unexpected organism is identified in the sequence data) this could indicate reagent contamination or a problem with the analysis pipeline and thus positive results from samples processed in parallel are invalidated and should be repeated. this having been said, of cases of encephalitis identified in this review (table ) , only included positive controls and included negative controls. the accuracy of a positive result is critical for patient management; however a negative result can also be useful to exclude infection, particularly where anti-inflammatory and immunosuppressive treatments are being considered. consequently prior to provision of a clinical service appropriate controls must be in place to ensure results are reliable and therefore clinically actionable. the use of controls is aptly demonstrated by mongkolrattanothai et al., who not only included positive and negative controls but also implemented defined criteria in their analysis pipeline that dictates any viruses detected in a clinical specimen should not be detected in the negative controls and, moreover, bacteria detected in a clinical specimen should only be reported as a significant finding if detected with a reads per million (rpm) ratio ≥ (rpm ratio = rpm sample / rpm negative control). this approach overcomes the common problem of reagent contamination with microbial nucleic acids; of cases in which the presence or absence of contaminating reads was reported, cases reported the presence of environmental bacteria, plant viruses, bacteriophages and/or avian retroviruses. , , , , , , [ ] [ ] [ ] [ ] , , , turn-around times only cases reported the time from specimen collection to pathogen identification; for these the turn-around time was - days, with a median time of days. due to limitations of currently available sequence library preparation methods and sequencing chemistries a sample-to-answer turn-around time as short as hours is only achievable with a fast downstream analysis pipeline; in the reported case analysis took only minutes compared to up to two days in other, computationally intensive, pipelines. nevertheless it serves as proof of principle that relatively short turn-around times are achievable, even though up to days is more common. the sample-to-answer turn-around time of specific realtime pcr, which is the current gold standard for diagnosis of viral infections, is often less than hours in a clinical laboratory and potentially less than hours without batch processing. consequently ngs cannot yet offer the same speed of result as pcr, which could delay the diagnosis in instances of encephalitis caused by well-known pathogens that are detectable by pcr. nonetheless with rapidly increasing library preparation and sequencing speeds, turn-around times are likely to significantly improve in coming years. metagenomics for pan-pathogen detection has the potential to revolutionise the diagnosis of encephalitis and other difficultto-diagnose infections; however, there are some limitations of the technique that should be considered. the sensitivity and limit of detection (lod) of metagenomics can be determined for model organisms that represent major pathogen groups, such as dna and rna viruses, gram positive and negative bacteria, fungi and parasites; however, the broad-range nature of the technique makes it impossible to determine the sensitivity or lod for every possible organism. this is also a recognised problem with pan-bacterial pcr detection which is used clinically, however can be confounded in metagenomics by differences between specimens and specimen types (for instance tissue biopsies versus csf) in the quantity of genomic material, the ratio of host:pathogen sequences and, depending on the sequencing chemistry and degree of specimen multiplexing, the sequencing yield. some of these limitations may be overcome by the careful use of processing and sequencing controls, as discussed elsewhere. whilst metagenomics will produce a sequence for every fragment of dna or rna in a specimen, only pathogens with homology to known organisms in the sequence database of choice will be identified. if an organism is missing from the database, or if the pathogen causing infection is novel with no homology to known organisms, it will not be identified. consequently, a negative result obtained by metagenomics, whilst reducing the likelihood of an infectious cause, cannot unequivocally exclude infection. sequencing total dna or rna will inevitably include sequencing host dna or rna transcripts which can result in > % of the sequence data generated mapping to the human genome. the consequence of this is wasted cost, as the sequencing reaction is dominated by host rather than pathogen sequences, and also has implications for the turn-around times and sensitivity of pathogen detection. in order to detect pathogen sequences, which can be as few as nine in million reads, vast sequencing read depths are required; very high throughput sequencing platforms with only a very limited number of samples sequenced in parallel are required to achieve this. to overcome this, depletion of host dna or rna prior to sequencing is required; however the options for this are currently limited and are not all suitable for detection of viral pathogens. improved methods for host dna and rna depletion would considerably reduce the cost and time to result and improve the sensitivity of metagenomics for diagnosis of encephalitis. finally it is important to remember that in some cases of encephalitis, the pathology is due to the immune response and the pathogen may be rarely detected if at all. for example in japanese encephalitis virus (jev) infection, the commonest cause of encephalitis in asia, the most sensitive rt-qpcr detects rna in less than % of cases, and the mainstay of diagnosis is serology. in these cases, ngs is unlikely to significantly improve the diagnostic yield. this systematic literature review and case series suggests there is preliminary evidence to support a role for ngs in the management of undiagnosed encephalitis. undeniably, the research is limited to case reports, with poor reporting of clinical case definitions of encephalitis, baseline tests performed, or adherence to clinical guidelines. nonetheless, current epidemiological data suggests that the cause of encephalitis remains unknown in - % of cases , ; and ngs has striking potential to identify undiagnosed pathogens and thus reduce the number of cases with unknown aetiology. ngs also has utility for pathogen detection in other clinical syndromes, such as respiratory infections, therefore the implementation of this technique in clinical laboratories would have wider implications for diagnosis of infection beyond encephalitis. notably, current turn-around-times prevent the replacement of routine methods, such as pcr, for the diagnosis of acute encephalitis. for these reasons, the role of ngs in clinical algorithms is still to be delineated. at this point in time, we suggest ngs is routinely applied for the diagnosis of acute cases of encephalitis for which no cause is found after targeted investigations using pcr. recommendations for firstline targeted testing are discussed in detail elsewhere but in the uk should include pcr for hsv, vzv and enteroviruses. however due to differing local epidemiology clinicians should consult the relevant national guidelines. , , , , in immunocompromised patients however, metagenomics ought to be considered earlier; % of case reports in this review involved immunocompromised patients. this is the population at most risk of infection with novel and unexpected organisms and, moreover, may present with a more chronic or insidious clinical history in which a one-week turn-aroundtime is more acceptable. given that the causative pathogen is not always detected in csf, in all cases of encephalitis in which diagnosis by ngs is being sought the preferred specimen type is brain biopsy. nevertheless csf samples are acceptable if it is the only specimen available. our recommendations for the use of ngs in diagnosis of microbial causes of encephalitis are summarised in fig. a , with the contrasting algorithm for targeted testing summarised in fig. b . this review was limited to pathogen detection by ngs in brain biopsies or csf. there may also be a role for testing other specimens, such as throat samples and urine. this was recently shown in an encephalitis case diagnosed by ngs of urine, identifying a case of japanese encephalitis virus. it is expected that over the next few years, the cost and time-to-result of metagenomics will reduce, and with this, it is foreseen that it will be possible to offer this as the first-line diagnostic test. this depends on the ability to deplete host dna and rna prior to sequencing, reduced read depth requirement and faster sequencing and bioinformatics technologies. the role of autoimmune encephalitis, in some instances triggered by an infection, is beyond the scope of this review. however, since up to % of cases of encephalitis are caused by autoimmune disorders, in which the immune system attacks specific host proteins, , a comprehensive diagnostic service should include antibody mediated, as well as infectious, causes of encephalitis. the clinical presentation of autoimmune (non-infectious) and infectious encephalitis are similar, however the treatment is often opposing. non-infectious causes may require immunosuppressive therapy ; however administering immunosuppressive therapy where the cause is infectious exacerbates the infection, with potentially fatal results. a conclusive diagnosis of the causative agent of encephalitis would improve differentiation between infectious and auto-immune causes thus appropriate management of immunosuppression. in addition to the direct impact on individual patients, improving the diagnosis of encephalitis will increase our understanding of the causes of encephalitis generally. this knowledge is critical for future development of fast point-of-care tests and to develop clinical algorithms that minimise the time to diagnosis and treatment, thus maximising the chances of recovery. tb and jrbrown conducted the systematic review and prepared the manuscript, figures and table. jbreuer contributed to manuscript preparation. case definitions, diagnostic algorithms, and priorities in encephalitis: consensus statement of the international encephalitis consortium causality in acute encephalitis: defining aetiologies the epidemiology of acute encephalitis challenge of the unknown: a systematic review of acute encephalitis in non-outbreak situations approach to the patient with central nervous system infection beyond viruses: clinical profiles and etiologies associated with encephalitis diagnostic 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pediatric autoimmune encephalitis the spectrum of acute encephalitis: causes, management, and predictors of outcome encephalitis, myelitis, and acute disseminated encephalomyelitis (adem): case definitions and guidelines for collection, analysis, and presentation of immunization safety data a clinical approach to diagnosis of autoimmune encephalitis jr brown is supported by a paediatric research grant from the great ormond street hospital children's charity ("diagnosis of encephalitis by deep sequencing", v ). jbreuer receives funding from the ucl/uclh nihr biomedical research centre.all research at great ormond street hospital nhs foundation trust and ucl great ormond street institute of child health is made possible by the nihr great ormond street hospital biomedical research centre. the views expressed are those of the authors and not necessarily those of the nhs, the nihr or the department of health.the corresponding author (jbrown) had access to all the data and had final responsibility for the decision to submit for publication. the authors declare no conflicts of interest. supplementary data related to this article can be found at https://doi.org/ . /j.jinf. . . . key: cord- -blc mnbj authors: bernard-valnet, r.; perriot, s.; canales, m.; pizzarotti, b.; caranzano, l.; castro-jimenez, m.; epiney, j.-b.; vijiala, s.; salvioni chiabotti, p.; anichini, a.; salerno, a.; jaton, k.; vaucher, j.; perreau, m.; greub, g.; pantaleo, g.; du pasquier, r. title: csf of sars-cov- patients with neurological syndromes reveals hints to understand pathophysiology date: - - journal: nan doi: . / . . . sha: doc_id: cord_uid: blc mnbj objective: coronavirus disease (covid- ) has been associated with a large variety of neurological disorders. however the mechanisms underlying these neurological complications remain elusive. in this study we aimed at determining whether neurological symptoms were caused by sars-cov- direct infection of by pro-inflammatory mediators. methods: we checked for sars-cov- rna by rt-qpcr, sars-cov- -specific antibodies and for cytokines/chemokines/growth factors (by luminex) in the cerebrospinal fluids (csf) +/- sera of a cohort of covid- patients with neurological presentation and neurological control patients (inflammatory [ind], non inflammatory [nind], multiple sclerosis [ms]). results: we found sars-cov- rna and antibodies specific for this virus in the csf of / and / covid- patients, respectively. the presence of sars-cov- antibodies was explained by a rupture of the blood brain barrier (passive transfer) in / ( %). an intrathecal synthesis of sars-cov -specific antibodies was present in / patients. of the four categories of tested patients, the csf of ind exhibited the highest level of chemokines (ccl , ccl , cxcl , cxcl , cxcl , and cxcl ), followed by the csf of ms patients (cxcl , and cxcl ). there was no significant difference between covid- and nind patients, even if some chemokines (ccl , ccl , cxcl , andcxcl ) tended to be higher in the former. interestingly, among covd- patients, the csf of those with a severe disease (encephalitis/encephalopathy) contained higher levels cxcl and cxcl than those with other neurological presentations. interpretation: our results do not show obvious sars-cov- infection of the central nervous system, but point to a mild inflammatory reaction reflecting an astrocytic reaction. methods: we checked for sars-cov- mrna by qpcr, sars-cov- -specific antibodies and for cytokines/chemokines/growth factors (by luminex) in the cerebrospinal fluid (csf) +/- serum of a cohort of covid- patients with neurological presentation and neurological controls (inflammatory, non inflammatory, multiple sclerosis). results: we found sars-cov- mrna and antibodies specific for this virus in the csf of / and / covid- patients, respectively. the presence of sars-cov- antibodies was explained by a rupture of the blood brain barrier (passive transfer) in / ( , %), but an intrathecal synthesis of sars-cov -specific antibodies was present in / .as compared to sars-cov- -negative nind patients, the csf of ind patients exhibited the highest level of chemokines (ccl , ccl , cxcl , cxcl , cxcl , and cxcl ), followed the csf of ms patients (cxcl , and cxcl ). there was no difference between covid- patients with neurological diseases compared to nind even if some chemokines (ccl , ccl , cxcl , andcxcl ) tended to be higher than nind. interestingly, among covd- patients, the csf of those with a severe disease (encephalitis/encephalopathy) contained higher levels cxcl and cxcl than those with other neurological presentations. interpretation: our results confirm the absence of obvious sars-cov- infection of the central nervous system and point to a mild inflammatory reaction reflecting an astrocytic reaction. corona viruses' outbreaks have been repeatedly associated with neurological disorders. indeed, human tropic coronaviruses seem able to reach the central nervous system and are found in brain necropsies and in cerebrospinal fluid (csf) of severe acute respiratory syndrome coronavirus (sars-cov) patients , . in mouse model, it has been shown that a strain of human tropic coronavirus is able to reach the olfactory bulb trough the cribriform plate and then to spread through a neuron-to-neuron transmission . in line with this neurological tropism, coronavirus disease (covid- ) has shown a large range of neurological complications that may be classified as followed: critical care-related neurological syndromes either central (sub-cortical deficit characterized by attention and executive dysfunction) or peripheral (critical care-associated polyneuropathies or myopathies ) ; anosmia/dysgueusia ; myelo-meningo-encephalitis , ; guillain-barré syndrome (gbs), and its variant affecting cranial nerves (miller-fischer syndrome) ; and cerebrovascular disease (strokes) . if data from previous outbreaks point to a neurotropism of the coronaviruses, the pathophysiology underlying sars-cov- -related neurological deficits remains elusive. indeed, to date, sars-cov- has only been rarely found in the csf, suggesting that direct brain infection is not obvious , . thus, the main hypotheses to explain neurological complications in covid patients point at mechanisms either related to low grade presence of the virus in the cns, to cytokine storm or to the presence of an auto-immune response, such as anti-neuronal antibodies by analogy to what occurs in autoimmune encephalitis. however, data firmly establishing one or the other hypothesis are still missing. yet, the fact that encephalitis/encephalopathies caused by sars-cov- may respond to corticosteroids , suggest involvement of immune mechanisms. in an attempt to decipher mechanisms underlying neurological symptoms, we looked at sars-cov- -encoding rna, sars-cov- -specific antibodies and at a panel of cytokines/chemokines/growth factors in the csf of study patients. seventeen of them were infected with sars-cov- , and were control sars-cov- -negative patients suffering from inflammatory, including ms or non-inflammatory neurological disorders. we found that sars-cov- patients tend to have signs of blood brain barrier opening and possible astrocytes activation, but no strong immune response in the csf or obvious cns infection by the virus. study population. all consecutive patients seen at lausanne university hospital (chuv) during the first wave of covid- (march to end of may ) with any neurological all rights reserved. no reuse allowed without permission. perpetuity. preprint (which was not certified by peer review) is the author/funder, who has granted medrxiv a license to display the preprint in the copyright holder for this this version posted november , . ; manifestations and for whom a lumbar puncture including sars-cov- pcr was performed, were included in this study. the control cohort consisted of patients who were diagnosed with inflammatory neurological disorder (ind), non-inflammatory neurological disorder (nind) or multiple sclerosis (ms) prior to the covid- wave, thus who were sars-cov- -negative by definition, and had been enrolled in the coolin'brain cohort between and . samples (serum and csf) for ms were all harvested during a clinical relapse of the disease before any treatment with corticosteroids. ethics. this study was approved by canton de vaud ethical committee (cer-vd) in the frame of coro-neuro study (authorization n° - ) and coolin-brain study (authorization n° - ). all patients included in this study signed specific informed consent. sars-cov- pcr. sars-cov- tests in csf specimens were performed using our automated platform with an in-house rt-qpcr targeting the e-gene with the primers and probe described by corman and colleagues . sars-cov- tests in respiratory specimens were performed either using our automated platform (at the beginning of the pandemic) or using the cobas sars-cov- test on the cobas instrument (roche, basel, switzerland), since th march . both methods were compared and exhibited . % of concordant results . anti-sars-cov igg specific to the native trimeric spike (s) protein were quantified using a multiplex bead assay as previously described graphical representation and statistical analysis. graphical representation and statistics were generated using prism software (version . . , graphpad software, la jolla, ca, usa). all rights reserved. no reuse allowed without permission. perpetuity. preprint (which was not certified by peer review) is the author/funder, who has granted medrxiv a license to display the preprint in the copyright holder for this this version posted november , . ; https://doi.org/ . / . . . doi: medrxiv preprint multiple group analysis were made using kruskal-wallis test with dunn's multiple comparison test. for analysis with group, mann-whitney test was used to determine statistical significance. heatmaps and k clustering were made using clustvis web-based tool . from march to may , patients benefited from a research of sars-cov- by rt-qpcr and had a concomitant lumbar puncture. among them, the diagnosis of covid- was established in based either on positive nasopharyngeal swab rt-qpcr ( %) or serology ( %). clinical description of sars-cov- patients is summarized in table . these patients presented with various neurological presentation including encephalopathy ( ), encephalitis ( ), myelitis ( ), optic neuritis ( ), guillain-barré syndrome ( ), mononeuritis multiplex ( ) and headache ( ) ( table .) half of them were admitted to intensive care unit (icu) and ( %) required mechanical ventilation. no prominent mri abnormalities were found, especially neither leptomeningeal enhancement nor diffusion restriction, in contrast with a previous report . all electroencephalographic (eeg) recording showed some abnormalities, consisting mainly in encephalopathic slowing but also irritative activity in one patient with encephalitis. several patients also exhibited alterations in nerve conduction, including mononeuritis multiplex, polyradiculopathy and critical myopathy/polyneuropathy. (table . ). covid- patients showed abnormal lumbar puncture characterised mainly by elevation of protein level and elevated albumin index. furthermore, oligoclonal bands were found in a minority of patients and consisted mostly in identical bands in the serum and the csf (type ; table ). these features point toward an opening of the blood brain barrier. pleocytosis was encountered only in few patients with encephalitic presentation. as expected, the csf profile was characterized by high protein level and pleocytosis in most ind patients and oligoclonal bands restricted to the csf (type ) in all ms patients ( table ). all rights reserved. no reuse allowed without permission. perpetuity. preprint (which was not certified by peer review) is the author/funder, who has granted medrxiv a license to display the preprint in the copyright holder for this this version posted november , . ; https://doi.org/ . / . . . doi: medrxiv preprint all covid- patients enrolled in this study undergone a rt-qpcr for detection of sars-cov- rna in the csf. it came back negative in all, despite positivity for most ( %) in the nasal swab (fig. c ). most tested patients ( / ) had positive serology for sars-cov- in the blood with high titers (positivity threshold > ). two patients with ind had sars-cov- -specific antibodies above the positive threshold in the blood, however the overall titer was low (fig. c ). of note, both patients presented with pathologies regularly associated with cross-reactive antibodies: sarcoidosis and paraneoplastic syndrome. antibodies against sars-cov- were detected in the csf of half of covid patients. of interest, there was evidence of an intrathecal synthesis of sars-cov- -specific antibodies in / patients (reiber index > ; data not shown). conversely, sars-cov- antibodies were not detected in the csf of any control study patients, except for the patient suffering from neurosarcoidosis, where it was also positive in the serum (fig. c) . first, the analysis of the cytokine panel in the serum of the sars-cov- -infected patients for whom it was available showed an increased cytokine production compared to the serum of sars-cov- -negative patients with other neurological disorders, inflammatory (ind) or not (nind) (fig. d ). as previously demonstrated il- , cxcl- and il- -ra were significantly elevated in sars-cov- -infected patients (fig. e ). contrary to previous works, increase of cxcl and tnfa were not observed in the serum of our patients ( fig. e and data not shown) , . while the cytokine profile of sars-cov- -infected patients with neurological conditions was inflammatory for some in the serum (especially for icu patients), this was not the case in the csf. indeed, the analyses performed in the csf revealed that sars-cov- -infected patients were mainly clustered with nind and ms ones, while the csf of ind patients exhibited a stronger immune signature, characterized by elevated levels of several cytokines, chemokines and growth factors ( fig. a) . this unbiased clustering was confirmed by individual cytokine/chemokine analysis with significant elevation of il- , ccl , ccl , cxcl , cxcl , cxcl , cxcl , g-csf and vegf-a in the csf of ind patients (fig. b and data not shown). of note, we observed an increased level of several chemokines, including cxcl and cxcl , and to a lesser extent ccl and ccl , in sars-cov- -infected patients compared all rights reserved. no reuse allowed without permission. perpetuity. preprint (which was not certified by peer review) is the author/funder, who has granted medrxiv a license to display the preprint in the copyright holder for this this version posted november , . ; https://doi.org/ . / . . . doi: medrxiv preprint to nind controls, but which did not reach significance (fig. b) . interestingly, this increase was more pronounced in sars-cov- -infected patients with a more severe involvement of the cns (eg. encephalopathy, encephalitis, myelitis), as compared to the ones with milder covid- associated neurological disorders (headaches without meningitis) or with predominant peripheral nerve involvement (fig. c) . even if they did not display strong immune signature, ms patients showed higher levels of cxcl , cxcl and g-csf in the csf (fig. b and data not shown) . in this study, we attempted to understand the pathogenesis of neurological impairments in the context of covid- disease. first, we did not detect sars-cov- rna in the csf of any of our patients (fig .b ). only few authors reported that, in severe encephalitis with neuronal and astroglial destruction, sars-cov- rna could be found in the csf but this remains an exception . interestingly, in vitro experiments using human induced pluripotent stem cells (hipsc)-derived brain organoids demonstrated the capacity of sars-cov- to infect neurons and astrocytes but at a very low yield , . thus the lack of sars-cov- detection in the csf does not rule out the presence of viral rna in the brain parenchyma, but does suggest that, if present, its concentration is too low to be detected. second, we found that half of our sars-cov- -infected patients exhibited virus-specific antibodies in their csf (fig .c) . two of them showed evidence of an intrathecal synthesis of antibodies against sars-cov- . these findings suggest that a humoral immune response against the virus may take place in the cns, but this mechanism may concern only a subset of patients. indeed, the fact that we found an increased permeability of the blood-brain barrier (increased albumin in the csf, type oligoclonal bands) in almost all sars-cov- -infected patients suggests that a large proportion of the virus-specific antibodies found in the csf may come from the periphery. nevertheless, wherever these antibodies come from, they may be instrumental in the antiviral response in the brain. supporting these findings, authors recently showed that the csf from a covid- patient displayed neutralizing antibodies able to prevent neuronal infection in an hipsc-derived brain organoid model . finally, our results bring an unprecedented insight on the inflammatory mechanisms involved in neurological complications of covid- . indeed, we first compared to inflammatory and non inflammatory neurological controls and have been able to show that, contrary to patient with inflammatory disorders, sars-cov- -infected patients do not display specific immune signature in the csf. interestingly, the sera of these sars-cov- -infected patients exhibited all rights reserved. no reuse allowed without permission. perpetuity. preprint (which was not certified by peer review) is the author/funder, who has granted medrxiv a license to display the preprint in our observation of blood brain barrier alterations in covid patients also favours the hypothesis of astrocytes activation. indeed, astrogliosis can lead to the disruption of the blood brain barrier via reduction of astrocytic gap junctions, among other mechanisms . in addition, astrocytes dysfunction, even in absence of a strong inflammatory milieu, can affect directly neuronal functioning and integrity, such as suggested by high neurofilament levels in the serum of covid- patients . to conclude, our results suggest that moderately severe neurological complications in sars-cov- -infected patients are not due to a major viral infection of the brain nor to a massive inflammatory response in this organ. however, the absence of such a massive response does not mean that the brain of covid- patients with neurological features is unharmed by inflammation. indeed, it is possible that the csf only imperfectly reflects the inflammatory mechanisms taking place in brain tissue. supporting this hypothesis, we can refer to our cohort of patients with active ms, whom we know exhibit a solid inflammation in the brain that needs corticosteroids to be tamed, but in the csf of whom only a slight elevation of few chemokines can be detected. precisely, others have reported that corticosteroids improve the neurological symptoms in covid- patients with neurological complications , . all rights reserved. no reuse allowed without permission. perpetuity. preprint (which was not certified by peer review) is the author/funder, who has granted medrxiv a license to display the preprint in the copyright holder for this this version posted november , . ; ( ) ( ) ( ) ( ) ( ) ( ) preprint (which was not certified by peer review) is the author/funder, who has granted medrxiv a license to display the preprint in the copyright holder for this this version posted november , . ; all rights reserved. no reuse allowed without permission. perpetuity. preprint (which was not certified by peer review) is the author/funder, who has granted medrxiv a license to display the preprint in the copyright holder for this this version posted november , . ; https://doi.org/ . / . . . doi: medrxiv preprint expression is represented in log scale. k-means clustering was used to determine patients clusters (cluster , n= ; cluster , n= ; cluster , n= ). cytokines/chemokines with no variations across all patients are not displayed. period refer if patient were sampled during covid- pandemy or before it (prior) (b) bar plot representation (mean ± sem) with log scale of ccl , ccl , cxcl , cxcl , cxcl and cxcl expression in the csf of patient with sars-cov- infection (red circles, n= ), ind (green circles, n= ) or nind (blue circles, n= ). statistical significance comparing to nind group calculated using kruskal-wallis with correction for multiple comparisons (adjusted p:* ≤ . *** ≤ . , **** ≤ . ). (c) ccl , ccl , cxcl and cxcl expression (mean ± sem) in sars-cov- infected patient according to neurological presentation either headache/peripheral nerve/else (white bar, n= ) or encephalopathies/encephalitis/myelitis (grey bar, n= ). representation corrected by detection limit. statistical significance was calculated using mann-whitney test (p:* ≤ . ). all rights reserved. no reuse allowed without permission. perpetuity. preprint (which was not certified by peer review) is the author/funder, who has granted medrxiv a license to display the preprint in the copyright holder for this this version posted november , . ; https://doi.org/ . / . . . doi: medrxiv preprint multiple organ infection and the pathogenesis of sars detection of sars coronavirus rna in the cerebrospinal fluid of a patient with severe acute respiratory syndrome axonal transport enables neuron-to-neuron propagation of human coronavirus oc severe neurologic syndrome associated with middle east respiratory syndrome 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disease we are grateful to mrs géraldine le goff for her help in collecting study subjects samples. this work was made possible by grants to rdp from the swiss national foundation - and from the swiss multiple sclerosis foundation. all rights reserved. no reuse allowed without permission. key: cord- -n zrfsp authors: carnevale, silvia; ghasemi, somayehsadat; rigatelli, anna; jaillon, sebastien title: the complexity of neutrophils in health and disease: focus on cancer date: - - journal: semin immunol doi: . /j.smim. . sha: doc_id: cord_uid: n zrfsp neutrophils are essential soldiers of the immune response and their role have long been restricted to their activities in defence against microbial infections and during the acute phase of the inflammatory response. however, increasing number of investigations showed that neutrophils are endowed with plasticity and can participate in the orchestration of both innate and adaptive immune responses. neutrophils have an impact on a broad range of disorders, including infections, chronic inflammations, and cancer. neutrophils are present in the tumour microenvironment and have been reported to mediate both pro-tumour and anti-tumour responses. neutrophils can contribute to genetic instability, tumour cell proliferation, angiogenesis and suppression of the anti-tumour immune response. in contrast, neutrophils are reported to mediate anti-tumour resistance by direct killing of tumour cells or by engaging cooperative interactions with other immune cells. here we discuss the current understandings of neutrophils biology and functions in health and diseases, with a specific focus on their role in cancer biology and their prognostic significance in human cancer. neutrophils are the essential players in the early response against pathogens and during acute inflammation [ , ] . in response to injury and inflammatory stimuli, neutrophils are rapidly recruited in the tissues [ ] where they engage multiple cross-talks with immune and non-immune cells [ ] . accordingly, neutrophils play an important role in the regulation of innate and adaptive immune responses and are part of the pathogenesis of numerous diseases [ ] . in addition, a growing number of studies showed that neutrophils are endowed with plasticity and heterogeneity which have long been underestimated [ ] [ ] [ ] [ ] . cancer-related inflammation has emerged as a hallmark of tumour biology [ , ] and the tumour microenvironment (tme) consists of both stromal and inflammatory cells, including neutrophils [ ] . the role of neutrophils in tumour biology has long been underestimated while particular emphasis has been placed on the role of other myeloid cells, such as macrophages [ , ] . however, recent findings showed that neutrophils are an important component of the tme and have highlighted their importance in tumour progression as well as in the orchestration of pathways leading to tumour resistance [ ] [ ] [ ] [ ] [ ] . these contrasting findings are likely the results of the previously underestimated plasticity and heterogeneity of neutrophils in cancer [ , , ] . in humans, the density of tumour-associated neutrophils (tans) can have prognostic significance for patient's outcome [ ] [ ] [ ] [ ] . here we review the current understandings on neutrophils biology from their development to the mechanisms of their trafficking and functions in health and disease, with a focus on their role in tumour biology and their prognostic significance in human cancer. abbreviations: -mca, -methylcholathrene; ackr , atypical chemokine receptor ; apc, antigen presenting cell; g-csf, granulocytes colony stimulating factor; g-mdscs, granulocytes-myeloid-derived suppressor cells; gm-csf, granulocytes-macrophages colony stimulating factor; gmps, granulocyte monocyte progenitors; inos, inducible nitric oxide synthase; m-mdscs, monocytes-myeloid-derived suppressor cells; mmp- , matrix metallopeptidase ; mpo, myeloperoxidase; nets, neutrophil extracellular traps; nk, natural killer; prrs, pattern recognition receptors; ros, eactive oxygen species; tans, tumour-associated neutrophils; tcr, t cell receptor; tils, tumour infiltrating leukocytes; tme, tumour microenvironment; trail, tnf-related apoptosis inducing ligand; trpm , transient receptor potential cation channel subfamily m, member ; utc, unconventional t cell. circulation requires a continuous production in the bone marrow (bm), which is mainly regulated by the production of granulocyte-colony stimulating factor (g-csf) and granulocyte-macrophage colony-stimulating factor (gm-csf) [ , [ ] [ ] [ ] . in humans, up to × neutrophils are produced daily during the process of granulopoiesis in the bm and released in the bloodstream [ ] . the process of granulopoiesis has been extensively studied under both steady state and emergency conditions [ , , ] . in the bm, hematopoietic stem cells (hsc) differentiate into lymphoid-primed multipotent progenitors (lmpps) or common myeloid progenitors (cmps) which can both give rise to granulocyte monocyte progenitors (gmps). gmps will commit to the generation of neutrophils upon receiving g-csf and gm-csf signals produced by bone marrow stromal cells [ , , ] . accordingly, g-csf − /− and gm-csf -/mice present a severe and chronic neutropenia [ ] . the process of granulopoiesis is regulated by the successive expression of specific transcription factors (tf) and deficiency in these tfs in mice has been associated with neutropenia [ ] [ ] [ ] [ ] [ ] . in particular, ccaat/enhancer-binding proteins (c/ebps) are a family of six tfs among which the members c/ebpα, c/ebpβ and c/ebpε are involved in neutrophil development [ ] [ ] [ ] [ ] . specifically, c/ebpα acts at the gmp stage of the process and its disruption in mice lead to a complete loss of mature neutrophils by blocking the transition from cmps to gmps [ ] . c/ebpβ is not necessary for the development of neutrophils in steady state but plays an important role in emergency granulopoiesis (see below) [ , ] . on the other hand, c/ebpε drives gmp to myelocyte transition and may be important in the terminal step of the granulopoiesis [ , ] . other tfs, such as the growth factor independent- (gfi- ) have also been implicated in the process of granulopoiesis [ , ] . transcription factors control gene expression profiles, that define the molecular signatures of immature cell subsets revealing their heterogeneity [ , ] . state of the art technologies, such as total and single-cell rna sequencing (scrnaseq) and mass cytometry by time-of-flight (cytof), have been employed to characterize neutrophil differentiation and distinct cellular phenotypes have been identified from gmp to mature neutrophils [ ] [ ] [ ] [ ] . in particular, a population of proliferative gr + cd b + cxcr hi cd int cxcr − neutrophil precursors (defined as preneu) with a potential to differentiate into non-proliferative immature and then mature neutrophils has been identified in mouse bm [ ] . recently, progenitors of the preneu population have been described as early committed neutrophil progenitors (proneu ), which gives rise to an intermediate progeny (proneu ) and then to the subsequent populations [ ] . these neutrophils progenitors are present in mice and humans and their phenotypic characteristics are outlined in table . a study previously described an early unipotent neutrophil progenitor (defined as nep), comprising different clusters of cells [ ] . it is conceivable that this heterogenous population represents a mixture of neutrophil progenitors described as proneu , proneu and preneu [ ] [ ] [ ] . the release of neutrophils in the bloodstream is a process controlled by the regulation of the expression of genes coding for cxcr and cxcr in neutrophil precursors [ ] . in steady state, cxcl is constitutively expressed by bm stromal cells which supports the retention of cxcr + neutrophils inside the bm. during their maturation, expression of cxcr on the surface of neutrophils is downregulated, while the expression of cxcr is increased, leading to their release in the blood via the interaction with cxc chemokines (e.g. cxcl , cxcl ) present in the circulation [ , , , ] . circulating aged neutrophils upregulate the expression of cxcr in order to migrate into the bm where they are eliminated by macrophages [ ] . in steady state, bm is the unique site of haematopoiesis, but during inflammatory conditions, such as cancer and systemic infection, the spleen can be involved in the generation of neutrophils [ , ] . in inflammatory conditions, expression levels of granulopoietic cytokines, such as g-csf, gm-csf and il- are increased, leading to the process of emergency granulopoiesis [ , ] . interestingly, despite that g-csf − /− and gm-csf -/mice present a severe neutropenia in steady state, the expression of il- in these mice can be sufficient for the production of neutrophils during emergency granulopoiesis [ , ] . however, the phenotype and effector functions of neutrophils produced in g-csf − /− and gm-csf -/mice remain to be fully elucidated. neutrophil development during inflammation is also controlled by the expression of specific transcription factors [ , , ] . increased expression of g-csf and g-csf receptor signalling triggers the phosphorylation of signal transducer and activator of transcription (stat ) which directly enhances c/ebpβ expression in progenitor cells [ ] . in a negative feedback loop, the phosphorylation of stat activates the suppressor of cytokine signalling (socs ), which in turn, by inhibiting the activation of stat , prevents an excessive production of neutrophils [ ] . elevated number of circulating neutrophils have been reported in inflammatory conditions, including cancer [ , , ] . accordingly, the production of cytokines and chemokines (such as il- , il- β, g-csf, cxcl , cxcl ) involved in the development and mobilization of neutrophils is upregulated in tumour-bearing host [ ] . tumour cells themselves or tumour-associated immune cells can produce these cytokines. for instance, tumour-associated macrophages (tams) produce il- β, which stimulates t cells to produce il- . in turn, il- increases the expression of g-csf and the release of neutrophils in the circulation (see also the paragraph . ) [ ] . the pressure created by the tumours on neutrophil mobilization can lead to the release of immature neutrophils in the circulation [ , , ] . in mouse models of cancer, the frequency of ly g high cd − immature neutrophils in the blood has been positively correlated with the tumour burden [ ] . in both human and mice, neps (see above), are reported to infiltrate the tumour tissue [ , , , ] . in particular, neps have been identified in the blood and tumour of melanoma patients and reported to promote tumour growth in mice through their immunosuppressive activities (see below) [ ] . studies described pro-tumoral capacity for immature neutrophils through their t-cell suppression ability [ , ] . however, not all immature neutrophils show immunosuppressive proprieties [ , ] and further investigations are needed to fully understand the exact mechanism in which these cells affect tumour growth. once in the circulation, neutrophils are ready to migrate into the tissues [ ] . different conditions provide chemotactic gradients for neutrophil recruitment. in particular, neutrophils express the chemokine receptors cxcr and cxcr and the presence of their ligands provides important chemotactic pathways for neutrophil recruitment to the tissues [ , ] . extravasation of neutrophils across the vascular endothelium is a multistep process orchestrated by adhesion molecules present on vascular endothelial cells and neutrophils. this process has been extensively studied and involves the following steps: capture, rolling, arrest, firm adhesion and transmigration [ ] . neutrophil capture and rolling on endothelium occur through the initial interaction of p-selectin glycoprotein ligand (psgl ) expressed on neutrophils with p-selectin and e-selectin expressed on endothelial cells. in addition, engagement of l-selectin expressed by neutrophils with sialylated ligands on endothelial cells is also involved in the rolling of neutrophils and activation of integrins [ , ] . lymphocyte function-associated antigen (lfa- ) is the principal integrin which mediates arrest and firm adhesion of rolling neutrophils. lfa- binds to its ligands intercellular adhesion molecule (icam ) and icam expressed on the endothelium [ ] . once arrested on the endothelium, neutrophils are ready to transmigrate from the vasculature to the tissue where they can play different roles depending on the tissue context or inflammatory situation (see below). in addition to their migration in tissues, evidences showed that neutrophils can migrate from the inflamed tissues to the circulation through the process of reverse migration, which has been associated with the disruption of endothelial cells tight junctions and an altered production of chemoattractant molecules [ ] [ ] [ ] . the process of reverse migration has been proposed to prevent excessive inflammation and tissue damage through the removal of neutrophils but could also act as a trigger of inflammation in a secondary site through the migration of activated neutrophils [ , ] . therefore, while neutrophil trafficking to inflamed tissues has been extensively studied, the mechanism and role of reverse migration remain poorly understood and future investigations are needed to decipher the different aspects of this process. as mentioned above, neutrophils express cxcr and cxcr , which can bind to their ligands cxcl , cxcl , cxcl , cxcl , and cxcl released by tumour cells, stromal cells and leukocytes in the tme [ , ] . in addition to chemokines, tumour cells and tumour-infiltrating leukocytes are reported to produce cytokines involved in the development, mobilization and recruitment of neutrophils. for instance, t cells are shown to produce gm-csf, tnfα, il- , cxcl and cxcl , which can directly or indirectly recruit neutrophils to the tme [ , , [ ] [ ] [ ] . tumour-associated neutrophils (tans) themselves produce cxcl and cxcl , which in turn enhance their recruitment in the tme [ ] . in different models of primary carcinogenesis, deficiency in cxcr has been associated with a dramatic reduction of neutrophil recruitment in the tme associated with reduction in tumour growth [ , ] . in addition to chemokines, tumour-derived oxysterols are also shown to trigger the recruitment of neutrophils to the tme through a cxcr dependent mechanism [ ] . recent investigations showed that neutrophils were present in the tme at the very early stages of tumour growth. indeed, intravital multiphoton imaging in transplantable tumour models of mice with neutrophil-specific fluorescence revealed that neutrophils were present in the tme as early as h after tumour cell injection and were present in the tissue up to days after tumour cell injection [ ] . here, intra-tumoral neutrophils showed lower motility compared to peri-tumoral neutrophils, suggesting that the tme can affect neutrophil motility and functions [ ] . the classical view of neutrophil roles has long been limited to their effector functions in the elimination of pathogens. however, a growing body of evidence showed the recruitment of neutrophils into naive tissues and their role in maintaining tissue homeostasis, such as in the lung where pulmonary endothelium retains cxcr + neutrophils through the expression of cxcl [ , , ] . under homeostatic condition, neutrophils are also found in liver, spleen and lymph nodes where they play different roles [ , , ] . indeed, liver infiltrating neutrophils can participate in lipid metabolism [ ] , whereas neutrophils present in the spleen provide helper signals to b cells through the production of b-cell activating factor (baff), a proliferation-inducing ligand (april) and il- [ ] . in lymph nodes, neutrophils are mostly located in proximity of t cells and natural killer (nk) cells. these neutrophils express high amount of major histocompatibility complex ii (mhcii), suggesting a role in cd + t cell activation [ ] . neutrophils have been also detected in other tissues, such as intestine, white adipose tissue, skin, and skeletal muscle but their role remains to be elucidated [ ] . neutrophils can detect a wide range of ligands expressed by microbes through their broad repertoire of cell associated and soluble pattern recognition receptors (prrs) and represent the first line of defence against invading pathogens [ , , ] . neutrophils can eliminate pathogens through phagocytosis and production of ros into the phagocytic vacuole, discharge of cytoplasmic granules containing microbicidal components, release of neutrophil extracellular traps (nets), or indirectly by secreting pro-inflammatory cytokines and chemokines (e.g. tnfα, il- , il- β and il- ) [ , , ] , which promote the recruitment and activation of other immune cells [ ] [ ] [ ] [ ] . in addition to their role in defence against pathogens, nets are reported to drive thrombosis [ , ] . in coronavirus disease (covid- ) patients, increased plasma levels of nets were correlated with the disease severity and in autopsy lung samples from patients, neutrophils undergoing netosis were shown to colocalize with platelets in structures consistent with blood vessels, suggesting the role of nets in covid- related thrombosis [ ] . these results suggest nets formation as a potential target for covid- treatment. in a more general view, these results highlight that an excessive presence of activated neutrophils in tissues can be deleterious for the host [ , ] . thus, an efficient resolution of inflammation is crucial for the restoration of tissue integrity and function after an infection. neutrophils are actively involved in this process through the production of pro-resolving lipid mediators (including resolvins and protectin d ). these molecules can block the infiltration of neutrophils in tissues and contribute to chemokines and cytokines scavenging [ , , ] . in addition, phagocytosis of apoptotic neutrophils changes the phenotype of macrophages to produce anti-inflammatory cytokines such as tgfβ and il- [ ]. a growing number of studies showed that neutrophils can be involved in different inflammatory and autoimmune diseases, such as in cardiovascular disorders, multiple sclerosis (ms), systemic lupus erythematosus (sle) and rheumatoid arthritis (ra) [ , [ ] [ ] [ ] [ ] . proteins stored in neutrophils granules, such as mpo and ll- have been associated with the process of atherosclerosis through the activation of endothelial cells and the recruitment of inflammatory monocytes, respectively [ , ] . nets have also been associated with cardiovascular diseases through different mechanisms, including activation of endothelial cells, plasmacytoid dendritic cells (pdcs) and macrophages [ , ] . accordingly, neutrophil depletion in mice decreased the number of monocytes in aortic lysates and reduced the plaque sizes [ ] . neutrophils have also been involved in autoimmune diseases [ ] . for instance, a population of low density neutrophils producing pro-inflammatory cytokines (e.g. ifnα, tnfα) and nets has been identified in sle patients [ ] . impaired clearance of apoptotic neutrophils by macrophages is also observed in these patients [ ] . interestingly, nets can activate pdcs, which in turn produce ifnα and il- to promote b cell differentiation into plasma cells [ , ] . neutrophils can also support the b cell response through the production of baff [ , ] . collectively, these data suggest that neutrophils, neutrophil-derived molecules and nets represent important targets for therapy in patients with inflammatory and autoimmune disorders. in steady state, both circulating and tissue neutrophils are endowed with functional and phenotypic characteristics [ , ] . accordingly, neutrophil heterogeneity has been associated with different parameters, such as maturation, ageing or activation states in response to signals from the tissue microenvironment, such as cytokines [ ] . the bm is the principal site of granulopoiesis and elimination of aged neutrophils. interestingly, in vivo studies revealed specialized subsets of neutrophils in the bm displaying niche-supportive and hsc-supportive functions. in particular, a subset of granulocytes expressing the histidine-decarboxylase and histamine was shown to support the quiescence of myeloid-biased hscs [ ] . moreover, bm neutrophils can secrete prostaglandin e which promotes the retention of hsc in the bm through the production of osteopontin by the preosteoblasts [ ] . neutrophils are released in the blood following a circadian rhythm and circulating neutrophils present different phenotypes over the course of the day in both humans and mice [ , , , ] . in particular, mature neutrophils freshly released from the bm are described as cd l + and cxcr + neutrophils. within ~ h in the circulation, neutrophils downregulate the expression of cd l and upregulate the expression of cxcr and cd b and acquire a hypersegmented nuclei. these cxcr + cd b + cd l low neutrophils exhibit increased integrin activation and a significant capacity to form nets in inflamed mouse venules [ , ] . these phenotypic changes were linked to a circadian transcriptional oscillation of the transcription factor bmal which controls the expression of cxcl by neutrophils. in turn, cxcl acts on cxcr to drive neutrophil ageing [ ] . heterogeneity and plasticity of tumour infiltrating leukocytes (tils) have been well characterized. macrophages served as a paradigm for this phenomenon with classically activated m macrophages and alternatively activated m macrophages exerting anti-tumour and protumour activities, respectively [ , ] . tans can also undergo two different polarization states and by analogy with the nomenclature of m and m macrophages, tans are classified into anti-tumour n neutrophils and pro-tumour n neutrophils. here, tgfβ presents in the tme drives the polarization towards the n phenotype [ ] . n tans have been characterized by their cytotoxic activity towards tumour cells, their capacity to produce t cell chemokines ccl , cxcl and cxcl and to sustain cd + t cells activation [ ] . in contrast, n tans showed a pro-tumour phenotype characterized by the release of proangiogenic factors (e.g. matrix metallopeptidase (mmp- )) and the inhibition of cd + t cells activation through secretion of arginase (arg ) [ , ] . neutrophil differentiation and maturation trajectories are profoundly altered in tumour-bearing mice [ , ] . in advanced neoplasia, immature myeloid cells endowed with immunosuppressive properties appear in the circulation, primary tumours and metastases, often referred to as monocytes or granulocytes-myeloid-derived suppressor cells (m-mdscs or g-mdscs) [ , , ] . g-mdscs are functionally characterized by their ability to suppress t cells proliferation and activation ex vivo [ ] . however, there is no consensus regarding their phenotypic characterization based on surface molecule expression. in human, g-mdscs are described as cd + cd b + cd dim hla-dr − rendering them indistinguishable from the other neutrophil subsets [ , ] . besides their heterogeneity in gene expression profile and surface molecule expression, neutrophils were classified by their sedimentation proprieties into low density neutrophils (ldns), normal density neutrophils (ndns) and high density neutrophil (hdns) [ , , ] . ldns were reported to include both immature and mature neutrophils and to accumulate in cancer [ ] . ldns were described to be generally endowed with immunosuppressive proprieties [ , , ] . however, immunosuppressive neutrophils (cd b + , cd + ) have been also observed in the hdn fraction [ ] , highlighting the urge for an accurate system to define the neutrophil subsets in cancer. cancer-related inflammation has emerged as a hallmark of cancer [ , ] and neutrophils were found to infiltrate different types of neoplasia, including non-small cell lung cancer (nsclc), colorectal cancer (crc), gastric cancer, hepatocellular carcinoma, melanoma, breast cancer and renal carcinoma [ , , [ ] [ ] [ ] ] . in the tme, neutrophils represent a source of cytokines, chemokines and growth factors that sustain tumour growth and progression including epidermal growth factor (egf), hepatocytes growth factor (hgf) and platelet-derived growth factor (pdgf) [ , ] . other neutrophil-derived mediators that influence tumour progression are found in neutrophil granules. for instance, ne has been shown to favour the proliferation of different cancer cell types in vitro, such as human oesophageal cell lines and mammary cell lines through the transactivation of the egf receptor (egfr) and tlr , and human prostate cancer cell lines through the activation of the mitogen activation protein kinase (mapk) pathway [ ] [ ] [ ] . in a genetically engineered mouse model of lung adenocarcinoma induced by oncogenic kras, ne-deficient mice displayed reduced tumour cells proliferation and tumour growth. mechanistically, in vitro experiments showed that ne acted by degrading the insulin receptor substrate (irs- ), which usually inhibits the phosphoinositide -kinase (pi k) in tumour cells. in turn, pi k can interact with the cytoplasmic domain of the pdgfr, which has a potent mitogenic action on human and mouse lung cancer cells [ ] (fig. ) . in addition to their role against microbial infections, nets were observed in different tumours (e.g. liver, breast and intestinal) and were associated with the presence of metastasis (see below) [ ] [ ] [ ] [ ] [ ] [ ] . a recent study showed that nets were able to interfere with lymphocytes cytotoxicity in the primary tumour. in vivo and in vitro experiments demonstrated that nets can form a protective layer on tumour cells that acts as a shield against the cytotoxic activity of cd + t cells and nk cells (fig. ) . interestingly, pharmacological inhibition of nets synergized with combination immunotherapy of anti-pd plus anti-ctla to delay tumour progression in mice injected with t breast cancer cell line [ ] . in tumour, neutrophil-derived ros have been associated with increased dna damage and genetic instability in epithelial cells [ ] suggesting that neutrophils were involved in the initiation stage of carcinogenesis (fig. ) . accordingly, neutropenic mice (csf − /− mice) showed reduced urethane-induced lung carcinogenesis and forcing the recruitment of neutrophils in csf − /− mice by g-csf was sufficient to restore tumour formation in the lung. the detrimental activity of neutrophils was linked to the production of ros, which acts as an amplifier of dna damage induced by urethane carcinogenesis [ ] . in apparent contrast, in -methylcholathrene( -mca)-induced sarcomagenesis, neutrophils were found to be crucial for the initiation of an early anti-tumour response. indeed, neutrophils amplified the production of il- in macrophages, which induced type polarization and ifnγ production in a subset of unconventional t cell (cd -cd -tcrβ + ) [ ] . these contrasting reports are likely related to the plasticity and heterogeneity of neutrophils in different tissue contexts and conditions. ros were shown to be a potent arm for neutrophil anti-tumour activity, through which they can directly kill tumour cells [ , ] . in vivo evidences showed that neutrophils can kill tumour cells through a cell-contact dependent mechanism and the generation of ros (i.e. h o ). cancer cells undergoing epithelial-mesenchymal transition (emt) upregulated the transient receptor potential cation channel, subfamily m, member (trpm ), an h o -dependent ca + channel, which induces a lethal influx of ca + in target cells [ ] (fig. ) . accordingly, reduced trpm expression in cancer cells protected them from the cytotoxic activity of neutrophils. moreover, the expression of trpm is associated with increased release of cxcl , a potent neutrophil chemoattractant. these data suggest that trpm has a dual role in tumour, by promoting the recruitment of neutrophils in the tme and the elimination of cancer cells [ ] . neutrophil-mediated killing of tumour cells can also be induced by the expression of tnf-related apoptosis inducing ligand (trail) by neutrophils [ ] and tnfα. tumour cell-derived tnfα induced the expression of the hepatocyte growth factor receptor (hgfr, also called met) in neutrophils [ ] . this induction is crucial for neutrophil transmigration through inflamed endothelium, the production of inducible nitric oxide synthase (inos) and no by neutrophils in response to hgf. in turn, no kills cancer cells [ ] (fig. ) . in contrast, the hgf-met pathway was also shown to drive an immunosuppressive phenotype in neutrophils associated with limited expansion of anti-tumour t cells [ ] . therefore, the impact of met expression on neutrophils and its possible therapeutic applications is still to be fully clarified. the accumulation of neutrophils in tissues distant from the primary tumour has been shown to contribute to the formation of the premetastatic niche. in the pre-metastatic lung, neutrophils were found to promote the formation of metastasis via the secretion of several mediators, such as the proangiogenic factors bv (also called prokineticin ) and mmp- [ ] , the chemoattractant molecules s a and s a [ ] , ne and cathepsin g that mediate the degradation of thrombospondin- (tsp- ) [ ] , the pro-inflammatory cytokine il- β and the leukotriene b (ltb ) [ ] . in particular, neutrophil-derived mmp- was shown to induce the liberation and the activation of vegf and the subsequent production of new vessels. in vivo studies have shown that bv , produced by neutrophils, was responsible for the poor efficacy of anti-vegf therapy. thus, the inhibition of il- \g-csf axis was shown to increase the therapeutic efficacy of anti-vegf treatment [ ] [ ] [ ] [ ] . the use of oncogene-driven cancer model has highlighted how the interaction between tumour cells and neutrophils was involved in metastasis formation [ , , , ] . in k cre ; cdh fl/fl ; trp fl/fl (kep) mouse model of breast cancer, the accumulation of neutrophils in the pre-metastatic lung with immunosuppressive features was associated with the formation of lung metastasis [ ] . further studies have demonstrated that tumour cells, through the secretion of wnt ligands induced a systemic pro-inflammatory cascade starting by the secretion of il- β by macrophages [ ] . in turn, il- β induced the production of il- by γδ t cells, leading to a g-csf-dependent expansion of neutrophils and their polarization in an immunosuppressive phenotype [ , ] (fig. ) . accordingly, il- derived from cd + t cells or γδ t cells drives most of the neutrophils-derived pro-tumour activities described above. studies showed that nets can support the formation of metastasis through their ability to trap disseminating cancer cells and to facilitate their seeding and proliferation in a distant anatomical site [ ] [ ] [ ] [ ] [ ] [ ] . in an in vivo model of lung metastasis, sustained lung inflammation and cancer cell-derived g-csf induced nets formation, which in turn promoted the proliferation of dormant cancer cells. accordingly, administration of dnase to eliminate nets reduced the formation of lung metastasis [ ] . in ovarian cancer, the formation of new metastasis in the omentum was also dependent on the formation of nets in the pre-metastatic niche [ ] . mechanistically, recent investigations have provided new insights regarding the molecular interaction between cancer cells and nets. in particular, cancer cells express the coiled-coil domain containing protein- (ccdc ), which acts as an extracellular dna sensor and can activate cell motility [ ] . therefore, the formation of nets in the liver or lung induced the migration of cancer cells and ccdc -deficient mice present a reduced formation of metastasis. moreover, in silico analyses showed that ccdc was expressed by several cancer cells in human and in cohorts of patients with breast cancer or colon cancer, the expression of ccdc was correlated with poor clinical outcome [ ] . in apparent contrast with the previous findings, neutrophils recruitment and activation in the metastatic niche was found to be important for the reduction of metastasis formation through the killing of cancer cells [ , ] . genetic ablation of the atypical chemokine receptor (ackr ) resulted in increased levels of chemokine receptors (i.e. ccr , ccr and ccr ) in hematopoietic progenitors and the release from the bm of activated neutrophils endowed with cytotoxic activity towards tumour cells and anti-metastatic potential [ ] . ackr deficient mice transplanted with t breast cancer cell lines or intravenously injected with b f melanoma cell lines showed protection against metastasis formation [ ] . thus, targeting ackr unleashed anti-metastatic potential of neutrophils and represents an interesting novel myeloid checkpoint for innovative therapeutic strategies. a recent study has demonstrated a mechanism that may explain, at least in part, why breast cancer patients develop metastasis [ ] . the injection of human breast cancer cells with low spontaneous metastatic potential in non-obese diabetic (nod)/severe combined immunodeficient (scid) mice, resulted in reprogramming of neutrophils in the pre-metastatic lung. breast cancer cells with low spontaneous metastatic potential secreted high amount of ccl compared to cancer cells with high metastatic potential. ccl induced the recruitment of ifnγ-producing ccr + monocytes in the pre-metastatic lung, leading to upregulation of the transmembrane protein (tmem , also called sting) on neutrophils and activation of their cytotoxic activity [ ] . collectively, these studies underlined the relevance of neutrophils in the tme, both in the primary site and in the metastatic niche. neutrophils with pro-tumour activities are associated to all steps of tumorigenesis, from the promotion of genetic instability, through the proliferation, dissemination and implantation of cancer cells to distal anatomical sites. however, when properly activated, neutrophils can kill tumour cells and participate to the anti-tumour response. thus, studies aimed at clarifying the pathways that trigger neutrophils cytotoxicity against cancer cells, may be useful to pave the way for new therapeutic strategies targeting neutrophils. once recruited into inflamed tissues, neutrophils can engage bidirectional interactions with non-immune (e.g. platelets, mesenchymal cells) or immune cells (e.g. dcs, macrophages, nk cells, b cells, t cells) [ ] . these interactions can occur in a contact-depend manner or via the secretion of soluble mediators. in these cross-talks, neutrophils receive signals that modulate their survival or activation status, while on the other hand neutrophils amplify, suppress or initiate the innate and adaptive immune responses [ , ] . these interactions have been shown to be relevant in the tme. for instance, tumour-entrained neutrophils can produce chemokines, including cxcl , ccl , ccl , cxcl and cxcl , responsible for the recruitment of t cells and other leukocytes [ , ] . in non-small cell lung cancer (nsclc), phenotypic analysis of neutrophils in early stage patients revealed that neutrophils can acquire antigen presenting cell (apc)-like features and activate cd + t cells and cd + t cells [ ] . these apc-like neutrophils derived from a subset of immature progenitors (cd b + cd hi cd -cd int/low ) that in response to gm-csf and ifnγ present in the tme increased the expression of mhcii and cd (fig. ) . on the same line, neutrophils isolated from colorectal cancer (crc) patients were able to enhance cd + t cell responsiveness to t cell receptor (tcr) triggering [ ] . neutrophils have been described to be important in orchestrating the establishment of an effective anti-tumour immunity in sarcoma [ ] . in a model of -mca-induced sarcoma, neutrophils were found to engage a tripartite interaction with macrophages and a subset of utc αβ . neutrophils, by amplifying the production of il- by macrophages, were essential to promote type i polarization and ifnγ production in utc αβ , which support tumour resistance in vivo (fig. ) . interestingly, in silico analyses indicated that this neutrophil dependent anti-tumour axis was relevant in selected human tumours, including undifferentiated pleomorphic sarcoma (ups), crc and ovarian cancer [ ] . as mentioned above, neutrophils in the tme can produce different mediators including ros, reactive nitrogen intermediates (rni) and arg involved in the suppression of both innate and adaptive immune cells effector functions. in a mammary transplantable tumour model, c-kit + immature neutrophils were shown to be sensitive to glucose limitation. in this condition, neutrophils use mitochondrial fatty acid oxidation to support napdh oxidase-dependent ros production which inhibits the t cells response [ ] . this mitochondrial fitness is driven by c-kit and was especially observed in splenic neutrophils [ ] . in response to tgfβ found in tme, neutrophils produced high amount of arg , which reduced the availability of l-arginine in the tme and lead to t cell dysfunction and suppression of the t cell-mediated anti-tumour response [ ] (fig. ) . notably, arg produced by neutrophils from renal carcinoma and nsclc cancer patients inhibited t cell response [ , ] . recent investigations showed an interaction between neutrophils and the microbiota in cancer [ , [ ] [ ] [ ] [ ] . microbiota can regulate the granulopoiesis through the activation of the il- /g-csf pathway in the intestine [ ] . in crc, neutrophils were reported to have a tumour suppressive effect through the expression of antimicrobial peptides, which limit the invasion of bacteria and the subsequent tumour-promoting inflammation [ ] [ ] [ ] (fig. ). in addition, the process of lung carcinogenesis induced by kras mutation coupled with p loss has been described to be associated with dysbiosis of the airway microbiota, which stimulates il- production by resident γδ t cells resulting in neutrophilia and tumour growth [ ] . neutrophils engage important bi-directional interactions also with innate lymphoid cells (ilcs), particularly with nk cells. for instance, neutrophils have been shown to promote the metastatic spread by preventing nk cells-mediated clearance of disseminating cancer cells [ , ] . in vivo experiments showed that in the absence of nk cells, neutrophils acquired a pro-tumour phenotype characterized by increased expression of vegf-a [ ] . these results suggest that nk cells can control neutrophils pro-tumour activities. neutrophils can express ligands of checkpoint receptors, such as pd-l and v-domain immunoglobulin suppressor of t-cell activation (vista) involved in the suppression of the t cell response by driving checkpoint engagement and t cell exhaustion (fig. ) . pd-l expressing neutrophils were found in hepatocellular carcinoma and gastric cancer and were associated with a bad prognosis [ , ] . in a murine transplantable model of melanoma, blockade of vista induced the production of il- in tumour associated monocytes and dendritic cells, via a myd -dependent pro-inflammatory response [ ] . however, the inhibition of vista on neutrophils did not alter their immunosuppressive phenotype, suggesting that combination therapy, with vista inhibition and reprogramming or depletion of neutrophils, should be considered in this model. collectively, these findings suggest that neutrophils can amplify or suppress the immune anti-tumour immune response, through different mechanisms. deciphering these mechanisms may provide new therapeutic approaches to reprogram neutrophils in an anti-tumour activation state. high density of tans or a high level of absolute neutrophil count (anc) in the blood are generally associated with a poor prognosis in cancer patients [ , [ ] [ ] [ ] . accordingly, blood neutrophil to lymphocytes ratio (nlr) has been proposed as a clinical biomarker in patients with cancer. this parameter is easy to obtain in clinical practice and can be monitored over time. a systematic review of electronic databases has been conducted to explore the significance of blood nlr in a variety of solid tumours. in , patients, with different types of cancer, blood nlr was associated with faster tumour progression and reduced overall survival (os) of patients in the majority of solid tumours [ ] . however, it is important to note that the validity of the nlr parameter has been questioned. indeed, nlr is a dynamic parameter that can undergo variations over time and can be affected by other clinical parameters, such as infections. therefore, it remains to establish an optimal and uniform threshold to define a low, normal and high nlr in cancer patients [ ] . beside circulating neutrophils, the density of tans has been also associated with outcome of patients with cancer. neutrophils represent a significant proportion of tumour-infiltrating leukocytes in different subtype of solid tumours, including nsclc, hepatocellular carcinoma, renal carcinoma, crc and gastric cancer [ , , , , , , [ ] [ ] [ ] . in most cases, density of tans has been associated with a bad prognosis, likely due to their immunosuppressive features and their tumour promoting functions (see above). for example, tans represent the dominant leukocytes population in nsclc and were found inversely associated with t cell infiltration [ ] . in other tumour contexts, such as in crc, endometrial cancer, invasive ductal breast carcinoma, low grade glioma and undifferentiated pleomorphic sarcoma (ups), density of tans appear to correlate with a good prognosis [ , , , , , ] . in crc patients, tans were frequently colocalized with cd + t cells and can enhance their responsiveness to tcr triggering [ ] . in patients with ups neutrophil infiltration and a neutrophil signature were associated with a type i immune response and a better prognosis [ ] . thus, currently available data on the occurrence and significance of neutrophils in human cancer suggest that circulating and tumour infiltrating neutrophils may have opposite prognostic significance in different tumour contexts. high levels of circulating neutrophils, nlr or density of tans have been associated with a poor response to different treatments, including chemotherapy, radiotherapy and immunotherapy, in the majority of solid tumours [ ] [ ] [ ] [ ] [ ] [ ] [ ] . some exception are crc, gastric cancer and ovarian cancer where higher levels of tans have been associated with a good response to therapy [ , , [ ] [ ] [ ] [ ] [ ] [ ] . for instance, high levels of cd b + neutrophils infiltration in stage (i-iv) crc patients were found to correlate with a better clinical outcome [ , ] and in a cohort of stage iii crc patients and gastric cancer patients treated or not with -fu-based chemotherapy, high levels of tans infiltration have been associated with a better response to therapy [ , ] . interestingly, similar findings have been observed in patients with ovarian cancer treated with a platinum-based chemotherapy [ ] . therefore, further investigations are needed to define the molecular and cellular mechanisms by which neutrophils can improve the efficacy of chemotherapy in specific cancer subtypes. the role of neutrophils has long been considered restricted to the elimination of invading pathogens and to the acute phase of inflammation. however, a large number of studies have challenged this view and neutrophils are now recognized for their role in the activation and orientation of both the innate and adaptive immune responses. moreover, neutrophils are endowed with a previously underestimated plasticity and can engage complex cross-talks with immune and nonimmune cells, depending on the tissue and inflammatory contexts. accordingly, neutrophils can participate in the pathogenesis of different disorders such as infections, chronic inflammation, autoimmunity and cancer. the role of neutrophils in cancer has long been considered insignificant and was neglected. however, neutrophilia is observed in cancer and neutrophils represent an important component of the tme. therefore, the clinical significance of circulating neutrophils, nlr and tans has been proposed in human cancer and mostly associated with a poor prognosis. neutrophils play a dual role in cancer and can support both protumour and anti-tumour mechanisms. neutrophils can sustain genetic instability, tumour cell proliferation and metastasis and can suppress the immune response. in contrast, neutrophils can kill tumour cells and can be part of the anti-tumour immune response. this dual role is probably related to the diversity and plasticity of neutrophils in different tumour contexts, leading to the formation of different subtypes of neutrophils with opposite functions. therefore, a better dissection of tans diversity in different cancer subtypes is required to define a comprehensive nomenclature of neutrophils in cancer and to identify the pathways that shape neutrophils in an anti-tumour activation state. we assume that further therapeutic approaches targeting neutrophils in cancer represent a new frontier in cancer immunotherapy. ministero dell'istruzione, dell'università e della ricerca (miur) (prin k fsybto sj, ministero della salute (gr- - to sj) and fondazione airc per la ricerca sul cancro (airc ig- to sj) are gratefully acknowledged. neutrophil recruitment and function in health and inflammation neutrophils in the activation and regulation of innate and adaptive immunity neutrophils instruct homeostatic and pathological states in naive tissues social networking of human neutrophils within the immune 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and disease leukotriene b -neutrophil elastase axis drives neutrophil reverse transendothelial cell migration in vivo reverse migration of neutrophils: where, when, how, and why? the junctional adhesion molecule jam-c regulates polarized transendothelial migration of neutrophils in vivo neutrophil migration in infection and wound repair: going forward in reverse getting tanned, how the tumor microenvironment drives neutrophil recruitment different cytokine profiles released by cd + and cd + tumor-draining lymph node cells involved in mediating tumor regression the tumor-promoting actions of tnf-alpha involve tnfr and il- in ovarian cancer in mice and humans granulocyte-colony stimulating factor promotes lung metastasis through mobilization of ly g+ly c+ granulocytes tumor-associated neutrophils: friend or foe? inhibition of cxcr profoundly suppresses inflammation-driven and spontaneous tumorigenesis cxcr -expressing myeloid-derived suppressor cells are essential to promote 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beyond expression conquering neutrophils how neutrophils kill microbes neutrophil extracellular traps kill bacteria neutrophil extracellular traps: the biology of chromatin externalization neutrophil activation and netosis are the major drivers of thrombosis in heparin-induced thrombocytopenia understanding the roles of cytokines and neutrophil activity and neutrophil apoptosis in the protective versus deleterious inflammatory response in pneumonia update on neutrophil function in severe inflammation resolution of acute inflammation and the role of resolvins in immunity, thrombosis, and vascular biology apoptotic neutrophils and t cells sequester chemokines during immune response resolution through modulation of ccr expression tissue microenvironments define and get reinforced by macrophage phenotypes in homeostasis or during inflammation, repair and fibrosis diverse novel functions of neutrophils in immunity, inflammation, and beyond human neutrophils in auto-immunity single-cell sequencing of mouse heart immune infiltrate in pressure overloaddriven heart failure reveals extent of immune activation understanding the multifaceted role of neutrophils in cancer and autoimmune diseases neutrophils in atherosclerosis: from mice to man kettritz, beta integrinmediated cell-cell contact transfers active myeloperoxidase from neutrophils to endothelial cells auto-antigenic protein-dna complexes stimulate plasmacytoid dendritic cells to promote atherosclerosis neutrophil extracellular traps and endothelial dysfunction in atherosclerosis and thrombosis hyperlipidemia-triggered neutrophilia promotes early atherosclerosis netting neutrophils induce endothelial damage, infiltrate tissues, and expose immunostimulatory molecules in systemic lupus erythematosus increased apoptotic neutrophils and macrophages and impaired macrophage phagocytic clearance of apoptotic neutrophils in systemic lupus erythematosus plasmacytoid dendritic cells induce plasma cell differentiation through type i interferon and interleukin neutrophils activate plasmacytoid dendritic cells by releasing self-dna-peptide complexes in systemic lupus erythematosus baff overexpression is associated with autoantibody production in autoimmune diseases neutrophils contribute to excess serum baff levels and promote cd + t cell and b cell responses in lupus-prone mice neutrophil diversity in health and disease bone marrow myeloid cells regulate myeloid-biased hematopoietic stem cells via a histamine-dependent feedback loop g-csf-induced sympathetic tone provokes fever and primes antimobilizing functions of neutrophils via pge a neutrophil timer coordinates immune defense and vascular protection circadian regulation of human peripheral neutrophils neutrophil ageing is regulated by the microbiome tumor associated macrophages and neutrophils in cancer defining the emergence of myeloid-derived suppressor cells in breast cancer using single-cell transcriptomics coordinated regulation of myeloid cells by tumours myeloid-derived suppressor cells coming of age the nature of myeloid-derived suppressor cells in the tumor microenvironment survival of residual neutrophils and accelerated myelopoiesis limit the efficacy of antibodymediated depletion of ly- g+ cells in tumor-bearing mice human neutrophils in the saga of cellular heterogeneity: insights and open questions on the origin of low-density neutrophils er stress regulates myeloid-derived suppressor cell fate through trail-r-mediated apoptosis mature cd (+) and immature cd (-) neutrophils present in g-csf-treated donors display opposite effects on t cells mouse versus human neutrophils in cancer: a major knowledge gap distinct functions of neutrophil in cancer and its regulation the serine protease inhibitor elafin maintains normal growth control by opposing the mitogenic effects of neutrophil elastase infiltrating myeloid cells exert protumorigenic actions via neutrophil elastase neutrophil elastase induces cell proliferation and migration by the release of tgf-alpha, pdgf and vegf in esophageal cell lines neutrophil elastase-mediated degradation of irs- accelerates lung tumor growth neutrophil extracellular traps produced during inflammation awaken dormant cancer cells in mice neutrophil extracellular traps sequester circulating tumor cells and promote metastasis coagulation induced by c ar-dependent netosis drives protumorigenic neutrophils during small intestinal tumorigenesis neutrophils facilitate ovarian cancer premetastatic niche formation in the omentum cancer cells induce metastasis-supporting neutrophil extracellular dna traps neutrophil extracellular traps promote inflammation and development of hepatocellular carcinoma in nonalcoholic steatohepatitis perez-gracia, i. melero, cxcr and cxcr chemokine receptor agonists produced by tumors induce neutrophil extracellular traps that interfere with immune cytotoxicity genotoxic effects of neutrophils and hypochlorous acid early neutrophil responses to chemical carcinogenesis shape long-term lung cancer susceptibility tumor entrained neutrophils inhibit seeding in the premetastatic lung trpm mediates neutrophil killing of disseminated tumor cells trpm modulates neutrophil attraction to murine tumor cells by regulating cxcl expression neutrophil-derived tnfrelated apoptosis-inducing ligand (trail): a novel mechanism of antitumor effect by neutrophils met is required for the recruitment of anti-tumoural neutrophils reactive neutrophil responses dependent on the receptor tyrosine kinase c-met limit cancer immunotherapy lung inflammation promotes metastasis through neutrophil protease-mediated degradation of tsp- contribution to tumor angiogenesis from innate immune cells within the tumor microenvironment: implications for immunotherapy an interleukin- -mediated paracrine network promotes tumor resistance to anti-angiogenic therapy oncogenic ras pathway activation promotes resistance to anti-vegf therapy through g-csf-induced neutrophil 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inducible nitric oxide synthaseexpressed cd b+/cd (-)/cd +/cd + myeloid-derived suppressor cells and cd + t lymphocytes in patients with advanced-stage non-small cell lung cancer arginase i-producing myeloid-derived suppressor cells in renal cell carcinoma are a subpopulation of activated granulocytes gut microbiota, inflammation, and colorectal cancer cell-type-specific responses to interleukin- control microbial invasion and tumor-elicited inflammation in colorectal cancer adenoma-linked barrier defects and microbial products drive il- /il- -mediated tumour growth cross talk between neutrophils and the microbiota the microbiota regulates neutrophil homeostasis and host resistance to escherichia coli k sepsis in neonatal mice neutrophils suppress intraluminal nk cell-mediated tumor cell clearance and enhance extravasation of disseminated carcinoma cells nk cells control tumor-promoting function of neutrophils in mice peritumoural neutrophils negatively regulate adaptive immunity via the pd-l /pd- signalling pathway in hepatocellular carcinoma tumour-activated neutrophils in gastric cancer foster immune suppression and disease progression through gm-csf-pd-l pathway immunecheckpoint protein vista regulates antitumor immunity by controlling myeloid cell-mediated inflammation and immunosuppression neutrophil-lymphocyte ratio in metastatic breast cancer is not an independent predictor of survival occurrence and significance of tumor-associated neutrophils in patients with colorectal cancer neutrophils dominate the immune cell composition in non-small cell lung cancer peritumoral neutrophils link inflammatory response to disease progression by fostering angiogenesis in hepatocellular carcinoma neutrophils oppose uterine epithelial carcinogenesis via debridement of hypoxic tumor cells neutrophil infiltration is a favorable prognostic factor in early stages of colon cancer association between pretreatment neutrophil-to-lymphocyte ratio and outcome of patients with metastatic renalcell carcinoma treated with nivolumab baseline neutrophil-to-lymphocyte ratio (nlr) and derived nlr could predict overall survival in patients with advanced melanoma treated with nivolumab neutrophil to lymphocyte ratio is associated with outcome during ipilimumab treatment clinical significance of tumorinfiltrating lymphocytes and neutrophil-to-lymphocyte ratio in patients with stage iii colon cancer who underwent surgery followed by folfox chemotherapy neutrophil/lymphocyte ratio predicts chemotherapy outcomes in patients with advanced colorectal cancer baseline neutrophilia, derived neutrophil-to-lymphocyte ratio (dnlr), platelet-to-lymphocyte ratio (plr), and outcome in non small cell lung cancer (nsclc) treated with nivolumab or docetaxel association of neutrophil/lymphocyte ratio and platelet/lymphocyte ratio with er and pr in breast cancer patients and their changes after neoadjuvant chemotherapy tumorassociated neutrophils correlate with poor prognosis in diffuse large b-cell lymphoma patients the significance of tumor-associated neutrophil density in uterine cervical cancer treated with definitive radiotherapy high density of cd b in primary high-grade ovarian cancer independently predicts response to chemotherapy tumour-infiltrating neutrophils counteract anti-vegf therapy in metastatic colorectal cancer tumorinfiltrating neutrophils is prognostic and predictive for postoperative adjuvant chemotherapy benefit in patients with gastric cancer tumor-associated neutrophils recruit macrophages and t-regulatory cells to promote progression of hepatocellular carcinoma and resistance to sorafenib key: cord- -i j y authors: mejdoubi, anasse; khoulali, mohamed; raouzi, nabil; nasri, siham; mebrouk, yassine; oulali, noureddine; moufid, fayçal title: neurosyphilis revealed by compressive cervical spine syphilitic gumma: a case report date: - - journal: spinal cord ser cases doi: . /s - - - sha: doc_id: cord_uid: i j y introduction: neurosyphilis is a sexually transmitted disease secondary to the invasion of the central nervous system by the treponema pallidum. the spinal syphilitic gumma is rare. case presentation: we report a case of extradural cervical spinal syphilitic gumma revealed by spinal cord compression in a -year-old male. the epidural lesion was removed via a posterior approach. histological examination revealed syphilis. syphilis serologies were positive. brain mri showed an associated cerebro-meningeal syphilitic gumma. antibiotic regime based on aqueous penicillin g was introduced for days. discussion: currently, there is an increase in the frequency of syphilis and changes in its clinical manifestations. neurosyphilis can take atypical forms. spinal syphilitic gumma is a rare manifestation and its association with cerebral involvement is exceptional. diagnosis is based on serologies in the blood and cerebrospinal fluid. the place of imagery, especially magnetic resonance imaging, is essential. neurosyphilis should be discussed as a possible differential diagnosis in evaluation of spinal and cerebral lesions. neurosyphilis includes all neurological disorders secondary to the invasion of the nervous system by the treponema pallidum. it can appear during the primo-secondary (early neurosyphilis) or tertiary (late neurosyphilis) phases. the diagnosis of neurosyphilis is currently challenging because of the diversity of symptoms and atypical presentations. furthermore, the widespread use of penicillin has altered the clinical manifestations of neurosyphilis and serological profiles, making diagnosis even more difficult [ , ] . compared to cerebral involvement, spinal syphilis is rare, manifested by myelitis, tabes dorsalis, and gummas. spinal syphilitic gumma represents an exceptional entity; only a dozen cases have been reported in the literature [ ] [ ] [ ] . the association of spinal and cerebral syphilitic gummas is even rarer [ ] . in this article, we report an exceptional case of neurosyphilis combining an extradural cervical spinal and cerebro-meningeal syphilitic gumma revealed by spinal cord compression. a -year-old married male, with a -month history of progressive heaviness in the four limbs was admitted to a peripheral hospital. due to worsening of the neurological deficit and the appearance of genito-sphincter dysfunction, the patient was referred to our hospital center for management. physical examination in the emergency room revealed a conscious male, well oriented, without disturbance of the superior functions, or cranial nerve palsy. neurological examination revealed spastic tetraplegia with muscle strength / in the upper limbs and / in the lower limbs with hypoesthesia below the t level. spinal magnetic resonance imaging (mri) showed ( fig. ) posterior cervical epiduritis on the th cervical vertebra (c ), hypointense on t -weighted image, hypointense on t -weighted image, enhancing after injection of gadolinium, exerting a mass effect on the cervical cord. the preoperative biological assessment showed a lymphocytic hyperleukocytosis % (leukocyte = , , with normal level: - , /mm ) and c-reactive protein at . mg/l (normal level: . - . mg/l). an emergency posterior laminectomy of c was performed without osteosynthesis. a thick extradural lesion adhering to the dura was separated and extirpated (fig. ) . histopathological examination revealed a chronic inflammatory epiduritis rich in plasmocyte elements with russel's body evoking a syphilitic gumma. given this result, we performed syphilitic serology in the blood which showed a reactive veneral disease reagent laboratory (vdrl) test with a titer of / and highly positive ( +) treponema pallidum hemagglutination assay (tpha). the syphilitic serology in his wife also returned positive. screening for immunodeficiency virus antibodies immunoglobulin g (igg = mg/dl, with normal level: . - . mg/dl) and a normal white blood cell count ( element, with normal level < /mm ). vdrl and tpha test in the csf were negative while the fluorescent treponemal antibody absorption (fta-abs) test in the csf returned positive. a brain mri (fig. ) in search of a secondary lesion objectified a right temporal cerebro-meningeal lesion, hypointense on t -weighted image, heterogeneous hypointense on t -weighted image, homogeneous enhancement after contrast injection and associated with a wide range of perilesional edema on flair sequence evoking a cerebromeningeal syphilitic gumma with intraparenchymal extension. the patient was given iv million units of aqueous penicillin g per day for days. the follow-up at months shows a partial recovery of the sensory-motor deficit, mainly in the two upper limbs, with persistence of vesico-sphincter disorders which requires the use of a permanent urinary catheter. due to the covid- pandemic and the national containment that has restricted the circulation of the population, our patient is undergoing a reduced re-education and rehabilitation program. this will impact neurological recovery and require a longer follow-up period. syphilis is a systemic sexually transmitted infection that is worldwide spread caused by a spirochete, t. pallidum. it is a very old disease that had become rare in industrialized countries since the discovery of penicillin until the - s during which it resurfaced due to the outbreak of the hiv/aids infection [ , , ] . neurosyphilis is the invasion of the nervous system by t. pallidum; it complicates - % of untreated syphilis. the prevalence of neurosyphilis is particularly high in people with hiv coinfection [ ] [ ] [ ] . t. pallidum can infect different parts of the central nervous system such as the meninges, brain, brainstem, cerebellum, spinal cord, and nerve roots, as well as the cerebral and spinal vessels. the clinical forms of neurosyphilis are therefore diverse [ ] . neurosyphilis has two stages [ ] : ( ) early neurosyphilis: the initial stages of syphilis. most often asymptomatic (asymptomatic neurosyphilis), the main neurological manifestations are meningitis or meningoencephalitis and cranial pair palsy [ , , , ] . ( ) late neurosyphilis: it occurs one year after initial infection, and is often associated with vascular and/or parenchymal involvement (meningo-vascularitis, general paresis, myelitis, tabes dorsalis, and syphilitic gumma) [ , , , ] . currently, typical forms are becoming less common while there is an increase in the incidence of atypical manifestations such as psychiatric and cognitive disorders [ , ] . these changes make diagnosis more challenging, especially in the early phase. syphilitic gumma is the consequence of a cellular immune response, secondary to t. pallidum invasion, causing the formation of an inflammatory granulation "tumor-like" consisting of lymphocytes and plasma cells. this destructive lesion can occur in any part of the organism [ , ] . in the central nervous system, syphilitic gumma develops from the dura and pia mater [ ] . usually cerebro- meningeal, it may exceptionally be intracerebral posing a problem of differential diagnosis with a malignant cerebral tumor [ ] . in the spine, some cases of syphilitic gumma have been reported in the literature. they can develop in the intradural, intramedullary or extramedullary space, or the extradural space as in our case [ , ] . the association of cerebral and spinal syphilitic gummas is exceptional. the first case of this association was reported by shen [ ] . microbiological diagnosis of syphilis is based on a serological study in the blood which includes vdrl, tpha, and fta-abs. the biological diagnosis of neurosyphilis is based on nonspecific arguments (hypercellularity of the csf, hyperproteinorachia, increase of the immunoglobulins g in the csf) and serological tests in the csf (vdrl, tpha, tppa, and fta-abs). normal csf does not eliminate neurosyphilis [ , , , , ] . in our case, biochemical abnormalities were found in the csf (hyperproteinorachia and increased immunoglobulin g) with negative csf syphilis serology. neuroimaging plays a fundamental role in the management of neurosyphilis. it provides diagnostic elements, ensures radiological monitoring, and assesses therapeutic effectiveness. mri represents the most efficient technique in terms of sensitivity and specificity [ ] . the spectrum of neuroradiological lesions includes cortico-subcortical parenchymal atrophy, predominant in the frontotemporal regions, lesions of white matter and gray nuclei, and infarctions distributed in the vascular territories of the perforating arteries [ ] [ ] [ ] . syphilitic gumma presents on mri as a hypo or iso-intense nodule on t -weighted image and hyperintense on t -weighted image, with homogeneous enhancement after contrast injection, and may be accompanied by a linear enhancement of the dura mater [ , ] . in our case, the mri was hypointense on t weighted image, and hypointense on t -weighted image with homogeneous enhancement after contrast injection. however, it should be noted that the radiological features of neurosyphilis are as variable as the clinical manifestations; the lesions observed could be confused with other neurological pathologies [ , ] . imaging follow-up after antisyphilitic treatment can show neuroradiological progression in some cases including infarction lesions, mild to severe brain atrophy, and white matter demyelination suggesting that we need to continue imaging follow-up and treatment of these patients [ ] . first-line treatment for neurosyphilis in any form, including central nervous system gummas, is represented by highdose intravenous aqueous penicillin g. the recommended protocol is - million units, administered intravenously for - days [ ] . in the case of penicillin allergy, ceftriaxone is an alternative treatment at g per day in intramuscular or intravenous for days. azithromycin and doxycycline are other therapeutic alternatives for penicillin allergy [ , ] . administration of mg of prednisone during the first h of penicillin cure is recommended to prevent the jarisch-herxheimer reaction [ ] . the same treatment protocol is used for the patients with hiv co-infection; a prophylactic treatment based on intramuscular benzathine penicillin at a dosage of . million units for weeks or doxycycline mg twice a day for a month is recommended, with clinical and serological control for at least years [ ] . almost all of reported spinal syphilitic gumma cases were suspected as spinal tumors and they were undergoing surgery. on the other hand, most cerebral syphilitic gumma can be significantly reduced or completely resorbed after the antisyphilis treatment, and surgery may be unnecessary [ ] . neurosurgical treatment of syphilitic gumma should be reserved in case of signs of compression (spinal cord compression, and intracranial hypertension) or secondary neurological worsening [ , , ] . early diagnosis of neurosyphilis and appropriate antibiotic treatment can show clinical improvement and resolution of csf abnormalities [ , ] . however, neurologic deficits may fail to improve even with proper treatment [ ] . in patients with meningeal neurosyphilis, quick resolution of symptoms is the rule, whereas in parenchymatous forms, the resolution may not occur [ ] . resolution of csf abnormalities is the best measure of the adequacy of treatment. the cdc recommends that csf examination be repeated every months after completion of therapy until abnormalities resolve [ , ] . the follow-up of our patient at months of the treatment finds a partial recovery; however, a longer-term follow-up is necessary to judge the neurological outcome. neurosyphilis is a major complication of syphilis and still represents a public health problem, characterized by clinical, biological, and radiological polymorphism. atypical forms are now the most common; hence, the importance of screening and adequate treatment of all syphilis in the initial phase. conflict of interest the authors declare that they have no conflict of interest. publisher's note springer nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. incidence and clinical presentation of neurosyphilis: a retrospective study of cases neurosyphilis in the modern era: literature review and case series tertiary syphilis in the cervical spine: a case report and review of the literature diagnosis and treatment of spinal syphilitic gumma: a case report spinal cord syphilitic gumma presenting with brown-séquard syndrome: a case report and literature review multiple intracranial and spinal cord syphilitic gummas in a human immunodeficiency virus-negative man with untreated syphilis: a case report neurosyphilis: old disease, new implications for emergency physicians medullary involvement in neurosyphilis: a report of cases and a review of the literature early neurosyphilis presenting with multiple cranial nerve palsies: a case report of management by combined penicillin-corticosteroid treatment neurosyphilis: a historical perspective and review cerebral syphilitic gumma misdiagnosed as a malignant brain tumor solitary spinal dural syphilis granuloma mimicking a spinal meningioma neuroimaging findings in neurosyphilis neurosyphilis: mri features and their phenotypic correlation in a cohort of patients from a tertiary care university hospital neurosyphilis in hiv-positive and hiv-negative patients: neuroimaging findings cerebral syphilitic gummata: a case presentation and analysis of reported cases neuroimaging features, follow-up analyses, and comparisons between asymptomatic and symptomatic neurosyphilis key: cord- - lov f authors: benameur, karima; agarwal, ankita; auld, sara c.; butters, matthew p.; webster, andrew s.; ozturk, tugba; howell, j. christina; bassit, leda c.; velasquez, alvaro; schinazi, raymond f.; mullins, mark e.; hu, william t. title: encephalopathy and encephalitis associated with cerebrospinal fluid cytokine alterations and coronavirus disease, atlanta, georgia, usa, date: - - journal: emerg infect dis doi: . /eid . sha: doc_id: cord_uid: lov f there are few detailed investigations of neurologic complications in severe acute respiratory syndrome coronavirus infection. we describe patients with laboratory-confirmed coronavirus disease who had encephalopathy and encephalitis develop. neuroimaging showed nonenhancing unilateral, bilateral, and midline changes not readily attributable to vascular causes. all patients had increased cerebrospinal fluid (csf) levels of anti-s igm. one patient who died also had increased levels of anti-envelope protein igm. csf analysis also showed markedly increased levels of interleukin (il)- , il- , and il- , but severe acute respiratory syndrome coronavirus was not identified in any csf sample. these changes provide evidence of csf periinfectious/postinfectious inflammatory changes during coronavirus disease with neurologic complications. there are few detailed investigations of neurologic complications in severe acute respiratory syndrome coronavirus infection. we describe patients with laboratory-confirmed coronavirus disease who had encephalopathy and encephalitis develop. neuroimaging showed nonenhancing unilateral, bilateral, and midline changes not readily attributable to vascular causes. all patients had increased cerebrospinal fluid (csf) levels of anti-s igm. one patient who died also had increased levels of anti-envelope protein igm. csf analysis also showed markedly increased levels of interleukin (il)- , il- , and il- , but severe acute respiratory syndrome coronavirus was not identified in any csf sample. these changes provide evidence of csf periinfectious/ postinfectious inflammatory changes during coronavirus disease with neurologic complications. elisa with % sensitivity and % specificity for confirmed covid- against pre- controls. csf was serially diluted from : to : , and csf from case-patient who had hiv infection (hospitalized during march ) and from pre- healthy subjects ( ) were included for comparison. we measured levels of plasma igg against the receptor-binding domain of s by using a commercial elisa (genscript, https:// www.genscript.com) at a : dilution. we analyzed csf inflammatory proteins (milli-poresigma, https://www.emdmillipore.com) by using a luminex- platform and a modified manufacturer's protocol as described ( ) . these proteins include interleukin (il)- α, il- β, il- , il- , il- , il- , il- , il- , il- , il -p , il -p , interferon-gamma-induced protein (ip- ), monocyte chemoattractant protein (mcp- /ccl ), macrophage-derived chemokine (mdc/ccl ), fractalkine (cx cl ), and tumor necrosis factor α (tnf-α). we performed molecular testing for sars-cov- by using real-time quantitative reverse transcription pcr (qrt-pcr). we extracted total nucleic acid from µl of csf from each person by using the ez virus mini kit version . and the ez advanced xl instrument (qiagen, https://www.qiagen.com) after lysis with avl lysis buffer (qiagen). we performed a -step qrt-pcr by using -ncov_n or -ncov_n combined primer/probe mix (integrated dna technologies, inc., https://www.idtdna.com) in a roche lightcycler ii (https://lifescience. roche.com), an endogenous control, and an in vitro transcribed full-length rna of known titer (integrated dna technologies, inc.) as a positive control. we followed the same procedure for influenza a virus except using a primer/probe mixture ( ) and a mitochondrial cytochrome oxidase subunit dna endogenous control ( ) . we tested all samples in duplicate. patient , a -year-old african-american woman who had sickle cell disease (scd) and was receiving dabigatran for a recent pulmonary embolus, came to a community hospital after days of progressive dyspnea. an initial chest radiograph showed a right lower lobe infiltrate, and she was given a blood transfusion and antimicrobial drugs for presumed scd crisis and pneumonia. her breathing became more labored, and a repeat chest radiograph showed worsening bilateral infiltrates. a nasopharyngeal swab specimen was positive for sars-cov- and influenza a virus (negative for influenza b virus). she was empirically given hydroxychloroquine ( mg daily) and peramivir ( mg daily), but acute kidney injury and progressive hypoxemic respiratory failure developed. she was intubated and transferred to our institution on day . her paralysis and sedation were discontinued on day after improved oxygenation, but she remained comatose with absent brainstem reflexes on day . brain magnetic resonance imaging (mri) showed nonenhancing cerebral edema and diffusion weighted imaging abnormalities predominantly involving the right cerebral hemisphere, as well as brain herniation ( figure ). an occlusive thrombus was identified in the right internal carotid artery, and edema was also identified in the cervical spinal cord. the overall appearance was most consistent with encephalitis and myelitis, with superimposed hypoxic ischemic changes. csf showed high opening pressure of cm of water, nucleated cells/ml, , erythrocytes/ml, an increased protein level (> mg/dl), and a glucose level within a standard range (table) . her nucleated cell count remained strongly increased even after correction for the traumatic tap (≈ nucleated cell/ erythrocytes). given a grave prognosis, the family withdrew life-sustaining care and the patient died on day . patient , a -year-old african-american man who had hypertension, showed development of fever, shortness of breath, and cough. computed tomography of the chest showed bilateral, diffuse ground glass infiltrates. a nasopharyngeal swab specimen obtained on day showed sars-cov- . he was given a -day course of hydroxychloroquine, but hypoxic respiratory failure developed, which required intubation, followed by encephalopathy with myoclonus on day . his neurologic examination showed profound encephalopathy, absent corneal and gag reflexes, multifocal myoclonus involving both arms, and absent withdrawal to painful stimuli. electroencephalography showed diffuse slowing with a suggestion that the myoclonus was seizure-related. brain mri on day showed a nonenhancing hyperintense lesion within the splenium of the corpus callosum on fluid-attenuated inversion recovery and diffusion weighted imaging sequences ( figure ). csf showed high opening pressure of cm h o, no pleocytosis, erythrocytes/ml, a mildly increased protein level, and glucose level within the reference range. patient , a -year-old african-american man who had hypertension, showed development of cough, dyspnea, and fever with multifocal, patchy, ground glass opacities on chest computed tomography and a nasopharyngeal swab specimen positive for sars-cov- . his symptoms progressed to hypoxic respiratory failure requiring intubation, and his multifocal myoclonus began soon after starting to take hydroxychloroquine. his neurologic examination showed profound encephalopathy, absent oculocephalic reflex, multifocal myoclonus affecting bilateral arms and legs, absent withdrawal to pain, and diminished deep tendon reflexes. the resolution of his myoclonus coincided with fentanyl cessation, but it is not clear that the symptoms were related. a motion-degraded brain mri showed an equivocal nonenhancing area of fluid-attenuated inversion recovery abnormality in the right temporal lobe. csf obtained on hospital day showed a normal opening pressure; levels of nucleated cells, erythrocytes, and protein within reference ranges; and an increased glucose level (table) . his mentation began to improve on day , and he was subsequently discharged without major neurologic sequelae. plasma anti-s receptor-binding domain igg levels were increased for all patients, consistent with severe covid- (t. ozturk et al., unpub. data ). an indirect elisa for plasma showed an increased level of anti-s igm for patients ( : ) and ( : ), a highly increased level of anti-s igm for patient ( : , ); an increased level of anti-e igm for patients and ( : ), and a standard level of anti-e igm for patient . an indirect elisa for csf showed markedly increased levels of igm for sars-cov- s (figure , panel a) and e (figure , panel b) proteins for the most severely ill patient , and mildly elevated levels of igm for s only for patients and . the number of csf erythrocytes in patient suggested plasma contamination at an approximate dilution of : , , which still emerging infectious diseases • www.cdc.gov/eid • vol. , no. , september placed these csf igm levels higher than those for patients and . csf from patients and underwent detailed inflammatory protein profiling as described ( , , ) . when we compared historical and present control subjects who had normal cognition (no viral illness) ( ), we found that patients with covid- and neurologic symptoms had increased csf levels of il- , il- , il- , ip- , and tnf-α ( figure , panel c). levels of il- , il- , ip- , and tnf-α were also available for subjects who had hiv-associated neurocognitive disorders ( ) . increased levels of il- and il- appeared to be unique for neurologic complications of sars-cov- , and increased levels of ip- and tnf-α were common features between neurologic complications of sars-cov- and hiv. we used a real-time rt-pcr to test for sars-cov- and influenza a virus (tested because patient showed a co-infection). results were negative for all patients. we report patients who had severe covid- and showed development of various neurologic symptoms and findings in a us hospital. all patients had more severe symptoms affecting cortical and brainstem functions at the peak of their neurologic illnesses than a recent series of case-patients with milder illness in france ( ) . all patients were also co-incidentally given a short course of empiric hydroxychloroquine, although there was no temporal correlation between the medication and their neurologic manifestation. similar to the case-series in france, we did not isolate sars-cov- rna from csf, although such viral rna has been inconsistently identified in other cases ( ) . however, increased levels of csf anti-s igm and altered levels of csf cytokines are consistent with direct cns involvement by sars-cov- . because mri changes seen in these patients could be caused by hypercoagulability ( ) or metabolic encephalopathy ( ) , we propose that csf investigation can improve the distinction between neurologic involvement of sars-cov- (or neuro-covid) and neurologic symptoms caused by other covid-related causes. in health and many noninflammatory neurologic disorders, the intact blood-brain barrier prevents major central translocation by plasma immunoglobulins or cells that secrete them ( ) . increased levels of csf antibodies can thus result from disrupted blood-brain barrier, regulated migration of peripheral antibodysecreting cells into the cns, or de novo antibody synthesis within the cns. the relatively normal protein levels in patients and would argue against an unequivocal blood-brain barrier disruption. the lack of clear correlation between plasma and csf titers provides some support for an active cns process. the failure to detect csf sars-cov- rna does not diminish the likelihood of direct cns infection because it is only recovered from blood in % of the actively infected cases ( ) , and increased levels csf igm are also more commonly found as evidence for cns infection than viral recovery in other encephalitides, including those for infection with japanese encephalitis virus ( ) , dengue virus ( ) , human parvovirus ( ) , and rabies virus ( ) . at the same time, undetectable csf rna raises the possibility that mechanisms other than direct brain infection might account for the observed mri and clinical changes. these changes include peri-infectious inflammation (mediated by antibodies, complement, or both) ( , ), vasculopathy, and altered neurotransmission. until definitive neuropathologic studies or effective antiviral therapies are possible, infectious and peri-infectious etiologies need to be examined for neuro-covid. increased levels of csf multiple cytokines in these neuro-covid patients are consistent with earlier reports of cytokine analysis of blood ( ; m. woodruff et al., unpub. data). we additionally identified changes shared (and not shared) by sars-cov- and hiv. factors associated with increased levels of csf il- in patients infected with hiv should be investigated in future neuro-covid studies, and increased levels of csf il- might uniquely provide useful information on the pathophysiology of cns. we did not include plasma cytokine levels because their levels are much more influenced by demographic factors than their csf counterparts (w.t. hu et al., unpub. data). a larger cohort is necessary to better distinguish between csf and plasma cytokine alterations, and including patients without confounding disease (e.g., scd in patient ) or standard mri results can also determine the relative roles of noninfectious/inflammatory causes of encephalopathy, including hypoxia or hypercoagulability ( , ) . nevertheless, we demonstrated in these case-patients that sars-cov- antibodies are detectable in the csf for patients with neurologic complications and are associated with selective csf cytokine alterations. future investigations should align neurologic outcomes with csf infectious and immunologic profiles, such that an evidence-based treatment algorithm can be determined for preventing and treating neuro-covid- . dr. benameur is an neurologist and associate professor in the department of neurology at emory university school of medicine, atlanta, ga. her primary research interest is in neuroinflammatory changes related to covid- asymptomatic patients with novel coronavirus disease (covid- ) clinical characteristics of deceased patients with coronavirus disease : retrospective study possible central nervous system infection by sars coronavirus severe neurologic syndrome associated with middle east respiratory syndrome corona virus (mers-cov) nervous system involvement after infection 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metabolic encephalopathies: mri appearance immune regulation of antibody access to neuronal tissues detection of sars-cov- in different types of clinical specimens how many patients with anti-jev igm in cerebrospinal fluid really have japanese encephalitis? importance of cerebrospinal fluid investigation during dengue infection in brazilian amazonia region detection of human parvovirus dna in the patients with acute encephalitis syndrome during seasonal outbreaks of the disease in gorakhpur long-term follow-up after treatment of rabies by induction of coma human coronaviruses and other respiratory viruses: underestimated opportunistic pathogens of the central nervous system? viruses clinical features of patients infected with novel coronavirus in wuhan difference of coagulation features between severe pneumonia induced by sars-cov and non-sars-cov vaso-occlusive crisis and acute chest syndrome in sickle cell disease due to novel coronavirus disease (covid- ) key: cord- -j ltpaa authors: naser moghadasi, abdorreza title: encephalopathy associated with covid- in a patient with multiple sclerosis date: - - journal: j neurovirol doi: . /s - - - sha: doc_id: cord_uid: j ltpaa from the beginning of covid- pandemics, the involvement of patient’s nervous system with this virus is increasingly reporting. although various reports are published on affliction of multiple sclerosis (ms) patients with sars-cov- , no report has been published on brain involvement by this virus in ms patients so far. herein, a -year-old patient with ms who experienced the decreased level of consciousness and encephalopathy following covid- involvement has been reported. from the beginning of covid- pandemics in december , the involvement of patient's nervous system with this virus is increasingly reporting (ahmad and rathore ). ye et al. reported a man who hospitalized with a diagnosis of covid- developed loss of consciousness. the brain computed tomography (ct) scan was normal, but in the cerebrospinal fluid (csf), white blood cells were observed at . × /l by . g/l protein and . mmol/l sugar. although the result of the covid- test in csf was negative, the patient was treated with the diagnosis of covid- encephalitis. then, he was discharged in good general condition after further examinations and the rejection of other causes (ye et al. ) . also, rayan el-zein et al. reported a -year-old man without any risk factors and previous illness admitted with a diagnosis of covid- pneumonia and discharged after the treatment. however, he was brought to the hospital day after discharge with a decrease of consciousness level. magnetic resonance imaging (mri) and ct scans of brain were normal, but in the csf, white blood cells were detected with a lymphocyte preference of %. although the polymerase chain reaction (pcr) test of sars-cov- was negative in csf, the patient was treated with a diagnosis of the encephalitis caused by covid- due to the exclusion of other causes. intravenous immunoglobulin (ivig) was started for the patient. the patient's condition gradually improved. eventually, the patient was discharged in a good general condition (el-zein et al. ) . misayo hayashi et al. also reported a -year-old man with a history of alzheimer's disease who referred to the hospital with a complaint of tremor in his left hand and inability to walk and diarrhea in the same time. also, the ct scan of lungs showed the evidence in favor of covid- , confirmed by pcr testing. mri showed a unique lesion in the splenium of corpus callosum. unfortunately, he died during the hospitalization due to the pulmonary involvement, despite improving his neurological condition. due to improving the neurological conditions, the patient was diagnosed with mild encephalitis/encephalopathy with reversible splenial lesion (mers) caused by covid- (hayashi et al. ) . in addition to patients with encephalitis, the cases with encephalopathy are reported due to causes other than the direct invasion of sars-cov- . this encephalopathy can occur due to high inflammatory activity in covid- disease or hypoxia due to covid- -related lung involvement and pneumonia (berger ) . although the cases with encephalopathy are increasingly reported, no case of encephalopathy associated with covid- in patients with multiple sclerosis (ms) has been reported. herein, we reported a patient with ms who experienced the decreased level of consciousness and encephalopathy following covid- involvement. the patient was a -year-old woman with the established ms diagnosis for years. her disease had been * abdorreza naser moghadasi abdorrezamoghadasi@gmail.com multiple sclerosis research center, neuroscience institute, tehran university of medical sciences, tehran, iran initiated with lower limb paresthesia, and at the beginning, avonex was prescribed for her. subsequently, due to the frequent relapses of the disease, betaferon, and then dimethyl fumarate were administered, and finally treatment with rituximab was performed. however, despite all these medication changes, her disease has progressed. the brain mri revealed multiple confluent periventricular lesions with gadolinium enhancement (fig. a, b, c) . lumbar puncture (lp) was performed. csf analysis was normal. oligoclonal bands (ocbs) were present in csf. rituximab was discontinued, and the patient was treated with cyclophosphamide g per month along with mg oral prednisolone per day with a diagnosis of active progressive ms. before developing covid- , the patient fig. a, b brain mri revealed multiple confluent lesions with gadolinium enhancements. c cervical and brain stem involvements were seen in cervical mri. d chest ct scan revealed lung consolidation. e numerous old lesions were observed in the new brain mri only complained about the recurrent urinary tract infections which improved with oral antibiotics. the patient totally received cycles of cyclophosphamide. by passing days from receiving the last dose of injective cyclophosphamide, she complained of fever, coughs, dyspnea, generalized weakness, and nausea. in the first examination, the followings were measured: respiratory rate: , pulse rate: , temperature: °c, and bp: / . moreover, o saturation was % with no oxygen use. neurologically, the patient had complete time, place, and person orientation. in addition, she had imbalance during walk, and her reflexes had also increased. the muscle power of limbs was four-fifth, which had no difference with the examination performed before covid- . the chest ct scan showed bilateral lung involvement especially on the right side (fig. d) . so, the patient was hospitalized with covid- diagnosis. in the tests performed, white blood cell (wbc) count was , lymphocyte . %, c-reactive protein (crp) mg/dl, and lactate dehydrogenase (ldh) was . notably, nasopharyngeal and oropharyngeal swab real-time polymerase chain reaction (rt-pcr) test of coronavirus was positive. the patient received azithromycin and hydroxychloroquine. on day of hospitalization, the patient's fever increased up to °c, and o saturation was %. the patient found diminished consciousness level. afterward, she was localized in response to painful stimulus, and she also had neck stiffness. routine tests including blood sugar and biochemistry were normal. subsequently, ceftriaxone and vancomycin were administered for her, and then emergency mri was performed. in mri, numerous old lesions were observed in the periventricular area induced by ms, which had no enhancement in the injected view (fig. e) . moreover, lp was performed for her. the csf pressure was cm h o. in csf analysis, glucose was measured as , protein as , and wbc as . csf smear was negative in terms of bacteria. igg levels were low in the requested tests. due to the history of receiving rituximab and low igg levels, the patient was treated with the ivig mg/day for a -day period. complementary tests including tuberculosis, brucellosis, herpes simplex virus (hsv), and human immunodeficiency virus (hiv) were negative, and coronavirus pcr in csf was also negative. the patient health condition gradually improved as well as her consciousness conditions that returned to normal level. the patient's fever stopped, and she was discharged from the hospital with a good general status. as mentioned above, there are some reports on covid- -induced encephalitis and encephalopathy. the best diagnosis of this patient was covid- associated encephalopathy. covid- disease can cause brain involvement in different ways. firstly, coronavirus is a neurotropic virus, and through attaching to angiotensin converting enzyme (ace) receptor, which is located on cells surface such as astrocytes, can cause direct brain involvement. moreover, another route of involvement is through olfactory nerve and bulb. eventually, the hyper-inflammation induced by covid- can cause acute disseminated encephalomyelitis (adem)-like involvement in patients with covid- (abdi et al. ) . whether the ms disease itself or the drugs consumed (such as immunosuppressive drugs) can increase the chance of developing brain involvement of covid- is something that needs to be further explored. nevertheless, one cannot overlook is the effect of cyclophosphamide as an immunosuppressive drug on developing covid- associated encephalopathy in this patient. author contributions anm contributed to conception and design, described the case study, analyzed data and prepared the manuscript. conflict of interest the author declares that he has no conflict of interest. neurological manifestations and complications of covid- : a literature review encephalitis as a clinical manifestation of covid- covid- -associated meningoencephalitis treated with intravenous immunoglobulin covid- -associated mild encephalitis/encephalopathy with a reversible splenial lesion covid- and the nervous system the association of sars-cov- infection and acute disseminated encephalomyelitis without prominent clinical pulmonary symptoms key: cord- - dgmdtj authors: nan title: neurocritical care society th annual meeting: october - , sheraton denver downtown hotel denver, colorado date: - - journal: neurocrit care doi: . /s - - - sha: doc_id: cord_uid: dgmdtj nan ahrq guidelines for venous thromboembolism (vte) prophylaxis recommend risk stratification of patients and tailoring prophylaxis to that risk. while anticoagulation is a mainstay of optimal vte prophylaxis after trauma, little data exists to determine when tbi patients warranting neurosurgical intervention become candidates for such treatment. our group sought to determine the natural evolution of intracranial hemorrhage in these high risk patients and identify factors contributing to early radiographic stabilization. all tbi patients undergoing craniotomy and/or intracranial monitoring and surviving at least hours were followed prospectively from feb to nov . radiographic stabilization was defined as the time between injury and the final ct scan that showed no worsening during the hospital stay. kaplan meier (km) curves were used to compare time to stabilization by type of intervention. binary logistic regression was used to identify covariates contributing to stabilization within hours of injury. for the overall cohort (n= ), km curves showed no difference in time to radiographic stabilization by type of neurosurgical intervention. significant associations were found between stabilization at hours and higher presenting gcs (or: . , %ci . - . ), younger age (or: . , %ci . - . ), and male gender (or: . , %ci . - . ). subjects with a presenting gcs of > (n= ) had an % ppv for radiographic stabilization by hours after injury. the auc for the logistic regression model was . . sentinel headache refers to discrete thunderclap headache in the weeks preceding hospital admission for sah. a large proportion of these events are thought to represent aneurysmal bleeding events. repeat hemorrhages have been found to increase the extent of vasospasm in experimental models, and are often assumed to increase the risk of delayed cerebral ischemia (dci) in humans (the "double bleed effect"). cerebral performance category (cpc) is a standard outcome measure after cardiac arrest, but has limited ability to discriminate between mild and moderate brain injury. we hypothesized that many cardiac arrest survivors with good cpc scores would have significant deficits on blinded neurocognitive testing. patients initially comatose after cardiac arrest treated who awoke after therapeutic hypothermia (th) were evaluated by a neuropsychologist prior to hospital discharge with the repeatable battery for the assessment of neuropsychological status (rbans), a well-validated tool that assesses function in multiple domains compared to standardized normal values. patients admitted between nov and may awoke after th, completed the rbans evaluation after leaving the icu and ready for discharge. median age was yrs (range - ), % male, had initial rhythm vt/vf, median time to rosc was minutes (range - ). seven patients had a cpc of , patient had a cpc of , and patient had a cpc of . seven patients were discharged home and to acute rehab. attention and delayed memory were severely abnormal half of the patients (below th percentile), language and visuospatial domains were affected less often in % of the patients (below th percentile). on cumulative scores of all domains, all patients scored below the th percentile compared to age and education adjusted scores, regardless of cpc score. cardiac arrest survivors with cpc scores considered 'good' frequently had severely abnormal neurocognitive function just prior to hospital discharge. the cognitive domains most frequently affected were attention and delayed memory. more sophisticated testing with tools such as rbans may better identify components of cognitive dysfunction after cardiac arrest which may be targets for additional therapeutic intervention and be a more meaningful tool for long-term follow-up studies. introduction quantitative brain diffusion-weighted imaging (dwi) mri may help predicting the degree of functional recovery in patients ain volume with an apparent diffusion coefficient (adc) < x - mm /sec differentiated between cardiac arrest survivors who regained an independent lifestyle and those with impaired functional outcome. we aimed to validate this threshold in an external dataset. dwi mris of comatose post-cardiac arrest patients were obtained between - hours post-arrest. survivors who regained consciousness by day were assigned to one of two recovery groups: good recovery (discharged to home) and impaired recovery (discharged to a skilled nursing facility, rehabilitation facility or another hospital). the quantitative dwi data were obtained blinded to patient outcomes. the brain masks were semi-automatically created on the b images using medical image processing, analysis and visualization program (mipav). the adc values of each voxel within the brain were determined. data of patients from five us centers (columbia, mgh, mayo clinic jacksonville, northwestern, and stanford) with adequate mris were analyzed. of these, ( %) patients regained consciousness and survived to discharge: mean age ± years, % female, arrest duration ± minutes, % of patients received therapeutic hypothermia, mri obtained at ± hours post-arrest. the median (iqr) percentage of brain volume with adc< x - mm /sec was . % ( . - . ) in patients with good recovery (n= ) and . % ( . - . ) in patients with impaired recovery (p= . ). an adc< x - mm - ) sensitive and % ( %ci - ) specific for good recovery. the results of this validation study support earlier findings that quantitative dwi mri in comatose post-cardiac arrest patients is a sensitive prognostic test to predict the degree of functional recovery in post-cardiac arrest survivors. according to the universal determination of death act, death in the united states is determined in accordance with accepted medical standards, which can be national, regional, or local. as a result, significant variability in brain death (bd) determination has been reported among the best hospitals across the country. we tested the hypothesis that similar variability exists in individual states, such as michigan. michigan health and hospital association and gift of life of michigan (the local organ procurement organization) databases were reviewed for hospital bd policies. only hospitals with > beds and an intensive care unit were included. several bd determination process variables were extracted and analyzed with descriptive statistics. results / hospitals had bd policies, did not and in it was unclear. ten different combinations of physicians allowed to perform the exam were included. in . % there were no prerequisites to initiate bd and in . % no established cause mentioned. ten different temperatures to initiate bd exam were required. five different arterial blood carbon dioxide levels to establish positive apnea test were cited. a single bd exam was requested in . % of policies, a dual in . % and a single or dual in . %. confirmatory tests were optional ( %), recommended ( . %) or mandatory ( %). electroencephalogram was the most common confirmatory test ( %) and ct angiogram the least common ( %). we report significant variability in the bd hospital policies in michigan despite published guidelines from the american academy of neurology. if one accounts for additional variability in the strict implementation of these policies at the bedside level, the urgency for a uniform state-wide bd policy becomes even more obvious. intrathoracic pressure regulation (ipr) therapy is a novel therapy that non-invasively modulates pleural pressures to take advantage of the physiological benefits that occur by creating pressure differentials in the thorax. after each positive pressure breath ipr lowers intrathoracic pressure to subatmospheric levels relative to the rest of the body. this intervention enhances cardiac preload and output and decreases intracranial pressure (icp). we hypothesized that ipr therapy which has been previously shown to increase calculated cerebral perfusion pressures would also increase cerebral blood flow (cbf) in a porcine model of elevated icp. in this pilot study, four isofluorane anesthetized pigs ( . ± . kg) were subjected to a focal brain injury by epidural insertion of an french foley catheter into the left hemisphere which was slowly filled with saline to simulate a traumatic brain injury with elevated icp. in the right hemisphere, a thermal diffusion probe was used to measure cbf (hemedex, inc., cambridge, massachusetts) while a millar catheter was used to measure icp. once a stable elevated icp was confirmed, ipr therapy was applied at a level of - cmh o for minutes. end tidal co was held constant at mmhg by adjusting the respiratory rate during ipr use. tbi is a major risk factor for the development of alzheimer's disease (ad). in previous animal and human studies, an increase in the expression of amyloid precursor protein (app) after tbi was found to correlate with the disruption of neuronal activity, beta-amyloid plaque formation, cognitive decline, and even death.to date, no interventions used at decreasing amyloid plaque load after tbi have been identified. in this study, using the controlled cortical impact device we produced a severe head injury in month old xfad mice. at minutes and hours after injury, the xfad mice were treated intraperitoneally with either placebo or resveratrol (anti-oxidant; mg/kg). at month after injury, the animals were intracardially perfused with . % saline followed by % phosphate-buffered formalin. the whole brain was removed, sliced, and stained for beta-amyloid levels using immunohistochemistry. in addition, tunel+ cells were measured at the indicated time-points to determine the level of neural injury. in this study we found that treatment with resveratrol at minutes and hours post-injury resulted in a significant reduction in beta-amyloid plaque load near the injury zone (parietal cortex) (p< . ) and hippocampus (p< . ). also, the mice treated with resveratrol had reduced (p< . ) tunel+ staining. while a multitude of etiologies may lead to coma, treatments for coma remain elusive. the hypothalamic orexin pathway, critical in sleep/wake cycles, can stimulate multiple areas of the brain and provides a potential pharmacologic target towards improving arousal after coma. we used a post-cardiac arrest (ca) rodent coma model to assess whether postresuscitative orexin-a intracerebroventricular (icv) infusion after bolus injection would provide immediate and long term arousal after ca. seventeen adult wistar rats (male, - gms) were implanted with a icv cannula attached to an osmotic pump. one week later, rats underwent baseline eeg followed by -minute asphyxial ca. forty-five minutes after resuscitation, rats were randomized to either orexin-a (n= ) or saline (n= ) icv bolus and infusion. eeg was monitored continuously for hours after ca, and for minutes at hrs, hrs, hrs, and days post-ca. behavioral testing (neurologic deficit scale; nds) was also conducted at these times. eeg was quantitatively analyzed using information quantity (iq), an entropy based nonlinear previously established by us. rats receiving orexin-a almost immediately exhibited higher iq when compared to saline ( . ± . vs. . ± . ; p< . ). this acute improvement in iq appeared with slowest sub-bands (e.g.ð) improving first followed progressively by faster sub--ca. moreover, orexin--band at hrs ( . ± . -band at hrs ( . ± . vs . ± . ; p< . ). behaviorally, orexin-a allowed rats to perform significantly better on the nds at hrs ( . ± . vs. . ± . ; p< . ); hrs ( . ± . vs. . ± . ; p< . ); hrs ( . ± . vs. . ± . ; p< . ), and hrs ( . ± . vs. . ± . ; p< . ). heart rate variability (hrv) characteristics have been associated with outcome after traumatic brain injury. we sought to determine if hrv characteristics in the first hours after subarachnoid hemorrhage (sah) are associated with hospital morbidity and mortality. continuous ekgs recorded ( hz sampling) during the first hours post-sah was analyzed in of consecutively admitted patients between and . admission clinical scores, radiographic, surgical, ventilation and the pan-tompkins algorithm was applied to identify the qrs complex. fft calculations were generated for the following - . hz), low frequency (lf: . - . hz), very low frequency (vlf: . -nerated sample entropy and /f --minute (fft< . hz), or -minute individual multivariable logistic regression analyses of hospital morbidity and mortality controlling for admission hunt and hess grade, apache ii physiological sub-score, age, and mechanical ventilation status were conducted. dialysis disequilibrium syndrome (dds) is characterized by varying central nervous system manifestations secondary to cerebral edema that most often occurs after the first round of hemodialysis (hd). literature suggests that underlying brain injury may predispose patients to the development of dds. however, the pathophysiology has yet to be elucidated. herniation from hd is thought to be exceedingly rare with current dialysis methods and has not been reported in the era of modern neurointensive care. we present a case series of three patients with acute neurological injury undergoing hd in the intensive care unit that rapidly developed fatal brain edema, secondary to dds, even after several previous uneventful rounds of hd. three patients, ages , and years, with traumatic brain injury, hypertensive intracerebral hemorrhage, and ischemic stroke underwent hd in the intensive care unit. the number of dialysis sessions prior to the development of dds was , and . all three patients developed clinical signs of herniation within minutes to hours of hd. ct scans showed global cerebral edema with both transtentorial and tonsillar herniation. aggressive osmotherapy with mannitol and supersalt were ineffective in reversing the massive edema and all three patients died. two of the patients had a significant reduction of the bun ( % and %) while the third had only a modest reduction. our case series illustrates the potential dangers of hd in patients with acute neurological injury who have a high potential for worsening cerebral edema. it also reaffirms that dds with fatal cerebral edema can occur even after several rounds of hd and with current hd techniques. utilization of continuous veno-venous hemofiltration instead of hd may prevent the rapid shifts of osmoles and prove safer in neurologically injured patients. traumatic coma is believed to be caused by disruption of the ascending reticular activating system (aras), a complex network of arousal pathways projecting from the brainstem to the hypothalamus, thalamus, and basal forebrain. there is a critical lack of diagnostic tools for detecting which components of the aras network are disrupted in traumatic coma. we aimed to determine whether an advanced mri technique, high angular resolution diffusion imaging (hardi), can detect disruptions in the brainstem arousal network that are implicated in the pathogenesis of traumatic coma. we used hardi tractography to analyze neural network connectivity in two postmortem brains: one from a -year-old woman who died three days after traumatic coma, and one from a -year-old woman who died of non-neurological causes. both specimens were scanned as dissected blocks of the brainstem, hypothalamus, thalamus, and basal forebrain on a small-bore, high field ( . tesla) mri scanner. hardi tractography analyses were performed to compare the structural integrity of each component pathway of the aras network in the traumatic coma and control specimens. upon completion of imaging, both specimens were sectioned and stained for correlative histopathological analysis. hardi tractography revealed that specific components of the aras network, including known cholinergic, glutamatergic and noradrenergic projections connecting the brainstem to the thalamus and basal forebrain, were severely disrupted in the traumatic coma specimen, as compared to the normal specimen. these disruptions were consistent with histopathological tissue tears and axonal swellings. by contrast, connectivity between the brainstem and hypothalamus, and within the thalamus itself, was partially preserved in the traumatic coma specimen. hardi tractography can detect disruptions in specific components of the aras network that are implicated in the pathogenesis of traumatic coma. this advanced imaging technique may be used to elucidate the neuroanatomic basis of coma in individual patients. refractory intracranial hypertension (rich) is associated with high mortality rates and is the final pathway of many neurocritical entities, such as severe traumatic brain injury (stbi). objective: to determine modifications in intracranial pressure (icp) and cerebral perfusion pressure (cpp) following indomethacin (indo) infusion after rich secondary to stbi. indo was administered in a loading dose ( . mg/kg/ minutes), followed by continuous infusion ( . mg/kg/h) in patients with icp> mmhg for more than minutes who did not respond to first line therapies. changes in icp and cpp were observed. clinical outcome was assessed at -day according to glasgow outcome scale (gos). analysis of indo safety profile was also conducted. differences in icp and cpp values were assessed using repeated-measures anova with an a-level of p< . twenty-nine consecutive stbi patients ( men and women) with a mean age ±sd ± years wereincluded. median posresucitation gcs score at admission was (iqr: - ) with a predominance of grade iv in marshall ct classification. our findings support the effective and feasibility of indo in reducing icp and improving ccp in rich patients. future studies to evaluate different doses, lengths of infusion and longer-term effects together with effects on outcome are needed. hematoma expansion after acute intracerebral hemorrhage (ich) occurs most frequently in patients presenting within hours of symptom onset. therefore, most investigational therapies have been tested only in patients presenting ultra-early in their disease course. however, the majority of ich patients present outside this time window or with an unknown time of onset. we investigated the prevalence of hematoma expansion in these patients with delayed presentation and assessed the accuracy of the ct angiography (cta) spot sign for identifying risk of hematoma expansion. we performed a prospective cohort study. consecutive ich patients undergoing cta and follow-up head ct were enrolled over ten years. cta spot sign readings were performed by two experienced readers and hematoma expansion was assessed using semi-automated software. expansion was defined as an increase in volume of > ml or an increase of > % from baseline ich volume. hematoma expansion occurred in % of patients. when stratified by time from symptom onset to initial ct, hematoma expansion rates were: % within hours; % between - hours, % beyond hours (but with known onset), and % in patients with an unknown symptom onset time. of patients who developed hematoma expansion, only % presented within hours. the accuracy of the spot sign in predicting hematoma expansion was . for patients presenting within hours, . between to hours, . after hours and . for patients presenting with an unknown onset time. a substantial number of patients destined to suffer from hematoma expansion present either late or with an unknown time of symptom onset. the cta spot sign accurately identifies patients destined to expand regardless of time from symptom onset, and may therefore open a path to offer clinical trials and novel therapies to the many patients who do not present acutely. intraventricular fibrinolysis has been shown effective in clearing intraventricular hemorrhage (ivh) in small series of patients. we present our experience with using fibrinolytics over years. retrospective analysis of prospectively collected data of patients with ivh admitted to two neuro-icus and treated with rt-pa instillation (one patient with tenecteplase) via intraventricular catheter (ivc) until the rd and th ventricles were cleared of blood. all patients were treated by the same neurointensivist with the same instillation protocol but different doses of drug based on individual patient characteristics. the graeb and leroux semi-quantitative scales were used to measure the amount of ivh before and after the last dose. patients (mean age . years, . % male) were admitted with a median gcs . thirty-one had intracerebral and aneurysmal subarachnoid hemorrhage, brain tumors, head trauma, arteriovenous malformation.and primary ivh. t-pa was administered at a total dose of . ± . mg (individual doses ranging between . to mg), with st dose . ± . hours from admission and for a duration of . ± days. the pre-fibrinolysis graeb and leroux scores were . ± . and . ± . and decreased post fibrinolysis to . ± . and . ± . (p< . ). a significant correlation between total fibrinolytic dose and difference in pre-post amount of ivh was found for the leroux scale (pearson . , p < . ). three patients had small tract bleeds (< cc, with one bleeding profusely at the incision site requiring transfusion) and one had extension of ich in the upper midbrain. no patient developed ventriculitis. the total dose of t-pa was lower in patients who received shunt, compared to who did not ( . ± . vs . ± , p< . ). eighteen ( . %) patients had -up of . ± . days. in our large series of patients, intraventricular fibrinolysis significantly decreased ivh with minimal complications. distinguished poster ___________________________________________________________________________________ white matter lesions significantly impact on outcome after aneurysmal subarachnoid hemorrhage (asah). brain extracellular tau is indicative for axonal injury, associated with poor neurological outcome after severe traumatic brain injury, however has not been elucidated so far in patients with asah. twenty-five consecutive asah patients monitored with cerebral microdialysis (cmd) and brain tissue oxygen tension (p b to ) were included. cmd total tau, phospho-tau- and beta-amyloid -- ) levels were analyzed at a hours interval until d and -hours interval until d using an elisa-technique (innogenetics). statistical analysis was performed with non-parametric tests and a mixed effects model as appropriate. median age was y ( - y) and admission hunt&hess grade ranged from to . cmd-tau, phospho-tau-- were detectable in all patients. probe location in perilesional tissue revealed a higher overall cmd-tau level (p< . ) - and phospho-tau. cmd-tau positively correlated with cmd-lactate (r= . , p< . ). brain hypoxic (p b to were associated with increased cmd-tau levels (p< . ). no correlation was found between other variables besides a higher phospho-tau level and cmd samples categorized as brain hypoxic hyperlactatemia. patients with poor outcome -tau level during hospital-course (p< . ) but no difference in phospho-tau-- (adjusted for disease severity). cerebral tau is elevated after asah and associated with perilesional probe location and poor -months functional outcome. association with brain-morphological abnormalities and neuropsychological deficits need further investigations. neurocrit care ( ) :s -s to date only two studies have evaluated anemia status in acute intracerebral hemorrhage [ich] . on admission anemia [oaa] was associated with larger hematoma volume and lower hemoglobin levels during hospital stay were related to poorer outcome. it remains unknown whether anemia impacts outcome primarily through its effects on ich volume or itself has independent effects. this retrospective analysis included consecutive patients with spontaneous supratentorial intracerebral hemorrhage. clinical data including the pre-admission-status, neuroradiological, initial presentation, treatment, and outcome were evaluated through institutional databases, patient's medical charts and by mailed questionnaires. multivariate logistic and graphical regression analyses were calculated to evaluate associations of oaa with functional outcome and to determine independent effects of oaa. oaa was associated with larger ich volume ( . cm³ versus . cm³, p= . ), greater extent of intraventricular hemorrhage [ivh] (p= . ) and poorer neurological status on admission (p< . ). further, oaa showed a true positive and accurate association with larger hemorrhage volumes (roc: p= . ,auc> . ). multivariately, for all patients despite age, only oaa could be elucidated as independent predictor of unfavorable functional outcome (mrs > ) at days (or= . ;p= . ). comparison of separate multivariate models revealed: for oaa-patients no independent predictor could be identified, whereas in non-oaa patients ich volume demonstrated known independent effects on functional outcome (or . ;p= . ). within this study oaa was shown to be a significant predictor of an unfavorable functional outcome and has independent effects beyond its accurate association with larger hemorrhage volumes. oaa appears to be a very relevant and previously unrecognised predictor of functional outcome at days. the recognition of anemia and its treatment could possibly open up new therapeutic avenues to decrease the rate of functionally dependent patients after ich. this strongly supports the need of prospective interventional studies to evaluate the influence of anemia in patients with intracerebral hemorrhage. in patients with suspected subarachnoid hemorrhage and negative brain imaging, lumbar puncture is recommended. this test is frequently complicated by false-positive results due to a traumatic tap. we hypothesized that blood precipitating in the thecal sac following non-traumatic subarachnoid hemorrhage would be visible on mri. a prospective database for subarachnoid hemorrhage was searched for patients who received mr lumbosacral spine imaging during admission for subarachnoid hemorrhage. electronic chart review was completed. all mr studies were read and interpreted by a neuroradiologist. patients (n= ) with subarachnoid hemorrhage underwent delayed mri imaging of the lumbosacral spine an average of days (range - days) after the onset of symptoms. the median hunt-hess grade for this cohort was (range - ). the median fisher grade was . blood precipitating in the thecal sac was visible in out of patients ( %). the density of blood compared to csf was hyper-intense on t (bright) and hypo-intense on t (dark). the blood was most evident at l and s levels and layered in a dependent fashion. delayed ct head non-contrast obtained at the time of the mri ls spine demonstrated resolution of subarachnoid hemorrhage in / patients and a small amount of isodense intraventricular hemorrhage layering in the occipital horns was detected in / patients. delayed neuroimaging with ct head after subarachnoid hemorrhage has a high false negative rate. mr imaging of the lumbosacral spine detected persistent blood products settling in the thecal sac despite clearance of subarachnoid blood on ct head imaging. mr lumbosacral spine imaging could serve as a 'virtual lumbar puncture' in patients with suspected subarachnoid hemorrhage. stroke patients receiving iv tpa can be admitted to an icu or a stroke unit (su) but su admission may be more costefficient. we compared icu admission vs su admission in tpa-treated patients. during the initial years of this retrospective study, patients were admitted to the icu as we lacked a su. in the following years, patients were admitted to a new su. demographics, medical history, nihss, treatment interventions, neurologic and medical complications, and mortality were collected to determine if icu admission resulted in better outcome and less complications. categorical variables were analyzed with fishers exact test and continuous ones with proportion of the means test (t-test). we compared icu admissions and su admissions. icu admission included % males and su admission included % males (ns). median age for icu and su admission was and respectively (ns). admission nihss was for icu patients and for su patients (ns). the median length of stay in the icu was day (as per protocol) and the median su length of stay was days. intravenous anti-hypertensives (bolus) were used in % of icu patients and in % of su patients (p= . ) and continuous infusions in % of icu patients and % of su patients (p= . ). initial nihss scale of > predicted need for mechanical ventilation (p= . ). intracranial hemorrhage occurred in % of icu and % of su patients (ns). complications (pneumonia, venous thromboembolism, sepsis, or death ) did not differ. there was no difference in the proportion of patients with mrs of or less in the two groups ( % vs %). admission to the su resulted in savings of $ , per patients/day. patients receiving tpa can be safely admitted to a su resulting in significant cost savings. patients with nihss > are likely to need icu admission for mechanical ventilation. stroke patients with dysphagia have a high incidence of aspiration, which may lead to pneumonia. evidence suggests that ace inhibitor use may decrease the risk of pneumonia via their inhibitory effects on substance p degradation. the objective of this study was to investigate the association between ace inhibitor use and the development of pneumonia in hospitalized stroke patients. a retrospective case-control analysis was performed. eligible patients (n= ) were individuals admitted to saint louis university hospital with a diagnosis of acute ischemic stroke, spontaneous intracerebral hemorrhage, or non-traumatic subarachnoid hemorrhage between march st , and november th , . patients greater than years of age, who died or were discharged to hospice within days of admission, or who had a baseli excluded. cases were patients with an icd- code for pneumonia or antibiotic treatment course for at least days with a positive respiratory culture. controls were patients without pneumonia matched using primary diagnosis, baseline demographics, history of prior stroke, diabetes, hypertension, heart failure, and initial nihss scores. ace inhibitor use, length of stay, discharge disposition, and other pertinent data were collected and analyzed using descriptive statistics, chisquare, and logistic regression. there is growing evidence supporting the role of inflammation in aneurysmal subarachnoid hemorrhage (asah) pathophysiology and it is of great interest to elucidate which immune mechanisms are involved. methods asah patients (sahp) and healthy control subjects (cs) were enrolled prospectively. the protocol was authorized by the ethics committee of our hospital and all subjects (or patient next of kin) signed an informed consent. the median age of sah patients was years ( - ) and of control subject was years ( - ). we assessed leukocytes subpopulations and their activation status by multiparametric flow cytometry in cerebrospinal fluid (csf) and peripheral blood (pb) of sahp at the same time and in pb of cs. we found an increase in cd +-monocytes percentage (p= . ) in csf compared with pb in sahp and a decrease in pb of sahp compared with cs (p= . ). sahp also showed a marked increase in the expression of cd (activation antigen) in pb cd +t cells compared with cs (p= . ). additionally, csf cd +t cells showed a decreased expression of cd (p= . ) and cd (p= . ) (activation markers) compared to pb cd +t cells in sahp. similarly, pb cd +t cells in sahp showed an increased expression of cd compared with cd +t cells of cs (p= . ). csf cd +t cells showed a decreased expression of cd (p= . ) and an increased expression of cd compared with pb cd +t cells (p= . ). b and nk cells were decreased in sahp compared with cs (p= . and p= . respectively). as far as we know this is the first report that analyzes leukocytes subsets in csf and pb in patients with asah. our data suggests not only csf leukocytes recruitment (from the blood) but also an increase status of activation at this level. overall, these results indicate that asah probably stimulates both the innate and adaptive immune responses. subdural hematoma (sdh) is a common diagnosis in neurosurgical and neurocritical practice. comprehensive outcome data and management guidelines are lacking for non-traumatic sdh. thus, we aimed to determine factors associated with in-hospital mortality in a large sample of patients with non-traumatic sdh. using the nationwide inpatient sample, we included adults with a primary diagnosis of acute non-traumatic sdh (icd- code, . ) hospitalized in the united states between and . demographics, comorbidities, craniotomy treatment and discharge outcomes were identified. univariable and multivariable analyses were performed to identify predictors of in-hospital mortality. of patients with non-traumatic sdh, the mean age was . (sd . ) with % male, and . % admitted during the weekend. surgical evacuation was performed in . % of patients; . % ( . % of patients requiring surgical evacuation) required a second craniotomy. death during hospitalization occurred in . % of patients. factors significantly associated with higher in-hospital mortality included increasing age, female sex, comorbidities (congestive heart failure [chf] , coagulopathy, renal failure, liver disease), mechanical ventilation during the first days (mv), premorbid warfarin use, repeated sdh evacuation, and admission during the weekend. craniotomy was associated with decreased in-hospital mortality. in multivariable analysis, age (or . , % ci . - . ), female sex (or . , % ci . - . ), chf (or . , % ci . - . ), warfarin use (or . , % ci . - . ), mechanical ventilation (or . , ) and weekend admission (or . , % ci . - . ) were independent predictors of inhospital mortality. surgical sdh evacuation was a strong independent predictor for decreased mortality (or . , % ci . - . ). one in nine patients with non-traumatic sdh dies during hospitalization. of several predictors of mortality, the weekend effect and the decision for or against surgical evacuation are potentially modifiable factors. further investigation may lead to improvement of management practice and better outcomes. to determine the burden of structural damage of the central nervous system (cns) in patients who died in the setting of non-neurological critical illness. critically ill patients who died in the medical, surgical or cardiac icus over a year period and underwent autopsy were included. patients with known cns lesions, cardiac arrest, and those from neurological icus were excluded. brain specimens were reviewed by a neuropathologist and classified according to location and lesion type (infarct, hemorrhage, inflammation). acute brainlesions were found in of patients studied. mean gcs at admission was lower in patients with neuropathological findings ( . vs. . ; p= . ). the most common sites of injury were cortex ( . %) and hippocampus ( . %). infarcts ( . %), hemorrhages ( . %), and signs of inflammation ( . %) were the most frequent findings. patients with septic shock and ali/ards had more lesions than patients without these critical illnesses, albeit these differences were not statistically significant. ischemic brain injury is prevalent in patients dying from non-neurological critical illness and may occur secondary to cns hypoperfusion. efforts to optimize brain oxygen delivery during critical illness may be neuroprotective. after ca, microcirculatory reperfusion disorders develop despite adequate cerebral perfusion pressure. increased blood viscosity strongly hampers the microcirculation resulting in plugging of the capillary bed, arteriovenous shunting and diminished tissue perfusion. the rheologic properties of blood depend on hematocrit and plasma constituents, mainly acute phase proteins. the aim of the present study was to assess blood viscosity in relation to cerebral blood flow in patients after a cardiac arrest. we performed an observational study in comatose patients after cardiac arrest. patients were treated with mild therapeutic hypothermia for hours and passively rewarmed to normothermia. blood viscosity was measured ex-vivo at , , , , , and hours after admission using a contraves ls viscometer. mean flow velocity in the middle cerebral artery (mfv mca ) was measured by transcranial doppler (tcd) at the same time points. the median viscosity on admission was . ( . - . )mpa.s, remained stable at . ( . - . )mpa s and . ( . - . )mpa s at and hrs respectively (p= . ). from hrs after admission viscosity decreased significantly to . ( . - . )mpa s (p< . ). median mfv mca was low ( . ( . - . )cm/s) on admission, and significantly increased to . ( . - . ) cm/s at hrs (p < . ). there was a significant association between the viscosity and the mfv mca (p= . ). median hematocrite was . ( . - . )l/l on admission and subsequently significantly decreased to . ( . - . ) l/l at hrs (p < . ) in contrast, acute phase proteins such as crp and fibrinogen increased during admission (from . ( . - . )mg/l to ( - . )mg/l and ( - )mg/l to ( - )mg/l respectively (p < . ). viscosity decreases in the first days after cardiac arrest and is strongly associated (correlated) with an increase in cerebral blood flow. since viscosity is a major determinant of cerebral blood flow, repeated measurements may guide therapy to restore cerebral oxygenation after cardiac arrest. initial hemorrhage burden is an independent predictor for delayed cerebral ischemia (dci) in patients with aneurysmal subarachnoid hemorrhage (sah). among the different definitions of blood burden, cisternal plus intraventricular hemorrhage volume (cihv) has been regarded as the most sensitive blood volume definition in predicting dci. however, it is not clear whether clot clearance is associated with dci. quantitative analysis of hemorrhage volume and clot clearance was made in consecutive patients who were scanned within hours from onset. cistenal plus intraventricular hemorrhage volume (cihv) was calculated for clot burden analysis. serial cihv was measured up to days after sah onset. clot clearance was calculated up to days as a percentage of residual clots compared to the initial scan. initial clot burden and clot clearance were compared in patients with and without dci. included patients were . ± . years old with female preponderance ( . %, ( / )). dci was developed in patients ( . %). conventional risk factors were not different between patients with and without dci including age, sex, ht, dm, smoking, admission h&h scale and apache score. patients with dci had higher cihv ( . ml, ) compared with those without dci ( . ml, iqr ( . had higher odds for dci (or . , % ci ( . - . , p = . ). however, clot clearance rate was not different between patients with and without dci (day : . % vs. . %, p = . , day : . % vs. . %, p = . , day : . % vs. . %, p= . ). quantitative clot clearance rate using cihv is not associated with the development of dci while initial cihv is an independent predictor for dci. the majority of patients who die from subarachnoid hemorrhage have withdrawal or limitation of care and a focus on comfort at the end of life. ethnic disparities at the end of life has been examined in general critical care settings but not specifically in brain injured patients. patients with aneurysmal subarachnoid hemorrhage were prospectively followed in an observational database from august to january . demographic information including ethnicity was collected from medical records and self reported by patients or their family. significant in-hospital events including care withheld or withdrawn (comfort measures only, cmo) and mortality was recorded prospectively. included were patients of white, black or hispanic race. patients were included in our analysis: whites, blacks and hispanics. age was the only baseline characteristic that was different between groups. whites ( ± years) were older than blacks ( ± years) and hispanics ( ± years). no difference in morality was seen: % in whites, % in blacks, % in hispanics. cmo was more commonly ordered for whites ( %) than blacks ( %) and hispanics ( %) (p= . ). in multivariate analysis controlling for age and initially hunt-hess grade hispanics were less likely to have cmo orders than whites (or, . ; %ci, . - . ; p= . ). of the patients who died % of whites had cmo orders compared to % of blacks and % of hispanics (p< . ). in multivariable analysis controlling for age and hunt-hess, blacks (or, . ; %ci, . - . ; p< . ) and hispanics (or, . ; %ci, . - . ; p< . ) were less likely to die with cmo orders than whites. multiple assessment measures are used to evaluate post-aneurysmal subarachnoid hemorrhage (asah) outcomes / complications. the use of a common measure has not been established, thus choosing which measure to control for becomes difficult when conducting multivariable analysis in clinical research. we compared odds ratio (or) and positive predictive value (ppv) to determine measures with strongest associations with post-asah complications / outcomes. subjects (n= ) with asah were recruited from an ongoing study with measures were assessed on admission: hunt and hess (hh), fisher, claassen, glasgow coma scale (gcs), world federation of neurological surgeons (wfns), and nih stroke scale (nihss). dependent variables were measured as follows: delayed cerebral ischemia (dci) was defined as clinical deterioration due to cerebral ischemia, moderate/severe vasospasm was diagnosed using sonography/angiogram, infarction was diagnosed via head ct scan. three and month outcomes were assessed by barthel index and modified rankin scale (mrs). logistic regression and spearman correlation were used. when predicting vasospasm and dci (controlling for age, gender, clipping/coiling), fisher scale had the largest ors ( . and . ), with a ppv of . % and . % (p<. ), respectively. when predicting infarction, hh had the largest or ( . ) with a ppv of ( . %); p=. . all scales were significantly associated with poor mrs ( - ); p<. . for and -month poor mrs, fisher scale had the largest or ( . and . ) with a ppv of . % and . %, respectively. admission nihss had the largest correlation coefficient (-. ) with -month barthel index while wfns had the largest correlation coefficient (-. ) with -month barthel index (p<. ). fisher scale has the strongest association with vasospasm, dci and mrs, while hh has the strongest association with infarction. we recommend clinical studies control for fisher when investigating vasospasm, dci, and mrs and for hh when investigating infarction to determine independent risk factors. to date, there has been a shortage of evidence-based quality improvement initiatives that have shown positive outcomes in the neurosurgical patient population. a single-institution prospective intervention trial with continuous feedback was conducted to investigate the implementation of a urinary tract infection (uti) prevention bundle to decrease the catheterassociated uti rate. all patients admitted to the adult neurological intensive care unit (neuro icu) during a -month period were included. the study consisted of two -month pre-intervention observation periods (approximately catheter days) followed by a month intervention phase ( , catheter days). a comprehensive evidence-based uti bundle encompassing avoidance of catheter insertion, maintenance of sterility, product standardization, and early catheter removal was enacted. the urinary catheter utilization rate dropped from % to . % during the intervention phase (p < . ) without any increase in the rate of sacral decubitus ulcers or other skin breakdown. the rate of catheter-associated uti was also significantly reduced from . to . infections per catheter days (p < . ). there was a linear relationship between the decreased quarterly catheter utilization rate and the decreased catheter-associated uti rate (r = . , p < . ). this single-center prospective study demonstrated that a comprehensive uti prevention bundle along with a continuous quality improvement program can significantly reduce the duration of urinary catheterization and rate of catheterassociated uti in a neuro icu. continued efforts to reduce ca-uti beyond the study resulted in sustained reductions when all components of the bundle were in place and daily foley rounds were maintained as a nursing intervention. matrix metalloproteinases (mmps) are extracellular proteolyic enzymes that may modulate the neuroinflammatory response to brain injury. we sought to determine the effect of mmps on pro-inflammatory cytokine production following severe traumatic brain injury (stbi). as part of a prospective cohort study, adults with stbi underwent multimodal monitoring with high cutoff, cerebral microdialysis and arterial and jugular venous bulb catheters. the concentration of mmps and pro-inflammatory cytokines were measured in microdialysate and blood over -days. interleukin- -alpha (il- ), il- -beta, il- , il- , and tumor necrosis factor-alpha (tnf-alpha) concentrations were initially high in microdialysate and then declined to low levels. the microdialysate concentration of il- also declined after first being high, but then increased between -and -hours. with the exception of il- , il- , and tnf-alpha, the cytokine blood concentration was low to undetectable. using generalized estimating equations, we observed a positive change in the microdialysate concentration of il- [( . pg/ml)/(pg/ml); % ci, . to . ] with an increase in the mmp- microdialysate concentration. in contrast, a significant increase in the microdialysate concentration of mmp- was seen with an increase in il- -alpha [( . pg/ml)/(pg/ml); % ci, . to . ] and il- -beta [( . pg/ml)/(pg/ml); % ci, . to . ]. in blood, a significant change in mmp- occurred during an increase in the levels of il- -beta [( . pg/ml)/(pg/ml); % ci, . to . ] and il- [( . pg/ml)/(pg/ml); % ci, . to . ]. although il- levels were higher in cerebrospinal fluid (csf), no major difference in mmp or cytokine concentration was observed between arterial and jugular venous blood or, for the three patients who were also fitted with csf drainage catheters, between cerebral microdialysate and csf. stbi is associated with a substantial central cytokine or neuroinflammatory response, which may influence or be influenced by production of mmps. severity classification of traumatic brain injury (tbi) has traditionally been based on the glasgow coma scale (gcs), with mild tbi being defined as - . however, there is often a subset of "mild" tbi that requires surgical intervention. the current study examines this subgroup to decipher any symptomatology that may be helpful in identifying who these patients may be. this observational cohort study included consecutive adult patients presenting with a tbi. independent variables included vomiting, seizure, loss of consciousness (loc), alteration of consciousness (aoc), and post-traumatic amnesia (pta); these were tested for correlation with surgical intervention, the dependent variable. data were entered into redcap, a clinical data capture system housed in our center for translational science institute. the z-test for proportions was used to determine significance of symptomatology. statistical analyses were performed in jmp . for the mac. of the total mild tbi cohort (n= , ), % were male. the median age was (iqr: - , r: - ). thirty seven patients required surgical intervention. symptoms significantly associated with surgical intervention on univariate regression included vomiting (p= . ), and aoc (p . ). multiple regression analysis revealed that time (length of) loc (p= . ) and pta (p< . ) were also significantly correlated with surgical intervention. age was also a statistically significant predictor of surgical intervention (p< . ). these pilot data suggest that older patients, as well as patients who present with vomiting, loc, or pta, have a significant likelihood of requiring surgical intervention. this calls attention to proactively seeking these data and ensuring adequate neuroimaging for all patients with tbi, regardless of gcs score at presentation. the prevalence of chronic subdural hematoma (sdh) is expected to increase with an aging population and increased use of anticoagulants. we aimed to develop a tool to predict mortality after sdh. a prospective study was conducted between - of patients with chronic subdural hematoma (n= ) admitted to a tertiary neuro-icu. three-month mortality data was collected. after testing admission demographic, radiographic and - , - , ; p= . ) and herniation (p= . ) were found to be independent predictors of death in multivariate logistic regression analysis. a score was composed ( - ) with each variable weighted based on its independent strength of association with mortality (b value) as - = , gcs - = , herniation= point. overall, % of patients died and -month mortality increased with each point of the sdh score ( = %, = %, = %, = %, = %). the sdh score predicted death (or . , % ci . - . , p< . ) with an area under the curve of . , sensitivity . %, specificity . %, ppv % and npv %. the hosmer and lemeshow and nagelkercke r for this model were . and . , respectively, indicating a strong model. sdh evacuation reduced the odds of death by % when added to a multivariate model including age, gcs and herniation (adjusted or . , % ci . - . , p= . ). the sdh score allows for a reliable prediction of mortality for patients with chronic sdh. this score may help risk stratify patients for surgical treatment. we developed a novel method capable of determining the degree of conformance of observed morphological changes of intracranial pulses with their expected patterns associated with global vasodilatation and vasoconstriction, respectively. these patterns were formed as a template consisting of pulse morphological changes during co tests that were consistent for multiple subjects. we used this novel pulse morphological template matching (pmtm) algorithm to study ) the incidence of cerebral vasoconstriction/vasodilatation associated with lpr increase episodes; ) how likely cerebral vasoconstriction/vasodilatation could lead to or lag behind lpr increase. we studied microdialysis data samples collected in an average interval of . hours from severe tbi patients. the lpr increase episodes were automatically identified using a moving time-window of hours. the pmtm algorithm was applied to the continuous intracranial pressure (icp) signal time-synched to the identified lpr episodes. across all subjects, more than half of the lpr increase episodes are not associated with any detectable cerebral vasoconstriction or vasodilatation (p = e- ). comparing lpr episodes with either vasoconstriction or vasodilatation, it was more likely that vasoconstriction rather than vasodilatation occurred during an lpr increase episode (p = . ). also for out of subjects with dominant number of vasoconstrictive lpr episodes, a causality relationship between vasoconstriction and lpr increase were observed, i.e., vasoconstriction occurred in one hour before lpr increase started. across the tbi subjects studied, the incidence of either vasoconstriction or vasodilatation associated with lpr increase was low. however, about percent of subjects had a dominant number of lpr increase episodes associated with cerebral vasoconstriction. furthermore, cerebral vasoconstriction occurred within one hour preceding lpr increase. placement of an intracranial pressure (icp) monitorto guide the management of severe traumatic brain injury (tbi) patients has been historically performedby neurosurgeons. trials have suggested decreased morbidity and mortality with timely resuscitationand rigorous treatment of intracranial hypertension. we hypothesize that icp monitors can be placed by non-surgeon neurointensivists, with placement success and complication rates comparable to neurosurgeons. we retrospectively reviewed the medical records of tbi patients who required insertion of parenchymal icp monitors from may to december in a large level i trauma center. monitor placement was performed by neurointensivists (board certified by the abim in critical care medicine and by the ucns in neurocritical care). patient data recorded are age, gender, ct findings, icp monitor placement location and length of placement, complications related to the icp monitor, and patient outcomes. twenty seven ( ) these findings were comparable to published outcomes from neurosurgeon placements. we believe that insertion of parenchymal icp monitors should be considered a core skill for neurointensivists and should be included in neurocritical care fellowship training. insertion of icp monitors by neurointensivists is safe and may aid in providing prompt monitoring of patients with severe tbi. use of computers at the bedside for trending primary signals like icp or cpp brings obvious advantages in neuro-critical care unit. software can be extended to calculate secondary indices reflecting underlying pathophysiological phenomena, like disturbance of cerebral compensatory reserve and vascular reactivity. during - more than severe tbi patients were monitored using icm+ software. various modalities were used, including icp, abp, pbtio , nirs, tcd blood flow velocity, brain temperature, etc. from icp and abp waveforms secondary indices were extracted. compensatory reserve was assessed using moving correlation index between slow changes in pulse amplitude and mean icp (rap). pressure reactivity index (prx) was calculated as moving correlation between mean icp and abp. 'optimal cpp' (cppopt) was estimated as cpp corresponding to the best cerebrovascular reactivity within the period of past hours. trending compensatory reserve showed that usually it is good (rap around ) in the first few hours after admission (rap around ), with gradual deterioration triggered by aggravating brain edema. in most cases rap stayed close to + (impaired reserve). it decreased to negative values (exhausted reserve) on top of plateau waves and in refractory intracranial hypertension, indicating critical icp. prx proved to be highly variable, responding to changes in abp, icp and ventilation. it deteriorated on top of plateau waves, and at extreme values of cpp. in cases of refractory intracranial hypertension, deterioration of reactivity seemed to preceed the elevation of icp above mmhg. cppopt fluctuated during the monitoring period. absolute distance between current cpp and cppopt was strongly associated with outcome. too low cpp (below cppopt) correlated with greater mortality rate (p< . ) and too high cpp -with greater rate of severe disability (p< . ). individual observations of secondary indices calculated by icm+ software help in better interpretation of primary signals in intensive care of tbi patients. financial support: the software for brain monitoring icm+ is licensed by the university of cambridge (cambridge enterprise). authors ps and mc have a financial interest in a part of the licensing fee. to determine the differences in hospital outcomes among adult mild traumatic brain injury (tbi) patients where the severity of tbi is defined by glasgow coma scale (gcs) score. this is a retrospective chart review of consecutive adult who came to the ed department of a tertiary care hospital in north central florida. the tbi severity was classified according to gcs score, with patients with gcs score of - categorized as having mild tbi. outcome variables such as admission status, icu admission status, in-hospital death and -month death among patients with different mild gcs scores of , , and . we had a total of mild tbi patients in the specified period of time. the majority of this cohort had a gcs of ( or . %). this was followed by a gcs of ( or . %) and gcs score of ( or . %). there was a statistically significant difference between mild tbi with gcs , , (p< . , anova) with the outcomes of hospital admission ( % vs. % vs. %), icu admission ( % vs. %, vs. %), in hospital death ( % vs. % vs. . %), and month death rate ( % vs. %, vs %). there is a % increase in hospital admission rates for each point decrease in gcs score. the -month death rate nearly doubles with each incremental decrease in gcs score. there is a significant difference in outcome within "mild" head trauma across the continuum. to characterize the patterns of presentation of children with head trauma to the pediatric emergency department. this is an observational cohort study that sought to collect injury and outcome variables with the goal of characterizing the very early natural history of pediatric traumatic brain injury in children over the age of years. statistical analyses were performed using jmp. cohort (n= ) . similar multivariate model showed that as children grew older, they were more likely to be admitted in hospital because of a tbi as a result of recreational activities (p= . ) and traffic accidents (p< . ), and less likely due to sport tbis (p= . ) with adjr = %. % of the children who were admitted ended up in icu with mean icu-los of day with an iqr of - . one percent had an in hospital death. kids with amnesia were significantly more likely to be admitted to the icu (p= . , r = %). children who got admitted to icu (p= . ) and were older ( . ), were significantly more likely to be readmitted to the hospital within days. these preliminary data suggest that pediatric brain injury is not without significant morbidity. the objective of this study was to identify pre-hospital markers of in-hospital mortality in traumatic brain injury (tbi) patients due to fall. this study was an observational cohort study performed at a level- trauma center. study subjects included all adult arriving in emergency department with history of tbi due to fall over a period of months. study variables were symptoms such as vomiting, seizure, loss of consciousness (loc), alteration of consciousness (aoc) & post-traumatic amnesia (pta), glasgow coma scale (gcs) scores, vitals, pre-hospital glucose. jmp for windows & z-test of proportions were used to perform statistical analysis. the study cohort comprised of adult (median age of yrs and iqr of - ). in-hospital death (ihd) was observed in % (n= ) of the total cohort, with male ihd= ( %) greater than female ihd= ( %). pta ( %,p< . ),loc ( %, p< . ) & aoc ( %, p< . ), higher pre-hospital glucose (p= . ) were individually found to be much more significantly associated with ihds versus the whole cohort. multivariate regression analysis showed significant correlation with ihds with: ) higher age (p< . ) when adjusted for severe gcs. ) vomiting (p= . ) and longer duration of loc (p= . ), when adjusted for rest of the symptoms. % of patients presenting to ed with vomiting (p= . ) had gcs score= , and % of that sub-group suffered ihd patients presenting to emergency department with higher blood glucose and symptoms such as pta, aoc, longer duration of loc & vomiting, were more likely to have worse outcome of in-hospital deaths compared to rest of the patients. hence identifying these symptoms in fields might help to make key decisions for providing intensive care and improving the overall patient outcomes. to determine which symptoms affect severity in pediatric traumatic brain injuries (tbi). study design-this observational cohort study was performed at a level one trauma center that has a dedicated pediatric emergency department. consecutive patients age - were included. the age cutoff of years was used because it was decided that younger children may not be ale to report their symptoms, particularly to endorse aoc (alteration of consciousness) or pta(post-traumatic amnesia). the dependent variables were vomiting, seizures(sz), loc (loss of consciousness), aoc, and pta at the time of the head injury. the independent variable was ed tbi severity based of the glasgow coma score, with mild being defined as - , moderate as - , and severe as less than . the median age of the cohort was with an interquartile range of to . % were boys. in the univariate model, all symptoms except vomiting were statistically significant: seizures (p= . ); loc (p= . ); aoc (p< . ), pta (p< . ). multiple regression analysis of these factors revealed all of variables to retain statistical significance. the r coefficient of determination was %, which means that almost one-third of the variance can be explained by just these five factors (symptoms), suggesting that our multivariate model is a robust one. symptoms at time of head injury in children including seizure, loc, aoc and pta were statistically significant predictors of the severity of tbi. this data allows clinicians to judge the severity of the tbi depending on the symptoms at presentation. these pilot data may be useful in designing clinical care algorithms. icp dynamic system of an injured brain is susceptible to various acute changes disturbing the system homeostasis that should be detected by icp monitoring. such a capability is particularly useful for comatose patients. our aim was to demonstrate a novel approach to detect acute deviation from steady state of an icp dynamic system without involving significant mean icp changes. steady state of icp dynamic systems is reflected as icp pulses of similar mean icp resembling each other. therefore, a steady state indicator can be calculated by quantifying inter-pulse distances after matching their mean icp. besides euclidean distance and pearson correlation, geodesic distance was introduced as a novel metric. these different metrics were evaluated on three types of continuous icp: ) those between two consecutive imaging studies showing new acute ventricular enlargement for slit ventricle syndrome patients undergoing a trial of shunt externalization and clamping (svs+); ) those between consecutive brain imaging studies from patients under the same trial without ventricular enlargement (svs-); ) overnight recordings from patients with suspected normal pressure hydrocephalus (nph). it was expected that both svs-and nph recordings represent steady state. we observed that only geodesic distance correctly differentiated between svs+ and svs-and between svs+ and nph while avoiding discriminating between svs-and nph. it was also found that % svs+ cases, none of svs-, and . % of nph cases had a multimodal geodesic distance histogram. pulses with a large number of distant pulses at similar mean icp for the five multimodal-histogram svs+ cases fell in short time windows indicating that acute ventricular changes may have occurred in these confined time windows during which no significant changes of mean icp occurred. geodesic inter-pulse distance is a promising metric to quantify distance intrinsic to the underneath geometric structure of icp signals. patients with severe traumatic brain injury have multiple causes for acute respiratory decompensation. computed tomography pulmonary angiography (ctpa) is being used extensively to evaluate acute cardiorespiratory changes. we reviewed the use of ctpa in critically ill patients with traumatic brain injury to evaluate the results and their impact on patient care. all adult trauma patients with traumatic brain injury who were admitted to our level trauma center intensive care units for greater than hours, were identified (january -december ). those who underwent ctpa for acute respiratory decompensation were reviewed to determine the findings of these studies and the resulting interventions. we identified patients that met these criteria [ admitted to neurosurgery/neurocritical care(ncc) , admitted to trauma service(ts)]; of these patients underwent ctpa studies for acute physiologic changes (ncc- , ts- , p= . ). ts patients were significantly younger with higher severity of injury and longer length of stay. pertinent clinical finding were identified in of the ( %) studies; and included atelectasis/collapse ( %), pleural effusion ( %), pneumonia ( %) and pulmonary embolus ( %). these results prompted targeted interventions, most frequently consisting of modifications of ventilator therapy ( , %), a change or initiation of antibiotic therapy ( , %), mini-bal ( , %) bronchoscopy ( , %), vena cava filter ( , %), and anticoagulation ( , %). no change in patient management occurred after studies. agreement, for different findings, between chest x-ray and ctpa ranged from - %. patients admitted to a ts are more likely to undergo a ctpa evaluation. ctpa is a useful tool in the evaluation of critically ill patients with acute physiologic decompensation beyond the diagnosis of pe. the results of these studies provide significant insight into the underlying pathology in this patient population and offer an opportunity to direct subsequent patient care. somatosensory evoked potentials (ssep) provide valuable information of the neurophysiological state of the patient throughout a surgery and the errors in the surgical procedure are easily noticed. it is hence important to analyze and monitor the ssep during scoliosis surgery in a minimum amount of time. the study uses pca-walsh algorithm to analyze posterior tibial nerve ssep and compare with the conventional signal averaging method in twelve surgical procedures. the tibial ssep from twelve different subjects were recorded and assessed throughout the respective surgeries using a unique pca-walsh algorithm by using only trials at a time and compared the extracted ssep information with conventional method. the ssep were recorded in two bipolar channels c -c and c z -f z throughout the surgery and analyzed remotely using an automated software pca-walsh algorithm. the results are compared with the actual clinical information and presented with the merits. in all the twelve cases, the algorithm results presented consistency throughout the surgery with an average accuracy of . % when compared to the conventional method, which takes several hundred trials. the average variation in time latency was . % and in amplitude was . %, well within the limit of % following the clinical criteria. the pca-walsh algorithm is capable of automated extraction of the tibial ssep during a surgery using a minimum number of trials. the analysis using the algorithm was successful and proved conclusive to the clinical information through the different surgical procedures. the faster recording and analysis of ssep signals provides a much better perspective for neurophysiological monitoring through the surgical procedure. the authors appreciate the support provided by the national science foundation under grants cns- , hrd- , cns- , and cns- . the authors are also thankful for the clinical suppo certain admission characteristics are known predictors of adverse outcomes in moderate-severe traumatic brain injury (mstbi) patients, but explain only / of outcome variability. retrospective studies suggest that non-neurologic organ failure may contribute to / of all deaths after mstbi, but actual incidence rates of intensive care unit (icu) complications and their impact on outcome are not known. we examined the incidence rates of pre-specified medical and neurological icu complications, and their impact on in-hospital mortality and functional outcome at hospital discharge. in a prospective observational study, consecutive mstbi patients from a single level i trauma center between / - / were analyzed. poor outcome was defined as glasgow outcome scale - .multivariable logistic regression was utilized to adjust for admission characteristics and icu length-of-stay. the mean age was years, % were men, and the median glasgow coma scale and injury severity scores were and , respectively. the five most common medical icu complications were: hyperglycemia ( %), fever ( %), hypotension requiring vasopressors ( %), systemic inflammatory response syndrome ( %), anemia requiring transfusion ( %). neurological icu complications were: intracranial pressure crisis (icp; [ % of n= with icp monitor in place]), brain edema ( %), herniation ( %), intracranial rebleed ( %), clinical seizure ( %). among medical complications, hyperglycemia was associated with poor outcome (or . ; % ci - . ]) while cardiac complications (e.g. cardiac arrest, arrhythmia, acute myocardial infarction) were associated with death (or . ; % ci . - . ). when combining medical with neurological icu complications, brain edema (or . ; % ci . - ) was associated with poor outcome, while cardiac complications and brain edema were associated with death (or . ; % ci . - . and or . ; % ci - , respectively). icu complications are very common after mstbi. we identified specific potentially modifiable predictors of adverse outcomes after mstbi. confirmation of our findings in a larger cohort is warranted. too much oxygen may increase oxygen free radical production, possibly triggering cellular injury and apoptosis. although laboratory investigations support the potentially detrimental effects of hyperoxia exposure after tbi, clinical data are lacking. we retrospectively identified tbi patients admitted to our neuro-icu between july and february . we identified a total of patients with complete data including gcs, apache ii, age, gender, abg within hours of injury, and outcome (glascow outcome scale-gos at discharge from the hospital). patients were divided into groups defined a priori based on pao on the first abg values obtained after injury. hyperoxia was defined as pao of mm hg or greater, and normoxia as pao between and . poor outcome is defined as gos of - . the patients in the normoxia group (n = ) and the hyperoxia goup (n = ) were matched on baseline characteristics, age ( among a small number of patients admitted to the neuro-icu following traumatic brain injury, patients with arterial hyperoxia had a trend towards worse outcome compared with patients with normoxia. this provides scientific rationale for large prospective clinical trials of controlled oxygenation in tbi patients. elevated intracranial pressure (eicp) contributes to secondary injury in stbi, therefore its control is paramount. boluses of hypertonic solutions are usually used to reduce icp but the impact of early continuous infusions has not been widely explored. we conducted this study to compare the effect and security of hypertonic saline % (hs %) infusion vs normal saline. all stbi patients arriving to the emergency room within hours of trauma were enrolled to receive an isovolumetric infusion of hs % or normal saline (placebo) during hours. icu physicians and investigators were blinded to the sodium levels during the trial. main endpoint: number of eicp episodes (> mmhg). secondary endpoints: neurologic outcome (gos, mrankin), electrolyte and osmolality levels, and adverse events (ae). twenty non-penetrating shbi patients were included. median age was . years (iq - : . - . ). median iss was (iq - : - ). we didn´t find significant differences for the total number of episodes of eicp at h between groups ( , iq - : - vs. , iq - : - , p= . ); however, when we analyzed patients with at least one episode of eicp we found a significant low number of eicp episodes in hs % group ( , iq - : - vs. , iq - : , - , ; p= . ). we found a sodium plateau at h of infusion (hs %: . ± . vs. control: . ± . meq/l, p= . ) which lasted until the beginning of weaning from hs %. the most frequent ae was hypokalemia and no patient had renal failure. the sixmonth gos and mrankin scores had a non-significant tendency towards better outcomes in hs % group. an early infusion of hs is feasible and seems to be safe in stbi patients. serum sodium kinetics showed a plateau after h of hs % infusion with no consequences in renal function and no rebound effects after tapering. hs % continuous infusion could reduce eicp episodes and it could conduct to better neurologic outcomes at six months. traumatic brain injury causes diffuse shearing of long fiber tracts. this can be detected by quantitative dti imaging even in patients who have primarily localized contusions. in our population the cingulum, cotricospinal tracts and external sagittal striatum were preferentially affected compared to age and gender matched controls. these findings support the use of advanced mri to assess the degree of injury and inform prognosis and goals for rehabilitation. neurocrit care ( ) :s -s most deaths following severe traumatic brain injury (tbi) are associated with a decision to withdraw life-sustaining therapies (wlst)( ). however, the incidence and the impact of wlst in clinical trials is unknown. this systematic review was performed to assess if and how wlst are dealt with in clinical trials involving patients with severe tbi. we searched medline, embase, cochrane central, biosis and cinahl databases and references of included studies. all randomized controlled trials (rcts) published over a -year period (january (january - , in one of selected journals in general medicine, critical care medicine and neurology/neurosurgery were considered for eligibility if ) and reporting data on mortality. our primary outcome was the assessment of wlst. secondary outcomes were the timing of evaluation, justification for wlst, proportion of wlst among deaths, factors that may have influenced the wlst and risk of bias of rcts. two reviewers selected rcts and collected data independently using a standardized case report form. from citations retrieved, rcts were included (n= , , ranging from to patients). were single center rcts and were multicenter. the incidence of wlst was reported in studies ( . %). three studies reported crude numbers of patients, studies reported the timing of wlst and studies reported the justification for the decision to wlst. studies were considered at high risk of bias, study at low risk of bias and studies did not give enough information to conclude on the risk of bias. wlst was rarely reported in rcts involving patients with severe tbi over the last decade. considering the variation of wlst in clinical practice, we suggest that wlst should be systematically reported in rcts performed in tbi. reference : . turgeon et al. cmaj . previous pediatric brain injury studies have considered fevers as discrete events instead of as a "temperature dose." we sought to evaluate the population size difference captured at various fever thresholds in severely brain-injured pediatric patients, considering fever burden in terms of degree-hours; and to compare fever burden in pediatric traumatic brain injury (tbi) vs. cardiac arrest (ca). charts from brain- y, admitted in - within hrs of admission were included. no temperature modulation protocols existed in the pediatric icu during this period. -day core temperatures were used to generate areas-under-the-curve (auc) above fever thresholds of . , , . , and o c. these were normalized for different lengths of stay. charts met inclusion criteria, with mean patient age . y (range d - y). diagnoses distributed (non-exclusively) as ca, accidental tbi, non-subarachnoid hemorrhage (sah) intracranial bleeds, sah, strokes, non-accidental tbi, ca after tbi, and other cns pathologies. cohort mortality was %, with % suffering brain death. fever burdens were measurable in % of patients over . o c, in % over o c, and in % over o c. normalized fever burdens at these thresholds were statistically different by -way anova (p< . ), with all fever burdens being statistically less than at . o c. remarkably, a shift in threshold from . to o c resulted in a % reduction in measured fever burden. fever burdens fell from a peak of . ± . o c-h on day to . ± . o c-h on day after admission. accidental tbi (n= ) and ca (n= ) patients did not experience different fever burdens above . o c. measured fever burden is markedly affected by shifting the threshold from to . o c. tbi and ca appear to induce similar fever burdens. pediatric fever burden reference values will allow more quantitative comparisons in severely braininjured children. little is known about the natural history of function after traumatic brain injury. our objective was to track the stability of drs scores over time and to identify factors associated with worsening drs scores. we collected disability rating scale (drs) scores, which capture the cognitive ability to perform activities of daily living such as communication, motor response, feeding, toileting, overall functioning and employability, longitudinally on severely brain injured patients in neurosurgery clinic. multivariable logistic regression was used to identify patient factors that were independently associated with changes in drs score over time. patients with severe brain injury had more than one drs score collected. of these patients, had worsening scores over time. changes in scores ranged from - to (mean - . , standard deviation . ). this represented a change from partial/no disability to moderate disability for patients and from moderate to severe disability for patients. patients improved from moderate to partial/no disability while only one patient improved from severe to moderate disability. using multivariable logistic regression, there were no patient factors that were associated with worsening drs scores including gender, age, comorbidities, race, insurance status, mechanism, injury severity score, gcs or final disposition. while half of worsening drs scores were seen within the first days after discharge, were seen months or more after the hospital stay, with one seen over a year after hospital discharge. for the most part, drs scores were stable over time. a group of patients were identified who experienced significant decline in function as far out from discharge as a year. this preliminary study highlights the need to identify those at risk for decline and to set up mechanisms for long-term follow-up for those patients in need. the identification of traumatic axonal injury (tai) lesions that undergo neuronal recovery could improve prognostication in patients with traumatic brain injury (tbi) and facilitate the development of novel therapies for preventing secondary axotomy. we aimed to determine whether diffusion tensor imaging (dti) detects neuronal recovery after tai. we retrospectively identified tbi patients ( severe, moderate, mild) who underwent at least acute-to-subacute dti scans and who had at least tai lesion in the corpus callosum (cc), as defined by hyperintensity on dwi or t flair. the median number of days from tbi to image acquisition was (range - ) for the first dti scan and (range - ) for the second scan. tai lesions were manually outlined on the acute dwi datasets and then coregistered to the subacute datasets to measure longitudinal changes in lesional fractional anisotropy (fa) and apparent diffusion coefficient (adc). "neuronal recovery" within a tai lesion was defined on the final scan by mean lesional fa within standard deviations of published normal fa values for the cc. initial fa and adc values in lesions with and without neuronal recovery were compared (unpaired t-test). eleven cc tai lesions ( splenium, body, genu) were identified. fa recovered in splenium lesions ( . +/- . [mean+/-sd]) and genu lesion ( . ) on the final scan. three of these lesions were flair hyperintense, were associated with gre microbleeds, and were initially adc hypointense. splenium lesions with neuronal recovery did not differ significantly from lesions without recovery for fa ( . +/- . vs. . +/- . , p= . ) or adc ( +/- vs. +/- x ^- mm^ /s, p= . ) on the initial scan. dti may detect neuronal recovery within tai lesions, as indicated by subacute normalization of fa. acute dti biomarkers of tai reversibility were not identified in this preliminary analysis. increased intracranial pressure (icp) in patients with traumatic brain injury (tbi) is associated with higher mortality and poor outcome. mannitol and hypertonic saline (hts) have both been used to treat high icp, but it is unclear which one is more effective. we compared the effect of mannitol and hts on lowering cumulative icp burden after severe tbi. the brain trauma foundation tbi-trac® new york state database was used for this retrospective study. a total of patients with severe tbi who received only hts were identified. patients who received only mannitol were matched for age, pupillary reactivity, occurrence of hypotension on day . univariate analysis was performed to compare icp burden, cumulative hyperosmotic doses, number of icu days (wilcoxon signed rank test), and two-week mortality (mcnemar test). icp burden was defined as the total number of days with icp spikes (icp> mmhg) expressed as a percentage of total number of days of icp monitoring. cumulative hts and mannitol doses were converted to osmolar doses for comparison. the mean age and gcs were similar in the two groups ( . vs. . years; . vs. . ; hts vs. mannitol, respectively) . patients received % hts and received . % hts. all patients in the mannitol group received % mannitol. there was no difference in number of days of icp monitoring (p= . ) or number of icu days (p= . ) in the two groups. icp burden was significantly lower in hts group vs. mannitol group ( . % vs. . %. p= . ). there was no significant difference in the cumulative dose of hts and mannitol (p= . ), and two-week mortality in the two groups was similar (p= . ). hts is more effective in lowering cumulative icp burden after severe tbi compared to mannitol. this did not translate into reduction in two-week mortality, possibly due to the small sample size. spreading depressions (sds) have been consistently associated with hypoglycemia in animal studies. the frequency of these depolarization events, while influencing infarct size, also appears to be influenced by the plasma glucose concentration during experimental ischemia. low cerebral dialysate glucose have also been correlated with sd events in humans. we hypothesized that low serum glucose should be associated with an increase in the frequency of sd events in human acute brain injury. to determine the relationship between serum glucose and cortical spreading depolarizations (sds) after traumatic brain injury (tbi), subdural electrode strips were placed on peri-contusional cortex in patients from centers who underwent craniotomy following tbi. prospective electrocorticography was performed during neurointensive care with retrospective analysis of hourly serum glucose data. patients were divided into those with sds and those without and the distribution of glucose values among these two groups were compared using the -way kolmogorov-smirnov method. in patients ( %), sds (spreading depressions and peri-infarct depolarizations) were observed. the probability of a depolarization occurring increased significantly as a function of rising serum glucose (p< . ). median glucose values in patients with and without sds was . and . mmol/l, respectively. among patients with sds, glucose values recorded within minutes of the onset of an sd were higher than those occurring < minutes before an sd (p< . e- ) ( figure ). serum glucose does not appear to affect the generation of sds as it does in animals but paradoxically may be elevated. this may reflect a stress response to the initial acute brain injury and critical illness or a physiologic mechanism to increase glucose supply during sd events in which cerebral glucose utilization is increased. overall, the data suggest that plasma glucose is being managed within appropriate levels in this study group. to determine difference in tbi severity and abnormal radiologic findings in different age groups. this was an observational cohort study on all adult patients (> yrs) arriving to the emergency department, with a history of traumatic brain injury as a result of "fall" at a level trauma center in the southeastern united states. data collected included ed gcs score and head ct results. abnormal ct scans have the presence of either an intra-cranial bleed and/or cranial fracture. there were patients in the cohort with history of fall with median age of yrs (iqr of - ). we divided them into two age groups: group a: - yrs( %) & group-b: > yrs( %). group a comprised mostly of males ( %) compared to females, meanwhile group b was equivocal in gender composition ( % male). out of the whole cohort of patients, % patients had head ct performed. out of these, . % (n= ) showed an abnormal head ct. age group b ( %) had a significantly greater percentage of abnormal ct scans compared to group a ( %) (p< . ). among abnormal ct scans, group a ( %) had a significantly greater percentage of skull fractures than group b ( %) (p= . ). among patients with mild and moderate tbi (gcs> ), group b ( %) was more likely to have an abnormal ct scan than group a ( %) (p< . ), however, there is no significant difference between likelihood of abnormal head ct between group a and b for severe tbi. younger adults are at a higher risk of cranial fractures after a fall related tbi, probably due to severe mechanisms of injury. on the other hand elderly population with mild tbi mostly due to ground level falls had worse outcomes on ct scans. accumulating pre-clinical data suggests that matrix metalloproteinase (mmp) expression following cerebral trauma contributes to brain injury. we sought to characterize the temporal mmp response to severe traumatic brain injury (stbi) in humans and its relationship with outcomes. we conducted a prospective cohort study that included adults with stbi. high-cutoff, cerebral microdialysis and arterial and jugular venous bulb catheters were used to measure the concentration of mmps and other markers over -days. the concentration of mmp- was initially low in microdialysate and blood, but increased between -and -hours. mmp- blood levels were high and stable throughout the study while blood levels of mmp- were initially low and then gradually rose. in microdialysate, mmp- and - increased and then peaked between -and -hours. mmp- also increased in microdialysate following stbi while its levels were low and stable in blood. mmp- and - were initially high in microdialysate and then slowly decreased over time. while the concentration of mmp- was also initially high in blood and then progressively declined, the mmp- blood level increased with time. among the patients that also had cerebrospinal fluid (csf) drains, marked and sometimes opposite concentration trends were observed for mmp- in microdialysate versus csf. generalized estimating equations suggested that significant changes in mean microdialysate concentrations of mmp- , - , - , and - and mmp- , - , - , and - occurred with increases in microdialysate glucose and the lactate pyruvate ratio, respectively. moreover, the mean microdialysate level of mmp- increased with intracranial pressure (icp) [( . pg/ml)/mmhg; % confidence interval, . to . ] while that of mmp- decreased with cerebral perfusion pressure (cpp) [(- . pg/ml)/mmhg; % confidence interval, - . to - . ]. monitoring of mmps following stbi is feasible, and their expression may be associated with cerebral metabolism, icp, and cpp. to determine significance of laboratory markers for in-hospital death after fall related adult traumatic brain injury. this was a consecutive cohort observational study done at a level- trauma center serving surrounding counties. cohort consisted of all adult patients (> yrs) arriving to the ed with a history of fall. study variables were lab values of the following parameters on ed admission: sodium, potassium, bicarbonate, lactate, blood glucose, inr, aptt, wbc, rbc, platelets; along with pre-hospital glucose values in the field. study cohort comprised of adult subjects arriving at ed with history of fall with median age of yrs (iqr of - ). in-hospital death (ihd) was observed in % (n= ) of the total cohort, with male ihd= ( %), and female ihd= ( %). older age groups [> yr] ( %, n= ) had higher incidence of in-hospital deaths compared to younger age group - yr ( %, n= ) with p= . (ci= . - . ). in a univariate regression model higher levels of: pre-hospital glucose (p= . ), ed blood glucose (p< . ), lactate (p= . ), inr (p= . ), aptt (p< . ) and wbc (p< . ) were significant individual predictors of in-hospital death. while lower levels of bicarbonate (p< . ) and rbc (p< . ) were significant individual predictors of in-hospital death. the following multivariate regression models showed statistical significance with higher probability of in-hospital death: ) higher: ed blood glucose (p= . ), aptt value (p= . ) | lower: bicarbonate (p= . ), rbc (p= . ) with adj.r = % ) higher: aptt value (p= . ) and wbc count (p= . ) ) higher: aptt value (p= . ), older age (p= . ) | lower rbc (p= . ) and gcs scores (p< . ) with adj.r = % lab parameters such as ed blood glucose, rbc count, wbc count, bicarbonate level & aptt level are individually or simultaneously important predictors of in-hospital death in adult tbi patients with history of fall. traumatic brain injury (tbi) is an epidemic with severe consequences. brain tissue oxygen tension (pbto ) monitors detect secondary injury and direct clinical therapies to mitigate damage. blood transfusion is one therapy often used, however its effect in tbi patients is not well defined. we studied pbto data in patients who received transfusion after tbi. sixty-nine severe tbi patients were consecutively admitted to a neurocritical care unit and received pbto monitoring as part of standard clinical care for this unit. data were collected from electronic medical records as entered by the bedside nurse. patients were managed according to the brain trauma foundation guidelines. transfusions were identified through nursing comments. hourly pbto values were analyzed for up to six hours after starting transfusion. other factors were also analyzed for their potential influence on pbto readings. of patients, received a total of transfusions in the setting of pbto monitoring. two groups were identified: transfusions that led to an increase in pbto and those that did not. six transfusions resulted in increased pbto , with an average increase of . mm hg. twenty-two transfusions did not: of these were unchanged and decreased. the groups did not differ in age (mean . and . , respectively), maximum temperature (mean . and . ), minimum cerebral perfusion pressure (mean . and . ), or initial glasgow coma scale (gcs) (mean . and . ). blood transfusion is often used in the critical care setting. the effect of transfusion on brain tissue oxygen tension is variable. age, temperature, cerebral perfusion pressure, and initial gcs were not useful in distinguishing patients who benefited from transfusion. pbto only rises in a minority of patients; therefore additional prospective studies are needed to evaluate which patients are likely to benefit from transfusion. mannitol use in patients with traumatic brain injury can lead to acute renal failure and may worsen outcome. the purpose of this study is to determine the rate of acute renal failure (arf) among patients treated with mannitol and its impact on outcome in a multicenter review. we analyzed a one-year data ( ) ( ) from the premier database, a nationally representative hospital discharge database in the united states. we compared baseline and clinical characteristics of patients with traumatic brain injury (tbi) treated with mannitol in the first days of admission who developed arf to those who didn't. length of stay, cost of hospitalization and discharge status were ascertained. from a total of admissions with a diagnosis of traumatic brain injury requiring mannitol within the first days of admission, % (n= ) of patients had arf. arf is a common complication of tbi treated with mannitol. it is associated with longer length of hospital stay and increased rates of in-hospital mortality. the result highlights the importance of using alternative therapy to hyperosmotic agents such as hypertonic saline in treatment of tbi patients at risk for acute renal failure. cervical spine immobilization (csi) is a relative contraindication for percutaneous dilatational tracheostomy (pdt) because of the inability to extend the neck, making tracheal puncture at the correct level more challenging. patients with csi routinely undergo pdt at our institution, however, with both traditional bronchoscopic as well as with real-time ultrasound (rtu) guidance. our objective was to review the incidence of complications related to pdt in patients with csi versus patients without csi. we reviewed the records of consecutive patients who underwent pdt performed by a single operator at our neurocritical care unit between / - / . all patients requiring tracheostomy are screened for eligibility for pdt by the attending neurointensivist on service. we recorded the percentage of patients who successfully underwent pdt vs requiring conversion to surgical tracheostomy, the specific guidance used (bronchoscopy, rtu) and all short-and long-term complications including placement of the tube above the first tracheal ring. a total of patients underwent pdt performed by a single neurointensivist. all patients screened by the operator underwent an attempt at pdt, and all patients successfully completed the procedure without conversion to surgical tracheostomy. ninety-eight of ( %) did not require csi and ( %) required csi. in the csi group, bronchoscopy alone was used in / ( %) and bronchoscopy plus rtu in / ( %). no complications occurred in the csi group. in the no-csi group, there were ( %) complications (one tracheal granuloma and two tube dislodgments within days). no other short or long term complications were recorded. all tubes were placed below the first tracheal ring. it is feasible to safely perform pdt in patients with cervical spine immobilization using bronchoscopic and real-time ultrasound guidance. following traumatic brain injury (tbi), increased serum biochemical marker levels reflect the extent of neurological damage, prognosis and clinical outcomes. effective tbi management strategies are lacking. despite the neuroprotective effects of therapeutic hypothermia after cardiac arrest, its tbi use remains controversial. delays in achieving target temperatures in human trials taking - hours (nabish-i; nabish-ii) may have contributed to the lack of benefit. we hypothesized prompt, rapid induction of hypothermia, immediately following tbi would lower predictive serum biomarkers of brain injured swine. sixteen domestic cross-bred pigs ( - kg) were subjected to a atm ( ms) fluid percussion tbi. eight injured animals were cooled to °c within minutes of injury and maintained for hours using transpulmonary hypothermia. eight control animals were maintained at °c using similar doses of inhalational and intravenous general anesthesia. brain temperature was monitored with camino.® serum markers of tbi: s- calcium binding protein b (s- b), neuron-specific enolase (nse), glial fibrillary acidic protein (gfap) and phosphorylated axonal form of the neurofilament subunit nf-h (pnf-h) were measured prior to injury and seven times over hours. surviving animals were euthanized and necropsied five days post-injury. at , , and hours, s- b, nse and pnf-h, were lower in the hypothermia group vs. controls. gfap levels were decreased at hours. after injury, peaks and troughs of the biomarkers occurred at various intervals. s- b levels were reduced in both groups during the initial hours post-injury, with control levels increasing at hours. early initiation and rapid cooling of brain temperature to - °c for hours was associated with attenuated s- b, nse, gfap and pnf-h levels in swine. general anesthesia was associated with early mitigated s b levels. prompt therapeutic hypothermia and prolonged anesthesia may offer neuroprotection after tbi. mild traumatic brain injury (mtbi) from blast exposure represents a significant threat to military personnel. until now there has been no way of knowing what the individual service member experienced during an exposure. we report the first individual measurements recorded during combat operations and how those readings were used to assist evaluation of the injured service member. the nato role- hospital, kandahar afghanistan received the index case of a service member (sm) exposed to an improvised explosive devise (ied) blast while wearing a blast dosimetry system composed of blast gauges placed on the back of the helmet, chest, and shoulder. the gauges include status lights that allow immediate feedback for injury risk via colored lights: green = negligible (< psi peak), yellow = moderate (between and psi), and red = severe ( psi and above). in addition, time traces of the overpressure and -axis acceleration are recorded and available for download through a micro-usb port. the sm's gauges were initially checked hour minutes after the blast, demonstrating a yellow status light. the blast data downloaded from the gauges demonstrated a consistent exposure of . msec composed of a primary flow immediately followed by a secondary wave. the head gauge recorded a peak overpressure of . psi and impulse pressure of . psi-sec. there was msec of sustained pressure above psi from the primary flow. all gauges demonstrated similar blast profiles, including a secondary reflective wave. these measurements are firsts in both the recording of an individual's exposure during a blast related attack and the use of that data for patient triage and medical evaluation. blast gauges measure environmental exposure and do not diagnose mtbi, however; they do provide clinicians with important information in the evaluation of patients subjected to blast. to consider the definition of initial signs and symptoms to compare outcomes after "severe" traumatic brain injury regard to mechanism of injury. design-this study included all adult patients who presented to ed at a level- trauma center with severe (gcs score< ) traumatic brain injury. from the total cohort(n= ), % suffered tbi because of "fall" and % due to traffic accident(mvc). significant proportion of each sub-group was comprised of males ( % in-mvc with median-age= ; % in-falls with median-age= ) · for all the patients arriving to ed after a traffic accident with severe gcs: % had loc, % had aoc, % had pta, % got admitted to hospital, % had an abnormal head ct (bleed/fracture), % got admitted to icu, % had some neuro-surgical intervention and % patients died in hospital. · for all the patients arriving to ed after a history of fall with severe gcs: % had loc, % had aoc, % had pta, % got admitted to hospital, % had an abnormal head ct (bleed/fracture), % got admitted to icu, % had some neuro-surgical intervention and % patients died in hospital. · decrease in systolic blood pressure (p= . ) and increase in diastolic blood pressure (p= . ) are more likely to have a fracture after a traffic accident in severe tbi. increasing of blood pressure p= . ) and decreasing of pulse (p= . ) is significantly associated with icu admission after a fall. comparing data for two most common mechanism of injury in severe tbi suggest that some vital signs and symptoms have significant impact with outcomes depends on mechanism of injury. these observations should be studied in larger cohort to find more significant association between mechanism and outcomes. cerebral edema is the one of the most significant predictors of poor outcome after traumatic brain injury. it is still unclear what the pathophysiological and cellular mechanisms and predictors of post-traumatic edema are. the exponential growth in genetic information has opened an avenue for investigation in traumatic brain injury and implicated specific genes in the pathophysiology of post-traumatic injury edema. two examples are the aquaporin- and cacna genes, which respectively encode water and calcium channels. the aquaporin- gene on chromosome q . - . encodes the aquaporin- protein (aqp ) water channel. aqp is one of the bidirectional high capacity water channels that is primarily expressed in astrocytic foot processes in the central nervous system at the blood-brain barrier and is thought to be critical for brain water homeostasis. experimental studies showed that aqp deficient mice had significantly reduced cerebral edema and better survival in a water intoxication model. the cacna gene on chromosome p encodes the a a subunit of a neuronal calcium channel. patients with familial hemiplegic migraine and delayed fatal cerebral edema and seizures from minor trauma have been found to have mutations in cacna , which are hypothesized to enhance development of cytotoxic edema. a missense mutation is reported to enhance risk of delayed fatal cerebral edema. hypothesis: the cacna gene missense mutation s l and aqp polymorphisms will be over-represented in patients with post-traumatic cerebral edema. to perform full exon sequence analysis of these two genes in well-defined cases of excessive cerebral edema. our long term goal is to systematically investigate genetic variants as determinants of risk of excessive cerebral edema. patient recruitment is currently ongoing. it is hoped that this will further elucidate secondary mechanisms of injury specifically in the formation of post-traumatic edema and lead to targeted therapies in the future. microwave occurs when improvised explosive devices was exploded. however, the effect for brain by microwave has not been clarified. under general anesthesia, s-d rats were irradiated by head-focused microwave by microwave fixation system (model mmw- / muromachi kikai co., ltd.), which were classified in three groups ( . kw/ . sec (i), . kw/ . sec (ii), . kw/ . sec (iii), and sham group) by intensity (n= in each group). vital signs were evaluated, arterial blood gas was examined, and we checked pathologic findings by hematoxylin-eosin (he) stain immediately after microwave irradiation, post hours, hours, hours, hours, weeks, and weeks in each group. blood pressure was elevated transiently immediately after irradiation, and recovered in short period. pao was unchanged in post-irradiation phase, except in group i. in he stain, neuron was degenerated and left out especially in cerebral cortex and hippocampus, microglia cells were accumulated in these regions. these pathological changes were observed frequently and earlier, when irradiation was intense. the result was firstly reported that head-focused microwave irradiation induced brain injury in s-d rats, and this brain injury was related with intensity of microwave. pathological change was impressive because it was occurred gradually and progressive. further study will be required, whether this type of brain injury is similar with traumatic brain injury, or cerebral ischemia or not, and the study of behavioral effects of microwave irradiation is necessary, especially when the intensity of irradiation was not severe. the efficacy of decompressive craniectomy (dc) in the treatment of moderate-severe traumatic brain injury (mstbi) is a topic of debate in neurocritical care. despite the recently published randomized dc in diffuse tbi (decra) trial, it is still unclear when and for which tbi patients this procedure should be considered. in order to assess the utility of dc in evidence-based clinical practice, we present a matched case-control study that compares surgical and non-surgical outcomes among patients with mstbi. we conducted a retrospective analysis of mstbi injuries treated at a single level i trauma center from to . twenty mstbi patients aged between and years, who underwent dc, were enrolled. paired controls that underwent medical therapy only were selected according to glasgow coma scale (gcs) score and age. primary lesion type, pupil reactivity, hypotension, hypoxia and icp crisis were secondarily considered in matching cases with controls. we focused on mortality, glasgow outcome score (gos) score upon hospital discharge and gos score at months as the primary measures of outcome. in the dc group, we found that % of patients died; % had a favorable outcome at discharge ( or higher on gos); and % had favorable outcome at months. in the control standard-care group, we found that % of patients died; % had favorable outcome at discharge; and % had favorable outcome at months. pupil reactivity and gcs score on admission were the variables highly correlated with mortality. statistical analysis will be available at the meeting and presented for the first time. in this cohort, undergoing dc did not seem to confer a mortality benefit to patients with mstbi. good recovery after mstbi was observed in a larger percentage of the non-surgical group, which is consistent with the findings of the decra trial. each year in the united states, over . million patients present to emergency departments as a result of traumatic brain injury (tbi). severity classification of tbi is based on the glasgow coma score (gcs), with severe tbi being a gcs score between and . there is always a subset of "severe" tbi that requires surgical intervention. the current study examines this subgroup to decipher any symptomatology that may be helpful in identifying who these patients are. the objective is to determine which if any factors predict the need for surgical intervention in patients with severe traumatic brain injury (tbi). this study is a subgroup analysis of the larger cohort of consecutive adult tbi patients that presented to the ed. our sample included only severe tbi patients (gcs< ). besides descriptive analysis, logistic regression analysis was done to determine the significant predictors of surgical intervention in this subset of patients. lab values (sodium, potassium, bicarbonate, lactate, blood glucose, wbc, rbc, platelets, inr, aptt) and symptoms (such as-seizures, vomiting, loss of consciousness, alteration of consciousness, post-traumatic amnesia) were the dependent variables compared with surgical intervention (independent variable). of the total severe tbi cohort (n= ), % required surgical intervention. presence of abnormal head ct (bleed in % of the total cohort) is significantly associated with surgical intervention (p= . ). vomiting (p= . ), lactate (p= . ), higher wbc (p= . ) and lower platelet count (p= . ) individually showed significant association with surgical intervention on a univariate regression model. these data suggest that abnormal head cts, particularly those that result from bleeding, as well as lactate, platelet count, wbc count and vomiting are significantly associated with surgical intervention. the association of lab values with likelihood of undergoing surgical intervention is an interesting future research point. to study the potential usefulness of initial vital parameters and laboratory evaluations to predict short term prognostic this is an observational cohort study of all adult patients who came to the emergency department(ed) of a tertiary care hospital, in a month period during - . for the purpose of analysis, we considered initial vitals and lab values available for all patients. we individually compared vitals (pulse and mean arterial pressure-map) and laboratory values [sodium(na + ), potassium(k + ), bicarbonate(hco -), glucose, wbc, rbc, platelets, inr) for the following prognostic variables: abnormal head ct finding(yes/no), hospital admission (yes/no), icu admission, in-hospital death, hospital length of stay(hlos), and -month mortality using t-tests and correlations. the significant variables were then entered into a logistic regression model (for categorical variables) and a multiple regression model (for continuous variables) simultaneously to determine significant predictors of prognostic outcomes. significance level was set at p= . . increase in glucose(p= . ) and wbc(p< . ) lead to a higher likelihood of having an abnormal head ct, when controlling for map and hco -; increase in glucose (p= . ) and wbc (p= . ), and decrease in hco -(p= . ) and platelets(p= . ) increases the likelihood of getting admitted, when controlling for map, k + , and rbc; increased glucose(p< . ), decreased hco -(p= . ) and decreased platelets(p= . ) increases chances of in-hospital death, when controlling for wbc and inr; increased glucose(p= . ), increased wbc(p< . ), and decrease in rbc(p= . ) increases hlos, while controlling for pulse, k + , and hco -. the study indicates that some initial vitals and lab values can help to determine the prognostic outcomes in adult traumatic brain injury. though our study is limited by a single-site patient population, the interesting findings warrant further research efforts in this specific area. ultrasonic assessment of optic nerve sheath diameter (onsd) as a non-invasive measure of intracranial pressure (icp) has been evaluated in the literature as a potential valid technique for rapid icp estimation in the absence of invasive intracranial monitoring. the technique can be challenging to perform and little literature exists surrounding intra-operator variability. in this study we propose an examination of onsd utilizing a variety of novel ocular models, to both define the ability of the ultrasound linear array probe to capture different known onsd, and to assess intra-operator variability with the technique. here we present the model and data. we designed ocular models composed of gelatin spheres and variable three dimensional printed cylinders, which simulate the globe of the eye and variable onsd's respectively. these models will then be suspended in a gelatin background. operators will then utilize the linear array ultrasound probe on these models in order to determine onsd of sizes, with measurements each in order to assess intra-operator variability with the technique. our optic nerve sheath model offers ultrasound images comparable to in vivo, and is quick to manufacture. analyzing the data, we removed the first two measurements from the series of ten. we defined those as "practice attempts" with the technique. for the onsd models, the means were: . mm with sd of . mm ( % ci of . ), . mm with sd of . mm ( % ci of . ), . mm with sd of . mm ( % ci of . ). utilizing the standard linear array ultrasound probe for onsd measurements in our model provided reliable results with minimal intra-operator variability across variable sheath sizes. knowing this, we can further apply this novel model of onsd to us teaching and training courses with confidence in its ability and the techniques ability to produce consistent results. the objective of this study was to identify factors (signs and symptoms after injury, vital parameters, and glucose) that can be used as predictors of an intracranial bleed. this will improve identification and treatment of patients who present to the emergency department (ed) with tbis. this is an irb approved observational cohort study done at a level trauma center, and included all adult patients presenting to the ed following a tbi. data for patients presented during study variables included age, loss of consciousness (loc), seizure (sz), vomiting, alteration of consciousness (aoc), post-traumatic amnesia (pta), glucose level, pulse, and blood pressure (bp). all variables were tested for association with intracranial bleeding using chi-square tests of independence, t-tests, and then significant variables were included in a regression model. limitation of study were chart review and a single ed. the cohort consisted of , patients, of which % (n= ) had a ct scan of head. % of total patients had an abnormal head ct, and % of those had an intracranial bleed. statistical analysis indicated that loc (p= . ), aoc (p= . ), pta (p< . ) and advanced age (p= . ) were significantly correlated with having a bleed. vomiting and sz were not statistically significant. among patients who had head cts, both pulse and systolic blood pressure decreased between the first and second measurements; both pulse(p= . ) and bp(p= . ) decreased significantly less in patients with bleeds compared to those without bleeds. additionally, higher ed glucose level was associated with having a bleed (p< . ) on head ct. these data indicate that older age, loc, aoc, pta, and elevated glucose levels can be used as predictors of intracranial bleeds. sustained elevation of pulse and systolic blood pressure may also be indicative of a bleed in tbi patients. patients with moderate-severe traumatic brain injury (mstbi) commonly die from withdrawal of support, likely as a consequence of an unfavorable outcome prognosis provided to the family by the treating physician. it is unknown whether prognostication may lead to self-fulfilling prophecies, and whether the presence of intensive care unit (icu) complications may accentuate possible provider bias. in this study, we surveyed clinicians caring for patients with mstbi to examine the variability of outcome prognostication and the influence of icu complications on these predictions. we conducted an anonymous electronic survey of clinicians, including faculty members (neurology, neurosurgery, trauma, anesthesia/critical care), neurology house staff, icu affiliate practitioners and neuroicu nurses at a single level i trauma center. the survey included three tbi case vignettes and their respective icu courses. questions were designed to assess the utilization of known tbi prognostic models, relative importance of icu complications for outcome prognostication and aggressiveness of care recommended by the survey participant. a total of surveys were distributed by email or paper, and have been returned. so far, we have found that % of participants consider medical icu complications as very important in tbi prognostication. age, icu course and head ct findings are the prognostic variables considered most important to outcomes. % of non-critical care neurologists are uncomfortable providing tbi prognostication. case responses suggest that clinicians tend to recommend aggressive care (surgery), but predict unfavorable outcomes. the survey is ongoing, but complete results will be available at the meeting and presented for the first time. we have discovered great variability in outcome predictions made by clinicians with different levels of experience in treating mstbi. self-fulfilling prophecies may exist among mstbi outcomes. outcome studies should focus not only on admission variables, but also on icu complications in order to guide clinicians in providing prognostication. the objective of this study was to identify pre-hospital factors that are associated with worse severity of head injury in order to help physicians identify when tbi treatment may be necessary. this is an observational cohort study that included adult patients presenting to the(ed) following a motor vehicle collision. study variables included age, gender, seatbelt use, loss of consciousness (loc), seizure, vomiting, alteration in consciousness (aoc), and post-traumatic amnesia (pta). severity of tbi was classified according to the glasgow coma scale, with mild defined as - , moderate being - , and severe being anything less than . the gcs was obtained both in the pre-hospital and ed settings. the cohort of was % male. the median age was (iqr: - , r: - ). the breakdown of severity in the prehospital setting (n= ) was % mild, % moderate, and % severe. in the ed (n= ), the breakdown was % mild, % moderate, and % severe. pre-hospital factors significant for the z-test included seatbelt (sb) use, loc, aoc, pta, and gender. males, patients who did not wear seatbelts, and patients who had a positive loc, aoc, or pta were more likely to sustain a moderate or severe tbi. having a seizure was also significantly associated with increased tbi severity (p= . ). (see table ) additionally, the data show that the likelihood of having an abnormal head ct increases with age (p< . ). although vomiting was associated with greater tbi severity, the results were not statistically significant. early symptoms such as loc, aoc, seizures, and pta are early predictors of worse severity in patients who sustain a head injury during their motor vehicle collision. age, male gender, and lack of seatbelt use also correlate with greater tbi severity. identifying crucial symptomatic predictors of icu admissions, icu length of stay and mortality rates in traumatic brain injury (tbi) patients with history of fall. retrospective chart analysis was performed on all adult patients arriving to emergency department with history of fall at a level one trauma center for parameters like vomiting, alteration of consciousness (aoc) & loss of consciousness (loc) after tbi; post-traumatic amnesia (pta) and history of seizures before or after injury, along with outcomes such as icu admission & icu length of stay. from the total cohort (n= ), % (n= ) of patients were admitted to icu, most of them were males( %,p= . ). aoc was found to be strongly associated with icu admission ( %, p< . )[including the patients who had brief loss of consciousness of < mins( %)], and month mortality rates(p= . ) when adjusted for mild gcs scores. · icu length of stay was higher in patients admitted to icu with aoc (p= . ) and pta ( . ). icu admissions had higher day readmission (p= . ), in-hospital death (p< . ) and month mortality rate ( %, p< . ). · % of patients were found to have intra-cranial bleed when presented to ed with aoc(p= . ), and % of these patients were admitted to icu. on a multivariate regression model analysis, patients who had abnormal head ct with mild gcs on ed presentation had higher month mortality rates (p= . ) when adjusted for age. patients with symptoms such as alteration of consciousness and post-traumatic amnesia after traumatic brain injury as a result of fall are more likely to be admitted to icu with significantly longer icu length of stay. mild traumatic brain injuries in fall patients should not be overlooked in daily practices because of significant mortality rates. cardiovascular disturbances remain a leading cause of morbidity and mortality in patients with acute spinal cord injury (asci). asci patients often develop symptomatic and potentially life-threatening bradycardia. our practice has been to use albuterol elixir prophylaxis in asci patients, taking advantage of its side effect profile associated with a typical dose of mg tid or qid, to prevent further symptomatic bradycardia. evidence of efficacy with this regimen is, however, lacking. we set out to determine whether treatment with oral albuterol would decrease the frequency of bradycardic episodes in patients with asci. we retrospectively identified adult patients admitted to university of new mexico hospital between - who sustained an asci and received oral albuterol therapy. the frequency of bradycardic events (hr < bpm) before and after initiation of albuterol was collected. we compared the number of bradycardic events before and after albuterol within each subject using the wilcoxon signed rank test. bootstrap methods were used to further validate our findings. we identified asci patients who had evidence of symptomatic bradycardia before the initiation of the albuterol therapy, including hypotension and in cases bradycardic cardiac arrest. the median number of bradycardic events was ( . , iqr) before albuterol and was ( , iqr) after albuterol. we found that patient's had a significantly lower number of bradycardic events after the initiation of albuterol (p = . ). ten patients experienced less bradycardic events. the median difference was less bradycardic episodes. bootstrap estimates of the median difference were consistent with our initial analysis. albuterol appears to be an effective means of treating bradycardia in patients with acute spinal cord injury. severe traumatic brain injury (tbi) is frequently associated with eeg changes like epileptiform discharges, seizures; periodic lateralized epileptiform discharges pleds or paroxysmal delta activity. we report a case of tbi with generalized hz spike and wave pattern that did not represent seizures a y old girl without epilepsy history presented after being involved in a motor accident. initial gcs was and remained the same over the next days. ct showed contusions with small left subarachnoid hemorrhage. phenytoin was started for seizure prophylaxis. on day , she improved clinically, however, on day she had fluctuating consciousness and continuous eeg monitoring was initiated. various antiepileptic medications were tried over the next several days including lacosamide, valproate, topiramate, levetiracetam and ethosuximide (eth) without significant change clinically or on eeg. she started improving clinically on day but became extremely drowsy on day , all meds except eth were weaned. she showed improvement and was discharged to rehab on day . a prolonged eeg after months was normal and eth was weaned off. she continues to do well almost one year after and is maintaining her school grades at pre-injury level. the patient's initial eeg (day post injury) showed generalized hz spike and wave pattern occurring every - seconds which continued for days despite treatment with various anti epileptics as described. on day eeg pattern changed to generalized rhythmic delta activity( - hz) especially during arousal. mri during the stay showed micro hemorrhages in both frontal lobes and right temporal lobe reflective of diffuse axonal injury. a hz spike and wave pattern mimicking absence seizures can be seen on eeg transiently after tbi, however its clinical significance is unclear. whether it needs to be aggressively treated or not cannot be conclusively established but the longterm prognosis appears to be benign. free radical-induced lipid peroxidation (lp) has been demonstrated to lead to the formation of isoprostanes from arachidonic acid and neuroprostanes from docosahexaenoic acid. lp is common after traumatic brain injury (tbi) and constitutes one of the key mechanisms of pathology related to secondary injury after tbi. one of the consequences of lp is the compromise of neuronal calcium (ca ++ ) homeostasis, leading to ca ++ overload and activation of the proteolytic -spectrin. the purpose of this project is to characterize the concentration--spectrin degradation after tbi. this study is a prospective, single-center study of adult moderate to severe tbi patients. inclusion criteria are age > yo, closed head injury, within hours of tbi, and glasgow coma score (gcs) < . serial samples from urine, blood, and cerebrospinal fluid (csf, when available) are obtained for up to weeks after injury. demographic data and pertinent clinical information are also collected. the biomarkers ( & f t -isoprostanes, f -isofurans and f -neuroprostanes) are measured via -spectrin breakdown products (sbp) by western blot analysis. we have enrolled fifteen patients to date. preliminary results suggest that the study population is typical of tbi (mean age . years, % male, median admission gcs ). serum and csf & f t -isoprostane values are above published values for normal individuals, with csf values peaking at hours after tbi. sbp are also measured in elevated amounts in csf compared to non-tbi controls (in whom they are not measurable). preliminary data suggests that serum and csf isoprostane values are elevated after tbi. continued patient accrual, further sample analysis, and comparison to control groups is needed to more precisely define the effect of tbi on the time course of lp biomarkers. traumatic intraventricular hemorrhage (tivh) is generally considered to be associated with moderate to severe traumatic brain injury and a significant mortality rate. there exists, however, a rare subset of individuals who manifest with isolated traumatic intraventricular hemorrhage and have a good prognosis and outcome. we present a case of an -year old female who suffered polytrauma and an isolated ventricular hemorrhage following a traumatic fall while mountain climbing. her history indicated mild transient confusion and amnesia occurring around the time of the fall. her glasgow coma score was , her neurologic exam was normal and she had no neurologic complaints other than positional lightheadedness and nausea. a comprehensive exam was notable for a right hip dislocation, nasal fracture, l vertebral body fracture, right apical pneumothroax and pulmonary contusion. computed tomography of the head showed an acute hemorrhage in the left lateral ventricle prompting concerns for traumatic brain injury. no additional pathology was noted on a follow-up magnetic resonance imaging. repeat ct scan showed mild interval decreases in the size of her ventricular hematoma. the patient was discharged one week after admission and had developed no neurologic complications. she was diagnosed with concussion and isolated intraventricular hemorrhage. isolated intraventricular hemorrhage is a rare complication of traumatic head injury that can have a good prognosis and outcome. the case shows the difficulty in categorizing this particular condition within the current spectrum of traumatic brain injury and specifically highlights shortcomings with classification systems that utilize neuro-imaging abnormalities to determine severity of injury. traumatic intracranial aneurysms (tias) are distinctly uncommon, comprising fewer than % of all cerebral aneurysms. tias that develop following blunt head injuries present the clinician with both diagnostic challenges and clinical difficulties. the natural histories of giant intracranial aneurysms are generally grave owing to mass effects, severe hemorrhage, and distal thromboembolism. case report. we present the case of a -year-old male was involved in an accident in which he suffered severe head injury from a falling heavy iron hammer. the immediately unenhanced head computerized tomography showed hemorrhagic contusions, subarachnoid hemorrhage, skull fracture and basal fracture. he had been in a deep coma ever since. the computed tomographic angiography (cta) revealed a giant aneurysm of right internal carotid artery about one month after the blunt head injury. the aneurysm was measured . cm at its maximal diameter on image. of note, the patient failed to improve the following day and died on the fiftieth hospital day. giant tias are very rare but fatal complications of blunt head injury probably related to effects of vessel wall trauma and possibly a combination of neurological deterioration. in our case, the involved mechanism was suspected to be related to skull base fractures or resulted from stretching of the artery across the process during the impact. cta has a high sensitivity of about . % and a high specificity of about . % for diagnosing cerebral aneurysms (including traumatic aneurysms). apart from this, cta permits -dimensional visualization of aneurysms and assesses surrounding intracranial structures that are not visible on dsa. therefore, although -dimensional digital subtraction arteriography is currently the diagnostic gold standard in cerebral aneurysmal disease, fast and noninvasive cta may be preferred in the acute setting of tias. julio cabrera , corina puppo major burnt patients require large volumes of fluid replacement due to a generalized increase in permeability and edema caused by cytokines. fifty percent of the administered fluids produce edema in "preserved" tissues. multiple organ edema follows fluid replacement. escharotomy is frequently performed to decompress limbs and thorax, but not neck. our objective was to describe and diagnose neck-head compartment syndrome in patients with neck circumferential burns and/or neck edema by ) suspectng it and ) confirming diagnosis with the help of transcranial doppler (tcd) ultrasonography, searching for a high resistance pattern in cerebral blood flow velocity at basal cerebral arteries. tcd examination was performed before and after escharectomy in two both patients presented a neck-head compartment syndrome, evidenced by the cerebral hemodynamic repercussion of neck compression: hypoperfusion with an increased resistance pattern in dtc. p : secondary compartment syndrome due to massive fluid replacement; without circumferential burn. p : compartment syndrome in circumferential neck burn. tcd confirmed the clinical suspicion of cerebral hypoperfusion, guiding the decision to perform surgical decompression to treat it, and helped to assess the results of the decompressive surgery. introduction . % hypertonic saline is used for the treatment of increased intracranial pressure (icp) and in the prevention and reversal of brain herniation syndromes. the use of hypertonic saline in the management of combat related penetrating and severe traumatic brain injury is described. . % hypertonic saline effectively managed icp with decreased risk of hypovolemia and secondary hypotension compared with mannitol. . % hypertonic saline also preserved cerebral blood flow, decreasing the risk for secondary cerebral ischemia in acute neurotrauma patients, where hyperventilation is contraindicated. the nato hospital, kandahar afghanistan treated eleven ( ) patients with twenty-seven ( ) doses of . % saline from -march to -april . hypertonic saline was used to treat acute elevation in icp, as well as to maintain an elevated serum sodium concentration during periods of cerebral edema. all patients were treated with initial conservative icp management. external ventricular drains were placed and drainage of - cc of csf was performed in an attempt to maintain icp before using hypertonic saline. patients with life-threatening clinical signs of elevated icp secondary to brain edema or acute neurologic deterioration were potential candidates for . % hypertonic saline therapy. - ml of . % sodium chloride was administered via a central line infusion. . % hypertonic saline was successful in acutely reducing icp. a ml bolus of . % saline predictably increased the serum sodium levels allowing reliable titration and maintenance of serum sodium levels and efficient management of the patient's volume status ( cc of . % = cc of %). penetrating and severe closed head injuries have the potential to lead to neurologic emergency as a result of brain edema associated with primary tbi or following neurosurgical intervention. in a combat tbi population, . % hypertonic saline demonstrates a clinical benefit over alternative treatments by decreasing the risk of secondary cerebral injury during the management of elevated icp and was well tolerated. unintentional death was the ninth leading cause of death among elderly patients. given their comorbidity profile, many of them are also on antiplatelets or anticaogulants. we sought to characterize the burden of "pro-bleeding" medications such as antiplatelets and anticoagulants in the population aged over who sustain a head injury. this observational cohort study was conducted at a level one trauma center that has a county catchment area serving over million. the trauma acuity is high, with over % of our patients haveing iss scores over . the age cutoff of for "elderly" is based on our trauma alert activation criteria. thirty-nine percent of the cohort was on at least one type of anticoagulant or antiplatelet, as follows: warfarin %, aspirin %, clopidogrel %, asa+dipyridamole %, heparin/lmwh %.a third of the cohort required icu admission. icu length of stay ranged from - days. patient in particular, on warfarin had a significantly longer icu length of stay (p= . ) when adjusted for inr level. the median inr for the whole cohort was . with an iqr of . to . . the median inr amongst those on warfarin was . with an iqr of . to . . patients on an antiplatelet or anticoagulant agent were significantly more likely to have an abnormal head ct (p= . ). % of the patients who were on warfarin needed some sort of anti-coagulant reversal to minimize bleed. patients on warfarin were more likely to undergo neurosurgical intervention (p< . ) when compared to cohort not on warfarin. antiplatelet and anticoagulant drugs can confer additional morbidity to persons who sustain a tbi. it may be important to recognize this early, and prepare for higher level care needs. introduction therapeutic hypothermia (th) is know to cause immune suppression. determining the degree of immune suppression at the bedside is often difficult or impossible. immune cell function (icf) measures the concentration of atp from circulating cd cells following in vitro stimulation with phytohemagglutinin (pha) as an indicator of immune cell function. icf is often used in solid organ transplant programs to modulate the immunosuppressive treatment. we propose the use of ifc to determine the degree of immune depression in the patient treated with th. immune cell function, cylex inc, columbia, md was obtained in three populations of patients: group : patients treated with th, ifc obtained while at target temperate, degrees c group : patiients that were admitted to the care of the neurocritical care team, requiring icu care. group : patient from sanford renal transplant program with stable immunosuppressive therapy. the average icf of group were , of group , and of group , . patient being treated with th have a profoundly depressed icf. the level of immunosuppression is equal to if not greater that those with solid organ transplants. according to the cylex data a level of less than represents an immune suppressed state. this does not appear to be a phenomenon of the critically brain injured patient since those without th had a normal icf while further studies are in process, this data has effected out practice. we now treat patients on th as immunosuppressed patients. very early prediction of neurological outcome after cardiac arrest (ca) remains challenging. several single center studies have suggested that bispectral index (bis) can predict outcome for patients treated with therapeutic hypothermia (th). we evaluated the ability of bis to predict outcome in a multicenter study. medical centers prospectively enrolled comatose ca patients treated with th. outcome was defined as good (go) if cerebral performance category (cpc) score was - , and poor (po) if cpc - at hospital discharge (hd) and at months ( m). bis data was assessed blind to outcome for initial value after first dose of neuromuscular blockade (nmb -bisi) and at hours post-rosc (bis ). patients were enrolled with a mean age of (sd ) years, % were male, % witnessed, initial rhythm was vt/vf in %, pea in %, asystole in %, and time to rosc was . ( ) minutes. at hd, ( %) had go with similar age as po but shorter median time to rosc at (iqr - ) mins vs ( - , p= . ). go patients also had more vt/vf as initial rhythm and witnessed ca (p< . ), and more males (p= . ). on roc curve comparisons, both bisi (auc . ) and bis (auc . ) performed better than time to rosc (auc . ) or age (auc . ) -p< . for all comparisons. among ca-th treated patients, this is the first multicenter trial to confirm that bispectral index values after first dose of nmb and at hours post-rosc predicted outcome better than time to rosc, rhythm, or age. bis appears promising as a tool to predict outcome very early after ca, and may be helpful during clinical trials to stratify the severity of brain injury sustained during ca. hypotension negates the cerebral protective effect of therapeutic hypothermia (th). myocardial depression, "cold-induced diuresis," and hypokalemia can lead to refractory hypotension during the maintenance phase of th. intravascular volume replenishment and inotropic infusion are effective but cause wide swings in heart rate, blood pressure, cardiac output and acid-base status. we propose the use of vasopressin as a physiologically appropriate agent to correct hypothermiainduced hypotension. hypothesis: in swine, the investigators tested the hypothesis that an infusion of vasopressin would restore blood pressure to normal levels during th. six domestic cross-bred pigs ( - kg) were subjected to a atm fluid percussion injury to the brain followed by systemic hypothermia ( °c) for hours. the animals were turned side to side and to sternal recumbency every six hours. during phase i (first hours), the blood pressures were maintained in the normal range with intermittent doses of epinephrine and fluid boluses. during phase ii (second hours), continuous vasopressin infusion ( . ug/min) was added to maintain blood pressure. the number of episodes of hypotension (map < mm hg), the volume of fluids (liters), and the total dose of epinephrine (mg) used during both phases were compared using student's paired t-test (p> . ). in all animals, the infusion of vasopressin effectively mitigated the occurrence of hypothermia-induced hypotension. the episodes of hypotension ( . ± . v . ± . ), the total volume of fluids ( . ± . v . ± . ), and the total dose of epinephrine ( . ± . v . ± . ) administered were significantly reduced during phase ii. in order to maximize the benefits of th, hypotension must be avoided. animal studies show that despite hypothermia, hypotension causes cerebral cortical tissue depletion of atp and phosphocreatine and an increase of lactate and nadh levels. the infusion of a low dose of vasopressin reverses these anomalies and effectively mitigates hypotension. hypotension, hyperoxia, and hypoxia early after the return of spontaneous circulation (rosc) are each associated with increased mortality, while early hypertension is associated with good outcome. we assessed these variables and their relationship to outcome in cardiac arrest (ca) survivors treated with therapeutic hypothermia (th). with irb approval, we reviewed prospective and retrospectively collected data in a single-center database of patients undergoing th after ca. demographics and clinical factors were compared among patients with cpc - (good outcome) and cpc - (poor outcome) in a bivariate model. various definitions of hypotension, hypertension, hypoxia, and hyperoxia were evaluated. we constructed logistic regression models including potential confounders and the variables of interest. among patients, age, vt/vf rhythm, shorter time to rosc, witnessed arrest, bystander cpr, and stemi on initial ecg were each strongly associated with good neurological outcome, as were a lower peak neuron-specific enolase level and higher bispectral index (bis) score after neuromuscular blockade. hyperoxia (pao > mmhg) was common (present in . with good and . with poor outcomes, respectively) as were hypoxia (pao < mmhg) and hypotension. none of these factors was a predictor of outcome. logistic regression models intended to adjust for the potential confounding influences of age, time to rosc, heart rhythm, witnessed arrest, and bystander cpr, also did not identify a relationship between the variables of interest and outcome. our data did not confirm the previously described relationship between post-resuscitation factors and outcome. this may reflect an inadequate sample size, but it is also possible that post-resuscitation hemodynamic and biochemical factors are minimally important to outcome, compared to the duration and type of the arrest. further investigation in larger data sets is warranted. determining the presence of an infectious process during therapeutic hypothermia (th) can be difficult. in addition, differentiating central vs systemic fever is difficult in the brain injured patient. procalcitonin (pct) was been used to guide the use of antibiotics in sepsis and pneumonia in patients that are critically ill. we propose the use of pct to predict the presence of a systemic infection in patients during th. all patients treated with th had pct measured at the start of th. all patients were cooled with the medivance arctic sun . when the water temperature was < degree c, pct and two sets of blood cultures (bc) were drawn. sputum cultures (sc) were obtained if there was a change in sputum or during bronchoscopy. antibiotic use was determined by the neuro-intensivist results patients were evaluated; ich, tbi, cva and cardiac arrest (ca). a total of pcts were obtained. one patient ( %) had positive bc, pct of . ; patients ( %) had positive sc. remaining patients had negative bc and sc. all ca patients had increased pct > . (normal < . ) of which ( %) had positive sc and none had positive bc. of the remaining without positive bc ( %), ( %) had positive sc, all had pct < . . of the ( %) patients without positive sc, all had pct < . pct is a reliable method to exclude an infectious process in patients being treated with th that have not had a ca. while further studies are warranted, a pct < . appear to exclude both pulmonary and blood infections, while a pct < . appears to exclude a blood stream infection. from this data, pct is not a good marker for infection in the ca patient. therapeutic hypothermia (th) has become widely accepted practice for neuroprotection and improved mortality in comatose survivors of out of hospital v-fib cardiac arrest. evaluation for appropriateness of th is now part of acls algorithm. its use in non-shockable rhythms such as pea and asystolic arrest is less well established. we present our center's experience with th after cardiac arrest and review the clinical and electrophysiological parameters that may impact prognosis. this is retrospective review of medical charts including patients undergoing th after cardiac arrest at a single center from through the first quarter of . demographic and clinical data were collected. continuous eeg results were reviewed by two independent epileptologists who were blinded to the outcome of the patients. eegs were graded based on the synek scale for grading severity of eegs. patient's neurologic outcome will be assessed by grading cerebral performance category (cpc) score at the time of discharge. multivariate regression analysis will be performed on the data to identify parameters that would affect prognosis in cardiac arrest after cooling. fifty-eight patients were identified from our database. the overall rate of survival to discharge was %. the survival rate for v-fib arrest was % whereas the survival rates for asystolic arrest and pea arrest were % and %, respectively. results from the multivariate analysis will be forthcoming. our results affirm the predominant view that th indeed improves outcomes after cardiac arrest. in particular with ventricular fibrillation and pulseless ventricular tachycardia arrest, we have seen very encouraging results. patients with pea/asystolic arrest fared worse but outcomes are still improved compared to historical control. since , mild therapeutic hypothermia (mth) has been the standard of care when spontaneous circulation returns after a witnessed, out-of-hospital ventricular fibrillation arrest[ ]. at our institution, we have initiated mth for approximately fifty patients since february . a knowledge, attitude, and practices survey was conducted querying neurology residents and attendings, emergency medicine (em) residents and attendings, and internal medicine (im) residents. our aim was to identify areas of weakness so that we could strengthen the overall awareness of the utility and benefit of mth. the survey consisted of nineteen multiple choice questions, ranging from asking how many times the participant had initiated mth; to parameters for the protocol; to how it impacts survival. the surveys were completed by: ten neurology residents and five neurology attendings; twelve em residents and two em attendings; and twenty im residents. all of the neurology residents and em physicians surveyed had been the primary provider for a post-arrest patient who underwent mth. the neurology residents unanimously agreed that mth after resuscitation from a shockable rhythm is standard of care, however only % of em physicians and % of im residents agreed. % of em physicians and % of im physicians answered that mth may be initiated in cases presenting after either a shockable or a non-shockable rhythm. % of the participants acknowledged that ventricular fibrillation portends the most favorable outcome. nearly % of participants agreed that ideal rosc is less than thirty minutes. three-quarters of physicians indicated the goal temperature as - °c; however, half of the neurology residents and % of neurology attendings answered this incorrectly. in conclusion, this survey has revealed a general understanding of mth, however, each specialty has its deficiencies. we can now educate each subset of physicians in a problem-focused manner. early quantitative assessment of non-contrast brain computed tomography (ct) using specialized software correlates with outcomes of cardiac arrest survivors. the proposed algorithm compared hounsfield units (hu) in the putamen (pu) to the posterior limb of the internal capsule (plic), but the work has not been validated in patients treated with therapeutic hypothermia (th) or using standard software and equipment. we included ca survivors treated with th who underwent ct in the first h after resuscitation (rosc). hu were averaged bilaterally at two levels in the pu and plic, and the pu/plic ratio calculated by a board-certified radiologist using a ge lightspeed vct slice scanner and agfa pacs system. receiver-operator characteristic (roc) curves were constructed, evaluating pu or pu/plic to predict poor outcome (cpc - ) at hospital discharge (hd) and months ( m). patients had median age years, % male, % out-of-hospital ca, % witnessed, % vt/vf, % pea, and % asystole. median (iqr) time to rosc was ( - ) minutes. / ( %) patients had po. when stratified by outcome, ct performed . ( . - ) hrs after rosc showed similar hu measurements for plic ( . po vs . go, p= . ) but lower hu in pu ( . vs . , p= . ) and pu/plic ( . vs . , p= . ). hu values for pu and pu/plic both predicted outcome: roc area under the curve (auc) for pu = . ( %ci . - . ) and pu/plic = . ( . - . ). among patients with m outcome data, pu predicted outcome ( . po vs . go, p= . ) with auc = . ( . - . ), but pu/plic did not. early after ca, hounsfield unit measurements in the putamen, and the pu/plic ratio were lower among patients with poor outcome, but the magnitude of the differences was small, and clinical utility uncertain. additional study is warranted. global cerebral edema following aneurysmal subarachnoid hemorrhage (asah) is associated with % in-hospital mortality. therapeutic hypothermia (th) is recommended for reduction of intracranial pressure (icp) based on class i evidence; however safety in prolonged states remains poorly studied. we retrospectively reviewed all cases of refractory icp elevation at the mayo clinic florida neurointensive care unit (nicu) from - who received adjunct th for more than hours. primary safety endpoints were qtc prolongation, development of bacteremia, and coagulopathy. additional outcomes included in-hospital mortality, hospital/nicu length of stay, and functional status at months. patients with asah and/or intracerebral hemorrhage underwent adjunct th. median age was ; were male. on admission, median apache was , and wfns was higher than in , all being modified fisher - . required barbiturates in addition to sedation, paralysis and hyperosmolar therapy. th was initiated on a median of hospital day and continued for a median of days (minimum= , maximum= ). mean icp over hours prior to th was . mmhg(sd= . ; range . - . ), decreasing to . mmhg(sd= . , range . - . ) over the first hours of th. patients had external ventricular drains placed and required decompressive craniectomy on average day hospital stay (range - ). safety data showed torsades-de-pointes in , mean qtc prolongation of with mean lengthening of aptt by . . patients had bacteremia on admission with new infections (urine, sputum, blood) documented in during th. overall, ( %) survived to discharge. median nicu/hospital length of stay was / . average modified rankin score at follow up was . . hypothermia greater than hours as an adjunct to standard icp reducing therapies appears feasible in patients with refractory intracranial hypertension. however, definitive safety of prolonged th would require direct comparison with similar cohort. refractory raised intracranial pressure (ricp) secondary to intracerebral hemorrhage (ich) and severe subarachnoid hemorrhage (sah) is a life threatening condition. treatment for ricp typically induces hypothermia (th) and decompressive hemicraniectomy (hct). however, direct comparison of the efficacy of these two therapies is lacking. data from this study may help determine the sequence of therapies that might improve outcomes in this patient population. in the present study using retrospective design, we tested the hypothesis that for patients with ricp, th is as effective in reducing icp as hct, using functional outcome at discharge as defined by modified rankin scale (mrs) as the primary outcome. we retrospectively reviewed all adult patients admitted to the neurointensive care unit from to with sah and ich with resultant elevated icp, who survived the first hours after admission. exclusion criteria included: pupillary anisocoria, limitation of care within hours of admission; or hemicraniectomy or craniotomy with clot evacuation prior to icp monitoring were excluded. initial review included patients (th= and hct= ). based on univariate analysis, admitting gcs score was higher with hct ( vs , p= . ), but other baseline demographic and clinical characteristics were similar. th group had longer icu los ( vs ), los ventilation ( vs ), and higher cost. however, discharge mrs ( vs ,p= . ) was similar. our initial analysis indicates longer icu care and overall cost with th, but similar functional outcomes at discharge. subsequent analysis will include inclusion of additional patients, icp comparison and adjustment for baseline characteristics. malignant middle cerebral artery(mca) infarction is devastating ischemic stroke, which the mortality rate is up to %. therapeutic hypothermia is one of the most promising neuro-protective therapies. successful result of hypothermia for cardiac arrest renewed interest in therapeutic hypothermia for stroke. the purpose of this study was to assess whether therapeutic hypothermia can reduce the cerebral edema and can improve the functional outcome in patients with malignant mca infarction. we reviewed retrospectively patients with malignant mca infarction presented within hours of symptom onset in a single center hypothermia registry. after informed consent, patients who had refused decompressive hemicraniectomy were treated with therapeutic hypothermia and monitored in the neurocritical care unit for complications. a modified rankin scale(mrs) and national institutes of health stroke scale(nihss) were obtained at months after symptom onset. eleven patients with a mean age of ± years and an nihss score of . ± . were treated with therapeutic hypothermia( ± ). seven of eleven patients were mca infarction, and four was ica t-occlusion. the mean time from symptom onset to initiation of hypothermia was . ± . hours and the total duration of hypothermia was . ± . hours. noncritical complications included shivering(n= ), bradycardia(n= ), hypertension(n= ), pneumonia(n= ), and arrhythmia(n= ). electrolyte imbalances were common during the hypothermia (hypernatremia;n= , hypokalemia;n= , hypophosphatemia;n= ). mortality rates was %(n= ) and the mean nihss at discharge was . ± . . the mean mrs at months was . ± . in all patients and . ± . in survivals. this result shows that therapeutic hypothermia can prevent the progression of cerebral edema and improve functional outcome in acute malignant mca infarctions and ica t-occlusion. long duration hypothermia more than days appears feasible and safe in these patients. therapeutic hypothermia may be a good alternative therapeutic option to early decompressive hemicraniectomy. large clinical trials are needed whether hypothermia will be a best treatment to improve functional outcome. therapeutic hypothermia (th) is know to cause immune suppression. determining the degree of immune suppression at the bedside is often difficult or impossible. immune cell function (icf) measures the concentration of atp from circulating cd cells following in vitro stimulation with phytohemagglutinin (pha) as an indicator of immune cell function. icf is often used in solid organ transplant programs to modulate the immunosuppressive treatment. we propose the use of ifc to determine the degree of immune depression in the patient treated with th. immune cell function, cylex inc, columbia, md was obtained in three populations of patients: group : patients treated with th, ifc obtained while at target temperate, degrees c group : patiients that were admitted to the care of the neurocritical care team, requiring icu care. group : patient from sanford renal transplant program with stable immunosuppressive therapy. group , patients, average icf: group : patients, average icf: group : patients, average icf, . patient being treated with th have a profoundly depressed icf. the level of immunosuppression is equal to if not greater that those with solid organ transplants. according to the cylex data a level of less than represents an immune suppressed state. this does not appear to be a phenomenon of the critically brain injured patient since those without th had a normal icf while further studies are in process, this data has effected out practice. we now treat patients on th as immunosuppressed patients. therapeutic hypothermia (th) has become a first-line therapeutic modality in patients suffering from traumatic brain injury and cardiac arrest. shivering induced by th reduces the ability of the cooling device to achieve target temperature. this can lead to increased intracranial pressure (icp) and increased metabolic demand. the bedside shiver assessment score (bsas) has been validated in identifying and grading shivering. however, the bsas cannot identify microshivering which is visually undetectable shivering that is thought to have the same detrimental physiologic consequences as shivering. continuous channel eeg (ceeg) can detect microshivering but is labor intensive, requires specialized training to interpret results and is expensive. we propose that the philips eeg with compression spectral array lead (philips ) can be utilized to detect microshivering as effectively as ceeg but is more cost effective. the philips was placed by the bedside nurse. the lead placement varied depending on underlying injuries. patients were assessed utilizing the bsas and the philips . if high frequency activity increased on the philips , the patients were assessed using the bsas. if the bsas was then - mg of vecuronium was given to intubated, sedated patients. both patient temperature and water temperature were recorded. two patients with tbi were evaluated. the water temperature decreased and the patient's temperature increased during the periods of high frequency activity on the philips . after vecuronium, the high frequency activity ceased, water temperature increased and core temperature returned to the previously set level. the philips is a relatively low cost device when compared to ceeg that can be applied and monitored by the nursing staff to detect microshivering. additionally, we were able to validate that control of microshivering improved the th device's ability to achieve and maintain the patient's temperature goal. therapeutic hypothermia is widely accepted as a standard of practice for out of hospital cardiac arrest (ohhca). however, its implementation is still highly variable in different hospital settings. most of the current data comes from centers of excellence. we wanted to evaluate performance of implementation of "hypothermia protocol" (hp) including its complications and outcomes in our large referral community based hospital. we conducted retrospective chart review of patients who underwent hp from - . data collected included demographics, time of cardiac arrest, time of arrival to er and time to induction of hp, methods used for induction, complications and outcomes. out of the patients, patients ( %) had pulse less electrical activity (pea), ( . %) patients had ventricular tachycardia/fibrillation, and ( . %) had complete heart block as the initial rhythm. average time to arrive to er was minutes. almost % of patients had ht induction in ed, % (%) in icu and . % outside of the hospital. average time to initiate ht from the initial event was hour and minutes. average time to achieve the target temperature from the initial event was hours. inner cool was the most common modality used in . (%). lactic acidosis ( . %) was the most common complication encountered, followed by hypotension ( %), coagulopathy ( %) and seizure ( %) trend of improved outcomes with less renal failure, coagulopathy, seizure was observed with shorter induction times. time to achieve target temperature had no effect. initial rhythm, age and gender also had no impact on the outcome. shorter induction time appears to decrease complications and improve outcomes. using multiple cooling modalities also appeared to have better outcomes. however larger studies are needed to confirm this observation. earlier induction of mild therapeutic hypothermia improves survival and neurological outcome and decreases incidence of some of the complications. introduction secondary brain injury after aneurysmal subarachnoid hemorrhage (asah) is a major cause of mortality. mild hypothermia ( - c) may protect against cerebral ischemia and edema in asah patients. the aim of this study is to describe the use of ct perfusion (ctp) characteristics to initiate re-warming in patients with secondary brain injury after asah. we performed a retrospective review of all patients admitted to a large comprehensive stroke center between and with asah who were treated with hypothermia and received ctp imaging. mild hypothermia ( - c) was started because of severe vasospasm, increased intracranial pressure or cerebral edema. baseline characteristics, including clinical severity grading by hunt hess (hh) and fisher scales, were collected. clinical outcomes were measured by discharge modified rankin score (mrs) and disposition. ctp was performed with a -slice scanner. twenty patients fulfilled inclusion criteria. in / ( %) patients, re-warming was based on favorable ctp characteristics and in / ( %) based on favorable tcd findings. the mean duration of hypothermia was . days. five patients were re-warmed due to normal ctp, despite tcd findings suggesting moderate to severe vasospasm. patients, re-warming was initiated given improving tcd findings and despite less favorable ctp data (most showing "matched" abnormalities of decreased cbv, cbf and increased mtt). clinical outcomes were worse in this group; mrs better outcome was seen in all patients in whom re-warming was initiated based on normal ctp. in these patients, there was a discrepancy between ctp and tcd data. poor outcome was associated with abnormal ctp regardless of tcd findings. ctp may be a useful tool to guide treatment of asah patients receiving hypothermia. diagnosis of pediatric brain death (pbd) continues to be a significant challenge. new guidelines for pbd diagnosis were published in pediatrics in . we recently conducted a mailed survey to assess current understanding of these new guidelines and general perspectives about pbd among a convenience sample of midwest usa physicians. we developed a item survey. items included demographic questions, question about familiarity with the guidelines, and questions concerning perceived discrepancies and other attitudes toward the guidelines. we mailed our survey to physicians at university hospitals: pediatric intensivists, neonatologists, adult neurointensivists, and pediatric neurologists, three weeks after the initial mailing, we followed up with a reminder by mail and/or phone. we performed fisher's exact test to assess statistical significance of responses among different specialties. after weeks, we had a % response rate. respondents included pediatric neurologists, neurointensivists, pediatric intensivists, and neonatologists. twenty percent of respondents were unfamiliar with the new pbd guidelines (neonatologists were least familiar). twenty-three percent stated they were 'not comfortable' making a pbd diagnosis and % deemed it was either preferable or essential to obtain a neurointensivist or pediatric neurology consultation for pbd assessment. there was general agreement that the current intervals for the required exams were appropriate in children (delineated by age). interestingly, % allowed patients to remain ventilated for a significant period of time after pbd declaration. we found that a significant number of pediatric physicians are not familiar of the new pbd guidelines and there remains some variability in the assessment of these patients. pediatric neurologists or neurointensivists are still considered an important part of the process of pbd determination. the mid-position fixed pupil (mpfp) is an imperfect reference to the mid-size pupil that occurs with the complete loss of neural influence from devastating midbrain injury (primary or secondary) and death (brain and cardiopulmonary). for this reason, proper recognition and interpretation of the mpfp is critical to the neurological localization/diagnostic process and a vital element to the clinical verification of brain death. while the description of the size range of the mpfp has been dogmatically passed down from numerous classical texts ( - mm) for decades, it has not been accurately quantified. modern pupillometry offers accurate quantification of pupil size. using a portable infrared pupillometer (forsite, neurooptics inc., irvine, ca), within hours after death, we evaluated the pupil size of dead patients who did not have any previous eye surgery, known eye disease, or use of eye medications. pupils were evaluated in dead patients (mean age ) an average of hours after death. the pupil size range was . - . mm, with a median size of . mm (sd of . mm). / pupils ( %) were < mm and none were > . mm. / patients ( %) had a side-to-side difference of at least . mm. thankfully none were reactive! the mpfp is generally smaller than classically described and % fall between . and mm. % of mpfp's are less than mm. we never found any mpfp's more than . mm. subtle but frequent side-to-side asymmetry (> . mm) existed in approximately % of the dead patients. with our continued work we can finally achieve a more quantitative description of the important finding of the mpfp so that it can be incorporated into our definitive texts, enveloped into our understanding, and applied to our clinical practice. brain death diagnosis is clinical in uruguay. it is defined as the irreversible loss of brain stem functions. ancillary tests are needed as confirmatory tests in selected cases: ) impossibility or contraindication to perform clinical testing (barbiturates, facial trauma, etc.); ) non demonstrable structural lesion; ) unknown coma etiology; ) difficulties to wait for a second clinical test. the most used confirmatory test is transcranial doppler (tcd) ultrasonography. objectives: to study ) the clinical characteristics of patients in whom brain death could not be diagnosed clinically; ) tcd ultrasonographic patterns; ) number of cases in which tcd aided in management. epidemiologic and observational study. patients included: those in who brain death was suspected but the clinical examination of brain stem reflexes and/or apnea test could not be performed for different reasons. period: from to . the variables studied were demographic and clinical characteristics, tcd sonographic patterns. cerebral circulatory arrest was diagnosed when the patterns found were systolic spikes, reverberating flow, and no-flow (if a previous study had demonstrated ultrasound permeability of skull windows) in bilateral anterior sectors and posterior sector. continuous flow or systolic peaks were negative for the diagnosis of cca. patients in who the clinical diagnosis of brain death was not possible or needed to be confirmed. % adults. % were men, with an average of y.o. in adults, and y.o.in children; structural etiology %. etiology: traumatic %; vascular %; anoxic-ischemic %, infectious %, toxic-metabolic %, other %. cca was confirmed in %, systolic spikes in %. cca was discarded in %. in this group the study was repeated in %, confirming cca in %. it was not concluding in %. dtc helped in the decision to how to continue the management of the patient in % of the cases, diagnosing cca in %. there is an awkward physician and cross-institutional variability in the approach to brain death (bd) diagnosis and all of its ramifications; physiological, logistical, and psychosocial. physician variability is related, in part, to a basic knowledge deficit and inexperience. however, public confidence in the reality of bd relies on consistent and accurate diagnosis and the physician's facility with the management of its implications. our full-day ( hour) brain death simulation workshop (bdsw) was designed to enhance confidence with bd diagnosis and management. it included a didactic lecture and seven learning stations: case study analysis (recognizing brain death mimics), a high fidelity mannequin simulation (bd examination including cold water calorics and apnea testing, hemodynamic management, and diabetes insipidus management), family discussions with professional actors trained to provide feedback, and four relevant content stations. each participant was observed by a neurocritical care expert, each receiving one-on-one and group feedback. physicians participants from continents participated in the bdsw with expert faculty. all participants felt much more confident with brain death diagnosis and management. at least % were humbled by the station on "discussion of brain death with families", recognizing their need to practice communicating about brain death effectively. % felt better equipped to contemporize their local policies and advocate for enhanced uniformity of practice. our bdsw provides a model comprehensive training experience that had a favorable impact on trainee confidence and their interest and capacity to advocate for better uniformity and training of peers. . the bdsw can be part of a future tiered approach to credentialing experts in this important clinical area. . we are conducting the nd bdsw on november , with improvements based on the st workshop. neurocritical care experts must embrace the primary responsibility for preserving the integrity of bd diagnosis and educating our colleagues. the use of carbogen in apnea testing to declare brain death may facilitate achieving the prerequisite pco needed to confirm apnea testing by establishing a target end point that is typically reached faster and has been shown to limit adverse effects. as the use of extracorporeal membrane oxygenation (ecmo) in adults increases, so does the need to perform apnea testing while on ecmo. however, traditional apnea testing on critically ill patients is compounded by lung derecruitment and hemodynamic instability rendering an aborted apnea test or worse, cardiac arrest and death. the literature on apnea testing of patients on venous-arterial (va)-ecmo is minimal. per hospital protocol, a carbogen mixture ( % oxygen and % carbon dioxide) was delivered through the ventilator for an apnea test on a year old female on va-ecmo. the ventilator's mandatory rate was set at breaths/minute to adequately deliver the carbogen mixture through the artificial airway. a carbogen formula was used to calculate a target end-point of an etco of mmhg for a positive apnea test. an abg was drawn prior to the apnea test and again once the target etco was achieved. pre-apnea abg: . / / / %. the etco goal was reached within minutes and the post-apnea abg was drawn: . / / / %. the patient remained hemodynamically stable throughout the apnea test which was confirmed as a positive apnea test. the use of carbogen in apnea testing on a patient receiving va-ecmo demonstrates the possibility of performing a successful apnea test for declaration of brain death. although more investigation is needed, this case demonstrates the ability to perform apnea testing on critically ill and unstable patients while maintaining hemodynamic stability which preserves the option for organ donation. drowning victims have historically been eliminated from consideration for lung donation as aspiration may cause direct pulmonary toxicity, often confounded by significant neurogenic pulmonary edema. a significant minority of these patients ( - %), aspirate only minimal amounts of water into their lungs, protected by severe-persisting laryngospasm (dry drowning), but progress to brain death due to significant anoxic injury. historically, even with limited evidence of aspiration,transplant centers do not consider evaluating drowning victims as lung donors. however, as the division between the number of eligible recipients and available donor organs continues to grow, criteria for acceptable donor organs are expanding. once an absolute contraindication for lung donation, this practice has persisted on a per case basis but is reported infrequently with somewhat mixed results. we analyzed the unos registry of donors for lung and heart-lung transplant from january , to december , (n= ), and then examined survival outcomes from lung transplant recipients from donors who suffered drowning between to recipients (n = ) to outcomes previously reported from lung transplant recipients during that period. for recipients of lungs from donors with drowning as cause of death, unadjusted survival at one drowning victims, even when initially resuscitated, often suffer significant anoxic injury and death by neurologic criteria. while the management of drowning victims as organ donors may present additional challenges, with proper donor selection, the use of lungs recovered from carefully screened donors after drowning appears to be a safe option for the expansion of the donor pool. racial disparity in health care utilization and outcomes is an area of substantial concern. a study performed in the 's in our neuro-icu found that nonwhites were half as likely to withdraw life-sustaining therapy (wlst). this may be explained by differences in socioeconomic status (ses), cultural preference, lack of end-of-life planning, or trust in the health-care system. to better understand the basis and evolution of this disparity, we analyzed it over two more recent epochs (determining whether it has improved over time), while specifically accounting for ses. we extracted data from a prospective neuro-icu database on all ventilated patients with gcs of or less between and . we analyzed how the rate of wlst was affected by age, race, gender, insurance and socioeconomic status (quintiles based on median household income of residence zip code), marital status, receipt of surgical/icu interventions, gcs and apache ii. we then compared ses-adjusted disparity for wlst (non-whites vs. whites) in - with - . non-whites accounted for of patients ( %) and were younger, less likely to be married ( % vs. %), insured ( % vs. %), and reside in upper-income zip codes (all p< . ). rate of wlst was lower in non-whites ( % vs. %, p< . ), despite comparable overall hospital mortality. after controlling for ses and other confounders, non-white race was still associated with lower odds of wlst (aor . , % ci . - . ). this disparity was prominent in the earlier epoch (aor . , . - . ) while race was no longer a statistically significant marker in the more recent cohort (aor . , . - . ). race appears to influence the likelihood of wlst in severely brain-injured patients independent of ses. this disparity, which has been attenuated over the past decade in our icu, may be related to cultural differences or barriers relating to end-of-life planning or trust. multiple parameters have been associated with outcome in comatose post-cardiac arrest patients. anecdotal observations suggest that patients who are cooler upon ed arrival tend to have poorer outcomes; if arrival temperature correlates with outcome, it may serve as an additional tool for patient prognostication. we performed a retrospective analysis of a prospectively collected data set from comatose post-cardiac arrest patients to determine if a relationship exists between arrival temperature and outcome. of the patients, patients ( %) with out-of-hospital cardiac arrests and with arrival temperatures recorded prior to initiation of hypothermia treatment were included and divided into those with good outcomes ( subjects; mrs => ) or poor outcomes ( subjects; mrs =< or death) at months; subjects ( poor outcome survivors and who progressed to brain death) remained when patients whose poor outcome (death) was due to withdrawal of care were removed from the poor outcome group. analysis using a two-tailed unpaired t-test on subjects with good versus with poor outcomes demonstrated a significant difference in temperature on ed arrival: mean temperature of patients with good outcomes was . o c (sd= . o c), while that of patients with poor outcome was . o c (sd= . ), p= . . when patients who died due to withdrawal of care were included in the analysis, a strong trend in difference between the two groups remained, but was not statistically significant (p= . ). low body temperature upon ed arrival correlates with poor outcome in post-cardiac arrest patients and may serve as an additional prognostic variable. cooler temperatures may merely reflect longer lapsed time before return to normal circulation; alternatively, they may be a result of poor temperature regulation in more severely brain injured patients. further investigation of this issue with a larger patient pool is warranted. diencephalon injury (di) has been described in neurocritical care. consciousness alterations (ca), dysnatremia, hemodynamic instability, fever, muscle dystonia are signs of di. these symptoms are non-specific. the goal of the study was to describe structure of acute diencephalon dysfunction syndrome (adds) on the model of isolated acute di. this retrospective study evaluated all patients operated in - . inclusion criteria: adult patients in stable preoperative condition; sellar region tumors (srt); complicated postoperative period. exclusion criteria: intra-cranial complications, not related with direct di (epi-, subdural hematomas, brain ischemia). organ dysfunctions and dysnatremia were registered. patients were included, excluded. all had ca and dysnatremia. hemodynamic dysfunction developed in patients, respiratory dysfunction in patients, ileus in patients, thrombocytopenia in patients, renal dysfunction in patients, hepatic dysfunction in patient. there were groups. first (n= ) had ca, dysnatremia. icu los was . days. glasgow outcome scale (gos) had patients; gos : patient. second group (n= ) had ca, dysnatremia, one somatic organ dysfunction (sod). icu los was days. gos had patients, gos : patients. third group (n= ) had ca, dysnatremia, two sod. icu los was . days. gos , had patients; gos : patients; gos : patients. fourth group (n= ) had ca, dysnatremia, sod. icu los was days. gos , had patients; gos : patients; gos : patients. fifth group (n= ) had ca, dysnatremia, sod. icu los was . days. gos had patients; gos : patient; gos : patients. sixth group (n= ) had ca, dysnatremia, sod. icu los was . days. all died. adds consists of ca, dysnatremia, and at least one sod. severity of adds depends on number of sod. intracranial pressure (icp) monitoring is widely used in the management of patients with traumatic brain injury. icp monitoring may also be useful in other situations characterized by high icp, including cardiac arrest survivors (cas) after return of spontaneous circulation (rosc). however, no prospective study has examined the incidence of raised icp among cas. this pilot study will examine the feasibility of screening for elevated icp in cas admitted to the toronto western hospital (twh) in -using the non-invasive technique of optic nerve ultrasonography (onus) --to identify patients with elevated icp, who might benefit from invasive icp monitoring to optimize their management after they survive cardiac arrest. evidence of elevated icp will be examined by blinded ultrasonographers(usf) who will measure the optic nerve sheath diameter (onsd) in both eyes of all cas every hours from rosc. all findings will be defined in a dichotomous method (elevated/not elevated). primary outcome: incidence of major protocol violations, defined as the inability to attain of onus recordings during first hours at the specified time point (every hours) by each usf. for every major protocol violation, an audit will be done to understand the reason for the violation and tailor the protocol to improve compliance in future studies. advances in resuscitation medicine have demonstrated an improvement in patient outcomes in cas by the implementation of th. the exact mechanism of action of th is not well understood and has been postulated to partially involve a decrease in icp. no prospective data currently exists linking th with icp. using onus as a non-invasive modality, we have designed a single centre feasibility study to assess the ability of onus to measure icp in cas, as well as to aid in sample size calculations for a larger multicentre prospective cohort study. a preliminary study demonstrated that > % of whole brain volume with an apparent diffusion coefficient (adc) < x - mm /sec identified poor outcome (death/vegetative state) with % specificity and % sensitivity. we aimed to validate this threshold in an external dataset. a multicenter retrospective observational study of dwi mris of comatose post-cardiac arrest patients obtained between and hours post-arrest was performed. poor outcome was defined as death or persistent coma at day . imaging was processed in a blinded fashion using medical image processing, analysis and visualization program (mipav). the brain was semi-automatically outlined on the b images using a levelset algorithm. the adc values of each voxel within the brain were determined. outcomes were assessed blinded to quantitative dwi information. treating physicians were not blinded to the mri scans, but they were unaware of the quantitative dwi analysis. data from patients from five us centers were included: mean age was ± years, % female, arrest time ± minutes, % of patients received hypothermia, and mris were obtained at ± hours post-arrest. thirty-two percent had a good outcome. the median (iqr) percentage of brain tissue with adc< x - mm /sec was . % ( . - . ) in good and . % ( . - . ) in poor outcome patients (p< . ). an adc< x - mm /sec > % was % ( % ci - ) specific and % ( % ci - ) sensitive for poor outcome with a positive predictive value of % ( - ) and a negative predictive value of % ( - ). the odds ratio of having a poor outcome if > % of brain had an adc< x - mm /sec was ( %ci - ). quantitative dwi mri in comatose post-cardiac arrest patients holds great promise as a prognostic adjunct between and days after arrest. predicting outcome for comatose post-cardiac arrest patients is challenging and compounded by the use of therapeutic hypothermia and sedative agents. brain mri is a potential attractive prognostic adjunct not affected by drugs or metabolic derangements; however, most proposed methods require image post-processing. we assessed the prognostic value of color apparent diffusion coefficient (cadc) maps. consecutive post-cardiac arrest patients remaining comatose after resuscitation were prospectively enrolled. cadc maps were created by assigning adc values to colors ranging from red to blue. the treating teams did not see these maps. two raters independently and blinded reviewed the cadc maps and predicted month outcome as poor (glasgow outcome scale (gos) - ), or good (gos of - ). both raters were "trained" by viewing examples. the agreement between raters and the predictive performance of the cadc maps were assessed. cadc maps of patients ( % with poor, % with good outcome) were reviewed: age ± years, % females, % underwent therapeutic hypothermia, median (iqr) arrest duration min ( - ), and time between the arrest and mri hours ( - ). kappa for agreement on predicting favorable vs. unfavorable outcome was . . for the two reviewers, the sensitivity for predicting poor outcome was . ( % ci . - . ) and . ( . - . ), the specificity . ( . - . ) and . ( . - . ), and the true positive predictive rate % ( - %) and % ( - %), respectively. for mri scans acquired between - hours after the arrest (i.e. the time-interval when adc changes are most apparent), the specificity improved to . ( . - . ) and . ( . - . ), respectively. mri color adc maps are easy to interpret and may be useful for predicting outcome of comatose post-cardiac arrest patients in the first days after the arrest. color adc maps do not require post-processing and can be created in realtime. there are few reports of outcome in patients with fat embolism syndrome with diffuse mri abnormities. we report the outcome of patients with fat embolism syndrome. case a -year-old previously healthy gentleman had a right femur fracture from a motor vehicle accident. he had acute respiratory failure hours later requiring intubation. chest x ray showed bilateral lung infiltrates. neurological examination showed patient comatose with intact brainstem reflexes and extensor posturing. on day , he had fever, tachycardia, profuse sweating, and diffuse petechial rash. mri brain showed diffuse restricted diffusion lesions. he started to open his eyes in weeks and underwent tracheostomy and feeding tube placement. at month follow up he only had mild memory problems. case a -year-old previously healthy gentleman had a gun shot in the left foot. over the next hours he became stuporous. x ray showed multiple fractures including calcaneus, soft tissue swelling and subcutaneous emphysema. over the next hours he worsened and displayed extensor posturing. mri brain showed diffuse innumerable tiny infarcts. patient was noted to have episodic fever, profuse sweating, and severe tachycardia. patient had spontaneous eye opening next day and underwent tracheostomy and gastrostomy. he was transferred to a long term facility. patient improved substantially and at months follow up he was independently living at home with minor neurologic deficits. substantial improvement may occur in comatose patients with fat embolism syndrome despite paroxysmal sympathetic hyperactivity syndrome and significant mri abnormities. malignant pertussis is a rare life-threatening illness characterized by severe respiratory failure, extreme leukocytosis, and pulmonary hypertension. during , an outburst of whooping cough was experienced at montevideo, uruguay. we present the cases of two infants, and months old, suffering malignant pertussis, admitted to a university pediatric intensive care unit (picu) for severe acute respiratory failure associated with severe leukocytosis. both children showed signs of profound coma and bilateral arreactive dilated pupils while being aggressively treated. both of them showed a transcranial pattern of cerebral circulatory arrest (cca) on transcranial doppler (tcd).to our knowledge, a pattern of cca has not been previously reported like mode of death secondary to neurologic injury in this disease. both cases were very similar: a -month-old boy, incomplete vaccinatinon, malnourished. a month-old girl, vaccinated. both had suffered at one and months-old, severe bronchiolitis caused by respiratory syncytial virus, both needed days of mechanical ventilation. both were admitted to icu with cough, fever, increased work of breathing, hypoxemia and were mechanically ventilated. they presented respiratory acidosis, hipoxemia, extreme leukocytosis greater than , bilateral hyperinsuflation in chest x-ray. echocardiography: pulmonary hypertension, - mmhg spap, circulatory failure, anuric renal failure. bordetella pertussis was diagnosed with pcr of airway secretions treatment: blood exchange transfusions, milrinone, maximum dose inotropic drugs, peritoneal dialisis. after one week arreactive dilated pupils and profound coma were evident. brain death was suspected, sedation and muscle blockers were interrupted. neurologic exam confirmed brain death. tcd showed sysytolic spykes in bilateral middle cerebral arteries, basilar artery, confirming cca. necropsy performed in case showed bilateral pneumonia, small pulmonary artery branches thrombosis, neuronal necrosis, with brain edema, and renal tubular necrosis. the mode of death in these two cases was brain death, with cca. the probable pathophysiologic mechanisms were related to hyperviscosity and cardiac failure. davf's can be associated with benign or aggressive symptoms based on location and venous drainage. cerebral venous ischemia is a reversible process emphasizing the importance of early recognition and treatment of davf's. in a geographically isolated region with limited neuroscience intensive care unit (nsicu) capacity, neurointensivists are often challenged to allocate resources and triage intracerebral hemorrhage (ich) patients. we sought to assess the factors impacting the neurointensivists' triage decision for nsicu admission after ich. consecutive patients hospitalized for ich between and at a tertiary center that has the only -bed nsicu for the state, geographically isolated from the nearest nsicu (> , miles away), were studied. multivariable logistic regression models were used to test for predictors of nsicu admission, adjusted for each component of the ich score, transfer from another hospital, initial systolic blood pressure (sbp) > mmhg, and early do-not-resuscitate (dnr) order. among a total of consecutive patients hospitalized for ich, patients ( %) were admitted to the nsicu while patients ( %) were admitted to a non-nsicu unit. the ich patients were more likely to be admitted to the nsicu if they had hematoma volume > cm (or . , % ci . - . ), intraventricular hemorrhage (or . , % ci . - . ), glasgow coma scale (gcs) score of - (or . , % ci . - . ), gcs score of - (or . , % ci . - . ), infratentorial hemorrhage (or . , % ci . - . ), transfer from another hospital (or . , % ci . - . ), and sbp > mmhg (or . , % ci . - . , % ci . - . ) and early dnr order (or . , % ci . - . ). the triage decisions for nsicu admission after ich were based on clinical severity, age and early dnr status. a prospective study is needed to help establish a safe triage algorithm for ich patients in a region with limited neurocritical care capacity. using a semi-automatic threshold based volumetry algorithm. neurological status (nihss) was recorded daily and outcome was assessed at discharge using the modified rankin scale (mrs). the difference of phe volumes between day and day - , representing the edema growth (phe delta ), correlated significantly with the mrs at discharge (p= . ; f= ). this correlation was still significant, when ich volume on admission was controlled. other factors that showed a significant association with outcome at discharge were nihss (anova: p> . , f= . ) and ich volume (anova: p> . , f= . ) on admission. in a multivariate regression model only the initial nihss remained a significant predictor of functional outcome. phe growth showed a weak trend towards significance (p= . ). phe growth at the first days after symptom onset may influence early functional outcome after spontaneous ich. treatment strategies aimed at reduction of phe burden after ich may take advantage of this finding. assess the use of a -factor prothrombin complex concentrate (pcc, profilnine®), compared to fresh frozen plasma (ffp) in establishing hemostasis in warfarin associated intracranial hemorrhage (ich). dmitted to unc health-systems between / / and / / that received pcc, ffp, or both in conjunction with phytonadione for the treatment of warfarin associated ich. patients who received a factor product other than profilnine®were excluded. data collection included hematoma expansion, achievement of inr reversal (inr < . ), -day mortality and endpoints related to safety (thromboembolic events, infection, and transfusion related acute lung injury). of the patients included, patients received pcc alone, patients received pcc plus ffp and patients received ffp alone. hemorrhage expansion occurred in of patients ( %) in the pcc group, of patients ( %) in the pcc plus ffp group and of patients ( %) in the ffp group (pcc versus ffp, p= . ; pcc plus ffp versus ffp, p= . ). inr reversal occurred in % of patients in the pcc alone group, % of patients in the ffp alone group and of patients ( %) in the combination group. this study assessed the impact of profilnine®, ffp, or the combination, on achieving hemostasis based on hematoma expansion. profilnine® achieved inr reversal but appeared to be less effective than ffp in preventing hemorrhage expansion. fever after ich is common and associated with poor outcome. however, the impact of therapeutic temperature modulation (ttm) to treat fever after ich is unclear. we performed a case-control study of ttm in ich patients with fever. patients undergoing ttm with advanced temperature modulating devices were prospectively enrolled in our ttm database from - (ttm group). target temperature was c in all cases. controls were matched in severity by ich score and retrospectively obtained from a period ( ) ( ) ( ) ( ) before our routine use of ttm for ich. primary outcome was discharge modified rankin score. we enrolled patients in each group. median ich score was (range - therapeutic normothermia is associated with increased length of mechanical ventilation and nicu stays, but is not associated with improved discharge outcome. spontaneous intracerebral hemorrhage (sich) is a dynamic process with significant growth in over one-third during the first hours. catheter-based evacuation of sich plus recombinant tissue plasminogen activator (rtpa) is a novel surgical approach for which optimal timing of stereotactic catheter placement and clot aspiration are not known. we investigated factors associated with significant ich expansion (> % or . cc volume increase) on prerandomization ct scans of patients meeting criteria for the mistie trial, a multi-center phase ii clinical trial, evaluating safety and efficacy of minimally invasive surgery plus thrombolytic to treat ich. subjects randomized to surgery underwent stereotactic clot aspiration followed by injections of rtpa through the hematoma catheter every hours, up to doses, or until a clot reduction endpoint. median diagnostic ich volume was . cc (iqr . ). overall, . % of patients exhibited significant hematoma expansion at a median of . (iqr . ) hours from symptom onset. predictors of hematoma growth were smaller diagnostic ich volume (or . ; p= . ), longer interval from symptom onset to pre-randomization ct (or . ; p= . ), non-lobar location (or . , p= . ), lower initial platelet count (or . ; p= . ), and lower initial hematocrit (or . ; p= . ). age, gender, admission blood pressure, initial coagulation parameters, hematoma shape and density scores did not predict hematoma expansion. end of treatment hematoma expansion occurred in / ( . %) patients of whom had early ich expansion and underwent surgical intervention. stabilization of hematoma growth can be anticipated within hours of symptom onset in patients considered for minimally invasive surgery using the mistie protocol. smaller initial ich size, deep location and lower hematocrit and platelet counts were independent determinants of significant ich expansion before surgery. patients with early expansion may represent a group at higher risk for re-expansion with clot aspiration and thrombolytic therapy. financial support: daniel f. hanley received funding from nih grant r ns . thin-section noncontrast ct (ncct) provides a measure of thrombus composition based on hounsfield units (hu) and may predict resistance to thombolytics in acute ischemic stroke. hematoma composition may affect thrombolytic efficacy of tissue plasminogen activator (tpa) in acute intraventricular hemorrhage (ivh). we assessed the value of hematoma hu quantification as a predictive marker of ivh clearance after intraventricular tpa administration. serial ncct was performed on patients who received intraventricular tpa as part of the clear ivh trial (clot lysis: evaluating accelerated resolution of ivh) and controls with ivh treated with external ventricular drainage (evd) alone. a blinded investigator calculated hu values for ivh volumes on admission, day - and day - ncct for each patient. median ivh volume on admission for tpa-treated patients was . (iqr . )cc, and decreased to . ( . )cc at day - . mean(sd) hu for ivh was . ( . ) on presentation and decreased significantly to . ( . ) on day - , and to . ( . ) on day - . ivh hu count was significantly correlated with ivh volume at all ct timepoints: admission:p= . ; day - :p< . ; day - :p< . . there was no correlation between admission serum platelet count, fibrinogen level or hemoglobin and clot hus. only csf protein was positively correlated with ivh hu (p= . ). total ivh hus were significantly lower in tpa-treated (vs. control) patients at day - (p= . ), but not at day - . change in ivh volume from admission to day - was positively correlated with higher initial hu in tpa-treated patients (p= . ), but hu was not significant after adjustment for ivh volume and tpa treatment. hounsfield unit counts of ivh decrease significantly over the first week on ncct and the decrease is greater in tpatreated patients. unlike thrombus hus in large intracranial vessels, ivh hus are not associated with erythrocyte or platelet concentrations. higher hu is not an independent predictor of success of intraventricular thrombolysis. although neurocardiogenic myocardial injury is well described among patients with spontaneous intracerebral hemorrhage (sich), it has not been investigated systematically in patients with acute subdural hemorrhage (asdh). we sought to investigate the prevalence and characteristics of myocardial injury in patients with asdh. consecutive adult patients with a diagnosis of asdh admitted to the rush university neurosciences intensive care unit were analyzed. myocardial injury, defined as troponin i elevation (> . ng/ml) on admission or during hospital course, was identified. electrocardiograms (ecg) and echocardiograms, obtained within the first hours and read by a cardiologist blinded to the patient's history, were retrospectively reviewed. a total of patients were admitted with asdh between / and / . the mean age was years (sd years), and % were male. comorbidities included hypertension ( %), diabetes mellitus ( %), coronary artery disease or prior myocardial infarction ( %), congestive heart failure ( %), coronary artery stent or bypass procedure ( %). ecgs were normal in %. non-specific st-t morphologic changes, qrs changes or sinus arrhythmias were seen in %. st-elevations suggestive of myocardial infarction were not seen. of patients with elevated troponin, had known severe cardiac disease, and had severe medical complications (sepsis, renal failure, hepatic failure and acute lymphoma crisis). diffuse ecg changes ("cerebral t waves") and echocardiographic findings suggestive of neurogenic stunned myocardium (reversible wall motion abnormalities, apical ballooning) were not seen. although we found ecg changes to be common after asdh, myocardial injury was only observed in the context of concomitant cardiac or medical disease. classic neurogenic cardiac findings (cerebral t waves, neurogenic cardiomyopathy) were not observed. while myocardial injury in sich often is attributed to neurocardiogenic causes, these are unlikely prominent mechanisms in patients with asdh. other cardiac or medical causes are common and should be excluded. prognostication in intracerebral hemorrhage is complex and mortality remains high. while tools such as the ich score have been developed to assist with prognostication, physicians clearly use additional parameters in clinical practice. though do-not-resuscitate orders do not indicate the withholding of any treatment other than cpr, they are associated with increase risk of death in patients after ich. we sought to understand early dnr (< hours) designation in patients with hope of moving toward more precise tools for prognostication. patients admitted to the neurological intensive care unit from july to december with a diagnosis of supartentorial ich were identified. data for all patients were collected retrospectively. patients without a dnr order throughout their admission were compared to patients who received a new dnr order in the first hours of admission. patients with pre-existing dnr orders were not included. subjects were included in the study with . % made dnr within hours of admission to our nicu. factors showing a significant correlation with a new dnr order included advanced age, caucasian race, or residence in a skilled nursing facility. history of malignancy, atrial fibrillation, current use of antihypertensives or warfarin, or alcohol abuse predicted a dnr order. ich resulting in a low admission glasgow coma score, high ich score, intraventricular extension and blood volumes greater than ml correlated with a new dnr order. while individual elements of the ich score correlate with a new dnr order in this population, other characteristics were also associated with an early dnr order. early dnr orders may create a self-fulfilling prophecy if limitations of support are instituted without a clear understanding of who may benefit from aggressive care. thus, identification of factors that providers believe to be life-limiting may serve as a starting point to avoid early limitations in aggressiveness of care. intracranial hypotension is caused by low cerebrospinal fluid (csf) pressure, clinically distinguished by orthostatic headaches and neurologic signs. subdural effusions and even hemorrhage can be a secondary effect. known causes include dural punctures as well as spontaneous csf leaks. treatments are guided towards repairing the cause of the hypotension. a year-old man on coumadin for a mechanical aortic valve was transferred to our institution for evaluation of bilateral sdh. he presented twenty-four hours earlier with sudden-onset severe headache and normal neurological exam. upon transfer, he was noted to be drowsy, with a left third cranial nerve palsy. he endorsed a postural headache that worsened upon standing. mri of the brain showed small subdural effusions with subacute hemorrhage and minimal mass effect, as well as severe distortion of the midbrain with narrowing of the ventricular system, crowding of the basal cisterns and extensive pachymeningeal enhancement. mri of the spine did not demonstrate a csf leak but showed a small perineural cyst at t . the patient's headache and exam findings initially improved with lying flat. a trial of elevating his head of bed failed, with the patient further developing decreased level of arousal, frontal release signs, and recurrent left third nerve palsy. two attempts at lumbar epidural blood patch (ebp) did not lead to sustained improvement, but a thoracic-directed ebp led to durable and complete resolution of the headache and neurologic deficits. intracranial hypotension should be considered as a cause for subdural hemorrhages in the absence of trauma. clues include postural headaches and clinical evidence of brainstem dysfunction and radiographic evidence of brainstem distortion out of proportion to the size of the sdhs. ebps directed at suspected csf leaks can be effective when nondirected lumbar ebps fail. multiple guidelines recommend the reversal of oral anticoagulation when a patient experiences an intracranial hemorrhage (ich). both activated prothrombin complex (apcc) and recombinant factor vii activated (rfviia) have been utilized to reverse warfarin-associated coagulopathy. however, there have been no direct comparisons of these agents. this was an irb approved, retrospective cohort analysis of patients with ich. patients were included if they received either apcc (at least units) or rfviia (at least mg), if they had a discharge diagnosis of intracranial hemorrhage, and if they received warfarin prior to admission. patients were excluded if they were less than , or did not have documented pre-treatment and post-treatment inrs. the primary endpoint for this study was the change from pretreatment inr and post-treatment inr in the apcc group and rfviia group. secondary endpoints included change in ct measured hemorrhage volumes, icu length of stay (los), hospital los, mortality, icu discharge gcs, and thromboembolic adverse events. a total of patients were included in the analysis. of those, received apcc and received rfviia. baseline demographics were comparable; however, patients in the apcc group had a higher rate of atrial fibrillation ( % vs. %; p= . ). when compared over time, both apcc and rfviia significantly reduced the inr (p< . ); however, there was no difference in the amount of change observed between the two groups (p= . ). in addition, we saw no significant differences with regard to icu los, hospital los, mortality, icu discharge gcs, or thromboembolic adverse events. over time, an increase in ich volume was observed in both groups. in patients with intracranial hemorrhage, apcc and rfviia are associated with rapid reversal of warfarin-associated coagulopathy; however, these agents may not slow ich growth. accurate prognostication of patients with intracerebral hemorrhage (ich) is critical because it may affect aggressiveness of care and patient outcome. ich prediction models help stratify patients according to their chance of a good or poor outcome. we compared the accuracy of neurointensivists' prediction of functional outcome to outcome prediction by the ich score. adult spontaneous ich patients with an admission gcs > were prospectively enrolled. the treating neurointensivist predicted the -month modified rankin scale score (mrs) within days of hospital admission. none of the neurointensivists used the ich score routinely to help predict outcome. patient outcomes were dichotomized to good (mrs - ) and poor (mrs - ). neurointensivists' predictions were compared to the ich score using the actual -month mrs as the reference. of prospectively enrolled patients, were included: withdrew consent and were lost to follow-up. neurointensivists' overall accuracy was %, which was higher than the accuracy of the ich score at a cut-off of > ( %; p= . ) or > ( %; p= . ). at a cut-off > , the sensitivity for poor outcome prediction did not differ, but the neurointensivists' specificity for poor outcome was greater ( % vs. %; p< . ). conversely, at an ich score cut-off > the specificity for poor outcome prediction did not differ, but the neurointensivists' sensitivity for poor outcome was greater ( % vs. %; p< . ). the results were similar if, instead of the original ich score, a modified ich score was used as the comparison that had been developed on the same patient cohort. neurointensivists at our institution predict ich outcome overall with % accuracy. generally, predictions for poor outcome are more accurate than those for good outcome. outcome predictions for the individual patient by the treating neurointensivist are more accurate than those based on ich prediction models. there is continued controversy regarding glycemic control and its effect on outcomes for patients with ich as well as other icu patient populations. the relationship between ichsize and glycemic control has not been clearly defined. a retrospective review of patients with supratentorial ich and no history of diabetes between and was performed. admission blood glucose (bg) as well as bg at , and hours was measured while all patients were maintained on the same sliding scale insulin regimen. statistical analysis was performed to compare admission ich size to admission bg and subsequent bg control. bg> mg/dl (mean ± ) and mean ich size . ± . . average bg levels over average bg levels over hours were ± mg/dl. elevated admission bg was significantly correlated with admission ich size (p= . ). average bg levels over hours trended towards, but were not significantly correlated with admission ich size (r = . , p= . ). in nondiabetic patients, elevated admission glucose is significantly associated with ich size. poor outcomes associated with elevated glucose may be associated more with extent of cerebral insult than with glycemic control. the benefits of marriage on health have been known for over years. more recently, married couples have been found to have a lower risk of cancer, dementia, and heart disease. we aimed to explore the effects of marital status on outcome after intracranial hemorrhage. a prospective study was conducted between - of patients with subarachnoid hemorrhage (sah, n= ), intracerebral hemorrhage (ich, n= ) and subdural hemorrhage (sdh, n= ), admitted to the neuro-icu at a tertiarycare academic hospital. marital status was coded as married versus single, widowed, divorced or separated. modified rankin score and barthel index were compared between the two groups at -months using multiple logistic regression analysis. of patients, ( %) were married, ( %) were single and ( %) were widowed, divorced or separated. women were less likely to be married, and smoking was less common among the married (both p< . ). there was no difference in age, insurance or employment status, race, education, days to diagnosis, or history of heart disease, diabetes, hypertension, trauma or coagulopathy. after adjusting for age, admission gcs, apache physiologicalsubscore, gender, tobacco and bleed type, marriage was significantly protective against death or severe disability (mrs - ; adjusted or . , %ci . - . , p= . ) and predicted better activities of daily living (barthel index), (aor . , %ci . - . , p= . ). there was no difference in discharge disposition, length of stay or hospitalization costs. marriage is protective against death or severe disability and predicts better activities of daily living among patients with intracranial hemorrhage. warfarin associated intracranial hemorrhage leads to poor outcomes. we studied the influence of a standardized emergent warfarin reversal protocol incorporating prothrombin complex concentrates (profilnine sd®) on time to achieve the protocol was implemented in . sixty three patients ( pre and post protocol) from - with intracranial -protocol patients received recombinant factor a (rfviia); post protocol patients with inr . - and > received and - units/kg of profilnine sd® respectively. hemorrhage volumes were measured on consecutive ct scans using mipav semi-automated software. groups were similar for baseline median inr ( . vs . ), nihss ( vs ), follow-up ct time ( . vs . hours) and hemorrhage volumes ( . vs . cc) but differed in hemorrhage type: ich ( % vs %) and sdh ( % vs %), p= . . treatments also differed: vitamin k ( % vs %, p= . ), profilnine sd® ( % vs % p=< . ), rfviia ( % vs % p= . ) and number of plasma units ( vs , p= . ).time to target inr was similar ( . vs . hours) driven by pre-protocol rfviia use (rfviia used vs not, . vs . , p= . ) and this led to inr rebound in < hours. excluding rfviia, the post protocol group normalized inr faster ( . vs . hours, p= . ). the post protocol group had less absolute ( . vs cc p= . ) and relative hemorrhage growth ( % vs % p= . ) without any thrombotic events. despite comparable mortality ( vs %), post-protocol survivors more frequently achieved mrs - ( vs % p=ns). a standardized emergent warfarin reversal protocol is not only safe but leads to faster inr normalization, less hemorrhage growth, plasma conservation and possibly better neurological outcomes. perihemorrhagic edema (phe) after intracerebral hemorrhage(ich) may exceed the initial hematoma volume by to % respectively and thereby lead to increased intracranial pressure (icp), clinical detoriation or even herniation. intravenous hypertonic saline (hts) has been shown to reduce phe formation after ich. clinical data suggest that hts may be superior to mannitol in lowering icp. eusi and asa guidelines recommend the use of intravenous mannitol up to a serum osmolality (so) of mosmol/kg or hst in order to reduce elevated icp. we aimed to investigate the effect of mannitol and so on the evolution of phe after ich. nineteen patients with supratentorial spontaneous ich treated with % intravenous mannitol solution ( - ml every h) for - days and controls who did not receive mannitol or any other osmotic agents during the course of treatment were identified retrospectively from our institutional ich database. patients treated with mannitol and controls were matched for ich-volume (± ml). phe volume was calculated on ct scans using a semiautomatic threshold based volumetric algorithm. diagnostic ct scans and follow-up scans performed on days , - , - , - and - were analyzed. so, concentration of sodium and glucose were obtained from patient records. the matching resulted in similar ich-volumes in both groups (mannitol: . ± . ml, controls: . ± . ml). mean age was ( - ) years in the mannitol group and ( - )years in controls (p= . ). initial relative phe did not differ significantly in both groups (mannitol: . ± . , controls: . ± . , p= . ). there was no effect of mannitol treatment on the course of phe (f= . ,p= . ). there was no significant correlation between so and relative phe at any timepoint of follow-up. we found no effect of mannitol use and so on the evolution of phe. other underlying mechanisms may explain the shortterm effect of mannitol boluses on icp in patients with spontaneous supratentorial ich. when operating at maximum intensive care unit (icu) bed capacity where allocation of critical care resources is required, physicians may be pressured to initiate do-not-resuscitate (dnr) orders in patients with intracerebral hemorrhage (ich). we sought to assess the relationship between early (< hours from admission) dnr orders and neuroscience intensive care unit (nsicu) bed capacity in patients admitted with acute ich. we retrospectively studied consecutive patients hospitalized for ich between and at a tertiary center that has the only -bed nsicu for the state, geographically isolated from the nearest nsicu (> , miles away). multivariable logistic regression models were used to test for predictors of early dnr orders, adjusted for each component of the ich score and nsicu bed census on admission. nsicu bed census was dichotomized to full (all beds occupied) vs. not full (at least available bed). among maximum icu bed capacity on admission is not associated with the decisions to initiate early dnr orders in ich patients. this suggests that physicians were not preferentially initiating early care limitation when critical care resources were becoming scarce. dural arteriovenous fistulas (davf) are rare, acquired cerebrovascular lesions consisting of abnormal vascular connections between arteries that normally supply the dura and veins that drain the brain parenchyma -that is to say, arteries not associated with the brain parenchyma manage to drain via the dural venous sinus system. the clinical consequences of these lesions are typically hemorrhage, seizure, or venous congestion. venous congestion may present acutely with hemorrhage or subacutely with signs and symptoms such as progressive cognitive decline, seizures, or encephalopathy. parkinsonism, tinnitus, and intracranial hypertension have also been described. case report with review of literature. we describe a -year-old man with no past medical history who developed subacute onset dementia with bithalamic t hyperintensity on mri without restricted diffusion. subsequent intraventricular hemorrhage resulted in emergent transfer to our institution's neurocritical care unit for an emergent diagnostic cerebral angiogram of a borden ii/cognard iib davf with immediate angiographic embolization and obliteration. davfs are lesions with significant risk of aggressive neurologic devastation related to venous congestion and subsequent hemorrhage. the severity of davfs requires clinicians to be aware of these lesions and of their common and uncommon presentations. little is known about the ability of prognostic scores to predict outcome in patients with secondary intraparenchymatous intracranial hemorrhage (iph). our objective was to describe the clinical characteristics, ich scores at presentation and prognosis in patients with secondary iph. we performed a post-hoc analysis of prospectively collected data of consecutive patients admitted to a tertiary hospital with iph. the characteristics of patients with secondary iph were compared to those of patients with spontaneous iph. patients with secondary iph had either a positive underlying vascular lesion identified as the iph etiology or impaired coagulation at presentation (a platelet count < , per cubic mm, an inr > . , or an aptt > than seconds). a total of patients with iph were admitted to our hospital from january- to january- . of those, patients ( %) had a secondary iph ( cavernomas, arteriovenous malformations, dural fistula, reversible vasoconstriction syndrome and sacular aneurysm patients with secondary iph had lower ich scores at presentation and lower in-hospital mortality than patients with spontaneous iph. despite lower ich scores at admission, patients with secondary iph had similar functional outcomes when compared to patients with spontaneous iph. larger studies should focus on specifically developing better prognostic tools in such patients. a large number of studies in traumatic brain injury patients have shown efficacy of hypothermia for control of icp and if used for prolonged duration, has shown to improve mortality and functional outcome. for other neurologic catastrophes, due to a risk of rebound edema during re-warming, medical complications and other factors, it has either not been commonly used or been used when most of other options are exhausted. this is a retrospective analysis of patients with massive ich (blood volume of > ml), of non traumatic etiology, dominantly in brain parenchyma. all patients had intracranial pressure monitoring via external ventriculostomy catheter. hypothermia was induced and maintained at target temperature via non-invasive, surface cooling pads. modified rankling score (mrs) was recorded at months after the ictus in all survivors. patient ages ranged from to years. cause of ich was hypertension in patients and ruptured aneurysm in one patient. duration of treatment ranges from - days. target temperature required to adequately control icp ranged from - c. two patients ( %) survived with good recovery (mrs of ), one ( . %) with moderate disability (mrs of ), two ( %) with moderately severe disability and three ( . %) died. most common side effect of hypothermia was hypotension requiring pressors in five ( %), electrolyte imbalance in ( %), pneumonia in ( %), thrombocytopenia in ( %) patients. all complications were successfully treated and major complications of treatment (bleeding diathesis, septic shock syndrome, death) were not observed. controlled hypothermia for up to days is safe and feasible for the treatment of cerebral edema and intracranial hypertension in young patients with massive (> ml of blood volume) non traumatic ich. however, prolonged duration of treatment may be required for definitive control of icp. this study serves as a template for future efficacy trials. intracerebral hemorrhage (ich) accounts for % to % of strokes and is associated with substantial morbidity and mortality. it remains controversial whether surgical intervention or a conservative approach is the best option for treating ich. we assessed the hypothesis that early surgical intervention in patients with primary supratentorial ich may serve to improve -day outcome. a total of patients with primary supratentorial ich, in whom surgical intervention was indicated for hematoma removal according to the guidelines, were admitted to our hospital during a continuous -month observation period. patients with the consent to the surgical intervention (n= ) underwent surgery within hours of symptom onset and the others (n= ) were given the conservative treatment. the outcome was the proportion of patients who had an unfavorable outcome (persistent vegetative state or death), as assessed on the basis of the glasgow outcome scale (gos) at days. the -day mortality rate was . % (standard error, . %). there was no significant difference in outcome between the two treatment groups. after adjustment for other significant covariates, although a lower unfavorable outcome was found in surgical group but the difference was not significant (odds ratio = . ). among the confounding factors, presence of intraventricular hemorrhage (ivh) and low glasgow coma scale (gcs) score on admission were independently associated with poor outcome days after ich (both p < . ). we found no benefit for early surgical intervention over conservative treatment in patients with primary supratentorial ich. presence of ivh and low gcs score were strong predictors of poor outcome in these patients. given the high morbidity and mortality associated with intracerebral hemorrhage (ich), family members and healthcare providers base early supportive management decisions, at least partly, on expected prognosis. in the comatose patient with ich, this short term prognosis is most overtly characterized by regaining of consciousness. a retrospective consecutive cohort of patients, between and , with ich and admission glasgow coma scale that were associated with regaining of consciousness after coma in ich. variables associated with awakening in univariate analysis were tested in multivariable logistic regression. the group that awakened had higher initial gcs scores, smaller ich volumes, and less ivh, but was similar in other baseline characteristics. early dnr orders, in the first hours, tended to be used more frequently in patients who ultimately remained comatose, but the difference was not statistically significant. admission gcs, volume of ich, and presence of ivh identified in univariate analysis were tested along with age and gender as potential confounders of outcome in multivariable analysis. higher admission gcs score was associated with an increased likelihood of awakening from coma (or . [ %ci . - ] per category, p= . ). % of patients with initial gcs of - , % with initial gcs of - , and % with initial gcs of - regained consciousness. awakening from coma, in the cohort of patients who regained consciousness, occurred in % of patients by day , % by day , and % by day . gcs score is the single most important predictor of early awakening in patients who present in coma after ich. patients who regained consciousness typically did so within the first days of hospital admission. intracerebral hemorrhage (ich) is an infrequent but severe complication in pregnant women with hypertension, it accounts for % of all deaths related to cerebral complications in this group. a- -year-old female, g p at weeks of gestational age, with prenatal care, no relevant past medical history, presented for a follow-up visit. she was admitted with bp / mmhg, and treated with iv labetalol; the preeclampsia work-up was negative, bp range between / and / mmhg. approximately h after admission, she complained of diffuse headache, nausea, vomiting, and epigastric pain. headache symptoms increased follow by focal seizure and progression to generalized tonic-clonic seizures. magnesium sulphate and phenytoin were administered to control the seizure, immediate blood analysis revealed dic. the diagnosis of eclampsia was made, and emergency csection followed. the airway was secured with rapid sequence technique; a healthy infant was delivered under general anesthesia. the patient remained comatose hour after surgery with gcs , minutes later she demonstrated a decerebrate posture with non-reactive pupils. a non-contrast ct-scan revealed an intracerebral hematoma. dic was treated, and neurosurgeon performed a right frontotemporal craniotomy. a postoperative ct scan confirmed the resolution of the ich. the patient opened her eyes and started responding to commands by the third day, on day she was extubated and the gcs was . by the rd week, the patient was transferred to rehabilitation, where she remained for weeks. at years, she regained a full cognitive recovery. this case emphasizes that even short time hypertension should be treated aggressively to prevent ich. the prompt intervention of a multidisciplinary team (obstetric, neurosurgery, and anesthesiology) is required to ameliorate the devastating effects of eclampsia and ich. although hypertension is the commonest cause of non-traumatic intracerebral hemorrhage (ich), it is important to rule out other causes. most patients with ich have an elevated bp on presentation but many are unaware if they have longstanding hypertension. echocardiographic abnormalities may be revealing in such circumstances. we studied the incidence of echocardiographic abnormalities and their usefulness in determining the etiology of ich in these patients. we conducted a retrospective study of echocardiographic abnormalities in ich patients admitted to a tertiary university hospital between jan to oct who also had a cerebral angiogram. subjects with and without underlying vascular location (categorized as typical hypertensive location or not), history of hypertension and the presence of the following echocardiographic abnormalities: left ventricular hypertrophy (lvh), diastolic dysfunction (dd), systolic dysfunction (sd), hyperdynamic ventricular function (hvf), wall motion abnormalities, atrial enlargement (ae) and valvular abnormalities using chi-square test and fisher exact test. we then conducted a multivariate logistic regression analysis including variables with a p< . in the univariate analysis. a total of subjects were admitted with an ich. subjects had an echocardiogram and of these, also had an angiogram (conventional angiogram: , ct angiogram: , mr angiogram: ). the echocardiogram was abnormal in . % ( . % with a history of hypertension p= . ). common abnormalities were: lvh ( . %), dd ( . %), hvf( . %), sd ( . %) and ae ( . %). of these, only dd (p= . ) was significantly associated with absence of underlying vascular abnormalities on a univariate analysis. on multivariate analysis, none of the echocardiographic abnormalities showed a significant association. echocardiographic abnormalities, mainly lvh and dd are commonly seen in ich patients, however none of these abnormalities are independently associated with an absence of underlying vascular anomalies. stroke in the hiv+ population is a growing problem, though it is unclear whether hiv is an independent risk factor. we describe a series of hiv+ patients with intracerebral hemorrhage (ich). we reviewed records of all patients with diagnoses of ich and hiv/aids admitted to an academic, inner-city hospital between and . patients with traumatic hemorrhage, ischemic stroke with hemorrhagic conversion, hemorrhagic neoplasms, toxoplasmosis with hemorrhage, subarachnoid hemorrhage, and extra-axial hemorrhages were excluded. we reviewed demographics, risk factors, laboratory tests, and neuroimaging. outcomes at days were determined by modified rankin scale (mrs). six hiv+ patients ( % male, mean age ) met inclusion criteria, with patient having recurrent hemorrhages; % were black, % hispanic, and % of other racial groups. all patients met criteria for aids. risk factors included: prior stroke ( %), diabetes ( %), hypertension ( %), smoking ( %), and illicit drug use ( %). only patient was taking antithrombotic medication. the co-prevalence of hcv was %. admission blood pressure was > / in / patients. laboratory evaluation demonstrated patient with a prolonged inr (> . ) and patients with thrombocytopenia (< ). the hemorrhages were lobar in / and deep in / . only patients had vessel imaging; one had an avm and none demonstrated aneurysm or vasculitis. at days, four patients were deceased and the two survivors had mrs of and . in this cohort, hiv-associated ich occurred only in aids patients. outcomes were uniformly poor, with % of patients having a htn and unexpected predominance of lobar hemorrhages in younger patients, suggesting a distinct mechanism of ich. in gregory call and marie fleming reported four patients with what appeared to be a reversible form of cerebral vasoconstriction. since then a number of authors have reported reversible cerebral vasoconstriction syndromes (rcvs), often in association with potential etiological precipitants. the major complication of rcvs is ischemic stroke, but hemorrhagic strokes can also occur, eventually leading to permanent sequelae and even death. recent reports and case series have suggested that intracranial hemorrhages may be frequent in rcvsand its presentations may range from cortical subarachnoid hemorrhages to intracerebral hemorrhages and subdural hemorrhages. we report two cases of rcvs in middle age women, with hemorrhagic strokes caused after the prescription of dipirone, isometheptene and anhydrous caffeine, with putaminal hemorrhage, and lobar frontal hemorrhage. both cases showed complete reversion of arterial vasoconstriction weeks later by the transcranial doppler. despite the reversibility of the vascular constrictions that characterize rcvs, brain lesions are observed in up to % of the patients.most of these lesions are of ischemic nature; however hemorrhagic phenomena are not uncommon and have only been reported in %- % of the cases. isometheptene has been described as a trigger for rcvs in only a handful of patients, all of whom were women in the postpartum period. even though rcvs diagnosis demands evidence of complete reversibility of the vasospasms, differential diagnosis with sah can be made by the identification of classic rcvs triggers and assessment of the vascular patterns brain arteries. magnesium (mg) has been hypothesized to have a neurprotective effect against cerebral ischemia. several ongoing studies are examining the effect of exogenous magnesium in reducing disability and maintaining normal cerebral function. we examined initial endogenous mg levels in patients with spontaneous intraparenchymal hemorrhage (iph), in order to determine if higher mg blood serum levels would confer neuroprotective benefit. this is a retrospective study on patients admitted to a university affiliated community hospital. demographic data were obtained from a prospectively collected registry database. initial magnesium levels were gathered retrospectively from the registry database. we included all patients with iph in our analysis. for evaluating the severity and outcome of the patients with iph we used the university of california san francisco intracerebral hemorrhage (ucsf ich) score on admission / hours to quantify stroke severity and mrs on discharge to measure outcome. we employed correlation coefficients (spearman's rho) and the mann whitney test for analysis of the data. spss version was used for data processing. our review identified patients with a diagnosis of iph. the serum mg levels in patients with iph negatively correlated to ucsf ich score on admission (p= . , r= - . ) and at hours (p= . , r= - . ). there was a trend towards better outcomes at discharge in patients with higher mg levels (p= . , r= - . ). higher levels of endogenous serum mg were found to confer reduction in iph severity and progression. initial serum mg levels could serve as an early predictor of iph severity. a larger prospective study is warranted to study the effect of endogenous mg on outcomes in patients with iph. spinal dural arteriovenous fistulas (davfs) account for % of all vascular spinal malformations. the incidence is - /million/year in the general population although it is generally under-diagnosed. men are affected five times more often than women and the mean age at the time of diagnosis is - years. spinal davfs generally do not present acutely and are very rarely located in the cervical region. we present a case of atypical acute spinal cord infarct secondary to a cervical davf. case report and extensive literature search carried out to understand spinal davfs. this year old gentleman presented to our neurocritical care unit with bilateral upper extremity weakness and right lower extremity weakness proceeded by upper back and neck pain. the patient rapidly deteriorated to near quadriplegia and respiratory failure requiring prolonged artificial ventilation. initial studies included normal mri of the brain and ct angiogram of the head and neck. mri of the spine revealed abnormal signal intensity within the anterior cervical cord from c -c levels in the distribution of the anterior spinal artery. there were no flow voids to suggest dilated perimedullary vessels. however, given the clinical picture, a spinal angiogram was obtained and demonstrated a cervical davf supplied by a dural branch vessel originating from the left vertebral artery. understanding spinal vascular anatomy is important for diagnosis of spinal davfs. our case is unusual because ) acute evolution of quadriplegia and respiratory failure, ) lack of any abnormal vessels seen on mri, and ) ischemic changes restricted to the anterior spinal artery distribution. the case emphasizes the importance of proceeding with spinal angiography if the clinical suspicion of davf is high. early detection and management can lead to improved functional outcome. although coma is a syndrome commonly associated to catastrophic brain injury, this patient population remains poorly characterized. the chief goal of therapy is aimed at reversal of coma. despite this urgency, there is paucity of data regarding the factors that predict emergence. we characterize a population of patients with new onset of coma in the neuro-icu and describe clinical and structural factors that predict emergence. prospective longitudinal consecutive cohort of patients, enrolled in an intensive care setting. three hundred patients met investigation enrollment criteria between may and july . a brain lesion was identified at the onset of coma in most patients ( %). frequent etiologic factors were cerebrovascular ( %), seizures ( %), trauma ( %), cns infection ( %), or other ( %). the most frequent cerebrovascular factors were any ich ( %), ivh ( %) and sah ( %), either alone or in combination. emergence from coma was predicted by a higher initial gcs (emergence= [ - ] vs. no emergence= . [ - ] p< . ), seizures as presenting disorder (emergence= % vs. no emergence= % p= . ), and a trend to lesser frequency of ich component (emergence= % vs. no emergence= %, p= . ). the importance of mass effect as measured by midline shift reversal and cisternal compression resolution is presented in a separate poster. mortality in this cohort is %. the population of patients with acute coma is highly heterogeneous. however clinical and structural factors predict emergence. a higher initial gcs predicted recovery of coma. structural cerebrovascular lesions with less ich component had a tendency toward higher rates of recovery. non-structural treatable causes of coma such as seizures were associated with higher rates of recovery. mortality and disability remain dismal in this population. optimal blood pressure (bp) control in intracerebral hemorrhage (ich) patients remains controversial. aggressive bp reduction may limit hematoma expansion, but may also cause hypoperfusion and ischemia. we investigated the relationship bp lowering in the first hours and the presence of diffusion weighted imaging (dwi) lesions on mri. we prospectively enrolled consecutive patients presenting with an acute spontaneous ich. brain mris were reviewed for the presence of lesions with reduced diffusion attributable to tissue compression, vessel compression, or hypoperfusion ipsilateral to the hematoma. bps were recorded on hospital presentation, and at , , , and hours. of eligible patients, met inclusion criteria: age: ± years; hematoma volume: ml (iqr - ); admission nihss: (iqr - ); ich onset to maximal bp reduction hours (iqr - ); and ich onset to mri: hours (iqr - ). dwi lesions were detected in % of patients: % of patients had lesions attributed to tissue compression, % to vessel compression, and % to hypoperfusion (some patients had multiple lesion types). dwi lesions were associated with larger hematoma volumes ( vs. ml, p < . ); higher admission mean arterial pressures (map) ( vs. mmhg, p= . ); and greater average map reductions ( vs. %, p= . ). after controlling for ich volume using logistic regression: for every % of map reduction, the risk of dwi lesions increased (or . , % ci: . - . ); for each % reduction in map the risk of dwi lesions more than doubled (or . , % ci . - . ). the proportion of patients with dwi lesions increased as the maximum percent map reduction increased in a dose dependent fashion. ischemic brain lesions in patients with spontaneous ich are common and associated with hematoma volume and bp lowering. aggressive bp lowering may contribute to ich associated ischemic lesions. financial support: sources of funding: this research was supported by the nih (r ns ) to cacw, and the stanford school of medicine medical scholars program to jtk. coma is a major cause of death, disability and economic burden to the health care system. acutely comatose patients with primarily neurologic injury are at risk to develop neurologic and systemic complications. in this study, we seek to identify the timing of medical complications and their impact on mortality in acutely comatose patients admitted to neurocritical care unit. one hundred patients with acute coma for at least hours or longer were enrolled prospectively in the study from may to jan . major neurologic and systemic complications were identified prospectively and the frequency and timing of each major complication was established. of the patients studied, mean age was ± . years and % were females. a mean of . ± complications occurred. in this cohort of patients with coma, there were more non-neurological ( %) versus neurological ( %) complications. most complications ( %) were noted in the - day interval. further characterization of these complications is essential to the care of comatose patients in the nccu. pathophysiology of brain dysfunction associated with sepsis is still poorly understood. our purpose was to study the metabolic alterations and mithocondrial dysfunction in a clinically relevant model of septic shock. twelve anesthetized, invasively monitored, and mechanically ventilated pigs were allocated to a sham procedure (n = ) or sepsis (n = ), in which peritonitis was induced by intra-abdominal injection of autologous faeces. animals were studied until spontaneous death or for a maximum of hours. in addition to global hemodynamic and laboratory assessment, intracranial pressure and cerebral microdyalisis were assessed , , and hours after sepsis induction. after death, brain were removed and brain homogenates were studied to assess mithocondrial dysfunction. all septic animals developed a hyperdynamic state associated with organ dysfunction. in the septic animals, there was a progressive increase in l/p ratio and glycerol, as well as a progressive decrease in brain glucose concentration during the study period. the comparison between control and septic animals and the analysis of brain homogenates are undergoing. in this model of peritonitis, cerebral metabolism was derranged, with increasing levels of l/p ratio and decreasing levels of brain glucose during study period. these alterations may play a role in the pathogenesis of sepsis-associated encephalopathy. at sanford usd medical center, neuro critical care (ncc) patients are frequently treated with continuous infusions of % sodium chloride. it has been observed that this patient population often develops iatrogenic hyperchloremic metabolic acidosis, frequently managed with intravenous sodium bicarbonate. upon notification of a nationwide intravenous sodium bicarbonate shortage (march th , ), our ncc providers were forced to explore other potential options for managing this acidosis. it was decided that our ncc patients would be initiated on enteral sodium bicarbonate at the time continuous % sodium chloride was started. a retrospective chart review of ncc patients years and older, initiated on continuous % sodium chloride with enteral sodium bicarbonate tablets from march th , to june th , were evaluated. data collected included demographics and the following while in the intensive care unit (icu): baseline serum sodium, chloride, and bicarbonate; type of injury; acidosis defined as serum bicarbonate level < , or lower than baseline; and volume of % sodium chloride and bicarbonate administered. of the patients identified, . % developed iatrogenic metabolic acidosis during icu stay. average duration of continuous % sodium chloride infusion was days (range - days) with an average volume of % sodium chloride dispensed of , mls (range - , mls). three patients evaluated developed an acidosis during icu stay, of which were hyperchloremic at the time of acidosis. only patient required intravenous sodium bicarbonate, however the patient had been off hypertonic saline for more than day. enteral sodium bicarbonate appears to be an effective method at preventing iatrogenic hyperchloremic metabolic acidosis when initiated along with continuous infusions of hypertonic saline in ncc patients. this may be a method to conserve intravenous sodium bicarbonate during drug shortages. further studies are needed. fever is common in neurocritically ill patients. it can be from central causes, inflammatory, infectious, and other conditions. a method to differentiate infectious from non-infectious fever would allow for appropriate initiation of empirical antimicrobial therapy. apart from avoiding unnecessary antimicrobial usage, this approach can save health-care costs and limit the development of antimicrobial resistance. procalcitonin is a peptide precursor of the hormone calcitonin. procalcitonin levels rise as a proinflammatory response to bacterial infections. numerous studies have evaluated procalcitonin levels utility in the initiation and discontinuation of antibiotics in the inpatient setting; however, there is a paucity of studies regarding the use of procalcitonin levels in neurologically ill patients. this study examines the effectiveness of a procalcitonin-guided algorithm in a neurocritical care unit. a modified prorata trial procalcitonin algorithm was developed and utilized prospectively. patients that met criteria of ) admission into the neurocritical care unit ) age > years ) temperature > f in the last hrs ) no obvious source of infection were enrolled. depending on the procalcitonin level and the presence of new sirs criteria antibiotics were initiated per our algorithm. radiographical, microbiological, laboratory, and clinical outcomes were recorded to determine the accuracy of the procalcitonin algorithm in the decision to initiate or modify patient's antibiotic therapy. results from the first enrolled patients found the procalcitonin algorithm had % sensitivity and % specificity in predicting bacterial infections as the etiology of fever with a positive predictive value of %. the study population included intracranial hemorrhage ( %), ischemic stroke ( %), and others ( %). % of the study population had infectious fever while % had non-infectious fever. interim results suggest a procalcitonin-guided algorithm may be a valuable tool in differentiating infectious from noninfectious fever in the neuro-icu. further research is needed; data collection is ongoing. posterior reversible encephalopathy syndrome (pres) is defined by acute neurologic symptoms caused by vasogenic cerebral edema. recurrence of pres is thought to be rare and has not been well described. patients prospectively diagnosed with pres from - were pooled with retrospectively identified patients diagnosed with pres from - at an academic referral center. detailed clinical information and radiologic imaging results were collected. patients without clinical or radiographic resolution between episodes and patients without brain imaging available for review were excluded. of a total of patients with pres, ( %) had recurrence. one patient had four episodes, one patient had three, and ten patients had two episodes each, resulting in total pres episodes. seven patients ( %) had an autoimmune disorder. the average time between episodes was months. acute hypertension was present in of episodes. of these, mean blood pressure was / mmhg. etiologies of pres included hypertension (n= ), cytotoxic medications (n= ), sepsis (n= ), and multifactorial (n= ). renal failure was present in / episodes, and was acute in . clinical symptoms included headache (n= ), seizure (n= ), visual disturbances (n= ), encephalopathy (n= ) and focal deficits (n= ). only one patient ( %) had the exact same clinical symptoms with recurrence. ten patients had mri at each episode of pres. vasogenic edema affected the same brain areas at each episode in patients. in the rest, some affected regions were similar, but additional regions were different between pres episodes. none had entirely new areas of involvement. pres recurred in approximately % of our patients. in the majority, clinical symptoms differed at recurrence compared to the initial episode. in all patients, radiologic patterns of vasogenic edema in the repeat episode were similar to those in the initial pres episode, but also affected other brain regions in approximately %. ventilator-associated pneumonia (vap) is a common complication in comatose patients. diagnosis in this population is unreliable despite physician training and validated criteria leading to potential misdiagnosis and inappropriate antimicrobial use. we investigated clinical features associated with misdiagnosis of vap and excess antibiotic days (ead). ventilated comatose patients (glasgow coma scale motor score < ) suspected of having vap were prospectively identified in a neurocritical care unit in . vap was retrospectively diagnosed using centers for disease control (cdc) criteria by two neurointensivists and an infection control practitioner. appropriateness of the nccu team's vap diagnosis and therapy was performed using clinical, microbiologic and radiographic data. of comatose patients, cases were treated as possible vap by the nccu team. of these, patients had vap by cdc criteria. vap and non-vap groups did not differ in age, admission gcs, total ventilator days or mean total antibiotic days ( . ± . (vap) vs. . ± . (non-vap); p= . ). clinical features significantly associated with vap (vs. non-vap) were change in sputum character, tachypnea, oxygen desaturation, persistent infiltrate on chest xray and positive sputum microbiology. two-thirds ( . %) of non-vap patients received pneumonia targeted antibiotics for > days vs. . % of vap patients (p= . ), contributing eads, including vancomycin days, piperacillin-tazobactam days and cephalosporin days. median days from intubation to starting antibiotics was (non-vap) vs. (vap) days (p = . ). no pre-specified factors were associated with inappropriate continued vap treatment. inappropriate diagnosis and treatment of vap resulted in a cumulative eads in one year in the nccu. clinical differences between patients without vap who had antibiotics continued or discontinued were minimal, suggesting that clinician behaviors contribute to unnecessary prescribing. strategies to improve the diagnosis of and antibiotic use for vap in comatose patients is needed. management of hyponatremia in patients with acute brain injury can be challenging. the oral vasopressin receptor antagonist has been studied extensively in other disease process but not in acute brain injury. we report our experience regarding the efficacy and safety of tolvaptan, an oral vasopression v -receptor antagonist, for the correction of hyponatremia in acutely brain-injured patients. tolvaptan for the correction of euvolemic or hypervolemic hyponatremia. baseline serum sodium concentration was . ± . meq/l. seven patients received mg of tolvaptan once (singledose-users), and patients received another mg on the next days (double-dose-users). hours after tolvaptan administration, serum sodium concentration increased by . ± . meq/l in single-dose-users (p = . ) and . ± . meq/l in double-dose-users (p = . ). hours after administration of first dose of tolvaptan, serum sodium increased by . ± . meq/l in single-dose-users (p = . ) and . ± . meq/l in double-dose-users (p = . ). during four days of observation, the increases in the average area under the curve of the serum sodium concentration was . ± . meq/l in single-dose-users (p = . ) and . ± . in double-dose-users (p = . ). urine output increased by . ± . l during the first hr in single-dose-users (p = . ). no significant changes in fluid balance, serum creatinine and glasgow coma scale were observed. of four patients who underwent neuro-monitoring, intracranial pressures, cerebral perfusion pressures and mean arterial pressure did not change significantly compared with their baseline values. tolvaptan was effective and well-tolerated for the correction of hyponatremia in patients with acute brain injuries. validation can be done with further studies. patients with acute brain injury but normal lung function often undergo intubation and subsequent tracheostomy for the concern of airway protection. we previously described patients with primary brain injury and encephalopathy who fail extubation demonstrated signs of disrupted ventilation usually with periods of prolonged hypoventilation. we examined the clinical characteristics of patients with a tracheostomy who are readmitted to the icu with respiratory decompensation. retrospective review of patients admitted to the neurocritical care unit (nccu) of a tertiary care hospital who underwent tracheostomy from september to june . of patients who received tracheostomies during their admission to the nccu, ( %) were successfully transferred to the floor, ( %) were readmitted to the icu, and ( %) had other dispositions such as discharge to rehabilitation and withdrawal of care. there were a total of readmissions, due to respiratory decompensation and due to cardiopulmonary arrest. hypoventilation is commonly seen in neurological patients who receive a tracheostomy. potential predictors of respiratory decompensation and readmission of these patients include their brainstem reflexes and respiratory patterns as assessed by the four score as well as their duration of mechanical ventilation. twenty-two percent of neurocritically ill patients may become hypernatremic. moderate-severe traumatic brain injury (mstbi) patients may develop hypernatremia possibly from diabetes insipidus (di) or osmotherapy for brain edema treatment. retrospective studies suggest that hypernatremia (serum sodium [sna] > mmol/l) may be associated with an increased risk of death. however, these studies failed to adjust for di and the use of osmotherapy. we examined the impact of mean and peak sna on in-hospital mortality and functional outcome at hospital discharge, adjusted for these important variables. in a prospective observational study, consecutive mstbi patients from a single level i trauma center between / - / were analyzed. poor outcome was defined as glasgow outcome scale (gos) - . multivariable logistic and ordinal regression was utilized to adjust for admission characteristics, injury severity, icu length-of-stay, brain edema, osmotherapy (mannitol/hypertonic saline), and di. firth's method was used in logistic regression models to accommodate small sample sizes. the mean age was years, % were men, and the median glasglow coma scale and injury severity scores were and , respectively. higher mean and higher peak sna were significantly associated with worse outcomes, both when using the dichotomized (or . ; % ci . - . for mean and or . ; % ci - . for peak sna) and ordinal gos (or . ; % ci . - . for mean and or . ; % ci . - . for peak sna). for every mmol/l increase in mean sna and every mmol/l increase in peak sna, patients worsened by one gos category. our results suggest that higher sna values are associated with worse neurological outcome, independent of osmotherapy, brain edema and di. it will be important to determine which sna might be "too high". while autonomic instability occurs as part of anti-n-methyl d-aspartate (anti-nmda) receptor encephalitis, anti-nmda receptor encephalitis is not a recognized cause of the clinical syndrome of paroxysmal sympathetic hyperactivity (psh). we present a case of anti-nmda receptor encephalitis in which psh was a cardinal feature. a -year-old woman had a generalized tonic-clonic seizure, and then developed progressively worsening neuropsychiatric symptoms, including mania, hallucinations, echolalia, and suicidal ideation. diagnostic work-up revealed anti-nmda receptor antibodies detected in the serum and in the cerebrospinal fluid (csf). one week after symptom onset, the patient experienced intermittent episodes of sinus tachycardia, hypertension, tachypnea, diaphoresis and extensor posturing. the episodes were both spontaneous and stimulus responsive (for example, during endotracheal suctioning). the episodes, consistent with psh, were initially treated with dexmedetomidine, which was titrated to effect. gabapentin and propranolol were added later for symptom control, but eventually weaned off as her symptoms abated approximately six weeks into the illness. we believe that the autonomic instability associated with anti-nmda receptor encephalitis may often be psh. psh often goes unrecognized in patients outside of the setting of tbi, thus specific psh management strategies may be overlooked in other contexts. anti-nmda receptor encephalitis may represent the functional companion to the structural lesion encountered in tbi. recognition of psh in this setting is important to guide the management of the autonomic instability, but may also have mechanistic implications. a -year old male with history of motor vehicle accident s/p frontal sinus surgery was admitted with streptococcus pneumoniae meningitis and altered mental status. upon admission, he was febrile with leukocytosis. head ct showed left sinus opacification and csf studies were consistent with bacterial meningitis. despite broad-spectrum antibiotics and interval improvement in his head ct and csf studies, his mental status continued to decline. shortly after icu admission, he became lethargic with a new right-sided hemiparesis. cta revealed diffusely narrowed intracranial arteries most compatible with vasospasm and mri was consistent with multiple areas of infarction. his neurological exam continued to deteriorate necessitating intubation for airway protection. tcds showed bilateral mca vasospasm. initially, vasospasm was managed with nimodipine, hypertension, and euvolemia. systolic blood pressure was artificially elevated with vasopressors, inotropes, and ultimately with methylene blue. despite aggressive medical management, there was little improvement clinically. therefore, he received four sessions of angiography with intra-arterial verapamil. after the final intra-arterial verapamil treatment, he demonstrated angiographic and clinical improvement. we conclude that patient's cerebral vasospasm was a direct complication of streptococcus pneumonia meningitis. intra-arterial verapamil appears to be effective in treating pneumococcal meningitis induced symptomatic cerebral vasospasm. however, there is limited data to predict its vasodilatory sustainability and optimal treatment intervals. pneumococcal meningitis is the leading cause of bacterial meningitis beyond the neonatal period. clinical and experimental research had demonstrated that vascular alterations are common in bacterial meningitis and are associated with stroke. despite the introduction of the pneumococcal vaccine, availability of effective antibiotics, and advances in adjunctive strategies, mortality and morbidity rates associated with arterial complications secondary to pneumococcal meningitis remain high. this case is noteworthy because to our knowledge this is the first reported case of pneumococcal bacterial meningitis induced vasospasm that has been successfully treated with intra-arterial verapamil. xuemei cai , osmotic myelinolysis is a life threatening problem associated with rapid correction of chronic hyponatremia. the brain cannot readily restore organic osmolytes; thus rapid correction of serum osmolality leads to cellular shrinkage causing axonal dissociation from myelin sheaths. current guidelines state that serum sodium (sna) should be corrected at a rate not exceeding - meq/l/day but when extracellular volume depletion is the cause, vasopressin suppression after saline treatment increases risk of rapid overcorrection. there is no standard of care that directs treatment once osmotic myelinolysis occurs. we report a case of a patient who developed clinical symptoms of osmotic myelinolysis syndrome who was successfully treated with re-induction of hyponatremia which led to complete neurological recovery. a -year-old woman on thiazide treatment for hypertension developed protracted vomiting and diarrhea for several days followed by confusion and lethargy. in the emergency department, sna was meq/l. she received isotonic saline and over the next hours, sna rose meq/l. on hospital day two, her neurological condition deteriorated rapidly with development of mutism, increased tone in all extremities, hyperreflexia. osmotic myelinolysis syndrome was diagnosed on clinical grounds. she was given desmopressin with % dextrose in water (d w) to rapidly lower her lower her sna. her neurological status improved at a sna of meq/l. thereafter, sna was slowly uptitrated with desmopressin and % normal saline. she made a complete neurological recovery. mri performed at discharge and one month later showed no abnormalities. overcorrection of sna in chronic hyponatremia is a common iatrogenic problem which can lead to osmotic myelinolysis syndrome, a highly morbid and oftentimes fatal neurological condition. our case supports immediate re-induction of hyponatremia in patients with symptoms suggestive of osmotic myelinolysis at a time when imaging may be unremarkable and complete neurological recovery is achievable. posterior reversible encephalopathy syndrome (pres) is manifested by acute neurological findings with evidence of vasogenic edema on brain imaging possibly due to cerebral vascular endothelial dysfunction. the epidemiology of pres in pediatric critical care has not been well described and it may be under recognized and thus prompt treatment delayed. we performed a retrospective review of all patients with diagnosis of pres over month period (january to june ) in a pediatric critical care unit (pccu) at a tertiary care university hospital. data from hospitalization and month follow up were reviewed. there were admissions to pccu and neurology service consultations during the study. six patients were diagnosed with pres (incidence - in pccu admissions) with median age years (mean±sd; . ± . years). all patients presented at onset with generalized tonic-clonic or clonic type seizures that lasted up to hrs and returned to baseline mental status in - days. other clinical features were headache and visual impairment. risk factors preceding the onset of pres included anemia [hemoglobin . ± . g/dl], azotemia, hypertension, hypernatremia, hypocalcemia, hypomagnesemia, and recent use of chemotherapy (azathioprine, cyclophosphamide, tacrolimus and mycophenolate mofetil). brain mri demonstrated increased t /flair signal within the parieto-occipital white matter in all patients, frontal lobe changes in patients and vertebro-basilar system changes in patients. no regions of restricted diffusion were seen on diffusion weighted imaging. at month follow up, no patients had residual neurological deficits from pres and neuroimaging revealed significant resolution of white matter signal changes. pres is associated with multiple disease states including systemic lupus erythematosus, sickle cell disease, sepsis, recent use of cytotoxic medications and renal failure. knowledge of the risk factors associated with pres, its clinical presentation, and characteristic mri findings may lead to more rapid recognition and treatment. adults with neurological injury are at increased risk for tracheobronchial foreign body aspiration. this report will present a case of silent foreign body aspiration in a patient who presented to the emergency department with status epilepticus. case report and review of the literature. an year-old african american man presented to the emergency department with status epilepticus. seizures were controlled with intravenous lorazepam and fosphenytoin, and the patient was intubated for airway protection. on day four following admission to the neurosciences critical care unit, a routine magnetic resonance imaging (mri) scan demonstrated susceptibility artifact from a metallic focus which completely obscured the spine structures at c -c . upon review of the patient's previous imaging, numerous abnormalities were reported on daily chest x-rays and a foreign body was identified within the trachea on a thoracic ct from admission. a bronchoscopy was performed which revealed a watchband within the trachea and right mainstem bronchus. tracheobronchial foreign body aspiration should be considered in patients with unexplained respiratory symptoms, and a high degree of clinical suspicion should be maintained in patients with neurologic impairment. abnormalities on chest xray and computed tomography should prompt an early pursuit of the diagnosis in high-risk patients. mri, although generally considered to be a safe imaging modality, could be potentially harmful to patients with unidentified foreign bodies. hypokalemic periodic paralysis (hypopp) is a disease characterized by muscle weakness or paralysis secondary to low serum potassium levels. neurogenic diabetes insipidius (di) is a condition where patient excretes large volume of diluted urine due to low level of anti-diuretic hormone (adh). here, we report a case of hypopp in a patient with neurogenic di. a year-old right-handed hispanic male was admitted for seizures after developing a dental abscess. this patient had a history of pituitary adenoma resection at the age of with subsequent pan-hypopituitarism for which he was on hormonal supplementation. on hospital day three, he developed sudden onset of quadriparesis with motor strength in upper extremities / bilaterally and / in both lower extremities and absent deep tendon reflexes throughout. his routine laboratory studies showed severe hypokalemia of . meq/dl. nerve conduction study (ncs) revealed absent compound motor action potentials with normal sensory potentials. electromyography (emg) revealed no abnormal insertional activity or spontaneous activity. some muscles demonstrated no volitional motor units and a few others had decreased recruitment in distal small motor units. following aggressive correction of the hypokalemia he regained his full strength and repeat emg showed normal motor units, normal recruitment, but no myotonic discharges. ncs showed return of compound motor action potentials in all nerves tested. hypopp remains an important differential in an acute case of paralysis and acute management is important. we report a case of a -year-old caucasian male who presented to a community hospital with complaints of flu-like symptoms. he underwent pulmonary-vein isolation for chronic atrial fibrillation thirty days prior to admission. his history includes left frontal and right parietal ischemic infarcts, mitral valve repair, coronary artery bypass grafting, patent foramen ovale closure, and coronary artery disease. approximately hours prior to arrival, he developed nausea, vomiting, fatigue and confusion. he was febrile and appeared encephalopathic. a telemedicine stroke consultation recommended transfer to a tertiary care facility. while the initial concern was for acute cerebral ischemia, he did not meet exclusion criteria for thrombolytic therapy. the patient received aggressive initial hydration and broad spectrum intravenous antibiotics for coverage of meningitis. blood cultures, complete blood count, comprehensive metabolic panel, urinalysis, stool culture and a lumbar puncture were performed. interestingly, his blood cultures remained persistently positive for gram positive cocci in chains and clusters. occult stool was positive and his oral gastric tube demonstrated bloody drainage. the remainder of his laboratory work was unremarkable. ct scan of his head revealed old ischemic infarcts without hemorrhage or hypodensity. the patient continued to decompensate in the neurointensive care unit where he eventually required intubation. a ct scan of the chest was highly suspicious of a left atrial-esophogeal fistula. cardiothoracic surgery was notified of the atrio-esphogeal fistula and he was taken to the operating room for a right thoracotomy with repair of the fistula and intercostal muscle flap. post-operative mri brain demonstrated innumerable air emboli and diffuse areas of ischemic infarction. atrio-esophageal fistula is a very rare complication following pulmonary vein isolation, and because prognosis is dependent upon prompt surgical correction, neurointensivists should be aware of this entity financial support: none propofol infusion syndrome (pris) is a rare but devastating complication of high dose administration of diprivan in children and young adults which presents with metabolic acidosis, rhabdomyolysis and fatal cardiac dysrhythmias. we report a case of pris in a -year old, previously healthy, postpartum female who received a high dose diprivan infusion for hours at an outside institution for the treatment of presumed refractory convulsive status epilepticus. patient received diprivan mcg/kg/min for the first hours. diprivan was increased to mcg/kg/min to achieve burst suppression on the electroencephalogram. diprivan was stopped after hours due to lactic acidosis. subsequently patient developed renal failure and elevation of ck up to , .she was transferred to our institution for continuous hemofiltration and possible extracorporeal membrane oxygenation (ecmo).after transfer she developed atrial fibrillation, ventricular tachycardia and fibrillation. an ecmo catheter was placed when she was in ventricular fibrillation for minutes. after starting ecmo the patient developed asystole for hours, requiring a transvenous pacemaker. her cardiac dysfunction improved rapidly and ecmo was discontinued after days. the patient started to follow commands consistently at days after the onset of fulminant pris. mri of the brain showed a subacute right posterior cerebral artery infarct attributed to cardiac embolism. the patient left intensive care unit after weeks. close metabolic and cardiac monitoring should be applied when a patient is on high-dose of diprivan(> mcg/kg/min). diprivan should be stopped as soon as unexplained metabolic acidosis, rhabdomyolysis and cardiac dysrhythmias are noticed, and transfer the patient to a center with continuous hemofiltration and ecmo capabilities should be considered. ecmo can be a lifesaving intervention in patients with fulminant pris. postpartum cerebral angiopathy (pca) is a rare pregnancy complication. pca is often a benign condition that resolves spontaneously, but can lead to stroke or death. the purpose of this case study is to describe events that transpired in the care of a patient with severe persistent pca, for whom unconventional treatment was initiated because conventional treatment failed. retrospective and current chart reviews were conducted, including relevant medical history. objective data related to the patient's condition were reviewed. we examined the evolution of medical and nursing care as the patient's condition deteriorated despite aggressive conventional therapy, and reviewed ensuing events: multidisciplinary collaboration to search for other viable treatment options, consultation with colleagues from another major medical center regarding their experience with nicardipine and recommendations on off-label use for pca, and decision-making including the family about whether or not to administer intraventriuclar nicardipine. multiple disciplines (i.e., doctors, nurses, and pharmacists) and family members contributed to the complex decision to initiate unconventional treatment. we administered mg intraventricular nicardipine every eight hours for seven days. using transcranial dopplers, cerebral arteriograms, and clinical assessment data, we evaluated the effectiveness of this unconventional treatment. after seven days, we discontinued the nicardipine, while continuing standard treatment to maintain hypertension and hypervolemia. currently, the patient is expected to make a full recovery with few residual stroke deficits. a multidisciplinary approach, including the family in the decision-making process, enabled creative problem-solving for a challenging clinical situation. when conventional methods failed, our team collaborated to think outside the box and take a calculated risk, altering the course of our patient's condition from critical toward survival and recovery. the objective was to determine the diagnostic yield and safety of brain mri in critically ill patients with icu-acquired acute brain dysfunction. patients in the medical and surgical icus who developed acute brain dysfunction and underwent brain mri were included. patients with preexisting brain disorders and those from neurological icus were excluded. mri scans were analyzed by three specialists trained in neuroimaging. outcome variables included glasgow outcome scale at discharge ( - categorized as unfavorable and > as favorable) and death. patients underwent brain mri for evaluation of encephalopathy, seizures, focal deficit. signs of parenchymal brain abnormalities were detected in patients ( %) including white matter hyperintensities in . % and acute cerebral infarcts in . %. results from brain mri led to modification of diagnosis and treatment in % of cases. patients with mri defined lesions were more likely to have an unfavorable outcome. there were no adverse events from transportation to the radiology site or from mri performance. in icu patients with acute brain dysfunction, mri is a safe noninvasive diagnostic tool that often leads to substantial modification of diagnosis and treatment. structural brain injury contributes significantly to the pathogenesis of cerebral dysfunction during critical illness and should be taken into account even if other reasons for encephalopathy are presumed. central nervous system (cns) and intraventricular infections are a devastating complication for patient admitted to an intensive care unit. the use of intrathecal (it) antibiotics for the treatment of cns infections has been reported in small case studies. our purpose was to report patients who have received it antibiotics for intraventricular infections in our facility and discuss our findings. retrospective case series of patients who received intrathecal antibiotics in combination with systemic antibiotics for treatment of intraventricular cns infection over the past years. basic demographic and clinical measures were collected from the hospital data base. seven patients received it antibiotic therapy for cns infection. admitting diagnoses were head trauma ( ), intracranial hemorrhage ( ), and subarachnoid hemorrhage ( ). one patient had an infected ventriculoperitoneal shunt. all of the patients received an external ventricular drainage device during admission prior to developing cns infection. time from hardware placement to first positive csf culture for patient was days; patients were positive with first csf; were within days; and had his vp shunt in place for days prior to positive cultures. pathogens cultured from csf included klebsiella pneumoniae, acinetobacter baumannii and vancomycin-resistant enterococcus faecalis in patients each, and methicillin-resistant staphylococcus aureus in patient. the intrathecally instilled antibiotics were colistin, streptomycin, tobramycin and vancomycin. two of the patients cleared csf cultures in day, patient cleared in days, patients cleared in days and took days to clear csf. based on this small case series we found it antibiotic adjunct therapy as a viable option for treating cns infections as most of our patients cleared csf within days of treatment initiation. further studies are warranted to support our findings. we report a case of an esthesioneuroblastoma or olfactory neuroblastoma (onb) presenting with frontal lobe dysfunction and hence depression with rapidly declining mental status resulting from hydrocephalus and stroke meningitis. this is a year old man who presented with fever, headache and ams. he had months history of progressive headache, face pain, rhinorrhea, nasal congestion and depression. ct head showed destruction of the cribriform plate by a mass arising from the right nasal cavity with extension into the right inferior frontal cranial fossa. an evd was placed emergently for elevated icp. he was also found to have multiple strokes in the right basal ganglia and corpus callosum. an incidental mycotic aneurysm was seen at the right posterior cerebral artery. labs showed wbc of , sodium of , potassium of , bicarbonate is . a lumbar puncture was performed which showed evidence of bacterial meningitis. a diagnosis of onb was established by histopathology and confirmed by immunohistochemistry. on staging, the mass was classified as a kadish stage c tumor. he underwent coiling of the pseudo aneurysm of right pca and maxillary embolization, followed by bifrontal craniotomy and endovascular resection of tumor onb is a rare malignant tumor of neuroectodermal origin and is thought to arise from the olfactory epithelium. symptoms are related to nasal obstruction, orbital extension, invasion of thecribriform plate, paraneoplastic syndromes with hypercalcemia and hyponatremia and can cause frontal lobe dysfunction. physical examination generally reveals a vascular, polypoid mass located in the nasal cavity. mri helps to differentiate tumor from other causes of nasal obstruction. they typically stain for neuron-specific enolase (nse). there has been no standardized rct done due to rarity of the tumor but traditionally the mainstay of treatment in such locally advanced patients is combinedotolaryngologic and neurosurgical craniofacial resection followed by adjuvant radiotherapy. we describe a case of delayed ptld in a year old diabetic patient with esrd several years after multiple solid organ transplants; a successful pancreatic transplant and a rejected renal transplant. she initially presented with mild left hemiparesis and was found to have enhancing and non-enhancing both supra and infra tentorial lesions, without evidence of disease in the graft, skin or bone marrow. the histological diagnosis of ptld was made after a right frontal brain biopsy. she had intercurrent worsening of left hemiparesis post biopsy due to hemorrhagic transformation of one of the lesions. the patient initially responded to a decrease in immunosuppressive medications which included tacrolimus and cellcept however, she eventually also required rituximab and whole brain radiation to maintain remission. in this case report we highlight the manifestations of cns ptld, dilemmas in diagnosis and various strategies for management. this can be a fatal complication of solid organ transplants if not recognized and treated early. dysautonomia has been well associated with guillain barre syndrome (gbs). the dysautonomic effects of gbs may cause a variety of reversible clinical syndromes associated with sympathetic dysfunction including pres and takotsubo cardiomyopathy. pres can be a presenting feature following gbs treatment with intravenous (iv) immunoglobulins or may present later in recovery. dysautonomia resulting from gbs is the most likely explanation for this assocication while another possible mechanism can be the influence of cytokines, produced in the context of gbs, on the permeability of blood brain barrier. in this abstract we highlight a self limited case of pres presenting as an early complication of gbs. case: our patient was a year old female with hypertension who presented to an outside hospital with alteration in mental status. she had developed bilateral lower extremity weakness and difficulty ambulating for - days prior to admission. she reportedly had an upper respiratory infection about weeks prior to presentation. at the time of transfer to our hospital the patient had a generalized tonic clonic seizure and was started on keppra. she had a fluctuating mental status from being awake to stuporous. bilateral lower extremity power was / in all muscle groups. initially, deep tendon reflexes were + in lower extremities but after a few hours she became areflexic in lower extremities with + reflexes in upper extremities and downgoing plantars. mri brain t /flair images showed lesions consistent with pres. csf showed cyto-albuminologic dissociation and diagnosis of gbs was made. she was started on a day course of iv ig. she was discharged to a rehab facility with some improvement in her paraparesis and no recurrent seziures. this case report illustrates that patients can develop pres as a complication of gbs perhaps due to dysautonomia but pres may be self limited in this setting. data exists describing the outcomes of critically ill patients with specific conditions in specialty intensive care units (icu) versus general icus. severe sepsis and septic shock(ss/sh) outcomes have not been robustly evaluated in community hospitals between specialty icus. we chose to evaluate whether patients admitted to icus with ss/sh would have higher mortalities in neuroscience (ns) and cardiac (cards) icus versus general medical surgical icus (msicu). intensivists. the variables collected include age, time to antibiotics, intravenous fluids given, central line placements, code status, vasopressor requirements at hours and mortality. chi-square analysis was used to compare mortality rates. icus who were directly admitted from the ed were ns % (n = ), cards %(n= ), msicu % (n= ) p=. . the mortality rate for patients admitted with ss/sh was similar independent of the type of the icu the patients received care in. a multivariate analysis needs to be done to confirm these outcomes neurocrit care ( ) :s -s s thromboembolism is a known and feared complication of administering prothrombin complex concentrates (pcc) but the true incidence is unknown. most data is in regards to mi, dvt, pe and dic with little reported on ischemic stroke. this is the first known report in the literature of acute basilar thrombosis after reversal of anticoagulation with pcc. we present a year old women with acute basilar thrombosis after reversal of anticoagulation using pcc (profilinine sd). she was admitted with a hemodynamically stable lower gi bleed with a supratherapeutic inr of . she was taking coumadin for a recent pulmonary embolus. anticoagulation was reversed using profilnine sd units ( u/kg) and vitamin k mg intravenously. hours later she developed left facial weakness, quadraparesis and anarthria. ct brain showed no early ischemic changes. ct angiogram showed occlusion from the mid-basilar to the basilar apex with normal vertebral arteries from the origin to the site of occlusion. factor ii activity was elevated with normal activity of factor vii, ix and x. tte showed normal wall motion and ejection fraction without evidence of thrombus or shunt. pcc protocols for reversal of anticoagulation are used with increasing frequency, even in non-emergent situations. thromboembolism is a known complication of administration, even with modern formulations of pcc which include anticoagulants. risk of thromboembolism increases with doses above u/kg and with repeated dosing. the cause of thrombogenicity remains uncertain. accumulating data indicates the importance of factor ii (prothrombin) which has a linear relationship with thrombin generation. our case suggests that given potentially fatal thromboembolic complications, pcc administration should be weighed against the need for rapid correction of coagulopathy. more discussion is needed regarding complications of pcc administration, optimal dosing and uniform production of pcc products on the market. endovascular reperfusion reduces infarct volume to improve clinical outcome; however treatment effect may be diluted by subsequent care. an exploratory analysis was done to determine if discharge disposition impacted day mrs after definitive reperfusion therapy. in our study, patients discharged to snf & ar after thrombectomy have similar medical & neurological severity at admission and similar final infarct volumes at discharge. despite these similarities, patients discharged to snf had a significantly lower probability of achieving a good neurological outcome. further study is required to determine if ar should be considered in more patients to improve clinical outcomes. patients with acute ischemic stroke develop respiratory failure due to airway compromise from loss of protective reflexes or cerebral swelling. in such patients, traditional weaning parameters poorly predict successful extubation. failure of extubation increases complications, prolongs hospitalization and increase cost of care. we hypothesize that predictive factors can be identified in determining ischemic stroke patients with respiratory failure who can be successfully extubated. between january to december , consecutive patients admitted to a metropolitan academic stroke center with acute ischemic stroke and were mechanically ventilated within hours of admission were reviewed after irb approval. patients who were intubated for procedures only, extubated within hours, or placed on comfort measures were excluded, leaving patients for analysis. statistical analysis was done using sas . and univariate or multivariate logistic regression was performed when appropriate. of the included patients, the average age was . ± . years, and ( . %) were male. the median admission nihss was . and majority of patients had cardioembolic ( ) or large vessel atherosclerotic ( ) strokes. patients had posterior circulation stroke ( . %). eleven patients failed extubation ( . %). acute basilar occlusion was found to be a strong predictor of extubation failure (or= . %ci: . - p= . ) when adjusted for age, stroke severity and duration of mechanical ventilation. increasing age and higher nihss showed trend toward increased risk for extubation failure but did not reach statistical significance. hospital length of stay doubled, icu length of stay tripled, and total hospital cost doubled in patients who failed extubation. patients with respiratory failure due to acute stroke from basilar occlusion were more likely to fail extubation. patients who fail extubation had longer icu and hospital stay doubling the cost of care. further studies are needed to determine whether preemptive tracheostomy may be beneficial in this group of patients. early detection of patients likely to develop malignant middle cerebral artery (mca) infarction (mmcai) is essential to enable timely decision for promising interventions (e.g., decompressive hemicraniectomy). this study was designed to evaluate whether quantitative eeg (qeeg) could predict mmcai within hours of stroke onset. this prospective, observational cohort study enrolled patients with a mca infarct. all of them underwent eeg monitoring within hours after symptom onset. subsequently, their raw eeg data were quantitatively analyzed and the qeeg parameters including (delta+theta) / (alpha+beta) ratio (dtabr) and brain symmetry index (bsi) were computed based on the power spectral density. patients were classified in the mmcai group if they had decline of consciousness with radiological signs of space-occupying brain edema, whereas the others were allocated into the non-mmcai group. for the groups, we compared the above qeeg parameters, and clinical and imaging variables. univariate and multivariate discriminant analysis was used to determine the most accurate predictors of mmcai. of the patients included, developed mmcai. univariate analysis showed that the values of dtabr and bsi, the nihss scores on admission and a hypoattenuation on admission cerebral computed tomography (cct) scans > % mca territory were significant predictors of mmcai. the further logistic regression analysis identified bsi > . (odds ratio [or] . , % confidence interval [ci] . to . ; p = . ) and the infarct size > % mca territory on cct scan at admission (or . , % ci . to . ; p = . ) as independent predictors, and bsi > . was the better predictor, which achieves a positive likelihood ratio (lr) of . ( % ci . to . ) and a negative lr of . ( % ci . to . ). quantitative eeg allows the early prediction of mmcai, and can help in the selection of patients for decompressive hemicraniectomy. financial support: none the modified rankin scale (mrs) is a -level outcome scale used to assess level of function in neurological disease. its utility is underscored by widespread use in stroke outcomes assessment, but the basic levels of function encoded by the mrs are not specific to stroke. still, poor interobserver reliability and the requirement for expert and face-to-face interviews are problems in determining an mrs score. we have developed a question "yes/no" questionnaire, the mrs- q, and an online mrs calculator to quickly and accurately determine the mrs. we hypothesize that ( ) the mrs- q has acceptable interobserver reliability, ( ) the mrs- q can be administered equally well in person or over the telephone, ( ) the mrs- q can be administered accurately by personnel without clinical expertise, and ( ) the mrs- q allows application of the mrs to a broad range of neurological conditions. the mrs- q was administered by form or telephone. a web-based tool calculated the mrs and performed error checking. part compared the mrs- q to an mrs structured interview (n= ). part compared mrs- q administration by telephone and by paper form (n= ). part compared administration by an expert interviewer with administration by a non-expert (n= ). part examined reproducibility over weeks (n= ). agreement was very good in all study parts. in part (mrs- q vs. mrs-si), k was . and k w was . . in part (telephone vs. paper), k was . and k w was . . in part (expert vs non-expert), k was . and k w was . . in part (reproducibility), k was . and k w was . . the mrs- q can reliably determine the mrs by paper survey or over the telephone. importantly, the mrs- q survey does not require the participation of trained experts-excellent results are obtained when non-medical study personnel administer the survey. potentially inappropriate medications (pims) are medications that may increase cognitive burden and impact clinical outcomes in elderly icu patients. this study evaluates the use of pims and outcomes in elderly stroke patients. this is a retrospective study of p july . number of pims, length of stay (los), and changes in gcs and rass scores were evaluated. fisher's exact test was used to compare groups. of a significantly longer nsicu los and worse outcomes. introduction ais patients often have acutely elevated bp requiring iv antihypertensives (ivah). previous work shows aha/asa recommended antihypertensives used to reduce bp in ais commonly results in polypharmacy and its consequences: overshoot hypotension and increased mortality. this study evaluates the association between ivah polypharmacy and both clinical and economic outcomes in ais. premier, a us hospital administrative database. patients with ms-drgs to and a primary ais icd- code ( .x or .x ) were included. patients were matched in a : fashion utilizing propensity score methodology controlling icu admission, baseline characteristics, and pre-existing conditions. from january to december , study patients received at least one ivah on day one or two of hospitalization and . % of those received more than one ivah. after matching, patients remained in each group. patients in gp had a lower mortality rate than gp ( . % vs . %, p= . ), lower vasopressor use ( . % vs . %, p= . ), shorter los (median days vs days, p< . ), and lower total hospital costs (median $ , vs $ , , p< . ). t-pa use was similar between groups ( . % vs . %, p= . ). polypharmacy to treat acute hypertension is associated with worse clinical and economic outcomes in ais regardless of tpa administration. recent evidence suggests precise and reliable bp control is critical during the entire stroke pathway of care. currently recommended ivah do not reliably manage bp as single agents. in order to avoid polypharmacy and improve outcomes and costs, the ideal ivah drug needs to reliably manage and maintain precise bp control as monotherapy. financial support: authors are employees of the medicines company which markets an iv antihypertensive agent. mean corpuscular hemoglobin concentration (mchc) is a red blood cell indicie that is obtained as part of a complete blood count (cbc). mchc values reflect individual red blood cell (rbc) hemoglobin (hgb) content, and are directly affected by changes in hgb production and dna synthesis. recently another hematologic indicie: the red cell distribution width (rdw), has been shown to be an independent predictor of outcome in patients with stroke. we sought to determine if mchc on admission could be predictive of clinical outcome. this is a retrospective study on patients admitted to a university affiliated community hospital. initial mchc data were gathered retrospectively from the registry database. we included both ischemic and intraparenchymal hemorrhage (iph) stroke patients in our analysis. for evaluating the severity and outcome of the patients with ischemic strokes we used nihss on admission and mrs on discharge respectively. in iph patients, we utilized the university of california san francisco intracerebral hemorrhage (ich) score on admission / hours to quantify the severity of the stroke and mrs on discharge to measure the outcome. we used correlation coefficients (spearman's rho) and the mann whitney test for analysis of the data. spss version was used for data processing. our review identified patients with a diagnosis of iph and with a diagnosis of ischemic stroke. the mchc values in the iph group positively correlated to ucsf ich score on admission (p= . , r= . ) and at hours(p= . , r= . ), as well as to mrs at discharge (p= . , r- . ). the mchc levels for ischemic stroke patients correlated weakly and negatively to nihss on admission (p= . , r= - . ) and d-mrs (p= < . , r= - . ). mchc levels on admission correlate significantly with clinical measures of stroke severity and disability. mchc could serve as an early predictor for outcome in different stroke subtypes. in the absence of specific guidelines, there is considerable variance in pre-procedural intubation practices for endovascular treatment of acute ischemic stroke. the purpose of this study is to understand and characterize the variance in pre-procedural intubation practices and identify the reasons that influence the choice of pre-procedural intubation practices among treating physicians. we selected random cases from a prospective database of patients undergoing endovascular treatment for acute ischemic stroke and prepared a case summary providing pertinent demographic, clinical, and imaging data. twenty clinicians independently reviewed the case summaries and responded to whether they would intubate any of the patients and identified the reasons for their choices. clinicians were also asked to identify their training background (neurology, neurosurgery or radiology trained endovascular specialist, vascular neurologist or neuro-intensivist). reasons for intubation and agreement between clinicians for each case were ascertained. the decision to intubate the patient was made in of total clinical scenarios. the major reasons identified by the physicians for pre-procedural intubation were high national institute of health (nih) stroke scale scores on admission . % (n= ), labored breathing or desaturation . % (n= ), less than optimal respiratory status of patients combined with drowsiness or reduced level of consciousness . % (n= ), inability to follow command such as due to aphasia . % (n= ), seizures . % (n= ), and no reason . % (n= ). overall agreement between clinicians regarding decision of pre-procedural intubation among the case scenarios was . the decision of pre-procedural intubation varies widely among clinicians. due to recent data that suggests that decision of pre-procedural intubation may impact on patients' outcomes, better standardization of such practices is required. hyperglycemia has been shown to be associated with worse outcomes, increased hemorrhage rates, and increased mortality in patients with acute ischemic stroke (ais). we evaluated the effect of admission hyperglycemia on -day functional outcome, mortality, and hemorrhage rates in patients undergoing multimodal endovascular therapy (met) for ais. retrospective review of glucose on admission was performed in patients undergoing met between and in a tertiary care academic medical center. demographic data, diabetic status, nihss score, radiologic studies, and recanalization timi grade were analyzed, amongst other known predictors of hemorrhage and poor outcome. mean age was . + . and mean nihss . + . . hyperglycemia was present in ( admission hyperglycemia in patients undergoing met is associated with poor -day functional outcome and higher rates of in-hospital death and hi. in non-diabetic patients, hyperglycemia was only associated with increased mortality and hi. despite equivocal results for induced normoglycemia, this data justifies a prospective trial for moderate glycemic control in this patient population. previous studies suggest that low cholesterol levels are associated with higher rates of hemorrhage after acute ischemic stroke (ais). we studied the effect of serum lipoproteins and premorbid statin use on the rate of hemorrhage in ais patients treated with multimodal endovascular therapy (met). retrospective review of statin use and lipoprotein levels on admission including ldl, hdl and total cholesterol (tc) was perfomed in patients undergoing met between and in a tertiary care academic medical center. demographic data, nihss score, radiologic studies, and recanalization timi grade were analyzed, amongst other known ldl < mg/dl was associated with a higher incidence of ht (or . , % ci . - . , p= . ). hdl > was associated with higher rates of ph (or . , % ci . - . , p= . ). tc levels and premorbid statin use were not associated with higher rates of hemorrhage. statin use, ldl, hdl and tc were not independently associated with functional outcome at months. patients with hemorrhage and tc < had significantly higher rates of good functional outcome compared to those with tc > (or . , % ci . - . , p= . ). there was no significant association between statin use and rates of hemorrhage or functional outcome in patients presenting with ldl < . low ldl and high hdl levels are associated with increased rates of hemorrhage after met for ais. statin use had no effect on post-intervention hemorrhage or functional outcome regardless of admission lipid levels. despite the association between low ldl and hemorrhage, statin use in patients with a low ldl was not associated with poor outcomes. this data justifies further study of the effect of continuation and early initiation of statin therapy in this patient population. mexican americans (mas) have shown lower post-stroke mortality compared to non-hispanic whites (nhws). limited evidence suggests race/ethnic differences exist in intensive care unit (icu) admissions following stroke. our objective was to investigate the association of ethnicity with admission to the icu following stroke. cases of intracerebral hemorrhage and acute ischemic stroke were prospectively ascertained as part of the brain attack surveillance in corpus christi (basic) project for the period january, through december, . logistic regression models fitted within the generalized additive model framework were used to test associations between ethnicity and icu admission and potential confounders. an interaction term between age and ethnicity was investigated in the final model. a total , cases were included in analysis. mas were younger, more likely to have diabetes, and less likely to have atrial fibrillation, health insurance, or high school diploma than nhws. on unadjusted analysis, there was a trend toward mas being more likely to be admitted to icu than nhws ( . % versus . %; or= . ; % ci . - . ; p= . ). however, on adjusted analysis, no overall association between ma ethnicity and icu admission (or= . ; % ci . - . ) was found. when an interaction term for age and ethnicity was added to this model, there was only borderline evidence for effect modification by age of the ethnicity/icu relationship (p= . ). no overall association between ethnicity and icu admission was observed in this community. icu utilization alone does not likely explain ethnic differences in survival following stroke between mas and nhws. the medicines company, parsippany, nj, usa the relationship between blood pressure variability and inpatient outcomes and costs following ais is not well understood. using data from > us hospitals (cerner health facts®), we identified all admissions between / / and / / of - -cm diagnosis codes .x , .x ). in patients with principal diagnoses of ais, time of initial clinical presentation was designated "index time"; for those with secondary diagnoses of ais, index time was in-hospital onset of stroke symptoms. we calculated blood pressure variability (bpv) as maximum difference (md) (i.e., highest -lowest recorded bp) in the -hour period following index time. patients were igh"] vs <median value ["low"]). we examined rates of hemorrhagic transformation, in-hospital mortality, and total in-hospital charges in the two groups. a total of admissions satisfied all selection criteria (high bpv: ; low bpv: ); patients were comparable with respect to age (mean age, years), gender, and comorbidities. patients with high bpv were nominally more likely to experience hemorrhagic transformation ( . % vs . %; p= . ) and to die in hospital ( . % vs . %; p= . ), respectively. they also had significantly higher total in-hospital charges ($ , vs $ , ; p< . ). highly variable blood pressure during the hours following ais is associated with increased total in-hospital charges. further research is needed to better understand the degree to which better early hypertensive management may improve patient outcomes. financial support: our research was funded by the medicines company. mr. berger, mr. moynahan, and dr. oster are employed by policy analysis inc., an independent contract-research organization with previous and ongoing previous work from our group revealed an association between lower end-tidal carbon dioxide (co ) values with worse outcomes in intubated, mechanically ventilated patients undergoing intra-arterial procedures for acute ischemic stroke. based on that observation, we hypothesized there might also be an association between lower arterial co and outcomes in this patient group. we retrospectively reviewed the charts of participants in a national, multi-center mechanical thrombectomy trial. all patients were intubated and mechanically ventilated at the time of their intra-arterial intervention per protocol at our institution. arterial blood gasses (abg) were typically obtained post-procedurally at admission to the icu as part of admission laboratory panel. demographics, hospital course data, arterial blood gasses and outcomes ( -day modified rankin score) were collected. descriptive statistics (median and intraquartile range for continuous variables; counts and percentages for categorical variables) were used to describe clinical data. categorical variables were compared using fisher's exact tests, and ordinal comparisons were made with the wilcoxon-mann-whitneytest. significance was set at : mrs - ; vs. unfavorable: mrs - . seventy-five patients were identified as part of the registry, though were excluded from this analysis ( had no recorded abgs, lost to follow up). patients with an unfavorable outcome were older ( [ - ] v. [ - ] , p= . ). in the unfavorable group, median pco levels were statistically significantly lower than those with a good outcome ( [ - ] v. [ - ] , p= . ). our results suggest that there may be an association between lower pco values and outcome in intubated, mechanically ventilated ischemic stroke patients. one possible explanation is that hypocapnea facilitates cerebral vasoconstriction, worsening ischemia and possibly enlarging the ischemic penumbra. this association should be further interrogated in a prospective, controlled trial. although ischemic brain tissue should appear particularly dependent on the main oxygen carrier hemoglobin (hb), optimum hb and hematocrit (hct) levels in neurointensive care unit (nicu) patients with severe ischemic stroke (is) are still unknown. research thereof and of the best red blood cell (rbc) transfusion regimen is neglected. here, we aimed to investigate associations of hb/hct dynamics and transfusion activity with nicu variables and outcome in nicu patients with ischemic stroke. patients with is were retrospectively assessed if admitted to the nicu for at least days and mechanically ventilated. assessment involved clinical characteristics, rbc transfusion events, and laboratory parameters such as hb, hct, creactive protein, blood count, creatinine. mortality and functional outcome (by mrs) were assessed prospectively by telephone interview at , and months after discharge. statistics were descriptive and explorative, i.e. involved correlation / variance tests for associations between hb, hct levels, transfusion events and outcome (linear regression analysis, t-test). patients were analyzed. % had anterior circulation, % posterior circulation ischemic stroke with a median nihss of and a median apacheii of . median nicu stay was days, median ventilation time hours. baseline hb and hct were g/dl and . , mean decrease over nicu stay was % leading to minimum hb and hct of g/dl and . , respectively. mrs at day was ( %), ( %), ( %) and (=dead, %). a first preliminary analysis did not reveal any consistent associations between severity of anemia or transfusion activity and outcome. anemia is frequent in nicu patients with ischemic stroke. at present, this retrospective analysis does not support a more aggressive rbc transfusion practice. more detailed subgroup analysis results will be presented at the time of the meeting. although not clinically validated, physicians frequently use imaging to help predict neurologic and cognitive outcome after hypoxic-ischemic injury to the brain following cardiac arrest. we systematically reviewed the current literature investigating the accuracy of imaging in predicting outcome in adult patients who are comatose after cardiac arrest. the electronic databases of medline via pubmed, embase via ovid, and the cochrane database of systematic reviews were searched using combinations of the search terms "cardiac arrest," "cardiopulmonary resuscitation," "brain hypoxia," and "neuroimaging." eligible studies were reviewed in detail and classified by level of evidence (loe) and methodological quality as defined by the international liaison committee on resuscitation (ilcor). articles were identified, of which met inclusion criteria and were reviewed in detail. articles found imaging to be helpful in accurately predicting outcome: were categorized as loe p ( good, fair, poor quality), loe p ( good, fair, poor quality), and loe p ( good, fair, poor quality). articles found imaging to not be helpful in accurately predicting outcome: were categorized as loe p ( good, fair quality), loe p (all poor quality), loe p ( good, fair, poor quality). article was found to be equivocal, categorized as loe p (fair quality). no loe p trials have been performed and although two loe p studies exist, they do not compare different modalities and have limited generalizability in the clinical setting. there is insufficient evidence to recommend routine use of imaging to predict outcome in comatose cardiac arrest patients. further studies are needed to determine the utility of imaging in this setting, and should include comparison with already validated methods of prognostication such as the clinical exam and electrophysiology. in acute ischemic stroke, factors affecting blood pressure (bp) levels in the first few hours after onset are not well known. the rise in bp levels can be a mechanism to compensate for vessel occlusion and to ensure the viability of penumbral brain tissue (pbt). between january and december , data of consecutive acute ischemic stroke patients admitted to florence nightingale stroke unit within the first hours and evaluated with stroke mri were analyzed. potential early correlates of bp levels were analyzed with multivariate logistic regression technique. age, gender, hypertension (ht) history, treatment for ht, onset to door time, nihss, level of consciousness, stroke type and stroke etiology, pwi/dwi mismatch, index proximal vessel occlusion (ipvo), were included in the analyses. a pwi/dwi mismatch, a potential indicator of pbt, was considered as present if estimated at least to be approximately % by eyeballing the lesion. index pvo was defined as symptomatic occlusion or - % stenosis of carotid or vertebrobasilar artery system vessels. high bp level was defined as sys> mmhg or dys> mmhg at admission. two hundred-six patients ( male; mean age ; range - years) were evaluated. hundred and four patients ( , %) had high bp at admission. in univariate logistic regression analysis, women (p: , ), age (p: , ), tacs (p: , ), hypertension history (p: , ), ipvo (p: , ) were associated with high bp values. only tacs (or: , ; % ci: , - , ) was independently associated with high bp readings at admissions in multivariate analysis. we did not find any argument to state that high admission blood pressure is a compensatory response following brain tissue ischemia. intravenous recombinant tissue plasminogen activator (iv r-tpa) has revolutionized the management of acute ischemic stroke. however, symptomatic intracranial ( %) and severe systemic ( . %) hemorrhagic complications after thrombolysis remain a concern. we present a rare complication of r-tpa and underscore the importance of close monitoring after thrombolysis. the clinical history, laboratory, and imaging studies were reviewed. a year old man with psoriasis and morbid obesity presented with acute aphasia and right hemiplegia. he had fallen as a result, striking his right eye. his examination demonstrated right periorbital ecchymosis without ptosis, expressive aphasia, leftward gaze deviation and corresponding hemianopsia, and right facial weakness and hemiplegia. his summated nih stroke scale (nihss) was . initial cranial imaging demonstrated no blood, though did show an abnormal hyperdensity within the proximal left middle cerebral artery territory. he received iv-tpa hours from symptom onset with significant neurological improvement within minutes of thrombolysis. minutes after initiation of iv r-tpa, he rapidly developed periorbital edema with ecchymosis leading to complete ptosis of the right eye. repeat cranial imaging showed an enlarging retro-orbital hematoma. an emergent lateral canthotomy was performed of the right eye to rapidly decompress the optic nerve. within days of thrombolysis and successful orbital decompression, as visualized on repeat cranial imaging, he made near full neurologic and visual recovery. to our knowledge, this is the first reported case of a near catastrophic hemorrhagic ocular complication after iv r-tpa therapy for acute ischemic stroke. despite the suspected trivial nature of injury, thrombolytic treatment should proceed with caution in the setting of any trauma. this report highlights the importance of careful inspection and maintaining a high index of suspicion and vigilance for unanticipated complications after thrombolytic therapy. in the setting of acute or evolving stroke, outcome may be dependant on the urgent re-establishment of cerebral perfusion.options for restoring cerebral blood flow include the intra-venous or intra-arterial administration of thrombolytic agents, mechanical thrombolysis, and urgent carotid endarterectomy. there is very limited experience with emergency extracrannial-intracranial (ec-ic) bypass in this setting. we reviewed the medical records and neuroimaging studies of consecutive patients who underwent urgent ec-ic bypass in the face of acute cerebral ischemia. none were considered appropriate candidates for endovascular therapy. ages ranged from to years, average . years. average follow-up was . years. preoperative angiographic evaluation identified critical narrowing of the supraclinoid ica in , the m segment of the middle cerebral artery in , and the cervical/petrous ica in . all had progressive, refractory symptoms associated with enlarging areas of ischemic changes on diffusion-weighted mri despite maximal medical therapy including anticoagulation and antiplatelet agents, blood pressure elevation, and fluid resuscitation. all patients underwent urgent sta-mca anastomosis. in every case, bypass resulted in stabilization of the progressive ischemic symptoms; in cases, revascularization was followed by rapid, dramatic improvement of preoperative deficit. five patients awoke with transient worsening of their preoperative neurological deficit which improved over - hours. no patient demonstrated a significant new area of ischemia on mr imaging. emergency ec -ic bypass for acute ischemic injury was both safe and effective in our experience. this population was characterized by relatively young patients with severely limited collateral circulation. in this series of carefully selected patients, bypass was successful in arresting ongoing ischemic symptoms, and in some cases, resulted in rapid neurological improvement. the ability for clinicians to predict outcome is of paramount importance when treating and counseling stroke patients and families. the dragon score is used to predict outcome in patients with anterior circulation strokes that have received intravenous tpa. we sought to determine if the dragon score could be applied to patients undergoing endovascular stroke therapy. charts for patients with interventions performed by a single operator (mfs) from january to march were reviewed. presenting symptoms were used to derive the dragon score. outcome predictability was compared to the findings in the original dragon score paper. twenty-four patients underwent endovascular stroke treatment; fourteen patients presented with anterior circulation ischemic strokes. five patients had only endovascular treatment, and patients had both ivtpa and endovascular treatment. the total average time from onset to termination of the endovascular procedure was minutes. in the endovascular alone patients, patients survived with a mean dragon score of . and mean discharge mrs of . . of the patients who received both intravenous and endovascular therapy, survived with a mean dragon score of . and mean discharge mrs of . . four of the surviving patients had greater than % specificity for poor outcome (mrs - ) based on the original paper. these patients however demonstrated a good recovery with an average mrs of . . despite the extended window for treatment and recanalization, patients who receive acute endovascular stroke therapy appear to have similar outcomes to the predicted outcome using the dragon score. furthermore, our study showed that patients who were expected to have poor outcome had the potential to improve clinically. this study reinforces the benefit of endovascular stroke therapy. intracranial arterial stenosis are relatively common findings of stroke patients in asia area. we reviewed stroke database to investigate clinical risk factors related to intracranial arterial stenosis, including carotid disease, and peripheral arterial disease which reflects advanced atherosclerosis. acute stroke patients at the national health insurance corporation ilsan hospital from january to december with available transcranial doppler(tcd) examination, carotid ultrasound and ankle-brachial indexes(abi) formed the analysis cohorts. retrospective review was performed. a total of patients were included during that period, patients with incomplete tcd study due to poor insonation windows were excluded( %). according to tcd criteria, groups of intracranial arterial stenosis are defined: vessel stenosis is in patients( %), - vessels in patients( %), more than vessels in patients( %). as the arterial number of intracranial stenosis increased, abi is decreased(p= . ) and the size of carotid artery plaque is increased(p= . ). among the risk factors, diabetes, age, past stroke history are increased(p= . , p= . , p= . ) and hdl cholesterol showed tendency of decrease(p= . ). however hypertension, smoking, total cholesterol, ldl cholesterol, triglyceride and sex are not correlated with intracranial arterial stenosis. among the acute stroke patients, about a half of them have intracranial arterial stenosis and these patients tend to have higher burden of advanced atherosclerosis as evidenced by a higher prevalence of diabetes, large sized plaques of carotid artery and peripheral arterial occlusive disease. dedicated neurocritical care service in an acute-icu setting with specialized neuro nurses and physicians improves the quality of care and patient outcomes. we aimed to find out the impact of specialized focused neurocritical service as compared to a general surgical/medical icu setting in a community hospital. we retrospectively reviewed data from - , on patients who received endovascular treatment (iatpa, thrombolysis, mechanical thrombectomy, with or without intra and extra cranial stenting) in order to achieve recanalization. patients were divided into two groups: group a (n= ) general med/surg icu care in - and, group b (n= ) focused neurocritical care - . functional outcome data (mrs days) between the groups was compared through patient records. group a patients were cared for with general surgical/medical icu care nurses while group b patients were cared for by a specialized core group of - nurses specifically trained in neurocritical care. both groups were comparable in terms of age, sex, admission nihss and co-morbidities (hypertension, hyperlipidemia, diabetes, ccf, a.fib, other). group a mrs - (n= ) %, group b mrs - (n= ) %. group a mrs - (n= ) %, group b mrs - (n= ) %. group a mrs (n= ) %, group b mrs (n= ) %. mortality for both groups was comparable at % (group a n= ; group b n= ). functional outcomes of fully independent patients (mrs - ) improved from % to % when a focused neurocritical care nursing service was implemented as compared to standard medical icu nursing care. strict adherence to neurocritical care protocols and proper attention to co-morbidities is the key to improved outcomes in critically sick acute stroke patient populations. in-hospital strokes remain a significant source of morbidity for patients. paradoxical embolism has been implicated as a potential source for these strokes. to date, there is only minimal literature regarding paradoxical embolus as a cause for stroke in the hospitalized patient. over a one-year period we studied in-patient stroke alerts and their etiologies at our institution. the hypothesis of this study is that strokes in hospitalized patients are caused by paradoxical emboli. this is a retrospective analysis of prospectively collected in-hospital stroke team calls (n= ) over a one-year period. we excluded patients on the stroke service or on neurologic floors including the neurological intensive care unit. we further excluded patients that were found to have stroke-mimics by consensus. from these patients, we collected demographic information and results of transthoracic echocardiograms (tte) and lower extremity (le) duplex. the categorical data was analyzed using chi-square on jmp . . a confirmed acute ischemic stroke was found in ( %) of the in-hospital stroke alerts. the majority of stroke alerts in our institution were from the cardiology and cardiothoracic services ( . %). a tte and le duplex were available in . % and . %, respectively. two patients were identified with a patent foramen ovale (pfo) and nine with a deep venous thrombosis (dvt). one patient was found to have both a dvt and pfo which was presumed as the source of embolus. overall, there was no significant association of in-hospital stroke and presumed paradoxical embolus. the present study shows no association of in-hospital strokes and paradoxical emboli. this study is limited by the infrequent ordering of le duplexes in this at risk population but is strengthened by the available tte results. posterior circulation stroke (pcs) is associated with high mortality and poor outcome. this single centre, retrospective analysis evaluates long-term mortality and functional outcome in pcs patients treated with/without revascularization therapy (rt). between january and december , dataof consecutive pcs patients admitted to florence nightingale stroke unit within the first hours were analyzed. after evaluation with mri, eligible patients with pwi/dwi mismatch selected with eye-balling technique were treated with rt. ninety days modified rankin score (mrs) and mortality were the main outcome. eighty-two patients ( male; mean age ; range - years) were evaluated. the mean onset to door time was minutes (sd: ). seventy-eight patients were examined with mri while patients were examined with ct. twenty-one patients received rt; intravenous thrombolysis in , endovascular multimodal revascularization in and bridging therapy in patients. mean nihss score was (range: - ) [treated group (tg): ( - ); untreated group (utg): ( - ) p: , ]. arterial occlusion was present in ( %)(tg: , %;utg: , %; p: , ). mean door to treatment time was minutes (sd: ).mean onset to treatment time was minutes (sd: ).mean discharge nihss score was (range: - ) [tg: ( - ); utg: ( - ) p: , ]. discharge mrs - ratio was , % (tg: , %; utg: , %; p: , ). the inhospital mortality rate was , % (tg: , %; utg: , %; p: , ). first month (tg: , %; utg: , %) and rd month (tg: , %; utg: %). mrs - ratio (p: , vs. , respectively) also th month (tg: , %; utg: , %) and rd month (tg: , %; utg: , %) mortality (p: , vs. , , respectively) were similar between groups. in posterior circulation stroke, despite severe clinical manifestations at admission and hospital discharge, after long term follow up, the outcome in patients treated with revascularization therapy is similar to those patients with benign outcome and not necessitating any revascularization therapy from the outset. we present a case series that highlights the feasibility of decompressive hemicraniectomy (dhc) in pediatric patients with ischemic stroke. a retrospective chart review identified cases of ischemic stroke at texas children's hospital between - where dhc was performed for high intracranial pressure (icp) after standard medical therapy failed to lower icp. information was obtained about patient characteristics on admission, radiological features of the stroke, surgical procedures, complications of the dhc and cranioplasty, and clinical outcomes. we also surveyed published literature on dhc for pediatric patients with ischemic stroke. there was no mortality in this case series. case had a modified rankin score (mrs) of at a follow up visit after months. case had mrs of at a follow up visit after months. cranioplasty was complicated by epidural abscess in his case. case had mrs of at a months follow up. review of literature identified other published case series consisting of cases of dhc in pediatric patients with ischemic stroke. detailed analysis of these cases is presented in the tabular form. this case series highlights the fact that dhc can be performed safely and effectively in pediatric patients with ischemic stroke with potential lifesaving and improved functional outcome. decompressive hemicraniectomy should be considered as a therapeutic option for refractory elevated icp following large hemispheric strokes in the pediatric population. basilar artery occlusion (bao) is a devastating neurological disease that can be difficult to diagnose due to its protean manifestations, and the initial ct will often not reveal an acute infarction. we present a patient with bao who was initially diagnosed with lyme disease. a y/o female presented with neck pain, an unsteady gait, partial facial paralysis, and mild dysarthria. she was noted to have an erythematous area on her neck that contained a tick. the initial head ct was negative. lyme disease was diagnosed and ceftriaxone and doxycycline were initiated. within hours, her symptoms progressed to hemiparesis and aphasia. a stat mri demonstrated the absence of flow in the basilar and left vertebral arteries with restricted diffusion in the pontine and mid-right parietal regions. the patient was transferred to a primary stroke center, but she was outside the window for stroke rescue. acute lyme disease is characterized by lymphocytic meningitis, cranial neuropathy (particularly facial palsy) and radiculoneuritis. though these symptoms usually take weeks to occur, the initial tick bite may not be recognized thus precluding an accurate evaluation of the time course. bao may present with a similar constellation of symptoms including headache, facial paralysis, and transient paresis called the "herald hemiparesis" of bao. the fluctuating course of early bao may be confusing and a high index of suspicion is required. intra-arterial lytic therapy, mechanical thrombolysis, or a combination is recommended up to hours of symptom onset. recanalization is paramount to preserving neurologic function. unfortunately, she arrived at our institution outside the window for invasive therapy. her symptoms continued to progress to a locked-in state and she was transferred to a ltac facility. the neurological manifestations of bao may be confused with other diagnosis and a high index of suspicion is required. metabolic abnormalities negatively influence outcome in patients with traumatic brain injury, subarachnoid hemorrhage, hemorrhagic stroke and ischemic stroke with or without thrombolytic therapy. the prognostic value of many potentially correctable physiologic markers in stroke patients receiving thrombolysis is unknown. twenty-one consecutive acute ischemic stroke patients treated with tissue plasminogen activator (tpa) were retrospectively studied. multiple metabolic and physiological variables including blood urea nitrogen, creatinine, sodium, potassium, chloride, calcium, phosphorous, magnesium and body temperature were analyzed. independent t test was used to compare mean scores of these variables and determine their effect on outcome. functional status at discharge was the primary outcome measure, being fully or partially independent determined as good outcome and fully dependent or dead as poor outcome. secondary outcome was the presence of hemorrhagic conversion. seventeen patients had good outcome, mean age , while patients had poor outcome, mean age . hyperthermia and admission acute physiology and chronic health evaluation (apache) ii score were associated with poor outcome (p< . ). hemorrhagic conversion occurred in patients and was associated with hyperthermia, higher simplified acute physiology score (saps) ii score and hyponatremia (p< . for all). this single-center, retrospective study suggests that mild hyperthermia, hyponatremia and higher apache ii and saps ii scores are associated with poor functional outcome and hemorrhagic conversion in patients with acute ischemic stroke treated with tpa. further study is required to determine if correcting these variables influences outcome. alterations in electrolyte balance and other basic physiologic indicies such as glucose have been implicated in the pathophysiology of coronary heart disease. however, the relationship between the electrolyte levels and other physiologic indicies measured immediately after an acute ischemic stroke has not been clearly delineated. objective: the aim of the present study was to test whether changes in a patient's basic metabolic panel modify the severity or outcomes of acute ischemic stroke. the study is a retrospective study on ischemic stroke patients admitted to a university affiliated community hospital. demographic data were collected from the data registry. values were obtained within one hour of presentation for serum sodium (na), potassium (k), glucose (gluc), chloride (cl), magnesium (mg), bicarbonate (hco ), bun, and creatinine (cr). as well glomerular filtration rate (gfr) and temperature values were also recorded. severity and outcome were measured using the nihss on admission and the mrs on discharge respectively. correlation coefficients (spearsman's rho) and the mann whitney test were employed in the analysis of the data. spss version was utilized for data processing. results consecutive acute ischemic stroke patients met the study criteria. serum ca (p= . , r= - . ) and gluc levels (p= . , r= . ) were significantly correlated with the mrs. serum cl, ca, bicarbonate, temperature and bun were significantly correlated with nihss on admission measurements (p= . , . , . , . ; r= . , - . , ; . ; . respectively). mg showed a negative trend of correlation with the nihss on admission as well (p= . ; r=- . ), suggesting a protective effect of higher mg levels. the study shows that initial metabolic parameters, such as serum mg, ca, hco , bun, and temperature may potentially allow for early prediction of the severity and outcome in patients with ischemic stroke. hypersomnolence is not typically appreciated as a focal neurologic finding, though bilateral thalamic infarcts may present with hypersomnolence as the only neurologic manifestation. a year old man presented with acute onset confusion, somnolence and slurred speech. his neurological examination was notable for somnolence, bilateral ptosis and dysarthria. routine laboratory investigations and csf analysis were unremarkable, aside from a urine toxicology screen which was positive for opiates. initial head computed tomography (ct) with ct angiography of the head and neck were unrevealing. magnetic resonance imaging was contraindicated as the patient had an automatic internal cardiac defibrillator (aicd). a working diagnosis of opiate intoxication was made in light of the urine toxicology results. because the patient failed to improve over the ensuing hours, a repeat head ct was obtained which revealed bilateral medial thalamic infarctions. while hypersomnolence is often associated with toxic-metabolic disorders, it may rarely be the result of acute arterial stroke. in the context of stroke, hypersomnolence can be accompanied by other symptoms including weakness, paresthesias, memory impairment, sectoranopsia, and personality changes. the feature of hypersomnolence is usually the result of an infarct of perforators arising from the posterior cerebral artery, specifically the paramedian,and tuberothalamic arterial branches, which are involved in irrigation of the reticular, and intralaminar nuclei of the thalamus that are involved in arousal. concomitant neurologic signs may not be present or may be difficult to elicit in this setting as patients are often unable to participate in the neurologic exam. acute stroke should therefore be considered in the differential diagnosis of hypersomnolence. failure to consider stroke as a potential eitiology may lead to delay in acute or secondary stroke prevention. metals play key roles in epigenetic events in living organisms. zinc, cadmium, lead, selenium, calcium, magnesium, sodium, and potassium have been found to be associated with stroke risk in nhanes and other studies. the central hypothesis of this pilot study is that metals and metalloproteins may determine and distinguish stroke phenotype (ischemic vs. hemorrhagic). stroke patients at the university hospital emergency department (ed) were enrolled in a plasma banking project. after irb approval and informed consent, blood draws were performed in the ed, and demographic and clinical information recorded. we analyzed plasma samples collected within hours of symptom onset. we used the proteomic techniques of affinity chromatography (to remove the abundant proteins albumin and igg), followed by size exclusion chromatography (sec -to eliminate low molecular weight compounds and fractionate the proteins), inductively coupled plasma mass spectrometry (icpms -to identify differentially expressed metalloproteins in plasma) and electrospray mass spectrometry (to identify the tryptic peptides known to represent specific proteins in the plasma). the areas and standard deviations of the chromatograms for the metalloproteins for stroke mimics (n= ), ischemic (n= ) and hemorrhagic (n= ) stroke patients were calculated using origin software. differences between sec-icpms peak areas of the metalloproteins for the ischemic, hemorrhagic and mimic samples were examined using two-sample t-test and box chart statistics. mg, al, mn, cu, zn, se, mo and pb were studied. significantly different metals were mg, al, mn, cu and se. box chart statistics performed for the sec-icpms metalloprotein peak area data revealed significant differences in all metalloproteins except al. tryptic peptide mapping identified significant differences in metalloproteins. sec-icpms detected differences in fractions of specific metal containing proteins in the plasma of stroke patients and patients who presented with a stroke mimic. ongoing efforts are aimed at identifying potential biologically relevant stroke biomarkers from the current list of differentially expressed proteins. retrospective chart review of patients admitted to the neurocritical care unit from august to august who developed icp crises (> mm hg for > minutes) and were treated with . % hts. only data for the first ever treatment with hts were collected. patient demographics, onset and duration of action, lowest icp achieved and use of adjunctive therapies were recorded. descriptive statistics and correlation analysis were performed. complete data were available for patients. ten subjects ( %) were female, the mean age was + years. glasgow coma scale (gcs) was + and ( %) patients concomitantly received therapeutic hypothermia and pentobarbital coma. a + . % reduction in icp following administration of . % hts was observed (absolute change: + . mmhg). the mean time to icp < mmhg was + minutes and time to rebound icp > mmhg post-hts administration was minutes in % of our cohort. following treatment the mean improvement in gcs was + . a dose-response curve was generated. . % hts was associated with a % reduction in icp values in critically ill neurology/neurosurgery patients. time to clinical endpoint of icp < mmhg was minutes and in % of patients the duration of action was minutes. an improvement of points in gcs was also observed. the first description of a dose-response curve for . % hts in humans is reported. over a ten year period, we accumulated a prospective dataset of severely brain injured patients with multimodality monitoring (brain tissue oxygen monitoring and outcomes project). patients' data existed within individual excel files with heterogeneous fields. as different research subprojects arose, additional excel files were created to support new data extraction from clinical records. several issues were apparent: ( ) merging and querying data was time-consuming and rate-limiting in research productivity; ( ) users were unable to make uniform changes to all files; ( ) different users could not simultaneously enter data, ( ) auditing data entry was difficult. our goal was to convert the dataset into a relational database, to enhance clinical research efficiency. microsoft access was used to build a database with a relational backend structure and a graphical user interface (gui) frontend. a reporting tool was built for analysis, preview, printing, and customized queries. extract-transfer-load functions were programmed to create seamless data integration between the access database and the enterprise-wide clinical data warehouse (e.g. laboratory values, radiology results). it took approximately man-hours to audit existing excel data, and to load distilled data from excel files into structured database tables. it took approximately man-hours for application implementation and testing. the gui supported multiple simultaneous users' during data auditing, enforced validation rules that corrected data entry in realtime, and centralized user account management. we have provided research queries to date. excel has limitations as a tool for clinical research informatics. a relational database that is built with pre-defined rules, fields, and tables dispenses with the time-consuming step of merging and cleaning data and makes large dataset queries and analyses more efficient. it allows straightforward integration with other relational databases such as enterprise-wide clinical data warehouses, enabling expansion of queries into other clinical information systems. financial support: elsa lin is a data analyst whose salary was partially supported within the past twelve months by a grant from integra (brain tissue oxygen monitoring) for the specific purpose of creating a relational objective of this case study is to report a case of central nervous system (cns) histoplasmosis presenting as an ischemic pontine vasculitis and chronic basilar meningitis. histoplasmosis, a disease caused by fungus histoplasma capsulatum, primarily affects immune-suppressed patients and commonly involves the lung but occasionally can have variable cns presentations. a thirty-five year old caucasian immune-competent male came with worsening of aphasia and confusion after having presented four weeks prior with dysarthria, gait ataxia and bilateral upper extremity weakness. he was diagnosed with bilateral pontine ischemic strokes secondary to small vessel vasculitis and but had limited response to high dose steroids. cerebral spinal fluid (csf) examination showed elevated protein, low glucose and elevated cells suggestive of meningitis and he was started on empiric antibiotics and trials of repeat intravenous (iv) steroids. follow-up imaging revealed obstructive hydrocephalous and he underwent successful ventriculo-peritoneal (vp) shunt placement. his csf culture came back positive for h. capsulatum. csf histoplasma antigen and urine antigen were also positive. he was initially treated with ambisome but changed to voriconzaole secondary to renal insufficiency and was eventually continued on itraconazole. at one year, the patient good clinical improvement and follow-up cultures were negative. while pulmonary involvement of histoplasmosis in immune-suppressed patients is common, systemic presentation of this fungal infection in immune-competent patients is exceeding rare. clinicians should consider cns histoplasmosis on the differential diagnosis in atypical stroke cases, particularly those with chronic basilar meningitis. there is increasing incidence of dengue fever in our country and encephalopathy is the most common neurological manifestation of severe infection. however, recent studies have shown that there is increasing evidence for dengue viral neurotropism. dengue encephalitis, a distinct clinical entity have been found to be associated with the neurovirulence involving serotypes den- and den- . the objective of this study is to report the clinical course, laboratory, and radiographic findings of dengue encephalitis that did not go through the usual state of dengue fever. management of this specific viral infection will likewise be discussed. case presentation and report with literature review. a -year old filipino male, methamphetamine and marijuana user was admitted to our hospital because of seizures preceded by headache and fever. he was managed as a case of viral meningitis supported by cranial mri findings and csf studies. after nearly days, he clinically deteriorated initially from a very agitated, restless and combative state progressing to frank stupor. body temperature was uncontrollably high. repeat csf studies revealed elevated pressure, lymphocytosis, normal protein and sugar, and positive igm dengue virus. serum study for dengue virus igm capture elisa was also positive. other significant tests ruled out malaria, hiv and nmda antibody as source of encephalitis. after intravenous steroids were started, on top of antipsychotics, clinical symptoms were noted to eventually resolve. we theorize that dengue encephalitis should be considered in the differential diagnosis of acute viral meningoencephalitis though the classical manifestations of dengue may not exist. while dengue infection may be endemic in asian countries, this should be considered in other parts of the world especially when patients rapidly deteriorate in the course of the disease. immunecompetence definitely play a vital role in the recovery. steroid therapy may be life saving in very severe cases. intracranial monitors can help guide the care of patients with severe brain injury. the devices are invasive and so may be associated with complications. furthermore, accurate interpretation of the monitors' data is needed to be of potential benefit. in this study we asked whether experience influences "device failure" or interpretation. retrospective analysis was performed on a prospective database that included patients (median age ; range - ) with severe brain injury and who received intraparenchymal multimodality monitoring through a triple lumen bolt (licox imp#). a total of triple lumen bolts were placed during an -year period. device failure was defined as: ) broken or bent (n= ; . %); ) improper placement (n= , . %); and ) ineffective (no response to o challenge n= , . %). there were ( . %) devices thought to provide "incorrect" data but subsequently were found to be accurate, i.e. improper data interpretation. there was a decline in device failure over the entire study period. each calendar year was divided into quartiles. device failure incidence was %, %, % and % per quarter, i.e. was greatest during the third quarter during the time of academic and staff changeover (or . ; p = . ). in addition, improper data interpretation was greatest during the rd quarter. our data suggest that experience with multi-modality monitors is associated with a reduced incidence of device failure or improper data interpretation. educational efforts may reduce the need for device replacement. financial support: peter leroux funding from integra for research. while efforts to "go green" and promote sustainability are well-established in many sectors, there has not been an adequate push toward such practice in the healthcare and medical fields. healthcare accounts for % of all commercial energy use, bil pds of waste, and % of greenhouse gas emissions in the us. these figures requires significant for efforts to be implemented; we each subscribe to, "first, do no harm" demands that these negative environmental impacts be addressed and mitigated immediately. the intent of this report is to investigate and analyze the opportunities the healthcare industry has to embark on sustainable practices. we analyzed green architecture for new healthcare campuses and renovation of outdated facilities, submit efficiency and cost analyses of disposable versus reusable textiles, and offer observations on innovative technologies being developed to promote sustainability. this study was conducted after an extensive review of published literature, verified statistical reports presenting the cost-effectiveness and improved efficiency of pursuing an sustainable model of healthcare delivery. in cluded is a cradle-to-grave analysis of multiple facets of the healthcare/sustainability field, and addresses a number of specialist-specific avenues, including critical care and anesthesiology. energy-efficient building options -including rooftop gardens and alternative power sources -can cut energy consumption by %. healthcare providers in all fields are making efforts toward lowering the carbon footprint of hospitals by reducing greenhouse gas emissions and utilizing resourcing, second use and extensive recycling techniques and efforts. extensive life cycle assessments (lcas) prove that reusable medical textiles and tools are dramatically less expensive environmentally and financially than their disposable counterparts. while efforts are being made to promote sustainability in healthcare, more must be done. the evidence is clear: environmentally-conscious endeavors save money and help lessen the stress placed on the environment. for such a heavy-hitting culprit of consumption, the healthcare industry simply must begin implementing "green" practices based on already-present data. standard metabolic prediction equations have been validated in general critical care populations, but have not been well studied in the neurologically critically ill. we sought to determine whether: ) standard prediction equations accurately predict caloric requirements in neurocritical care patients; ) variation in resting energy expenditure (ree) exists among different subpopulations of neurocritical care patients; and whether the same factors influence ree among different neurocritical care subpopulations. indirect calorimetry measurements were retrospectively reviewed for mechanically-ventilated patients admitted to the neuro icu from january to june . the measured ree data were compared to the predicted basal energy expenditure (bee) calculated with the modified penn state university (psu-m) equation. patients were classified into neurological subtypes, stroke (n= ), status epilepticus (n= ), and other (n= ). traumatic brain injury (tbi) patients were not included. of the entire cohort, median measured ree was (iqr - ) kcal/d and median predicted bee was (iqr - ) kcal/d. the predicted bee correlated well with the measured ree (coefficient . ; p< . ) in the overall cohort. there was no significant difference in the predicted calorie requirement for stroke or status epilepticus. however, there was a suggestion that patients with status epilepticus were relatively hypometabolic (defined as ree < % of the predicted bee) compared to other subgroup populations [or= . ; % ci ( . - . ); p= . ]. factors significantly associated with ree include: maximum hour temperature, administration of intravenous sedation, body mass index (bmi) and sex. age and hospital day of ree were not predictive of energy expenditure. the psu-m predictive equation accurately estimates caloric needs for patients with non-tbi neurological injury. patients with status epilepticus may be hypometabolic relative to other neurologically injured patients, which may be due to use of multiple sedatives in this subpopulation. further research is needed to confirm these findings. the american society of anesthesiology provides guidelines for preoperative fasting for healthy patients undergoing elective procedures. these guidelines are often extrapolated to the critically ill population for procedures and extubation. we tested the hypothesis that npo practice differs between subspecialty, institution and practitioner-type. after irb approval, we conducted surveys of the memberships of the society of critical care medicine (sccm), neurocritical care society (ncs), and american burn association (aba) regarding their npo practice in critically ill patients. survey questions included frequency of use of nasogastric (ng) vs. nasoduodenal (nd) tubes, npo time prior to procedures, and npo time prior to extubation. responses were analyzed with stata . , using a one-way analysis of variance by ranks. we received a total of responses ( % response rate) encompassing practitioners from medical, surgical, neurosurgical ( responses), pediatric, cardiac, burn, trauma, and multidisciplinary icus. respondents ( . %) report % use of ng tubes, whereas ( . %) report % use of nd tubes. excluding responses from pediatric icu practitioners, the npo practice in nicus for intubated and non-intubated patients with nd tubes undergoing procedures is similar to respondents from other icus except the burn icu (p< . ). there is no difference in npo practice of patients with ng tubes undergoing procedures across all icus. nicu respondents report the most commonly used npo time prior to procedures is hours for intubated patients with nd tubes ( . %) and hours for those with ng tubes ( . %). for burn icu respondents, the most commonly reported npo time for intubated patients with nd tubes prior to procedures is hours ( . %), while hours is reported for those with ng tubes ( . %). npo practice in critically ill patients varies across the subspecialty units. further research is necessary to develop evidence-based guidelines for npo practices in the critically ill patients. patients intubated for primary neurological reasons represent a unique critically-ill population. extubation failure rates in primary brain injury (pbi) patients are - % compared to - % in the general critical care population. these populations have never been directly compared. we hypothesized that intubated pbi patients would have higher rates of extubation failure compared to non-pbi patients. retrospective cohort of intubated patients admitted to the medical intensive care unit or the neurocritical care unit in a tertiary-care university hospital between october , and september , . extubation failure was defined as requiring endotracheal intubation at hours, hours and one week. of the . failing extubation at hours did not put patients at increased risk for vap. total ventilator days were similar between pbi and non-pbi patients. pbi patients who failed at hours did not have a significant increase in ventilator days, intensive care unit days or mortality. our data indicates pbi patients are at increased risk for extubation failure compared to non-pbi patients. future prospective study is warranted to determine predictors of extubation failure at hours in pbi patients. peripherally inserted central catheters (picc) is been routinely used instead of central venous catheter (cvc) in our intensive care unit (icu) patients, that includes critical neurologic/neurosurgical patients. there are a number of studies has been done to evaluate risks of picc placement in general medical and surgical icus. a retrospective analysis to determine risk of large vein thrombosis due to picc in neurologic sub-population of patients in a general medical/surgical icu. charts and venous ultrasound studies of patients admitted to icu primarily for neurologic condition were reviewed. out of consecutive patients, underwent picc insertion. ( . %) had clinical and ultrasound evidence of large venous thrombosis attributed to picc. the presence of a picc line conferred a relative risk of . for the development of a dvt. patients with picc lines had a longer duration of stay in the icu (mean days = . +/- . ) when compared to patients without picc lines (mean days= . +/- . ) t( ) = . , p <. . routine placement of picc instead of cvc is associated with increased risk of thrombotic events in large veins in neuro critical sub population of a general icu, which may be associated with longer icu stay. more caution should be exercised before routinely using picc instead of cvc. there are many potential obstacles to guideline adoption and compliance in clinical practice. the purpose of this research was to develop a computer-readable format for clinical pathways, guidelines, and research protocols such that they could be rapidly distributed, displayed at the bedside, and driven by patient context. the goal is to increase guideline compliance and reduce errors made at the bedside. we collected institutional clinical practice guidelines from the abstract authors, guidelines from professional societies (including the neurocritical care society), and one multi-center research protocol (boost-ii). we analyzed each to look for common constructs that would form the basis of a computer-readable care path "language". we also reviewed previous attempts at computer-readable guidelines to discover what might be applicable to our system. the analysis showed considerable variation in the way guidelines are put to practice at the bedside. despite this, we found a set of generalized patterns that were used to develop a care path representation (language) that could encapsulate the content of the guidelines. structured goal-oriented steps, alarm and time couplers, and a "monitoring cycle" were designed and represented in an xml-based language. a scripting method for decision logic also was developed. software was written to read the xml script, display the care path "flow-chart", provide interaction with the health care provider, and links to related instructional content. integration with real-time multimodal monitoring data allows the care path to be driven by the context of the patient. this abstract outlines the first part of a larger project to develop an open-standard guideline format and display software that will decrease the time to adoption of neurocritical care guidelines and increase compliance in clinical practice. financial support: funding received by moberg research from nih/ninds and us army/tatrc to carry out this work. one of the authors (r moberg) is president and owner of moberg research. the objective of this study was to develop empiric treatment guidelines for patients admitted to the neurosciences intensive care unit based on unit specific antimicrobial surveillance. a prospective chart review was performed from october to april of all adult patients admitted to the neurosciences intensive care unit with positive cultures from any site. in addition to culture data and antimicrobial sensitivities, time of admission, diagnosis, placement of an external ventriculostomy device (evd), duration of cefazolin prophylaxis and risk factors for healthcare-associated infections (hai) were collected. hospitalization within days, residency in an extended care facility or hemodialysis at the time of admission were considered hai risk factors. cultures were analyzed as those occurring before or after day of nicu stay. patients residing in the unit as a result of overflow were excluded. a total of patients and positive culture results were included and analyzed by duration of icu stay < days (n= ) or > days (n= ). evd placement and cefazolin prophylaxis were present in % of patients for a mean of . days. at < days, methicillin-resistant staphyloccous aureus (mrsa) was the most common pathogen in patients with risk factors for hai. at < days without risk factors, the most common pathogens were methicillin-sensitive staphylococcus aureus (mssa) (n= ) and enterobacter (n= ) in the sputum and enterococcus (n= ) in the urine. a further analysis revealed theseisolates emerged after day of admission in patients receiving cefazolin prophylaxis. beyond days, sputum isolates predominated and consisted of gram negative pathogens (n= ), mssa (n= ) and mrsa (n= ). selective pressure from cefazolin prophylaxis was apparent in unit surveillance and emerged at or after days. based on these results, institutional empiric antibiotic treatment regimens were adjusted to cover these pathogens after day of nicu stay. the direct thrombin inhibitor dabigatran etexilate is approved for the prevention of stroke and systemic embolism in patients with non-valvular atrial fibrillation. despite the clinical benefits of dabigatran, hemorrhage remains a feared complication due to the lack of reversal agent and limited experience with interventions to reverse dabigatran's anticoagulant effect. in addition, reliable laboratory tests to measure the degree of anticoagulation associated with dabigatran are not widely available. an interprofessional team developed institutional protocols for the management of dabigatran and dabigatran-associated hemorrhages. clinical and neuroimaging data was collected from four patients with dabigatran-related subdural hematoma, subarachnoid hemorrhage and/or intracerebral hemorrhage who were treated between november and april . data collected includes age, gender, past medical history, renal function, coagulation and hematology parameters, computed tomography findings, blood products or clotting factors administered, and hemodialysis parameters, if applicable. the patients ranged in age from - years. all patients were inappropriately prescribed dabigatran due to age over years, renal insufficiency, increased bleeding risk, and/or an unlabeled indication. serial evaluation of each patient's coagulation assays was conducted in order to quantify the degree of anticoagulation. three of the four patients received emergent hemodialysis and one patient received recombinant activated factor vii. two patients received blood products, including ffp and platelets, with no observed clinical change. all patients survived to hospital discharge. though this case series is small, it demonstrates the importance of thoroughly evaluating a patient's renal function, bleeding risk, and concomitant medications to determine the appropriateness of dabigatran therapy. it is imperative for clinicians to understand dabigatran's pharmacokinetics and recognize the major factors that increase dabigatran exposure. increased age and renal insufficiency seemed to play a significant role in the hemorrhagic cases we encountered. post-surgical cerebral venous sinus thrombosis is extremely rare. the management of this complication is challenging for neurointensivists; since anticoagulation may increase the risk of bleeding after craniotomy. a previously healthy, year-old male was found to have a right cerebellopontine angle mass on brain magnetic resonance imaging (mri) during headache evaluation. he underwent a prolonged surgery with retrosigmoid craniotomy and resection of an acoustic schwannoma in left lateral decubitus position. immediately post-operatively, the patient had a seizure. brain ct-scan showed hyperdensity in the right transverse sinus suggestive of thrombus. cerebral angiogram confirmed occlusion of the superior sagittal sinus (sss) torcula and bilateral transverse sinuses. intravenous heparin was initiated; however, due to further deterioration with brain herniation, endovascular administration of tissue plasminogen activator (rt-pa) assisted with thromboaspiration with penumbra catheter was performed, followed by continuous infusion of rt-pa ( mg/hr) via microcatheter in the sss for days. a repeat angiogram showed near complete recanalization of the sinuses. heparin was continued, but he developed heparininduced thrombocytopenia, and was switched to bilvalirudin. his hospital course was complicated with intraventricular hemorrhage, acute respiratory distress syndrome, methicillin-resistant staphylococcus aureus (mrsa) bacteremia, and takasubo's cardiomyopathy. he had residual right facial nerve palsy and hemiparesis related to pontine ischemia. his prothrombin gene mutation was positive for one copy. he was ambulating with assistance prior to discharge to acute inpatient rehabilitation. cerebral venous sinus thrombosis is a rare complication of retrosigmoid resection of cpa tumor. aggressive treatment with endovascular rt-pa administration into the venous sinuses may be life-saving, but carries significant risks in the fresh post-operative period. many lives have been lost due to the loss of the airway in critically ill patients. the introduction of the video laryngoscope has been a useful tool that has saved many lives in recent years. one of the limitations of the video laryngoscope is that despite being able to see the vocal cords and the airway beyond it may be difficult to advance the endotracheal tube into the airway. we present a novel approach using a bougie to simplify this problem. a cohort of ten patients in a community critical care unit with difficult airways were intubated using this new technique in a nonrandomized fashion over a period of six months. a video laryngoscope was used in each case with patients sedated and paralyzed with usual agents used for rapid sequence induction. historically, the bougie when used with standard laryngoscopes is introduced into the airway by line of sight and the endotracheal tube is advanced over the bougie. with a video laryngoscope a direct line of sight is not available and passing the bougie is challenging because of a degree angle from the open mouth to the airway. this new technique involves lubricating both the metal stylet with the degree turn and a french x cm bougie and advancing them to the end of the endotracheal tube. the bougie is then advanced into the airway through the endotracheal tube under direct vision through the video laryngoscope. the endotracheal tube is then advanced into the airway over the bougie. all ten patients were intubated without difficulty and without complication. this new technique should be considered as an option in securing the airway in critically ill patients. further validation testing by other investigators is warranted regarding this new technique to determine if a randomized controlled trial is justified. francis r. ventilator-associated pneumonia (vap) remains a problem in traumatic brain injury and high-risk surgery patients. we use early non-bronchoscopic broncho-alveolar lavage (screening-bal) in the surgical intensive care unit (sicu) to identify ventilated patients with bronchiolar bacteria prior to hours. we reviewed results of these screening-bals in neurotrauma patients from / to / . all ventilated patients in the sicu underwent screening-bal - hours after intubation; quantitative cultures (> cfu/ml) were used to identify positive specimens. clinical pneumonia was defined as clinical pulmonary infection score (cpis)> and subsequent positive diagnostic-bal. continuous and dichotomous data were compared from the screening-bal results and clinical diagnosis of pneumonia. screening-bals were performed in neuro-trauma patients (mean iss . ± . ) with an average head abbreviated injury score (hais) of . ± . . thirty-three of these were positive for organisms ( %). twenty-four clinical pneumonias were diagnosed and in of these patients the causative organism identified was the same organism in the screening-bal ( . % agreement; kappa . ; p = . ). one patient with a negative screening-bal developed clinical pneumonia. the median day to develop pneumonia was . ( , ). the hais was higher in patients with a positive screening-bal ( . ± . vs. . ± . ; p = . ). there were no significant differences in the age, icu length of stay, iss, or hais in patients with a positive screening bal vs. the patients that developed a clinical pneumonia. positive screening mini-bal results are associated with the development of vap by the same organism. screening bal in neuro-trauma patients may be a mechanism to identify patients who are at-risk for developing pneumonia later in their hospitalization and early identification of the causative agent. further studies are warranted to determine if intervention on these results changes clinical course. human rabies is a relatively rare disease in the united states, with approximately cases diagnosed annually. the most common exposure in the u.s. relates to bats, however canines and other animals have also been implicated. the typical incubation period from exposure to development of symptoms is - months, while periods of up to years have been described. we present an atypical case of human rabies presenting after a prolonged incubation period in the united states. we describe a case of a year old brazilian man without prior medical history who presented with progressive sensory symptoms leading to encephalopathy and ultimately death. extensive workup revealed no other causes of his symptoms, and brain tissue samples sent to the cdc at the time of his death confirmed a diagnosis of rabies by direct fluorescent antibody testing. in addition, sequencing of the virus confirmed a variant found in canines in brazil. the patient had not traveled to brazil in over years, and had no confirmed exposure other than an encounter with a wild dog in brazil without reported bites or scratches before immigrating. because the viral genotype has not been previously identified among animals in the united states, this case represents the longest confirmed human rabies incubation in the united states to date. characterization of the illness revealed loss of evoked potentials, electroencephalography amplitude attenuation, mr spectroscopy changes of the deep nuclei, and an atypical inflammatory response on pathologic testing. we speculate that either an atypical immunologic response or the patient's recent anabolic steroid use may have mediated delayed progression. this case underscores the importance of keeping human rabies in the differential diagnosis of rapidly progressive encephalomyelitis, even without an exposure history, or with a remote exposure history. the full outline of unresponsiveness (four) score has been validated as an alternative to the glasgow coma scale (gcs) in the evaluation of stuporous and comatose patients and predicts long-term outcomes. the utility of serial four score and gcs by nurses in detecting changes in neurologic exam in the neurocritical care unit (nccu) and whether high frequency monitoring after the first assessment is beneficial has not been studied. the electronic charts of consecutive patients with surgical and non-surgical brain pathology admitted to a nccu were reviewed, yielding observations of gcs, fourscore, and cranial nerve assessments. changes in neurologic exam promoting notification of a provider were abstracted from nursing notes. of patients (m:f: : , age: + yrs), had semi-elective neurosurgery, -ischemic/hemorrhagic stroke, encephalopathy/infection, -subarachnoid hemorrhage, -traumatic brain injury, -seizures, -other. admission median gcs was (iqr- ); median fourscore was ( ). comparison of q - h fourscore vs. qdaily fourscore readings showed no significant difference in fourscore by frequency of measurement (p= . ). in occurrences of change in neurologic exam resulting in provider notification, changes in mean fourscore and gcs from hours prior to the event were . (sd- . ) and . ( . ) respectively; p= . ). from hours prior to event, changes in mean fourscore and gcs were . ( . ) and . ( . ) respectively; p= . ). in one cerebral herniation event, neither scheduled fourscore nor gcs changed. use of the fourscore for serial monitoring and early detection of worsening of neurologic condition performs similarly to gcs and is less sensitive than subjective assessment of trained nccu nurses. the utility of incorporating the fourscore into the on-going nursing assessment paradigm of all nccu patients requires further evaluation. there may be subsets of patients or conditions (with lower sumscores than in our cohort) for which daily or more frequent monitoring has predictive value. a technique for real time, non-invasive blood flow monitoring would be a major asset to clinicians in neurocritical care. we studied the ability of a new hybrid technology employing ultrasound tagged near infrared spectroscopy (ut-nirs) to detect changes in cerebral blood flow (cbf) as compared to measurements by xenon single photon emission computer tomography ( xe-spect). twelve healthy volunteers were enrolled in the study. a cerox monitor (ornim medical ltd. israel) provided continuous ut-nirs monitoring of regional tissue oxygen saturation (sto ) and regional cerebral blood flow index (cfi). xe-spect (ceraspect; dsi, waltham, ma, usa) was then used to measure cbf at baseline, minutes and minutes after acetazolamide injection. ten subjects completed the study. significant increases in cbf as measured by both ut-nirs cfi and xe-spect cbf were noted minutes after acetazolamide injection. at minutes following injection, xe-spect cbf had returned to baseline while ut-nirs cfi remained elevated compared to baseline. a significant correlation between ut-nirs cfi and xe-spect cbf values was found at minutes but not minutes after acetazolamide injection. specificity and sensitivity for detecting an increase in cfi following acetazolamide injection were calculated using a receiver operating curve (roc), with an area under the curve of . (+/_ sem . ). no statistically significant changes in ut-nirs sto were noted following acetazolamide injection. ut-nirs cfi can detect increased cbf following acetazolamide injection, correlates with a gold standard, xe-spect, and the roc curve analysis demonstrates excellent discrimination. the difference in the measurements at minutes may be explained by different ratios of gray matter to white matter in the regions of interest as assessed by the two techniques. ut-nirs cfi can be more sensitive to changes in cerebral perfusion than simple regional tissue oximetry. financial support: dr gress is a member of the scientific advisory board of ornim medical ltd and holds stock options in the company. level of coma has traditionally been measured clinically (e.g. glasgow coma scale, four score, etc.), or with neurodiagnostic tests (e.g. eeg). developing more objective, longer term measures of coma could improve quantitation of arousal and modification of response to therapy. we used a post-cardiac arrest (ca) rodent coma model to test -d bodily acceleration as a wireless, continuous measure of early movement during coma arousal, and compared it to eeg based markers validated previously. five adult wistar rats (male, - gms) underwent eeg electrode implantation wk prior to asphyxia-induced min ca. four hours after resuscitation, rats were attached to a wireless eeg-accelerometer system. wideband and sub-band eeg were analyzed to yield iq, an entropy based and previously validated measure of coma arousal. we defined activity as the variability in -d acceleration as quantified by the standard deviation of acceleration. we found a significant positive linear correlation between accelerometer activity and full band eeg iq (r= . ± . , mean ± sd). when eeg sub-bands were divided into two categories ( . - hz and - hz), accelerometer activity had better correlation with higher frequency sub-bands (r= . ± . vs. r= . ± . ). during individual sub-band analysis, we were able to find a moderate correlation with the higher frequency iq - hz (r= . ± . ). these results suggest that -d acceleration based activity, measuring early subtle movements during coma arousal, correlates with eeg iq. this relationship was stronger for higher frequency sub-bands. this suggests that subtle motor activity quantitated by an accelerometer may be an acceptable indirect measure of arousal. such accelerometer-based systems also have the advantage of being more objective and affordable while also offering longer term monitoring. therefore, accelerometer-based monitoring for coma arousal may have clinical applicability in intensive care units. recent literature emphasizes the impact of vancomycin concentrations on patient outcomes, especially in serious infections such as central nervous system infections (cnsi) and pneumonia. achieving adequate concentrations is challenging in the critically ill due to changes in volume of distribution and clearance. we investigated the impact of a pvds in our neurologic units. retrospective chart review comparing outcomes of pvds (rph-group) to pre-implementation control group (md-group). adult inpatients receiving vancomycin on neurologic units (neuro icu and floor) were included in a month pre/post period. rph-group patients receiving vancomycin not consulted to pvds were excluded. outcomes evaluated number of vancomycin levels and proportion within goal range ( - mcg/ml). in md-and rph-groups, and patients were enrolled, respectively. rph-group had a higher percentage of patients with weight > kg and crcl > ml/min. icu patients accounted for % and % of the md-and rph-groups, respectively. common indications were cnsi and pneumonia in both groups. levels were drawn in md-group versus levels in rph-group. a higher percentage of levels were within goal range in rph-group ( %) versus md-group ( %, p = . ). amongst patients with cnsi, rph-group had a higher percentage of levels within goal range ( % vs. %, p = . ). icu patients in rph-group had a higher percentage of levels within goal range ( vs. %, p = . ). in icu patients, younger age (p = . ) and crcl > ml/min (p = . ) trended toward initial subtherapeutic levels despite receiving ~ mg/kg/day of vancomycin. implementation of the pvds in neurologic units resulted in higher attainment of therapeutic concentrations. in icu patients, addition of a loading dose or higher daily doses of vancomycin may need to be employed by the pvds to ensure achievement of target concentrations. intraventricular therapy (ivt) with polymyxin b (polyb), an antibiotic with similar pharmacological action to colistin (polye), by external ventricular derivation (evd) has the main goal of offering major bioavailability of the drug, since its use by intravenous and direct action are restricted by the blood-brain barrier, with penetration of only %. the patient of the present report had arterial venous malformations followed by hemorrhagic stroke, which caused elevated intracranial pressure. the objective is to show an example of the effect of ivt polyb in a patient with meningoencephalitis infection by multidrug-resistant gram-negative bacteria (a. baumannii and p. aeruginosa), that are common in the icu. a literature review was made on the subject of therapy with polyb about the pharmacological characteristics, nephrotoxicity and neurotoxicity. a comparative table of the resistance profile of the strain treated in this study was created, with the intrinsic resistance of the species. also, the development of liquor evolution (culture and routine) of the patient before the treatment was monitored, until negative liquor. the effectiveness of evd, the colonizer germ and monitoring of the serial aspects of the liquor were analyzed. the patient was treated with intravenous and intrathecal administration of polyb (ivt) from november th to november th. on / / , therapy with intravenous polyb was started: ui( . ui /kg/d) once a day, on every day of treatment; and ivt by evd: ui in solution once a day during the first three days, and on alternate days during all the treatment. as a result of the use of intrathecal polyb associated with intravenous, effectiveness was proven in the routines of liquor negative for such germs, not showing any reports of neurotoxity and nephotoxity. ivt polyb proved to be very efficient on treating meningoencephalitis quickly. no toxic effect was associated with the drug. enhancing the level of alertness in comatose patients after acute brain injury is a very challenging problem. the use of alerting agents like modafinil is reasonably established for tbi patients in the chronic phase but not in the acute settings. we retrospectively reviewed the use of these agents at our center over a five year period to determine efficacy and use patterns in the acute brain injury settings. a chart review for patients who were admitted to the nicu at dumc during ( ) ( ) ( ) ( ) ( ) and treated with an enhancing agent (modafinil, methylphenidate) for decreased level of alertness secondary to an acute brain injury. electronic records were then reviewed to confirm the intended use of the agent, and a number of clinical data elements was recorded. patients were found to meet study criteria and data elements were extracted. patients received modafinil, received methylphenidate. the average gcs was on admission and at discharge. average delay in trialing alerting agents was ( . ) days and in most cases the agents were used within a few days of withdrawal of care or discharge to hospice. outcomes varied widely with ( . %) going to nursing home, ( . %) going to rehab, . % going home and . % to hospice or death. sah was the most common injury ( . %) followed by ich ( . %), sdh ( . %) and tbi ( . %). review of documented gcs during acute hospitalization showed no significant changes during the period of alerting agent trial for any diagnosis other than tbi. in tbi a significant points improvement was seen on average. our data showed that starting methylphenidate or modafinil for the purpose of improving the level of consciousness in acute brain injury patients is not effective except for patients with traumatic brain injury. based on these observation alternative agents like l-dopa should be explored. nurses in the neurocritical care unit (nccu) are responsible for performing serial neurological exams to establish baseline and potentially detect patient deterioration. nurses spend considerable time doing frequent neurological checks but the current neurological exam is open to subjectivity. we want to quantify the agreement between nurses doing these exams. over the course of one week we tracked the neurological exams of patients admitted to the neurocritical care unit. we compared exams between the off-going and on-coming nurses. each exam consists of single elements, loc, orientation, right and left pupil size, reaction and description, characteristics of speech/communication and motor response in all four extremities. grouping right and left pupils gave element-groups. we examined change of shift (cos) opportunities. when there was more than one variation of an element-group a thorough chart review was performed to identify clinical indicators, such as medications given, to determine if there was a true clinical explanation for the variation. cos exams were the same between nurses % of the time whereas % of exams had a single variation and % contained or more single variations. of the cos opportunities with multiple variations in element-groups only exams showed a clinical reason for the change. that leaves exams with multiple unexplained variations. this accounts for % of overall total exam opportunities. nearly % of the time nurses do not agree on the neurological exam of a patient when examined before and after cos. inconsistency in terminology and methods between nurses may hinder accurate communication. a comprehensive literature search did not reveal a standard neurological exam for nccu nurses. further discussion needs to take place between neuro-nurses across the nation with the goal of defining terms and developing a national standard for the serial neurological exam performed by nurses. electrical impedance spectroscopy (eis) is novel, portable, easy-to-implement device that aims to provide rapid, affordable point-of-care detection, assessment, and monitoring of acute brain injury. an adaptation of "passive" electroencephalography (eeg), eis relies on non-invasive measurement and modeling of the conduction of minute electrical currents applied transcranially across a spectrum of frequencies. our purpose was to test of the feasibility of eis to distinguish the impedance differences between normal subjects and brain injury attributable to acute/subacute intracranial hemorrhage or subacute ischemic stroke. we performed a prospective, observational, proof-of-principle study of patients admitted to our neurosciences intensive care unit for ischemic stroke or intracranial hemorrhage, and healthy volunteers. -minute eis recordings were obtained for each patient. the eis device delivered a small "white-noise" alternating current through a pair of stimulation electrodes; voltages were recorded across three bilaterally symmetric electrode pairs in an eeg montage. log-log plots of impedance (y-axis) as a function of current frequency (x-axis, range hz- khz) were produced for each set of electrodes per patient. mean age was years (range - ); % ( / ) were female. of these brain-injured patients: (a) among all patients with subacute hemorrhage (days old), impedances dropped at higher current frequencies; (b) among all patients with subacute ischemic stroke (days old), impedances increased at higher frequencies; and (c) in one patient with acute hemorrhage (hours old), impedances were not significantly different at higher frequencies but evolved to the subacute hemorrhage pattern (a) at a day- follow-up recording. all brain-injured patients were distinguishable from normal control volunteers. eis is a noninvasive, portable diagnostic modality that has potential for clinical applications in multi-modal neuromonitoring and far-forward battlefield/ambulance arenas for diagnosing and monitoring acute and subacute brain injured patients. future development requires clinical validation, standardization, hardware and software optimization, and graphical user interface development. financial support: this work is supported by national institute of biomedical imaging and bioengineering point of care center for emerging neurotechnologies (poc-cent), subaward u eb - and by an "innovation gra hypertonic saline (hs) improves cerebral edema, blood flow, and is inexpensive. however, use of hs is complicated by reports of induced renal dysfunction and associations with increased blood-stream infection. we hypothesize hs alters renal perfusion leading to a state of relative renal insufficiency. with institutional review board approval, we retrospectively reviewed our hospital's use of hs since march of , and prospectively since october . comparisons were made between admission diagnoses, changes in creatinine (cr), and formulation of hs received ( % nacl, % nacl/sodium acetate mix, and . % nacl) to patients receiving normal saline or lactated ringers. intervariable associationswere calculated between using pearson's correlation coefficients. patients of the retrospective portion were identified. the data presented represents the first patients with data. there were significant differences in the apache ii scores and glasgow coma scale (gcs) scores between the different formulations of hs. the overall correlation of chlorine (cl -) and sodium (na + ) with creatinine (cr), and within each of the saline types, were not significant. when dichotomized by the diagnosis, significant correlations appear. traumatic brain injury (tbi) patients demonstrated moderate correlation between na + & cr of . . stroke patients demonstrated small correlations between na + & cr, and c l-& cr ( . for both). patients receiving hs outside the neurocritical care unit (nccu) demonstrated a small but significant correlation between cl and cr at . . patients receiving hs have lower gcs and higher apache ii scores. elevations of na + or cl in stroke, na + in tbi, and cl in non-nccu patients correlating with elevations in cr. as reductions in renal function predict mortality, therapies precipitating kidney injury are concerning. cl -, a potent renal vasoconstrictor, reduces renal blood flow. prospective comparisons of hs formulation and renal function are needed to further assess if formulation affects outcome and cost. first recognized after rapid initiation of nutrition in prisoners of war during world war ii, refeeding syndrome (rs) is the manifestation of fluid and electrolyte disturbances precipitation systemic dysfunction. here we report a case of rs in a patient with duchenne's muscular dystrophy (dmd). a case report and literature review. a -year-old male with past medical history of dmd, chronically ventilated and feed via a percutaneous endoscopic gastrostomytube, presented with pneumonia, sepsis, and status epilepticus. he was treated with broad spectrum antibiotics, early goal-directed therapy, and hours of electrographic seizures suppression with a midazolam infusion. admission labs demonstrated a minimally low albumin ( . g/dl), mild hypokalemia ( . mmol/l), and the presence of urinary ketones. enteral nutrition was started post-admission day (pad) one. pad found elevated serum glucose and precipitous drops in potassium, phosphate, calcium, and magnesium refractory to replacement. pad three attempts to wean the patient to his home ventilator setting failed, and he remained encephalopathic. enteral nutrition was changed to a more elemental, peptide-based formulation, and multivitamin with thiamine was added. electrolyte abnormalities persisted. pad , it was learned the family had reduced the patient's daily enteral nutrition by approximately half over six months to have him fit within his wheelchair. learning this, enteral feeds were reduced by half, advanced at a reduced rate reaching goal in days, and electrolyte abnormalities resolved commensurately. over the next three days, the patients mental status returned to baseline and ventilation improved. no cardiac or hemodynamic complications occurred, but his infections resolved slowly. a significant concern in the critically ill, the constellation of problems associated with refeeding syndrome have systemic implications. these are centered on increased cellular uptake of phosphorus following the reintroduction of carbohydrates. the role of dmd in refeeding syndrome is uncertain, and has not been previously reported. to determine hospital mortality and complication rates associated with surgical clipping and endovascular coiling of cerebral aneurysms in children, and to evaluate the trend of utilization of these procedures over the recent years in various us hospitals. from the kid's inpatient sample database for the years through , we identified a cohort of children admitted with the diagnoses of intracranial aneurysms and aneurysmal subarachnoid hemorrhage. hospital-associated complications and in-hospital mortality were compared among the clipping and coiling treatment groups. a multivariate logistic regression analysis was used to identify independent variables associated with hospital mortality. cochrane-armitage test was used to assess the trend of hospital utilization of these procedures in various hospital subtypes. after data cleansing, children were included in the analysis. two hundred ( %) children had aneurysm clipping and ( %) had endovascular coiling procedures. the coiled group was younger ( . ± . versus . ± . )and had even gender distribution. hospital mortality was higher in the clipped population, . % versus . % (adjusted odds ratio . ; % ci . , . ; p = . ). in addition, hydrocephalus, status epilepticus and pulmonary complications were higher in the clipped population (p < . ). lastly, the length of hospital stay as well as the hospital charges was higher in the clipped population (p < . ). the rate of hospitals' use of the endovascular coiling has increased in various types of hospitals over the years included in this study (p < . ). the trend in mortality rates among the clipped population remained higher ( . %- . %) compared to the coiled group ( - . %). endovascular coiling of cerebral aneurysms in children is associated with fewer deaths and complications, shorter hospital stay, and less hospital charges compared to clipping. the trend of hospitals' utilization of coiling procedures has increased during the recent years. understanding and managing complex physiologies is a critical, but difficult, problem in the neruologicial-icu. most of the information that must be assimilated in the icu exists at the level of raw data, individual test results and observations, and individual clinician notes. this mass of data obscures a holistic view of the patient, hides the development of trends, makes it difficult for clinicians to notice interactions between different variables. graphical displays and patient summaries enhanced or outperformed traditional text displays in numerous studies (elson & connelly, ; balas et al. ) , but this work hasn't yet been extended to support intracranial pressure (icp). the aim of this effort was to develop an interactive icp-specific data visualization using cognitive engineering principles. the visualization is designed to transform and consolidate complex multimodal physiological data into integrated interactive displays. we have developed a drill-down interactive visualization to enable clinicians to manage icp and identify blood pressure target goals that will ensure adequate cerebral perfusion and thereby create and maintain an optimal physiologic environment for the comatose injured brain to heal. using high-resolution physiologic monitoring data, this drill-down screen depicts the status of cerebral autoregulation using methods well described in the clinical literature (czosnyka, smielewski et al. ; jaeger, schuhmann et al. ) additionally, the drill-down provide graphical display of bloodpressure, intracranial pressure, and brain oxygen tension over time. with this interactive visualization, along with medication and lab data, the clinician can determine the target brain oxygen tension for a specific patient and whether to intervene on blood pressure, intracranial pressure or a combination of both in order to achieve a brain oxygenation goal (i.e., goal-directed therapy). the next step in this project is to conduct an experiment comparing this visualization against standard methods. nicom is a novel technique of monitoring hemodynamic status which is based on bioreactance technology. ventricular outflow causes changes in the phase of radiofrequency waves as they cross chest. measuring the phase shift enables calculation of flow. technique is entirely non-invasive. retrospective analysis of collected data. we describe the use of nicom in a tertiary care neuroscience intensive care unit. patients were monitored on the nicom from january until june for an average of days. diagnoses of patients monitored on nicom were: sah - , ischemic stroke - , ich , tbi - , sdh- , brain tumor- , spinal surgery- and others. % of patients were on mechanical ventilation, % were treated with pressors. in the first hrs of monitoring, there were plr (passive leg raising) tests and fluid challenges performed to measure fluid responsiveness. patients ( %) were fluid responsive and ( %) had an intervention. selected cases will be presented nicom system is safe and can be useful in the neuroicu setting. it can be used in intubated patients with sepsis, unexplained hypotension, hypertensive therapy in sah or during hypothermia therapy. it is also useful in non intubated, alert patients, were fluid status has to be monitored closely. although nicom is a seemingly simple-to-use technology, there were multiple clinical challenges including education of the staff, proper test performance and consistent charting. inconsistent machine calibration, use of compression stockings during a plr, and untimely sensors changes were the main problems. in the neuroicu patients with increased icp, use of fluid challenge can be safer than plr. repeated staff training resulted in more consistent data. limited information is available regarding the current state of informatics in various ncc units. we sought to assess the current state and needs for informatics infrastructure to help determine priorities and future directions of informatics research in neurocritical care. a survey instrument was developed and with the support of the neurocritical care research consortium chair, distributed to the participants/registrants of the nd neurocritical care research conference. a response rate of % ( of ) was achieved. most responders worked in an academic medical center ( . %), level trauma center ( . %) and/or mixed multi-bed (mean= . ) neuromedical/neurosurgical icu ( . %), commonly treating ich ( . %), sah ( . %), ischemic stroke ( . %), and traumatic brain injury ( . %). acquiring, integrating, storing and analyzing mm data in a comprehensive informatics architecture for clinical and research use is stated as important but is rarely achieved due to financial and technical barriers. a centralized dissemination of technical assistance and a societal statement prioritizing informatics to advance ncc research may help facilitate future adoption. access to neurocritical care units (nccus) in the mountain west is geographically limited. we evaluated practice patterns among providers in this region and hypothesized that hospital size and distance from nccus impact decisions to transfer patients with critical neurological illness. surveys were sent to hospital providers with varying degrees of access to nccus in the mountain west, to examine what factors influence decisions to transfer patients with critical neurological illness. the survey queried location, hospital size, locally represented specialties, patterns of transfer, frequency of illness presentation, influences for and against decisions to transfer such as timeframes and perceived futility, and awareness of nccus and services they provide. responses were received. responses were grouped by distance from the closest nccu and by hospital size. results showed that futility in outcome has a strong influence on decisions against transfer for smaller hospitals and hospitals that require air transport (p< . ). notably, distance required to transfer is not a strong factor in the decision to transfer patients (p= . ). for larger hospitals and hospitals within ground transport range of a nccu, patient condition, patient risk during transfer, and specialized intensivist support are less influential in transport decisions. patient transfer for critical neurological illness originates from hospitals with varying size and geographic access to nccus. while distance required to transfer does not appear to be a significant limitation, perceived futility in outcome is a strong influence against deciding to transfer. among providers in smaller hospitals at greater distance from nccus, significantly more providers have never heard of nccus or services provided. these findings suggest that therapeutic nihilism regarding critical neurological illness in smaller hospitals at greater distances from nccus influences patient outcomes. patients and providers in these locations may be significantly impacted by further education about neurocritical care and implementation of tele-neurocritical care services. neurocritical care is a multidisciplinary specialty whose participants originate from diverse medical backgrounds. review of the growing body of literature is essential for clinicians and strategies for continuing education may be expected to be unique for this field. this exploratory survey aims to define how the neurocritical care team (ncct) educates itself. a fifteen question survey was sent to all neurocritical care society members and responses were gathered over a one month period. basic statistical analyses of rates and comparisons of response rate proportions were conducted. surveys were returned ( %). % of respondents were physicians, % were non-physician team members, and % were physicians in training. regardless of background or training, individuals seek published literature through a combination of electronic-print media outlets ( %) rather than a singular approach. however, % spend the most time reading journal articles. % of ncct members review the same journals monthly and allocate individual manuscript time contingent upon interest. neurocritical care ( %), critical care medicine ( %), and new england journal of medicine ( %) are the most commonly reviewed journals. % of ncct members do not attend a journal club. academic neurointensivists ( %) and fellows ( %) are most and nurses are least ( %) likely to attend. participation in ncc subspecialty ( %) or general critical care ( %) clubs is more common than neurology ( %) or neurosurgery ( %). responders rate national meetings ( %) as their most influential educational experience. attending physicians ( %) are more likely than trainees and non-physicians ( %) to consider personal literature review most valuable (p < . ). % of all ncct members attended last year's ncs annual meeting, compared to sccm ( %) and regional conferences ( %). ncct members infrequently attended (< %) general topic neurological or neurosurgical national conferences. despite diverse backgrounds, ncct members seek continuing medical education through common subspecialty specific methods. financial support: none the contributions and perceptions of staff regarding nurse practitioners (nps) and physician assistants (pas) in neuroscience icus throughout the country are not well known. the objectives of this study were to determine the impact of neuroscience nps and pas and assess demographics of icus. all members of the neurocritical care society were asked to complete a survey to obtain their perception regarding the addition of nps and pas to the icu team. participants rated the abilities of nps and pas to promote a team environment, anticipate or prevent neurologic deterioration, address patient or staff concerns in a timely manner, safety, and communicate effectively on a - likert scale. in addition, members were asked to provide basic demographics and background information on the type and size of icu, type of providers in charge, and the role of nps and pas in their icu, including procedures performed, documents written, and number of patients per provider. both quantitative and qualitative data was collected and analyzed. a mantel-haenszel chi square and ordinal logistic regression model were used to determine the relationship between the background information and the perception of the abilities of nps and pas. the study cohort composed of % of ncs members. additional responsibility of nps and pas was associated with higher scores in safety, ability to promote a team environment, address patient or staff concerns, communication, and most importantly the ability to anticipate or prevent a neurologic deterioration (p< . for all). number of nps and pas, number of years of employment of nps and pas, number of procedures, and amount of documentation also positively affected safety. additional responsibility of nps and pas has strong potential to improve staff, patient, and family satisfaction, safety, and prevent neurologic deterioration. nps and pas should be utilized to the full extent of their role. we conducted a survey study in an academic, co-managed neuro icu to explore family satisfaction regarding the care of their surviving loved ones and compared results with concurrent data from the hospital's closed medical icu (micu). over days, we administered the family satisfaction-icu instrument to neuro icu and micu patients' families at time of icu discharge. those whose loved ones passed away during icu admission were excluded. the capture rates of families from the neuro icu and micu were . % ( surveys) and . % ( surveys in our neuro icu, patients' families could be more satisfied with several aspects of care. further study is needed to determine ( ) whether a closed neuro icu model improves family satisfaction and ( ) whether instituting a system in which the neurointensivist team regularly meets with all available families daily improves perceptions of shared decision making, even in routine situations. non-funded prospective patient registries at any given institution rely largely on volunteer clinical personnel. presupposing that an all-inclusive database would be self-defeating in this type of environment, we designed and implemented a quality improvement (qi) database with intentional iterative design. neurointensivists identified by consensus the injury/disease related events and procedures that were most important to track for qi and for judging clinical intensity of our unit. we compiled a list of syndromes that were either commonly studied by principal investigators or were common primary diagnoses in our unit. for each syndrome, we identified commonly accepted grading or intensity scores. the clinical and translational science awards electronic data entry module, redcap, facilitated data collection. consecutive patients in our icu were entered upon discharge. weekly meetings served to adjudicate disease classification, grading scores (frequently based on consensus imaging review), and discharge disposition. opportunities to enter free-text items were allowed to enhance the intentionally iterative design. in quarterly reviews, we removed items that were consistently left blank and added standardized items corresponding to consistently annotated free-text items. since its implementation in january , the neurocritical care qi patient registry has accrued separate entries. consensus-driven iterative changes to the registry have resulted in complete data entry. participation at weekly registry meetings has been consistent and enthusiastic, routinely drawing - physicians ( - fellows, - attendings). qi projects have been enabled to date. resource limitations may be a practical hindrance to achieving all-inclusive databases outside of funded clinical studies. an iterative design driven by consensus in the described approach can result in a rich database with complete data entry and continued volunteer participation. future incorporation of supplemental information sources via enterprise-wide clinical data warehouses may achieve more complete databases that comply with standardized ideals such as the common data elements. many neurology residency programs have begun implementing mandatory rotations through neurocritical care (ncc) as part of the curriculum. the added experience was thought to be beneficial for residents after graduating the program; however, we wondered how it might affect residents and patients during residency. we thought to survey residents about their programs and the amount of time they spend in ncc rotations. we also wanted to know how they felt the extra time spent in these rotations affected their consulting habits, and therefore their ability to manage cases on their own. all neurology residents in the united states were the target population for this survey. a list of neurology residency programs was obtained from the american medical academy's freida database. the names and email addresses of program directors were generated, and they were contacted by email with a link to an online survey. the respondents were neurology residents ( pgy- , pgy- and pgy- residents). of the respondents, . % stated that ncc was a mandatory rotation in their program while the remaining . % said that it was not. of those who had mandatory ncc rotations, . % said they were for - weeks duration, while . % agreed they should be - weeks long. when asked how often they ask for consultations from other specialties, residents who had mandatory rotations through ncc were more likely to say they usually do not consult other specialties, while those who did not have mandatory rotations were more likely to consult for all non-neurological issues. the survey results demonstrated that neurology residents who have mandatory rotations in ncc are more confident in their abilities to manage their own patients. this is thought to promote continuity of care and may reduce medical errors as well as healthcare cost. a botulism epidemic in a maximum-security prison cell-block posed numerous logistical dilemmas for which telemedicine served as a bridge to management. inmates in a high-security prison cell-block brewed batches of "pruno" by fermenting fruit, raw potato, and granulated sugar in reusable bags that were passed throughout the cell-block. one of the batches was contaminated with type a botulism. twenty-nine inmates were potentially exposed, but the actual exposures were initially indeterminable due to the inmates' fears of incrimination. the index case developed nausea, emesis, diplopia, and ptosis approximately six hours after exposure and presented to the emergency department (ed) two days later with generalized weakness, dysarthria, dysphagia, hypophonia, and dyspnea. he required intubation and was admitted to the neurocritical care unit (nccu). four additional inmates presented with similar symptoms within several hours of the index case. two required intubation and all were admitted to the nccu. within twenty-four hours of admitting the first five cases, nine additional inmates developed symptoms. five were evaluated in the ed; three were admitted to the nccu and two were discharged to the prison infirmary and monitored using telemedicine. two patients were initially evaluated and monitored with telemedicine at the prison. the remainder of the cell-block was evaluated by prison infirmary staff. botulinum toxin type a was confirmed with bioassay and cultures in these patients, but classic electrodiagnostic findings were absent. the eight inmates admitted were treated with hepavalent botulinum antitoxin (h-bat). obtaining the antitoxin required collaboration with the cdc for transport from several sites around the country. inmates were followed post-discharge using telemedicine and showed improvement. this botulism epidemic presented a logistical logjam. initial telemedicine evaluation and subsequent monitoring played a key role in managing nccu access and optimizing security resources for the prison, ed, and nccu. intrahospital transport of neurocritical care unit (nccu) patients is associated with accidental line removal, unplanned extubation, and hemodynamic instability. further, because patients must be accompanied by a nurse during intrahospital transport, there is an inherent reduction in home unit staffing which reduces direct patient care and monitoring for other nccu patients. the purpose of this project was to assess the impact of a neurocritical care transport nurse (ntrn) on patient safety, improved direct patient care time and improved staff satisfaction. the -month ntrn pilot program was initiated in our bed nccu. for three months, the ntrn worked five -hour shifts per week. the ntrn accompanied patients during intrahospital transports, assisted with admissions, functioned as resource nurse in the nccu, and relieved nurses for meal breaks. data was collected in real time and included time-inmotion data, adverse event records, and a pre-post work-flow surveys. the ntrn completed intrahospital transports with were zero safety events. the mean length of time for intrahospital transport prior to the pilot was significantly greater than transport by the ntrn ( vs. minutes; p<. ). the mean time it took nurses to stabilize a new admission/post-op patients was reduced from minutes to minutes. staff surveys were overwhelmingly positive with % of nurses reporting the ntrn saved them time; % reported increased opportunity for meal breaks, and % attributed reduced overtime due to the ntrn program. individual nurses reported that the ntrn program saved them an average of . minutes each shift ( . hours per shift). the ntrn pilot program was associated with fewer safety events, increased staff satisfaction, more rapid attention to patient needs and reduced overtime. the program should be implemented full time and evaluated for potential costsavings. many factors are associated with time delays to reperfusion in endovascular treatment for acute ischemic stroke (ais). we assessed if a prototypical neurointensive care unit layout where both the angio suite and ct scanner are inside the unit can reduce times to reperfusion. we compared time from ct to groin puncture (gp) in patients that were transferred from outside hospitals (osh) directly to the nicu versus those who went through our emergency department (ed). we retrospectively reviewed patients from a prospectively maintained database from october -june who underwent endovascular therapy for ais. a univariate analysis was performed to compare the patients' characteristics between the two populations and to identify differences in time intervals between ct imaging and gp. a total of patients were included in our analysis. ( %) patients were from osh. patient characteristics in both groups were similar except for osh patients had significantly less history of hypertension ( % vs %, p< . ) but had longer time intervals from last known normal to gp (median mins vs. median mins, p < . ) and lower pretreatment aspects ( % < vs. % < , p < . ). patients' transferred from osh had significantly lower times from inhouse ct to gp as compared to patients from the ed ( . +/- mins vs. +/- mins). although there was an increased number of non-contiguous multimodal imaging studies performed on ed patients compared to those from osh ( % vs %, p< . ), exclusion of these patients still resulted in a significant shorter time frame between ct to gp ( . +/- mins vs. . +/- . mins) among osh transfers. the design of an integrated biplane angio suite within the nicu reduces the times from ct imaging to gp, thereby lowering the times to reperfusion, and potentially, patient outcome. sepsis is a challenge for the intensive care unit (icu), being the main cause of death during hospitalization. it was performed a longitudinal and individualized intervention authorized by the hsja ethics committee applying the campaign 'simple actions save lives' in which educational adhesives worked as a guide for washing hands and flags for high contaminated locations. a decontamination routine of monitors, control panels, ventilators and infusion bombs was established every hours; and continued education for the health team was intensified during the intervention. two groups were created, patient enrollments in periods of days before and after the intervention, more than hours of hospitalization: group a with patients and group b with patients. the hospital infection incidence decreased by % and vap by . %. urine culture was positive in , % of those patients (n= ) in group a and in . % (n= ) in group b (a . % decrease ). the cultures of catheter tip were positive in . % (n= ) of catheters in group a, which used catheter in total, and none in group b, which used catheters. the sepsis incidence decreased by . %. septic shock was detected in . % (n= ) of patients in group a. there was a drop of the costs between groups (r , . , . %). the cost of campaign material was r$ . . this intervention was a simple form to decrease the related number of infections in the neurovascular icu, having spent irrelevant values when compared to treatment of these clinical tables. intracranial pressure (icp) management guidelines have been established; however there is no data documenting actual icp management practices in the united states, or the degree to which clinicians comply with existing guidelines. the primary aim of this study is to explore nursing and medical practice patterns associated with icp monitoring and management. a prospective multi-center non-randomized observational design was used.the study sample was composed of consented nurse/patient dyads, with dyads enrolled per study site. study patient subjects included were over age , had icp monitoring in situ, and were diagnosed with intracranial pathology. nurse subjects included were those assigned to the patient, who routinely worked in the unit, and had completed their orientation training. each dyad consented to a hour observational period, where data was collected on nurse interventions for icp management. dyads (n= ) were enrolled at hospitals between august and may . patients were primarily male %, mean age of years, and non-hispanic. nurses were primarily female %, non-hispanic, and a mean of . years of critical care experience. we observed distinct nursing/medical interventions hypothesized to reduce icp. although csf diversion and limiting stimulation were the most frequently used interventions, there was not a consistent hierarchical approach to initiating icp reduction interventions. wide variances in nursing and medical treatment patterns were observed for icp treatment threshold, first-line therapy, and the order in which interventions were initiated. despite established guidelines, variability exists throughout the u.s. in how physicians and nurses monitor and manage icp. more research is needed to compare intervention techniques to determine the impact these differences have on outcomes in patients requiring icp management. administrative data are being increasing being used to measure quality of care, for public reporting, and in pay for performance. administrative data are inexpensive, readily available, and target clinical outcomes. the aim of this study was to evaluate the use the use of administrative data in identifying potentially preventable events and iatrogenic complications in patients admitted to an academic medical center with a primary diagnosis of acute stroke. administrative data for all adults patients (> years of age) with a discharge diagnosis of stroke , . , . , . , . , . , . , . , . , . , . , . , and ) were evaluated from january -december for complications based on administrative data by looking at secondary diagnoses that were not present on admission using data from the university healthsystem consortium database. both the agency for healthcare research and quality (ahrq) quality indicators (qis) for inpatient conditions and known codes for other complications such as catheter associated urinary tract infection, pneumonia, and deep vein thrombosis or pulmonary embolus (not associated with surgery) were used to identify potential events. of the cases reviewed, many cases had at least one complication. the leading cause of potentially preventable events were related-to-infection (central line associated bloodstream infection, sepsis, catheter associated urinary tract infection, and aspiration and/or hospital-acquired pneumonia). the ahrq qi only captured a fraction of the events. patients with subarachnoid hemorrhage had the highest mortality, followed by intracranial hemorrhage. several of the deaths occurred in low risk patients and had at least one potentially preventable complication. when reviewing flagged records, a small number of events reflected opportunities to improve documentation and/or coding, with the majority of remaining events associated with opportunities for improvement. administrative data may be a useful adjunct to quality improvement efforts. financial support: co-deputy project lead for the ahrq qi project (ahrq sub-contractor). continuous video-eeg (cveeg) monitoring is often utilized in the evaluation of impaired consciousness. nonconvulsive seizures may be distinguished from metabolic disorders when triphasic waves (tw) are recorded. however, rhythmicity detected on cveeg may call into question the presence of electrographic seizures. the following case describes the transient resolution of rhythmic tws after acute administration of glucose in a patient with hypoglycemic encephalopathy. case report reviewing clinical, laboratory and electroencephalographic features of a patient with metabolic encephalopathy secondary to hypoglycemia. a -year-old woman with type- -diabetes and treated osteomyelitis of the foot presented with altered mental status. she was intubated and stuporous. cranial nerves were intact. all four extremities withdrew to noxious stimulation. plantar responses were flexor. mri brain exhibited leptomeningeal enhancement consistent with meningitis. serum bg= mg/d, and csf glucose= mg/dl. month after antibiotic treatment, she was following commands. repeat mri revealed complete resolution of leptomeningeal enhancement. during recovery, she developed sudden onset stupor with left facial movements, and underwent cveeg monitoring. eeg showed generalized, polymorphic delta/theta slowing intermixed with tws, without electrographic correlate of facial movements. during cveeg, tw activity increased in rhythmicity and frequency, coinciding with worsening hypoglycemia, with a nadir bg= mg/dl. electrographic activity was not induced or exacerbated by stimulation. administration of ml of d ( g d-glucose) resulted in transient resolution of tws within minutes, which corresponded to a bg= mg/dl. however, background slowing remained on cveeg, with gradual reemergence of infrequently occurring tws despite normoglycemia. mental status returned to baseline approximately hours after bg stabilization. rhythmic triphasic wave activity due to hypoglycemia may be distinguished from electrographic seizures after acute correction of bg, with corresponding transient resolution of triphasic waves. however, clinical response to correction of metabolic dysfunction may be delayed for up to hours. continuous-iv-midazolam (civ-mdz) is recommended for treatment of refractory status epilepticus (rse) but doses are controversial. here we compare a historical cohort (n= ) treated with low dose to a subsequent cohort of patients treated with high dose civ-mdz for rse. following the analysis of the historical cohort ( - neurology , ( ) : - ) we changed our protocol for rse allowing for higher civ-mdz doses and collected consecutive cases ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) . exclusion criteria: cardiac arrest; prior treatment with a different civ-aed. we collected data on baseline characteristics, civ-mdz doses, seizure control, complications, hospital course, and outcome. high dose was compared to low dose civ-mdz on an intention to treat basis using logistic regression analysis with the significance level set at p< . . baseline characteristics were similar between groups. median maximum civ-mdz dose was . mg/kg/h (iqr . , ) for the high and . mg/kg/h (iqr . , . ) for the low dose group (p< . ), but duration of civ-mdz was the same between the two groups (median hours). "withdrawal seizures" (within hours after civ-mdz discontinuation) were less frequent in the high dose group ( % vs %, or . ; %-ci . - . ). "breakthrough seizures", "ultimate civ-mdz failure", and complications were not different. discharge mortality was lower in the high dose group ( % vs %, or . , %-ci . - . ) after controlling for age, etiology, and apache- scores. at months, mortality was similar between the two groups. lower death or vegetative state rate for those treated with high dose civ-mdz was seen at months ( % vs %; or . ; %-ci . - . ), but this finding is limited by missing -month functional outcome data in both groups. high dose civ-mdz treatment for rse can be performed safely in an icu setting and may be more efficacious in controlling seizure activity. outcome data are promising and warrant further prospective study. the clinical utility of free valproic acid (vpa) levels is unclear, and the actual free fraction (ff) of vpa in hospitalized patients is not well established. our goal was to assess and compare the total levels, free levels, and ff of vpa in inpatients and outpatients and to determine factors that may influence the ff. retrospective chart review of paired total and free vpa levels in inpatients and outpatients. demographical, laboratory, and concomitant interacting medication data were collected and analyzed. paired total and free levels were categorized based on their status in regards to the therapeutic range (i.e., subtherapeutic, therapeutic, or supratherapeutic) and whether the paired levels were concordant or discordant (e.g., both levels in therapeutic range, or mismatched). linear regression was used to assess the impact of variables on the ff. logistic regression was used to determine if variables predicted the likelihood of having discordant paired levels. inpatients had a significantly higher median ff compared to outpatients ( . % vs. . % respectively; p < . ). total levels were found to be a poor predictor of free levels (r = . ) in hospitalized patients. inpatient free levels were discordant with the therapeutic status indicated by the total level % of the time. in a linear regression model, albumin (p < . ), total protein (p < . ) and co-administration of phenytoin (p= . ) and carbapenems (p= . ) were found to significantly and independently impact the ff. multiple logistic regression indicated albumin as a significant predictor of the total and free levels being discordant in regards to therapeutic status (or . [ %ci . - . ], p< . ). inpatients had a significantly higher ff compared to outpatients. inpatient free levels were frequently discordant with the total levels in terms of the therapeutic status. decreasing albumin was a significant predictor of discordance between the free and total levels. increased continuous eeg utilization in the icu has generated an interest in faster acquisition and interpretation of eeg data. limited electrode arrays (lea) coupled with quantitative algorithms have been leveraged for this purpose. however, previous studies with lea's have suggested an inherent error rate produced by a reduced number of electrodes. the aim of the current project was to test a novel lea and determine if multiple montages could correct any error rate. with approval from our irb, short de-identified eeg segments were retrospectively collected from clinical ceeg archives. segments contained one of five primary findings: normal, diffuse slowing, periodic epileptiform discharges (peds), seizure and burst suppression. all files were reformatted into an electrode array containing a lateral chain and central electrode bilaterally. segments were distributed to four experienced neurophysiologists in two phases. in phase , segments were interpreted in a single anterior-posterior bipolar montage and compared to the original read. in phase , fifty frequently misread segments from phase were reinterpreted using four additional montages. in phase , eeg interpretations were reviewed yielding a sensitivity of % for seizure and % for peds, burst suppression, and normal. the specificity was greater than % in all cases. the sensitivity and specificity for diffuse slowing was % and %. in phase , eeg interpretations were collected with no significant improvement noted in the detection of any eeg finding. in agreement with past studies, this trial suggests that leas contain a base error rate engendered by the reduced number of electrodes. this error rate is maintained regardless of the number of available montages. the implication of these findings suggests that studies examining the use of lea's for use in seizure detection and neurophysiologic algorithms should calculate an error rate specific to the electrode array before algorithm testing. the incidence of nonconvulsive status epilepticus (ncse) and other electrographic features in comatose post-cardiac arrest syndrome patients treated with therapeutic hypothermia (th) is still under investigation. the objective of this study is to determine the incidence of ncse and other electrographic features and correlate with neurologic outcome and survival. review of consecutive subjects treated with th and receiving continuous eeg (ceeg) monitoring between may and december . demographic data, survival, and functional outcomes using cerebral performance category (cpc) scale were prospectively recorded. forty eight patients were included, with mean age of years (sd ), majority were males (n= , %) and experienced out-of-hospital cardiac arrest (n= , %). ventricular fibrillation was the initial cardiac rhythm in patients ( %). all patients received th. twenty seven patients ( %) died. seventeen patients ( . %) had good neurologic outcome (cpc or ). ncse occurred in patients ( . %), both of whom died. periodic epileptiform discharges occurred in patients ( . %), ( %) of whom had poor neurologic outcome or death (cpc - ) compared to % poor outcome in whom periodic epileptiform discharges did not occur (nonsignificant). burst suppression occurred in patients ( . %), all ( %) of whom had poor neurologic outcome or death compared to . % poor outcome in whom burst suppression did not occur (p < . ); and severe background attenuation occurred in patients ( %), ( %) of whom had poor neurologic outcome or death compared to % poor outcome in whom severe background attenuation did not occur (p< . ). ncse occurred in . % of post-cardiac arrest patients undergoing therapeutic hypothermia. outcomes are poor in postcardiac arrest patients undergoing therapeutic hypothermia with ncse, burst suppression or severe background attenuation. larger prospective studies are needed to further evaluate and characterize ceeg findings in comatose postcardiac arrest patients undergoing th. encephalopathy is a frequent occurrence in the critical care setting. previously, we have shown that patients with a primary neurologic injury and encephalopathy are at high risk for ceeg seizures. patients with a presumed metabolic etiology of encephalopathy have been poorly characterized. the purpose of this study was to identify the frequency and underlying etiology of ceeg seizures that occur in critically-ill patients with a presumed metabolic etiology. we retrospectively reviewed prospectively collected ceeg and clinical data on consecutive patients monitored from january , , to december , . we identified those patients with ceeg seizures (n= ) and included in this study only those patients with metabolic etiologies. eeg seizures were defined as evolving rhythms in frequency, distribution, and/or morphology at hz or greater for more than seconds duration. statistical analyses were performed with jmp . . sixty-six ( . %) patients were identified as having metabolic causes for ceeg seizures with the most common etiology being sepsis ( . %) which linearly increased (r = . ) in detection from in to in . other etiologies included liver failure ( . %), posterior reversible encephalopathy syndrome (pres; . %), electrolyte/glucose derangement ( . %), drug overdose/withdrawal ( . %), and renal failure ( . %). . % of the ceeg seizures were without clinical signs. a linear increase in ceeg seizures occurred with a decrease in level of consciousness (r = . ). the majority ( . %) of patients were eventually discharged for rehabilitation, but . % expired prior to discharge. this retrospective study shows an increase of ceeg detected seizures in patients with a presumed metabolic etiology from to . this increase in seizures is likely due to increased targeted monitoring. this highlights the value of using ceeg database information to target at risk populations. our results should guide the use of ceeg monitoring in the metabolic patient particularly those with septic encephalopathy. medically induced burst suppression on eeg is often seen in critically ill patients who are sedated for treatment of status epilepticus, cerebral edema, and in patients with anoxic brain injury or post cardiac arrest undergoing hypothermia treatment. previous studies have demonstrated that the majority of these patients have poor prognosis. we decided to investigate if specific eeg patterns during burst suppression in these patients would correlate with different outcomes. we retrospectively identified patients with medically induced burst suppression out of patients who had continuous eeg monitoring (ceeg) from january through december in our neuro icu. neonates and children were excluded. all eeg tracings were independently reviewed by two electroencephalographers and classified into discrete seizures, status epilepticus (se), interictal epileptiform discharges (ied), burst suppression, and epileptic bursts defined as burst suppression with ied within the burst activity. primary outcome was cerebral performance categories (cpc) at hospital discharge. of the patients, were identified to have epileptic bursts-one se, eight anoxic brain injury, two ischemic stroke, hemorrhagic stroke, five other medical conditions. the mortality rate of patients with epileptic bursts was % compared to % for those without. only % in each group had good neurologic recovery defined as cpc score of - . patients with epileptic bursts on average had longer duration of monitoring ( days versus ) due to refractory seizures and, subsequently, increased number of aeds ( . vs . ) used. similar to previous findings, the patients in our study had poor prognosis. our findings additionally show that epileptic bursts in this patient population correlated with more refractory seizures and a higher mortality rate. the presence of epileptic bursts may be used as an adjunctive indicator for prognosis in patients who are in medically induced burst suppression. larger population study is underway. epidemiologic studies in epilepsy using large administrative databases depend on accurate icd- -cm classification. we sought to determine the accuracy of icd- -cm code . (grand-mal status) for diagnosing status epilepticus (se) after hospital admission. a case-control study at an academic institution was conducted. twenty-one subjects with discharge icd- -cm code . (grand-mal status) and consecutive admissions without the code of interest were randomly selected. se was defined as neurologist documentation of continuous clinical seizure activity for five minutes or longer and/or two or more discrete clinical seizures without inter-ictal return to baseline (clinical diagnosis) and/or eeg consistent with se by board certified neurophysiologist interpretation (eeg diagnosis). all cases and none of the controls met our pre-defined criteria of se. therefore, the sensitivity, specificity, positive predictive value (ppv), negative predictive value, and accuracy of the code was %. when the diagnosis relied on clinical criteria alone, the sensitivity decreased to % with ppv of %. when the diagnosis was made by eeg criteria alone the sensitivity decreased to % and ppv of %. the icd- -cm code . is both accurate and specific for the diagnosis of se after admission at an academic institution. clinical definitions of se and the prevalence of the disease may affect the sensitivity and ppv of icd- -cm code . for the diagnosis of se. the results of our study require further validation in other cohorts. refractory status epilepticus (se) has been linked to significant morbidity and mortality. when pharmacological treatment fails, ketogenic diet has shown to suppress seizure activity in children and is gaining acceptance as an adjunctive treatment in adults. while case reports exist, there are no standard guidelines for implementing ketogenic diet in adult neurocritical care patients. the purpose of this abstract is to demonstrate a standard guideline for ketogenic diet utilization in a neurocritical care unit. a performance improvement project was undertaken to standardize ketogenic diet administration in enterally fed neurocritical care patients with medically refractory se. the guidelines include patient selection, team communication, patient monitoring, family education, patient transitions out of intensive care and measures for patient outcome from this treatment. patients admitted with se are initiated with standard pharmacologic treatment; if treatment does not result in se cessation, then ketogenic diet is reviewed. discussion with the patient's family is required to review long-term implications and potential lifestyle choices related to diet after critical illness. a standard checklist within the guidelines assures communication to all necessary organizational departments including appropriate consults. daily monitoring and discussion in patient rounds evaluates daily patient progress. team communication is focused on diet tolerance, medication carbohydrate content, concurrent pharmacologic se management and patient progression. since , after implementation of the standardized guideline, seven adult se patients have been treated with ketogenic diet. mean age was . years; range - years; two patients were male. ketosis was achieved in six of seven patients and five of six patients sustained resolution of se after ketosis was achieved. our organizational experience indicates that coordinated team care, family education, goal planning and a standardized guideline contribute to successful implementation of ketogenic diet. further research is needed to determine overall effectiveness of this therapy. status epilepticus (se) is a potentially life-threatening condition that is frequently under-recognized, may be refractory to initial treatments, and often requires admission to general intensive care units (icus) we hypothesized that admission of patients with se to the neurosciences icu (nicu) vs the medical icu (micu) might correlate with surrogates for improved patient outcome. we performed a single-center, retrospective cohort study of patients with se admitted to the nicu vs the micu in our institution between - . admission to either icu depended on bed availability and emergency medicine preference. clustering methods were used for analyses, taking into account multiple visits of the same patient. there were visits for patients with definite or probable se [ ( %) in the nicu and ( %) in the micu]. apache ii scores were significant higher in the micu group ( . vs . , p= . ). more continuous eegs were ordered in the nicu ( % vs %, p< . ). ceeg was ordered more frequently in complex partial/non-convulsive and less in convulsive clinical presentations. the nicu had a higher rate of complex partial/non-convulsive se and the micu of generalized convulsive se ( % vs % and % vs %, p< . ). admission diagnoses differed, with the nicu having a higher rate of stroke and the micu a higher rate of toxometabolic etiologies ( % vs % and % vs %, p< . ). after adjusting for covariates, no difference was found in the icu or hospital length-of-stay and modified rankin scale at discharge. management differences occurred in micu vs. nicu-managed se, possibly based on variabilities in presentation and etiology. however, no reduction in length-of-stay or different discharge outcomes between the icus was found. hongki song , taechon kang , dongjin shin although levetiracetam(lev, s-(oxo- -pyrrolidinyl)butanamide, keppra®, ucb pharma) has been reported to be well tolerated and effective in se refractory to benzodiazepine (bdz), there was little preclinical or clinical data concerning the outcomes of lev in comparison to dzp, and vpa in se-induced neuronal death. to address this relevant lack of information, we have performed the preclinical study to investigate the effect of diazepam (dzp), valproate (vpa), and lev alone, and the efficacy of lev as an add-on treatment with dzp on the se-induced neuronal death. dzp and vpa. however, it is noticeable that lev as an add-on drug with dzp could not alleviatese-induced neuronal damage as compared to effective to protect neuronal damages from se, as compared to dzp. in contrast to lev, vpa( and mg/kg) as an add-on drug with dzp significantly reduced se-induced neuronal damage as compared to dzp alone, and showed the similar effect of vpa ( mg/kg) alone. these findings indicate that, unlike vpa, lev may negatively interact with dzp, and suggest that lev may be more effective to prevent se-induced neuronal death as a first line drug than as a second line therapy after bdz treatment, and that lev as an add-on drug with bdz may not provide any additional benefit to outcome of se. temkin and colleagues found that phenytoin exerted a beneficial effect by decreasing the rate of seizures by % during the first week after a traumatic brain injury. the purpose of this study was to determine the need for monitoring and titrating to therapeutic free phenytoin levels in patients receiving phenytoin for prophylaxis within days following a traumatic brain injury. this was a retrospective study of patients for a traumatic brain injury (tbi), who met the inclusion criteria and received phenytoin for seizure prophylaxis for days following injury. eligible patients were divided to two arms: patients with phenytoin levels (n= ) and patients without levels (n= ). the primary outcome measure was the incidence of seizures in those that were monitored for free phenytoin levels and those that were not monitored for free phenytoin levels. the secondary outcome measure was the appropriateness of phenytoin dosing in regards to initial loading and maintenance dose. a total of seizures occurred in the entire study population. both seizures transpired in patients with phenytoin levels. patient was diagnosed with a seizure event on day , with free phenytoin obtained on day at a therapeutic level of . mg/l. patient had a witnessed seizure on day , with free phenytoin level obtained on day also within therapeutic range at . mg/l. there was no incidence of seizure in patients who were not monitored for phenytoin levels. inconsistent phenytoin loading and maintenance doses were identified. this study suggests that monitoring phenytoin to therapeutic levels for seizure prophylaxis did not demonstrate a decrease in the occurrence of seizures. we are unable to make recommendations given the inherent limitations of our study. a large prospective, randomized trial is needed to clarify the need for monitoring phenytoin to therapeutic levels. seizure prophylaxis for nontraumatic intracerebral hemorrhage (ich) and aneurysmal subarachnoid hemorrhage (sah) is common practice in the intensive care unit(icu). typical antiepileptics include phenytoin (ptn) and levetiracetam (lvt). previou studies have suggested worse long term outcomes with icu ptn use, but such data is lacking for lvt. in addtion, few studies have compared lvt to ptn for seizure prophylaxis in ich or sah patient in the icu setting. we hypothesize that seizure prophylaxis with lvt, as compared to ptn, for patients admitted with ich and sah will result in similar outcomes at hospital discharge as measured by the modified rankin scale (mrs). this study is a single center retrospective review from - , to ultimately include approximately adult patients with the diagnosis of sah or ich who received seizure prophylaxis with either lvt or ptn. basic demographic, past medical history, severity of illness scales; length of mechanical, icu and hosital length of stay; seizure occurrence, use of continuous electroencephalogram, data will be collected, in addition to other variables. patients with prior seizure history or seizure on presentation, do-not-resuscitate hours within hours of icu admission, will be excluded. to date, our analysis includes patients (lvt = and ptn = ). comparing ptn to ltr, univariate analysis of demographics, baseline clinical characteristics and outcomes were similar between the two groups (all p> . ). in our initial univariate analysis, functional outcome at discharge was similar between ptn and lvt when used for seizure prophylaxis in patients admitted with ich or sah. subsequent analysis will include additional patients (approximately ) with multivariate adjustment. cerebral microbleeds (cmbs) are commonly found in patients with microvascular pathology such as primary intracerebral hemorrhage, cerebral amyloid angiopathy, and ischemic stroke. however, to our knowledge, there have been no reports of cmbs or their acute appearance in patients with status epilepticus (se). here we describe two patients admitted to our neuro-intensive care unit with generalized tonic-clonic seizures. laboratory tests were unremarkable except for mild pleoc onset and did not showed abnormal findings. seizures continued despite multiple anti-epileptic drugs including phenytoin, valproic acid, topiramate, clonazepam, pregabalin, lacosamide, phenobarbital, levetiracetam, and continuous infusion of propofol, ketamine and midazolam (up to . mg/kg/hr in the first patient and . mg/kg/hr in the second patient). followup . -tesla susceptibility-weighted imaging revealed new cmbs ( lobar [ frontal, parietal, temporal, occipital, and insular], deep [ corpus callosum and deep/periventricular white matter], and infratentorial [ brainstem and cerebellum]) in the first patient (performed days after initial imaging) and new cmbs ( lobar [ frontal, parietal, temporal, and occipital], and deep [ corpus callosum and deep/periventricular white matter]) in the second patient (performed days after initial imaging). multimodal neuromonitoring was available between initial and follow-up imaging in the second patient and suggested metabolic distress (lactate-pyruvate ratio > ), cerebrovascular dysautoregulation (pressure reactivity index > . ), brain tissue hypoxia (brain tissue oxygen partial pressure < mmhg), and fluctuations of blood pressure (variance, mmhg) and cerebral perfusion pressure (variance, mmhg). cmbs may develop acutely in patients with refractory se, which may point towards microvascular disturbances in refractory seizures. further prospective studies are necessary to explore the pathophysiology and clinical implications of new cmbs in se. synthetic cannabanoids, often sold as "spice" and various other labels, are a popular product sold in incense shops and through the internet. when inhaled, consumers often report experiences similar to marijuana use, and have thus become a popular street substitute for marijuana. unfortunately, with increasing use, there has been an increase in the number of patients presenting to emergency departments due to toxic effects of these products. we describe a year old gentleman with history of bipolar disorder but no history of neurological disease who presented to the emergency department with altered mental status and tachycardia who subsequently had a witnessed tonic-clonic seizure. patient received appropriate workup for his potential toxicity. we also performed a literature search on "spice" incense found in his backpack on presentation. patient had admitted to smoking "spice" incense on questioning. patient's negative drug screen, negative workup, as well as symptomatic improvement on phenytoin supported the source of his seizure as the toxic effect of inhaled "spice". we also on literature review discovered several other cases similar to this patient's case. "spice" or synthetic cannabanoid-induced toxicity is an emerging etiology of new-onset seizure and does not appear on conventional drug screens. critical care professionals should be aware of this product to recognize and appropriately treat this toxicity. refractory status epilepticus (rse) is associated with high morbidity and mortality. etiological heterogeneity and refractoriness to treatment remain a challenge for the treating intensivist. here we present a patient with rse and folic acid (fa) deficiency. brain metabolism was hourly analyzed using cerebral microdialysis (cma -analyzer; cma -catheter). fa concentrations of brain extracellular microdialysate (famd-ec) and serum (faserum) were analyzed using elecsysfolateiii® -assay. in vitro recoveryof fa was calculated using cerebrospinal fluid (csf). a -year-old male was referred to our neurocritical care unit with se refractory to levetiracetam ( g/d) valproic-acid ( . g/d) and , mg/kg bw/h midazolam continuous infusion. the patient had a history of short bowel syndrome (sbs) after small intestine resection five months prior. admission electroencephalography showed continuous rhythmic epileptiform activity over the right hemisphere despite adding ketamine continuous infusion ( , mg/kg bw/h) and lacosamide ( mg/d). neuroimaging demonstrated diffusion-weighted-imaging (dwi)-hyperintensities over the right hemisphere. csf was normal, common causes of rse were unlikely after extensive laboratory and csf studies. fa serum was found to be lower ( . μg/l; . - . μg/l) at day two of rse. after thiopental anesthesia ( hours) and parenteral fa substitution ( mg/d), the patient was successfully weaned without electrographic or clinical seizures. repeated imaging of the brain at day showed improvement of dwi-hyperintensities. glutamate levels in md ec decreased overtime. the patient could be extubated and fully recovered to the functional level before rse. fa serum increased by % to . μg/l, post hoc analysis of fa md-ec revealed an increase by % (from . μg/l to . μg/l). in vitro recovery of fa was %, therefore calculated fa brain / fa serum ratio was initially , which is comparable to previous animal studies. brain extracellular folic acid can be measured using cerebral microdialysis. although causality cannot be proven, fadeficiency may have influenced the course of rse in our patient. the management of inter-ictal eeg patterns such as sirpids (stimulus-induced rhythmic, periodic or ictal discharges) in comatose intensive care unit (icu) patients remains poorly understood whether these are secondarily injurious to brain or simply a of marker of underlying brain injury. we describe cases of brain-injured patients with sirpids with ictal spect imaging and in regards to aggressive neuroicu management and patient outcomes. case series, n= . case # -a -year old female suffered a cardiac arrest and remained comatose after days. continuous icu eeg demonstrated nonconvulsive seizures (ncsz) and status (ncse) with up to hz maximal bilateral centroparietal head spike and wave by day # which was refractory to initial iv levetiracetam, iv lacosamide, iv phenytoin but finally responded to iv phenobarbital load ( mg/kg) and propofol infusion. sirpids were noted despite these medications with any form of tactile or auditory stimulation. we performed ictal (stimulation provoked sirpids) and interictal technetium- -spect which was negative for hyperintense focus. case # -a year old female was admitted comatose for subarachnoid hemorrhage secondary to aneurysm rupture. she received a left-sided hemicraniectomy with operative clipping of the aneurysm and drainage of a small left subdural hematoma. on postoperative day (pod) # , ceeg showed left frontotemporal sharp waves. she was placed on leviteracetam, lacosamide, benzodiazepine, propofol infusion, and phenytion. by pod # , ceeg revealed left frontal sharply countoured discharges when the patient was stimulated by nail bed pressure on examination, consistent with sirpids. by pod # an ictal spect scan showed broad areas of hypoperfusion in the left hemisphere due to infarcts but there were no findings suggestive of a seizure focus scintigraphically. spect-scan negative sirpids may be helpful in terms of deescalating aggressive brain-metabolic suppressive therapies such as propofol and barbiturates, but larger, outcome-based studies are needed. thromboelastography (teg) is point-of-care test that allows for rapid global assessment of coagulation. teg analyzes whole blood, not plasma, which better accounts for the effects of cellular components on hemostasis. we sought to determine whether there is evidence of hypercoagulability by teg and whether it correlates with discharge outcome after aneurysmal subarachnoid hemorrhage. ten patients with moderate-to-severe sah were prospectively enrolled in an irb-approved observational study of serial thromboelastography. teg analysis, using kaolin activated citrated samples, was performed on post-bleed days , , , and . thrombus velocity curves, including the maximal rate of thrombin generation (mrtg), time to maximal rate of thrombin generation (tmrtg), and total thrombin generation (ttg), were plotted for each patient. a hypercoagulable state was defined a priori as a g value of > dynes/cm or a maximum amplitude (ma) of greater than mm. secondary outcome measures included discharge disposition. mean age of patients was . +/- . years. / patients were women and / were discharged home. the mean g parameter was within the normal range ( . dynes/cm ) on day , demonstrated a hypercoagulable profile on day ( . dynes/cm ), peaked on day ( . dynes/cm ), remained hypercoagulable on days ( . dynes/cm ) and day ( . ). the day g value was significantly different from the day value (p= . thromboelastography may identify a transient hypercoagulable state that peaks around post-bleed day in patients with sah. this state reflects accelerated thrombin generation and correlates with discharge disposition. defining a hypercoagulable state in patients with sah may lead to better risk stratification and novel therapeutic interventions. financial support: this study is supported in kind by haemonetics. they supply teg machines, kits and reagents. they have neither participated in study design nor are they aware of these preliminary results. intravenous sedation has been associated with impaired cognitive recovery following critical illness but its influence on recovery following asah remains unknown. data from consecutive patients with asah admitted to columbia-presbyterian hospital and enrolled into the shop database between / - / were analyzed after exclusion of deaths and unemployment prior to hemorrhage. employment status at year was obtained through self report or through patient surrogate and trichotomized (same level, decreased level, unemployed). proportional odds models were used to test the association between the use of continuous intravenous sedation with employment and cognitive function at year after controlling for baseline demographics (age, race, occupational level, admission hunt hess grade) and hospital complications (pneumonia, infarction from vasospasm). proportional hazards model was used to examine the association of sedation with time to return to work. patients who had the primary outcome data of employment status at year were analyzed. in multivariate analysis, exposure to continuous intravenous sedation was significantly associated with worse employment status at one year (or= . , ci= . - . , p= . ). poor judgment (or= . , ci= . ,- . , p= . ) and apathy (or= . , ci= . - . , p< . ) at one year were significantly associated with worse employment status but not with sedation exposure. with multivariate proportional hazards model, sedation was a significant risk factor of unemployment (hr= . , ci= . - . , p= . ). among those who returned to work within year, patients who received intravenous sedation returned to work significantly later than those who did not (median vs. days, p= . ). patients who received continuous intravenous sedation following asah had worse one year employment status and returned to work later. although poor judgment and apathy was associated with worse employment status, they were not associated with sedation exposure. future studies should investigate the effects of intravenous sedation exposure on cognitive and functional recovery following brain injury. despite an improvement in mortality, many survivors of asah still have significant disability and impairment in quality of life. we investigated predictors of unemployment at year among survivors of asah. data from consecutive patients with asah admitted to columbia-presbyterian hospital enrolled into the shop database between / - / were analyzed after exclusion of deaths and unemployment prior to hemorrhage. employment status at year was obtained through self-report or through patient surrogate and trichotomized (same level, decreased level, unemployed).pre-morbid occupational level was trichotomized (full time, part time, housewife). proportional odds models were used to test the association between baseline demographics, pre-morbid and discharge functional status with employment status at one year. proportional hazards model was used to test the association of these factors with time to return to work. a total of patients had the primary outcome data of employment status at year. patients ( %) remained unemployed, patients ( %) worked at a decreased level, while patients ( %) were employed at the same level. after controlling for age, modified fisher scale, and discharge functional status, ethnicity (p= . ) and pre-morbid occupational level (p< . ) were significantly related to employment status. hispanics (or= . , ci= . - . ) were less likely to be employed than other minority groups with caucasian as the reference group. caucasians working full time pre-morbidly provided the greatest odds for employment (or= . , ci= . - . ) over part time employees (or= . , ci= - . ) and housewives (reference) among those who returned to work at year follow-up, patients who were employed at the same level returned to work sooner that those employed at a decreased level (median: vs. days, p= . ). unemployment among survivors of asah remains problematic, especially among certain underrepresented minorities. future studies should investigate modifiable factors which impede successful reintegration to the work force. cerebral vasospasm after aneurysmal subarachnoid hemorrhage (sah) remains a major cause of death and disability. delayed cerebral ischemia (dci) after sah is likely multi-factorial, but eventually leads to altered cerebral blood flow (cbf) and cerebral infarction. neurointerventional treatment is used for medically refractory vasospasm, but with limited data on efficacy and impact on cbf and clinical/dci outcomes. patients with sah scheduled for neurointerventional treatment of refractory vasosasm were consented for intraprocedural cbf monitoring. we measured regional cbf using two sodium iodide scintillation scalp detectors approximating the cortical vascular territory of the treated vessel. a . ml saline bolus of - mci of -xe is injected through the coaxial catheter immediately before and after endovascular treatment. tracer washout is recorded under stable physiologic conditions for . minutes. cbf is calculated using the initial slope index, the monoexponential slope of tracer washout from - seconds after isotope injection. data were analyzed including standard corrections for remaining activity and physiologic parameters (cortexplorer cbf a, ceretronix, denmark). mean arterial blood pressure, paco , serum hemoglobin, and delivery of anesthetic agents were monitored. we calculated change in cbf expressed as a mean + standard deviation using repeated measures anova before and after endovascular treatment. a total of sah patients with refractory vasospasm were enrolled in the study. moderate to severe angiographic spasm was reported in % of subjects. treatment included ia verapamil in ( %), angioplasty only in ( %), and both in ( %). mean change in cbf was + ml/ gm/min, an average of % change in regional cbf. in our prospective study of patients with endovascular treatment for refractory vasospasm, we detected a mean change of % in quantitative cbf using the intra-arterial -xe washout method. without significant radiographic evidence of large vessel change at the time of measurement, increases in cbf may be related to the microcirculatory effects of treatment. early detection of cerebral vasospasm (vs), a common complication of subarachnoid hemorrhage (sah) enables prompt initiation of treatment. screening and detection of vs is done by repeated neurological examinations and transcranial doppler (tcd) monitoring, while angiograms are used for definitive diagnosis. this study aims to test the ability of a novel nirs based cerebral-oximetry method to detect vs in the post sah period. -hess score of - were enrolled. patients underwent neurological examinations, tcds and had - minute nirs monitoring sessions daily. whenever vs was suspected, angiography was performed. clinical event was defined as the combined endpoint of angiographically proven vasospasm, flow velocity > m/s over mca or aca territories, or neurologic deficit manifested rformed using the cerox , utilizing ultrasound tagged light (utl). pathologic cerebral oximetry was defined as having cerebral saturation below % for more than % of recording time and aut > second%. patients were analyzed, of whom had angiographic vasospasm. these were correctly detected by both nirs and tcd. of combined events over the aca territory, nirs detected / events. nirs also detected desaturations in / remaining cases, when no clinical or imaging event was detected. of combined events over mca territory, had an increase in desaturation auc, and / cases with no event had increase in desaturation events. both cases of angiography proven vasospasm were detected by nirs as an increase in desaturation auc, and by tcd as increase in flow velocities. cerebral oximetry using utl based nirs is comparable to tcd in detecting cerebral vasospasm, and may be superior in early detection of clinical neurologic worsening. extracellular fluid volume (ecfv), the main determinant of total circulating blood volume, is determined by the mass balances of na+ plus k+ (mbnk). in patients with aneurysmal subarachnoid hemorrhage (asah), diminished ecfv and reduced circulating blood volume are risk factors for worsened neurologic outcomes. maintenance of a normal ecfv based on nurse entered fluid balance (fb) has been reported to be difficult. the purpose of this study was to describe the time course of fluid and electrolyte mass balances over days in a cohort of patients receiving hypervolemic or normovolemic therapy. data from a randomized trial were secondarily analyzed. the intensive management of pressure or volume expansion in subarachnoid hemorrhage trial randomized patients to receive either a normovolemic or hypervolemic fluid management protocol. the standardized fluid management protocol included maintenance iv fluids with rate adjustments or boluses based on -hourly fluid balance and cvp (when available) with a target net positive fluid balance of - l in the hypervolemia group, and < . l in the normovolemia group. mbnk was calculated using published formulae. fb and estimated mbnk were compared between groups using random-effects generalized least square regression. baseline characteristics were similar between groups. fb was higher in the hypervolemia group than in the normovolemia group (mean difference: ml/day, %ci: - , p= . ). mbnk was also higher in the hypervolemia group (mean difference: meq/day, %ci: - , p= . ). average daily fb did not reach the target in the hypervolemia group. mbnk was negative on / days in the hypervolemia group, and / days in the normovolemia group. hypervolemic therapy resulted in higher net fb and mbnk compared to normovolemic controls, but was relatively ineffective at generating a consistently positive fb or expanded ecfv. our results support the notion that hypervolemia is difficult, if not impossible, to maintain in asah patients. exposure to hyperoxia is commonly seen but it is largely unknown whether hyperoxia is beneficial or harmful in patients with subarachnoid hemorrhage (sah). we hypothesized that hyperoxia may be associated with increase in the risk of delayed cerebral ischemia (dci) and poor -month outcome after sah. we analyzed data from single center, prospective, observational cohort database between and . patient nical ventilation, and ) arterial partial pressure of oxygen (pao ) measurements. patients expired within two weeks were excluded. hyperoxia was defined as the highest quartile of an average area under the curve of pao until the development of dci (pao mmhg) or until the post-bleed day (pao three months. of patients, no baseline characteristics were clinically contributing to hyperoxia. ninety-seven ( . %) patients developed dci. outcome data were available in patients, and poor outcomes were observed in ( . %) patients. the hyperoxia group had significantly higher incidence of dci (p = . ) and poor outcome (p = . ). after adjusting for modified fisher scale, hyperoxia was independently associated with dci (adjusted or, . ; % ci, . - . ; p < . ). after adjusting for age, smoking, alcohol consumption, previous stroke, previous heart disease, hunt-hess scale, aneurysm size, acute physiology and chronic health evaluation ii score, serum glucose, hyperoxia was found to be independently associated with poor outcome measured at months (adjusted or, . ; % ci, . - . ; p = . ). our data suggest that exposure to hyperoxia after sah is associated with dci and poor -month outcome. exact mechanism and the clinical implications can be explored by further investigations. advances in management of aneurysmal sah (asah) including refinement of neurosurgical techniques, availability of endovascular options and evolution of neurocritical care have led to improved outcomes following aneurysmal sah. we evaluated outcomes in asah patients admitted to our institution(s) over the past decades. prospectively collected data of aneurysmal sah patients admitted to the johns hopkins medical institutions between - was reviewed. we compared surivavl to discharge and functional outcomes at first clinic appointment post discharge ( - days) in patients admitted between - (phase =p ) and - (phase =p ) respectively using dichotomized gos (good outcome: gos - ). consecutive asah patients were included in the analysis (p . %; p . %). there were higher rates of poor grade hunt & hess (p %, p %; p< . ), admission gcs < (p : %, p %, p< . ), known medical comorbidites (p %, p %; p= . ), associated intraventricular hemorrhage (p %, p %, p< . ) and an older population in phase (p : . , p . ; p < . ) admissions. overall in-hospital mortality was low ( . %) and there was no significant difference between the periods in survival to discharge (p> . ). good outcomes were more common in phase ( . %) compared to phase ( . %); this difference was statistically significant after correction for other confounding factors following multivariate analysis (p< . ) with -fold greater adjusted odds of good outcomes in phase . our institutional experience over decades confirms that patients with asah have shown significant outcome improvements over time. hyponatremia in hospitalized patients has been associated with increased mortality, while chronic mild hyponatremia may impair attention and gait. hyponatremia after aneurysmal subarachnoid hemorrhage (sah) is common, yet its effect on cognitive outcome remains unclear. we aim to demonstrate the domain-specific cognitive effect of hyponatremia on patients after sah. we retrospectively analysed data from consecutive patients enrolled in our columbia university sah outcomes project between april and november . subjects were excluded if withdrawal of care of death occurred in the first three days. hyponatremia was defined as a sodium level < meq/l at any time during hospitalization. univariate and multivariate analyses were performed by a poisson regression, and a preset alpha of < . was set for statistical significance. a total of were included in the study. hyponatremia developed in subjects ( %). their mean age was years (sd+/- ), and subjects were men ( %). median time to onset and nadir of hyponatremia were (iqr - ) and days . univariate analysis associated hyponatremia with worsened modified rankin scale at discharge (rr= . , ci . - . ), three-month telephone interview of cognitive status (tics) (rr= . , ci . - . ), three-month barthel index (rr= . , ci . - . ), and three-month lawton instrumental activities of daily living (rr= . , ci . - . ). after adjustment for age, gender, hunt and hess grade, rebleeding, delayed neurologic ischemic deficit, and generalized cerebral edema, hyponatremia was associated with worsened three-month tics (rr= . , ci . - . ). by one year, hyponatremia was not associated with either functional or cognitive impairment. hyponatremia-related injury after sah appears to be associated with cognitive rather than functional impairment at three months. early and aggressive reversal of hyponatremia may expedite cognitive recovery among survivors of sah. financial support: dr ortega is supported by the spotrias fellowship funded by the national institute of neurological disorders and stroke (ninds)-p ns .dr mayer consults for actelion pharmaceuticals.there are no studies have shown that decreased quality-of-life (qol) after sah is a significant problem. the factors that predict poor qol after sah remain unclear. we sought to identify predictors of a poor quality of life months after sah. we prospectively studied -month qol in a cohort of patients consecutively admitted with sah between july and may . admission clinical scores, radiographic, surgical, and acute clinical course was documented during hospitalization. twelve months after sah qol was assessed using the sickness impact profile (sip). reduced qol was defined as two standard deviations below population-based normative values on the sip. univariate statistics were used to identify candidate predictors of poor qol, and to identify significant concurrent symptoms. backwards stepwise logistic regression was used to generate multivariable models of reduced qol. at months, % of survivors who participated in the follow-up survey ( / ) reported reduced qol. univariate admission factors associated with reduced qol were non-white race/ethnicity, high school education or less, poor clinical grade, loss of consciousness, hydrocephalus, pneumonia, and cerebral infarct from any cause. multivariable analysis revealed that poor hunt-hess grade (or . ; ci % . - . ), non-white race/ethnicity (or . ; ci % . - . ), and years or less of education (or . ; ci % . - . ) were significant admission risk factors for poor qol. common significant co-morbidities associated with poor qol at months included greater unemployment, not currently driving, more financial difficulties, current symptoms (e.g., headaches), marital difficulties, fear of recurrent sah, and dissatisfaction with rehabilitation. poor qol affects as many as one-third of sah survivors, and is predicted by poor admission clinical grade, non-white race/ethnicity, and lower educational status. further research is needed to determine if improved access to support and rehabilitation services for high-risk patients groups can improve qol after sah. biochemical mediators alter cerebral perfusion potentially resulting in neurological decline and delayed cerebral ischemia (dci); a significant cause of morbidity and mortality following aneurysmal subarachnoid hemorrhage (asah). estrogens (estrone-e and estradiol-e ) are mediators that have demonstrated neuroprotective properties that could play a role in dci however few studies have evaluated the impact of estrogens on outcomes in humans following asah. this study sought to examine the association between cerebrospinal fluid (csf) e and e levels and dci following asah. csf samples were collected after hemorrhage on adult asah patients [ -males, -pre and -post-menopausal females) admitted to the nv-icu enrolled in a nih study (ro nr ). up to csf samples per patient were selected for analysis representing days - after hemorrhage. samples were analyzed for e and e using liquid chromatography-tandem mass spectrometry. dci was operationalized as radiographic/ultrasonic evidence of impaired cerebral blood flow accompanied by neurological deterioration. statistical analysis using sas(v . ) included group based trajectory and multiple logistic regression. e was detected in more csf samples than e ( % vs %). group based trajectory identified distinct populations over time for both e ( % e high) and e ( % e high) values using censored normal model. non-weighted chisquare analysis identified differences between e trajectory groups by hh (p=. ) and dci (p=. ). using log metabolite levels, higher csf e measurements were associated with higher hh (p=. ) and fisher (p=. ) scores. csf e levels were not associated with dci (p=. ). there were no differences between csf e and severity of injury or dci.there was a significant relationship between csf e and e concentrations (p<. ). these findings provide evidence that estrogen metabolites are measureable in csf and may be associated with severity of injury. future studies are warranted to further explore these findings and their association to outcomes. high-grade spontaneous subarachnoid hemorrhage (sah) patients are monitored in the icu for up to days, as they are at risk for complications. the diagnosis of treatable complications such as vasospasm of cerebral arteries, cardiac arrhythmias and neurogenic stress cardiomyopathy is often delayed by the limitations of monitoring capabilities. we hypothesized that changes in heart rate variability (hrv) would correlate with the onset of these conditions following sah. we applied computational methodology to a cohort of sah patients in a single neurointensive care unit, examining hrv profiles to identify biomarkers of vasopasm, cardiomyopathy and impending respiratory failure. hrv was quantified for individual min epochs of the electrocardiogram waveform ( hz). qrs complexes were identified and the interbeat (rr) interval time series was constructed. mean, standard deviation and coefficient of variation of rr intervals, as well as the ratio of low frequency to high frequency power spectral density and standard poincare statistics were quantified. vasospasm occurred in ( %), stress cardiomyopathy in ( %) and respiratory failure in ( %) of patients. in a sah patient with takotsubo's cardiomyopathy and respiratory failure, we found a decrease in hrv that predated the discovery of cardiomyopathy as well as the onset of respiratory distress by several hours. the early clinical detection of vasospasm, cardiomyopathy and impending respiratory failure from on-line ekg hrv analysis would be of tremendous clinical value. in the face of changing autonomic influences in the critically ill postaneurysmal subarachnoid hemorrhage patient, the finding of an early signal prior to clinical detection of respiratory failure is encouraging. a larger and more highly annotated dataset may be required to increase the signal to noise ratio to realize the clinical potential of hrv-based biomarkers. retrospective analyses have found an association between transfusion and vasospasm, medical complications and mortality in subarachnoid hemorrhage (sah) patients. yet, none of those studies assessed the timing of transfusion, whether it occurred before, or, after vasospasm or complications. we sought to clarify whether transfusion could be considered a cause or consequence of vasospasm and complications. this interim analysis indicates that transfusion is not associated with vasospasm or infection when timing of transfusion is considered; fluid overload was more common after transfusion. the most dismal sequelae of aneurysmal subarachnoid hemorrhage (asah) are the development of cerebral vasospasm and consecutive delayed infarctions. their severity is linked to the clinical grade of the initial hemorrhage and the amount of blood in the basal cisterns. together, they represent the major cause of unfavorable clinical outcome and death in asah patients. from retrospective data, a promising method to reduce the incidence of vasospasm is the use of a lumbar drain to remove the blood from the subarachnoid space. the recently completed lumas trial addressed the safety of this approach in good-grade asah patients ( ). however, so far prospective data from subjects being at high risk for vasospasm and delayed infarction is lacking. we present the protocol of the earlydrain study, a prospective randomized multicenter trial comparing an intervention group with early continuous lumbar csf drainage to a control group receiving standard neurointensive care only ( ). eligible for participation are adults suffering from asah of all clinical grades who receive aneurysm treatment within hours of ictus. primary endpoint is the modified rankin score at six months. secondary endpoints include mortality, angiographic vasospasm, cerebral infarction, transcranial doppler sonography (tcd) mean flow velocity and rate of shunt insertion at six months after hospital discharge. the earlydrain study had recently been launched and, at abstract submission, patients of planned were enrolled. interim safety analysis did not reveal any concern on the use of lumbar drains after aneurysmal sah. up to now, ten centers in germany, switzerland and canada are participating. interested centers willing to join and contribute are still much appreciated. patients with aneurysmal subarachnoid hemorrhage (asah) require management in centers with neurosurgical expertise necessitating emergent interhospital transfer (iht). our objective was to compare outcomes in asah ihts to our institution with asah admissions from our institutional emergency department (ed). data for consecutive patients with asah admitted to johns hopkins medical institutions between and were analyzed from a prospectively obtained database. we compared in-hospital mortality and functional outcomes at first clinical appointment post-asah ( - days) using dichotomized glasgow outcome scale (good outcome: glasgow outcome scale - ) in ed admissions with ihts. a total of consecutive patients with asah were included in analysis (ed . %, iht . %). direct ed admissions had a higher incidence of poor hunt and hess grade ( / ) and major medical comorbidities, with no significant differences between the groups in age, intraventricular hemorrhage, and hydrocephalus. in-hospital mortality for ed admissions ( . %) was significantly lower than that for ihts ( . %), with . times greater adjusted odds of survival after multivariate analysis (p = . ). emergency department admissions had nearly -fold greater odds of good outcomes (odds ratio, . ; p b . ) after multivariate analysis. our institutional ed sah admissions had significantly better outcomes than did ihts, suggesting that delays in optimizing care before transfer could deleteriously impact outcomes. left ventricular (lv) systolic abnormalities occur commonly after subarachnoid hemorrhage (sah). cardiomyopathy associated with sah can include either predominate apical lv systolic dysfunction (cm-apical) or predominate basal lv systolic dysfunction (cm-basal). we aimed to determine if outcomes and diastolic function were different between patients with various forms of lv dysfunction after sah. patients hospitalized for sah between and were eligible for our study. those patients with a history of heart failure, myocardial infarction, or a documented acute coronary process were excluded. echocardiograms were reviewed and a wall motion score was provided for each of lv segments. patients were classified as cm-apical if the average wall motion score for apical segments was greater than the average wall motion score for basal segments. patients were classified as cm-basal if the average wall motion score for basal segments was greater than the average wall motion score for apical segments. of patients with sah had an echocardiogram. patients had normal lv function, cm-apical and cmbasal. the in-hospital mortality was not different between those with no echocardiogram or those with an echocardiogram who had normal lv function, cm-apical or cm-basal. patients with cm-apical were more likely to have shock and pulmonary complications, whereas patients with cm-basal were more likely to have sepsis. during a median follow up of . years, patients with cm-apical had the worse survival. patients with cm-apical and cm-basal had impaired lv relaxation as compared to those with normal lv systolic function. in-hospital mortality is not different between those patients with normal lv systolic function, cm-apical, or cm-basal. cmapical is associated with shock and pulmonary complications and a worse long term survival. further work evaluating the response to medical intervention and the differences in hemodynamic profiles of patients with cm-apical and cm-basal is warranted. therapy using sodium nitroprusside (snp) intrathecal (intraventricular) aims for a more effective approach for prophylaxis and treatment of cerebral vasospasm associated to a subarachnoid hemorrhage (sah). qualitative study whose objective was to analyze clinical cases to specific approach for cerebral vasospasm related to sah. two patients, the first one is a years old female with aneurysm rupture of the left posterior communicating artery, sah fisher iii, hunt hess . the second one is years old male with artery rupture of the middle cerebral artery, sah fisher iii, hunt hess , both were submitted to embolization, leading to acute hydrocephalus, in which external ventricular drainage (evd) was established. through the evd, a prophylactic intrathecal protocol was instituted ( ml snp with , ml of normal saline , % solution applying ml nps through the evd each hours for hour by infusion pump). patients evolved well with no neurologic or motor sequel and with a modified rankin scale = . the third patient was a years old male with aneurysm rupture in anterior communicating artery, sah fisher iii, hunt hess , severe vasospasm per operative in the left middle cerebral artery (mca), treated by angioplasty with balloon. starting the treatment protocol of cerebral vasospasm by lombar catheter: dosage mg ( ml) snp, solution with ml snp at ml of normal saline , % applying ml through the lombar catheter each hours for hour by infusion pump. patient without complication with modified rankin scale = . the cost for prophylactic therapy for days was u$ , ; if the patient had developed clinical vasospasm, the cost for a day treatment would be an average of u$ . , , having a great impact on morbidity, mortality and cost of hospital stay. angiography does not reveal a source of bleeding in - % of those with subarachnoid hemorrhage. these patients usually have a benign course and favorable outcome, especially those with a perimesencephalic pattern of bleeding (pm-sah); more diffuse bleeding has been associated with higher risk of vasospasm and neurological disability. we evaluated whether amount or pattern of bleeding better predicts risk of neurological complications and outcome. methods angio-negative sah patients were prospectively studied over seven years. six were excluded when a vascular etiology was identified on repeat angiography. pattern of bleeding, amount of cisternal (hijdra score) and ventricular blood (ivh score), and ventriculomegaly (bicaudate index) were assessed. neurological outcomes included hydrocephalus, angiographic vasospasm, and delayed ischemic neurological deficits (dind, based on clinical deterioration). functional outcome was assessed at -year using the modified rankin scale (mrs). bleeding was perimesencephalic in ( %), diffuse in ( %), cortical in and ct-negative in . patients with diffuse bleeding had higher hijdra ( [iqr - ] vs. [ - ]) and ivh scores ( [ - ] vs. [ - ]), and bicaudate index ( . [ . - . ] vs. . [ . - . ]) than those with pm--v ( % vs. %, p= . ) and require ventriculostomy ( % vs. %) and shunt placement ( % vs. %, both p= . ). moderate-severe angiographic vasospasm developed in % diffuse vs. % pm-sah (p= . ), while dind only occurred in those diffuse bleeding ( %). neither hijdra nor ivh score was higher in those developing vasospasm, across or within bleeding patterns. those with diffuse sah were less likely to be discharged home ( % vs. %, p= . ) or achieve minimal disability (mrs - , % vs. %, p= . ). angio-negative sah can result in hydrocephalus, vasospasm, cerebral ischemia, and residual disability. this is more likely in those with diffuse bleeding, a disparity not explained by a greater volume of cisternal or intraventricular bleeding. independent of the cholesterol lowering effects of hydroxymethylglutaryl conenzyme a reductase inhibitors(statins), there has been much debate about their protective effect against delayed cerebral ischemia (dci). various ongoing trials are aimed at assessing their effectiveness against dci after primary subarachnoid hemorrhage (sah). there is scanty literature on dci in patients who were on statins prior to the occurrence of sah. a retrospective chart review was done after approval from the institutional review board. data was collected from july to april using the icd code for sah. patients with sah secondary to avm, trauma and surgery were excluded. demographics, baseline characteristics and occurrence of clinical dci were collected. admission home medication list was used to identify patients on statins prior to admission. all statistical analysis was done using sas. a total of patients with primary sah were included. out of patients, ( . %) were on home statin. only ( %) patients within this group developed dci while ( . %) patients in the statin naive group developed dci (p= . ). this difference persisted even after correcting for age (p= . ), sex (p= . ), race (p= . ), smoking (p= . ), history of diabetes (p= . ), stroke/tia (p= . ), peripheral vascular disease (p= . ), hypertension (p= . ), hyperlipidemia (p= . ), home calcium channel blocker use (p= . ) and fisher grade (p= . ). a multivariate logistic regression analysis with backward selection further confirmed that the only significant factor affecting vasospasm was prior statin use (p = . ). the above findings suggest that prior statin use reduces the rate of dci after sah. though the known confounders were taken into consideration, the possibility of unknown confounders cannot be completely excluded. a larger prospective study may be required to verify these effects. the potential clinical implication of this would be to put patients with unruptured and untreated aneurysms on long-term statins. patients sometimes report that surviving a near-death experience results in enhanced appreciation of the preciousness and joy of life. we sought to determine how frequent the "stroke of insight" phenomenon occurs after sah. we prospectively enrolled sah patients between and and followed up survivors with a telephone interview at and months. patients were asked "do you enjoy life more, about the same, or less than you did before your brain hemorrhage?" surrogate responses were not analyzed. global functional outcome was evaluated with the modified rankin scale (mrs) and qol with the sickness impact profile (sip). of survivors who responded to the survey, the majority ( %, n= ) reported that they enjoyed life more since the hemorrhage, whereas only % enjoyed life less. enhanced life enjoyment was associated with female gender and white (versus non--hess grade. patients with enhanced life enjoyment were more likely to report improved marital status ( % versus %, p< . ), and were less likely to have rumination on their illness (p< . ). improved life enjoyment was associated with better sip qol scores ( . ± . versus . ± . , p< . ), but had no relationship with concurrent disability on the mrs (p= . ). remarkably, % of those reporting that they enjoyed life more were unable to walk without assistance (mrs or ). the majority of sah survivors enjoy life more after their hemorrhage. increased life enjoyment has no relationship with physical disability and handicap, but is associated with improved qol. informing patients of the "stroke of insight" phenomenon may be a simple and effective way to set positive expectations and promote recovery after sah and similar life-threatening illnesses. parenteral diclofenac infusion is commonly used in neurocritical patients and has been shown to effectively decrease body temperature after aneurysmal subarachnoid haemorrhage (asah). hemodynamic side effects and in specific the effect on brain homeostasis are understudied. twenty-one asah patients with multimodal neuromonitoring of intracranial pressure (icp), brain tissue oxygen tension (p b to ), and cerebral metabolism (microdialysis, md) receiving parenteral diclofenac infusions were analyzed in a prospective observational cohort study. mg diclofenac diluted in cc normal saline was administered at the discretion of the attending neurointensivist. we analyzed core body (cbt) and brain temperature (bt) over hours and hemodynamic (cardio-, cerebrovascular) and cerebral metabolic parameters over hours after intervention. ten-minuteaverage files of cardio-and cerebrovascular parameters and hourly files of md datasets were analyzedusing a generalized estimating equation.a pre-intervention baseline was calculated for every parameter. one-hundred-twenty-three parenteral diclofenac infusions over min (iqr - min) were analyzed. cbt and bt decreased to a minimum of . ± . °c and . ± . °c, h and h after diclofenac infusion (baseline . °c± . °c and . ± . °c, respectively, p< . ). hemodynamic side effects included a % reduction of map (by ± mmhg) and cpp (by ± mmhg) resulting in increased use of vasopressors in % of interventions (p< . ). p b to significantly decreased from ± mmhg baseline by % (p< . ) resulting in brain tissue hypoxia (p b to < mmhg) in % of interventions and % (n= ) of patients. in none of the interventions with baseline p b to above mmhg, brain tissue hypoxia was observed. baseline-p b to below mmhg was independently associated with brain tissue hypoxia during intervention (p< . ). there was a trend towards higher brain tissue lactate-pyruvate ratio and lower pyruvate after parenteral diclofenac after sah is associated with hemodynamic side effects and may result in brain tissue hypoxia without significantly affecting brain metabolism. the impact on outcome needs further investigation. delayed cerebral ischemia (dci) is a complication of subarachnoid hemorrhage (sah) with significant mortality/morbidity. digital subtraction angiography (dsa) can detect cerebral vasospasm which is a surrogate marker for dci. there is emerging data that perfusion computed tomography (ctp) is useful in detecting dci. we have compared the utility of ctp and dsa in detecting dci. patients with primary sah admitted to two academic institutions between july and april were identified. patients with clinical dci who underwent dsa or ctp (image processing through vitrea®) were included. the area of perfusion abnormality was traced out to generate cerebral blood flow (cbf), mean transit time (mtt) and cross sectional area. abnormal cbf and mtt values were compared to normal symmetrical areas in the opposite hemisphere. dsa reports were reviewed to identify radiologic vasospasm. out of patients, had clinical dci( . %). in those with dci, / patients that underwent ctp had abnormalities ( . %) compared to / patients that had vasospasm on dsa ( . %; p= . ). median abnormal cbf was . ( . - . ) ml/ gm/sec compared to . ( . - . ) ml/ gm/sec in area of normal perfusion (p= . ). median abnormal mtt was . ( . - ) seconds compared to the normal area of ( . - ) seconds (p< . ). median interhemispheric cbf and mtt difference was . ( . - . ) ml/ gm/sec and . ( . - . ) seconds respectively. median area of abnormal perfusion was . ( . - . ) cm . seventeen patients underwent ctp and dsa. a normal ctp excluded vasospasm on dsa. perfusion abnormalities involving an area of less than . cm did not have vasospasm on dsa. ctp is a useful indicator of dci and is comparable to dsa. in patients with clinical dci and a normal ctp, dsa is unlikely to pick up vasospasm. as the area of perfusion abnormality increases (greater than . cm in our subset of patients), dsa is more likely to show vasospasm. aneurysmal subarachnoid hemorrhage (asah) is more common in women than in men. current knowledge on potential gender differences after an asah occurred is sparse, albeit of clinical relevance. retrospective cohort study including patients with asah admitted to a neurovascular center at a major academic center at the university hospital of bern, switzerland. patients below age and with non-aneurysmal sah were excluded. we included consecutive patients with asah between january , and february , . women were older than men (median age years [interquartile range [iqr] - ] versus [iqr - ], respectively, p= . ), and progressively overrepresented with increasing age ( . % of women for the whole cohort). of note, in the swiss population the proportion of both genders between and years is similar, with women being slightly overrepresented at older ages. global disease severity at admission, measured by the acute physiology and chronic health evaluation (apache) ii score, was higher in women than in men (median score points versus [iqr - ], p= . ) even after correction for age. the apache ii score independently predicted an unfavourable outcome and mortality as opposed to gender. we found no differences between genders in the adopted aneurysm-securing strategy, intensive care interventions (administered drugs, rates of endotracheal intubation, tracheostomy, length of mechanical ventilation and placement of an external ventricular drainage). women and men with asah confirmed to be similar in terms of medical history, clinical / radiological severity of asah, complications and outcome. in conclusion, this study confirms that women with asah outnumbered men, especially at higher age. global disease severity on admission is higher in women and predicts, independently from gender, unfavourable outcome and mortality. finally, this study finds new relevant similarities between genders. complications of aneurysmal subarachnoid hemorrhage (asah) may include hypertension and neurogenic myocardial stunning. subsequent management often involves beta blockade. high fisher grade asahs may also be complicated by cerebral vasospasm, which could have pathophysiologic influence from sympathetic nervous system stimulation or inhibition. we investigated any relationship of beta blockade to the incidence of radiographic vasospasm in asah by retrospectively examining adults admitted to the sicu at loma linda university medical center between / and / , excluding those who expired within days of admission because of inability to assess outcomes. three groups were isolated relevant to beta blockade: were never beta blocked (no/no), were started on a beta blocker after admission (no/yes), and were continued on their home beta blockers (yes/yes). records were analyzed for the development of vasospasm with or without resultant infarction, death, and discharge status. outcomes were evaluated using multivariate analysis through logistic regression and adjusted for potential confounders. odds ratios were calculated setting the or for no/no patients to . one hundred and forty five patients had vasospasm, consequently infarcted, and died or required care in a longterm facility. patients in the no/yes group had significantly increased radiographic vasospasm ]. however, despite increased incidence of vasospasm, these patients had significantly fewer deaths or need for long term care [or . ( . - . )], with decreased tendency for infarcts ]. in the yes/yes group, there was a trend toward increased vasospasm ] that led to infarction )], with decreased mortality or need for long term care in a facility [or . ( . - . )]. the use of beta blockers in asah is associated with increased incidence of radiographic cerebral vasospasm. however, despite the increased rate of vasospasm, the use of beta blockers was associated with improved discharge characteristics. patients with subarachnoid hemorrhage (sah) frequently undergo continuous electroencephalography (ceeg) monitoring in the icu. we describe commonly encountered eeg patterns in sah patients with clinical correlation. patients with primary sah admitted to two academic institutions between july and april were identified. records were reviewed to note the presence of intraventricular hemorrhagic extension (ivh), intracerebral hemorrhagic extension (ich), location of subarachnoid blood, occurrence of delayed cerebral ischemia (dci), patient outcomes and length of stay (los). eeg reports were reviewed and classified as to the presence of arrhythmic continuous slowing (acs), rhythmic and periodic slow activity of triphasic morphology (tw), epileptiform activity (ea), and coma pattern. patients with metabolic causes for tw were excluded. of patients, had a routine eeg or ceeg monitoring.thirteen ( . %) exhibited non-metabolic tw, ( . %) had ea, ( . %) had acs, patient had coma pattern and had normal eeg. the presence of subarachnoid blood around the basal cisterns did not influence eeg patterns.in patients with ivh, the presence of tw patterns was significantly more common than other patterns ( . % vs. . %;p= . ). ea was associated with dci ( . %) as compared to non-epileptiform patterns ( . %;p = . ).ea was more common in patients with ich without statistical significance( . % vs. . %;p= . ). median los in patients with tw, ea and acs were ( - ), . ( - ) and . ( - ) days respectively without significant difference. patient outcomes were similar among all groups. non-metabolic tw are scantly reported in the literature and typically associated with diencephalic and brainstem lesion. in patients with sah, the presence of ivh and not cisternal blood was associated with non-metabolic tw. dci was significantly associated with the generation of epileptiform activity and the presence of ich seemed to favor an epileptiform pattern. eeg patterns did not influence los or outcome in our subset of patients. adenosine is an endogenous purine nucleoside that causes transient heart block in the av node when administered parenterally. we describe our experience with cases of severe intraoperative aneurysm rupture in which adenosine was administered to allow for control of the intraoperative bleeding. over a year period, we have treated approximately aneurysms with open microsurgery. two-thirds were unruptured. severe intraoperative aneurysm rupture that could not be readily controlled occurred in cases. of the aneurysms had recently bled, case was an unruptured aneurysm. in all cases, the amount of bleeding precluded safe application of temporary clips. an intravenous infusion of adenosine ( mg) was given in all cases. in , there was significant bradycardia and hypotension culminating in a brief cardiac pause ( - seconds), allowing for rapid dissection and clipping of the aneurysm. in cases, there was bradycardia and hypotension, but no cardiac arrest. in cases, there was limited bradycardia and hypotension, and a second dose ( mg) was required to slow the heart enough to allow for aneurysm treatment. in such cases, the adenosine allowed us to clear the field adequately to apply temporary clips in a precise fashion, and then to clip the aneurysms properly. poor response to the initial dosing was not related to patient size or other identifiable factor. adenosine has been used safely in our experience to allow for management of severe intraoperative aneurysm rupture. in most cases, there is a meaningful cardiac pause. in some instances, patients are less sensitive, and the dose must be repeated to achieve the desire effect. no adverse cardiac or pulmonary events were associated with the use of adenosine in our series. intraventricular hemorrhage (ivh) is an established independent predictor of poorer outcome in subarachnoid-and intracerebral-hemorrhage. though, limited knowledge exists regarding the pathophysiologic mechanisms that may lead to cerebral injury and poorer outcome. this is the first report presenting in vivo data on cerebral perfusion and brain tissue metabolism during the occurrence of ivh and after intraventricular fibrinolysis (ivf). a -year-old woman with severe subarachnoid hemorrhage (sah), hunt&hess grade , modified fisher scale , was admitted to our neuro-critical care unit. within the first hours an extraventricular drainage was placed and a left-sided mca aneurysm was coiled. after obtaining informed consent from the legal attorney, the patient received invasive multimodal neuro-monitoring, consisting of a cerebral blood flow (cbf)-and microdialysis-probe placed into the ipsilateral frontal white matter. within hours after probe placement we observed a significant drop of cerebral blood flow (cbf below ml/ g/min) and an increase in l/p-ratio without significant changes in cerebral perfusion-or intracranial-pressure. imaging revealed a re-hemorrhage into the ventricular system with blockage of the foramina of monro and acute hydrocephalus. consequently, therapeutic ivf was undertaken with mg of rtpa which lead to sufficient clot resolution. after ivf we normalization of cerebral perfusion and metabolism. this is the first report on ivh and its potential mechanisms that may contribute to secondary injury in the human brain. a decrease of cerebral blood flow and disturbance of cerebral metabolism was documented during the occurrence of ivh, supporting existing hypotheses of global impairment. moreover, we could document profound treatment effects of ivf leading to a restored cbf and a stable aerobic metabolism in the investigated brain tissue. many patients with aneurismal subarachnoid hemorrhage (sah) present with acute, labile, hypertension and may be at risk for rebleeding. clevidipine, a novel, ultra-short acting dihydropyridine has been used in cardiac surgery, acute hypertensive emergencies and patients with intracerebral hemorrhage, but not in sah patients. the clash study (clevidipine in aneurismal subarachnoid hemorrhage) is a prospective evaluation of the efficacy and safety of clevidipine in controlling systolic blood pressure (sbp) before the aneurysm is secured. the primary endpoint is the number of patients achieving sbp target within minutes. post-hoc, sbps pre-infusion, during-infusion and postinfusion were compared using a generalized estimating equation. we present the first patients enrolled: men and women, mean h&h and fisher . , aneurysms coiled and clipped. mean sbp upper and lower goals were ± . and ± . mmhg. analyses included , sbp data points. all patients reached sbp target within . ± min using an infusion rate of . ± . mg/hour. the mean preinfusion, during-infusion and post-infusion sbps were . ± . , . ± . and . ± . mmhg (pre-infusion vs during-infusion p < . , pre-infusion vs post-infusion p < . , during-infusion vs post-infusion p < . ). after the st sbp control readings, sbp was above the upper target goal . ± . % and below the lower . ± . % of the time. icp did not increase during infusion (n= ). no patient rebled. in one patient the infusion was stopped temporarily times due to sbp below the target range. there were ( . %) sbp values < mmhg and none < mmhg. clevidipine controlled sbp in all patients with sah in < min and kept sbp within the selected range in . % of the time without any patient rebleeding. financial support: research grant from the medicines company to conduct this study. aneurysmal subarachnoid hemorrhage (asah) is associated with numerous adverse sequelae. patients who survive the initial hemorrhage are at high risk for delayed secondary brain injury, including cerebral infarction, neuronal cell death, white matter abnormalities, and hydrocephalus. resulting in focal neurological deficits, cortical dysfunction, and both longterm cognitive and psychosocial deficits referred to as sah-induced "delayed neurological deficits" (dnds). review of the literature revealed that heparin had previously been advocated to reduce complications of asah. here, we report on our favorable experience with the use of heparin prophylaxis in the management of patients who are at a high risk for developing sah-induced dnds. a retrospective chart review of patients that presented to the university of maryland medical center were reviewed between january and may . inclusion criteria were patients with fischer grade iii sah due to rupture of a true saccular aneurysm and were treated by surgical clipping within hours of the patient's ictal event. exclusion criteria were patients who had a localizing deficit related to an intracerebral hematoma from the ictal event. included in this study were patients that were started on an intravenous infusion of heparin and an additional patients that served as matched controls. none of the patients exhibited heparin-induced thrombocytopenia (hit). the heparin regimen used appeared to be safe. patients administered low-dose iv heparin experienced significantly fewer occurrences of ischemia-related ct hypodensities as well as symptomatic vasospasms than case controls. retrospective analysis of our clinical experience with constant iv infusion of low-dose heparin in patients at high risk for sah-induced dnds indicates early use of low-dose iv heparin infusion may be safe and perhaps beneficial in patients having undergone surgical clipping. further study with a double-blind placebo-controlled trial is warranted to establish the role of heparin in the prevention of sah-induced dnds. subarachnoid hemorrhage patients (sah) may experience cardiac biomarker elevation in serum troponin and b-type natriuretic peptide (bnp). we hypothesized that elevations in these cardiac biomarkers after sah are predictive of increased patient mortality. we retrospectively reviewed the medical records of all non-traumatic sah patients admitted from march to march including medical history, modified fisher scale on initial head ct scan, initial glasgow coma scale (gcs), serum troponin t and bnp within hrs of admission. survival data was dichotomized as either alive or dead by chart follow-up. values (> pg/ml) versus normal values against alive or dead status. we identified sah patients, with initial measured troponin, and with initial measured bnp.the mean age was (range - ) and % male. modified fisher grade was - in %, and grade - in %. the initial gcs mean was (range - ), % of patients had intracranial aneurysm, while % were 'angiogram-negative' sah. twenty sah patients died, with a mean of days post sah (range - ), six from cardiopulmonary or multiple organ failure, from sah, and unknown/other. elevated troponin was seen in % ( of ) with a mean = . (range, . - . ), and elevated bnp in % ( of patients) with a mean = (range, - ). patients with elevated levels of troponin had a greater chance of death (p= . ). patients with elevated levels of bnp also had a higher mortality (p= . ). the data demonstrate a statistically significant association with elevated cardiac biomarker elevation and risk of subsequent death after sah, which occurs not only during the immediate post sah period but after initial hospitalization. delayed cerebral ischemia (dci), length of stay and glasgow outcome scale (gos) following angiogram-negative sah (ansah) are infrequently and inconsistently described in the literature. furthermore ansah are generally considered to have a better prognosis than aneurysmal sah (asah). ansah subgroups include benign perimesencephalic sah (pmh) and aneurysmal-type or diffuse sah. we report and compare outcome data of patients presented with diffuse ansah and diffuse asah. a retrospective chart review of patients who presented to academic institutions between july- and april- who met the criteria for diffuse spontaneous sah were reviewed. the patients were further divided into ansah (n= ) and asah (n= ). delayed cerebral ischemia rates, length of stay and discharge gos were compared and analyzed between two groups using sas statistical software. discharge gos scale was dichotomized in good outcome (gos - ) out of patients, a total of ( . %) patients meet the criteria of diffuse ansah and ( . %) meet the criteria of diffuse asah. demographics and baseline characteristics including age, sex, race, hypertension, diabetes, gcs on presentation, hunt & hess score and fisher grade among two groups were comparable. overall % (n= ) of ansah and % (n= ) of asah showed dci (p= . ). mean length of stay was days in nasah and days in asah. good outcome was seen in % (n= ) in nasah and % (n= %) in asah groups (p= . ). in our patient cohort of ansah, % of patients had dci. even though it is less then asah group it is considerably higher then previously reported in the literature. furthermore length of stay and discharge gos between two groups were comparable. this study indicates that diffuse ansah is not a 'benign' condition and warrants a low index of suspicion for complications with a multidisciplinary approach to management. transcranial doppler (tcd) is a common method used to measure cerebral blood flow velocities and estimate flow resistance related to intracranial pressure (icp). we present the case of a patient with subarachnoid hemorrhage and clipped aneurysm, who, while undergoing tcds, rebled. a year old man presented with sudden-onset severe headache and neck pain. ct of the head showed a subarachnoid hemorrhage (sah) with intraventricular extension and obstructive hydrocephalus. an anterior communicating artery (acom) aneurysm was found and clipped and a ventriculostomy was placed. after surgery there was an interval decrease in the sah. eight days after the original event the patient re-bled during a tcd test because of clip failure. tcd waveforms were captured before, during the bleed and post treatment with mannitol and csf drainage from the ventriculostomy. prior to the bleed. icp was mm hg, the left mca flow velocity was cm/sec and the pulsatility index (pi) . . during the bleed the icp increased to and pi to . - . , with the waveform showing a narrow peak and decreased diastolic and mean velocity. mannitol g was given and the ventriculostomy was opened to drain. within minutes the icp decreased to mm hg, the pi improved to . , the waveform widened and the velocities returned to previous levels (video will be provided with the abstract showing the tcd changes). repeat ct of the head showed increased sa blood and extensive new intraventricular hemorrhage; catheter angiogram a malpositioned clip. the acom aneurysm was coiled successfully. we present this unique case of tcd capturing the dynamics of a real-time intracranial aneurismal bleed with significantly elevated icp. our data demonstrated the tcd pi, flow velocities and waveforms changed dramatically during the rebleeding and improved quickly with treatment. transcranial doppler (tcd) is the least invasive method to detect cerebral vasospasm but is unable to interrogate vessels beyond the circle of willis and is highly operator-dependent. we tested a novel technique whereby we record the miniscule pulsation of the skull gated with cardiac contraction and compared it to tcd in patients with subarachnoid hemorrhage. skull accelerometry was performed using a prototype device designed by jan medial, inc. (mountain view ca). the device has highly sensitive accelerometers that couple through plastic feet to the patient's scalp, arrayed with detectors over the forehead, at midline occiput, each over the temporal bones, and on the patient's vertex. they are held in place with a plastic strap. paired tcd recordings and accelerometry epochs (typically minutes of recording) were compared in patients with and without spasm. a total of accelerometry recordings were obtained in subjects with subarachnoid hemorrhage who had paired tcd recordings. this allowed distinct pairings of data sets (right, left, posterior). a unique signature was identified by a fast fourier transform waterfall technique revealing a shift in accelerometry signals to higher frequencies (representing a "bruit" of sorts) in patients with tcd identified vasospasm. an analytic model was created based on the first recordings, and validated using the remaining recordings. this revealed % sensitivity and % specificity for detection and localization of spasm. highly sensitive skull accelerometry detects a shift toward higher vibration frequency in patients with vasospasm-a cranial "bruit". this technique may be a highly sensitive tool for the detection of cerebral vasospasm following subarachnoid hemorrhage. a prospective, blinded validation study is on going to measure this novel tool's performance characteristics in a larger sample of patients. financial support: research grant from jan medical, inc. j.n. is year-old hispanic male prisoner previously healthy presented to our institution altered due to diffuse subarachnoid hemorrhage (fisher grade iv) and a bi-lobed "mickey mouse" right m middle cerebral artery (mca) ruptured aneurysm. initially, j.n.'s hunt and hess grade level of on arrival, but declined to a in the ed. j.n. was intubated and an external ventricular device was placed. the anatomy of the aneurysm was complex in nature measuring mm in maximal dimension with the superior lobe measuring . mm and the inferior lobe measuring . mm. based on the complex anatomy of the aneurysm, a -vessel angiogram was planned to treat the aneurysm with a trans-arterial coil-embolization approach. a x mm septal balloon was used with a synchro microwire, with the balloon been placed across the neck of the inferior aneurysm. the superior aneurysm was accessed with a sl- microcatheter and coiled in the usual fashion. the sl- microcatheter was then re-directed to the inferior aneurysm and coiled similarly. post-angiographic images showed complete obliteration of the aneurysm with a small neck residual to protect en passé branches. evaluation of the literature is scant with reports of bi-lobed aneurysm with the classic description of "mickey mouse" or "mirror" aneurysm. trans-arterial coil-embolization provided a safe, rapid, and effective method for coiling a complex bilobed aneurysm with no major thrombo-embolic events. trans-arterial coil-embolization is a procedure used in the treatment of gross hematoma and fistula in human and the veterinary population. to our knowledge, there is no report of trans-arterial coil embolization for the treatment of bi-lobed aneurysm posted within the usual medical research engines. our institution is presenting a novel endovascular technique in the treatment of a classic bi-lobed mickey mouse aneurysm. j.n. was able to recover fully and eventually discharge to the infirmary in federal prison. the routine practice of therapeutic hypothermia is advocated in the management of comatose survivors of out-of-hospital cardiac arrest (ohca), particularly if ventricular fibrillation is the initial rhythm. potential benefits of hypothermia were evaluated for comatose survivors after ohca due to aneurysmal subarachnoid hemorrhage (sah). following return of spontaneous circulation (rosc), therapeutic hypothermia was induced for comatose sah patients except for those with devastating brain damage on brain ct and cardiac arrest over minutes. immediately after diagnosis and evaluation of cardiac function, cooling was promptly initiated by nasogastric lavage with iced water and surface cooling under general anesthesia. the ruptured aneurysm was obliterated by surgical clipping with wide decompressive craniectomy. core temperature was maintained at -urokinase was injected via cisternal drain and nicardipine and fusdil hydrochloride were intravenously administered to prevent cerebral vasospasm. clinical outcome was assessed according to the glasgow outcome scale (gos) months later. six women, aged between and years, were eligible during the past years. their glasgow coma scale was after resuscitation. electrocardiogram on arrival was asystole in and pulseless electrical activity in patients. myocardial stunning was detected in patients by echocardiogram. surgery and hypothermia treatment were uneventfully conducted. postoperative mri revealed extensive cerebral ischemia in and vasospasm-related ischemic lesion in patient. their gos was good recovery in , severe disability in , persistent vegetative state in , and death in patients. therapeutic hypothermia was feasible for ohca patients due to sah. since neurogenic stunned myocardium could be a possible cause of cardiac arrest in sah, beneficial effects of induced hypothermia are expected just like cardiogenic cardiac arrest. appropriate prognostication methods are warranted for decision making to treat or not. autonomic shift (as), characterized by increased sympathetic nervous system activation, has been implicated in neurologically mediated cardiopulmonary dysfunction and immunodepression following stroke. however direct measurement of autonomic nervous system dysfunction is difficult to obtain routinely in critically patients. we investigated the prevalence of as defined by readily available clinical parameters and determined the association of as with subsequent infection in a cohort of patients with aneurysmal sah (asah). data were obtained from a single center cohort study of asah patients admitted from january , through april , . as was defined as at least one early routine clinical marker of neurologically mediated cardiopulmonary dysfunction (based on electrocardiogram, echocardiogram, cardiac enzyme testing or clinical diagnosis of neurogenic pulmonary edema). exclusion criteria were beta-blocker treatment a known pre-existing abnormal electrocardiogram. multivariable logistic regression models were developed to evaluate the association between as and subsequent infection after adjusting for other covariates. a total of patients were included (mean age , % male). autonomic shift was seen in / ( %), and infection was seen in / ( %). autonomic shift was associated with subsequent infection on unadjusted analysis (or= . , % ci . , . ). however, on multivariable analysis adjusting for other predictors of infection, there was no significant association between as and subsequent infection (or . , % ci . , . ). age, clinical grade, aneurysm location and presence of ich were all identified as independent predictors of infection following asah. we identified evidence suggestive of as based on readily available clinical markers in % of patients with asah. however, as defined by these clinical criteria was not an independent predictor of infection. additional studies may be warranted to determine the optimal definition of as and to determine the clinical significance of this finding. we have previously studied the effects of falling temperature on the incidence of asah at our institution over days observing , asah. we previously reported that every degree decrease in temperature was associated with . % increase in risk of asah [relative risk (rr), . , p = . ]. we looked within the same data using other metrics to identify patterns in temperature changes which might result in physiological stress that increases the incidence of asah admissions at our institution. we developed a mathematical equation based on the premise that degrees fahrenheit is the ideal external temperature for humans. our formula measured the variation above or below ° as a percentage of ° for every day of days of observation. the relationship of absolute differences between tmax and tmin was examined to see if daily temperature variation was associated with increasing incidence. the odds ratio for incident asah relative to ° was . (ci . - . ) p= . . likelihood of incident asah increased as the ratio of tmax to ° fell below zero (i.e. experienced colder temperatures). intraday variation as measured by the absolute difference between tmax and tmin was strongly associated with increasing incidence, p= . , or . , ci ( . - . ). a smaller, not larger, difference between tmax and t min was associated with increased likelihood of asah admission. colder daily maximum temperatures relative to ° f, and smaller intraday temperature fluctuations are associated with increased asah admissions at our institution. smaller daily temperature ranges correspond to seasonal periods with the least daylight in this region, and may represent sudden arrival of cold weather in warm months. both metrics support the hypothesized increased likelihood of asah with falling environmental temperatures. these new methods may assist in the development of new algorithms for asah predictions based on temperature. near-infrared spectroscopy (nirs) is a noninvasive means of measuring cerebral regional mixed arteriovenous (av) brain oxygenation. we hypothesized that frontal nirs would correlate against more established modes of vasospasm monitoring and systemic variables for severe aneurysmal subarachnoid hemorrhage (asah). case report we describe a year old male who presented with coma (gcs= , e m v t) after severe asah (modified fisher ) from a ruptured giant basilar aneurysm ( . cm x cm) who developed severe diffuse vasospasm with no change on clinical examination. frontal nirs monitoring was applied in addition to map, cpp, cbf (hemedex tm ), cardiac output (co), spo , core temperature, continuous quantitative eeg (qeeg) with alpha delta ratio (adr) monitoring, along with daily tcd. the patient developed severe diffuse vasospasm and underwent angioplasty of the mca, aca, and pca arteries and received intra-arterial verapamil. pearson's correlation coefficient was used to analyze trends in variables pre-and post intervention. values were recorded over a four-day period. calculated correlation coefficients revealed invasive cbf to right nirs r= . (p= . ) and left r= . (p= . ) but was contralateral to the cbf probe, co to right nirs r= . (p= . ) and left r= . (p= . ). coefficients with weak or negative correlation included arterial map to right nirs r=- . (p= . ) and left r=- . (p= . ), noninvasive map to right nirs r=- . (p= . ) and left r= . (p= . ), spo to right nirs r=- . (p= . ) and left r=- . (p= . ). noninvasive map to arterial map r= . (p= . ), noninvasive map to cpp r= . (p= . ), and arterial map to cpp r= . , (p= . ). nirs correlates with ipsilateral invasive cbf values (r= . , p= . ) and trends with cardiac output. nirs did not correlate with map, cpp, spo , tcd or qeeg adr data. larger prospective studies are needed to validate these preliminary results. this case report describes the use of intraventricular nicardipine in a pediatric patient for the treatment of severe cerebral vasospasm following sah from traumatic pica dissection. intraventricular nicardipine has been suggested as an adjuvant to standard therapies in adults with aneurysmal sah but its use has not been described in pediatric patients. a year-old boy was transferred from an outside hospital for treatment of severe sah following sports related head injury. he was found to have a dissecting pica psuedoaneurysm which was treated endovascularly. bilateral ventricular drains had been placed for hydrocephalus. his neurological examination declined on hospital day and ct angiogram demonstrated severe vertebrobasilar vasospasm. intraventricular nicardipine was administered in addition to treatment with transluminal balloon angioplasty, induced hypertension and nimodipine. the patient received mg intraventricular nicardipine twice daily for days and the dose was then increased to mg every hours for a total of days. both ventricular drains were clamped for min following administration. he tolerated doses without hemodynamic effects, elevations in intracranial pressure or evidence of ventriculitis. after improvement in clinical examination and mean cerebral blood flow velocities by tcd, intraventricular nicardipine was stopped. he was discharged to acute rehab and was ambulatory and preparing to restart school at age appropriate grade level at month follow up. intraventricular nicardipine was safely administered in this year-old patient with severe vasospasm following sah with a good outcome. intraventricular nicardipine should be considered as an adjuvant to standard therapies for vasospasm in pediatric patients, though further studies are needed to evaluate safety and efficacy in both pediatric and adult patients. the benefit of early tracheostomy has been well described. patients with aneurysmal subarachnoid hemorrhage (asah); however, represent a distinct population to which traditional weaning parameters may be difficult to apply. the purpose of this study is to identify admission characteristics of asah patients that predict need for tracheostomy. this was a retrospective cohort analysis of consecutive asah patients. we excluded patients with a history of symptoms longer than hours prior to transfer, expired within hours, or no ct scan available prior to cerebral angiography. we collected data including: demographics, co-morbidities, neurologic exam, labs, ejection fraction % on echocardiogram, modified fisher scale, and hijdra scale. chi-square or wilcoxon tests were performed where appropriate with subsequent multivariate analysis of statistically significant variables. the data set included tracheostomy patients and non-tracheostomy patients. [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] ,p=< . ). the modified fisher and all components of the hijdra scale were significantly higher in the tracheostomy group. the bicaudate index was significant ( . vs. . ,p= . ); however, presence of hydrocephalus using this index was not. in the multivariate analysis older age, lower albumin, higher pco and presence of ventricular blood by hijdra scale remained significant predictors. neurologic status on admission, advanced age, burden of systemic illness, and intraventricular hemorrhage are associated with increased risk of tracheostomy. further research in this patient population on the benefits of early tracheostomy (lower mortality, less ventilator days and less intensive care unit days) is warranted. patients with subarachnoid hemorrhage(sah) have variable outcomes, some of these leading to major disability. established guidelines advocate administration of nimodipine to patients with sah. several recent trials have investigated the utility of statins and magnesium, however there has not been much data showing clinical benefit. we present data of patients with primary sah who had therapy with magnesium, nimodipine and simvastatin for prevention of delayed cerebral ischemia (dci). patients with primary sah admitted to two academic institutions between july and april were identified. all patients received therapy with magnesium, nimodipine and simvastatin for dci prophylaxis. outcomes were categorized as good in those with glasgow outcome scales (gos) of - and poor in those with gos - . chi-square analysis was used to compare outcomes between age, sex, race, hunt-hess scores ( - vs - ), presence of vasospasm and glasgow coma scale (gcs) on presentation (below or above ). of patients identified with primary sah, . % had a good outcome. the mean age for patients with a poor outcome was . (sd . ) when compared to . (sd . ) in patients with a good outcome (p= . ). among those with a gcs - on admission, . % had a good outcome while in those with less than only . % had a good outcome (p< . ). when comparing hunt-hess scales, . % of those with grades between - had good outcomes, while . % of patients with grades - had poor outcomes (p< . ). among those who developed dci, . % had a good outcome as compared to . % had poor outcomes (p= . ). age and race failed to show any difference in patients with good and poor outcomes. we present our data on therapy with nimodipine, magnesium and statin for prophylaxis against dci. age, admission gcs, hunt-hess scale and occurrence of dci were predictors of patient outcome. intraoperative rupture during the surgical treatment of a previosuly unruptured intracranial aneurysm is a rare event. we describe our experience with intraoperative aneurysm rupture in this setting. we reviewed all cases of unruptured aneurysms treated by a single surgeon from july, to june, and identified those patients who suffered intraoperative aneurysm rupture. of unruptured aneurysms treated during this period, there were instances of intraoperative aneurysm rupture ( . %). in our experience, rupture occurred during dissection of either a perforator ( cases) or a major efferent vessel ( cases) from the aneurysm dome or of the dome from adherent overlying cortex ( case). in one instance, the aneurysm ruptured during removal of the anterior clinoid process. in cases, blunt rather than sharp dissection was being employed. in cases, bipolar electrocautery and gentle tamponade successfully sealed the rupture point. in cases, a clip placed across the bleeding site well up on the dome of the aneurysm controlled the bleeding and allowed for completion of the dissection and proper clipping. in the last case, the administration of adenosine was utilized to stop the bleeding and allow for proper clip placement. intraoperative angiography confirmed adequate aneurysm obliteration in each case. there were no clinical consequences associated with these intraoperative ruptures. intraoperative rupture during elective surgery for a previously unruptured aneurysm is uncommon. in our experience, rupture was typically associated with blunt dissection on the dome of the aneurysm. the use of bipolar electrocautery, clipping of the bleeding point, or intravenous adenosine infusion were successfully used to control bleeding in our cases. the neurovascular surgeon should be prepared to address this unlikely event, should it occur. aneurysmal subarachnoid hemorrhage (asah) is a life-threatening form of hemorrhagic stroke which is more common in women than men, typically between ages - . over the course of our nursing practice, we have observed a trend of pre-menopausal asah female patients who experience the onset of their menses during the initial week of hospitalization. we became curious as to whether there is a correlation between asah and an earlier onset menses than a normal - day cycle. retrospective, single-center review of the medical record of pre-menopausal females ages - years who were admitted to our neuroscience intensive care unit with the diagnosis of asah. chart review was specific to documentation of the onset of menses during the first week of hospitalization, medical/gynecological history with regard to last menstrual period, usual menstrual cycle characteristics, contraceptive use, past surgical history, and pertinent medications. over a month period (june -june ), we identified asah patients with being female. of the female asah charts screened, we found study patients. nine of ( %) females had documentation of starting their menses during their initial week of hospitalization for asah, much earlier than a normal range of the menstrual cycle of - days. one patient had menses documented on hospital day . this small retrospective study suggests that asah may disrupt the "normal" menstrual cycle of pre-menopausal females. to our knowledge, this is the first description of this gender and disease specific phenomenon. a prospective study is planned to better understand the role asah has on hypothalamic-pituitary-ovarian-uterine physiology. introduction - % of patients with spontaneous subarachnoid hemorrhage can have normal cerebral angiogram. vasospasm, hypoperfusion or thrombosis may hide the aneurysm. dynact is a promising new technique which may help in these cases. we present a case report that highlights the ability of dynact in identifying a thrombosed aneurysm that was undetected with routine cerebral angiogram. a year old female presented with the worst headache of her life. ct scan of the brain showed subarachnoid hemorrhage (sah) in suprasellar cistern, extending into the anterior interhemispheric fissure, bilateral perisylvian, prepontine cistern and right perimesencephalic cisterns along with extension in to the third and fourth ventricles. after the placement of an external ventricular drain, the patient was immediately taken to angio suite where a biplane cerebral angiogram showed - mm saccular aneurysm at right middle cerebral artery bifurcation and an unremarkable vasculature otherwise. repeat imaging using dynact showed the presence of a mm ruptured and thrombosed aneurysm at the right mca bifurcation. the thrombosed aneurysm was visualized and clipped surgically. this case report highlights the promising utility of dynact in identifying the culprit aneurysms. treatment of severe cerebral vasospasm in subarachnoid hemorrhage remains challenging. with failure of noninvasive therapy, endovascular modalities may be undertaken, albeit with limited efficacy; balloon angioplasty can be used only for proximal, focal spasm and intra-arterial calcium-channel blocker (ccb) bolus infusion has transient vasodilatory effects. we present a patient with severe vasospasm after subarachnoid hemorrhage, who demonstrated significant angiographic improvement with continuous infusion of intra-arterial verapamil over hours. a female in her mid- 's with sickle cell anemia presented with a hunt and hess , fisher grade iv subarachnoid hemorrhage secondary to a ruptured right posterior communicating artery. on initial assessment, the patient was localizing with only her upper extremities. the aneurysm was completely coil embolized and standard triple-h therapy maintained. on post-bleed day , the patient developed left-sided hemiplegia. angiography demonstrated critically severe, diffuse right anterior and posterior circulation vasospasm. angioplasty could not be performed due to microwire and balloon inaccessibility of stenosed anterior and posterior circulation vessels. subsequently, two microcatheters were positioned with their respective tips in the petrous right internal carotid artery (ica) and v segment of the right vertebral artery for continuous machine controlled intra-arterial verapamil infusion. dosing consisted of administering mg/hr verapamil into the right vertebral artery and mg/hr into the right ica. the patient was placed on a heparin drip and taken to the neurointensive care unit for monitoring. after hours of continuous ia verapamil infusion, angiography demonstrated significant improvement in right anterior and posterior circulation vasospasm, with only residual diffuse moderate stenosis. unfortunately, no corresponding clinical improvement was noted. prolonged infusion of intra-arterial ccb's may provide extended angiographic improvement in severe vasospasm refractory to conservative treatment and unsuitable for balloon angioplasty. with systematic study of such techniques, optimal agents and dosing for sustained vasodilation and clinical optimization may be defined. vasospasm remains a significant cause of morbidity after subarachnoid hemorrhage (sah), inducing delayed ischemic events. sah typically results in numerous complications including severe, treatment-refractory headache. fioricet® (acetaminophen mg/butalbital mg/caffeine mg) is a commonly used analgesic medication for the treatment of headache in sah. caffeine has been shown to reduce cerebral blood flow. the purpose of this study was to determine if there is an association between fioricet® administration and early vasospasm. a retrospective, medical record review was conducted, and patients were identified using the university health consortium (uhc) database. patients were included if they had an aneurysmal sah with a presenting hunt and hess grade of i-iv. data points included occurrence of clinical vasospasm, daily amount of fioricet® and other analgesics, daily pain scores, and patient demographics. a univariate analysis was performed to determine the association between extent of fioricet® exposure and early vasospasm (within the first days) after sah. a multivariate analysis was performed accounting for amount of fioricet® use, patient age, and hunt and hess grade. the population characteristics were typical of the sah population. patients who experienced clinical vasospasm received more fioricet® than those who did not have vasospasm (mean . + . tablets/day versus . + . tablets/day (p= . )). the odds ratio for vasospasm with regards to fioricet® use when controlled for age and hunt and hess grade was . ( % ci . - . ). the multivariate analysis did not yield any statistically significant associations with vasospasm. there was a significant association between fioricet® exposure and vasospasm in our univariate analysis. however, when correcting for age and sah severity, the association is not significant. thus, the data do not currently support a clear causal association. this preliminary data will be used to support a comparative study investigating headache treatment in sah. isolated complete third nerve palsy (tnp) in the setting of a subarachnoid hemorrhage (sah) is most commonly seen secondary to a posterior communicating artery (pcom) aneurysm. however, this same clinical picture with a negative angiogram and otherwise negative imaging studies becomes extremely rare. although trauma has been described as one of the most common causes of isolated tnp, concomitant post-traumatic sah and late onset isolated complete tnp has never been reported. we report a case of a delayed onset complete tnp after traumatic sah. case report. a year-old male with type- diabetes mellitus presented to the emergency department with painless diplopia and left eye ptosis three days after sustaining a fall with closed-head injury without loss of consciousness. his non-contrast head ct scan showed a fisher grade subarachnoid hemorrhage. upon arrival and throughout his hospitalization, the patient had a glasgow coma scale (gcs) of . his neurological exam revealed findings consistent with isolated complete thirdnerve palsy (tnp) involving the pupil. his neurological examination was otherwise normal. diagnostic digital subtraction angiography (dsa) was negative as it was his brain mri for aneurysm or vascular lesion. mri did however show traumatic sah pattern and small subdural hematomas consistent with trauma. laboratory findings (esr, crp, ace, c-anca and p-anca) did not raise suspicion of secondary vasculitic or ischemic causes of tnp. the patient was discharged five days after admission with no further complications but without any improvement of tnp signs and symptoms. this case illustrates an atypical presentation of traumatic sah with delayed-onset, isolated complete tnp. to our knowledge, this is the first case with these features described in the literature. his atypical presentation may represent the combination of both diabetes and traumatic injury to the cranial nerve iii in the subarachnoid space, rather than either etiology alone. diringer section of neurocritical care improved clinical outcomes after aneurysmal subarachnoid hemorrhage (asah) have been demonstrated for patients treated at high volume centers. these centers treat only % of all asah. it is common for asah patients to be transferred to high volume comprehensive stroke centers after presentation to a community hospital. this study aims to determine if the hospital of presentation has impact upon asah outcomes. a -year retrospective analysis of asah treated in a comprehensive stroke center was undertaken. the comprehensive stroke center consisted of a neurocritical care unit, dedicated vascular neurosurgeons, and endovascular and neurocritical care specialists. demographic and outcome data were collected on all asah patients who had a confirmed and secured aneurysm, survived > days from admission, and completed tcd monitoring and observation for complications of vasospasm. univariate and multivariate analyses were evaluated for differences in mortality, complications, incidence of vasospasm, discharge disposition, and length of stay. patients were included ( direct and transfer). baseline parameters known to influence outcome (age, medical complications, glasgow coma scale, fisher and hunt and hess grade) were similar between the two groups. transferred patients developed ultrasound defined vasospasm more frequently ( % versus %; p< . ) and had a greater delay in time to surgery ( . versus . days; < . ). adjusting for key predictors, direct admit patients spent . fewer days in the icu compared to transferred patients (t=- . , p= . ). multivariate analysis showed that the likelihood of vasospasm was significantly higher for transfer patients (or . , ci: . - . , p = . ). longer in-hospital stays and decreased rates of home discharge were observed in transferred patients (p< . ). mortality rates were not statistically different (transfer . %, direct . %, p= . ). asah patients admitted directly to a comprehensive stroke center have better outcomes than those transferred from lower acuity facilities. numerous advances have been made in the management of subarachnoid hemorrhage (sah) and its complications, including symptomatic vasospasm. however, the optimal management of vasospasm in patients without neurological deficit remains uncertain. we performed an electronic survey of members of the neurocritical care society (ncs) to elucidate clinical practice in this regard. an electronic survey with ten questions about different aspects of sah management was formulated. our institutional review board and ncs approved the survey. three scenarios were presented for good grade sah patients without evidence of delayed cerebral ischemia (dci): those with either normal tcd values, vasospasm on tcd, or vasospasm on angiography. members answered the survey (response rate of %). up to % of respondents utilized transcranial doppler (tcd) measurement to diagnose vasospasm, while % ( % ci, - %) used clinical examination and % ( % ci, - %) used angiography (ct or catheter). in good grade sah patients with no evidence of dci, % ( % ci, - %) of respondents indicated using nimodipine in all three scenarios. in the subset with normal tcd values, % ( % ci, - %) recommended use of hypervolemia, % ( % ci, - %) hemodilution and % ( % ci - %) induced hypertension. however, in the subset with vasospasm on angiography and no referable clinical symptoms, % ( % ci - %) recommended the use of hypervolemia, % ( % ci, - %) hemodilution, % ( % ci, - %) induced hypertension and % ( % ci, - %) endovascular therapy with intra-arterial vasodilators, angioplasty or stents. from the sample above, it appears that good grade sah patients without neurological deficit but radiological vasospasm are treated aggressively. this is not supported by current literature or guideline recommendations, which imply little benefit of aggressive therapy in such patients. further studies are needed on the optimal management of this subset of patients, in whom the effects of vasospasm remain unclear. key: cord- -jyk zphp authors: bonaventura, aldo; vecchié, alessandra; wang, tisha s.; lee, elinor; cremer, paul c.; carey, brenna; rajendram, prabalini; hudock, kristin m.; korbee, leslie; van tassell, benjamin w.; dagna, lorenzo; abbate, antonio title: targeting gm-csf in covid- pneumonia: rationale and strategies date: - - journal: front immunol doi: . /fimmu. . sha: doc_id: cord_uid: jyk zphp covid- is a clinical syndrome ranging from mild symptoms to severe pneumonia that often leads to respiratory failure, need for mechanical ventilation, and death. most of the lung damage is driven by a surge in inflammatory cytokines [interleukin- , interferon-γ, and granulocyte-monocyte stimulating factor (gm-csf)]. blunting this hyperinflammation with immunomodulation may lead to clinical improvement. gm-csf is produced by many cells, including macrophages and t-cells. gm-csf-derived signals are involved in differentiation of macrophages, including alveolar macrophages (ams). in animal models of respiratory infections, the intranasal administration of gm-csf increased the proliferation of ams and improved outcomes. increased levels of gm-csf have been recently described in patients with covid- compared to healthy controls. while gm-csf might be beneficial in some circumstances as an appropriate response, in this case the inflammatory response is maladaptive by virtue of being later and disproportionate. the inhibition of gm-csf signaling may be beneficial in improving the hyperinflammation-related lung damage in the most severe cases of covid- . this blockade can be achieved through antagonism of the gm-csf receptor or the direct binding of circulating gm-csf. initial findings from patients with covid- treated with a single intravenous dose of mavrilimumab, a monoclonal antibody binding gm-csf receptor α, showed oxygenation improvement and shorter hospitalization. prospective, randomized, placebo-controlled trials are ongoing. anti-gm-csf monoclonal antibodies, tj and gimsilumab, will be tested in clinical trials in patients with covid- , while lenzilumab received fda approval for compassionate use. these trials will help inform whether blunting the inflammatory signaling provided by the gm-csf axis in covid- is beneficial. coronavirus disease is caused by severe acute respiratory syndrome coronavirus (sars-cov- ) with a clinical spectrum ranging from asymptomatic/paucisymptomatic forms to severe pneumonia leading to respiratory failure, need for mechanical ventilation, and death ( ) . to date, no specific treatment is approved for covid- , and management is supportive. severe covid- pneumonia seems to be mediated by a cytokine storm ( , ) . therefore, therapies that target hyperinflammation may be effective. in a recent report, patients with covid- needing intensive care unit (icu) admission showed a cytokine profile similar to that of secondary hemophagocytic lymphohistiocytosis with increased levels of several inflammatory cytokines [interleukin (il)- , il- , granulocyte-colony stimulating factor (g-csf), granulocyte-monocyte stimulating factor (gm-csf), interferon-γ-inducible protein (ip- ), monocyte chemoattractant protein (mcp- ), macrophage inflammatory protein -α (mip -α), and tumor necrosis factor-α (tnfα)] ( ). additionally, increased levels of ferritin and il- have been shown to correlate with a worse prognosis [ ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ; supplementary table ]. these observations underline that covid- is a complex disease capable to combine different patterns of inflammatory biomarkers. indeed, most of the infections can trigger the release of il- β from the inflammasome ( ) followed by the production of il- that increases the circulating levels of c-reactive protein, the prototypical acute-phase reactant ( ) . in viral infections, including covid- , elevated levels of the pro-inflammatory cytokine il- , that derives from the inflammasome as il- β, are found along with high levels of ferritin ( ) , thus replicating the events commonly observed in the macrophage activation syndrome ( ) . altogether, these findings support the hypothesis that a maladaptive hyperinflammatory response to the virus orchestrated by il- , il- β, and eventually gm-csf-referred to as cytokine storm-rather than the virus itself may drive the lung damage leading to hypoxia and acute respiratory failure. immunomodulation may be beneficial in the treatment of hyperinflammation-associated conditions. data supporting the role of hyperinflammation in sepsisrelated acute respiratory distress syndrome (ards) are derived from a sub-group analysis of a phase randomized controlled trial of il- receptor antagonist (anakinra), which showed significant survival benefit in patients treated with anakinra compared to placebo ( ) . il- β is an upstream pro-inflammatory cytokine that is released following activation of the inflammasome in response to infection and/or injury ( ) . il- is a pleiotropic cytokine that influences several processes, such as acute-phase protein generation, inflammation, and antigen-specific immune responses ( ) . in the innate immune response, il- is produced by myeloid cells [e.g., macrophages and dendritic cells (dcs)] following the recognition of sterile or non-sterile stimuli through toll-like receptors at the site of infection or tissue injury. in the adaptive immune response, il- is a critical modulator of plasma b-cell differentiation and antibody production ( ) . a deregulated il- expression is involved in the pathogenesis of several disorders, such as chronic inflammatory diseases, autoimmune diseases, and tumor development ( , ) . cytokine release syndrome (crs) represents an on-target effect of chimeric antigen receptor (car) t-cell therapy and consists of a systemic inflammatory response due to a massive cytokine release, including il- , gm-csf, and interferon-γ, following the in vivo activation of car t-cells ( , ) . the incidence of crs after car t-cell therapy ranges from to % with - % of patients having severe crs ( ) . tocilizumab, an il- receptor blocker, has been approved for the treatment of severe crs after car t-cell therapy in light of its association with a rapid improvement of clinical manifestations and a decrease in the aforementioned cytokines along with a low toxicity for car t-cells ( ) . different trials are recruiting patients with covid- pneumonia to test whether il- receptor blockers (tocilizumab, sirukumab, and sarilumab: chictr , nct , nct , nct ; nct , nct , and nct ) and an il- receptor blocker (anakinra, nct , nct , nct , nct , nct , nct , nct , and nct ) improve covid- pneumonia outcomes. the identification and treatment of hyperinflammation using existing therapies with understood safety profiles that are either in clinical development or approved for other indications represent a valid option to cope with the immediate need to reduce the rising mortality of covid- . in an attempt to approach hyperinflammation upstream of both il- and il- and to target neutrophils as well as macrophages, gm-csf may be considered as an appealing mediator. gm-csf is generally perceived as a pro-inflammatory cytokine and is produced by many cells, including macrophages, t-cells, fibroblasts, endothelial cells, epithelial cells, and tumor cells ( ) , with most of the production occurring at sites of inflammation ( ) . gm-csf signals are mediated by the gm-csf receptor (gm-csf-r) consisting of a specific ligand-binding α-chain (gm csf-rα) and a signal-transducing β-chain (gm csf-rβ) ( figure a) . downstream signaling of gm-csf-r includes janus kinase (jak )/signal transducer and activator of transcription (stat ), nuclear factor kappa-light-chain-enhancer of activated b cells (nf-κb), extracellular signal-regulated kinase (erk), and the phosphoinositide -kinase (pi k)-akt pathway ( ) ( ) ( ) ( ) . importantly, erk activity is responsible for gm-csf-mediated human monocyte survival in vitro ( ) . interferon regulatory factor (irf ) is a hemopoietic-specific transcription factor that has been involved in the induction of dc-like properties in monocytes treated with gm-csf ( , ) . recently, achuthan gm-csf is involved in the differentiation of alveolar macrophages, thus enhancing the clearance of respiratory microbes through an increase in phagocytosis and release of pro-inflammatory cytokines (il- β, il- , and tnf-α) in a feed-forward inflammatory loop. based on previous experiences, the early administration of a rhgm-csf, like sargramostim, may improve the initial response against viruses, including sars-cov- . (c) mavrilimumab prevents gm-csf from binding to the α-chain of its receptor, while gimsilumab, lenzilumab, and tj directly bind gm-csf with the final common result of blocking the intracellular signaling. based on the current knowledge, these agents can be used to reduce the hyperinflammation caused by sars-cov- in the course of the disease. differently from rh-gm-csf, these agents should be considered later in order to not negatively impact the favorable effects of gm-csf on the immune response. apc, antigen presenting cell; dc, dendritic cell; gm-csf, granulocyte-macrophage colony-stimulating factor; sars-cov- , severe acute respiratory syndrome coronavirus . this figure has been partially created using servier medical art templates, which are licensed under a creative commons attribution . unported license; https://smart.servier.com. et al. found that gm-csf is capable to up-regulate irf expression via jumonji domain-containing protein d (jmjd ) demethylase in monocytes/macrophages ( ) . increased levels of irf are responsible for the production of chemokine (c-c motif) ligand (ccl ), which is involved in inflammation and tissue remodeling, as occurs in arthritis ( ) . the gm-csf-irf signaling was also described to up-regulate major histocompatibility complex (mhc) class ii expression in mouse bone marrow cultures and macrophages ( , ) . gm-csf levels are low or undetectable in normal conditions; however, any immune trigger can rapidly increase concentrations, as it has been seen in the lungs of patients with asthma or within the synovial fluid of patients with arthritis ( ) . bacterial endotoxins and inflammatory cytokines (e.g., il- β, il- , and tnf-α) potently induce gm-csf ( ) . indeed, increased mrna expression for tnf-α, il- β, and il- were reported in monocytes/macrophages treated with gm-csf ( ) . these findings led to hypothesize that gm-csf is part of the inflammatory milieu of some inflammatory/autoimmune reactions. gm-csf would work as a co-regulator along with tnf-α, il- , and il- , as part of a positive feed-forward inflammatory loop involving monocytes/macrophages, fibroblasts, and endothelial cells ( ) ( ) ( ) , but also dcs and th cells ( ) ( ) ( ) . il- , however, was found to induce intestinal and splenic production of gm-csf ( ), thus promoting systemic effects, like an increase in splenic macrophage precursors. the importance of il- β and the il- receptor/myeloid differentiation primary response (myd ) signaling axis appears of importance in the regulation of gm-csf by cd + and γδ t cells ( ) . il- β, together with tnf-α, can also promote monocyte viability via gm-csf while not inducing any specific macrophage polarization ( ) . increased levels of gm-csf have been found in the bronchoalveolar fluid of patients with ards compared with healthy controls ( , ) . higher levels were observed in the early phases ( - days) with a progressive decrease in late stages (day ) ( ) . gm-csf may indirectly contribute to ards by the suppression of neutrophil apoptosis ( , ) as activated neutrophils play a major role in the microvascular damage contributing to lung damage ( , ) . limited evidence describes a regulatory role for gm-csf through the promotion of dc differentiation to a tolerogenic profile, thus increasing the number and function of regulatory tcells ( ) . this can also lead to t-cell hypo-responsiveness and/or anergy ( ). the mechanisms underlying pro-inflammatory and immunomodulatory phenotypes of gm-csf are not fully understood and need to be further investigated. these properties are hypothesized to depend on the dose and the presence of other cytokines in the setting of the immune response. at lower doses, gm-csf stimulates the tolerogenesis of myeloid cells involved in the regulatory t-cell homeostasis ( ) , while at higher doses gm-csf causes myeloproliferation, leading to a sustained immune response ( ) . gm-csf-derived signals are critically involved in the differentiation of macrophages and in the proliferation and activation of other immune cells. alveolar macrophages (ams) are essential to clear respiratory microbes ( , ) , and their depletion has been associated with increased disease severity in murine models of influenza infection ( , ) . therefore, several pre-clinical studies reported that the intranasal administration of gm-csf prior to inducing an experimental viral infection conferred resistance to respiratory pathogens through an increased proliferation of ams [( , ); figure b ]. this is probably due to an enhanced clearance of the virus, thus limiting the direct damage provided by the virus itself. recently, a subset of ams, the nerve-associated interstitial alveolar macrophages (nams), have been identified and characterized in human and murine lung ( ) . nams seem to originate from the yolk sac and, differently from the other ams, require colonystimulating factor (csf ) and not gm-csf for development and maintenance in adulthood. mouse models of influenza virus infection on selectively nam-depleted animals suggest a central role for nams in the negative regulation of virusinduced inflammation, whereas the other gm-csf-dependent ams display a pro-inflammatory profile ( ) . in addition, gm-csf receptor activation triggers stimulation of multiple downstream signaling pathways, including jak /stat , the mitogen-activated protein kinase (mapk), and the pi k, all fundamental in activation and differentiation of myeloid cells [( , ) ; figure a ]. along with its key role in inflammation, gm-csf is critical in lung physiology. this has been clearly highlighted by gm-csfdeficient and gm-csf receptor-deficient mice which develop pulmonary alveolar proteinosis (pap) because ams require gm-csf to differentiate ( ) . the poor differentiation of these macrophages is responsible for the accumulation of surfactant proteins, saturated phosphatidylcholine, and cholesterol, leading to pap. indeed, local expression of gm-csf in the lung is able to restore normal surfactant homeostasis and clearance in the setting of pap ( ) . additionally, gm-csf-deficient mice show a persistent, low-grade inflammation resulting from inappropriate responses to commensal microbes. this chronic inflammation predisposes mice to develop different kinds of tumors ( ) . to date, no function-altering gm-csf mutations have been identified in humans. however, an autoimmune form of pap can develop in humans and is associated with high levels of neutralizing gm-csf autoantibodies that inhibit gm-csf signaling ( ) . a congenital form of pap ending up with a complete inhibition of the macrophage clearance of surfactant has also been described and is caused by mutations in csf ra or csf rb, the genes encoding the gm-csf-rα and gm-csf-rβ chains ( ) . increased circulating levels of gm-csf have been recently described in patients with covid- compared to healthy controls ( ). a paper from china appearing on the preprint online platform biorxiv reported that in patients with covid- , especially those admitted to the icu, cd + t lymphocytes were rapidly activated in the lung to pathogenic t helper (th) cells and generated gm-csf and il- . this potent pro-inflammatory environment strongly induced cd + cd + monocytes, which also released gm-csf and il- , further worsening the cytokine storm. these aberrant and numerous gm-csf + -il- + cells may enter the lungs and explain the detrimental actions provided by hyperinflammation in the most severe and even fatal cases ( ). in light of the results in animal studies following the intranasal administration of gm-csf in the setting of respiratory infections, two human recombinant gm-csf (hrgm-csf), sargramostim and molgramostim, were investigated in humans ( ) ( ) ( ) . sargramostim was tested in a randomized, doubleblind, placebo-controlled clinical trial in patients with acute lung injury/ards ( ) . the drug was administered as an intravenous infusion once daily for days at a dosage of µg/m . the study showed no significant difference in the number of ventilator-free days, organ failure-free days, and -day mortality between the hrgm-csf and placebo groups; there was also no difference in the number of serious adverse events ( ) . a randomized, double-blind, placebo-controlled phase ii study tested the effects of low-dose hrgm-csf (molgramostim, µg/kg daily) for days in patients in addition to the standard of care in critically ill patients with severe sepsis and respiratory dysfunction ( ) . the study found that hrgm-csf was associated with an improvement in gas exchange and functional activation of pulmonary macrophages; however, there was no improvement in -day survival ( ) . in another randomized, double-blind, placebo-controlled clinical trial in patients with bacterial and fungal abdominal sepsis, molgramostim µg/kg daily for days was administered in addition to standard of care. the treatment group had a reduction in the rate of infectious complications and in the length of hospitalization ( ) . in the early phases of viral infections, gm-csf's role may be protective as it helps limit virus-related injury. for this reason, an inhaled formulation of sargramostim is being tested in patients with covid- -related acute hypoxic respiratory failure (nct ). in later stages of covid- , the severity of the illness appears to be driven by the inappropriate release of several cytokines, such as il- and gm-csf. these mediators are involved in the inflammatory lung injury, predisposing patients to respiratory failure and eventually ards. therefore, inhibition of gm-csf signaling may be a reasonable treatment in this stage of disease. this is supported by pre-clinical data in crs showing that gm-csf blockade reduced car t-cell therapy-related toxicity by preventing crs development without affecting its therapeutic activity ( ) . mavrilimumab is a high-affinity monoclonal igg antibody against gm-csf-rα [( ); figure c ]. the efficacy and safety of mavrilimumab have been studied in rheumatoid arthritis (ra) and showed promising results. in a phase b multicenter placebocontrolled study, patients with moderate-to-severe ra were randomized to receive different dose levels of mavrilimumab ( , , and mg subcutaneously every weeks) or placebo. mavrilimumab at a dose of mg subcutaneously every weeks was the most effective in improving clinical and laboratory disease activity ( ) . no substantial differences in adverse events or severe adverse events were observed between groups ( ). these results on safety and efficacy were confirmed in a phase double-blind randomized trial evaluating the use of mavrilimumab at a dose of mg subcutaneously every other week in long-standing ra patients ( ) . a post-hoc analysis of these studies has shown that the administration of mavrilimumab was associated with a significant downregulation of the macrophage-derived chemokine c-c motif chemokine ligand (ccl ) and il- ( ), related to a direct inhibition of the proinflammatory cytokine release from myeloid cells. mavrilimumab also showed a decreased expression of il- /il- -associated transcripts, the latter suggesting an indirect suppressive effect of mavrilimumab on t cell activation ( ) . moreover, a sustained suppression of serum markers of disease activity, such as c-reactive protein and il- , was observed in ra patients treated with mavrilimumab ( ) . mavrilimumab is currently under investigation for the treatment of giant cell arteritis (nct ). a prospective interventional single-center cohort study tested the efficacy and safety of mavrilimumab in patients with severe covid- pneumonia and evidence of hyperinflammation in italy ( ) . thirteen non-mechanically ventilated patients with severe covid- pneumonia and hyperinflammation were treated with a single intravenous dose of mavrilimumab mg/kg upon admission to the hospital. twenty-six non-mechanically ventilated patients with severe covid- pneumonia and hyperinflammation and with similar baseline characteristics were evaluated as a control-group. all patients received similar standard of care therapy, including antivirals and antibiotics. over the course of the -day follow-up period, mavrilimumabtreated patients experienced earlier and improved clinical outcomes than control-group patients, including earlier weaning from supplemental oxygen and shorter hospitalizations. death occurred in % (n = / ) of mavrilimumab-treated patients by day compared to % (n = / ) of control-group patients ( ) . these data are consistent with the hypothesis that excessive host immune response driven by t cells and monocytes may have a central role in the pathogenesis of covid- pneumonia. a randomized controlled trial is being designed and is now active (mavrilimumab in severe covid- pneumonia and hyperinflammation , nct ). five monoclonal antibodies targeting gm-csf (gimsilumab, otilimab, namilumab, lenzilumab, and tj ) are in development and are currently under investigation mainly for the treatment of ra. the principal clinical trials both completed and ongoing are described in table ( , ) . recently, tj (also known as tjm ) obtained the us food and drug administration (fda) clearance to start a clinical study for covid- associated crs (i-mab) . additionally, lenzilumab has received fda approval for compassionate use in covid- patients (fda) , while a phase study is ongoing. a clinical trial has also been approved for gimsilumab for the treatment of covid- and is now enrolling patients in the us (nct ) (figure c ). in addition, csl is a monoclonal antibody targeting the gm-csf-rβ, common to gm-csf, il- , and il- . a phase trial is evaluating the safety and tolerability of this drug in patients with asthma ( table ) . because gm-csf is a key mediator in pulmonary homeostasis, there is the theoretical concern that inhibition of gm-csf signaling by either binding to gm-csf or blocking the receptor may result in dysfunctional ams, leading to pap and development of new infections. fortunately, there has yet to be a case of pap reported with the use of anti-gm-csf monoclonal antibodies. this may be due to the fact that patients with autoimmune pap have to reach a "critical threshold" of neutralizing antibodies to develop the disease, and the doses currently being utilized in clinical trials may not reach this threshold ( ) . this may actually be true for the chronic use where the low level of lung penetration of the - mg subcutaneously every weeks may not provide the level of necessary inhibition ( ) . however, in the case of covid- pneumonia and hyperinflammation, the lung penetration of the drug may be critical. this is the reason why the dose has been increased from . - mg/kg subcutaneously to - mg/kg intravenously. this means that pap should not necessarily be an issue in the covid- treatment in that a single intravenous dose is being given and it will wear off in a month, while pap is a disease caused by chronic inhibition over years. as covid- pneumonia is likely to be aggravated by a cytokine storm, immunomodulation gained importance as a possible therapeutic strategy to this disease. a wealth of il- and il- blockade trials are ongoing and results are awaited. i-mab announces ind clearance from fda for tjm to treat cytokine release syndrome (crs) associated with severe coronavirus disease . available online at: https://www.biospace.com/article/releases/i-mab-announcesind-clearance-from-fda-for-tjm -to-treat-cytokine-release-syndrome-crsassociated-with-severe-coronavirus-disease- -covid- -/ (accessed april , ). however, an approach targeting hyperinflammation upstream of il- and il- as well as neutrophils and macrophages may be envisioned through gm-csf signaling. gm-csf is an immunomodulatory cytokine that may help to clear respiratory microbes by stimulating ams. a clinical trial with a hrgm-csf, sargramostim, will be conducted in covid- patients with the rationale that it may help clear the sars-cov- earlier in the disease course. however, in the later phase of covid- lung injury, the marked elevation in gm-csf levels as part of the cytokine storm during the onset of covid- pneumonia suggests that gm-csf may actually be deleterious at this stage of the disease. blocking gm-csf signaling could therefore be an effective therapeutic strategy by reducing the cytokine storm, which leads to the progression of acute respiratory failure in patients with hyperinflammation. multiple clinical trials with inhibition of the gm-csf pathway are either ongoing or under development. ab, av, and aa conceived the manuscript. ab and av drafted manuscript, figure, and tables. tw, el, pc, bc, pr, kh, lk, bv, ld, and aa critically revised the manuscript. all authors approved the final version. clinical characteristics of coronavirus disease in china cardiovascular considerations in treating patients with coronavirus (covid- ) covid- : consider cytokine storm syndromes and immunosuppression clinical features of patients 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anti-gm-csfralpha antibody enables therapeutic dosing that limits exposure in the lung the supplementary material for this article can be found online at: https://www.frontiersin.org/articles/ . /fimmu. . /full#supplementary-material key: cord- -glcq qp authors: bodro, marta; compta, yaroslau; llansó, laura; esteller, diana; doncel-moriano, antonio; mesa, alex; rodríguez, alejandro; sarto, jordi; martínez-hernandez, eugenia; vlagea, alexandru; egri, natalia; filella, xavier; morales-ruiz, manuel; yagüe, jordi; soriano, Álex; graus, francesc; garcía, felipe title: increased csf levels of il- β, il- , and ace in sars-cov- –associated encephalitis date: - - journal: neurol neuroimmunol neuroinflamm doi: . /nxi. sha: doc_id: cord_uid: glcq qp nan a national outbreak of severe acute respiratory syndrome coronavirus (sars-cov- ) emerged in december in wuhan, china, rapidly evolving to the coronavirus disease (covid- ) pandemic. neurologic complications of covid- include headache, confusion, hyposmia, and dysgeusia, with encephalitis being rarely reported. coronaviruses can potentially invade the cns through trans-synaptic propagation via nasal entry, likely causing hyposmia. alternatively, cns dysfunction may result from the systemic hyperinflammatory response to the virus. we report patients supporting this hypothesis. a -year-old healthy man presented with -day history of headache, left-sided paresthesias, and ipsilateral paresis progressing within hours to confusion and agitation. his axillary temperature was . °c. brain ct and mri scans were normal. csf showed lymphocytic pleocytosis and increased proteins. he was started on iv acyclovir, ampicillin, and ceftriaxone, which were discontinued when csf cultures and pcr ruled bacterial or viral etiologies. pcr of sars-cov- was negative in csf but positive in the nasopharyngeal swab. on day , he fully recovered except for amnesia of the previous days. a healthy -year-old man presented with fever, myalgias, and dry cough lasting week. a few hours after admission, he developed difficulty naming objects, temporospatial disorientation, confusion, and agitation. a thoracic ct scan showed bilateral peripheral opacities suggestive of covid- pneumonia. brain ct and mri scans obtained days later were unremarkable. csf showed lymphocytic pleocytosis and increased proteins. the patient was empirically started on acyclovir, ampicillin, and ceftriaxone, which were discontinued once csf cultures and pcr returned negative. pcr for sars-cov- was positive in the nasopharyngeal swab, but negative in csf. three days later, he was back to normal except for amnesia of the previous days. none of the patients developed severe respiratory problems or required intensive care support. *both these authors contributed equally to this work. go to neurology.org/nn for full disclosures. funding information is provided at the end of the article. the article processing charge was funded by the authors. "hospital cĺ ınic infecto-covid- " group and "hospital cĺ ınic neuro-covid- " group coinvestigators are listed in appendix at the end of the article. this is an open access article distributed under the terms of the creative commons attribution-noncommercial-noderivatives license . (cc by-nc-nd), which permits downloading and sharing the work provided it is properly cited. the work cannot be changed in any way or used commercially without permission from the journal. for the latest articles, invited commentaries, and blogs from physicians around the world npub.org/covid clinical features, serum, and csf characteristics including cytokines and angiotensin-converting enzyme (ace) profile from both cases are shown in the table. these patients suggest that encephalitis may be the first or dominant manifestation of covid- . for patient , the focal neurologic deficits were the first symptom manifestation; his young age, absence of risk factors, and comprehensive studies ruling out other etiologies suggest a link between the neurologic symptoms and systemic sars-cov- infection. by contrast, patient presented with typical covid- symptoms, but confusion, disorientation, and aphasia rapidly dominated the clinical picture. three previous case reports of cns involvement in covid- suggest different pathogenic mechanisms: direct cns infection demonstrated by detection of sars-cov- rna in csf, recrudescence of symptoms related to previous lesions (e.g., brain infarction) in the context of systemic infection, and inflammatory-mediated mechanisms resulting in acute hemorrhagic necrotizing encephalopathy. this is a rare complication of viral infections, usually influenza, considered to result from severe systemic inflammation associated with elevated cytokine levels, such as interleukin abbreviations: ace = angiotensin-converting enzyme; il = interleukin. a hypertension, respiratory chronic disease, cardiovascular disease, diabetes, cancer, chronic hepatopathy, or immunosuppression. csf il- β and il- were considered increased when greater than . pg/ml and pg/ml, respectively. as for ace, the normal range was - . u/l. (il)- and tumor necrosis factor-α. no information on serum or csf cytokine levels was provided for any of these patients. in our patients, we cannot completely rule out a direct infectious mechanism, despite the negative csf testing of sars-cov- , but the rapid recovery in less than days makes it unlikely. alternatively, and in keeping with the abovementioned cytokine-mediated systemic inflammation, there is evidence that patients with coronaviruses can develop a cytokine storm syndrome with increased il- and il- among other inflammatory mediators. hence, in a study of children with acute encephalitis-like syndrome, serum anti-human coronavirus-oc immunoglobulin m antibodies were present in % of patients and levels of il- , il- , monocyte chemotactic protein- , and granulocyte macrophage colony-stimulating factor were increased in their csf. the elevated csf levels of il- in our patients, and il- β in of them, are in line with those studies. biological anti-il treatments targeting il- (anakinra) or il- (tocilizumab and siltuximab) are useful to treat symptoms of cns involvement related to the cytokine storm triggered by chimeric antigen receptor t-cell therapy. our patients improved spontaneously, but these treatments could be considered in more severe cases of covid- -associated encephalitis with increased csf levels of ils. finally, our patients also had increased csf levels of ace. it has been postulated that sars-cov- enters the cell using the ace receptor. although it is difficult to interpret the meaning of increased csf levels of ace in these patients, they could be linked to the postulated alteration in the ace pathway in covid- . the main implication of these patients is that physicians should be aware of covid- infections presenting or predominantly manifesting as encephalitis, likely resulting from activation of inflammatory pathways with increased ils and ace in csf. the authors acknowledge the patients for their generosity and to all other people suffering the covid- pandemics, as well as to all the health professionals and other staff crucially involved in their care and the handling of this unprecedented global crisis. no targeted funding reported. the authors report no disclosures relevant to the manuscript. go to neurology.org/nn for full disclosures. received epidemiological and clinical characteristics of cases of novel coronavirus pneumonia in wuhan, china: a descriptive study a first case of meningitis/encephalitis associated with sars-coronavirus- covid- -associated acute hemorragic necrotizing encephalopathy: ct and mri features neurological complications of coronavirus disease (covid- ): encephalopathy coronavirus infections in the central nervous system and respiratory tract show distinct features in hospitalized children clinical care of chimeric antigen receptor t-cell patients and managing immune-related adverse effects in the ambulatory and hospitalized setting: a review covid- and angiotensin-converting enzyme inhibitors and angiotensin receptor blockers key: cord- -gxs wit authors: ashhurst, thomas myles; vreden, caryn van; niewold, paula; king, nicholas jonathan cole title: the plasticity of inflammatory monocyte responses to the inflamed central nervous system date: - - journal: cellular immunology doi: . /j.cellimm. . . sha: doc_id: cord_uid: gxs wit abstract over the last three decades it has become increasingly clear that monocytes, originally thought to have fixed, stereotypic responses to foreign stimuli, mediate exquisitely balanced protective and pathogenic roles in disease and immunity. this balance is crucial in core functional organs, such as the central nervous system (cns), where minor changes in neuronal microenvironments and the production of immune factors can result in significant disease with fatal consequences or permanent neurological sequelae. viral encephalitis and multiple sclerosis are examples of important human diseases in which the pathogenic contribution of monocytes recruited from the bone marrow plays a critical role in the clinical expression of disease, as they differentiate into macrophage or dendritic cells in the cns to carry out effector functions. while antigen-specific lymphocyte populations are central to the adaptive immune response in both cases, in viral encephalitis a prominent macrophage infiltration may mediate immunopathological damage, seizure induction, and death. however, the autoimmune response to non-replicating, non-infectious, but abundant, self antigen has a different disease progression, associated with differentiation of significant numbers of infiltrating monocytes into dendritic cells in the cns. whilst a predominant presence of macrophages or dendritic cells in the inflamed cns in viral encephalitis or multiple sclerosis is well described, the way in which the inflamed cns mobilizes monocytes in the bone marrow to migrate to the cns and the key drivers that lead to these specific differentiation pathways in vivo are not well understood. here we review the current understanding of factors facilitating inflammatory monocyte generation, migration and entry into the brain, as well as their differentiation towards macrophages or dendritic cells in viral and autoimmune disease in relation to their respective disease outcomes. monocytes, macrophages (mu), and dendritic cells (dc) are part of the 'mononuclear phagocyte system', also known as the 'reticuloendothelial system', found throughout the body. in normal tissues most mu and dc are considered to be 'tissue-resident', populating the tissue early in life, often specifically named. in the cns, the resident mu are microglia. they originate from the yolk sac [ ] and are renewed in situ [ ] . during inflammation, however, monocytes can migrate from the bloodstream into affected tissues, including the cns, where they differentiate into ''infiltrating'' mu or dc. whilst these resident and infiltrating cells may play prominent roles in the cns during viral or autoimmune disease, the methods by which the inflamed cns induce the mobilisation of monocytes in the bone marrow is poorly understood. moreover, the signalling events responsible for alternate monocyte or dc differentiation in the local inflammatory environment of the cns are not well described. as these signalling events dictate the nature and progression of the immune response to cns pathologies, an understanding of the mechanisms involved is crucial to identify novel targets for immune modulating therapy in these diseases. monocytes are one of the mononuclear cell types circulating in the blood that are produced in hemopoietic tissues of the bone marrow (bm) throughout life. they can be identified in human and mouse by flow cytometry, using a combination of cell surface markers. under normal conditions, the entire monocyte population is identified as cd + (including cd lo and cd hi subpopulations) in humans, and cd + cd b + in mice and humans. in both species, two principle populations and an 'intermediate' population are identifiable. classical (also termed 'inflammatory') monocytes are cd hi cd À in humans and ly c hi (cd lo ccr hi cx cr lo ) in mice and are the major monocyte population in the blood [ , ] . non-classical (also termed 'patrolling') monocytes, represent a much smaller subset (approximately %) of blood monocytes and are cd lo cd hi in humans, and ly c lo (cd hi ccr lo cx cr hi ) in mice [ , ] . the intermediate group is cd hi cd hi in humans and ly c hi cd hi population in mice. there are transcriptional similarities between humans and mice comparing their respective subsets, but functionally, mouse classical monocytes appear to be more related to human intermediate monocytes, based on their pro-inflammatory roles [ ] . during normal hematopoiesis in mice, the mu/dc progenitor (mdp) gives rise to a pre-dc [ ] and a recently-described common monocyte progenitor (cmop), distinguished from the mdp by its downregulated cd and upregulated ly c, although it still lacks cd b. the cmop differentiates into c-kit À cd + cd b + ly c + monocytes and in turn into c-kit À cd + cd b + ly c À monocytes [ ] . ly c hi monocytes are generated in the bm and during homeostasis they likely emigrate, but eventually give rise to ly c lo (cx cr hi ) monocytes [ ] . under homeostatic conditions, ly c lo cx cr hi monocytes patrol the luminal side of vascular endothelium in a programmatic, albeit peripatetic manner [ ] . during inflammation, however, ly c hi monocytes emigrate from the bm along the ccr -ccl axis into foci of tissue inflammation, differentiating into inflammatory mu, tipdc, or inflammatory dc, which may then migrate to the draining lymph node (dln), presumably transporting antigen acquired on the way [ , ] . while microglia are normally self-renewing [ ] , they may be supplemented and/or replenished by infiltrating ly c hi monocytes during cns infection and/or irradiative inflammation [ , ] and these immigrants become ly c lo on entry into inflamed tissue [ , ] . ly c lo monocytes are also recruited in later stages of inflammation where they are involved in tissue repair. these cells typically differentiate into m , i.e., anti-inflammatory, mu, supporting healing in the injured spinal cord [ ] . however, the developmental connection between these phenotypically similar but often temporally disparate populations is not completely clear. the mechanisms by which cns inflammation induces monocyte mobilisation in the bm are not well understood. the recruitment of bm monocytes during inflammation appears to depend on two initiating events: induction of emigration of existing bm monocytes into the circulation, and generation of new monocytes in the bm to replace the diminished population, which subsequently contribute to the emigrating monocyte population. monocyte generation relies on two processes, the 'pull' of a diminished downstream population, and/or the 'push' or direct stimulation of hematopoietic stem cells (hsc) or other progenitors. this process has been reviewed in depth elsewhere [ ] , but relevant factors are considered here. ccl is crucial for ly c hi inflammatory monocyte emigration from the bm [ ] . recently, bm stromal cells, but not hsc, were shown to secrete ccl in response to low levels of circulating toll-like receptor (tlr) ligands [ ] . this induced monocyte migration towards the vascular sinuses, and was dependent on myeloid differentiation primary response protein (myd ) (involved in responses to tlr ligand binding in all but tlr- [ ] ), but was independent of tnf and type-i interferon (ifn) expression. as the ccl -expressing cells also expressed various tlr, it was suggested that these cells function to detect infection and rapidly induce monocyte emigration into the circulation. whilst this provides some insight into the mechanisms of monocyte emigration, it does not explain the 'push' signal for monocyte production. this signal is presumably provided by direct inflammatory stimulation of hsc or other progenitors, inducing increased differentiation of self-renewing hsc into downstream progenitors [ ] . direct inflammatory modulation of hsc (which are ccr + ) by circulating tlr has been described, inducing such differentiation [ ] . furthermore, soluble immune-mediators may induce bm changes; seo et al. showed that ifn-a signalling to hsc was required for the generation of ly c hi monocytes in a model of viral pneumonia [ ] and type i ifn, produced in the spleen in response to infection with listeria monocytogenes, promoted monocyte emigration from the bm [ ] . interestingly, mice deficient in either myd or ifn-a receptor (ifnar) still had significant monocyte migration from the bm to the site of inflammation, whereas mice deficient in both did not [ ] . interestingly, in these studies, ifn-c did not play an important role in monocytopoiesis. however, in a model of chronic mycobacterium avium infection, ifn-c, but not ifn-a, was found to activate hsc, resulting in differentiation into downstream myeloid and lymphoid progenitors replacing the diminished populations [ , ] . on the other hand, in experimental autoimmune encephalomyelitis (eae), gm-csf produced by cns cells triggers monocyte mobilisation and emigration from the bm [ ] . in cns-initiated mobilisation of the bm, it is unclear if there is a difference in initiating events that predisposes to the preferential differentiation of monocytes towards a mu or dc phenotype, prior to entering the cns. the fact that tlr, type-i and -ii ifn, and gm-csf each induce monocyte emigration and the production of monocytes from progenitors in different situations may explain some differences in the manner of bm mobilisation by different cns pathologies. monocyte infiltration during viral encephalitis is rapid, with lethality in animal models occurring within days of initial infiltration [ , ] . the pathogenesis of eae, on the other hand, is chronic and/or relapsing and depends on t cell responses against myelin proteins that are initially induced by dc [ ] . nevertheless, a potential connection exists between viral and auto-immune causes of cns infiltration by monocytes. in multiple sclerosis (ms), there is evidence to suggest that reactivity against myelin proteins may occur following cns viral infection, if anti-viral responses cross-react with myelin proteins [ ] . the initial presence of viral rna or dna in the circulation could certainly induce monocyte emigration from the bm, via binding of intracellular rna by tlr within bm stromal cells [ ] , and tlr stimulation or direct infection of hsc could induce downstream differentiation of these cells [ ] . however, although tlr ligands may be present initially, recurrent episodes of ms would have few if any virus-associated tlr ligands. it is also possible that whilst type i ifn (ifn-a/b) and type ii ifn (ifn-c) are involved in both cns diseases, the innate differential production of ifn-a/b, crucial for virus control in the early response against cns viruses [ ] , by a variety of cell types during infection [ ] may induce monocyte mobilisation differently from that seen in ms/eae. high levels of ifn-c, which play a role in viral disease pathogenesis [ ] , is associated with the later development of the adaptive immune response, and may contribute here to the continued recruitment of monocytes. in contrast to acute viral infection, in eae that progresses as a relapsing or chronic condition, ifn-c production occurs over a long period of time, similar to the role of ifn-c in hsc stimulation in chronic infection of the lung [ , ] . as such, the differences in type-i and type-ii ifn production may differentially skew the monocyte phenotype towards a mu profile in acute infection and a dc profile in a chronic noninfectious setting. however, this has not been investigated experimentally. the separation of the cns from the peripheral circulation by the blood-brain barrier (bbb), limits the access of soluble factors and leukocytes to the cns. however, during inflammation various changes enable the recruitment of leukocytes into the brain. the process of infiltration involves detection of local chemokine gradients by susceptible migratory leukocytes in the vicinity of the affected cns parenchyma secreting the chemokines, rolling followed by firm adhesion to the local endothelium, and finally diapedesis and transmigration into the brain parenchyma [ ] . chemokines produced at the site of inflammation mediate chemotaxis of leukocytes passing through the neighbouring blood vessels to that site. the chemokine receptors expressed on monocytes include: ccr , cx cr , ccr , ccr , ccr , ccr , ccr and cxcr [ ] . of these, ccr , ccr , and ccr appear to be among the most important for migration. the ccr -ccl axis is the best-studied chemokine pathway in monocyte migration and infiltration into the cns. ccr , which is upregulated on monocytes in a variety of cns pathologies, binds to ccl (or ccl ), which is produced at high levels by infected neurons in animal models of wnv encephalitis [ ] and likely by glial cells in eae [ ] . ccl neutralisation in wnv encephalitis [ ] or ccl (or ccl ) deletion in bacterial infection [ ] result in reduced monocyte recruitment. whilst this resulted in higher bacterial loads, in the latter case, reduced monocyte infiltration during wnv encephalitis led to extended, but not permanent survival in mice [ ] . this highlights the severity of the immunopathology induced by infiltrating monocytes in the cns. ccl is highly upregulated in a variety of viral cns infections [ , ] and binds to ccr (expressed on ly c hi monocytes) and ccr . in ms, infiltrating monocytes express both ccr and ccr , with their ligands being expressed in the inflamed cns [ , ] . ccl and ccl are both upregulated in cns infection with wnv [ , ] . in eae, mrna and protein levels of cx cl and cx cr are elevated in the dorsal root ganglia and spinal cord [ ] . whilst these studies were focused on neuropathic pain, they highlight the potential for recruiting cx cr + non-classical monocytes to the inflamed cns during eae/ms. interestingly, in atherosclerosis, ccr lo monocytes did not rely on cx cr to enter plaques, instead using ccr to some extent, while ccr hi monocytes used cx cr , ccr , and ccr [ ] , emphasising the ability of monocytes to adapt in different disease settings. the entry of monocytes into the cns requires their margination and initial binding to endothelium, followed by firm adhesion to enable transmigration across the bbb into the cns parenchyma. ly c hi monocytes express a variety of cell surface molecules involved in adhesion to vascular endothelium in the cns, including l-selectin (cd l), p-selectin glycoprotein ligand (psgl ), lymphocyte function-associated antigen- (lfa- ), macrophage receptor- (mac- ), platelet endothelial cell adhesion molecule- (pecam- ), and very late antigen- (vla- ). these are reviewed in more detail elsewhere [ ] . cd l is required for entry into the inflamed peritoneum and is also critical for the migration of monocytes into the dln through high endothelial venules [ , ] . the high expression of cd l on ly c hi monocytes appears to be relevant for their entry into the cns in eae [ ] . this not only suggests a role for cd l in tissue entry, but possibly that monocytes predisposed towards a dc phenotype retain cd l expression when they migrate to the dln as efficient apc. during murine wnv encephalitis, the infiltration of pathogenic ly c hi monocytes correlates with the upregulation of vcam- and icam- on cns vascular endothelium [ ] , implicating vla- and lfa- , respectively, in ly c hi monocyte infiltration. vla- antibody blockade reduced monocyte infiltration by $ % and increased survival by up to % in infected mice. not surprisingly, as vla- antibody blockade is used in ms to reduce t cell infiltration, this treatment also reduced t cell infiltration into the cns in these animals, however, importantly, this was not sufficient to abrogate virus clearance. on the other hand, despite reducing monocyte infiltration by > % in these experiments, lfa- blockade had no effect on survival [ ] . it has been shown that patrolling of the luminal side of vascular endothelium by ly c lo monocytes is mediated by lfa- [ ] . this raises the possibility that interfering with lfa- -mediated interactions might prevent ly c lo , potentially m monocytes, from entering the cns, which may abrogate normal anti-inflammatory processes in viral encephalitis and enhance immunopathology mediated by infiltrating ly c hi monocytes. however, this was not explored in these studies. this study highlights the differential function of monocyte subsets using different adhesion molecule-integrin receptor pairs, and the importance of vla- use by ly c hi monocytes in cns invasion. moreover, this was the first study to demonstrate in vivo that carefully timed suppression of specific elements of the innate immune system during an acute lethal neurotropic infection could enhance survival by reducing immunopathology without interfering with the generation of immunity. inflammatory monocyte migration and subsequent differentiation into dc or mu are hallmarks of several immunopathogenic cns diseases, but the factors directing this differentiation have not been defined clearly. it has been suggested that mu and dc, as well as undifferentiated monocytes may cause immunopathology. however, the reported studies have not always distinguished unambiguously between monocytes and mu, which makes drawing firm conclusions difficult. invasion of the cns by a replicating virus results in local activation of resident microglia and astrocytes, with obvious migratory responses by these cells within the brain parenchyma, as well as the immigration of a range of leukocytes from the blood stream. this infiltrate typically contains monocytes, which differentiate into mu or activated microglial phenotypes in the brain, and these have been implicated in several diseases. thus, inflammatory mu infiltration precedes the onset and peak of disease symptoms in neurotropic coronavirus infection [ ] . in lymphocytic choriomeningitis virus (lcmv), monocytes (as well as neutrophils) play a highly pathogenic role [ ] . theiler's encephalomyelitis virus (tmev) infection results in major inflammatory monocyte infiltration into the cns within h and ultimately induces severe monocyte-dependent cns damage, with subsequent differentiation into activated mu linked to the development of cns lesions [ ] [ ] [ ] . several other neuroinvasive viruses, such as neurotropic mouse hepatitis virus (mhv), tick-borne encephalitis (tbe) and wnv, are associated with mu infiltration into the cns [ ] [ ] [ ] [ ] . inflammatory (ly c hi ) monocyte differentiation into ly c hi mu and/or activated microglia upon entry into the cns is a key feature of wnv encephalitis and plays a significant role in the pathology (and lethality) of this disease [ ] . inflammatory monocytes also play a role in mediating cns damage in autoimmune diseases such as amyotrophic lateral sclerosis (als) and ms [ ] . in eae, the mouse model widely used to study t cell-mediated autoimmune disease in general and ms specifically, inflammatory monocytes have a major impact. breaking peripheral tolerance to myelin proteins, leads to activation of myelin-specific t cells in secondary lymphoid organs. once these t cells arrive in the cns they become re-activated by apcs, resulting in the expression of pro-inflammatory cytokines, ifn-c, il- , gm-csf and tnf, as well as chemokines by t cells. the circulating ly c hi monocyte population, which expands exponentially before eae onset, represents a major proportion of the inflammatory cells in the eae cns and are dc precursors. although mu are observed in the cns of eae mice, dc are more efficient apc and activated dc co-localize with cd + t cells responsible for demyelination in the cns, implicating dc, rather than mu, in the amplification of responses [ , , ] . while both dc and mu may be present in the diseased cns in autoimmune or viral encephalitis, mu appear to have a more prominent role in viral encephalitis, while dc are more common in autoimmune diseases. interestingly, however, dramatically reducing immigration of inflammatory monocytes into the cns at particular timepoints in either of these diseases using negatively charged microparticles, which mediate sequestration and apoptosis of these cells in the spleen, abrogates the symptomatology and in the case of wnv encephalitis, results in up to % survival with immunity in an otherwise lethal disease [ ] . this suggests that these cells are very similar, if not the same, in the blood stream in both diseases, and that their differentiation is mediated in the cns, presumably by the prevailing milieu, despite their different fates there. alternatively, it is possible that separate mu and dc precursors may share a common receptor that mediates particle uptake, resulting in both being sequestered by the spleen with the same apoptotic outcomes. historically, monocyte-to-dc differentiation in vivo was hypothesized to be restricted to inflammatory scenarios, however, studies using fluorescent latex bead uptake to track circulating monocytes, indicated that during steady state conditions ly c hi monocytes differentiate into cd + dc, whereas ly c lo monocytes give rise to a cd b hi dc subtype [ ] . a separate study utilizing microspheres as monocyte markers, also suggests that the decision of monocytes to differentiate into mu or dc might not solely be determined by cytokines. the authors found that adding microspheres to phagocytic monocytes travelling to the lymph node (ln) induced differentiation into dcs, while monocytes staying at the site of activation became mu [ ] . the differentiation of monocyte-derived dc (mddc) in the absence of inflammatory stimuli is likely a result of basal levels of signaling factors present in this environment. these studies, while useful, do not account for the confounding possibility that the microspheres themselves, once phagocytosed, may have their own influence on the subsequent differentiation of monocytes [ ] . granulocyte-macrophage colony-stimulating factor (gm-csf) and macrophage-colony-stimulating factor (m-csf) are well known to influence the differentiation of monocytes in culture. gm-csf, also known as csf- , is secreted by a broad range of cells upon stimulation with cytokines, microbial products and/or antigens, and regulates survival, differentiation, and activation of target cells such as neutrophils, monocytes, mu and dc [ , ] . m-csf, also known as csf- , through its receptor, cd , influences several cell types and its effects include mediating the regulation, development, survival, proliferation and differentiation of mu [ ] . culturing human peripheral blood mononuclear cells (pbmc) with m-csf alone favors the differentiation of monocytes to mu. the outcome of pbmc culture with gm-csf, on the other hand, depends on the density of gm-csf receptors (gm-csfr) on the surface of the monocytes; low gm-csfr expression is associated with mu differentiation, higher gm-csfr density with differentiation towards dc. the addition of il- , which has a m-csf inhibiting function, overrides the effect of different levels of gm-csfr and blocks differentiation towards mu, thus favoring a dc phenotype [ , ] . gm-csf and il- function by downregulating cd on pbmc at a transcriptional level [ , ] . culturing human pbmc with gm-csf, ifn-c and il- skews the differentiation of monocytes to functional dc, which are not terminally differentiated but capable of presenting antigen to t cells [ ] [ ] [ ] [ ] . human monocyte-enriched pbmc cultured with only m-csf differentiate into immature mu but, with the addition of gm-csf and il- , convert to immature mddc. these dc lose their phagocytic ability and become potent apc [ ] . however, removal of these factors convert mddc back to a mu phenotype, indicating that the plasticity enabling differentiation into either mu or dc remains intact for some time. though culture with m-csf or gm-csf can both result in mu differentiation, mu obtained with different treatments exhibit different morphology. m-csf-stimulated mu (m-bmm) exhibit a spindle-shaped morphology, while gm-csf-derived mu (gm-bmm) appear more rounded [ , ] . the fate of monocytes in an inflammatory milieu is markedly different from their fate under homeostatic conditions. this can be seen with adoptively-transferred ly c hi monocytes, which give rise to regulatory mu in the non-inflamed colon, but become inflammatory dc capable of priming t cells and producing il- , il- , il- and tnf in colitic mice [ ] . the fate of monocytes in inflammatory scenarios such as eae or viral encephalitis is difficult to predict due to the intricate interplay between cytokines, chemokines and receptor-ligand interactions. as would be expected, simulating an ''inflammatory'' environment by adding pro-inflammatory factors alters the differentiation outcome. in m-csf-and gm-csf-directed differentiation of bm monocytes (bmm) to mu, lps induced high levels of pro-inflammatory cytokines, il- , tnf and il- , from gm-bmm, whereas m-bmm produced higher levels of il- and ccl . this raises the possibility that gm-csf-induced mu represent differentiation down the classical/m activated pathway and m-csf-induced mu represent differentiation down the alternative/ m regulatory pathway associated with il- production. these two mu populations were not terminally differentiated, as the addition or removal of m-csf or gm-csf to the relevant cultures results in an interchange between mu phenotypes [ ] . inflammatory stimuli, such as lps, combined with gm-csf and il- result in the maturation and irreversible commitment of mddc to mature dcs [ , ] . addition of tnf to immature dc in vitro reduces their apc capacity, possibly making them functionally mature [ ] . culturing human pbmc with m-csf, il- and il- shifts monocyte differentiation from dc to mu. however, these factors do not have the capacity to convert immature dc to mu or monocytes [ ] . current dogma suggests that gm-csf is crucial for the differentiation of mddc from inflammatory monocytes, and although this is true for several in vitro experiments, the in vivo scenario is markedly different. mddc differentiation in the absence of gm-csf was examined in several disease models in vivo, including infection with influenza a, streptococcus pneumonia, salmonella typhimurium, l. monocytogenes, lps stimulation and eae [ ] . csf- rb À/À and csf- rb À/À mice in all these inflammatory scenarios had similar inflammatory dc numbers compared to wild type (wt) mice. these dc were identified as fully functional tipdc, indicating that gm-csf is not required for monocyte accumulation and differentiation into dc during these inflammatory conditions. the authors did however detect high levels of m-csfr and m-csf levels on dc and in inflamed tissues, respectively. removing the m-csf signal by antibody blockade of m-csf or the excision of m-csfr allele did not alter the number of ly c hi monocytes infiltrating inflamed tissue. however, mddc numbers were significantly reduced, indicating that m-csfr signaling is likely critical for the differentiation of mddc in vivo but not for the recruitment of inflammatory monocytes [ ] . the importance of m-csf in dc differentiation was also confirmed in an inflammatory skin model. uv irradiation resulted in homing of circulating gr hi (ly c/g complex) monocytes to the inflamed dermis and epidermis, which differentiated into langerhans cells (lc) upon entry. m-csfr-deficient mice exhibited impaired lc differentiation and tissue mu development. although these mice had similar monocyte numbers homing to inflamed skin, they possessed significantly fewer lc, indicating that m-csfr is directly involved in monocyte-to-dc differentiation in the skin [ ] . during infection, inflammatory stimuli are not only derived from pathogens (e.g. lps from bacteria) but cells from the host also contribute factors changing the microenvironment, which directly influence monocyte differentiation. the differentiation of monocytes derived from the blood occurs once they reach target tissue [ ] and cross the endothelium [ ] . stromal cells like fibroblasts have been shown to impact upon this differentiation. although monocytes cultured with gm-csf and il- yield dc [ ] , the coculture of these cells with fibroblasts in steady-state conditions, skews the differentiation to mu. monocytes, which become activated when they cross the endothelium, secrete m-csf and stimulate the release of il- from fibroblasts. il- in turn increases the expression of functional m-csfr responsible for transducing the m-csf signal and thereby initiates mu differentiation [ ] . however, co-culturing these human pbmc with fibroblasts, il- , gm-csf and tnf, induces a terminally differentiated dc phenotype. this occurs because il- facilitates the utilization of m-csf, whereas tnf induces the internalization of m-csfr resulting in monocytes being unresponsive to autocrine il- and m-csf. these results suggest that the balance of the tnf/il- may be crucial in determining the fate of monocytes during inflammation [ ] . this is further supported by the overexpression of ccl in the brain induced by adeno-associated virus, which results in microglial activation and elevated il- and gm-csf levels [ ] . il- , which is involved in monocyte-to-mu differentiation, is elevated in the cerebrospinal fluid of jev-infected patients [ ] . activated murine microglia in jev and hsv infection release pro-inflammatory cytokines il- and il- b [ ] [ ] [ ] . this release of pro-inflammatory cytokines from microglia likely occurs via a rig-i-mediated pathway [ ] . infiltrating mu are also a major source of il- in tmev infection of mice; along with the il- produced by microglia this might induce monocyte to mu proliferation through an autocrine loop [ , ] . high levels of il- are present in the wnv-infected brain although it is not clear which cells produce it [ ] . other resident cells influencing monocyte differentiation include endothelial cells in the bbb, astrocytes, microglia and oligodendrocytes, which secrete tgf-b and gm-csf in inflammatory conditions. these factors promote human pbmc (cd + ) differentiation to cd + cd + (dc-sign + ) dcs, which secrete il- p , tgf-b and il- [ ] . another factor influencing monocyte differentiation is the presence of apoptotic cells, for example in influenza virus infection. these actively dying cells release soluble mediators, directing monocyte differentiation towards mu [ ] . the activation of the caspase pathway associated with apoptosis, has also been identified as playing a role in the fate of monocyte differentiation. caspase- deletion arrested the m-csf-induced differentiation of bm-derived monocytes to mu [ ] . caspase- and caspase- were specifically activated in human pbmc stimulated with m-csf to become mu but not by gm-csf and il- . treatment with a broad-spectrum caspase inhibitor induced a switch from mu differentiation to death, further implicating these proteins as crucial factors in the pathway of monocyte differentiation [ ] . human cytomegalovirus-stimulated monocytes relied on caspase- rather than the caspase- activation seen in m-csf-induced differentiation [ ] . this raises the possibility that infected neurons undergoing apoptosis may skew the differentiation of infiltrating monocytes towards a mu phenotype. in addition to resident infected cells, the pathogen itself also influences monocyte differentiation. differentiation of monocytes into either mu or dc in vivo can be triggered indirectly by the recognition of microbial ligands by pattern recognition receptors (prr) or directly by cytokine activation. prr are a very diverse group of receptors that function by signalling from a cell membrane or cytoplasmic location, or following endocytosis of pathogens in order to destroy them. tlr are important and abundant prr on monocytes that contribute crucially to the activation of innate and adaptive immune responses. binding of microbial or viral products by tlr results in dimerization of the receptors and the subsequent triggering of intracellular signalling pathways that operate via nf-jb and map kinase pathways and result in the production and release of cytokines potentially inducing mu or dc differentiation. both tlr / and il- b receptor signalling have been implicated in monocyte differentiation through the common myd signalling pathway. impairing tlr- signalling by using tunicamycin-induced er stress to suppress nf-jb activation, markedly suppressed the ability of lps-stimulated monocytes to differentiate into mu [ ] . tlr / -induced differentiation of monocytes to mu or dc relied on specific cytokine-receptor interactions, with il- and gm-csf inducing cd + mu and dc, respectively. il- b activation favored the proliferation of mu over dc, while cd + mu proliferating from il- b-activated culture showed enhanced phagocytosis of mycobacteria compared to tlr / -induced mu in culture [ , ] . in leprosy, lesions from patients with tuberculoid leprosy (t-lep) contain both cd + mu and cd b + dc, while in lepromatous leprosy (l-lep), lesions contain only cd + mu [ ] . in this disease, where tlr / becomes activated, it is the form of the developing disease that influences which effector populations differentiate from monocytes. dengue virus rna has been shown to co-localize with tlr- in a human monocyte cell line, which results in il- and ifn-a/b release. tlr- also plays a role in wnv, by restricting infection in neurons; its importance was confirmed in myd À/À mice, which show much faster viral spread in the cns than control animals. szretter et al. showed that monocyte-derived mu (and t cell) recruitment to the cns was reduced in the absence of myd [ ] . the production of tlr-pathway inhibitors by pathogens is a widespread immune evasion method; wnv ns- protein for instance blocks tlr- -induced nf-jb nuclear translocation and thereby prevents il- production, which plays a role in monocyte-to-mu differentiation [ ] . even in eae, in the absence of pathogens, tlr have been found to influence potential mediators of monocyte differentiation. tlr can be activated by endogenous ligands, which, for example, come from dying cells and thus contribute to autoimmunity and neurodegeneration [ ] . in mice immunized with mog peptide, treatment with , (oh) d resulted in a reduction of symptoms, inflammatory cell infiltrate and tnf, ifnc and il- expression. these findings correlated with a reduction of eae-induced tlr expression in the spinal cord of mice after , (oh) d treatment, especially of tlr . testing of , (oh) d effects in a human monocyte cell line confirmed the reduction in tlr expression and indicated lower mrna levels of tnf and il- b [ ] . activation of nod , an intracellular prr, by its ligand (nodl), also stimulates monocyte responses. netea et al. showed that il- directly induced the differentiation of monocytes to a cell type that exhibited the morphology and functionality of a mu but possessed some dc-specific markers [ ] . interestingly, nod l activation primarily induced the differentiation of human pbmc to cd b + dc, whereas tlr / activation resulted in both mu and dc populations. the dc derived from nod l-activated cells were superior apc to tlr / -induced dc. transfection with sirna resulting in the knockdown of il- rna, subsequently blocked the nod l-but not tlr / -mediated differentiation of pbmc into cd b + dc. in this study, increased il- mrna and nod expression in patients with t-lep correlated with higher cd b + dc numbers present in lesions. this identifies nod l-induced il- as a distinct pathway of dc differentiation in humans [ ] . thus, il- b seems to be a more potent stimulator of monocyte-to-mu differentiation, whereas tlr / and especially nod favor differentiation to dc. interferon regulatory factors (irf), which are activated by tlr and rig- -like receptors, are transcription factors playing a crucial role in host defense mainly by controlling the production of ifn [ , ] . increased irf expression can be found in several cns pathologies associated with mu or dc infiltration, although the extent to which these factors may be involved in modulating the monocyte differentiation in the cns has not been examined. irf- is crucial for the control of wnv infection and spread to the cns [ ] and along with irf- and - is upregulated in lymphocytic choriomeningitis infection [ ] . irf- is necessary to control viral replication and il- production in tmev [ ] and flaviviruses such as jev and dengue virus can induce irf- , - and - in culture, respectively. although in the periphery irf- is mainly expressed in pdc, in the cns irf- can be upregulated on neurons during viral encephalitis and correlates with type i ifn production [ ] . however, increased irf- gene expression is also present in eae cns disease progression, with the absence of irf- associated with increased severity of disease and mu infiltration [ ] . irf- is well known for its role in the differentiation of non-monocyte-derived dc and maturation of myeloid dc [ ] [ ] [ ] , as well as the normal differentiation of microglia [ ] . there are several examples of irf- and - directing monocyte differentiation to mu in vitro [ , ] . interestingly, irf- has been shown to negatively regulate tlr , which is constitutively expressed upon monocyte-to-dc differentiation. irf- and irf- compete for binding, which induces tlr promoter activity on mddc [ ] . irf- , in turn, has been associated with monocyte-to-mddc differentiation in vitro [ , ] . although no established link has been made between irf in the cns and monocyte differentiation, in vitro studies suggest that these factors might form a link in the sequence of events steering monocyte differentiation during viral infection or autoimmune disease. other effector cells recruited to the inflammatory milieu are stimulated to secrete factors influencing monocyte differentiation. ifn-c, which is secreted by nk cells and activated t cells, can suppress the differentiation of monocytes to dc by inducing m-csf and il- production from monocytes in vitro [ ] . however, ifnc has also been implicated in mu induction. nk cells isolated from the blood of patients suffering from rheumatoid arthritis and psoriatic arthritis induced differentiation of monocytes into dc in a cell contact-, gm-csf-and cd -dependent manner. these mddc differ phenotypically from dc obtained from in vitro culture of monocytes with gm-csf and il- , but still efficiently presented antigen and activated cd + t cells, which were polarized toward th [ ] . ly c + monocytes in trypanosoma brucei brucei-infected mice gave rise to tnf-and inos-producing inflammatory tipdc (cd À ). il- treatment significantly reduced this differentiation of dc from inflammatory monocytes [ ] . inflammatory monocytes expressing ly c and producing tnf and il- are crucial for the defense against intestinal infection by toxoplasma gondii (t. gondii) [ , ] . activation and function of this subset was severely impaired in t. gondii-infected cxcr knockout (ko) mice and disease symptoms were exacerbated. cxcr ko mice also show impaired recruitment of cd + t cells and production of ifn-c by these cells. cxcr was identified as important for cd + t cell trafficking and consequent ifn-c production in inflamed intestine. adoptively transferred ifn +/+ cd + t cells but not ifn À/À cd + t cells restored ly c + monocyte function and activation [ ] . nk cell-produced ifn-c may also regulate the differentiation of monocytes to dc, and il- production during intraperitoneal infection with t. gondii. these experiments identify ifn-c, produced by either nk or cd + t cells, as a key player in monocyte activation and differentiation to dc during inflammation [ , ] . in the autoimmune scenario represented by eae, cxcr ko mice also presented with exacerbated disease; however, in contrast to the effect of cxcr deletion in t. gondii infection, the number of t cells trafficking to the cns was not affected. in eae and ms, infiltrating activated t cells expressing cxcr are attracted to cxcr ligands, cxcl , cxcl and cxcl produced in and around the perivascular space and are thus restricted to this location. deletion of cxcr leads to uncontrolled spread of t cells throughout the cns but also reduces the recruitment of foxp + t cells and effector t cell interaction, and therefore results in more severe autoimmune-mediated tissue damage [ ] . non-activated inflammatory monocytes and inflammatory mddc are apc capable of stimulating t cells, which in turn produce gm-csf, inducing the differentiation of inflammatory monocytes to activated inflammatory dc. activated inflammatory dc are potent apc, capable of stimulating large numbers of antigen-specific t cells, which secrete more gm-csf, tnf and ifn-c. the resulting activation of inflammatory monocytes and dc leads to increased nitric oxide (no) production. no production by inflammatory monocytes, activated by a combination of ifn-c, gm-csf and lps, has been shown to suppress cd + t cells cultured with mog-peptide in vitro [ ] . however, it should be noted while no clearly has antiviral efficacy [ ] , sustained levels in vivo may cause disease and result in substantial bystander damage during wnv encephalitis either directly [ ] or indirectly, probably via ifn-c stimulation of inflammatory mu [ , ] . another effector function of cd + t cells in eae was revealed by co-culture of myelin specific cd + t cells with monocytes from eae mice; this resulted in monocyte upregulation of mhc-ii, cd c, cd and cd , and downregulation of ly c, indicating a shift toward dc phenotype. this was further confirmed by adding cd + t cells isolated directly from the spinal cord of eae mice to monocyte culture, resulting in monocytes from healthy animals upregulating dc markers and co-stimulatory molecules, becoming more granular, larger and forming dendrites [ ] . nkt cells are a group of regulatory immune cells of emerging importance, which mainly recognize lipids and glycolipids presented by cd d. their capacity to rapidly release an array of different cytokines allows them to influence the direction of the immune response. nkt cells secrete gm-csf, il- and ifn-c when they bind cd d [ ] , which is expressed on monocytes, inter alia. as a consequence, monocytes isolated from human blood are capable of inducing nkt cell cytokine production, which then drives monocytes to differentiate into dc [ ] . a more recent study suggests that during acute neuroinflammation in eae, monocyte differentiation is skewed to m mu by invariant nkt cell (inkt) activation with cd d induction and il- production. the switch from m mu to m mu resulted in improved disease outcome [ ] . although the factors determining monocyte fate are not completely understood, studies to date strongly suggest that multiple factors at the site of differentiation, including those secreted by resident and immune effector cells, play crucial roles in this process. clear differences between mediators in an infectious setting, such as viral encephalitis, and the autoimmune response, which drives eae, likely determine the outcome of monocyte differentiation into mu or dc, respectively. these differences may also affect the mechanisms by which the cns mobilizes monocytes in the bone marrow. as monocyte differentiation can have both protective and immunopathological outcomes in cns disease, it is crucial to gain better insight into defining factors that govern differentiation to inform more tailored approaches for intervention in these diseases. fate mapping 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coronavirus replication and enhanced encephalitis via impaired ifnalpha/beta induction in macrophages myelomonocytic cell recruitment causes fatal cns vascular injury during acute viral meningitis hippocampal protection in mice with an attenuated inflammatory monocyte response to acute cns picornavirus infection ccl transgene expression in the central nervous system directs diffuse infiltration of cd (high)cd b(+) monocytes and enhanced theiler's murine encephalomyelitis virus-induced demyelinating disease ifngamma influences type i interferon response and susceptibility to theiler's virus-induced demyelinating disease visualization of central european tick-borne encephalitis infection in fatal human cases inflammatory response in human tick-borne encephalitis: analysis of postmortem brain tissue infiltrating macrophages are key to the development of seizures following virus infection maturation and localization of macrophages and microglia during infection with a neurotropic murine 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infection rig-i mediates innate immune response in mouse neurons following japanese encephalitis virus infection the bacteriostatic protein lipocalin is induced in the central nervous system of mice with west nile virus encephalitis the blood-brain barrier induces differentiation of migrating monocytes into th -polarizing dendritic cells differentiation of monocytes to macrophages induced by influenza virus-infected apoptotic cells caspase- serves both apoptotic and nonapoptotic roles specific involvement of caspases in the differentiation of monocytes into macrophages human cytomegalovirus stimulates monocyte-to-macrophage differentiation via the temporal regulation of caspase er stress induced impaired tlr signaling and macrophage differentiation of human monocytes il- beta triggers the differentiation of macrophages with enhanced capacity to present mycobacterial antigen to t cells tlr activation triggers the rapid differentiation of monocytes into macrophages and dendritic cells the innate immune adaptor molecule myd restricts west nile virus replication and spread in neurons of the central nervous system west nile virus nonstructural protein inhibits tlr signal transduction targeting toll-like receptors: emerging therapeutics for multiple sclerosis management qin, , -dihydroxyvitamin d suppresses tlr expression and tlr -mediated inflammatory responses in monocytes in vitro and experimental autoimmune encephalomyelitis in vivo interleukin- induces the differentiation of monocytes into macrophage-like cells nod triggers an interleukin- -dependent human dendritic cell program in leprosy differential regulation of human interferon a gene expression by interferon regulatory factors and irf family of transcription factors as regulators of host defense interferon regulatory factor irf- induces the antiviral alpha interferon response and protects against lethal west nile virus infection differential regulation of interferon regulatory factor (irf)- and irf- gene expression in the central nervous system during viral infection irf helps control acute tmev infection through il- expression but contributes to acute hippocampus damage following tmev infection neurons produce type i interferon during viral encephalitis interferon regulatory factor- modulates experimental autoimmune encephalomyelitis in mice icsbp/irf- retrovirus transduction rescues dendritic cell development in vitro icsbp is essential for the development of mouse type i interferon-producing cells and for the generation and activation of cd alpha(+) dendritic cells essential role for icsbp in the in vivo development of murine cd alpha + dendritic cells ifn regulatory factor is a key constitutive determinant of the morphological and molecular properties of microglia in the cns icsbp directs bipotential myeloid progenitor cells to differentiate into mature macrophages monocyte differentiation to macrophage requires interferon regulatory factor critical role of irf- in negative regulation of tlr expression by src homology domain-containing protein tyrosine phosphatase- activity in human myeloid dendritic cells differential expression of ifn regulatory factor gene in human monocyte-derived dendritic cells and macrophages irf- expression in the human myeloid lineage: up-regulation during dendritic cell differentiation and inhibition by alpha, -dihydroxyvitamin d natural killer cells trigger differentiation of monocytes into dendritic cells il- dampens tnf/inducible nitric oxide synthaseproducing dendritic cell-mediated pathogenicity during parasitic infection nk cell-derived interferon-gamma orchestrates cellular dynamics and the differentiation of monocytes into dendritic cells at the site of infection cxcr -dependent cd (+) t cells are required to activate inflammatory monocytes for defense against intestinal infection cxcr signaling reduces the severity of experimental autoimmune encephalomyelitis by controlling the parenchymal distribution of effector and regulatory t cells in the central nervous system plasticity of ly- c(hi) myeloid cells in t cell regulation functionally distinct subsets of cd d-restricted natural killer t cells revealed by cd d tetramer staining nkt cells direct monocytes into a dc differentiation pathway activation of invariant nkt cells in early phase of experimental autoimmune encephalomyelitis results in differentiation of ly chi inflammatory monocyte to m macrophages and improved outcome tma is supported by and australian postgraduate award, cvv is supported by a university of sydney international scholarship, pn is supported by an international postgraduate research scholarship. this work was supported by nh&mrc project grant to njck. key: cord- -almkb zd authors: tan, donald t.h.; fong, allan title: efficacy of neural vision therapy to enhance contrast sensitivity function and visual acuity in low myopia date: - - journal: journal of cataract & refractive surgery doi: . /j.jcrs. . . sha: doc_id: cord_uid: almkb zd purpose to evaluate the efficacy and safety of neural vision enhancement technology (nvc, neurovision, inc.) to improve visual acuity and contrast sensitivity function in eyes with low myopia. setting singapore eye research institute, singapore, singapore. methods this noncomparative interventional case series comprised asian adults between and years of age with low myopia (cycloplegic spherical equivalence [se] from − . diopter [d] to − . d in the worst eye; astigmatism not exceeding . d in either eye; uncorrected visual acuity [ucva] ≤ . logmar) who had nvc treatment. the main outcome measures were distance ucva, uncorrected contrast sensitivity, refraction, accommodative amplitude, and safety. results all eyes had improvement in ucva and contrast sensitivity. after treatment, the mean distance ucva improved by a mean of . lines on the early treatment diabetic retinopathy study logmar chart. the mean contrast sensitivity improved over a range of spatial frequencies on sine-wave contrast sensitivity chart testing ( . to cycles per degree). follow-up data up to months posttreatment showed that the gains were retained. treatment did not alter refraction (mean spherical equivalent) or accommodative amplitudes. no adverse effects were reported. conclusion preliminary evidence suggests nvc treatment is safe and improves ucva and uncorrected contrast sensitivity in adult patients with low myopia. the term perceptual learning describes a process whereby practicing certain visual tasks leads to an improvement in visual performance. several studies of this phenomenon suggest that learning occurs as a result of modification of neural processes at the primary visual cortex in adults. [ ] [ ] [ ] [ ] [ ] the human visual system consists of a highly sophisticated optical processing system that begins when the cornea and lens conduct an optical image onto the retina and leads to a hierarchy of progressive levels of visual processing, starting from light detection and transduction in the eye by photoreceptors through several stages of spatial integration. each stage forms receptive fields of increasing complexity. contrast is one of the most important parameters activating cortical cells involved in vision processing. responses of individual neurons to repeated presentations of the same stimulus are highly variable, with signal and noise levels imposing a fundamental limit on the reliable detection and discrimination of visual signals by individual cortical neurons. [ ] [ ] [ ] [ ] neural interactions determine the sensitivity for contrast at each spatial frequency, and the combination of neural activities make up an individual's contrast sensitivity function (csf). the relationship between neuronal responses and perception are mainly determined by the signal-to-noise ratio (snr) of the neuronal activity, and the brain pools responses across many neurons to average out noisy activity of single cells, improving snr and leading to significantly improved visual performance. several studies show that the noise of individual cortical neurons can be modulated by appropriate choice of stimulus conditions. , polat et al. [ ] [ ] [ ] [ ] [ ] showed that contrast sensitivity at low levels can be increased through control of stimulus parameters. the typical building block of these visual stimulations is the gabor patch (figure ), which efficiently activates and matches the shape of the receptive field in the visual cortex. polat et al. found that contrast sensitivity in adult human subjects at low levels can be increased by a factor of through specific control of the gabor patch parameters. this stimulation-control technique is called lateral masking, in which collinearly oriented flanking gabors are displayed in addition to the target gabor image. when subjects are practicing contrast modulation under a very precise and subject-specific stimuli regimen, a significant improvement in contrast sensitivity is achieved. it is these neural modifications that are the basis of brain plasticity, which relates to the ability of the nervous system to adapt to changed conditions, sometimes after injury or stroke, but more commonly in acquiring new skills. brain plasticity has been shown in many basic tasks, with evidence pointing to physical modifications in the adult cortex during repetitive performance. several studies show that the plasticity of neural interactions resulting from repetitive performance of specific visual tasks lead to improved visual performance, with retention up to years of retesting. an increased range of neuronal excitatory interactions and reduced inhibition were observed in subjects with normal vision and in monkeys. these studies , pointed to activity-dependent plasticity of the visual cortex, where the specific connections activated throughout repetitive performance are modified, leading to improved performance, thus underpinning the concept of perceptual learning. work on perceptual learning by polat and others has been adopted for clinical use in the form of a computerized, internet-based perceptual learning program developed by neurovision, inc. the neurovision (nvc) correction technology probes specific neuronal interactions, using a set of patient-specific stimuli that improve neuronal efficiency and induce improvement of csf due to a reduction in noise and increase in signal strength, resulting in improved spatial resolution or visual acuity. initially developed for the treatment of adult amblyopia, nvc has been shown to be effective in inducing a sustained improvement in csf and visual acuity in patients who were part of a prospective randomized clinical trial of adult unilateral amblyopia. in this u.s. food and drug administration (fda) monitored prospective masked controlled study (fda (k) approval given in august ), adult amblyopic patients (mean age years) were randomized to amblyopic nvc treatment or a placebo vision-training program. pretreatment visual acuity in both study arms was . logmar, and this improved by . lines to / in the nvc treatment group, with no improvement in the control group. this increase in acuity was corroborated by a commensurate increase in csf to within the normal range. these improvements in acuity and csf were sustained after months. the efficacy of nvc treatment may be ascribed to the fact that amblyopia is characterized by several functional abnormalities in spatial vision, including reduction in visual acuity and csf that occur as a consequence of optical defects such as anisometropia or strabismus, which nvc treatment directly addresses. the reduction in csf, pronounced at high spatial frequencies, is believed to result from a low snr. a low snr limits performance on letter identification. generally, it is known that csf (especially at higher spatial frequencies) is closely related to resolution (visual acuity). patients with amblyopia also have abnormal neural interactions, reduced excitation, and increased inhibition, an effect that underlies deficient contrast response. finally, there is mounting evidence that neural plasticity persists in adult amblyopia , [ ] [ ] [ ] [ ] as in several studies of visual improvement in adult amblyopia in which visual loss in the good eye resulted in improved vision in the amblyopic eye. this challenges the current dogma that adult amblyopia is irreversible and untreatable. the nvc treatment has also been adapted to treat nonamblyopic cases of visual blur or optical defocus in individuals with low myopia. in simple myopia, in which a ''front-end'' optical defect in emmetropization develops after the critical period, there is often a mismatch between the optical defect and the neural connections formed during early childhood. the neuronal connectivity has developed normally and is capable of processing images efficiently; however, the visual input is subnormal and limited by optical defocus. the visibility of high spatial frequencies is perceived as low contrast even when their physical contrast is high. thus, csf is reduced at the high spatial frequencies, resembling the amblyopic csf, which, as a consequence, degrades visual acuity. in particular, reduced signal strength is expected to degrade letter identification, as demonstrated by solomon and pelli's study. in this study, the level of noise was systematically increased; this was followed by a parallel degradation in letter recognition. activation of neurons in the visual cortex is directly related to signal strength (contrast); when the effective contrast is low, neurons are weakly activated. thus, the snr is low, which limits performance on letter identification (visual acuity), as indicated in the previously mentioned study. we performed a prospective noncontrolled preliminary clinical study as a prelude to a formal randomized clinical trial to evaluate the efficacy and safety of nvc correction technology in improving uncorrected visual acuity (ucva) and csf in adults with low myopia. study subjects consisted of adults with low myopia of cycloplegic spherical equivalence (se) within the range of À . diopter (d) to À . d in the worst eye and with astigmatism not exceeding . d in either eye. inclusion criteria included a stable refractive state with no increase beyond . d over the past months, ucva better than . logmar, and best spectacle-corrected visual acuity (bscva) better than . logmar (early treatment diabetic retinopathy study [etdrs] logmar charts). exclusion criteria included ocular condition or cause of reduced visual acuity other than simple myopia and/or astigmatism, myopia-related ocular complications, diabetes mellitus, previous ocular surgery, pregnancy, and altered cognitive or emotional state that might impair the subject's ability to perform treatment. written informed consent was obtained from all study subjects before the study was initiated. the study and protocol conformed to the tenets of the declaration of helsinki and were approved by the singapore eye research institute review board. patients were chosen from healthy volunteers at the outram medical campus. study phases comprised a baseline screening and enrollment visit, nvc evaluation and initiation of treatment, an end-of-treatment (eot) examination, and follow-up visits for up to year. complete medical and ophthalmic histories were taken. this was followed by a baseline examination that included manifest subjective and objective refractions, cycloplegic subjective and objective refractions, accommodative amplitude (raf rule), distance bscva and ucva using m distance etdrs charts with a self-illuminating light box with a background luminance of to cd/m , uncorrected and corrected csf using a wall-mounted sine-wave contrast test (swct) chart (stereo optical co.) at m with controlled room lighting ( cd/m , within the range of to cd/m specified by the manufacturer), near visual acuity, slitlamp biomicroscopy, and fundus evaluation by binocular indirect ophthalmoscopy. neurovision technology involves automated proprietary algorithms delivered to treatment workstations. the neuro-vision data center was located in a secured hosting facility in singapore and is composed of a cluster of computer servers and databases, storing all relevant clinical and treatment data. for the treatment of low myopia, the nvc lm algorithm was used. algorithms are patient-specific and adaptive to subject response, thus providing analysis of each subject's dysfunctions, resulting in individualized treatment algorithms. improved visual function is achieved through repetitive performance of interactive visual perception tasks (vpts), in which subject-specific stimuli are administered on a treatment workstation. the nvc treatment may be clinic based or home based and adapted to the performance of each subject. in this study, patients were offered the option of home-based or clinic-based treatment. the primary stimulus used for treatment is the gabor patch. gabors are local gray-level gratings with spatial frequencies of . to . cycles per degree (cpd) modulated from a background luminance of cdm À (figure ). they are widely used in visual neurosciences and have been shown to efficiently describe the shape of receptive fields of neurons in the primary visual cortex. in all experiments, the standard deviation of the gabor is maintained equal to the wavelength (s z l). a philips multiscan p color monitor was used to project the interactive vpts using a standard personal computing system (hp compaq evo d or equivalent with intel p processor, mb sdram or more, nvidia vanta mb x agp graphics card or better, and windows xp operating system). the effective size of the monitor screen is cm  cm, which at a viewing distance of cm subtends an angle of degrees  degrees. subjects have treatment sessions in a dark cubicle or room in which the only ambient light is from the display screen. they wear headphones to hear auditory responses and to reduce external ambient noise. for home treatments, study investigators ensured similar conditions prevailed. before initiation of the training exercises, evaluation sessions were performed to measure the subject's specific basic spatial functions such as contrast sensitivity and spatial interactions, the latter representing degrees of cortical suppression and facilitation. from these sessions, initial treatment training parameters were selected. training sessions involved determination of contrast threshold attained, which was measured by a procedure in which the study subject is given a vpt and is required to choose between forced-choice alternatives. a typical vpt comprises consecutive displays; of the pair displays the target gabor the subject should identify. the target is presented in the first or second of images, each lasting to ms, at an interval of ms. the subject is seated . m from the screen and uses a -button mouse to activate the presentation of each pair of images at his or her own pace. with subjects instructed to keep both eyes open at all times, detection of the target gabor stimuli in the first of the images requires a ''left click'' response with the mouse, while detection of the target gs in the second of the images requires a ''right'' click for the correct response. subjects are informed of a wrong response by an auditory response. depending on the level of ucva, at the start of treatment, standardized myopic undercorrection in the form of training glasses of À . d or À . d or no glasses are prescribed for the treatment sessions. these glasses are subsequently reduced in power or removed as visual acuity improves. in some instances, to focus treatment visual training on the worst eye, a neutral density filter to fog vision over the better eye is used in the training glasses. subjects are presented with a series of gabor stimuli with the following parameters dynamically controlled: number of gabors, spatial arrangement, global and local orientation, target-flankers separation, exposure time, contrast, spatial frequency ( figure ) . treatment sessions involved contrast detection tasks with gabors, with and without flanking collinear high contrast patches. through the treatment sessions, the spatial frequency and orientation of the stimuli were changed, starting with lower spatial frequencies and progressively moving to the higher ones, with varied orientations at each size. thresholds for the contrast-detection task were measured with a oneup/three-down staircase (with steps of . log units), which was used to estimate the stimulus strength at the % accuracy level. after each treatment session was completed, the session results were automatically transmitted to the neurovision data center, where they were automatically analyzed. if the subject was progressing (ie, the performance was within a certain range), the next session parameters were generated toward increased levels of difficulty. otherwise, repetition might occur, but with a new set of parameters enabling the subject to perform fundamental tasks within normal ranges. in this way, subsequent sessions were determined on an individual response basis using the automated expert system algorithms and were dependent on the study subject's performance during the previous session in relation to a standard performance of emmetropic subjects with normal vision. the total number of treatment sessions was determined by the patient's visual response during the course of treatment. treatment sessions were approximately minutes in duration and were administered to times a week. after every sessions, the ucva and csf were tested to continuously monitor the subject's progress; treatments were continued until no further visual improvement occurred. maximum improvement is subject dependent, typically achieved in approximately to sessions over a course of months. constant monitoring of each study subject by the algorithms at the neurovision data center ensures real-time feedback with regard to the individual subject's compliance and performance. study noncompliance, which could result in study termination, included subjects absent for more than weeks and subjects who were performing fewer than treatment sessions per month. all study measurements and assessments were performed in an unmasked manner by institutional research optometrists at the singapore eye research institute myopia research clinic who were not directly involved with the study. the primary outcome measure was ucva measured by etdrs charts and the secondary outcome measure, csf using swct charts. subjects would have treatment termination according to any of the following eot criteria: . failure of treatment defined as no improvement in the ucva of . logmar or more than % of the required improvement from baseline in either eye at any of the first visual acuity tests (ie, after sessions). . improvement in visual acuity ceases: ucva no longer improves more than . logmar in either eye in the last visual acuity tests from visual acuity test onward. . subject has completed treatment sessions. following the eot decision, the subject was scheduled for the eot examination, which was a repetition of the baseline examination minus the cycloplegic refraction. if the eot manifest refraction differed from the baseline manifest refraction by more than . d, cycloplegic refraction was performed. subjects were requested to complete a quality of vision-quality of life (qov-qol) questionnaire upon termination of treatment. subjects visited the clinic every months for up to year posttreatment. at each visit, the subject repeated the eot examination with cycloplegic refraction performed at the -year visit. subjects were requested to complete a final qov-qol questionnaire at each follow-up visit. twenty asian adults ( men, women) with a mean age of . years (range to years) were recruited for the study. the mean subjective cycloplegic se was À . d in all eyes, with À . d in the worst eye, and c . d in the best eye. of the subjects, all of whom completed treatment, were available for the -month follow-up visit and returned for the -month visit; a major reason for the loss to follow-up related to the ongoing severe acute respiratory syndrome (sars) crisis in singapore that occurred during the course of the study. the sars outbreak precluded study patients from returning to the clinic due to severe infection control and isolation restrictions on patient mobility between hospital institutions during this period. at the baseline examination, the mean distance ucva in study patients was . logmar ( % confidence interval [ci], . - . ). at the eot examination, the mean ucva had improved to . logmar ( % ci, . - . ), a mean improvement of . log-mar lines (figure ). all eyes had gains in ucva, but to a varying extent (range . to . logmar lines and to letters). further analysis of the data showed that although all eyes showed an improvement in ucva, the eyes with worse ucva at baseline had greater improvement than eyes with better baseline ucva ( table ) . sixteen subjects completed months of follow-up after eot. ninety percent of the visual improvement in ucva was maintained (from . logmar before treatment to . logmar immediately after treatment and . logmar after months) at . logmar (figure ) . eleven subjects completed the full months of follow-up, with retention of % of the ucva improvement (from . logmar before treatment to . logmar immediately after treatment and . logmar after months) (figure ). mean csf improved posttreatment over a range of spatial frequencies by the swct charts. this improvement was sustained at the -month visit ( figure and table ). subjects were given subjective qov questionnaires at the baseline and at the end of the treatment. analysis of the answers showed that almost all subjects ( . %) reported less dependence on glasses or contact lenses after the treatment, with many of them ( . %) not using glasses or contact lenses at all (figures and ) . the mean se was not significantly altered after treatment. the mean baseline cycloplegic se and mean baseline manifest se were À . d and À . d, respectively. the eot mean manifest se was À . d. accommodative amplitude also did not alter significantly posttreatment (mean amplitude . d at baseline and . d eot). no complications occurred during the study. no treated eye had a drop in ucva or bcva, and no subject reported adverse effects. manifest and cycloplegic refractions remained essentially unchanged for the duration of the study. the study reports the efficacy and safety of nvc vision correction technology, a noninvasive perceptual learning computerized program to enhance the ucva and uncorrected csf in individuals with low myopia (!À . d). the mean improvement of . lines in logmar ucva was of sufficient magnitude clinically. if we assume measurement noise to be a difference of log-mar unit, the measured improvement of a mean of . logmar units would appear to be real and beyond measurement variability. for patients with relatively good ucva with initially low myopia, a recorded improvement is clinically important. in addition, this visual improvement was supported by the subjects' subjective impression as the qov questionnaire showed less dependence on optical aids after treatment. the increase in csf further corroborated the visual acuity improvement (table ) . it supports the original hypothesis of how the treatment works, and we are not aware of any other currently available treatment that can improve csf. also important is the consistent retention of effect of up to months. in retrospect, this is not that surprising in view of the ''memory effect'' of higher cortical learning programs. after all, one never quite forgets how to ride a bicycle. that compliance plays a role in the efficacy of treatment may also explain why some study subjects responded less effectively to treatment, and this remains a limitation to treatment efficacy. variability in efficacy may also be a reflection of different individuals' ''final cortical potential'' that could be achieved through the perceptual learning process, which in turn is dependent on the state of inherent neural plasticity. this study supports mounting evidence that neural plasticity is retained in the adult brain. despite the prevailing belief that adult brain cells do not grow and that whatever limited plasticity exists in the adult brain does not involve structural remodeling, recent cortical imaging studies using -photon laser scanning microscopy techniques are challenging traditional concepts. lee et al. confirmed the presence of dynamic structural remodeling and dendritic arbor growth occurring in gaba-positive nonpyramidal interneurons in the visual cortex of mice. holtmaat et al. recently reported dendritic spine growth in the mouse adult neocortex as a direct response to novel sensory experience by whisker trimming, providing evidence that sensory input might modulate structural and functional synaptic changes in specific neocortical circuits. as with all learning processes, the results will vary with the time and effort put in as well as the innate limits of each individual. compliance and concentration issues are important. follow-up studies may be designed to address these issues and find the optimal ''exposure dosing.'' however, individual effort and motivation will be expected to be different and have a resultant impact on variability of final result. limitations of this study include a small sample size, the absence of a comparative control group, and relatively subjective efficacy parameters. care was taken to reduce the memory effect of vision testing, which included rotating logmar charts used over a -month period and the use of sine-wave grating csf charts as apposed to letter-based charts. in conclusion, this study of nvc treatment in subjects with low myopia supports the hypothesis of perceptual learning in its ability to improve ucva and uncorrected csf. the results are highly encouraging and warrant further studies. beyond this study, we have begun a randomized placebo-controlled trial of adult subjects with low myopia recruited from military personnel in the singapore armed forces and have also initiated studies to assess nvc treatment efficacy in children with early myopia progression. studies of the utility of this treatment in enhancing vision and contrast sensitivity of postrefractive patients, patients with early presbyopia, and patients with mild to moderate degrees of foveal pathology are also in progress. these studies will further evaluate this perceptual learning technology and its prospective role in the enhancement of visual potential. baseline end treatment figure . dependency on glasses/contact lenses (''how many times a week do you wear your glasses/contact lenses?''). perceptual learning: learning to see perceptual learning reflects external noise filtering and internal noise reduction through channel reweighting mechanisms of perceptual learning the neural basis of perceptual learning learning to see: experience and attention in primary visual cortex striate cortex of monkey and cat: contrast response function the statistical reliability of signals in single neurons in cat and monkey visual cortex noise, neural codes and cortical organization bayesian analysis of identification performance in monkey visual cortex: nonlinear mechanisms and stimulus certainty the variable discharge of cortical neurons: implications for connectivity, computation, and information coding functional architecture of long-range perceptual interactions visual cortex neurons in monkeys and cats: detection, discrimination, and identification lateral interactions between spatial channels: suppression and facilitation revealed by lateral masking experiments the architecture of perceptual spatial interactions spatial interactions in human vision: from near to far via experience-dependent cascades of connections collinear stimuli regulate visual responses depending on cell's contrast threshold colinear facilitation promotes reliability of single-cell responses in cat striate cortex plasticity of spatial interactions in early vision santa fe institute studies in the science of complexity isolating excitatory and inhibitory nonlinear spatial interactions involved in contrast detection improving vision in adult amblyopia by perceptual learning abnormal long-range spatial interactions in amblyopia perceptual learning improves contrast sensitivity and visual acuity in adults with anisometropic amblyopia the visual filter mediating letter identification improvement of visual function in an adult amblyope visual improvement during psychophysical training in an adult amblyopic eye following visual loss in the contralateral eye visual recovery of the amblyopic eye in an adult patient after loss of the dominant eye prediction of improved vision in the amblyopic eye after visual loss in the non-amblyopic eye dynamic remodeling of dendritic arbors in gabaergic interneurons of adult visual cortex experience-dependent and cell-type-specific spine growth in the neocortex [letter] key: cord- -chwk bs authors: nan title: abstracts: poster session date: - - journal: ann neurol doi: . /ana. sha: doc_id: cord_uid: chwk bs nan an immune etiology has been postulated for acute cerebellar ataxia of childhood (acac) since it frequently follows viral infections. we analyzed serum and cerebrospinal fluid (csf) from acac patients for antibody cross-reacting with cerebellar neurons. serum and csf were obtained within days of onset of pancerebellar ataxia from subjects aged . to years. varicella infection preceded cases. results of enhanced cranial ct scans were normal; csf demonstrated - cells/mm with sterile cultures. serial dilutions from : of serum and undiluted csf were screened for antineuronal antibody by indirect immunofluorescence (iif) using frozen, unfixed normal human cerebellum. serum ( : ) was examined further for antineural antibody by western immunoblotting using purified cerebellar neuronal extracts as antigen. serum from age-matched, neurologically normal pediatric inpatients served as the control group for iif and immunoblot experiments. in acac patients, no antineuronal immunoreactivity was observed by iif. immunoblots demonstrated no consistent pattern of immunoreaction when comparing acac to controls, though patient exhibited distinct bands at kd (neurofilament protein) and kd. although antecedent infection suggests an immune etiology for acac, our preliminary results do not support a humoral mechanism for this disorder. ingrid taff; joseph zito, robert gould, and steven pavlakis, great neck and manhasset, ny in a -month period we studied patients between the ages of weeks and years with magnetic resonance angiography (mra). studies were performed on a . t magnet (siemens magnetom sp) with a circular polarized head coil. a three-dimensional time-of-flight technique was utilized. occasionally, images were obtained after gadopenetate dimeglumine infusion. two-dimensional projection images were calculated using a maximum intensity projection algorithm and recorded on laser film. sixty-seven patients also had routine mri. a sampling of vascular lesions was demonstrated. nineteen patients had clinical and mri evidence of stroke. mra revealed intracranial vascular occlusion in patients, diminished focal cerebral flow in the affected area in , and generalized ipsilateral underdeveloped cerebral circulation in . a moya-moya vascular pattern was found in and sickle-cell vasculopathy was found in patient. seven mras were normal. seventeen vascular hamartomas were demonstrated including vene of galen malformations, arteriovascular malformations, and venous angiomas. three aneurysms were found. thirty-one mras were normal. we find m u to be a valuable adjunct to routine mr imaging in the evaluation of pediatric patients with potential cerebrovascular disease. it demonstrates a spectrum of pathology, is noninvasive, and allows for serial follow-up examinations. angiography in pediatric cerebrovascular disease p . thalamic change in acute encephalopathy of adult rats. twelve weeks after grafting, clinical and histological studies were performed. we developed a protocol for evaluating functional deficits that follow spinal cord injury in the rat. the survival, growth, differentiation, and parenchymal integration of the graft were documented histologically on semi-thin section. animals that received the transplants demonstrated qualitative and quantitative improvements in several parameters of locomotion. donor tissue integrated most often with the host spinal cord at interfaces with host gray matter; however, some implants also exhibited sites of fusion with damaged host white matter. we suggest embryonic rat spinal cord transplantation may be a useful treatment of spinal cord injury and a possible therapeutic strategy in human spinal cord injury and amyotrophic lateral sclerosis. the basic neuropathophysiology of hemineglect after unilateral cerebral lesions is still not clear. one theory holds that degraded perceptual processing occurs in the damaged hemisphere due to intrahemispheric deficits. another holds interhemispheric interaction at fault, with the intact hemisphere actively inhibiting spatial cognitive processes in the damaged one. we tested adult macaca fascicuhris with acute neglect on a task in which the whole visual surround was restricted to degrees from central fixation, and a second in which an opaque lens occluded the eye either ipsilateral (ipsi) or contralateral (contra) to the lesion. using paired t tests, in the first task there were no differences in reaction time to the ipsi and contralesional hemifields. in the second, there was no change in extent of the ipsilesional field (obtained with the contralesional eye occluded), as compared to its extent without occlusion. the contralesional field, however, improved significantly ( p < . ) with the ipsilesional eye occluded. since reducing sensory input to both hemispheres leads to no worsening of hemineglect, but reducing sensory input to the intact hemisphere alone leads to improvement of hemineglect, we conclude that adverse interhemispheric interactions play a major role in the pathophysiology of hemineglect. we assessed the sensitivity and applicability of a new, cornbined cognitive and mood screening battery for multiple sclerosis (ms). sixty consecutive, untreated clinically active ms patients, each relapsing-remitting and chronic progressive, underwent the battery and head mri upon entering concurrent treatment trials. the battery combines the faust-fogel brief cognitive screen and visual analogue dysphoria scale, both previously validated in other neurological diseases. cognitive domains tested were immediate and delayed sentence and word-pair recall, verbal fluency, and conflicting response suppression. patients marked ''usual mood" along a "happy-sad" cartoon continuum. relapsing patients were program and abstracts, american neurological association younger ( . vs . yr mean), with shorter ms durations ( . vs . yr), and had lower kurtzke disability scores ( . vs . ). modified qualitative mri grading (lesion burden, confluence, localization) was compared. half as many relapsing patients ( % vs %) scored ''abnormal'' cognitively, despite similar "sadness" rates ( % vs %). subjective dys-~ phoria in both groups correlated with denser periventricular lesion burdens. the battery was well tolerated and easily administered within minutes without special equipment. this combined cognitive and mood screening battery is sensitive and convenient for clinically active ms. alternate forms of the battery are needed for repeatability. questionnaires may be reliable and valid supplements to laboratory tests for brain-damaged patients, as they can be applied to situations for which laboratory testing is not possible. we investigated the usefulness of informant-based data in alzheimer's disease (ad) by comparing caregivers' subjective evaluations of probable a d patients' performance on an abbreviated version of the memory self-report questionnaire to objective evaluations derived from an extensive battery of neuropsychological tests and to clinicians' evaluations. similar information was obtained from healthy agematched controls. caregivers' subjective appraisals of patients' memory correlated significantly with objective measures of secondary memory, with all cognitive variables, measures of activities of daily living, and clinicians' evaluations of dementia staging. scores were independent of clinical indicators of depression. the abbreviated memory questionnaire showed good reliability, internal consistency, and external validity. its positive predictive value is . and its negative predictive value is close to %. results suggest that ( ) informant-based questionnaires may be useful for obtaining valid information on cognitive ability outside of laboratory settings; ( ) the scale reflected more than just memory functions; and ( ) the scale may be promising for screening cognitive difficulties in epidemiological or clinical settings. although neglect along the horizontal dimensions of extrapersonal space is well recognized, there are only a limited number of observations documenting neglect along the vertical and radial spatial dimensions. we report an investigation of neglect along the principal dimensions of extrapersonal space in a patient with bilateral mesial temporo-occipital infarctions. neglect was assessed by asking the patient and controls to bisect lines of lengths oriented in directions with respect to the body: horizontal, vertical, and radial. our patient showed significant neglect of upper vertical and far radial space, as well as neglect of left hemispace. his line bisection errors were consistently in a direction opposite the slight directional biases shown by controls for all line orientations ( p < . ). the magnitude of the patient's bisection errors increased by moving the lines toward the neglected sectors of -dimensional space. neglect of upper vertical and far radial space was also evident on line cancellation tasks. our results suggest that following focal brain injury, neglect may be observed along all dimensions of extrapersonal space. these findings provide further empirical support for functional specialization within inferior and mesial temporooccipital regions for attending to upper vertical and far visual space (previc, ) . p . posterior cortical atrophy: degenerative disease with primary visuospatial and visuosemantic deficits a. kertesz, m . polk, and a. kirk, london, ontario, and saskatoon, saskatchewan, canada posterior cortical atrophy is a recent, and heidenhahn's disease is an old, label for a miscellaneous group of patients with imaging or pathological and clinical evidence of visuocognitive deficits and cortical atrophy localized to the posterior cortex. the extent of this cortical localization and the nature of the pathological findings are not fully agreed upon, but spongiform degeneration and alzheimer pathology have been described. detailed examination of patients who are representative of the problem and have uniquely specific deficits is presented. one patient had visual associative agnosia, prosopagnosia, and transcortical sensory aphasia. lexicosemantic experiments of categorization, word retrieval, and comprehension of auditory and visual stimuli showed a specific impairment of visuoverbal semantics. a striking preservation of phonological, orthographic and visual structural input, and intercategory dissociations was demonstrated. consistency of errors argued for specific loss of semantic knowledge. another patient with apraxia, primary visuospatial deficit, agraphia, and amnesia at the beginning had predominantly right-sided posterior cortical atrophy, demonstrating further fractionation of the entity and the striking specificity of visuospatial function. the behavioral specification of degenerative disease is clinically and theoretically important. permanent neurological deficits after ischemic stroke are mainly determined by the location and size of the infarct. clinical recovery also depends on the functional state of adjacent brain tissue, where both neuronal loss and deactivation without gross morphological damage may affect flow and metabolism to a varying degree (g. mies et al, stroke ; - ) , and where the ability to respond to stimulation by appropriate neuronal recruitment may be impaired. therefore, degree of resting hypometabolism and of responsiveness to functional activation may provide a measure of prognosis. in patients (age . . yr) with aphasia consequent to ischemic stroke of the dominant hemisphere, regional cerebral metabolic rate of glucose (rcmrgi) was measured at rest and in of them also during spontaneous speech, using positron emission tomography (pet) of -(f )-fluoro- -deoxy-~-glucose (fdg). the pet study and a standardized neuropsychological test battery to assess the main aspects of language were performed around the fourteenth day after the stroke, and the language functions were assessed again to months later. performances in various dimensions of language weeks and to months after stroke were related to rcmrgl in topographically meaningful areas at rest and during activation using wilcoxon-rank program and abstracts, american neurological association sum test and multiple regression analysis. severity of aphasia was assessed by the token test, which showed a bimodal distribution to slight and severe, and a lower representation of moderate cases. global and all regional cmrgl at rest and during activation were significantly correlated to scores in token test at first and second examination, with the highest correlation coefficients ( - . to - . ) for broca's, wernicke's, and left temporoparietal regions. for performance after to months, the relationships were still significant with lower coefficients. verbal fluency also was correlated to kmrgi, but with lower coefficients that slightly increased for the recovery state. language performance at different stages in the course after ischemic stroke was significantly related (r = . for token test, r = . for verbal fluency). however, there exists a high variability in recovery that may be explained by stepwise regression of metabolic values. significant effects were observed only for cmrgl of the left hemisphere outside the infarct (partial r' = . ) at rest and for cmrgl within the infarct ( . ), the contralat-era mirror region ( . ), and broca's region ( . ) during activation, with a sum of all partial weight factors of . at rest and . during activation. our results furnish indicators for recovery of aphasia: the resting metabolism of the left hemisphere outside the infarct, and the activated metabolism in residual tissue within the infarct and in languagerelated areas. although the hemispheric metabolism at rest might be related to neuronal loss and thereby to the brain's reserve capacity, the extent of metabolic activation indicates neuronal recruitment and the capability of neuronal networks for functional recovery. heparin therapy for acute myocardial infarction: the timi-i pilot and randomized trial combined experience m . a. sloan, t . r. price, m . l. tevrin, and s. forman for the timi investigators, baltimore, m d of , myocardial infarction (mi) patients treated with rt-pa and heparin, ( . %) developed ischemic cerebral infarcts (ci). all ci patients had detailed neurological evaluations and ( %) had c t scans. age range was to years (mean yr), were male, and were caucasian. electrocardiographic location of mi was anterior in ( %) and nonanterior in ( %). six cis occurred within hours; between and hours; between and hours; between and hours; during the second week; and others distributed over the weeks after study entry. six of cis did not involve cerebral cortex; ( %) had multiple cis. of cis thought to be embolic in origin, had at least cardiac abnormality (mural clot, wall motion abnormality, aneurysm, or transient atrial fibrillation) known to be associated more specifically with embolism than just the diagnosis of myocardial infarction. eight of ( %) with ct scans had hemorrhagic conversion of varying degrees. the time of occurrence and sites of ci after rt-pa and heparin therapy for acute mi are similar to those reported in the prethrombolytic area. nancy futrell andjeanne m. riddle, detroit, m i photochemical irradiation of the carotid artery of rats has been used to induce endothelial damage, producing a nonocc h i v e thrombus (that apparently embolizes spontaneously) thrombi and emboli and multiple cerebral infarcts. evidence for embolism generally has a presumptive component. to document further that cerebral infarcts in this model are indeed due to embolism, we studied the ultrastructure of the carotid thrombi and the presumed cerebral emboli using scanning and transmission electron microscopy (sem, tem). the right carotid artery of wistar rats was irradiated with a laser ( nm, mw/cm , min) following the injection of the photosensitizing dye photofrin , . mgikg. rats were sacrificed from to hours later. endothelial damage with formation of a fragmenting thrombus, composed mainly of platelets and erythrocytes (with no fibrin in most areas), was present in the carotid arteries of all rats by sem. sem was done on cerebral vessels, containing peripheral blood elements, with single ( ) and aggregated ( ) platelets (causing occlusion in ), single ( ) and aggregated ( ) erythrocytes (without occlusion), and single ( ) and aggregated ( ) leukocytes (without occlusion). tem demonstrated that the platelet aggregates did not adhere to the cerebral endothelium. the endothelial surface of all cerebral vessels was normal, which provided additional evidence that the mechanism of cerebral infarction in this model is embolism. model of repetitive ischemia: this effect is significantly enhanced when combined with mild hypothermia ashfaq shuaib and elisabeth sechocka, saskatoon, saskatchewan. canada there is considerable evidence that glutamate release resulting in activation of postsynaptic receptors (especially nmethyhaspartate) is a major mechanism of ischemic neuronal injury. in vivo experiments have shown that a more severe release of glutamate may be responsible for the excessive damage seen with repeated ischemic insults. we have shown that in cell cultures the effect of brief repeated insults is more severe than a single insult of similar duration. in the present study, we tested the protective effects of cgs- in a cell culture model of single ischemic and multiple-insult paradigm. in the multiple-insult paradigm, in some cultures cgs- was combined with mild hypothermia to see if this would offer additional protection. cgs- offered a dose-dependent protection in cell cultures exposed to a single ischemic insult. cgs- was protective to cultures exposed to repeated ischemic insults. the protective effects were enhanced significantly when they were combined with hypothermia, resulting in almost complete protection of the cultures. the combination of therapies appears to be a valuable strategy in neuronal protection during cerebral ischemia. toby i. gropen, i . prohovnik, t . k. tatemirhi, z. sharif; and m. hirano, new york, n y although a rare syndrome, mitochondria encephalomyopathy, lactic acidosis, and stroke (melas) may offer a unique insight into stroke mechanisms. we report novel observations in a patient with melas studied with serial and quantitative cerebral perfusion after stroke using """tc-ceretec spect and ' ixe rcbf. a -year-old man with melas presented with left-sided headache, generalized seizures, fluent aphasia, and right hemianopia. serial ct and mri showed infarction of the posterior left hemisphere in a multiterritorial distribution. spect performed days after stroke showed to % greater flow in the infarct than in normal brain, which reversed days after stroke. quantitative rcbf (m isi, reflecting mostly gray matter), when corpatients died. we recommend ct evaluation in all patients who have a seizure or lose consciousness during the peripar-tum period. despite intensive management, mortality is high. seizures should not be attributed to eclampsia without careful neurological assessment. when prenatal care is sought, women should be counseled about the dangers of cocaine to themselves as well as to their babies. changes in circulating blood volume following stephan a. mayer, matthew e. fink, laura lenniban, louise m. klebanoff; auis beckford, lsak prohounik, william young, and robert a. solomon, new york, n y reduction of blood volume (bv) has been implicated as a risk factor for delayed cerebral ischemia (dci) due to vasospasm after aneurysmal subarachnoid hemorrhage (sah). volume expansion guided by target filling pressures has gained popularity as a means of preventing or reversing dci; however, the adequacy of central venous pressure (cvp) as a reflection of bv in this setting remains unclear. we measured bv and cvp concurrently in patients ( males, females; mean age yr) day after craniotomy (mean . days after sah) and an average of . days later. the mean bv (mllkg) measured using chromium '-labeled red blood cells (rbcs) fell from . to . (normal range - ), a reduction of % ( p = . , paired student's t test). despite this, mean cvp (mm hg) remained unchanged ( . vs . ). similar reductions of plasma volume ( %) and rbc volume ( %) accounted for no change in mean hematocrit ( . vs . ). bv fell . % among grade iiiliv patients (n = ) compared to . % among grade / patients (n = ). a moderate correlation between bv and cvp ( r = . , p = . ) was found only with the first set of measurements. time-related alterations in venous capacitance, myocardial contractility, or systemic vascular resistance may explain our findings. axel rosengart, louis r. caplan, michael s. pessin, atherostenosis of the extracranial vertebral artery (ec-va) has rarely been studied systematically in series of patients with acute vertebrobasilar strokes or transient ischemic attacks. we identified by conventional angiography and neuroimaging (ct, mri, ultrasound, mr angiography) patients with ec-va disease among patients with posterior circulation ischemia. patients with cardiac sources of emboli were excluded. the probable etiologic mechanisms were: group a: vertebral artery origin (vao) atherostenosis with embolism- patients; group b: vao atherostenosis with hemodynamic spells- patients; group c: patient with vao atherostenosis and both intra-arterial embolism and hernodynamic spells; group d: ec-va dissection- patients ( unilateral, bilateral); patient had perioperative compromise of the ec-va with presumed intra-arterial embolism; group e: vao disease in addition to other distal vascular lesions- patients ( with intracranial va and with basilar artery [baf occlusive disease). ec-va disease is not always benign. vao atherostenosis and dissection of the ec-va are sources of intra-arterial emboli. hemodynamicrelated ischemia occurs with bilateral and unilateral va disease but is often transient. vao atherostenosis is often accompanied by severe occlusive disease of the intracranial va and ba. program and abstracts, american neurological association sebastian e. ameriso, vicky l. y. wong, andvas gruber, hidemi ishii, and mark fisher, los angeles and la jolla, c a , and kanagawa, japan hemostasis abnormalities are associated with ischemic stroke. these changes typically are demonstrated in antecubital venous blood samples and may not necessarily represent changes within the vasculature of the brain. the purpose of this study was to identify potential differences in hemostatic profile from samples of cranial versus noncranial venous sites in patients with acute ischemic stroke. eight patients were studied within days of acute brain infarction. some patients were studied on separate days. blood was drawn from the external jugular vein and immediately thereafter from an antecubital vein without the use of tourniquet. we measured hematocrit, leukocyte count, platelet count, fibrin d-dimer (cross-linked fibrin fragment), plasminogen activator inhibitor- (pai- , an important antifibrinolytic protein), and anticoagulant proteins thrombomodulin and activated protein c. a jugular-to-antecubital ratio was calculated for each paired blood sampling. thirteen paired samples were obtained from the eight patients. external jugular-to-antecubital ratios (mean to-antecubitat ratio for pal was significantly different from ( p < . ), with higher concentrations in jugular samples. in conclusion, levels of hemostatic proteins measured from cranial venous blood may differ from antecubital samples in patients with acute ischemic stroke. in animal models of transient cerebral ischemia, the effects of repetitive insults are more severe than a single ischemic episode of similar duration. we used the cell culture model of ischemia to determine if the effects of repetitive ischemia are similarly more severe in this model of ischemia. for cell culture, we used fetal mice cortical astrocytic and postnatal cerebellar (glutamatergic) granular neurons and cerebral gamma-aminobutyric acid (gaba)ergic cells. lactic dehydrogenase (ldh) (activity per gram protein) release in the medium was used as a measure of cellular damage. compared to a single insult, there was a large increase in ldh release during repetitive ischemia in astrocytes ( vs , p < . ) and granular cells ( vs , p < . ) (highly significant) and a modest (but significant) increase in the cortical neurons ( . vs . p = . ). the demonstration that repetitive ischemia produces more severe damage in cell culture would suggest that the mechanisms are not predominantly vascular. cell culture could prove useful to study the mechanisms of neuronal damage with repetitive ischemia. we studied the spontaneous recovery of neurological function after acute ischemic stroke using a standardized stroke nih stroke scale scale ( n i h stroke scale) to assess the extent of improvement, differences in stroke types, and early predictors of later outcome. we performed serial neurological assessments on admission; , , and hours after admission; and to days and > days after admission. twenty-six patients had presumed embolic occlusion of the middle cerebral artery (mca) and had a clinical diagnosis of lacune. admission score was better in the lacune group compared to the mca group. the mean scores for all patients improved by the to -day and the > -day examination, but the degree of improvement was greater in the mca group than in the lacune group at > days ( p < . ). the degree of change at to days correlated with the change in score at hours ( r = . , p < . ) and hours ( r = . , p < . ). most patients improve after acute ischemic stroke, but to variable degrees and at different rates. david w. desmond, thomas k. tatemichi, miguel figueroa, dew'itt t . cross, and yaakov stern, new yovk, n y to investigate the effects of lacunar infarction (li) on cognitive function, we examined li patients months after stroke (age = . ? . yr; education = . . yr) and stroke-free nondemented control subjects (age = . k . yr; education = . k . yr) with a battery of neuropsychological tests. li was defined as a presenting infarct of cc and a mean volume of any additional subcortical infarctions of cc on ct scan. using multiple regression analyses, with significance set at p < . to minimize the risk of type i error, we considered the role of li as a correlate of performance in multiple cognitive domains. controlling for the effects of demographic factors, vascular risk factors, alcohol use, and depression within the multivariate models, li was a significant independent correlate of deficits in memory (( = -. , p = . ), verbal (p = -. , p = . ), visuospatial (p = -. , p < .oool), abstract reasoning (p = -. , p = . ), and attentional skills (p = -. , p < . ). we further investigated the effects of infarct number, volume, and location, as well as atrophy, on global cognitive function within the li group. the only significant independent correlate of global cognitive performance was a preponderance of left-hemisphere infarctions (p = -. , p = , ). these results suggest that li may produce dysfunction in multiple cognitive domains, particularly when the left hemisphere is differentially involved. p . increased intracranial atherosclerotic stroke in hispanics and blacks from northern manhattan ralph l. sacco, christina zamanillo, t . shi, andj. p. mobr, new york, ny intracranial atherosclerosis has been found to be more frequent in blacks compared to whites, whereas hispanics have rarely been characterized. among consecutive patients from northern manhattan over age hospitalized at the presbyterian hospital from to , cerebral infarction occurred in whites, blacks, and hispanics. all patients had at least one c t scan, % had duplex doppler, % transcranial doppler, and % angiography. strokes were classified as atherosclerotic (ath), cardioembolism, lacunar, and as infarcts of undetermined cause. ath was further subdivided into extracranial (eath) or intracranial (iath) . overall, the frequency of ath was similar in the three racelethnic groups (white , black ' , hispanic ) . the distribution of the atherosclerosis, however, was different in whites compared to blacks and hispanics. whites had more eath stroke than blacks and hispanics (white %, black % , hispanic lo%), while iath was similar in blacks and hispanics and greater than in whites (white , black , hispanic %). nonwhites have more iath stroke than whites. the similarity in the distribution of atherosclerosis between blacks and hispanics argues for shared environmental risk factors, rather than genetic differences. ethnic differences in stroke risk factors may help explain differences in infarct subtype. we studied mild to moderate alzheimer's disease (ad) patients with a series of " water bolus positron emission tomographic (pet) activation studies, and compared them to similar studies in age-matched normal controls. for each group, pet images were mapped onto the subjects' mri scan, and results of a particular activation condition were averaged across the group. naming a series of pictures (line drawings of animals) minus counting abstract designs as a baseline produced strong activation of the anterior cingulate gyrus only in the ad group. silent reading of words minus viewing a baseline series of "xs" similarly showed strong activation of the anterior cingulate gyms in the ad subjects but not the normals. naming block (activation condition) of % unnamed pictures, minus a second block (baseline) of easily named pictures, demonstrated much greater cingulate activation in the ad patients, for naming of the more difficult pictures. we conclude that this cingulate activation may reflect the greater involvement of an attentional network (of which the anterior cingulate is a part) in tasks requiring a higher degree of "mental work" on the part of ad patients. dementia in alzheimer's disease j. w. pettegrew, k. panchalingam, w. e. klunk, and r. j alzheimer's disease (ad) predominantly affects the brain, resulting in the loss of multiple cognitive abilities. some studies suggest the membranes of peripheral cells are involved in the disease. to investigate erythrocyte membrane molecular dynamics in ad patients and age-matched controls, we investigated erythrocyte membrane molecular motion at the surface (fluorescamine), aqueous-hydrocarbon interface (dppe-ans), and hydrocarbon core ( j-as; ppc-dph) by steady-state fluorescence anisotropy measurements of probable ad patients ( males; females) and ( males; females) age-matched controls. cognitive function was assessed by the mini-mental, mattis, and blessed scales. we found that intergroup comparisons revealed decreased motion at the surface ( p = . ) and aqueous-hydrocarbon interface ( p = . ) and increased motion in the hydrocarbon core ( p = . ) of the moderately to severely impaired ad patients compared to the controls. in the ad patients, there were significant correlations between decreasing membrane surface motion and worsening blessed scores (males p = . ; r = . ; femalesp = . ; r = . ). these findings suggest that molecules are being produced in the brain of ad patients that gain access to the circulation. these molecules insert into the erythrocyte membrane and secondarily alter erythrocyte membrane molecular motion. the production of these molecules correlates with the dementia and could contribute to the molecular pathophysiology of the disease. parkinson's disease (pd) and alzheimer's disease (ad) are common disorders of old age and may therefore coexist. the prognosis in demented pd patients is poor and early recognition of such cases is therefore desirable. the objective of this study was to identify characteristics that distinguish pd + ad from pd patients during early stage. all patients were clinically evaluated over a -year period . clinical diagnosis of dementia was made only when unequivocal clinical evidence of progressive decline in memory and cognitive function was documented, and pathological diagnosis of ad and pd was made using standard criteria. twentysix patients who had only pd or pd + ad were identified; had no dementia and at autopsy had pd. six patients had clinical evidence of parkinsonism and dementia and at autopsy had distinct pathological findings-pd and ad. these cases could be classified as having simultaneous or sequential evolution of pd + ad. those with sequential onset had pd before age years but were inexplicably functionally disabled early on, whereas those with simultaneous onset manifested pd after age years. pd + ad patients had rapid disease progression, shorter survival, poorer drug response, and more side effects of levodopa than pd patients. to study the prevalence ofwhite matter lesions in the general elderly population, and to investigate whether white matter lesions were relatively frequent in subjects with classic vascular risk factors and with hemostatic risk factors, magnetic resonance scans were obtained of participants, aged to years, of the rotterdam elderly study. the subjects for the imaging study were a random sample from the general population, stratified by age and gender. t -weighted images were obtained in the axial plane. white matter lesions were considered present when moderate or severe periventricular hyperintensities or when more than small focal lesions or focal confluent lesions were found. overall, % of subjects had white matter lesions. the prevalence and severity of le-program and abstracts, american neurological association sions increased with age. history of stroke or myocardial infarction, presence of peripheral arterial disease, factor viic activity, and fibrinogen level were each significantly and independently associated with the presence of white matter lesions. significant relations with actual systolic as well as diastolic blood pressure, with a history of hypertension, and with plasma cholesterol were observed only for subjects between and years. this study suggests that white matter lesions in the elderly may be related not only to the classic cardiovascular risk factors. but also to hemostatic factors. joan m. swearer, paula nelligan, hanno muelher, beatrice woodward, and david drachman, worcester, ma although behavioral disturbances occur frequently in alzheimer's disease and other dementing disorders, little is known about the factors that predict their development or predispose to their occurrence. in the present study we examined sets of possible predictive/predisposing factors retrospectively for behavioral disturbances in mildly to severely demented, community-dwelling patients. the factors examined included: individual distinguishing features (age, gender, age of onset, premorbid personality traits, prior psychiatric history) and dementia severity (dependence in activities of daily living [adls) and self-care, duration of dementia, global disease severity). spearman correlations and t tests were used to assess the relative influence of these factors on the occurrence of types of aberrant behaviors: aggressive behaviors, disordered ideation, and motor abnormalities. forty percent of the patients exhibited aggressive behaviors, % exhibited disordered ideation, and % had motor abnormalities. neither a prior history of psychiatric disorders nor premorbid personality traits were associated with the occurrence of the target behaviors. dependence in adls and self-care and greater global severity were associated ( p < . ) with the frequency and severity of aggressive behaviors, disordered ideation, and motor abnormalities. these results suggest that severity of dementia is a consistent and reliable factor in the development of aberrant behaviors, whereas preexisting personality traits are not. dementia of the alzheimer t y p e w. j. burke, a. ranno, w. h . roccafrte, s. p. wengel. b. l. bayer, and n . k. willcockson, omaha, ne l-deprenyl is an irreversible inhibitor of mao-b that has been reported to cause modest improvements in short-term memory and behavioral symptoms in persons with dementia of the alzheimer type (dat). thirty-eight subjects meeting research criteria for mild d a t were enrolled in a placebo-controlled, double-blind trial of r-deprenyl at a dose of mg twice a day. subjects underwent extensive clinical and neuropsychological assessments at entry, and at and months. after months, subjects taking both l-deprenyl and placebo showed a significant decline in their scores on the mini-mental state examination, the clinical dementia rating (cdr) scale, and the sum-of-boxes score derived from the cdr. when the change in scores on these clinical measures was examined across the groups, there was no significant difference. there were no significant differences within or between groups on several behavioral measures including the brief psychiatric rating scale and the cornell rating scale for depression in dementia. neuropsychological testing demonstrated no significant differences berween groups based on mean score change. l-deprenyl did not affect cognition or behavioral symptoms of dat in this -month study. k. marder, m-x. tang, r. ottman, l. cote, y. stern, and r. mayeux, new york, n y the etiology of dementia in parkinson's disease (pd) is probably multifactorial but there may be a shared susceptibility for p d and alzheimer's disease (ad). reliable risk factor interviews were conducted with informants of nondemented p d patients (pd-d) and demented p d patients (pd + d) enrolled in a longitudinal community study of pd. p d + d were older ( . yr) than pd-d ( . yr) and had later age at onset of motor signs ( . yr) than pd-d ( . yr) ( p < , ). the frequency of smoking, alcohol use, head injury, and family history (fh) of pd did not differ but fh of ad was significantly more frequent in the pd + d group (or . , ci . - . ). using stepwise logistic regression, only age of onset of motor signs (or . ), education < years (or . ), and the interaction of age of onset of motor signs and fh of a d (or . ) were independent predictors of dementia in pd. to address variable years at risk for development of dementia, life table analysis revealed the cumulative risk of a d to age in first-degree relatives of p d + d was . , and . in pd-d relatives ( p < . ). cox proportional hazards analysis controlling for the differences in ages of the relatives of both groups yielded a rate ratio of . (ci . - . ) for the development of a d among p d + d compared to pd-d relatives. we conclude that a genetic susceptibility to a d may raise the risk for dementia in patients with pd. differentiated from alzheimer's disease? john c. mowis, elizabeth grant, rita canfield, eugene rubin, and daniel mckeel, jr, st louis. mo vascular dementia (vd) is believed to account for to % of all us cases of dementia; however, pathologically confirmed cases are quite rare. this discrepancy suggests that current diagnostic criteria lead to the clinical overdiagnosis of vd. twenty v d subjects (mean age . yr; men, women) were diagnosed solely on the basis of the presence of dementia, a history of stroke(s), and a documented relationship of stroke to onset andlor course of dementia; ischemic scores (is) and neuroradiographic findings were not used for diagnosis. compared with subjects (mean age . yr; men, women) with dementia of the alzheimer type (dat), there were no significant group differences for comparable clinical dementia rating stages of dementia for measures of language, activities of daily living, or general cognition. the vd group scored significantly higher than the dat group on the modified is (f [ , ] = . , p < , ). all autopsied d a t subjects had verified alzheimer's disease (ad); also had cerebral infarctions. the autopsied v d subjects had , , and cc of brain tissue affected by stroke; ( cc) also satisfied histological criteria for ad. we conclude that ( ) the clinical features of vd and a d overlap considerably; ( ) diagnostic criteria based on the temporal association of stroke with dementia may have predictive value for vd; and ( ) the frequent coexistence of a d and strokes indicates that refinement of criteria is needed to distinguish "mixed" and "pure" vd. clinicopathological correlation remains essential for any study of putative vd. left-handedness has been proposed as a marker for decreased survival in the general population, but possible effects of handedness on longevity in alzheimer's disease (ad) have not been examined. we hypothesized that left-handed ad patients would evince more rapid deterioration and therefore die at an earlier age than right-handed patients. subjects were demented patients consecutively confirmed at autopsy to meet nincds-adrda criteria for "definite" ad. handedness was determined from structured interviews with primary caregivers and validated for most subjects with the edinburgh inventory of handedness. age at onset of dementia symptoms retrospectively determined by caregivers was used to calculate the duration of illness at the time of death. because of reported gender differences with regard to longevity, we first partialled out effects of gender before using hierarchical regression procedures to test the hypothesis. four of men and of women with definite ad were left-handed. the mean age at onset did not differ significantly between handedness groups (f [ l,loo] = . ), but the mean duration of symptoms ( alterations in the optical properties of brain can be used to detect pathological changes in patients with alzheimer's disease (ad). using time-resolved spectroscopy (trs) and phase-modulation spectroscopy (pms), we measured the absorption (ua) coefficient, scattering (us) coefficient, and mean photon pathlength (pl) of red light directed through the base of the frontal lobes of patients with ad and age-matched control subjects. the measured values and the asymmetry index (ai) (an indication of the symmetry of the measurements between the left and right side of the brain) were correlated with the severity of disease as determined by mini-mental state score. there were significant differences between the ad and control group for ua, us, pl, and the standard deviation of ai. there was no correlation between the mms score and ua, us, or pl. however, the highest asymmetry index values were seen in moderately impaired patients , which suggests that the asymmetrical nature of the pathological process detected by optical spectroscopy is most marked during this stage of the illness. this noninvasive technique may provide a convenient method to detect and monitor the pathological changes that occur in the brain of patients with ad. disease p . memory impairment in very mild alzheimer's a memory impairment is often the earliest indication of alzheimer's disease (ad). we investigated components of disease learning and recall to determine which aspect of memory function is impaired the earliest in incipient ad. using the mayo clinic alzheimer's disease patient registry, which is a longitudinal prospective project on ad and normal aging, we identified patients with very mild ad (i.e., with a mini-mental state score of or greater) and age-and sex-matched controls. we assessed performance on memory measures: the rey auditory verbal learning test and the buschke free and cued selective reminding test (fcsrt). the parameters evaluated included a measure of acquisition, total learning over trials (tl), and delayed recall (dr). on the fcsrt, an index of facilitation of performance with semantic cues (sc) was assessed. results indicated that all indices, tl., dr, and sc, were capable of separating the mild ad group from the controls ( p < , ). using a linear discriminant analysis with stepwise variable entry, the measure that assessed the patient's ability to use semantic cues (sc) was the most sensitive parameter for separating the groups ( f = . , p < . ), and the acquisition parameter (tl) was also useful at adding some additional predictive power (f = . , p < . ). the delayed recall measure, however, did not add anything to the previous measures. it appears that very early ad can be detected using appropriately structured memory tasks, and these procedures can be helpful in identifying at-risk individuals. alzheimer's disease (ad) has an insidious onset that is difficult to date reliably. we developed a standardized interview to provide objective criteria for dating the onset of different symptoms (memory complainr, performance problems, language deficits, disorientation, depression, behavior problems, and psychosis), yielding an estimated disease onset date. inrerrater reliability (icc = . ;p < , ) and interinformant reliability (icc = ; p < . ) for the onset of first symptom was high. interrater agreement for the order in which symptoms appeared was high (icc = . - . ) as was interinformant reliability for all symptoms except memory complaint. the interview was administered to patients with ad. mean estimate duration of illness was . years k . years and correlated significanrly with problems in instrumental activities of daily living. sixty-six percent had memory complaint and % had performance problems as their initial symptom. this technique provides a reliable characterization of disease onset. longitudinal studies will determine if particular onset symptoms differentially predict disease progression. the purpose of this study was to determine whether there is an excess of white matter disease (wmd) in alzheimer's disease (ad). brun and englund ( ) reported an excess of wmd in brains of patients with ad vs age-matched controls. there have been reports both confirming (bowen et al, ; fazekas et al, ) and refuting (leys, ) these findings using ct and mri in patients with clinically diagnosed ad. postmortem t -weighted mri scans and program and abstracts, american neurological association neuropathology were graded on brains of pathologically confirmed ad subjects and brains of age-matched neuropathologically normal controls. white matter lesions were scored on a to scale (none, mild, moderate, severe) separately for periventricular (pvl) and deep white matter (dwm) areas in mri scans and lux fast blue (lfb)-stained brain sections. correlations between mri and neuropathology were good ( r = . for pvl, r = . for dwm). pvl scores were higher in ad than in normal subjects on mri (ad: . l . vs controls: . rfr . ; p < . ). on pathology the difference in scores did not reach significance (ad: . . vs controls: . * . ; p = . ). similarly, dwm scores were higher in ad subjects than normals on mri, but not neuropathology. in conclusion, ad brains have a significant excess of wmd on mri compared to controls. although the pvl and dwm scores for pathological sections are not different in the groups, mri is much more sensitive than lfb-stained sections for wmd. thirteen autopsy cases of progressive supranuclear palsy (psp) were investigated for clinical-neuropathological correlations and heterogeneity. we reviewed clinical records of men and women aged to years (mean age yr) with disease duration ranging from to years. most patients had classic features of psp including ophthalmoplegia, postural instability, and extrapyramidal signs. dementia was eventually observed in of the patients ( %). six of patients ( %) on whom adequate initial documentation was available presented with memory loss or behavior change. five of the patients ( %), including with an initial presentation of memory loss, were diagnosed clinically as having alzheimer's disease (ad) rather than psp; neuropathological diagnoses in these cases varied: i had combined ad-psp; had ad-psp combined with parkinson's disease (pd) changes; had psp-pd; and had "pure" psp. the patients with concomitant pd changes showed lewy bodies in the substantia nigra, locus coeruleus, nucleus basalis, and neocortex. the remaining patients were clinically diagnosed as having psp; neuropathological diagnoses in these cases included with "pure" psp and with psp that also met neuropathological criteria for ad (psp-ad). these findings emphasize the clinical and neuropathological heterogeneity in psp. the neuropsychological battery developed for the consortium to establish a registry for alzheimer's disease (cerad) is currently used in many research studies to index the cognitive impairments of alzheimer's disease. in spite of its widespread use, normative informarion on the battery, important for interpretation of performance, has not been available. we report norms for the cerad battery based on a large sample of elderly control subjects (n = ; white men and women; ages - yr) enrolled in the national study of cerad. performance on the neuropsychological measures was examined separately for subjects with high ( yr) and low (< yr) education. distribution of scores and basic descriptive information (means and sd) for each measure were determined. significant age and sex effects were observed on most cognitive measures in the highly educated group. in contrast, no significant age effects were observed in the low education group. effect of sex was not explored in this group due to the limited sample size (n = ). further exploration of cerad performance in normal controls from underrepresented groups including minorities, residents of rural communities, and individuals with low education is in progress. intraneuronal inclusions of cytoskeletal proteins appear in several neurological diseases; for example, the neurofibrillary tangles of alzheimer's disease contain a cytoskeletal protein, tau. because the previously described slowing of axonal transport in aged animals might lead to accumulation of cytoskeletal proteins in nerve-cell bodies and axons, we assessed the abundance of major cytoskeletal proteins in brain tracts of rats at age months or months. immunoassay was performed with monoclonal antibodies to alpha and beta tubulin and to nf-l (the core neurofilament protein) by published methods. samples were dissected in a standardized fashion and -mrn pieces of the following tracts were assayed: optic nerve, corticospinal tract (medulla), superior cerebellar peduncle, l dorsal root, and l ventral root. between months and months, the nf-l content approximately doubled in each brain site. tubulin substantially increased at of aged rats all sites except the fimbria-fornix. in contrast, tubulin did not change in the spinal roots. nf-l increased slightly in the ventral but not the dorsal root. this tendency of senescent brain neurons to accumulate cytoskeletal proteins in their axoplasm may predispose them to formation of intraneuronal inclusions in various degenerative diseases. we performed a prospective study of preoperative magnetic resonance imaging (mri) in consecutive patients with intractable partial epilepsy who underwent a stereotactic resection of an extrahippocampal temporal lobe foreign-tissue lesion, "lesionectomy," between june and january . interpretation of the mri studies was performed by an investigator blinded to the presurgical evaluation, surgical outcome, and pathology. hippocampal formation (hf) atrophy was assessed using mri-based volumetry (n = ) and visual grading of the h f (n = ). mri-detected hf atrophy has been shown to be a reliable marker of moderate to severe mesial temporal sclerosis (mts) (cascino gd, et al, ann neurol ; : - evidence from experimental animals indicates that endogenously produced platelet-derived growth factor (pdgf) is an important regulator of glial proliferation and differentiation. because of the striking degree of glial proliferation in chronic epilepsy, we sought to determine whether cultured glia from human epilepsy tissue would be responsive to pdgf. the effects of pdgf on dna synthesis, proliferation, and relative distribution of a b (+) glia were studied in a cell culture derived from temporal lobe white matter of adult epilepsy lobectomy tissue. by immunocytochemistry, glial fibrillary acidic protein (gfap) was detectable in % of cells in untreated or -day pdgf-treated ( ng/ml) cultures, which confirmed their astrocytic nature. in contrast, a b ( +) cells increased from to % in untreated cultures to % after pdgf treatment, which suggested that type astrocytes (a b [ + , gfap[ +]) had been elicited. dna synthesis of cells resembling oligodendrocyte-type astrocyte ( - a) progenitors occurred within hours after program and abstracts, american neurological association pdgf treatment as evidenced by nuclear incorporation of brdu in bipolar a b ( +) cells. these studies demonstrate that expansion of adult human gfap( +) astrocyte populations is sensitive to regulation by pdgf. further, these data imply the existence of pdgf-responsive - a progenitor cells in astrocyte-rich cultures derived from human epilepsy tissue. ( we have recorded vagal and esophageal-evoked potentials after electrical (e) and balloon (b) stimulation in epileptic patients who had vagal stimulators for the control of intractable epilepsy and the results were compared with esophagealevoked potentials in healthy controls and diabetic patients. the vagal and esophageal-evoked potentials showed similar configurations with major positive and negative potentials. the amplitudes of the responses habituated rapidly over trials at per second up to per seconds. latencies were shorter from the upper esophagus ( cm above lower esophageal sphinctereles]) cf. lower esophagus ( cm above les) yielding conduction velocities of to meters per second but conduction was significantly slower in the diabetics. the vagal-evoked potentials have validated the use of esophageal-evoked potentials as a practical method of assessment of the integrity and speed of conduction in vagal afferent pathways in man. ronald e. kramer and neil l. rosenberg, englewood. co seizure disorders were analyzed in patients in whom toluene was the sole or major drug of abuse. toluene abuse is increasing; therefore, physicians should gain experience with its neuropathological and ciinical sequelae. a retrospective chart review found patients meeting criteria. the average patient age was years; abuse onset averaged . years; abuse averaged . years; and patients were male. ten were daily, were weekly, and were intermittent users. seizures occurred in . one suffered a single generalized tonic-clonic seizure without recurrence and without treatment. his we characterized the clinical dose-response curves for relief of parkinsonism and production of dyskinesias as a function of plasma levodopa and - -methyldopa levels in patients with parkinson's disease (pd) and fluctuating responses to oral levodopa/carbidopa. dose response to graded intravenous levodopa was measured after overnight drug withdrawal on occasions, first after chronic, intermittent oral levodopa/ carbidopa and second after to days of continuous intravenous levodopa. continuous intravenous levodopa shifted the dyskinesia dose-response curve to the right, and reduced maximum dyskinesia activity, but did not significantly alter dose response for relief of parkinsonism. improvement in dyskinesia was apparent by the second day of continuous levodopa, during which ratios of plasma dopa/ - -methyldopa remained constant. our results support the hypothesis that relief of parkinsonism and production of dyskinesias occur by separate mechanisms. continuous dopamine-mimetic therapy should be sought as a therapeutic goal for advanced pd. dopa-responsive dystonia (drd) is a distinct subset of idiopathic dystonia with diurnal fl uctuation and a dramatically beneficial response to l-dopa. it has hitherto been considered an autosomal-dominant disease with reduced penetration (mckusick no. ) . we studied an arabic family of members with d r d spanning generations. we examined members and of their spouses. l-dopa was withheld for hours from patients in treatment. five family members had generalized dystonia with diurnal auctuation ( i male, in an arabic family females). dystonia started between the age of and years with gait difficulty and involvement of the legs. mri, eeg, evoked potentials, and screening for wilson's disease were negative. an excellent response to l-dopa was noted in all patients with continued long-term clinical stability for as long as years. the patients were the products of consanguineous marriages, and their siblings were normal. the patients were descendants of the same great-great-grandparents. this pedigree suggests an autosomal-recessive type of inheritance. we believe this is the first report of d r d with an autosomal-recessive type of inheritance. a. achiron, m . gornish, h . goldberg, i . ziv, r. djaldetti, y. zoldan, h. smka, and e. mekzmed, petah tiqva, israel freezing gait is an incapacitating symptom that occurs often in advanced parkinson's disease and also in other neurological disorders, eg., multiinfarct state, multisystem atrophies, and normotensive hydrocephalus. we evaluated, videotaped, and rated patients ( men, age k , - yr) who developed pure progressive freezing gait during . . , . - years. severity was mild in with sudden motor blocks mainly when confronted with obstacles; moderate in with gait arrests upon any attempt to initiate walking and changing direction, requiring a walking stick or partial external assistance; and severe in with total inability to start walking, requiring a walker, massive assistance, or a wheelchair. in all, freezing was associated with postural instability. they could mimic normal gait when seated or lying prone and could overcome arrests by the "walking over lines" maneuver. neurological examination was otherwise normal with no signs of dementia, parkinsonism, or pseudobulbar palsy. ischemic risk factors including ischemic heart disease, hypertension, and diabetes occurred in and previous strokes in . brain c t and mri were normal or showed mild cortical atrophy in and putative lacunae in only patients. none responded to levodopa or dopamine agonists. progressive pure freezing gait should be recognized as a separate nonparkinsonian neurological entity. it may be due to degenerative or ischemic non-nigral brainstem lesions. we describe patients with causalgia and dystonia, triggered by peripheral injuries in patients and occurring spontaneously in patients. the injury was often trivial. the mean age at presentation was . years. the legs were affected in patients, and the arm was affected in the remaining patients. all had burning pain, allodynia, and hyperpathia, along with vasomotor, sudomotor, and trophic changes. all developed typical dystonic muscle spasms in the affected part. the spasms typically were sustained, producing a fixed dystonic posture, in contrast to the mobile spasms characteristic of idiopathic torsion dystonia. dystonia always followed the causalgia and was painful. there was spread of the causalgia and of the dystonia from its initial site both in the affected limb and to other extremities, the latter in a hemiplegic, transverse, and triplegic distribution. all forms of conventional treatment failed to relieve either the pain or the dystonia. we suggest that functional changes in the corticobasal ganglia-thalamic system are responsible for this painful dystonic syndrome. program and abstracts, american neurological association holiday for parkinson's disease: a controlled clinical trial r. kurlan, c . m . tanner, c. g. goetz, j . sutton, p. carvey, c. deeley, l. cui, c. itvine, and m . mcdemzott, rochester, ny, chicago, il, and san jose, c a the efficacy and mechanisms of levodopa (ld) drug holiday for parkinson's disease (pd) remain controversial. we performed a double-blind, randomized study with advanced pd patients ( men, women; aged - yr) with entry criteria of inadequate response to ld plus dose-limiting ld-induced side effects (dyskinesias, hallucinations, and confusion). subjects were assigned to: ( ) % placebo for ld (complete drug holiday) or ( ) % ld and % placebo for ld ( % drug reduction) for days. after subsequent open-label ld dose optimization, subjects were followed to end point (defined as the time when entry criteria were again satisfied or a maximum of year). median survival time to end point was not significantly different for the complete drug holiday ( days) and % drug reduction ( days) groups ( p = ) . aspiration pneumonia occurred in complete drug holiday patients and no significant morbidity occurred with drug reduction. after a -mg dose of ld, clinical and pharmacological responses were no different before and after drug holiday ( p = . ) or reduction ( p = ). subject to the limitations of our small sample size, we conclude that complete drug holiday is associated with greater morbidity and confers no major advantage over % drug reduction. we found no evidence of significant alterations of pharmacokinetic or pharmacodynamic properties of ld after drug holiday or reduction. ( (pc) of the substantia nigra on t -weighted images. the narrowing of the pc signal has been attributed either to atrophy of the pc or to increased deposition of iron in this region. we have studied details of iron distribution in the midbrain of formalin-fixed human brains by scanning pixe analysis. three p d brains, juvenile pd brain, and control (amyotrophic lateral sclerosis) brains were studied. in the controls, the iron content of the pars reticulata (pr) was almost equal to that of the red nucleus (rn), but that of the pc was less than % of the pr. in parkinsonian brains, the iron content in the pc was significantly higher than in the controls. the iron content ratios of pc/pr and pc/rn in parkinsonian brains were significantly higher than in the controls. these findings suggest that iron deposition increases in the pc of parkinsonian brains. the difference in the pattern of iron content corresponds to the intensity profile pattern noted on mri. mri findings in parkinsonian patients may reflect a change in the iron distribution pattern. (jankovic and brin, nejm, ) . such resistance within exposures is not likely due to toxin antibody formation. of cd patients evaluated per protocol receiving or more botox tx under multichannel emg monitoring, ( . %) showed no benefit after the first and second tx, despite mild neck weakness on static muscle testing and, in some instances, emg signs of denervation. the responders ( men, women) had younger age onset cd (mean yr vs yr), but similar duration (mean yr vs yr) compared to the male and female nonresponders (in contrast to jankovic and schwartz, arch neurol, ) . both groups had similar degrees of severity and tx dose (mean iu, range . - ). although disparate group size prohibits statistical analysis, some interesting comparisons include: nonresponders were more apt to have extranuchal dystonic sites ( % vs %), antecollis ( % vs %), dark eyes ( % vs %), women with elevated antinuclear antibody titer ( % vs %), history of intracranial operation ( % vs o%), significant for age focal mri abnormality ( of vs of ). history of cervical operation ( patients) did not limit responsiveness. rates of prior remission, perinatal stress, antecedent trauma, left-handedness, and family history of movement disorder were similar for both groups. antecollis presents problems for optimizing tx to affected muscles, but central mechanisms may play a role in why some patients with focal dystonia do not improve with botox tx from the outset. enrico fazzini, new york, n y with parkinson's disease does deprenyl have a symptomatic effect on patients with untreated and l-dopa-treated parkinson's disease (pd)? once deprenyl is started, how long is it before another medication is needed to control symptoms of continued disease progression? there has been controversy over whether deprenyl has effects on delaying disease progression (nejm ; : ) and/or in alleviating the symptoms of pd. one hundred seventy-five patients already taking l-dopa (group ) and patients who had never taken l-dopa (group ) were treated with deprenyl mg/day. unified pd rating scale (updrs) scores were measured before and after deprenyl. patients were followed until pd symptoms progressed to the point of requiring additional medication. one hundred eighteen of ( %) patients in group (reduced updrs mean activities of daily living [adl) to , motor [mtr] to ) and / ( %) patients in group (reduced updrs mean adl to , mtr to ) reported symptomatic benefit. an average of months' duration was found in both groups before further medication adjustments were needed. deprenyl provides symptomatic benefit for an average of months in the majority of patients with p d regardless of whether or not they are being treated with l-dopa. yasuo iwasaki, masao kinoshita, toshiya shojima, and ken ikeda, tokyo, japan, and cleveland, oh in parkinsonian patients we measured fasting plasma amino acids in parkinson's disease patients and controls matched for age and sex. all patients were receiving l-dopa and they were free of any medications other than l-dopa. normal controls were free of any medication. there were no differences in diets between patients and controls. fasting blood specimens were collected in heparinized tubes and immediately were centrifuged at , g for minutes. analysis of plasma amino acids was performed by automated ion-exchange chromatography with lithium-based buffer and an amino-acid analyzer. parkinsonian patients had significant elevations of aspartate, glutamate, and glycine. the other amino acids were not significantly different from those in controls. n o correlation between severity or activity and degree of abnormality in plasma level of amino acids in patients was established. we conclude that excitatory amino-acid metabolism is altered in patients with parkinson's disease. bonnie e. levin, rachel tomer, and william weiner, miami, fl disease there is evidence linking obsessive-compulsive symptoms (ocs) to basal ganglia dysfunction. we investigated the presence and severity of ocs in a sample of patients with an unequivocal diagnosis of idiopathic parkinson's disease (pd) using the leyton obsessional inventory. ocs was found in the majority of the patients, with ( %) scoring above the normative cutoff for the symptom score and ( %) scoring above the normative cutoff for the trait score. when severity of oc symptoms was correlated with a battery of neuropsychological measures, significant relationships were observed between ocs and a preponderance of tests associated with right-hemisphere functions. these findings were observed especially on those tests with a strong frontal lobe component (block design: r = -. ; embedded figures: r = -. ; set shifting: r = -. ; and perseverative responses: r = . ; p < . for all measures). in all cases, the more severe oc symptoms, the poorer the performance. a similar trend was observed between the leyton trait scores and the cognitive measures. these findings suggest that ocs is present in a subgroup of pd patients, which may reflect greater compromise of right-hemisphere basal ganglia-frontal lobe pathways. intraclass correlations (iccs) were calculated for the total motor score and for each individual sign. results indicated excellent agreement (icc > . ) for the total motor score, resting tremor, gait, arising from a chair, and speeded, repetitive movements; good agreement (icc > . ) for rigidity, action tremor, posture, postural stability, and bradykinesia; and poor agreement (icc < . ) for speech and facial mobility. a factor analysis was then performed on updrs motor scores for pd patients from a community-dwelling cohort. three factors were extracted by principal components analysis with subsequent varimax rotation, accounting for . % of the total variance: factor -balance and stability (posture, postural stability, gait, arising from a chair, and bradykinesia); factor -rigidity and motor speed (rigidity, speech, facial mobility, rapid alternating movements, leg agility, hand movements, and finger tapping); factor -tremor (resting and action). these results indicate that the updrs motor examination is reliable between raters and measures the cardinal signs of pd. an open pilot study was performed to evaluate the efficacy of botulinum a toxin (botox) injections for disabling hand tremors. a previous report on the use of botox for hand tremors suggested that it was helpful, but relied on subjective clinical rating scales. the extent of normal clinical fluctuations or a placebo response could not be determined. to investigate these issues more objectively, patients with parkinson's disease and with essential tremor with refractory hand tremors underwent electromyographically guided intramuscular injections of botox into wrist flexors and extensors. patients without great medication-related tremor fluctuations were selected. results before and after botox were determined by comparing ( ) patient perceptions of functional improvement, ( ) clinical assessments using the unified pd rating scale for tremor and the webster rating scales, and ( ) physiological measurements using accelerometric analysis of hand tremors of tremor frequency, amplitude, and waveform characteristics. all patients reported some improvement, ranging from mild to marked with a mean of . on a to ( = marked) global rating scale. however, only / patients showed a significant improvement in the clinical rating scales, confirmed by > % reduction in tremor amplitudes. these findings show that most patients reported improvement not confirmed by the clinical or physiological measures. efficacy of botox injections for tremors is implied, but controlled trials are needed before this procedure can be generally recommended. christopher g. goetz and glenn t . stebbins, chicago, i l we tested whether hallucinations, motor disability, and cognitive decline were risk factors for nursing home placement in advanced parkinson's disease (pd) and whether these effects were independent or synergistic. between and , we identified patients admitted to long-term nursing homes. using case control methodology, we matched each for age, pd duration, and sex with control pd patients remaining at home. parkinsonism was assessed by the motor and activities of daily living subscales of the unified p d rating scale (updrs); hallucinations and dementia were determined by scores on the thought disorder and intellectual impairment items of the updrs. tests of synergy were based on a mantel-hentel model. hallucinations were a significant risk factor with odds ratio = . , x = . , p < , . motor impairment alone and cognitive impairment alone were not significant risk factors for nursing home placement (x for motor severity = , , p > . , and x for cognitive impairment = . , p > . ). furthermore, combined odds ratios for hallucinationslmotor severity and hallucinations/cognitive impairment showed no synergy of effect (x < . for both,p > . ). of the variables studied, hallucinatory behavior is the most prominent and independent risk factor for nursing home placement in these patients; the data suggest that aggressive control of hallucinations may be warranted to prevent nursing home admission. temperature-sensitive paramyotonia congenita phenotype* louis j . ptacek, philip mcmanis, hzrbert kwiecinski, alfred george, robert barchi, launce gouw. and mark leppert, salt lake city, u t , rochester, mn, warsaw, poland, and philadelphia, pa the periodic paralyses are a group of autosomal-dominant muscle diseases sharing a common feature of episodic paralysis. in one form, paramyotonia congenita (pc), the paralysis is temperature-sensitive, usually occurring with muscle cooling. electrophysiological studies of muscle from patients with pc have revealed temperature-dependent alterations in sodium channel (nach) function. this observation led to the identification of distinct mutations in an s segment of a skeletal muscle nach in unrelated pc families. we describe the use of the single-strand conformation polymorphism (sscp) technique to define a third allele specific to pc patients in an additional family. this aberrant pattern, though distinct from the first , occurs in the same exon of this nach gene. sequencing is currently underway to define the molecular alteration causing this aberrant pattern. two additional families with the pc phenotype have been sampled and do not demonstrate these sscp variants. we are currently searching for new mutations in these families to define further the molecular heterogeneity of this temperature-sensitive pc phenotype. parag mehta and roger w. kukz, brooklyn, n y abnormal accumulation of calcium (ca) in myofibers is thought to play a role in pathogenic myonecrosis. attempts at reducing intracellular ca content with ca channel blockers in duchenne muscular dystrophy (dmd) have been clinically unsuccessful. dantrolene, however, which acts at the sarcoplasmic reticulum to inhibit ca release from intracellular stores, has produced dramatic reductions in serum creatine kinase (ck) in dystrophic mice and more recently in dmd. we investigated the effect of low-dose dantrolene in a group of patients with limb girdle dystrophy (lgd), dmd, and other myopathic disorders. all subjects received dantrolene in incrementing doses from to mg daily over a to -week period. mean baseline ck was compared to ck with dantrolene treatment. dramatic reductions in serum ck levels averaging % were seen at to -mg doses in lgd patients ( ). dmd patients ( ) and patients with other myopathies ( ) showed a similar but less dramatic reduction in ck. three of the weakest patients complained of increased fatigue while taking mg, which suggests that higher-dose dantrolene may confound longer-term trials assessing clinical muscle strength and function. dantrolene in dosages well below conventional antispastic doses has a dramatic effect on serum ck and possibly myofiber necrosis in lgd, other dystrophies, and other muscle disorders. p . antigen-specific therapy in myasthenia gravis: myasthenia gravis (mg) is mediated by anti-acetylcholine receptor (achr) antibodies, believed to be t-cell dependent, and antigen-specific therapy would be preferable to current nonspecific immunosuppression. exposing mouse t-cell clones to mhc class i molecules complexed with relevant antigen on planar membranes induced proliferative unresponsiveness (quill and schwartz, ) , and soluble mhc class i molecules complexed with myelin basic protein (mbp) peptide resulted in unresponsiveness of specific tlymphocyte clones in vitro (sharma et al, ) . we have used our well-defined dr -restricted t-cell clone (ong et a [, ) isolated from an mg patient and specific for p - of the achr alpha subunit. overnight incubation of these t cells with a soluble p - : dr complex substantially inhibited the subsequent response to challenge with soluble antigen and presenting cells. in contrast, antigen response after preincubation with dr complexed to an irrelevant peptide (mbp - ), soluble dr alone, or an experimental approach studied in vitro p - alone (at equimolar concentrations) did not differ appreciably from that in untreated cells. the p - :dr complex had no effect on other non-achrspecific cell lines/clones. these results suggest that the use of soluble mhc-peptide complexes may be an approach to selective immunotherapy in mg patients. p . high-dose intravenous immunoglobulin in the shawke a. soueidan and marinos c. dalakas, bethesda, md inclusion body myositis (ibm) is a severe disabling inflammatory myopathy with characteristic clinical and histological features. it is commonly suspected when a patient with presumed polymyositis does not respond to available immunotherapies. the need for an effective treatment in patients with ibm prompted the present pilot study using high-dose intravenous immunoglobulin (hd-ivig), an apparently effective immunomodulating agent in several autoimmune neuromuscular disorders. we treated patients with muscle biopsy-proven ibm with up to monthly infusions of gml kg ivig. after the first infusion, of the patients showed definite functional improvement consisting of independent ambulation, fewer falls, and increased ability to lift weights. the muscle strength of the proximal and less atrophic muscle groups improved by one grade mrc scale (from to ), whereas the distal and atrophic muscles remained unchanged. the improvement, sustained up to months, was greater in patients with the most severe endomysial inflammation. we conclude that hd-ivig may be the first promising agent that can improve the strength of certain muscle groups in patients with ibm. because ivig is prohibitively expensive, the present encouraging results warrant a large-scale controlled therapeutic study. hays, and n . lutov, new york, n y , and milan, italy anti-myelin-associated glycoprotein (mag) antibodies from patients with neuropathy cross-react with the glycolipid -sulfated glucuronyl paragloboside (sgpg). among patients tested by enzyme-linked immunosorbent assay and western blot, had highly elevated antibody titers ( , ) to both mag and sgpg, had highly elevated titers to mag alone, and had highly elevated titers to only sgpg. immunostaining of normal nerve myelin by the antibodies correlated better with anti-mag than anti-sgpg activity. twenty-one of the patients, including patients in all groups, had predominantly sensory or sensorimotor neuropathy, and biopsy specimens revealed deposits of igm and complement on affected myelin sheaths. three patients presented with motor syndromes, all with antibodies specific for sgpg; had a predominantly motor demyelinating neuropathy, had upper and lower motor neuron signs and peripheral neuropathy, and had amyotrophic lateral sclerosis confirmed post mortem. all had deposits of complement on peripheral nerve myelin sheaths. these studies suggest the following: ( ) that anti-mag or sgpg antibodies may differ in their fine specificities and biological activities, ( ) that anti-sgpg antibodies also may occur in motor neuron diseases, complicating the clinical presentation, and ( ) that both mag and sgpg should be used as antigens in testing for autoantibody activity in peripheral neuropathy. david b. williams, john steele, ulla-katrina craig, sandra bryant, peter o'brien, and leonard kurland, newcastle, new south wales, australia, mangilao, guam, and rochester, m n continuing surveillance of neurodegenerative diseases in the mariana islands reveals changes in frequency and clinical characteristics since the s that resemble those in other known western pacific foci (kii peninsula, japan, and irian jaya, new guinea). recent surveys of patients years and older were conducted on rota, tinian, and yigo, guam. possible cases of dementia, parkinsonism, and amyotrophic lateral sclerosis (als) were identified by local trained personnel using a questionnaire, world health organization neurology test, and cognitive screening. those who failed the screening were examined by a neurologist. in the small populations of rota and tinian, there were no definite cases of als compared to i to cases present in previous surveys. the high prevalence of parkinsonism-dementia complex (pdc) was unchanged and dementia was increased compared to earlier surveys. in yigo, als and pdc continue to be prevalent; however, the als patients are predominantly long-term survivors (> -year disease duration). in areas of previous high prevalence of als/pdc, dementia (as pdc) was associated with extrapyramidal signs, whereas in areas of previously low prevalence of als/pdc, dementia alone, possibly of alzheimer type, predominated. these observations help to confirm previous reports of changing clinical patterns, but suggest that the geographic distribution of the (presumed) environmental etiological agent for als/pdc remains stable after almost years. p. v . fragokz, . frongillo, m. michisanti, g. antonini. derangements of the cardiac conducting system are the most common features of heart involvement in myotonic dystrophy (md). in view of chis patients with various grades of md ( males and females, mean age yr) underwent -lead ecg and holter monitoring. in patients ( %), almost all with a severe grade of md, or more conduction defects were found: first-degree atrioventricular block (i-avb) in cases, second-degree avb in case, right bundle branch block in cases, left anterior hemiblock in cases, left bundle branch block in cases, and trifascicular block in case (pacemaker implanted). an -year-old boy had a chronic atrial fibrillation with slow ventricular rate; he died suddenly while awaiting electrophysiological study. thirty-seven patients were followed over a mean period of months (range - mo). a -year-old woman experienced a myocardial infarction and was excluded from subsequent considerations. conduction defects de novo appeared in patients: i-avb in and i-avb plus -avb (mobitz i and i type) in . nine patients, all with i-avb at initial evaluation, showed deterioration of their defects' conduction: a bifascicular block was observed in cases and a trifascicular block in cases (pacemaker implanted). conduction defects may run a malignant course in md, mainly in patients with more severe grades of the neuromuscular disease; thus, a close cardiological evaluation is mandatory for a proper therapeutic approach in single cases. hiroshi mitsumoto, surest kumar, kevy h. levin, robert w. shields, jr, michelle secic, asa j . wilbourn, and rajendra g . desai, cleveland, oh, and santa ana, ca twenty patients with amyotrophic lateral sclerosis (als) entered a pretreatment study with monthly quantitative isometric muscle strength tests ( muscles in each extremity) and quantitative tufts scales including vital capacity, bulbar diadochokinetic rate, timed water drinking, timed rising from a chair, and timed walking meters. after to months of pretreatment observation, the patients received mg/kg intravenous immunoglobulin (ivig) (gamimune-n, miles) every month for up to months. during the study period, patients died and patients withdrew from the study. the slope of each variable's changes over time before and after the ivig treatment were compared statistically. none of the quantitative scales showed significant change with ivig. however, the slope of the upper extremity muscle strength revealed improvement with ivig treatment ( p = . and . , right and left, respectively). when all extremities were combined, the slope was also significantly improved with ivig ( p = . ). lower extremity muscle strength alone showed similar trends but no statistical significance. electrophysiological and immunological data were also analyzed. our results warrant a double-blind, controlled study with ivig for the treatment of als. leber's hereditary optic neuropathy (lhon) is a mitochondrial disorder with predominantly optic nerve abnormality. it can be associated with dystonia, ataxia, encephalopathy, cardiac abnormalities, and other less well-characterized neurological syndromes. we describe members of a family with lhon with a slowly progressive motor polyneuropathy. a -year-old man and his -year-old sister have had mild motor impairment since early childhood. a gait disorder and distal muscle weakness became evident at puberty. the girl, but not the man, also has blindness, distal numbness, type i diabetes mellitus, and short stature. physical examination showed in both: bilateral foot drop, limb hyperreflexia but absent ankle reflexes, distal sensory loss, and a slight brownish scaly skin discoloration over the forearms. only the girl had clonus and optic nerve atrophy. the man had peripapillary telangiectasias. the jaw jerk was normal in both. motor nerve conductions in both showed absent tibia and peroneal responses, whereas other motor and sensory nerve conductions were normal. emg revealed denervation, more so distally. muscle biopsy findings showed recent denervation and previous denervation followed by reinnervation. n o raggedred fibers were observed with the modified trichrome and sdh stains. brain mri was normal in both. cerebrospinal fluid in the girl was normal. blood samples from both patients and maternally related family members revealed a mitochondrial dna point mutation at position (d. c. wallace). in this family, only male had lhon; females had lhon and others, including the patients' mother, were asymptomatic carriers. this association of chronic motor neuropathy and hyperreflexia with lhon appears to be a distinct syndrome. the pathogenic mechanism by which the mitochondrial dna defect causes the neuropathy (and other neurological deficits) requires analysis. duchennelbecker muscular dystrophy henry j . kaminski, mazen al-hakim, r. john leigh, bashar katirji, and robert l. ruff; cleveland, oh fast-twitch extremity muscle fibers are preferentially affected in duchenne/becker muscular dystrophy (dbmd). since saccades are thought to be mediated by fast-twitch fibers, saccadic velocities would be expected to be decreased among these patients. to investigate involvement of extraocular muscle (eom) by dbmd, we studied with infrared oculography patients who were wheelchair-bound and able to perform only minimal activities of daily living. saccades were slightly slowed but were within % confidence limits of normal. all patients showed square wave jerk movements (swj). in patients, the frequency of the swj exceeded that of normal subjects, which suggested central nervous system dysfunction. clinical neuroophrhalmological examination of other dbmd patients was normal. this investigation is the first study of ocular motility in dbmd and demonstrates that eom function is relatively preserved even in far advanced patients. eom is composed of a heterogenous mix of fiber types that differ in anatomical and physiological characteristics from extremity muscle. study of eom in dbmd may prove to be useful in understanding why some muscles are resistant to dbmd and in characterizing properties that limit muscle degeneration. (supported by nih grants ey , ey , the department of veterans affairs, and the evenor armington fund.) since patients with myotonic dystrophy (mtd) exhibit a marked resistance to insulin effect on glucose uptake and an impaired handling of the insulin-sensitive amino acids, it is possible that muscle wasting in mtd may reflect a derangement of insulin action on muscle protein metabolism. increased muscle protein breakdown in mtd would be expected if the normal inhibitory effect of insulin on protein catabolism is impaired. the forearm perfusion technique combined with measurements of -methylhistidine ( -mh) arteriovenous (a-v) differences by high-performance liquid chromatography provides a unique method to investigate skeletal muscle myofibrillar protein degradation in vivo. we studied -mh (a-v) and efflux from the forearm muscles in men moderately affected with mtd and normal men. efflux values (q) were calculated as the product of -mh (a-v) times forearm plasma flow measured by the indicator dilution technique. forearm -mh release (estimated as {a-v} or q) of mtd patients did not differ significantly from normal controls. we conclude that myofibrillar degradation is not increased in mtd even when measured in a muscle compartment selectively affected by wasting. the possibility of an impaired anabolic action of insulin in mtd has yet to be determined. to study the relationship between the ragged red fibers (rrf) and age, we have reviewed muscle biopsy specimens prospectively. patients with well-established mitochondrial myopathy syndrome and with myopathies known to produce secondary rrf were excluded. the number of ragged red fibers (rrf) was counted under x (lpf) magnification. rrf were identified by the modified trichrome and sdh stain. for the final analysis, the sdh staining was used. the frequency of rrf was analyzed in relation to patients' ages. the frequency of cases with more than rrf increased with aging: % in the first decade, % in the fourth decade, and % in the eighth decade. the frequency of cases with more than rrf also increased with aging: % in the first three decades, % in the fourth decade, and % in the eighth decade. of patients with well-established mitochondrial myopathy syndrome, had more than rrf under lpf. the number of rrf in muscle increases with aging, indicating that mitochondrial activity in muscle is affected by aging. this finding may complicate the diagnostic criteria of mitochondrial myopathy in older individuals. ten rrf under lpf seems to be a reasonable cutoff point for the diagnosis of mitochondrial myopathy. schwartz-jampel, a rare autosomal-recessive syndrome characterized by short stature, myotonia, skeletal abnormalities, and peculiar facies, was reported by aberfeld in . the same sibship was earlier reported by schwartz and jampel in with emphasis on blepharophimosis. as of this writing about cases have been reported in the literature. most of the features of this syndrome are believed to be secondary to primary muscle disease. several peripheral electrophysiological studies showing features of myotonia have been reported. we describe patients with schwartz-jampel syndrome showing evidence of central conduction disturbance documented by somatosensory-evoked potentials (seps). median nerve seps showed normal latencies to erb's point and n- in all. interpeak latencies between n and n were prolonged in with complete block in . emg showed typical myotonic discharges in all. motor nerve conduction velocities, visual and brainstem auditory-evoked potentials, ct, and mri were normal in all. seps in the parents were normal. we believe this is the first report documenting evidence of central nervous system (cns) involvement in schwartz-jampel syndrome. schwartz-jampel syndrome and myotonic dystrophy may have similar cns ''lesion'' as sep abnormalities also have been shown in myotonic dystrophy. epidemiological studies have associated consumption of certain batches of l-tryptophan (lt) with development of the eosinophilia-myalgia syndrome (ems). , '-ethyledenebisltryptophan) (ebt or peak e), a derivative of lt, is a trace contaminant associated with implicated batches of lt. three female lewis rats received ebt, mg per gm daily, by intraperitoneal injection. four control rats received unimplicated lt. n o peripheral eosinophilia, rash, or weakness were observed in either group. one rat from each group died during the experiment (control-bowel infarct; ebt-death under anesthetic). after days, forelimb and hindlimb muscles of the remaining animals were frozen and fixed for program and abstracts, american neurological association ultrastructural and histological studies. two ebt rats had a myopathy involving soleus with a perimysial infiltrate containing lymphocytes, macrophages and sparse eosinophils, and necrotic fibers; the other showed few necrotic fibers in gastrocnemius. occasional eosinophils were seen in fascia in both animals given ebt but not in controls. fiber-type specific quantitative analysis of the microvasculature showed no decrease in the capillary index. ultrastructural examination revealed an increase in the size of microvessels in ebt animals. no denervation or reinnervation were demonstrated. the perimysial inflammation replicates an important feature of human ems and supports the epidemiological evidence that ebt is the causative agent of the disease. britta ostermeyer-shoaib, bernard m. patten, and tetsuo ashizawa, houston, t x five women (patients - ) developed motor neuron disease (mnd) years (range - yr) after receiving silicone gel-filled breast implants. at explant in patients, had both and had the left implant ruptured with silicone spilled into tissue. one woman (patient ) developed amyotrophic lateral sclerosis (als) years after numerous injections of free silicone into her face. biceps muscle biopsy specimens in all showed neurogenic atrophy. patient developed als with bulbar involvement and died years later of respiratory failure. she had anti-gm antibodies and autopsy findings confirmed the diagnosis of typical als. patient developed als, but also fatigue, myalgia, arthralgia, and skin rash. she had anti-gm antibodies, antisilicone antibodies, positive antinuclear antibodies (ana), and decreased serum igg, iga, and c , but increased igm and creatine phosphokinase (cpk) and chronic inflammation was revealed in muscle biopsy specimens. patient developed als, but also had hair loss, skin rash, fatigue, headache, sjogren's syndrome, and positive ana. patient developed als, but also had myalgia and arthralgia. patient developed lower mnd, but also fevers, arthralgia, and joint stiffness. she had anti-gm antibodies, positive ana, antimyelin antibodies, and decreased serum igg and iga with chronic inflammation shown in nerve biopsy findings. patient developed a steroidresponsive and steroid-dependent als with bulbar involvement. she had a monoclonal gammopathy in the cerebrospinal fluid and increased cpk. we suggest that silicone acts as an adjuvant that damages motor neurons via an indirect autoimmune mechanism. implants and silicone injections into the face p . silicone adjuvant breast disease: more forty-five women developed mixed sensory-motor neuropathy ( ), motor neuron disease ( ), multiple sclerosis ( ), multiple sclerosis-like syndrome ( ), or myasthenia gravis ( ) years (range mo- yr) after receiving silicone-gel breast implants ( ), saline-filled silicone-covered breast implants ( ), or direct injections of silicone into the breast ( ). most patients had, in addition, severe fatigability, myalgia, arthralgia, morning stiffness, skin rash, lymphadenopathy, sjogren's syndrome, and short-term memory problems. laboratory results revealed in most of the women decreased or increased serum immunoglobulins, autoantibodies, a serum monoclonal gammopathy, or oligoclonal bands in cerebrospinal fluid. at explantation in , had both and had implant neurological cases ruptured. biopsy of the fibrous implant capsule in most patients showed foreign-body giant cells containing refractile material consistent with silicone whether or not the elastomer shell was ruptured, indicating silicone bleed. the major finding on surd nerve biopsy was loss of myelinated fibers, on biceps muscle biopsy was neurogenic atrophy, and on pectoralis muscle biopsy was myositis with vasculitis and free silicone in some. we suggest that silicone may provoke damage to nerve and muscle, probably indirectly promoting autoimmunity. james f. howard, jr, m . kathleen donovan. and m. susan tucker, chapel hill, nc urinary symptoms of urgency and incontinence have been reported only rarely in patients with myasthenia gravis (mg) and then most often in association with myasthenic crisis. we report the case of a -year-old woman who in december had the onset of chest pain and was found to have a lymphocytic thymoma. in june she developed urinary incontinence, was found to have an open bladder neck. and underwent a suspension procedure for stress incontinence in january . eight months later she developed exertional fatigue and a diagnosis of m g was made. in july there was a recurrence of urinary incontinence. these symptoms clustered toward the end of the day and at trough mestinon dose. neuro-urophysiological studies demonstrated her previous open bladder neck, the inability to sustain a pelvic floor contraction, and increased bladder wall contraction. singlefiber electromyography (sfemg) recordings from the anal sphincter demonstrated a mean consecutive difference (mcd) of ysec, and % of fiber pairs had impulse blocking while recordings in the extensor digitorium communis muscle were normal. following a course of plasma exchange, there was significant clinical improvement with a reduction in the frequency of urinary incontinence, and improvement in anal sphincter sfemg studies (mcd, ysec with no blocking). this case demonstrates that in those myasthenic patients with predisposing bladder outlet dysfunction, urinary incontinence may be a manifestation of worsening mg. diana m. escolar, mohamed eldaly, and jaime rich, boston, m a debate still exists as to the role of antibody versus celmediated factors in the pathogenesis of guillain-barre syndrome (gbs). we describe a patient with increased proportion of circulating t cells and a t-cell lymphoma who developed gbs and responded to intravenous immunoglobulin (ivig). a -year-old man with t-cell lymphoma drveloped gbs by clinical, nerve conduction, and cerebrospinal fluid criteria. he had an elevated proportion of t cells and markedly reduced b cells with a normal cd /cd ratio. he responded rapidly to ivig, with return of nearly normal motor function in week. three weeks later, he relapsed and his vital capacity dropped. ivig was again administered and within hours he nearly recovered. a third relapse, days later, again responded to ivig. he has remained asymptomatic with ivig maintenance. the role of t-cell lymphocytes in initiating experimental autoimmune neuritis has been shown by adoptive transfer experiments. this patient with t-cell neoplasia may represent an analogous model in hu-guillain-barre syndrome mans supporting the role of t-cell (cell-mediated) autoimmunity in the pathogenesis of gbs. ivig therapy may act primarily by inhibiting the t-cell-mediated attack on myelin. p . sympathetic skin response: age effect it is frequently stated that the sympathetic skin response (ssr) can be elicited in all normal subjects, but the age of the investigated population usually is not considered to be a significant factor. we have examined the ssr in the upper and lower limbs of normal subjects, aged to years ( of them males). the ssr was elicitable in the lower limbs in all subjects under the age of years and in the upper limbs in all subjects younger than years. in contrast, it could be elicited in the lower limbs in only % and in the upper limbs in % of octogenarians. the amplitude of the response, though highly variable, showed a remarkable decline with age, both in the upper ( p < . , r = . ) and in the lower ( p < . , r = . ) limbs. these results indicate that age affects both the elicitability and the amplitude of the ssr. this has to be taken into consideration when evaluating the autonomic function in the elderly. we reviewed records of patients appearing to have motor neuron disease (mnd) to whom we recommended immunosuppression over years ( of m n d patients). atypical findings engendered hope rhat they might have treatable neuropathy. electrophysiological studies were mainly consistent with mnd, but also showed conduction block or other evidence of relatively mild peripheral nerve disease in . sensory symptoms were present in ; or more reduced or absent deep tendon reflexes in ; elevated cerebrospinal fluid protein in ; and nonspecific abnormalities on sural nerve biopsies in of . anti-gm levels were measured in (including who improved), but none was significantly elevated. immunosuppression included cyclophosphamide ( patients); prednisone ( ); plasma exchange ( ); intravenous gammaglobulin ( ); cyclosporine ( ); and total lymphoid irradiation ( ). seven treated patients died, worsened, remained stable for years, and improved. two patients declined treatment. one died and the other did not worsen in years. we conclude that immunosuppression by our methods is, at best, rarely effective in atypical mnd. we studied serum antiglycolipid antibodies by enzymelinked immunosorbent assay in patients with typical miller fisher syndrome (mfs), patients with atypical mfs who were lacking in some of the cardinal signs, patients with guillain-barrc syndrome (gbs) with ophthalmoplegia, patients with gbs without ophthalmoplegia, patients with multiple sclerosis (ms), and patients with other immunological disorders (oid) including systemic lupus erythematosus, polymyositis, and mixed connective tissue disorder. all patients with typical mfs had increased activity of igg antibody against ganglioside g q l b in the early phase, and it reduced with time. such anti-gqlb igg activity also was detected in of the patients with atypical mfs and in of the patients with gbs with ophthalmoplegia. in atypical mfs, the only patient without increased anti-gq b igg activity demonstrated normal eye movement with ptosis, whereas eye movement was impaired in the other patients. no patients with gbs without ophthalmoplegia, ms, or oid had increased anti-gqlb igg activity. these findings suggest the close association between increased anti-gql b igg activity and impaired eye movement in mfs and gbs. serum anti-gqlb igg activity possibly plays a role in impaired eye movement in mfs. our goal is to develop an assay that can be used to monitor a relevant immune effect of interferon p (ifnp) in multiple sclerosis (ms) patients during the course of ifnp immuno-' therapy, since recombinant ifnp is being tested in multicenter clinical trials. this report extends our prior studies of the inhibitory effect of ifnp on t-cell activation. peripheral blood mononuclear cells (pbls) from healthy donors and clinically stable ms patients were studied. pbl cultures were stimulated with cona, mab to cd , or with the phorbol ester pma in the presence of the calcium ionophore ionomycin. parallel cultures were studied in the presence of ifnp, uiml. t-cell activation was monitored by determining the percent cells positive for il- receptor (il- r) using facs analysis, or with a sensitive enzyme-linked immunosorbent assay for ifny. ifnp markedly inhibited il- r expression induced by cona, by mab to cd , or by pma and ionomycin, which activate t cells via different pathways. the results suggest that ifnp inhibits t-cell activation by actions independent of membrane receptors. we observed significant inhibition of cona-induced t-cell il- r expression in both ms patients ( . % inhibition, p < . ) and controls ( . % inhibition, p < . ). there was no significant difference in percent inhibition between ms and controls, but there was more variance among the ms patients. variability of biological effects of ifnp on t cells may relate to differential therapeutic responses to exogenously administered ifnp in ms patients. preliminary experiments suggested that ifnp inhibited ifn gamma secretion by pbl stimulated with cona. ifnp inhibits a number of events associated with t-cell activation, in both normal and ms t cells. response to ifnp appears more variable in the ms cases. immunological monitoring of t-cell activation in patients receiving ifnp may assist in understanding the observed therapeutic responses and planning clinical protocols. myelin destruction is associated with many central nervous system disorders, such as multiple sclerosis, head trauma, and ischemic injury. inflammatory cells, monocytes/macrophages, and polymorphonuclear leukocytes (pmn) may me-program and abstracts, american neurological association diate myelin injury, and lipid peroxidation may be an important mechanism. inhibiting myelin oxidation could have substantial benefit, so we evaluated the ability of a -minosteroid, u a, to inhibit myelin oxidation by monocytes and pmn. fresh rat brain myelin was harvested by multiple sucrose gradient, ultracentrifugation steps, and the final product was confirmed to be pure myelin by sds-page electrophoresis. human monocytes or pmn were obtained from healthy, unmedicated volunteers by gradient separation techniques. monocytes ( . x lo cells/ml) and pmn ( . x lo cells/ml), myelin ( pg protein/ml), and lipopolysaccharide ( pg/ml) were incubated for hours with or without wm u a in -ml wells. myelin oxidation was evaluated by a thiobarbituric acid reactive substance assay for production of malondialdehyde (nmol/ ml). myelin oxidation by monocytes was . +- . (mean standard error of mean) without u a and was reduced to . ? . by pm u a ( p < . ). pmn-mediated myelin oxidation was . _t . without drug and . ? . with drug ( p < . ). these results demonstrate that u a markedly inhibits monocyte-and pmn-mediated myelin oxidation and suggest that the aminosteroids may help disorders associated with inflammatory cell-induced myelin injury. microglia cells participate in the pathological reactions of the cns to multiple insults including trauma, inflammation, and neuronal degeneration. functional roles for these cells could include mediating tissue in jury, promoting repair, or modulating immune responses. with regard to the latter, we have observed that the majority of adult human-derived microglia express major histocompatibility complex (mhc) class molecules under basal culture conditions, in contrast to astrocytes derived from the same surgical biopsy specimens. all morphological subtypes of the microglia (ameboid, bipolar, and ramified) expressed mhc class i molecules, indicating a discordance between morphology and mhc antigen expression as markers of microglia activation. the microglia actively ingest myelin constituents, as assessed using fluorescein-labeled myelin basic protein and laser confocal microscopy. autologous t cells (e') freshly isolated from the systemic blood and cocultured with candida antigen underwent active proliferation in the presence of to % microglia, indicating the functional capacity of the microglia to serve as antigen-presenting cells. y-interferon augmented both mhc class expression and functional antigen-presenting capacity. these results indicate the potential of the adult human microglia to promote immune reactivity within the cns. protein (mbp) neutralize anti-mbp purified from multiple sclerosis cerebrospinal fluid active phases of multiple sclerosis (ms) are associated with increased titers of intrathecally produced antimyelin basic protein (anti-mbp). anti-mbp can be purified by antigenspecific affinity chromatography from csf igg of patients with acute relapses of ms. eighteen synthetic peptides of human myelin basic protein (h-mbp) containing between and amino-acid residues and covering the entire length of the molecule were synthesized by the fmoc method. purified anti-mbp was reacted with increasing amounts of h-mbp as well as each of the peptides in an initial liquid phase assay, and subsequently titers of f anti-mbp in all resulting mixtures were measured by a solid-phase radioimmunoassay . purified anti-mbp was neutralized by h-mbp and of the synthetic peptides containing overall residues corresponding to to of h-mbp. the remaining synthetic peptides covering both the amino and carboxyl terminals of h-mbp did not significantly react with purified anti-mbp from these patients. in conclusion, anti-mbp purified from csf of ms patients has affinity for epitopes located between residues and of h-mbp. in a double-blind study involving patients, we recently demonstrated that -aminopyridine ( -ap) is superior to placebo in the treatment of multiple sclerosis (ms) (ann neurol, in press). the related agent , -diaminopyridine (dap) also appears to be effective. to enable a preliminary comparison, patients, who in our previous study had not benefitted from -ap, were now treated ( wk) with dap (up to . mg/kg/day) for weeks in an open-label fashion. instruments for assessment and registration of side effects were the same as in the previous trial. the optimal dose of dap was . mg/day compared to . mg/day for -ap. significant changes in the edss ( . point or more) were not found, whereas significant improvements in neurophysiological parameters were found (no difference between -ap and dap, allp > . ). subjective side effects during -ap ( patients) mainly suggested cns-function disturbance (dizziness and gait disturbance) and during dap ( patients) mainly suggested peripheral nervous system-function disturbance (paresthesias). systemic tolerability clearly was diminished for dap compared to -ap, with patients withdrawing because of severe gastric complaints and developing liver function abnormalities. these data suggest that -ap is more valuable than dap in the treatment of ms. cy clophosphamide/methylprednisolone therapy in multiple sclerosis multiple sclerosis (ms) is a presumed autoimmune disease in which various forms of immunotherapy have been attempted. mri studies show the disease to be more chronically active than is clinically evident, thus a single treatment is unlikely to provide lasting benefit. recently, the northeast cooperative treatment group found that pulse cyclophosphamide ( mg/m every other month for years) slows progressive ms. we initiated a pilot study to determine the effect of a more intensive and prolonged pulse therapy regimen in both progressive and earlier stages of the disease. pulse therapy was given after induction with either iv cyclophosphamide/corticotropin ( mg/m x over days) or iv methylprednisolone ( gm x over days). patients received a single iv dose of cyclophosphamide ( - , mg/m ) adjusted to produce leukopenia plus gram of iv methylprednisolone monthly for a year, every weeks for the next year, and every months in the third year. another group received pulse methylprednisolone without cyclophosphamide. as of this writing, patients have been treated, of which have completed years. interim analysis shows that patients treated with pulse methylprednisolone were more likely to become treatment failures than those treated with pulse cyclophosphamide/methylprednisolone ( % vs %) independent of induction therapy. withdrawal due to toxicity, however, was higher in the pulse cyclophospharnide group. current regimens involve methylprednisolone induction alone followed by pulse cyclophosphamide/methylprednisolone, analogous to lupus nephritis pulse therapy. this regimen can be given solely on an outpatient basis, does not cause alopecia, and is more amenable for use in earlier stages of the disease. to determine the incidence of pathologically confirmed malignancy in multiple sclerosis (ms) patients in funded clinical trials of cyclophosphamide (ctx) and of azathioprine (aza), data were collected longitudinally using telephone interviews, written questionnaires, physical examinations, and medical records for ctx and aza patients from a community in-hospital and out-patient ms clinic in fargo, nd. in the ctx study (goodkin et al, arch neurol ; : - ) , clinically definite (cd), chronic progressive ms patients were enrolled. twenty-four were controls and received a mean induction dose of . grams. fourteen of the induced patients then received boosters every other month for months resulting in a mean total dose of . grams. no malignancies were detected. in the aza study (goodkin et al, neurology ; : - ) , c d relapsing ms patients participated. twenty-five were controls and received mgtkg of aza daily by mouth adjusted to maintain a white blood count of greater than , o/cmm for years (mean dose . mg/kg). two of aza patients developed resectable skin cancers: basal cell (bcc) at months and i squarnous cell (scc) at months. the incidence of developing a bcc or scc was not significantly increased after initiating therapies as compared to the untreated ms controls (aza: fisher exact testp value = . ). no other malignancy has been detected during to months of clinical follow-up. allergic encephalomyelitis paula dore-dufly, ruth washington, and robert h. swanborg, detroit, m i postcapillary endothelium at sites of inflammation undergoes many changes referred to as activation. activated endothelial cells (ec) exhibit increased surface expression of immunorelevant proteins (icam- ; ncam, elam, and mhc class i and class i antigens [ags)). the sequence of events that characterizes ec activation may be important in susceptibility, induction, and perpetuation of experimental allergic encephalomyelitis (eae). in this study we examine expression of ec activation antigens in central nervous system (cns) microvessels in response to interferon gamma (ifn-y). cns microvessels from sjl and bio.s mice were incubated for hours in ifn-y ( u/ml), fixed, permeabilized, and then stained with an antibody that recognizes class i, class i mhc antigens, icam- , and factor viii. relative fluorescence intensity was determined using a laser cytometer. results indicate that microvessels from all strains tested expressed no detectable icam- and class i ags. little class i antigen and transferrin receptors were expressed. upon stimulation with ifn-y, sjl microvessels exhibited increased surface expression of all ec activation ags. b o.s microvessels exhibited icam- and class i mhc but mhc class i ags were not upregulated. results indicate that there are strain differences in the ec response to ifn-y. resistance of b o.s mouse ec to activation by ifn may be a factor in decreased susceptibility or induction of eae, or both. protein-t-cell line-mediated experimental to investigate a possible pathogenic role of interferongamma (ifn-y) in experimental allergic encephalomyelitis (eae), an immunocytochemical study was undertaken to localize this cytokine in the spinal cord of lewis rats in which eae was produced by adoptive transfer of myelin basic protein-specific t cells. one pm-thick cryosections of spinal cord were labeled with monoclonal antibodies (mab) db- and db- recognizing different epitopes of rat ifn-y. in the spinal cord of naive rats, mab db-i, but not db- , stained processes of astrocytes, suggesting that astrocytes contain a protein with an epitope cross-reacting with ifn-y. in rats with at-eae, numerous ifn-y-positive cells stained with both mab db- -and db- -positive cells were present from days to after cell transfer and had disappeared on day . at day they entered the spinal cords predominantly through subpial vessels. ifn-y-positive cells could be identified as w / + leukocytes as well as ed -positive macrophages. as in naive rats, astrocytes in at-eae were labeled only with mab db- , but not db- . we never observed labeling of motor neurons with these mab. the transient presence of ifn-y in the rat spinal cord at the onset of at-eae suggests a pathogenic role of this cytokine in acute immune-mediated demyelination of the cns probably as a local stimulus for expression of mhc class i antigens and adhesion molecules, as well as for the release of tnf-a and toxic oxygen radicals from macrophages and microglia. immune responses to stress or heat shock proteins are implicated in the pathogenesis of several autoimmune diseases, including multiple sclerosis (ms). we examined the hypothesis that antigens in myelin cross-reacted with stress protein antigens. two techniques were used: immunocytochemistry and western blotting. frozen histological sections were prepared from normal human central and peripheral nervous system tissues. sections were incubated with murine monoclonal antibodies to different mycobacterial stress proteins. antibody binding was determined using avidin-biotin complexed antimurine antibody linked to alkaline phosphatase. program and abstracts, american neurological association a monoclonal antibody to the stress protein sp from m . leprae strongly stained both central and peripheral nervous system myelin. no myelin staining was noted with antibodies to sp or sp . proteins from purified central and peripheral nervous system myelin were separated by sds-page. western blots were prepared using a monoclonal antibody to sp and a polyvalent rabbit antibody to myelin basic protein (mbp) as primary antibodies. antibody binding was determined using antimurine or antirabbit igg antibody coupled to alkaline phosphatase. strong staining of central but not peripheral mbp by the anti-sp antibody was observed. the rabbit anti-mbp antibody stained both central and peripheral nervous system mbp. the presence of antigenic epitopes shared by a stress protein and the potential autoantigen, mbp, supports the hypothesis that immune responses to stress proteins may be involved in the pathogenesis of presumed autoimmune diseases such as ms. ( ). of patients with borderline or positive csf lyme titer, received parenteral ceftriaxone. all had later relapses consistent with ms. one patient received a month of oral doxycycline. the fifth patient had a negative western blot and was not treated. we conclude that an incidental borderline or positive lyme serology in an ms patient is unlikely to indicate neurological lyme disease. borderline serologies should be documented to rise on later testing; positive serologies should be confirmed by retest in a different laboratory or by western blot. csf abnormalities suggestive of neurological lyme disease (pleocytosis, protein elevation, intrathecal lyme antibodies) are distinct from those suggestive of ms (ogb, elevated igg index, mbp). in such patients antibiotic treatment may be appropriate, but will not alter disease course. when designing and analyzing therapeutic trials for multiple sclerosis (ms), investigators commonly compare the proportion of patients in the experimental and control groups who worsen one or more steps on the disability status scale (dss) or expanded dss during the study (typically years' duration). however, the intervals between the scores in the dss may not be equal. it may be easier to change by one or more steps in the lower end of the scale (e.g., dss = - ) than in the midportion of the scale (e.g., dss = - ). to evaluate this possibility, we compared the proportion of patients who worsened by one or more steps in the years after entering our program with dss = or with dss = . fifty-one percent ( ) natural history data may be useful for designing therapeutic trials for multiple sclerosis (ms). since , we have collected such data in a standard format on patients in the ucla multiple sclerosis research and treatment program. t o describe the course in our group, we have performed survival analysis (kaplan-meier) of patients with or more assessments who entered the clinic with disability status scale (dss) scores of to (n = ). an increase of one or more steps in the dss score persisting for more than months defines worsening. median times to worsening for a dds at entry of to were approximately years (range . - . ); but were . years for dss and . years for dss . for those starting at dss (n = ), only % worsened by year, % by years, and % by years. the percent worsening when starting at dss (n = ) were %, %, and %, respectively. these variable rates of worsening (i.e., time spent at each starting level) influence therapeutic trial design. including patients with dss or will increase the sample size and study duration. for testing nontoxic agents, we recommend enrolling patients with dss to . for more toxic treatments, we suggest dss to . (partially supported by usphs grant ns , the conrad n. hilton foundation, and various donors.) pi . cholinergic antagonists and p-adrenergic agonists inhibit experimental allergic encephalomyelitis in an additive manner mark a. jensen, avertano noronha, and bawy g. w. amason, chicago, il lymphoid organs receive a sympathetic (sns) and possibly a parasympathetic innervation. lymphocytes express padrenergic and cholinergic receptors and thus are sensitive to regulation by these neurotransmitters. the severity of experimental allergic encephalomyelitis (eae) is increased in sns-ablated animals. local parasympathectomy decreases plaque-forming responses in submandibular nodes. p,-adrenergic receptors are upregulated on cds t cells in progressive multiple sclerosis, as are m,-muscarinic acetylcholine receptors on cd t cells. we examined the effect of isoproterenol, a p-adrenergic agonist, and scopolamine, a cholinergic antagonist, on the course of eae in lewis rats. eae was induced by injection of . ml of incomplete freund's ad juvant containing guinea pig spinal cord ( % wlv) and m. tubercdosis ( mgiml) in one hind footpad. scopolamine ( . mglkg, twice daily) and/or isoproterenol(o. mglkg, twice daily) or saline were injected subcutaneously starting on the day of immunization. scopolamine or isoproterenol alone reduced severity ( p < . , t test) and duration ( p < . , t test) of disease compared to controls. the combination of scopolamine and isoproterenol further reduced disease severity compared to either agent alone ( p < . , x test), suggesting an additive protective effect of cholinergic antagonists and p-adrenergic agonists in eae. antigen-presenting cells a . conrad, v. sanders, p. schmid, and w. w . tourtellotte, los angeles, c a tissue is cryopreserved by the method of tourtellotte; this procedure minimizes or eliminates ice artifacts and preserves surface protein markers. the dissected plaques are lightly fixed in % paraformaldehyde and then suspended in % sucrose. blocks are mounted in oct, cryosectioned at p,m, and picked up on gelatinized slides. the activity of plaques is determined by the presence or absence of myelin debris (antimyelin basic protein stain or lux fast blue) or presence or absence of neutral lipids indicating myelin digestion as seen by oil red (oro) staining and the presence or absence of a class i major histocompatibility complex antigen (evidence for an antigen-presenting cell) on macrophages or microglia as seen by immunocytochemically staining for hla-dr (hb atcc). the following is our classification of the activity of multiple sclerosis (ms) plaques. type i, the most active, is defined as an area of hypercellularity, positive for hla-dr with no o r staining or staining for myelin debris. type , or active, is defined as an area of hla-dr-positive cells that stain mildly with o r at the plaque edge but positive for myelin debris; evidence for demyelinating activity < hours (prineas; raine). there is an inner area of plump cells that are positive for hla-dr and oro. type , or modestly active, is defined as a "shelf" of plump hla-dr-positive cells at the edge loaded with program and abstracts, american neurological association oro-stained neutral lipid but a paucity of or no myelin debris. a region of hypocellularity is observed at the center of plaque. type iv, least active or inactive, is defined as scattered hla-dr-positive cells at plaque edge with little or no o r staining and no evidence of myelin debris. the frequency of plaque types was determined from a random sample of ms tissue blocks from patients who died of ms. ten percent of the plaques were type i; %: were type ; % were type . the majority of plaques ( %) were inactive (type iv). accordingly, it is necessary to classify the demyelinating activity of ms plaques for protocols designed to investigate etiopathogenesis. do these results suggest that whatever causes ms can be eradicated in half of the demyelinating areas by the time of death? p . localization of g d l b ganglioside antigen in the human peripheral nervous system susumu kusunoki, atsuro chiba, tadashi tai, and lchiro kanazawa, tokyo, japan serum antibodies against ganglioside gm and/or g d l b frequently are detected in autoimmune neuropathies such as rnultifocal motor neuropathy, igm paraproteinemic neuropathy, and guillain-barre syndrome. some of them bind to gm or g d l b monospecifically but the others cross-react with both of the antigens. to investigate respective localizations of gm and g d l b antigens in the human peripheral nervous system (pns), immunohistochemical study of dorsal root ganglia (drg), dorsal roots, ventral roots, and sympathetic ganglia (sg), which were obtained from human autopsy specimens, was performed by using mouse monoclonal antibodies, each monospecific to gm and gdlb. ggr , monospecific to gd b, immunostained nerve cell somas and axons of drg, sg, and dorsal and ventral roots. ggrl also recognized some myelin, including paranodal areas. however, gmb , monospecific to gm , did not bind to either neuron or myelin. thus, serum anti-gdlb antibody can bind to neurons and some myelin in the human pns. further study is necessary to identify the localization of gm antigen, because previous biochemical studies have shown that gm is also present in the human pns. the purpose of this study was to compare t and t lymphocytes in migraine patients versus controls, and a review of the literature was done. fifty-six migraine patients, aged to , were tested, as were controls. the t :t ratio in migraine patients was : , compared to : for controls. suppressors (t ) were reduced from in controls to in migraine patients. helpers (t ) decreased from , in controls to in migraine patients, and total ?' lymphocytes decreased from , to , . previous studies have revealed similar differences in t lymphocytes, with higher t : t ratios being found in both migraine and tension-type headache patients. in food-induced migraine, an increase in circulating immune complexes was noted, with increased t levels. differences in serotonin binding to mononuclear cells have been noted in migraine patients. a decreased sensitivity of the lymphocyte beta-adrenergic receptor in migraine patients was suggested by one study. after lymphocyte incubation with il- , the natural cytotoxic response is augmented in cluster patients. the percentage of lymphocytes expressing receptors for il- was decreased in cluster patients in a fur-review of the literature ther study. this il- receptor defect was independent of whether the clusters were present. there is a loss of highaffinity binding sites for serotonin on lymphocytes in both episodic tension and chronic tension headaches. we used positron emission tomography (pet) to measure local cerebral blood flow in volunteer subjects while they performed tasks of memory-guided saccades and a visual fixation control. tasks were performed continuously for seconds during emission scans, after bolus injection of h lso. eye movements were verified with electrooculography. areas of significant increase in regional blood flow between tasks were matched to three-dimensional reconstructions of brain magnetic resonance images of each subject. compared to the visual fixation control, saccade tasks evoked bilateral activation in the posterior superior parietal lobule with extension into the inferior parietal lobule. activation was also seen bilaterally in an area of frontal cortex immediately rostra to the precentral gyrus extending from the superior frontal gyrus to the inferior frontal sulcus. the increased blood flow in the posterior parietal cortex most likely corresponded to enhancement of visual attention required during saccades, while that in the frontal lobe, which included the frontal eye fields, indicated activation for saccade motor output. pamela blake, alexander s. mark, martin kolsky, and jorge kattah, washington, dc fifty patients with third cranial nerve (cn) palsy underwent precontrast and postcontrast mri to assess the utility of this study in this clinical context. mri demonstrated an appropriate lesion in cases. six patients had brainstem lesions ( infarcts, mass lesion, cryptic vascular malformation, hemorrhagic shearing injury, with compound q toxicity). lesions of the cisternal segment of the nerve were present in patients ( aneurysms, lymphomas, ophthalmoplegic migraine, viral meningitis, coccidiodomycosis, nerve avulsion), with enhancement of this segment in patients. fourteen patients had cavernous sinus lesions ( lymphomas, nasopharyngeal carcinoma, tolosa-hunt syndrome, cavernous carotid aneurysms, pituitary apoplexy, aspergillosis). eighteen patients, all with history of diabetes or vascular disease, had normal mri results, suggesting microvascular infarction of c n . in patients with cn i palsy, mri can detect the presence of brainstem or cavernous sinus lesions and often can suggest their cause. mri with contrast enhancement can demonstrate involvement of the cisternal segment of cn i in patients with inflammatory or infiltrative processes that previously could not be radiographically demonstrated. our study suggests microvascular infarction does not cause nerve enhancement on contrast-enhanced mri. we describe patients with acute hyperglycemic, hyperosmolal, nonketotic stupor who had ocular flutter or opsoclonus clinically. these are the fourth and fifth adult patients reported with the acute onset of stupor and opsoclonus; all patients had nonketotic hyperglycemia and hyperosmolality (rapid eye movement sleep also causes stupor and saccadic eye movements). three of the patients had myoclonic jerks in addition to opsoclonus. in the , opsoclonus began when glucose and osmolality acutely increased, and completely resolved when glucose and osmolality became normal, showing that opsoclonus is a specific and reversible effect of the metabolic disorder and implying that either acute hyperglycemia or hyperosmolality directly causes opsoclonus. since acute hyperosmolality caused by nacl or sucrose can cause a similar syndrome in experimental animals (trans am neurol assoc ; : - ) and infants, hyperosmolality is probably more important. opsoclonus is thought to be due to abnormal activity of saccadic "burst" cells in the pons. acute hyperosmolality may cause spontaneous saccades by disinhibiting burst cells from normal "pause" cell inhibition or by directly activating burst cells. the combination of acute deterioration in mental status and either ocular flutter or opsoclonus should suggest acute hyperosmolality, in particular, nonketotic hyperglycemia. neural cell adhesion molecule (n-cam) and the related cell adhesion molecule l play significant roles in axon outgrowth and mediation of cell-cell contact in development, and after peripheral nervous system injury. to understand the role of these molecules after injury in the adult cns, we studied alterations in n-cam and l in the rat brain's response to entorhinal cortex (erc) lesion. post lesion, reactive synaptogenesis and axonal sprouting follow a well-defined temporal course in restoring the synaptic density of the dederented outer two-thirds of the hippocampal dentate gyrus molecular layer (ml) to near prelesion levels. we found striking regionand lamina-specific staining of n-cam and l in the normal hippocampus and marked alterations in these molecules after injury. embryonic n-cam, present in high amounts during development but expressed at very low levels in the adult hippocampus, was massively re-expressed in the denervated zone; the embryonic form was still heavily expressed days later, when synapse number returned to >so% of prelesion levels. l staining, normally evenly distributed through the ml of the dentate, was completely lost in the outer ml, which is denervated by the erc lesion. this staining had not returned by days after lesion. neural cell adhesion molecules play a role in specificity of neural connectivity both in development and after injury. in reactive synaptogenesis and axonal sprouting after injury, both ontogenetic (the reexpression of embryonic epitopes) as well as uniquely adult sequences of repair are utilized. following hemispherectom y" a. pascual-leone, h . t. chugani, l. g. cohen, j . p . brasil-neto, e. m . wassermann, j . and m. hallett, betbesh, md, and los angeles, ca we studied subjects, aged months to years, who underwent hemispherectomy months to years earlier for intractable epilepsy. all had a spastic hemiparesis contralateral to the resected hemisphere, which was present presurgically. we used focal transcranial magnetic stimulation to map the areas of the preserved hemisphere targeting the abductor pollicis brevis (apb), the biceps, and the deltoid ipsilaterally and contralaterally. in subjects who had hemispherectomy after age , the same area targeted ipsilateral and contralateral muscles. in the remaining subjects, who were more functional, areas targeted ipsilateral muscles. one area coincided with the contralateral representation, but stimulation induced motor-evoked potentials (meps) of lower amplitude and longer latency in the ipsilateral muscles. the other area, to cm anterolaterally, targeted exclusively ipsilateral muscles and stimulation induced meps of normal amplitude and latency. this separate ipsilateral representation was more distinct in subjects studied a long time after the hemispherectomy and in those who were younger at the time of the operation. these results show evidence of motor reorganization after hemispherectomy. better motor function is associated with topographically differentiated ipsilat-era and contralateral representations, which may depend on age at the time of hemispherectomy and the time since then. cynthia l. comella, glenn t . stebbins, nancy brown-toms, and christopher g. goetz, chicago, il in a single-blind, crossover study, we evaluated the effect of an intensive outpatient physical rehabilitation program (re-hab) on the severity of parkinson's disease (pd). the re-hab program consisted of -hour sessions per week for weeks. sixteen patients completed phases, a rehab phase and a control phase, separated by months. the order of participation in each phase was randomized. all patients were evaluated using the unified pd rating scale with subscales for mentation (ment), activities of daily living (adl), and motor function (mot) by an investigator blinded to the rehab phase of the patient. all patients were evaluated immediately before and after each phase. pd medications were not changed during any phase. following the re-hab phase, there was significant improvement in adl score (pre-rehab , post-rehab , p = . wilcoxon) and in mot score (pre-rehab , post-rehab , p = , wilcoxon), but no change in ment score. after the con-trol phase, there was no significant change in any outcome measure. this is the first controlled, crossover study of an intensive rehab program in pd. it demonstrates that re-hab improves objective motor function and adl scores. sensitive and quantitative measurements of leg weakness, one of the most common deficits in multiple sclerosis (ms) patients, can be made using specialized extremity testing equipment. to determine whether such determinations could be used in clinical trials, ms patients with leg weakness that had been stable for at least months were evaluated every days over a -month period. each testing session was carried out at the same time of day and medication dosages and schedules were kept constant (only patient was taking baclofen and his dosing remained constant). quadriceps and hamstrings strengths were measured in isometric contraction in a mechanical testing apparatus (kincom). variability for each series of determinations was expressed as the standard deviation of the mean as a percent of the mean. the overall variability then was expressed as the mean of the individual variabilities. the mean variability for the strength determinations over months was . k . , and only one series of determinations out of had greater than % variability. these results suggest that quantitative strength determinations might be useful in clinical trials, and early experience in trials of aminopyridines will be discussed. polymyositis (pm) is an inflammatory myopathy of unknown cause, but the accumulating data strongly suggest an autoim-mune pathogenesis. the histological picture is of muscle fiber necrosis and inflammation, whereas in postviral fatigue syndrome (pfs), a disorder characterized by severe fatigue with myalgia and psychiatric symptoms, the histological picture of muscle is essentially normal. enteroviruses have been implicated on epidemiological and serological studies in both. we have used the polymerase chain reaction (pcr) and an enteroviral-specific probe and found persistent enteroviral genomic material in both pm and pfs muscle biopsy specimens. furthermore, we used a radiolabeled full-length cdna probe derived from coxsackie b in an in situ technique to look for viral d n a in pcr-positive cases. coxsackie genome was clearly identifiable in the muscle biopsy specimens of patients with pm but negative in pcr enteroviral-positive cases of pfs. the virus excites an inflammatory reaction only in pm. a murine animal model for pfs developed in our laboratory showed positive muscle pcr using enterovirus probes and a conspicuous increase in interleukin within the brain. these results provide major clues in the search for the etiology of these two puzzling disorders. human t-lymphotropic virus type i (htlv-i) is a cause of adult t-cell leukemia and tropical spastic paraparesis. in a specific population of iranian jews originating from the city of mashad, there is a high incidence of htlv-i infection ( . %) and associated t-cell leukemia. we evaluated the incidence of possible correlation between htlv-i infection and spastic paraparesis in israeli mashadi-born jews. we have examined mashadi-born immigrants in a mashadi community center ( men, women, mean age t_ . yr) and non-mashadi iranian-born jews. blood samples were tested for htlv-i antibodies by particle agglutination test. the polymerase chain reaction (pcr) was used to amplify htlv-i sequences of d n a from peripheral blood mononuclear cells. twelve mashadi-born immigrants ( %) were seropositive for htlv-i. in of those serologically positive for htlv-i ( %), neurological examination revealed spastic paraparesis of varying severity. none of the non-mashadi iranian jews were seropositive for htlv-i or had clinical signs of spastic paraparesis. these results support other studies of htlv-i-associated myelopathy. the high incidence of htlv-i-associated spastic paraparesis in the mashadi community might be related to their unique history of a high rate of intermarriage among members of this ethnically segregated group. further epidemiological studies are underway to evaluate the incidence of htlv-i in mashadi families as well as in mashadi-originating jews born in israel, to identify whether infection might be genetically transmitted. lymphotropic virus t y p e i-associated acute t-cell leukemia/lymphoma william j . harrington, jr, william a. sheremata, susan snodgrass, and mark raven, miami, fl human t-cell lymphotropic virus type i (htlv-i)-associated acute t-cell leukernia/lymphoma (atl) is thought to produce important c n s disease infrequently. we wish to correct this impression by presenting neurological findings in patients seen between and the present. all had positive western blots to htlv with polymerase chain reaction confirmation of htlv-i infection. only had a concomitant human immunodeficiency virus infection. all were black, aged to years, were men and women. all but americans came from the caribbean nations of haiti ( , dominican republic (l), jamaica ( ), and trinidad ( ). sexual transmission was the risk factor for htlv-i for all except for intravenous drug user. all patients were systemically ill. three had preceding neurological abnormality ( , months, and yr) and had major neurological disease concomitantly. two of these had tumor masses demonstrated in brain and in the spinal canal. one had a minor facial sensory abnormality; only had no deficits. we conclude that cns disease is commonly associated with atl, and that in the us atl occurs principally in caribbean natives. a. nath, v . hartloper, and m . furer, winni$eg, manitoba, canada microglia and astrocyte cultures were established from human fetal brain. microglia were infected with human immunodeficiency virus (hiv) strains, hiv,, or hiv,,,, resulting in a rising titer of p antigen in the supernatants. multinucleated giant-cell formation, vacuolar changes, and rising levels of lactate dehydrogenase in the supernatants were seen, indicating a cytopathic infection of microglia. astrocytes were infected with free virus or cocultivated with an hiv-infected lymphocyte cell line (hut- ). after a productive phase (rising titers of p antigen and detection of hiv antigens by immunocytochemistry), the cells went into a latent phase where hiv could be detected only by dna polymerase chain reaction. a -fold increase in astrocytes staining for hiv antigens was seen after cocultivation with hut- cells. lymphocytes adhered to astrocytes by hours of cocultivation. no adhesion was seen to microglia. fusion of plasma membranes was seen on electron microscopy. infected astrocytes did not show cytopathic or morphological changes. cell-to-cell contact may be important in viral transmission to astrocytes. hszao-huei chen, steven b. stein, wing kong, and raymond p. roos, chicago, i l one of our goals is tq delineate molecular determinants for disease phenotypes produced by theiler's virus (tv), a mouse picornavirus. the identification of these genes and gene products may clarify viral pathogenesis and also lead to the identification of genes that are important in normal cns function and nonviral cns disease. members of the gdvii subgroup of tv cause an acute, fatal neuronal infection, whereas members of the to subgroup are less neurovirulent and produce a demyelinating persistent infection. our studies of infectious tv cdna clones have demonstrated that the gdvii b(vp )- c segment is critical for neurovirulence. several other areas of the genome, including the ' untranslated region (s'utr), also affect tmev-induced disease. the 'utr of poliovirus, another picornavirus, has a critical role in paralysis; this effect on neurovirulence is believed to result from an altered translational efficiency related to bind-region ing of neural cell proteins. tv 'utr has an unusual predicted secondary structure, even for picornaviruses. our studies demonstrate that the tv 'utr affects translational efficiency and has a distinctive protein-binding pattern. investigations of the tv 'utr may clarify features of translational regulation of cns genes in general. p . coronaviruses infect primate brain from g a y f. cabirac, ronald s. murray, galen cai, kristen hoel, and kenneth soike, englewood, co, and covington, la recently, we described finding coronavirus (cv) rna and antigen in active demyelinating plaques of multiple sclerosis (ms) brain tissue (murray et al, ann neurol, in press). molecular analysis showed the cv rna to be more closely related to murine cvs than to human cvs. we then demonstrated that, following intracerebral inoculation, the murine cv jhm and the putative ms isolate cv-sd could infect and cause demyelination in primate brain (murray et al, virology, in press). we now have data showing that murine cv can infect primate brain following intranasal or intravenous routes of inoculation. standard virology, histopathology, and the molecular analysis of viral cytotropism will be presented. we conclude that cvs related to murine cvs can infect primate cns from peripheral routes and warrant consideration as potential human pathogens. probes (gag, pol, or enw). eleven were white and were black. a history of transfusion was obtained in , and sexual risk of transmission was present in but not in others. fulminant disease occurred in transfused men months to years later but such disease was only seen in woman (in month). in contrast, of women with multiple sexual partners had rapid progressive disease. the male-to-female ratio was : for transfusion association and : for those at sexual risk but : for unknown risk, transfusion is an important risk for tsp/ham and the diagnosis must be considered in all gait problems, regardless of the "diagnosis."transfusion association should decrease with regular testing of blood donors, but this will not affect the risk of sexual transmission. spastic paraparedhtlv-i-associated myelopathy (tsp/ ham). it is unclear why htlv-i infection causes atl in some individuals and tsplham in others. differences in the genome of viral isolates or immunological and host factors have been hypothesized to play a role in disease expression. at present there are no animal models of tsplham and no suitable animal models of htlv-i infection that can address these issues. we transplanted severe combined immunodeficient (scid) mice with peripheral blood mononuclear cells (pbm) from tsplham patients in an attempt to produce htlv-i disease. two weeks after transplantation of pbm from tsplham patients, we detected anti-htlv-i igg in serum samples of of scid mice by enzyme immunoassay using sonicated whole virus. five weeks after transplantation, we detected anti-htlv-i igg to p , p , gp , or gp in serum samples of of scid mice by immunoblot of disrupted virus. these findings suggest that scid mice may be valuable in the study of molecular and pathological determinants of htlv-i-induced disease. theiler's virus produces an encephalomyelitis in susceptible mice. during the course of the disease, specific regions in the cns become infected. compared to the immunocompetent mouse, the nude mouse provides a useful model where viral dissemination can be studied in the absence of functional t lymphocytes and antibodies. we investigated the distribution and spread of the da strain of theiler's virus in the cns of nude mice. by immunohistochemistry, the hippocampus, arnygdaloid nuclei, entorhinal cortex, cingulate cortex, thalamus (anteroventral nuclei), midbrain, and spinal cord all contained viral antigens by weeks after infection. in addition, the olfactory nuclei, mamillary body, hypothalamus, basal ganglia, and nucleus raphe dorsalis often were involved. in the brain, the limbic system was the site commonly infected by theiler's virus. the time course of virus dissemination varied depending on the site of initial virus infection, though the final distribution of virus was the same. olfactory bulb injection, which is a direct inoculation into the olfactory pathway, resulted in more rapid spread than did cortex injection. we demonstrated the constant presence of viral antigen in the limbic system and a different kinetics of viral dissemination between the two different routes of intracerebral inoculations. these results suggest that limbic structures and their connections are important to the dissemination of theiler's virus. -associated myelopathy in a northwest native indian d. foti, d. werke, g. dekaban, g. p. a . rice, andj. oger, vancouver, bc, and london, ontario, canada a -year-old indian of the oweekeno tribe was admitted for investigation of a myelopathy. initially symptomatic with midthoracic radicular pain, she developed progressive spastic paraparesis over year with mild sensory symptoms and urinary retention. mri of the cord and brain were normal. csf showed elevated protein ( mgll), lymphocytes, and weak oligoclonal banding with evidence of intrathecal igg synthesis. antibodies to human t-lymphotropic virus type i (htlv-i) were positive by enzyme-linked immunosorbent assay and western blot on both serum and csf. presence of htlv-i was demonstrated by polymerase chain reaction of blood lymphocytes. htlv-i-associated myelopathy, or tropical spastic paraparesis, is endemic in southern japan, the caribbean basin, and several tropical islands but has not been reported in natives of northwest canada. this patient's only risk factors for htlv-i infection were blood transfusions years previously. ongoing familial and epidemiological studies as well as virus sequencing should indicate if this case represents an indigenous or an imported infection. caused by herpes simplex virus type lawy blankensbz) and herbert . newton, columbus, oh myeloradiculitis occasionally occurs secondary to herpes simplex virus type (hsv ) infection, but rarely has been reported after herpes simplex virus type (hsv ) infection without encephalitis. we describe a -year-old man who developed cervical myelopathy and radiculitis, never developed symptoms of encephalitis, and had positive hsvl spinal fluid cultures. h e initially developed extremity weakness and incoordination, numbness, paresthesias, and neck pain. evaluation was negative except for mri results, which showed a high-signal lesion centrally within the cervical cord. the weakness, sensory loss, and radicular pain progressed over several months. subsequent mri showed extension of the high-signal abnormality and mild enlargement of the cervical cord. symptoms stabilized briefly with dexamethasone but soon worsened, and were accompanied by paroxysmal kinesiogenic dystonic episodes of his arms and right leg. repeat evaluation was unrevealing except for the spinal fluid, which grew out hsv . the patient was treated with dilantin and a i-month course of intravenous acyclovir, with slow improvement of neurological status and resolution of the dystonic episodes. this case illustrates that hsvl can cause a myelopathy with a subacute and protracted course, requiring serial was more frequent in the middle-aged group ( p < . , p = . ). history of hypertension, previous strokes, diabetes mellitus, and antithrombotic treatment was similar, as were sex ratio, qualifying event type (transient ischemic attack or nondisabling stroke), and angiographic features; stenosis severity in either side and presence of ulceration were all similar. in elderly patients, ecad is associated with symptomatic cardiac disease (scad or af), whereas in middleaged patients it is associated with precursors of generalized atherosclerosis (smoking and hyperlipidemia). to identify the anatomic factors correlating with dementia in patients with lacunar infarctions, we examined digitized ct data on elderly patients (mean age = . yr; education = yr) who presented with acute lacunar infarction. dementia was diagnosed in patients ( . %) based on neuropsychological tests given months after stroke onset. the following ct variables were assessed: infarct location and number, total infarct volume, brain parenchymal and csf areas at levels, and width of the frontal horn, third ventricle (tvw), and lateral ventricle plus their ratio to the intracranial width. atrophy and leukoaraiosis were rated semiquantitatively using a standard scoring method. in the group overall, mean infarct volume was . cc and mean infarct number was . . in univariate analyses, dementia was significantly related to infarct volume, number, tvw, bilaterality, and infarct predominance on the left side, but not to leukoaraiosis or atrophy. in a regression model adjusting for demographic factors, the ct variables correlating independently with dementia status were infarct number (p = . , p = . ) and the presence of left cerebral infarcts (p = . , p = . ). we conclude that the most important ct variables related to dementia in lacunar stroke are lesion multiplicity and the presence of lesions on the left side. brain ischemia results in potassium (k+)-induced voltageregulated presynaptic calcium (ca") accumulation, which may contribute directly to neuronal in jury presynaptically, and also promote excessive release of excitatory neurotransmitters leading to cell damage postsynaptically. k+-induced depolarization of brain synaptosomes may be used as an in vitro model to study the therapeutic potential of pharmacological agents to alter ischemia-induced presynaptic ca + accumulation. we preincubated gerbil cerebral cortical synaptosomes in r (janssen pharmaceutical) at concentrations of to lo-' m, subsequently depolarized the synaptosomes with k + , at concentrations of to mm, and measured intrasynaptosomal ca ' ([ca +]) with the fluorescent indicator fura . r had no effect on ecaz'i in nondepolarized synaptosomes, but significantly (<. , student t ) depressed depolarization-induced {ca +] at to lo-' m in a dose-dependent fashion. the greater the degree of depolarization employed, the greater degree of depression in [ca +] was seen at each dose of r . since r had no effect on [ca +] when utilizing ca +-free incubation media, it was demonstrated that r prevents depolaritation-induced [ca ' ] increase by blocking voltage-regulated influx. because r appears to block voltage-regulated presynaptic ca ' accumulation, it should be further evaluated as a potential therapeutic agent after cerebral ischemia. sneddon's syndrome (ss) is a focal and diffuse arthropathy affecting mainly the vascular wall of the skin and cerebral arteries. etiology is not determined. we studied patients with ss ( females, males), aged to years. clinical, radiological, and immunological studies were done. all patients developed cerebrovascular disorders: ischemic stroke in %, transient ischemic attack (tia) in %, and ischemic stroke and tia in %. cerebral scan showed small and medium (less than cm) ischemic lesions in %. these lesions were superficial in the cerebral cortex ( %); deeper in the centrum semiovalis, internal capsule, and basal ganglia ( %); and both superficial and deeper ( %). ultrasound and angiogram studies revealed obstruction of intracranial arteries in %, and of extracranial arteries in %. partial or complete improvement of cerebrovascular symptoms was observed in %. immunological studies showed increased content of b-cell lymphocytes ( p < . ), increased levels of igm ( p < o.oool), and circulating immunocomplexes ( p < . ). anticardiolipin antibodies were increased ( %) and lupus anticoagulant detected ( %). cerebrovascular disorders in ss that lead to small ischemic cortical lesions have a good prognosis. pathogenesis of this syndrome may be related to antiphospholipid antibodies. the cheiro-oral syndrome is characterized by pure sensory deficit limited to the hand and mouth. this syndrome has been described in diverse lesions of the parietal operculum and brainstem, but occurs most commonly due to lesions of the contralateral thalamus, and suggests a humuncular representation of sensation in the ventral posterior lateral (vpl) and ventral posterior medial (vpm) nuclei. we describe a -year-old hypertensive woman who presented with sudden onset of numbness of the left side of her body. examination revealed a left hemisensory deficit to all modalities that spared the hand and peri-oral regions. cat scan and mri demonstrated an acute infarction of the posterolateral right thalamus, with a rim of preservation adjacent to the internal capsule. pure hemisensory loss with sparing of the hand and mouth ("inverse cheiro-oral syndrome") has not been reported previously, and complements previously published studies of the cheiro-oral syndrome in demonstrating somatotopic sensory representation in the thalamus. to examine the relationship between depression and dementia after stroke, we administered the -item hamilton depression rating scale (hdrs) and neuropsychological tests to elderly patients months after ischemic stroke. using dsm- -r criteria, we found dementia in ( . %). hdrs score was . -t . overall, and higher in demented compared to nondemented patients ( . . vs . t . , p = . ). the frequency of depression (total hdrs score > ) was also higher with dementia ( . % vs . %, p = . ). however, demented patients did not differ from nondemented patients on ratings of depressed mood. instead, hdrs items for psychomotor retardation, reduced work activities, and impaired insight best distinguished the groups. in multiple regression analysis, stroke severity (p = . , p = . ) was the most important correlate of hdrs score; dementia status was not correlated independently. mean scores on mini-mental state examination and neuropsychological tests assessing memory, orientation, verbal, spatial, attentional, and abstract reasoning skills did not differ by depression status. although weakly related to intellectual impairment, hdrs score in our stroke sample was most importantly associated with stroke severity. higher scores on hdrs in demented stroke patients may be explained by physical and cognitive symptoms that are expected with dementia. these findings do not support a causal link between depression and dementia, and argue against the importance of "depressive pseudodementia" as an explanation for intellectual decline after stroke. to investigate the pathophysiological mechanism of the white matter lesions in progressive subcortical vascular encephalopathy (psve) of binswanger type, we measured regional water partition coefficient (pc) (reflecting water content) and effective p h (pht) (weighted average of intra-and extracellular ph) with dynamic positron emission tomographic technique using - co,, o,, and c- co,. si-multaneously, regional cerebral blood flow (rcbf) and regional cerebral metabolic rate of oxygen (rcmro,) were evaluated. eight subjects ( normal, psve, multiple infarction) were examined. in the white matter lesions in psve, corresponding to high-intensity areas in t -weighted images of mr, the pc increased and pht was unchanged or decreased, whereas both rcbf and rcmroz declined. the ratio of white matter pc to gray matter pc was . in psve and . in normals. in the frontal gray matter, the pc decreased in psve, whereas rcbf and rcmroz also decreased. these results suggest that tissue water content increases in the white matter lesions in psve reflecting the edematous status of the damaged regions. elevated pht with high pc may reflect the increase of extracellular water of the tissue. measurement of pc and pht provides useful information about the pathogenesis of psve. stroke has been the second most common cause of death in korea but its risk factors (rfs) have not been studied intensively, so the rfs or causes were investigated prospectively in , consecutive stroke patients who were admitted to the chungnam national university hospital, taejon, korea, between and . they included cases of cerebral ischemia (ci) ( . %), cases of intracerebral hemorrhage (ich) ( . %), and cases of subarachnoid hemorrhage (sah) ( . %). control data were obtained from healthy spouses of the patients. multivariate analyses showed that hypertension (ht) was the strongest rf for all stroke types and was followed by old age, diabetes mellitus (dm), smoking, high-density lipoprotein cholesterol, and fibrinogen. the rfs for atherothrombotic ci included old age, ht, dm, smoking, fibrinogen, and cholesterol. for lacunar infarcts, ht, dm, old age, fibrinogen, alcohol abuse, smoking, and female sex were the significant rfs. ht was the cause of ich in ( . %) and alcohol abuse ( , . %) and vascular anomalies ( , . %) followed. most alcoholassociated ichs occurred characteristically in the posterior fossa. frequencies of hospital admission of ich patients correlated positively with diurnal variation of temperature ( r = . , p < . ). aneurysmal rupture was the cause of sah in patients ( . %). three major sites of aneurysms identified on cerebral angiograms were the anterior communicating artery ( , . %), the middle cerebral artery ( , . %), and the posterior communicating artery ( , . %). thirty-nine patients ( . %) had no identifiable cause of sah. in korea curvilinear subinsular lesions have been noted on ct but the underlying pathology is unknown. we reviewed the cranial mr scans ( . tesla ge machine) of serial patients over the age of who had no cause for mr hyperintensities (hi) other than age or vascular risk factors. seven ( %) had linear subinsular h i (sihi). four of ( %) patients with sihi and of ( %) without sihi had marked periventricular h i compatible with subcortical arteriosclerotic encephalopathy (pvhi) ( p < . ). patients with sihi were older ( . yr) than patients without sihi ( . yr) ( p < . ). four of ( %) patients with sihi had hypertension program and abstracts, american neurological association or vascular risk factors. a further patient with diabetes, hypertension, and the opercular syndrome (bilateral facial, pharyngeal, and lingual weakness) had subinsular lesions on ct. the brain arteries were injected postmortem with a lead/ gelatin suspension, and mr of the whole brain and x-ray films of the brain slices were taken. bilateral sihi were seen on mr and corresponded with linear cavitary infarction pathologically, which on microangiograms was found to lie in the border-zone between cortical and basal penetrating arteries. we conclude that sihi are associated with older age and pvhi, and can be due to infarction in a deep watershed territory and can be associated with clinical deficits. hemodilution-treatment results in consecutive cases james l. frey, phoenix, az because precipitous neurological deterioration occurred during blood pressure reduction in a seminal case of lacunar infarction, subsequent patients with partial or evolving lacunar deficits were treated with hemodilution and blood pressure nonintervention to test the hypothesis that lacunar strokes represent perfusion failure. isovolemic hemodilution was performed using hetastarch with target hematocrit of to . results of pretreatment ct brain scans, carotid ultrasound, and echocardiograms were normal. nine patients recovered normal neurological function, and regained complete functional independence in close temporal correlation with hemodilution. mri brain scans demonstrated appropriate single white matter lesions in cases. no specific risk factor combination could be identified. n o patient has had recurrent stroke in follow-up from to months. response to hemodilution suggests a hemodynamic pathophysiology. successful treatment requires ( } blood pressure nonintervention and ( ) hemodilution prior to severe clinical deterioration. the hemodynamic classification for the carotid-cavernous sinus fistula (ccf) is important for the implication of prognosis and therapy, but satisfactory objective criteria for such differentiation is still lacking. retrospectively, we studied the application of extracranial duplex sonography in cases of ccf with emphasis on the hemodynamic parameters of resistivity index and flow volume. a correlation was made with the angiographic findings in an attempt to evolve an objective hemodynamic classification by this noninvasive method. the alterations in membrane metabolism and structure could be the primary etiological event in alzheimer's disease (ad) that results in the clinical and neuropathological findings. to investigate in vivo brain membrane phospholipid and highenergy phosphate metabolism in probable ad patients and control subjects, the building blocks (pme) and breakdown products (pde) of membranes and the high-energy phosphates pcr and atp were measured noninvasively by in vivo brain p mrs in probable ad patients ( males; females) and controls ( males; females). all subjects were assessed by mini-mental, mattis, and blessed scales. we found that, at clinical onset, ad females had elevated pme ( p = . ), decreased pcr ( p = . ), and decreased atp ( p = . ). similar changes were not seen in ad males at clinical onset, but the severely demented ad males had increased atp ( p = . ). correlation analysis for the ad patients revealed that increasing dementia was associated with decreasing pme ( p = . ; r = . ), increasing pde ( p = . ; r = . ), increasing pcr ( p = . ; r = . ), and increasing atp ( p = . ; y = . ). similar metaboliccognitive correlations were not seen in the controls. these results demonstrate alterations in membrane and energy metabolism at the earliest clinical stages of ad. increasing dementia correlates with markers of membrane degeneration and decreased utilization of high-energy phosphates. both of these findings suggest the dementia in ad is secondary to membrane changes resulting in synapse loss. alzheimer's disease: postmortem mri and histological correlates fen-lei f. chang, j. e. purisi, c. r. jack+ jr, and r. c. petersen, rochester, mn hippocampal atrophy, defined by mri-derived volumetric measurement, has been useful in differentiating alzheimer's disease (ad) patients from normals. since several studies have demonstrated anatomical and functional gradients along the rostral-caudal (r-c) axis of the hippocampus, it is tempting to speculate on the existence of a differential distribution of morphometric changes along this axis. the hippocampi from patients with clinically and pathologically confirmed ad were studied by postmortem mri and by reconstruction of serial histological sections. there was good correspondence between these two methods. the r-c length of the hippocampus in ad was preserved compared to normal. this length was longer on the left, both in normals and ad. the adassociated atrophy was due primarily to the reduction of the coronal cross-sectional area of the hippocampus. with increasing hippocampal atrophy, volume reduction was more prominent in the rostral area (pes hippocampus). this nonuniform reduction in volume may be associated with different connectivity patterns between rostral and caudal hippocampus. the purpose of this study was to determine the neuropathological validity of nincds-adrda criteria (nac) for probable and possible ad. mckhann et al ( ) provided clinical criteria for the categories probable ad and possible ad. the validity of these categories has not yet been reported. a retrospective, blinded evaluation of the complete neurological history, examination, neuroimaging, laboratory, and psychometric data was done for subjects from the state of florida brain bank. pathological classification, which was blind to clinical diagnoses, was in categories: pure ad, ad + , and other dementias. ninety-three percent of probable ad (n = ), whereas only % of possible ad (n = ) patients, had ad or a d + ( p = . ). pure ad was found in % of probable ad and % of possible ad patients ( p = . ). pathological evidence of coexisting parkinson's disease was present in % of all ad brains. these results suggest differential predictive power of nac for probable and possible ad, as suggested by the labels. nac are, therefore, usefui for research and clinical purposes. our prior study of falls in the elderly had shown significantly more white matter low attenuation (wmla) on ct scan among fallers than nonfallers, but no association between wmla and cognitive impairment among nondemented subjects. as these subjects were followed over time, it appeared that fallers were becoming demented at a more rapid rate than nonfallers. as a result, we analyzed the relationship between wmla and rate of change on the blessed test of information, memory, and concentration (bimc) in a combined cohort of subjects from the falls study and from a longitudinal study of dementia and normal aging. we selected of these subjects whose initial bimc score was less than (i.e., not already severely impaired) and who had at least yearly bimc evaluations. ct scans were scored on an -point ordinal scale for hemispheric wmla. linear regression was used to summarize the rate of change for each subjects' test scores. the rate of change on bimc was . points per year with standard error (se) of . among subjects who eventually became demented; nondemented subjects declined at . points per year with se of . . multiple regression analysis was performed with initial bimc score and wmla as the independent variables and rate of change of bimc as the dependent variable. wmla accounted for % of the variance (partial r = . , t = . , p < . ). these data suggest that elderly subjects with wmla may be at increased risk for rapid cognitive decline. such as g proteins and adenylate cyclase. the activities of adenylate cyclase, and of the g protein-associated enzyme activity, low-km glutamyl transpeptidase (gtpase), were assayed in membranes prepared from the postmortem brains of alzheimer-diseased and age-matched control subjects. both basal and fluoroaluminate-stimulated adenylate cyclase activities were significantly reduced in ad frontal cortex compared to control subjects ( p < . ; two-tailed student's t test). in addition, a significant, though smaller, reduction in basal gtpase activity also was detected in ad frontal cortex ( p < . ). in contrast, no significant change in the activity of either enzyme was detected in the hippocampus. the stimulation of gtpase activity by muscarinic and gababreceptor agonists was not altered significantly by the presence of alzheimer's disease. however, the degree of stimulation was much lower in human tissue compared to that observed using fresh rat brain, suggesting that the ability of receptors to activate g proteins declines post mortem. these results suggest that alzheimer's disease causes alterations in some key components involved in signal transduction. the association of lobar hemorrhage (lh) with cerebral amyloid angiopathy (caa) and that of caa with alzheimer's disease (ad) are well known. to determine how frequently lh and caa occur in ad, we reviewed patients with cerebral or cerebellar hemorrhage and caa. five patients were treated surgically, none was demented, died, and came to autopsy. eight patients died of lh and came to autopsy. of the autopsied patients, had ad, both clinically and neuropathologically. they comprised . % of cases of autopsy-confirmed ad in our laboratory. none of the other patients were known to be demented. five had senile plaques, with or without neurofibrillary tangles, in the hippocampus, and occasional senile plaques in the cortex, but none met the consortium for establishing a registry for alzheimer's disease neuropathological criteria for ad. ad patients were , , and years old and the ages of the nondemented patients ranged from to (mean = . yr). seven were younger than years of age. despite the frequent occurrence of caa in ad, we found that lh was uncommon. in addition, most of our patients with lh and caa were not demented and tended to be younger than ad patients with lh. these results indicate that the hf is involved primarily in acquisition or leaning processes and less so in retrieval of previously learned information. these findings relate to memory and structural brain changes found in normal aging. alzheimer's disease y . stern, l. stricks, g. alexander, . prohovnik, and there is an inverse relationship between parietotemporal cerebral blood flow and years of education in alzheimer's disease (ad) patients matched for clinical severity, which suggests delayed clinical manifestation of ad in patients with higher education (stern et al, soc neurosci abs ). we classified the lifetime primary occupations of ad patients using the dictionary of occupational titles of the us department of labor and derived factor scores describing intellectual, interpersonal, and physical job demands. after controlling for age, education, age at onset, illness duration, and dementia severity (mental status and activities of daily living), relative perfusion in the parietotemporal region (assessed using -xenon inhalation) showed significant correlations with job complexity (pl: r = -. , p < . ) and interpersonal (p : r = -. , p < . ) factor scores. in a stepwise multiple regression, job complexity and interpersonal skills increased explained parietotemporal flow variance by . % ( f = . , p < . ) over that explained by demo-graphic and severity indices; physical demands then accounted for another . % of the variance ( f = . , p < . ). we conclude that occupational demands, similar to but independent of education, may provide a reserve that delays the clinical expression of ad. richard mayeux and ming-xin tang, new york, n y risk factors for alzheimer's disease (ad) were collected from patients with ad and healthy elderly controls in an urban community population consisting of ethnic groups: black, hispanic, and white. advanced age (> yr) (or = . ; % ci . - . ) and head injury with loss of consciousness (or = . ; . - . ) were associated with ad, controlling for all known putative risk factors. factors such as low education (< yr) (or = . ; . - . ) and family history of ad (or = . ; . - . ) were not found to be significantly related to ad. head injury occurred in . % of the patients and .?% of controls. most ( %) head injuries in the patients with ad occurred after age , prior to disease onset. in controls with head injury, had experienced a head injury before age . the duration of unconsciousness was consistently longer in patients with ad than in the controls. the overall effect in each ethnic group was similar (or,, . ; . - . ). these results confirm and strengthen the previously described putative relationship between head injury and ad. we also conclude that both the severity and the timing of the head injury as well as the frequency of head injury in the population at risk may be important factors in understanding the causal relationship between head injury and ad. the purpose of this study was to determine how native language affects the cutoff scores in screening tests for dementia. there is a paucity of data o n this issue at present. screening tests used in the study were: folstein mini-mental state (mms); clockdrawing (clock); preparing a letter for mailing (mail); -item grocery list (list); and hamilton depression scale (ham). the subjects included ( demented) native english speakers (eng) and ( demented) native spanish speakers (spa) with memory complaints. diagnosis of dementia was determined by neurological, neuropsychological, and psychiatric evaluation. age and gender were unrelated to mms. in spa only, education was positively related to mms. ham scores were higher in de- and . (spa); c d and list did not add discriminative power to mms for eng but did so for list in spa. mms discriminates between demented and nondemented far better in english than in spanish speakers. only mail adds discriminative power to mms. a. heyman, g. fillenbaum, s. mirra, and participating cerad neuropathologists, durham, nc, and atlanta, ga the clinical diagnosis of alzheimer's disease (ad) has become more accurate in recent years due to the application of specific clinical criteria, wider use of neuroimaging procedures, and greater expertise among physicians. we report the frequency of clinical misdiagnosis of ad among patients who at autopsy were found to meet the rigorous clinical diagnostic criteria imposed by the consortium to establish a registry for alzheimer's disease (cerad) study. these patients included men and women (mean ages and yr, respectively) who were among the group of patients who died in cerad medical centers in the us between and . the clinical diagnosis of ad was neuropathologically confirmed in ( . %) of the cases. of these cases, varying degrees of concomitant cerebrovascular disease were present in % and coexisting parkinson's disease changes were found in %. in of the patients without neuropathological evidence of ad, the diagnoses were: lobar atrophy, diffuse lewy body disease, nonspecific neurodegenerative changes, and mesocorticolimbic dementia, respectively. the fifth patient showed no morphological abnormalities. on the basis of these results, it would appear that application of strict diagnostic criteria, as well as the use of brain scans and detailed clinical and neuropsychological tests by experienced clinicians, cannot yet distinguish some types of primary degenerative dementias from alzheimer's disease. we designed a scale that measures an underexplored facet of functional decline in alzheimer's disease (ad): the patient's dependence on others for supervising or performing activities. two hundred twenty-three informants for patients with mild ad (clinical dementia rating [cdr] = for ; cdr = for ) were interviewed and dependence was staged from to . interrater reliability was assessed by separate interviews of informants; agreement was % for dependence stage. dependence stage differed significantly at the cdr levels (chi square = . , p < . ) and correlated significantly with modified mini-mental state (mmms) ( r = -. , p < . ) and blessed dementia rating scale-part (bdrs) ( y = . , p < . ). seventy-eight percent of patients in a health-related facility were at stage or higher vs % of patients living at home. in a multiple regression model, both the bdrs and dependence scale accounted for unique portions of the variance in mmms, suggesting that they assess unique aspects of functional ability. dependence increased significantly in patients retested at year. we conclude that the dependence scale is reliable and relates to both disease severity and progression. formal assessment of dependence should prove useful for studies of the natural history of ad as well as for clinical trials. cortical-basal ganglionic degeneration (cbgd) is a disorder characterized by an asymmetrical akinetic-rigid syndrome and cortical signs such as apraxia, alien limb phenomena, and cortical sensory loss. dementia has been present in many cases, but always as a late manifestation. we report cases pathologically consistent with cbgd, presenting as primary degenerative dementia, fulfilling nincds-adrda criteria for probable alzheimer's disease (ad). the first patient presented with changes in memory and personality, language dysfunction, decreased verbal output, and shuffling gait. follow-up examinations over years showed progressive dementia, wide-based gait, and frequent falls. the second patient presented with complaints of memory loss. neuropsychological examinations showed progressive deficits in memory, attention, calculations, and visuospatial functioning. no movement disorder developed over years of follow-up. at autopsy, both patients had typical changes of cbgd and lacked pathological features of ad. definitive diagnosis of cbgd rests on both clinical and pathological criteria. cbgd should be considered in the differential diagnosis of patients with dementia resembling that found in ad, especially if extrapyramidal signs are present. a quick, easily administered and scored test for praxis is desirable in evaluation of neurological patients. during months in , each patient seen in the outpatient setting by the principal investigator (e. k.) received of praxis screening batteries. thirty-six patients were tested with a short battery. eleven had normal cognition based on neurological history, examination, and a short test of mental status. twenty-five had cognitive decline (cd). handedness, male/ female ratio, education, and mean age were similar in both groups. mean time of completion of the test was seconds in normals and * seconds in patients with cd. total scores (maximum ) were . * . in the normals and . in the cognitively declined patients. twenty-five other patients, with cd and with normal cognition, were tested with a longer battery containing oral/ facial, upper and lower limb, axial, sequential, and imitation subtests (maximum score ). normals completed the battery in i , and cognitively declined patients completed the battery in ? seconds. of the various subtests, tests of sequential praxis were performed most poorly by patients with cd: . * . in normals vs . * . in patients with cd. oral/facial praxis was least affected by cd: olivopontocerebellar atrophy (opca) is generally understood to be a nondementing neurodegenerative disorder affecting the cerebellum, lower brainstem, and spinal cord. one of us (s. k.) recently reported that postmortem cerebral cortex from patients with dominantly inherited opca shows a widespread reduction of cholinergic markers similar to that observed in alzheimer's disease (ad). we were interested to determine the status of other neurotransmitter systems in opca postmortem cerebral cortex. samples of frontal, parietal, temporal, and occipital cortex were dissected from confirmed cases of opca and age-matched controls. after processing, neuropeptide levels were measured by radioimmunoassay. concentrations of somatostatin were significantly reduced by to % in of the cortical areas of opca brain that were examined. the area that was spared was the inferior temporal gyrus, a region in which somatostatin levels are markedly reduced in ad. levels of neuropeptide y were normal in all areas, while concentrations of cholecystokinin, vasoactive intestinal polypeptide, and substance p were significantly increased in of the areas. these data show widespread neuropeptide changes in the cerebral cortex of opca postmortem brain. in contrast to cholinergic markers, the pattern of neuropeptide changes is different from what is observed in ad. it has been postulated that demise of the corticomotoneuron is the initial event in amyotrophic lateral sclerosis (als) and that the anterior horn cell dies as the result of antegrade glutamatergic excitotoxicity (muscle nerve ; : ). excitability of the corticomotoneuronal system can be tested by measuring threshold-to-cortical magnetic stimulation and the motor-evoked potential (mep)/compound muscle action potential (cmap) ratio, which estimates the number of corticornotoneurons stimulated. cortical threshold and mep/ cmap ratio were measured in patients early in the course of als. the mean time interval from onset of first symptoms was . months. mean threshold and mepicmap ratio measured . f . % and . rfr . %, respectively. in ( . %) patients, threshold was paradoxically low (< %, mean . +. . %) and in ( . %) patients there was no response. there was a significant ( r z = . ) inverse power relationship between cortical threshold and mep/cmap ratio given by . x mep/cmap-'j. six months later, / ( . %) patients still had low thresholds but the mean mep/ cmap ratio had dropped to . ? . % and in . % there was no response. we conclude that early in als the corticomotoneuronal pathways are abnormally excitable. this may explain early cramping and fasciculation, which characteristically diminishes as als progresses. one of the distinct clinical features in patients with amyotrophic lateral sclerosis (als) is loss of elasticity of skin. however, little is known concerning the biochemical nature of skin elastin in als. in our study, cross-links unique to elastin, desmosine and isodesmosine, were measured and compared in skin tissue (left upper arm) from patients with als and from age-matched controls. the contents of desmosine and isodesmosine were decreased significantly ( p < . and p < . , respectively) in patients with als (mean * sd, . ? . and . * . nmol/mg dry weight; range . - . and . - . nmol/mg dry weight, respectively) as compared with those of controls (mean sd, . * . and . . ;range . - . and . - . , respectively), and were negatively and significantly associated with duration of illness in patients with with amyotrophic lateral sclerosis als ( r = - . , p < . , and r = - . , p < . , respectively). the ratio of desmosine and isodesmosine was constant ( : ) in all samples analyzed. the decline in skin desmosine and isodesmosine is more rapid in als than in normal aging. thus, cross-linking of skin elastin is affected in als. (supported in part by n i h grants de , de , de , ar , ar , and nasa grant nag- - .) pl . natural history of amyotrophic ( ) collection of large numbers of twin pairs in disease of low prevalence is difficult. to circumvent this, we devised a new approach termed the "death discordant twin pair" method. eleven thousand deaths from motor neuron disease (mnd) were extracted from the office of population censuses and surveys during to . birth indexes from onward were searched for possible twins. for each twin so identified ( pairs), the national health service central registry located the relevant family practitioner committee and thence the co-twin's general practitioner. the search produced: ( ) living co-twins; ( ) embarked; ( ) dying as adults or infants; ( ) not mnd; and ( ) validity of the accuracy, sensitivity, and specificity of the world federation of neurology (wfn) subcommittee on motor neuron disease working group criteria for the clinical diagnosis of amyotrophic lateral sclerosis (als) has been tested against neuropathological criteria in autopsied patients (neurology, in press). integration of clinical and electrodiagnostic data to meet wfn criteria for possible, probable, and definite als was studied in this samegroup. patients received . ifr . (mean * standard deviation) electromyograms (emgs) per patient and included . . emg levels (bulbar, cervical, thoracic, lumbar) per patient. proportionately fewer emgs were performed as the level of diagnostic certainty at presentation increased: suspected ( . %), possible ( . %), probable ( . %), definite ( . %). in only . % of all patients studied did the first emg alone change the level of diagnostic certainty of the diagnosis of als. only . % of patients presenting with suspected als alone or with possible or probable als were associated with a change in level of diagnostic certainty following or more emgs. however, although increasing the number of emgs performed per patient may be associated with an increasing chance of increasing the level of diagnostic certainty ( emgdpatient = . %; emgdpatient = . %), selection of the level of emg analysis was more crucial. the "el escorial" criteria emphasize the importance of emg evidence of lower motor neuron involvement in a limb with clinical upper motor neuron signs. our analysis of emg studies in autopsy-confirmed als patients suggests that complete evaluation of bulbar and thoracic levels for lower motor neuron changes and complete evaluation of motor unit recruitment patterns are important for the integration of emg data with clinical data in the application of the "el escorial" criteria for the diagnosis of possible, probable, and definite als. sibships on guam annette grefe, john steele, linda flares, and stephen waving, birmingham, al, and umatac and mangikao, guam reports in the s indicated that % of guamanian chamorro patients with amyotrophic lateral sclerosis (als) gave october a positive family history. subsequent investigators have inferred that purely genetic factors are not responsible for guamanian als or its clinical variant, parkinsonism-dementia complex (pdc). we report the first chamorro sibships selected in an ongoing study of familial aggregations. the basis for the initial selection was that the youngest patient in the sibship had als. age of onset was to years (mean yr). fourteen of persons in these sibships were affected. others in the sibship developed pdc, progressive supranuclear palsy, or pure dementia later in life (mean yr, range - yr). these cases developed to years (mean yr) after onset of disease in the first sibling. the age at onset of the first case and the intervals between earliest and latest cases in such sibships may help to determine minimal and maximal latency (i.e., interval between exposure to an exogenous agent and onset of symptoms). our observations suggest that exposure may have occurred early (before age ) with varying and often long latency (up to yr). we also find that variability of clinical expression may be correlated with the age at onset. concentrating on the study of familial cases on guam may enhance the identification of the etiologic agent(s) of this prevalent and tragic disorder. the spinocerebellar ataxias are an uncommon group of genetic disorders that have been well characterized in north america and europe. information concerning these conditions in africa and other parts is scant. to address this problem, a large-scale survey has been undertaken in the cape province of south africa. in this investigation, more than persons in affected families have been appraised and investigated and phenotypic features have been analyzed in detail. linkage studies have been undertaken in families with similar phenotypes in which the condition was transmitted as an autosomal-dominant trait. human lymphocyte antigen (hla) typing was carried out on members of the families and linkage analysis was undertaken using the liped program to analyze the data with a correction factor for age of onset. maximum lod scores were: family a: . ( = . ); family b: . ( = . ); family c: . ( = . ); family d: . ( = . ). these results indicate linkage to hla in out of families. these findings provide support for the concept of genetic heterogeneity in these phenotypically homogeneous families. pcr typing with the reportedly more closely linked d s locus is now being undertaken in these south african families. h.-p. hartung, g. f. hoffmann, and g. becker, wunburg, heidelberg, germany recently, l- -hydroxyglutaric acidemia has been described as a novel metabolic disorder in children. we report the occurrence of this disease in adults. one of brothers developed at the age of an abnormal gait and dysarthria; in the other , clumsiness and walking delay were noted at age . symptoms progressed and at the time of admission, when the patients were and years, neurological examination revealed a spastic ataxic gait, limb ataxia, dysmetria, dysarthria, dystonic posturing, and mental retardation. ct and mr imaging revealed subcortical white matter changes with loss of arcuate fibers, folial atrophy, and leakage in the cerebellar vermis, as well as atrophic changes in the cerebellar hemi-acidemia program and abstracts, american neurological association spheres. on biochemical screening, highly elevated concentrations of l- -hydroxyglutaric acid were found in csf, plasma, and urine. the pathological accumulation of l- hydroxyglutaric acid in these adults, along with the clinical picture characterized by cerebellar, extrapyramidal, and pyramidal symptoms and oligophrenia, and the neuroradiological findings of severe loss of myelinated arcuate fibers in subcortical white matter, conform with what previously has been described in the few neuropediatric cases. the biochemical abnormality underlying accumulation of this organic acid remains elusive. this study was undertaken to differentiate primarily affected areas from functionally suppressed areas due to remote effect in aphasic patients with focal brain degeneration, using an activation method with - water, in comparison with [sf] -fluoro- -deoxy-~-g~ucose (fdg) positron emission tomographic examination at rest. the subjects were patients with slowly progressive aphasia showing contrasted clinical symptoms (nonfluent type vs fluent type). regional cerebral metabolic rate of glucose (cmrglu) was measured with intravenous injection of mbq of f- fdg at rest. regional cerebral blood flow (cbf) was measured with intravenous bolus injection of . gbq of - water in different conditions: at rest, under repetition tasks, and under naming tasks. changes of cbf were evaluated between a resting condition and task-performing conditions. regional cmrglu was decreased focally in both broca's and wernicke's areas similarly in these cases in spite of the difference in clinical symptoms, whereas the patterns of regional cbf changes were different. the fluent patient showed prominent activation in the bilateral frontal areas on repetition tasks. the nonfluent patient showed, whereas the fluent patient did not show, focal activation in the left occipitoparietal area on a naming task. evaluation with an activation method can provide detailed information about the pathophysiological process and the location of primary lesion. defective complex i activity has been linked to huntington's disease (hd) and parkinson's disease (pd). intrastriatal injection of inhibitors of complex i reproduces the pathological features of hd, and the neurotoxin mpp+ kills dopaminergic neurons by inhibiting complex i. defects in complex i have been reported in hd and pd, but the distribution of this enzyme in the brain is unknown. to map complex i in brain quantitatively, we developed an assay using e h)dihydrorotenone to label the enzyme in tissue sections. this high-affinity binding is saturable and is displaceable by rote-in brain none and mpp+. using pm rotenone to define nonspecific binding, more than % of binding is specific. highest levels of binding are found in kidney, followed by myocardium. moderate levels of complex i are seen in striated muscle and some brain regions. within the brain, binding varies more than -fold and is heaviest in the cerebellar molecular layer and dentate gyrus. lower levels of binding are found in cortex and striatum and very low levels are located in substantia nigra. this assay may help to clarify the role of complex i in neurodegenerative disorders. ( in , patients with medically intractable parkinson's disease underwent autologous adrenal medullary-to-caudate transplants at the university of california-los angeles (ucla). these persons had been followed for several years before operation at -or -month intervals. at each visit, their disability had been rated on the quantified ucla scale and the hoehn and yahr stage of disease. these provided a longitudinal assessment of the progression of disease in each patient, which could be compared to the rate of progression in the cohort of cases followed at ucla for years. in addition, the unified parkinson's disease rating scale provided supplementary data for the immediate preoperative and subsequent postoperative evaluations. the hours "off" also were recorded for the preoperative and postoperative periods. after operation, the same evaluations were performed by the neurologist who previously had cared for the patients. the longitudinal postoperative data revealed that at to months after operation, all patients improved. after years, continue to be less disabled than their preoperative baselines. the progression of their disease, while still evident, is nonetheless proceeding at a slower rate than before transplant. the fourth patient had brief improvement shortly after operation, but then rapidly worsened to his previous level. his disease has continued to progress at a rapid pace, unchanged from progression before operation. individuals at risk for huntington's disease h . p. h . kremer, w. shtybel, b. snow, c. clark, j . theilmann, m . r. hayden, and w. in huntington's disease (hd), caudate hypometabolism as demonstrated by positron emission tomography (pet) is a well-established feature in symptomatic patients. in individuals at risk, however, conflicting findings are reported. since we have performed pet scans in asymptomatic persons at risk for hd (age - yr) . linkage analysis with independent dna probes or subsequent evolution to clinical hd (in patients) allowed a risk estimate for subjects. twenty were considered to be at increased risk (? %), at decreased risk (< %), and in individual no modification could be given. nine subjects were not tested. a pet scan was considered abnormal if either the caudate/thalamus ratio or the caudate/whole-brain ratio of rcmrglu was more than these criteria, scans in individuals were abnormal. none had received a decreased risk. comparison of the ratios of increased risk, decreased risk, unmodified risk, and control subjects failed to show statistically significant differences (analysis of variance). follow-up of persons with an abnormal scan showed conversion to symptomatic status within years after the first abnormal scan. this result suggests that an abnormal pet scan in a person at risk for h d heralds the onset of choreic movements. robitaille, m . el-awar, b. clark, l. scbut, m. ball, l. young, r. currier, and k. sbannak, toronto, ontario, and montreal, quebec, canada; pittsburgh, pa, minneapolis, mn, portkmd, or, and jackson, ms we measured the levels of dopamine in striatum of patients with end-stage dominantly inherited olivopontocerebellar atrophy (opca). on average, dopamine levels were reduced in putamen ( - , as compared with controls), caudate ( - %), and nucleus accumbens ( - %). however, individual patient values showed a wide variation (normal to - ), indicating that striatal dopamine loss is a common, but not constant feature of opca. seven patients had marked putamen dopamine loss (- to - ) but without corresponding severe substantia nigra cell damage; this suggests a "dying-back'' phenomenon in which nerve terminal loss precedes cell-body degeneration. in this regard, opca may offer the possibility of examining nigrostriatal dopamine neuronal degeneration at an early stage. although patients were found to have severe nigral cell loss with near total ( - to - %) striatal dopamine loss, none had depression in parkinson's disease (pd) has been correlated with low cerebrospinal fluid (csf) levels of -hydroxyindoleacetic acid ( -hiaa). l-dopa may precipitate or exacerbate depression in % of pd patients. to determine if l-dopa affects -hydroxytryptamine ( ht) metabolism, patients were studied. four had pd. of these, were taking l-dopa and both were depressed. the diagnoses in the remaining were progressive supranuclear palsy (psp), striatonigral degeneration (snd), normal pressure hydrocephalus, and pseudoseizures with depression. neuropsychological examinations and lps of all patients were done. three patients were started on l-dopa (the previously untreated pd patients and the psp patient) and retested days later. one pd patient started on l-dopa became depressed. mood in the others was unchanged. csf was analyzed for ht and -hiaa. ht could not be detected in the csf of patients who were not taking l-dopa, but was easily detectable in all of the patients who were taking l-dopa. -hiaa levels were low in the untreated pd patients, and also in the patients with psp, snd, and pseudoseizures with depression. -hiaa levels were even lower in the l-dopa-treated patients. the ratio of -hiaa: ht (an index of ht turnover) was lowest in the l-dopa-treated pd patients who were depressed. low csf -hiaa in untreated pd may reflect depletion of brain ht. l-dopa may induce depression by inhibiting ht turnover in ht-depleted brain. p . ventroposterolateral medial pallidotomy in the e. fazzani, m. dogali, a. ben;, d. eidelberg, j. gianutsos, t. kay, b. newman, s. loftus, d. samehon, and l. laitinen, new york, n y , and stockholm, sweden in patients with parkinson's disease (pd), as a consequence of low dopamine there exists an increase in inhibitory output from the globus pallidus. ten patients ( men and women) with pd received unilateral ( right, left) ventroposterolateral medial globus pallidotomies (vplmp). the average patient age was years (range - yr), and the average duration of disease was years (range - yr). patients fluctuated between "on" chorea and "off" parkinsonism. hoehn and yahr stage "on" was i in , in ; and "off" was i in , iv in , and v in patients. unified pd rating scale (updrs) score averages hours off medicines ( hom) were activities of daily living (adl): and motor (mtr): preoperatively (preop). capit score averages preop hom were pronation-supination (ps): seconds (s), finger tap (ft): s, board (b): s for the most affected contralateral side, and gait: s ( patients could not walk). re-examination was done to days after pallidotomy. updrs scores hom decreased an average of %. three patients had major bilateral improvement in bradykinesia. rest tremor, prominent in patients, also was diminished. capit scores hom decreased to ps: s, ft: s, b: s; the average gait of the patients who could walk preop improved to s. there were no side effects. vplmp leads to an immediate overall significant improvement in patients with pd. s. kish, y . there is a growing interest in the genetic aspects of parkinson's disease and other basal ganglia disorders. we have studied families whose ancestors immigrated to north america from contiguous regions of northern germany and southern denmark. the pedigrees contain , , and individuals spanning , , and generations with , , and affected members, respectively. autosomal-dominant inheritance is clearly present in families and probable in the third. typical levodopa-responsive parkinsonism with bradykinesia, rigidity, resting tremor, and impaired postural reflexes uniformly develops in affected individuals from all families. n o downgaze impairment, pyramidal signs, sensory disturbances, cerebellar dysfunction, or orthostatic blood pressure changes have been observed. dementia, however, has developed in a few elderly individuals, especially in family. laboratory studies are normal. mri shows moderately enlarged ventricles and cortical atrophy. -fd positron emission tomography demonstrated reduced striatal uptake in examined patient and normal uptake in l individual at risk. autopsy of only subject has been performed (in ). brain weight was , grams and there were no obvious gross abnormali- program and abstracts, american neurological association tuesday, october ties, but microscopic examination was limited. further research on these families is planned. electrophysiological study s. maurri, m . cincotta, a. ragazzoni, g. descisciolo, and f. barontini, florence, ltah many neurophysiological examinations were conducted of a -year-old woman with familial mirror movements. n o other neurological abnormalities were detected. examination included voluntary electromyographic (emg) activity from various muscles, f-wave as well as short-and long-latency reflex responses of the thenar muscles from electrical stimulation of the median nerve, mapping of motor-evoked potentials (meps) to transcranial magnetic stimulation, movement-related cortical potentials (mrcps), and somatosensory-evoked potentials (seps). emg documented mirror activity in the upper limbs, most marked in the muscles of both hands. onset latency of emg activity in response to an auditory stimulus was identical in active and mirror muscle. long-latency responses from median nerve stimulation were recorded on contralateral as well as ipsilateral thenar muscles. short-latency reflexes and f wave were strictly ipsilateral. unilateral scalp magnetic stimulation evoked bilateral responses at similar latencies in the thenar muscles; midline scalp stimulation activated no responses. scalp distribution of median nerve seps and of mrcps associated with self-paced thumb abduction were normal. our findings suggest that congenital inherited mirror movements in otherwise normal subjects can be generated by corticospinal fibers pro jecting to ipsilateral motoneurons of the spinal cord. we have previously identified dysphagia and constipation (both slow transit and defecatory dysfunction types) as common gastrointestinal (gi) problems in parkinson's disease (pd). since apomorphine has been shown to be capable of terminating off-periods when injected subcutaneously, we have evaluated the effects of apomorphine injection on objective parameters of dysphagia and bowel dysfunction in pd patients. nine subjects underwent the following battery of studies to characterize their pd features, swallowing, and bowel function: gi assessment survey, unified pd rating scale, videoesophagram, colon transit study, defecography, and anorectal manometry. specific abnormalities on the studies were noted and the most abnormal study was repeated after subcutaneous administration of mg apomorphine. all individuals were pretreated with domperidone mg four times daily for days prior to apomorphine administration. improvement in both esophageal motility and deglutition was noted in the individual in whom videoesophagram was repeated. for the other patients, defecography or anorectal manometry was performed. significant improvement in specific parameters was demonstrated after apomorphine administration, but individuals experienced syncope during radiographic procedures. we conclude that subcutaneous apomorphine administration holds promise as a potential therapeutic approach to dysphagia and, especially, bowel dysfunction in pd, but that further investigation and refinement are necessary. asymmetrical effect of unilateral thalamotomy or subthalamotomy on tremor in parkinson's disease nico diedericb, christopber g. goetz, glenn t . stebbins, harold l. klawans, k. nittner, a. kozrlosakis, p. sanker, and v . strum, cologne, germany, and chicago, il in the past, stereotactic operation was a regular treatment for unilateral tremor in parkinson's disease (pd). however, follow-up studies were usually short term and always unblinded. we examined pd patients in long-term follow-up (mean . yr after operation) who underwent unilateral thalamotomy for parkinsonian tremor. we used videotapes and the unified parkinson's disease rating scale to blindly compare tremor ipsilateral and contralateral to the side of operation. since the patients were specifically selected for stereotactic operation because of asymmetric tremor, we reasoned that a sign of long-term efficacy would be current postoperative reversal of tremor side predominance. upper extremity tremor was significantly better contralateral to the side of operation compared to the ipsilateral side ( z = . ; p < . ). for the lower extremities the difference was not statistically significant. in chronic follow-up, stereotactic operation improved the absolute magnitude of arm tremor or ameliorated its rate of progression. since asymmetric bradykinesia and dyskinesia were not prerequisites for the choice of surgical side, we cannot make any conclusion about longterm impact of operation on these features. subtle extrapyramidal signs resembles that of patients with parkinson's disease m . richards, k. marder, l. cote, y . stern, and r. mayeux, new york, n y to investigate the relationship between extrapyramidal signs (eps) and cognition, eps severity and neuropsychological function were assessed in normal elderly individuals and nondemented patients with idiopathic parkinson's disease (pd) from a community-dwelling cohort in new york city. multivariate analysis of variance (manova) indicated poorer neuropsychological performance ( p = . ) in pd patients on verbal memory, orientation, verbal fluency, visuomotor construction, and psychomotor speed, but not naming, abstract reasoning, or matching. controlling for eps severity abolished these differences. one hundred fourteen ( %) of the normal individuals had subtle eps (mostly postural abnormality, bradykinesia, or rigidity) but no identifiable neurological disorder. manova indicated poorer neuropsychological test performance ( j = , ) in these individuals than in normals without eps on verbal memory, orientation, abstract reasoning, naming, verbal fluency, matching, and psychomotor speed but not visuomotor construction. we conclude that: ( ) cognitive impairment in pd is specifically associated with eps, and ( ) a similar association occurs in individuals with subtle eps but no neurological disorder. whether this represents a preclinical stage of pd or ad is yet to be determined. disease in olmsted county, minnesota emre kokmen, fatma sibel ozekmekci, c. mary beard, and peter c. o'brien, rochester, m n , and istanbd, turkey there have been many studies of prevalence of huntington's disease (hd) in diverse populations around the world. to study the incidence, we took advantage of the availability of detailed health care records for the population of olmsted county, mn, from mayo clinic, its affiliated hospitals, olmsted medical group, county hospital, state hospital, records of solo practitioners, nursing homes, death certificates, and autopsy records. we reviewed all records with a diagnosis of hd, huntington's chorea, chorea major, and chorea otherwise unidentified, and sought evidence for progressive chorea, progressive cognitive and/or behavioral dysfunction, and family history compatible with autosomal-dominant inheritance with onset of symptoms in the period between january , , and december , , while the patient lived in the geographic boundaries of olmsted county. we found males and females who met these criteria. average annual incidence rate (age/sex adjusted to us white population) for hd for this -year period was . cases/ year/ , population. we also estimated prevalence by taking account of in-migration, out-migration, and deaths. the agelsex adjusted ( ) prevalence for - - was . , and for - - it was . / , . the small number of cases caused the instability of the prevalence rates, but our rates are similar to rates reported in other populations. alton e . btyant, , l. breeden hollis, john a. hamjian, john d. wooten, ill, and francis . walker, nc we described patients with unusual episodic movement disorders and normal diagnostic work-ups: a -year-old woman who had recurrent episodes of tonic jaw deviation and forced right-eye closure; a -year-old woman who developed unexplained pain and subsequent spells of tonic inversion of the left leg; a -year-old man who presented with a bizarre episodic right-arm tremor; and a -year-old woman who experienced intermittent abdominal undulations. examination of the affected body part provoked or enhanced symptoms in all patients. using suggestion and placebo activation in the form of a medicated patch, intravenous saline, or cervical massage, we first induced and then aborted typical episodes of their abnormal movements. postinduction discussions of the procedure led to a marked reduction in the frequency of attacks in patients. activation procedures are useful in diagnosing psychogenic disorders because they demonstrate that situational, not medical, factors govern the expression of the abnormal behavior. we speculate that patients who are refractory to simple suggestion may respond to induction because it offers the potential of validating their symptoms. as in the case of psychogenic respiratory distress or pseudoseizures, positive induction can assist in counseling and symptom control. had alzheimer's disease with parkinsonism (adp), had essential tremor (et), had cerebellar tremor in multiple sclerosis (ms), and had tardive dyskinesia (td). clozapine was used either to treat psychosis ( pd, adp, dys, td) or tremor ( pd, et, ms). two pd patients were retrospective analysis of patients counted twice, who was treated for psychosis and then tremor and who was treated on separate occasions for psychosis with different responses. all dys patients improved, with complete resolution of their dystonia on changing antipsychotic drugs. the patients with et ( mg) and ms ( mg) improved mildly but sedation and clumsiness caused drug discontinuation in the ms patient. one adp patient ( . mg) responded well and the other became sedated and confused ( mg). the p d responses for psychosis at a dose range of . to mg daily were good ( ), very good ( ), and excellent ( ), whereas were intolerant. pd tremor responses were good ( , very good ( ), excellent ( ), and poor ( ) at doses of . to mg daily. one patient died of unrelated causes shortly after initiation of the drug. adverse effects included sedation, weight gain, hypersalivation, fainting, clumsiness, transient granulocytopenia, and "spasms" necessitating discontinuation in patients ( pd, l td, and l ms). tourette's syndrome and attention-deficit disorder patients s. m. silverstein, p. g. coma, d. palumbo, l. west, and r. kurlan, rochester, n y impaired attention is a common comorbid behavioral feature of tourette's syndrome (ts) and a key clinical feature of attention-deficit hyperactivity disorder (adhd). however, the pattern of attentional impairments reported in adhd has not been observed in ts. we therefore compared ts patients ( male, female; mean age ? yr), adhd patients ( male, female; * yr), and normal controls ( male, female; ? yr) on specific neuropsychological (np) and computer-administered tasks of attentional ability. adhd, but not ts, subjects performed significantly worse than controls on the n p tasks (digit symbol, perceptual speed) and had a trend toward poorer performance on a computerized measure of attention. however, both the adhd and ts groups had significantly greater test performance variability on some, but not all, tasks and had more subjects with deviant scores. among ts patients, higher scores on an obsessive-compulsive disorder (ocd) inventory and a greater number of adhd symptoms correlated significantly with poorer performance on the attentional tasks. moreover, ts patients with observed tics during testing had greater attentional impairment than those without tics. these results suggest that: ( ) many adult ts patients do not have impaired attention; ( ) attentional impairment in ts differs from that observed in adhd; and ( ) attentional impairment in ts is associated with the full neurobehavioral spectrum of ts (i.e., tics, ocd, and adhd). frank r. sharp, cathleen miller, thomas rando, and steven greenberg, san francisco and palo alto, c a six patients are described with choreoathetoid movements and marked proprioceptive sensory loss. one patient had a traumatic injury to the right parietal cortex that produced severe proprioceptive sensory loss and choreoathetosis in the left arm. another patient had a left thalamic infarction that resulted in profound proprioceptive sensory loss and chorea on the right side of the body. two patients had cervical spinal cord disease, proprioceptive sensory loss, and diffuse choreoathetosis. another patient had dorsal root ganglionitis associ-a hypothesis program and abstracts, american neurological association ated with small-cell lung carcinoma that produced diffuse loss of all sensory modalities and chorea. the last patient had an ulnar sensory neuropathy and choreic movements of the fifth finger. lesions anywhere along the pathway that transmits limb proprioception may cause pseudochoreoathetosis. furthermore, choreoathetosis without sensory loss caused by focal lesions of striatum may occur because of disruption of cortical proprioceptive inputs to striatum-perhaps explaining why most focal lesions of striatum do not produce chorea. sporadic inclusion-body myositis (s-ibm) and autosomalrecessive hereditary inclusion-bod y myositis (h-ibm) are of unknown cause and pathogenesis. in both there are muscle fibers with rimmed vacuoles containing to -nm cytoplasmic tubulofilaments (ctfs) and denervation atrophy; in s-ibm, but not h-ibm, there is a varying degree of inflammation. vacuolated fibers contain ubiquitinated inclusions (askanas ) and congo-red positivity indicating amyloid (mendell ). because immunoreactive p-amyloid precursor protein (app) and p-amyloid protein (p-ap) are constituents of ubiquitinated senile plaques in alzheimer's disease (ad) brain, we studied immunolocalization of app and p-ap fibers in ibm muscle using antibodies against: ( ) non-p-ap fragments of app, viz. (a) c-terminus (residue - courtesy d. selkoe) and (b) n-terminus (residue - courtesy b. frangione and d. levartovsky); ( ) p-ap (sequence - , courtesy g. glenner, and sequence - courtesy d. selkoe); ( ) ub (chemicon). in of ibm patients, including one h-ibm, % of the vacuolated muscle fibers contained large or several small app and p-ap immunoreactive (ir) inclusions, which by double-labeling fluorescence were closely colocalized with each other and with ub-ir. none of control muscle biopsy specimens (including polymyositis) contained app-ir, p-ap-ir, or ub-ir inclusions characteristic of ibm. control experiments utilizing omitted, replaced, or absorbed primary antisera were negative. p-ap, a product of proteolytic cleavage of app, is receiving attention regarding the pathogenesis of ad. our study provides ( ) the first demonstration of app and p-ap accumulations in abnormal human muscle, and ( ) raises the possibility that in ibm muscle and ad brain rhey may form from similar cellular events. jeffrrey d. rothstein, lin jin, and ralph kuncl, baltimore, m d the pathogenesis of motor neuron death in amyotrophic lateral sclerosis (als) is unknown. accumulating evidence suggests that the disease is characterized neurochemically by a derangement in the control of neurotransmitter glutamate metabolism: csf levels of glutamate and aspartate are ele-of motor neuron degeneration vated and their high-affinity transporter is defective in brain and spinal cord. inefficient glutamate transport, and subsequent chronic increase in extracellular glutamate, could be responsible for selective motor neuron death. to test the hypothesis that chronic defects in glutamate uptake can produce motor neuron toxicity, we developed a tissue culture model employing organotypic rat spinal cord maintained under conditions of chronic glutamate uptake inhibition. slices ( pm) of lumbar spinal cord from -to -day-old rat pups were cultured on millicell membranes. chronic uptake inhibition was produced by culturing tissue in the presence of threohydroxyaspartate (tha) or pyrrolidine-dicarboxylic acid, both known to be specific inhibitors of glutamate transport. tha produced chronic elevation of glutamate in the medium and produced motor neuron toxicity after to days in culture using pm tha, and after days using pm tha, as determined by assay of tissue choline acetyltransferase (chat) activity and by histological analysis of -micron plastic sections. motor neuron toxicity was completely blocked by the non-n-methybaspartate (nmda) antagonists cnqx or nbqx, but not by the nmda antagonist mk- . this model demonstrates that the chronic loss of glutamate transport in als can produce motor neuron degeneration and that motor neurons appear to be susceptible to non-nmda-mediated glutamate toxicity. guillain last year, we described a distinct acute paralytic syndrome in children and young adults from northern china and differentiated it from guillain-barre syndrome (gbs) by epidemiological, clinical, and nerve conduction (nc) features. to distinguish chinese paralytic syndrome (cps) from gbs more clearly, we measured nc in cps patients (mean yr, range . - yr) and in gbs patients from johns hopkins (mean yr, range - yr). sensory nc was normal in all (n = ) but nerve of cps patients, whereas sensory nc was frequently abnormal in gbs patients: median nerve, %; ulnar nerve, %; and sural nerve, %. motor n c also differed between the groups. in all nerves, distal latency (dl) was significantly longer in gbs than in cps. for example, in the median nerve, mean dl was . ms (se ) in gbs and . ms ( . ) in cps ( p < . ). motor conduction velocity was significantly reduced in gbs median and ulnar nerves compared with cps nerves. f-wave latency was significantly longer in gbs median nerves than in cps nerves. these data support the distinction both clinically and electrodiagnostically between cps and north american gbs. the use of n c may be especially important in field epidemiological studies in separating the disorders. clinical manifestations and gene analysis of the first japanese kindred yoshihide sunada, teruo shimizu, lchiro kanazawa, and toru mannen, tokyo, japan familial amyloidotic polyneuropathy type iv (fap iv) has been clustered in the finnish population and only a few cases have been reported from the netherlands. denmark, and united states. we describe the first japanese family with fap iv. the family originates from nagano prefecture, a mountainous district in the middle part of japan, and has no relationship to the finnish population. this family has members in generations, and individuals are affected with slowly progressive cranial neuropathy and corneal lattice dystrophy. the genetic trait is autosomal-dominant. polarizing microscopy and immunohistochemistry show abundant amyloid deposits reactive to an anti-gelsolin monoclonal antibody. direct sequence analysis of a dna fragment spanning codon of the plasmagelsolin cdna from the propositus, and restriction analysis using a modified pcr from other family members demonstrate a single base substitution, g to a at the first base of codon , which is identical to the mutation of finnish fap iv. this suggests that the mutation causes the fap iv phenotype regardless of ethnic background. gene expression focal puncture injury has been used as a model to study degenerative and regenerative responses of skeletal muscle. previous studies have demonstrated the ultrastructural and metabolic effects of muscle injury. however, the early genomic response to focal injury is presently unknown. we asked whether the immediate early genes (iegs) or early response genes-~$ , c-jun, nur , and junb-are responsive to muscle injury. these iegs encode transcription factors and are expressed rapidly after cell-surface stimulation. we have previously shown that surgical denervation and neural stimulation of muscle induced differential patterns of ieg expression. in this study, we produced injury of mouse gastrocnemius muscle by injection of c . of normal saline. we used the contralateral (uninjected) muscle as a control and examined the milna levels of each of these iegs. we found that ~$ and junb levels were increased at and hours and returned to basal levels by hours. in contrast, mrna levels of nur and cjun remained unchanged. this pattern of ieg response is distinct from that seen after muscle stimulation or denervation. the selectivity of this pattern suggests that ieg expression may play a role in the response of muscle to injury. amyotrophic lateral sclerosis (als) is a degenerative disease that leads to the restricted loss of motor neurons (mn). the reason for the selective death of mn remains unknown. we hypothesize that mn-enriched or mn-specific genes are important for normal m n function and that their disturbance may play a role in the pathogenesis of als. we have produced clonal hybrid cells derived from embryonic and neonatal spinal cord m n for the study of m n gene properties. some of these hybrid mn clones express traits typical of mn, such as high levels of choline acetyltransferase enzyme activity and message, glycine receptor message, and neurofilament and neural cell adhesion molecule proteins. we are using molecular techniques to identify novel mn-enriched or mn-specific genes in these cells. with this strategy, we have identified several cdna clones preferentially expressed in mn hybrid cells but not in the parental neuroblastoma cells by differential hybridization of an embryonic mn hybrid cdna phage library. we are extending these observations by performing subtraction hybridization experiments. these results suggest that mn-enriched or mn-specific genes can be identified, and may lead to a greater understanding of the etiology of als. there is a syndrome of slowly progressive, mid-adult-onset fasciculating progressive muscular atrophy (pma) affecting upper more than lower limbs, without bulbar or corticospinal signs, more often in males, associated with igm monoclonal gammopathy, and no nerve conduction block. two such men, ages (a) years and (b) years, duration and and one-half years, csf protein and , had failed to achieve sustained improvement with: prednisone, cyclophosphamide, total-body irradiation, and multiple lymphoplasmaphereses in a; and interferon alpha a in b. intravenous immunoglobulin (ivig), . gm/kg/day, has provided dramatic benefit, sustained and increasing for > and > months to date. (there is a continuing base of depotestosterone, mg weekly, which initially alone provided very minimal improvement.) strength increase was evident at and days after the first course of daily ivig infusions. it further increased for to and one-half weeks after treatment, and then began to diminish. repeat -day treatment weeks after the first course resulted in summated improvement, now sustained and enhanced by an average of treatment per week. quantitated strength testing by a blinded observer has shown a -fold to >loo-fold gradually increasing muscle function in all limbs. patient a regained the ability to feed himself, get out of a chair, walk unaided, and go up steps; quantitated hip flexors increased -and -fold. patient b regained the ability to feed himself, take care of personal toilet needs, walk securely, and drive miles; quantitated hip flexors increased -fold, and biceps flexions increased from with no weight to > reps while holding -pound weights. bukhara jews: a new cluster with typical oculopharyngeal muscular dystrophy (opmd) is a rare, late-onset myopathy with autosomal-dominant inheritance. its ultrastructural hallmark is the finding in muscle fibers of intranuclear tubular filaments of . -nm outer diameter. most opmd cases were described among french canadians; in france, the homeland of their ancestors, the prevalence is / , (brunet et al, ) . in israel's central area live approximately , jews who have immigrated from the bukhara and samarkand regions in uzbekistan. they represent a homogeneous ethnic group with its own language and community life. among them we have identified opmd in families ( affected individuals). the inheritance, clinical, electrophysiological, and histological features of these pa-tients are similar to those described in other pdts of the world, with typical intranuclear inclusions seen on electron microscopy. the minimal estimated prevalence of opmd in this population is approximately : . this cluster of opmd among bukhara jews is the second largest in the world. because many bukharian families are large, they may be suitable for linkage genetic studies. human muscle during ontogenesis e . scarpini, g. conti, p. l. baron, and g. myoblast transfer has been proposed recently as a possible therapy for duchenne muscular dystrophy patients. because immune rejection can represent a major problem in myoblast implantation, immunological characteristics of human muscle should be investigated. previous studies showed that human muscle cells cultured in vitro can constitutively express human lymphocyte antigen (hla) class i, but not hla class . furthermore, human y-interferon induces the surface expression of hla class i on mononuclear myoblasts, but not on multinucleated myotubes. however, whether the cells produce and present the antigen by themselves or take this material from the environment, where it could be released by infiltrative cells, is not yet clear. in this study, we analyzed hla molecules at the protein level by immunocytochemistry with monoclonal antibodies against different hla-dr epitopes and hla-abc molecules on frozen serial sections of human muscle during development and at the adult stage. human muscle infiltration by macrophages and monocytesmacrophages also were studied with m and leum specific monoclonal antibodies at the same stages of development. our results show that during muscle development and maturation, hla-dr and hla-abc antibodies do not label muscle fibers but some m -and leum -positive cells within the muscle. these data can be useful to understand the role of infiltrating monocytes-macrophages in the muscle immune response. mounting evidence suggests that excitotoxicity, mediated via the glutamate receptor, is involved in the pathogenesis of amyotrophic lateral sclerosis (als), as well as in other neurological diseases. we therefore initiated an open label, phasen i trial of highdose dextromethorphan (dm), a noncompetitive, selective n-methybaspartate antagonist, in als. patients began with mg/kg/day, divided into doses, and incrementally escalated their medication to mg/kg/day or their maximum tolerable dose. thirteen patients, all extensive metabolizers of dm, were enrolled. total daily doses ranged from . to mg/kg. major side effects were lightheadedness ( ), slurred speech ( ), and fatigue ( ). no biochemical, hematological, or neuropsychiatric abnormalities occurred after up to months of maximal therapy, except for depression in patient. plasma kinetics of dextrorphan (dt) (the major metabolite of dm) were studied after an acute oral dose of . mg/kg dm. median elimination halflife was . hours. plasma dt concentration peaked at a median of hours, with a median cmax of . pm. median amyotrophic lateral sclerosis cerebrospinal fluid/plasma dt ratio was . . this study demonstrates the feasibility of long-term, high-dose dm therapy. we are now conducting a phase i study of highdose dm in als, designed to assess its efficacy. polyneuropathy: a chronic inflammatory demyelinating polyradiculoneuropathy variant? d. cros, k. h. chiappa, s. patel, and s. gominak, boston, ma, we describe patients ( men, woman) with a pure, adultonset sensory neuropathy. the course was chronic in all cases. three patients had a relapsing-remitting course over to years with several attacks every year; the onset was gradual and followed by a plateau in the fourth patient. all patients had positive and negative sensory symptoms, and had positive motor symptoms (fasciculations). in all patients, muscle power was normal at the time of peak deficit. all were areflexic and had large fiber sensory deficits, and patients had sensory ataxia. three patients had elevated csf protein, whereas the csf was normal in patient. mri demonstrated marked thickening of the lumbosacral spinal roots in patient. motor conduction studies were normal in all patients, and mild f-response abnormalities were noted in . neurophysiological investigations of the sensory pathways were abnormal in all. three patients had several studies over a -year period. sensory nerve action potentials were unobtainable in patients, and normal in the others. median and tibial somatosensory-evoked potentials showed conduction slowing consistent with demyelinating lesions affecting the peripheral sensory pathways, either globally or focally in the proximal segments. two patients appeared to respond to plasma exchange or intravenous immunoglobulin therapy, or both. glial fibrillary acidic protein cells in experimental motoneuron disease raul n . mandler, pam c. allgood, and james a. wallace, albuquerque, nm neuronal degeneration in human and animal motoneuron disease has been emphasized, but glial phenotype alterations have not been studied as extensively. we carried out a developmental and topographic study of astrocyte expression in the wobbler mouse model of motoneuron disease. wobbler mice and normal littermates were studied at , , , and weeks of postnatal development. anesthetized animals were perfused intracardially with paraformaldehyde. spinal cords were dissected and landmarks were identified carefully for systematic study. sections were stained with monoclonal antibodies against glial fibrillary acidic protein (gfap) neurofilament and neuron-specific enolase. cell quantitation was done with video-enhancing microscopy. in symptomatic animals, marked increases in gfap staining were found in rostral and caudal spinal cord areas. quantitation studies revealed a to -fold increase in gfap+ cells in the wobbler. we conclude that gfap+ cells are markedly increased in the wobbler mouse at cervical, thoracic, and lumbar areas. this cell may also be relevant in motoneuron disease pathogenesis. ( indirect evidence suggests that polio virus may persist in the human cns years after initial infection and may be a cause for the post-polio syndrome. to evaluate whether the polio virus genome can be detected in the cns of patients with previous polio infection, we identified patients who had died with autopsy findings and clinical history consistent with poliomyelitis. rna was extracted from paraffin-embedded sections of brain or spinal cord and subjected to reverse transcription followed by dna amplification by polymerase chain reaction (rt-pcr) using primers specific for heat shock protein (hsp ) and a conserved region of the polio viruses. hsp mrna could be detected in all specimens, indicating that amplifiable rna had been isolated. in no specimens could polio virus rna be detected. this study suggests that polio virus does not persist in the human cns in quantities detectable by the sensitive pcr method. with cmt type , seen at mayo clinic rochester between and , for the frequency of selective calf weakness in cmt type , the form of cmt most similar clinically to distal sma. anterior compartment weakness exceeded calf weakness in patients ( %); anterior and posterior involvement was equal in ( %). calfweakness exceeded anterior compartment weakness in patient ( %). selective calf weakness in distal sma thus helps distinguish this disorder from cmt type , and similarly from distal sma with weakness resembling cmt, in that we are unaware of the distributions in distal sma occurring in the same family. given the possibility of genetic heterogeneity, linkage studies of distal sma probably should include patient selection criteria such that the distribution of leg muscle weakness is homogeneous. p . conjugal amyotrophic lateral sclerosis: amyotrophic lateral sclerosis (als) is a sporadic neurodegenerative disorder of unknown cause. unusual cases may provide etiologic clues. we report a married couple, both of clue to etiology? whom developed als in year. the couple grew up in southeastern pennsylvania and attended the same schools. they married after high school and have healthy children. in september , a -year-old woman noted right-hand weakness and associated fasciculations that progressed to the entire right upper extremity. by january , the lower extremities were asymmetrically weak and fasciculating. she then developed left-arm weakness, d ysarthria, dysphagia, and emotional incontinence. she had hyperreflexia and bilateral extensor plantar responses. then, in may , her husband, aged , noted difficulty whistling, which progressed to frank dysarthria. later, he developed dysphagia, emotional incontinence, and weakness, wasting, and fasciculations in the upper extremities. hyperactive gag, jaw, and limb reflexes were present. in both, electrodiagnostic testing revealed widespread evidence of lower motor neuron degeneration. numerous laboratory tests were normal. although these cases may represent a chance association, the development of als in a young husband and wife suggests a possible environmental cause. the authors welcome suggestions about these cases from the neurological community. conduction block in demyelinating neuropathies usually is assessed from differences in the sizes of surface-recorded maximum m-potentials evoked by supramaximal stimulation at successively more proximal sites along the course of motor nerves. as the maximum m-potential is comprised of many bitriphasic surface-recorded motor unit action potentials (muaps), differences in the relative latencies between muaps may lead to phase cancellations, reducing the m-potential size and rendering any quantitative assessment of the extent of conduction block relative to phase cancellation difficult. cooling a muscle (not the nerve), however, by as much as °c increases the negative peak durations of muaps by as much as to times and moves the point at which maximum phase cancellation might occur to some theoretical point well proximal to the spinal roots. in cases of guillain-barre syndrome (gbs) studied to date, cooling produced little change in percent reductions in m-potential negative peak areas between successively more proximal sites of stimulation. this finding suggests that "true" conduction block rather than interpotential phase cancellation best explains reductions in m-potential size at successively more proximal sites of stimulation in gbs. associated with trimethoprim-sul famethoxazole rare cases of primarily motor polyneuropathy have been associated with the use of sulfonamides. the incidence of polyneuropathy has diminished substantially with the abandonment of earlier methylated compounds. we describe patients who developed allergic phenomena, including a skin rash and debilitating, painful sensory and autonomic polyneuropathy within days of receiving trimethoprimsulfamethoxazole. in patient, examination revealed resting tachycardia, marked blood pressure orthostasis and near-program and abstracts, american neurological association syncope, hyporeffexia, urinary incontinence, and reduced sensation distally in the lower extremities. his cerebrospinal fluid was acellular with a protein of mg/dl. the other patient showed a resting tachycardia, sluggish pupils, reduced distal vibration perception, and hyperpathia of hands and feet. conventional nerve conduction studies demonstrated normal motor results in both patients, absent or reduced sensory amplitudes in the first patient, and normal sensory results in the second. autonomic studies identified profound abnormalities in testing of sympathetic skin potentials, sinus arrhythmia, and valsalva's ratio. in both cases, nerve biopsy was not performed for fear of exacerbating the patient's hyperpathia. subsequent hemodynamic and electrophysiological testing showed improvement in autonomic function, paralleling the patients' clinical amelioration. although uncommon, a painful, sensory and autonomic, partially reversible polyneuropathy may develop after the use of trimethoprim-sulfamethoxazole. the remote effects of botulinum a toxin injections into vocalis muscles for treatment of focal laryngeal dystonia were investigated using single-fiber electromyography (sfemg). botulinum a toxin injections have been proven effective therapy for various dystonic disorders including focal laryngeal dystonia, blepharospasm, and torticollis. previous sfemg studies have demonstrated remote effects of the toxin in noninjected muscles after treatment for both blepharospasm and torticollis. these effects include an increase in fiber density, mean jitter (mcd), and percentage of fiber pairs with increased jitter. other researchers have postulated that the distant effects of this toxin may be related in part to the dose of botulinum toxin injected. to investigate this hypothesis we have studied patients treated for focal laryngeal dystonia because the amounts of toxin required are / th to / ooth of the doses used to treat other dystonias. using electromyographic (emg) guidance, bilateral injections of . or . mouse units of botulinum a toxin were injected into each vocalis muscle of patients. each patient had significant improvement in phonotory function within hours after injections and have been followed serially (usually within weeks and again at mo) after injections, with sfemg recordings of the left extensor digitorum communis and sternocleidomastoid muscles. five patients have had more than series of injections over the months since we began this study. sfemg studies have revealed no significant change in the fiber density, mean mcd, or percent of fiber pairs with normal jitter in either muscle. in conclusion, our studies support the hypothesis that the presence of remote effects of botulinum toxin may be related, in part, to the amount of toxin used. the c bl/ / a mouse exhibits the remarkable characteristic of prolonged survival of axons separated from their cell bodies (slow wallerian degeneration). previous work has demonstrated that the axon itself is responsible for the phenotype of prolonged survival. we investigated whether the lack of rapid axonal degeneration after axotomy in this substrain is due to an inability to break down cytoskeletal components, a process that is normally accomplished by activation of intrinsic calcium proteases. segments of desheathed sciatic nerves from normal and ola mice were incubated for hours under conditions that disrupt the axolemma (freeze/ thaw or in % triton x-loo), allowing external calcium free access to axoplasm. nerves were analyzed by western blot for neurofilament (nf) proteins and by electron microscopy. in high-calcium media ( mm caci,), nf immunoreactivity was lost and axoplasm was reduced to watery debris in both substrains, whereas in egta-buffered media, axoplasm was preserved. these results demonstrate that calcium-activated proteases are present and can be activated in ola nerves. the defect in these mice that allows for prolonged survival of transected axons is likely in the mechanism for calcium entry into the distal stump. the mechanism by which the analogue of adrenocorticotrophic hormone, acta - , prevents cisplatinum (cp) neurotoxicity is unknown. murine n e. neuroblastoma cells and neural crest-derived, squirrel fish erythrophore cells tuesday, have similar vesicular transport mechanisms to human neural cells. they were used to study the effects of cp and acth , on cellular transport. differentiated n e. cells were treated hour prior to observation with serum-free media (sfm, control); sfm/cp pg/ml; or sfm/cp pg/ml and ng/ml acth -,. organelle transport was studied ( neurites and - organelles per condition) using computer-enhanced video microscopy. mean fast anterograde ( . k . pmsec-' vs . * . pmsec-') and retrograde ( . . pmsec-' vs . +- . pmsec-') transport were decreased in cp-treated compared to control cells ( p < . ). in cp/acth ,-treated cells, mean anterograde ( . +_ . pmsec-') and retrograde ( . . pmsec-') velocities were greater than in cp cells ( p < . ). velocities in control and cp/acth , cells were not statistically different. erythrophore pigment granule transport was observed in a blinded study, using similar techniques. mean aggregation velocity was greater in control ( . k . msec-') and cp/acth , ( . f . msec-')-treated cells compared to cp ( . * . msec-') cells ( p < . ). incubation with cp for or hours affected velocities equally, but acute exposure was more easily reversed by control or acth,, containing media. there is striking inhibition by cp in cross-species models of organelle transport. this can be prevented by acth ,. erythrophores allow future study of individual transport components. neurotrophin to investigate signal transduction pathways involved in neurite growth, the cytoplasmic regions of p sngfr, the common neurotrophin receptor monomer, were searched for a motif analogous to the predicted secondary structure of the tetradecapeptide mastoparan. potential sequences were modeled using a semi-empirical molecular mechanical force field approach. the sequence rat ~ ~~~ - represents a highly conserved amphiphilic domain predicted to be involved in neurotrophin signal transduction via g-protein mechanisms. to test this prediction, peptides containing sequences homologous to p ngfr - were examined for effects on trophic factor-induced survival/differentiation responses of rat pc pheochromocytoma cells, chick embryo drg neurons, and chick embryo ciliary neurons. a peptide identical to ~ ~~~ - accelerated the neurite growth response to nerve growth factor (ngf) of pc cells and drg neurons in a time frame that paralleled uptake into cells, but mastoparan did not influence ngf-mediated neurite growth. millimolar mg+ + and benzalkonium chloride, known to block the actions of mastoparan, blocked the effect of the peptide on ngf-mediated neurite growth by pc cells. peptides mutated to alter cationic amino acid relationships or amphiphilicity were less effective than the peptide in accelerating ngf-mediated neurite growth. these observations complement and extend evidence suggesting a pivotal role of ~ ~~~ in ngf-mediated signal transduction. these studies complement and extend evidence suggesting a pivotal role of p sngfr in neurotrophin signal transduction and evidence that activation of intracellular signalling processes involving specific g-protein mechanisms are involved in neurotrophin-mediated neurite growth. crushing the hypoglossal nerve causes hypoglossal motor neurons to decrease expression of choline acetyltransferase (chat) and begin expressing p ngfr, the low-affinity ngf receptor. these changes are evident within days after the injury and continue for several weeks. inhibition of axonal transport by vincristine applied to uninjured nerves causes loss of chat expression without induction of ~ ~~" . we sought to determine if topical vincristine would alter p sngf' expression after nerve injury. hypoglossal nerves were surgically exposed unilaterally in anesthetized rats and crushed. one week later, rats were reanesthetized and the same nerves were re-exposed. vincristine or saline was applied at the crush sites by soaking a strip of cotronoid wrapped around the nerves. one week later, rats were anesthetized and perfused with aldehydes. frozen sections from the brainstems were stained by indirect immunoperoxidase to demonstrate chat and ~ ~~~' . saline-treated controls showed decreased chat and abundant ~ ~~" in hypoglossal motor neurons ipsilateral to the crush injury. vincristine-treated animals showed no chat and no p tngf'. we interpret these results as indicating that a signal originating from the injury site maintains ~ ~~" expression after nerve injury. catecholamines have been reported to be toxic to embryonic-derived rat neurons and glia via the formation of reactive oxygen species (rosenberg, ) . we tried to determine whether oligodendrocytes (ol) from adult -month-old rat brain are similarly susceptible. toxicity to ol was examined using light microscopy and galactocerebroside immunohistochemistry where the relative number of surviving ol and their extent of process formation were graded. five days of exposure to norepinephrine (ne) and epinephrine (epi) at and fm produced significant toxicity ( p < . , analysis of variance [anova)) to adult rat ol; this toxicity was evident by hours of exposure. treatment with catalase ( p,g/ml), a free-radical scavenger enzyme, completely prevented the toxicity of catecholamines. to ascertain whether astrocytes, which have free-radical scavenging capacity, could prevent the catecholamine-induced injury to ol, rat ol were seeded on neonatal rat astrocytes. under such conditions, the toxicity of n e and epi was reduced significantly ( p < . , anova). these findings suggest that impairment of this protective function of astrocytes may render ol and its myelin membrane susceptible to free-radical-mediated damage. lyme neuroborreliosis is an increasingly prevalent disorder, but the diagnosis generally has been indirect. thus, the pres-ence of other manifestations of the infection, consistent findings on neurological exam or lumbar puncture, or presence of csf antibody have been used rather than direct isolation or identification of the organism from the csf. we have previously developed polymerase chain reaction with hybridization (pcr/h) to identify borrelia burgdorferi in the blood and organs of infected mice, and found that the assay was equivalent or, in some cases, preferable to culture (ann neurol ; : ) . the assay used for primers oligos derived from a sequence of genomic b. burgdovfri d n a expressed on a plasmid by rosa and schwan. a two-stage nested pcr was performed on csf samples in which the d n a was isolated in a variety of ways. pcr products were subsequently hybridized with a digoxigenin-labeled internal probe by slotblot hybridization. the sensitivity of the assay was excellent, being <o. fg of purified b. burgdoorfevi dnaiml of csf, and to spirochetes per ml of csf when "spiked" csf was tested. the assay then was applied to a battery of csfs stored over the past years in patients with definite, probable, and possible lyme neuroborreliosis. the lack of pcr/h positivity in some patients with definite and probable disease signifies that the spirochete does not reside in the lumbar csf in all patients with lyme neuroborreliosis, and may be concentrated in the meninges or parenchyma. progenitor cell line on transplantation into the adult murine brain t . kitagwcbi, d.-h. chui, and t . tabira, tokyo, japan several multipotent neural cell lines were generated via retrovirus-mediated v-myc transfer into primary culture cells of the septum of embryonic murine brains (the retroviral vector, kindly provided by d r c. l. cepko, boston, ma). two of those cell lines, when transplanted back into to -week-old mice by stereotaxic operation, integrated into the septum in a nontumorigenic manner. the implanted cells, which had been labeled with pkh dye, could be identified in animals up to at least weeks after transplantation. immunocytochemical analysis demonstrated that the transplanted cells were seen assuming a round morphology and no processes. they did not stain with any glial or neuronal markers except for a b and hnk-i, in the same way as in vitro. interestingly, after weeks of implantation, the cells were located discretely in the transplanted site and displayed a small and round morphology with fine processes. most of the transplanted cells showed immunoreactivity with monoclonal antibodies directed against neuronal markers such as neurofilament and map . these data indicate that the immortalized cell lines might be useful in characterizing factors that regulate cell type differentiation in the mammalian nervous system. (supported by a grant from the science and technology agency of japan.) related to serum response factor in human brain and muscle dana leifer, dimitri krainc, racbael neve, roger bveitbavt, and stuart a. lipton, boston, m a we have identified cdna clones for a novel transcription factor that is expressed at high levels in human fetal brain and skeletal muscle, but not in a variety of other tissues, as demonstrated by northern blotting. sequence analysis indicates that the clones appear to have alternatively spliced forms and have extensive homology with human serum re-sponse factor (srf) and a family of related transcription factors that has representatives throughout eukaryotic evolution in species from yeast to man. srf is thought to have a role in regulation of immediate early genes and of muscle-specific genes. in gel mobility-shift assays, the srf-like proteins coded for by our clones bind specifically to the myocytespecific enhancer binding factor- (mef- ) regulatory element, a dna sequence that has so far been found to be functionally important in the promoter and enhancer regions of a variety of muscle-specific genes. moreover, our srf-like proteins specifically activate transcription of reporter genes containing the mef- element. given the abundant expression of our clones in human brain as well as in muscle, our results suggest that these proteins may have a significant role in development not only of muscle but also of brain. a host of quantitative eeg studies, most of which employed variations of the fast-fourier transform, have been made on isolated clinical entities such as the epilepsies, metabolic syndromes, and sleep with varying degrees of satisfactory correlations. using a time domain wave-by-wave computer program that simulated the logical approach of the clinical electroencephalographer, we have studied more than unselected routine neurological clinic and ward patients. the presenting problems ranged from headache to major cerebral involvement. employing the dichotomy of normal and abnormal, the computer-read eegs agreed with the routine eeg, the clinical manifestations (including imaging techniques), and neuropathological findings in % of all cases. this high level of correlation, which approached that achieved by welltrained eegers reading the same records, was obtained by solely utilizing the background eeg activity. such results became possible only when the computer program was so organized that the "raw" computed and original oscillograph records were made to match closely. the diagnosis, with topographically diagramed localization, was printed automatically as the final step of the program. our data strongly suggest that routine computer reading of the human eeg is feasible, reliable, and potentially useful. protein accumulation in damaged neurites and microglial cells abnormal accumulation or abnormal processing of amyloid precursor protein (app), or both, may be critical to the development of p-amyloid protein (bap) deposits. however, alzheimer's disease (ad) has another pathological hallmark, which is the appearance of neurofibrillary tangles. the question is, therefore, to clarify the possible relationship between altered app metabolism and neurofibrillary degeneration. one classic method for inducing neurofibrillary tangles is the administration of aluminum salts. although these tangles are morphologically different than those seen in ad brain, this phenomenon has given rise to the hypothesis that aluminum might be an environmental factor in the causation of ad. this theory is controversial, but the animal aluminum model remains one of the few ways that long-lasting neuroskeletal changes can be induced. to explore the effects of aluminum salts on app metabolism, we examined app immunodistribution in rat brain injected intraventricularly or intrastriatally with aici,. we found a long-lasting accumulation of app in affected neurites, as well as in activated microglia/macrophages. abnormal neurites also showed argentophilic changes, neurofilament accumulation, and alz- immunoreactivity. the results support the hypothesis that interruption of axoplasmic flow by aluminum salts, as by other means, can lead to both app accumulation and neuroskeletal alterations. p . nondemyelinating conduction slowing of myelinated fibers due to inactivation of n a channel takanori yokota, yukinobu saito, and tadashi miyatake, tokyo, japan in patients with acute or chronic inflammatory demyelinating polyradiculoneuropathy, the marked improvement of motor-nerve conduction velocity without change of amplitude or configuration of the compound muscle action potentials (cmaps) was observed in days. to investigate the mechanism of the rapid improvement of nerve conduction, the influence of na channel blockade on nerve conduction was studied. in healthy volunteers, the recovery of the cmap and sensory nerve action potential (snap) of median nerve stimulation were recorded after the intravenous infusion of mg of lidocaine. after the loading, cmap and snap were reduced rapidly in amplitude and were slowed in latency. after the recovery of amplitude of cmap and snap, the nerve conduction velocities were improved gradually in hours with the amplitudes and configurations of cmap and snap unchanged. this indicated that myelinated nerve conduction velocity could be slowed by the inactivation of na channels without demyelination or conduction block. the prolongation of rising time in depolarization of axonal membrane potential should be one of the mechanisms of this conduction slowing due to inactivation of na channels. paraneoplastic cerebellar degeneration (pcd) in gynecological and breast cancers frequently is accompanied by an autoantibody response (type i or "anti-yo") reacting with and -kd cytoplasmic antigens of cerebellar purkinje cells. sakai et al, using a cerebellar library (stratagene), recently have isolated a cdna clone expressing a -kd protein recognized by igg from a patient with pcd and uterine carcinoma (sakai et al, ann neurol ; : ) . this clone is believed to share some homology with the message encoding the -kd protein recognized by type i sera. because pcd was isolated using serum from a single patient, however, it is not known whether the expressed protein of pcd is recognized uniformly by all patients with type i antibody response. e. coli strain xl -blue transformed with pwr - containing the snabiihindiii fragment of pcd , pwrsbo-pcdl?sn, was induced using isopropylthiogalactoside. bacterial lysates were electrophoresed on % sds-page gels and analyzed by western immunoblot program and abstracts, american neurological association methods using sera and csf from patients with pcd associated with gynecological and breast malignancies who exhibited type i antibody response. the -kd expression product was labeled by sera and csf samples tested from antibody-positive patients but was not labeled by sera or csfs from controls. our studies demonstrate that the -kd protein expressed by pcd contains epitopes that are consistently recognized by both systemically and intrathecally produced antibodies from patients with pcd accompanying gynecological and breast malignancies. tax is a novel antitumor drug that has demonstrated impressive clinical activity in breast, ovarian, and lung cancers. although sensory neuropath y has been described secondary to both taxol alone and taxol-cisplatin combination, detailed neurophysiological and pathological studies have not been described. nineteen patients with solid tumors were treated with combinations of taxol ( - mg/m ), cisplatin ( - mg/m ), and granulocyte colony-stimulating factor (g-csf) ( pglkglday). sequential neurological examinations, nerve conduction studies (ncs), and quantitative vibration testing (qst) were performed during and after treatment. eighteen of patients developed sensorimotor neuropathy and developed myopathy. thirteen were symptomatic with numbness or weakness and had abnormal results on neuromuscular examination. ncs showed absent or prolonged h-reflexes ( ), reduced peroneal motor amplitude ( ), reduced sural sensory amplitude ( ), and myopathic motor unit potentials in proximal muscles ( ). qst results were abnormal in of tested patients. muscle biopsy specimen in a patient with myopathy showed features of a toxic myopathy. the neuropathy was worse with higher cumulative dosages of taxol (> mg), with higher single doses of taxol(> mg/m ), or with a preexisting neuropathy. we conclude that sensorimotor neuropathy and myopathy are dose-limiting neurotoxicities of combined cisplatin and taxol use, now that neutropenia can be controlled with neuropathy and myopathy g-csf. central nervous system lymphoma casilda balmacedz and lisa deangelis, new york, ny ally periventricular and may seed the csf by direct growth through the ependyma. we reviewed the csf profile of non-acquired immunodeficiency syndrome (aids) patients (pts) with pcnsl. all pts had lumbar puncture (lp) and had multiple samples from an ommaya reservoir. definite lm involvement was identified with a positive csf cytology, lymphomatous lm infiltration on a surgical specimen, or mri with gadolinium showing lm tumor. probable lm lymphoma was diagnosed in pts with suspicious or atypical csf cytology. there were women and men with a median age of (range - yr). at diagnosis, mean white blood cell count was /mm (range - , ); mean lumbar csf protein was mgldl (range - , ); and mean ventricular csf protein was mgldl (range - ). glucose was always normal. nineteen of pts sampled had oligoclonal bands, and / had elevated pz microglobulin. at diagnosis ( %) had an abnormal csf cytology: positive, suspicious, and atypical. one pt had pathological infiltration of the lm and had lm tumor on spine mri for a total of ( %) pts with definite or probable lm lymphoma. in pts with abnormal cytology, the abnormality was found in ( %) lps and / ( %) of the ommaya and ventricular specimens. in pts the lumbar cytology was the only abnormal specimen despite multiple ventricular samples, and in only the ventricular csf was abnormal. thirty-six of ( %) pts developed recurrent tumor afrer treatment. forty-two percent ( ) of all patients with relapse had lm recurrence. lm recurrence was accompanied by brain recurrence in pts, systemic in , ocular in , both systemic and ocular in , and isolated in . at diagnosis / patients received treatment directed against the lm. of these, ( %) had meningeal recurrence whereas of the ( %) patients who did not receive this treatment had lm recurrence. lm involvement by pcnsl is frequent, may be missed on a single csf sample, and requires specific therapy at diagnosis. sixty-three solid cancer patients with a single brain metastasis were prospectively randomized for neurosurgery and radiotherapy combined (arm ) or radiotherapy alone (arm ). they were stratified for lung or nonlung cancer and for active versus stable or absent extracranial disease. world health organization performance status was . age, sex, performance status, and location of brain metastasis were divided evenly over both groups. one-month mortality was % in arm and % in arm . median survival of months after combination therapy was significantly better compared to months after irradiation alone ( p < . ). it made no difference whether they had lung or nonlung cancer. the largest difference between both treatment arms was observed in patients with stable or absent extracranial disease ( vs mo, p < . ). when systemic disease activity was present, median survival was months irrespective of treatment arm. functional independent survival was to months shorter than overall survival and was significantly better for patients with stable extracranial disease after combined therapy. multivariate analysis showed that age was also an independent prognostic factor. patients older than years had a hazard ratio for dying of . (p. ). we will detail the type and pattern of neurological complications in t-cell non-hodgkin's lymphoma (nhl), and review how they differ from those associated with b-cell nhl, and the lymphomas in general. this study is the first step in a process to characterize these tumors to determine if special staging or cns prophylaxis are indicated in any of the subtypes of t-cell lymphoma. we recently have encountered women with breast cancer and an unusual sensorimotor neuropathy. the neuropathy was the major clinical problem. in women the initial symptom was severe itching, generalized and first localized to the involved breast and then generalized. all developed distal extremity numbness and burning that very slowly progressed proximally, and in became generalized. four complained of painful muscle cramps in the extremities ( ) and jaw ( ). all had mild extremity weakness, distal ( ) and proximal ( ) . three women developed symptoms up to months prior to cancer diagnosis, shortly after diagnosis, and years after diagnosis. four women had disease confined to the breast and regional lymph nodes, and had metastatic disease in remission. although annoying, symptoms were generally not disabling. three women stabilized or had slight improve-associated with breast cancer ment with cancer treatment, and continue to gradually progress while in cancer remission; required a cane to ambulate after years due to sensory ataxia. one who developed cancer relapse had concurrent neurological relapse. one woman treated with high-dose immunoglobulin did not improve. none had significant weight loss. laboratory abnormalities included elevated erythrocyte sedimentation rate ( - ) in , antinuclear antibody : in , csf with lymphocytic pleocytosis ( - white blood cellslmm) in / and elevated protein ( - mg/dl) in available. emgi ncv showed mild sensory-to-motor polyneuropathy in available. none had detectable antibodies against peripheral nerve or dorsal root ganglia. the etiology of sensorimotor neuropathy in these patients is unknown, but it may represent a distinct paraneoplastic syndrome that can herald the onset of, and parallel the course of breast cancer. our objective was to determine whether ceramide induces differentiation of anaplastic glioma cells. sphingomyelin hydrolysis resulting in ceramide production has been linked to differentiation of leukemia cells. t rat anaplastic glioma cells, seeded at x lo cells per well, were grown in serum-a glioma cell line program and abstracts, american neurological association free media. on day , cells were photographed, scored for processes longer than one cell body, and counted. c ceramide changed plump cells to flattened cells with many long processes: ceramide treatment increased the percentage of cells with processes from % (sd) to * % (n = , p < . ). control cells grew to . x lo cells/well . x lo cells; ceramide-treated cells grew to . x lo ? . x los (n = , p < . ). although one ceramide analog reproduced the c ceramide-associated changes, the optical isomer of this analog did not, demonstrating stereospecificity. cerarnide did not decrease cell viability by trypan blue. ceramide inhibits proliferation and induces process formation in a glioma cell line, causing it to assume a more differentiated phenotype. cerarnide or its analogs represent possible future therapeutic agents that would inhibit the growth and affect differentiation of anaplastic gliomas. [bashir, mod pathol ; ( ) : - )). this is similar to the pattern seen in ebv-infected human b cells and unlike the uniform latent infection seen in burkitt's lymphoma. we tested the hypothesis that long-term passaging of ebv-immortalized human b cells in immunodeficient mice leads to emergence of a uniform nonlytic pattern of ebv infection associated with appearance of the malignant profile. ebv-infected normal human b cells were serially passaged intracerebrally in severe combined immunodeficient cb mice ( x ' cells per mouse, mice per passage for a total of passages). frozen mouse brain sections from each passage were stained with vca antibody (ebv lytic cycle) and hybridized with biotinylated bamh -w sequence of ebv. all injected animals developed tumors as previously described (bashir, lab invest ; ( ): - ) . tumor cells continued to express vca and showed latent and lytic hybridization patterns with bamh,-w after passages despite exhibiting monoclonality (surface immunoglobulins) and random chromosomal changes. lytic infection of immortalized b cells with ebv is stable, resembling brain lymphomas in aids, and unlike the latent infection seen in burkitt's lymphoma. a previously healthy -year-old man developed diplopia and incapacitating, diffuse weakness over a period of weeks. examination showed a "one-and-a-half'' syndrome of horizontal gaze paresis, patchy severe weakness with atrophy and fasciculations, absent tendon reflexes in the legs and right biceps, and decreased vibration sense in the feet. csf contained a mild pleocytosis, elevated protein, and oligoclonal igg bands. electrophysiological testing indicated a generalized sensorimotor axonal neuropathy with diffuse denervation. small-cell lung carcinoma was diagnosed by bronchoscopy. prednisone produced mild subjective improvement. chemotherapy was begun but the patient developed fatal septicemia. serum was negative for anti-hu or anti-gm with paraneoplastic encephalomyeloneuritis antibodies. serum and csf contained high titers of igg antibodies reacting specifically with a protein antigen of approximately kd in immunoblots of human cerebral cortical neuronal nuclei or of human purkinje cells. this pattern of autoantibody reactivity was not present in sera from any of other patients with small-cell lung carcinoma, of whom had paraneoplastic encephalomyeloneuritis and anti-hu antibodies, nor was it present in many patients with other neurological disorders. the patient's serum has been used to probe a human cerebellum expression library and to isolate a cdna clone that is being characterized. acute encephalopathy is the problem in % of neurology consultations reported at mskcc. we studied patients ( prospectively and retrospectively) to determine clinical findings, causes, and outcome. fifty-five were women and were men, and the average age was years. all patients had cancer: lung ( %), gastrointestinal tract ( %), breast ( %), and others ( %), forty-two patients ( %) were delirious on admission and delirium developed an average of days later in %. encephalopathy occurred postoperatively in %. symptoms included confusion ( %), lethargy ( %), agitation ( %), hallucinations ( %), and seizures ( %). signs included deficits in attention ( %), memory ( %), language ( %), lateralizing signs ( %), and asterixis ( %). the average mini-mental status test (mms) score was ( = normal). a single cause for delirium was found in only % of patients with an average of etiologies per patient. metabolic abnormalities were found in % of patients, and were a primary cause in %; disseminated intravascular coagulation contributed to delirium in %. cns metastases were found in % and were a major cause of delirium in all. fifty percent of the patients had fever/ systemic infection, but sepsis was present in only %; only patient had cns infection. medication contributed to delirium in % but was a primary cause in only %. the -day mortality rate was % and delirium improved in % (average mms = ). patients with cancer have multiple, potentially treatable causes of delirium. delirium is associated with a high death rate, though patients generally improve. radiation therapy for brain tumors d. w. dodick, b. mokri, k. k. unni, g. m. miller, and e. g. shaw, rochester, m n osteosarcoma in a previously normal bone is a rare but recognized remote effect of radiation therapy. any bone in the field of radiation can be affected. involvement of cranial bones is exceedingly rare. we could identify only patients ( men and women) with postirradiation osteosarcoma of the calvarium seen at the mayo clinic over a -year period, from to . all had received radiation for brain tumor, osteosarcoma had appeared in the field of radiation in all, the interval from radiation therapy to the appearance of sarcoma ranged from to years, and diagnosis of sarcoma was confirmed histologically in all cases. the patients' age at the diagnosis of the brain tumor ranged from to years. the nature of the brain tumor was unverified in cases, was a low-grade ependymoma in the third case, and a pilocystic astrocytoma in the fourth case. one patient is still alive months after the diagnosis of the sarcoma. she received chemotherapy and subsequently underwent resection of the osteosarcoma. one patient died postoperatively after partial resection of the sarcoma. the other patients died months and months after the diagnosis of the osteosarcoma despite additional radiation therapy in the former and aggressive chemotherapy in the latter. element-la fusion gene as a potential marker of neural tumor differentiation lawrence recht, chiffon wu, and louis j. degennam, worcester, ma inducing cancers to differentiate into more benign differentiated tumors represents a novel oncological strategy. to establish a model that would permit assessment of this phenomenon at a molecular level, we created and have partially characterized a murine neuroblastoma line that has been stably transfected with a synthetic fusion gene containing the promoter element of the rodent synapsin i gene (synapsin i regulatory element isre)). in vivo, this promoter directs the neuron-specific expression of the synapsin i gene in normal adults. the gene also is expressed in varying amounts in neuronal tumors including neuroblastoma. in the synthetic fusion gene, the sre has been linked to the lacz gene that encodes bacterial p-galactosidase. a simple histochemical assay for p-galactosidase therefore provides a specific marker of the expression of the fusion gene. our preliminary experiments as of this writing have shown that it is possible to detect p-galactosidase activity in the transfected neuroblastoma cells both in vitro and in transplanted tumors. it appears possible therefore that this transfected neuroblastoma cell line can provide a useful model system with which to assess the effects of differentiation therapies. larger lesions (> cm ), and larger midline shifts (> mm). twenty-eight of ( %) patients with prior has had bt has compared to of ( %) patients without prior has. in patients, their bt h a was similar to their previous ha but was more frequent or severe. we conclude that has in bt patients are common but usually not severe. nausea, vomiting, an abnormal neurological examination, or a change in prior headaches warrant further investigation. cairncross and macdonald showed that procarbazine, lomustine, and vincristine (pcv) are effective for recurrent anaplastic oligodendrogliomas (ao) (ann neurol ; : - ) . pcv now has a major role in management of all forms of oligodendrogliomas ( ), but the biological basis for this response is unknown. to evaluate one subset of possibilities, we studied patients ( ao, ) with a ct method that permits measurement of blood-to-tissue transport (kj, tumor-to-blood transport (k ), and vascular volume (v,) (ann neurol ; : - ) . pcv was used to treat of the patients. k, (~ grr-' min-') values were highly variable for whole tumor, ranging from . to . (mean . k . ) with no difference between a and . k and v, were also highly variable. k, of tumor-free brain was . to . (mean . ? . ). in comparison to malignant astrocytomas, which have a mean k, in the range of . with some as high as . , a and appear to be much less permeable. this suggests that the efficacy of pcv may be due to factors other than capillary transport, such as tumor-cell sensitivity. frameless stereotactic localizer gene h. barnett, donald w. komos, and charles p . steiner, cleveland, oh extent of tumor resection has been shown to correlate with prognosis in malignant gliomas. although frame-based stereotactic techniques can provide information regarding tumor margin, they are often unwieldy and require expensive and elaborate computing systems. a frameless stereotactic neurosurgical localizing system was designed that overcomes these liabilities. this armless, frameless, stereotactic pointing device provides real-time three-dimensional localization information during operation. in addition to assisting in placement of a trephine craniotomy, it allows volumetric resection of the tumor with virtually complete excision of even large irregularly shaped tumors. mean error on localizing a point in space using this system has proven to be less than mm. a technical description of the system as well as surgical results are presented. to investigate the effect of age on response rate to chemotherapy and time to progression (ttp) and death ('itd) in patients with recurrent astrocytomas and malignant astrocytomas, we reviewed case records and scans of patients who received chemotherapy at the university of michigan with bischloroethylnitrosourea or procarbazine. three age groups were studied: ( ) < yr (n = ); ( ) - yr (n = ); ( ) > yr (n = ). tumors were grouped as grade r+ (recurrent grade plus grade , n = ) or grade (n = ). serial computed tomographic or magnetic resonance scans were analyzed in a blinded fashion and graded as progressive disease (pd), stable disease (sd), or partial response (pr, > % decrease in size). the pr rates for the age groups were %/ %/ % for grade r+ tumors ( p = . ) and %/ %/ % for grade tumors ( p = . ). median 'itp was weeks for grade r + and / weeks for grade . median ltd was / / weeks for grade r+ and / / weeks for grade . we conclude that age is an important prognostic factor with respect to likelihood of response to chemotherapy, duration of response, and survival irrespective of grade. cheryl p. harris and kurt a. jaeckle, salt lake city, ut intravascular malignant lymphomatosis (iml), a b-cell lymphoma confined to small venules and capillaries, often presents with neurological symptoms. this disease is uniformly fatal ( -month mean survival); no successful treatment has been identified. we observed marked reproducible neurological improvement after plasmapheresis in a -year-old woman with iml. presenting with a cauda equina syndrome, she progressed over year with neurological, hepatic, and hematological disease. persistent laboratory abnormalities included a high sedimentation rate ( mm/hr), coagulopathy, hemolytic anemia, and elevated liver enzymes. extensive evaluations for infectious, autoimmune, and neoplastic processes, including bone marrow examination, were inconclusive. because of neurological progression, empiric therapy with high-dose steroids followed by cyclophosphamide was initiated without response. plasmapheresis ( ml/kg in exchanges) effected resolution of encephalopathy and normalization of the coagulopathy and sedimentation rate. neurological progression recurred within weeks of pheresis; repetitive courses reproduced neurological response. finally, progressive dementia ensued, and a decision was made to cease pheresis; the patient died days later, months after presentation. autopsy disclosed diffuse intravascular cd- positive malignant lymphoma cells in small vessels of all organs. although the mechanism is unknown, the serendipitous discovery of response to plasmapheresis in this patient warrants further consideration. morphine is an effective analgesic in the rat after injection into a number of discrete brainstem regions, including the periaqueductal gray (pag), the locus coeruleus (lc), and the nucleus raphe magnus (nrm). early work with morphine gray and the nucleus raphe magnus established the existence of synergy between the brainstem and the spinal cord in rats. more recently, studies from our laboratory revealed synergy between two brainstem structures, the pag and the lc. in the current study, we explored the analgesic interactions between the pag and the nrm using indwelling cannulae. first, we established morphine dose-response curves and calculated the ed,, independently in the pag ( . pg) and the nrm ( . pg). we then simultaneously injected various morphine doses into both regions. injecting morphine at pg into either the pag or the nrm did not elevate tailflick latencies above baseline values. however, administered into both regions simultaneously, the -pg doses produced an % maximal response, corresponding to more than a threefold increase of baseline latencies. a fixed morphine dose of pg in the pag shifted the morphine dose-response curve fivefold in the nrm (ed,, . pg), whereas a fixed nrm dose of fg shifted morphine's dose-response in the pag approximately twofold. together, these results clearly show synergistic interactions for morphine between the pag and the nrm. the presence of synergistic interactions between brainstem nuclei as well as between the brainstem and the spinal cord underscores the complexity of opioid analgesic systems. p . the glutamate uptake inhibitor ~-trans- , -pyrrolidine dicarboxylate is neurotoxic in neonatal rat brain john d. e. barks and faye s. siloerstein, ann arbor, m i important evidence of the neurotoxicity of endogenous glutamate (glu) in mammalian brain was provided by the observation that dl-threo- -hydroxyaspartate, a high-affinity glutamate uptake (hagu) inhibitor, was neurotoxic in adult rodent striatum (j neurochem ; : ) ; however, the absence of neurotoxicity in neonatal brain was interpreted as evidence that immaturity of glutamatergic innervation limited the potential role of endogenous glu as a neurotoxin in the immature brain. yet, considerable data provide indirect support for the hypothesis that glu can be neurotoxic at this stage. to resolve this issue, we assessed the neurotoxicity of a novel, selective hagu inhibitor, ~-trans- , -pyrrolidine dicarboxylate (l-pdc) (j med chem ; : ), in postnatal day (pnd) rats (n = ). l-pdc (ph . ) was stereotaxically injected into right anterior striatum (str) ( nrnol, n = ) or through dorsal hippocampus into posterior str ( nmol, n = ; nmol, n = ). animals were killed days later, and neuropathology was assessed in cresyl violet-stained sections. after anterior injections, focal neuronal necrosis was evident in dorsal str; high-dose posterior injections caused prominent hippocampal lesions with pyramidal layer thinning and focal necrosis in dorsal thalamus, while nmol produced small foci of pyramidal cell loss. in both groups, focal cortical necrosis and callosal cysts were apparent adjacent to the injection track. l-pdc-induced brain injury provides direct support for the hypothesis that endogenous glu may be neurotoxic in the developing brain. (sax et al, ann neurol ; : a) . the signs and symptoms of of these patients lessened for to months. furthermore, a patient with a severe oral-lingual biting dyskinesia improved when taking doses up to milligrams per day for months. we noted no-significant adverse reactions, although patients had mild but labile elevations in sgop and sgpt. one patient receiving opioid analgesics for pain inadvertently failed to discontinue the naltrexone but noted no reduction in the pain-alleviating effects of the analgesic. although hyperkinesia, especially of midline functions, as well as quality of life improved for the hd patients, their cognitive deficits remained unaffected. these observations suggest that chronic naltrexone is a safe and effective agent to treat chorea, dysphagia, and oral dyskinesia in hd for periods longer than a year. furthermore, they indicate that naltrexone can be effective in ameliorating oral-lingual biting tardive dyskinesia. these findings support our previous hypothesis that endogenous opioids play a role in rhe modulation of the dopamine system in hyperkinetic stereotypic movement disorders. a. jon stoessl, elizabeth szczutkmuski, and hanna fydvszak, london, ontario, canada we previously have demonstrated (psychopharmacology ; : - ) that intraperitoneally (ip) administered cholecystokinin (cck)- s suppresses vacuous chewing mouth movements (vcms, a putative mode of tardive dyskinesia) in rats exposed to chronic neuroleptics. as cck is not thought to cross the blood-brain barrier in significant amounts, its site of action in this paradigm is unclear. other behavioral and neurochemical effects of ip cck are blocked by vagotomy. male sprague-dawley rats were administered fluphenazine decanoate (flu; mg/kg im) or its vehicle every weeks for approximately weeks. cck ( , , or ng intracerebroventricularly) had no effect on neuroleptic-induced vcms. another group of neuroleptic-treated rats was subjected to bilateral subdiaphragmatic vagotomy or a sham procedure. cck- s ( , , or pg/kg intraperitoneally) suppressed neuroleptic-induced vcms in shamoperated animals, which confirmed our previous results. in vagotomited animals, chronic flu failed to induce vcms and cck was without effect in vehicle-or neuroleptictreated animals. these data suggest that the effects of cck on flu-induced vcms may be mediated peripherally, and that vagal pathways may be important for generating this response. (supported by the ontario mental health foundation and the ontario ministry of health.) p . excitotoxic amino acids are not involved in dopaminergic neurotoxicity of m p t p eldad mehmed, jutta rosenthal, and avinoam reches, petah tiqva, tel aviv, and jerusalem, israel the dopaminergic (da) neurotoxicity of -methyl- phenyl- , , , -tetrahydropyridine (mptp) is mediated via its oxidation in cns to mpp + , which enters da neurons and poisons mitochondrial complex i. da neuronal damage induced by direct nigral mpp+ injection is prevented by pretreatment with n-methyl-d-aspartate receptor antagonists, which suggests that excitatory amino acids are involved in mptp toxicity. since local mpp + application may produce nonselective nigral damage, we examined whether excitotoxins have a role in toxicity of systemically administered mptp. c black mice were injected intraperitoneally, once, with mptp.hci( mg/kg) and decapitated days later. groups of animals underwent the following pretreatments: (i) decortication week prior to mftp; ( ) intracerebroventricular injections of the excitatory amino acid receptor antagonists -amino-phosphonoheptanoate and d-glutamyl-glycine; and ( ) intraperitoneal injections of the calcium channel antagonists nimodipine, diltiazem, and flunarizine minutes prior to mptp. mptp produced marked striatal da depletions. decortication, destroying glutamatergic corticostriatal projections, intracerebral amino acid receptor antagonists, and systemic calcium channel antagonists did not protect mice against mptp toxicity; mptp-induced striatal da decreases were similar to those given the neurotoxin alone. this study suggests that excitotoxins are not involved in the mechanism of mptp toxicity. scopolamine-induced cognitive deficits k . j . meador, m . e. allen, p. franke, e. e. moore, and d. w. loring, augusta, ga a unique neurophysiological role for thiamine in cholinergic systems has been suggested. total thiamine content in cholinergic nerve terminals is comparable to that of acetylcholine, and the phosphorylation state of thiamine changes with release of acetylcholine. thiamine binds to nicotinic receptors and may exhibit anticholinesterase activity. based on these observations, we investigated the effects of pharmacological doses of thiamine on the cognitive deficits induced by the anticholinergic scopolamine in healthy young adults using a randomized, double-blind, placebo-conrrolled, double crossover design. cognitive tests included the p eventrelated potential and free recall memory for a verbal paragraph. conditions included baseline (bl), thiamine gm by mouth and scopolamine . mg/kg intramuscularly (b + scop), and lactose by mouth and scopolamine (plac + scop). testing was performed hours post thiamine or placebo, and . hours post scopolamine. thiamine significantly reduced the adverse effects of scopolamine on p latency (f [i, = . , p . ) and percent recall memory ( f el, ) = . , p . ). means (+sd) and p latency (ms) were bl = ( ), b + scop = ( ), and plac neurol ; : - ) . we previously reported that cff improved in of ms patients ( %) given el- orally in divided daily doses ranging from . to . mg (stefoski et al, neurology ; : - . subsequent analysis revealed that in of the patients cff improvements and reversals followed in a phase-locked trend the rising and falling serum concentrations of el- , including patients whose changes remained below the % increase needed to qualify for the improved category. these results resemble the phase-locked effects of temperature on neurological function in ms. the cff changes, because they so closely reflect variations in serum concentration, suggest that el- tissue levels closely follow those in serum and that el- rapidly crosses the blood-brain barrier. efficacy of el- in ms is also predicted to have a close relationship to serum levels. p . development of a n internal standard detectable by proton and phosphorus- nmr and hplc w. e. klunk, k. panchalingam, r. j . mcclure, and j . w. pettegrnu, pittsburgh, pa comparison of quantitative results from different analytical techniques can prove difficult due to the peculiarities of the particular techniques and the lack of a common standard applicable to all of the techniques. recently, both in vivo and in vitro nuclear magnetic resonance (nmr) have been applied to the quantification of a large variety of metabolites. although nmr can be applied to the study of living tissue, the question arises of how this technique compares with more traditional techniques such as high-pressure liquid chromatography (hplc). although this question can be addressed partly by studying perchloric acid (pca) extracts, it is difficult to directly compare this in vitro nmr data with results from hplc. to address this question, we have developed an internal standard that can be quantified directly by in vitro phosphorus- nmr, proton nmr, and by -fluorenylmethyl chloroformate (fmoc) derivatization followed by separation by hplc. a variety of aminophosphonic acids were studied by 'p nmr, 'h nmr, and hplc. promising compounds were added to pca extracts of human brain. the optimal compound was found to be -aminopropylphosphonic acid (app, ho p-ch -ch,-ch -nh ). app is easily detectable by all techniques, falls well outside of the region of interest in phosphorus- nmr, and is well resolved by proton nmr at mhz. it is easily separable from the phosphomonoesters and phosphodiesters observable by hplc and from the amino acids that occur in brain in significant concentrations. app appears to be a useful internal standard in the study of phosphorus and amino acid metabolites by in vitro p nmr, 'h nmr, and hplc. theories of sentence comprehension hypothesize at least a grammatical component that establishes the relationship among words in a sentence, and a semantic component that determines the meanings of these words. we used positron emission tomography (pet) to quantify regional cerebral blood flow (rcbf) in neurologically intact subjects during their detection of a letter target, a grammatical target, or a semantic target in the same written sentences. a mixedmodel analysis of variance (anova) revealed significant main effects for region (f e , ) = . ; p < . ), condition (f , = . ;~ < . ), and a significant region times condition interaction ( f {go, = . ; p < . ), but there were no differences between individual subjects. subsequent anovas revealed increased rcbf in a unique set of brain regions during the subjects' response to a grammatical probe when compared to their response to a letter probe of the same sentences. a unique distribution of rcbf also distinguished response to the semantic probe from response to the grammatical probe and the letter probe. other brain regions apparently contributed to performance for several activation conditions. these findings support the hypothesized dissociation of specific linguistic components based on their unique cerebral topographical representation, and that a distributed network of brain regions subserves sentence comprehension. cerebral music processing-a comparison study of musically trained and naive individuals louis s. russo, jr, jachonville, fl we performed topographical mapping of brain electrical activity in right-handed symphony musicians and righthanded, musically naive individuals during various musical tasks; namely, listening to solo piano music, silent singing of familiar music, and silent reading of unfamiliar music. fast-fourier transform ( f r ) of electrocortical activity was carried out during task performance and the eyes-open resting state. data were analyzed using a computer-assisted model. increases in regional beta activity of greater than standard deviations from the resting state were considered significant of activation. during audition, the musicians showed activation in the right posterior parietotemporal region; the naive showed no change from the resting state. during silent singing, the musicians showed bitemporal activation, r > l; the naive showed activation in the right mid-and posteriortemporal regions alone. during silent sight reading, the musicians showed a major activation in both temporal regions, l > > r, the naive showed only a marginal change in the posterior temporal-occipital regions. these data suggest that music processing is primarily a right cerebral function in untrained individuals and a bilateral function in musicians. musicians, in contrast to the naive, show progressively more left brain activation as task complexity increases. the present study analyzes language profiles in patients who presented with primary progressive aphasia (ppa) without global dementia for at least years. language and cognitive impairment were evaluated using the western aphasia battery (wab) and the mattis dementia rating scale (drs). expressive language disability with reduced speech fluency and anomia, but preserved language comprehension and nonverbal cognition, were typical features in early stages. spontaneous speech was significantly more impaired in ppa than in anomic aphasia after left-hemisphere stroke and in language impairment in probable alzheimer's disease (ad) ( p = . ). the profile of aphasia suggests that ppa tends to affect anterior parts of the language-dominant cortex first. neuroimaging generally showed mild to moderate brain atrophy. in patients atrophy involved especially the left frontal progressive aphasia cortex. follow-up examinations that were done in patients or several years after the first assessment revealed continuous, most often rapid deterioration of language impairment. two patients died and years after the onset of ppa. neuropathological examination showed ad in patient and pick's disease in the other patient. beverly clarke, adrian upton, markad kamath, and helene griff;., hamilton, ontario, canada eight patients implanted with a cyberonics neurocybernetic prosthesis model to stimulate the vagus nerve were assessed for changes in cognitive performance. the patients had complex partial seizures for more than years, with more than per month. patients were years * . sd old. cognitive evaluation included response time to a randomized light signal appearing on a switch box (test a); test b, in which the signal appeared bilaterally; and test c, in which a response to the signal was required while the patient simultaneously ignored a second signal. data were collected and analyzed using an apple i e computer and switch pad. all patients were taking therapeutic levels of anticonvulsant medications and dosages were constant. testing occurred times during a day preoperatively (day l), weeks postoperatively with the stimulator on (day ), and months after turn-on (day ). patients were randomized into high-and low-frequency stimulation groups (hfg and lfg). hfg parameters were hz, so msec pulse width (pw), and lfg hz, msec pw. examiners were blinded as to group. student's t-test analyses of mean differences between groups and individual measurements showed a significant difference between hfg and lfg for test c ( p < . ). lfg showed a significant improvement for tests a, b, c between day and , and for test c between day and . no group effect was seen between day and in the lfg. individual measurements showed improvement for test b ( p < . ) for the hfg between day and , test b ( p < . s) between day and , and tests a and b ( p < . ) and ( p < . ) day vs . the lfg group improved between days and and and . between day and day , the lfg showed improvement only for test b ( p < . ). chronic stimulation of the vagus nerve improves cognitive function in epileptic patients and this improvement is more marked with low-frequency stimulation. a. guidotti, and j. d. rothstein, bologna, itah, washington, dc, and baltimore, m d we reported idiopathic recurring stupor (irs) in a patient with stuporous episodes without known causes and reversed by flumazenil, a specific benzodiazepine (bz) antagonist. ictal plasmdcerebrospinal fluid (csf) showed increased bz-like activity (ann neurol, in press). recently, an endogenous bzreceptor ligand (endozepine [ez]) has been purified from mammalian brain with properties similar to diazepam. it acts like diazepam to potentiate gamma-aminobutyric acidmediated postsynaptic inhibition. we hypothesized that irs might be due to an excess of this substance. irs was diagnosed in patients, and years old, who had recurring stupor or coma episodes lasting hours to days. ictal brain ct/mri, kidney, liver, heart, blood glucose, ammonia, and osmolality were normal. eeg showed fast -hz background activity while the patients were unreactive to stimuli, reversed by flumazenil. ictal serum or csf revealed an enormous increase of the ez in both patients, with levels as high as nm, compared to to nm in control serum/csf. interictal csf or serum in irs contained ez levels similar to control csf and serum. irs may be due to excess ez. the cause for increased ez is unknown. parkinson's disease: evidence from word learning murray grossman, jenger mickanin, barbara schaefr, kris onishi, matthew b. stern, steven gollomp, and howard hurtig, philadelphia, pa several reports have suggested that patients with parkinson's disease (pd) have intellectual impairments in several domains such as memory, but few studies have explored difficulties in language processing. we investigated the ability of nondemented pd patients with mild motor impairments to learn about the grammatical and semantic information represented in a new verb. the new verb was presented to patients in a sentence-picture matching context, and we probed their recall of the verb minutes later. a sentence judgment task assessed grammatical knowledge by asking patients to judge the new verb, known verbs, and pseudowords used appropriately or incorrectly in a sentence. we found that % of pd patients were significantly impaired in their grammatical appreciation of the new verb (f [i, = . ;p < . ). this was not related to their motor disorder or neuropsychological performance. a picture classification task used pictures illustrating specific aspects of the new word's meaning to evaluate semantic knowledge. pd patients were as accurate as controls at deciding whether a picture illustrated the meaning of the new verb (f ( , = .lo;p > . ). only pd patient ( %) had difficulty sorting pictures. selective difficulty recalling only grammatical aspects of a new word suggests that the word learning impairment in pd cannot be entirely explained by poor memory. instead, in agreement with other recent findings, pd patients may be impaired in some aspect of grammatical processing in language. we discuss the hypothesis that defects in the frontocaudate axis in pd underlie this impairment. neuropsychological performance on both verbal and nonverbal tasks is reported to differ between healthy men and women. some of these cognitive differences are postulated to reflect differences in interhemispheric and intrahemispheric cerebral organization. our preliminary study indicated that women with alzheimer's disease (ad) performed worse than men on a composite neuropsychological battery, even after effects of potentially confounding variables were considered (buckwalter et al, j clin exp neuropsychol ; : ) . to explore further the nature of gender-associated differences in ad, we analyzed data from a verbal and a nonverbal task (the boston naming test and drawings from the spatial quantitative battery supplement to the boston diagnostic aphasia examination) for men and women who met nincds-adrda criteria for "probable" ad. prior to ana-grip strength kg [range . - kg]). the electrophysiological examination showed improvement with reversal of conduction block in patients and was unchanged in . an apparent response to the placebo was seen in patients. improvement after ivig therapy was maintained for variable durations ( - wk) and reoccurred with subsequent infusions. an equally effective response was documented after infusion of a single ivig dose of gm/kg. we conclude that ivig therapy is effective in some patients with cidp, even after long duration of illness. the best responses were observed in patients with recent relapse. a single high-dose treatment may be equally effective. the object of this study was to examine whether borrelia burgdorjeri antigens could be detected in csf in the absence of detectable antibodies to b. burgdorjeri (the etiological agent of lyme disease). osp a is a -kd antigen that is specific for . duvgdorferi osp a was probed using western (immuno) blot and specific mouse monoclonal antibodies. polyclonal lyme antibodies were detected in csf using standard micro enzyme-linked immunosorbent assay. seven patients had osp a in csf without detectable lyme antibodies. there were men and women aged to years (mean yr). disease duration ranged from weeks to years. neurological syndromes included confusion with acute flulike illness, optic neuritis, hemiparesis with inflammatory brain lesion, encephalitis, headache with erythema migrans, bilateral facial nerve palsies, and encephalomyeloradiculitis. three patients had csf abnormalities. in patients csf parameters were otherwise completely normal. possible explanations for undetectable csf lyme antibodies included early infection ( patients), prior antibiotics ( patients), and prior steroids plus antibiotics ( patient). in patient there was no obvious explanation. we conclude that osp a, a specific antigen of b. burgdorferi, may be present in csf without a detectable humoral response. the diagnosis of neurological infection with b. bwgdorjeri should not require a positive csf serology. mollaret's meningitis: demonstration by polymerase chain reaction a -year-old man was seen in september for his fourth episode of aseptic meningitis over a -year period. the episodes conformed to criteria for mollaret's meningitis as published by bruyn, straathof, and raymakers, and subsequently by others; they lasted about week, and were characterized by fever, headache, meningismus, lymphocytic pleocytosis, elevated protein in cerebrospinal fluid (csf), and spontaneous resolution without residua. extensive prior evaluations had failed to uncover a cause. the patient was otherwise well and neither he nor his wife had any history of sexually transmitted diseases. suspicion of herpes simplex virus (hsv) arose due to a single transient, raised skin rash several weeks earlier that failed to yield virus on culture. the patient was treated with acyclovir, which resulted in rapid resolution of symptoms. though culture and immunological studies of csf and blood again were unrevealing, polymerase chain reaction (pcr) studies of csf confirmed the presence of hsv type . we suspect that herpes simplex virus is a more common cause of recurrent aseptic meningitis than current culture and immunological techniques would suggest. pcr offers increased diagnostic sensitivity for neurotropic viruses and should be considered in patients with recurrent meningitis of cryptic etiology. differentiation by inorganic lead: a role for protein kinase c j. lutewa, j. p. bressler, r. r. indurti, l. belloni-olivi, and g. w . goldstein, baltimore, m d microvascular endothelial function in developing brain is altered by inorganic lead. this may result from changes in protein kinase c (pkc) modulation. we examined the effects of inorganic lead on an in vitro model of neural endothelial differentiation. astroglial-induced endothelial differentiation into capillary-like structures was inhibited by lead acetate with % maximal inhibition occurring at . pm lead. inhibition was independent of effects on cell viability or growth. we examined the effects of lead on cellular pkc pools under conditions that inhibited capillary-like structure formation. membranous pkc increased in c astroglial and neural endothelial cells after exposure to lead acetate. exposing c cells to p,m lead for hours increased membranous pkc by % as determined by immunoblotting. membranous pkc increased in response to as little as nm lead and saturated at pm. phorbol esters were used to determine if pkc modulation was mechanistically related to lead's inhibition of capillary-like structure formation. -myristate -acetate ( nm) inhibited endothelial differentiation by * %, whereas -alpha-phorbol , didecanoate was without effect. these findings demonstrate that inorganic lead may induce cerebral microvessel dysfunction by interfering with pkc modulation in microvascular endothelial or perivascular astroglial cells. w. f. brown, b. v . watson, j . garland, g . c. ebers, and n . desai, london, ontario, canada gait difficulties in multiple sclerosis (ms) are commonly accompanied by fatigue and dyspnea. possible explanations for the latter include weakness andlor dyssynergia of the respiratory muscles, including possible abnormalities in central pathways regulating respiration. this study examined central and peripheral motor conduction to the diaphragm in ms patients whose gait was notably labored and accompanied by breathlessness. peripheral conduction was assessed by measuring the latency and size of the surface-recorded diaphragmatic maximum m-potential responses to supramaximal stimulation of the phrenic nerve in the neck, and central motor conduction by comparable measurements in response to magnetoelectrical stimulation over the vertex. peripheral motor conduction was normal. the most striking abnormalities were in central motor conduction. cortical stimulus-evoked diaphragmatic responses were absent on both sides in patients, and unilaterally in l patient, whereas in others the latency of the cortical stimulus-evoked response was increased and the size clearly reduced and entirely normal in only patients. these studies show that central conduction program and abstracts, american neurological association to the diaphragm is commonly abnormal and may play a role in the fatigue and dyspnea experienced by ms patients. six men ( - yr) developed myelopathy that progressed slowly over several months and was characterized by asymmetrical, incomplete spinal cord syndrome manifested at the sensory level at the trunk, mild spastic paraparesis, and urinary incontinence. the spinal cord lesions at appropriate levels were recognized by mri as enhancing lesions in of the men. coxiella burnetii infection was confirmed in the blood of all patients by immunofluorescence microscopic assay (ifa) and transmission electron microscopy (tem). in patients, we detected c. burnetti by tem and ifa using csf of the patients inoculated onto fresh peripheral blood lymphocytes. four patients who were treated with appropriate antibiotics responded with either partial resolution of symptoms or arrest of further neurological progression. in patients the lesion was shown on mri to have decreased in size. in summary, we report cases of transverse myelopathy associated with c. barnetti infection. this is the first report, to our knowledge, of coxiella-related chronic myelopathy. we present a series of patients with different labyrinthine lesions diagnosed by mri. twelve patients with sensorineural hearing loss were studied by gadolinium-enhanced mri, including -mm contiguous t -weighted images through the labyrinth. ten patients had enhancement of the cochlea or vestibule, or both. all patients with cochlear enhancement had severe neural sensory hearing loss. all patients with vestibular enhancement had severe vestibular symptoms. the patients' final diagnosis included viral labyrinthitis ( patients), syphilitic labyrinthitis ( patients), bacterial labyrinthitis ( patient), and vestibular neuromas ( patients). one patient had an acoustic neuroma extending in the basal turn of the cochlea. the enhancement in patients with vestibular neuromas was brighter and there was slight mass effect in comparison with the patients with inflammatory labyrinthine lesions. one patient had hemorrhage within the vestibule from an adjacent temporal bone hemangioma. one patient with ct-proven cochlear otosclerosis had pericochlear areas of enhancement on gadolinium mri. mri can diagnose a variety of labyrinthine lesions that correlate very well with the patient's clinical symptoms. gadolinium should be used routinely in patients with suspected labyrinthine disease. the diagnosis of meniere's disease and endolymphatic hydrops remains a diagnosis of exclusion. few radiographic findings have been correlated with the clinical symptoms of this entity. we describe patients with symptoms of hearing loss or vertigo, or both, who demonstrated enhancement of the endolymphatic sac on gadolinium-enhanced mri. no enhancement was noted in a series of controls with no symptoms of hearing loss and vertigo. enhancement in the brain correlates with inflammatory or neoplastic conditions. we thus can speculate that enhancement of the endolymphatic sac reflects an inflammatory process in this location that may interfere with the normal resorption of indulin and secondary hydrops. in addition to excluding an acoustic neuroma and a labyrinthine schwannoma (which clinically may be confused with meniere's disease), contrast-enhanced mri may provide objective evidence in favor of labyrinthine hydrops. it was intermittent ( %) but progressive. the ha was mild to moderate in severity; it was the worst symptom in only ( %) and the first symptom in ( %) patients. has were worse in the morning in ( %) and interfered with sleep in ( %) patients. unlike true tension-type has, bt has were worse with bending over in ( %), with valsalva's maneuver in ( %), and nausea or vomiting were present in ( %) patients. an abnormal neurological exam was found in ( %) patients with has and ( %) patients without has lyzing the effects of gender, we used a hierarchical regression procedure to control for possible effects of subject age, education, age at onset of dementia symptoms, dementia duration, and family history of dementia. significant gender effects were found for the verbal task ( p < . ) (mean boston naming test score of . for women and . for men), but not for the drawing task. we conclude that verbal abilities are more severely affected in women than in men with ad, a difference that may in part reflect premorbid gender-associated differences in cerebral hemispheric organization. hemispatial placement is known to affect line bisection in patients with neglect. whereas placing stimuli in neglected space increases bisection error, placing stimuli in nonneglected space attenuates error. the effects of hemispatial placement on line bisection were examined in patients with chronic neglect (over months after stroke). all patients had large (frontotemporoparietal), unilateral, right-hemisphere lesions. each patient bisected lines of different lengths ( , , , and cm) in hemispatial conditions ( cm left of midline, midline, and cm right of midline). like previous reports, when patients bisected lines in left hemispace, a consistent ( / trials) left-sided neglect was observed ( . cm). however, when lines were bisected in center space, misbisections occurred on either side of the midline; and, unlike previous studies, when lines were bisected in right hemispace, a consistent ( / trials) right-sided neglect was observed ( . cm). the magnitude and directional consistency of line bisection errors were significant. neither visual field defects nor limitations in reaching accounted for the results. recovery in chronic neglect may involve a realignment of limited attentional resources favoring the body's midline. consequently, performance in both hemispatial fields can be biased toward midline, resulting in neglect of opposite directions. despite agreement that depression is the most common neuropsychiatric symptom associated with multiple sclerosis (ms), many aspects of this emotional change are unclear. one of the more controversial issues concerns the relationship between severity of ms and depression. this relationship is used to evaluate whether depression is an integral or reactive symptom of ms. examination of this relationship is complicated by the presumed overlap between somatic features of depressive and neurological symptoms in ms. to clarify this situation, we examined the relationship between severity of ms and categories of depressive symptoms using the beck depression inventory (bdi). eighty-nine patients and normal controls were examined. for certain comparisons, patients were classified as mild (extended disability status scale of - ) or moderatelsevere ( ) ( ) ( ) ( ) ( ) ( ) . results indicated that total bdi scores and the depressive symptom categories (mood, self-reproach, vegetative, and somatic features) were elevated in patients with ms, but the extent of these elevations was not related to severity of disease. these results suggest that depression in ms is not a simple reaction to physical disability. furthermore, clinical examination of depressive symptoms is straightforward and not confounded by severity of ms. neurological involvement in wegener's granulomatosis was studied in consecutive patients diagnosed at the mayo clinic. one hundred and nine patients ( %) had neurological involvement. peripheral neuropathy was seen in ( . %), cranial neuropathy in , external ophthalmoplegia in , cerebrovascular events in , seizures in , and miscellaneous involvement in . the mean age and sex ratio did not differ in those with or without neurological involvement. among the patients with peripheral neuropathy, had multiple mononeuropathy, had distal symmetric polyneuropathy, and had unclassified peripheral neuropathy. multiple mononeuropathy was one of the major presenting symptoms in patients. kidney involvement was significantly higher in the patients with peripheral neuropathy compared to those without it (p < . ). among the cranial nerves, the second, sixth, and seventh nerves were affected most frequently. multiple cranial nerves were affected in patients. unusual neurological manifestations among the miscellaneous group included spastic paraparesis, temporal arteritis, homer's syndrome, and papilledema. this is the first comprehensive study on the frequency and distribution of neurological involvement in wegener's granulomatosis. chronic inflammatory demyelinating polyneuropathy: a double-blind placebo-controlled crossover study treatment with high-dose intravenous human immunoglobulin (ivig) has been reported to be beneficial in some patients with chronic inflammatory demyelinating polyneuropathy (cidp), yet most observations have been nonblinded. we examined the effect of ivig therapy in patients ( men, women) with cidp in a double-blind, placebo-controlled crossover study. disease was chronic progressive (n = ) or chronic relapsing (n = ) and of variable duration ( mo to yr). the diagnosis was confirmed by electrophysiological ( ) and nerve biopsy ( ) examinations. the trial consisted of two -day periods each. patients were randomly treated with ivig ( . mg/kg/day) or placebo on consecutive days and followed. function was assessed by a quantitative neurological disability score, functional grade, grip strength measurement, and electrophysiological examinations at the beginning and end of each treatment period. with ivig therapy, significant improvement was documented in / patients (improvement in neurological disability score mean key: cord- - rlotyz authors: bohmwald, karen; gálvez, nicolás m. s.; ríos, mariana; kalergis, alexis m. title: neurologic alterations due to respiratory virus infections date: - - journal: front cell neurosci doi: . /fncel. . sha: doc_id: cord_uid: rlotyz central nervous system (cns) infections are one of the most critical problems in public health, as frequently patients exhibit neurologic sequelae. usually, cns pathologies are caused by known neurotropic viruses such as measles virus (mv), herpes virus and human immunodeficiency virus (hiv), among others. however, nowadays respiratory viruses have placed themselves as relevant agents responsible for cns pathologies. among these neuropathological viruses are the human respiratory syncytial virus (hrsv), the influenza virus (iv), the coronavirus (cov) and the human metapneumovirus (hmpv). these viral agents are leading causes of acute respiratory infections every year affecting mainly children under years old and also the elderly. up to date, several reports have described the association between respiratory viral infections with neurological symptoms. the most frequent clinical manifestations described in these patients are febrile or afebrile seizures, status epilepticus, encephalopathies and encephalitis. all these viruses have been found in cerebrospinal fluid (csf), which suggests that all these pathogens, once in the lungs, can spread throughout the body and eventually reach the cns. the current knowledge about the mechanisms and routes used by these neuro-invasive viruses remains scarce. in this review article, we describe the most recent findings associated to neurologic complications, along with data about the possible invasion routes of these viruses in humans and their various effects on the cns, as studied in animal models. respiratory diseases caused by viral agents are one of the most critical problems in public health, as every year they are responsible for high rates of morbidity and mortality, mainly of young children, the elderly and immunocompromised individuals (talbot and falsey, ; tregoning and schwarze, ; englund et al., ) . the most common respiratory viruses that affect susceptible population are human orthopneumovirus-previously known as human respiratory syncytial virus (hrsv), influenza virus (iv), coronavirus (cov) and human metapneumovirus (hmpv; nichols et al., ) . the transmission of these viruses is mainly by contact with fomites or suspension droplets (kutter et al., ) . all these viruses have in common the ability to produce bronchiolitis and pneumonia, being responsible for large numbers of hospitalizations every winter season (nichols et al., ; talbot and falsey, ; tregoning and schwarze, ) . besides respiratory tract infections, these viruses have been associated with neurological clinical manifestations in patients with a severe occurrence of the respiratory disease (antonucci and fanos, ; akins et al., ; antonucci et al., ; desforges et al., a; fok et al., ; algahtani et al., ) . commonly, the invasion of the central nervous system (cns) and the subsequent pathology have been more studied in infection caused by japanese encephalitis virus (jev), varicella-zoster virus (vzv), measles virus (mv) and human immunodeficiency virus (hiv), among others (koyuncu et al., ) . nowadays, the interest in increasing the knowledge about the characteristics and mechanisms involved in their neurological manifestations has risen. neurological abnormalities found in patients with severe respiratory illness have a spread spectrum of clinical signs, being the most reported seizures (niizuma et al., ; li et al., ) , status epilepticus (sweetman et al., ; vehapoglu et al., ) , encephalopathies (antonucci and fanos, ; mizuguchi et al., ; niizuma et al., ; meijer et al., ) and encephalitis (ng et al., ; niizuma et al., ; fok et al., ; table ). the effects of each respiratory virus mentioned above in the cns infection will be discussed in detail later. for the proper functioning of the cns, it is essential to maintain homeostasis. both, the blood-brain and the blood-csf barriers play an important role in protecting the brain of free passage of unwanted molecules, pathogens and cells (mcgavern and kang, ) . the blood-brain barrier (bbb) is the first line of defense that prevents the entry of pathogens into the brain, and it is composed by cerebral microvascular endothelium, astrocytes, pericytes and extracellular matrix (mcgavern and kang, ; swanson and mcgavern, a) . importantly, the brain microvascular endothelium cells (bmecs) are a cell type found in significant proportions in the bbb; in between these cells are tight junctions (tj), which controls the barrier permeability (koyuncu et al., ; miner and diamond, ) . several routes of cns invasion can be used by viral pathogens, among these are included the hematogenous route-which is the infection of the endothelium or the ''trojan horse'' mechanism-and the peripheral nerves or olfactory sensory neurons (mcgavern and kang, ; swanson and mcgavern, b; dahm et al., ) . after primary infection, most neurotropic viruses can enter the bloodstream to reach cns, a process which is called viremia (gonzalez-scarano and tyler, ) . once inside the bloodstream, viruses can pass through the bbb by a transendothelial mechanism, which is transcytosis across bmecs and pericytes by endocytic vesicles (suen et al., ) . another transcellular entry method is the infection of endothelial cells, allowing the direct pass across bbb (koyuncu et al., ; suen et al., ) . besides, disruption low levels of tnf-α in csf. elevated il- and bdnf in csf correlates with brain damage. cappel et al. ( ) ; hirayama et al. ( ) ; ng et al. ( ) ; zlateva and van ranst ( ) ; otake et al. ( ) ; kawashima et al. ( kawashima et al. ( , and morichi et al. ( ) of bbb permeability by destabilization of tjs allow viral entry into the brain in a paracellular transmigration way (li et al., ; swanson and mcgavern, b) . this event is a consequence of a systemic infection that releases inflammatory mediators-such as cytokines and chemokines-besides the matrix metalloproteinase (mmp; roe et al., ) . finally, the ''trojan horse'' mechanism consists in the infection of bloodstream leucocytes-mainly monocytes/macrophages-which can transmigrate via paracellular route, across the permeable bbb into the cns (suen et al., ) . the correct working of the organism requires continuous communication between cns and peripheral tissues. in this process, neurons play an essential role, since these cells innervate the peripheral organs, that can be used by viruses as a gate to enter the cns (swanson and mcgavern, b) . neurons are polarized; this characteristic allows them to receive, process and transmit signals to other cells (koyuncu et al., ; swanson and mcgavern, a) . some viruses can infect and migrate through the nerve ending which can be sensory or motor (swanson and mcgavern, a) . for this purpose, viruses use the motor proteins dynein and kinesins-which are responsible for the retrograde and anterograde neuronal transport (swanson and mcgavern, b ). an alternative route for neuroinvasion is the transport through olfactory neurons (swanson and mcgavern, b) . this pathway is an excellent mechanism to access cns for viruses that enter the body intranasally (koyuncu et al., ) . olfactory nerve has the particularity to be in communication with the nasal epithelium and also with the olfactory bulb, the gateway to the cns (koyuncu et al., ; swanson and mcgavern, a) . this route is commonly used by respiratory viruses that infect the cns but is not the only one, as it will be discussed later in this review. the hrsv is an enveloped, negative-sense singled stranded rna virus, which belongs to the mononegavirales order and has recently been assigned to the pneumoviridae family and the orthopneumovirus genus (afonso et al., ; king et al., ) . accordingly, this virus has also been recently renamed human orthopneumovirus, but for the purpose of these publication we will refer to it as hrsv. the main and most studied pathologies caused by hrsv are bronchiolitis and pneumonia (antonucci et al., ) . however, in the past years, extrapulmonary manifestations have been associated with this virus (eisenhut, ) . notably, there is evidence that relates hrsv infection with pathologies such as myocarditis (esposito et al., ) , hyponatremia (hanna et al., ) , hepatitis (kirin et al., ) and encephalopathy (ng et al., ) . in wallace and zealley ( ) , in a study performed in children with a febrile status, hrsv was detected, and its infection was related to neurological damage. later, cappel et al. ( ) detected viral antibodies in cerebrospinal fluids (csf) of patients that have suffered symptoms of cns infection such as seizures, convulsions and neck stiffness (figure ) . one of the most significant findings from this report was that hrsv infection was associated with neurological abnormalities such as encephalitis (cappel et al., ) . a few years later, a case report of three preterm infants which were hospitalized by hrsv-induced bronchiolitis, also presented neurological abnormalities (morton et al., ) . despite these finding, a few years passed until, in hirayama et al. ( ) reported the case of a -year-old child that was hrsv-positive with clinical signs of ataxia. in the csf, a high number of leucocytes was found; however, they could not detect hrsv by polymerase chain reaction (pcr). the authors concluded that this child manifested a meningoencephalitis with cerebellitis associated with hrsv-infection (hirayama et al., ) . , ng et al. ( , performed a retrospective study where clinical data of patients with bronchiolitis by hrsv infection were evaluated. the results of this analysis showed that . % of the children exhibited visible clinical signs of encephalopathy, particularly seizures. another retrospective investigation that evaluated patients detected that were hrsv-positive and hsv-negative. in the hrsv-positive cohort, about a . % presented seizures; however, this number was similar for the one reported in the hrsv-negative cohort (kho et al., ) . in addition to this, it was found that . % of the patients exhibited apnea, but no differences were found when compared with the hrsv-negative cohort (kho et al., ) . importantly, these data support the idea that it is relevant to analyze other symptoms associated with hrsv bronchiolitis carefully. the first detection of hrsv rna in csf was from a -month-old boy hospitalized by pneumonia and febrile convulsion (zlateva and van ranst, ; figure ). in this study, the authors were able to identify that the hrsv strain found belonged to the serogroup b (zlateva and van ranst, ) . to achieve a better understanding of the effects of hrsv-infection in the cns, the csf of an -month-old boy that exhibited neurological abnormalities were analyzed, to evaluate the contribution of cytokines in this phenomenon (otake et al., ) . the results showed an increase of il- in the csf but not in serum, which suggests a local effect, implicating that cns cells-such as astrocytes and microglia-can be the source of these cytokines (otake et al., ; figure ). moreover, an increase of il- was also found in three cases of infants younger than -years-old in which it was possible to detect hrsv rna-serogroup a-in csf ; figure ). the authors suggest that their result support the idea of a direct invasion of the cns by hrsv . interestingly, the same group found in another cohort of children infected with hrsv the presence of viral rna, which correlated with low levels of tnf-α in csf (kawashima et al., ) . also, most of the patients showed an increase in the production of several chemokines such an il- , ccl and ccl which may play an essential role in this disease (kawashima et al., ; figure ). the encephalopathies caused by hrsv are classified in four groups: ( ) metabolic error type; ( ) cytokine storm type; ( ) excitotoxicity type; and ( ) hypoxic encephalopathies (morichi et al., ) . the encephalopathy caused by metabolic error is an abnormality of the brain function that can be reversible and involves an alteration of metabolites. remarkably, it was found in one of nine patients in this study (morichi et al., ) . in the second type of encephalitis, a high increase of several cytokines at systemic levels-which also affect other organs-can be detected. these were reported in only one of the patients (morichi et al., ) . in five of the total of patients, excitotoxic encephalopathy was found, which is characterized by febrile convulsion status epilepticus (mizuguchi et al., ; morichi et al., ) . two patients manifested encephalopathies associated with hypoxia, which is a condition that does not include any sign of the others classifications (morichi et al., ) . importantly, hrsv rna was found in csf in five of the nine patients analyzed, and the levels of il- were increased only in the patients who exhibited excitotoxic or cytokine storm encephalopathy type (morichi et al., ; figure ) . moreover, in all the patients the levels of nitric oxide (no) were significantly increased independently of the encephalopathy type (morichi et al., ) . these results are consistent with the previous report of these authors where they described the finding of hrsv rna in five of eight patients and also elevated no levels when compared to influenzaassociated encephalopathies (morichi et al., ). despite the low frequency of neurological complications associated with hrsv-infection, the cases reported exhibit similar profiles, which considers elevated levels of il- in csf (miyamoto et al., ) . related to this, morichi et al. ( ) examined molecular markers in csf as a prognostic indicator of encephalopathy severity -which includes no, brain-derived neurotrophic factor (bdnf) and il- ( figure ) . the analysis of these molecular markers was categorized by the encephalopathy type described earlier, in addition to the non-evaluated encephalopathy type (morichi et al., ) . although the authors analyzed a low number of cases, they found that in two patients with cytokine storm encephalopathy, il- and bdnf were significantly elevated, when compared to the control group (morichi et al., ) . moreover, in four patients with non-evaluated encephalopathy, the levels of no were significantly higher than in the control group (morichi et al., ) . the pediatric cerebral performance category scale (pcpc) score was used in order to correlate these results to the neurologic prognosis. only il- and bdnf correlated with pcpc scores, indicating that in more damaged patients, the secretion of these molecules is elevated (morichi et al., ) . these tools are nowadays an import advantage in the understanding of the neuropathies associated with hrsv-infection and help to prevent that severe cases lead to death, as was described recently (xu et al., ) . nowadays, the mechanisms involved in neurological complications due to hrsv-infection remains unknown. decades ago, researchers adapted the hrsv long to the brain of newborn mice to study the pathogenesis of this virus in a mice model (cavallaro and maassab, ; cavallaro et al., ) . these authors inoculated the virus intracranially several times and reported that animals exhibited clinical signs of lethargy, ataxia and tremors between the rd and th-day post inoculation (cavallaro et al., ) . interestingly, the authors also observed that in a few cases, mice manifested convulsions spontaneously and also died after one or days (cavallaro et al., ) . in addition to this, histological analyses showed an association between the extensive necrosis and the liquefaction in the brain with the clinical signs of encephalitis in the mice (cavallaro et al., ) . by intracranial inoculation, the authors described that hrsv was not found in others organs and that mice did not exhibit a pulmonary disease (cavallaro et al., ) . during years, there was no report about the relationship between hrsv-infections and cns pathologies. a few years ago, li et al. ( ) described, in a study that sought to assess the persistence of infection, the ability of the virus to infect sensory neurons that innervate the lung. these authors hypothesized that hrsv infects not only pulmonary neurons but also that the g-hrsv glycoprotein can interact with the chemokine receptor for cx cl (cx cr ) expressed in these cells (li et al., ) . according to this, they also studied the ability of hrsv to infect primary cortical neuronal cultures and observed, by immunofluorescence, co-localization of n-hrsv protein with neuronal markers. remarkably, this was not observed when the cx cr was blockade. these results suggest that hrsv can infect neurons in vitro at a low percentage ( %) and that it can also infect sensory neurons of the lungs, as reported in culture (li et al., ) . this work highlights the fact that hrsv can invade the cns and infects resident cells which may explain how this virus can cause neurological abnormalities in patients. to give more insights about this phenomenon, espinoza et al. ( ) evaluated the neuro-invasive ability of hrsv in a mice model performing an intranasal inoculation that differs in the methodology used in the s. importantly, the authors observed that viral genome and proteins could be detected in the brain of the infected mice, mainly in cortex, hippocampus and ventromedial hypothalamic nucleus (vmh) at days post-infection (espinoza et al., ) . later they evaluated a possible route of entry for hrsv into the brain-the trojan horse mechanism-by using a blocking antibody for cd d, which is expressed by leukocytes and is required for transendothelial transmigration of this cells (espinoza et al., ; figure ). the results obtained showed a decrease of viral load in the brain of hrsv-infected mice previously treated with anti-cd d, suggesting that this can be the entry route used by hrsv (espinoza et al., ) . interestingly, as hrsv proteins were found in the hippocampus-a central zone where the cognitive and behavioral process takes place-alterations in the normal function were evaluated. both hrsv-infected mice and rats were used for the evaluation of behavior and spatial learning, respectively (espinoza et al., ) . marble burying test was used to evaluate the mechanical digging behavior in rodents, and the data showed that a month after hrsv-infection, these mice exhibited an impairment in this behavior (espinoza et al., ) . moreover, hrsv-infected rats were submitted to the morris water maze test-which evaluates spatial learning-a month after the infection. the data shows that hrsv-infected rats exhibited a delay in their learning capacities, when compared to the control group (espinoza et al., ) . considering these results, it is suggested that hrsv-infection causes behavioral and cognitive sequelae, that have not been described yet in patients. recently, an in vitro study using neuronal n a cells as hrsv-infection model-which are a neuroblastoma cell line that can differentiate into cells that possess neuronal characteristic-was performed (yuan et al., ) . the data presented by the authors indicated that this virus infects these cells and that viral titers increased up until h post-infection, suggesting that hrsv replicates in this cell line (yuan et al., ; figure ) . additionally, they evaluated if toll-like receptor (tlr ) and nucleolin (c ) are able to recognize the f-hrsv protein in n a cells, as was reported in the literature. using confocal microscopy, they found that this interaction also occurs in the hrsv-infected cells (yuan et al., ) . according to this, they also observed that hrsv-infection increases the protein levels of tlr and c in n a cells (yuan et al., ) . to evaluate the contribution of infected neurons, in encephalopathies associated with hrsv-infection, the secretion of pro-inflammatory cytokines in the supernatant of n a-hrsv infected cells was assessed by elisa. the data obtained showed an increase of il- and tnf-α in n a hrsv-infected cells when compared to the control cells (yuan et al., ) . despite this new knowledge, there is no in vivo evidence that shows whether hrsv infects neurons or other resident cells. more research in this field is required to achieve a better understanding of the mechanism involved in the cns disease induced by hrsvinfection. iv is the etiological viral agent most relevant in respiratory tract infections. the influenza a (iav), b (ibv), c (icv) and d (idv) viruses belong to the orthomyxoviridae family and are the only members of their respective genus, within the unassigned order (bouvier and palese, ; resa-infante et al., ; su et al., ; king et al., ) . these viruses are enveloped, negative-sense, segmented-stranded rna and the subtypes of iav are determined by two structural proteins, hemagglutinin (ha) and neuraminidase (na; louten, ; su et al., ) . there are different ha subtypes (h -h ) described and at least subtypes of na (n -n ; louten, ) . based on this, any combination of ha and na proteins could be found, being relevant in human diseases the h , h and h which are transmitted between individuals (louten, ) . in addition to this, when iav from animals infects naïve human individuals, antigenic shift-a process in which the re-assortment of genes segments from two subtypes of virus-takes place, sometimes leading to iv epidemics, such as the recent h n epidemic (jang et al., ; louten, ; su et al., ) . the circulating iav that are more risk to human health are h n , h n , h n and h n , along with ibv (louten, ; skowronski et al., ) . associations to respiratory pathologies in iv infections have been described, with neurological complications in both children and adults (goenka et al., ; popescu et al., ; paksu et al., ) . accordingly, clinical signs that have been observed in patients includes encephalitis (newland et al., ) , myelitis (salonen et al., ; zlateva and van ranst, ; xia et al., ; ruisanchez-nieva et al., ) , meningitis (liang et al., ) , seizures (chiu et al., ) and guillain-barre syndrome (sivadon-tardy et al., ) . one of the first reports related to the pandemic h n infection in . therein, the authors describe that the main neurological symptoms were detected in the nerve centers and-time after the infection-manifestations such as depression and neuritis appeared (turner, ) . considering this background, years later a study of h n (asian influenza) showed that neurological complications incidence increased, when compared to h n pandemics and remarkably iav was isolated post-mortem from the brain of a patient (kapila et al., ) . a posterior retrospective study revealed neuromuscular manifestations in % of the patients, with a wide range of clinical signs spectrum (paisley et al., ) . moreover, iv was detected in the csf of one patient from this study (paisley et al., ) . years ago, the detection of iv in csf from patients with neurological manifestations was rare. nowadays, there is more and more evidence of the ability of iv to develop neurological damage. in a new h n pandemic was the causative agent of high mortality rates and exhibited increased reports of neurological complications. according to this, a retrospective study of the clinical files of patients infected with h n detected a % of visible neurological symptoms (asadi-pooya et al., ) . in this cohort, the most frequent neurological sign reported was headache − % of the patients-and a few were diagnosed with severe neurological complication − % of the patients (asadi-pooya et al., ) . another study performed in malaysia-that collected clinical data from pediatric hospitals during the pandemic-reported that . % of children under years old presented neurological manifestation, among which the . % of them manifested febrile seizures (muhammad ismail et al., ) . importantly, . % and . % of children exhibited influenza-associated encephalitis and acute necrotizing encephalopathy (ane), respectively (muhammad ismail et al., ) . however, there was no detection of iav genetic material in the four csf samples available, whereas brainimaging showed that a few patients exhibited alterations such as cerebral edema and ane, besides the three cases where the neurological sequelae were permanent (muhammad ismail et al., ) . according to this information, landau et al. ( ) described that in a cohort of hospitalized children, % of them presented neurologic complications mainly seizures. only one patient from this study was diagnosed with transverse myelitis and presented permanent sequelae (landau et al., ) . in addition to this, a fatal case attributed to the h n pandemic infection was reported, and the clinical finding showed that the cause of death was an intracerebral thrombosis and hemorrhage with presence of the virus in the brain, but not in lungs or csf (simon et al., ; figure ) . the knowledge not only came from epidemic iav, as these neurological signs also have been described for seasonal iav. the h n and h n seasonal iav have also been associated with neurological manifestations. according to this, a study described in patients of a wide range of age with neurological alterations showed that the primary clinical sign was encephalitis and about % of the patients have sequelae (steininger et al., ) . moreover, although in this study the detection methodology for iv genetic material detection in csf was improved, only one sample was positive (steininger et al., ; figure ). in another approximation to understand the etiologic agent causing myelopathy post-influenza-like syndrome, csf obtained from a patient with this disease was inoculated in several cell lines, previously reported to be permissive for the growth figure | influenza virus (iv) spreads from the lungs to the cns through the vagus nerve promoting an inflammatory state. upon infection of iv, the virus reaches the lungs and, from there, it can spread into the cns by transneural route, through the vagus nerve. once set on the brain, it induces the secretion of several pro-inflammatory cytokines such as il- , il- , il- and g-csf. viral rna has been detected in csf of infected patients, and also microglial apoptosis has been described. of this virus (paiva et al., ) . importantly, seasonal iav h n was detected in mdck cells, identified as the cause of the neurological symptoms of the patient (paiva et al., ) . besides the main symptoms reported for seasonal h n , h n and ibv, altered state of consciousness are consistently detected with seizures, in patients infected with pandemics iav subtypes (newland et al., ; popescu et al., ; paksu et al., ) . both in this and in others studies, it has been difficult to correlate clinical signs such as csf pleocytosis with neurological manifestation; mainly due to the low number of patients that exhibit pleocytosis (paksu et al., ) . however, the presence of neuroradiological diagnosis suggests that the phenotype observed in patients may be a cause of direct viral invasion into the cns (paiva et al., ; paksu et al., ) . besides the neurological signs described above, a study in japan reveals that patients with neurological manifestations also had mild impairment of consciousness, typically delirium or hallucinations and abnormal behavior among others, which belongs to the neuropsychiatric disorders (manjunatha et al., ; mizuguchi, ) . accordingly, there is still necessary to perform more studies about the incidence of these neuropsychiatric manifestations, besides the direct evidence of this association with iv infection. highly pathogenic strains of iav have been used as a model to test the mechanisms involved in the cns abnormalities caused by iv-infection in humans. using an h n iav that initially originated from a water bird and was adapted into chickens to increase its virulence, shinya et al. ( ) inoculated mice intranasally and evaluate its neurovirulence. the histological data collected exhibited that iav caused non-suppurative encephalitis. remarkably, they could also recover iav from the brain until day post-infection (shinya et al., ) . years later, this group performed experiments using the same virus to elucidate the route used for entry into the cns. mice were infected intranasally or intravenously, and only the first group exhibited bronchitis and viral detection in the mucosal epithelium of the trachea to the bronchiole (shinya et al., ) . later, when they evaluated the brain histology, they observed that in addition to the non-suppurative encephalitis, there was an infiltration of macrophages and lymphocytes (shinya et al., ) . finally, the most critical finding was that viral antigens were detected in the vagal and trigeminal ganglia at day post-infection (figure ) . this event was preceded by the early infection of the nasal cavity, trachea and lungs (shinya et al., ) . additionally, the hypothesis of the transneural invasion was corroborated in a study performed by matsuda et al. ( ) , which showed that iav reaches cns mainly via the vagus nerve (figure ) . moreover, through an in vitro assay-utilizing neuron cultures in a compartmentalized system-authors suggested that the mechanism of the neurotropic h n to reach cns is a retrograde axonal transport (matsuda et al., ) . another research using the h n iav (hong kong/ / ) also found that both rna and viral antigens were detected, first in the vagal and trigeminal ganglia, then later in the brainstem (park et al., ) . the next step in the research was to evaluate in more detail the effects of iv in the brain of challenged mice. in this context, jang et al. ( jang et al. ( , observed that mice challenged intranasally with h n (vietnam/ / ), viral detection in cns was positive at days post-infection and that the virus can infect neurons and microglia but not astrocytes (figure ) . in addition to this, they report that h n infection promotes microglial apoptosis, inducing an inflammatory state, which lasts up to days post-infection, similarly to the idiopathic parkinson's disease in human (jang et al., ) . moreover, the authors show a loss in dopaminergic neurons in about a %, that began as a local immune response that could contribute to cns disease as is described in humans (jang et al., ) . later research demonstrated that the recovery of the neuronal lasted until days post-infection and that, mainly in the substantia nigra pars compacta (snpc), the profile of cytokines was altered due to the h n infection (jang et al., ) . interestingly, il- showed an early induction in the acute phase of the viral infection and then decreased rapidly, to eventually increase after days post-infection (jang et al., ) . moreover, gm-csf, another cytokine, was not detected in the acute infection, but increased at the same time as il- increased, whereas cytokines such an il- , il- and g-csf, among others, were only detected until days post-infection (jang et al., ; figure ). all the data suggest that the local immune, response mediated mainly by microglia, promotes neurons death and protein aggregation, inducing the development of neurodegenerative diseases (jang et al., ) . this phenomenon was also observed for h n (ca/ ), which promotes the microglial activation mainly in the snpc and the hippocampal dentate gyrus, however, this virus is not neurotropic (sadasivan et al., ) . additionally, this virus is not able to induce the disruption of bbb which is consistent with the absence of immune cells infiltration into the cns (sadasivan et al., ) . the iv infection has also been related to neuropsychiatric disorders. yu et al. ( ) used the neonatal model to evaluate whether iv infection might cause alterations in normal brain functions. unlike other studies described above, in this one, iv was administered intraperitoneal, as the primary focus of the research was to evaluate the systemic spread of the mouseadapted h n (nws/ ; yu et al., ) . the results showed viral detection in the hippocampus, cerebellum and cerebral cortex among other zones. in addition to this, in infected brain zones, neurons and astrocytes underwent apoptosis, which is consistent with neuroinflammation accompanied by gliosis (yu et al., ) . moreover, viral rna was detected in csf from adult mice, but this does not discard the possibility that in neonates, this also occurs and that iav furthermore invades the cns by crossing the blood-csf barrier (yu et al., ) . according to these findings, specifically the detection of the viral rna in the hippocampus, hosseini et al. ( ) recently evaluated three different mouse-adapted iavs: two non-neurotropic virus h n (pr ) and h n (mahk ); one neurotropic virus h n (rsc m). as they expected, no viral particles were found in the brain of h n -infected mice, although a few amounts of viral particles were found in h n -infected mice and viral detection was evident in several brain zones in h n -infected mice (hosseini et al., ) . therefore, no signs of pathological changes were detected in the brain of h n -and h n -infected mice, but in the h n -infected mice, there was a moderate immune cell infiltration and zones with gliosis (hosseini et al., ) . interestingly, when they evaluated the effects of iav infections in the behavior, no virus has affected the mice anxiety or locomotor activity whereas at and days postinfection, however, h n and h n -infected mice showed an impairment of spatial learning and memory (hosseini et al., ) . this work proves that, unlike what is reported by jurgens et al. ( ) -which perform cognitive test and neuron morphology experiments during the acute phase of the infection-the h n infection does not lead to long-term impairment in spatial memory nor affects the neuron morphology (hosseini et al., ) . the h n subtype is not able to replicate in the cns; however, it is capable of increasing the levels of tnf-α in the hippocampus and also increase the number of microglia (hosseini et al., ; figure ). on the other hand, h n not only increases the level of ifn-γ and tnf-α in the cns but also alters the long-term potentiation (ltp) and disrupts the permeability of the bbb, promoting a stronger inflammatory immune response than h n (hosseini et al., ) . based on all these data, the most important conclusion is that it is necessary to know the immune response promoted by ivs, as it has been proven that long-term alteration can be caused without cns viral replication. importantly, all the knowledge that we have today about iv infection allow us to be more prepared to diagnose and treat more efficiently patients with this infection. cov is a group of viruses that belong to the coronaviridae family and the nidovirales order. accordingly, there are four genera of cov within the coronavirinae subfamily: alphacov (acov), betacov (bcov), deltacov (dcov) and gammacov (gcov; king et al., king et al., , . their name proceeds from their characteristic crown-shape and is responsible for a wide range of respiratory and enteric diseases in several hosts, such as rodents, cats, pigs and humans (desforges et al., b) . there are several human cov (hcov) described as pathogenic in humans, among which are included hcov-oc , hcov- e, middle east respiratory syndrome cov (mers-cov) and severe acute respiratory syndrome cov (sars-cov), all of them with their respective different genotypes (gaunt et al., ; cabeça et al., ; matoba et al., ) . remarkably, neurotropic and neuro-invasive capabilities have been described in several of their hosts, including humans among them, leading to symptoms such as multiple sclerosis (ms) and encephalomyelitis (lau et al., ; yeh et al., ; zlateva and van ranst, ; talbot and falsey, ) . however, the capacity of cov to infect cns in humans is not well characterized, with their detection in these samples performed mainly by detection of viral rna, exhibiting persistent infection (arbour et al., ; desforges et al., a) . covs are enveloped viruses with a positive non-segmented single-stranded rna genome of about kb of length, one of the largest among the rna viruses. they codify for four structural proteins-five in the case of some bcov-and several non-structural proteins comprised mainly on two orfs (orf a and orf b) that will eventually be cleaved into or proteins (desforges et al., b) . it has been described that non-structural proteins are the leading cause of host immune system modulation and they also play a role in the replication of the genetic material of the virus (gorbalenya et al., ) . as described in mice, viral entry is mediated through the interaction of viral spike (s) protein and cellular cecam- receptor, along with other co-receptors (williams et al., ; bergmann et al., ) . from there, the virus can replicate its rna and translate it into proteins. among the cells that are permissive to mhv infection are macrophages, microglia and astrocytes (bergmann et al., ; jacomy et al., ) . remarkably, the year st-jean et al. ( described the recovery of an infectious hcov-oc with neurovirulent capacities from a full-length cdna clone inserted in a bac, with the same phenotype as a wt virus, generating an interesting methodological approach for the study of this virus. despite hcov capacities to infect cns, it has been recently characterized, its presence in human cns-related samples date back as early as , where the first detection of this virus was performed in autopsy of patients with ms (burks et al., ) . following that, a few reports confirming the presence of this virus in samples from patients with ms was confirmed through several methods (murray et al., ; stewart et al., ) . the year , through research in an autopsy samples from patients with various neurological diseases (being ms most prevalent among them). showed that a % were positive for hcov (with hcov- e being twice as common as hcov-oc ; arbour et al., ) . moreover, the prevalence of oc in ms samples was statistically higher than in control patients, is the first report to provide a significant indication of the neurotropic capacity of these respiratory pathogens (arbour et al., ) . the first case of sars-cov infection with neurological manifestations was reported the year in a -year-old woman (hung et al., ) . she was first admitted with swinging fever, chills, productive coughing and diarrhea, which eventually lead to oxygen requirements, vomit, seizures and episodes of four-limb twitching. the respiratory failure continued until she was sedated, and ventilation was required (hung et al., ) . sars-cov infection was confirmed in both tracheal aspirates and csf samples, followed by ribavirin treatment, with no improvement in seizures persistence. with additional treatments, seizures were no longer detected, and she was discharged weeks after admission (hung et al., ) . the following year, another case of sars-cov infection with detection of genetic material in csf samples was reported in a -year-old woman (lau et al., ; figure ) . detection was also positive for stool specimens and peritoneal fluids. figure | human coronavirus (hcov) enters the cns through the olfactory bulb, causing inflammation and demyelination. upon nasal infection, hcov can reach the cns through the olfactory bulb, as ablation of this part of the brain restricts its neurotropic capacities in mice. once the infection is set, the virus can reach the whole brain and csf in less than days. accordingly, it has been described that this virus can induce demyelination. likewise, primary glial cultures have been described to secrete il- , il- p , il- , tnf-α, cxcl and cxcl upon viral infection. the patient was admitted in week of pregnancy and at days post-admission, mechanical ventilation was required. at day , she presents a sign of acute renal failure, and pregnancy termination was decided. through cesarean, a baby girl was born without further complications (lau et al., ) . at day the patient was still sedated and on mechanical ventilation, with convulsions and loss of consciousness. starting at day she was extubated and from there followed an uneventful recovery. she exhibited no further convulsions and no evident sequelae (lau et al., ) . organ dissemination of sars-cov in autopsy samples from patients that died of this disease was determined. the report indicates the presence of sars-cov-n protein and viral rna in the stomach, small intestine, kidney, sweat glands, parathyroid, pituitary gland, liver and cerebrum, further confirming the capacity of this virus to induce a systemic infection (ding et al., ) . a case report of hcov-oc detection in nasopharyngeal and csf samples from a child patient was performed the year (yeh et al., ) . the child exhibited acute disseminated encephalomyelitis, a low-prevalence cns disease that induces demyelination, being this the first case related with hcov. no other infectious agents were detected in any of the samples (yeh et al., ) . following this, a brief characterization of the cytokine profile in the cns, induced by sars-covinfection, was published. therein, the authors indicated that both chemokine induced by ifn-γ (cxcl , a cxc chemokines family member) and ifn-γ-inducible protein (cxcl ) were highly induced in brain samples from a deceased patient (xu et al., ; figure ) . however, there are reports about high levels of cxcl in sars-cov infected patients, with no neurological manifestations. therefore the authors suggest that cxcl could be closely related to cns infection (xu et al., ) . remarkably, they also showed there that peripheral blood lymphocytes and eosinophils counts in cns-hcov-infected patients were lower when compared with respiratory-hcov-infected patients, while the opposite trend was observed for neutrophils (li et al., ) . these differences in the recruitment of immune cells could be related to the immune response elicited by the virus, either it is respiratory-restricted or exhibits neurotropism capabilities (li et al., ) . hcov capacity to reach cns after the nasal infection has been described previously in mice, particularly for hcov-oc (st-jean et al., ) . st-jean et al. ( ) reported that upon infection, viral antigens are detected in the olfactory bulb days later, with no presence of virus in perivascular blood cells or any other part of the brain. after days, the virus is detected throughout the whole brain tissue, indicating that it can rapidly propagate once set in cns. this replication leads to a rapid death by acute encephalitis of infected mice. remarkably, ablation of the olfactory bulb prevented the spread of mouse hepatitis virus (mhv), upon nasal infection (perlman et al., ) . therefore, hcov exhibits an intrinsic capability to infect neural cells and spread from cns to the periphery via a transneural route, as has also been seen for mhv (perlman et al., ; barthold et al., ; figure ) . mice studies are mainly performed with mhv, a virus that belongs to the bcov genus and is genetically related to hcov-oc ; likewise, the disease at cns as elucidated by both viruses are similar, as they both induce demyelination (jacomy and talbot, ; bergmann et al., ; figure ). jacomy and talbot ( ) were among the first to describe a mouse model to characterize the cns disease in their publication the year . therein, they exhibit that balb/c and c bl/ mice could be infected through nasal instillation with mhv, although they chose to use intracerebral inoculation to favor cns infection (jacomy and talbot, ) . they also determined that viral rna could be detected in brain, heart, spleen, lungs, liver and muscles (jacomy and talbot, ) . likewise, the year glass et al. ( ) described a systemic non-lethal model of infection for sars-cov in c bl/ mice that eventually reached the brain. finally, in jacomy et al. ( ) described that hcov-oc could infect glial and neuronal cells of both rat and mice (figure ) . therein, they also showed that surviving animals exhibited decreased motor functions. recently, wheeler et al. ( ) described that microglia is essential for the regulation of mhv infection, as depletion of this cell type led to faster viral replication, enhancing its capacity to avoid adaptive immunity. according to this, glial primary cultures of mhv-a -infected cells showed an increase in the secretion of il- p , tnf-α, il- and il- compared with a non-neurotropic mhv, suggesting that the infection with a neurotropic virus activates glial cells and induces a pro-inflammatory state (li et al., ; figure ) . as described so far, covs are respiratory viruses that exhibit neurotropic capacities that not only allows them to achieve latency and avoid the immune response of the host, but also have neurological implications that can complicate the disease associated to its infection. although their mechanisms and routes to reach the cns have not been elicited yet, the detection of either viral proteins or genetic material in this issue has been confirmed thoroughly, branching the researcher's goals into acquiring new insights regarding this topic. so far, epidemiological reports have allowed to achieve this, but further work in animal models is required to fully comprehend the mechanisms that cov uses to reach cns and to achieve more suitable treatments to resolve this viral infection without an exacerbated disease. the hmpv is a new virus first reported the year in netherlands (van den hoogen et al., ) , and since its discovery, several epidemiological reports have placed it among the most prevalent respiratory viruses worldwide, although its disease burden has not been thoroughly characterized (hamelin et al., ; edwards et al., ) . it is responsible for respiratory illness mainly in newborns, infants and immunocompromised patients, although it can also infect healthy adults, with mild symptoms (edwards et al., ) . its genome is negative sense and about kb length, with nine structural proteins codified on it (van den hoogen et al., ; schildgen et al., ) . hmpv belongs to the mononegavirales order, pneumoviridae family and the metapneumovirus genus (amarasinghe et al., ; king et al., ) . since it is closely related to hrsv, both in its classification and its genome, their diagnosis has been usually mistaken (edwards et al., ) . currently, there are no effective treatments against this virus, neither vaccines nor specifics treatments, mainly due to the lack of thorough knowledge associated with its immune response and disease pathogenesis (zlateva and van ranst, ; schildgen et al., ) . in humans, there is a handful of reports associated with encephalitis and hmpv-infections. the first approach to this topic was a report described the year in china (peiris et al., ) . therein, the authors describe several cases of children with acute respiratory diseases, particularly patients, of which were positive for hmpv rna detection (peiris et al., ) . although the scope of this report was not to associate hmpv with any cns abnormality, they do report five cases of children with a febrile seizure, reaching levels comparable to the ones seen in influenza, the respiratory viruses most prone to induce seizures, as they state (peiris et al., ) . following this, the first case in which the presence of hmpv rna in brain samples of a patient with encephalitis was reported, was the year in germany (schildgen et al., ) . this report described the case of a -month-old boy that was received in a primary care hospital with a high fever and unresponsiveness to stimulus, either verbal or tactile. previous to its internalization, during the same day he reported seizures and no spontaneous eye movement (schildgen et al., ) . after days of hospitalization without further improvement, the child was considered to be dead and was extubated. autopsies revealed the presence of genetic material of hmpv in both brain and lungs, and no other virus, such as hrsv or hsv, were detected (schildgen et al., ) . concomitantly the same year, a case of encephalitis where hmpv rna was detected in nasal mucus and tracheal aspirates was reported (kaida et al., ) . from this point on, several cases were described where hmpv-infection was related to encephalitis, and in some cases, the detection of genetic material in different cns samples was detected. the year , the death of a -month-old girl was reported days after her admission to a healthcare unit (hata et al., ) . she exhibited generalized convulsions and was diagnosed with acute encephalopathy the day she was admitted, and h later she fell into a coma. although there was no detection of genetic material in csf, the presence of hmpv-f protein was confirmed by rt-pcr in throat swab and urine (hata et al., ) . two years later, arnold et al. ( ) described in an epidemiological report the presence of nine cases of hmpv-infection related to different spectrums of cns abnormalities, distributed in two different study groups. in the first group, composed of patients, samples were hmpv positive, and of those, four patients were reported with seizures. therein, they also expose data regarding hrsv, showing that of the samples were positive for this virus and only one of the patients was reported with seizures. through statistical analysis, they can confirm that the frequency of hmpv patients with seizures is statistically significant, unlike the one has seen in hrsv. the second group consisted solely of patients hospitalized with high fever or any cns abnormality, ranging from the age of -month-old to years old. in that group, they reported five patients with hmpv, where seizures were diagnosed in three (arnold et al., ) . they also described another patient with hmpv-infection and detection of genetic material of enterovirus in csf. remarkably, the presence of hmpv genetic material was not detected in the csf of any of the available samples. despite this, they indicate that a normal csf profile does not necessarily exclude a neurotropic mechanism, as seen for rabies encephalitis, in which only half of the csf samples are positive for csf pleocytosis (arnold et al., ) . likewise, the case reported above by schildgen et al. ( ) identified hmpv rna postmortem, despite normal csf cell count and a negative detection of the virus by pcr in spinal fluid (arnold et al., ). on the same line, the year the first report associated with the hmpv genetic material in csf was published (sánchez fernández et al., ). sánchez fernández et al. ( confirmed this detection through pcr-in a -year-old girl with signs of acute encephalitis-and then characterized the evolution of the disease and also included neuroimaging features therein. detection of other etiological agents, such as herpes virus and adenovirus, were negative. magnetic resonance imaging (mri) showed signs of acute encephalitis and demyelinating process mainly in the temporal and occipital lobes, but these lesions eventually spread to frontal and parietal lobes (sánchez fernández et al., ) . eventually, thanks to the treatment received, clinical improvement was noted and- days post hospitalization-she was discharged home, unlike many other cases, where these symptoms resulted in the death of the child (sánchez fernández et al., ) . however, some of the sequelae registered include inappropriate social abilities and infantile behavior, with severe attention-but no motor or memory-deficits (sánchez fernández et al., ) . more cases in which status epilepticus was reported-a single long-lasting seizure or several seizures in a specific time rangewere associated with hmpv. a . -year-old girl with hmpv respiratory disease was reported the year (niizuma et al., ) . the year , two cases were reported, a -month old and a -month-old girl, that reported hmpv infection and eventually developed respiratory failure (webster et al., ) . these three cases were discharged without apparent sequelae. the year , the first reported case of a child with refractory status epilepticus-a non-responsive condition with a worse prognosis than commonly responsive status epilepticus-was described (vehapoglu et al., ) . the -month-old boy was received and eventually transported to pediatric icu. csf pcr analysis was negative for hsv, and nasal scraps were only positive for hmpv. as stated above, treatment with antiepileptic drugs resulted in unresponsiveness. eventually, seizures passed, and days post-admission she was discharged. no evident sequelae were detected, though antiepileptic drugs were maintained for the following months (vehapoglu et al., ) . most recently, cases of adults with acute encephalitis and hmpv detection have been described. the first report was published the year in australia, in a -year-old man (fok et al., ) . the man was found unconscious after days of respiratory symptoms and immediately hospitalized. pcr testing of csf elicited no presence of classical cns pathogens (hsv, varicella zoster virus, enterovirus) and eventually nasopharyngeal aspirates were positive for hmpv and no other respiratory viruses, although at this point, not enough csf was left for further testing. no evident sequelae were detected during the following months (fok et al., ) . remarkably, lesions found in the mri analysis were similar to the ones described in the previously reported cases (schildgen et al., ; sánchez fernández et al., ) . then the year , two reports-one associating hmpv respiratory infection, the other one indicating the presence of hmpv genetic material in csf-were published. early during that year, jeannet et al. ( ) described the case of a -year-old swiss man with influenza-like symptoms, which eventually suffered from a headache and seizures. mri was inconclusive, and pcr analysis was negative for csf samples, although nasopharyngeal swabs were positive for hmpv and no other respiratory virus (jeannet et al., ) . then, later that year, the case of a -year-old man was reported, although the case dated from years before its publication. the man was admitted with unspecific low backache and fever, mri was normal, and no unusual symptoms were detected (tan and wee, ) . eventually, the patient de-saturated and respiratory failure was diagnosed, with posterior mechanical ventilation. bronchoalveolar lavages and csf pcrs were both positive for hmpv (tan and wee, ) . the patient was treated for week with ribavirin (unspecific antiviral) and eventually respiratory symptoms subdued, although he exhibited reduced cognition and intermittent agitation as sequelae. he began rehabilitation, but there was not a significant improvement throughout the following months (tan and wee, ) . remarkably, no studies have been performed describing the ability of hmpv to cause cns damage in mice. however, it has been described that hmpv can persist in the lung of mice after acute infection (alvarez et al., ; liu et al., ). this characteristic could be aiding the virus to eventually reach the cns, as the latency state is achieved through the infection of neuronal processes that innervate the lungs, as the genetic material of the virus can be detected in them (liu et al., ). moreover, as described by the authors, latent virus can be reactivated upon treatment with immunosuppressive drugs such as steroids. hmpv remains a viral agent that needs to be thoroughly characterized, as although most of its respiratory-related pathogenesis has been taken to spotlight, its capacity to achieve latency in cns cells is yet to be elicited. although there are extensive case reports that indicate neurological manifestations associated to hmpv-infection in humans, further studies are required in mice models to characterize this disease. considering that this virus was first described only years ago, there is plenty of work to be done in order to fully comprehend the impact that this virus may be playing in cns-related pathologies. respiratory viruses are the leading cause of bronchiolitis and pneumonia throughout the world, affecting varying ranges of ages, but being more aggressive in children, elderly and immunocompromised individuals. most prominent respiratory viruses are hrsv, iv, cov and hmpv. remarkably, extrapulmonary symptoms have been described in these viruses, highlighting their capacity to cause neurological complications. febrile seizures, loss of consciousness, convulsion, ataxia, status epilepticus, encephalitis, myelitis, neuritis and ms are among the several extra-pulmonary symptoms that have been described. case reports of children, elderly and even adults exhibiting these symptoms have been described throughout the years for all of these viruses, bringing to spotlight the urgency to describe their neuroinvasive capacity. moreover, the detection of genetic material and even viral proteins in cns samples, such as csf or brain, is a recurrent fact described in several case reports. several ways to achieve cns have been described, including transneural and hematogenous pathways. however, the specific mechanisms responsible for dissemination of each of these viruses with neurotropic capacities into the cns, have not been thoroughly characterized yet. for instance, hrsv has been reported to reach cns via the hematogenous pathway, although other routes cannot be discarded entirely. iv can reach the brain via the transneural route, mainly by retrograde axonal transport, reaching first vagal and trigeminal nerves. likewise, cov has been described to reach the brain via olfactory bulb, spreading from this point onward into the cns and the periphery. since hmpv is 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( ) ; salonen et al. ( ) we want to thank trinidad celis for her help in the design of the figures. key: cord- - zaoxn authors: nan title: publication only date: - - journal: bone marrow transplant doi: . /sj.bmt. sha: doc_id: cord_uid: zaoxn nan ischemic myocardial damage is an increasing cause of heart failure in the western world and has long been considered irreversible because adult cardiomyocytes are terminally differentiated and do not proliferate. stem cells are undifferentiated cells capable of self-renewal, proliferation, and differentiation into multiple lineages permitting tissue regeneration. a number of types of stem cells are now recognised, as well as partially differentiated progenitor cells that are capable of proliferation and differentiation to multiple lineages. reversal of heart failure would require myocardial revascularization, remodelling of the left ventricle and replacement of damaged myocyte. we investigated the safety of transplanting un-manipulated autologous bone marrow into infracted myocardium in two patients. these patients underwent coronary bypass using the pi-circuit technique and external reshaping of left ventricle in off-pump surgery. we evaluated the efficacy of this combined technique in the improvement of cardiac function. autologous bone marrow ( ml) was obtained by bilateral posterior iliac bone aspiration at the time of surgery. bone marrow mononuclear cells were isolated by means of a density ficoll-paque gradient. then the cells were exhaustively washed and resuspended in a normal saline solution containing % human serum albumin. cell count, viability and cultures were appropriately performed. following the operation the bone marrow mononuclear cells ( ml) were injected directly to the myocardium of the left ventricle. no significant complications were observed. the left ventricular ejection fraction at rest was improved significantly in both patients from and % to and % respectively, three months following the operation. furthermore, we observed significant reduction of the end diastolic volume of the left ventricle and improvement in the inferior-posterior wall motion, this area was not revascularized, in comparison to the previous one before the operation. these findings suggest that transplantation of unmanipulated autologous bone marrow into scar tissue of the human heart is safe and enhances cardiac function, when used in combination with myocardial revascularization and remodelling of the left ventricle. this benefit can be seen after months of the bone marrow transplant and is maintained after months of follow-up. a. symeonidis, m. tiniakou, a. spyridonidis, m. karakantza, e. triantafyllou, a. kouraklis-symeonidis, v. pesli, p. matsouka, n. zoumbos univers of patras med school (patras,gr) the haematopoietic sct unit in patras university was established in , aiming to cover the requirements of the area of peloponese and south-western greece. since then autologous and allogeneic stem-cell transplants have been performed in patients. auto-transplanted patients were male and female (median age , range - years), diagnosed as non-hodgkin's lymphoma (nhl= ), multiple myeloma (mm= ), hodgkin's lymphoma (hl= ), acute myelogenous leukemia (aml= ) chronic myelogenous leukemia (cml= ) and chronic lymphocytic leukemia ( ) . nine mm patients received tandem auto-transplants, while auto-and allo-were second transplants. at transplantation patients were in cr, in pr and had refractory disease. bone marrow (bm) grafts were used in cases and peripheral stem-cell grafts (pbsc) in . patients were conditioned with melphalan ( ), cbv ( ), beam ( ), busulfan-based regimens ( ) and thio-tepa-based regimen ( ). engraftment was achieved in / cases. three patients died early post-transplant. after a median follow-up of months, patients are alive ( %), of them disease-free, and have died, due either to transplant-related complications ( ), or after an early (< year post-transplant) (nhl= , aml= , mm= ), or a late relapse (> years post-transplant) (mm= , hl= , nhl= ). for all allo-transplants the donor was a matched sibling. patients were male and female (median age , range - years), diagnosed with aml ( ), acute lymphoblastic leukemia (all= ), myelodysplastic syndrome (mds= ), aplastic anemia ( ), cml ( ) and myelofibrosis (mf= ). excluding patients with aa, disease status at transplantation was st cr for patients, nd cr for , and active residual disease for four. stem-cell source was bm in and pbsc in . a standard conditioning was used in cases and a reduced intensity one in . engraftment was achieved in all cases. one patient died early, due to uncontrollable vod, and (all= , aml= ) due to disease progression, which was early (< months) in cases, and late (at and months post-transplant) in . five patients developed acute gvhd grade i-ii, and one grade iv. after a median follow-up of months, twelve patients are alive and disease free (aml= , mds= , aa= , cml= , mf= ) but of them have manifested chronic gvhd, (extensive in ). in one patient chronic gvhd emerged after dli, administered for smoldering relapse. activation of plts by collagen, measurement of hypotonic shock response (hsr) and extent of shape change (esc) were tested in addition. in os-pc and c-pc plt yield was . ± . and . ± . x /unit (recovery . % and . %, p= . ), residual erythrocytes were < . x /unit, residual leucocytes < . x /unit in all pc. on d values for po in os-pc and c-pc were . ± . and . ± . mmhg, pco . ± . and . ± . mmhg (p< . ), hco . ± . and . ± . mmol/l (p< . ), respectively. on d values for po in os-pc and c-pc were . ± . and . ± . mmhg, pco . ± . and . ± . mmhg (p< . ), hco . ± . and . ± . mmol/l, respectively. results (mean±sd) of metabolic and activation markers and morphologic features on d and d are shown in the table. in both groups functional parameters revealed a sufficient capacity for aggregation during storage. compared to c-pc os-pc were significantly more efficient in recovery of plts, whereas cd p and cd were significantly higher due to a higher extent of plt activation in the automatic system. a possible difference in clinical outcome of transfusion of os-pc compared to c-pc has to be investigated in further studies. objectives: recently the investigation of differentiation potential of bone marrow stromal cells becomes important because of their implementation in transplantation. but the structure of the stromal cells pool is still not fully known. the aim of the study -to investigate the differentiation ability of bone marrow clonogenic fibroblasts and to elaborate the standart assays for clonogenic proliferation and adipogenic and osteogenic differentiation (ad and od) of bone marrow fibroblasts in children. methods: the material -bone marrow aspirates from healthy donors, patients with acute lymphoblastic leukemia (all) and patients with acute myeloblastic leukemia (aml). bone marrow stromal fibroblasts were cultured accoding a.fridenstain in our modification. od was induced by: b-glycerophosphat x - m; dexametasone x - m; ascorbic acid x - m. for ad were used: dexametasone x - m and insulin x - m. results: optimal conditions for maximum cloning efficiency were following: isolation bone marrow mononuclear cells; density by explantation - x /ml cells; culture medium: medium with % human serum of any blood group. osteogenic induction increased the proportion of fibroblasts colonies from normal bone marrow with alkaline phosphatase activity from , % to , %; the adipogenic inductors increased the proportion of colonies with lipid-rich vacuoles (detected by sudan) from , % to , %. the cloning efficiency of stromal precursors (the number of colonies per x explanted cells) in the patients with aml did not differ from normal donors ( , and , ) and was much lower in all patients ( , ). stromal cells from leukemic patients showed decreased (in comparison with normal donors) potency for od in the presence of inductors (from , % to , % for aml; from , to , for all patients). the ability of stromal cells to ad did not differ in patients with acute leukemias and normal donors. conclusion: standardization of stromal fibroblasts clonogenic cultivation assay is necessary for the evaluation of bone marrow stroma state. the using of osteogenic and adipogenic inductors demonstrate the possibility of in vitro differentiation of stromal progenitors and showed the differences between normal and leukemic stromal cells. initiation of leukapheresis for peripheral blood stem cell collection at a low level of circulating cd + cells l.k. tan, t.g. soh, b. tan, j. mah, t.c. liu, c.s. chen, p. law national university hospital (singapore, sgp) objectives: most patients or donors undergoing leukapheresis (lp) for autologous or allogeneic peripheral blood stem cell (pbsc) collection require multiple lp to achieve a sufficient cd + cell dose (e.g., ≥ . x /kg). lp is initiated when peripheral blood (pb) cd + reached a certain level (e.g., ≥ per microliter). the aim of this retrospective analysis is to summarize our institutional experience of initiating lp at a lower pb cd + cells level of per microliter and to investigate the merits of this practice. methods: all patients or donors underwent lp (using cobe spectra or baxter amicus) processing times the blood volume. a total of procedures ( autologous and allogeneic) was performed in patients or donors between jan and oct . autologous patients were mobilized with chemotherapy and g-csf ( mcg/kg) while allogeneic donors with g-csf ( mcg/kg) alone. a "good" lp is defined as having ≥ x cd + cells/kg in the collection so that a minimum dose of x /kg can be achieved in sessions. cd + cells were measured by sequential gating (staining with antibodies against cd and cd together with forward and side-scattering). results: each lp contained . x wbc/kg (median, range: . - . ) and . x cd + cells/kg (median, range: . - . ). cd + cells/kg in lp were correlated to pb cd + cell counts (r = . ). as shown in table , initiating lp at higher levels of pb cd + cell (≥ per microliter) increased the proportion of "good lp", whether "all" collections or only the first collections were considered. however, a substantial number of "good lp" (≥ %) would be missed if lp was initiated at or cd + cells per microliter in pb (table ), but almost none at cd + cells per microliter. conclusion: the result demonstrated that initiating lp at pb cd + cells per microliter is helpful to some patients /donors. additional criteria may need to be adopted to determine whether lp should be discontinued if mobilization is adequate to minimize resource utilization. g-csf mobilised peripheral blood progenitor cells (pbpc) grafts are widely used in haematopoietic transplantation, but the effect of this cytokine on the cellular content of the graft is not fully understood. apart from the cd +cells, t lymphocytes, nk cells and dendritic cells present in pbpc grafts play a crucial role in the haematopoietic and immune reconstitution following allogeneic transplantation. the aim of this study was to evaluate and compare cell populations present on g-csf mobilised pbpc collected on the th day of mobilisation and on peripheral blood (pb) prior to mobilisation of healthy adult donors with a median age of (range - ). the cell populations studied were t lymphocytes (cd and cd ), nk cells (bright and dim) and dendritic cells (myeloid and lymphoid). total nucleated cells (tnc) were determined by haematological counters, cell phenotypes were evaluated by flow cytometry using colour staining, and each cell subset was determined using specific markers. t lymphocytes were divided in cd +cd + or cd +cd +; nk cells into nkdim (cd -cd +) and nkbright (cd cd ++); dendritic cell were considered lin-and hla-dr++ and then divided into lymphoid (ldc) cd ++ (high) and myeloid (mdc) cd c+. in both pb and pbpc collections t lymphocytes were the main cell population present. with the mobilisation procedure there was a variable fold increase of cell populations (table ), namely fold increase for tnc, while t lymphocyte, dc and nk populations showed , and fold respectively. despite the differences in number, the t lymphocyte, dc and nk sub-populations kept the same ratio. there was a significant correlation between the pb tnc concentration and the fold increase of tnc, cd + cells and nk cells in the pbpc graft. there was no correlation with the increase in dc and any pb parameter evaluated. in summary, g-csf mobilisation while increasing the tnc number (up to fold) does not affect nk, dc and t lymphocytes sub-populations ratio in the grafts from healthy adult donors. further studies are required to determine if the development, maturation and functional activity, namely cytotoxic and immune capacity of these cells are affected by the procedure. toxicity and efficacy of donor lymphocyte infusion after haematopoietic stem cell transplantation s. roncon, m. bini-antunes, f. campilho, i.l. barbosa, a. avila, s. ferreira, h. leal, c. pinho vaz, a. campos, r.b. ferreira, p. pimentel, a. carvalhais instituto portugues de oncologia -crop (porto, p) relapse remains one of the main complications after allogeneic haematopoietic stem cell transplantation (hsct). donor lymphocyte infusion (dli) is a therapeutic approach that is able to mediate antitumour effects and restore prolonged remissions. this antitumour effect has been well established in chronic myeloid leukaemia (cml) and less in other malignant diseases. we retrospectively analysed patients ( f/ m) who relapsed or were at high risk of relapse after hsct between december and september . patients median age was years ( - ). they had the following diagnosis: cml , acute myeloid leukaemia (aml) , acute lymphoid leukaemia (all) , multiple myeloma (mm) , hodgkin disease (hd) , aplastic anemia (aa) , non-hodgkin lymphoma (nhl) , myelodisplastic syndrome (mds) . graft source was g-csf mobilized peripheral blood in patients and bone marrow in . conditioning was myeloablative in patients (in of them graft was t cell depleted in vitro) and of reduced intensity (ric) in the remaining patients. eight patients underwent a nd allogeneic transplantation and of them later received dli. the indications for dli were relapse/disease progression in patients and pre-emptive in . the median interval between transplantation and the first dli was months . the number of dli was one for patients, two for and three for patients. the median dose of cd + lymphocytes infused was x /kg ( , - x /kg). a complete response was observed in % of patients ( / acute leukaemia, / mm, / lmc) and a partial response in ( mm and smd). there was no remission in % of patients ( / hd, / aa, / cml, / la, / mm). in two patients there was no available information. all the five patients relapsed after prophylactic dli. eight patients developed graft versus host disease (gvhd) "de novo" after dli. seven patients with cml did not achieve molecular remission after dli but did it when imatinib mesylate was associated. at the last follow up, patients remained alive and of them established full donor chimerism. in summary, complete remissions induced by dli were inferior to reported in literature. gvhd developed just in an acceptable number of cases and was controlled. further studies need to be performed to evaluate which patients really benefit from dli. report of stem cell transplantation in solid tumours in iran k. alimoghaddam, n. mahdavi, s. samiee, m. jahani, a. mousavi, a. ghavamzadeh horc (tehran, ir) introduction: the prognosis for many pediatric and young adult patients with solid tumors that have metastasized at the time of diagnosis or have relapsed after therapy remains very poor the lack of efficacy of chemotherapeutics, radiotherapy and cytokine-based immunotherapy for many patients with metastatic cancers has catalyzed, at least in part, enthusiasm for exploring allogeneic-based immunotherapy against solid tumors. while there is little doubt about the potential for its application in oncology, extensive use of nonmyeloablative hematopoietic cell transplantation (nmhct) in the treatment of solid tumors will likely remain limited until both the safety and efficacy of the approach are improved. materials and method: we collect the data of patients who had undergone stem cell transplantation by reviewing their records from the date of their transplantation up to the date of last contact. analysis of the data has done via using spss software. results: of all the patients ( . %) were male, and ( . %) were female. the main diagnoses before transplant are brought in the following table. (table- ) the median age was years old; ( - y/o) respectively. ( . %) patients received autologous and ones ( %) received allogeneic stem cell transplantation. ones ( . %) had peripheral blood and ( %) ones had bone marrow as graft type. the median duration of hospitalization for autologous transplanted patients was days which was days for allogeneic transplanted ones. transplant mortality rate in the first days was %. the median follow-up duration was days, with minimum and maximum of and days respectively and during this period the overall survival (os) is . % and the disease free survival rate (dfs) was . %. palifermin for oral mucositis prophylaxis in autologous transplantation n. miranda ( ) , p. santos ( ), t. mendonça ( ) , i. ferreira ( ) , a. guimarães ( ) , j.l. , m. abecasis ( ) ( )instituto português de oncologia (lisbon, p); ( )hospital egas moniz (lisbon, p) objectives: oral mucositis is a near universal complication of intensive chemotherapy. the patient discomfort often require i.v. narcotics and is the main cause for total parenteral nutrition. some authors have associated the duration and the severity of mucositis with transplant outcomes. until very recently no effective treatment or prophylaxis was available. palifermin is the recombinant human keratinocyte growth factor and a previous study have shown its ability to decrease the length and severity of mucositis in conditioning regimens including total body irradiation (tbi). from august to november we have used palifermin for the prophylaxis of mucositis in adult patients (pts) under autologous bmt with conditioning regimens without tbi. methods: nine patients ( females and males) with a median age of . (range - ) have been included in an open label study included in an extended access program. the predominant diagnosis was non hodgkin's lymphoma ( ). the conditioning regimen was beam in , vp + melphalan in and carboplatinum + vp + thyotepa in . palifermin was given according to the manufacturer instructions micrograms/kg body weight on days before chemotherapy and on days starting on day after stem cell infusion. we analysed severity and length of mucositis, narcotic need, haematological reconstitution and side effects of palifermin. we have compared the results with a historical cohort matched for diagnosis, age, conditioning regimen and number of cd + cells transplanted. results: there was only one case of discontinuation of treatment due to toxicity (generalized skin oedema with severe hypotension). severe pruritus was present in pts and generalized rash in all but one patient. a statistical significant increase in weight was observed in pts treated with palifermin on day + (median + kgs versus - kgs without palifermin). the number of days under iv narcotics was lower on palifermin group ( . vs . ) and more patients did not require narcotics at all ( out of nine versus in ). the mean of the number of days without any oral nutrition was lower on palifermin group ( . versus . ). all this differences did not reach statistical significance. the haematological recovery, antibiotic need, bacteriological isolates and length of stay on hospital were equivalent in both groups. conclusion: in our series palifermin decrease the severity and duration of mucositis and was associated with significant skin toxicity. clinical experiences with the new keratinocyte growth factor palifermin in patients treated with high-dose chemotherapy followed by autologous stem cell transplantation m. fillitz, e. koller, e. schlögl, e. pittermann-höcker hanusch-hospital (vienna, a) oral mucositis is a common side effect of chemotherapeutic and/or radiotherapeutic treatment in malignant diseases resulting in pain, diarrhoea, malnutrition, gastrointestinal bleeding complications, local and systemic infection leading to prologation of hospitalization duration and increased medication and treatment costs. this side-effect of cancer treatment has to be expected in up to % of all treated cancer patients and the percentage is even higher in the hematopoietic stem cell transplantation setting. as the functional role of different cytokines such as tumor necrosis factor-alpha and different interleukines and probable protective factors like secretory iga in the pathological pathway leading to mucositis is not yet well understood, neither a widely accepted prophylaxis nor a therapy is available. we report our data of prophylactic application of the recombinant human keratinocyte growth factor palifermin in autologous stem cell transplantation recipients(female ; male ). in cases diagnosis was relapsed non-hodgkin´s lymphoma, patients were suffering from multiple myeloma. the patients age ranged from to years, karnofsky status was % in all. palifermin was given as intravenous bolus injection on consecutive days , third dose at least hours before administration of high dose chemotherapy. second cycle consisted of another doses that were given on consecutive days starting with the day of reinfusion of the stem cell harvest product. the single injection doses ranged from . to . mg according to the producer´s mcg/kg/d recommendation. we experienced only one case of who-grade -mucositis which could not be distinguished from mucosal hyperproliferation due to the medication, all other patients showed no sign of oral mucositis. diarrhoea was maximum who-grade . treatment related side effects consisted of mucosal oedema (vagina, tongue, palate, lids), erythema of the face and upper body,dysphagia and disturbances of taste and sensibility. in one patient excessive proliferation of the oral mucosa lead to detachment from palate and tongue, in another patient mild temporary dyspnoea occurred. altogether those findings were well manageable and resolved without additional measure within days after last injection. we observed no septic event. the hospitalization duration was slightly reduced to former comparable patients even though the first cycle of palifermin led to a days prologation of the pre-transplant phase. prevention of oral mucositis in patients undergoing radiochemotherapy and allogeneic stem cell transplantation with recombinant human keratinocyte growth factor (palifermin): a single-centre experience w. rabitsch, p. kalhs, w. köstler, s. wöhrer, a. schulenburg, v. supper, m. mitterbauer, h. greinix bone marrow transplantation (vienna, a) objectives: oral mucositis is one of several common adverse effects of myeloablative therapy. it is particulary frequent in patients receiving high-dose chemotherapy with or without total-body irradiation for hematopoietic stem cell transplantation (hsct). palifermin is the first agent to be approved for the prevention of oral mucositis induced by myelotoxic therapy. so far, data on ist use in allogeneic hsct are rare. patients and methods: we analyzed the efficacy of palifermin on consecutive patients (median age: , range - years) undergoing allogeneic peripheral blood stem cell transplantation (sibling donor; n= ; unrelated donor, n= ) at our institution. all patients received a conditioning therapy with cyclophosphamide and total body irradiation. palifermin was administered intravenously in a dosage of µg/kg/day days before myeloablative therapy and days after stem cell infusion. mucositis was assessed daily and graded according to the who-scale. results: one patient died on day + after transplantation due to gram-negative sepsis and multi-organ failure. the other patients were eligible for assessment of mucositis. the eligible patients experienced only who grade mucositis. the drug was generally well tolerated without severe side effects. one patient developed erythema on the face, which resolved -hours after palifermin administration without the need of medication. in the other patients no side effects were observed. only patient experience acute graft-versushost disease (gvhd) of the skin grade ii. currently, of patients are alive, , and months after hsct. conclusion: palifermin represents the first drug approved by the us fda for reduction of incidence and duration of oral mucositis in a specific patient cohort. in our cohort of patients we observed no serious side effects and only low grade mucositis. the effect of palifermin on gut epithelia or gvhd incidence and severity has to be assessed in larger patient cohorts. pegfilgrastim in comparison with filgrastim after allogeneic stem cell transplantation c. lutz, g. massenkeil, i. tamm, t. terwey, s. neuburger, b. doerken, r. arnold university hospital charité (berlin, d) purpose: after autologous and allogeneic peripheral blood stem cell transplantation (pbsct) filgrastim (g-csf) is given daily to enhance neutrophil recovery and prevent risk of infection. peg-filgrastim (polyethylen-glykol-g-csf) has been approved in . since the clearance of peg-filgrastim is mediated by neurophils, in neutropenia activity and serum concentration is prolonged and elevated. the aim of this study was to evaluate for the first time the efficacy of a single fixed dose ( mg) in patients after allogeneic pbsct from matched unrelated donors. methods: on day + after allogeneic pbsct five patients ( aml, all, cml; median age y) received mg peg-filgrastim s.c. the neutrophil and platelet recovery was compared to patients (n= ; all, aml, mds, cml, mm; median age , y) treated with daily filgrastim ( µg/kg i.v. over h). definition of neutrophil recovery was ≥ , /nl, platelet recovery ≥ /nl on three consecutive days and without transfusions respectively. g-csf serum levels were measured by elisa (r&d systems). results: peg-filgrastim was well tolerated. in patients treated with peg-filgrastim the neutrophil engraftment was days versus days in the filgrastim group. the platelet recovery in the peg-filgrastim group was days and days in the filgrastim group. these differences were not significant. g-csf levels were measured after the first administration of filgrastim or peg-filgrastim respectively. the results showed a neutrophil mediated clearance of peg-filgrastim with a peak serum level on day + that dropped in parallel to neutrophil recovery. the peak level of peg-filgrastim was about ten fold higher than the peak level of filgrastim ( ng/ml vs ng/ml). conclusions: our data suggest that a fixed dose of mg peg-filgrastim used after allogeneic pbsct is effective and gives comparable results to daily administration of filgrastim. larger prospective studies are needed. use of pegfilgrastim after high-dose melphalan and autologous peripheral blood stem cell transplant in multiple myeloma patients ( ) , p. iacopino ( ) ( )azienda ospedaliera bmm (reggio calabria, i); ( )policlinico universitario (messina, i) single dose of pegfilgrastim is equivalent to daily filgrastim after standard dose chemotherapy in decreasing the duration of neutropenia. daily filgrastim started within - days after autologous peripheral blood stem cell transplant (apbsct) leads to decrease in time to neutrophil engraftment. we undertook a study of pegfilgrastim after high-dose melphalan (hdm) and apbsct. in all, patients with multiple myeloma (stage iii durie-salmon classification), eligible to undergo hdm and apbsct, were enrolled. patients were conditioned with hdm at a dose of mg/m² intravenously on day - .the day of apbsct was termed day . the median stem cell dose infused was x /kg (range - ). patients received a single dose of mg pegfilgrastim subcutaneously h after apbsct. there were no adverse events secondary to pegfilgrastim. neutrophil engraftment was defined as first of consecutive days of an anc equal or greater than . x /l after a previous nadir. platelet engraftment was defined as platelet count of equal or greater than x /l. all patients engrafted neutrophils and platelets with a median of days (range, - ) and days (range, - ), respectively. the incidence of febrile neutropenia was . % ( / ). the median duration of febrile neutropenia was days (range, - ). the mean number of platelet and packed red blood cell transfusions were + . u and . + . u. it's interesting to note that / patients didn't required packed red blood cell transfusion. one patient died for cerebral bleeding after engraftment, on day + ; patients experienced persistent reversible neurophilia. neutrophil and platelet engraftment were compared with a cohort of patients (same diagnoses, method of stem cell collection, conditioning regimen and stem cell dose) treated at our institute, who received filgrastim at mg/kg subcutaneously daily, starting at day + or day + and no statistical difference were shown (p= ns). in conclusion, pegfilgrastim given as a single fixed dose of mg appears to be safe after hdm and apbsct. pegfilgrastim may be convenient to use in outpatient transplant units. the concept of treating chemosensitive leukemia, lymphoma and myeloma patients with high dose chemotherapy followed by reinfusion of peripheral stem cell has been implemented in the set-up of bone marrow transplantation, and often considered as transplantation procedure. with the use of growth factors to stimulate bone marrow and allow the harvest of peripheral stem cells with an apheresis machine haemoneticsr mcs p and cryopreservation, the procedure of autologous peripheral stem cell transplant (apst) became feasible in hematology departments, and not necessarily in bone marrow transplant units. for this reason, we decided eight years ago to set up such a system in our institute. our team included three md hematologists, one nurse and one apheresis technician. we equipped our institute with the necessary separator machine and after a period of ten months trial of investigation of the harvest quality, we started to treat our patients in our institute, instead of referring them to a transplant unit. our institute is a part of a -bed university hospital and covers a population of inhabitants. in this abstract we would like to present our results. using two haemonetics, approximately harvests have been performed with achievement of an excellent yield of cd stem cells (median, . x /kg). the harvest was performed at the day care unit and then cryopreserved in liquid nitrogen. in most patients myeloablative chemotherapy was also given ambulatory. following the reinfusion of the harvest, patients were hospitalized in isolation rooms. the median time of recovery (neutrophils > . x /l) was days. at the end of the year (eight years activity), auto-transplantations were performed, including tandem. the diagnoses were: multiple myeloma in , non-hodgkin's lymphoma , hodgkin's disease , aml , all , amyloidosis and cll cases. treatment related mortality was . %. we believe that such endeavor should be encouraged and advised for more hematology centers. hematologists in training and senior hematologists have the benefit of keeping their patients under close supervision with the challenge of further therapies, increasing the clinical level, the motivation and the interest in the field of hematooncology. h. dimitriou, e. linardakis, g. martimianaki, e. stiakaki, a. fillipidi, m. kalmanti university of crete (heraklion, gr) mesenchymal stem cells (mscs) are multipotent progenitor cells within the bone marrow (bm) capable of differentiating into various tissue specific cells. mscs form an integral part of the bm stroma, have immunomodulatory functions and play an important role in the support of hematopoiesis. their multipotentiality and ease of ex vivo expansion has raised great interest in the clinical use of mscs for tissue repair and gene therapy. in order to evaluate if malignant and non malignant hematological diseases quantitatively and qualitatively affect bm derived mscs, bone marrow from children with acute lymphoblastic leukemia (all diagnosis n= , different phases of treatment n= , end of therapy n= ), idiopathic thrombocytopenic purpura (n= ), autoimmune neutropenia (n= ) and control patients (solid tumors without bm involvement, n= ) was harvested and the mononuclear cell (mnc) fraction isolated. mscs were expanded in amem supplemented with % selected fcs, characterized and compared in terms of their phenotypic characteristics, clonogenicity and ability to differentiate into adipo-(a), osteo-(o) and chondrocytes(c). mncs at day expressed high levels of cd , cd , cd and cd , and very low levels of cd , cd and cd . expression of hematopoietic markers on cells at passage (p ) and thereafter progressively diminished while expression of cd , cd , cd and cd increased approaching %. cell doubling time ranged from to days at all passages. high clonogenicity was observed in all samples at all passages as shown by the presence of cfu-f colonies (> cells) with the exception of all samples at diagnosis which showed impaired proliferation and clonogenicity that returned to normal since remission was achieved at the following phases of treatment till the end of therapy. at p or p , mscs were differentiated towards the a, o, and c lineages by using specific induction media. differentiation was assessed by histochemistry and rt-pcr (lpl and ap for a, osteoprotegerin, osteocalcin and alp for o, aggrecan and col ii for c). p or p mscs from all groups exhibited bi-or trilineage differentiation. preliminary cloning experiments showed that msc population is composed of cells with differing proliferation potential and clonogenicity. these results indicate that blood diseases of childhood do not affect the characteristics of mscs which could have clinical applications particularly in hematopoietic reconstitution following transplantation. pegfilgrastim after autologous stem cell transplantation c.r. rinaldi, c. becchimanzi, a.m. risitano, n. marra, b. rotoli, g. de rosa university federico ii (naples, i) recombinant hemopoietic growth factors are not added routinely after autologous stem cell transplantation (asct) in our institution. in myeloma patients, a multicentric protocol to which we partecipate indicates granulocyte growth factor after re-infusion, at the dose of ug/kg, from day + to hematological recovery. because of equivalence between a single peg-filgrastim (peg) dose and daily filgrastim doses in decreasing the duration of neutropenia after standard dose chemotherapy, we used peg after asct in patients affected by myeloma or lymphoma. from february to november , we enrolled patients, suffering from myeloma ( undergoing single-asct and tandem-asct) and suffering from lymphoma ( non-hodgkin, hodgkin), conditioned by mel and beam, respectively. all patients received a single dose of mg pegfilgrastim subcutaneously h after autologous stem cells infusion. all patients engrafted neutrophils and platelets with a median time of and days, respectively, (regardless the underlying disease and type of conditioning). the incidence of febrile neutropenia was % ( / ) with a median duration of hours. we observed no adverse events secondary to peg injection. no patient had clinically significant mucositis. we compared this cohort of myeloma patients with an historical group of autotransplanted myeloma patients treated by standard daily doses of filgrastim and of lymphoma patients transplanted without g-csf administration. time to neutrophil and platelet recovery was identical in both groups of myeloma patients, and appeared sensibly reduced in lymphoma patients treated with peg as compared to no-g-csf patients (days and in nhls, and in hls) . we conclude that a single dose of pegfilgrastim after asct is safe, well tolerated and accelerates neutrophil recovery, thus decreasing time of hospitalization it seems equivalent to daily dose of filgrastim. we also documented no differences between our cohort of patients and historical groups in order of febrile neutropenia and proved infections. since peg disappearance from the circulation is not due to renal or hepatic clearance, but only to uptake on granulocytic cells and their precursors, this drug can be given even early after stem cells infusion, and will be utilized as soon as the engraftment occurs. mesenchymal stem cells are able to stimulate alloreactive immune cells l. fang, c. lange, m. engel, a.r. zander, b. fehse university medical center eppendorf (hamburg, d) bone marrow-derived mesenchymal stem cells (msc) have been suggested to be "immune-privileged" while exerting a strong immune-modulatory function as "third-party" cells in an hla-independent manner. therefore msc are interesting candidates for cell and gene therapeutic applications. however, a better understanding of the mechanisms underlying their immune-modulatory potential would be very important for msc application in clinical settings. we investigated the interaction of msc with allogeneic immune cells by co-culturing them with un-matched pbmcs and using them as third party in mixed lymphocyte cultures. we present data demonstrating that the immune-privileged state of msc results from an interplay of stimulating and suppressing factors. the directly stimulating activity leads to both active lymphocyte proliferation and secretion of pro-inflammatory cytokines by allo-reactive lymphocytes. stimulation is, however, dominant only at low msc:effector cell (< . under our experimental conditions), but outweighed with higher msc numbers by the suppressing activity. allogeneic mixed lymphocyte cultures as well as msc-mediated stimulatory effects are efficiently suppressed by the addition of mscconditioned medium underlining the important role soluble factors play in msc-mediated immune modulation. in conclusion, based on our data we suggest that the "immuneprivileged status" of msc reflects a sensitive balance of mhcmediated immune activation and the suppression of immunological reactivity largely conferred by soluble factors. circulating endothelial progenitor cells in children with non-malignant diseases following allogeneic bone marrow transplantation m. massa, v. rosti, r. campanelli, e. bonetti, v. meli, d. poddighe, t. mina, d. pagliara, d. lisini, f. locatelli irccs policlinico san matteo (pavia, i) bone marrow-derived endothelial progenitor cells (epcs) circulate in the peripheral blood (pb) of healthy subjects (hs). epcs seem to play an important role in maintaining vessel wall homeostasis, in the neo-angiogenetic processes and in the re-endothelization of the wall of injured vessels. the aim of the study is to assess the number and origin of circulating epcs in children with non-malignant diseases who received allogeneic bmt from an hla-identical sibling or a matched unrelated donor. we studied patients with thalassemia major (n= ), fanconi anemia (n= ), sickle cell disease (n= ), mucopolysaccharidosis (n= ), diskeratosis congenita (n= ), acquired aplastic anemia (n= ), or chediak higashi syndrome (n= ). we evaluated pb samples at , , , , and days after transplant. the number of epcs was evaluated as cd +vegfr- + or cd +cd +vegfr- + cells by cytofluorimetric analysis, and by in vitro culture. the analysis of pb samples from age matched donors (hs) was included in the study. donor or recipient origin of epcs was assessed on at least individually picked endothelial colonies by micro-satellite analysis. in patients tested days after transplant the percentage of circulating cd +vegfr- + cells (median . %, - . ) and the percentage of cd + co-expressing the cd and vegfr- antigens, representing a restricted subset of immature epcs (median . %, - . ), were comparable to those found in hs (median . %, . - . ; median . %, . - . , respectively). the number of epc derived colonies was also comparable in patients tested at days after transplantation (median / mononuclear cells, - ) and in hs (median . / mononuclear cells, - ). neither the percentage of circulating cell subsets, nor the number of epc derived colonies, showed significant modifications during day follow up (by anova test). microsatellite analysis was performed on the epc derived colonies of patients, tested at time points ranging from to months. in patients, all the analysed colonies were of donor origin; in the third patient all the analysed colonies were of patient origin (hematopoietic engraftment donor/recipient %/ %). circulating epcs are detectable in patients given allogeneic bmt from days up to months after transplantation. further studies are needed to definitively conclude their origin and to assess whether their recovery can be correlated to the clinical outcome of the transplanted patients. a. spiropoulos, e. goussetis, m. theodosaki, k. stefanaki, i. peristeri, v. kitra, e. petrakou, s. graphakos "aghia sophia" children's hospital (athens, gr) bone marrow and peripheral blood of adults contain a subset of progenitor cells, which are able to differentiate into mature endothelial cells, thus contributing to re-endothelialization and neo-vascularization. the number of these cells in healthy subjects is rather low; almost . % of total mononuclear cells and a variety of factors may further influence their number. to investigate how allogeneic stem cell transplantation (sct) influence circulating endothelial progenitor cells (cepcs), we obtained peripheral blood samples from transplant recipients at different time points (ranging from months to years after transplantation). peripheral blood mononuclear cells were separated by ficoll density-gradient centrifiguration and were seeded to fibronectin-coated well dishes containing endocult medium (stem cell technologies). in order to remove monocytes and mature endothelial cells, non-adherent cells, at day two of culture, were harvested and further cultured for an additional three days to allow formation of endothelial colonies. the phenotype of the cells that emerged in culture was characterized by immunohistochemistry, and their origin was determined using a polymerase chain reaction (pcr)-based assay for polymorphic short tandem repeats (strs). all samples gave rise to epcs colonies in days. the mean number of epcs colonies/ cells was ± . (range; - ) and it didn't seem to correlate with the post-transplant time. the cultured cells expressed typical endothelial markers such as cd and vwf. for each patient and at all time points, str-pcr analysis showed that cultured cells came exclusively from the donor. these results demonstrate that cepcs are detectable after sct and that their number is independent of post-transplant time. early cd + cells recirculate after autologous peripheral blood stem cell transplant and peg-filgrastim administration for haematological malignancies s. de matteis, n. piccirillo, s. de vita, f. sorà, m. tarnani, l. laurenti, p. chiusolo, g. reddiconto, s. sica, g. leone università cattolica del sacro cuore (rome, i) cd protein is widely accepted as reliable marker for identifying hemopoietic stem or progenitor cells in bone marrow and in peripheral blood. cd + cells represent a heterogeneous cell population consisting of primitive uncommitted and pluripotent progenitors as well as committed stem cell. previous studies showed that these progenitors, mainly myeloid-committed subsets, are detectable in the early phase following infusion of autologous/allogeneic stem cell at day + (albo et al, haematologica ) . based on these findings, we investigated the kinetics of appearance of cd + cells after autologous peripheral blood stem cell transplant (apbsct) and administration of pegfilgrastim mg at day + , and its correlation with haematological engraftment. we studied in consecutive patients (pts), affected by haematological malignancy and treated with apbsct (table ) , the percentage of cd + cell in peripheral blood every other day from day + until patient discharge. these cells were detectable starting from day + after transplantation. the peak of the cd + cells was at day + (range - ), at this day wbc were /mmc (range - /mmc) and the number of cd +/mmc were . (range . - ). there was no correlation between total number of cd +/kg infused and day, absolute number and percentage of cd + peak. statistical analysis demonstrated a significant negative correlation (r- . , p= . ) between age of pts and peak of cd +, while a significant positive correlation (r . , p= . ) between age of pts and day of cd + peak. when haematological reconstitution after apbsct we observed a significant positive correlation between day of cd + peak and time to absolute lymphocyte> . x /l(alc) (r . , p= . ), pmn> . x /l (r . , p= . ), plt> x ³/mmc (r . , p= . ), length of hospitalization (r . , p= . ). this data seems to link up the appearance of cd + cells with the bone marrow reserve: younger pts release higher number of cd + in peripheral blood after apbsct and these cells are detectable sooner than in the older pts. furthermore, time to alc> . x /l and pmn> . x /l recovery, plt> x ³/mmc and length of hospitalization are longer in pts that release later cd + cells, i.e. the older pts. early appearance of cd + cells after apbsct and peg-filgrastim might be considered as surrogate marker of bone marrow reserve. further analysis of cd + subset are ongoing to confirm these data and to clarify their significance. imbalance of osteoprotegerin/receptor activator of nuclear factor-kb ligand in bone marrow plasma and microenvironment after allogeneic stem cell transplantation c. selleri, l. tauchmanovà, p. ricci, a.m. risitano, m.c. martorelli, g. cerciello, i. imperatore, a. casale, t. musella, g. lombardi, a. colao, b. rotoli federico ii university (naples, i) persistent decrease in bone mineral density (bmd) is a known complication after allogeneic stem cell transplantation (allo-sct) due to the transplant procedure, gonadal failure, immunosuppression and deficit of osteoblast precursors in the marrow microenvironment. in addition, transplanted patients may develop chronic endocrine and immunological disorders, including chronic graft versus host disease, that can further affect bone turnover. osteoprotegerin (opg) plays a pivotal role in bone remodelling, by neutralizing the effect of rankl on differentiation and activation of osteoclasts. we investigated the relationships between densitometric values, circulating opg and interferon-gamma (ifn-gamma) levels; moreover, opg and rankl levels were measured in marrow plasma and in conditioned medium of long-term cultures (ltc) of marrow mesenchymal-derived osteogenic cells. thirty six allo-sct patients ( females; age: ± yrs) were enrolled and compared to controls matched for age, gender and body mass index. bmd was measured at lumbar spine and femoral neck by the dexa technique. lumbar and femoral bmd were lower in patients than in controls ( . ± . vs . ± . , p< . ). serum opg and ifn-gamma were significantly (p< . ) higher in patients than controls. patients' serum ifn-gamma correlated with opg levels (r= . ; p= . ). by contrast, opg levels were lower in patients than in controls in marrow plasma (p< . ) and in conditioned media after one (p= . ) and months (p= . ) of ltc of marrow mesenchymal-derived osteogenic cells. rankl values were similar between patients and controls, being lower in conditioned medium than in marrow plasma (p< . , both groups). the rankl/opg ratio in ltc was significantly higher in patients than in controls, although this ratio progressively increased with the time of ltc in both groups (p< . ). there was no correlation between serum, in situ opg levels and densitometric values. in conclusion, our findings suggest that after allo-sct: ) the paradoxically high serum opg levels, which correlate with ifn-gamma, are likely due to deranged immune system; ) low marrow and microenvironment opg levels confirm persistent quantitative and qualitative deficit of osteoblastic precursors; ) the increased microenvironment rankl/opg ratio may negatively affect bone remodelling. mesenchymal stem cells (mscs) are endowed with multilineage potential and immunomodulatory ability, these properties rendering them attractive for tissue engineering and immunotherapy. however, it is still a matter of debate whether donor mscs have a sustained engraftment in the host bone marrow (bm) after allogeneic hematopoietic stem cell transplantation (hsct). in particular, studies on the fate of mscs transplanted with cord blood (cb) are lacking. the aim of this study was to analyse the donor/recipient origin of mscs in pediatric patients receiving an allogeneic hsct. thirty-six patients undergoing allogeneic hsct for either malignant ( cases) or non-malignant disorders ( cases) were enrolled in the study; patients received cb transplantation (cbt, from a related and from an unrelated donor) and patients bm transplantation (bmt, from a related and from unrelated donor). results were also compared with those obtained in adults given hsct for either malignant ( cases) or non-malignant ( cases) disorders. mscs were grown from bm aspirates taken - months after hsct. msc samples at the third-fourth passage were phenotipically characterized and resulted to be positive for cd , cd , cd , cd , cd (> %) and negative for cd , cd , cd (< %). donor/recipient origin of mscs was assessed by amelogenin assay (in case of male recipient/female donor) and microsatellite analysis. mscs were grown from pediatric patients; in samples ( after bmt and after cbt) a confluent layer of cells did not grow, leading to an insufficient quantity of mscs for chimerism analysis. molecular analysis on mscs demonstrated a full recipient chimerism in / and in / of the assessable pediatric patients given bmt and cbt, respectively. a mixed msc chimerism with donor cells was observed in patients transplanted with bm cells and in children given cbt. chimerism analysis performed on peripheral blood mononuclear cells (pbmcs) of the same patients, showed a full donor chimerism in all children given bmt but one, while a mixed chimerism was detected in out of children given cbt. a full recipient msc chimerism was observed in all adult patients, who also displayed a full donor pbmc chimerism. these data suggest that bm soil of pediatric patients might be more favourable than that of adults for the engraftment of transplanted mscs and that mscs able to engraft in the host can also be transferred with cb. graft engineering r cryopreservation of peripheral blood progenitors for autologous transplantation in haematological malignancies with different concentration of cryoprotectant -five-year single-centre experience a. pivkova, l. cevreska, n. siljanovski, z. stojanoski, o. karanfilski, s. genadieva stavrik, i. panovska, s. krstevska balkanov, s. trajkova, b. georgievski clinical center (skopje,mk) in this study we present our five year center experience with cryopreservation of pbsc and autologous transplantation in patients with hematological malignancies treated in a period - at department of hematology, skopje. material and methods: diagnosis of patients were ( aml, nhl, mm, hd) and median age at transplant was years ( - ). mobilization of pbsc was provided with etoposide (vp- ) + g-csf mcg/kg in aml patients, and high dose cyclophosphamide - gr/msq+g-csf mcg/kg or alone g-csf mcg/kg in patients with limphoproliferative diseases. collected pbsc were cryopreserved in solutions with % dmso in patients and % dmso in patients, computer programmed until - c, and stored different period in liquide nitrogen on - c. autologous transplant was preformed wid conditioning consisted of myeloablative highdose chemotherapy, bucy in aml patients, high dose mel in mm patients, beam or hd ice in nhl patients and beam in hd patients. cell viability was assessed by fluorescence microscopy using acridine orange dye exclusion. results: a total of pbsc cryopreservation procedures were preformed in our group of patients with median ( - ) apheresis procedures. median period from storage of cryopreserved pbsc grafts until thawing was days ( - ). total number of infused cd +cells was between , - x /kg and median number of mononuclear cells was , x /kg ( , ) . the amount of infused dmso solution ranged between - ml (median ml) with dmso concentration ranging ml- ml (median ml) in a group preserved with %dmso and - ml (median ml) in % dmso cryopreserved grafts. the viability of the fresh harvests before storage vas median % (range , - , %). the poorer viability was associated with harvest cell count. bellow x /l the median viability was % and only / cases had < % viable cells. harvests count above x /l the median viability was % ( , %- %). in a group of patients that received pbsc grafts preserved with % dmso, also revealed signs of mild dmso infusion related toxicity ( %vs %). hematopoietic recovery was similar in both groups, sor ne> , x /l on day + ( - ), plt > x /l on day + ( - ). our results confirm that the infusion of cryopreserved autologous pbsc in hematological malignancies revealed successful engraftment in all patients and good cell viability. we did not registered "hard to mobilize" patients and graft failure. thermogenesis axp™ and bioarchive™ systems for automated cord blood banking l. dobrila ( ), s. jiang ( ), j. chapman ( ), d. marr ( ), k. kryston ( ), p. rubinstein ( ) ( )national cord blood program at new york blood center (new york, usa); ( )thermogenesis corp (rancho cordova, usa) background: good tissue practices (cgtp) in cord blood banking require product uniformity and reproducible mononuclear cell recovery and viability, suggesting that automation could be critical to facilitating cgtp-compliance for cord blood banks. processes that lend themselves to automation are cord blood volume reduction, controlled rate freezing, storage and retrieval that avoids unnecessary transient warming events. we have evaluated the autoxpresssystem (axp),that allows for automated volume reduction in a closed system. the autoxpress consists of a microprocessor-controlled device and a disposable closed blood bag set that provides for the separation of cb into a freezing bag, an erythrocyte bag and an excess plasma bag, the mononuclear cell product is concentrated into a uniform volume in the freezing bag, ready to be cryoprotected and fully compatible with the bioarchive system,a system that allows the controlled-rate freezing, liquid nitrogen storage and retrieval of , cbus. study design: the efficiency with which cord blood hematopoietic progenitor cells can be concentrated into the freezing bag of the autoxpress bag-set was determined using the cd cell marker and colony-forming unit (cfu) counts as principal indices. the product was cryoprotected with % dmso, frozen in the bioarchive system, stored for - weeks under the liquid n level and then retrieved and thawed using the standard clinical protocol. twenty-three consecutive cord blood units were evaluated for cell recovery by measuring the collection and product volumes, the hematocrit and the counts of total nucleated cells (tnc), mononuclear cells (mnc), cd + cells and colony-forming units (cfu),before and after axp processing. we also determined these indices after freezing, storage in the bioarchive system and thawing. results: results are presented as the mean s.d. (n= ) for all values. the axpprocess achieved mnc fraction volumes of . . ml with a final average hematocrit of . . %. the post-processing recovery of cd + cells was . . % and those of cfu . . %, of mnc . . % and of tnc . . %. less than % of tnc were lost into the excess plasma bags. granulocytes, accounting for % of tnc and less than . % of the cd + cells were lost into the red cell bags. post-thaw the recoveries of cfu and viable cd + cells were . % and . %, respectively. conclusions: the axpefficiently and reproducibly separates cord blood mononuclear and cd + cells into a consistent, uniform volume. these cells retained their viability post bioarchive freezing, storage and retrieval (> %). thus, axpcoupled with the bioarchive system supports a very high quality standard for automated cord blood processing. the influence of cryopreservation and the duration of frozen storage k. nowak, s. giebel, m. krawczyk-kulis, j. wojnar, j. holowiecki silesian medical university (katowice, pl) among the factors which enable successful transplantation, the ability to store and subsequently recover sufficient viable hematopoietic stem cells to reestablish hematopoiesis is critical. the goal of this study was to evaluate the impact of freezing procedures and cryopreservation in liquid nitrogen into - °c on cell viability, wbc, cd + cells recovery and clonogenic capacity. in the retrospective study samples derived from patients with hematological malignancies (n= ) and healthy donors (n= ) were analysed. median duration of the product storage was . years ( day - . years). the viability (%), wbc (g/l), and cd + (%) all decreased significantly after cryopreservation (p< . ) with median relative changes of - . %, - . %, and - . %, respectively. after thawing the viability equaled . % ( - %), wbc . g/l ( . - . g/l), and cd + cells . % ( . - . %). in multivariate analysis the following factors were associated with poor recovery: ) viability: presence of malignant disease (p= . ), use of cyclophosphamide (ctx) for mobilisation (p= . ), ) wbc: presence of malignant disease (p= . ), older age (p= . ), ) cd + cells: presence of malignant disease (p= . ), storage duration (p= . ). after thawing the median number of clones/ cells was as follows: ) cfu-gm on day : ( - . ), ) cfu-gm on day : . ( - . ), ) bfu-e: . ( . - ), ) cfu-gemm: . ( - . ). the clonogenity was negatively influenced by: ) cfu-gm on day : presence of malignant disease (p= . ), chemotherapy-containing mobilisation regimen (p= . ), ctx for mobilisation (p= . ), ) cfu-gm on day : older age (p= . ), presence of malignant disease (p= . ), chemotherapy-containing mobilisation regimen (p< . ), ) bfu-e on day : presence of malignant disease (p= . ), chemotherapy-containing mobilisation regimen (p< . ), cfu-gemm on day : diagnosis other than cml (p= . ). we conclude that both diagnosis and mobilisation regimen have impact on recovery and clonogenity of cryopreserved hematopoietic stem cells. on the other hand, even long-term storage enables preservation of vial cells with high clonogenic potential, which may be used for transplantation. immune reconstitution after hla-haploidentical transplantation using unmodified marrow and cd depleted blood stem cell: not different from hla-identical transplantation p. rujirojindakul ( ) immune reconstitution (ir) is a key role of allogeneic hematopoietic stem cell transplantation (hsct) not only because persistent immune defects is related to posttransplant infectious morbidity, but also because it may influence the risk of relapse and the development of secondary malignancies after hsct. many factors have an impact on ir, especially the degree of genetic differences between donor and recipient. several studies have shown extreme slow ir in patients receiving t-cell depleted graft from human leukocyte antigen (hla)-haploidentical (hap) donor. differently, we have used unmodified marrow on day and cd -depleted mobilized blood cells (mbc) on day for haploidentical transplantation. cd -depleted mbc are devoid of cd -positive cells, they contain cd -positive cells, nkcells and a minority of cd -positive cells. to compare the ir during the first year post transplant between hap and hlaidentical (id) recipients, we have carried out a prospective, longitudinal analysis of ir in hsct patients in each group. we analysed reconstitution of naïve, memory t cells, b cells, natural killer cells ,dendritic cells and monitored thymic output by using tcr rearrangement excision circles (trecs). surprisingly, reconstitution did not differ between the groups. this study reveals that ir in hap transplantation using unmodified marrow and cd -depleted mbc is not worse than id group. background: autologous graft versus host disease (gvhd) is a novel self-limited autoaggression syndrome, encountered in the autologous pbsct (peripheral blood stem cell transplantation) setting with spontaneous occurrence in patients receiving cd + enriched autografts. aim: to present the case report, concerning the occurrence of a rather rare form of gvhd in a patient with cd + enriched auto-pbsct for ewing sarcoma. case report: we present the case of a years old female patient with ewing sarcoma of the scull and right hip who underwent autologous pbsct with a cd + enriched graft. the preparative regimen consisted of busulfan and melphalan. engraftment occurred on day + for neutrophils and on day + for platelets. at day + signs of acute gvhd involving only the skin occurred initially in the axillae, flexion sites of the elbows and popliteal region and shortly afterwards involving the face, neck, trunk and abdomen. beside maculopapular exanthema, bullae formation and large epidermal descuamation were observed, an image corresponding to stage iv of acute gvhd. the patient remained feverless, without pruritus or other signs or symptoms. at the time of onset the investigation of immune reconstitution presented marked lymphopenia (abslolute values per microliter):cd + = ,cd + = ,cd +/cd + = ).no other modified biological parameters could be found. skin biopsy revealed lymphoid, predominantly cd + infiltration. in days the lesions progressively disappeared, with the maintenance of residual hyperpigmentation and nail keratosis. conclusion: autologous gvhd, confined to the skin without any clinical-biological evidence of internal organ disease is a possible self-limited complication of hct cd +cell enrichment of the graft could be in our case a predisposing factor.the impact on the evolution will remain an issue to be assessed. why will an amotosalen-based protocol for extracorporeal photopheresis be valuable? j. e. hearst ( ) , f. heshmati ( ), s. talib ( ) ( )university of california (berkeley, usa); ( )hopital cochin (paris, f) bone marrow transplantation has the potential of providing a complete cure of the disease symptoms of hematologic malignancies and, ultimately, with an appropriate safety profile, the symptoms of hemoglobinopathies as well. even in the case of related fully matched bone marrow transplantation, there is morbidity and mortality associated with graft-versus-host-disease (gvhd). successful application of mismatched (related-haploidentical and unrelated) bone marrow transplantation (bmt) in patients with leukemia or lymphoma requires that improved overall outcomes of bmt be obtained, coupled with the avoidance or successful treatment for the gvhd now experienced in existing mismatched bmt protocols. these two goals may require use of reducedintensity conditioning (ric) in support of the transplantation. extracorporeal photopheresis (ecp) has proven to be effective as a treatment modality for gvhd following transplantation and is gaining popularity as a treatment protocol. yet, there remain significant technical challenges in the application of ecp. as a solution to many of these problems, we propose the development of a more simple and less expensive process for the psoralen photochemical treatment of the transplant patient's autologous leukocytes. this process will define the therapeutic dose of photochemically treated cells required for clinical responses equivalent to those which have been observed to be most effective using presently approved ecp instrumentation and protocols. however, this new process uses amotosalen, a more photochemically efficient psoralen, and it uses lymphocytes collected by the conventional methods of blood banks. the greater photoefficiency of the psoralen compound results in far shorter exposures of the target leukocytes to uva light, leading to less non-specific damage of the cells, leaving them metabolically active, although unable to divide. it is our premise that these are the ideal properties that photochemically treated lymphocytes must have in order to s maximize the positive immunological responses associated with a successful photopheresis treatment. uva phototherapy as a treatment for sclerodermic chronic graft-versus-host disease refractory to immunosuppressive therapy g. marotta, m. pellegrino, s. sammassimo, m. tozzi, c. miracco, m. fimiani, f. lauria azienda ospedaliera universitaria senese (siena, i) chronic graft versus host disease (cgvhd) is the most common late complication affecting long-term survivors of allogeneic hsct. several organs are targets of cgvhd but the skin is the tissue mainly affected. two subtypes of involvement, cutaneous lichenoid and sclerodermoid, have been described, based on clinical and histopathological examinations. chronic gvhd is usually treated with immunosoppressive drugs which, however, are not effective in about - % of patients. in this setting of refractoty/resistent patients, extracorporeal photochemotherapy and ultraviolet a (uva) radiation often show a positive clinical outcome, but the results are limited by the low number of treated cases. in our institute, uva phototherapy has been used for treating patients with advanced cutaneous cgvhd (generalized sclerodermoid skin involvement) resistant to conventional immunosuppressive therapy. patients enrolled gave fully informed consent and they underwent skin biopsies before and after uva phototherapy. moreover, the cutaneous elasticity has been evaluated by means of an elastometer. uva radiation ( - nm) was emitted by a gp- h irradiation unit with a dose of j/cm². irradiance was measured with a spectroradiometer and found to be mj/cm² at skin level. patients were treated times per week for a total of weeks (total j/cm²) and the cutaneous lesions were carefully inspected and palpated before starting every treatment. patients were conditioned with a reduced intensity regimen and received unmodified g-csf mobilized pbsc from matched related donors. gvhd prophylaxis consisted of csa and mtx. all patients had failed to respond to at least three lines of immunosuppressive therapy. at the end of sessions the clinical response was assessed subjectively and objectively, and it was graded as good (obvious softening), moderate (mild softening) and poor (no change). two patients had a good response and one a moderate response reporting remarkable softening of skin lesions after uva phototherapy without side effects. clinical responses were associated to an improvement of histopathological findings. the evaluation of skin elasticity showed a significant increase in resilience, hysteresis, and distensibility. our results demonstrate the efficacy of uva phototherapy. it appear, in the setting of cutaneous cgvhd, as a procedure well tolerated and effective particularly for patients who do not respond to standard immunosuppressive treatments. rituximab in chronic graft-versus-host disease of the lung i. von olshausen, a. spyridonidis, h.c. bremer, w. windisch, h. bertz, j. finke university hospital freiburg (freiburg, d) we report about a year old male patient in whom severe obstructive chronic graft-versus-host-disease (cgvhd) of the lung improved with rituximab, an anti-cd antibody. the patient had undergone hla-matched unrelated allogeneic peripheral stem cell transplant for his acute myeloid leukaemia in august . with discontinuation of cyclosporine after day + mild cgvhd of the skin and liver occurred, but improved spontaneously within a few weeks. in january the patient presented with severe dyspnoea associated with an afebrile upper respiratory tract infection. the work-up revealed severe obstruction on lung function tests, nodular infiltrates in computed tomografy scan (ct), acute bronchitis in bronchoscopy, but no infectious agent in bronchial lavage. initial and long term treatment with various antibiotics, antimycotics and several immunosuppressants (prednisone, rapamycin, mycophenolatmofetil, cyclosporine a, cyclophosphamide) was unsuccessful. after a single dose of rituximab ( mg/m²) dyspnoea improved for a few days, but symptoms reappeared. we started a regular weekly rituximab application in july resulting in marked clinical improvement, slight improvement on lung function (peak exspiratory flow, pef) and regression of the nodular infiltrates in ct scan despite tapering off other immunosuppression and intermittent respiratory infections. the patient was able to participate in household thresholds again. rituximab therapy was discontinued in november because of patient's admission with spontaneous pneumothorax. the patient is now being evaluated for lung transplantation. like in our case, monoclonal anti-cd antibody has recently shown efficacy in cgvhd and several autoimmune diseases, probably due to elimination of b cells which may act as antigen presenting cells for t cells and as a source of autoantibody production. conclusion: we propose regular weekly rituximab application as a treatment option in cgvhd of the lung. are peak level measurements of cyclosporin a useful in allogeneic stem cell transplantation? c. scheid, c. heinz, u. holtick, k. hübel, v. göde, t. zander, a. engert, m. weihrauch, m. hallek university of cologne (cologne, d) peak level plasma levels of cyclosporin a (csa) or hours after drug administration (c or c ) correlate with the degree of calcineurin inhibition and with the auc much better than trough levels (c ). in solid organ transplantation dosing according to c levels rather than c led to reduced rejection rates. in allogeneic stem cell transplantation csa doses are still adjusted according to c levels. to investigate whether c and c levels would be useful in csa dosing after stem cell transplantation these measurements were performed in consecutive patients in addition to c levels. if intravenous csa was given, peak levels were assessed after the end of a hour infusion. so far serial measurements were undertaken in transplant patients ( aml, all, saa, myeloma, cll). there was only a weak correlation between trough and peak levels of csa and between dose and plasma levels irrespective whether the drug was administered orally or intravenously. although all trough levels were within the target range of - ng/ml peak levels were all below ng/ml. there was no evidence of so-called "late absorbers" as all c levels were lower than the c levels. two patients showed evidence of microangiopathic hemolytic anaemia, however peak and trough levels of csa were not different from other patients. all but two patients showed signs of acute gvhd. in view of the csa doses given and the trough levels, the peak levels were considerably lower than expected from results in liver or renal transplantation. this was even more surprising as most patients received itraconazole, a drug known to increase csa levels by interfering with csa metabolism in the liver. in summary c monitoring is feasible and demonstrates lower peak levels than expected from solid organ transplantation. these levels should lead to a lesser degree of calcineurin inhibition. whether this translates in increased rates of gvhd remains to be seen with a larger patient number. if this holds true csa dosing with higher c target levels might decrease acute gvhd after stem cell transplantation as previously shown for rejection rates after liver transplantation. clinical and histopathological features of oral chronic graft-versus-host disease following allogeneic reducedintensity conditioning haematopoietic stem cell transplantation: a single-centre blind study f. demarosi ( ) introduction: oral involvement of chronic graft-versus-host disease (cgvhd) occurs in % of patients suffering from cgvhd and the oral cavity may be the primary or even the only site of cgvhd involvement. lichen planus-like lesions are the most distinctive oral changes of cgvhd. the histopathologic changes of oral cghvd include epithelial atrophy, apoptotic bodies, hydropic degeneration of the basal cells, and a mononuclear subepithelial cell infiltrate with lymphocyte invasion. aim: the aim of this blind study was to compare clinical and histological features of oral mucosa in patients who underwent allogeneic reduced intensity conditioning hematopoietic stem cell transplantation (ric hsct). patients and methods: this study enrolled adult patients who consecutively underwent allogeneic ric hsct for hematological malignancies between october and october . all patients were assessed for the presence of oral cgvhd by oral examination performed on the day + at the unit of oral pathology and medicine, university of milan. clinical lichenoid changes of the oral mucosa were regarded as positive for cgvhd. following informed consent, an incisional biopsy was taken from oral mucosa of all patients, either with or without oral cgvhd lesions. biopsies were examined by a pathologist who was unaware of clinical aspect of the mucosa (blind). results: biopsies were taken from patients with clinical evidence of oral cgvhd and patients with apparently normal oral mucosa. biopsies were performed at a time point ranging from to days (median . days) after transplantation. sites of biopsies were buccal mucosa and gingiva. histological cgvhd changes were detected in all the patients having also clinical evidence of oral cgvhd, and in out of patients with apparently healthy mucosa. conclusions: while histological changes of oral mucosa without corresponding clinical changes are not sufficient to make a definite diagnosis of oral cgvhd, their detection might be of considerable help to predict the onset of the disease following ric hsct. a longer follow up of patients showing histological changes with no clinical counterpart, will possibly elucidate whether such changes are indeed predictive of the occurrence of clinically evident lesions. evaluation of safety and tolerability of extracorporeal photochemotherapy in paediatric patients affected with graft-versus-host disease c. del fante, c. perotti, gl viarengo, p. bergamaschi, d. romano, l. salvaneschi policlinico s. matteo (pavia, i) objectives: extracorporeal photochemoterapy (ecp) is a therapeutic option for treatment of acute and chronic graft versus host disease (agvhd and cgvhd) resistant to standard drug therapy. in the pediatric context, ecp procedure has some technical limitations when compared to adult subset. low body weight, venous accesses, hypersensitivity to hypocalcemia, fluid balance and transfusion demand may represent a limitation for ecp treatment in children. we report our experience in very low body weight children (≤ kg) affected with agvhd and cgvhd in terms of safety and tolerability of ecp. methods: ecp consists of distinct steps: ) collection of mononuclear cells by spectra cobe (version . ) cell separator device processing blood volumes; ) ex-vivo dilution with saline and addition of -mop to the bag, transfer of the buffy coat in a uv-a permeable bag and irradiation at °c; ) reinfusion of the cells to the patient after hours to avoid hypotermia. patients, median age . years (range: - ) median weight . kg (range: - ) were treated. a central venous catheter was positioned in all patients. our treatment protocol consisted in procedures per week in agvhd and procedures per week in cgvhd, both followed by procedures every weeks for - weeks then by procedures per month. patients were affected with agvhd (grade ii-iv with skin, liver and gut involvement); patients had extensive cgvhd (skin, mucosal, liver, and lung).the cell separator device was primed every time with filtered and irradiated rbc. calcium gluconate was continously administered by pump during the procedure. the acd/whole blood ratio was always setted at : . all patients were monitored for blood pressure and heart rate during the entire procedure. results: the total number of ecp procedures performed was , with a median of (range: - ) procedures/patient. / ( . %) patients experienced mild hypotension, / ( %) moderate hypotermia and / ( . %) hypocalcemia (nausea and vomiting); no procedure was discontinued. the transfusion demand did not augment during all the course of treatment; no life-treathening infections were recorded. conclusion: we demonstrated that ecp is feasible and safe even in very low body weight patients on condition that ecp is performed adopting some simple precautions. our experience broadens the cohort of patients who can benefit of this therapeutic option. silesian centre for cellular transplantation, poland k. suchnicki, a. lange lower silesian center for cellular transplantation (wroclaw, pl) haematological patients -malignancies (cml: n= ; aml:n= ; all:n= ) and saa (n= ); children (n= ) and adults (n= ) were transplanted with bm (n= ) and pbpc (n= ) from hla-identical sibling donors in years to . all leukemia patients received myeloablative conditioning (bucy or bucyvp);saa patients were conditioned with cy or cyatg. chimerism was tested and proved to be complete in all but cases (death before day + ). during years of follow up: patients died of transplant related causes (regimen related toxicity:n= , rrt/agvhd:n= , infection: n= , agvhd/multi organ failure/inf:n= , cgvhd/mof/inf:n= ) or relapse (n= ). agvhd was seen in , % cases (grade = , % of patients with hematological recovery, grade = %, grade> = , %). severe agvhd (grade> ) was seen only in leukemia patients, who received toxic conditioning, but not in saa conditioned only with cy+-atg. agvhd was more frequent and severe in pts with a high grade of toxicity (among patients with low rrt there were % cases of agvhd= and no cases of agvhd> ; among patients with high rrt there were % agvhd= and % agvhd> ). in addition to toxicity infections and inflammation seemed to aggravate agvhd as shown by the presence of an elevation of serum crp at the advent of severe agvhd. apparently the presence of toxicity and agvhd had an additive negative effect on survival. the incidence of cgvhd among all patients living>day+ was % and increased with :i) diagnosis: saa( , % of saa cases),aml ( , %),all( , %),cml( , %),ii)previous agvhd: all surviving patients with agvhd> developed cgvhd. cumulative survival is better in following :i)saa vs aml/all/cml (p= , ),ii)all, cml early stage vs advanced (p= , ),iii) conditioning bucy vs bucyvp (p= , ), iii) age: children vs adults (p= , ),iv) gender: female recipient from male donor better than all other pairs (p= , ),v) rrt: toxicity grade - (sum of who degrees for toxicity of skin, mucous, liver, gastro-intestinal tract)vs grade - vs grade - (p= , ), vi) crp: serum level of - vs level> (p= , ),vii) agvhd grade - - vs grade - (p= , ), viii) cgvhd grade lim better than no cgvhd better than extcgvhd (p= , ) which reflects gvl surveillance of gvh cells (less relapses) without overt, life threatening reaction. this observation documented that the outcome of transplantation in sibling setting depends largely on toxicity, infections and agvhd which in turns aggravate themselves. the role of atg in reducing acute gvhd in related and unrelated donor transplants y. zalyalov, a. ganapiev, s. alexeev, a. smirnova, n. stancheva, n. ivanova, a. alyanskiy, b. afanasyev pavlov's state medical university (st. petersburg, rus) background: acute graft versus host disease (gvhd) remains the major complication of allogeneic haemopoietic stem cell transplantation (allohsct) with an incidence of - % and mortality of up to - %. atg is commonly used in the conditioning regimens for donor transplants to reduce the risk of rejection and gvhd. patients and methods: since up to date we have performed allohsct ( ( %) with atg and ( %)without atg) for the following patients -aml (n= ), all (n= ), cml (n= ), non-hodgkin's lymphoma (n= ), aa (n= ), hodgkin's lymphoma (n= ), mds (n= ), kostmann's syndrome (n= ), hypereosinophilic syndrome (n= ). the average age was years . dose of atg was mg/kg.b.w. ( - ). myeloablative conditioning regimen consisted mainly of bu-cy ( pts ( %), nonmyeloablative conditioning treatment -flu-cy ( pts ( %). the gvhd prophylaxis was short term mtx and csa. results: rate of gvhd grade -ii in pts with atg was % ( pts), gvhd grade iii-iv - % ( pts). rate of gvhd -ii in group pts without atg was %( pts) and gvhd iii-iv - % ( pts). tmr in group with atg was % ( pts), without atg - %( pts). os in pts with atg was %, without atg - %. conclusions: the use of atg in conditioning regimen reduces the incidence of acute gvhd grade iii-iv and increases overall survival in patients after allohsct (p= . ). intracellular markers of eosinophils and mast cells in patients with lymphoproliferative and myeloproliferative disorders associated with high-grade eosinophilia l. komarova, y. zueva, n. mikhaylova, b. afanasyev, a. totolian pavlov state medical university (st. petersburg, rus) objectives: distinct eosinophilia rarely occurs in chronic graftversus-host disease (gvhd) after allogeneic hematopoetic stem cell transplantation (hsct), but is often seen in hematological malignancies, including lymphoproliferative diseases. a hypothesis exists that marked eosinophilia in chronic gvhd is mainly of non-clonal origin and, in general, is associated with favorable prognosis. the aim of this study was to investigate the serum levels of secretable eosinophil and mast cells proteins in the patients with chronic gvhd and other hematological malignancies. patients and methods: six post-transplant patients with chronic gvhd with eosinophilia were followed up after allo-hsct. thirty hematological malignancies in patients with marked eosinophilia (> . x /l) were also observed, including cases of lymphoproliferative (n= ) and myeloproliferative (n= ) diseases. eighteen patients with bronchial asthma (ba) comprised a reference group for polyclonal eosinophilia. high content of peripheral blood eosinophils served as the major criterion of patients' selection and was confirmed by flow cytometry. eosinophil cationic protein (ecp) and tryptase were measured in serum by fluoroimmunoenzyme assay (pharmacia, sweden). results: the percentages of circulating eosinophils in patients with lymphoproliferative (median: . %; range, - %) and myeloproliferative diseases (median . %; range, - %), and in cases of chronic gvhd after allo-hsct (median . %; range, - %) were significantly higher than in ba patients (median . %; range, - %); Ð= . . the levels of total ecp were markedly increased in the patients with lymphoproliferative diseases (median: . ng/ml; range, - ng/ml) compared to ba (median: . ng/ml; range, . - ng/ml); p= . . however, the serum levels of tryptase and ecp in total group of hematological patients (median: . ng/ml; range, to . ng/ml) were not increased, as compared with asthma cases (median: . ng/ml; range, . to . ng/ml); p= . . likewise, in chronic gvhd with eosinophilia, the serum contents of both ecp and tryptase did not differ from those in ba. conclusion: the significant differences in ecp levels in blood serum in the patients with lymphoproliferative diseases and absence of differences in ecp and tryptase levels in blood serum of chronic gvhd and ba patients suggests intact functions of mature eosinophils in gvhd, thus reflecting nonclonal genesis of this feature in both states. natural killer cell activity in the early phase after allogeneic stem cell transplantation: impact of conditioning strategies l. fischer, o. penack, c. gentilini, e. thiel, l. uharek charité campus benjamin franklin (berlin, d) background: research into the role of natural killer (nk) cells in allogeneic stem cell transplantation (sct) has been greatly expanded recently. nk cells may contribute to the gvl reaction possibly without exerting a gvhd effect. using a novel flow cytometric assay, which detects the lytic granule membrane protein cd a as a marker for nk cell degranulation, we investigated the effect of in vivo t cell depletion and the type of conditioning on nk cell function in the early phase after allogeneic sct. methods: peripheral blood mononuclear cells were collected at day + and + after allogeneic sct and incubated with the nk sensitive cell line hl ( hours at °c, e:t ratio : ). pe-cy conjugated anti-cd a antibody was added prior to incubation, monensin ( . mm) was added after hour of incubation. finally cells were further stained against cd , cd and cd and the proportion of cd a positive nk cells was measured by flow cytometry and the absolute number of degranulating nk cells was calculated. results were compared to values from healthy controls. results: twenty two patients were investigated of whom had been treated with a conventional dose conditioning regimen and had received a reduced dose regimen. at day + , the proportion of nk cells with cytotoxic activity (cd a+/cd +) was significantly reduced as compared to normal donors ( . % vs. . %, p< . ). at day + the percentage of degranulating nk cells in five evaluated patients was within normal range (mean . %). the predominant proportion of degranulating cells was in the cd dim/cd -subpopulation (mean . %). at day + the percentage of cd a+ cells averaged . % after conventional conditioning compared to . % after reduced intensity conditioning (p= . ). the absolute number of degranulating nk cells was significantly reduced after conventional conditioning ( . /µl vs. . /µl, p= . ). neither the percentage ( . % vs. . %, p= . ) nor the absolute number ( . /µl vs. . /µl, p= . ) of cd a+ nk cells differed significantly in patients with and without atg induced t cell depletion at day + . conclusion: according to our data cytotoxic activity of nk cells is reduced after allogeneic sct. the absolute number of nk cells with cytotoxic activity is significantly higher after reduced intensity conditioning which may impact on the outcome of this strategy. we saw no significant influence of antibody mediated in vivo t cell depletion by atg on nk cell activity during the first two months post sct. it is known that nk alloreactivity is involved in the control of neoplastic cells in the setting of aploidentical bone marrow transplantation (bmt) in patients with acute myeloid leukemia (aml). the role of nk alloreactivity in the setting of marrow unrelated transplant (mud) in lymphoid neoplasia is still controversial. in a series of patients ( acute lymphoid leukemia, non-hodgkin's lymphoma l, severe aplastic anaemia), we investigated whether nk alloreactivity is involved in the control of lymphoid neoplastic cells. in addition, using monoclonal antibodies (moabs), we evaluated the expression of killer immunoglobulin-like receptors (kirs), killer lectine-like receptors (klrs), and natural cytotoxicity receptors (ncrs) in patients under study. according to hla-cw alloreactivity, patients were separated into two groups: a group of patients with potential nk alloreactivity (group a: all, nhl, saa), a second group of patients lacking nk alloreactivity (group b: all, nhl). the mean follow up was months ± months. % of group a patients were alive, whereas % of group b were still in remission, indicating a role for alloreactivity in lymphoid neoplasia. concerning expression of nk receptors, group a was characterized by the high expression of kirs, potentially involved in alloreactivity in out of patients. klr was represented by cd /nkg a in out of and in one case by cd /nkg c. this last case showed a recovery of cd /nkg a phenotype after months. ncrs and nkg d were usually expressed, with exception of nkp . group b was characterized by an heterogeneous pattern of expression of kir, whereas cd /nkg a was expressed in out of cases and cases expressed nkg c. interestingly, one of these two cases relapsed during follow-up. ncrs were usually expressed, with the only exception of nkp . our data indicate that nk alloreactivity might be an advantage for survival in patients affected by acute lymphoid neoplasia. no correlation could be demonstrated with expression of natural killer receptors (nkrs) and clinical behaviour, suggesting that analysis at clonal level is mandatory to get insights into the mechanism involved. this study was supported by a grant from fondazione città della speranza. a rare complication after allogeneic stem cell transplantation: acute axonal neuropathy a.d. moicean, d.n. colita, v.n. mirea, a.m. dumitrescu, t. puscariu, z. varady, a. tanase, i. constantinescu fundeni university institute (bucharest, ro) despite considerable progress in the management of allo-hsct, infection remains an important cause of morbidity and mortality after transplant. cmv is frequently involved in the infectious pathology after hsc transplantation. we present a -year old women with high-risk acute lymphoblastic leukemia who underwent matched sibling pbsc transplantation. she received standard conditioning regimen (endoxan mg/kgc and tbi gy). gvhd prophylaxis received was with csa and methotrexate. she developed grade ii gvhd (cutaneous and intestinal) on day + with resolution under corticotherapy. starting with day + she had weakness, osteoarticulary and muscular pain with functional impairment. neurological examen and the electrophysiological study showed an acute axonal neuropathy. she had more than cmv copies in the blood pcr. she received mg bid i.v ganciclovir for days as induction therapy and than mg/d for more weeks with complete remission of the neurological symptoms a year old female patient was diagnosed with cml in chronic phase and stem cell transplantation (sct) was planned from her one antigen mismatched sister. pre-transplant routine investigations revealed a high antibody titer against toxoplasma gondii (ift : , kbr : , igm negative) indicating the history of a previous infection. cerebral magnetic resonance imaging (mri) ten days before transplantation showed no abnormalities. the patient was conditioned with busulfan ( mg/kg) and cyclophosphamide ( mg/kg). because of the hla-a mismatch rabbit atg (fresenius, mg/kg) was added. graftversus-host disease (gvhd) prophylaxis consisted of cyclosporine a and mtx. pbscs containing . * /kg cd pos stem cells were transfused. engraftment occured rapidly on day + . on day + the patient developed severe headache and hours later generalized seizures. computered tomography showed multiple patchy hypointense lesions in the subcortical white areas with corresponding nodular signal enhancements in t weighed mri analysis suggesting an infectious process. eeg revealed pronounced focal abnormalities in the left hemisphere extending from frontal to the temporal regions. liquor analysis revealed no causative infectious agent. assuming reactivation of toxoplasmosis we initiated a specific quatruple therapy consisting of pyrimethamin orally, sulfadiazin orally, clindamycin i.v. and tmp-smx i.v. clinically the patient improved rapidly. two weeks later cerebral mri showed regression of the temporoparietal white matter lesions with further improvement during the following weeks resulting finally in complete resolution. antibody titers against toxoplasmosis slowly regressed during the months following transplantation under continuation of oral specific therapy. the patient is now more than one year after stem cell transplantation alive and well without signs of toxoplasmosis and without neurological sequela. toxoplasmosis is a rare but dreaded complication usually developing between and months after stem cell transplantation with a high mortality rate. cml patients seem to be at increased risk for reactivation of toxoplasmosis after stem cell transplantation. the course of our patient, however, is unusual because of the very early manifestation after stem cell transplantation, the rapid course and prompt response to treatment. mucositis is a common side effect of chemotherapy and radiotherapy with no effective treatment. it occurs when cancer treatment destroys the rapidly dividing epithelial cells, particularly in the oral cavity, leaving the mucosal tissue open to ulceration and infection. the aim of this study was to assess the state of oral mucosa in a patient after allo-pbsct who has received palifermin, a recombinant human keratinocyte growth factor. materials and methods: a -year-old male was treated in the department of haematology of the medical university in warsaw due to the aml. conditional chemotherapy was applied, according to the bucy + atg regimen and allogeneic haematopoietic cells transplantation from an unrelated donor. he was receiving palifermin ( microg/kg/d) intravenously for consecutive days immediately before the initiation of conditioning therapy (on days - , - , - ) and after allo-pbsct (on days + , + , + ). on day + the oral mucous membrane was pale and swollen, with linea alba visible on cheeks. superficial glossitis and viral pharyngitis were noted. beginning with day + /+ proliferative gingivitis was observed. on day + gingival contour was altered and the gingiva covered nearly completely tooth crowns of all teeth. the gingiva were whitened, as if covered by thick epithelium. slight gingival hyperplasia was still observed on day + . during the forming of gingival hyperplasia the patient had a subjective "membrane growing" sensation with tingling and itching. he reported an oral cavity pain score of in the -point pain scale. since day + /+ skin rash coexisted, spreading over hairy head skin, face, dorsum and chest. disseminated papulopustular (acne-like) lesions were observed. some of them were related to the hair follicles. skin changes were present till day + . neutropenic fever was noted on day + (absolute leucocytosis . g/l). concomitant medications: orungal x mg p.o., heviran (aciclovir) x mg p.o., tazocin x . g i.v. on days + and + , maxipime x . g i.v. on days + till + , vancomycin x g on days + till + , metronidazole x mg i.v. on days + till + , neoral x mg i.v. since day + and x mg since day + , zyrtec tablet/d since day + . conclusions: palifermin is an efficient pharmaceutical in mucositis prevention in patients after allogeneic pbsc transplantation. transient complication of hyperplastic gingivitis with a concomitant skin eczema of a papulopustular nature arose. progressive fatal respiratory failure due to pseudomembranous aspergillus tracheobronchitis following allogeneic stem cell transplantation p. sedlacek, p. hubacek, k. zdrahalova, p. pohunek, o. nyc, p. pavlicek, j. stary university hospital motol (prague, cz) invasive fungal infections are frequent and often fatal in patients following allogeneic hematopoietic stem cell transplant (hsct). long-term immunosuppressive therapy for graft versus host disease (gvhd) seems to be the most predisposing factor. we present a year old girl with paroxysmal nocturnal hemoglobinuria (pnh). due to unfavourable course (severe attacks of hemolysis requiring hemodialysis, budd-chiari syndrome) she was indicated for hsct. she consequently failed to engraft two unmanipulated grafts from hla mismatched (b, cw alleles) unrelated donor. first time bone marrow was infused with cd x / /kg, conditioning consisted of treosulfan, cyclophosphamide and atg. second time peripheral blood stem cells (pbsc) were used with cd x / /kg after fludarabine, cyclophosphamide and atg. after months lasting aplasia third allogeneic graft (pbsc -cd , x / /kg) was given with no conditioning surprisingly followed by rapid and full trilineage engraftment. she than developed severe acute and chronic extensive gvhd, treated with cyclosporine a, steroids, tacrolimus, mycophenolate, sirolimus and antihymocyte globuline. during post-transplant course she suffered from bkv hemorrhagic cystitis, repeated cmv reactivations and colitis, drug induced nephropathy and steroid diabetes. she had a long-term and lasting preemptive voriconazole prophylaxis. for acute hemodynamic instability due to severe gastrointestinal bleeding months after hsct she was transferred to icu and electively intubated. at that time she was also heparinized for acute vein thrombosis. early on she started to desaturate, chest x-ray showed unilateral atelectasis. bronchoscopy revealed whitish membranes, plugs and casts causing extensive obstruction of both lungs. cultures grew aspergillus fumigatus. therapy including caspofungin, nebulized amphotericin b and repeated mechanical removal of obturating membranes failed to stop progression and patient died due to isolated respiratory failure days upon arrival to icu. autopsy confirmed fungal involvement of trachea, larynx and bronchi. pseudomembranous mycotic tracheobronchitis may be a rapidly progressing complication in heavily immunocompromised patients. complex therapy including even combination of potent antifungals may fail to overt fatal course. even in patients on antifungals this complication must be actively looked for with early use of bronchoscopy and exact identification of pathogen. systemic candidiasis is a rare but life threatening complication in immunosuppressed patients undergoing allogeneic sct. combination of new antifungal agents might improve outcome in these patients. here, triple anti-mycotic therapy is described in an all patient in urgent need of allogeneic bone marrow transplantation. the patient with t-cell acute lymphoblastic leukemia of thymic differentiation achieved remission after treatment according to the gmall / protocol. two months after the consolidation therapy relapse resistant to treatment regimens containing fludarabin, cytarabin, etoposide and amsacrine occured. even after claeg (cladribine, etoposide, cytarabine and campath- h) the patient still had % leukemic bone marrow blasts requiring high dose chemotherapy with allogeneic stem cell transplantation. one day after start of the conditioning regimen the patient showed mycotic skin manifestations and blood cultures became positive for candida cruzei despite fluconazol prophylaxis. because of the limited sensibility of fluconazol resistant candida species to liposomal amphotericin b and the high mortality rate in patients with systemic candidiasis voriconazol was added immediately to liposomal amphotericin. subsequently, the patients body temperature increased and caspofungin was added. since the mycotic skin manifestation responded to this triple anti-mycotic combination allogeneic peripheral blood stem cell transplantation from an unrelated donor could be performed. altough the fever resolved later the patient showed signs of a septic shock requiring intravenous administration of dopamine. with the unchanged triple antifungal therapy the patient became afebrile, skin manifestations showed complete resolution and cultures became negative. three months after the onset of systemic candidiasis the patient was fully active with no signs of fungal infection and in haematological and molecular remission. this case shows, that the entire intense conditioning chemotherapy could be administered and allogeneic stem cell transplantation is feasable in patients with systemic candidiasis when such combined antifungal treatment is given. i. vrelust ( ), a. gadisseur ( ), e. steel ( ), w. schroyens ( ), a. van de velde ( ), z.n. berneman ( ) ( neutropenic patients, especially following aggressive chemotherapy, are at high risk for infectious complications. these are an important contributary factor to the treatment related morbidity and mortality (trm). because neutrophils represent the first line of host defense, granulocyte transfusion therapy is a tempting therapeutic approach. although such therapy has been employed sporadically for several decades, clinical benefit has been compromised by technical problems and low granulocyte yields resulting from inadequate donor stimulation. the discovery of granulocyte colony-stimulating factor (g-csf) as a means to elevate blood neutrophil counts in healthy donors has rekindled interest in granulocyte transfusion therapy. we describe a -year old female patient with an acute myeloid leukaemia (aml) who underwent remission induction chemotherapy. in absence of repopulation of the peripheral blood the bone marrow showed persistent aml. despite antifungal prophylaxis the patient developed an invasive pulmonary aspergillosis. voriconazole, g-csf and caspofungin were added, and granulocyte infusions were started because of the continuing neutropenia. the granulocytes were collected from donors with a compatible blood group through leucaferesis after stimulation with g-csf and dexamethasone ( mg), and then irradiated. the transfusions were given three times a week. conditioning for a reduced intensity (ric) allogeneic peripheral blood stem cell transplant (pbsct) with a hla-identical brother was started. in total granulocyte infusions were given; of which after transplantation, the last on day + after pbsct. they were discontinued when the anc count reached . x /l. all granulocyte infusions were tolerated very well. four months after transplantation she is in a complete hematologic remission without signs of graft-versus-host-disease (gvhd), and without signs of active pulmonary infection. the precise role of donor granulocyte infusions remains to be delineated, partly because of the lack of defined clinical trials. we conclude that granulocyte transfusion therapy may be useful for neutropenia-related fungal infections in patients with hematologic malignancies. the use of granulocyte infusions during a ric-pbsct procedure does not seem to lead to an increased risk of gvhd or hamper engrafment itself. a. kerguelen, m. canales, a. lopez, m. martin, t. cobo, d. hernandez, j. g-bustos, f. hernandez-navarro university hospital la paz (madrid, e) introduction: patients with haematological diseases previously diagnosed with invasive fungal infection (ifi) are considered to be at high risk of suffering reactivation of the infection during subsequent intensive chemotherapy. methods: in the last years patients with haematological diseases ( aml and acquired aplastic anaemia) and previous invasive aspergilosis have undergone allogeneic haematopoietic stem cell transplant in our centre. all patients received as primary antifungal therapy combination of liposomal amphotericin b (ambisome) and caspofungin displaying complete or good partial radiological resolution of the infection. itraconazole and voriconazole was continued as secondary prophylaxis. conditioning regimen consisted of busulfan and cyclophosphamide in patients with aml and atg and cyclophosphamide in the aplastic anaemia patient. cyclosporine in combination or not with methotrexate was the regimen administered to prevent ghvd. results: in a patient with aml no clinical or radiological signs of reactivation of fungal infection were observed through the transplant procedure. in the other patient with aml itraconazole was changed by liposomal amphotericin because of unresponsive fever. in the patient with aplastic anaemia combined therapy with amphotericin and caspofungin was initiated because of radiological worsening, but galactomannan antigen was negative in all analysis performed. no patient has died because of infectious complication during or after transplant. conclusion: availability of new antifungal agents does allow pre-transplant therapy of previous ifi, aiming to achieve a clinically undetectable state of infection, and an adequate antifungal treatment during transplant to diminish risk of reactivation of fungal infection in allografted patients. however, the optimal use of antifungal agents or their combinations remains to be determined and more studies are necessary to confirm our experience. the frequency of invasive fungal infections, in particular infections due to aspergillus and other moulds, has increased over the past two decades. whereas invasive aspergillosis mainly involves the respiratory tract, lung or sinus, the lower gastrointestinal tract is rarely affected, most often in the frame of secondary dissemination. primary invasive aspergillosis of the gut is a rare event associated with high mortality and has not been reported to date in patients after autologous stem cell transplantation (sct). we report on a -year-old boy who developed isolated intestinal aspergillosis soon after autologous stem cell transplantation for pnet of the central nervous system. the boy received broad-spectrum antibiotics because of neutropenic fever, and antimicrobial prophylaxis included trimethoprim-sulfamethoxazole, metronidazole, acyclovir, fluconazole and topical amphotericin b. antimycotic therapy was started because of persistent fever and abdominal pain, and rising serum levels of galactomannan and the isolation of aspergillus fumigatus from the stool suggested invasive aspergillosis. the boy underwent enterostomy on day + , and diagnosis of intestinal aspergillosis was pathohistologically confirmed. no other site of invasive aspergillosis was evident. the patient was treated with antimycotic combination therapy consisting of liposomal amphotericin b, voriconazole and caspofungin. the clinical condition slowly improved over the next months. enterostomy was removed on day + and antimycotic treatment has been stopped soon after. currently, on day + , the boy is at home without major gastrointestinal complaints. we conclude from this case that primary intestinal invasive aspergillosis can occur in patients undergoing autologous stem cell transplantation, and therefore, this diagnosis has to be considered in this setting in patients suffering from fever and abdominal pain. in case of positive galactomannan antigenemia, an extensive search for invasive aspergillosis should be performed and, at the same time, early antifungal therapy should be started. mycobacterial disease is a rare and difficult diagnosis in hematopoietic transplant recipients. we report the case of a twenty-year-old woman who underwent a second matched unrelated donor (murd) hsct for a null all in second complete remission. conditioning regimen consisted on cyclophosphamide mg/msq; busulfan mg/kg and thiotepa mg/msq. on day + , while receiving acyclovir prophylaxis, and a previous cotrimoxazole prophylaxis, she developed an acute ascending paraparesis with anaesthesia and reflex loss and loss of sphincter control. mri imaging showed gadolinium contrast enhancement on cauda equina roots. csf analysis showed elevated cell count ( /mm³, normal range nr: - /mm³), elevated csf protein concentration ( mg/dl: nr: - mg/dl), and no hypoglucorraquia ( mg/dl with simultaneous plasma concentration of mg/dl). flow cytometry analysis showed % t lymphocytes, . % non clonal b lymphocites, , % monocytes and , % neutrophils. bacterial, fungal and mycobacterium presence was ruled out and empirical antiviral treatment consisting on foscarnet ( mg /kg /day), immunoglobulin ( . g/kg/ hours) was started. metilprednisolone g per day for tree days flowed by mg per day for three consecutive weeks. no clinical response was observed and neurotrophic viral infection was excluded by pcr technique. a second lumbar puncture was performed days later showing a lower number of lymphoid cells ( /mm³) but with an increase in protein concentration ( mg/dl) without hypoglucorraquia; moreover auramine positive rods were present. antituberculous treatment with rifampicin ( mg/d), isoniazide ( mg/d), ethambutol ( . g/d) and pyrazinamide ( g/d) were started, with no clinical improve but no worsening. hepatic toxicity was developed and isoniazide an rifampicin were suspended after a month of complete treatment and ofloxacin ( mg/ h, ethambutol and pyrazinamide was started as a second-line therapy. this is to our knowledge the first reported case of transplantationrelated tuberculous arachnoiditis. pentastomiasis of the liver in a patient following unrelated stem cell transplantation with estimated hepatic acute graft-versus-host disease j. dahlke ( ) , r. kobbe ( ), a.a. oyekunle ( ) , f. ayuk ( ) , n. kröger ( ) we report a case of human hepatic pentastomiasis (armillifer armillatus) in a -year old man with aml who immigrated to germany from togo in and died as a result of relapse, days post hla-matched unrelated stem cell transplantation. at day + post transplantation, diarrhoea occurred and gastroscopy with excisional biopsy revealed an acute graft versus host disease of the stomach and upper small bowel. for treatment we started with a tapering schedule of methylprednisolone ( mg/kg body weight/day). three days later we observed a hyperbilirubinemia and slightly increased liver enzymes. the abdominal ultrasound showed an increased size of the liver and an increased portal perfusion. the findings were interpreted as a hepatic involvement of the acute graft versus host disease (agvhd) and a salvage treatment with mycophenolat mofetil was initiated. the patient died as a consequence of intracerebral bleeding in a cerebral infiltrate of the leukaemia. at autopsy, in addition to the cerebral findings, we found multiple pentastomides (documented by photographs) up to cm length (spec. armillifer armillatus) subcapsular, in the parenchymatous tissue and in the portal veins. no agvhdlike inflammatory infiltrate in the liver was observed. late effects and quality of life r trilineage hypoplasia after initial neutrofil engraftment in patients with acute myeloid leukaemia undergoing autologous transplantation with bu/cy conditioning: frequency, outcomes and prognostic significance (case report) a. pivkova, l. cevreska, n. siljanovski, z. stojanoski, s. genadieva stavrik, i. panovska, s. krstevska balkanov, s. trajkova, b. georgievski clinical center (skopje, mk) myeloablative conditioning with autologous stem cell transplantation (asct) is a treatment option for aml patients with lack of sibling donor. hematopioetic engraftment may be prolonged depending on the type of myeloablative regimen, mobilization regimen, dose of mnc and patients age. in a year period ( ) ( ) ( ) ( ) ( ) ( ) we realized a total of autologous transplanations with cryopreserved peripheral blood cells (pbsc) and busulfan and cyclophosphamide conditioning. we present two cases ( %) of aml (standard risk) patients (two females and years at transplant) that underwent autologous transplantation at department of hematology, skopje. mobilisation of pbsc was preformed with vp- mg/msq + g-csf µgr/kg in one patient and hd-arac gr/mscx , idarubicine mg/msqx +g-csf µgr/kg in the other patient. a minimum of , x /kg mnc and , x /kgmnc respectively were collected and preserved in % dmso solution. we registered engraftment for ne> , x /l on day + and + and for plt> x /l on day + and + . patient were followed up in outpatients and pancytopenia was registered one month after transplant with plt< x /l with mild haemorrhagic complications plt transfusions dependable, hb < g/dl and red cell transfusion dependence (on two week interval), wbc < , x /l with cytokine treatment (twice a week g-csf µgr/kg). bone marrow biopsy revealed trilineage hypoplasia, no signs of organomegaly, no microbiological and viral findings. one patient has recovered completely months after transplantation and the other is still in good physical condition but present pancytopenia months posttransplant. we conclude that prolonged pancytopenia is due to bu/cy conditioning, the age could be a significant factor for starting myeloablative conditioning prior autologous transplant as well as minimum mnc dose could prolonge immune reconstitution. neurological long-term follow-up after allogeneic bone marrow transplantation p. sostak, c. padovan, m. holtmannspötter, g. ledderose, h. kolb, a. straube klinikum großhadern (munich, d) to improve our knowledge about the neurological outcome after allogeneic bone marrow transplantation (bmt), we have started a prospective study already years ago. so far we could show that within the first year after transplantation a significant proportion of patients ( %) had developed neurological sequelae. besides well-defined neurological complications more than half of the study population suffered from new neurological abnormalities of unknown origin predominantely affecting the peripheral nervous system. in a small subgroup of patients already central nervous signs, cognitive deficits and white matter lesions could be detected and this was in relation to chronic graft-versus-host disease (gvhd)/immunosuppression. to determine whether central nervous system (cns) involvement during gvhd might manifest at a later time in more bone marrow recipients, the follow-up period now was extended and patients again received a neurological examination, underwent a neuropsychological test battery and standard mri sequences. long-term follow-up results will increase the insight onto the spectrum, incidence and etiology of neurological sequelae after allogeneic bmt. they will be discussed in relation to retrospective and experimental data, which suggest involvement of the cns during gvhd. keratoconjunctivitis sicca with recurrent calcium deposition in the cornea and severe visual loss due to gvhd p. keslova ( ) we present severe keratoconjuctivitis sicca with recurrent calcium deposition in the cornea after keratoplasty in a patient with extensive chronic graft versus host disease (gvhd). a -years-old girl with myelodysplastic syndrome (raeb) underwent stem cell transplantation (sct) using peripheral blood stem cells of two alleles mismatched unrelated donor (b, cw). conditioning regimen consisted of busulfan, cyclophosphamide and melphalan, for gvhd prophylaxis combination of rabbit antithymocyte globulin (atg fresenius) and cyclosporine a (csa) with short term methotrexate were used. due to renal toxicity csa was early switched to tacrolimus and corticosteroids were started at day+ . week after she developed steroid resistant acute gvhd grade iv with skin and gut involvement. for that she was successfully treated with combination of steroids, mycophenolate mofetil and sirolimus (day+ through day+ ). during period of acute gvhd severe keratoconjuctivitis sicca with deep corneal defects has been developing and visual acuity had deteriorated. there were increasing pancorneal calcium deposits (calcareous degeneration). serum calcium and phosphate levels were normal at several times points. longterm immunosuppression improved symptoms of gvhd but healing of corneal defects was not reached despite local therapy of antibiotics and corticosteroids. at day+ patient underwent removal of calcium deposits and both eyes were covered by amniotic membrane. at day+ keratoplasty in the left eye was performed with transient improvement of visual acuity. two weeks after keratoplasty calcified plaques have recurred in the transplanted tissue and fast visual loss reappeared. we suggest that ectopic corneal calcifications are probably associated with persistent epithelial and stromal defects and keratoconjunctivitis sicca as a symptom of persistent active chronic gvhd. combined immunosuppressive therapy should therefore continue. we plan to repeat surgery at the later time under better control of gvhd. patient is now one year after sct with no signs of leukaemia relapse with full donor hematopoiesis, but prognosis of vision still remains at this moment uncertain. supported by vz fnm mzo thyroid dysfunction is an important problem in patients receiving bone marrow transplantation. however there was no case of hashimoto encephalitis in a patient after stem cell transplant reported. we describe the case of year old female patient who underwent allogeneic bone marrow transplantation and who developed hashimoto encephalitis. aml patient was subjected to allogeneic stem cell transplant with reduced intensity conditioning (flag-ida) in october . early posttransplant period was complicated by reactivation of cmv infection and prolonged peripheral cytopenia. months after transplant the patient chimerism analysis revealed graft rejection without aml relapse. months after transplantation the patient developed fatigue, loss of appetite, vomiting but also psychiatric symptoms: hallucinations and paranoid ideations. ct scans does not revealed anything specific. typical psychiatric treatment was not efficient. later patient experienced episodes of epilepsy and developed cerebellar ataxia and progressive unilateral paresis with impaired consciousness. in accessory investigations -in csf elevated level of proteins and eeg abnormalities were noted, blood and csf cultures were negative, thyroid hormones level were slightly decreased, tsh and atpo antibodies titers were elevated. results of mri confirmed disseminated pathologic changes in white matter. hashimoto disease with encephalitis was diagnosed and the patient was treated with high dose methylprednisolone i.v. for days with rapid improvement noticed within first h of the treatment. the neurological state normalize within one week. maintenance treatment with decreasing dose of oral prednisone is carried out. occurrence of hashimoto encephalitis in described patients seems to be connected with chronic graft rejection process and impaired prolonged regeneration of immunity after transplant. the role of different infections mainly viral should also be investigated. bmt: care about donors a. tellier, c. bauchetet, z. marjanovic, j.-p. marie, b. rio hotel-dieu paris (paris, f) purpose: bmt improvement must take in consideration ethical issues regarding donors. based on donors refusal cases report, our analysis underlines the significance of identifying «unwilling donors». moreover, we are concerned about the process of obtaining informed consent of family membres to undergo hla histocompatibility tests in order to prevent psychological consequences of peripheral blood and bone marrow donation. background: studies about psychological issues of bone marrow donation show that donors may be worried about their health status (switzer et al., ; molassiotis et holroyd, ) . switzer et al. correlate donors difficulties to their hesitation during donation decision process and consider that screening donors motivations is useful to a psychoprophylaxis approach. other authors note that families donors are even more exposed to psychological problems (chang et al, ) . directly concerned by recipient reactions to bmt, family donors (wollcott et al, ) may feel unconscious guilt in cases of unsuccessful or complicated bmt (futterman and wellisch, ) . deeper psychodynamics of bmt process indicates that donor can confuse biological (hla) and psychological identity and be anxious about being ill. (alby, (alby, , ascher, ascher, , . topall-rabanes et al. ( ) notice that about % of studied family donors are classed as «reluctant» : for these subjects, donation decision is not considered as a real choice. they suffer from health problems and feelings of regret even longtime after donation. methods and results: we reviewed the records of donor refusal cases representing % of allografts performed in our twenty years bmt practice. in-depth analysis of patients psychosocial situation, quality of social support, family members relationships, patient-family-staff communications modalities, in particular regarding to donation information process. we will present these cases and retrospective analysis to introduce ethical questions on hla histocompatibility testing. conclusion: regarding to our experience and to our data, it seems that information process of family membres about bone marrow donation may lead to an impossible choice. appointed by hla testing as donor, family member is moved to suffer of biological determination and to become «unwilling donor». protecting these persons is a medical responsability. more research is necessary on this subject. there are patients with paranoiac reactions (persecution, sensitive, litigious, invention) were possible to allocate among the mental disorders at our patients. results: paranoiac reactions with ideas of persecution (n = ) are the most often in our sample. in such cases patients suspect those around them, especially medical personnel, in preconceived attitude to them and, possibly, to conspire for damnification of patients. such ideas have a systematic character and are resistant for treatment. sensitive paranoiac reactions (n = ) are joined with perception of physical handicap and characterized by sensation of slighting attitude and mockery those around patient and by confidence in dissemination of detractive rumours. litigious paranoiac reactions (n = ) are characterized by lowsystematized and insufficiently well-founded requests and multiple joined with hematological malignancies such as requests to compensate the prejudice applied by disease or laying claims to medical personnel are at fault, in opinion of patient, in the prejudices joined with disease. invention paranoiac reactions (n = ) in our sample are characterized by elaboration of self-treatment methods of his/her disease. treatment of examined states was significantly resistant and included antipsychotic and anxiolitic medications. conclusions: pr at the patients with hemato-oncological disorders after bone marrow transplantation are the separate problem demanding the special attention both the hematologist and the psychiatrist, especially relative to its treatment. b.v. afanasiev ( ), n.v. stancheva ( ), l.s. zubarovskaya ( ), e.v. semenova ( ), m.a. ovsyannikova ( ), t.i. ionova ( ), t.p. nikitina ( ), a.a. novik ( ) ( )st. petersburg state medical university (st. petersburg, background: quality of life (qol) is increasingly used as a treatment outcome along with traditional clinical outcomes in children with cancer undergoing bone marrow transplantation (bmt)/stem cell transplantation (sct). the aim of our study was to estimate qol parameters and symptoms in survivors of childhood blood cancer after bmt/sct. patients and methods: fifteen survivors were evaluated - years (median, years) after allogeneic bmt/sct for acute leukemia ( ), chronic leukemia ( ) and myelodysplastic syndrome ( ) . median age at transplantation - yrs (range . - . ), girls/boys- / . pedsql™ generic core scales and sf- questionnaires were used for qol assessment in the group younger than yrs at the time of the survey and in the group yrs and older, respectively. nj children cancer symptom inventory and md anderson symptom inventory were used for symptom assessment in the younger and older groups, respectively. for comparison healthy controls ( for younger group; -for older group) matched to survivors by age and gender were included in the study. results: no significant differences in qol parameters (physical, psychological and social functioning) between survivors and control group were revealed. only school functioning for children younger than yrs was lower in the group of survivors ( vs , p< . ). seven survivors experienced moderate or severe symptoms ( to scores on - scale). four of them had pronounced psychological symptoms. other pronounced symptoms were chronic pain, fatigue, lack of appetite, shortness of breath, drowsiness and nausea. six survivors had at least two moderate or severe symptoms. qol parameters, namely, physical, psychological and social functioning in long-term survivors of childhood blood cancer post allogeneic bmt/sct is comparable to healthy people. however, nearly half of survivors experience different symptoms in long-term period after transplantation. this confirms the importance to monitor and control late related symptoms in order to preserve qol of long-term survivors of childhood blood cancer. differential diagnosis of t-cell lymphoproliferative disease after allogeneic haematopoietic progenitor cell transplant s. koschmieder, m. stelljes, a. schmidt, g. silling, t. spieker, g. köhler, c. renne, w.e. berdel, j. kienast university of muenster (muenster, d) two cases are presented of a rapidly developing cervical lymphadenopathy days after allogeneic peripheral blood stem cell transplant for acute myeloid leukemia. patient # , a year-old female, developed painful bilateral cervical adenopathy (up to cm) overnight, fever ( . °c), and a fine maculopapulous rash. no further enlarged lymph nodes were found by computed tomography, and the right cervical mass was removed. histology showed infiltration of the lymph node by atypical lymphoid cells that expressed cd and cd . molecular analysis revealed monoclonally rearranged tcr gamma chains. histology of the skin showed a leukocytoclastic vasculitis without any signs of acute gvhd. ebv pcr was consistently negative in peripheral blood and lymph node. the diagnosis of t-ptld was made, and the patient received a -day course of methylprednisolone. with this regimen, the patient´s condition rapidly improved, and the fever, lymphadenopathy, and rash resolved completely within a few days. patient # , a year-old female, developed painful acral bullous erythematous lesions on her arms, legs, and in her mouth on day post transplant which progressed to generalized maculopapulous rash. histology showed acute graft versus host disease (gvhd) of the skin. on day + , the patient presented with painful bilateral cervical adenopathy (up to . cm), and abdominal ultrasound and mri showed no other regions of lymphadenopathy. histology of one removed lymph node demonstrated a highly proliferating, diffuse infiltrate of cd positive t cells with % of t cells expressing cd and % expressing cd , respectively, as well as scattered cd positive cells. molecular analysis detected a polyclonal pattern of tcr rearrangement and an oligoclonal immunoglobulin receptor rearrangement. ebv pcr was negative in peripheral blood and lymph node. the clinical scenario was interpreted as concomitant lymphadenopathy associated with acute gvhd, and the patient received methylprednisolone for days and completely recovered. this report highlights the necessity to remove suspiciously enlarged lymph nodes, developing after allogeneic transplant, in order to distinguish between t-ptld and lymphadenopathy accompanying acute gvhd. the evaluation of t-cell receptor gamma gene expression for prognosis of relapse development after stem cells transplantation a. sipol, d. butlitskiy , u. vorobjeva , a. aljanskiy , m. zarayskiy , l. zubarovskaya, b. afanasyev st. petersburg state med pavlov university (st. petersburg, rus) background: the hematopoetic stem cells transplantation (hsct) in patients with hematological disorders is the most radical method of the therapy. the early diagnostics of relapse in post-hsct period could improve significantly the therapy effectiveness. the purpose of this study was to develop the minimal residual tumor cells detection method by studying the mediated mechanisms of organism's reaction to tumor clone. materials and methods: nineteen patients with different hematological malignances, who were undergone the hsct, have been included in the research. total mrna was extracted from peripheral blood leukocytes, sampling in the time of conditioning regime completion, just prior to stem cells transfusion. we performed the rt-pcr with primers specific to v-j-gamma junctions (tcr-gamma gene). specific signal was detected in % agarose gel. results: absence of tcr-gamma gene expression at day- were significantly more often in group of the patients who were staying in remission after hsct (p= . ). in group of the patients with myeloablative pre-transplant conditioning the significantly differences in tcr-gamma gene expression depending on the fact of relapse has not been revealed. in group of the patients with reduce intensity conditioning regime the positive correlation between the presence of tcr-gamma gene expression at day- and relapse in post-hsct period was observed (p= . ). conclusion: the definition of tcr-gamma gene expression before hematopoietic stem cells infusions is the method of a tumor process activity estimation during the therapy, especially in case of using the reduce intensity conditioning regime. we suggest this criterion as the early prognostic factor for the relapse developing in post-hsct. mrd-directed adoptive immunotherapy following allogeneic stem cell transplantation fails to permanently revert disease progression in childhood acute lymphoblastic leukaemia between viii/ and vii/ we performed allogeneic haematopoietic stem cell transplantation (hsct) in consecutively children with acute lymphoblastic leukaemia (all). we analysed prae-and post-transplant minimal residual disease (mrd) levels using quantitative rq-pcr targeted to immunoglobulin and/or t-cell receptor rearrangements in of them with available targets (with adequate sensitivity and specificity according to esg-mrd-all criteria). seven of patients with detectable mrd prior hsct (n= ) relapsed after transplant and one died in ccr before day + due to transplant related complications. in the group of prae-hsct mrd negative patients (n= ), only one relapse appeared. in a total of patients, there was a time-frame for an attempt to avert the disease progression detected by rq-pcr. in one bcr/abl+ patient, imatinib mesylate dose was increased and doses of dli ( . x /kg cd + cell) were administered. despite this, patient relapsed + days after hsct. second bcr/abl+ patient developed molecular relapse despite chronic gvhd. therefore, immunosuppression was quickly discontinued and imatinib mesylate was administered. he achieved temporary molecular remission but months later died of cns disease progression. third patient developed mrd positivity + days after hsct and therefore immunosuppression was quickly tapered. gvhd reactivation required steroid and csa treatment. after gvhd resolution immunosuppression was ceased and three escalating doses of dli ( x , x , x cd +/kg) were given. nevertheless, this did not avert haematological relapse. this patient after high-dose chemotherapy achieved second complete remission (cr) with mrd negativity prior to second hsct and now is months after hsct in continuous cr and mrd-negative. the fourth patient (bcr/abl+) developed relapse despite three dlis and imatinib mesylate treatment given for positive mrd + after hsct. adoptive immunotherapy (rapid cessation of immunosuppression, infusion of dli in to weeks interval and/or use of imatinib mesylate in bcr/abl+ all) after the transplantation was not successful in our cohort in relapse prevention and might only postpone the manifestation of relapse and facilitate further efficacious chemotherapy and retransplantation. we are confident that all effort should be aimed to a better control of the pre-transplant mrd levels. grant support: fnm , mz - , gauk / , mz and msm . the dynamics of chimerism evolution determines the differential outcome of various transplant settings i. buño, p. balsalobre, g. iglesias, d. barroso, c. manzano, r. carrión, d. serrano, a. gómez-pineda, j.l. díez-martín hosp. g.u. gregorio marañon (madrid, e) background: several factors such as the intensity of the conditioning regimen, the t-cell content of the graft or the gvhd prophylaxis, influence the degree of chimerism after sct. objective: to evaluate the dynamics of chimerism after different sct settings (ablative, reduced intensity conditioning -ric-and t-cell depleted -tcd-) and its influence in the success of the procedure. patients and methods: sct: ablative (including from mud), ric and tcd (including from haploidentical donors and from mud with hla disparity). chimerism analysis was performed by fish or str-pcr (sensitivity %). samples: bone marrow (bm) and peripheral blood (pb) on days + , + , + , + and once a year thereafter, as well as pb and leukocyte lineages (t lymphocytes cd +, b lymphocytes cd + and myeloid cells cd + isolated (purity > %) by immunomagnetic means, automacs, miltenyi biotec), every weeks, starting on day + and until complete chimerism (cc) was achieved. results of chimerism follow-up were censored once the diagnosis of relapse or rejection was established. results: incidence of mixed chimerism (mc) on day + (ablative: bm %, pb %; ric: bm %, pb %, cd %; tcd: bm %, pb % , cd %), as well as its dynamics (mc on day + : ablative bm %, pb %; ric bm %, pb %, cd %; tcd bm %, pb % , cd %) were different in the three sct settings. moreover, the percentage of recipient cells (%r) was significantly higher after ric and tcd than after ablative sct, as well as in t lymphocytes than in bm or pb ( / cases with simultaneous studies showed mc in t lymphocytes and cc in pb). all ric sct evolved to cc by day + while tcd sct showed persistent mc ( patients with stable mc after one year). the incidence of rejection was greater after ric ( / ) and tcd ( / ) than after ablative sct ( / ). all these patients showed mc, mainly in t lymphocytes, which allowed early diagnosis and successful treatment with immunosuppression withdrawal and donor leukocyte infusion. patients with cc in pb/t lymphocytes on day + had a higher incidence of gvhd>i than those with mc. in the present series, however, a relationship between chimerism and relapse, disease free survival or overall survival, was not observed. conclusions: sct with greater incidence of mc (ric and tcd) favor immune tolerance between donor and recipient which reduces the risk/severity of gvhd at the expense of a higher incidence of graft rejection. reliable quantification of haematopoietic chimerism after allogeneic stem cell transplantation by real-time quantitative pcr analysis a. picardi ( ) introduction: increasing mixed chimerism (mc) represents a poor prognostic factor after allogeneic stem cell transplantation (sct). moreover, to define the best timing of immune-suppression withdrawal and donor lymphocytes infusion, a strict monitoring of donor hemopoiesis is needed. methods: we evaluated donor/recipient pairs using a quantitative real-time pcr (qrt-pcr) with the aims ) to evaluate the informativeness of this chimerism assay and ) to compare qrt-pcr analysis with standard methods such as fluorescence in situ hybridization (fish) for mismatched sex pairs or variable nucleotide tandem repeats (vntr) for matched sex pairs. qrt-pcr (lightcycler . , roche) was performed on bone marrow and peripheral blood samples collected monthly, using eleven biallelic dna genetic system located on chromosomes , , , , , , , x and y. glyceraldeyde phosphate dehydrogenase (gapdh) gene was used as active reference. before quantification, donor and recipient dnas were genotyped using primers and probes specific for all genetic markers. patients had a median age of . years (range - ) and were affected by acute leukemia (n= ), or lymphproliferative disorders (n= ). standard regimen was used in cases, reduced intensity conditioning in , while patients underwent an unrelated sct. median follow-up of the patients was . months (range . - . ). results: both qrt-pcr and fish detected donor/recipient differences in % of pairs, while vntr was not informative in % of sex matched pairs. mixed chimerism was observed in / patients ( . %) using qrt-pcr and in of the patients ( . %) evaluable with standard methods. overall, / patients ( . %) relapsed; before relapse, mixed chimerism was observed in all patients by qrt-pcr and in / by fish/vntr. qrt-pcr detected mixed chimerism days (range - ) earlier than standard methods. in cases in which vntr was either not informative or not predictive for relapse, the interval between detection of mixed chimerism by qrt-pcr and relapse was and days, respectively. conclusions: chimerism determination using qrt-pcr is more informative than standard methods and may represent an useful tool for the follow up of allogeneic sct. reduced intensity transplant programme. a single-centre experience a. bonini, a. tieghi, f. merli, l. gugliotta asmn (reggio emilia, i) from we introduced a reduced-intensity conditioning regimen for allogeneic transplants(allo). this regimen is tailored for old patients (pts) or for those who have previously received an allo or autologous bmt. at now we have transplanted pts; of them received previously an autologous and an allo bmt. they were males and females and the donors were males and females. the mean age of the pts was yrs (range - yrs).the diagnoses were: hd, nhl, mds, renal cancer, sarcoma, myeloma and cml; the solid tumors were all metastatic, pts were in pr, in ii cr, in iii cr and the one with mds at the onset. the abo compatibility was:complete for , minor for and major for . the cmv status was: donor/pt positive for and positive donor/negative pt for . the conditioning regimen consisted of thiotepa, fludarabine and cyclophosphamide except for the pt with cml who received busulphan and fludarabine. the source of stem cells was peripheral blood in and bone marrow in and the stem cells were cryopreserved after the harvest. the gvhd prophylaxis consisted of csa and short course mtx. the pts who received peripheral stem cells the mean cell dose was x /kg cd + cells, for the one who received bone marrow was . x /kg mnc. the mean time to reach pmns > . x /l and plts > x /l was respectively days(range - days)and days (range - days). the mean number of transfused rbc and plt units was respectively (range - )and (range - ). the mean grade of mucositis (according to the who classification) was . the major complications during neutropenia were fuo, gram + bacteremias and cerebral aspergillosis. no cases of vod of the liver were observed. pts had agvhd( grade and grade ) and limited cgvhd. two pts developed cmv reactivation after allo; the complication was cured with gancyclovir. nine over pts died: for disease (the solid tumors but for the with renal cancer a response even if transient was observed and nhl), for acute gvhd and for cerebral aspergillosis. the chimerism was complete for all the evaluable pts at days. none needed dli. six patients are in cr with a median follow up of year ( were in partial remission and in ii chronic phase at transplant) and relapsed. this pilot study demonstrated an acceptable regimen-related toxicity(according to the age and previous chemotherapy including transplants), the possibility to reach a very early full-donor chimerism and to cure high risk pts. introduction: autologous stem cell transplantation (autosct) is usually preferred to allosct, due to its widespread availability, lack of the immunological problems intrinsic to the development of graft-versus-host disease (gvhd), and the infrequent bone marrow involvement present in hodgkin's disease(hd), for patients undergoing high-dose chemotherapy/radiotherapy. allosct has been associated with a high transplant-related mortality (trm) in patients with hd due to a high incidence of gvhd and of fatal infectious events after transplantation. the poor outcome of these patients after allosct may reflect in part the advanced status of the disease at transplantation and the poor performance status of the patient population allografted. furthermore, the high trm present in the conventional allosct setting has never allowed a proper evaluation of a possible graft-versus-hodgkin's effect. in an effort to reduce the trm associated with allosct, low-intensity regimens have been developed; the curative potential of these protocols would rely on the graft-versus-leukemia effect of the allogeneic infusion more than in the conditioning regimen per se. case: in our hospital a total of patients with relapsed hodgkin's disease underwent reduced-intensity conditioning (ric) allogeneic stem cell transplantation (allo-sct) from an hla-identical sibling. we explored reduced-intensity allografts using fludarabine-melphalan conditioning and early withdrawal of immunosuppression as an alternative to palliative chemotherapy. graft-versus-host disease (gvhd) prophylaxis was mini-methotrexate and ciclosporine with weaning at day . the age of patients was , and years. the time from initial diagnosis was , and months and from autograft was , and months. the patients were in refractory relapse. time to neutrophil recovery (absolute neutrophil count ≥ /microl) was days for patients and days for the other. time to platelet recovery ( platelet count ≥ /microl) was , and days.the -day mortality was . none of them developed acute gvhd. one patient developed mild chronic gvhd. as of november patients remain in complete remission. conclusion: although the number of hd patients allografted with reduced-intensity protocols is low and the follow-up still short, it seems that the reduced-intensity allogeneic stem cell transplantation is effective in relapsed and refractory hodgkin's disease where autografts have failed. remarkable reduction of acute gvhd and infectious complications after reduced-intensity conditioning and low-dose ( mg) campath- h p. reményi, Á. bátai, b. kapás, a. sipos, s. lueff, i. bodó, m. réti, v. goda, t. masszi national medical centre (budapest,hun) during the four month-period from may, to september, we performed reduced intensity (ric) allogeneic transplantation for five patients with their hla indentical sibling donors. the median age of the female and male patients was . years ( . - . ). two patients had chronic lymphocytic leukemia (cll), one hodgkin's disease (hd), one follicular nhl grade i (fl) and one myelodysplastic syndrome (mds). the hd patient relapsed after previous autologous stem cell transplant. the fl patient was in complete remission, the others were in partial remission before transplant with low tumour burden. the conditioning regimen consisted of x mg campath- h and fludarabine x mg/m² for all patients, adding melphalan ( mg/m²) for the lymphoid malignacies, and busulphan ( mg/kg) for the mds patient. for gvhd prophylaxis mg/kg cyclosporin a (continuously) + mg/m² methotrexate was given on days , , . all patients engrafted. one patient developed grade i acute gvhd. two patients had febrile neutropenia, one developed central venous line infection. no one had cmv reactivation or disease. after a median of day-follow up ( - days) all patients developed full donor chimera tested by vntr pcr. the two cll patients are in remission proven by flow cytometry (less than % cd /cd + cells). the fl patient is in cr and received four courses of mabhtera ( mg/m²) as maintenance therapy. the mds patient is in cr according to bm histology, but still thrombopenic ( g/l). the hodgkin's patient has active disease with minimal tumor burden with mixed chimerism at day , now waiting for dli. conclusion: low dose ( mg) campath- h+ csa/mtx gvhd prophylaxis is a well balanced regimen regarding the incidence and severity of acute gvhd, infectious complications and gvl effect after ric conditioning. these preliminary results -especially concerning the late infections complications -compare much better to those we observed in our previous series of ric transplants with higher doses ( or mg) of campath- h, or that we read in the literature. with the publication of these preliminary results we would like to underline the message that less is better regarding campath- h in reduced intensity conditioning. conditioning regimens consisted of fludarabine mg/m²/day x days plus melphalan mg/m²/day x day (n= ) or plus oral busulphan mg/kg/day x days (n= ) or plus cyclophosphamide mg/kg/day x days and globuline antithymocyte . mg/kg/day x days (n= ). the patients were grafted with bone marrow (n= ), cord blood (n= ) or pbsc either unmanipulated (n= ) or cd + selection (n= ) or cd /cd depletion (n= ). gvhd prophylaxis was performed with csa+ mtx (n= ), csa only (n= ) and csa + steroids (n= ). donors were either related (n= ) or unrelated (n= ). the median number of cd + cells infused was . x /kg recipient bw (range . - . ). results: there were a rapid recovery of neutrophils (median days; range - ) and platelets (median days; range - ). the median length of hospital stay was days ( range - ). with a median follow-up of months (range - ) the incidence of agvhd and cgvhd were ± % and ± % respectively. the probability of trm was ± %. patients grafted with manipulated pbsc had the lowest trm ( ± %). the relapse incidence was ± %. high number of infused cd + cells (p= . ) and cgvhd (p= . ) was associated with a lesser ri. the event-free survival was ± %. nine patients died of relapse or progressive disease (n= ), agvhd (n= ), cgvhd (n= ) and other (n= ). conclusion: fludarabine-based ric provide a good alternative to myeloablative conditioning for allogeneic transplantation either malignant or non-malignant disease in children. induction: treatment related mortality is the trade off of allogeneic transplants. although the probability at two years has been reduced to - % following non-myeloablative (reduced intensity conditioning) allogeneic transplant it remains a barrier to administer these transplants. we desired to identify mechanisms that compromise safety or would enhance safety. methods: the different outcome parameters in transplantation were defined as safety or efficacy parameter and per safety and efficacy parameter the literature was searched for investigational method, prophylactic, supportive or therapeutic measurement. furthermore the toxicity scales were reviewed and known side effects were listed and include in the search. results: outcome parameters defined as safety parameter included engraftment, disease recurrence; side effects of major concern, acute and chronic graft versus host disease, infections, death as well as life threatening side effects defined by the safety scales. more that first complete remission was considered and adverse safety parameter. ) engraftment failure was noted after inadequate cytoreductive treatment such as cgy tbi and cyclophosphamide or underdosing of fludarabine (< mg/m²); adequate dosing of cytoreductive and immune suppressive treatment appeared critical for rapid trilineage engraftment. ) tbi appeared associated with higher risk of graft versus host disease and conditioning with drugs only. ) in vivo t-cell depletion by f.e anti-thymocyte globulin was associated with high risk of cytomegali virus reactivation. ) t-cell depletion of the graft is associated with increases risk of relapse ) b-cell depletion of the graft seems to reduce the risk of chronic graft versus host disease ) cmv positivity is not a prerequisite for cmv reactivation ) a combination of immune suppressive agent reduces the risk of acute graft versus host disease discussion: based on these and less stringent criteria we defined a practice guideline and make recommendations for reduction of side effects and treatment related mortality. the method developed model will be presented. monitoring of mixed chimerism by single nucleotide polymorphism analysis in two children supported with granulocyte transfusions after allogeneic stem cell transplantation w. schwinger, p. sovinz, h. lackner, h. dornbusch, m. benesch, a. moser, g. lanzer, c. urban medical university graz (graz, a) objectives: neutropenia is a major risk factor for early transplant related mortality in children undergoing haematopoietic stem cell transplantation (hsct). in case of infection, refractory to antibiotic therapy the combination with allogeneic granulocyte transfusions is a logical approach to manage this problem. these patients are chimeras of at least three different cell populations (recipient -stem cell donorgranulocyte donor). two patients where followed closely by single nucleotide polymorphism (snp) analysis to reveal the duration and percentage of leukocytes derived from granulocyte donors. patients and methods: one patient suffering from evans syndrome and was transplanted with cord blood of a mmud after myeloablative conditioning with busulfan, thiotepa, etoposide and atg. the child received one neutrophil transfusion at day + . the second patient was diagnosed with aml (fab m ) and was transplanted in persistent bm-aplasia after induction chemotherapy with bm of her hla-identical mother after reduced intensity conditioning (ric) with fludarabine, melphalan and atg. this child was transfused with eight granulocyte transfusions of three different allogeneic donors starting at day - until day + with two three days intervals. snp analysis was performed the day after each granulocyte transfusion and once weekly until stable allogeneic engraftment was achieved. results: both patients showed allogeneic engraftment of > % donor chimerism at day and respectively. third party leukocyte dna could be detected from day + until day + in the first patient and from day - until day + in the second patient. in the second child up to five genetically different leucocyte dna populations where detectable and could be quantified. conclusions: granulocyte transfusions of third party donors after allogeneic stem cell transplantation are increasingly used. snp analysis allows precise monitoring of donor chimeras even in case of two or more donor cell populations after transplantation. this could be important especially in case of ric transplantations in which precise follow up of engraftment kinetics is very important. patients are alive and without disease. one year overall survival and disease free survival (dfs) is % ( - %) and % ( - %), respectively. transplant related mortality (trm) was the cause of death in patients, months and one year cumulative incidence (ci) of trm were % ( - %) and % ( - %), respectively. eight patients relapsed (median time: days ( - ); one year ci % ( - %). twelve patients developed acute graft versus host disease (agvhd); ci % ( - %) fourteen of evaluable patients developed chronic gvhd; ci % ( - ). the development of cgvhd was associated with better dfs: % ( - %) vs. % ( - %) p= , . conclusion: ric allo-sct is a curative option in patients with advanced age diagnosed of amd/mds with an acceptable trm. age should not be an exclusion criteria in patients with aml/mds for allo-sct. cgvhd is associated with better dfs. finaced in part by g / , /xt . allogeneic stem cell transplant (allo hsct)is a curative approach for patients with hematologic malignancies but it is associated with high treatment related morbidity and mortality. because transplant related mortality increases with advanced age, advanced disease and unrelated donors, patients older than - years may be excluded from this procedure. reduced intensity conditioning and new preparative regimens are therefore explored to allow hsct to a wider patient population. the aim of this study was to evaluate efficacy and toxicity of the combination treosulfan (water soluble alkylating agent, busulphan derivative) and fludarabine as preparative regimen for allo-hsct in patients receiving match sibling or unrelated donors for advanced heavily pretreated hematologic malignancies. since july to november patients ( all, aml, cml, mm ) entered this study. mean age was years (range - ). conditioning consisted of treosulfan gr/m² for days, fludarabine mg/m² for days, cyclosporine plus short mtx and anti-thymocyte globulin (thymoglobulin) at a total dose of mg /kg. all patients engrafted; mean time to neutrophil recovery > x /l was (range - ) days , to platelets > x /l was ( range - ) days. no conditioning regimen related deaths was observed. three ( ) patients experienced gi toxicity ( grade , grade ), patients had grade liver toxicity. no acute gvhd was observed. all patients are alive with a follow-up ranging from to days. despite the short follow-up, in this preliminary report we underline that treosulfan-fludarabine -atg conditioning is characterized by reduced toxicity; long term follow-up is necessary to evaluate os, dfs and relapse in these patients. background: prognosis of patients with aml relapsing within a year of allografting is poor. management options are limited and response to donor lymphocyte infusion(dli) is poor due to disease kinetics. second allografting using conventional conditioning is unpopular due to high transplant related mrotality(trm).there is paucity of data on second allografting with reduced intensity conditioning(ric) as a salvage for relapses post-allografting. herein, we report such patients who were successfully salvaged and achieved a durable complete remission(cr) with ric and second allografting. case : a year old male with normo-cytogenetics aml received an allograft from a hla identical sibling after myeloablative conditioning. he relapsed months after transplant with loss of donor chimerism. he was salvaged with flag regime followed by serial dlis and achieved cr which lasted months before relapsing again. he was reinduced with flag regime followed by pbsc infusion from the original donor. he received no post-transplant immunosuppression with the development of grade graft versus host disease(gvhd). he has since achieved % donor chimerism and remains in cr for more than months. case : a year old male with myelofibrosis transforming into aml associated with del( q) received his first allograft from a hla identical sibling after non-myeloablative conditioning. donor chimerism declined after months and blast counts continued to rise despite serial dlis. he was reinduced with idarubicin and cytarabine, followed by a second allografting from the original donor with ric (fludarabine mg/m² d- to - , melphalan mg/m² d- to- ). full donor chimerism ensued and he remains in cr for more than months. immune suppression was tapered with development of grade ii gvhd. conclusion: remission durations after the second allografting exceeded those after the first. a ric regime with second allografting is a more effective modality than dli in the treatment of relapsed aml. a ric regime lessens the trm associated with second transplants, while allowing for engraftment of infused pbscs with execution of gvl effect. immune modulation by aggressive manipulation of posttransplant immune suppression also appears to be a key element in successful second allografting. this salvage approach offers a practical, well-tolerated and potentially curative treatment for patients who in most circumstances, would have been precluded from further active management. reduced intensity conditioning (ric) allogeneic stem cell transplantation (allo-sct) can reduce the frequency of transplant-related toxicities, at least in the early period after allo-sct. here, we analyzed the profile of platelets recovery and transfusion requirements in the first months after ric in a single institution series of consecutive patients receiving allo-sct from an hla-identical sibling. patients and graft characteristics were: age y. (range, - ), diagnoses: myeloid malignancies ( %), lymphoid malignancies ( %), and metastatic solid tumors ( %). pts ( %) received a fludarabine, busulfan and atg-based ric, while pts ( %) received a low dose irradiation-based ric. all patients received a pbsc graft. pts ( %) developed grade - acute gvhd. platelets recovery (> . /l) was observed at a median of days (range, - ). the kinetics profile of platelets recovery is shown in the figure below. in the whole study population, the nadir was observed around day + after allo-sct, and a plateau was reached by the end of the first month. in this series, patients needed a median of unit (range, - ) of filtered and irradiated donor apheresis platelets. of note, pts ( %) did not require any platelets transfusion during the follow-up period (median follow-up: days). among the patients ( %; %ci, - %) who received at least one transfusion of platelets, were not transfused beyond day + after allo-sct. when comparing these patients, to the group of patients who were never transfused, platelets count prior to ric allo-sct (median count . /l vs. . /l; p= . ) and the occurrence of severe acute gvhd (p= . ; % of patients with grade - acute gvhd were transfused) were the parameters significantly associated with platelets transfusion needs. in this cohort, pts could be assessed for platelets recovery at day + : among them, ( %) had a platelet count > . /l. at day + after allo-sct, a diagnosis of myeloid malignancy (aml, cml or mds) was associated with a delayed platelet recovery (p= . ). overall, these observations show a significantly lower rate of platelets transfusions and a quicker kinetics of platelets recovery after ric allo-sct. in addition, the low level of myeloablation observed after ric, may offer a window of opportunity for testing of megakaryocytic growth factors, towards further improving the safety and outcome of ric or nonmyeloablative allo-sct. fungal infections have become the major cause of infectious morbidity and mortality in patients undergoing bone marrow transplantation (bmt). in many cases invasive aspergillosis infections create a major therapeutic dilemma and contraindication to marrow transplantation. we report on a years old boy with secondary acute myeloid leukemia, who underwent unrelated donor peripheral blood stem cell transplantation (pbsct) with previously diagnosed pulmonary aspergillosis and successfully recovered from the infection. probable invasive pulmonary aspergillosis (ipa) was diagnosed in the patient acc. to eortc-criteria. a large diffuse wedge-shaped infiltration was observed in thorax ct scans two months before pbsct and throughout the early posttransplant period. liposomal amphotericin b (l amb) mg/kg/d i.v. and voriconazole p.o. x - mg/kg/d were administered for the whole peritransplant period. after conditioning regimen incl. treosulfan x g/m², melphalan mg/m², atg fresenius x mg/kg the patient received pbsc ( . x cells cd +/kg recipient bw) from the unrelated donor, who was mismatched at a*-allel. csa, mtx and atg were used as gvhd prophylaxis. a rapid and sustained allogeneic engraftment (neutrophils > . g/l on day + , thrombocytes > g/l on day + ) was observed. the posttransplant period was uneventful except for weeks long lasting exhausting morning cough and subtle breathing difficulties requiring passive oxygen therapy. three weeks after pbsct, bronchoscopy with broncho-alveolar lavage revealed no pathogens. nevertheless it was decided to continue treatment with l amb mg/kg every second day. in control thorax ct the infiltration was considerably smaller and no indication was found to perform an open lung biopsy. the patient received further treatment with voriconazole p.o. for months. regularly performed thorax cts revealed a continuous regression of pulmonary changes. the patient remains alive and well in cr months from transplant without any pulmonary abnormalities, except for a slight thickening of the interlobar groove and is given itraconazole p.o. this report demonstrates that administration of full-dose antifungal therapy and shortening the neutropenia period due to peripheral blood stem cell transplantation allow the successful outcome, even in high-risk patients with previous aspergillosis. post-transplant pulmonary complications are not rare and their mortality is still very high. we present -old year girl with high-risk acute lymphoblasic leukemia after allogenic hematopoetic cell transplantation (hct) from matched related sibling donor. conditioning regimen consisted of fractioned total body irradiation ( gy) and high-dose etoposid ( mg/kg). as graft versus host disease (gvhd) prophylaxis cyclosporine a was used. graft contained , x /kg cd + cells. on day + persistent fever occurred, antibacterial and antifungal treatment were administrated. on day + ug/kg g-csf was added. we observed symptoms of respiratory failure. on day + wbc was /ul, cutaneous gvhd grade iii appeared and steroids were administrated. on day + she has all symptoms of pulmonary oedema, was intubated and mechanically ventilated. simultaneously rapid hematopoesis reconstitution was observed: leukocytes> . g/l and granulocytes> . g/l on day + , and platelets> g/l on day + . after few days of the gradual improvement, her status suddenly deteriorated. chest x-ray showed fluid in the alveolar space in both lungs. pentamidine was added to treatment. severe but stabile status (opportunity to controlled ventilation, fio above %) lasted about months. she was treated with wide-spectrum antibiotics and antifungal drugs (liposomal amphotericin, voriconazole) although her blood cultures were still negative. in this time we also treated her with anti-tnf-alfa antibodies. because of probable invasive aspergillosis and candidiasis we introduced treatment with caspofungin. gradual improvement was observed: significant decrease of the requirement for oxygen, possibility to reduction of controlled pressures and respiratory rates and simultaneous improvement of radiologic changes. after few days of continuous positive airway pressure ventilation right pneumothorax appeared and continuous suction drainage had been used for three weeks. simultaneously she was detoxicated from thiopental and morfine using fenobarbital and methadone. on day + mechanical ventilation was discontinued. requirement for passive oxygen therapy gradually decreased. nowadays chest tomography shows mild pulmonary fibrosis and bronchiectasia. the patients is alive, in good general status, under intensive physical and s pulmonary rehabilitation with stable full donor chimerism without immunosupression. nevertheless regular long-term pulmonary follow-up is still required. cns symptoms and their severity strongly correlate with outcome of patients with familial haemophagocytic lymphohistiocytosis (fhl). here we describe a case of a patient without primary cns involvement related to fhl who developed progressive cns damage of unknown aetiology after allogeneic stem cell transplantation (sct). the patient was diagnosed at the age of . years with fhl, mutation in perforin gene was not proved, no signs of reduced perforin expression were observed. he was treated according to protocol hlh and then transplanted in clinical and haematological remission of fhl from his haploidentical father (peripheral blood stem cell -cd positive selection; clinimacs) because no matched donor was available. myeloablative conditioning consisted of busulfan, cyclophosphamide and ratg. soon after engraftment he suffered from cmv reactivation and he consequently experienced acute graft rejection. following okt and steroids further t cell depleted graft sct was infused days after st sct. early after second engraftment he developed severe acute encephalopathy and syndrome of inadequate adh secretion (hyponatremia, hypothermia, neurologic seizures,…), hhv variant b was at that time detected in cerebrospinal fluid (csf) and blood. this was early followed by marked ebv and b cell proliferation treated with rituximab. after engraftment, complete donor haematopoiesis was confirmed with exception of almost full autologous recovery of t lymhocytes (split chimaerism). at day+ all t lymphocytes ( , . /l) were activated (expressing hla dr) and were of host origin. eleven months after sct hhv was repeatedly detected in peripheral blood and later frequent pharmacologically almost uncontrolled epileptic seizures started initiating progressive mental retardation. throughout this whole period repeated mri scans and examinations of csf, blood and marrow failed to document involvement of cns due to uncontrolled fhl. one year after sct cytototoxic assays showed significantly decreased activity of t cells. despite the number of donor origin t cells started to predominate the host ones only years after sct, there were no clear clinical signs of fhl. unfortunately the patient developed irreversible cns damage. we speculate that multiple herpetic infections were responsible for proliferation of activated autologous t lymphocytes that contributed to severe cns damage in this patient. supported by grants of mh cr no. , and consecutive twenty seven patients with inborn errors: four inborn metabolism: three metachromatic leukodystrophy, and one mucopolysacaridosis type : hurler´s syndrome; also one osteopetrosis, two mayor thalassemia, two fanconi anemia, and eighteen inmunodeficiencies: twelve severe-combined immunodeficiency (scid), two wiskott-aldrich syndrome and four hemophagocytic lymphohistiocytosis, were transplanted in our centre during the last nine years. we report two cases of scid: scid type jak deficiency (t-, nk-, b+) and major histocompatibility complex (mhc) ii deficiency which were treated with double haploidentical parental donor positively selected hla-mistmached cd + progenitor cells were isolated from peripheral stem cells, after mobilization with granulocyte colony-stimulating factor (g-csf) at standard dose, by the isolex (baxter)and clinimacs(miltenyi)systems selection devices respectively. the first case was a male, . months years old with jak deficiency (t-, nk-, b+) who was undergone to transplant without conditioning regimen; value of peripheral cd + cells infused was . x /kg and the mean cd + cells number was . x /kg; and . log t-cell depletion. he had graft rejection at day + and therefore was undergone to second haploidentical from the same family donor: father, this time with no-myeloablative conditioning with fludarabine (f) + melphalan + anti-thymocyte globuline (atg) and prophylaxis for graft-versus-host disease (gvhd) with cyclosporine (cya), he died ten days after the second transplant by heart insufficiency and metabolic failure. the second patient was a female, months years old with mhc class ii deficiency, she was undergone to haploidentical transplant with myeloablative conditioning based f+ busulfan and ciclophosphamide and atg. the value of peripheral cd + cells infused was . x /kg and the mean cd + cells number was . x /kg, and . log t cell depletion; she had graft rejection at day + and mixed chimerism therefore was underwent to second haploidentical transplant from the same parental donor: mother, the conditioning regimen was based okt- and dexametasone, she died five days after the second transplant by lung bleeding. despite poor prognosis at diagnosis of these cases, and the engraftment failure regardless mega-doses of cd +; this approach could be a feasible therapeutic option for patients lacking a suitable donor. introduction: mobilized peripheral blood stem cells (pbsc) represent the most important source for autologous stem cell transplantation, even in children with malignancy. the current practice is administration of g-csf alone or in combination with chemotherapy. recently, a polyethylene glycol (peg)conjugated form of g-csf (pegfilgrastim) has been licensed. preliminary data indicate it has the same effects of filgrastim in terms of elevation of absolute neutrophil count, mobilization of pbsc, and reduction of duration of chemotherapy-induced neutropenia, with the obvious advantage that these effects could be sustained for several days from a single injection without added toxicity. in a recent experience the efficacy of a single dose ( microgr) of pegfilgrastim, in combination with salvage chemotherapy, was tested in an open-label phase ii study of pretreated patients. the authors concluded that pegfilgrastim as an adjunct to chemotherapy is a predictable and highly effective mobilization regimen in pretreated lymphoma patients (isidorins ). very limited experience is available on the use of pegfilgastrim in children. in the only two available report it was used to shorten the duration of severe neutropenia after cytotoxic chemotherapy in five children with ewing sarcoma (te poele em ) and in seven pediatric cancer patients (wendelin g ). we are not aware of any report on the use of pegfilgrastim for mobilization in children. patients: we treated children, males, aged , and years, affected of rhabdomyosarcoma, ewing sarcoma, and b-nhl. all received chemotherapy with vp and edx and than a single subcutaneous dose ( microgr) of pegfilgrastim. two patients had a good response, with the peak cd + count ( and ) on day + and + , while the failed to mobilize and to collect a sufficient number of cd + cells even after conventional g-csf and bone marrow harvests. the only patient who so far has completed the treatment program underwent, according to the treatment protocol, autologous transplant repeated three times and engrafted (pmn > /microl) on day + , + , and + . conclusion: mobilization with peg-filgastrim was safe and efficient in our patients; failure to mobilize was observed only in an heavily pre-treated patient with ewing sarcoma. stem cell transplantation (sct) is the treatment of choice for patients suffering from severe aplastic anaemia (saa) who do not respond to immunosuppressive treatment (ist). patients transplanted from an hla-identical sibling have an excellent prognosis compared to patients who were grafted from alternative donors. t-cell depletion by cd positive selection of peripheral stem cells reduces the risk of gvhd considerable. this, however is followed by an increased risk of graft rejection. most recently, it could be shown, that changing the graft processing from cd positive enrichment to depletion of cd and cd positive peripheral cells does facilitate and improve engraftment in children transplanted from hla-haploidentical parents. consequently, also patients with saa who are at highest risk for graft rejection might benefit from such a new graft processing technique. a -year-old girl who failed to respond to ist received an allograft from mud. the conditioning regimen consisted of fludarabin ( x mg/m²), thiotepa ( x mg/kg), melphalan ( x mg/m²) and okt ( x . mg/kg). g-csf mobilized peripheral blood stem cells (pbsc) were purified using anti-cd /cd microbeads (miltenyi). recovery of cd + progenitor, cd + nk cells and cd + monocytes was more than % each. residual t-and b-cells were < . and . %, respectively. in total, . x cd + cells; . x cd + cells; . x cd + cells and . x cd + cells per kg were administered. without g-csf, engraftment occurred on day + for leucocytes and both neutrophils and platelets on day + . acute toxicity was mild (grade i). post transplant immunosuppression was performed using mmf. gvhd grad i was observed, which responded to prednisolone. noteworthy, although t-cells were severely depleted, t-cell regeneration was fast with more than /µl cd and cd positive cells detectable already on day + . chimerism analysis showed complete donor chimerism. at day , good immune recovery with /µl cd +, /µl cd +, /µl cd + and /µl cd + cells was detected. in conclusion, cd /cd depleted peripheral stem cells from a mud in combination with a reduced intensity conditioning regimen could be a promising option in the treatment of patients with aplastic anaemia not responding to immunosuppressive treatment and lacking a matched sibling donor. idiopathic myelofibrosis (imf) comprises myelofibrosis, extramedullary haematopoiesis, hepatosplenomegaly and pancytopenia. in adults imf represents a poor prognosis, progressive fibrosis and leukaemic transformation are frequent. allogeneic hematopoietic stem cell transplantation (hsct) is a treatment option but is connected with high risk of graft failure and toxicity. in children the disease is rare and variable, stable course or spontaneous remission has been reported. we describe two cases of imf in children. in a girl mild anaemia and thrombocytopenia were first documented at the age of years. at years pancytopenia was found, trephine bone marrow (bm) biopsy revealed normocellular haematopoiesis with myelofibrosis. she remained in a good clinical state with a stable blood count but further bm biopsies showed decreased cellularity with myelofibrosis. . years after the diagnosis her blood count dropped off, hepatosplenomegaly was noted and bm biopsy revealed marked myelofibrosis. months later at the age of years hsct was therefore performed (matched unrelated donor, flu+mel+ratg). anc engrafted on day , platelets on day , complete donor chimaerism achieved on day . corticosteroids started on day for mild extensive gvhd. bm biopsy at day remained hypocellular with myelofibrosis, however blood count was normal. -year-old male presented with pallor, quickly developed pancytopenia with blasts. bm biopsy showed myelofibrosis, increased blasts, trisomy + . hsct from a hla identical sibling was performed months later (flu+mel+ratg), anc engrafted on day , platelets on day . complete donor chimaerism was achieved on day , no gvhd. at day bm biopsy did not display myelofibrosis, peripheral blood count was stable. clinical deterioration started months post-hsct with fever, weight loss, hepatosplenomegaly, mixed chimaerism, thrombocytopenia, blasts and extramedullary infiltration of breast. trephine and breast lump biopsy confirmed relapse of imf in transformation to aml-m with trisomy + and trisomy + . second hsct from the same donor was carried out months after the first hsct (bu+cy+mel). gvhd grade ii treated with steroids manifested on day . anc engrafted on day , platelets not engrafted. despite artificial ventilation diffuse alveolar haemorrhage resulted in death on day . reduced intensity conditioning composed of fludarabine and melphalan is preparative regimen of choice for hsct in children with imf. there are no studies on the connection between graft versus host disease (gvhd) and angiogenesis. however, chen et al have shown in nat med ( ) that the vascular endothelial growth factor-c (vegf-c) -vascular endothelial growth factor receptor- (vegfr- ) -axis has an effect on alloimmunity, and that the blockade of vegfr- signaling is immunosuppressive. both vegf-c and angiopoietin (ang ) are important in angiogenesis and lymphangiogenesis. in this study we measured the levels of these two factors in children after urd-sct (unrelated allogeneic stem cell transplantation). patients and methods: nine patients aged - yrs were included, six of whom developed significant acute gvhd (agvhd, gr ≥ ) while three did not. the diagnoses of the agvhd patients were all (acute lymphoblastic leukemia) (n= ) and saa (severe aplastic anemia) (n= ). the non-gvhd patients also had all (n= ) and saa (n= ). gvhd prophylaxis consisted of cyclosporin and short methotrexate. the serum concentrations of vegf-c and ang were analyzed by elisa at - days posttransplant, - samples/patient. results: the vegf-c and ang concentrations (median, range) were ( - ) pg/ml and ( - ) pg/ml, respectively. the presence or absence of agvhd did not make any difference in the levels. neither did we find correlation between hemoglobin, white blood cell count, bilirubin, crp or sedimentation rate and these two angiogenic factors. the vegf-c levels were significantly lower than in our previous study on all patients at diagnosis. the individual maximal ang levels correlated with survival (≥ mo follow-up, p= . ). both absolute lymphocyte count and platelet count correlated with vegf-c levels, probably because these cells are known to produce vegf-c. conclusion: our results did not support the hypothesis about correlation of the levels of angiogenic factors with gvhd. instead, there was a novel finding about low concentration of ang being predictive of a good outcome. our findings pose important questions on the emerging role of angiogenic factors in the evaluation of the pathogenesis of gvhd. background: hematopoetic stem cell transplantation (hct) does appear to be a therapeutic option for children and adolescents suffering from shwachman-diamond syndrome with severe cytopenias and/or myelodysplastic syndrome. the paucity of experience with children undergoing hct for sds has been the major obstacle for recommendations regarding time point, transplant regimen, and patient subgroup benefiting most from hct. most but three reported patients received a preparative regimen either consisting of bu/cy or bu/tbi. however, severe early toxicity with cardiomegaly, myocardial fibrosis, and cyclophosphamide associated cardiomyopathy have been described. therefore, we tested the feasibility of a cyclophosphamide free protocol using fludarabine, treosulfan, and melphalan as a conditioning regimen. methods: between and two children with sds were enrolled. age at transplantation was and years. both patients received conditioning with fludarabine ( mg/m²/day x ), treosulfan ( g/m²/d x ), melphalan ( mg/m²/d x ), and campath- h. all children received a non manipulated fresh bone marrow graft. the first patient from a hla-identical sibling, the second from a / locus matched unrelated donor. mean cell doses transplanted were . x nucleated cells/kg bw ( . x /kg and . x /kg). results: both patients achieved donor derived engraftment, no gvhd exceeding grade ii was observed, and both maintained donor chimerism at %. all patients developed grade iii mucositis. on patient experienced a cerebral seizure early after transplant most likely caused by csa toxicity. gvhd prophylaxis was switched to mmf and the patient fully recovered from this single event. apart from this, no rrt > grade ii was seen. all patients are alive after a follow up of and month. conclusion: a fludarabine based, cyclophosphamide free conditioning regimen seems to be a feasible approach for matched sibling and matched unrelated hct in children and adolescents with sds. larger numbers and a longer follow-up are needed to make these results more comparable to traditionally used preparative regimens. a prospective comparison of immune reconstitution after autologous haematopoietic stem cell transplantation in children v. wiegering, b. winkler, m. eyrich, p.-g. schlegel university of wuerzburg (wuerzburg, d) introduction: autologous haematopoietic stem cell transplantation (auto hsct) has become an established therapy for numerous advanced paediatric solid tumours. after haematopoietic stem cell transplantation all recipients experience a period of immunodeficiency. regeneration of adequate t-cell numbers and repertoire diversity are key elements in the recovery of immune competence. patients: immune reconstitution was studied in children ( transplants; median , years; range m- y; female, male). blood samples were drawn before auto hsct, on days (take), , , , and and > months. methods: we analyzed lymphocyte subpopulations using flow cytometry. intracellular cytokines (ifngamma, il , tnfalpha, il , il , and il ) were determined by facs after in vitro stimulation with pma, ionomycin and brefeldin for h. additionally, we measured il , il and tgfbeta in unstimulated sera by elisa. additional we measured trec´s and spectatypes. results: as to lymphocyte subpopulations after auto hsct, nk-cells were the first to regenerate. in t-cells, an inverted cd -cd -ratio could be detected during the first year. in cd + t-cells the memory phenotype (cd ro+) predominated. as to cytokine levels in unstimulated sera, we saw high levels of il shortly after transplantation, levels decreased to pretransplant values during one year. tgfbeta levels increased during the first year and decreased thereafter. ifngamma levels remained stable. in stimulated t-cells, ifngamma and tnfalpha increased in the first year and went down afterwards. interestingly, high ifngamma levels after transplantation correlated significantly with a better survival. non-irradiation containing conditioning regimen for children with fanconi's anaemia m. jarrar, m. sarhan, m. milhem, f. abdel-rahman, r. rihani, s. sharma, i. na'em king hussein cancer center (al-jubeiha, jor) fanconi anemia is an inherited disorder that leads to progressive bone marrow failure. the only curative treatment of the severe aplastic anemia that ultimately develops in these patients is allogeneic stem cell transplantation. patients with fanconi anemia have increased chromosomal fragility. as a result they are prone to both short and long term complications when conditioning regimens containing radiation are used. we report on patients ( males and females) with fanconi anemia who were transplanted from matched siblings without using radiation as a part of conditioning. median age at time of transplant was years ( - years). none of the patients had mds changes or leukemia prior to transplant. conditioning regimen consisted of fludarabine mg/kg, cyclophosphamide mg/kg and rabbit atg mg/kg. peripheral blood was the source of stem cells in patients, while bone marrow was the source in patients. gvhd prophylaxis consisted only of cyclosporine. at a median follow up time of days ( - days), patients are alive with normal hematopoesis. one patient failed to engraft. he was transplanted again with a different conditioning; however he had late rejection and died of sepsis days after second transplant. median cd + cell/kg infused was . million ( . - . million). median time to neutrophil and platelet engraftment was days and days, respectively. two patients had fever with atg; one patient had bacteremia during the neutropenic period. two patients developed fungal infections after engraftment. two patients had mild vod. three patients received vod prophylaxis consisting of urisidiol and spironolactone. cmv reactivation occurred in patients and was treated with gancyclovir. grade - skin and liver acute gvhd occurred in patients. limited chronic gvhd occurred in patients. one patient developed extensive chronic gvhd. conditioning without radiation is well tolerated in fanconi anemia patients and results in prompt engraftment. infectious complications appear to be high. autologous peripheral blood stem cell transplantation in low weight paediatric patients: methods and clinical outcome a. galmes, m. canaro, m. guibelalde, m. morey, l. bibiloni, a. vila, a. gutierrez, j. besalduch son dureta hospital (palma de mallorca, e) peripheral blood stem cell (pbsc) collection may be difficult in low weight body pediatric patients, with technical and clinical problems related to vascular access, low total blood volume, citrate toxicity, high extracorporeal volume, and patient's tolerance. methods: we present our experience with consecutive children weighing ≤ kg, diagnosed with acute leukaemia ( ) and solid tumors ( ), which were collected and transplanted between september and february at our centre. patients mean body weight was kg (range - ); median age was years (range, - years) ( table ).harvesting of pbsc was started after days of cytokine alone (g-csf mcg/kg/ hs. s.c.). procedures were performed using a baxter cs- plus separator primed with a mixture of irradiated and white cell-depleted red cells resuspended in % albumin and diluted with saline to match the patient's haematocrit. heparin and acd-a was used for anticoagulation (heparin ui in ml acd-a) in patients weighing < kg. the median number of leukapheresis was (range - ), processing . volemia in each session. the platelet count decreased significantly after each procedure without requirement of platelet transfusions. special monitoring of toxicity was done. the children were no sedated and showed no serious side-effects. all pbsc were cryopreserved with dmso % and stored at - ºc in mechanical freezer. results: the median time from cryopreservation to transplantation was . ( - ) days, and the median number of infused mononuclear cells and cd + cells were . ( . - . ) x /kg and . ( . - . ) x /kg, respectively. the median number of infused post-thawing cfu-gm was . ( . - . ) x /kg. all patients showed a safe and sustained engraftment. median time to reach and neutrophil per microl was ( - ) and ( - ) days. median time to and platelets level per litter was ( - ) and ( - ) days (table ) . long-term hematopoietic recovery at , and months was achieved in all cases (table ) . conclusion: our experience shows that our pbsc collection and cryopreservation method is a safe and efficient procedure in children weighing less than kg., with sustained haematopoietic reconstitution. an approach to retrospective validation and performance monitoring of pbsc collection and other white cell procedures using the cobe spectra cell separator: "cells collected" as a function of "cells processed" by different methods k.w. douglas, j. sinclair, m. mcgarvey, a. mcphelim, s. taylor, m. drummond s.n.b.t.s. clinical apheresis unit (glasgow, uk) validation of cell separator machines for a specific apheresis procedure poses a number of challenges. in particular, for white cell collection procedures it is not feasible for individual centres to carry out a prospective validation process prior to introduction of the procedure, because there is no way of performing a "dummy run" of a white cell procedure without actually putting a donor on the machine. we therefore attempted retrospective validation of our cobe spectra cell separator machines for pbsc collection using the mononuclear cell (mnc) procedure by estimating efficiency of the mnc procedure for each of the individual machines, in terms of the total mononuclear cell dose achieved in the apheresis product as a function of the number of mononuclear cells processed by the machine. there should be a direct correlation between total mononuclear cells processed by the machine and the final mnc dose in the product. the difficulty is in estimating "total mononuclear cells processed by the machine", which will only ever be an approximation. we estimated "cells processed" using different methods: method : the donor's peripheral mononuclear cell count (lymphocytes plus monocytes) multiplied by the run time; method : the number of total blood volumes processed multiplied by the donor's peripheral mononuclear count. retrospective audit was performed on pbsc collections carried out on five spectra machines over an -month period. total mononuclear cell dose in the apheresis product was graphed as a function of "cells processed" as calculated by both methods above. both methods showed a clear, statistically significant linear correlation, but there was less scatter and a lower p value using method (r= . , p< . ). it was noted that the gradient of the trend line is a measurement of the efficiency of pbsc collection. the data was therefore subdivided depending on which of the spectra machines had been used for collection, and individual data analysis was performed for each machine. this showed that the five machines all performed the mnc procedure with very similar efficiency (gradients of trendlines . to . ). the same process can easily be applied to ongoing performance monitoring of the five machines, and our aim will be to do this annually from now on. m. miorin, e. brunetta, e. scquizzato, s. varotto, c. messina, s. cesaro, g. binotto, i. baesso, e. calore, m. facco, e. ave, m.v. gazzola, r. destro, g. semenzato, r. zambello, l. trentin university of padua (padua, i) allogeneic bone marrow transplantation (abmt) is one of the powerful therapies for several hematological malignancies. multiple mechanisms contribute to the graft success, such as the entity of primary disease, donor bone marrow availability, minimal residual disease (mrd) and complications including infections and acute graft-versus-host disease (gvhd). gvhd represents a major complication of abmt and is the main cause of morbidity and mortality. the present study takes into account the recovery of the t cell-compartment before and after abmt in pediatric patients affected by different hematological malignancies. to evaluate the pattern of accumulation of t cells, we investigated the usage of t cell receptor (tcr-beta) chain variable regions (tcrbv) and the complementarity-determining region (cdr ) up to year follow-up after abmt. increased expression of some tcrvb families were observed in patients during the months after abmt. in the following months after abmt, we confirmed the presence of the same t cell clones and, sometimes, we showed the appearance of a new tcrbv family subset. we observed a random distribution of overexpressed tcrbv families and we did not show a preferential expression of a peculiar tcrbv. in order to clarify whether cells expressing a tcrbv region were clonally expanded, we performed cdr spectratyping and sequencing analysis. a predominant polyclonal pattern was observed in donors and patients before transplantation while at and months after bmt some clonal subsets were identified. in the majority of patients the presence of these subsets persists until month after abmt. a skewed tcrbv repertoire and oligoclonal/monoclonal subsets we observed may explain the post-bmt immunodeficency detectable after transplantation and may reflect responses to pathogens or alloantigens in correlation with clinical gvdh. this study was supported by a grant from fondazione città della speranza. s. ganepola, k. rieger, c. loddenkemper, j. maul, a. muessig, e. berg, h. stein, e. thiel, r. duchmann, l. uharek university medicine berlin, charite (berlin, d) introduction: tregs are involved in the control of immune responses to foreign antigens and play an important role in the pathophysiology of gvh-reactions. they are characterized by expression of cd +, cd + and the transcription factor foxp . although tregs are of emerging interest in allogeneic cell therapy, their precise enumeration in heterogeneous cell products has been extremely difficult. using monoclonal antibodies (moabs) against foxp and immunenzymatic labelling at the single-cell level in paraffin-embedded tissues, we have investigated different techniques to identify tregs in cellular products and tissue biopsies. methods: µl of the anti-human pe conjugated foxp antibody (clone pch , ebioscience) were used for intracellular labelling of x cells of cd depleted, macs sorted cd +/cd + cell fractions of peripheral blood mononuclear cells. the abcam goat polyclonal foxp antibody was used for double immunoenzymatic labelling of foxp /cd and foxp /cd of pbmc cytospins, of macssorted cd +cd + selected cell fractions and of paraffinembedded tissues. results: nuclear staining of the foxp and measurement by flow cytometry showed bright results in macs-sorted peripheral blood cells as well as in cytospins. gating on cd + cells of the cd depleted, cd enriched cell fraction, % of the cd + cells are positive for the foxp marker. the frequency of foxp + treg in the peripheral blood lymphocytes of healthy individuals ranged between - % of total lymphocytes, like previously described. in double labelling cytospin analyses of foxp and cd , we confirmed co-expression of cd on all foxp positive cells. counting high-power fields, % of the cd +/cd + cells are foxp +. in healthy individuals pbmc-cytospins we found % cd +/cd +/ foxp + cells. in double labelling analyses of foxp and cd in paraffin embedded tissues, we confirmed co-expression of cd on all foxp positive cells. only a few weakly cd -stained cells were negative for foxp staining, indicating a preferential staining of the cd high cell population. conclusion: direct staining of cellular products with moabs against nuclear foxp and subsequent flow cytometry can give similar results as nuclear foxp -staining in cytospin preparations or the assessment of cd +cd + cells by flow cytometry. these new techniques allow straightforward identification and quantification even of very low numbers of treg in peripheral blood subsets or other tissues. patient age and granulocytic contamination of apheretic harvests are important factors for adverse events after infusion of cryopreserved hsc g. milone, a. strano, s. mercurio, s. coppoletta, k. battiato, s. leotta, m. poidomani, r. giustolisi ospedale ferrarotto (catania, i) adverse events (ae) after cryopreserved cellular infusions are frequent and seldom can be life-threatening . dmso is considered important in their pathogenesis, however other factors could well play a role. we have prospectively collected data on ae presenting after hsc infusions following high dose chemotherapy performed in our centre during a y. period in patients affected with haematological neoplasm. stem cell source was pbsc in cases while bone marrow in . in all cases an endotoxin-free dmso was used. one or more ae was registered in / infusions ( . %). gastrointestinal complains were reported in % of all infusions, skin rashes in . % of cases, shaking in %, cough in . % , fever in . %, shortness of breath in . %, dizziness in %, headaches in . %. in univariate analysis patient age over was significantly associated to a higher incidence of ae, in fact incidence of ae was % below years of age, % in - decade and % over years (p= . ). frequency of ae was higher after pbsc than after bm ( % versus . %,chi-test: p= . ). in univariate logistic regression factors found important for ae in pbsc group were total number of cells infused /kg ( p= . ) and volume/kg of dmso infused (p= . ). adverse events were more frequent also when total number of granulocytic cells/ present in pbsc harvest was > . x /kg in respect to infusions containing a total number of granulocytic cells below this threshold ( % versus %, chi test: p= . ). all aforementioned factors were evaluated in multivariate logistic regression and age of patient (p= . ) and granulocytic contamination over . x /kg (p= . ) were still significant while the total volume of infused dmso loose importance (p= . ). no cardiovascular events were recorded during infusions, however we registered a statistically decrease of blood pressure and a statistically decrease of cardiac frequency. a significant correlation existed between reduction of cardiac frequency and volume/kg of dmso infused (r: . , p= . ). in conclusion while non cardiovascular ae are dependent from patient age and from granulocytic contamination of apheretic harvests, cardiovascular changes are influenced only by volume/kg of dmso infused. as far as non cardiovascular adverse events are concerned, particular attention should be paid in infusions of hsc in patients over the age of years and when the grafts have a granulocytic contamination over to . x /kg. haematopoietic stem cell transplantation for haematological malignancies: a -year single-centre experience b. georgievski, l. cevreska, n. siljanovski, a. stojanovic, o. karanfilski, z. stojanoski, s. genadieva stavrik, i. panovska, s. krstevska balkanov, a. pivkova clinical center (skopje,mk) hematopoietic stem cell transplantation (hsct) is widely used therapeutic method in the treatment of patients with hematological malignancies. in this study we present our results in years experience in transplantation for hematological malignancies. in a period - a total of transplants have been realized, allogeneic sibling and autologous transplantations. in the group of patients treated with allogeneic sct, % of patients were in active disease prior transplantation, with diagnosis ( aml; cml; aa; nhl; cll; all); ratio males:females = : , median age years ( - ). bone marrow (bm) as source of hsc was used in patients and were preformed with peripheral blood stem cells (pbsc) with donor sex ratio : ( / ). conditioning was provided with bu/cy ( pts), bu/cy+mel ( pts), beam ( pts), hdice( pts), nonmyeloablative flag/ida( pts), flu/mel( pt). the amount of infused fresh bone marrow was ml( - ml) with mnc . x /kg( . - , ) and pbsc , x /kg ( , - . ). median number of transfused blood products was for er median doses ( - ) and plt doses ( - ). engraftment for ne> . x /l and plt > x /l was recognized on day + ( - ). acute graft versus host disease (gvhd) gr iii/iv was noticed in patients and chronic extensive gvhd in patients. in the second group of autologous recipients, patients with diagnosis ( aml; nhl; hd; all) received fresh bm as source of hsc and the other group of patients ( aml; all; hd; nhl; mm) received pbsc previously mobilized with g-csf+chemotherapy. median age was years ( - ), males:females= : . % of patients with limfoproliferative diseases were with refractory/relapsed disease and other patients were in complete remission before sct. conditioning regimens consisted of high-dose chemotherapy mainly bu-cy, beam, ice high-dose, melphalan. the amount of infused fresh bm was ml( - ml) with mnc , x /kg( . - . ) and pbsc . x /kg ( . - . ). median number of transfused blood products was for er median doses ( - ) and plt doses ( - ). engraftment for ne> . x /l and plt > x /l was recognized on + ( - ). median follow up for both groups was months ( - ), trm in allogeneic recipients was patients ( , %) with survival of patients transplanted in cr of , %, and in the autologous group trm was patients ( , %) with survival of , %. s. genadieva-stavrik, l. cevreska, a. pivkova, z. stojanoski, b. georgievski clinical center (skopje, mk) introduction: the administration of a combination of chemotherapy and cytokines g-csf is associated with a significantly increased efficacy of stem cell mobilization compared with either modality alone. method: the aim of this study was to evaluate the efficacy of g-csf preceded by chemotherapy (cyclophosphamide g/m sq for dose) for hematopoetic progenitor cell mobilization for lymphoma and myeloma patients. we started g-csf as a fixed dose mu sq every day as soon as the leukocyte counts began to rise after chemotherapy induced myelosupression. leukapheresis was commenced at the time when leukocyte count rose up to /ul, and repeated for - consecutive days until target number of cd + cell, at least x /kg was collected. results: thirty-five patients (male to female, : , age range - , lymphoma , myeloma ) underwent a total of courses of leukapheresis for hematopoetic progenitor cell collection prior to autologous transplantation from april through may . the target amount of marrow was harvest in all patients. all the patients achieved good engraftment after autologous transplantation. the mean days required for wbc count to be over , /ul was - days. patient's age, sex, underlying malignancy, exposure to chemotherapy before mobilization did not show any statistically significant correlation. conclusion: we can conclude that chemotherapy followed by g-csf administration is an effective way for mobilization of hematopoetic progenitor cell and verified itself as a good mobilization method. cyclophosphamide + gcsf as mobilising schedule in multiple myeloma and malignant lymphoma patients: factors associated with mobilisation efficiency r. carrion, j. anguita, m. calderon, d. serrano, p. balsalobre, i. buño, a. gomez-pineda, jl. diez-martin hospital gregorio marañon (madrid, e) backgroung: cyclophosphamide (cy) at dose of . grs/m² and gcsf is commonly used to mobilize blood stem cells to support high dose therapy in patients (pts) with multiple myeloma (mm) or malignant lymphoma (ml). however, - % of pts do not achieve the minimum stem cells dose needed for a rapid hematopoietic engraftment and risk factors for poor mobilization are not well known. methods: pts diagnosed of mm or ml and candidates to autologous stem cells transplant (asct) between october and march were treated with cy . grs/m² (d ) followed by gcsf mcg/kg/d from d+ . a first apheresis procedure was planned on d + . all pts were treated as outpatients and all of them were naïve to mobilization procedures. we analyzed some clinical factors, in addition to cd + cells count, wbc and platelet count on the first day of apheresis. we defined failure as: preapheresis cd count less than /microl, or cd yield ( st day) lower than . x /kg, or cd yield ( st round, three days) lower than . x /kg. results: male/female / ; median age (min , max ); mm/ml / ( , hodgkin disease). according to diagnosis (mm vs ml) mm pts had: a higher proportion of women ( % vs %, p= . ), older (median vs , p= . ), with a lower number of previous courses of chemotherapy ( vs , p= . ). asct did not performed in pts: due to poor mobilization ( pts by disease progression, by graft tumoral contamination, and by a second neoplasm), mm pts and ml pts. twelve pts ( / , %, mm pts and ml pts) had to undergo another mobilization program (most of them with gcsf alone) to achieve sufficient stem cells. as a whole, pts ( %) would be considered as a failure to get an adequate stem cells collection. a median of days was necessary to get a minimal preapheresis cd count. this interval was not different between mm and ml pts(p= . ). significant correlations were seen between preapheresis cd + counts, cd yield ( st day) and total ( st round) (p= . ). some covariates were selected to explain failure of mobilization (bone marrow involvement in ml pts, number of chemotherapy courses, and preapheresis wbc and platelet). in contrast with other studies none factor associated with mobilization failure was found by logistic regression analysis. conclusions: ) a combination of cy + gcsf is a safe, predictable and effective for the most of mm or ml pts ( %). s ) we have not identified any premobilization factors predicting poor mobilizer pts. k. ali moghaddam, s. samiee, n. mahdavi, l. nedaeifard, m. jahani, a. mousavi, m. iravani, b. bahar, a. ghavamzadeh horc (tehran, ir) introduction: umbilical cord blood (ucb) may be an alternative source for allogeneic transplantation for the treatment of hematological malignancies and genetic disorders in patients without suitable donors particularly in ethnic minorities. early experience with umbilical cord blood transplantation (cbt) demonstrated a lower incidence of graftversus-host disease (gvhd) even though the procedure was performed with hla-disparate grafts. the major drawbacks of cbt are slow hematopoietic recovery and a high incidence of graft failure, as a result of a lower number of progenitors infused. materials and method: we collect the data of patients who had undergone cord blood transplantation by reviewing their records from the date of their transplantation up to the date of last contact. analysis of the data has done via using spss software. results: patients received cbt which in our center during the past years, consist of talassemia, severe combined immune deficiency (scid), aml-m , mps- (hurler syndrome). of all the patients ( . %) were male, and ( . %) were female. the median age was years old; ( months - ys) respectively. donors were hlaidentical siblings in ( . %) patients and unrelated in ( . %) patients. the conditioning regimen for . % of patients was busulphan, and cyclophosphamide. patients had not gvhd, ( of them died in first days), had only gi gvhd, had gi and skin gvhd, and had gi, skin and also liver gvhd. the median duration of hospitalization for transplanted patients was days. transplant mortality rate in the first days was %. the median follow-up duration was days, with minimum and maximum of and days respectively and during this period the overall survival (os) is . % and the disease free survival rate (dfs) was . % conclusion: cord blood is a considerable alternative source for hematopoietic stem cells for allogeneic transplantation for malignant or nonmalignant hematopoietic disorders. as our cases were few we can not conclude definitely about the advantages or disadvantages, but in our study cord blood recipient from hla-identical siblings had lower gvhd and mortality than unrelated donors. peripheral blood stem cell collection: from outsourcing to in-service process. an opportunity to optimise the procedure. results after one year at eio -milan d. laszlo, a. agazzi, a. alietti, l. orlando, m. cassatella, l. roveda, g. vasaturo, m. venturino, a. lettiero, c. massaro, c. rabascio, a. cocquio, g. martinelli european institute of oncology (milan, i) peripheral blood stem cell (pbsc) collection by leukapheresis (laf) represents the standard method to obtain blood stem progenitors. this procedure is usually referred to transfusion centers joined with transplant units. a possible pitfall of this kind of management could be the impossibility to perform "ad hoc" the procedure in any time. from a transplant program with pbsc collection is ongoing at the eio; until june the laf has been performed by the transfusionist team outsourcing and then governed by the medical-nurse staff of our division. aim of the study was to evaluate the outcome of this shift in management. methods: firstly we considered what kind of laf-related variable could be examined to optimise the service for the patients in terms of harvest quality and assistance and for the institute in terms of reduction of time and cost of the procedure. secondly we organized a special training with the aim to perform the laf in-service using our know-how and resources. successively a comparative analysis of the data after one year of our management has been performed and compared to the data collected during one year of the previous management. in particular all the following variables of the procedure have been analyzed by a specific data-base: a)quality of the stem cell product; b)comfort for the patient; c)time related to procedure; d)costs. results: from july to july we performed consecutive laf. the staff was operative hours every day saturday and sunday included. the two populations of patients were matched for age, sex, diagnosis, stage of disease and previous treatment. the collection target were> . x /kg and > . x /kg cd + for donors and patients respectively. the table shows the collection results obtained. the best ratio optimal collections/patients obtained with our management ( %vs %) could be attributed to the optimisation of the procedure timing and to the excellent cooperation between medical doctors and the nurses in the in-service experience. concerning the time for an optimal collection, no difference was observed into the two managements (only % of the patients needed three or more procedures). the activity of the internal staff has permitted to cut down significantly on expenses in charge of the institute for the transfusionist's performance. . conclusions: thanks to the constitution of an internal dedicated team, we were able to perform the pbsc collection with similar results and lower cost in comparison with the outsource management. ( ) ( )leningrad regional clinical hospital (st. petersburg, rus) background: mobilized peripheral blood stem cells (pbsc) have become the main source for autologous transplantation in patients with haemathological malignancies or solid tumours. the aim of the study was to establish the influence of diagnosis, sex, age, number of previous courses of ct, mobilization regimen, bone marrow involvement on the outcome of pbsc mobilization. patients and methods: patients with haemathological malignancies and solid tumors were included in the study (hodgkin's lymphoma, hl (n= ), non-hodgkin lymphomas, nhl (n= ), multiple myeloma, mm (n= ), acute leukaemias, al (n= ), solid tumors, st (n= )). ( %) were females, ( %)males. ( %) patients were > years of age, ( %) patients were an age of less than years. patients ( %) were mobilized with g-csf mcg/kg for days, a combination of ct and g-csf ( mcg/kg) has been used in patients ( %). patients ( %) received more than six courses of ct and ( %) less than six respectively. bone marrow (bm) involvement was diagnosed in ( %) patients. apheresis was performed on 'spectra'v. . (gambro). the following criteria for the mobilization outcome were used: non-mobilizable patients < ± cd +/kg; poorly mobilizable patients > ± xcd +/kg; mobilizable patients > ± xcd +/kg (s.fu et al., ) . results: patients ( %) were non-mobilizable; patients ( %)-poorly mobilizable; ( %)-mobilizable patients. according to the diagnosis we observe the following results: hl- . ± . x cd +/kg, nhl . ± . x cd +/kg, mm- . ± . x cd +/kg, st- . ± . x cd +/kg, al- . ± . x cd +/kg. comparing all groups between each other we found no significant differences. there were also no influence of age and sex on stem cell mobilization (p= . , . respectively). bm involvement does not seem to be an independent factor with significant adverse influence on pbsc mobilization (p= . ). in patients received less than six courses of ct stem cell yield was significantly higher ( . ± . x cd +/kg against . ± . x cd +/kg (p= . )). better outcome was seen in patients mobilized with ct plus g-csf than g-csf alone ( . ± . x cd +/kg against . ± . x cd +/kg (p= . )). conclusions: better stem cell yield was seen in patients received less than courses of ct and in patients mobilized with g-csf combined with ct than g-csf alone. diagnosis, age, sex, bm involvement doesn't influence outcome of pbsc mobilization. inroduction: the phenomenon of nonspecific cell aggregation (cell clumping) can be observed in nucleated cell preparations obtained from bone marrow, peripheral blood and umbilical cord blood (ucb) following thawing. such preparations containing populations of both mature as well as immature hematopoietic progenitor cells are obtained by processing whole cord blood and freezing. different techniques may be applied for the thawing of such preparations. object: to evaluate the phenomenon of cell clumping, the influence on it was examined at increasing densities of nucleated cell suspensions which had been extracted from cord blood and then frozen. the two selected techniques of thawing were also evaluated and their influence on cell clumping. methods: the fraction of nucleated cells from the ucb was obtained by sedimentation. samples containing the suspensions of these cells ( , , , mln/ml)were cryopreserved. in vitro cultures of the colony forming cells (cfc) were performed before freezing and after thawing. results: the intensity of the cell clumping increased simultaneously with the increasing density of the cell suspension. it increased from approx. % in the and mln/ml groups to approx. % in the mln/ml group, when thawed accordingly to "the classic" technique. if the solution containing dextran (rubinstein's technique) was applied post thaw to dilute the cell suspension, the clumping phnenomenon was markedly inhibited. it didn't exceed approx. % in any density group. independently to the intensity of the aggregation process, the number of the cfc among the whole pool that remained suspended after thawing (per cells), maintained a quite stable level of approx. % of the pre-freezing value. conclusions: the intensity of the cell clumping phenomenon is directly influenced by the probability of the cell contact. neverthless, the substances like dextran may markedly inhibit this process. this phenomenon is not selective process and affects both early hematopoietic cells (cfc) as well as mature cells, independly of the initial density of the frozen suspension. it seems in order to protect thawed nucleated cell suspensions from clumping and preventing the associated looses, future studies will have to concentrate on the composition of adequate freezing mediums containing clumping inhibitors. centre of slovenia, ljubljana d. domanovic, z. dovc, a. nunar-perko, p. mali, m. cukjati blood transfusion centre of slovenia (ljubljana, svn) collection of peripheral blood stem cells (pbsc) by aphaeresis is safe and reliable procedure that is generally well tolerated. beside the adverse reactions associated with g-csf or gm-csf mobilization, some adverse effects related to the aphaeresis procedure can occur. in the period from january to the end of october we retrospectively analyzed pbsc collections in autologous and allogeneic donors. pbsc collections were performed in the blood transfusion centre of slovenia, ljubljana, by standard volume aphaeresis procedures (two blood volumes processed) on the amicus blood cell separator (baxter). the targeted number of collected cd + cells was > x /kg of recipient's body weight (bw) and > x / kg bw in plasmocytoma donors. peripheral antecubital vein access was established in ( %), femoral catheter in and subclavia catheter in collections were placed without documented side effects. we have identified ( . %) adverse effects and ( . %) instrument troubleshooting. in ( . %) collections, the adverse effects occurred during the establishment of venous access (repeated phlebotomy %, unsuccessful phlebotomy %). during the collection we documented ( . %) adverse reactions. the citrate reaction in collections ( . %), hematoma in collection ( . %), fatigue in collections ( . %) and heart arrhythmia in collections ( . %). citrate reactions were mostly present as significant paresthesias and cured by oral calcium tablets and aphaeresis continued. there were no significant post donation decrease of platelet count and platelet transfusions were not necessary. the one troubleshooting was due to the unrecoverable stop ( . %) an four were during the disposable set instalation ( . %). the data confirmed the presence of mild but relatively common adverse effects in the collection of pbscs by apheresis especially in the autologous donors. the serious adverse reactions were not documented. predictive factors that affect the mobilisation of cd + cells in healthy donors treated with recombinant granulocyte colony-stimulating factor m. martino, i. callea, a. pontari, g. praticò, t. moscato, e. massara, g. console, e. quartarone, g. irrera, g. pucci, a. condemi, a. dattola, p. iacopino azienda ospedaliera "bianchi-melacrino-morelli (reggio calabria, i) no specific characteristics have been identified as predictors of peripheral blood stem cells (pbsc) mobilization in healthy donors. in this study, clinical characteristics and laboratory data for healthy donors who underwent apheresis on day of treatment with recombinant granulocyte colony-stimulating factor (g-csf) were retrospectively analyzed for correlations with cd + cell mobilization. the variables that were analyzed included age, sex, body weight, basal complete blood count and maximum white blood count (wbc) before apheresis, g-csf type and dosage. median age and body weight were . years (range - ) and . kg (range - ), respectively. by univariate analysis, male sex (p= . ), body weight (< vs. > kg, p= . ) and donors age (< vs. > years; p= . ) were correlated with the number of cd + cells mobilized. by multivariate analysis, donor's age and male sex were the only two variables that significantly predicted a high cd + cell level. in conclusion, our data suggest that male sex and younger age are the only factors that significantly affect cd + mobilization in healthy donors. hla-a is a "broad" specificity, which includes a few serologically defined antigens (spits): a , a , a , a , a , a . the hla-a occurs at a rather high frequency in most human populations, and the a splits are found at differing frequencies in different ethnic groups and populations. the distribution of a splits is very important for transplantation medicine, especially for bone marrow transplantation. incorrect assignment of a splits is one of a major problems in using serology for typing hla-a antigens. sometimes serology hardly discriminates a splits and the low resolution dna typing is required. the data of a split distribution in russians are few and ambiguous. the objective of our study was to determine the distribution of a splits in russian donors typed in research center for hematology (moscow) in . methods: donors were typed. serological typing was performed using commercial sera. in case of hla-a or homozygosity in hla-a locus revealed by serology the samples of donor blood were retyped using pcr-ssp ("protrans" or kit of research center for hematology, moscow). results: by serological typing hla-a was found in donors ( . %). the a splits were defined in . the hla-a* (by pcr-ssp) was detected in donors ( %). the most frequent among hla-a splits was a* (n= ; . %). a* and * were found with the equal frequency (n= ; . %). a* was observed only in cases ( . %) and a* in cases ( . %). in our donors we didn't reveal a* . in one case serologically defined a was not confirmed by pcr-ssp. conclusion: these results confirm the studies that dna typing for hla class i improves the typing quality. serological typing is insufficient for hla-a split identification. objectives: the availability of unrelated donor hematopoietic stem cell transplantation(hsct) could be increased with the use of allele mismatch donors if the status of hla mismatching were better understood. with the use of different transplant and immunosuppression regimens according to the patient-risk grouping will allow donor selection criteria to be refined to meet the needs best of the individual patient. methods: we conducted a hla matched or mismatched unrelated donor hsct based on the hla high-resolution typing for consecutive cases( male, female) of adult acute myeloid leukemia(aml) patients. the median age was (range, - ) and the median follow-up duration was months(range, . compared to the group of patients received hsct from hla perfect matched( / ) unrelated donor(cohort ), twentyfive( % of total population as cohort ) mismatched unrelated donor at the level of hla-cw antigen(n= ) or - alleles(n= ) at the hla-a, b, and dr loci were considered. two cases were mismatched at the hla-cw combined with allele level. fortyeight patients were in st cr and were in impending relapse status and most of them had a very poor cytogenetic profiles. the majority of patients(n= ) had intermediate or unfavourable cytogenetic features. the main conditioning regimen consisted in cyclophosphamide plus tbi(n= ) with our standard gvhd prophylaxis containing fk- plus short course methotrexate. some of cohort patients(n= ) received additional mycophenolate mofetil from the day posttransplant. the majority of patients received bone marrow as a stem cell source. the two groups had similar pre-transplant characteristics. results: all, except (cohort ), transplanted patients were engrafted. the incidence of acute gvhd( % vs %, cohort vs cohort ) was significantly different in the two cohort, but not in the chronic gvhd( % vs %). five( %) patients were relapsed in the cohort . the -year non-relapse trm was % vs %. the estimated probability of dfs and efs at -year was % vs % and % vs %, respectively. conclusion: these data suggest that allele and antigen mismatches can elicit more detrimental gvhd together with even a chance of graft failure that lead to increased trm. analysis of large hsct populations with a diversity of mismatches is necessary to define a tolerable mismatched unrelated donor so that we can enlarge the available allogeneic unrelated hsct donor pool. the majority of haematopoietic transplantation are currently performed with peripheral blood progenitor cells (pbpc). in donors, it is very important to optimize pbpc harvesting to obtain the target cd + cell dose required for transplant (> . x /kg body weight of the recipient) with a reduced number of apheresis. in the present study we retrospectively analysed data from pbpc collections of healthy adult donors (n= , m / f) with a median age of years (range - ) performed between august and october . donors were mobilized with daily administration of g-csf ( - mg/kg) for days, with generally mild to moderate side effects. total nucleated cells (tnc) and cd + cells present on pbpc grafts were evaluated with haematologic counters and flow cytometry respectively. on the collection day, the peripheral blood from most adult donors contained > cd + cells/µl, previously established as a marker of a good mobilisation. healthy donors had standard apheresis collection using the cobe spectra, with few adverse consequences, mainly related either to vascular access or to metabolic or hemodynamic changes. the majority of donors (n= %) underwent one apheresis, whilst the remaining % and % of donors had and apheresis respectively. for adult donors the pbpc collected yielded a median . tnc x /kg ( . - . ) and . x cd + cells/kg ( . - . ) body weight of the recipient and only in collections the target number of cd + cells was not reached. we found a correlation between age of the donor and the number of cd + cells collected per kg bw of the recipient, being in the older donors more difficult to achieve the required cd + cell dose. according to gender, male donors underwent one apheresis, had significantly higher pb cd + cell count prior to aphaeresis and cd + cell/kg bw recipient collected in comparison to the female donors who had a median of apheresis and had less cells (pb cd +cells: vs and cd + cell/kg bw recipient . vs . for male and female respectively). there was no correlation between the g-csf dose used in the mobilization and pb cd + cell count prior to apheresis or the total cd + cell collected. our results show that g-csf mobilisation and pbpc collection in adult healthy donors is feasible and safe for the harvesting of the graft for allogeneic haematopoietic transplantation and the main factors affecting collection are age and gender of the donor. thrombophilic screening in healthy donors treated with recombinant-human granulocyte-colony stimulating factor for mobilisation of peripheral blood stem cells m. martino, t. moscato, g. console, g. irrera, g. messina, e. massara, g. praticò, e. quartarone, c. mammì, f. luise, a. piromalli, p. iacopino azienda ospedaliera bmm (reggio calabria, i) the granulocyte-colony stimulating factor (g-csf) induces an activation state of endothelial cells and coagulation system increasing thrombotic risk. laboratory testing for the identification of heritable trombophilia in high-risk subject groups have become common practice. the objective of this study was to evaluate the effectiveness of thrombophilia screening in healthy donors prior to use g-csf to mobilize peripheral blood stem cells (pbsc). thrombophilia screening comprised of testing for factor v leiden (fvl) g a, prothrombin (ptm) g a, thermolabile variant (c t) of the methylene tetrahydrofolate reductase (mthfr) gene, protein c (pc), protein s (ps), factor viii (fviii) and homocysteine (hcy) plasmatic levels, antithrombin iii (atiii) activity, and acquired activated protein c resistance (apcr). we investigated prospectively healthy donors, men and women, with a median age of years (range - ). five donors ( , %) were heterozygous carriers of fvl g a; two healthy donors had the heterozygous ptm g a gene mutation; c t mutation in the mthfr gene was present in ( , %) donors in heterozygous and in donors ( , %) in homozygous.apcr was revealed in donors of the study ( . %). the pc plasmatic level was decreased in donors ( . %); the ps level was decreased in donors ( . %). an elevated fviii dosage has been showed in donors ( . %) and hyperhomocysteinemia in donors ( . %). concentration of atiii was in the normal range for all study group donors. the fvl mutation was combined with the heterozygous ptm g a in two cases and with ps deficiency in one case; two healthy donors presented an associated deficiency of pc and ps. the basal screening of thrombofilia permitted to identify healthy donors with a higher risk of thrombotic events. a careful monitoring should be considered in these cases before administer g-csf. during g-csf therapy, we administered low-molecular-weight heparin in all donors and folic acid, vitamins b and b in healthy donors with c t mutation in the mthfr gene and a hyperhomocysteinemia plasmatic level. our strategy of prophylaxis was correlated with the absence of short-and long-terms thrombotic and hemorrhagic side effects. no complications known to be related to the anticoagulant occurred in this cohort of healthy donors. differential levels of chimeric tolerance in a single patient. how much change in quantitative chimerism values is enough for clinical significance? d. kristt, j. stein, r. narinski, h. or, t. klein, d. kristt rabin mc (petach tikvah, il) chimerism monitoring based on strs is frequently used following sct. it is best implemented in terms of long-term tracking since engraftment is a dynamic process. when viewed this way, trends and fluctuations in the chimerism values can be observed. one fundamental question raised by such observations is what is the minimal change in chimeric status that has biological and/or clinical significance. as an indirect approach to this question, we present a case of thalassemia major which we were fortunate to follow over a year multi-sample course with two scts each with its own stable % chimerism level. following the first sct the patient developed a stable chimeric tolerance of approximately % [ - %]. however, he remained transfusion dependent, necessitating a second transplant two years ago. once again his chimerism stabilized but at a higher plateau of approximately % [ - %]. he is currently healthy, and no longer requires transfusions. in conclusion, this interesting case afforded us the opportunity to compare two different, but stable levels of chimeric tolerance, differing by approximately - %. since over the long-term, str-platform error is approximately - %, the patient is likely to have sustained a stable elevation of approximately % chimerism that freed him from transfusion dependency. the case raises a number of important questions regarding the biological implications of chimerism values, such as: ) is % a relative or absolute figure; ) is there a minimum/threshold chimerism value for functionality of the graft in these metabolic diseases; ) do all patients have the same % chimerism interval/differential and threshold? novel aspect of long-term chimeric tolerance in a patient with severe combined immune deficiency: in utero stem cell transplant suppressed by subsequent paternal iatrogenic transplant t. klein, i. yaniv, b. garti, d. kristt rabin mc (petach tikvah,il) severe combined immune deficiency (scid) is often treated with stem cell transplantation (sct). the successfully treated patient usually lives in a state of mixed graft-host chimeric tolerance. this relationship, however, may not be entirely static. we report here a case of a child in which a transfusion of maternal stem cells occurred in utero. this was documented by demonstrating a mixed chimerism in the child based on an evaluation of the child's blood using hla tissue typing and molecular dna methods. at age mo the patient manifested scid, which necessitated an exogenous sct from the father. following the paternal sct, three dna sources were demonstrated, with four hla haplotypes. however, in the ensuing period progressive loss of the maternal component of the patient's tolerance state was observed. now, after yr post-paternal sct, the patient is healthy, but there is no significant maternal dna signal demonstrated in the child, although he does have a stable chimeric level of - %. in conclusion, the three-way mixed chimerism was not tolerated, and the in utero maternal stem cell component was ultimately suppressed by the subsequent paternal sct. this case suggests that even a tri-valent tolerogenic state may be regulated by the dynamic interactions between the host and graft, enabling one set of graft-host interactions to become dominant. successful treatment of graft rejection with immunosuppression withdrawal and/or donor leukocyte infusion after early diagnosis based on t-cell mixed chimerism i. buño, p. balsalobre, g. iglesias, d. barroso, c. manzano, d. serrano, r. carrión, a. gómez-pineda, j.l. díez-martín hosp. g.u. gregorio marañon (madrid, e) background: graft rejection is a serious and difficult to manage complication after sct. objective: to evaluate the usefulness of chimerism monitoring for the early diagnosis of graft rejection in different sct settings, as well as for the follow up after treatment with immunosuppression withdrawal (isw) and/or dli. patients and methods: sct ( ablative, ric, tcdincluding haploidentical-). chimerism analysis was performed by fish for the sex chromosomes or str-pcr (sensitivity %). chimerism was analyzed in pb and leukocyte lineages (t lymphocytes cd +, b lymphocytes cd + and myeloid cells cd + isolated (purity > %) by immunomagnetic means, automacs, miltenyi biotec), every weeks, starting on day + (except in ablative), and until complete chimerism (cc) was achieved. results: after initial engraftment in all patients, graft rejection was diagnosed in ( ( %) ablative, ( %) ric, ( %) tcd), either established (severe pancytopenia and bm aplasia) or incipient (progressive decrease in pb and bm cell counts) a median of . days (range - ) after sct. all patients showed mixed chimerism (mc) in bm and pb with higher percentages of recipient cells (%r) in pb. in / patients studied, t cells showed persistent mc with high %r (> % in / ; < % and > % in / ). b cells showed mc in / patients studied, with lower %r (< % in / , > % in / ). only patients showed mc, transient in one of them, in myeloid cells. patients were not treated due to concurrent multiorgan failure and subsequently died. reduction of is in patients obtained response (normal pb and bm counts, and cc). the other patients underwent isw but no further response was obtained. one of them received a second sct while the other were treated with dli, and all of them responded. the last patient (transplanted from a haploidentical family donor) who was not receiving is, responded to treatment with dli. time from therapeutic intervention to response was variable with a median of months (range - ). patients developed gvhd>i, which was the cause of death in one and was controlled in the other three. one patient died from sepsis in complete remission (cr) months after the transplant. patients are alive in cr a median of months after sct (range - ). conclusions: the observation of mc, mainly in t lymphocytes, together with a decrease in pb and bm cell counts, allowed early diagnosis and successful treatment ( / patients) of graft rejection. immunosuppressive effects of nucleic acids -or how to learn from artefacts? t. yang, h.j. kolb, i. steinmann, m. svihla, r. buhmann gsf (munich, d) defibrotide, a single-stranded nucleic acid (ssna), was already shown to mediate immunosuppressive effects. in the current survey we investigated whether randomly chemically synthesized ssnas of different length and composition could provide similar effects. for this purpose, purified t-cells were stimulated in the presence of dntp´s or ssna with allogeneic, irradiated pbmc´s, pha or anti-cd /cd dynabeads. cellular proliferation was assessed by incorporation of tritiumlabelled thymidine (³h) thymidine), respectively (³h)damp or by staining with cfda (carboxyfluorescein diacetate, succinimidyl ester). after h or h of incubation, the incorporation of (³h)thymidine, or (³h)damp as well as the cfda distribution was assessed. cell viability was measured by trypan blue exclusion. t-cell activation was measured after h by quantifying the number of cd + t-cells expressing the activation markers cd and cd . cellular uptake of cy labelled nas was detected by fluorescence microscopy. moreover, the interference of different nas or singular nucleotides with nucleoside analogues on t cell proliferation was tested by cfda-assays. na of different length, composition or concentrations (up to mm) did not cause cytotoxic effects to lymphocytes. but the incorporation of (³h)thymidine or (³h)damp was competed by na. these effects were found to be dependent on length, concentration and base-composition of the na. the proliferative capacity of the t-cells, as assessed by cfda-staining, seemed to be unaffected. moreover it could be shown, that nas interfere with nucleoside analogues and antagonized the antiproliferative and cytotoxic effects of these drugs. the standard approach to detect cellular proliferation by incorporation of tritium-labelled nucleotides or derivatives is not useful to assess changes in cellular metabolism or proliferation in context with nas. even more important, treatment approaches using nucleoside analogues like fludarabine, cytarabine e.g. in context with nas might be critical and diminish the efficacy of these drugs. e. elli, f. colnaghi, a. colombo, m. parma, v. rossi, e. terruzzi, l. verga, a. biondi, e. pogliani ospedale s. gerardo (monza, i) introduction: chimerism status (cs) analysis is useful for evaluation of donor (d) cells engraftment after allogeneic haematopoietic stem cell transplantation (hsct). adverse events are often described as associated to a loss of chimerism, but a strict correlation between cs and residual disease is still controversial. pcr-based assays analyzing polymorphic short tandem repeat (str) markers are actually the more employed methods in cs monitoring, even if a standardized specific panel is not still available. objective: we tested a semi-quantitative method, based on multiplex pcr amplification of str markers using a commercial kit (powerplex system promega), in order to evaluate its informativity in cs monitoring and the correlation between cs and some clinical variables. methods: the informativity of the assay was tested on peripheral blood samples from allografted patients (pts) and their related sibling donors; perspective evaluation of cs was performed on pts at , , , , , , and months after hsct. pts who showed no evidence of recipient (r) cells were considered to have a complete chimerism (cc), pts who presented each d and r cells were defined as mixed chimerism (mc). results: the multiplex assay gave at least one high informative marker (range: - , median ) in all the pts. we analyzed blood samples from pts, ( , %) presented cc, ( , %) mc and none autologous reconstitution. some pts were defined as having an increasing mc (imc) when they shifted from cc to mc or when in a mc setting the r amount increased in two or more consecutive controls. we evaluated if there was a correlation between cs and some clinical variables: r/d gender, diagnosis, r/d sex mismatch, abo system incompatibility, conditioning regimen, cd + and cd + cell dose infused, disease status at hsct, stem cells source, acute and chronic graft versus host disease (gvhd), marrow relapse. chi-square analysis demonstrated a significant correlation between icm and a brief time marrow relapse, moreover a low incidence of chronic gvhd, male d and diagnosis of acute leukemia seem to be associated with increasing level of r dna. conclusion: pcr amplification of a panel of str loci is an informative method to evaluate cs in pts after hsct. imc seems to be useful to predict marrow relapse; some clinical conditions such as male donor and acute leukemia diagnosis seem to limit a complete d engraftment; chronic gvhd is favorable for a stable cs. non-haematopoietic tissue repair r gmp production of autologous cd + cells for intracoronary administration after acute myocardial infarction r. giordano ( ) subjects affected by acute myocardial infarction (ami) with absent angiographic myocardial blush (mb) and lack of st segment elevation resolution after primary angioplasty, have short-and long-term poor clinical prognosis. we recently started a phase i/ii randomized controlled study based on the hypothesis that, in this target population, after primary angioplasty and stenting, intracoronary injection of cd + cells from bone marrow (bm, group a) or mobilized peripheral blood (mpb, group b) could enhance endothelial regeneration and improve heart function compared to controls treated with standard pharmacological therapy alone (group c). the study started in june and it is expected to enroll patients ( per each randomization group). up to november , patients have been included. in group a (n= ), bm was processed within hours of collection. in group b (n= ), the administration of g-csf ( µg/kg/day for - days), started from day - after ami and leukapheresis was performed following standard procedures. an automated cd + stem cell selection was performed with the clinimacs® plus instrument (miltenyi biotec) in our class b -iso facility. the mean (±sd) number of cd + cells after immunoselection was . x (± . ) in bm samples and . x (± . ) in mpb samples respectively, with a purity of % (± ) in the group a and % (± ) in group b. the percentages of viable cells (propidium iodide) in the post-selection samples were (± ) in group a and (± ) in group b, respectively. the sterility tests for bacteria and fungi on the purified samples were negative. purified cd + cells were injected in the culprit vessel using a well-sized over-the-wire angioplasty balloon within three minutes of occlusion. no adverse events have been reported during and immediately after the cell administration. this results show that cd + selection is feasible and safe also in ami patients. the short and long term efficacy of this cell therapy approach in preserving the myocardial viability and function after ami is currently under investigation using pet-based techniques and echocardiography. defibrotide (df) is a polydisperse mixture of % singlestranded polydeoxyribonucleotides with anti-thrombotic, profibrinolytic and anti-apoptotic functions. df is already successfully used in the treatment of hepatic veno-occlusive disease in allogeneic stem cell transplantation (sct). our observation that df can also protect endothelial cells (ec) from conditioning (fludarabine)-mediated apoptosis ( ) prompted us to apply it prophylactically to patients (pts) at risk for endothelial complications. pending on the magnitude of risk, pts received - mg every h in h-infusions, usually from day (d) - until d+ post sct. circulating ec (cec) as a marker of conditioning-mediated endothelial toxicity ( ) were detected by magnetic bead separation of cd + cells from edta blood of sct pts ( df, non-df) and co-staining with ulex europaeus antigen lectin . cec maxima until d+ post sct were compared between the two groups. df pts showed significantly lower maxima of cec than untreated pts ( [± ] in the df treatment group vs. [± ] cec/ml in non-df pts, respectively, p= . ). similarly, when cec maxima were compared in the time period of df prophylaxis, again, df pts had less cell counts ( [± ] vs. [± ] cec/ml in control pts, respectively, p= . ). in an overlapping cohort of pts ( non-df, df) serum was assayed for its induction of apoptosis in a human microvascular endothelial cell line (hmec), a monitoring approach that had been found to correlate with episodes of gvhd and severe microangiopathy ( ). apoptotis was determined by flow cytometric analysis of the cellular granularity of propidium-iodide-negative indicator hmec. similar to the cec measurements apoptosis inducing maxima until d+ were compared between df and non-df pts and turned out to be significantly different (apoptosis by pts´ sera normalized to untreated control hmec: . [± . ] in df pts vs. . [± . ] in non-df pts, p= . ). these preliminary analyses suggest the protective efficacy of df prophylaxis in the course of sct. the final proof of principle is to be validated in long-term clinical follow-ups. .g. eissner et al., blood , - ( ). .a. woywodt et al., blood , - ( ). .a. ganster et al., bone marrow transplant. , - ( . treosulfan, cyclophosphamide and anti-thymocyte globulin for allogeneic haematopoietic stem cell transplantation in severe aplastic anaemia s. giebel, j. wojnar, m. krawczyk-kulis, m. markiewicz, i. wylezol, t. kruzel, m. kopera, j. holowiecki silesian medical university (katowice, pl) graft rejection is the major cause of failure after allohsct in severe aplastic anemia (saa), when cyclophosphamide is used as a single agent for conditioning. to reduce the risk of this complication we decided to intensify the preparative regimen by adding reduced dose of treosulfan -an alkylating agent possesing both immuno-and myeloablative properties. between between - years) were treated in a single institution with allohsct from either hlaidentical sibling (n= ) or an unrelated volunteer (n= ). conditioning regimen consisted of treosulfan g/m²/d on days - , - , cyclophosphamide mg/kg/d on d. - , - , - , - , and anti-thymocyte globulin (thymoglobulin, genzyme) mg/kg/d on d. - , - , - . each bone marrow and peripheral blood was used as a source of stem cells in cases. all patients engrafted with the median time to neutrophil > . x /l and platelet > x /l recovery of ( - ) days and . ( - ) days, respectively. complete donor chimerism was achieved on day + in all cases. none of the patients developed grade iii-iv acute gvhd, one patient experienced grade ii acute gvhd. at one year the cumulative incidence of extensive chronic gvhd equaled %, overall cgvhd - %. at the median follow-up of . ( - ) months all patients remain alive and disease-free with complete donor chimerism. at one year the karnofsky index equaled % in patients, % -in one case. we conclude that treosulfan + cyclphosphamide + antithymocyte globulin is a well-tolerated preparative regimen and allows stable engraftment in saa patients. the use of treosulfan allows intensification of the conditioning without providing an additional non-hematological toxicity. clinical outcome in adults with severe aplastic anaemia. a retrospective single-centre analysis s. buchholz, e. dammann, c. koenecke, e. mischak-weissinger, m. stadler, m. eder, j. krauter, b. hertenstein, a. ganser hannover medical school (hannover, d) introduction: allogeneic bone marrow transplantation (bmt) is the treatment of choice in young patients suffering from severe aplastic anaemia (saa). due to improved, less toxic conditioning regimens and advances in prophylaxis against graft-versus-host-disease (gvhd) survival has improved steadily. nonetheless, long-term side effects, such as chronic gvhd, occur in up to % of patients requiring treatment and leading to an increased mortality. here we present the analysis of for patients with saa comparing. patients and methods: between and , twenty one patients ( male, female) with saa and one patient (female) with paroxysmal nocturnal haemoglobinuria (pnh) were transplanted in our centre. the median age at transplantation was . years (range - ). fifteen patients were transplanted with stem cells from their hlaidentical related donor, patients from hla-identical matched unrelated donors and patients were transplanted from a syngeneic donor. in cases stem cell source was bone marrow (bm), patient received bm and peripheral stem cells (pbsc) and were transplanted with pbsc. conditioning regimen consisted of cyclophosphamid (cy) alone (n= ), or in combination with either total nodal irradiation (n= ), or with fludarabin (flu, n= ). one patient was treated with cy and total body irradiation (tbi) while patient received flu, cy and tbi. gvhd-prophylaxis was cyclosporin (csa) and methotrexate (mtx) in all but the patients transplanted from syngeneic donors. all patients engrafted. acute gvhd developed in patients ( %) and was readily controlled by immunosuppression. chronic gvhd occurred in patients ( %; limited, extensive) within a median follow-up of years (range . - . years) . twenty one out of patients are alive and free of haematological disease, one patient died because of toxoplasmosis before day + . conclusion: the incidence and severity of acute and chronic gvhd is similar other studies. our data suggest that bmt is a favourable therapy for young patients with aplastic anaemia, showing good engraftment, controllable complications and a good clinical outcome. stem cell transplantation and immunosupressive treatment of severe aplastic anaemia: single-centre experience l. tukic, d. stamatovic, o. tarabar, v. glavicic, m. elez, l. simic, s. marjanovic, m. malesevic military medical academy (belgrade, cs) background: stem cell transplantation (sct) from an hlaidentical fully mached sibling donor (msd) is the best treatment option for severe aplastic anaemia (saa). patients without suitable msd should be treated with immunosuppressive therapy (ist). patients and methods: between / and / patients with newly diagnosed saa were treated either with sct from msd ( patients) or with ist ( patients). there were performed allogeneic sct in patients. all donors were hla-identical sibling ( donor was identical twin). source of stem cells was bone marrow in (one with second transplant) and peripheral stem cell in scts. conditioning regimens were based on cyclophosphamide (cy) with atg in and fludarabine with cy and atg in scts. patients received combined ist with antithymocyte or antilymphocyte globuline (atg/alg), cyclosporine a and steroids and patients atg with steroids. the median interval from diagnosis to ish was days (range to ). results: engraftment was documented in patients with allogeneic sct ( patients died without engraftment). one patient developed acute gvhd grade - and died after days, and the other had pneumonitis interstitialis (cmv+) and died after days. till november of patients ( %) are alive with sustained engraftment. median survival from sct is months (range to ). concidering ist, of patients ( . %) achieved response ( had two or tree cycles of ist). one patient relapsed year after ist. two patients from ist group died, major causes of death were infection and hemorrhage. overall survival in the ist group is . % ( / patients) after a median follow up of . months (range to ). conclusion: our results confirm significant improvement in outcome of saa patients during last decades in due to modern induction front line therapy including allogeneic autoimmune diseases r successful treatment of autoimmune thrombocytopenic purpura after bone marrow transplantation with anti-cd antibody: a case report b. giannini, c. bosi, m. stanzani, g. bandini, f. bonifazi seragnoli (bologna, i) we describe a case of persistent, severe autoimmune thrombocytopenia, refractory to prednisolone but responsive to chimeric monoclonal antibody anti-cd ( rituximab). a patient transplantated for hodgking disease (upn ), developed autoimmune thrombocytopenia with severe bleeding, days after unrelated bone marrow transplantation: at the same time there were no signs of graftversus-host-disease, cytomegalovirus infection, sepsis or microangiopathic process. high titer of antibody against platelet antigens was found. during treatment with prednisolone mg/kg, for two days, the platelet count remain below /µl. in spite of increasing the dose of cyclosporine and methilprednisolone ( mg/kg/daily) with addition of intravenous immunoglobulin for five days, only a transitory partial response ( platelet /µl) was observed. after four days from last dose of immunoglobulin, the platelet count fell again below /µl; antiplatelet antibodies still highly positive. after two weeks of therapy with steroid, we started with anti-cd antibody with first dose of mg/m² followed by dose of mg/m² once weekly for weeks. complete response was achieved after weeks from therapy initation, with a complete normalitation of platelet count and with disappearance of antiplatelet antibody in peripheral blood. no apparent toxicity, or side effects that could be attributed to rituximab were observed. the patient is in complete response mounths after therapy with anti-cd . rituximab induced complete response in approximately % of the patient with immune thrombocytopenic purpura refractory to prednisone or splenectomy; it's also effective in patients with secondary itp. to our knowlege, only few cases of itp after bone marrow transplantation have been successful treated with rituximab. we suggest for an early use of anti-cd in similar cases. treatment of a malignant form of multiple sclerosis with immune ablation and autologous stem cell transplantation v. kimiskidis, i. sakellari, c. smias, k. kapinas, a. anagnostopoulos, a. kazis, a. fassas g.papanicolaou (thessaloniki, gr) malignant forms of multiple sclerosis (ms) are rare cases characterized by very aggressive demyelination and rapid progress to disability leading to death within years from onset despite treatment, which fails to control the disease. we report a case of a young male patient of years old with aggressive ms who was treated with a high-dose immunosuppressive regimen (hdis) using myeloablation followed by autologous blood stem cell transplantation (asct) that has induced a dramatic and long-lasting remission of the disease. the patient was diagnosed with ms in june . at that time he had minor disability, his edss score being was . , and active disease on mri showing gadoliniumenhacing (gd+) lesions. he was put on steroids and interferon-beta which, however, had no effect, while disability was rapidly accumulating. by february , i.e. within months, the edss score rose to . and the patient was unable to walk unaided for more than metres. on mri, gd+ lesions increased to , as did their volume. in march it was decided to treat him with hdis and, in order to mobilize blood stem cells, he received cyclophosphamide (cy) g/m² plus gcsf ug/kg. six days after cy infusion he had a disease flare with worsening of edss score to . , and further increase in the number and volume of gd+ lesions. the patient was treated with steroid pulses for days and showed some improvement which allowed the continuation of g-csf and subsequent stem cell collection. two months after cy infusion, he underwent asct with busulfan mg/kg over days plus antithymocyte globulin . mg/kg for conditioning. one month post asct the edss score dropped to . and no gd+ lesions were detected. the patient continued to improve over the following years. the last assessment at months after asct showed nearly absent disability (edss score: . ) and, again, no gd+ lesions on mri. the spectacular responses of this case and also of the three similar cases reported in the literature support the role of asct in rapidly evolving, so-called malignant, forms of ms. an obvious amelioration in a case of scleromyxedema after successful double autologous peripheral blood stem cell transplantation followed by thalidomide and bortezomib consolidation s. ataergin, f. arpaci, a. ozet, m. demiriz, s. komurcu, b. ozturk, o. kuzhan gata (gulhane) objective: scleromyxedema is characterized by cutaneous deposition of mucin, dermal fibrosis and monoclonal gammapathy. the response to treatment has been variable after several treatment modalities including high-dose treatment (hdt) and autologous stem cell transplantation (asct). methods: we report a case of scleromyxedema who achieved amelioration after double hdt and asct, followed by thalidomide and bortezomib consolidation. a -year-old male patient was admitted with persistent pruritis. he was treated with anxiolytic and antidepressant drugs with diagnosis of neurodermatitis; however, the skin was thickened and papular lesions appeared. a skin biopsy revealed scleromyxedema. he was treated with topical corticosteroids and retinoic acid preparations. however, no amelioration were noted. topical cyclophosphamide, systemic corticosteroids were used for three months, then the patient has discontinued all the medications. low-dose interferon-a treatment ( . mu) was initiated for three times weekly; however, the treatment was stopped due to an acute and severe rhabdomyolisis after the third administration. on his admittance in our department, he had papular lesions of . mm without pain, erythema or desquamation all over his body. he had also some nodular formations of . x . cm, on his face and neck. the whole blood count and biochemical analysis were normal. erythrocyte sedimentation rate, crp were within normal limits and all viral markers were negative for hepatitis, ebv, cmv, toxoplasma, hiv as well as rheumatologic markers. serum immunoglobulin (ig) levels were within normal ranges except for ig g and ig light-chain lambda. the peripheral blood smear and bone marrow aspiration and biopsy revealed no pathology. he then underwent an asct after conditioning with melphalan ( mg/m²) and a second transplant was done four months later using the same conditioning regimen ( mg/m²). results: after the transplantation, the immunoglobulin levels have partially regressed. physical appearance has been ameliorated and the skin biopsy revealed a regression in mucin deposition in dermis. consolidation treatment was initiated with thalidomide followed by bortezomib. he is still on his follow-up without any progression for three years. conclusion: hdt and asct may be an alternative treatment in the amelioration of lesions related to scleromyxedema. ( ) the depletion of autoreactive lymphocyte populations in the graft is mandatory for the success of autologous transplantation in patients with severe autoimmune disease. clinical grade cd + selection can be used to obtain lymphodepletion in autologous leukapheresis products. we report on the implementation of a gmp production process of autologous purified cd + cells from mobilized peripheral blood of patients affected by systemic sclerosis (ss) in our institution. for cd + cell mobilization, patients ( male, females, aged years ± ) with ss refractory to standard immunosuppressive therapy, received cyclophosphamide (cy) g/mq and g-csf (filgastrim, ug/kg/day s.c.) starting days after the last cy administration until stem cell collection. leukapheresis was performed when wbc and cd + cell count were at least x /l and /ul respectively, using a fresenius hemocare as (c y) instrument. the automated selection (clinimacs, miltenyi) was performed in hours of leukapheresis collection. the selection procedure and the preparation of the final product for the cryopreservation were performed in our class b -iso facility. the cells, resuspended in normal saline solution and human serum albumin, were cryopreserved in % final dmso. for quality control of the cryopreserved product, the cells from a satellite segment of the cryopreserved bag were thawed and their viability evaluated by trypan blue exclusion. the mean (±sd) wbc, cd + and cd + cell content of the pre-selection products were , x (± , ), x e (± ) and , x (± , ) respectively. after immunoselection we obtained a mean (±sd) cd + and cd + cell number of x (± ) and . x (± . ), with a purity of % (± ). the mean percentage of viable cells (propidium iodide) in the post-selection samples was (± ). the sterilty tests for bacteria and fungi on the purified samples were negative. the viability of the post-thawed samples was % (± ). the conditioning regimen consisted of cy mg/kg/day for days i.v. and rabbit anti-thymocyte globulin . mg/kg/day for days. neutrophil engraftment was reached at day + (± ), and all patients are alive, with stable or improved clinical conditions after months (± ) after transplantation. these results demonstrate that gmp production of cd + cells for autologous transplantation is feasible, safe and could efficiently support a transplantation program for patients affected by ss. high-dose immune immunoablative therapy and autologous stem cell transplantation in severe resistant crohn's disease: profound response for months followed by treatable relapse y. sorour, k. robinson, p. hurlstone, k. el-ghariani, a. lobo, j.a. snowden sheffield teaching hospitals (sheffield, uk) we report the case of a year old female with severe resistant crohn's disease (cd) treated with high-dose immune immunoablative therapy (hdit) and autologous stem cell transplantation (sct). diagnosed with cd at age , initial disease control had been achieved with azathioprine and steroids, but, at age , colectomy and ileostomy were performed for a severe flare. from age , increased disease activity was unsuccessfully controlled with azathioprine, steroids, infliximab, methotrexate, combination rifabutin/metronidazole/clarithromycin, thalidomide and tacrolimus. from nov -jan , surgical episodes had resulted in resection of . m small bowel. dietary modifications had included an elemental diet, but from the patient was dependent on home total parenteral nutrition. severity of symptoms had resulted in recurrent and prolonged inability to work and to warrant care under a palliative medicine specialist. based on poor quality of life, risk of life threatening complications, and inability to control the disease effectively, the option of autologous transplant was pursued after examination of the case and proposed treatment protocol by the local research ethics committee, review by two independent gastroenterologists and one transplant haematologist, and obtaining informed written consent. treatment commenced in sept with mobilisation using cyclophosphamide (cy) g/m² and g-csf. in nov the patient was treated with cy mg/kg, rabbit atg mg/kg and methylprednisolone gx followed by . x /kg isolex enriched cd + cells. treatment was complicated by neutropenic sepsis, oropharangeal and stomal mucositis. engraftment time was within normal limits. discharge was on day+ . pre-and months post-treatment data are summarised in the table. cd remained inactive until march with the development of increased stoma output and abdominal pain. relapse was confirmed by ileal biopsy. in contrast to pre-sct, disease control has been achieved with immunosuppressants and surgery, permitting ileal reanastomosis, pouch formation and reversal of ileostomy in sept . the patient remains stable as of nov . in conclusion, hdit and autologous sct may be an effective therapy for medium term control of severe, treatment resistant cd. it remains to be seen whether post-sct relapse is easier to control than cd activity before sct, as suggested by data in other autoimmune diseases. in addition to randomised trials, future studies could look at means of prolonging responses, such as maintenance treatments. hyperkalemia is one of the side effects of cyclosporin (csa). the mechanism of hyperkalemia is unclear. apparently many factors in pathogenesis of hyperkalemia may be involved. nephrotoxicity of csa that significantly impairs renal perfusion, glomerular filtration reduced k+ excretion and secondary hypoaldosteronism seemed to be the reasons for csa-associated hyperkalemia. there are publications about hyperkalemia in patients after renal transplantation but only few reports devote this phenomen in bone marrow transplantation patients. we report about cases of hyperkalemia with bradyrhythmia during csa administration in patients with cml in cp, male and years old undergoing bmt from hla -identical siblings. hyperkalemia ( , - , mmol/l) and bradyrhythmia ( - /min) in - days after transplantation was observed. at that time a serious worsening in renal function (increase serum creatinine and serum urea, decrease filtration rate) and in serum csa level rise was found in both. after discontinuation of csa treatment all these symptoms disappeared. these observations suggest that csa may be a cause of hyperkalemia associated with bradyrhythmia in bone marrow transplantation patients. careful monitoring of csa level in blood and renal function may be important in prevention these complications. objectives: allogeneic haematopoietic stem cell transplantation (allo-hsct) is the only curative therapy for patients with chronic lymphocytic leukaemia (cll). however, transplant-related mortality remains relatively high and relapse is still a major problem. there are only few anecdotic reports of the use of thalidomide, an immunomodulating agent, in such patients. case report: -year old patient with resistant cll is presented. he was treated unsuccessfully with several cycles of different agent combinations, started with chlorambucilmethylprednisolone then fludarabine-cyclophosphamide and also with monoclonal antibody rituximab. as he has hla matched sister we proposed allo-hsct. on admission before transplantation patient was presented with massive lymphadenopathy, anemia (hb g/l), wbc . x /l with absolute lymphocytosis (lymphocytes %) and thrombocytopenia x /l. pre-transplant conditioning consisted of high-dose cyclophosphamide and total body irradiation. combination of monoclonal antibody alemtuzumab (campath- h), cyclosporine a and short methotrexate were given for the prevention of acute graft versus host disease (gvhd). the patient received . x /kg stem cells. there were no serious complications in post transplant period. lympadenopathy completely disappeared. patient was discharged weeks after transplantation, without gvhd and with normal complete blood counts (cbc). on bone marrow examination there was still residual leukaemic infiltration ( %). two months later acute gvhd developed, with skin involvement only. at that time lymph nodes massively enlarged again and high wbc with absolute lymphocytosis reappeared. cyclosporine immunosuppressive therapy was stopped and thalidomyde mg/day was introduced combined with methylprednisolone mg three times per week. during period of months treatment he was readmitted once for lung aspergillosis which responded well to voriconazole. improvement appeared slowly, with regression of lymphadenoapthy. after months he still has residual leukaemic marrow infiltration -about %, but normal cbc without absolute lymphocytosis. conclusion: our patient is new evidence that thalidomide may have significant antileukaemic effect in refractory cll. due to this and anti gvhd effect perhaps in the future it could be incorporated as a first line gvhd prophylaxis regimen in patients transplanted for cll. intensive combination therapy and autologous pbpct for blast crisis cml revisited d. heim ( ) imatinib is the most active therapy for chronic myeloid leukaemia (cml) in all phases of the disease. overall survival of patients with blastic phase cml treated with imatinib monotherapy however is months only. the reason for imatinib resistance in advanced phase cml is mostly due to bcr-abl independent mechanisms. therefore a combination therapy of imatinib with conventional high dose chemotherapy is often used for remission induction and allogeneic stem cell transplantation is performed if a suitable donor is available. autologous stem cell transplantation has drawn new attention in the treatment of cml since sufficient numbers of peripheral stem cells (pbpc) can be mobilized under concomitant treatment with imatinib and collection of bcr-abl negative autologous peripheral stem cell transplants has been reported. we tested in a pilot trial of patients the feasibility of a treatment consisting of imatinib mg qd combined with cycles of cytarabine mg/m² x d in patients with cml in blast crisis (bc) who do not have a hla-matched stem cell donor. pbpc were mobilized after the th cycles of cytarabine with g-csf (filgrastim) ug per day. pbpc were cryopreserved and reinfused after a conditioning therapy with either cyclophosphamide mg/kg and busulfan mg/kg (bucy)(table : patient + ) or cyclophosphamide mg/kg plus gy tbi (cy/tbi)(table : patient ) the stem cell products of all patients contained sufficient numbers of cd + cells after mobilization with g-csf after the th cycle of high dose cytarabine and imatinib given through. the autologous pbpc graft of one of the patients was bcr-abl negative, a second graft had detectable bcr-abl at low level in the q-rt-pcr (table ). no data on the presence of bcr-abl is available for the third graft. engraftments of the transplants were in the expected range. no excessive toxicity was recorded. treatment of cml in bc with imatinib combined with high dose cytarabine and autologous pbpct after high dose chemo-/radiotherapy is feasible and may result in sustained complete molecular remission. alemtuzumab and autologous sct in cll, experience in a small centre e. zappone, e. ortu la barbera, u. coppetelli, c. ciabatta, f. ciccone, s. nardelli, a. centra, g. potente, a. de blasio ospedale s.m. goretti (latina, i) alemtuzumab (al) as a single agent or in combination with chemotherapy is an effective treatment for chronic lymphocytic leukemia (cll) in refractory or relapsing patients (pts) and has been shown to induce complete molecular responses. autologous stem cell transplantation (sct) induce prolonged and durable remission in many hematological malignancies but its role in the treatment of cll is controversial. we included al in the treatment of refractory, relapsed or high risk cll pts prior to stem cell collection and high dose chemotherapy consolidation of the response obtained. we treated pts with binet stage iii cll in partial remission (pr) or stable disease (sd) after to lines of chemotherapy, including fludarabine and pt with advanced b-lymphocytic lymphoma in pr. chemotherapy debulking prior al was necessary in pts with high disease burden and large nodal involvement. disease status before al treatment was complete remission (cr) , pr , and sd . al was given subcutaneously, treatment duration was - weeks and total dose was - mg. results after al were cr and pr , all pts had below normal lymphocytic counts, and lymphoid marrow infiltration below %; out of cll pts had cd /cd expression in the marrow below %. after al out of pts were mobilized: with cyclophosphamide - gr/mq and with additional chemotherapy followed by aracytin; mobilization is planned in the last pt. stem cell collection was adequate: - cd x /kg, in - apheresis procedures. two pts were not mobilized after being treated for cytomegalovirus (cmv) infection at the end of al treatment, they maintain cr without further treatment and months after al. four pts undergo sct: engraftment and clinical course were normal, pt progressed before transplant. of the transplanted pts are in ccr at , and months post sct, pt progressed months post sct and was retreated with al with minor response. cmv reactivation occurred in out these pts and antiviral therapy was necessary in . al was effective in inducing significant clinical response in these high risk pts, stem cell collection was feasible and autologous sct could be performed without significant early or late complications. cmv reactivation occurs in the majority of al treated pts and must be carefully monitored. the results in this very small group of patients are encouraging but we can not draw any conclusion about the general application of this program in cll. at the time of imatinib era in cml treatment hsct became a disputable issue. in this study we compared the outcome of unrelated donor transplantation to that in sibling donor setting in cml patients receiving reduced toxicity conditioning. patients received hsct from matched sibling donors, patients from unrelated donors. reduced toxicity consisted of: : busulfan mg/kg, fludarabine mg/m² (allo sib) or mg/m² (mud) and atg. donors for unrelated transplant were matched for specificities at following resolution levels : loci a, b, c at intermediate or high resolution and dr at high resolution level only. patients were stratified into groups : i) allo-sib ( hla matched ; n= ) , ii) mud fully matched ( / match at intermediate or high resolution level for hla class i and high resolution for class ii ; n= ) , iii) mud mismatched ( at least allele mismatched ; n= ). cumulative proportion survival was: i) % at the end of y follow up period in sib hsct , ii) % in patients transplanted from mud donors fully matched in specificities and iii) % in patients transplanted from donors mismatched in at least one allele. (figure) our results document, that optimal matching in five loci benefit the outcome of transplantation, which in unrelated donor transplantation can be similar to that obtained with sibling donors providing / matched donor at high resolution level. refractory graft-versus-host disease after stem cell transplantation in thalassaemia: pentostatin is safe and effective treatment g. leopardi, g. visani, c. giardini, g. sparaventi, f. d'adamo, g. nicolini, b. guiducci, s. barulli, m. lucesole, l. malerba, a. isidori san salvatore hospital (pesaro, i) toxicity, graft rejection with return of the thalassemia and graft-versus-host disease (gvhd) are the main causes of failure after stem cell transplantation (sct) in thalassemic patients, particularly in those at poor prognosis (i.e. class and ). steroid refractory gvhd is associated with a not negligible non-relapse mortality. few data are actually avalaible on the use of pentostatin for refractory gvhd in thalassemic transplanted patients. we analyzed four children, females and males, aged - (median . years), transplanted for beta-thalassemia major ( class , class , class ). two patients (class and class ) received unrelated sct ( bone marrow, pbsc). the two other children (both class ) were transplanted from hla identical sibling after previous transplantation procedures, having rejected twice and once, respectively. three patients ( unrelated and hla identical sct) developed refractory acute gvhd grade iii-iv with multiple organ involvement at + , + and + days, respectively. one patient had chronic extensive gvhd. in the patient receiving the third allogeneic hla identical sct both acute and chronic refractory gvhd occurred. patients affected by acute severe gvhd failed to respond to primary treatment with cyclosporine a and methylprednisolone at doses varying from to mg/kg/day and thus received salvage therapy with pentostatin . mg/sqm for consecutive days. one presenting with acute multiorgan gvhd (skin, liver and gastrointestinal tract) had short term response. two patients responded: one developed a secundary, chronic extensive, gvhd. this child as well as the other with chronic severe extensive gvhd (involving skin, liver, eyes and oral mucosa in one case and skin and mouth in the other) starting at + and + days from transplant were treated with pentostatin ( mg/sqm i.v. every weeks) for and months. they are alive with significant improvement of skin and mouth symptoms and tapered concurrent immunosuppressive treatment. no toxicity or impairing chimerism due to pentostatin were observed. pentostatin thus appears as safe and effective treatment for acute and chronic severe gvhd after sct for thalassemia. supported in part by ail pesaro onlus pure red cell aplasia after mud stem cell transplant in thalassaemia major: successful treatment with rituximab m. lucesole, g. visani, c. giardini, b. guiducci, g. sparaventi, g. nicolini, f. d'adamo, g. leopardi, s. barulli, l. malerba, a. isidori hematology and transplant center (pesaro, i) pure red cell aplasia (prca) is a not infrequently observed complication of allogeneic sct performed across the abo complex and often refractory to standard strategies. the peculiar recovery of erythropoiesis after sct in thalassemia major could be a possible factor confounding for a correct diagnosis, as well as for the therapeutic choice. a young male patient was transplanted from abo incompatible (donor b rh+; recipient rh+) hla-identical unrelated donor for beta thalassemia major. after standard myeloablative conditioning regimen (bu-cy-thio), he received cyclosporine a and mtx as gvhd prevention prophylaxis. the post-transplantation course was characterized by an incomplete haematological recovery. a poor graft function was observed at day + . the bone marrow was hypoplastic with the apparent absence of erythroid precursors, and a possible rejection of the graft was suspected; nevertheless, fish analysis of chromosome y for the engraftment showed % of donor cells, with a normal count of the beta chains ( %); no evidence of haemolysis was recorded. all dna virus were negative. the diagnosis was prca. epo . u/every other day was started by day + ; additionally we submitted the patient to plasmapheresis ( procedures, last at day + ) without hematologic response ( - units rbc/weeks, - platelets for week). at day + a first dose of rituximab (rtx) ( mg/sm) was administered; we observed a progressive increase in reticulocytes and platelet counts (respectively /mm³ and /mm³) days after the infusion of rtx. insorgence of cystitis and pielonephritis (p.aeruginosa) delayed the administration of a following dose of rtx with a progressive, increasing transfusion dependency. after the resolution of the infective complications, a second dose of rtx was administered on day , and a third dose on day + . two weeks after the last dose of rtx a response was observed: reticulocytes count increased, and the level of hgb slowly normalized .the patient is now in complete remission months after transplantation, and with a complete hematologic take. rituximab could thus be a promising agent for treatment of not infrequent cases of prca, in transplanted thalassemia patients, refractory to standard therapies. a successful t-lymphocyte engraftment achieved by megadose cd +selected peripheral blood stem cell s transplantation in a t-b-nk+ scid case without using conditioning regimen a. ikinciogullari ( ), c. aytekin ( ), f. dogu ( ) , m. yuksek ( ), a. yildiran ( ) severe combined imunodeficiency (scid) is a genetic disorder characterized by profoundly defective or absent t and b cells functions. allogenic stem cell transplantation (sct) is to date the only curative therapeutic option for scid. here we report a t-b-nk+scid patient who was transplanted by megadose peripheral blood stem cells (pbsc) collected from his father without conditioning regimen. case: a months old boy referred to us with the diagnosis of t-b-nk+scid. his physical examination showed disseminated hyperceratotic papular skin lesions. immunohistochemical investigation of the skin biopsy revealed hsv infection. he was treated with acyclovir and foscarnet combination, but skin lesions didn't resolve. thus he received a megadose cd + selected (clinimacs, miltenyi biotec) pbsc ( x cd +cells/kg) transplantation from his father without conditioning regimen. he received cyclosporine for gvhd. he engrafted at posttransplant day . detection of chimerism performed by str (short tandem repeat) pcr analysis and whole blood samples showed mixed chimerism with % donor t cells. acute gvhd grade i developd at day rapidly resolved with systemic corticosteroid treatment. his chronic hyperceratotic papular herpetic lesions completely recovered at second month after sct. he is doing well with successful immunoreconstitution five months after sct. to our knowledge he is the first successfully engrafted scid case with t-b-nk+ phenotype following uncoditioned haploidentical pbsc transplantation. myeloid-related proteins (mrp ) and (mrp ), both s proteins, are the major calcium-binding proteins expressed in phagocytes during specific stages of differentiation. they form stable complexes and are present in circulating neutrophils and monocytes, representing the first cells invading inflammatory lesions. the protein complex is found in inflammatory fluids in distinct inflammatory conditions, including rheumatoid arthritis, allograft rejection, inflammatory bowel disease, and lung disease. prerequisite for its secretion is the contact of phagocytes with extra-cellular matrix proteins or inflamed endothelium, resulting in elevated intracellular calcium levels and activated protein kinase c. mrp /mrp is thereby released specifically at inflammatory sites and leads to increased serum levels in correlation with the degree of inflammation, indicating an extra-cellular role of these molecules in inflammatory processes. we report a year-old girl with: a) severe anemia, b) neutropenia, c) inflammation and d) severe growth failure. bone marrow examination showed moderate dyserythropoiesis. we did not detect hemolysis, iron deficiency, hemoglobinopathies, immunological diseases or any autoantibody. serum levels of copper and ceruloplasmin were within normal range, although serum zn concentration was markedly increased ( µg/dl). urinary zn excretion and erythrocyte zn concentrations were within normal range. family studies demonstrated normal zn and cu plasma levels. patient's plasma calprotectin concentration showed a -fold increase ( mg/l) compared to normal values. calprotectin concentration is known to be elevated in many inflammatory conditions but is generally below mg/l and thus far below the levels reported in this patient. we describe this case as an inborn error of zinc metabolism caused by dysregulation of calprotectin metabolism, which mainly presented with the features of chronic microcytic anemia and inflammation. we suggest that bone marrow transplantation could be the best clinical intervention for this new disease. sequential autologous peripheral blood stem cell transplantation with beam conditioning as salvage treatment for refractory high-grade lymphoma e. van hul, a. gadisseur, e. steel, w. schroyens, a. van de velde, z.n. berneman antwerp university hospital (edegem-antwerp, b) t( ; ) mature b-cell (burkitt's) lymphoma/leukaemia (bl) is classified as one entity in the world health organisation (who) classification. bl is a poor-risk, aggressive non-hodgkin lymphoma. despite significant improvements in the treatment of bl, outcomes of adults are generally inferior to those of children. further intensification of the chemotherapy regimens and the inclusion of up-front, high-dose therapy and autologous peripheral blood stem cell transplantation (asct) has significantly improved the duration of response and survival. strategies to improve survival in these poor-risk patients also include sequential asct, and asct followed by non-myeloablative allogeneic transplantation. we present a -year old male who was diagnosed with bl with a double translocation t( , ) and t( , ). he was in remission after cycle of the hovon study protocol (prednisone, vincristin, daunorubicin, asparaginase) but relapsed after the second cycle (ara-c, mitoxantrone). he was then treated according to the hoelzer protocol but after initial good response proved progressive after the second block, with increasing abdominal mass, acute renal failure and metabolic encephalopathy. salvage therapy was initiated with an autologous peripheral blood stem cell transplantation (pbsct) with beam (carmustine, cytarabine, etoposide, melphalan) conditioning resulting in a very good partial remission, which was then consolidated at day + by a second autologous pbsct after beam conditioning. he was planned for an haploidentical allogeneic pbsct according to the perugia protocol on day + after the second pbsct but died unexpectedly of complications of an acute gastrointestinal bleeding before the conditioning (tbi, thiotepa, fludarabin, atg) could be started. we present what appears to be the first reported case of tandem asct with beam conditioning in an adult patient with burkitt's lymphoma/leukaemia . this intensive therapy proved feasible and relatively well tolerated, certainly in view of the bad condition of the patient at the start of the first conditioning. the use of palifermin (keratinocyte growth factor) in the future could further increase the tolerability of this regimen. beam has been proved to be a highly effective treatment in lymphoma but provokes a severe mucositis. nevertheless, tandem beam and autologous pbsct should be further developed as a salvage regimen in the treatment of patients with poor-risk aggressive lymphomas. an association between anbioimmunoblastic t-cell lymphoma and hcv infection: therapeutic difficulties g. mihailov, p. ganeva, n. vasileva national center of haematology and transplantation (sofia, bg) angioimmunoblastic t cell lymphoma (ail) is a rare lymphoproliferative disorder characterized by systemic lymphadenopathy, hepatosplenomegaly, fever, liability to s infections, skin eruption, polyclonal hypergammaglobulinaemia, hemolytic anaemia. clinicaly the disease runs a fatal course in the majority of patients even after multiagent chemotherapy, interpheron á, cyclosporine a, corticosteroids, danazol and recently purine analogues. fewer than % of patients survive years after diagnosis. highdose chemotherapy (hdct) followed by autologous bone marrow transplantation represents a promising new treatment modality for patients with this type of lymphoma. we present a case of year-old woman with association of ail and hcv+ infection with high replication of virus and interesting clinical course of her disease (cns infiltration and complication with bacterial meningitis). it is widely thought, but not yet explained that there might be a pathogenetic link between the infection of hepatitis c virus and onset of b non hodgkin's lymphoma (nhl). in our case we have association with t cell lymphoma. we discuss our treatment difficulties ( courses imvp- , course for brain type lymphoma), following by fludarabine. there is an evidence that ail is susceptible to hdt and autologous stem cell transplantationshuold be considered to the patient. recent data suggest that high dose chemotherapy (hdct) and autologous stem cell transplantation (asct) may be of benefit in patients with aids-related lymphoma (arl). we report on a patient with refractory arl successfully salvaged by hdct and asct. in august a -year-old man presented with a rightlower quadrant abdominal mass. he was known to be hivpositive since (cdc category c ). highly active antiretroviral therapy (haart) was initiated in . his latest cd cell counts was /µl and his hiv-load < copies/ml. abdominal ct-scanning confirmed a lesion of x cm in greatest dimension localized in the right iliac fossa. the spleen was enlarged at . x . cm. a biopsy revealed a stage iia diffuse large b-cell lymphoma. from august to december the patient received courses chop resulting in a partial remission. because a pet-scan indicated residual active lymphoma more courses r-choep (rituximab, cyclophosphamide, doxorubicin, vincristine, etoposide, prednisone) were applied and a complete remission (cr) was achieved. months later he relapsed with an isolated tumor localized in the right iliac fossa. the lymphoma increased in size under courses of bendamustine/rituximab. in july the patient received courses of cisplatin/cytarabine/dexamethasone (dhap) supported by g-csf. a sufficient number of peripheral blood stem cells were harvested after the st cycle ( . x /kg cd + cells). the patient experienced stable disease and received another course ifosfamide/etoposide/epirubicin (iev). however, progressive disease caused an incomplete paresis of his right leg. in september hdct (beam) was administered followed by asct ( . x /kg cd + cells). haart was continued throughout the transplant period with the hiv viral load remaining negative. the patient developed neutropenic fever, a central venous catheter-related infection (koagulase-negative staphylococci), bacteremia (klebsiella pneumonia) and toxic enteritis who grade . broad spectrum antimicrobial therapy was given for weeks. the granulocyte count reached . x /l on day + and platelets x /l on day . a ct-scan performed weeks after asct showed a partial remission. moreover, the paresis of his right leg has disappeared. the patient is doing well and currently undergoes consolidating radiotherapy to the initial tumor bulk. hdct and asct may be of benefit in selected cases of refractory arl. angioimmunoblastic t-cell lymphoma: treatment in two cases a. lópez de la guía, m. martin-salces, a. kerguelén, r. de paz, t. cobo, d. hernandez, j. g-bustos, m. canales, f. hernandez-navarro h.u. la paz (madrid, e) introduction: angioimmunoblastic t-cell-lymphoma (ail) is one of the mature t-cell neoplasm defined in the real-and who-classifications. although patients with angioimmunoblastic t-cell lymphoma (ail) have a poor prognosis with conventional treatment , there are no generally accepted treatment guidelines of proven effectiveness because of low frequency. in that way, it would be necessary to determinate new lineage-treatment to improve unfortunately evolution. methods: between and we reported the good development with high-dose chemotherapy (hdct) and autologous haematopoietic stem cell transplantation in two patients with refractory in our centre. results: the age at transplantation was and years-old respectively. treatment prior to bone marrow transplantation in one case was initially prednisone alone and cladribine, cyclofosfamide and prednisone for cycles, and the other one was chemotherapy based of increased dose of schedule epoch; in this case was necessary secondary treatment with ifosfamide and etoposide ife for cycles. cd + selected autologous peripheral blood stem cell transplantation was given like third line of treatment in the two cases. the regimen for the mobilization of peripheral blood stem cells (pbsc) included ifosfamide, etoposide and g-csf. in one case the median yield of pbsc was , x cd +cells/kg and . x cd +cells/kg and the other one was , x cd +cells/kg and . x cd +cells/kg. the conditioning treatment consisted of beam regimen. there was none treatment-related death. post-taspe complications were herpes zoster infection and biclonal gammapathy igg kappa and igg lambda in one case, and no evidence of acute complications in the second patient. the patients remain in complete remission after a following time of and months respectively and there is no evidence of relapsed. conclusions: our cases confirm previous experience that ail is susceptible to high-dose chemotherapy and cd + selected autologous peripheral blood stem cell transplantation, but more cases and longer observation time as well as better selection of patients with refractory ail would be necessary to determinate the standard treatment. ( )imperial college (london, uk) introduction: we describe the novel use of beam campath conditioning in an autologous setting. we used alemtuzumab ( campath h ) in combination with beam conditioning in two patients who underwent autologous peripheral blood stem cell transplantation for aggressive t cell lymphoma. case a: a year old lady of carribean origin presented with htlv- driven atll. she was treated with courses of chop + dacluzimab with good pr. she received dhap salvage therapy with stem cell collection followed by a beam campath h autograft. she developed cmv reactivation treated with valganciclovir. her day + re-staging showed cr with significant reduction in htlv- proviral load from copies to . copies/ pbmcs. case b: a year old south american presented with massive splenomegaly & pancytopenia. he was diagnosed with gd hepatosplenic lymphoma and was started on chop chemotherapy regimen with initial response followed by progression after cycles. he received dhap chemotherapy followed by autologous peripheral blood stem cell transplant with beam campath h conditioning. post transplant, he developed cmv reactivation managed with valganciclovir. he received splenic irradiation weeks post transplant and at months post transplant, he remains in complete remission. role of alemtuzumab (campath h)-campath h targets all lymphocytes expressing cd . this includes gd t cells. htlv- predominantly infects cd + t cells ( % to % of the proviral load). the expression of cd on htlv- infected cells has not been studied in humans but in nod/scid mice developing htlv- positive tumours, the 'leukemic' cells express cd to a high level. conclusion: the use of beam campath h conditioning regimen in an autologous setting is a novel approach in selected patients with aggressive t cell lymphomas. it merits further investigation. unrelated bone marrow transplantation in a child with high-risk anaplastic large cell lymphoma: case report g. tezcan, v. hazar, v. uygun, a. kupesiz, a. yesilipek akdeniz university school of medicine (antalya, tr) the use of allogeneic stem cell transplantation in non-hodgkin lymphoma patients is not yet clearly defined, especially in children and adolescents. for patients who are in need of bmt, to have a chance for human leucocyte antigen (hla) identical sibling to serve as an allogeneic donor is only %.. for others, transplant from a matched unrelated donor (mud) is an alternative option but with higher risk for gvhd and graft rejection as well as infectious complications and organ toxicity. here, we describe a case of alcl treated with unrelated cord blood transplantation. year old boy admitted with the complaint of mass in right medial thigh region was diagnosed as alcl and chemotherapy was started. at the beginning of second course, because of progression, chemotherapy was changed and daclizumab was added. by the beginning of third chemotherapy course, he was in remission and chemotherapy was continued while an unrelated donor was being searched since hla matched sibling donor was not available. at the end of forth course, one antigen mismatched cord blood was found and following marrow recovery, he was referred to our center for stem cell transplantation while he had been still in remission. the patient was conditioned with total body irradiation ( total cgy) given in six fractioned doses in association with etopside ( mg/kg) and cyclophosphamide ( mg/kg). he was infused with . x /kg mnc and . x /kg cd + cells. cyclosporine, metotrexate and atg were used for gvhd prophylaxis. engraftment was achieved for neutrophil and thrombocyte on posttransplant day and , respectively. on posttransplant day , venooclusive disease developed and it was treated with defibrotide. he has not been experienced any sign of gvhd and he has been still in remission by the end of posttransplant three months. he was the first pediatric alcl case to our knowledge who was treated with unrelated cord blood transplantation. the increasing number of volunteer stem cell donors and although in very limited cases successful results published in the literature regarding the unrelated stem cell transplantation in this group of patients, make this therapeutic option acceptable in the treatment of high risk or treatment failure patients although more data and long term follow up in larger series are needed. high-dose chemotherapy and autologous stem cell transplantation for malignant lymphoma: influence of disease status at time of transplant s. genadieva-stavrik, l. cevreska, a. pivkova, z. stojanoski, o. karanfilski, b. georgievski clinical center (skopje, mk) high-dose chemotherapy with autologous stem cell transplantation (asct) could improve the survival of patients (pts) with malignant lymphoma, but the optimal time of asct is still not clear. this therapeutic method is considered as the optimal strategy in high risk patients in remission and relapsed malignant lymphoma patients. however the potential role in chemoresistent disease and primary refractory relapse is still controversial and optimal timing and indication is still to be assessed. methods: we analyzed consecutive adult patients with malignant lymphoma who underwent asct in a period - . m.hodgkin (hd) was diagnosed in pts, non-hodgkin lymphoma (nhl) in pts. the graft was non-purging, peripheral stem cell. the median follow-up was months (from to months). in hd group the conditioning regimen was beam in all cases. status at asct was complete remission (cr) in cases, chemo sensitive relapse in cases and primer refractory disease in cases. patients in complete remission are patients with adverse prognostic factors for hodgkin disease. in nhl group the conditioning regimen was beam in cases, and other chemotherapeutic regiment in cases. high grade histology was diagnosed in all patients, diffuse large bcell pts, anaplastic large t-cell , lymphoblastic pts. status at asct was complete remission (cr) in cases, chemosensitive relapse in cases, and refractory disease in cases. according to the international prognostic index (ipi) which is validated scoring system predictive of survival in various types of de novo aggressive non-hodgkin lymphoma patients with cr has been evaluated as high risk patients. results: permanent complete remission was achieved in out of pts ( %) with cr at asct in out of pts ( %) with chemosensitive relapse. complete remission could not be achieved in refractory disease. conclusion: the retrospective analysis of our data showed that the asct in patients with malignant lymphoma is an effective salvage therapy. status of the disease at the time of asct is prognostic factors which strongly influenced the outcome of asct. the study suggests that the best results are achieved if asct is performed in complete remission compared with refractory/relapsed disease. patients with malignant lymphoma and initial high risk prognostic factors should be transplanted in first complete remission. autologous stem cell transplantation after radioimmuntherapy with ibritunomab-tiuxetan (zevalin) followed by high-dose cyclophosphamide is feasible in lymphoma patients with end-stage renal disease w. stein, f. gottschalk, o. knigge, u. aurich, m. wernicke, m. kiehl clinic frankfurt/oder (frankfurt/oder, d) we report on a year old male patient suffering from follicular lymphoma grade ii, stage iiia, first diagnosed in . initial therapy consists of cycles r-chop (rituximab, cyclophosphamide, doxorubicine, vincristin, and prednisolone) with partial response. in april patient became symptomatic with abdominal pain and a ct scan demonstrates enlarged lymph nodes paraaortocaval and a left hydronephrosis necessitating ureter splint. after verification of lymphoma relapse two cycles of dexabeam (dexamethasone, bcnu, etoposide, ara-c, melphalan) chemotherapy were initiated followed by stem cell harvest and as lymphoma shows progression we added one cycle of r-dhap (rituximab, dexamethasone, high dose ara-c, cisplatin) again without any s response. due to further lymphoma progression patient became haemodialysis dependent as creatinine level increase to µmol/l and urea level to . mmol/l. furthermore, he suffers from oliguria, ascites and severe edema of the legs, scrotum, and abdomen. as no standard chemotherapy with a clear chance of response was feasible we decided to start a high dose chemotherapy followed by autologuous stem cell transplantation. conditioning regiment consist of radioimmunotherapy with ibritunomab-tiutexan ( , gbq) days prior to high dose cyclophosphamide ( mg/kgbw/day) on days - and - . on day , x cd + cells per kg bw were given. platelet engraftment was observed on day + ( . /µl) and day + ( . /µl), respectively, as neutrophil exceed /µl on day + . patient did not show any problems associated with either radio-chemotherapy or neutropenia. as edema and ascites disappear during neutropenic phase kidney function resolve weeks post transplant. mrt scan shows a very good partial response with residual lymph nodes paraaortocaval. from this case we conclude that in refractory follicular lymphoma radioimmunochemotherapy followed by high dose cyclophosphamide as conditioning regiment even in the case of renal failure necessitating haemodialysis is feasible and highly effective. twenty patients (male: female= : , ages ranging - with a median of ) received high dose chemotherapy followed by peripheral blood stem cell transplant (pbcst) at inha university hospital. their diseases consisted of diffuse large b cell lymphoma (dlbcl, n= ), peripheral t cell lymphoma (ptcl, n= ), lymphoblastic lymphoma (lbl, n= ), anaplastic large cell lymphoma (n= ), burkitt lymphoma (n= ), nasal type extranodal nk/t cell lymphoma (n= ), mycosis fungoides (mf, n= ), and nodal marginal zone lymphoma (mzl, n= ). most of them were in stage via (n= ) or vib (n= ), and others were in stage ie (n= ), bulky iia/bulky iiae (n= ), or iiia (n= ). three patients had disease extended to cns at the very beginning of the treatment. chemotherapy including chop, r-chop, promace/cytabom, copblam-v, dhap, high dose methotrexate, or regimens for acute lymphoblastic leukemia (patients were exposed to a median of courses of chemotherapy ranging - ) yielded cr (n= ), pr (n= ), cr (n= ), cr (n= ), and pd (n= ) right before pbsct. a total of ( allogeneic and autologous) pbsct were performed for patients. allogeneic pbsct was carried out in case of disseminated mf, recurrent ptcl after autologous pbsct, and recurrent ptcl in cr after tandem autologous pbsct. a patient with lbl received double autologous pbsct. the conditioning regimen was cbv (cyclophosphamide,bcnu, etoposide) for most autologous pbsct, cytbi (cyclophosphamide and total body irradiation) for mf, fludarabine based chemotherapy in other allogeneic settings. radiotherapy was given before or after pbsct in patients (brain in , abdomen in , mediastinum in , and nasal cavity in ). a fatal veno-occlusive disease developed in mf patient who died even after orthotopic liver transplant. fatal septicemia in patients at immediate post-pbsct period hampered proper evaluation of the treatment efficacy. a median disease free survival duration was months (range ~ +), and overall survival duration . months (range ~ +). all the patients died of disease who had metastatic disease in their brains. as of this writing, of ( %) are alive disease free at a medial of months (range ~ ). it is of note that, among them, a ptcl patient who received triple pbsct is alive disease free at months post-transplant, a patient with dlbcl in cr at months, a patient with disseminated mzl in cr at months, and a patient with ptcl in cr at months. a. mousavi, s. samiee, m. iravani, b. bahar, m. jahani, k. ali moghaddam, a. khodanbandeh, i. bibordi, a. ghavamzadeh horc (tehran, ir) the non-hodgkin's lymphomas (nhls) are cancers of the cells that populate lymph nodes. it is classified according to its histology, its immunophenotype, cytogenetic and molecular biology. most nhls are cancers of b-lymphocytes. although some of patients with nhl are cured with chemotherapy with or without radiotherapy, the ones who relapse and those with primary refractory disease have poor outcomes with salvage regimen. over the past years, several clinical trials using high dose chemotherapy or chemoradiotherapy with autologous stem cell transplantation or allogeneic stem cell transplantation in this setting have been reported. approximately % of patients appear to be cured using this approach. high dose therapy/autologous stem cell transplantation is standard therapy in two scenarios, in relapsed or refractory non-hodgkin's lymphoma and in patients with refractory aggressive lymphoma whose disease is shrinking with second-line chemotherapy. here we report patients with nhl, who have undergone high-dose chemotherapy (hdct) followed by hematopoietic cell transplantation (hct). of all the patients ( . %) were male, and ( . %) were female. the median age was years old; with minimum and maximum of and y/o respectively. the majority of patients who had immunophenotype study were diagnosed with b-cell lymphoma ( . %). patients ( . %) received autologous stem cell transplantation and ones ( . %) had peripheral blood as graft type. before (hct) . % of patients were in first complete remission. (including cr with salvage therapy) the conditioning regimen for the majority of patients ( . %) was ccnu, etoposide, ara-c (cytarabin) and melphalan. the median duration of hospitalization for autologous transplanted patients was days which was days for allogeneic transplanted ones. transplant mortality rate in the first days was %. the median follow-up duration was days, with minimum and maximum of and days respectively and during this period the overall survival (os) is . % and the disease free survival rate (dfs) was . %. zevalin therapy of a non-hodgkin relapsed lymphoma patient following an autologous peripheral stem cell transplantation l. rejto ( ) the yttrium- ( y) -labelled ibritumomab tiuxetan (zevalin, idec-biogen, san diego ca) is an accepted therapy for relapsed, or therapy-refracter b-cell non-hodgin lymphomas, but it is not officialy recommended in patients who have failed an autologous stem-cell transplant. patients with recurrent lymphoma following an autologous transplant have limited treatment options. cases so far only have been described in the literature whose relapse following autologous peripheral stem-cell transplantation (apsct) has been treated with zevalin. in the present paper the authors discuss the case of a year-old male patient, who underwent lymph-node biopsy due to generalised lymphadenomegaly. the histology test proved positive for cd follicular lymphoma. following an cycle chop (cyclophosphamide , doxorubicine, oncovin, prednison), a cycle fnd (fludara, mitoxantron, dexamethasone), later a cycle dhap (dexamethasone, high-dose ara-c, cisplatin) therapy, autologous peripheral stem-cell transplantation was performed. six months later due to a relapse a cycle r-cepp (rituximab, cyclophosphamide, etoposide, prednison, procarbasine), then a cycle hyper-cvad therapy was applied. the patient (had no compatible sibling) proved to be therapy-refractory, therefore zevalin treatment was started (after a preparatory rituximab therapy mbq zevalin was applied). the zevalin treatment did not caused any unusually serious side effect. during the months since the start of the zevalin therapy the patient has been in a complete haematological remission. in nhl, in case of a relapse following apsct zevalin therapy has proved to be a good alternative. the use cd- monoclonal antibody in the treatment of bcell non-hodkin lymphoma with autologous stem cell transplantation o. tarabar, l. tukic, d. stamatovic, z. tatomirovic, b. balint, b. cikota, z. magic medical military academy (belgrade, cs) introduction: recent trials have shown that cd- monoclonal antibody, rituximab (r) may be effectively associated with autologous stem cell transplantation (asct) in the treatment b cell non-hodgkin's lymphoma (nhl). aim: to analyze the efficacy of the incorporation of r at different steps of autologous transplantation (r-asct) programs in patients (pts) with high-risk nhl. methods: between march to december r+asct was applied for the treatment of pts with b cell nhl ( low grade; diffuse large cell lymphoma -dlcl). asct were performed after chop induction chemotherapy (ct) with r (r-chopx ) in pts or without r in pts. in the time of asct complete remission (cr) had pts and others pts had partial remission (pr). regimen mobilization were g-csf with cy ± vp- in pts, eshap ct in pt, r+cy in and megar-choep in pt. a single infusion of r ( mg/m² ) used as in vivo purging days prior cy or day of therapy megar-choep. the average number of colected mobilized cd + cells was , x /kg bm (range , - , ). all pts received cvb conditioning regimen. the posttransplant immunotherapy consisted of a single dose r every months (m) started m following asct in pts. sequential monitoring of minimal residual disease (mrd) during and after treatment was performed by pcr. five of seven pts are available for mrd. results: at a median follow-up of months ( - ), pts are alive. after r+asct treatment, out of available pts became pcr negativ (low grade lymphoma- ; dlcl- ). four pts with dlcl are still in complete remission (cr) and two of them in molecular remission (mr) and months (m). the both pts with low grade lymphoma relapsed and m after transplantation, the one of them never attained pcr negativity and second pt reverted to pcr positivity after m. this therapy is well tolerated, with no adverse effects on hematological recovery of incidence of infections. conclusion: this therapy was effective in the subset of pts with high risk dlcl. r+asct treatment is able to eliminate mrd, whereas pcr positivity is associated with a high risk of relapse. double syngeneic transplantation in plasma cell leukaemia c. barrenetxea, m. callis, s. iraheta, e. sanchez, v. pons vall d´hebron (barcelona, e) introduction: plasma cell leukemia (pcl) is a rare disorder, characterized by circulating clonal plasma cell. it accounts for less than % of all plasma cell dyscrasias and has a fatal prognosis. it can be primary or secondary, when there was a previously diagnosed plasma cell dyscrasia. the median survival is - moths in the first case and moths in the second. case: we present a years old man, diagnosed in november , with multiple myeloma iga kappa, bence jones +, that presented weight loss, retinal hemorrhages, respiratory distress, hepatomegaly, splenomegaly, osteolytic lesions, the cariotype showed hyperploid, chromosome monosomy, translocations t ( , ) and t ( , ). first, he was treated with tree cycles of a drug's combination with melphalan, carmustine, vincristine and dexametasone with no response, therefore, was changed to cyclophosphamide, adriamycin, vincristine methotrexate y citarabine. after two cycles, the patient got complete remission. the patient had a twin brother, and we decided, to do a double transplantion to consolidate the response. the first transplantation was condicionated with carmustine, etoposide, citarabine and melphalan (beam), and the second with cyclophosphamide and total body irradiation; the patient remained in complete remission. eight months after second transplant was admitted to the hospital with disorientation, bradipsiquia, headache, and sensoriomotor loss of lower extremity. laboratory examination showed differential count of leukocytes, haemoglobin and platelets were normal, ldh increase, absence of monoclonal gammophaty in blood and urine by inmunofixation, and the brain's computerized tomography showed multiple intraparenchymatous lesions, with peripheral edema, in both cerebral hemispheres, confirmed by magnetic nuclear resonance, all of that suggested a neoplastic disease. these lesions were biopsied with the result of multiple myeloma lambda. the patient died one day after biopsy because intracranial hypertension. conclusion: plasma cell disease has poor prognosis, and transplantation could be a good option for some patients, but in our case we only achieve to extend life a few months. delayed engraftment following autologous stem cell transplantation: risk factors apart from stem cell dose r. fineman, n. haddad, t. zuckerman, i. avivi, t. faibish, m. markovitz, l. dan, d. abu zemach, l. akria, j.m. rowe rambam medical center (haifa, il) background: the use of mobilized peripheral blood stem cells results in prompt engraftment of all three cell lines in both autologous and allogeneic transplantation compared to bone marrow. the stem cell (cd +) dose is known to be a major factor for bone marrow recovery. although reinfusion of ≥ x cd + cells/kg is considered more than adequate in terms of rapid engraftment, there are still patients who engraft after day , despite sufficient cd + cell dose. we looked for other factors contributing to delayed engraftment apart from the stem cell dose. methods: retrospective data on lymphoma and multiple myeloma patients with delayed engraftment (absolute neutrophil count > /ul after day ) after autologous transplantation were analyzed. factors including stem cell dose, infectious complications and outcome were evaluated. median age at hsct: ( - ), pts > . diagnosis: mds= , saml= . twenty-seven pts received a median of ( - ) chemo cycles before hsct; pts received only red cells transfusions allo, haplo, mud). hsct source: pbsc %) pts; , , and after auto, allo, haplo and mud, respectively. causes of death: infection , disease progression , ards , other . all pts evaluable for disease status after hsct (n°= ) were in cr at first marrow examination median os from hsct is ( - ), median. at last update pts ( . %) are alive in cr after a median follow-up of days haplo and mud, respectively. r results of high-dose chemotherapy followed by autologous stem cell transplantation in children with advanced neuroblastoma in paediatric bone marrow transplant centres in poland from wojcik ( ), k. kalwak ( ), e. gorczynska ( ), d. turkiewicz ( ), a. dyla ( ) pl) purpose: postransplant morbidity and clinical outcome in children with advanced neuroblastoma (nbl) who underwent high dose chemotheraphy followed by autologous stem cell transplantation were investigated. patients: the total children with stage iii/iv neuroblastoma treated in five bone marrow transplantation units in poland from to were analysed. median age of childen was , years (range - , years). children were transplanted in complete/partial remission (cr/pr), in patients megachemotherapy was a part of the treatment of relapse (> cr/pr). aphaeresis was done in patients. bone marrow was collected in pts; bone marrow and stem cells were transplanted in patients melfalan + etoposide + carbo in pts.; melphalan in pts.;thiotepa + ctx + carbo in patient and thiotepa + topotecan + carbo in patient. results: ( , %) children are still alive at median observation time months. ( , %) children died dfs) is , ; expected -year os and dfs is , and , respectively. os at months in the group of children transplanted in cr/pr was , ; dfs , . os and dfs expected at years were , and , respectively. in children transplanted > cr/pr estimated os was , , dfs , at months and expected -year os and dfs were , and , respectively. conclusions: megachemotherapy followed by autohsct in patients with advanced neuroblastoma has not many adverse effects. estimated -year os and dfs are higher in the group of children results: a total of patients underwent an autologous stem cell transplantation for lymphoma and multiple myeloma at the rambam medical center between the years and ( and respectively). indications for transplantation included a chemosensitive relapse in lymphoma high risk patients and rarely also for refractory patients. in myeloma auto transplants were performed for patients achieving good response upon initial chemotherapy. patients with lymphoma received the beac/beam conditioning and patients with myeloma were given melphalan mg/m². patients showed a delayed engraftment, of them were patients with multiple myeloma and only with lymphoma. median time to engraftment was days (range - ). in patients the cd + cell dose was > x /kg and in one patient it was x /kg. patients died of severe infections, one with lymphoma on day without engraftment, the other with myeloma although engrafted on day . the outcome of other patients was uncomplicated. there were no detectable differences in the clinical course or toxicity among those who engrafted eraly or the late engrafters. conclusion: the use of mobilized peripheral stem cells shortens time to engraftment. using high doses of stem cells is safe, but there remains a significant risk of delayed engraftment in % in myeloma compared with < % in lymphoma patients. considering the homogenicity of the groups and the high stem cell dose reinfused, it is likely that bone marrow microenvironment, known to be impaired in multiple myeloma, has a role in fascilitating engraftment. a single-centre experience in autologous stem cell transplantation for patients with multiple myeloma h. kasparu, j. könig, o. krieger, m. girschikofsky, d. lutz elisabethinen hospital (linz, a) from oct. to aug. we performed autologous stem cell transplantations (abct) in multiple myeloma (mm)-patients (age (median: - ) years; female: , male: ). following conventional chemotherapy patients (transplanted between - or patients not eligible for a tandem transplantation concept due to late infections (n= ) or toxic side effects (carditoxicity ( ), neurotoxicity ( ), dermatitis ( ), smds ( )) underwent a single course of abct and patients multiple courses of transplantations ( double and tripple abct). no significant differences between both groups were seen according to age, sex distribution or stage of disease at time of abct. more patients who relapsed after conventional treatment ( vs. pts) were included in the single abct group. the conditioning chemotherapy consisted of mel mg/m² for single and double abct and mg/m² for tripple abct. all patients were transplanted with peripheral stem cells. the time to granulocyte recovery > , g/l lasted - days (median: ) and to platelet recovery > g/l - days ( median: ) without a difference between the consecutive numbers of transplantation. all patients but one in each group responded to transplantation: cr pts., pr pts., trm pt. (single abct group) and cr pts., pr pts., failure pt.( multiple abct group), resp. in the single abct group pts. ( %) relapsed after - months (med: mo) , in the multiple abct group pts. ( %) relapsed so far after - months (med: months). the median observation time is shorter in the multiple abct group ( vs. months). the median pfs and os in the single abct group is and months, resp. in the multiple abct group median pfs lasted months, the median os is not reached yet. both transplantations were well tolerated even in older patients. there is a trend to longer pfs in patients after multiple abct, but due to different observation periods no final conclusions can be drawn concerning os. autologous haematopoietic stem cell transplantation for the treatment of multiple myeloma o. tarabar, l. tukic, d. stamatovic, m. elez, v. glavicic, l. simic, b. balint, s. marjanovic medical military academy (belgrade, cs) high dose therapy with autologous stem cell transplantation (asct) is the treatment of choice for patients (pts) with multiple myeloma (mm). we report here our centre experience in pts with mm who have undergone asct. between december and september pts ( m/ f) with a median age of years (range - ) were transplanted. most pts ( %) were in stage iii (durie-salmon). before transplantation, pts have received line of chemotherapy ( - cycles vad). at the time of autograft % pts were responders (partial remission or very-good partial remission) and the others had minor response/refractory disease. all pts received peripheral blood stem cell support after conditioning with melphalan ( pts) or melphalan associated to cyclophosphamide and busulfan (by/cy +m). three months (m) after asct pts received interferon ( mu/s.c. t.i.w) and pts thalidomide ( - mg daily) as maintenance therapy. after transplantation a complete (cr) or very-good partial response was achieved in / pts ( %); all other treated pts experienced a reduction of m-component > %. with the median follow up of , m (range - ), % pts were alive. to date, in cr are still pts with the median duration of remission from asct of m. seven pts relapsed or progressed during to m after asct. two pts died from transplant related complication. asct is safe and effective procedure not only in chemotherapy sensitive pts with mm but also in resistant cases. our date confirm that asct is the current gold standard therapy for many pts with mm. dose intensity and efficacy of treatment in patients with multiple myeloma e. darskaya, s. bondarenko, a. smirnova, b. afanassyev pavlov state medical university (st. petersburg, rus) mm patients were included in our study. induction therapy was vad-d-d or idad-d-d. patients with mm were undegone intensification dexabeam. patients received cycle dexabeam. was sensitive to the -st line therapy, dexabeam resulted in ( %) cr, ( %) ncr and ( %) pd. patients was unsensitive and the resulte was ( %) cr, ( %) pr, ( %) sd, ( %) na. cycles dexabeam received patients, of them were in pr after the first line therapy, -in sd, -in progression of disease. ( %) sensitive patients achieved ncr, patients, resistant to the first line therapy achieved ( %) cr, ( %) pr, ( %) -sd. patients of mm underwent asct, patients ( , %) after conditioning regimen of melphalan mg/m², patients ( %) -melphalan mg/m², patients ( , %) - mg/m². patients with mm received tandem asct. -years dfs in sensitive to induction and intensification therapy patients was %, dfs in group of the patients with progression or relapse before asct was no longer that . year. p = . . the -years duration of a plateau phase of the patients, sensitive to the first line therapy and intensification, after -st asct was %, the duration of a plateau phase of the patients with progression before asct, was no longer that . year. p = . . the -years duration of a plateau phase of the patients, who received melphalan mg/m² as conditioning regimen, was %. in patients, received mg/m² melphalan and less it was no longer that . years.s years dfs in group of the patients with tandem asct was % in comparison with % in group of the patients with single asct. p = . . the conclusion: our preliminary results are that the increasing of dose intensity improves the efficacy of treatment of patients with multiple myeloma. successful myeloablative allogeneic haemopoietic stem cell transplantation in a patient with end-stage renal failure on haemodialysis a. alfred ( ) introduction: end stage renal failure (esrf) has conventionally been considered a relative contraindication to hsct. although increasing numbers of patients undergoing autologous procedures with hd are being reported, documented experience in the allogeneic hsct setting remains extremely limited. to the best of our knowledge only three previous case reports have been published. case report: a year old male, treated with regular hd for esrf from , was diagnosed with myelodysplastic syndrome associated with monosomy (ipss int- ) in . after extensive counselling with the patient, careful consideration of donor issues, and collaboration between bmt and renal teams, a decision was made to proceed with allogeneic hsct with curative intent. in april , the patient underwent myeloablative conditioning with total body irradiation gy and cyclophosphamide mg/kg followed by transfusion of g-csf mobilised allogeneic pbsc from his hla and abo-matched sister (cd cell dose = . x /kg). recipient and donor cmv serology was negative. during conditioning, hd was performed on a daily basis to optimise biochemistry. the patient was closely monitored for cyclophosphamide cardiotoxicity with ecg and echocardiography. oral mesna was used to prevent haemorrhagic cystitis. hd was subsequently maintained at the regular thrice weekly schedule. graft-versus-host disease (gvhd) prophylaxis was ciclosporin and methylprednisolone. ciclosporin levels were little affected by hd and there were no significant problems maintaining the therapeutic range. otherwise careful consideration was given to drug dosing in relation to hd. regimen related toxicity was no more than expected in a patient with normal renal function. engraftment was prompt and discharge was on day + . mild acute and chronic ghvd has been managed with additional corticosteroids. there have been two inpatient re-admissions for post-transplant complications. bone marrow examination at three months post hsct confirmed trilineage engraftment and % female karyotype with no evidence of monosomy . the patient is presently stable, in remission and on reducing immunosuppression at over months post-hsct. conclusion: this case highlights the feasibility of myeloablative conditioning and allogeneic hsct in carefully selected patients with esrf on hd. ethical considerations should incorporate the additional impact on the donor of a potential future histocompatible living donor renal transplantation. liver complications in hematopoietic transplantation (hct) setting may be life threatening, and hepatitis b virus (hbv) infection increases the risk of hepatic complications in patients undergoing hct. we describe a year old white women who was treated years before with chemotherapy for non-hodgkin disease achieving complete remission. she acquired hbv infection because of a blood red cell transfusion. five years after complete remission she developed anemia, piastrinopenia and leucopenia. bone marrow biopsy histology excluded lymphoma relapse but revealed multilineage myelodisplasia with excess of blasts. cytogenetic examination detected qdeletion. according to ipss score myelodisplasia was classified at high risk. since an high viral b load ( copies/ml), despite normal alt and ast values, the patient underwent lamivudine prophylaxis. hct was considered and his brother resulted full hla match. he was succesfully mobilized with g-csf. before transplantation viral b load did not decrease so antihepatitis b prophylaxis was changed from lamivudine to adefovir dipivoxil that was also employed as the only antiviral prophylactic treatment along all the procedure. patient underwent reduced conditioning regimen: e.v. busulfan ( , mg/kg weight) plus fludarabine ( mg/m²) and rabbit atg fresenius ( mg/kg).graft versus host disease (gvhd) prophylaxis consisted in cyclosporin and metotrexate. , x /kg cd and , x /kg monocleated were infused. neutrophil engraftment was at + , while platelet engraftment at + . the patient developed steroid sensitive cutaneous grade ii acute gvhd. complete chimerism was achieved at + . at day she developed abrupt alt and ast increase (> iu/l), without bilirubin increase and a concomitant viral b load more than copies/ml. lamivudine was reintroduced and adefovir was continued. one week later transaminases began to decrease and at day both alt and ast were under iu/ml, while viral b load was x and the patient is well at day . a reduced conditioning regimen with fludarabin and e.v. busulfan together with rabbit atg revealed to be safe and efficacious in this year old patient. moreover lamivudine and adefovir association was able to control hbv replication. adefovir alone successfully avoided every other viral infection (hsv, cmv etc.)along transplant procedure and it did not impair engraftment. haematopoietic stem cell transplantation in poor prognosis mds and saml: prolonged patients survival is achievable, but refining of infections management and reduction of treatment-related toxicities is required to improve patients outcome m. tassara, a. crotta, l. camba, f. lunghi, m. marcatti, j. peccatori, m. bregni, f. ciceri, m. bernardi san raffaele scientific institute (milan, i) introduction: allogeneic hematopoietic stem cells transplantation (hsct) is the therapy of choice for poor prognosis mds and saml patients (pts); a hla identical, related (allo) or unrelated (mud), or haploidentical familial (haplo) donor is available for most pts. autologous (auto) hsct is an alternative for pts without a donor. prolonged overall survival (os) and disease free survival (dfs) are reported in a minority of mds/saml pts after hsct. major drawback of allo, haplo and mud hsct is the high incidence conclusions: prolonged os and dfs are achievable with hsct in poor prognosis mds and saml pts, also in the elderly. prevention of pts contamination before hsct, mainly from aspergillus, and reduction of early trm, mainly in the haplo subset of pts, could improve pts survival. trials for primary/secondary anti-fungal prophylaxis are ongoing and reduced-toxicity conditioning regimens are under investigation at our institute. auto in cr is an alternative if hsct from a donor is not feasibile; to reduce the relapse rate after auto an experimental maintenance treatment should be proposed. high-dose chemotherapy and autologous peripheral blood stem cell transplantation followed by a successful extremity sparing surgery in a case of osteosarcoma arising in osteogenesis imperfecta s. ataergin, f. arpaci, k. erler, b. demiralp, a. kaya, a. ozet, m. basbozkurt gata (gulhane) faculty of medicine (ankara,tr) objective: osteosarcoma (os) arising in osteogenesis imperfecta (oi) is reported as only nine proven cases in the english literature; among these one case had a limb sparing surgery, but none underwent a high-dose chemotherapy (hdc) and autologous peripheral blood stem cell transplantation (apbsct). methods: we recently demonstrated the effectiveness of hdc and apbsct in localized osteosarcomas. this case is the first one of os occurring in oi who underwent a hdc and apbsct. results: a- -year male patient had been followed-up since his birth with type i oi and had recurrent traumatic bone fractures. he was admitted to orthopedics clinic with complaints of bone pain, edema and swallowing in right proximal tibia. an incisional bone biopsy revealed conventional osteosarcoma. the stage was iib and two cycles of neoadjuvant chemotherapy consisting with cisplatin mg/m², adriamycin mg/m², ifosfamide . g/m² and mesna . g/m² were administered in three consecutive days, every three weeks, according to our institutional treatment protocol for osteosarcoma. fifteen days later, stem cells were mobilized using g-csf ( µg/kg/day in two doses) and collected by cobe spectra cell separator (cobe bct inc., lakewood, co). the amount of cd + cells was . x /kg. hdc (ifosfamide g/m², carboplatin . g/m², etoposide, . g/m² and mesna on a dose of - % of the total ifosfamide dose were given in dividing doses on six consecutive days) and apbsct were performed thereafter. extremity sparing surgery (wide en-bloc resection and reconstruction prosthesis) was applied after the neutrophil and platelet engraftment. postoperative tumor necrosis rate was % (by histopathology) and the same induction chemotherapy regimen was given for three additional cycles as consolidation chemotherapy. conclusion: the patient is still disease-free with a good functional score and no fracture has occurred in months after the last follow-up. improved survival in neuroblastoma by autologous peripheral blood stem cell transplantation: a single-centre experience h. kook, j.y. kim, h.j. baek, d.k. han, h.r. yi, h.j. kim, t.j. hwang chonnam university hwasun hospital (hwasun, kor) objectives: neuroblastoma is the most common extracranial solid tumor of childhood, and its outcome in advanced diseases has been very poor. in this study, the author evaluated the treatment outcome and prognostic factors in advanced neuroblastoma. methods: the study group comprised of patients who were diagnosed and treated with neuroblastoma at chonnam national university hospital from january, to may, . data were obtained from the retrospective review of the medical records. patients were classified according to the evans group. the conventional treatment including surgery, radiotherapy, pre-and post-operative chemotherapy was given to stage i, ii, and iii patients. for stage iv, relapsed patients, high-dose chemotherapy followed by autologous peripheral blood stem cell transplantation (pbsct) was administered. the chemotherapy consisted of cisplatin, doxorubicin, etoposide, and cyclophosphamide (ccg , ). conditioning for pbsct was modified vamp-tbi(cisplatin, doxorubicin, etoposide, melphalan, and total body irradiation). all patients who completed cytotoxic therapy were then either received no further therapy or treated with -cis-retinoic acid for six months. results: among patients, were males and females. the median age at diagnosis was . months (range, - months). the primary sites were the adrenal glands in patients, followed by retroperitoneum in , and thoracic cavity in . most of the patients were in advanced stages: stage iii in ; stage iv in . autologous pbsct was done in cases. the -year event-free-survival (efs) rate was % in all study patients with % for stage i, % for stage ii/iii, % for stage iv, % of iv-s. in cases with stage iv neuroblastoma, the efs rate at years after diagnosis was better among the patients who underwent autologous pbsct than among the patients who received chemotherapy ( % vs. %; p = . ). also, efs was better in patients who received -cis-retinoic acids after pbsct than those who did not ( % vs. %; p < . ). conclusion: treatment with high dose chemotherapy and autologous pbsct improved efs among children with advanced neuroblastoma. in addition, treatment with -cisretinoic acid was beneficial for patients who underwent transplantation. prospective randomized study is warranted to further improve survival for subset of advanced patients who might fail to current management strategies. key: cord- -f w fw q authors: nan title: abstracts presented at the neurocritical care society (ncs) th annual meeting date: - - journal: neurocrit care doi: . /s - - - sha: doc_id: cord_uid: f w fw q nan external ventricular drain (evd) management after subarachnoid hemorrhage (sah) is thought to influence patient outcomes and complications. evidence from single center randomized controlled trials suggest that an early clamp trial is safe and associated with shorter icu stay and fewer evd complications. however, a recent survey revealed that most neuro icu's across the us still adopt a gradual wean and continuously draining evd strategy. therefore, we sought to determine the optimal approach at our institution. we reviewed consecutive patients admitted to our institution from to with nontraumatic sah requiring an evd. in , our neurocritical care unit revised our internal evd management guideline from a gradual wean to an early clamp trial approach. we performed a retrospective multivariate analysis to compare outcomes before and after our guideline change. patients that were gradually weaned after institution of the new guideline were also included in the early clamp trial group. we observed a significant reduction in ventriculoperitoneal shunt (vps) rates after changing to an early clamp trial approach ( % early clamp vs % gradual wean, p= . , or= . on multivariateanalysis). there was no increase in delayed vps placement at months ( . % vs . %, p= . ). an early clamp trial approach was also associated with a shorter mean evd duration ( . vs . days, p< . ), shorter icu length of stay ( . vs . days, p= . ), shorter hospital length of stay ( . vs . days, p< . ), lower rates of non-functioning evd ( % vs %, p= . ), and fewer ventriculostomyassociated infections ( . % vs . %, p= . ). we found no difference in symptomatic vasospasm rates between the groups ( . % vs . %, p= . ). an early clamp trial approach is associated with fewer complications and shorter length of stay compared to a gradual evd wean. prospective multicenter studies are needed to provide further insight into the best strategy. autoimmune encephalitis refers to rare sometimes paraneoplastic conditions in which the immune system attacks the brain, leading to altered function. delayed diagnosis and treatment potentially leads to permanent neurological injury or death. the primary objective of this study was to analyze the admission and discharge modified rankin scale (mrs) assessments among patients diagnosed with autoimmune encephalitis, and to identify any effectiveness of immunosuppressive therapy on a subset of these patients. through retrospective chart review we identified patients that met currently accepted clinical and serological criterion for autoimmune encephalitis. clinical data was obtained on these cases and a modified rankin score mrs was assessed on both hospital admission and discharge or subsequent 'best clinical' visit. assessment of "improvement" from initial therapy was based on any decrease in mrs score and clinical neurological functional improvement in accordance with physician and patient affirmation by the time of discharge. seventy-seven patients met criterion for clinical or serological autoimmune encephalitis. of these patients, had cancer and did not have known cancer. fifty-seven ( %) patients underwent immunosuppressive therapy with corticosteroids, ivig, and/or plasma exchange and patients experienced a decrease in mrs score. improvement from initial treatment was %, %, %, and % for admitting mrs scores or through respectively. the p-values for improvement from initial immune therapy based on an mrs of , , or compared to an mrs of were . , . , and . respectively. immunosuppressive therapies for patients with an initial mrs score of , or may have a higher yield than for those with an mrs of . these therapies are generally reserved for those with an mrs of or greater. further study is needed to assess functional improvement in those with autoimmune mediated encephalitis treated with immunosuppressive therapies. many patient, family and hospital factors have been associated with obtaining consent for organ donation after brain death (bd). we evaluated potential factors that played a role in the consent rate in a large tertiary hospital over a period of . years. we evaluated all declarations in our hospital's bd registry between january and june regarding consent for donation. we cross-matched the hospital electronic medical records with the records of the local organ procurement organization to identify this population. patients were included in the registry ( . % african american) and were approached for donation. there was a . % consent rate for organ donation. there was no significant relationship between sex, admission diagnosis, icu (neuro vs. medical vs. surgical), physician specialty (neurology vs. other), time from event to bd declaration or religion and decision to donate. families were more likely to consent to donation if the patient was non-aa ( % vs % for aa, p< . ), was younger ( . vs . , p= . ), had a lower creatinine at the time of death ( . ± . vs . ± . mg/dl, p= . ), and had an apnea test completed ( % vs %, p= . ). in a logistic regression model, only aa race and pao independently predicted refusal of donation (odds, %ci, . , p< . and . , p= . , respectively) . although the majority of bd patients in this large series were aa, their families were times less likely to consent for organ donation than non-aa families. there is an urgent need to explore the reasons for low donation rates in this population. post-anoxic myoclonus is seen in up to % of patients who remain comatose, and historically was felt to be a poor prognostic sign. little distinction has been made in the literature between epileptic (cortical) vs subcortical myoclonus. from consecutive cardiac arrest patients that did not return to baseline (may -may ) we identified % (n= ) patients with clinical myoclonus. basic demographics and characteristics of their arrest were collected and eeg reports were reviewed. raw eeg including video was reviewed by two epilepsy-trained neurologists, whenever available. myoclonus was subcategorized into subcortical and cortical based on the presence of a preceding eeg correlate. jerk-locked eeg back-averaging was performed on two representative patients. the average age of patients with myoclonus was +/- years, and % (n= ) survived to discharge. cortical myoclonus was twice as likely as subcortical myoclonus ( % vs %, respectively). compared with patients without myoclonus, patients with myoclonus were more likely to have longer, more severe arrests. patients with subcortical myoclonus were at risk for electrographic seizures, although at a lower rate than those with cortical myoclonus ( % vs %, respectively). mortality rates did not differ between patients with cortical and subcortical myoclonus ( % vs %). patients with cortical myoclonus were more likely to be discharged in a vegetative state compared to those with subcortical myoclonus ( % vs %, respectively (or . ; %ci . - . ). amongst survivors, good functional outcome at discharge did not differ between cortical vs subcortical myoclonus ( vs %, respectively). jerk-locked eeg back-averaging was useful in distinguishing subcortical from cortical myoclonus. myoclonus is seen in every sixth patient with cardiac arrest. cortical and subcortical myoclonus cannot be distinguished using clinical criteria. both may have good outcomes when managed with targeted temperature management and an aggressive antiepileptic regimen. intoxication by central nervous system (cns) depressant drugs can lead to anoxic brain injury by cardiac or respiratory arrest. we tested the hypothesis whether intoxication by these drugs contributes to mortality in acute anoxic brain injury we utilized healthcare cost and utilization project databases (nationwide inpatient sample and kids' inpatient database) to obtain patients admitted with diagnosis of anoxic brain injury. patients with drug intoxication (opioid, alcohol, sedative/hypnotic drugs) were identified. regression analysis was used to assess relationship between drug intoxication status to in-hospital mortality. the regression model was adjusted for age, gender, chronic medical comorbidities, presence of cardiac arrest and hospital characteristics. we analyzed a total of , patients with anoxic brain injury out of which ( . %) had drug intoxication and % were reported to have cardiac arrest. median age was years and % patients were males. in-hospital mortality was %. among the survivors, % underwent feeding tube placement and % had tracheostomy. drug intoxication was a significant positive predictor of inhospital mortality with adjusted odds ratio . ( . - . ), p= . . cns depressant drug intoxication is associated with higher in-hospital mortality in patients with acute anoxic brain injury. cardiac arrest affects approximately , individuals every year and is the third most common cause of mortality in the us. currently, there is no way of reliably risk stratifying survivors of cardiac arrest. identifying early predictors of outcome is vital for triaging and clinical trial enrollment. we proposed to identify key clinical and laboratory parameters that can reliably predict long-term outcomes among comatose survivors of cardiac arrest. this was a retrospective chart review of comatose survivors of cardiac arrest. we gathered data regarding several clinical (age, pre-arrest mrs, gcs on admission, and hours, presence/absence of shock and respiratory failure) and laboratory parameters (troponins, lactate, creatinine, and alt at admission, and peak values within the first and hours) as well as characteristics of the cardiac arrest (duration, arrest rhythm, location, and bystander cpr). we used a dichotomized gos ( - vs - ) at months as the primary outcome. we performed univariate and multivariable analysis to identify predictors of poor outcome. a total of patients were enrolled. on univariate analysis, higher age, higher pre-arrest mrs, lower gcs at hours, non vf/vt arrest rhythm, in-hospital arrest location, absence of bystander cpr, and shock were statistically significant (p < . ) for poor outcome. in multivariable analysis, only higher prearrest mrs and lower gcs at hours were independent predictors of poor outcome; no bystander cpr demonstrated a trend for being an independent predictor. none of the early laboratory data achieved statistical significance for predicting poor outcome. we identified several clinical predictors of poor outcome in our small cohort of comatose survivors of cardiac arrest. the above variables need to be analyzed among a larger cohort that includes all survivors of cardiac arrest in order to develop an injury severity score that can help risk stratify cardiac arrest survivors. after cardiac arrest, somatosensory evoked potentials (sseps), eeg characteristics, and mri are routinely used to evaluate comatose patients. the relationship between structural hypoxic injury, absent cortical potentials and the generation of background reactivity or epileptogenic potentials is unclear. here we evaluate a consecutive series of patients with cardiac arrest that were studied with sseps and evaluate clinical, eeg, and mri measures to study the dissociation between hypoxia-induced thalamic disconnection and spontaneous cortical activity. in this retrospective cohort study, all comatose patients post-cardiac arrest who received sseps were identified and reports were reviewed. patients were found; one patient with a high cervical cord injury was excluded. we recorded presence of cortical (n ) and subcortical evoked responses (p ), whenever available. based on the closest available eeg (maximum days from ssep recording) we determined reactivity, background characteristics (diffuse suppression or burst-suppression versus all other backgrounds), and presence of generalized periodic discharges (gpds) or seizures. diffusion weighted or t flair abnormalities in the thalamus were evaluated based on available mris. chi-square and fisher's exact test were applied as applicable. of patients with ssep, ( %) had absent n s, and % of those (n= ) had absent p . eeg reactivity was possible, albeit less common, in patients with absent n s ( % vs %, p< . ), but none of the patients with absent p s had a reactive eeg. those with absent n s were more likely to have diffusely suppressed or burst-suppressed background ( % vs %, p= . ) and to have abnormal thalamic signal on mri ( % vs %, p= . ). gpds, stimulus-induced gpds, and seizures were equally common in those with and without n s. the integrity of the somatosensory thalamo-cortical pathway does not appear to be necessary for presence of reactivity or generation of periodic epileptiform discharges. all families of patients who have become brain dead (bd) should be offered the choice of donation. this does not always happen and the factors that lead to approaching them or not are not known. our objective was to evaluate which factors influence the donation coordinators (dc) working for an organ procurement organization approach families after brain death we evaluated all declarations in our hospital's bd registry between january and june regarding consent for donation and cross-matched the hospital electronic medical records with the records of the local organ procurement organization. in order to refine neurologic prognosis in cardiac arrest patients we sought to incorporate heart rate variability into a multimodal prediction model. heart rate variability has been shown in animal studies to be preserved in survivors of cardiac arrest. in our preliminary study, we retrospectively analyzed patients admitted to the university of virginia who had undergone a cooling protocol following cardiac arrest. analysis of heart rate variability for each patient was done in the frequency domain using the fast fourier spectral transform with spectral bands at . - . hz for high frequency (hf) and low frequency (lf) power within the frequency band . - . hz. the unit-less lf/hf ratio was considered a measure of balance between sympathetic and parasympathetic tone. over a -year period, a total of patients were cooled. patients ( %) had ceeg, ( %) had routine eegs and ( %) had sseps performed. numerous patients ( , % of all arrests or % of all eegs performed) had malignant patterns, defined as burst suppression, severe suppression, or generalized periodic discharges. of the sseps, had an absent n (none survived to discharge) and had an n that was present ( survived to discharge). patients with absent n s and malignant eegs had lower lf/hf ratios when compared to survivors with present n s ( . vs. . ). the trend towards parasympathetic dominance following a severe neurologic injury and loss of normal sympathetic tone in those patients with absent n s and malignant eegs and may serve as an additional marker of poor prognosis following cardiac arrest. physicians often struggle with the intricacies of brain death determination and communication about end-of-life care. in an effort to remedy this situation, we introduced an educational initiative at our medical school to improve student comprehension and comfort dealing with brain death. beginning in july , students at our medical school were required to attend a -minute brain death didactic and simulation session during their neurology clerkship. students completed a test immediately before and after participating in the initiative. of the students who participated in this educational initiative between july and june , ( %) consented to have their data used for research purposes. students correctly answered a median of % of questions (iqr - %) on the pretest and % of questions (iqr - %) on the posttest (p< . ). comfort with both performing a brain death evaluation and talking to a family about brain death improved significantly after this initiative ( % of students were comfortable performing a brain death evaluation before the initiative and % were comfortable doing so after the initiative, p< . ; % were comfortable talking to a family about brain death before the initiative and % were comfortable doing so after the initiative, p< . ). incorporation of simulation in undergraduate medical education is high-yield. at our medical school, knowledge about brain death and comfort performing a brain death exam or talking to a family about brain death was poor prior to development of this initiative, but awareness and comfort dealing with brain death improved significantly after this initiative. this initiative was clearly a success and can serve as a model for brain death education at other medical schools. early withdrawal of life support (ewls) is a major factor in deaths following hypoxic ischemic injury after cardiac arrest (ca) in patients receiving targeted temperature management (ttm). appropriate timing of prognostication, and subsequent withdrawal of life support is recommended in recent guidelines, but is not always followed in clinical practice. we describe the impact of ewls in a multicenter registry database. using data from the international cardiac arrest registry (intcar), we defined ewls as withdrawal in the first three days of hospitalization. among all patients treated with targeted temperature management, we developed a logistic regression model to predict ewls. we then performed a propensity score and evaluated the incidence of good outcome between deciles of risk for ewls. patients entered into intcar from - from different hospitals were included. mean age was (± ) years, mean cpr duration was (± ) minutes, ( %) had a shockable rhythm, and ( %) received bystander cpr. support was withdrawn in , with ( %) events classified as ewls. ( % of total cohort). among patients with support withdrawal, older age (p= . ), nonshockable rhythm (p= . ), increased ischemic time (p= . ), and shock on admission (p< . ) were associated with ewls. among propensity matched patients grouped into deciles of probability for ewls, survival with good functional outcome occurred in % ( th decile), % ( th), % ( th), % ( th), and % ( th decile). early withdrawal of life support after cardiac arrest occurs frequently, and is associated with age, duration of cpr, a non-shockable rhythm, and shock at the time of admission. a cohort of patients propensity-matched to those with ewls had - % survival with favorable neurologic outcomes. these data support that in most patients receiving ttm, conservative and delayed prognostication after cardiac arrest is appropriate. brain herniation (bh) is a deadly event that requires immediate central venous access for infusion of hyperosmotic agents, especially . % nacl. traditional venous catheters, whether peripheral or central, takes several minutes to place, and requires skill for successful placement, thus delaying critical treatment. intraosseous (io) cannulation has been shown, at least during cardiac arrest, to be a secure and rapid means of central vascular access that requires limited training. however, limited data exists on the use of io in bh for administering . %. the aim of study of this study is to measure changes in serum sodium and bh reversal after administering . % via io. retrospective chart review of patients with acute neurologic injury requiring . % and io placement due to a lack of central access. demographics, diagnosis, gcs, sodium (na+), and pupillary reactivity, and immediate and delayed complications were collected. results patients included: males, age range - yo. diagnosis include intracerebral hemorrhage ( n= ), extra-axial hematoma (n= ) and sah (n= ). gcs ranged to . all patients were intubated. most patients were co-treated with hyperventilation, nabicarb, mannitol, and propofol. io was placed in tibia ( ) or humerus ( ); all placed correctly on first attempt. comparing hr post- . % nacl treatment to pretreatment: na+ level increased in of ; gcs improvement in of ; and returned pupillary reactivity in of . no adverse events reported, such as shock, cardiac arrest, tissue or limb injury. preliminary data suggest that during bh, io cannulation results in safe and timely . % administration in patients with no central access. additional safety data is needed, particularly with regards to the potential for myonecrosis. however, if safe, io cannulation should replace central line placement as the initial route of central venous access during bh. the pupillary light reflex is associated with outcome after cardiac arrest as a dichotomous variable (present/absent) at various time points following resuscitation (rosc). infrared pupillometry provides quantitative measures including pupil diameter (pd), and neurological pupil index (npi) which ranges from (nonreactive) to (brisk) and reflects velocity and degree of pupil constriction in response to a standardized light stimulus. these measures may provide early prognostic information to guide therapy. comatose adult survivors of cardiac arrest treated with targeted temperature management were monitored with the neuroptics npi- pupillometer. outcomes were defined as good (go) if discharge cerebral performance category score was - , and poor (po) if - . data are presented as median (iqr). groups were compared using non-parametric statistical tests. fifty-one patients were enrolled; the median age was ( . - . ), and ( %) were male. initial rhythm was vt/vf in %, asystole in %, and pea in %. outcome was good in ( %) patients. the initial pd did not differ between outcome groups [ . ( - . ) po vs . ( - . ) go]. the initial npi was lower in poor outcome patients [ . ( . - ) vs . ( . - . ) go, p= . ] measured . ( . - . ) hours after rosc. npi dropped below in more poor outcome patients [ ( %) vs ( . %) go, p= . ], and to zero in ( %) poor vs ( %) good outcome patients (p= . ). receiver operator characteristic curves confirmed that initial npi predicted poor outcome better than pupil diameter (auc . vs . , p= . ). a low neurological pupil index predicted poor outcome - hours after resuscitation from cardiac arrest, and dropped to abnormal levels (< ) and to zero (reflecting a non-reactive pupil) more often in patients with poor outcomes. additional research is needed to define potential confounders, optimal timing, and thresholds for different levels of neurological risk with pupillometry. prediction of death in a timely manner after withdrawal of life support (wls) is essential during organ donation after cardiac death (dcd). we aimed to develop a modified version of the recently develop dcd-n score to improve the specificity of prediction and test it in a specific group of patients with catastrophic brain injuries referred for dcd. we analyzed prospectively collected data by our local organ procurement agency on all consecutive adults with severe neurological injury evaluated for dcd across centers in the usa from march to may . we analyzed three variables used in the dcd-n score (corneal reflex, cough reflex and oxygenation index) and substituted the fourth variable for vasopressor support. a total of patients, mean age (sd± ) years were included in the final analysis. anoxic brain injury was the most common cause of death ( %) followed by stroke ( %). in multivariate logistic regression analysis adjusted for age and cause of death, absent corneal reflex (or . , % ci . to . , p = . , points), absent cough reflex (or . , % ci . to . , p = . , point), vasopressor support at high doses (or . , % ci . to . , p < . , points) and o ind ci . to . , p = . , points) were associated with the likelihood of death within minutes of specificity % and auc . . the modified dcd-n score has a greater specificity in predicting death within minutes of wls and is developed specifically from a cohort of patients evaluated for dcd. future prospective studies are needed for further validation of this scoring system. significant number of the patients with anoxic brain injury have poor neurological recovery. this created a significant anxiety in families who often request early prognostication. this study was conducted to evaluate possible ultra-early prediction of good neurological recovery in patients undergoing hypothermic protocol for anoxic brain injury retrospective chart review of the patients with anoxic brain injury was conducted. all patient underwent standard evaluation and management in the early stages of icu care, including initiating of hypothermic protocol with intravenous cooling device. all patients underwent evaluations with eeg and ssep within first day after admission during hypothermia phase and mri brain after re-warming. total of charts were reviewed. patient had normal ssep and normal mri. all patients had good neurological recovery, except for one patient who died secondary to severe cardiac failure eeg did not have any predictive value for the good neurological outcome when it was done during hypothermic protocol. normal ssep may a reliable predictor for good neurological recovery. apnea test is the essential component to confirm brain death by stimulation of respiratory center in brainstem. guidelines recommend that apnea testing should meet the criteria of disconnection from mechanical ventilator, and oxygen supplying by catheter. however, during the apnea test under this technique, disconnection from ventilator may induce hypoxia due to abrupt decruitment of alveoli and pulmonary barotrauma such as pneumothorax. there are some studies that suggest continuous positive airway pressure can be effective for patients with hemodynamically instability and respiratory impairment. we suggest a novel method of apnea test by using ambu bag with positive end-expiratory pressure (peep) valve to avoid abrupt change of peep during the apnea test. apnea testing was performed by using ambu bag with peep valve to adult brain death patients. ambu bag was not bagging during the testing and just connected to endotracheal tube with l/min of % oxygen. peep valve was applied the same peep of previous mechanical ventilator. on the apnea testing, vital signs and ekg were monitored. arterial blood gas analysis were measured and minutes after disconnection from mechanical ventilator. there were no significant differences in mean pao between before and after apnea test ( ± and ± , p= . ). mean arterial blood pressure were ± mmhg and ± mmhg before and after the test, respectively. during the intervention and following observation, arrhythmia or pulmonary complications had not occurred. we suggest a novel method of apnea testing which is a simple and easy technique by using ambu bag with peep valve to minimize decruitment of alveoli. this method shows vital signs and respiratory oxygenation of the patients remained stable during the test. declaration of brain death based on clinical exam has been plagued with challenges. as a result, ancillary testing such as nuclear scintigraphy cerebral blood flow (cbf) studies have been recommended. we present a case in which the apnea test could not be completed due to hemodynamic instability and where the nuclear scintigraphy cbf study resulted in a false declaration of brain death. a y/o male was admitted with bilateral hearing loss and confusion. on day two, the patient developed blurred vision, and an mri confirmed bilateral cerebellar and pontine infarctions. by day four, he had developed diffuse cerebral edema in the cerebellum, brainstem, and bilateral occipital lobes, and we proceeded with the clinical exam for brain death. all cranial reflexes were absent; however the apnea exam could not be completed due to blood pressure instability. a nuclear scintigraphy cbf study revealed the complete absence of radiotracer activity, and the patient was pronounced dead. two hours later, the patient regained a gag reflex. on the following day, the clinical exam, with the exception of apnea testing, was again consistent with brain death. a cerebral angiogram was performed and demonstrated normal blood flow to the anterior circulation. the patient was ultimately pronounced dead on day eight after two separate complete clinical brain death exams, including apnea testing, were performed. cerebral angiography showed essentially normal blood flow where nuclear scintigraphy showed no blood flow. nuclear scintigraphy cbf studies are commonly recommended when apnea testing cannot be completed. given the dramatic differences in the results observed between these modalities, a reevaluation of this practice should be considered. patients' perceptions of recovery moderate outcomes, however studies exploring the specific cognitive, functional, and psychological domains associated with subjective perceptions of recovery at hospital discharge after cardiac arrest (ca) are lacking. this is a prospective, observational cohort of patients admitted to columbia university medical center after ca, and survived to hospital discharge between / - / . patients with sufficient mental status to perform a neuropsychological exam and a questionnaire at discharge were included. subjective perceptions of recovery were assessed via responses to the forced-choice dichotomized question, "do you feel that you have made a complete recovery from the arrest?"objective outcome measures of recovery included: repeatable battery for neuropsychological status (rbans), modified lawton physical self-maintenance scale (l-adl), barthel index (bi), cerebral performance category scale (cpc), center for epidemiological studies-depression scale (ces-d), and post traumatic stress disorder-checklist (ptsd-c). chi-square, wilcoxon-rank sum, and logistic regression were used to compare the respondents, and determine factors associated with subjective perceptions of recovery. patients were included with mean age of ± years; % were men and % were white. % responded not having made a complete recovery. no significant differences were found between respondents in terms of demographics, charlson comorbidity index, arrest-related variables, rbans, l-adl, bi, pre-or post-arrest cpc scores. those responding that they had not made a full recovery had higher rates of ptsd-c ( % vs %, p< . ), and depression ( % vs %, p= . ). moreover, everyone that screened for ptsd (n= ) reported not having made a complete recovery. patients with higher ptsd scores were more likely to report not having made a complete recovery (or . ; p< . ) after adjusting for age, gender and depression scores. presence of post traumatic stress disorder symptomatology at discharge, and not neurocognitive or functional status, is highly associated with post-cardiac arrest patients' subjective perceptions of recovery. early eeg background reactivity is a strong predictor of neurological recovery after hypoxic-ischemic brain injury despite hypothermia and sedation. unfortunately, expert interrater-agreement on visual scoring of eeg background reactivity ranges from - %. recent studies indicate that machine-learning approaches using quantitative eeg (qeeg) might yield equivalent or superior performance to current eeg reactivity assessment practices, however its ability to predict outcomes has not been tested. we hypothesized that a qeeg reactivity method can predict long-term functional outcome in hypoxic ischemic brain injury. we retrospectively reviewed clinical and eeg data of cardiac arrest patients managed with hypothermia at two university hospitals. eeg reactivity was tested daily using a structured exam consisting of auditory, tactile, and visual stimulation. our quantitative eeg method evaluated changes in eeg spectra, entropy, and frequency features during seconds before and after each stimulation-step ( qeeg features used). only the first eeg reactivity assessment for each subject was used in the final analysis. good outcome was defined as cerebral performance category of - at six months. a penalized multinomial logistic regression was utilized for feature selection and a random-forest classifier was employed in the training and validation sets. model performance evaluation metric was the area under roc curve (auc). outcome and eeg data was available for a total subjects, and cases were excluded due to presence of burst-suppression, periodic epileptiform discharges, or eeg artifact. forty-seven subjects were included in the final analysis. mean age was . (standard deviation . ) years and . % had good outcome. the combination of four features provided best outcome prediction performance with an auc of . (kolmogorov-smirnov test, skewness, two-group test, and renyi entropy). early qeeg reactivity is predictive of good outcome at six months. a quantitative approach to eeg reactivity analysis might facilitate accurate and individualized prognostication in hypoxic-ischemic brain injury. hypoxic-ischemic brain injury is the leading cause of morbidity and mortality following cardiac arrest, and the ability to predict neurologic recovery in comatose cardiac arrest survivors is limited. functional mri measures brain network connectivity and resting-state network connectivity can be measured in comatose patients. the default mode network (dmn) is one resting state network that has been correlated with consciousness. we hypothesized the degree of connectivity in the dmn and other resting-state networks would correlate with consciousness recovery in post-cardiac arrest coma. consecutive patients with hypoxic-ischemic coma were enrolled. functional mri was obtained on all patients on post-arrest day - on an inpatient tesla mri. the connectivity in multiple resting-state networks was analyzed using pearson's correlations between component maps for each subject and previously defined standard network maps. connectivity in the default mode network and in additional resting-state networks was correlated with outcome. good outcome was defined as consciousness recovery at any point in the acute hospitalization. patients were included in this study. the mean age was ± years ( - ) and were male. patients survived with good outcome. the primary arrest rhythm and the duration of cardiac arrest did not differ between groups (primary rhythm as vt/vf: % vs %, good vs poor, p= . ; cardiac arrest duration: . ± . minutes vs . ± . minutes, good vs poor, p= . ). patients with good outcome had significantly higher mean network connectivity ( . ± . vs . ± . , good vs poor, p= . ). dmn connectivity showed a trend towards significance ( . ± . vs . ± . , good vs poor, p= . ). in comatose patients following cardiac arrest higher fmri measured resting state connectivity correlated with consciousness recovery. functional connectivity may be developed as a prognostic biomarker. sedative and analgesic infusions and neuromuscular blockade agents (nmba) are commonly used for comfort, suppression of shivering, and reduction of metabolic activity during targeted temperature management (ttm) after cardiac arrest. the optimal sedation and analgesia regimens are unknown. we sought to describe variability in sedation and shivering management practices at us and european cardiac arrest receiving centers. international cardiac arrest registry (intcar) centers were surveyed regarding sedation protocols for ttm after cardiac arrest. the survey was administered via redcap with a response rate of %. ten united states and european centers completed the survey. shivering is measured at ( %) of centers and recorded at ( %) centers. ten centers use nmb to control shivering prophylactically, centers use nmb only if shivering occurs, and centers increase opioids or sedatives when shivering occurs, but do not use nmb. the most common sedative was propofol ( / centers), followed by midazolam ( / ) and the most common analgesic was fentanyl ( / ) followed by remifentanyl ( / ). , , and centers report having a sedation target of light, moderate, or deep respectively. a sedation scale is used at ( %) centers, targeted to patient comfort at ( %) centers. daily sedation lightening is protocolized when rewarming starts at ( %) centers, when normothermia is reached at ( %) and not specified in the remainder of groups. of patients who awaken, centers report that they expect this to occur at ( centers), ( centers) and ( centers) hours respectively. among cardiac arrest receiving centers internationally, there is significant variability in ttm sedation and shivering management strategy. our hospital policy allows an optional sbd (with an apnea and a cerebral blood flow test) or a dbd (with an apnea test). we have evaluated the adoption of and reason for performing a single brain death exam (sbd) vs two (dual) brain death exams (dbd) and their impact on organ function and consent for organ donation. we evaluated our hospital's bd registry between january and june regarding sbd or dbd. we also cross-matched our electronic medical records with the records of the local organ procurement organization. of bd declarations, ( %) were sbd and ( %) dbd. during the st five years, % of all bd exams were sbd and during the second %. patients with sbd were older ( . ± . for sbd vs . ± . years for dbd, p= . ), had a primary neurologic diagnosis ( % vs %, p< . ) and were admitted to the neuro-icu ( % vs %, p< . ). during the nd exam, . % patients were on equal or higher dose of pressors. sbd patients had lower k+, bun, creatinine and heart rate, but higher peak na+ and apnea pao (for all p< . ), although apnea ph and paco were similar. the time between injury to bd pronouncement was shorter in sbd by . hours. there was no difference in consent rate between sbd and dbd ( % vs %, p= . ). at our institution, bd declaration was more often done by dbd exams, although the primary diagnosis and the unit of admission influenced the decision. an increased adoption of sbd exams was noted after the aan bd guidelines, supporting sbd exam, were published. although the number of exams did not affect rate of consent for donation, surrogate markers indicated better function of organs after sbd, while dbd patients stayed in the icus over a day longer. there are no data supporting better numbers or function of organs in donors after brain death (bd), if there is a shorter waiting period (as expected with single brain death exam [sbd] ) from the time that bd is declared to the time the patient arrives at the operating room (or). our goal was to find if the number of brain death exams, either sbd or dual (dbd), had any impact on the number of organs recovered and transplanted we evaluated our hospital's bd registry between january and june regarding sbd or dbd and cross-matched our electronic medical records with the records of the local organ procurement organization out of bd declarations, led to consent, of which ( . %) after sbd and ( . %) after dbd. there was a trend for longer consent to or time for dbd ( . ± . hours vs . ± . for sbd, p= . ). there was no difference in the number of organs recovered or transplanted based on the number of exams ( . ± . vs . ± . organs/patient recovered and . ± . vs . ± . transplanted for sbd vs dbd, respectively, p> . ). there was a trend for more lungs to be transplanted after sbd exam ( % vs %, p= . ), but this was not found with kidneys, heart, liver, pancreas or intestines. in multiple logistic regression models, adjusting for variables pertinent to each individual organ function (for example, bun or creatinine level for kidneys, blood gases for lungs etc), the number of exams was not an independent predictor for successful transplantation conclusions sbd exam led to similar numbers of organs transplanted compared to dbd exam in this single center registry analysis. more rapid brain death declaration, as with sbd, is not a factor that influences organ transplantation the glasgow coma scale (gcs) is a standardized and commonly used way of assessing important aspects of neurological condition for critically ill patients. while it is a validated tool for prognostication, it is unclear whether serial measurements add value to this prognosis. we used a large set of serially collected gcs measurements to assess the impact of gcs score on the trajectory of neurological recovery as well as factors affecting score variance. gcs total and subscores ( , time points from , patients) recorded hourly by registered nurses in the neurosurgical intensive care unit (nsicu) between january, and may, were analyzed retrospectively. k-means clustering provided groups with similar progression characteristics during nsicu stay. k-means clustering provided groups with similar progression characteristics during nsicu stay. descriptive features for each cluster were binned into histograms and evaluated for similarity using and kruskal-wallis tests. linear correlations of the sub-scores were very high (eye-verbal: . , eye-motor: . , verbal-motor: . ), while compositional variance was low for aggregate scores. hour-to-hour variance in gcs correlates to significant nsicu activities such as nursing shift changes. among patients with similar minimum gcs scores during their stay, those that recovered were significantly less likely to have deteriorated in the hospital ( , p<< . ). for patients with a minimum gcs<= , those that arrived at their minimum score (i.e., did not deteriorate in nsicu) were . % more likely to recover than those who deteriorated in-hospital (kw, p<< . ) . patients that experienced recovery show significantly greater improvement as early as hours after their minimum score (kw, p<< . ). the gcs is unnecessarily complex for most nsicu patients and can be represented by fewer variables. serial gcs measurements do provide value for prognosis and may be able to distinguish patients with potential to recover early in their hospital course. stroke is a major cause of death and disability, and common admission to neurological intensive care units. preferences for cardiopulmonary resuscitation (cpr) are often discussed, but there is limited understanding of cpr outcomes among stroke patients. systematic review and meta-analysis of published literature from to among stroke patients undergoing in-hospital cpr. preferred reporting items for systematic reviews and meta-analysis, metaanalysis of observational studies in epidemiology, and utstein guidelines were used to construct standardized reporting templates. detailed searches of pubmed and cochrane libraries were supplemented with hand-searched bibliographies. primary data from studies meeting inclusion criteria at two levels were extracted, i) survival to hospital discharge after cpr, and stroke as a primary admitting diagnosis, and the less restrictive, ii) survival to hospital discharge after cpr with stroke listed as a comorbidity, were meta-analyzed to generate weighted, pooled estimates of survival to hospital discharge. of articles screened, there were articles ( %) that underwent full review. three articles met primary inclusion criteria, specifically identifying patients with stroke as a primary admitting diagnosis. twenty additional articles met secondary inclusion criteria, listing stroke as a comorbidity. there was an % ( % confidence interval (ci) . , . ) rate of survival to hospital discharge rate from a combined sample of patients that received in-hospital cpr. among the more heterogenous population of inpatients with stroke listed as a comorbidity, there was % ( % ci . , . ) rate of survival to hospital discharge. adherence to utstein reporting guidelines was poor, and neurological outcomes were measured in ( %) of studies. survival to hospital discharge among stroke patients is lower relative to general hospital populations. these preliminary findings highlight the need for improving the quality of evidence to inform patient and provider discussions of cpr among stroke patients. there is often a tendency to treat patients with traumatic brain injury (tbi) and a glasgow coma scale (gcs) score of on presentation less aggressively because of low expectations for a good outcome. based on the crash trial database, a prognosis calculator has been developed for the prediction of outcome in tbi patients. our aim was to investigate whether the crash calculator can be used for prognostication in patients with tbi and gcs of on presentation. we performed a retrospective review of patients with tbi and a gcs score of from / to / . the crash calculator has been validated to estimate mortality at days and death and severe disability at six months (glasgow outcome scale-gos - ). the calculator uses country of origin (usa in our dataset), age, gcs, pupils reactivity to light, presence of major extracranial injury, and findings on ct scan of brain (petechial hemorrhages, obliteration of the third ventricle or basal cisterns, subarachnoid bleeding, midline shift, and non-evacuated hematoma). the individual prognosis for mortality at days and unfavourable outcome at months was calculated and compared with the actual outcomes. a total of patients were included. a tend toward underestimation of the risk of mortality at days was found (estimated mortality was % compared to actual mortality of %; difference of %, p = . ). however, the estimation of outcome at months was accurate (estimated gos - was . % compared to actual of . %, p = . ). the crash prognosis calculator underestimated the risk of mortality, but accurately predicted unfavourable month outcome in patients with tbi and gcs of on presentation. pending larger studies to validate our findings, we believe that crash calculator can only support -not replace -clinical judgment. there are no nationally enforced standards regarding brain death. few data exist on how brain death is determined across the u.s. we used claims data from - from a nationally representative % sample of medicare defined as icd- -cm code . . the primary outcomes were evaluation by a neurologist or neurosurgeon, defined as a physician evaluation-and-management claim associated with the medicare provider specialty codes for neurology or neurosurgery, during the dates of the hospitalization. cpt codes were used to ascertain ancillary testing: brain radionuclide imaging, transcranial doppler ultrasound, or electroencephalography for brain death determination. exact binomial confidence intervals (cis) were used to report proportions. we identified patients with a brain death diagnosis. common associated neurological diagnoses were stroke ( patients; . %), cardiac arrest ( ; . %), and traumatic brain injury (tbi) ( ; . %). head ct or brain mri was performed in . %; this was true of . % of cases of stroke or tbi versus . % of cardiac arrests. neurologists were involved in the care of patients ( . %; % ci, . - . %). they were more commonly involved in the care of stroke ( . %) or cardiac arrest ( . %) than tbi ( . %) or other conditions ( . %). neurosurgeons were involved in cases ( . %; % ci, . - . %), mostly after tbi or stroke. two hundred patients ( . %; % ci, . - . %) were seen by a neurologist or neurosurgeon. twenty-nine patients ( . %; % ci, . - . %) underwent any ancillary testing. two hundred and nine patients ( . %; % ci, . - . %) were seen by a neurologist or neurosurgeon or underwent ancillary testing. in a nationally representative cohort of elderly patients, one-third of patients with a brain death diagnosis were not evaluated by a neurologist or neurosurgeon or by using ancillary tests. traumatic brain injury (tbi) is a major cause of death and disability in the us. recent advances in d illustration ( di) can precisely quantify intracranial pathology on computed tomography (ct). the current standard of measurement, abc/ , demonstrates variability in precision with bleed phenotype. the aim of this project is to assess accuracy automated di and compare it to standard abc/ measurements. baseline ct scans collected during the protectiii multicenter clinical trial (n= ) were retrospectively reviewed by a central neuroradiologist. subdural and epidural hematomas were identified (n ). the radiologist calculated abc/ score using osirix (mac) and radiant (pc) workstations. in a blinded fashion, research assistants concurrently generated di using the following methods: dicom data were resampled to . mm thickness slices and symmetrized using image analysis software (aquarius terarecon inc, ) . lesions were then compiled into single volumetric regions of interest ( d slicer v . , ) . hemorrhages were divided into two groups for analysis: group . volume of hemorrhage bland-altman analysis. this study was irb approved. there is a significant difference between the results of the di and abc/ methods. in group . the estimated relative bias between the two measurements (after transformation) is . (sd . ; pvalue . ; % ci . , . ). in group , the relative bias is - . , sd . , pvalue < . , % ci (- . , - . ). the di method calculates detailed surface area measurements in large and small volume hemorrhages, while abc/ averages cross-sectional area. the abc/ estimates vary by bleed phenotype and offer less topographical precision than di. this is particularly true in extra-axial hemorrhages, which are numerous studies have shown a significant association between hypotension and poor outcome in patients with head injuries. prior investigations have demonstrated that generation of negative intrathoracic pressure (itp) in ventilated patients with brain injury improves mean arterial pressure (map) and lowers intracranial pressure (icp). we hypothesized that augmentation of negative itp by breathing through an impedance threshold device (itd) with cmh o of inspiratory resistance would improve mean arterial pressure in a porcine model of intracranial hypertension. six spontaneously breathing female pigs ( . ± . kg), anesthetized with propofol, were subjected to focal brain injury through inflation of an french foley catheter placed in the epidural space. once a stable injury was obtained, baseline data were collected for minutes followed by minutes of itd use. results are reported as mean ± sd. the itp without the itd during inspiration was - . ± . mmhg, compared to - . ± . mmhg with the itd, p< . . following brain injury, map (mmhg) was significantly higher during itd use ( ± vs. ± ; p< . ). cerebral perfusion pressure (mmhg) was also significantly higher during itd use ( ± vs. ± ; p< . ). icp (mmhg) was not significantly different between groups ( . ± . vs. . ± . ; p= . ) although end tidal carbon dioxide levels (mmhg) were significantly higher during itd use ( ± vs. ± ; p< . ) presumably due to lower respiratory rates during itd use ( ± vs. ± ; p= . ). contralateral cerebral blood flow (ml/ gm/min) was similar between groups ( ± vs. ± ). in this porcine model of intracranial hypertension, spontaneous respirations through an itd significantly improved map and cpp. this approach could be utilized to prevent hypotensive episodes in the setting of brain injury. the impact of applying nanotechnology and biomedical engineering to improve the management of patients with spinal cord injuries (sci) is still not accurately described, nor understood. a systematic review of the literature was conducted, according to prisma criteria, to identify publications revolving around "sci+nanotechnology" and "sci+biomedical engineering" indexed on pubmed in the period - . furthermore, the database of clinicaltrials.gov was searched to highlight the stage of translation of this research into clinical practice through randomized clinical trials (rct). finally the uspto database was interrogated to identify the number of pertinent patents filed in northamerica in the same timeframe. the literature on bioengineering and nanotechnology contributions to sci is exponentially growing, with almost % of articles published between and . its quality and the interest of the scientific community are high, as confirmed by the average impact factor ( . ) and the average number of citations ( ) of articles published in the last two years. this field still represents a niche of sci research: the articles reviewed represent only . % of all articles on sci published in the same decade. this trend is confirmed on clinicaltrials.gov: out of rct on sci only few focus on the application of those technologies, furthermore out of articles spurring from the rct identified were published after , and % after . interestingly, with patents registered by the uspto, the interest in the commercial application of this research seems vivid. currently, the most promising areas of research are: nanofabrication/nanoscaffolding for structural repair, nanodrugs for regeneration, and design of neural interfaces for functional therapies. this review showed that both universities and independent research institutions (mostly from usa, china and european union) are driving this research race; the figures provided above suggest its potential to become a successful example of translational medicine. there are no neuroprotective and neuroregenerative treatments available for traumatic brain injury (tbi). clinical trials investigating potential treatments such as therapeutic hypothermia and progesterone have failed. pre-clinical studies indicate there may be a role of stem-cells in promoting neuroprotection/neuroregeneration in-vivo in animal models of tbi. we aim to provide a pre-clinical literature review into stem-cells as a potential therapeutic option in tbi-animal models. a literature search was conducted on pubmed and google scholar using the terms "traumatic brain injury", "stem-cell", "preclinical", and "animal studies". studies were included if there was an in-vivo animal model of tbi with either intravenous or intra-cortical stem-cell transplantation, along-with a control group, and investigated either motor or behavioral outcomes, or a combination. twenty-seven studies (n= animals) satisfied the criteria. / ( . %) animals were investigated for outcomes. studies harvested stem-cells from human-source, whereas harvested stem-cells from animal-source. bone-marrow stromal-cells (bmsc) were used in studies, neural stemcells (nsc) in , and miscellaneous in . / ( . %) animals received any stem-cell transplantation, whereas were controls. of animals receiving stem-cell transplantation ( ), ( . %) showed significantly better outcomes relative to control animals in each individual study, with exception of one study. amongst transplanted animals, functional outcomes did not differ significantly when grouped by stem-cell type (p= . ), transplantation route (p= . ), and source (p= . ). animals were followedup until week (n= studies), weeks (n= ), weeks (n= ), or > -weeks (n= ). this pre-clinical data demonstrates that stem-cell transplantation may have treatment potential in tbi as shown by improvement in functional outcome in as many as three-quarters of all animals that were treated with stem-cells. this data provides a foundation for the design of clinical translational studies. age of trauma patients including those with asdh is increasing as stated by national trauma registers. we were especially interested if age > years significantly influences outcome compared to younger patients and if other factors like initial gcs have an influence too. methods midline shift, if asdh was surgically removed, additional contusions, comorbidities and intake of anticoagulants. outcome was analyzed using the glasgow outcome scale (gos) at hospital discharge (gos ) and if possible months after discharge (gos ). uni-and multivariate analysis (cox regression model) was performed using the sigma stat softwar . . adverse outcome p= . . in addition, all patients > years with an initial gcs died whereas only % of younger patients with initial gcs died (p< . ). this was the only significant result in the multivariate analysis the monovariate analysis of our data showed a significantly higher risk for adverse outcome after asdh whe it should be considered if it is reasonable to transfer them from local hospitals to a specialized neurosurgical clinic, especially in times of limited resources. reported incidence of pulmonary edema in isolated head injury varies from - %. lung sonography is a potentially useful non invasive technique to detect extravascular lung water(evlw). this study aimed to identify the presence of evlw using lung ultrasound (b lines > per lung field) in chead injured patients admitted to icu . secondary objectives were to compare diagnostic accuracy and time to identification of evlw using chest x ray versus lung ultrasound. association of evlw with duration of mechanical ventilation (mv)and icu stay were observed after ethical clearance (iec no. int/iec/ / ), patients with head injury requiring mv and critical care were enrolled in the study. daily routine chest x ray and bedside lung ultrasound were done from the day of icu admission until the patient was on mechanical ventilator support. four inter costal spaces (ics) were scanned in semi recumbent position; third and sixth ics on either side of sternum till mid clavicular line. evlw was reprted as > b lines per lung field scan sonographically. details of mv and icu management were noted . evidence of evlw at the time of admission using sonography and cxr was recorded in and patients respectively. during icu stay . % patients showed evlw using lung usg (vs patients on cxr). mean delay in detection of evlw on cxr after detection on ultrasound was . ± . days. patients with low gcs, s. albumin, pao /fio ratio and greater apache ii and saps ii had significantly higher incidence of evlw. duration of weaning, mechanical ventilation and icu stay was significantly longer in patients with presence of evlw (p < . ) conclusions: lung ultrasound appears promising in detecting evlw earlier than chest x ray and may aid to minimize the duration of mechanical ventilation, weaning and icu stay . antiepileptic drugs (aeds) are recommended by guidelines for prophylaxis of early post-traumatic seizures (pts) associated with traumatic brain injury (tbi). there has been an increased use of both phenytoin and levetiracetam for this indication. the purpose of this study is to determine the incremental cost-effectiveness of phenytoin compared with levetiracetam for early pts prophylaxis in tbi patients. a cost-effectiveness study was conducted comparing phenytoin and levetiracetam for early pts prophylaxis during the days post-tbi. patients were included if they were years or older, received a study drug, and had a diagnosis of tbi. patients were excluded if they had a history of epilepsy, did not sustain a recent tbi, were initiated on both study drugs concurrently, or were switched to pentobarbital for elevated intracranial pressure. data was collected via retrospective chart review using electronic medical records and publically reported costs. effectiveness was measured as having a successful seizure prophylaxis regimen (sspr), which was defined as ) no clinical or electrographic seizure, ) no discontinuation of study aed, ) no cross-over of study aed to different aed, or ) no addition of aed during the days of therapy. the costs included costs of the study drugs, phenytoin level, and eeg. the data was used to calculate the primary endpoint, the incremental cost for the incremental change in sspr or the incremental cost effectiveness ratio (icer). the phenytoin regimen (n= ) cost $ . and had an sspr of . %. the levetiracetam regimen (n= ) cost $ . and had an sspr of . %. the icer was $ for each % increase in sspr with levetiracetam. the sspr of phenytoin and levetiracetam were similar. because patients who received phenytoin may differ from those who received levetiracetam, further analysis is needed prior to drawing any conclusions about the cost-effectiveness of levetiracetam relative to phenytoin. augmented renal clearance (arc) has been reported in up % of critically ill tbi patients and may impact therapeutic drug concentrations. improved predictors of arc are needed. serum cysc, a validated marker of glomerular filtration, has not been examined as a marker for arc in critically ill tbi patients. this pilot study tested the hypothesis that serum cysc concentrations are lower than reference values following tbi. adult tbi patients enrolled in the ukccts-unctracs prospective study of arc effects on drug clearance, were eligible. cysc serum concentrations (elisa -r & d cysc) were measured daily for up to days and compared to reference values. descriptive statistics and student t-test for continuous measures (patient vs. reference lower range cysc) were calculated. the first ten patients [ m/ f, mean age= . years ( - y/o), median gcs= (iqr - )] provided a total of serum cysc for analysis. each patient provided at least samples (range - ) for up to seven days. measured serum cysc concentrations were below the reference range in of samples. the overall mean cysc concentration was . + . mg/l vs expected mean of . + . . (ns) measured values fell below the lower reference range in patients ( m/ f) for the first study days (mean = . + . vs . + . p< . ). the mean difference between measured concentration and reference value was . + . mg/l. after days, four patients ( m/ f) remained below reference values with a mean difference of . + . mg/dl. preliminary results show cysc was not consistently below reference ranges in all tbi subjects. a subset of subjects showed significantly lower cysc within seven days of injury. the relationship between cysc and arc needs to be further examined as analysis continues. functional connectivity of the default mode network (dmn) is believed to be necessary for recovery of consciousness after coma. however, dmn connectivity has not been comprehensively studied in patients with acute severe tbi. we hypothesized that dmn connectivity in patients with acute severe tbi is associated with level of consciousness. we prospectively enrolled patients admitted to the intensive care unit for acute severe tbi and performed resting-state functional mri (rs-fmri) as soon as safely possible. dmn functional connectivity was assessed by rs-fmri analysis of the blood-oxygen level dependent (bold) signal using a seed-based approach. pearson's correlation coefficients were calculated between the mean bold time series within dmn nodes and all other regions in the brain. level of consciousness was assessed at the time of the scan using the coma recovery scale-revised (crs-r). two-sample t-tests were performed to identify brain regions with connectivity differences between conscious and unconscious subjects. we then tested for associations between level of consciousness and dmn connectivity within these regions. we enrolled patients ( male, mean+/-sd age +/- years) and matched controls ( male, age +/- years). rs-fmri was performed . +/- . days post-injury. at the time of rs-fmri, patients' levels of consciousness were coma (n= ), vegetative state (vs; n= ), minimally conscious state (mcs; n= ), and post-traumatic confusional state (ptcs; n= ). connectivity within the medial prefrontal cortex and posterior cingulate was selectively reduced in unconscious patients (coma and vs) compared to conscious patients (mcs and ptcs; false discovery rate-corrected p < . ). when these regions were further interrogated, connectivity correlated with crs-conclusions dmn functional connectivity correlates with level of consciousness after acute severe tbi. traumatic brain injury (tbi) is a substantial source of death, disability, and healthcare utilization. many older tbi patients present to community hospitals and are transferred to trauma centers for further care; however, little is known about the provision of care and patient outcomes at the final receiving hospital. we described trauma center care among geriatric transfer patients with tbi. we conducted a secondary analysis on a sub-cohort from a prospective multi-center study focusing on ambulance and emergency department (ed) care of injured older adults transported via ambulance. the current analysis focused on tbi patients transferred to the region's level i trauma center from another hospital. transfer paperwork from the originating hospital was reviewed and we conducted a detailed medical record abstraction, including computed tomography (ct) findings, procedures, length of stay (los), and ed disposition. data were collected on transfer patients. thirty had confirmed abnormalities on head ct ( . %). the mean age was years (range: - ), % female, and the most frequent mechanism of injury was falls ( %). average los was . days (range: - , median los . ), with patients staying one day or less. ct findings included subdural hematoma ( %), subarachnoid hemorrhage ( %), and intraparenchymal hemorrhage ( . %). five patients required neurosurgical intervention ( %), eight required icu admission ( %), two were discharged from the ed ( %), and two transitioned to inpatient hospice ( %). tbi is a frequent cause of transfers to trauma centers. in our sample, admission occurred in the majority of patients, but neurosurgical intervention was less common. however, for appropriately selected patients, strategies such as telemedicine may reduce transfers thus saving resources and improving continuity of care for patients and their families. this is an area in which future research is warranted. the prospects and timing of decannulation may affect surrogate decision making regarding tracheostomy for traumatic brain injury (tbi) patients, yet predictors of decannulation are unknown. methods tracheostomy admitted to an affiliated acute rehabilitation hospital between january and december . patients who had life-sustaining measures withdrawn were excluded. admission data, including injury characteristics and presence of lung injury on initial chest x-ray, and inpatient complications were compared. patients were followed throughout rehab and to the point of decannulation. patients lost to follow up were eliminated from analysis. time of decannulation was verified by inpatient physician notes. a cox proportional hazards model was created to determine factors associated with the time to decannulation and reported as hazard ratios (hr). there were tbi patients admitted to the icu during study period and ( % men, mean yearsold, median gcs ) underwent tracheostomy after ± days of intubation, of which were followed throughout rehabilitation. overall cannulation time was ( - ) days. ( %) patients had their trach removed prior to discharge from rehab after ( - ) days of cannulation. in a cox proportional model adjusting for sex, reintubation, aspiration pneumonitis, and presence of lung injury on admission chest x-ray; a higher hospital discharge gcs was associated with a shorter time to decannulation (hr, . ; % ci, . - . ; p =. ) while patients who required inpatient dialysis had a longer time to decannulation (hr: . ; % ci, . - . ; p = . ). the majority of tbi patients that require tracheostomy will be decannulated prior to discharge from rehab. longer durations of tracheostomy cannulatio hospital discharge and those that receive inpatient dialysis. goal directed therapy (gdt) is thought to be associated with outcome after traumatic brain injury (tbi). our team applied gdt to standardize care in patients with moderate to severe tbi, who were enrolled in a large multicenter clinical trial. physiologic goals were defined a priori in order to standardize care across sites participating in the protect iii trial. data were collected hourly for all randomized subjects (n= ). hours where gdt were not achieved were classified as "transgressions". these included: map . ; platelets mg/dl; and sbp mmhg. the proportion of hours spent in transgression was calculated for each parameter and grouped by quartile. poor outcome was defined via stratified dichotomy of the gos-e. data were adjudicated electronically and via expert review. for each parameter, the association between outcome and either ( ) occurrence of transgression or ( ) cumulative duration of transgression was estimated via logistic regression model, and backward selection was used to identify the physiologic parameters associated with outcome. subgroup analyses were performed in subjects with intracranial monitoring (ticp, n= ) . parameters significant at alpha . are reported. prolonged duration of transgression was associated with poor outcome when: glucose> mg/dl (p= . ); hgb mg/dl (p= . ) and inversely associated with map mg/dl (p= . ) or and was inversely associated with map< mmhg (p= . ). the protect iii clinical trial rigorously monitored compliance with gdt after tbi. multiple significant associations between physiologic transgressions and patient outcome were found. the data suggest that reducing physiologic transgressions is important to minimizing patient morbidity after tbi. the measurement and management of intracranial pressure (icp) is a key component in the care of severe head injury. extracranial ventricular drains (evd) have remained the standard due to the ability to lower icp with the drainage of cerebrospinal fluid (csf). placement of an evd is a more invasive procedure than intraparenchymal icp monitors (ipm) and it is unclear if the use of an evd improves outcomes. we hypothesized that early placement of an evd, in adult patients with severe head injury, would not affect outcomes. utilizing data from the citicoline brain injury treatment (cobrit) trial, a prospective multicenter study, we identified patients who met the inclusion criteria; ) placement of an icp monitoring device, ) glasgow coma score (gcs) less than , ) evd placement prior to arrival or within hours of arrival at the study institution. primary outcome was glasgow outcome score-extended (gose) at days post injury. secondary outcomes included neuropsychological evaluations at days post injury, mortality, and length of icu stay. logistic regression with forward-stepwise predictor adjustment and propensity score adjustment was performed to assess the independent association between evd placement and outcomes. patients who received an evd prior to or within hours of arrival at the study institution had worse gose at days ( . ± . vs . ± . , p= . ), higher in hospital mortality ( % vs %, p = . ), and did worse on out of neuropsychological measures at days. there was no difference in icu length of stay ( . ± . vs . ± . , p= . ). early placement of evds in severe adult head injury is independently associated with worse outcomes and higher in hospital mortality. goal directed therapy (gdt) is thought to be associated with outcome after traumatic brain injury (tbi). our team applied gdt to standardize care in patients with moderate to severe tbi, who were enrolled in a large multicenter clinical trial. physiologic goals were defined a priori in order to standardize care across sites participating in the protect iii trial. data were collected hourly for all randomized subjects (n= ). hours where gdt were not achieved were classified as "transgressions". these included: map . ; platelets mg/dl; and sbp mmhg. the proportion of hours spent in transgression was calculated for each parameter and grouped by quartile. data were adjudicated electronically and via expert review. for each parameter, the association between outcome and either ( ) occurrence of transgression or ( ) cumulative duration of transgression was estimated via logistic regression model, and backward selection was used to identify the physiologic parameters associated with mortality. subgroup analyses were performed in subjects with intracranial monitoring (ticp, n= ). parameters significant at alpha . are reported. mortality was . % and . % in the full and ticp cohorts. prolonged duration of transgression was associated with increased mortality for: hgb . (p mg/dl (p mg/dl (p= . ), and sbp . (p . (p= . ). covariates inversely related to mortality included single occurrence of map mmhg (p< . ). the protect iii clinical trial rigorously monitored compliance with gdt after tbi. multiple associations between physiologic transgressions and mortality were observed. the data suggest that maintaining physiologic measures within gdt guidelines may be important in preventing deaths. current outcome models in moderate-severe traumatic brain injury (mstbi) include only admission characteristics. yet, mstbi patients commonly have prolonged intensive-care-unit(icu)-stays with high risks to develop icu complications, lending to the hypothesis that these may be additionally associated with outcomes. the objective of this study was to examine the incidence rates of pre-specified medical and neurological icu complications, and their impact on post-traumatic in-hospital mortality and month functional outcomes. we analyzed mstbi patients consecutively enrolled in the prospective observational optimismstudy at a level- trauma center between / - / . poor outcome was defined as glasgow outcome scale - . multivariable logistic regression was employed to adjust for admission characteristics and icu-length-of-stay. the mean age was ± years, % were men, and median motor glasgow-coma-scale and injury-severity-scores were (iqr ; ) and (iqr ; ), respectively. the three most common medical and neurological icu complications were: hyperglycemia ( %), systemic inflammatory response syndrome ( %) and fever ( %); intracranial pressure crisis (icp; [ % of n= with icp-monitor]), brain edema requiring osmotherapy ( %), herniation ( %). multivariable models were adjusted for age, marshall-ct-classification, motor glasgow-coma-scale, pre-admission hypotension, icu-length-of-stay and injury-severity-score. after adjustment, in-hospital mortality was significantly associated with in-icu-cardiacarrest (or ; %ci . - . recent studies suggest benefits for early tracheostomy in patients with traumatic brain injury (tbi), yet data regarding who will require tracheostomy is lacking. ad lifesustaining measures withdrawn were excluded. admission and inpatient variables were compared. multivariable logistic regression analyses were used to assess admission and inpatient factors associated with receiving a tracheostomy and to develop models predictive of tracheostomy. there were patients ( % men, mean years-old, median gcs ) meeting study criteria with tracheostomy performed in ( %). admission predictors of tracheostomy included gcs, marshall score, injury mechanism, pao /fio ratio, and number of quadrants on chest x-ray with consolidation. inpatient factors associated with tracheostomy included the requirement for an external ventricular drain (evd), number of operations, pneumothorax, inpatient dialysis, aspiration, reintubation, and the presence of hospital acquired infections. multiple logistic regression analysis demonstrated that the development of hospital acquired infection (adjusted odds ratio [aor], . ; % confidence interval [ci], . - . ; p < . ), number of operations (aor, . ; % ci, . - . ; p < . ), pneumothorax (aor, . ; % ci, . - . ; p = . ), reintubation (aor, . ; % ci, . - . ; < . ), penetrating tbi (aor, . ; % ci, . - . ; p= . ) and placement of evd (aor, . ; % ci, . - . ; < . ) were independently associated with patients undergoing tracheostomy. a model of inpatient variables only was more strongly associated with tracheostomy than one with admission variables only (roc auc . vs. . , p< . ) and did not benefit from addition of admission variables (roc auc . vs . , p= . ). potentially modifiable inpatient factors have a stronger association with tracheostomy than do admission characteristics. existing traumatic brain injury (tbi) guidelines are designed primarily for the evaluation and management of tbi in tertiary care centers with advanced neuroscience capabilities. military special operations medical providers, however, are often required to treat and sustain patients in austere environments with limited resources for up to hours. tbi management guidelines directed specifically toward the care of these patients are needed. a review of recent operational experiences involving tbi and a survey of military special operations medics prompted a multidisciplinary expert panel to develop draft clinical practice guidelines/recommendations for prolonged field management of tbi. the panel conducted an in-depth review of literature on tbi and related topics and adapted existing and emerging therapies to address the unique challenges encountered in prolonged field care. tbi management while optimal management of pbto is not fully established. the objective of this -coeur arterial blood gas was drawn (icp, cpp, hemoglobin, temperature, pco and pao ). probes were localized in normal appearing white matter. we used a was calculated. a total of data sets were collected from patients (mean age . ± . , median gcs , mortality range from to ). mean pao for the group as a whole was mmhg (± ) and mean cpp was mmhg (± ). mean duration of pbto monitoring was . days (± . ). taking into account all determinants of pbto and using a protocolized approach to correct pbto , the mmhg for a few days. high pao values are possibly required due to the fact that oxygen delivery to the brain is rate-limited by diffusion and impaired by oedema or microvascular ischemia. it should be noted that pulse oximetry is not sensitive to detect pao below this level. traumatic brain injury (tbi) and stroke are extremely common causes of acute brain injury (abi), which cause long term disability and permanent neurological impairment. coma and stupor are common manifestations of abi, due to interruptions of the ascending reticular activating system (aras). neuro stimulants can improve functioning of the aras. despite decades of research there is a paucity of prospective high-level evidence utilizing neuro stimulants to help with earlier awakening from coma and stupor in abi. we reviewed the literature using the grade level of evidence (loe) methodology. we performed a preliminary literature search of the national library of medicine (nlm) using search terms abi and stimulants. within the literature we searched for timing of stimulant use among abi studies and included all forms of abi such as tbi, stroke, and anoxic injury. we retrieved total results, of which we excluded since they did not meet grade high loe criteria or were "n of " studies or aggregates. only high loe randomized studies or meta-analyses were found. among these various stimulants were investigated including methamphetamines such as methylphenidate and lisdexamfetamine, caffeine, armodafinil, galantamine, and amantadine. methylphenidate had randomized trials and a meta-analysis in subacute tbi but reported only attention as a main outcome. we were unable to draw broad-level recommendations about optimal timing, best stimulant, and patient centered outcomes from this data. there is insufficient data to recommend optimal stimulant, timing, and dosing among heterogeneous abi disease models. we propose conducting future homogenous abi neuro stimulant trials in for safety, tolerability, dose-finding, optimal timing, and outcomes based efficacy. neuro stimulants could play a role in earlier awakening and extubation in abi which could improve outcomes similar to sedation/vacation bundles in icu's currently if studied adequately. tbi remains the leading cause of death and disability in young adults in the us and europe. thus far, pharmacological and non-pharmacological intervention studies did not confirm benefits on functional outcomes. the inducible enzyme nitric oxide synthase (inos) is upregulated in response to brain injury, causing excessive production of no, a key driver of secondary injury after tbi. the antipterin vas is a structural analogue of the endogenous nos cofactor and a potent in-vivo selective inhibitor of inos. a randomized, placebo-controlled phase study examined dose levels of vas in patients with acute moderate or severe tbi. cerebral microdialysis showed pharmacologically relevant drug concentrations close to the injury and a tendency for vas to increase the arginine/citrulline ratio, an indirect marker of nos inhibition (stover et al., j neurotrauma ). vas conferred a significant benefit on the extended glasgow outcome scale interview (egos-i) at and months after injury. no changes in systemic blood pressure or partial brain oxygen pressure were noted. a recent pharmacokinetics and pharmacodynamics study further corroborated the selective inos inhibition by vas . the confirmatory nostra phase trial (eudract no. - - ; clinicaltrials.gov identifier nct ) was initiated in . adult patients with a nonrequiring intracranial pressure monitoring, are randomized : to vas or placebo, administered in addition to standard of care, as intravenous continuous infusion for hours, starting between and hours post tbi. the primary efficacy endpoint is egos-i at months post injury. additional endpoints include the daily therapy intensity level and tbi-specific quality of life measures. continuous safety monitoring is performed by an independent committee. nostra iii, the only ongoing registration study in acute moderate and severe tbi, is sponsored by vasopharm gmbh, and plans to recruit patients by q . a glasgow coma scale (gcs) score of on presentation in patients with traumatic brain injury (tbi) portends a poor prognosis. consequently, there is often a tendency to treat these patients less aggressively because of low expectations for a good outcome. we performed a retrospective review of patients with tbi and a gcs score of . demographics, apache iv scores , pupillary reactivity to light, intracranial pressure (icp), icp burden (the number of days with an icp spike > mm hg as a percentage of the total number of days monitored), and outcome (mortality and glasgow outcome scale-gos at months, with good outcome defined as gos of - ). patients were divided into groups: group (gos = - ) and group (gos = - ). a total of patients were included. the overall mortality rate was . %. at -month, patients ( . %) achieved a gos - . compared to group (n = ), group (n = ) had higher average apache iv score ( ± vs ± , p = . ), more patients with bilateral fixed pupils ( % vs %, p = . ), and higher icp burden ( ± vs ± , p = . ). gos score - was achieved in % of patients presenting with bilateral reactive pupils versus . % of patients presenting with bilateral fixed pupils (p = . ). . % of patients with tbi and a gcs of at presentation achieved a good outcome at months. apache iv scores, icp burden, and pupillary reactivity were significant predictors of outcome. we believe that patients with severe tbi who present with a gcs of should still be treated aggressively initially since a good outcome can be obtained in a significant proportion of patients. elevated circulating catecholamine levels are independently associated with functional outcome and mortality after isolated traumatic brain injury (tbi). we assessed the ability of peripheral catecholamine levels to improve the prognostic performance of the crash and impact-tbi models. prospective, observational, multicenter cohort study, conducted at three level trauma centers in canada and usa. epinephrine (epi) and norepinephrine (ne) concentrations were measured in the peripheral blood at admission (baseline), , and h after trauma. outcome was assessed at months and dichotomized into favorable [extended glasgow outcome scale (go -tbi models, which identified core prognostic markers of severe tbi. the baseline model (m ) included age, gcs and pupillary size/reactivity. the model (m ) included m + hypoxia, hypotension and marshall ct classification. model and included m + epi levels, and m + ne levels, respectively. the risk models performance was assessed by comparing receiver operating characteristic (roc) curves, and by the use of integrated discrimination improvement (idi) index. m had significantly higher roc and idi than the baseline model (m ), to predict mortality. m had a roc = . ( . - . , p = . ) and idi = . (p = . ). the prediction of mortality was not improved by including ne [m = roc = . ( . - . , p = . ) and idi = . (p= . )]. the integrated discrimination improvement index indicated the prediction of unfavourable outcome by the baseline model was improved by including epi (idi = . , p= . ), and ne (idi = . , p= . ) in the models. catecholamine levels improved risk models performance to predict mortality and unfavorable outcome after traumatic brain injury. following traumatic brain injury (tbi), depression is common and may influence recovery. small trials demonstrated that various drugs are beneficial in managing depression following tbi, but no large, definitive study has been conducted. we performed a meta-analysis to estimate the potential benefit of anti-depressant medications following tbi. multiple databases were searched using the terms "anti-depressant tbi," and "depression treatment tbi" to find prospective pharmacologic treatment studies of depression following tbi. studies were excluded if they did not measure depression as an outcome. effect sizes for anti-depressant medications in post-tbi patients were calculated for within-subjects designs that examined change from baseline after receiving medical treatment and treatment-placebo designs that examined the differences between anti-depressants and placebo groups. a random effects model was used for both analyses. of titles screened, studies were included, with total patients. medications evaluated included selective serotonin reuptake inhibitors, monoamine oxidase inhibitors, and tricyclic antidepressants. pooled estimates showed significant reduction in depression scores for individuals after pharmacotherapy (mean change [mc] - . , % confidence interval [ci]: - . to - . ) and significant difference in reduction of depression scores between medications and placebo in the pooled estimate (standardized mean difference of four trials [smd] - . , % ci: - . to - . ); however only one of the four treatment-placebo studies found medications significantly reduce depression scores more than placebo. this meta-analysis found a significant benefit of pharmacotherapy for treatment of depression in patients with tbi. however, there was a high degree of bias and heterogeneity regarding tbi severity, time since injury, depression severity, and demographics. larger prospective studies on the impact of anti-depressants on post-tbi depression are warranted to better understand treatment effects and the relationship of post-tbi depression and outcome more broadly. pleural effusion (pe) has been reported in % of medical icu. there is little published data on the prevalence and clinical significance of pe in mechanically ventilated patients with traumatic brain injury. head injury patients admitted to icu for mechanical ventilation (mv) within - hours and gcs > were assessed for eligibility. presence of pe was assessed by serial cxr on daily basis and volume of effusion was estimated and recorded. in case there was no evidence of pe on cxr, a bedside sonography in semi recumbent position was done within h of icu admission. pleural fluid volume was estimated based on -point classifications on sonography. details of mechanical ventilation and icu management were recorded. successful weaning was defined as ability to breath spontaneously for h. primary aim was to observe prevalence of pe in mv head injured patients. as secondary measure; impact of pe on duration of mv, weaning and length of icu stay were compared. study enrolled patients. three baseline cxr showed pe. total of ( %) patients developed pe in icu. patients had evidence of pe on both cxr and usg. patients had only sonographic evidence of pe, which were not detected on cxr. significantly more minimal effusions were detected on sonography ( / , p= . ). duration of mechanical ventilation and duration of icu stay were significantly more in patients with pe. (p= . , mann whitney rank sum test) there was no significance difference in duration of weaning in patients with and without effusion ( . ± . , . ± . , p= . ). chest ultrasonography increased the detection rate of pleural fluid. patients with pe had longer duration of mechanical ventilation. early detection may be associated with shorter period of mechanical ventilation and icu stay spine surgery can trigger a systemic inflammatory response syndrome and lead to hypotension requiring vasopressors. as sepsis is a major differential diagnosis in the post-operative period, the objective of this study is to understand the prevalence of a true systemic infection in this setting. retrospective review of all consecutive adults with post-operative shock requiring vasopressors following spine surgery in an academic tertiary medical center. a total of patients, median age years (iqr - ), were included in the final analysis. comorbidities included a median bmi of (iqr - ), coronary artery disease ( %) and diabetes mellitus ( %). median estimated blood loss was cc (iqr to cc). circulatory volume was adequately replaced in a total of % patients within hours post-op. all patients received crystalloids, and an additional % received multiple (> ) units of prbcs transfusion. adequate urine output was confirmed in ( %) of the patients. the maximum median rate and duration of each vasopressor infusion was as follows: phenylephrine mcg/min (iqr - , n = ), hours (iqr - ), norepinephrine mcg/min (iqr - , n = ), hours (iqr - ), epinephrine mcg/min (iqr - , n = ), hours (iqr - ) and vasopressin . units/min, hours ( - , n = ). of the patients, ( %) met at least sirs criteria. infection was confirmed in a total of patients; positive respiratory or blood cultures in ( %) patients and positive urinalysis or urine culture in ( %). two patients ( %) were diagnosed with myocardial infarction. no patients had pulmonary embolism. our study suggests that the risk of infection and sepsis in patients with persistent shock following spine surgery is small but not negligible. larger multicenter studies are needed to confirm our findings and to identify the predictive factors. ischemic and hyperemic injuries may occur unnoticed after severe traumatic brain injury (tbi) and contribute to additional brain damage. maintaining an adequate cerebral perfusion is considered crucial in preventing such injuries, as deviations from autoregulation-guided optimal cerebral perfusion pressure (cppopt) are associated with greater mortality and disability. this makes reliable estimation of cppopt an interesting diagnostic and treatment tool for monitoring. cppopt is defined as the cerebral perfusion pressure (cpp) at which the pressure reactivity index (prx) is minimal. the leading method for estimating cppopt automatically, by aries et al. ( ) , fits a parabola to pairs of prx and cpp data. the method uses preset heuristics to reject the fit as unreliable, namely when the parabola is too "shallow" or does not cover a certain cpp range. as a result, the cppopt estimates could be generated only about - % of the time. moreover, the manually set heuristics potentially restrict the generality of the model. here, we propose an alternative method based on bayesian inference. treating prx at each time as a function of cpp corrupted by noise serves as a "forward model" that can be inverted to yield, for a given data set, a temporally evolving posterior probability distribution over cppopt. the mean of this distribution is a bayesian estimate of cppopt; we find that these estimates are generally consistent with those obtained from the classic method. importantly, the width of the distribution at a given time serves as a metric of uncertainty about cppopt estimation. we find that this uncertainty tends to be large at time points where the classic method with preset heuristics rejects the fitted parabola. our method makes manually setting rejection criteria unnecessary. bayesian estimation of cppopt holds promise as a tool for providing additional decision support in the care of individual tbi patients. quantitative parameters derived from continuous eeg (ceeg) have been useful to understand evolution of traumatic brain injury (tbi) and the impact on regional networks. these parameters are often interrogated at a global level rather than region-specific. the regional evaluation of quantitative eeg parameters may provide an objective assessment of regional network function, and be of predictive value for prognostication continuous eeg was performed in patients with tbi, and mri imaging was obtained during acute and chronic time points post injury (within days and months, respectively). the extended glasgow outcome scale (gose) assessed clinical recovery at months, with good recovery defined as gose score - and poor as gose score - . volumetric measurements of selected brain regions, both cortical and subcortical, were obtained at acute and chronic time points. quantitative parameters derived from ceeg, such as percent alpha variability (pav) and hemispheric symmetry, were calculated continuously and anatomically (frontal, temporal, occipital) through the acute hospitalization course. we hypothesized that persistent regional variation in alpha power post injury would lead to brain regionspecific atrophy and may predict outcome at months acute pav within the first hours post injury was poor in patients with poor outcome. in addition, patients with poor outcome had significantly more atrophy in the thalamus, hippocampus, and temporal and occipital lobes. asymmetry of the hemisphere pav values correlated with both brain atrophy and clinical outcome regional asymmetry of pav within the first hours post injury correlates with chronic brain atrophy and clinical outcome after tbi after moderate and severe traumatic brain injuries (tbis), individuals are often admitted to an intensive care unit (icu), and later require intensive rehabilitation. many neuro-icus engage therapists and physiatrists for rehabilitation and therapy during a patient's icu admission. however, the optimal timing, intensity, and components of rehabilitation needed while in the icu are not known and practice patterns are highly variable. the goal of this study is to describe the rehabilitation practices to identify whether there is consensus on best practices. an electronic survey asking participants to describe tbi rehabilitation practices in their icu was distributed via redcap through the neurocritical care society (ncs) and american congress of rehabilitation medicine (acrm) websites. potential respondents were first asked if they cared for patients with tbi in the icu, and if they answered "yes," they were invited to complete the survey. two email reminders were sent to each group for completion. after weeks, the data were extracted and analysis completed. there were respondents who reported that they cared for patients with tbi in the icu ( attending physicians, advanced care practitioners, therapists, nurses, fellows, and other). of these, % recommended early rehabilitative care. the most common reasons to wait for the initiation of physical therapy and occupational therapy were normalization of intracranial pressure (icp) ( % and % respectively) and hemodynamic stability ( % and % respectively). speech therapy was typically recommended after extubation ( %) and normalization of icp ( %). the majority of clinicians caring for patients with tbi in the icu support early rehabilitation efforts, typically after a patient is extubated, intracranial pressure has normalized and the patient is hemodynamically stable. prospective studies evaluating the merits of these self-reported rehabilitation initiation criteria are warranted. high-dose methylprednisolone (hdmp) has been studied as a potential therapeutic option for acute sci, with mixed results regarding efficacy and consistent suggestion of complications. we conducted a retrospective cohort study of acute sci patients extracted from the medical information mart for intensive care iii (mimic-iii) database to evaluate the hypothesis that steroid-related adverse drug events (ades) occur less often than in published clinical trials using hdmp. three groups of patients coded for acute sci were identified from mimic-iii from june to october : hdmp recipients per nascis ii/iii protocols (hdmp, n = ), patients who received some steroids but not per nascis ii/iii protocols (non-hdmp, n = ), and patients who did not receive steroids (no steroids, n = ) . demographics and data on complications of steroid therapy were extracted. one-way anova or student's t test were used to evaluate continuous variables; chi-squared or fisher's exact test were used for nominal or categorical variables. there were no differences in steroid-related ades between the three groups. there were higher average blood glucose readings in recipients of any steroids compared with the no steroids group, and more variation in blood glucose readings in hdmp recipients compared with the other two groups. there was a higher icu los and ventilator time in the hdmp group compared with the other two groups. compared with three other trials examining similar use of hdmp in acute sci, there were higher rates of pneumonias overall, though lower rates of urinary tract infections, skin & soft tissue infections, pressure ulcers, and superficial thromboemboli/thrombophlebitis. the results of this study are consistent with previous works related to the potential for harm regarding the use of hdmp or any steroids in acute sci. changes in selected adverse event profiles may be due to standardization of icu supportive care over time. cervical spinal immobilization and clearance protocols are important steps in the minimization of secondary spinal cord injury. patients with primary neurologic diseases are frequently found down and placed in rigid cervical collars despite sustaining minimal-to-no cervical injury. in these patients, neurologic dysfunction can complicate and delay cervical clearance. decreasing time spent in cervical spinal immobilization could improve patient care by allowing greater access to / range-of-motion of the neck, increasing patient comfort, and decreasing skin breakdown. through retrospective chart review over a -month period, we collected the following: the rationale behind each mri, any mri evidence of cervical instability, the result of any ct imaging, and the basic mechanism of any trauma. for patients that were simply found down, any evidence of trauma either by history or physical exam was recorded. during the study period, there were instances where an mri of the cervical spine was performed. of those mris, ( %), were performed for cervical spinal clearance. sixty-one ( %) of mris were ordered without any ct imaging first. of the patients with a normal ct, six ( . %) were found to have mri evidence of cervical instability. notably, of the patients who were found down, there was only one instance where the mri demonstrated instability. that patient had extensive facial injuries suggestive of an unwitnessed fall. in the patients that were found down with no history or evidence on physical exam of trauma, there was no mri instability. for patients that are found down without any history or evidence on physical exam of trauma, a ct of the cervical spine is likely sufficient for cervical spinal clearance. acute subdural hematoma (asdh) represents a major clinical entity in severe traumatic brain (stbi), approximately % are accompanied by various extents of asdh. stbi has been reported to cause cerebral circulatory disturbances at an acute stage and had the worst circulatory disturbance among stbi. in this study, we focused on the cerebral circulation of asdh, evaluated the absolute left-right difference between cerebral hemispheres and compared the cerebral circulation between the favorable outcome group and the unfavorable group. we retrospectively reviewed patients with asdh. they were admitted to our hospital from to . in these patients, we simultaneously performed xenon-computed tomography (xe-ct) and perfusion ct to evaluate the cerebral circulation on post-injury days - . we measured cbf using xe-ct and mean transit time using perfusion ct and calculated the cerebral blood volume (cbv). a significant absolute difference in cerebral circulation between the hemispheres among different types of tbi was observed in mtt. there was no significant difference in these parameters between left-right hemispheres with asdh among the favorable outcome group and unfavorable group. although there was no significant difference in age, gcs at the onset of treatment, cbf and cbv, there was significant difference only in mtt between the favorable outcome group and unfavorable group. the circulatory disturbance in patients with asdh occurs diffusely despite the focal injury. additionally, in unfavorable patients, the circulatory disturbance is worse than in favorable patients. because asdh suffered ischemia more than other types of stbi, we had to perform not only removal of the occupying lesions, but also neurointensive care, including whole-body management and hypothermia therapy for the ischemic brain after surgery. we have to adopt a treatment strategy appropriate to the pathophysiology of the different tbi types. kcentra is -factor prothrombin complex concentrate that is fda approved for reversal of warfarin. there is limited research describing the use of kcentra for coagulopathy in the setting of traumatic intracranial hemorrhage. here, we show the largest ever retrospective review for the use of kcentra in the setting of traumatic intracranial hemorrhage. retrospective chart review was performed from - for patients with intracranial hemorrhage who presented to the r adams cowley shock trauma center. patients who received kcentra were identified. basic clinical information was obtained including cardiac/stroke history, blood pressure, glasgow coma score, medication history, and categorization of hemorrhage. pre and post inr level was assessed. hemorrhagic expansion was assessed with ct scan up to up to hours. disposition and thromboembolic events were recorded. forty-four patients were identified as receiving kcentra in the setting of traumatic intracranial hemorrhage. pre and post kcentra dosing inr was found to be significantly different (p< . ) across the two groups assessed (warfarin and tbi/noac coagulopathy). seventeen patients ( . %) had hemorrhagic expansion as determined on ct scan. disposition (home vs rehab vs death) was found to have three significant variables: history of stroke, hemorrhagic expansion, and admission glasgow coma score. eight patients ( . %) were found to have thromboembolic events. here, we show the largest retrospective review describing the clinical use of kcentra for coagulopathy reversal in the setting of intracranial hemorrhage. overall, kcentra is shown to be a safe and effective drug for the reversal inr. importantly, our reported hemorrhagic rate of . % is lower than established rates reported in the literature for warfarin/coagulopathic patients with intracerebral hemorrhage ( - %). the prognostic importance of hemorrhagic expansion was highlighted in the disposition analysis which showed that zero patients were discharge home if there was recorded expansion. despite the impact of post-traumatic amnesia (pta) duration on long-term functional outcome after traumatic brain injury (tbi), radiologic predictors of pta duration are lacking. we hypothesized that the number of traumatic microbleeds (tmbs) detected by gradient recalled echo (gre) magnetic resonance imaging (mri) in neuroanatomic regions that mediate memory correlates more strongly with pta duration than does the number of global tmbs. using a prospective outcome database of patients treated for mild-to-severe tbi at an inpatient rehabilitation hospital, we retrospectively identified patients who underwent acute mri with gre. pta duration was determined by the galveston orientation and amnesia test, orientation log or chart review. a rater blinded to pta duration identified tmbs on the gre datasets globally and in neuroanatomic regions that mediate memory, including the hippocampus, fornix, corpus callosum, thalamus, and the temporal lobe. associations between global and regional tmbs (in the mentioned locations) and pta duration were tested using spearman rank correlation coefficients. the cohort was comprised of % ( hippocampus and corpus callosum tmbs are associated with pta duration, and thus may have greater utility for predicting functional outcomes than global tmb number. validation of these findings in larger prospective studies is indicated. using a large two-center cohort of penetrating traumatic brain injury (ptbi) patients, we previously developed the survival after acute civilian penetrating brain injuries (spin) score, a logistic regressionbased parsimonious risk stratification scale for estimating survival after civilian ptbi. the objective of the present study was to externally validate the spin-score. our multicenter validation cohort comprised ptbi patients retrospectively identified from three u.s. level- trauma center registries. the spin score variables (motor gcs [mgcs], sex, pupillary reactivity, self-inflicted ptbi, transfer status, injury severity score [iss] and inr) were collected from the trauma registries supplemented by chart review. using the spin-score multivariable logistic regression model from the original study, receiver-operating-characteristic area-under-the-curve (roc-auc) analysis and hosmer-lemeshow goodness-of-fit testing was performed. the mean age was ± years, and patients were predominantly male ( %), with % white and % black. in-hospital mortality was %, and -month mortality of discharge survivors was . in this multicenter external validation study, the full spin-model predicts in-hospital survival after ptbi with excellent discrimination and calibration. after removing inr from the model, discrimination remained excellent, but model calibration diminished. the full spin-score may provide important information to guide families and physicians after civilian ptbi. limited data has described alterations in vancomycin pharmacokinetic (pk) parameters in traumatic brain injury (tbi) patients that have resulted in sub-therapeutic concentrations. the primary objective of this study is to evaluate the pk parameters of vancomycin in tbi patients to determine if using the common clinical practice of capping creatinine clearance (crcl) to ml/min in determining dosing impacts achievement of therapeutic concentrations. this was a single-center, retrospective study of patients at least years of age with tbi who received vancomycin and one reported steady-state vancomycin serum level from april to december . predicted pk parameters based on population data using actual and capped creatinine clearance (crcl) at ml/min were compared with calculated pk parameters based on serum trough concentrations at steady state. the difference was assessed using a two-sample wilcoxon rank-sum test where p < . was considered statistically significant. when using actual crcl [median ml/min patients with tbi experienced crcl that were greater than predicted. based on the results of this study, actual crcl is more accurate at predicting vancomycin pk than the common practice of capping crcl at ml/min. therefore, actual crcl should be used when determining vancomycin dosing regimens in patients with tbi to achieve desired therapeutic concentrations. neurocritical care is traditionally provided within institutions in urban centers while access in rural communities has been limited. transport to urban centers is not always favorable for a variety of reasons including critical patient condition, family wishes, weather, and geography. our hypothesis is that tele-neurocritical (tele-ncc) can extend access to this service with meaningful impact on icu outcomes. a tele-ncc pilot study was initiated within intermountain healthcare. starting / / , the study included all ischemic stroke patients admitted to the icu of one primary stroke center in utah. tele-ncc consultations were provided by ncc physicians at our flagship hospital located three hundred miles from the spoke site. tele-ncc consultations occurred via an existing telehealth platform developed inhouse. primary outcomes for this pilot study were icu and hospital lengths of stay (los). secondary outcomes include stroke complication rates and results on a provider satisfaction questionnaire. to date, tele-ncc consultations have been performed with median hospital los = days (iqr . - . ) and icu los = . days (iqr - ). in the months prior to the pilot, there were admissions to the icu for ischemic stroke with median los = days (iqr . - . ) and icu los = . (iqr - . ). for this small sample size, the p-values for comparison of hospital and icu lengths of stay before and after tele-ncc are . and . respectively. tele-ncc care can have significant impact on icu outcomes by expanding access to critical support from neurocritical care specialists. tele-ncc expands access to not only consultation on critical neurological emergencies, but also on when to de-escalate from the icu or in end of life discussions with which general icu teams may not be comfortable. these impacts could be measured as important decreases in hospital and icu los. hospital readmissions increase health care costs, increase patient exposure to nosocomial disease, and imply patients were not stable for discharge. because readmissions are a target for hospitals and payers, several centers have developed predictive readmission scores in order to identify high-risk patients. we contend that these general readmission scores are not suitable for neurocritically ill patients and that specific predictive score must be developed to identify high-risk patients. we conducted a retrospective chart review of consecutive patients admitted to our neuroscience critical care unit. we recorded the readmission scores, reason for admission, length of stay,and if they were readmitted. we then compared the median readmission scores between the two groups. after removing patients without readmission scores or died at the end of the original admission,we analyzed the records of patients. patients were more likely to be readmitted if they were initially emergently hospitalized or had malignancy. readmitted patients had a longer original hospital length of stay. we found no difference median readmission score between those who were readmitted, and those who were not. most readmitted patients ( . %) had an original "low-risk" readmission score. we found that our center's score was poor in predicting readmission for neurocritical care patients and that several components of the score do not apply to our patient population. we propose that to accurately predict readmission,centers should create their own unique readmission scores for more homogeneous admission populations. clinical evaluation of the level of consciousness in non-communicative patients can be very challenging. in this study, we aimed to evaluate the nurses and nursing assistants' (nas) perception of the consciousness on patients suffering from disorder of consciousness (doc). through their activities, nurses and nas have an extended observation time of patient's behavior, and make repeated implicit assessments of patients' clinical state of consciousness. we hypothesized that even in the absence of a structured and explicit evaluation of consciousness (in contrast for instance with the coma recovery scale revised -crs-r), nursing expertise could be a valuable measure to improve assessment of state of consciousness in doc patients. this was a prospective observational single-center study. our primary objective was to correlate the nurses and na's assessment of doc-patients' consciousness quantified through an analogic visual scale (the "doc-feeling score") with the results of the standard methods (including crs-r, fmri, electrophysiology). the secondary objective was to identify elements which correlate with this assessment and/or with the expert's diagnosis (such as visual pursuit, patient's participation to nursing, motor responses to verbal command or adapted reactions to painful care). . linear regression reveals a good correlation between the "doc-feeling score" and the crs-r gold standard (r = . , p-value < . , figure ). global assessment of the level of consciousness by all the caregivers interacting with the patient using the "doc-feeling score" is reliable and can improve assessment of state of consciousness in doc patients. investigating causes of deterioration in neurological patients is important to anticipate these complications and improve outcomes. this is a prospective observational study performed at an academic tertiary care trauma, stroke and neurorehabilitation center. data was collected over a year from rapid response system activations (rrsa). in one year, our center had admissions. rrsa were performed on patients. most common admission diagnosis was ischemic stroke ( %). most common rrsa organ system involvement was respiratory system (n= , . %). the only predictors of death or new limitation of care in those patients who had rrsa were age ( years vs years, p < . ) and history of cancer ( %) vs ( %) p= . . . % (n= ) of rrsa happened during day shift and . % (n= ) during night shift. . % (n= ) of rrsa happened around shift change and were more likely to result in an unplanned icu admission. . % (n= ) of rrsa happened within hours of admission and were more likely to result in unplanned icu admissions. the most common reasons for in hospital decompensation in neurological inpatients are nonneurological. most common organ system involvement responsible for rrsa is respiratory system. the only predictors of death or new limitation of care were history of cancer and age and older. rrsa activations were more frequent during day shift. however, there was no different in the outcomes we evaluated between day and night shifts. rrsa happening around shift change wew more likely to result in unplanned icu admission. rrsa within hours of admission showed an increased risk of unplanned icu admission when compared to rrsa happening after hours of admission. neurocritical care is a growing field with an increasing number of dedicated neuroscience intensive care units. in this dynamic context, it is unclear which types of physicians provide neurocritical care across the united states. we performed a retrospective cohort study using claims data from a nationally representative % within analyzed critical care procedures . the primary outcome was physician specialty, defined by medicare provider specialty codes. in a sensitivity analysis, we excluded claims for services on the day of admission and claims associated with a diagnosis of cardiac arrest, since these activities may often occur outside of neuroscience intensive care units. we identified between and , neurologists were responsible for approximately one-quarter of neurocritical care services among a nationally representative cohort of elderly patients. critically-ill patients on mechanical ventilation (mv) cannot verbally communicate. research suggests several phenomena occur in patients during mv because of impaired communication including anxiety, loss of control, loneliness, and compromised interaction (schou and egerod, ) . for neurocritical care patients, this can be especially profound when coupled with cognitive and motor/sensory deficits. currently, the blom® speaking valve (sv) is the only approved product available that allows phonation in ventilator-dependent patients with tracheostomy. sv trials are known to facilitate vent-weaning. the current standard of care (passy-muir speaking valve, minute trials), is contra-indicated in patients who cannot tolerate cuff deflation. as such, the blom® sv was evaluated for clinical and quality efficacy. we retrospectively evaluated clinical outcomes associated with blom® sv on mv during a trial in a neuroicu of a large tertiary center between / / and / / . baseline demographics, diagnoses, physiologic, sedation, delirium, mobility and swallowing parameters, length of stay, ventilator modes and settings, ventilator days, work of breathing and presence of pneumonia were abstracted along with patient, family and interdisciplinary staff satisfaction survey results. patients were recommended for blom® tracheostomy. patients received sv trials. of the trials were performed, % ( ) were optimal/completed ( + minutes); % ( ) were suboptimal/completed trials ( - minutes); % ( ) unable to complete. satisfaction results from patients/families were positive compared to the interdisciplinary team survey results. remaining parameters currently in analysis with results pending, to be completed by end of august, . impaired communication during mv is suboptimal for neurocritical care patients. our clinical experiences with blom® sv showed positive and negative outcomes. positive benefits were enhanced patient/family engagement and family satisfaction. unanticipated obstacles included significant increase in patient fatigue during sv trials, often delaying ventilator weaning. further study is needed to determine efficacy in this population. patients with clinical signs of cerebral herniation require immediate intervention known as a "brain code". in our neurosciences critical care unit (nccu), a rapid response program is in place to ensure the safety of . % hypertonic saline's use (high risk medication) and to expedite its delivery to the bedside given the emergent need for this medication when ordered. our institution, however, is lacking a more holistic and structured approach to cerebral herniation syndrome that include components of tiers zero to three emergency neurological life support elevated icp or herniation protocol. the neurocritical care communication council consists of bedside staff nurses, nursing leadership, advanced practice providers (nurse practitioners and clinical nurse specialist), pharmacists, respiratory therapists and physicians. the council identified processes during neurological brain codes that could be improved as a result of using a bedside debriefing tool. the unit leadership council of the nccu reviewed literature on hospital debriefing tools and referenced this organizations current resuscitation debriefing tool. from these sources, a brain code debriefing form was constructed. a clinical tool was developed with the expectation of standardizing the brain code process in this nccu. the brain code debriefing form will be piloted to determine unit and system wide value. pre-and postimplementation data will be collected to discover areas of improvement for optimized patient care. through the development of this debriefing tool, it was ascertained that a clinical practice guideline for impending cerebral herniation would be beneficial to further guide and direct evidence-based care. thus, a preliminary algorithm for identification and emergency treatment is in process. the americas medical center is a -bed tertiary hospital complex, located in the city of rio de janeiro. the center was elected by the international and the brazilian olympic committees as the referral hospital for the olympic family (of), comprised of athletes and their crews, support and technical personnel, credentialed media and credentialed governmental representatives from the participating countries. the neurology and neurocritical care teams were selected to head a comprehensive program of acute emergency neurology, including a -bed neurocritical care unit (nicu), and - emergency neurology service. we hereby describe our experience during the olympic games in rio de janeiro, brazil results neurological assessments were conducted in patients from the of, of these involving athletes from countries. the most common reason among athletes were traumatic brain injuries (tbi), with politraumas (all involving cycling), mild tbi ( of boxing, of field hockey, of rowing and of cycling) and moderate tbi (cycling and water polo). three patients were admitted to the nicu: ischemic strokes and politraumas with tbi. motor vehicle accidents with associated tbi involving the of were surprisingly frequent, with assessments, none requiring admissions. finally, ct scans of head, ct scans of the cervical spine and mri scans ( brain and spine mri) were performed to assess the patients. of note, cases of seizures, functional deficits, multiple sclerosis flare and psychiatric complaints were observed affecting the of. not only that multiple sports-related injuries were observed, cases of diverse acute neurological issues were reported involving members of the of. olympic games are complex events mobilizing thousands of people, and a comprehensive and detailed plan for neurological emergencies is of extreme importance the term "handoff" has been defined as the "transfer and acceptance of responsibility for patient care that is achieved through effective communication, passing patient-specific information from one caregiver or team of caregivers to another to ensure the continuity and safety of the patient's care" (patterson and wears, ) . the joint commission reported that two-thirds of sentinel events occur at the time of patient handoff, which led to a national patient safety goal, requiring standardized process for handoffs (the joint commission on accreditation of healthcare organizations, ) . to support this npsg, a nccu specific handoff tool and timeout process were created to support the transition from or to nccu. nccu postoperative handoffs were identified as an area to enhance staff satisfaction and patient safety. baseline data to evaluate the frequency of neurosurgery report was performed in may . using a qualtrics survey in june , staff satisfaction with current ns or report was obtained from nccu rns, nps, and fellows evaluating whether they felt they received: accurate medical history, accurate information about performed procedure, sufficient handoff for patient care, specific patient goals, recent pharmacological intervention, anticipated concerns regarding diagnosis/procedure, estimated blood loss, blood/fluid intake, airway concerns, complications and overall satisfaction with handoff. a taskforce of rns, nps, neurointensivists, and neurosurgeons was established, ending with the creation of a handoff tool and timeout process. the new tool and process were initiated and two months later, a repeat survey was sent to evaluate staff satisfaction and perceived effectiveness of the new process and handoff tool. currently being tabulated at time of submission. using standardized, open communication techniques including handoff tools and a timeout throughout the perioperative period is crucial to positive outcomes and can improve perioperative care in the nccu patient and increase satisfaction and collaboration of all team member during or handoff. in the age of the healthcare reform and rising costs, it is important for strategic service lines to explore cost saving and care efficiency strategies. beginning in september , physician and administrative leadership within the duke neurosciences intensive care unit (neuroicu) began investigating per patient cost to explore opportunities to decrease direct cost to the neuroicu, cost to the patient, and reduce redundancy of care. with assistance from health system finance, the team assessed the following data points within each cost group and compared these values to that of our peers within the us news and world report top honor roll: · number of units · direct cost per unit · total direct cost our performance according to our peers in the following cost areas was: .pharmacy-ranked th out of .laboratories-ranked th out of .radiology-ranked th out of .cardiovascular-ranked th out of . based on these performance metrics, neuroscience administrative and medical leadership developed a project grid of prospective initiatives and identified the following for each cost area: ·stakeholder-led teams inclusive of providers, nursing, and administration ·duration or impact of each initiative: short, medium, or long ·activity phases based on duration the stakeholder-led groups would propose and validate projects based on scope and duration. at each group's meeting, members reviewed charge level financial data by activity code for the group's respective cost area to develop applicable initiatives. multiple initiatives are currently underway including those within the cost areas of pharmacy, laboratories, and radiology. included among these initiatives is a change in routine resistant organism screening and cervical spine clearance. other initiatives will be target intravenous anti-hypertensive treatment and laboratory frequency. the total cost savings from these initiatives can only be estimated at this point but will likely be in excess of $ , for the calendar year. it is uncertain whether dedicated neurocritical care units are associated with improved outcomes for critically ill neurologically injured patients in the era of collaborative protocol-driven care. we examined the association between dedicated neurocritical care units and mortality, and the effects of standardized management protocols for severe traumatic brain injury. we surveyed trauma medical directors from centers participating in the american college of surgeons trauma quality improvement program (tqip) to obtain information about icu structure and processes of care. survey data were then linked to the tqip registry, and random-intercept hierarchical multivariable modeling was used to evaluate the association between dedicated neurocritical care (ncc) units, the presence of standardized management protocols and mortality. we performed three sensitivity analyses reclassifying ncc units by restricting to closed units, under ucns director leadership, and exclusion of neurotrauma units. data was analyzed from , adult patients with isolated severe traumatic brain injury admitted to tqip centers between to . fifty icus were dedicated neurocritical care units, whereas were general icus. rates of standardized management protocols were similar comparing dedicated neurocritical care units and general icus. care in a dedicated neurocritical care unit was not associated with improved risk-adjusted in-hospital survival (or . ; ( % ci . - . ; p= . ). the results from the model were robust in our sensitivity analyses. the presence of a standardized management protocol for these patients was associated with lower risk-adjusted in-hospital mortality (or . ; % ci . - . ; p= . ). compared to dedicated ncc models, standardized management protocols for severe traumatic brain injured patients are low-cost process-targeted intervention strategies that may improve clinical outcomes. understanding the differences in processes of care within the context of icu structure is necessary to better understand mortality differences observed between centers, and may help in the design of future trials for severe tbi patients. complex multidisciplinary care of patients in the neurocritical care unit requires reliable and effective communication to minimize medical errors. we implemented a structured rounding process that incorporates ahrq-endorsed team strategies and tools to enhance performance and patient safety (team stepps) to improve interprofessional collaboration between team members. we convened a project team of physicians, advanced practice providers (apps), nurses, respiratory therapists, and pharmacists in a -bed nicu. we defined structured rounding processes and implemented team stepps strategies to promote closed-loop communication between team members during daily rounds. the assessment of interprofessional team collaboration scale (aitcs-ii) was administered to team members at baseline and months post-intervention. impact on overall team collaboration and subscale domains of team partnership, cooperation and coordination was assessed. the possible range of the overall collaboration score is to ; higher scores indicate better collaboration. the survey was completed by ( %) staff at baseline, and ( %) staff post-intervention. overall team collaboration scores improved significantly pre and post-intervention ( . ± vs . ± , p < . ), as did subdomain scores of team partnership ( . ± . vs . ± . , p < . ), collaboration ( . ± . vs . ± . ), and coordination ( . ± . vs . ± . ., p < . ). perceived shared understanding of patient daily goals between nurses and providers (physicians/apps) increased from % to % (chi-square . ; p < . ). % of staff reported that the intervention shortened or did not affect the duration of rounds. of those who reported longer duration of rounds, % responded that the intervention was worthwhile. interprofessional team collaboration can be enhanced by structured rounding and communication workflows. by promoting closed-loop communication during daily rounds, shared understanding of patient daily goals between team members is increased, and may optimize quality and safety of patient care. advanced practice providers (apps) are increasingly utilized to provide clinical care within neurocritical care units (nsicu) . despite the complex issues in this patient population, the specific educational and orientation needs of these providers have not been established. to meet the demands for rapidly and effectively training apps to provide advanced neurocritical care (ncc), a structured educational curriculum was developed and integrated within the standard orientation and on-boarding process for newly-hired app within our nsicu. this curriculum was designed with measurable learning goals, objective assessments of goal achievement, and opportunities for additional education and remediation at multiple steps within the program. the curriculum has three phases with distinct goals and assessments. phase i covers basic triage and resuscitation issues for the acutely-decompensating patient. phase ii covers general critical care principles in significantly greater depth. phase iii provides detailed experience and exposure to specific ncc issues. each phase incorporates relevant reading assignments with a tailored study guide, as well as a multiple-choice question post-test to demonstrate knowledge acquisition. phases ii and iii also include an oral exam incorporating hypothetical patient scenarios to allow the app to demonstrate comprehension and application of the goals for each phase. each phase lasts approximately to weeks with the expectation that the entire orientation curriculum will be completed within six months of employment. in addition to the educational curriculum, phases i and ii include working alongside a more senior app preceptor and providing bedside care for a progressively increasing number of patients. apps not meeting minimum established standards on any aspect of the curriculum are provided additional remediation and instruction by the preceptor and ncc faculty based on an individualized learning plan. a standardized educational curriculum provides a structured learning environment for new apps in the field of neurocritical care. reimbursement changes from the centers for medicare and medicaid services and value based purchasing systems have made quality improvement linked to clinical outcomes more crucial than ever. in one neuroscience icu, providers and nurses collaborate to address key infection parameters that impact patient outcomes. quality metric data in one neuroscience icu was collected over a period of fiscal years. outcome measures, consisting of glycemic and temperature control, and ventilator weaning strategies, were obtained after certain parameters were enforced over two years and then compared to the initial year. the urinary catheter utilization has decreased by over %, with catheter associated urinary tract infections decreased by % in years (p-value < . ). central line utilization decreased by %, with a % decrease in central line associated blood stream infections in years (p-value . ). new ventilator weaning strategies were put into place utilizing adaptive support ventilation mode, which decreased total ventilator days by days/year . successful weaning and extubations resulted in no recorded ventilator-associated pneumonia in the last years. this neuroscience icu maintains glucose below mg/dl more than % of the time. regarding temperature control data, a normothermia protocol was implemented that utilizes aggressive temperature control coupled with bromocriptine administration. as a result, % of patients had a temperature less than °c. all of the quality initiatives that have been implemented have improved the observed/expected mortality ratio by . %. this study shows that by optimizing infection control, temperature management, glycemic control and ventilation strategies, there is an overall positive impact on the patient's morbidity and mortality. as evidenced by these results, this institution is now a top performer when compared in a national clinical database. this presentation will share the pragmatic strategies to create a culture of quality improvement in any neurocritical care unit or patient care organization. health care records are not accessible universally at point of care delivery. in developing countries like thailand a large proportion of health care records are still paper based. patients may not able to convey relevant information about their own medical problems and medications during patient-physician encounters or in the event of emergency. our purpose was to create a simple platform for recording relevant basic healthcare information through a system that can be securely accessed even in countries like thailand. our vision is to improve healthcare communications and leverage social media in thailand and other developing countries, particularly for patients with lower levels of education or socioeconomic status. we created a cloud-based personal healthcare information platform 'meid' that uses a qr code scanned from wristbands and other products like stickers to access patient information. conventional methods require a treating team to request medical records from a patients' prior hospital visits including visits at different medical facilities. time lost during this process can potentially cause delay in treatment decisions. we also aim to improve health literacy in thailand. application name 'meidth' is available in both apple store and google play. we launched meid in thailand in april of . we have more than , active users and have sold more than wristbands. the meid thailand facebook page has received , likes. there are at least two patients that have already benefited from this product: one of these patients received intravenous tissue plasminogen and had a good outcome. timely access to his past medical history and medications via meid was a key in this case. our cloud based personal healthcare information platform using qr codes from wristbands and stickers can help increase health literacy, decrease times to appropriate treatment, improve patient safety and decrease healthcare costs. clinical pharmacists have become an integral part of multidisciplinary medical teams. expanding the role of pharmacists in the neurocritical care units has the potential to positively impact the quality of patient care and provide costs savings. this study examines these potential benefits at one neurocritical care unit. we reviewed patient medication profiles and had formal rounds with a pharmacist four times per week. for the purposes of this study, the focus was on minimization of a select number of high expense drugs. nine months of baseline data was compared to three months of post intervention data. interventions were performed at the time of rounding, which involved timely conversion to enteral formulas, changes to alternative medications or discontinuation of medications. we then performed a cost-benefit analysis to assess the net amount of money saved by reducing inappropriate pharmacy drug use following the interventions. average cost of nicardipine was $ , pre-intervention, compared to $ , post-intervention (pvalue . ). the cost of iv levetiracetam usage on average was $ , pre-intervention and $ , post-intervention (p-value . ), while the cost of iv dexmedetomidine was $ pre-intervention compared to $ post-intervention (p-value . ). average expense per month was reduced by approximately $ , per month compared to the average expense per month from the previous months (p-value . ). appropriate use of stress ulcer prophylaxis was also positively impacted; patient days/month on famotidine was reduced by approximately % from baseline, patient days (pre-intervention) vs days post-intervention. pharmacist interventions within a neurocritical care unit are known to be beneficial clinically for patients, however this study also shows that their interventions offer substantial cost benefits and should justify creating collaborations between pharmacists and neurointensivists. multi-disciplinary rounds have been shown to improve patient outcomes. the objective of this study is to observe the effect on patient care, team dynamic, and nursing satisfaction before and after the implementation of a nursing-led rounding model in the neurological icu. prior to the implementation of the nursing-led rounds quality initiative, nurses in the neurointensive care unit (nicu) were asked to answer a brief survey on basic demographics and perceptions of team dynamics and satisfaction in the nicu. a multidisciplinary systems-based rounding sheet inclusive of the abcdef bundle and previous nicu checklist was created and revised with extensive bedside and senior nursing educator input. while rounding, nurses presented and clinicians were to in real-time come up with an assessment and plan and relay these to the nurse and other team members. any questions, educational pearls or concerns by the clinician team or the bedside nurse were encouraged during these rounds. nurses completed a month post-implementation survey. of the full-time nicu nurses ( %) responded to both the pre-implementation and postimplementation surveys. a bimodal distribution of nursing experience was noted with % new nurses (< year) and % experienced nurses ( years+). more than half of the nurses ( %) reported doing both night and day shifts as opposed to being exclusive to only day or night. there was an increase in the nursing perceptions of participation during rounds as well as education during rounds. nurses felt significantly more involved with patient decision making and felt that they were able to give input into the patients care. the implementation of a nursing-led rounding structure may be beneficial to communication, education and overall patient care. as the project continues, we hope to further examine common icu objective measures as well as other subjective measures such as patient satisfaction scores and communication perceptions. with increased elective and non-elective volume, directing the flow of admissions has become essential to the efficient operation of inpatient strategic service lines. this is especially true in the neurosciences where widespread acute ischemic stroke intervention has placed an especially high demand on comprehensive stroke centers. as a result, an important collaboration was formed at duke between the health system, transfer center and neurosciences to create an algorithm-driven multi-hospital triage and pre-hospital care system called phast (pre-hospital acute services team). in this abstract, we present the formation and current state of this service. this effort was formally begun in the spring of with an initial focus on centralizing the admission process into the duke neurosciences intensive care unit (neuroicu) by an icu physician. after some initial success, it was clear that the service line would benefit from a more formalized process. as a result, a successful multidisciplinary collaboration with a core group of physicians and administrators was formed to develop algorithms and to overcome multiple administrative and legal hurdles. over a period of months, multiple algorithms were developed to systematize neuroscience admissions including acute ischemic stroke and vascular and non-vascular neuroscience emergencies. in an effort to decrease door-to-intervention times as well as effectively mitigate the impact of limited bed-space availability, this system now serves hospitals including with acute neurointerventional services and the rd with a burgeoning neurohospitalist program and incorporated rehabilitation services. in addition to systematizing the transfer and admission process, quality assurance, improvement, and educational processes are in a place. the current state of phast is that of a young but maturing and now essential service for duke neurosciences. extubation involves removal of an endotracheal tube (ett) and is a common intensive care unit (icu) procedure. extubation failure occurs in - % of icu patients and can be difficult to predict accurately. we hypothesized that a multivariate re-intubation scale calculation (risc) model could predict extubation failure better than a single variable like rapid shallow breathing index (rsbi). after irb approval, we conducted a retrospective review of data on mechanically ventilated icu patients above years of age who were not receiving mechanical ventilation through a tracheostomy tube from january , , through december , at mayo clinic rochester. various data points were gathered on these patients via electronic medical records search, and reintubations within hours of extubation were identified. univariate and multivariate logistic regression models were used to predict reintubation after extubation and construct a risc estimate. we included a total of patients which were randomly divided into a derivation set (n= ) and validation set (n= ). in the derivation set, patients had a mean age of ± years, and % were men. three hundred and ninety three extubation failures occurred within hours. predictors of extubation failure included underweight status, gcs score>= , mean airway pressure at minute= ml and total mechanical ventilation days>= in the final multivariable model. risc score was calculated using the validation set and ranged from to . logistic model result shows that, as risc increased by , the odds of having extubation failure was . fold higher (c-index= . ). roc analysis shows that the best cut off for risc was >= vs. < , which demonstrated a sensitivity of . , specificity of . and auc= . . the current risc model warrants further exploration in a prospective study to help critical care providers to decide when extubation can be done more safely. this report presents results of the nd nationwide survey concerning neurocritical care units (ncus) in china. this is an observational cross-sectional survey and close-ended self-reported questions were used. the questionnaire was sent to different provinces (autonomous regions and municipalities) across china from october st, to january st, . basic information, equipment and device information, and staffing and organization information were investigated. in total, questionnaires from ncus at hospitals in regions were received. most of the hospitals with ncus were large-scale (average hospital beds: ), teaching ( . %), and tertiary hospitals ( . %). the average number of ncu beds was , occupying . % of the total number of beds in their department. most of the equipment and devices ( / ) were available in over % of the ncus. however, some devices were centralized by hospital and operated with assistance from other departments. a total of full-time doctors and full-time nurses were employed at the ncus. a few of the ncus achieved a doctor-to-bed ratio of . : ( . %) and a nurse-to-bed ratio of : ( . %). and respiratory therapists, clinical dieticians, clinical pharmacists, and physiotherapists were present in . %, . %, . % and . % of the ncus. the number of ncus increased, the availability of ncu equipment became more sufficient, and the staffing of ncus improved. however, we should pay attention to the management of specialized ncu equipment, the shortage of ncu staff, and the need of ncu training. automated devices collecting quantitative measurements of pupil size and reactivity are increasingly used for critically ill patients with neurologic disease. however, there is limited data on the effect of ambient light conditions on pupil metrics. to address this issue, we we tested the range of pupil reactivity in healthy volunteers in both light and dark conditions. we measured quantitative pupil size and reactivity in seven healthy volunteers with the neuroptics- pupillometer in both bright and dark ambient lighting conditions. bright conditions were created by overhead led lighting in a room with ample natural light. dark conditions consisted of a windowless room with no overhead light source. the primary outcome was the neurologic pupil index (npi), a composite metric ranging from - in which > is considered normal. secondary outcomes included resting and constricted pupil size, change in pupil size, constriction velocity, dilation velocity and latency. results were analyzed with multi-level linear regression to account for both inter and intra-subject variability. seven subjects underwent ten pupil-readings in bright and dark conditions, yielding total measurements. mean resting pupil sizes were . v. [ . - . ], p< . ). all additional secondary outcomes except latency were also significantly different between conditions. we found that ambient light levels impact pupil parameters in healthy subjects. however, changes in npi are small and more consistent in varying lighting conditions than other metrics. further testing of patients with poor pupil reactivity is necessary to determine if ambient light conditions could influence clinical assessment in the critically ill. practitioners should standardize lighting conditions to maximize the reliability of their measurements. neural stem cells (nscs) are known to have anti-inflammatory effect in strokes in previous studies. however, the mechanism of anti-inflammatory effect in direct co-culture with nscs in hemorrhagic stroke remains unclear. the aim of this study was to investigate whether direct co-culture with nscs modulates hemolysate-induced inflammation in raw . cells. we stimulated raw . cells with hemolysate to induce hemorrhagic inflammation in vitro. hemolysate-activated raw . cells were co-cultured with hb .f directly for hours. following direct co-culture, the production of cycloxygenase- (cox- ), interleukin-signal regulated kinase (erk) were assessed by western blotting, and tumor necrosis factor (tnfevaluated by enzyme-linked immunosorbent assay (elisa). hemolysate generates an activation of inflammatory response in raw . cells. direct co-culture with hb .f significantly suppressed the phosphorylation of erk / in hemolysate-activated raw . cells. the expression of inflammatory mediators such as cox- , il-by direct co-culture with hb . f cell. in addition, the expression of cox- , il-attenuated by erk inhibitor (u ). our results demonstrated that direct co-culture with hb .f cells reduced the inflammatory responses in hemolysate-activated inflammation via suppressing erk / pathway. this suggests that nscs treatment can suppress the inflammatory response in hemorrhagic stroke. no pharmacological intervention improves outcomes after primary intracerebral hemorrhage (ich). we developed a novel therapeutic approach based on known biological function of endogenous apolipoprotein e (apoe). apoe is a key mediator of neuroinflammatory responses and modifies recovery from a variety of acute and chronic brain injuries. unfortunately, intact apoe holoprotein does not cross the blood brain barrier (bbb) and cannot be administered as a neurotherapeutic. we created apoemimetic peptides that cross the bbb and down-regulate neuroinflammatory responses in vitro and in vivo. cn- , our lead candidate, is a -amino acid apoe-mimetic peptide derived from apoe's receptorregion. cn- retains anti-inflammatory and neuroprotective effects of intact apoe, was well-tolerated in preclinical studies, readily crosses the bbb, and demonstrates excellent pharmacokinetic, safety, and tolerability profiles in phase studies. this is a multicenter, open-label phase a trial of cn- in patients with acute primary supratentorial ich. a total of participants between the ages and years across study centers, with a confirmed radiographic diagnosis of spontaneous, primary supratentorial ich. patients will be evaluated for eligibility within hours of symptom onset. eligible participants will receive cn- intravenously over --minute infusion every hours up to day maximum. participants will be monitored daily throughout the treatment phase and receive standard-of-care treatment for the duration of the study. primary: to assess safety of cn- administration in primary ich. secondary: to evaluate effects of cn- administration on --day mortality and functional outcomes. exploratory: to investigate feasibility of radiographic surrogates of clinical outcomes using perihematomal edema measurements on serial brain ct and mri, and investigate feasibility of serial biochemical markers of neuroinflammation as surrogate measure of perihematomal edema and clinical outcome. cn- represents a first-in-class agent now entering phase clinical trials in patients with acute ich. novel oral anticoagulant (noac) associated intracranial hemorrhage is a life-threatening condition for which activated prothrombin complex concentrate factor eight inhibitor bypassing activity (feiba) may be used for reversal. few studies report its use in spontaneous or traumatic intracranial hemorrhage. our institutional protocol is reversal with feiba units/kg and escalating doses as needed. the safety and efficacy of this protocol was assessed. we performed a retrospective review of adult patients presenting to a level trauma center between - with spontaneous or traumatic intracranial hemorrhage while on a noac . we evaluated the medication they presented on, indication for anticoagulation, location and size of the hemorrhage, presentation gcs, dosage of feiba recieved, change in size of hemorrhage on serial imaging as well as time between serial images, complications from reversal, and need for blood product transfusion. we identified patients with an acute intracranial hemorrhage while on noacs. patients underwent a baseline head ct documenting acute intracranial blood, were reversed with feiba ( u/kg), and underwent repeat imaging hours later per protocol. ten ( %, / ) patients had no increase in hematoma volume on repeat imaging. two underwent neurosurgical procedures (aneurysm coiling, sub-occipital craniectomy) without intra-operative bleeding complications. five ( % / ) patients had clinically insignificant increase in size of hemorrhage. of those, one underwent a subsequent neurosurgical procedure, which was already anticipated. two ( %, / ) patients had clinically significant hematoma enlargement. of those, one underwent urgent craniectomy (indicated based on initial presentation) and one required a ventriculostomy for hydrocephalus. two patients had no repeat imaging. adjusted dose feiba ( units/kg) may be an effective alternative to standard dose ( unit/kg) for reversal of noacs in acute intracranial hemorrhage. our experience showed clinically significant hematoma expansion in % of patients and no increase in unplanned neurosurgical procedures after reversal with feiba. here we sought to determine if there is an association between recanalization success and rate of hemorrhagic transformation amongst patients who have undergone intra-arterial thrombectomy for ischemic stroke secondary to anterior circulation large vessel occlusion (lvo), many treated at extended time from last seen well (lsw) after mri assessment. stroke patients with anterior circulation lvo treated with thrombectomy between april, to june, were studied. group-wise comparisons were made between patients with post thrombectomy hemorrhage (as confirmed by a single, blinded neuro-radiologist reviewer) and patients without hemorrhage. failed or incomplete recanalization was defined as mtici < b. symptomatic intracranial hemorrhage (sich) was defined as validated hemorrhagic conversion or parenchymal hematoma plus point decrease in nihsss. pertinent baseline characteristics were recorded and analyzed. sich was more prevalent amongst patients with tici< b recanalization (or . [ ci . - . ]). interestingly there was a low rate of sich amongst patients with tici= recanalization ( / [ . %]). although many patients were treated at advanced time lsw no excess rate of sich was observed. baseline characteristics including age, presentation nihss, and presentation aspects were similar among the two groups. rates of sich are low after successful mri seleted thrombectomy regardless ot time lsw. patients with poor recanalization show increased rates of sich in keeping with past literature. our data suggest that thrombectomy after mri selection may be safe and effective for patients at extended time lsw of tor patients with unknown lsw. cta spot sign is associated with hematoma growth, a common complication of intracerebral hemorrhage (ich) that portends worse outcomes. magnesium and calcium are cofactors in the clotting cascade and for platelet aggregation. we tested the hypothesis that magnesium and calcium levels are associated with the presence of the cta spot sign. patients with spontaneous ich presenting to northwestern memorial hospital were identified from a prospective observational registry. inclusion criteria included cta obtained within hours of symptoms onset and admission magnesium and calcium levels. cta spot sign (active contrast extravasation on ct angiography) was identified by a board-certified neurointensivist or neuroradiologist. variables suggesting association with spot sign at p< . were assessed for inclusion in a logistic regression model, and a parsimonious predictive model for ct spot sign was developed using backward stepwise variable selection. patients (age ± . years, % male, median ich score [iqr - ]) were included. seventeen ( . %) patients with cta spot sign were identified. admission magnesium was . +/- . and calcium was . +/- . . lower magnesium (or . , % ci . - . , p . ), lower calcium (or . , % ci . - . , p . ), and higher ich score (or . , % ci . - . , p . ) were independently associated with ct spot sign. magnesium and calcium level on admission are associated with the presence of a cta spot sign in patients with ich. magnesium and calcium supplementation may be attractive therapeutic targets for preventing harm from hematoma growth. cerebellar intraparenchymal hemorrhage (iph) is a rare and likely underreported complication of subdural hematoma (sdh) evacuation. we present two cases of post-operative iph and review the literature. case . an -year-old man underwent craniotomy for evacuation of a chronic right frontoparietal sdh. post-op ct showed pneumocephalus. the patient was extubated and clinically improved. three days post-operatively, he became lethargic and a ct brain revealed a cc right cerebellar iph. he was unable to safely swallow, declined a feeding tube and died under hospice care nine days later. case . a year-old man underwent craniotomy for evacuation for a left convexity sdh. routine post-op ct revealed an incidental left cerebellar iph. he returned to baseline one month later. only four such cases have been reported in the literature ( - ). two cases led to death within one week and two recovered, one with significant deficits. five more occurred following burr hole drainage of sdh and two others following drainage of subdural hygromas ( , - ). the incidence of cerebellar iph following supratentorial craniotomy has been reported in up to . % of cases with significant morbidity or mortality ( ). it occurs irrespective of age, pre-existing coagulopathy or arteriovenous malformations. size of insult and amount of csf loss do not correlate to iph, despite the fact that cerebral blood flow imaging shows over-drainage of cerebrospinal fluid (csf), causes intracranial hypotension and subsequent damage to dural veins ( , ). iph also occurs independently of operating room position, even though having the head turned is thought to compress venous drainage in the neck and cause congestion ( ). cerebellar vasculature may be more sensitive to changes in intracranial pressure, though why this does not lead to complications more routinely is not clear. cerebellar iph should be considered in cases of neurological decline after sdh evacuation. intracerebral hemorrhage (ich) location predicts outcome, but most studies have examined differences between deep, lobar, and infratentorial locations. this study aims to characterize specific deep ich locations in a diverse cohort. the ethnic/racial variations of intracerebral hemorrhage (erich) study is a multi-center, prospective, u.s.-based study. subjects with supratentorial deep ich, known ich volume, and three-month follow-up data were included. logistic regression was used to evaluate the association between location and poor outcome (mrs > ). receiver operating curve (roc) analysis was performed to identify ich volumes specific for poor outcome by location. thalamic, putaminal, and caudate ichs were included. median ich volume was largest in putamen ( ml), followed by thalamus ( ml) and caudate ( ml, p<. ). intraventricular hemorrhage (ivh) was most prevalent in caudate ( %), followed by thalamus ( %) and putamen ( %, p < . ). subjects with thalamic ich were older ( vs vs years, p < . ) and more likely hypertensive ( % vs % vs %, p= . ) than those with putaminal and caudate ich, respectively. compared to thalamic, putaminal ich had more ich expansion ( % vs %, p < . ) and surgery ( % vs % p = . ) but fewer external ventricular drains ( % vs %, p < . ). thalamic location predicted poor outcome (or . , % ci . - . ) at days after adjustment for age, sex, premorbid disability, ich volume, ich expansion, ivh, and admission gcs. roc analysis identified ml for thalamic and ml for putaminal ich without ivh as having % specificity for poor outcome. there are significant differences in characteristics and outcomes within deep ich. specificity estimates for the identified ich volume thresholds require external validation. these findings may have implications for prognostication and clinical trial design. racial differences in outcome after intracerebral hemorrhage (ich) among asians, native hawaiians and other pacific islanders (nhopi) have been inadequately studied since these racial groups have been historically aggregated into a single racial category. a multiracial prospective cohort study of ich patients was conducted from to at a tertiary center in honolulu, hi to assess racial disparities in come after ich. favorable outcome was defined as month modified rankin scale (mrs) score £ . patients with no available -month functional outcome, race other than asians and nhopi, and baseline mrs > were excluded. multivariable analyses using logistic regression were performed to assess the impact of race on favorable outcome after adjusting for the ich score, early do-not-resuscitate (dnr) order and dementia/cognitive impairment. a total of patients ( asians, nhopi) were studied. overall, ( . %) achieved favorable outcome at months. nhopi were younger than asians ( . ± . vs. . ± . years respectively, p< . ), and had higher prevalence of diabetes ( . % vs. . % respectively, p= . ), obesity ( . % vs. . respectively, p< . ), and lower prevalence of early dnr order ( . % vs. . % respectively, p= . ) and advance directive presence ( . % vs. . % respectively, p= . ). nhopi race was a predictor of favorable outcome in the unadjusted model (or . , % ci: . , . ) and after adjusting for the ich score (or . , % ci: . , . ) but not in the final model (or . , % ci: . , . ) . in the final model, the ich score remained as the only independent negative predictor of outcome (or . , % ci: . , . per point). nhopi are more likely to achieve favorable functional outcome after ich compared to asians even after controlling for ich severity. however, this association was attenuated after adjusting for dnr status and baseline cognitive factors. intracerebral hemorrhage (ich) patients often require continuous antihypertensive infusions. we sought to identify clinical and care process predictors of anti-hypertensive infusion duration, and tested whether infusion duration independently predicts intensive care unit length of stay (icu los) after adjusting for validated measures of ich illness severity. we identified spontaneous ich patients admitted / - / to a tertiary center, excluding those transitioned to comfort care within hours. we abstracted demographic and clinical variables from the medical record. we calculated the total duration each patient received continuous infusion of an antihypertensive medication. we categorized glasgow coma scale score as - ; - ; or < . two reviewers independently classified ich location and etiology. we determined univariate associations of clinical variables to anti-hypertensive infusion and performed regression analysis to determine the effect of continuous infusion on icu los. we identified spontaneous ich patients and excluded for early comfort care. in the remaining , mean age was [ - ] years, % were female, median ich score was [ - ], and % had lobar hemorrhages. continuous infusion included nicardipine, clevidipine, labetalol and diltiazem. a total of ( %) patients received anti-hypertensive infusions, mean hours. mean time to enteral antihypertensive administration medication was . hours, and mean icu length of stay was . days [ . - . ]. predictors of longer antihypertensive infusion duration were male gender (p= . ), non-lobar ich (p= . ), non-caucasian race (p< . ), younger age (p< . ), higher initial systolic (p= . ) and diastolic bp (p= . ), worse gcs category (p< . ), and longer time to first enteral medication (p< . ). anti-hypertensive infusion duration independently predicted icu los (p< . ) after adjusting for age, race, gcs category, time to enteral antihypertensives, and ich score. worse gcs category, younger age, non-lobar ich location, and race are significant independent predictors continuous iv antihypertensive infusion duration, which is significantly associated with longer icu stay. patients with sich have a high risk of vte. pharmacological prophylaxis such as unfractionated heparin(ufh) has been demonstrated to reduce vte. however, published datasets exclude patients with recent ich out of concern for hematoma enlargement. aha/asa guidelines recommend ufh - days after hematoma stabilization while the eso has no recommendations on timing of ufh. there are few data for patients who received ufh before hours. our institutional practice is to begin ufh following sich after hours of clinical and radiographic stability. we examine the impact of this practice on risk of hematoma expansion. we performed a retrospective cohort analysis of sich patients admitted in - to a single us university hospital. demographic and clinical characteristics were abstracted. ich was measured via d volumetrics for an admission ct, a hour follow-up, and a follow-up prior to discharge. percent hematoma growth between -hour ct and discharge ct was calculated. risk factors for expansion > %, including early heparin use, were analyzed via oneway t-test and chi-squared tests. results sich patients analyzed had a median ich score of (iqr - ) and median admission gcs of (iqr - ). %( / ) patients received early ufh. %( / ) suffered hematoma expansion > %. overall mean hematoma growth was higher with early ufh (ufh hr - %,p= . ). in multivariate analysis, ich score, gcs and initial hematoma size did not predict > % hematoma expansion. early vte prophylaxis at hours from sich had a statistically significant increase in hematoma size, but this increase is clinically insignificant. in this cohort, early ufh did not increase risk of significant hematoma expansion. further prospective trials are warranted, given the high risk of vte in this population. antiplatelet therapy at the time of spontaneous intracerebral hemorrhage (sich) may increase the risk of hemorrhage expansion and mortality. current guidelines recommend consideration of a single dose of desmopressin in sich associated with cyclooxygenase- inhibitors or adenosine diphosphate receptor inhibitors. this study sought to compare outcomes in patients that received desmopressin for antiplatelet reversal in the setting of sich to similar patients that did not receive desmopressin. this retrospective chart review of the electronic medical record included adult patients admitted for sich that were on antiplatelet agents at the time of diagnosis. patients that received desmopressin were compared to similar patients that did not receive desmopressin. exclusion criteria included traumatic brain injury, active coagulopathy and thrombocytopenia. the primary outcome was the incidence of hematoma expansion. additional outcomes included average increase in hematoma volume, in-hospital mortality and functional outcome at hospital discharge. overall, patients ( received desmopressin, did not receive desmopressin) were included for analysis. incidence of hematoma expansion was not different between groups ( % with desmopressin vs % without desmopressin, p= . ). average largest increase in hematoma volume on follow-up imaging from baseline was not different ( . ± . ml with desmopressin vs . ± . ml without desmopressin, p= . . in-hospital mortality was significantly higher in the desmopressin group ( % vs % without desmopressin, p= . ) as well as the incidence of a modified rankin score of - at discharge ( % vs % without desmopressin, p= . ). administration of desmopressin for antiplatelet reversal in sich does not appear to reduce the incidence of hematoma expansion. further studies assessing temporal relation of desmopressin administration and hematoma expansion are needed to confirm the results of this single-center retrospective study. clinical outcome after intracerebral hemorrhage (ich) remains poor. definitive phase- trials in ich have failed to demonstrate improved outcomes with intensive systolic blood pressure (bp) lowering.we sought to determine whether other bp parameters-diastolic bp, pulse pressure (pp), and mean arterial pressure (map)-showed an association with clinical outcome in ich. we retrospectively analyzed a prospective cohort of patients with spontaneous ich and documented demographic characteristics, stroke severity, and neuroimaging parameters. consecutive hourlybp recordings allowed for computation of systolic bp, diastolic bp, pp, and map. threshold bp values that transitioned patients from survival to death were determined from roc curves. using inhospital mortality as outcome, bp parameters were evaluated with multivariable logistic regression analysis. patients who died during hospitalization had higher mean pp compared to survivors ( . ± . mmhg vs. . ± . mmhg; p= . ). the following admission variables were associated with significantly higher in-hospital mortality (p < . ): poorer admission clinical condition, intraventricular hemorrhage, and increased admission normalized hematoma volume. roc analysis showed that mean pp dichotomized at . mmhg, provided a transition point that maximized sensitivity and specific for mortality. the association of this increased dichotomized pp with higher in-hospital mortality was maintained in multivariable logistic regression analysis (or . ; %ci . - . ; p < . ) adjusting for potential confounders. widened pp may be an independent predictor for higher mortality in ich. this association requires further study. a national confidential enquiry into patient outcome and death (ncepod) report concerning management of aneurysmal subarachnoid haemorrhages in the uk suggested up to half of patients received suboptimal consideration for organ donation. as demand for organs continues to increase, so does the need to pursue all potential sources of donor organs. subarachnoid haemorrhages have an estimated mortality of % and can potentially provide younger donor organs with less chronic pathology. this is a comprehensive picture of donation rates within a tertiary centre. retrospective data regarding all deceased patients on the neuro-intensive care unit during with aneurysmal subarachnoid haemorrhage as the cause of death was obtained from the nhs blood and transfusion team. the local audit committee provided ethical approval. data regarding organ donation was extracted and compared to national data, then analysed using fisher's exact test. referral rates were %. this is greater than the national average of . % (p= . ), yet only . % of referred patients proceeded to organ donation. consent was withheld in . % of potential donors. nationally . % of donors are lost due to non-consent (p= . ). . % of consented patients were unable to donate organs, similar to national figures (p= . ). referral rates within this centre are excellent; consent remains the main obstacle. consent rates can be improved using a long contact model where specialist nurses in organ donation establish relationships with relatives prior to any discussion of donation. the ideal discussion is a pre-planned collaboration involving a senior doctor and a specialist nurse. early brain stem testing may facilitate earlier acceptance of death by relatives whilst reducing the duration of the multi-systemic effects of the associated hyperresponsive cascade on donor organs. neurosurgeons should be encouraged to suggest organ donation when declining referrals. further work is needed to assess the barriers to instituting these measures and inspiring change. spontaneous brainstem hemorrhage has been historically associated with high mortality. however, updated data on the frequency and outcome of spontaneous brainstem hemorrhage is scarce vis-a-vis advances in neuro-critical care. the purpose of this study was to investigate the frequency and outcome of spontaneous brainstem hemorrhage. records of consecutive intracerebral hemorrhage (ich) patients presenting to an urban academic medical center from january though december were reviewed. cases with brainstem hematomas were isolated for analysis. data on demographics and outcomes were collected and analyzed. sub-group analysis was also done to look at outcomes based on location of hemorrhage in the brainstem. of consecutive spontaneous ich patients, ( . %) presented with brainstem hemorrhage; ( . %) were pontine, ( . %) mesencephalic, and ( . %) were located in both the pons and the midbrain. the average age was . years and ( . %) were men. median glasgow coma scale on presentation was . . thirty-day mortality rate was . %, with in-hospital deaths and deaths post discharge. two and patients were discharged home or a rehabilitation facility, respectively. in subgroup analysis, thirty-day mortality for midbrain, pons and combined pons/midbrain hemorrhage was %, % and %, respectively. spontaneous brainstem hemorrhage remains an uncommon but highly fatal clinical entity. more than one-half die within days. only a minority are discharged to rehabilitation or home. in sub-group analysis, location of brainstem hemorrhage was shown to influence outcome, with % mortality in case of combined pons/midbrain hemorrhage, and more than % mortality with pontine hemorrhage. midbrain hemorrhage was associated with good outcome with % survival. patients with intracerebral hemorrhage (ich) frequently present with hypertension. it is unclear whether this is due to preexisting hypertension (prhtn) causing the bleed, an effect of the bleed, or both. we retrospectively analyzed a single-institution cohort of ich patients presenting between and . data included home antihypertensive use; asbp; tte, and ekg and imaging results; and nicardipine administration. the primary objective was to assess the relationship between prhtn and asbp, while the secondary objectives were to assess the relationship between prhtn, imaging and acute antihypertensive requirements. ich patients met inclusion criteria. in our assessment for prhtn, we found that % of patients were on antihypertensives, % had lvh on ekg, and % had lvh on tte. there was a significant relationship between lvh on tte and lvh on ekg (p< . ), but not between home antihypertensive use and presence of lvh using either modality. asbp was higher for all patients with markers of phtn, but this was only significant for patients with lvh on tte ( mmhg, iqr - vs. mmhg, iqr - , p < . ) and patients with lvh on ekg ( mm hg, iqr - vs. mm hg, iqr - , p< . ). all patients with markers of prhtn were more likely to require nicardipine, but this was only significant for patients with lvh on tte ( % vs. %, p= . ) and patients with lvh on ekg ( % vs. %, p= . ). all patients with markers of prhtn were more likely to have deep bleeds (p= . for patients with lvh on ekg vs. those without lvh on ekg). there was no relationship between any markers of prhtn and the presence of a spot sign. in patients with ich, prhtn is related to higher asbp, deep bleed location, and increased acute antihypertensive requirements. all spontaneous intracerebral hemorrhage (sich) patients, including those with low severity are uniformly admitted to the intensive care unit (icu) at our institution. many may not benefit from this high-intensity observation and leave the icu within hours without experiencing any complications. identifying low-risk characteristics could aid in triaging such patients to stroke units instead. retrospective data collection of all sich patients admitted to our institution from june , -june , included ich score, need for surgical interventions, medical complications, and icu/hospital los. we analyzed variables predicting short (< hour) icu los among low severity (ls-ich) patients (defined as those with ich score - ). ( %) of sich patients had ich scores of - , of which just under half ( ) had icu los hr. they also spent significantly fewer days in hospital ( vs . , p< . ). we could not identify a clear ich score cutoff that was sensitive enough to predict short icu los. however, requirement for antihypertensive infusion and early clinical deterioration correlated strongly with longer icu los p< . . there appears to be a subset of mild ich patients (ich score - ) who do not require icu observation. a risk assessment score incorporating gcs and ich volume may be able to delineate this low-risk population who could instead be admitted to a stroke unit, with the potential for significant cost saving and hospital efficiency. obesity has been linked with relative longevity in several disease conditions. this relationship has been termed the "obesity paradox." in this study we sought to evaluate the impact of obesity on short-term outcomes in patients with intracerebral hemorrhage (ich). patients admitted with a diagnosis of ich were selected from the - nationwide inpatient sample (nis) database, using icd- codes. patients with ich were dichotomized based on the presence of obesity as a coexisting diagnosis based on icd- codes and diagnosis related groups. the primary outcome measure was in-hospital mortality. length of stay and total charges were also examined as resource utilization measures. of obesity is a major health care burden as evidenced by higher resources utilization. counterintuitively, obesity appears to be associated with lower in-hospital mortality in ich patients. one possible physiological basis for this could be that the higher ldl levels on presentation result in a lower likelihood of hematoma expansion. recent short-term outcome analysis indicates association of spontaneous intraventricular hemorrhage (ivh) related hydrocephalus with incontinence and gait dysfunction. we explore the association of hydrocephalus scores, intraventricular alteplase and clinical variables with these outcomes at long term follow up in survivors from the clear iii trial. clear iii, a randomized, multi-center, double-blinded, placebo-controlled trial was conducted to determine if pragmatically employed external ventricular drainage (evd) plus intraventricular alteplase improved outcome, in comparison to evd plus saline in patients with ivh causing obstructive hydrocephalus. we assessed hydrocephalus scores on survivors at diagnosis, days and . incontinence and dysmobility were defined using -month barthel index subscores (< for bladder and < for mobility, respectively). outcome measures were predictors of incontinence and gait dysmobility at year after ich. this prospective observational study analyzed consecutive ich-patients (n= ) treated at the neurological and neurosurgical departments of the university-hospital erlangen, germany over a month inclusion period ( / - / ). we analyzed the influence of patient characteristics, inhospital measures and functional status on treatment recommendations and on oac initiation during -month follow-up. clear treatment recommendations by attending stroke physicians seem necessary to ensure oac initiation after ich. oac showed beneficial associations; however data here suggests the presence of an indication bias introduced by treatment recommendations and outpatient care during follow-up. therefore, observed association with age and functional status might affect unadjusted analyses. although recently, non-vitamin k antagonist oral anticoagulants (noacs) therapy in patients with non valvular atrial fibrillation have half the incidence of intracerebral hemorrhage (ich) compared to warfarin. however, it would be still controversial subject that outcome of noac-associated ich (nich) might be worse or better than warfarin-associated ich (wich). in this study, we investigated clinical outcome and radiological finding of ich between two different anticoagulation treatments. retrospective review of medical records was performed for , patients who admitted with ich from to in seoul national university bundang hospital. clinical characteristics, functional outcome, location and volume of ich, and all-cause mortality within days were analyzed. among those patients, patients with wich and patients with nich were included. lesion location was common in supratentorial deep area ( . %, . %), lobar area ( . %, . %) and brainstem and cerebellum ( . %, . %) in the nich and wich group, respectively. no significant difference found in initial nihss ( . vs ), discharge nihss ( . vs ), mrs ( to ) at discharge ( . % vs . %), mrs ( to ) at discharge ( . % vs . ), mrs ( to ) at days ( . % vs . ) and mrs ( to ) at days ( . % vs . ) in nich and wich group. we did not find any difference between nich and wich for allcause mortality at discharge ( % vs %), days ( . % vs %), and year ( % vs %). median baseline ich volume was not significant difference in two groups ( . vs . ). in our study, functional outcome, mortality, and baseline ich volume were similar following nich and wich. because of low statistical power due to small sample size in our study, further studies with prospective larger patient cohorts will need to be conducted. novel oral anticoagulants (noac) are increasingly used as an alternative to vitamin-k antagonists (vka) such as warfarin for anticoagulation and have shown lower rates of intracranial hemorrhage in several randomized clinical trials. it has been suggested that noac-iphs might be particularly dangerous, yet the literature regarding hematoma characteristics and outcomes between noac-iphs and vka-iphs is inconclusive. given the lack of standardized reversal strategies and lack of information on outcomes following noac-associated iph, the aim of this meta-analysis was to compare ) mortality; ) hematoma volume, and ) risk of hematoma expansion in patients who developed an iph on noacs versus vka. a meta-analysis of the literature through december was conducted using pubmed, embase and cochrane databases in accordance with prisma guidelines. pooled risk ratios (rr) were calculated for mortality and hematoma expansion and pooled mean difference (md) was calculated for hematoma volume (ml) using random-effect (re) and fixed-effect (fe) models. noac-iph was not associated with increased mortality (re and fe: rr: . ; %-ci: . ; . , i = . %, p-heterogeneity= . ; studies) and hematoma expansion (re and fe: rr: . ; %-ci: . ; . , i = . %, p-heterogeneity= . ; studies) compared to vka-iph. the hematoma volume of noac-iph was smaller than vka-iph (re: md: - . ml; %-ci: - . ; - . , fe: md: - . ml; %-ci: - . ; - . ; studies), but with considerable heterogeneity that could not be alleviated (i = . %, p-heterogeneity . ). noac-iph was not associated with increased mortality or hematoma expansion compared to vka-iph and may be associated with a smaller hematoma volume. controversy exists regarding blood pressure (bp) reduction and perihematomal ischemia (phi). we investigated the association of acute bp reduction and presence of qualitative and quantitative phi in a large prospective cohort of intracerebral hemorrhage (ich). consecutive patients from the prospective nih funded dash study (> years, primary spontaneous ich) were included. phe volume was outlined on t /flair and ich volume on gre; these and adc were co-registered. tissue characteristics was defined as: ce = adc x - mm /sec. the association of clinical, radiographic factors and bp at baseline and hours with qualitative perihematomal and/or remote ischemia (i.e. dwi bright adc dark) and quantitative ce on adc were determined. patients ( % female) with mean age ± , and nihss (iqr , ) were included. mri time was . hours (iqr , ). % had lobar ich. ich volume was cc (iqr , ). % had perihematomal ( %) or remote ischemia ( %). % of patients had areas of perihematomal adc cc) was associated with higher absolute ( ± mm hg, p= . ) and relative ( % ± % vs % ± %, p= . ) map reduction, younger age (p= . ), h/o tia/stroke (p= . ) and larger ich volumes ( vs cc) (p< . ). in multivariate analysis, map reduction was not significantly associated with ce whereas ich volume was (p= . ). perihematomal and remote ischemia is frequently seen after ich, but the severity of phi is small and of unclear significance. bp reduction may be associated with phi but this was not an independent predictor. introduction: patients with left ventricular assist devices (lvads) receive anticoagulation and antiplatelet therapy to prevent pump thrombosis. consequently, neurological events including intracranial hemorrhage (ich) are one of the most feared causes of morbidity and mortality in these patients. there is little evidence to guide initiation of anticoagulation after such ich events. methods: this is a retrospective, single academic center analysis of lvad patients from - . the electronic medical record was reviewed after irb approval for the physiologic, laboratory, and radiographic data of these patients as well as survival or cause of death by days or by discharge. results: during the analysis, patients were reviewed, of which ( . %) had intracranial hemorrhage. one patient was excluded from analysis after care was transitioned to hospice, thus follow-up scans were not obtained. the remaining patients were receiving both aspirin ( - mg daily) and warfarin ( - mg daily) with an inr of . - . (mean= . ) at the time of ich. aspirin ( - mg daily) was resumed within - (mean= . ) days post ich. warfarin was resumed - (mean= . ) days post ich at - mg (mean= mg) with goal inr ( . - )-( - ) depending on device. there was death due to withdrawal of life support in setting of multiple comorbidities, though follow-up scan days post warfarin resumption revealed no evidence of rebleed. the remaining patients showed no evidence of rebleed on ct scans at months post warfarin resumption and were subsequently discharged to rehab facilities or home with modified rankin scores - (mean= . ). conclusion: in this review of lvad patients, about % suffered ich, and of those survivors aspirin was safely resumed within days and warfarin was safely resumed as early as days post-event. further studies are needed in order to establish safe practice guidelines and risk factors to prevent ich. intracerebral hemorrhage (ich) remains a devastating form of stroke, and perihematomal edema worsen outcomes after ich. recent studies have demonstrated the safety of minimally invasive surgery (mis) for hematoma removal, but the efficacy of mis in the treatment of ich is controversial. this study aimed to evaluate the effect of mis compared with medical treatment for basal ganglia ich. we retrospectively analyzed the clinical outcomes of prospectively collected data from two stroke centers. the treatment strategies of the two stroke centers for basal ganglia ich are different; one stroke center underwent mis and the other stroke center medically treated according to the current guidelines. we hypothesized that mis could reduce perihematomal edema and improve functional outcomes compared to medical treatment. primary outcome of this study was a modified rankin scale (mrs) at months after ich occurrence. a total of patients with basal ganglia ich were treated with different treatment strategies; patients underwent mis and patients received medical treatment. no statistically significant differences were found in age, sex, hematoma volume, and glasgow coma scale scores between the groups. a better functional recovery (mrs < ) at months was found in the medical treatment group than the mis group ( . % vs . %, p < . ). no significant differences were observed between groups in terms of mortality. our findings suggest that the best medical treatment improves functional recovery after basal ganglia ich compared to mis. these results are contrary to other studies of ich, and further randomized trials are required. perihematomal edema (phe) after intracerebral hemorrhage (ich) is thought to be predominantly vasogenic. the presence and extent of cytotoxic edema (ce) is controversial. we investigated phe diffusivity (phed) and factors associated with ce. consecutive patients from the prospective nih funded dash study (> years, primary spontaneous ich) were included. phe volume was outlined on t /flair and ich volume on gre; these and adc were co-registered. tissue characteristics was defined as: ce = adc x - mm /sec. clinical and radiographic factors associated with ce were determined. cytotoxic edema is detected in the perihematomal area, early after ich and is associated with younger age, larger ich and prior h/o tia/stroke. its clinical significance needs to be studied further. hemorrhagic stroke carries a high mortality rate and determining prognostic factors during initial presentation can aid redirecting intensive care unit (icu) management. we described the physiological profile and clinical outcomes of hemorrhagic stroke patients in a colombian icu. we retrospectively reviewed all hemorrhagic stroke patients admitted to our icu from - . clinical characteristics, outcomes, available laboratory values and hourly vital signs from the first hours in the icu were retrieved and analyzed. our primary stroke center admitted patients, ( %) were hemorrhagic. out of these, required icu management, representing % of the total icu admissions during this time frame. intracerebral hemorrhage (ich) was present in patients while subarachnoid hemorrhage (sah) was seen in . the latter had a median fisher score of . for all patients, the most common risk factors were hypertension ( . %), dyslipidemia ( . %) and smoking ( . %). icu mortality was . % ( . % with ich and . % with sah). mean sequential organ failure assessment (sofa) score was significantly greater in patients who died ( . vs. . , p< . ) and mean glasgow coma scale was significantly lower ( . vs. . , p< . ). vasopressors were required in patients ( . %), mechanical ventilation in ( . %), and half of the patients requiring either support therapy died. only patients ( . %) had fever in the first hours and all died. mean coefficient of variation for systolic, diastolic and mean blood pressure was significantly lower in patients who survived. mortality cases were more likely to have hypokalemia and hypomagnesemia than surviving patients ( . % vs. . % and . % vs. %, respectively). icu-admitted hemorrhagic stroke patients have a poor prognosis. sofa and gcs are accurate predictors of mortality. certain electrolyte disturbances, fever and a higher variation of blood pressure during the first hours were associated with a worse outcome. the association between worsening cerebral edema and unfavorable outcome in ich patients has been described in rcts. the objective of this analysis was to compare hospitalized spontaneous ich patients with and without perihematomal edema (phe) expansion and to evaluate relationships between hypertonic saline (hts) use, peak serum na, phe expansion, and short-term outcomes. we conducted a cross-sectional study of consecutive spontaneous ich patients admitted to a single center from / - / . head cts during the first week of admission, use of hts, and phe (using abc/ method) were evaluated. phe expansion of % or more was considered worsening edema. outcomes of interest included time to peak na, poor disposition (not home or inpatient rehabilitation), discharge mrs - , and in-hospital death. of ich patients, % experienced worsening phe. there was no difference in age, race, sex, arrival bp arrival, or vascular risk factors in patients with or without worsening phe. however, for each mm of midline shift (mls) present on initial head ct, odds of phe expansion was decreased by % (or . , %ci . - . , p= . ). mls on initial head ct was the best discriminator of phe expansion (auc . ( %ci . - . ). although hampered by small sample size, our data indicates that finding that ich patients with degree of mls on initial head ct is the best radiographic predictor of had lower odds of phe expansion. those without mls at presentation may be at risk of phe expansion, and counterintuitively may be those most in need of aggressive medical management. may suggest a role for intensive osmotherapy in patients with favorable imaging at presentation. intracerebral hemorrhage (ich) is a devastating stroke with high mortality rates. previous studies have shown a potential role of immune cells as a prognostication method. a high neutrophil to lymphocyte ratio was associated with poor outcomes after ich. we sought to determine whether absolute lymphocyte count(alc) at admission was predictive of outcomes in patients with ich. we performed a retrospective chart review of all patients admitted to our hospital with a diagnosis of ich from january to december .we collected baseline demographic characteristics, medical history, ich scores, differential leucocyte, platelet and total leucocyte(tlc) counts at admission. the functional outcomes after ich were measured using modified rankin scale (mrs) at discharge. mrs of and were considered poor outcomes. statistical analysis was done after grouping lab values into higher and lower groups with respect to the normal reference ranges a total of patients with ich were admitted to our center during the study period. patients were included in the study and the rest were excluded due to lack of differential leucocyte counts at admission. % ( of ) had poor outcomes. univariate analysis using fisher's exact test showed significant association between low alc levels ( . ) were also found to be significantly associated with worse outcomes (p = . , . , . , respectively). however, after multivariate analysis, only low absolute lymphocyte counts retained significant association (p = . ). intracerebral hemorrhage patients with low absolute lymphocyte counts at admission have a higher probability of poor outcomes at discharge. further studies are required to confirm our results. intraventricular hemorrhage (ivh) is a significant predictor of poor outcome after intracerebral hemorrhage (ich), and may differentially predict hydrocephalus and mortality among blacks vs. nonblacks. we aimed to confirm these findings in a separate cohort of spontaneous ich patients with severe ivh. the cleariii-ivh trial was a randomized, multi-center placebo controlled trial examining the effect of intraventricular alteplase versus saline, on outcomes in patients with spontaneous ivh. we retrospectively analyzed data on all patients, including self-reported race/ethnicity, medical comorbidities, presentation characteristics and functional outcomes. represented race/ethnic groups with > subjects per group were ( . %) white/non-hispanic (wnh), ( . %) white/hispanic (wh), ( . %) black/african american/non-hispanic (bnh), and ( . %) asian. bnh were significantly younger than rest of the cohort with median age [interquartile range] [ , ] years, had more hypertension( %, p= . ), and significantly higher rates of antihypertensive medication non-compliance ( . %, p= . ). wnh had more frequent coronary artery disease ( . %, p< . ), use of vitamin k antagonists ( . %, p= . ) and elevated inr on presentation ( . %, p= . ). bnh had significantly more frequent hydrocephalus on presentation ( . %, p= . ), and a higher rate of ventriculoperitoneal shunt placement ( %, p= . ). neither ich nor ivh volume at enrollment, nor ivh remaining at end of treatment differed significantly between race/ethnic groups. however, bnh patients were more likely to have greater than % ivh reduction, a recognized endpoint for better functional outcomes in cleariii ( . % vs. %-wh; . %-wnh; . %-asian; p= . ), and this difference persisted in those who received intraventricular alteplase (p= . ) and after adjustment for diagnostic ivh volume (p= . ). race/ethnicity was not an independent predictor of mortality or poor outcome at or days on multivariable logistic regression. although functional outcomes did not differ significantly among race/ethnic groups, differences in risk factors, hydrocephalus/shunting post ivh and response to thrombolytic therapy warrant further exploration. investigators from the randomized trial of unruptured brain arteriovenous malformations (avm) trial (aruba) reported in that interventions to obliterate unruptured avms resulted in greater morbidity and mortality compared to medical management. we investigated whether patterns of avm treatment changed after aruba's publication. we used inpatient and outpatient claims data from - from a nationally representative % sample of medicare beneficiaries. unruptured brain avms were identified using icd- -cm code . . the date of first avm diagnosis was coded as occurring before or on november , (online publication of aruba) versus after. outcomes were referral to a neurologist or neurosurgeon, and interventional treatment. interventional treatments were identified using cpt codes - , - , , , or - . the likelihood of outcomes after versus before aruba was compared using survival analysis with log-rank tests and cox proportional hazards models adjusted for age, sex, race, and the charlson comorbidity index. we censored patients at diagnosis of intracranial hemorrhage. we identified , patients with a mean . (± . ) years of follow-up after diagnosis of unruptured brain avm. diagnosis was most often by neurologists ( . %), neurosurgeons ( . %), and internal medicine specialists ( . %). after aruba publication, there were no changes in -year cumulative rates of referral to a neurosurgeon ( . % after, . % before; p = . ) or neurologist ( . % after, . % before; p = . ), but there was an increase in avm treatment ( . % after, . % before; p = . ). after adjustment for demographics and comorbidities, there was an increased likelihood of interventional management (hr . ; % ci, . - . ) after aruba's publication. in a nationally representative cohort of elderly patients, we found an increase in interventional avm management after publication of aruba. this is notable given that our data pertain to older patients who are generally seen as less suitable surgical candidates. elderly patients with severe intracerebral hemorrhage (ich) are often projected to have future functional dependence but unclear degree of cognitive recovery. surrogates for such patients frequently weigh multiple concerns when facing the difficult decision of whether to prolong life with tracheostomy and gastrostomy tube insertion versus pursue comfort care. we aimed to characterize distinct groups of surrogates in these situations, based solely on how they prioritize their concerns. subjects recruited from a probability-based us population sample completed an online best-worst survey that presented the above scenario and asked the respondent to prioritize concerns as the patient's surrogate. clusters were identified with latent class analysis after weighting data to match the us census demographic distribution. class solutions were replicated times from random starting seeds, with the solution chosen after factoring in akaike's information criterion. we identified distinct decisional groups among respondents (response rate = . %). all groups reported multiple concerns as important, but group ( . %) was more concerned than any other that the patient was too old to live with disability. group ( . %) focused on ensuring agreement among other family members. group ( . %) was concerned that the patient might suffer if tube feeding and iv fluids were stopped and that the prognosis could be incorrect. group ( . %) had numerous considerations that were comparably important but prioritized paying for long-term care. groups varied in whether they would actually request prolonging care for the patient (group = . %, g = . %, g = . %, g = . %, p< . ). in a multivariate model, religious affiliation and frequency of attending religious services were the only variables independently predicting group membership. we identified distinct profiles of decisional patterns for surrogates of severe ich patients with uncertain prognosis. these data will inform development of strategically tailored decision aids. cerebral venous sinus thrombosis (cvst) represents an important cause of both ischemic and hemorrhagic strokes in young people. while recent guidelines recommend management in a stroke unit, the impact of neurocritical care in this condition has not been studied. we aimed to assess whether the introduction of a neurocritical care program influenced clinical outcomes in cvst patients. we retrospectively reviewed electronic medical records of adult patients admitted to yale new haven hospital's neuroscience icu (nicu) between and with a diagnosis of cvst. demographics, vascular risk factors, comorbidities, length of stay and discharge modified rankin scale (mrs) were collected. patients were excluded for age hours of presentation. we compared two time periods, before (epoch , - ) and after (epoch , - ) the introduction of continuous staffing of cvst cases by neurointensivists in the nicu. univariable and multivariable logistic regression were utilized to model the odds of poor outcome (dichotomized mrs - vs - ). fifty-three patients with cvst met the inclusion criteria during the study period (mean age (+/- ) years, % female). patients were identified for epoch and patients for epoch . overall, patients ( %) had a good (mrs - ) outcome. for epochs and , good outcomes were observed in ( %) and ( %) patients, respectively (p= . ). in both univariable and multivariable regression analysis (adjusted for age and sex), admission during epoch was associated with a significantly reduced odds of a poor outcome (or . , ci . - . ; p = . ) and (or . , ci . - ; p= . ), respectively. in this small, single-center cohort of patients with cvst, most patients experienced a good outcome. the institution of continuous neurointensivist coverage was independently associated with better outcomes. further validation in prospective, multicenter cohort studies is needed. thrombelastography (teg) provides a dynamic assessment of clot formation, strength, and stability. we examined relationships between teg parameters and outcomes from intracerebral hemorrhage (ich). we prospectively enrolled patients with spontaneous ich between to . teg was performed at the time of admission. we divided patients into two groups based on the presence or absence of hematoma expansion (he). clinical characteristics, baseline teg values, and outcomes were compared between the two groups. multivariable regression analysis was conducted to compare the differences of teg components between the two groups after adjusting for potential confounding effects. we included patients, ( %) with he and ( %) without he. patients with he were more often male and had higher rates of aspirin use, lower incidence of intraventricular hemorrhage, and larger baseline hematoma volumes. after controlling for potential confounders, mean r time was independently associated with he ( . ± . vs. . ± . mi significantly higher risk of he with or . ( % ci: . , . ), p=< . . patients with hematoma expansion were more likely to have poor neurological outcome (mrs - ) at discharge ( % vs. %, p= . ) and had higher mortality rates ( % vs. %, p= . ). overall, patients ( %) died in the hospital. following multivariable analysis, patients who died had significantly lower mean delta ( . ± . vs. . ± . mins.; p= . ) and smaller angle ( . ± . vs. . ± . degrees; p= . ) than those who lived. hematoma expansion and mortality from ich are independently associated with slower clot formation on teg. baseline teg identifies significant coagulation disturbances which may predict poor outcome and represent potential targets for therapeutic intervention. intracerebral hemorrhage (ich) patients often present with acute hypertension requiring intravenous and enteral antihypertensive medications. we performed a cohort study to determine clinical predictors of time to enteral antihypertensive medication and its effect on icu length of stay (icu los). we identified consecutive spontaneous ich patients admitted from / to / to a tertiary center, and excluded those transitioned to comfort care (cmo) within hours of admission. we calculated time from hospital admission to first enteral (oral or feeding tube) antihypertensive. we abstracted demographic and clinical variables. two reviewers examined medical records and classified ich location and etiology. we determined univariate and adjusted associations of clinical variables to time to enteral antihypertensive medication and performed regression analysis to determine effect on icu los. we identified patients and excluded for early transition to cmo. endotracheal intubation (p= . ), higher ich score (p< . ), no outpatient antihypertensive use (p= . ), and non-lobar ich location (p= . ) predicted longer time to starting enteral antihypertensive in adjusted analysis. presenting systolic or diastolic bp, time of icu admission (day vs. night), sex, and race were not significant predictors of time to enteral antihypertensive. time to enteral anti-hypertensive is the strongest predictor of icu los (p< . ) after adjustment for age, gcs, ich score, sex, race, and duration of iv antihypertensive infusion. patients with higher ich scores, intubation, no prior antihypertensive use, and non-lobar ich are at risk for increased time to enteral antihypertensive administration. timely enteral antihypertensive administration is an important and potentially modifiable predictor of icu los in acute ich. overall mortality from intracerebral hemorrhage (ich) represents a combination mortality from a potentially fatal disease as well as practice variation around treatment withdrawal of care. early do-not-resuscitate (dnr) rates are independently associated with in-hospital mortality and may serve as a proxy for withholding aggressive care. the american heart association (aha) guidelines recommended that dnr orders should not be applied before hours out of a concern that less aggressive care would lead to a self-fulfilling prophecy and excess mortality. we performed a retrospective analysis of temporal trends among primary ich patients presenting to all nonfederal emergency departments in california from to using data from the office of statewide health planning and development (oshpd). demographic information, clinical covariates (such as mechanical ventilation, craniotomy), and early dnr status within hours were collected and analyzed using segmented regression to evaluate for differences in linear trends from - compared with - . over a use of early dnr orders for ich patients has steadily decreased over the last years, even after adjusting for age and disease severity. the pace of this downward trend did not significantly change around the time when recommendations on early dnr use for ich in aha guidelines were revised in . spontaneous intracerebral hemorrhage (ich) is a common form of stroke that often results in severe morbidity or death. for most ich, there are no proven therapies for acute management. evidence suggests minimally invasive surgical evacuation of ich may result in improved patient outcomes. the enrich clinical trial is designed to determine the efficacy and economic impact of early ich evacuation using minimally invasive, transulcal, parafascicular surgery (mips) compared to standard guideline-based management. in this abstract we present the trial design and rationale at the foundation of the enrich clinical trial. enrich is an adaptive, prospective, multi-center clinical trial designed to enroll up to patients with acute ich. patients are block-randomized based on hemorrhage location (lobar vs basal ganglia) : to mips or standard management. included patients are - years, gcs - , baseline mrs , presenting within hours from last known well and found to have a spontaneous, cta-negative, supratentorial ich ( - ml). primary efficacy will be determined by demonstrating significant improvement in the mean utility-weighted mrs at days after enrollment. economic effect of mips will be determined by quantifying the cost per quality-adjusted life-years gained at pre-determined time points. the rationale for early intervention is to interrupt the time-dependent ich related pathophysiology caused by mechanical pressures and the pro-inflammatory secondary cascade that leads to worsened cellular injury and edema formation. the planned enrichment strategy acknowledges that hemorrhages in varied locations may have a differential response to mips. study adaptation, in the form of enrichment, may occur if pre-determined futility rules are met for the primary outcome in either of the two locations. enrich is designed to establish the clinical and economic value of early mips in the treatment of ich. enrollment was initiated in december . early seizures (< days) after intracerebral hemorrhage (ich) may be associated with the presence and degree of perihematomal cytotoxic injury. we explored the association between perihematomal diffusivity (phd) and early seizures after ich. consecutive patients from the prospective nih funded dash study (> years, spontaneous ich) were included. all patients had multimodal mri within weeks. perihematomal edema (phe) volume was outlined on t /flair and ich volume on gre; these and adc were coregistered to analyze phd. eeg monitoring was performed for clinical suspicion of seizure. mean adc values of phe and the percentage of phe volume were compared between the seizure and no-seizure groups, with adc values as vasogenic edema. results ( %) of a total of patients had early seizures at a median of day post ich. mean adc in the phe region was higher in the seizure group (mean: +/- vs +/- , p= . ). ich, absolute, and relative phe volumes were not different between groups. the phe of the seizure group had a lower percentage of cytotoxic edema ( % vs %, p= . ) and a higher percentage of vasogenic edema ( % vs %, p was the most predictive of seizure with auc = . , though adc thresholds between - had largely similar auc's. phe volume of > % (of adc > ) identified patients with seizure with sensitivity of . , specificity of . , and remained significant in multivariable analysis. patients with early post-ich seizures have higher mean perihematomal adc and a larger percentage of vasogenic edema in the perihematomal region. vasogenic edema due to bbb breakdown and perihematomal inflammation rather than cytotoxic injury is associated with early post ich seizures. novel neuroprotective treatments hold the promise to improve patient outcomes by broadening time windows of intervention and reducing hypoperfusion and reperfusion injury in the era of mechanical thrombectomy for acute ischemic stroke. hibernating species, such as arctic ground squirrels (ags), demonstrate remarkable resilience to ischemic and reperfusion injuries. bioinformatic analyses of genomes of hibernating species reveals signatures of convergent evolution in genes regulating stability and formation of mitochondrial respirasomes. hypoxia pre-conditioning (hpc) also leads to improved survival upon subsequent exposure to hypoxia, and is associated with increased stabilization of respirasomes. the respirasome is a macromolecule consisting of oligomers of complex i, iii, and iv. cox a l is a key mediator of respirasome stability via interactions with complex i and iii. in this study, we explored the role of cox a l in mediating respirasome stabilization in ags neural stem and progenitor cells (nsc/npcs) as well as mouse nsc/npcs exposed to hpc. respirasome stability was assessed using blue native gel electrophoresis and mitochondrial metabolism assessed by measuring oxygen consumption in vitro (seahorse metabolic analyzer). exposure to mild hypoxia and induction of hif leads to stabilization of respirasomes, upregulation of hif, and modulation of mitochondrial metabolism. interestingly, overexpression of the ags isoform of cox a l, which has amino acid substitutions in residues mediating respirasome stability, recapitulates the effects of hypoxia on respirasome stability and mitochondrial metabolism without altering hif expression. targeting respirasome stability by modulating cox a l is a potentially novel neuroprotective target for treatment of ischemic injuries. testing of these hypotheses in pre-clinical models of stroke is on-going. acute stroke symptoms need timely diagnostics in order to ensure best outcomes. as a non-academic, community-based center located in rural western nc, where we are the regions only comprehensive stroke center, we developed a process to intake stroke patients quickly directly from ct to interventional radiology when applicable. a smooth transition reduces the quantity of time from imaging to interventional suite, ultimately reducing the time it takes to prepare to actively treat a patient. interventional radiology value stream mapping started in june . multidisciplinary team worked in multiple work groups to design and create "code ir stroke now". flow chart created to show multiple moving parts simultaneously, to streamline transition from er (sometimes this includes triage from the region also) to ir. an ir "ready" criteria was made, er and ir checklists, followed by post procedure debrief and treatment plans/order set to standardize care and documentation. first mock code ir was done / / , this was critiqued/perfected. "go live" date: / / . we continue process improvement today. in first three months, patients have gone through this process. average compliance for goal door to puncture < min went from . % to %. door to groin times reduced from minutes to minutes. our performance is minutes quicker than other comprehensive stroke centers ( m avg gwtg database). saved an average of million neurons per patient. total of million neurons saved on average since / / ! door to groin times can be reduced with streamline approach to care. multidisciplinary team approach, including house supervisors, anesthesia, switch-board in addition to the bedside staff and providers can make a smooth transition from the time a large vessel occlusion is identified to getting the patient to the interventional suite. activation of "code ir stroke now" page activates this team / . it is unknown whether antithrombotics for secondary stroke prevention in patients with acute ischemic stroke (ais) due to infective endocarditis (ie) reduce the rate of secondary ais or increase major bleeding. we conducted a multi-center, retrospective cohort study from - of patients with ais secondary to left-sided ie, separated into two groups (antithrombotic vs no antithrombotic). antithrombotics included antiplatelets and/or therapeutic anticoagulation. the primary outcome was a composite of recurrent ais and major bleeding. secondary outcomes included ais and major bleeding individually. a binary logistic regression model adjusted for age and native vs prosthetic valve involvement was used for outcome evaluation. the final analysis included patients ( antithrombotic vs no antithrombotic). median age was years and ( %) patients had prosthetic valve infections. infecting organisms were mostly methicillin sensitive s. aureus ( %) or streptococcus spp. ( %). valve repair/replacement occurred in ( %) patients. aspirin with or without another antithrombotic ( %) was the most common antithrombotic treatment. the primary outcome occurred in . % vs . % of patients with antithrombotics vs no antithrombotics, respectively (or . ; % ci . to . ). ais ( . % vs . %; or . ; % ci . to ) and major bleeding ( . % vs . %; or . ; % ci . to . ) were similar between groups. a subgroup analysis of aspirin monotherapy vs no antithrombotic yielded similar results for the primary outcome ( . % vs . %; or . ; % ci . to . ) and ais ( . % vs . %; or . ; % ci . to . ). major bleeding was increased, however ( . % vs . %; or . ; % ci . to . ; p= . ). antithrombotics after ais secondary to ie were not associated with a decrease in recurrent ais or an increase in major bleeding. aspirin monotherapy was associated with an increase in major bleeding without any reduction in ais. malignant hemispheric stroke (mhs) represents between - % of all hospitalized ischemic stroke in the united states. pooled analysis of european studies has demonstrated that decompressive hemicraniectomy (dchc) for mhs reduces mortality compared with conservative medical management and may also improve functional outcomes. these trials however, excluded patients with major medical comorbidities that might confound clinical outcomes. apache ii and sofa scores are validated icu scoring systems to help characterize disease severity and estimated hospital mortality. this study aims to evaluate apache ii and sofa scores in predicting outcomes for patients undergoing dchc for mhs. this is a single center retrospective analysis of patients who underwent early dchc for mhs between may through january at unc chapel hill. apache ii and sofa scores were calculated for the date of admission or date of first presentation to neurologic care. outcomes included mortality at discharge, mortality at day, and functional outcome at last follow up, up to one year. multivariate analysis included timing of surgery, age, laterality, presence of midline shift, hemorrhage or multiple territory infarction. we identified patients who met inclusion and exclusion criteria. the median age was ( to ), -nine percent of patients received surgery by hospital day . full statistical analysis is pending. our hypothesis is a positive correlation between icu severity scores and mortality. given apache ii and sofa scores capture the effects of acute and chronic disease that would affect patient recovery, we hope to provide a more comprehensive prognostication of outcomes following surgery to help guide physicians and family members of these patients in their decision-making process. we conducted this study to investigate the effects of decompressive craniectomy (dc) combined with hypothermia on mortality and neurological outcomes in patients with large hemispheric infarction. within hours of symptom onset, patients were randomized to one of the following three groups: dc group, dc plus head-surface cooling (dcsc) group and dc plus endovascular hypothermia (dceh) group. we combined the data of the dcsc and dceh group to dch group during analysis. the primary endpoints were mortality and modified rankin scale (mrs) score at months. there were patients in the dc group, patients in the dcsc group and patients in the dceh group. for all patients, the mortality at discharge and after months was . % ( / ) and . % ( / ), respectively. the dch group had lower mortality, but the difference was not statistically significant (at discharge, . % vs. . %, p= . ; months, . % vs. . %, p= . ). after months, patients survived, and . % of the surviving patients had good neurological outcomes (mrs score of - ). the dch group had better neurological outcomes, but this difference was also not statistically significant ( / , . % vs. / , . %; p= . ). the total number of patients experiencing complications in the dc group and the dch group was ( . %) and ( . %), respectively. treatment with hypothermia led to decreased mortality and improved neurological outcomes in lhi patients who received dc. a multi-center rct is needed to confirm these results. destiny ii investigated hemicraniectomy in patients -years and older for the treatment of malignant cerebral edema. we sought to describe the treatment effect of early hemicraniectomy in destiny ii, using number needed to treat to benefit (nntb) and benefit per hundred (bph) treated at and months. as an mrs of is generally undesirable, we also present nntb and bph excluding this outcome. for all possible dichotomizations of the mrs, net nntb was derived by taking the inverse of absolute risk difference, and net bph by multiplying absolute risk difference by . for benefits simultaneously across all disability transitions on the mrs, nntb, and bph, estimates were derived using joint outcome tables: ) algorithmic minimum and maximum and ) four independent experts. the expert data is presented as geometric mean. the algorithmic nntb was . (range . - . ) at -months and . ( . - . ) at -months, while bph was . ( - ) and . ( - ). the expert nntb was . ( . - . ) at -months and . ( . - . ) at -months, and the bph was . ( - ), and . ( - ) respectively. excluding mrs the algorithmic nntb was . (range - . ) at -months and . ( . - . ) at -months, while bph was ( - ) and . ( - ). the expert nntb was . ( . - . ) at -months, and . ( . - . ) at -months, and bph was . ( - ) and . ( - ) respectively. early systematic hemicraniectomy improves outcome (including mrs ) for every - patients treated. excluding patients with mrs , hemicraniectomy improves outcome for every . patients treated. the algorithmic range provides bounds to the data, while the expert geometric mean provides the most accurate point estimate. these data provide a powerful tool to describe the potential treatment outcomes to families during the first day following a malignant middle cerebral artery infarction. background cerebral bypass surgery is performed to restore, or revascularize blood flow to the brain. previous studies have not shown whether emergency surgical reperfusion therapy may be effective in acute ischemic stroke patients with large artery occlusion and hemodynamic deterioration. objective to evaluate the effect of emergency sta-mca bypass surgery on the outcome of hemodynamic compromised patients who had progressive or fluctuating stroke despite best medical treatments. we retrospectively reviewed the clinical and radiological data of consecutive patients treated by both emergency bypass surgery ( cases, . %) and elective bypass surgery ( cases, . %) due to large artery occlusion at a single center. the effect of surgical therapy was measured with the modified rankin scale (mrs) at months. clinical severity was evaluated by the national institutes of health stroke scale (nihss) between pre-and post-operative state. major perioperative complications were defined as any hemorrhagic stroke, myocardial infarction and death. results occlusive sites were the cervical internal carotid artery in ( . %) patients and the middle patients in emergency surgery group and ( . %) patients in elective surgery group. emergency bypass surgery improved nihss (preoperatively, [ - ] ; weeks postoperatively, [ - ]). major perioperative complications in days were happened in three patients ( . %) after emergency bypass surgery, and four patients ( . %) after elective bypass surgery. emergency revascularization surgery may be effective alternative treatment for acute ischemic stroke patients with hemodynamic deterioration refractory to maximal medical treatments without significant complications. larger randomized clinical study is needed to evaluate the effect of emergency revascularization surgery in acute hemodynamic deterioration. multiple studies have reported lower mortality rates in obese patients with various cardiovascular disorders, a phenomenon called as the 'obesity paradox'. such relationship has been largely unreported in patients with neurological pathologies especially stroke. this study reports the effect of obesity on prognosis in patients with ischemic stroke. analysis of national inpatient sample data ( - ) showed a total of , , patients discharged with primary diagnosis of is, icd- code .xx and .xx. patients with obesity were identified using agency of healthcare research and quality (ahrq) criteria. we used binary regression to compare inhospital mortality between obese and non-obese patients with ischemic stroke. from - , , , patients with ischemic stroke were identified of which . % were found to be obese. obese patients with ischemic stroke were more often younger, female, and african american as compared to caucasian. after risk adjustment for demographics, and baseline comorbidities, obese patients with ischemic stroke had lower observed in hospital mortality as compared with non-obese patients with ischemic stroke ( . % vs %, or: . ci= . - . p< . ). from an eleven year nationwide cohort of patients with ischemic strokes, we observed a significant protective effect of obesity and better prognosis including a lower mortality rate. more prospective studies are warranted to further analyze this counter-intuitive trend. very early mobilization of critical care patients improves outcome, length of stay, and patient satisfaction. data for efficacy of very early mobilization for stroke patients have been mixed, and there is limited outcomes data for patients mobilized within hours of receiving intravenous alteplase (iv tpa). the objective of this retrospective observational study was to determine if patients receiving iv tpa who were mobilized earlier were more likely to discharge home. medical records of ischemic stroke patients who received iv tpa between and at two urban facilities were reviewed for mobility protocol activities. patients who received endovascular treatment, were placed on comfort care day zero or one, mobilized after the first hours, and transferred out or left against medical advice were excluded. multinomial regression was used to determine if there were significant differences in patients' discharge status by time first mobilized, adjusting for stroke severity using the national institutes of health stroke scale (nihss), age and gender. of the patients included, . % (n= ) were female, mean age was . (± . ), and the median admit nihss was . [iqr: . , . ]. the median time first mobilized was . hours [iqr: . , . ], . % (n= ) of patients were discharged to home, . % (n= ), a skilled nursing facility (snf), . % (n= ), an inpatient rehab facility (irf), and . % (n= ) hospice or expired. there was suggestive, but inconclusive evidence for a relationship between time first mobilized and discharged to snf versus home (p=. ). for every one hour increase in time mobilized, patients were . ( % ci= . - . ) times more likely to be discharged to snf than home. this study reveals very early mobility is potentially efficacious after iv tpa. longer time to first mobility was associated with discharge to skilled nursing facility, although this was not statistically significant. medical management of cerebral edema after large volume stroke varies greatly across institutions. hypertonic saline has emerged as a common treatment strategy to attempt to reduce edema and theoretically prevent the need for decompressive hemicraniectomy. there is no established protocol for hypertonic saline administration and there have been concerns regarding safety. in a single-center, retrospective analysis we identified patients who received hypertonic saline for malignant edema after an ischemic stroke involving the entire hemisphere or diffuse middle cerebral artery (mca) territory. we compared patients who received continuous infusions of % or % hypertonic saline to those who received continuous infusions with boluses of . %. the primary endpoint was time to goal sodium ( ). secondary endpoints included the need for surgical decompression and adverse events. we included patients who received only continuous infusions of hypertonic saline and patients who received a combination of continuous infusions and bolus doses. we found no significant difference between number of patients who reached goal sodium ( vs respectively, p= . ) or time to goal sodium ( hours vs . hours, p= . ). there was a significant difference in the number of patients who underwent surgical decompression ( vs , p= . ). there was not a significant difference in the rate of acute kidney injury or development of acidosis between groups ( vs. , p= . ). both hypertonic strategies appear to be safe. bolus dosing, on review, was more often instituted during clinical deterioration, accounting for the higher rate of surgical intervention. we feel we can safely be more aggressive earlier in the clinical course to potentially avoid surgical decompression. furthermore, we may need to look more closely at our target sodium, evaluating whether it should be based on the patient's baseline sodium or a universal value. even though recanalization is strongly associated with improved functional outcomes and reduced mortality, clinical benefit from thrombolysis is reduced as stroke onset to treatment time increases. in the recent study, endovascular treatment(evt) has been demonstrated to improve functional outcome in patients with acute ischemic stroke (ais) within the time window of onset to or hours. however, beyond usual thrombolysis time window, early neurologic deterioration(end) related with proximal artery occlusion is not uncommon in ais. with this, we report ais case series treated with evt because of end related proximal artery occlusion. from january through march , all patients underwent iat for ais with anterior circulation stroke. among them, twenty-four patients underwent evt due to end. at admission, all twenty-four patients showed near to complete occlusion of a proximal artery and had diffusion-perfusion mismatch. mean age was . initial median initial national institutes of health stroke scale (nihss) was and nihss after end was . all patients had diffusion-perfusion mismatch over %. seven patients treated with iv-tpa before evt. good recanalization (tici b/ ) was achieved in . %. the hemorrhagic complication was seen in the follow-up computed tomography scan in of cases: three were hemorrhagic transformation, another was the subarachnoid hemorrhage. the thromboembolic mortality case. in our report, evt in ais with end achieved safe and successful recanalization. and successful recanalization was associated with good clinical outcome. we think evt could be a useful method in case of end in ais patients with proximal artery near to complete occlusion, even beyond usual to hours time window for evt. jugular bulb venous monitoring can provide information about cerebral hemodynamics and metabolism. we investigated the feasibility and clinical application of jugular bulb venous monitoring in acute ischemic stroke patients at neurocritical care unit. from march to june , we conducted jugular bulb venous monitoring in patients in a tertiary referral hospital. five patients were excluded; without ventilator care and other diseases than stroke. jugular venous catheters were placed in internal jugular vein by ultrasound-guided method. lactate, venous oxygen saturation (sjvo ), and arteriovenous oxygen saturation differnece (avdo ) were monitored every hours. metabolic derangement was defined when lactate level was more than . mmol/l. patients were divided according to presence of clinical deterioration. for long-term prognosis, modified rankin scale - at months were defined as poor outcome. twelve patients ( . %) showed metabolic derangement and they experienced more frequent clinical deteriorations compared to patients without metabolic derangement (n= , . % vs. n= , . %, p= . ). clinical deterioration was noted in patients, and lactate level was significantly higher in the presence of clinical deterioration group ( . ± . vs. . ± . mmol/l, p= . ). adjusting other potential variables (age, baseline stroke severity, sjvo , and avdo ), metabolic derangement was an independent factor associated with clinical deterioration (or . , % ci . - . , p= . ). meanwhile, poor outcome group (n= ) showed no difference on lactate level, but avdo were higher in poor outcome group ( . ± . v. . ± . , p= . ). avdo remained an independent factor for poor outcome after multivariable logistic regression analysis (or . , % ci . - . , p= . ). this study showed that lactate was associated with clinical deterioration during neurocritical care, whereas venous desaturation contributed to long-term prognosis. jugular bulb venous monitoring is a feasible tool in patients with acute ischemic stroke at neurocritical care unit. swift recognition of stroke symptoms, immediate access to testing and timely treatment plays a vital role in functional outcomes (middleton et al., ) . delays can postpone treatment and complicate recovery. delays at this facility included registration, order entry times, and imaging. pi included evaluating and eliminating interruptions, with a goal of reducing the time to treatment. process improvement (pi) utilized an evidence-based algorithm to improve performance metrics and treatment of acute strokes. setting was a suburban, ancc magnet recognized primary stroke center with beds in the ed that experiences , ed visits and , admissions per year. patients included in the acute stroke protocol presented with signs and symptoms of stroke and last known well within hours of symptom onset. participation included ed staff, and staff working in areas impacted by stroke care. code stroke was initiated for patients who fit the criteria. an overhead page was implemented notifying the team throughout the hospital. radiology would prioritize ct and call the ed as soon as ct was ready. in the meantime, ed team continued assessments. with ct resulted, the physician would determine whether the patient was eligible for tpa. the acute stroke protocol included a list of inclusion/exclusion criteria for tpa administration. other treatment requirements included reminders for frequency of vital signs, neuro checks and assessments. implementation began in may and the team began to see a significant decrease in ct times and better compliance of dysphagia screening and nih assessments. ct tat completed within minutes increased from % to %. nih stroke scale completion rose from % to %. compliance with completing dysphasia screening increased from % to %. results stem from a commitment to excellence from the entire team. pi continues to further improve care for stroke patients. induced hypertensive therapy (iht) has used to enhance cerebral perfusion pressure in subarachnoid hemorrhage and stroke, but there is no established indication for iht in ischemic stroke. we report the usage of iht in acute ischemic patients with hemodynamic instability caused by steno-occlusive disease of a main cerebral artery. we reviewed acute ischemic stroke patients with cerebral perfusion deficit due to intracranial and extracranial steno-occlusive disease. iht was applied for early neurological deterioration and maintained until hemodynamic instability was stabilized over hours or neurointervention including angioplasty and extracranial intracranial arterial bypass surgery were performed. patients were analyzed. territories of stroke were of anterior circulation of intracranial vessels, of posterior vessels, and of extracranial vessels. mean duration of ih therapy was . minutes. pre and post nihss score of ih therapy was . and . , respectively. patients ( . %) were showed improvement and patients ( %) were stabilized without further aggravation. patients revealed bradycardia. there was no fatal complication of therapy. patients were performed further treatment include bypass surgery, angioplasty, and stenting after ih therapy. at months follow up, patients showed good outcomes (modified rankin scale , , and ). iht may be safe and effective for the neurologic deterioration or progression of acute ischemic stroke with hemodynamic instability due to severe steno-occlusive disease of major cerebral artery. large randomized trials are needed to confirm this result. most patients with progressive stroke have a poor prognosis. the aim of our study was investigate the factors related with progressive neurologic deficit (pnd) in the patients receiving recanalization therapy for acute ischemic stroke. -month period, were enrolled. blood pressures (bps) at , , and hours after admission and bp variation (bpv) for the first hours were collected. variables associated with pnd were analyzed. among enrolled patients, patients showed pnd. the patients with pnd had higher systolic bps at , , and hours after admission and higher bpv than the others (p < . ). posterior circulation stroke was more prevalent in the patients with pnd (p < . ). in logistic regression analysis, pnd was independently associated with posterior circulation stroke [odds ratio (or) = . , p < . ] and systolic bp at hours after admission (or = . , p = . ). pnd may be associated with elevated systolic bp for the first hours after admission in the patients receiving recanalization therapy for acute ischemic stroke. telestroke has revolutionized stroke care delivery in the modern era. massachusetts general hospital (mgh) uses the most common model, the hub and spoke. the demonstration of superiority of endovascular therapy (et) with intravenous tpa over tpa alone for acute stroke patients with large vessel occlusions prompts a thorough assessment of telestroke's role in the delivery of et, particularly in terms of transferring patients to hubs capable of et. our primary objective was to examine associations between transfer time and clinical outcomes. patients were selected from the get with the guidelines-stroke registry who were transferred to mgh from jan to oct who had nihss> and last known well< h on mgh arrival (n= ). we excluded patients for whom we could not calculate the primary predictor, transfer time (defined as the mgh arrival time minus the telestroke consult answered time, n= ). several clinical outcomes were explored by linear and logistic regression to determine association with transfer time. of the patients in the study, ( %) were transferred by ambulance, ( %) by helicopter, and ( %) underwent et at mgh. median transfer time was min, and median aspects decrease was during transfer. longer transfer time was associated with decreased likelihood of undergoing et (p= . ). however, transfer time was not significantly associated with aspects decrease during transfer. for those patients undergoing et, transfer times bore no association to day mrs. this study identifies an association between longer transfer time and decreased likelihood of undergoing et. reasons are varied, and are not clearly related to imaging progression alone. only % of transferred patients underwent et. more efficient spoke triage and transfer may improve the ratio of patients treated with et. these data provide an important perspective during this period of stroke triage evolution. intra-arterial thrombectomy (iat) has been approved for acute treatment of ischemic strokes (is). with the advent of several new devices for iat, this procedure has become more widely utilized with better outcomes. we performed this analysis to evaluate trends and predictors of utilization of iat over an year period. analysis of nationwide inpatient sample data ( to ) showed a total of , patients discharged with a primary diagnosis of is, icd- code .xx, and .xx. iat was ascertained by icd- procedure code . . independent predictors of iat were studied using binary logistic regression. the predictors included in the model were age, sex, race, teaching status, and insurance type. results or . % of is patients received iat. mean age of patients receiving thrombolysis was . years. percentage of is patients receiving iat has consistently increased from . % in to % in . we also observed significant year to year decrease in mortality among patients receiving iat. in , . % of iat patients died as compared to . % in . using binary logistic regression, the statistically significant independent predictors of iat utilization were age (or= . , p= . ), female gender (or= . , p= . ), insurance type as compared to medicare (private insurance or= . p= . , and self-pay or= . p= . ). as compared to caucasians, african americans were less likely to receive treatment (or= . p= . ). also, a teaching hospital was found to be more likely to administer iat as compared to a non-teaching hospital (or = . , p= . ). is patients with younger age, female gender, private insurance and patients admitted to teaching hospitals are more likely and african americans are less likely to receive iat. this study showed that iat utilization has increased significantly since with a steep decline in the in-hospital mortality. this may point to improved iat devices and better patients' selection. telestroke plays an integral role in stroke care. nationally the most common model is the hub and spoke, which is used at our institution. understanding telestroke's role in the transfer of candidate patients for endovascular therapy (et) is critical to minimizing delays. our primary objective was to evaluate predictors of transfer delay. patients were selected from the get with the guidelines-stroke registry who were transferred to mgh from jan to oct with nihss> and last known well< h on mgh arrival (n= ). we excluded patients for whom we could not calculate transfer time (the mgh arrival time minus the telestroke consult answered time, n= ). ideal time was calculated using google maps incorporating date/time information for ground transfers and straight line distance at mph for helicopter transfers. ideal time was subtracted from actual time to calculate delay, accounting for distance, mode of transport, weather, and traffic. analysis of covariance was used to explore possible predictors of delay (night vs. day, weekend vs. weekday, tpa delivery at spoke). of the patients in the study, ( %) were transferred by ambulance, ( %) by helicopter, and underwent et. a significant proportion of the variation in delay was explained by the predictors (f= . , p< . ). nocturnal transfer ( - hrs) was associated with significantly longer delay ( . additional minutes relative to daytime transfers, p< . ). weekend vs. weekday transfer and tpa delivery at spoke hospital did not contribute significantly to model variance. our findings highlight the importance of refining protocol approaches. nocturnal transfers were associated with substantial delay relative to daytime transfers. in contrast, delivery of tpa was not associated with delays, underscoring the impact of effective protocols that are in place. metrics and protocols for transfer, especially at night, may have a positive impact on transfer times. the use of anticoagulant therapy in the acute stage of ischemic stroke is controversial. novel oral anticoagulant (noac) is effective in preventing recurrent embolism in patients with non-valvular atrial fibrillation (nvaf), but the risk of hemorrhagic transformation is the major concern for its early use in ischemic stroke. we aimed to study the use of noac in patients with acute ischemic stroke and nvaf. patients with acute ischemic stroke and nvaf, who were admitted to our acute stroke unit from to , were recruited in this single-centre cohort study. the timing of initiation of noac is at the discretion of the treating physician based on the stroke severity and infarct size. nvaf attributed to . % ( / ) of all ischemic stroke cases. the early recurrent embolism rates were . %, . % and . % at one week, two weeks and one month respectively. noacs were prescribed in patients. noacs were initiated within one week in patients ( . %). the median time to noac initiation were five days (iqr . - . ), nine days (iqr . - . ) and days (iqr . - . ) for patients with no/small-sized infarct, moderate-sized infarct, and large-sized infarct respectively. at one month, two patients had recurrent ischemic stroke despite treated with noac. only one patient, who had a large-sized infarct, developed symptomatic hemorrhagic transformation. early use of noac in ischemic stroke appears to be safe. further large prospective studies are required to evaluate the risk and benefit of noac use in acute ischemic stroke. osmotherapy (hypertonic saline or mannitol) is the mainstay of available therapy to counter cerebral edema that can develop after large hemispheric infarction. in a post-hoc analysis of the games-rp trial, we hypothesized that patients with large infarction, treated with intravenous glyburide, might require less osmotherapy than placebo treated patients. games-rp was a multi-center prospective, double blind, randomized, placebo controlled study which enrolled patients with large anterior circulation infarction. patients were randomized to iv glyburide administration (biib ; n= ) or placebo (n= ) with target time from symptom onset to drug infusion decompressive craniectomy (dc), or both. total bolus osmotherapy dosing was quantified by an "osmolar load" (volume in l * osmolarity in mosm/l). of the subjects, the percentage of patients who received bolus osmotherapy did not differ between the glyburide and placebo treated subjects ( % v. %; p= . ). there was no difference in mean total osmolar load received (mosm) or hours from drug bolus to osmotherapy administration. overall, subjects received osmotherapy. the baseline dwi lesion volume (ml) was significantly larger in the osmotherapy treated group ( . ± . v. . ± . ; p= . ). the presence of adjudicated malignant edema on imaging was more common in the osmotherapy group ( % v. %, p= . ), as was dc ( % v. %; p< . ). among patients with adjudicated clinical neurologic deterioration from edema, % (n= ) did not receive osmotherapy. treatment with iv glyburide was not associated with less osmotherapy, possibly due to a ceiling effect resulting from the large infarct volumes. however, osmotherapy use was associated with larger infarct volumes, malignant edema, and higher incidence of dc. use of osmotherapy did not always follow the appearance of clinical or radiographic malignant edema. acute ischemic stroke patients receiving intravenous alteplase (iv-tpa) are placed on bedrest for hours or longer due to provider fear of worsening stroke symptoms from decreased cerebral perfusion. this is based on medical uncertainty and lack of robust studies, despite american stroke association (asa) recommendations for mobilization when hemodynamically stable. this retrospective observational study evaluates very early mobility in acute ischemic stroke patients post iv-tpa while evaluating for change in nihss. medical records of ischemic stroke patients who received iv-tpa between and at two urban hospitals were reviewed for mobility protocol activities. patients who were given endovascular treatment, placed on comfort care on day zero or one, mobilized after the first hours, transferred out or left against medical advice were excluded from the analysis. multiple linear regression was used to determine if those patients mobilized earlier saw a greater change between nihss at admit and hours post iv-tpa administration, adjusting for age and gender. of the patients included in the final analysis, . % (n= ) were female, mean age was . the multiple linear regression results showed no significant relationship between change in nihss from admit to hours post iv-tpa and earlier mobilization, after adjusting for age and gender (ß= - . change in nihss points per hour; p= . ). this study reveals early mobility does not worsen stroke symptoms or severity based on nihss. this suggests that very early mobility of patients after iv-tpa is safe as recommended by asa. interhospital transfers to a stroke center following iv-tpa administration are increasingly common. however, no studies have evaluated icu needs in these transfer patients and such understanding may have a significant impact on resource utilization. the aim of this study is to compare the frequency, timing, and nature of icu-level needs in post-iv-tpa patients that were transferred versus those who present directly to the admitting hospital. retrospective chart review of consecutive, tpa-treated ischemic stroke patients admitted to the icu at a comprehensive stroke center servicing a large telestroke referral network from / to / was performed. we evaluated patient demographics, stroke characteristics, and icu needs between transfer and non-transfer patients before and after icu admission. results patients were admitted to the icu post-tpa. patients ( . %) were transferred from an outside hospital, of which patients had icu needs ( . %). this frequency of icu needs was no different when compared to the non-transfer patients ( / , . %, p = . ). similar icu needs were observed for each specific icu intervention between transfer and non-transfer patients (iv antihypertensive, vasopressor requirement, iv rate control, respiratory support, ia therapy, icp monitoring, hypertonic therapy, and neurosurgical intervention, all p > association with icu needs (or . in transfer patients, or . in non-transfer; both p < . ). transferring post-iv-tpa patients is not associated with increased icu needs. about one-half of post-tpa patients do not have icu needs, and these patients typically have milder stroke severity. our data supports the safety of transferring post-tpa patients, and to potentially monitor a subgroup of these patients in a non-icu setting. the ability to appropriately triage post-tpa patients may lead to more efficient and cost-effective stroke care. stroke patients requiring decompressive craniectomy remain at high risk of prolonged mechanical ventilation as well as ventilator associated pneumonia (vap). early tracheostomy placement may provide a reduction in the duration of mechanical ventilation however prediction of those who ultimately require a tracheostomy remains a clinical challenge. a preoperative assessment of tracheostomy dependence may help to guide decision making. the authors compare key outcome data after early versus late tracheostomy and develop a preoperative decision-making tool to predict postoperative tracheostomy dependence. a subsequent validation utilizing a decision tree analysis applied prospectively is ongoing and will be presented. we performed a retrospective analysis of prospectively collected registry data and developed a propensity weighted decision tree analysis to predict tracheostomy requirement utilizing factors present prior to surgical decompression. outcomes include probability functions for icu los, hospital los, and mortality based on data for early ( day) tracheostomy. a subsequent validation of the decision tree is being applied prospectively to evaluate its predictive value. a total of surgical decompressions were performed on patients with acute ischemic or spontaneous hemorrhagic stroke between - . forty eight patients ( . %) required a tracheostomy, whereas ( . %) developed vap, and ( %) survived hospitalization. mean icu and hospital los were . and . days respectively. utilizing gcs, sofa score and hydrocephalus presence, our decision tree analysis provided a % sensitivity and % specificity for tracheostomy prediction. early tracheostomy conferred significantly fewer ventilator days (p< . ) and shorter hospital los (p= . ) with similar vap and mortality rates between groups. early tracheostomy shortens duration of mechanical ventilation and length of stay following surgical decompression for stroke, however without a demonstrable impact in mortality or vap rates. a preoperative decision tree awards a practical tool that may provide insight to guide preoperative decision-making with patient families. patients suspected acute stroke are critical in time delay of endovascular or intravenous thrombolytic therapy. prehospital notification from emergency medical services (ems) may shorten the door to recanalization time. the 'brain saver', web-based prehospital notification system could reduce the time interval from symptom onset to recanalization. beginning in march , stroke team consisted of stroke specialized doctors, nurses and radiologists of multi departments received direct alarms via smart phone application from paramedics of ems about transport information of patients with suspected stroke. we compared baseline characteristics and prehospital/ in-hospital delay time in stroke patients treated with intravenous thrombolysis or endovascular treatment for months with and without ems use brain saver protocol. patients ( patients with protocol and patients without protocol) were enrolled in this program. the patients who used brain saver had shorter median onset-to-arrival times ( minutes versus minutes, p < . ) and in in-hospital delay time ( minutes versus minutes, p<. ). prehospital notification by brain saver was associated with shorter median door-to-imaging time ( minutes versus minutes, p<. ), door-to-needle time ( minutes versus minutes, p <. ), door to puncture time ( minutes versus minutes, p < . ) we found that prehospital notification was associated with faster door-to-imaging time, door-to-needle time and door-to-puncture time in patients presenting within hours of symptom onset. close collaboration between stroke team in hospitals and the ems system gives stroke suspected patients an in-time emergency care system. infection is a common complication in the acute phase after ischemic stroke. furthermore, malnutrition is associated with unfavorable outcome in patients with stroke. therefore, we investigated that premorbid undernutrition identified by objective and quantitative method, nutritional risk index (nri) was related to the risk of infection after ischemic stroke. a consecutive patients who were admitted within days after ischemic stroke onset between october and october were included. we assessed initial nutritional status using nri, and nri formula as follows: nri = ( . × serum albumin, g/dl) + { . × present weight (kg)/ideal body weight (kg)}. the patients were categorized into three groups on the basis of nri [no risk (nri > . ), moderate risk (nri . - . ), and severe risk (nri < . )]. we compared the clinical characteristics and nri according to the presence of infection. among the included patients (mean age, . years, male, . %), ( . %) patients experienced infection during hospitalization. the rate of pneumonia was . % (n= ), and the rate of urinary tract infection was . % (n= ) among total infection. the proportion of lower nri patients (moderate risk and severe risk) was significantly greater in the infection group ( . % vs. . % and . % vs. . %, p < . ). moreover, higher nri patients were less likely to be admitted to the intensive care unit ( . % vs. . % vs. . %, p = . ). a multivariate analysis revealed that lower nri groups had a higher risk of infection [odds ratio ( % confidence interval); moderate risk . ( . - . ); severe risk . ( . - . ), p for trend = . ]. our study demonstrated that the lower nris predicted infection complications and severe infections after ischemic stroke. this suggests that assessment of nutrition depletion could be a useful predictor and a modifiable risk factor for infection following stroke. cyp c plays a major role in the metabolism of the clop[idogrel. cyp c generates an active oxidized metabolite of clopidogrel that exerts antipl;atelet activity by inhibiting p y reeceptor. the major alleles of the cyp c gene are * , * , * and * and approximately % of caucasians and % of asians have one or more loss of function alleles in this study, patients with at least two * or * allels were classified as poor metabolizer(pm), those with one * or * allele were classified as intermediate metabolizer(im), and those without a * , * or * alleles were classified as extensive metabolizer. in addition. those with (* /* or * /* ) were classified as unknown metabolizer. stroke patients were enrolled for this trial. the mean age was years, and % were women. % had a history of hypertension, % of dm and % of dyslipidemia. of the participants, % were classifies as em, % as um, % as im, % as pm and % as unknown metabolizer. % had good genotype for clopidogrel metabolism and % had poor genotype. there were no significant diffirences in the demographic and clinical findings between the good and poor genotype groups the prevalence of cyp c polymorphisms is different according to the ethnicity. the racial difference in platelet function may lead to diffrerences in the treatment as well as new targets for antiplatelet therapy the social brain hypothesis is an evolutionary theory proposing that the number of contacts in a primate's social network is proportional to neocortical volume. we tested the hypothesis in a patient population with social network data before and after vascular events. we studied whether social network indices would decrease after stroke, but not after myocardial infarction (mi), as anticipated by the theory. we examined trajectories of the lubben social network scale score (range - , higher values indicating larger network) before and after vascular events in participants from the cardiovascular health study. we used a repeated measures design with linear mixed models to compare the change in social network score before and after events in persons with ischemic stroke and with mi. over a mean of . years of follow-up for stroke and . years for mi, we examined an average of social network scores for each participant. we controlled for socio-demographics, baseline cognitive function, and comorbidities. social network scores declined significantly after stroke (an additional - . points every year, % ci - . , - . , p= . ), but not after mi (- . , % ci - . , . , p= . ) compared to the baseline slope in fully adjusted models. social network score declined more steeply after stroke than after mi, even after adjusting for potential confounders. these findings support the social brain hypothesis but do not address mechanism. shrinkage of the social networks may be a specific target for interventions to optimize recovery in vascular diseases, particularly stroke. emergency neurological life support (enls) protocols are an essential component to assessment and management of patients within the first hours of the neurological emergency. with increasing focus on emergent endovascular treatment for large vessel occlusion (lvo) in acute ischemic stroke our institution incorporated stroke van assessment as part of the enls acute stroke initial assessment protocol. stroke van screening tool was taught to all nurses in the emergency department (ed) who triage stroke. all patients who presented to the ed with suspected stroke had a van assessment completed prior to ed physician evaluation and ct imaging. patients with weakness in addition to visual changes, aphasia, or neglect were considered van + and triaged immediately to ct angiogram head/neck with immediate notification to the neurointerventionalist. a sample of patients presenting to the ed as a stroke alert over an month time period were utilized. using the stroke van assessment tool was found to improve time to identification of lvo by reducing time from arrival to cta for van positive patients from minutes pre-intervention (n= ) to minutes post-intervention (n= ). this was a significant decrease in time to identification of patients presenting with lvo (p< . ), improving time to endovascular treatment. incorporating stroke van as part of the acute stroke assessment protocol improved identification of patients presenting with lvo, decreased time to cta imaging and improved time to endovascular treatment which is well documented with improved neurological prognosis. time is essential in neurological emergencies. the van assessment is quick and easy to perform, requires no scoring or calculations, and is the only lvo screening tool tested in the ed by ed nurse and physicians. we suggest incorporating stroke van to the enls acute stroke protocol as a way to improve identification of lvo and improve time to endovascular treatment. elevated blood pressure (bp) is known to be related to hemorrhagic transformation (ht) after ischemic stroke. however, the effect of bp variation on the ht remains unclear, especially in patients with successful recanalization after mechanical thrombectomy. therefore, we investigated the relationship between bp and ht after mechanical thrombectomy following ischemic stroke. a consecutive patients with acute ischemic stroke and successful recanalization (tici b or tici ) were included for the analysis between january and november . the information on bp was obtained over the first hours using various parameters including mean, maximum (max), minimum (cv), and successive variations (sv) for systolic, diastolic bp, and mean bp. we defined major ht as a parenchymal hematoma type (ph ). among the included patients (age, . ; and male, . %), patients ( . %) developed major ht over the first hours after successful recanalization. systolic bp max-min was significantly increased in patients with major ht compared to those without major ht ( . mmhg vs. . mmhg, p = . ) while other bp parameters were not. in addition, systolic bp max-min was significantly associated with symptomatic ht (n= , . %, p = . ). after adjusting for confounders, systolic bp max-min was independently associated with major ht (odds ratio, . ; % confidence interval, . - . ). our results demonstrated that absolute change of systemic bp over the first hours was associated with major and symptomatic ht after successful mechanical thrombectomy after acute ischemic stroke. this suggests that maintaining stable systolic bp is an important factor in possibly preventing major ht after successful recanalization. the benefits of intravenous tissue-plasminogen activator in acute ischemic stroke are highly timedependent. however, there are so many cross-departmental tasks to eligible patent that many stroke centers have difficulty achieving the guideline recommended -hour door-to-needle (dtn) time. we have developed web based visual task management system called "task calc. stroke" (tcs) by using information and communication technology. herein, we performed a trial installation and preliminary evaluation of tcs. the application software of tcs was designed to run on the google cloud platform. tcs alerts the relevant hospital staff to the patient's arrival condition and time, and displayed tasks to be performed and its treatment status by changing color in real time on networked wall-mounted smart devices in the several relevant departments. we started a trial installation of tcs during the daytime from august . we compared lead times before (august to july ) and after (august to july ) trial installation of tcs. trial installation of tcs in our hospital showed successful information sharing. a total of patients included (pre: , post: ) . after the installation, significant reductions occurred in the median time from door to complete blood count time [ . vs. . min, p < . ] and a trend toward a reduction from door to needle time [ . vs. . min, p = . ]. tcs may be useful tool to reduce the lead times of acute stroke patients. tcs is a new approach that has the potential to promote efficiency for acute stroke care. prior history of intracranial hemorrhage (ich) has been considered a contraindication to administration of intravenous recombinant tissue plasminogen activator in acute ischemic stroke, per the original activase fda label and aha/asa guidelines. however, limited data are available on the risks of lysis in patients with prior ich. we performed a cross-sectional study of adult patients who received thrombolysis, using administrative claims data on admissions to acute care hospitals in california between - . diagnosis codes were used to identify patients who received thrombolysis, and to ascertain ( ) a prior diagnosis of ich, including intraparenchymal hemorrhage (iph), subarachnoid hemorrhage (sah), subdural hematoma (sdh), or epidural hematoma (edh); and ( ) relevant comorbidities, including hypertension, smoking, diabetes, heart failure, atrial fibrillation, renal disease, malignancy, and demographic data. we used univariable and multivariable logistic regression to model the odds of in-hospital mortality as a function of prior ich, after adjusting for potential confounders. , patients received thrombolysis during the study period (mean age [sd ], female count , [ %]). of these, patients ( . %) had a documented diagnosis of prior ich on admission. inhospital mortality was % overall, . % for patients without prior ich, and . % for patients with prior ich. in multivariable analysis, all prior ich subtypes remained independently associated with in-hospital mortality, including iph (or . , ci . - . , p < e- ); sah (or . , ci . - . , p < e- ); and sdh (or . , ci . - . , p= . ). only patients had edh and testing was not possible. . % of patients who received thrombolysis during the study period had prior diagnosis of ich. prior ich was found to be significantly associated with in-hospital mortality regardless of ich subtype. we evaluated the association between early neurological improvement (eni) after ert and time spent from symptom onset to recanalization, according to the degree of collateral circulation measured using multiphase cta. patients with anterior circulation occlusion who underwent ert based on a non-contrast brain ct and multiphase cta were evaluated. collateral status was evaluated using a pial arterial filling score, which was developed into a six-point scale. eni was defined as equal to more than %, or as an -point decrement in nihss from baseline. neurological statuses at day and at day (or discharge) were determined by a certified neurologist using nihss. the collateral circulation degree measured by multiphase cta was inversely correlated with baseline stroke severity (p= . ). the proportion of eni at day was significantly lower in patients with poor collateral status (score ~ ) according to the time from symptom onset to recanalization ( - , . %; - , . %; > , . %; p= . ). however, the proportion was similar in patients with a good - , . %; - , . %; - , . %; > , . %; p= . , day or discharge; - , . %; - , . %; - , . %; > , . %; p= . ). collateral status was the best predictor for eni after ert. eni was achieved in only ( . %) patients with poor collateral status, and their time from symptom onset to recanalization was more than minutes. the time window for ert might differ according to baseline collateral status measured by multiphase cta. the current time window for ert within hours from symptom onset to groin puncture could be atrial fibrillation (af) is the most common cardiac arrhythmia among adults. despite of the proven advantage in primary and secondary stroke prevention in patients with af, antithrombotic therapy has been reported to be still underused in many countries. however, there is a little data about the incidence of af and any changing pattern of antithrombotic therapy among patients with af over the past decade in korea. data source for this study were obtained from the nationwide sample cohort comprising , , individuals ( % of entire population in korea) which were established by nationwide health insurance system. during a -year follow-up period, there was , developed af ( . %). the incidence of patients with af remained relatively constant during study period ( . % in vs . % in ). the proportion of patients with antithrombotic therapy increased from . % in to . % in significantly (p for trends < . ). however, the proportion of patients with antiplatelet agents was higher than with oral anticoagulation. af steadily increased over recent years in korea. however, only . % of af patients were receiving antithrombotic therapy. our study demonstrated that there was huge gap between the clinical practice and treatment guideline in antithrombotic medication for af patients in korea over the past decade. ohiohealth (oh) possesses one of the nation's largest neuroscience programs and is the leading volume provider of stroke care in ohio. oh is comprised of hospital-based sites, primary stroke centers, comprehensive stroke center, and a virtual health (vh) stroke network that serves hospitals throughout the state. in august , stroke services at ohiohealth were restructured to enable dedicated clinical time for vh providers, require expertise, training, and quality review participation for stroke responders, streamline activation algorithms to limit hand-offs, and eliminate identified barriers to vh consultation. a month interim analysis was planned to assess the impact of these changes (termed "stroke . "). comparative analyses were performed between the first months of stroke . and the similar time period of the year prior to restructuring. pre-defined metrics included consultation volume, vh response time, iv-tpa time to treatment, research enrollment volume, endovascular referral rate and time to treatment, ischemic stroke (is) observed : expected (o:e) mortality data, and patient retention rate at associate vh sites. during the first months of stroke . , encounters were seen (historical ) with a mean activation to vh log in of . minutes. both volume of patients treated with iv-tpa ( vs. , p< . ) and mean treatment times ( vs. minutes; p< . ) were significantly reduced. mean time to endovascular intervention was less during stroke . ( vs. minutes, p < . ). system-wide o: e mortality was reduced after restructuring ( . vs. . , p< . ), accounting for additional lives saved. acute stroke research enrollment doubled ( vs. ) during this same period. transfer rates to vh hub were unchanged ( vs. %, p = . ). strategic changes in staffing, expertise, vh structure, and access can have profound and positive changes on a well-functioning stroke system. strokes due to cns fungal infections (scfi) are often misdiagnosed. retrospective study of electronically-extracted records in patients with strokes & positive fungal studies, from cerebrospinal fluid (csf) or brain biopsy. other stroke etiologies were excluded. thirteen patients had scfi by a priori exclusion & inclusion criteria. nine were males. mean age was + years. symptoms were mild [nihss ( , . ) (median and iqr)]. focal deficits & headaches (both . %) were common. seventy-percent were immuno-compromised (medications, malignancy, transplant recipients). clinical course was indolent in . %. seventy-percent had poor outcome ( -ltac, -snf, -dead). ninety-two percent had csf pleocytosis (range: - ) while % had csf glucose less than mg/dl (range: - ). seventy-five percent had lymphocytic predominance. seven strokes were from yeasts ( -cryptococcus, -coccidiomycosis, -histoplasma, -candida) and from molds ( -zygomycetes, -aspergillus). sixty-two percent had posterior circulation involvement ( . % yeast vs % molds). there was lepto-meningeal enhancement in % of yeast vs. % of molds infections (p= . ). the basal ganglia (bg) was involved in % of intravenous-drug users (ivdu) vs. % of non-ivdu (p= . ). one had abnormal cns vessel imaging directly attributed to the ischemic lesions. in this series, patients were young, immunocompromised or ivdu. stroke sizes & clinical deficits were modest with no angiographic evidence of vasculitis. majority had csf pleocytosis & hypoglycorrhachia. posterior circulation involvement was typical. lepto-meningeal meningitis was only seen in yeast infections. the bg was spared in non-ivdu but common in ivdu. mechanism of stroke in yeast infections is probably from meningitis & secondary involvement of small perforating branches. mechanism in mold infections in immuno-competent ivdu is probably direct angio-invasiveness in small vessels of the bg. outcomes are poor in spite of therapy. scfi should be considered in selected cases of cryptogenic (recurrent or progressive) strokes with clinical, csf and mri features described. life-threatening bleeding requires prompt reversal of factor xa (fxa) inhibitors. their anticoagulant effects can be reversed with the antidote andexanet alfa. the efficacy of andexanet to reverse bleeding in an apixaban anticoagulated porcine trauma model was investigated. after ethical approval, male pigs (n= ) were given apixaban for days ( mg daily); the sham group (n= ) received placebo. standardized polytrauma by blunt liver injury and bilateral femur fractures were inflicted. minutes post-trauma, animals were randomized (n= per group) to a single andexanet bolus ( , mg), a bolus ( , mg) + infusion ( , mg over hours) regimen, or vehicle (control). blood loss (bl) and hemodynamics were monitored over hours or until death and analyzed by anova (mean±sem). apixaban anti-fxa levels were ± ng/ml with no differences between anticoagulated groups prior to injury. bl in the sham animals was ± ml minutes after injury (total bl ± ml at "x" hours; % survival). anticoagulation with apixaban significantly increased bl minutes after injury ( ± ml; p< . ). controls exhibited a total bl of , ± ml with % mortality (mean survival time = minutes). treatment with a bolus or bolus+infusion of andexanet was associated with a significant reduction in bl versus sham (p< . ) and % survival. two hours after injury, apixaban anti-fxa levels in bolus animals were ± ng/ml, whereas the bolus+infusion regimen resulted in levels of ± ng/ml (p< . ). hemodynamic parameters (e.g., cardiac output) and markers of shock (e.g., lactate) recovered to pre-trauma levels in andexanet-treated groups. clinically and macroscopically, no adverse events were observed. in this study, andexanet effectively and safely reversed apixaban anticoagulation and reduced bl induced by severe trauma under anticoagulation. the bolus alone had a similar impact on survival and bl as the bolus+infusion regimen in this lethal porcine model. current guidelines for management of pain, agitation, and delirium in mechanically ventilated patients in the intensive care unit (icu) recommend an analgesia-first approach to sedation management. however, these guidelines are derived from non-neurologic patient populations leaving uncertainty in their generalization to this population. the purpose of this study was to evaluate implementation of an analgesia-first sedation clinical pathway in the neuroscience icu. a single-center cohort study was performed within the neuroscience icu including patients mechanically ventilated for greater than hours over a time period of three months before and after clinical pathway implementation. providers were educated on the pathway with emphasis on frequent assessment of richmond agitation-sedation scales (rass), critical care pain observation tool (cpot), and confusion assessment method-icu (cam-icu) scores and systematic de-escalation of sedatives through adequate pain and delirium management. outcome measures included frequency and magnitude of rass, cpot, and cam-icu scores, analgesic and sedative medication prescription/administration per day of mechanical ventilation (mv). a total of patients met inclusion criteria ( pre-pathway and post-pathway). there was no statistically significant difference in the median frequency of rass ( . vs. . ) and cpot ( . vs. . ) assessments per day of mv or in median rass (- vs. - ) and cpot ( vs. ) scores. mean acetaminophen usage increased from . % to % (p< . ) post-pathway implementation. there was no statistically significant difference in mean opioid or propofol usage, however a trend toward increased morphine and decreased propofol usage was observed post-pathway. analgesia-first sedation pathway implementation trended towards increased opioid analgesic and decreased sedative use, however only increased acetaminophen usage was significant. this highlights challenges in changing unit-based practices and future directions include focus on the frequency and reliability of pain, agitation and delirium assessment. interdisciplinary coordination and communication remains necessary for effective unit-based practice changes. andexanet alfa (andexanet), a modified, recombinant human factor xa (fxa) molecule, binds and sequesters fxa inhibitors. in a phase study of apixaban, rivaroxaban, edoxaban, and enoxaparin in healthy volunteers, andexanet rapidly reversed pharmacodynamic markers of anticoagulation. here, the ability of andexanet to reverse the anticoagulant activity of betrixaban was investigated. in a randomized, double-blind, phase study in healthy subjects, andexanet (n= ) or placebo (n= ) was administered intravenously following mg po qd betrixaban to steady state ( days). in cohort (andexanet bolus only), subjects (n= ) received a -mg andexanet bolus hours after the last betrixaban dose (day ) or placebo (n= ). in cohort (andexanet bolus plus -hour infusion), subjects (n= ) received a mg andexanet bolus hours after the last betrixaban dose, followed by a -hour infusion of andexanet ( mg/min) or placebo (n= ). endpoints included safety and pharmacodynamic markers of anticoagulation reversal. following treatment with betrixaban in cohort , andexanet rapidly decreased anti-fxa activity from . ± . to . ± . ng/ml, while the anti-fxa levels following placebo were largely unchanged ( . ± . to . ± . ng/ml). unbound betrixaban plasma concentration decreased from . ± . to . ± . ng/ml with andexanet, but remained constant following placebo administration ( . ± . to . ± . ng/ml). similar results were observed in cohort following andexanet bolus ( minutes after bolus), and the effects were maintained during the -hour infusion of andexanet. for cohort , thrombin generation was restored in / ( %) and / ( . %) of andexanet-administered and placebo subjects, respectively. for cohort , thrombin generation was restored in / ( . %) of andexanet subjects versus / ( . %) of placebo subjects. andexanet was well tolerated; there were no thrombotic events or serious/severe adverse events. andexanet was well tolerated and rapidly reversed anticoagulation effects of betrixaban in healthy subjects. these and other studies indicate that andexanet could be a universal antidote for fxa inhibitors. andexanet alfa (anxa), a recombinant human fxa molecule, reverses the anticoagulant activity of fxa inhibitors. in studies of healthy volunteers, anxa showed dose-dependent reversal of direct and indirect fxa inhibitors in tissue factor (tf)-initiated thrombin generation (tg). we compared rivaroxabaninduced inhibition of tg initiated via the extrinsic pathway (tf) versus intrinsic pathway (non-tf). tf-initiated tg was measured using a calibrated automated thrombogram (cat) and ppp-reagent. non-tf-initiated tg was measured using cat and actin fs. anti-fxa activity was measured using an anti-fxa chromogenic assay. pooled plasma was spiked with rivaroxaban or rivaroxaban+anxa; tg, anti-fxa activity, and clot formation were measured. for low tf-initiated clot formation, thromboelastography profiles were measured. anxa alone had minimal effect on endogenous thrombin potential (etp). anxa fully reversed rivaroxaban-induced anticoagulation in the actin fs assay, independent of anxa-tfpi interaction. modulation of tf activity was assessed by correlating etp versus anti-fxa activity with rivaroxaban or rivaroxaban+anxa. rivaroxaban dose-dependently inhibited tf-initiated tg as anti-fxa activity increased. at similar anti-fxa levels, rivaroxaban+anxa had higher etp than rivaroxaban alone, but not in the actin fs assay. clot formation was studied in plasma using thromboelastography without rivaroxaban. anxa did not affect thromboelastography parameters, with/without recombinant tissue plasminogen activator (rtpa). when low tf initiated clot formation without rtpa, anxa reduced the thromboelastography-r parameter, but not maximum amplitude. the fibrin clot was lysed at low rtpa, resulting in well-segregated coagulation and fibrinolysis. with the optimal rtpa, fibrin clots formed at each tf concentration were compensated by the fibrinolytic activity of rtpa. without a fxa inhibitor, anxa had minimal effect on tf or actin fs-initiated tg with no direct effect on rtpa function. anxa dose-dependently and completely reversed rivaroxaban-induced inhibition of tg initiated by intrinsic or extrinsic pathways, but had different effects on etp due to the anxa-tfpi interaction. there is a growing body of evidence relating poor outcomes to off-hour management. studies investigating the effect of overnight extubation (oe) have produced mixed results, and limited data is available for brain-injured patients. there may also be tendency to limit oe due to decreased staffing levels at night. we sought to determine the safety of oe and risk factor profiles associated with extubation failure (ef) in this cohort. we conducted a retrospective review of mechanically ventilated patients admitted to a single-center in-house database. exclusion criteria included limitations in care, tracheostomy placement, selfextubation, and death prior to extubation. the primary outcome was ef defined as non-elective endotracheal intubation within hours. ef rates were compared between daytime ( am - : pm) and overnight ( pm - : am) extubation cohorts. in-hospital mortality served as a secondary outcome. amongst identified patients, ( . %) underwent daytime extubation (de) and ( . %) oe. ef was indifferent between de and oe ( . % and . % respectively; p= . ). however, multivariable adjustment for clinical severity indicators suggests higher ef for oe (or: . , ci: . - . ; p= . ). compared to de, oe was more likely performed in elective post-operative patients ( . % vs . %; p= . ) with lower apache-ii scores (median vs ; p= . ), and shorter durations of mv (median . vs . days; < . ). higher apache-ii score, longer duration of mv, and admission diagnoses of acute vascular injury or neuromuscular disease were associated with ef. there was no difference in mortality (p= . ). in our cohort, oe was not associated with increased ef or mortality. our results suggest that oe can be performed safely if standard extubation criteria are met in low-risk patients. these data provide a basis for subsequent more robust studies. case series have reported reversible left ventricular dysfunction, also known as stress cardiomyopathy or takotsubo cardiomyopathy, in the setting of acute neurological diseases such as subarachnoid hemorrhage. the nature of the association between various neurological diseases and takotsubo remains incompletely understood. we performed a cross-sectional study of all adults in the national inpatient sample, a nationally representative sample of u.s. hospitalizations, from - . our exposures of interest were primary diagnoses of acute neurological disease, defined by icd- -cm diagnosis codes. our outcome was a diagnosis of takotsubo cardiomyopathy. binary logistic regression models were used to examine the associations between our prespecified neurological diagnoses and takotsubo cardiomyopathy after adjustment for demographics. we identified , , adults with a primary acute neurological diagnosis and , , patients admitted to the hospital without a primary acute neurological diagnosis. among neurological diagnoses, subarachnoid hemorrhage (odds ratio [or], . ; %ci, , status epilepticus (or, . ; % ci, . - . ), transient global amnesia (or, . ; % ci, . - . ), and meningoencephalitis (or, . ; % ci, . - . ) were most strongly associated with takotsubo cardiomyopathy. weaker associations were present for ischemic stroke (or, . ; % ci, . - . ) and migraine headache (or, . ; % ci, . - . ). intracerebral hemorrhage and guillaine-barre syndrome were not significantly associated with takotsubo cardiomyopathy. in our multivariable model, female sex was significantly associated with takotsubo (or, . ; % ci, . - . ). we found associations with takotsubo cardiomyopathy for several acute neurological diseases besides subarachnoid hemorrhage. gram-negative meningoventriculitis (gnmv) causes significant morbidity and mortality. in addition to intravenous antibiotics, intra-thecal (it) or intraventricular (iv) antibiotics may be used to treat central nervous system (cns) gram-negative infections, including multi-drug resistant gnmv. there are limited studies on the effect of direct cns administration on cerebrospinal fluid (csf) cultures, csf routine parameters and other clinical outcomes. we conducted a retrospective chart review of all patients who received it or iv antibiotics for gnmv since . demographics, source of illness, severity of illness (sofa), intravenous and it/iv antibiotic choice and csf microbiological, drug level and routine analysis were collected. time to pathogen clearance from csf culture was also measured. there were inpatient encounters where iv/it antibiotics were given for gnmv during our study period, of which were cared for in a neurosciences intensive care unit. antibiotics utilized were: gentamicin ( ), colistimethate sodium ( ), amikacin ( ), and tobramycin ( ). the most common pathogens were p. aeruginosa ( ), k. pneumoniae ( ), enterobacter sp. ( ) and e. coli ( ). prior to dosing, median csf white blood cell (wbc) count, protein and glucose was /ul, mg/dl and mg/dl, respectively. it/iv antibiotics were dosed a median of times per patient and clearance of csf culture occurred in a median of days. there were significant changes in csf wbc (p< . ), protein (p<. ) and glucose (p<. ) between the first and last dose of iv/it antibiotics. twenty-five ( . %) patients survived to discharge, ( . %) were confirmed alive at months. patients who survived to discharge went to rehabilitation ( ), home ( ), long-term acute-care ( ) and skilled nursing facility ( ). it and iv antibiotics significantly improve csf wbc, protein and glucose profiles and clear csf cultures in patients with gnmv. it and iv administration may provide additional benefit to systemic therapy. gram-positive organisms are the most common cause of meningo-ventriculitis. systemic antimicrobial therapy may fail to achieve adequate cerebrospinal fluid (csf) concentrations, particularly against organisms with higher minimum inhibitory concentrations, such as mrsa and vre. direct intraventricular (iv) or intra-thecal (it) administration may be beneficial as they can facilitate high csf levels at the site of infection. there are limited studies on the effect of direct central nervous system (cns) administration of antibiotics on csf cultures, csf routine parameters and other clinical outcomes. we conducted a retrospective chart review of all patients who received it/iv antibiotics for grampositive meningo-ventriculitis since . demographics, source of illness, severity of illness (sofa), intravenous and it/iv antibiotic choice and csf microbiological, drug level and routine analysis were collected. time to pathogen clearance from csf culture was also measured. there were inpatient encounters where iv/it antibiotics were given for gram-positive meningoventriculitis during our study period, of which were cared for in a neurosciences intensive care unit. antibiotics utilized were: vancomycin ( ) and daptomycin ( ). the most common pathogens were staphylococcus sp. ( ), enterococcus sp ( ), and streptococcus sp ( ). prior to dosing, median csf white blood cell (wbc) count, protein and glucose was /ul, mg/dl and mg/dl, respectively. it/iv antibiotics were dosed a median of times per patient and clearance of csf culture occurred in a median of days. there were significant changes in csf wbc (p< . ), protein (p<. ) and glucose (p=. ) between the first and last dose of iv/it antibiotics. twenty-nine ( . %) patients survived to discharge, ( . %) were confirmed alive at months. it and iv antibiotics significantly improve csf wbc, protein and glucose profiles and clear csf cultures in patients with gram-positive meningo-ventriculitis. it and iv administration may provide additional benefit to systemic therapy. use of prothrombin complex concentrate (pcc) for urgent reversal of anticoagulant associated coagulopathy is increasing, and at the university of illinois hospital (uih), an anti-thrombotic reversal guideline was developed in may in order to assist licensed practitioners in choosing the appropriate reversal agent, optimal dosing, and improve timely administration pcc. the current study examined the safety and efficacy of pcc used for the urgent reversal of anticoagulant associated coagulopathy before and after the development of the anti-thrombotic reversal guideline. this was a retrospective chart review of adult patients who received pcc as the only hemostatic agent at the uih from jan to april . the primary endpoint was hemostasis and secondary endpoints included thromboembolic events and time to pcc administration. there were and patients who received pcc before and after the anti-thrombotic reversal guideline, respectively. frequent cause of coagulopathy was warfarin ( % and %, respectively), and frequent indication for pcc was acute intracranial hemorrhage ( % and %, respectively). -factor pcc was more frequently used before the guideline and -factor pcc was more frequently used after the guideline. in patients presenting with warfarin induced major bleeding, target inr < . was achieved in % and % of these patients before and after the guideline, respectively. clinical assessment of bleeding cessation from direct oral anticoagulant (doac) therapy was difficult to assess. thromboembolic event was observed in % and % of the patients, respectively. median time to pcc administration from its initial order was minutes and minutes, respectively. hemostasis was similarly observed in the warfarin group before and after the development of reversal guideline, but more thromboembolic events were observed before the reversal guideline. in order to further reduce the pcc administration time, a change in workflow has been made to administer pcc in timely manner. dexmedetomidine, a selective alpha- adrenoreceptor agonist inhibiting sympathetic neuronal activity, is a mild sedation agent. two recent case reports showed reduced norepinephrine (ne) requirement in septic shock with clonidine, a less selective alpha- agonist. increased vasopressor responsiveness (vr) was also observed with dexmedetomidine in cardiovascular surgical settings. sympatholytic effects of the alpha- agonists reverse vascular desensitization due to high levels of sympathetic activity in sepsis. depletion of intra-neuronal catecholamines with reserpine has shown to increase vr. in septic sheep infused with escherichia coli, clonidine reduced renal sympathetic tone and restored vr. additionally, alpha- agonists have shown to decrease pro-inflammatory cytokines and reduce mortality, improve capillary perfusion deficit, and lower arterial lactate in animal sepsis models. a prospective trial in human septic shock is in the pipeline. we report decreases in vasopressor requirement with initiation of dexmedetomidine in two patients with brain injury. a -year-old woman presented with a high-grade subarachnoid hemorrhage and concomitant reverse takatsubo cardiomyopathy. her clinical course was complicated by septic shock secondary to aspiration pneumonia at admission. when dexmedetomidine was started after hours of ne infusion, a steady decrease in ne dosage was observed until its discontinuation. increased vr was also observed in a year-old man being treated for new onset refractory status epilepticus. on hospital day , the patient continued to have stimulus-induced seizures on ketamine, midazolam and pentobarbital infusions and required ne to maintain an adequate mean arterial pressure. when dexmedetomidine was added, a decrease in ne infusion was observed within an hour and continued for six hours until the patient no longer required vasopressor therapy. these findings are consistent with aforementioned reports of restored vr by alpha- agonists in septic shock, and warrant further investigation of possible beneficial effects of attenuated hyperadrenergic state conferred by alpha- agonists in various neurocritical care settings. decreasing the amount of time a patient remains intubated has been shown to reduce multiple negative outcomes. by extubating these patients earlier, risk of infection, prolonged immobility, and delirium are reduced. in early , this nsicu was chosen to participate in the society of critical care medicine's icu liberation collaborative. the collaborative was focused on implementation of the abcdef bundle or icu liberation. the successful implementation of the bundle led to a decrease in the amount of time neurocritically ill patients were intubated. the bundle elements began to be rolled out in june (end of st quarter). included in the bundle's roll out was the creation of a respiratory clinical specialist role to help the interprofessional team with the respiratory components of the bundle. this role was a full time respiratory care practitioner who was dedicated to the nsicu and helped to ensure standards were being met. additionally, as a part of the bundle's implementation, a spontaneous awakening trial and spontaneous breathing trial algorithm was developed and initiated. this algorithm relied on interprofessional collaboration between nursing and respiratory therapy with communication to the provider and was rolled out in september (end of rd quarter). ventilator o/e for : st quarter- . , nd quarter- . , rd quarter- . , th quarter- . ventilator o/e for : st quarter- . , nd quarter- . the bulk of the research conducted that proved the benefits of the bundle elements has been completed in medical and/surgical patient populations. the neurocritical care patient population is very specialized and has several nuances that may impact the way the various elements need to be implemented. through this process, we have found that the techniques suggested within each element can positively impact the neurocritical care patient population. the cognitive reserve hypothesis refers to inter-individual differences in the ability of patients to cope with brain pathology. cognitive reserve can be measured by surrogate markers such as education and occupation and has shown to be an important predictor of outcomes in alzheimer disease, multiple sclerosis and traumatic brain injury. in this prospective longitudinal cohort study we determined whether cognitive reserve measured as number of years of education and employment status predicted -month functional outcome of ncc patients. demographic and clinical data, including number of years of education and occupational status, were collected. at three months after discharge, glasgow outcome scales (gos) were collected via telephone from patients or surrogate respondents. gos scores were categorized into 'good' or 'poor' outcome (gos - ). from march to july , / patients with -month follow-up data were included. mean age was ± years, ( %) were male, with stroke as the predominant admitting diagnosis.the two groups with good vs poor outcomes did not differ in age, gender or race in univariate analysis although employment status was statistically different in the two groups. in multivariate logistic regression neither employment nor education was a significant predictor of good vs poor outcome (p = . , p = . ). prognostication in neurocritical care patients is difficult. the effect of cognitive reserve needs to be studied further. our current sample size is small and as enrollment continues, we will determine the relationship between cognitive reserve and -month functional outcome. fever commonly occurs in patients with spontaneous intracerebral hemorrhage (sich). however, it is non-infectious in the majority of cases. blood cultures (bcx) are often obtained as part of a fever workup, yet their utility may be limited and false-positive results may potentially compromise patient care. we hypothesized blood cultures in the first hours would more likely be false-positive. we performed a retrospective chart review of patients admitted to a tertiary medical center with a diagnosis of spontaneous intracerebral hemorrhage. patients with secondary causes of ich as well as institution of comfort measures only were excluded. data obtained included demographics, clinical parameters of ich and blood culture results. blood culture results and charts were reviewed for adjudication of false-positive and true-positive cultures. of included patients with sich, patients ( %) had blood cultures obtained. cultures were positive, of which were classified as false-positive and as true positive. false positive results were more common in the first days ( vs. ), while true positive results were more common after the first hours ( vs. ) (p= . ). early blood cultures in patients with sich are more commonly non-infectious. in line with prior published data, our results demonstrate the high cost and limited yield for blood cultures within the first hours. predictive energy expenditure (pee) equations are commonly used in lieu of indirect calorimetry (ic) due to cost and limited resources; however, these equations may not be as accurate as ic in estimating resting energy expenditure (ree) in critically ill patients. the purpose of this study is to compare pee and measured energy expenditure (mee) in critically ill adults with acute brain injury. this was a retrospective review of adult patients admitted with acute brain injury between may st, and april st, who had ic performed. three predictive equations (pe), harris benedict (hbe), penn state university, and mifflin st jeor (msj), were used in comparison to ic results. subgroup analyses included a modified aspen weight-based equation, stratifying patients based on bmi and type of acute brain injury. patients met inclusion criteria. comparing the pee estimated by the three predictive equations to the mee from ic found no significant difference. high degrees of interpatient variability were discovered in each anova analysis, with standard deviations ranging from - %. despite no difference found among pee and mee, pearson's correlations indicated weak associations when hbe, penn state, and msj were individually compared to mee (r-values = . , . , and . , respectively). in patients with a bmi < kg/m , a significant difference was found (p-value= . ) with pee underestimating the ree. additionally, in aneurysmal subarachnoid hemorrhage a significant difference was observed between pee and mee( p-value= . ). the results of this study highlight the importance of using ic whenever feasible due to the interpatient variability of the ree of critically ill patients with acute brain injury. although predicative equations appear to have similar estimations as ic, interpatient variability warrants more accurate measurement with ic to optimize nutrition in patients with acute brain injury. introduction -factor prothrombin complex concentrate (pcc) should be administered as soon as possible for reversal of anticoagulation in the setting of life-threatening bleeding or urgent procedures. limited information is available on the safety, efficacy, and time to administration of pcc when administered at high infusion rates. on march , grady health system implemented a rapid pcc administration strategy while attempting to reduce times from order entry to administration as a quality improvement initiative. this irb-approved, retrospective evaluation includes pcc administrations days pre-and post-protocol implementation. after protocol implementation, pcc doses were prepared in up to four, -ml syringes, dependent on the ordered dose. each syringe was administered over minutes, not exceeding a rate of iu/minute. the primary objective of this study is to evaluate the safety of a rapid administration strategy for pcc. secondary objectives include turn-around times and effectiveness of inr reversal in patients previously on warfarin. results unique pcc administrations were identified: administrations in the pre-cohort and in the postimplementation cohort. most pcc administrations were in the setting of spontaneous or traumatic intracranial hemorrhage. there were no infusion-related adverse events documented with the exception of a possible pcc infiltration post-implementation which resolved with supportive care only. the median order entry to administration time was higher in the post-implementation group ( vs. minutes). administrations in the pre-cohort and administrations in the post-cohort were for warfarin reversal. a greater percent of patients previously on warfarin reversed to an inr < . in the post-cohort compared to the pre-cohort, . % vs . %, respectively. this retrospective evaluation suggests that rapid intravenous push administration of -factor pcc is safe and effective. time to administration was longer after implementation of rapid pcc administration and may have been due to operational limitations. icu readmission is defined as a return to the icu during the same hospital admission. there are multiple studies related to medical and general surgical recidivism, however there is limited data on icu readmissions following spine surgery. the aim of this study was to evaluate factors associated with icu readmissions following spine surgery. patients requiring icu admission following spine surgery from june to june were studied. variables included age, gender, icu and hospital disposition, icu and hospital length of stay, bmi, comorbidities, surgical location, number of previous surgeries and vertebra manipulated, estimated blood loss, post op blood transfusions, and cause of readmission. a : matched control group based on age, bmi and location of surgery was identified. thirty-two patients required readmission following spine surgery during the study period. there was a higher prevalence of preoperative atrial fibrillation in the readmission group ( % vs. %, p= . ). ebl ( vs ml, p= . ) and lowest maps ( vs . mmhg, p= . ) were not significantly different in the two groups. we found a higher mortality rate ( % vs %, p= . ), longer icu ( . vs . hours, p= . ) and hospital los ( . vs . days, p= . ) in the readmission group. respiratory distress ( %) was the most common reason for readmission followed by cardiovascular instability ( %). discharge rates to inpatient rehabilitation and nursing facilities were similar for both groups; however % of the control group went directly home as opposed to % of the readmission group. complex spine patients who experience icu recidivism have a longer hospital stay and incidence of death within years of their index procedure. they are less likely to be discharged home. preoperative a-fib correlates with increased incidence of readmission to icu post-operatively. further studies are needed looking at post operative fluid and pain management. to demonstrate the feasibility of exenatide infusion for hyperglycemia following acute brain injury. adult patients with acute brain injury and having two blood glucose concentrations > mg/dl and was administered within hours of admission and continued per protocol for a maximum duration of hours. the primary endpoint was feasibility (< % of subjects experiencing severe hypoglycemia (< - mg/dl). descriptive endpoints were also collected. data is presented as medians [interquartile range] or percentages. a total of eight patients received exenatide (age . years [ . , . ], . % male, . % caucasian, . % history of diabetes, a c . % [ . , . ]). admitting diagnoses were intracerebral hemorrhage (n= ), acute ischemic stroke (n= ), subarachnoid hemorrhage (n= ), and subdural hematoma (n= ). glascow coma score was . [ . , . ] and sequential organ failure assessment was . [ . , . ]. based upon predefined criteria, feasibility was met with % of subjects experiencing severe hypoglycemia, . % achieving the blood glucose goal, and % experiencing nausea requiring discontinuation. blood glucose was controlled during the -hour exenatide infusion ( intravenous exenatide infusion is feasible for the treatment of hyperglycemia following acute brain injury. extubation failure remains a common complication in critical care patients, and is associated with increased intensive care unit and hospital length of stays, hospital costs, morbidity and mortality. the most common cause of reintubation is laryngeal edema, often identified by the presence of a high pitched inspiratory whistling sound known as post-extubation stridor (pes). providers in the neurocritical care unit (nccu) at a large urban academic medical center noted higher than normal rates of pes. to reduce the rates of pes and reintubation without delaying extubation, a clinical pathway was created by an interdisciplinary team. the purpose of the pathway was to aid in the identification of patients expected to develop pes and guide prophylactic treatment. prior to project implementation, all providers in the nccu completed hands on training with practice in completing the pathway in the form of a checklist. during the week implementation phase, checklists were completed on all intubated patients daily during rounds. during the week trial, there were a total of ventilator days. there were completed checklists, yielding an . % compliance rate for utilization of the clinical pathway. of the patients who were extubated during the trial, had a checklist completed, generating . % compliance on the day of extubation. a chi-square analysis was performed to evaluate outcomes following all non-palliative extubations during the week pre-implementation (n = ) and post-implementation (n = ) periods. implementation of the pathway was associated with a statistically significant reduction in rates of pes ( , n = ) = . , p< . , reintubation ( , n = ) = . , p< . and reintubation due to pes, ( , n = ) = . , p< . . the clinical pathway implemented in our nccu was safe and effective in reducing rates of pes, reintubation and reintubation due to pes. agency for healthcare research and quality (ahrq) identified postoperative deep vein thrombosis (dvt) or pulmonary embolism (pe), also commonly referred to as venous thromboembolism (vte), as one of the complications acquired in the hospital and thus developed a mechanism to report its rate using administrative data. postoperative vte rate reduction became top priority for the university of illinois (uih) due to its high yearly rate, especially among patients in the neurosciences intensive care unit (nsicu). therefore, a quality improvement team in the nsicu implemented vte bundle and analyzed its effect on the vte rate. the vte bundle was initiated on all neurosurgery and neurology patients admitted to the nsicu since march . vte bundle included lower extremity doppler ultrasound within hours of admission, vte education provided to patient or family member within hours of admission, and daily surveillance on proper use of mechanical sleeves and the mechanical device, low-dose heparin initiation and maintenance therapy, and documentation of activity status. the nursing staff were encouraged to follow the early mobilization protocol. mean vte rate was . per cases approximately -year before and . per cases approximately -year after the implementation of vte bundle. the rate of compliance was high on all aspects of vte bundle, especially on correct placement of ipc sleeve > %; functioning ipc device > %; low-dose heparin > %; documentation of activity status > %. no adverse effects were noted (i.e., skin breakdown, major bleeding) during the study period. this was the first time in years at uih, the postoperative vte rate was reduced among nsicu patients based on the ahrq reports. the reduction may partly be attributed to the implementation of vte bundle; however further evaluation need to be performed to determine the effect size of vte bundle. increasing evidence suggests that large volume infusions of . % sodium chloride (nacl) for resuscitation are associated with hyperchloremic metabolic acidosis and renal vasoconstriction leading to an increased risk of acute kidney injury (aki). in patients with neurologic injury, hypertonic ( . % or %) nacl or sodium acetate (naacetate) may be required for therapeutic hypernatremia, treatment of cerebral salt wasting or elevated intracranial pressure. the primary aim of this study was to determine the incidence of aki in neurologically injured patients receiving intravenous hypertonic nacl and in those who were switched to hypertonic naacetate based on provider preference. this single-center, retrospective study compared patients that received only hypertonic nacl to patients that were switched to naacetate. data was collected to assess renal function, hyperchloremia, and metabolic acidosis. a total of patients were screened and of those were included. the patients who were switched from nacl to naacetate (n= ) had a greater incidence of aki ( % vs. %, p< . ) and hyperchloremia ( % vs. %, p = . ) compared to patients who received only nacl (n= ). the incidence of metabolic acidosis was increased but not statistically significant ( % vs. %, p = . ). on average, hypertonic nacl was switched to hypertonic naacetate on day of treatment with a mean chloride of . meq/l at the time of the switch. there was no statistical difference in the administration of nephrotoxic antibiotics, mannitol, vasopressors, or contrast dye between the two groups. the receiver operating characteristic (roc) analysis demonstrated that if a patient received greater than meq of chloride over days they were more likely to develop aki (sensitivity %, specificity %, p= . , auc . ). neurologically injured patients receiving hypertonic sodium therapy requiring a switch to hypertonic naacetate had an increased incidence of hyperchloremia and aki. in-hospital complications following acute neurological injury has been a topic of extensive research to help reduce the morbidity and mortality among the patients. however, the incidence and prevalence of in-hospital infections following an acute neurological injury at the national level has never been studied. the aim of our study is to determine the frequency and prevalence of in-hospital complications among different patient groups admitted following acute neurological injury. we identified patients with primary diagnosis of ischemic stroke (is), subarachnoid stroke (sah), intracerebral hemorrhage (ich), status epilepticus (se), meningitis, encephalitis and traumatic brain injury (tbi) from nationwide inpatient database ( - ) through using the respective icd- codes. common in-hospital complications among the above-mentioned diagnoses through using their respective icd- codes patients with primary diagnoses of is (n= ), sah (n= ), ich (n= ), se (n= ), meningitis (n= ), encephalitis (n= ), tbi (n= ) were identified. in-hospital events such as myocardial infarction (mi), sepsis, pneumonia, deep venous thrombosis (dvt), pulmonary embolism (pe), urinary tract infections (uti), and gi bleed were identified and compared among different patient groups. patients with se were noted to experience higher systemic complications, mi ( . %), sepsis ( . %), pneumonia ( . %), dvt ( . %), uti ( . %), gi bleed ( . %). patients admitted with meningitis had a higher incidence of sepsis ( . %), pneumonia ( . %), dvt ( . %), pe ( . %) and uti ( . %) compared to the other groups. uti was the most common in-hospital complication observed. based on our analysis, we report a higher incidence of urinary tract infections among all patients admitted following acute neurological injuries. patients with primary diagnosis of status epilepticus experienced more systemic complications compared to the other diagnoses. macroglossia is a phenomenon that has been documented in association with prolonged neurosurgical procedures, brainstem injury, phenobarbital administration, and venous/lymphatic congestion of the tongue. however, exact causation of this condition in the neurocritical care population remains unclear. patients with macroglossia face significant risk for airway compromise. no interdisciplinary patient safety and management protocol exists. patients admitted to two neuro icu's within a single health system between - were reviewed. twenty-five patients with macroglossia were identified. an interdisciplinary patient management protocol was created, instituting airway safety standards, oral care directives, and interventions to promote symptom resolution. early consultation to oral and maxillofacial surgery and consideration of early tracheostomy was recommended. seventeen patients ( %) were women. age ranged from - years. the majority ( / ) of patients were african american. primary diagnoses included status epilepticus ( / ) and stroke ( sah, ais, ich). nineteen patients received antiepileptic medications before diagnosis. average gcs at symptom onset was . [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] and at time of discharge was . [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] . median symptom onset was hospital day [ - ]. twenty patients ( %) required tracheostomy. nine ( %) experienced symptom resolution by hospital discharge. two patients received botulinum toxin injection; both experienced symptom resolution. lingual massage was performed in two patients; in both patients, tongue swelling resolved. tongue lacerations occurred in / patients ( %), although most were observed following macroglossia onset, ruling out trauma as an inciting event. chlorhexidine oral rinse was discontinued for all except five patients due to concern for angioedema. endotracheal tube was dislodged in two patients, complicating reintubation, although successful. no trend in pre-existing allergies or antibiotic regimen was apparent. macroglossia is a relatively uncommon but high-risk condition in the neuro icu that warrants further study. care of patients with macroglossia should be standardized in order to ensure airway safety. an interdisciplinary approach is recommended. one of the biggest challenges of magnetic resonance imaging (mri) examination is the acquisition of high-quality diagnostic images, as it requires the neurological intensive care unit (nicu) patients to keep still for a significant time. in situations with poor patient cooperation, unplanned sedation is inevitable, which can lead to complications such as desaturation and hypotension. we investigated the incidence and factors related to complicated mri examinations (mri-c) in patients admitted to the nicu. we designed a retrospective study to review the data of patients who had an attempt to undergo brain mri during stay in the nicu between july and august . the mri-c group was defined when a patient met one of following criteria: ) required sedation for mri examination due to irritability mmhg or required inotropic agents, ) developed cardiac or respiratory arrest. of patients, ( . %) developed mri-c. the most common cause of mri-c was unexpected irritability at the mri room. among patients with mri-c, ( . %) patients required unplanned sedation; , desaturation; , hypotension; none, cardiac or respiratory arrest. higher apache ii scores (p = . ) and lower gcs scores (p = . ) on admission and use of sedative agents during critical care in the nicu were associated with mri-c (p < . ). in addition, patients with mri-c had longer mri scan time than those without mri-c (p = . ). many of neuro-critically ill patients undergo unsafe mri scans. our findings suggest that severity of illness and use of sedative agents during management in the nicu were factors related to mri-c. introduction: fulminant hepatic encephalopathy (fhe) with diffuse cerebral edema has dismal prognosis if transplantation is not performed. novel therapeutic interventions may change this outcome. we reviewed all cases with fhe admitted to our hospital since . in , we developed a multidisciplinary management protocol, mandating transfer of patients entering grade from other icus to the neurosciences-icu (nicu) for intracranial pressure (icp) management. multiple interventions were utilized including coagulopathy reversal with factor vii and prothrombin complex concentrate (pcc, kcentra), icp device placement, osmotherapy, aggressive ammonia lowering regimen with lactulose and rifaximin, early renal replacement therapy, mild hypothermia for refractory icp, in conjunction with liver transplantation candidacy investigation. results: twenty-four patients ( women, mean age of all patients years) were admitted; seven were managed in the micu/sicu and in the nicu. the etiology of fhe was acetaminophen toxicity in % of patients. the model for end-stage liver disease (meld) admission scores and liver enzymes between the micu/sicu and the nicu were not different (mann-whitney test). although the nicu admission ammonia level was higher than the micu/sicu ( . vs . , p = . ), the lowest achieved ammonia was lower in the nicu ( . vs . , p = . , mann-whitney). patients received icp monitoring (all in the nicu plus in the sicu) and the highest icp recorded was mm hg. the preand post-coagulation reversal inr were . and . , p= . , wilcoxon test). seven patients in the nicu received hypothermic treatment. mortality in the micu/sicu was . % ( / ) and in the nicu . % ( / ), p = . (chi square test). conclusion: a multidisciplinary approach centered around anti-cerebral edema protocol-driven management based on novel interventions may improve the outcome of patients with fhe. catheter-associated urinary tract infections (cauti) are among the most common health-care associated infections (hcais), (gould, ) . neurological patients in the critical care setting are particularly at risk for cauti due to cognitive, motor, and sensory deficits. in the neuro intensive care unit, despite following recommended cauti reduction bundle guidelines, cauti rates continued to rise over the last five years with rates reaching . per catheter days. in january of , the unit implemented a cauti taskforce to perform a literature review of best practices and subsequent : peer training and education targeting cauti reduction. in an analysis of organisms causing the infections, e. coli and enterococcus bacteria accounted for more than % of cautis on the unit. the taskforce (comprised of staff nurses) focused on fecal management, proper cleaning technique, and proper indications of indwelling urinary catheter necessity. using training videos, indwelling urinary catheter care checklists, and real-time feedback on technique, the taskforce performed : training with bedside staff over four weeks. to ensure undivided attention, the taskforce worked in pairs enabling one trainer to teach and observe the staff member receiving training while the second trainer provided the necessary clinical duties for the trainee's patients. after implementation, the cauti rate decreased to . for january-march and . for april-june , lowering total cauti events to for fy compared to for fy . implementing a : training program focused on fecal management, cleaning techniques, and appropriately timed catheter removal can reduce cauti rates in the neuro critical care setting. brain aneurysms can be treated with coil embolization or flow-diverting devices. thromboembolism is a major complication of aneurysmal coil embolization, with an incidence as high as % . new flowdiverting devices have been designed to have a mesh with high coverage area and high flexibility to facilitate the redirection of blood flow. these features can induce blood stagnation and thrombosis. to reduce the risk of thrombosis, the common but unproven practice of dual antiplatelet therapy with aspirin and clopidogrel has been implemented from the cardiac literature. despite some favorable outcomes, clopidogrel, "non-responsiveness" has been reported to be present in as low as % to as high as % making this agent not optimal. this will leave practitioners with other oral p y alternatives such as prasugrel and ticagrelor that have not been studied widely in this setting. it is therefore likely that controversy exists among practitioners regarding the use of optimal antiplatelet agents in neurointerventional procedures. we hypothesized that practices in regards with the use of oral antiplatelets in neurointerventional procedures are likely heterogeneous and different from state to state. by using an electronic survey, we would like to identify different practices surrounding the use of oral anti-platelets in neuro-endovascular centers in the united states. an electronic survey will be distributed via the web using survey monkey (seattle, wa). the survey will be posted on the neuro-critical care society (ncs) web page. all practicing neuro icu or stroke physicians, pharmacists, physician assistants, or nurse practitioners are eligible to respond to this survey. this survey is approved by the johns hopkins hospital irb and the ncs research committee. centers have completed the survey at this point. the results will be analyzed after the closing date of survey ( / / ). to be completed myasthenia gravis (mg) crisis and guillain-barre syndrome (gbs) are immune mediated diseases that may require mechanical ventilation as part of their management if severe. comparative analysis of outcomes in terms of length of stay, disability, and mortality between these two disease entities at national level is not reported mechanically ventilated patients with primary diagnosis of guillain-barre syndrome and myasthenia gravis were identified from the nationwide in-patient sample (nis) database for the years to mechanically ventilated mg patients (n= , mean= +/- . years) were older compared to gbs patients (n= , mean= . +/- . years, p= . ). medical co-morbidities were significantly higher in mg patients (diabetes mellitus, congestive heart failure, coagulopathy, chronic lung disease and dyslipidemia) whereas significantly higher nicotine dependence and alcohol abuse were noted in gbs. significantly higher in hospital complications of pneumonia and urinary tract infection were noted in gbs. disease severity measured by apdrg severity index and rate of treatment with intravenous immunoglobulin and plasma exchange was comparable. length of stay ( . ± . days , p < . ); hospital charges ( $ . ± . vs . ± . p = . ) ; moderate to severe disability ( . % vs . % p < . ) were significantly higher for gbs patient compared to mg. inhospital mortality was comparable ( . % gbs vs . % mg, p = . ). in multivariate analysis after adjusting for confounders including treatment, myasthenia gravis patients had significantly less disability (or . ( % ci . - . ) and shorter length of stay (or . , % ci . - . ). mechanically ventilated gbs patients have higher in-hospital complications, length-of-stay, and disability compared to mg. this may reflect a delay in diagnosis of gbs at admission and poor response to immunotherapy in certain gbs variants. betrixaban is an inhibitor of factor xa (fxa) for prophylaxis of venous thromboembolism (vte) in at-risk patients hospitalized for acute medical illness. a phase trial (apex) compared extended-duration anticoagulation with betrixaban to enoxaparin in acute medically ill patients; the effect of patient characteristics on population pharmacokinetics and exposure-response relationships is analyzed here. patients received betrixaban ( - days; n= , ) or enoxaparin ( ± days; n= , ). the primary efficacy and safety endpoints were composite occurrence of vte events and incidence of major bleeding, respectively. betrixaban dose was mg po qd ( mg po qd for patients with severe renal insufficiency/requiring concomitant p-glycoprotein inhibitor). pharmacokinetic samples were collected - hours or - hours after the most recent dose of study medication. patient characteristics included age, sex, race, region, body weight, crcl category, and specific p-glycoprotein inhibitor. , pharmacokinetic samples were analyzed. at mg, the projected concentration was . ng/ml at hours post-dose and . ng/ml at hours post-dose, showing a stable daily concentration. coadministration of p-glycoprotein inhibitors on the day of sampling more than doubled betrixaban concentration to ~ ng/ml at hours post-dose. at mg, the projected concentration was . ng/ml at hours post-dose, indicating a greater-than-dose-proportional exposure relationship. patient age, sex, weight, crcl category, p-glycoprotein inhibitors, and region were significant covariates affecting betrixaban pharmacokinetics. the exposure-response relationship for the primary efficacy endpoint was not significant, but the relationship between betrixaban concentration and major/clinically relevant nonmajor bleeding was significant in multivariate testing (p= . ). the betrixaban pharmacokinetic profile exhibited stable serum concentrations with qd dosing. several covariates had a %- % effect on betrixaban concentration, but no effect on efficacy/safety. betrixaban dose should be adjusted to mg for patients taking amiodarone or clarithromycin, but not other p-glycoprotein inhibitors. andexanet alfa is being investigated for reversal of anticoagulation by factor xa (fxa) inhibitors. a pharmacokinetic/pharmacodynamics model, developed in healthy subjects, predicted the andexanet regimen required to reverse anticoagulation by fxa inhibitors. the current analysis validated the pharmacokinetic/pharmacodynamic model using interim data from the annexa- study in patients with acute major bleeding. in annexa- , an ongoing prospective, open-label study, bleeding anticoagulated patients received iv andexanet bolus ( or mg) followed by -minute infusion ( or mg/min). anti-fxa activity was measured before andexanet administration (baseline), at end of bolus (eob), at end of infusion, and at , , and hours after infusion. the relationship between baseline anti-fxa activity and reversal in healthy subjects was derived from the pharmacokinetic/pharmacodynamic model and used to predict percent reversal for patients with acute major bleeding. from the first interim analysis of annexa- , patients (apixaban, n= ; rivaroxaban, n= ) had plasma levels available for model qualification, although did not meet criteria for inclusion into safety and did not meet criteria for efficacy analysis. the mean observed percent reversal of anti-fxa activity for rivaroxaban and apixaban was well predicted by the healthy subject pharmacokinetic/pharmacodynamic model; the point estimates fell within the % confidence intervals of predicted values. the percent reversal at eob for rivaroxaban and apixaban were . [ . - . ] and . [ . - . ], compared with . and . predicted by the model. the predicted reversal closely fit the observed confidence intervals through the first hours for rivaroxaban and apixaban, and extended through all evaluated time points for rivaroxaban and slightly outside of post- -hour time points for apixaban, possibly due to higher baseline anti-fxa activity levels for apixaban. the pharmacokinetic/pharmacodynamic model in healthy subjects closely predicted the extent of reversal of anti-fxa activity for apixaban and rivaroxaban in patients with major bleeding. risk factors and methods to predict extubation failure are well established for patients in medical icus and surgical icus. literature on patients who fail extubations in neurological icus is limited. the intention of this study was to collect descriptive information from patients with neurological injuries who failed liberation from mechanical ventilation. retrospective review of all patients with acute neurological injury who were admitted to our neuro icu and who required reintubation within hours of discontinuation of mechanical ventilation between january -february . we identified patients intubated primarily due to neurological pathology who required reintubation within hours after initial extubation over a -year study period. the majority of reintubated patients (n= ; . %,) had a positive fluid balance prior to failed extubation. twenty-six of the reintubated patients had a concurrent underlying chronic cardiac and/or pulmonary disease. five patients were placed on noninvasive ventilation post extubation. low glascow coma scale and absence of basic brainstem functions (gag and cough reflexes) was only minimally predictive of extubation failure. most of our reintubated patients did not have significant supratentorial midline shift nor an insult to the posterior circulation or dominant hemisphere. in patients with primary brain injury who required reintubation, a positive fluid balance prior to extubation may confer a lower rate of successful extubation. lesion location and supratentorial midline shift may not be tightly associated with extubation success. overall, our reintubation rate is quite low. early tracheostomy may play a small but significant role in the low rate of reintubation. further studies may be useful in creating a scoring system to identify the likelihood of extubation success in patients with neurological injury. surgical prophylaxis guidelines for evd insertion recommend peri-procedural antibiotics rather than prolonged antibiotic administration for the duration of evd placement. several small studies have shown that prolonged systemic antibiotic use does not reduce the incidence of catheter related ventriculitis. prolonged use is also associated with a higher rate of multi-drug resistant (mdr) infections. this study aims to show that prolonged antibiotic administration following evd insertion is potentially harmful. this is a single center, retrospective, chart review. all patients admitted to our hospital who had an evd placed from january to march were identified. patients with preceding infections, incomplete data or uncertain infection diagnosis were excluded. sixty-nine patients were analyzed. documented variables included demographics, comorbidities, indications for evd, duration of antibiotic therapy, infections and organisms' sensitivities. eight patients ( %) did not receive any antibiotic therapy; the rest received cefazolin following evd insertion. infections occurred in of ( %) patients; of ( %) were mdr bacteria. ventriculitis occurred in ( %) patients, and of these were resistant to cefazolin (mdr). ventriculitis was not associated with the use or duration of antibiotic therapy. graphical analysis showed that the probability of any infection decreased during the first days of antibiotic prophylaxis. after days, the longer patients remained on prophylactic cefazolin, the higher the probability of infection (spearman rank correlati patients who received antibiotics for > days were . times as likely to develop mdr infections ( % ci, . to . ; p= . ). cefazolin may prevent infections for the first days after evd insertion. however, prolonged administration increased the risk of mdr bacterial infections. a randomized study comparing periprocedural ( hrs) antibiotic use is needed to resolve this controversy. each year more than , deaths are associated with urinary tract infections. eighty percent of all utis are associated with an indwelling catheter. neuroscience intensive care (neuro-icu) units have the highest rates of catheter associated urinary tract infections. catheter associated urinary tract infection (cauti) increases morbidity rates, length of stay, and costs among hospitalized patients. at an urban academic medical center, our neuro-icu had the highest cauti cases among our icus. the purpose of this project was to reduce our cauti cases by %. this quality improvement project used several strategies: ( ) formed a multidisciplinary cauti task force that included nurses, physicians, infection control, management and supply chain personnel; ( ) developed an action plan to update standard of practice by conducting a review of the literature and pilot testing new products; and, ( ) educated staff using huddles, a bedside guide, and email blasts with cauti facts starting in august . additionally, cauti prevention was discussed during patient handoffs among nurses and physicians. data were collected for all neuro-icu patients from fiscal year (fy) - . cauti cases are determined by utilizing cdc's national healthcare safety network. analysis included evaluation of trends across time. we reduced our number of cauti cases from in fy to in fy . as of the beginning of fy , we have not had a cauti for days. a comprehensive approach with a strong commitment by clinicians is critical for sustaining a reduction in cauti. we reduced our cases and exceeded our goal. our efforts to provide evidence-based care are ongoing as we continue to monitor the research and upcoming supplies aimed at making hospitalacquired cauti a never event. isophane insulin (nph) is a commonly prescribed basal insulin to manage hyperglycemia in critically ill patients on continuous tube feeding due to its intermediate duration of action. however, the incidence of hypoglycemia may be higher given the duration of nph can last between - hours and because of the potential for unexpected interruption in feeding. using scheduled regular insulin (ri) instead of nph may reduce this risk given its shorter duration of action. it may also improve glycemic control due to more frequent titration. this was a single-center, retrospective, observational, cohort study from december to may . patients on continuous tube feeding who were prescribed scheduled ri were compared to those prescribed nph. all patients continued to receive an insulin sliding scale. choice of agent was determined by the bedside team. the primary endpoint was incidence of hypoglycemia while secondary endpoint assessed efficacy. in our patient population, a higher incidence of hypoglycemia was seen in those that received nph. hypertonic saline bolus (hsb) is a proven intervention for neurological emergencies arising from cerebral edema and increased intracranial pressure. safety of hsb administered via central venous catheters is well established. however, infusion of hsb through peripheral intravenous access raises concern for complications related to caustic nature of the solution. we aim to assess the safety of peripherally administered boluses of hypertonic saline ( % sodium chloride) at a regional level trauma and comprehensive stroke center. we performed retrospective chart review of patients who received hsbs from january , to january , as part of a quality improvement project. we identified instances of hsb administration. the cases were individually reviewed for iv gauge, location of the iv, whether central access was present at the time of administration, documentation of iv removal, and volume of boluses. patients were excluded if there was concurrent central venous access catheter present at the time of hsb administration or unrelated death within hours after administration of hsb. adverse events were defined as line infiltration, erythema, or swelling at the site of hsb administration. charts were excluded from the study because of presumed administration of hsb through central venous access, not peripheral iv. two patients had adverse events ( . %). none of the patients progressed toward limb threatening complications. the majority of patients ( / ) did not experience erythema or infiltration of the iv. hsb administered through peripheral intravenous access does not pose significant risk of severe complications and may be safely used in emergency situations in the absence of central line access. routine screening of high risk asymptomatic trauma or surgical patients for venous thromboembolism(vte) is controversial. studies suggest against screening while others recognize that some patients at high risk may benefit. the purpose of this pilot study is to evaluate the outcome of routine screening in patients who underwent neuro-surgical interventions. all adult patients admitted to a neuro-intensive care unit with a primary diagnosis of brain injury requiring surgical interventions were included. data from april-june, were retrospectively collected on all subjects who had either spine or cranial surgery. data collected include: incidence of vte, number of times duplex ultrasonography and computed tomography of the chest was performed. on july st, prospective data collection began by screening for presence of deep vein thrombosis(dvt) on day , and from admission or surgery day. all patients received pharmacologic and mechanical vte prophylaxis within - hours post-operatively. a total of (pre-pilot, n= and post-pilot, n= ) subjects were included in the study. in the pre-pilot group, the ages ranged from - and most were male. majority, / ( %) had either craniotomy/craniectomy while / ( %) had spine surgery. about / ( %) were admitted with primary diagnosis of traumatic brain injury. of the subjects, had duplex screening for dvt and had screening for pulmonary embolism(pe). the incidence of vte was confirmed in / ( %); (dvt- % and pe- %). median hospital length of stay was (iqr - ) days. / ( %) were discharged home and / ( %) death rate was attributed to pe. in the post-pilot group, one incidence of pe was identified on day post surgery screening. the rest of the results are still pending. in this preliminary report, post surgical patients have a higher incidence of vte. routine screening might benefit to lower the incidence of mortality caused by pe. epsilon aminocaproic acid (eaca) is an antifibrinolytic agent that crosses the blood-brain barrier and has shown benefit in decreasing bleeding in patients acutely. its use in intracranial hemorrhage has uncertain benefit. we aimed to describe the administration and impact of eaca in a single-center neurosciences intensive care unit (neuroicu) over one year. we performed a single-center retrospective study of neuroicu patients undergoing intravenous eaca administration over a one-year time period. inclusion criteria included eaca administration over hours for a diagnosis of acute traumatic hematoma. the dose and duration of eaca infusion was collected. we additionally collected and compared pre-administration and post-administration prolonged thromboplastin time (ptt) hematology assays and neuroimaging. clinical outcomes were reviewed for survival at hospital discharge. over a -month period (april -may ), patients each received a -hour infusion of eaca. the most common indication for eaca was to prevent worsening of intracranial hemorrhage in patients in traumatic coma (gcs < ). % of patients underwent neurosurgical management. ptt assay values showed a significant difference before and after eaca administration. (ptt . +/-sd vs. . +/-sd; student's t test p< . , n= ). stability of the intracranial hematoma burden was evident following eaca in % of patients. % of patients who received eaca survived to discharge. patients receiving eaca showed a significant reduction in ptt assay values hours after completing their dose. ct neuroimaging demonstrated stable intracranial hemorrhage burden in most patients receiving eaca despite a high prevalence of acute operative neurosurgical management. however, only a modest number of patients receiving eaca survived to discharge. these results suggest that eaca may acutely reverse hematologic abnormalities and enable emergent neurosurgical management in patients with severe, acute traumatic hemorrhage, despite a limited role in affecting survival outcomes in these patients. prognostication is difficult for patients admitted to a neurocritical care unit (nccu). can serum biomarkers obtained as part of routine admission lab work help predict outcomes among patients in this prospective cohort study, the following biomarkers were measured at admission: c-reactive protein (crp), arterial lactate, neuron specific enolase (nse), lactate dehydrogenase (ldh), albumin, and brain natriuretic peptide (bnp). we collected information about demographics, comorbidities, hospital procedures and complications and -day mortality. we compared these serological biomarkers in patients who were alive versus those who had died at days. a total of patients were enrolled over months from june to september , of which whom ( . %) died within days of admission. there were no statistically significant differences in age or gender between the two groups. the -day mortality group had a higher mean charlson comorbidity index (cci) ( . vs . , p= . ) as well as mean nse ( . vs . ug/l, p= . ) and bnp levels ( . vs . pg/ml, p= . ). mean crp, lactate, and ldh were also higher in the -day mortality group ( . vs . mg/l, . vs . mmol/l, and . vs . u/l) while mean albumin was lower ( . vs . g/dl), although these differences were not statistically significant (p< . ). cci and serological biomarkers may have utility in predicting -day mortality among patients admitted to the nccu. as we continue enrollment, we plan to develop a predictive model for -day mortality on admission for patients admitted to the nccu using serological biomarkers, cci and admission characteristics. among hospitalized acutely ill medical patients, the risk for venous thromboembolism (vte) is high. the goal was to examine vte prophylaxis of at-risk patients and vte risk during hospitalization and in the outpatient continuum of care. acutely ill medical patients were identified from the marketscan commercial and medicare databases from / / to / / . inclusion criteria were hospitalization for heart failure, respiratory diseases, ischemic stroke, cancer, infectious diseases, and rheumatic diseases; months of continuous insurance coverage prior to (baseline period) and after (follow-up period) the index hospitalization. outcomes included the proportions of patients receiving inpatient and/or outpatient vte prophylaxis, and the risk for vte events. years, and . % were female. patients were hospitalized for infectious diseases ( . %), respiratory diseases ( . %), cancer ( . %), heart failure ( . %), ischemic stroke ( . %), and rheumatic diseases ( . %). mean hospital length of stay was . days. in total, . % (n= , ) of patients did not receive any vte prophylaxis, and . % (n= , ) received both inpatient and outpatient vte prophylaxis. during hospitalization, . % (n= , ) received vte prophylaxis (enoxaparin, . %; warfarin, . %; enoxaparin and warfarin, . %; a direct oral anticoagulant (doac), ~ %). following discharge, . % (n= , ) received outpatient vte prophylaxis (warfarin, . %; doac, . %; enoxaparin, . %; enoxaparin and warfarin, . %). among the entire study population, the vte event risk remained elevated up to - days after hospital admission. among hospitalized acutely ill medical patients, the risk for vte was present in both the inpatient and outpatient settings, with significant vte risk extending into the post-hospitalization period. only a small portion of at-risk patients ( . %) received vte prophylaxis in both the inpatient and outpatient continuum of care, suggesting an unmet medical need for vte prophylaxis in the post-hospitalization. brain edema is a good research target in various forms of neurologic injury. a real time measurement of brain edema is possible using thermal conductivity methods. however, this technique might be hard to apply in small rodents, which are commonly used as experimental brain edema models. we developed a new approach method for applying thermal conductivity methods in rodent brain edema model. a -week-old spraque-dawley rats were used for brain edema model. qflow probe was inserted through a suboccipital burr hole, located mm left from the midline, then was advanced anteriorly mm from the occipital bone margin until probe place assistance value indicates valid values (ranging from to . ). probe was fixated using adhesive glues and tagging suture. in vivo brain water content was continuously calculated using thermal conductivity values. for validation, calculated brain edema was compared with standard methods (dry/wet brain weight ratio) in water intoxication models (intraperitoneal injection of distilled water, % of body weight) and drying effect of mannitol was validated in streptokinase induced intracerebral hemorrhage (ich) models. calculated brain water content was . ± . % in thermal conductivity method and . ± . % using dry/wet weight ratio methods (p= . ). in water intoxication model, brain water content started to increase minutes after injection and reached up to . ± . % at hours post injection. on wet/dry weight method, edema was measured as . ± . % (p= . ). in ich model, brain water content started to drop minutes after administration of mannitol ( . mg/kg) and drifted back hours after injection of mannitol. thermal conductivity method in assessing brain edema is applicable in rodents using suboccipital approach through burr hole. this method may better reflect dynamic changes of brain edema. in patients with critical brain injury, alterations of brain physiology with dialysis initiation are poorly understood. from a consecutive series of brain-injured patients undergoing invasive multimodality monitoring between and , patients that underwent continuous veno-venous hemodialysis (cvvh-d) and patients that underwent intermittent hemodialysis (ihd) were identified. changes in mean arterial pressure (map), intracranial pressure (icp), and brain tissue oxygenation (pbto ), and microdialysis lactate-pyruvate ratio (lpr) were compared six hours prior to and twelve hours following dialysis initiation. high-resolution data was collected every seconds, with the exception of lpr collected hourly. data were normalized to patient maximum values, analyzed by fitted segmented regression, and checked for slope change-points by davies' test. values prior to dialysis initiation were averaged as a baseline for comparison. median values for patients undergoing cvvh-d were map +/- . , icp . +/- . , pbto . +/- . mmhg (n= ), and lpr . +/- . (n= ). normalized median values for patients undergoing ihd were map +/- . , icp +/- . . for the cvvh patient segmented regressions with normalized data, there was no change in map (slope . ) during the twelve hours. however, we found a change-point in icp at . hours (ci . - . , slope change . to . ) and pbto at . hours , slope change . to - . ). lpr increased through cvvh (slope . +/- . ). median values for patients undergoing ihd were map +/- . , icp +/- . . there was no identified change-points in map or icp in ihd patients, further parameters were limited by small sample size. initiation of cvvh in patients with neurologic multimodality monitoring showed change-points in icp and pbto in setting of stable map, with slight decrease in icp and pbto . initiation of hd in showed no change-points in icp. data on the cerebral effects of antihypertensive agents are limited but potentially important in patients requiring blood pressure reduction in neurological emergencies. our objective was to measure the effect of rapid-acting antihypertensive agents on cerebral blood flow (cbf) in patients with acute hypertension we conducted a prospective, quasi-experimental study of patients with a sbp > mmhg and planned rapid-acting antihypertensive treatment in the emergency department. patients < years or pregnant were excluded. non-invasive hemodynamic and transcranial doppler measurements of the middle cerebral artery mean flow velocity (mfv) were obtained prior to and post treatment. analysis included descriptive statistics and generalized linear modeling to test the effect of four categories of antihypertensive agents on mfv. categories included clonidine, iv labetalol, iv hydralazine and combination therapy. we enrolled patients ( % female) with a mean age of ± years. eight ( %) patients received clonidine, ( %) iv labetalol, ( %) iv hydralazine and ( %) combined therapy. the mean baseline sbp was ± mmhg and mfv ± cm/sec. the mean percentage fall in sbp by medication was: clonidine - ± %, labetalol - ± %, hydralazine - ± %, and combination - ± %. the overall change in mfv was - ± %, and by medication was: clonidine - % ( %ci - to - %), labetalol - % ( %ci - to - %), hydralazine + % ( %ci - to + %), and combination - % ( %ci - to - %). adjusting for baseline bp, hydralazine caused less change in mfv compared to other medications (difference between means + %, %ci - to + %, p= . ). in this study with modest bp reductions, rapid-acting antihypertensive medications had comparable effects on cerebral blood flow. these results hint that cerebral blood flow may be more stable with hydralazine administration, but further testing of hydralazine and infusions such as nicardipine is required. studies exploring correlations between non-invasive (oscillometric) blood pressure (nibp) and intraarterial blood pressure (abp) have excluded neurocritically ill patients with continuous infusion of vasoactive medications. compared to abp, nibp monitors generally tend to over-read at low values and under-read at high values. this study examines the relationship between simultaneously measured nibp and abp recordings in these patients. following informed consent, prospective observation of patients (n= ) admitted to a neurosciences icu, with simultaneous abp and nibp monitoring and continuous vasopressor (n= ) or antihypertensive (n= ) infusion. paired nibp/abp observations along with covariate and demographic data were abstracted via chart audit. analysis was performed using sas v . . , paired nibp/abp observations from subjects ( % male, % white, mean age years) receiving vasopressors (n= ) or antihypertensive agents (n= ). t-tests show significant difference between paired readings: ([sbp: m= vs mmhg respectively; p<. ], [dbp: m= vs mmhg respectively, p<. ], [map: m= vs m= mmhg respectively, p<. ]). the paired differences for specific medications were tabulated, with - % of the differences < mmhg, and - % of the values with < mmhg difference. bland-altman plots for map, sbp, and dbp demonstrate good intermethod agreement between paired measures (excluding outliers) and demonstrated marked nibp-abp sbp differences at higher blood pressures. pearson correlation coefficients for paired measurements show strong positive correlation for map (+ . ), sbp (+ . ), and dbp (+ . ). despite a statistically significant difference between nibp and abp readings for patients on vasoactive medications, there may be no clinical significance. the relatively positive and linear correlation between paired values guide providers towards not being forced to use one over the other. the final manuscript will aim to detail whether there is a clinical significance in particular vasoactive medications. pathological activity in continuous electroencephalogram (ceeg) data of icu patients is conventionally categorized into a small number of named rhythmic and periodic patterns. we aimed to develop a valid method to automatically discover a small number of homogeneous pattern clusters, to facilitate efficient interactive labeling by ceeg experts. we extracted time and frequency domain features from + hour ceeg recordings from different icu patients. after removing artifacts, we applied principal component analysis ( % variance retained), then separated the data into clusters (k-means). from each cluster we took random samples plus the most central one, rendering samples in total. three expert electroencephalographers independently categorized all samples into one of standard pattern categories (seizures, gpds, lpds, lrda, grda, burst suppression, other). we compared two methods for labeling clusters: ( ) "labor intensive labeling" (lil): assign the most frequent of expert-provided labels; ( ) "labor efficient labeling " (lel): assign the most frequent of the expert labels for the central sample. we compared interrater agreement (ira) among experts vs. between each expert and consensus labels using lil vs. lel. finally, we used laplacian eigenmaps (le) to visualize the data. this research suggests that large ceeg datasets can be automatically clustered into a small number of patterns described by standard icu eeg pattern labels. we demonstrated efficient cluster labeling by inspecting only the central-most representative of each cluster. furthermore, le visualizations support the hypothesis of an interictal-ictal continuum. real time measurement of cerebral oxyhemoglobin (oxyhb) and deoxyhemoglobin (deoxyhb) using near infrared spectroscopy (nirs) may help us better understand the status of cerebral oxygenation and possibly cerebral blood flow (cbf) in patients with acute brain injury. we developed multichannel functional nirs (fnirs) system and evaluated its role in patients with acute brain injury. a channel fnirs system (nirsittm) was used for measuring cerebral oxyhb and deoxyhb in patients with brain injury. measurement protocols were as follows; baseline measurement for minutes with activation stimuli (nipple pinching for seconds). patients groups were categorized as follows; ) global cerebral ischemia with profound cerebral injury (n= ), ) large ischemic stroke or decrease in cbf in the frontal lobe due to severe stenosis in the middle cerebral artery (mca) or internal carotid artery (ica) (n= ), ) high grade subarachnoid hemorrhage with a risk of vasospasm (n= ), control groups did not have either cerebral lesion or cbf abnormality (n= ). global ischemia with good functional outcome group had better oxyhb level (rso ) compared to those with poor outcome ( . % vs. . %, respectively, p = . ). patients with poor perfusion in the mca territory had low oxyhb level compared to mirror lead in the contralateral hemisphere. oxyhb level in patients with decreased vasomotor reactivity on diamox spect had improved after carotid stenting. three patients who underwent superficial temporal artery-middle cerebral artery bypass surgery had transient hyperperfusion syndrome. oxyhb and total hb were elevated in the affected area. patients with sah and vasospasm had blunted oscillation pattern of oxyhb compared to those without vasospasm. bedside multichannel measurement of oxyhb and deoxyhb using fnirs might be useful in understanding hemodynamic changes occurring in patients with acute brain damage at the real time. multimodality monitoring (mmm), brain tissue oxygenation (pbto ) and microdialysis (md) in sah may be important to the treatment of delayed cerebral ischemia (dci). our hypothesis was that concordance between pbto and md occurs in the tissue bed displaying angiographic vasospasm. this retrospective observational study includes patients with sah. the extent of angiographic vasospasm for each vessel was graded on angiography by the on call neuro-interventionalist and quantified as (no spasm) to (severe spasm). pbto and md probes were placed in the frontal lobe white matter. the severity of vasospasm was estimated by the weighted average of ( x aca + x mca + x ica) / . cases with score of or more were considered to have clinically relevant vasospasm. using a within-subjects design, epochs of baseline mmm were compared with during spasm using daily mean for pbto , lpr, glucose, icp and cpp. given the limited number of observations the simplifying assumption was made that the observations from all epochs are independent. the measurements from all patients were divided in the two groups with and without spasm and were compared using a twotailed non-paired student t-test. sixteen sets of baseline and vasospasm epochs were evaluated for pbto and for md. compared with baseline values, the average pbto was significantly lower ( . vs . mmhg, p= . ), lpr was non-significantly higher ( . vs . , p= . ), and glucose was similar ( . vs . mmol/l, p= . ) during vasospasm epochs. there was no difference in icp ( . vs . mmhg, p= . ). these differences were unaffected by induced hypertension, when cpp was augmented for treatment of dci ( . vs . mmhg, p= . ). mmm during angiographic vasospasm after sah suggests discordant changes in brain oxygenation and metabolism. these data suggests that dci may be related to metabolic factors other than tissue oxygenation. multimodal monitoring including brain tissue oxygenation (pbto ) is increasingly used for the management of acute tbi patients. the optimal management of pbto is not fully established. increasing fio is efficacious to correct pbto but may mask other oxygen delivery mechanisms which may be deficient. the objective of this study was to explore the clinical utility of a pbto /pao ratio to detect overtreatment by fio . retrospective cohort stud were collected simultaneously whenever an arterial blood gas was drawn (icp, cpp, hemoglobin, temperature, pco and pao ). causes of cerebral hypoxia (pbto < mmhg) were noted. pbto /pao ratio < . was considered abnormal and plotted over time for each patient individually. data sets were collected from patients (mean age . ± . , median gcs , mortality %). . % of the time and associated with a mean pao of mmhg. measures within the low pbto -low ratio category had significantly lower cpp ( vs mmhg), higher pao ( vs mmhg) than patients with normal pbto or normal ratio respectively. various causes of hypoxic pbto were reported when the ratio was abnormal: hypocapnia, low cpp, low cardiac index, long equilibration time... four patterns of evolution of the ratio over time were identified and associated with different mortality rate: . %, . %, . % and %. conclusions associated with increased pao and decreased cpp. this suggests clinicians often used fio to compensate for deficient cerebral oxygen delivery. indeed, various causes of hypoxia besides low pao were identified and corrected. pattern of temporal evolution of the ratio seems to correlate with mortality. pupillary light response (plr) evaluates cranial nerves ii, iii, and midbrain function. bedside quantitative infrared pupillometry provides reproducible assessment of the plr, reported as the neurological pupillary index (npi). increased intracranial pressure results in decreases in npi. intracranial hypotension (ih) can also cause brainstem distortion. we therefore hypothesized that similar changes in npi could be seen with ih. here, we describe sequential changes in npi in ih before and after treatment. we identified four patients monitored with pupillometry for clinical care during ih diagnosis and treatment. ih was diagnosed with a compatible history, exam, and characteristic neuroimaging findings. patients' npi at baseline, during symptomatic ih, and after treatment were compared using related samples friedman's two-way anova and wilcoxon signed ranks tests. two patients were male; causes of ih were csf leak following lumbar instrumentation (n= ) and basilar skull fracture (n= ). mean baseline npi was normal (defined as > ) and declined in one or both eyes concurrent with clinical deterioration in the - hours preceding definitive diagnosis. all patients underwent treatment for csf leak with epidural blood patch or fracture repair, with return of npi > within hours of treatment. the baseline, symptomatic and post treatment npi's differed significantly ( . ± . vs . ± . vs . ± . , mean +/-sd, pre-treatment vs nadir vs post-treatment, p= . ). both baseline and post treatment npi's differed from the npi nadir (p= . ) but there was no difference between baseline and post-treatment npi (p = . ). impairment of the plr, as measured by npi, occurred during symptomatic ih and resolved after treatment. because management of intracranial hyper-and hypotension differ markedly, our results emphasize the importance of evaluating the clinical context before attributing pupillary/npi changes to increased icp. automated pupillometry provides a non-invasive, bedside tool for monitoring progression and treatment of intracranial hypotension the correlation of optic nerve sheath diameter (onsd) as seen on ultrasonography (us) and directly measured intracranial pressure (icp) has been well described. nevertheless, differences in ethnicity and type of icp monitor used are obstacles to the interpretation. therefore, we investigated the direct correlation between onsd and ventricular icp and defined an optimal cut-off point for identifying increased icp (iicp) in korean adults with brain lesions. this prospective study included patients who required an external ventricular drainage (evd) catheter for icp control. iicp was defined as an opening pressure over mmhg. onsd was measured using a mhz us probe before the procedure. linear regression analysis and receiver operator characteristic (roc) curve were used to assess the association between onsd and icp. optimal cut-off value for identifying iicp was defined. a total of patients who underwent onsd measurement with simultaneous evd catheter placement were enrolled in this study. thirty-two patients ( . %) were found to have iicp. onsd in patients with iicp ( . ± . mm) was significantly higher than in those without iicp ( . ± . mm) (p . mm disclosed a sensitivity of . % and a specificity of . % for identifying iicp. onsd as seen on bedside us correlated well with directly measured icp in korean adults with brain lesions. the optimal cut-off point of onsd for detecting iicp was . mm. impaired cerebral autoregulation following neurological insult has been established as a strong predictor of clinical outcome. hypothermia may offer autoregulatory protection in these patients, although the effect of body temperature on autoregulatory status is unclear. retrospective analysis of data from an ongoing prospective study to evaluate multimodal monitoring using near infrared spectroscopy (nirs) for bedside measurement of autoregulation. ninety-one comatose patients (gcs < ) were continuously monitored for up to three days. nirs derived cerebral oximetry index (cox) was used as a marker of autoregulation. cox was calculated as a moving, linear correlation coefficient between regional cerebral oxygenation saturation and map. autoregulation improves as cox values approach , and is impaired as values approach . patients were grouped by trend in temperature seen over the monitoring period: no change (< oc temperature change, n= ), increase (n= ), decrease (n= ), increase followed by decrease (n= ), decrease followed by increase (n= ), and fluctuating (n= ). we performed multivariable logistic regression analysis to assess the association between temperature and outcomes. the association between hourly temperature and cox was assessed using mixed random effects models with random intercept. in patients showing a sustained increase or decrease in temperature, a linear relationship between temperature and cox was seen; for every oc increase or decrease in temperature, cox changed by . ± . (p< . ) and - . ± . (p= . ), respectively, after adjusting for pco , haemoglobin, map and temperature probe location. mean temperature changes over the monitoring period for these groups were . ± . oc and - . ± . oc, respectively. cox did not change significantly in other groups. there was no significant difference in mortality or poor outcome (mrs - ) at discharge and , , or months between patients in each group. in acute coma patients in the neurocritical care unit, increasing body temperature is associated with worsening cerebral autoregulation as measured by cox. the historical tradition of examining the pupillary light reflex (plr) required the examiner to score the size and reactivity of the pupil. a change in the plr from brisk to sluggish or fixed may be a marker of a pathological process and a need for intervention. the plr has been difficult to quantify and has poor inter-rater reliability. handheld pupillometry provides several novel measures, such as the neurological pupillary index™ (npi) and constriction velocity (cv) that may be more quantifiable than the plr. the purpose of this analysis is to examine the relationship between cv and npi in neurologically injured patients. the end-panic registry is a prospective registry of pupillometer values and variables associated with intracranial dynamics (e.g., icp). this analysis from adult (over years) patients from hospitals includes , pupillometer readings; left eye ( , ), and right eye ( , ). subjects had a mean age of . yrs and . % were male. the primary admission diagnosis included neoplasm ( ), ischemic stroke ( ), sah ( ), ich ( ), tbi ( ), and other ( ). the left eye mean/s.d. cv ( . / . ) npi ( . / . ) and size ( . / . ) were similar to the right eye cv ( . / . ) npi ( . / . ) and size ( . / . ); statistically significant difference related to large sample size. the correlation between left eye cv and npi (r = . , p< . ) was significantly improved after controlling for size (r = . , p< . ). the correlation between right eye cv and npi (r = . , p< . ) was significantly improved after controlling for size (r = . , p< . ). constriction velocity is highly dependent on size of the pupil. further studies need to be undertaken to determine the sensitivity and specificity of abnormal npi and cv in detecting pathological processes such as midline shift or rd nerve compression that effect pupillary reactivity. cerebral injury is increasingly described in adult recipients of extracorporeal membrane oxygenation (ecmo) therapy. we describe the association between regional brain tissue oxygenation (rso ) measured by near infrared spectroscopy (nirs), survival, and cerebral injury on neuroimaging. a single-center retrospective chart review was conducted of adult patients who underwent veno-arterial (va) ecmo from april to october . all patients had received nirs monitoring during ecmo therapy. baseline demographics, in-hospital complications, and mortality were recorded. desaturations of rso , defined as decline > % below baseline or absolute value < , were recorded and analyzed. desaturation burden was calculated by area under the curve analysis and measured by rso *seconds. eighteen va ecmo patients ( females) underwent nirs monitoring during the study period. eleven patients experienced desaturations, while did not. patients with desaturations tended to be younger ( . vs. . years old), more likely female ( vs. ), had lower ejection fraction ( . % vs. . %) and experienced liver dysfunction ( patients vs. ). patients with desaturations were more likely to have abnormalities on ct scan ( vs. ). eleven of the patients survived to discharge. survivors tended to be younger ( . vs. . years old) and had lower initial ecmo sweep ( . vs. . ). survivors had lower baseline rso values at the beginning of nirs monitoring (right - vs. , left - vs. ), fewer desaturation events ( vs. ), lower desaturation burden, and spent less overall time desaturating ( : vs. : hours). desaturation on nirs may be correlated with cerebral injury in the adult va ecmo population and may have utility in triggering clinical investigation or determining prognosis. further studies in larger patient populations are needed to determine its reliability and accuracy. pressure reactivity index (prx) is the most validated index to measure cerebrovascular reactivity in patients after traumatic brain injury. the aim of this study is to identify the natural history of cerebral autoregulation measured by prx in various forms of brain injury to monitor restoration or not of cerebral vasomotor reactivity in the acute phase. retrospective analysis of data from ongoing prospective study to evaluate multimodal monitoring using prx for the measurement of cerebral autoregulation at the bedside. thirty comatose patients (glasgow coma scal used as a marker of autoregulation. prx was calculated as a moving, linear correlation coefficient between icp and map. impaired cerebral autoregulation has been pre standard maximal medical therapy was implemented to treat elevated icp, cerebral edema, etc. patients with withdrawal of care in the first hours or brain death on neurological exam were excluded. thirty comatose patients from acute brain injuries ( intracerebral hemorrhage, tbi, aneurysmal subarachnoid hemorrhage, intraventricular hemorrhage, hypoxic ischemic encephalopathy) were studied. the average prx upon starting neuromonitoring using prx was . ± . (impaired), whereas the average prx at the end of day of neuromonitoring was ± . (restored). one third of the patients had icp crisis during monitoring. the average opening icp= . , average highest recorded icp= . . impaired cerebral autoregulation has been implicated as a predictor of clinical outcome. aggressive medical therapy instituted by the neurocritical care team (icp and cerebral edema management, blood pressure control, etc.) may result in restoration of cerebral vasomotor reactivity measured by prx by intensive care day - . restoration of cerebral vascular reactivity may be a necessary but not sufficient for favorable outcome. elevated intracranial pressure (icp) is an important cause of death following acute liver failure (alf). while invasive icp monitoring (iicpm) remains the gold standard, the presence of coagulopathy increases the risk of bleeding in alf. measurement of optic nerve sheath diameter (onsd) using optic nerve ultrasound (onus) may accurately detect elevated icp. our goal was to study the ability of onus to detect sustained intracranial hypertension following alf, and to predict death and therapeutic intensity level (til), a quantitative measure of the intensity of treatment required to control icp. consecutive patients with alf admitted to our institution in a -year period underwent onus. blinded measurement of onsd was performed from deidentified onus videos. patients underwent iicpm on the basis of an institutional protocol for selection of appropriate candidates, coagulopathy reversal and insertion of an intraparenchymal monitor. the til-basic for management of icp during the icu stay was recorded. the ability of highest onsd to predict concurrent icp> mmhg at the time of measurement, sustained icp elevation > mmhg at any time and til-basic> was assessed in patients who underwent iicpm, while prediction of death was assessed in all patients. receiver operating characteristic (roc) curves were constructed for the outcomes of interest. thirty-nine patients with alf were admitted during the study period, / ( %) underwent onus, / ( %) underwent iicpm and ( %) died. of patients who underwent iicpm, ( %) developed sustained icp elevation and ( %) had a til-basic> . the roc area under the curve (auc) of onsd for prediction of concurrent icp> mmhg was . ( % confidence-interval . - . , p= . for null hypothesis of auc= . ), sustained icp elevation at any time was . ( . - . ,p= . ), death was . ( . - . ,p= . ) and til> was . ( . - . ,p= . ). in patients with alf, onsd measurement performed poorly for detection of icp elevation, and was a poor predictor of til and death. limited literature exists regarding the neurochemical and physiologic events that occur as brain death develops. using intracranial multi-modality monitoring, we identify physiological changes that signal the onset of brain death. we measured intracranial pressure (icp), brain partial oxygen tension (pbto ), cerebral blood flow (cbf), and biochemical correlates of cerebral metabolism in patients with diffuse hypoxic ischemic brain injury after cardiac arrest during the development of brain death. monitoring probes were inserted into cerebral white matter through a burr hole using a ct compatible multi-lumen bolt. brain tissue energy-related metabolites (lactate, pyruvate, glutamate, glucose, glycerol) were measured using a bedside microdialysis analyzer. pbto and temperature were measured via a licox catheter. cerebral perfusion was measured with a hemedex bowman perfusion monitor. brain death was confirmed in accordance with institutional guidelines. a characteristic pattern of physiologic and neurochemical findings emerged as brain death occurred. absolute loss of cerebral autoregulation, with a near perfect correlation between icp and map was followed by equalization of map and icp resulting in progressive drop in cpp to zero, followed by a progressive decline in pbto that became unresponsive to a % fio challenge. cerebral perfusion decreased in tandem with pbto . lactate/pyruvate ratio (lpr), glutamate, and glycerol all increased precipitously, with lpr consistently > . brain temperature decreased despite maintenance of a normal core temperature. finally, intracranial compliance, while initially very low (evidenced by marked increase in the p component of the icp waveform), appeared to paradoxically re-normalize as the recording continued. continuous neuromonitoring reveals a characteristic pattern of cerebrovascular physiologic changes that accompany brain death. these changes are consistent with a progressive cessation of cerebral perfusion caused by diffuse cerebral edema. although not currently a part of the formal brain death determination process, such monitoring could be helpful to identify when brain death has truly occurred. automated devices that collect objective quantitative data on pupil size and reactivity are increasingly used for critically ill patients with neurologic disease. however, there is limited data on the normative range of pupillary reactivity in the critically ill, and the relationship between reactivity and traditional monitoring metrics. to determine pupil characteristics in this population, we prospectively collected quantitative pupillometry data in patients admitted to the neuro icu with an expected stay of at least hours. trained nursing staff measured pupillary reactivity with the neuroptics- pupillometer device every -hours. measurements included the neurologic pupil index, (npi) a composite metric ranging from - in which > is considered normal, resting and constricted pupil size, constriction velocity, dilation velocity and latency. these recordings were compared with averaged intracranial pressure (icp) and glasgow coma scale (gcs) assessments within the same hour. we used univariate and spearman's rank tests to explore associations between pupil characteristics and clinical variables, followed by multi-level linear regression to account for intra-and inter-subject variability. one-hundred patients underwent paired observations. fifty-five patients had at least one recorded episode of anisocoria, had low npis in more than % of recordings, and had normal pupil reactivity. average and minimum npi was correlated with average and minimum recorded hourly glasgow coma score (gcs) (p values < . ). increased asymmetry in both pupil size (p= . ) and dilation velocity (p= . ) was associated with increased intracranial pressure (icp). anisocoria was associated with hyperosmolar therapy (p= . ). the presence of low npis in more than % of total pupil measurements was associated with death at discharge (p= . ). the range of pupillary metrics varies among critically ill neurologic patients and correlates with gcs and icp. further study is needed to establish whether change in pupil metrics predict specific clinical events. near infrared spectroscopy (nirs) provides a non-invasive measurement of regional cerebral oxygen saturation (rso ) that may be able to detect seizure activity. in this study, we explored the hypothesis that rso is lower ipsilateral to seizures or epileptiform activity compared to the contralateral side in comatose patients. five patients ( men and women; mean age ) underwent continuous electroencephalography (ceeg) monitoring and nirs recording. ceeg data were classified as baseline, epileptiform activity or seizure, slowing, or burst suppression at hourly intervals over the course of the recoding period (mean duration . hours, range to hours). three patients had idiopathic status epilepticus, two had intracranial hemorrhage, and one had a temporal meningioma. the relationship between rso and epileptiform discharges was explored using scatterplots. the association was assessed using mixed random effects models with a random intercept. an independent within-subject residual structure was used. there were measurements with ceeg and nirs from patients. one patient was excluded as the nirs sensors were potentially reversed. epileptiform activity or seizures were observed in a median of % of the measurements (iqr - %). rso was significantly lower on the side ipsilateral to seizures - . , p < . ) after adjusting for map. all patients only had partial seizures with no generalization. partial seizures and/or epileptiform discharges were not associated with impaired autoregulation. we found a significant lower cerebral oxygen saturation ipsilateral to seizures and/or epileptiform activity. the association was observed in patients with various etiologies of coma, and with either convulsive and non-convulsive seizures. decreases of regional cerebral oxygen saturation at the bedside may alert the clinician of ipsilateral seizures. elevated intracranial pressure (icp) and cerebral edema are common causes of mortality in neurocritical-care patients. key monitoring techniques for icp-elevation include neuroimaging and invasive icp-measurement. examination of the pupils is routinely performed to determine disturbances within cerebral physiology but shows high inter-rater variability. portable infrared pupillometry is increasingly used for accurate measurements. the benefit of these technique remains to be established in patients with elevated icp. aim of this study was identify pupillary parameters associated with icpcrisis in neurocritical-care patients. we prospectively enrolled critically-ill patients (subarachnoid hemorrhage/intracerebral hemorrhage/stroke/bacterial meningitis) admitted to our neurointensive care unit( / - / ) who required placement of external ventricular drains. we recorded serial pupillometer readings [i.e. maximum/minimum apertures(mm), constriction/dilation velocities(mm/sec.), latency period(sec.)] and corresponding icp values every hours after admission. neurological pupil index(npi), an algorithm that compares above mentioned pupillary parameters to a normative model of pupil reaction to light, grades pupil-function on a scale of (nonreactive) to (normal). receiver operating characteristic(roc) curve analysis was performed to investigate associations between pupillary parameters and presence of icpcrisis(icp> mmhg). in our data suggest a relationship between non-invasively detected changes in npi, cv or mcv and icpcrisis. yet, clinical benefit of these parameters is subject to future studies. lung injury is frequently observed in patients with severe, acute brain injury. while these patients often require mechanical ventilation, a lung protective ventilation strategy has not been extensively studied. this may be due, in part, to concerns that elevated positive end-expiratory pressure (peep) could adversely affect intracranial pressure (icp) or cerebral perfusion pressure (cpp). we were interested in exploring this relationship as a first step towards understanding whether mechanical ventilation resulted in a transmission of pressure to the brain. ) and received both mechanical ventilation and icp monitoring were enrolled in this pilot study. an esophageal balloon was inserted to measure their transpulmonary pressure (ptp). fluid responsiveness was assessed prior to the intervention. subjects underwent a step-wise increase in peep (increments of five) from to cmh o. airway pressure, ptp and icp were measured at each peep interval. of the planned twenty, three patients have been enrolled to date. primary diagnoses included aneurysmal subarachnoid hemorrhage and intraparenchymal hemorrhage with a median gcs of . patients were ventilated using either volume control or pressure support ventilation; median fio was . . two patients were on vasopressors and the same two patients were determined to be fluid responsive. at baseline (peep ), mean icp, cpp, and ptp were mmhg, mmhg, and - . cmh o, respectively. when peep was increased to cmh o, the average change from baseline in icp and cpp was - . % and - . %, respectively. when increased to cmh o the change from baseline in icp and cpp was . % and . %. during the intervention icp did not exceed mmhg, nor did any patient experience hypotension. preliminary data suggests that intrathoracic pressure is not directly transmitted to the intracranial compartment. continued enrollment is needed to confirm these findings. neurocritical care after severe traumatic brain injury (stbi) is focused on detecting and preventing secondary brain injuries. in addition to intracranial pressure (icp), measures of brain tissue oxygen (pbto ), regional cerebral blood flow (rcbf), and electrocorticography (deeg) may provide critical clinical data. few studies have assessed the safety of such an approach and the reliability of data that is gathered. we describe here the placement, complications, and reliability of multimodality monitoring (mmm) data from a novel, single burr hole approach using a four-lumen bolt at our institution. we included consecutive adult stbi patients admitted to the neuroscience intensive care unit at the university of cincinnati from april to march who underwent mmm as part of standard clinical management per institutional protocol. data was obtained regarding device placement and complications. all data was visually inspected for errors and gaps in data. patients were included. the mean age was +/- and % were men. bolts were placed a median of . (iqr . - . ) hours from injury. no clinically significant complications occurred, although . % had minor complications (e.g. small tract hemorrhage or pneumocephalus). suboptimal placement of probes was noted in %. we monitored patients a median . (iqr . - . ) hours. icp data was the most reliable, with data available . % of the total monitoring time and only . % error time. pbto and deeg data were reliable for > % of total monitoring time with < % error time. rcbf provided data for % of total monitoring time and had . % error time. mmm in stbi may be carried out via a single burr hole without significant clinical complications and reliably yields continuous data to facilitate clinical decision making. this supports the feasibility of our approach as an alternative to icp monitoring alone. intracranial hemorrhage patients with non-compliant ventricles may have high intracranial pressure (icp) despite normal ventricle size. we aimed to assess the incidence of elevated icp among those with no radiographic evidence of intracranial hypertension. prospectively enrolled primary intracranial hemorrhage patients (sah, sdh and iph) admitted to two tertiary-care centers between / - / were retrospectively reviewed. among patients with external ventricular drainage (evd), admission head ct (hct) scans within h prior to evd placement were reviewed for evidence of elevated intracranial pressure (icp) including: ventricle size (bicaudate index, temporal horn size), basal cistern effacement, midline shift and global cerebral edema. when all of these features were absent, patients were classified as having normal-icp hct. the incidence of elevated icp (> mmhg) at the time of evd placement and during hospital stay were recorded. of intracranial hemorrhage patients enrolled, ( %) had evd. / ( %) had a normal-icp hct. of these, / ( %) had elevated opening pressure at the time of evd placement, and / ( %) had elevated icp during their hospital stay. among normal-icp hct patients with icp> mmhg, % had sah, and the median gcs and hunt-hess scores were (range - ) and (range - ). the positive and negative predictive values of normal-re % %, respectively (auc . , p= . ) the only radiographic feature that was associated with elevated icp was global cerebral edema (or . , % ci . - . , p< . ). approximately half of intracranial hemorrhage patients without radiographic features of elevated icp had icp> mmhg at the time of evd placement and additional patients had elevated icp during their hospital stay. radiographic findings should not be relied upon to exclude the possibility of elevated icp. the measurement of intracranial pressure (icp) is a cornerstone of intensive care management following severe traumatic brain injury (stbi). it has been only recently that the time integral of icp has been quantified in relation to outcome; the time integral of brain tissue oxygen (pbto ) has not been studied. we gathered time-locked intracranial monitoring data on s tbi patients at the university of cincinnati over years. clinical management of all patients followed national standards. raumedic pto probe was used to measure icp and pbto ; accuracy was verified by visual inspection with automated data cleaning. normalized data was mapped based on correlation with glasgow outcome scale scores at - months. we studied patients aged +/- years (mean+/-sd); % were male. initial post-resuscitation glasgow coma scale score was median (interquartile range: . - ). / underwent craniectomy prior to monitoring. among those with good (gos - ) and poor (gos - ) outcome, the average icp was . +/- . mmhg and . +/- . mmhg (p= . ); the average pbto was . +/- . mmhg and . +/- . mmhg (both n.s.). the correlation with outcome was dependent on both icp and time: an icp > mmhg for > minutes was associated with poor outcome, whereas an icp < mmhg was associated with poor outcome only after hours. in contrast, the pbto level, but not the duration, correlated with poor outcome in those without craniectomy at a pbto < mmhg, and particularly below mmhg. pbto burden was less reliable in those following craniectomy. we replicated the effects of icp/time in a cohort of patients with severe tbi, both with and without craniectomy and subsequently demonstrated the burden of brain tissue hypoxia in those without craniectomy. the time integral of multimodality monitoring data may provide more accurate measures of secondary insult burden with implications for clinical care and prognosis. neurologists who work in neurocritical care (ncc) as neurointensivists may have critical care (cc) charges rejected for payment unless they are classified per centers for medicare services (cms) taxonomy codes in their systems as critical care providers. the neurocritical care society and cms created a new ncc code a x to fix this issue. we polled the aan ccen section members for awareness of this problem. we conducted a six question google forms survey using the aan ccen synapse community website to assess knowledge of the ncc taxonomy code: we received anonymous responses by the time we closed the poll on / / . question (q ) and (answers, a ): are you a neurology or neurosurgery back grounded intensivist who does neurocritical care at your hospital? y/n (yes/no). a : % reported being neurologists. q : were you aware of the new cms neurocritical care taxonomy code a x ? y/n a : % were aware of the taxonomy code. q : are you aware why the ncc taxonomy code was created? y/n a : % of respondents were unaware why this code was created. q : what is your primary department for revenue collection? a : % reported neurology, % neurosurgery, % critical care, and % blend. q : are you aware that medicare can reject critical care charges ( and ) can be rejected unless you are listed as a cms 'critical care provider' or as a neurocritical care provider? a : % reported rejected charges at their centers. q : are you aware of rejection of critical care charges happening at your own institution due to this misclassification? y/n a : % of respondents reported rejected charges at their center. although limited in sample size, this survey revealed almost half of the respondents were unaware of the ncc code. we believe a larger study is warranted. arterial subdural hemorrhage (sdh) is a rare but potentially devastating neurologic entity. it has been associated with ruptured aneurysms. we report a case-series of five patients with arterial sdh and their outcomes. a retrospective chart review of our institute's vascular database was conducted using a pre-defined search strategy including the terms "aneurysm", "arterio-venous malformation", "subdural hemorrhage", and "dural arterio-venous fistula" (dural-avf). amongst patients in the database, five cases were identified with ages ranging from to (four females). four had sdh due to aneurysm (two internal-carotid, one middle-cerebral, and one posteriorcerebral artery; one had parieto-occipital dural av-fistula. no patient had preceding head-trauma or anticoagulation. of aneurysmal patients, one had no sah. on admission ct-head imaging, the mean-sdh size was . mm (sd . ; range . - mm), and mean midline-shift (mls) was . mm (sd . ; range - mm). the mean ratio between sdh-size and mls was . (sd . ). in a historic cohort of acute subdural hemorrhage of non-arterial etiology ; the mean size of sdh was . mm and the mean mls was . mm. ratio of mls: sdh size was . . in our series, three patients with aneurysms had decompressive-craniectomy, one had mini-craniotomy for sdh evacuation; the patient with dural-avf had coiling and mini-craniotomy for sdh evacuation. four patients had died during hospitalization, whereas patient with dural-avf recovered to baseline functional-status at -month follow-up. arterial sdh is a rare entity and may present without subarachnoid hemorrhage and any preceding head-trauma. the degree of midline-shift is usually out of proportion to sdh size. there should be a low threshold to obtain vessel imaging in cases of sdh with no clear trauma history. mls: sdh ratio may be a useful screening tool for possibility of arterial sdh especially in absence of trauma and may reflect rate of bleeding. neurostimulant medications (amantadine and modafinil) are sometimes prescribed after acute nontraumatic brain injury to facilitate wakening and rehabilitation participation; the safety and effectiveness of this practice is unknown. following a retrospective evaluation of our experiences, we characterized anticipated challenges to designing a prospective randomized trial of neurostimulant medications to promote rehabilitation participation after acute non-traumatic brain injury. retrospective chart review of patients over with subarachnoid hemorrhage (n= ), intracerebral hemorrhage (n= ) and ischemic stroke (n= ) who received neurostimulant medications over a period of months. data regarding clinical course and potential confounders to assessing response were collected. continuous data are reported as median and interquartile range. neurostimulant medications were initiated in patients at a median of ( - ) days after hospital admission, for hypersomnolence ( %), not following commands ( %), lack of eye opening ( %), and/or low gcs ( %). thirty-nine ( %) patients were receiving sedatives or opioids at the time of neurostimulant(s) initiation. twenty-two ( %) patients received newly prescribed sedatives or opioids after neurostimulant(s) initiation. potentially sedating antiepileptic medications were prescribed to ( %) of patients. twenty-two ( %) patients were intubated at the time of neurostimulant initiation confounding the gcs-v. potentially confounding clinical factors included hydrocephalus ( %), vasospasm ( %), and seizures ( %). twenty-eight ( %) of patients had temporary cerebrospinal fluid diversion in place at the time of neurostimulant initiation. initiation and titration of neurostimulant medications after acute non-traumatic brain injury was common, but timing and indications varied widely. confounders to assessing effectiveness included concomitant sedating medications, variable pathophysiology related to the type and location of the stroke, and clinical factors like seizures, vasospasm, and hydrocephalus. these confounders are likely to make prospective evaluation of neurostimulant medication effectiveness difficult in the period of initial therapy following acute non-traumatic brain injury. brain small vessel disease can cause cognitive impairment via ischemic or hemorrhagic mechanisms. current imaging modalities, specifically magnetic resonance imaging allow for easier detection of different intracranial pathological processes including cerebral microhemorrhages (cms). research demonstrated that the number and location of cms correlate with the type of cognitive impairment (memory, processing speed, executive function, and motor speed). a retrospective analysis of patients (age to ) seen at our neurology outpatient clinic from to who were identified by linguamatics software to have "microhemorrhage" in their radiology mri report. additional information included age, sex, cognitive examination, presence of cardiovascular risk factors, mri, and the number and location of cms. cognitive function was determined by mini mental state examination (mmse) score and diagnosis by a cognitive neurologist. patients were grouped by presence of cm or greater ( to ) and regression was used to determine a relationship with mmse and vascular risk factors. the number of microhemorrhages per patient were ( patients), ( patients), ( patients), ( patients), ( patients), ( patients) and ( patients). vascular risk factors included hypertension ( patients), diabetes mellitus ( patients) and smoking history ( patients). regression analyses indicated that the presence of more than cm correctly predicted mmse lower than at % (p= . ). age was the only factor that influences this finding and increased this prediction to %. this study provides novel evidence that the presence of multiple cms on brain images predicts the presence of cognitive impairment. this study raises the need for more investigations. point-of-care ultrasound is a valuable tool in critical care, allowing timely and frequent beside assessment of clinical questions. neurocritical care has long utilized transcranial doppler but is still early in the adoption of other critical care ultrasounds. this study looked at the comfort level and competency of the participants at the point-of-care ultrasound workshop at the neurocritical care society annual meeting. the workshop comprised of didactics and hands-on small group practice using live models. topics covered included ultrasound physics, lung, cardiac, optic nerve sheath ultrasounds, as well as case studies in neurocritical care. participants were asked to complete an anonymous pre-and postworkshop assessment on a volunteer basis. a total pre-workshop and post-workshop assessments were completed. the mean age of the participants was . ± . years. there were ( . %) attending physicians, ( . %) advance practice practitioners, ( . %) fellows, ( . %) residents, and ( . %) research scientist. participants had limited ultrasound experience prior to the workshop, with ( . %) reported none, ( . %) reported < year, and ( . %) reported to years. on a - scale on comfort using ultrasound with being very uncomfortable and being very comfortable, participants reported a median score of (iqr - ) pre-workshop with an improvement to (iqr - ) post-workshop. for matched pre-and post-tests, all participants had an improvement in their ultrasound knowledge. while the majority of the participants at this workshop had prior ultrasound experience, many are still uncomfortable with their ultrasound competency. the format of didactics and hands-on small group practice improved the comfort level as well as overall ultrasound knowledge of these participants. additional opportunities for point-of-care ultrasound training should be considered in neurocritical care fellowships and meetings. event related potentials (erps) allow assessment of cognitive processing in unconscious brain-injured patients. here we explored the diagnostic and prognostic value of erps obtained shortly after brain injury. we prospectively collected a comprehensive erp paradigm labeled "local global paradigm" from a consecutive series of unconscious patients with acute brain injury. this auditory paradigm allows the assessment of: ) cortical responses, ) unconscious cognitive processing, ) unconscious focusing of attention, and ) conscious processing of sounds. levels of consciousness assessed with the coma recovery scale-revised (crs-r) at the time of recording were correlated with the presence/absence of each erps component and functional connectivity/complexity measures. we tested the prognostic value of each measure for recovery of consciousness prior to discharge. we analyzed recordings from patients (median recordings per patients [iqr , ]). recordings were made [iqr . , . ] days after brain injury and all patients were unconscious at the time of the initial recording. underlying etiologies included ich(n= ), sah(n= ), tbi( ) and other (n= ). there were trends for higher crs-r scores in patients with preserved erp components. crs-r scores correlated with the functional connectivity indexed (rho= . ; p= . ) but not with complexity measures. five ( %) patients regained consciousness (within to days from brain injury). one of these patients had to be excluded due to poor quality recording. all the ( %) remaining patients had the three type of non-conscious responses preserved on at least one recording in comparison to only ( %) among patient who did not recover consciousness (fischer p-value = . ). similarly, connectivity was greater in patients who regained consciousness ( . vs . ; p= . ) but the complexity was similar. simple bedside erp responses indexing cognitive processing during the local global paradigm obtained shortly after brain injury correlate with the level of consciousness but, more importantly, the probability to recover consciousness. over a decade ago, the institute of medicine introduced family-centered care (fcc) as a vital aspect of quality health care by strongly recommending that family members of intensive care unit (icu) patients be actively involved in decision-making. while there are many resources to help icu staff conduct meetings and provide information to families, the latest society of critical care medicine guidelines for fcc recommend the implementation of communication and decision support tools for family members to use. electronic decision support tools such as my icu guide have been effective in pilot studies at allowing family members to customize information delivery and communicate their preferences to icu staff. we sought to integrate a decision support and communication tool for families into an electronic patient portal. we developed an electronic patient and family engagement checklist for the neurointensive care unit (nicu) using doctella (patient doctor technologies, sunnyvale, ca), an existing patient engagement application. checklist components included: identifying a spokesperson, developing an advance directive, understanding diagnosis and prognosis, access to helpful resources, and a family meeting guide and planner. we presented the checklist to our hospital's patient and family engagement steering committee for the icus and received useful feedback. the checklist will also be vetted by the hospital's patient and family advisory council. usability testing will also be conducted. a family engagement checklist using an electronic patient portal is a novel strategy to enhance communication in the nicu. further validation of the tool is needed. applying painful stimuli to brain injured patients is a time-honored practice assumed to provide valuable clinical information for diagnosis, prognosis, and potential guidance for therapeutic interventions. however, there is limited literature that has evaluated and discussed the benefits and potential adverse effects related to repeated painful stimulation during bedside neurological examinations. though providers intend to do no harm, the practice of repetitive painful stimulation can unintentionally damage patient's skin, muscle, and bone, as well as inflict emotional duress. in conjunction with basic ethical principles used to justify painful stimulation during patient examinations, we propose a revisiting of the practice of routine repetitive painful stimulation in neurologic bedside assessments. . discuss the current literature regarding the use of painful stimulation and its beneficial and damaging effects, . describe alternative strategies for neurologic assessments, . propose guidelines to optimize accurate neurologic assessments while avoiding unnecessary repeated painful stimulation, . propose the development of a graded methodology for delivering painful stimulation when necessary for neurologic assessments. a summary of the literature will be outlined and discussed focusing on the ethical considerations and justification for the use of painful stimulation in the neurologic patient and the perception of pain in coma and minimally unconscious patients, . alternative strategies will be presented to minimize bodily and emotional injury, . a proposed outline with a companion flow diagram "easing the pain guidelines" implemented in a tertiary care neurocritical care unit will be presented. there has been little attention paid to the burden of painful stimulation inflicted on patients in the neurocritical care unit. the guiding principle of nonmaleficence (do no harm) morally obligates clinicians to evaluate current practice standards using repetitive painful stimulation in routine neurologic assessments. implementing standardized guidelines will limit unintended harm to patients without compromising accurate neurologic assessments. plasmapheresis is utilized in anti-n-methyl-d-aspartate receptor (nmdar) encephalitis to remove autoantibodies. antiepileptic drugs (aed), such as valproate, are often used to control seizures which may complicate anti-nmdar encephalitis. it is important to prevent rapid reductions of aed levels to ensure proper seizure management in this setting. we obtained total and free (active drug, unbound to plasma proteins) valproate levels intermittently throughout two -day courses of daily plasmapheresis. during the first course, trough levels were obtained. during the second course, levels were obtained before, during, and after plasmapheresis. the patient was a year old female, weighing kg. albumin was . g/dl. her valproate regimen ranged from mg to mg every hours ( to mg/kg/day), given intravenously or enterally. prior to the first plasmapheresis, valproate dose was mg every hours, resulting in a total level of mcg/ml (reference range: - mcg/ml). free valproate was mcg/ml (reference range: - mcg/ml); free fraction was % (reference range: - %). four days later, prior to the th plasmapheresis, the total valproate level was . mcg/ml. two days after the th plasmapheresis the total level was unchanged at mcg/ml; free valproate was mcg/ml and free fraction was %. during the second course of plasmapheresis, valproate total levels, free levels, and free fractions were mcg/ml, mcg/ml, and % before, mcg/ml, mcg/ml, and % during (valproate dose given upon initiation of plasmapheresis), and mcg/ml, mcg/ml, and % after plasmapheresis, respectively. valproate serum levels were not markedly influenced by plasmapheresis. free valproate levels and the free fraction were highly elevated throughout the patient's hospital course, however. future studies should evaluate critically ill patients' clinical response and toxicity correlations as the free fraction of valproate appears to be elevated in this setting. the purpose of this study is to assess knowledge retention of emergency neurological life support (enls) after participation in the course via a prospective observational study. study subjects seeking enls certification consented for study participation (enls-vs) from the ncs website then took a closed-book, multiple-choice question pre-test within hours of enls course participation. after completion of the enls course, participants took the same closed-book, multiplechoice question test (post-test). these tests consisted of novel questions from material presented in the course. questions were not repeated from the enls certification exams. thirty participants enrolled in the study with completing both the pre-test and immediate post-test. all participants' scores improved on the post-test as compared to the pre-test. the mean percent correct on the pre-test was . % with a median of . % (range . - . %). of the participants who have completed both pre-and immediate post-test, the mean pre-test score was . % with a median of . % (range . %- . %). the mean post-test score was . % with a median of . % (range . %- . %). the improvement of scores was statistically significant on the post-test compared to the pre-test ( . % vs. . % %, p< . ). all participants in the emergency neurological life support course showed improved test scores immediately after participation in the standard enls course. this study will assess knowledge retention at -months following training, and is actively enrolling new participants. augmented renal clearance (arc, defined as a creatinine clearance of > ml/min) has been demonstrated in neurocritical care disease states such as traumatic brain injury, intracranial hemorrhage, and subarachnoid hemorrhage. arc may result in increased elimination of renallyeliminated medications, thereby reducing drug exposure with standard doses. the overall prevalence of arc is not well described. the purpose of this study was to estimate the overall prevalence of arc in a neurocritical care population. this was a retrospective cohort study of adults > years of age admitted to the intensive care unit on the neurosurgery service. demographic and pertinent laboratory data were collected for patients admitted from january , thru december , . an arctic score was calculated for each patient ( or greater suggests arc). parametric data was compared using one-sample student's t-test, nominal data was compared using fisher's exact test (alpha = . ). statistical analysis was conducted using ibm spss version . present in a total of . % of patients. a broad spectrum of neurocritical care diagnoses was present. the mean age in years was significantly lower in patients with arc [ . ( sd)] than without arc [ ( sd), p = . ], as was the serum creatinine [with arc . mg/dl ( . sd) vs without arc mg/dl ( . sd), p < . ]. mean hospital length of stay was greater in patients with arc than without [ . ( sd) vs . ( sd), p < . ]. arc occurs commonly in neurocritical care patients and likely merits proactive screening or direct measurement of creatinine clearance in select patients. pharmacokinetic studies of commonly used renally-eliminated medications may be needed to establish population parameters in the neurocritical care population. education surveys demonstrate gaps in resident neurocritical care education and training. we assessed junior residents' baseline knowledge of neurologic emergencies, procedural competency, knowledge of available resources, and the impact of pre-rotation orientations. junior residents (neurosurgery pgy s and neurology pgy s) who had not previously rotated in the neuroicu were surveyed. a three-part survey was administered: part i, knowledge of icu structure and personnel; part ii, procedural competency; part iii, comfort with common neurocritical care emergencies. the survey was comprised of selection responses. after the survey but prior to starting the rotation, each resident was oriented to the unit by a neuroicu attending and nursing director. this orientation reviewed rotation goals, icu structure, personnel and rounding expectations. a survey was repeated to evaluate the orientation. of residents who had not rotated in the icu, ( . %) responded. none of the residents understood their specific role within the icu team. % did not understand the role of the resource nurse and were unaware of where to find procedure equipment. % of residents were not comfortable placing an a-line; % were not comfortable performing a lumbar puncture. over half of respondents said they "didn't know and could not easily find" the indications for hemicraniectomy after malignant mca ischemia, the indications for icp monitoring, or the initial workup of autoimmune encephalitis. residents responded to post orientation surveys. % felt the orientation was helpful in explaining the roles of team members. % felt it was at least "somewhat helpful" in understanding the role of the resource nurse. % felt the orientation was "helpful" and % felt it was "somewhat helpful" in identifying the goals of the rotation. these baseline measures underscore the importance of structured interventions, both before and during the neuroicu rotation, to improve junior resident comfort and preparedness in managing neurologic emergencies. physician-staffed helicopter emergency medical services (hems) are a well-established component of prehospital care in japan. however, there has been no report on hems and neurocritical care patients. we studied characteristics of neurocritical care patients who were transported by hems. we retrospectively evaluated neurocritical care patients who were brought to our emergency and critical care medical center (eccmc) by hems between january, and march, . we excluded patients in whom the outcome was unknown, those who were transported to other hospitals or between facilities. of the most important role of hems is rapid transportation of a flight medical team to the scene to provide immediate, lifesaving medical treatment. we found that half of patients admitted to our hospital by hems were neurocritical care patients. as proposed in the enls of neurocritical care society, hems is considered useful to allow neuro-emergency patients to receive the best care in the first hour. optic nerve sheath diameter (onsd) measurement using ocular ultrasound has been shown to accurately detect elevated intracranial pressure (icp), but does require specialized training. variations in the optimal onsd threshold for detection of elevated icp in the literature limits clinical utility, and may reflect heterogeneity in manual measurement techniques. our objective was to develop, and validate against expert measurement, an image-analysis algorithm for onsd measurement to facilitate standardization and ease of use of this technique. consecutive patients with acute brain injury admitted to the neurointensive care unit underwent ocular ultrasound with a multipurpose point-of-care ultrasound machine. a -second video was recorded from each eye in the axial plane and downloaded in dicom format. the onsd measurement algorithm was as follows. an average of images was calculated using non-overlapped segments of the image sequence. a line integral was performed to estimate the border of the region of interest (roi), the globe. the roi orientation and globe point of the segmented region were established, then a point mm posterior to the globe point identified. the onsd was measured at this point. manual onsd measurement was performed separately from the dicom videos by an expert blinded to the algorithm measurement. an intraclass coefficient (icc) was calculated for absolute agreement between highest onsd measured by the algorithm and expert manual measurement. a total of patients with acute brain injury underwent ocular ultrasound. the icc for absolute agreement between algorithm (median . , interquartile range . - . ) and manual expert (median . , . - . ) highest onsd measurement was . ( % confidence interval . - . ). an algorithm for automated measurement of onsd was developed and demonstrated good inter-rater agreement with expert measurement, although further refinement is required. automated measurement may help standardize and simplify a promising noninvasive bedside tool for the detection of elevated icp. after transition to electronic health record (ehr), transition to inpatient hospice required a separate encounter to account for change in insurance payer in our neuroicu. this negatively affected completed transitions and hence patient-centered care. the focus of this quality improvement project was to define the new process, improve outcomes, and identify further opportunities. the quality improvement method "plan-do-study-act" was employed for this work within a -bed neuro-icu at a large academic medical center. we assessed the current state (not enabling transition to inpatient hospice) and the desired state (enrollment in hospice during inpatient stay). a new process was created using an ehr discharge navigator, coordinating all relevant stakeholder groups (patient/caregiver, nurse, pharmacist, bed control, physician,). in addition, standard methodology for unit-based education, in-service, just-in-time training, and booster education was employed to identify process, outcome, and improvement opportunities. after rollout of the new discharge navigator, % of all patient-facing staff successfully completed the inpatient hospice training. process improvements lead to increase in palliative care consults by % ( to annually) and inpatient hospice discharges up to % ( to annually). furthermore, there was a statistically significant improvement in the vizient mortality index r = . , f( , ) = . , p < . and length of stay index r = . , f( , ) = . , p < . within the study population and period. ability to transfer patients to inpatient hospice is often limited and complicated. this study shows how employing standard quality improvement as well as education and implementation methods can result in improved process and outcome measures with sustainable success. opportunities remain in further analyzing and optimizing 'time to palliative care consult', 'time to admission to hospice and withdrawal of artificial support'. arnp and pa's are a rapidly growing part of the critical care workforce. proper selection among a pool of app candidates for the job is necessary to ensure the "right fit" and optimize patient safety. conventional interview techniques may not be adequate when selecting critical care app's. we hypothesized that use of a simulation center could be used to help select app candidates based on their critical thinking skills in conjunction with contemporary interviewing skills. from to , we performed conventional interviews for app's to staff critical care and neurocritical care patients. in we changed to an interview process consisting of the conventional interaction with the interviewee followed by simulation. after narrowing down the initial candidate pool, each was taken to the simulation center where they participated in a simulation of a decompensating patient. proctors were able to view the simulation in a separate room and direct the simulation mannequin. during this time proctors were able to evaluate the interviewee's patient interaction, assessment and interventions. an evaluation tool was to grade app candidates for their decision making skills, communication and thought. from to , we screened candidates before selecting for interviews and finally of those for simulation. over this timeframe, our center hired app's. comparing the ratio of screen applicants to employment was . and ratio of interviewed to hired candidates was . . these ratios show the competitive process and potential use of simulation in selecting apps. compared to the time period of applicants prior to simulation to after, retention went from to %, and disciplinary action for practice deficiencies went from % to %. the use of simulation based interviews for critical care app's in our institution improved retention and decreased the number of disciplinary actions compared to conventional interview methods. the contraindications for lumbar puncture (lp) in the setting of cerebral mass effect remain debatable. limited retrospective data advocate its potential safety. yet, high-quality guidelines specifically addressing this topic are not available. specific patient populations (post-instrumentation & immunosuppressed) may benefit from csf studies. we reviewed consecutive patients who underwent lp and cerebral imaging a week before or after lp from - . all individuals with evidence of brain herniation, a component of midline shift, or mass effect were included. all subjects received a low volume lp ( - cc of csf). there were patients with radiological increased icp. midline shift (average = mm) was present in patients. we also observed herniation: uncal (n= ), subfalcine (n= ), and a combination of both (n= ) , ventricular effacement (n= ) and cisternal compression with partial occlusion: quadrigeminal cistern (n= ), cerebellar-pontine-angle cistern (n= ), ambient cistern (n= ), crural cistern (n= ), prepontine cistern (n= ), suprasellar cistern (n= ), basal cistern (n= ), suprachiasmatic cistern (n= ), cisterna magna (n= ), interpeduncular cistern (n= ), medullary cistern (n= ). all patients tolerated the lp without complications. most survived a week after the procedure (n= , %). notably, four individuals deteriorated for reasons unrelated to the lp and expired within a week because of withdrawal of care. as brain compliance cannot yet be accurately determined radiologically, we believe anatomical involvement should drive decision-making regarding lp safety. our data suggest that a low volume lp ( - cc) might be safe in individuals with subfalcine herniation, midline mass effect < mm at foramen of monro level, and partial cisternal effacement. we believe that while lps might be safer in patients with supratentorial mass effect, individuals with posterior fossa involvement may tolerate it as well. these promising findings need further verification in larger sample populations. the importance of neurocritical care has recently been recognized in japan. however, to date, there has been no neurocritical care training program. we developed the neurocritical care hands-on seminar as a proposed training module, and here we report the satisfaction of participants. we prepared a post-course questionnaire about participants' degree of satisfaction. the main concept of our seminar was "how to maintain cerebral oxygen demand and supply balance." beginning with a short lecture about this concept, participants joined four hands-on scenarios: post-cardiac arrest syndrome (pcas), subarachnoid hemorrhage (sah), traumatic brain injury (tbi), and states epilepticus (se). in the pcas scenario, participants learned how to trouble shoot regarding targeted temperature management, especially in regard to the management of shivering. in the sah scenario, they learned about perioperative management, including delayed cerebral ischemia. in the tbi scenario, starting with actual insertion of an intracranial pressure (icp) monitor in the simulator, they learned about icp management through a scenario-based simulation. in the se scenario, they learned about se management, with actual continuous electroencephalogram monitoring. this seminar was held twice in . most participants were middle-aged intensivists; % were in their twenties ( / ), . % were in their thirties ( / ), . % were in their forties ( / ), and . % were in their fifties ( / ). most of the participating physicians were specialists in emergency or intensive care medicine ( . %; / ); nurses ( . %; / ) and a clinical engineer ( . %: / ) also participated. most participants ( . %; / ) were satisfied with the seminar, and almost all ( . %; / ) improved their self-confidence in the ability to carry out clinical practice in neurocritical care. we received positive, satisfied reactions from the japanese intensivists who participated in our seminar. for further improvement, we need to collect objective data to assess the utility of our neurocritical care hands-on seminar. lumbar puncture in the presence of mass effect? ciro ramos-estebanez uhcmc, case western reserve university/neurology, cleveland, ohio, usa introduction ) we propose an international consortium that would prospectively confirm the safety of low volume lumbar puncture (lp) in the presence of mass effect in selected scenarios. ) welcome peers and advisors to join the effort. lp may be clinically necessary in the presence of cerebral mass effect. while empirical antibiotic therapy is generally successful, specific groups such as post-instrumentation patients and immunosuppressed individuals may benefit from cerebrospinal fluid (csf) studies. in the absence of high-quality clinical recommendations, uncontrolled retrospective literature suggests that a small volume lp ( - cc) might be without complications in specific situations. nevertheless, the ethical principle of non-maleficence and the liability risk prevent clinicians from performing lps. in this scenario, an extended length of stay, poor outcomes, or a higher cost of care are legitimate concerns. synthesize an external peer reviewed methodology to maintain rigour and transparency. . seek appraisal, approval and endorsement of national and international quality improvement committees. . generate and assimilate the most current clinical evidence through: a. systematic review and meta-analysis. b. prospective randomized controlled clinical trial. . construct a protocol to inform decision making amongst healthcare and non-healthcare personnel. . dissemination and implementation. . schedule updates and/or revision. centers across the globe (north america = , south america= , europe= , and asia= ) have agreed to establish an lp consortium so far. retrospective analyses suggest low-volume lp's relative safety in the presence of increased icp. thereby, an expert consortium trusted with prospective verification would potentially benefit specific patient populations. patient centered decision making in the nccu requires family members understanding of their loved one's preferences and values as well as the complexities of their medical condition and treatments. family-centered care (fcc) is essential so that family members are actively involved in decision-making. stakeholders have reported their preference to receive prognostic information in smaller packets and recapitulated in different venues including rounds, bedside and care conferences. we examine implementation of a multimodal communication strategy on clinician utilization, family engagement and satisfaction in the nccu. an interdisciplinary team convened to develop a plan implementing a multimodal communication strategy. pre-implementation survey of clinicians (mds, nps, rns, etc.) and patient families was completed to determine the level of family engagement already in place in the nccu. four interventions were implemented: family communication boards were installed in patient rooms; family engagement pamphlets developed; a script and schedule for family care rounds was developed; nursing and provider staff were educated on inviting families to participate in patient care team rounds. family involvement on patient care rounds and family conferences was compared before and after the implementation of the best practice initiatives. additionally, pre and post implementation patient satisfaction survey results were also compared to evaluate the project's success. pre-implementation data was collected from october -november . sixty-one clinician surveys and forty family surveys found that family more consistently participated on daily rounds. baseline and postimplementation surveys demonstrated families feeling supported during the decision-making process. the implementation of a multimodal communication framework to achieve consistent family engagement and communication has led to an appreciable change in utilization by clinicians. its use is supported by consistent positive family attitudes towards communication and availability of information in the nccu. neurocritical care society undertook initiatives to integrate social media in member engagement activities and initiated a twitter journal club (#ncstjc) in with the first journal club conducted in february . articles were chosen by a subgroup of the communications committee in consultation with dr. eelco wijdicks, chief editor, neurocritical care journal. these articles were chosen based on their overall importance and the interest bound to generate amongst the journal club attendees. the journal club occurs bimonthy over an hour and is unique in the participation of the authors. the journal club is registered with healthcare hashtag project. each article chosen for #ncstjc is made available free to download weeks before and after the scheduled date for the journal club courts springer. analytics data on usage on article discussed in #ncstjc was obtained from sean beppler, editor, clinical medicine. between feb and apr , sessions were held with data available from . the ncc articles discussed had higher than average altmetric scores (measuring social media activity). these articles represented % of all ncc articles discussed on twitter since feb but . % of all tweets. total usage (number of times an article html page is accessed, or a pdf is downloaded ) was , ( mean , n= ) representing . % usage of all neurocritical care articles, a total of citations and downloads ( mean ) . the upper bound of the audience as assessed by the publisher was total of , for all articles (mean , per article) twitter is becoming an emerging platform for dissemination of information in medical education and academic activities. while the exact impact of the initiative on member engagement or outreach in enhancing journal impact or citations is hard to determine, we saw trends in enhanced engagement. neurostimulant medications have been studied in patients with traumatic brain injury, but few studies describe their use in patients with acute non-traumatic brain injury. our objective was to describe neurostimulant medication prescribing patterns, clinical response, and potential adverse effects in this patient population. retrospective database review of patients with acute ischemic stroke, intracerebral hemorrhage, or subarachnoid hemorrhage who received amantadine or modafinil from december through june . neurostimulant selection, dosing regimen, and indication were recorded. patients were classified as responders if they met two of the following three criteria within days of neurostimulant initiation: ) increase in average daily gcs of greater than points, ) neurological improvement documented in provider progress notes, or ) increased participation in rehabilitation therapies documented in physical or occupational therapist progress notes. safety data included need for a new anxiolytic or sleep aid or seizure. continuous data are reported as median with interquartile range. eighty-eight patients received neurostimulants: intracerebral hemorrhage (n= ), ischemic stroke (n= ), subarachnoid hemorrhage (n= ). median age was ( - ) years and ( %) were male. amantadine (n= ), modafinil (n= ), or both (n= ) were initiated a median of ( - ) days after hospital admission. the median initial daily dose of amantadine and modafinil were mg and mg, respectively. reasons for initiation included somnolence ( %), not following commands ( %), lack of eye opening ( %), and low gcs ( %). forty ( %) patients were responders, occurring at a median of ( - ) days after neurostimulant initiation. twenty-three ( %) patients required new prescription of an anxiolytic or sleep aid. four ( %) patients developed seizure. neurostimulant medications may increase wakefulness and participation in rehabilitation therapies in patients with acute non-traumatic brain injury, with tolerable adverse effects. the role of neurostimulants in this population should be defined in prospective studies. difficulty in obtaining peripheral intravenous (piv) access often necessitates central venous access placement in many critically ill patients. central line placement exposes patients to potential complications such as pneumothorax, hemorrhage, catheter-related infection or deep venous thrombosis. ultrasound-guided piv placement has become common practice in emergency departments, but there is no systematic program to train and support routine use of ultrasound-guided piv access in icus. we have developed a systematic program to train and support icu nurses in becoming experts and clinical leaders in ultrasound-guided piv placement. we hypothesize that implementation of this program will increase nurse confidence and chances of successful piv placement subsequently decrease central line-related complications in -bed neurocritical care icus. we have developed a video didactic training program for the neurocritical care nursing staff. the program discusses use and maintenance of the ultrasound machine and guided-technique for piv placement including the short-and long-axis approaches. the training video is followed by a hands-on simulation session using mannequins. standardized-surveys are administered to nurses before training and then at and months post training. we are prospectively collecting data on nurse comfort level with ultrasound-guided piv placement, total iv attempts, patient central line associated bacteremia (clab) rates and total number of patient central line days. we will compare this data for months pre-and post program implementation. comparisons will be made using t-test and chi-square analyses or non-parametric equivalents depending on data distribution. central-line related complications are an important clinical problem in all icus. we have developed and implemented a systematic training program to support nursing-led ultrasound-guided piv placement. we will determine if this program reduces the overall number of central lines placed, duration of indwelling central lines, and clab rates in a neurocritical care, and subsequently expand to additional icus and beyond. ultrasound measurement of optic nerve sheath diameter (onsd) is a sensitive and specific non-invasive ultrasonographers. despite clinical applications in the icu, er and outpatient settings, neurology residents lack experience and training. the aim of our project was to provide neurology residents with foundational skills in ocular ultrasound and onsd measurement. we designed a two-part workshop for neurology residents covering ultrasound basics, measurement of onsd, and ultrasound appearance of papilledema. workshop was a minute lecture and demonstration followed by minutes of hands-on practice. two weeks later, workshop included additional minutes of practice to consolidate learning. the practical portions were facilitated by emergency medicine attendings and residents with experience in performing ocular ultrasounds. neurology residents tracked the number of practice ultrasounds performed. they also completed anonymous pre-and posttests to assess their knowledge of ocular ultrasound and their comfort level and likelihood to perform future procedures using a -point likert scale. prior to the workshop, the majority ( / ) of neurology trainees had never performed an ocular ultrasound. one ( / ) was able to answer two basic questions about the procedure correctly, which increased to % on the posttest (n= ). trainees performed an average of ultrasounds total during the workshops. resident self-assessment of comfort performing the procedure increased from a median of "very uncomfortable" to "moderately comfortable" on the -point likert scale (p= . ). resident likelihood to perform the procedure in the future increased from a median of "very unlikely" to "moderately likely" on the -point likert scale (p= . ). this session successfully increased basic knowledge, comfort, and likelihood to perform ocular ultrasound among neurology residents. future directions include follow-up to gauge magnitude of practice changes and accuracy of procedural skills. reaching patients by telephone is a common method of assessing functional outcome, cognitive function, and quality of life after hospital discharge. however, when patients do not answer the phone, missing data creates bias and warrants strategies to increase follow-up rates. we hypothesized that we would have less follow up with patients discharged to long-term care facilities and sought to examine other potential sources of lost data. between / and / , we identified all patients admitted to the university of cincinnati neuroscience intensive care unit (nsicu). we excluded those with recurrent admissions, boarders, and those admitted < hours or for uncomplicated post-op care. telephone follow-up was attempted for each patient. univariate analysis was used to identify associations with patients who did not answer the phone. critically-ill patients were included. average age was . +/- . and % were men. the average hospital length-of-stay was . +/- . days. major diagnoses were: ischemic stroke ( %), intracerebral hemorrhage ( %), traumatic brain injury ( %), seizures/status epilepticus ( %) and subarachnoid hemorrhage ( %). ( %) died in the hospital; ( %) died by follow-up. survivors were assessed . +/- . days following admission. calls were answered, were not. there were no associations between rate of answered calls and age, gender, race, hospital length-of-stay, diagnosis, or hospital disposition. there were no differences in between morning vs. afternoon calls. only the number of attempts differed: the probability of an answered call was % on the first attempt but declined to % by the third attempt (or . ; p< . ). our outcome assessment strategy captured data on % of neurocritically ill patients. those who answer the phone are most likely to answer with the first call; the probability of a patient answering after a second phone call may not justify resources needed to continue calling these patients. posterior reversible encephalopathy syndrome (pres) typically presents with vasogenic edema on neuroimaging. a subset of patient, however, can have "atypical findings" including restricted diffusion and intracranial hemorrhage. these atypical findings all suggest acute vascular injury, and may mark a distinct pathophysiological subtype of pres. however, it is unknown whether atypical imaging findings are associated with differences in precipitating factors or outcome. patients with evidence of restricted diffusion, frank hematoma, microhemorrhage, or subarachnoid hemorrhage were classified as having atypical imaging findings. the demographics, risk factors, clinical outcomes, and degree of vasogenic edema for patients with typical (n = ) vs atypical pres findings of vascular injury (n = ) were analyzed. patients with atypical pres had a longer hospital stay ( . vs . days; p = . ) and were less likely to be discharged home ( . % vs . %; p = . ). severity of vasogenic edema (graded using a standardized radiologic scale) was also higher in patients with atypical imaging findings (severe edema: . % vs . %; p = . ). restricted diffusion and hemorrhage are features of acute vascular injury that may mark a unique pathophysiological subtype of pres. pres patients with these atypical imaging features had longer hospital stays, greater degree of vasogenic edema, and were less likely to be discharged home. this may be due to the fact that bleeding and infarction lead to irreversible brain injury, prolonging hospital stay and contributing to overall disability. in , deaths occurred in the neuro-oncologic critical care unit (nccu). the impact of this event is significant for patients, families, and the staff that care for them. brain and spinal pathology can be incredibly debilitating causing a rapid and impromptu decline of the patient's status. in order to better support patients, family, and staff throughout the dying process, the nccu staff created formalized endof-life care interventions. these interventions include educational pieces and supportive approaches to aid all involved through the dying process. several interventions were created to help transition the family members during the dying process. these include the creation of: dnr-cc closet, homemade blankets, condolence cards, hygiene bags, educational packets, word clouds, and aromatherapies and massage items. once the patient's code status is changed to comfort care, a blanket is given to the patient. family members are provided with a bag of toiletries for those that remain at bedside. education on the dying process are given to family members. multidisciplinary resources are provided, such as religious support focused on patient/family beliefs, palliative care for symptom management, and dietary provision of light snacks to the family. education for the physicians, nurse practitioners, nurses, and patient care associates was provided for those who wished to attend to further understand this end of life care program. a item tool was created to collect before and after data on staff satisfaction and comfort with the end of life process. results from this process are currently in process. creating specific end-of-life care interventions for the nccu have enabled staff to better care for patients and families. through the creation of the interventions and utilization of the dnr-cc closet, this unit has been able to better provide comprehensive education and supportive pieces to patients and family members during such a difficult time. delirium is a neuropsychiatric syndrome, characterized by disturbance in awareness, with reduced ability to sustain attention, impaired cognition, perception, tends to fluctuate in severity during the day; in critical care is associated with longer stay and increase mortality. this study aimed to determine the incidence, prevalence, predictors, risk factors and outcomes of delirium in critically ill adult. a historical cohort study was conducted in adult patients hospitalized in a polyvalent icu from january until december . delirium was diagnosed using cam-icu. a bivariate and multivariable risk were analyzed and presented as odds ratio (or) and % confidence interval (ci). a total of patients were enroll. delirium developed in patients.the incidence was . %. three independent predictors for delirium were identified, sedation (or ( % ci, p < , ); alcohol dependence (or , ; %ci, p < , ) and glasgow coma scale < (or , ; % ci, p < , ). delirious patients had a significantly apache ii ( ( - ) vs ( - ), p < . ), higher sofa, ( ( - ) vs ( - ), p< . ) and higher saps iii ( ( - ) vs ( - ), p< . ). other risk factors were hyperlactatemia ( p< . ), and hypotension (map< mmhg),(p< . ). patients required prolonged mechanical ventilation, p< . ), and prolonged icu-hospital stay. the incidence of delirium in the period from january to december , was . % in a polyvalent intensive care unit. exposure to sedative medications, alcohol dependence, and decrease glasgow coma scale minor are independent predictors for the development of delirium. similarly, the icu stay was longer in the group that developed delirium; however, mortality was not affected by the presence of this condition. it has been previously reported that the course of hsv- in the cns is significantly more benign that hsv- , and that it rarely causes encephalitis or significant morbidity in immunocompetent adults. the aim of our study was to investigate the claim that hsv- cns infections are typically benign, and to assess for predictors of poor outcome in those patients who do suffer significant morbidity from hsv- cns infections. restrospective chart reviews were completed on patients with a positive hsv pcr at our institution from july until july . patients with a hsv pcr positive for hsv- were selected in our analysis. multiple clinical variables were evaluated in these patients and we assessed outcome in the patient population, dichotomizing outcome into two categories at the time of discharge: good outcome defined as home or inpatient rehabilitation versus those with poor outcome defined as death, hospice, or placement in a long term acute care facility. patients with hsv- positive pcrs were identified. their charts were evaluated for demographics, laboratory values (serum and csf), imaging results, and outcome. there were patients with poor outcomes. it was noted that they were all female, their mean age was . (vs . in the good outcome group) and two of the three were immunocompromised ( % vs % in the good outcome group). statistical analysis was performed however due to the small sample size no statistical significance was found. however, age, sex, clinical presentation consistent with encephalitis and immune status seemed to have a trend towards poor outcome in this pilot study. future study with larger sample size is warrented to further assess this trend, as hsv- may not be as benign as previously reported. there is a high prevalence of non-traumatic illness in patients presenting to emergency departments as trauma team activations (tta). we sought to determine the prevalence of neurologic emergencies within a population of patients receiving a tta. this was a retrospective review of prospectively-collected registry data capturing all ttas in a highvolume, urban, academic level i trauma center. records from june through june were reviewed to identify patients found to have a diagnosis of ischemic stroke, intracerebral hemorrhage (ich), subarachnoid hemorrhage (sah) or status epilepticus. further demographic, clinical, and outcomes data was then abstracted from the electronic medical record. a proportion of abstracted charts were reviewed by an independent reviewer to ensure data quality. there were , trauma activations in the registry during the study period. patients ( . %) were found to have a nontraumatic neurologic emergency and were included in the analysis. of these patients, there were ischemic stroke ( %), ich ( %), sah ( %), and status epilepticus ( %) patients. the mean age was , and patients ( %) were male. the mean gcs on presentation was . about half of these patients ( %) were intubated in the emergency department. all patients received a head ct scan. patients ( %) received intravenous thrombolysis. neurologic emergencies such as ischemic stroke, ich, sah or status epilepticus were common diagnoses in this population of trauma activation patients. clinicians caring for patients in these settings must maintain a high index of suspicion for non-traumatic illnesses, and act quickly to mobilize appropriate resources when a diagnosis is made to avoid delays in care. further research is needed to examine ways to improve both time to diagnosis and quality of care in this patient population. formalized communication strategies decrease post-traumatic stress disorder (ptsd) symptoms in caregivers in the intensive care unit (icu). in one study, only % of family meetings met all shared decision-making criteria. however, much of the research has focused on family meetings, ignoring less formalized communication. the decision maker (patient or caregiver) was interviewed for all patients admitted to the medical (micu), neurosciences (nsicu), surgical (sicu), and cardiothoracic icu (cticu) for greater than hours. subjects who stated significant decisions had been made were asked to report on aspects of shared decision making on a -point scale. they identified the lead provider, who was subsequently approached to complete the same questionnaire. overall, eligible decision makers were identified, ( %) in the micu, ( %) in the nsicu, ( %) in the sicu, and ( %) in the cticu. of these, ( %) were unable to be contacted, ( %) had insufficient english, and ( %) reported no decisions made, with ( %) enrolled. nineteen ( %) provider interviews were completed. topics most reported covered "well" or "thoroughly" by caregivers were assessment of understanding ( , %) and the nature of the decision ( , %), while those least covered were need for input from others ( , %) and the context of the decision ( , %). topics reported most covered by providers were the nature of the decision ( , %) and opinions about the treatment decision ( , %), while those least covered were patient's values and preferences ( , %) and their preferred role in decision making ( , %). eighteen ( %) caregivers and ( %) providers described all topics covered "well" or "thoroughly." these results demonstrate differences in perception of shared decision making by decision makers and providers. further qualitative investigation is underway to elucidate the nature of these inconsistencies. organ donation is a life-saving medical intervention. the effect of race, insurance and economic status on organ donation and recipients has not been studied at a national level. in our study, we analyzed nationwide in-patient (nis) database of years - to select donors and recipients. baseline demographics (i.e., age, gender, race), insurance status and socio-economic status was compared between two groups. we identified donors (n= ) and recipients (n= ) from - . recipients were significantly older (mean age ± sd, . ± . vs . ± . , p< . ). donors had higher ( . % vs . %, p< . ) proportion of women compared to recipients. both groups had a higher proportion of whites compared to other races ( % and . % respectively). insured patients were largely represented in both groups with private insurance predominating in donors ( %) and medicare in recipients ( . %). interestingly, self pay represented . % of donors but only . % of recipients. race, insurance and socioeconomic status seem to be evenly similarly represented in donors and recipients. interestingly self pay insurance has a higher distribution among donors than recipients. central line-associated bloodstream infections (clabsis) are a common health care associated infection accounting for , infections annually in the intensive care and acute care areas (cdc, ). according to the center for disease control, clabsis result in thousands of deaths yearly and upwards of billions of health care dollars spent on preventable hospital acquired infections. intensive care patients, especially the neurocritical care population, have an increased need for centrally placed catheters related to inadequacy of peripheral access, need for caustic iv medications, and fluid resuscitation. our neuroicu's goal was to decrease utilization and subsequently reduce number of clabsis. in february , we initiated a patient-centered quality improvement effort with this goal in mind. the neuroicu clinical nurse leaders conducted rounds daily to evaluate the necessity and management of central lines. the neurocritical care team and clinical nurse leaders collaborated in exploring alternatives if central lines were present. in addition to daily rounding, clabsi bundles based on cdc guidelines for clabsi prevention were initiated. our neuroicu developed checklist "buster cards" in september of , prompting staff to the bundle interventions. the intent of the cards was to enhance nurse to nurse dialogue of bundle elements. the cards were evaluated monthly for trends in care. from august -june , there was a % reduction in neuroicu utilization of central lines. in addition, the mean number of clabsis per month decreased from . to . . trending of unit buster cards did not show care variances during this time period. implementing daily clinical nurse leader rounds with enhanced team communication significantly reduced the neuroicu's utilization of central lines and thereby decreased the rate of clabsis. percutaneous dilatational tracheostomy (pdt) is one of the most commonly performed procedures on critically ill patients. many studies showed the safety and feasibility of pdt, but there is limited data of pdt in neurocritical care units. we have described our experience of pdt performed by neurointensivist. pdts were performed by neurointensivists at bedside using the griggs guide wire dilating forceps technique. to confirm a secure puncture site, pdt was done under fiberoptic bronchoscopic guidance. from september to may , procedural data were prospectively collected. the patients' demographic and clinical characteristics were retrospectively reviewed. we analyzed immediate complications of pdt as the primary outcome. pdts were performed for patients; the mean age was . years, ( . %) were male, and mean acute physiology and chronic health evaluation ii score was . ± . . overall, the procedural success rate was % and the mean procedural time was . ± . min. periprocedural complications occurred in patients; had minor bleeding and had tracheal ring fracture. there were no serious periprocedural complications of pdt. from our experience, pdt performed by neurointensivist was safe and feasible and was implemented without serious complications. the neurocritical care unit (nccu) is a fast paced setting with a multitude of providers and team members requiring optimal communication. it is also a high cost/high utilization environment, dictating the need for patients to be moved thru appropriate levels of care efficiently. all of this must be accomplished while providing support and opportunity for collaboration and decision making on the part of the patient/ family unit. there is great discussion in the case management world about the benefits of a unit based verses service based case management model. we looked at outcomes following the implementation of a unit based case manager in the nccu. a dedicated case manager (cm) was implemented in the nccu to maximize assessment, advocacy, communication, education, identification of resources, and facilitation of services. processes to support maximal contributions were created.interventions included use of a discharge planning worksheet, implementation of a morning huddle, and space for the case manager to be physically available on the unit. los of patients discharged from nccu decreased from . to . . alos for patients that passed thru the nccu during their hospitalization decreased from . days to . . there was a % increase in discharges from nccu from to . average time from admission to cm assessment decreased from hours to . hours. progress notes indicating intervention and/or communication of the plan increased from to . staff questionnaire indicated increased awareness of los and dc plan needs. in this midwestern, academic medical center, integrating a dedicated, unit based cm resulted in improved los, increased discharges and improved staff awareness of dc plans. high throughput genotyping technologies and large collaborative consortia have revolutionized the field of medical genetics. open data access is the final barrier to be overcome to capitalize fully on the opportunities currently available in stroke genetics research. the international stroke genetics consortium (isgc) has created the cerebrovascular disease knowledge portal (cdkp), a comprehensive web-based resource to explore and freely access genetic data related to cerebrovascular diseases. funded by the nih, the cdkp has been jointly developed by the isgc and the american heart association (aha) institute for precision cardiovascular medicine. the cdkp seeks to democratize access to genomic data and potentiate stroke genomics research by providing open access to genetic, phenotypic and imaging data on stroke. within the cdkp, data are aggregated, integrated, and harmonized according to a pre-specified standardized pipeline. any institution or investigator working with stroke genomic data is welcome to deposit their data or use available data. the cdkp houses two types of data, each meeting different regulatory and analytical needs: summary level data and individual level data. the cdkp offers three main features: ( ) a web-based graphical user interphase that allows the exploration of stroke genomics information through a wide menu of integrated tools for analysis and data visualization; ( ) a repository of full sets of genome-wide summary statistics produced by published landmark studies in the field, available with a single mouse click ; and ( ) a repository of individual level data, accessible through a secure cloud working space provided by the aha platform for precision medicine. the cdkp can be accessed at www.cerebrovascularportal.org. the cdkp advances the isgc's goal of liberal data sharing in stroke genomics and other areas of cardiovascular research that may benefit from genomic analyses. in the future, phenotypic datasets can be added to further enrich sharing of non-genetic data as well. hyperosmolar therapy using hypertonic saline is common in patients admitted to the neurocritical care unit (nccu) for the management of different type of cytogenic cerebral edema or increased intracranial pressure (icp). vancomycin is commonly prescribed in nccu as empiric antimicrobial therapy. the purpose of the study is to evaluate the effects of hypertonic saline therapy on the pharmacokinetic parameters of vancomycin in critically ill patients with generalized or compartmental icp. this was a retrospective, observational study of adult patient consecutively admitted in the nccu between february and february who received hypertonic saline ( % sodium chloride) and vancomycin dosing protocol managed by the pharmacist. patients with serum creatinine > . mg/dl were excluded from the study. the estimation of vancomycin trough levels was done by using published pharmacokinetic equations and then compared to the measured trough levels with the paired t test. the study protocol was approved by our institutional review board. of forty-four patients who met the inclusion criteria, twenty-one patients ( . %) were diagnosed with intracerebral hemorrhage, nine ( . %) ischemic stroke, seven ( . %) subarachnoid hemorrhage, four ( %) subdural hemorrhage, two with brain tumors, and one patient with chiari malformation. the mean dosing regimen was . ± . mg/kg every ( - ) h. the mean measured trough level was lower than the predicted trough level ( ± . vs. . ± . mcg/ml; p < . ). the mean serum sodium level was ± meq/l and the mean serum osmolality was ± mosm/kg. critically ill patients with cerebral edema or high icp who were treated with hypertonic saline achieved a subtherapeutic vancomycin level that may lead to lower through level and possibly poor clinical response. further research is warranted to evaluate the clinical response of vancomycin in this patient population. unnecessary telestroke activations are costly to emergency departments (ed), telestroke providers, and patients. therefore it is important that ed nurses are well trained to effectively recognize stroke symptoms, and decrease the rate of false-positive stroke code activations. the nursing-driven acute stroke care (nas-care) study aims to determine if implementing a standardized ed stroke program decreases door-to-needle times in emergency departments utilizing telemedicine. the nas-care intervention consists of ed nursing education including mock codes, nihss certification, and implementation of a standardized flow sheet. in this interim analysis from the first (of ) nas-care study hospitals, we examined ed admission and discharge diagnoses at each site for months of blinded baseline data collection ("control") and months after standardized training ("intervention"). false-positive encounters were defined as stroke code activations for which the patient diagnosis on leaving the ed was not stroke. although hospitals trended toward a reduction in false-positive stroke code activations after implementation of the standardized stroke education, mock stroke codes, and flow sheet, none of the values were statistically significant. further research is needed to determine whether intensive ed nursing education can improve telestroke resource utilization. pharmacist-driven intravenous (iv) to oral (po) conversion protocols result in greater compliance, improved cost-savings, and better patient outcomes related to length of stay, re-admission, and duration of intravenous therapy. this study aims to determine the cost-savings and patient impacts of such a conversion protocol for anti-epileptic drugs (aeds) including lacosamide, levetiracetam, phenytoin, and valproic acid. a retrospective, observational phase was conducted to determine usual practice patterns concerning conversion to oral therapy between / / and / / . the conversion protocol was approved in december and implemented in january . a second retrospective phase observed conversion practices beginning / / and ending / / . length of intravenous and oral therapy, date eligible for conversion, and date of conversion were recorded. hospital acquisition costs were utilized for medication expenditure calculations. this information was used to determine financial impact of the protocol and is presented as descriptive endpoints. adverse drug events were collected via an institutional incident reporting system. a total of encounters were identified, resulting in encounters in the pre-cohort and postcohort encounters. looking at the pre and post cohorts respectively, both cohorts had similar median lengths of stay ( days vs. days), -day readmission rates ( . % vs . %), and rates of conversion from oral back to intravenous therapy ( . % vs . %). the median length of intravenous therapy was days prior to protocol implementation and decreased to days in the post-cohort. the average cost per day of aed therapy was $ . in the pre-cohort but decreased to $ . in the post-cohort. median missed opportunity costs, defined as the cost savings if conversion occurred at the earliest possible date, also decreased between the cohorts from $ . to $ . . pharmacist involvement in aed conversion had a positive financial impact without compromising patient care. the national institute of neurological disorders and stroke (ninds) established the nih strokenet to facilitate the rapid initiation and efficient implementation of multi-center exploratory and confirmatory clinical trials focused on promising interventions in stroke prevention, treatment, and recovery. strokenet was initiated in the fall , and involves over hospitals across the us. the network is anchored by regional coordinating centers (rccs), along with the national coordinating center (ncc) at the university of cincinnati and national data management center (ndmc) at the medical university of south carolina, as well as active participation by the ninds. one of the primary goals of the strokenet is to serve as the primary infrastructure for conducting stroke clinical trials and pipeline for new potential treatments. to maximize the impact of nih strokenet, it is important for the larger community of stroke researchers and clinicians, including the neurocritical care specialists, to know its structure and the process and timeline by which stroke trials are developed and implemented. since the inception of the network, * proposal concepts have been submitted to the strokenet and are in different development stages. among those evaluated to obtain ninds permission to submit a grant application, have submitted and are in the process. every application has been prepared and submitted for peer review within months of the ninds permission. two* funded strokenet trials are now underway with brisk enrollment rates, and another is awaiting study initiation. (*as of abstract submission date) the nih strokenet has become a stable infrastructure and offers several distinct advantages to developing competitive clinical trial proposals, including scientific input from the strokenet working groups, comprehensive feasibility assessments (including site enthusiasm and patient availability), assistance with grant budgeting, and other requirements for grant submission that are likely to help refine and improve the application. the modified early warning score (mews) is a physiological scoring system, validated in adult medicalsought to determine the value of mews to identify clinical deterioration or occurrence of sepsis in neuroicu patients. we retrospectively reviewed all patients admitted to the neurological intermediate care unit (imc) or neuroicu of a large tertiary care center from / presentation/during admission. baseline characteristics, diagnoses, physiologic parameters, infections, treatment with antibiotics, neurological worsening and mortality were abstracted from the electronic medical record. outcomes were defined as escalation of care and discovery of a new infection or sepsis. of p were male. % were intubated, and in-hospital mortality was % (versus % for all admissions). ( %) were already treated with antibiotics for a known infec diagnosed in %. in reaction to the elevated mews score, antibiotics were added or broadened in %, and level of care was escalated in . % from imc to icu. in . %, there was neurological worsening, most frequently associated with increasing cerebral edema ( %) and midline shift/herniation ( %). the mews score is not a valuable screening tool in the neuroicu population. it preferentially was triggered in known high acuity patients with ongoing or present infections with no change of management in the majority of patients. while associated with high mortality, its ability to indicate new infections or sepsis was poor. in out of patients, the mews score was associated with neurological worsening known at that time of the score. other screening tools should be explored for early warning in the neuroicu. introduction: it is challenging to maintain neurosciences critical care nursing expertise in an environment of a rapidly expanding knowledge, changing evidence-based practices and technological advancements. to address the needs for neuroscience nursing expertise in a mixed critical care unit, our institution developed a core group of nurses, known as "neuro champions", who have additional training and expertise in neurocritical care. methods: nursing participation was voluntary and recruitment was via unit-wide announcements. the goal was to improve patient care by developing a core group of nurses who serve as resources and educators for all things neurosciences related. to develop content expertise, the nurses initially completed a set curriculum including: neuro anatomy and pathophysiology, cerebral hemodynamics and multimodal monitoring, pupillometry, eeg interpretation, temperature management, evds, and quality indicators. bi-monthly meetings continued ongoing education, with content including clinical case studies and review of processes and protocols. additionally, beds staffed by neuro champions were designated as critical neurological care unit ("cncu") beds to co-localize the highest acuity neurosciences patients. the neuro champions are responsible for educating and sharing neuro related practices with the entire icu nursing staff. results: as a result of the implementation of the neuro champion role, our icu has benefited from: ) dedicated co-localized beds for the highest acuity neuro patients; ) increased number of enls certified nurses; ) improved collaboration between the medical team and nurses; ) promoting care uniformity to maintain comprehensive stroke center certification; ) integration of multimodal monitoring advancements, all of which supports advances in patient care and research. conclusions: the neuro champion role has provided a platform for neurosciences-specific nursing expertise in a mixed critical care unit and has facilitated education dissemination to the entire staff via a core group of nurses. this expanded knowledge has improved the care of the neurologically critically ill patients. the rate of cerebrovascular complications in patients treated with extracorporeal membrane oxygenation (ecmo) is about %. transcranial doppler (tcd) can be used to noninvasively monitor cerebral blood flow velocities (cbfvs) in patients undergoing ecmo. the aim of this study is to describe tcd-cbfv patterns in patients undergoing venovenous (vv) and venoarterial (va) ecmo. a neuro-surveillance protocol among ecmo patients was initiated as part of a quality improvement project at our institution. daily neurological exam, daily tcd, brain-ct on days one and three and -hr continuous eeg were performed in all patients undergoing vv and va-ecmo. demographics, clinical and imaging data were collected for the duration of ecmo support. cbfvs, lindegaard ratios (lr), pulsatility index (pi) and resistance index (ri) on tcd were collected. total of patients were included in the study [ female ( %); caucasians ( %)]. mean age was years. ( %) patients received va-ecmo; ( %) vv-ecmo; ( . %) received both va and vv-ecmo. median days on ecmo was days. median number of tcd studies performed was (mean, . we observed an overall pattern of low-normal flow cbfvs and reduced pulsatility in patients on va-ecmo. nurse practitioner (np) and physician assistant (pa) roles continue to expand in the critical care setting. single and multisite studies have examined various aspects of app practice, but none have focused on role implementation within the neurologic critical care unit (nccu). the purpose of this study was to obtain foundational knowledge about how nccu apps are implementing the role nationally this was a voluntary, cross-sectional, descriptive study of nurse practitioners (np) and physician assistant (pa) practicing in the us. apps were invited to participate in this voluntary, item survey. distribution occurred initially through email inquiry via multidisciplinary, professional organization listservs (ncs, aacn, aann) followed by snowball effect circulation. enrollment occurred from march to june . data was collected in redcap and analyzed using spss with descriptive statistics for demographic, institutional, practice, role characteristics of the sample and for each survey data element app participants completed the survey: % np, % pa, % other. the majority of respondents were master's prepared ( . %) acute care trained, ( . %) and hospital employed ( . %). participants were either early in their career ( . % - years as app) or experienced ( . % > years). % work in a direct care role with % providing total care for their patients with an average daily caseload of . + . patients. % of providers believed - patients was a reasonable caseload for total care. in addition to the nccu, % of participants care for patients in step-down or emergency department ( %) with % routinely bilingl for their work. this study is the first to provide information regarding how ncc apps are implementing the role in the united states. this study provides benchmarking data which may guide future research with this population as well as serve as a template for evaluation of other app specialty roles. despite advances in treatment, the median survival for high grade gliomas (hgg) remains poor. there is a growing body of research showing that palliative care improves quality of life and survival in patients with advanced malignancies. we sought to examine our own practices in the neurologic intensive care unit (nicu) regarding palliative care consultation in this population. we hypothesized that the incidence of palliative care consultation is low and associated with a clarification of patient's wishes, measured by a change in code status. we conducted a retrospective cohort review of patients with previously diagnosed hgg admitted to the nicu from - with a length of stay (los) greater than hours. the primary outcome was the incidence of patients with an advanced directive or inpatient palliative care consult (pcc). secondary outcomes included intensive care unit los, change in code status and location of death. patients were identified with hgg. the mean age was . years ( - years), % were male, % were white. zero patients were admitted with an advanced directive on admission. pcc was obtained in patients ( %). pcc was associated with increased nicu stay ( hrs vs hrs p= . ), a change in code status to do not resuscitate ( % vs % p= . ), and an increased likelihood to not die in the hospital ( % vs % p= . ). at our large academic tertiary care facility intensivists underutilize palliative care services for hgg patients. patients with fatal brain tumors are not having end of life discussions prior to admission, indicating a need for early palliative care intervention. patients are six times more likely to change their code status and there is a trend towards dying outside of the hospital if they receive a palliative care consult. hypertonic saline (hts), a hyperosmolar solution, is typically administered using a central venous catheter (cvc) due to concerns of extravasation, but a cvc is rarely readily available. in emergent situations, intraosseous (io) access is used when peripheral intravenous access is not available. existing literature does not address the administration of hypertonic saline using io access for adult patients with brain injury. the administration of hts is often delayed due to the time taken to obtain a central venous access. insertion of an io needle is typically much faster than a cvc. we report the safety and tolerability of hts using io route. a prospective pilot study on the safety and tolerability of % hts via io is currently underway. data on local complications at the site of injection, pain during insertion and during infusion, and serial serum sodium levels were collected. additionally, we report a case of successful administration of . % hts using the io route. preliminary data demonstrated that % hts was well-tolerated, with no reports of severe pain, infections, extravasation, soft tissue injury or local infectious complications in our sample of patients with brain injury. indications for use of hypertonic saline included patients with cerebral edema and mass effect from intracerebral hemorrhage. an appropriate rise of serum sodium levels by approximately mmol/l/hr in was observed. in the case where ml of . % hts, no local complications were observed and serum sodium levels rose appropriately. administration of hts using io route appears to be safe and feasible. utilizing io access for urgent administration of hts may reduce the lag time to administration of the initial bolus, reduce the need for emergent placement or eliminate the placement of cvc in certain cases. optic nerve sheath diameter (onsd) measurement is an emerging bedside tool to assess intracranial pressure (icp) non-invasively in brain injury patients. multiple studies demonstrate onsd width from . mm to . mm correspond to an external ventricular device (evd)-measured icp > mmhg. we sought to create a low cost, -d constructed, re-usable osnd teaching model to train neurology, neurosurgery, and critical care advanced practice providers and physicians. we searched the national library of medicine using terms "optic nerve sheath diameter ultrasound" with combinations of "simulation" and "model." the literature was used in conjunction with a human non-contrast head ct head model to make an eye ball model which was then tested in our simulation center and compared to a live human model. we identified articles, of which were associated with models and two with simulation. one gelatin model was reported, upon which we based our initial design. we could not validate the visual findings of this model. however, following construction of multiple beta models, the design most representative of human eye anatomy was a globe made of ballistics gel with either a mm, mm or mm -d printed "optic nerve" attached to a platform composed of ballistics gel and psyllium powder with a hollowed out core for ultrasound gel the globe rests upon. this model was taught to learners at a continuing medical education event prior to teaching osnd on a live human model. a -d printed skull from ct head data is being created to incorporate this model. a simple -d ballistic onsd model allows learners to learn proper hand placement, basic landmarks, onsd measurement, and practice proper pressure on human eyes. this model can be replicated and utilized in a sustainable fashion given that the globe and platform are composed of ballistics gel. pressure measurements using pressure guide wires is an invaluable diagnostic tool in the management of many endovascular revascularization therapies. its role is well established in coronary artery disease management such as use of fractional flow reserve (ffr) as a standard diagnostic tool to determine need for stenting, angioplasty or bypass. renal fractional flow reserve remains an integral physiologic parameter used in endovascular revascularization therapy of renal artery stenosis. despite the wide spread use of pressure wires in endovascular therapies, its application in the management of cerebral venous diseases remains vastly unexplored. we sought to evaluate the safety and applicability of pressure guide wires in several cerebral venous diseases. patients undergoing diagnostic angiography for possible venous outflow obstruction had pressures measured by pressure guide wires (volcano verrata® or prestige primewire®) across the following vessels: superior sagittal sinus, torcula, right and left transverse sinus, right and left sigmoid sinus, and right and left internal jugular vein. venous pressures were also collected from patients undergoing venous thrombectomy, stenting, or an arteriovenous malformation embolization (avm). five patients who underwent diagnostic angiography for pseudotumor cerebri showed no major variability in their pressures across the cerebral venous architecture which was confirmed by lack of stenosis or thrombi on intravascular ultrasound (ivus). four patients had a pressure difference above which was suggestive of a stenosis and later confirmed by ivus. patients undergoing pressure measurements that had evidence of stenosis or thrombosis by ivus showed improvement in pressure gradients post stenting or thrombectomy. no variability in pressure gradients were noted in a patient that underwent avm embolization. pressure measurements using pressure guide wires can improve diagnostic accuracy and guide management of several diseases of the cerebral venous system. further studies are necessary to understand the applicability of this approach in the management of venous disease. monitoring metrics is imperative for quality assurance and ongoing improvement in a developing clinical unit. a new neurocritical care unit (nccu), specializing in the treatment of critically ill, neurologicallyinjured patients opened in july . this study examined quality metrics that correlate with the development and growth of a neurocritical care program. data from patients with principle diagnoses of ischemic stroke (isc), subarachnoid (sah) or intracerebral (ich) hemorrhage, seizure, or brain tumor, admitted to nccu in and were used in the analyses. quality metrics included overall and individual complication rates per , patient days of pneumonia, venous thromboembolism, pulmonary embolism, sepsis, septic shock, pulmonary edema, gastrointestinal bleeding, and catheter associated urinary tract infection, as well as hospital mortality and length of stay (los). chi-squared and mann-whitney tests and poisson regression were used to compare metrics between and . patient volumes increased by . % ( to ) from to . the overall complication rate declined significantly from . to . per , patient days (p= . ). the highest complication rate in and was pneumonia ( . and . per , patient days, respectively). the proportion of patients who expired decreased from . % (n= ) in to . % (n= ) in , though not significantly (p= . ). there were no significant differences in los among patients with isc, brain tumor or seizure. however, those with sah or ich had significantly shorter stays in (median [interquartile range] = . [ . , . ]) versus ( . [ . , . ]) (p= . ). data suggest that over the initial -year period, complication rates among patients in the nccu improved. los did increase for hemorrhage patients; however, this may be related to greater severity of illness in the patient population over time. further analyses will be conducted to account for severity and other factors. delirium is a frequently seen but underestimated problem in critical care settings. delirium screening is considered time consuming, which is one of the factors leading to under diagnosis. the cam-icu screening tool for delirium has been validated in medical and surgical icus. among neurological patients, it has been validated in stroke patients but not in general neurocritical care population. this study was designed to validate cam-icu flow sheet in neurointensive care unit. a prospective cohort study was conducted in a bed neurointensive care unit of a university hospital. patients meeting the inclusion criteria (all nicu patients) and exclusion criteria (comatosed, aphasic, psychotic, prior diagnosis of neurocognitive disease, persistently vegetative state, sedated) were screened for delirium by ( ) a nurse practitioner using confusion assessment method (cam-icu) and ( ) a physician reference rator using delirium screening criteria in diagnostic and statistical manual of mental disorders- . assessments were done daily monday through friday for the icu stay. paired assessments were done less than hours apart. the study enrolled patients ( male, female). daily assessments were done. mean age of the patients was . and mean sap score was . admitting diagnoses were ich ( ), sah ( ), ischemic stroke ( ), tumor ( ), spinal surgery( ), neurological infections( ), seizures( ),elective angiograms( ), hydrocephalus( ), transverse myelitis( ) and av dural fistula( ). using dsm- criteria, the reference rator identified delirium in out of ( %) patients during the icu stay. out of assessments were positive for delirium according to dsm- and according to cam-icu. cam-icu flow sheet had sensitivity of . % ( %ci . % - . %) and specificity of . % ( %ci . %- . %). cam-icu has high sensitivity and specificity for diagnosing delirium in critically ill neurological population. it is a valid tool for diagnosing delirium. a value stream mapping event (vsm) for general neurology inpatients, revealed multiple barriers related to videofluoroscopy swallow studies. there was a high volume of patients requiring instrumental swallow assessments, a limited number of radiology appointments, and transportation delays that were delaying feeding plans, discharge recommendations and goals of care discussions. an operations engineer involved in the vsm event started the process by collecting observational data regariing timing. after meeting with the chief operating officer, director of patient transport, director of radiology, speech pathology manager, neuro intensive care unit manager and the operations engineer, a pilot program was agreed upon. the results for the three week pilot program were successful, and resulted in a permanent change in procedure. the pilot data showed a decrease in test time by minutes, a decrease in transport delays by minutes, and a decrease in length of stay by . days. the number of patients waiting for the study dropped from . to . per week. by annualizing this data, the change has created new available bed days, additional patient encounters and an incremental annual contribution margin of $ , . with the appointment time consistent, the nurse is able to plan patient care around the study, and ensure the patient is prepared and not delayed for the study. it has also allowed, if deemed safe for the patient to swallow, medications to be changed from the intravenous route to the oral route earlier, and earlier determination of safe feeding and diet restrictions. we previously reported outcome for children with refractory and super-refractory status epilepticus in a cohort of patients. mortality was %. % of survivors required new tracheostomy and/or gastrostomy tubes. the majority of surviving patients experienced some degree of disability at discharge as determined by the pediatric cerebral performance category scale (pcpc). here, we aimed to identify patient factors in this cohort that were associated with a decline in functional neurologic outcome at discharge. retrospective chart review of children age - years who received pentobarbital infusion for status epilepticus in the pediatric intensive care unit of a large tertiary children's hospital from - . outcome was defined using pcpc at admission and discharge. potential factors associated with outcome were evaluated using fisher's exact test and wilcoxon rank sum test. children were included. pcpc score at admission (p= . ), etiology of status epilepticus (p= . ), new tracheostomy (p= . ), and new gastrostomy tube (p= . ) were all significantly associated with children were more likely to have normal baseline neurologic function and more likely to have febrile encephalitis, stroke/trauma, or hypoxic ischemic encephalopathy as the etiology of status epilepticus. duration of pentobarbital infusion (median = days vs. days) (p= . ) and duration of hospital admission (median = . months vs. . months) (p= . ) were both longer in patients who had an admission pcpc score, etiology of status epilepticus, new tracheostomy and gastrostomy tube as well as longer duration of pentobarbital infusion and longer hospital stay were significantly associated with a decline in functional neurologic outcome at hospital discharge in children with refractory and superrefractory status epilepticus. status epilepticus (se) is the most common pediatric neurological, and super-refractory se is a lifethreatening form of se that continues or recurs for more than hours despite multiple therapeutic interventions. this population-based study investigated pediatric se and srse admissions in germany. pediatric (age - years) admissions between - were identified in the arvato health analytics database. se, epilepsy, and febrile seizure cases were identified using a modification of a previouslypublished algorithm based on icd- diagnosis codes (g , g , and r ) and coding for ventilator and intensive care unit use. based on primary diagnosis, prior epilepsy status, and ventilation se was subclassified as non-refractory, refractory (rse), and super-refractory (srse). inpatient mortality, costs, length-of-stay (los), and discharge disposition were assessed overall and for rse and srse. the algorithm identified , seizure-related admissions and classified % as se, of which . % were rse and . % were srse. the rse frequency was highest among ages - . the incidence of cases classified as srse peaked among newborns (age< year), decreasing between ages - years. cases classified as se accounted for . % of total costs associated with seizure-related hospitalizations. srse exhibited the highest per case cost (mean € , ), amounting to . % of all se costs, and these costs correlated with the highest los (median . days). srse was associated with greater mortality ( . %) cases classified as srse accounted for . % of all pediatric seizure-related costs, despite representing only . % of admissions. srse was associated with the highest los and mortality rate. these results highlight the burden of illness associated with srse and suggest that optimization of srse management has the potential to improve outcomes and reduce costs. despite its more routine use and the recognition that mri provides superior detection of traumatic brain injuries, there has been little written about how mri might affect the acute management of trauma patients. we sought to describe mri findings in a cohort of children admitted to the picu with tbi and to extend comparisons between ct and mri in acute trauma. a secondary aim was to quantify in what ways mri findings influenced clinical management in this cohort. we retrospectively identified patients admitted to the picu with an acute head injury between september and may who underwent head mri within the first hrs. we compared mri with ct findings, using the nih common data elements definitions of injury type. we determined by chart review the indication for mri and if there was documentation that mri led to a change in management, defined as either an escalation or a de-escalation of care. seven patients had mri only, and mri identified additional lesions in of the patients who had first undergone head ct. of these, patients had new intra-parenchymal lesions, had new extra-axial lesions, and had both a new intra-parenchymal and a new extra-parenchymal lesion identified. the most frequent new lesions were contusions and traumatic or diffuse axonal injury. acute management was influenced by mri in a majority of patients, leading to an escalation of medical or surgical management in nearly one third and a de-escalation of care in half. early mri may have a beneficial role in the acute management of pediatric traumatic brain injury. mri frequently identified clinically important lesions not appreciated on ct, and findings influenced management decisions. future studies will assess whether early mri improves patient outcomes or provides cost/benefit by reducing length of stay. while adverse outcomes of decompressive hemicraniectomy (dh) including infection, disturbances of the csf compartment, and sunken flap syndrome are well documented, there is a dearth of literature assessing outcomes related to the timing of cranioplasty. while adverse outcomes of decompressive hemicraniectomy (dh) including infection, disturbances of the csf compartment, and sunken flap syndrome are well documented, there is a dearth of literature assessing outcomes related to the timing of cranioplasty. we identified patients who received dh, of whom underwent reconstructive cranioplasty at our institution. the post-cranioplasty complication rate was %, which was due in part to hemorrhage, infectious complications, or csf compartment disturbances. patients receiving early cranioplasty developed an increased rate of hemorrhagic complication ( % vs %; p = . ), increased median hospital length of stay (los) ( vs days; p = . ) and increased median icu los ( vs days; p = . ). of the patients who received dh surgery related to malignant cerebral edema from an acute ischemic stroke, total complication rates trended down for early compared to late cranioplasty surgery ( % vs %; p = . ). patients receiving dh surgery for any cause who underwent early reconstructive cranioplasty, experienced higher rates of hemorrhagic complications and increased hospital and icu los. however, among those patients receiving dh surgery for the specific indication of malignant cerebral edema from acute ischemic stroke, significant differences did not exist between the early and late cranioplasty groups. the total complication rates in these patients trended lower in the early group. another important and mainly unpublished finding is that a majority of dh patients are lost to surgical follow up and may therefore impact the complication rate of this not so benign surgery. postoperative antibiotics (pa) are often administered to patients after instrumented spinal surgery until all drains are removed to prevent surgical site infections (ssi). this practice is discouraged by numerous medical society guidelines, so our institutional neurosurgery quality improvement committee decided to discontinue use of pa for this population. we retrospectively reviewed data for patients who had instrumented spinal surgery at our institution for seven months before and after this policy change and compared the frequency of ssi and development of antibiotic related complications in patients who received pa to those who did not (non-pa). we identified pa patients and non-pa patients. discontinuation of pa did not result in an increase in frequency of ssi ( % of pa patients vs. . % of non-pa patients, p= . ). growth of resistant bacteria was not significantly reduced in the non-pa period in comparison to the pa period ( % vs. %, p= ). the cost of antibiotics for pa patients was $ , . , whereas the cost of antibiotics for the non-pa patients was $ . on a per patient basis, the cost associated with antibiotics and resistant infections was significantly greater for patients who received pa than for those who did not (median of $ . with iqr $ . -$ . vs. median of $ with iqr $ -$ ; p< . ). after discontinuing pa for patients who had instrumented spinal procedures, we did not observe an increase in the frequency of ssi. we did, however, note that there was a non-significant decrease in the frequency of growth of resistant organisms. these findings suggest that patients in this population do not need pa, and complications can be reduced if pa are withheld. the development of flow-diverting stents has allowed for new treatment options for giant vertebrobasilar aneurysms. however, the expertise required to perform these procedures safely and concerns about complications continue to limit their use. we sought to identify common complications of this treatment that can be anticipated by neurointensivists, to optimize management in the postoperative period. we retrospectively reviewed our hospital database of treated aneurysms to identify those with giant vertebrobasilar aneurysms. medical and neurological complications were recorded. six patients ( male, female) underwent treatment of giant vertebrobasilar aneurysms with pipeline embolization devices. five received adjunctive coiling. frequently reported pre-procedure symptoms were dysphagia (n= ), diplopia (n= ), dysarthria (n= ), facial weakness (n= ), hemiparesis (n= ), gaze palsy (n= ), and nystagmus (n= ). five patients ambulated normally. due to concerns about necessary procedures after stenting when on antiplatelet therapy, three patients received prophylactic ventriculoperitoneal shunts, two underwent gastrostomy, and two underwent tracheostomy. angiography confirmed successful aneurysm embolization in all patients. postoperatively, all patients developed new or worsened symptoms attributed to brainstem edema, including hemiparesis (n= ), facial weakness (n= ), dysphagia (n= ), diplopia (n= ), nystagmus (n= ), gaze palsy (n= ), and dysarthria (n= ). neurological symptoms were treated with steroids, with most symptoms subsiding by discharge. five patients had medical complications, including pneumonia (n= ), respiratory failure (n= ), gastrointestinal bleeding (n= ), arrhythmia (n= ), urinary tract infection (n= ), and myocardial infarction (n= ). two patients were re-intubated, three underwent gastrostomy, and one underwent tracheostomy. functional status at -months was available for five patients. three achieved modified rankin scale scores between - , one regressed to a , and one died. the treatment of giant vertebrobasilar aneurysms presents significant challenges. practitioners should anticipate temporary postoperative neurological worsening and various medical complications. prophylactic shunt placement, gastrostomy, and/or tracheostomy should be considered in patients anticipated to likely need these procedures after treatment. ventriculostomy-related infection (vri) remains a major complication of external ventricular drain (evd) placement. historically, prophylactic antimicrobials are utilized to decrease the incidence of vri after evd placement. recent guidelines for the insertion and management of evds recommend a single preoperative dose prior to evd insertion and urges against the use of duration antibiotic prophylaxis. prior to the publication of this guideline, we hypothesized that significant variations existed among institutions with respect to antibiotic prophylaxis practices in this setting. the purpose of this practice survey was to determine trends in antimicrobial prophylactic strategies utilized by various healthcare institutions for evd placement prior to publication of the neurocritical care society (ncs) evidence-based guidelines for the insertion and management of evds. a seven-question practice survey on antimicrobial prophylaxis for evd placement was distributed to active pharmacist members of the ncs by email and open for response from / / to / / . the following information was collected: antimicrobial prophylaxis regimen utilized, pharmacologic class, utilization of impregnated catheters, and institution guidance. survey results were analyzed for trends in antimicrobial prophylaxis in the setting of evd placement. respondents ( / , % response rate) from institutions completed a seven-question evd management survey. most institutions initiate a single dose of antibiotics prior to evd insertion ( / , %). periprocedural antimicrobial therapy is the most common prophylactic strategy utilized by respondents ( / , %). of respondents who do not continue antimicrobial prophylaxis for the duration of evd placement, % ( / ) utilize antimicrobial-impregnated catheters to reduce incidence of vri. the importance of antimicrobial prophylaxis to prevent infectious complications associated with evd placement is widely accepted. prophylactic strategies vary between institutions. periprocedural antimicrobial therapy is the most common prophylactic strategy utilized by survey respondents. antimicrobial-impregnated catheters are commonly utilized in institutions using periprocedural antimicrobial prophylaxis. the postoperative course seen in critically ill neurosurgical patients is known to vary depending on the timing of the surgical procedure. this study seeks to compare the clinical characteristics, complications, and outcomes between elective or urgent surgery patients admitted to the intensive care unit (icu). retrospective review of a two-year neurosurgical patients' cohort. the pre and postoperative conditions and outcomes were compared between elective (group a) and emergency (group b) surgery patients. a total of patients were evaluated, in group a and in group b. the most common diagnosis was intracranial tumor. the mean american society of anesthesiology (asa) score was significantly higher in group b than in group a ( . vs. . , p< . ). mean sequential organ failure assessment (sofa) score on admission was higher in group b ( . vs. . , p< . ). these patients were more likely to require mechanical ventilation (or . , p< . ) and vasopressors (or . , p< . ) . group b had a higher probability of rebleeding (or . , p< . ), intracranial hypertension (or . , p< . ), hydrocephalus (or . , p< . ), and reintervention (or . , p= . ). post-operative nausea and vomiting were less likely in group b ( . % vs. . % and vs. . %, respectively). mean hospital and icu los were shorter in group a than in group b ( . vs. . and . vs. . , p< . respectively). mortality rate during icu stay was higher in group b ( . % vs. . %; or . , p< . ). the preoperative glasgow coma scale (gcs) in patients who died, was below in only a minority of them ( . % in group b; % in group a). in this cohort of neurosurgical patients, emergency, compared to elective operations, were associated with higher post-operative complications and mortality rates. emergency surgery was associated with a higher severity of illness measured by the sofa and asa scores. intraprocedure rupture (ipr) is a rare but potentially serious complication of endovascular coiling of intracranial aneurysms. potential complications include hemorrhage, ischemic stroke, vasospasm and hydrocephalus which can lead to increased morbidity and mortality. the clinical course for these patients is not well studied and characterized. we performed a retrospective review of prospectively collected data for all unruptured aneurysms treated with endovascular coil embolization between july and march at a large universitybased hospital. out of cases of all unruptured aneurysms coil embolizations, ( . %) patients had ipr. we reviewed baseline data, procedure notes, clinical course, and outcomes at discharge and at , and months. among the ten patients, the location of the aneurysms included: basilar apex, internal carotid artery anterior communicating artery, posterior cerebral artery, and posterior communicating artery aneurysm. patients were monitored in the icu for variable lengths of time and daily transcranial doppler ultrasound detected no significant sonographic vasospasm. the large majority of the patients ( / ) were discharged to home at their baseline functional status assessed by modified rankin scale. one patient was discharged to inpatient rehabilitation for cognitive deficits from ipr of a basilar apex aneurysm. they were subsequently discharged home with supervision. there was a single mortality in a patient receiving retreatment of a proximal ica aneurysm with prior stenting and coil embolization who developed massive subarachnoid hemorrhage with diffuse intraventricular hemorrhage with external ventricular drain placement. the incidence of ipr is very low and potentially serious complications occur rarely in these patients. the location and factors associated with ipr are highly variable and without clear associations. outcomes of such complications are overall favorable. a short observation period in the hospital is likely warranted with a benign clinical course the most likely outcome. the standard treatment of cerebral venous-sinus thrombosis (cvst) is anticoagulation. however some patients clinically deteriorate secondary to mass-effect from infarct or intracerebral-hemorrhage (ich). the role of decompressive-craniectomy (dc) in this patient population is unknown. we elucidate the baseline characteristics of patients treated with dc, and report their outcomes. a retrospective chart review of our institutional database identified patients with cvst who were treated with dc. demographic and clinical data were collected. imaging variables collected from ct-head or mri-brain immediately before dc were intracerebral-hemorrhage volume (ich-v), combined volume of mass-effect from infarct/ich and peri-lesional edema (me-v), midline-shift at level of pinealgland (mds-p), midline-shift at cranial-most portion of corpus-callosum (mds-cc), and herniation-type. favorable outcome was defined as glasgow-outcomes scale of - upon last-known follow-up. a total of patients (females= ) treated with dc were identified with mean-age . (+/- . ), mean glasgow-coma scale (gcs) before surgery (+/- . ), mean-ich-v . ml (+/- . ), mean-me-v . ml (+/- . ), mean-mds-p . mm (+/- . ), and mean-mds-cc . mm (+/- . ). transverse-sinus was most commonly involved (n= ). / patients had any herniation, most commonly cingulate (n= ). meanchange in gcs from admission to before-surgery was - . (+/- . ). ten patients were anticoagulated before surgery. on last-known follow-up, / patients had a favorable outcome. four had died. on chisquare analysis, superior-sagittal sinus thrombosis was associated with unfavorable outcomes (p= . ), and mortality (p= . ). on univariate binary-logistic regression, there was a non-significant trend towards unfavorable outcomes (p= . ) and mortality (p= . ) with every-point decrease in mean-gcs before surgery. the predictive-value of other factors towards outcomes is unknown given limited sample-size. decompressive-craniectomy might improve outcomes even in patients with cvst who have developed coma, cerebral herniation, have failed treatment with anticoagulation, and have large-volume masslesions causing midline-shifts of > mm. a prospective multi-institutional observational-cohort would poster presentations better delineate outcomes in comparison to matched-patients who are not treated with decompressivecraniectomy. meningiomas are often benign and mostly asymptomatic, and the treatment approaches may include open surgical resection, radiosurgery, and/or watchful waiting. reported morbidity and mortality rates for elderly patients undergoing meningioma resection vary widely. we sought to investigate mortality rates for elderly patients undergoing craniotomy for meningioma resection using the nationwide inpatient sample (nis). the nis datasets from to were used to identify patient admissions for meningioma resection based on the icd- -cm code . . age categories were defined as years of age. primary outcomes were in-hospital mortality, poor outcomes (defined as death or discharge to a facility other than home), cost and length of hospitalization. a total of , patients were identified who underwent meningioma resection during - of which . % were elderly (> years). each of the primary outcomes was heavily influenced by the advancing age. in-hospital mortality was higher in the elderly as compared to the younger patients ( . % vs % p< . ), as was the rate of a poor outcome ( . % vs . %, p< . ). elderly patients also had a higher cost ($ vs $ , p= . ) and increased length of hospitalization ( . vs . days, p< . ). in our study, age > was strongly associated with adverse outcomes after meningioma resection. this increased risk should be taken into account when considering surgical intervention in this subgroup. based on this study, closer perioperative monitoring may be warranted in the elderly patient subgroup. treatment with anticoagulation improves outcomes in cerebral venous-sinus thrombosis (cvst). however patients who develop extensive infarcts and/or intracerebral-hemorrhage with mass-effect resulting in comatose-state are at risk of poor outcomes, and may benefit from decompressive craniectomy (dc). we evaluated the role of dc in the management of malignant cvst and its impact on outcomes. literature-search was conducted on pubmed and google-scholar using terms "craniectomy", and "cerebral venous-sinus thrombosis". we included studies that described any number of patients with cvst who underwent dc after clinical deterioration and reported their outcomes. a similar search strategy identified patients from our institute. outcomes were reported as modified-rankin scale (mrs) or glasgow-outcomes scale (gos) and were classified as favorable (mrs - ; gos - ), or unfavorable (mrs - ; gos - ). a total of patients (females= ; males= ; unknown= ) who underwent dc for malignant-cvst were identified from studies (n= ) and our institute (n= ). age and gcs (before-surgery) were only available from patients, with mean-age . (+/- . ) and mean-gcs . (+/- . ). patients ( . %) had favorable-outcomes, while patients ( . %) died. in the multi-variate binarylogistic regression-model, every point-drop in gcs decreased the odds of favorable-outcomes by . times (p< . ; %ci= . - . ), and survival by . -times (p= . ; %ci= . - . ). thrombosis in internal-jugular vein (ijv) (or= . ; %ci= . - . ; p= . ) and deep-cerebral veins (dcv) (or= . ; %ci= . - . ; p= . ) predicted unfavorable-outcomes. ijv-thrombosis (or= . ; %ci= . - . ; p= . ) and dcv-thrombosis (or= . ; %ci= . - . ; p= . ) also predicted mortality. interestingly, cortical-vein thrombosis was associated with lower odds of unfavorable outcomes (or= . ; %ci= . - . ; p= . ). data regarding anticoagulation and long-term follow-up were not uniformly available. for patients with malignant-cvst, craniectomy could potentially improve outcomes. factors such as gcs before-surgery and cvst location can help predict outcome following dc and aid the decision-making process. a multi-institutional observational cohort should be designed to prospectively evaluate predictors for, timing of, and outcomes after craniectomy in cvst. the external ventricular drain (evd) is commonly used in the neurocritical care unit to help monitor intracranial pressure (icp) with the added advantage of therapeutically treating elevated icp by diverting cerebrospinal fluid (csf). placement of an evd can be complicated by hemorrhage surrounding the catheter insertion tract, which in some cases may prove to be fatal. this retrospective study was designed to look at the rate of tract hemorrhages after evd placement that were performed at our institution as well as associated outcomes. we conducted a retrospective review of all patients who underwent evd placement during a year period using our institutional database. postinsertion computerized tomography (ct) scans of the head were analyzed independently by physicians to identifying tract hemorrhages. data on primary diagnosis, age, sex, length of icu stay and mortality were collected and analyzed. a total of patients were identified as having had an evd placed during their hospital course. patients were excluded as there were no images of evds present in their records. patients were analyzed, of which % were male. mean age was . years. % of patients had a diagnosis of subarachnoid hemorrhage, % with intraparenchymal hemorrhage and % with ischemic stroke. mortality was % among all evd patients. the rate of tract hemorrhages among all patients with evd images was %. asymptomatic tract hemorrhages occurred in patients ( . %) with patient ( . %) dying due to the tract hemorrhage itself. among patients with tract hemorrhages mortality was . %. the rate of tract hemorrhages was noted to be % with the majority being asymptomatic. there was no difference in mortality among patients with evds who developed tract hemorrhages compared to patients with no tract hemorrhages. verapamil is a phenylalkylamine calcium channel blocker that blocks the calcium ion influx through slow channels into conductile and contractile myocardial cells and vascular smooth muscle cells resulting in vascular relaxation and vasodilatation. symptomatic hypotension and/or extreme bradycardia/asystole are often seen with intravenous verapamil administration requiring pharmacologic treatment. in neuroendovascular field verapamil is mainly being used as a vasodilator agent. current lack of pharmacokinetic/pharmacodynamics data of intra-arterial verapamil often makes very challenging to neurointerventionalists during endovascular procedures. the purpose of this study is to observe acute hemodynamic effects of intra-arterial verapamil administration as well as the safety of higher dose of the medication during endovascular treatment. ten patients who underwent endovascular treatment for acute ischemic stroke were evaluated pre and post procedure with vital signs. the dosage of intra-arterial verapamil was documented and tabulated along with the pre and post heart rate and systolic blood pressure. the dose of intra-arterial verapamil varied from to mg in each internal carotid or vertebral artery, total dose per patient per procedure varied from . to . the average dose of intra-arterial verapamil administered was . ± . mg or . ± . mcg/kg that were infused over to minutes. at the baseline before administration of intra-arterial verapamil, the mean systolic blood pressure (sbp) was . ± . mm hg and the mean heart rate (hr) was . ± . bpm. after administration of intraarterial verapamil, sbp decreased by mean of . ± . mm hg but we observed no symptomatic hypotension requiring any pharmacologic treatment. hr changed only by mean of . ± . bpm post intra-arterial verapamil. we observed no acute significant changes in hemodynamic parameters with administration of verapamil in carotid or vertebral arteries. this may represent its safe use during neuro-endovascular therapy. growing evidence suggests inflammation is critical in epileptogenesis. endogenous brain apolipoprotein e protein (apoe) modulates neuroinflammatory responses to injury through downregulation of glial activation and secondary neuronal injury. we created a amino acid peptide (cn- ) mimicking the binding face of apoe. cn- downregulates the inflammatory response in vitro and in vivo and improved histologic and clinical outcomes across several injury models in mice. we hypothesized that downregulation of inflammation by administration of cn- will reduce the development of epilepsy after pilocarpine induced status epilepticus in mice. c bl/ mice were intraperitoneally injected with pilocarpine to induce status epilepticus. following induction of status, animals were randomized to receive two doses of cn- or vehicle at minutes and hours. status was terminated by injection of benzodiazepine at minutes. epidural eeg leads were surgically placed at weeks and continuous video-eeg (cveeg) monitoring was performed for several days in a row at - weeks post status to determine spontaneous seizure development and frequency. at - weeks following induction of status epilepticus, administration of . or . mg/kg cn- reduced the development of epilepsy by approximately % compared to vehicle treated animals. further, cn- treated animals that did develop seizures had significantly fewer seizures than vehicle mice. similar results were seen with daily doses of mg/kg starting at minutes. importantly, cn- is not an anticonvulsant as cveeg monitoring during status induction clearly demonstrated that seizures were not stopped or reduced by injection of cn- . these results are consistent with the hypothesis that inflammation plays an important role in the development of epilepsy after injury and demonstrates treatments that target inflammation, like cn- , can prevent and/or reduce the development of epilepsy. this represents the first therapy to prevent the development of epilepsy that has entered into clinical trials. to determine the speed of brain entrance of the antiepileptic drugs (aeds) brivaracetam (brv) and levetiracetam (lev) after single intravenous dosing in humans. brv and lev both bind to synaptic vesicle protein a (sv a), but brv has more rapid brain entry than lev in mice and monkeys [ ] . sv a can be quantified in the living human brain using pet imaging with [ c]ucb-j[ ]. pet scans (n= ) were performed with [ c]ucb-j administered by a bolus-infusion protocol in healthy volunteers (n= ). therapeutic dosages of brv ( mg, n= ; mg, n= ; or mg, n= ) or lev ( mg, n= ) were administered as -minute intravenous infusions minutes after the start of the first pet scan. tracer displacement half-times were determined by subtracting the radioligand clearance halftime from the radioligand displacement half-times estimated by exponential fitting of the post-aed drop in distribution volumes (vt). data were also analyzed using an advanced mathematical model that described the relationship between brain [ c]ucb-j pet data and time-varying aed plasma curves to directly estimate brain entrance (k ) of both aeds and [ c]ucb-j, free fraction of [ c]ucb-j in the brain, and vt values. the radioligand clearance half-time was minutes. tracer displacement half-times were . and . minutes for brv mg, and ± minutes for lev mg. lower brv doses had longer half-times, but values were misleading as they assumed % sv a occupancy. the advanced compartment model described well -dose scans. using the advanced model, the brv uptake rate (~ ul/min/cm ) was found to be at least -fold higher than that of lev (~ ul/min/cm ). the results demonstrate that brv enters the human brain faster than lev. the potential therapeutic benefit of this has yet to be determined. while intravenous anesthetic therapy (ivat) represents the gold-standard for treatment of refractory status epilepticus (rse), the optimal depth and duration of therapy is not known. the goal of this retrospective observational study was to describe the relationship between the depth of burst suppression and the ability to successfully wean ivat during rse treatment. fifty patients were identified with rse who underwent continuous electroencephalography. using persyst, the suppression ratio (sr) was calculated up to hours prior to weaning ivat. the type and duration of ivat was recorded, as well as complications. we compared these variables between successful and unsuccessful weans. the mean sr for all patients was . ± . %, with a mean treatment duration of . ± . hours. there was no difference in treatment duration between successful and unsuccessful weans(p= . ), but sr was significantly lower in successful weans ( . ± . % vs . ± . %, p= . ). the receiver operating curve (roc) for the sensitivity and specificity of the mean sr to predict a successful weaning attempt did not identify a threshold to predict weaning success. the use of pentobarbital was associated with a significantly higher sr when compared to midazolam ( . ± . % vs . ± . %, p < . ). patients failed ivat weaning a mean . ± . hours after initiating the ivat wean, which occurred after a mean decrease in the midazolam infusion rate of ± %. depth of sr was not associated with infection risk (p= . ), but was associated with the need for tracheostomy ( . ± . % versus . ± . %, p= . ). vasopressors were required in . % of patients while on ivat. unsuccessful weaning of ivat was associated with a higher depth of sr, which is likely a marker of disease severity. depth of sedation was not associated with increased risk of infection, but was associated with the need for tracheostomy. vasopressor requirements are common. the primary objective of this study was to determine the sensitivity and specificity of real-time neuro icu nurse interpretation of quantitative eeg (qeeg) trends in the identification of recurrent nonconvulsive electrographic seizures in adult patients admitted to the neuro icu. thirteen adult patients admitted to the neuro icu that had nonconvulsive seizures on continuous eeg (ceeg) monitoring were included in the study. neuro icu nurses consented for their participation and underwent a brief, standardized qeeg training session. a -hour qeeg panel (rhythmicity spectrogram, left/right and amplitude-integrated eeg, left/right) printout containing the marked sentinel seizure(s) was displayed next to the bedside ceeg/qeeg monitor. at one-hour intervals, the nurses logged the number of seizures seen in the past hour based on their qeeg interpretation for the duration of their shift. their answers were compared with the gold standard of neurophysiologist interpretation of seizure occurrence on raw eeg. a total of hours of qeeg data was reviewed for patients. average length of data collection was . hours. for the neuro icu nurses' ability to detect the presence of seizures on real-time qeeg the sensitivity was . % ( % ci, . - . %) and specificity was . % ( % ci, . - . %). the positive predictive value for seizure detection was . % ( % ci, . - . %) and the negative predictive value was . % ( % ci, . - . %). the false-positive rate was . /hr. a simplified panel of qeeg trends can be used by neuro icu nurses to screen for recurrent electrographic seizures in critically ill patients with a reasonable sensitivity, an excellent specificity and a very low false-positive rate. this may facilitate earlier identification of recurrent electrographic seizures by notifying the neurophysiologist who is not present in the icu and not able to perform real-time ceeg interpretation. nonconvulsive status epilepticus (ncse) is an indicator of poor outcomes in neurocritical care settings. however, because of unfamiliarity with continuous electroencephalography monitoring (ceeg), the diagnosis and treatment of ncse remains challenging, and its clinical impact and prognostic factors have not been sufficiently reported in japan. we performed ceeg for adult patients in our neurocritical care unit with coma or unexplained altered mental status from april to september . we reviewed all ceeg records according to the american clinical neurophysiology society's terminology ( version), and diagnosed patients with ncse when the ceeg revealed spatiotemporally evolving or fluctuating periodic or rhythmic discharges and after considering clinical information based on the modified salzburg consensus criteria. patients with ncse were aggressively treated with benzodiazepines, fosphenytoin, and levetiracetam. they were divided into a generalized convulsive status epilepticus (gcse) group and a non-gcse group. we compared mortality and outcomes between the two groups after months using fischer's exact test. outcomes were defined as poor when the glasgow outcome scale score was worse at the -month follow-up than at admission. we excluded cases undergoing supportive care or lacking of follow-up. of cases in the study, cases were diagnosed with ncse, including cases with accompanying gcse and cases without. mortality rates at the -month follow-up were significantly higher in the non-gcse group than the gcse group ( % vs. %, respectively; p = . ). the rate of poor outcomes was significantly higher in the non-gcse group than in the gcse group ( % vs. %, respectively; p = . ). this study suggests that the absence of gcse is associated with increased mortality and poor outcomes among ncse patients. limitations of this study include its retrospective design and small number of ncse patients. further studies are necessary to identify additional prognostic factors. super-refractory status epilepticus (srse) is a life-threatening condition in which status epilepticus recurs or continues for over hours despite first-, second-, and anesthetic third-line agent (tla) medications. no treatments are currently approved for srse. a randomized, double-blind, multi-center, placebo-controlled phase trial evaluated brexanolone (usan; formerly sage- injection), a synaptic and extrasynaptic gabaa receptor positive allosteric modulator as adjunctive therapy for srse (nct ; "status trial"). enrolled subjects underwent a qualifying tla wean after at least hours of seizure-or burstsuppression. srse subjects failing the qualifying wean were randomized : to a blinded infusion of brexanolone or placebo as adjunctive therapy following resumption of one or more tla infusions. subjects were administered the blinded infusion for days, during which attempts were made to wean off tla infusions. clinical standardization guidelines (csgs) facilitated standardization across sites by outlining eeg patterns for which tla weaning should be continued, paused, or discontinued. an on-call clinical standardization team provided real-time support. safety was assessed via adverse events, laboratory testing, vital signs, and ecg parameters. the primary endpoint was defined as successfully super-refractory status epilepticus (srse) is a life-threatening neurological condition characterized by status epilepticus persisting over hours despite treatment with first-, second-, and third-line agents (tlas) or upon the weaning of tlas. currently, there is no consensus around treatment protocols for srse. this study aims to describe srse treatment patterns and related outcomes in a us population. we retrospectively identified srse cases in cerner healthfacts®, a large, de-identified, us electronic health record database, using records from - . cases were classified as srse using a modified version of a previously published algorithm using icd- and procedure coding for status epilepticus ( . , . , . x, . , . , . , and . ) , ventilator support, pharmacotherapies. descriptive and univariate statistics were used to evaluate anesthetic treatment, anti-epileptic medications, and the association between glasgow coma score (gcs) and mortality. using our algorithm, srse cases ( patients) were classified. multiple tlas were received in % of cases, and in %, > concurrent tlas were received. the first post-admission tlas were propofol, lorazepam and midazolam, respectively, in %, % and % of cases. median anesthetic duration was . days. mortality was higher in - ( . vs. . days; p< . ). srse patients identified in our analysis underwent variable treatment patterns, reflecting lack of co days of tla treatment. nonconvulsive seizures (ncs) and nonconvulsive status epilepticus (ncse) occur in approximately % of neurologically critically ill patients. the most effective antiepileptic drug (aed) regimen to treat ncs and ncse is unknown. this study was designed to determine the efficacy of add-on clobazam, a unique , -benzodiazepine with favorable pharmacokinetic properties, in the treatment of ncs and ncse. a retrospective chart review was performed on adult patients who were admitted to the neurological intensive care unit between january , and june , , were diagnosed with ncs or ncse by continuous eeg monitoring and received clobazam as add-on therapy. the primary efficacy endpoint was defined as clobazam being the last aed added before ncs/ncse cessation, regardless of latency between dosing and ncs/ncse cessation. of the patients included in this study, ( %) had ncs vs. ( %) with ncse. the most common etiologies were autoimmune (n= ) and cns tumor (n= ), with patients ( %) having pre-existing epilepsy. clobazam was the last aed added before cessation of ncs/ncse in of ( %) subjects. clobazam was chosen as the rd to th line agent. clobazam was started at a median of days from the onset ncs/ncse (range - days). the median total daily dose of clobazam was mg (range - mg). this study suggests that clobazam may be effective at various time points in the treatment of ncs/nsce and may prevent the need for addition of intravenous anesthetic drugs to control seizures. however, a prospective study is warranted to determine efficacy and optimal dosing. continuous electroencephalography monitoring(ceegm) with international - system is essential for detect nonconvulsive status epilepticus (ncse). in japan, both ceegm systems and human resources are lacking, and few facilities are able to conduct such advanced monitoring. the ceegm headset, described in this report, is a novel and easy-to-use technology. we attempted to validate the novel ceegm headset by comparing it with a conventional, international - ceegm system (conventional ceegm). we completed this study at a single center, eight-bed neurocritical care unit, between january and june . the new, ceegm headset features eight electrodes (f, c, t, o), and is capable of simultaneously transmitting eeg data by bluetooth. patients with disturbed consciousness, of unknown etiology, underwent ceegm headset followed by conventional ceegm. we verified the concordance rate of the two systems for detecting eeg morphologies (e.g. periodic discharges, rhythmic delta activity, spikes and waves), and diagnosing ncse. eeg morphologies were appreciated according to "american clinical neurophysiology society's standardized critical care eeg terminology: version" and diagnosis of ncse were done according to modified salzburg consensus criteria. among this period, we enrolled thirty patients. three patients were excluded because of not satisfying protocol. final analyses included verified data from patients. the mean age was years old (range: - ), % were male, mean acute physiology and chronic health evaluation (apache) ii score was (range: - ), and mean full outline of unresponsiveness (four) score was (range: - ). we appreciated concordant eeg morphologies, and ncse, in % ( / ), and % ( / ) of patients, respectively. this easy novel ceegm headset may be useful in settings with limited resources or access to conventional ceegm technology. further study is needed to validate the actual diagnostic ability of this novel headset. the traditional approach to interpreting eeg requires physicians with formal training to visually assess the waveforms. this approach is less practical in critical settings when a trained eeg specialist is not readily available to diagnose subclinical seizures, such as non-convulsive status epilepticus, in patients with altered mental status. we have recently invented an algorithm for sonifying eeg, and in the current study, we explored whether individuals without eeg training can detect ongoing seizures by simply listening to one channel of sonified eeg. we sonified eeg samples ( -second long) that represented various conditions commonly seen in the icu ( seizures; lpd, gpd, or burst suppression, and normal or slowing). medical students and nurses were asked to indicate each audio sample as "seizure" or "non-seizure". we then compared their performance with that of eeg experts [epilepsy attendings with > years of experience (n= ) and epilepsy fellows (n= )] and some of the medical students (n= ) who also diagnosed the same eegs on visual display. non-experts listening to single-channel sonified eegs detected seizures with remarkable sensitivity (students: ± %; nurses: ± %) compared to experts or non-experts reviewing the same eegs on visual display (attendings: %; fellows: ± %; students: ± %). if the eegs contained seizures or seizure-like activity, non-experts listening to sonified eegs rated them as seizures with high specificity (students: ± %; nurses: ± %) compared to experts or non-experts viewing the eegs visually (attendings: ± %; fellows: ± %; students: ± %). our study confirms that individuals without eeg training can detect ongoing seizures or seizure-like rhythmic periodic activity by merely listening to short duration of sonified eeg. while sonification of eeg cannot replace the traditional approaches to eeg interpretation, it provides a meaningful triage tool for fast assessment of patients with suspected subclinical seizures. super-refractory status epilepticus (srse) is a life-threatening form of status epilepticus (se) that continues despite, or recurs after, hours of therapeutic interventions, including continuous intravenous anesthetic third-line agents (tlas). no therapies are approved for srse, leading to substantial variation in both management and determination of treatment response. for the phase trial of brexanolone as adjunctive therapy for srse involving up to international sites, we developed and implemented clinical standardization guidelines (csgs) for real-time support of tla administration, weaning, and outcome assessment under eeg neuromonitoring. a clinical standardization team (cst), including investigators and se experts, developed consensus csgs defining acceptable eeg patterns for continuation, termination, or pausing the weaning of tlas. csg implementation was facilitated by training and cst call centers staffed internationally by physicians with critical care eeg expertise. in cases of disagreement, the local site retained final decision-making authority. a "traffic light" system defined: )"green" tolerated eeg patterns (improving background, seizures within hours, discharges > hz, or discharges - . hz with evolution and no improvement over hours), and )"amber" eeg patterns not meeting the above, for which tla weaning should be paused while optimizing anti-epileptic medications and monitoring for transitions to green/red eeg patterns. the initial cst consultations yielded % csg compliance; % of eegs underwent cst review. few cst consultations lasted > minutes ( %); most lasted < minutes ( %). this phase trial demonstrates the feasibility of applying neuromonitoring csgs for tla weaning in srse patients, to ensure better consistency of clinical care and reliability of the primary outcome measure in clinical trials. csgs were well accepted by investigators and may serve as a framework for future clinical trials or clinical therapies in srse. severe brain trauma is a leading cause of death and disability worldwide. post-traumatic epilepsy (pte) is a chronic complication that occurs in up to % of cases (frey, ; najafi et al., ) . drugs and other interventions to prevent epileptogenesis would likely be most effective early after traumatic brain injury (tbi), but cannot be given indiscriminately. there is a critical need for tools that quantify those at high risk for pte. abnormal neural activity, in the form of ictal-interictal continuum abnormalities(iicas) are increased acute brain injuries, and appear to differentiate patients at risk for secondary brain injury (e.g. kim et al., ) . we hypothesized that iicas acutely following tbi may be a marker of posttraumatic epilepsy risk. we evaluated continuous eeg data from moderate to severe tbi patients who did and did not develop pte, (any seizure - months post-tbi; n= ). seizures < month post-tbi were classified as symptomatic, not pte. conventional - scalp electrode placement was used and eegs were reviewed by standard visual analysis, by the mgh neurophysiology service. daily eeg reports were scored for the presence of iicas and seizures. demographic data including gender, age, tbi severity and type of brain injury were recorded. univariate and multivariate regression analyses were performed to determine which iica and demographic features correlated with pte. gcs (p= . ) and tbi severity (p= . ) were significantly associated with pte, as expected. seizures (p= . ), epileptiform discharges (p= . ), generalized periodic discharges ( . ) and lateralized rhythmic delta activity (p= . ) independently predicted risk for post-traumatic epilepsy. epileptiform discharges, in particular, were more prevalent acutely post-tbi in pte patients. increased iica prevalence is significantly associated with pte and may be a predictive marker for identifying patients who may benefit from anti-epileptogenesis trials. rapidly obtaining eeg signals in the ed and icu for at-risk patients can enhance diagnosis accuracy and speed, while cutting down time until treatment. ceribell inc has developed a portable eeg data recorder and electrode headset with rapid setup (~ min) technology without any eeg technician required to overcome the inaccessibility of eeg in urgent situations when seizures are suspected. the purpose of this study is to evaluate the signal quality and performance of the ceribell system compared to a reputable clinical eeg system. we collected eeg samples in the laboratory and at stanford university medical center. laboratory collections on healthy volunteers included simultaneous collection of eeg using ceribell and nihon kohden systems, and a split-signal that recorded eeg to both data recorders from the same electrodes. in the icu, eeg was recorded with the ceribell system on patients and subsequently with the clinical eeg system. data was filtered and spectral densities, mean frequency (mf), spectral entropy (se), and % spectral edge frequency (sef ) were computed. in the split-signal test, the waveforms consistently appeared similar by visual inspection. the analysis of ceribell data revealed (mf = . hz, se= . , sef = . ) similar to the commercial system (mf = . hz, se = . , sef = . ). in the simultaneous test, the ceribell system produced (mf = . hz, se = . , sef = . ) similar to the commercial system (mf = . hz, se = . , sef = . ). in the clinical setting, the ceribell system showed spectral density distributions comparable with the commercial system. our results indicate that the signal quality of the ceribell system is similar to a commercially available eeg used widely in the clinical setting, while requiring less setup time and allowing more portability. status epilepticus (se) is a life-threatening condition characterized by prolonged seizures without regaining consciousness between seizure events. when se continues or recurs hours or more after treatment with third line anesthetic agents, it is considered super-refractory se (srse). there are few population-based studies on the descriptive epidemiology of srse at a national level. the objective was to estimate the incidence of srse in canada in - . we analyzed standardized national administrative record-level data covering all provinces across canada as provided by the canadian institute for health information. srse episodes were classified from two databases for acute care admissions (discharge abstract database) and emergency visits (national ambulatory care reporting system) over fiscal years ( / to / ). cases were identified as srse using a modification of a previously published algorithm using icd- -ca diagnostic codes for epilepsy (g ), status epilepticus (g ), or convulsions (r ) plus an intensive care unit stay of days or more with mechanical invasive ventilation. using our algorithm, from - , the mean annual number of cases classified as srse was , ( . / , persons per year). the annual incidence was higher in males ( . / , per year) than females ( . / , per year). the highest rates were in the age group - years: . and . per , per year for females and males, respectively. the mean age of srse patients was years (sd= years), with % males. the most common comorbidities for srse included metabolic disturbances ( %), sepsis ( %), toxic withdrawal state ( %), cardiovascular disease ( %), and head trauma ( %). in-hospital mortality for srse was %. this is the first study reporting estimates of srse incidence in canada. these results suggest that srse is associated with a substantial disease burden. interventions that improve patient outcomes and reduce mortality are required. new-onset refractory status epilepticus (norse) is a condition characterized by prolonged pharmacoresistant seizures in a previously healthy individual with no identifiable etiology during initial evaluation. typical magnetic resonance imaging (mri) findings include bilateral limbic and neocortical t -weighted hyperintense lesions. fluorodeoxyglucose (fdg)-positron emission tomography (pet) findings have not been previously reported. this study sought to describe fdg-pet and mri characteristics in patients with norse. methods patients were retrospectively identified amongst a database of autoimmune-mediated encephalitis from - , meeting diagnostic criteria for norse and having undergone mri and pet over the course of their illness. imaging findings were confirmed with a board-certified neuroradiologist. nine patients were autoantibody positive: three n-methyl-d-aspartic acid (nmda) receptor, two glutamic acid decarboxylase (gad), three voltage-gated potassium channel (vgkc)-complex with two having leucine-rich glioma-inactivated protein igg positivity, and one gamma-aminobutyric acid (gaba) b receptor. all patients had identifiable abnormalities on fdg-pet. hypometabolism was most common, with of patients having diffuse, bilateral, or unilateral frontal, parietal, or occipital cortical hypometabolism. nine patients also had bilateral ( ) or unilateral ( ) mesial temporal hypermetabolism. two patients had multifocal hypermetabolism with bilateral or unilateral frontal abnormalities in addition to mesial temporal findings. of the nine patients with fdg-pet hypermetabolism, concurrent mri scans failed to show corresponding t -weighted hyperintense lesions in the mesial temporal and medial frontal regions in two patients. fdg-pet findings in norse include bilateral or unilateral mesial temporal or mesial frontal hypermetabolism with diffuse, bilateral, or focal cortical hypometabolism. hypermetabolism may reflect regions predominantly involved in acute epileptogenesis. fdg-pet may improve sensitivity when compared to mri alone. while seizures are uncommon but reported in primary intraventricular hemorrhage (ivh), little evidence is available on the prevalence of hyperexcitable patterns on long term eeg monitoring. we sought to determine the prevalence of hyperexcitable patterns and seizures in patients with primary ivh who were extracted from a cohort consisting of patients with spontaneous intracerebral hemorrhage (sich) who underwent continuous electroencephalogram (ceeg) monitoring between january and december at yale-new haven hospital. indications for ceeg monitoring included fluctuation of or depressed mental status, abnormal movements and a limited clinical exam. we recorded demographics, radiologic hydrocephalus, duration of eeg recording and eeg findings. hyperexcitable patterns comprised generalized, bilateral independent or lateralized periodic discharges (pds), lateralized rhythmic delta activity (rda), brief potentially ictal rhythmic discharges (b(i)rds), and spike-and-wave discharges (sw). of adults with sich who had ceeg performed, patients had primary ivh. hydrocephalus was present in patients ( %). patients were monitored for a mean duration of . (± . ) hours. patients had hyperexcitable patterns and/or electrographic seizures ( %): electrographic seizures and co-existent hyperexcitable patterns were captured in of patients ( %) and hyperexcitable patterns without seizures in of patients ( %). hyperexcitable patterns included periodic discharges (pds) ( ) (generalized, lateralized and bilateral independent, with and without rhythmicity), rhythmic delta activity (rda) ( ) (both lateralized and generalized, with and without sharps), brief potentially ictal rhythmic discharges(b(i)rds) ( ) and spike-and-wave discharges (sw) ( ). there was no significant difference between patients with and without hydrocephalus and hyperexcitability or electrographic seizures (p= . ). both electrographic seizures and/or patterns of hyperexcitability on eeg are common in our selected cohort of primary ivh patients. this underscores the importance of continuous eeg monitoring in this patient population, since the detection of non-convulsive seizures may offer an opportunity for therapeutic intervention. patients with aneurysmal sah (asah) frequently have ictal-interictal continuum (iic) eeg patterns. while seizure burden can worsen outcomes, less is known about iic burden. we investigated the impact of iic burden and anti-epileptic drug (aed) treatment on asah outcomes. we included patients with asah undergoing continuous eeg (ceeg) from - . patients with nonaneurysmal sah or %, - %, - %, - %, < %. age gender, admission gcs, apache ii score, fisher and hunt and hess (hh) scores, aed dosing and discharge gos were ascertained by chart review. presence of iic patterns in asah independently predicts worse neurologic outcome, although maximum burden does not. although nearly half of these patients receive aed treatment, our data suggest that aed treatment may not influence outcome. prospective studies may further delineate the clinical risks and benefits of aed treatment. refractory status epilepticus (rse) is defined by failure to control epileptic activity after the administration of st and nd line antiepileptic agents. mortality associated with rse has been estimated to be around - % at hospital discharge. we conducted this study to analyze trends in the frequency and management of rse. we conducted a cross-consortium (uhc) database from to . this is a database from academic medical centers and their affiliated hospitals in the united states and consists of a sample of , , patients. data including age, sex, antiepileptics (aed) and length of stay was collected. total mean age was . years and females were . %. there was an increasing trend of using lorazepam as the first line aed ( . % in to . % in ) and a decreasing trend was noted of using midazolam as the first line aed ( . % in aed ( . % in to . % in . leviteracetam was the most common second line aed used throughout all years which was followed by propofol followed by phenytoin/fosphenytoin. mean length of hospital stay was . days. between to , the proportion of hospitalized patients in the united states diagnosed with rse has increased. lorazepam and leviteracetam have been the most common aeds used. mean length of hospital stay has not changed. status epilepticus is associated with high risk of multi-organ dysfunction. ketamine for the treatment of super refractory status epilepticus (srse) has the benefit of a different mechanism and lack of cardiac depression when compared with other anesthetic agents. this study evaluated the improvement in sequential organ failure assessment (sofa) score in patients treated with ketamine for srse. this is a retrospective study of patients with srse from to . the timing and dosage of anesthetic agents used in their treatment were abstracted. sofa scores at admission and for the first days after initiation of ketamine were calculated. the presence of shock prior to initiation of ketamine included septic shock and cardiogenic shock. outcomes including mortality, organ failure, and hospital associated infections (hais) were also recorded. a total of patients were treated with ketamine after failure of seizure control using other anesthetic agents. seventeen ( . %) had an improvement of their sofa score while ( . %) did not. the median sofa score on admission was (iqr - ) for those who had an improvement and (iqr - ) for those who did not (p= . ). cardiac arrest was the etiology of srse for ( . %) patients who improved vs. ( . %) patients who did not (p= . ). patients required to vasopressors for hemodynamic support, with less needed for those who had an improvement (p= . ). there was a higher rate of hais in patient who did not have an improvement of their sofa score (p= . ). there is a subset of patients treated with ketamine for srse who have an improvement in their sofa score, require less vasopressor support, and have a lower rate of hais. further studies are needed to better understand which patient population may most benefit from the use of ketamine for treatment of srse. the ceribell eeg system (ces) is a novel channel eeg device with instant sonification and visual display capability that can be set up quickly without an eeg technician. we hypothesized that by using ces, we can decrease time to eeg acquisition and improve diagnosis and treatment decisions in suspected nonconvulsive seizures (ncs). adult icu patients (gcs < ) who had continuous eeg (ceeg) as part of clinical care were enrolled. once ceeg was ordered, consent was obtained and ces was placed by the treating physician (n= ) who listened to the left/right hemisphere signals for seconds each. suspicion for seizure ( =low, =high) and decision to treat (yes/no/not sure) were rated pre-and post-sonification. three blinded epileptologists compared accuracy of sonification with visual ces eeg. outcomes were difference in time to eeg acquisition, change in suspicion for seizure and decision to treat, and ease of use ( =challenging; =easy). patients (mean age +/- , median gcs of (iqr - . ) were enrolled from : am to : pm. start of eeg acquisition was significantly faster for ces ( minutes (iqr - ) vs minutes (iqr - ) p< . ), median difference minutes (iqr - ). one patient had ncs during sonification and this was accurately identified and treated. low suspicion for seizure ( ) was more likely postsonification ( % vs %, p= . ). treatment decision changed in % after sonification, and this was in the correct direction % of the time. inappropriate decision to treat decreased from % to % (p= . ). negative predictive value was % ( % ci - %). ces was consistently rated easy to use. the ceribell eeg system is easy to use, speeds eeg acquisition, accurately identifies ncs, and enables appropriate treatment decisions. it has the potential to greatly enhance timely diagnosis and treatment of ncs in critically ill patients. the aim of the study was to understand the efficacy of ketamine in refractory status epilepticus and identify the underlying factors affecting the effectiveness of ketamine. moreover, we also studied the rate of complications in patients who underwent continuous midazolam ketamine dual therapy for treatment of refractory status epilepticus. this is retrospective cohort study evaluating the efficacy of ketamine in patient with refractory status epilepticus in total of patients admitted to university of maryland medical center in either neuro intensive care unit /micu during the last five years between ( - ). we established a standardized algorithm for managing refractory status epilepticus. electrographic and clinical control of seizures was classified into four groups: likely response, possible response, permanent response and no response reviewed by a team of epileptologist and neuro intensivist. the effective doses of ketamine to abort rse were studied. complications intensive care unit stay while on therapy were reviewed. of the patients, were male, were female. % of the patients had cardiac arrest as an etiology of seizures. median loading dose was . mg/kg, median maintenance dosage was mg/kg/hr. % of the patients had no response to ketamine. % were responsive to ketamine of which, patients had likely response to ketamine, patients had possible response. . % of the patients had permanent response to ketamine. % patients had hospital acquired infections, % patient had metabolic acidosis, % had ards. this is one of the largest single center study illustrating the efficacy of ketamine in aborting rse. further study should address the difference in incidence of complications in patients with usage of ketamine versus groups alternative therapies. this study also demonstrates the etiology of seizures and its influence on efficacy of ketamine in aborting rse. acute cardiopulmonary complications are frequently observed in convulsive status epilepticus but mechanism is poorly understood. complications include tachy-arrhythmias, myocardial ischemia, takotsubo cardiomyopathy and neurogenic pulmonary edema. herein, we mapped evolution of cardiac dysautonomia as function of sequential electrographic stages of se in four subjects admitted to icu. we hypothesize pathological co-activation of both arms of autonomic system contributes to cardiac complications. heart rate variability (hrv) is considered a proxy for ans tone on heart. we analyzed hrv in time and frequency domain, complexity measure (lempel ziv-lz) during se and mapped changes as function of stages of se as determined by scalp eeg. conventional scalp eeg recording and lead i-ekg (sampled at hz) were analyzed using kubios hrv software . . cardiac vagal index (cvi) and cardiac sympathetic index (csi) were calculated using geometric lorenz-plot method. parasympathetic activity is expressed in rmssd, pnn, cvi, and hf power four adults (range - ; m= ) were admitted to icu following convulsive se. ictal hrv changes initially reflected high sympathetic system activation (high csi) and reduced vagal tone (low hf, rmssd) as reported previously with convulsive seizure. earlier stages of se (stage i and ii) were marked by dual activation of the ans with sympathetic predominance (lower cvi/csi ratio). later stages of se (stage iv and v), demonstrated progressive increase in parasympathetic activity (hf power, rmssd, cvi, cvi/csi ratio). hf power and rmssd at stage v se was three times higher than during discrete seizure. lz complexity measure downtrended with the loss of fluctuations in late stages of se. in one subject se terminated with asystole this case series highlights dynamic changes in sympatho-vagal imbalances with progressive se. dual activation of sympatho-parasympathetic system and loss of complexity measures are associated with increased cardiac complications. therapies directed towards stabilization of cardiac dysautonomia might minimize complications super-refractory status epilepticus (srse) is a life-threatening neurological condition that is characterized by status epilepticus that persists for hours despite treatment with first-, second-, and third-line agents (tlas) or upon the weaning of tlas. srse is associated with limited treatment options, and high morbidity and mortality. this study aims to describe and quantify inpatient srse treatment and its associated outcomes in the us. srse cases were classified retrospectively using a modified version of a previously published algorithm applied to a large, de-identified, us electronic health record database (cerner health facts®) covering > hospitals ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) . cases were classified utilizing icd- and procedure coding for status epilepticus ( . , . , . x, . , . , . and . ) , ventilator support or with los> days or missing age were excluded. univariate statistics were used to describe mortality, hospital los, icu los, and discharge disposition. our algorithm classified cases as srse ( patients). most cases ( %) were to large ( + beds) and/or teaching hospitals ( %). mean hospital los was . days, and icu los was . days. both los and icu los were significa average mortality rate was . %. mortality rates increased with number of tlas used ( - tla= . %; -rged home ( % with tracheostomy), while % (n= ) were discharged to another facility. treatment of srse requires acute, intensive management in the hospital setting. los and mortality rates were high and increased with increasing use of tlas. while good outcomes remain possible even after srse, additional interventions are needed that enable seizure control, liberation from anesthetic and ventilator management, and improved mortality. refractory status epilepticus (se) carries an exceedingly high mortality and morbidity, often warranting an aggressive therapeutic approach. initially used in childhood epilepsies, ketogenic diet (kd) has also accumulated supporting evidence in the treatment of pediatric se. recently, the implementation of kd in adults with refractory and super-refractory se has been shown to be feasible and effective. we describe our recent experience with a new onset refractory status epilepticus (norse) patient and the unexpected challenge of achieving and maintaining a ketotic target. practical advice, a comprehensive review of offenders jeopardizing ketosis commonly used in the neurocritical care unit and alternatives is provided. a previously healthy -year-old woman was admitted with cryptogenic norse following a febrile illness with a course complicated by prolonged super-refractory se. a comprehensive work-up was notable only for mild cerebral spinal fluid (csf) pleocytosis, elevated non-specific inflammatory serum markers, and edematous hippocampi with associated diffusion restriction on magnetic resonance imaging (mri). repeat csf testing was normal and serial mris demonstrated resolution of edema and diffusion restriction with gradually progressive hippocampal and diffuse atrophy. she required an aggressive approach including high anesthetic infusion rates, anti-seizure drug trials (in various combinations), empiric partial bilateral oophorectomy, and immunosuppression. enteral ketogenic formula was started on hospital day , however, sustained beta-hydroxybutyrate levels > mmol/l were only achieved days later following a careful comprehensive adjustment of the care plan. notably, a significant response to kd was only achieved with beta-hydroxybutyrate levels > . mmol/l. there are hidden carbohydrates in commonly administered medications for se, antibiotics, and even electrolyte repletion formulations and solutions used for oral care -all challenging the use of kd in this setting. tailoring comprehensive care and being aware of possible complications of kd are important for the successful implementation and maintenance of ketosis. early seizures are estimated to occur in - % of patients with moderate to severe traumatic brain injury (tbi) (herman , vespa ). continuous eeg (ceeg) is essential for detection of nonconvulsive seizures (claassen ) the university of california davis protocol for tbi includes ceeg on a case by case basis, which we reviewed. a retrospective review of patients admitted to icu for tbi from / / - / / was performed for demographics, icu length of stay (los), and ceeg. patients with ceeg were assessed for demographics, tbi severity, gcs, ceeg indication and findings. patients were identified. twenty-one were monitored on eeg. median age was , % were female. indications for ceeg included seizure prior to admission (n= ), altered mental status (ams) (n= ), ams with paroxysmal events (n= ). seizures were recorded in patients. median duration of ceeg was . , . , and . hours among the groups. those with seizures prior to hospitalization were connected to ceeg earliest (median . hours) but had the longest median icu los ( . hours), followed by ams ( . and . hours) and ams with paroxysmal events ( . and . hours). median gcs was , , and respectively. median los for patients without seizures or interictal epileptiform activity (iea) was . hours, . for those with iea only, and . for those with seizures. median gcs was . , , and among the eeg groupings. our data suggests seizures prior to hospitalization, ceeg recorded seizures, and iea predict longer icu los. associated lower gcs likely indicates more severe injuries. tbi patients with ams may have delay to seizure detection and treatment. our rate of seizure detection is lower than expected. a more consistent protocol for ceeg will likely improve seizure detection. prospective studies are needed to determine if ceeg can predict and influence outcomes. status epilepticus is a serious neurologic emergency. although many studies have been published on incident status epilepticus, there are few data on the risk of recurrent status epilepticus. we performed a retrospective cohort study using administrative claims data to identify all patients hospitalized with status epilepticus in california, new york, and florida between - . our primary outcome was a recurrent hospitalization for status epilepticus. survival statistics were used to calculate the cumulative rate of recurrence at days, year, and years. in subgroup analyses, we compared rates of recurrence according to age, gender, race, and etiology (stroke, traumatic brain injury, acute and chronic central nervous system (cns) infections, brain tumors, dementia, autoimmune cns disease, or unspecified etiology). we identified , patients with status epilepticus. during a mean follow-up of . (± . ) years, , ( . %; % ci, . - . %) developed recurrent status epilepticus. the cumulative rate of recurrence was . % ( % ci, . - . %) at days, . % ( % ci, . - . %) at year, and . % ( % ci, . - . %) at years. the -year cumulative rate of recurrence was . % ( % ci, . - . %) in women versus . % ( % ci, . - . %) in men, . % ( % ci, . - . % ( % ci, . - . %) in patients < , and . % ( % ci, . - . %) in white patients versus . % ( % ci, . %- . %) in non-white patients. the -year cumulative rate of recurrence was highest for status epilepticus associated with autoimmune cns disease ( . %; % ci, . - . %) and chronic cns infection ( . %; % ci, . - . %). approximately in patients with status epilepticus experienced a recurrent episode within years. recurrence was most often seen in younger patients, non-white patients, and patients with underlying autoimmune cns disease or chronic cns infection. super-refractory status epilepticus (srse) is a rare, life-threatening form of status epilepticus (se) refractory to multiple therapies including anesthetic third-line agents (tlas). enrollment in a srse clinical trial is challenging because patients may present urgently before srse is confirmed or may dynamically improve before randomization. pivotal clinical trials in srse require patient selection criteria accurately identifying srse at randomization. in this phase trial of brexanolone as adjunctive therapy for confirmed srse, the enrollment scheme enabled operationally confirming srse prior to randomization during a qualifying wean (qw) under real-time eeg neuromonitoring. informed consent was obtained for all subjects ) admitted in se having failed first-and second-line therapies; ) transferred on tlas in seizure-or burst-suppression; or ) transferred without seizure-or burst-suppression or not receiving tlas. subjects were required to achieve seizure-or burst-suppression for hours through continuous administration of one or more tlas, followed by a post-enrollment qw of tlas. enrolled subjects failing the qw were randomized to concomitant brexanolone or placebo following reinstitution of one or more tlas. subjects not randomized after a successful qw underwent a -week follow-up. the qw protocol and criteria for qw failure were developed and implemented utilizing eeg neuromonitoring to confirm srse after enrollment using the definition of shorvon and colleagues. a qw was performed on over evaluable subjects across international sites to enable enrollment of patients with confirmed srse. subjects with a successful qws who were not randomized provided insight into outcomes associated with se and avoided the randomization of patients who did not meet srse criteria following enrollment. the use of neuromonitoring-guided diagnosis during a structured qw helped confirm srse, facilitating the enrollment of appropriate patients into this phase trial in a rare, critically ill, and dynamic srse patient population. autoantibodies to the kda isoform of gulutamic acid decarboxylase (gad ab), commonly found in t dm patients, have been associated with drug resistant epilepsy. ketosis prone diabetes is a heterogenous syndrome encompassing various forms of beta cell dysfunction culminating in diabetic ketoacidosis. rates of epilepsy in patients with ketosis prone diabetes are not known. we compared the prevalence of epilepsy in patients with ketosis prone diabetes in a multi-ethnic population with the prevalence of epilepsy in the type diabetes population as well as the general population in a metropolitan medical center. our study design is prospective review of retrospectively collected sera of patients admitted for diabetic ketoacidosis (defined as ph < . , bicarb < , with ketonemia or ketonuria) for the presence of gad ab. all these sera were assessed separately for autoantibody presence or absence at dr hampe's lab in washington, seattle. we also reviewed patients medical records for neurological diagnoses. this done in a blinded fashion by two separate reviewers. out of our patients with ketosis prone diabetes, . % also had epilepsy. this is higher than the published rate in type diabetics ( . %) and the general population in the surrounding area (< . %). antibody testing revealed % of patients with ketosis prone diabetes were gad ab positive with a rate of epilepsy of %. a two-tailed t test between the gad ab + group and gad ab -group showed no statistically significant difference in prevalence of epilepsy in these two groups. while prevalence of epilepsy is higher in the ketosis prone diabetes population than the general population of houston, the difference is not related to titers of gad ab, and must be due to some other unknown factor in these patients management of refractory status epilepticus commonly involves the induction of seizure-or burstsuppression using anesthetic agents. however, the duration and endpoints of these therapies are not well defined. specifically, weaning anesthetic agents is complicated by the emergence of eeg patterns on the ictal-interictal continuum (iic), which have uncertain significance, given that iic patterns may worsen cerebral metabolism and oxygenation, have a dissociation between scalp and depth eeg recordings, and indicate a late stage of status epilepticus itself. determining the significance of iic patterns in the unique context of anesthetic weaning is important to prevent the potential for unnecessarily prolonging anesthetic coma. we identified a series of patients who underwent over hours of burst-suppression therapy, multiple weaning attempts, and continued weaning despite the initial emergence of iic patterns. patients who experienced anoxic brain injury were excluded from the series. we report cases of patients who underwent successful weaning despite initial emergence of iic patterns. eeg patterns following anesthetic weaning (including lateralized periodic discharges approaching hz frequency and lateralized rhythmic delta activity) as well as terminal eeg patterns are described in detail. in these patients, continuing weaning of anesthetic agents despite the emergence of iic patterns did not result in relapse to status epilepticus. while the metabolic impact of these patterns on brain activity is uncertain, weaning strategies that treat iic as a surrogate of recurrent status epilepticus risk further prolonging anesthetic management and its known toxicity. we speculate that iic patterns are transitional and may have a context-specific association with status epilepticus relapse, with less risk conferred when these patterns are observed during the weaning of anesthetic agents after prolonged burst-suppression therapy. other electrographic features aside from this clinical context may discriminate the risk of status epilepticus relapse, such as eeg background activity. brivaracetam (brv) is approved as adjunctive therapy for focal (partialyears) with epilepsy. brv is available as oral tablets, oral solution, and an intravenous (iv) formulation. the formulations are interchangeable. this abstract reports the safety and tolerability of iv brv. during clinical development, participants received iv brv. we report pooled safety findings from participants receiving brv - mg doses. the therapeutic range of brv is - mg twice daily. in n , healthy volunteers received iv brv as a -minute infusion or mg/min bolus ( , , , or mg single doses; n= in all groups). in ep (nct ), healthy volunteers received iv brv mg as a single -minute bolus injection or oral tablets. in n (nct ), patients received days of brv oral tablets mg twice daily or placebo, and then . days of iv brv mg twice daily either as a -minute bolus or -minute infusion for nine doses in total. treatment-emergent adverse event (teae) data were pooled. data reported are for iv brv - mg (n= ). most frequent teaes were somnolence . %, dizziness . %, fatigue . %, headache . %, dysgeusia . %, euphoric mood . %, feeling drunk . %, and infusion-site pain . %. infusion-site pain was specific to administration route. most teaes were mild or moderate and occurred mostly in healthy volunteers. iv brv was well tolerated, with an ae profile consistent with oral administration except for routespecific injection-site aes, dysgeusia, euphoric mood and feeling drunk. the interpretation of these data was complicated by the difficulty of pooling disparate studies involving healthy volunteers and epilepsy patients with heterogeneous medical histories and concomitant antiepileptic drug use. further clinical trials or real-world experience are needed to understand potential clinical impact. ucb pharma funded refractory status epilepticus (rse) is a challenging condition that requires multiple antiepileptic drugs (aed) to treat. during rse, the brain is under excessive excitation, which results in an increase in glutamate receptors such as alpha-amino- -hydroxy- -methyl- -isoxazolepropionic acid (ampa) and nmethyl-daspartate (nmda).. perampanel (per), a novel, noncompetitive ampa-receptor antagonist, may have a role in the treatment of rse and there are positive results in different animal models with rse. we identified adults patients over a month period who were treated with per for different forms of rse. one was excluded as the etiology of rse was anoxic brain injury and care was transitioned to comfort only within hours of initiating per. three patients had a definite response to per, which we defined as resolution of ictal patterns on electroencephalogram (eeg) within hours of per without adding a new aed. one had a possible response with significant improvement in eeg findings; however, there was some eeg improvement predating the initiation of per. in observed several treatment factors that may have increased response to per. those who responded had it used earlier in the treatment cascade (sixth or seventh vs. ninth or tenthaed ), higher initial dose ( mg vs mg), and were escalated to maximum dosage within hours. they were also more likely be receiving continuous ketamine and midazolam, suggesting a possible synergy with per. there were no documented adverse effects in any patient prior to discharge. one patient did experience a decline in phenytoin levels, which could be related to per as there are reports of enzyme-inducing properties. we observed efficacy of per in several patients with focal and generalized rse without a significant adverse effect profile. further studies are needed to clarify the dosing, timing and appropriate indications in rse treatment. topiramate is a potent broad-spectrum anti-epileptic drug (aed) with several mechanisms of action including blockage of the inotropic glutamatergic ampa receptor, voltage-gated sodium channels, antagonism of non-nmda glutamate receptors and enhancement of gaba mediated chloride conductance. we hypothesize that topiramate is an effective adjunctive therapy in rse and srse due to multiple mechanisms of action. we performed a retrospective analysis of patients admitted to the intensive care unit with status epilepticus (se) at a tertiary referral center from - . we reviewed demographics, age, seizure type, etiology, prior aed/topiramate exposure, time to response to treatment, eeg reports and neuroimaging results. rse was defined as failure of benzodiazepine and another conventional second line aed to stop se. srse was defined as se that continues or recurs hours after being treated with an anesthetic agent. ( %) were male, ( %) had a history of seizures; mean age of patients with se was . years. of treated patients, ( %) had focal non-convulsive se (ncse), ( %) had myoclonic se, had myoclonic, followed by generalized ncse, ( %) had generalized ncse, and ( %) had focal and generalized nonconvulsive se, prior to administration of topiramate. ( %) patients were treated with aeds, ( %) patients with aeds prior to topiramate. electrographic seizures improved in ( %) patients after receiving topiramate. resolution of electrographic seizures occurred within hours in ( %) patients, hours in ( %) patients, hours in ( %) patients and hours in ( %) patients. our findings suggest that topiramate could be an effective adjunctive treatment in rse and srse. however, prospective studies, including larger number of patients are needed to confirm these findings. patients with refractory status epilepticus (se) require multiple antiepileptic drugs (aeds) to abort seizures, and often barbiturates. there is a paucity of data on how to wean aeds safely once seizures are controlled while minimizing medication side-effects or withdrawal symptoms. a retrospective review of patients admitted to mayo clinic in rochester, minnesota for se between and was performed. patient demographics, se type (focal versus generalized, convulsive, and refractoriness), seizure etiology, aeds in admission and at outpatient follow-up, aed side effects from use and withdrawal, and functional outcomes in terms of modified rankin scale were recorded. of ( . %) patients had refractory se, ( . %) patients had refractory non-convulsive status epilepticus (ncse), ( . %) patients had convulsive se, ( . %) patients had ncse, and ( . %) patients had epilepsia partialis continua. of the patients with outpatient follow-up (ranging to weeks following hospital discharge with . % patients following-up within one month), patients were on an aed regardless of etiology. patients were on a median of aed in both refractory and nonrefractory se at follow-up. ( . %) patients had withdrawal seizures after aeds were weaned ( had a prior stroke, traumatic brain injury, idiopathic, multifactorial). none of the patients completely weaned off a barbiturate had seizure recurrence at follow-up. -month mortality in refractory se was / ( . %) and / ( . %) in non-refractory cases. favorable functional outcome at follow-up was achieved in / ( . %) patients with refractory se versus / ( . %) in non-refractory se. we found a low rate of late seizure recurrence after weaning aeds in refractory and non-refractory se, particularly in the case of barbiturates. spreading depolarizations (sd) are strongly associated with secondary brain injury after aneurysmal subarachnoid hemorrhage (sah). however, studies to understand whether sds play a causal role in secondary injury are hindered by existing sd induction methods which are invasive, cumbersome, and cause primary tissue injury. we developed a method to study the role of sds after experimental sah using commercially available transgenic optogenetic mice which express channelrhodopsin (chr ) in cortical neurons. we used in vivo laser speckle and doppler flowmetry, intrinsic signal imaging, and local field potential (lfp) and extracellular potassium shifts to detect sds. we optogenetically induced sds with light through intact and unaltered skull in multiple regions without causing primary brain injury. we found regional differences in thresholds for optogenetically-induced sds (from lowest to highest threshold): ( ) whisker barrel, ( ) motor, ( ) sensory, and ( ) visual cortex. lower thresholds were associated with higher chr tissue expression. changes in lfp and increased extracellular potassium concentrations at the site of stimulation preceded precipitation of an sd. finally, we induced and detected sds in the setting of sah over several days through chronically implanted glass coverslips non-invasive optogenetic light stimulation can reliably induce sds in the setting of sah. longitudinal optogenetic induction of sds in chr transgenic mice is a potentially useful tool to study the role of sds in the pathogenesis of secondary brain injury after sah. aneurysmal subarachnoid hemorrhage is a devastating neurologic injury with significantly prolonged hospital courses and high morbidity and mortality. when aneurysms are detected, they often require securement either via surgical clipping or endovascular techniques. a subset of intracranial aneurysms, given location, poor surgical approach, and wide neck are amenable to flow diversion which promotes thrombosis through redirecting of blood flow within an aneurysm leading to slow obliteration. approximately % of treated aneurysms with flow diversion do not obliterate after months, but currently there is no validated way to predict treatment failure. computational models of blood flow of flow diverted aneurysms predict a significant difference in the hemodynamic energy loss across aneurysms between cases that resolve and those that do not. energy loss could be estimated clinically during angiography, however, this hypothesis needs to be validated experimentally because computer models often over estimate hemodynamic parameters, poorly predict flow through stents, and may not have the resolution to fully describe intra-aneurysmal blood flow. in this pilot study, four cases of giant fusiform intracranial aneurysms will be selected --two with resolution following flow diversion treatment, and two without resolution. models of each vessel geometry will be fabricated using additive manufacturing techniques. under fluoroscopy, within the model vessel, flow diverting stents will be placed within the aneurysm in the same configuration that was achieved clinically. model blood, containing tracer particles will be pumped through model aneurysms and using particle image velocimetry, energy loss will be calculating within model vessels following treatment. energy loss between aneurysms successfully and unsuccessfully treated with flow diversion will be compared experimentally. hemodynamic energy loss may be a clinically measurable value which could predict treatment failure after flow diversion. additive manufacturing techniques can be used to test patient specific hemodynamics to improve understanding of flow-diversion treatment success or failure. the national institute of neurological disorders and stroke (ninds) and the national library of medicine (nlm) initiated development of unruptured cerebral aneurysms and subarachnoid hemorrhage (sah)specific common data elements (cdes) in as part of a joint project to develop data standards for funded neuroscience clinical research. through the development of these data standards, the ninds and nlm sah joint cde initiative strives to improve sah data collection by increasing efficiency, improving data quality, reducing study start-up time, facilitating data sharing/meta-analyses and helping educate new clinical investigators. the sah cde working group (wg) consisted of international members with varied fields of sahrelated expertise and was divided into domains such as subject characteristics and assessments and exams. the wg developed a set of sah-specific cde recommendations by selecting among, refining and adding to existing field-tested data elements, especially established stroke cdes. wg cde recommendations were drafted into the nih cde repository. following an internal review of recommendations, the sah cdes were vetted during a public review on the ninds website for weeks and later posted on nlm and ninds websites. version . of the sah cdes was available on the ninds cde website in april . these new sah cdes and recommendations include those developed for unruptured intracranial aneurysms and long-term therapies. the website provides uniform names and structures for each data element, as well as guidance documents and template case report forms using the cdes. the ninds encourages the use of cdes by the clinical research community in order to standardize the collection of research data across studies. the ninds cdes are a continually evolving resource, requiring updates as research advancements indicate. these newly developed sah cdes will serve to be a valuable starting point for researchers and facilitate streamlining and sharing data. subarachnoid hemorrhage (sah) represents % of stroke admissions in the us. aneurysmal hemorrhage represents the most dangerous etiology, however - % of sah have negative digital subtraction angiography (dsa). there is variation in practice with regards to repeat diagnostic studies and timing of such studies. it is not uncommon to repeat dsa in - days of the initial assessments. this study aims to describe the costs associated with prolonged icu stay and repeat diagnostic studies this patient cohort. retrospective review of all patients admitted for spontaneous sah between january and april at our single institution. patients with at least one negative initial angiogram for suspected spontaneous sah were included. patients were categorized into diffuse patterns of sah and nondiffuse. cost estimates were based on standard costs as provided by our financial department and cdc estimates for costs of hospital acquired infections. one hundred fifty-four patients were identified with initial negative dsa. second angiograms were performed in % of patients, and potentially positive causal findings in / ( . %). icu los for angiogram negative diffuse sah and non-diffuse were . and . days respectively. other indications for icu stay included vasospasm ( . %), evd placement ( . %), and intubation ( %). the excess cost estimates per patient for angiogram negative diffuse and non-diffuse sah were $ , and $ , respectively. hospital acquired complications were an additional total $ , for the cohort. this is the first study to our knowledge attempting a cost analysis of the diagnosis and management of patients with angiogram negative sah. we had a high frequency of patients requiring icu admission for other indications, which should continue to dictate the level of care. however, there may be a cohort of lower risk patients in which de-escalation would not harm, and be of benefit in the reduction of morbidity and cost. purpose: to evaluate the feasibility and potential role of bedside optical coherence tomography (oct) as a diagnostic protocol in terson's syndrome (ts) in patients with acute subarachnoid hemorrhage (asah). background: % of sah patients become permanently legally blind. the average cost of lifetime support and unpaid taxes for each blind person is approximately $ , . ts presents as ocular bleeding commonly associated with asah. it can be diagnosed by fundoscopy, yet retinal haemorrhages, detachments and macular holes may be undetected. early ts identification is critical since untreated it may lead to legal blindness, limit rehabilitation and impair quality of life. pilot study: sah patients were screened for ts with dilated fundoscopy and then with oct. mood assessments (phq- , hds), quality of life measures (nih-promis) and subjective visual function scales (vfq- ) were performed. there was a . % (n= ) incidence of ts. dilated retinal fundoscopy significantly failed to detect ts (n= , . % missed cases). ivh was significantly more in ts ( . % vs. %). no participants experienced any complications from oct examinations. neither decreased quality of life visual scores nor a depressed mood correlated with objective oct pathological findings at weeks follow-up after discharge. there were no significant mood differences between ts cases and controls. oct is the gold-standard in retinal disease diagnosis. this pilot study showcases its bedside feasibility in asah. in our series, oct was a safe procedure that enhanced ts detection by decreasing false negative/ inconclusive fundoscopic examinations. it allows early diagnosis of macular holes and severe retinal detachments, which require acute surgical therapy to prevent legal blindness. besides, oct aids ruling out potential false positive visual deficits in individuals with a depressed mood at follow up. a comprehensive study is underway to understand the impact oct might exert on blindness prevention and quality of life. fever is common in patients with aneurysmal subarachnoid hemorrhage (asah), and blood cultures are commonly sent to diagnose etiology. several studies have shown a low incidence of positive blood cultures, but no studies have assessed blood cultures in patients with asah. we performed a retrospective analysis of patients admitted with asah between january to december . blood cultures were adjudicated as true positive (tp) or false positive (fp) based on speciation, time to positivity, number of cultures positive, and repeat culture results. tp patients were compared to all other patients. age, gender, hunt hess, modified fisher, aneurysm treatment, incidence of delayed cerebral ischemia (dci), length of stay (los), and neurological outcomes were analyzed. patients with asah were included. blood cultures were sent on ( %). sixteen were positive. eleven were adjudicated tp and fp. thus, . % ( / ) of patients had true bacteremia, and blood culture yield for true infection was . % ( / ). fp rate was . % ( / ). eight tps were gram negative ( %), and all contaminants were staphylococcus non-aureus. median post-bleed day for tp results was . only patients were tp within the first week of admission ( . %). tp patients had higher admission wfns (p=. ) and ivh score (p=. ), but age, gender, aneurysm treatment, and fisher score did not differ. tp patients had longer icu and hospital los and higher incidence of dci ( % vs %, p=. ). mortality did not differ in the two groups either. the yield of blood cultures in asah patients is low. even with a contamination rate under %, % of positive blood cultures are fp. future studies should evaluate factors to identify patients at higher risk of bacteremia to reduce costs and improve care. intra-arterial verapamil therapy reduces cerebral vasospasm after aneurysmal subarachnoid hemorrhage (sah). there is little literature that quantitatively describes its safety, required dosing, or efficacy. as a result, therapeutic outcomes need to be subjectively analyzed by experienced radiologists during the intervention and clinically correlated by cerebral perfusion pressure, intracranial pressures and transcranial dopplers. we present a novel imaging analysis to quantify cerebral perfusion in realtime and apply this technology to patients undergoing therapy for vasospasm. we developed software to evaluate changes in contrast flow dynamics for digital subtraction angiography (dsa) scans performed pre-and post-intra-arterial therapy for vasospasm. performing signal intensity curve deconvolution on a voxel by voxel basis provides quantitative d perfusion parameters including: time to peak, time to drain, area under the curve, root mean transit time, arrival time, tissue concentration, arterial input functions and cerebral blood flow at each voxel. after aligning perfusion studies, our software then displays and automatically creates regions of interests for changes in perfusion to visualize the effects of interventions. our software quantitatively measures perfusion from dsas and can normalize two dsas accounting for differences in volume and speed of contrast administration. two applications of this technology are demonstrated. the first subtracts perfusion from pre-and post intra-arterial interventions quantifying exact changes in perfusion at each voxel. the second compares two dsa studies of the same patient at different dates to contour the territories susceptible to delayed cerebral ischemia. we compare this analysis to mri imaging when applicable demonstrating ischemic changes aligning to the susceptible territories outlined by our analysis. dsa based perfusion is an effective study to quantify the need for and the precise effects of endovascular interventions. quantitative thresholds and analysis based on dsa perfusion may assist with real-time assessment of treatment efficacy for patients undergoing intra-arterial verapamil therapy. we aim to characterize the clinical predictors of ventriculoperitoneal shunt (vps) placement in aneurysmal subarachnoid hemorrhage (asah) patients. there has been no clear consensus as to effective measures of predicting vps placement in these patients. we reviewed the clinical data of patients with aneurysmal subarachnoid hemorrhage (asah) who were treated at our institution between - . we eliminated patients who died or had withdrawal of care during admission. we recorded patient demographics and clinical predictors including admission/discharge glasgow coma scale (gcs), hunt hess score, aneurysm size/location, modified fischer score, modified rankin scale (mrs), intracranial pressure (icp) values during evd clamp trial, and incidence of vasospasm requiring intra-arterial therapy. there were patients included in this study and % of patients required vps (n= / ). vps patients had significantly worse mrs functional scores at discharge ( . vs . ; p= . ), but this began to balance at year ( . vs . ; p= . ). aneurysms were significantly larger in vps patients ( . cm vs . cm; ci: . to . ; p= . ). a greater percentage of vps patients had posterior fossa aneurysms, but this was not found to be statistically significant ( % vs %; p= . ). vps patients had significantly lower gcs scores at admission ( . vs . ; p= . ), and discharge ( . vs . ; p= . ). there was no difference in modified fischer score (p= . ) or hunt hess (p= . ), but both variables were higher in the vps cohort. there was no difference in the frequency of vasospasm in the vps cohort (p= . ), or icp values (p= . ). patients presenting with large aneurysms and poor gcs scores had a significantly higher likelihood of requiring vps during admission. these patients had significantly poorer mrs scores at discharge but not at year. subarachnoid hemorrhage (sah) affects a young population and results in death or disability in the majority of those who experience it. this epidemiology is very different from other forms of stroke. consequently, patients with sah and their families may have different priorities for recovery. involving patient perspectives is encouraged in research and is often accomplished using patient-reported outcome measures (proms). however, whether proms reflect patient and family priorities is unclear given that (a) proms are often developed without their input; and (b) generic proms may not apply to specific conditions. we aimed to systematically review the sah literature that has: a) involved patient, family or caregivers in evaluating existing outcome measures, b) developed novel outcome measures by incorporating their perspectives (including co-development), or c) described outcomes important to patients, families, or caregivers. we searched embase and ovid medline from inception to december , . study eligibility and data extraction was performed independently and in duplicate. for each eligible citation, we abstracted the following: study population, design, type of patient involvement, and outcome measure(s), as applicable. we planned a qualitative summary of all included studies. our search yielded unique citations. only four articles have met our eligibility criteria. in each, patients (n= ) self-report impairments resulting from sah and their impact on their lives (aim c). none involve the evaluation of prom applicability. additionally, we found articles that, although they did not meet our a priori eligibility criteria, discuss collecting proms (n= ), using proms to predict health outcomes (n= ), and comparing prom applicability without patient perspectives (n= ) in sah populations. based on our findings, there is alack of patient, family, or caregiver involvement in selecting or identifying outcomes after sah with direct relevance to them. sah research may be overlooking outcomes that are important to patients. early brain injury (ebi) after aneurysmal subarachnoid hemorrhage (asah) is defined as brain injury occurring within hours of aneurysmal rupture. although ebi is the most significant predictor of outcomes after asah, its underlying pathophysiology is not well understood. we hypothesize that ebi after asah is associated with an increase in peripheral inflammation measured by cytokine expression levels and changes associations between cytokines. methods asah patients were enrolled into a prospective observational study and were assessed for markers of ebi: global cerebral edema (gce), subarachnoid hemorrhage early brain edema score (sebes), and huntassays to determine levels of pro-and anti-inflammatory cytokines. pairwise correlation coefficients between cytokines were represented as networks. cytokines levels and differences in correlation networks were compared between ebi groups. of the patients enrolled in t associated with high grade sebes. correlation network analysis suggests higher systematic inflammation conclusions ebi after sah is associated with increased levels of specific cytokines. peripheral levels of il , il and ession levels of individual cytokines may offer deeper insight into the underlying mechanisms related to ebi. few recent studies have evaluated health resource utilization and patient outcomes in aneurysmal subarachnoid hemorrhage (asah) in the united states. empirical evidence implicates asah as one of the highest cost diseases treated in the hospital. we identified asah patients to determine hospital charge, length of stay (los) and patient disposition associated with care in u.s. hospitals using claims data from the national inpatient sample (nis). patients within the international classification of disease, th revision (icd- ) diagnosis code were identified; a secondary analysis of the nis ( ) was conducted utilizing icd- clinical modification codes excluding patients with traumatic and non-aneurysmal sah. population size, patient outcome, average charge and average los were calculated using subgroups including: aneurysmal clipping or endovascular coiling (n= , ), aneurysmal clipping or coiling with external ventricular drain (evd) (n= , ), use of evd only (n= , ), other surgical procedures (n= ) and medically managed (n= , ). analyses were survey-weighted and adjusted for patient and hospital characteristics. in , asah resulted in an average per patient hospital charge of $ , , an average los of days, an average mortality of % and total, annual hospital charges of $ . billion. the highest average charge per patient ($ , ) and hospital los ( days) were attributed to clipped or coiled patients with evd, and highest mortality ( %) found in medically managed patients. these data support the conclusion that asah is a high cost illness managed in u.s. hospitals, and help raise awareness to the potential economic benefits resulting from developing safer, more effective therapies. additional analyses with updated datasets including lifetime burden of asah (e.g. physician fees, long term medical and care costs, hospital re-admission impact, quality of life, productivity loss, caregiver burden) should be explored to understand the full economic burden of asah and the potential cost effectiveness of new therapies. external ventricular drain (evd) placement is a mainstay of treatment for patients with aneurysmal subarachnoid hemorrhage with hydrocephalus or elevated intracranial pressures, but the optimal strategy for evd management is still unclear. the goal of this study was to compare the impact of evd clamping at three different levels on the duration of drain placement and the intensive care unit (icu) length of stay. we performed a retrospective analysis of patients admitted with aneurysmal subarachnoid hemorrhage to the neurological icu from december to january and included all patients who had an evd placed. patients who died were excluded from the study. patients were divided into three groups: patients whose evd was clamped at mmhg, patients whose evd was clamped at mmhg, and patients whose evd was clamped at mmhg. duration of drain placement in days and icu length of stay in days was compared among the groups using an analysis of variance (anova). outcomes were adjusted for presenting hunt-hess score, modified fisher grade, gender, and age. there were patients who had their evd clamped at mmhg, who had their evd clamped at mmhg, and who had their evd clamped at mmhg. there was no difference in duration of evd placement among the three groups (adjusted p-value . , unadjusted p-value . ) nor in icu length of stay (adjusted p-value . , unadjusted p-value . ). evd clamping at three different levels did not affect drain duration nor length of stay in icu. this study was limited by the small number of patients enrolled. further studies are need to clarify optimal strategies for evd management in the icu. headache is a presenting complaint in majority of patients with asah and is known to persist long after initial icu care. various medications have been used for control of headache with major emphasis on opiate use. history of a prescription for an opioid pain medication increases the risk for overdose and opioid use disorder. we looked at prevalence of opiate use at discharge and its associated factors. chart review of all patients admitted in a tertiary care center between jan and march was carried out. along with baseline demographic data, information about use of pain scores, csf diversion, use of opiates, average morphine equivalent doses, use of opiates at discharge and destination at discharge was collected. analysis was carried out using microsoft excel. the study was approved by hospital irb. patients were admitted with asah in above period ( % female, average age: yrs). ( % home, % snf) survived to discharge. among survivors, % required csf diversion for hydrocephalus. all people complained of pain on presentation and were prescribed opiates during hospital stay. average oral morphine equivalent doses used was mg per day. ( %) patients were prescribed opiates on discharge. alternative regimens included ( patients: tricyclic antidepressant (tca), opiate + tca, acetaminophen, dexamethasone, tca and opiates). most common prescribed form of opiate was oxycodone. there was no significant association between opiate use/morphine dosing and age, gender, final disposition and csf diversion, opiate prescription at discharge is common in patients with asah. no clinical characteristic seem to predict analgesic need at discharge. little data exists about better alternatives leading to variety of treatment approaches. further controlled trials are needed to decrease opiate use and prevent adverse effects delayed cerebral ischemia (dci) in sah has been associated with vasospasm-dependent and vasospasmindependent phenomena. for more than years isolated hemostasis disorders have been reported in these patients. the objective of this systematic review is to describe the natural history of hemostasis in sah. we systematically reviewed the medline, embase, cochrane and lilacs databases using controlled language and the prisma statement and included studies on spontaneous sah analyzing any hemostasis parameter. we screened titles, of which observational were included. evidence was evaluated following the strobe statement. no meta-analysis was attempted because of the methodological nature and heterogeneity of the studies. hemostasis is profoundly altered during the first hours after bleeding, with several alterations noted including a hypercoagulable state concomitant with increased fibrinolysis activation and reduced clot stability. direct and indirect coagulation markers show a trend towards normalization of hemostasis in the first to days. platelet count decreases with a nadir to days after bleeding and a recovery in the following weeks. a later nadir is associated with dci. platelet aggregability is consistently decreased in the first few days, regaining its normal function around the second week after bleeding. in addition, the persistence of these alterations or the presence of a second peak in pro-coagulatory activity is associated consistently with dci and worse functional outcomes. the hyperacute phase of sah is characterized by a profound activation in hemostasis with reduced clot stability, probably due to an increase in the fibrinolytic pathways. on the second day post-bleeding, a slow trend towards normalization takes place, except in patients evolving towards dci. further research on the pharmacologic manipulation of hemostasis in sah might be warranted to decrease dci and improve outcomes in this population. hypertonic saline(hts) is a treatment for sah-related cerebral edema, administered to improve cerebral perfusion and reduce brain injury. hts a supra-physiological chloride concentration that can contribute to acute kidney injury which can lead to a poor outcome. in a previously published single-center cohort of , l sah patients, . % developed acute kidney injury (aki). hyperchloremia, but not hypernatremia, was correlated with an increased risk to develop aki (o.r. . ). aki was correlated with increased mortality. a secondary analysis of the aforementioned sah patient cohort ( ) ( ) ( ) ( ) ( ) ( ) , was analyzed. trends of acute kidney injury were evaluated in relation to the burden of exposure to intravenous chloride, as well as serum levels of sodium and chloride. the proportion of patients developing aki with a maximal serum chloride concentration of (p , will be randomized into one of two treatment groups: standard hypertonic saline solution (nacl . %) versus a solution of nacl/na-acetate. we hypothesize that by reducing the iv chloride burden(baseline compared to post randomization exposure), the delta serum chloride level will decrease, and will subsequently reduce aki occurrence (acetate trial, clinicaltrials.gov nct ). aki is common in sah patient population, and associated with worse outcomes. serum chloride concentrations are a significant risk factor for the development of aki. a prospective randomized clinical trial now underway examining the relationship between the hypertonic solution composition and serum chloride concentration, and to the development of acute kidney injury in aneurysmal sah. spontaneous spinal subarachnoid hemorrhage (ssah) is a rare but serious condition that can lead to a variety of medical complications. literature to this point primarily includes isolated case reports, and none have looked at hyponatremia as a complication. patients were identified from the electronic medical record database at the mayo clinic in rochester, minnesota. the advanced cohort explorer tool was used, searching from january to december . inclusion criteria were spinal subarachnoid blood products due to hemorrhage into the spinal subarachnoid space not due to ( ) redistribution of blood from intracranial subarachnoid hemorrhage, ( ) trauma, ( ) medical procedures, or ) predominant hematomyelia who experienced symptoms and received treatment at our facility. eight patients (median age years, range - ) were identified as meeting the study criteria. five of these eight patients experienced hyponatremia during hospitalization with a median value of meq/l. all of these patients were treated with free water restriction and one patient briefly received . % sodium chloride solution; in all cases the hyponatremia improved after free water restriction. in all cases the hyponatremia improved with fluid restriction, and there was no documentation of increased urine output, suggesting that it was likely due to siadh. cord compression and hyponatremia were present together in two patients, and in these cases treatment of the hyponatremia was particularly useful to avoid worsening edema. to our knowledge this is the first compilation of cases of spontaneous ssah highlighting hyponatremia as a complication. there is significant morbidity and mortality associated with aneurysmal subarachnoid hemorrhage (sah) and only about % of patients survive and resume their previous lifestyle after - months. many randomized clinical trials (rcts) have been conducted yet no treatment definitively improves outcome from sah. outcome is strongly related to baseline factors, yet imbalances are common in early trials. we developed a technique to identify promising treatments at early phase using a pooled control arm model (ppredicts: kent, shah, mandava neurology ) that compares early studies at their own baselines. we applied this method to sah to develop a multi-dimensional model (ppredicts-sah). models for functional outcome and mortality (dependent variables) were developed based on baseline variables (eg: wfns grade - % and age) using methodology developed for ischemic stroke (mandava, kent, stroke ). the outcome model is a -dimensional surface bounded on either side by +/- . prediction interval surfaces. these prediction interval surfaces incorporate statistical variability to assess whether a treatment differs from expected outcome. treatment arms from rcts and single arm trials, of various treatments of sah were compared against the pooled controlled arm. the best model fit was for good outcome (modified rankin score - equivalents) based on % patients with wfns - and age (r = . ; p< . ). seven trials of known negative drug tirilazad were superimposed on the model and fall within the +/- . prediction interval surfaces confirming futility. three trials were neutral and within the prediction interval surfaces while case series using implanted prolonged release nicardipine and a low dose heparin study were above the +p= . surface showing promise. models were also developed for mortality (r = . , p=. ). outcome models based on percentage of high grade wfns and age were successfully developed. this approach may be useful to prioritize treatments worthy of further study. oral nimodipine is recommended to improve outcome in treatment of aneurysmal subarachnoid hemorrhage (asah). fda approved nimodipine liquid oral solution (nos) in to reduce complications associated with administering nimodipine capsules (nc) to patients with impaired swallow. experience with nos at our center has been complicated by increased liquid bowel movements (lbm) prompting unnecessary testing for infectious diarrhea and exposure to invasive fecal management devices. study approved by local qualtiy improvement review committee. data was collected prospectively in consecutive patients diagnosed with asah during intensive care unit (icu) course. formulations of nimodipine available were generic nc (heritage pharmaceutical) and nos (arbor pharmaceuticals). we examined total icu days exposed to nos, icu days with lbm, infectious diarrhea investigations, and fecal management device use. all statistical tests were performed using minitab. patients were studied from / / to / / ; patients exposed to nos for icu days, icu days with lbm, infectious diarrhea investigations, and required fecal management devices. patients exposed to nc for icu days, icu days with lbm (all cases were also received nos), no infectious diarrhea investigations, and no fecal management device requirements. odds ratio for lbm with exposure to nos was . ( % ci . to . , p < . ). the high incidence of lbm with nos resulted in more infectious diarrhea testing and fecal management device use. uncontrolled diarrhea may increase risk for dehydration and delayed cerebral ischemia, although this is not explored in the current study. nos can mitigate risks associated with needle aspiration of nc, however these issues coupled with higher cost may limit benefit of its use. possible solutions may include compounding nc into a liquid formulation by pharmacists or pharmacy technicians. possible safety and cost benefits require further investigation. headache (ha) management after subarachnoid hemorrhage (sah) is challenging and lacks standardization. we hypothesized that inadequate inpatient ha pain management leads to the development of chronic ha (cha) after sah. prospective, observational study of non-traumatic hunt and hess (hh) grades i-iii sah patients admitted from / to / . after informed consent we recorded demographics, clinical and radiographic features, analgesic and steroid doses, hospital course and inpatient pain scores using numeric rating scale (nrs, - ) before (nrs-pre) and after each analgesic administration over post-bleed days - . a phone survey administered - months after admission evaluated cha burden. inpatient ha control effectiveness was evaluated by percent pain resolution from initial pain score, using nrs-pre. the percentage of administrations yielding full pain resolution was compared between those with and without cha. chi-square and t-tests were used for statistical analyses. patients, % female, mean age . ± . years with hh grade i ( / ), ii ( / ), and iii ( / ) sah were enrolled with lost to follow-up. at follow-up, . % patients ( / ) reported daily ha, . % ( / ) occasional ha, and % ( / ) no ha. full pain resolution after analgesic administration was associated with less cha ( [ . %] vs. [ . %], p= . ). mean daily inpatient opioid dose (morphine equivalents) for patients with and without cha was . mg and . mg, respectively (p= . ). mean nrs-pre were . vs . for patients with vs without cha, respectively (p= . ). inpatient analgesia for sah-related ha is inadequate and may be associated with the development of chronic ha. patients with cha had higher mean inpatient pain score and fewer analgesic administrations resulting in complete pain resolution. inpatient opioid dose per day was higher in cha group, although not statistically significant. additional research is needed to characterize the relationship between inpatient headache management and chronic headache after sah. subarachnoid hemorrhage (sah) remains a significant cause of neurological morbidity and mortality with few interventions to prevent delayed cerebral ischemia. hypocapnia has been associated with worse outcomes in brain injury. sah patients may be particularly susceptible to hypocapnia induced vasoconstriction. this study aims to describe the incidence of iatrogenic and spontaneous hyperventilation in sah patients. a descriptive analysis was performed on a retrospective cohort of adult sah patients admitted to beth israel deaconess medical center icus between and with gcs < who were treated with mechanical ventilation and an extraventricular drain, and had at least one abg. patients on chronic ventilator support were excluded. the lowest paco per icu day was analyzed. patients were included with days with at least one documented paco . mean gcs on admission was . (sd . ). . % of patients survived to hospital discharge. . % of patients were exposed to severe hypocapnia (paco mmhg, those with severe hypocapnia had similar pao and pao /fio ratios, but mildly increased leukocytosis ( . vs . ). . % of paco s < mmhg occurred during spontaneous ventilation or over-breathing. prior studies have shown that hypocapnia causes decreased brain tissue perfusion and is associated with worse outcomes in sah patients. these recent data demonstrate that severe hypocapnia is common in patients with sah severe enough to warrant intubation, and is associated with both iatrogenic and spontaneous hyperventilation. hypocapnia is not primarily compensatory or hypoxia driven, as suggested by mean ph and pao . confirmation of this association and potential future interventions require further study. although delirium is associated with higher rates of hospital complications among critical care patients, limited data exist on risk factors for delirium in aneurysmal subarachnoid hemorrhage (sah). a previous study identified older age, high hunt hess grade, intraventricular hemorrhage (ivh), and hydrocephalus as risk factors for delirium. we sought to identify risk factors for delirium during admission after sah. retrospective review was performed of prospectively collected data for consecutive sah patients enrolled into the university of maryland recovery after cerebral hemorrhage (reach) study. baseline data and clinical complications during each admission, including delirium, were recorded. statistical analysis was performed using univariate and multivariate logistical regression. sah patients from july to january were reviewed. while age was not singly associated with delirium during icu admission, higher hunt hess grade, ivh, hydrocephalus, hospital-acquired infection, elevated troponin, and intubation were significantly associated with delirium on univariate analyses. upon stepwise multivariate logistic regression, ivh (or . , p= . ) and intubation (or . , p= . ) remained significantly associated with delirium. ivh and intubation predicts delirium during icu admission for sah. further analyses are needed to determine if the relationship between ivh and delirium is primarily explained by risk of hydrocephalus, risk of fever, medication exposure, or through independent mechanisms. stroke triage scales are very important in order to expedite acute evaluation, assure quick door to neuroimaging time and decrease door to needle time in patients with ischemic stroke eligible to intravenous thrombolysis. subarachnoid hemorrhage (sah) is associated with a high mortality in the acute phase due to a particular risk of early and devastating re-bleeding. therefore patients with sah also need urgent assessment. the performance of classic triage stroke scales in the identification of patients with sah was not previously evaluated. the objective of our work was to evaluate the performance of the los angeles prehospital stroke screen (lapss) in identifying patients with sah admitted to a tertiary hospital. we evaluated consecutive patients admitted to a tertiary hospital with sah from january to may . at hospital admission, lapss was applied by trained nurse personnel to all noncomatose patients with complaints suggestive of neurological disease. a total of with sah patients were evaluated (mean age . +/- . ), . % females). lapss was applied to patients. lapss was positive in only patients ( . %). patients with a positive lapss had higher nihss stroke score at admission ( , [ , ] versus , p< . ), lower glasgow coma scores ( [ , ] versus , p< . ) and a significant shorter door to neuroimaging time (p< . ). in patients with sah and mild symptoms, lapss was not a sensitive screening tool in our series. hospital and pre hospital services using lapss for triage of patients with stroke should be aware of this limitation and include in triage flowcharts specific questions evaluating sah specific symptoms. spontaneous subarachnoid hemorrhage (sah) is a neurological emergency, which despite current advances in management strategies and advent of institutional protocols, remains with significant rates of mortality due to poorly understood causes. our objectives were to characterize in-hospital mortality by evaluating the primary cause of death and externally validate the hair score, a clinical score that prognosticates mortality. in this retrospective cohort study, we reviewed all sah patients admitted to our neuro-icu between april , and march , . univariate and multivariate logistic regressions were performed to identify predictors of in-hospital mortality, our primary outcome. to validate the hair score, the model's predictors were hunt and hess score at treatment decision, age, intraventricular hemorrhage, and re-bleeding within hours. discrimination was assessed by visualizing the receiver-operating curve and calculating the area under the curve (auc). among sah patients with a median age of years (interquartile range, - ), . % females, inhospital mortality was . % (n= ). of those, ( . %) had a neurological cause for death or withdrawal of care and ( . %) had a cardiac death. median time from sah to death was days. the main causes of death were the primary effects of the initial hemorrhage, re-bleeding and refractory edema. factors significantly associated with in-hospital mortality in the multivariate analysis were age, hunt and hess score, and intra-cerebral hemorrhage. maximum lumen size was also a significant risk factor among aneurysmal sah patients. the hair score had a satisfactory discriminative ability, with an auc of . . our in-hospital mortality is lower than previous reports, attesting to the continuing improvement of our protocolized subarachnoid hemorrhage care. the major causes are the same as previous reports. the hair score showed good discrimination and could be a useful tool for predicting mortality. so far, scientific and therapeutic efforts mainly focused on the prevention of rebleeding and ischemic complications(dci) in patients with subarachnoid hemorrhage(sah). however, data regarding the impact of parenchymatous hemorrhage(ph) on longterm outcome in these patients is limited. all consecutive patients with atraumatic sah admitted to our hospital over a -year-period( - ) were retrospectively analyzed. extent of sah as well as presence, localization and volume of ph were evaluated. functional and health outcome were assessed after months using the modified rankin scale (unfavorable: - ) and the eq- d. propensity-score(ps)-matching was performed to minimize potential bias due to confounding variables between sah-patients with and without ph. of overall patients with atraumatic sah, ( . %) patients had ph on initial imaging. ph-patients had a worse clinical condition on admission (wfns: ph ( - ) vs. Øph ( - );p< . ) and a greater extent of sah (modified fisher: ph ( - ) vs. Øph ( - );p= . ). median ph-volume was . ( . - . )ml with largest volumes in patients with ruptured )ml). after successful ps-matching (parameters: age, wfns, modified fisher and graeb score) patients with ph had worse functional and health outcome after months compared to those without ph (mrs - : ph / ( . %) vs. Øph / ( . %);p= . ; eq- d: ph ( - ) vs. Øph ( - ); p< . ). in multivariate analysis presence of ph was the strongest independent predictor of unfavorable outcome after months followed by the occurrence of dci (risk-ratio( %ci): ph . ( . - . ); p< . ). parenchymatous hemorrhage is frequent and associated with functional and subjective impairments in patients with atraumatic sah. aneurysmal subarachnoid hemorrhage (asah) is associated with early and delayed brain injury. insulin growth factor (igf ) is a potent cellular growth-promoting factor with demonstrated independent neuroprotective actions in stroke and neurologic disease but has not been well characterized after asah. this study sought to examine the relationship between plasma igf levels and outcomes after asah. this cohort of asah patients was . years (sd . ) and female ( %) with a mean hh ( %), wfns ( %) and fisher ( %). initial and peak plasma igf concentrations were measured in plasma samples from a banked biorepository using a commercial sandwich solid-phase elisa kit. delayed neurological deterioration (dnd) and delayed cerebral ischemia (dci) were determined using radiologic and clinical information. igf levels were log transformed due to non-normality. anova, t-tests, pearson correlations and logistic regression analyses were completed using spss and sas. older age was significantly associated with lower initial and peak plasma igf levels (r=. , p<. ; r=. , p<. ). men had higher initial and peak plasma igf levels than women (p<. ; p=. ), and premenopausal women had higher initial and peak plasma igf levels than post-menopausal women (p=. ; p=. ). lower peak plasma igf levels were associated with increased clinical severity by wfns (p=. ) and fisher grade (p=. ) as well as the development of dnd (p=. ; p=. ). lower peak igf levels were associated with the presence of dci (p=. ). controlling for age and fisher grade, log peak plasma igf levels remained significantly associated with the presence of dnd (p=. ; or . ; ci: . -. ) and dci (p=. ; or . ; ci: . - . ). igf levels have not been well characterized after asah. these results suggest lower plasma igf are associated with clinical severity and outcomes after asah and provide impetus for future work to further examine these relationships. induced hypertension (ih) is the mainstay of medical management for delayed cerebral ischemia (dci) after subarachnoid hemorrhage. however, using vasopressors to raise systemic blood pressure well above normal levels may be associated with systemic and neurological complications, of which posterior reversible encephalopathy syndrome (pres) has been increasingly recognized. however, the frequency and risk factors for ih-induced pres have never been systemically evaluated we identified patients treated with ih from sah patients admitted over a three-year period. pres was diagnosed based on clinical suspicion (i.e. unexplained deterioration), confirmed by imaging. we conducted retrospective extraction of data on ih therapy, including baseline and highest target mean arterial pressure (map) and vasopressor dose/duration. we compared those with pres to ihtreated controls and also described the clinical features and sequelae of all pres cases. five sah patients were diagnosed with pres, with median time from initiation of vasopressors to diagnosis of . days (range - days). baseline map did not differ between pres and ih controls, but highest target map was greater ( vs. mm hg, p= . ). magnitude of ih was similarly greater ( vs. mm hg above baseline, p= . ). all cases presented with lethargy, three had new focal deficits, and one had a seizure. one died from cardiac complications but the other four patients had complete resolution with ih discontinuation, without infarction or residual disability. pres was diagnosed in % of patients undergoing ih therapy and was most likely when map was raised well above baseline to levels exceeding the traditional limits of autoregulation ( - mm hg). high clinical suspicion for this reversible disorder appears warranted when aggressive ih targets are maintained for several days or in the presence of unexplained neurological deterioration. other interventions may be preferable for refractory dci when moderate degrees of ih have been attempted. patients with aneurysmal subarachnoid hemorrhage (asah) may receive significant exposure to potentially harmful ionizing radiation exposure (phire) from diagnostic tests and therapeutic procedures during their initial hospitalization. we hypothesized that risk factors to detect excessive phire are present at the time of admission. following irb approval, all patients admitted to our institution with documented asah over a -year period were retrospectively evaluated for inclusion and exclusion criteria. patients were excluded if they died prior to discharge. all study data, including sah-specific and patient-specific risk factors, were obtained from the electronic medical record. the total effective dose of ionizing radiation (tedir) per patient was calculated from previously published radiation exposure data. phire was considered to have occurred if tedir was greater than msv, the annual phire limit for radiation workers. logistic regression models were then fit to the dataset to evaluate clinical variables that significantly the risk of phire in these patients. data were collected from patients ( . % of all asah patients evaluated). the mean tedir in these patients was . msv. forty-two ( . %) of patients met criteria for phire. in multivariate logistic regression modeling, male gender (or= . , ci= . - . ), posterior circulation aneurysms (or= . , ci= . - . ) and ventriculostomy (or= . , ci= . - . ) were significantly associated with an increased risk of phire. in this study, approximately % of asah patients received phire. male gender, posterior circulation aneurysms and ventriculostomy were significantly associated with increased risk of phire. these factors may serve as important predictors of patients who require additional or complex care necessitating repeated diagnostic or therapeutic procedures during their hospitalization. alternative diagnostic or therapeutic modalities should be considered for patients with these risk factors to limit the risk of phire. future research should also evaluate the effect of phire on neurologic outcomes in these patients. it remains unclear whether patients with unruptured intracranial aneurysms (ica) should be treated. vessel wall enhancement (vwe) in high-resolution magnetic resonance vessel wall imaging constitutes a promising marker of aneurysm instability in this population. to find risk factors for aneurysm instability, we sought to identify predictors of vwe in patients with unruptured icas. we conducted a retrospective analysis of prospectively collected data on patients with unruptured ica evaluated by a single provider. all patients were evaluated using a previously validated algorithm to ascertain vwe using high-resolution magnetic resonance vessel wall imaging. two different raters, blinded to the study data, categorized all observed aneurysms as vwe-positive or vwe-negative. kappa statistics were used to evaluate the reproducibility of this approach. univariable and multivariate logistic regression modelling was utilized to identify factors associated with vwe after adjusting for potential confounders. patients with unruptured ica were included in the analysis (mean age [sd ] , female sex [ %]). of these, ( %) were vwe-positive and ( %) were vwe-negative. inter-rater reliability for vwe ascertainment was excellent (kappa . , %ci . , . ). out of ( %) patients presenting with cranial nerve palsy were vwe-positive. in univariable analysis, age (p= . ), headache on presentation (p= . ), and size (p< . , per additional millimeter) were associated with vwe-positive status. in multivariable analysis, headache on presentation (p= . ) and size (p= . ) remained independently associated with vwe. cranial nerve palsy is an established clinical marker of aneurysm instability; consequently, our results confirm the role of vwe as a marker of aneurysm instability. headache on presentation and aneurysm size are independently associated with vwe; these risk factors for aneurysm instability could be used to select patients with unruptured icas that may benefit from vessel wall imaging. prognostication in subarachnoid hemorrhage (sah) patients presenting in coma is crucial for surgical decision making. indications for aggressive aneurysmal treatment are unlikely for those not demonstrating signs of neurological improvement chronologically or after ventricular drainage. early neurological evaluation is, however, challenging in critically ill sah patients requiring anesthesia and intubation for airway protection. in this single-center retrospective study, we applied continuous amplitude-integrated eeg (aeeg) monitoring using a subhairline montage for wfns grade v patients who did not undergo emergency aneurysm treatment. monitoring was initiated soon after admission to the icu. patterns of aeeg findings were classified according to rundgren, et al. as follows: flat (f); suppression-burst (sb); electrographic status epilepticus (ese); and continuous (c). based on the aeeg findings, indications for aneurysm treatment were reevaluated. outcome was assessed at six months using the glasgow outcome scale. twenty-three patients, men and women, aged . ± . years (mean ± sd), were eligible since december . all patients underwent prophylactic intravenous sedation. the population represented % of all grade v patients including those resuscitated after cardiac (n= ) or respiratory (n= ) arrest. the glasgow coma scale scores were (n= ), (n= ), and (n= ), respectively. aneurysms were located in the posterior fossa in patients ( %). aeeg monitoring was initiated . ± . hours median . , . - . hours after arrival. all patients showing early f (n= ) or sb patterns (n= ) died. one patient demonstrated ese remained in a persistent vegetative state. five out of patients with a c pattern underwent aneurysm treatment; clippings and coil embolization. moderate disability was attained in and severe disability in . two patients undergoing conservative therapy died. continuous aeeg provided useful prognostic information for identifying salvageable sah patients undergoing sedation in the early phase. delayed cerebral ischemia (dci) may result in focal neurological deficits and cerebral infarction after subarachnoid hemorrhage. while global cerebral blood flow (cbf) may be variably reduced, dci is more likely related to regional impairments in cbf below critical perfusion thresholds. we applied volumetric methods to assess the proportion of brain exhibiting hypoperfusion (pbh) in those with clinical dci and in the symptomatic hemisphere of those with focal deficits. methods patients with aneurysmal sah underwent o-pet and ct imaging during period of risk for dci (median days after sah, iqr - ). we measured pbh as proportion of voxels with cbf < ml/ g/min, after excluding regions of infarction/hematoma on ct. we compared pbh in patients with vs. without dci at time of pet and, in those with focal deficits, we compared hypoperfusion between affected and unaffected hemispheres. pbh was greater in the ( %) with dci compared to those without dci ( %, ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) p= . ) despite higher mean arterial pressure (map) and most being on active hemodynamic therapies. global cbf was also lower in those with dci ( . vs. . ml/ g/min, p= . ) but did not differ between those remaining symptomatic and those whose deficits had resolved. while mean hemispheric cbf was not lower in the affected hemispheres of with lateralizing deficits ( . vs. . ml/ g/min, p= . ), there was greater pbh in the symptomatic hemisphere ( % vs. %, p= . ). sah patients with dci have a greater proportion of brain with hypoperfusion despite active hemodynamic therapy and higher map. there was also larger proportion of the symptomatic hemisphere with hypoperfusion despite no asymmetry of hemispheric cbf. such measurements of hypoperfusion may better reflect the regional pathophysiology of dci than global averaged measures of cbf. further studies should determine whether burden of hypoperfusion correlates with tissue and patient outcomes. patients who survive aneurysmal subarachnoid hemorrhage (asah) are often burdened with lasting cognitive impairment due to a combination of sequelae including neuro-cardiac injury. the impact of neurocardiac injury after asah is poorly understood. this study sought to examine if neurocardiac injury detected by global longitudinal strain (gls) is associated with poor performance in neuropsychological np memory impairment after asah. we studied asah patients at months and at months (sahmii study r nr ) after hemorrhage. speckle tracking gls from apical views were assessed days - from bleed from transthoracic echocardiograms. neuropsychological (np) outcomes covering domains were completed at and months after hemorrhage by trained personnel. memory tests included controlled oral word association (cowa), wechsler memory scale (wms) and rey auditory (r-aud) and complex figure (reyc). anova and kruskal-wallis, pearson and spearman correlations and logistic regression were completed using spss and sas. there were ( %) patients with abnormal gls (defined as >- %) and ( %) in the and months groups respectively. gls groups had similar age, gender and fisher grade. abnormal gls was associated with higher hh at (p=. ) and (p=. ) months. abnormal gls was significantly associated with decreased performance in r-aud memory domains at months (p=. ) and months (p=. ) after asah and even when controlling for age and hh at months (p=. ). gls<- was associated with poor memory performance months after asah in cowa (p=. ) and the wms (p=. ) even after adjusting for age and hh, cowa (p=. ) and wms (. ). neuro-cardiac injury detected by gls was associated with decreased performance in memory domains of np function at and months after asah. while these relationships require further examination, neurocardiac injury may contribute to long term np impairment after asah. delayed cerebral infarction (dci) is a frequent complication following high-grade aneurysmal subarachnoid hemorrhage (asah). management of dci includes maintaining hypertension, which is challenging in heavily sedate patients. ketamine is a hemodynamically stable, analgesic sedative not studied in this population. we hypothesize that ketamine infusion (k), as compared to traditional sedatives (control), will safely improve the hemodynamic profile in high grade ventilated asah patients retrospective review of asah patients admitted / to / requiring mechanical delayed cerebral infarction (dci) is a frequent complication following high-grade aneurysmal subarachnoid hemorrhage (asah). management of dci includes maintaining hypertension, which is challenging in heavily sedate patients. ketamine is a hemodynamically stable, analgesic sedative not studied in this population. ventilation > hrs, and without dnr within hrs from admission. we assessed demographics, hemodynamics, pressor, dci at weeks, ventilator and icu los, and mortality. fisher exact, wilcoxon, and paired t-test applied. comparing k (n= ) vs control (n= ), median (q , q ) results for: age ( , ) vs ( , ); hunt and hess ( , ) vs. ( , ); mpm- day estimated mortality . % vs. . %; and gcs ( , ) vs ( , ) . ketamine initiated on day ( , ); icu los ( , ) vs. ( , ); and vent los ( , ) vs. ( , ) . mean (sd +/-) for hours before and after ketamine: map ( ) vs ( ), p . , except where noted. ketamine infusion, as a second line sedative, had no effect on mortality or icp, and improved map. however, there was a nonsignificant increase in dci as well as vent los, without a greater rate of tracheostomy. prospective studies are needed to study the effect on dci and long term outcomes. seizures are a well-known complication of aneurysmal subarachnoid hemorrhage(asah) and occur most commonly in the immediate post-hemorrhagic period. most commonly used antiepileptic drugs (aeds) for seizure prophylaxis in asah include phenytoin and levetiracetam. there is no reliable data available on the safety and efficacy of restricting aed prophylaxis only till the aneurysm is secured. we retrospectively chart reviewed patients admitted to our neurosciences intensive-care-unit with asah during the last two years. seizure incidence was studied in patients treated with phenytoin versus levetiracetam and in patients treated for to days versus those where aed was discontinued immediately after aneurysm was secured. in patients aed prophylaxis was discontinued immediately after the aneurysm was secured, and in patients it was continued for to days. of th phenytoin was used in patients and levetiracetam was used in patients. in patients receiving aed prophylaxis for to days, phenytoin was used in cases and levetiracetam was used in cases. none of these patients had seizures reported during hospitalization or at three month follow-up. stopping the aed prophylaxis immediately after aneurysm coiling is not associated with increased risk of seizures. seizures at presentation in patients with asah are not associated with development of epilepsy at months. both phenytoin and levetiracetam are well tolerated in patients with asah when limited to the immediate post-hemorrhagic period. the main preceding factor of delayed cerebral ischemia (dci) in asah is cerebral vasospasm (cvs). anticipating dci can have major impact on patient outcomes. studies have attempted to predict dci in patients with asah by using various imaging modalities that measure cvs, ranging from transcranial doppler ultrasonography, ctp, and mr perfusion. few compare these imaging modalities to the accepted gold standard of dsa. we propose that mri using asl imaging can be used as a sensitive and specific measure of cvs and can be used as a marker to identify patients with asah who are at risk for developing dci. to support our hypothesis, we compare asl results in patients with documented cvs on dsa who developed dci. patients in the academic years to with the diagnosis of asah were admitted to our nicu. the inclusion criteria for the patient population was the presence of asah confirmed by dsa, diagnosis of dci by a neurointensivist, mri with asl, and a repeated dsa during the hospitalization after dci was suspected. all patients underwent mra with asl on day in an attempt to capture the peak time of cvs. nine patients were included in this study. all cases with perfusion defects on asl sequences had confirmed cvs on dsa except for one. the outlier in our cohort developed dci with asymmetry on asl that was not demonstrated on dsa. to our knowledge, no studies have compared the specificity of asl with dsa in detecting cvs. this study highlights the utility of asl in detecting cvs in patients with asah. our limited data suggests asl can be utilized for detection of dci and cvs with greater confidence than the conventional modalities. we also suggest that asl approaches the utility of dsa in the detection of cvs. blood glucose dysregulation following aneurysmal subarachnoid hemorrhage is associated with serious complications and poor clinical outcome. an influence of hyperglycemia on the occurrence of delayed cerebral ischemia (dci) is assumed, nevertheless the exact mechanism remains unclear. the goal of the present study aims to investigate the influence of systemic blood glucose level on cerebral perfusion measured by dynamic perfusion computed tomography (pct) and outcome. daily serial blood glucose levels and pct data sets of patients treated at our neurointensive care unit after asah were retrospectively analyzed. serial pcts were performed between six hours and days after aneurysm repair. mean average of mean transit times (mtts) was calculated for each perfusion scan. the maximum mean transit time (maxmtt) and outcome assessed with glasgow outcome scale were correlated with defined blood glucose ranges as followed .) > mg/dl (hyperglycemia) .) - mg/dl (elevated glucose level) .) - mg/dl (strict glucose control) and < mg/dl (low glucose level). hyperglycemia (> mg/dl) was associated with prolonged maxmtt (p< . , rs = . ) and was linked to an increased risk of infarction (p < . ) whereas strict glucose control ( - mg/dl) correlated significantly negative with maxmtt (p < . , rs = -. ). strict glucose control was also associated with a lower occurrence of cerebral infarction and good outcome (p < . , rs = . ). in contrast, elevated blood glucose levels ( - mg/dl) and hyperglycemia showed a negative correlation with good outcome (p < . , rs = -. , rs = -. ). the present analysis supports for the first time the assumption that dysregulation of blood glucose balance influences cerebral perfusion and thus may contribute to the occurrence of dci and poor outcome. therefore careful monitoring and prompt treatment of blood glucose levels after asah should be highly valued to avoid cerebral perfusion deficits correlated with poor outcome. the aim of this study was to determine the correlation between transcranial doppler (tcd) velocities and angiographic vasospasm after subarachnoid hemorrhage (sah). methods patients with sah were evaluated with spencer technologies tcd power m mode from - days, following the sah. both the temporal windows were insonnated to determine flow velocities in the middle (mca) and anterior cerebral arteries (aca) and the suboccipital widow was used to determine flow velocities in the vertebral (va) and basilar arteries (ba). the middle cerebral artery/ipsilateral extracranial internal carotid artery velocity ratio (lindegaard ratio) was also correlated with vasospasm ct angiography and conventional cerebral angiography was used to confirm tcd findings suggestive of vasospasm. the sensitivity, specificity, likelihood ratios for positive and negative tcd results, positive there was males and females and with mean age . +- . years. % were aneurysmal sah. delayed ischemic neurological deficits (dind) developed in / patients ( . %). interobserver ue of cm/s were useful (likelihood ratio for negative result = . , likelihood ratio for positive result = . ). lindegaard ratios correlated well with vasospasm. tcd diagnosis of vasospasm was more often present in the mca, followed by aca and basilar arteries. tcd is a good non invasive method to detect vasospasm and predict the occurrence of dind. very high angiographic vasospasm. tcd is also useful to follow up patients with angiographically proved vasospasm. aneurysmal subarachnoid hemorrhage (asah) is a significant cause of morbidity and mortality. the mortality rate approaches %. nearly half of the survivors remain unable to care for themselves . dci occurs in % of these patients . when present, it doubles the risk of poor outcome. -several methods have been used to treat cerebral vasospasm and dci, which is a major cause of morbidity and mortality in patients with aneurysmal subarachnoid hemorrhage (sah). milrinone safe and, potentially, effective treatment of dci as reported in low level of evidence literature . however, the efficacy not compared in a randomized way to placebo. we will examine the effectiveness and safety of intra-venous injection of milrinone for the treatment of dci following aneurysmal sub-arachnoid haemorrhage. our intension is to study the outcome of using milrinone as an addition to current therapies such as hypertensive therapy are not effective enough, yet can not be replaced as it is standard of care. as intravenous milrinone was not yet shown to have an affect in dci in a randomized controlled trial. this pilot trial is a step towards that study. the study is a pilot trial of a randomized placebo-controlled double blind trial testing the potential beneficial effect of milrinone, a phosphodiesterase inhibitor, on clinical neurological outcome in patients with dci after aneurysmal subarachnoid hemorrhage. the study drug will be given along with the standard therapy when dci occurs. the administration of milrinone increases cerebral blood flow most likely as a result of cerebral vasodilation. as intravenous milrinone was not yet shown to have an affect in dci in a randomized controlled trial. this pilot trial is a step towards that study. milirinone promising treatment for delayed cerebral ischemia following aneurysmal sub-arachnoid haemorrhage .particulary by using standardized protocol as a finding suggestive of good prognosis fever in the neurocritical care population is very common and is strongly associated with increased mortality and poor outcome. fever is aggressively treated in the icu due to its deleterious effects. yet despite best efforts with standard antipyretic agents and even with aggressive cooling measures with endovascular cooling catheters some patients may still have refractory fevers. celecoxib, a cyclooxygenase- (cox- ) inhibitor, has been used as an adjunctive antipyretic agent. this is a retrospective analysis to evaluate the effectiveness of celecoxib in lowering temperatures in patients with refractory fevers. this is a retrospective chart review of patients admitted to a neurointensive care unit at a single institution with fevers (> . c) that do not respond to convention treatment with acetaminophen, endovascular cooling catheters and ibuprofen. patients with severe traumatic brain injury, subarachnoid hemorrhages and intracerebral hemorrhages were included. patient temperature recordings were obtained in the period of hours before and hours after administration to the first dose of celecoxib. the mean temperature of the before and after periods were compared and temperature difference was calculated. patient records were included. the average of the mean temperatures in the before periods and after periods were . c (+/-sem . ) and . c (+/-sem . respectively. there was a significant difference on mann-whitney-wilcoxon rank sum test (p< . ). one average there was a drop of . (+/-sem . ) degree celsius of the mean temperature after the start of treatment. in neurocritically ill patients with fevers that are refractory to conventional treatments adding celecoxib, a cox- inhibitor seems to be effective at lowering the core body temperature. further study is warranted to evaluate for adverse effects such as risk of cardiovascular events. achieving and maintaining normothermia (nt) after subarachnoid hemorrhage (sah) or intracerebral hemorrhage (ich) often requires temperature modulating devices (tmd). shivering is a common adverse effect of tmd's that can lead to further costs and complications. we evaluated a new esophageal tmd, the ensoetm (attune medical: chicago, il), to compare nt performance, shiver burden, and cost of shivering interventions with existing tmd's. patients with sah or ich and refractory fever were treated with the ensoetm. patient demographics, temperature data, shiver severity, and amount and costs of medication used for shiver management were prospectively collected. control patients who received other tmds were matched for age, gender, and body surface area (bsa) to ensoetm recipients and similar retrospective data was collected. all patients were mechanically-ventilated. fever burden was calculated as areas of curves of time spent above . or c. demographics, temperature data, and costs of ensoetm recipients were compared to recipients of other tmd's. eight ensoetm recipients and controls between october and november were analyzed. there were no differences between the two groups in demographics or patient characteristics. no difference was found in temperature at initiation (p = . ) and fever burden above °c (p = . ). ensoetm recipients showed a non-significant trend in taking longer to achieve nt than other tmd's (p = . ). ensoetm recipients required fewer shiver interventions than controls (p = . ). ensoetm recipients incurred fewer costs than controls per day (p = . ). the ensoetm achieved and maintained nt in sah and ich patients and was associated with less shivering and lower pharmaceutical costs than other tmd's. further studies in larger populations are needed to determine the ensoetm's efficacy in comparison to other tmd's. targeted temperature management is an important aspect of care in neurologically impaired patients. however, achieving optimum temperature for a specific patient can be challenging; a patient's size, body composition, metabolism, and hypothalamic function contribute to his or her response to a given temperature management modality. the purpose of this study is to evaluate patient response to esophageal temperature management when continuously applied for at least h. deidentified core temperature data for patients (a total of measurements) were obtained from three hospital sites where esophageal temperature management was used for at least h (range - h). indications for active temperature management included: cardiac arrest ( ), refractory fever ( ), subarachnoid hemorrhage ( ), intracranial hemorrhage ( ), and traumatic brain injury ( ). goal temperatures ranged from - °c and initial patient temperatures ranged from - °c. deviation from goal was calculated by subtracting target temperature from actual temperature for each measurement which allowed the calculation of the mean and standard deviation for each time point across all temperature management protocols. across time points, representing an average treatment time of . h, . % of mean deviations from goal were within ± °c and . % were within ± . °c. in interpreting these results, several limitations must be considered. this dataset reflects a wide range of temperature management protocols and clinical scenarios. for example, a larger than average deviation in measurements recorded in the - h period was related to rewarming in cardiac arrest patients who rewarmed slowly. also, the later time points were dominated by sah, ich, and refractory fever patients who often experience more pronounced fever spikes. this analysis indicates that esophageal temperature management is a feasible option for patients who require active temperature management for or more hours. the role of therapeutic temperature management (ttm) in neurocritical care is uncertain. one question that has been inadequately addressed is the diversity of practice across multiple neurocritical care units (nccu) throughout the world. a barrier to understanding this practice variance is a data collection method that would provide adequate understanding of how ttm is implemented in various nccus. the purpose of this pilot study is to test the efficacy of a data collection method that would provide unitlevel data on ttm practice. the design of this study was prospective, observational, and cross-sectional study using quality assurance methodology. the study received institutional review board approval. to reduce the risk of loss of confidentiality and promote privacy, individual patients were not consented. data on temperature management was collected each day for consecutive days. completed data was available for days. mean daily census of patients included the following mean number of patients with sah ( ), ich ( ), ischemic stroke ( ) and other ( ). of those, ttm was provided to at least one patient during of days ( . %). the most common ttm method (tylenol) was used on patient days; surface cooling was used on patient days. ttm was initiated for fever management ( patient days) and normothermia ( patient days). the most common associated complication was hypocalcemia ( ) and hypokalemia ( ). the data collection form was easily and quickly filled completed on a daily basis, but provides limited data. although the form captured a significant number of events surrounding the use of ttm, the primary limitation noted is the inability to link specific events (e.g., hypokalemia) to specific patients or diagnoses. this pilot study demonstrates the efficacy of data capture and provides insight towards refining a prospective observational study to describe ttm practice. brainstem tumors are exceedingly dangerous due to its proximity to the structures responsible for basic human survival in the neurocritical care setting. these lesions may cause autonomic dysregulation. we report on a rare case of a female with a past surgical history of ventriculoperitoneal shunt with a brainstem mass of müllerian type epithelial tissue. methods year old caucasian female presented to our hospital status-post fall after episodes of lightheadedness, as well as, episodes of decreased respirations in her sleep. mri showed a medullary contrast enhancing mass with calcifications measuring . x . x . cm and a small calcified lesion in the right lateral ventricle. suboccipital craniectomy for biopsy and decompression was performed. intraoperatively, the heart rate and blood pressure dropped transiently due to the mass being firmly adhered with calcification to the medulla. the neuropathologist diagnosed the tissue as mullerian type epithelium with estrogen receptors. post-operatively, our patient encountered several instances of cardiac pauses on monitoring that required the need for cardiology to place a permanent pacemaker. the above is a rare case of a calcified heterogeneously contrast enhancing brainstem mass that underwent neurosurgical biopsy. histopathology results indicated müllerian type epithelial tissue which is tissue that gives rise to female reproductive organs. the origin of a brainstem lesion from an embryologically gynecological site could be speculated to have traveled retrograde via the ventriculoperitoneal shunt catheter. patient required postoperative cardiac management and intervention with a pacemaker for encroachment or mechanical conflict of the mass onto the rostral ventrolateral medulla. oncology recommended pet ct scan and further consideration for tamoxifen chemotherapeutic regimen. this case is a reaffirmation of the importance of brain tumor location and tissue diagnosis for the purpose of adjuvant treatment of neurosurgical lesions in the neurocritical care setting. tranexamic acid (txa) has been used off label in cardiovascular and orthopedic surgery, as well as in trauma resuscitation. the use of txa has increased since the publication of crash- ( ) and matters ( ), demonstrating its efficacy in trauma patients to reduce bleeding. there remains concern about the thrombotic risk as well the reduction in the seizure threshold after txa administration. case description: we present a case of a -year-old female admitted to the surgical icu after a motor vehicle accident with multiple traumatic pelvic and extremity fractures and soft tissue injury. she subsequently developed extensive arterial and venous thromboses with bilateral acute ischemic strokes with superimposed posterior reversible encephalopathy syndrome after txa administration. a second case involved a -year-old female who had a fall from standing and given txa in the field by ems. shewas admitted to the neurocritical care unit with status epilepticus and suffered a complicated course with cardiogenic shock due to stress induced cardiomyopathy. discussion: the risk-benefit balance of txa administration is generally considered acceptable in severe bleeding. the cases presented here suggest the neurological risks of txa administration may be poorly understood and demonstrate the need for better patient selection and heightened awareness for early identification and management of complications given the possible severity of neurologic sequelae. conclusion: txa is an anti-plasmin drug that is increasingly used in the areas of trauma and postoperative bleeding. we aim to educate clinicians in the potential neurological complications that can arise with its use. cryptococcus neoformans is normally an opportunistic infection known to cause meningoencephalitis and can present with stroke like symptoms. in imaging, cns vasculitis can be identified, which can lead to cerebral infarcts. when involved, these cerebral vessels are small sized leading to lacunar infarcts. we present a case that involved a large vessel territory leading to patient mortality. initial treatment with glucocorticoids, though beneficial in other meningoencephalitidies, may actually be harmful in fungal cns infections. case: a year old male with a presents with weeks slurred speech and worsening headache. an enhancing lesion on brain mri in left temporal lobe was concerning for vasculitis. patient was treated with glucocorticoids, with a negative rheumatologic workup and discharged home. patient subsequently presented days later with worsening symptoms, with ct imaging showing completed infarcts. blood cultures were positive for cryptococcus neoformans; patient died due to diffuse right mca territory edema and brain herniation syndrome. discussion: it is important to consider cns infection even in immunocompetent patients who present with any of the following: fever, nuchal rigidity, mental status change, and headache. cns vasculitis in association with infection is caused by basilar meningeal exudates. these cause traversing vessels to become inflamed, leading to distal inflammation and subsequent thrombus and infarction. we present a right mca territory infarct , presumed to be based on the aforementioned vasculitic process. when acute infarcts are associated with opportunistic cns infections, they are usually not associated with large vessel infarction. we also examine the adjunctive use of glucocorticoid therapy for treatment of fungal cns infections. this is an infrequent case of cryptococcus neoformans causing a cns infection in an hiv-seronegative patient not on chronic immunosuppressive medications. this case presents a unique complication of cryptococcal infections, a cns vasculitis leading to infarction in a large vessel territory. we describe the baseline characteristics, continuous intravenous midazolam doses, seizure control, hospital course and outcomes in patients who received high dose continuous midazolam infusion for refractory status epilepticus in this retrospective case series study, we evaluated adult patients with refractory status epilepticus treated with high continuous intravenous midazolam doses in an academic neurocritical care unit between august and june . four patients were identified. the maximum midazolam dose for each patient was: withdrawal seizures (occurring within hours of discontinuation of continuous iv midazolam) occurred in patient b. "ultimate continuous iv midazolam failure" (patient requiring change to a different continuous intravenous antiepileptic drug despite maximum optimized dose) was not observed in any of the four patients. hospital complications occurred in patient a and b due to infections. hypotension related to continuous infusion midazolam occurred in patient a. three out of four patients discharged alive to a skilled nursing facility; after a goals of care discussion with the family, the fourth patient had withdrawal of care due to the severity of his brain injury. in this case series, we report the use of high dose continuous iv midazolam for treatment of refractory status epilepticus. there were no midazolam-related deaths. neurologic complications in infective endocarditis (ie) occur up to % and are independent predictors of mortality. infectious intracranial aneurysms known as "mycotic aneurysm" (ma) are rare constituting - %. hemorrhaging rate is %. mortality is % with rupture. ruptured ma poses significant management conundrum due to lack of available solid prospective data guiding the order (cardiac vs neurosurgical) or timing (early vs delayed) of surgery. a y/o male iv drug abuser presented with acute hypoxemic respiratory failure secondary to pneumonia and suspected meningitis. gsc intubated on iv antibiotic. hemodynamic instability prompted tee showing large aortic valve vegetation. blood cultures positive mssa fulfilled criteria for ie. tests showed kidneys infarctions. ct brain showed r mca territory infarct with sah.cta head revealed small ma along the distal r mca m branch confirmed with cerebral angiogram. acute heart failure and arrhythmia led discussion on cardiothoracic surgery for valve replacement. due to ruptured ma, decision to secure it was made prior to cardiac surgery. after failed endovascular intervention, patient underwent surgical clipping. post operative mri brain showed new infarcts suggesting recurrent embolization. due to risk of intracranial bleeding, cardiac surgery was postponed for at least weeks initially then to weeks. patient underwent avr after completed weeks of antimicrobial therapy with st jude mechanical valve and discharged on anticoagulation with a modified rankin scale of . this case reflects on how urgent surgical intervention should take place.safety period between neurological event and cardiac surgery is largely debated because of lack of controlled studies. there has been no consensus on how to approach those cases as paucity of robust evidence. given their rarity the best management modality remains unclear. this case stress the importance of multimodal therapy in achieving good outcome although the timing of surgery remains a matter of debate. we present a patient with vertebral cerebral artery embolism (cae) following blunt trauma. case presentation: a year-old male was admitted with a right vertebral artery dissection and occlusion with intraluminal air, widespread pneumocephalus, bilateral pneumothoraces, a pulmonary laceration, and multiple fractures including ribs, c transverse foramen (with normal alignment), and femur following a motor vehicle collision. his pupils were initially nonreactive, and he experienced one hour of witnessed generalized seizure activity on arrival despite aggressive treatment. management: midazolam infusion, levetiracetam, and fosphenytoin were initiated for seizure control. targeted temperature management to celsius was initiated on arrival out of concern for hypoxic brain injury. computed tomography at hours demonstrated resolution of vertebral and intracerebral air, diffuse edema, and diffuse loss of gray-white matter differentiation, thus a hypertonic saline infusion was initiated. the following day, an mri demonstrated diffusion restriction in the areas adjacent to the air, including c - and diffusely throughout bilateral cerebral hemispheres. prognosis was thought to be poor. however, the following day, the patient awoke. by day four he followed commands. he was discharged to skilled nursing on day . at three months he had only minimal residual right hip weakness. discussion: there are only three case reports of cae following blunt trauma, and only one involving the vertebral artery. air migrates to the arterial circulation due to a positive gradient from low central venous pressure or high airway pressure. pulmonary venous air then embolizes to cerebral vasculature. as little as ml of arterial air emboli can be fatal with the major cause of death being circulatory obstruction and arrest from air trapped in the right ventricular outflow tract. conclusion: this patient developed pneumocephalus and cae due to a pulmonary laceration. as the cerebral air reabsorbed, his seizures resolved and his exam improved. petrous ica aneurysms are extremely rare - and difficult to treat surgically, due to the inherent challenges of microsurgical access to the carotid canal of the petrous bone - . endovascular approaches may also prove challenging, typically as the consequence of therapeutically-unamenable morphology, but occasionally due to size considerations as well. a -year-old male presented with headache and vertigo for the past weeks. the patient was hivpositive with medication noncompliance and denied any history of trauma or head injury. head ct identified a . x . cm heterogeneous soft tissue density lesion in the right petrous bone. ct angiography revealed a . x . x . cm lobulated giant aneurysm of the right petrous ica. mri/mra was performed to rule out thrombosis and showed giant partially thrombosed right petrous ica aneurysm. the decision was made to treat using flow diversion. the patient underwent catheter angiography, confirming a giant x . cm right internal carotid artery petrous segment aneurysm. we proceeded with flow diversion pipeline endovascular device, placement of two pipeline endovascular devices (flex x and x ) successfully. final angiographic runs showed significant stasis within the aneurysm and demonstrated the flow-diverter construct was well placed both proximal and distal to the aneurysm neck with no sign of endovascular leak. the patient was discharged home well. we suggest that flow diversion is an ideal treatment for petrous ica aneurysms, specifically un-ruptured lesions of complex morphology. other options for treating petrous ica aneurysms challenging, not possible, less effective, and/or carry substantial risks. second, several of the disadvantages of pedocclusion of side vessel branches and preclusion of future coil embolization, do not apply to the petrous segment of the ica. lastly, use of ped in petrous ica aneurysms has proven effective in the vast majority of reports. the spot sign is a focus of enhancement within the hematoma on ct angiogram (cta) with unique characteristics. it has a spot-like appearance within the margin of a parenchymal hematoma without connection to an outside vessel. it should measure greater than . mm in diameter in at least one dimension. its contrast density (hounsfield units, hu) is at least double that of the background hematoma. finally, there should be no hyperdensity at the corresponding location on non-contrast ct. it is a strong predictor of hematoma expansion and poor prognosis in intra-parenchymal hemorrhage. the pathogenesis of spot sign remains unclear. some studies showed an association with faster rates of contrast leakage which indicates continued bleeding. a spot sign has not been reported with isolated intraventricular hemorrhage (ivh) before. a case report of a -year-old man with a past medical history of hypertension who got admitted to the er with acute encephalopathy and right-sided weakness. head ct-scan (hct) revealed isolated ivh. cta was notable for a spot sign. it measures . mm in diameter and hu in density (surrounding hematoma measures hu). it lies within the hematoma without connections to any adjacent vessel. a follow-up hct after four hours showed expansion of the ivh. although seems uncommon, looking for a spot sign in isolated ivh can also anticipate expansion of the hemorrhage. a further study is needed to validate this observation and calculate the prevalence of the spot sign in isolated ivh. west nile neuroinvasive disease may present with nonspecific physical exam and imaging findings. to our knowledge, this is the first report of wnnd involving the temporal lobe in adults with neuroimaging suggestive of limbic encephalitis. our patient presented in winter and developed autonomic instability and sensory deficits, which are all rare findings in wnnd. -year-old texan with dm presented with acute confusion and seizure in november. patient complained of headache, fever, diarrhea and lower extremity weakness after a fishing trip. patient was febrile with mosquito bites on his arms. neurological exam was significant for comatose state, absent brainstem and deep tendon reflexes, and flaccid paraparesis. he developed autonomic instability with labile blood pressures. lp revealed wbc/mm (monocyte predominance), rbc/mm , glucose mg/dl, elevated protein of mg/dl, and a positive west nile virus (wnv) igm antibody; gram stain, hcv pcr, and the paraneoplastic and autoimmune panels were negative. eeg showed severe diffuse brain slowing. mri brain had t flair and dwi changes in right hippocampus and posterior limb of internal capsule. emg described severe subacute sensorimotor axonal polyneuropathy without prolonged distal latencies and normal conduction velocities. he received days of ivig without improvement and was terminally extubated. our patient presented with both clinical entities of west nile: wn fever and wnnd (present in less than % of cases). our patient had axonal polyneuropathy with paralysis which is due to inflammatory changes in the white matter tracts affecting spinal sensory pathways. sympathetic ganglia involvement caused the autonomic instability, another very rare manifestation of wnnd. november presentation was due to warmer texas winter. recognize that west nile fever and west nile neuroinvasive disease may present together in winter. recognize that west nile neuroinvasive disease can present with rare temporal lobe neuroimaging, sensory involvement, and autonomic instability. intracerebral hemorrhage (ich) is a common pathology seen in the neurocritical care setting that can be associated with significant morbidity and mortality. the use of sympathomimetic agents containing phenylpropanolamine (ppa) have been associated with ich in the past which lead to the drugs' removal by the fda as an over the counter medication in . we report a case in which ppa was the etiology for a spontaneous ich in a patient who was taking an appetite suppressant. case report and review of the literature we report a case of a year old female with no prior medical history, who presented with sudden onset left sided hemiparesis and hemianesthesia found to be due to a right striatocapsular intraparenchymal hematoma. systolic blood pressures at presentation and throughout the hospital course were normal. extensive work up including multiple ct scans of the head, mri brain, ct angiography, mr angiography and digital subtraction angiography were performed with no evidence of any vessel abnormality. etiology of the ich was attributed to the use of ppa. in young patients with no known comorbidities, ppa use should be considered a primary etiology of ich when no intracranial vessel abnormality can be detected. seizures have been known to cause sudden death, but reports in the literature of only cardiopulmonary failure in cases of sudden unexpected death in epilepsy (sudep). we present the case of a patient who presented post-seizure and developed sudden progressive and fatal cerebral edema within hours after a second seizure. a year old female with a history of down syndrome and epilepsy presented to the emergency department after a prolonged convulsive seizure. she received doses of mg lorazepam and levatiracetam . mg/kg with cessation of seizure activity and return to baseline neurologic status within hours of the initial event. head ct showed lack of sulci throughout the cerebral hemispheres and basilar cistern effacement despite being at her baseline neurologic status. hours after presentation the patient had another seizure, vomited, was intubated and an additional mg/kg of levatiracetam given. hours after presentation, the patient was admitted to the neuroicu with absent brainstem reflexes and repeat head ct with worsened cerebral edema and tonsillar herniation. formal brain death testing was performed approximately hours after the patient's initial presentation. seizures are known to cause a hypermetabolic state in the brain. uncontrolled neuronal firing leads to hyperemia, failure of na+/k+ atp pump, increased levels of neuronal chloride, and inability for cells to maintain homeostasis. in this case, the patient's initial head ct showed cerebral edema, likely from prolonged seizure activity. once the second convulsive seizure occurred, a period of pre-intubation hypoxemia coupled with post-intubation hypotension allowed for progression of cerebral edema in an already compromised brain; similar to what is seen in post-cardiac arrest and traumatic brain injury. this case illustrates the importance of controlling for factors that can contribute to secondary brain injury in seizure patients. posterior reversible encephalopathy syndrome (pres) is a clinico-radiographic syndrome characterized by seizure, headache, encephalopathy and neuroimaging findings of symmetric white matter edema in the posterior cerebral hemispheres. cerebellar and brainstem involvement occurs rarely. here, we report a patient who presented with severe pres complicated by diffuse cerebellar edema and obstructive hydrocephalus requiring decompression with ventriculostomy placement. this is a case report from a tertiary medical center. a -year-old woman with a history of migraine presented to the emergency room with -day history of fever, right upper quadrant abdominal pain, nausea and vomiting. on day two of hospitalization, the patient developed worsening headache, dizziness and lethargy and her blood pressure was elevated to / mmhg. ct of the brain showed cerebellar edema and bilateral occipital lobes with effacement of the fourth ventricles and associated hydrocephalus involving the lateral and third ventricle. mri obtained post-operatively revealed t -weighted/flair diffuse hyperintensities in the parietal, occipital lobes and cerebellum. there was no mass lesion or restricted diffusion in diffusion weighted images (dwi) suggestive of acute infarction. cerebellar edema with compression of the fourth ventricles with hydrocephalus was slightly improved status post interval ventricular drain placement. ventriculostomy was weaned off over the course of seven days. follow up mri showed improvement of the hydrocephalus with decreased in t -weighted hyperintensities in posterior parietal and occipital lobes as well as within the cerebellum. severe cerebellar edema with obstructive hydrocephalus is an exceedingly rare complication of pres; however, prompt recognition and surgical decompression in addition to usual medical management is critical to achieve a favorable outcome. while obstructive hydrocephalus may be successfully treated with medical management and blood pressure reduction, this case emphasizes that clinical evidence of brain herniation should prompt immediate consideration for emergent ventriculostomy placement or surgical decompression to redirect cerebrospinal fluid and reduce intracranial pressure. one of the biggest uses of qeeg is the alpha delta ratio (adr). adr drops of % from baseline are associated with vasospasm (vsp/dind). we describe a case in which subtle qeeg adr change occurred in a poor grade sah patient over a number of days, making it challenging to detect an acute adr drop. this is a case report and literature review. this study also compared hemispheric adr values against the mca values by tcd, dsa, cta and clinical exam. a year old female with hunt hess iii, wfns iv, came in comatose with a ruptured ica aneurysm. over six days, she developed refractory vsp/dind. the patient's adr was gradually declining but their increased icp required propofol sedation, which itself lowers adr. re-analysis over multiple days had to be performed, and that re-analysis showed a gradual adr decline preceding the vsp/dind. when looking at our cases, we found a sensitivity and specificity of ( , %) when using the adr nadir compared to cta/dsa. recent publications have shown the adr method has less than ideal sensitivity and specificity of ( , %). qeeg adr is a useful multimodal monitoring parameter in neuroicu patients with relatively good baseline adr. however, its ability to detect vsp and dind in poor grade sah patients who have adr values that are already low (< . ) is challenging, particularly given the confounders in this population, such as eeg artifact which artificially raise adr values, and sedation (e.g., propofol) which suppress adr values. based on this information, we would suggest neuroicu centers carefully use continuous eeg monitoring for other indications such as nonconvulsive seizures, unless they have sophisticated bedside protocols about sedation vacation (baseline daily adr that is not) and eeg department resources (technicians who can fix eeg electrode artifacts). hypoxic-ischemic brain injury is a severe consequence of global cerebral hypoperfusion following cardiac arrest. brain ct findings may include diffuse sulcal effacement, loss of cisternal spaces, poor differentiation of grey/white matter, and decreased densities in the basal ganglia and watershed territories. the connection between aggressive resuscitation, as seen with in-hospital cardiac arrest, and cerebral edema is unclear. here we present the case of a hemodynamically unstable patient who developed transient reversible cerebral edema believed secondary to aggressive resuscitative efforts and pressor therapies. a year old female with a past medical history significant for diabetes and hypertension presented to the emergency department with headache and non-bilious vomiting. workup revealed isolated ventricular hemorrhage secondary to a ruptured left posterior inferior cerebellar artery (pica) aneurysm and cerebellar arteriovenous malformation, which underwent subsequent embolization. during her early hospital course she remained intubated due to pulmonary factors, but awake and alert with a non-focal neurologic examination. her course was subsequently complicated by a severe metabolic acidosis requiring several doses of bicarbonate boluses and continuous infusion, cvvhd, intravenous crystalloids, hydrocortisone and multiple pressors to maintain stability. over a hour period she received liters of volume while maintaining a mean arterial pressure above mm hg and o saturations above %, without requiring cpr. subsequent progressive encephalopathy developed, with a ct brain revealing diffuse sulcal effacement prompting hyperosmolar therapy. gradually her encephalopathy began to improve, with repeat imaging showing improvement of cerebral edema and return of grey/white matter differentiation. this case highlights a potential etiology of reversible cerebral edema that may confound early prognostication in patients with hemodynamic instability such as multi-organ failure and in-hospital cardiac arrest. further investigations are warranted. langerhans cell histiocytosis (lch) is a rare disease with an incidence of . - . cases per , children under years of age. frequency in adults is unknown. the hypothalamic-pituitary manifestations of lch (commonly diabetes insipidus) and hypernatremia are well known complications. here we present a case where a patient presented with poor mental status and the etiology remained unknown initially despite extensive testing. electronic medical record was reviewed regarding hospital course, sodium trends, and radiology images. this patient is a year old female with history of langerhans' cell histiocytosis with biopsy-confirmed suprasellar metastases (complicated by pan-hypopituitarism) who was transferred to our institution for hypernatremia and hydrocephalus. she had undergone two cycles of chemotherapy, most recently one week prior to presentation, and five rounds of radiation completed three months earlier. her presentation to the community hospital from a nursing facility was with unresponsiveness and she was intubated on arrival. her sodium was at that time; and had been three days prior. sodium was corrected from to over the course of four days with a drop from to within the first ten hours. her mental status improved to the point where she was awake and following commands; however still remained intubated. when she presented to our institution her sodium was and subsequently became unresponsive with a poor neurological exam limited to cranial nerve function only. she was evaluated with eeg monitoring and mri brain; however both were unrevealing for a cause. she had an external ventricular drain placed for concern for hydrocephalus that did not change her exam. one week later repeat mri brain revealed extrapontine myelinolysis. this case highlights the complications associated with intracranial lch and the need for repeat imaging in patients with rapid sodium correction to identify effects of osmotic demyelination. cangrelor is a rapid-acting, intravenous p y platelet receptor inhibitor with a plasma half-life of - minutes and full platelet recovery achieved within one hour after discontinuation. because it is rapidly reversible, cangrelor is commonly used to bridge patients with recent coronary stents to cabg surgery. oral p y inhibitors, such as clopidogrel, have a delayed onset and offset with platelet recovery occurring over - days, making their use challenging perioperatively or in the setting of an acute bleed. safety and efficacy data of cangrelor in noncoronary stents are lacking. we present two patients in whom cangrelor was used to maintain internal carotid artery (ica) stent patency acutely. both patients presented with an ischemic stroke secondary to acute occlusions of the left ica and left middle cerebral artery (mca) and were taken emergently to the neurointerventional suite for carotid artery stenting (cas) and mechanical embolectomy of the mca clot. heparin and eptifibatide were administered intraoperatively. post-procedure dynact demonstrated intracranial hemorrhage complications. dual antiplatelet therapy (dapt) with clopidogrel and aspirin, typically initiated following cas, was deferred given the difficulty of reversing their antiplatelet effect in hemorrhage expansion. instead, cangrelor was initiated to maintain carotid stent patency at . mcg/kg/min in one patient and . mcg/kg/min in the other patient and infused for . and hours, respectively. platelet reactivity was trended with the verifynow® assay and used to adjust cangrelor dosing. serial imaging was obtained to monitor hemorrhage expansion. one patient was transitioned to oral dapt and discharged while the other patient deteriorated neurologically from malignant cerebral edema and expired. cangrelor may be useful following cas complicated by intracranial hemorrhage when the need to maintain stent patency must be balanced with the risk of hemorrhage expansion. further research is warranted to determine its safety and efficacy in noncoronary stents. cerebral amyloid angiopathy (caa) although has been described in the literature, the different categories of this entity and its recognition and subsequent treatment are still elusive. it is important for neuro intensivists to recognize its variable presentation . we describe a single case report and perform a systemic review. caa depending on pathology can be categorized as inflammatory-caa where perivasculitis is seen on biopsy. this causes a non-destructive perivascular inflammatory infiltration and amyloid deposition pattern. on the other hand, amyloid beta related angitis (abra) results in a vasculitis and there is predominantly granulomatous angio-destructive inflammatory mediated disease affecting leptomeningeal and cortical vessels characterized by meningeal lymphocytosis and abundant amyloid-beta deposition within the vessel walls. caa on the other hand results in no inflammation of vessels but rather just deposition of amyloid deposition in the walls of vessels. we report a case of a year old man with an extensive cardiac history, who presented with syncope. initial computed tomography (ct) of head was negative. during admission, he acutely started having trouble answering questions including his name, and was unable to communicate his needs. repeat ct head showed hypodensity in left frontal region which was attributed to a stroke. he than developed complex partial seizures requiring intubation and seizure management. lumbar puncture showed mild pleocytosis. mri brain showed edematous changes of the left subcortical and deep white matter frontal lobe region which on repeat imaging subsequently worsened. biopsy was eventually performed which confirmed inflammatory cerebral amyloid angiopathy. he was treated with steroids and immunosuppression with gradual improvement. month follow up in clinic with continued improvement to independence. recognize the various subtypes of caa in their pathology, presentation and potential treatment. in acute emergency situations, intraosseous vascular access represents an alternative route of vascular access when peripheral vein insertion is difficult. we present the first documented case of intraosseous alteplase (tpa) administration in a patient with acute ischemic stroke symptoms. methods year old male with past medical history of hypertension, end stage renal disease, and diabetes mellitus presented to the hospital with sudden onset expressive aphasia and right sided numbness minutes prior to ed arrival. nihss was and code stroke was activated. patient blood pressure was / . ct head did not show any acute intracranial hemorrhage. it was decided to proceed with thrombolytic therapy. one peripheral venous access was obtained through which nicardipine drip was started to lower the blood pressure however second peripheral venous access was attempted multiple times but was unable to be obtained. tpa is more effective the faster it is administrated, and there was no known contraindications to administering tpa via intraosseous access (io). we report the first known case of successful and safe administration of fibrinolytic therapy through the intraosseous route in a patient with acute ischemic stroke symptoms. intraosseous access has been considered to be more invasive than intravenous (iv) and carries theoretical risk of bleeding however we were able to demonstrate tpa administration through io without any local or systemic complications. the bioavailability of alteplase through io access has not been studied however it is considered to be close to iv infusion in case of morphine and vasopressors. no studies negate or support the use of intraosseous access in stroke patients. contraindications are few and complications are uncommon. the findings of our case report suggest that intraosseous cannulation may be safely used for fibrinolysis in acute ischemic stroke patients with difficult peripheral venous access in in-hospital or out-of-hospital setting. tufts medical center, boston, massachusetts, usa we report a case of a pregnant patient with bilateral ovarian teratomas who presented with treatment refractory nmda receptor encephalitis despite removal of bilateral teratomas, successfully treated with rituximab. case report and discussion of treatment and outcome. year old weeks pregnant female with known ovarian cysts who presented with one week of confusion and subsequent status epilepticus. she was started on empiric treatment with ivig while undergoing workup. nmda receptor antibody was confirmed. left oophorectomy and right ovarian cystectomy were performed, both of which confirmed ovarian teratoma. she was given high dose steroids. her worsening condition prompted consideration of additional agents. plasma exchange and rituximab were initiated and then she was continued on rituximab alone. she improved dramatically over six weeks and delivered at full term via spontaneous vaginal delivery. at one year follow up, the child was healthy and meeting appropriate milestones. we report the use of rituximab for safe and successful treatment of nmda receptor encephalitis in a gravid female. neovascular glaucoma (nvg) is a known complication of carotid endarterectomy in patients with carotid stenosis. there are no previous reports of acute nvg refractory to medical treatment following carotid artery stenting (cas). we report a patient who needed surgical treatment for acute exacerbation of nvg following cas. a -year-old man with hypertension, diabetes, and hypercholesterolemia presented with recurrent transient weakness in his right hand. fifteen days before presentation, he had experienced acute loss of vision on the left side because of central retinal artery occlusion. magnetic resonance imaging of the brain was unremarkable. conventional angiography showed an occlusion of the left proximal internal carotid artery. ophthalmological evaluation before cas showed neovascularization of the iris and a normal intraocular pressure (iop) of mm hg in the left eye. cas was uneventful, but the following morning, the patient developed pain in the left eyeball with an iop of mm hg. anterior chamber paracentesis followed by intraocular injection of bevacizumab, panretinal photocoagulation, and medical treatment failed to reduce the iop below - mm hg. eighteen days following cas, an ahmed glaucoma valve was implanted in the left eye to treat the refractory nvg. iop decreased to mmhg and his ocular pain resolved completely post implantation. although nvg is a rare complication of cas, it should be suspected in patients who develop acute ocular pain following cas. nvg may respond to anterior chamber paracentesis, panretinal photocoagulation, and bevacizumab, but surgical treatment, such as implantation of an ahmed glaucoma valve, should be considered in cases with refractory nvg. background: cerebral amyloid angiopathy is a common cause of spontaneous lobar intracerebral hemorrhage. convexal subarachnoid hemorrhage can be a manifestation of cerebral amyloid angiopathy. whether focal amyloid burden predicts future hemorrhage is unclear. case: an -year-old man presented with transient left arm weakness and paresthesias in the setting of previous cognitive decline. mri showed a convexal subarachnoid hemorrhage of the right central sulcus, as well as susceptibility weighted imaging findings consistent with superficial siderosis. lumbar puncture revealed normal cell count with a mildly elevated protein. he had spontaneous resolution of his symptoms after several hours. one year later he presented with sudden onset confusion and imaging again showed a convexal subarachnoid hemorrhage over the posterior right frontal lobe. susceptibility weighted mri revealed hemosiderin over the right posterior frontal and anterior parietal lobes. an amyloid-pet, obtained one year prior to his first spell as a research participant, demonstrated asymmetric amyloid deposition in the right temporo-parietal region. years after his initial episode he presented again with confusion, headache, and decreased level of alertness. a ct scan demonstrated a right-sided temporo-parietal intracerebral hemorrhage in the area of asymmetric amyloid deposition on pet. his family opted for comfort measures only, and he was discharged to hospice. autopsy revealed severe amyloid angiopathy, as well as alzheimer disease, braak stage vi. discussion: this case illustrates the clinical course of a patient with amyloid angiopathy, including recurrent convexal subarachnoid hemorrhages, and superficial siderosis. of importance, the amyloid pet scan predicted the location of his intracerebral hemorrhage years later. the case of a -year-old man, presenting with a past medical history of migraine headaches, dipola, vertigo, with symptoms later progressing to lethargy and confusion for days. brain mri revealed a peripherally enhancing mass within the left thalamus with central restricted diffusion, which is consistent with a cerebral abscess. case report of congenital heart disease when discovered in adulthood is an interesting entity, especially when it is the source of brain abscesses. detailed history taking, physical examination and appropriate imaging can usually reveal the anomaly. the diagnosis of brain abscess should promote the clinician to consider right to left shunts as a possible predisposing condition for brain abscess management of acute cerebral embolism in patients with implanted ventricular assist devices (vads) is particularly challenging, since chronic anticoagulation often precludes the use of intravenous tissue plasminogen activator (iv-tpa). we describe a vad patient who suffered cerebral embolization, and was successfully treated with thrombectomy, emphasizing the nuances particular to this clinical scenario in the context of limited historical experience. a year-old man with heart failure (ejection fraction %) and heartware ii vad implantation about months prior, was found at the scene of a car accident with expressive aphasia, right homonymous hemianopia, extinction and right hemiplegia, with a national institutes of health stroke scale (nihss) score of . upon arrival, his ct was unremarkable, but cta revealed occlusion of the left middle cerebral artery (m segment). since his inr was . , he underwent emergent thrombectomy with the solitaire device, resulting in complete revascularization (tici = ) minutes from onset, with rapid deficit resolution (nihss = ). the procedural and clinical success was accompanied by lack of evidence of infarction in subsequent ct studies, and a modified rankin score of upon discharge. the removed thrombus displayed early organization, suggesting unexpected firmness, and underscoring the potential importance of mechanical removal rather than chemical lysis in vad patients. our case has attributes that set it apart from those previously reported: ) the use of a hybrid (i.e. retrieval plus aspiration) endovascular retrieval technique, ) the lack of concurrent use of thrombolytic drugs, and ) the rapid, sustained and optimal clinical improvement. the utilization of vads continues to grow, yet the literature regarding endovascular techniques for managing these types of patients remain scarce. however, the increasing availability of centers capable of delivering this type of treatment, suggests that thrombectomy should be strongly considered in vad patients with acute cerebral embolism. extreme cerebral oxygen changes has not been reported via monitoring of partial brain tissue oxygen levels. here we present an asah patient with brain tissue oxygen (pbto ) monitoring, who developed cerebral hypoxia due to cerebral vasospasm, then went on to develop cerebral hyperoxia with associated cerebral infarction. methods yo female with hh fg sah with initial gcs of t underwent coiling of a ruptured basilar tip aneurysm. a pbto monitor was inserted to guide therapy. this patient had multiple episodes of low pbto ( mmhg). this corresponded to infarction on follow up head ct and mri with preservation of local arterial vessels on mra, consistent with diagnosis of dci. in the present case, high pbto is more likely resulted from a combined effect of ) increased cbf from co-administration of ketamine at the time of milrinone infusion; ) decreased cerebral metabolic demands in already infarcted left frontal lobe, resulting in reduced oxygen uptake; ) accelerated reperfusion and thus hyperemia with milrinone. restoration of flow with milrinone may have been too late to reverse the prolonged period of vasospasm induced ischemia, resulting in perfusion of infarcted tissue, or luxury perfusion. clinicians utilizing pbto monitoring for dci management should be cautious of high pbto values, as it may herald cerebral infarction. further studies are needed to better elucidate the mechanism of reperfusion injury and potential treatments. patients with acute brain injury, especially those with intracranial hemorrhages are at a higher risk for hemorrhage while on therapeutic anticoagulation. unfractionated heparin (ufh) is frequently used as it is easily reversible and has a short half-life. activated partial thromboplastin time (aptt) is traditionally used to monitor its effect. several disadvantages with aptt monitoring include inability to reach therapeutic goal, over-or under-dosing and its associated complications. anti-xa level is reported to have better correlation with actual degree of anticoagulation using ufh. retrospective chart review of patients with acute brain injury who required initiation of early therapeutic anticoagulation and monitored with anti-xa level. case - year-old-man with intracerebral hemorrhage (ich) developed lower extremity deep vein thrombosis (dvt) and required therapeutic anticoagulation. patient became therapeutic within six hours of titrating infusion based of anti-xa levels and remained therapeutic. asymptomatic rectal bleeding associated with fecal management system was noted. case - year-old-man with cerebral venous sinus thrombosis presented required therapeutic anticoagulation. ufh infusion was initially monitored using aptt levels which had widely varied lab results, thus monitoring was switched to anti-xa levels which provided a more consistent therapeutic range. however, patient developed thrombocytopenia in the setting of inflammatory bowel disease. therefore, ufh infusion was changed to argatroban infusion. case - year-old-man with lower medullary acute ischemic stroke due vertebral artery dissection required therapeutic anticoagulation to prevent recurrence. patient became therapeutic within hours of titrating based of anti-xa levels. to monitor therapeutic anticoagulation, anti-xa level appears to achieve target anticoagulation level faster and without serial variation as compared to aptt. however, anti-xa level estimation is costlier as compared to aptt and not widely available. by restricting it to special populations like those with acute brain injury might justify its use and underscore cost-effectiveness. neurological admissions presenting to the icu benefit from a dedicated neurocritical care team but many community hospitals lack this subspecialty expertise. with an aging population and a neurointensivist shortage, more patients are transferred to designated neurocritical care units which increases healthcare spending and resource utilization. recognizing this obstacle, we describe the management of a patient in status epilepticus via our novel "eneuro-icu" consult program in which a 'sub-hub' of the northwell health tele-icu was set up at the only hospital out of in our health system that is staffed / by neurointensivists. a -year-old man with history of a left frontal meningioma presented with multiple seizures to a hospital within our healthcare system. he received mg of lorazepam and levetiracitam in the emergency department and was admitted to the icu for further monitoring. there he was witnessed to have recurrence of clinical activity concerning for ongoing seizure. levetiracitam was increased and phenytoin was added. neither immediate neurological consult nor continuous eeg was available, thus an "eneuro-icu" consult was obtained. in this model, the bedside provider contacts the tele-icu that facilitates a conference call with the neurointensivist. av technology was used to provide consultations and follow ups. the neurointensivist determined the patient was rapidly returning to baseline and recommended a head ct, lab studies and continuation of the anti-epileptic drugs. the eicu team monitored the patient overnight. by leveraging the infrastructure in place for management of critically ill patients remotely, an additional level of subspecialty care was offered in a timely manner and allowed the patient to remain at their local facility. based on the success of the initial program we are currently in the process of extending the virtual consult service to various community hospitals' eds/icus to improve outcomes for patients who would benefit from neurocritical care services. hypoglycemic encephalopathy is a potentially life-threatening manifestation of hypoglycemia, it is usually caused by metabolic change, hypoglycemic agents, and malignancy. here, we report a patient with hypoglycemic encephalopathy caused by sleeve gastrectomy a -year-old woman was admitted due to unconsciousness of acute onset. she showed normal corneal and vestibulo-ocular reflex but sluggish pupil light reflex and decerebrated posture by painful stimulation. she has taken severe medications for weight control including orthosiphon powder and hydrochlorothiazide after bariatric surgery. laboratory studies showed significantly low blood glucose level ( mg/dl) with normal liver enzyme and creatinine. there was no evidence of adrenal insufficiency. electroencephalography showed no epileptiform discharge. initial and follow-up brain magnetic resonance imaging revealed diffuse high signal intensity on white matter expanded to cortex, corpus callosum and posterior limb of the left internal capsule, suggesting hypoglycemic encephalopathy. in abdomen-pelvic ct, there is no mass lesion like carcinomas or insulinoma. the clinical diagnosis of hypoglycemic encephalopathy followed by sleeve gastrectomy was made by given history of bariatric surgery and the lack of evidence of hypoglycemic agent overdose, adrenal insufficiency, endogenous hyperinsulinism or malignancy. there are several hypotheses that sleeve gastrectomy can encourage hypertrophy of beta cells, hypersecretion of glucagon-like peptide, glucagon abnormality and increased insulin sensitivity, that may induce hypoglycemia. we suggest that clinicians should consider sleeve gastrectomy itself as a possible cause of profound hypoglycemia pulmonary embolism (pe) is a fatal complication in neurological conditions with plegic extremities. clinical presentations and supportive testing can be variable. we present a case of pe which presented with st segment elevations weeks after spontaneous intracerebral hemorrhage (sich). case report and review of the literature we present a case of a year old female with a history of a recent sich with resultant left hemiplegia who presented with a syncopal episode and chest pain. on physical examination, she was noted to be tachypneic and tachycardic with an unchanged neurological exam. pulmonary embolism can present with a variety of ekg abnormalities including st elevations after sich and the treating physician should be aware of these idiosyncrasies. anticoagulation should be cautiously initiated in such cases. infectious intracranial aneurysms (iia) are rare neurovascular lesions associated with infective endocarditis. we present a case of a large iia which developed within hours of a negative ct angiogram and ruptured despite weeks of appropriate antibiotic treatment. a year-old woman presented with fevers and malaise. her initial workup revealed an aortic valve mass and blood cultures grew out streptococcus. three days after intravenous penicillin therapy was initiated for bacterial endocarditis, she developed a new headache and right hemianopsia. a head ct demonstrated a left occipital lobe stroke with hemorrhagic transformation. further workup with ct angiography revealed a mm outpouching along of the distal branch of the left pca, consistent with an infectious intracranial aneurysm. on repeat imaging, this aneurysm demonstrated growth despite medical treatment, and required coil embolization/occlusion. aortic valve replacement was planned after weeks of antibiotic therapy because of continued severe aortic insufficiency and persistent valve vegetation. on the day of surgery, she developed acute word-finding difficulty followed by a rapid neurologic deterioration resulting in coma. a head ct demonstrated a new left frontal intraparenchymal and subarachnoid hemorrhage associated with the rupture of an mm x mm irregularly shaped aneurysm in the region of the left mca bifurcation, which had been absent on a prior surveillance ct angiography just hours prior. she underwent emergent coil embolization, extraventricular drain placement, and decompressive hemicraniectomy. despite these measures, her exam did not improve. she was transitioned to comfort measures and life-sustaining therapies were withdrawn. the development of iia can occur despite appropriate medical treatment. these aneurysms may rapidly expand and rupture within hours, as shown by our case. even with prior exonerating imaging, clinicians should have a high suspicion for iia development in all infective endocarditis patients. the corneomandibular reflex, also known as wartenburg reflex or von solder phenomenon, is a rare pathological reflex signifying severe supranuclear trigeminal injury. it presents as contralateral jaw deviation to corneal stimulation. etiologies include upper brainstem lesions, large hemispheric lesions with brainstem compression, as well as advanced amyotrophic lateral sclerosis and multiple sclerosis when corticobulbar pathways are affected. this clinical finding is useful in differentiating structural and metabolic causes of coma, as this examination finding would not be present in metabolic phenomena. a middle aged man presents with a hypertensive right thalamic hemorrhage and a four score of e m b r . the patient's cornea was stimulated with a cotton swab. the cornea was tested bilaterally to determine any lateralizing features. recording on video was performed with patient's family written consent as patient was comatose. upon stimulation of the patient's cornea a contralateral jaw jerk was appreciated. this was replicated contralaterally. this case describes a common patient with a rare physical examination finding. there is utility in recognizing this finding as it will aid in determination of the underlying cause of a comatose state. the corneomandibular reflex present at presentation rules out a metabolic cause. a structural cause was validated by imaging studies (shown). the reflex arc was researched and has been independently artistically rendered (shown), which demonstrates the pathway beginning with the afferent limb of the corneal stimulus (v ) which travels to the main trigeminal sensory nucleus via the trigeminal ganglion. severe supranuclear trigeminal lesions will inhibit inhibitory interneurons within the mesencephalic nucleus, leading to activation of the motor nucleus of the trigeminal nerve. this causes activation of the ipsilateral external pterygoid muscle which produces a contralateral jaw jerk. overall this patient fared poorly and expired several days after admission. pneumocephalus is when air enters and is contained inside the intracranial compartment. when intracranial pressure increases causing neurological decline, patients can experience nausea, vomiting, seizures, dizziness, and altered mental status. here we present three cases of postoperative pneumocephalus which resolved quickly with humidified oxygen delivery via high-flow nasal cannula. we follow the cases with a review of the mechanisms and pathophysiology of pneumocephalus and its treatment, as well as future directions in management. case series of patients with post-operative pneumocephalus who were treated with high-flow nasal cannula. case describes a -year-old woman who underwent hemicraniotomy for removal of meningiomas, with focal postoperative neurological signs and mm of midline shift on head ct due to pneumocephalus. case describes a -year-old woman who underwent right anterior temporal lobectomy for seizures, who developed postoperative focal prefrontal lobe signs and mount fuji sign on head ct. case describes a -year-old man with bilateral subdural hematomas, status post bilateral burr hole evacuation. he was excessively somnalent postoperatively with bilateral pneumocephalus. with high-flow nasal cannula, they all returned to clinical, and near radiographic baseline within , , and hours, respectively. recognizing the limitations of a small case series, we believe these cases support use of high-flow nasal cannula when treating patients with symptomatic pneumocephalus. thsee patients showed more rapid clinical and radiographic improvement after implementation of hfnc oxygen therapy than previously described using other methods. high-flow nasal cannula may help washout nitrogen from the lungs, allowing a downward gradient from the nitrogen in the intracranial air bubble out the lungs. in addition, high-flow nasal cannula is more comfortable for the patient, allowing for more consistent treatment. randomized studies are needed to confirm our findings. the neurotoxin produced by clostridium botulinum is the most lethal toxin known by weight. early recognition and treatment of botulism are crucial for full recovery. we present a case of progressive paralysis secondary to botulism toxemia following a gunshot wound (gsw). a -year-old man suffered a gsw to the right lower extremity. he was treated in the emergency department where the wound was irrigated and closed. some bullet fragments could not be retrieved due to close proximity to popliteal vessels and surrounding nerves. he returned ten days later with diplopia and nausea. he denied consumption of canned foods or illicit substances and had no preceding upper respiratory or gastrointestinal illnesses. on examination, he exhibited ptosis and symmetric bilateral motor weakness with diminished deep tendon reflexes. the gsw showed no signs of infection. progressive respiratory insufficiency resulted in intubation and mechanical ventilation. a lumbar puncture revealed normal opening pressures and cerebrospinal fluid analysis was unremarkable. titers for acetylcholine receptor and anti-muscle specific kinase antibodies were negative, as was a tensilon test. blood toxicology analysis showed no evidence of illicit substances or heavy metal poisoning. a high suspicion for wound botulism led to consultation with the regional poison center and cdc. blood and anaerobic wound samples were obtained for toxin bioassay and culture. empiric intravenous penicillin g therapy was started. equine heptavalent antitoxin (h-bat) was obtained and administered on hospital day . serum toxin bioassay tested positive for botulinum neurotoxin type a. the patient required a gastrostomy tube due to persistent dysphagia. after one month of hospitalization, he was discharged home and continues outpatient physical therapy. wound botulism from traumatic injury is exceedingly rare with only one to two cases reported annually. our case is the first reported incidence of wound botulism from a single gunshot wound. hyperammonemic cerebral edema (hce) with brain herniation carries a dismal prognosis historically despite aggressive treatment. however, we report a case where a patient with severe hce and herniation returned to her neurological baseline after aggressive medical management. a -year-old woman became acutely comatose with a blown left pupil and required intubation several days after admission for encephalopathy. head ct demonstrated diffuse cerebral edema with central and bilateral uncal herniation. profound hyperammonemia ( ug/dl) was implicated, though hepatic function was normal. her intracranial hypertension was ultimately controlled using hyperventilation, sedation, and osmotherapy, resulting normalization of her brainstem reflexes and improvement in her coma and imaging. continuous veno-venous hemodialysis (cvvhd) normalized her ammonia and encephalopathy that was initially refractory. multiple porto-hepatic shunts were identified on hepatic ct angiogram as the cause of her hyperammonemia, and were embolized. she was eventually weaned off cvvhd and extubated, without residual neurological deficits. our case demonstrates that, with contemporary management, clinical and radiographic reversal of hce and herniation is possible and prognosis is not uniformly poor. therefore, neurological prognostication in these patients should only be performed after assessing the clinical trajectory following cerebral resuscitation and ammonia reduction. furthermore, our case provides an example of how cvvhd can be used to reduce refractory hyperammonemia quickly until the cause of the hyperammonemia can be ascertained and addressed. finally, this is the first case reported of hce secondary to primary portosystemic shunt in absence of hepatic disorder; vascular imaging of the liver should be considered in the work-up of patients with hyperammonemia. a good neurological prognosis is possible for patients with hce and cerebral herniation with aggressive management that includes reduction of icp and ammonia. ccvhd is a useful adjunct to treat refractory hyperammonemia. a porto-systemic shunt should be considered as an etiology for hyperammonemia. cerebral venous sinus thrombosis (cvst) often presents with intracerebral hemorrhage and seizures. extensive involvement of the cerebral sinuses can lead to comatose state due to cerebral edema and associated intracranial hypertension. if not reversed with early therapeutic anticoagulation, then mechanical thrombectomy and decompressive hemicraniectomy (dhc) may be necessary as life-saving measures. however, etiological diagnosis of associated hypercoagulable state is needed for successful long-term treatment. case report of a patient presenting with cvst requiring anticoagulation, dhc and total colectomy (to treat underlying ulcerative colitis) as treatment with full anticoagulation was associated with lifethreatening hematochezia. twenty-five year old man with one week history of diarrhea presented with left sided weakness. imaging studies confirmed extensive cvst with minimal venous drainage through bilateral cavernous sinuses as well as right hemiparesis secondary to left post cingulate intracranial hemorrhage. patient subsequently developed loss of vision and became encephalopathic, despite initiation of anticoagulation with heparin. hence, mechanical thrombectomy was attempted but was unsuccessful. he also developed consumptive thrombocytopenia for which his anticoagulation was switched to argatroban. progressive neurologic deterioration necessitated dhc. his neurological examination progressively improved upon re-initiation of anticoagulation resulting in restoration of vision and resolution of left hemiparesis. later in the disease course, he developed symptomatic hematochezia associated with his primary disease, ulcerative colitis and required total colectomy. subsequently he was transitioned to oral anticoagulation and transferred to inpatient rehabilitation facility due to deconditioning from prolonged hospitalization. cvst can be life-threatening unless early treatment is initiated. appropriate and timely treatment including etiological diagnosis can lead to favorable patient outcomes. adverse effects of intrathecal non-ionic contrast during myelography are rare but can include seizures and encephalopathy. to our knowledge, cerebral edema has only been reported in the literature in two previous cases. we report a case of malignant cerebral edema following intrathecal administration of non-ionic contrast who developed seizure like activity with radiographic evidence on a head computerized tomography (ct) scan of acute diffuse cerebral edema. an year-old male underwent an elective spinal ct myelogram using mm of isovue m non-ionic contrast to evaluate chronic lumbar pain related to spinal stenosis. no complications were reported intra-procedurally and the patient was discharged home. the patient began to complain of progressive worsening headaches. the following morning he started complaining of nausea/vomiting, lost consciousness with posturing vs seizure like activity. a head ct revealed extensive brain edema and swelling with crowding of the brainstem and herniation ( fig. ). this patient was intubated and given an iv mannitol, . % hypertonic saline followed by an infusion of % hypertonic saline infusion. serial cts revealed complete resolution of his cerebral edema hours after admission ( fig. and ) . the patient's mental status improved, was extubated, and then was discharged home days after admission. while significant adverse effects of non-ionic contrast following spinal myelography are rare, the potential life threatening severity of these incidents warrants further patient education following this routine outpatient procedure. we recommend close neurological monitoring after intrathecal administration of contrast media. patients should be provided with detailed instructions about the potential side effects of non-ionic contrast and how to seek medical attention if symptoms of cerebral edema are noted post procedurally. a large acute traumatic subdural hematoma with brain compression and midline shift is typically considered a neurological emergency necessitation surgery. spontaneous resolution of a large subdural hematoma is considered a rare phenomenon with a few case reported in the literature. to our knowledge, we present the first case of spontaneous resolution of a traumatic acute subdural hematoma with brain compression and midline shift on dual antiplatelet therapy. a year-old patient initially presented after being found down and unresponsive in his home. the patient was on aspirin and clopidigrel. he was found to have altered mental status, wasn't following commands, and had a glascow come scale score of < . the patient's initial head ct revealed a large left acute subdural hematoma (sdh) measuring . cm in diameter. neurosurgery was consulted upon arrival for possible emergent evacuation. the patient's repeat head ct showed a decreased sdh to . cm in diameter. given the rapidly resolving sdh, surgery was postponed. another repeat head ct the following day revealed a decrease in size of the sdh to mm in diameter. several theories have been proposed for the rapid resolution of an acute sdh including csf leaking into the sdh through a tear in the arachnoid membrane with rapid reabsorption, redistribution of the hematoma in the subdural space, and acute fluctuations in icp driving the spontaneous resolution of the sdh. close neurological and repeat imaging may be helpful in managing these patients. as seen in our patient and others, a low density band in the subdural hematoma may indicate csf and be a predictor for spontaneous resolution of an acute sdh. the features of this atypical case offer points of discussion regarding the surgical or non-surgical approach of these patients. early post-hypoxic myoclonus -or myoclonic status epilepticus -develops within hours of the initial anoxic injury and is associated with poor outcomes per current aan practice guidelines. late posthypoxic myoclonus -or lance-adams syndrome -develops > hours after the anoxic injury, consciousness is regained, and is associated with relatively good outcomes. the patient is a yo man with a history of alcohol and cannabis use disorder, bipolar disorder, pnes who presented after attempted hanging for up to minutes. intial rhythm was pea; he had rounds of cpr, received mg epinephrine, and was intubated prior to rosc. myoclonic jerks were noted within hours post arrest. hypothermia protocol was initiated as gcs was t. ct head showed subtle loss of grey-white differentiation. eeg initially showed that his generalized myoclonic jerks correlated with cortical activity. he was started on versed gtt, keppra, vpa with improvement in the frequency of jerks. on post-arrest day , mri brain showed mild cerebellar edema. mri c-spine was negative for significant myelopathy, arguing against myoclonus as a spinal reflex. mentation gradually improved; on post-arrest day he opened his eyes to command. eeg evolved to show gpeds and sirpids and oxc and tpm were added. on post-arrest day a paralytic challenge resolved electrographic spikes, suggesting subcortical origin of myoclonus. he continued to improve cognitively, but despite clonazepam, vpa, home oxc he continues to have severe intention myoclonus. despite the presumed poor prognosis, the patient's family pursued aggressive measures and his mentation gradually improved. early post-hypoxic myoclonus carries a poor prognosis, however, in this case, the patient survived with a good cognitive outcome likely owing to his age and relatively few comorbidities. further studies are needed to differentiate early-onset lance-adams from myoclonic status since prognosis differs greatly. posterior reversible encephalopathy syndrome (pres) can occur from multiple etiologies and often presents with rapid-onset headache, altered consciousness, seizures and/or visual disturbances. vasogenic edema involving predominantly cerebral white matter is a key finding on imaging studies. although seizures are a frequent presenting symptom of pres, refractory status epilepticus (rse) requiring multiple antiepileptic medications is very rare. a case report of a patient presenting with pres and clinical course complicated with rse necessitating use of intravenous anesthesia, ketamine, and newly-available brivaracetam. -year-old woman with history of congestive heart failure, chronic iron deficiency anemia and uncontrolled hypertension was admitted for severe encephalopathy and convulsive status epilepticus (cse) for longer than minutes necessitating propofol and midazolam infusions. her admission systolic blood pressures were in the s, and mri brain revealed bilateral parieto-occipital t /flair hyperintensities consistent with pres. despite adequate control of hypertension following admission, patient remained encephalopathic and continuous electroencephalography (eeg) demonstrated nonconvulsive status epilepticus (ncse). the patient's ncse continued despite use of maintenance antiepileptics (fosphenytoin, lacosamide, levetiracetam) and high-dose infusions of midazolam and propofol. ketamine infusion was started to maximize nmda receptor blocking properties, and burstsuppression pattern on eeg was easily achieved with bolus infusions followed by continuous infusion. addition of brivaracetam was used to replace levetiracetam and allowed patient to remain seizure-free when iv anesthetics were weaned off. patient required prolonged hospitalization with gastrostomy tube placement and tracheostomy, which was later decannulated prior to patient's discharge to home with family. high index of suspicion is necessary to identify patients in ncse with prolonged encephalopathy that have pres. early use of ketamine along with a benzodiazepine may result in rapid achievement of burstsuppression to treat se. brivaracetam may be a useful agent to treat rse. diagnosis of diabetes insipidus(di) includes polyuria, hypernatremia and low urine specific gravity. we present two patients, receiving hyperosmolar therapy for intracranial hypertension (iht), in whom using low urine specific gravity to diagnose di lead to delayed treatment. this is a retrospective case series. criteria used to diagnose di at our institution include polyuria, sodium < mosm/kg and urine to plasma osmolality ratio < . case : -year-old male with subdural hematoma, iht on hyperosmolar therapy, developed polyuria. sodium rose from to meq/l. urine specific gravity was . excluding di. eventually, sodium rose to meq/l. specific gravity remained . but urine osmolality was mosm/kg and urine/plasma osmolality ratio was . , consistent with di. vasopressin was initiated, however the patient had already developed lactic acidosis and renal failure due to hypovolemia. case : -year-old female with intracerebral hemorrhage and iht on hyperosmolar therapy, developed polyuria. sodium rose to meq/l, specific gravity remained > . but urine osmolality was mosm/kg and urine/plasma osmolality ratio was . consistent with di. vasopressin was initiated. hyperosmolar therapy increases urine osmoles and raises urine specific gravity. this interferes with diagnosis of di which requires low urine specific gravity. while specific gravity measures the weight of particles, osmolality measures particles independent of their weight and thus accurately measures urine tonicity in the presence of heavy particles like mannitol. moreover, urine/plasma osmolality ratio is able to demonstrate relative hyposmolarity of urine when compared to serum assisting with diagnosis of di even when urine specific gravity is elevated. we conclude that urine specific gravity does not reliably detect di in patients receiving hyperosmolar therapy. urine osmolality and urine/plasma osmolality ratio may detect di earlier and prevent dehydration and kidney injury. these findings should be validated prospectively. endovascular intervention in the treatment of cvt(cerebral venous thrombosis) is an alternative strategy when cases deteriorate despite best medical management or develop refractory intracranialhypertension. we present a patient with cvt due to heparin-induced thrombocytopenia(hit), with intraparenchymal hemorrhage(iph) and refractory intracranial-hypertension, who was managed with systemic anticoagulation, continuous intra-sinus infusion of rtpa and mechanical thrombectomy(mt) resulting in excellent outcome. case report: a -year-old woman with left parafalcine meningioma s/p cyberknife was started on subcutaneous heparin for radiation necrosis days prior to admission. she presented to the hospital with new onset headaches and nausea. ct head showed increased edema with mid-line shift around the meningioma, for which steroids were started. within days her headaches worsened and repeat imaging demonstrated right temporal iph. emergent hematoma evacuation was performed. mri brain showed right cerebellar infarct and mra head showed extensive cavernous sinus thromboses, from right internal jugular vein and into sigmoid and transverse venous sinuses. she tested positive for hit and was switched to argatroban drip. patient however continued to deteriorate due to refractory intracranial-hypertension. intra-cavernous rtpa injection and mt was done but the thrombosis was noted to recur on repeat angiogram hrs later. an intra-sinus catheter was left in place for continuous infusion of rtpa at mg/hr. for hrs was done while argatroban drip was continued. the patient's intracranial pressure returned to normal. repeat venogram showed resolution of cvt. patient tolerated the therapies well, without any further hemorrhagic complications. modified rankin score at month follow-up was . this case features successful aggressive endovascular interventions including in-situ rtpa infusion, mt and concomitant systemic anticoagulation for cvt due to hit, complicated by intracranial hemorrhage and refractory intracranial hypertension. the paucity of high quality evidence related to safety, efficacy and modality of endovascular treatment lead to making therapeutic decision on individual basis. acute brain injury may be followed by encephalopathy marked by electroencephalographic features along the ictal-interictal continuum (iic). the use of perfusion imaging to co-localize radiographic features of known malignant eeg patterns may add an important context to guide treatment escalation or de-escalation. this is only the second report in which widely available ct or mr perfusion techniques were favored for this application over more cumbersome metabolic imaging such as pet. retrospective analysis was performed on records for patients admitted to a neurosciences icu, exhibiting encephalopathy, with eeg features on the iic, who underwent perfusion imaging. studies included ct perfusion, mr perfusion, arterial spin labeling, or spect. these studies were obtained for unrelated purposes. escalation or de-escalation of anti-convulsant and sedative medication, hospital course, and patient outcomes were extracted. perfusion imaging data was juxtaposed with eeg patterns along the iic, and patient outcomes are described in narrative form. seven cases were identified. four cases occurred in the context of intraparenchymal hemorrhage, of which one was secondary to meningioma resection. two cases occurred after treatment for subdural hematoma, and one case was related to ischemic stroke. anti-convulsant and sedative management was escalated or de-escalated relative to the presence or absence of radiographic co-localization of hyperperfusion in all but one case. emerging data indicates that some iic eeg patterns may merit aggressive treatment. metabolic signatures of secondary brain injury as measured by cerebro-oximetry or microdialysis have associated these patterns with unfavorable outcomes. we report case studies in which information gleaned from basic perfusion imaging may suffice to distinguish between benign iic patterns and those that should be regarded as near-ictal. the cases hint at novel ways to conceptualize treatment of encephalopathy following acute brain injury and suggest a dimensional shift in thinking towards electroperfusive status epilepticus. sudep has classically been a diagnosis of exclusion. recent studies have shown, however, that similar genes -and even genes within the same family -are associated with sudep and brugada. this suggests that perhaps the cardiac irritability of brugada syndrome exists on a spectrum with epileptic sudden death. a yo man with a history of presumed seizure disorder presented as a transfer from another hospital after being found to have anoxic brain injury following cardiac arrest. he had been shopping with his wife when he was thought to have one of his typical seizures. he was non-responsive for about minutes. on arrival ems found him pulseless. cpr was started en route and continued for minutes in the ed where he was defibrillated three times before achieving rosc. he completed the therapeutic hypothermia protocol. cardiac catheterization was clean. eeg showed diffuse slowing with no epileptiform discharges. imaging showed diffuse anoxic brain injury. after nearly two weeks without clinical improvement he was made comfort care. . of note, previous seizure workup failed to identify epileptiform activity. he was given an aed prescription which he never filled. further chart review showed that he had previously presented to the ed after a "seizure" episode which lasted minutes. his neuroexam was non-focal. ct head was negative. review of his ekg at that time showed type brugada syndrome pattern with an elevated jpoint and t-wave inversions in v and v . his sudden cardiac arrest is most likely a result of symptomatic brugada symptomatic brugada is important to identify early since deaths such as the one discussed above may be prevented by an implanted defibrillator. this case highlights the need for heightened awareness and more effective testing for brugada in the setting of seizure or pseudoseizure. patients with cerebral air embolism (cae) often exhibit more severe symptoms than those typically associated with the number of air emboli and size of infarcts on brain images. however, this discrepancy between symptoms and imaging findings has not been sufficiently explained. we report a case of cae in which disruption of the blood-brain barrier (bbb) and perfusion defects were identified via brain magnetic resonance imaging (mri). a -year old man with a lung mass was admitted to our hospital. percutaneous needle aspiration of the mass was performed in the left lower lobe of the lung. the patient developed sudden confusion and irritability after the procedure. during neurological examination, he could follow only simple commands and exhibited symptoms of left-sided weakness and neglect (medical research council grade ). noncontrast computed tomography (ct) of the brain revealed a few small air emboli in the right frontal subcortical area. multimodal mri of the brain was performed minutes after the onset of symptoms. t -weighted gradient-echo imaging revealed only a few small air emboli in the right frontal area, and diffusion-weighted imaging findings were unremarkable. in contrast, time-to-peak imaging revealed widely distributed perfusion defects in the right hemisphere, while contrast-enhanced t -weighted imaging revealed prominent leptomeningeal enhancement, suggestive of bbb disruption in the right hemisphere. magnetic resonance angiography revealed no steno-occlusive lesions. the patient was treated with % oxygen via a high-flow nasal cannula. his weakness subsided the next day, although his confusion persisted for days. follow-up mri performed five days after the onset of symptoms revealed resolution of the abnormal findings. our findings suggest that disruption of bbb and perfusion defects may develop in patients with cae. extensive impairments of the bbb and perfusion may explain the mismatch between severe neurological symptoms and small air emboli/infarcts. co-existence of cerebral salt wasting and diabetes insipidus is an extremely rare entity that has only been described in adult case series and a paediatric series. due to the complex nature of diagnosing this entity, mistreatment may ensue and lead to high morbidity and mortality rates. we report a case of a patient who was admitted to the neurosurgical intensive care unit after sustaining a subarachnoid haemorrhage secondary to a ruptured anterior communicating artery aneurysm. a -year old lady presented with sudden onset of severe headache and nausea. gcs was (e v m ) with no focal neurological deficits. she underwent endovascular coiling and embolisation of the aneurysm under general anaesthesia and had a left external ventricular drain inserted. in the immediate postoperative period, she was found to be polyuric, with the initial workup suggestive of diabetes insipidus. desmopressin was administered with initial good effect. however, her polyuria recurred and persisted despite desmopressin. the repeat workup revealed the presence of concomitant cerebral saltwasting. she was then treated with fludrocortisone and sodium chloride supplementation. careful monitoring of her serum sodium levels and overall fluid balance allowed close titration of the desmopressin, fludrocortisone and sodium chloride supplementation. she was eventually weaned off treatment and discharged well with normal sodium levels and with no neurological deficits. this case highlights the difficulty encountered in managing concomitant cerebral salt wasting and diabetes insipidus in critically ill neurosurgical patients and the need to for a high index of clinical suspicion, early intervention and close monitoring. levetiracetam is a commonly used antiepileptic drug (aed) used to treat epilepsy. this agent was approved by the fda in , is available in oral and intravenous formulations, and offers advantageous pharmacokinetics, minimal drug interactions, and a favorable side effect profile. the purpose of this case report is to describe a case of severe, asymptomatic rhabdomyolysis exacerbated by levetiracetam administration. the medical record was reviewed and data was collected to describe a case with a pertinent review of the literature. a -year-old african-american male with a history of hypertension presented to the emergency department following a tonic-clonic seizure. baseline labs were drawn and revealed a ck level of , iu/l, negative urine myoglobin and normal renal function. levetiracetam therapy was initiated and no further seizures were noted. the patient's ck continued to trend up throughout his stay despite aggressive fluid resuscitation with a positive myoglobin on hospital day . the ck reached a peak of , iu/l on hospital day . after a literature review and evaluation of his medication list, six casereports were identified linking elevated ck and rhabdomyolysis to levetiracetam administration. at that time levetiracetam was discontinued and the ck rapidly declined to , iu/l on hospital day . the patient never had muscle pain or kidney injury and was discharged on hospital day . this case-report describes rhabdomyolysis associated with levetiracetam administration with a naranjo probability scale score of indicating a probable adverse drug reaction. the adverse effects of generalized pain and neck pain are described in the package insert with an incidence of - %; however, it is not reported that ck levels were monitored. due to the frequent use of this aed and given the rare yet serious adverse effect of rhabdomyolysis, ck levels should be monitored upon initiation. acute toxic leukoencephalopathy (atl) is a potentially reversible disturbance to white matter caused by exposure to toxins. we report the first case of a patient with atl in the setting of a fentanyl overdose and reviewed the literature. a year-old man with a history of opiate abuse was found unconscious, last seen well nine hours prior. he was known to have purchased mg of fentanyl that day. he was intubated and briefly required blood pressure support. he was initially hypoglycemic and suffered fulminant liver damage, acute kidney injury, rhabdomyolysis, and stunned myocardium. comprehensive toxicology screen was positive for cannabis and fentanyl. mri of the brain showed pronounced bilateral restricted diffusion in the high frontoparietal subcortical white matter with radiographic stability five days later. he remained intubated and neurologic exam poor with fluctuating brainstem reflexes and posturing despite improvement in end-organ function. atl has been reported in a -month-old girl and an -year-old man with exposure to transdermal fentanyl, both of whom had favorable outcomes ( , ). one case has been reported following oral oxycodone ingestion ( ). of cases of atl secondary to inhaled heroin, % were fatal ( ). preferential white matter injury has been seen in cases of hypoxic ischemic encephalopathy (hie) ( , ). it was initially thought to be secondary to wallerian degeneration following grey matter damage, but post-mortem pathology has shown direct insult to axons ( ). atl has been reported in one case of hypoglycemic coma ( ) and one case of uremia ( ). it has never been reported in isolated hepatic encephalopathy, secondary to seizure, or with cannabis use alone. based on our review of the literature, the most likely causes of this patient's atl are fentanyl or hie. fentanyl should remain on the differential as a previously unreported cause of atl. autonomic dysregulation is a common complication of acute spinal cord injury (sci). subsequent hypotension may worsen central nervous system injury as well as neurologic and mortality outcomes. to help mitigate this occurrence, consensus guidelines recommend maintaining patients' mean arterial pressure (map) > mmhg within the first seven days based on evidence from limited clinical trials. limited data exists describing the use of midodrine, an alpha- agonist and the previously only available enteral vasopressor, for blood pressure (bp) augmentation in this setting. the use of midodrine is limited by cardiovascular side effects such as bradycardia. droxidopa, a novel enteral precursor of norepinephrine that works independently of the central nervous system, may serve a role in sustaining map in acute sci. we describe a novel case of droxidopa use in a -year old male who sustained a spinal cord contusion secondary to severe stenosis at the fourth cervical vertebrae following a ten-foot fall. droxidopa was used to facilitate vasopressor wean in the setting of neurogenic shock as a complication of acute spinal cord injury. to sustain adequate cns perfusion (map goal > - mmhg) and facilitate patient transfer to a lower level of care, droxidopa mg three times daily was initiated after five days of continuous infusion of intravenous norepinephrine. daily assessments of hemodynamic parameters were performed, including blood pressure, heart rate, map, and an electrocardiogram. successful wean of norepinephrine was achieved within hours of droxidopa initiation, with an average map sustained above mmhg. the patient was transferred to a lower level of care within hours of droxidopa initiation. no cardiovascular side effects were observed. droxidopa was well tolerated and facilitated transition from norepinephrine infusion to an enteral option. droxidopa may be a viable option in stable neurocritical care patients who require vasopressors to sustain adequate cns perfusion. traumatic brain injury is acute and sometimes rapidly aggravated during or after surgical treatment. imaging study is most important and computed tomography (ct) is the golden standard in tbi. however the patient should be transfer to ct room or relatively high cost mobile ct scanner may be used. ultrasound is not expensive and also does not produce radiation exposure. we studied the effectiveness and advantages of intra-operative ultrasound examination in traumatic brain injury patients intra-operative ultrasound was used after decompression of injured brain from june to april . the ultrasound device was the affiniti (philips ultrasound inc, usa) and . mhz transducer was used. the transducer was covered by thin transparent sterilized vinyl with ultrasonic gel with aseptic manner. to protect brain injury by the ultrasonic probe, a saline soaked gauze was applied on the cerebral cortex. the axial images were captured and then stored in pacs system promptly. ultrasound images were compared to postoperative ct scan. there were male and female patients were examined by ultrasound during there surgery. ipsilateral hemisphere, especially cortical layer was slightly distorted to identification. brain stem area was visible in most cases. contralateral hemisphere was seen in unilateral craniotomy and craniectomy cases. in bilateral craniectomy cases, both hemispheres were observed well. parenchymal hemorrhage was also identified and confirmed for removal using ultrasound. in severe brain swelling cases, arachnoid space was seen increased echogenicity. ultrasound image was compared to postoperative ct scan. intra-operative ultrasound is effective in real time inspection of brain during surgery and may helpful detect opposite or parenchymal hemorrhage before closure and leaving operation room. to describe a rare case of a varicella zoster virus (vzv) meningitis with progressive multiple cranial nerve deficits in the absence of cutaneous zoster rash. a young woman with idiopathic thrombocytopenic pupura on steroids presents with horizontal diplopia in the setting of seven days of intractable headache. she had no meningeal signs, fever, leukocytosis or cutaneous rash. within three days into hospitalization, she developed bilateral cn vi, cn iii, right cn v and right cn vii palsies in a progressive fashion. csf analysis revealed cell count of , /mm , a protein of mg/dl and glucose mg/dl. cytology, tuberculosis, bacterial and fungal cultures, ace and hiv testing were negative. vzv-dna was detected in csf in high titers vzv quant: . million. contrasted brain mri revealed mild diffuse leptomeningeal enhancement in the basilar region. she recovered almost all cranial nerve function within days of treatment with acyclovir and high dose steroids. a diagnosis of polyneuritis cranialis with zoster sine herpete (zsh) was made given pcr positive vzv-dna in csf. vzv reactivation with a wide array of neurological deficits can present without rash making diagnosis challenging. zsh should be in the differential for acute cranial nerve deficits as prompt treatment with acyclovir can lead to rapid recovery. stress-induced cardiomyopathy or neurogenic stunned myocardium is a well-documented cardiac complication following aneurysmal subarachnoid hemorrhage (sah). onset is usually immediate, within hours after aneurysm rupture, and is characterized by left ventricular dysfunction with pulmonary edema and elevation in cardiac biomarkers. this can often be mistaken for an acute myocardial infarction or ischemia. the pathogenesis appears to be the result of elevated catecholamine levels following injury leading to myocardial contraction band necrosis and cardiac dysfunction. this syndrome occurs more commonly in patients with severe or "high-grade" sah. we review a case of delayed cardiac dysfunction coinciding with the onset of vasospasm. a -year-old female presented with a h&h , mf sah. she appeared to have lost consciousness prior to arrival and was reporting worst headache of life. she had an evd placed upon arrival with opening pressure at . she underwent endovascular coiling of a ruptured aneurysm of her anterior communicating artery aneurysm. initial echocardiogram demonstrated normal wall motion with ef of %, and minimal troponin i elevation at . ng/ml. on post-bleed day the patient became more somnolent and developed chest pain with an ecg demonstrating st-elevation in all anterolateral leads concerning for acs. she was taken for cardiac catheterization where she had non-obstructive vessels with no vasospasm seen. her ef was reported at - % with apical ballooning present. her repeat echocardiogram also demonstrated a new apical akinesis with ef %, and troponin peaked to ng/ml. her tcds at the time were suggestive of vasospasm with bilateral lr > , but no focal deficit present. it appears that regardless of timeline, stress-induced cardiomyopathy or neurogenic stunned myocardium occurs after sympathetic or catecholamine surge and may occur after the onset of vasospasm in patients with aneurysmal sah. the rapid neurological assessment of critically ill patients with neurologic disease is paramount when determining a course of action. neuromuscular blockade is often used during critical care transport and in the emergency department. unfortunately, this can delay examination and assessment leading to unnecessary testing and procedures. historically, neuromuscular blockade reversal was accomplished using a combination of neostigmine and glycopyrrolate. however, this can lead to incomplete reversal and unwanted side effects from these medications. sugammadex is a cyclodextran injectable compound that has been fda approved in the united states since for rapid reversal of rocuronium induced neuromuscular blockade. sugammadex works by forming a complex with rocuronium and rendering it unable to bind to nicotinic cholinergic receptors at the neuromuscular junction. sugammadex can reverse neuromuscular blockade without the unwanted side effects of cholinesterase inhibitors. this is a case report of the successful use of sugammadex to reverse the effects of neuromuscular blockade in an intracerebral hemorrhage patient. a year old male with a history of atrial fibrillation and a supratherapeutic inr presented via aeromedical ambulance with a ml left frontal intracerebral hermorrhage causing a mm midline shift. he received a mg bolus of rocuronium prior to arrival and had a gcs of upon presenting to the neurosciences icu. a train-of-four revealed / twitches. he was given mg/kg of sugammadex with a return of / twitches within seconds. a more accurate neurological examination was then obtained demonstrating that his brainstem reflexes were intact, he could open his eyes spontaneously and reacted purposefully to painful stimulation. this allowed a non-operative course to be taken. sugammadex can reliably and quickly reverse neuromuscular blockade allowing for the immediate assessment of the neurocritical care patient. it is a useful tool with minimal side effects. piperacillin-tazobactam is commonly deployed as empiric antibiotic therapy. piperacillin-induced hematologic laboratory test abnormalities were rare in pre-marketing studies, and whether these alterations are of clinical significance has been studied little. aberrations in platelet function have not been implicated. in the present case, we discuss a patient presenting with hypertensive intracerebral hemorrhage (ich) who sustained two additional hemorrhages in distinct locations after routine removal of intracranial monitors and an external ventricular drain (evd). these significant bleeding events occurred exclusively during piperacillin-tazobactam therapy and were correlated with new abnormalities in the patient's platelet function assay (pfa) results. a -year old vietnamese male with hypertension presented for treatment of a left basal ganglia ich. epinephrine/collagen and adenosine diphosphate/collagen pfas at the time of evd and quad-lumen bolt placement were normal, and imaging showed no hemorrhage after placement. hospital course was complicated by aspiration pneumonia requiring empiric piperacillin-tazobactam administration. after removal of the quad-lumen bolt and evd on separate days, both follow-up ct scans showed new hematomas in the devices' tracts, with significant intraventricular hemorrhage. repeat pfas were abnormally prolonged, representing a distinct change from baseline. a trend toward normalization of pfas was observed after discontinuation of piperacillin-tazobactam with progression toward baseline thereafter. the present case is unique in that the significant bleeding that occurred was attributable to objectively confirmed platelet dysfunction rather than thrombocytopenia. other possible innate causes of bleeding were less likely as the patient demonstrated normal platelet count, von willebrand multimers, platelet morphology, and clotting factors. this is the first reported case of intracranial (periprocedural) hemorrhage potentially related to piperacillin-tazobactam; further research into this drug's impact upon qualitative platelet function is needed. the life-saving potential of extracorporeal membrane oxygenation (ecmo) has been well recognized since the s. modern advancements of research and technology have allowed ecmo to be accepted as a dependable intervention for patients with severe pulmonary or cardiac failure. however, with increased use, associated complications that detract from the benefit of ecmo are surfacing as well. this case report describes a case of diffuse intracerebral hemorrhage (ich) after prolonged ecmo resulting in cerebral edema, mass effect, and eventual brain herniation. the patient is a previously healthy year old female who presented with fever, chills, and myalgia. when evaluated at urgent care, she was noted to be hypoxic and was sent to an outside hospital where her monospot test was positive. upon arrival, the patient was placed on venovenous ecmo (vv-ecmo) due to severe hypoxia. she was also in acute renal failure requiring continuous renal replacement therapy (crrt). she had an episode of hypotension with bradycardia. subsequently, her pupils were noted to be fixed and dilated. a stat ct head then showed diffuse bilateral hemorrhages at the graywhite junction as well as diffuse edema. labs showed thrombocytopenia likely due to disseminated intravascular coagulation (dic). her exam was consistent with brain death. it has been estimated that up to % of patients who were placed on ecmo as a last resort for respiratory failure have neurological complications including ich. there is no stereotypical pattern of bleeding but diffuse hemorrhage has been seen, which is consistent with the pattern seen in our patient. notably, those with ich have significantly higher rates of mortality. thrombocytopenia, dic, and platelet dysfunction that develop as a result of ecmo are thought to play a role in the development of ich. to present a case report of syndrome of the trepheined (sot) and paradoxical herniation without craniectomy. sot is reported when a constellation of positional neurological symptoms arise following large craniectomy, resolving in a delayed fashion following cranioplasty. paradoxical herniation may occur in extreme cases.the pathophysiology is incompletely understood however proposed mechanisms include compression of underlying brain by the flaccid skin flap due to the gradient between atmospheric and intracranial pressure exacerbated by upright pressure, changes in cerebral blood flow, and csf fluid. a middle aged woman with a history of mood changes eight months preceding admission presents with worsening left hemiplegia over one week. mri revealed a x mm right frontal cystic mass. hyperosmolar therapy and steroids were initiated for midline shift and brainstem compression. her immediate post operative course after tumour excision was uncomplicated. on post-operative day two, she developed uncontrolled hypertension, worsening anisocoria, and decerebrate posturing requiring urgent intubation. head ct revealed uncal and subfalcine herniation despite a large resection cavity. an external ventricular drain was placed and removed due to lack of drainage. within hours of trendelenburg positioning, she improved both clinically and radiographically. she did not undergo an intraoperative csf reduction and no preadmission history (back pain, orthostatic headache, trauma) to support an occult csf leak. she had a recurrence of symptoms on post-operative day eight which also resolved upon lying flat for hours. she was ultimately discharged to acute rehab and tumor pathology returned as glioblastoma (who grade ). this novel case of sot in the absence of craniectomy demonstrates the complex and poorly understood consequences of slow growing massive tumors, csf dynamics and exertional force on static cns structures. this case also illustrates the benefits of a collaborative, multidisciplinary approach to patient care in the neuroicu. to present a lesser known leukoencephalopathy that occurs when patients overdose on inhaled heroin vapor 'chasing the dragon" is a method of inhaled heroin vapor that is different from smoking or snorting heroin. heroin powder is placed on aluminum foil, which is heated by placing a flame underneath. the white powder turns into a reddish-brown gelatinous substance that releases a thick, white smoke, which resembles a dragon's tail. the fumes are "chased" or inhaled through a straw or small tube. currently the us is facing a growing epidemic of heroin use making this leukoencephalopathy more pronounced. a -year-old female with history of drug abuse presented to the emergency department with altered mental status. the boyfriend informed staff that she likely smoked heroin. on arrival, she was drowsy but easily arousable. her brainstem reflexes were intact but she was grossly dysmetric. urine drug screen was positive for opiates only. initial ct of the brain demonstrated extensive loss of gray-white differentiation within the cerebellar hemispheres and bilateral lucency in the globus pallidus and developing hydrocephalus. patient was placed in the neurointensive care unit to monitor and was managed medically with hypertonic therapy to combat her cerebral edema. an mri was done which demonstrated a distinctive pattern of symmetrical white matter t hyperintensities in the cerebellum, hippocampus and internal capsule bilaterally characteristically known as "chasing the dragon" sign. the patient gradually improved with supportive treatment, but continued to have mild ataxia upon discharge. we present a case of leukoencephalopathy that was generally rare to see, but now that heroin use is now at a year high within the us, this phenomenon may become more prominent. heroin inhalation leukoencephalopathy should be suspected in all patients with a history of chasing the dragon when they present with neurological abnormalities. the use of intra-venous (iv) thrombolysis for the treatment of acute ischemic stroke is now the standard of care. this is typically followed by endovascular thrombectomy if patient is eligible does not improve . we present a rare acute ischemic posterior circulation stroke that had progression of the stroke despite receiving both intra-venous thrombolysis and endovascular thrombectomy. case report: a years old african-american gentleman with past history of obesity, sleep apnea and prostatic hyperplasia, presented with acute onset left hemiparesis, with limb ataxia, who then progressed to altered sensorium in the emergency room needing endotracheal intubation. his initial nihss was . he was given iv thrombolysis, with subsequent vascular imaging that showed a top of the basilar clot, that was removed via endovascular intervention. a sister and one of the aunts reported a history of 'clots' when asked about family history. despite initial improvement, the patient deteriorated clinically after about hours from symptom onset, and was found to have extension of stroke into the brainstem, with simultaneous acute loss of brainstem reflexes . the patient was started on palliative withdrawal of care by the family about days from the initial onset of symptoms. his thrombophilia work-up revealed later that he was homozygous for methylenetetrahydrofolate reductase (mthfr) gene mutation, c >t. this case with a poor outcome due to extension of the ischemic stroke despite receiving the standard of care therapy, highlights the need for considering the use of anticoagulation within hours postthrombolysis and thrombectomy in cases with underlying thrombophilia. the current guidelines do not support this aggressive approach. there is a dire need for randomized controlled trial about such cases to provide evidence based care to avoid a repetition of a similar poor outcome. barbiturate therapy has shown benefit in reducing intracranial pressure (icp) in patients who are refractory to other treatment modalities. however, severe adverse drug effects can accompany barbiturate use when used at the high doses required for icp management, such as hypotension, hepatic/renal dysfunction, and infection, among other deleterious consequences. dyskalemia has been reported infrequently in the literature with most of the cases involving patients on thiopental. there remains little guidance for management of this adverse effect. we present a case of severe dyskalemia induced by high-dose pentobarbital therapy and experience with management of this rare but life threatening effect. the patient was a -year-old male with traumatic brain injury and subdural hematomas complicated by refractory icp elevations. after hyperosmolar therapy, sedation, and csf drainage failed to control icp, and he was deemed to not be a candidate for surgical decompression, high-dose pentobarbital was started. after initiation of pentobarbital, his initial potassium of . mmol/l decreased to a nadir of . mmol/l over the next hours despite aggressive repletion with a total of meq of oral and intravenous potassium chloride. upon down-titration and discontinuation of pentobarbital, the serum potassium rapidly rebounded to . mmol/l with st-segment elevations on ekg. pentobarbital was restarted in an attempt to stabilize escalating icps and elevated serum potassium. subsequently a slow taper was utilized to mitigate dyskalemia during barbiturate discontinuation. dyskalemia associated with high-dose barbiturate therapy presents a significant dilemma to practitioners as both severe hypo-and hyperkalemia can be life threatening. published literature provides little guidance on how to safely manage patients who experience this adverse effect. patients receiving barbiturate therapy should have frequent potassium monitoring especially in the initiation and discontinuation phases. potassium repletion should be approached with caution, especially preceding discontinuation of barbiturate therapy. diffuse astrocytoma (formerly known as 'gliomatosis cerebri') may present with seizures or symptomatic raised intra-cranial pressure. this is typically followed by a few months of relatively stable phase (with treatment) and then possible subsequent development of glioblastoma multiforme. we present a rare case of a previously healthy caucasian lady who had new onset seizures, that showed glioblastoma multiforme already present on a background of diffuse astrocytoma. case report: a years old caucasian lady with no significant past medical history was admitted with new onset focal seizures with secondary generalization, needing intubation and propofol for airway protection. brain imaging showed left frontal ring-enhancing mass, with a smaller satellite lesion in the left insular cortex, on a background of diffuse infiltrative lesion involving left fronto-temporal lobe and a smaller area of right parafalcine frontal lobe. biopsy of the left frontal mass revealed it to be glioblastoma multiforme. this is a rare situation when a previously healthy patient presents with new onset seizures and already has glioblastoma multiforme on a background of diffuse astrocytoma (or 'gliomatosis cerebri'). her post operative imaging revealed disease progression with increase in the size of the left insular cortical lesion. she was discharged home with plan for radiotherapy and chemotherapy. diffuse astrocytoma with glioblastoma multiforme within can remain asymptomatic till late in the disease course. diffuse astrocytoma (or 'gliomatosis cerebri') is a rare disease and even more rare is to have this remain asymptomatic till the development of glioblastoma multiforme within. this particular case highlights the need for vigilance about such a possibility, as this aggressive brain tumor carries a grave prognosis, especially when it develops on background of a diffuse astrocytoma. subdural hygromas (sdg) are cerebral spinal fluid collections in the subdural space that may occur following trauma. decompressive craniotomy may increase the risk for acute sdg or other forms of external hydrocephalus along the surgical plane. while these are traditionally benign and resolve spontaneously, they may in rare cases cause clinical deterioration. we report three cases. cases and were alcoholic men aged and , respectively, who suffered severe traumatic brain injury (tbi) following falls while intoxicated. they had early clinical deterioration prompting emergent hemicraniectomy for left-sided sdh with midline shift (mls). case clinically worsened on postoperative day (pod) with posturing, decreased pupillary responses, and new-onset seizures. new bilateral, extensive subdural hygromas were noted, enlarging over serial ct scans up to -cm with progressive mass effect. uncal herniation and downward brainstem displacement occurred by pod despite external ventricular drainage. case deteriorated on pod with fluctuating exam and newonset seizures. imaging revealed new subgaleal fluid collection measuring . -cm and a contralateral sdg. on pod , hemicraniectomy was performed for new mls from enlarging fluid and hemorrhage in extradural component. both died shortly after withdrawal-of-care. case was a year-old man with dural arteriovenous fistula who presented with spontaneous left-sided sdh and underwent left hemicraniectomy. on pod , he had new-onset seizures and new bilateral sdg measuring . -cm on the left and . -cm on the right without mass effect. two days later; the right sdg grew to . -cm causing significant mass effect. he recovered after burr-hole evacuation and temporary subdural drain placement. sdg following sdh evacuation can have a malignant course, causing clinical deterioration without prompt recognition and csf diversion. all patients had large volume sdh and two were alcoholic; larger prospective cohorts are required to identify risk factors. seizures may be an early clinical sign. moyamoya disease is an intracranial vasculopathy that results in stenosis of bilateral internal carotid arteries with subsequent development of extensive collateralization. the diagnostic criteria for moyamoya disease are well established and generally accepted, yet reaching the diagnosis can be challenging in some cases. herein, we present an unusual case of progressive cerebral vasospasm triggered by pituitary apoplexy that led to a delay in the underlying diagnosis of moyamoya disease. case report. a -year-old female with hyperlipidemia presented to the emergency department with a bifrontal headache, right-sided weakness, and dysarthria. ct angiogram showed extensive multifocal narrowing of the bilateral supraclinoid icas, proximal aca/mcas, and posterior circulation. mri brain revealed a left insular stroke as well as a sellar mass with a central hemorrhagic component. mr perfusion demonstrated decreased perfusion in the right hemisphere. lumbar puncture and extensive vasculitic workup was unremarkable. endocrine studies were notable for elevated prolactin with low fsh and lh levels. despite protracted blood pressure augmentation strategies, the patient continued to experience progressive infarcts in the left mca/aca territory. repeat ct angiogram showed progression of vasculopathy, and transcranial doppler studies demonstrated worsening vasospasm of the right mca and left pca arteries. the patient received corticosteroids given concern for apoplexy, and was maintained on aspirin and verapamil. given the aggressive nature of her vasculopathy, the patient underwent conventional angiography two weeks later, which revealed bilateral suzuki grade iii moyamoya. following this diagnosis, she received bilateral sta-mca bypass surgeries. it is important to revisit the differential diagnosis of cerebral vasospasm when the clinical course does not conform to expectations. this case highlights moyamoya as the causative agent in progressive vasculopathy likely masqueraded by pituitary apoplexy and concomitant vasospasm. moyamoya is an important diagnosis to consider in patients with a fulminant vasculopathy refractory to traditional treatment of vasospasm. visualization of intracranial structures by ultrasound in adults is limited by the presence of skull, though ultrasound imaging can occur through temporal windows. point of care ultrasound allows assessment of midline shift, brainstem, and ventricles. doppler allows visualization of cerebral perfusion patterns. patients with a hemicraniectomy have better temporal windows available since a portion of their skull has been removed. in such patients, ultrasound can provide a non-invasive method to serially assess midline shift, intracranial hematomas, and focal ischemia at the bedside. we present images of a cranial ultrasound that shows remarkable anatomical details that correlate well with computed tomography (ct) head. a year-old male presented with right-sided weakness and confusion and was found to have a left parietal intraparenchymal hemorrhage with cerebral edema and left-to-right midline shift on ct head. increase in cerebral edema and expansion of the hematoma caused clinical neurological decline necessitating a left-sided hemicraniectomy with clot evacuation. a cranial ultrasound was performed two days after surgery to assess for progression of cerebral edema and intracranial hemorrhage. a transtemporal approach in axial plane was used to visualize intracranial structures through the craniectomy window. physiological structures like the falx cerebri, lateral ventricles, midbrain, mammillary bodies, choroid plexus, splenium of corpus callosum, thalami, and circle of willis were visualized with incredible anatomical detail. pathology such as intracranial hemorrhage, focal ischemic areas, vasogenic edema as well as encephalomalacia were identified with close correlation to the noncontrast head ct. the patient is currently recovering in the neurocritical care unit with supportive care. cranial ultrasound has potential applications in point of care assessment of intracranial pathology in neurocritical care patients. this application has promising use in directing therapy in patients who are otherwise unstable for transport or are unable to undergo neuroimaging secondary to positioning needed for management of cerebral edema. cerebral mucormycosis is a rare infection caused by fungi found in soil and decaying vegetation. the rhino-orbital-cerebral type is classically associated with aids, diabetes, malignancy and immunosuppression. we observed a series of young immunocompetent patients who presented with a fulminant form of isolated cerebral mucor associated with severe meningoencephalitis, parenchymal necrosis and symptomatic cerebral edema. six patients with histopathological diagnosis of cns mucormycosis admitted to the university of cincinnati neurocritical care unit between and are presented. patient ages ranged from - (median ). none had diabetes or hiv. drug use (intravenous and intranasal) was confirmed in patients. they presented with altered mental status ( ) and focal neurologic deficits ( ). four patients presented with fever and leukocytosis. mri revealed lesions in the basal ganglia ( ) or cerebellum ( ) which were characterized by t hyperintensities with patchy restriction and susceptibility signal. contrast enhancement was present in patients. mass effect ( ) and midline shift ( ) were prominent. mechanical ventilation was required in four patients. all but one patient received amphotericin b. three died from intractable intracranial pressure (icp). one patient eventually gained functional independence, one still requires high level of care, and one was lost to follow-up. csf analysis was negative for mucor in all cases. fulminant cerebral mucormycosis should be considered in every young patient presenting with rapid onset meningo-encephalitis and necrotized cerebral lesions, especially if located in the basal ganglia. history of ivdu should raise further suspicion. these patients should be monitored in intensive care settings as they can rapidly develop malignant cerebral edema and increased icp. antifungal therapy should be initiated upon presentation as it has been shown to improve morbidity and mortality. the incidence of acute ischemic stroke in the immediate post-partum period ranges between - % and is considered a serious cause of morbidity and mortality. pregnant or postpartum women are less likely to receive iv tissue plasminogen activator (tpa) primarily because of pregnancy, ongoing peripartum bleeding and/or recent delivery. the fda classifies tpa as a category c drug and current recommendations consider pregnancy a relative contraindication for receiving tpa. we present two cases of peripartum ischemic strokes with varying ischemic stroke time windows requiring aggressive revascularization therapy (endovascular and pharmacologic). a y g p presented to an outside hospital days post-partum with new onset of facial droop and left upper extremity weakness (nihss ). imaging showed right m cutoff and occlusion of several m branches. the patient was not a candidate for tpa given ongoing vaginal bleeding. the decision was made to proceed with mechanical thrombectomy when her exam worsened to nihss . the thrombectomy was successful with tici c reperfusion. she was discharged home days later with a nihss of zero. a y g p presented days post-partum with new onset of left facial droop and slurred speech (nihss ). imaging showed right m cutoff with reconstitution, but with significant associated penumbra. acute worsening of exam post tpa triggered a push for mechanical thrombectomy achieving a tici recanalization. post procedure the patient's only symptom was decreased sensation in left fingertips. at -day follow up the patient had returned to her baseline with a nihss of zero. endovascular and pharmacologic revascularization therapy should be considered on an individual basis in the peripartum population. current literature is limited to case reports /case series. larger multicenter trials are warranted and anticipated in the near future. while the optimal duration of burst suppression for status epilepticus (se) has not been established, burst suppression poses significant morbidity that may be dependent on the amount of time spent in burst suppression. herein, we report a case of se that resolved after ultra-short burst suppression. case report. a year-old female was admitted to the neuro-intensive care unit after experiencing several brief tonic-clonic seizures characterized by right-sided shaking and left-sided head turn. despite lorazepam and levetiracetam administration, the patient did not return to baseline and was transferred to our unit. on presentation, her workup revealed a leukocytosis and a glucose level > mg/dl. lumbar puncture showed a mild pleocytosis for which broad spectrum antibiotics were initiated. on initial examination, she was unresponsive and was not following commands. electroencephalogram (eeg) demonstrated frequent sharp and slow discharges in the right posterior quadrant with generalization (~ seizures/hour) with minimal improvement following levetiracetam and phenytoin administration. given the refractory nature of seizures, the patient was intubated and treated with general anesthetics. using propofol, burst suppression was achieved (consisting of - s bursts with intermixed suppressions) and was continued for < hours. following weaning, the patient had no further evidence of seizures, and eeg showed lateralized periodic discharges in the right occipital lobe. mri did not demonstrate an occipital focus, but did reveal cortical diffusion restriction in the bilateral posterior hemispheres. the patient was extubated the following morning, and was transferred to the neurology floor two days later. this case provides evidence that in certain situations, relatively brief periods of burst suppression in se can serve as a "reset switch", allowing for resolution of seizures while minimizing toxicities associated with prolonged burst suppression. further studies to determine which patients may benefit from ultrashort burst suppression are warranted. there are two systems of facial control, voluntary and emotional; these are independent up to the level of the facial nucleus. we described a case of a patient who presented with isolated emotional facial palsy after intracerebral hemorrhage (ich). retrospective review of a case admitted to the neurocritical care unit (nccu) of the johns hopkins hospital. a year-old woman with history of migraines who presented to the emergency room after a colleague noticed she was not moving the left lower side of her face when she smiled. head ct showed a large right frontal ich involving the medial frontal lobes and anterior thalami. on review of an old mri done, an underlying developmental venous anomaly with an associated cavernoma was seen. her exam was notable for a flattened emotional affect, no facial palsy when asked to activate on command, but a facial droop that occurred in the context of her smiling to jokes and other humor. her nccu course was complicated with significant brain edema requiring osmotherapy up to weeks out from the initial insult with self-limited episodes of brain herniation characterized by extensor posturing, dilated pupils, hypertension, hyperventilation and tachycardia. these were initially dismissed as sympathetic storming vs seizures as she will come out of those to her baseline (awake with mild left sided weakness) many times without therapy. she eventually required a hemicraniectomy two weeks after presentation. conclusions solated emotional facial palsy can be the presenting sign after ich when the hemorrhage involves the contralateral thalamus, of the striato-capsular region or the medial frontal lobes. in this case, transient icp elevations were leading to dilated pupils, tachycardia and hypertension -highlighting that heart rate changes can be variable with elevated icps and that in young patients, brain herniation episodes can be self-resolved with hyperventilation. yo female with no pmh developed fever, headache, and neck pain. she presented to outside hospital day after ct head was negative, patient was discharged. symptoms did not improve and she went to her pcp on day and was instructed to go to the ed. she presented to osh and underwent a lp that was indicative of viral meningitis with wbc cells/mm and protein mg/dl. patient admitted and treated with acyclovir. on day , she developed generalized body aches. on day , she was trying to stand with assistance and she became rigid. parents report a total of seizures and was intubated for airway protection. she underwent another lp on day with an opening pressure of cm h o. csf was sent for paraneoplastic panel. csf analyses and blood cultures were negative. evd placed for icp pressures of - cm h o. history obtained from mother and father who reported the patient had been hiking weeks prior. results mri brain showed meningeal enhancement scattered throughout the supratentorial and infratentorial brain and most compatible with inflammatory sequela of meningitis. patient continued on keppra, high dose steroids, antibiotic, antiviral, antifungal therapy until cultures resulted. additional treatments included ivig therapy followed by plasmapheresis, and finally rituximab. continued workup with brain biopsy showed demyelinating process and possible necrotizing encephalitis. mri four weeks after initial presentation showed white matter demyelination and deep gray nuclei lesions consistent with adem. four score of on admission improved to (e , m , b , r ) weeks after patient presented from osh. diagnosis of adem vs ms variant made based on the above data. case provides information for the clinician diagnosing and treating adem. potential for further studies with treatments described above and their effect on meaningful neurological outcomes. dengue is a flaviviruses transmitted via mosquitos and prevalent in south east asia. neurological complications are rare but can involve encephalitis, myelitis, neuromuscular dysfunction and neuroophthalmological problems. we describe an interesting case of dengue encephalomyelitis. retrospective review of a case admitted to the neurocritical care unit (nccu) of the johns hopkins hospital a year-old ship filipino captain with no significant past medical history but an extensive exposure to heavy metals, travel throughout the pacific, who presented with progressively worsening fevers, encephalopathy, urinary retention and tremors. he was transporting iron ore and other metals in a cargo ship from russia through south-east asia through to bermuda. while passing through the pacific, he began to experience malaise, myalgia, and fever. he was treated with amoxicillin but became worse, developing urinary retention, periods of confusion, and word finding difficulties. he was initially hospitalized in bermuda and then transferred to our hospital for further workup. given his rapid deterioration, he was initially in the nccu. his exam was notable for mild expressive aphasia, paratonias, right-sided weakness with hyper-reflexia, and a low amplitude tremor. his csf was notable for lymphocytic pleocytosis, elevated protein, low glucose. mri brain showed flair hyper-intensities in the frontal lobes, and diffusion restrictions in the bilateral basal ganglia and thalami. mri spine showed extensive flair hyper-intensity lesions. an extensive workup evaluated for heavy metal toxicities, autoimmune disorders and infectious workup. csf analysis came back positive for dengue igg and igm, leading to a diagnosis of acute dengue fever and encephalomyelitis. with supportive care in the nccu, he improved considerably over - weeks and was discharged home to the philippines. dengue encephalomyelitis is a rare infection but should be considered in patients living in endemic areas. treatment includes supportive care with fluid resuscitation, neurological monitoring and monitoring for hemorrhage. posterior reversible encephalopathy syndrome (pres) is known to cause altered mental status and leukoencephalopathy in the setting hypertensive emergency. we present a novel case of severely asymmetric pres due to a concurrent right transverse sinus dural arteriovenous fistula (davf). a year-old woman with hypertension, non-compliant on medication, had fatigue and weeks of intermittent left sided weakness when she presented to an outside hospital for evaluation. initially upon arrival her glascow coma scale (gcs) was . her mental status deteriorated over hours, eventually requiring intubation. her peak blood pressure was / . outside ct demonstrated scattered intracerebral hemorrhage (ich) and she was transferred for higher level of care. on admission her gcs was . review of her outside ct was remarkable for extreme right-sided white matter hypodensity, moderate left white matter hypodensity, and small scattered ich. workup including infectious, inflammatory, and neoplastic processes were excluded through serum, csf studies, and mri. conventional angiogram demonstrated a right transverse sinus davf with reflux into cortical veins, which was subsequently embolized. her white matter t -weighted hyperintensities improved on follow-up mri, and her gcs was at the time of discharge. our case highlights the possibility of asymmetric pres due to abnormal venous congestion due to the right-sided davf. venous hypertension likely caused the patient's intermittent left sided symptoms in the weeks prior to admission. few cases of unilateral or asymmetric pres have been reported following induced hypertension for treatment of subarachnoid hemorrhage or in the setting of vascular malformation. to our knowledge, this is the only case of severely asymmetric pres and preceding stroke like symptoms due to a davf. the most common pathology associated with an intraluminal carotid thrombus is underlying atherosclerosis. in rare cases it may be associated to thrombocytosis. currently there are no clear recommendations for the treatment of ischemic stroke associated with thrombocytosis. our case describes the use of plateletpheresis for the acute management of thrombocytosis complicated by an internal carotid artery thrombus resulting in a right mca stroke. a -year-old female with past medical history of menorrhagia who presented complaining of left face, arm and leg weakness with associated shortness of breath. upon arrival her nihss was and the initial head ct was unremarkable. laboratory results revealed a hemoglobin . mg/dl, hematocrit mg/dl, and platelet count of x /ml. she was not a candidate for thrombolytic therapy due to the time window. soon after admission she had acute worsening of symptoms with an nihss of . a cta of the head and neck showed acute ischemic infarction involving the right mca territory with non-occlusive intraluminal thrombus within the right carotid bulb. asa mg and heparin infusion were initiated promptly. after a thorough work-up for thrombocytosis, reactive thrombocytosis secondary to iron deficiency anemia was diagnosed. plateletpheresis as well as oral ferrous sulfate were started. after one plateletpheresis cycle the platelet count stabilized at x /ml. complete thrombus resolution was confirmed on follow-up cta on day of admission without need for surgical revascularization. the role for plateletpheresis is not well established in secondary thrombocytosis. in cases with extreme thrombocytosis immediate surgical thrombectomy may be contraindicated due to high risk of rethrombosis. urgent cytoreduction with correction of the putative mechanism for thromboyctosis should be undertaken for optimal management. plateletpheresis is safe and efficient in reducing the platelet count to decrease the risk of clot progression or further clot formations which could worsen patient outcome. hyperpyrexia is an elevated core body temperature secondary to an elevated hypothalamic set temperature. hyperthermia is an elevated core body temperature beyond the normal hypothalamic set temperature. intracranial hypotension can present with a wide variety of symptoms ranging from orthostatic headache up to coma. it has never been reported to present with fever, namely hyperpyrexia. a case report of a year old female patient with a history of depression, diabetes mellitus, hypertension, and angiogram negative subarachnoid hemorrhage status post ventriculo-peritoneal (vp) shunt placement six years ago who was complaining of worsening headaches and slurred speech for the past three months but acutely decompensated one morning. she suddenly became confused and agitated but became obtunded. initially, she was given haldol. she was found to be febrile (rectal temperature of . f). she was given dantrolene and bromocriptine for suspected malignant neuroleptic syndrome with no effect. creatine phospho-kinase was not elevated. she underwent infectious work up which later came negative. cooling measures like external cooling, peripheral iv cooling, tylenol and nsaids were also not helpful. fever responded to central intravascular cooling but encephalopathy did not. several expert attempts of lp and shunt tapping failed to obtain csf. brain imaging showed bilateral chronic symmetrical hygromas, diffuse pachy-meningeal thickening and enhancement, slit-like ventricles and slumping of the midbrain with closure of the mammillary pontine distance. following shunt setting adjustment, the encephalopathy markedly improved and the fever did not recur after stopping the cooling measures and antimicrobials. intracranial hypotension might present with hyperpyrexia, likely secondary to hypothalamic dysfunction. in our case, hyperpyrexia was reversible as the intracranial hypotension was emergently treated. nevertheless, spontaneous intracranial hypotension might be difficult to diagnose especially if it presented with non-classical symptoms like fever. complex emotions about critical illness can affect families in the icu. rightfully, we put focus on how they are impacted, but we also need to pay attention to how it can affect providers and our decision making. a poignant case from my training was a -year-old girl struggling with lupus. she had now developed lupus cerebritis and had massive intracranial hemorrhages. despite aggressive efforts to manage cerebral edema, she repeatedly herniated brain matter out of old craniotomy scars with incredible force. it was the most horrifying thing i've ever seen. other organs were also failing, with four consulting services working to salvage them unsuccessfully, prompting numerous procedures. this went on for a month. the therapies that we can offer have limits from a physiological standpoint which we must recognize and respect. we struggle with reconciling the interventions we feel compelled to implement versus what is realistic. i remembered the most valuable advice that i once received: "only do something to someone if it does the complexity of the neuro icu is amplified by the nature of intracranial catastrophes and poor recovery (in contrast to pure medical illness). providers cling to what is technically indicated while families cling to hope, but neither is enough and concurrently too much. we lose our autonomy to grieving families telling us to "do everything" losing sight of the bigger picture. we lose our autonomy to one another by pushing onwards, which can unintentionally push each other into the territory of doing more harm than good. something for them". all services began to share this view, thus slowly dialysis, steroids and immunosuppression stopped. eventually, her heart stopped. my experiences have reiterated a simple paradigm: to do no harm. through this, i can empower myself to take control of each situation by first taking control of myself. we report a case of an hiv positive patient who presented with cryptococcus gattii meningitis who then developed acute respiratory distress syndrome (ards) secondary to pneumocystis jirovecii pneumonia (pjp) that required ecmo support. ards in immunocompromised hiv positive patients is associated with extremely high mortality. ecmo can improve oxygenation in patients without increasing alveolar pressure and therefore avoid mechanical lung damage with ventilation. we present a patient with newly diagnosed aids with cryptococcus gattii meningitis and course complicated by pjp that progressed to severe acute respiratory distress syndrome (ards) for which veno-venous ecmo was initiated. patient is a year old male who presented to the emergency department with new onset seizures. lumbar puncture in the ed overflowed the manometer and demonstrated wbc , rbc , protein , glucose , positive yeast gram stain positive for yeast with pcr and ag positive. his cultures later grew out cryptoccoccus gattii. he was admitted to the nsicu and we placed a lumbar drain and an intraparenchymal ipc monitor that demonstrated elevated icps to the - mmh but improved with drainage. the day of admission he acutely desaturated and required emergent endotracheal intubation. chest x-ray demonstrated bilateral infiltrates. bal was positive for pj. five days following presentation and respiratory failure he was started on veno-venous ecmo. two days following initiation of pjp treatment with bactrim his chest x-rays and lung compliance began to improve. he remained on ecmo for a total of days before decannulation. he underwent induction chemotherapy for four weeks for meningitis. this case report demonstrates the use of ecmo in a complicated and critically ill patient with aids, pjp, and cryptoccous gattii meningitis. to our knowledge, few cases of ards secondary to pjp are reported and none are reported with concurrent cryptococcus gattii infection. sympathetic storming occurs during the acute care of patients following severe brain injury. cannabinoid cb receptors (cb r) mediate the effects of delta( )-tetrahydrocannabinol (thc), the psychoactive component in marijuana. expression of cb r is widespread in the central nervous system and includes the hypothalamus, which is thought to mediate the hypothermic inducing effects of cannabinoids. dronabinol is a synthetic analogue of thc we present a novel therapeutic use of cannabinoids in a case of super-refractory sympathetic storming following coccidioidal meningitis and extensive bilateral subcortical stroke a -year-old previously healthy man was transferred from an outside hospital for treatment of meningitis, vasculitis, and hydrocephalus requiring placement of a ventriculostomy. workup subsequently revealed coccidioidal meningitis. during hospitalization the patient had severe vasospasm, elevated intracranial pressure, diabetes insipidus, cerebral salt wasting, and severe sympathetic storming. intermittent storming episodes with high fever persisted for over weeks despite treatment with bromocriptine, dantrolene, tylenol, ibuprofen, phenobarbital, and sinemet. due to its mechanism of action, a trial of dronabinol mg divided twice daily was tried. the storming episodes ceased and within hours the average temperature decreased by about . degree centigrade. temperature over the next several days was better controlled with a substantial reduction in use of anti-pyretics, surface cooling measures, and other storming medications our case highlights a novel therapeutic use of cannabinoids in super-refractory sympathetic storming related to brain injury. dronabinol may be an alternative pharmacotherapy with unique mechanism of action in difficult to control sympathetic storming patients with poor grade subarachnoid hemorrhage(sah) commonly present with significant mental status changes that preclude reliance on neurologic exam for screening for neurologic deterioration. jugular venous oximetry monitoring has been suggested for use in guidance of hyperventilation therapy, barbiturate coma, and vasospasm monitoring. no studies are found in literature validating its use in sah. milrinone has been using for the treatment of vasospasm in sah in an established protocol in the montreal neurological hospital. this study was performed using multiple methods of monitoring, but not jugular bulb oximetry. we report one case with high grade subarachnoid hemorrhage complicated by vasospasm treated with milrinone using jugular bulb monitoring for dose titration. methods years old female presented with thunderclap headache and subsequently became comatose. noncontrast head computer tomography showed posterior fossa subarachnoid blood. she was intubated, external ventricular drain (evd) was placed and she was admitted to neurosurgical intensive care unit (nsicu). angiogram showed left posterior inferior cerebellar artery aneurysm and was successfully coiled. her hospital course was complicated by refractory symptomatic vasospasm. angiogram showed basilar artery vasospasm treated with intra-arterial verapamil. post procedure patient was not able to tolerate norepinephrine due to tachycardia and could not maintain hypertension on phenylephrine. milrinone was then started. jugular bulb catheter was place because the area at risk was not amenable to invasive multimodality monitoring. oximetry was monitored and her milrinone rate was titrated to goal of venous oximetry in the range of - %. on day , angiogram showed no more evidence of vasospasm. her exam was back to her prior poor baseline. subsequently, she was discharged to long term care facility. our case demonstrates the benefit of using jugular venous oximetry monitoring guidance for milrinone dose titration. further, it may be an effective tool is research studying treatments of cerebral vasospasm repetitive transcranial magnetic stimulation (rtms) is increasingly used in treatment of various conditions including depression, chronic pain, and movement disorders. the use of rtms for chronic management of medically refractory epilepsy has grown substantially in the last years. however, little literature exists on use of rtms for acute status epilepticus. the exact antiepileptic mechanism of rtms remains unclear, but may be secondary to inhibition of cortical excitability. we report promising response to rtms in a case of super-refractory focal status epilepticus. the study is a case report. a daily dose of pulses of hz rtms was applied to the left occipital lobe. treatment course was divided into periods of - consecutive days each for a total of days of treatment over days. a -year-old woman with recent hemiarthroplasty complicated by wound infection presented with acute unresponsiveness and right gaze deviation, evolving into fluctuating encephalopathy, word finding difficulty, and right hemineglect. eeg revealed persistent left posterior quadrant lateralized periodic discharges (lpds), at times evolving into electrographic seizures, and positron emission tomography demonstrated a co-localized hypermetabolic focus. mri revealed subtle bilateral occipital t hyperintensity without diffusion restriction, which later resolved; cerebrospinal fluid was noninflammatory. seizures continued despite treatment with multiple aeds, burst suppression, and empiric trial of high dose corticosteroids. the patient demonstrated abrupt electrographic and clinical improvement after rtms initiation. previously unseen brief periods of lpd resolution were observed within minutes after first tms session with further improvement in eeg background correlating with improvement in encephalopathy and clinical findings over subsequent days. given excellent safety profile, rtms may be useful transitional therapy in management of some cases of status epilepticus. durability of efficacy, patient selection, and optimal treatment schedules remain important unresolved questions. further study is required. central pontine myelinolysis (cpm) occurs due to rapid osmotic shifts causing demyelination in white matter, typically due to rapid correction of hyponatremia mostly in setting of alcoholism, malnutrition, and/or liver/renal dysfunction. sequelae may include cranial neuropathies, quadriparesis, seizures, and encephalopathy. no specific treatment exists; literature reports indicate favorable outcomes in only - % of patients. our patient is a year old male with hypertension, tobacco and alcohol abuse, admitted with severe aortic stenosis, complicated by alcohol withdrawal, pneumonia, and acute kidney injury. he was treated with benzodiazepines, broad spectrum antibiotics, and fluid resuscitation. on hospital day (hd) , he had to be intubated for airway protection due to acute confusion and quadriparesis. his blood work was notable for wide fluctuations in serum sodium, from on admission to on hd to on hd . otherwise, laboratory evaluation was remarkable only for mildly elevated ast and serum creatinine. mri brain days after symptom onset (hd ) showed dwi and flair hyperintensities around central pons bilaterally crossing midline. eeg showed severe generalized slowing. diagnosis of cpm was made and intravenous immunoglobulin (ivig) ( . g/kg/day for days) was initiated within days of symptom onset, on hd . after initiation of ivig, patient showed rapid improvement, first noted in the bilateral upper extremities. by hd i.e., days after initiation of ivig, he was able to be successfully extubated; and he had regained - / strength in all extremities. neuropsychology testing at month demonstrated intact cognition. we describe a case of rapid clinical improvement in cpm following treatment with ivig. in addition to ours, about similar cases have been reported, in which beneficial outcomes were demonstrated following prompt initiation of ivig. one proposed theory would be through reduction of myelinotoxic antibodies, thus promoting remyelination. few cases have reported central neuronal hyperventilation (cnh) secondary to infiltrative malignancy or autoimmune disease. the lesion is usually located at the pontine tegmentum and interrupts the fibers between the respiratory centers in the pons and those in the medulla. we report a case of a year old female with multiple comorbidities who was admitted to the neurocritical-care unit after intra-operative rupture of a mm distal basilar aneurysm while being electively coiled. an external ventricular drain (evd) was placed due to early signs of ventriculomegaly. the postoperative exam showed progressive encephalopathy, left > right hemiplegia progressive tachypnea (rate and depth) despite being on assisted mode ventilation leading to severe hypocapnia ( . mmhg) and compensatory renal acidosis (bicarbonate = . mmol/l) to maintain normal ph. attempt to sedate the patient led to severe metabolic acidosis. intraventricular nicardipine was started and the patient ventilator settings were changed to bi-level pressure control. transcranial doppler (tcd) showed markedly improved vasospasms. the patient respiratory rate and, to a lesser extent, the tidal volumes improved after several days. sedation was weaned off successfully. evd was successfully weaned off and removed. tcd and ct angiogram showed severed basilar artery vasospasm while mri done later showed bilateral tegmental midbrain ischemia. one case has reported acute central neuronal hyperventilation following left thalamic bleed while another reported chronic neuronal hyperventilation that was attributed to old bilateral lacunar thalamic strokes by exclusion. our case is the first to report central neuronal hyperventilation following aneurysmal subacrachnoid hemorrhage that got complicated by bilateral tegmental midbrain strokes. while respiratory centers are known to exist in the medulla and the pons, more recent articles have described networks that regulate breathing extending to the midbrain peri-acquiductal grey and possibly the thalami. our unique case supports this hypothesis. serotonergic and atypical antipsychotic drugs are often used in the critically ill in the treatment of posttraumatic depression and anxiety disorders. hyperactive delirium may mask serotonin syndrome, which carries high morbidity and mortality if left untreated. we describe a case of serotonin syndrome in a critically ill patient in the setting of surgical and neurocritical intensive care unit. a -year-old male with remote trauma presented with left upper abdominal pain. a ct-scan of abdomen showed left diaphragmatic hernia. he underwent left thoracotomy and repair of diaphragmatic hernia. his postoperative course was complicated by sepsis, ileus, and aspiration pneumonitis. he was started on sertraline and quetiapine for stress-induced anxiety disorder, depression and agitation. despite increasing doses of sertraline, patient became agitated, tremulous, and confused. physical examination included fever, tachycardia, hypertension, diaphoresis, dilated pupils, hyperactivity, and clonus. initially considered to be due to hyperactive delirium, these manifestations did not improve with haloperidol. neurocritical care was consulted. due to presence of hyperactivity, fever and clonus, serotonin syndrome was strongly suspected. sertraline and quetiapine was discontinued and cyproheptadine added. within -hours his symptoms improved and cyproheptadine was tapered over days. serotonin syndrome, a potentially life-threatening syndrome, is manifested by triad of mental status changes, neuromuscular and autonomic hyperactivity. a multitude of drug combinations can result in serotonin syndrome. serotonin syndrome is a diagnosis of exclusion, based on history and neurological examination in a patient taking serotonergic drug. ht- a receptors are most commonly incriminated along with high levels of norepinephrine.the keys to management include discontinuation of all serotonergic agents, supportive care, and cyproheptadine. cyproheptadine, a potent ht- a antagonist, is effective in ameliorating symptoms. a high suspicion for diagnosis is important for reducing morbidity and mortality associated with this neurologic syndrome in the critically ill. ruptured cerebral mycotic aneurysm as consequence of infective endocarditis (ie): a management qeeg adr in poor grade sah: is it really useful? recognize the various subtypes of cerebral amyloid angiopathy bilal butt baylor college of medicine -hour development of a giant infectious intracranial aneurysm: a case report catherine albin intra-operative ultrasound in traumatic brain injury patients namkyu you syndrome of the trepheined (sot) and paradoxical herniation without craniectomy elysia james spectrum health neurosciences -icu division stephen a. trevick , andrew naidech , leah tatebe patients were included. median age was years. % were female, % smokers, % hypertensive and % diabetic. % had a history of cad or mi and % had hyperlipidemia. in the multivariable analysis, the odds ratio for unfavorable outcome, defined as mrs score of - , was . ( %c.i: . - . ) and . ( %ci: . - . ) for the intermediate-grade(iii) and high-grade(iv and v) hh groups respectively, when compared to the low-grade(i and ii) hh group. age, hypertension and diabetes were found to be negatively associated with mrs, while hyperlipidemia was positively associated. gender, race, smoking and history of cad/mi were not significantly related to mrs. a positive trend for better mrs outcome was observed across years (p= . ). this trend was not related to hh grade on admission, (p= . for interaction between hh grade and year). hh scale on admission is associated with the mrs outcome upon discharge for patients with nontraumatic sah. models predicting the probability of a good mrs outcome could be created based on the hh grade on admission, age, hypertension, diabetes and hyperlipidemia status. the data suggest a trend toward improvement in medical and surgical care for this patient population across years. ciro poor-grade subarachnoid hemorrhage (sah) is associated with high mortality rates. although death rates have decreased in the last three decades, the exact mechanisms of demise are still to be determined in this patient population. a retrospective study of consecutive poor-grade sah patients (world federation of neurosurgical societies grades iv and v) aggressively treated in two academic high-volume centers, one in the netherlands (amc) and one in canada (smh). the primary outcome was in-hospital mortality. the main reasons of death were evaluated. a total of poor-grade sah patients were admitted between and , to amc and to smh. ( %) patients died, and ( %) of those patients died before having the culprit aneurysm treated. the median interval between hospital admission and death was three days (iqr - ).withdrawal of life support was the main reason of death in both centers (total of deaths - %), cardiopulmonary causes, aneurysm rebleeding, refractory intracranial hypertension, and other extracranial causes), represented less than %. extensive review of patients chart for all the data collection including literature search for similar cases if reported before. although rare, there are multiple case reports and series of nkh and clinical findings of hemichorea-hemiballism (hc-hb). there are few case reports of nkh with unilateral signal changes in the caudate and putamen. our patient presented with acute right basal ganglia ich. despite the typical imaging findings of nkh, work-up and management of ich took precedence over control of bg. mri findings were different in our patient given presence of positive gre and dwi/adc in areas other than t hyperintensity, which is known to be associated with nkh. we hypothesize an association between ischemia and hemosiderin deposition with hyperglycemia. the selective vulnerability of unilateral involvement of basal ganglia and caudate is unclear and needs more research. identification of neuroimaging findings in nkh in absence of focal neurological deficits (hc-hb) is important, especially for a first responder. early recognition can prevent icu admission, provide efficient patient care and allocation of resources. although most metabolic diseases affect basal ganglia bilaterally; nkh is associated with specific unilateral neuroimaging findings even in absence of movement disorders or focal neurological deficits. a year old male with a history of seizure disorder due to mesial temporal lobe sclerosis, presented with altered mental status after a lamotrigine overdose. he had consumed . gm of the drug. he was awake and alert at presentation. urine toxicology was negative. initial creatine kinase (ck) was iu/l and peaked at iu/l; his creatinine was . mg/dl. lamotrigine level went from mcg/ml to . mcg/ml after hours. four days after admission it was mcg/ml. a head ct at admission was negative. despite initial alertness, he developed profound encephalopathy with agitation and rigidity, requiring heavy sedation, induced paralysis, and intubation. this in turn lead to hemodynamic instability, which along with persistently elevated lamotrigine levels, prompted initiation of continuous veno-venous hemodia-filtration (cvvhdf) on hospital day . the lamotrigine level declined to . mcg/ml within hours, the encephalopathy and rigidity resolved, and he was extubated. to our knowledge, this is the first reported case of lamotrigine toxicity managed with cvvhdf. overdoses up to g have been reported and can even result in death. while cleared hepatically, the half-life of lamotrigine is approximately twice as long when patients have chronic renal failure. in a small series of patients with renal failure, approximately % of lamotrigine was reported to be removed by hemodialysis. we applied this principal to our patient. our experience suggests that augmenting drug clearance with dialysis may help reduce the time on mechanical ventilation, need for higher doses of sedatives, and improve time to discharge. cvvhdf should be considered a supplemental treatment option for lamotrigine toxicity. traumatic brain injury (tbi) complicated by percutaneous coronary intervention (pci) remains a significant clinical dilemma. dual anti-platelet therapy (dapt) is standard after pci, but may contribute to progression of tbi. novel antiplatelet drugs with ultra-short half-lives, such as the p y -adenosine receptor antagonist, cangrelor, may provide added clinical flexibility in avoiding tbi-associated hematoma progression, particularly in the absence of reversibility options. case report. we report a year-old female who presented to the ed after a syncopal episode with a fall down a flight of stairs. an ekg was obtained demonstrating inferior wall stemi. signs of head trauma included facial and scalp contusions, and bloody otorrhea. initial gcs was . a non-contrast head ct demonstrated tsah and contusions of bilateral frontal lobes and left temporal lobe, and a non-displaced fracture of the left temporal bone. neurosurgery, interventional cardiology and critical care were consulted. the patient developed signs of cardiogenic shock related to stemi and was taken emergently to cath lab. successful revascularization of proximal rca occlusion was achieved. heparin was given per protocol, and aspirin and cangrelor administered post-pci. cath lab was complicated by tonic-clonic seizures requiring intubation. repeat head ct demonstrated blossoming of bifrontal contusions, trace subdural hematoma development and increased tsah conspicuity. dapt infusion was continued, and subsequent imaging was stable, allowing transition to asa and clopidogrel. she survived with only minor disability. newer generation p y inhibitors can be administered intravenously with reliable platelet inhibition similar to older p y receptor inhibitors. with rapid reversibility upon discontinuation, their utilization should be considered any time pci complicates tbi. cerebral air embolism (cae) is a rare but potentially fatal entity with high morbidity and mortality, commonly seen secondary to iatrogenic causes like neurosurgical procedures, vascular surgeries, etc. as also deep sea diving. cae after esophagogastroduodenoscopy (egd) is extremely uncommon. we present a rare case of cae post egd resulting in diffuse cortical infarction. an year old man underwent an elective (egd) for esophageal stricture with biopsy and balloon dilatation. patient did not wake up after procedure. on initial exam, patient was comatose, glasgow coma scale t with decerebrate posturing. computed tomography (ct) revealed multiple foci of cerebral air embolism. ct angiogram of the brain was negative. diffusion weighted imaging and apparent diffusion coefficient imaging sequences in magnetic resonance imaging (mri) showed diffuse, global bi-hemispheric cortical infarction. ct chest showed pneumomediastinum. only cases of cae from egd have been reported in literature prior to this case. received hyperbaric oxygen therapy(hbo). patients had a documented patent foramen ovale (pfo) or some form of arteriovenous (av) shunt. presence of av shunts/ pfo, therapeutic endoscopic procedures providing vascular communication as well as providing pressure gradient are all factors facilitating air embolism associated with egd. hbo therapy has been shown to improve outcomes in cae patients, initiating therapy > hours after insult and early and significant ischemic changes seen on ct/ mri prior to starting therapy were strong predictors of poor outcomes. our patient did not have a documented echocardiogram with a shunt study prior to the egd. cae after egd causing global cerebral bi-hemispheric ischemia as seen in our case is extremely rare. hbo has been shown to improve outcomes. time to treatment > hours and early ct/ mri changes suggest poor outcomes. studies do not recommend benefit of screening for pfo or av shunts prior to every egd. key: cord- -pol qm authors: nan title: third international congress on the immune consequences of trauma, shock and sepsis —mechanisms and therapeutic approaches date: journal: intensive care med doi: . /bf sha: doc_id: cord_uid: pol qm nan this issue of the journal contains the abstracts for the third international congress on the immune consequences of trauma, shock and sepsis -mechanisms and therapeutic approaches. we hope that the information contained in this special issue will stimulate you to participate in the congress, to contribute to the knowledge being developed in this field and to use this information to help you in providing better care for your patients. we thank the editors and the editorial board and publishers of the journal for their interest and support in preparation of this special issue. we also, on behalf of the scientific committee, welcome you to the third international congress in munich on - march . when, in the mid- s, we thought of having a worldwide congress, we hoped to bring together investigators to discuss this theme. the explosion of knowledge occurring around that time provided an excellent background against which the first conference in provided stateof-the-art information and consensus on factors involved in injury and sepsis. in , the second congress was held at the time of another resurgence of research, study and information on injured and operated patients. it seemed then that there would be a lull in the development of new information and therapy, and that another state-of-the art conference might not be necessary until or . however, the explosion in molecular biology has continued. the wonderful world of cytokines has gone from ill to il- to il- , il- and il- and beyond. the vast amount of information about mediators and their importance in disease is impressive. this has all suggested a magic bullet that might be used to alter or block inflammatory responses. this has not happened, however, and the question is "why not"? our science is powerful, but our therapy is still weak. what are the issues, then, in , to be dealt with at this symposium and congress? ( ) proposals for new terminology. there have been a number of proposals for new terminology and new classifications of injury, sepsis, inflammation and various other problems related to human illness. the question is whether this is the way to go. will this contribute to better clinical trials, information basis and better research? the pros and cons of this development will be reviewed by those making the proposals and those questioning the need for and wisdom of this effort. ( ) magic bullets: the prospect of a magic bullet to deal with inflammation in injury and infection seemed highly promising earlier. many preclinical trials and a lot of animal research suggested the possibility of a great breakthrough in clinical care. what has become, then, of all the expensive and extensive multi-institution randomized, placebo-controlled, double-blind clinical trials of agents that block mediators and endotoxin. many such studies have yielded equivocal, marginal or negative results. the reasons for this and the future of clinical research will be the subject of presentations and discussions to set the stage for further work. ( ) should future clinical trials be based on new classifications of illness such as mods, sirs, apache iii, sap ii, mrm, etc., or should trials be dedicated to specific diseases -urinary tract infections, pneumonia, trauma patients, cardiac surgery and other specific problems, rather than generalized problems of sepsis, the sepsis syndrome and other classifications? in other words, should we now begin to have clinical trials on specific diseases with causes that are known and can be attacked? the causality of disease becomes an important consideration in this regard. ( ) a multitude of potential therapeutic agents has been proposed on the basis of animal studies. how should we decide which of them should be brought to clinical trial? the possibilities are endless as we develop new clinical information about the mechanisms and pathogenesis of human disease. ( ) information on the pathogenic mechanism of disease states and of injury continued to emerge in an explosive fashion, and in light of our gathering knowledge we can look forward to working out a cohesive system of response to injury. ( ) additional information will be provided in plenary sections, many symposia and free communication sessions and posters, which will update the participants on a variety of relevant topics presented by many of the leading in-iv vestigators in these fields. topics will range from molecular mechanisms, such as signal transduction, through the explosive growth of information on the role of cytokines and pathophysiology, to practical considerations in the design of immunomodulatory therapeutic regimens. these merely touch on a few areas, from the basic to the clinical, which will be the subjects of those symposia. all this information will fit into the jigsaw of this exciting area and its stimulus to further research study. this promises to provide an exciting, educational programme with experts and participants from all over the world. we hope it will set the stage for many years to come and will increase our understanding of trauma, shock and sepsis and help us to provide better therapy for those of our patients who are affected by such problems. a. the clinical syndrome of mods versus mof will be reviewed in detail by those who have made these proposals. b. an extensive review of the design and interpretation of clinical trials in patients with shock and injury will be provided. the reasons why so many clinical studies in the recent past have been negative will be reviewed. the therapeutic strategies that are being developed for the treatment or prevention of mods or mof will be the subject of another panel discussion by experts who have been involved in and contributed to this area. a consensus conference or controversy conference will be presented about various aspects of mods or mof, including the benefits of supernormal oxygen delivery, bacterial translocation, parenteral nutrition, the immune response and other aspects. the successes and failures of completed clinical trials will be presented by those who are involved in these clinical trials, with a refreshing review of the problems related to that injury. there will be late news about studies just being completed at present or after the beginning of and where they stand. c. the mechanisms and biochemical profiles of specific organ dysfunction or failure will be reviewed. what are the definitions? what are the mechanisms? how can organ dysfunction and/or failure be defined? an extensive review of the biological mechanisms involved in production of injury by mediators will be presented. a session will be devoted to how future ongoing trials might be better designed and what can be done about the studies recently completed, many of which are negative. d. the immunological or inflammatory pathways resulting in organ injury will be reviewed in detail in presentations and a panel discussion. we look forward to welcoming you to an exciting and rewarding conference, which undoubtedly possesses the potential to become a landmark event and major reference point for any scientific discussion about the complex of host defense dysfunctions following trauma, shock and sepsis. studies over the past years have established that the contact system, which forms bradykin/~, is gax important mediator in hypotensive septicemia. in addition to hradyk{nln, another product of the contact system, kailikrein, can mediate inflammation by virtue of its chemotaetic mad neutrophj/activating properties. using functional and immunochemical tech~ ques, we have demonstrated activation of the contact system in the adult respiratory distress syndrome in typhoid fever and clin/cal sepsis. we have also been able to inhibit the hypotension but not the disseminated intravaseular coagulation in a model of primate sepsis by the use of a monoclonal antibody directed agsi~st factor xii, the initiating protein of the contact system, in volunteers given e. coil endotoxin, who did not develop hypotension, we were also able to demonstrate activation of the contact system with a rise of alpha- macrogiebulin-kalllkrein complex. we have also examined, j~ an i~tensive care situation, patients with sirs. we found that serial measuremezzts of the contact system were useful in eva~u~ting prognosis+ these studies suggest that inhibition of kalllkrein a~d l e r bradykinin actions might be useful i~ obviating many .of the features seen in sepsis and septic shock. dextran sulfate (dxs) activates the contact system and, in vivo, produces transient hypotension. in order to better define the mechanisms underlying the dxs-induced hypotension, we investigated the effects of either the plasma kallikrein inhibitor, des-pro -iarg] ]aprotinin (bay ) or the b kinin antagonist, hoe on the hypotensive response to dxs. in the first study, anesthetized miniature pigs ( pigs/group, randomly assigned) were given one of the following treatment protocols: ) dxs ( mg/kg), - ) dxs plus bay ( , , , or rag), or ) saline. dxs alone produced a profound but transient systemic arterial hypotension with a corresponding reduction in plasma kinin-containing kininogen. circulating kinin levels, complement fragment c adesarg and fibrin mom)mer were all increased. bay produced a dose-dependent delay or attenuation in these effects with the highest dose completely blocking dxs-induced hypotension and elevations of kinin, c adesarg and fibrin monomer levels. thus, the effects of dxs are solely dependent on contact system activation and this activation is sensitive to bay . llowev~:r, contact system activation is known to produce changes in a variety of vasoactive mediators, all of which can affect blood pressure. in a second study, two groups of pigs ( /group) were given either dxs alone ( mg/kg) or dxs minutes after a bolus injection of hoe ( #g/kg). dxs alone produced transient hypotenmon. this response was completely blocked by hoe pretreatment. both groups had identical reductions in kinin-containing kininogen. we conclude that dxs-induced hypotension is produced by activation of the contact system which results in the production of bradykinin. liberation of bradykinin is both necessary and sufficient to produce all of the hemodynamic changes observed. dr. matthias siebeck, department of surgery, university of munich, klinikum lnnenstadt, nussbaumstrasse , d- munich, germany in experimental animals exposed to i.v. injection of endotoxin accumulation of leukocytes in various organs as lungs and the liver is a prominent feature. as a part of these morphological changes damages of endothelial ceils are regularly seen. this process, which is a part of endothelial-cellular interaction, leeds to exposure of the sub-endothelial basement membran. the basement membran is known f r its capacity to activate the contact system of plasma. during this cascade activation, coagulation factor xii is converted to the active factor xii. this activation might produce increased plasma kallikrein activities and thereby give release of the vasoactive substance bradykinin. using a porcine model we have noticed that endotoxin infusion ( , mg/kg) induces elevated plasma kailikrein activities within two hours after the start of the infusion. this enzyme activity remained increased during the next hours and reached value of up to u/ . in patients with sepsis we also have observed elevated plasma kallikrein activities with enzyme activities up to u/ . in order to further elucidate the significance of these elevated enzyme activities, we prepared human plasma kallikrein and injected it intravenously in anaesthetized pigs ( ). when very small plasma kailikrein activities ( , u/kg bodyweight) were given intravenously a % decrease in arterial blood pressure was seen in the animals. in the patients with sepsis also decreases in prekallikrein values and functional plasma kallikrein inhibition are frequently seen. furthermore, degradation of high molecular weight kioinogen is found in these patients indicating formation of bradykinin. these experimental and clinical studies underline that contact activation in sepsis might results in the release of very powerful mediator substances which can be of pathophysiological importance in this disease. a number of pathological disorders as reperfusion injury, bone marrow transplantation, polytrauma and septic shock are associated with capillary leakage. as the activation of the complement system and the contact phase play a major role in these diseases we investigated whether cl-lnhibitor (c -inh), which inactivates cl-esterase, kallikrein and clotting factors xii and xl, could abolish vascular leakage. a capillary leakage was induced in rats by the administration of interleukin- ( x iu/kg). the increased vascular permeability was monitored for one hour as the extravasation of fitc marked rat serum albumin from a mesenterial vessel by a video-image processing system. ci-inh (berinert®, behringwerke) given as a single i.v. bolus in concentrations of , or u/kg dose-dependently prevented the capillary leakage. carrageenaninduced inflammation in the rat leads to vascutar leakage and to edematous swelling of the paw. ci-inh in this model leads to a dose-dependent decrease in paw edema formation. finally, we investigated the effect of ci-inh (infusion ( - u/kg x h) on a lps-induced shock in the rat by combination therapy with the antithrombotlc agents antithrombin ill (kybernin®) or rec. hirudin (both substances from behringwerke). in this animal model mortality was % in the untreated control. both antithrombotic agents decreased mortality rates by inhibiting formation of dic; a further significant improvement of survival was achieved by the treatment with ci-inh. thus+ it could be concluded that c -inh has a beneficial effect in diseases associated with a vascular leakage. iclb and laboratory for experimental and clinical immunology, university of amsterdam, the netherlands; thrombosis research center, temple university, penn., usa; oklahoma medical research foundation,. ok. city, usa. to evaluate the contribution of the contact system to activation of other mediator systems in an experimental model of sepsis, we investigated the effect of mab c b which inhibits activation of factor xli, on activation of complement and fibrinolytic cascades and activation of neutrophils in baboons suffering from a lethal sepsis. activation of the complement system was assessed by measuring circulating levels of c b/c and c b/c, and a significant reduction was observed in animals that had received a lethal dose of e. coli together with mab c (treatment group), compared to animals that had received a lethal dose of e. coil only (control group). activation of the fibrinolytic system as reflected by circulating plasmin-= antiplasmin complexes and tissue plasminogen activator, and activation of neutrophils, assessed by measuring circulating elastase-=l-antitrypsin complexes, was also significantly less in the treatment group. we conclude that activation of the contact system protein factor xll during the inflammatory response to a lethal dose of e. coil in this baboon model, modulates directly or indirectly activation of the complement and fibrinolytic systems and that of neutrophils. in a prospective study, plasma levels of c a, c , and c a were measured in patients from an internal intensive care unit. patients were clinically septic defined by the criteria of bone et al.(l) . the remaining patients were critically ill but didn't fulfill the clinical criteria of sepsis. from both groups of patients blood samples were taken over a l days period. during the first days blood samples were drawn every h, on day - every h and the last days once daily. mean plasma concentrations of c a within the first h after clinical onset of sepsis were + pg/ml, whereas non-septic-patients exhibited mean values of only +_ p_g/m/. c levels were lower for septic-patients ( + lag/ml) than for non-septic-patients ( _+ lag/ml). the most profound difference between both groups was found, when the c a/c ratio was compared ( . + . for septic-patients and . _+_ . for the control group). no significant differences between both patient groups were observed in c a plasma levels ( . + . ng/ml in septic-patients vs. . _+ . ng/ml in control patients). in of cases of clinically defined sepsis causative organisms like bacteria, protozoa or fungi could be cultured from blood, bronchoalveolar lavages and/or section materials. application of the complement parameters to survivors (n= ) and non-survivors (n=l ) within the septic-group revealed, that the c a/c ratio could also be used as a prognostic parameter for clinical outcome. the possibility of rapid and easy measurement of c a and c in only - minutes ( ) and the significant difference of the c ajc ratio between the septic and non-septic group renders this parameter a good candidate for early diagnosis of sepsis in the intensive care unit. hirudin, a single polypeptide chain composed of amino acids with cysteine residues (mr daitons), is the most potent and specific thrombin inhibitor, which is now available as a genetically engineered product (rec. hirudin -hbw , behringwerke; marburg). the aim of our study was to establish a rabbit model of tissue factor (tf) induced activation of the extrinsic pathway of coagulation and to evaluate the therapeutic efficacy of rec. hirudin. coagulation was induced in female nzw rabbits by infusion of . p.g/kgxh thromboplastin for hours. development of disseminated clotting was manifested by a decrease of fibrinogen and platelets to . % and , % respectively, and by an increase of fibrin monomers from . to > . ~tg/ml. we administered rec. hirudin to rabbits in different concentrations ( . , . and . mg/kg); treatment started simultaneously with the infusion as an i.v. bolus. rec. hirudin significantly prevented the decrease of fibrinogen, platelets and the increase of fibrin monomers. this effect was dose dependent and long lasting, even hours after the administration of rec. hirudin, clotting was still significantly reduced. as could be drawn from the plasma levels, rec. hirudin had been cleared from plasma at this time. in a post-treatment study we administered rec. hirudin ( . , . and . mg/kg i.v. bolus) as late as hours after the start of tf infusion. at this time there was already a prominent activation of coagulation. even in this post-treatment regimen rec. hirudin significantly prevented disseminated clotting. hence, it was concluded, that rec. hirudin by inkihiting thrombin could be effective in the prevention of coagulation disorders including disseminated intravascular clotting (dic) induced by a septic disease. research laboratories of behringwerke ag, marburg, germany $ novel protease inhibitory activities of the second domain of urinary trypsin inhibitor (r- ) and its effect on sepns-lnduced organ injury in rat atsuo murata , hitoshi toda , ken'ichi uda , hidewaki nakagawa , takesada mori , hideaki morishita , tom yamakawa , jiro hirese , atsushi ni~ , nariaki matsuura osaka university medical school, osaka, mochida pharmaceutical co. ltd. tokyo, wakayama medical schoof, wakayama, japan inhibitory-activities of the second kuntz-type inhibitor domain of human urinary trypsin inhibitor (uti) and its effect on sepsis-induced organ injury in rat were investigated by using the recombinant protein. uti is a glycoprotein with a structure in which kunitz-type inhibitor domains are linked in a row. we isolated the gene encoding the second kunitz-type inhibitor domain of uti, and then constructed expression plasmids by ligating it to the e. coli phoa signal peptide gene. these plasmids expressed the second domain in e. coil strain je which lacks the membrane lipoprotein. the recombinant second domain (r- ) innb[ted trypsin, plasmin, neutrophil elastase and chymotrypsin. in addition it inhibited blood coagulation factor xa and plasma kallikrein in a concentration dependent and competitive manner. the in vivo effect of the recombinant r- was investigated in a rat model of septic shock induced by cecal ligation and puncture. the administration of r- significantly improved the survival rate of the rats and attenuated the pathological changes of lung and iiver. we found out the novel protease inhibitory activities of the second domain of uti and its protective effects on sepsis-induced organ injury. macrophages are known to secrete lysosomal proteinases,mainly cathepsin b and cathepsin l, and also ~-proteinase inhibitor (pi),related to acute phase proteins.disturbances of proteinases/ proteinase inhibitors correlates with inflammatory process,leading sometimes to noncontrol "pathglogical" proteolysis (jochum et ai., ) . the cathepsin l-like and cathepsin b-like activity were measured in serum of patients with chronic bronchitis ( -with obstructive, -with nonobstructive bronchitis),acute bronchitis ( ) and healthy persons.simultaneously the level of~pi was determined in the same groups.cysteine proteinases were measured with help of fluorogenic substrates,as was presented earlier (korolenko et ai., ) , ~pi with help of immune enzyme method. it was shown increase of cathepsin l-like and cathepsin b-like activities during aggravation of chronic bronchitis comparatively to the controls ( - fold) .after treatment there was a tendency to normalization of indices,but the increase was about - % more than the control values.~pi level in this group was also increased (two-fold),in patients with acute bronchitis - - -times more comparatively to the control.it is possible to conclude that chronic bronchitis induced increased secretion both cysteine proteinases and d{pi into blood. some peculiarities of ratio were noted in patients with emphysema. endotoxins are microbial products derived from the outer cell membrane of gram negative bacteria. the active component of endotoxin is lipopolysaccharide (lps), a complex macromolecule consisting of polysaccharide covalently bound to a unique lipid, termed lipid a. now recognized to embody the endotoxic principle of lps, lipid a consists of a/ - diglucosamine backbone, both ester and amide linked fatty acids, some of which are acyloxyacylated, and charged constituents such as phosphate, phosphorylethanolamine and amino arbinose lps, exerts its biological effects in vivo by noncytotoxic interactions with a variety of host inflammatory mediator cells, primarily the mononuclear phagocyte and the endothelial cell, although other host cells also participate. these interactions are modulated by lps-specific binding proteins found in plasma, including lps-binding protein (lbp) scd and perhaps other proteins as well. specific receptors for lps have been identified on mammalian cells which mediate signal transduction via multiple pathways. lps-activated host cells are stimulated to secrete or express multiple proinflammatory mediators, including tnf-a, illa, il- / , ifn-a, il- , il- , il- , paf, pge, ltb and procoagulant activity. the overproduction of these proinfiammatory mediators results in the manifestations of endotoxemia, observed experimentally as fever, hypotension, disseminated intravascular coagulation and death. modulation of activity of these mediators protects animals against lethality. similar pathways are thought to be operative in gram negative sepsis, and control studies with human volunteers support such conclusions. immunotherapeutic approaches in clinical gram negative sepsis have, to date, been less successful. in vitro experiments and studies in animal models have recently shown that several proteinaceous bacterial exotoxins can evoke cytotoxic effects that ultimately lead to cardiovascular collapse and shock. since the possible relevance of bacterial exotoxins in the pathogenesis of septic shock has received very little attention in the past, an attempt will be made here to provide a brief overview of this generaily neglected topic. protein toxins act intracellularly or they dz~nage the integrity and function of the plasma membrane. major representatives of the former group are the adenosine diphosphate (adp)-ribosylating toxins, e.g. cholera and cholera-like toxins, diphtheria toxin), and the neurotoxins. most medically relevant toxins of this category have been studied in great detail. although often responsible for severe and sometimes fatal disease, their association with septic shock is rare. in contrast, experimental evidence is accumulating for a role of membrane<lamaging toxins in the pathogenesis of inflammatory lesions. formation of transmembrane pores is probably the most widespread mechanism via which such physical membrane perturbation can occur ( , ), the present discussion will be restricted to this category of toxins. the author shall first discuss basic properties of pore-forming proteins, and consider the factors that direct their attack to specific targets. thereafter, attention will be drawn to diverse functional consequences that may follow membrane damage so that a general concept of how pore-forming toxins may contribute towards the development of shock will evolve. i. bhakdi, s. and tranum-jensen. j. gram positive bacteria produce exotoxins that, at least in part, exhibit the unusual properties of superantigens. superantigens (sag) activate v/ selectively t cells to produce lymphokines including i - and tnf/lymphotoxin. systemic application of the sag staphylococcal enterotoxin b (seb) to d-galactosamine sensitized mice causes lethal shock within hours. seb reactive v/ + t cells accumulate within - min mrna for the ii- , i - , tnfai~ and ifn- ,. parallel in time high concentrations of bloodborne i - and tnf are noted. anti tnf t~// neutralizing mab protect mice against seb induced t cell dependent lethal shock thus indicating a key role of tnf in effecting lethal toxicity. within - h distinct levels of tolerance to seb become discernable on ligand reactive v/ t ceils, dependent of the dose of seb used. these levels include anergy, t cell receptor downregulation, loss of cd , cd , cd accessory molecules and programmed cell death. m.freudenberg, y.yaegashi, p.nielsen, c.galanos. the sensitivity towards endotoxin (lps) is genetically determined, however it may be increased under different experimental conditions. these include treatment with hepatotoxic agents such as d-galactosamine, and with live (infection) or killed bacteria. it is well established now that d-galactosamine sensitizes to lps by increasing the susceptibility of the host to toxic activity of lps-induced tnf~. sensitization by bacteria, especially by gram-negative once is of especial interest because it implies that infecting microorganisms not only produce lps, but als sensitize the host to its lethal activity. sensitization to lps by bacteria proceeds in all lps-sensitive (lps n) mouse strains that have been investigated so far. it also proceeds in lps-resistant (lps d) c h/hej mice. the absence of sensitization to lps in lps d c bl/ sccr mice was identified as being due to their inability to produce interferon- (ifn?). administration of exogenous ifn? to these mice conferred sensitivity to lps. conversely, administration of anti-ifn? to lpsn; mice inhibited the development of sensitization'by bacteria. it may be concluded therefore that ifn is the mediator of sensitization to lps by bacterial infection. the ifn? defect of c bl/ sccr mice concerns only the ifn response to bacteria, since the response to the t-cell mitogen con a and to cd monoclonal antibodies was not impaired. the ifn?producing cells themselves were shown to be intact. on closer investigation the impaired ifn? response was identified as the result of a defective accessory function of macrophages. macrophages of c bl/ sccr mice are incapable of producing interferon-~ (ifn~) which we could show to be obligatory for the production of ifn? in response to bacteria. although antibiotics are required to clear bacteremia, they do not reverse evolving shock, because endotoxin free in the bloodstream or exposed on the surface of circulating bacteria continues to activate the production of inflammatory mediators, furthermore, antibiotics have been shown to induce release of endotoxin from gram-negative bacteria during the treatment of severe infection. in an in-vitro study, using live escherichia coil, we have observed the release of endotoxin upon treatment with several antibiotics like cefuroxim, imipenem, ceftazidime and aztreonam. incubation with imipenem or ceftazidime induced an early lyeis and a mild rise in endotoxin levels, whereas incubation with cefuroxime or aztreonam resulted in the formation of filaments with a late but dramatic rise in endotoxin levels. in a second study we studied the influence of different antibiotics on the tnf-= production of e.coli stimulated human monocytes. we found again great differences in the production of this important cytokine among the different antibiotics according to their capacity to induce liberation of endotoxin. these differences in the amount of endotoxin release are probably caused by penicillin-binding-proteins (pbp) specificities of the antibiotics with resultant differential morphological changes, in a rat sepsis model treatment with imipenem or ceftazidime resulted in a transient increase in il- levels, whereas treatment with aztreonam resulted in progressively increasing levels of il- and a significant increase in mortality. the increased mortality in pbp- specific aztreonam treated rats might have been the result of filament formation in the abdominal cavity of the septic rats. the endotoxin sequestered at local sites may account for the precipitation of gram-negative shock. finally, we've also demonstrated that in patients under treatment for septic shock endotoxin release can be related to the administration of antibiotics. carbapenem-and cephalosporin-lnduced release of lps from smooth and rough pseudomonas aeruginosa: in-vivo relevance j. jackson and h. kropp, merck research laboratories, rahway, nj b-lactam (cell-wall active) antibiotics are generally deemed the class of antibiotics most responsible for the release of free endotoxin (lps) from gram-negative bacteria due to their cell lytic actions although all antibiotic classes studied thus far induce release of endotoxin. we have recently shown in-vitro that antibiotics within the b-lactam class (i.e. imipenem , meropenem and ceftazidime , with equivalent mics) differ in their propensities to release excessive amounts of lps from both smooth-(s-) and rough-(r-) lps laboratory and clinical and antibiotic-sensitive and -resistant p. aeruginosa isolates and that lps release is independent of cell lysis. additionally, variations in the induction of endotoxin release is antibiotic concentration dependant and correlate with antibiotic penicillin-binding protein (pbp) affinities which result in morphological changes. these studies also show that lps differences measured via chromogenic lps (c-lal) assay correlate with lps lethality in lps-sensitive mice. release of lps was compared to changes in cfus, cell mass, morphology and antibiotic pbp-specificity. corresponding in-vivo studies in cd mice against s-lps p. aeruginosa isolates show that, despite equivalent in-vitro protection, antibiotic efficacy in mice differ as much as -to lo -fold. to establish a possible in-vivo role for antibiotic-induced release of free lps we compared antibiotic efficacy results in mice challenged with virulent parent s-lps and its relatively avirulent r-lps mutant (lacking only its side chain) p. aeruginosa isolates. results show the relevance of quantity and physiochemical composition (bioreactivity) of free lps to virulence and in-vivo antibiotic efficacy. in other in-vivo studies chemical agents that destroy cells (m~) which mediate lps lethality in-vivo were used to pretreat mice prior to antibiotic therapy and bacterial challenge with wild type pseudomonas. we conclude that circumstances in which antibiotic-induced filamentation occurs are also conditions that yield excessive lps release, the in-vivo effects of which are shown through the requirement of larger amounts of antibiotic to afford equivalent protection. bacterial endotoxins have been reported to play a significant role in the pathogenesis of gram negative sepsis by their interaction with, and stimulation of, host inflammatory mediator cells to produce cytokines, biologically active lipid intermediates, and procoagulant activity (tfa). although both microbe-associated and free endotoxin are capable of stimulating mediator production, studies from our laboratory suggest that free endotoxin may be the more potent stimulus for tnf and tfa. further, our studies suggest that the majority of the proinflammatory activity released from antibiotictreated e. coli coisolates with lps by two different fractionation techniques. to assess whether actions of endotoxin directly contribute to lethality in gram negative sepsis, an experimental model was developed in which mice were made hypersusceptihle to the lethal effects of endotoxin by treatment with d-galactosamine (dgaln). challenge of cf- dgaln-treated mice with e. coli olll:b resulted in an lds of approximately . x cfu. resolution of the peritonitis and bacteremia by treatment with cell wall-active antibiotics resulted in an increase in the ldso. ceftazidime and imipenem, which differ in their mechanism of action and in their capacity to effect endotoxin release in vitro, also differed in their in vivo capacity to provide chemotherapeutic protection ( fold vs fold respectively, p< . ). parallel studies with endotoxin hyporesponsive c h/hej mice indicate significantly greater levels of protection with imipenem (> fold vs saline controls). collectively these data suggest that endotoxin may contribute directly to the pathogenesis of experimental gram negative sepsis. bacterial lipopolysaccharides (lps) are the endotoxins of gram-negative bacteria and represent their major surface antigens. lps is made up of three chemically, biologically and genetically disctinct regions, i.e, the o-chain, the core region and the lipid a moiety whereby the latter represents the endotoxic center. it is our current understanding that lps is responsible for many of the pathophysiological events observed during gramnegative infections and that one of the major mechanisms leading to shock and death is the lps-induced activation of macrophages resulting in the production and release of lipid and peptide mediators, among which tumor necrosis factor seems to be the most important. therefore, in the fight against the lethal outcome of gram-negative infections, modern strategies, in addition to antibiotic treatment, aim at i) the neutralization of tumor necrosis factor, ii) the inhibition of the production of tumor necrosis factor or iii) the neutralization of the activation potential of lps for macrophages by monoclonal, preferably human antibodies. the latter approach, to be effective against a broad spectrum of gram-negatives, must be directed against common structures of lps (lipid a and core region). the molecular basis of this approach and the controversy in this field will be discussed. passive immunotherapy has been used since , when von behring described the administration of immune horse serum to treat a patient with diphteria infection. even if this therapy was sometimes successful in bacterial infections, it has been largely replaced by antibiotics. however, antibiotics have their limitations, especially in critically-ill patients. to improve outcome, adjunctive therapies such as immunotherapy with polyclonal and monoclonal antibodies particularly against endotoxin are again considered. the role of humoral immunity in host defenses against bacterial infections is weu known. for instance, tile importance of antibodies in the defense against gramnegative infections has been established clinically by studies relating the outcome of patients with gram-negative bacteremia to tilers of antibodies directed at the offending pathogens at the onset ofbacteremia (mccabe ; pollack ) . ever since we know the role of endotoxins in the pathophysiology of sepsis, antibodies against the s-and r-lps have also been detected in sepsis patients. the aim of the administration of iv/g to the sepsis patient is as follows: ) enhancing of opsonization and phagocytosis(antibactericidai activity) ) synergistic effects with [ - actam antibiotics ) neutralization of endotoxin, the main pathogenic mediator of gram-negative sepsis ) modulation and/or inhibition of cytokine release the enhancement of opsonic-and phagocytic-activity especially with igg via fc and c receptors has been well documented. monoclonal antiendotoxin antibodies, proven in clinical studies, do not appear to neutralize endotoxin in vitro and are not reproducibly protective in animal models of sepsis. also they can not suppress endotoxin-induced tnf-~, il- release in mice (baumgartner , corriveau and danner ) . in conlrast, recent studies of a polyclonal immunoglobulin preparation, containing high levels of antibodies against gram-negative bacteria and their o-antigen of lps in igg, igm and iga classes (pentaglobin®) provide evidence to neutralize endotoxin. this effect is demonstrated in vitro (berger (berger , , in animal models (stephan , berger and also in prospective, randomized, controlled clinical trials (schedel , poynton , behre . furthermore mortali b' was reduced statistically in patients with septic shock and endotoxemia by using this preparation, as has been demonstrated by sehedel. anti-core lps monoolonal antibodies: binding specificity and biological properties f.e. di padova, r. barclay, e.th. rietschel. bacterial lps and cytokines are responsible for the pathological processes of gram-sepsis and are suitable targets for therapeutic interventions. chemical characterization and structural analysis of different lps have revealed common features. the inner core region of lps shows a high degree of similarity among e. coli, salmonella and shigella. among a large number of broadly cross-reactive murine anti-core lps mab one of these igg ak) has been selected and chimerized into a human igglk (sdz - ). in elisa and in immunoblots on purified lps both sdz - and wni - show a strong reactivity with all smooth lps from e. coli and salmonella. reactivity with all the known complete core structures from e. coli and salmonella (ra) is evident. reactivity with re structures or free lipid a is not observed. this mab cressreacts with all clinical e. coli isolates from blood, urine and feces and with other enterobacteriaceae. sdz - and wni - have biological activity as they inhibit the lal assay and the secretion of monokines (il- and tnf) by mouse and human macrophages. moreover, sdz - and wni - inhibit the release of il- and tnf in vivo. in vivo sdz - as well as wni - neutralize the pyrogenic activity of e. coli lps and protect mice from lethality in d-gain-sensitized mice. the possibility to use wni - as a capture antibodies in the immunolimulus assay opens the possibility to differentiate the origin of the lps in patients with endotoxemia. franco di padova, sandoz pharma ag, ch basel, $chweiz $ presentation of lps to cd by lps binding protein peter s. tobias, julie gegner, katrin soldau, lois kline, loren hatlen, douglas mintz, and richard j. ulevitch. the activation of myeloid cells by lipopolysaccharides (lps) has been shown to require the serum glycoprotein lps binding protein (lbp) and binding of lps to membrane bound cd (mcd ). other cells such as human umbilical vein endothelial cells (huvec), smooth muscle cells, and some epithelial cells, which do not express mcd but nevertheless respond to lps in the presence of serum, have receptors for complexes of lps with the soluble form of cd (scd ). these complexes of lps with scd are only formed efficiently in the presence of lbp. we have begun to characterise the mechanisms by which lbp enables lps to bind to cd , either soluble or membrane bound. with the use of fluorophore and radiolabelled reagents we have developed procedures for quantitative measurement of the association of lps with lbp and of lps-lbp complexes with cd . these results show that the delivery of lps to scd is catalysed by lbp, i.e., lbp is not included with the lps-scd complex. in contrast, on the surface of cells, lbp does not dissociate from the cells after lps binds to mcd . the kinetics, equilibria and stoichiometry of these reactions will be discussed in the context of models for cellular activation by lps and cellular uptake of lps. supported by nltt grants gm , ai , ai , gm , and assistance from the pharmaceutical research institute of johnson and johnson. the scripps research institute, imm- , n. torrey pines rd. la jolla, ca usa . modulation of endotoxin-induced cytokine production by lps partial structures h.-d. flad, h. loppnow, t. mattern, and a.j. ulmer department of immunology and cell biology, forschungsinstitut borstel, d- borstel lipid a constitutes the active moiety of endotoxin (lps) of gramnegative bacteria. it activates mononuclear phagocytes to produce cytokines, such as tnf, i _- , and il- , which are the major mediators of the endotoxic effect of lps in vivo. lipid a precursor la (synthetic compound ) does not induce cytokines, but is able to specifically antagonize lps-or lipid a-induced mediator production in human mononuclear cells, vascular endothelial cells, and smooth muscle cells. furthermore, we present evidence for the first time that t-lymphocytes proliferate in response to lps and express mrna for interleukin- and interferon-~ and that these responses are also antagonized by synthetic lipid a precursor la. when comparing the agonistic and antagonistic activity of lipid a and different partial structures at the functional and binding level, the number and length of the fatty acids and the number of phosphoryl groups were pound to be of crucial importance. unexpectedly, lipid a precursor la, although biologically inactive, turned out to be both the most potent antagonist and competitor in inhibiting the binding of lps. taken together, our results provide evidence for a model in which lipid a partial structures compete with lps for specific cell surface receptor(s). in this sense, biologically inactive lipid a analogues may be good candidates as therapeutic agents for the prevention of gram-negative septic shock. two mammalian lipid a-binding proteins have been identified that are believed to have important roles in mediating the host response to endotoxin: lipopolysaccharide-binding protein (lbp) and bactericidal/ permeability-increasing protein (bpi). human lbp shares a % amino acid sequence identity with human bpi. despite the sequence homology, the two lipid a-binding proteins have very different functional activities. lbp is an acute phase serum protein that markedly potentiates the proinfiammatory host response to gram-negative infection by a mechanism which involves binding of the lbp-lps complex to cd receptors on monocytes, neutrophils and endothelial cells. in contrast, bpi is a neutrophil granule protein with potent bactericidal and lps-neutralizing activities. the divergent functional properties of these two lps-bindlng proteins can be explained by the inability of bpi-lps complexes to bind to cell-surface cd receptors. a recombinant protein (rbpi ), corresponding to the amino terminal kd fragment of human bpi, has been shown to retain the potent biological activities of the hdlo protein and may represent a novel therapeutic agent for the treatment of gram-negative infections, sepsis and endotoxemia. for therapeutic effectiveness in many clinical situations, rbpi will have to successfully compete with relatively high serum levels of lbp ( - ~g/mi) for binding to endotoxin and gram-negative bacteria. to evaluate this issue, experiments were conducted to compare the relative binding affinities of rbpi and human recombinant lbp (rlbp) for lipid a. the binding of both proteins to iipid a was specific and saturable with apparent kd's of . nm for rbpi and nm for rlbp. in a competition assay format rbpi was approximately -fold more potent than rlbp in inhibiting the binding of nsi-rlbp to lipid a. these results demonstrate that rbpi has a significantly higher affinity for endotoxin than does rlbp and may explain the potent inhibitory activity of low concentrations of rbpi in a variety of in vitro functional assays for lps activation of cells despite the presence of high lbp levels. for example, rbpi at . ~tg/mi was able to totally inhibit lps-induced tnf release from monocytes despite a -fold weight excess of rlbp over rbpi . and for heparin binding. three separate domains which inhibit the lal reaction to lps and bind to heparin were identified in amino acid regions - , - and - . a single synthetic peptide ( - ) was bactericidal. these results suggest that rbpi contains three separate functional domains which may contribute to its high affmity interaction with gram-negative bacteria and heparin. the individual activity of each domain and the cooperative interaction among domains provide the basis for developing rbpi analogues with increased biologic efficacy. a considerable body of experimental data has accumulated implicating tumour necrosis factor (tnf) as a principal mediator of the pathophysiological features of septic shock. these data prompted the development of clinical strategies designed to limit excess (inappropriate) tnf production. monoclonoal antibodies (mobs) were developed and a phase ii dose escalation trial in patients confirmed that the mab was safe, and suggested that it was having a beneficial effect on certain parameters. preliminary results of a large phase iii study indicated that (a) the mob was safe; (b) that it was of no discernible benefit in non-shocked patients; (c) that it reduced mortality in shocked patients, especially during the first days. an alternative strategy was to take advantage of the high binding affinity of soluble receptors for tnf (stnfr). stnfr-iggfc constructs were made for both the p and p receptors. both were effective in animal models of lps challenge, but when a clinical trial was done with the p stnfr-fc there was unexpected mortality in the treated arm. using an animal model of live e.coli sepsis, we have shown that this may have been due to the release of bound tnf from the construct. plasma enhances while bpi inhibits lps-induced cytokine production from peripheral blood mononuclear cells (pbmc). pseudomonas species produce cytokine-inducing substances which are different from lps as indicated by the fact that polymyxin b blocks only % of the cytokine-inducing activity of these pyrogens. we now tested the effect of plasma and bpi on the il- [ -inducing activity of pseudomonas maltophilia -derived pyrogens (pmp). bacteria were cultured to the log phase and filtered ( kd) to obtain prop. dilutions of pmp or lps were added to pbmc alone or to pbmc in % plasma +/-bpi ( ng/ml). pbmc were incubated for hours at °c and total il-i~ was measured by ria. results: il-i[~ in ng/ml (n= , mear~+sem, *p< . vs control). control . _+ + bpi . + % plas. . _+ + bpi . _+ pmp (ng/ml) lps (ng/ml) . _+ . _+ . _+ . _+. . +. . _+. . _+. " _+ " . _+ " . _+ " . _+. . + _+ _+. * _+ " . +. " . + -+ . -+ " . _+. " cba, c bl/ , balb/c, akr, dba, swiss mice, guinea pigs, rabbits have been used in research work. the toxicity, immunogenicity, mitogenic and immunomodulating activity of lps have been studied. the possibility of reduction of the toxic activity of lps on macroorganism by bioglycansimmunomodulators obtained from sea invertebrates anymals (crenomytilus grayanus, stromhus gigas) have been investigated too. lps has been shown to induce specific antibody response of laboratory animals. cba mice are high responsive to lps. lps stimulates humoral immune response of mice to tdependent and t-independent antigens and suppresses intensity of the delayed hypersensitivity. the small doses of lps stimulate functional activity of macrophages, the large doses of lps -decrease one and show the cytotoxic effect. the bioglycans enhance the resistance of mice to the lethal effect of lads and provide protection - % of mice. one opens possibility to use of bioglicans for reduction of toxinemia in generalizated forms of pseudotuberculosis. thus, lps from y.pseudotuberculosis is immunogen and immunomodulator wich has influence on humoral and cellular factors of immunity and plays the important role in immunopathogenesis of infection. endotoxaemia is implicated in the pathophysiology of obstructive jaundice. the lirnulus lysate (lal) assay is the gold standard method for measuring endotoxin concentrations, but inherent biochemical and technical problems limit the usefulness of this assay. the endocab elisa is a novel assay which measures endogenous antibody (igg) to the inner core region of circulating endotoxins (acga). objectives we evaluated the significance of endotoxaemia in biliary obstruction using the endocab assay and subsequently the specificity of the humoral response to endotoxin compared with an exogenous antigenic challenge [tetamls toxoid (tt) ]. materials and methods in experiment i three groups of male wistar rats ( - g) were studied [no operation (n= ) , sham operation (n= ), and bile duct ligation for days (bdl)(n= )]. plasma was collected and assayed for bilirubin, endntoxin(lal) and acga(endocab). in experiment ii rats were actively immunised with tetanus toxoid ('it) and then randomised to have no op(n= ), sham op(n= ) or bdl(n=i ). blood was taken at this time (to) and days later(t at sacrifice for acga concentrationslendocab] and igg produced to tt(ttab) [elisa] . antibody concentrations are expressed as % increase from control values.results in bdl rats, acga concentrations were significantly increased compared with controlslp< . , mann-whitney]. endotoxin concentrations were sporadically elevated in the jaundiced rats but the rise was not significant. in experiment [i there was no difference between the acga or ttab concentrations in the fllree groups at to, bdl rats had a significant rise in acga concentrations by t [p< , ,paired t-test] and humoral response to tt was significantly impaired in bdl rats compared with control groupslp< . , paired ttest data plasma endotoxin was measured by means of an endotoxinspecific endospecy test after pretreatment of the plasma with a new perchloric acid method that we developed. the normal value of plasma endotoxin is less than . pglml. polymyxin b was administered at a dose of , u every hours. plasma endotoxin rapidly decreased to the normal range in of the patients. body temperature fall significantly. apache ii scores were also significantly improved. tumor necrosis factor-o~ and interleukin decreased in survivors, while in high values tended to persist in patients died. no side effects were observed in any of the patients. in conclusion, intramuscular injection of minute of polymyxin b was useful in the treatment of endotoxemia. - uchimaru, morioka , japan. l e v a n t g r a m n e g a t i v organisms. m e t h o d s : u n d e r general anesthesia, n o r w e g i a n b r e d landrace pigs ( - kg) of either sex, pr group, u n d e r w e n t t r a c h e o s t o m y a n d w e r e v e n t i l a t e d on a / air a n d o x y g e n m i x t u r e a i m e d at m a i n t a i n i n g a n o r m a l p h a n d a isocapnic level. ventilation w a s not readjusted d u r i n g the observation period. the anesthesia w a s k e t a m i n e . m g / k g h a n d d i a z e p a m . m g / k g h i n t r a v e n o u s l y . h e m o d y n a m i c m o n i t o r i n g of m e a n aorta, p u l m o n a r y artery, central v e n o u s a n d p u l m o n a r y capillary w e d g e pressures w a s p e r f o r m e d w i t h a f s w a n -g a n z catheter a n d an aorta catheter. a continous infusion of r i n g e r ' s acetate ( m l / k g h ) w a s g i v e n intravenously. w h e n stabilised, the a n i m a l s w e r e g i v e n . x l cfu of e colt intraperitoneally as a bolus in ml saline, the a n t i b o d y g r o u p received in a d d i t i o n m g / k g e a n t i e n d o t o x i n i n t r a v e n o u s l y over h o u r via a n infusion p u m p at the start of the observation period. the a n i m a l s w e r e observed for hours. results : a t a n d hours, the o x y g e n c o n s u m p t i o n increased by % in the a n t i b o d y treated g r o u p w h e r e a s there w a s a significant fall of % in the sepsis group. in the a n t i b o d y group, the arterial p h a n d the cardiac index were also significantly h i g h e r at the s a m e p o i n t s in time. there w a s no significant difference in arterial po . in severe bacterial infections it would be beneficial to neutralize the plasma endotoxin content with complex forming compounds. the phenothiazines are able to form complexes with endoto×in and the existence of these complexes were already shown in differential speetrophotometry and animal experiments, however, the mechanism of partial neutralization was not clarified. therefore some representative phenothiazines and structurally related compounds were tested for anti-endotoxin activity. the endotoxin neutralizinb effects of several benzophenothiazines were investigated in differential speotrophotemetry, tnf induction and in the conventional limulus test. in animal experiments some beneficial effect of complex forming compounds was found. the benzophenothiazines were not able to inactivate the biological effect of endotoxin in the limulus test. the recent findings indicates that a multifocal effect can be responsible for "anti-endotoxin action in vivo". effects of tnf inducing effect of endotoxin in leukocytes and bypotensiv action in experimental animals were reduced by some phenothiazine derivatives. monophosphoril lipid a was without effect. of microbiology, albert szemt-gydrbyi medical university, odm t~r lo, h- szeged~ hunbary involvement of streptococcus pyogenes erythrogenic toxins in the induction oflstreptococcal toxic shock syndrome heide mgller-alou~* , joseph e. alouf , die [er gerlach , ~atherine fitting., and jean-marc ca~aillon . unit des toxines microbiennes and "unit d'immuno-allergie, institut pasteur, , rue du docteur roux - paris (france) ; institut f~r experimentelle mikrobiologie, jena (germany). superantigen erythrogenic toxin a (eta) is thought to be involved in toxic shock syndrome in humans by inducing massive release of cytokines by patient immune cells. the cytokineinducing capacity of eta w~:s £:ompa~ed to that of lps, a gram-negative bacterial cell wall component. eta elicited weak production of il- d and ~, tnf ~ and il- in purified human monocytes whereas lps stimulated the production of high amounts of these cytokines. in the presence of t cells, eta elicited the production of significant amounts of il-i~, il-i~, il- and il- . however, the most preponderant cytokine was tnf~, which peaked at i ng/ml after stimulation with i ~g eta. comparable amounts of tnfd (ca ng) were induced by .i ~g eta and .i ~g lp$. in contrast to lps, eta was a strong inducer of tnf~ which was produced only in marginal amounts by lps. these results suggest that the septic shock induced by gramnegative bacteria (lps) and by gram-positive bacteria {extracellular superantigens) follows different pathogenic pathways. lps-induced shock is mainly mediated by monocytes and monocyte-produced cytokines (il-i and tnf). the eta-induced shock is mediated by t-cells or depends on t cell help for the production of monocyte-liberated cytokines. production of t cell cytokines such as tnf~ and interferon in addition to the other cytokines contribute very likely to the severity of the toxic-shock resulting from s. auzeus and s. pyogenes infections in humans. the present study was utidertakc~l to cvalu~tlc the effect of soluble chemically modified giucan during septic shock. carboxylnethyl-b-i, -glucan (ram ) was injected twice and h before the shock i.v. in a dose of ing/kg. shock was induced in u~?esthetizcd (sodikm~. l)mntobarbital) rats by i.v. injection of endotoxin of escherichia colli bs, mg/kg. aiiofcmg pretreated ruts survived during first haher ¢ndotoxine, while in controi shock group the lethality was %. the concentration of ~col)terin in serum was significantly elevated hafterthc second cmginjection (appare~tly % if compare with the control rats), but didu't chartged rain and s rain after endotoxin injectjom cardiac output in cmogroup was higher a* the i and min after endotoxine onset ( i % trod ~, respectively of initial level) than in the control shock group ( % and % at the same time). pretreatment of rals with soh~ble giucan w~ts associated with beneficial effects o~ the hepatic c~ergy $ia[tls after h after challenge of endotoxiae: the tissue level of lactale was ahnost twice lower than in the control ruts, me~mthne the tissue atf in cmg pretreated group was higher at %. twice injected macrophage stimuhttor soluble glucan can prevent the endotoxic shock, and extremely ir~creased survival rate after endotoxine injection. the national committee of surgical infections of the spanish association of surgeons have produced a computer program for the collection and analysis of information on surgical infections. the program is suitable for ibm compatible hard disk personal computers and works through the ms-dos system. the main menu is called up on the screen when the operating disk has been installed; it reads as follows: i. new record; . modify records; . erase records; . searches; . reports; . configure; o. ouit. if you ask fdr a new record the screen will prompt you to enter the number of case, record number, hospital, age and sex. the next screen will come up and the words "topographic diagnosis" will flash. a menu of areas or organs will be displayed. then, the words "type of pathology" (inflammatory, neoplastic, traumatic and other). days of postoperative period. type of surgery (programmed and emergency). type of operation (clean, clean contaminated, contaminated and dirty). duration of surgery. this is followed by "order of operation" and the "type of anaesthesia (general, regional or local). you are then required to supply the "diagnostic code of who" (icd ) and the "procedure code of who. analytic and concurrent illnesses (total proteins, albumin, haemoglobin, haematocrit, leucocytes, red corpuscles, glucose and bilirubin). the next screen asks for "risk factors" (obesity, uraemia, neoplasia, malnutrition, urinary catheter, distant infection, artificial valve, immunosuppressive drugs, over years and anergy. this is followed by a screen headed "postoperative complications". "evolution" (the questions asked are drainage, systemic antibiotics, and on each ocasion a choice of antibiotics is displayed), local antiseptics, reoperation, etc. under "microhiology" is a choice of organisms and the chance of identifyin organisms. finally, "sepsis score". our recent work had shown that renshen-fuzi-chaihu mixture could increase the survival rate in experimented study. the purpose of this study was to determine the effect of combined administration of renshen-fuzi-chaihu mixtuer and antibitics (sa) in patients with septic shock. the result showed that, in sa group ( cases), the total effective rate was , %, in the contral group (combined administration of gentamycin and dexamethasone, cases) the total effective rate was %. however the obviously effective rate in sa group % was significantly higher than in contral group % (p<o.os). sa group appeared to be more obvious than the contral group in raising arterial pressure, recovering consciousness and pulse, improving cold lilmbs and reducing fever (p<o.o ). it was found that sa could inhibit the pathogeneses changes of septic shock such as the decreases of superotide dismutase (sod) and glutathione peroxidase (gsh-px) in erythrocytes and the increases of plasma free hemoglobin (phb), plasma malondialdehyde (mda) and -glucuronidase ( -gc) occured in septic shock. sa had stronger effect than gd in inhibiting the changes mentioned above. hence, these results suggest that sa has beneficial effect in the treatment of septic shock. sa could diminish the decrease of the antioxdase activities and inhibit lipid peroxidization and stabilize cellmembrance. this may be one of the principal mechanisms of the beneficial effect of sa in the treatment of septic shock. donna l. lehman, heather a. childers, irshad h. chaudry and alfred ayala. the thymus is thought to be a privileged sight for tcell generation. here the t-lymphocytes are either selected for their potential to recognize and react competently to foreign antigens or de-selected, due to their potential to react to auto-antigens, de-selected. tcells with this latter capacity are thought to be deselected through a process referred to as programmed cell death or apoptosis. while it is known that thymic apoptosis is elevated by a variety of stressers associated with infection and inflammation, little or no information is available concerning its presence in polymicrobial sepsis. it was, therefore, the aim of this study to determine whether thymocytes exhibit increased apoptosis during sepsis. to assess this, thymocytes were harvested from c h/hen mice at i and h following cecal ligation and puncture (clp; to induce sepsis) or sham-clp (sham). thymic yields were assessed and the extent of apetosis determined by staining the cells with propidium iodide (a dna intercalating dye) for facs analysis or by examining the extracted dna electrophoretically for the endogenous endonuclease activity the results (n= /grp) indicate that at i h post-clp there was no marked changes in any of these parameters. however, at h the thymic cell yield from clp mice was significantly decreased (sham: . ! . x l vs clp: . i . x * cells; *,p< . ), the % of cells apoptotic by facs analysis increased from . i . % in sham to . ± . %* in clp, and the septic mouse genomic dna exhibited dna degradation these results indicate that clp, but not simple laparotomy, induces marked thymic apetosis, which may contribute to the lymphocytic immune deficiency seen in these mice the purpose of this retrospective study was to evaluate effectiveness of irrigation to treat septic knee joints from longterm results. from to patients with septic knee joints have been treated. treatment in acute cases started with asp#at[on of the effusion to evaluate for cultures. without awaiting the result treatment was continued with immediate joint irrigation under arthroscopic control. three redon tubes ( ch ) were introduced for continuous irrigation and for intermittent distention of the joint cavity. in case of chronic joint infection additionally all foreign and synthetic material was removed. systemic antibiotics were given. patients could be re -examined with a mean follow-up of years. re-examination showed that immediate arthroscopic lavage started at the onset of septic sings had best results (less signs of osteoarthr[tis, less synovitis, less pain while loading and less range of motion loss ). patients ( %) out of patients with s tre_=.tsd acute se.~tic knee joint had excellent functional recovery, whereas all patients with chronic septic knee joints showed poor results. additionally, in of these patients with chronic septic knee joint after irrigation a further procedure was necessary due to recurrent septic knee joint. the concept that bacteria can translocate across the intestinal mucosa under a variety of circumstances is well established. however, due to the inherent limitations of in vivo studies, the cellular mechanisms underlying the process of bacterial translocation (bt) across the epithelial barrier are poorly understood. this is especially true in those circumstances in which there is no merphologic evidence of mucosal injury. consequently, we have utilized an in vitro cell culture model system to investigate the cellular events involved in the translocation process. in this model system, a polarized monolayer of intestinal cells (caco- cell line) is grown on a micron filter in a two compartment system. after tight junction integrity is established, bacteria are placed in the apical surface chamber and bt across the caco- monolayer is measured by culturing the basal chamber. the results of our studies utilizing the caco- system indicates that; l) several strains of non-pathogenic bacteria will translocate across the caco- monolayer in a time-dependent and dose-dependent fashion. however, the incidence and magnitude of bt are highest for those strains of bacteria (gram-negative enterics) that are most commonly cultured in vivo. ) caco- cells are actively involved in the transcellular passage of bacteria across the caco- monolayer, since inhibition of caco- microtubule or microfilament function decreases the magnitude of st. ) lectin but not integrin receptors are involved in bacterial translocation of e. col~ across the caco- cell monolayer. ) caco- cells have the ability to ingest and kill certain strains of bacteria. the mechanisms involved in caco- bactericidal activity appear similar to that of pmns to the extent that both are oxidant but not nitric oxide mediated. these in vitro studies suggest that caco- cells can function as "nonprofessional" phagocytes and that both bacteria and enterocytes are involved in the translecation phenomenon. bacterial translocation due to gut barrier dysfunction is considered to be a major cause of septic complications and multiple organ failure following trauma, burn injury, hypoxia, shock and shock-like states. the invasion of bacteria into the circulation from the gastrointestinal tract or even from other sources, however, should not necessarily result in systemic infection or organ cohinisation, as long as the humoral and cellular defense mechanisms are not impaired. thus, a reduced res clearance capacity and hepatic clearance function of bacteria and toxins can be observed under the same conditions which enable bacterial translocation from the gut. in order to evaluate the influence of circulatory, metabolic and humorul disorders on the elimination of bacteria out of the blood circulation, a series of experiments were performed in anesthetized and ventilated rabbits which received defined numbers ( . x colony-furming units) of escherichia coli as a bolus injection. in unaffected control animals the intravenously injected bacteria are eliminated about . % within the first minutes and are totaiy cleared within minutes. % offue total cfu counted in the cultures of the organ homogenates were found in liver and spleen, whereas only very low numbers could be detected in the lungs and kidneys. systemic injury of the animals by hemorrhagic shock, endotoxinemia, hypoxia, coagulation disorder, systemic vasoconstriction by norepinephrine and other types of injury reduced the elimination rate of bacteria and changed the pattern and degree of their dissemination and tissue distribution. the bacterial clearance was delayed mostly in rabbits subjected to hypoxemic hypoxia in which about cfu of viable e.eoli per ml blood could still be counted hours following their injection. the number of viable bacteria was some ten powers higher in organs of hypoxic rabbits in comparison to control ardmais, and an excessive colonisation of the lungs and kidneys with % respectively % of the total cfu of the cultared organ homoganates was observed. unilateral iscbemia of the kidney prior to the intravenous injection of e.coli only moderately delayed the bacterial clearance, bowever, caused an intense eohinisation of the affected reperfused organ. in contrast to this, inhalation injury did not promote the accumulation of bacteria in the lungs. conclusion: shock, systemic and local affections reduce the clearance of bacteria from the blood circulation and enable their dissemination and accumulation in vital organs. the degree of this reduction and the organ pattern of the bacterial distribution varies with the type of injury. thus, these factors seem to be essentially involved whether or not bacterial translocation and multiple organ failure will follow the invasion of bacteria into the circulation as in the ease of gut barrier dysfunction. movement of enteric baaterla from gut to extralumeaal sites (bacterial tra~sloeation) m~y play a role ~. nraltipl~ orga~ failure. traumatic stress (shock, hffectiort, and im~lammation) and severe illness are common alateeedents. tnt~st~aal lleus is e common proximme causal factor. we have exanlined the hypothesis that bacterial ttanslecation from the gut in expe~me~tal paaerestitis is due to bacterial overgrowth as a cuas~ucnee of a deere~ae in intestinal transit. ne~rotit_.~g pancreatitls fbliowlng bile duct iigation in the opossum is associated with colonization of the pa,se ~re, aa by gut derived organisms or salmonella of biljary origin in infected animals (previously reported). to ti~rther define the mechanism of transloemdon in panereati* inflammation, pancteatitis was induced hy iigatlon of t~e lower bile duet distal to the pancreatic duets m the rat. intestinal transit, enteric bacteriology, and tromsloeatien of bacteria to mesanterie lymph nodes, blood stream and splanelmie organs wen deletmhaed at (n~ ), g (n ~ ), and (n~ ) hours with the fallowing results: ly~testinai,..transfi". geometric mean of fluorescent marker - % at hour~ with return to % of gut leugth (similar to ~m) at hour~. bacterial overgrowt_hff -increase in total bsctetia ~ad gram negative rod.~ at ,; hours with return to sham control at hours ( log cfu/g veraus log cfu/g ia ileum). translocation to mesenteric nodes - , , and percent at , , and fi hours compared to b in sham controls (n=l of ). conclusion -bacterial translocation following panetoatieobiliary duct ljgation induced panereatitls ha the rat is tighlly linked to intestinal trausit and bacterial overgrowth within the gut lumen. speclrjlation -the ilens in in/~arm~ation may be related to activation of eytokiaes and mediated by nitric oxide. preliminary evidence for this notion will be discussed. in a series of studies we investigated: a) the time course of bacterial translocation (bt), b) the kinetics of lps and cytokine appearance (tnf, il- , soluble tnf receptor [stnf-r] ) in the circulation, and c) the role of lps and/or tnf with regard to haemorrhagic shock (hs) related pathophysiologie alterations and mortality in baboons and/or rats. method: baboons were subjected to femur fracture, soft tissue trauma (acute experiment), and hs ( mmhg mean arterial pressure: map for - h terminated at an accumulated oxygen debt of - ml/kg followed by resuscitation). a chronic model of hs was set up by administration of zymosan activated plasma (zap) before and after hs but without trauma. hs was induced in rats by bleeding the animals to map of - mmhg - rain followed by resuscitation. results and conclusion: in rats, lps increased in the systemic circulation, plasma tnf levels were found to be significantly elevated at min and were still markedly increased at rain postshock. plasma tnf, il- , and stnf-r levels increased already during the shock period in baboons, while tnf was not detectable. our data suggest that haemorrhagic shock may lead to early bt in the intestinal wall (at min following resuscitation in rats subjected to hs for min, similarly at h after the end of oxygen debt period in baboons ) and transient access of gut-derived lps into the circulation (levels became significant at min, while in baboons higher levels up to pg/ml were found at the end of shock and h after reinfusion, partially accompanied by bacteremia). in addition, since the treatment of rats with anti lps measures or anti tnf therapy significantly improved the -hour survival rate it can be assumed that endogenous lps and lps-induced mediator(s), in particular tnf, may lead to organ injury and mortality following haemorrhagic shock. alteration of the mesenteric blood flow and the consequent ischemia / reperfusion injury to the intestine appear to be one of the earliest events in the systemic inflammatory response following severe thermal injuries. the causative relationship between the subsequent disruption of the intestinal integrity and the potentiation of the translocation of indigenous bacteria and/or endotoxins to remote body organs and the systemic circulation has been deeumented in several studies. among other effects, endotoxemia solely or acting synergistically with thermal injuries has been shown to promote bacterial translocation. as these sequences continue without adequate intervention, multiple organ failure and eventually death may become inevitable. hence, the crucial need of treatment modalities with the capacity of modulating the intestinal blood flow and preventing the manifestations of these pathological incidents. although the underlying mechanisms of this process have not yet been fully elucidated, there is accumulating evidence that various interacting vasomediators are involved. the actions of these mediators can probably be modulated by either nonspecific or selective agents. among the nonspecific agents, the composition, volume and timing of the resuscitation fluids appear to play an important role in this process. nitropmsside, a nonspecific vasodilator, has been shown to prevent the decrease of the postbum mesentefic blood flow when selectively infused in the mesenteric artery. the nonspecificity of various vasodialators with their possible undesirable systemic effects emphasizes the importance of the identification and modulation of selective vasomniiators. thromboxane a (txa ) and angiotensin ii (a-ii) appear to be the primary mediators of the postburn vasoconstriction. both mediators were found to be elevated following thermal injuries. in a previous study, pretreatment with oky- , a thromboxane synthetese inhibitor was found to attenuate the postbarn mesenteric vasoconstriction. in a bum/sepsis porcine model the efficacy of dup , an a- receptor antagonist as a posthum treatment modality was evaluated. treatment with dup prevented the early posthum and the late posteodotoxin elevation in the mesenteric vascular resistance and subsequently abrogated the fall in the mesenteric blood flow. the incidence of burn & endotoxin induced bacterial translocation was significantly reduced by dup from % to % (p< . , fisher's exact test). the indigenous flora of the gastrointestinal tract exerts a potent influence on the developmental maturation of normal systemic immunologic responsiveness. moreover, both oral and intraportal administration of experimental antigen is associated with a systemic state of immunologic hyporesponsiveness, known as oral tolerance. since trauma and critical illness are associated with changes in the flora of the gut and with altered systemic immune reactivity, we have hypothesized that the interactions of an altered gut flora with immune tissues in the gut and liver play a role in the pathogenesis of the immunologic derangements of critical illness. prolonged feeding of two clinically relevant killed organisms-pseudomonas and candida-to a rat results in significant impairment of cutaneous delayed hypersensitivity (dh) reactivity. conversely, measures directed against gram negative bacterial overgrowth in experimental peritonitis result in partial restoration of impaired dh reactivity. to ascertain the role of the liver in the pathogenesis of these alterations, we studied the differential immunologic sequelae of portal versus systemic (ivc) infusion of killed bacteria. experimental infusion of live or killed gram negative organisms into the portal vein produces dh suppression in rive, and profound suppression of mitogen-driven lymphocyte proliferation in vitro; systemic administration of the same organism has no effect on these phenomena. suppression is tgf -dependent, and mediated by a soluble factor released by fixed tissue macrophages of the lung and spleen. the suppressive influence appears to be mediated at the level of interactions between il- and its high affinity receptor. release of this factor can be induced by a second circulating factor present in the plasma of portally-, but not systemically-infused animals two hours after bacterial administration. these studies suggest that the release of inducible immunoregulatory factors from the liver, and possibly from the gut, may play a role in the pathogenesis of the profound derangements of systemic immune homeostasis that accompany trauma and critical illness. acad.hospital st. radboud,postbus , lib nijmegen introduction. the central question being tested in this study was whether liposome encapsulated dichloromethylene-diphosphonate (ci mdp) mediated elimination of liver and splenic maorophages would influence zymosan induced systemic toxicity (st) and bacterial lranslocation (bt). materia|s and methods. male swiss (icr) mice were used weighing - g elimination of liver and spleen macrophages was accomplished by intravenous injection of / suspension of ci mdp-liposome suspension. control mice received an i.v. injection with pt phosphate-buffered saline (pbs) . two days later all mice were challenged with an i.p. injection of zymosan suspended in paraffin (z) at a dosage of either mg/g, . mg/g or . mg/g body weight (n= in each group), signs of st and bacterial translocation bt were measured hours later the st was assessed by blindly grading the degree of lethargy, conjunctivitis, diarrhea, and ruffled fur of each mouse on a four point scale. bt to the mesenteric lymph nodes (mln) and systemic organs (liver, spleen, lung and blood) was tested by culturing on blood and macconkey's agar at the time of sacrifice sections of the distal ileum were taken for histology. an additional two control groups (n= in each group) were tested to verify that neither paraffin nor pbs or ci mdpqiposome suspension alone induced bt or st. furthermore survival over a -day period was assessed in a control and macrophage-depleted group with a dose of d mg/g z. results. neither the paraffin nor ci mdp-tiposome suspension induced bt or st the incidence of bacterial translocation to the mln was similar between macrophage depleted and control groups. however, macrophage depletion was associated with a significantly higher incidence of systemic spread of bacteria. data expressed as positive tissues/total tissues tested: / vs. / at . mg/g zymosan (p~o. ); vs. at mg/g zymosan (p~q ); / vs. / st . mg/g zymosan (p~o.o ).the magnitude of bt however did not show any differences. although systemic bt was greater in the macrophage depleted groups, the systemic toxic response was significantly less at doses of . mg/g zymosan ( . -+ . vs. . -+ , p~o. ) and . mg/g zymosan ( . -+ . vs. . -+ . , p<_.o. ) . the day mortality after . mg/g zymosan was % in the control group and % in the macrophage depleted group (p=o. ). histology did not show any differences between the control and macrophage depleted groups. conclusion. the lethal and toxic effects of zymosan in this model appear to be more related to the excessive activation of macrophages than to the systemic spread of bacteria. bacterial translocation (bt) in hemorrhagic shock was studied using a porcine model. in this study three groups were randomized: one control (n= ) and two experimental (n= each). a portal vein catheter was placed and remained in situ hrs. a femoral artery and vein catheter were in place hrs. the arterial eaanula was used for monitoring mean arterial pressure (map) in all animals and to bleed the experimental animals and the venous cannula to repelfuse with ringer's lactate (rl) or autologous blood (ab). baseline samples and values were taken in this period. control animals were observed for a sham shock period of . hrs., given more hours of anesthesia, and studied for further hrs. % of the estimated blood volume was withdrawn from each experimental animal over min. these animals were kept in shock for . hrs. at the end of the shock one group was perfused with rl and the other with ab. two hours later they were extubated and further studied for hrs. samples from portal blood for cultures and measurement of leukotriene b (ltb ), prostaglandin e (pge ), and nitric oxide (no) metabolites were taken from all animals at intervals beyond baseline up to hrs. after hrs. under general anesthesia a second laparotomy was performed and samples for culture from mesenteric lymph nodes (min), liver and spleen were taken; as well as samples for histologic study with light and scanning electron microscopy were taken from jejunum, ileum, and colon. animals were then euthanized. results: significant shock was induced as indicated by map and arterial blood gases. significant bt to min was observed in all groups. no significant bt was observed in cultures from liver and spleen in any of the groups. increased values for ltb , pge and no metabolites were found during the period of shock. these results suggest that the isehemic phenomenon triggers a significant inflammatory response, and that bt to min may be an epiphenomenon without apparent significant influence on the inflammatory response. objective: to study the potential effects and mechanisms of action of systemic administration of monoclonal antibodies anti-endotoxin (ha- a) in a animal model of gut-origin sepsis. materials and methods: exoeriment a. balb/c mice were transfused with allogeneic blood (from c hb-iej mice). five days post-transfusion the animals were gavaged with lxl viable escherichia coil and randomized into groups (n= /group) to receive a sham burn (sb group) or a % thermal injury immediately followed by the systemic administration, via a tail vein, of monoclonal antibodies ( mg/kg) (ha- a group) or a % burn injury and administration of equal volume of sterile saline (c group). all groups were resuscitated by intraperitoneal injection of . ml of saline. the animal survival rate was observed for days post-burn. experiment b, transfusion and burn procedures were identical to experiment a but the mice (n= /group) were gavaged with viable e.coli labeled with mmndium oxine. four hours after burn the mesenteric lymph nodes (mln), liver, lungs and blood were harvested to determine plasma endotoxin levels and the magnitude of transtocation of labeled bacteria as measured by the residual radioactivity (dpm) in the organs. circulating endotoxin levels were determined by limulus colorimetric assay. diamine xidae(dao) is an enzyme existing in the mucosa of small intestines, but its activity is low in the plasma. it is evoked in the blood with the intravenous injection of much heparin. it is well known that da declines under the chronic mueosal injury when the small intestines are cut off or the mucosae become atrophied. aim of this paper is to confirm that da goes up to the measureable level by the intravenous injection of a small amounts of low molecular heparin(lmh objectives: the aims of this study is to investigate the inhibitor" effect of the prolease inhibitor, urinastatin, on endotoxin induced bacterial transleeation in mice. materials and methods:lcr mice were divided in six groups as the fotlows, group i: control mice receiving intraperitoneal injections (ip) of saline . and hr prior to sacrifice, group ih treated mice receiving ip of utinastatin . hr and endotoxin ( mg/kg) hr prior to sacrifice,group ilk sham reated mice receiving ip of saline . hr and endotoxin ( mg/kg) hr prior to sacrifice,group iv: saline control mice receiving ip twice during rain intervals,group v: mice received ip of utinasmtin, and min later they were received ip of endotoxin ( mg/kg), group vi: mice received ip of saline,and min later they received ip of endotoxin ( mg/kg). in group l,ii and ill ,the mice were killed by cervical dislocation.using stetile techniques,a middle-line incision was made and the mesenteric lymph nodes were harvested in order to culture on selective agars for quantitation of bacterial trauslocation. in group iv, v and vi, survival was recorded for days. unnastatin pretreatment could rednce mortality significantly. first surgey, shiga university of medical science, seta ,ohtsu - ,japan increased gut permeability correlates with inflammatory response in multiple trauma aptients. pape, h.-c. dwenger, a. grotz, m. regel, g. purpose: disturbances of intestinal permeability have been advocated as a pathogenetic factor of posttranmatic multisystem organ failure (mof). a close relationship with impairment of the stationary reticulocndothelial system (res) and a systemic inflammatory response (sir) was discussed. these tactors were investigated prospectively in patients with multiple trauma showing posttraumatie complications (multiple organ failure, mof). methods: polytranmatized patients were studied prospectively. according to a mof-score (goris) those with posttrattmatic complications were selected (> points at days), others were excluded. every second day gut permeability according to the ratio of urine concentrations of lactulose and mannitol (l/m) was evaluated (enteral application). at parallel time points res clearance capacity (k-value, invasion constant, normal range . - . mind) was studied after i.v. injection of mbq rotehuman albumin. liver perfusion was calculated from these data, total serum bilirubin (/zmol/l) was documented. serum elastase (#g/l) levels were determined enzymatieally. results . + + liver perfusion did not ehangu, bilirubin showed progressive worsening indicating mof. a positive correlation was present between l/m and k (r= . ) and between l/m and ela (r= . ). conclusions: there is a positive correlation between the time pattern of intestinal permeability dysfunction and res hyperactivity as well as between intestinal permeability and the systemic intlammatory response (elastase levels). the results speak in favor of an interaction between intestinal and extraintestinal inflammatory systems, which in eombiuation are likely to be responsible for post~anmafic complications. endotoxemia, il- release and consecutive acute phase reaction are observed as a host response to surgical trauma. as well vasodilative prostaglandins (pg) and thromboxane (tx) are released after abdominal meaenteric traction (mt). the following hypotension and acute hypoxeraja are duo to prostacyelin (pgiz) arm can be avoided by perioperative cyclooxygenase inhibition. we therefore focused on the effect of pg and tx liberated following mt on the induction of endotoxemia. methods: in a prospective, randomized double-blinded protocol patients, who were scheduled for major abdominal surgery (pancreatic or infrarenal abdominal surgery), were studied. ibuprofen ( mg i.v.) or a placebo equivalent was administered minutes before skin incision. mt was applied in a uniform fashion. baseline values were obtained before induction of anesthesia. further measurements followed before the incision of the peri[onenm (tl) and , , , min, . the plasma concentrations (,pc) of -keto-pgft,, txb: and-ki- -pgf ~ (stable metabolites of pgi , txa and pge~) were determined by ria. we measured endotoxin pc by limulus-amoebocyte-lysate test and il- levels by elisa. data are given as mean+sem (* p< . placebo vs. [ibuprofen] ). results: endotoxin plasma levels increased before incision of the peritoneum tl both in the ibuprofen pretreated and in the placebo group. peak pc were observed minutes after mt. endotoxin pc were significantly higher in the ibuprufen treated group (t . + . e[ . + . ] eu/ml). il- pc demonstrated an increase continuously from t to t (t + [ + ] ng/l) in both groups. after intentional abdominal mesenteric traction we observed a marked increase of -keto-pgf~,, pc up to h after mt in untreated patients with a peak of *[ ] ng/ at tl. also txb: and kh pge pc showed a considerabe increase up to h after mt in the placebo group. in ibuprofen pretreated patients the pg and tx pc remained within the normal range. discussion: our data clearly indicate a significant endotoxemia and il- release following major surgical trauma which is not initiated either by prostaglandin or thromboxane release. moreover endotoxemia is accentuated by ibuprofen pretreatment. therefore we hypothesize that in major abdominal surgery prostacyclin release-after mt may play a crucial physiological role in maintaining splanclmic microcirculation and thus preserving gut mucosal barrier function. objectives of the study it has been shown recently that parenteral and certain euteral diets promote the translocation of gut flora to the mesenteric lymph nodes (mln) and systemic organs, a process termed bacterial translocation (bt). in chow fed rats bt usually does not occur without further promoting factors. the goals of the present study were to determine whether the provision of defined amounts of standard lab chow during iv-tpn administration wotfld redane the incidence of bt, materials und methods male spf spragnle-dawley rats were divided into groups. group received standard laboratory chow feeding ad lib. in group a central venous catheter was placed, ligated and secured by a spring coil tether attached to a swivel allowing free movement in the housing cage and chow was fed ad lib. in group % of the calculated daily required calory intake (drci) ( /kcal/kg) was given by iv-tpn ( % glucose, , % amino acids) and % by limited chow administration. groups and received % and % of the drci by i.v. tpn and % and % respectively by chow feeding. group received iv-tpn only. after days the rats were sacrificed and the mln, liver, spleen and cecum removed aseptically, homogenized and cultured for bt samples of distal ileum were taken for light microscopy. the group with the least amount of chow shown to be protective against bt received the amount of non-fermentable fiber of that chow regimen during iv-tpn feeding and bt was studied. , + , , - , , / + ~ " , -+ , , -+ ~ - , / +~ + _+ , + , , - , -+ + , ~ , , -+ ~ conclusions: the administration of % of drci by chow feeding during iv-tpn significantly reduced the incidence of bt and maintained gut barrier function. the addition of the respective amount of dietary fiber of this group did not prevent iv-tpn-indueed bt. dr. med. m naruhn., dep. of general surgery, eberhard-karls-university, hoppe-seyler-str. previous experimental studies have suggested that a disturbed ecology of the enteric bacterial population might contribute to the development of bacterial translocation from the gut in acute liver failure (alf). in the present study, the effect of oral administration of lactobacillus reuteri r lc and oat fiber on bacterial overgrowth and translocation was investigated in rats with acute liver failure induced by subtotal ( %) liver resection. the oatmeal soup base was anaerobically inoculated with lactobacillae and fermented for hours, after which the animals were fed with either fermented or unfermented oatmeal or saline daily for days prior to the operation. bacterial translocation to mesenteric lymph nodes (mln) and the systemic circulation was determined, as well as the intestinal bacterial flora and enterocyte protein content. the incidence of bacterial translocstion to the systemic circulation was nit in rats subjected to sham operation and saline treatment and % in animals subjected to % bepatectomy and lreatment with fermented oatmeal, while - % and - %, respectively, in rats subjected to hepatectomy and treatment with either saline or unfermented oatmeal. only one rat with fermented oatmeal demonstrated bacterial growth in mln (p < . vs hepatectomy and treatment with saline or unfermented oatmeal). the enterocyte protein content significantly decreased (p < . ) in salinetreated animals following % hepatectomy, while there was no significant difference between bepatectomized animals with oral administration of fermented or unfermented oatmeal. the number of anaerobic bacteria, gram-negative anaerobes and lactobacillus significantly decreased and the number of e.cnli increased in the distal small intestine and colon in hepatectomized animals with enteral saline or unfermented oatmeal as compared with animals subjected to sham operation or bepatectomy with fermented oatmeal. our results thus show that the occurrence of bacterial translocatiou from the gut in % hepatectomy-induced alf could be prevented by enteral administration of fermented oatmeal, maybe partly due to a positive effect on the enteric bacterial ecology. _+ " +_ " . " data=mean_+sd, * stats anova p< . vs control. l+air and lap groups, both exposed to exogenous i.ps shnwm:t m significant increase (p<. ) in lps gut translocation compared to control and l+co . this correlated with a significant increase in peritoneal inflammatory responses (o -,tnf) above that of the control and l+co groups, while mac- and cr opsonized phagocytosis were significantly impaired. the absence of significant differences between l+air and lap groups indicates that lps rather than wound factors is the principle mediator. thus, lps plays a significant role in regulating peritoneal responses in the early post-operative period dept of surgery, rcsi, beaumont hospital, dublin , ireland brlke e, berger d, staneseu a, buttenschsn k, vasilescu c, seidelmann m, beger hg in patients undergoing a colonoscopy, endotoxin, endotoxin neutralizing capacity (enc), thromboxane b o (stabile metabolite of tbmomboxane ~), -keto-prostaglansin, leueotriene c , interleukin and the incidence of bacteremia were determined before and then every five minutes during the procedure. twenty-one of patients showed a significant increase of endotoxin plasma levels during colonoscopy (p= . ), whereas only one patient had a positive blood culture with bacteria obviously derived from the gastro-intestinal tract. the enc decreased significantly five minutes after the beginning of eolonoscopy and was diminished further thereafter. the baseline values were reached after hours. ~hromboxane b o levels also increased after five min. from to pgyml peaking at min. with pg/ml. -keto-prostaglandin,leucotriene c , ii- and crp remained unchanged. a control group of i volunteers who were not subjected to endoscopy, were prepared for eolonoscopy by orthograde lavage. the blood sampling procedure remained identical. no differences were seen in all described parameters for the controls. these data show that the gut barrier can be compromised by mininml invasive procedures, at least, concerning bacterial products. living bacteria, on the contrary, do not pass the gastro-intestinal wall. endotoxin, when determined by enc, is more sensitive than the conventional limulus-amebocyte-lysate test. no acute-phase reaction was induceri by the observed endotoxin translocation. it can be speculated from the dramatically enhanced thromboxane b levels, together with its hemodynamie effects, that the thromboxane release may support translocation of bacterial products. sepsis is common after hemorrhagic shock. this study aims to demonstrate that hemorrhagic shock alone can promote translocation of gut bacteria from intestinal tract to its regional nodes and subsequently to blood. one hundred twenty mice, divided into groups were subjected to , and minutes of %, % and % of hemorrhagic shock. on the specified time, blood cultures were taken and mice were sacrificed. the intestinal tract were histologically examined for any changes which allows translocation and its regional nodes were quantitatively cultured for translocated bacteria. there was a direct relationship between duration and degree of hemorrhagic shock and incidence of translocation (p . ). there was a high incidence of gut bacterial translocation to the mesenteric and mesocolic nodes in all degrees of shock (p . ). bacterial growth in the regional intestinal nodes increased and blood cultures were positive in direct proportion to degree and duration of shock. histologic evaluation of segments of git showed submucosal congestion to allow bacteria normally contained within the gut to cause systemic infections. translocation of gut bacteria in untreated hemorrhagic shock is clearly shown in this study on animal models. in this study, guotobiotic rats with known species of bacteria were subjected to total parenteral nutrition(tpn) and subsequent hemorrhagic shock. the purpose of the study was to observe the impairment of gut barrier function following tpn and hemorrhagic shock and to study the mechanism of enterogenic infection induced by tpn and shock.the results were as follows: .long term( - days) tpn induced impairment of gut barrier function, evidenced by atrophy of intestinal mucosa, significant decrease in diamine oxidase activity of intestinal mucosa and blood, and marked microecologic imbalance of the intestinal mucosa flora with dorminant growth of aerobes and relative decrease in anaerobes. the degree of mucosal damage were proportional to the duration of tpn. .in tpn+shock groups, failure of gut barrier function was found. ri,~ere were further damage in the mucosa, with a large number of gramnegative organisms invading mucosa and submucosa and a significant decrease in dao activity as compared with each relative tpn groups. these changes were significantly correlated with enhanced bacterial translocation, elevation of lps and mda levels in the plasma. these findings suggested that long term standard tpn impaired the gut barrier function, precipitating posttraumatic gut barrier failure. thus infec. fion following shock might be oi'iginated from the gut and it was obviously related to the impaired gut defence resulted from antecedent tpn. the determination of plasma dao activity might provide a valuable tool for the ear. ly diagnosis of gut injut;y during tpn and after trauma. in our earlier studies we have investigated the dynamics of granuloayte infiltration of the ischemic/reperfused s~all intestine (g. illy~s, j. hamar int. j. exp. athol. . . .) . there was a increasing infiltration of the mucosa c m~nating at the d to th hours of reperfusion. in the present series we have studied sc~e of the conseqn/ences and the possible role of this cellular reaction. ~in isehemia was followed by a hour reperfusion in the anesthetized rat. arterial ~/ad mesenteric venous blood samples were collected at m_in, i, ~ , and hours of reperfusion. elastase and lactate concentrations were determined and hamoculture was carried out from the blood samples, and tissue pieces from the heart, lung, liver and kidney were collected for histological analyses at the above mentioned times of reperfusion. all blood samples were free of cell bacteria. staphylococci appeared only occasionally at the th hour in the arterial blood .and at the d and th hours in the venous blood, respectively. arterial and venous elastase activities were high throughout the reperfusion, venous concentrations being higher at all times. lactate concentrations of the arterial and mesenteric venous blood samples increased during shock. ~ranuloeyte infiltration of all organs studied appeared during the d hour and it increased at later times of reperfusion. it is concluded that heavy infiltration of the intestinal mucosa can block bacterial translocation in most of the cases during reperfusion. granulocytes activated either by the reperfused area or by the released cytokines infiltrate other organs contributing by this way to the mesenteric shock s!rndrc~e. intestinal motility plays an important role for maintaining nutrient transport and absorption and for balancing the enteric bacterial population. disturbances of intestinal motility may be one of the earliest notable changes in intestinal function. in the present study, we aimed at determining early alterations in intestinal transit time following ischemia-reperfusion injury induced by occlusion of the superior mesenteric artery in the rat. intestinal ischemia was induced for and minutes by applying a microvascular clip on the superior mesenteric artery followed by reperfusion , and hours after clip removal. intestinal transit time was measured by the propulsion of a radiolabelled solution (cr ). light microscopy was performed on intestinal samples. macroscopical pathological changes were not observed. however, microscopically, mucosal epithelial oedema, degeneration or slight ulceration occurred in rats hours after reperfusion in ischemia- rain group and and hours after reperfusion in the ischemia- rain group. delayed small intestinal transit time was seen from hours and on after intestinal ischemia for both and rain ischemia followed by reperfusion. the distribution of radioactivity demonstrated that most radioactivity was accumulated in the first two segments following intestinal ischemia and reperfusion, significantly differing from what was seen in animals subjected to sham operation (p < . ). the distribution of radioactivity in segments and in the group with repeffusion hours after intestinal iscbemia for rain was significantly higher than that noted in the group with repeffusion hours after intestinal ischemia for min (p < . ). q'he results indicate that a delayed intestinal transit time may be one of the earliest pathophysiological alterations noted, associated with duration of gut ischemia, and a potential factor for the development of bacterial overgrowth, gut barrier failure and bacterial translocation, in hypovolemic conditions. bacterial infections still constitute a major cause of morbidity and mortality in patients with acute liver failure. the present study aimed at evaluating the effect of ethylhydroxyethyl cellulose (ehec) on bacterial translocation following surgically induced acute liver failure. acute liver failure was induced by subtotal hepatectomy ( %) in the rat. water-soluble ehec was administered orally and hours prior to hepatectomy. the incidence of bacterial translocation from the gut to mesenteric lymph nodes (mlns) and systemic and portal circulation was evaluated and the number of isolated bacteria from these samples and from intestinal content were determined. intestinal transit time, bacterial adherence onto the intestinal surface, intestinal mucosal mass, bacterial growth and dna synthesis, bacterial surface characteristics (hydrobiology: hydrophobicity, hydrophilicity and neutrality; surface charges: positive, negative and neutral) were also determined. hepatectomized animals showed a - % translocation rate to mlns or blood and hours after operation, while only - % of rats subjected to sham operation or animals with % hepatectomy and pre-treatment with ehec (p < . ). bacterial overgrowth, increased bacterial adherence onto the intestinal surface as well as decreased intestinal mucosal masses were observed in animals with subtotal liver resection alone, alterations that were prevented by enteral ehec treatment. a delay in intestinal -hour transit time occurred in both groups with subtotal liver resection, with or without enteral ehec. ehec inhibited bacterial growth and dna synthesis, and altered bacterial surface properties following hour incubation with bacteria. in conclusion, the findings in the present study imply that ehec alters enterobacterial capacities for metabolism, proliferation and invasion by effects on e.g. bacterial surface characteristics. furthermore, ehec seems to possess a trophic action on the intestine, rather than exerting its effect by enhancing intestinal motility. department of surgery, lund university hospital, s- lund, sweden disturbances in intracellular calcium signalling can potentially result in impairments of cellular responses vital to the functional integrity of both immune and non-immune cells, and thus contribute to a decrease in host resistance against infection and to multiple organ system failure during sepsis. studies in our laboratory have focused on assessments of intracellular ca ÷ regulation and ca~+-depended cellular responses in the liver, skeletal muscle and splenic tlymphocytes harvested from rats subjected to gram-negative intraabdominal sepsis. cytosolic ca + concentration, [ca *]i, and ca + fluxes were measured by the use of fluorescent ca + chelating dyes (fura- or indo- ) and ca respectively. to assess sepsis-related changes in ca + dependent cellular responses, we measured the acute phase protein response in the liver, the regulation of protein and sugar metabolism in the skeletal muscle, and the proliferation response in the splenic tlymphocytes. altered ca + i signalling with sepsis was correlated with an exaggerated inappropriate acute phase protein response ( % ¢) in the liver, and a blunted insulin mediated sugar utilization ( % ) and increased proteolysis ( % ~) in the skeletal muscle. in t-lymphocytes, a decrease in mitogen induced elevation of [ca +]i by - % was correlated with a significant depression in their proliferative capacity. these studies clearly suggest that altered calcium signalling is correlated with disturbances in cellular responses in both immune and non-immune cells during sepsis. the altered cellular responses adversely effect the outcome of the septic injury. (supported by nih grant gm ). alfred ayala, ping wang and irshad h. chaudry. changes in macrophage capacity to respond to foreign pathogens are thought to be central to the developing immunosuppression associated with traumatic injury. in this respect, the suppression seen in m~ functions following hen (a common component of traumatic injury) may be mediated by the direct or indirect inhibition of their capacity to perceive external stimuli (e.g., opsonized & non-opsonized bacteria, and their cellular components, etc.} due to the breakdown of the receptormediated signal transduction system. results of a number of studies by our laboratory and others indicate that this inability to respond to external stimuli is in part due to the loss of cell surface receptors. decreases have been documented for not only la antigen, but also c b, fc, and tnf receptors following hem in mice. furthermore, studies which have examined second messenger generation in these cells indicate that m~ derived from the peritoneum and spleen exhibit a decreased capacity to mobilize ca + from intracellular stores. this protein kinase dependent process of [ca+ ] i mobilization appears to be linked to the inability to synthesize inositol triphosphate. of interest, the depression in ca + signal generation appears to be inversely related to presence of elevated levels of camp in m~ from hen mice. we have reported that m~ priming agents, such as ifn- (which exhibits salutary effects on m~ function following hem), appear to restore cell signal transductive capacity while reducing the levels of camp. nonetheless, the extent to which depressed receptor signal transduction in hem, is due to receptor loss~dysfunction or elevated antagonistic second messenger levels remains to be determined. conclusions: significant impairment of calcium signaling occurs at all time-points prior to and following pha stimulation in trauma patients. tcell activation failure can, in part, be explained by the inadequacy of this essential intracellular second messenger system. restoration of immunocompetence following trauma will have to address strategies to better assess and restore this vital step in the activation sequence leading to proliferation during the antigen recognition process. patrick a. bseuerle institute biochemistry, albert-ludwigs-university, hermann-herder-str. , d- freiburg, germany the active form of the transcriptional activator nf-~b is a heteredimer composed of a and kda polypeptide. in this form, nf-'<b can bind with high affinity to decameric sequence motifs (consensus: "-gggpunnf'ypycc- ") found in enhancers and promoters of many genes activated upon a wide variety of pathogenic conditions, including viral and bactedai infections, acute phase response, oxidative stress and uv and gamma irradation. clinically important target genes for nf-k:b encode inflammatory cytokines (il- b, tnf, [l- , il- , gm-csf etc), cell adhesion molecules (icam- , elam- , vcam- ), acute phase proteins and immunoregulatory ceil surface receptors. in most cell types, nf-~:b is sequestered in the cytoplasm and, therefore, unable to initiate transcription of these genes. the cytoplasmic form of nf-~:b contains one additionai subunit, referred to as h,;:b. i~b can reversibly suppress dna binding and nuclear uptake of nf-~b by virtue of its association with p . upon stimulation of cells, ikb is proteolytically destroyed, allowing nf-kb to enter the nucleus and activate genes upon binding to transcriptional enhancers. we have demonstrated that various antioxidative compounds inhibit the activation of nf-~b in response to many inducers. moreover, nf-~:b was shown to be activated upon treatment of cell cultures with p.m-amounts of hydrogen peroxide. these data suggested that nf-~b prinicipaliy is an oxidative stress-responsive transcnption factor. the notion is supported by numerous reports on the induction of oxidative stress by many nf-nb-inducing agents. nf-~b activition can likewise be suppressed by protease inhibitors. these drugs apparently inhibit a yet unidentified protease responsible for the inducible decay of inb. we propose to use nf-nb inhibifors as novel drugs with a very broad application under acute phases el inflammation, injury and infection. northern blot experiments revealed that expression of the recently discovered lipopolysaccharide binding protein (lbp), a / kd glycoprotein, in the liver rises substantially during the acute phase response. experiments with an in-vivo acute phase model using new-zealand-white rabbits and also in-vitro experiments employing stimulated hep-g cells showed that lbp transcripts could be induced to ford within hours after induction with cytokines. by using a newly established nonradioactive nuclear run-on technique we show that induction of lbp during the acute phase is transcriptionally regulated. furthermore we screened a human genomic library and were able to clone the lbp gone, containing the promoter . kb upstream. several liver-specific and "acute-phase-typical" potential binding sites were found fn the promoter region and reporter gone assays were set up. several caat-boxes, the il- responsive elements hstf-hsp . and h-apf- rs as well as the transcription factor hnf- and the glucocrticoid-responsive elements oct- -d and gcre were found, which correlates with the induction pattern of lbp mrna in hep-g cells by il- , il- and dexamethasone. pcr-based analysis of the exon-intron pattern of the lbp gene revealed similarities with the genes of two other lps binding proteins, namely bpi and cetp. further analysis of this novel acute phase protein, that also has an important role in endotoxin recognition and immunomodulation will help in understanding the complex acute phase mechanism and potentially lead the way to new therapeutic strategies. lps activation of nucle?/r fa-ctor:~b " (nf:~bj"in cd .transfected fi'broblasts does not appear to require tyrosine kinase activity d. t. golenbock, r. delude, r. savedra, s. vogel and m. j. fenton lipopolysaccharide (lps) is the major constituent of the outer leaflet of gram-negative bacteria. during the course of serious infection, shock may occur as a result of the interaction of lps with macrophage lps receptors. cd , a kda glycosyl phosphatidylinositol (gpi)-linked protein, functions as an lps receptor. a critical issue in lps activation concerns the mechanism by which cd , which has no transmernbrane domain, transduces a sig,nal after lps binding. evidence exists which suggests that cd may signal cells after lps binding by interacting with an lps signal transducer with tyrosine kinase (tk) activity. wild-type chinese hamster ovary fibroblasts (cho) normally do not respond to lps, but can be activated following transfection with cd (cho/cd ). stimulation of cho/cdi with as little as ng of lps/ml resulted in the release of h-arachidonic acid ( h- : ) into the culture supernatant. in addition to h- : release, we examined lps-imiuced activation (transl cation) of the transcription factor nf-~b. similar to lps-induced h- : release, these responses were observed only in cho cells lransfected with cd closely resembling the same response in the marine macrophage cell line raw . . maximum nf-~cb expression occurred at minutes. in contrast to lps-induced h- : release, dexamethasone, cycloheximide, and actinomycin d had no effect on activation of nf-r,.b in cho/cd , suggesting that nf-r,b activation begins early after lps binding to cd and does not require transcription or translation. we then examined cells for lps-induced tk activity by western blotting cellular ly~ates with anti-phcsphot-'rosin:~ anl',bodie~" although tk activity was seen in lps-stimulated raw cells and phorbol ester stimulated cho/cd , lps-induced tk activity in cho/cd was not observed. although the tk inhibitor herbimycin inhibited the release of h- : in cho/cd and raw cells, neither herbimycin nor genestein effectively blocked nf-r,b activation in either cell type. these results suggest that tk activity is involved in a portion of the signaling pathway that is distal to initial signal transduction. the absence of observable lps-induced phosphotyrosine residues in cho/cd cells as well as the failure of tk antagonists to inhibit lps-induced nf-~cb activation suggest that the proposed cd -related lps signal transducer in cho/cd is not a tymsine ldnase. dimished cardiac inotropic function and response to -adrenergic receptor ( -ar) stimulation are frequently seen in septic patients, these cardiac defects are also seen in animal models of entoxemia. to determine the characteristics of the decreased transmembrane signal associated with the decreased beta adrenergic response we measured the force and rate of contraction of atria harvested from sprague-dawley rats exposed to endotoxin ( : gm/kg). to stimulate various components of the transmembrane signal cascade of -ar was isoproterenol (i -ar), acetylcholine (m ach receptor), naf(gs and gi proteins), forskolin (adenylate cyclase), and calcium (intracellular contracttile signal) were used. to eliminate the transmembrane control of calcium and test the intrinsic contractile response, hypothermia ( °c) was used the results showed alterations in the inotropic function with no significant difference in chronotropic response. the inotropic reponse for the endotoxin exposed atria and controls is shown below these results show that there is a transmembrane signal defect both for the i~-ar signal and for ca +. these data indicate that the level of the defect appears to be at the g proteins. ischemia-reperfusion of the rat intestine produces an injmry both to the intestine and to distal organs, notably the lungs and liver. prior studies have shown that h of intestinal ischemia leads to a nemtrophil independent reperfusion injury of the gut. thus, rats rendered neutropenic by pl~l antiserum have been shown to express a severe local gut injury when subjected to ischemia and reperfusion. this gut injury is postulated to he mediated at least in part by the complement system. the soluble (s) cri receptor which binds to c b and cdb was given to rats at a dose of mg/kg by intravenous bolus, min before reperfusion of the ischemic gut. a control group was given pbs buffer. at h of rmperfmsion the animals were sacrificed. pbs treatment led to a significant activation of both classical and alternate complement pathways while scri inhibited both pathways (fall to zero of chbo ). intestinal mucosal injury score, assessed by histological grading was reduced by scri ( . ± . to . _+ . ,p< . ) . intestinal neutrophil sequestration estimated by assay of myeleperoxidase was also reduced ( . ± . to . ± . u/g). c was detected in the gut hy i~unohistochemistry. remote organ injury was modified that is lung permeability (ration of concentration of radiolabelled albumin in broncho-alveolar lavage fluid to that in blood) was reduced ( . ± . x i to . ± . x , p<o. ). however, neutrophil sequestration by the lungs was unaffected. this remote protection is thougth to be related to prevention of ic b deposition on imng andotheli~ which activates adherent neutrophils. these findings support the hypothesis of complement mediated local and remote injury. we postulate that complement fragments may be deposited either as a result of the ischemia induced loss of membrane bound complement regulatory proteins, such as decay accelerating factor or membrane cofactor protein, which allow complement fragments to accumulate on the endothelial cell surface. alternatively, increased expression of p-selectin on the endothelial cell surface may, by means of its complement binding domains, act as a lattice for complement deposition. indeed, p-selectin has been detected on the surface of endothelial cells ans platelet-neutrophil aggregates recovered from the intestine of these animals. ischemia ( hr) followed by reperfusion (dhr) of rat hind limbs resuits in local (muscle) injury as well as in remote (lung) injury, which is characterized by increased vascular permeability (leakage of j sl labeled albumin) and neutrophil accumulation (tissue content of myeloperoxidase, mpo). within minutes of reperfusion following ischemia, tumor necrosis factor-o~ (tnfc , interleukin-i ( l-i) and il- plasma levels increased significantly, reaching maximum levels after hours ofreperfusion. polyclonal antibodies to tnfet and l-i provided significant protection against muscle and lung injury. muscle and lung vascular permeability decreased in anti-tnfct treated rats by % and % respectively and mpo was reduced by % and %. antml- yielded a protection in muscle and lung vascular permeability of . °, and . % respectively whereas muscle mpo was reduced by . % and lung mpo by . °, . these results were confirmed by the use of soluble tnf~ receptor and il-i receptor antagonist (il-ira). when icam-i expression in vivo was examined using mabeled antmcam-i, constititive expression of icam- was evident only in lung but not in muscle. during reperfusion icam- expression increased by . %. treatment with anti-tnfcx or il-ira reduced icam-i upregulation by . °, and . % respectively. frozen sections of normal rat lung revealed no evidence of e-selectin expression, whereas lung sections obtained after ischemia and reperfusion immunhistochemically demonstrated expression of e-sdectin. lungs from rats pretreated with anti-tnfct were negative for e-selectin staining. our data indicate a role for cytokines in the upregulation of adhesion molecules in ischemia/reperfusion injury. adherence of neutrophils to the coronary endothelium is associated with significant myocardial injury following min of ischemia (mi) and . h of reperfusion (r). p-selectin is expressed by activated endothelial ceils and platelets within min and tethers polymorphonuclear leukocytes (pmns) to the endothelium. we examined the cardioprotective effects of a monoclonal antibody (mab) designated pb . to p-selectin in a feline model of mi+r. pb . ( mg/kg) administered rain post-occlusion ( min prior to reperfusion) significantly attenuated myocardial necrosis compared to a non-blocking mab (nbp . ) for p-selectin ( + vs + % of area-at-risk, p< . ). endothelial dependent relaxation to acetylcholine was also significantly preserved in ischemic-reperfused coronary arteries isolated from cats treated with pb . compared to nbp . ( + vs + , p< . ). furthermore, pmn adherence to ischemic-reperfused coronary endothelium was significantly attenuated in pb . treated cats ( + vs + pmns/mm z, p< . ). also, normal coronary endothelium activated with thrombin ( u/mt) had increased pmn adherence under conditions of shear stress, an effect which was significantly (p< . ) blocked by pb . , but not by nbp . . furthermore, p-selectin is markedly upregulated - min post-reperfusion in coronary arterial and venous endothelinm. similar results were obtained in a rat model of mesenteric ischemia/reperfusion, including upregulation of p-selectin on mesenteric vascular endothelium. these results demonstrate that tethering of neutrophils to endethelium by p-selectin is an important early consequence of reperfusien injury, and a specific monoclonal antibody to p-selectin exerts significant endothelial preservation and cardioprotection in ischemia and reperfusion states. as recent interest has increased in the role of extracellular matrix proteins in inflammation, we examined the role and relationship of laminin and fibronectin to the processes of cell infiltration in a rat model of heart isografts. changes occurring in this system are on a nonimmunological basis and may be an effect of the initial surgical manipulation and the warm and cold ischemic times of transplantation. lewis (lew) donor hearts were transplanted into lew recipients, naive lew acted as naive controls (nc). grafts were harvested , , , , , , , and h after transplantation (n= /time point) . snap frozen sections of organs were stained for cd- (w / ), cd- (ox ), t-lymphecytes (tl) (ox- ), macrophages (ed , ed ), neutrophils (pmn) (mom), icam-i (iap ), laminin and fibrinogen. results were evaluated visually by counting cells/field of view (cf) for w / , ox , ox , ed , ed and mom, staining for icam , lain and fib was assessed visually using a scale (ve= - ). as early as h after transplantation, fibrinogen and laminin expression increased in i, peaking h after transplantation (ve= . ; nc: ve=i. ) . at and h in i, on both vessels and interstitial cells laminin and fibrinogen expression had declined to baseline (ve=i. ) but rose again at h, peaking at h (ve= ) and remaining elevated thereafter (ve= ). the high expression at h correlated well with the maximal pmn infiltration at that time (cf= ) in i returning to baseline at h (cf= . ) and thereafter. the second peak at h correlated with the onset of macrophage and t-lymphocyte infiltration. thus, laminin and fibrinogen seem to guide leucocyte infiltration following graft ischemia during the time of reperfusion. background. reperfusion of ischemic kidneys with xenogeneic blood leads to activation of endothelial cells and circulating leucocytes with the final consequence of microvascular thrombosis. in concordant systems, the mechanisms of rejection can be studied due to cross-reactivity of mediators with anti-human monoclona~ antibodies. aim of the study. to obtain information about the kinetics of proinflammatory cytokines and production of soluble adhesion molecules in the acute phase of reperfusion, eight kidneys from rhesus monkeys were perfused ex-vivo with human blood (group b/o) for one hour in a closed system. blood levels of il-lb, il- , tnf-a, soluble icam and e-selectin were measured in elisa-technique under steady-state conditions. results. cytokine levels rose significantly within the minute interval (il-lb: , __+ , to , __+ , pg/ml; il- : . - , to , __+ , pg/ml; tnf-a: , __+ , to , + , pg/ml; p< . ). immediately after the beginning of reperfusion, soluble icam- and selectin levels were abnormally high and constantly rising throughout the observation period, reaching significance at minutes. discussion. high levels of proinflammatory cytokines may lead to an induction of adhesion molecules, thus upregulating the leukocyte-endothelial interaction in an complement-independent mechanism. specific pretreatment with monoclonal antibodies against icam- , lfa- or other soluble mediators may be useful in downregulating hyperacute rejection in transspecies transplantation. prolonged ischemia has been associated with later dysfunction of solid organs as it may cause upregulation of adhesion molecules, production of cytokines on vascular endothelium and migration of host cells into the graft. these early events may lead to irreversible organ injury. this study evaluates the effects of global ischemia on early immunohistological changes in cardiac grafts transplanted in rats. isografted hearts (lewis->lewis) were were divided into ischemic and non-ischemic groups (n= /group). all donor hearts were flushed immediately with cold saline. non-ischemic hearts were then transplanted within rain, ischemic hearts were stored in cold ringer's solution for hours before revascularization. representative grafts were removed after . , hrs, and days, and evaluated immunohistologically (cells/field of view=c/f). restitution of ventricular activity was significantly delayed in ischemic grafts ( vs rain). after hrs, all ischemic grafts exhibited an extensive interstitial edema, declining slowly thereafter. at the same time, numbers of pmn peaked ( vs c/f in non-ischemic grafts), whereas edl+macrophages ( vs c/f) and tnfe expression peaked by hrs. by hrs t-lymphocytes began to enter ischemic myocardium and icam- was moderately increased. after days cellular infiltration had returned to baseline, and no differences were seen among both groups after days. global myocardial ischemia inhibits initial graft function, and engenders a brisk inflammatory reponse, primarily pmn and macrophages, with increased mhc class ii and cytokine expression. leukocyte -endothelial interactions are the result of endothelial activation, leukocyte activation or combination of both, which are accompanied by nee-expression, upregulation or shedding of adhesion molecules (selectins, inlegrins). such interactions differ with regard to the stimulus (e.g. thrombin or histamine for p-selectin, endotoxin or tnf/il- for e-selectin), the time course of response (minutes versus hours) and the localisation in different organs. recently assays are available for circulating soluble fragments of the cell bound adhesion molecules e.g. se-seleetin was found to be increased in plasma concurrent with high circulating endoloxin and cytokine levels. the importance of adhesion molecules for the sepsis event is evident, while effectiveness of anti-adhesion inolecu]e therapy is controversial e.g. beneficial anti-e-selectin therapy in baboon bacleremia but deleterious effects of amti-cd treatment in the same model. in other species similar controversial results with anti-cd therapy in sepsis were reported. steven l. kunkel,theodore standiford* and robert m. stricter. the migration of leukocytes to the lung during endotoxemia is dependent upon the coordinated expression of lung vascular adhesion molecules and the subsequent production of appropriate leukocyte chemotactic proteins. in experimental animals, neutrophils accumulate within the lung soon after the administration of endotoxin, while mononuclear cell infiltration occurs in a more distal manner. a kinetic analysis of lung leukocyte levels revealed a -fold increase in neutrophil numbers associated with dispersed lullg tissues hours after lps treatment, while macrophage levels increased by -fold at the hour time point. thus, the recruitment of different leukocyte populations to the lungs during endotoxemia is likely directed by different mechanisms. recent studies have identified a supergene family of small inducible chemotactic cytokines (chemukines) which possesses chemotactic and activating properties for neutrophils. the prototype of this family is interleukin- (il- ). interestingly, a related supergene family has been identified which possesses activity for recruiting mononuclear cells. examples of this group of inflammatory chemukines are monocyte chemotactic protein-i (mcp-i) and macrophage inflammatory protein-i alpha (mip-i). in initial in viva studies we examined whether mip-i was expressed systemically or in a compartmentalized fashion post lps challenge. assessment of plasma cytokine levels revealed maximal tnf levels occurred i hour post lps administration, returning to baseline by hours, while mip-i levels were maximal at hours ( , ng/ml), with a second peak at hours after lps challenge. interestingly, aqueous extracts of liver homogenates from lps treated animals demonstrated no mip-i levels, while aqueous extracts of lung revealed a -fold increase in mip-i levels over control lungs. immunohistochemical analysis of the lungs from hour lps treated animals demonstrated the alveolar macrophage was a rich source of mip-i protein. cell-associated mip-i was also expressed by blood monocytes adherent to the pulmonary vascular endotheliun, however the expression of monocyte-mip-i was observed by hours post lps administration. immunohistochemical analysis also demonstrated that mip-i antigen is associated with the extracellular matrix on the interstitial side of the endothelium. this suggests that the extracellular matrix, which is produced during inflammation, can bind mip-i and this may serve as a depot for the prolonged presence of nip- . in additional studies we have demonstrated that the intratracheal instillation of rmui [ip-l(loong) activation of polymorphonuclear leukocytes by inflammatory stimuli may contribute to the development of multiple organ failure in septic patients. thereby pmnl are proposed to avidly adhere to vascular endothelium causing damage by the subsequent release of toxic agents. as cellular adhesion is primarily mediated by -integrins and lselectins, the present study compares the expression of these adhesionmolecules on pmnl in septic patients and healthy volunteers. methods: expression of -integrins and l-selectins on pmnl was measured in whole blood by flow cytometry using the monoclonal antibodies ib and dreg , baseline values were determined immediatley after drawing blood. in addition cells were incubated min at °c to allow for spontaneous regulation of adhesion molecules. blood specimens from septic patients were obtained during the course of their illness. control values were determined in healthy volunteers. results: baseline expression of -integrins and l-selectins was not signifcantly different in septic and in healthy subjects. in contrast, there was a significant upregulation of g -integrins and shedding of l-selectins of pmnl in septic patients (sp) compared to healthy volunteers (hv). the local or systemic production of inflammatory cytokines, such as tumor necrosis factor alpha (tnfc~), can serve to modulate multiple aspects of neutrophil function. the ability of neutrophils to leave the circulation and migrate to areas of infection is one essential component of host defense. l-selectin, a leucocyte-associated adhesion molecule, is responsible for the initial reversible contact between neutrophils and endothelium and the subsequent roiling action of neutrophils along the vessel wall. in contrast to other adhesion molecules, l-selectin expression is rapidly down-regulated after neutrophil activation. the loss of l-seleclin may thus be a critical determinant of how neutrophils become unbound from their endothelial attachments and enabled to proceed towards an underlying extravascular area of infection. we hypothesize that the shedding of l-selectin is a strictly controlled process, occurring primarily at localized sites of inflammation, which may be modulated by tnf~, a flow cytometric method of staining neutrophhs by monoclonal antibodies in whole blood is described whereby the kinetics of l-selectin shedding may be followed in real time. the dose response and time course of in-vitro l-selectin shedding by neutrophils from normal human subjects was assayed after exposure to n-formyl-methionylleucyl-phenylalanine (fmlp) and tnfc~. either singly or in combination, our results show that l-selectin shedding can be reliably followed over time. a significant percentage of cells shed l-selectin after exposure to pg/ml tnfc~ or nm fmlp (but not at pg/ml tnfc~ or nm fmlp). greater numbers of cells were able to shed their l-selectin when fmlp and tnf~x were presented in combination rather than alone. high levels of tnfc~ did not appear to alter the threshold concentration of fmlp required to induce shedding, we conclude that the extent and rapidity of l-selectin shedding may be modified by different combinations of ligands and that shedding, by vidue of the high concentrations of cytokines or chemotactic factors required, is a process localized to sites of infection or inflammation. we prospectively studied patients with severe sepsis syndrome; group a : septic shock with or without adult respiratory distress syndrome lards) (n = , bacteremia = ); group b : sepsis syndrome without septic shock (n = , bacteremia = ). serial plasma samples obtained on day , , , , and , were assayed using elisas method (british biotechnology), normal control levels of soluble icam- and e-selectin, obtained from healthy volunteers, were respectively ± . ng/ml and ± . ng/ml (mean _+ se), acute lung injury was quantified dally on a tour-point score system (murray, am rev respir dis, ) . compared to control mean values, initial levels of groups a and b were significantly higher for icam- (p < - ) and e-selectin (p < - ). comparisons of group a and [] (* = p< . ; ** = p< . t) soluble icam- levels of group a enhanced significantly (p< . ) during the first hours, and a sustained high levels was of bad prognosis ( % of survivors at day ). the evolution of soluble icam- and e-selectin levels were significantly correlated with murray's score (spearman test : p < . ). conclusion: these results suggest that endothelial adhesion molecules are released into the plasma of patients with severe sepsis syndrome. soluble icam- and e-selectin are correlated with endothelial lung damage, and loam- seems to be a better indicator of the severity of endothelial injury. introductory remarks to anti-adhesion molecule strategies as a therapeutic modality ch wortel, repligen corporation, one kendall square, building , cambridge, ma , usa. the development of antimicrobial therapy represented a major breakthrough in the struggle against disease. it strengthened the notion that disease could be overcome by eliminating foreign invaders threatening the host. this paradigm has proven to be very successful, the threat of many infectious diseases has significantly changed, some have even been eradicated. nevertheless, sepsis has remained a severe condition, increasing in incidence while mortality remained very high. more recently, it has become increasingly clear that besides the nature and treatment of an exogenous agent, the reaction of the host defense itself plays a pivotal role in the outcome of the event. endogenous mediators, such as tnf, il-i, il- and il- , govem many of the actions of the host defense system. while the expression of these cytokines more often than not benefit the host, (over)-expression can cause severe damage. based on this hypothesis,anticytokine strategies, such as those targeted against tnf or il- , have been evaluated for the treatment of sepsis. results of these early studies have not yet indicated success in improving the outcome of the disease. it has been difficult to define a patient population where a benefit could be reproducibly shown. furthermore, it has been documented that synergy between cytokines occurs, but detailed knowledge of the cytokine network is not yet available. it is conceivable, that neutralization of one cytokine prompts the induction of another which will evoke the intended response in the host. recent data obtained in human endotoxemic volunteer models seem to confirm this. if this turns out to be the case, neutralizing a single cytokine may not be a successful approach. cytokines in tum, induce various adhesion molecules, such as icam- . such molecules regulate for instance the neutrophil-endothelial cell interactions, which are thought to play an important role in the pathogenesis of systemic organ injury. the potential for monoclonal antibodies to adhesion proteins to reduce vascular and tissue damage has been studied in a large number of experimental models. protective effects have been observed in a wide variety of inflammatory, immune, and ischemia-reperfusion injuries. thus, altering the host response by modulating the function of adhesion molecules may attenuate the inadvertent injury caused by inappropriate behavior of host defense cells. targeting cellular surface interactions has been added to the efforts to change the outcome of disease. modulation oftheseprocesses seems very promising, but may temporarily leave the host without effective defense mechanism. great care therefore, must be exerted when studying this powerful two-edged sword in a clinical setting. our knowledge of the role of adhesion molecules in the intlammatory response has increased rapidly due to the availability of new reagents and mice geneticly deficient in adhesion molecules. these molecules are important in interactions of leukocytes with endothelial cells, other leukocytes, platelets, and epithelial cells. when these molecules are engaged, they can also play a role in activating leukocytes and their effector functions. in the venules of the systemic circulation, adhesion often occurs through a series of sequential interactions. initial interactions are mediated by members of the selectin family to loosely associate the leukocytes with the endothelium and are followed by firm adhesion requiring members of the integrin and immunoglobulin family. later interactions with endothelium may require pecam. adhesion molecules are usually required for leukocyte emigration in response to extravascular stimuli and for neutrophil-mediated endothelial cell injury. they are critical for host response in many diseases including infections. however, when the inflammatory response results in damage to host tissues, patients may benefit from blocking the leukocyte response. anti-adhesion molecule agents are an important potential antiinflammatory therapy. the focus of anti-adhesion therapy may be at any step of the sequence. diseases where anti-adhesion molecule therapy may benefit patients include ischemia/reperfusion injury in many organs, ards and mof, and transplantation, both to protect the donor organ from ischemia/reperfusion injury and to inhibit graft vs host disease. many strategies have been considered and include: ) blocking the ability of adhesion molecules to recognize their ligand using antibodies that have been humanized or soluble receptors linked to igg to prolong their circulating halflife, ) blocking the ligands for adhesion molecules using soluble adhesion molecules, peptide analogues, or oligosaccharides, and ) blocking the production of the adhesion molecule using anti-sense oligonucleotides. because the synthesis of adhesion molecules is usually regulated by cytokines, inhibiting the action of cytokines is another potential site for interrupting the adhesion process. although important issues of safety must be evaluated, the potential for modulating the inflammatory response make this an exciting area of improvement in health care delivery. claire m. doerschuk, m.d.; riley hospital for children, room ; barnhill drive; indianapolis, in usa. modulation of neutrophil-endothelial cell adhesion with anti-cdl i/cd monoclonal antibodies as a therapeutic modality. ch wortel, repligen corporation, one kendall square, building , cambridge, ma , usa. the central role of inflammatory cells in the pathogenesis of lung and systemic organ injury is well recognized. binding of neutrophils to endothelial cells and migration into the parenchyma are largely regulated by complementary adhesion molecules. the leukocyte integrins are glycoproteins expressed on the neutrophil surface and in the cytoplasmic granules. integrins consist of a common beta or cluster differentiation (cd) chain covalently linked to one of three different alpha chains (cdlla, cdllb, cdilc) and exist on the cell surface as three distinct heterodimers. cdlla/cd is expressed on all leukocytes, whereas cd b/cd and cd c/cd . are restricted to cells of myeloid origin. cd i / cd interacts with intracellular adhesion molecule- (icam-i), its ligand on endothelial cells. the potential for monoclonal antibodies to adhesion proteins to reduce vascular and tissue damage has been studied in a large number of experimental models. protective effects with anti-cd antibodies have been observed in a wide variety of inflammatory, immune, and isehemia-reperfusion injuries, such as arthritis, burns, endotoxic shock, bacterial meningitis, autoimmune diabetes, nerve degenemrion, allograft rejection, allergic asthma, acute lung inflammation, skin lesions, and ischemia-reperfusion models of the intestine, myocardium, lung, skeletal muscle, and central nervous system. protective effects have also been observed in animals resuscitated following hemorrhagic shock. blockage of cd , however, would affect all leukocytes, as would antibodies to cdlla/cdi . targeting cdllb/cd would affect cells of the myeloid lineage only, which could prove to be beneficial. cd b/cd is not only involved in transendothelial migration, but is also implicated in adherencedependent formation of reactive oxygen species. blocking cd lb/cd may therefore not only reduce the numbe r of leukocytes accumulating in the tissue, but also attenuate the oxidant stress of infiltrated neutrophils. anti-cd b treatment has been used effectively to reduce tissue injury initiated by ischemia-reperfusion, complement activation and endotoxemia. altering the host response by modulating the function of adhesion molecules may attenuate the inadvertent injury caused by leukocytes, but may also temporarily leave the host without effective defense machinery. overall, animal studies suggest that it may be safe to inhibit neutrophil adhesion for a limited period of rime. these observations will have to be confirmed in carefully designed clinical trials. c, arbobydrams are ubiquizom constir~uts of cell sv.rfaees, and possess many c~xssfies ttm~ m~,e ~em ide~. canaidates for r~ognifioa mole~ule& in m~y systems whe,~ cer udhesioa ~lays a critical ro~ car~hydram l:~dtag ~otegas have been shown to b~ad tocell surfa~ earbohydzaxes ~nd pzrl~pate in cell-ceil lumtaefion& such sys,.ems include ~rti~za~io=, deveaopmeat, l~thoge~-hcet reeog--ition ~d i~zmmadon_ in particular, tb.z recent di%~ve~ of lhe selec~ and th~ impo.~a~c~ in teukccy~udo~lelium adh~ion has -~f~m av.c~on ok l~in m~ted cell adhe~on. s~vere/poten~s/cs.rbohydr~ l~ga~s hrve ~e~l ~u~ilied for ~he s~lcc~ins. the,~ c~u be broadly di,,sded la~o ~wo m'oups -sibyl l~wis x m~ mh~.~l oligo~chadd~s, ~d sf/~ ca~ohydmma, all ~:~ ~l~dns bind m siflyl l~wis x (sie$ o!igos~ccb.e.rkms, zlthou~ w~ differing avi~re~. 'we have i~¢n~ed the functional g~oups a s~ex ~n~ med/a~ ~he b~u ~di~g of ~h~ c~b hydmm = e-se/sedm we have used ~hat iv.formation to sya~esize sle ~ '~mt gs r.he, t focus on replacing slslic ~sd ~nd fuc s¢ wi~ simpler, more stable strunt~es. a[~ou~a ~ proeer~ is ongoing, we hve been ~ucee,.~ful a~ rep~aein t.ke si~ic a~id. residue wi~ std.fzte. ~ce~ or la~c amd groupa we t'we ex aninad &e ten, bunion of ezed~ hydroxyl group of the fizeose residue ~ billding of e-, l-~nd p-selees..u. we have also found m~fi~fio~ of the reducing end ~¢.cha'i~ ~z increase mtagovsst activity. the, m¢ond. group of figs,rids a.r eontzin su~a~ u a ea.rbohydr~t¢ support,, und seem to bi~.d to t~e sele~ti~s wi~ dlf:ferem characteristics c .an does sle:, s=h compounds are m ogniz~d by l-selects. md p-selectia, bur., in genera/, not e, selecti~ these dam may mdicam r.hat l-and p-s~ ¢at~ h~d via o, second ~te thaz operates lu~.ead of, or in conjunction with ~tc sle" b~ding ~iite. dam rela~&~g to ±e, se two types of ,ml~ liga~ds have beam t~ed to desig~ potential the ~peutics for i~fi~anmat ry disease. lr:rng maimai models of acute lung lu ury we can demo~trate that eompmmds that inhibit seleetiu birding ~ ~i~o hzve ber~ficial effects when uc~d in rive. progressive microvascular damage in the tissue adjacent to a cutaneous burn injury results in extension of burn size. the role of leukocytes in the pathogenesis of microvascular injury was investigated by inhibition of their adherence to the microvascular endothelium using monoclonal antibodies directed to leukocyte cdi or its endothelial ligaud, intercellular adhesion molecule- (icam- , cd ). a model of thermal injury was developed using new zealand white rabbits. two sets of three full-thickness burns separated by two x -mm zones were produced by applying brass probes heated to °c to the animals' backs for sec. cutaneous blood flow determinations carried out with a laser doppler blood flowmeter were obtained for hours. there were five experimental groups: controls given saline alone; animals given monoelonal antibody to the cd r . prior to burn injury (pre-r . ); animals given r . min after burn injury (post-r . ); animals given a monoclonal antibody to icam-i, r . prior to burn (pre-r . ); and animals given the r . min postburn injury (post-r . ). blood flow in the marginal "zone of stasis" between burn contact sites was significantly higher in the antibody-treated animals. administration of the antibodies min after injury was as effective as preburn administration in preserving blood flow. at hr post-burn all antibody -treated animals had blood flow in the areas at risk for progression (i.e., the zone of stasis) at or above baseline levels while the control animals had levels equal to . _+ % of baseline (p < . by analysis of variance and mann-whitney u test). these results indicate that leukocytes play an important role in the pathogenesis of burn wound progression, and that this progression can be attenuated by moduiating adherence to endothelial cells. a wealth of information now supports the hypothesis that inhibition of cell adhesive mechanisms will nter the course of immunologicand inflammatory processes. what remains unclear is whether inhibition of specific mechanisms wfl[ be of therapeutic benefit in any specific human disease. current data derived from animal models are not inconsistent with the hope of therapeutic benefit, but techniques for inhibition (e.g., antibodies, antisense oligonucleotides, inhibitory peptides, inhibitory carbohydrates, smaii synthetic inhibitors, etc), tissue and species differences in the relative contributions of adhesion molecules to the inflammatory process, and the cascade model of adhesive interactions are all confounding issues, making predictions of therapeutic benefit in any specific human disease process very difficult. additional concerns involve the potential roles of adhesive mechanisms in host resistance to infection. as human therapeutictdals are initiated, more exact information on the roles qf specific adhesion molecules in human disease should emerge. inhibition of leukocyte adherence to endothelial cells can represent a novel therapeutic approach to septic shock. we performed a pilot study to evaluate the safety and tolerability to cy- , a monoclonal antibody against human e-selectin, in patients with septic shock. septic shock was defined by clinical signs of sepsis, a documented source of infection, and fluid-resistant hypotension requiring the use of vasopressors. eleven patients entered the study, but patients who died during the first hours were excluded, as this was part of the protocol. cy- was administered as a single intravenous bolus of . mg/kg (n= ), . mg/kg (n= ) or i mg/kg (n= ) mg/kg. the antibody was well tolerated. none of the patients died during the day follow-up period. organ failure was assessed for organs (cns, lungs, liver, kidneys and coagulation). the mean number of organs failing, which was initially . ± . , decreased to . ± . at the end of the study (p<o.o ). these results are therefore encouraging. administration of an antibody to e-selectin in patients with septic shock requires further study. the importance of cytotoxic t lymphocytes (ctl) in the host defense mechanism against viral as well as parasitic and bacterial diseases is becoming increasingly appreciated. the design of vaccines to generate cytotoxic t ceils from nonviable or subunit constructs, however, poses challenges due to the unique characteristics of antigen processing and presentation by class i major histocompatibility complex (mhc) molecules. since the final antigen recognized by ctl are short peptides capable of high affinity binding to class i mhc molecules, we have designed strategies: a) to define the structural characteristics of peptides that are required for their high affinity binding to mhc; and b) to formulate these peptides into immunogenic vaccines that are capable of generating cytotoxic t lymphocytes. the strategy and results to accomplish these two goals will be discussed. amnon altman and erich gulbins ras proteins represent essential intermediates in receptor-mediated growth and differentiation pathways. they couple signals initiated by receptor or nonreceptor protein tyrosine kinases (ptks) at the cell surface to cytoplasmic targets which coordinate signal transduciion to the nucleus. ras also participates in t lymphocyte activation initiated by the antigen-specific t cell receptor (tcr)/cd complex, which leads to lymphokine production and proliferation. receptor ligation causes activation of associated ptks of the src and syk families as the earliest identifiable event in this pathway. the importance of ras in t cell activation is indicated by the findings that an oncogenic ras protein activates the interleukin- (il- ) gene promoter; conversely, a transdominant inhibitory ras mutant inhibits this event and the activation of map kinases. the rate-limiting step in ras activation is the exchange of bound gdp for gtp, which is catalysed by guanine nucleotide exchange factors (gefs). we identified the sh /sh domain-containing vav protein, which is selectively expressed in hematopoietic cells, as a ras-specific gef coupled to several hematopoietic receptors. vav can be activated by two pathways, i.e., by ptk-mediated phosphorylation or by binding of diglyceride second messengers to a cysteine-rich, protein kinase c (pkc)homologous vav domain. these pathways are independent as demonstrated by structure/function analysis and the use of pharmacological inhibitors. the two activation pathways may enable vav to integrate signals from distinct hematopoietic cell receptors, and couple them to ras. t cells express another, ubiquitous ras gef, i.e., sos, which is known to be coupled to activated tpk receptors. t cell activation leads to membrane association of a signaling complex consisting of sos and two other signaling proteins, grb- and shc, via direct binding of the shc sh domain to the tyrosine-phosphorylated chain of the tcr/cd complex. thus, multiple receptors, ptks and ras activators participate in signaling pathways that lead to hematopoieitc cell growth and differentiation. the interactions and function of these various signal transducers will be reviewed. phagocytes. immunity is mediated by specific t lymphocytes, cytokines produced by these specific t cells activate antimicrobial capacities in mononuclear phagocytes, thus contributing to protection. other immune mechanisms also participate in the acquisition of resistance. on the other hand, t cells are also crucial for many pathologic sequelae of intracellular bacterial infections. although ifn-y is central to macrophage activation, synergistic and antagonistic effects with other cytokines occur. the cd/ t cells, representing the major t cell population in experimental mice, are the main mediators of antibacterial immunity; an auxiliary role for y/~ t cells appears likely. the c~// t cell population further segregates into cd t cells which recognize microbial peptides in the context of gene products of the major histocompatibility complex (mhc) and cd t cells which see their peptide in the context of mhc class i. the -y/~ t cells are generally double-negative. knock-out mice in which the genes for various t cell receptor chains, for mhc class i or mhc class ii molecules, for cytokines or cytokine receptors had been deleted, have provided extremely helpful tools for analyzing the role of t cell subsets and cytokines in antimicrobial immunity. using these mutant mice, the role of distinct t cell subsets and cytokines in bacterial elimination and granuloma formation was analyzed in detail. these studies underline the central role of a// t cells and ifn-y in antibacterial immunity and, furthermore, show a distinct function of /$ t cells. moreover, these studies show that the relative contribution of cd or cd t cells to antimicrobial immunity varies in response to different pathogens. apoptosis is an active form of cell death in which the cell participates in its own demise, providing the biochemical pathways that trigger and mediate the complex events of the process. although this phenomenon has been studied for many years, it is only recently that significant progress has been made towards the elucidation of the molecular mechanisms that control apoptosis. once such mechanism came as something of a surprise: in a number of eases, apoptosis can be promoted by the action of the c-myc protein, a protooncogene product that had previously been associated only with cell proliferation (and presumably, survival) . expression of c.myc in cells can cause them to enter an apoptotic pathway or proliferate, mad this "decision" depends upon additional signals, such as growth factors that inhibit apoptosis and allow the ceils to proliferate. thus, c-myc directs cells out of rest and the choice of proliferation or death depends upon the presence or absence of survival signals. other survival signals are generated by oncogene products that can cooperate with my¢, including bcl- and abl there are implications of this interplay between apoptotie and anti-apoptorlc signals in cell iransformation and the resistance of some tumor ceils to therapeutic agents capable of inducing apoptosis. oncogene interactions therefore control the life or death of transformed ceils but also have important roles in normal, development processes. in the immune system, developing t ceils are "screened" for potential self-reactivity by a process of negative selection, in which activation via the t cell receptor (tcr) induces apoptosis. this process can be mimicked in t cell hybridomas, and appears to depend upon the expression of the c-mye protein. evidence that this represents a requirement for the myc/max heterodimer will be presented. as above, these cells are presented with the "choice" to proliferate or die and this depends not only upon myc but also other signals. thus, expression of functional v-an kinase in these ceils can render them resistant to the activation of apoptosis via tcr ligation, surprisingly, bcl- does not appear to be capable of blocking this form of apoptosis, and the reasons for this will provide new insights into the development of the immune system and the process of apoptosis. although the oncogene products discussed here probably do not directly participate in the biochemical process of apoptosis, they represent molecular controls on this complex mechanism. as such, they gave us new ways to look at the life and death of a cell recent attention has focused on macrophage release of cytokines, such as il- , il- and tnf as important mediators of septic shock. however, serum cytokine levels have correlated poorly with outcome of septic shock. the purpose of this study was to compare the functional status of macrophages to serum cytokine levels in early sepsis by an analysis of splenic macrophage protein synthesis during abdominal sepsis. macrophage total protein distribution and biosynthetic activity was assessed by large format -d page, autoradiography of s-labeled proteins and computer assisted densitometric analysis. sepsis was induced in sprague dawley mts by cecal ligation and puncture (clp). untreated and sham operated rats served as controls. splenic macrophages were harvested at and hrs. following clp. serum il- levels were determined at , , and hours by the tdi bioassay. by hrs. % of the clp mls exhibited multi-microbiaj bacteremia. control or sham operated rats did not show bacteremia. clp resulted in a significant % elevation (p < . ) in serum il- levels at hrs. there was no difference in il- at and hrs. when compared to the control groups. d page studies examined splenic macrophage total protein distribution and biosynthetic activity at and hrs. post-clp. at hrs. macrophages from clp rats showed a substantial decrease in total protein pattern ( protein spots vs protein spots) when compared to non-septic control macrophages. a decrease in the number of lower molecular weight proteins (< kd) was observed. at hrs. post-clp, splenic macrophages from clp rats showed a substantial increase in the synthesis of low molecular weight (< kd) proteins, when compared to non-septic controls. we conclude that: l) fulminate abdominal sepsis induces an early ( hrs.) staining with cd and cd mabs will identify two monocyte subpopulations in human blood: a major population of regular monocytes, which strongly expresses the cd antigen (cd ++) and a minor population with weak expression of cd and expression of the cd antigen lcd +/ cd + cells). in healthy control donors(n= ) the latter cells account for + cells/mm and %+ % of the monocytes. in sepsis patients, the cd] +/cd + cells can become a major population with more than % of all monocytes in of patients and with more than cells/mm in of cases. there was no correlation of cd +/ cd + cells to any clinical parameter except for cd +/cd + percentage and body temperature (p= . ). the cd ++ monocytes showed a substanttial decrease in cd antigen density in of patients. three-color-if shows that the cd +/ cd + cells in sepsis patients when compared with the cd ++ monocytes exhibit a higher level of class ii antigen and a lower level of cdiib and cd antigens, consistent with a more mature nature of the cd +/cd + cells. levels of il- were increased in sepsis patients: of patients with high numbers of cdi +/cd + cells ( /mm ) had high levels of il- ( u/ml). these data suggest that septicemia may lead to substantial changes in blood monocyte composition and this may be related to elevated levels of cytokines such as il- . human peritoneal macrophages were exposed to increasing doses of lipopolysaccharide (lps) or a synthetic lipid a analogue (mrl ) and production of the cytokines il-lb, il- , tnf-a and g-csf was assessed at the protein-and mrnalevel. cells were also stimulated with low doses of lps and mrl to study their "adaptation" to a secondary challenge with high doses of lps. tnf-a production after stimulation with lps could be almost completely relieved by preincubation of cells with low doses of lps and similarly, il- production was suppressed. surprisingly, however, "adapted" cells were able to synthesize even larger quantities of of g-csf and il-lb when exposed to a secondary lps challenge. the changes in tnf-a, il- and g-csf protein synthesis could a/so be detected on the mrna level, whereas transcript levels for il-lb remained unaltered as assessed by northern blot analysis. high doses of the synthetic lipid a analogue mrl were also able to "adapt" macrophages for a secondary lps challenge by downregulating tnfa-and il- -production, while priming secretion of g-csfand il-lb as well. taken together, here we describe for the first time the discordant adaptation of human peritoneal macrophages to a secondary lps stimulus in vitro. these findings appear to bear ramifications for the in-vivo endotoxin response during inflammation and gramnegative septicemia. shock states with inadequate cellular oxygen delivery may contribute to macrophage (mo) activation or dysregulation. in this study we compared the effect of transient hypoxia and endotoxin pretreatment (lps ) on mo mediator release with a second endotoxin stimulus (lps ). methods: in vitro cultures of marine peritoneal exudate mo were exposed to hr. of hypoxic or normoxic conditions, then incubated hr under identical normoxic conditions _+ ng/ml of lps pretreatment. during the final hours all m~l were exposed to a range of lps concentrations. m~i supernatants were assayed for tnf, il- , il- , pge , nitric oxide (no), and superoxide release. results: lps markedly inhibited mo tnf release by lps but hypoxia had no effect on lps -triggered tnf release. hypoxia increased mo il- production in the absence of lps , but inhibited lps augmentation seen under normoxic conditions. pretreatment with lps increased no production from lps under normoxic conditions, but bypoxiainhibited this effect. pretreatment with lps signifcantly augmented mo release of il-i and pge , but hypoxia had no significant effect (data not shown) superoxide production was increased by lps under normoxic conditions, but hypoxia significantly inhibited superoxide release (not shown a. lepape, c. docile, f. arpin, m. c. gutowski, v. banssillon and j. bienvenu. i aim of the study. increased levels of tnf are inconsistently correlated with the severity of sepsis. one possible explanation is a decrease ability of ceils to produce eytokines. the objectives of this study were m compare at different levels of of clinical severity the responsiveness of cytokine producing cells. this was evaluated by the response to a stimulation by lps as well as to the inhibitory effect of ptx. the technic used is a whole blood ex-vivo model, as described by desch et al. ( ) . h materials and methods. different groups of icu patients, postoperative (n= ), sepsis (n= ), septick shock (n= ), and healthy control (n= ), were studied. blood was drawn in endotoxio free heparinized robes. stimulation was achieved by addition nf pl oflps ( ng/ml) to id of whole blood in presence of various concentrations of ptx ( , " , - , " , i " m). after hrs incubation at °c, the tubes were centrifuged and supematants frozen at - °c. tnfa and/l were measured by elisa (medgenix r and immunotech r respectively). monocytes were quantified on a technicon h . the results are expressed as median values. non parametric tests are used for testing differences between groups. hi results, tnftx and , , corrected for monocytes count, are given as % of their initial value, the baseline before stimulation or inhibition being %, ) stimulation by lps. the control group is much more stimulated (> % for il , > % for tnfa). blood samples taken postoperatively and in patients with simple sepsis are significantly less stimulated (> % for il , > % for tnfa ). the lowest stimulation was observed in patients with septic shock (median = %), some patients being not stimulated at all. )effects of ptx.the inhibitory effect of ptx on tnftx production is effective in all groups at - m (reduction to less than '¼ of the median values), and is almost complete at " m. the septic shock group has a decreased sensitivity to ptx. il production exhibits a lesser reduction at - m (~ 'a to ½ of the median values), further increased at - m. the septic shock group is again less sensitive to ptx. iv conclusion: the reduced ability of circulating monocytes to produce cytokines during severe infections is confirmed here. ptx is able to reduce significantly tnfc~ at - m and the inhibition is nearly complete at - m. surprisingly, there is a lesser, but significant suppressive action of ptx on il , not found in experiments using purified monocytes. one possible explination could be the interplay between cytokines production. ( ) lymphokine research ( ) cdna sequencing constitutes a powerful method of measuring steady-state mrna levels for all genes transcribed in a given cell or tissue at a particular stage of differentiation. by comparing transcript abundance both prior to and following differentiation, individual genes can be identified whose transcription is regulated both positively and negatively. in order to examine monocyte activation, the human monocyte line thp- was induced with phorbol ester ( h) and activated for h with lipopolysaccharide (lps) after which polya + rna was purified. the rna from control and lps-treated cells were each used to construct a cdna library under identical conditions, and all resulting clones were selected for cdna sequence analysis. each clone sequence was evaluated by matching with both genbank and our own gene databases. very different patterns of gene expression were seen in the two libraries, the latter reflecting very high levels of known inflammatory mediators such as il- and tnf. a second set of libraries were made from umbilical vein endothelial cells (huvec), both with and without lps stimulation, and were analyzed in a similar fashion. the effects of lps induction on specific gene transcription in both cell types will be discussed. t. tadros, md, th wobbes, me) phd, rja goris, md phd to investigate whether the preactivation of regional macrophuges by liposomes containing muramyl tripeptide (mtp-pe) can counteract the detrimental effect of blood transfusions on both anastomotic repair and host susceptibility to infections. methods eighty lewis rats received lmg/kg of either empty or mtp-pe encapsulated liposomes, intraperitoneally (ip). twenty-four hours thereafter, the animals underwent resection and anastomosis of both ileum and colon, and received ml of either saline or blood from brown norway donors,iv. the animals were killed or days after surgery and examined for septic complications and anastomotic repair. the average anastomotic strength, as assessed by bursting pressure (+sd), was significantly diminished in the transfused animals, as compared to the non-transfused animals (ileum;day ; -+ vs + , p< . ). transfused animals pretreated with mtp-pe encapsulated liposomes showed a significant improvement of their anastomotic bursting pressure ( + , p< . vs transfusion). pretreatment with mtp-pe encapsulated liposomes decreased significantly the incidence of anastomotic abscesses in transfused animals ( from % in ileum on day to %, p< . ). conclusions preactivation of regional macrophges by intraperitoneal administration of mtp-pe encapsulated liposomes prevents the detrimental effects of transfusions on anastomotic repair and reduces the incidence of intraabdominal sepsis. academic hospital nijmegen, dept of general surgery, pb i, hb nijmegen, the netherlands. leukemia cell line, teip- . robin s. wa, gner*, perry v. halushka "~, and james a. cook*, departments of physiology , pharmacology "l" and medicine "t, medical university of south carolina, charleston, s.c. . adherence of monoeytes to endothelium and extracella/ar matrix proteins is essential for accumulation at sites of inflammation. txa , an arachidonic acid metabolite, inhibits human monocyte chemotactic responses suggesting that txa may alter monocyte adhesiveness. we selected the thp- cell line, a human monocytic leukemia cell line to further investigate the effect of txa on adhesion. we tested the hypothesis that txa alters lpsinduced adhesion of thp- cells and that txa exerts its effect on adhesion via a camp dependent mechanism. thp-i cells were exposed to s. enteritidis endotoxin (lp.g/ml) _+ the cyelooxygenase inhibitor lndomethacin (in), the txa mimetic i-bop ( . .tm,) or txa receptor antagonists bms and l ( ~m). cells were allowed to adhere for hours and adherent protein/well was determined. lps-induced a significant (p< . ;n= ) increase in adherence of thp- cells (basal, . + . gg protein/well; lps, . +_ . p.g protein/well). the amino acid glutamine is an essential compound for synthesis of purine and pyrimidine basis and therefore necessary for rna-and dna synthesis. in human plasma the concentration of glutamine is between . - . mm, and is reduced in septic patients up to % ( . - . mm). monocytes play a central part in the inunune system and it was of interest, whether glutamine is involved in the modulation of cell surface markers and phagocytosis of these cells. human peripheral blood mononuclear ceils were obtained from ml heparinized blood of apparently healthy donors by ficoll-paque density gradient and isolated by counterflow elutriation. the puritiy was more than %. subsequently cells were cultured in phenolred-free rpmi medium with various concentrations of glutamine ( . , . , . , . , . , , mm) in teflon-fluorinated ethylene propylene bottles to exclude cell adhesion and possible cell activation. aider seven days culture, cell viabilty was determined by trepan blue exclusion and varied between and %, independent of glutamine concentrations. cell surface markers were detected by flow cytometry, noaspecifie phagoeytosis was measured with latex beads and specific phagocytosis with opsonizied e.eoli using a facscan. lower concentrations of glutamine decreased the expression of hla-dr and icam- /cd on monocytes in a dose-dependent manner. the receptor for fc'/rucd as well as the receptors for complement cr /cdllb and cr /cdllc were down-regulated. cr /cd which is only slightly expressed on monocytes was not influenced. furthermore, no effects on the expression of cdi , the receptor for transferrin cd and fc'friii/cd were seen. our data indicate, that lower concentrations of glutamme influence the phenotype of monocytes. we are now interested to study whether glutmnine influences non-specific phagocytosis, or whether specific phagocytosis correlates with the decreased expression of fc'/r and complement receptors. we investigated immunologically more than patients who were admitted to icu because septic syndrom during the last four years. patients were immunologically followed up - times per week until release from icu. the expression of hla-dr antigen on monocytes turned out to be the best prognostic parameter. the persistence (> days) of low hla-dr expression (< %) predicts fatal outcome (mortality > %). the altered phenotype was associated with a functional deactivation of monocytes (diminished apc, ros formation, cytokine secretion). we called this phenomenon "immunoparalysis". ifn-gamma and gm-csf were able to restore the altered phenotype and function in vitro. however, addition of autologous plasma from septic patients with "immunoparalysis" to these cultures prevented the cytokine-induced restitution. the inhibitory activity could not be removed by dialysis. therefore, we started a study to prove the therapeutic efficacy of plasmapheresis. indeed, [ of patients recovered from "immunoparalysis" following repeated plasmapheres; of them survived ( %). patients recovered temporarely and patients did not respond (all died). the survival rate in the control group of septic patients with persistent "immunoparalysis" was of ( %; p< , ). in summary, plasmapheresis in association with immune monitoring may be an alternative strategy to improve survival rate in severe sepsis. taurolidine, a synthetic taurine-formaldehyde derivative has antiadherent, bactericidal and anti-lps properties functioning primarily through binding of the lipid a region of the lps molecule. the active derivative of taurolidine, taurine, modulates calcium channel activity, critical to the initiation of a number of immunostimulatory pathways. we hypothesised that taurolidine may have direct immunostimulatory activity. the aim of this study was to investigate the immune effects of taurolidine on peritoneal macrophage (pmo) function and then determine the role of taurine in this response. study : in vivo stimulation:cd- mice (n= ) were randomized to receive taurolidine ( mg/kg bw/i.p.) or saline cor~trol. peritoneai cells were harvested after hours and were assessed for pm function [superoxide anion generation (o -), nitric oxide (no), tumor necrosis factor (tnf), fc/cr -mediated phagocytic function (phago) study : control pm were harvested and cultured in vitro with taurine ( . mg/ml for hrs), after which time they were assayed for -and tnf release. in vivo stimulation with taurolidine taurolidine has specific immunological effects on m . release of the inflammatory mediators -and tnf, and fc/cr -mediated phagocytosis were significantly increased, while release of the endothelial relaxing factor no was significantly reduced. in addition, the amino acid taurine, which is released as a byproduct of taurolidines breakdown has an immunostimulatory effect on pmo and may be the active moeity of the compound tanrolidine. in sepsis, a number of mediators which affect vasomotor tone and cardiovascular function are produced. inasmuch as sepsis causes decrease in systemic vascular resistance (svr), attention is usually focussed on vasodilators such as lactate, tumor necrosis factor, interleukin-i & , and nitric oxide. but injury and inflammation als cause production of several vasoconstrictors whose effect may not be evident in changed svr, but may significantly affect organ blood flow or function in the paracrine environment. endothelin (et) is a amino acid peptide vasoconstrictor produced by ischemic or injured endothelial cells (ec's). et is also a potent constrictor for renal mesangial and coronary vessels, an endocrine regulator, and a negative cardiac inotrope. systemic et levels increase significantly in hypoperfusion and ischemia. while et is principally produced by ec's, we asked if human monocytes might also produce et and thereby regulate vasomotor tone in areas of inflammation. monocytes from healthy donors were separated on ficoll, resuspended in rpmi + % fetal calf serum and stimulated with i ug/ml endotoxin (lps). et was measured by radioimmunoassay. lps-stimulated monocytes produced ! fm of et/ cells (vs. unstimulated controls of < ). this calculates to - % of the amount of et observed in patients with low cardiac output, sepsis or ischemia. we conclude that et is a cytokine produced by both ec's and monocytes with potent effects on numerous cells and organs in the critically ill. wuppertal , germany we and other authors showed that fatal outcome in septic disease is associated with a decreased capacity of peripheral blood monocytes for the in vitro production of proinflammatory cytokines, especially tnf-alpha. we found that this monocytic deactivation is completed by a persistent and marked decrease of hla-dr expression on monocytes (< % hla-dr+ monocytes) and a diminished antigen presenting activity whereas the capacity to form the antiinflammatory il- receptor antagonist remains high. in order to evaluate the in vivo situation and to determine at which level tnfproduction/secretion is altered we assessed the tnf-alpha mrna expression in freshly isolated peripheral blood mononuclear cells (pbmnc) from septic patients. tnf-mrna was onty rarely detected by semiqaantitative polymerase chain reaction in pbmnc's from septic patients with monocyte deactivation. meanwhile, it was found in almost all pbmncs from septic patients without monocytic deactivation. we wondered, whether il-i , which ,is known to depress monocytic proinflammatoly response and mhc class ii expression, could be one of the mediators in fatal sepsis. in fact, we found that il- message in pbmncs of septic patients peaked in the beginning phase of monocytic deactivation. in further investigations we found that tnf-administration can induce monocytic deactivation in a murine model/n vivo and provoke il- message in human pbmncs in vitro. these results support our hypothesis that an excessive delivery of proinflammatory cytokines in a first phase can induce an overwheiming inhibitory feedback, mediated by immuninhibitory mediators like il-l , which leads to often fatal monocytic deactivation in a second phase. interferon-gamma which is known to counteract il- production and the effects of il- on monocytes restores the function and phenotype of monocytes from septic patients with monoq, te deactivation in vitro and could be a possible therapeutic agent in otherwise fatal sepsis. our laboratory previously reported that lps dependent macrophagederived tnf-a production can be enhanced by pretreatment with lps at substimulatory lps priming doses coincident with a suppression of lps dependent nitric oxide (no) production (zhang and morrison, j. exp. med : , ) . in order to extend the characterization of these lps priming effects in mouse macrophages, we examined the capacity of substimulatory lps to modify lps dependent il- production. macrophages were obtained from peritoneal exudate of thioglycollate treated c heb/fej mice and cultured in rpmi medium containing % fetal bovine serum. macrophages were pretreated with various subthreshold stimulatory concentrations of lps (olll:b ) for hours, washed three times, and then stimulated with the effective stimulatory concentration of lps for hours. the amount of il- in the supernatant was measured by il- dependent cell line (b and td ) proliferation assay. il- was produced by macrophages at lower threshold doses of lps than those required for tnf-o~ or no production. subthreshold doses of lps modulated il- production in a biphasic manner characterized by an initial suppression and then potentiation. higher doses resulted in secretion of il- during the initial incubation with lps and subsequent desensitization. il- , like tnf-~ and no, is, therefore, also affected by lps pretreatment. moreover, tnf-a and il- shared the similar potentiational pathway, but differed by the fact that only il- was inhibited. (supported by r ai and po a .) department of microbiology, molecular genetics and immunology and the cancer center, wahl east, university of kansas medical center, kansas city, ks - . korolenko t.,urazgaliev k.,and arkhipov s. the role of macrophage (mph) stimulation in mechanism of protective effect of new immunomodulators yeast polysaccharides -heteropolysaccharide cryelan and homopolysaccharide mannan rhodexman (both produced by petersburg chem.-pharm. inst.) was studied. in vitro according to nst test incubation of murine peritoneal mphs with cryelan or rhodexman, ~g/ml, min was followed by increase of potencial microbicidic activity of mphs. in vivo mph stimulation by immunomodulators studied included increase rate of carbon particles phagocytosis during single i.v. or i.p. mode of administration to mice - days after (peak at nd day for i.v. and th day for i.p. mode of administration of the same dose of mg/ g b.w.).the preliminary injection of cryelan ( mg/ g, or h before) to mice with acute cold stress (- ° c, h) revealed protective effect restorating the value of depressed phagocytosis up to the normal level;the positive effect on ultrastructure of hepatocytes was noted also.there was no changes of plasma corticosterone level between group with acute cold stress and mice with cryelan + acute cold stress (several fold increase comparatively to the control mice).as was suggested, the mechanism of protection can include mph stimulation and secretion of some acute phase proteins responsible for positive effect of immunomodulators. new yeast polysaccharides cryelan and rhodexman can be used for macrophage stimulation,especially in pathological states. immunomodulators were shown to increase production and secretion of lysosomal enzymes (like zymosan). secreted enzymes,especially cysteine proteinasescathepsins b and l -involve in the process of inflammation;however, excessive release of these enzymes may lead to noncontrolled proteolysis followed by tissue degradation (assfalg-machleidt et al., ) .the effect of zymosan,bcg and new immunomodulator carboxymethylglucan (cmg), second fraction on secretion of lysosomal enzymes by murine peritoneal macrophages was studied. zymosan increased the secretion of n-acetyl-~-d-glucosaminidase and ~-galactosidase into the culture medium ( - fold); bcg possessed similar effect.cmg in the same concentrations ( /~g/ml) increased release of these enzymes only saightly ( . times).it's known that zymosan-induced secretion reflects the enzyme release from formed lysosomes (warren, ) .it was suggested that cmg activated macrophages via interaction with scavenger-receptors,followed by weak secretion of lysosomal enzymes and as a result decrease of tissue damage. in vivo zymosan induced stimulation of mononuclear system of phagocytes followed by increase of cysteine proteinases activity in liver at the th day. in the same time in blood n-acetyl-~-d-glucosaminidase and n-acetyl-~-d-galactosidase activity increased - fold. it was concluded that in drug design it's possible to select such immunomodulators,e.g. cmg,which can activate mononuclear system of phagocytes and do not damage tissue. endothelin-i (et-i) is produced by injured/ ischemic endothelium, mobilizes intracellular ca ++ and is a potent vasoconstrictor. it is also a ca ++ agonist for anterior pituitary or renal mesangial cells and monocytes. et-i causes monocytes to produce interleukin-l, , , prostaglandin e , and substances which trigger neutrophil superoxide production. et-i levels increase in shock and et may play a role in activating leukocytes post shock causing reperfusion injury. but blood flow experiments suggest splanchnic circulation changes more profoundly in shock than peripheral circulation. we therefore asked if et- (or vic), the et which predominates in splanchnic vessels, had any effect on monocyte cytokine production. human monocytes from health~ blood donors were separated on ficoll. . x ucells/ ml in rpmi + % fcs were incubated i min., & hrs. with - m et-i, - m vic or i ug/ml of lps. supernatants were assayed by elisa. we have shown that low dose endotoxin pretreatment (lps ) for hrs markedly inhibits the macrophage (mo) release of tumor necrosis factor (tnf) and increases interleukin- (il-i) in response to a subsequent endotoxin stimulus (lps ). in this study we examined the kinetics of lps inhibition of tnf and augmentation ofil- . methods: murine peritoneal exudate mo from balbc mice were exposed in vitro to medium or ng/ml of lps for intervals of to hours. culture medium was then replaced with , or ng/ml of lps for hrs. tnf and il- in mo supernatants were measured by specific bioassays. during sepsis endotoxin (lps) activates macrophages (mo) to release mediators such as tumor necrosis factor (tnf), interleukin- (il- ), interleukin-i (il-i) and prostaglandin e (pge ). we showed that preexposure to lps (lps ) alters the response of murine m~i to subsequent lps stimulation (lps ). we hypothesized that in vitro cytokine release by lps in human monocytes (mo) is also be altered by preexposure to lpsi. methods: human peripheral blood mo were obtained from healthy volunteers (n= ), cultured in vitro hrs, then pretreated hr _+ lps -cultures were then stimulated with lps and mediators in mo supernatant measured: tnf, il-i, and il- by specific bioassays, pge by immunoassay kit. serum cytokine levels (specific elisa kits) were compared to in vitro supernatant levels. data is expressed as % control_+sem, lps = ng/mh the table shows that all mediators were increased, in the absence of lps . pretreatment with lps resulted in complete inhibition of lps -triggered tnf release. in contrast, lps significantly increased mo secretion of il- , il- and pge (data not shown). serum cytokine levels were as follows: tnf _+ , il-i + , and il- . -+ . ng/ml. these serum levels were low, showed an extremely wide variation, and did not correlate with in vitro lps -triggered mediator production. conclusion: human monoeyte mediator production is differentially regulated by preexposure to lps . provocative in vitro testing of monocytes may ultimately be clinically useful to identify prior in vivo lps exposure or mo macrophages release numerous secretory products involved in host defense and inflammation. activated macrophages with cytokines produced have been implicated in tissue damage in sepsis and multiple organ dysfunction. aimed to elucidate the organ-association phenomena,this study is to compare peritoneal macrophage(pm),alveolar macrophage(am), and kupffer cells(kc) during sepsis in terms of cellular protein contents as symbol of activation by flow cytometry analysis. sepsis were produced by cecal ligatien and perforation (clp) in wistar rats weighing - g.pm were obtained by peritoneal lavage,am by bronchial lavage and kc by incubating the collegenase digested liver with pronase-e. leukocytes have been implicated as a mediator of the microvascular dysfunction associated with reperfasion of ischemic tissues. a role for ieukocytes is largely based on observations that rendering animals anutropenic with anti-neutrophil serum or preventing leukocyte adhesion with monoclonal antibodies attenuates the increased fluid and protein leakage from the vaseulature that is normally observed in postischemic tissues. we have recently undertaken studies designed to determine the relationship between leukocyte-endothelial cell adhesion and albumin leakage ia rat mesenterlc venules exposed ~o ischemia-reperfusion (i/r). leukocyte adherence and emigration as well as albumin extravasafion were monitored in single postcapillary venules using iatravital fluorescence microscopy, lschemia was induced by complete occ!usion of the superior mesenteric artery and ~dl parameters were monitored at various intervals following reperfusion. the magnitude of the leukocyte adherence and emigration, and albumin leakage elicited by i/r was positively con-elated with the duration of ischemia. the albumin leakage response was also highly correlated with the number of adherent and emigrated leukocytes. monoclonal antibodies against the adhesion glycoproteins cd , cdllb, icam- and l-selectin, but not p-or e-selecdn, reduced i/r-induced leukocyte adherence and emigration as well as albumin leakage. phauoidln, an f-aetin stabilizer, largely prevented the emigration (but not adherence) of leukocytes and greatly reduced, the raicrovascular protein leakage. plateletleukocyte aggregates were formed in postischemic vemdes; the number of aggregates was reduced by antibodies against p-selecdh, cdilb, cd , and icam- , but not e-selectin or lselectin. a significant fraction of the mast ceils surrounding the posteapillary venules degranulated in response to ischemia/repeffusion, but mast cell stabilizers did not afford protection against the albumin leakage elicited by i/r. these results indicate that reperfusloninduced albumin leakage is tightly coupled to the adherence and emigration of leukocytes in posteapillary venules. this adhesiomdependent injury response is primarily mediated by cdllb/cdi on activated neutrophils and icam- on venular endothellum, and appears to require l-selecda dependent leukocyte rolling. mast cell degranulation does not appear to conwibate to the vascular pathology associated with i/r. m.d. rod=iek, boston, ma, usa the polymorphonuclear neutrophil (pmn) has long been known to pa~tlcipats in the inflammatory rebpons~ as a phagocyte and killer of invading organisms, but little attention has been given to its potential as a participant in the in~une interaction of lymphocytes and macrophages. we and others have shown that the pmn may have i~m~/nomcdulatory effects both in vitro and in vlvo. more recently it has been proven that the pmn can make mrna for and secrete the proinflammatory oytokines illa, il-ib, tnfs, il- and il- as does the other major circulating phagocyte, the monocyte/macrophags. furthermore it has been shown to make the potentially autoregulatory oytokines gcsf and gmcsf. these functional capabilities suggest that the pmn is not an wend cell ~, but one which has a potential role in regulation cf ~he immune response and that this potential ~cle should no longer be ignored when considering the immune abnormalities existing in patients following majo~ injury or surgery. we have investigated the proinflaznmatory oytokine secretion patter~ by pmn in patients following major ~hermal or tra~matic injury and in volunteers fellowinq endotoxemia. ?ollowing major injury there is variable pmn secretion of these cytokines when stimulated in vlero. following endotoxemia in a group of human volunteers pmn showed a hypo=esponsivenesa to lps hrs following endotoxin infusion followed at hre by an overshoot. pretreatment with steroids modulated this overshoot phenomenon, suggesting that receptors for steroids are involved in the regulation of cytokin® secretlon by fmn. these results sugges~ that the pmn, the most numerous cell in the circulation and the first to respond to an ins~l~ may be a so~rce of the prolnflammatory cytokine cascade following injury that has been recognized as significant in the process which often leads to multiple o;gan failure, the immunosuppresslon which occurs following major thermal injury may predispose these individuals to infection and sepsis, which remain a significant cause of morbidity and mortality. included among the many immune aheratlons are the p integrln (cdlla, b,c/cd ) dependent activities of adhesion, chemotaxls, diapodesls, and phagocytosls. our investigations indicate that, following major thermal injuries, the expression of the [~ integrlns, but not cd , is significantly decreased on neutrophlls (pmns). it remains unclear if pmns from thermally injured patients respond normally to lps, the effects of treatment in vitro with lps and f-met-leu-phe (fmlp) on the expression of cdtlb was examlned on pmns from the peripheral blood of healthy volunteers and non-septic burn patients (> ~; total body surface area, >ls~ full thickness), the pmns were incubated with lps (]ng- p.g/ml) or f'mlp ( " to " m) et oc for mln, in ~; human ab serum, the expression of the ]ntegrins was detected using monoclonat antibodies and flow cytometry. lps and f'mlp resulted in a slight increase ( fold) in the expression of cd b on pmns from burned patients compared to an and fold increase, respectively, on pmns from healthy individuals. this inability of lps or fmlp to increase cd b expression was not due to the amount of lps bound to the two cell populations. because the same defect is seen after either lps or fmlp stimulation, it is speculated that the defect must be in the amount of preformed cd ] b or its transport to the plasma membrane. platelet-activating factor (paf) and neutrophils have been implicated in the patbophysiology of ischemia-repeffusion injury, in addition, paf stimulates neutrophi[ (pmn) oxidative metabolism in vitro. the present study examined the potential role of paf in repeffusion injury in an in viva rabbit model. eight anesthetized rabbi~s underwent retroperitoneal exposure of the infrarenal abdominal aorta after percutaneous insertion of a catheter through the jugular vein into the infrahepatic inferior vena cava. doppler flow probes were placed around the abdominal aorta and the right common femoral artery to assess flow through these vessels. an occlusive ligature was placed around the abdominal aorta (superior to the flow probe) at t = and total occlusion of blood flow to the lower extremities was maintained for g mins., after which the ligature was released allowing for reperfusion of the ischemic lower limbs. effluent blood from the ischemic hind-limbs was collected through the ivc catheter at the times indicated below and assayed for paf by a direct radioimmunoassay. in addition, neutrophil h production was determined by a previously described ' '-dichlorofluorescein flowcytametric assay. _+ amean _+ s.e.m, pg/ml blood; brelative fluoresenee (% of baseline); caortic and femoral artery flow (% of baseline); *p < . vs. baseline; "p < . vs. baseline. a significant elevation of paf was observed in ischemic hind-limb effluent blood at min. after release of the aortic ligature during the repeffusion phase, as compared to baseline levels. in addition, pmn h production was increased by . -fold above baseline values by hour after ligature release during the reperfusion phase. both of these elevations were transient and returned toward baseline by hours post-isehemia. tatar occlusion of hind-limb flow was achieved as evidenced by the absence of aortic or femorat flow at rain. post-ischemia, however after release the ligature a significant reactive hyperemia was observed by mln. into the rapeffusion phase. histolog[c examination of reper[used gastrocnemius muscle revealed moderate pmn infiltration into the interstitium. in conclusion, these data indicate that paf is released into the circulation during repeffusion, and is likely involved as a mediator in the observed pmn oxidative burst activity, thereby contributing to reperfusien injury. following thermal injury and infection granulocyte function ts abnormal. to elucidate the mechanism by which thermal injury and infection affect the granulocyte's ability to polymerize and depolymedze actin, we serially measured f-actin levels in granulocytes from burned patients (mean age , +_ . years, mean burn size . % _+ . %) during the first s weeks post injury. six of the patients had infections during the course of the study, (septicemia, wound invasion and pneumonia). actin levels in granulocytes from eleven healthy volunteers (mean age years) were measured repeatedly and served as controls. lysecl white blood cell preparations were brought to c and incubated with n-formyl-met-leu-phe (stim) or with dulbecco's phosphate unbuffered sellne (unstim). the cells were concomitantly stained and fixed with formaldehyde, lysoleclthln and fiuoresceln phafioidin. actin depolymedzation (depol) was measured by incubating stimulated cells at °c before the stain-fixative was added. baseline (base) f-actln levels were assessed by adding stsln-fixatlve to icecold unstimulated cells. fluorescence was estimated in a facscan and expressed as ilnesr mean channel fluorescence_+ sem (mcf). figure displays granulecyle fectln levels in infected and uninfected patients as compared to controls. f-actln levels were consistently lower in control cells than in those from burned or burn-infected patients under all measured conditions. granulocytes from infected burned patients demonstrated a significant decrement in their ability to depofymerlze f.actin compared to both uninfected burned patients and controls, while there were no significant differences between infected and ,~ uninfected patients in the baseline, unstlmuleted and stimulated conditions. those results indicate la that grsnulocytas from burned and bum-infected patients contain higher levels of polymerized actln than ~ , s control cells. in order to study tumor necrosis factor (tnf) receptor sensitivity in septic critically ill patients we investigated blood samples of such people in reaction of leucocyte migration inhibition. migration of their polymorphonuclear leucocytes (pmns) was studied with stimulation with human recombinant tnf in concentration of . u/ml (recommended by manufacturer is the range of - o/ml) and without such. ten healthy blood donors formed control group. the results obtained showed diminished pmn reactivity to tnf in patients (migration inhibition was absent) oscaring with significantly increased migration ability of their pmns ( . % of that in control group). at the same time normal pmns in control group did show migration changes upon tnf stimulation. considering all the above we come to a conclusion that externally added tnf fails to activate pmns in critically ill patients more than they are by their endogenous tnf. moreover, this tnf no longer serves a positive chemotactic factor for such pmns. these findings may suggest that in critically ill septic patients reactivity of pmns to tnf is deeply altered. tnf receptors of pmns are either exhausted as such by excessive stimulation with endogenous tnf or further transmission of their message is impossible due to "fatigue" of the cell's activation mechanisms. we express our gratitude to reanal factory of laboratory chemicals for generously providing us with a tnf com~rcial sample. ~-sanguis medical, ekaterineburg russia; s-urals med.lnst. activated neutrophils infiltrating the local site of inflammation following trauma release high amounts of destructive lysosomal enzymes into the extracellular space. cytokines were discussed to be involved in regulation of this early process. the task of this investigation was to evaluate the possible regulatory role of interleukin- (il- ) and its potential immunosupressive opponent, the transforming growth factor-&, in regulation of neutrophil degranulation. we analysed the concentration of the al-proteinase-inhibitor complex of the lysosomal elastase as marker for the degranulation of neutrophils as well as the levels of il- and tgf- in the plasma probes of patients undergoing multiple trauma and severe surgeries. the time courses of il- and elastase were found to be highly correlated, wheras the concentrations of the cytokine tgf-e~ were found to be not significantly altered in comparison to the control group. this close temporal correlationship was confirmed by investigation of fluids derived from sites of inflammation. interstingly, the inhibitory potential (~zcproteinase inhibitor, antithrombin iii) was dramatically reduced in the early inflammatory phase. to prove this in vivo findings, the effects of il- and tgf-i~ on the degranulation of isolated human neutrophils of healthy donors was investigated in vitro. pathological high concentrations of rhll- up to u/ml (as detected in fluids derived from local inflammatory site) were found to be capable to induce a significant release of lysosomal elastase in a concentration-dependent manner, whereas the degranulation of neutrophils was uneffected by tgf- . in conclusion, these data suggest a contribution of il- in regulation of neutrophil activation at sides of inflammation. the immunosuppressive cytokine tgf-i&~ seems to have no direct regulatory effect beside its described chemotactic function on neutrephils. postirradiation chan~es of adhesive properties arid supercoiled nucleoid dna structure of blood leukocytes were studied in macaca nemestrina andrats. the dynamics of membrane chan~es after nonlethal irradiation of rats demonstrated the temporary increase of the leukocyte adherence at h followed by return of this parameter to normal levels at h. after lethal irradiation of both animal species the increase in adhesive leukooytes fraction was detected as early as at h. this hi~her index persisted until the end of experiments ( days). the early ( - h) temporary loosin~ of supercoiled dna structure was demonstrated in the leukocytes of nonlethally irradiated animals. this phenomenon seems to be connected with the lymphocyte fraction chan~es. this process was not dependent on altered adhesive properties of leukocyte membranes. the membrane chan~es of leukocytes preceded decondensation of supercoiled dna after lethal irradiation of animals, in this case loosin~ of supercoiled dna pro-~ressively increased at h and at the later terms of postirradiation period. the systemic inflammatory response syndrome (sirs) involves many inanunological reactions of the host including acfivatinn of inflammatory mediator cascades and depression of cellular reactivity in t-lymphecytes ( ). there are reports of nentrophil dysfunction in inflammatory disorders of the skin ( ), are there dysfunctions concerning the unspecific host defense in sirs, as well? in this study, we examined the reactivity of neutrophil granolocytes from patients suffering from sirs. twenty-one patients (apache ii-score ± ) with diagnosis of sirs entered the study. granulocytes were prepared as reported previously ( ) . in parallel, granulocytes from healthy individuals were tested. two granulocyte functians were studied in vitro: . migration of the ceils in a boyden chamber through a filter matrix following stimulation with different receptor dependent stimuli (c a, intefleukin- , platelet-activating-factor, leukotrien b , fmlp). . release of glucuronidase following stimulation with the aforementioned activators. the results demonstrate, that the release of -glucuronidase in patients suffering from sirs was comparable to the enzyme release of granulocytes prepared from healthy individuals. each stimulant induced release of p-glucuronidase in a characteristic dose dependent fashion. all granulocyte preparations from the healthy donors showed a positive chemotaxis response in the migration-assay. in contrast, only ten out of twenty-one patients had granulocytes migrating after stimulation. the two groups of patients displaying reactive or non-reactive granulocytes differed clinically: the nonreactive group consisted of patients with multiple organ failure ( / ) and nonsurvivors ( / ), whereas / patients in the reactive group survived. thus, the in vitro chemotaxis of granulocytes is impaired in a subgroup of patients with sirs. this defect of the non-specific host defense may contribute to poor prognosis and outcome of these patients. dermatol. : - , klinik ffir an~isthesiologie und operative intensivmedizin der cau kiel, schwanenweg , kiel, germany. objectives of the study: major emphasis has been given to the analysis of interactions of antibiotics with microorganisms. effects of antibiotics on cells of primary host defense mechanisms, such as the neutrophils, are less well known. therefore, attention has been focused on clindamycin, a member of the lincoseamide family. materials and methods: the effect of clindamycin (i -i ~g/ml) on granulocyte functions (healthy volunteers) such as random migration, chemotaxis (agarose method), ingestion (radiometric assay), superoxide (cytochrom c reduction) and hydrogen peroxide production (phenol red oxidation), lucigenin-and luminol-amplified chemiluminescence (luminometry) and degranulation (turbidometry with micrococcus lysodeicticus) were investigated in vitro. results: motility and degranulation were inhibited, ingestion of saccharomyces cerevisiae, zymosan-induced lucigenin-and luminol-amplified chemiluminescence, superoxide and hydrogen peroxide production were stimulated in a dose dependent fashion. conclusion: clindamycin has granulocyte function modulating properties. recognition of immunomodulating effects of antibiotics may have therapeutic significance, especially in patients with long-term antibiotic therapy or immune deficiencies. the intense muscle activity (ea) of rats resulted in increase of neutrophil influx in muscles during the recovery. we investigated neutrophil proteinases involvement in neutral proteinases balance of skeletal muscles by na. the rats were submited to swim with the load ( % of body mass) till exhaustion. immediately after na the neutrophil antiserum was injected i.p. to rats of experimental group. saline was injected to control animals° injections were repeated in h of the recovery and cytosol proteolytic activity (ph . ; fitc-casein) was determined. isolated soleus muscles were incubated also in vitro and proteolytic activity of incubation media was measured. it was found that there was - -fold proteinases activity increase in cytosols of all investigated muscles (soleus, white and red portions of quadriceps) of control animals by h of the recovery (the comparison was done with the sedentary rats). in h cytosol proteolytic activity decreased and then increased again by h of the fast. antiserum injections resulted in relible decrease of the proteolytic activities at every investigated time. when incubating m. soleus in vitro the activities of proteinases in incubation media turned out reliably less if soleus muscles were isolated from the animals to which antiserum was injected. the conclusion is that neutrophil proteinases can be involved in the balance of rat skeletal muscle neutral proteinases after ~a. a lot and new clinical problems complicating the outcome of polytrauma, burn and septic patients in surgical intensive care units, have arisen as the care improvement prolonged the patient's survival: a progressive degradation of organ and system functions often develops, usually making its first clinical appearance by ards, followed by the other organ failure (mof) and sepsis symptoms. the clinical picture is polymorphic, the end result of a complex systemic pathophysiological reaction trigg~ed off by trauma consequences (tissues disruption, hypo~xygenatiun and necrosis). nowadays there is not a preventi~ or specific therapy to lower the mortality rate ( - %) and-'mdy-a~ early, aggressive surgical approach .-evacuating haematomas, stopping bleeding, toileting all septic, necrotic foci and restoring anatomic continuity-, seems to be of some help this complex clinical entity has not an univocal denomination yet. the proper labelling of an illness should come from the full understanding of its pathopysiology and suggest the proper treatment choice. clinical and experimental studies demonstrated that pathophysiologic mechanisms involved in the past-traumatic illness, share the same anatomo-pathological elemem: the interstitial edema, due to a generalised endothelial micro circulatory injury. this alteration, as constantly seen in polytrauma patients, develops in a few hours after trauma as a consequence of the deregulation of the homoeostatic and immune mechanisms. in fact the overproduced oxygen free radicals and r~ombinam cytokines (il ,tnf), together with the complement degradation fragments, the proteolytic enzymes and many other mediators are all strongly h~l ~ ,_he e,,j,yheha! ceils. our~osect, atim~,-bnsed on examination of autopsical specimens from polytraanm patients, showed that such endothelial damage, supporting the interstitial edema, is widely and simultaneensly distributed, ensues shortly arer trauma and shows its effects in different organs at different times, only because each apparatus has different fimctienal reserves: the lung is the first organ to fail just because its ah, celocapillary membrane is one of the most delicate bodily structure, and its function is irroplace~le. we think it will be of a great help, in planning a preventive therapy, to chose a denomination focusing the physician's attention on the earl)" generalized endothelial injury and its effects, as in trauma patients it is present -even if latenflysince the first few hours. we would like to see the generalised endothelial microcircolatory injury properly highlighted when considering the best definition and the optimal nomenclature for the post-traumatic s mdrome. the presence of interleukin (il)- in bronchoalveolar lavage fluid of critically ill patients correlates clinically with the development of the adult respiratory distress syndrome lards), and inhibition of il- in animal models can attenuate lung injury. collectively, evidence to date suggests that il- attracts and activates neutrophiis (pmn), which are then responsible for the capillary leak of ards. however, an alternative explanation is that il- is directly toxic to the endothelial cell (ec). in this study, we have hypothesized that il- can disrupt endothelial integrity independent of pmn. meth ods: human umbilical vein (huv) ec monolayers were cultured to confluency on collagen-coated micropore filters. to assess ec integrity, .albumi n leak was quantitated by measuring the counts which crossed the monolayer, using a gamma counter. il- (lpg/ml) was incubated in the culture medium with .albumi n for hrs. the il- dose was not cytotoxic. to determine the involvement of protein synthesis in this process, selected monolayers were pretreated with cycloheximide (ch) prior to .- addition. statistical analysis was performed using anovmfisher plsd. we have previously shown that platelet activating factor (paf) enhances cdt expression and primes pmn's for subsequent generation. both are important steps in pmn mediated injury and are assumed to occur in concert. following major trauma non-specific pmn inflammation is activated, however, unbridled systemic pmn activity needs to be minimized. since circulating catecholamines are high early post-injury, we hypothesised that they downregu/ate cd expression and pmn priming via the [ adrenergic signal transduction pathway. methods: normal human pmns were primed with paf ( ng/ml for min) or pre-treated with - m of isoproterenol (i) or forskoklin (f) for rain and then primed with paf. cd expression was measured by flow cytometry (fig.l) and -generation in response to -rm fmlp was determined as sod inhibitable reduction of cytochrome c ( fig. holler** and georg w. bornkamm* lymphocyte-endothelial interactions are crucial for various immune responses, including cytokine driven inflammatory processes. protein kinase c (pkc)-inhibitors on the other hand are discussed as potential cytokine antagonists. in the present study we investigated the influence of the pkc-inhibitor gf x on cytokine-and endotoxin induced expression of intercellular adhesion molecule (icam- ) and on adhesion of lymphocytes to cytokine activated endothelial cells. we found that tumor necrosis factor alpha (tnfo -and lipopolysaccharide (lps)-induced icam- expression on human endothelioma celts (eahy ) were unaffected by the pkc-inhibitor and thus appeared to be independent of pkc activation. in contrast, gf x significantly reduced icam- expression induced by interferon-y (ifn-?) and interleukin- (il- ). the functional relevance of these findings was evaluated in an adhesion assay using human umbilical vene endothelial cells (huvec) and peripheral blood mononuclear cells (pbmc). in fact, the ifn-? and il- induced adhesion of pbmc to cytokine treated huvec could be downregulated by the pkc-inhibitor, whereas tnfc~-and lps-mediated adhesion was not influenced. additionally, the il- driven icam- expression on eahy cells as well as the il- induced adhesion of pbmc to huvec was found to be tnf-dependent, since both effects could be inhibited by an anti-tnf monoclonal antibody ( f) . these in vitro data further support the idea of examining pkc-inhibitors, such as gf x, for their biological relevance in cytokine related dysregulations. seiffge, d., bissinger, t., laux, v., during inflammation there are some key processes, which occur in the microcirculation: the release of mediators from various cell types, the migration of inflammatory cells towards a chemotactic stimulus in the tissue, the expression of adhesion molecules on different cells, and the extravasation of plasma proteins. the aim of the present study was to elucidate the mediator induced interaction of leukocyte adhesion and plasma leakage in postcapillary venules. using an analogous video-image analysing system we have studied the effect of different mediators on leukocyte adhesion and macromolecular permeability in the mesentery of the rat. the increase in permeability was measured as changes in optical density. we found that topical administration of leneotriene b (ltb , x " tool/l) or intravenous injection of interleuldn- (il- , - iu/kg b.w.) and lipopolysaccharide (lps, mg/kg b.w.) resulted in a significant extravasation of fitc-labelled rat serum albumin (fitc-rsa) in venules but not in arterioles. we could correlate the changes in vascular permeability with a locally increased number of rolling and sticking leukocytes in venules. both effects were dose dependently inhibited by different drugs. pentoxlfylline inhibits lps-indueed fitc-rsa extravasation and leukocyte adhesion at a dose of mg/kg b.w., superoxid-dismutase (sod, . iu/kg b.w.) was able to decrease the ltb effect, and the immuumodulating drug leflunomide (hwa ) exerted inhibitory effects on il- -induced permeability at a dose of mg/kg b.w.i.v. the obtained results demonstrate that lps, ltb or il- induced extravasation of fitc-rsa is mediated by activated leukocytes and can be deminished following administration of different drugs. platelet-endothelial cell adhesion molecule-i (pecam-i), a member of the immunoglobulin superfamily, is constitutively expressed at high levels on the endothelial cell surface. in vitro data have suggested that pecam-i functions as a vascular adhesion molecule, specifically in neutrophil transmigration across the endothelium. this current work is the first demonstrating the in vivo role of pecam- in neutrophil migration. blocking antibodies to human pecam- , in which the antibodies are crossreactive with rat pecam- , were able to block the movement of neutrophils into the rat lungs after igg immune complex deposition. furthermore, when human foreskin was transplanted into mice with severe combined immunodeficiency and the site injected with tnf-alpha, anti-pecam-i blocked neutrophil emigration into the dermal interstitium. it has already been established that neutrophil recruitment is dependent upon selectin mediated rolling, followed by firm adherence that is icam- / integrin mediated. these data suggest, for the first time, that a third endothelial adhesion molecule (pecam-i) is involved in the coordinated recruitment of neutrophils in vivo. to test whether trauma causes generalized activation or priming of pmns, cdi adherence receptors were measured with iinmunomonitoring in whole blood after lps stimulation ex vivo. anesthetized (fentanyl) mongrel pigs ( - kg) were subjected to % arterial hemorrhage + soft tissue injury and after liar, resuscitated with all the shed blood + supplemental fluid. blood was collected at hr intervals from unanesthetized animals with indwelling catheters, pmns were counted, and lps was added ( , , , i.tg/ml) ex vivo. after hr incubation at - °c, %cd (+) pmns were determined with fitc-ib and flow cytometry from mean channel fluorescence histograms. ± # p< . vs baseline * p< . vs sham $p< . vs no anesthesia these observations provide direct evidence for time-dependent changes in pmn priming following major injury because cd expression was depressed for at ]east hr after trauma relative to sham but by hr, was enhanced, relative to sham, and because fentanyl anesthesia at hr had a greater effect on cd expression in trauma vs sham. neutrophil (pmn) adhesion to vascular endothelial cells (•c) is a key element in the inflammatory response and tissue injury. inflammatory mediators such as lps (exogenous) and tnf (endogenous) can promote pmn-ec interaction which is believed to be responsible for capillary leakage and subsequent organ injury. however, the mechanism of this injury remains unclear.we hypothesised that the mechanism of tissue injury is due to ec necrosis with release of toxic products and that activated pmn are responsible. human pmn were obtained from healthy donors, separated by density gradient, and activated with lps ( ng/ml), tnf( ng/ml), and lps/tnf( ng/ ng/ml). cultures of the human ec tine(ecv- ) were used as surrogates of the microvasculature, were exposed to either lps, tnf, lps/tnf and pmn activated with lps, tnf, lps/tnf and incubated for , , , and hrs. ec necrosis was assessed by a cr release cytotoxicity assay. pmn activation was assessed by cd lb receptor expression and respiratory burst activity hr _+ . -+ -+ . _+ _+ . _+ _+ . _+ . hr + . _ _+ . _+ _+ _+ " +_ +-- . " lghr - . _+ +_ - " o:fo , " ~ +- . * hr _+ . - -+ +_ * _+ _+ * _+ _+ " data = ec % necrosis mean_+sd stats: student's t-test with significance (*) set at p< . vs control. ( our previous studies have indicated that despite the increased cardiac output and maintenance of tissue perfusion, hepatoceliular dysfunction occurs during early sepsis. nonetheless, it remains unknown whether vascular endothelial cell function (i.e., the release of endothelium-derived relaxing factor/nitric oxide) is depressed under such conditions and, if so, whether endothelial cell dysfunction also occurs at the microcirculatory level. to determine this, rats were subjected to sepsis by cecal ligation and puncture (clp), following which these and corresponding shams received ml/ g bw normal saline. at hr after clp (hyperdynamic sepsis) or sham operation, the thoracic aorta was isolated, cut into rings, and placed in organ chambers. norepinephrine (ne, xi - m) was used to achieve near-maximal contraction. responses for an endothelium-dependeut vasodilator, acetylcholine (ach, via nitric oxide), were determined. in additional studies, the small gut was isolated at hr post-clp. after pre-contraction of blood vessels in the isolated gut with xl m ne, vascular responses to ach ( x m) and an endotheliumindependent vasodiiator, nitroglycerine (ntg, xl - m), were determined. total vascular resistance (tvr, mmhg/mi/min/ g) was then calculated as pressure/ perfusinn rate. ach-induced relaxation (%, n= /group) in the aortic rings were: ach lxl i~s, st-in ~ ~ significantly at hr post-clp (i.e., increased *p(o vs. sham; n- per group. tvr) in the absence of any changes in ntginduced relaxation (fig. a) . thus, the vascular endothelial cell dysfunction observed in the aorta in early sepsis also occurs at the microcirculatory level. introduction: the cytokine-mediated adherence of leulcooytes to vascular endothelium is considered as an early step in the cascade of pathologic reactions culminating in the "systemic inflammatory response syndrome" (sirs); the purpose of this study was to evaluate the influence of interleakin- on leukooyteendothelial cell-interactions and microoirculation in the liver after hemorrhagic shock by means of intravital microscopy. methods: in anesthetized female sprdrats co.w. - g) shook was induced by fractionated withdrawl of arterial blood within rain and maintained for h (map at mm hg, cardiac output % of baseline). rats were adequately resuscitated with % of shed blood and twice the volume in ringer's solution additionally. following h of reperfusinn (map > mm hg, co > % of baseline) the microcirculation in liver lobules was examined by intravital fluorescence microscopy after labelling of leukocytes. continuous administration of il-lra (synergen, boulder, colorado, mg/kg/h) was started at different time points in a randomized and blinded manner. the animals in group p (n= ) received the il-lra as pretreatment beginning min prior to shock induction. in the group t (n= ) the application of il-lm started at the beginning of the reperfusion period with a bolus injection of mg/kg and was followed by continuons administration of mg/kg/h. the control group c (n= ) received equal volumes in nac , %, the sham-operated group s (n= ) was not exposed to shock. results: macrohemodynamics were comparable in all shook groups. the increased percentage of permanendy adherent leukocytes after hemorrhagic shook (s: , % + , %; c: , % _+ , %) was significantly reduced by pretreatment or treatment with il-lra (p: , % -+ , %; p< . , t: , % -+ , %, p< . , anova). temporary adhesion of leukocytes was unaffected by application of il-lra. liver microcirculation measured by volumetric blood flow in liver sinusoids and sinusoidal diameters was impaired after hemorrhagic shock in all groups and was not affected (c: iam /s + um /s, p: llm /s + }am /s, t: ams/s -+ lam /s, s: am /s -+ am /s). di.seu~sinn: the results demonstrate that permanent adherence of leukocytes to endothelium is in part regulated by il- . pathological adherence could be reduced by application of illra, even given at die time of resuscitation. the effect of ll-lm on permanent adhesion is a specific event and might be caused by reduced expression of specific receptors on sinusoidal endothelial cens and leukocytes. objectives of the study. the adhesion of activated neutrophils (pmn) to endothelial ceils (ec) and the concomitant production of reactive oxygen metabolites (rom) initiates organ damage after trauma, sepsis, shock and organ reperfusion. aien of this study was to investigate the effect on adhesion and rom production of the highly water-soluble, membrane-permeable and physiological ascorbic acid (asc). materials and methods. adhesion of pmn to nylon fiber (cell count) and simultaneous rom production (chemiluminescence-cl-response) were measured up to retool/ asc as well as adhesion, rom production and ec damage (lllln-release from labeled ec) of endotoxin-activated pmn to cultered ec moanlayers. in an in vivo animal model (sheep with lung lymph fistulas) the effect of asc ( g/kg bw bolus, followed by . g/ kg-h infusion) on the endotoxin-induced ( . ixg/kg bw) neutropenia (cell count), lung capillary permeability damage (lung lymph protein clearance) and rom production of neutrophils (zymosan-induced cl response) was measured. results. asc scavenged rom dose-dependently during adhesion of pmn to nylon fiber (p< . at mmol/l asc), adhesion itself was unchanged. during the activated pmn/ec interaction asc scavenged rom (p< . at mmol/l asc) and reduced the adhesion dose-dependently (p< . at mmol/l asc); ec damage was also reduced (p< . at retool/ asc). in the in rive model asc increased the endotoxin-induced blood pmn decrease (p< . ), decreased the protein clearance (p< . ) as well as the zymosan-induced rom production (p< . ), indicating the asc-mediated reduction of adhesion, rom production and lung tissue damage processes. conclusions. by in vitro and in rive experiments ascorbic acid reduced the adhesion-and rom production-initiated tissue damage. therefore, i.v. administration of ascorbic acid is recommended for oxidative stress-associated states after trauma, sepsis, shock and organ reperfusion. for neut rophi l-accumulat ion and activation. we investigated the influence of or to the activation and the expression of lecam-i and cdiib,cdi on neutrophils and lymphocytes. methods: from blood samples (n= ) all white blood cells (wbc) and neutrophils (nc) were isolated and cultured. or were produced via the xanthine oxidase/hypoxanthine system. after , , , , and minutes a giemsa-staining to determine the granulation of neutrophils (n: normal, r : reduced ) and a facs-analysis with monoclonal antibodies detecting cdiib,cdi and lecam-i was performed. results: under the influence of or a degranulation of neutrophils starting at min was observed in wbc-cultures (n/r: min / , min / , min / , min / , min / ). these data were confirmed in the dot-plots of facs-analysis. only in wbc-cultures or induced a significant increase of lecam-i expression on neutrophils up to min followed by a decrease to normal values at min. lecam-i on lymphocytes disappeared totally during the observed period. cdllb,cdl -expression was not altered. conclusion:increased lecam-i expression on neutrophils due to or could enhance the 'rolling' of neutrophils along the endothelium which is a prerequisite for neutrophil sticking and migration. further or are able to activate neutrophils without endothelium. these changes seem to be mediated by other wbc. introduction. multiple organ failure (mof) has been hypothesized to be the result of an excessive uncontrolled autedestructive inflammatory response. since the complement system is an important mediator and initiator of the inflammatory response, interruption of this cascade could theoretically lead to an attenuation of mof. in order to test this hypothesis we evaluated the response of c -delicient mice in a model of zymesan indt~ed mof. materials and methods. c -deficient b d /oid and c -sufficient b d /new mice were used in this study. on day all mice received an intraperitoneal injection with zymosan suspended in paraffin in a dose of mg/g body weight. between day and , biological parameters (temperature, body weight and clinical condition) were measured daily and mortality was monitored. clinical condition was assessed by blindly grading the degree of lethargy, conjunctivitis, diarrhea, and ruffled fur of each mouse on a two point scale (maximum score= ). on day all surviving mice were sacrificed and relative organ weights of lungs, liver, spleen and kidneys (relative organ weight= (organ weight/body weight)x ) wore calculated. earlier experiments with our model have shown a good correlation between histological organ damage and relative organ weights. statistical analysis of biological parameter was performed using the koziol curve analysis. analysis was divided in an acute phase (day - ) and a late phase (day - ). relative organ weights were analyzed using wilcoxon's test and mortality rate using fischor's exact test. results. all zymosan injected mice showed a typical triphesic illness. deterioration of the clinical condition as indicated by the symptom score and the decrease in temperature and body weight in the acute phase were all significantly lass severe in c deficient mice (all p< . ). in the late phase no differences could be noticed in the courses of biological parameters. overall mortality was / ( %) in c deficient mice and / ( %) jn c sufficient mice (p= . ), a difference mainly due to a difference in the acute phase. organ damage assessed as the relative organ weights did not show any statistical differences for any organ between both strains. conclusion. complement factor c appears to play an important role in the acute hyperdynamic septic response in this model but deficiency of c could not prevent organ damage in the late mof phase. this suggests that other factors could be more important in the development of the inflammatory response leading to mof. proinflammatory cytokines are thought to play a critical role in the pathophysiology of multiple organ failure (mof). in mice, zymosan-lnduced generalized inflammation (ztgi) leads to mof. therefore we performed a sequential study into plasma levels of, and macrophage production capacity for, four cytokines during the development of mof in the zigi model. male young-adult c bl/ mice received zymosan ( mg/g body weight) intraperitoneally. groups of animals were killed after , , , and h and subsequently at each day until day . plasma was collected and peritoneal macrophages were isolated and cultured overnight with or without lipopolysaccharide (lps). interleukin -ct, and - (il-lc~,~,), and tumour necrosis factor-o~ (tnf-c were measured in plasma and culture fluid by means of a ria (detection limit . ng/ml). interleukin- (il~) levels were assayed using the b hybddoma cell proliferation assay. zymosan induces a three-phase disease in mice. after an acute phase the animals recover. around day , they start to develop clinical signs which resemble mof. plasma tnf-~ peaked within h after zymosan injection and disappeared within h. from day onwards, tnf levels started to rise again. plasma il- behaved almost similarly in the acute phase, but in the mof phase plasma il- remained low. no circulating il- could be detected at any time point. macrophage lps-stimulated production of il-lcq il- ~ and tnf--c~ was suppressed immediately after zymosan injection. production of il- and tnf-~ was normalized within h, while production of il-lc~ remained lower than that in macrophages from untreated control mice. only at day did production of il-i~ reach control values. il- production was higher than control values from day onwards. il production was similar to that of ili-il the production of tnf-ct was strongly elevated between days and and again during days to . the development of mof-like symptoms during zlgi in mice is accompanied by increased plasma levels of tnf-ct without enhanced il- or il- . also, the ability of macrophages to produce excessive amounts of il- and tnf--~, as well as the suppressed capacity to produce il-lcq could be important mechanisms in the pathophysiology of mof. when conjugated to an asialoglycoprotein, dna and oligonucleotides are specifically taken up by the hepatocytes via the asialoglyccprotein receptor which is unique to the liver. human asialoglycoprotein (~ -acid, asgp) was derivatized with low molecular weight poly(l)lysine(pll) and complexed with antisense dna's (as) complementary to the ' region of the il- gpl receptor. the antisense were '-agtttagggatgagg- ' (asl), '-atcttcatcttctgaat- ' (as ), '-aagtgaatgattaaaacact- ' (as ), '-aaacctttataggcg- ' (as ), and '-cgttctacaactgcaacgt- ' (as ). using hepg , the biological effects of these antisense complexes on the high affinity il- receptor were evaluated by scatchard analysis, cellular proliferation, and acute phase protein expression by radioimmunoprecipitation and two dimensional gel electrophoresis. scatchard analysis demonstrated that high affinity receptor expression was inhibited by incubation of cells with asgp-pll-asi for h. underivatized asl was less effective and the complex, asgp-pll-as , had minimal effects on high affinity binding. when the cells were treated with the conjugates and stimulated with il- (i units) asgp-pll-asi alone showed a dose dependent ( .i- . ~m) inhibition of ss fibrinogen synthesis. two dimensional gel electrophoresis showed that expression of other acute phase proteins was also blocked. these results indicate that the targeted delivery of antisense molecules via conjugates recognized by the asialoglycoprotein receptor can block the cytokine stimulated acute phase protein response in hepatocytes, this approach may be relevant to the therapeutic management of patients with severe injury and sepsis. it has been established that immune cells are able to express neuropeptide genes and to release products that were considered to be of neuroendocrine origin. we have shown that proenkephalin (penk), a neuropeptide encoding gene, is expressed in lymphoid cells in culture. to study the physiological significance of these observations we have used the model of experimental endotoxemia. in this model, a disease state is induced by bacterial lipopolysaccharide (lps), that activates the immune system, the adrenocortical axis and the nervous system. we found that the expression of penkmrna is markedly enhanced in vivo immediately after lps injection both in the adrenal glands and in the lymph nodes. in situ hybridization analysis combined with immunohisto-chemistry indicated that the induced penk expression is confined to macrephages within the lymph nodes and chromaffin cells in the adrenal medulla. furthermore, this expression in lymph nodes is modulated by ligands of the adrenergic system. our results strongly support the notion that immune derived opioids participate in the bidirectional communication between the nervous and immune systems. of neurology hadassah university hospital, jerusalem , israel. objectives of the study: multiple-organ-failure is recognized as the most severe, and often lethal, complication after multiple trauma. however there is no adeqate animal model available. our goal was to develop an animal model, in which reproducable irreversible failure of parenchymal organs is achieved in the late phase after insults in the early phase (trauma). materials and methods: l female merino-sheep were included (mean weight: kg). day : hemorrhagic shock (mean arterial pressure (map) mmhg for hrs.), closed femoral nailing (ao-technique), day - : bolusinjection of endotoxin (et) ( , ~tg/kgbw) und zymosan-activated plasma (zap) ( ml) every hrs., day - : observation. bronchoalveolar lavage (bal): day , , . the course of representative parameters of organ function was documented: cocardiac output (i/min), svr -systemic vascular resistence (dyn ~ s cm- ), pap -putm.art.pressure (mmhg), pap -arterial oxygen pressure (mmhg), bill -bilirubin (;xmov ), crci -creatinin clearence (ml/min) statistics: data as means+sem, *significant from baseline (wileoxon test; p< ) results: baseline day day day day heart: co , _+ , , _+ , , _+ , , _+ , * , _+ , * svr _+ + _+ +_ " +- " lung: pap , _- , , _+ , " , +- , " , + , " , +- , ' pap , + , , +- , , _+ , , +- , , +_ , * liver: bill , _+ , , _+ , ' , _+ , ' , _+ , " , _+ , " kidney:crcl , +_ , , [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] , _+ , , + , , + , histologic specimens showed all signs of fulminant mof. combination of hemorrhagic shock, femoral nailing, et und zap (insults in the early phase) lead to an irreversible organ failure in the late phase. prostaglandin e (pge) levels are elevated by trauma, shock or sepsis and can profoundly affect the immune response. pge is produced by many cell types including fibroblasts, macrophages, monoeytes, follicular dendritic cells, and epithelial cells and is induced by il-i, bacterial lps, components of the complement cascade, tnf, il- and crosslinking of surface fc receptors for igg, iga and ige. our research has shown that pge inhibits b cell activation (specifically enlargement, class ii ~c and fc~ rii expression), proliferation, igm and igg responses, t cell proliferation, and il- synthesis in the mouse model. in contrast, pge greatly promotes class switching to ige,the isotype responsible for type i allergic hypersensitivity. thus, our model mirrors th~ general immunosuppression and elevated ige titers of the trauma or sepsis patient. pge increases the number of cells secreting ige and iggl, acts on surface igm positive b cells, synergizes with il- and lp$ to induce preswitch germline transcripts, and induces more rapid expression of mature vdj~ mp~a than in eontro~ pge intracellular signalling occurs through cyclic adenosine monophosphate (camp) levels and can be mimicked by camp-inducing agents and blocl~ed by an inhibitor of campdependent protein kinase a. pge action requires de novo protein synthesis and candidate pge-inducible regulatory proteins have been identified by d gel eleetrophoresis. thus, pge inhibits a number of immune mechanisms while promoting ige production. a deeper understanding of pge immune regulation may lead to more effective treatment of immune perturbations as sequelae of trauma, shock or sepsis. during infrarenai aortic surgery mesetueric traction (re.t.) results in prostacyclin (pgi:) release and consecutively in hemodynamic disturbances (decreased systemic vascular resisteace, mean arterial pressure; increased cardiac output, heartrate). these symptomes are bypassed by cyclooxygenase inhibition. hemodynamic symptoms vanish after - rain even without cyclooxygenase inhibition although pgi levels remain elevated. to study the endocrine vasopressor system in a prospective double blinded protocol, we investigated patients undergoing major abdominal surgery as compared to ibuprofen ( rag, i.v.) pretreated (ibu) patients. the surgeon applied m.t. in a uniform fashion. we chose a general anesthesia combined with a supplemental thoracic epidural anesthesia. at the points in time , , , , , , , rain after and before (to) mesentzrie traction we determined the plasma concentrations (pc) of -keto-pgf~o~pr~, epinephrine, norepinephrine, dopamine, renin, aldosterone, adh and cortisol. pc of -k-pgf~,tp~, peaked minutes after m.t. ( _+ , ibu: _+ , to: +i ng/l) and declined monotonously over h ( +_ , ibu: _+ ng/ ). catecholamine pc "s did not exceed the reference range during the observation period. reninpc peaked after rain ( _+ , ibu: + , to: -+ /~u/ml); aldosteronc also presented a maximum after rain ( + , ibu: -+ , to: +- pg/ml), whereas cortisol demonstrated irrespectively of circadian rhythms a maximum h after m.t. ( +_ , ibu: -+ , to: +_ ~g/ ). adh pc peaked min after m.t. ( + , ibu: -+ , to: +_ pg/ml) and showed analogously to -k-pgft~j~ pc a monotone decline over the observation period. our data demonstrate a counteractive reaction to pgiz mediated vasodilation via adh secretion. the second regulative is the renin-angiotensin-aldosterone system (raas), which is activated min after m.t., the aldosterone pc does not paratlel the cortisol pc, which peaked post operafionem in both groups, probably due to the end of anaesthesia. a regulative release of catecholamines could not be documented. the activation of adh and raas after mt is not a hormonal response primaryly related to surgical trauma and/or stress but a counterregulation to systemic vasoditafion induced by prostacyclin. although adh and raas support systemic circulation, angiotensin and vasopressin may compromise local organ blood flow (e.g. splancimic vascular bed). insfitut f. klin. chemic, anaesthesiologie ~, chirurgie l*, univ. ulm, elm, expression of c-fos protein in rat brain following occlusion of superior mesenterie artery. takanobu there is general agreement that neurologic abnormalities are seen in sepsis. the aim of this study is to examine what effect does the brain receive in case of sma occlusion by immunohistochemistry using antibody to c-fos, an immediate early gene, which is recently recognized as a genetic marker of activated neurons. moreover, we investigated the correlation between c-fos induction in the brain and plasma endotoxiu level. rats of them received sma clipping and others wee used as control. control and treated rats at , , , hours were perfused and fixed. the brain were sectioned at pm and stained by abc method using c-fos antibody. plasma endotoxin level of rats were measured at , , , , hours after the treatment by chromogenic limulus method. immunohistochemical study showed scarcely no immunoreactivity in control rat brain. in treated rat brain, the significant expression of c-los was detected in specific nuclei including the habenula, some hypothalamie nuclei, amygdala, locus ceruleus and nucleus tractus solitarii. such immunoreactivities were increased in time curse, which well corresponded plasma endotoxin levels. the mean plasma endotoxin level of , , , , hours after the treatment were . ± . , . _- - . , . _+ . , . ± . and . ± . pg/ml, respectively. the results indicate that limbic and hypothalamic-brainstem systems are involved in sma occlusion, and suggest that such neuronal actival.jon may precede the elevation of plasma endotoxin icy.el. systemic vascular resistance and increased cardiac output accompanied presumingly by a increased pulmonary shunt (qs/qt). this response is induced by prostacyclin (pgi ). we examined oxygen transport after traction on the mesentery root and the transpulmonary prostacyclin levels in a prospective placebo controlled study with intravenous ibuprofen. methods: with approval of the human [nvestigadon review board we studied patients in a prospective, randomized double-blinded protocol who were scheduled for major abdominal surgery. ibuprofen ( mg i.v.) or a placebo equivalent was administered minutes before skin incision. pulmonary artery thermodilution and radial artery catheters were placed after induction of anesthesia. mt was applied in a uniform fashion. baseline values preceded the incision of the peritoneum (to). fulther assessments followed , , , , . tile plasma concentrations (pc) of -keto-pgft, (stable metabolite of pgi ) were determined in arterial and mixed venous blood by radioimmunoassay. at all points in time we measured arterial and mixed venous blood gases. qs/qt was calculated by standard formula. data are given as median (p < . placebo vs. [ibuprofen] [ ] mmhg (*p< . i). these changes were accompanied by a marked increase of -keto-pgf~ pc up to rain after mt in arterial and mixed venous blood of untreated patients with a peak of *[ ] ng/l tl (*p< . ol). there was no difference between arterial and mixed venous pc. ibuprofen pretreated patients (n=zr) demonstrated stabile qs/qt and pao while -keto-pgf~ pc remained within the normal range. discussion: our data clearly indicate that mesenteric traction response includes a critical rise in qs/qt followed by significant decrease of paov stable oxygen transport determinants following cyclooxygenase inhibition signify an action mediated by prostacyclin. an indicative transpulmonary gradient for -keto-pgft~ was not detectable. a splanchnic vascular source for pgi release seems to be likely, but could not be proved by our current data. department of anesthesiology, cliu. chemistry * and surgery*; university clinics uim, prittwitzstral]e , ulm, germany it is unclear whether injuries like bums, in general, directly result in alterations of cell-mediated immunity that, in turn, promote endotoxic and bacterial translocation or, alternatively, whether these conditions allow increased bacterial invasion that, in turn, inhibits cmi. aim: to determine whether infectious challenge, as clp alone or combined with ti causes further immune abnormalities in the days following clp. study plan: on day , two groups of n= week old aj mice were subjected to either a % scold burn (ti), or were untreated (c) n= . on day , mice (ti+clp) and mice (clp) were subjected to clp. the two other groups (ti and c) were untreated. at days , and after thermal injury splenocytes (sp) were harvested and cultured with cona for an assay of il- and adherent splenocytes (as) were cultured with lps for il- , tnf, il- and pge . results: either ti + clp or clp alone result in significantly decreased secretion of all cytokines tested. in the ti group almost every cytokine production determined was elevated in comparison to ti + clp and prosmcyclin (pgi ) has been implicated in the pathophysiology of septic shock. however, pgi~'s role in the inflammatory response to sepsis is not well-defined. the purpose of this study was to identify which acute septic events are mediated by pgi during graded bacteremia. methods: eleven ~nesrhetized, hemodynamically monitored adult swine were infused iv with aeromonas h. ( /ml) at rates increased incrementally from . to . mi/kg/hr over hours. animals were studied in two groups: septic control (sc), graded bacteremia only (n= ); pga (n= ), graded bacteremta plus anti-pgiz antibody, ml/hr iv, beginning at hours. mean systemic (map) and pulmonary arterial (pap) pressures and arterial po , mmhg, cardiac index (ci), l/min/m , oxygen delivery index (do i) and consumption index (vozi), ml/min/m , and oxygen extraction (er), %, )latelet aggregometry (plt), %max., plasma pg -keto f alpha ; in the first instance~ peak values of lt ~ after i~ hrs post infarction were times higher than in the controls and excess leucocyte infiltration was noted at the infarction zone. in second instance two levels of lt b led to weak infiltration of the infarction zone by leucocytes. a. mo~e~o, in~.~p~siolo~,d~t.e~.cardiolo~,bogotsolets , ~ev , ukrmne systemic lesion$of erythron in traumatic disease and possibilities of their regulation by opioid peptides. redkin y. v., fominih s. g. using clinical ( patients) and experimental material( rats and dogs) we revealed general regularities of erythron lesions after hard mechanical trauma of various genesis as well as some mechanisms of development of posttraumatic anemia and possibilities of its correction with preparations of opioid peptides. the condition of central and peripheral compartments of erythron was studied with unified morphologic, immunogematological, biochemical and radiological methods. it was revealed that irrespective of the experimental animal species (dogs, rats) or in clinical experiments (patients) and irrespective of the injuring factor type (skeletal trauma, craniocerebral trauma, loss of blood) in erythron can be observed one-directed unspecific reaction realized by the considerable lowering of hemoglobin concentration, erythrocytes number and hematocrit. in the initial period ( - days) in the system of erythron prevail processes of distraction and elimination of er~zthrocytes relatively to the general production of stimulated erythropoiesis. the primary alterating factor is the prolonged intensification of peroxydation of membrane iipids of erythrocytes with simultaneous lowering of reserves of reduced glutathione. the distraction of erythrocytes is supported by the developing phenomena of autoallergization of organism that becomes apparent by the appearance of sensitized t cells and antierythrocyte antibodies. the intensified production of erythropoietin rules to the realization of he program of fetal and terminal (reserved) erythropoiesis. failure of erythropoiesis function is supported by disturbances of the processes of the injuring of cell metabolic apparatus. using of dalargin ( microgram per kilogram of body mass intrap'eritoneally within days after the trauma) showed the precise pharmacotherapeutic effect revealed by the diminishing of anemia of experimental rats, more . fiberbronohoscopic procedures are known to produce "peep-like" effects and to increase pulmonary artery (pa) resistance [ ] . peep can affect rv function by reducing preload and ejection fraction (ef) [ ] . since changes of rv function during bronchoscopy in septic patients are not reported, we measured rv parameters before, during and after fiberoptic bronchoalveolar lavage (bal). method: this -year-old patient (apache-ii: ) developed a hyperdynanlic septic state due to staphylococcus aureus (blood culture). we inserted a "fast response" thermistor pa-catheter (baxter-edwards) to evaluate rv performance [ ] . the therapeutic procedure included volume replacement, vasopressors (dopamine , dobutamine gg/kg/min. iv) and analgosedatior/. before bronchoscopy (olympus bf- , od= mm) the patient received pancmonium for muscle relaxation. ventilation was not changed during the procedure (endotracheal tube: id= ram, bennett a, pressure controlled mode, pm~x= mbar, peep= mbar, i:e=i:i, fio = . ). we measured rv enddiastolic volume (edv), stroke volume (sv), ef, heart rate (hr), cardiac index (ci) and mean pa pressure (mpap gerlach h, gerlach m, clauss m, falke kj renal hypoxia and/or ischemia initiates the development of a deteriorated medullary perfusion based on fibrin deposition in the peritubular capillaries, vasoconstriction, and perivascular edema, which is followed by a swelling of the tubular epithelial ceils, intraluminal tubular obstruction, and a backleak of fluid through the injured tubules into the renal interstitium, finally leading to an acute tubular necrosis (atn) [ ], clinically diagnosed as acute renal failure (arf). one important pathway for induction of enhanced vascular procoagulant activity and permeability is based on the synthesis and expression of macrophage-derived cytokines, which bind to specific endothelial cell surface receptors. we recently described the identification and purification .of a new , dalton polypeptide, which is synthesized and expressed by murine macrophages after stimulation with lipopolysaccharide, and exerts procoagulant activity on cultured endothelial cells [ ] . in the presented study, we demonstrate that the new polypeptid is also synthesized by macrophages under hypoxic conditions. the protein binds to specific receptors, which are expressed by endothelial cells dependent on the environmental oxygen tension. animal studies were performed after approval by the local committee for animal safety; the animals were anesthetized, treated and supervised in accordance with the guidelines of this committee. in contrast to other authors, who performed long-term hypoxia experiments in awake animals, we preferred to implement the studies under anesthesia for ethical reasons, although regulatory functions for ventilation might be influenced. animal studies demonstrated that the intravenous injection of the polypeptide initiates fibrin formation in the peritubular vessels. keeping the animals under hypoxic conditions induces similar effects, which are reduced by a rabbit-antiserum against the new protein. in conclusion, the new polypeptide obviously contributes to the pathogenesis of acute renal failure by tubular necrosis during and after hypoxic events. the use of verapamil as cardioprotective agents for management of patients with acute ischemic/reperfused heart is based on the assumption that the increased intracellular ca+ level is a key factor in causing cell death. our in vitro study was designed to focus on effects of verapamil on the metabolic potential of cardiac slices after reversible ischemia in rats. the material consisted of two main groups : group a (non ischemia/reperfusion group) and group b (ischemia/reperfusion group), each is subdivided into two subgroups (a and b). each subgroup included rat hearts. group aa is the control group, group ab is verapami] added group. group ba is ischemia group without verapamil. group bb is verapamil added group. ischemic cardiac slices were obtained from rats subjected to min. haemorrhage to induce reversible global ischemia. both nonischemic and ischemic cardiac slices were placed in well oxygenated krebs ringer phosphate buffer containing mg% glucose & gm% bovine albumin and incubated in dubnoff shaking water bath for min at °c the results revealed that there was an enhancement in release of free fatty acids (ffa) ( %) and lactate ( %) and in glucose uptake ( %) in group ba as compared with group aa. these metabolic alternations produced by ischemic cardiac slices were reversed by verapamil addition ( ml%) but in group ab verpamil did not alter the release of ffa & lactate from non-ischemic cardiac slices, whereas it inhibited glucose uptake from these slices by %. the improvement of the metabolic intervention of ischemic myocardium indicates that verapamil may be of importance in reducing the extent and severity of acute myocardial ischemic injury in acute haemorrhage. severe endothelial dysfunction occurs following injury to carotid arteries which is characterized by a decreased ability of these arteries to dilate when challenged with ach or a , but not with a direct vasodilator (nano ). this failure to relax to ach and a reflects an inability of endothelium to generate edrf, but relaxation recovers gradually to control values by weeks. exogenous no donors (e.g., c - or spm- ), accelerate the recovery of the injured endothelium in rat carotid arteries. intravenous infusion of an no donor ( p.g/day) with an implanted osmotic pump significantly accelerated the recovery of regenerated endothelium to produce edrf at days. rat carotid artery rings relaxed only + % and + % to gm ach in vehicle treated rats and in inactive no donor treated rats respectively days following injury compared with + % in no donor rats (p< . ). relaxation to gm nan was normal in all groups indicating that the differences in relaxation were not the result of damage to vascular smooth muscle. contraction to l-name ( mm) was markedly reduced by injury, but was protected by no donors (p< . ). thus, exogenous no donors enhance the ability of the endothelium to regenerate and to release edrf in response to endothelium-dependent vasodilators. this may be due to an anti-proliferative and anti-mitogenic effect of no on vascular smooth muscle cells, allowing the endothelium to regenerate without intimal thickening. no also has been shown to inhibit platelet aggregation, and to attenuate neutrophil adherence and activation. the superoxide scavenging effect of no is not the basis for these effects since hsod is inactive in preserving endothelial function in injured arteries. thus, no exerts a variety of cytoprotective effects which may be of importance in protecting against vascular injury. much evidence has now accumulated to show that the excess production of the vasodilator nitric oxide (no) in sepsis is an important contributor to the hypotension and multiorgan failure characteristic of this condition. various cytokines play an important role in this process through their ability to induce the production of one of the enzymes responsible for no synthesis, the inducible no synthase (inos). we have studied the effects of cytokines on the induction of this enzyme both in vitro using vascular smooth muscle cells, and in a murine model of gram-negative sepsis. tn smooth muscle ceils, the cytokines il- , ifnq', and tnf-oc show strong synergy with one another in the production of inos. in order to define the molecular basis for this synergic effect, we have linked the promoter of the inos gene to a "reporter" gene, chloramphenicol acetyl transferase (cat), and transfected these constructs into vascular smooth muscle cells. assays of cat activity reflect the activity of the promoter in this system, and by generating sets of deletion mutants of the promoter sequence we have been able to define the area within the promoter which mediates the synergic effect of these cytokines. in addition to stimufatory effects on inos production, certain cytokines are able to down-regulate the production of inos in vascular smooth muscle cells, and the effects of these counterregulatory cytokines will be discussed. the interaction of these cytokine effects in the whole organism has been studied in a murine model of gramnegative sepsis. widespread induction of inos occurs in this model as assayed by enzyme activity and through use of specific antisera to inos. neutralizing antibodies to tnf-~ and tfn-y are both able to prevent death in this model, but it is only the anti-ifn-y which attenuates the induction of inos assayed in the liver. clearly there is some redundancy in the effects of cytokines on the production of inos in sepsis, and greater understanding of the most important factors in inos production is required in order to target anti-cytokine therapy most appropriately. effects of nitric oxide on hepatocyte metabolism in inflammation. j. stadler, department of surgery, tu mqnchen, frg hepatocellular nitric oxide (no) synthesis is induced by proinflammatory mediators such as tumor necrosis factor, interleukin- and interferon gamma or by bacterial toxins such as lipopolysaccharide. stimulation of the hepatocytes (hc) with a combination of these agents leads to an output of no in quantities which are not seen in any other celltype. it has been demonstrated by various investigators that important effects of these cytokines and bacterial toxins on hc metabolism can be attributed to the action of no. in contrast to other celltypes hc seem to be relatively resistant to suppression of basic metabolic functions such as energy metabolism by no. therefore, cell damage has not been described to a significant extent following exposure to no. however, no does inhibit total protein synthesis. the exact biochemical mechanism of this phenomenon has not been uncovered yet, but it has been demonstrated for some specific proteins that their production is inhibited at a posttransscriptional level. as in many other celltypes cgmp generation is elevated in hc by no through activation of the soluble guanylate cyclase. cyclic gmp may possibly exert a plethora of metabolic functions, but it is interesting to note that most of the cgmp seems to be transported out of the cell. some very specific effects of no on hc metabolism include the inhibition of the glyceraldehyde- -phosphate dehydrogenase (gapdh) and the cytochrome p (cyp) enzymes. inhibition of gapdh activity is mediated through nitrosylation of critical domains of the enzymes by no which enhances auto-adpribosylation. this effect on gapdh activity might be responsible for the inhibition of gluconeogenesis by no, which has been described recently. finally, no-mediated inhibition of cyps may help to explain the suppression of hiotransformation processes which is a characteristic featur,'~ r ~ "~flamed liver. nitric oxide (no) is an endogenous inhibitor of polymorphonuclear leukocyte (pmn) adhesion which limits pmn-endothelial cell interactions under normal conditions. we have previously demonstrated that following ischemia, no production by the vascular endothelinm is dramatically reduced. accordingly, we investigated the effects of no-donors on pmn accumulation and tissue injury following hemorrhagic shock and ischemia. hemorrhagic shock was induced in anesthetized rats by bleeding to mmhg for hours followed by reperfusion. segments of superior mesenteric artery (sma) were isolated and suspended in organ baths. in rats receiving saline sma relaxation to acetylcholine (ach, nm) was reduced by % compared to control sma segments (p< . ) while relaxation to sodium nitrite ( gm) was unaffected. in addition, mesenteric tissue pmn accumulation as determined by myeloperoxidase (mpo) activity was significantly elevated compared to controls (p< . l). interestingly, treatment with the no-donating agent, s-nitroso-n-acetylpenicillamine (snap) significantly preserved sma relaxation (p< . ), attenuated mesenteric mpo (p< . ) activity, and significantly improved survival compared to saline vehicle. in anesthetized, open-chest dogs we investigated the cardioprotective actions of a novel no-donor, spm- (schwarz pharma), following regional myocardial ischemia ( hour) and reperfusion ( . hours) . treatment with spm- ( rim) significantly reduced myocardial necrosis by % (p< . ) compared to an no-deficient analog of spm- , spm- . furthermore, mpo activity within the ischemic-reperfused zone was also significantly (p< . ) reduced following treatment with spm- compared to spm- ( . + . vs. . + . u/ mg tissue). these data strongly suggest that no is a potent inhibitor of pmn-mediated tissue injury following hemorrhagic shock as well as in acute myocardial ischemia-reperfusion injury. overproduction of nitdc oxide (no') may contribute to sepsis-induced hypotension. during septic shock, excess no" is produced by an isoform of nitric oxide synthase (nos) which is induced by inflammatory mediators. nonselective nos inhibitors have been proposed as a new therapeutic approach to treating hypotension in septic shock. we studied the differential hemodynamic effects of n~-methyi-l-arginine (l-nma), a nos inhibitor, in normal canines versus those challenged with endotexin (lps) and compared the activity of this drug across the venous, pulmonary and systemic vascular beds. awake canines were challenged with lps ( mg/kg, n= : mg/kg, n= ; or mg/kg, n= ) and treated with l-nma ( , , , , mg/kg/hr) for hours following a , , or mg/kg loading dose. animals were resuscitated with iv ringers solution ( ml/kg/hr). hemodynamic data were collected at , , , , , and hours using intravascular catheters and radionuclide heart scans and analyzed by anova. in both normal and endotoxemic animals, l-nma at all doses studied similarly increased mean arterial pressure (p= . ), and systemic vascular resistance index (p= .ol) and decreased cardiac index (p= . ) and oxygen delivery index (p= . ). in contrast, the effect of l-nma on mean pulmonary artery pressure, central venous pressure, pulmonary capillary wedge pressure, and pulmonary vascular resistance index was greater in lps-challenged canines compared to normal animals (p< . ), but this differential effect on the venous and pulmonary circulation occurred, > hours after lps challenge. l-nma did not significantly increase survival rates or times at any of the doses studied ( , , , or mg/kg/h) in either the low ( mg/kg) or high dose ( mg/kg) lps-challenge groups. a nonsignificant (p> . ) trend toward a beneficial effect on survival ol low dose l-nma ( mg/kg/h) in animals given the mg/kg lps-cha[lenge was not enhanced by increasing the lethality of the model or by administering higher l-nma doses. at the highest l-nma dose used in this study ( mg/kg/h), survival time decreased significantly for both the low and high dose lps-challenge animals (p< . ). this increased mortality was not explained by changes in plasma concentrations of either lps or tnfc~. thus, l-nma did not have a greater effect on the systemic arterial circulation in endotoxemic compared to normal canines. however, in the venous and pulmonary vascular beds, the effect of l-nma increased with time after endotoxin-challenge these data suggest the induction of nos activity by endotoxin in canines may be relatively greater in venous and pulmonary vessels compared to systernic arteries. l-nma, a nonselective nos inhibitor, did not decrease mortality in endoloxemic canines and the highest dose studied was harmful. pulmonary hypertension (ph) and arterial hypoxemia are characteristic features of the adult respiratory distress syndrome (ards). reducing pulmonary vascular pressures may promote the resolution of pulmonary edema. intravenously infused vasodilators lower ph in ards, but, as a result of their general vasodilatatory effects, systemic mean arterial pressure may also decrease. furthermore, blood flow may be increased to non-ventilated or poorly ventilated lung areas resulting in a rise of intrapulmonary shunt, thus causing a further fall in pad . recently, short term inhalation of low concentrations of the gas nitric oxide (no), an endogenous endothelium derived relaxing factor, which is rapidly inactivated in blood by hemoglobin, was reported to decrease ph without causing systemic vasodilation in sheep [ ]. similar changes have been observed in patients with severe ards during repeated short term inhalation of no ( and ppm), which rapidly and selectively decreased the mean pulmonary artery pressure (pap) and, in contrast to intravenously infused prostacyclin, induced a remarkable increase of pad [ ] . this improvement in oxygenation was caused by a redistribution in blood flow away from intrapulmonary shunt areas to normal ventilated lung regions. continuous no inhalation ( - ppm) consistently lowered the pap and augmented the pao /f.o for up to days. no negative side effects were observed during the whole time span examined. in particular methemoglobin levels always remained below . %. following these investigations, it could be shown that these effects may also occur using concentrations in the parts per billion range [ ] , which may reduce possible toxic side effects. however, in the same study it was demonstrated that the dose-response curves for pa and pap have different patterns. whereas pap presented a continuous dose-dependent downward tendency with an eds o of approximately - ppm, the improvement of oxygenation had a maximum at ppm and, at higher doses, drifted back towards the baseline data. the ed~o was estimated at approximately ppb, i.e. more than ten times lower than for the reduction of pap. in conclusion, inhalation of no by patients with severe ards may result in persistent and reproducible decreases in pap associated with an evident improvement in pad , thus allowing reduction of the f.o . no inhalation should be performed using low concentrations which are less toxic, although any possible risks still have to be considered carefully. dose-response studies for the individual patients are recommended urgently. finally, controlled randomized studies are required to demonstrate that additional no inhalation is able to reduce mortality of ards. inhibition of the activity of glyceraldehyd- -phosphate dehydrogenase (gapdh), an enzyme of the glycolysis/gluconeogenetic pathway, through adp-ribosylation is promoted by nitric oxide (no). since no is produced in the septic liver and hypoglycemia is a major problem of late sepsis, it was investigated whether no interferes with gluconeogenesis of hepatocytes. hepatocytes (hc) were isolated from sprague-dawley rats using a collagenase perfusion technique and differential centrifugation. exogenous no was applied by incubation with the no-donors s-nitrosyl-acetylpenicillamine and sodium-nitroprusside. endogenous no synthesis was induced by incubation with cytokines (tnfcq il- , ifnj and lipopolysacchafide (lps). hrs later the incubation medium was changed to a solution containing lactate, ornithine, lysine, ammoniumchloride and glucagon for optimal conditions of gluconeogenesis. after more hrs glucose and nitrite levels were determined spectrophotometrically. gapdh activity was measured by the nadh-dependent conversion of , -diphosphoglycerate to glyceraldehyde- -phosphate. incubation of hc with no-donors led to a concentrationdependent inhibition of gluconeogenesis and gapdh activity. however, gapdh activity was about times more sensitive to the inhibitory effect of exogenous no. incubation of hc with cytokines and lps induced nq synthesis as measured by an increase in nitrite concentrations. endogenously produced no suppressed gluconeogenesis by _+ %. in contrast to exogenously applied no, the effect of endogenous no synthesis was less on gapdh activity resulting in an inhibition of only _+ %. in conclusion, exogenous and endogenous no inhibited gluconeogenesis as well as gapdh activity. however, there was no correlation between the extent of inhibition of these two parameters of hepatocellular glucose metabolism. we have shown that inhibition of hepatocyte (hep) synthesis of nitric oxide (no) potentiates cell injury in a model of acetaminopheninduced oxidative stress and the extent of damage was paralleled by depletion of reduced glutathione (gsh) stores. to clarify the role of no in modulating the redox state of hep, we studied the effect of inhibition of cytokine-mediated no production on hep gsh stores, in a system of isolated rat hep in primary culture, no synthesis was induced (stim) by exposure to il- , tnf, ifn, and lps for hours. , , and ~m of n-monomethyi-l-arginine (nmma), a specific inhibitor of no synthesis, was added. cells incubated in media alone served as controls (cont). the no metabolite (no ); aspartate aminotransferase (ast), an indicator of cell injury; and gsh were assayed. (data presented as mean + sem; n= .) gsh (nmovma orotein) ..~ (nmol/ma orotein) cont . + . + . # stim . + . + stim+ o tzm nmma . + . + . # stim+ ~m nmma . _..+ . * + . # stim+ pm nmma . + . * + . # stim+ )lm nmma . + . * + . # anova , . (* p < . versus stim, # p < . versus stim; anova with neuman-keuls) gsh in cont+ i~m l-nmma was equivalent to that of cont ( . vs. . ). ast release was equivalent in all treatment groups. these data show that inhibition of hep synthesis of no depletes intracellular stores of reduced gsh. we conclude that hepatocyte no production modulates cellular gsh homeostasis and as a result, may be hepatoprotective in oxidative injury. nitric oxide (no) is a modulator of immune response and may be involved in the changes in immune reactivity after major trauma and operations. we investigated no-generation in rat and mice spleen cells (sc) after partial hepatectomy (ph). c bl/ mice and lew rats underwent a % and % ph, respectively. sc were prepared - days after ph and plated at to x ecells per well. after h incubation at °c, no-production was measured as nitrite levels (griess reagent). normal mouse sc did not produce no, neither basal nor in response to lps or con a starting at the second day after ph, we found a substantial production of no. in rats, also sc from control animals were able to generate no; both basal and stimulated no-generation were further enhanced after ph (table, values expressed as mean --se). after shame operation, there was only a modest elevation of noproduction in rat and mouse sc. in first experiments we could demonstrate no-production also in phagocytes from a patient days aider liver partial resection ( . nmol nitrite/ cells) enhanced no-production in macrophages may contribute to the changes of immune reactivity after partial hepatectomy. nitric oxide (no) is recognized as an important mediator in endotoxemia and sepsis. increased synthesis of no has been demonstrated in septic humans and animals, and no inhibitors have been used in the treatment of septic shock. recent reports have, however, suggested that this form of therapy may cause serious organ damage. in the present investigation circulatory and metabolic changes in the liver were studied during treatment with the no-synthase inhibitor n-nitro-l-arginine-methyl ester (l-name) in endotoxemia. methods: juvenile pigs were randomized to one of the following treatment groups: ) encletoxin and l-name, ) endotoxin, ) naci and l-name, ) nach preliminary results from groups (n= ) and (n= ) are presented. catheters for pressure measurement were introduced into the aorta, hepatic and portal veins and ultrasonic transit time flow probes were placed on the hepatic artery and portal vein. a catheter was introduced into the pulmonary artery. endotoxin ( . gg/kg/h) was given as a continous portal infusion over the entire observation period of hrs. l-name ( mg/kg) was given as a bolus after hrs. of endotoxemia. results: endotoxin transiently reduced portal vein flow (pvf) by %* and hepatic artery flow (hal e) by %*, while l-name caused a further and lasting reduction in flow (pvf %, haf %)*. transhepatic (portal-hepatic vein) vascular resistance increased to times baseline value during endotoxemia while l-name caused a further marked increase in resistance to times initial value. portal oxygen saturation (so ) decreased by %* during endotoxemia. l-name caused a reduction in portal so by %*. arterial so was unchanged in both groups. hepatic oxygen uptake was not changed by endotoxin, but was markedly reduced after addition of l-name. endotoxin caused a % reduction in cardiac output (co). the addition of l-name reduced co by a total of %*. *: p < . . conclusion: is the present model of endotoxemia treatment with the nitric oxide synthase inhibitor l-name markedly reduced liver perfusion and portal oxygen supply. this might explain the increased liver damage reported in previous studies using no-inhibitors. the increase in transhepatic resistance found after l-name treatment will tend to cause pooling of blood in the splanchnic veins, resulting in reduced filling of the heart and thus contribute to the observed reduction in cardiac output. institute for surgical research, rikshospitalet, the national hospital, university of oslo, oslo, norway. we have investigated the role of tumour necrosis factor (tnf) and interleukin-i (il-i) in the induction of nitric oxide synthase (nos) by bacterial endotoxin (lipopolysaccharide; lps; mg kg -i i.v.) in vivo. in anaesthetized rats, pretreatment with a monoclonal antibody for tnf (tnfab; mg kg -i s.c., at h prior to lps) or with an il-i receptor antagonist (il-ira; mg/kg bolus and . mg/kg/h infusion) ameliorated the fall in mean arterial blood pressure (map) at - min after lps. for instance, endotoxaemia for min resulted in a fall in map from -+ (control) to -+ mmhg (p< . ; n= ). in contrast, animals pretreated with tnfab or il-ira prior to lps injection maintained significantly higher map at min when compared to lps-control: -+ mmeg (n= ) and -+ mmhg (n= ), respectively (p< . ). three hours of endotoxaemia significantly reduced the contractile effects of noradrenaline (na) in the thoracic aorta ex vivo. the hyporeactivity to na was partially restored by in vitro treatment of the vessels with ng-nitro-l-arginine methyl ester (l-name, min, x - m). pretreatment of rats with tnfab or il-ira significantly (p< . ) prevented the lps-induced hyporeactivity of rat aortic rings ex vivo. l-name did not alter or only slightly enhanced the contractions of aortic rings obtained from tnfab or il-ira treated lps-rats, respectively. at min after lps there was an induction of calcium-independent nos activity in the lung ( . -+ . pmol citrulline/mg/min, n= ), which was attenuated by tnfab and !l-ira by -+ % and -+ %, respectively (n= ; p< . ). thus, the production of both tnf and il-i contributes to the induction of nos by lps in vivo. the protective effect of agents which inhibit the release or action of tnf or il-i in shock may be, in part, due to inhibition of nos induction. neal garrison, md objective: sepsis is often accompanied by organ dysfunction, in part due to impaired microvascular perfusion. recently, nitric oxide (no) has been described as an important mediator of the hemodynamic changes of sepsis, and no synthase (no-s) inhibitors have been advocated for treatment of septic shock, but their visceral microcirculatory effects are inadequately characterized. we postulated that no-s inhibition would exacerbate the impaired organ perfusion of sepsis. methods: six groups ofdecerebrate rats were studied. bacteremia was induced with live e. coli, which consistently increased cardiac output - % above baseline (bl). the no-s inhibitor nm-nitro-larginine methyl ester (l-name, mg/kg iv), prevented this increase and elevated map by - %. in the first groups, total hepatic blood flow (thbf, ml/min by time transit flowmetry) and microvascular perfusion (mi-ibf, ¼ bl by laser doppler flux) were measured. in the other groups, in vivo videomicroscopy was used to observe renal microvascular responses (ila=interlobular artery, aff=afferent arteriole, eff=efferent arteriole; % bl for all). results: data are rains after e. cob. n= - /group. * p< . vs bl by remanova and § p< . vs e. coli alone by anova. ec+l-name -+ - _+ " § - _+ * § - _+ * § - + * - + * § conclusions: l-name administration in controls decreased renal blood flow, indicating no contributes to basal renal tone. bacteremia decreased mtlbf but not thbf, and mi-ibf was further impaired by no-s inhibition. e. coli caused renal preglomemlar, but not postglomerular constriction and reduced flow. l-name exacerbated these e. coli-induced alterations and caused eff constriction. these data indicate that no-s inhibition exacerbates bacteremia-induced impairment of renal and hepatic blood flow, suggesting that no is an importam compensatory dilator mechanism in these organs during sepsis. irf (iron responsive factor) is the central regulatory protein of intracellular iron metabolism able to bind to responsive rna elements (ires) present atthe 'untranslated region (utr) of ferritin mrna and 'utr of transferrin receptor mrna. binding of irf to ires results in repression of ferritin mrna translation and increased stability of transferrin receptor mrna leading to enhancement of transferrin receptor translation. we describe here that either tetrahydrobiopterin dependent stimulation as well as cytokine (ifn-~)/lipopolysaccharidemediated induction of nitric oxide synthase activates irf, which is due to direct interaction of nitric oxide with the iron-sulphur-cluster of irf. this was shown by gene expression studies using a plasmid containing a ferritin ire and a cat indicator box which was transfected into k myelomonocytic cells, which were shown to have a constitutive form of nitric oxide synthase (nos). furthermore, the increased binding of re to irf due to irf activation of irf by nitric oxide was demonstrated by gel shift assays. irf activity was much more increased in cellular extracts from murine macrophages (j ) where a cytokine inducible form of nos has been characterized earlier as compared with irf activity in k cells, where nos was stimulated by increasing the availability of the essential nos cofactor , , , -tetrahydrobiopterin. we then demonstrated that activation of irf by nitric oxide is accompanied by alterations in ferritin translation as checked by metabolic labeling and immunoprecipitation. these results suggest a reasonable mechanism for the regulation of iron disturbances under chronic inflammatory disorders, characterized by increased concentration of immune activation parameters like ifn- or neopterin and low serum iron and hemoglobin concentrations. taken nitric oxide, no, the putative endothelial derived relaxant factor, edrf, has been shown to be a potent inhibitor ofplatelet aggregation in vitro. in vivo evidence however, is scarce. accumulation of platelets in the lungs has been shown to occur during extracorporeal circulation. the aim of the present study was to investigate the effect of inhaled no on this reaction. materials and methods: the animals were divided into two groups, each consisting of pigs. platelets were selectively labelled with luln-oxine. dialysis was instituted via catheters in the femoral vessels. in group , no, ppm, was added to the inhaled gas from the start of dialysis. in group no was not given. the activity over the lungs was followed dynamically with a gamma camera. central hemodynamics was monitored via a swan -ganz catheter. results: the activity was significantly lower in group , from minutes after start of dialysis and onwards, indicating diminished accumulation of platelets in the lungs. parallel to this the hemodynamic response in terms of increased pulmonary artery pressure and pulmonary vascular resistance was blunted in this group conclusion: inhaled no in this model seems to affect pulmonary platelet sequestration. an associated attenuation of the changes in central hemodynamics was also seen. previous studies from our laboratory have demonstrated that vascular contractility decreased in endothelium-intact blood vessel rings in early and late stages of sepsis. although endothelium removal in early sepsis restored vascular contraction, the depressed smooth muscle contractility observed in late sepsis was not restored by endothelium removal. this indicates that impairment of smooth muscleper se may be responsible for such dysfunction in late sepsis. the aim of this study, therefore, was to determine whether or not smooth muscle-derived nitric oxide (no) plays a role in producing vascular smooth muscle dysfunction during late stages of sepsis. to study this, rats ( - g, n= - /group) were subjected to sepsis by cecal ligation and puncture (clp). septic and shamoperated rats then received rrd/ g bw normal saline. the animals were killed at , , or h post-clp ( h post-clp=early sepsis; - h post-clp=late sepsis), and thoracic aortic rings were prepared for contraction studies using organ chambers. the complete removal of endothelial cells was tested by the absence of any significant acetylcholine-induced vascular relaxation. contractile responses to norepinephrine (ne, to - m) were determined in the aortic rings without intact endothelium. ng-monomethyl-l-arginine (l-nmma, /~m, an inhibitor of no synthase) was then added to the organ chamber and ne-induced peak contraction was determined before and after the addition of l-nmma. the peak contraction (rag/rag tissue, mean_+sem) is shown below: the results indicate that the addition of l-nmma did not significantly affect ne-lnduced peak contraction in endothelium-denuded vessel rings at and h after clp. in contrast, l-nmma administration produces an % increase (p< . ) in peak contraction during late sepsis. therefore, the vascular smooth muscle contractile dysfunction observed at h post-clp is partially due to smooth muscle-derived no over-production. thus, unlike macrophages in which inducible nitric oxide synthase (inos) is observed in early sepsis, the inos in vascular smooth muscle appears prominent only in the late stages of sepsis. in three cases of human septic shock in which ng-monomethyi-l-arginine, (l-nmma) a nitric-oxide-synthase-inhibitor was applied, we isolated three completely different types of pathogens: candida, pseudomonas aeruginose and multiresistant coagulase-negative staphylococci. this observation suggests that endotoxin alone is not the main factor triggering hypotension in septic shock by the nitric oxide pathway. in a -years-old woman in severe septic shock due to a candida and pseudomonas aeruginosa infection complicated by adult-respiratorydistress-syndrome conditions deteriorated despite adequate conventional therapy. in this trial, effects of l-nmma on cytokin-levels were investigated. the study-protocol was approved by the ethical committee of the department of surgery. after two boll of mg of l-nmma, a continuous infusion was installed ( . mg/minute and kg body weight l-nmma). as expected mean arterial blood pressure rose ( to mmhg}, heart rate stayed stable ( + b/rain), systemic vascular resistance increased ( to dyne.sec/cm ), cardiac output decreased ( to . l/rain), and cardiac index declined ( . to . l/min/m }. before and after minutes while the infusion of l-nmma, blood samples for immunological measurements were taken and processed together. pulmonary-shunt-volume was observed before the application of l-nmma, after one hour and after matutes. neopterine increased from . to . ng/ml, tumour-necrosis-factor-a increased from . to . pg/ml and intedeukin- increased from . to . pg/ml. immunoglobulines a, g, and m ( . to . , . to . , . to . g/i), complement factor c- c and c- ( . to . , . to . g/i), alpha-l-antitrypsine ( . to . g/i), c-reactive-protein ( . to . rag/i), interleukin- ( pg/ml) and soluble interleukin- ( to units/ml) did not change significantly. pulmonary-shuntvolume decreased from . % to . % within one hour and to . % after minutes. in septic shock blocking nitric oxide as an intervention at the end of a not ~,et ful!y understood cascade might have important influences on pulmonary-shunt-volume and inter-cell-communication. department of surgery, pharmacy* and immunology**, university hospital of zurich, r~imistrasse , zurich, switzerland we previously reported that hypoferremic cba mice had an increased resistance to salmonella infection, and that injection of ammonium ferric citrate (afc) to these mice led to enhanced infection (ganthier et at. . microbiol.immuno : ) . because nitric oxide (no) is involved in the antimicrobial activity of routine macmphages towards various inttacellular pathogens, we investigated the influence of iron on the bactericidal activity of cba mouse macrophages towards s.typhimurium and on the production and activity of reactive nitrogen intermediates (rni). peritoneal macrophages hum cba mice were cultured in the presence (or not) of afc ,um, ifn-,/ u/ml, lps fig/m/, ngmonomethyl-l--arginine (mmla) ram. nitrite (no -) content of the supematants was determined by a standard griess reaction, and h release was measured by the peroxidese dependant oxidation of phenol red. for intracellular killing, macrophages monolayers were infected, and, at various intervals, lysed by triton x- , and surviving bacteria enumerated by colony counting on agar. for in vivo experiments, mice were infected ip with . ml of a suspension of . ~" s.typhimurium, strain c , and injected with aminoguanidine (ag) mg/ml in saline. our results show that the rn[ inhibitor ag strongly accelerates the mortality of infected mice, the survival rate decreasing from % in the control group to % in the treated group, days after challenge. correlatively the rni inhibitor mmla induces in vitro a decrease in the rate of bacterial killing, fxom % to %, in macrophages triggered with ifn-? + lps. the cultivation of macrophages in the presence of afc leads to a decreased no -accumulation, . nmole/well v.s. nmole/well. conversely h production is enhanced from nmole/well up to , nmole/well. nevertheless, macrophages cultivated in the presence of afc exhibit an increased tale of intracellular killing, % in iron exposed macrophages v.s, % in control macrophages. when triggered with ifn-~, alone, macrophages have a reduced antibacterial activity ( % v.s. %) whereas the addition of afc to these macrophagas restores an elevated ( %) rate of killing. in conclusion, the results show that bactericidal activity of cba macrophages towards s.typhimurium depends on the production of no by these macrophages ; but they also demonstrate that no is not the only reactive species involved in the intracellular kil/ing of s.thyphimurium ; indeed afc which strongly inhibits rni production, stimulates h release by these macrophages and increase their bactericidal activity in vitro. nevertheless afc may promote bacterial growth in vivo. crssa. unit de microbiologie. bp . la tronche cedex france. henning jahr, ulrike noack, karin braun the large amounts of no produced by the inducible no synthase in rat macrophages have direct antimicrobial effects, but inhibit the activation of the lymphocyte-dependent host defense system. the aim of this study was to investigate if complement activation influences no-generation. spleen cells from lew rats were incubated at °in tcm- / % fcs, with or without additional rat serum. after h, nitrite (end product from no metabolism) was measured by oriess reagent. in rat spleen cell preparations, most of the no is produced by macrophages. complement activation in vivo was carried out by i.v. injections of u cobra venom factor/kg b.w. at days and . significantly higher (p<o.o ) lps-stimulated no-generation was found in spleen cells prepared at day ( . - . nmol nitrite/ cells, n= ) compared to spleen cells from non-treated animals ( . ± . nmol, n= ) complement activation was effective also in vitro. when incubated in % zymosan-activated rat serum, both basal and lps-stimulated noproduction were enhanced in spleen cells (table, values increased production of nitric oxide (no) is associated with sepsis, however, the effect of no inhibition on survival during sepsis is unclear. we examined the effect of no inhibition on host's ability to eliminate bacteria and survival in mice sepsis model. sixty female balb/c mice were injected with e.coli ( qbody) into peritoneal cavity. the treated group received n-ultro-l-arginine-methyl-ester (l-name), an inhibitor of nos, one hour before bacterial challenge. thirty mice were observed for survival after e.coli challenge and the other mice were sacrificed at hours. blood was obtained by cardiac puncture and peritoneal lavage fluid (plf), liver and lung were harvested. the number of peritoneal exudative cell (pec) was counted and colony forming units (cfld of bacteria in the tissues, blood, and plf were also determined. in addition, pec was cultured in vitro with or without lipopolysaecharide (lps). tnf levels in the blood, plf, and supematants of cultured pec were measured by l bioassay. survival rate (%) qrql/,p n hr hr dav control (normal saline . ml/body i.p.) n (l-name mg/kg i. administration of l-name before e.coli injection increased the number of bacteria in plf and tnf level in plasma, the result suggests that nos inhibitor may depress the host's ability to kill the injected bacteria and that it may induce greater systemic tnf production. we conclude that nos inhibitor is detrimental to animals in sepsis. the hypothesis that sod prevents reperfusion injury to the liver by protecting the endogenous endothelium derived vascular relaxing factor (no) from being inactivated by oxygen free radicals should be investigated. male wistar rats were subjected to min of partial liver ischemia ( %) and min of consecutive reperfusion in situ. some rats were pretreated with the no synthase inhibitor nitro-l-arginin (nla: i mg/kg). sod, when used, was given as mn-containing preparation at a dose of u. i.v. i min prior to ischemia. results (sod/ nla+sod/ ni~,, respectively): bile flow (~i/g/min) : . ± . "#/ . ± . / . ± . . alt (u/ ) • ± ~#/ ± / ± . gldh (u/l): . ± . e#/ . ± . #/ . ± . . in absence of nla, sod enhanced postischemic bile flow and reduced leakage of alt and gldh into the plasma, while the effects of sod disappeared, when endogenous ~synthesis was inhibited by nla. nla had no ~ffect on untreated animals submitted to the same protocol. these results may suggest, that oxygen free radicals interfe~:e with the endogenous vasodilator no and that the benefit of sod can be attribuated in large part to a protection of no during reperfusion. nitric oxide (no) reduces pulmonary vascular resistance and increases oxygenation by inhalation in ards patients [ ] . animal studies on the endogeneous no production proved that no is exhaled after synthesis in the respiratory tract [ ] . with approval by the hospital ethics committee, we studied healthy volunteers and ventilated patients by measurement of inand exhaled no by no/no -chemiluminescence in segments of the upper respiratory tract. we found that no is synthesized predominantly in the nose and in the upper nasopharynx, leading to inspiratory tracheal no concentrations of . - . parts per million in non-smoking volunteers; data during mask ventilation per nose were as follows: non . . . . , parts per million (ppm) no, mean, n = ; ~ p < . (t-test) between smokers and non-smokers; p < . (t-test) between in-and expiration. about % of no is resorbe.d by the lung, and the exhaled no -in contrast to former presumptions -is the remaining part of the autoinhaled no. intubated and ventilated patients are deprived of no autoinhalation, and the exhaled no in the trachea decreases more than % {o. ppm before intubation vs. . ppm after intubation, ~, < . ), whereas the nasal no concentration increases by cumulation. hence, low-dose no inhalation in ards patients might be considered as a no replacement, especially since the tracheal no concentrations we measured in volunteers are similar to those which could be demonstrated to be effective for ards in former studies [ ] . the data demonstrate that no is synthesized in the nose, and inhaled and resorbed by the lung. this phenomenon of no autoinhalation, which is reduced in smokers and in ventilated patients, does possibly contribute to the regulation of the ventilation-perfusion ratio in the lung. kikuchi , yoshihiro inoue , masao yoshida critical care and emergency center, and department of bacteriology, iwate medical university. nitric oxide (no) is produced by macrophages and vascular endothelial cells. it is thought to be the true form of endotheliumderived relaxing factor, and to be involved in the fall of blood pressure during septic shock. we have reported previously that endotoxin (lps), tnf-c~ and il- contribute to the occurrence of septic shock (circ shock : ; . the present study investigated the in vitro effects of lps, tnf-o~, il- and ifn-'i on the production of no by macrophages. peritoneal macrophages were cultured with lps, il- , tnf-~ and ifn-¥. in culture with lps, no was produced in a dose-dependent manner, and the production was suppressed by a no production inhibitor, l-nmma. ifn-y, il- and tnf-c~ used alone did not promote the production of no, but helped lps to facilitate no production. a combination of il- and tnf-c( enhanced lps-facilitated no production to a greater extent than il- or tnf-c~ alone. the above results suggest that these cytokines are involved in endotoxin shock through the mechanisms that facilitate no production. - uchimaru, morioka . japan. t. yolk , , i. ioannidis , w.j. kox and h. de groot objectives: nitric oxide (no.) is known to play an important role in neurotransmission, vasoregulation, and bactericidal as well as tumoricidal cellular defense systems. by means of no-donating agents we studied the mechanism of no-mediated cytotoxicity against rat liver microvascular endothelial cells. materials and methods: -morphoiinosydnonimine-n-ethylcarbamide (sin-l) and sodium nitroprusside (snp) were used as no. donators, ldh release and trypan blue exclusion were applied to indicate cell viability. results: sin- ( mm), which releases both no. and o -., caused a time-dependent cytoreduction of % and % after and hrs, respectively. this effect was not inhibited by superoxide dismutase (sod), which removes --to form h and . in contrast, the addition of catalase (cat), which removes h to form h and , diminished the cytotoxic effect ot sin- to %, equivalent to high concentrations of snp ( mm), which solely releases no.. in addition, the amount of h formed by mm sin- equaled the amount ot enzymaticany produced h by mu/l glucose oxidase (god). whereas god in this concentration and snp in low concentration ( mm) alone were not toxic to endothelial cells, the combination caused an % reduction of viability, comparable to the effect observed with sin- ( mm) alone. moreover, toxicity mediated by high concentrations of snp ( mm and ram) was reduced by % under hypoxic conditions indicating synergism with no-and . conclusions: we conclude, that no.-induced toxicity against endothelial cells is enhanced slightly in the presence of oxygen and is not due to an interaction with -. however, toxicity increases dramatically in the presence of h . this may possibly occur either by a chemical formation of more potent reactive hydroxyl radicals or by independent actions on different cellular targets. although it is becoming increasingly clear that nitric oxide (no) is associated with otxan dysfunctions in septic patients, little is known as to die kinetics of no production in these patients to clarify these kinetics, we have investegated serum arid monocyte associated no in septic patients. five patients who had undergone gastrointestinal surgeries mid were complicated postoperatively with severe sepsis served as the subjects in this study (sgroup), using twelve healthy volunteers as a control group (c group). serum no and no levels were measul*d sliectrophotometricallymonocyte cultures were done for itrs with or without lps+ifn-t stimulation mid no and no levels in the supernat,'utts were also measured spectrophotometrically. furthermore,using an no selective electrode,time course of the lps+ifn-t induced no production by monocytes was studied. l) serum no levels in s group were slightly lfigher than dmse in c group,bnt there were no significoatt differences between the two. )both no levels in the supematmlts of monocytes cultured with lps+ifn-t mid no , no levels ill lhe supernatants of monocytes cultured without lps+ifn-y were significantly higher in s group. further,the time required for the no production by enltnred monoeytes was siglfificantly shorter also in s groap. conclusions l)in severe septic patients, monocyte no productions wei~ not only primed, but also activated concomitantly, suggesting that these monocyte activations m'e strongly associated with organ dysfunctions in sepsis. )based on the restdts of serum no nteasuremeuts, it can be deduced that no p~'oduced by monocytes in septic patients affects tissues and org~s ioeoregiomflly. objectives of the study: nitric oxide (no) is an important messenger which accounts for a wide spectrum of physiological and pathophysiological processes, e.g. mediating vasodilation in endoloxin shock investigating the signal lransducfion pathways involved in the regulation of the inducible nitric oxide synthase (inos), we report the involvement of phosphatidylcholinespecific phospholipase c (pc-plc) and proteinkinase c (pkc). materials and methods: no-production was induced in the murine macrophage cell line j with lipopolysaccharide (lps) [lljg/ml] and interferon ;f (ifny) [ u/ml]. after hours of incubation no-release was determined as nitrite (no ) by using a colorimetric assay based on the griess-reaetion. results: preincubation of cells with the pkc-inhibitors staurosporine (stp), chelerythrine, bisindolylmaleimide (bim) and d , that recently has been identified as a selective inhibitor of pc-plc, diminished significantly lpsand ]fnt-induced no -production (p<o, ). although we observed no toxic effect on cell viability, no -production was related to protein concentrations to exclude any inhibitory effect on celt growth. celts preincubated with stp [lo nm] released % less no in response to lps and ifn,[ compared to control cells cultured without inhibitor. chelerythrine [ }jm] and bim [ioijm] reduced the no -formation by % and %, respectively. interestingly, the most potent inhibitor of no-production turned out to be the xanthate d cells pretreated with d [ ~g/mt] released % less no than stimulated controls the inhibitory effect on no production decreased the later the inhibitore were added after stimulation; e.g. bim added and hours after stimulation reduced no -production only by % and %, respectively. accordingly, inqs activity present in stimulated cell lysates supplemented with l-arginine and co-factors was not suppressed by the inhibitors tested. conclusions: our findings suggest, that the induction of inos but not its activity is a pkc and particularly pc-plc dependent process. dr. k tschaikowsky, institut for anaesthesiologie, universital erlangen--nqrnberg, krankenhausstr , erlangen interleukin- (il- ) affects nearly every cell type by increasing the expression of a variety of genes associated with the promotion of inflammatory processes. these include upregulation of cyclooxygenase and nitric acid synthases. the il- ri transmits a signal(s) through the cytoplasmic domain which is structurally related to the fruit fly toll protein. we have recently cloned the promotor most proximal to the ' utr of the human il- ri gene. the genomic sequences reveal a lack of tata and caat boxes but rather a considerable ( %) gc rich region. the translation of the type i il- r appears under a significant suppression and may limit the biological activities of il- by low level of expression. on the other hand, the type ii il- r, lacking a significant cytoplasmic domain, does not transmit a signal and acts as a decoy receptor preventing the binding to the type i receptor. there are several approaches to reducing il- activity; inhibition of il- converting enzyme which cleaves pro-il-l[ , anti-il- ri, the il-i receptor antagonist (il- ra) and soluble (extracellular) il- ri and il- rii. il- ra is structurally related to il- and binds at °c to the il- ri with near equal affinity as that of bona fide il-i; however, il- ra does not transduce a signal. il- ra has been given to humans in pharmacologic levels (mean plasma levels of gg/ml) without evidence of agonist activity. in addition, there has been no affect on normal homeostatic parameters, suggesting that il-i does not play a role in health. in healthy humans given intravenous endotoxin, il-ira reduced endotoxin-associated neutrophilia by % and completely reversed endoloxin-iuduced immunosuppression, il-tra is produced naturally and is elevated in the circulation in several diseases. in a variety of diseases, plasma levels of il- ra are in -fold molar excess to those of il-i [ . it is unclear whether these endogenous levels of il- ra are sufficient to block il- activity in disease. a single injection of ]l- or ifna in humans does not induce il- but rather high levels of il- ra ( ng/ml). prof.dr.l.a. aarden interleukin (il ) is a multifunctionai cytokine with a central role in host defense mechanisms and consequently in inflammation. il is thought to be involved in the pathogenesis of various diseases. therefore, specific inhibitors could have therapeutic value. a human-mouse chimeric monoclonal antibody to il has been applied in a phasei clinical trial in patients with multiple myeloma and in primate models of sepsis. no toxicity was observed and the most remarkable outcome of this study was the build up in the circulation of il -anti-il complexes, suggesting that plasma il measurements represent a gross underestimation of il production in the patient. until now no natural antagonists of il have been described. the biological activities of il results from binding to a lowaffinity il -receptor (il r). the il /il r complex induces disulfide-linked homodimerization of the signal transducing receptor component, gp . studies on the structure-function relation of il revealed that disruption of the gpl -interaction site on il gives rise to a mutant il that, by virtue of its intact binding to the il r, acts as a potent antagonist on human hepatocytes and on human b cells. in vivo studies using this molecule will be initiated in the near future. the evolution of infla~ation involves a dynamic series of cellular events controlled by specific signals which are important to the initiation, maintenance, and final resolution of the inflammatory response. the various phases of inflammation are influenced via the expression and subsequent regulation of a number of key mediators which play a predominant role during each particular stage of the developing lesion. for example, the initial elicitation of blood born leukocytes to on inflamed area is a complex system that is dependent upon the expression of early response cytokineso these proximal mediators, including both interleukin-i (il-i) and tumor necroisis factor (tnf), are important in the initiation phase by activating both immune and non-immune cells and establishing a series of cytokine networks, thus, the above proximal mediators can stimulate endothelial cells, epithelial cells, smooth muscle cells, and fibroblasts to generate more distal cytokines. these latter cytokines include interleukin- (il- ), macrophage inflammatory protein-i (mip-i), and monocyte cbemoattractant protein-i (mcp-i). the importance of the above chemukines can be found in the role they play in leukocyte elicitation, as well as wound repair. numerous investigations have shown the diverse cellular sources of il- and the ability of il- to elicit neutrophils in vitro at pm to nm concentrations, however, recent studies have sho%~ an additional role for il- during the resolution phase of the inflammatory process. using corneal pocket animal models of angiogenesis/neovascularization, il- was found to be a potent angiogenic factor at concentrations ranging from - ng/ml. sequential fluorescein angiograms demonstrated that il- induced neovascularization by days, which regressed by weeks. in further analyses, both conditioned media recovered from either inflamed human heumatoid synovial tissue macrophages or lps-stimulated peripheral blood monocytes were found to possess potent angiogenic activity, which was effectively blocked by neutralizing antibodies directed against human il- . in addition, an il- antisense nligomunclentide blocked the expression of a functionally active angiogenic factor from lps treated monocytes. the above data demonstrate that il- has multifumctional activities during the initiation, maintenance, and resolution of a local inflammatory response. inhibits specific t-cell responses to soluble protein antigens and alloantigens. the proliferative responses of th , thl and th cd ÷ t-cell clones and cytokine production by these t-cell subsets are equally well inhibited. the inhibitory effects of il- are indirect and related to inhibition of antigen-presenting capacity and accessory function of the antigen-presenting cells (apc). however, il- also directly inhibits t-cell proli/eration induced by cross]inked anti-cd or anti-cd mabs (e.g. in the absence of professional apc), by specifically inhibiting il- gene transcription and il- protein production. il- also inhibits the production of the pro-inflammatory cytokines il-l-a, ~, il- , and tnf-a and the chemokines il- and mip-i-u, which are strong chemo attractants for neutrophils and macrophage, respectively. in contrast, il- enhances the production of the il- receptor antagonist, a cytokine with anti-inflammatory activity. il-l produced by monocytes downregulates class ii mhc expression and il- production by these cells in an autoregulatory fashion. collectively, these in vitro data indicate that il- is a powerful suppressor factor for immune-and inflammatory responses and therefore may have potential clinical utility as an anti-inflammatory agent. this notion is supported by recent studies which indicated that il- also prevents lethal lps-induced toxic shock in mice. five of the chamekines (hip-i~,era~tes. ip-io and il ) also chemoattraet t lymphocytes, while others act on mast cells. we have studied the effaces of these chemokines on t cell subsets. il preferentially chemoattracts uns~imulated t cells. raises ~ttracte resting as well as activated cd and cd memory t cells. ip- chemoattracts only preactivated (not resting) cd or cd t cells. hip-l~ preferentially chemoattracts gd t calla, while hip- more readily attracts the cd t call subset. the chemoklnes and ip-io were also sho~rn to promote the adherence of ~he same subsets of anti-cd activated t cells to ili activated human umbilical vein endothelial cells (hitvec). this was not associated with a~y i~crease in the number of lymphocyte adhesion molecules, bu~ could be inhibited by neutralizing monoclonal antibodies re lfa-i and vla- . these chemokines also rapidly increased ~ha adhesion of resting t l~phoeytes to plates coated wi~h integrlns (i-cam or v-cam) or matrix proteins such as f~bronectln. this induction of adhesion began wiehln ', peaked by hr and was completed in hr. this suggests that chemoklnee rapldly increase the binding affinity of adhesion prote~ns on t cells. we also recently observed ~hat mcaf/mcpi and rantes chemoattract unatimulated mast calls. furthermore, igs actlvared mast calls were chemoattraeted by mcaf, ra~tes, pf and mip.i~. however. ~he chemoklnee did not induce mast cells ~o release histamines. presumably, chemokines as regulators of the adhesion, migration and homing of all kinds of leukocytes participate in many types of inflammatory diseases. natural killer cell stimulatory factor or interleukin- (il- ) is an heterodimerie cytokine formed by two covalently linked glycosylated chains of kd and kd. il- was originally purified from the supermatant fluids of ebv-transformed human b ly~phobiastoid cell lines. however, in addition to b cells, phagocytic cells, i.e. monocytes, macrophages, and neutrophils, have been identified as the major physiological producers of il- . phagocytic cells produce il- in response to bacteria, bacterial products such as lps, and intracellular parasites. the cell types on which il- exert biological activity are t and nk cells. the major direct biological function of il- on these cell types are the induction of cytokine production, primarily ifn-y, the enhancement of a generation of cytotoxic cells, and a mitogenic effect on antlgen-activated lymphocytes. in part thromgh its ability to induce ifn-y production, il- , produced in response to infections, represents an important functional link between effector cells of innate resistance, phagocytic cells and nk cells, and effector cells of adaptiye resistance, t and b lymphocytes. the ability of il- to induce ifn-y production from circulating. nk cells and at least a proportion of t cells determines a rapid, antigen-independet production of ifn-y during infections. ifn-y acts as a potent enhancer of phagocytic and bacteriocidal activity of phagocytic cells, participating early in infection to activate some of the inflammatory mechanisms that represent the first innate resistance against infection. this antigem-independet activation of phagocytic cells that involves, in addition to il- , other monocyte derived cytokines such as tnf and il-l, has been shown to be important in experimental animals for the resistance against infection by microorganisms such as limteria monocytogens and toxoplasma gondii. in addition to this role, early in infection in the antigen-independet phagocytic cell activation, il- has a major effect in the ensuing antigenspecific admptive immune response by facilitating the generation of t helper type (th-i) response and suppressing a th- response. the presence of il- during antigen stimulation in vitro using human peripheral blood lymphocytes, or both in vivo and in vitro in experimental animals, induces generation of th-i cells producing ifn-y and il- , and inhibits the generation of th- cells producing il- . il- induces a direct differentiative effect on th-cells, priming them for high ifn-y production. ~is priming effect is stable and maintained even when t cell clones are cultured for extended periods in the absence of il- , however, il- needs to be present during the first few days after stimulation with antigen or mitogen and established th clones are resistant to the priming effect of ifn-y. unlike the generation of high ifn-y producing clones, the il- -mediated inhibition of il- producing cells in probably due to selective mechanisms, which may include a selective mitegenic effect of il- for thl cells and an inhibitory effect of ifn-y on th cell proliferation. in several experimental systems, it has been shown that the enhancing effect of il- on th-i responses is dependent on production of if~-y and on the presence of nk cells, possibly needed for the early production of ifn-y. the importance of phagocytic cells and nk cells in determining the characteristics of the th response during antigenic stimulation indicated that the mechanisms of innate resistance have a determining influence on the subsequent antigen-specific immune response: il- appears to have a key role in mediating the interaction between phagocytic cells. nk cells, and t lympocytes in these proccessem. trinchieri, g. , interleukin- and its role in the generation of t e cells, imm~ol. on peripheral blood monocyte/macrophages, il- behaves like il- in a variety of assays, and shows both similar and contrasting, activties with either il- or ifn-y. il- reduces inflammatory monokine and il- production. it reduces the pyrogen-induced expression of tissue factor (herbert et el, , febs lett. , ) . it mpregulates manmose receptor and ~c class ii expression, while reducing cdi expression. il- produces morphological changes (extensive cell processes, aggregation, giant cell formation) which have previously been observed with il- . it inhibits niv-i production (montaner et el, , i. exp. mad. , ) , and interferes with hiv-induced cell fusion. the activities of il- and il- differ on t-ly•ocyte and large granular lymphocyte population. in particular il- does not exhibit the suppressive effects of il- on thl-type-helper functions (ifny production) and cytotoxic functions (perforin production). data will be presented showing that il- and il-l share a common receptor on a nu~er of cell types, but not on t-lympocytes (see also zmrawski st el. , embo j. . - ), explaining both thai common and divergent activities. the levels of il- ~a in activated peripheral blood lymphocytes are greater than or equivalent to those of il- mp~ja and the two ml{nas are expressed by different t lympocyte population: in particular, t cytotoxic lymphocytes express markedly higher levels of il- ~rna. in vivo, il- may thus be contributing, significantly to some of the activites previously ascribed to il- il- . recently, we cloned the human cdna encoding interleukin- (il- ). human il- is a non-glycosylated protein of aa with a molecular mass (mr) of kd and has biological activities on monocytes and b cells, il- , like il- , strongly enhanced the expression of class ii mhc antigens on monocytes and induced expression of cd (fc~rii). furthermore, il- , like il- and il~ , inhibited the production of pro-inflammatory cytokines il- , ]l- , tnfo~ and chemokines miplc~ and tl- by lps activated monocytes. both il- and il- enhanced the production of il-lra by monocytes. in contrast, il- lacked the t cell stimulating activities of il- . the responses of monocytes to il- and il- were not additive or synergistic, supporting the hypothesis that il- and il- receptors are complex and share a common component which is important for signal transduction, taken together, these data indicate that il- , il~ and il- have important anti-inflammatory activities on human monocytes. in addition, these results indicate that il- is unique in that it can modify inflammatory reactions without stimulating (as does il- ) or inhibiting (as does il- ) t cell responses. transforming growth factor beta i (tgf-fll) belongs to a family of proteins that have potent immunoregulatory properties ranging ftom effects on the growth and dfflerentiafion of primitive stem cells to the differentiated functions of immune system effector cells. tgf-i~i is synthesized as a large secretory precursor polypeptide of amino acids that is inactive until processed to a disulfide linked homodimeric molecule of t amino acids each. recently it has been found that tgf-[~i can have autocrine as well as paracrine effects on the immune system indicating that immune cells can activate the inactive secreted form of tgi~-~i. in addition, tgf-i~i has differential intraceuular effects on cell surface receptor modulation, tyrosine phosphorylation and cytokine gene transcription as well as cell mediated cytotoxicity. the systemic administration of tgf- has proven beneficial in a variety of animal models such as septic shock, allograft rejection and experimental forms of autoimmunity including collagen induced arthritis and experimental autoimmune encephalomyelitis. moreover the increased levels of tgf-ill and other tgf- isoforms found in several disease states associated with immunosuppression such as different forms of malignancy, chronic degenerative diseases and aids implicate the involvement of tgt~-i~ in the pathogenesis of some diseases. hopefully, well designed clinical trials will define the potential roles of the tgf-[ family of proteins in the treatment of diseases of man. patient development of systemic inflammatory response syndrome (sis) after severe trauma is accompanied by a wide range of immune aberrations and altered cytokine production. in particular, t lymphocyte proliferation and ifn, t production is suppressed while monocyte (mo) tnfc~, il- , pge and tgf~ production is massively increased concomitant to decreased antigen presenting capacity. recently, two new cytokines, il- and il- , have been characterized as critical in regulation of mo tnfa and il- , as well as in induction of t lymphocyte proliferation and ifn production. in this study, peripheral blood leukocytes (pbl) from severe trauma patients (injury severity score > ) were analyzed for their il- levels, their in vitro proliferation to mitogen (pha) and their response after il- addition. since il- produced either by mo or by t lymphocytes can depress m~ antigen presenting capacity, inhibit t cell ifn,/production and directly diminish t cell proliferation, it might be suggested that immunosuppressed patients' mo and/or t lymphocytes would have increased il- levels. increased patient il- production might also be resulting from the high levels of tnfa a known stimulator of il- . conversely, since il- augments mo antigenpresenting capacity, thl induction and proliferation, post-trauma leukocytes might be il- deficient. pbl of trauma patients were compared to normals' pbl, either unstimulated or ptta induced, and their levels of il- found to be dramatically and significantly reduced. patients' isolated m~, either stimulated with the bacterial cell wall analogue, mdp, or unstimulated, also had depressed il- production concomitant to elevated tnfa production when compared to normals' mo. mechanisms for the depressed patients' mo il- were explored. increases in tgf[ may have partially contributed to the patients' depressed il- level, but elevated pge had no effect. addition of il- to patients' pbl significantly increased their mitogen responses. these data imply that sis is characterized by disruption in the interactions between mci and t lymphocytes so that patients' m~i produce excesses of some mediators (tnfa, il- , pge ) and a dearth of other monokines (il- , il-io). t lymphocytes are not activated and, therefore, unable to function in both immune defense and monocyte regulation. it is known that lge receptor-mediated or ca-ionophore-induced activation of mouse bone marrow-derived mast cells ( mmc) may result in the production of different cytokines including the interleukins (il) , , , and as well as gm-csf and tnf-a. in the present study we analyzed the effects of exogeneously applied pro-inflammatory cytokines (il- , l- , tnf-c as well as various mast cell growth factors (il- , il- , il- , il- , ngf, kl (kit ligand)) on cytokine production in primary mouse bmmc using a standard activation protocol (lxl bmmc/ml; ll.um ionomycin; - h). the actixdties of bmmc supernatants were assessed in specific biological (il- , il- il- , l- ) and/or elisa assays (il- , il- ). here we show that homogeneous populations of bmmc (> %alcian blue+/safranln-; in vitro age: weeks) generated in the presence of recombinant (r) rail- from normal balb/c mice produced modest amounts of l- and low or undetectable levels of il- , - , and - after induction with lp.m ionomycin only. however, a dramatic increase ( -to -fold) of these cytokine activities was noted, when in addition to ionomycin also human ( ) rll-la was provided during the induction period. this il- effect was dose dependent with a maximgm at - u/ml hrll-la and specific, as pre-incubation (lh) of bmmc with ng/ml hrll- receptor antagonist abolished the action of u/ml hrll-lcc similar effects were noted with hrll-lg or rurll-lb (lng/ml, respectively), but not with rhll- or rmtnf-~. both mrll- and hrll- substantially enhanced ionomycin-induced l- production of bmmc in the absence or presence of il- . il- significantly enhanced il- and il- production while decreasing il- activities to abont - % of control levels, when il-i was provided in the presence of il-l/ionomycin. a monoclonal anti-nfil-t antibody (ascites : ) abrogated the effects of mrll- . other mast cell-active cy~okines (] ,- , il- , l- , ngf, or kl) added to ionomycia-or l- /ionomycin-treated bmmc had no major effects on cytokine production. il- and il-i did not induce significant cytokine release in the absence of ionomycin suggesting tlmt cadependent signalling was required. at doses of " m, dexamethasone, corticosterone, or hydrocortisone almost completely abolished ionomycin/il- /ll- induced cytokine production. the inducer cocktails per se did not interfere with the cytokine bio-assays. in case of il- inducibility of this cytokine in bmmc was confirmed at the mrna level by northern blot analysis. hence our data show that activated mast cells are a source of il- previously recognized as a product of th type lymphocytes only. moreover, our study reveals novel functional roles for i-l-i, il- , and ghicecorticoids in the regulation of cytoldne production in mast ceils. accumulating data suggests that cytokines, peptides involved in regulation of both physiological and pathological immunological responses, predominantly are produced at the local site of antigen stimulation. a new method was used to detect cytokine-producing cells in haman tissue at the protein level. single-cell production of different httman cytokines, ilia, ill [ , illra, il , il , il , il , il , ils, ill , gm-csf, tnfa, ifn and tgf[ . , was identified by indirect immunohistochemical staining procedures and use of carefully selected cytokine-specific mab's. frozen sections were fixed with % paraformaldehyde and permeabilized by . % saponin treatment, eluting cholesterol from the membranes. the intracellular presence of all cytokines except ill, illra (late) and tfg[ _ , could be demonstrated by a characteristic perinuclear configuration in producer cells. in addition, the immunoreactivity extended over a large extracellular area encompassing the producer cell. a localization of the cytokine to the golgi-organelle was established by use of two culour staining including a haman golgi complex specific mab. this staining pattern was only evident in producer cells because injection of recombinant human cytgkines into the tissue caused a membraneous and extracellular staining pattern. both the extra-and the intracellular types of staining reaction could, however, be blocked by preincubating the cytokine specific mab with pure human interleukins. oxygen radicals (or) directly induce lipid peroxidation, indirectly they trigger adhesion and activation of pmn leukocytes. we investigated whether or also lead to a release of acute-phase response cytokins such as tnf-alpha, il-i beta or il- in whole blood cultures to maintain the induced inflammatory reaction. methods: blood samples from healthy volunteers (n= ) were incubated at °c. or were produced by the xanthine oxidase (xo)/ hypoxanthine (hx) system. after , , , , and minutes plasma levels of tnf-alpha, il-i beta and il- were determined with elisa kits. results: under the influence of or tnf-alpha plasma levels increased from , pg/ml at min to pg/ml, pg/ml, pg/ml after , and min. il-ibeta ( , pg/ml, , pg/ml, , pg/ml, pg/ml and pg/ml after , , , and min) and il- ( , pg/ml, l,lpg/ml, , pg/ml, pg/ml and , pg/ml after , , , and min) plasma levels were increased min later than tnf-alpha. summary: these data suggest that or do not only play an important role in initial accumulation and activation of pmn leukocytes but also lead to a stimulation of monocytes to produce the acute phase reaction cytokins tnf-alpha, il-i beta and il- to maintain and strengthen the inflammatory reaction. department of general surgery, steinhsvelstr. , ulm, germany jan k. horn md, greg a. hamon md, robert h. mulloy md, greg chen bs, rebecca chow bs, and christof birkenmaier md. transforming growth factor-i~l (tgf- ) is released from inflammatory ceils following injury and in sepsis. in vitro experiments have confirmed that low concentrations of tgf- ( . - . ng/ml) are chemoattractive for monocytes, whereas higher levels of tgf- (> . ng/ml) potentiate production of the immunedepressive prostaglandin e . other investigators have shown that tgf-] can cause the appearance of cd (fc immunoglobulin receptor) on monocytes exposed to ng/ml of tgf-[~i for hours. monocytes also express on their surface a glycoprotein that binds complexes of lipopolysaceharide (lps) and lpsbinding protein (lbp). such binding is associated with generation of proinflammatory cytokines such as tumor necrosis factor alpha. we have shown that cd is depressed in septic patients and therefore we hypothesized that tgf- could account for the down-regulation of cd observed in these individuals. we incubated normal human monocytes with platelet-derived tgf-[ for and hours at °c and examined ceils for cd and cd expression using flow cytometry after immunnfluoreseent staining with appropriate monoclonal antibodies. monocytes were selected on the by usual criteria for size and granularity. non-viable ceils were excluded with the use of propidium iodide. two populations of monocytes could be found afcer incubation at °c alone. one displaying high density of cd had increased fluorescence over the homogeneous expression of cd in cells maintained at °c (baseline). the other population displayed decreased cd expression relative to the baseline cells. tgf-i~i ( - ng/ml) caused a shift of ceils from the high density into the low density cd population. this trend was observed within hours of incubation and was complete by hours. we observed a net decrease in cd expression f % for all subjects studied (p< . vs controls). phorbol myristate acetate ( ng/ml) also caused down-regulation of cd to a similar degree as tfg-i~i. we also confirmed that monocytes could be induced to express cd after incubation with tgf- ( ng/ml) for hours. these studies demonstrate that monocytes incubated with immunodepressive levels of regulation of cd by tgf- deplete their surface expression of cd while generating cd . this down-regulation of cd by tgf- correlates with our clinical observations of lower cd expression on monocytes obtained from septic patients. for over years, activated t lymphocytes have been considered to be the cellular source of mif. we recently isolated and cloned the murine homolog of mif after identifying the specific secretion of this protein by lpsstimulated pituitary cells in vitro and in vivo. however, further experiments showed that mif protein is detectable both in t-cell deficient (nude) and hypophyseetomized mice, suggesting that yet additional cell types may produce mif in vivo. since monocytes/macrophages are a major source of the cytokines that appear in response to lps administration, we examined the possibility that mif also is expressed in cells of the monocyte/macrophage lineage. we found that mif is expressed constitutively in the murine macrophage-line raw . and in thioglycollate-elicited peritoneal macrophages. significant amounts of mif mrna (rt-pcr) and protein (western blotting) were observed in cell lysates. in raw . cells, mif secretion was induced by as little as pg/ml of lps (e.coli l:b ), peaked at ng/ml, but was not detectable at lps concentrations > txg/ml. similar data were obtained with elicited macrophages, but higher lps concentrations were required, unless the cells had been preincubated with ifn . production of mif by lps-stimulated (l ng/ml) macrophages peaked at hr. expression ofmif mrna and tnf mrna by lps-stimulated raw . macrophages was investigated by rt-pcr. as expected tnf mrna expression increased over the range of lps concentrations ( pg/ml to p_g/ml). in contrast, levels of mif mrna correlated inversely with lps concentration. by competitive pcr, mif mrna was observed to increase approximately -fold after lps induction ( pg/ml). mif secretion also was induced by tnfoc ( ng/ml) and ifn? ( iu/ml), but not by il- and il- (up to ng/ml). lps and ifn had additive effects in inducing mif secretion. in separate experiments, macrophages stimulated with recombinant mouse mif ( gg/ml) were found to secrete bioactive tnf~ (> pg/ml by l cytotoxicity). we conclude that the macrophage is an important albeit overlooked cellular source of mif in vivo. mif secretion is induced by lps, tnfc~ and ifn?. mif also stimulates macrophages to secrete tnf. taken together with previous observations that anti-mif antibody protects against lethal endotoxemia, these data implicate mif as a critical mediator of inflammation and septic shock. inflammation is characterized by an exacerbation of proinflammatory cytokine production. cytokines such as il- , il- , and tgf , have been identified as anti-inflammatory mediators thanks to their ability to down regulate the production of il- , il- , il- , tnfc~ by activated monocytes / macrophages. however, other cells, including polymorphonuclear cells (pmn) do contribute to the release of pro-inflammatory cytokines. we investigated the capacity of the so-called anti-inflammatory cytokines to control the release of il- by activated neutrophils. human pmn were purified following glucose-dextran sedimentation and ficoli-hypaque centrifugation. the cells were cultured at °c for h in the absence or presence of lipopolysaccharide (lps) or tnfa. il- release was measured in the supernatants using a specific elisa. among tested cytokines, il- was the most efficient inhibitor of il- production by lps-activated pmn. il- was also active, whereas no down regulation was noticed with tgfp~i. when tnfa was used as a triggering agent, none of the cytokine could prevent il- production. northern analysis are under investigation to precise the level of the il- -and il- -induced inhibition of il- production by pmn. our data illustrate that il- and il- possess the capacity to down regulate the production of il- by both monocytes and pmn, whereas tgfb has a more limited inhibitory activity. ciliary neurotrophic factor (cntf), a member of the il- superfamily, has recently been shown to promote axonal growth and neuronal healing. cntf production is also increased during neuronal and muscle damage, associated with soft tissue injury or trauma. we postulated that production of cntf may explain the loss of skeletal muscm protein that occurs in inflammation. female, wistar ( - gm) rats received either or pg/kg bw s.c. injections of recombinant rat cntf for seven days, or received sham injections and were freely-fed. additional animals were pretreated with mg/kg ibuprofen lp prior to pg/kg bw cntf. rats treated with ,ug/kg bw cntf lost . _+ . gms bw as compared to freely-fed controls which gained . _+ . gms (p<o. by anova and lsd), and ibuprofen treatment had no effect on cntf-induced weight loss (- . _+ . ). animals pair fed to the high dose cntf gained body weight ( . -+ . ), although weight gain was less than seen in freely-fed controls. rats treated with pg/kg bw had reduced body weight gain (+ . +_ . gin). food intake was also reduced by % in pg/kg cntf (from . +_ . gm/day to . _+ . gm/day; p<o. ) and was also unaffected by prior ibuprofen treatment ( . +- . gm/day). despite the loss in body weight noted in the high dose cntf animals, there was no appreciable hepatic acute phase response, since liver weight ( . + , gms vs. . _+ . gins), total protein ( . +- . gms/l vs . +- . gins/l) and albumin ( . + . gms/l vs. . +_ . gms/l) were not different between cntf and freely fed animals. we conclude that cntf causes anorexia, and increases body weight loss in excess of the associated anorexia. unlike the cachexia associated with il- and tnf infusions, which is associated with an acute phase response, cntf acts predominantly on food intake and carcass weight through a prostaglandin-independent mechanism and has only minimal acute phase inducing properties, n. joseph espat, md. university of florida, department of surgery, j-box , gainesville, fl. . of cytokines by neurotrophic factors, the neuroendocrine system and phenotiazines. cytokines, including tnf and il- , have a pathogenetic role in various diseases related to infection and inflammation. the action and/or production of cytokines can be regulated by drugs or endogenous mechanisms that involve the cetral nervous system (cns) we found that the antipsychotic chlorpromazine (cpz) potently inhibits tnf production and protects mice against endotoxic shock. cpz also inhibits brain tnf production in mice intracerebrally injected with endotoxin, whereas cpz analogs that do not cross the blood-brain barrier inhibit only perypheral, not brain, tnf. tnf production and susceptibility to the toxicity of endotoxin, tnf and il- are increased in adrenalectomized mice, indicating that endogenous corticosterone (cs) controls cytokine production and toxicity, in a feedback fashion since endotoxin and cytokines increase serum cs. increased tnf production was also observed in mice where the hypothalamus-pituitary-adrenal axis was blocked by chronic administration of dexamethasone, following a two-day washout. administration of cytokines acting through the gp signal transd,uction protein, including il- and ciliary neurotrophic factor, cntf potentiated il-l-induced elevation of cs. il- and cntf also induced hepatic acute-phase proteins. thus il- and cntf might have antiinflammatory activity, by potentiating the feedback responses of the neuroendocrine axis. these data indicate how complex can be the interregulatory relationships between the brain and the immune system, how they can be used to pharmacomodulate cytokine action and how their disregulation might exacerbate il- -and tnf-mediated pathologies. lipopolysaccharide (lps) constitutes a well established activator of monocytes/ macrophages (mizi) and murine b lymphocytes. the hydrophobic part of lps, termed lipid a, represents the endotoxic principle of lps. we now demonstrate that lps is also capable of inducing proliferation of human t cells at a dose of to pg/ml. the specificity of this stimulation was analysed by the following experiments: i) the synthetic hexaacyl escherichia coiltype lipid a (compound ) also stimulated human t cells; ii) the synthetic tetraacyl lipid a precursor (compound ) or the phosphono-oxyethyl analogue pe- , structures known to antagonize lps-induced monokine production in human mz, also inhibited lpsinduced t-cell proliferation. the t-cell proliferation expressed the following features: i) cd ÷-as welt as cd ÷ t cells proliferate in response to lps, ii) limiting dilution analyses reveal that the frequency of responding cells was between / to / cells; iii) purified t cells alone cannot be stimulated by lps, but need the help of monocytes. this help cannot be replaced by b cells or fixed monocytes. furthermore, for activation a direct cell-to-cell contact between t cells and monocyte is neccessary; iv) t cells stimulated with lps express mrna for il- and ifnf, but not for il- or il- (determined by pcr). in supernatants, ifnf was detectable (elisa); v) lps-activated cd ÷ as well as cd + t cells express cd , the high affinity il- receptor. our results indicate that in contrast to previous reports human t cells respond to lps with il- receptor expression, lymphokine production, and proliferation. the reactive cells appear to belong to the t, cell type, the finding that human t cells can be activated by lps is of important relevance for the understanding of the pathomechanisms of infections by gramnegative bacteria. this may lead to new strategies for the therapy of sepsis. we have recently shown that human eosinophils produce mrna for interleukin (il)- when stimulated with calcium ionophore (eur. j. immunol. ( ), ). we now present evidence that human eosinophils contain preformed il- protein although they do not express mrna for il- as measured by rt-pcr. il- protein expression was determined using specific anti-human il- monoclonal antibodies by western blotting, facs analysis and immunohistochemistry. moreover, preformed il- was re/eased into supernatant by % pure eosinophils after priming with gm-csf or il- and subsequent -min stimulation with paf, rantes, ionomycin or pma, however not with il- or il- , as measured by elisa. this observation implies that activation of protein kinase c and/or intracellular calcium mobilization are necessary events for il- release in human eosinophils. the determined amounts of preformed il- protein was higher in patients with asthma compared to normal individuals suggesting a role for eosinonhi! derived il- in asthma. since the eosinopnit is the ,,r~:=z~mmant cell in the asthmatic airways, we determined il- concentrations in bronchoaiveolar lavage (bal. ~ids from normal !i~.~ijvtduals and asthmatic patients. indeed, il- concentrations in bals from both groups were similar to those seen in the supernatants released by activated eosinophils. the role for il- in asthma remains to be determined. based on a well established rat model named activity-induced ulceration (asu) or activity based anorexia (aba), we have developed a rat model for chronic stress. male rats were exposed to a feeding schedule in order to reduce body weight by - % within days. while one group of rats was kept sedentary, the other had access to a running wheel. food restricted rats allowed to exercise developed highly increased activity as compared to ad libitum fed rats in wheels (ca. versus kin/day). they developed elevated plasma corticosterone levels ( _+ gg/dl) at their circadian peak, increased adrenal weight and decreased thymus and spleen weight as compared to control rats and weight matched sedentary rats, establishing this paradigm as a model of chronic stress. no differences in plasma acth were seen among the various groups, suggesting increased sensitivity of the adrenal glands to acth. the effect of chronic stress accompanied by hypercorticism on baseline and endotoxin-induced cytokine levels was studied in this model. under baseline conditions,splenic il- a was suppressed in both food-restricted groups. this was more pronounced in rats with access to a running wheel. il- and tnf activity in plasma was not affected. after administration of .tg/kg lipopolysaccharide (lps), tnf activity in plasma was suppressed by food restriction but there were no differences between sedentary or exercising rats. in addition, no differences were found among groups for corticosterone, spleen il-lc~ and plasma il- activity. we conclude, that the food restriction-induced hyperactive rat model is a useful model for studies on the effects of chronic stress. in this model, under basal conditions il-le~ in tissues may be subject to downregulation by glucocorticoids, whereas after lps-stimulation of cytokine production only tnf activity is regulated by fast acting feedback loop between cytokines and the hypothalamo-pituitary-adrenal axis (hpa-axis). this is supported by strong correlations between plasma tnf activity and corticosterone in stressed rats. cytokine antagonists modulate cytokine actions and it appears that a balance between production of cytokines and cytokine antagonists is important to control of host responses. recent observations show that during myocardial infarction there is an increase in circulating cytokines such as tumor necrosis factor (tnf) and interleukin (il- ). we tested the idea that compensatory increases in cytokine antagonists likewise occur and investigated changes in plasma concentrations of tnf and interleukin i (il- ) and those of their specific antagonists interleukin- receptor antagonist (il- ra) and soluble tumor necrosis factor receptor type i (s tnf r-i). we also measured plasma concentrations of ~-melanocyte-stimulating hormone (~-msh), an endogenous neuropeptide that occurs in pituitary, brain, skin, gut and other tissues. when administered to laboratory animals, this peptide has potent antiinflammatory and antipyretic activity, perhaps by mimicking an endogenous modulatory influence on cytokine actions. samples were obtained from patients with acute myocardial infarction at presentation in the coronary unit, every three hours for hours, and daily thereafter until discharge. we found elevations in the plasma cytokines il- and tnf only in a proportion of subjects whereas cytokine antagonists c~-msh, il-lra, and stnfr were elevated in all patients, o~-msh was elevated at presentation but it decreased progressively in successive tests, reaching normal values within hr whereas stnfr and l-lra peaked at - hr or later. there were significant correlations between plasma concentrations of cytokine antagonists and enzymes released from injured myocardium, including creatine kinase (ck), aspartate serum transferase (ast), and lactate dehydrogenase (ldh). the data show that cytokine antagonists increase in the circulation of patients with acute myocardial infarction likely to modulate prninflammatory effects of cytokines. objects of the study: interleukin- (il- ) is a multifunctionai cytokine known to be involved in important homeostatic processes. il- levels are increased in many pathological situations, and correlates with disease activity and patient outcome. to exert its effect il- binds to its receptor on the membrane of its target cell. this receptor is also present in a soluble form in plasma and in urine. in this study we quantitatively measured the availability of soluble il- receptor (sll- r) in biological fluids in healthy volunteers. materials and methods: blood, urine, cerebrospinal fluid (csf), and synovial fluid (sf) are obtained from healthy volunteers, sil- r and il- are measured using elisa's. both elisa's are tested for interference of sil- r mid il- . the effect of sll- r in the il- bio-assay (b ) is tested. results: baseline levels are (mean + sd): . -+ . ng/ml in serum, . -+ . ng/ml in urine, . _+ . ng/ml in csf and . + . ng/ml in sf. there is no interference of sll- r in the il- elisa or bio-assay and no interference of l- in the sil- r elisa. conclusions: in all investigated biological fluids sll- r can be found. these data provide baseline values for investigations concerning pathological situations. both elisa's described are useful tools to do these investigations. trauma-associated immune aberrations coincide with augmented release of immunoregulatory and proinflammatory cytokines. the present study examines the effect of thermal trauma on the capacity for specific (receptormediated) response of lymphoid cells to interleukin (il ) and to turnout necrosis factor ~x (tnfc . methods. bum patients (n= , -> % total body surface area) were studied weekly up to days post-injury. the limulus amoebocyte lysate (lal) test was used to measure plasma endotoxin levels. the percentage of il ~-and tnfcz-binding t(cd ) lymphocytes was assessed by flow cytometry analysis. levels of il receptor antagonist (il lra) in patients' plasma and cultures of peripheral blood ceils (pbc) were determined by immunoassay. results. plasma endotoxin concentrations were significantly (p< . ) increased up to weeks post-bum (means . + in non-surviving and . + . u/ml in surviving patients vs < u/ml in the control). within weeks of bum, the percentage oft ceils expressing receptors for tnfa and il [~ constitutively was elevated (by - fold). in contrast, the capacity for de novo receptor expression by activated pbc was reduced. serum levels of il ira were significantly increased (range . - x j pg/ml vs < . x j pg/ml in the control). in all patients, high concentrations of il lm were released spontaneously in unstimulated cultures of adherent ceils (range - x - pg/ml vs - x j pg/ml in the control). however, its secretion was decreased in lps-stimulated parallel preparations. conclusions. in the bum patient, susceptibility to the immunoregulatory effect of tnfcz and tl ~ may be modulated by infection-related products. alterations in the capacity for receptor expression and secretion of l lra may affect il -regulated biological responses including specific immune reactions. while studies suggest that il- is an important lymphokine involved in cell-mediated immunity, little is known about this mediator's role in hem-induced immunesuppression. our aims, therefore, were to determine: i) if il- contributes to depressed t-cell responses seen following hem; and ) how other agents, known to play a role in hem, effect il- release. to study this, c h/hen mice were bled to and maintained at a map of mmhg for h and then adequately resuscitated. mice were killed h post-hem to obtain splenic t-cells (nylon-wool purified). il- 's immunosuppressant role was demonstrated by the ability of monoclenal antibody (mab) to il- to markedly improve the t-cell proliferative response [ . #g the marked increase in capacity of t-cells from hem mice to produce il- was significantly reduced by treatment with either ibu or mabs. since ibu, tgf-~, as well as il- are all reported to directly/indirectly influence prostanoid synthesis, this implies that eicosanoids play a major role in inducing il- release by t-cells following hem which depresses t-cell function. the mechanisms underlying immunosuppression induced by thermal injury and alcohol ingestion are in part due to cytokine dysregulatinn. il- down-regulates production of eytokines by maerophages and may be an important regulator of the initiation of the immune response. il- has also been demonstrated to inhibit the production of no by macrophages. this study examined the alterations in eytokine production and effect of inhibition of no production on immunologic function in a routine thermal injury model. methods: balb/c mice (n= ) were randomized to groups: saline-sham(ns-sham), alcohol-sham(etoh-sham), ns-bum, etoh-bum. animals received % etoh or ns daily for days by gavage. a % full thickness bum was induced hrs after the last dose of etoh or ns. animals were resuscitated, then sacrificed days post bum. splenic lymphocytes were cultured for days with lps, and lps with two concentrations of n-monomethyl-l-arginine, a nitric oxide inhibitor (l-nmma . ug/ml, ug/ml). splenocyte production of il- , interferon-gamma, il- , pge were measured, and lymphocyte proliferative response examined. results: il- production was significantly suppressed in thermal injury. exogenous l-nmma normalized the suppression of .- in a dose-dependent manner, indicating nitric oxide may modulate il- and interferon-gamma production in thermal injury. il- production is normal in etoh-burn animals. conclusion: il- and interferon-gamma production is altered in this murine thermal injury model, and may contribute to this injury-induced immunosuppression. inhibition of no synthesis normalizes il- production and should be investigated further as an immanomodalator in thermal injury. surgery, infection and inflammation results in the production of pro-inflammatory cytokines which mediate metabolic and immunologic host responses. the aim of this study was to characterise the elaboration of cytokine release following a variety of surgical procedures. twenty one patients undergoing elective intermediate, hip, knee and major gastrointestinal surgery were studied. levels of interleukin- (i - ), interleukin- (i - ), the interleukin- receptor antagonist (i - ra) and the acute phase c-reactive protein (crp) were measured in bloods drawn , , , , , , and hours following operation. a portion of the results are shown (mean -+ sem). + -+ _+ one and two factor anova; *p< . , #p< . , §p< . , ¶p< . , for differences between groups i - was not detected at any time point. both ii-ira and i - increased after surgery. maximum responses occurred following major git and hip surgery, minimal responses were seen after intermediate and knee surgery. ii-ira levels increased within two hours and remained elevated for hours; the b-ira increase was a thousand fold greater than the rise in i - levels. i - levels increased up to hours after surgery. crp levels reflected maximum ii-ira and i - levels (r =. , p< . and r =. , p< . respectively). high ii- ra and i - levels reflect major surgery, however the ii-ira response is more rapid and of greater magnitude. the strong i - ra correlation with crp may indicate that this regulatory cytokine is itself a mediator of host responses to surgery. dept. of surgery, meath/adelaide hospitals, heytesbury st., dublin , ireland. change of il- and soluble il- receptor levels after surgery s. hisano, k. sakamoto, s. mita, t. ishiko, m. ogawa [objectives] under surgical stress, il- plays a main role in producing acute phase proteins and contributes to host defense mechanism. soluble il- receptor (sll- r) is considered to be agonistic to il- , unlike other soluble type receptors of cytokines. here we measured il- and sll- r levels in the serum and drain fluid from surgical field in order to investigate the changes of il- and sll- r after surgery and their origins. [materials and methods] serum and drain fluid samples from cases ( of esophagectomy and of gastrectomy ) were serially collected before and after surgery. il- and sll- r levels were measured by elisa. [results] ( ) serum il- : all cases reached the maximum level on pod-l, more precisely - hours after operation. ( ) il- in the drain : maximal il- levels in the drain were recognized - hours after operation, at almost the same time as serum il- . furthermore the il- values in the drain were much higher, about times, than those in serum. ( ) sll- r in the serum : all cases reached minimum levels - hours after operation and recovered to the preoperative levels a few days later (decrease ratio : . + . ~,, range : - ~'). ( ) sll- r in the drain : sll- r levels in the drain showed almost the same value and change as serum sll- r. [conclusions] ( ) il- is produced from the cells gathering around operative fields whereas sll- r is considered to be produced in the cells which do not gather around the operative fields. ( ) there may be a mechanism that down-regulates sll- r in the early stage of surgery. [objectives] il- plays an important role in host defense in the early stage after surgery. in the present study, we examined changes in il- concentration after major thoracoabdominal surgery and elucidated the effect of surgical trauma and factors influencing postoperative elevation of serum il- . [materials and methods] thirty-eight patients undergoing elective surgery of the thoracoabdomen were classified into groups according to the location of the operation. bloods and drain fluids were serially obtained and samples were frozen until measured, keukocytes were simultaneously collected for northern blot analysis. concentration of il- was measured by elisa and il- mrna was detected by northern blotting after total rna was extracted by the acid guanidium phenol chloroform method. [results] ( ) serum il- levels reached the maximum concentration on the st postoperative day in all patients. ( ) the il- peak was significantly correlated with surgical trauma as defined by the operation length and the volume of blood loss during operation (r= . , p< . , r= . , p< . , respectively). ( ) the peak concentration of serum il- in patients undergoing esophagectomy was significantly higher than in those undergoing pancreaticoduodenectomy (p< . ), despite a similar degree of surgical trauma. ( ) peak l- concentration observed in a patient who underwent esophagectomy was about fold greater in the drain fluid of thorax than in the peripheral blood. ( ) il- mrna was demonstrated in leukocytes from thoracic and abdominal exudate at , and hours after surgery. in contrast, il- mrna could not be detected in leukocytes from the peripheral blood. [conclusion] il- is mainly produced in the operative field and subsequently enter the peripheral blood to induce cytokinemia. the operation length, volume of blood loss and thoracotomy are factors influencing the concentration of cytokine in the blood. zaragoza spain age may be an important factor influencing the function of immunocompeteut cells releasing cytokines after both accidental and surgical trauma the aim of the present paper is to ascertain if patients (pts) over years old show a different serum level cytokine pattern than pts under after a standard surgical procedure considered as a "medium strength trauma". patients and methods: pts( females males)with gallstone disease were perspectively studied, pts were allotted in two groups: gr.a: pts under years(mean age: . +- )gr.b: pts over years(mean age: . _+ ). all pts underwent cholecystectomy and cholangiography. pts in gr.a and pts in gr. b underwent common duct exploration. spbintercctomy was performed in each group. on the day of surgery (pre) and on the st and th postoperative day(leo, po) : percentages of cd , cd , cd , cd and cd cells we measured by means of flow cytometry using moab. and levels of il- , il- , il- and tnf "in vivo" by elisa using moab. results: ere: cd % was . _+ in gr.a and . objectives of the study. after surgery for esophageal cancer multiple organ damage has been reported to be caused by polymorphonuclear leukocyte (pmn)-mediated injury. we measured serum granulocyte colony-stimulating factor (g-csf) and interleukin (il- ) levels to determine a role of g-csf and il- in pmn function after surgery for esophageal cancer. materials and methods. peripheral pmn counts, peripheral pmn chemiluminescence, serum g-csf levels, and serum il- levels were measured before and after surgery in patients with esophageal cancer (ec), and patients of gastric cancer (gc). esophagectomy with thoracotomy and laparotomy were performed for patients with ec, while subtotal gastrectomy with laparotomy were performed for patients with gc. results. peripheral pmn counts (p< . ) and peripheral pmn chemiluminescence (p< . ) of patients with ec were significantly decreased compared to those of patients with gc at and hours after surgery. serum g-csf levels of patients with ec were significantly (p< . ) increased compared to those of patients with gc at and hours after surgery. serum il- levels of patients with ec were significantly (p< . ) increased compared to those of patients with gc at , and hours after surgery. significant inverse correlations (p< . l) between peripheral pmn count and serum g-csf and il- levels were seen at hours after surgery. conclusion. these results suggest that many circulating pmns, which are excessively activated by g-csf and il- , may adhere to the endotherial cells and then migrate into the tissues, and cause multiple organ damage after surgery for esophageal cancer. immunnogical changes in patients with severe brain trauma receive increasing attention since morbidity and mortality ere still high. interleukin- (il- ) was previously detected in the cerebrospinal fluid (csf) during different pathologies of the nervous system ( , , ). in our study we monitored il- and nerve growth factor (ngf) production in the csf after human brain trauma. since astrocytes within the brain constitute one of the major cell type contributing to the inflammatory response through the release of cytokines and other factors after injury, we investigated the functional relationship of il- and ngf on a single cell niveau using cultured astrocytes. methods csf was obtained from patients with severe brain injury (glasgow coma score (gcs) < and ct abnormatities or gcs < over hours) after implantation of intraventricular icp monitoring device for therapeutic purpose and collected over hours csf and serum. il- and ngf were assayed by elisa. astrocytes were isolated from neonatal mouse brain as described ( ) . ngf production by cultured astrocytes was measured by elisa in the presence of csf, il- and il- antibody. astrocyte migration was tested in a chemstaxis chamber. results head trauma patients were included in this study (approved by the university hospital medical ethics board) and the csf was obtained through intraventricular catheters. high levels of il- were detected in the csf of these patients when compared to serum during the first days after brain trauma. furthermore ngf could be found inside the intracerebral compartment. csf containing high levels of il- could stimulate ngf production in cultured astrocytes. this effect could be [nhibited partially by il- antibodies, purified il- exposed to cultured astrocytes in vitro, stimulated the migratory activity of these cells in a dose response fashion. il- was found in the csf of brain injured patients, suggesting a role for this cytokine in the pathophysiology of brain injury. since astrocytes are involved in maintaining the homeostasis of the brain, we further investigated the possible role o il- on astrocyte functions, il- promoted ngf production in vivo and in vitro, thus contributing to neuronal cell survival and regeneration. furthermore il- stimulated astrocyte migration in a dose response fashion, potentially contributing to astrocytosis following brain injury and inflammation, these results show that il- represents a key cytokine in traumatic human brain injury with possible systemic effects, which are at preserlt under investigation. we studied a) the role of tnf and b) the therapeutic effect of a mab to tnf with regard to haemorrhagic shock (hs) related ,pathophysiologic alterations and mortality in rats. method: a prolonged hs was induced by bleeding to a blood pressure of - mmhg for pin followed by reinfusion of shed blood (sb) and resuscitation with two times of sb volume of ringer's lactate over rain. animals received a bolus dose ( mg/kg) of tnf mab (celltech, berkshire, uk) at min after resuscitation (tn ). the control group (n = ) was treated similar to the tn group but received ringer's lactate (con). results: at min the prolonged hs resulted in a metabolic acidosis indicated by a significant decrease of blood ph ( . + . ), hco -( . ___ . mm), and base excess (- . + . ram) values with pco ( . + . mmhg) and po ( . + . mmhg) in the tn with no difference to the con group. immediately after resuscitation ( min) plasma endotoxin levels were found to be increased in both groups ( . + . in tn vs . _ . pg/ml in con group) . prior to the treatment with tnf mab ( min) there was also no difference between plasma tnf levels of the two groups ( . + . in tn vs + . pg/ml in con group). treatment with the tnf mab at rain post-hs improved the hour survival rate to . % as compared to . % in the control group. macropathologic evaluations revealed frequency of intestinal bleeding in oniy animals in the tn vs in the con group. no bleeding in the kidneys was found in the tn but in rats in the con group. the significant increase in lung wet weight observed in non-survivors in the con (n = ) was prevented in animals which died in the tn (n = ) group (( . +_ . vs . +_ . g/kg). conclusion: our data suggest that tnf formation induced by hs in rats is an important mediator for pathophysiologic alterations leading to multi organ failure and lethality. antibodies to tnf might be a useful agent in the treatment of haemorrhagic shock related disorders. -+ n=ll*$ -+ n= _+ n= * * p< . vs baseline :~p< . no anesthesia vs anesthesia thus ) tnf production increased - fold by - hrs following trauma in unstimulated blood, but was reduced or not changed after lps stimulation, so circulating leukocytes are probably not an important source of tnf post trauma; ) anticd had no obvious effect on tnf production in unstimulated or lps stimulated blood, relative to vehicle, which suggests that the protective mechanism of anticd does not involve tnf suppression; ) fentanyl anesthesia at hrs following trauma unexpectedly decreased lps-evoked tnf production, which suggests that anesthesia alone can influence an inflammatory response. proinflamrnato~ cytokines have been shown to play a signific~t role in the pathogenesis of sepsis, which is a very common occurrence in born injury. tnfa is infrequently detected in the blood of burned patients, the ability to detect the shed receptors of stnfg has not been determined. serial serum mmples from burn patients were collected from the time of admission until death from septic shock. these samples were analyzed using an enzyme-linked immunosorbent assay (elisa) for stnfr, l-ira, tnf-a, and il-ib. the patients ranged in age from to yeas of age. the percentages of bum ranged from % - %. cytokine concenlrntions vmled from patient to padent irrespective of bum size. tnfa levels were consistentiy in the range of pgjml - pg/ml. peaks in the tnfa values were above pg/ml and were also associated with a peak in the stnfr levels. these levels began at < , pghnl within the in,st ins of injury and gradually increased with time. clinically. ti~ appearance of eytoklnes was independent of positive wound, blood, or respiratory cultures however peak values in tnfa and stnfr were ~ialed with a fluid requirnmenl levels of il-i ra were also elevated independent of clinical findings as well as extent of injury. in pl there is a significant corresponding peak in il-trn (> ~ /ml) at the same time as t/~:a and stnfr levels. we aimed to characterise the pattern of secretion of interleukin- beta l-ii ), intefleukin- (il- ) and tumour necrosis factor alpha (tnfa) in multiply injured patients and to relate these results to their clinical condition and outcome. two hourly blood samples were taken from ten patients from the time of injury until hours. cytokine levels were measured using sandwich enzyme-linked immunosorbent assays (elisas). injury severity scores (iss) were calculated and haemorrhage was assessed from the blood transfusion requirement over the hours. patients' ages ranged from to years. iss varied from to and transfusion requirement from to units. five patients died after the study period. ] ,- was raised in / patients (max level , pg/ml) but was unrelated to condition or outcome. / showed a rise in il- b (max level pg/ml) which was negatively correlated to iss (i=- . , p< . ). tnfa was raised in / (max level pg/ml). peak tnfc~ was positively correlated with iss ( = . , p< . ) and haemorrhage (i= . but p< . ). il-ib and tnfa production was mutually exclusive. there was no common cytokine profile for these patients. unlike elective surgery there was no correlation between peak ,- and severity of injury: tissue damage may not be the stimulus for the cytokine response to multiple injury. periods of ischemia or hypoxia produce endothelial damage in peripheral organs. tumor necrosis factor-alpha (tnf) plays a central role for regulation of endothelial physiology during septic events, taking influence on vascular permeability and coagulant activity [ ] . animal experiments demonstrated a synergism between hypoxia and septic shock on letality, leading to the hypothesis that low oxygen tension leads to enhanced sensitivity of target cells for tnf [ ] . radioligand binding studies with ~ odid-tnf on cultured human endothelial cells were performed after incubation in several environmental oxygen tensions (pc ) for hours. data were achieved by nonlinear regression of an idealized saturation curve according to the equation: b = n " k./( + k,); b = totally bound tnf; k,: association constant (concentration for half-maximal binding); n: number of binding sites per cell. p_o o (mm h¢i): _k, (nm}: n (molecules/cell): - . ± . _+ - . ± . + - , ± . -+ - . + . -+ presented are calculated values on the idealized curve + % percentiles. hypoxia induces enhanced binding of tnf to specific receptors on the endothelial cell surface in a time-and dose-dependent manner by a mechanism, which is not dependent on oxygen radicals, as shown by additional protocols with radical-scavenging drugs. with respect to former findings about a correlation between growth and tnf receptor affinity [ ] , these data lead to the hypothesis that enhanced tnf binding during hypoxia is due to a biochemical conversion of the receptor protein from the low affinity to the high affinity state, possibly by posttranslational phosphorylation of the binding protein by intracel)ular kinases. the proposed involvement of tnf-dependent pathways in pathogenesis of organ dysfunction and multiple organ failure after hypoxia/ischemia may provide a basis for understanding the initiation of hypoxic vascular injury, as manifested by increased permeability and prothrombotic tendency, and, thus, merits further attention. the levels of activity of circulating cytokines (ill, il- and tnf-alpha) which are believed to play important regulatory role in response to trauma are determined (by hioassays and respective anti-cytokine antibodies) in mice and rats subjected to scald injury ion c, see, ° v bsa, ld ) and ( c, see, ~ b ~^)~ , respectively. biphasic increase of cytokine activity was noted in mice: initial increase of il-i and il- , - hr following injury and of try activity hr after scald, followed by elevated levels of il-i and il- at hr, with tendency of decrease of activity at later time points. increased activity of tnf was noted hr following injury, in rats, initial, short-lived increase of il-i and tnf activity was detected lhr following injury, folowed by increase on days i and postburn. il- increase peaked - hr after scalding and levels remained elevated - days following injury. similar kinetics of appearance of proinflammatory cytokines (il-i and tnf-alpha) both in lethal and ncnlethal injury concomitant with differential profile of circulating il- activity (early,short-lived increase and later slow decrease of activity in lethal burn injury) with late persistent high levels of activity in nonlethai injury demonstrated in the present study highlight the need for investigation the relationship of these cytokines in burn-injury induced inflammation. zikica jovicic,lnstitute for medical research, mma,crnotravska , belgrade~yu. asadullah k ( ), woiciechowsky c ( ), liebenthai c ( ), doecke wd ( ), volk hd ( ), vogel s ( ), v. baehr r ( ); depts. of med. immunology ( ) and neurosurgery ( ) , medical school (char#d), humboldt university berlin, frg in patients after polytrauma or major abdominal surgery a hyperinflammatory phase seems to be followed by the development of a phase of monocyte inactivation. the latter is charaeterised by a decrease of monocytic hla-dr expression and a shift to anti-inflammatory cytokine production. as shown, by us and others, this phenomenon indicates severe immunodepression with a high risk of infection. however, the mechanisms leading to monocyte inactivation in the above mentioned syndromes may be multiple. to elucidate the influence of a selective, sterile trauma to the central nervous system (cns) on immune reactivity the neurosurgieal patient is an interesting model. initially, patients who developed a systemic inflammatory response syndrome following neurosurgery were analysed. in all of them a marked decrease of monocytic hla-dr expression was observed soon after the operation. these results suggest that neurosurgery alone can induce immunodepression and lead us to conduct a prospective study, in which we closely monitored l patients undergoing neurosurgery from the first preoperative day until at least day after the operation. hla-dr expression was decreased hi all patients to various extent only hours after surgery. in one patient only we found a persistently reduced hla-dr expression and this was the only patient to develop sepsis syndrome. this suggests that a prolonged, postoperatively decreased hla-dr expression is predictive of infection following cns trauma. in order to assess, whether a decrease of hla-dr expression was associated with a preceding inflammatory response, local cytokine release in the cns was compared with systemic cytokine release. for this purpose, paired samples of earebrospinal fluid (csf) from a vantricle drainage and peripheral blood plasma were obtained. in the csf extremely elevated futerleakin (il)- levels, peaking already a few hours after the operation were found. in plasma, by eontrast, il- ( and tnf-alpha) was detectable not until days later and only if infection was present. the antiinflammatory ili-ra, on the other hand, was also present in csf but peaked after il- and was detectable in peripheral plasma too. we believe there is an association between the inflammatory response in the cns and the following depression of hla-dr expression on peripheral blood monocytes. our results suggest that even a sterile cns-trauma by itself may contribute to general immunodepressinn leading to septic complications. the aim of this study was to evaluate the effect of haemorrhagic shock (hs) a) on total capacity of the host, and b) the circulating blood cells to produce tnf immediately after bleeding. in vivo studies: baboons were subjected to a limited oxygen deficit ( - ml/kg) hypotension phase (mean arterial pressure = map of - mmhg for - hours followed by adequate resuscitation). rats subjected to hs (map of - mmhg for rain followed by reinfusion of shed blood and fluid resuscitation) were challenged with endotoxin ( ~g/kg i.v.) at the end of shock (rhs group). the control group (rco) received the same dose of endotoxin as rhs group but without prior bleeding. in vitro studies: whole blood (wb) obtained from both baboons and rats before and at the end of hs were incubated with endotoxin ( ng/ml) for hrs at °c. results: at min post-lps challenge we found significantly higher plasma tnf levels in rats that were subjected to hs prior to the endotoxin challenge as compared to the control group ( _+ vs + pg/ml) . after hs the tpc was significantly decreased in in vitro stimulated cbc of both rats ( + post-hs vs + ng tnf/ml pre-hs) and baboons ( ± post-hs vs ± pg tnf/ml pre-hs). in contrast, the il- productive capacity was increased in baboons cbc (not yet analysed in rats) stimulated at the end of hs ( ± pre-vs ±_ pg il- /ml post-hs). conclusion: from our data we suggest that despite of down regulation of the cbc to produce tnf the overall tpc is enhanced at the early stage of i-is. with regard to the related literature (chaudry's group) it can be assumed that among the macrophage/monocyte populations, as the main source only the kupffer cells (kc) exhibit enhanced tnf production capacity following haemorrhage. the mechanisms of down/up regulation of cytokine response of cbc and/or kc following hs remain to be examined. d. eg~er, s. geuenich °, c. dertzlin~er °, e. schmitt*, r. mailhammer, h ehrenreich #, p. drrmer, and l. h mer gsf-instimt fox experimentelle h~znatologie, °medizinische kliulk iii, klinikum groghadern, munich, *institut for immunologic, johannes gutenberg universit/it, malnz, and #psychiatrische k/in& der georg-aagust-universi~t, grttingen, germany. it has been shown previously (ehranreich et al., , new biol. : ) that mouse bone marrow-derived mast cells (bmmc) synthesize and secrete endothelin- (et-i) and express eta-type endothelin receptors (eta). so far, however, no functions of et- /et a in bmmc have been described. in the present study we investigated the effect of exogeneously administered et- on the release of histamine, serotonin, and leukotriene c (ltc ) by primary mouse bmmc (in vitro age: weeks) caltured with different recombinant mttrine cytokines (interleukin (il- ) and/or kit ligand (kl) in the presence or absence of il ) for two weeks prior to activation. et- ( x - to lxl - m) induced an extremely rapid (_<lmin) and dose-dependent release of histamine and serotonin (up to % and % of total mediator content, respectively, comparable to ige/ag-indueed mediator release) from bmmc cultured with il- and il- . only when cultured in the additional presence of il- rather than in medium containing kl or kl plus il- alone, bmmc released histamine and serotoaln upon challenge with et- .furthermore, et- stimulated ltc biosynthesis up to -fold in bmmc cultured in the presence of il- . in contrast, the peptide was without major effect on ltc biosynthesis in bmmc culturd without il- . the release of histamine and sereotonin was not altered if bmmc were preincubated for i h with il- prior to activation with et-i, suggesting that a differentiation process rather than a functional priming effect had been initiated by ]l- . et- ( " m) -induced histamine and serotouln release from bmmc was inhibited by an eta-specific antagonist (cyclic id-asp-pro-d-val-leu-d-tel ) in a dose-dependent manner, with a complete inhibition observed at an antagonist concentration of - m. crude peritoneal cells (containing - % serosal mast cells) obtained from normal balb/c-mice released % histamine upon challenge with et- ( - m; rain). taken together, these resu/ts demonstrate that et- can directly function as a histamine and serotohin secretagogue and as a stimulator of ltc production in mast cells. il- appears to be involved in the differentiation of immature mast cell prec~trsors towards an et-l-reactive functional phenotype. when inflammatory reactions are advanced, type ii phospholipase a (type ii pla ) is produced in high levels by various cells at the site of inflammation. cytokines such as tnf-a and il-i activate type ii pla and promote the production of eicosanoid, which is one of the mechanisms by which they enhance the inflammatory reaction. in the present study, the blood levels of type ii pea , endotoxin, tnf-c~, il- and il- were determined in patients with sepsis to examine the relationship between these levels and the patients' pathological conditions. the levels of type ii pla and tnf-a in these patients were . ± . ng/ml and . ± . pg/ml, respectively, the two parameters being significantly correlated (r = . , p = . ). there were also a significant correlation between the level of type ii pla an il- (r = . , p = . ). there was no significant correlation between the level of type ii pla and il- . these in vivo findings suggest that tnf-a may be involved in the production of type ii pla . a. filzmaver, g. reisbach, e. schmitt °, r. mailhammer, and l. hiittner. gsf-iustitut ff~r experimentelle h~imatologie, munich, and °iustitut fttr immunelone , johannes gutenberg universit~t, mainz, germany the product of c-kit, a transmembrane tyrosin ldnase receptor, and a corresponding kit ligand (kl) are critically involved in the control of different hemopoietic cell lineages including the proliferation, maturation, migration, and function of mast cells. it has been reported previously that interleukin (il)- down-regulates kit receptors in human primary myeloid leukemic cells and in a human mast celt line (sinaber et al. , j. immunol. : ) . transforming growth factor (tgf) gl, was also found to down-modulate c-kit mrna and kit receptor proteins in human aml blasts, a mechanism probabely contributing to the anti-proliferative effect of tgfih on kl-stimulated aml ceils in vitro (de vos et at. , cancer res. : ) . in porcine endothelial cells transfected with a retroviral construct carrying the human (hu) e-kit gena, exogenous hu kl transiently down-regulated kit receptors (blume-jeusen et al. , embo j : ) . in the light of these previous findings we analyzed the effects of these exogenously applied cytokines (il- , tgfgl, kl) on kit receptor expression and kl-mediated proliferation and chemotactic migration of primary mouse bone marrow-derived mast cells (bmmc) (in vitro age: weeks). our results show that in bmmc cultures initiated with recombinant (r) marine (mu) il- the additional presence of rmull- for or days did not change the expression of kit protein (assessed by facs analysis with the anti-mouse c-kit antibody ack- ) nor kl-mediated proliferation or chemotaxis. in contrast, il- syeergistically increased kl-stimulated growth of bmmc in a short-term proliferation assay (mtt-tes . pre-incubation of bmmc with rhutgfl~ ( . , . , or . ng/ml) for h had no effect on kit receptor expression, although tgffil at concentrations of . - . ng/ml completely suppressed the proliferative action of kl on these ceils. similar anti-proliferative effects of tgfg were observed in various factor-dependent mast cell lines stimulated with different mast cell growth factors (il- , i,- , il- , and/or il- ). moreover, pre-incuhation ( h) of bmmc with tgf-gl (> pg/ml) significantly enhanced spontaneous undirected cell movement (chemokinesis) and synergistically increased il- -or kl-induced chemetaxis. when bmmc were preancuhated with rmukl ( ng/ml) for , . or days, a transient down-modulation of kit receptors with a maximum effect on day was demonstrated by facs analysis and correlated well with a decreased chemotactic response of these cells. in conclusion our results show that neither il- nor tgfi affect expression of kit receptors in primary murine bmmc. it is reasonable to suggest that c-kit expression is controlled in a cell type-specific manner.interestingly, tgfgl is obviously able to dissect the proliferative from the migrational signal transducted by kl in these cells. objectives of the study: antisense strategies using dna-otigonucleofides (odn) to modulate the cytokine response are presently under investigation. odn are thought to act very specifically with little or no relevant negative side effects. we now report that odn unspeeifically protect wehi cells from tnf-mediated cytolysis. material and methods: wehi subclone ceils ( x ), that are highly sensitive to the cytolytic activity of tnf, were grown on -well culture plates in rpm medium. after hours, phosphorothioate(ps)and partially ps-modified-odn as well as phesphodiester-odn ( - bp) were added ( . , and pm). four hours after incubation with odn, ce(i lysis was induced by recombinant murina tnf. after hours the plates were washed and stained with crystal violet cell lysis was determined by reading the absorbance (abs) at nm. results: wehi ceils incubated with tnf ( - ng/ml) were completely lysed after hours ( % abs). interestingly, wehi cells incubated with tnf and odn resisted complete lysis, eg cells incubated with . ng/ml tnf and jm odn showed still % of the absorbance observed in control ceils without tnf ( % abs). the protective effect of odn started at . pm, reached a maximum at ,um, and diminished at jm. with increasing amounts of tnf the protective effect of qdn decreased and no protection was detectable at ng tnf per ml conclusions: dna-oligonucleotides were found to unspecifically inhibit tnf-induced cytolysis. we hypothesize, that this protective effect of qdn results from an inhibition of the binding of tnf to its receptor, or from interference of odn with the subsequent signal transduction mechanisms. as a consequence, to discriminate the specific effect of odn in biologic systems, several control odn should be used. secondly, whether dna released by degradation of tumor cells or leukocytes can significantly impair tumor-and immune-defense mechanisms merits further investigation dr. med. michael meisner, institut for anaesthesiologie der universitat erlangen-nqmberg, krankenhausstral~e , d- erlangen. in this study we investigated the involvement of serine protease and free radical generation in the systemic release of tumor necrosis factor-alpha (tnf) and interieukin i(il- ), in the sepsis model of lipopolysaccharide (lps, mg/kg i.p.) induced hepatitis in galactosamine (gain, rag/mouse, i.p.) sensitized mice. treatment of gain-sensitized mice with lps (gain/lps) led to dramatic increase in serum cytokine (tnf and il-i) ievels and transaminase activity at hr and hr respectively. pretreatment of serine protease inhibitor, c~jantitrypsin (a j-at, mg/kg i.p.), rains prior to gain/lps treatment, fully protected the animals against the hepatotoxic challenge with significantly reduced serum tnf and il- levels. in order to block and scavenge superoxide generation, the mice were pretreated with xanthine oxidase inhibitor, allopurinol (al, x mg/kg i.p.) and pyran polymer-conjugated superoxide dismutase (sod, x unit/mouse i.v) r spectively. pretreatment with al and sod ( and hr prior to gain/lps) prevented gain/lps hepatitis and blocked lps induced released of tnf and il- into serum of the mice. the protective agents like cq-at or al/sod did not protect the mice against th~ hpp~totoxi£ ch~llpn-e indllee b'~ th~ recombinant mmlse tnf-o' ( . ~/rno~e j.p.) ~d oi~lps ~ caln-.~dlfa%aed mlce. it-l cett~aged la tnf (x/gain treated mjde was not detectable in animals pretreated with oq-at or al/sod. our study suggests that a serine protease sensitive to cq-antitrypsin is responsible in regulating tnf release, possibly by proteolytic cleavage of a tnf-precursor or membrane bound tnf. in addition our evidence suggest that the balance of extracellular protease/antiprotease activity may be regulated by free radical generation, possible superoxide anion, resulting in inactivation of the antiprotease. il- release may be subsequent to tnf release. objective: during sepsis one can observe a dramatically impaired production of proinflammatory cytokines like the tumor necrosis factor alpha (tnf-a), interleukin i-alpha (il-la), intedeukin i-beta (il-i&) and interferon gamma (if~) upon in vitro stimulation of circulating cells. however there is also evidence of a decreased ability to produce cytokines in other immuno-deficient states. in this study we compared the capacity to secrete proinflammatory cytokines upon in vitro stimulation of patients in severe sepsis and patients with malignant tumors. methods: heparinized blood samples of ten patients ( + years) in severe sepsis (sepsis score > according to e}ebute and stoner) were drawn at onset of disease, from fifteen patients with solid growing carcinoma ( + years) blood was drawn at diagnosis prior to any therapy. controls were obtained from fifteen healthy volunteers. pl of whole blood were incubated either with / of a standard medium or with pl of a standard medium and pl of phytohemagglutinin (pha) a potent mitogen. after an incubation period of hours plasma concentrations of tnf-a, il-la, il- and if-~ were determined by elisa. comments: our results suggest that down-regulation of cytokine secretion or of cell responsiveness to non-specific mitogens during sepsis has occurred. we observe a similar phenomenon for the group of carcinoma patients vs control significant for stimulated tnf-a and stimulated if-t. sustained immunological interactions between tumorcells and cytokine producing cells could effect responsiveness of the latter, a general increased immuno-tolerant state in patients with carcinoma has to be discussed. however we found significant differences between sepsis and cancer concerning the in vitro capacity of responsable cells to produce il-la and il-i#. the dramatically decrease of the ability to produce il-i upon in vitro stimulation could be more sensitive for a septic state than stimulated tnf-a or if- ,. objective: tumor necrosis factor alpha (tnf-a) has been implicated as a central mediator of sepsis and its sequelae. increased systemic levels of this cytoklne seem to be correlated with severity of sepsis and outcome. however mechanism of action and metabolism of tnf-g are not fully understood. in most studies blood samples for tnf-a determinations are obtained either by peripheral venipuncture, a central venous catheter or by an indwelling arterial catheter. very often blood samples are taken in different manners within the same study. in this study we measured circulating tnf-a and the amount of tnf-a released upon in vitro stimulation in arterial and central venous blood. methods: heparlnized arterial and central venous blood samples of ten patients ( males, females, mean age +_ ) with severe sepsis (sepsis score > , elebute and stoner} were drawn on day , , , , and of disease. blood was immediately placed on ice and processed within hour. pl of whole blood were incubated with pl rpmi-medium supplemented with antibiotics and l-glutamlne or with pl of rpmi-medium and pl phytohemagglutinin (pha) a potent mitogen. after an incubation period of hours samples were centrifuged and plasma was harvested and stored at - ° celsius before assessment of tnf-a concentration by elisa. statistical analysis was performed with the paired student-t-test. results: we found a significant difference (p < , ) for circulating mean arterial tnf-a concentration ( pg/ml _+ sem} and central venous tnf-a ( pg/ml +_ sem). upon in vitro stimulation there was also a significant difference (p < , ) between released arterial tnf-~' { pg/ml _+ sem) and venous tnf-a ( pg/ml +_ semi. conclusions: these results are difficult to interprete but could reflect the influence of pao and sao on tnf a release. it could also be the result of different concentrations of tnf-o release influencing factors like for example endotoxin, interferon-f or prostaglandin. a possible pulmonary and/or a hepatic metabolism of tnf-n and tnf-a producing cells cannot be ruled out. however for better interpretations of tnf-a release in septic states it is necessary to use either arterial or venous blood samples. early inflammatory processes following trauma and/or infections were found to be associated with the secretion of high amounts of proinflammatory cytokines. besides intedeukin-t (il- ), tumor necrosis factor-a (tnf-c and interleukin- (il- ) the multifunctional cytokine intedeukin- (il- ) was described to be a central regulatory element of the primary cellular and humeral defence reaction. the previously described close temporal correlation of pathologically elevated il- -concentrations and the extracellulary release of lysosomal enzymes from activated pelymorphnuclear neutrophils suggests, that il- may be a potential substrate of these preteases. the serine preteases elastase (ec . . . ) and cathepsin g (ec . . . ) derived from the azurophilic granules were assumed to be mainly involved in unspecific proteolysis at sites of inflammation by cleavage of structural as well as soluble proteins at random sites, if the inhibitory potential is decreased. the possible proteolytic activity of elastase and cathepsin g toward the proinflammatory cytokine interleukin- (il- ) was investigated. the addition of purified neutrephil elastase and cathepsin g to recombinant human il- leads to a rapid sequential degradation in vitro. at least two intermediate products could be detected by silver staining and western blotting following protein separation under reducing conditions. the serine protease inhibitor g-anitrypsin was shown to prevent the proteolytical degradation of intedeukin- . furthermore the loss of the biological activity of both, recombinant and natural human il- , was demonstrated by determination of the capacity of protease-treated il- to stimulate hybddoma growth ( td bioassay). these data suggest a possible downregulation of pathologically elevated il- levels by proteolytic activity of extracellulary released enzymes at sites of inflammation. the aim of the study was to compare circulating levels of three cytokines -il- , il- , _- -between critically ill subjects who developed gram-negative sepsis and who did not. materials and methods: the patient population consisted of patients admitted to an intensive cars unit, with different underlying diseases. sepsis diagnosis was given according to pre-estabilished cdteda. nineteen cases were enrolled in sepsis group, twenty in control group. serum sampling was collected in sterile tubes at study entry and every three days until study dismissal. serum concentrations of il- , _- and il- were measured using commercially available test kits, based on the dual immunometric sandwich principle. results: the causative patogens of sepsis were: pseudomonas aeruginosa, acinetobacter, eseherichia co~i, serratia marceseens, proteus mirobilis and citrobacter freundl the time of observation was equal to days, for a total of four tests performed (to, tl, t , t ). i .- was not detected in any samples. the serological profiles of the two cytokines .- and _- were similar; augmented levels were found at study entry and throughout the observation period, peaking at t and decreasing at t . however, in patients with sepsis, il- and _- concentrations were significantly higher in respect to control group. conclusion: our observations shown that in icu patients increased il- and il- release may be induced by cdtical illness; however, in subjects in which sepsis occurred, il- and il- production appears more significantly elevated, suggesting a role of il- and _- in the pathophysiology of sepsis. the fact that ii. objective: to check whether continuous veno-venous haemofiltration (cvvh) could remove the cytokines, namely tumour necrosis factor alpha (tnfc and interleukin (il- ) from the circulation of critically ill patients with sepsis ad multiple organ failure (mof). setting: the intensive therapy unit of the medical school teaching hospital. patients: nine critically ill patients with sepsis and mof treated with cvvh. methods: blood samples were collected before the cvvh had been started. then, blood and ultrafiltrate samples were collected simultaneously after hours and every hour. tnfct and il- levels were measured using the bioassays with cell lines wehi- ci and td , respectively. other data were recorded from the patient notes and intensive therapy unit charts. results: no measurable concentrations of tnfct were detected in either blood or ultrafiltrate samples. il- was found in all the patients' plasma samples and five patients' ( . %) ultrafiltrate samples. the il- blood level ranged from . to . u/ml (mean . , sd . ). the il- level in positive ultrafiltrate samples ranged from . to . u/ml (mean . , sd . ). conclusions: our preliminary results suggest that il- is present in bloodstream of septic patients. we assume we could not detect tnfa in any sample because we usually started observations when septic state had developed. cvvh could extract cytokines from the circulating blood. it remains under discussion, whether that extraction may be beneficial to patients with mof. the pattern of some significant cytokines tnf, il- and il- and their pharmacomodulation were evaluated in an experimental model of polimicrobial sepsis induced in cd- mice by cecal ligation and puncture (clp) in order to understand their roles. this model of sepsis, which resembles the clinical situation of bowel perforation, was also compared with that induced by administration of pure endotoxin (lps). tnf was detectable in serum and tissues during the first h with a peak h after clp at a significantly lower level than after lps. il- was measurable in serum only after h, significantly increased in spleen and liver after and h and in mesenteric lymphonodes from to h after clp compared with shammice. il- was significantly increased in serum throughout the first h after clp. pretreatment with dexamethasone (dex), ibuprofen (ibu) and nitro-l-arginine (n-arg) significantly reduced the survival time while chlorpromazine (cpz) and tnf did not affect it. only the antibiotics and pentoxifylline (ptx) significantly increased the survival in clp. however cpz and dex protected from lps-mor~ality. in conclusion, by inhibiting tnf with dex, cpz, ptx a reduced, unchanged and increased survival time was observed and by increasing tnf with ibu and tnf administration the survival was decreased or unchanged respectively suggesting that the modulation of this cytokine does not seem to play a significant role in clp unlike lps_ moreover the negative effects of ibu and n-arg suggest an important and protective role by prostaglandins and no in clp. to gain more insigths on the contribution of tnf~, il-i~ and if to lps toxicity, we explored the time-course of the cytokine production in ealb/c mice given different doses, from the lethal (= ld ) to the sublethal (= / ld ) of three different lps (e.coli oiii:b and :b ; p.aeruginosa r ) endowed with different degree of toxicity cytokines were measured in serum and organs with specific elisas up to i h after lps administration. results demonstrate that i) circulating and organ levels of tnf~ do not reflect lps toxicity. in fact, the lethal dose of lps :b induced as much tnf~ as the sublethal dose of lps :b ; furthermore, lps r , whose cytokine inducing capability is far lower than that of lps from e.coli, induced higher tnf~ levels at the sublethal than at the lethal dose. in addition, policlonal anti tnf ab, that were able to protect mice from e.coli lps induced mortality, failed in mice treated with lps r ) circulating il-i~ levels are generally low and increase significantly only in muribond animals. on the contrary, in spleen and lung very high levels of il-i~ are persistent from i to h post lps administration moreover, the treatment with mgr of neutralizing policlonal anti il-i~ ab, did not modify survival in lps challenged mice. ) circulating and organ levels of if are proportional to the dose and degree of toxicity of all the administered lps even if lps r was again a less efficient cytokine inducer than lps from e.coli. csa is an immunos~ppressive drug, able to inhibit gene expression for many cytokines, including if . to study the effect of cytokines modulation on lps toxicity, csa was administered to mice twice at the oral dose of i mg/kg before the challenge with lps. mice were monitored in terms of mortality and tnf~, il-i~ and if production. together with the total ablation of if , the strong reduction of tnfu and unmodified il-i~ levels, a significant increase of lps toxicity was also observed. these results suggest the hypothesis that the numerous factors that jointly mediate lps toxic effects, can also be protective, the final outcome depending on their relative ratio rather than on the absolute amount interleukin- (il- ) mediates the septic shock syndrome and affects intestinal secretion in vitro. we studied the intestinal production of il-t and its effects on diarrhea during endotoxic shock. cd- mice were randomized to mg/kg e.coli :b lps or saline infusion (i.p. or i.v.). diarrhea invariably occurred following lps infusion. mice were sacrificed at , ', lh, . h, h, h, h, and h ( mice/group/time-point). the small bowel was compressed and the intestinal contents were weighed and expressed per g sb weight. the small (sb) and large bowels (lb) were eventually frozen, weighed, and homogenized for either cytosolic protein or total rna. il-i~ (cell-associated agonist) was measured with a radioimmunoassay specific for mouse il-l~ (detection limit pg/ml) and expressed as ng/g weight + sem (lowest detectable amount ng/gwt). northern analysis of total rna and in sfu hybridization of paraformaldehyde-fixed frozen tissue were done with [ ~- p]-iabeled mouse il-lc~ cdna probes. only sb had il-i~ constitutively present ( . + . ng/gwt). lps i.p. or i.v. induced elevation of il-lc¢ in both organs in a biphasic pattern; lps i.v. induced -fold more il-i~ than lps i.p. following lps i.p., il-i~ in sb was . + . ng/gwt at lh, reached maximal levels at . h ( . -+ . ng/gw-i) and returned to baseline at h. saline controls maintained their constitutive il-i~ levels. sb had fold more il- ¢ than lb and identical kinetics, but lb showed a clearer doseresponse. northern analysis of sb-total rna showed induction of il-i~ mrna by lps in correlation with il-lc¢ kinetics. il-i~ mrna producing cells were mononuclear cells in the lamina propda and epithelial cells at the bottom of the crypts of ueberkuhn. mucus and fluid were increased in the small bowel post-lps in correlation with intestinal il-lc~ kinetics (r = . ). separate mice were pretreated with saline i.p. orthe il- receptor antagonist (irap, mg/kg bolus i.p.) and were challenged rain later with . mg/kg lps i.p. or saline i.p. specific blockade of il- by irap decreased intestinal secretion at h and h post-lps challenge (p<_. . , student's-t-test). these data indicate that local (intrinsic) intestinal il-i~ mediates sepsis-induced intestinal changes. inflammatory cytokines initiate the host response to endotoxemia, causing severe physiological and hemodynamic changes which may lead to septic shock. among the regulatory systems that play an important rote in controlling host inflammatory responses is the pituitary. it has been known for many years for example, that hypophysectomized animals are extremely sensitive to lps lethality. while investigating the possibility that protective, pituitary mediators might explain this phenomenon, we identified the cytoldne mif to be a specific secretory product produced by pituitary cells in vitro and in vivo after lps challenge. analysis of serum mif levels in control, t-cell deficient (nude), and hypophysectomized mice revealed that pituitary-derived mif contributes significantly to the rise in serum mif that occurs after lps administration. of note, pituitary mif content ( . % of total pituitary protein) and peak serum mif levels ( - ng/ml) were determined to be within the range observed for other pituitary hormones that are released after pituitary stimulation. to investigate a possible beneficial role for mif in septic shock, we co-injected mice with purified, recombinant murine mif (rmif) together with lps ( mg/kg). surprisingly, rmif markedly potentiated lps lethality compared to control mice that were injected with lps alone ( % vs. %, p = . ). to confirm these results, mice were treated with anti-rmif antibody prior to injection of a high dose of lps ( . mg/kg). anti-rmif antibody fully protected mice against lps lethality, increasing survival from % to % (p = . ). serum levels of tnf,~, the first cytokinc that appears in the circulation after lps challenge, were reduced by . _+ . % in anti-rmif-treated mice. we conclude that pituitary derived mif contributes significantly to circulating mif in the post-acute response in endotoxemia and may act in concert with other pituitary mediators to regulate both pro-and antiinflammatory effects. moreover, mif may play a critical regulatory role in the systemic host response in septic shock. our results suggest that anti-rmif antibody might be of potential therapeutic use in the treatment of septic shock. although anti-interleukin- (il- ) antibodies and il- receptor antagonist have been shown to improve survival in animal models of endotoxemia and abrogate the lethal effects of tnf, the presence of il- in the serum does not correlate well with outcome. we hypothesized that this may be because il- acts mainly in a paracrine fashion and is metabolized before it diffuses into the circulation. methods: we measured the il-i~ mrna expression with the differential reverse transcription polymerase chain reaction (rt-pcr) using g-actin as internal standard in the peritoneal macrophages and lung tissue in normal controls and mice after cecal ligation and puncture (clp). clp resembles human intra-abdominal sepsis in that it is characterized by very slight elevations of serum il- levels. results: il-lg mrna levels after clp are expressed as % of normal (mean+sem, n= in several experimental models of infection exacerbation of disease was observed, when infected animals were depleted of tuajor necrosis factor (tnf). after sublethal cecal ligation and puncture (clp) leading to peritonitis and sepsis the survival of mice also critically depends on tnf as demonstrated in earlier studies, when clp-treated mice injected with anti-tnf antibody died, whereas mice injected with a control antibody survived after clp (echtenacher et al. , j. inununol. : ) . from a panel of different cell types (macrophages, neutrophils, t lymphocytes, natural killer cells, mast cells) able to produce tnf upon activation~ the mast cell is apparantly the only one capable of storing in cytoplasmic granules preformed tnf-ct which is rapidly released following challenge. in the present study-we analyzed serum tnf after lps injections as well as the outcome of clp in severely mast cell deficient mutant mice (wav v) as compared to syngeaeic wild-type littermates (+/+). we proposed that concentrations and/or kinetics of serum tnf should be different between wavv mutants and wild-type mice, if mast cell-derived tnf significantly contributes to the rise in serum tnf levels following systemic stimulation with endotoxin. although similar levels of increased tnf were detected in the sera of both genotypes after and hours of lps injection ( btg/ . ml / mouse i. p.), mast ceil-deficient mice indeed showed decreased serum tnf levels iron after injection amounting to only to % of the concentrations observed in the corresponding sera of normal wildtype mice. in the clp model of septic peritonitis we found that mast celldeficient mutant mice were dramatically more sensitive to clp than syngeneic normal mice resulting in % mortality in w/w v versus % mortality in +/+ mice . days after initiation of clp. further experiments with w/w v mutants selectively reconstituted with cultured bone marrow-derived mast cells from normal syngeneic wild-type mice and the use of an antibody specifically blocking the action of tnf tn vivo should clarify a potential protective function of mast cells in this model of septic peritonitis. interleukin- (il- ) inhibits cytokine production, including tumor necrosis factor (tnf), by lipopolysaccharide (lps)-aetivated maerophages. we recently observed that lps injection (e.coli :b , gg ip) into balb/c mice induces the rapid release of circulating il- ( ± u/ml at min). blocking endogenous il- using monocional antibody (jes - a , mg, h before lps) resulted in a massive increase in tnf production ( ± in lps+anti-il- treated mice vs ± ng/ml in lps alone, p< . , n= to mice per group) and an enhanced lps-induccd lethality ( % vs % in anti-il- +lps or lps alone respectively, p= . , n= mice per group). irrelevant igg rat monoclonal antibody (lo-dnp) did not influence neither tnf production nor lethality associated with endotoxin shock. this led us to study the production of il- during human septicemia. plasma samples were obtained from patients with gramnegative (gns, n= ) or gram-positive septicemia (gps, n= ) and from healthy volunteers. among these patients, suffered from septic shock at the time of sampling. il- levels were measured by elisa (detection limit: i pghrd). we found that patients ( %) had increased il- plasma levels (range to pg/nd). patients with gps had il- levels similar to the ones observed in gns (median: vs . pg/m, respectively). patients with septic shock had higher il- values (median: pg/ml) than septicemic patients without shock ( pg/ml, p= . ). no il- was detected in plasma from healthy volunteers. we conclude that il- is produced daring human septicemia. our experimental data suggest that il- might be involved in the control of the inflammatory response induced by bacterial products. dr arnand marchant, immunology department, hopital erasme, route de lennik, brussels, belgium. to provide information about the role of tnf in sepsis and mods we measured tnf and stnfr-i levels in septic patients and investigated if there is a relation between plasma concentration of these molecules and the severity of sepsis evaluated by two scores (apache i and sss). patients and melhods: septic patients fullfilling sepsis criteria of american college of chest physician and society of critical care medicine were studied. tnf-cc and stnfr-i ( kda) were measured by enzyme immuneassays (norms values = + pg/ml and . _+ a ng/ml respectively). results: the mean tnf and stnfr-i values for each patient (mean+sd) were + pg/ml and . + . ng/ml respectively. these values are approximately seven and ten times greater than those observed in normal healthy volunteers (p< . ). mean tnf concentrations for each patient were significantly greater in non survivors ( + vs _+ pg/ml p< . ); stnfr-i levels also were greater in this group, but the difference was not statistically significant ( . + . vs . _+ . ng/ml). plasma tnf and stnfr-i concentrations were significantly correlated (r = . p< . ). mean tnf levels were significantly correlated with apache ii (r = . p< . ) and sss (r = . p<o. ); stnfr-i levels were likewise correlated with both scores (r = . p< . and r = . p< . respectively). tnf and stnfqgi levels increase with the number of dysfuctional organs; in particular tnf and stnfr-i increase when hemodynamics deteriorate and kidney failure ensues. conclusions: the correlations between tnf and stnfr-i levels and sepsis severity scores suggests that tnf is involved in sepsis and may influence the occurrence of mods and finally clinical outcome. tnf and stnfr-i are in fact especially increased when mods occurs. in particular their concentrations are elevated when cardiovascular system and kidney failure ensue, suggesting that tnf can have a direct role in hemodynamic derangements caused by sepsis and that kidneys are involved in tnf and stnfr-i excretion. lif is a pleiotropic cytokine that has been implicated in the pathogenesis of sepsis in animal models. we conducted a prospective study in septic patients to correlate lif plasma levels with patient's clinical and biological data (among them il- ). plasma was drawn daily from day one to .ten and lif presence was determined with a specific elisa and with the dai,a bioassay (sensitivity of pg/ml and ngml respectively). risk factor associated wftt~ with elevated lif plasma level was determined by an univariate analysis. a multivariate stepwise logistic regression analysis was subsequently performed with a bmdp statistical software. lif was detected with the elisa and with the bioassay in and out of the patients, respectively. lif peak plasma levels were statistically associated with high creatmin levels, temperature and il- . multivariate regression analysis showed that high serum creatinin level was the major independent risk factor associated with elevated lifplasma levels. this correlation was also found for il- . peak plasma levels of both lif and il- correlated inversely with patients survival duration. cox's analysis for plasma levels of lif > pg/ml yelded a hazard ratio of [exp ( . )= . ]. our study indicates that lif levels were associated with clinical and biological parameters of illness severity and significantly increased (cut-off value pg/mi) in patients with fatal outcome. current consensus exists about the central role of tumor necrosis factor (tnf) alpha in initiating the systemic inflammatory response syndrome (sirs). a correlation with sirs has inconsistently been found. tnf effects its pleiotropic reactions upon two distinct cellular receptors. soluble extracel]ular fragments of the human kda tnf receptor (stnfri) and the kda receptor (stnfrii) are detectable in the circulation. the kinetics of these endogenously produced tnf-inhibitors were measured to evaluate their role in patients with sirs. fourteen patients of an operative icu were included with the diagnossis of sirs (mean apache ii score: points). serial blood samples were obtained within h after diagnosis of sirs, every hrs for the first hrs and every hrs thereafter until patients died or recovered. soluble tnfri and stnfrii were assayed by an enzymed-linked immunological binding assay. soluble tnfri and ii could be detected in all samples with a significantly higher level (p<o,o ) compared to healthy controls (normal value: tnfrh , ng/ml; tnfrii: , ng/ml). the serum concentration of tnfri ( , ng/ml) as well as tnfrii ( , ng/ml) were significantly higher in nonsurvivors (n= ) compared to survivors ( , and , ng/ml; n= ) at the onset of sirs and during the course of the inflammation response (p< , ). a positive correlation exists between both receptors (r- , ; p < , ). increasing levels and maximal values of stnfri ( ng/ml) and i ( ng/mi) were detected only in patients who died. the serum concentrations of patients who recovered did not change and are remaining at the initial level. tnf alpha bioactivity was detected in out of patients. no significant correlation exists between the concentration of both receptors and tnf alpha as well as the apache ii score at the onset of sirs. elevated serum concentration of stnfri and are found in patients with sirs. these naturally occuring antagonists reflect the inflammatory process. the measurement of soluble tnf receptor levels may be useful in monitorina sirs and in the prediction of outcome. - is given to patients with advanced melanoma or renal carcinoma. however, this therapy is accompanied by severe side effects, including the vascular leak syndrome (vls) that closely resembles septic shock. to investigate the involvement of cytokines and phospholipase a z in the pathogenesis of the vls we collected serial plasma samples from patients during the first hours after administration of il- . in these patients we monitored temperature, blood pressure and heart rate and assessed levels of the cytokines tnf, il- and il- using immuno assays. in these plasma samples we also measured phospholipase a activity using an enzyme activity assay, since this enzyme is beleived to play a major role in the generation of lipid mediators. we demonstrate that during il- therapy the cytokines tnf, il- , and il- are produced and that the release of these cytokines is followed by an induction of phospholipase az activity in the plasma of these patients, we further discuss the role of these mediators in the development of the vascular leak syndrome observed in patients receiving il- . objectives of the study: sepsis caused by candida albicans (ca) is no longer a clinical rarity but a common consequence of immunosuppression and surgery, and is associated with high mortality. tumor necrosis faetor-c¢ (tnf-<~), interlcukin-lg (il-lg) and interleukin- (il- ) are thought to play an important role in the pathogenesis of the sepsis syndrome. we therefore studied the in vitro production of tnf-<x, il-ib and il- by human peripheral blood mononuclear cells (pbmc) stimulated by ca compared to stimulation by bacterial lipopolysaccharide (lps). materials and methods: pbmc were isolated from venous blood of healthy volunteers and cultured at a concentration of . • ~ eells/ml in culture medium with a dose-range of either heat-killed ca or lps. supernatants were harvested after hours. using elisa, tnf-c~, il-ib and il- concentrations were measured. ca was isolated from a patient with ca sepsis and cultured under sterile conditions. results: ca and lps stimulate the production of tnf-~t, il- ] and il- by human pbmc. we observed a dose-dependent synthesis of these three cytokines in human pbmc. tnf-c¢, il-ib and il- in ng/ml as mean _+ standard error. ca/ml . . ( ) we constructed a single-chain fv-fragment (sc-fv) from the variable domains of a neutralizing antibody to human tumornecrosisfactor-alpha (tnf) because sc-fv should have some advantages in vivo. the sc-fv has a similar affinity ( , x - m) as the fab-fragment ( , x - m) but bind the antigen with a eightfold lower avidity compared to the parental mab ( , x - ). the parental mab as well as its fragments can compete the binding of rhutnf to both tnf-receptors. this was shown by competitive binding to the cell line hl- and to immobilized rtnf-receptors. in the nutralization assay on the cell line, l , the sc-fv can neutralize tnf but in comparison to the mab or its fabfragment, the capacity was three-fold lower as expected from the molar ratio of the dissociation constants. the in rive-neutralizing capacity was tested in a mice receiving toxic dose of rhutnf. the sc-fv showed a -fold lower neutralizing capacity in comparison with the fab-fragment. this could be explained in different manners: i) partial instability of scfv at c, ii) shorter half-life because sc-fv but not fab-fragments are eliminated via kidney, iii) elimination of tnf-immunocomplexes may be improved by opsonization mechanisms which requires some structures of the constant region (as in fab or mab). in summary, despite the encouraging in vitro experiments, the in vivo data suggest that sc-fv are not the right strategy for neutralizing tnf in vivo. tnf production in endotoxin non-responder mice, effect of a glucocorticoid antagonist g. iazgtr jr, e. duda, j. ol~th, e. husztik, g. iazar glucocorticoids inhibit lipopolysaccharide (lps)-induced tnf production and tnf can stimulate pituitary adrenocorticotropin secretion, resulting in the release of corticosterone. earlier studies ( ) reveales that the glucocorticoid antagonist ru , nufepristone, sensitizes both endotoxin responder and nonresponder, c h/hej, mice to endotoxin lethality. we have also shown that the glucocorticoid antagonist ru sensitizes endotoxin-tolerant (endotoxin-pretreated) and normal mice to the lethal effect of septic and endotoxin shock. on this basis, we set out to study the influence of ru on tnf production induced by endotoxin in endotoxin responder and non-responder mice. one and hrs following lps injection, ru significantly increased the tnf concentration in the serum, liver and spleen of treated animals as compared with the control and methylprednisolonetreated groups. in tissue cultures, ru induced the tnf synthesis of myeloid cells and increased the tnf production of genetically modified hela cells, which synthesize tnf constitutively. normal and tumor cell cultures exhibited an increased sensitivity toward tnf in the presence of ru . however, the same dose of lps did not induce tnf production in the serum, liver and spleen of endotoxin non-responder mice and this was not influenced by concomitant ru treatment. these studies suggest that ru aggravates lps lethality via factors other than tnf in endotoxin non-responder, c h/hej mice. the cytokine response to a novel exnacellular mitogenic factor, mf, produced by the group a streptococci (gas), and to the streptococcal pyrogenic exotoxins (spe) a and b, was determined by in vitro stimulation of peripheral blood mononuclear cells (pbmc) obtained from healthy blood donors. the induction and kinetics of different cytokines was studied at single-cell level using cytokine specific monoclonal antibodies and intracellular immunofluorescent staining. all three mitogens induced a massive ifny and tnfi response in - % of the pbmc after - hours of cell stimulation. in contrast, il and tnfc~ containing cells was only detected in - % of the pbmc. the induction ofil , il , il , and ill , was weak or completely absent. thus, the response indicates activation of th cells. however, illc~, illl , illra and il , were consistently found and gradually produced, peaking at hems in approximately - % of the pbmc. mf induced an equally extensive cytokine as well as proliferative inducing capacity, as did spea and speb, which suggests that also mf is a superantigen. a marked inter-individual variation could be noted between lymphocytes isolated from different individuals, both in the toxin induced proliferation and cytokine induction. this may be one explanation for the varying clinical severity noted in patients after infection with clonal gas strains. introduction -tumour necrosis factor (tnf) is released by mononuclear cells in response to inflammation and sepsis. it has been implicated as a possible mediator in inflammatory bowel disease (ibd) but its pathogenic role remains uncertain. this study assessed the relationship between tnfct and disease activity by measuring plasma and faecal tnf concelnmtions in an experimcntai model of ibd. methodologo' -colitis was induced in male wistar rats by intmcolonic instillation of rag , , -trinitrobenzenesulphunic acid in . ml % ethanol (n= ). control animals received an isovohlmetric instillation of normal saline (n= ). faecal pellets were collected before induction of colitis (day ) and throughout the experiment. after days the colon was excised, ueighed and the colon macroscopic score (cms) assessed plasma tnf (ptnf) samples were assayed by bioassay and elisa. faecal samples were vortcxed in water and the supernatant removed for estimation of protein and faecal tnf (itnf) content (elisa). there is a significant increase in itnf on day -#p= alld the total ftnf measured during the study correlatcs with the cms oll day -p= . there is no correlation between ftnf and ptnf. conclusions -therc is evidence of coloific tnf excretion by macroscopically normal animals and following induction of colitis there is increased faecal tnf which correlates with disease actmty. there is no significant elevation in bioactive or imnmnoreactive plasma tnf in colitie animals this data would suggest that faecal tnf is a marker of colonic inflamrnatiort and serial tnf assessment inay be useful as an indicator &severity in ibd. to study the effect of in-vivo hypoxia/reoxygenation on cytokine elaboration by routine peritoneal macrophages. materials and methods: adult male cba mice ( - g) were primed intraperitoneauy with either lipopolysaccharide (lps) ug]anlmal or saline. five days later, the animals were subjected to hypoxia ( % ) for , followed by of normoxia. harvested peritoneal macrophages were plated in-vitro, culture supernatants were collected at , , and and assayed for tumor necrosis factor (tnf), prostaglandin e (pge ) and nitric oxide (no). control animals underwent the same procedures, but were maintained in normoxia ( %o disease , berlin, germany the precise mechanisms of reactivation of latent human cytomegalovirus (cmv) infection are still unknown. apart from the permissive effect of diminished cellular immunity there is evidence of a cytokine mediated cmv reactivation as it is known for other systemic virus infections (e.g. hiv). we found that tumor necrosis factor-alpha (tnf) -in contrast to all other cytokines tested -can stimulate the activity of the cmv-ie-enhancer/promoter region in the human monocytic cell line, hl . we wondered whether our in vitro results were actually reflected by the incidence of cmv reactivation in diseases which go together with high tnfalpha levels. cellular immunodeficiency as well as at least temporarily increased tnf levels are known to occur in septic disease. we tested for the presence of cmv infection in peripheral blood mononuclear cells (pbmnc) by means of h centrifugation culture, immunocytological detection of different cmv antigens (apaap-technique), and pcr technique (cmv-ie dna). in striking contrast to healthy controls almost all examined septic patients were positive, irrespective of the method for cmv-ddtection applied, including the less sensitive immunocytology. follow-up studies showed that cmv-antigen positive pbmnc ceased to be detectable in patients with good outcome once septicaemia had been overcome. to further back up our hypothesis, we looked for a coincidence of enhanced tnf-alpha serum levels and cmv antigenaemia (immunocytology) in some other diseases which are known to have either enhanced tnf serum levels or increased incidence of active cmv infection and found the majority of patients with b-cell chronic lymphocytic leukemia, liver cirrhosis, and common variable immunodeficiency to be positive for these two parameters. we now wondered whether, apart from cmv reactivation, tnf-alpha could also cause cellular immunodeficiency this way promoting cmv replication and spreading and eventually causing cmv disease. in summary, we were able to show that a diminished cellular immune response results from/n vitro or in vivo methylprednisolone (mps) is used to suppress both inflammatory and immune responses. il- receptor antagonist (il-lra) and tnf binding protein (tnfbp) are naturally occuring anti-cytokine proteins, possibly modulating inflammatory and immunological responses. to define mps modulation of cytokine and anticytokine responses, we examined effects of mps on il-lra production and tnfbp generation as well as il-i~ and tnfa production by human peripheral blood mononuclear cells (pbmc) stimulated with lipopolysaccharide (lps). pbmc were purified from the blood of healthy volunteers, then incubated with lps ( ng/ml) supplemented with different concentrations of mps ( , pg/ml, mg/ml). after hrs incubation, the supernatants were collected for rias of secreted cytokines. the cell lysates obtained by cycles of freeze-thaw, were also collected for r ias of cell-associated cytokines. both the supernatant for secreted cytokines and the cell lysates for the cell-associated cytokines were subjected to rias for il-i~, tnfa, il- ra, and tnfbp-i (tnfsrp ). il-la, il- ra, and tnfbp were produced mainly as cell-associated forms in response to lps, whereas most tnfa was secreted into the supernatant of lps-stimulated pbmc. mps supplement resulted in reduction of il-la, il-lra, and tnfa production. il-la production was reduced up to . + . %( mg/ml) by mps in a dose dependent fashion, whereas about % decrease in tnfc, and il-lra production was observed at both high and low dose mps supplement. however, total tnfbp generation was not reduced significantly by mps supplement. furthermore, tnfbp secretion was increased times more in the supernatant of lps-stimulated pbmc with gg/ml of mps. these data suggest that mps could effectively block tnf activity by both reducing its production and up-regulating tnfbp generation and that mps works as an anti-inflammatory drug as well as an immunosuppressant by modulating cytokine and anticytokine responses. in severe gram-negative sepsis, excess of proinflammatory cytokines is associated with increased morbidity and the mortality. we have found that immunocytes possess functional -adrenergic receptors (~-ar) which, when activated, down regulate the lipopolysaccharide (lps) induced gene expression and subsequent secretion of tnf-~ in vitro. in the present study, we used a lethal endotoxic model in mice to test the hypothesis that -ar stimulation will down regulate the gene expression thus decreasing the circulatory levels of proinftammatory cytokines and improve the survivals in severe endotoxicosis. the animals (iso group) were injected times with ! -ar agohist isoproterenol ( . mg/kg i.m. and . mg/kg s.c.) , and hours prior to a lethal dose of lps injection ( mg/kg i.p.). control group (ctl) received same volume of saline at the same times before the lps insult. at the appropriate time points after lps injection animals were sacrificed and venous blood was collected from the inferior vena cava. the plasma levels of tnf-cc and il-loc were determined by elisa. in the mortality study, each grou the data showed that isoproterenol significantly decreased the mortality and the circulatory levels of tnf-c~ and il-lcc (fig. ) . these data suggest that -ar activation of immunocytes down regulates cytokine gene expression, decreasing circulatory eytokine levels thus reducing endotoxicosis mortality. these results provide a new pharmacological approach to reduce the excess of proinflammatory cytokines and mortality in sepsis. introduction: cytokines released from monocytic cells are thought to play an important role in the pathophysiology of sepsis. during the last few years several investigations have been performed aimed at modulating this mediator response. in the present investigation using a full blood model we have studied the effect of a standard immune globulin and an antitipopolysaccharide (lps) immune globulin preparation and a monoclonal anti-lps antibody in modulating the endotoxin induced cytokine response. methods: citrated blood from healthy human donors was incubated at °c in rotating polystyrene tubes. samples were taken before addition of x ng/l e. col± endotoxin and after hours. plasma was assayed for the cytokines minor necrosis factor alpha (tnf-~) and interleukin- (il- ) using elisa methods. the eodotoxin concentration was assayed by lal and end-point chromogenic substrate technique. the blood was preincubated with standard immune globuline (human igg from pooled plasma, gammagardr, baxter) or with an anti-lps immune glubuline (human anti-lps igg obtained by screening of blood donors, novo nordisk) in the concentrations of mg/ml or mg/ml or with the monoclonal lipid a igm antibody ha- a for ten minutes before the endotoxin was added. results: two hours after endotoxin addition markedly elevated tnf-~ and il- values were found.in the plasma. cytokine values at hours: tnf-ec pg/ml (mean) and l- pg/ml (mean). preincubalion with mg/ml standard immune globulins reduced il- values by % (mean) while there were no effects on tnf-c~ values at hours. a slight reduction in il- values was also found with the lower concentration, mg/ml, but this was not statistically significant. preincubation with anti-lps immune globulins had no effect on il- nor on tnf-cc values. preincubation with the monoclottal lipid a igm antibody ha- a in variable concentrations had no effect on cytokine release in this model. in this full blood in vitro model standard immune globuline reduced endotoxin induced l- release while tnf-cc values were unchanged.the anti-lps immune glubuline preparation and the monoclonal lipid a igm antibody ha- a had no effect on the il- or tnf-ct release. the effect of anti-tnf treatment in severe haemorrhagic/traumatic shock was investigated in a conscious rabbit model. the jugular vein (for administration of substances and blood withdrawal) and right femoral artery (for measurement of bp) were cannulated and an aortic thermistor probe inserted into the left; femoral artery for the measurement of cardiac output (co). the following day, upto % of the estimated blood volume was withdrawn over - rain, to reduce bp to - mmttg and co by / mad withheld for rain. shed blood ( %) was then reinfused followed by lactate solution to give a total potential dreulat%ag fluid volume of %. zymoeen activated plasma ( , gg/ml) was given rain prior to haemorrhage and during blood r~n%eion, all ~,~ir, ak were killed at h and o~gane removed for histology. rabbita received either monoclonal antirabbit tnf (r , rat igg , mgkg i.e. n= ) or emine (nffi ), rain prior to haemorrhage. the cardiovascular and biochemical changes during the haemorrhage and hypotensive phase (is. haemorrhagic stimulus) were similar in both groups. upon reinfusion, haematccrit and white cell count remained sigutfia~utly (p< . ) higher in saline compared to r -treated animals, indicating a relative haemoconcentration (i,e. reduced circulating volume) in saline animals. the lettkocytosis persisted in the saline animals upto h. plasma glucose, lactate and asparteto .m~-o transferase all rose similarly in both gorups but plasma ¢rea~!~+-e levels were markedly (p< . ) higher in ealine ve r animals at and h indicative of kidney damage, using a macro and micro. pathological scoring system, major organ damage was seen in the lung, liver and kidney which was significantly (p< . ) attenuated in r treated animals. in the hmg and liver, this was primarily due to a reduced bleeding and pmn infiltrate and to an additional maintenance of tubular integri~:y in the kidney. hence, in tlais model anti-tnf treatment markedly reduced the biochemical and histological indices of severe ,, ~gan damage during liaemorrhage and ranerfn~ion. thermal injuries and systemic bacterial sepsis produce a wasting diathesis with anorexia and marked loss of lean tissue and body fat. endogenously produced cytokines, including interleukin- (il- ), are thought to mediate many beneficial as well as pathologic host changes that occur during burn injury and infection. to evaluate whether bluckade of il- in vivo would affect survival and attenuate the anorexia and cachex[a associated with a thermal injury and chronic fulminant gram negative infection, sprague-dawley rats were studied. anesthetized rats were divided in groups, implanted with alzet r minipumps and randomized to receive the following treatment for days:i: gg/kgbw/min of il-lcc; ii: ~tg/kgbw/min of il- receptor antagonist (il-ira); iii: buffered vehicle. the animals were subjected to a % bsa, full thickness scald burn and the wounds then infected with + cfu/ml pseudomonas aeruginosa. eight additional rats were anesthetized, but were not burned, and served as healthy controls. . il-hx reduced food intake transiently but was otherwise without effect however, il-lra significantly attenuated weight loss over the first days and improved food intake between days and . we conclude that blockade of il- after a thermal injury with superimposed infection does not adversly affect the progression of the disease, but does attenuate the anorexia and weight loss associated with this model. these data indicate that il-i receptor blockade may offer a means to minimize the morbidity associated with catabolic illness. tumor necrosis factor (tnf) and its downstream induced mediators have pivotal roles in the pathogenesis of sepsis and septic shock. the suppressive effect of pentoxyfillin (ptx) on tnf production may be of clinical importance. when peripheral white blood ceils were stimulated with either lps or staphylococcus aureus, pentoxyfillin inhibited tnf production. in contrast, no inhibitory effect was observed on the induction of il- . ptx inhibited not only the tnf production of monocytes but also the tnf secretion of both granulocytes, and unseparated whole blood. the facs analysis revealed that the expression of cd antigen on monocytes was not influenced by pentoxyfillin. the in vitro tnf and il- producing capacity in septic patients were higher than in the control group, but decreased on subsequent days especially in fatal cases. administration of ptx ( mg/day) in septic patients resulted in tnf and il- productions similar to those found in control donors. it also subsequently led to an improvement of the clinical status. a further improvement in apache score could be achieved by administration of pentaglobin ° (biotest)( ml/kg/day ). the prevention of lpsinduced in vitro tnf and il- production by pentaglobin ° could be demonstrated in in vitro experiments involving the use of whole blood rather than purified lymphocytes, very probably because of the presence of lps binding protein in the plasma. the level of soluble icam- in sera of septic patients was significantly higher ( - ng/ml) than in normal individuals ( - ng/ml), but it decreased following the ptx and pentaglobin ° therapy. it is presumed that ptx and pentaglobin ° can antagonize eytokine production at different levels, resulting synergistic action being beneficial in the treatment of sepsis. albert szent-gy rgyi medical university, institute of microbiology, szeged, hungary h- ddm t. . e~i~im~/tii, septic sh ~ '. tnf alfa/pge, and somatostatln lands ji., alvarez j., gram m., llanos k., alcalde j., sanchez ja., balibr~d jl. taurine, a semi-essential sulphur-containing t:~-amian acid, promotes neutrophil phagocytic activit ' against yeasts and enhances intracellular killing of e.coli. paradoxically it protects against hypochlorous acidmediated biomembmne oxidatio~ and abrogates the pyrogenic effects of intracerebral endotoxin. it is therefore possible that taurine mediates some of these effects by modulating the cytokine cascade. aim to assess the cytekine responses in a rat model of intraperitoneal sepsis, pretreated with supplenmntary taurine, by measuring tnf and il- concentrations. methodology female wistar rats (wt - g) (n- per group) were prefed with % tanrine, . % glyciuc ( in tap water) or tap water (tw) for days prior to caecal ligation aud puncture (clp). normothermia was maintained intraoperatively and the animals received . % saline subcutaneously ( mls/ g) post-procedure. animals were venosected by direct cardiac puncture at or hours post-operation and the blood placed in endotoxin-free tubes. centrifilgation and storage of plasma at - °c was completed within hours. tnf and il- were measured using wehi and b bioassays respectively. results (means ± sem) bioactivc tnf concentrations were not significantly different between clp or sham groups. however plasma il- estimation revealed a statistically significant reduction at hours in tanrine-treated animals compared to glycino and tw controls ( objective: to evaluate the effects of allogeneic blood transfusion, thermal injury and bacterial garage on interteukin (il- ), tumor necrosis factor alpha (tnf) production and host mortality and to study if the administration of thymopentth (thy) could affect these events. materials and methods: balb/c mice were transfused with allogeneic blood (from c h/hej mice) and treated daily with thy (i mg/kg)(t+thy) . control animals were transfused but not treated (t). after days systemic blood was harvested to determine the circulating levels of il and tnf. a second group was treated with thy for days and then received a gavage wide lxl escherichia coli and a % burn injury (bg+thy). one hour post-bum, blood was collected to measure cytokins. control animals received the same treatment but thy (bg). a third group was transfused, treated with thy for days and then received gavage and burn (tbg+thy). one hour post-burn, blood was collected to measure cytokins. control animals received the same treatment but thy (tbg). all treated and control groups had mice/group. in separate groups (n= /group), receiving the same treatments, mortality was observed for days post-burn or transfusion. the production and release of oxygen radicals by phagocytic leukomytee is one of the most relevant and important functions of these substances in biology. when neutrophilic granulocytes or certain macrophages phagocytize they produce super oxide and hydrogen peroxide and form secondary products of these activated species. all of these substances are released in to the phagosome. it has been appreciated since that the amount of oxygen consumed by phagocytes in producing free radicals is prodigious. what has not been totally appreciated is the significant contribution this consumption contributes to total body oxygen consumption. our understanding of increased oxygen consumption following sepsis is compounded by a paradox of increased cardiac index and a narrow av difference. it was mclean in a study reported in that showed an increase in cardiac index and a narrowing of the avo z difference in humans following sepsis. other studies documented similar changes in humans and it has recently been speculated that increasing o~ delivery might have a favorable impact on outcome. at least three different explanations have been put forward as to why there are possible alterations in energy metabolism during sepsis. these include decreases in oxygen supply, peripheral shunts and direct action o~ cellular mediators on o~ delivery, a provocative review by hodgkiss and nark in shewed that in experimental animals sepsis did not cause any evidence of bioenergatic failure in muscle i liver, heart or brain tissue. they further showed that the increase in serum lactate is mot necessarily due to cellular hypoxia. they conclude that cellular hypoxia and defects in energy producing metabolites are not the cause of metabolic abnormalities in sepsis. we set out on a series of experiments to try to explain the paradox in cellular metabolism and whether or not white cells might contribute to total body oxygen consumption during sepsis. the first set of experiments involved growing rat cardiac myocytes on tissue culture. pyruvate was added to the medium as substrata and physiologic levels of peroxide were also added simultaneously, carbon labeled co~ production was measured as was nucleotides and degradation products from the cell. the peroxide induced a significant inhibition of pyruvate dehydrogenase. in addition, there was a loss of cellular atp and a corresponding rise in the release of inosine and adenine from the cell. we concluded from this first sst of experiments that physiologic levels of peroxide are a potent inhibitor of pyruvate dehydrogenase in isolated cardiac mitochendria as well as the cultured myocyte. we further showed that pyruvate dehydrogenase inhibition blocks the aerobic oxidation of glucose and leads te adenine nucleotide metabolism with adenosine and inosine release from the cell. other enzymes within the tca cycle did not appear to be specifically blocked by peroxide. this would explain the increase in lactate and the ability of the cell to continue to make high energy phosphate by entry of metabolites in to other entry points of the tca cycle, the second set of experimbnts was designed to estimate the magnitude of whole body reactive oxygen generation via nadph oxidase following granuloeyte activation invivo. using a guinea pig model baseline oxygen consumption was determined. ~iaphenyl iodinium (dpi) was used as a specific inhibitor of granulocyte nadph oxidase. animals were divided into two groups; those that received dpi and those that served as control, all animals were then injected with phorbcl myristate acetate (pma) intravenously. pma is a potent stimulus for granulocyte activation and subsequent reactive oxygen generation. whole body oxygen consumption measurements were then repeated after the p~la injection. in addition, arterial blood gas amalysis was performed, circulating neutrophils were collected and assayed for their ability to generate hydrogen peroxide and the r~ght lung was removed for wet and dry weight measurement. total body oxygen consumption increased by percent after pma injection. arterial oxygen tension fell significantly in the control animal. neutrophil hydrogen peroxide generation rate doubled and lung water in the untreated animals increased by percent. in this animal model we could show that granulocyte activation substantially increases whole body oxygen consumption to approximately percent of control values. the nadph oxidase inhibitor, dpi, decreases granulocyte hydrogen peroxide generation invivo and prevents the pulmonary injury induced by pma. using this animal modal it is possible to transpose this to the human septic state. if we assume there are , neutrophils per cubic millimeter of blood, a kg man would have ~ . xi u circulating neutrophils. when activated ixl ~ neutrophils generate one nanemole of hydrogen peroxide per minute. t~erefore, the circulating neutrophil pool could generate - . xi nanomoles of hydrogen peroxide per minute. this corresponds to an oxygen consumption of - . ml/ojmin by the circulating neutrophil pool alone. this does not take into account the production of oxygen by macrophages or the amount of oxygen consumed to produce nitric oxide. manipulation of oxygen delivery in human sepsis may or may not be beneficial. irshad h. chaudry, ping wang, and alfred ayala life threatening blood loss, due to soft tissue and bony injuries, is a frequent complication in trauma victims. although numerous organs and cells are adversely affected by hemorrhage, the focus here will be to discuss whether or not there are any differential alterations in splenic and hepatic immune functions. in particular, m~ antigen presentation (ap) and associated processes will be described since one of the important functions of the m is to activate resting t-cell lymphocytes by processing and presenting foreign antigens in a mhc class ii-restricted fashion. studies have shown that splenic m and kupffer cell (kc) ap was significantly depressed following hemorrhagic shock and remained so for at least h alter resuscitation. the presentation of antigenic fragments associated with mhc class ii antigen itself was not decreased following hemorrhage, but the catabolism of antigens in antigen-presenting cells was decreased. this is likely due to a significant loss of cellular atp. although m ap was depressed in splenic, as well as kc, only kc showed an enhanced capacity to produce inflammatory cytokines, il- , il- , and tnf. this difference in response between kc and splenic md may be due to differences in the microenvironment in which these cell populations are found, as well as potential differences in the effects that hemorrhage has on the environment from which these cells are obtained. splenic m from hemorrhage-resuscitated mice exhibited a significantly reduced cytotoxic response, whereas kc showed an enhanced cytotuxic capacity. the increased kc cytotoxicity is probably mediated through the increased expression of cell-associated tnf and the release of reactive nitrogen. the enhanced production of reactive nitrogen may play an important role in the clearance of microbes translocated from the gut following hemorrhage. however, excessive release of reactive nitrogen by kc may have deleterious effects on the adjacent hepatncytes. such an effect could, in part, explain the reduction in active hepatocellular function, which is seen following hemorrhage-resuscitation. thus, hemorrhage induces differential effects on splenic and kc m populations; it decreases splenic m but increases kc capacity to mount a cytotoxic response. the depressed splenic md and kc ap was, however, significantly improved by posttreatment of animals with atp-mgcia, chloroquine, diltiazem or interferon-'/. these agents also decreased the susceptibility to sepsis following hemorrhage. thus, the use of atp-mgci~, dfltiazem, chloroquine or interferon- offers new therapeutic modalities in the treatment of immunosuppression and for decreasing the susceptibility to sepsis, which is observed following severe blood loss. the production of macrophages from bone marrow precursor cells is a dynamic and highly regulated process. the differentiation of myeloid lineage-restricted progenitor cells is initially controlled by interleukin- , which drives the progenitors along a maturation pathway that leads to their response to specific lineagerestricted colony-stimulating factors(csfs) we have previously hypothesized that thermal injury can initiate a self perpetuating cycle of production of defective immune cells: thermal injury can initially affect circulating immune cells. then these cells, by unbalanced production of colony stimulating factors and other mediators, can cause a derangement of hematopoiesis resulting in an abnormal production profile of tnf, il-i, and il- and cytotoxic activity of bone marrow derived cells. the results of our studies so far have supported parts of this hypothesis. il- and il- +m-csf treatments increased the production of tnf in burn day macrophages and progenitor cells compared to normal cells. il- +m-csf+il- increased the production of il- by burn progenitor cells compared to normal macrophagee. preliminary results for the ratios of tnf/~ actin mrna indicate that il- +m-csf increased the mrna for tnf in the -day macrophages derived from burned animals compared to macrophages derived from normal animals. ratios of il- /~ actin mrna indicated that il- , il- +m-csf, and m-csf increased the il- ~rna in progenitor cells from burn animals compared to progenitor cells from normal animals. these preliminary results support our hypothesis that bone marrow derived macrophages and progenitor cells from burned animals act differently compared to cells from unburned animals regarding the production of inflanunatory cytokines. more specifically, the results suggest that the different cell types are regulated in different ways by cytokines, and cells from burned animals are regulated differently than cells from unburned animals. it i,~ to ~ssume that the translneatinn is due to a ixx~h,v functioning ~astroiutestlnal surface pro|ection system, which leads to an unacceptably high load ¢ln the imrsunc dcfcncc system, partlcul~ly the local system in the gastrointestinal wall, leading tu exh~ustiou of ihi~ system. tlae g&~lrointesliual i]'ac( can be regal'deal a.s a t~ servojf of appr m ~ separating i~ eucayotic ceils of the hosl ttom l ~ bacterial culls. in the human body the the myriad of cndngan¢nts micrix)rganistns, exogenous itl,~ttdittg micro~ganisms altd taxies m-'e sepia"areal li'otll lhe rest of the body by a simple epithclhd layer. the barrier function is heavily tlcpcnding tm =~ proleclive layer cmtsisting ie. surl~:emts, nluclts, probiotic bacteria, and ,~ub~tances with strong antioxid~lt, antipfotease al~d other i~rotective eft'ecru, the surfaet~ml htycr consists at tile besl if i(.) bimolecular layers, fl is thus very iltil~, the epilhelial layer is renewed every hours , and tile tanuwer of the mucus is ~so fa,~t, all attdphy of die epithelial layei induced by g&sttointestinal st~'vation does also include the mu~usprndocing cells, gnhlet cells, which leaduhg lu slmnage aad luwer quality of gi mucus, altered viscoelas,jc properties and reduced protection, ]~'obioiic bactcrla keeps the ppm's low, produce nutrients for the intestinal mllcosa, eliminates tuxia~ and stimulates the local immune system, the gl surface is conslanlly under hc wv wearing by ingested bacteria and their enzymes, ingested substaucas, chefftie,'ds at~d toxins. 'll~e n~tective flora is reduced in disease, by stress and by cm'clcss antibiotic therapy. wc have made attempts tn recondition the i,,.astroiniesli!l~d. tnucosa by rcstlpply of surfactants or sttrfactanfliko glyc~lipids, gels with pseudomuein [until.oil, and probiotl¢ laetobaeilli. by supply of glycolipids we have bt:cn able m prevent and heal intlammatory and tlleerallve injuries (and io p)'cveut microbe transhteatkln) in uppe~' and lower (. u'acl, this c~al be fu~'ther augmentented by shnultaueos supply el' a p.~uedomucin,likc gel like pectin. ()at contain~ one hundred times more ot membrane lipid$ th~a =nay other food, much of fiber, ~spceially watcrsnlnhlc betaglucaal~, at~d has an close to ideal amino acid cornpo~ition, <')at, termented by species-specific lactobacilli, has a strol~ ability to prevent local inflammatory and ulcerative chunges,transh)cadnn and sepsis in experimeut~d attimals mid to pl~ven~ revert mof in ba,nan.~.lt seems us, that it the mucos~lnucus/probiotic bacterial filnclinns cat'= %' restated by mucosa reconditioning, even in severely sick iudividuals) the hx:~d iuuuuue ~y,~teme will be capable tu pr~)tec! the ix~y ag~lls~ sepsis ~nd mof. the impaired immune response associated with multiple system organ failure (msof) has been most widely studied following surgery for inflammatory disease and trauma. the majority of late deaths following trauma are due to infection and msof. not all patients with multiple organ failure have concomitant infection present, yet most have been septic at some time in their hospital course. their generalized inflammatory condition often persists whether or not organisms have been recovered. immune failure probably precedes msof with or without overt infection and indeed may perpetuate such widespread sepsis. macrophage tumor necrosis factor-alpha (tnf) production is thought to represent an important pathogenic mechanism by which this is mediated. cecal ligation and puncture (clp) is a clinically relevant murine model of intra-abdominal infection and sepsis. serum tnf and endotoxin levels, and peritoneal macrophage tnf production are only slightly elevated in this model even in endotoxin sensitive animals. mortality in this model, therefore, does not appear to be attributed to overwhelming endotoxemia and/or tnf production. it has therefore been postulated that tnf and other cytokines may act in a paracrine fashion to cause local injury. tnf messenger rna (mrna) expression was therefore measured and found to be increased in the lung and liver following clp. a different pattern of expression was seen after endotoxin administration, characterized by a more rapid rise and fall, and enhanced tnf mrna expression in the spleen as well. route of spread of infection as well as the type of stimulus may determine the organ specific cytokine response. these data support the concept of locally produced tnf as a contributing factor in tissue damage and multiple organ failure during sepsis. the estimation of histamine's role in sirs changed over the years based on increased knowledge in shock pathogenesis, evidence later found to be faulty, and the discovery of other, more fashionable mediators. over the decades, the prevailing view has been that histamine is a noxious mediator contributing to the fatal outcome of shock. the analysis of experimental data on exogenously administered, endogenously released and/or newly formed histamine in septic/endotoxic shock suggests that histamine contributes to the early cardiovascular changes via hi-receptor vasoconstriction thus excerbating (directly and indirectly) the effects of catecholamines. in the later phase of septic shock (low out-put, high resistance states), however, it can be assumed that histamine exerts strong vasodilator and positive inotropic effects via h -receptors. these effects are considered beneficial in counteracting or attenuating the effects of sympathetic responses of catecholamines and other mediators. thus, a blockade of the early reaction with hi-antagonists and a stimulation of the h -receptors by h -agonists are worthwhile therapeutic approaches. shock produc~s ~ij alterations, an encrgy crisis and eventual c~ll damage, however, shock in itse|f do~ not lead freqoenfly to r~mote organ damage. in animal ~xpcrhnents and clinical expariez:ces in korea and vietnam, shock i~r s¢ was not associated with lung damage, renal failure or ards. the same is true with g.l bleeding. however, when shock is due to trauma or associated with tlssus injmy, then organ malfunction m~d damage is frequent. shock is bclleved to increase the frequency of infection, but clinical evidcnc~ for this is scant. hemonhsgio shock akme is an unusual o~orrenee dinica[ly. immune dysfolletion dons occur witl~ experhnental hypovolemic shock and in pailcnts with hypotcnsion or after receiving blood ~ansfosions. tbc most significant factor affecting reoorrcnce of resented ca,lorectal liver mclastasis was hypotensivc episodes during operation. blood transfosions depress immune function, improve transplant ~raft survival and increase ttll'rmr rec~r'tcnos ill co[eli and head and nc~k cancers. in experimental hemorrhagic shock, we found decreased response of [ymphocytes to mitogens, el:clad mixed lymphocyte reactilln and i[,- decreased. othe~.x hsvc found decreased macrophage and t and b ¢¢lt function, deci~ased re,ease of/l~i, increased poe v depressed macrophagc antigen presanlatiota, increased ien y, decreased il- , and , and shared mscrophags funclion with loss nr f and c b r~ccpto~s. this is inhibited by chlornqoine, ibuprofen, dihiazem, and atp. shock may activate ncutrophils contributing to ilappitlg in cspillarics, no refluw, increased adhesion, cytotoxicity and organ damage. this may be related to decreased clearance of bacteria after shock. t~lls shock, hypotcnsion and decreased microcirculatory blued flow initiate or set the stage for immune dysfmtction. they, along with tissue injury trigger inflammatory cascades. this also contributes to immunologic dysfonctitm, which is associated with increased lufoction. thus altered bh,od flow and mediator cascades are bolh involved, the question now is can wc rapidly improve microcirculatory blood flow, and dg'masa inflsrmnatory fosponss, which is also necessary for survival the effe~ of ~nterferon ~amma @n wour~ healing was ewaluated ~n a routine mo~el. ~mma ~n~erferon we g~ven in doses of ~ . to ~ , u~%$ synchronous with ereatio~ of a pa~splnal woumd then daily for is ~lays. ~mteet~on ~amma impaired tensile strength a% all ~oses relative to control. m zphology suggested ~reas~ ¢oll~gen deposition and r~du~-~ neovag~ulaeit¥ ~n t~eat~ animals, interferon ~amma was a so ~valuated in a murine mo~el of cecal llgatlon and punneure. one hundred to ,s uni~ vere ~iv.n following ~nju~ an~ than daily. interferon qamma was associated with inco:ea~ed mortalit~ and earlier death a~/a~n ~n a dose dependant manner (p< , ). afmlnistra~ion the proinfiammatory cytokines tumor necrosis factor-c~ (tnf-c , interleukin- (il-ii ), interleukin- (il- ), and interleukin- (il- ) have been implicated in the pathogenesis of the multiple organ dysfunction syndrome (mods) following shock and trauma. these mediators which are released in high concentrations by activated macrophages, endothelial cells, and connective tissue cells cause a systemic inflammatory response syndrome (sirs), thus leading to a shock-like state and tissue destruction. recently, a number of proteins have been characterized in in vitro and in vivo studies demonstrating potent anti-inflammatory properties. these latter cytokines include interleukin- (il- ), interleukin- (il= ), transforming growth factor- (tgf-j ), and the newly sequenced interleukin- (il-i ). they are considered antiinflammatory, since in vitro studies using purified macrophage cultures or whole blood assays revealed an inhibitory effect of these antmnfiammatory cytokines on ~he synthesis and release of tnf-cl and il-u . in addition, they reduced the severity of disease and reduce plasma levels of tnf-c~ and il-ii when administered to animals with infection or inflammation. furthermore, naturally occurring substances have been described that specifically neutralize the bioactivity of il- b and tnf-a. the il- receptor antagonist (il-i ra) is related structurally to il-i and binds to the same cell-surface receptors, but does not stimulate the cell. in animal models ore. cob-induced shock, inflammatory bowel disease, and peritonitis, injection of il-lra significantly reduced the severity of disease. the cytotoxicity of tnf-c~ can be inhibited through two types of soluble tnf receptors (stnfr) type i (p ) and type ii (p ). these stnfrs are proteolytic cleavage products of the cell-bound tnfreceptors. when given to baboons to combat e. coil-induced shock, soluble receptm's to the tnf type [ receptor decreased the severi~ of the shock-like state. it has been well accepted that shock and severe injury result in an increased release of proinflammatory cytokines with a high incidence of sirs and mods. our studies investigating plasma levels of anti-inflammatory mediators (il- , il-lra, stnfrs) in patients after severe injury further suggest that shock and injury lead to an activation of anti-inflammatory processes with altered plasma levels of these mediators. however, while plasma levels of il- and l-lra were significantly increased in trauma patients compared to healthy volunteers, stnfrs did not differ from control samples. thus, shock and severe injury result in different activation patterns of anti-inflammatory processes. m,l. rodrick, boston, b~usa following severe thermal injury significant suppression of the immune response has been demonstrsfed. these changes are associated with increased susceptibility not only to co,on human pathogens, but to organisms not normally pathogenic in the uncompromlsed host. early observations included prolonged graft rejection and skin test anergy in patients following burn injury. work from our laboratory and that of numerous others has shown that the~e is a profound £mmunoeuppresslon following severe thermal injury. i~mune deficiency has been identified in these patients hyz ii lack cf t nell responses to mitogens, ) early decreases in total t and helper t cells in the peripheral bloo~ } lack of response to tetanus toxoid i~uniza~io~ in spite of increased non-speoifi~ i~k~unoglobulin praduction and ) severe and prolongei deficiency of interleukin- (il- ) prcductlon by peripheral blood mononuclear cells (pbmo). this il- deficiency could be an underlyin~ cause of all the afo~ementloned immune defects by failing to activate both helper and suppressor t cell functions. mo~a recent work has focused on the molecular mechanisms of this il- deficiency end shown that the defect lies post-transcrlptionally since mrna for il- is also depressed following thermal injury. we have also investigated the potential of a defect in ii- receptor status on peripheral blood mononuclear uolls from patients fqllowing burn injury and in a murine model. in both oases no decrease in il- r was found either by phenotypic markers or by funational assays. another hallmark of thermal injury is hyperao~ivatlon of proinflammatory ~ytok~ne secretion, which may play a significant role in multiple organ failure following burn injury. therapy o~ major burn injury may reasonably be aimed, therefore, at up-regulating t cell ~unction and down-regulating hyperactive secretion of proieflaam~ntory cytokines. the majority of patients dying of bums succumb to sepsis which coincides with dysfunction in the specific and non-specific host defences. generally, concepts relating to the mechanism(s) of specific immune failure in thermal trauma imply that inhibition of t (cd +) cell responses is imposed by the injury or infection-related factors. however, the same factors elicit strong biological reactions within the lymphoid compartment. recent evidence indicates that systemic activation is inherent in the burn patient. to establish the relation of molecular and cellular events to the development of post-bum anergy we examined the state of and the capacity for t cell activation with regard to: ) occurrence of activation-induced cell death (aicd), ) activation-related phenotypic and functional_ diversity in the pool of peripheral cd + t cells. initially, (up to days post-bum), peripheral blood lympliocytes from severely injured (> % total body surface area) patients exhibited high levels of constitutive expression of surface receptor for ]l (cd ) and spontaneous blastogenesis. the presence of activation-related t cellproducts in bum plasma was also apparent. subsequent impairment of the t cell receptor (tcr)-regulated t cell responses in vitro was accompanied by significantly increased dna fragmentation that is associated with cell death by the mode of apoptosis. using molecular markers we established that flesh peripheral blood ceils from immunosuppressed patients also contain large numbers of apoptotic cells. fluctuations in the number of viable (pi-) peripheral blood lymphocytes involved primarily cd +/cd ro+ (memory) subset of t ceils. the above observations suggest that thermal trauma-associated t cell anergy develops through aicd, a phenomenon commonly associated with the tolerogenic activity of bacterial superantigens. persistence of staphylococcal infections in the burn patient may support this assumption. response following trauma jane shelby, ph.d. the immune system is integrated with other physiologic systems, and is exquisitely sensitive to changes in nervous and endocrine systems changes following traumatic stress challenge. the immune, nervous and endocrine systems interact via both direct and indirect pathways which utilize neuro and endocrine hormones, neurotransmitters, neurepeptides and immune cell products. it is now known that the immune system may be affected by all of the neuroendocrine products produced during a stress response, with evidence for innervation of iymphoid organs, lymphoid cell receptors for neuroendocdne products, and leukocyte production of chemicals which are virtually identical to certain neuroendocdne peptides (acth, endorphins). trauma induced alterations in the equilibrium of various neuropeptides and neuroendocdne hormones have a significant impact on immune response potential, affecting control of proliferation, differentiation and function of immune cells. for example, the neurohormone melatonin is thought to be a natural antagonist to counteract glucocorticeid associated immunosuppression resulting from stressful challenges, such as surgery and trauma, plasma melatonin levels are known to be significantly reduced in burn patients. the administration of exogenous me[atonin improved cellular immune response following burn injury in an animal model. melatonin was also shown to have in vivo cytokine regulatory activity, increasing the potential for il- secretion and downregulating excessive il- and ifn~ in burn injured, stress susceptible mice. the regulatory interactions between the immune, nervous and endocrine systems provide mechanistic pathways for trauma associated immune dysfunction. increased knowledge of these interactions will enhance the potential for the design of novei clinical interventions to improve immune response and decrease the risk for infection in trauma and surgical patients. . animals receiving e were given a single dose daily of either . g/kg of e in a % solution by garage (ge), or . g/kg of sterile ive in saline. four hours following the last dose, bum animals were subjected to a % body surface area bum injury to their dorsum. twentyfour hours following injury, the animals were sacrificed and spleen cells were harvested for assessment of lymphocyte function. splenocytes were prepared by mincing the spleen, followed by incubation on glass petri dishes to remove adherent macrophages. non-adherent cells were then tested for proliferative response to t-cell mitogen concanavalin a (con a) and b-cell mitogen lipopolysaccharide (lps). data were analyzed by anova. results: chronic alcohol exposure and burn injury independently inhibit lymphocyte response to con a but not to lps. the combination of e plus bum injury, however, pmfouedly decreases this response to both con a and lps as outlined in the this data clearly identifies the synergistic impairment of immune function produced by ethanol and bum injury. it is furthermore apparent that ibis effect is gut mediated and that gastrointestinal exposure to alcohol is necessary to produce this effect. further studies will work to identify cellular and subcellular mechanisms to explain this effect. in experimental animal studies and investigations on human volunteers endotoxin infusion is mgulary accompanied by the release of the cytokine tumor necrosis factor a (tnf-~) determined by elisa technique. in patients with menigococcal sepsis also elevated tnf-a values have been found using a functional assay. we have studied the role of tnf-et in surgical icu patients with sepsis. using functional technique, we were not able to detect tnf-~ activities in the patient plasmas. when this cytokine, however, was determined by immunochemicai technique (el sa) elevated tnf-e~ values where frequently oberserved. in order to further elucidate these observations, we studied shedding of tnf receptors in the patients. in these studies, we noticed that shedding of tnf receptors oecured regulary in the patients. at the time of diagnosis, soluble tnf receptor p and p were both - fold higher than values found in plasma samples obtained prior to die diagnosis of sepsis. we also observed that the sepsis patients revealed higher maximum values of p and p during the icu stay compared to values found in surgical icu patients without sepsis. these observations indicate that soluble tnf receptors are available in sufficient amounts to bind tnf-ot which is released in surgical patients developing sepsis. this mechanism may explain why functional tnf-c~ was not detected in the patients. institute for surgical research, rikshospitalet, the national hospital, university of oslo, oslo, norway. decker, d., sch ndorf, m., bidlingrnaier, f., hirner, a., yon rfcker, a. the advantage oflaparoscopic cholecystectomy over conventional open surgical approaches in the treatment of symptomatic cholelithiasis has been shown convincingly by clinical studies. in order to facilitate comparisons of different surgical approaches, we evaluated the cell biological characteristics of tissue trauma by measuring changes in various cell surface markers on leukocytes and eytokines in plasma as a possible means to assess tissue trauma in choleeystectomy. patients recruited into our study had experienced at least one typical bifiary colic, had ultrasound-proven cholelithiasis (stages -ii according to me sherry), were - years old, and presented for elective choleeysteetomy. patients could choose between laparoscopic and conventional eholeeystectomy after being informed about the advantages and disadvantages of each procedure. cell surface markers on leukoeytes were determined using whole blood techniques with the help of commercially available fluorescent monocloml antibodies and flow cytometry. shed cell surface markers in plasma and cytoldnes were measured with the help of sandwich-elisa kits. blood samples were drawn h before surgery, immediately before incision (after anaesthesia), h and h after incision. seventeen cell surface markers were examined on different cell populations and cellular subsets in laparoscopic and open-surgery patients. three soluble cell surface markers and six cytokines were monitored. by statistical analyses (multivariate regression analysis, student's t test, wilcoxommann-whituey's rank sum test) the six markers/cytekines that best distinguished open surgical from laparoscopic procedurea were determined. these were . the interleuldn- receptor and im soluble form (cd /scd ); . the activation antigen fd- and its soluble form (cd /scd ), a member of the nerve-growth-factor receptor family; . the cd ro epitope which characterizes t memory ceils; . the trausferrin receptor cd ; . the soluble adhesion molecule icam- ; and . the cytokines interieukin- and interleuldn- . on the basis of these results, a tissue trauma activation (tta) index was calculated by combining the marker/cytoldne measurements by simple multiplication. anaesthesia and pre-ineision maneuvers did not significantly change cell marker or cytokine levels in either surgical approach as compared to h before surgery. h after incision the tra index in open cholecystectomy showed a distinct - fold increase, whereas in laparoseopic surgery a mere - fold increase was noted. h after incision, the tra-index returned to near pre-surgery levels. in conclusion, our results demonstrate that changes in cell surface markers and cytokines can help evaluate the magnitude of tissue trauma in diffei'ent surgical approaches. the relationship between lymphocyte subpopulation changes after thermal injury and the increased susceptibility of burned patients to infection is unclear. in this study, we have attempted to correlate such subpopulation changes with the presence of infection in burned patients. peripberal blood from patients was monitored for lymphocyte subpopulation changes three times weekly for three weeks postburn and weekly thereafter for three additional weeks. mean bum size was . % (range %- %) of total body surface and mean age was years. infection was diagnosed by carefully defined clinical and laboratory criteria and its presence or absence noted each time blood was drawn. samples taken when patients had wound infection, bacteremia, or pneumonia were compared with samples taken in the absence of systemic infection. whole blood samples were stained with four monoclonal antibodies, the red blood cells lysed and the leukocytes fixed and analyzed by flow cytometry. for each patient sample, the proportion of lymphocytes falling within the light scatter gates was determined as the percentage of cells negative for cd and most strongly positive for cd . this percentage was used to correct each sample for the presence of debris or nonlymphocytic cells. the proportion of cd and cd positive cells was slightly greatc~ in the samples from infected patients, while the proportion of b cells (cd +) was unchanged and nk (cd +) cells were decreased by ahnos[ % compared to sampie~ li'om uuiuleclcd patients. the percentage of cells positive for cdilb (c~ integrin) decreased sharply and cd ro (memory cells) decreased slightly in samples from infected patients while the expression of the lymphocyte homing receptor and cd were unchanged. cd (il receptor) and cd (early activation marker) were significantly increased in the samples from the infected patients while hladr was unchanged. these changes in lymphocyte phenotype correlate with the presence of infection. if they closely precede or occur during the early development of infection they may be valuable clues to the mechanism of susceptibility following thermal injury. trauma patients are subjected to an immediate massive impact on their host defense integrity due to the combined effect of tissue trauma, shock and endotoxemia. cytoldnes are playing a crucial role within the course of an impaired cell mediated immune response (cmi) resulting from a disruption of intact m%/tcell interaction. the current study was undertaken to further elucidate the mechanisms of dysfimctional cmi following major burn and mechanical trauma -via comparative analysis of mrna expression and protein release. the major regulatory levels for different cytokines were determined in mitogen, respectively lps stimulated peripheral blood mononuclear cell (pbmc) cultures of trauma patients on consecutive days ( ) t, , , and post injury. we analyzed the cumulative data for interleukin- beta (il-i[ ), il- , il- as well as tumor necrosis factor alpha (tnf-~) and saw a considerable impairment of the protein release in the stimulated pbmc cultures until d post-trauma and recovery thereafter. *p < . , ** p < . vs control comparing the autoradiographies of the specific cytokine mrna expression with the protein release in the supernatants, we saw a good correlation between mrna signal intensity and protein synthesis for il- and ,- , suggesting that for these cytokines the main regulatory mechanisms are located at the pre-/transcriptional level. for the other cytokines investigated one has to suppose posttranseriptional mechanisms. the analysis of our data clearly indicates a severe impairment of forward regulatory immune mechanisms following trauma. most likely the regulatory mechanisms, that are involved are greatly different among the cytokines investigated. it may be concluded, that depressed cmi responses post-trauma are partly due to an impaired pro-inflammatory cytokine production. the severity of the injury (iss) correlated with the development at multiple organ failure (mof-score; r= . ). the levels of mediators and markers of the inflammatory response were generally higher in the more severely injured group (iss> , n= ). i - , - , g-csf, fpa, and c a -levels differed significantly (p< . ) between the iss-groups (>-< iss ) at the time of admission, whereas on day tnfa, c a, - , and ealpi showed significant differences. beyond the first week, major differences were restricted to pge and c a. the formation of two groups with respect to later multiple organ failure (mof < ; mof > n= ) yielded similar results. leukocyte-facs analysis revealed significant differences mainly in the cd (monocytes), cd /cd (i - r + t-cells), and cd /cd (th calls) populations. summarizing our findings we were able to detect some alterations in the surface antigens of immunocompetent cells. the inflammato d response, however, seemed to be more pronounced and correlates wi~ the further clinical course. using an experimental bum model in rodents, we have demonstrated that administration of a full thickness, scald burn involving % or more of the total body surface area (tbsa) elicits systemic responses which are characterized by numerous alterations in t-ceu function (i.e., lymphokine production and contact hypersensitivity (ch) responses) plus an enhanced susceptibility to bacterial infection. in the present study we questioned whether the apparent systemic effects mediated by large burns would be elicited as site-specific alterations in immune function following administration of small area burn trauma ( % tbsa). following a % tbsa burn, ch responses to contact sensitizing antigens were found to be altered. the depression in ch responses could be induced independent of the site used for topical skin sensitization. following a % tbsa thermal injury, development of ch responses were affected in a site-specific manner. immunization of % tbsa thermally injured mice in a site near the position of the burn resulted in depressed responsiveness, whereas immunization through a contralateral site resulted in responses that displayed both the intensity and kinetics of a ch response equivalent to sham-bumed mice. similar systemic and site-limited changes in lymphokine production were observed with % and % tbsa thermal injuries, respectively. a % tbsa injury affected the lymphokine producing potential of all cells regardless of which lymphoid tissue the cells were isolated from. the effect of a % tbsa burn was significant but site-specific. thus, ceils from lymph nodes receiving drainage from thermally injured tissue were specifically affected, whereas lymphokine production by cells from lymphoid organs receiving drainage from unaffected skin was normal. it was concluded that modulation of lymphokine production and cellular immune responses may be a normal consequence of burntrauma regardless of the size of the burn. changes in immune competence can be mediated either regionally or systemically in direct proportion to the area of skin exposed to the burn injury. this work is supported by phs grant gm and the office of navy research n - -j- . division of cell biology and immunology, department of pathology, university of utah school of medicine, salt lake city, ut . post spleneetomy septic sequelae may be fatal, but the mechanisms remain unclear. the objectives ef this study were to assess the mortality from concomitant splen-'etomy and ]~eritoneal bacterial challenge and to elucidate the local cetkdar responses. cd- mice were randomised to receive laparotomy and sham splenectomy (l) or splenectomy (s) with simultaneous ca'-cal ligation and "):mcture and the survival patterns assessed. subsequently, cd- mice were randomised into control (c), l or s groups and peritoneal cells studied at hours for bacterial phagocytosis and killi:~g, superoxide ( -) and tumour necrosis factor (tnf) production and macrophage activation vsing mac-i(cd- b) receptor in~.ensity expressed es mean channel of fluorescence (mcf). these resides indicate that sf!enectomy predisposes to nrortal~ty from bacterial sepsis ia the early pos~ operative period compared to sham operated animals. failure ~f p'.acrophages to kill bacteria in the splenectomv group '~:cured in t?~e absence of impairment of oxygen freeradical or tnf pred:~ctien. the macrovh~ge ac!ivotion marker mac- was significantly reduced in both l and s groups and impaired phagocytosis of bacteria oceured in both operative groups compared to controls. laparotomy a!one reduces macrophage activity in terms of surface re:eptor mac- expression and !ingestive capacity. splenectomy however s~gnificantiy ~mpairs r-acrophage-wediated l~,acterial killing and this qefect rttav co~tribut~ sig~ifjcav'ly to th-~ dissemination of local infection and to n':ortalit). depts of haem~ tology & surgery, beaumont hosoital, dub!in ,eire. introduction: loss of cell membrane integrity appears to be a common pathway of injury to tissues subjected to high-voltage electrical shock. the cell membrane is the most heat labile structure in the cell, and is also the most vulnerable to externally-imposed electrical forces. skeletal muscle and nerve cells are particularly susceptible to electroporation by clinically relevant electric fields. restoration of membrane integrity is essential for cell survival in victims of electrical shock. we have studied the effect of non-ionic triblock copolymers ( poloxamer class) on the transport properties of isolated rat skeletal muscle cells following electroporation-induced membrane disruption. - mm long adult skeletal muscle fibers were isolated by enzymatic digestion from the rat flexor digitorium brevus and maintained under standard culture conditions. they were loaded with the calcein-am dye and placed in a ,c chamber for recording by real-time video confocal microscopy. the cells were subjected to msec, v/era, a field pulses with a low duty cycle to allow thermal relaxation. peak temperature rise was , .c. the uye content of the cell was monitored in real time. experiments were carried out in calcium-free phosphate buffered saline, with mm mg%. experiments were repeated with mm neutral dextran ( the aim of the present paper is to ascertain if thuracotomy induces a different pattern of variations of cytokines, immunocompetent cells and antibodies from laparotomy in the early postoperative period. patients ( males females,mean age: . _+ ) with gallstone disease and with non neoplastic pulmonary disease were studied. none of these patients received blood transfusion, biological response modifiers, radiotherapy or surgery for at least months before being included in our study. anaesthetic procedures were similar in all patients and none were matnourished. on the day of surgery and on the st and th postoperative days (pre, lpo, po) percentages of cd , cd , cd , cds, cdi were measured by means of flow cytometry using moab., and levels of ig a, lgg, igm, ige. by nephelometry cytokine levels in peripheral blood(il- , il- , il- , il- , tnf) were measured in pts. of each group by means of elisa using moab. _r. esults:variations of il- and il- were not s.s.. il- increased but differences between groups were not statistically significant (s.s). il-i decreased on po and increased on po in both groups but were only s.s. in the th.g., and therefore, the differences between groups were s.s (p< . ).tnf decreased in the l.g. and increased in the th.g. on the po, the difference was s.s(p< . ); on po, tnf decreased in the l.g. and decreased in the th.g. but these variations were not s.s. cell percentages decreased an lpo and increased on po, except for %cd cell that increased on lpo and decreased on po ,in both groups of pts. differences were not s.s. ig a, igm decreased and ige increased in both groups (p< . i), but differences between them were not s.s. in contrast, igg decreased on po (p< . ) and increased on po in both groups, but the decrease iu the th.g. was greater than in the l.g. twenty male children,aged from six months to years,admitted for elective inguinal operation were studied. the operations were performed under balanced combined anaesthesia (fentanyl,thiopemtone,vecuronium, % nitrous oxide in oxygen) and blood samples were collected before flunitrazepam premedication,after anaesthesia, and hours after anaesthesia. cells from the wound were collected with cellstick sponge which was removed from the wound or hours after anaesthesia. the study was approved by the local ethical committee. the percentage of neutrophils was increased and that of lymphocytes was decreased in perpheral blood after the operation.the values in the wound were close to the values found in peripheral blood. the percentage of t-lymphocytes (cd ) and helper-t-cells (cd ) decreased in peripheral blood being lower in the wound than in peripheral blood after the operation. the percentage of t-eytotoxic cells (cd ) also decreased in peripheral blood and was similar to that in the wound. b-lymphocyte (cd ) percentage was increased in pe~pheral blood after the operation and was higher than in the wound. the percentage of activated t-cells (cd +hla-dr-positive cells) in peripheral blood increased while that of natural killer cells (cd +cd +leu -pos) was increased just after anaesthesia being decreased at g and hours after the operation. spontaneous lymphocyte proliferative responses didn't change while phytohemagglutinin a and concavalin a induced responses were decreased in peripheral blood samples hours after the operation with recovery at hours.pokeweed mitogen induced lymphocyte proliferative responses were decreased at hours (p<o. ) and hours after the operation we previously reported that plasma ige increased after traumatic injury. in this study, we measured plasma ige ievels in trauma patients on hospital admission and x weekly in the intensive care unit to evaluate ige kinetics. patient characteristics were: mean age -+ years ( m, f), mean injury severity score (iss) + , sepsis ( patients), sepsis syndrome ( patients), ards ( patients), renal failure ( patients). ige increased in patients ( %). the mean increase was ng/ml (range to ng/ml; % ci was to ng/ml). increase in plasma ige was significantly greater in patients with sepsis syndrome (p= . ) and renal failure (p< . ). although mean plasma ige was higher with ards, pneumonia, hepatic dysfunction, and shock, these differences were not significant (p> . ). plasma ige increase was not related to severity of injury by iss score (p = . ). the mean day to highest ige was . -+ . . the day sepsis was first observed preceded the day of highest ige by . + . days. there was a significant association between the day of sepsis onset and the day of highest ige (p= . ). eight of nine patients with sepsis syndrome had > % increase in plasma ige from admission. one patient's ige levels were normal ( - ng/ml) for days and then increased to ng/ml over the next days, after onset of sepsis syndrome. changes in ige plasma levels may reflect the action of cytokines, such as il- , which concurrently regulate production of ige and il- receptor antagonist in a response to sepsis. sepsis remains a leading cause of late mortality in trauma and hs. although hs-induced bacterial translocation is supposed to be the major cause of sepsis and mof, depression of the res increases susceptibility to infection after injury. the purposes of this study were: a) to evaluate the res in the lung, spleen and liver after hs and subsequent hypertonic saline (hsl) treatment, and b) to document the patterns of phagocytic activity in these organs during hrs. adult male wistar rats ( +_ gin) were submitted to hs (sbp tort) and after t hr (shock i hr) and hrs (shock hrs) hsl (nac . %, . ml/kg) treatment, e. coli (i ) was injected into the portal vein ~tci (n_> ). twenty minutes later, the lungs, spleen and liver were harvested and scintilographic counts obtained. data is depicted as mean_%+sem * p< . , ~" p< . and statistical analysis was performed by analysis of variance and wilcoxon tests. one hr after treatment, lung uptake was increased and liver and spleen uptake were reduced compared to sham. twenty four hrs after treatment, all organs, except lung uptake, returned to normal values. radioautographic histological analysis revealed radiolabeled particles inside phagocytic cells of all organs. we conclude that pulmonary phagocytic activity increases after hr of hs hsl reatment, diminishing by hrs although still above normal values. in contrast, res suppression occurs in liver and spleen after hr hs hsl treatment, returning to normal values by hrs. these results may explain lung complications and immunosuppression after trauma. infusion of endotoxin as well as major surgery is followed by lymphopenia in peripheral blood. the purpose of this study was to investigate to which tissues the lymphocytes are redistributed in response to endotoxaemia and major surgery. in addition changes in lymphocyte subpopulations and expression of mecii was measured. lymphocytes were isolated from peripheral blood of rabbits, labelled with indium-tropolene and reinjected intravenously into the rabbits, i rabbits received an infusion of escherichia coli endotoxin ~g/kg, while i rabbits were subjected to a major sham operation and i rabbits served as a control group. the redistribution of lymphocytes were imaged with af gamma camera, and calculated with an interfaces computer before, and , and hours after major surgery or infusion of endotoxin or saline. interleukin-l~ and serum cortisol were measured. in addition we followed cd , cd , cdlla/b, cdis, cd , cd , mhcii and cd /cd ratio. following endotoxaemia interleukin-lf~ increased significantly, following endotoxaemia as well as major surgery serum cortisol increased significantly. following major surgery as well as endotoxaemia there was significant lomphocytepenia in peripheral blood with a decreased cd /cd ratio while the cd positive subpopulation increased. in addition there was a decrease in the expression of mhcii on the lymphocytes peripheral blood. the radioactivity of the lymphatic tissue in and around the intestine increased to % of initial values following endotoxaemia and to % following major surgery. the results indicate that endotoxaemia as well as major surgery induces redistribution of lymphocytes from peripheral blood to lymphatic tissue. among the lymphocytes staying in peripheral blood there was a decreased expression of mhcii and a relative decrease in cd cells compared to cd positive lymphocytes. in order to analyze the effects of immune suppressive substances on expression of mrna of interleukin- (il- ) and interleukin- reeeptor(il- r), this study was carried out. twenty male rabbits with comminuted fracture were used in the study. ten ml blood were taken at , i, , , days after injury. the sera were tested for the effects on lymphocyte blastogenesis and induction of il- stimulated by concanavalin a(con a): the sera from the rabbits days after injury were analyzed with sds-page gel eleetrophoresis, and divided into three groups by ultrafiltration (ufpi ttk, kd,milipore; centricon- , kd,amicon), that are less than kd, between i and kd, and more than kd. each group of the substances also was tested for the expression of il- and il- r by the dot blot hybridization. the results showed that: i) all sera from the rabbits after injury had significant suppression on lymphocyte proliferation and secretion of il- by the con a-stimulated splenocyte in mice; ) the sera from the rabbits days after injury had more profound suppression than other injured sera; ) there was a marked band at about kd in sera from the rabbits days after injury, but nothing at the same position in normal sera analyzed with electrophoresis; ) the substance with molecular weight of about iokd had more obvious suppressive action on expression of mrna of il- and il- r than other groups substances, of which molecular weights are more than kd. it is concluded that: i) the sera from the injured rabbits can reduce immune response; ) there is kind of substance, of which molecular weight is about kd, it is probable the main factor involved in the pathogenesie of postinjury suppression immune; } the substance can depress the expression of mrna of both il- and il- r. research institute of surgery daping, chongqing, p. r. china acute ethanol uptake prior to injury modulates monocyte tnfo~, production and mononuclear cell apoptosis. g. szabo, b. verma, p. mandrekar, d. catalano monocytes (mo) have been shown to contribute to immunosuppression after both major injury and alcohol consumption. we reported that acute ethanol exposure of m( results in decreased antigen presentation, induces tgf- and pge while inhibiting inflammatory monokine production. we also showed that post-trauma immunosuppression is mediated by hyper-elevated mo tnfc~ and il- . consequently, here we investigated rnonokine production in trauma patients (n= ) who had elevated (>o.lmg/dl) or had no blood alcohol level (n=t ) at the time of emergency room admission. none of the patients had chronic alcohol use history. met tnfc~ production from trauma patients with prior alcohol uptake was undetectable during days - post-injury in contrast to patients without alcohol exposure. furthermore, decreased tnf~x levels were found in alcoholic patients' mci after mdp or ifny + mdp induction. however, mcl tnfc~ levels during the - days post injury period became higher in alcoholic trauma patients. furthermore, over days post-injury, alcoholic trauma patients showed significantly elevated mci tnfo~ production after adherence isolation, mdp, or ifn+mdp stimulation compared to patients without alcohol. these results suggest that acute ethanol uptake prior to injury decreases tnf(x inducibility in the early post-trauma period, but these patients' mo produce hyper-elevated tnfa levels later post-injury, thereby prolonging their cytokine shock risk. tnf ng/ml - days post-injury days post injury stimulus ale. pt. pt . . . . immunosuppression might also be increased by the elevated apoptotic activity found in trauma patients' mononuclear ceils, which was even greater in alcoholic trauma patients' cells. in non-alcoholic trauma patients' preactivated mo, in vitro acute ethanol ( - mm) exposure resulted in a significant down-regulation of tnfc~ (p< . ) and il- (p< . ) production. in contrast, in vitro ethanol exposure increased the production of inhibitory monokine, tgfi]. these results provide both in vivo and in vitro evidence for the effect of acute ethanol exposure increasing immunosuppression and cytokine shock. the 'systemic inflammatory response syndrome' (sirs) with consecutive septic multi-organ dysfunction represents the major cause of late death following major mechanical and burn trauma. systemic hyperinflammation and concurrent depression of cell mediated immune response (cmi) render the traumatized host anergic, resulting in profound susceptibility to opportunistic infection. monooytes/macrophages (mo) play a central role within the host defense system in developing and manifesting states of injury, shock and sepsis. the mechanistic scrutiny of the synthesis patterns of crucial cccytokines appears to be a helpful tool to further analyse mo behaviour in the compromised individual. the objective of this study was to further dissect the characteristics of cytokine regulation in pbmc under stressful conditions, via analysis of the expression of cd + receptor, the proinflammatory mediator il- , the macrophage activating factor ifn- ,, and neopterin (npt) a metabolite of activated mo. we investigated pbmc's on consecutive days , , , and after mechanical trauma of and after bum trauma of patients (mean age ~ years; mean iss ± pts). in trauma patients we saw a massive increase of pha induced neopterin synthesis compared to controls. however, when discriminating the npt levels in the supernatants for the amount of mo stimulated, the npt output of the individual cell was lower compared to mo of nontraumatized individuals. interestingly there was a contrary coarse in the cumulative protein release patterns of il- and ifn- in mechanical versus burn trauma patients. wheras in burn patients ifn-y was decreased significantly ( + u/ml) compared to controls ( + u/ml) as well as mechanical trauma ( + u/ml). il- showed a significant suppression following mechanical trauma ( + u/ml) vs control ( + u/ml) and bum patients. the rt~,na signal intensity for beth eytokines was in concurrence with the protein release in more than % of the individual patients investigated. from these data we can conclude that the inadequate low npt synthesis predominantly in bum patients appears to be a sign of cellular immaturity and is probably partly due to low t-cell ifno t signals. in addition we could state that the quality of trauma is apparently responsible for the different synthesis patterns of ]l- and ifn-q,. it has been postulated that bacterial invasion or endotoxemia are necessary for cytokine production following burn injury. we studied the organ distribution and kinetics pattern of il-fc~ (cell-associated il- agonist) in eutrophic rats subjected to either % tbsa cutaneous scald injury (bi), muscle scald injury of equivalent % tbsa (mbi), sham muscle bum (resection of skin only, up to % tbsa) (smbi), and sham cutaneous burn (sbi), followed by saline resuscitation ( mukg i.p.). separate rats were infused with mg/kg e.coli :b lps or saline lv. unmanipulated rats were baseline normal controls. liver, lung, spleen, ileum, thymus, kidney, skin, and plasma were harvested at various time-points within the first h. tissues were frozen, weighed, homogenized, the homogenates centrifuged and the supernates assayed with a radioimmunoassay specific for rat il-l(z (detection limit pg/rnl). il-lc~ was expressed as ng/g weight + sem (lowest detectable amount . ng/gwt). il-lo~ was constitutively present only in the skin ( + . ng/gwt). cutaneous burn and sham cutaneous bum induced biphasic elevations of il-lcc in the liver and lung only, with maximal levels at . h (in the liver, bi = . _+ . ng/gwt, sbi = . + . ng/gwt, p _< . ; in the lung, bi = . + . ng/gwt, sbi = . + . ng/gwt, p -< . ). of note, both bi and sbi rats had detectable il-i~ in the liver at timepoint already ( min real-time). these levels increased in parallel until min and became eventually different by log at - . h. all other organs as well as plasma were below detection limits. muscle burn injury and sham muscle burn (skin resection) induced similar elevations of il- ~ in the liver at lh, indistinguishable from each other and from cutaneous burn. in contrast, lps challenge induced dramatic elevation of il-t~ in all organs tested except for the kidney; the spleen was the most responsive organ to lps-induced il-lo~ production. these data indicate that thermal or mechanical injuries induce very early and organ specific production of il- c~ in vivo by mechanisms other than endotoxemia. injury-induced complement and platelet activation may be involved as well as the neuro-endocrine axis, which may explain the low levels of il-lo~ induction observed in all rats at the very early time-points. trauma services, massachusetts general hospital, and department of surgery, harvard medical school. fruit, st, boston, ma . j. f. schmand *#, a. ayala* and i. h. chaudry* studies indicate that i.v. infusion of the colloid hes in normal animals does not adversely affect non-specific immunity. it remains unknown, however, if lies affects cell mediated, specific immune functions after trauma and hemorrhage (hem). to study this, non-heparinized c h/hen mice underwent midline laparotomy to induce trauma and were then bled to and maintained at a bp of mmi-ig for rain. the animals were then resuscitated with either times (x) the shed blood vohune as lactated ringer's solution (lrs) or x lrs + lx % lies. sham mice were neither hemorrhaged nor resuscitated. at or hours post hem serum, peritoneal (pm~) and splenic macrophages (sm~) were obtained. bioassayes were employed to assess the levels of ii-l, il- ( alternatively pmqb showed no differences in il- release between all groups at and h, while sm~ from the lrs + hen group showed a depression at h. tnf production by pm~ was depressed in all groups at h and remained so in the lrs + hes group at h. sm~b showed decreased tnf release values in both hem groups at and h. in summary, the levels of inflammatory cytokines (particularly the values of circulating il- ) after trauma/hem are positively influenced by the administration of hes. this might be due to a protective effect on pmqb and sm~, but also on other cytokine producing cells, e.g. kupffer ceils. we conclude that hes is not only a safe, but also beneficial agent in the resuscitation of patients atler trauma/bemorrhagic shock. this study investigated endotoxemia and consecutlve immune response in patients with multiple trauma (median injury severity score = , ). blood samples.were collected shortly after injury and after , , , , s and l days. endotoxin was measured with limulus-amebocyte lysate test and the specific antibody content (sac) against endotoxins of the classes igg, igm and lga by elisa-technique. five antigens were used: lipopolysaccaride (lps) of e.coli (ec), lipid a of e.coli (la), lps of pseudomonas aerog. (pa), lps of vibrin cholerae (vc) and cx-hemolysin of staphylococcus anreus (oth). a nephelometer indicated the total concentrations of igg, igm and iga. differences were checked with wilcoxon-test and p< , s was considered significant. cross-reactivity was calculated with rank correlation coefficients. results: endotoxemia peaked shortly after injury ( - h) at , eki/ml (median), decreased thereafter to , eh/ml at day s and remained on this level. sac oflgmclass increased to all endotoxins and peaked at day revealing the lfighest level to la followed by pa (= % of la-sac), ec (= % of la-sac) and vc (= % of la-sac). lga antibodies increased as well but only slightly and not significant (exception: sac to la was elevated significantly at day ). igg antibodies increased similar to iga class only slightly and again only sac to la was significantly higher at day and . however sac to (xh of all ig-classes remained continuously on the same level troughout the observation time. correlation analysis revealed strong cross-reactivity (r> , ; p< , ) most often between antibodies of igm-elass ( %) followed by igaclass ( %) and lgg class ( %]. conclusions: multiple trauma is associated with temporary endotoxemia. endotoxins probably translocated from the gut cause specific increase of anti endotoxin antibodies in blood of the igm-class. endotoxins cause no increase of antibodies to gramposilave bacteria. igm antibodies are most unspecific. during cardio-pulmonary bypass, as well as postoperatively, high levels of endotoxin, interleukin- (ii- ) and c-reactive protein (crp) were measured in patients. i female and male, ageing from to with a median age of . blood sampling was done preoperatively, immediately after induction of anaesthesia, after thoracotomy, after cannulation of the aorta and right atrium after the first half of the reperfusion phase, after closure of the thorax, and hours after the operation and then every morning until the th postoperative day. blood was drawn into heparinized tubes (i iu/ml) which were free of endotoxin. crp levels were determined through the use of the behring nephelometer. - levels were measured by using commercially-available elisa test. the endotoxin level was determined by a chromogenic modification of the limulus amebocyte test. the statistical analysis was done using the wilcoxon ranks test and correlation analysis. a significant increase {p . ) in endotoxin plasma occurred during surgery, culminating in a peak (median value of . eu/m!) during reperfusicn. plasma levels of endotoxin continued to be slightly raised till the th day after surgery, whereas those of interleukin- rose at the end of the operation and were at their highest hours later (median value of . pg/ml). crp levels were also high postoperatively with a median value of mg/l, and were markedly raised on day ( mg/l). a definite, statistically significant correlation between the plasma levels of endotoxin and - during the operation was establisthed (p . ), leading us to conclude that the endotoxin liberated during cardiac surgery acts as the main trigger in the releasing of - , and thus induces the postoperative acute phase reaction. there was no evidence of a correlation between crp and endotoxin or - plasma levels. impaired immune function is well described following trauma and hemorrhagic shock (hs). prior studies have utilized peripheral blood or spleen cells to index immune function following hs. however, changes in mucosal immunity are not weii characterized in this setting. gut origin sepsis is thought to be an important cause of organ failure and death following trauma. a rodent model was utilized to allow comparison of mucosal-associated immune function vs, systemic compartments after hs. fischer rates underwent hs (map ± mm hg) for minutes followed by resuscitation with shed blood and lr. sham animals were instrumented only. rat tears were collected at and hours following hs for quantitation of slga by ria. animals were sacrificed at hours and spleen (spl), peripheral lymph nodes (pln), and mesenteric lymph nodes (mln) harvested for cell population analysis using flow cytometry and mitogen stimulation analysis. cell marker expression analysis revealed no changes in t or b ceil populations following hs. mitogen mucosal immune function appears relatively spared following hs. the mechanism(s) for this variability in immune function requires further investigation. we have found that transplantation of bone marrow in a hind-limb graft to syngeneic lethally irradiated recipient is followed not only by rapid repopulafion but also overpopulation of bone marrow cavities. the question arises whether this unexpected phenomenon could be the result of stimulation of stem cells by factors (cytokines) released from surgical wound at the site of anastomosis of graft with recipient. aim of the study was to investigate which tissues damaged during the procedure of limb transplantation may be a potential source of humoral factors accelerating in vivo bone marrow proliferation. methods. experiments were carried out on lew rats in groups. in group i, the hind limb was transplanted orthotopically to a syngeneic recipient; in group ii, sham operation was performed; in group iii, a four-cm long cutaneous wound was made on the dorsum; in group iv, limb skin was harvested, fragmented and implanted into peritoneal cavity; in group v, bm from femur and tibia was implanted intraperitoneally. bm, lymphoid tissues and blood were sampled and days later for cell concentration and phenotype evaluation. results. the yield of nucleated cells from tibia was on day in the control . + . , in group . + . , in group ii . + . , in group iii . + . , in group iv . _+ . , in group v . _+ . x ( ). the evident increase in bmc yield in all groups continued until day . increase in weight and total cell count of spleen and mesenteric lymph nodes in all but group iii was also found. no differences in percentage of maturing erythroid cells, but higher of mature myeloid cells and lower of lymphocytes were observed. conclusions. trauma of skin, muscles, and bone brought about an increase in bone marrow cellularity and acceleration of maturation of myeloid lineage. transplantation of bm ceils alone did not produce this effect. transplantation of bm in limb graft is a good model for studies of natural factors reaulatin~ bm hemormesis. this study sought to determine a relationship, if any, between the degree of hypochclesterolemia upon trauma patients' admission and their subsequent outcome. all blunt and penetrating trauma patients admitted to a level i facility from through , and who had serum cholesterol assayed during the first hrs were retrospectively studied for development of death or significant organ dysfunction. the mantel-kaenzel chisquared test was used to determine significance of data at the p< . level. results: trauma patients were admitted during the four-year period who had serum cholesterol assays performed in the first hrs. patients had cholesterol levels less than mg/dl; of these ( . %) died, ( . %) developed ards, ( . %) developed acute renal failure, and ( . %) developed multisystem organ dysfunction; hypocholesterolemia in these patients was not due to liver injury or massive fluid administration. the risk of death was times greater and risk of multi-organ failure times greater in this group than in those with a normal serum cholesterol (>if mg/dl; patients; p< . ). conclusions: admission serum cholesterol level in the trauma patient serves as a powerful marker for those at risk of subsequent organ failure or death. hypocholesterolemia in this setting may result from organ hypoperfusion and humeral mediator release. lung tissue contains many immunocompetent cells. resection, therefore, is expected to activate extensively inflammatory mediators such as pmn-elastase, pmstanoids and pteridines. in a prospective clinical study we compared patients (pts) undergoing either thomcotomy with or without lung tissue msectioh and tboracoscopic lung resection concerning activation of inflammatory response. material & methods: group a pts (n= ) had thoraantomy but no lung tissue injury; group b pts (n=ls) had thoracotomy and lung tissue resection due to benign diseases; group c (n= ) represents group b tissue resection but using a thomcoscopic procedure. the following parameters were determined pre-, peri-, and postoperatively: elastase and crp as indicators of activation of pmn-leukocytes and injury severity; prostacyclin (pgi ) and thromboxane (txa~) as parameters of lung endothelial response; prostaglandin f ~ (pgf~) and pgm representing pulmonaly metabolic activity; pge a and neopterin as proof of macmphage activation. statistics were performed using analysis of variance for repeated measures. results: group b pts revealed postoperatively an increase in crp (p< . ) indicating a higher injury severity in comparison to the thoracoscopic procedure (c). both, controls (a) and group c pts did not show pmn-activation, whereas group b demonstrated a reversible increase in elastase. surgical trauma caused in all groups a release of pgi z and txa which was more pronounced in c (p< . ) and most in b (p< . ). similar results were found for pge~ and pgf =. there was no activation of maerophages since neopterin did not increase. apparently, metabolic lung function was not impaired because there was no marked rise in pgm except in b (p< . vs. c). discussion: our results demonstrate that lung tissue injury aggravates the mediator release induced by thoracic traum. these mediators among others are able to increase capillary pressure and hence lung edema formation. impairment of lung function, however, seems dependent on the extent of the liberation. therefore, the maximal release reactions occured in group b and c after lung tissue resection, whereas the controls showed the highest levels immediately after the incision. we conclude that thoracoscopic procedures are superior in reducing the resection trauma per se and hence might prevent severe mediamr-induced (pulmonary/systemic) sequelae. in a prospective study we investigated patients using radiochemical method according to sch~dlich (s) and photometric method according to hoffmann (h). serum of severly traumatized patients was withdrawn directly after admission at our emergency room and in narrow time intervals during first hours after trauma. follow up control samples were taken daily until day ten. whereas no elevated pla-ca was found during first hours, a peak was regularly observed around day four. there was high correlation between pla-ca and iss (r= . , p<o.o ) except patients suffering from thoracic trauma, thoracic trauma {tt) provoked similar gons-score-and pla-ca-values compared to multiple trauma (mt) despite significantly lower iss: pla-ca (h) mt . + . tt , +_ , n.s. pla-ca(s) mt . +_ . tt . _+ . n.s. goris mt . - . tt . _+ . n.s. iss mt - tt - p< . we found delayed elevation of pla-ca after severe trauma, however detection of delayed pla-ca in the serum of traumatized patients cannot give reliable information about the exact role of pla in the inflammatory reaction after trauma as latest results show that pla is secreted early but bound at lipophilic sites of vascular membranes. hern~mdez m j, amiguet ja, monreal ji, vid~n jr, jim nez f j, l pez-vivanco g acute phase reactant proteins increment in serum of patients with traumatisms and inflammation has been previously reported. alpha- antitrypsin (aat), alpha- macroglobulin (amg) and ceruloplasmin (cp) are evaluated in the following of patients, males and females (mean age: years), with compound fractures. % of them were located in tibia and fibula. intercurrent infection developed in patients and internal fixation rejection in one. measurements were made by radial immunodiffusion on days , , , , , and . a control group of healthy volunteers was also evaluated. aat and cp showed increased values from day which kept elevated untill the end of the study. amg elevated from day . when infection developed, aat and cp values were even higher whereas amg values decreased. fixation rejection caused aat, amg and cp increment. aat and cp increment in non complicated fractures might be due to accompanying inflamation, by means of cytokines release and aprps hepatic synthesis induction. therefore, increment is higher when infection or rejection develop. amg behaviour at early stages of evolution is secondary to hiperconsumption, considering mean life shortening after proteases ligation. amg modifications in fixation rejection remain obscure. aprps increment modulates inflammation and bony callus formation by means their antyprotease and antioxidant properties. clinically, aprps might be useful parameters in determining complications onset in fractures evolution. hemorrhagic shock (hs) is a common complication of trauma, as well as some major surgical procedures. the alteration of the immune system by hs is thought to contribute to the increased septic morbidity and mortality seen in these patients. multiple mediators are released following hs. il- , il- , tnf, pge and tgf-b have all been implicated in these immune system changes. in vitro, pge causes many of the immune system changes following hs, yet the time course of pge release has not been well deliniated. the aim of this study was to determine the time course of pge release by peritoneal and splenic macrophages following hs. c h/hen mice were bled to a mean bp of + mm hg for minutes. the animals were resuscitated with the shed blood and normal saline. control animals were cannalated, but blood was not withdrawn. the animals were sacrificed at , , , , and hours following hs. peritoneal and splenic macrophages were harvested from each animal and cultured with lps for hours. the supernatants were collected and frozen until assayed. pge was assayed using an elisa (cayman chemical). results are shown below for the peritoneal macrophages: pge release by peritoneal macrophages was significantly elevated at , and hours over control. it was maximal at hours, but by hours it had started to decline. splenic macrophage pge release was much more variable. (data not shown.) there was less consistency between time points and no clear trend was seen. in conclusion, pge is significantly elevated at hours following hs, and reaches a peak at hours. pge release may be responsible for the immune system changes seen in the first few days following hs. splenic and peritoneal macrophages respond differently in their release of pge following hs; which may be due to differences in their milieu. there is an obvious association between the altered immunocompetence of patients following traumatic injury of various types and the increased riskto develop complications. the present investigation was undertaken in order to analyze the value of both neopterin (n) and c-reactive protein (crp) in monitoring disease course in patients with multiple trauma. we were interested to compare nconcentrations as marker of t-cell/macrophage activation with crp levels as indicator of the inflammatory acute-phase response daily serum concentrations(n and crp) were measured in patients ( male, female, mean age . ), admitted to our icu following serious trauma (mean iss= ) and in control subjects. the clinical course of each patient was evaluated with apacheii score according to knaus. mean stay in icu was . (range - ). patients didn't develop organ failure(nof-group), patients survived, developing mof (s,mof-group), patients died with mof (ns, mofgroup). three patients(one s and two ns) showed signs of septicemia and septic shock. the serum samples were measured for neopterin (immu-test-ria, henning-berlin) and for crp (immunoturbidimetric method) -at the university hospital for internal medicine, innsbruck, austria. the highest crp levels were measured h after trauma, but they were not accompanied by significant increase in n-values. we found significantly elevated n-concentrations from the days - . n and crp levels showed a parallel release peaks on the days - and the values discriminated significantly among the three outcome groups(the second crp peak is lower in comparison to the first posttrauma peak). by the ns-group we found constant and parallel increasing crp and n levels, reaching extremly high values - h and preceding clinical signs of mof and sepsis. the values increased dramatically before death. the serum levels by all control subjects were upper the normal limit for n and crp.. the parallel occured peak changes in n and crp concentrations showed a significant correlation with clinical status, using a disease activity score (apac h eli). our findings suggest that both-cellular and humoral mediated immune mechanisms are activated after multiple trauma and that simultaneous determination of n-crp may be of advantage as diagnostic and prognostic parameter in polytrauma-patient monitoring panel. it is apparent, that measurement of very high levels may be of value as a srong indicator in developing of mof or septic complications and thus offer the possibility for earlier therapeutic intervention. the modification of t-cell/macrophage and acute-phase responses may be potentially useful in the treatment of icu patients, resuscitated after severe trauma, and suggest that n-crp may be relevant indicator for testing experimental immunomodulating therapies. although several in vitro studies suggested the catalytic action of iron in oxygen free radical generation, few data are available concerning iron-metabolism following ischemia-reperfusion injury and hemorrhage in vivo. aim of the present studies was to evaluate iron metabolism following mild and severe hemorrhage in the presence and absence of intraperituneal hematoma. methods. male lewis rats ( g) divided in groups (observation time hrs): a: mild hemorrhage by sequential blood sampling ( . ml each)( , , , , , , , , hrs). b: a with hemoperitoneum (hp)( ml/ g), c: hemorrhagic shock: blood withdrawal of ml/ g over min and resuscitation with ringer's lactate ( x shed blood) and isogenic donor blood; d: c with hp. parameters: serum iron ([g/dl], ferrozin-method); fe labeling of erythrocytas (application of ci fe i.a. into a donor rat, days later bleeding of the rat and injection intraperitoneally in experimental rat; plasma transferrin ([g/ml], ria). t-tests and mann whitney. data are expressed as mean + sem. results. compared to baseline, mild and severe hemorrhage caused a significant increase in serum iron at and hrs, respectively ( + vs. + ; +_ vs. +_ ). hp reduced this increase ( +_ vs. + ; + vs. +- , p<. ). i-ip significantly increased hemoglobin between to hrs in mild ( . +_. vs. . +. ) and severe hemorrhage ( . +. vs. . +. ). in mild and severe hemorrhage comparable maximal resorption ( fe labeled erythrocytes in circulating blood) of intraperitoneal hematoma occured at hrs (mild: +_ vs. severe: +- counts/rain). hp increased survival-rate following hemorrhagic shock (no hp: / ; hp: / ). in mild hemorrhage no alterations of plasma transferrin concentrations. in shock group without hp, there was at hrs a significant higher transferrin level than with hp alone ( . +_. vs. . + . ). in conclusion, hemoperitoneum with hemorrhage prevents increase in plasma iron levels by increasing hemoglobin through resorption of erythrocytes and thereby suppressing iron mobilization from endogenous iron resources, e.g. liver. this suggests that intraperitoneal hematoma not necessarily promotes iron-mediated oxygen free radical production, especially since survival in these groups was improved. increased transfarrin levels in group c at the end of the experiment suggest increased iron turnover which was not observed in shock groups associated with hemoperitoneum. primary immune response to thymus-dependent (srbc) and thymus-independent (vi-polysaccharide) antigens was determined during days after cct. antigenic challenge was made on day i, , or . number of spleen plaque forming cells (pfc) and serum haemagglutinin (ha) titre were studied on day after immunization. cct stimulated immune response to t-dependent, but not to t-independent antigen. enhanced response was noted when srbs were given in posttraumatic day i, it was higher than preceding one after injection on day and reached a maximum when antigen was presented on day after trauma (pfc number . times exceeded the level of immunized, but nontraumatized rats). the rise of pfc number in spleen correlated with increased ha level. thymectomy had been performed days before cct completely abolished posttraumatio stimulation of immune response to t-dependent antigen. standard thoracotomy also enhanced humoral response when srbc were administered on day after the operation. thymus trauma modeled by pincett squeezing of its right lobe and performed at the same time with thoracotomy fully abrogated as well as thymectomy stimulation of immune reaction. thus, different experimental chest traumas are able to increase antibody production via thymus-dependent mechanisms. endothelin is a amino acid peptide produced by ischemic or injured endothelial cells. in vitro and in animal studies, et is also a potent constrictor for renal mesangial and coronary vessels, a negative cardiac inotrope and an endocrine regulator. our lab has shown that et is an agonist for monocyte production of interleukins i, & , prostaglandin e (pge ) , and substances which trigger superoxide production. systemic et levels increase in hypoperfused and septic patients, and et may be yet another important cytokine in critical illness. but while et has been shown to be a potent vasoconstrictor in healthy dogs, systemic vascular resistance does not increase in critically ill patients with high et levels. (we hypothesize that this might be due to pgez-mediated vasodilation predominating over et-mediated vasoconstriction.) we next asked what happened to systemic et levels in patients with major burns (> %.) ten hemodynamically stable patients resuscitated by a modified parkland formula to a urine output > cc's per hour had et levels drawn on admission, at i, , , and hrs. et levels were measured by radioimmunoassay. mean levels were elevated at ± pg/ml at all time points versus levels in healthy controls of ± . in summary, systemic et levels increase significantly in patients with major burns. et may be yet another cytokine playing a significant role in the immune, inflammatory and multiorgan dysfunction observed with major burns. restoration processes in an organism after ischemic damage are realized through ~n~lammatory mechanisms~ the intensity of which is significantly defined by blood levels of neuropeptides. myocardial infarction (mi) was chosen for studyin these processes since it eradicates the influence of infectious factc~rs. dogs~ in whom mi underwent different forms o¢ healer, g; bhn~ed ~h~t during the acute phase of the disease there was a characteristic rise of ne!~ropeptides in the blood. these neuropeptides had nociceptive and antinociceptive effects. particularly substance p and -endorphins triggered off the development of compensatory and adaptive mechanisms and defined the intensity of inflammatory reaction at the zone of ischem~t: damage-notable fall in substance p levels after an ~nitial increase, while the ~-endorphins stayed high was an important condition for non complicated healing of mi. on the other hand high levels of substance p with low ~-endorphin concentrations lead to increased infiltration o~ neutrophils into the infarction zone and weakened the activity of synthetic processes~ thereby leading to left ventricular aneurysm. at the same time low intitial levels of substance p slowed down the development of necrotic processes which lead to delay in refunctioning of the heart and complicated the healing process. thus, regulation of the levels of neuropeptides in the blood in trauma forms a perspective method of its treatment. of laparascopic versus open choleocystectomy c. schinkel, s. zimmer, v. lange, d. fuchs, e. faist the impairment of immune function due to surgical trauma may be followed by deleterious septic sequelae. compared to open abdominal surgical procedures (lap), laparaseopic surgery (lsc) is associated with a decrease in hospital stay and in accelerated patient recover. the aim of the study was to evaluate the sensitivity of the immune sermn parameters of il- , saa and neopterin, the percentage of cd + cells, the in-vitro il- synthesis after mitogen stimulation and lymphocyte proliferation, in order to purposefully discriminate differences in the severity of trauma. we investigated the blood of patients with cholecystolithiasis undergoing either laparascopic ( ) or open (i ) cholecystectomy on consecutive perioperative days - , , and . there was no significant difference between the two groups concerning age and sex. patients with clinical signs of acute cholecystitis were excluded from the study. operation time and hospital stay were obviously longer in lap patients ( versus minutes, versus days) compared to the lsc group. concerning the unspecific acute phase reaction we could show no difference in the increment of senun amyoid a (saa) synthesis in the lsc group (d-i + lng/ml, d + ng/ml) versus lap group (d- + ng/ml, d + ng/ml), while in serum il- levels we saw a less steep increment in the lsc group ( -fold from d- to d ) compared to the lap group ( -fold from d- to d ). the analysis of cd + receptor expression and serum neopterin did not reveal any difference between the groups. lymphocyte function showed an impairment of proliferation to antigen stimulation in lap (d - : . + . cpm, d : . + . cpm) compared to the lsc group (d -h . + . cpm, d h . + . cpm). in both groups il- synthesis was decreased post-operatively. our data indicate that laparascopic cholecystectomy reusults in a less distinct unspecific acute phase reaction post-trauma compared to that following lap. neopterin serum levels and cd receptor expression show that these parameters apparently are less useful markers to detect differences of surgical trauma severity while it appears that the impact of lap is reflected most impressively on the lymphocyte compartment. trauma alters the host resistance of organism and is accompained by appearence of excgenic and endogenic proteins in the body. to understand the molecular mechanisms of host resistans disorders in trauma, as a first step, the genetic regulatory mechanisms of immune response after antigen injection has been studed. the appearence of specific protein factors ( - and kda), in the nucleus of rat splenic and brain cells, accordingly, was shown after immunization with sheep erythrocytes. the stimulatory effect of these factors on the il- mrna and il- production was detected. the nucleotide sequences of the human il- gene regulatory region bounding by the splenic nuclear proteins were determined between + - b.p. the il- trans-factors shows the affinity to splenic and thymic lymphocytes in vitro. thus, the antigen causes the appearence of specific protein factors in the cells,which act on the gene level,stimulate il- production and the host resistance. these results cause the next step of experiments using the same model, but after trauma. these investigations will let us verify the hypothesis that the protein il- gene trans-factors may play a definite role in the decrease of the cell immune responce after trauma. confronted with the routine procedure of prophylactic treatment of candidates for surgery in a rural african hospital, we initiated studies on the fre'quency of post-surgical malaria. in tanzania non-pregnant patients from rural areas were followed. of preoperative patients % had a parasitaemia and those maintaining it showed no increase or complaints. nine percent of patients without detectable parasitaemia before surgery came down afterwards and one-third had malaria-like complaints. spinal and general anaesthesia were equally applied in these last patients. in burkina faso we studied patients of which % had a parasitaemia on admission and % had postoperative malaria. half of the surgical patients came from rural areas, whilst only % of those with malaria lived in the city (with much less exposure and immunity). % underwent major surgery and % minor. bloodtransfusions ( % with parasites) never evoked a parasitaemia in recipients. post-surgical malaria is thus a reality in about % of the adult cases, both in east and west africa. surgery evokes a cascade of factors, varying from cortison to interleukines and acute phase proteins; immune responses may temporarily be suppressed. clinical attacks of malaria in otherwise immunes could be evoked by one of these factors. though malaria can easily be cured, the differential diagnosis is difficult because of post-surgery fevers; we found that % was treated without justified indication. the involvement of "student-doctors" a. this study examines glucose uptake and hexose monophosphate (i~ip) shunt activity in normal human peripheral lymphocytes and polymorphonuclear leukocytes (pmn). glucose uptake was determined by measurir,g the uptake of tritiated deoxyglucose, a non-metabolized glucose analogue. adsorption of co derived from [i- c] glucose was used to determine knp shunt activity. in vitro assays were carried out in hormone concentrations approximating normal and elevated trauma blood levels. (normal -cortisol . ~g/ml, glucagon #g/m , epinephrine ~g/ml, insulin t~u/ml; traumaeortisol . ~g/ml, glucagon /*g/ml, epinephrine ~g/ml, insulin ~ij/ml. analysis of twenty subjects showed a reduction of ° ~mp shunt activity by lymphoeytes and a ] % reduction in glucose uptake by p~n in normal vs. trauma hontc,nes p < . . lymphocyte glucose uptake was also reduced by trauma hormones p~ . . it ha~ be.ea~ suggested thgt idiopatno pulmonary fibrous (y.pf) [s a consequence of severe alveolar epithelial injury and is associated with an nveolar irnammamry reactio~ and the presence f.neutr phils. there~bre, neutr pk~ chemoattra~ant~ are probably important in the genegs oft.he infial lesions of ipf. the obse,"wson that stimulated macrophages are or~n histologically promin~t in fibmfio [-~gs ~.nd am capable of p~oducmg a v~dery f flbrogenic pep'ides also a~gues for their role ~n the pathogenic prc~e~ oflpf. the observation that stimume~ maerophages ere often histologica[iy prominent in fibrotio lungs and ~re ~pable of producing a varie~, offibroge.~e peptide~ also argues for tkek role in the pathogenic process, therefore, we ha-~e tested the potentn for iater!eukln- (i ..- ) and mo~tocyte chemotacde pop, de (x¢cp- ) to induce neutro~hil ~d mononuclear phagocyte accumuhdon in lungs of pafient~ with pulmonary .~r~idosis and i~f. brenet~o.alveolar lavabo (bal) fluids from ipf and sar~qidosis patient were conexntratea by reversed-phase chromatography, ~d ii. arid mcp-i asso.~ed by ells& ehemotaxis mad enzyme-reieasing ~ssas's on msnocyte~ and neatrophiis. elisa revealed significenfly elevated b al-eoneentrations o£mcp-i ( . ng]mg aibumm) in purisms with p~monary sarcoidodis artd in ipf ( . ng!mg) in comparises to . normal individuals ( . ng/mg) and to patients w~th obreic bronentis (cb) (~, rig/rag). similarly, chemota*dc ac~a~' for monocles (mcp- e.qu/va]ent) was strongly increased in sareoidosis ( . ngjmg) as well as ~n f pag,nts ( . ng/mg). norra.al indlvidu~s and cb patiants hzd a . or -fold lower ~cn%i~y, re~peefively. patients with ipf and sarcoidosi~ also h~l eievated il- ievei~ ( . and . rig/rag, respe~veiy; nomzls: . rig/rag; cb: . ng/mg) mad nvatropmi ohemotax~ ( . ~'~d . nnmg, res!z~ztiveiy; aormals: . ng,'mg; cb: l ngmg). these data suggest that increased ievels of born mcp. ~d il- may be oharacted~tie for ~arcoidosis or ipf_ it appears iikely that both ehernoattraetants ~ontribute to the influx ofmonocytes and neutrophils into the pulmonary alveoius and interstit~um in these dlsea~es. we have recently shown that the combined administration of noninjurious doses of lps and paf in the rat produce ards-like lung injury characterized by neutrophil adhesion to lung capillary venules, neutrophil accumulation in lung parenchyma, pulmonary edema, and increased protein and neutrophil count in bal fluid. this new paradigm of lung injury was associated with elevated serum tnfc~ and pretreatment with anti tnfa mab dose-dependently prevented these responses. also, the combined administration of lps and paf induced lung mrna levels of tnfe~ ( fold vs. lps or paf alone), ll-lg ( fold), kc ( fold) and il- . taken together, these data suggest that this new paradigm of lung injury is cytokinemediated and that lps/paf in vivo can functionally couple to the activation of gone expression of a multi-cytokine network system, all of which may be involved in the pathogenesis of ards. materials and methods. the sheep model included hemorrhagic shock and closed femoral nailing at day , hourly injections of e. coli endotoxin and zymosan-activated autologous plasma at clays - and further observation and measurements at days - . from venous blood and bronchoalveolar lavage(bal)fluid of ten merino sheep (mean weight kg) neutrophil counts ( e pmn/ml blood or epithelial lining fluid-elf-), the elf/ plasma ratio of albumin (r), and the zymosan-induced (stim) and non-induced (spont) chemiluminescence response (cl) of blood ( e cpm/ , pmn), and of blood-and bal-isolated pmn ( e cpm/ , pmn) were measured. for statistical calculations the wilcoxon test was used. data of the changes in polymorphonucleur leukocyte (pivinl) metabolism have been suggested to play a pivotal part in the post-traumatic systemic inflammatory response syndrome. the underlying cellular mechanisms which control this response are not yet completely understood. since the 'ca + second messenger'-system has been shown to be involved in regulation of pmnl-'respiratory burst', we investigated changes in pmnl-ca z÷ regulation in relation to oxygen free radical mediated injury. methods. in polytranmatized patients (mean injury severity score = ) arterial and venous blood samples during days. daily evaluation of horowitz-quotiant (po /fio ), plasma lactate (mg/dl) and body temperature ( results. body temperature peaked at day and (day : +. ; day : . +. ). plasma lactate was significantly increased at day l ( + ) and day ( . + ). hurowitz-quotient (day : + ) was low at day ( + ) and day to ( + )(p<. ). at day a substantial rise in venous pmnl-superoxide production (day : . +_. , day : . +. , day : . +_. ), oecured with significant increase in plasma lipid peroxidation (day : . + . ; day : . + . ). pivin~-myeloperoxidase activity was high at day ( . +--. ) and then continuously declined (day : . +. ). plasma antiexidant activity (glutathione pemxidase) was reduced by % at day (day : . +. ; day : . +_. ; day : . +. ). whereas basal ca + concentration remained unchanged (day : +_ , day : +_ ), fmlp-stimulated cytosolic ca + mobilization increased at day (day : + , day : , day : + ). conclusion. the present study in polytraumatized patients shows, that seven days after injury the agonist-induced pmnl ca + mobilization is significantly enhanced. at the same time, pmnl-oxygen free radical release and phagocytotic activity, systemic fever response and lactate concentrations were maximal. these observations were accompanied by post-tranmatic respiratory failure and in some patients by clinical signs of multiple organ failure. preliminary data from an ongoing study using hes-and dextran-infusions in these patients show attenuation of this inflammatory response. stefan rose, m.d., trauma surgery, univ. of saarland, homburg/saar donnelly sc, haslett c, dransfield i, robertson ce, grant is, carter c, ross ja, tedder tf. dept's of respiratory medicine, accident & emergency, intensive care, surgery, university of edinburgh, scotland and dept. tumor immunology, dana farber cancer institute, boston. the selectins are a family of adhesion molecules (l-selectin, e-selectin, pselectin), all of whom are implicated in inflammatory cell transendothelial migration. they, as a family can be proteolytieally cleaved from their parent cell and exist in a soluble form within the circulation. ards is a disease state in whic neutrophils and neutrophil transendotheliat migration have been implicated. in this study we wished to investigate whether the levels of these circulating soluble receptors from patients at-risk of ards at initial hospital presentation, correlated with subsequent ards progression. eighty-two patients were enrolled (pancreatitis (n= ), perforated bowel (n= ), and multiple trauma (n= )), of whom progressed to ards. assays for soluble l,p & e-selectin were performed on collected plasma samples via a sandwich elisa. (ns = not significant, **** = p<o. ) we have found a highly significant inverse correlation between a low circulating soluble l-selectin (and not soluble e & p-selectin) and subsequent ards. these results further our understanding of neutrophilendothelial interactions in the early stages of ards pathogenesis and offer the promise of sl-selectin in combination with other markers of inflammatory events being used to identify individuals at particularly high risk of ards progression on initial hospital presentation. it has been suggested that polymorphonuclear leukocytes aggregate in the lung capillaries of patients developing the adult respiratory distress syndrome (ards) and account for the endothelial damage while releasing toxic metabo-iites such as o.a. free oxygen radicals. among many antioxidants which have been investigated in in vitro systems and in animal models, n-acetylcysteine (nac) has been established to be useful as a free radical scavenger in vivo. to assess the influence of n-acetylcysteine (nac) on the activation of neutrophils in patients undergoing cardiopulmonary bypass (cpb) surgery with extracorporeal circulation (ecc), we evaluated the plasma levels of elastase and myeloperoxidase (mpo) throughout the operation and up to hours after surgery. four hours after surgery also a bronchoalveolar lavage was performed. a spectrophotometric method was used for the detection of plasma elastase activity and mpo levels were measured using a radioimmunoassay. we found that pretreatment with intravenous nac prevented the increase in elastase activity ( _+ pmol/i vs _+ ; p - . ), but not the release of neutrophil related mediators ( . _+ . ng/i vs . _+ . ; p= . ). besides, nac had the capability to prevent the enhancement of neutrophil numbers in lavage fluid as is normally seen during cpb ( . + . % vs . _+ . ; p = . ). although not significant, nac prevented also the increase in elastase levels in lavage fluid ( . + . pmol/i vs . + . ; p = . ). it is concluded that nac is able to protect against the inactivation of a -proteinase inhibitor, the main inhibitor of elastase activity, and that nac interferes with adhesion and migration of neutrophils. therefore nac may be useful in the prevention of tissue damage. this study is supported by inpharzam belgium. sodium nitroprnsside (snp) has previously been shown to attenuate the neutrophil induced lung injury associated with limb ischaemia and repeffusion. glyceryl trinitrate (gtn), already widely used in aortic surgery, also increases nitric oxide but has a less marked splanchnic "steal" effect. we examined the effect of gtn on lung injury, neutrophil infiltration and activation in a model of aortic occlusion ( rain) and repeffusion ( min). sprague dawley rats were randomized into: control (n=i ); ischaemia repeffusion (ir) (n= ); and ir treated with gtn ( ug/kg/min) during repeffusion (n= ). myeloperoxidase activity (mpo) measured pulmonary neutrophil influx. pulmonary endothelial permeability was measured by wet to dry weight ratio (w/d), broncboalveolar lavage protein (bal) and neutrophil counts (bal pmn). neutrophil superoxide release (mean channel fluorescence mcf) was measured by flow cytometry in a further ir v gtn experiment (n= per group). control _+t "* _+ _+ ** bal pmn/mm l+- " + # _~ *p< . , #p< . v control/gtn;**p< . v ir. students t test the significant increase in mpo activity produced by ir was attenuated by gtn. the increase in pulmonary microvascular leakage after reperfusion was also prevented by gtn. neutrophil superoxide release increased significantly from . + . to . _+ . mcf after minutes repeffusion (p<. ). this increase in superoxide was prevented in the gtn treated group. these results indicate that gtn inhibits neutropbil superoxide release during limb reperfusion, thus preventing pulmonary vascular leakage and neutrophil infiltration. these data suggest that the beneficial effects of gtn in aortic surgery may be due in part to a protective effect onpulmonary microvasculature. department of surgery, beaumont hospital, dublin , ireland. lipton adult respiratory distress syndrome (ards) is marked by increased vascular permeability of the lung to white blood ceils (wbc). indeed, influx of wbc into the lung is believed to be the central mechanism in acute lung injury of ards. evidence is developing that the neuropeptide c~-melanocyte stimulating hormone (c~-msh), a proopiomelanocortin derivative, inhibits inflammatory reactions in animai models of inflammatory responses in humans. to test the influence of a-msh on experimental ards, the peptide was administered to rats after intratraeheal infusion of endotoxin (s. typhosa, #g in . ml saline). three groups (n= each) received: intratracheai infusion of endotoxin plus ip injections of o~-msh ( ~tg in . ml saline) at , , and hr post-endotoxin treatment; intratracheal endotoxin infusion plus saline injections; control intratracheal saline infusion plus saline injections. the bal data showed overall differences among groups (f , = . , p<. ), o~-msh treatment inhibited endotoxin-induced wbc migration into the pulmonary tree (p<. ). circulating wbc counts were markedly lower in both groups given endoroxin than in the control group (p<. ), but there was no difference in wbc count between these two endotoxin-treated groups. the results indicate that c~-msh can reduce wbc migration in experimental lung injury. in further experiments we tested the hypothesis that c~-msh, administered alone or in combination with an inhibitor of gram-negative bacterial growth, increases survival in a model of peritonitis/endotoxemia/septie shock. cecal ligation and puncture were performed under sodium pemobarbital anesthesia ( mg/kg, i.p.) in female balb/c mice ( - g) that were then randomly assigned ( - mice per group) to receive at time and hr later: control saline thjectioas (i.p.), injections of ~-msh ( ~tg), gentamicin sulfate (i mg/kg), or both ~-msh and gentamicin. one ml of normal saline was given s.c. to each animal for fluid replacement. both ix-msh and gentamicin treatments administered singly improved survival; when given in combination survival was even greater these findings suggest that the anticytokine ot -msh molecule might be useful for treatment of systemic inflammation, perhaps particularly when used in combination with agents that inhibit bacterial growth. previous studies showed a close relationship between xanthine oxidase activation, membrane damage and postischemic cardio-respiratory failure following aortic clamping and reperfusion. aim of the present study was to evaluate alterations of polymorphonuclear leukocyte (pmnl)-metabolism and signal lransduction systems during ischemia/reperfusion induced by aortic clamping in man. methods, in patients ( female, + years) with elective reconstruction of infrarenal aortic anearysms, arterial (a) and venous (v) blood samples were obtained before clamping and declamping, and min after declamping. arterial pla concentrations were higher than venous ( + vs. + ). while cytosolic basal pmnl ca + concenlxation was not altered at the end of ischemia (v: . + ; a: _+ ) and during reperfusion (v: + ; a: + ), fmlp-stimulated cytosolic ca + mobilization showed a significant arterial increase as compared to venous ( _+ vs. + , p < . ). these presented data indicate that the reperfusion syndrome after infrarenal aortic clamping is characterized by ) pulmonary membrane damage possibly due to pmnl-degranulation, ) activation of pmnl-superoxide radical production with release of activated pmnl in arterial circulation, and ) a significant arterial increase of pmnl cytosolic ca + mobilization after fmlp-stimulation parallel to pmnl-activation. in conclusion, ischemia/reperfusion in man induces pulmonary damage and activation of pmnl superoxide production possibly due to an altered pmnl-ca + messenger system by inflammatory mediators (e,g. ii- , tnf, paf). the septic lung diseases (i.e. ards) generally show distinct alveolar damage and surfactant disturbances. it is thought that alveolar macrophages are involved in specific release products like h and cytokines like tnf. in this pathway the type ii alveolar epithelial cell (p cell) is not only an important target, but as recently shown it is also capable of producing h . the aim of this study was to investigate the influence of endotoxin on h release of p cells. we obtained p cells from lungs of female wistar rats and investigated the effect of lipopolysaccharid (lps) on h release of fresh isolated cells and on cells cultured for days in a fibronectin matrix. furthermore we conditioned isolated alveolar macrophages for hours with p.g/ml lps and tested the conditioned medium (mcm) on cultured p cells. p cells h ex vivo were _> % pure, _> % vital and had an basal h release of . _+ . nmol h per hour and million cells. adding p.g/ml lps to the incubation medium the h release decreases slightly but significantly to . _+ . nmol. adding . p.g/ml phorbol myristate acetate (pma) to the basal incubation medium the h release increased -fold to . _+ nmol. pma induced h release decreased to . + . nmol after addition of p.g/ml lps. after culture days the p cells were _> % pure and showed a pma inducible h release of . _+ . nmol addition of p.g/ml lps had the inverse effect as on freshly isolated cells as it increased the h release up to . _+ . nmol. addition of mcm to cultured p cells increases pma-stimulated h release to . +_ . nmol. the release decreased to . _+ . nmol when an murine anti-tnf-alpha antibody was added. vitality of cultured cells was > % in all experiments. the results show that lps has an direct effect on p cells cultured on fibronectin. we conclude that the observed additional stimulatory effects of mcm seems to depend on tnf-alpha. the induction of h release of p cells could be important for generating internal oxidative stress in p cells before external oxygen radicals exceed. the produced h did not necessarily damage p ceils, but it can effect surfactant metabolism, especially when extracellular h release of alveolar macrophages following an immune response is increasing. introduction: primary stabilization of femoral shaft fractures in patients with multiple trauma is beneficial. however, in patients with associated lung contusion we have found an increased incidence of ards, apparently associated with primary reamed femnral nailing (rfn). previous animal studies revealed, that perioperative disturbances of lung ftmetion appear to be related to the reaming procedure, ix~ssibly due to pulmonary embolizafion of bone marrow fat. in a prospective clinical analysis we compared effects of intrameduuary nailing with and withont reaming on parameters known to be related to ards-pathoganesis. in order to gain further insight into the role of endotoxin and cytokines in the pathogenesis of the adult respiratory distress syndrome (ards), we enrolled patients with severe lung injury after sepsis ( ) or polytrauma ( ) and obtained multiple blood samples ( days) for endotoxin, tumor necrosis factor e (tnfa), interleukin (il- ) and interleukin (il- ) determination. to evaluate the cytokine releasing capacity of the blood, plasma concentrations of tnfe, il-l and il- were also determined after the "in vitro" stimulation of the whole blood samples with lipopolysaccharide (lps, . ng/ml) for hours at c (stimulated values). the difference among stimulated cytokines levels and the basal plasma concentrations were defined as "delta values", an expression of the cytokine releasing capacity of the blood. the pao /fiao quotient was used as an index of the severity of lung injury (sli). the endotoxin plasma level was significantly higher in patients with sli < ( . ± . eu/ml, mean values ± sem) versus the patients with a sli > ( . ± . eu/ml, p<o. ). the basal plasma concentration of ll- also correlates with the sli index (p<o.o ). the delta tnfe and delta il- values were s~gnificantly decreased in patients with a severe lung injury. compared with the survivors (n: ), the deceased patients (n= ) showed higher endotoxin level, a reduced tnfa and il- releasing capacity of the blood and higher basal il- levels. we can conclude that endotoxemia, high il- plasma level and a decreased capacity of the blood to release tnfa an il- under endotoxin stimulation associates with the severity of lung injury. [objectives] experimental and clinical findings implicate the involvement of inflammatory cytokines in the pathogenesis of the decreased oxygenation in the lung after major surgery, though the mechanism remains unclear. in the present study, we elucidated the involvement of il- in the pathogenesis of lung injury defined by deterioration of pulmonary oxygenation after esophagectomy. [materials and methods] seventeen patients underwent subtotal esophagectomy through a right thoracotomy. in all patients, tbe alveolar-arterial oxygen tension difference (aado ) was measured everyday and bronchoalveolar lavage fluid (balf) samples were obtained from a single segment ($ or $ ) of the right and left lung through a bronchoscope on days l, , and days after surgery. they were then examined for cell analysis, measurement of cytokines by elisa, and measurement of neutrnphil elastase (ne) by elisa. [results] aado unexceptionally deteriorated postoperatively and the mean aado in patients reached the maximum (mean ± sem; _+ mmhg) on the rd postoperative day. the mean concentrations of il- , ne and neutrophil count in balf also increased postoperatively and reached their maximum level ( + pg/ml, -+ gg/l, and ± x cells/ml, respectively) on the rd postoperative day. these increases in balf were recognized in both the right and left lung. a highly significant correlation was observed between il- and ne levels in balf (r= . , p= . ). also, significant correlations between aado and the neutropbil count, and the concentration of ne or l- were observed. in contrast to il- , neither il- , il- nor tnfa was detected in balf. [conclusion] major surgical trauma such as esophagectomy may induce recruitment of neutrophils to the lung and cause lung injury. il- increased in the lung is an important mediator of neutrophil recruitment and activation in the lung. phorbol myristate acetate (pma) is commonly used to cause edema and other tissue damages in the lung. lung injuries induced by the administration of pma has been shown to be mediated mainly by neutrophils. neutrophils recruited to the lower respiratory tract may damage lung tissues by releasing reactive oxygen species, neutral proteases and lysosomal enzymes. the present study was conducted to investigate whether c~tocopherol, entrapped in dipalmitoylphosphatidylcholine liposomes and delivered directly to the lung, could counteract some of the pma-induced lung injuries. plain liposomes or c~tocopherol-containing liposomes ( mg c~-tocopherol/kgbody wt.) were instilled into the lungs of rats h prior to pma exposure ( pg/kg, intratracheally) and treated rats were killed h later. lungs of control animals exposed to pma developed increases in lung weight and lipid peroxidation as well as decreases in lung angiotensin converting enzyme (ace) and alkaline phosphatase (akp) activities. pma treatment also caused an increase in myeloperoxidase (mpo) activity in the lung, suggestive of neutrophi] infiltration. pretreatment of pma-treated rats with plain liposomes had no effect on pma-induced injuries. in contrast, pretreatment of rats with liposomal c~-tocopherol, h prior to pma administration, resulted in a significant elevation of pulmonary a-tocopherol concentration, accompanied by a concomitant reduction in mpo activity and reversal of pmainduced changes in lung edema, lipid peroxidation, ace and akp activities. these results appear to demonstrate that the intratracheal administration of a liposome-associated lipophilic antioxidant, such as a-tocopherol, can significantly ameliorate the toxic effects of reactive oxygen species, released from pmastimulated pulmonary target cells and infiltrating neutrophils. jk wojcik, g palasiewicz, w roszkowski immunology department,institute of tuberculosis and lung diseases~ warszawa, poland, m~larhospital, eskilstuna, sweden. introduction:the critical point in neutrophil migration to the alveolar space in the course of ards is the attachment between giyeoproteins of neotrophil membranes(sialyl lewis x carbohydrate epitope containing u-l-fucose) and lectin domains of endothelial p and e selectins (gmp- ,elam-i). a potential beneficial therapeutic strategy may be designep to suppress neutrophil recruitment to the lungs by preventing their rolling and attachment to the pulmonary endothelial cells. objeclive:to investigate if tz-l-fueose prevent experiments pulmonary hypertension in ards as effective as metylpredniselone. methods:only healthy rabbits (n- ) weighing .d- . kg oaselinepao> kpa and mean pulmonary arterial pressure (mpap)<i. kpa were included in this study. anaesthesia was induced with thiopental - mg/kg bw and "blind" jntubation was performed. a f swan ganz catheter was introduced via the left jugular vein into the pulmonary artery. pma, as was used in our experiments activates neutrophils and produces acute respiratory failure with pulmonary hypertension and pulmonary edema. eight rabbits serving as control animals received pma ug/kg bw iv only. sixteen other animals were divided into two groups receiving either ~-l-fucose mg iv or metylprednisolone mg iv min before pma admininstration. results:after pma administration notable physiological changes including marked increase in mpap( . -+o.~ kpe ..lere found. the increase of npap was observed to be completely blocked in both groups of animals which received ~-l-fucose or metylprednisolone. conclusion:in aur rabbit model ards cz-l-fucose pretreatment prevents pulmonary hypertension after pma administration. the effect is comparable with high dose metylprednisolone. the possible explanation is that g-l-fueose molecules block iectin domains of gnp- or elam-i preventing the adhesion of the neutrophils to the pulmonary endotheliom. bartens c, elmadfa i, steltzer h, fischer m, druml w introduction: various micronutrients and vitamins are particulary effective in modulating immune functions and host defense. the nutritive ant±oxidants vitamin c, e and b-carotene, as well as selenium lower the burden of reactive free radicals and protect the structural integrity of cells and tissues of the immune system, as well as other systems in the body. certain cells of the immune system like polymorphonuclear leucocytes (pmn's) use free radicals as a weapon to destroy invading pathogens. these reactive molecules, when released into the surrounding area, mediate lipid peroxidation and tissue injury. there is growing evidence that pmn's are activated in vivo by complement or immune complexes in disorders such as ards. the respiratory host defense mechanisms of patients with adult respiratory distress syndrome (ards) may be defective. the aim of this study was ta evaluate the status of the free radical scavengers and their activity in ards and to investigate the respiratory burst of ards patients. methods: seven ards patients ( sepsis, trauma) with an fie of . + .( , a peep of . ± . , and a murray score of . ± . were included in this study. healthy adults served as controls. superoxide anion production in granulocytes was measured photometrically, and the hydrogen peroxides by fluorimetric assay. vitamin a, e, and g-carotene concentrations were assessed by hplc, and plasma vitamin c photometrically. plasma selenium was determined by dectrothermal aas. plasma lipid peroxidation pruducts, expressed as malondialdehyde (mda), were determined by thiobarbituric acid assay and hplc. results: mda-plasma (/xmol/l) . ~: . * . ± . *= < . , **=p< . condusion: ards patients showed significantly decreased plasma concentrations of vitamin c, e and g-carotene, as well as selenium. these nutritive ant±oxidants are consumed during increased oxidative stress. mda, a final product of lipid peroxidation, is increased. however, a difference in rates of release of hydrogen peroxides and superoxide-anion of pmn could not be detected. therefore, oxygen radical accumulation is more likely a result of excessive inspiratory oxygen concentrations (f.io ), pro-oxidant drugs, and a high settlement of pmn in the lungs, and not an increased release of free radicals of the single pmn. kd glycoprotein secreted by the alveolar type ii cells. recent study showed that sp-a exists in the sera from normal healthy adults and that the significantly elevated serum sp-a levels were observed in the patients with interstitial pneumonia and pulmonary alveolar proteinosis. these findings means there may be a mechanism that the sp-a produced in the alveoli escapes into the bloodstream. here we examined the serum sp-a levels in the patients with critical illness including sepsis and ards. ]clinical subjects & methods] a total of serum samples were collected from patients hospitalized in the division of critical care medicine(icu), sappom medical college hospital. serum sp-a levels were measured by an enzyme-linked immunosorbent assay using monoclonal antibodies to human sp-a. ]results] there was no significant difference between the septic (n= ) and non-septic (n= ) patients. patients with ards (n= , . + . ng/ml) and with respiratory disease other than ards (n= , . +- . ng/ml) showed significantly higher levels of serum sp-a than those with non-respiratory disease (n= , . _+ . ng/ml). as to the ards patients, it was likely that the serum sp-a levels were getting higher in their clinical courses. however, there was no significant correlation between the serum sp-a levels and the pa%/fio ratio. ]discussion] at the present time, the exact mechanism of the escape of sp-a into the bloodstream remain to be unclear. our results may suggested that there was some relation between this leakage of sp-a and the alveolar-capillary permeability because of the higher sp-a levels observed in the ards patients. however it is also likely that higher serum sp-a level represents the proliferation of alveolar type ii cells as the regeneration of damaged lung. further studies are now being done. neutrophils contain a number of enzymes within intracellular granules,potentially damagingto lung tissue.release of these enzymes into the lung tissue from the sequestred activated neutrophils is felt to play significant role in lung injury in the course of aros. using -hours acute animal model of aros the activity of cathepsins,beta-glucuronidase and acid phosphatase was determined in the lung tissue. results of this research were compared with neutrophil proteases activity in serum and broncho-alveolar lavage.also hemodynamic,respiratory and biochemical investigations were performed before beginning of experiment,and in a two-hours time intervals of its duration.after hours was determined total and free activity of cathepsins,beta-glucuronidase and acid phosphatase in full homogenate of the lung tissue,mitochondriallysosomal fraction and the final supernatant. in the course of our experiment we observed an increase ( .i- . %) both total and free activity of neutrophil proteases in all fractions of the lung tissue.generally accepted current pharmacological treatment of aros only attenuated this increase but never caused reversion to the level before beginning of our investigations. the presence of aros was histologically proved.an activity of acid phosphatase in serum and the lung tissue fractions was compared with histochemical pictures of the lung. results of our research indicate that neutrophil proteases are a very important mediators in aros and they are a cause of the lung injury. in addition neutrophil-derived proteases can amplify the inflamatory process by enzymatitally activating of many other mediators and they also may to increase an oxidant induced injury by imparing of antioxidant defenses. oepartment of general surgery, sk~odowskiej a - ~ia~ystok, poland, tel/fax + . in vitro studies demonstrated that paf activates polymorplionuclear leukocyte (pmn) - ipoxygenase, but the generation of leukotrienes (lts) is critically dependent on exogenous arachidunic acid (aa) disposal (f. grimminger et at., mol. pharmacol. : , ) . we investigated the features of paf-evoked lt synthesis in a model of pulmonary microvascular leukostasis, in the presence and absence of intravascular n- -and n- -prescursor fatty acid supply. human neutrophils were infused into isolated, ventilated and perfused rabbit lungs to achieve microvascular sequestration of ligand-responsive leukecytes. leukotrienes (lt) and hydroxyeicosatetra(penta)enoic acids (het(p)e) were quantified by itplc techniques. intravascular application of paf ( um) or arachidonic acid (aa;ioum) provoked the generation of limited quantities of -series lts and -hete (total sum of - ipoxygenade products rd. and rd. pmol/ml.; both in pmn-preloaded and non-preloaded lungs). combined administration of pat r and aa caused amplification of - ipoxygenase product formation with predominance of cysteinyl-lt synthesis both in lungs without (total sum rd. pmol/ml) and, much more strikingly, with pro-application of neutrophils (total sum rd. pmofml). eicosapentaeooic acid ( um) elicited exclusive generation of -series lts and -hepe (total sum rd. pmol/mi). dual stimulation with paf and epa provoked amplification of epa-derived - ipoxygenase product formation, again with predominance of cysteinyl-lts, in lungs without (total sum rd. pmul/ml) and in particular in those with preceding microvascular pmn entrapment (total sum rd. pmol/ml). we conclude that the paf-evoked - ipoxygenase product formation in the neutrophil-harbouring lung capillary bed is critically dependent on intravascular precursor fatty acid supply, with epa representing the preferred substrate as compared to aa. pmn-related transcellular eicosanoid synthesis is suggested to underly the predominant generation of cysteinyl-lts. these findings may be relevant for lipid mediator profiles provoked by infusion of n- -versus n- enriched lipid emulsions in diseases with pmn-related inflammators events. adult respiratory distress syndrome (ards) involves damage to the alveolar epithelium and microvascular endothelium. products released from neutrophils (pmn) are thought to be among the chief causes of this damage, while the role of mononuclear cells (mnc) is uncertain. we studied patients at risk of or with acute ards. we measured the concentration of myeloperoxidase (mpo) and elastase (el) within circulating pmn as an index of pmn activation, elastin degradation products (edp) in the plasma as an index of tissue damage, as well as the cytotoxici~y of pmn and mnc to endothelial cells (ec) in vitro. cells and plasma were prepared from venous blood of healthy controls; or systemic arterial blood of patients on ventilators in intensive care but not at risk of ards; at risk of ards because of sepsis; at risk because of multiple transfusion/major trauma; or with acute ards. lung injury, multi-organ failure and apache h scores were recorded. to measure cytotoxicity, human umbilical vein ec were labelled with cr- , incubated with either pmn or mnc for hours, and the supernatant counted. the adherence of the pmn and the mnc to the ec was also measured. standard assays were used to measure mpo and el in the pmn, mad edp in the plasma. the mnc cytotoxicity did not differ between the groups. the pmn cytotoxicity was higher in the transfusion/trauma patients compared to each other group, which did not differ from each other. there was no correlation between cytotoxicity and adherence for any group. the mpo and the el concentrations were significantly decreased in the pmn from each of the patient groups, including the patient controls, compared to healthy controls. the edp were only increased in patients at risk of or with ards. thus pmn degrannlation occurred in patients not at risk of ards, but tissue damage as measured by increased edp only occurred in patients with or at risk of ards, suggesting that factors additional to pmn activation and degranulation are required for the progression to ards. there was no correlation between the parameters measured and the indices of disease activity, or outcome. these measures of pmn and mnc activation and function are not of value as prognostic indicators in patients with or at risk of ards. the liver is made up of several different cell types which subserve distinct functions in vivo. in addition to the different functions of the distinct cell types, it is now evident that even within the population of hepatocytes substantial functional heterogeneity exists. this heterogeneity occurs in a very organized fashion in the normal liver based upon the position of the hepatocyte in the acinus. although it has been proposed that the acinar heterogeneity of hepatocytes arises from migration of immature hepatocytes from the periportal region to the perivenous region where they become senescent and die, many of the heterogeneous responses can be accutely reversed by reversal of the oxygen gradient via retrograde perfusion. under normal conditions in which the acinus is exposed to unidirectional flow, the periportal hepatocytes are exposed to relatively high levels of oxygen, substrates and hormones. as these substances are extracted along the aeinus their concentration decreases leaving relatively low concentrations for the perivenous hepatocytes. in the normal liver, the heterogeneity of response is attenuated by extensive communication via gap junctions. the major liver gap junctions are made up of hexamers of connexin (cx ) which forms channels between cells capable of pas.~ing any molecule smaller than d. coupling is sufficient to allow diffusion of molecules such as atp or camp from one hepatocyte into > adjacent hepatocytes within seconds. during stress conditions such as endotoxemia or zymozan inflammation, expression of cx is markedly decreased while the secondary gap junction protein cx is either unchanged or even increased. while cx readily effects electrical coupling, molecules > d pass only very slowly. this would result in restriciton of transmission of moecules the size of atp or camp. since inhibition of gap junctions also attentuates metabolic response to hormone or nerve stimulation, it is evident that modulation of hepatocyte hetereogeneity by gap junctions must be considered in determining the mechanisms of metabolic alterations during stress. already minor haemorrhage decreases portal venous blood supply to the fiver and the reduction in portal blood flow becomes more pronounced with more profound btood loss. severe hacmorrhagic hypovolemia also reduces hepatic arterial blood supply which, however, is maintained over a vide range of haemorthage. the net effect of blood loss is a reduction in liver oxygee supply and this reduction is in proportion to the vulume iossed. however, oxygen supply to the liver exceeds the demands of the normal liver and this is the ca~ stilt following reduction of % of blood volume. the situation in sepsis is more complicated. po~l venous supply to the liver is redur.~i fairly early following normovolemic sepsis while hepatic arterial blood supply is maintained at le,~t initialiy, oxygen saturation might be maintained in arterial blood but may also be slightly reduced during sepsis, oxygen saturation of portal venous blood is significantly reduced during sepsis due to increased extraction of the intestines. therefore oxygea delivery to the liver during sepsis becomes sigalfkzntly reduced. at the s,~ne time and for mai.v.ly unknown reasons the need for oxygen becomes significantly increased in the ~-~ptic liver. as a consequence liver oxygen consumption becomes flow dependent and the liver is likely to suffer from ischemia during septic conditions. $ although liver failure is well recognized in sepsis, it is generally thought to be a late complication following pulmonary and renal failure. jaundice, hypoglycemia, encephalopathy and bleeding secondary to low levels of liver-synthesizing clotting factors are, however, signs of rather severe end-stage hepatic failure. furthermore, elevated liver enzymes (sgot and sgpt) represent hepatucyte damage and not hepatocellular dysfunction. in view of this, a more sensitive indicator of hepatic function is desirable in order to detect early hepatic abnormality. in this respect, indocyanine green (icg) is a tricarbocyanine dye that possesses several properties which makes it particularly valuable inthe assessment ofhepatic function. this dye is bound m albumin and is cleared exclusively by the liver through an energydependent membrane transport process and is nontoxic at lower doses. we propose that maximal velocity (vm~,) of icg clearance is a valuable measure of active hepatocellular function, since the total concentration of functioning receptors is directly proportional to vm~. we have utilized a fiber optic catheter and an in vivo hemoreflectometar to continuously measure the administered icg in vivo and consequently determine its clearance without the need of blood sampling. using this technique, we have found that in the early stages of sepsis (i.e., and h following cecal ligation and puncture), the vm~ and kinetic constant (k=) of icg clearance was significantly depressed. it should be noted that at this stage of sepsis, there was no elevation in serum enzyme levels. furthermore, hepatic blood flow and cardiac output increased at the above mentioned time points. thus, the extremely early depression in active hepatocellular function in sepsis, despite the increased hepatic blood flow and cardiac output, may form the basis for cellular dysfunctions leading to multiple organ failure during sepsis. additional studies indicated that following hemorrhage, active hepatocellular function was markedly depressed. this returned to prehemorrhage levels after ringers lactate resuscitation, however, this function was not maintained and decreased significantly after fluid resuscitation. nevertheless, the depressed active hepatocelinlar function following hemorrhage was markedly improved by post-treatment of animals with either atp-mgci , peutoxifylline or diltiazem. thus, the use of icg clearance provides an early sensitive indicator of hepatic abnormality during sepsis and following hemorrhage and this method should be used, not only experimentally, but also in the clinical arena for the early detection of hepatocellular abnormality. although multiple organ dysfunction syndrome (mods) remains a major cause of mortality and morbidity in intensive care units, very little is known about the mechanisms that precipitate its development. since an episode of inadequate tissue oxygenation is considered to be the trigger for mods, we have proposed that a primary localized injury such as ischemia/reperfusion may be sufficient to cause a change of gene expression of remote and apparently unaffected organs. such modulation of remote organ gene expression may decrease the organ's tolerance to a subsequent stress contributing to the development of mofs. to test this hypothesis, rats were subjected to hepatic regional ischemia by clamping the blood flow (hepatic artery and portal venous inflow) of the left and median liver lobes. intestinal congestion was prevented by allowing flow through the smaller right and caudate lobes. after minutes of ischemia, the clamp was removed and the blood flow restored. the animals were allowed to recover for , and hours. kidneys were removed, total rna was isolated and poly(a) ÷ selected by affinity chromatography on oligo(dt) columns. message was in vitro translated using rabbit reticulocyte iysates in the presence of radioactive amino acids. the gene products (radiolabeled polypeptides) were fractionated by two dimensional gel electrophoresis, and visualized by fluorography. analyses of the two dimensional fluorograms indicate that there is a dramatic change in the electrophoretic pattern of in vitro translated products in samples corresponding to kidneys obtained after minutes of hepatic ischemia and hours of reperfusion with respect to kidney samples obtained after sham operation or from control rats. the latter were not subjected to any surgical manipulation. these studies suggest that the gene expression of the kidneys is specifically modified after a remote organ injury (hepatic ischemia/reperfusion). we speculate that this change of gene expression in kidneys after an indirect injury may be part of the early events leading to the development of mods. a priming event, e.g. local ischemia, in combination with a second insult, e.g. sepsis, may amplify a host's response and lead to multiple organ failure. to better understand the mechanisms involved in the pathophysiology, male fischer rats were subjected to min of hepatic ischemia followed by reperfusion (rp) and injection of . mg/kg salmonella enteritidis endotoxin (et) at min of rp. et injection potentiated the postischemic liver injury as indicated by histopathology and an increase of plasma alt activities from + u/l (i/rp only) to + u/l at h rp. inhibition of kupffer cells (kc) with gadolinium chloride ( mg/kg) attenuated liver injury in this model by %, however, monoclonal antibodies (cl , wt ) directed against adhesion molecules ( integrins, cd ) on neutrophils had no effect on the injury despite the substantial accumulation of neutrophils in the liver at that time ( + pmns/ hpf; baseline: + ). isolation of kc and neutrophils from the postischemic liver indicated a -fold increase of the spontaneous superoxide formation only in the kc fractions [ . + . nmol o -/h/ %elts (kc ); . _+ . (kca) ] at h rp compared to control cells. in addition, stimulation with phorbol ester or opsonized zymosan revealed a substantial priming of kc for reactive oxygen formation. in contrast to the short-term experiments ( h), the antibody wt ( mg/kg) attenuated liver injury by % at h of rp and improved survival. conclusion: liver injury during the early rp phase is mediated mainly by kc generating excessive amounts of reactive oxygen while neutrophils are primarily responsible for organ damage during the later rp period. (es- and gm- ) tumor necrosis factors (tnf) are cytokines which are cytotoxic towards some tumors in vivo and certain tumor lines in vitro. moreover, these polypeptides are powerful immunomodulators and have been found to be distal mediators in several models of septic shock and septic organ failure. one of the best-characterized experimental systems is the hepatitis caused by lps or tnf in galactosamine (galn)-sensitized mice. here we describe a cell culture system, in which the direct toxicity of tnf towards mouse hepatocytes was examined. the toxicity of tnf, as determined by ldh-release or formazan-formation, was dose-and time-dependent. the threshold of toxicity was ng/ml, which corresponds to serum concentrations found in mice after lpsinjection. toxicity was only observed in hepatocytes sensitized with transcriptional inhibiters such as galn, actinomycin d (actd) or cxamanitin. sensitization was neither observed with different translational inhibitors nor with various other metabolic inlaibitors or toxins. inhibitors of protein synthesis or protein processing such as cycloheximide, puromycin, tunicamycin and ricin protected actdsensitized hepatocytes from tnf-induced cytotoxicity. tnf induced apoptotic changes and dna-fragmentation in sensitized hepatocytes which is in line with the above findings that cell death is dependent on protein synthesis. thus tnf may be a trigger of programmed cell death during inflammatory organ damage. with the purpose of studying the role of complement activation in tissue injury after ischaemia and reperfusion we blocked the complement cascade in a model of rat liver isehaemia and reperfusion, either by administration of soluble human complement receptor type (scri), mg/kg iv after vascular occlusion (n= ) or by depleting the complement system using cobra venom factor (cvf), . mg im, and hours before ischaemia (n= ). non-ischaemic rats (n= ) and ischaemic non-treated rats (n= ) were used as controls. the experimental procedure consists of the temporary interruption of arterial and portal blood flow to the left lateral and medial lobes of the liver during minutes, followed by reperfusion, recording the liver blood flow and haemoglobin saturation with a laser doppler flowmeter and photometer during one hour after declamping; alt levels were assayed and immunoperoxidase stainings for c and c were performed. there were statistically significant differences between the experimental ~roups and the untreated ischaemic control group in terms of post-isehaemic blood flow (p< . ) and haemoglobin saturation (p< . ). c and c were present in the endothelium of the ischaemic control group. no deposits of c or c were found in the cvf group. few c and no c were found in scri treated rats. these results show that the effect of reperfusion injury in the rat liver is ameliorated either by depleting complement with cvf or by regulating complement activation with scri. hepatic dysfunction, a major cause of mortality following hemorrhagic shock, has not yet been well characterized. the present study was designed to assess the effects of liver blood flow and cytokine levels on hepatic function following resuscitation from severe hemorrhagic shock in normal and cin-hotic rats. methods: aftor pentobarbltal anesthesia, control and cirrhotic sprague-dawley rats were subjected to severe hemorrhage to reduce their systolic blood pressure to + mm hg. this level of hypotension was maintained until the skeletal muscle transmembrane potential (era) depolarized by %.; the animals were then resuscitated with ringer's lactate solution in three times the volume of the shed blood. serial blood samples for tumor necrosis factor (tnf) determination (a modified flow-cytomeuic wehi cell bioassay) were obtained at baseline, during hemorrhage and following resuscitation. liver blood flow measurements by low dose galactose clearance (glc) and functional bepatocyte mass (fhm; defared as galactose elimination capacity [gec] from the zero order portion of the plasma disappearance curve following an intravenous galactose bolus [ mg/kg], divided by liver weight) were measured before shock and after resuscitation. results: higher survival rates (p < . ) were observed in control as compared with cirrhotic rats. shock produced a significant reduction in gec (to < . ); fhm ( < . ); and liver blood flow (p < . ) in normal and cirrhotic rats. decreases in gec and fi-im were greater (p < . ) in cirrhotic rots. tnf levels were higher (p < . ) in cirrhotic rats at baseline and during induction of shock. pre gap junctions provide pathways for metabolic signals between cells. in the liver, the majority of gap junctions are composed of connexin (cx ) polypeptide subunits, and are regulated by gluconeogenic hormones. since sepsis and other inflammatory states alter hepatic glucoregulatory control, we have evaluated the contribution of gap junctional conductance to the metabolic dysregulation in the liver. an acute inflammation was induced in rats by injection with e. coli endotoxin (lps lmg/kg). northern blot/hybridization analysis of total rna isolated from livers after endotoxin injection show a decrease in the steady state transcript levels of cx to % of sham controls. immunostaining of liver sections using anti-cx revealed punctate fluorescent staining on the plasma membrane at regions of call-cell contact in saline injected animals, whereas, staining was only observed in cytoplasmic vesicles hrs after animals were treated with lps, suggesting the internalization of cx without replacement on the cell surface. the staining was quantitated and expressed as % of pixels above threshold. at hr post injection . % ofpixels exceeded threshold, compared to . % in sham controls. functional gap junctional communication was assessed by dye coupling using lucifer yellow in an isolated perfused liver under intravital fluorescence microscopy. dye diffusion was markedly decreased hr after endotoxin injection. this suggests that decreased metabolic coupling after lps injection results from decreased gap junction abundance. the present data suggest that metabolic dysregulation during sepsis may arise in part from changes in intercellular communication caused by a decrease in gap junctional expression and communication. given the marked metabolic heterogeneity of hepatocytes with respect to acinar location, metabolic signaling via gap junctions most likely serves to moderate this heterogeneity, contributing to a coordinated metabolic response. altered cellular ca ÷ regulation might be a critical step in organ dysfunction during sepsis and ischemia/reperfusion events. the aim of the present study was to evaluate hepato-ceuular ca ÷ regulation in isehemiah'eperfusion after hemorrhage and to assess effectiveness of tnfc~-monoclonal antibody (tnfo~-moab). methods. male sprague-dawley rats ( g, n>_ /group; pentobarbital mg/kg) with hemorrhage for rain at mm hg. reperfusion by ringer's lactate ( x maximal bleed out/ min) and % of citrated shed blood. tnfcz-moab (tn , ceutech, mg/kg in . % nac ) infused during flrst min of reperfusion. at baseline, end of ischemia and min of reperfusion, hepatecyte isolation by liver collagenase perfusion. " hepatocyte incubation ( mg w.w./ml) with caci ( . + + + mbq/ml) for rain (ca influx [slope, /mini; ca uptake [nmol ca /mg protein]) w/ and w/o epinephrine (epi, nm). hepatecyte resuspension in radioisotope-free medium and farther incubation (exchangeable ca + (ca +ex) [nmol ca +/mg protein]; ca + membrane flux [nmol ca +/mg protein'min]). during incubation, aliquots ( ~tl) were centrifuged through oil/lanthanum gradient and acivity measured by scintillation counting. statistics: anova. mean + sem. results. hepatocyte ca +ex and membrane ca + flux were significantly increased at both, the end of ischemia ( . +. ; . +. ) and reperfusion ( . +. ; . +. ), as compared to sham-operated animals ( . +_. ; . +. )( <. ). tnfc~-moab treatment significantly prevented reperfusion-induced increase of ca +ex ( +. ) and membrane ca + flux ( . +. )(p<. ). fast ca + influx was significantly increased by epinephrine in hepatecytes from sham-operated rats ( . +. vs. epi: . +. , p< . ). this hormone effect was not observed in isehemia ( . +. , epi: . !-_. ) or reperfusion (untreated: . +. , epi: . +. ; tnft~-moab: . _+. , epi: . +. ). conclusion. the present study clearly demonstrated hepato-cellular ca + overload in ischemia and reperfusion as a result of hemorrhagic shock. analysis of membrane ca + fluxes and hormone ca + mobilization suggests disturbances of membrane ca + transport mechanisms, e.g. through ca +-atpases. reperfusion-induced oxygen free radical generation which affect exchange kinetics of cellular ca + buffering compartments might also be operative. prevention by tnfct-moab indicates the pivotal role of tnf as an early inflammatory mediator of hepatocellular alterations in signal transduetion mechanisms and cellular homeostasis. although the precise mechanism has not yet been elucidated, bacterial translocation and endotoxin absorption have been frequently shown after burn, and have been postulated to be one of the underlying processes of sepsis. the purpose of the current study is to define the hemodynamic response of the liver to endotoxin release in burns, in correlation to bacterial translocation. twelve female minipigs, weighing - kg, underwent a laparotomy & transition time ultrasonic flow probes were positioned on the portal vein, the common hepatic artery, and the superior mesenteric artery. . fr catheters were inserted in the superior mesenteric vein and the left hepatic vein. a jejunostomy was also performed. after five days all animals were anaesthetized and randomized to receive % of tbs a third degree burn. eighteen hours after burn. gg/kg e. coli lps was intravenously administered over rain. ali animals were studied for additional hours and then sacrificed. several recent data suggest that in severe injuries, such as shock state, the gradual activation of kupffer cells and the excessive release of destructive and immunosuppresive products from macrophages may contribute to the development of "multiple organ failure". in in vivo experiments in mice, the effect of kupffer cell phagocytosis blockade on the correlation between the tissue distribution of lps, endotoxin sensitivity and lps-induced tnf production was investigated. to depress the activity of the kupffer cells, gadolinium chloride (gdc ) or carrageenan was used. th~e studies indicate the dissociation of tissue localisation of cr jllabelled endotoxin and endotoxin lethalithy. both gdc and carrageenan depressed kupffer cell activity, but endotoxin sensitivity was enhanced only by carragenan treatment. however, there was a close correlation between the sensitivity to lps and lps-induced tnf production as measured in the serum, since lpsinduced tnf production was enhanced only by carrageenan treatment. on the other hand, gdc pretreatment significantly increased tnf production in the spleen. these results support our earlier findings that gdc -indueed kupffer cell phagocytosis blockade leads to activation of the spleen, and may explain some of the immunological effects of gdc . inositol(l, , ) triphosphate (ip ) has been proposed as a second messenger for calcium mobilization. the addition of ip at low concentration has been shown to cause calcium release from intracellular microsomal store in rat hepatocytes. the effects of sepsis on the ip binding from microsomal fraction of rat hepatocytes during sepsis were investigated. sepsis was induced by cecal ligation & puncture (clp). control rats were sham-operated. three microsomal fractions (rough, intermediate and smooth) were isolated from rat liver. study of ip receptor binding was performed with tridium label ip . the results shewed that the ip binding was significantly depressed by - % (p< . ) during late sepsis ( hrs after clp), but not in early sepsis ( hrs after clp). the ip binding depression during late sepsis was most significant on rough and intermediate endoplasmic reticulum (p< . ), but not on smooth subfraction. since ip binding plays an important role in the regulation of intracellular calcium homeostasis in hepatocytes, an impairment in the calcium release due to depressed ip binding on smooth and intermediate endoplasmic reticulum during late sepsis may have a pathophysiological significance in contributing to the development of altered hepatic metabolism during septic shock. septic organ failure is currently recognized as an overactivation of the nonspecific immune system by bacterial stimuli giving rise to proinflammatory mediators. little is known about the mechanisms of the resulting cellular injury. here, a synergism is described between tnf as a major mediator of septic organ injury released by macrophages and hydrogen peroxide (h ) as a representative of reactive oxygen species as formed by e.g. neutrophils. rat hepatocytes are only slightly sensitive to either agent alone. when treated with a conbination of tnf and h# a stronq synergistic toxicity was found, especially w~e~ tnf-treatment preceeded challenge with h~o~. we have recently described a coculture model bfzrat liver macrophaqes and hepatocytes where lps induces hepatocyte cell death partially mediated by macrophage tnf release. when h was also employed in fhis more complex cellular system a similar synergism was found: the ecc~ of lps was consecutive patients with liver cirrhosis admitted to the department of surgery over a year period from january to december were studied for their complement profiles in relation to other parameters of liver function, the aim of the study was to determine if a direct correlation existed between low complement levels and end stage liver cirrhosis. cirrhotic patients were divided into child's a, b and c categories using child's classification. complement levels (c , c ) were measured and functional assay for complement (ch ) were performed in each of these groupings in addition to normal blood donor controls. these results show that the qualitative c , c and the functional chs complement assays have good predictive values in assessing deteriorating liver function• in particular, the functional assay for complement (ch ) showed marked impairment in child's c patients (p< . ) confirming the impaired immunological status of these patients. sera from this group of patients (child's c) were titrated with pig red blood cells (rbcs) in a haemolytic assay. the results showed that there were significantly less haemolysis of pig rbcs in these patients (p= . ) as compared to the controls. this findings strongly support an impaired immunological status in child's c liver cirrhosis and may explain the high incidence of sepsis as a terminal event in these patients. aim:kupffer cells(kc) have an importamt play to cause hepatocellular injury in sepsis, because these cells release many kinds of substances. we reported that oxygem radicals released by kcs stimulated by lipopolysaccharide (lps) caused hepatocellular injury. aim of this study is to investigate the relationship between imtracellmlar calcium(ca) concentration of cultured rat kcs stimulated by lps and release of oxygen radicals, and effect of prostaglandin e~ (pge~) on imtracellular ca concentration. production of acute phase proteins (c-reactive protein, crp, transferrin, tf) and £erritin (f) in rat hepatocytes (hps) and its dependence on extracellular matrix components were studied. hps isolated from the liver by collagenase perfusion were cultured at ~o per . ml medium fi +dmem ( : ) with % fetal calf serum for days on uncoated or type i collagen coated plastic surface or in the presence of dextrane sulphate in the medium. hps were stimulated by conditioned medium (gm) from i~ia-p or e. coli lps preineubated human blood mononuclear cells. production of crp, tf and f by hps was detected by elisa. it was found that both cms decreased tf synthesis in hps by - % (p<o.os). the level of f synthesis didn't change or only slightly decreased. the addition of cms led to the enhancement of crp synthesis more than . times. the most reproducible results were obtained when plastics had been coated with collagen. dextran sulphate markedly increased crp synthesis. thus using this model of an acute phase reaction in vitro we found the enhancement of crp synthesis and the decrease of tf synthesis. our data correspond with nublished materials on the acute phase in vivo and point to relevance of the proposed model. metabolism of hepatocytes under traumatic illness has not been sufficiently investigated. therefore we have applied absorption and fluorescent cytophotometry techniques to study the content of rna, glycogen and its fractions in hepatocytes of patients with severe mechanical trauma, both with and without endointoxication at various stages. for these purposes slides were prepared from the repeated aspiration biopsy material according to special techniques (kudryavtseva et al., ) . slides were stained by gallocyanin-chromalum to measure rna or underwent fluorescent pas-reaction to measure glycogen and its fractions (kudryavtseva et al., (kudryavtseva et al., , the ability of metabolites derived from the xanthine-xanthine oxidase system to inhibit mitochondrial function was examined using freshly isolated rat liver mitochondria. under uncoupled conditions, mitochondria exposed to free radicals exhibited a significant decrease in consumption supported by nad+-iinked substrates, but showed almost no change in consumption in the presence of succinate and ascorbate. the activity of electron transfer complex i (nadh oxidase and nadh-cytochrome c oxidoreductase) and the energy-dependent reduction of nad+ by succinate were unaltered by oxidative stress. exposure to free radicals also had an uncoupling effect at all three coupling sites. the degree of mitochondrial swelling was closely correlated with the inhibition of state oxidation of site i substrates and with the increase in state oxidation of succinate. the immunosuppressive agent cyclosporin a (cya) completely prevented the mitochondrial damage induced by oxygen free radicals (swelling, ca + release, sucrose trapping, uncoupling, and selective inhibition of the mitochondrial respiration of site i substrates). the same protective effect was found when ca + cycling was prevented, either by chelating ca + with egta or by inhibiting ca + re-uptake with ruthenium red. these findings suggest that the deleterious effect of free radicals on mitochondria in the present experimental system was triggered by the cya-sensitive and ca ÷-dependent membrane transition, and not by direct impairment of the mitochondrial inner membrane enzymes. correspondence should be addressed to: naoshi takeyama the aim of this study was to clarify the critical role of reduced glutathione (gsh) on the progression of lipopolysaccharide (lps)induced liver damage of mouse. the conditions of gsh-depletion and -richness were produced by intraperitoneal administration of buthionine sulfoximine (bso), a specific inhibitor of gsh synthetase, and of gsh-monoethyl ester (gsh-me), respectively. gsh-me, which was esterified the caboxyl group of the glycine residue, is readily transported into cells and is hydrolyzed intracellularly; this leads to greatly increased the cytosolic and mitochondrial levels of gsh. bso and gsh-me were administered intraperitoneally mmol/kg and mmollkg, respectively, and lps ( mg/kg) given h later. hepatocytes were isolated by in situ perfusion of the liver with collagenase h after lps injection. the degree of hydrogen peroxide (h ) synthesis and mitochondrial membrane potential were measured by flow cytometry using fluorescence probe dichrolofluorescein diacetate and tetrametyl rhodamine ethyl ester, respectively. the degree of mitochondria membrane permeability transition (mmp) was assessed by [ ]sucrose entrapment. we found that lps treatment results in a decrease in hepatic gsh level and mitochondrial membrane potential, and an increase in h synthesis and mmp. lps administration to bsq-pretreated mouse worsened at[ these parameters. in contrast, gsh-me pretreatment ameliorates. all together, gsh may acts an important role in cellular defence against lps-mediated liver damage, and mmp may result in the decrease in mitochondrial membrane potential. it has been shown, up to now, that disturbances of enzymatic processes in the cell organella are the basic factor of the changes taking place during endotoxin shock it has been observed that lysosomes are biochemically changed as result of the effect of lipopotysacharides of gram negative endotoxic bacteria. the purpose of the experiment was: .evaluation nfthe course of the ees due to the biochemic changes .determination of the influence of the ees on the activity of lysosomal enzymes in liver cell .deterrmnatian of the influence of h and d on the activity of some lysosomal enzymes in liver cell during ees material and method: examinations ware carried out on dogs. the shock was evoked by i.v. administration of endotoxin e.coli. the dogs were divided into groups: icontrols, i[ -dogs with untreated shock, iii-dogs in shock treated with h, ivdogs in shock treated with d, v -dogs in shock treated with h and d. the following parameters were determined: .activity of acid phosphatase in the venous blood serum. - .total and free activity of acid phosphatase and catepsins in the full liver cell homogenate, in mitochondrial-and-lysosomal fraction, and in the superuatunt (all after hours of the experiment). conclusions . essential increase of activity of lysosomal enzymes was observed in the ees. . increased activity of lysosomal hydrolases in the liver cell homogenate corresponds, as a result of the endotoxin effect, with increased enzymatic activity in the peripherial blood. extensive investigations from our laboratory of the pathophysiology of hepatic no-flow ischemia ( min) -reperfusion injury demonstrated substantial kupffer cell activation with reactive oxygen formation during the first few hours of reperfusion as indicated by increases in plasma glutathione disulfide (gssg) levels in vivo (am. j. physiol. : g , ) and enhanced superoxide formation by kupffer cells (kc) isolated from the postischemic livers (free radic. res. commun. : , ) . the early kc-induced injury initiated the later, primarily neutrophilmediated reperfusion injury (faseb j. : , ). to test whether or not similar mechanisms are operative during hepatic ischemia induced by hemorrhagic shock, male fischer rats ( - g) were subjected to rain of hemorrhage (mean arterial pressure mm hg) followed by resuscitation (rs) (reinfusion of shed blood). hepatic atp levels declined from . + . txmol/g to . _+ . ( min shock) indicating severe ischemia, and recovered to . -- . at min rs. to test for potential oxidant stress during rs, plasma gssg conc. were measured. although gssg levels increased by % compared to baseline levels ( . !-_ . i.tm), there was no significant difference to shamoperated controls. spontaneous superoxide formation of isolated kc was . + . nmol o -/min/ ecells (kc ) and . + . (kc ) in controls and did not change significantly after shock and rain rs. addition of phorbol ester or opsonized zymosan to isolated kc did not indicate a priming of these cells. conclusion: in striking contrast to our previous findings with hepatic no-flow ischemia, there is no evidence for a significant kc activation after min of hemorrhagic shock and up to rain of resuscitation. objectives-this study investigates morphometric changes in kc nltrastmcture which might account for this diminution in phagacytosis. materials and methods -three groups of wistar rats were studied: control, sham-operated [sham] and bile duct ligated [edl] . after weeks animals were sacrificed and livers were "perfusion fixed" with % glntaraldehyde and processed for transmission electron microscopy and image analysis. results -in the the bdl group kupffer cell numbers were greatly increased. biliary ductular proliferation was evident and sinusoidal spaces were compromised. eleven nuclear and cytoplasmic parameters were measured and statistically significant results are summarised in the results: ascorbic acid levels were slightly elevated after the hs and returned to basal values after rain. glutathione concentrations were increased significantly after the hs and the gsh levels kept rising continuously to as much as -fold of basal levels at the end of min. mda showed no significant variation before and after the hemorrhagic shock. plasma concentrations of ratine, teucine, isoleucine, phenylalanine, proline, threonine and serine showed significant increase over basal levels during the whole ischemic period. glutamic acid was slightly elevated at the onset of hs and remained the same for the rest of ischemic period. hydroxyproline was significantly below the basal value at the mid-point of ischemic period. alanine, glycine, histidine and aspartate did not change significantly throughout experimental time course. conclusions: ( ) oxidative stress might not be severe in the systemic hemorrhagic shock for pigs since no significant variations were observed for ascorbic acid and mda ( ) the increase of gsh might be that it was released from hepatocytes when the animal was under systemic ischemia. ( ) celzain amino acids might be acting as transmitters in the event of ischemia. however, fm"lher investigations are needed to clarify the detailed mechanism in regard with antioxidants and amino acids. dr. fang-ku p'eng taichung veterans general hospital talchung, taiwan , r.o.c. jia shi yong, huang zhi oiang and men xian jun. in patients underi~ent major hepatic surgery,obstructive jaundice has been implicated with fnoreased susceptibility to sepsis and liver failure. jaundice was said to he associated with derangement of iamune function and result in expression of pr,:.inflar...aatory cytoki~es that cause hepatooellular damage. this study is ~.o investigate the effects ,.,t lip,~polysaccharide(lps)rats. jaundice were produced in healthy ~istar rats of either sex weighing - m~ by common hi l~ duet l igation(bl)l).seven days post l igation, rats were given lps( .gg. ) . rag/kg intraperitoneally. at the end ,:,f l,gand hours after lp$ ehanllenge, liver were removed and prepared for forzen sections immediately. i)emonstration of inf in liver parenchyna were performed by method of abu i~munohistochemistry using r~onoolonal antibody against tnf, leve of mrna for tnf ~ere measured by in-situ hybridization using biotin-labeled edna probe. results sho'~ed that tnf stained granules appeared in kup~'fer eell~ and polymorphonuclear leukocytes(pnnl,but not in hepatoeytes nor endothelial eel is. they ~'ere located within cytoplasms and peaked at - hours after lps ohal lenge. there was vocomitant tnfmrna expression but peaked earlier hour post lps challenge. tnfmrna vcere detected notably in necrotic area and barely in bdl rats ~ithout lps challenge. it is therefore postulated that production of tnf by inflammatory effeetor cells-kupffer cell and phn in site, in the obstructive jaundice rats during sepsis may atribute to the hepatocellular damage. it also provide evidence for the beneficial effects of early release of biliary obstruction. in the present study, an animal model of a c in wlstar rats was developed by tigatlng the common bite duct and injecting d. mt solution of e. goli % b, (g× ' efm/mt) into the bite duct. the mortality rate was ~ at h after aoc. at , , , h after aoc, kcs were isotated and cultured atone or cocuttured with hepatocytes to evaluate the modulation effect of kgs on hepatoeyte protein synthesis. the results showed that tactodehydrogenase leakage was increased progressively as the injured k~ function and structure developed. tnf and il-i tn the supernatant were detected with the peak values at h after aoc. at h after aoc, ~-teueine incorporation was obviousty decreased in the normal hepatocytes eocuttured with stimulated kcs compared to the uormai hepatocyte cultured group (p< . ), but it was slgnificantiy increased in the group cocultured with normal kcs. in the a c group, 'h-teuclne ineorporatien was decreased progressively in the injured hepatoeytes cocuttured with stimulatedkgs and it was markedly elevated when cocuttured wlthnormat kcs at h after a c (p< . og). it is concluded that the impaired hepatocyte proterin synthesis was directly mediated hy the stimulated kc function during aoc. such a mechanism possibly may be involved in the patho ensis of hepatic dysfunction and m f following h . " the project supported by nsfc. in the past few years it has become evident that cytokines are implicated in various pathophysiological mechanisms. therefore measurement of cytokines and their potential inhibitors in biological fluids may be helpful in diagnosing and monitoring of diseases. however, dependent on the type of assay used investigations performed in different laboratories have often yielded conflicting results. in order to assess reliability and reproducibility of cytokine measurements two comparative studies for the determination of cytokines in biological fluids were launched by several investigators interested in this field: one national austrian ~tudy and one european study in the context of the european workshop for rheumatnlogy research. serum and synovial fluid samples were distributed by the organisers, and participants had to measure one or several of the following cytokines in all samples: il- , il- , l- , i , tnf-alpha, ifn-gamma, gm-csf, and the soluble receptors for il- and tnf; both commercially available immunoassays and home-made assays were allowed. so far, the main conclusions emerging from these preliminary studies are: ( ) the same immurzoassay (elisa) yielded comparable results in different laboratories; ( ) immunoassays from different manufactureres usually measured the highest concentrations in the same samples, yielded similar relative values but disparate absolute values; ( ) assays for soluble receptors yielded less discrepant results; ( ) some assays seem to be more suitable for measuring cytokines in biological fluids than others. the mean retrieval of exogenous recombinant human cytokines was approximately % for most cytokines tested. however, it must be borne in mind that natural and recombinant cytokines may behave differently in immunoassays. this raises the question as to the necessity of setting up international standards for all cytokines. possible interferences by serum components such as (lipo)proteins, complement, rheumatoid factors, etc., which may either decrease assay sensitivity or yield false positive results, must also be considered. in addition, natural cytokine binding proteins (e.g. soluble receptors) might interfere with cytokine determination in immunoassays. the problem of immunoassays versus bioassays has not yet been approached, but is without any doubt worth indepth investigation. thus, these studies are a promising first approach to deal with the difficulties of cytokine analysis in biological samples, and it is hoped that they will help to overcome some of the major methodological problems in the near future. tumor necrosis factor (tnf) is a pleiotropic cytokine which plays a key role in a number of immunologically mediated disorders like septicemia or cachexia. tnf receptors are found on the surface of a variety of cells, where they provide molecular signals for the cytokine effect. there exist two types of soluble tnf receptors (stnf-rs), tnf-r p and tnf-r p . these stnf-rs can compete with the cell surface receptors and block their activity. the expression on and shedding from the cell surface of stnf-rs is enhanced by interferon gamma. increased concentrations of stnf-rs can be found in patients with infectious as well as malignant disorders. in patients undergoing immune stimulatory therapy with interleukin- and interferon alpha a simultaneous increase of tnf and its soluble receptors can be seen. in patients with hiv infection a strong correlation exists between the levels of stnf-rs and markers of immune activation like neopterin or beta- -microglobulin. likewise patients with hematological disorders show elevated stnf-r levels. patients with weight loss have higher concentrations than patients with stable weight. the final value of stnf-rs within the complex network of cytokines is not yet clear. however, there exist striking parallels between infectious and malignant disorders pointing to a key role of endogenous immune activation. biochemicatly, d-erythro-neopterin derives from guanosine triphosphate. in vitro studies show that large amounts of neopterin are produced by human monocytes/macrophages stimulated with interferon-j (ifn-j). neopterin is biologically stable, and its halflife in the blood seems to solely depend on renal excretion. specimen for neopterin determination can be stored without special precautions. increased concentrations of neopterin have been described in body fluids of patients suffering from diseases which are apparently associated with an activated cellular immune response and with enhanced endogenous formation of ifn-~', e.g,, ) increasing neopterin levels predict rejection episodes in allograft recipients ) virus infections induce increased neopterin concentrations, and the degree of neopterin elevation predicts prognosis in these patients which is particularly true in hiv- infection ) in autoimmune disorders such as systemic lupus erythematosus or rheumatoid arthritis neopterin levels reflect activity of the disease ) increased neopterin concentrations in patients suffering from certain types of cancer indicate poor prognosis. significant associations between changes of neopterin and decrease of hemoglobin as well as the development of weight loss and cachexia, e.g., in cancer patients and in patients with hiv- infection, support the concept that endogenously formed cytokines such as ifn-~" and tumor necrosis factor-~( are involved in the pathogenesis of such symptoms. in conclusion, neopterin monitoring is a sensitive tool to detect the activation status of the cell-mediated immune system in vivo. repair and healing or perpetuation of acute inflammation is characterized by a complex network of interacting cellular and humoral defence mechanisms. of the numerous inflammatory mediators/effectors investigated hitherto, the proteolydc lysosomal enzyme pmn elastase has turned out to be highly destructive when released extracellularly thus contributing considm:ably to organ dysfunctions in severe posttraumatic and postoperative courses. besides various cytokines, elastase in complex with its main antagonist c¢ -proteinase inhibitor (a pi) is also supposed to induce the shedding of intercellular adhesion molecules from inflammatory cells (pmns, monocytes/macrophages, endothelial cells). these soluble adhesion molecules may modulate the inflammatory process in many different ways, e.g. via acting as chemotaxins, blocking neutrophil activation or competing with the membrane-bound forms in cell-cell adhesion. thus, measuring circulating or local levels of elastase-a pi and soluble adhesion molecules (icam, e-selectin,-pselectin, l-selectin) may be a helpful tool in judging the severity of an acute inflammatory process. in several clinical studies on surgical patients suffering from multiple trauma and/or sepsis we could demonstrate that the measurement of these parameters in consecutive plasma samples and local body fluids was highly valuable for early diagnosis and prognosis of multiple organ failure. prof. dr. m./ochum, institut fdr klinischechemie und biochemie, lmu miinchen, nufibaumstrage , m nchen, germany procalaitonin (proct) a aa peptide is dramatically increased in the blood of patients with various sepsis and infections. the increase begins as soon as hours after the andoto:edn release and readied maximal level with a to b-fold increase h after li~ edrmrdstration. prcc, alcitorfin response was temporally different from these of tnf~ and ii- , that reached peak levels at h and h respectively. at the opposite of eytokine (tnfcz, ild, j, is), proct is a very stable molecule. the half iife of proct in ~he blood is surprising if we consider the half llfe (about nine mirtute) of mature ¢alcitonin (co. we have found that it is due to the binding of proct with a protein on the n-termktal part. thin complex of about daltons is unable to cross the kidney and the c_n barriers and the half life is at least elghteen hours. this long half llfe is very useful in the evaluation of a sepsis. a very sensitive and specific sandwich in'ununoassay has been developped. the results cart be delivered in two hours. at time, we know that in the healthy adults, the values are less thau . ng/ml. it is also clear that proct is not increased ~n patients wlth inflammatory dleeasea without lnfectlon. for instance, proct is not signlffcat vely increased in purpura rhumatom, arthritis, crohn's disease, rectocolitis, also in various cancer patients with inflammatory components. am other interesting finding, is the absence of increase or a very low increase in the viral infections. at the opp site, in all the patients with bacterial sepsis or in the malaria pat/ants, progt was dramatically increased, from i to fold. p~oct can be useful to the follow up of patients with sepsis. actually, we don't know the origin of this proct production, in the united states, will also be discussed. the septest portion of this study will eventually enroll over patients tentatively diagnosed as septic. the results of septest will be compared with commonly accepted symptoms and criteria associated with sepsis in order to determine the clinical utility of this endotoxin assay. preclinical results of patients and normals be presented as well as data on the time course of endotoxemia and clinical outcome of selected septic patients. preparation of dna libraries clifford w. schweinfest, ph.d. a fundamental tool of the molecular biologist today is the ability to work with purified dnas representing genes of interest with respect to the investigator's field of study. originally applied almost years ago to the genetic dissection of simple organisms such as viruses and bacteria, molecfllar cloning was rapidly applied to study the more complex genetics of the mammalian cell. today, virtually any study of cell physiology which depends upon specific gene expression is approachable using the tools of the molecular biologist. therefore, stress induced gene expression (such as the heat shock gene, hsp ) or the regulation of the nitric oxide synthetase gene or the induction of cytokines and growth factors as a result of trauma or injury lend themselves to investigation at the level of the gene. during these workshops, we will discuss the means by which cdnas and genes are isolated, amplified and identified. we will discuss the strategies and methods for cloning cdnas and genomic dnas, for organizing such cloned libraries, for finding specific genes and for characterizing those genes. this speaker will focus on the techniques and considerations involved in the synthesis of cdna libraries. topics include rna isolation, mrna quality, reverse transcription, choice of cloning vectors and library quality. genomic dna library synthesis will also be discussed with respect to the vector/host systems available and applications which are specific to genomic dna. finally, polymerase chain reaction (pcr) will be discussed briefly with regard to its impact on dna cloning. the identification of the genes or gene products that have a role in cellular processes is fundamental to our understanding of genetic control and methodologies to achieve this aim are currently available. all levels of the various signal transduction pathways can b~ molecularly analyzed to define the mechanism by which critical steps in each pathway are achieved. the questions that are unresolved in each area of investigation serve to direct the scientist towards analyses of gene products or gene regulation of the gene itself. thus, selection of the type of library (genomic or cdna) to be used is dependent upon the specific goals of each investigator. to facilitate resolution of this question, an overview of the uses for the different types of libraries will be presented. at least four methodologies are currently available for screening a library, which are dependent upon specific interaction between nucleic acids, nucleic acids and proteins, antibodies and antigens or between different proteins. the use and advantages of each of these methodologies will be discussed along with a description of probe preparation. sequence methodologies required to begin characterization of gene clones will be presented. hopefully, the participants in the workshop will help define areas of emphasis and allow time for directed interaction to discuss specific applications based upon their own experimental systems. alterations of physiological conditions, such as those occurring after shock and sepsis, induce changes in gene expression of cells composing the major organ systems. the challenge is to detect and characterize these changes in geae expression and relate them to the injury. the development of cloning techniques has emerged as the methodology of choice. changes in gene expression are usually characterized by variations in the concentration of cellular messages because synthesis of the final product "the polypoptide" is usually proportional to the concentration of mrna. due to the rapid regulation of gene expression the cellular concentration of messages changes very quickly. this is commonly referred to as steady-state levels, a balance between transcription and degradation. steady-state levels of mrna can be detected in a single cell (in situ hybridization) or in total rna isolated from cells or organs and separated in agarose gels (northern blots). although analysis of mrna steady-state levels are very useful, they do not provide information about the mechanisms that regulate gene expression. changes in gene expression primarily occur at the level of transcription; characterization of the rna species being synthesized at a given time is performed by the nuclear run-off technique. when there is no a priori information about the gene that may be regulated after a particular situation such as sepsis, the total pool of messages present in cells at given time can be characterized by a combination of in vitro translation of the cellular messages and fractionation of the in vitro translated products by two dimensional gel electrophoresis. such approach permits the visualization of the general pattern of gene expression, a "finger print", of the physiologic state. in summary, researchers now have access to a great variety of techniques to identify and characterize genes expressed in response to a physiological condition that may be utilized as "molecular tools" to study the organism's responses to shock and sepsis. the acute phase proteins are a set of plasma proteins with greatly increased plasma concentrations during acute inflammations and after tissue trauma, e.g. after major surgery. the proteins counteract the source of injury, facilitate clearance of infectious particles by phagocytes, assist immune responses and initiate wound healing. the corresponding genes are expressed in liver hepatocytes and regulated by the inflammatory mediators interleukin and interleukin (ill, il ). class acute phase genes are mainly controlled by ill and by combinations of ill plus il ; class genes mainly by ii, . the human c-reactive protein(crp), serum amyloid a(saa) and complement c genes will be discussed as examples of class genes, and rat c~ macroglobulin as a prototypical class gene. both classes of genes use different types of ¢ytokine response elements in their promoter-proximal transcription control regions; class genes use the transcription factor nf-il or c/ebp as the key factor to mediate cytokine responsiveness; class genes use a different, so far unidentified factor to achieve responsiveness to il . ongoing efforts to purify this factor, called the il -response factor (il -rf), from rat liver nuclear protein extracts will be described. the il -rf apparently mediates the effects of il in a broad range of cell types, including b lymphocytes and other hematopoietic cells. it is therefore likely to be of equal general importance for gene regulation by il as nf-il . the importance of transcription factors as potential targets for novel bioactive substances and possibly therapeutic agents will be discussed with the help of several recent examples. new methods for altering the germ lines of mice provide powerful tools for understanding the roles of individual genes in normal and pathological processes, and for reproducing specific genetic mutations that result in congenital disease. three approaches will be discussed. conventional transgeulc mice are frequently constructed using artificial transgenes that express genes from a promoter other than the normal gene promoter. this paradigm is used to produce very high levels of a gene product, for example, with a viral promoter, or to attempt to regulate gene expression using an inducible promoter. alternatively, normal promoter sequences can be hooked to a gene whose expression is lethal to the cell (e.g., diptheria toxin a chain) to ablate all cells expressing that gene. another approach uses an intact gene with its normal regulatory sequences. here, an elevated level of the gene product is delivered from the correct cells in the correct tissues at the correct developmental stages or in response to natural signals. this approach has been refered to as "genetic pharmacology," and provides a precise delivery of the gene product to the target. however, to be most effective, the transgene should contain arl regulatory sequences as welt as the entire genomic segment containing gene coding regions. the introduction of conventional transgenes is limited to roughly kilobases, while genes containing all regulatory sequences frequently stretch over dozens or even hundreds of kilobases. to overcome this problem, procedures have been developed recently to introduce yeast artificial chromosomes (yacs) into mouse embryonic stem (es) cells. yacs can include over mb of human dna, large enough to encompass the largest known human genes. es cells are also useful for targeted gene deletions and mutations. homologous recombination can be targeted to any known gene in es cells. when these modified cells are injected into a mouse blastocyst, they can be incorporated into all tissues of the resulting mouse, including germ cells, so subsequent generations will pass the genetic modification as an inherited trait. assessment of the effects of a null allele on development can provide valuable insights into its normal role. ultimately, these technologies may be adapted to human cells -not for embryonic intervention, but to provide modified stem cells for various tissues (e.g., gut, eye, hematopoietic system) which could then be applied to somatic therapies. with major illness/injury that respirswy faihne fi'equently followed sepsis, tilney el al nse, d the term "soqueutial syste~ failure" for patients with renal failure after solmir of ruptured abdominal aortic ~aeurysms, i then reviewed our experiences after inju~ and oporstion and suggested that multiple, progsesslve or sequential systems or organ failure was a syndrome to be reckoned with in the, 's. eiseman et al then wrote about "multiple organ failure", especially with liver failure. polk el m found that renmte orgau failure often signalled occult anita-abdominal infection. fry et al ampbasized the role of uncontrolled infection in organ failure, border et ai described metabolic alterations with mof and used the term multiplesystems organ feilmo (msof). other early conltibutots to our knowledge of mof include faist, trunkey and miller, marshall and dimic&, cassone, catlleo, meakins and marshall, (}otis et at., mater and many others. mof or msof were used eommoifly. cerra coined the terms "post-trsumatic septic sfndromc" and "hyper metabolism otgau failure complex", and border et al, used lhe ex ~ression "gut origin seplic states" to illdioate important eurrem aspects of abe gt.'nerlc problem of organ failure and death, other terms include acute organ-system failure, multiple organ injury syndrome, post-traumatic multi-system organ failure nod post-~aumatic organ system infection s~dmmc (fi~sis), i bdieve the latin "maltiple organ failure" is clean, neat and says it all, now there is a proposal for a new set of torminolugios -mods and sirs. will they be helpful in the study and esro of patients? our speakers in this session will review thcsa proposals and the evidence as we strlvo for eoasensns. it has been well demonstrated that the administration of pro-inflammatory cytokines, and especially tumor necrosis factor (tnf) can result in a picture similar to septic shock with secondary multiple organ failure. it is also clear that tissue hypoxia can lead to organ damage. we believe that it is the interaction between the two phenomenons that can lead to mof. on the one hand, the inflammatory response is amplified in the presence of hypoxia. on the other hand, the release of the mediators of sepsis can increase tbe oxygen demand ef the tissues, alter their oxygen extraction and decrease myocardial contractility, and the combination of these elements accounts for the development of septic shock. this hypothesis is supported by two lines of evidence. first, mof is usually preceded by an episode of acute circulatory failure (even though frank circulatory shock may not be evident). second, recent experimental and clinical studies indicated that immunotherapy (especially antibodies to tnf) is particularly effective in the most severe forms of sepsis, associated with circulatory shock. hence, an effective way to prevent mof may be a combination of aggressive cardiovascular management and immunotherapy. development of the multiple organ dysfunction syndrome (mods) in the critically ill follows an heterogeneous group of stimuli including infection, sterile inflammmion, extensive tissue injury, ischemia, intoxication with a variety of substances, and immune system activation. whether the resulting physiologic abnormalities reflect a common pathologic process, or are simply a non-specific manifestation of tissue injury is unknown. moreover, the lack of clearly-defined functional criteria for the characterization of the syndrome on the one hand, and of reliable biochemical markers of its evolution on the other, has made attempts to define its epidemiology and natural history difficult. using a numeric scoring system to quanfimte the clinical severity of mods, we demonstrated that, although mods is more common in patients who have significant infection at the time of icu admission, a much stronger correlation is evident between the severity of mods and the subsequent development of nosocomial icu-acquired infection. furthermore the severity of mods correlates strongly with the severity of the clinical septic response, and with the degree of immunologic compromise evident on delayed type hypersensitivity skin testing. to determine the relative importance of the initial stimulus, and the host response to that stimulus, to the subsequent emergence of mods, we undertook a matched cohort study of critically ill patients admitted to the icu with infection, matched by age, sex, and apache ii score to uninfected controls. organ dysfunction scores were higher in patients admitted with infection, but were also significantly associated with the expression of a septic response at the time of icu admission, independent of the presence of infection. by stepwise regression analysis, the magnitude of the septic response was the most powerful predictor of the severity of mods in both infected and uninfected patients. these data suggest a model of mods in which a stimulus (eg infection) and the host response to that stimulus (eg the septic response) result in a state of altered systemic homeostasis (manifested in this example by immunologic dysfunction). moreover they suggest that the principle determinant of the emergence of mods is the response of the host, rather than the nature of the stimulus, and are consistent with the hypothesis that a stereotypie systemic response to an heterogeneous group of injurious stimuli underlies the pathogenesis of mods. arthur e. baue, m.d, ever since the attorapt build the tower ef babel, as desclibcd in the old testament of the biblc, it has been difficult to got agreement o;x common definitions or language, although mof has served well, there will always he now classifications proposed with good reasons for them, thus mods and sirs are not the final oxpressinns in the lexico~aphic sweepstakes. attcmpts to develop standard animal shock and scpsis models (hemorrhage, endotoxin, foes[ pellets, cecal ligation and puncture, e. colt hlf sioi to evaluate therapy have net been suoca.ssf~. clinical syndromes of mof, sepsis, sepsis syndrome and athers have not been aeacpte.d by all. our problem is that we arc tryittg tu d¢ffino a non-emily. mof or mods or sirs is net a disease or oven a syndrome. it is a series of complications of injury, disease and intensiva care which loads to death for many patients in intensive care units, it is not a fixed entity, but an ewilving picture. we have lumped (ugethet for therapy and definition a number of diseases and problems according to clinical manifestatieos rather than the eause of ihe problem, the search for unified mechanisms has not bean successful. will we benefit oleo from a more precise classification and definition of a non-outjty, a hodgu-podge of human disorders which have charsmeristic.s and manifestations of tissue injury, inflammation and infactian? re.hi clinical trials of potential "magle ballets" suggest that we should go in a different direction--instead of lumpors, we should be splitters, to seek effective therapy, we must fucus on specific disorders such as trauma, specific infections, inflammatory disease and chronic or acute organ damage (copd -renal failure, etc.), i will say more aboet this later ha the me.ethag. thcre is cue potentially important pert of the mods proposal, and that would be do~mcntation of organ dysfunction before fsiiure occurs and before tile need for external supporl dovelope. this could lead to hatter methods of preventing organ failure. preveution is ultimately the only answer to organ failure, m mof, mods and sirs. have wo reached a consensus?--only that we arc dealing wilh a diffl~x:lt probtolr looking for solutlons. acute lung injury (ali) and its most severe form, acute respiratory distress syndrome (ards) characterized by severe hypoxemia, diffuse infiltration of both lungs, a reduction of compliance and a wedgepresure lower than mm hare related to direct or indirect injury. when aliresults from direct injury (toxic agents, lung contusion, pneumonia), the lesions of epithelial barrier and the activation of lung macrophages are the first events leading to the release of inflammatory mediators wh:ch are responsible for alteration of the membranar permeability and for invasion of the alveoli by fluids, proteins and cells. an inflammatory reaction develops, with injury to endothelial cells, adhesion of polymorpbonuclear leucocytes (pmn) and the release in blood stream of intlammatory mediators dispersing the inflammatory reaction to other organs and tissues. the failure of lung function also results into hypoxia in other organs, leading to tissular ischemia, triggering an inflammatory reaction leading to organ dysfunction. frequent complications of direct lung injury are septic pneumonia with endotoxin release, phagocytes activation and cytokine production disseminating inflammation. by this way, direct lung injury can be at the origin of the multiple organ dysfunction syndrome (mods), the frequence of which having been estimated to % and is increased when all has occured in patients alreadypresenting an organ dysfunction (immunodepresslon, cancer...). when ali results from redirect or extrathoraclc injury (trauma, infections, hypovolemic shock, acute pancreatitis...), inflammatory mediators and micro-aggregates released in particular organs or tissues reach the lung via the blood and lymph streams, are filtered in the organ or trapped in the lung capillaries, and are responsible for a local inflammatory reactaon (endothelial cell activation, pmn trapping and adhesion, platelet aggregation, release of mediators). sepsis acts by the way of endotoxiu and sequential eytokines release (tnf, ill,...) while trauma with important blood loss acts especially by the way of hypoperfusionreperfusion phenomena leading to a disseminatedinflammatory reaction and to a possible bacterial transloeation when intestinal hypopeffnsion is present. in these conditions of indirect injury, all (or ards) is a iocal early (often the first) manifestation of a general phenomenon of altered membrane permeability and inflammatory reaction, easily and rapidly recognized by its clin~cai signs. the iung is one of the numerous organs participating to the mods, in these conditions, the mortality rate is high, reaching more than % when sepsis is present or when at least organs are implicated in mods. by direct or redirect injury, lung is so a target organ for mods and injured lung can be the cause of mods or simply a participant to this syndrome. jorge cervts-navarro main determinants for brain dysfunction are breakdown of the blood-brainbarrier and raise of intracranial pressure (icp), in second place decrease of systemic blood pressure, disorders of blood coagulation and respiratory function. the blood-brain-barrier of cerebral blood vessels can be opened by stroke, cerebral trauma, inflammation, convulsions, acute hypertension and changes in the blood osmolarity. the consequence is brain oedema, increase of lop and decrease of the perfusion pressure. a close correlation between intraventricular pressure and the fate of patients with severe brain injuries has been established. regional cbf values of to ml/ g/min are reflected in isoelectric eeg, and values about t ml/ g/min, result in loss of somatosensory evoked potentials. for ionic pump failure the threshold has been determined by values of about ml/ g/min and is reflected in massive release of intracellular potassium. in stroke and in brain trauma when the oedematogenous condition is initially localized the tissue pressure variance within and between the hemispheres lead to tentorial and falciform herniations are the common cause of death even before the critical threshold of intracranial pressure is reached. the increase of the icp over the level of the perfusion pressure leads to the arrest of the cbf. if this persists {or periods exceeding seconds of global brain ischemia loss of conciousness and developing seizures appear. if it exceeds min irreversible injuries of the brain appear. following cerebral ischemia and after severe head injury a tendency to increased coagulation can be detected. vascular occlusion may develop either as a result of local thrombus formation or from emboli caused by circulating platelet aggregates. the formation of microthrombi is triggered by the plateletactivating factor a mediator in central nervous injury also responsible for aggravation of posttraumatic brain edema. acute hemorrhagic leucoencephalitis and brain purpura are known to arise as complications of acute viral infections and following gram-negative septicaemia. institute of neuropathology, free university of berlin, hindenburgdamm , berlin, germany undoubtedly, attempts to quantify as objectively as possible the degree of dysfunction of the various organs is very valuable. however, cardiovascular failure has a peculiar place in the context of sepsis. the development of acute circulatory failure (shock) is of paramount importance as a cause rather than as a consequence of organ failure. it is therefore essential to clearly separate patients with and without circulatory failure. once this has been done, attempts to list the cardiovascular system among the other systems have been unconvincing. in a number of studies, cardiovascular complications are assimilated to a low systemic vascular resistance (svr), but since svr is basically the ratio of blood pressure over cardiac output, and since hypotension is already included in the definition of shock, then a low svr is basically equivalent to a high cardiac output, which is a characteristic rather than an ominous complication of severe sepsis. development of a low cardiac output unresponsive to fluids is usually a terminal event. it is not advisable to list other problems such as severe arrhythmias, myocardial infarction or tamponade as complications of severe sepsis. host defense dysfunction following trauma, shock and sepsis e. faist, g. f. babcock * major accidental, burn and operative injury often precipitates a profound multicentric immunologic depression that is believed to predispose patients to become anergic and thus to develop towards a higb probability of infection. anergy or a lack of immunologic reactivity stands for a total elimination of skin test reactivity and recall antigen responses in the inununocompromised patient. anergy following overwhelming trauma or major septic conditions results from a massive impairment of cell mediated immunity (cmi) together with a whole body malignant inflammationiike state. we and others have demonstrated clearly that the skeration of cmi following trauma is mainly due to the disruption of intact monocyte (moo) / t-cell interaction. within this phenomenon we see a shift of the cell ratio in the compartment of peripheral blood mononuclear cells (pbmc) with a considerable increase of prostaglandin e synthesizing m~ and a simultaneous decrease of functionally competent cd + and cd + lymphocytes. t-cell dysfunction in states of profound stress is characterized by impaired synthesis of two crucial lymphokines -interleukin- (il- ) and gamma-interferon (y-ifn). the inability to produce adequate amounts of il- , most likely attributable to alterations in the transmission of signals from the cell membrane to the nucleus, results in incomplete proliferative t-cell responses to antigenic stimuli, while a lack of ?-ifn results in inadequate m~ antigen processing and presentation. the role of the two functionally distinct t-helper subsets, t-helper- (thl) secreting principally il- and ?-ifn and t-helper- (th ) acting principally in inducing antibody formation with a secretion of il- , il- , il- and tnf-[ has still to be established within the context of major trauma. antibody formation to common protein antigens is impaired, the predominant finding is a shift from igm to igg synthesis. although excessive secretion of prninflammatory cytokines (il-u , tnf-c~, il- , il- ) has been considered to be deleterious for the host in states of major inflammation and infection, in-vitro and whole blood investigation of me function has clearly revealed that there is initially a marked suppression of moo from septic patients to synthesize and secrete proinflammatory cytokines to endotoxin. this probably will result in immunodeficiency of traumatized as well as septic patients, since these cytokines in low concentrations are involved in the upregulation of the essential humoral and cellular immune functions. abnormalities of pmn function noted after serious injury have been interpreted by some investigators as signs of unresponsiveness of this cell population, others have demonstrated signs of pmn byperactivation. latest findings revealed that there is a clear pmn dysfunction in circulating blood: % of all pmn's in this compartment show downregulated or non existent ~-integrins within hours posttrauma -these cells do not phagocytose, have a reduced respiratory burst and appear to be completely degranulated. restoration of these cell functions within days post-trauma is significantly correlated to survival of traumatized patients. other reports however stress increased expression of cr + receptors (cdll/cd complex) on circulating pmn's, increasing the ability of these cells to adhere to intercellular adhesion molecules, thus contributing to play a major role in inducing the pulmonary capillary leakage. our knowledge about the perturbation of host defenses associated with serious injury and sepsis is continuously increasing and represents the precondition for the design of effective therapeutic measures to prevent and counteract the resistance to invading microorganisms. dept. of surgery, klinikum grosshadern, ludwig-maximilians-university, munich, germany. * dept. of surgery, university of cincinnati, cincinnati, ohio, usa lg thijs~ ce n~ck sepsis is the most common cause of acute disseminated intravascular coagulation (dic) and coagulation disorders as well as abnormalities of fibrinolysis are common in septic patients. some clinical and experimental studies indicate that dic is a pathogenetic factor for the development of multiple organ failure. endotoxin and various mediators (tnf, il-i) enhance tissue factor and decrease expression of thrombomodulin on endothelial cells in vitro. also, tpa activity is suppressed and release of pai-i is stimulated. in such a way the endothelial surface becomes procoagulant whereas fibrinolysis is inhibited. following administration of endotoxin to normal volunteers levels of prothrombin fragments (fi+ ) and thrombin-antithrombin (tat) complexes increase, indicating thrombin generation. also the fibrinolytic system is activated as evidenced by a rise of levels of tpa and plasmin-antiplasmin (pap) complexes but this is counteracted by a subsequent release of pai-i. in severely ill septic patients levels of tat complexes are increased and concentrations of the natural inhibitors of coagulation (at iii, protein c) are usually decreased. also, tpa levels snd pap complexes as well as concentrations of pai-i are elevated. the severity of many of these alterations is related to mortality. thus, both coagulation and fibrinolysis are activated in sepsis, but not in a balanced way, thereby promoting coagulation. one of the hallmsxk pathological alterations associated with sepsis is the development of microvascular thrombosis, an entity ~lmre~erizod by microvas~tlar plugbing with leukoeyies (predominately nentrophils) and fibrin deposits. preranably, ischemia remlt~g fibm ve~-'ttlac ow,.luwion contributes sisni.ficantly to end orgen darnaje and consequent dysfatlctlon. p, er~t srldies haw focused pmdominately on the role of adhesion molecules in the pathogenesis of neu~ophil sequestration during infection and s~sis. this preparation will review the mechanisms underlying intravak-'v.ler i~brin deposition and its contribution to organ injury. the normal endothelial cell sure is generally conddered o be anticos~am. this state is maintained by two major anticoag~lam molecules on the cell so,ace: hepax'an su,lfale and thmmbomodulin. studies performed over the past decade have do~mentod a general shii~ towards a procoa~qtlant staw during gram negative hffeodon, aft e~| mediated mainly by changes in the endothalial cell surface. antt-tf antibodies have been shown to be pro~ective during l~'ml endotexemia. in summary, ~erova~'~ler fibrin deposition, in ~rt mediated by ~rcgulation of cellular tf, is a consistemt patholo~cal flndin~ during sepsis and contributes to organ injury. strategies designed to abrogate this event may n~nimjze the magnitude of erg~ dysftm~on. n, v, christou md phd, department of surgery, megill university, montreal, quebec, canada interactions between immune calls and the endothelium vie adhesion molecules serve to modulate immune cell traffic between the blood strum and the extrmvascular space, these families of molecules acting in a cascade and closely integrated with the oytoklna network, blood coagulation and the complement system, are responsible for the homing of leukocytes to areas of inflammation and the realrculatlon of tymphocytes. there are three types of immune ceils that must exit the blood stream into the extravascular space: lymphocytes ; polymorphonuclear leukocytes; and non-lymphocyta mononuclaar cells (monooytes, basophlls, aoslncphils). b lymphocytes exit the blood stream and recircu}ate mostly via the high endothelial venule (hev) in lymphatic tissue, rarely, in chronic }nfectlon=, they may recirqulate vie hev in non-lymphoi:t tissue. t-lymphocyte recircutation on the other hand can occur via hev in lymphoid tissue, as well as, the endothalium of the skin, returning to peripheral lymph nodes via the lymphatic channels. lymphocyte recirculation is the mechanism which allows ¢ontaot between the immune repertoire and pathogens, and homing delivers these cells to the appropriate environment for activation and for effeotor function, pmns exit the blood stream through endotbelium in close proximity to the site of inflammation after appropriate endothelial membrane receptor expression. they must reach the site of pathogen tnveslon quickly in order to be effective. their exudation to the inflammatory site can be modulated by their intravascular pdmlng, which results from appropriate receptor expression. because endotoxin leak from the gut has been postulated as an important trigger mechanism for the systemic inflammatory response syndrome seen after serious injury, the immunologic and metabolic effects of bacterial endotoxin infusion into normal volunteers can be expected to shed considerable light on the validity of this hypothesis. we and ethers have used tbis model to show that endotoxin causes a temporary paralysis in the ability of circulating monocytes to prudce the proinflammatory cytokines interleukin-i (il-i) and tnf-a. endotoxin, however, causes increased production of pge by the same cell population. endotoxin infusion causes significant suppression of circulating t-cell numbers and a profound suppression in the ability of the circulating t-cell population to proliferate and to produce interle~in- (il- ) upon in vitro stimulation. after endotoxin circulating t-cells are also more sensitive to the inhibitory action of exogenous pge . administration of cyclooxygenase inhibitors at the time of endotoxin infusion largely abrogates the effect of endetexin on t-cell proliferation and il- production. endotoxin infusion also causes the release of increased levels of circulating catabolic hormones including cortisol. administration of cortisol alone or cortisol along with endetowin to volunteers has allowed dissection of cortisol effects from those of endotoxin itself. cortisol infusion alone causes a diminution in the circulating t-cell population but the remaining ceils continue to produce il- after appropriate stimulation. cortisol infused along with endotoxin blocks the overshoot in monocyte proinflammatory cytokine production seen - hours after endetoxin infusion. at present, one may conclude that administration of sublethal doses of bacterial endetoxin to human volusteers produces alterations in cytokime production and tlymphocyte activation which are both similar and dissimilar to those seen following serious injury. hemodynamic support is based first on intravenous fluids and second on vasoactive agents. colloids are usually preferred to crystalloids, for their more rapid hemodynamic effects and their lesser passage in the interstitial space. in the presence of hypotension, the pharmacological profile of dopamine makes it the first agent of choice. it is only when high doses of dopamine are insufficient to restore a sufficient tissue perfusion pressure that norepinephrine should be added. even when cardiac output is not decreased, a dobutamine infusion is often indicated to counteract the myocardial depression, prevent vasoconstriction, and increase oxygen delivery to the tissues. the benefit derived from the administration of "renal" doses of dopamine is doubtful, but stimulation of dopaminergic receptors may increase blood flow also to the gut. in any case, none of these catecholamines has been shown to significantly improve the oxygen extraction capabilities of the tissues. agents with antiinflammatory properties but also with some vasodilating properties, such as n-acetylcysteine, prostaglandin el or pentoxifylline represent more attractive options to increase oxygen extraction. cardiovascular support should aim not only at the restoration of a sufficient arterial pressure but also at the maintenance of an optimal oxygen delivery to the cells. it should thus be guided also by measurements of cardiac output, mixed venous oxygen saturation and oxygen extraction, and indexes of cellular hypoxia, such as blood lactate levels, gastric intramucosal ph or vanearterial pc gradients. supranorma[ delivery contributes to the prevention of tissue hypoxia tissue hypoxia is considered to be the common final pathway in the pathogenesis of multipte systems organ failure. it may directly contribute to cellular injury and organ dysfunction as well as enhance further mediator activation and release by a positive feed back mechanism. prevention of tissue hypoxia, is therefore, one of the most important aims in the supportive therapy of critically ill patients, especially in those with sepsis and septic shock. mismatch of cellular o= supply and demand in these patients may resuit from the impairment of the cardiocircu[atory system on all levels. . myocardial depression with a drop in delivery (do~) . redistribution of blood flow between the different organ systems . inadequate nutritive blood flow due to tissue edema and endothelial damage (gic, leucocyte swelling etc.) these pathological changes may go along with increased metabolic needs. cellular and organ supply may therefore be inadequately matched with cellular needs in patients with normal and even supral normal doz. hyperdynamic circulation is often present in adequately volume resusucitated patients. it is most likely that one of the bodys main compensatory mechanisms would maintain cellular o= supply in the face of increased metabolic needs and impaired oz extraction capabillities. this pathophysiology may explain the findings in several clinical investigations that patients with normal or even supranormal doz can have signs of inadequate regional tissue oxygenation. it is also consistent with the results of different studies which demonstrated that the achievement of supranormal doz levels, either spontanuously or due to adequate supportive therapy, resuits in better patient outcome. any attempt to prevent or treat tissue hypoxia in these patients, however, has to take into account the effects of therapy not only on global dgz but especially on regional and microcirculatory blood flow. r. rossaint, d. pappert, k. lewandowski, h. gedach, k. slama, k.j. falke klinik for anaesthesiologie und operative intensivmedizin, ukrv, freie universit §t berlin, germany important contributory factors to the high mortality of severe acute respiratory distress syndrome lards) are the aggressive therapies required, such as mechanical ventilation with high inspiratory oxygen concentrations and airway pressures. as specific therapies for reducing or preventing the general inflammatory reaction of the lung resulting in severe hypoxemia is unknown, today's therapy is limited to procedures which predominantly support or maintain pulmonary function, i.e. pressure limited ventilation with peep and permissive hypercapnea, avoiding or treatment of fluid overloading, and positional maneuvers. extracorporeal lung assist combined with low frequency positive pressure ventilation has been recommended, when conventional therapy fails. today, extracorporeal gas exchange may be carried out using a system internally coated with covalently bound heparin. this allows for a lower level of systemic heparinzation reducing the risk of severe hemorrhagic complications of extracorporeal respiratory support. from april to august patients, aged from months to years, were transferred to our intensive care unit (icu) for treatment of severe ards. all fulfilled at least the slow entry criteria of the u.s. national ecmo study. due to hypoxemia patients required veno-venous extracorporeal membrane oxygenation (ecmo) immediately after transfer to our icu. the other patients were first treated with pressure controlled mechanical ventilation, permissive hypercapnea, prone position, and dehydration, if necessary. in out of these patients, in which the gas exchange did not improve in the following days, veno-venous ecmo with heparin-coated systems was additionally performed. heparin was given to achieve a partial thrombin time between - s and an activated clotting time between - s. if surgery was performed during ecmo, heparin infusion was interrupted. no severe bleeding complications related to anticoagulation for the extracorporeal bypass could be observed, however, in some patients, after three weeks of ecmo thrombi in the drainage cannula were observed. a problem of of membrane leakage shortened the life span of the membrane lungs to a mean of about four days. in the conventionally treated group % survived and in the additionally with ecmo treated group % survived, representing a % survival rate in patients whose risk of death is considered more than %. on the basis of these experiences, we conclude that the described strategy, including veno-venous ecmo with heparin-coeted systems, may improve the survival in patients suffering from severe ards. abnormally high skeletal muscle po in septic patients and its normalization during recovery: evidence against tissue hypoxia but for impaired oxygen metabolism of skeletal muscle? p. boekstegers, k. werdan department of medicine i, gro hadern university hospital, munich, germany a pathological oxygen uptake supply dependence was suggested to indicate tissue hypoxia in sepsis-a hypothesis which is discussed controversially. because the detection of reduced oxygen transport to tissue and &tissue hypoxia would have important implications for therapy, skeletal muscle oxygenation was studied in patients with sepsis. intermittent and continuous determination of skeletal muscle po by polarographic needle or catheter probes provided no evidence for skeletal muscle hypoxia even in severe stage of sepsis (boekstegers et al., crit care med ) . in contrast, mean skeletal muscle po was found to be abnormally high in patients with sepsis ( _+ , mmhg, n= ) compared to patients with limited infection ( , + , mmhg, p< . , n= ) , to patients with cardiogenic shock ( , + , mmhg, p< . , n=ls) and to healthy subjects ( , _+ , mmhg, p< . , n= ). furthermore, serial measurements in patients with sepsis showed that skeletal muscle po was higher ( , _+ , mmhg) on days with severe sepsis than on days with intermediate state ( , _+ mmhg) and than on days without sepsis ( , _+ , i mmhg) . thus, a decrease of abnormally high skeletal muscle po was related to improvement of sepsis. this was also demonstrated by comparing the decrease of abnormally high skeletal muscle po with the decrease of apache ii score or elebute score as well as interleukin- serum levels in a separate study (boekstegers et el., shock, in press). in summary, our data provide no evidence for skeletal muscle hypoxia in patients with sepsis but support the assumption &impaired oxygen metabolism in severe sepsis. neutropnlic emigration from the vascular space into the extracellular matrix is important for the response to tissue injury or contamination by providing a large recruitable pool of tissue-based phagocytes. the consequences of neutrophil exposure to intravaseular chemoattractants, likely relevant for il- and c a in sepsis, trauma, and in burn injury, are suppression of neutrophil attachment to endothelial cells and matrix proteins. it is probable that proteoglycan-bound attractants are of considerable biologic relevance, and may result in enhancement of responses to subsequently encountered cytokines. in the presence of connective tissue proteins expressing integrin-specific binding ligands, neutrophils respond to tnf-cq gm-csf and other cytokines by producing large amounts of hydrogen peroxide. this response is likely important in the digestion of contaminated or injured tissue by facilitating activity of neutrnphil granular proteases and inactivating plasma anti-proteases. this matrix-dependent oxidative response is important in defining potential novel targets for therapeutic intervention. the response appears to involve signalling by integrin-linked tyrosine kinases. the net effect of matrix protein injury through this mechanism is enhancement of the fibrotic response which underlies much of the prolonged ventilator dependence of patients with the adult respiratory distress syndrome. this adherence dependent response has also been implicated in direct hepatocyte injury, and may represent an early component of the syndrome of multisystem organ failure. study purpose: in this patient (pat.) population with a considerable sepsis morbidity and mortality, parameters proposed for sepsis assessment (elebute sepsis score [ele], sirs) were investigated with regard to their use in the early classification of pop. pat. at risk for developing sepsis. patients and methods: in a previous observational study on consecutive pat. after elective open-heart surgery, we had determined ele daily for at least pop. days (in pat. with a longer stay: until icu discharge). after publication of the newly proposed sirs definition, these criteria were retrospectively calculated and compared to ele. results: on the total of n = patient-days, both ele and sirs showed significant (p< . ) correlations with parameters reflecting the general and sepsis-related severity of the pop. clinical course. compared to sirs, the ele correlations were better (icu mortality: . vs. . ; icu treatment days: , vs. . ; days on mechanical ventilation: , vs. . ; days with vasopressor requirement: . vs. . ; number of microbiological findings: . vs. . ), the optimal classification criteria for the mainly sepsis-related outcome gave maximal youden indices of . for ele (ele >_ on >_ days, accuracy: %) compared to . for sirs (sirs present on > days, accuracy: %). accordingly, ele roc curve areas for both overall ( . ) as well as sepsis-related prognostic evaluation ( . ) were significantly (p< , ) larger compared to sirs ( . and . , resp.), this higher overall accuracy of the ele criterion was primary due to a more valid assessment already on the first and second pop. day, where sirs still had a high false positive classification rate ( % and %, compared to % and %, resp.). conclusion: in the early postoperative course after cardiac surgery, the sirs definition displayed a high false-positive classification rate (low specificity) for subsequent sepsis-related mortality compared to better classification results obtained by the elebute sepsis score. from the departments of medicine i and of "cardiac surgery, grosshadern university hospital, marchioninistr. , d- munich, frg. correlation between physiological and immunological parameters in critically ill septic patients. ma rogy, h oldenburg, r trousdale, s coyle, l moldawer, sf lowry a relationship between physiological parameters of severe sepsis and immunological function has not been established. in an effort to assess such a relationship we prospectively evaluated nine severely ill septic patients. physiological risk was assessed by the apache iii score , while one component of immunologic function was evaluated by peripheral blood mononuclear cells (pbmc) eytokine production after in vitro lps stimulation . four of the nine patients died. apache iii scores at h were lower in survivors (s) than in non-survivors (ns), ( -+ vs -+ p< . ), while apache iii scores at admission were not significant different between s and ns ( -+ vs -+ ). down regulation of cytokine production by pbmc upon lps stimulation was a transient event in s. while s demonstrated an fold increase of tnf-a bioactivity with[r~ hours, ns did not demonstrate any increase at all. a similar pattern was demonstrated for il- [ and il- immunoactivity. tnf was measured by wehi bioactivity, il- [~ and il- immunoactivity were determined by elisa. the sensitivity was pg/ml for tnf, pg/ml for il-ll and pg/ml for il- , respectively. in conclusion, both physiological as well as immunological functions of severe critically ill septic patients demonstrate predictive value for ultimate survival. while patients biological status seems to be more predictable by apache iii at day , p< . , the pattern of cytokine production by pbmc upon lps stimulation over the first h might be a reliable predictor as well. introduction: therapy of sepsis and its sequelae depends largely on its early recognition. many studies have investigated the change of certain mediators during sepsis and their potential to predict multiple organ failure and outcome. it was the objective of this study to investigate whether the onset of sepsis can be predicted by alterations of levels of interleukin- (il- ), tumour-necrosis-factor (tnf), pmn-elastase and c-reactive protein (crp). materials and methods: over a one year period, polytraumatized patients were prospectively studied (mean age y, % male, iss ). serum and edta-plasma samples were taken in h intervalls until the patient left the icu. il- , tnf, elastase, and crp were determined immunologically. sepsis was defined according to the criteria of 'systemic sepsis' (veterans" administration study, ) with at least of clinical signs: ( ) tachycar-dia> /min, ( ) temperature > , °c, ( ) blood pressure < mmhg, ( ) mechanical ventilation, ( ) leukocytosis > . /ml, ( ) thrombocytopenia < . /ml and ( ) presence of an obvious septic focus. clinical parameters, sepsis severity and serum levels were documented on a daily basis, beginning on day after trauma. results: of patients developed a systemic sepsis ( . %), and died. all mediator levels were elevated under septic conditions. the clinical severity of sepsis correlated well with the respective levels of mediators. in patients, who developed a sepsis the following day, il- ( vs. ng/l; p= . ), crp ( vs. mg/l; p= . ) and tnf ( vs. ng/l; p= . ) were significantly increased as compared to those patients who remained non-septic. elastase levels were considerably elevated but did not reach the level of significance. we conclude that il- , tnf and crp appear to be sensitive markers for prediction of septic complications in polytraumatized patients. objectives of the study: the assessment of liver function in polytraumatized patients who are at risk of developing mof is too inaccurate and late by using conventional biochemical parameters. methats: the injury severity of the patients (n= ) was determined by the injury severity score (iss). lidocaine is given at a dose of mg/kgbw over rain. i.v. and is metabolized in the liver by a cytochrome p- mechanism to monoethylglycinexylidide (megx). the metabolite is measured by a fluorescence polarization immunoassay. serial determinations of the test were performed between the ~t and the ~ day after trauma and were compared with other liver function tests (bilimbin, gldh, alt, ast). the systemic inflammatory response syndrome (sirs) is still a challenge concerning early diagnosis, therapy and prognosis. therefore, evaluation of inflammatory and disease activity becomes more important. c-reactive protein (crp) is a well established acute phase protein in chronic inflammatory diseases. recent reports suggest an induction of crp by interteukin- (il- ), a cytokine involved in the mediator cascade of sirs. on the other hand, tumornecmsisfactor alpha (tnfcx) is a very early released mediator in sirs removed very rapidly from circulation. in addition, soluble tnf receptors (stnfr~ , stnfr ) are released into circulation in the acute phase response. this study examines the kinetics of five acute phase proteins (crp, il- , tnfot, stnfr , stnfr ) in patients suffering from sirs. eighteen patients entered the study after diagnosis of sirs. blood samples were drawn every six hours during the first two days and every twelve hours thereafter. crp was measured in an routine turbimetric assay. il- was detected in an biological assay using the/l- dependent -cell line / . detection of tnfc~ was performed in an elisa system using a monoclonal antibody" for tnfo~. soluble tnf receptors were also measured by elisa. crp levels were elevated (> mg/l) in all patients and at all time points. crp values did neither differ significantly in patients with ( ± mg/l) or without ( a: ) multiple organ failure (mof) nor in survivors ( ± ) or non-survivors ( :t: ). in contrast, l- was elevated in patients wilh mof (mean pg/ml, range - pg/ml). il- levels correlated especially with lung dysfunction. tnf(x levels were consistently elevated in patients with mof. crp, il- and tnfoc did not correlate with each other. in contrast, levels for both stnfr showed a positive correlation (r= . ). patients could be divided into two groups by values for stnfr~ and stnfr : the group with higher soluble tnf receptor levels showed increasing values combined with a poor prognosis. the group with lower levels of soluble tnf receptor consisted of patients surviving mof or without mof. in conclusion, crp does not monitor the course of sirs adequately. in contrast, il- correlates with mof and episodes of high disease activity. high stnfr levels may indicate poor prognosis. klinik f r an/isthesiologie and operative intensivmedizin der cau kiel, schwanenweg , kiei, germany. ch. waydhas, md; d. nast-kolb, ivid; m. jochum, phi); l. schweiberer, mi) objective: to evaluate the irfflarranatory response after different types of orthopedic operations and compare them with the systemic effects of accidental trauma of varying severity. patients: in consecutive patients with multiple injuries (iss . ) the inflammatory response to trauma was prospectively studied. the patients were divided into groups according to their iss points. additionally, the alterations after secondary operations (> hr) were determined (msteosynthesis of the femur (n= ), pelvic girdle (n=ll) and spine (n= ), facial reconstruction (n= ), smaller osteosynthesis (n= ) and others (n= )). methods: specific and unspecific parameters of the inflammatory response were determined in the trauma patients every h, beginning on admission of the patient to the emergency room for a period of hr, and in the operative patients on the morning of the operation, at the end of the procedure and every hr during the first two days. results: lactate, neutrophil elastase, heart rate, po /fio -ratio, and other parameters discriminated significantly between the injury severity groups during the first hr (kruskal-wallis-test, p<. ). the degree of postoperative changes differed significantly (kmskal-wallis-test, p<. ) between the types of operations for lactate, heart rate, po /fio -ratio, nitrogen excretion and showed a strong discriminating tendency for neutrophil elastase and c-reactive protein. the extent of changes were highest after operations of the pelvic girdle, followed by procedures on the femur, spine, smaller bones, and the facial region. the postoperative changes after osteosynthesis of the femur or pelvis were comparable to the alterations noticed after smaller (iss to ) or moderate (iss to ) accidental trauma for neutrophil elastuse, heart rate, po /fio -ratio and parameters of the coagulation system. conclusions: there is a considerable inflammatory response to operative procedures that varies with the type of surgery. large operations cause changes in the body homeostasis that resemble those after multiple injuries. it remains to be established whether the inflammatory sequelae of surgical trauma are additive to the changes caused by accidental trauma. objective of the study: we retrospectively compared characteristics of elderly patients (~ years) and yeunger patients admitted to a surgical {sicu) and a medical intensive care unit (micu). we further studied the relations between advancing age, chronic disease, sepsis, organ system failure (osf) and mortality in the elderly group. material and methods: during a -year period, patients were consecutively admitted into the icu; and during a -year period, patients were consecutively admitted to t~mich. criteria for chronic disease, sepsis, osfsi.e. cardiovascular (cf), pulmonary (pf), renal (rf), neurological (nf), haematological (hf), hepatic (lf), and gastrointestinal failure (gf)-were derived from the literature. results: patients from the sicu and~cu were similar in age, number of osf, and length of stay. however, when compared to sicu patients, micu patients had more cf (p<o.o ), sepsis (p<o.o ), and mortality rate ( % vs. ii~, p<o.ool). in contrast, sicu patients had more hp and nf, and prior chronic disease (all, p<o.o ). of the total group of , patients, there were ( %) patients years of age or older. elderly and younger patients had comparable length of stay in the icj, but elderly patients had significantly more chronic disease, sepsis, and number of osf (all, p<o.o ). all osfs, except hf and nf, were more ~o~mon i~ mdeljl, eompard to you-ger patients. ~he mortality is also higher in elderly patients ( % vs. % in younger patients). after multiple logistic regression, only number of osf increased mortality by a factor of . ( % confidence limits, . - . ), age did not increased [odds ratio . (i. i.i )], while admission to the sicu reduced risk of death by a factor of . ( . - . ). conclusions: based on these results, we conclude that age does not have any impact on icu outcome in the elderly. the only prognostic factor is the extent of acute disease, as measured by the number of osf. hence, prevention of oaf may influence outcome in elderly patients with critical illness. the lower risk of death in sicupatients may be explained by the large proportion of postoperative elective patients with relative mild chronic disease, and the lower incidence of sepsis. department of surgery, free university hospital, de boelelaan , ~v amsterdam, the netherlands. septic shock and low output syndrome h.miyakawa, t.noguchi, s.hattori, a. mizutani, m. mori and n.honda objectives: cytckines are thought to cause the acute phase reactions under several critical conditions. we had investigated the relationships between cytokines network which included il- , l- ,acth and cortisol,and the outcome of the patients with septic shock or low output syndrome (los). materials and methods: in the septic group and los group,nine and four patients were enrolled respectively. furthermore, the patients in the septic group were divided into two groups,survival (n= ) and non-survival group (n= ).tn these patients,ll- , l- ,acth and cortisol had been measured every day after diagnosis of septic shock or los.the total number of measurements were points in survival septic group, l l points in non-survival septic group and points in los group.statistical analysis was used student's t-test to compare the mean of each group,and the changes were reported as clinicat significant if p< . . results: il- levels in non-survival septic group were higher than in both survival septic and los group.ll- levels in non-survival septic group were more significant than in both survival and los group.otherwise,there were no differences with respect to acth and cortisol among three groups. conclusions: we assumed that los would make several organs dysfunction as well as septic shock. but our results showed septic shock, especially non-survival,had different cytokins network mechanism for organs failure from los. lasson ,~, g/ ransson j, jijnsson k. eady diagnosis of complications after trauma is imperative to decrease morbidity and mortality. for this purpose an easy, cheap and fast diagnostic method is needed. the aim of this study was to analyse if complications after surgical trauma of various extent could be diagnosed earlier using preoperative or daily postoperative measurements of various plasma proteins than by using climcal signs of complications. material and methods: two acute phase proteins (c-reactive protein and antichymotrypsin), lbur main plasma protease inhibitors (alpha- -maeroglobulin, c estemse inhibitor, antithrombin ill and alpha- -antiplasmin) and one collagen precursor (procollagen iii peptide) were measured preoperatively and then once dally for days postoperatively. for the plasma protease inhibitors immunorcactive as well as functional levels were measured. haemoglbin, albumin and the white blood cell count were also measured. measurements were done preoperatively in patients and continued postoperatively in patients, % of the patients were operated on for malignancy. resulls: preoperative plasma c-reactive protein levels were higher in patients developing postoperative complications. this discrimination increased when plasma was sequentially analysed postoperatively, when a remaining high level ora second increase in plasma levels depicted a complication - days earlier than clinical signs. high levels were seen both in patients developing local as well as in patients developing general complications. there was no clear correlation between plasma protease inhibitory levels and complications, neither pre-nor postoperatively. procollagen iii paptide levels were slightly (but not significantly) higher postoperatively in patients developing complications. contusions: the acute phase response, especially concerning c-reactive protein, predicts postoperative complications better than both plasma protease inhibitors or collagen precursors. preoperative plasma c-reactive protein levels indicate the risk of postoperative complications, while daily postoperative measurements more readily depicts a complication, usually preceeding the clinical signs of complication by - days. dept. of surgery m'alm general hospital, s- i malmr, sweden. mof is a major threat to the extensive burned patients and associated with a high mortality rate. the role of endotoxemia in the pathogenesis of mof in burned patients remains controversial. in this study, we examined the relationship of plasma endotoxin levels to development of mof, and outcome in patients with thermal injury. methods: a prospective cohort study of patients admitted with burns covering more than per cent of body surface area was undertaken. circulating endotoxin concentrations were measured by modified limulus amebocyte lysate assay in serial samples of plasma. results: out of burned patients developed mof according to multiple criteria. the plasma endotoxin concentrations of patients with mof were . -- . eu/ml, significantly higher than that of patients without mof ( . - . eu/ml) on , , and days postburn (p< . -- . ). significantly more incidence of positive endotoxin test (>_ . eu/ml) was found in patients who developed mof as compared to that of non-mof during the observation period (p< . ). as the mean endotoxin levels increased, the prevalence of mof and death also increased (see table below), persistent endotoxemia carried a poor prognosis. conclusions: the present investigation provide further evidence that endotoxemia in severely burned patients commonly occur. cimulating endotoxin has also been found to be strongly associated with development of mof and mortality following major burn injury. multiple hemostatic changes occur in sepsis mad multiple organ failure (mof). to evaluate the role of platelcts in patients with sepsis and mof, we examined changes in surface glyeoproteins on circulating platelets of t patients with suspected sepsis and mof. the severity of sepsis and mof was assessed by eiebute and apache i scoring system, respectively.using flow cytometric techniques and platelets specific monoclonal antibodies, platelet surface expression of fibrinogen receptor on gpiib-iiia, ofvon willebrand receptor gpib, and of granula glycoproteins (thrombospondin, gmp- , and gp ) was measured. receptor density of gpiib-illa mad gpib on circulating platelets was not affected by sepsis or mof. in septic patients surface expression of activated fibrinogen receptor (libs expression) was significantly elevated (p< . ) and correlated well with severity of disease (f . ). no significant change in surface expression ofthrombospondin, gmp- or gp was noted in septic patients. in contrast, degranulation ofgraanle glycoproteins was significantly elevated in mof (! < . ) that correlated well with severity of mof (gmp- , r= . ; thrombospondin, r= . ).we speculate, that platelets in sepsis circulate in a hyperaggregable (fibrinogen receptor activation ) but still reversible state that results in increased risk of microthrombotic events. in the course of the disease, irreversible platelet degranulation might occur and may play an important role in development of mof. abdominal sepsis is still associated with high morbidity and mortality. the present study aimed at evaluating patients with abdominal sepsis treated at our surgical intensive care unit during a -year period with the aim of identifying potential prognostic factors, bacteriological cultures, diagnostic procedures, treatment and outcome. during the period - i patients with abdominal sepsis were treated at the icu at our university hospital. patients were women and men with a mean age of ( - ) years. in cases, the abdominal sepsis occurred as a postoperative complication. the patients were scored according to apache ii and bacteriological cultures and the occurrence of organ failure were noted. the patients were hospitalized in median for (- ) days out of which (- ) in the intensive care unit. out of patients ( %) died in median after ( - ) days. the primary cause of mortality was multiple organ failure ( / ; %). apache ii scoring could not predict a fatal outcome. abdominal bacterial cultures were dominated by bacteria of enteric origin ( %) and in % cultures grew multiple bacteria. patients bad organ failure and multiple organ failure. / patients ( %) had abdominal sepsis due to diffuse peritonitis despite a morphologically intact gastrointestinal tract and the absence of localized abscess formation. mortality in this group was significantly higher as was the percentage of positive blood cultures and the occurrence of multiple organ failure. abdominal sepsis is still associated with a high mortality, predominantly caused by multiple organ failure. abdominal culture findings are dominated by bacteria of enteric origin. in about / of patients with severe abdominal sepsis a diffuse peritonitis with intact gastrointestinal tract without localized abscess formation was found. in this group the mortality was increased as well as the risk of developing multiple organ failure. during the period from january to september patients, mean age + years were referred to our department of resuscitologywith the diagnosis of eclampsia. all the patients were delivered by cesarian section and were mechanically ventilated for . _+ . days. diagnosis of sepsis was confirmed in cases by clinical and microbiological methods. patients were divided in two groups: lnon septic patients, -patients with sepsis, the control group consisted of patients after cesarian section without symptoms of eclampsia or infection. we determined plasma concentrations of immunoglobulins a,g,m(a,g,m), complement factors (c ,c ), alphal-antitrypsin (aat), trausferrin (trf) and albumin (alb) using beckman (usa) analyzer,protein concentration, using kone (finland) analyzer. a(mg/dl) g(mg/dl) m(mg/dl) c (mg/dl) c (mg/dl) k +- + _+ + +- -+ " -+ * _+ " -+ ' _+ " +_ '* -+ ** -+ "* -+ "* _+ " in a prospective study we investigated serum of severly traumatized patients withdrawn directly after admission at our hospital (tr i). follow up controls were taken daily until day ten after trauma (tr ii). two control groups were performed: serum of healthy volunteers (co, n = ) was investigated as. well as serum of patients undergoing elective herniotomy (n= ) hours before (op i) and hours after operation (op ii). serum bactericidal index (sbi) was determined using a hemolytic e.coli strain :k :h . / suspension with a final concentration of - cfu were incubated with l oopl serum. after overnight incubation sbi was calculated according a special formula. results: co . _+ . opi . _+ . opii . _+ . * tri . _+ . "* trii . + . ** (*:p< . ; **:p<o. ) sbi represents a simple and reproducible method to detect immunobiological alterations after trauma and elective surgery. patients undergoing elective surgery showed slight but significant diminuation of sbi h after operation. deterioration of sbi was observed in serum of traumatized patients directly withdrawn after admission at our hospital. ten days after trauma significant improvement of sbi was found. further investigations have to be done to proof sensitivity and prospectivity of this simple but reliable test. a sepsis like syndrome (sls) occurs in i % of our patients following cardiac surgery. sls is characterized by a severe decrease in systemic vascular resistance requiring the use of vasopressors to maintain adequate hemodynamics in the presence of negative blood cultures. in order to determine whether preoperative factors predict the occurrence sls we retrospectively studied patients with sls (group i: age . ~ . years); they were compared to control patients (group : age ± . years). treatment of sls consisted of aggressive fluid replacement and norepinephrine infusion. rydrocortisone ( mg/ hrs) was given on the first day. antibiotics were switched prophylactic to cover gramnegative bacteria in pts. preoperatively pts in group revealed a significantly lower cardiac index ( . ± i/min/m ) compared to group ( . ± . i/min/m ; p < . ) higher left atrial pressure (group i: . ~ . mmhg; group : . ~ . mmhg; p < . ) and lower ejection fraction (group i: . z . %; group . ± . %; p < . ). bypass time, minimum and mean blood pressure on bypass and aortic clamp time were not different between the groups. immediately postoperatively (pop), the white blood count was elevated (group i: , ± , /ui; group : , ~ , /ui; p < . ) and core temperature hours pop was higher (group i: . . °c; group : . ~ . °c; p < . ). serum lactate was already elevated on arrival on the icu pop (group i: , z . mmol/l, group : . ~ . mmol/l) but dropped within hours (p < . ). icu stay (group i: . ! . ; group : . ± . days; p < . ) and mortality over months (group i: . %; group : . %; p < . ) were significant higher in group i. sls is observed more frequently in patients with preoperative cardiac congestion and results in longer icu stay and higher mortality. although intraoperative hemodynamics are similar in both groups, the white blood count and serum lactate levels suggest an intraoperative cause for sls. further investigations will focus on possible intraoperative causes of sls. the objectives of the study were to estimate the problem of stress ulcers in critically ill patients and to compare ranitidine and omeprazole to determine their efficacy in the prophylaxis of stress ulcers. patients who were admitted to the intensive care units between january and july ' with polytrauma, sepsis or those requiring ventilatory support for more than hours were selected for the study. the patients were randomized into groups to receive ranitidine, omeprazole or a placebo. the changes in gastric ph were monitored hourly and the patients underwent an upper gi endoscopy hours after the start of the study to look for stress ulcers and hemorrhage. the drugs were withdrawn one week after the start of the study. of the patients selected for the study, had undergone cardiac surgery, had polytrauma and had sepsis. the change in average pre and post drug gastric ph was + . in the omeprazole group, + . in the ranitidine group and -). in the placebo group (p < . ). endoscopic evidence of stress ulcers was seen in % of patients not on prophylaxis, . % of those on omeprazole and . % of those on ranitidine. while all the patients in the placebo group who had stress ulcers showed endoscopic evidence of hemorrhage, % of the patients with ulcers in the ranitidine group and none of the patients with ulcers in the omeprazole group showed hemorrhage (p < . ). in critically ill patients the incidence of stress ulceration is high. an alkaline gastric ph is protective against stress ulcers. omeprazole when used for prophylaxis against stress ulcers in critically ill patients or those on short term ventilation will reduce the incidence of formation and hemorrhage from stress ulcers. the early and accurate diagnosis of sepsis is of key importance for critically ill patients survival. many studies have shown that tumor necrosis factor ~x (tnf ~) and interleukin- (il- ) are mediators of the inflammatory response in sepsis. bacterial antigens interact also with antigen specific t cell receptors and the activation of t ceils takes place. activated t cells release soluble interteuldn- receptors (sil- r). the aim of this study was to evaluate: the significance oftnf c~, il- and sll- r in the diagnosis of bacterial sepsis and to evaluate the correlation of clinical and laboratory parameters with serum levels of tnf ~x, il- and sll- r. we examined patients with clinical signs of sepsis treated in the intensive care units of university medical centre ljubljana. venous blood was drawn from each patient within hours after the first clinical signs of sepsis. clinical data (body temperature, blood pressure), laboratory dam (peripheral white blood cell count with differential, platelet count, c-reactive protein), and quantitative blood cultures were recorded. serum levels of tnf c~, ,- and sil- r were measured in septic patients and in adult healthy controls. tnf ct, il- and sil- r serum levels were measured by commercially available ria and elisa (amersharn and eurogenetics) kits. the differences in serttm levels of tnf ~, il- and sil- r between healthy control and patients and correlation between clinical and laboratory parameters were calculated. we found that only sll- r serum levels were significantly (p< . , student t test) increased in patients in comparison with healthy controls. of all indicators of sepsis that we compared, only hypotension was significantly correlated with tnf ~x levels and leukocytosis with ] ,- levels. we conclude that only the increased serum sil- r levels were predictive of bacterial sepsis within hours after the first septic signs. tnf c~ and il- levels were not significantly increased at that time in lifts small group of patients. in order to create high precision prognostic system for patients with sepsis and septic shock we have made prospective and retrospective analis of cases of sepsis. patients were included in this analis according bone's criterions of sepsis. comparing with apache-h, we have excluded such phisiologic variables as:temperature,na+ ,k+ ,}it, ph,which had a little influence on prognos of illness. we have added the other, more important variables: biilirubin, plateles,pti. we took into account: the seat arrangement, methods of respiratory support, comorbid conditions as: caneer, diabets,obersity. in general new score has headings: -physiologic variables, -age, -seat arrangement, -chronic comorbid conditions, -methods of respiratory support. roc -analis have showed a greater precision of our score, compared with apache-ii score. there is significant difference between the roc-curve of apache-ii scare. the k.p.tit~v.,i.e.gurmanchuk. objectives of the study. sepsis is a severe complication of the local infection, th e fever and the systemic inflammatory response syndrome may take place in such a case as manifistations of the inflammation induced by bacteria. the systemic fever observed in infection is known to develop due to the action of certain cytoldnes. other systemic manffistation of inflammation are the production of acute phase reactants, acute phase proteins, the activation of the complement system. ivratertals and methods. we observed children with light and children with severe course(l- class of serionuess) of the sepsis. the ftmetional activity of the complement system (ch , level of c -c , factors b told d, c a), level of c-reactive protein (crp} and tumor necrosis factor (tnf) were investigated, results. the level of crp was increased in the children with more severe class of the septic process in both group ( %- st and ioo%- nd}( with light and hard eours of disese) of patients. parameters of the complement system -the activity of cl, c , c components, activity of b and d factors-were increased in the seram of children with light course of sepsis. in children with severe course of sepsis these parameters were signffieantty decreased, especially in the group of chiidrellwith higer level of crp. we found the negative correlation of the tnp-alfa concentration with the functional activity of classical pathway components (cl~ ) and the positive onewith the funfional activity of the alternative pathway factors b-and d of the complement system. conclusions. thus, in children with different course of sepsis certain changes in levels and functional activity of an acute phase o[ inflammation proteins -crp and the complement system ones -&ire characteristic. the relationship between the tnf-alfa level and the activity of classical and alternative pathways proteins indicates on its role in the complement proteins genes expression. schr der j, braun j, rennekampff ha, schr der dw various alterations of the immune response are known in trauma, but the course will not be complicated by multiple organ dysfunction syndrome (mods) in all patients. phenotyping of cells offers a rapid system of evaluation certain aspects of the immune response. different subsets of lymphocytes and neutrophils were determined to evaluate their prognostic role in developement of mods. in trauma patients with an injury severity score (iss) > (mean iss = ; mean age years) lymphocyte and neutrophil phenotypes cd (t-cells), cd (t-helper cells), cd (t-suppressor cells), ratio cd /cd , cd b (receptor for cr ) and cd (fcriii) were measured on day , , , , and post trauma. the expression of class ii histocompatibility antigen (hladr) on monocytes (hladr+ cd ) and il -receptors on t-helper cells (cd /cd were determined as well. the percentage of cells was monitored by immunofluorescence using monoclonal antibodies and three color cytometry. the percentage of hladr+ cd were significantly lower an day , , and in patients who developed mods (p< , ) compared to patients without mods and a healthy control (p<o,o ). the expression of il receptors on cd was significantly different only on day . no differences could be measured in lymphocyte phenotypes cd , , and cd /cd -ratio as well as in expression of cd b and cd on neutrophils. class ii histocompatibility antigen (hladr) on monocytes offers the best ability to reflect cellular immunosuppression in trauma. this parameter allows the best differentiation of patients with and without mods. we retrospectively studied the relative importance of age, prior chronic disease, sepsis and organ system failure (osf) to determine outcome in critically ill patients with acute renal failure (arf). arf was defined as serum creatinine > /zmol/i, a twofold creatinine rise in prior renal insufficiency or the need of acute renal replacement therapy. definitions for prior chronic disease and other osfs -i.e. cardiovascular (cf), pulmonary (pf), neurological (nf), haematological (hf), hepatic (lf), and gastrointestinal failure (gf)-were derived from the literature and described previously. of the consecutively admitted patients to a surgical and a medical intensive care unit during -ye r period, ( %) had arf. arf mortality was %. ninety-eight percent had other osf. overall, cf, pf, gf, and nf was significantly more common in nonsurvivors than in survivors (all, p<o, ). although both the number of osf before and after arf was higher in nonsurvivors than in survivors (p< . ), the number of osf before was higher compared to after arf in both groups of patients (p< . ). furthermore, age, cf and pf before the ontset of arf, nf thereafter, and renal replacement therapy were related to mortality (all, p< . ). however, prior chronic disease was not related to mortality and chronic renal insufficiency was inversely related to outcome (p< . ). after multiple logistic regression, advancing age, cardiopulrnonary failure and sepsis before arf, and nf after arf remained significant. these results show the high prevalence of arf in critically ill patients and that arf rarely occurs alone. the prognosis of arf in critically ill patients is poor, especially if associated with advancing age, additional osf, particularly cardiopulmonary failure and sepsis before arf, and nf after arf. hence, prevention of cardiopulmonary and sepsis before the outset of arf may influence outcome in arf in patients with critical illness. the search cominnes for a magic bullet ta help critically ill petienta, bat recent elini ',.ml rials of agents that showed ixomise in animal studies raise questions about such trials. expcrlmemai uvidan~ :in anlmats and human volanteet~ for concepts, mechanisms and treatment of inju~ ur illness can be substentlal, l~rauasive and exciting but the positive effects of potential therapy suggested by such animal and ctinieal research may be difficult to prove in sick patients. efficacy of a new txeatment can be difficult to prove became elinie.al situations, hi spi~e of important biologic phenomena, are v~iable and conrplex. there is redundancy and overlap of mediators ~d problems of timing of therapy. a majt~r cause of this dilemtns is not with the trials or hypotheses, but rather the promem of the cause or the causability of the human dise~.se that is trebled. this is/hu "causa nets" or '*specific cause" as described by stehbens, when prevention or tre.atmcnt is based em the sauna nero, extinetiem of the disease is ix~ssible as with saul/ pox. only elimination of the cause wl cure the disease. careful animal studit~s evaluate single pcrturbmiuns for survival or other changes. an ldso preparation may be infiuancad aignificantly by a ~mall clangs which may be insignific~lt ht pstiants, most human experiments ate carried out in normal volunleers with a single porlurbation, such as a low-level, non-lethal dose of a substance. such studies are huportant in defining mechanisms attd mediators of disease, in our world of injm'ed, operated, infected and inflslned patients, there are many diseases, mof is not one of lhem. it isn't even a syndrume, it is the final pathway for a number of diseaaes~ each of which has a cause. mecormock believes thal a "multi-causal" etiology is a euphemism for ignoranc# or a synonym fo~ unknown etiology. admission iv sn icu, a high apache seer% the sepsis syndrome, mof, muds, or sirs and having predictors indicating potential mortality, are not diseases. the inmpors are getting negative results in trials of agents on eiek or septic icu patients and aru now becoming splitters, dafining subgaoups at higher risk for death. such an approach may produce positive results if the diseases are identified and the treatmeul is specific for them, a major challenge in deterraintng the efficacy of new therapies for sepsis nnd shock is identifying patients whose e#temtc inflammatory response is appropriate from those in whom the mediator| are contributing to end organ system damage end death, traditional and recently revised categorical patient descriptions such as sepsis syndrome, sepsis syndrome with shock, multiple organ system failure, or the recently proposed systemic inflammatory response syndrome (sirs) may not be sufilcient since they identify individuals at varying levels of risk for short term mortality, the efficacy of new lmmunotherapy may be directly related to the patient's severity end risk of mortality at entry to the study. we recently reported (jama ; : z - ~ ) a comprehensive risk assessment approach for patients with sepsis syndrome, a common eategarlcal description used as an entry eriterlon for many trials. by screening s database of , recent admissions to hospitals in the united states and western europe, we identified , patients who met sepsis syndrome criteria but were not enrolled tn clinical trinh, we then developed a survival model end severlty assessment method to estimate their g.day mortality risk. k..ente physiologic abnormalities were the most important prognostic factors ( % of total chl square) influencing outcome. the specific disease resulting in ice admission and the days the patient spent in the hospital and icu before qualification were independent risks, as were age and a clinical history of cirrhosis. the predictive model was cross-validated within the original , patients with a receiver d_perator characteristics (roc) area of . and in two independent databases, the first independent database contained hospitalized patients with gram-negative infection meeting criteria for sepsis syndrome (roc= . ); in the second, the placebo patients withtn the recently completed phase ill e~aluatlan of il- ra (rocffi . ). in all databases the risk model was able to accurately risk stratify patients throughout the range of risks that varled from a very low % to a very hlglt %. by drawing on the ready avatlabillty of large clinically aeuurate, current databases and using the power of modern statistical techniques to model the bodfs response to sepsis, we are able to produce very aeettrate pre-treatment risk estimates, these prospectively defined and continuous risk estimates reduce reliance on multiple subgroups and data dredging that can compromise a stady's integrity. they cart provide a nnlform method for patient description across clinical trials of different attempts at immmlotherapy. risk calculations can also be useful in developing entry criteria for phase ii evaluations in order to initially test efficacy on a limited number of high risk patients, fonowing sueeessinl completion of a definitive trial, the risk estimates can be used to develop specific |ndlcattons for s drug's use and can monitor the impact of both new and multiple compounds on patient outrome, when dealing with therapeutic trials for the treatment of sepsis, there are ways of checking the comparability of the groups (placebo and treated groups) -to use several description items such as age, sex, preexisting disease, number and nature of organ failures, physiology score, such as saps il ( ) -to use a model for risk of death either from a score (apache ii, lii, ( ) saps ii) or directly (mpm ii system ( )) before using the model for risk of death in septic patients, is it mandatory to validate it (by calibration, i.e. goodness of fit, and discrin'dnation, i.e., roc curves) first on a data base using the same definition for sepsis as in the trial and secondly on the placebo group of the trial ? or is it better to use a specific model for each trial ? the general models usually do uot fit for septic patients. there are methods to make them fit : either to add variables to the original score (model for sepsis using the apache iii system ( )) or to customize the model. for saps ii the customization is applied to the equation regression, using the same saps ii score. for mpm i each component of the model can be customized. several remarks : only one model is published to be applied at the icu entry ( mpm ii ) before any therapy. -most of the published scores are calculated from the lsl icu day. applying these scores to the let day of sepsis could have a very different significance. -is the accp classification of sepsis useful at the bedside to select oatients ? ( the pathophysiology of sepsis involves a dynamic interaction between the host and the infecting microorganism(s). a multitude of biochemical and hemodynamic interactions occur which function in concert to determine the ultimate outcome of the septic episode. the complexities of the septic process preclude outcome analysis by in vitro systems or computer models, animal studies have become indispensable in evaluating new therapeutic agents in the treatment of sepsis. these studies provide valuable information on safety, pharmacokinetics, relative efficacy and dosing strategies for potential therapeutic agents in the treatment of sepsis. there are three basic types of animal models: ( ) toxicity models with injection of bacterial endotoxin or exotoxins; ( ) live or killed bacterial challenge models; ( ) actual infection models. all models employ some form of external manipulation under controlled conditions to induce sepsis and to analyze potential therapeutic efficacy. these experimental requirements may limit the ability of animal models to accurately predict the clinical value of new agents in human sepsis. four major end points are analyzed in the various animal models: ( ) hemodynamic parameters; ( ) modulation of host-derived inflammatory mediators; ( ) resolution of end organ injury; or ( ) lethality. each animal model has certain advantages and limitations. species-specific differences in physiologic and immunologic features make it difficult to directly extrapolate the results of animal experiments into clinical medicine. while no ideal animal model exists, consistent benefit of a new immunotherapeutic agent in multiple animal systems provides the most compelling preclinical evidence of its therapeutic potential in septic patients. consensus-assisted development of a study protocol on sepsis: an important difference from previous randomized trials there have been several, and perhaps too many metaanalyses of cliuical trials on surgical infections, but their results have only rarely been incorporated into the design of new randomized trials in sepsis. metaanalysis is retrospective. it seems more important to analyse and to criticize the design of tria/s before they are ongoing. incorporation of that into development of a study protocol is the aim of the following procedure: the time-schedule of about two years should include further cell culture and animal experiments after the first successful studies showing effectiveness. exhaustive evidence for a dose-dependent cost-banefit and near complete explanations of the pathomechanism should not be awaited for development of the protocol of the clinical study. however, a discussion forum of experts should be performed in a recently published, almost perfect trial in order to see the frontiers of the present research in cell and molecular biology, clinical pharmacology, statistics and decision making. this should be followed by a metaanalysis of at least study designs on sepsis with methods of formalized medical decision making (decision tree). clinical algorithms -developed by objective methods including nominal group processes -should be developed to standardize any concomitant treatment in the centers considered for a multinentre trial. the latter is usually necessary in the field of sepsis. finally, a pilot study should be performed to demonstrate not only feasability, but to learn about all possible types of interventions which modify endpoints as response variables. especially mortality is not free from such effects. the time for the individual phases of this consensus-assisted process of protocol development have to be shortened by their temporal hiterloeldug. due to the fast discovery of new chemical factors in sirs it wilt otherwise be impossible to come up with results of randomized trials which are fascinating enough to be incorporated in daily routine treatment. severity scoring systems are intended to provide a population-based estimate for the risk of an unwanted outcome resulting from some disease process. in the area of infections and the "septic" response, this has routinely been defined as death. for anti-infective trials, there has been a reasonable correlation of severity (apache ll) with outcome measured as persistent or recurrent infection, but this is not as robust as the association with mortality. it is expected that non-lethal failure would not immediately correlate with disturbed physiology: the basis for antiinfective failure depends on a variety of factors not measured in severity scoring, such as type of infecting organisms, density and susceptibility to administered antimicrobia[s, and anatomic features of infection (e.g., pancreatic). severity scoring is important in differentiating these "categorical" variables from continuous (physiologic) variables. there are, additionally, a series of problems related to the cause of death in relation to severity scoring. in those tdals in which cause of death is analyzed, high severity scores did not routinely predict death as an immediate sequelae of sepsis/septic shock. many of the predicted deaths occurred remotely of progressive background diseases. other important problems with severity scoring have to do with lead-time bias: hew long patients were ill before entry into the study. statistical techniques for condensing various categorical and continuous variables, as well as factoring in information requiring the pharmacodynamics of agents which affect outcome, are being developed to further refine deviation from predictive equations as an outcome measure. stepwise logistic regression analysis has identified the presence of shock, intm-abdominal or unknown source of infection, hospital acquired infection, age greater than years, neutropenia and inappropriate antimicmbial therapy as independent variables associated with increased mortality in sepsis. maldistribution or" these variables is a concern in targe randomized clinical studies. it should be noted that only the selection of antimicrobiai therapy could be addre.~'sed and altered at the time of enrollment in a sepsis clinics] trial. inappropriate antimicrobial therapy has been noted in %- % of septic patients. well designed, randomized sepsis trials do not assure the absence of imbalance between treatment and control groups with regard to inappropriate antibiotic therapy, especially with subgroup analysis. statistical manipulation to correct for any imbalance is appropriate but avoiding imbalance is ideal. selection of antibiotics in septic patients requires such considerations as clinical setting (history, physical examination and underlying disease), appropriate laboratory tests (such as gram stain), identification of source and associated bacterial flora, antibiotic susceptibility patterns for that hospital and likelihood of resistant organisms. determination of inappropriate antibiotic therapy is made retrospectively after culture results are available. in some circunmtances the organism and antibiotic sensitivities cannot be predicted, while in others it can. leibovici et al identified hospital acquired infection, antibiotic treatment before a bacteremic episode, endotracheal intubetion and thermal trauma as independent variables which predicted isolation of a multiresistant organism. the same authors developed a predictive index for resistant organisms, which when compared to prescriptions of attending physicians, improved antibiotic selection in critically ill patients. including a standard antibiotic regimen for all septic patients in a clinical trial is impractical. there is too much interhopsital variability in flora and antibiotic susceptibility. in addition, the aggressive broad spectrum coverage necessary for some patients is inappropriate for others. a protocol which guides a prescribing physician through a logical chain of reasoning might improve antibiotic selection in critically ill patients and could be included in the design of a clinical trial in sepsis. although this approach might minimize potential for maldistribution of inappropriate antimierobial therapy, it would likely create multiple problem areas. will the patient's physicians agree to the protocol choice? what is the availability of speciftc antibiotics at a participating hospital? what is the aplpiieability of study results in typical clinical situations? if there is agreement that thin is an appropriate antibiotic protocol, should it not be used in all seriously ill septic patients in the hospital? in other conditions such as ards, the success of protocol therapy is dependent upon measurable goals such as pao and highest plateau pressure, while measurable goals are not as clear in choosing antibiotics. based on the above confounding issues, a complex and extensive algorithm covering many potential possibilities of error and specific antibiotics seems impractical; however, an algorithm focusing on three to four major common errors in antibiotic selection and dealing with classes of antibiotics is plausible for inclusion in sepsis clinical trials. in this session we will attempt to outline the methodology of such a future study, emphasizing the immense difficulties involved in its proper conduction including: design, selection of patients, surgical considerations, supportive treatment, the "human factor" and ~nd points. only a close cooperation between a few dedicated surgeons, obsessively studying a large number of well selected and stratified patients over years, could make such a study possible. algorithmis have frequently been mistaken for the graphic format in which they are presented. however, an algorithm is neither a flow chart nor a list of instructions; it is a step-by-step approach to solving a problem. likewise, a clinical algorithm is a step-by-step approach to solving a clinical problem. cas are deterministic, which does not mean that they are rigid. they are practical rather than mathematical algorithms, that tan be used only with clinical judgment, and must be tailored to each patient. there are a number of types of, or uses for, cas that can improve sepsis management, including: diagnostic or severity scorm cas, a management ca for managing sepsis constructed according to recently published standards for use in teaching research protocol algorithms that must be used to collect data or guide implementation of a clinical trial aimed at validating one or more dicisions in the management ca; finally, protocol-style cas drawn from the management ca and should be validated using matching outcome studies. organisational problems peculiar to multicentre trials the organisation of any randomised clinical trial is fraught with difficulties, and that of a multicentre trial particularly so. there are problems associated not only with the principles on which the investigation is based but also with the detailed organisation and analysis. are all the participants truly unbiased about the relative merits of the regimens being studied? is like being compared with like -are all the end points and the standards for measuring them clearly defined so that they cannot be misunderstood? are all eligible patients being studied, and their results reported? is truly informed consent being sought that will satisfy not only the patient's right to choose what happens to him, but also the law of the land? and, finally, how can the participants be sure that the trial will be stopped at the right time, to ensure that no treatment is given to any patient that might be harmful? these questions may seem insoluble, but until we tackle them we shall never be certain. dr.m. ev~s, scar~mu~ hospital, scarborough, nonhyo~s~ y ql, u.k. increasing knowledge of the pathogenesis of sepsis and septic shock has led to the development of many new therapies and technologies capable of preventing, blocking or even reversing the deleterious cycle of events. recent results of subsequent multicenter trials testing some novel therapeutic drugs, however, did not confirm the promising sometimes overwhelming effects obtained experimentally. it is postulated that this is largely due to inappropriate design and conduct of multicenter trials as currently performed. one of the shortcomings emphasized in this paper is the "treatment mix" problem. multicenter trials in sepsis are performed because no single center is able to recruit the necessary number of eligible subjects within a reasonable time. each single center (icu) has its own individual policy which cannot be standardized for the trial (treatment mix). even if we ascribe that each icu did the "best" for their patients, it is highly probable that the outcome differs between the icus. this translates to the effect of the new therapy under investigation. ]t is therefore worthwhile to consider that icus must first demonstrate a certain standard based on the patients' outcome (audit) before becoming accepted as a participating center. this prerequisite ensures comparable quality between participating centers, led to the reduction of "noise level" and increases the probability of positive study results. the riyadh-icu program based on standard mortality ratios is recommended as an audit instrument. during the last decade most clinical trials of potential therapeutic agents in systemic sepsis have failed to demonstrate benefit from the drug under study. although in many instances it is entirely possible that the agent in question does not have clinical efficacy, an equally plausible explanation for the multitude of negative results is that the studies were improperly designed and thus could not show therapeutic benefit, if it existed. problems which have been identified in these various trials include inappropriate or unrealistic definitions of response to the drug; inadequate sample size, with attendant insufficient statistical power to demonstrate a difference, if it existed; insufficient standardization of customary therapeutic maneuvers in septic patients; imprecise criteria for patient entry into the study, which creates unacceptable inhomogeneity between control and treatment groups and invites unjustified post-study subgroup analysis; the lack of a formal sequential monitoring procedure for interim data analysis, which could potentially affect patient safety; and a primary analysis of study results that excludes protocol deviants and is not according to intent-to-treat. these and other problems have impacted upon the validity of the various trials conducted to date and may well have prevented the resolution of controversies surrounding the use of certain agents in the treatment of septic patients. the complexity of bacterially-induced septic shock involves a cascade of events that evolve over time, beginning with the proliferation of offending organisms and followed by the release of toxic mediators from the bacteria. endotoxins [e.g. lipopolysaccharides) from gram-negative bacteria initiate septic shock by precipitating a systemic inflammatory response syndrome (sirs) through release of a broad spectrum of hostderived mediators. over the last century, hundreds of these endogenous mediators have been proposed to serve as pathophysiological substances in this "alphabet soup" of cytokines, eieosanoids, biogenic amines, kinins, peptides, ions and hormones. an evaluation of the literature on septic shock leaves the investigator with a sense that, although many pieces of the septic shock puzzle have been presented, no clue was provided to indicate how these pieces fit together, in an attempt to assess objectively the causal role of individual mediators, we recently completed a collective meta-analysis of mediators that were individually reviewed and subjected to koch-dale analysis of causality by distinguished authorities in the field ( ). in summary, our attempt analyze available evidence to support a cause-and-effect relationship for putative mediators of septic shock revealed that only eight of the mediators analyzed could be strongly inferred as playing a causal role in septic shock. evidence supported the involvement of endotoxln, endorphins, complement, interleukins, tumor necrosis factor, eieosanoids, platelet activating factor and calcium. other mediators either lacked adequate supportive evidence or were not conclusively involved. posttraumatic outcome may be reduced by an amplified stress response mediated by neural and humoral stimuli. the classical hormonal catabolic response is predominantly mediated directly or indirectly by neural stimuli, while most changes in inflammatory responses such as leukocytosis, acute phase proteins and changes immunofunction are mediated by humoral mediators. clinical experience from controlled studies suggest afferent neural blockade and modification of stress response to improve organ function and postoperative outcome, and limited experimental studies also suggest neural blockade to be of beneficial value in shock states. of special value may be the improved gastro-intestinal motility after neural (visceral) blockade. no outcome studies are available from patients with major truma, multiple organ failure or sepsis, conditions where humorai mediators may be more important to modify than neurally mediated responses. future studies should investigate the clinical outcome effect of combined neural and humoral mediated blockade, as well as the potential role of an initial neural blockade in elective trauma (first hit) to improve the metabolic scene and outcome if a postoperative complication (second hit) should occur. neutrophil elastase is the predominant protease of the human neutrophil. this enzyme, and a variety of other enzymes, including neutrophil collagenase, are released by activated neutrophils in the setting of high concentrations of reactive oxygen metabolites. in addition, in this extracellular environment, the normal antiprotease defense mechanisms of the organism may have been severely inactivated oxidatively especially along capillary endothelial cells. accordingly, release of neutrophil elastase which mediates inflammation and degrades most extracellular matrix proteins, including elastin, fibronectin and many forms of collagen, may contribute to tissue injury and organ failure in a variety of diseases ranging in severity and acuity from pulmonary emphysema to the adult respiratory distress syndrome (ards). as a result, significant efforts by the pharmaceutical industry have been directed not only toward the development of human neutrophil elastase inhibitors, but also toward their characterization in a variety of animal models of neutrophil mediated diseases, and their evaluation as potential therapeutics for the treatment of a variety of human clinical conditions. data from animal models of organ injury and failure along with data collected from a series of patients at risk for ards and with ards will be presented and discussed as a rationale for inhibition of neutrophil elastase. parameters that need to be monitored to obtain success in future clinical studies will also be presented in this context. have turned out to initiate and maintain organ dysfunctions in severe posttraumatic and postoperative courses. such proteolysis-induced disorders are especially rendered possible by the concurrently arising consumption of inhibitory regulators (e.g. a vproteinase inhibitor=a pi, antithrombin iii=at iii) via cofnplex formation and/of proteolytic/oxidative inactivation. besides the well-known destructive potency against protein substrates, proteinases such as elastase or thrombin (active or in complex with the serpin inhibitors ~ pi or at iii) are also supposed to increase the inflammatory reaction by inducing cytokine release or shedding of soiuble adhesion molecules from various inflammatory cells (pmns, monocytes/macrophages, endothelial cells). those cell-derived mediators may provoke further cell activation through an autocrine/paracrine loop thus increasing significantly the proteinase burden at the inflammatory focus. therefore, this noxious circle might be interrupted by a convenient proteinase inhibitor therapy to ameliorate the inflammatory process. to verify this hypothesis, high dosis of at iii (mean plasma levels up to %) were applied in first controlled randomized studies on surgical patients suffering from multiple trauma or severe sepsis. in control patients we could clearly demonstrate the sequential appearance of proteinase/serpin-complexes (fa pi, tat), cytokines (il- , il- ), and soluble intercellular adh sion molecules (icam, elan, p-selectin) in the circulation. these factors turned out to be a helpful tool for early diagnosis and prognosis of forthcoming organ dysfunctions. interestingly, m at iii-treated patients a significantly reduced release/production of inflammatory mediators became obvious concomitant with the improved clinical course in comparision to an increasing deterioration in control patients. soluble mediators of inflammation are cytokines (e.g. il- , il- and tnfe) as well as breakdown products of complement proteins (e.g. c a and terminal complement complex). therefore, attenuation of both, release and generation of these synergistically functioning mediators together with stimulation of release of antagonists including soluble forms of receptors are potentially beneficial in all kind of inflammatory diseases. intravenous immunoglobulins (ivigs) are known to have an anti-inflammatory effect in humans (nih consensus conference on wig, ) . the mechanism of action becomes more and more clear. in single cells stimulated by anti-cd mab wig was able to down-regulate production of il- , il- , ifn% and tnfj~ significantly. igg coated solid-phase stimulates release from monocytes of interleukin- receptor antagonist (il-lra) but not of il- ; this observation is very much in contrast to the effect of endotoxin in the same in vitro system. furthermore, naturally occurring anti-cytokine antibodies can be demonstrated in apparently healthy individuals and in wig prepared from plasma pools. some of these antibodies can be detected by antigen/antibody reactions in fluid phase (anti-ll-le, anti-il- ), some only by solid-phase detection systems (anti-ll- , anti-ifn% anti-tnfc . infusion of wig to patients suffering from inflammatory disorders have resulted in a decrease in il- in circulation. during infusion wig might induce an acute but transient rise of tnfo~, il- , and il- . self limitiation of this process might be due to demonstrable instant release of il- ra and soluble tnfo~ receptors. in vitro ivig has also been shown to attenuate complement activation via the classical pathway. furthermore, acid haemolysis of erythrocytes in paroxysmal nocturnal haemoglobinuria could partially be prevented by ivig. ivig was able to attenuate forssman shock in guinea pigs. we have seen a patient with congenitally elevated turnover of alternative pathway of complement who repeatedly showed an increase of haemolytic titres after administration of ivig. thus, wigs apparently have multiple potencies including attenuation of soluble mediators of inflammation and might therefore be of benefit in trauma, shock, and sepsis. significance of sinusoidal liuing cells and of humoral factors on hepatic function following sepsis and major surgery hirata k, katsuramaki t, yamaguchi h, mukaiya m, yamashiro k. regeneration of the liver after hepatic necrosis or partial removal has been extensively studied, but uncontrolled mechanisms to irreversible hepatic failure following sepsis or major surgery of liver or with hepatic dysfunction have so far not been well documented. we suggested the importance of the intrasinusoidal aggregation between sinusoidal lining cells and intrasinusoidal running ceils in the mechanisms of hepatocyte dysfunction. thus, the purposes of this study was to investigate the changes in each sinusoidal lining cell during this process and the effect of cytokine on hepatocyte under various oxygenation. [ materials and methods ] ( ) clinical study : thirty two patients with histologically proved malignant tumor underwent major hepatectomy were devided in two groups, i_e. the cirrhotic liver group and the non-cirrhotic [aver group. most of the former were the patients with hepatocellular carcinoma and most of the latter were the patients with metastatic carcinoma of colorectal cancer. five patients were complicated septic conditions with hepatic failure. following operation in each patient, a verous blood sample was taken for measurement of serum il- , il- , tnf and c-reactive protein concentration. and hepatic biopsies were sometimes undergone in patients with anomalous hepatic function. ( ) experimental study : kupffer ceils, sinsoidal endothelial cells and hepatocytes were separated from mouse ( c hhen ) liver. and also polymorphonuclear cells and platelets were purified from blood and peritoneal macrophages were from peritoneal cavity of mouse. co-cultures between or among these cells with iipopolysaccharide were performed. cytokine concentrations in media and hepatocytic function were compared. [ results and conclusion ] our findings in above experiments demonstrated the cleanly different concept for the mechanism of hepatocyte dysfunction following sepsis or major surgery. namely, the effect of hepatoeytie function by cytokines or superioxide were significantly affected under low oxygenation, but not significant under good oxygenation. hirata m.d.,nishi ,hokkaido,japan $ immune complexes as .mediators of sepsis and immune dyafumcffon laa k horn, chxistof birkenmaier, and greg h mon departmen~ of surgery, san frandsco general hospital, urdversr , of calif(~rrda, san frandsco. immune complexes (ic) a;:e commonly found circulating in parians during infection and sepsis; and following traumm, bums, and massive l~ensfusion. clearance of c occurs by phago~'tmsis of esll-bou_nd or opsonized p~tlcles. monocyte activation du~jng phmgocytos/s could lead ~o release of cytokilies ieletexiot~ to the hosh to examine tb/s phenomenon, we formed insoluble ic by precipitating iow-endotoxin bovine serun~ albumin (bsa) with rabbit ant/-bsa igg. we have ~town that these ic are ingested by monocytes and concomitantly stimv/ate re~pizatory bu~t activity measuxed by assay of in%racellul~ peroxldaffon. we have shown that ic ingesffon produces signlqcant el,era,lore in the monoey~e phenotype. spedacally, cdi is down-regu/ated, along wl~ cd and cd . thus responsiveness to lipopolysacchmdde ( ) a~xd i=m~unoglobulin fe st~mu/mtlon is reduced. in cert,+rest, the complement rote.p tot cdc is u~-regulamd, indicaling mcremsed re~ponalx=~ness to opsonized pat,rotes, we examined monocyte superna~n~s for production of hzmor necrosis factor alpha (tnfg) following ic stimulation and showed that ic are capable of provoking ~elaase of hrmaunl)reac~ve tni~. j[c also increase mono=yte produchon of prnstaglandin e . in summary, ~/~ese results demonstrate lhmt ic are capable of indudng production of ¢ytokines and the imrau-nosuppressive prostaglandin pge . ic also allez ~he surface expression of important receptors involved in actlvaffon by lp~ and phagocyiosis. thus, ic aze competent for indud/on of the mepl/e s! otlse. by examining m n cyte phenotypms, it may be possible to d~mrmkne extent ohn'u~u~e complex activation and predict immune depression iu criffcally ill pa~ents. a great deal of experimental work has focused on preventing and/or treating sepsis and organ failure following injury. many of these strategies have tried to attack mediator substances released during the systemic inflammatory response following injury. sugermann has demonstrated the improvement of pulmonary function, but not eappillary leak using [buprofen in the porcine model. in a clinical trial, faist demonstrated the effectiveness of indomethacine in amelierating the post trauma of cellular amenity using in vitro parameters. morbidity and mortality, however, were not different between control and treatment groups. baue und chaudry have suggested that atp magnesium chloride may have protective effect in renal failure um kupfer cell dysfunction seen after shock. pentoxiphyllin has been found to be effective in improving tissue oxygenation and oxygen consumption following hemorrhage, and there may be promise for this drug in the ischemia reperfusion injury. monoclonal antibodies against endothxin and inflammatory mediators seemed promising at first glance; however, preliminiary clinical data of the treatment of sepsis with either ha-ia or e monoelonoal antibodies against bacterial cell wall have been disappointing. initial studies with il-i receptor antagonist also appeared encouraging, but no difference was seen in the most recent trial. currently studies are ongoing with monoclonal antibodies to tumor necrosis factor which may have more efficacy given the fact that it is a macrophage mediator released by endotoxin. although we seem closer to understanding and preventing the problems of sepsis and organg failure after trauma, we must continue to appreciate the complex interrelationships and delicate balances inherent in the host defense system. overzealous attempts at restoring immunity in the absence of understanding pathophysiology may be just as dangerous at post-traumatic immlmospremsion. severe acute pancreatitis was caused by different etiologic factors, but once pancreatic tissues were infected, patients show a similar pattern of aggravation and develop organ failure. remarkable elevation of serum il- was unexceptionally observed in patients with severe acute pancreatitis. the serum levels were more than pg/ml (normal: less than pg/ml) during the first one or two days after onset. there was a significant correlation between the peak serum il- level and the number of organs showing failure (r= . ). tnf-a production by isolated peritoneal macrophages following lipopolysaccharide (lps) stimulation was significantly increased in cerulein-induced pancreatitis rats. serum tnf-a activity was elevated following intraperitoneal administration of lps as the septic challenge both in pancreatitis rats and in control rats, being significantly higher in the former (p< . ). histological findings and liver function tests revealed that lps induced more severe liver damage in pancreatitis rats than in control rats. these results indicate that increased tnf-a production by peritoneal macrophages in acute pancreatitis augmented lps-induced liver injury. organ failure in severe acute pancreatitis could be caused, at least in part, by increased cytokine production. it is clear now, that in mods, organ injury is not directly due to exogenous factors, such as bacteria or toxins, but instead is largely a consequence of the host's own endogenously produced mediators. there is an extensive body of literature on the inhibition of mediator production, release and action in different cascade systems by glucocorticoids. they can serve as a host protection against overactive defense reactions if used adequately. a new understanding of the regulation of cytokine synthesis, especially tnf, may lead to an insight of the conflicting findings between the benefits of glucocorticoid therapy in animals and its current failure in recent clinical trials. glucocorticoid shares its role with other novel therapeutic approaches also shown to be successful only in experimental settings. a mete-analysis of steroids in sepsis, however, revealed a beneficial effect in the subgroup of patients with gram-negative infections. there is now a series of arguments to reevaluate the indication for steroid thera-py and/or prophylaxis in mods and sepsis. honjo i- - , kumamoto , japan in septic shock j. briegel, m. halter, h. forst, w. kellermaun, m. bittl, k. peter imrodnetlea and methods: hydrocortisone (hc) at an infusion rate similar to the maximal secretory rate of cortisol in man improves hemodynamics in human septic shock ( ). we hypothesized that the hc-induced hypercortisolemia prevents a harmful overshoot of immune reactions. to test this hypothesis we prospectively investigated patients admitted to the icu in refractory septic shock. after resuscitation (mechanical ventilation, fluid resuscitation, titration of vasopressors, and initiation of antimicrobial therapy) hc was started with a loading dose of mg/ rain, followed by a continuous infusion ( mggh). with hemodynamic improvement (dopamine < gg/kg/min) the infusion rate nfhc was tapered over a period of days. phospbolipase a (pla ), c-reactive protein (crp), and elastase-proteinase inhibitor complex (e-pi) were deierminated daily. results: according to our previous results hemodynamics improved during hc infusien. after days dopamine requirements decreased to less than gg&g/min in all patients. this corresponded to an attenuation of the systemic inflammatory response syndrome (sirs) indicated by the abrogation of fever and the decrease in heart rate and inflammatory mediators. pla activity and e-pi concentrations decreased to near normal values and crp concentrations decreased as well. after cessation of hc infusion (between day and ) a reinforcement nfthe sirs was observed despite cortisol levels within the normal range. this was first indicated by e-pi, followed by fever, increases in heart rate, crp, pla , and finally by the relapse of septic shock in four patients on days , , and o. a second infusion of hc again resulted in shock reversal and in a decrease in pla , crp, and e-pi in the patients under study. discussion: there is growing evidence that the endogenous steroid hc and proinflammatory cytokines integrate a complex immunoregulatory feec~ack circuit. the elaboration of tnf, il- , il- , and il- is attenuated by- rag hc prior to endotoxin challenge in humans ( , ) . cytokines like tnf, il- , il- , and il- are potent proinflammatory signals for acute reactants (--, clip), neutrophil degranulation (--, e-pi), and pla synthesis and secretion. our data provide evidence that hc at an infusion rate similar to the maximal secretory rate of cortisol in man attenuates the systemic inflammatory response in human septic shock. to evaluate the feasibility of a nigh-dose regimen of antithrombin iii (at iii) treatment in patients with multiple injuries regarding blood levels of antithrombin iii and undesired side effcts and to analyse effects of the initibitortherapy on the systemic inflammatory response. patients and methods: patients with an iss nf more than points who could be treated with at iii administration within rain after trauma were randomized to receive either at iii or placebo. at iii was given to reach an antithrombin iii inhibitory capacity in plasma of % by using the following calculation: at iii to administer = ( % -at iii measured) x body weight. at iii was given every six hours for a period nf hours and on the th day. patients were followed up throughout their stay in the icu but at least days. in this abstract the data of the first patients are included. results: the patients' median injury severity score was points with a median age of years. the patients were equally distributed between verum and placebo groups with respact to age , iss, prehospital treatment, blood pressure, hemoglobin and at nmevel on admission, and time from the accident until the test solution was given. the patients of the at iii group received , units of the inhibitor on average during the first days. with our regimen, the at iii levels could be kept between % and % of normal, whereas control patients an at hi concentration of % to % was observed. patients with at ni treatment required less red blood cells bur received the same amount of ffp and fluids. there were no differences with respect to platelet count, partial thromboplastin time, l~rothrombin time and prothrombim. we did not observe bleeding disorders or any other side effect with peak concetrations of up to % of normal. white blood count, pmn--elastase, interleukin and and c-reactive protein tended to be lower in the at iii group. there were no differences with respect to renal and hepatic function parameters but the duration od mechanical ventilation was shorter in the at ii group ( . vs. . days) conclusions: we conclude that our treatment protocol (a) was able to restore at iil levels to the desired range of %, (b) did not lead to coagulation disorders, (c) did not increase transfusion requirements, (d) did not reveal other undesired side effects, and (e) showed a tendency towards reduced posttranmatic inflammation and mechanical ventilation requirements department of trauma surgery, essen, germany antithrombin-lll (at-ill) acts as an inhibitor of thrombin, further proteases and the alternative pathway of the complement system. inhibiting thrombin, at-ill works as a functional antagonist of paf~ therefore, in state of hypevolemic-traumatic shock, continuous supranormal serum -and tissue -concentrations of at-ill may be efficient to reduce the local posttraumatic reactions resulting from complement and pmn-activation. patients with severe multiple trauma ( treatment [at-ill] -- controls [c]) were investigated. study cdteda were age > and < years, injury severity (iss) > points and glasgow-coma-scale > points; randomization and treatment has to be started within hours after trauma. permission for the clinical study was given by the local ethic committee. bradykinin (bk) and related kinins are potent inflammatory peptides which possess the ability to induce, vasodilation, increased vascular permeability and hyperalgesia. cp- , a novel homodimer bk antagonist has previously been shown to increase survival in rat and rabbit models of lethal endotoxin shock and is now in clinical trials for sepsis. we have now evaluated the effect of cp- in other models of inflammation. male rats were precannulated with a catheter in the carotid artery. h later bk was injected ia and the pain score ranked from (no responses) to (vocalization). cp- at . umoles/kg completely inhibited the pain responses for a period of . - h. cp- at . umoles/kg s.c. was also found to inhibit the increase in paw volume and hyperalgesia induced in rats over a - h period by an intraplantar injection of . % carrageenan. the abdominal constriction response o an intraperitoneal injection of kaolin was inhibited in a dose-dependent manner by cp- . when ul of . % formalin was injected into the paw of a mouse a characteristic licking response was observed which was biphasic in nature. cp- significantly inhibited both the first ( - min) and second ( - min) phase responses. ]n a rat burn model, where the hind paw is immersed in water at °c for sec the increase in paw volume was significantly reduced by pretreatment with cp- , . umoles/kg s.c. finally cerebrai edema was induced in rats by applying cold (- °c for sec) to the dural surface following a craniectomy. cp- at . umoles/kg s.c. produced a significant reduction in the amount of edema compared with sham controls h later. these data suggest that bk is an important mediator of inflammation and hyperalgesia and that the bradykinin antagonist, cp- , may be useful in the treatment of such inflammatory, hyperalgesic disorders. partial hepatectomy in humans is associated with a considerable morbidity due to hemodynamic and metabolic derangements, which increase the risk for organ failure and mortality. we hypothesized that endotoxemia may play a pivotal role in these complications. we therefore, investigated whether peri-operative infusion of rbpi , a recombinant protein of the human neutrophil bpi with bactericidal and endotoxin-binding capacity, could prevent postoperative derangements following partial hepatectomy. male wistar rats ( - g.) received a % liver resection (phx) or a sham operation (sh), and a continuous intravenous infusion of either . mg/kg/hr rbpi (phx-bpi, n= ; sh-bpi, n= ) or the (iso-electric, iso-kd) control protein thaumatin (phx-con, n= ; sh-con, n- ). various parameters were measured h after the resection or sham operation. mean arterial pressure, cardiac output and heart rate were significantly decreased in phx-con rats compared with sh rats, which effects were not observed in phx rats treated with rbpi . blood ph was significantly decreased in the phx-eon group, whereas the leucocyte count, hematocrite and il- levels were significantly increased compared to sham levels. in the phx-bpi group, these parameters were restored to near sham levels. in vitro experiments with rat plasma and human mononuclear cells (mncs) revealed that plasma of phx-con rats is highly capable of activating mncs, accompanied by the release of cytokines. this activation is attenuated with phx-bpi plasma. in vitro added acd or polymyxin b was able to reduce the activation by phx-con rat plasma to the levels of phx-bpi rats thus, these data suggest that systemic endctoxemia, possibly of gut origin, is a major cause of postoperative hemodynamic and metabolic derangements following phx and that rbpizz can prevent these changes. more recently we reported a transient appearance of both endotoxin and tnf in the circulation of rats subjected to the haemorrhagic shock (hs) already at - rain. similar to bpi, recombinant bpi was found to bind lps and inhibit tnf formation in vitro. the aim of this study was to investigate the effects of rbpi (kindly provided by xoma corporation, berkeley, ca) against haemorrhage related endotoxemia and mortality in rats. method: a prolonged hs was induced by blood withdrawal to a mean arterial pressure of - mmhg for rain followed by reinfusion of shed blood (sb) and resuscitation with two times of sb volume of ringer's lactate over rain. rbplg. was administered at a total dose of mg/kg i.v. ( . mg/kg at the -eginning followed by two doses of . mg/kg each at end of shock and the end of resuscitation). the control group was treated similar to the bpi group but received thaumatin as a protein control preparation at the same dose as rbpi . results: imrffe?diately after resuscitation ( min) the detected plasma endotoxin levels in the control group (mean = , range = - pg/ml) were almost neutralized by rbpi treatment (mean = , range = - pg/ml) . plasma tnf levyis were not significantly influenced by rbpi treatment at the two time points and min of experiment (means: and in bpi vs , pg/ml in the control group). the -hour survival rate was improved from / ( . %) in the control to / ( %). conclusion: these data suggest that haemorrhagic shock may lead to bacterial translocation and/or transient endotoxemia with concomitant cytokine formation that may play an important role in the pathogenesis after shock and trauma, rbpi might be a useful therapeutic agent against endogenous bacterfal/endotoxin related disorders in hemorrhagic shock. morbidity and mortality after hypoxia of the vital organs had been correlated to the production of oxygen radicle which is mediated by xanthine oxidase activity, in this study we have evaluated the survival rate after allopurinol. rabbits weighed + grams divided into two groups. group i included tabbits were treated with allopurinol mg/kg for seven days before induction of haemorrhage. group ii as a control included rabbits. all rabbits were subjected to % arterial blood loss through the central ear artery for one hour then resusciatation was done by the heparinized withdrawn blood through a marginal ear vein. during the experiment blood pressure and heart rate were monitored through the central ear artery. also uric acid, lactic acid, glutathione activity were estimated. animal survival was followed for days. postmortem vital organ histochemistry and histopathology examinations were done. in group i the survival after three days was out of while in group ii it was two out of . our conc|usion, allopurinol had increased the survival in aiiopurinol pretreated rabbits which may indicate the value of allopurinol premedication for patient prepared for elective bloody surgical intervention . h receptor antagonists are commonly used for stress ulcer prophylaxis, but their actions on the septic response are largely unknown, in an experimental model, pigs were first anesthetized, then injured with joules of energy to the posterior thigh, then hemorrhaged - % of their blood volume. after i hr of shock, all the shed blood plus x the hemorrhage volume as lactated ringers was infused. following resuscitation, ranitidine ( . mg/kg iv twice daily) or saline placebo was begun. the treatment group was randomly assigned in a blinded fashion. after hrs, a septic challenge was administered ( bg/kg of e. coil endotoxin (lps)). serial gastroscopy, gastric ph, hemodynamics, abg's, physiologic dead space ventilation, leukocyte counts, and tumor necrosis factor (tnf) levels were recorded for min. baseline values and units were cardiac index _+ ml/min/kg (ci), arterial po + mmhg(pao ), base excess . -+ meq (be), physiologic dead space fraction +_ % (pds), and tnf . + . units/ml. baseline gastric ph was . -+ . and . _+ . in the placebo and ranitidine groups, respectively. the gastritis following hemorrhage was marginally attenuated in the ranitidine group. following lps infusion the following were obtained: ci pao * be* gastric* pds* peak* rain rain rain ph min tnf ranitidine _+ _+ - . ± . bum injury results in hypermetabolism, fever and nitrogen wasting. endotoxin (lps) has been proposed to mediate these effects, either directly or via activation of macrophages to produce cytokines such as interleukin- (ii- ). this study was designed to clarify the role of lps and - in the metabolic response to bum injury. twenty-five burn patients ( -+ %; + % ft bsa burn; _+ years old) were studied serially for three weeks post bum. patients underwent partitional calorimetry to assess metabolic rate and compartmented heat loss. nitrogen was assayed using chemiluminescence. lps and i - were measured with limulus amebocyte lysate assay and elisa. patients were excluded if they suffered smoke inhalation, showed any sign of sepsis or failed to rapidly meet their nutritional needs via the enteral route. ten patients received intravenous polymixin b ( , u/kg/day to bind lps). these patients did not differ for the remainder. all patients were hypermetabolic and febrile in proportion to the size of their bum wound but were not endotoxemic ( . +_ . pg/ml; normal < pg/ml). i - did demonstrate a significant correlation with cole temperature (tr~ = . + . ogi - , p= . ) and with nitrogen excretion (nou t = - . - . ogi - + . tr, p= . ). administration of polymixin b had no effect on metabolic rate, temperature or i - levels but did reduce nitrogen excretion resulting in more positive nitrogen balance ( .t grn/day vs. - . gm/day, p= . ). although bum injury does not produce an obligatory endotoxemia, i - does appear to play a role in the fever and nitrogen wasting seen with such injuries. the effect ofpolymixin b on nitrogen excretion suggests that lps may play a role either locally or in the portal system. introduction: there is substantial evidence that release of inflammatory mediators by activated kupffer cells contribute to the course of a systemic inflammatory process, e.g. after shock or lrauma. besides the systemic effects of mediators such as tnf, paf or interleukines, local actions on hepatic microvasculature and hepatic inflammatory response have to be considered. our aim was to assess the role of tnf and paf by blocking their effects using anti-tnf monoclonal antibody, pentoxifylline and a paf antagonist. methnds: in anesthetized sprd-rats, hemorrhagic shock was induced by withdrawl of arterial blood within rain and shock state was hold for h at a map of mm hg (cardiac output of %). following adequate resuscitation with % of shed blood and twice of this volume as ringer's solntion, animals recovered to map > mm hg and co > %. hepatic microcirculation and sinusoidal leukocyte-endothelium interactions were examined by intravital epi-fluorescence microscopy at , , or hours after resuscitation. in a blinded fashion, a rat-specific monoclonal anti-tnf antibody [ mg/kg, celltech, uk) , pentoxffylline (ptx, mg/kg, hoechst, d), and a paf antagonist (web , boehringer, ingh., d) were given either as pretreatment or at the time of resuscitation (n= - group bolla. k*., duchateau, j., hajos, gy., mbzes, t., hern~di, f. prevention of temporary/secondary immune deficiencies or reduction of their severity and/or duration as well as the reduction of the perifocal inflammatory processes belong to the rational targets of posttraumatic/pedsurgical medication. such a targeted medication can result in less frequently occurring nosocomial infections, and in reducing the duration of the intensive care and convalescence period. the results of in vitro studies performed with the amino acid sequence - of thymopoietin, i.e., with thymocartin in whole blood and peripheral mono-nuclear celi(pbnc) cultures clearly show some characteristic effects of this immunomodulator. preincubation with the tetrapeptide significantly (p<o.ol) and concentration-dependent reduced the endotoxin(lps)-induced tnfalfa production from , to about pglml, but did not abolish it. the inhibition of the lps( . ug/ml)-induced tnf production occured already in presence of , pg peptide/ml and peaked at the concentration of , ng/ml. higher concentrations did not increase this effect. in contrast to the other two small thymic peptides (tp- and tp- ), thymocartin did not enhanced the pwm-induced pge production in pbmc cultures up to a concentration of ng/ml, inclusive. the selection of thymocartin (tp- ) for perisurgical medication and/or for treatment of trauma-induced temporary immune depression has been strongly supported also by targeted animal experiments. thus, mortality of about % registered in mice infected with pseudomonas aeruginosa after bum injury could be reduced with the tetrapeptide treatment to a level which did not differ significantly from that of the infected non-burned controls. moreover, observations gathered first of all with the inflammatory model of air poach/croton oil grenuloma (selye) clearly showed, that a very definite, antiexudative effect can be achieved with this thymic peptide. as to this effect, thymocartin has been confirmed, e.g., to be equipotent with locally administered fluocinolone acetonide and/or s.c. injected prednisolone. the interim results of two explorative double-blind clinical studies (involving more than patients after polytrauma and hip-fracture untill now, respectively) correspond to the expectations. the treatment significantly reduced the occurence of nosocomial infections, the days spend in intensive care and on antibiotics as well as it shortened the bed-out time and reduced the additional medication. objective of this prospective randomized study was to further delineate the mechanisms leading to these alterations and to investigate the effects of immunomodulatory intervention. patients and methods: patients (pts) formed control group a. group b pts received x mg indomethacin (indo) from the first (dl) to the fourth postoperative day (d ) intravenously to block prostaglandin e induced suppression of cmi response as well as mg thymopentin (tp- ) subcutaneously preop., on d and d to augment cmi reactivity. in vitro investigations preoperatively, on dl and d included measurements of the percentage of cd + t-helper (th) cells, pbytohemagglutinin (pha)induced synthesis of interleukin (il)- as one cytokine primarily produced by thl-cells, and pha-induced synthesis of il- as one cytokine primarily produced by th -cells. delayed type hypersensitivity (dth) skin response to an antigen skin test battery and specific antibody (ab) production following tetanus vaccination preoperatively and on d were used as in vivo tests for thl-and th -induced immune response respectively. results: postoperatively, group a pts showed a persistent significant reduction of cd + th cells, il- /il- ratio, and dth skin response as compared to baseline values (bl), while ab production increased (p< . vs.bl). in group b pts no change in cd + th cells, il- /il- ratio, or dth skin response was observed (p< . vs.group a), and postoperative ab production increased significantly (n.s. vs. group a). conclusions: .these results clearly indicate a significant suppression of th induced cmi response, while th induced response remains normal following ohs. .these alterations may gain clinical significance whenever a postoperative infection requires a th response and may explain the susceptibility to opportunistic microorganisms observed in pts after ohs. the aim of this study was to find out whether the establishment of administration of thymostimulin (tp-i) in jaundiced and anergic rats would return the rats to the state of immunocompetence. material and methods. male wistar rats weighing - g were injected with haemocyanin (klh). all rats with a positive response were included in the study and we evalu ated the t cell subpopulations and il- . they underwent laparotomy and the common duct was ligated and divided. those rats whic become jaundiced and anergic one week after the operation, were divided in two groups: control group ( objective of study. the goal of this study was to prove the necessity of thymus derived immunomodulators treatment in acute period of infantine sepsis, materials and methods. immune status was investigated in o infants. clinical conditions of children were defined by syndrome of multiple polyorgan]c insufficiency. the heaviness nf condition was estimated as the sum of points according to efforts needed to restore of basic living functions -respiration, hemodynamjcs, hemocoagulatlon, metabolism, functions of liver and kidneys. it was expressed in form of clinic condition classes from to . results.it was found that in % of infants with - classes of heaviness immunode[iciency took place simultaneously with ii and iii stages of hemocoagulattve syndrome. amounts of t cells and t helpers were decreased more then times, ratio th/ts was reduced to . or lower. concentration of lgg and igawas reduced almost a hull in comparison to control. phagocytic and metabolic activities nf neutrophils were depressed. complement system state was changed: the functional activity of the alternative pathway factors b, d and the level or c a subcomponent were increased but the level of c , c ,, c and c components of the classic pathway were decreased. during the development of coagnlopathy the direct correlation between chso, levels of plasminogen and antithrombine iii was found. in greater extent the function of immune system was disodered in accordance with point metabolic violations and hemocoagulative disturbances considered as disseminated inmtravaseular clotting syndrome on ii or ili stages. conclusions. the rehabilitation of the immune system functions was promoted by taetivine and thymaline in doses of i- mcg/kg per day and mg/kg per day accordingly during a week in children with - classes el heaviness. aiter - days tactivine had advantage. when eoagulopathy took place thynaaljne promoted restoration of ( - )-~-d-glucan (bgc) is a major cell wall compornent of pathogenic fungi and shows many immunopharmacological activities on experimental animals. lps is also derived from gram-negative bacteria and have in~ttnomodulating activities on immune systems positively or negatively. because of difficulty to cure contaminating lps from bgc preparations, it is very hard to evaluate the exact activities of bgc. in this study, we compared the immunological activities of lps ans highly purified bgc on murine system. materials and methods: c h/hen or c h/hej mice, to wkolds of both sexes were used through experiments. highly purified ( - )-{ -d-glucan (gurdran; bgc)was courteous gift from seikagaku kogyo co. tokyo, japan and resolved in endotoxin-free ultra-pure water. escherichia cell lps was purchased from sigma chemicals, usa. mitogenic activities of bgc or lps on splenic cells were measured by mit dye assay. in vitro activation of peritoneal exudate macrophage (pen) have moniotored by phagocytosis and intraphagocytic killing of pseudomonas aeruginosa (z~b- . induction of cytokine productions from pem or spleen cells induced by bgc or lps were detected by cytotoxic assay. results and (bnclnsions: i ) in vitro cultures of pem with bgc have induced enhanced phagocytic and bactecidal activities in c h/hen and c h/hej mice, whereas lps only induced such activities in c h/hen mice. most effective dose of bgc was ug/ml. ) bgc-dependent proliferative response of c h/hen splenic cells was not detected under the culture condition used. ) induction of tnf~ prodction was apparently observed in bgc-stimulated c h/hej pd cultures, but not in lps stimulated one. these findings indicate that activation mechanisms of pr and splenic cells by bgc are different from that by lps. to investigate the bsc-induced p~ activation, intracellular signaling pathways of p~ by bgc-stimulation are under consideration. yamagami k, uedono y, kawakami k, kitazawa y and tanaka t according to the latest reports of use of il- in a burned-sepsis model, the dose was too high to adjust for human therapy. adoptive transfer of l~ymphokine-activated killer (lak) cells has been demonstrated as a means of enhancing therapy in malignant tumors. we therefore attempt to use lak therapy on burned-infected mice instead of il- . under anesthesia, -weekold male c h-hej mice were subjected to a % scald or sham burn and then resuscitated. sham burned mice served as controls. scald burn mice were subjected to peritonitis by intraperitoneal innoculation of organisms of p. aeruginosa hr after burn. burned-infected-mice were divided into two groups. one group had no further treatment, and the other group received lak cells ( x ) intraperitonelly after septic challenge. the mice were scored for survival daily. on day after burn, using moabs dual-color flow cytometry, we studied lymphocyte expression in mice with use of blood and spleen. simultaneousely we studied the alteration of il- and il- in their serum using immunoassy kits. sham burned animals had no mortality. the mortality of the lak-treated mice was significantly reduced when compared with that of the burned-infected mice. the most consistent changes observed were depressions in percentages of thyl, positive cells and a remarkable depression of nk cells in spleen. after lak cell treatment, those two percentages of cells significantly increased. all the data of assay of ill and [l were not detected on sham burn mice serum. due to burn and septic challenge the levels of illand il (n= ) were raised to . _+ . and _+ pg/ml, while the levels in lak treated mice were reduced to . _+ . and _+ pg/ml, respectively. the results reported here demonstrate that lak therapy is able to improve cell-mediated immunity in burned-infected mice. this is associated with a significantly improved survival rate, since ill has been demonstrated to mediate immune and inflammatory responses. also a cause effect relationship between elevated endetoxin levels and increases of [l has been recently established. the aim of our present study was to investigate the effect of parenteral indomethacin on the immune system of patients under major operations. our study included operated patients, of which received mg indomethacin before and , and days after surgery (group a) and patients received no antiinflammatory therapy (group b). we measured total t-cells helper (cd ), suppressor (cd ), nk-cells and interleukin- and tnf production by pha or endodoxin (lps) stimulated peripheral blood mononuclear cells at day and , and days after operation. in group a we detected a significant (p< . ) increase in cd /cd ratio on day while no differences were ~oted in nk-cells or cytokine production in both groups (table ) . it seems, therefore, that parenterai indomethacin may have a benefitial effect on the immune system of patients under major operations by increasing the t-helper /t-suppressor cell ratio while having no effect on the production of cytokines by peripheral blood mononuclear cells. this study was undertaken td dete~nnine the role of prostaglandin synthesis inhibitor on survival post traulna sepsis. analysis of the effects of prostaglandin synthesis inhibitor on survival of four groups of mice each were made after laparatomy and intravenous administration of live e.coli. post trauma sepsis, group a received no treatment; group b received antibiotics im; group c received prostaglandin synthesis inhibitor im and group d received both antibiotics and prostaglandin synthesis inhibitor im. survival time was longest in group treated with prostaglandin synthesis inhibitor and antibiotics post trauma sepsis. the singular use of either antibiotic or prostaglandin synthesis inhibitor did not alter the outcome on survival. mortality rate was lowest in the group treated with combined prostaglandin synthesis inhibitor and antibiotics. use of this combination showed a reduction in bacterial growth in peritoneal and blood samples, mild morphologic changes secondary to sepsis in the heart, lungs, liver, kidney and stomach and marked enhancement of mean level of activity. the study indicates that addition of prostaglandin synthesis inhibitor in treatment of trauma-sepsis has clear beneficial effects. interferon-? (ifn-y) is a potent immunostimulant which has been shown to be detrimental when administered during septic shock. blockade of this cytokine however is beneficial during the acute septic response. the aim of this study was to evaluate the cellular effects of this cytokine and its blocking monoclonal antibody (moab) in lethal sepsis following injury. female cd- mice were randomized into two groups: septic=lps vs injury=hind limb amputation (hla vs igg. nstats= anova=p< . vs lgg ifn-y was detrimental when given to control septic animals. however its blocking moab was protective (p< . ). conversely ifn-y was proteetive in injured septic animals compared with anti-ifn-? (p< . ). these findings demonstrate that the immune stimulant ifn-? has therapeutic potential in the immunosuppressed injured host predisposed to sepsis, while blockade of this cytokine is required for protection in the septic host with a normal immune response. dept of surgery, rcsi, beaumont hospital, dublin , ireland. oxidants play a role in physiology. the potential noxious oxidant biochemistry is restrained by chemically distinct antioxidants. these antioxidants, which may reside in different cellular compartments, can act synergistically. in normal physiology an oxidant/antioxidant bai&nce exists. unbalance in favour of the oxidants is called oxidative stress. oxidative stress has been implicated in many pathologies including lung diseases(e.g, doxorubicin induced cardiotoxicity), ischemia/reperfnsion damage and central nervous system trauma. transition metals such as iron are involved in the early events leading to oxidative damage in these pathologies. this has been underlined by the finding that postischemic cns pathology closely resembles that caused by a chemical oxidative insult (e.g. iron microinjection). moreover, a protective effect of oxygen-radical scavening agents or compounds that inhibit lipid peroxidation (antioxidants) in brain ischemia/trauma is apparent. some known drugs (e.g. anti-arrhythmics or histamine h -antagonists may act as non-specific antioxidants. howe~er, recently, interesting new membrancespecific antioxidants (e.g. -aminosterdids) have been designed. subt&e effects on iron redox chemistry contributes to the potent antioxidant activity of these aminosteroids. since a few years, one area that greeted enthusimastlcally in clinical medicine is that of reoxygenatlon injury or ischemia-repeffusion, a phenomenon accepted to make a majo:" contribution to organ dysfunction in trauma, shock and sepsis through the formation o( oxygenated free radical species. however, thei detection in vivo always remains a difficult challenge. efforts have been made recently to directly establish the formation of free radicals in biological samples as complex as blood, plasma or tissue with the use of electron spin resonance (esr) spectroscopy. using spin trapping agents, the presence of reactive carbon-and oxygen-centered radicals has been demonstrated in animals blood submitted to heart, renal and intestinal ischemia-repeffusion or to liver transplantation. recently, the in vivo formation of free radicals in the cerebra; extracellnlar fluid during cerebral isehemia-repeffusion has been assessed by the spin trapping technique coupled to brain microdialysis. esr investigations have also been conducted to detect endotoxin-induced free radical generation in rat or baboon liver and oxygen free radicals (ofr), produced both at intra-and extra-cellular levels, seem to play a major role in the pathophysiology of tissue injury and organ dysfunction in sepsis, through induction of lipid pemxidation in cell membranes. oxidative damage can result both to an ofr-overpmduction or to a depletion of endogenous antioxidant defenses, which are represented by scavenger enzymes (e.g. sod), hydrosoluble and liposoluble antioxidants (e.g. ascorbate, vitamin e), and other mechanisms such as metal-ion sequestration. in animal models, oxidative damage has been proved by detection of increased levels of lipoperoxidative products, and a depletion of antioxidant defenses was found both in plasma and tissues. in the few clinical studies a similar results have been reported, and oxidative damage appears to correlate with dic, with organ dysfunction and with red blood cell deformability. in a group of critically ill septic patients we found increased levels of conjugated dienes (cd) (bioproducts of lipoperoxidation) and decreased plasma levels of alpha-tocopherol and coenzyme qlo (coqi )(endogenous liposoluble antioxidants). a relationship between cd and uric acid (p< . ) was found, may be related to xantine-dehydmgenase to xantine-oxidase conversion. antioxidant depletion is due both to overconsumption and to a nutritional deficiency, even if a reduced synthesis can also be present. in fact one more relationship between coqi and plasma cholesterol (p< . ) demonstrates the commun hepatic biosynthetic defect. is there any role for antioxidative therapy in sepsis? can it achives a significant improvement in the septic course, organ dysfunction, and final outcome? several antioxidant drugs, have been evaluated in experimental and clinical sepsis: scavenger enzymes, natural antioxidants, alloporinol, -aminosteroids or lazaroids, have been proved to reduce lipopemxidative damage, to protect organ dysfunction, and in some cases to improve survival. several questions must be addressed in evaluating safety and utility of antioxidative therapy. more specific and standardized methods must be applied to detect and quantify the lipoperoxidative damage. antioxidant drags must act selectively on ofr-production, without interference with other than that are beneficial for the organism. antioxidants must be able to achive in adequate amount the tissue or cellular compartment where their action is required. many antioxidants have also a pro-oxidative effect which can he detrimental in some conditions. the timing of administration, the dosage used and the association wih others antioxidant drugs must be defined. nutrition plays an important role as antioxidative therapy, since it supplies all compounds needed to maintain adequately levels of endogenous antioxidant or to support their synthesis. thiobarbituric acid reactive substances (tbars) concentration in serum has been determined in a large population of healthy subjects and in patients suffering acute hepatitis and chronic cases of hepatitis c, as well as several other processes. the correlation with different physiological and clinical parameters in these populations is also presented. treatment with interferon of the chronic active hepatitis c patients, x u three times a week during two months, led in those patients whose sgpt activity normalized in serum, to a concomitant decrease in serum tbars content. the possible theoretical involvement of peroxidation and antioxidants in this beneficial effect of i~terferon in hepatitis c patients is discussed. the results presented confirm the value of tbars as laboratory test in the management of disease and as a useful tool for the study of pathogenic and/or therapeutic mechanisms. -hydroxyalkenals are among the different substances measured by the tbars assay. these compounds show several biological effects that have been demonstrated mainly in different experimental models. we have studied the capacity of different tissues to conjugate these compounds patients surviving the initial subarachnoid hemorrhage (sah) from a ruptured in~raeranial aneurysm are prone to develop cerebra] ischemia from vasospasm which is associated with significant morbidity and mortality. among the many treatments that have been prol~osed to treat this condition, none has been shown to be satisfactorily effective. recently, a new drug, namely the -aminosteroid tirilazadmesylats, has been studied in a large, prospective, multicentor trial for its effectiveness to improve the outcome of patients with aneurysma! said. the study was conducted in europe and australia in neurosurgical centers and included patients. all patients received presently accepted standard treatment, including nimodipine. patients were randomized to four different groups: placebo and groups of tirilazad in escalating dosage ( . - mg/kg/day). the clinical outcome was compared for these groups and cerebral a~giography was performed in % of patients on day - follovang sah, to study the effect f'the dflf~ off cerebral vasospasm. the study reached its planned endpoint months ago. preliminary results ~how a significant improvement with regardto mortality in those patients receiving the highest tirilazad dose as compared to placebo and the two lower dose groups. additional beneficial effects were seen in the rag/day groups with regard to the occurence of symptomatic vasospasm and good clinical outcome. although the final analysis of all data is still pending, these results suggest that tirilazad-mesylate could represent a major improvement for the treatment of patients with sail. experimentally lipidperoxidation products (lpo) are increased in acute pancreatitis (ap) due to oxygen radicals (or). the purpose of this clinical study was to determine the antioxidant status and lpo in patients suffering from ap and to evaluate the effect of antioxidant treatment with sodium selenite (se) thus improving the function of the se dependant glutathione peroxidase which is the major detoxifying system for or. materials and methods: in z patients sufferin s from severe ap (c-reactlve protein > me/l) we determined on day and day after admission the lpo ma!ondialdehyd (mda), conjugated dishes (cd), reduced (gsr) and oxidized (gssg) glutathione, the vitamins a,c,e and se. moreover the patients were evaluated clinically using the ranson and the apache ii score. i patients were randomly treated with ug/day of se for days. results: all patients suffered from a severe depletion of antioxidants,especially a low concentration of se (only / of normal). thereby the increase in lpo correlated with the clinical course. during se treatment lpo decreased and the levels of antioxidant vitamins improved. se had no influence on leth-slity the lenl or the chan in rs or ap ii. background: since reperfusion injury occurs when oxygen is reintroduced into ischemic tissue, the ideal timing for administration of therapeutic compounds aimed at ameliorating oxygen radical mediated injury is at the time of initial fluid resuscitation. currently used colloid or crystalloid preparations do not provide optimal, or even significant, anti-oxidant protection. systemic iron chelation affords protection against the iron catalyzed components of oxygen and lipid radical mediated tissue injury. the conjugate resulting from chemical attachment of the clinically approved iron chelator, deferoxamine (dfo, desferal ®, ciba), to hydroxyethyl starch (hes) represents a novel approach to colloid based fluid resuscitation. hes-dfo contains % hes and % chemically bound dfo. the polymer-drug conjugate has a lower molecular weight than that of hes in order to allow more rapid excretion. results: preclinical and initial clinical trials indicate that hes-dfo is well tolerated, even at high doses. in animal studies, fluid resuscitation with hes-dfo does not significantly improve central hemodynamic recovery beyond that observed with hes, but hes-dfo seems to afford better protection of microcirculation in organs at risk (lung, liver and gut), possibly by decreasing neutrophil sequestration. in a burn model, total fluid requirements are lower and oxygen utilization higher in hes-dfo treated animals compared to hes controls, suggesting decreased vascular leak and improved tissue perfusion. conclusion: hes-dfo represents a means by which potent antioxidant protection can be administered at resuscitation. iron has been suggested to play a pivotal role in oxygen flee radical mediated tissue injury. in vitro experiments indicated its critical role as a katalyst in hydroxyl free radical generation fenton-reaction). since iron chelator deferoxamine administered in shock alone demonstrated severe side effects, a hydroxyethylstarch (hes)daferoxamine (dfo)-conjugute was used to modulate oxygen free radical injury during the ischemia/reperfi~ion syndrome induced by hemorrhagic shock. methods. female lewis rats ( - g, n> ; pentobarbital anesthesia mgjkg), in hemorrhagic shock ( the aim of the study was to elucidate ( ) whether the generation of or would affect lung and kidneys as primary shock organs in the very early phase of sepsis and ( ) whether dfo-hes could prevent this tissue damage. methods: in rats sepsis was induced by cecal ligation puncture (clp) peritonitis. the animals were randomly assessed to groups: one group was treated with ml dfo-hes ( mg/kg iv), the other rats received solely ml of the carrier starch solution. , , , and min after induction of sepsis respectively, the animals were sacrificed, the organs collected, and tissue contents of glutathione (gsh), malondialdehyde (mda), myeloperoxidase (mpo) and conjugated dienes (cd) determined. plasma samples were obtained for analyses of endotoxin (chromogenic lal test). blood pressure (map) was measured via a carotid artery catheter. results: clp caused sepsis with high (> . eu/ml) endotoxin levels. map in both groups decreased slightly but significantly during sepsis regardless any treatment. in the lungs mpo concentration was increased (p< . ) in the lies group already min after sepsis induction. concomitantly, tissue gsh level decreased and lipid peroxidation was pronounced as shown by elevated mda and cd levels. dfo-hes diminished tissue pmn accumulation and mpo concentration. moreover, at each time point lung mda and cd levels were lower (p< . ). histomorphological examination showed marked micro-atelectases, destruction of the alveolar septa, and splicing of the basal membranes in the lies group. in contrast, in dfo-hes treated rats the alveoli remained well-ventiiated and only some enlarged reticular fibers without splicing were observed. almost similar results were found for the kidneys. mpo levels differed neither within nor between both groups. the slight decrease in gsh levels seen after min in the dfo-hes group seems to demonstrate an oxidative stress to a lesser degree. the most impressive effect of iron chelation, however, was revealed by the lipid peroxidation products. at each time point, mda and cd levels were lower (p< . ) compared to the hes group. light and electron microscopic examination disclosed tubulotoxic and mitochondriat damages while dfo-hes lxeatment prevented that alterations. conclusion: both the biochemical and histological results of this study reveal an early and remarkable generation of or in peritonitis-induced sepsis. thereby, these or obviously cause pulmonary and renal tissue damages, intravenous application of dfo-hes may, however, benefit by preventing early lipid peroxidation of the tissue. the proteolytic irreversible conversion of xanthine dehydrogenase (xd) to xanthine oxidase (xo) is triggered by calcium flux. the aim of our study is to clarify ~he link between intracellular ca + levels and xo activity determined by uric acid release, and to evaluate the efficacy of verapamil, on the generation of hydrogen peroxide associated with reperfusion by assaying lactate & pyruva~e release and the levels of cytosolic free nad /nadh ratio. experimental protocol consisted of :(a) non ischemic/reperfused experiment in which normal cardiac slices of rats were perfusated with oxygenated kreb's ringer phosphate buffer containing glucose ( mg%) and bovine albumine ( gm%) for min at °c.it composed of groups, group aa (control group), and groups ab & ac (perfusate supplemented with verapamil in the dose of loo& mi% respectively). (b) ischemic reperfused experiment in which ischemic cardiac slices were obtained from rats subjected to min ~aemorrhage.lt was also divided into two groups; group ba and bb (verapam~/ mi% added to perfusate}. verapamil stimulated uric acid release from normal rat cardiac slices were % in group ab and % in group ac(dose related). rates of uric acid release is enhanced by verapamil in group bb. moreover, rates of uric acid release in groups ac & bb are insignificant. in verapmil added groups (group ab, ac & bb), increase uric acid release is associated with an enhancement in pyrurate release and with increase levels of cytosolic free nad+/nadh ratio, although it is not evident ~ ischemic group (group ba).it is concluded that the conversion of xd to xo is calcium independent. eicosanoids like thromboxane a , leukotriene b and leukotriene c are known as promoters of initial inflammatory reactions. we investigated whether oxygen radicals (or) are able to induce a release of these eicosanoids in whole blood. blood from healthy volunteers was incubated with xanthine oxidase/hypoxanthine to generate oxygen radicals. after , , , and minutes plasma levels of thromboxane b (txb ), leukotriene b (ltb ) and leukotriene c (ltc ) were determined via elisa technique. another volunteer had taken mg aspirin one day before taking the blood sample (no ). results: txb plasma levels increased from pg/ml at min to pg/ml, pg/ml, pg/ml and pg/ml at , , and min (p< , ) . ltb and ltc plasma levels showed an increase during the first few minutes (ltb : min: llpg/ml, min: pg/ml; ltc : min: pg/ml, min: pg/ml (p< , )) followed by a decrease to normal values at min. in the sample no the cyclooxigenase-pathway was completely inhibited, the txb plasma-levels did not alter at all, whereas ltb and ltc -plasma levels weren't affected. opallogeneic blood transfusion jane shelby, ph.d., and edward w, nelson, m.d, there have been numerous investigations dudng the last two decades examining the effect of surgery, anesthesia, blood loss and transfusion on vadous immune parameters in humans and animal models. there appears to be concurrence among several well controlled studies that transfusion of whole blood (containing leukocytes), has regulatory effects on immune ceil function which include decreased cell mediated immune response, and inhibition of il- secretion. these effects occur following transfusion alone and in con.cart with the distinct immune effects of surgery, trauma and anesthesla, the clinical consequences of this immune modulation by transfusion include decreased allogeneic response to transplanted organs, which has been exploited clinicelly in renal transplant patients. additionally, there is evidence for a strong association with increased risk for infection in transfused patients following surgical procedures. aiiogeneio blood transfusions have been shown to inhibit cellular anti.bacterial mechanisms, causing increased susceptibility to bacterial pathogens, in humans and in animal models. there is also concern that allog~neic transfusion may adversely affect cancer patients, resulting in decreased disease-free survival. several stategies have been proposed to minimize the adverse effects of blood transfusion. there is evidence that the risk of immune mediated infectious complications associated with transfusion may be greatly minimized wlth the use of autologous blood and leukocyte free allogeneic blood.products in surgical and trauma patients, it also appears that the inhibition of cellular immune response and il- productiorl following atlogeneic blood transfusion may be mediated by increased prostaglandin e secretion, and that immune response may be preserved in allogeneio whole blood transfused subjects receiving c lc~oxygenase inhibitors such as ibuprofen. among these are various alterations in immune function. efforts have therefore been made to utilize alternatives to homologous transfusions. these include the use of autologous predonation, supplemental iron therapy, and recombinant human erythropoietin. although initially considered innocuous, these therapies are now recognized to have potential deliterious immune sequelae. erythropoietin, by its ability to lower serum iron levels, can impair both lymphocyte and nk cell activity. autologous donation impairs nk cell function. finally, supplemental iron therapy can stimulate bacterial growth and increase the rate of infectious complications. this talk will present a discussion of these factors as well as a weighting of their importance. r.l rutan, rn;bsn, shriners burns institute and the university of texas medical branch, galveston tx, usa the serious sequelae of homologous blood transfusions have resulted in vigorous efforts at identifying alternate therapies for correcting red blood cell (rbc) deficits. erythropoietin (epo) was hypothesized to exist in the early th century, however the protein was not isolaled until . the human gene was identified and cloned in , which permitted the production of epo through recombinant techniques. the earliest clinical trials were performed in anemic end-stage renal failure palients on hemodialysis. treated patients experienced increases in erythropoiesis with normalization of hematocrit and hemoglobin levels, cessation of lrans-fusion requirements and improvement in general wellbeing. these studies, however, identified side effects of epo treatment such as hypertension, seizures and ee deficiency. volunteer trials have established that the hypertension is not a direct pressor effect but rather the result of abnormally rapid increases in red cell mass in the face of the incompetent volume-controlling mechanisms of the end stage renal failure patient. lower doses of epo and the subsequent gradual increases in red cell mass are associated with significantly lower incidences of hypertensive complications of epo therapy. likewise, seizure activity is not the result of a direct epileptogenie effect but parallels the incidence of hyper-tensive-related sequelae during high.dose epo treatment. in cross-over designed studies, pre-existing iron deficiency has been demonstrated to decrease or negate stimulated erythropoiesis but effective-hess can be restored with appropriate fe supplementation. exogenous epo is effective whether given by iv or sq routes and dose response curves do not vary with route of administration. increases in rbc mass are directly related to the dose of epo, both in amount and frequency of administration although there is a - day time lag between the first epo dose and laboratory indications of its action (i.e. increase in the number of reticulceytes in peripheral wood). epo is currently labelled for use in the treatment of anemias associated with end-stage renal disease and aids. however, its use in the surgical population has been explored because of its unique direct dose-response, epo has been used to effectively increase the blood harvest amounls in autologous pre-donation, significantly increase hematocrils in children following thermal trauma and successfully increase red blood cell mass following essential surgical procedures in patients with religious aversion to transfusion. by blood transfusion in colorectal cancer surgery mm heiss md, ch delanoff md, r stets md, j hofinann, e faist md, kw jauch md, fw schildberg md allogeneic blood transfusions are associated with an increased risk for postoperative infections in colorectal surgery when compared with autologous blood transfusions. attribution of this effect to immunomodulation was suspected in our previous study (lancet ; : - ) . task of the recent investigations was to analyze which specific effector systems were affected in-vivo by this transfusion-associated modulation. for global in-viva assessment of cell-mediated immunity (cmi) multiple recall skin-reactions were applied prior and post-operative. the specific humoral immune mechanisms were investigated by applying tetanus-toxoid one day preoperatively and deterimnating the quantitative igg-response. for indication of macrophage stimulation in-vivo tnf-levels were determinated by bioassay. dth-responses were significantly suppressed (p< . ) in patients receiving allogeneic blood (n= ) or operated without blood transfusions (n= ). dthresponses were not suppressed and tendentiously increased in patients with autologous blood transfusions (n= ). in contrast, specific igg-levels increased sigmficantly (p< . ) in patients receiving allogeneie blood (from . + . to . _+ . ie/ml) whereas in patients receiving autologous blood a smaller increase (from . + . to . + . ; p= . ) was observed. tnflevels demonstrated a similar pattern with a higher increase in patients receiving allogeneic transfusions (l . + . to . + . u/ml) compared to those patients with autologous blood ( . + . to . + . ). in conclusion these data indicate that allogeneic blood transfusions lead to a remarkable macrophage/rhs stimulation. this is corroborated by the boostered humoral igg-response which was initiated before onset of surgical trauma and blood transfusion. concerning cmi this caused a substancial suppression probably due to a stimulated secretion of immunosuppressive monokines. objective: firstly, to analyse the concentrations of the cytokines tumor necrosis factor (tnc), interleukin- (il-i), interleukin- (il- ) and coagulatioo/fibrinolysis parameters in postoperatively retrieved blood from a surgical area, secondly to characterize the correspanding cytokine patters in the patients and thirdly to study cytokine concentrations in the initial portion of drainage blood from a surgical area. materials and methods: blood retrieval was performed in a closed-loop system without anticoagulant during - hours after surgery in patients undergoing arthroplasty ( hips and knee). kf, il- , it- , thrembin-antithrombin complexes (tac) and antithrombin (at) ~ere determined in shed blood. patient plasma tn v, il-i and il- concentrations ~ere analysed at the beginnlqg and end of the - hour blood retrieval period. in a separate study ( hip arthroplasties) f~f, il-i and il- ~ere determined in the initial portion of drainage blood. cytekine analyses ~re performed usiog ipmuooassays. an omidolytic method was used for at determinaf.ion and tac was analysed by elisa. n~n-poram~tric tests was used for the statistical comparison. results: the patient plasma il- coocemtratiems rose from a median value of to pg/ml, p<o. , during the blood ret.rieval period. in c was detectable in patients, range: - pg/ml. il-i was not detectable in the patients. in retrieved blood tag was > mg/ml in all samples (ref:< . mg/ml) and at was . - . units/ml (ref:o. - . ) . the il- concentrations in retrieved blood was > pg/ml in all samples. tn v or il-i was not detectable. in the separate study, (n= ), characterlzing eytokine content in the initial portiere of drainage blood, in= (range: - pg/ml) and il-i (range: - pg/ml) ~re present in all samples but ii- (range:o- pg/ml) was detectable in o.qly one semple. conclusion: theses findings indicate that hypereoagulability and hic~ ccrcentratioos are present in retrieved blood. the cytokine pattern in the initial portion of blood from a surgical area differed from these observed in retrieved blood and in the systemic circulation. to identify the role of both autologous and homologous blood on postoperative infections in elective cancer surgery. materials and methods: patients with colo-rectal cancer submitted to curative elective surgery were prospectively studied. on hospital admission the following nutritional measurements were assessed: serum level of albumin, cholinesterase, delayed hypersensivity response , total lymphocyte count and weight loss, as were age and sex, duration of operation , operative blood loss, amount and type of blood given, pathological dukes' stage of the disease and the attending surgeon were also recorded. results : eighty-four patients ( . %) were perioperatively transfused. thirty-six ( . %) patients were given autologous blood , while ( . %) received homologous blood. no patients received both autologous and homologous blood. twenty eight ( . %) patients developed postoperative infections. non transfused patients had a . % infection rate , those receiving autologous blood had a . % infection rate, whi]e in the homologous blood group the infection rate was . % (p < . ). univariate analysis showed that infections were significantly related to operative blood loss (p< . ), length of operation (p< . ) blood transfusion (p< . ) and attending surgeon (p< . ) . multivariate analysis identified homologous blood transfusion as the only variable related to the occurrence of postoperative infections , while the other variables failed to reach statistical significance. blood transfusion (bt) remains an essential life-saving treatment for surgical patients. however, besides the beneficial short-term impacts, negative longer-term effects are observed, which include various alterations in the immune responsiveness. in surgical patients these alterations may contribute to the increased risk for infections and cancer recurrence. since relatively few data demonstrate immunologic changes occurring in other lymphoid compartments than blood after bt, we studied the effect of et on the frequency and responsiveness of immune cells in bone marrow (bm), spleen (spl) and blood (b) in a rat model. normovalemic, month old rats were transfused intravenously with syngeneic heparinized venous blood ( x ml, every other day), and , and days after the last transfusion bm cells ( leh is an experimental oxygen-carrying resuscitation fluid. since leh is cleared from the circulation primarily by the mps, its effect on the development of sepsis and the nature of its relationship with the mps remain a major concern. preliminary in vivo data from our laboratory failed to show any leh effect on the hemodynamic and hematologic responses to endotoxin lipopolysaccharide (lps) in the rat. in contrast, leh exacerbated the lps-induced tnfa production and early mortality. the exacerbation of early mortality by leh was attenuated by pretreatment with the tnfu synthesis inhibitor rolipram. ex vivo, peritoneal macrophages from rats treated with leh and lps have shown increased il-lg mrna signal as compared to lps alone. also, leh increased tnftx production by peritoneal macrophages in response to lps stimulation in vitro. additionally, recent pilot studies indicate that leh attenuates pma-induced superoxide production from rat peritoneal macrophages and that leh augments fmlp-induced migration of human monocytes. taken together, these data strongly support possible interactions of leh with the mps and therefore the nature of such interactions should be further explored. over the last decade, we have developed liposome encapsulated hemoglobin (leh) as an artificial oxygen carrying fluid, or blood substitute. our efforts have focused on studies to define the safety and efficacy of this resuscitative solutions. leh consists of distearoyl phosphatidylcholine, cholesterol, dimyristoyl phosphatidylglyeerol, and alpha tocopherol in a : : . : . mole ratio and can encapsulate hemoglobins of different origin (bovine, human, recombinant human). leh is fabricated using hydrodynamic shear to create an average particle size of . microns. leh can be lyophilized using disaccharides and stabilized in the dry state and easily reconstituted before administration. histopathology and clinical chemistries indicate that leh rapidly accumulates in tissue resident macrophages in small animals injected in the tail vein, principai y in the liver and spleen. the consequences of accumulation in the reticuloendothelial system are manifest by transient increases in liver transaminases (ast, alt), bilirubin, and bun over - hours with no change in biliary function (ggt, ap) . clearance through the liver and spleen is observed over the course of - -weeks. more recent attention has been focused on secondary consequences of leh administration especially with regard to inflammatory eytokines. leh does not elicit expression of tumor necrosis factor in vivo and in isolated macrophage cultures, but does result in a transient increase in serum il- . we have also examined the interaction of leh with lps in vitro macrophage culture to further understand how this blood substitute may effect the immune system. we have labeled leh with technetium- m ( mtc) to study the biodistribution of leh non-invasively in anesthetized rabbits. rabbits were infused with a % topload of leh ( mg of phospholipid, . g of hemoglobin per kg of body weight) and imaged continuously with a gamma camera. at hours, images were again acquired. animals were then sacrificed and tissue counts obtained, images revealed an initial rapid uptake bythe liver, % at minutes and % by hours. the spleen accumulated activity at a slower rate, % at minutes and % at hours. at hours, autopsy biodistribution studies revealed that approximately . % of the dose is in the blood pool, . % in liver, . % in spleen, . % in lungs, . % in muscle and . % in urine, with trace levels in kidney, brain and heart (< °/o). in a hypovolemic model, rats were % or % exchange transfused with mtc-leh. in the % exchange model, mtc-leh was rapidly taken up by the liver and spleen with minimal activity in the circulation at hours. with the % exchange, % of the leh was in circulation at hours. the interaction of leh with platelets labeled with indium- was also studied. after infusion of leh, the labeled platelets rapidly moved from the circulation to the lungs and liver. over the next minutes, the platelets gradually returned to circulation. this effect was not seen with iiposomes of the same lipid composition but containing no hemoglobin. non-invasive imaging is proving to be a very useful tool for the investigation of leh. the need for a safe, efficacious and commercially viable blood substitute is unequivocal. of the several strategies pursued to invent an adequate blood substitute, liposome entrapped hemoglobin (leh) has been already established as a leading possibility. major advances in liposome technology have already resulted in liposome preparations compatible with clinical use for drug delivery. recent technological advances made by the u.s. naval research laboratories resulted in the capacity to entrap hemoglobin into liposomes in a way which secludes hemoglobin from interacting freely with biological systems. the leh produced has already been tested in in vivo systems and was foun.d to be well tolerated. moreover, the leh originally produced as a solution can be transformed into a lyophilized form which can be reconstituted and delivered as a fresh solution. while important milestones in leh development for a practical blood substitute have been achieved, several issues remain to be explored. most notably, the long term consequences of leh on host defense mechanisms and, in particular, immune cell function. in addition, it is important to understand more fully the metabolic fate and repercussions of leh delivered at clinically relevant dose/schedule regimens. finally, while leh is a highly promising strategy for a blood substitute, the present formulations consist of human hemoglobin derived from human blood, to improve the safety profile, a recombinant preparation for liposome entrapment will be much desired, aa-ginine, a semi-essendai dietary amino acid, possesses several unique and potentially pharmacologic properties. argirdun is a potent secretagogue for pituitary growth hormone and prolacfin and for pancreatic insulin and glueagon; it modulates host protein metabolism by increasing nkmgen retention and enhancing wound collagen synthesis. it also is a potent t call function regulator. ait of these effects coupled with its relative lack of toxicity and safety make it an a~antive nulritionai pharmacologic agem (t). rodents fed supplemeutal arginine exhibit increased thymsc weight which is due to increased numbers of thymic lymphocytes present in the gland. thymic lymphocytes from animals fed supplemental ar~e demonstrate increased blastogenesis in response to coma. and pha ( ) . peripheral blood lymphocytes from humans given supplemental arginine also have heightened mitogunic responses to mitogen or antigens ( ) . in postsurgery padents supplemental arginine abrogates or diminishes the deleterious effects of trauma on lymphocyte responsiveness and restores peripheral blood lymphocyte responses much faster than observed in controls. overall host immunity is also enhanced by arginine. allograft rejection is enhanced and septic animals survive longer when given supplemental arginine ( ) . tumor bearing urginine-supplemented animals have decreased tumor growth and enhanced survival (i). lastly, asgmine can induce t cell maturation and t cell mediated responses in athyrnic nude mice. arginine also has remarkable effects on host nitrogen metabolism post-injury. in increases nitrogen retention in healthy human volunteers and in surgical patients. this beneficial effect on overall nitrogen metabolism is accompanied by a unique effect on the healing wound. supp]emental arginine increases wound collagen synthesis which also translates into increased wound breaking strength ( ) . arginine has no effect ou epithelialization. douglas w. wilmom, m.d. boston, ma gintamine is the most abundant amino acid in the body, but it has long been considered a nonessential amino aeid because it is synthesized in many tissues. fohov~g st,~'vation~ injury or infection, skeletal muscle pmteln inoresses its net tale of degradation and releases amino acids into the blunds~mm at an aocelerared rate. app~o)~mately one-third of the amino nitmgea is ghitamine, which is metabolized by the kidney where it parth:~pates in acid-base homeostasis, is the primly ~ for lymphocytes, mac~optmgcs and untexocyms, and contm'butcs to the synthesis of giumth~une. olmamine degrades slowly while in ~olu~ou, especially at usual room teml~mtums. because giulamine was considered nonessential, it has beer absent r'om nil intravenous and most gluts.mine should be considered a cendittona]ly essential nutrient for individuals with serious ilinesses, uspccially those confoanded by infcctinn and inflammation. over the uc~:t - years, glutamine will be incorgorated into most feeding formulas designed for patients with critical illness. o]~ga- pufa there continues to much interest in the application of the mega- pufa in clinical nutrition. the basic principle has been that the mega- pufa will displace arachidunic acid and result in a decrease in eic san id production. in addition these changes in pufa will after the physical characteristics of the membrane including flujdity, receptor function and transmembrane signals. animal studies have shown that there is omega- incorporation with continuou~ enteral feeding both in control and endotoxic animals within days. this includes the liver, spleen, circulating and alveolar marc phages and the lung. this incorporation resuls in significant changes in the eicosan id production including pgf and ket -pgflalpha. there is improvement in the cardio-vascular reep nse of these animals with ~ecreamed lactic acidosis and improved cardiac contractility. as well there is improved immune function with improved t cell response to mit gens. the ~ of a mumber of pharmacological agents blocking cicosanoid production can enhance the cell effects of mega- pufa. clinical studies using short term entsral nutrition with mega- either alone or with other enteral supplements in a number of clinical settings have shown significant mesa- incorporation and decreased eicosan id production. these positive results must be discussed with the additional evidence that long term omega- supplementation decrease eic san id production but als induce a state of immune suppression that is capable of increasing transplant sunvival. these ng te~ inune effects may benefit clinical conditions including rheumatoid arthritis and cr hn' disease early enteral nutrition instituted i~mediately afte~ injury will decrease the entry of bacteria into the intestinal wall and decrease the number of bacteria that translocate into the portal blood. these reductions are associated with & decreased catabolic response, decreased plasma cortisnl levels, end decreased vma excretion in the urine and prevention of mueosal atrophy. sdecific nutrients also affect the transloeation process. addition of arginlne to the diet significantly improves the ability to kill translocated organisms. however. translooetion across the gastrointestinal barrier is not affected. in contrast, glutamine diminishes the rate of translooation across the imtestinal barrier and also improves killing of the beetarla that do translooate. the omega fatty acids in the form of fish oil slightly decrease the rate of translocation but more significantly increase the ability of the animal to kill translo~ated organisms, all three dietary additives, i.e. argini~e, glu=amine and fish nil. significantly improve survival, hut adding glyoine or medium chain triglyeeridem do not, combinations of srginine and glutamlns, glutamine and fish oil, and fish ell end arginine each improve survival, and to a greater degree than a combination of all three. these studies add further evidence that translocation is an important determinant of survival after injury, early feeding with immunonutrlent enriched dices will improve survival and dsarease transloeation to varying degrees, depending upon the nutrients provided. objectives: we studied effects of supplementing a commercial enteral diet, impact r (imp, sander nutr lnc), with fiber (imp/fib) or alanyl-glutamine (imp/ag, exogenous glutamine (gln) gms/l) on influencing the incidence of bt to mesenteric lymph nodes (mln) in burned mice. fiber has been shown to improve gi integrity under certain stress/treatment conditions. the dipeptide ag is a water-stable source of gln, which is a specific fuel for many cells including enterocytes. traumacal (trcal), a high-protein, high-fat enteral diet (mead johnson iuc), was also studied, as well as rodent chow (harlan teklad inc), which contains very high protein & fiber. methods: anesthetized cf- mice aged - wks received % tbsa fullthickness dorsal burns & were resuscitated with cc ip saline. diets were allowed ad lib; caloric intakes were comparable in all gps except fasted gp (fast hrs, chow hrs). at hrs postburn mln were sterily removed, homogenized and plated on heart brain infusion agar; cfu/g mln tissue were determined. bt was analyzed by fishers exact test, cfu/g by anova-bonferroni. * p< . , ** p< . compared to imp and burn-fast gps. background. infectious complications following trauma, major operation, or critical illness adversely affect hospital cost and length of stay (los). some key nutrients have been shown to possess immune enhancing properties. this multicenter trial was conducted to determine if early administration of an enteral formula supplemented with arginine, dietary nucleotides and fish oil can decrease los and infectious complications in icu patients. methods. this was a prospective, randomized, double-blind study of adult icu patients who required enteral feeding for > days. patients entered the study within hr of the event, were stratified by age and disease, and were randomized to receive either the supplemented formula (impact®) or the conventional formula (osmolite ® hn). feedings were initiated at full strength and advanced to at least ml/hr by hr after event. results. both groups tolerated administration of formula well. for patients fed > days, the median los was % shorter (p=o.ol) for the--supplemented group ( days) compared to the conventional group ( days). the incidence of most infectious complications was lower in the supplemented group, but this difference reached significance only for urinary tract infections (p=o.o ). the supplemented group had a significantly shorter los from onset of infectious complication until discharge for patients with pneumonia ( vs. days) and skin/soft tissue infection ( vs. days). conclusions. administration of the supplemented formula was safe and well tolerated. when fed > days, it reduced the incidence of most infectious complications, and significantly reduced los. materials and methods: twenty-seven patients were randomised into groups ( n= each) to receive either a standard enteral formula, the same formula enriched with arginine, rna and omega fatty acids (enriched group) or isonitrogen, isocaloric parenteral nutrition. early enteral nutrition was started within hours following surgery ( ml/hour). it was progressively increased reaching a full regimen on day . on hospital admission and on post-operative day and , the following parameters were assessed: serum level of transferrin , albumin , prealbumin, retiool binding protein (rbp), cholinesterase. delayed hypersensitivity response, igg, igm, iga, lymphocyte subsets and monocyte phagocytosis ability were evaluated on admission and on post-operative day , , . the three groups were comparable for sex, age, cancer stage, type and duration of surgery, intra-operative blood loss and amount of blood transfused . in all groups a significant drop in all the nutritional and immunological parameters was observed on postoperative day . comparing post-operative day versus day a significant increase of prealbumin (p< . ) and rbp (p< . ) was found only in the enriched group. with respect to immunological variables an increased phagocytosis ability (p< . ) and a significant recovery in delayed hypersensitivity response (p< . ) was observed only in the enriched group. conclusions : these data are suggestive for a more effective post-operative recovery of both. nutritional and immunological status in cancer patients fed with enriched enteral formula. gastrointestinal intolerance was equivalent ( % in each group) and laboratory screening confirmed that both diets were safe. when analyzing clinical outcome for all patients, there were no significant differences in septic complications (immun-aid = % vs vivonex ten = %), mean mof score (immun-aid = l.b vs vivonex ten = . ), or mortality (immun-aid % vs vivonex ten = %) . kowever, when analyzing the subgroup of patients with severe injury (iss or ati _> ), patients receiving immun-aid appeared to have fewer septic complications ( % vs %) and their mean mof was significantly lower ( . _+ . vs . + . , p = . , student's t-test) . these preliminary data indicate that immun-aid is tolerated well when aggressively delivered immediately postinjury. the ultimate affect on clinical outcome appears ~avorable for immun-aid, but needs to be confirmed in larger patient groups. kemp?n, m., neumann, h.a., he i[michh b: as both increased, normal and reduced phagocytic capabilities of polymorphonuclear leukocytes (pmn) and monocytes in acute batterial infections have been reported, the role of phagocytes in patients with severe sepsis is less clear.we examined pmn and monocytes from patients in septic shock and heailhy votunteers for phagocytic function. phagocytosis was determined by flow cytometry (facscan) and was measured by the ability of pmn and monocytes to phagocytose e.coli marked with fluorescent antibodies. a septic shock was defined by the presence of a ~ource of i, nfoctiqn with a known bacteriology, distinct signs of a systemic response and defined minimum scores in icu scoring systems indicating the presence of a multiple organ failure. additionally we examined how phagocytosis is influenced when a new enteral diet formulation containing substrates suggested to improve immune function or arginine, one of its major compononts, is added in vitro in defined concentrations and incubated for minutes. pmn (p{o, ) and monocytes (p<o, ) of the septic patients showed a significantly enhanced phagocytosis compared tolhe phagocytes of the healthy group. the incubation with the enteral diet in vitro was followed by a higher dose-related rate of phagocytosis, especially in the healthy group, that was not statistically significant. after addition of l-argintne we atso observed an iqcrease in phagocytosis, that was lower than in the group with the complete diet. using a modern immunefluorescence-cytometric essay we founfl an improved phagocytic function in septic shosk. there are signs for an influence of nutrient substrates on phagocytosis which seems to be complex and should be further investigated. arg and gln were lower in the %-bcaa vs %-bcaa group; arg was related directly to arg dose and inversely to bcaa dose (p<o.o for all). clearances of bcaa increased with increasing bcaa dose (p<o.o ); arg and gln clearances were directly related to the bcaa clearances (rz=o. , p<o.o i). relationships between arg, gln and other aa seem to be ruled by patterns involving specific intracellular aa transport systems. regulation of arg and gln clearances through bcaa administration may be related to an increased flux of aa into synthetic processes. intestinal segments from rats immunized by infection with the nematode trichinella spiruus undergo intestinal anaphylaxis or type i hypersensitivity when challenged in vitro with parasite antigen. immunized rats exposed to t-radiation ( gy) and sustained on rat chow,fail to fully express type i hypersensitivity up to days post irradiation (dpi). we hypothesized that enteral nutritional support immediately following irradiation may be effective in preserving mucosal immune responsiveness or in reducing the recovery time after radiation-induced injury. rats were infected with x t.spiralis larvae. thirty to fifty days later rats received gy t-radiation from a co source as total abdominal irradiation (tai). immediately following tai, rats were fed an elemental amino acid diet (eaad) and at various dpi sacrificed and tested in ussing chambers for local anaphylaxis, expressed as antigen-induced ci secretion. the normal ci secretory response to antigenic challenge which is suppressed after irradiation,was restored by dpi when rats were placed on the eaad. antigen-induced c[ secretion is dependent on the interaction o~ antigen with specific ige antibodies on the surface o mucosal mast cells and their subsequent degranulation. mast cell-derived -ht,histamine and pg, together effect ci secretion.in irradiated rats on rat chow,the failure to respond to antigenic challenge appears to be associated with the destruction of mast cells. they are undetectable with standard staining procedures and mucosal histamine levels are drastically reduced. also,el secretion can be induced by direct stimulation of epithelium with cr secrctagogues and circulating ige levels are normal. despite a more rapid recovery of immune responsiveness in irradiated rats receiving eaad,mast cells were not restored. we conclude that the eaad contributes to an adaptation that supports the expression of local anaphylaxis in the absence of mast cells. total parenteral nutrition (pn) and bowel rest may influence the host response to infection. this study examined the different host response to infection in parenterally and enterally fed animals. forty-three rats were divided into pn group and total enteral nutrition (en) group. they received identically constituted isocaloric isonitxogenous diets for seven days. animals were then challenged intraperitoneauy with xl(y escherichia coli and survival were observed. in other experiments, they were sacrificed before and two hours after bacterial challenge. peritoneal cavity was lavaged with ml saline and peritoneal exudative cells (pec) were harvested and cultured in vitro for hours with and without lipopolysaccharide (lps). colony f(m, ning units of bacteria (cfu-b) in the peritoncal lavaged fluid (plf) were determined and tnf in the plf, serum and pec culture supernal,ants were measured by l bioassay. twenty-fonr hour survival rate in en group was significantly greater than that in pn group ( % vs %). the data suggest that local immune responses of pn-treated animals may be depressed with consequent disturbance in the clearance of bacteria. this may lead to a greater systemic cytokine response and resulting in a poor survival after bacterial challenge in pn group. the effects of intragastfic tube feeding of diets enriched with m- polyunsaturated fatty acids (pufa) from safflower oil (so) or (o- pufa from menhaden oil (mo) on eicosanoid production, and onthe membrane phospholipid fatty acid composition were investigated in a severe acute septic shock model. the percent calories from fat maintained at % in the diets was adjusted to provide % each of saturated, monounsaturated, and pufa using olive oil and palm oil as filler fats. after feeding for days, lipopolysaccharide (lps, mg/ g) was injected through the tail vein. the percent of animals surviving in the mo group was % ( / ), and did not differ significantly compared to % ( / ) in the so group. the plasma concentrations of tnf ct for the groups of animals fed so ( . + . ng/ml) and those fed mo ( . + . ) did not differ significantly. the levels of prostaglandin(pg)e , -keto-pgflc~ and tumor necrosis factor-ct (tnf-c were determined min after lps injection. the fatty composition (relative mole%) of the liver was determined before (lps-) and h after the lps administration (lps+). the data (mean_+sd; n= ) are as follows. group these data indicate that a marked reduction in the plasma levels of poe and -keto-pgflct for rats fed mo diet was associated with a reduction in aa which was displaced by epa in the membrane phospholipids. further, in these rats, we observed that there was a . % reduction in the percent of epa following lps challenge compared to the basal levels. however, for the group of rats maintained on so diet, the liver membrane phospholipid fatty acid composition before and after lps administration was unaltered. these finding suggest that although a reduction in aa was associated with a decrease in the production of pro inflammatory eicosanoids after days of continuous mo feeding, there was no marked effect on the survival following lps challenge. animal models have been used to examine the effects of various nutrients and nutrient combinations upon the immune system. we studied the effects of standard and specialized enteral formulations on the response of mice to infectious challenge with listeria monocytogenes, influenza a or candida albicans in order to test the efficacy of specialized ingredients. cf- outhred female mice ( - g) were fed purina # chow, commercially available osmolite hn ®, perative ® or impact*. mortality and tissue burden of pathogen were examined during the d period following induced infection. the results indicate that there were no differences between the groups fed the liquid formulas with regards to mean survival time or % survivors in these models of infection. examination of spleens in the groups challenged with l. monocytogenes and kidneys in the groups challenged with c. albicans revealed no differences in tissue burden of pathogenic organisms. alterations in the length of pre-feeding from to days prior to infection did not affect these results. these data demonstrate that, contrary to previous reports, the use of specialized nutrients did not improve resistance to disease in mice challenged with l. monocytogenese, influenza a or c. albicans as compared with mice fed osmolite hn. animals fed standard chow tended to be more resistant to infectious disease than those fed the liquid formulas perhaps due to the form of diet or complexity of ingredients. the results indicate that these animal models of infectious challenge may be of limited use to distinguish effects of modified nutrient composition of enteral formulas. laboratories the beneficial effects of sesame oil (ses) have been attributed to the presence ofsesamin, a non-fat constituent which is claimed to iultibit a- desaturase activity. we investigated the effects of ad libidum feeding of ses enriched diet on fatty acid composition of phospholipids from plasma, liver, on eicosanoid production, and on the protective effects in mice after cecal ligation and puncture (clp) and compared with safflower oil (so) diets. olive and palm otis were added as filler fats to the so diet to maintain the ratios of saturated, and unsaturated fatty acids similar to ses diet. thus, while the total fat remained at wt%, the only difference between the two diets is the presence ofsesamin in ses diet. the animals were fed for weeks. the incorporation of dihomo-y-linolealc acid (dgla: : o)- ) in the phospholipids from plasma and from the liver for the group of mice fed so diet ranged between - % compared to the undetectable levels for those fed so diet. there were no significant differences in the incorporation of other fatty acids either in plasma or in the cell membranes between the two groups. following an intraperitoneal administration oflps ( p.g/kg body wt), the plasma levels of both pge , and txb , were significantly decreased (p< . ) by nearly % for the group of animals maintained on ses diet compared to those maintained on so diet. these findings suggest that sesamin inhibited the release of arachidonic acid (aa) which upon accumulation is retroconverted to dgla which could reflect a modest increase in the content of dgla. failure to decrease the formation of aa could mean that the effects of sesamin on chain elongation process may be after aa is formed, and that sesamin may not inhibit a- desaturase activity. on the other hand, our data suggest that sesamin present in sesame oil inhibited the activity of cyclooxygenase which converts aa to its metabolites), or alternatively could block the activity of phosphalipase a (pla ) (which releases aa from membrane phospholipids) as is evident from a marked decrease in eicosanoid production. the percentage of animals surviving after cecal ligation and puncture in rite group of mice fed ses diet was % ( / ) which was markedly higher (p< . ) compared to only % ( / ) for the so group. increase in survival in the group of mice fed ses diet was associated with nearly % reduction in the production of tnf in response to lps i.p. challenge, for ses group compared to so fed animals. our data suggest that sesamin, present in sesame oil, decreased the production tnf~, inhibited the activity ofpla and consequently offered a significant protection against clp. thus sesamin or sesame oil appear to be novel antiinflammatory agents which are present naturally in many edible products. [dept. surgery, ~e. deasoness hosp.,harvard medical school, boston, usa] stress causes alterations in the homeostasis of an organism with major changes in various organs, lifespans of cells, protein turnovers, metabolic pathways of activation or stimulation, energy mobilization, etc. punctual changes have been described in several organs and various cell types of the nervous, endocrine and immune systems. many stored or newly synthesized proteins are released to the bloodstream to be transported to target organs or to be disposed of. as stress alters protein synthesis and requirements in the targets, the bloodstream is a useful system for monitoring the relevant changes. we present here the pattern of newly synthesized or released serum proteins in c bl/ mice that have been stressed by immobilization. the proteins have been labelled with ssmethionine in vivo and the study was performed by two dimensional gel electrophoresis based on the method originally described by o'farrel. the electrophoretic run was carried out in an isodalt apparatus. a molecular dynamics laser scanner and the kepler image analysis system (lsb, rockville, usa) were used for modelling the radiofluorographic images. the radiofluorographic images of the gels from serum samples obtained from mice injected with i mci of ss-methionine reveal about protein spots, from which a small fraction -about are altered in both qualitative and quantitative manner. the major changes are in the range of to . d. the analysis displays a highly reproducible pattern, where clear differences between stressed and non-stressed conditions are shown. differences between patterns attributed to chronic vs. acute stress will be presented. patients with severe trauma or major surgery are known to acquire alterations in neutrophil functions which contribute to their enhanced susceptibility towards microbial infections. we analyzed neutrophil functions from fourty patients admitted for major surgery days and days preoperatively and on the ist and th postoperative days (study-days i, , , and ) in a randomized placebo-controlled double-blind study. patients were divided into two groups, one ~eceived days preoperatively an enteral nutrition enriched with omega- fatty acids, arginiee, and rna (impact), the other an isocaloric, not enriched control nutrition (placebo). meutrophils were isolated from the peripheral blood and stimulated with the ca-ionophor a ( . pm). the capacity of the respective neutrophils to generate leukotrienes was analyzed by rp-hplc. neutrophils from impact group generated significantly higher amounts of ltb (mean values . ng/l x i~ cells) compared to the placebo group ( . ng) at study-day [p, . ). in contrast, the capacity of neutrophils to produce ltb was not significantly different in both patients populations at study-days and . the omega-oxidation of ltb to its biologically less active metabolites oh-ltb and cooh-ltb was not significantly different in both groups. however, neutrophils from group f patients generate more ltc at study-day (p( / ). the capacity of the patients'neutrophils to generate reactive oxygen radicals upon stimulation with fmlp, pma or the caionopbore a exhibited patient dependent differences but no correlations to the repective nutritional supplementation as was measured by luminol dependent ohemiluminescense= these data indicate that an enteral nutrition enriched in omega- fatty acids lead to alterations in distinct parameters of neutrophil functions. clinical patient characteristics and mean caloric intake were similar between the two groups. both formula were tolerated well and the incidence of diarrhea was low. the number of infectious and wound complications as well as the apache ii score was evaluated for the two study groups. although the number of complications in the group supplemented with arginine, rna and omega- fatty acids was lower no significant difference in the occurance of infectious and wound complications has been observed between the two groups. however, the apache ii score indicated a significantly improved clinical outcome in patients with supplemented diet. in conclusion, early enteral nutrition with an arginine, omega- fatty acids and rna supplemented diet helps to recover more rapidly after surgical total parenteral nutrition (tpn} is associated with intestinal atrophy and dysfunction attributed to the absence of the non-essential amino acid glutamine in current parenteral nutrition solutions. in this animal study with male wistar-rats we tested the hypothesis that glutamine-dipeptide supplementation during tpn for days would restore small bowel atrophy and tpn induced dysfunction. a conventional tpn solution ( kcai/kg bw) was compared to an isocaloric and isonitrogenous tpn ( , g n/animal/d) supplemented with alanin-glutamin (ala-gin) dipeptide ( , g n derived from ala-gin = % gin-n). an enteral fed cqntrol group was included. mucosal thickness, villous height and architecture, absorption of water and glucose as well as dissacharidase activity of maltase and alkaline phosphatase were evaluated. total parenteral nutrition in rats causes villous atrophy, a significantly reduced absorption rate and a decreased activity of villous enzymes compared to an enteral fed control (p<o, ). addition of ala-gin to parenteral nutrition solutions prevents intestinal atrophy and restores functional alterations with a significantly increased rate of water and glucose absorption as well as a higher dissacharidase activity compared to the standard tpn group (p< , ). there was no significant difference found between the enteral fed control and the dipeptide supplemented animals concerning intestinal structure and absorption, the enzyme activity was significantly decreased in the ala-gin supplemented group compared to the enteral fed control (p< , ). in this rat model supplementation of parentera] nutrition solutions with ala-gin dipeptide restores tpn induced intestinal atrophy and dysfunction. boston. ~ usa injury, infection and major o ~'ations s~nulate a variety of factors whi~ initiate the body's response to th~ st~..sses. 'i"hese reactions are m~liated by a cbmage ia the neta'al hormonal, eac.ranmemt, by the elaboration of eytokines and tl~ougb the stimu/ation of other inflammatory mediators. this eatabolio setting zesulis in body protein loss, which g-taduai y erodes both fimetianal and stm~ tissue. wiu _b the normally nom'ished individual can withslzod "d~ response for a brief period of time, ircolonged eatabollsm l~ associated with ~,nmunosuppmssion, poor wound healing, and proioagcd convalescence. surgeons have rej ed on intraveaous or eater~ feedings to ~everse this process. a va~ety of data now indicates that it is extremely di~edt to replete protein-containing tissue d~g the eataholie state of illness; the umutt response to hypercaloise feedings in this setting is the incxeased accretion of body fat md water. administration of gxow~ ho~moae ~cesults i.a n shift of the body's oxidative machinery to fat rather than eazbohydmte utiq~afic~; ¢oncomitaatiy, protein synthesis is stimulated. this metabolic state may have clinical utility in p~e.n.ts who need to heal large wotmds, in those who need to gain strength in order to be w~ed ore veatilatory support, or those patients who ~ve mulfiorgan failure and nee~ to ealaaace protein syatsesis to in.suze recovery. als are now in progress to dcterm~e the benefit of growth hormone in these and other disease state.s, rn the fature, the increased use of growth hormone, will occur;, this and other growth factors will serve to su~ po~ the host and shorten convalesceace following catabolic diseases. our objective was to study effects of pre-trauma growth hormone (gh) treatment on liver and skeletal muscle metabolism in a trauma model in piglets. twenty-four piglets were randomized to three treatment groups; one group was pretreated with gh iu daily three days before the experiment (gh- ), another group treated with gh ie at the start of the surgical procedure (gh- ) and one group served as untreated controls (con). they underwent a standardized surgical procedure to place sampling cathethers in the portal, hepatic and femoral veins and doppler flow probes around the portal vein, the hepatic and the femoral arteries. all pigs recieved a primed constant infusion of u- c-glutamine. substrate fluxes were measured one (lh) and five ( b) hours after termination of the surgery. nitrogen excretion was significantly decreased in the gh treated animals (gh- : . + . mg/kg, gh-i: . +_. . mg/kg, con: . +- . mg/kg, p= . ). in the hindieg, there was reduced net glutamine efflux due to decreased absolute glutamine release (gh- : . + . gmol/min at lh and - . + . in.tool/rain at h, gh-i: - . + . i.tmol/min at lh and - . + . pmol/min at h, con: - . +_ . g mol/min at lh and - . + . /amol/min at h, p= . , p= . ), whereas the absolute uptake of glutamine in hindleg was unchanged (p= . ). glucose uptake in the hindleg was decreased (p= . ). net glutamine uptake in liver was attenuated (p= . at h), whereas net glutamate release from liver was increased (p= . ). thus, pre-trauma treatment with gh improved nitrogen economy, attenuated net glutamine loss from hindieg due to decreased absolute release, and attenuated hindleg glucose uptake. liver net glutanaine uptake was decerased and net glutamate release increased. patients with major abdominal surgery exhibit a considerable catabolic response with negative nitrogen balance and protein breakdown, which may have detrimental effects on outcome. we investigated the effects recombinant human growth hormone on substrate metabolism in parenterally fed patients. metabolic healthy patients were randomized to receive either placebo or hgh in a dosage of , ; , or , i.u. per kg bw. substrate oxidation rate, energy expenditure as well as short life proteins were measured daily. six patients were excluded from analysis as drop outs, due to surgical complications. we could find a dose-dependend effect of growth hormone on resting energy expenditure, carbohydrate oxidation, fat and proteine oxidation. with increasing growth hormone resting energy expenditure increased from . calories per kg body weight to . calories. this was mainly due to an increased carbohydrate and fat oxidation, while proteine oxidation decreased. this was in accordance with a decreased urea.production rate and an improvement in cumulative nitrogene balance, which increased from minus . to plus . g n / days. in consequence short life proteins were also increased. the only negative side effect was an increased blood glucose concentration in some of the patients, making insuline administration mandatory in patients. our results are in accordance with other studies showing improvement of nitrogen balance, maintainance of lean body mass and muscular strength. if growth hormone administration may have any impact on morbidity, mortality and length of hospital stay in these patients it should be evaluated in a larger number of patients. in patient after liver transplantation (oltx) the effect of recombinant human growth hormone (rhgh) on protein metabolism and hormonal changes was studied. patients and methods: the underlying disease in all patients was end stage liver cirrhosis, non of the patients included in the study was suffering from malignancies. the patients were randomly allocated to receive either u of rhgh bid (sc) or no alternative medication. the study period lasted days. from daily hrs udne collection urinary nitrogen loss and daily loss of urea were determined. body compostion analysis (bca, bioimpedance, akern-ltaly) was performed preoperativly and at the postoperative days and to evaluate changes in body water and body cell mass (bcm). the effect on resting energy expenditure (ree) was analysed using indirekt calorimetry wrhin the same intervalls (metabolic monitor, datex). blood levels of gh, igf and igf , igfbp were measured daily during the study period, on day a hour profile of gh was.performed. samples were collected each minutes. results: cumulative udnary nitrogen and urea loss were not signifcantly different for the whole study period but for the first study days. bia indicated for the rhgh-group a loss of bcm to a lesser extent than for the controls (n.s.), changes in bodywater where similar for both groups. ree changes (kcal/kg bcm) were not significantly different for the two groups. blood levels of gh and igf differed significantly between the groups for the whole study period, but not igf and igfbp- . the circadian rhythm of endogenous gh-excretion had not been altered by gh, but absolute levels of gh were increased. conclusion: during the first days after oltx we could proof the protein sparing effect of gh treatment. although the differences between the two groups were not significantly different for the whole study period the results showed on all days a less catabolic situation for the rhgh treated patients. to possibly improve protein balance after major abdominal surgery we studied the effects of gh on protein metabolism after ltx. excluding malignant diseases, candidates for ltx were randomized to either postoperatively receive the usual treatment (co=control group, n= , age range - yr, bmi . + . kg/m ) or for days additional gh ( x i.u.s.c.) (gh group, n= , - yr, . + . kg/m ). metabolic studies (calorimetry and lsc-leucine infusion) were performed (test a) and days (test b) after surgery. tracer analysis was done by gas chromatography-mass spectrometry. during the -week study clinical parameters did not differ significantly between groups; however, there was a tendency for increased glucose levels, insulin need, and energy expenditure at the time of test b in the gh group. leucine oxidation (x+se) during test a;b was + ; + (co) and + ; ± (gh) pmol/kg ffm/h (n.s.). protein breakdown was ± ; + (co) and + ; + (gh) pmol/kg ffm/h (n.s.). nonoxidative-leuine disposal was ± ; + (co) and ± ; + (gh) pmol/kg ffm/h (p< . for the rise in the gh group). tracer data were supported by cumulative urea nitrogen excretion (days - : gh + vs co ± g; p< . ). we conclude that after major abdominal surgery gh lowers nitrogen excretion by increasing total body protein synthesis. it is not known which organs contribute to this effect. in several investigations it has been shown that the protein saving effect of recombinant human growth hormone (rhgh) in the postoperative state is accompanied by raised igf- levels in plasma. it was concluded that the anabolic effects of rhgh were probably, at least in part, mediated by igf- . this study was aimed at detecting the efficacy of igf-i on modulation of the protein metabolism in the catabolic state. patients and methodes: patients (both sexes, age: - years, bmi - kg/m ) with major upper gastrointestinal surgery (gastrectomy or partial gastrectomy) received ug rhlgf- /kg body weight/twice daily or placebo during treatment days beginning on the first postoperative day in a double-blind, randomized study. all patients received hypocaloric and hyponitrogenous total parenteral nutrition ( g chng, o. g aa/kg) troughout the whole study period. cumulated nitrogen balance, -methyl-histidin exretion in urine as well as serum-igf- levels have been determined. the two tailed wilcoxon test was used for statistical analysis results: first results will be presented previous studies have suggested that growth hormone (gh) potentiates various activities of leukecytes. however, it is not clear whether gh can improve survival of animals with gram-negative sepsis. we investigated the effects of gh on host response to infection in septic mice model. methods balb]c mice were randomized to groups (n= /gronp): gh-high ( + mg/kg/day), gh-iow ( a mg/kg/day) or control (saline). following subcutaneous treatment (every hours for days) with gh or saline, mice were challenged i.p. with e.coli (lxl /body). survival rate was recorded every hours. in the next experiment, gh-high and control animals were sacrificed hours after bacterial challenge. peritoneal cavity was lavaged with ml saline and the peritoneal exudative cells (pec) were harvested and counted. colony forming units (cfu) of bacteria in the peritoneal lavaged fluid (plf), liver, lung and blood were determined. pec were cultured in vitro for hours with lipopolymccharide ( p.g/ml in normotensive adults growth hormone (gh) rises when clonidine challenge is performed. recently evidence was shown for gh to accelerate wound healing. while gh secretion patterns are inconstant the gh-dependant insulin like growth factor (igf- ) and the insulin like growth factor binding protein (igfbp- ) reflect the integrated gh secretion over days. since we administer clonidine to patients with acute alcohol abuse we determined plasma levels of igf- and igfbp- . materials and methods: patients sheduled for esophagus resection were investigated once they had given written informed consent. patients showed acute alcohol abuse and were treated with clonidine postoperatively• patients with no history of acute alcohol abuse served as controls. besides liver enzymes igf- and igfbp- were determined. results: both groups had a comparable hepatic capacity of synthesis with decreased cholinesterase levels as to be expected after a major operation. regression analysis of igf- and igfbp- levels showed no differences between the groups. discussion: igf- and igfbp- plasma levels are not increased in normotensive patients who are treated with clonidine for prevention of postoperative alcohol withdrawl syndrom. further studies are required to discriminate whether our results are due to impaired postoperative liver function or if clonidine has not any impact on the gh-igf-axis in patients with alcohol abuse. recent studies have revealed that igf-i has several immanoregulatory roles. however, little is known about its effects on septic animals. we tested whether igf-i could increase the survival o f mice challenged intraperitoneally (i.p.) with e. coli. m~thqd$ balb/c mice received either high dose igf-i (n= , mg/kg/day), low dose igf-i (n= , . mg/kg/day) or saline ( = ) every eight hours subcutaneously for six days. animals were then challenged i.p. with lx e. coli. survival was observed every two hours. in the other experiment, mice that received high dose igf-i or saline were sacrificed four hours after bacterial challenge. peritoneal cavity was lavaged with ml saline and the peritoneal exudative ceils (pec) were harvested and counted. colony forming units (cfu) of bacteria in the peritoneal lavaged fluid (plf), liver, lung and blood were determined. in addition, pec were cultured/n vitro for hours with lipopolysaccharide ( ).tg/ml). tumor necrosis factor (tnf) in the supernatants of pec culture, plf and serum were measured by l bioassay. results igf-i improved the survival rate at a dose of mg/kg/day. severe chronic neutropenia (scn) is a disorder characterized by severe neutropenia secondary to either a maturational arrest of myelopoiesis at the level of promyelocytes (kostmann-syndrome; scn) or regular cyclic fluctuations in the number of blood neutrophils with a median absolute neutrophil count (anc) of less than /~tl (cyclic neutropenia). patients suffering from scn have an impaired acute inflammatory response to injury and an increased susceptibility to infections, i.e. bacterial or fungal infections, resulting in chronic oral inflammation, severe pneumonia, abscess formation and bacteraemia. we have treated patients ( scn, cyclic neutropenia) with r-methug-csf (filgrastim). thirty of patients with scn and all cyclic neutropenia patients responded to this treatment with an increase of the median anc to greater than /~tl. the dose of r-methug-csf needed to achieve and maintain the response varied between . and ~tg/kg/d. long-term treatment did not exhaust myelopoiesis: the anc remained stable after up to years of treatment and antibodies to r-methug-csf were not detected. the increase of anc was associated with dramatic clinical responses: significant reduction of severe bacterial infections, reduction of intravenous antibiotic treatment, and reduction of hospitalizations. no severe bacterial infections occurred in any of the r-rnethug-csf responders during longterm treatment. severe adverse events, most likely due to the underlying disease, included the development of mds/leukemia in two patients, and osteopenia/osteoporosis in patients. these results demonstrate the beneficial effects of r-methug-csf treatment in severe chronic neutropenia patients. the newborn is predisposed to mortality during infections due in large part to developmentally immature host defenses. recently cytokines have been identified that regulate the development of these defenses. one such cytokine is specific for neutrophils and is termed granulocyte colony stimulating factor (g-csf). in the studies to be discussed, we have attempted to impact hematopoiesis in the fetus using exogenous rhg-csf delivered through the pregnant dam. our rationale was that an enhanced myeloid system may result from such in utero treatment leading to, perhaps, enhanced protection to microbial insult. rhg-csf crossed the placenta and reached peak fetal serum concentrations hours after administration. these levels were -fold lower than the levels detected in the adult dams serum. white blood cell counts were elevated approximately - -fold over control newborn blood. this increase was due to circulating numbers of neutrophils. the marrows from these pups were hyperplastic consisting of predominately immature and mature neutrophilic cells. no changes were seen in the thymus or livers. pups born to mothers treated with rhg-csf since day of gestation (parturition in rodents occurs on approximately day ) survived a lethal challenge of group b- hemolytic streptococcus. in contrast their untreated yet infected counterparts did not survive. recent clinical developments have led to trials of rhg-csf in certain neutropenic states including cyclic and chronic neutropenia. we have identified several of these patients who were pregnant during treatment with rhg-csf. biologically and anfigenically identifiable rhg-csf was detected at increased levels in the cord serum of these neonates. we will discuss our findings with these patients in light of our animal model of neonatal myeloid development. we have previously demonstrated decreased g-csf production and g-csf mrna expression from activated mononuclear cells from newboms compared to adults (cairo, pediatr res : , ) . we have also recently observed that administration of a single dose of rhg-cse to one-day-old newborn rats induced significant neatrophilia, while administration for days induced neulrophilia and increased the bone marrow (bm) neutrophil storage and progenitor pools (cairo, blood : , cairo, pediatr res : , ) . we embarked on a phase i trial exploring the safety, pharmacokinetics, and biological efficacy of g-csf in newborns with presumed sepsis. infants < hrs old with a diagnosis of presumed sepsis were stratified into three groups: very preterm ( - wk), preterm ( - wk), and term (> wk) and randomized to receive either a placebo or , , and gg/kg/qd or and p.g/kg/bid of rhg-csf infused by pump over hour for consecutive days. cbcs were obtained at , , , , and hrs. tibial bone marrow aspirations were performed hrs after study entry and differential counts and cfu-gm pools were determined. c bi expression was determined at and hrs after rhg-csf, and g-csf pharmacokinetics were performed after the first dose of rhg-csf utilizing a sandwich elisa. a significant increase in the anc was observed at , and hrs following administration of both and ~tg/kg/d of rhg-csf. the maximum increase in the anc occurred hrs after and ~tg/kg/d ( - %) (p< . ) and ( % -+ %) (p< . ), respectively. there was a significant dose-dapendeat increase in the bm neutrophil storage pool ( _+ % vs. + %) (p< . ) (placebo vs. ~tg/kg/d). there was no significant difference in the nantrophil proliferative pool. an increase in cfu-gm and cfu-gemm was seen at all doses tested, compared to placebo ( . _+ . vs. -+ ) (colonies/l(p cells/plate). c bi expression was significantly increased hrs after bg/kg/d of rhg-csf ( + % vs. +- %) (p< . ). peak serum g-csf levels occurred at hrs and were dosedependent. the half-life of rhg-cse was . + . hrs. most importantly, there was no observed toxicity from g-csf in all patients studied. of patients were on ventilators prior to administration of rhg-csf and there was no increase in pulmonary toxicity. these preliminary data suggest that rhg-csf is well tolerated at all gestational ages in newborns with presumed sepsis. a multi-center phase ii/iii randomized double-blindad placebo controlled trial is required to determine the efficacy of rhg-csf in this clinical setting. we investigated the effects of recombinant canine granulocyte-colony stimulating factor (g-csf) on survival, cardiopulmonary function, serum endotoxin levels and tumor necrosis factor (tnf) levels in a canine model of lethal bacterial septic shock (clinical research. : , ) . methods: awake ylo beagles had serial cardiopulmonary and laboratory studies before and for up to days after intraperitoneal placement of an e. celi infected clot. nine days before and daily until days after clot placement, animals received high (n= ) or low dose (n= ) g-csf or protein control (n= ) subcutaneously. results: survival in high dose g-csf animals ( / ) was significantly improved compared to low dose ( ) and controls ( ) (p< . wilcoxon). high dose g-csf also improved cardiovascular function evidenced by a higher mean left ventricular ejection fraction (day after clot, p< . ) and mean arterial pressure (day , p< , ) compared to low dose and controls. high dose rcg-csf increased (p< . ) peripheral neutrophil numbers both before and after clot implantation ( hours to days) compared to low dose and controls. in addition, high dose rcg-csf produced a more rapid (p< . ) rise (day ) and fall (day ) in alveolar neutrophils determined by bronchoalveolar lavage compared to low dose and controls. lastly, high dose rcg-csf decreased serum endotoxin ( to h, p< . ) and tumor necrosis factor (tnf, h, p< . ) levels compared to low dose and controls. discussion: these data suggest that therapy with g-csf sufficient to increase peripheral neutrophil numbers during peritonitis and septic shock may augment host defense and endotoxin clearance, reduce cytokine levels (tnf) and improve cardiovascular function and survival. the use of g-csf in sepsis prophylaxis in neutropenic patients is well established and has been ascribed to accelerated recovery in granulccyte counts. here, an additional sepsis-prophylactic property could be demonstrated in healthy volunteers: eleven volunteers were employed in a sinqle-btind, controlled study and were given uq g-csf or saline placebo via subcutaneous injection. blood was withdrawn immediately before and or hours later. lps-inducible tnf, il- , stnf-r p and il-lra were assessed in the supernatant of whole blood incubations stimulated with ug/ml lps from salmonella abortus equi. similarly to previous animal studies, lps-inducible tnf was attenuated by about % hrs. after treatment. the same was true of il-lb. in contrast, lps-inducible stnf-r p which was indetectable in blood incubations from untreated donors increased dramatically hrs. after g-csf treatment. il-lra found after lps challenge was increased tenfold by g-csf treatment. it is concluded that g-csf treatment switches peripheral leukocytes to an antiinflammatery state characterized by an attenuation of il-i and tnf releasing capacity and an augmentation of the release of cytokine antagonists. this findinq minht offer a novel concept in septic shock prophylaxis. objective.the aim of the study was to investigate the effect of recombinant human g-csf (rhg-csf) on survival, bone marrow neutrophil myelopoiesis, neutrophil counts, levels of bacteria and some important sepsis mediators in a model of rat abdominal sepsis. lethal peritonitis was induced with a mm coecal perforation (cp) in male wistar rats. rhg-csf was administered as /.tg/kg iv every h, first dose at sepsis induction. bone marrow neutrophi] progenitors were determined as blast colonies, cfu-gm and cfu-g. neutrophils and bacteria were determined in peripheral blood and peritoneal fluid. lps, tnf, endothelin and lactate were measured in blood from femoral vein. mortality rates were registered with g-csf treatment starting either or days before or hours after cp. results. mortality was reduced from % to about % with rhg-csf intervention and there was no difference between the pretreatment and treatment groups. bone marrow blast colonies were not influenced while neutrophil myelopoiesis was augmented at the stages of cfu-gm and cfu-g. neutrophils in blood and peritoneal cavity were enhanced and numbers of bacteria in the same compartments were substantially reduced. circulating lps, tnf, endothelin and lactate were attenuated the first hours after cp. neutrophil myelopoiesis is augmented with increased number of neutrophils in blood and peritoneal cavity, resulting in enhanced clearance of pathogens. lps, tnf, endothelin and lactate are suppressed the first hours during sepsis course. a. wendel, j. barsig, g. tiegs gm-csf stimulates the proliferation and differentiation of granulocytic and monocytic progenitor cells. in addition the hemopoietic cytokine activates the inflammatory response in mature leukocytes. the priming effect of gm-csf towards lipopolysaccharide (lps)-induced cytokine production in vitro has been described, but little is known about proinflammatory gm-csf effects in vivo. we detected gm-csf in plasma of lps-challenged mice with kinetics similar to tnf, reaching peak levels h after lps administration. gm-csf pretreatment ( ~tg/kg i.v.) enhanced mortality in mice challenged by a sublethal dose of lps. plasma levels of tumor necrosis factor (tnf) and interleukin- (il- ) were significantly enhanced. a monoclonal antibody, which neutralizes gm-csf bioactivity, rendered mice less sensitive towards lethal lps-challenge. tnf-and il- -tevels were reduced in these mice compared to control animals without antibody treatment. in addition, severalfold potentiation of lps-induced cytokine release by gm-csf was observed in vitro in murine bone marrow cell cultures. these data demonstrate the proinflammatory capacity of gm-csf and suggest that the hemopoietic cytokine plays also a role as an endogenous modulator of lps toxicity. immune dysfunction, developing in the wake of multiple trauma, overwhelming infection and other forms of critical surgical illnes% is associated with increased infections, morbidity and mortality. the mechanisms responsible for alterations in immune regulation are incompletely understood but monocyte appear to play a central role. polymorphonuclear leukocytes (pmn) are known to play a central role in the inflammatory response of the host toward invading microrganisms. reports of defects in all the aspeots of pmn function have been accumulated in recent years. the possible role of gm-csf in modifing the state of immuno suppression detected in severe intraabdominal infected pt~. inspite of surgical appropriate procedures and in reducing the expected mortality is investigated. the safety of rh-gm-csf administration in sepsis is also evaluated. a double blind randomized study is proposed. this study include icu patients who do not exhibit signs of shock and/or ards, with clinical signs and symptoms of abdominal infection. immunodepressed patients-aids, chronic chemotherapy or chronic steroid administration do not partecipate to the study. patients will receive rgm-csf (l~g/kg/day) or placebo in hs. continuous infusion for days. safetyandefyieacy will be assessed till to day . the apache ii score is adopted for risk stratification of patients because it is reliable and validated, objective and composed of information that is indipendent of diagnostic criteria. patient's entry criteria is apache ii > (score corresponds to expected mortality rate of %).in this protocol the surgeons report the judgement of the efficacy of surgical procedure to remove or not the focus of infection. objectives: infections and subsequent septic responses remain the leading cause of death among surgical intensive care (sicu) patients despite tmprovetaunts in supportive care and brond-epectrum antibiotics. usually invading bacteria are efficiently cleared by neutrophil granulocytes. however, during sepsis various neatrophil dysfunctions have been demonstrated, leading to impaired host defense. granulocyte colony-stimulating factor (g-csf) induces a sustained increase in circulating neutrophils and enhances various noutrophil functions. it was the purpose of the present study, to evaluate the safety and efficacy of g-csf (filgrastim) in sicu patients at risk of sepsis. materiel a.d methods: the study was designed as an open-label phase-ll study of filgrastim. ten consecutive slcu patients, with a therapeutic interveotion score greater than , were included in the study. filgrastim was given by daily continuous intravenous infusion for days or discharge from the sicu. apache ll-score, multiple-organ-failure (mof) score, definitions of infections, sepsis, systemic inflammatory response syndrome (sirs), and acute respiratory failure were applied daily. a response to filgrastinl th_erapy was defined as an improvement in disease severity quantified by a decrease of > apache i score points on day after onset of treatment. results: none of the patients developed a sepsis or mof later on and no patient died during hospitalization. specific postoperative complications occured in one patient ~jth a leekage of the oesophagou-gastric anastomosis after oesophageus resection. at study entry the leucocytes amounted to . + . /~tl (mean + sem) and reached a level of . +_ . /tal at day after onset offilgrastim therapy. the apache ii score initally was + . (mean + sem) and as an indicator of filgrastim response a decrease of points ~dthin days oceured in out ot patients. filgrastim was well tolerated, side effects were not noted. growth of solid tumors might be modulated by the activity of inflammatory and/or immune effector cells of undefined specificity. in this study patients undergoing surgical treatment for gastric (n= ) or colorectal (n= ) cancers were evaluated for endogenous serum levels of granulocyte colony-stimulatingfactor (g-csf) during a pre-and postoperative time period. from the same blood specimens mononuelcar cells (mnc) were prepared. the release of ifn-%, and il- , which are secreted by thl cells, were stimulated in vitro by pha during a cell culture period up to hours. the patients were further classified for their immunreactivity by responses in dth skin testing to seven different antigens (e.g. tetanus toxoid, ppd, diphtheria toxin, trichophyton, streptococcus, candida and proteus antigens). dth testing has been repeated in each patient two remarkable results were obtained. the serum levels of endogenous g-cse showed a biphasic increase with maximum values of pg/ml (preoperative < pg/ml) on day and day to after surgical treatment. similar patterns of g-csf production were found in both groups of patients with gastric or colorectal cancers. high serum levels of g-csf were significantly (p < , ) correlated with infectious complications in patients whh gastric cancer (n= / ). secondly patients could be arranged into two groups according to an anergic (n= ) or normergi¢ (n = ) responsiveness in dth testing. the frequency of anergi¢ responsiveness was similar in both patients with gastric (n= / ) or colorectal (n= / ) cancers. interestingly we found a significant correlation (p < , ) between low serum levels of g-csf and anergy during the postoperative period in both groups. stimulation of mncs from anergic patients (n= ) within the pre-and postoperative period resulted in reduced mean values (about %) for ifn-ff release (preoperative means llo pg/nfl), if compared to patients with normergic dth (n= , preoperative means pg/ml). similar, but less significant results were obtained for il- secretion. our results confirm a correlation between infectious complications and g-csf in the postoperative period, however elevated levels were also found in some patients without any signs of infections. more interestingly there might be an association between cytokine (c~csf, ifn-% and il- ) release and dth, which is known to be mediated by activated thl calls. to recognize anergic dth as a possible higher risk in the postoperative outcome of cancer patients extended periods of observation are needed. objectives of the study effects of recombinant huraan granulocyte colony-stimulating factor(rhc-csf)a galnst severe septic infections were investigated by its single use or by its corn b{nation with cephera antibiotlcs.we examined its effects on the mortality,and circulating blood neutrophyis counts and functlons,such as phagocytic activity and h production using the rat severe septic model. rats were subcutaneously administsrd rhc~csf(s orl o ~ g/k~ body wt)after on set of peritonitis brought about by cecal ]igation and one puncture withe -gaug e needle once a day for three days.in addjtlon,cefmetazol na(cmz)( m$/k bo dy wt)was injected intrarnustularly to the rats tv~ce a day for three days. cirehlatlng blood neutrophyls counts were determoned electronically with a hem ocytometer,and blood smears stained with may~runwaldm.qlemsa~taln. neutrophyls functions in vltro,such as phagocytic activity and h producti on using the rat severe septic model was analyzvd by automated flow cytometri c single cell-analysis methods. the reortallty rate after weeks was significantly decreased by administratlon of rh~-csf(p< , ).ln addjtion,a combination therapy of rhg-csf wlte cephern ant~biotics(cmz)showed a significantly survive] advantage and the rate had b een reached . %. nextly,treatn%ent wlth rhg-csf(s ~ $/k body wt)increased the nuzaber of the peripheral blood neutrophjls slgn[fieantly(p< . ). iv~oreover,functions of neutrophlis which were phagocytic activity and h p roduction were remarkably enhanced by admlnlstratlon of rhg-cs~( ~ /ks b ody wt) (p< .( ). these findings suggest that combination therapy of rhcrcsf with cephern antib iotlcs(cmz)is an efficient regime against severe infectlons.and the increased ne utrophils counts and enhanced neutrophiis functions were played a important ro le about the survival advantage. granulocyte macrophage colony-stimulating factor (gm-csf) is a haematopoietic growth factor active on neutrophils and macrophages. leukopenia often occurs following renal transplantation and can be associated with infection and/or the myelosuppressive effect of azathioprine. aim: we report the use of gm-csf in renal allograft recipients with leukopenia. nonglycosylated recombinant gm-csf was obtained from e. coli transvected by human gm-csf gene. m~terial ~,nd methods : written informed consent was obtained from all patients. patients were suffering from toxic neutropenia (neutrophils < /mm ) with medullar hypocellularity on bone marrow aspiration, or leukopenia (neutrophils < /ram ) with cytomegalovirus infection requiring ganciclovir administtation. gm-csf was given subcutaneously at a dally dose of to mcg/kg/day, according to renal function. results : in all cases, neutrophil counts returned to normal levels within to days. in most of them, spectacular correction was observed within hours, with a single injection. adverse events due to gm-csf at this dose were mild and easily managed ( cases of bone pain treated with paracetamol). one acute rejection episode was observed after correction of leukopenia. conclusion : on the basis of this study, it appears that gm-csf at a dose below mcg/kg/day is an effective treatment for renal transplant recipients with leukopenia associated with cmv infection or toxic neutropenia. department of nephrology, , rue de s~vres, hopital necker, paris, france. changes in serum g-csf and il- after surgical intervention hitoshi toda , atsuo murata , hidewaki nakagawa , takesada mori , nariaki matsuura osaka university medical school, osaka, wakayama medical school, wakayama, japan we measured serum immunoreactive interleukin (il- ) and granulocyte colony-stimulating factor (g-csf) levels of the patients undergoing major thoraco-abdominal surgery for esophageal cancer. serum samples were collected from eight patients on the day before surgery, at the time of operation, and thereafter at suitable intervals for one week. il- and g-csf were measured by means of enzyme linked immunoassay. the normal range of serum ]l- was less than pg/ml and g-csf less than pg/ml. values between groups were compared with linear regression analysis. both serum g-csf and il- levels reached their maximal levels at the first postoperative day and decreased thereafter. the correlation between g-csf (y) and il- (x) was y= . x+ . (r= . , n= , p< . ), showing a significant correlation. in the case who suffered from aspiration pneumonia and ards at the second postoperative day, the peak level of il- was pg/ml and g-csf pg/ml respectively. the estimated value of g-csf was pg/mi by the regression equation. this means the real g-cse level was less than half of the estimated value. it suggests that low responsiveness of g-csf is one of the reason of immunodeficient state after the major surgery, neutrophils from injured patients ingest and kill bacteria less efficiently as compared to those of healthy individuals, probably reflecting the suppression in respiratoly burst which occurs after severe trauma. one of the main mechanisms of killing bacteria by neutrophil granulocytes is production of oxygen radicals (respiratory burst). granulocyte colony-stimulating factor (g-csf), a kilodalton cytokine, leads to a sustained, dose-dependent increase in circulating neutrophils. thus, it was investigated whether filgrastim (recombinant human granulocyte colony-stimulating factor, rhg-csf) therapy fits for prophylaxis of sepsis in postoperative/posttraumatic patients, and whether, besides an expected increase in neutrophil count, filgrastim would also augment neutrophil function. material and methods: this study was designed as an open label, prospective phase ii study of filgrastim and performed in a surgical intensive care unit (sicu) (university hospital). postoperative/post-traumatic patients with a therapeutic intervention scoring system (tiss) score greater than were treated with filgrastim ( . - l.tg/kg/day) for prophylaxis of sepsis on days or until discharge from the sicu. production of oxygen radicals can be quantified by analysis of fmlp-and zymosan-induced chemiluminescence. neutrophil oxygen radical production was tested by fmlp-and zymosan-induced chemiluminescence by the polymorphonuclear cells (pmn) of these patients in multiple blood samples over a period of up to days. results: none of the patients treated with filgrastim for prophylaxis of sepsis developed sepsis. in vitro fmlp-induced ( - reel/l) neutrophil oxygen radical production was significantly increased under therapy with filgrastim by a maximum of % +- % ( % - %) compared to pretreatment values of %. tapering of filgrastim resulted in a reduction of fmlp-induced neutrophil oxygen radical production within hours. in contrast, zymosan-induced neutrophil oxygen radical production was not affected by filgrastim treatment. conclusions: besides its quantitative effect on neutrophil counts enhanced neutrophil function, documented here as increased fmlp-induced oxygen radical production, may account for the beneficial effect of filgrastim for prophylaxis of sepsis in posttraumatic/post-operative patients. granulocyte colony stimulating factor (g-csf) and granulocytemacrophage colony stimulating factor (gm-csf) have been recently introduced in the treatment of chemotherapy-induced neutropenia. effects of these csfs on cellular immune system were evaluated in neutropenic gynecological cancer patients during chemotherapy. g-csf and gm-csf were equally able to induce a rapid recovery of white cell count within one or two days. g-csf treatment resulted in a significantly higher concentration of leukocytes measured in the peripheral blood although by gm-csf a sufficient effect was achieved (p< . ). before initiation of csf treatment urinary neopterin was similar in both groups of patients ( +/- and +/- lamol/mol creatinine for gm-csf and g-csf respectively expressed as mean +/-one sd). in g-csf treated patient only a marginal induction of neopterin was observed. on day the mean value was about % above the basal level (p< . ). on the other hand gm-csf treated patients were characterized by a pronounced increase in urinary neopterin levels. in comparison with the basal level a more than fold induction was noted and the difference between g-csf and gm-csf was highly significant (p< . ). this effect was confirmed in vitro by investigating the effects of these csfs on interferon-gamma mediated pathways in thp- human myelomonocytic cells. results suggest activation of immune effector cells by gm-csf which may help the organism to overcome infections. however, activated macrophages produce several growth factors which may increase malignant proliferation, and augmented neopterin production as sign of macrophage activation has also been associated with poor prognosis m several malignancies. more data are therefore necessary to clarify whether csf mediated immune activation is beneficial or deleterious for cancer patients but considering our results caution in applying csfs in oncology seems advised. from a historical perspective, the development of humoral immunity to bacterial endotoxin has assumed a prominent position in the spectrum of therapeutic approaches which have been explored for the treatment of gram negative septic shock. predicated upon the fact that rough strains of bacteria manifest lps containing exclusively conserved structural features common to lps from all gram negatives, specific antibodies were elicited which conveyed cross protective immunity in experimental models of bacteremia and endotoxemia. such studies culminated in a well-conducted, randomized, double-blind placebo-controlled clinical trial using passively administered human polyclonal antiserum to treat patients with suspected gram negative sepsis. the efficacy of treatment established in that trial spurred efforts to develop monoclonai reagents which, to date, have not been uniformly successful in reproducing those earlier studies with polyclonai antibodies. nevertheless, the numerous successes which have been documented in experimental models of endotoxemia continue to foster promise for this immunotherapeutie approach. several recent studies with human polyclonalimrnunoglobulin preparations containing antibodies reactive with lps and lipid a have yielded promising results in treatment of patients with sepsis. in addition, the recent development of an antiidiotypic monoclonal antibody which reflects an internal image of a kdo specific monoclonal antibody has provided an alternative experimental approach to generate anti-lps antibody. immunization of mice with the antiidiotype provides significant protection against subsequent lps lethality consistent with the development of circulating immunoglobulin specific for lps. thus, the use of polyclonal immunoglobulins contrives to provide an alternative and potentially cost effective method for the treatment of endotoxin shock. supported by r a and pot ca . john holaday, anne fortier, shawn green, glenn swartz, john madsen, carol naey, and jan dijkstra entremed, inc.. rockville, md, . at the time of diagnosis, the signs and symptoms of septic shock are an indication that the systemic inflammatory response is well underway; thus, it has been argued that the endotoxin "cat is out of the bag", and that subsequent passive immunization may be too late to achieve therapeutic benefit. our approach has been to evaluate active immunization as a prophylax~s against sepsis. mice were inoculated twice (two weeks apart) with liposomes containing dmpc[i. ], dmpg[ . ], cholesterol [ . ] , and monophosphoryl lipid a [ - gg/txmole phospholipid] by several routes (i.p., i.m.), and serum was collected - days after each inoculation. after a single injection, highest tilers of ab were produced in mice inoculated i.p., but mice inoculated by all routes produced anti-lipid a ab. following the second injection. ab levels were roughly equivalent in mice inoculated by all routes, regardless of lipid a concentration. mice vaccinated i.p. with liposomes containing , or gg lipid a were treated with cyclophosphamide to produce neutroperda and then challenged with e. cole in an infection model of gram negative sepsis. the lds for control (liposomes with no lipid a) mice was x bacteria; ld for mice vaccinated with p.g was x ( -fold increase in resistance) and with ~tg was x bacteria ( -laid increase in resistance). mice vaccinated as before were also treated with actinomyein d to increase sensitivity to lps (salmonella minnesota) challenge in an endotoxemia model of grain negative sepsis. the ld for control (liposomes with no lipid a) mice was ng lps; the ld for gg lipid a was rig lps ( -fold increase in resistance) and for xg was ng lps ( -fold increase in resistance). mice were similarly vaccinated and challenged with an aggressive gram negative pathogen, francfsella tularensis. the ld of franciseua in normal mice or mice inoculated with liposomes without lipid a was - bacteria. in contrast, mice vaccinated with liposomal lipid a ( ggl survived challenges as high as , bacteria, ( logs of protection). the impressive protective capacity of this vaccine did not correlate with ab liter in any of the sepsis models, nor did it correlate with classic nonspeeific events, such as macrophage activation. maerophages harvested from the peritoneum of mice vaccinated and protected against sequelae of gram negative infections did not spontaneously kill the bacteria in vitro, but could be activated by ifn-y for antimicrobial activity equivalent to that of macrophages from unt#eated mice. research is underway to defme the protective mechanism(s) activated by this liposomal-lipid a vaccine. intervention by monophosphoryl lipid a in septic shock jon a. rudbach, ribi immunochem research, inc., hamilton, montana, usa monophosphoryl lipid a (mla), the clinical form of which is called mpl®-immunostimulant, has been tested extensively as an intervenient material in septic shock. mla is protective when given to experimental animals prior to a live microbial challenge or challenge with lethal doses of microbial products or certain cytokines. this is shown with gram negative and gram positive bacteria, gram negative bacterial endotoxins, and gram positive bacterial exotoxins. furthermore, animals treated with a regimen of mla which results in a refractory state to a lethal dose of gram negative bacterial endotoxin concomitantly display increased resistance to a live bacterial challenge. thus, both endotoxin tolerance and nonspeciflc resistance to infection can be manifested simultaneously. also, prophylactic doses of mla do not interfere with other therapies given subsequently; an additive or a synergistic protective effect can be demonstrated with certain combinatorial treatment regimens, such as mla followed by antiendotoxin monoclonal antibodies. the preclinical studies were extended to human trials wherein the safety of agonistic doses of mla was verified. furthermore, when mla was administered to human volunteers hr before challenge with a pharmacologically active dose of reference endotoxin, febrile, cardiac, tnf, il- , and il- responses were all decreased significantly as compared with the responses of subjects pretreated with a control solution and challenged with endotoxin. human trials with mla are being extended into patient cohorts which have high probabilities of developing septic shock; this will expand the safety base and establish clinical efficacy for mpl®-immunostimulant. a considerable body of in vitro evidence supports the concept that the effects of lps on cells of the immune/inflammatory systems are controlled by interactions of lps with cd . to evaluate if blocking lps-cd interactions has potential as a therapeutic in septic shock we have evaluated the effect of anti-cdi monoclonal antibody (mab) on lps-induced cytokine production and physiologic changes in an experimental model of endotoxin shock performed in cynomolgus monkeys. a novel model has been established where animals were treated with interferongamma for three days prior to infusion of highly purified lps over an eight hour period. in this model lps challenge resulted in marked release of eytokines in the blood, substantial hemodynamic changes, release of liver enzymes and alteration in lung permeability observed over a hour period. to evaluate the effect of treatment with anti-cd mab, animals were given either nothing, an isotype control or anti-cd mab ( mg/kg) rains, prior to the beginning of the lps infusion. evaluation of physiologic changes including mean arterial blood pressure and cardiac output, quantitative analysis of eytoldne levels including tnfct, il- , i,- , il- and il- , and liver enzymes during a hour period revealed that treatment with anti-cd mab markedly attenuated all parameters of injury including decreased mean arterial blood pressure, increased cytnkine levels and the release of liver enzymes observed in animals given the isotype control mab or those not treated. administration of anti-cd mab to interferon-gamma treated animals not challenged with lps did not induce any detectable physiologic changes or increases in cytoldnes. these studies suggest that strategies to block lps-cd interactions will have utility in diseases such as septic shock or ards where lps plays a central role in initiating injury. preclinical studies with recombinant bactericidal/permeability increasing proteins (rbpi and rbpi ). p.w. "frown, dept. of preclinical science, xoma corporation, berkeley, california, usa. bactericidal/permeability increasing protein (bpi), from neutrophils, binds to and neutralizes lipopolysaccharide (lps); it also specifically kills gram-negative bacteria (gnb). these properties, which reside in the n-terminal half of the molecule, indicate potential therapeutic application in the treatment of gram-negative sepsis. the gene for human bpi has been cloned and recombinant holoprotein (rbpi) and a kd n-terminal fragment (rbpi; ) have been produced in sufficient quantities for preclinical studies. both rbpi and rbpi bind to lipid a and neutralize the biological activities of lps derived from a variety of organisms, rbpi has equivalent antibacterial activity to bpi against rough gnb but is up to x more potent than bpi vs. serum-resistant and smooth gnb. rbpi and rbpi compete with lps-binding protein (lbp) for binding to lps under physiological conditions. consequently, both rbpi and rbpi block the cd -dependent lpsinduced synthesis of the cytokines tnf, il- , el- and il- in vitro. rbpi has also been shown to inhibit the lps-induced synthesis of reactive metabolites, endothelial adhesion molecules and the procoagulant molecule tissue factor. in animals, rbpi has been reported to increase survival of endotoxin-challenged rats and mice, to inhibit the dermal schwartzman reaction in rabbits and to increase survival of neutropenic rats with pseudomonas bacteremia, rbpi increases survival and decreases cytokine production in endotoxin challenged mice and rats. it normalizes lps-induced changes in hemodynamic, pulmonary and/or metabolic parameters in lps-induced rats, rabbits and pigs. treatment with rbpi also increases survival and decreases cytokine production in bacterial challenge models in rats and mice. rbpi was not toxic to rats after daily consecutive i.v. doses of mg/kg. this combination of properties indicate that recombinant bpi may be useful in the treatment of sepsis. phase i/ii clinical trials of rbpi have begun. the discovery of lps binding protein (lbp) and subsequent identification of cd as a receptor for lps or lps-lbp complexes has resulted in a new understanding o£ how lps responsive ceils are stimulated. cd is found either as a glycosylphosphatidyl-inositol (gpi)-anehored membrane glycoprotein (mcd ) of myeloid cells or as a soluble serum protein (scd ) lacking the gpi-anchor. binding of lps to mcd triggers cell activation while binding of lps-scd complexes to cells such as endothelial or epithelial cells that normally do not express mcd activates these cells. these pathways are shown in schematic form below. ~di mcd plays a crucial role in presentation of lps to additional membrane components that make up a functional lps receptor. an immediate consequence of engagement of this functional receptor is protein tyrosine phosphorylation. the molecular mechanisms leading to these events will be discussed. understanding of these pathways will lead to the development of new therapeutic approaches to controlling host responses to lps. pretreatmen t posttreatment (before or after tnf peak) d) with different antibody dosages: mg/kg --- . mg/kg pretreatment with anti-tnfab prevented death in most model situations (except peritonitis), but also posttreatment up to h after sepsis induction was successful in the few studies performed. there is additional evidence that low-dose tnfab is partially effective. especially baboon anti-tnfab studies provided many insights into the pathophysiological sequences of sepsis induction, due to crossreactivity with human reagents. those events include the cytokine sequence with tnf-dependent il-i, il- , or il- , but also il-lra or stnf receptor release. granulocyte as well as endothelial cell activation were shown to be partly tnf related, and the procoagulatory response was influenced by anti-tnf treatment. from many animal studies the concept that tnf plays a pivotal role in sepsis is clearly evident and therefore anti-tnf therapy is a major candidate tbr clinical studies. the beneficial or harmful effects of tnf-mediated inflammatory responses depend on the clinical context. decreasing exaggerated tnf-mediated inflammatory responses may be useful in some patients with organ failure. tnfr:fc (immunex, seattle, wa) is a recombinant human protein composed of two identical extracellular p tnf receptors linked by the fc region of iggl. it neutralizes tnf with an affinity for tnf<z of - m and has a t / of • h in normal humans. methods: normal volunteers were randomized to receive either tnfr:fc vehicle (n= ), or low dose (ld) tnfr:fc ( mg/m iv, n= ), or high dose (hd) tnfr:fc ( mg/m iv, n= ) infused over rain prior to endotoxin (e) (escherichia coil : ; ng/kg iv) administration. plasma cytokines were measured using elisa and tnf bioactiv'dy. systemic hemodynamics were evaluated with indwelling arterial and pulmonary artery catheters and radionuclide heart scans and compared before and after fluid loading in subjects given vehicle with hd tnfr:fc. results: endotoxin-related symptoms were unchanged after ld or hd. peak temperature occurred at a later time point ( - h) in subjects given tnfr:fc compared to vehicle ( h) (p=. ). at h, h (post fluid load ), and h, subjects given vehicle developed increased cardiac index and heart rate and decreased systemic vascular resistance index (all p=. ). hd tnfr:fc did not alter this hyperdynamic cardiovascular response. ld and hd inhibited the e-induced teukopenta at i h post endotoxin (p<. ) and later leukocytosis (p< . ). peak tnf bioactivity in subjects given vehicle was + pg/ml and < pg/ml in the ld or hd tnfr:fc groups (p<. ). two immunoassays for tnf were performed because the detection of receptor bound tnf varies with elisa. tnf (biosource) was decreased in ld vs vehicle or hd (p=. ) whereas tnf (r&d systems) was increased after hd or ld vs vehicle (p<. ). decreased levels of il- (p=. t), granulocyte colony stimulating factor (p=. ), and il- receptor antagonist (p=. ) were found after ld or hd vs vehicle. il- levels were lower after ld vs vehicle or hd (p=. ). il- levels were similar among subjects given vehicle, ld, and hd tnfr:fc. conclusions: ld and hd tnfr:fc delayed the febrile response to e but did not alter the early hyperdynamic cardiac response. this suggests that mediators other than tnf play an important role in the initial cardiovascular response to e in humans. tnfr:fc attenuated the release of some e and tnf-induced cytokines. ti~e antiinflammatory responses of tnfr:fc may be useful in some patients with disease processes resulting from excessive tnf-mediated inflammatory responses. the biosynthesis of tnf within macrophages is regulated both at the transcriptional and the translational levels. the ' flanking region of the tnf gene contains several transcriptional control elements, including two kb enhancers similar to those of immunoglobulin genes and a "y box" similar to that of the mhc class ii promoter. these elements are critical for the enhancement of transcription noted after stimulation of macrophages with lps. despite these and other elements, transcriptional regulation alone accounts neither for the absence of tnf protein during normal homeostasis nor the profound increasein tnf production following stimulation with lps or other secretagogues. the translation of tnf mrna into protein is also tightly regulated via mechanisms involving the octameric "ttatttat" motif present in the 'untranslated region of tnf, human inducible no synthase, and several other cytokine genes. this region not only destabilizes mrna, but also confers a translational blockade so that tnf protein is not normally synthesized despite leaky basal transcription. lps stimulation releases translational blockade and, together with enhanced trancriptional rate, accounts for significantly increased tnf secretion. opportunities for inhibition of tnf production are available at each of these two levels. agents which increase intracellular camp (pentoxif¥ ine, amrinone) inhibit accumulation of tnf mrna; in contrast, corticosteroids act primarily at the level of translation, inhibiting lps induced translational activation. understanding the regulation of tnf biosynthesis may assist in clinical strategies designed to regulate tnf secretion. the serine protease thrombin is formed by enzymatic conversion from its proenzyme form after coagulation activation and may enhance the inflammatory response in various ways. in the last step of the coagulation cascade, thrombin cleaves its substrate fibrinogen, thus forming fibrin. the fibrin clot may prevent phagocytosis of bacteria and promote local bacterial growth. thrombin can also stimulate a specific th.rombin receptor that exists on a variety of cells. for example, minute amounts of thrombin ma), stimulate platelets to develop tissue factor and boost the activation of more thrombin, thus initiating a positive feedback loop. other cellular effects of thrombin receptor stimulation include increase in intracellular calcium of platelets and monocytes, contraction of smooth muscle cells, increase in endothelial permeability, thromboxane generation by neutrophils and lymphocytes, pulmonary vasoconstriction, and enhanced cytokine production. animal experiments have been conducted with a variety of thrombin inhibitors including heparin, antithrombin ili, hiradin, and hirudin-like peptides. an alternative approach is the inhibition of the coagulation cascade at an earlier point in the cascades, or administration of the anticoagulant enzyme protein c work from our own group in a porcine model of lipopolysaccharide-(lps)-induced shock with disseminated intravascular coagulation has shown that prelreatment with recombinant desulfatohirudin (r-h) or with a preformed complex of human donor antithrombin iii with heparin or post treatment with r-h are able to block the degradation of fibrinogen and the formation of fibrin monomer. in addition, after pretreatment with r-h it was found that lps-induced lung injury was reduced. since both natural hirudin as well as r-h were well tolerated by healthy volunteers, adminislration of r-h may be a promising therapeutic approach in patients with sepsis and sirs. corticosteroids have been used in the management of various shock syndroms including sepsis. the data concerning the effects of this therapy is, however, conflicting and in large prospective studies, the use of early administration of high doses of corticosteroids have been found to give no beneficial effects in patients with sepsis. we have particularly been engaged in studies on possible effects of corticosteroids on the plasma cascade systems and on the mediator release from leukocytes. in in vitro studies, high doses of methylprednisolone were found to facilitate endotoxin induced generation of plasma kallikrein. furthermore, plasma prekallikrein and kallikrein inhibitor values decreased together with formation of kallikrein-c inhibitor complexes ( ). in this in vitro plasma model we also found that methylpredoisolone alone in doses of mg/ml induced contact activation with the formation of kallikrein-c inhibitor complexes. methylprednisolone was also found to have an additive effect on endotoxine induced decreases in kallikrein inhibitor functions. in the same experimental model on endotoxemia methylprednisoione was found to give an additive effect on activation of the initial part of the complement cascade ( ) . activation of the terminal part of complement, however, was inhibited by the same dose of methylprednisolone. addition of methylprednisolone alone to plasma was also found to activate the initial part of complement. using a full blood model, we studied the effect of methylpredoisolone in modulating the endotoxin induced cytokine response. when ng/l e. coli endotoxin was added to citrated blood from healthy human donors preinkubation with gg/ml methylprednisolone significantly reduced the release of tumor necrosis factor et ( %) and interleukin- ( % ) at two hours after exposure to endotoxin. in conclusion, these studies show that corticosteroids might facilitate contact activation of plasma and reduce the function of major protease inhibitors. furthermore, we also observed that this drag in in vitro conditions facilitated activation of complement. on the other hand, corticosteroids were found to reduce endotoxin induced cytokine release from leukocytes in whole blood. these studies also showed a dose dependence of the effects of corticosteroids on endotoxin induced cellular and cascade system activation. different predisposing conditions like extensive trauma, infection or pancreatitis result in a remarkably similar inflammatory response. although thls inflammatory response primarily aims at the removal of devitalized tissues, destruction of bacteria and reestablishment of the integrity of host tissues, it may potentially become unregulated and generalized and thereby producing deleterious effects to vital organs or the body" as a whole. cytokines play an important role in the development of the systemic inflammatory response. most of their biological effects, however, are not directly mediated but exerted through other mediator systems. when the inflammatory response results in the formation of capillary leak syndrome and refractory hypotension, the unregulated activation of complement and contact systems appears to be the cause. both systems are almost exclusively regulated by c - nhibitor. am unbalanced degradation of the inhibitor protein was found to be associated with the onset of capillary leakage in various conditions such as bone marrow transplantation, severe burns, and septic shock. these observations suggest a relative deficiency of biologically active c - nhibitor in these septic shock like syndromes. therapeutic intervention studies were initiated using high doses of c - nhibitor concentrate. first results are promising, as in most patients the administration of the drug was followed by a quick and marked improvement of the patient's hemodynamics. whether the administration of c - nhibitor alone or in combination with other drugs is also sufficient to improve long-time survival has to be investigated in double-blind, placebocontrolled studies. salutary effect of human soluble complement receptor type- in models of adult respiratory distress syndrome g. feuerstein, m. dimartino, and *r. rabinovici, smithkline beecham pharmaceuticals, king of prussia, pa, usa, and *jefferson medical college, philadelphia, pa, usa adult respiratory distress syndrome (ards) is a severe pulmonary insufficiency syndrome associated with diverse diseases and injuries. ards is believed to be the consequence of a massive acute inflammatory reaction consisting of activated neutrophils infiltration into the lung. neutrophil activation is likely the result of multiple factors of which complement is believed to play a major role. a potent complement regulatory system in the form of the complement receptor type- (cr- ) exists on many cells where protection against complement injury is provided by inhibition of both the alternative and classical pathways for c /c convertase formation. we have recently purified the human recombinant cr- in a truncated form (brl , tp-io, scr- ) and tested this soluble protein for protective effects in several models of ards. specifically, we have used the following rat models: ) endotoxin-induced ards in paf primed animals; ) .- induced ards; ) thermal injury ( % body surface)-induced ards; ) acid aspiration-induced ards. ards-like features included edema (increase in lung wet weight and water content), neutrophil accumulation (histological inspection and myeloperoxidase assay) and increase in cells and protein in the bronchoalveolar lavage (bad. scr- , - mg/kg, i,v., given as prophylactic (and in some cases, therapeutic) treatment significantly inhibited edema formation and leukocyte infiltration and, in some cases (e.g., endotoxin/paf or il- ), virtually completely. these comprehensive studies strongly support the therapeutic potential of scr- in ards due to diverse injuries. the xanthine derivative pentoxifylline (ptx) and several analogs of ptx have been evaluated for their protective effect in various animal models of septic shock. data from these in vivo studies demonstrate that ( ) ptx suppresses lps-induced tnf release from activated macrophages; ( ) ptx prevents tnf-induced pmn activation; ( ) ptx attenuates lps-or tnfinduced acute lung injury as evidenced by prevention of increased pulmonary vascular permeability and detoriation of gas exchange; ( ) ptx exerts its .protective effects even when administered following initiation of sepsis. the sum of these actions suggest that administration of ptx in sepsis may have therapeutic potential. hans hoffmann md, dept. of surgery, klinikum grosshadern, ludwig-maximilians-universit~.t, marchioninjstrasse , d- m nchen, germany. pentoxifylline [pof] and related xanthins are drugs of known haemorrheologieal activity and widely used clinically. recently, new pharmacological aspects of these established drugs could be demonstrated. it was found that pof selectively inhibits the formation of tnf but not il- most likely by causing accumulation of intraceliular camp via inhibiting phosphodiestersse activity, and, secondly, by inducing prostacyclin in different cell types. furthermore, pof is able to counteract tnf-mediated adherence of neutrophils to vascular endothelium and to inhibit fmlp-indueed respiratory burst in neutrophils. we and others have, therefore, studied its effect in different animal models. it was found that pof protected from increased pulmonary vascular permeability and sequestration of neutrophils into the lung in different animal models of acute lung injury. moreover, in pigs with induced faecal peritonitis the drug improved haemodyrmmle variables as well as pulmonary compliance and reduced the number of pulmonary neutrophila and lymphocytes. consequently, as a general outcome, pof could be found to improve survival in different models of haemorrhagie and endotoxie shock. the protective effect of pof was dose-dependent and observed even when pof was given up to hours after endotoxin. in order to evaluate these pharmacological effects of pof in humans, we studied pof under controlled conditions of endotoxlnemia in volunteers. we found that pof selectively inhibits the lps-indueed endogenous formation of tnf without affecting il- and il- . moreover, pof counteracts the lps-indueed initial leukocytopenia by interfering with the adherence of neutrophils in the microvasculature. meanwhile the inhibition of endogenous formation of tnf by pof could be demonstrated under different clinical conditions of acute and chronic cytokine release syndromes, {okt first-dose reaction, cancer-or tuberculosis-induced cachexla}. it appears worthwile and promising to investigate wether pof exhibits beneficial effects also in septic patients. objectives: to determine the specific role of paf in both lethal primate bacteremia and nonlethal human endotoxemia. materials and methods: two double-blinded placebo controlled studies were performed. first, ten baboons ( . +. kg), were randomized to a control group receiving ° cfu/kg of intravenously administered e. co/i (n= ) and a treatment group (n = ), pretreated with . mg/kg of paf receptor antagonist ro - two hours prior to e. co/i infusion. in a second study, ten healthy male volunteers were randomized to a control group receiving ng/kg of intravenously administered endotoxin (lps) (n= ) and a treatment group (n= ) pretreated with mg of ro - eighteen hours prior to lps administration. physiologic parameters and cytokine levels were measured continuously in both studies for hours. results: baboons pretreated with the paf antagonist had improved oxygenation ( + mm hg v -+ in controls, p < . ) and creatinine clearance hours post e.coli ( . + . ml/min v . _+ . in controls, p < . ). similarly, volunteers pretreated with the paf antagonist experienced fewer symptoms, including rigors and myalgias at hour post lps. this was in concert with a diminished peak cortisol ( +_ mmot/l v +- mmol/l in controls, p < . ), epinephrine secretion ( + nmol/l v + nmol/l in controls, p < . ). hemodynamics and circulating tnfa levels (data not shown) were unaffected in either study by prior paf antagonism. concluslons: paf influences some components of the multi-organ failure syndrome in lethal gram negative sepsis and the counter-regulatory hormonal system in human endotoxemia through tnf-independent mechanisms. paf antagonists may serve as adjuncts to cytokine blockade in the treatment of gram negative sepsis. the mediator role of platelet-activating factor in gramnegative septic shock pierre g. braquet, david hosford and matyas koltai institut henri beaufour, le plessis robinson, france considerable evidence has been accumulated to support the concept that a paf/cytokine autogenerated feedback network is responsible for priming and amplification of inflammatory mediator release in septic shock. cytokines, such as tnf~x, il- and other interleukins interact with paf which then amplifies cytokine production, therefore, paf may be the principle mediator in endotoxin shock. a single factor identified as tnf is suggested to play a pivotal role in the fatal phase of septic shock. presumably excess tnf release is the result of amplification process primarily triggered by paf. one may assume that the high tnf concentration in the blood of 'non survivors' is the consequence rather than the cause of cell death. the dubious results of clinical trials with anti-tnf antibodies and il- ra, a naturally occurring il- antagonist protein, have risen debate around the exceptional role of cytokines in the pathomechanism of septic shock or systemic inflammatory response syndrome (sirs) induced by gramnegative microorganisms. there are similar problems with the interpretation of the results obtained in clinical trials of monoclonal igm antibodies against e. coli endotoxin, ha- a and e . future studies will answer the question as to the effectivity of inefficiency of this treatment. perhaps when the plasma concentrations of lps and cytokines exceed a critical level, treatment fails to save the patients life. with regard the causal role of lps in septic shock, the putative role of bacterial porins may have to be taken into consideration. the marked release of paf induced by endotoxin leading to excess txa production may be responsible for the microvascular failure and death in septic shock. antagonists of paf markedly protect against the release of txa , and may interrupt the vicious circle of amplification process. no produced by the inducible no synthase plays a pivotal role in causing vascular paralysis in lps-induced sirs. to explore the relationship of paf to no synthesis will provide better understanding of the pathomechanism of septic shock. several paf antagonists, including bn , have undergone phase ii and phase iii clinical trials. the strategy in the treatment of sirs, however, remains multifactorial, since little is known about the sirs caused by microorganisms other than gram-negative bacteria. evidence has begun to accumulate which suggests that in sepsis excess production of lps and cytokines can lead to uncontrolled production of inos and that this might in part explain the severe unresponsive hypotension seen in some septic patients. a number of strategies have been explored in an attempt to limit excess no production. a favourite target has been competitive inhibition of nos by arginine analogues such as l-nmma. in animals, some investigators have shown that the haemodynamic effects of lps challenge can be attenuated by l-nmma, but there is a narrow toxic-therapeutic ratio. in a model of live e. cell sepsis, we have been unable to reproduce these findings. localisation of no production by immunohistochemistry suggests that no production is compartmentalised, and more subtle methods of regulation may be required if this approach is to have clinical valuc. anti-adhesion molecule strategies ch wortel, repligen corporation, one kendall square, building , cambridge, ma , usa. the neutrophil has been implicated as a pivotal player in the pathogenesis of multiple organ dysfunction. an important first step in the process of neutrophilmediated organ injury involves the binding of neutrophils to endothelial ceils. this process is largely regulated by complementary adhesion molecules, some of which are present constitutively on the cell surface and others that can be upregulated in response to chemotactic and pro-inflammatory stimuli. several different adhesion molecules have been described. l-selec tin is expressed on the plasma membrane of neutrophils and is shed from the surface early in the inflammatory process. it is therefore thought to mediate the initial interactions with endothelial cells. e-selectin and p-selectin are endothelial adhesion molecules. e-selectin is not constitutively expressed but is upregulated by inflammatory mediators, particularly il- and tnf. p-selectin is present in the weibel-palade bodies within the endothelial cytoplasm and can be upregulated rapidly in response to thrombin, histamine, and oxidants. all selectins recognize oligosaccharides, particularly sialylated lewis x antigen. this carbohydrate moiety is expressed on many proteins, including the selectins themselves. the leukocyte integrins are glycoproteins expressed on the neutrophil surface and in the cytoplasmic granules. integrins consist of a common beta or cluster differentiation (cd)i chain covalently linked to one of three different alpha chains (cd a, cd lb, cd lc) and exist on the cell surface as three distinct heterodimers. cd l a/cdi is expressed on all leukocytes, whereas cd l b/ cd and cd lc/cd are restricted to cells of myeloid origin. cd i/cd interacts with intracellular adhesion molecule- (icam- ), its ligand on endothelial cells. icam- is a member of the immunoglobulin family of adhesion molecules. it is constitutively expressed on endothelial cells and can be upregulated by cytokines. the potential for monoclonal antibodies to adhesion proteins to reduce vascular and tissue damage has been studied in alarge number of experimental models. protective effects have been observed in a wide variety of inflammatory, immune, and ischemia-reperfusion injuries such as arthritis, bums, endotoxic shock, bacterial meningitis, autoimmune diabetes, nerve degeneration, allograft rejection, allergic asthma, acute lung inflammation, skin lesions, and ischemia-reperfusion models of the intestine, myocardium, lung, skeletal muscle, and central nervous system. protective effects have also been observed in animals resuscitated following hemorrhagic shock. since modulating the function of adhesion molecules may temporarily leave the host without effective defense machinery, safety evaluations are of utmost importance in evaluating this therapeutic approach. treatment of gram-negative septic shock with an immunoglobulin preparation: a prospective, randomized clinical trial ingolf schedel, ursula dreikhausen; birgit nentwig; marion hockenschnteder, dirk rauthmann, salim balikcioglu, rolf coldewey, helmuth deicher, md endotoxin may play a major role in the pathogenesls of muitiorgan failure in the context of the toxic process in septicemia induced by gram-negative pathogens. the hypothesis which has led to a prospective clinical study consisted in the idea of inhibiting the biological effects ofendotoxin during the early phase of septic process, and thereby attampting to attain a positive effect on the clinical course of the disease. -objecave: to evaluate the effectiveness ofa polyclonal immunoglobulln (ig) preparation containing igg, lgm, and iga as an adjunctive therapy for septic shock. -desigru prospective, randomized clinical trial. patients: fifty-five patients w~th septic shock were randomly allocated to two groups according to criteria of septic shock. -intervention: one group of patients (n = ) received a commercially available immunoglobulin preparation (containing high titers of antibodies specific for determinants to bacteria endotoxin) during the first days after inciosion in the study. the other randomized group (n = ) did not receive any immunogiobulin preparation. -measuremems and main resmts: during the period of < wks after the beginning of clinically apparent septic shock, death related to the septic process occurred in one ( %) of patients who received immunoglobulln. by comparison, nine ( %) of control group patients died during this period (p <. ). within the first hrs after onset of the clinically apparent septie process, significaptly increased activity of circulating endotoxin and simultaneously decreased specifie igg serum titers to lipid a were detected in the group of nonsurvivors. the rationale for the use of polyvalent intravenous immunogiobulins (ivig) to treat severe infections stems from in vitro and animal studies documenting that high-dose igg enhance opsonization and phagocytosis of pathogenic bacteria. moreover, recent clinical studies have shown that in septic patients the phagocytic function of circulating polymorphonuclear neutrophils (pmn) is defective; the degree of such impairment correlates to the severity of the septic state, the beneficial effect of administering high dose igo in septic patients can be two-fold: i) ivig provides large quantities of antibodies against invading pathogens; the igg bound to the pathogens can opsonize their phagocytosis; ) the opsonization of phagocytosis by exogenously administred polyvalent igo would be of special importance in those patients who present a significant defect of their phagocytic function, when they are challanged by a large bacterial invasion. a prospective, randomized, double-blind study was carried out comparing the administration of ivig (sandoglobulin@) vs, paeebo (human albumin) in severely septic surgical patients, only patients with gepis score >_ (meaning a mortality risk > %) were accepted into this protocol. patients were randomized to receive . g/kg of ivig or placebo on day (when they reached sepsis score> ), repeated on day + and + . at the beginning of icu treatment, the two groups of patients were similar for severity of sepsis, age, concomitant disease, type of surgical procedures, antra and perioperative procedures, antibiotic administration. the results of the study indicated a significantly reduced mortality in patients with severe surgical sepsis treated with ivig as compared to placebo control patients (mortality: % vs, % respectively; p< , ). in conclusion, the results of our study in patients with severe surgical sepsis were the following: ) ivig plus multimodal treatment of sepsis, including antibiotics, reduce mortality significantly', ) the reduction of mortality seems to be due to a decreased incidence of lethal septic shock. despite substantial clinical research, the avallable data regarding the effectiveness of supplemental immunoglobulin (ig) treatment in sepsis in adult patients do not yet allow definitive conclusions. in view of the persistently high sepsis mortality there is a need to continue clinical investxqations regarding supplemental sepsis treatmen~ in general, as well as concerning ig administration in particular. we present and discuss the protocol of the ongoing ,,score-based-immuneglobulin therapy of sepsis (sbits)" study. the protocol (theoret surg ( ) - ) of this multicenter, randomized, prospective and double-blind trfal relies on the results of an observational trial on i.v. igg treatment in patients with sepsis and septic shock (infection ~ ) - ), carried out as a prerequisite for the present trial. using microcomputer-based bedside routine score monitoring, we regard quantitative measures of severity of disease and sepsis: only patients with a certain degree of both severity of disease (apache ii score - ) and severity of sepsis (elebute sepsis score - ) will be included. by observing these previously validated inclusion criteria, this trial snould iqentify a priori and include patients with potentially optimal response to therapy, consisting o~ either placebo ( .i % albumin) or polyglobin n" - ml ( . g)/kg on day and ml ( . g)/kg on day i. with an anticipatedpopulation size of patients the study should comply with the statlstical requirements (estimated mortality: %, with a % reduction in -day mortality in the treatment groupl to prove or disprove the question of igg effectiveness in sepsis in terms of improved prognosis. up to november , more than patients had been included; patient enrollment will be finished in . previous studies have demonstrated rhll-i ra, a naturally occurring antagonist of il- , increases survival in animal models of andotoxemia and eschehchia coli bacteremia and attenuates the decrease in mean arterial pressure resulting from challenge with both gram-negative and gram-positive bacteria. previously, in patients, rhll-lra was demonstrated to increase survival in patients with sepsis syndrome and septic shock in a dose-dependent manner. methods: a randomized, double-blind, placebo-controlled, malticenter, clinical trial enrolled patients at academic medical centers in europe aad north america. eligible patients received either placebo (vehicle) or rhil-lra (anakinra) . or . mg/kg/hr by continuous intravenous infusion for hours. the presence of organ dysfunction (i.e., ards, dic, renal, and hepatic) at study entry was determined prospectively by a clinical evaluation committee using definitions which were developed a-priori. survival time was evaluated over days utilizing a linear dose-response model, assuming a log-normal distribution. results: patients had one or more sepsis-induced organ dysfunction(s) at study entry. a dose-related increase in survival time was observed with rhll-lra compared to placebo in patients with ards, dic, and renal dysfunction (p --< . endotoxin infusion releases platelet-activating factor (paf), a potent phospholipid mediator which leads to an autocatalytic amplification of cytokine release. bn (ginkgolide b), a natural paf receptor antagonist, has provided significant protection against sepsis in different animal models• a randomized, placebo-controlled, double blind, multicenter trial on efficacy (mortality at d ) and tolerance of bn ( iv infusion of mg x /day over days) in severe sepsis has enrolled pts. the day mortality rate was % for the placebo group and % for the bn group (p = . ). the efficacy of bn was greater in pts with gram-negative sepsis: the -day mortality rate was % for the placebo group and % for the bn group (p = . ). bn also reduced mortality among pts with gram-negative septic shock (mortality was % for placebo vs % for bn ; p = . ). using statistical adjusments for pronostic factors, the relative risk of death of the bn group was . ( . - . , % confidence interval; p = . ). this risk corresponds to an adjusted reduction in mortality of % for pts receiving bn . no differences in mortality rates were found between the placebo and the bn groups in the absence of gram-negative sepsis• there were no differences in adverse events between the placebo and the bn groups. bn is a safe and promising treatment for patients with severe gram-negative sepsis. a confirming study, focused on gram negative sepsis, is in progress. v~ lliam a. kanus m.d. and the rhll-lra it has been traditional within the field of infection and sepsis to think in terms of specific indications for drugs based on the type of infecting organisms, advances in antibiotic therapy now control or ltnflt the growth of bacteria. the majority of deaths are now caused by either an initial overwhelming response to infection or subsequent multiple organ system failure attributed, in part, to the effects of intrinsic biologic responses of the host. type of organism, therefore, may not be as critical as determining the exact severity of the host's severity or risk of death from infection. we also know that both the relative benefit of a new treatment across groups and its absolute benefit for an individual patient will vary with their risk in a predictable fashion. we recently iuve~iguted the relationship between one measure of host response, the acute risk of death as prospectively estimated by u comprehensive risk mode[ for -day mortality (jamb. ; : , - ) , by its retrospective application to the results from the phase in evaluation of recombinant human intcrlenkin- receptor antagonist (rhll. ira). we found that there was a significant interaction between the patient's predicted risk of mortality at the time of entry to the study and the ability of rhil-lra to prolong survival time (x = . , p [] . , log.normal) for all patients in the trial• survival benefit began st approximately % baseline risk of -day mortality. for the $ patients with a predicted risk > %, there was a % reduction (p= , $ log normal). when we examined the variation in patients above and below the % risk level with hazard functions, i.e., their daily risk of death during the study period, we found that placebo patients with < % risk had lltile acute daffy risk during the hlltial two days follawh~g study entry and this risk was little affected by rhil-lra, in contrast, patients with > % risk had high daily mortality risks during the tuttlal two days that high dose rhtl-lro substantially reduced. these results are compatible with our current understanding of outcome from sepsis and the proposed mechanism of action o£ immunotherapy, the earliest deaths from sop sis are secondary to an immediate inflammatory response followed closely by deaths secondary to multiple organ system failure, later deaths (after days) are not as closely related to the acute effeete of the inflammatory cascade. because of the timing and action of most proposed tmmunotherapy, they may be capable of preventing mortality primarily in these initial two phases. in this study, an independent predicted risk of mortality reflected this mortality pattern ned illustrated the potential benefit of immtmotherapy. use of a predicted risk of mortality in the design and analysis of clinical trials could improve our understanding of the clinical benefit of these new therapeutic approaches. the systemic inflammatory response syndrome (sirs) is a term recently proposed to describe patients with systemic inflammatory responses to insults such as infections (sepsis), trauma, burns, pancreatitis, and other initiating events. patients with sirs may have similar activation of inflammatory mediators and similar outcomes independent of the initiating event. these outcomes include organ dysfunction and failure, shock, and death. challenges to the successful conduct of clinical trials in sirs include the complexity of illness in these patients and the important--but limited--clinical benefits of novel compounds that may be limited to selected patient subsets. addressing these challenges will require new tools and approaches. these will include more sensitive and appropriate endpoints, and the use of methods such as baseline risk adjustment, to allow detection of drug risk interactions not captured adequately by categorical definitions, such as sepsis syndrome. on the basis of supportive preclinical and phase i safety studies, we have initiated phase ii clinical trials of a novel bradykinin antagonist, cp- , in four sirs subcategofies: sepsis, multiple trauma, burns, and pancreatitis. each of these studies is designed to measure the effect of cp- on mortality, organ dysfunction and failure, and activation of mediators. in addition to investigating rates of organ failure using standard definitions--a new endpoint--a continuous summary measure of organ dysfunction (the acute physiology score of apache tm iii) is being used to quantify the degree of organ dysfunction and the speed and pattern of recovery of physiologic stability. in the sepsis study, another new approach--a study specific risk model based on the apache ill database--has been developed which will be used to assign a pre-treatment baseline risk to each patient enrolled. the primary outcome variable will be risk adjusted survival time to days. this type of risk-adjusted analysis may allow for more efficient and powerful trials and more accurate and useful indications for use. study purpose: in post-cardiac surgical patients (pat.) at risk for sepsis, the efficacy of early i.v. immunoglobulin (ig) treatment was compared to a matching historical control (con.) population. postoperative risk assessment: using apache ii scores lap) (first postoperative [pop.] day) in a pilot study phase, we were able to differentiate between the large population ( . %) of pop. low-risk pat. (ap< ; mortality: %) and the small groups of pop. pat. at risk lap= - ) and high risk lap_ ) with a significantly higher mortality ( % and %, mainly due to sepsis). subsequently, among consecutive pop. pat. we prospectively identified and treated these pat. iq treatment reqimens: first study period (n = ): (gg (psomaglobin n a, tropon biologische pr~parate, cologne, frg, day : ml/kg, day : ml/kg). second study period (n= ): iggma (pentaglobin r, biotest, dreieich, frg, ml/kg on days to ). results: ig pat. and con. were comparable in demographic data, operation characteristics and baseline disease severity lap and elebute sepsis scores). in contrast to con. (risk: n= , high-risk: n- ), the ig pat. showed a marked improvement in disease severity (fall in ap), especially in the high-risk group (igg, n= : p<o. ; iggma, n= : p: . ). in this group, ig led to higher (p< . ) response rates, defined as an ap score decrease -> within four days (igg: %, iggma: %; con.: %), and reduction in mortality (igg: %, iggma: %; con.: %), statistically significant (p< . ) for ig treatment as a whole (igg and iggma). conclusion: given the good comparability of the study groups, our results indicate, despite the non-randomized design, that early supplemental ig treatment can improve disease severity and may improve prognosis in prospectively apache ii score-identified high-risk patients after cardiac surgery. objective. elevated plasma levels of endothelin (et) have been demonstrated in both experimental and human sepsis. et has been proposed as a sepsis mediator leading to vasoconstriction with tissue hypoperfusion and organ failure. the aim of the study was to determine the effects of sepsis treatment with volume resuscitation, antibiotics and the anti-lps monoclonal antibody es® on big et and active, aminoacids et (et ) in rat abdominal sepsis. methods. lethal peritonitis was induced with a mm coecal perforation (cp) in male wistar rats. plasma levels of big et and et were determined with amersham tm endothelin rias , and h after sepsis induction. experimental groups: . cp control, . volume replacement (vr); , % saline ml/kg/h continous iv infusion started after h, . antibiotic; imipenem mg/kg iv after h, . e ®; mg/kg iv after h, . vr + imipenem + es® after h. results. high concentrations of both big et and et could be demonstrated after h and lasting for h after cp. neither volume replacement nor imipenem did influence the elevated plasma et. e ® significantly reduced et both , and h after sepsis induction, but did not reduce big et. when es® was combined with vr and imipenem, reduction of et was the same as for e ® alone. these results strongly suggest that bacteria and hypovolemia per se are not decisive stimuli for et production during sepsis. e ® reduces circulating lps and tnf which is the probable mechanism of the suppressed et synthesis. the unaltered big et fraction after e ® treatment indicates conversion of big et to et as the site of action responsible for reduced et . conclusion. lethal peritonitis in the rat is followed by elevated plasma levels of big et and et . e ® anti-lps antibody significantly reduces plasma et while volume resuscitation and antibiotics failed to do the same. es® did not reduce plasma big et. pmx treatment on severe endotoxemia with multiple organ failure was safety and effect in prognosis, and sepsis related parameters. it was certified that reduction of plasma endotoxin was effective in severe endotoxemia. a. lechleuthner,s. aymaz, g. grass, c. stosch, s. dimmeler, m. nagelschmidt, e. neugebauer. ii. dept. surgery, university of cologne, germany. introduction: the cardiovascular therapy of hypodynarnic shock states is a challenging problem. in clinical as well as experimental studies beneficial functions of a new hg-agonist bu-e- in congestive heart failure has been demonstrated aumann, ). therefore, we investigated the effect of bu-e- in hypodynamic shock in pigs. materials and methods: pigs (deutsches hausschwein, pitrain, [ ] [ ] [ ] [ ] [ ] [ ] were anesthesized with fentanyl/dormicum, ventilated (n :o = : ) and cardiovascular parameters were monitored with a complete icu-eqnipment. the hypodynamic model was established in a pilot study ( animals) to evaluate the effective concentration of bue- in healthy and endotoxin (lps)-treated animals. endotoxic shock was induced by continous infusion of ~g lps/kgkg/h ( :b , fa. difco). the hypodynamic state was defined as a decrease of cardiac output by % of steady state levels. a wedge pressure of - mmhg was kept constant by volume resucitation during the experiment. in a subsequent randomized controlled trial (rtc) groups with animals per group were studied. the groups were treated as follows: group i, lps and , % nac ; group ii, lps and bu-e- ( #g/kgkg/h); group iii, famotidine (h -blocker) pretreatment ( mg/kgkg), lps and bu-e- . results: the pilot study in healthy pigs revealed, that bu-e- had positive inotropic effects. these effects were inhibited by the h antagonist famotidin. bu-e- however had no beneficial effects in the hypodynamic phase of endotoxic shock in the rct. cardiac index (ci) and the oxygen delivery (do ) were not significantly influenced by bu-e- application (group i versus group ii). bu-e- did not ameliorate the negative inotropic effect measuring left ventricular stroke work (lvsw) in hypodynamic shock phases. on the contrary, bu-e- led to a further significant decrease of lvsw (p < , ). famotidin pretreatment did not affect the response (group iii versus group ii). conclusion: in hypodynamic shock states the h -agonism seemed to have no beneficial effect under these experimental conditions. receptor down regulation or changes of signal transduction under septic conditions may be responsible. cellular studies may help to identify these mechanisms. objectives. antithrombin iii inactivation of proccagulant proteases is so far the only inhibitory therapeutic approach to disseminated intravascutar coagulation (dic). we therefore set out to investigate whether cll substitution reduces coagulation activation in an endotoxin induced rabbit dic model. materials and methods. male rabbits chbb:hm(spf) were randomty assigned to one of the following groups. group k : naci . % (control without endotoxin, n= ). group e : endotoxin tjg kg " bolus i.v. + naci . % (control with endotoxin, n= ). group c : endotoxin pg kg - bolus i.v. + cll u kg - bolus + u kg " h "~ i,v. (treatment group, n= ). all animals were anesthetized and mechanically ventilated. blood samples were drawn prior to endotoxin administration (m ) and after (m ) and rain. (m ). thereafter, lung and liver tissue samples were taken intravitatly in a standardized fashion for h&e microscopic fibrin quantification using a triple score (fibs). from all blood samples the prothrombin time (pt), activated partial thromboplastin time (aptt), fibrin monomers (fm), and d-dimers (dd) were measured. for statistical significance of differences between the groups anovas and the wilcoxon test (fibs) were performed. results. fibs for lung/liver were significantly different (p< . ) between group e (lung , liver ) and c (lung , liver ) (group k : lung , liver ). , a synthetic serine proteinase inhibitor, has an anticoagulant activity in the absence of" antithrobim iii. gabexate has been reported to be useful in the treatment of disseminated intravascular coaguiation due to neoplastic diseases. in this study, we investigated gabexate therapy for the treatment of dic due to sepsis in the postoperative critical patients. materials and methods: from july to june , patients in the surgical intensive care unit met the criteria of dic or pre-dic. eleven were male and four were female with the mean age of . years. all these patients suffered from some complication of operations which led to the development of sepsis. foy was administered at the rate of mg/kg/hr untii the coagulation profile retumed to normal or the patient died. the coagulation parameters were monitored before and on the st, rd, th and th day. results: fourteen of these fifteen patients died despite transient improvement of the coagulation parameters in five patients. these patients suffered from sepsis resulting from surgical complications which could not be well controlled. the only survival was a case of recurrent intrahepatic duct stone with biliary tract infection complicated with sepsis and dic. after choledocholithotomy and the use of foy, the patient recovered gradually. conclusion: dic is a late manifestation of sepsis in the critical surgical patients. the most important thing is to eradicate the cause of sepsis. if the underlying septic focus cannot be controlled, dic will persist despite the use of gabexate mesilate. emergency surgery, taipei veterans general hospital, taipei, taiwan. there are main types of bradykinin (bk) receptor, namely bk~ and bk z. the bk receptor is constitutive. the bk receptor is also constitutive but in the majority of cases is inducible and involved in chronic inflammatory syndromes such as sepsis, hyperalgesia and airways hyperreactivty in animals. the mechanism(s) involved in the upregulation of the bk receptor is unclear, however a variety of agents including lps, e coil and ill are particularly efficacious in vitro and in vivo. ill and bradykinin acting at their respective receptors are believed to be involved in sirs/sepsis. we have investigated the effect of antagonists at ill (antril), bk (bradycor [cp- ]),bk~ (cp- ) and bkz/bk (cp- ) receptors on the de novo generation of bk~ receptors (reflected by hypotensive responses to a bk agonist) in the lps-treated ( ug iv) rabbit. in lps treated rabbits hypotensive responses to bk~ but not bk agonists increased with time and at time min appeared maximally induced. constant iv infusions of cp- blocked bk but not bk~ and cp- bk~ but not bk responses. cp- ,cp- +cp- and antril+cp- blocked both bk and bk~ responses. antril alone had no effect on bk or bk~ responses. within - min after stopping the infusions of antagonists the responses to bk~ and bk z agonists were the same as those in nonantagonist infused rabbits. these results indicate, at least in the lps-treated rabbit, that neither bk ,bk ~ or ill receptors alone or in combination, are involved in the de novo generation of bk receptors. in vitro studies demonstrated that beth bradycor and cp- (but not antril) were antagonists at both bk z and bk~ receptors. if both bk z and bk receptors are significantly involved in chronic inflammatory situations in man such as sirs/sepsis then the rationale for the use of compounds such as bradycor or cp- is clear. infection is a major cause of or contributor for morbidity and mortality in liver transplant recipients. effectiveness of prophylactic and therapeutic protocols is important for the success of liver transplantation ( olt ). sdd is used as prophylaxis for reduction of infection caused by gram negative or fungal microorganisms. between september and july olt's in patients were performed at our department. the actuarial -year patient survival is %. infection prophylaxis is started with sdd and ciprofloxacin once the patient is accepted as an olt candidate. perioperatively metronidazol, tobramycin and cefotaxim, postoperatively cotrimoxazol are prescribed additionally. the table shows pneumonia, peritonitis, major wound and urinary tract infection are common nosocomial infections following severe injury. in a series of severely injured patients from the university of louisville hospital, pneumonia was the most common infection followed by peritonitis, intra-abdominal abscess formation and burn wound infection. pneumonia is actually the leading cause of death from nosocomial infection. these are defined as occurring from to hours after hospital admission. this definition has important implications for antibiotic therapy because the likely pathogens and their respective sensitivities are different for community acquired pneumonia. the diagnosis of nosocomial pneumonia is difficult following major injury as many patients will have pre-existing fever, leukocytosis, tachypnea, and chest x-ray changes. reliance on sputum gram stain and culture is important and best obtained by a bronchoalveolar lavage or protected specimen brush during bronchoscopy. predisposing risk factors include severe head injury, emergent intubation and shock, and such patients have been shown to benefit by early tracheostomy. staph aureus has been the most common pathogen isolated from the sputum and the remainder gram-negative organisms with pseudomonas aeruginosa, and klebsiella pneumonia predominating. bacteria recovered by site as well as by intensive care unit is published in the six month antibiogram which also includes recent antibiotic sensitivities. this aids in empiric antibiotic selection against such nosocomial organisms. in a series of severely injured patients (iss - ), mean temp. was . f, leukocytosis was k, pan was , fin was . , and peep was . at the time of diagnosis (ards excluded). there was marked reduction in class ii histocompatibility antigen (hla-dr) density on peripheral and bal monocyte/macrophages which recovered over time with resolution of pneumonia. immune suppression occurred prior to development of pneumonia, was especially localized to the infected tissue, but recovered with clinical improvement. specific immune modulation targeted to pulmonary white cells may hasten clinical recovery and minimize pulmonary dysfunction. -clinical experience j. tnllemar amphntericin b remains the drug of choice for many systemic fungal infections. its advantages include a broad spectrum of activity and intravenous administration. the major disadvantages of amphoterlcin b is its severe side-effects, especially the nephrotoxicity. to decrease the toxic side..cffccts various liposomal amphoteficin b formulations have been produced. it was found that these liposemal formulations were as effective as amphotericin b but in contrast had a low incidence of toxicity. at present there are three ~different variations of lipid formulations under assessment: amphotericin b lipid complex (ablc), amphotericin b coloidal dispersion (abcd) or true liposomes. the ablc has a ribbon like structure. it has been shown to have a reduced toxicity and an efficacy ranging from being as effective to four times less effective that conventional amphotericin b. regarding abcd the particles have a disk-like structure with a diameter of around t am and a thickness of nm. the ami-fungal efficacy is - times less than that of conventional amphotedcin b. both ablc and abcd are presently investigated in phase ii/iii studies in the us. ambiseme is currently the only commefieally available true lipesome. ambiseme is a spherical small unilamellar lipesome with a diameter less than nm with a mutina ld of > mg/kg. it has been used in dosages up to mg/kg/day in compassionate based studies with good tolerability. the mycological efficacy range from a % response rate for invasive candida infections to % response rate for aspergillosis. ambisomc have been evaluated as anti-fungal prophylaxis in randomized trials in bone marrow (bmt) and liver transplant (ltx) recipients. it was well tolerated. in bmt recipients the incidence of proven fungal infections was % among placebo treated patients compared to % for the ambisome treated patients (ns). in ltx recipients ambisome prophylaxis was effective, significantly reducing the incidence of deep fungal infections from % to % ill placebo and ambisome treated patients respectively (p< . ). prospective randomized trials comparing these various amphotericin b preparations with conventional amphotericin b is needed to determine their future place in the therapeutical arsenal. two patlentgroups ere particularly at risk to develop serious cmv disease: cmv seronegative transplant recipients of seroposltlva donors and those patlants treated for rejection with anti t-ceil preparations, we have evaluated the value of prophylactic anti-cmv immunoglobulin (cytotect", biotest pbarma gmbh, dreieich, frg) administration in high risk heart and kidney transplant recipients, in a double blind placebo controlled study kidney transplant recipients, treated for biopsy proved re)action with rabbit atg, received globullntplacebo infusions. the preparatlons were given i,v, in a dose of mg/kg at day , , , , and after the initiation of anti = rejection therapy, passive immunization completely prevented cmv related death, although it did not reduce th~ incidence of cmv isolation, viraemia or disease, this effect was mainly observed in cmv saronegativa recipients of a serop sitive donorktdney. seroposltive recipients did not benefit from treatment and seronegatlve recipients of a seronegetlye donor were not et risk for cmv infection at e!l. in a open study the incidence of cmv infection and disease was evaluated in consecutive i~eart sllograft recipients. sixty-five patients were cmv seronagatlve and they all received passive immunlzation according to the dosage schedule used in the kidney patients, but starting on the day of transplantation, this scheme resulted in median snti-cmv igg titers of elisa units during months. cmv infection occurred in / ~eronegetlve and in / seropositive recipients (n,s,), in ssronegetive donor-recipients pairs the incidence was significantly lower ( / ] , the passively immunized seronegstive recipients of e seroposltlve donorheart showed comparable incidence of cmv infection f t ) vs the seropositive recipients. primary infection more often resulted in disease than secondary infection ( v / ), but no difference in incidence of disease ( vs / ) or severity in symptoms was noted between the immunoglobulln treated serone(]ative patients and the seropositiva recipients. apparently passive immunization induces anti-cmv immunity which crossly resembles naturally acquired resistance. abdulkadirov k.,chebotkevich v., moiseev s. the incidence of infection is still high in patients underwent bmt. this complication is the major cause of mortality if it is not recognized and treated promptly and properly. our data showed that from patients with different types of leucemia after autologous and allogenzc bmt had the episodes of fever. in the ma i ority of these episodes the bacterial etiolog$ gram negative bacflli and gram positive cocci) can be proved. on the other hand, in % of the fever cases we detected also viral respiratory (corona-, adeno-, rs-and other) infection. our previous investigations showed that even in healthy persons the viral infection has influence on antibacterial immunity, in the cases of model experimental reaction in volunteers we found the decrease of delayed hypersensitivity - days after intranasal inoculation of influenza virus a (h n - ) to bacterial (staphylococcal, streptococcal and pneumococcal) and ~iycoplasma pneumoniae antigens in the leucocyte migration inhibition test. these results showed that respiratory viruses may be the important pathogenic factor in the development of bacterial infection in posttransplanted period. we consider the constant control of latent and visual respiratory viral infection in bmt patients to be very important. ficcb the ~ter£~li of the nation~l institute of trad/~atoloqy in budapest . consecutive cases of revision hip grafting were carried out arthroplasties wlth hemoloquous bone between the years and . in the same period of time pri~ total hlp replacen~nts were performed under i entieal technical conditions. the average septic rate for the 'total hip althroplasties was less than %. in the selected i cases the septic rate was % indicating the role of bone grafting° homografts were prepared by deep freezing~ it .is recognized that the cells of the hl~grafts become destroyed by the ium~unological, response of the host~ and the patients develop ~ti-hl~, ar~tib'o~ies. the dead ~trix, however, has a bone-inducing capacity that stimulates host osteoblasts to recolonize the *i~/trix which serves as scaffolding. the sequence of events favours the infections. for this reason, beside preventive perioperative systemic ant/biotic treatment, local ~ntibioties were also applied in the form of antibiotic-//npregnated cement. the role of age and the .immune status of the patients .is discussed.. the purpose of this study is to evaluate the rate of toxemia in patients with acute panereatitis and to find this coudition to the activation of cascade systems that are encountered in the subsequent complications of the disease. we studied a series of patients with acute pancreatitis, the severeness of which was evaluated by the ranson's criteria and the apach-ii scoring system. all of them were considered to have severe acute puncreatitis. the determination of toxemia was made using the limulus test (lal test). we also determined the levels of the third (c ) and fourth (c ) complement components as weu as the coagulation factors, iibrinolysis faeters and kimns by serial measurements. the severity of the disease was serially determined by the apach-ii scoring system. it was found that complement activation ( which was also assessed using a graphically illustrated method by a aggregometer ) was followed by an increase of morbitity and mortality .we also detected that toxemia (positive lal-test) was closely correlated with complement activation and more of the ranson's criteria. a clear relation existed between the number of ranson's signs and the enmplieations' rate ( "= - . , p < . ). the documentation of toxemia and the complement activation cannot predict the kind and the severity of complications. the study of coagulation, fibrinolysis and kinms systems didn't reveal any results with statistical significance. necrotizing pancreatitis still represents a life-threatenthg disease. infectious complications dominate among the causes of death. differences in the individual immune response could possibly explain different clinical courses even in patients with comparable pancreatic morphology. to explore the inflammatory response in acute pancreatitis, the following investigation was performed. methods: peripheral-venous blood was withdrawn on admission and furthermore twice weekly in as yet patients with acute pancreatitis and tested for the parameters mentioned below. in parallel, polymorphounciear granaiocytes were isolated using density gradient centrifugation and assessed for superoxide anion and hydroxyl radical producing capacity using electron spin resonance techniques. results: total leukocyte cotmt and total lymphocyte count did neither reflect the clinical course nor predict complications. this comes tree also for serum igg, igm, iga, c , c , crp, alpha-l-antitrypsin and neopterth as well as for plasma il-la, il-ib, il- ra, il- , il- r, il- r, tnf-ct, tnf-~r (p ) and icam- . in contrast, pmn-elastase, il- and il- closely correlated to the clinical course. isolated pmn's in vitro capacity to produce oxygen radicals depended on the respective radical species and was slightly elevated (superoxide anions) or decreased (hydroxyl radicals), respectively. patients with a cd +/cd + ratio below i were seen at risk of developing septic complications. in contrast, a percentage of monocytes of % or more among total mononuclear cells indicated an uncomplicated course, in general. conclusions: the immune status of the individual patient may significantly influence the course of acute pancreatitis. the cytokine pattern in peripheral blood is very complex and most parameters are of little use for the clinician. the pmn-elastase, il- and il- , however, closely correlate to the clinical course and may prove valuable for follow-up. the cd +/cd + ratio was found the best predictor of septic complications, but it failed in non-septic patients. a percentage of % or more of monocytes among total mononuclear ceils indicated a rather mild course. the reduced ability of the pmns to produce hydroxyl radicals may help to explain the frequent development of septic complications in severe necmtizing pancreatitis. peroxidation of membrane lipids contributes to ceil injury in pancreatitis. overwhelming release of toxic metabolites by infiltrating neutrophils is regarded a major pathogenetic factor, too. as yet little is known about the mechanisms by which oxidative stress and leukocytes damage pancreatic cells. the present study examines (i) the susceptibility of pancreatic acinar cells to attacks by oxidants and leukocytes and ( ) the potential of antioxidants to prevent such damage in order to better understand the cellular mechanisms of pancreatic injury in inflammatory states. methods: freshly isolated rat pancreatic acinar ceils were exposed to a model system of oxidative stress consisting of mu/ml xanthine oxidase (xod), mm hypoxanthine (hx), mm fec and mm edta. in a second set of experiments, acinar cells were exposed to excess autologous neutrophils or neutrophils obtained from patients with acute pancreatitis. neutrophils were stimulated by zymosan a, pma, and il- . cell viability was assessed by both cellular uptake of trypan blue (tb) and by release of ldh. results: the xod/hx system caused a time-dependent acinar cell injury. this injury was effectively prevented by catalase (cat) and gfutathione peroxidase (gpx). in comrast, superoxide dismutase (sod) enhanced cell injury. addition of both sod and cat abolished the damage seen with sod alone. the non-enzymatic scavengers mannitol, dmso, dmtu and the iron chelator deferoxamine were not protective and at a higher concentration even accelerated cell decline. the newly developed antioxidants of the lazaroid type effectively prevented oxidative acinar cell damage. stimulated neutrophils, both autologous and heterologous, did not damage healthy acinar cells but had even protective effects. conclusion: pancreatic acinar ceils are very susceptible to oxidative injury. a combination of catalase and sod prevented cell damage effectively. sod when given alone may rather damage than protect aelnar cells when h is generated in concentrations overwhelming the capacity of endogenous catalase. therapeutic approaches to pancreatic disease using antioxidants should, therefore, include combinations of protective substances. the lazaroids seem to be candidates for clinical use as antioxidants in pancreatitis. the results argue against direct toxic effects of stimulated neutrophils to pancreatic acinar cells. are ch~act~z~ by the presence of a polymicrobial flora, the pmtotyi~ cffthese inf~ons is secend~,y bacterial pedtonitlw, whereby a pathololoeal process in the ~trointesfimd tract r~ful~ in tim disrup~on ofi~ inteffrlty and ¢ollseqtlent sptl]nge of inte~.i,o~.l gontents into the peritoneal c~iry. the ensuing infection invariably contains a mixtm~ of gt~m negative enteric bacilli, gram positive b~eria and anaerobe& experimental and clinical =t~ies have de~ed the eantrlbution of each of th¢~ components to ti~ ovemu virulence of these in~ons, gram negative enteri~ such as f.veher~chla coil ere endowed with a virulent l~l~x~lyse~haride ptill~ly t~sponsible for lethality, by contrast, bacteroldes sl~cles, which rarely c~se death, prornot~ abscess fonllation, a uniqm~ capsul~ polyseccluu'ide, particularly on b.j~ogiljs slrai~, oontributes to tjtis erect, several mecltanims have bccn pml~ed whereby or~ microorganism mi~t interact with its microbial ~net to augment the overall virulence of a r~xed im~edan. these include: l) provision of nutrients by one apexes which stimulates the growth of its ~opathoge& ) inhibition of host deletes by one of the migroorganisms so that the other microbes might persist and exert their virulence, ) the trant~ of vim.©n~e traits between ~renr~a.,dsms and ) the ~.mizatian d the mi~oe~vironmental con~tion$ by one d the baetez'isl pa#, so that the other might persist. exampl~ for each of these m~banisms imv~ been provided by experimental ttudies i~stigating e.co!l-b.p~flls synergistic in~ra~ons. byproducts ofg.coli metabolim l~¢ovide essential short ebath fatty acids £~ optimal b,frosili~ ga'owth. fm-ther, oxygen ¢ons~tmption by kcelt lowers oxygen tension end redox potantial to levels eomlucive to b#a#lts gro~h. coawr~ely, b,~agtlis rolea~s proteases and fatty acids wl~¢h impair pl'tsgocy~¢ ~lt rmctlon tnd permit f-..¢oli proliferation and expression of its intrinsic virulent. in summaxy, interactions among the separate microbial cemponents of mixed infections heighten the overall virttienee of these lafectiot~, this knowledge provides ~r rationale for targetting of antibiotic therapy against the knowa eantributors of these synergistic pro~¢sses, intraabdominal abscess formation and the macrophage william g. cheadle, m.d., department of surgery, university of louisville school of medicine, louisville, ky inflammation of the peritoneal cavity following bacterial contamination has been classified into primary, secondary and tertiary, the last two relating to bacteria originating from the gastrointestinal lumen. the natural history of such infection is either resolution without clinical sequelae, which is uncommon, abscess formation, or generalized peritonitis, which occurs as a result of failure of peritoneal host defenses. early clearance of microorganisms by peritoneal fluid circulation and filtration througti subdiaphragmatic lymphatics into the thoracic duct and systemic circulation occurs as well. simultaneously peritoneal macrophages and the omentum approach the area of inflammation and lead to neutrophil influx and abscess formation adjacent to the affected viscus. we have found a shift in peritoneal macrophage function from antigen presentation to proinflarnmatory cytokine production that occurs early after experimental peritonitis produced by cecal ligation and puncture. this is also reflected by reduced class ii histocompatibility antigen expression on peripheral blood mononuclear cells and peritoneal macrophages. this is accempauied by an influx of both neutrophils and macrophages into the peritoneum and subsequent abscess formation. interestingly, there is little serum endotoxin or tnf seen in this model despite tnf mrna expression in peritoneal macrophages. we believe this model is more clinically relevant than other models of endotoxemia or bacteremia in which different patterns of cytokine expression are seen. newer agents aimed at reduction of systemic manifestations of sepsis originating from intra-abdominal infection such as monoclonal antibodies against cytokines or il- receptor antagonists may need to be directed against remote organ macrophage populations while preserving peritoneal macrophage function. inflammation is a complex process involving microcirculatory changes, extravasation of fluid and a cellular influx in the affected body area. in our communication, we will only consider the regulation of the cellular infiltrate which plays a major role in the defense of the peritoneum against microbial invasion. until recently, it was thought that the influx of leukocytes in the abdomen was induced by bacterial products, local humeral factors and secretions of resident macrophages. there is now increasing evidence that this view is too simplistic. many other cell types present in the abdominal cavity or composing the peritoneal membrane (mast-cells, mesothelial cells, fibroblasts) are able to release or secrete vasoactive or chemotactic substances such as histamine, prostagtandines, or cytokines. they are most likely to play a role in the regulation of intraperitoneal inflammatory reactions. the emigration of leukocytes towards the abdominal cavity is also modulated by a previous contact with gram negative bacteria. in the rat, this intriguing phenomenon is long lasting, cannot be transferred by serum and seems independent from t lymphocytes. the clinical relevance of these various regulating mechanisms has still to be determined. kinnaert paul, h pital erasme, route de lennik , bruxelles belgium generalized response in secondary peritonitis the clinical course of an intraabdominal infection may depend on a variety of variables including the capacity of host defense mechanisms and the degree of the inflammatory response. if local defense mechanisms fail to restrict the inflammation to the abdominal cavity a generalized inflammatory reponse will result. in a first stage generalized signs of a local inflammation become detectable whereas the second stage comprises the overwhelming systemic inflammatory response. the extent of this systemic response determines the outcome. sometimes it may appear to be unrelated to the severity of the intraperitoneal findings. the activation of plasma systems and cellular elements leads to a fast release of cytokines, inflammatory mediators and other substances. these parameters precisely reflect the degree of the generalized response. inflammation of the peritoneum causes significant morbidity. objektives: to test the hypothesis that peritoneal mesothelial cells play a role in regulating inflammatory responses within the peritoneal cavity, we examined neutrophil-chemotactic activity (interleukin ) and monocyte-chemotactic cytokine (mcp) release by sytokine-etimulated mesothelial cells. confluent human peritoneal mesothelial cells were exposed to varying concentrations of phorbolmyristate-acetate (pma) and the cytokines tumorneerosis factor a (tnf a) and interleukin i~ (il-i~). the supernatant was examined for il- by elisa and for mcp by investigating the ehemotactic activity for isolated human monocytes. mesothelial cells express low levels of il and monocyte chemotactic activity when cultured. these activies were significantly increased ( -fold) after stimulation with either tnf a or il-i~. additionally macrophage inflammatory protein was detected. these observations provide a probably important mechanism whereby peritoneal mesothelial cells respond to imflammatory stimuli released during peritonitis and how leucocyte recruitment by liberation of chemotactic cytokines is regulated. the perioperative course of lps, tnfa and il- in patients with bacteriologic proven abdominal infection (intraabdominal abscess , diffuse peritonitis , pancreatic necrosis , pancreatic abscess ) was followed prospectively and evaluated for possible correlation with septic state and organ function. methods: patients were studied in a to hours period during their first surgical intervention because of intraabdominal infection. all were monitored for their cardiovascular, respiratory, hepatic and renal function. plasma samples for lps. tnfa and il- determination were drawn preoperatively, intraoperatively, and until h postoperatively in regular intervals (min /pat), results: preoperative apache ii was in median (rain , max ). patients fulfilled the criteria of sirs. of them were in septic shock.there was a significant correlation between preoperative tnfa and apache ii (p= , i, spearman coefficient). preoperative cardiovascular (systol. rr< mmhg) and respiratory (pao < mm hg) dysfunction were associated with significantly elevated tnfa (cardial: p= , i, wilcoxon; pulmonal: p= , ) and il- (cardial: p= , ; pulmonal: p= . ) overall, lps, tnfa and il- values varied considerably during the observation period. however, tnfa was markedly higher in patients with sirs and septic shock (group a: n= i , mean pg/ml) than in those who did not fulfill these criteria (group b; n= , mean pg/ml; p= , i, wilcoxon). il- was significantly higher in group a (mean pg/ml) than in group b (mean pg/ml; p= , o i wilcoxon). conclusion: perioperative tnfa and il- were shown to correlate significantly with preoperative organ function, apache ii and the severity of sepsis. these results could help to define patients that might benefit from further therapeutic strategies, e.g. antibody administration. department of surgery, university vienna, akh wien, wahringer gurtel - , wien. aim of the study: the purpose of this pilot study was to establish and to prove a standardized reproducible animal model of intraperitoneal sepsis induced by e.coli-endotoxinaemia in lew.lw-rats in order to investigate early immunoserological responses to find a mediator based evaluating system of peritonitis sepsis. materials and methods: in lew. lw-rats, diffuse peritonitis was induced by intraperitoneal injection of a mixture of e.coli (khu +) and autogenous haemoglobin solution. in the control animal group (n= ) an intraperitoneally injection of physiological saline solution was done. blood samples were obtained by heart puncture after hours. stastistieal calculations were performed on a personal computer with the spss programm vers. . (correlation with pearson's r, mann-whitney-u-test, descriptives statistics, discriminant analysis). results: in contrast to the sham treated rats, the peritonitis animals showed significant differences in the concentrations of endotoxin, interferon-gamma (wn-y), the pteridin derivate biopterin and serum pla -activities [endotoxin range from . eu/i, sd= . to . eu/ , sd- . (p < ), ifn-¥ levels, range from . pg/ml, sd- . , to pg/ml, sd= (p < . ), circulating pla -activities range from . , sd= . to . u/ , sd= . (p < . ) and biopterin range from . nmol/l sd= . to . nmol/l, sd= . (p < . )]. for the peritonitis group we found strong correlations between the degree of endotoxinaemia to elevated levels of ifn-'~ (rp = . , p < . ) and bioptefin synthesis (rv= . , p < . ). the increase of ifn-t levels was correlated to the regulatory synthesis of biopterin (r = p < . .. p • , . . ) and to the pla -actwtues (rp = . , p < . ). the biopterin synthes~s correlates slightly with the pla -actn,ities (rp= : . ; p < . ). using the para, meters of endotoxin, ifn-y levels, biopterin and the pla~ -activities only, the statistical procedure of the linear discriminant analysis makes it possible, to distinguish between non-septic animals and septic animals correctly at a rate of %. anaerobes were found in . %, anaerobes were isolated in . %. there were aerobic and anaerobic associations in . % and microflora was not found in . % of the cases. express method of anaerobes discovering let to receive information on - days early than in generally accepted nethods. intraaotal transfusion of oxygenate blood and laser irradiation of blood reduces the duration of anaerobic sow, disminishes intoxication and accelerate the patients recovery. patients with abdominal sepsis are subject to long periods of hospitalization and high associated morbidity and mortality rates. this category of patients is thus consuming extensive facilities and costs. as the age-related outcome of abdominal sepsis is not fully known, the aim of the present study was to investigate abdominal sepsis in the elderly. out of patients with abdominal sepsis treated at the surgical intensive care unit during a -year period, ( %) had an age of years or more. were women and were men, a sex distribution not differing with patients younger than years. the patients were scored according to apache ii and septic severity score (sss) upon arrival to the intensive care unit. bacterial cultures, the occurrence of organ failure, hospitalization and outcome was noted. in median two operations were performed for both "younger and elderly" patients. the median time of hospitalization in the elderly was (- ) days including in median days in the icu. figures in patients less than years of age were comparable ( (- ) days out of which in median days in the icu). apache ii and sss-scores did not significantly differ ( . vs and . vs . , respectively), between the groups. neither did the incidence of organ failure differ ( / vs / ). however, the incidence of multiple organ failure was significantly lower in elderly patients ( / vs / (p < . )). the mortality rate, however, did not differ between the groups ( / vs / ). in conclusion, severe abdominal sepsis in the elderly was not associated with an increase in mortality, incidence of organ failure or hospital stay. with the help of light transmissional scanning electron microscopy morphology of erythrosytes of peripheric blood was studied in patients with different stages of diffuse peritonitis before and after intravascu!ar irradiation of blood with heliun-neon laser. peritoneal morphology was investigated in patients who died from peritonitis, it was established that in all phases of peritonitis occured stomatocytoric and echinocytoric transformation of erythrocytes which progressed simultaneously with increase of intoxication. it combined with strongly pronounced vessels variability of microcirculatory peritoneal bed which displaied by erythrocytes aggregation, stasis and microtrombogenesis. in intravascular laser irradiation of blood number of erythrocytes which underwent to stomatocytoric and echinooytorie transformation was lower than in patients without laser irradiation. it indicated that the intravascular irradiation of blood with helium-neon laser can prevent development of severe alterations of rheological property of blood and consequently variability of microcirlatory peritoneal bed in patients with diffuse peritonitis. abdominal sepsis is still associated with high morbidity and mortality rates, frequenfly caused by multiple organ failure. it has been reported that changes in capillary permeability play a role in the pathogenesis of multiple organ failure. the present study aimed at evaluating the influence of intraabdominal sepsis induced by cekal ligation and puncture on capillary permeability in multiple organs and tissues. adult male sprague-dawley rats were subjected to laparotomy with separation of the cekum (sham operation) or induction of intraabdominal sepsis by cekal ligation and puneatre (n-- in each group). at , , , , and hours (n= /timepoint), the animals were evaluated concerning mortality and capillary permeability as determined by the passage of : i-labelled albumin from capillaries to the peritoneum, the proximal and distal small intestine, cekum, colon, spleen, kidneys, lungs. the mortality rate in rats with intraabdominal sepsis was % both at and hours. capillary permeability in the peritoneum, cekum, colon and kidneys significantly increased from hours and on in rats with intraabdominal sepsis. in septic animals, capillary permeability in the lungs and spleen increased from hours and on and in the proximal and distal small intestine from hours and on. different types of alterations in capillary permeability seem to appear: ) a temporary short increase e.g. in the proximal small intestine and spleen; ) a temporary longer increase e.g. in the colon and kidneys; ) a persisting increase e.g. in the peritoneum, cekum, distal small intestine and lungs. we conclude that experimentally induced intraabdominal sepsis induces early alterations in capillary permeability in multiple organs and tissues. such changes may contribute to explain the development of sepsis-induced multiple organ failure. despite a number of significant advances in the care of burn and non-burn traumatic injury, infection and sepsis remain major causes of morbidity and mortality. the severe immunosuppresslon often seen in patients with severe trauma or large burns may predispose these patients to life threatening infections. included among the many immune alterations are changes in the functional capabilities of neutrophlls (pmns). we have examined the expression of the p integrins (cd l a, b,c/cd ), and the fc'?r (cd , cd , and cd ), as well as several functional parameters, on pmns from thermal and non-thermal traumatic injury, pmns were obtained from patients sustaining severe trauma (initial apache ii score > ) or thermal injury (> ~ total body surface area, % full thickness), and healthy controls. the expression of cd b and c and to a lesser degree cdi a was significantly reduced on pmns. the expression of cd and cd but not cd was also significantly reduced. pmns displaying this reduction in receptor expression have a significantly reduced ability to phagocytose bacteria and undergo the oxidative metabolic burst response. thermal and traumatic injury result in global reduction in the expression of integrins and for which may lead to decreased functional capabilities, these abnormalities may in turn account at least in part for the increased rate of infection in these patlems, institute, dept. of surgery, ~ ethesda ave, cincinnalt, oh, usa, - s b, antibiotic-phagocytic cell interactions: their effect on endotoxin release. c g c-emmet , dep[baeteriolog.z, univer_sitv of glasgow, scotlan~_d increasingly it is recognised that pathogenic bacteria are capable of surviving intracellularly within phagocytic cells in addition to their capacity to produce disease whilst in the extracellular milieu. as well as providing protection from certain antibiotics which fail to penetrate the phagocyte, such intraceltular bacteria may be transported from the initial site of infection to a distant more vulnerable body site wherein they may proliferate. it is also known that some antibiotics are capable of becoming concentrated within phagocytic cells mid displaying bioactivity therein. such bioactivity might be responsible for the release of endotoxia #orn gram-negative bacteria which when liberated from the celt could ~gger the cytokine cascade. anfib,.'otic-induced damage to the ultrastructure of bacteria can also occur when the target bacteria are exposed to low (sub-mic) concentrations of certain drugs. such bacteria may present quite altered surface components m host-defense cells as well as releasing biologically active ceil wall components such as endotoxin. the nature of these interactions at the cellular level as well as the consequences for the host will be discussed. new jersey medical school: umd, newark, nj a technique of physiologic state classification has been developed based on the m~itlvariable analysis of patient derived data sets of seventeen physiologic variables. these multivariable data sets obtained from critically ill patients requiring intensive care, were aormallsed by the mean and the standard deviation of recoverin~ trauma patients who were not critically ill, the resulting normalized seventeen variable sets were then clustered. seven independent data groupings were developed. the normal stress response hyperdynamic state seen post-trauma and in compensated sepsis (a stets)/ metabolic insufficiency seen in septic decompsnsation (b stste}; early (c,) and late (e ) respiratory insufficiency associated with ards; cardlogenlc dscompensation (n state); post-trauma hyvolemla without shock (r stats). the stats closest to a new patient's values allows patient classifi atlon with regard to his previous physiologic state. classifying observations f~om patients who lived or died who fell into these physiologic states enables a probability of death (p death) to be obtalned. utilizing this criteria for the staging of severity in recent trauma patients the physiologic states accurately and significantly predicted the likelihood that the patient had an increased circulating level of the eytoklnes tnf and il- . the probability of death (p death) as well as the cytoklne levels appear to be a function of the physiologic b state with the highest levels being seen in the b state of metabolic insufficiency and the c~ state of oombined respiratory and metabolic insqffioienoy characteristic of septlc ards. the increase in the magnltude of metabolic abnormalities associated with the transition from non-sepsls to septic a, septic b, or septic c z states was associated with an increasing probability of death (p denth)(mean a state =. , mean b state = . , mean ~ state = . ). the accuraay of this estimate was prospectively analyzed in this group of m~itlple patients of whom % had sepsis and % had ssptlo ards. the survivors had a mean p death of . and the deaths had a mean p death of . . the severity of post-trauma sepsis can be quantified by probability analysis and stra~ifie~ by physiologic state. serologic tests have not been extensively tes'~ed in surgical patients but seem to be of limited value. we use nystatin as the main form of chemoprophyhxis. patients "~'ith signs of infection who do not rapidly improve with antibacterial therapy are candidates for anti-funsal therapy, amphoteradn b remains the first llne of therapy although combination therapy '~'ith flueonazole is use;l with increasing freque~;c)', the recovery of c~dida from an antra-abdominal site represents a challenging problem, anti~ngal therapy in such patients depends on the underlying disease, the nature of the infected material and overall patient risk. role of neural stimuli and pain principles and practice of anesthesiology effect of combined prednisolone, epidural analgesia and indomethacin on the systemic response after colonic surgery arginine: biochemistry, physiology and therapeutic irnplications immunosfimulatory effects of arginine in normal and injured rats arginine stimulates lymphocyte immune response in heahhy humans rote of arginine in trauma, sepsis and immunity arginine enhances wound healing in humans if labrecque t, gv campion t, and the rhll-lra phase i//sepsis syndrome study group the cleveland clinic foundation a murine-anti-human tnf-monoclonal antibody known as cb was the first anti-tnf mab which was studied in a phase ii multinational trial in the treatment of patients with severe sepsis.this was an open-label, dose-escalation trial consisting of patients who were enrolled into one of four treatment groups: ( ) . mg/kg of anti-tnf mab, ( ) . mg/kg, ( ) mg/kg or ( ) . mg/kg at study entry and the second dose hours later. the small sample size in each group (n= ) precludes detailed statistical inference in this study. nonetheless, a considerable amount of useful information was obtained from this investigation. irst, this study demonstrated the clinical feasibility of specific anticytoldne therapy in septic patients. second, the measurement systemic levels of tnf proved to be an elusive target; interleukin- may prove to be a more useful indicator of cytokine activation. third, immunologic reactions including tnf: anti-tnf mab immune complexes and human anti-routine antibodies were frequently found in these patients. despite their apparent lack of overt toxicity in this study, these immunologic reactions may complicate this form of anticytokine therapy. additionally, the potential benefits of anti-tnf mab therapy occur within the first hours of therapeutic administration in these septic patients. infecting organisms differ in their potential to induce tnf in vitro and these differences correlate with circulating tnf levels observed in septic patients. rapid methods to define those patients most likely to respond to anticytokine therapy are needed to determine the ultimate therapeutic potential of these agents in clinical medicine. wherry, j., abraham e., wunderink r., silverman h., perl t., nasraway s., levy h., bone r., wenzel r., balk r., allred r., pennington j. and the tnfa mab sepsis study group.tnfa mab (bay x ) is a murine monoclonal antibody raised against human tumor necrosis factor. tnf~ mab has been shown to reduce morbidity and mortality in animal models of septic shock and has been safely administered to septic and non septic patients.to evaluate the efficacy and safety of tnf~ mab in patients with sepsis syndrome, a prospective, multicentered, double-blind, placebo-controlled trial was conducted in hospitals in north america. patients were prospectively stratified into shock or nonshock groups and then randomized to receive a single intravenous infusion either of mg/kg tnf~ mab, . mg/kg tnf~ mab or placebo ( . % human albumin).patients received standard aggressive medical/surgical care during the day post dosing period.the three treatment arms were well balanced with respect to demographics, apache ii score and other parameters. for all infused sepsis syndrome patients, those who received tnf~ mab had slightly reduced day all cause mortality compared to placebo. among shock patients there was a more pronounced trend towards efficacy at day post dosing with lower mortality rates in both active treatment arms. among nonshock patients tn~ mab did not appear beneficial. the initial clinical experience with a chimeric anti-tnf monoclonal antibody, ca , was undertaken in septic patients. the objectives of the study were to determine the safety, pharmacokinetics and effects on cytokine levels of ca . as a single infusion or in combination with ha- a in septic patients. the study was conducted with the intent to progress to an efficacy trial based on the information collected.the trial was conducted in three stages. stage was an open label trial in which groups of patients each with the clinical diagnosis of sepsis received ascending doses of ca ( . , , , mg/kg). stage was a randomized, double blind study in which patients received a single dose of ha- a ( mg) and placebo or one of doses of ca ( , , mg/kg). stage was a randomized, double blind study in which patients received a single dose of placebo or one of doses of ca ( . , , mg/kg). in addition to usual laboratory tests, the following assays were performed: chimeric anti-tnf concentration, anti-chimeric antibody, endotoxin, tnf, il- , and il- levels.a total of patients were enrolled from clinical sites ( in stage , in stage and in stage ). primary analyses were performed on patients in stage and . there were patients who received ca exclusively and patients received placebo. administration of ca was well tolerated at doses up to mg/kg. no patient discontinued treatment due to adverse events. human anti-chimeric antibody responses were positive in % ( / ) of evaluated patients. mean cma × and auc increased proportionally with increasing doses of ca . the mean half-life was - hrs ( - hrs). a dose related decrease in tnf concentration was observed hr post infusion of ca . tnf is considered to be one of the central endogenous mediators for the inili'ation of the pathophysiological changes in patients with sepsis and septic shock. high tnf levels were demonstrated to correlate with patient outcome. blocking or neutralising tnf with specific antibodies was effective in preventing death in some animal modets of sepsis. in a placebo controlled prospective randomized study we tested the mur~ne derived antibody mak f. it is a f(ab') fragment. the fragment rather the complete antibody was selected in order to reduce the potential immunogenicity and to facilitate tissue penetration. patients with severe sepsis or septic shdck were enrolied in the study, three different doses of mak f or placebo were administered ( , ; , and i mg/kg) over a perid of hours in random order. the patients were evaluated for side effects, hemodynamics, organ dysfunction, cytokines (il , il and tnf), and outcome. at this time only an interim analysis of patients is available i indicating that mak f in all dosage groups resulted in a decrease in il . this contrasted to a further in crease of il in the placebo patients. no serious side effects have been reported so far. a more detailed analysis on all patients in the study will be presented and discussed.$ s staubach,k.h., otto, v., kooistra,a,, rosenfeid,j.a., bruch, h.p., univ. lfibeek, germany once endotoxinemia occurs in sepsis a vieieus cycle with translocation of et can be established. increasing the clearance capacity for et would therapeutically be the ulimate aim. we developed a new et on-line adsorption (ad) system in whole blood by means of polymyxin b (pb) coupled eovalently to a matrix (acrylic particles) via a atom-chain spacer. the detoxification capacity was ug[et/ml column material. the biocompatbility resulted in ~ platelet recovery. the column contained ml of admaterial and was sterilized by high steam autoclave, anticoagulation was achieved by heparine . iu/h in the inflowline after bolus injection of . iu. hp was performed on pigs at a rate of ml/min by means of a roller-pump until the animals succumbed (h). animals served as controls (c). serum et levels rose from . pg/ml to , pg/ml after hours in the c and from . pg/ml only to pg/ml in the h group after hours whieh was highly significant. survival time could be extrended from to min. results are listed in the following l. blinzler, p. zaar, m. leier, r. b( rger, d. heuser clinic of anaesthesiology , city hospital nuremberg, germany sepsis and multiple organ failure (mof) are still related with poor prognosis inspire of pharmacological and technical progress. impressed by revealing reports about blood purification the continuous veno-venous hemofiltration (cvvh) was used as supporting treatment beside the critical cam basic therapy of mof. from to consecutive patients were treated by cwh. mof was caused by hemolrhagic-traumatic noxa in °, and by septic-toxic event in %. all patients required mechanical ventilation (fio > , ) . ° showed hyperdynamic shock. % had renal and % hepatic failure. medium appache ii score amounted to , points. cvvh was performed in postdilution mode with a polyamide membrane (fh ) and high volume exchange ( l/die). anticoagulation was done with heparin. hemofiltration in mof was installed, when critical cam basic therapy including adequate respiratory and hemodynamic management, pamnteral nutrition, antibiotic treatment, etc., failed to stabilize organ functions. during consequent application of cvvh most of these patients showed improvement of their clinical course. pulmonary stabilization was seen in %, hemodynamic in % and renal in % of the cases. % of the patients survived and were discharged from hospital. of non-survivors ( %) died because of fatal mof within h after admission to icu. patients with early application of cvvh in mof showed a better survival rate.mediators of mof, i.e. products of the complement cascade measured in blood and nitrafiltrate by elisa, were partially removed by cvvh. the testing ultrafiltrate by hplc demonstrated decreasing spikes ofpolypeptides during hemofiltration. mof seems to be generated by cascade-activation of immune competent cells and plasmatic mediators (e.g. bmdykinin, eicosanoides, cytokines, anaphylatoxins, etc.). therapeutic approaches aim to inactivate or eliminate single substances. cwh with high-flux membranes in combination with high-volume exchange allows elimination of many mediators with different molecular weight and therefore may contribute to improve the prognosis of mof. other significant advantages of this teqalnique like adequate nutrition, optimized fluid balance and control of body temperature should not be negicctod. introductioni pseudomonas (p) aeruginosa has to be considered an important pathogen of nosocomial pneumonia and septic organ failure. the lung seems to be the predominant target organ for the pore-forming p. aeruginosa cytotoxin, thus inducing microvascular injury. with respect to therapeutical consequences, the potential protective effects of paf-antagonist (web ), cyelooxygenase inhibitor (diclofenac) and specific and unspecific antibodies on cytotoxin-induced pulmonary vascular reaction and mediator release were studied in the isolated perfused rabbit lung. methods: cytotoxin ( p_g/ml) was administered into the perfusion fluid in all groups, either in the absence of inhibitors (n= ), or after pretreatment with web ( xl -gm, n= ), or diclofenac ( #g/ml, n- ). furthermore, the application of specific antitoxin (mg/ml, n= ) was tested in comparison with the unspecific immunoglobulins (venimmun®, behring, . mg/ml) (n= ) and the combination of immunogiobulins, web and diclofenac (n= ). six experiments without toxin served as controls. the arterial pressure mad the weight gain as an indicator of edema formation were continuously monitored during the three hour peffusion phase. arachidonic-ucid metabolites, as well as lactate dehydrogenase (ldh) and k + concentrations were determined at rain intervals. results: cytotoxin caused a gradual increase in pulmonary arterial pressure, reaching a maximum value of . times higher than the control, starting after min and a delayed onset of edema formation resulting in a mean weight gain of g after min. this was paralleled by a significant increase in prostacyclin generation and a continuous release of k + and ldh. thromboxane synthesis exceeded about times that of controls in the toxin treated lungs. pretreatment with web or diclofenac significantly attenuated the pressure response and edema formation evoked by cytotoxin. the addition of the unspecific immunognbulin preparation alone induced a transient pressure increase within the first minutes, but mean values remained below those of the cytotoxin group in the continuing observation period. mmost complete inhibition of the pressure reaction, the edema formation and the metabolic alterations was achieved mainly by the combination of immunoglobulin, web and diclofenac and to lesser extend by the specific toxin antibody. conclusion: the current results point towards the crucial role of paf and aa-metabolites as mediators of cytotoxin induced microvascular injury. the systemic or local application of cytotoxin antibodies or even unspecific immunoglobolins in combination with paf-antagonist and diclofenac appears to be a promising therapeutic approach in the case of infection with cytotoxin-preducing strains. cytokines have long been shown to be of particular importance in the metabolic derangements occurring in lps-induced shock. recent studies strongly imply the involvement of platelet aggregating factor (paf) in the pathogenesis of gram-negative bacterial sepsis. an autocatalytic feedback network has been postulated to exist between paf and tumor necrosis factor (tnf), a key cytokine involved in septic metabolic cascade, leading to an uncontrolled amplification of inflammatory mediator release. we have previously shown that st ( -n,n,n trimethylammonium-(r)- -isovaleroyloxy-butanoic acid z- -( -chlorphtalidiliden) ethyl ester bromide) was quite effective in inhibiting the "in vitro" binding of h-paf (ki= . x - m) to rabbit platelets. the present study shows that pretreatment of c bl/ mice with st , administered by different routes, dose-dependently and significantly reduces the lethality induced by endotoxin (e.coli :b injected at mg/kg intraperitoneally). very interestingly, st administered at the same doses as above (i.e. . , . , and mg/kg body weight) results to be significantly effective in reducing the endotoxin-induced release of serum tnf. the reported dual activity of st (i.e. paf antagonism and decreased circulating tnf levels) may turn out to be greatly beneficial, in combination with current therapies, in the treatment of diseases that involve overproduction of tnf and paf such as septic shock. introduction: recently, we reported that prophylactic whole body hyperthermia ( . °c) induces heat shock protein ('asp) and increases smvival - fold in a mouse endotoxin model (am. j. physiol. in press). other investigators reported that prophylactic pharmacologic induction of hsp- by sodium arsenite improves survival in a rat sepsis model (abstract a am. rev. resp. dis. vol. , ) . the effects of heat are complex and in addition to formation of lisp- include release of cytokines, changes in cellular ph etc. thus, the protective mechanisms of heat may differ from those due to pharmacologically induced . the purpose of this study was to compare the protection of heat vs the protection of pharmacologically induced hsp- in a mouse endotoxin model to determine if different protective mechanisms were likely to be involved.. i%'lethods: both sodium arsenite ( mg/kg) and ethanol ( ~ of % ethanol) caused marked induction of hsp- in lung, gut, kidney, and liver, which was comparable to heat-induced hsp- . female nd mice weighing - gms were pretreated with arsenite or alcohol hours prior to challenge with escherichia coli endotoxin (-ld ) and survival was compared to control mice. results: survival at hrs. for arsenite treated and alcohol treated mice was % and % respectively and was statistically different from the % survival for control mice. (p< . ) (n= mice per group). however, at days post endotoxin, there were no differences in survival in the groups, i.e., ~ % survival for all groups. in contrast, the protective effect of hyperthermia remains present at days, i.e., ~ % survival vs % survival control. conclusion: the protective effect of heat is probably due to other factors such as the effect of hyperthermia to release il-lc~ and is not due solely to hsp- formation. it was the aim of the study to examine whether bacteria play a causative role in the pathogenesis of anastomotic insufficiency following gastrectomy in man.the study was carried out in form of a prospective, randemised, double-blind, multicenter trial. primary endpoints were the rate of anastomotic insufficiencies, infectious-and uncomplicated postoperative courses. all pat. received a periop, i.v. prophylaxis with cefotaxim. identical numbered vial either contained placebo or polymyxin b, tobramycin, vancomycin and amphotericin b . the vials were administered x per day from the day be ~ fore the operation until the th postop, day. insufficiencies were detected by gastrographin swallow and recorded by x-ray on day postop.. evaluation was carried out on an "intention to treat'basis. statistical analysis was done with the pearson's chi square and fisher's exact tests~ results: interim analysis was carried out in / after pat. had been recruited. along with a significant reduction of s.aureus and enterobacteria there was a reduction in the rate of anastomotic insufficiency of the esophago-jejunostomy from . % in the placebo-group to . % in the treatment group. the difference was not yet significant. the rate of nosocomial infections (e.g. respiratory tract infection and uti) were significantly reduced from . % in the placebo-group to . % in the treatment-group (p ~ . ;fisher's exact test). in march final results with more than patients will be presented for the first time. (= po < mm hg, b s-creatinin > mg%). respiratory insufficiency was the most frequent systemic complication followed by sepsis and respiratory insufficiency. etiology of pancreatitis and initial serum increase of pancreatic enzymes predicted neither complications nor outcome. only of deaths occurred during the st week, all other deaths occurred late (after - weeks), generally as the consequence of septic complications and multi-organ failure. high levels of crp were correlated with a compliacted course and a fatal outcome. although same cytokines (e.g. -- ) were found increased in severe disease, the predictive value of these markers was not better than the combination of ctinical scores (ranson, imrie, apache ii) with gt or crp. conclusions: intensive care medicine can often control the inital shock situation in severe pancreatitis. thus. only % of deaths today occur eady in the course of the disease, whereas this percentage varied between - % just years ago. nowadays, most deaths are caused by late septic complications and multi-organ failure. ranson-and ct-scores as well as serum crp predict a course with systemic complications; they are less helpful for prediction of sepsis and late mortality. it is doubtful whether measurements of cytokines will help to better predict the late outcome. as yet, only careful and continuous monitoring of patients (e.g. by apache scores) may help to early identify those who develop septic complications and multi-organ failure. the classic description of severe acute pancreatitis has hinged upon the release of large volumes of activated enzymes into the peritoneal cavity and thertce the lymphatics and blood stream. these activated enzymes escape from the pancreas due to disruption of cells with associated ischaemia and occasional infarction of tissue. for to years it has been postulated that the bocly's defence system to activated pancreatic enzymes required supplementation iu the form of anti-protease support either in the vascular space or in the peritoneal cavity. all controlled studies have shown that this is either impracftcal or unnecessary.hore recently release of a large number of cytokines from monocytes, macrophages and neutrophils have been considered to be harmful to the body and various agent~ which oppose the action of tnf alpha, paf and similar cytokines are being examined in experimental anim~is and certain clinical trials, it has clearly been shown that higher levels of cytokines are released in the patients with objectively graded severe acute pancreatitis than in those with milder disease. we now seem to be moving into an exciting phase of potentially beneficial therapy in acute pancreatitis which has had no specific effective therapy through studies utilising aprotinin, gabexate mesilate and fresh frozen plasma. inflammation cascades may play a role in the pathogenesis of acute pancreatitis. to evaluate the status of the cellular immune system we examined serum concentrations of immune activation markers in patients with acute pancreatitis ( males, females; median age: years, range: - years). concentrations of neopterin, serum soluble tumor necrosis factor receptor (stnf-r) and serum soluble intercellular adhesion molecule type (slcam- ) were determined using immunoassays (henning, bender, t cell sciences). / had increased concentrations of stnf-r compared to the th percentile obtained in healthy controls (> . ng/ml), and / patients had increased neopterin (> . nmol/i), / presented with elevated slcam- (> u/i). all patients with increased neopterin also had increased stnf-r, patients had concentrations of all three markers outside the normal range. there existed a significant correlation between neopterin and stnf-r (rs = . , p < . ). weak associations between age and stnf-r (rs= . , p=o. ) or neopterin (rs= . , p = . ) were also found. our results demonstrate activation of the cell-mediated immune system taking place in a sub-group of patients with acute pancreatitis. the finding of increased neopterin and stnf-r levels implies that activated monocytes/macrophages are involved in the pathogenesis of the disease. further data are necessary to evaluate potential associations between changes of marker concent-rations and the course of the disease. pancreatic injury after heart surgery was reported as soon as ( , ) and characterized by increased serum or urine amylase levels (in about % of patients) in the fi~t postoperafi.'ve days. this pancreatic injury, which sometimes led to acute pancreatitis, was atreaay at~buted to inappropriate perfusion of this organ. in the ffs, studies were published dealing with pancreatic suffering alter heart surgery, in large series of patients, concluding ~n~at panc~a~c injury (with a low incidence of pancreatifis) is more common than previously recognized and is a potential source of complication after camliac surgery ( , , ) . in a recent study ( ), evidence of pancreatic cellular injury was found in out of patients undergoing cardiac surgery, with out of these patients presenting abdominal signs or symptoms and developing severe pancreafitis. this injury was associated w~th preoperative renal insufficiency, valve surgery, ~..stoperalive hytxxension, calcium administered periopuratively and length of bypass. we studied patients submitted to cardiopulmunary bypass (cpb) for heart surgery and used the measurement of un:~sin, pancreatic iso-amylase and lipase in plasma for biochemical characterization of pancreatic cellular injury. blood samples were obtained before surgery, directly aller surgery (return to inte~ve care unit), hours alter surgery and in the folfowing days alter surgery (days , , , and ). computed tomography scan of pancreas was performed in patients presenting hi~ levels of amylase on day . we measured abnormal levels of trypsin and pancteatic iso-amylase in % of patients and observed simultaneous releases of these enzymes, the fi,'st one in the hours after surgery and the second more intense from day and pa~icularly on day after smgery. this second release was concomitant with abnormal levels of llpase. these biochemical observations were accompanied by radiological and clinical signs of pancreatic injury in about % of our patients : pancrealic abnormalities were revealed by scan in patients and acute pancreatitis in i patient. more pronounced pancreatic suffering was observed in patients undergoing valve replacement than in patients undergoing coronam-anrtic bypass grafm~g. analysis of trypsin and pare're, tic so-amylase are sw.cific of pancreatic cellular injury and their simultaneous ir~rease in plasma alter cpb in our padents confirms the presence of an exocrine pancreatic injury. the presence of a simultaneous peak of lipase mcaezse~ the specificity of overt pancreatic injtu diagnosis. the precise cause of th/s injury could he related to hypoperfnsion leading to ischemic injury of foe splancbnic area, pancreas being largely sensible to hypoperfnsion ( ). this hypoperfosion could he responsible for the ftmt release of pancrealac enzymes observed in our patients and would contribute to the deterioration of other organs leading to an inflammatory reaction developing in the following days and responsible for the second release of pancreatic enzymes observed in our patients. patients with necrotizing pancreatitis show a heigh rate of pulmonary, renal and septic complications, whereas the course in acute interstitial pancreatitis is generally very mild. we have prospectively analysed the value of endotoxin, interleukin- (il- ) and transferrin in compare with c-reactive protein(crp) for the early assessment of the severity of acute pancreatitis. patients aud methods: the values of endotoxin(measured by limulus-lysate-test), ii- (elisa), transferrin and crp (nephelometry) were analysed daily along the first i days of hospitalisation by patients with acute pancreatitis admitted to our hospital from / to / . it was judged whether the patients have either interstitial (aip) (n= ) or necrotizing (anp) (n=lg) pancreatitis. patients with anp have died during the course of pancreatitis (mortality= . %). results: -severity o~ pancreatitis: signifcant differences (p<o. , mann-whitney test) could be calculated between aip and anp for endotoxin, il- , transferrin and crp during the i days of monitoring. -mortality: except of il- on days , , and after ad-mission there were no significant differences between the values of analysed parameters in patients who survived and those values in patients who died. -organ failure: the patients with pulmonary, renal or multiorgan failure have significantly heigher values of endotoxin, il- and crp along the i days of monitoring in compare with those patients without an organ failure. conclusions: endotoxin, il- and transferrin, an important endotoxin carrier, are promising parameters to differentiate between the severe and mild form of pancreatitis comparable with crp. this parameters may be helpfull in the early clinical assessment of patients with acute panereatitis. the determination of cytokines may elucidate the pathophysiologic mechanisms that produce early systemic complications in acute interstitial (i) or necrotizing (n) pancreatitis (ap). the appearence of respiratory insufficiency (ri) is used to appraise the early systemic complications and is compared to the results of cytokine blood levels. in a prospective clinical trial from / - / , patients with ap were recruited and blood samples taken for cytokine determination with commercial elisa-kits. the differentiation between i (n= ) and n (n=l ) ap was made through ct-scan in all and laparotomy for drainage and necrosectomy in patients. the etiology of ap was gallstones in , alcohol abuse in , hyperlipidemia in and other or unknown causes in patients.five of patients with nap died early (n=l) or of late septic complications. none died of iap.the peak of cytnkine level in the first three days of hospitalisation was used for calculation. ri was diagnosed in the first week of hospitalisation, if oxygenmask or iutubation for at least days was nescessary to treat arterial hypoxemia. for statistical analysis u-test of wilcoxon, mann and whitney was applicated.the il- concentration in blood reached up to pg/ml in the first days depending on the severity of ap and dropped to almost zero in the next days, independently of the clinical course. the differentiation of i-versus nap, using a cutoff-line of pg/ml was correct in patients ( %, sensitivity (se): %= specificity (sp): %, (z: , ). high levels of il- over pg/ml correlated well with the prognosis ifri in the first week (accuracy: %, se: %, sp: %, c~: , ). the blood levels of il- reached a maximum of pg/ml in the first days, depending on the severity of ap, and showed a correlation to the clinical course in the following days. the peak of l,- blood levels indicated correctly the severity of ap in out of patients using a cut offtevel of pglml (accuracy: %, se: %, sp: %, ~: , ). there was no correlation between cytokine blood levels and mortality, also there seemed to be no correlation between the levels of il- and il- . in the bloodsamples of five patients with i-or nap no c~-tnf was detectable.the blood levels of il- , and to a lesser extent of [l- , can predict the severity and early systemic complications of ap in men. the excessive rise of cytokines can be explained by the stimulation of immunological cells ( macrophages, lymphocytes and endothelial cells) in the course of ap. objectives: in an opossum model, ligation of the sphincter of oddi or separate ligation of the bile and the pancreatic duct together leads to severe haemorrhagic pancreatffis while single ligation of the pancreatic duct causes only an exocdne atrophy. since bo has been discussed to be a contdbutor t¢, res dysfunction, this may depdve the organism of a potent defensive mechanism thus promoting the course of pancreatitis. the present study wa,,; carded out tc develop a new method for measuring dynamic liver res func.. tion and to test the hypothesis that bo causes a res dysfunction. material & methods: opossums were randomly placed in three groups. all received a central venous line. after midline laparotomy, a specially designe tourniquet was placed around the common hepatic duct above its junction with the pancreatic duct (group a, n= ), around the pancreatic duct (group b, n= ) or around both ducts (group c, n= ). after one week, the tourniquets were closed. using a scintillation camera, the liver uptake of nanocoll, a mtcalbumin colloid with a diameter of nm, was measured before, , and days after the obstruction. the curves were analysed by a computer and two parameters (r, s) were calculated using natural logarithmic regression. as a common measure for res function, the corrected granulopexic index (a) wa,,; determined, after day , the pancreas of each opossum was searched for necrosis by morphometdc methods. results: all animals survived till day . the opossums in groups a & had a macro-and microscopic normal pancreas, while those in group c showed a severe hemorrhagic pancreatitis. the granuinpexic index showed a significan~ change to the worse starting at day in group a & b (p<o, ) and at day it~ group c (p< ,ol). at day , res function in group c was significantly worse than that in group a & b (p< , ). the regression parameter r showed no significant change in groups a & b, but a highly significant decrease in group c starting at day (p< . ), thus showing a dysfunction of the hepatic res. conclusions: obstruction of the hepatic or pancreatic duct alone does no{ result in immediate dysfunction of the hepatic res, while obstruction of beth ducts does. because only ligation of both ducts is followed by a severe hereof.. rhagic pancreatitis, res dysfunction could be an important factor in the pathogenesis of pancreatitis and resulting organ failure.logarithmic regressinrl has proven to be a valuable tool to analyse dynamic hepatic res function. (b ) in lewis rats weighing - g. there were five groups: group a (n= ) were untreated controls; group b (n= ) were given isotonic saline intraperitoneally and observed for hours; group c (n= ) ml x e. coli intraperitoneally and observed for hours; group d (n= ) were given ml x e. coli and observed for hours; group e (n= ) ml x i e. coli and observed for hours, the rats were than killed, the spleens were removed and heparinised blood taken for immunological tests. total t-ceils, helper t-ceils, suppressor t-cells, monocytes, and the interleukin receptor were determined by monoclonal antibodies, indirect immunfluorescence, and flow cytometry (facs analyser ). statistical analysis was by the t test on rank transformed data.in group b (isotonic saline) the total t-ceils increased in the blood (p= , ) and spleen (p= , )com pared with the untreated rats (group a), in the groups with peritonitis (c, d, and e) we found highly significant rises in suppressor t-cells in the blood and spleen (both p< , ) compared with the non-infected control groups a and b. this increase was significantly higher in group d than in groups c and e. as well as other alterations, we found a uniform increase in the number of t suppressor ceils in our model of acute peritonitis that was both time and dose dependent, department of surgery, university vienna, akh wien, w~hringer gurtel - , wien. d~'na~'aics of som~ l~mnunologic indices in children with abdo;~tinal shock v.z.i~ioskalenko, s.f.vesiol$,t.v.p~bina serum (iga,:~i,g, circulating imaune co!~plexes, co:apleaentary serum activity, antimesothelialvisceral and parietal antibodies) and cellular (i-l~q~hocytes, ~s/th,b-lyaphoeytes,i nlaunoleukosis to aesothelial allergens; spontaneous blast cell transformation to phytohemaaglutinin and .nesoth<-~lial ~,togen; phagooftic neutrophil activity) im:~une indices v.'ere studied in children operated v:ith symi~to'as of abdo~ainal shock. ,'~!-~ of them had appendicnlar( - ocalized jo-diffuse, -~eneralized) and -hemoperitoni-~is (dull abdominal trau;na with injayy of 'the spleen- , the spleen and liver-q), flo patients tjere operated on for co~iplete iieus (~-intussnsc.sption, -com:lissural ileus). he follo~'~ing serum factors: cireula:;ing im~aune complexes and ~).esothelial antibodies are the aost i~portant ~ar~ers of the abdominal shock course. :yith the progress of the process the indices increase irresponsive of t.l~e cha±.aeter of pathology. in case of ileus regress and hemoperitonotis an abrupt drop in ~he titer of anti~aesothelial antibodies is noted. in bacterial peritonitis a high ~iter of anti:aesothelial parietal antibodies in great dilutions ("+++","++++"-q ; ) persisted even in the period of clinical recovery. cellular factors cham'acterize dfnaaics of the abdominal shock coulees less specifically llast cell transfor~aation and phagocyue neut~ophil activit can inderectly characterize transition of shock f~orn reversible into irreversible phases. the past so years have brought a number of major advances in burn care. the initial advance was the discovery that burn victims required large volumes of resuscitation fluid in the initial hours to maintain hsmodynamic stability, this was followed hy the dsvelopmsnt of topical antimicrohlal agents to prevent end treat infections. next, was the reporting that early excision of burn wounds, with autografting of the excised wounds was possible.finally, was the identification of the importance of aggrsssive nutritional support for the hypermstabolic burn patient.these advances have markedly increaesd survival rates for given burn mimes, s~ch that burns which would previously have been considered to be aniformly fetal, are now readily survivable.thsre remain two unsolved problems in burn care, which are limiting survivability cf burn patients.the first ie ths treatment of inhalation injury. ths sscond is how to obtain coverage of ths maaaivsly burned patient, who has limited skin graft donor sites available.this ssssion will focus on the new treatments being developed to obtain permanent coverage of burn wounds.these include the use of various tcplcal growth £aotore which can speed the rate of reepithelialization of wounds, both the beneficial and dstrimental effects of using cultured karatinooyte preparations, to obtain in excess of one squats meter of a pseudoskin from a single fifteen square centimeter biopsy of unburned skin, will be discussed, finally, the used for artificlal dermal preparations will be discussed. integumentary wound healing consists of simultaneous epithelializalion and contraction. not long ago, it was thought that healing proceeds at an optimum fixed rate and could only be delayed. the recent explosion in molecular biology and recombinant gene techniques have produced information on the biological activity of compounds previously believed to be biologically inert. many current attempts to modulate the rate of wound healing center on the topical application of various growth factors produced by integumentary cells or agents designed to modulate growth factor expression or response. epidermal growth factor (egf), plateiet derived growth factor (pdgf), transforming growth factor (tgf) and fibroblast growth factor (fgf) have been demonstrated, both in vitro and in vivo, to stimulate the proliferation and diferentiation of their designated cells types. egf and pdgf have been clearly demonstraled to increase the rate of wound closure in partial and full thickness wounds. pdgf also has demonstrated efficacy in the closure of recalcitrant pressure sores, whereas tgf increases the tensile strength of incisiona[ wounds and fgf stimulates angiogenesis. it appears that epidermal keratinocyte migration during wound healing depends on integrin-mediated interactions with compounds and ceils extracellular matrix. additionally, the activities of extracellular glycoproteins such as fibronectin, fibrinogen, laminin and thrombospondin are coordinated by multiple integrins with specific distributions and binding affinities. currently, agents which incorporate specific protein sequences essential for the interaction of the glycoproteins with the cells of the extracellular matrix are being investigated. it is has been clearly established that the rate and nature of wound healing can be influenced by the application of various agents and that sufficient quantities of these agents can be manufactured. the future challenge is to develop techniques to now the objective evaluation of the clinical efficacy of these various agents and to employ the appropriate agents in a cost-effective manner. transplantation; and ) the immune status/manipulation of the host.multiple interactive variables will determine the effect of employing allogeneic cells and tissue in wound coverage design and a batter understanding of the dynamics of the wound-graft microenvironment will facilitate the dove opment of clinicauy beneficial graft composites. cadaver allograft skin (cas) is the "gold standard" for wound coverage, but has limitations including varied availability & quality, immunogenicity leading to rejection, & potential for disease transmission. our development of a temporary living skin replacement (tlsr) addresses these issues; the tlsr consists of highly screened human neonatal fibroblasts (hf) cultured in biobrane a, a bilayer dressing consisting of nylon mesh laminated to a thin silicone membrane which controls water-vapor transmission. studies in our lab indicated that "fresw ~ tlsr grafts reproducibly close full-thickness wounds in mice & perform better than bb controls. we studied wound adherence & structural/molecular properties of fresh & cryopreserved (cryo) tlsr. methods: tlsrs were prepared by seeding /co hf in bb nylon mesh in dmem. hf quickly attached to the nylon mesh & were allowed to proliferate - wks. fibroblast mitochondrial activity was assayed by mtt assay. matrix proteins were identified by immunostaining, mrnas for specific growth factors were identified by reverse-transcription polymerase chain reaction amplification, & quantitated by gel scans. bb structure was studied by scanning electron microscopy & stretching measurements pro/post cryo. grafts were cryo'd in % dmso, quick-thawed & sutured onto x cm excised full-thickness dorsal wounds on athymic mice. "fresh" grafts were similarly placed. controls grafts were cas and aeellular bb. adherence was quantitated by a device which peeled grafts from wounds at intervals following placement, using a serve motor and a strain gauge. results: postcryo, storage and subsequent thawing tile tlsrs maintained > % cell viability by the mtt assay, indicating continued mitochondrial activity, and bb structure & stretchability were maintained. multiple matrix proteins secreted and deposited in the bb nylon mesh (types l/iii collagen, decorin, fibroneetin) were identified by specific immunostaining. growth factor mrnas in the tlsrs (afgf, bfgf, kgf, tgf~,p~,) were present in - , x higher levels in fresh/cryo tlsrs than in adult hcs. grafts adhered to wounds on mice through days of followup. histologic exams on days - showed excellent vascular ingrowth and minimal inflammation. adherence of tlsrs to wounds was >cas adherence. burn wound coverage in the massively burned patient remains a difficult problem. although cultured keratinocytes have been utilized for burn wound coverage, their impact on the patient with burns greater than % total body surface area has not been spectacular, with poor graft take and unstable epithelium.current investigations have been directed toward dermal replacement beneath either very thin split-thickness autografts (stag) or utilizing cultured keratinocytes. current products include: collagen dermal replacement with thin stag (burke, et al). collagen dermal replacement with cultured keratinocytes and fibroblasts (boyce, et ai). allograft dermis with cultured keratinocytes (cnno, et al). allograft dermis with thin stag (life cell). polyglactin acid mesh and neonatal human fibroblasts with thin stag (hansbrnngh, et al).investigations regarding culture media, use of growth factors, topical nutrients and antibiotics, and melanocytes for pigmentation as well as safety and efficacy are needed before any of the current products become viable options for coverage of the massively burned patient. the~ is a growing world-wide problem with the ujc of cadaver tissues and ocgans bae, au~ of the tren~m~s~km of dilemma such a; cmutzfeldt.jukob disease and iiiv as we ] as ready availability of urdform lis~ue~. on dec~mt~r , , the fda assumed control of as tissue bar~s in the uldtod st=tea in an attempt to bflng ~s difficult problem of dise~s~ transmission under ¢onlrol. in europe, ~om¢ of the governments are consldofll~ a c~mplcte bat) on the use of cadaverlc fissu~s such as ddn, 'this |ncroam in regulation of cadavefle ~s,quct will incmar¢ the difficulty of obtain~g and dlslflbulmg them. however, thc nc~ for these tissues contlnue~ m incrcaso, we will discuss ~'l¢ solulion to this important pmbl~n: tissue engineering. tlssu~ engineering is an in~rdisdpllnary field that applies pdnclplc~ of angin~edng and die life sclcnce~ reward the development of ~olok~¢al sub~dtute,~ ih= mslom, maintain, or improve tissue function, " ssuc ongln~cdng can provide ~ho nccassary tlssuoa for wound repair ~d ibe assuranoe fl'~t the lissuos are d.ls¢~¢ free. in addition, a ds~uo-cng~ne~n~l wound covering will bo u~lvemally acceptable and evntlublc as "off g~o shell", consis~t products, them are several approaches to restating thls function in a large wound, 'l'nosc i~elud~ tmmcdiete long term coverage, short t=nn coverage, uandtl~el coverage and compost= dssu¢ coverage, "flssuo onglncrcd wound coverings that meet those vaflous ne,.cds will he r~vlowod.cllni~:sl and experimental d~la in venous ulcer, dlabctl¢ ulcers, prossur~ ulcers and bum wounds wgj be mvlcw~, a~ welt as new approacl~s u~ csrtilag¢, bone, liver and bone marrow it~suos. c oomplon, k nadirs, w press, g wetland, j fallen iv, shrtners burns institute and massachusetts general hospital, boston, ma~schusetts, usa the clinical "take" rate o? cultured epithelial autografts (cea) has been observed to increase with transplantation to allodermls, but the reasons for the improved clinical performance have not yet been defined. the aim of this study was to determine the biological impact of normal human dermis on cea differentiation and maturation, biopsies of cea transplanted to engrafted and de-opldermlzed human homograft dermis have been compared to nopsles of cea transplanted to granulation tissue in tullthickness burn wound beds on the same patient, each patient serving as hls or her own control. paired test and control biopstes from six patients have acquired from as early as one week postgrafting to as late as years postgrafting (one patient) and analyzed histopathologlcally, ultrastructurally and immunoh[stochemloally, results demonstrate more rapid normalization of differentiation markers (e,g., involucfln, fllaggrln, cytokeratln profiles) in the cea transplanted to allodermls compared to their corresponding controls by in all patients, the proliferation rate within the basal layer ot the epidermis as determined by ki- (proliferation-associated antigen) is seen to norh~altze more quickly in the cea transplanted to allodermls in every case, persistence of allodermal matrix can be dooumented in all patients by elastic tlssue-trichrome stain, allowing visualization of the dermal elastin network. the popu;atlon densities ot intraepldarmal langerhans cells are conslstently and signlflcantly higher in cea transplanted to ,allodermls, possibly reflectlng an immunologlcal reaction to the underlying allogenlc tissue. overall, these preliminary results indicate that transplantation to a normal human dermal matrix accelerates the maturation of cea-deflved epidermis, wound closure continues to be a major problem in patients who have sustained a major thermal injury, cultured epidermal autografts (cea) have been utilized extensively since when galllco et el reported theh'use in two brothers with greater than % total body surface area burn. unfortunately, cea take rate varies widely and the resultant skin coverage is often fragile and the cosmetic results are less than optimal however the overall take rate and durability of the coverase can be markedly improved by using nn allodermls base as the recipient bed. a review of cea applications performed by physicians using cultured outologens epithelium obtained from blusurfaoe teclmology, inc. shows a marked discrepancy in the results obtained utilizing different methods of wound bed preparation. tgf-b is an important modulator coordinating complex physiological events associated with growth and development. it is assumed that tgf-b is also involved in the well-coordinated process of cutaneous wound healing by regulating proliferation, differentiation, chemotaxis and matrix deposition. the purpose of our study was to analyze the spatial and temporal pattern of tgf-b expression during granulation tissue formation in patients with accidanutl surgical trauma (monotraumata mid polytraumata) and bum wounds. after debridement (day ), the full thickness wounds were covered with epigard, a synthetic dressing until day . after this time the granulated wounds were closed by transplantation of mesh graft. biopsies of the wound center were taken from patients at the beginning of surgical treatment (day ) and after , , and days. cryosections were stained with antibodies against tgf-fi s using the apaap technique and -for standard histology -with hematoxylin-eosin. for identification of the cell type expressing tgf- , double staining immunofluorescence experiments were conducted using antibodies specific for monocytes/macrophages, polymorphoanclear neutropkils and fibroblasts. the results showed a characteristic pattern of tgf-t~ distribution during wound development. tgf-fi appearence was mainly cell-associated znd the absolute and relative number of cells that were positive increased with lime. infiltrating cells and developing blood vessels were most prominently stained; epithelial and t-cells showed no immuno-reactivity. a delay of emergence for tgf-b during the time course could be seen in one patient group. this might reflect various regulation patterns depending on the type and severity of injury.( ) pharmatec gmbh, frankfurt ( ) institut fiir immonologie and serologic, heidelberg ( immune cells extravasating specifically in skin recognize and eliminate the invading antigens (bacteria, viruses, etc.) either in situ or transport them to regional lymph nodes. they also participate in the process of skin wound healing. cells which traffic through the skin can be harvested from efferent lymph drained from a given area of skin. the type of migrating cells changes after trauma, heating and infection. we have developed a method for collection of human afferent lymph in lower limbs. the method allows obtaining immune cells from normal and injured skin and their characterization. aim of the study was to characterize skin immune cells in situ and in skin lymph with use of immunohistological methods (staining, facs). results. group , cells migrating through skin: + % t lymphocytes (cd ), + % langerhans and dendritic cells (cdla, hla dr, s ), + % cd , + % cd , no b cells (cd , ), % cd r (memory cells), + % il r. approximately % cells possessed cdlla and antigens. cd lc was expressed only on large cells. the frequency of all phenotypes was different from the blood populations. group , cells in skin: langerhans cells were found only in epidermis, cd , and , cd r , rb, ila/ cells around venules, cd (macrophages) uniformly dispersed, no il r and b cells. hla dr positive were endothelial and some dispersed mononuclear cells. group , one, three and thirty days after surgical wound (simple varicous vein extirpation): high density of epidermal langerhans cells, hla dr positive keratinocytes and all endothelial ceils, few il r cells, perivenular infiltrates of cd , r but less cd cells, high density of cdlla/ cells. classic staining of isolated and in situ located ccl!s with mgg or he did not allow to follow kinetics of changes. conclusions. this study presents the first in the literature quantitative data of immune cell traffic through normal and injured human skin. in the controlled release of biological response modifiers for soft tissue regeneration. alan s. rudolph, helmut speilberg, mariam monshipouri, and florence rollwagen, and barry j. spargo. we have employed lipid microstructures as controlled release vehicles for the delivery of growth factors in wound repair. traditional liposomes as well as novel lipid based microcylinders have been examined for their in vitro kinetics of the release of transforming growth factor beta (tgf-b). in vitro reiease has been examined by setting up models with examine the physical release of iodinated tgf-b as well as a cell based bioassay (based on the ht bioassay). the hollow lipid microcylinders ( microns in length and i micron in diameter) show an initial burst ( - ng) followed be zero order kinetics which result in the release of approximately i ng tgf/day. this release behavior can be modified by temperature based on the phase behavior of the lipid bilayer which comprises the microcylinder.we have also examined the cellular response to lipid microcylinders applied in vivo. the lipid microcylinders are mixed in agarose and implanted as a composite hydrogel block under the flank of a mouse. the blocks are removed , , and days following implant and the cells analyzed by facs sorter analysis. the observed pattern of ceil recruitment to the blocks mimics that seen in a local inflammatory response. cell surface phenotype studies included the determination of cd and cd , mac-l, and ig bearing cells. we have also begun to examine the change in cell surface phenotype and kinetics of recruitment following the inclusion of tgf-beta in the lipid microcylinders.center for biomolecular science and engineering, code , naval research laboratory, washington, dc. - . expression pattern of heat shock proteins in acute, good healing and chronic human wound tissue. abstract: wound healing is a complex biologic process that is well characterized at the histological level, but its molecular regulation is poorly understood. after clot formation, inflammatory cells are rapidly drawn into the wound, followed by migration of fibroblasts and epithelial cells that divide and repopulate the wound area. during the last decade peptide growth factors and cytokine are thought to play a key role in initiating and sustaining the phase of tissue repair. these factors which are released from different cells appear to initiate the cascade of events that lead to healing. different studys described the rapid activation of a family of proteins,named heat shock proteins (hsp) in differnt tissue that were exposed to various forms of stress (heat, toxic agents, mechanical). in this context hsp's have the ability to regulate protein folding and assembly, to transport proteins across cytoplasm and membranes, to disrupt protein complexes, to stabilize, degrade and regulate the synthesis of proteins and to take part in dna replication and repair. we now attempted to find out if hsp-gene activation is also involved in injury and wound healing, which likewise resemble a stress situation for cells. therefore we collected tissue samples during operation and single biopsies from chronic wounds (decubitus for example) and granulation tissue. after rna preparation from these samples we used rna-pcr and nothern analysis to study the expression of objectives of the study chronic, non-healing cutaneous tflcers are a challenging clinical and socioeconomic problem. several animal studies have shown that cytukines (e.g. egf, pdgf, fgf, tgfb) accelerate the healing process and tissue repair in general. results from first clinical trials indicate a promising value of cytokines in the treatment of chronic non-healing diabetic and venous ulcers. recent reports in the literature indicate that the biological activity of the solution of platlet derived wound healing formula (pdwt~) released from c~-granules (mainly pdgf & tgfi~) is greater than the activity of the recombiant single factors like e.g. pdgf-bb (robson, lancet ) . the aim of our study was to determine whether a correlation exits between the concentration of tgfi~ & pdgf and the time course of wound healing. materials and methods pdwhf was prepared from ml of auto]ogous patient blood and diluted with a special buffer to a final concentration of ng/ml g-thromboglobulin. the concentrations of pdgf and tgfg were determined by elisa-tests developed in our laboratory. patients with chronic non-healing ulcers have been evaluated alter treatment by topical application of pdwhf. pdfg and tgff~ concentrations of the topical solution were measured and two patient groups formed for analysis the time course of wound healing was regularly and meticulously documented and evaluated by photography and casting. the time from initiation of treatment instil o wound volume reduction to go of the origional size (t %) was noted• results: healing of extensive burn wounds can be accelerated by grafting cultured autologous or allogeneic keratinocytes. the stimulation of granulation tissue formation and reepithelialization is presumably based on growth factors and cytokines released by keratinocytes. we wanted to prove this hypothesis by investigating the bfgf expression during wound development, bfgf is mainly described as an angiogenic protein with mitogenic activity on various mesodermal and ectodermal cell types pointing to its stimulating potential in wound heating. in the present study we compared the pattern of human bfgf m-rna expression and the localization of bfgf protein during the first days of wound healing. biopsies were taken from juvenile human bum patients, immediately after wound debridemerit mad on day after transplantation of cultured allografts. biopsies were snap frozen and cryosected. the pattern of bfgf expression was assessed by in situ hybridization of the bfgf m-rna with a digoxigenin-labelled antisense-rna and the parallel detection of the mature protein with an anfi-bfgf monoclonal antibody. our study revealed typical patterns of bfgf-m-rna-expression and intense bfgfprotein deposition during granulation tissue formation and reepithelialjzation of healing bum wounds. 'it, is known that major thermal injuries cause early impairment of wound healing followed by decreased influx of granuiocytes st. the site of injury. the role of granuiocytes in the process of wound healing is not ~"~ "" elucidated, it is now assumed that they are not merely phagocytic cells but active participants in ~n~*' ~.,.,a+~o~: processes secreting_ a number of various cvt-;kines, in order to investigate the effect of there is accumulating evidence that neuropeptides could be involved in the pathogenesis of several inflammatory reactions. vasocactive intestinal polypeptide (vip) and substance p (sp) have been detected by immunohistochemistry in normal as well as inflammed skin mostly in perivascular and periglandular location. both vip and sp are involved in vasodilatation, mast cell degranulation and irnmunomodulation.we determined the influence of sp and vip on the proliferation of lymphocytes in patients with psoriasis and healthy individuals. peripheral blood t-lymphocytes of psoriatics and healthy controls were isolated by density gradient centrifugation and passage over nylon wool. cell enrichment was controlled by facs analysis, lx t-lymphocytes were then incubated alone or in coculture with x irradiated autologous lymphocytes in culture medium containing - mol/i sp or vip. cell proliferation was measured semiquanfitatively by tdr uptake in a betacounter. significance was tested by the wilcoxon signed-rank test.our results show that sp and vip exert only an effect on unstirnulated t-cells. in healthy individuals but not in patients with psoriasis sp increases significantly proliferation of t-cells. vip, however stimulates significantly the blastogenesis of t-lymphocytes only in psoriatics.our results confirm the psychoneuroimmunologic component in inflammatory reactions and vip and sp could be partially implicated in their pathogenetic mechanisms. moreover psoriatic lymphocytes show an altered reaction to sp and vip. this might be due to a preexisting (genetic?) or more likely to an epiphenomenal receptor defect. the adhesive interactions between endothelial cells and circulating ~enkocytes in shock and innammatory vondltions is mediated by several distinct families of ce -surface determinants. of particular importance are the leukocyte integrins cdib / cdlla-c. in this study monoclonal antibodies to two of the u chains (cdlla & cdiib) and the common [~ chain (cdib) have been used to investigate leukocyte-dependent and leukocyte-independent plasma leakage in tee skin of rabbite. plasma leakage was measured as the local accumulation of t si-hsa over a rain period, the chemotac~c peptide imlp ( . . ng) and bradykinin were used to induce cell.dependent and cell- ndependent leakage respectively, the antibodies used were . e (cdis), nri (cdlla) and antibody (cdllb). ]ntradermal in~ections of bradyklnin and ~dlp both caused a dose dependent increase in plasma extravasatien ( .~. ffi . p.l to . z b.bttl and . ,- . ~ to . z . d respectively. . e ( . - . mf,/k~ iv) caused a dose dependent inhibition of imlp-induced but not bradyldnin.inducecl plasma exudation. at . mk/kg, the plasma leakage was completely inhibited, antibody nr produced similar results, treatment with antibody did not cause inhibition o£ plasma leakage due to either tnedi~tor. in vitro, the irmnune system ex~nination in persons with bone, chest and abdominal traumatic injury (i group . patients without infectious coz~lications and group - patients with wound infections development) was carried out. to restore found immunity disorders and host defense to infection patients of the group were treated with thymalin-the biologically active peptides prepared from bovine thymus. the examination on t~e i- days after injury revealed a considerable decrease of lymphocytes, ed ",$d ~ and cd cells amo~it in the blood, cd /cd ratio and indexes of let~ocyte migration inhibition test in both groups of patients. the imm~lity disorders recovered to norm on the - days in pateents of+the i group. but stable ~eple$ion of cd and cd cells amount, lower cd /cd ratio and indexes of leukocyte migration inhibition test in patients of the group were observed~ besides that, these persons showed higher cd cells amount and ig level in the blood. after thymalin therapy valid ii~rovement of inun~e status was discovered. also good clinical effect of immunotherapy and best wo~id healing observed in % of cases. these results allow us to propose that the thymus involution and the reduction of cell-mediated immunity responsiveness with disturbances of immu_uoregulatio~ on the level of restriction of activated cd tho cells play the most important role in the pathogenesis of wound infections development in persons with traumatic injury.dept. of immunology, military-nedical academy, lebedeva str. , , st.petersburg, russia a severe impairment of neutrophil (pmn) function often occurs following severe thermal or non-thermal traumatic injury. our laboratory has previously reported that following severe burn or non-burn traumatic injury the expression of the p integrlns (cd a,b,c/cd ) and the fw receptors (cd , and cd ) were significantly decreased on pmns, in this study, the effects of gm and g-csf on the expression of the f~ r and the ~ integrln family on pmns were examined, pmns were obtained from severe trauma (initial apache ii score ;z ) or thermal injury (> ~; total body surface area, > ~ full thickness) and incubated /n v/tro with gm or g-csf. the j integrins or fcyr were detected with monoclonal antibodies and flow cytometry. gm end g-csf induced a sllght increase in the percentage of pmns expressing cd lb, cd , and cd while gm bur not c-csf induced an increase in the percentage expressing cdi a, cd lc, and cd , gm-csf and to a lesser extent g-csf induced an increase in the density ( , fold) of the ~ integrlns on pmns from normal, burn, and trauma patients, these data suggest that cytoklne modulation with csfs could have a role clinically in certain situations. institute, dept. of surgery, bethesda ave, cincinnati, oh, usa, - . funl~al infections after solid organ transplantatlon(sot) lewis flint, md and ed,~-afd e. etheredge, me) dept. of surgery tullrte univ. school of medicine new orleans. louisiana infections contribute to increased gra loss and mortaliw following sot. pr~isposing facton include diabetes, hepatitis, leukopenia, cc.¢xistem infection, and intense, especially triple drug, immunosuppression. funga] infections occur ~s isolated conditions in % and in association with bacterial infection(l %), viral infection( */.), and combined infections(it%), candida sp. is the most common fungus recovered but aspecgillus, coccidiodies, cryptococcus, histoplasma, mueor~ ghizopus, tinea, and toruiop~is s?. also are pathogens. clinical syndromes vary among orga.aizms or may be variable with a single p~tthogen, for ~ample, with aggressive immunosuppression, candlda my be localized esophagitis or cystitis or systemically iavaslve with an associated high mortality. aspergilius presents ~ a diffuse pneumonia while cryptococcus causes pulmonary and centrad nervons sy'stem infection, clinical examination, ct scanning and aggressive sampling for c'ultures a.s wall as serologic tests contribute to diagnosis. empiric the~py is ind',cated where there is a high level of suspicion. preventlon of ca.adlda izfection is ~ci~itated by early remov-a. of central }ants, ca~hetess and stents as well as by the use of oral nystatin. amphotericin ]~ remains the drug of choice for treatment of in.save fungd infection, surgical resection of infectious loci in the lung and brain is indicated in selected patients. the main problems of diagnosis in lower respirator-), tract infection are the differentation of infection from colonization or contamination, and the isolation of a reliable and true pathogen. expectorated sputum may be unreliable in pneumonia, because of contamination by oropharyngeal flora. although blood cultures may be negative, they provide a precise diagnosis and should be obtained in all pneumonias. other more invasive procedures are transtracheal needle aspiration, fibrobronchoscopic techniques including protected specimen brush and bronchoalveolar lavage with quantitative culturing and cytological analysis, transthoracic needle aspiration, thoracoscopy -guided biopsy and open lung biopsy. recently m. e -ebiary, a. torres et al, reported quantitative cultures of endotracheal aspirates for the diagnosis of ventilator-associated pneumonia offering reliable results in these patients and should be further investigated. any invasive procedure in a severely ill patient should be carefully directed weighing the risks as well as the benefits, whilst taking the underlying diseases and expected survival into consideration. -current therapeutic approach is based mainly on monotherapy with broad spectrum antibiotics. combination therapy is apparently indicated only in p. aeruginosa infections and severe s. aureus pneumonia. graft infection can lead to fulminant graft failure or rapid progressive cirrhosis. for prevention of graft infection immunoprophylaxis, i,e. administration of human polyclonal anti hbs hypedmmunoglobutin (hig), starting in the anhepatic phase during operation, has proved to be at least partially succesful when performed on a long term basis.from a total of olt in adult patients olt were performed for hbsag positive liver disease (cirrhosis n= , fulminant liver failure n= , retransplantation n= ) in pat. all pat. received . u hig in the anhepatic phase and . u/per day for the first week. a small group of pat. received hig only for i week (short term immunoprophylaxis), in all other pat. hig is administered on a long term basis to keep anti hbs serum levels above uii or until graft infection occurs (long term immunoprophylaxis);one-year survival rates are % in pat. who were transplanted for fulminant hepatitis, % in pat. with cirrhosis and long term prophylaxis, and % ir~ pat. with short term prophylaxis. all fatalities were related to hbv graft infection. the total rate of graft infection was % under short term prophylaxis and was independent from preoperative hbv dna status, under long term prophylaxis graft infection occurad in % in pat, negative for hbv dna. in hbv dna positive pat. infection rate was %, the total rate of reinfection for all pat. with long term prophylaxis was %the results of liver transplantation in hbsag positive pat. are comparable to other indications, graft infection with hepatitis b virus ist the major risk factor for these patients. under long term therapy with hig the rate of graft infection can be significantly reduced. the crucial cellular element for mods-mof: monocyi'f_./m acrophaoe ronald v. meier, m,d., f.a,c,s. the severely :injured or crldcally ill surgical patient is at high risk for immune dysfunction. a major consequence of this immune dysfunction is multiple organ dysfunction and failure leading to death, the underlying etiology is now recognized to be an uncontrolled, unfocused, disseminated activation of the host normally protective inflammatory. ,, cascades.. the resultant "mahgnant' systemic" inflan'a'natlon produces d~ffuso multiple organ bystander injury !eading to progressive organ dysfunction and failure. systemic malignant inflammation involves diffuse actlvatton of all components of the humoral and cellular inflammatory host response. of these various components, the macropha~e is the crucial central cellular element. the tissue fixed macrophage is ideally located diffusely throughout the various organs injured to orchestrate the inflammatory process. the macrophage is long-lived and highly metabolic, the macrophage regulates both the extent and the dissemination of the inflammatory processes. the macrophage is an exu'emely active c¢ capable of producing and releasing not only directly eytotoxlc agents, s irnil~, to the neutrophil, including oxidants and numerous proteases out also the multitude of other cytokines and initiators of the interacting inflammatory cascades. the macrophage is the central source for ehemotactic agents (il- , ltb , c a) for neutrophils and other inflammatory cells, production of vasoaetive arachidonie acid metabolites (tx, pgi , poe, lt's), complement components (c a, csa), thrombotic agents (pca, tx), metabolic and physiologic modulators (il, , il- or tnf), and immunosuppressivc agents (poe , il- ). these products of the macrophage are highly effective in enhancing and augmenting the inflammatory response. disseminated activation otthe macrophage is critical to the induction of the long-term diffuse activation of inflammation necessary to induce multiple organ injury and failure. our ability to elucidate the molecular mechanisms that control the macrophage will lead to our ability to conu'ol the maerophage response and prevent mods-mof.flarborview medical center, - th ave za- , seattle, wa usa key: cord- -ilhr iu authors: nan title: isev abstract book date: - - journal: nan doi: . / . . sha: doc_id: cord_uid: ilhr iu nan introduction: primary tumours secrete large amounts of extracellular vesicles (evs), which play critical roles in preparing distant sites for a pre-metastatic niche formation, thereby promoting metastasis and even determining metastatic organotropism. whether biogenesis, secretion rates and organotropism of evs are linked remains unknown. we have recently shown that ral gtpases control evs secretion in nematodes as well as in mouse mammary tumour cells (hyenne et al. jcb ) . since both rala and ralb are overexpressed or over-activated in various human cancers, we aimed to investigate the mechanisms by which these two gtpases control evs secretion and to determine how this affects metastatic progression, with a focus on breast cancer. methods: we used t mouse mammary carcinoma cells knocked down for either rala or ralb and determined their ability to induce orthotopic tumours and metastasis in a syngeneic mouse model. in vitro, we investigated ev secretion mechanisms using confocal and electron microscopy (em). evs were isolated either by uc or sec and characterized by nta, em, rna sequencing and mass spectrometry. the function of evs was assessed using a transwell assay. finally, we tracked the organotropism of fluorescently labelled evs and their capacity to induce pre-metastatic niches in mice. results: we show that rala and ralb promote lung metastasis of breast cancer cells in mice without affecting their invasive behaviours. we found that rala and ralb control the biogenesis of exosomes, by acting on the formation of multi-vesicular bodies though the phospholipase pld . as a consequence, knock down of rala or ralb reduces the levels of secreted evs and modifies their rna and protein contents. these differences alter the pro-tumoural function of evs, as demonstrated with an in vitro permeability test. importantly, we show in vivo that evs from rala or ralb depleted cells have a decreased lung organotropism and, as a consequence, are less efficient in priming lung metastasis. finally, we show that high expression of rala or ralb is associated with a bad prognosis in human breast cancer patients. summary/conclusion: altogether, our study identifies ral gtpases as central molecules linking the mechanisms of evs secretion, their dissemination and their capacity to promote metastasis. nuclear proteins are recruited into tumour-derived extracellular vesicles upon expression of tetraspanin tspan introduction: tetraspanin tspan is a transmembrane protein that exhibits a unique expression pattern, being overexpressed in many cancer types, but undetectable in most healthy tissues. although there is increasing evidence of an effect of tspan in invasion, metastasis, and regulation of extracellular vesicle cargo, the molecular mechanisms of tspan are yet not fully understood. methods: to study the function of tspan , we have established a fibrosarcoma model consisting of the parental cell line (ht ) and its derivatives expressing tspan (ht -tspan ) either fused with different fluorescent tags or tag-free. life imaging, sted and storm microscopy were used to determine the intracellular localization of tspan . co-immunoprecipitation from nuclei lysates was performed to detect direct and indirect interacting partners of tspan . small evs were purified from cell-conditioned media using sec and subjected to mass spectroscopy and ngs for a comprehensive comparative analysis of the proteome and transcriptome of the evs. results: the results of the proteome analysis showed a strong effect in the protein cargo of evs upon tspan expression. remarkably, among of the most regulated targets, several histones and ribosomal proteins were enriched in the evs derived from ht -tspan cells. in line with this finding, life imaging and super-resolution microscopy revealed that, while a majority of the intracellular tspan is located on the cell membrane or intracellular membranes, -as it is known for other tetraspanins-, a portion of tspan is located on the nuclear envelope. in fact, several histones co-immunoprecipitated with tspan , indicating their interaction. summary/conclusion: our data show that the expression of tspan in the tumour cells greatly impacts ev cargo. moreover, localization of tspan on the nuclear envelope, together with the enhanced recruitment of nuclear and ribosomal proteins to the evs, suggests a new mechanism of action of tspan . introduction: extracellular vesicles (evs) modulate tissue development, regeneration and disease through the transfer of proteins, nucleic acids and lipids between cells. currently, the mechanism of cytosolic delivery of ev cargo is largely unknown. here, we unravel how evs release their cargo in recipient cells. methods: evs were isolated from gfp-cd and cd -rfp expressing hek t cells by ultracentrifugation. gfp-cd and cd -rfp evs were added to hek t cells stably expressing anti-gfp fluobody and fluorescently tagged galectin- , respectively. clem and fluorescence microscopy were employed to visualize fluorescent markers in recipient cells. bafilomycin a and u a were used to inhibit endosomal acidification and cholesterol export from lysosomes, respectively. results: fluorescent galectin- which binds to betagalactosides present at the luminal side of endosomes was used to detect endosomal permeabilization. the absence of galectin- recruitment to endosomes in presence of cd -rfp evs showed that endosome permeabilization is not the mechanism behind ev cargo release. gfp-cd ev addition to cells expressing anti-gfp fluobodies resulted in the formation of fluobody punctae, reflecting cytosolic exposure of ev cargo. subsequent clem of the fluobody punctae revealed endosomes as the underlying cellular compartments from where cargo release takes place. neutralization of endosomal ph and accumulation of endosomal cholesterol blocked cargo release, showing that ev cargo release is dependent on endosomal ph and cholesterol level. summary/conclusion: we show that genetically encoded cytosolic probes and clem offer an excellent approach to study both the mechanism and efficiency of ev cargo release in cells. we provide experimental evidence that ev cargo release occurs from endosomes. funding: the research was supported by dutch technology foundation ttw and netherlands organization for scientific research nwo, de cock-hadders stichting, and erasmus mundus namaste scholarship. healthy humans. to determine cut-off values for diagnoses, reference intervals of evs in plasma are needed. to establish such reference intervals, ( ) a significant number of healthy donors should be included, ( ) the presence of non-ev particles, residual platelets, lipoproteins, and haemolysis should be quantified, ( ) flow cytometry signals should be in si units. the long-term aim of this study is to determine reference intervals of ev concentrations in human plasma within known dynamic ranges of the detectors. methods: ( ) to establish a clinical reference, we collected blood from healthy volunteers and prepared platelet-free plasma. ( ) we performed quality control measurements including residual platelet count, serum index, and lipid spectrum. ( ) we measured all samples by flow cytometry (apogee a -micro) and used custom software (matlab r b) to automate calibration of all signals and data processing. scatter signals were calibrated in comparable units of scattering crosssection (nm ) and diameter (nm). fluorescence signals were calibrated in units of molecules of equivalent soluble fluorophores (mesf). results: the quality controls showed that most residual platelet concentrations ranged from ^ to ^ per ml except for one outlier, while the serum index and lipid spectrum were normally distributed. preliminary results of the first donors analysed, show that within the ev size range of - , nm, the median concentration of cd + evs is . • ^ per ml (apc> mesf), cd p+ evs is . • ^ per ml (pe> mesf), cd a+ evs is . • ^ per ml (pe> mesf), and cd + evs is . • ^ per ml (apc> mesf). summary/conclusion: we have developed reliable procedures for establishing reference intervals of ev concentrations, within a well-defined size and fluorescence intensity range, in human plasma by flow cytometry. we are currently applying these procedures to samples to obtain, for the first time, ev reference intervals for human plasma. funding: pol, e. van der is supported by the netherlands organisation for scientific research -domain applied and engineering sciences (nwo-ttw), research programmes veni . introduction: the use of extracellular vesicles for diagnostic and therapeutic applications has seen a major interest increase in recent years because of their capacity to exchange components such as nucleic acids, lipids and proteins between cells. isolation of a pure population of evs is the first step in studying their physiological functions since contamination of ev preparations with non-ev proteins can lead to incorrect conclusions about their biological activities. we have developed a new method termed tangential flow for analyte capture (tfac) using ultrathin nanomembranes to purify extracellular vesicles from pure, highly complex biological fluids such as blood plasma, resulting in a new method for extracellular vesicle purification. methods: the tff microfluidic devices are assembled through a layer stack process using patterned polydimethylsiloxane (pdms) sheets with the membranes sandwiched between top and bottom channels. undiluted plasma was tested in both normal flow filtration (nff) and tangential flow filtration (tff) modes on ultrathin nanomembranes. we have utilized a pore patterning technique called nanosphere lithography (nsl) that uses close-packing of nanoscale beads to pattern pores in an ultrathin membrane. results: nff of undiluted plasma resulted in a protein cake of~ μm on the membrane, which prevented further transport across the membrane and evs were buried in the formed cake that were impossible to identify. however, tfac as a modified version of tff, led to capturing cd positive evs on the pores of the membrane with little evidence of protein fouling. nsl allows us to fabricate nanopockets (bowls with a single pore at the base) with various diameter, depth and pore diameter. using nsl, we further utilize nanopocket membranes to purify ev samples in tfac devices. this nanomanufacturing technology will allow us to pattern nanopockets with various diameter, depth and pore diameter which increases the efficiency of capturing of evs. furthermore, nanopockets can be modified and coated by specific ev markers to capture different subpopulation of evs based on size and affinity and further allows identifying the phenotypic subsets of evs by combining both size and affinity-based techniques. summary/conclusion: we have developed a method for the capture and release of nanoparticles such as evs called tfac using ultrathin nanomembranes. nsl technology can be applied to fabricate nanopockets with different physical and biochemical properties. utilizing nanopocket membranes in tfac system will allow us to separate different subpopulations of evs based on size and affinity. funding: this project was supported in part by the national science foundation (iip ) to j.l.m and t.r.g., department of defence (ca ) to j. l.m., and the national institutes of health (r gm ) to t.r.g. the addition of a size exclusion chromatography step to various urinary extracellular vesicle concentrating methods reveals differences in the small rna profile introduction: urinary extracellular vesicles (evs) and their rna cargo are a novel source of biomarkers for various diseases, however non-vesicular rna (e.g. associated with proteins) is also present within urine. this study aimed to identify the optimal method for isolating and enriching evs from human urine prior to small rna analysis. methods: three ev concentration methods, ultracentrifugation (uc); a precipitation-based kit (pk); and ultrafiltration (uf), were compared using ml aliquots of pooled healthy volunteer urine. evs were then separated from protein contaminants by size-exclusion chromatography (sec). presence of evs was confirmed by transmission electron microscopy and western blotting, and evs were quantified using nanoparticle tracking analysis (nta). small rna content of concentrated urine and fractions obtained by sec (evs and proteins) were evaluated with the agilent bioanalyzer small rna chip. results: ev recovery following sec of concentrated samples was - %, however particle: protein ratio (indicating ev purity) was approximately x greater after sec, regardless of the concentrating method used. uf+sec yielded the highest number of evs (per ml of urine) compared with pk+sec and uc+sec. small rna analysis from uf-concentrated urine (prior to sec treatment) identified peaks at nucleotides (nt) and nt. following sec, rna analysis indicated that ev fractions contained mostly small rna of~ nt, whereas the protein factions contained small rna of nt in size (consistent with mirnas). summary/conclusion: uf+sec provided the best balance between ev recovery (per ml urine) and particle: protein ratio. these data indicate that most of the nt sized rnas, presumably mirnas, are not within evs in urine. ev preparations obtained after uc, pk and sec (regardless of concentrating method) contain pre-dominantly~ nt sized small rna. these data outline the importance of removing non-vesicular proteins and rna from urine ev preparations prior to small rna analysis. funding: this research has been funded by petplan charitable trust. the use of rev for the optimization of ev separation and characterization by af introduction: the reproducibility of extracellular vesicle (ev) research has been hampered by the infinite number of separation and measurement techniques and the lack of appropriate reference materials (van deun et al., nat methods, ) . recombinant extracellular vesicles (rev) were developed as a biological reference material to overcome these limitations (geeurickx et al. nat comm ) . since rev have ev-like physical an biochemical characteristics and as they are trackable and distinguishable from sample ev they can be used as a spike-in material for data normalization and method development, and as a quality control. we used rev to optimize ev separation by asymmetrical flow field-flow fractionation (af ). methods: an af long channel column with a frit inlet driven by the eclipse system (wyatt) was coupled to a uv detector (shimadzu), mals dawn helios-ii (wyatt) and fluorescent detector (agilent). a spacer of µm and a regenerated cellulose membrane of kda were used. pbs supplemented with . % nan was used as a running buffer. light scatter profiles and uv profiles were analysed as well as the fluorescent emission spectrum as the rev are gfp positive. fractions were collected and analysed by nanoparticle tracking analysis (nta) and western blot. we also estimated the repeatability and reproducibility of the af technique by light scatter and fluorescence profiles as well as the recovery efficiency by nta. results: in a first step * ^ rev isolated from conditioned medium by a velocity gradient were injected in the af system to optimize the ev characterization protocol. later concentrated conditioned medium was spiked with * ^ rev and injected in the af column to optimize ev separation from non-ev contaminants. the most optimal separation protocol was obtained by varying detector and cross-flow settings. this protocol shows elution of monodisperse particles at each time point and size distribution estimations by af correspond to size determination by nta and electron microscopy. summary/conclusion: we were able to optimize the af protocol for characterization of ev by af as well as for separation of ev from crude conditioned medium samples by using rev. we demonstrate that rev are suitable for method development and that af has high potential as an ev separation technique. comparative evaluation of ev isolation methods for ev subpopulation analysis in human urine, plasma and cell culture media liang dong, richard zieren, kengo horie, sarah amend and kenneth pienta the brady urological institute, johns hopkins university school of medicine, baltimore, usa introduction: extracellular vesicles (evs) are membrane-enclosed particles of variable sizes that are released by any cell types to the extracellular space and are identified in all body fluids. a shortcoming in ev research is the lack of standardized isolation protocol for various sample types, resulting in heterogeneous outcomes in downstream analyses. in this study, we compared the ev isolation purity and efficiency among ultracentrifugation (uc), precipitation, sizeexclusion chromatography (sec) and a microfluidic tangential flow filtration device (exodisc) in human plasma, urine and cell culture media (ccm). methods: all evs were isolated by different isolation methods and characterized per misev guidelines. single-particle interferometric reflectance imaging senor (sp-iris) with optional fluorescence and nanoflow (nfcm) were used for single particle analysis. results: in ccm, total particle yield of exodisc was about times higher than those of the rest three methods. size distribution differed per sample, but the ranges were comparable between the different isolation methods. the total protein amount of sec, precipitation and exodisc were similar which were - times higher than that of uc. uc had the highest particle-toprotein ratio followed by exodisc. precipitation and sec had low ratios. when loading ug of total protein for western blot, cd , cd , cd and flot could only be detected in uc and exodisc samples, but not precipitation or sec. sp-iris and nfcm demonstrated consistent purity findings. in urine, total particle yields of exodisc and sec were about times higher than those of the rest two methods. the total protein amount of precipitation was times higher than exodisc and sec, times higher than uc. sec had the highest particle-to-protein ratio followed by uc and exodisc. precipitation had low ratios. in plasma, total particle yields of exodisc and precipitation were times higher than those of the rest two methods. and so were the total protein amount. sec had the highest particle-to-protein ratio followed by uc. exodisc and precipitation had low ratios. western blot, sp-iris and nfcm demonstrated consistent purity findings in urine and plasma. to evaluate particle capture efficiency, we spiked a known number of density-gradient uc purified evs to each method and the recovery rate of uc, precipitation, exodisc and sec was . %, %, . % and %, respectively. summary/conclusion: the order of ev isolation purity in ccm is uc, exodisc, sec and precipitation. in urine it's sec, exodisc, uc and precipitation. and in plasma, this order is sec, uc, exodisc and precipitation. exodisc and sec have similar high isolation efficiency followed by precipitation. uc has low efficiency for ev capture. a capillary-channelled polymer (c-cp) fibre spin-down tip approach for the isolation and biomarker characterization of extracellular vesicles of ovarian cancer origin kaylan d. kelsey, rhonda r. powell, terri f. bruce and r. kenneth marcus clemson university, clemson, usa introduction: extracellular vesicle (evs) profiling has shown promise for disease detection through less invasive sampling (liquid biopsies). current diagnostic tools for ovarian cancer are invasive or only semiinformative. thus, use of evs could prove useful in early disease detection. demonstrated is a hydrophobic interaction chromatography (hic)-based capillarychannelled polymer (c-cp) fibre tip spin-down process for the isolation of ovarian cancer evs for use in diagnostics. methods: polyester c-cp fibre micropipette tips are employed in the isolation of evs from biological matrices including cell culture media, urine, and blood plasma in a spin-down solid-phase extraction (spe) approach. evs were isolated from standards of healthy urine origin and from skov cells (human ovary adenocarcinoma). the c-cp fibre isolation method (taking less than mins and μl sample volumes) preserves the morphology and functionality of evs as confirmed by sem, tem, and confocal fluorescence microscopy. results: the dynamic binding capacity of ev standards on a cm pet c-cp fibre tip was found to be~ e particles ( %). the release of evs was confirmed using dot blot analysis for cd , cd , and cd tetraspanin proteins. immobilized evs were subjected to immunolabeling to allow the positive identification of a profile of ovarian cancer biomarker proteins (her , cd , egfr, epcam, ca ). summary/conclusion: this new ev isolation method introduces a simple capture mode, allowing for direct immuno-characterization and imaging on the fibre surface. this offers a unique and cost-effective opportunity for clinical analyses related to early detection and diagnosis of ovarian cancers (and others). the longterm goal is the creation of a rapid ev isolation and biomarker detection platform. funding: support from the national science foundation, eppley foundation for scientific research, gibson foundation, prisma health system and itor biorepository are gratefully acknowledged. development and optimization of purification method of exosomes by tangential flow filtration and ion-exchange chromatography approach tek lamichhane, ali navaei, sandeep choudhary, yonatan levinson and senthil ramaswamy cell & gene therapy r&d, lonza inc, rockville, usa introduction: extracellular vesicles (evs) such as exosomes have significant therapeutic potential, however, translation of ev-based therapies has been slowed down because of the biomanufacturing challenges. the isolation of evs, especially exosomes, is inherently challenging due to their small size, and heterogeneity in the mixture. the current isolation methods either have low recovery rate, aggregation, damaging the structure, time consuming or co-precipitation of contaminants. specially, it is difficult to process larger sized samples by centrifugation-based or immunoaffinity based methods because of the time and cost associated with these methods. methods: to overcome these roadblocks, we developed and optimized alternative purification techniques to isolate evs with higher purity and yield by using tangential flow filtration (tff) coupled with ion-exchange chromatography. we used bioreactor platform to produce evs from serum-free medium using bm-msc and hek s cells. bm-mscs were cultured on stirred tank bioreactors using microcarriers which provide a high surface area to volume ratio for the optimal cell growth and evs production. impellers were used to enhance mixing and maintain homogeneous culture conditions that can be easily monitored and controlled. results: depth filtration was applied for clarification of conditioned medium. we screened different types of filters during depth filtration for the best recovery of evs. tff membranes with different pore sizes were used to optimize the purity and yield of evs. because of the negatively charged nature of evs, anion exchange chromatography was chosen to capture and separate tff purified vesicles by their surface charge characteristics. we compared monolith based and membrane-based anion exchange columns to remove contaminants and purify exosomal fractions. the purity, size and presence of exosomal markers in isolated evs at each step of purification was evaluated by f-nta, nano-fcm and tetraspanins based elisa kits. summary/conclusion: in summary, our optimized methods improved the speed of isolation and purity of evs to the clinical grade. the production and isolation methods of exosomes that we developed here will be easily expandable to support large-scale and cgmp compatible bio-manufacturing in the future. use of an alternating current electrokinetic microelectrode chip to positively identify oncology, neurology, and infectious disease samples through plasma extracellular vesicle analysis juan pablo hinestrosa, jean lewis, david searson, orlando perrera, alfred kinana, heath balcer and rajaram krishnan biological dynamics, inc., san diego, usa introduction: cancer, neurological, and infectious diseases are leading causes of death, with early detection needed to improve outcomes. extracellular vesicles (evs) in the blood contain disease biomarkers, but current methods do not allow rapid analysis, and are often limited to one biomarker type. methods: we developed methods using alternating current electrokinetics (ace) to isolate evs from bloodbased samples and analyse the evs in situ with downstream assays for protein and nucleic acid biomarkers. we investigated if we could identify tuberculosis (tb) donor samples, protein and nucleic acid biomarkers in evs derived from cancer cell lines, and alzheimer's disease (ad) protein biomarker levels. results: ev isolation was confirmed by positive identification of the proteins cd , cd , and cd and measurement of ev mrnas using a direct rt-ddpcr assay. different disease models were analysed following method development. tb was used as a model for infectious disease, with tb positive and tb negative samples isolated on ace chips and analysed for levels of lipoarabinomannan and ag . using a cut-off above the negatives, the auc of roc curves were . and . , respectively. for oncology, cancer cell lines were cultured and evs isolated from supernatants were spiked into human plasma for analysis. levels of pd-l or glypican- on evs were able to be measured following ace capture. additionally, dna and rna mutations known to be present in the cell lines were able to be detected using ngs and qrt-pcr, respectively. using ad samples as a neurological disease model, tau and phospho-tau t (p-tau t ) in human donor plasma were detected. in ad and healthy donor samples, p-tau t signal increased % in diseased versus healthy donors. summary/conclusion: ace chips are an innovative ev isolation and analysis platform that allow rapid disease sample detection in a wide range of studies with high sensitivity and specificity. introduction: colorectal cancer (crc) is one of the most frequent causes of cancer-related death. in the majority of crc patients, mutation in the apc gene is among the first genetic events. it leads to uncontrolled activation of the wnt pathway, and thus, to adenoma formation. some of these adenomas may then further progress to crc with the accumulation of other mutations. the d organoids maintain the cellular and genetic heterogeneity of in vivo tissues and haves proved to be so far the best ex vivo model of human cancers. here we analysed the ev-based communication between cancer cells and fibroblasts by i) identifying factors that substantially increase ev release from intestinal cancer cells and ii) by determining cargo components of evs that enhance tumour cell proliferation. methods: we used commercially available and patientderived fibroblasts and crc organoids. the medical research council of hungary approved all experiments with human samples and informed consent was obtained from patients. evs were studied by using antibody-coated beads, trps, nta, tem and western-blotting. we introduced apc mutation into wild type murine small intestinal organoids by crispr-cas . results: we found that in crc patient-derived organoid cultures, small evs were preferentially secreted. we observed that apc mutation and the accumulation of the extracellular matrix component collagen critically enhanced ev secretion in intestinal organoids. furthermore, we showed that amphiregulin, present on fibroblast-derived ev, contributed to the maintenance of the intestinal stem cell pool and to cell proliferation in epidermal growth factor-dependent crc organoids. summary/conclusion: by proving the key role of mutations, collagen deposition and ev-bound amphiregulin in the release intensity and functions of the evs, we identified novel mechanisms in the progression if crc. funding: this work was funded by otka-nn , by the national competitiveness and excellence program nvkp_ - (national research, development and innovation office, hungary) and by the national excellence program in higher education (ministry of human resources, hungary). prostate cancer-derived evs induce a pro-inflammatory phenotype in the stroma blandine f. victor a , dolores di vizio b , andrew chin c , tatyana vagner b , javier mariscal b , mandana zandian a , catherine grasso a , roberta gottlieb a and helen goodridge a a cedars-sinai medical center, los angeles, usa; b cedars-sinai medical center, west hollywood, usa; c cedars-sinai, los angeles, usa introduction: since % of patients with metastatic prostate cancer (pc) develop bone metastasis, identifying the mechanism that drives this process is essential. most ev research has been focused on the role of exosomes in mediating the pre-metastatic niche formation. however, most of these studies do not separate exosomes from large evs. our preliminary studies have demonstrated that a subclass of evs known as large oncosomes (lo) can reprogram prostate fibroblasts, at the primary tumour site, promoting angiogenesis and enhancing the migration and invasion of pc cells in vitro and tumour growth in vivo. the bone marrow is the initial site of entry into the bone microenvironment for disseminating tumour cells (dtcs) and is a rich source of nutrients that houses various cells types including bone marrow derived mesenchymal stem cells (bm-msc) and immune cells such as neutrophils, which have been implicated in metastasis. here we investigate the role of lo in reprogramming bm-mscs and driving bone metastasis in pc. methods: differential centrifugation, density gradient centrifugation, trps, rna sequencing, qpcr, migration assay, invasion assay, chemotaxis assay. results: we report that pc-derived evs induce distinct gene expressions changes in bm-mscs. rna-seq analysis identified inflammatory and immune regulating cytokines as top differentially expressed genes (deg) in bm-msc. moreover, lo induced a more potent response in bm-msc in comparison to exo and to non-treated controls. the genes enriched in lo treated bm-msc were associated with tumour cell motility. in agreement with the gene expression data, lo-treated bm-msc attracted migration and invasion of significantly more pc cells than exo -treated bm-mscs. in addition, the top deg expressed in ev treated bm-msc were identified as potent neutrophil chemoattractant proteins. in line with the rnaseq findings, the lotreated bm-msc demonstrated enhanced chemotaxis of neutrophils towards them in comparison with exo or vehicle-treated bm-msc. finally, we show that the observed differences in bm-msc's response to lo and exo may be mediated by distinct molecular pathways. summary/conclusion: the results from this study provide novel insight into how tumour derived evs alter the bone marrow microenvironment and how they may drive bone metastasis in prostate cancer. the αvβ integrin in cancer cell-derived small extracellular vesicles enhances angiogenesis introduction: prostate cancer (prca) cells crosstalk with the tumour microenvironment by releasing small extracellular vesicles (sevs). sevs isolated from prca cell media, express the epithelial-specific αvβ integrin, a surface receptor for fibronectin and vitronectin. the αvβ integrin is not detectable in healthy prostate tissues but is highly expressed in prca. in this study, we hypothesized that αvβ in cancer sevs plays a crucial role in angiogenesis. methods: the sevs isolated from prca cell media were characterized by nanoparticle tracking analysis, iodixanol density gradients and expression of sev markers. the αvβ -negative endothelial cells (hmec ) were incubated with αvβ -positive sevs from prca cells to evaluate the transfer of αvβ by immunoblotting (ib) and facs. the effect of αvβ -positive sevs on motility, tube formation and angiogenic signalling were assessed by boyden chamber, angiogenesis assays and ib in hmec . results: we demonstrate for the first time that the αvβ is de novo expressed on endothelial cell surface by sevmediated protein transfer. prca cell-derived αvβ -positive sevs, significantly promote the motility and the formation of nodes, junctions and tubules by hmec . mechanistically, we demonstrate that hmec treatment with sevs from pc cells that endogenously express αvβ , decreases pstat (y ), a negative regulator of angiogenesis, while upregulating survivin, an inducer of angiogenesis. hmec treatment with sevs isolated from pc cells harbouring crispr/cas -mediated downregulation of β , or shrna-mediated downregulation of β , results in increased levels of pstat (y ). this sev treatment also results in a decrease of survivin in sevs and hmec . summary/conclusion: overall, our findings show that αvβ in prostate cancer sevs regulates a novel proangiogenic signalling pathway. funding: this study was supported by nci r - (lrl); p - (lrl and dca). introduction: advanced prostate cancer (pca) is asso-introduction: extracellular vesicles (evs) are secreted from cells, and carry bioactive proteins and rna cargoes. increasing numbers of studies have identified key roles for exosomes in driving aggressive tumour behaviours, including metastasis. however, the detailed mechanisms and responsible factors in the ev cargo are still unclear. recently, immune system has been considered as an important factor in establishing and maintaining metastasis. our goal is to identify the role of head and neck squamous cell carcinoma (hnscc) derived small evs (sevs) in tumour metastasis from the study analysing the effects of sevs on metastasis and tumour immunity. methods: sevs were collected from the conditioned media of hnsccs and purified through cushioned density gradient ultracentrifugation. an orthotopic mouse model was used for the assessment of tumour angiogenesis and metastasis. moc (inflammationinducing rarely metastasizing murine hnscc line) and moc (highly metastasizing murine hnscc line) were used for this study. moc and moc cells were transplanted into mice tongues orthotopically, and moc /moc derived sevs or pbs were injected into the tumour twice in a week. two weeks after tumour transplantation, mice were sacrificed and tumours were sectioned for pathological analysis and facs analysis. in facs analysis, the number and species of tumour-infiltrated immune cells were measured. results: injection of sevs from moc into moc tumours suppressed frequency of lymph node metastasis. on the other hand, injection of sevs from moc into moc tumours didn't promote metastasis. cd positive t-cell distribution in moc tumour was significantly changed by moc sev injection. t-cell deprivation treatment using anti-cd antibody increased the frequency of metastasis in moc -sev treated moc tumours. from the result of proteomics analysis on moc and moc sevs, immune-regulated proteins and metastasis-suppressing proteins were observed in moc sevs. summary/conclusion: we find that low aggressive hnscc sevs affect metastasis of highly metastasized hnscc, and also find that changing immune cell distribution may be related to the result. this mechanism and finding contributes to understanding the possible role of hnscc sevs on metastasis as well as on the tumour immune microenvironment. funding: this work was supported by the nih under award numbers r ca and r ca to aw. desmoglein enhances squamous cell carcinoma tumour development through extracellular vesicles in an il- /mir- a-dependent mechanism introduction: the cadherin dsg is a stem cell marker that is upregulated in many different cancers, including sccs, and its expression correlates with poor prognosis. dsg activates mitogenic signalling and plays a key role in cell proliferation, migration, and survival. we recently showed that dsg enhances ev release, but the mechanism by which these evs modulate tumorigenesis is not fully understood. methods: we established scc cell lines stably expressing wildtype dsg or a palmitoylation deficient mutant, dsg cacs. evs were isolated by sequential ultracentrifugation, iodixanol gradient separation, or qev izon column, and analysed by nta and bca. tumour xenografts were established by subcutaneous injection of cells in scid mice and monitored up to weeks. cytokine profiling was determined by antibody array. mirna expression was analysed by rnaseq and confirmed by qpcr. results: dsg enhanced ev release by % and promoted a~fivefold increase in tumour size in xenograft models. tumour growth was increased when control cells were treated with a single µg dose of evs. loss of palmitoylation, which altered membrane trafficking of dsg , reduced ev release (~ %) as well as tumour development. plasma evs from xenograft mice reflected in vitro particle counts from scc cell lines. a cytokine array analysis was performed revealing that dsg -evs were enriched with pro-inflammatory cytokines including il- , a potent chemotactic and angiogenic factor. most importantly, il- was surface-bound on evs. furthermore, rnaseq revealed mir- a, a negative regulator of il- , to be significantly downregulated in response to dsg . treatment with mir- a mimic or mir- a inhibitor decreased or increased, il- expression in scc cells, respectively. summary/conclusion: in summary, dsg plays a key role in scc tumour development by increasing ev biogenesis and downregulating mir- a, which in turn upregulates il- synthesis and release which can promote invasion, angiogenesis and metastasis. funding: nih r introduction: stem-and progenitor cell transplantation therapy holds great promise for regenerating damaged heart tissue. several lines of evidence suggest that its efficacy is mainly caused by secreted extracellular vesicles (evs). indeed, cardiac progenitor cell (cpc)-derived evs have been shown to protect the myocardium against ischaemia/reperfusion injury in several preclinical models. however, the underlying mechanisms for cpc-ev-mediated cardioprotection remain elusive. here, we utilized the proteomic composition of cpc-evs released during different culture conditions, to unravel protein-mediated effects of cpc-evs on the endothelium. methods: cpcs were stimulated with calcium ionophore (ca ion-evs) or vehicle (control-evs) for hours and evs were isolated from serum-free conditioned medium using size exclusion chromatography. ev concentration and size was assessed using nta. evs were functionally characterized based on endothelial cell activation by western blotting and an endothelial cell scratch assay. the proteomic composition of both ev conditions was profiled using mass spectrometry. cpc-ev knockouts for specific proteins were generated using crispr/cas technology. results: we found enhanced phosphorylation of erk / and akt in endothelial cells and increased wound closure after stimulation with control-evs, but not after stimulation with ca ion-evs. proteomic analysis identified a total of ev-associated proteins, with proteins uniquely expressed in control-evs. another proteins were revealed as candidate proteins, based on their relative enrichment in control-evs compared with ca ion-evs. go analysis demonstrated that differentially expressed proteins were involved in vascular endothelial growth factor signalling, extracellular matrix organization and angiogenesis. to investigate the involvement of the individual candidate proteins on endothelial cell activation, knockout evs of multiple proteins were generated and functionally characterized. summary/conclusion: a specific set of ev proteins is identified that may be functionally responsible for the activation of endothelial cells upon exposure to cpc-evs. generating knockout evs for each of these proteins will help to investigate their individual roles. this may lead to a better mechanistic understanding of the use of cpc-evs as therapeutics for cardiac repair. funding: erc- -cog- evicare grant. hypoxia enhances the therapeutic potential of human cd + stem cell exosomes in ischaemic hindlimb repair ischaemic cardiovascular disease. we have previously shown that human cd + cell-derived exosomes (cd exo) improve perfusion and function of the ischaemic tissues. hypoxia is shown to modulate the secretion and content of exosomes in both cardiovascular and cancer research. therefore, we hypothesized that hypoxia can modulate the content and regenerative efficacy of human cd exo. methods: cd exo were isolated from primary human cd + stem cells cultured under hypoxia ( . % o , or normoxia ( % o , n-cd exo) using density gradient ultracentrifugation. cd exo size was measured using trps, nta, and dls and surface protein expression was determined using imaging flow cytometry. function of cd exo was assessed using cell viability, migration and matrigel tube formation assays in vitro and a mouse hind limb ischaemia model (hli) in vivo. protein content of hypoxic or normoxic cd exo was evaluated via lc-ms/ms and -d -dige followed by lc-ms/ms. results: we did not observe any significant differences in size or in quantity of exosomes secreted from h-or n-cd cells. both h-and n-cd exo expressed cd , cd and cd surface markers. interestingly, h-cd exo significantly improved cell viability, migration and tube formation of huvecs in vitro compared to n-cd exo. in the same line, h-cd exo also significantly improved perfusion (ratio: . ± . v . ± . ) and prevented ischaemic limb amputation ( % v . %) as compared to n-exo (p < . ; n = - ) in a murine (balbc nude) model of hind limb ischaemia. flow cytometry and confocal microscopy indicated that h-exo was uptaken by endothelial cells in the ischaemic limb. remarkably, we detected several proteins (including a fragment of hemopexin) and mirnas (mir- ) that could be responsible for the proangiogenic and beneficial function of h-cd exo. we have also demonstrated that removal of surface proteins diminished the pro-angiogenic function of cd exo. summary/conclusion: hypoxia enhanced the proangiogenic and regenerative potential of cd exo, and thus, may represent a more efficient clinical strategy for cd exo therapy. our research is clinically important to improve therapeutic angiogenesis in diabetic and cardiovascular patients with compromised stem cell populations. hyun-ji park a , jessica r. hoffman b and michael davis b a emory university, decatur, usa; b emory university, atlanta, usa introduction: exosomes, a subset of membrane nanovesicles, transfer cellular information by passing proteins and nucleic acids between cells. exosomes have been implicated as the mechanistic unit in stem cell therapy, as inhibition of exosome synthesis abrogates the effects of cell therapy following cardiac injury. more importantly, increasing evidence indicates that mirnas (mirs) within exosomes serve as important signalling molecules to regulate inflammation, recruit stem cells, and repair diseased tissue. among exosomal mirs, mir- and − are known to decrease angiogenesis, cell migration, and increase inflammation in various types of cells. here, we investigated the inhibition of these negative mirs as a means to improve the reparative capacity of c-kit+ progenitor cell (cpcs) exosomes. methods: cpcs were isolated from three paediatric patients using magnetic-bead sorting. ʹ-o-methylated rna duplexes inhibited mir- and − expressions in cpcs. exosomes (inhexos) were isolated from mirinhibited cpc conditioned medium. mir expression in exosomes and cpc was quantified by qrt-pcr. migration and proliferation of mesenchymal stem cells (mscs) were assessed two days post-exosome treatment. for inflammation analysis, thp cells with/without tnfα exposure were treated with exosomes and the expression of il- , − , and − was quantified by qrt-pcr. finally, the angiogenic potential of inhexos was tested by tube formation of cardiac endothelial cells. results: inhibitor treatment of cpcs decreased exosomal mir- and − expression. treatment with inhexos enhanced msc migration and proliferation compared with normal cpc exosome (norexo). moreover, inhexos showed promising results for immune regulation, as tnfα-induced inflammation was decreased in thp exposed to inhexos for h. however, tube formation capacity is slightly decreased (~ %) by inhexo compared to norexo. summary/conclusion: exosomes from mir- and − -depleted cpcs may be a promising strategy for the treatment of various cardiac diseases, as they enhanced stem cell recruitment and proliferation, and regulated inflammation and angiogenesis. while other studies focus on boosting the reparative potential of exosomes by increasing positive mir and mrna cargo, the inhibition of negative mir in exosomes could be an overlooked strategy for the treatment of cardiac disease. endo-lysosomes as an alternative intracellular location for ev cargo delivery with disease relevance introduction: extracellular vesicles (ev) are lipidbilayer nanovesicles that carry macromolecules and act as paracrine vectors for cell-to-cell communication. the processes regulating ev biogenesis are largely known, whereas how ev cargo is delivered to recipient cells remains poorly understood. a simple mechanism proposed is direct ev fusion with the cell membrane that liberate cargo into the cytosol. in this study, we observed that cargo release occurs also at an alternative intracellular location and that this acquires a disease relevance. methods: ev were isolated by serial centrifugation and characterized. for uptake studies, ev were traced by labelling donor cells with a lipophilic dye or by overexpressing gfp-cd . uptake was assessed by cytofluorimetry or by live confocal imaging. co-localization studies were performed with ectopic marker expression or by immune staining. protein-protein interaction was analysed by bi-molecular fluorescence complementation (bifc). prion-like transmission was studied using a pro-fibrillogenic tau fragment in donor cells and full-length tau in recipient cells. for quantification of subcellular localization, an automated algorithm based on machine learning was developed. lysosomal stress was monitored by nuclear translocation of tfe and lysotracker staining. antibodies directed against pathogenic epitopes of tau were employed to assess prionlike transmission. results: ev were taken up by recipient cells through an endocytic process and accumulated in endo-lysosomes (el). when cells were exposed to ev carrying a profibrillogenic tau, recipient cells accumulated tau within el by an autophagic process. direct interaction of ev-tau and cellular tau in el favoured the appearance of pathological epitopes. cells displaying this condition showed an increased el stress and cytotoxicity. summary/conclusion: in this study, for the first time we report that el represent a critical subcellular location where transcellular prion-like transmission mediated by ev of a neurodegeneration-associated protein occurs. thus, the degradative pathway most likely involved in the recycle of ev and endogenous proteins is highjacked in disease. these findings represent a novel mechanism for ev acting as vector for transcellular propagation of tau, which opens up new therapeutic interventions trying to halt the disease. funding: supported by gelu foundation. anti-human fab fragment of cd antibody prevents the endocytosis of melanoma and colon cancer-derived extracellular membrane vesicles and nuclear transfer of their cargos introduction: interfering with the mechanisms regulating intercellular communication mediated by extracellular membrane vesicles (evs) may find relevance especially in oncology where cancer cell-derived evs have an implication in the malignant transformation of tumour microenvironment. our laboratories recently demonstrated a novel intracellular pathway in which a fraction of endocytosed ev-associated proteins is transported into the nucleoplasm of the host cell via a subpopulation of rab + late endosomes entering into the nucleoplasmic reticulum. here, we have investigated the effect of a monovalent fab antibody against the tetraspanin cd (referred hereafter as cd fab), on the internalization of evs and nuclear transfer of their cargo proteins. chair: david r f. carter -oxford brookes university chair: neta regev-rudzi -weizmann institute of science methods: to monitor the intracellular transport of ev-associated proteins, we used bioengineered fluorescent evs containing cd -gfp fusion protein from femx-i melanoma, sw colorectal cancer and bone marrow-derived mesenchymal stromal cells (msc) as donors and the same cell types as recipients. evs were enriched by differential centrifugation from h serum-free conditioned media and characterized by zetaview nanoparticle tracking analysis, zetapotential and immunoblotting. cd fab was prepared from h hybridoma cells using the pierce fab purification kit. results: we previously demonstrated that silencing cd both in evs and recipient cells strongly decreased the endocytosis of evs and abolished the nuclear transfer of their cargos. here we show that cd fab significantly reduced the cellular uptake of cd -gfp+ evs and the nuclear transfer of their proteins in melanoma, colorectal cancer and msc used as receptor cells in a dose-dependent manner. the effect on the nuclear transfer is probably a direct consequence of the endocytosis inhibition of evs. in contrast, the divalent, intact cd antibody stimulated both events. summary/conclusion: the effect of cd fab appears independent of the used ev-donor cell types or receptor cells, probably due to the widespread expression of cd both at plasma membrane and ev surface. in conclusion, by impeding intercellular communication in the tumour microenvironment, cd fab-mediated inhibition of ev uptake, combined with direct targeting of cancerous cells could lead to the development of novel anti-cancer therapeutic strategies. a bright, versatile reporter for multivesicular body trafficking and exosome secretion and uptake bong hwan sung, ariana von lersner, jorje guerrero, evan krystofiak, david inmann, roxanne pelletier, andries zijlstra, suzanne ponik and alissa weaver vanderbilt university, nashville, usa introduction: live imaging of exosomes is one of the required tools to understand the function of exosomes. our previous live-cell reporter, phluorin-cd allows dynamic subcellular monitoring of exosome secretion in migrating and spreading cells. however, there were some caveats to its use, including dim fluorescence and the inability to make cell lines that stably express the protein. methods: a stabilizing mutation, m r is incorporated in the phluorin moiety and now exhibits stable expression in cells and superior monitoring of exosome secretion. a dual-tag reporter was created by incorporating a further ph-insensitive red fluorescent protein, mscarlet to the c-terminus of phluo_m r-cd . cancer cells stably expressing the constructs were imaged using a variety of microscopy techniques in vitro as well as in vivo. purified small evs labelled with phuo_m r-cd were imaged using immunogold transmission electron microscopy (tem) and quantitated for the half-life in the blood circulation using flow cytometry. results: phluo_m r-cd and phluo_m r-cd -mscarlet are exclusively detected in exosomeenrich small ev preparations. immunogold tem visualizes the phluo_m r tag is located on the surface of small evs. live cell imaging reveals phluo_m r-cd -positive puncta left behind migrating cells suggesting the deposition consists of exosomes. those puncta and trails are not only positive for exosome markers such as cd , alix, and tsg but also correspond to small evs observed by a scanning electron microscopy. the dual-tag reporter allows visualization of the exosome lifecycle, including multivesicular body (mvb) trafficking, mvb fusion, exosome uptake and endosome acidification. summary/conclusion: using phluo_m r-cd construct, we demonstrate superior visualization of exosome secretion in multiple contexts and a role of exosomes in promoting leader-follower behaviour in collective migration by observing that exosomes are secreted at the front of migrating cells and left behind in exosome trails. the dual-tag reporter allows visualization of the entire exosome lifecycle. we anticipate that these reporters will be broadly useful to investigate regulation and functions of exosome secretion and uptake in diverse physiological conditions. funding: r gm , r ca , u ca - s , r ca . uncovering novel genes regulating ev-mediated functional rna transfer using a crispr/cas -based reporter system introduction: extracellular vesicles (evs) play a pivotal role in intercellular communication through functional transfer of bioactive cargo, including rna molecules. despite increasing interest in ev-mediated rna transfer, our understanding of the pathways and mechanisms regulating ev-mediated rna delivery and processing is limited due to a lack of suitable readout systems. we recently developed a novel crispr/cas -based reporter system that allows study of ev-mediated rna transfer at single-cell resolution. here, we further validate this system by studying the role of known targets involved in ev uptake and intracellular membrane trafficking, and subsequently employ this system to uncover various novel genes that play a regulatory role in functional rna transfer. methods: we employed a novel crispr/cas -based stoplight reporter system, in which egfp expression is activated upon functional delivery of targeting single guide rnas (sgrnas) stably expressed by donor cells. intercellular functional rna transfer was assessed by measuring egfp expression in acceptor cells using fluorescence microscopy and flow cytometry after direct co-culture, transwell co-culture, and upon addition of isolated evs. potential roles of various genes in intercellular rna transfer were assessed by rnaimediated target knockdown in acceptor cells, prior to co-culture experiments. rnai knockdown was confirmed by qpcr analysis. results: a significant activation of egfp expression was observed in acceptor cells after direct co-culture and transwell co-culture with donor cells expressing sgrnas, as well as after addition of evs from cells expressing sgrnas. reporter activation was substantially decreased after knockdown of multiple targets involved in ev uptake through endocytosis and/or intracellular membrane trafficking. based on these results, a potential role of various novel genes in intercellular rna transfer was studied in acceptor cells. these experiments uncovered various novel targets involved in ecm binding, endocytosis, intracellular membrane trafficking, as well as various rho gtpase interactors. summary/conclusion: we previously demonstrated a crispr/cas -based reporter system that allows the study of functional delivery of small non-coding rnas with single-cell resolution. here, we show that this novel approach allows the study of specific genetic targets and pathways in ev-mediated functional rna delivery, and unravel the regulatory pathways that dictate the underlying processes. quantitative characterization of extracellular vesicle uptake and content delivery within mammalians cells gregory lavieu a , emeline bonsergent b , eleonora grisard c and clotilde théry d introduction: extracellular vesicles (evs), including exosomes, are thought to mediate intercellular communication through the transfer of biomolecules from donor to acceptor cells. occurrence of ev-content delivery within acceptor cells has not been unambiguously demonstrated, let alone quantified, and remains debated. methods: we developed a cell-based assay in which evs containing luciferase-tagged cytosolic cargo are loaded on unlabelled acceptor cells. measurement of luciferase activity associated with acceptor cells revealed ev uptake efficacy. additional cell fractionation procedure that separates membranes from cytosol revealed the occurrence of ev-content release within the cytosol of acceptor cells. results: results from dose-responses, kinetics, and temperature-block experiments suggest that ev-uptake is limited ( % spontaneous rate at h), does not depend on bona-fide ev-receptor, at least for the tested acceptor hela cells. yet, further characterization of this limited ev-uptake, through cell fractionation that separates membranes from cytosol, revealed the occurrence of ev-content release within the cytosol of acceptor cells. cytosolic release is inhibited by bafilomycin-a and overexpression of ifitm proteins, which prevent virus content delivery. summary/conclusion: our results show that ev-content release requires endosomal acidification and suggest the involvement of membrane fusion. funding: anr -ce - - and arc pja and pga rf . introduction: glioblastoma is a highly malignant brain tumour with a poor prognosis. its ability to develop therapeutic resistance result in devastating clinical outcomes. to solve the intractable problem, we need highly sensitive diagnostics that can detect the molecular changes during treatments. extracellular vesicles (evs) can be a potential biomarker to monitor treatments and the host cell ev mapping can better reflect molecular changes in the tumour immune microenvironment. we have developed a droplet-based single ev protein sequencing platform that overcomes limitations of current bulk measurement technologies, which make it difficult to discover a rare ev population in the presence of high background. methods: we multiplex protein measurements to profile hundreds of proteins at a time by using an antibody-dna conjugate and sequencing. we barcode each ev in droplets and make amplicons that are comprised of both ev barcodes and antibody barcodes for sequencing. barcoded antibodies are made using tco-tetrazine click reaction and evs are labelled with these barcoded antibodies. the labelled evs are encapsulated into droplets with barcoded beads that serve as a template for ev barcodes. we then perform extension to make amplicons that contain both ev barcodes and antibody barcodes for sequencing. results: we successfully fabricated barcoded beads using a split-pool approach and validated by observing a fluorescence decrease of the sybr green after dna strand denaturation. we used a -channel droplet maker to encapsulate barcoded beads, single ev, and master mix into droplets. close packing of barcoded beads allowed > % encapsulation into droplets. both droplet and tube-based methods achieved a similar high amplification efficiency (ct < for evs). we confirmed the amplicon size by running a gel, which showed the right amplicon size (~ bp) from the droplet and tube prepared samples and no signal from the negative control. summary/conclusion: the droplet-based single ev profiling platform has the ability to identify rare immune ev subtypes in the peripheral blood, which would otherwise be impossible to detect due to the copresence of abundant normal evs. this cutting-edge technique has the potential to revolutionize treatment monitoring of high-cost immunotherapies, avoid unnecessary toxicities, and enhance personalized medicine capabilities. funding: schmidt science fellows, in partnership with the rhodes trust po ca , ro ca , r ca quantbio graduate student award at harvard university. introduction: in this study, we compared four orthogonal technologies for sizing, counting, and phenotyping of evs. the platforms were: single-particle interferometric reflectance imaging senor (sp-iris) with optional fluorescence, nanofcm nanoflow (nf), nanoparticle tracking analysis (nta) with fluorescence, and microfluidic resistive pulse sensing (mrps) . results from these platforms were compared with results from standard ev characterization techniques such as transmission electron microscopy (tem) and western blot (wb). methods: human t lymphocyte h (high cd , low cd ) and promonocytic u (low cd , high cd ) cells were chosen for their distinct tetraspanin profiles without abnormalities that might result from genetic manipulation. evs were isolated from culture conditioned medium (ccm) by differential ultracentrifugation (duc) and size exclusion chromatography (sec) and characterized per misev guidelines. synthetic particles (silica and polystyrene spheres) with known concentrations and mixed size distributions were also tested. results: particle counts from nf and mrps were consistent, while nta detected approximately one order of magnitude lower for ccm derived evs, but not for synthetic particles. sp-iris events could not be used to estimate particle concentrations. for sizing, nf, mrps, and sp-iris returned similar size profiles, with smaller sizes predominating (per power law distribution), but with sensitivity typically dropping off below diameters of nm. nta detected a population of particles with a mode diameter above nm. additionally, sp-iris, nf, and mrps were able to identify at least three of four distinct size populations in a mixture of silica or polystyrene nanoparticles. finally, for tetraspanin phenotyping, the sp-iris platform in fluorescence mode and nf were able to detect at least two markers on the same particle. summary/conclusion: based on the results of the study, we can draw conclusions about existing singleparticle analysis capabilities that may be useful for ev biomarker development and mechanistic studies. funding: this project is funded by mh and ug ca . importance to ev organotropism. yet, most techniques rely on bulk characterization, or are severely restricted by the diffraction limit. the exoview r (nanoview biosciences) combines interferometry, immunocapture, and immunofluorescence, introduced as an alternative technique to multiplex protein detection on single evs below the limit of diffraction. here, we use this technique to characterize tetraspanin multiplexing on evs and to identify spatial patterning of tetraspanins using steric hindrance of antibodies (abs). methods: evs were isolated from conditioned media from skov- cell culture or human serum. evs were incubated overnight on chips to allow immunocapture by anti-cd , anti-cd , or anti-cd . chips were then incubated with three fluorescent abs against the same epitopes and imaged on the exoview r . following concentration optimization, evs were tested after preincubating with carboxy-fluorescein diacetate succinimidyl ester (cfse) or fluorescent abs against tetraspanins. results: using different concentrations of evs, binding curves could be fit to characterize binding kinetics of abs. maximum concentration of evs could be identified that minimized fluorescent overlap. bright-field interferometry (detection limit~ nm) distinguished x fewer bound evs than fluorescent detection, while pre-labelling evs with cfse produced x more detectable evs than immunofluorescence. interestingly, evs captured by one tetraspanin did not necessarily show high fluorescent detection of the same tetraspanin. upon pre-incubating evs with a single ab, vastly different expression profiles were identified, indicating significant steric hindrance between abs. furthermore, pre-incubating evs with anti-cd ab significantly decreased detection of cd with less impact on cd . this discrepancy indicated possible spatial patterning of tetraspanins with cd and cd closely colocalizing on the ev surface. summary/conclusion: this combination of interferometry, immunocapture, and immunofluorescence produces unique information about size distribution of evs and single ev protein profile. this data corroborates that evs have distinct subpopulations of tetraspanins and indicates that tetraspanins may be spatially patterned. regulation of liver homoeostasis, regeneration and diseases by mesenchymal stem cell-derived apoptotic extracellular vesicles university of pennsylvania, philadelphia, usa introduction: billions of cells undergo apoptosis and produce apoptotic extracellular vesicles (apopevs) each day, whereas the roles of apopevs in regulating the organismal health and disease remain poorly understood. mesenchymal stem cells (mscs) emerge as critical contributors to tissue homoeostasis, while mscs suffer from apoptosis in regenerative transplantation. in this study, we investigated the function and mechanisms of msc-derived apopevs in regulating the organismal homoeostasis. methods: fas mutant (fasmut) and caspase knockout (casp -/-) mice were applied for apoptotic and apopev deficiency. mouse bone marrow mscs were cultured and apoptosis was induced by staurosporine (sts). msc-derived apopevs were collected by serial centrifuges and were infused into mouse circulation via caudal vein. tracing of apopevs were performed by radioisotope or fluorescent labelling. liver homoeostasis was evaluated at the histological and functional aspects. liver regeneration was induced by partial hepatectomy (phx). acetaminophen (apap) was used to establish acute liver drug injury. high-fat diet (hfd) was used to establish type diabetes (t d) and non-alcoholic fatty liver disease (nafld). results: after systemic injection, msc-derived apopevs migrate to liver and can be uptaken by liver macrophages and hepatocytes. fasmut and casp -/mice develop hepatomegaly with structural disorders, which particularly reveals hepatocyte polyploidization. furthermore, fasmut and casp -/-mice demonstrate liver glucose and lipid metabolic disorders. importantly, msc-derived apopev infusion significantly rescues structural and metabolic dysfunction in fasmut and casp -/-mice. mechanistically, apopevs use the soluble n-ethylmaleimide-sensitive fusion protein attachment protein receptor (snare) protein for interactions with recipient organelles thus transferring signalling molecules. moreover, msc-derived apopev infusion promotes liver regeneration after phx, prevents apap-induced liver injury, and ameliorates nafld in t d. summary/conclusion: msc-derived apopevs serve as crucial regulators of liver homoeostasis, regeneration and diseases. these findings indicate potential significant roles of apopevs in maintaining the organismal health and in developing therapeutics for diseases. (msc-sevs) mediate osteochondral regeneration in rats. however, the therapeutic effects of these msc-sevs/exosomes in restoring the mechanical competence of the repaired cartilage for joint function in a clinically relevant animal model remain to be addressed. to investigate this, we compared the structural and mechanical properties of the repaired cartilage in a rabbit model after intraarticular administration of msc-sevs and hyaluronic acid (ha) with that of ha alone, which is widely used as visco-supplementation. methods: bilateral osteochondral defects were surgically created on rabbits. immediately after surgery and at days and post-surgery, rabbits received ml injections of µg msc-sevs and ha in both knees, and rabbits received -ml injections of ha in both knees. at and weeks, macroscopic evaluation, histological scoring and compressive testing at different points on the repaired cartilage were performed. results: defects treated with msc-sev/ha showed improvements with time in macroscopic and histological scores and mechanical properties than defects treated with ha alone. in contrast, ha treated defects showed some repair at weeks, but this was not sustained, as evidenced by significant deterioration of histological scores and a plateau in mechanical properties from to weeks. by weeks, the msc-sev/ha repaired tissues demonstrated significantly better macroscopic score ( . vs . ; p < . ) and histological score ( . vs . ; p < . ). mechanical strength as measured by the young's modulus was significantly higher in the msc-sev/ha repaired cartilage than that in ha repaired tissues [defect centre ( . vs . mpa; p = . ) and overall periphery ( . vs . mpa; p = . ], and approximated that of the adjacent native cartilage. summary/conclusion: our findings demonstrated that msc-sevs and ha not only improved tissue morphology of the repaired cartilage but also promoted functional mechanical competence. this study establishes a clinically translatable protocol for use of msc-sevs for cartilage repair. introduction: mesenchymal stromal/stem cell (msc)exosome (mex) treatment has shown considerable promise in experimental models of bronchopulmonary dysplasia (bpd) and pulmonary hypertension (ph). mechanisms by which mex afford their beneficial effects remain incompletely understood and here, we embark into investigating them through assessment of mex biodistribution and impact on immune cell heterogeneity. methods: newborn fvb mice were exposed to hyperoxia (hyrx, % o ) at birth and returned to room air at postnatal day (pn) . mice received a bolus mex dose at pn . adoptive transfer studies were used to determine the role of mex-educated myeloid cells in vivo. mice were harvested at pn , , , or to characterize mex biodistribution and for assessment of pulmonary parameters. results: mex therapy effectively ameliorated core features of hyrx-induced neonatal lung injury, improving alveolar simplification, pulmonary fibrosis, vascular remodelling and blood vessel loss. exercise capacity testing and assessment of ph showed functional improvements following mex therapy. biodistribution studies demonstrated that mex localize in the lung, where they interact with lung monocytes/macrophages. whole lung mass cytometry (cytof) revealed that mex treatment promotes a pro-homoeostatic shift in lung immune cell apportion, replenishing the early hyrx-induced depletion in pulmonary cd + immune cells, restoring alveolar monocyte and macrophage populations and suppressing cellular inflammation. ex vivo and in vivo analysis showed that mex promotes a "pro-resolving" ccr -monocyte phenotype. notably, adoptive transfer of mex-educated bone marrow-derived myeloid cells (bmdmy), but not naïve bmdmy, restored alveolar architecture, blunted fibrosis, improved vascular remodelling and pulmonary blood vessel loss. summary/conclusion: mex treatment ameliorates core features of experimental bpd, restoring lung architecture, decreasing pulmonary fibrosis and vascular muscularization, ameliorating ph and improving exercise capacity. the beneficial actions of mex are associated with modulation of immune cell phenotypes, arising from mex-monocyte interaction. furthermore, adoptive transfer of mex-educated bmdmy rescued, at least in part, alveolar architecture, reduce fibrosis, improve vascular remodelling and pulmonary blood vessel loss. funding: this work was supported in part by an american thoracic society foundation grant (grw); the little giraffe foundation (grw); charles h. hood foundation major grants initiative (sk), nih r hl (sk) and united therapeutics research grant (sk and sam). immunomodulatory small extracellular vesicles derived from mesenchymal stem cells: a potential cell-free therapy for acute and chronic pulmonary vascular diseases introduction: vascular inflammation plays a critical role in acute respiratory distress syndrome (ards) and pulmonary arterial hypertension (pah). despite decades of research, there is no curative therapy for either condition. mesenchymal stem cells (mscs) have shown preclinical efficacy, mediated by release of extracellular vesicles. hence, msc-derived small extracellular vesicles (sevs) can harness the benefits of mscs with advantages in cost and safety. this study aims to evaluate the immunomodulatory effects of sevs in preclinical ards and pah. methods: msc-sevs were characterized by nanoparticle tracking analysis, electron microscopy and western blot. live fluorescence imaging measured in vitro and in vivo distribution of sevs. using a lipopolysaccharide (lps)-induced mouse model of acute lung injury (ali), a time course study of inflammatory response guided endpoint analyses. cell count and cytokines were measured in bronchoalveolar lavage fluid (balf) and histological lung injury was assessed. in ali mice, saline, mscs, msc conditioned media or sevs were administered . h post-lps. using a monocrotaline (mct)-induced rat model of pah, animals received saline or sevs at day . haemodynamic changes and right ventricular hypertrophy were evaluated at weeks. results: msc-sevs were nm in size with cd / expression. pkh -labelled sevs were taken up by endothelial cells. in the ali time course study, cell count and il b in balf peaked at h post-lps, whereas il peaked at h. histology showed significant intra-alveolar cell infiltrate at h. msc conditioned media attenuated il b in balf, whereas a trend towards reductions in il b and cell count were seen from delivery of mscs and sevs. using fluorescence imaging, lung accumulation of dir-labelled sevs was highest when administered h post-lps as compared to h, h or h. for pah rats, sevs reduced right ventricular systolic pressure ( . ± . mmhg) as compared to control ( . ± . mmhg; p = . ), whereas no changes were observed for right ventricular remodelling. summary/conclusion: these findings demonstrate the potential of msc-sevs to be used as a cell-free immunomodulatory therapy for acute and chronic lung vascular diseases. additional live and ex-vivo biodistribution studies will determine optimal timing of sev administration, tissue distribution and clearance in both ali and pah. changes in extracellular vesicle protein cargo after pro-inflammatory priming of umbilical cord mesenchymal stem cells (ucmscs) have been shown to suppress inflammatory responses in studies of autoimmune diseases. these therapeutic effects can be attributed to paracrine signalling, by which extracellular vesicles (evs) are one of the essential components. this study looks at how the culture conditions of ucmscs affects the type of evs they secrete. it also aims to identify an ev population with an anti-inflammatory potential for the treatment of autoimmune diseases. methods: ucmscs were isolated and culture expanded in a quantum® cell expansion system, then grown at %o , %o and primed with a pro-inflammatory cocktail. evs were isolated from ucmsc conditioned media by differential ultracentrifugation using a sucrose cushion and characterised by transmission electron microscopy and nanoparticle tracking analysis. ev markers were analysed using a europium-based immunoassay, macsplex exosome detection kit and immunoblotting. a proximity-based extension assay was used to identify inflammatory proteins in the evs. results: there was no difference in evs cultured at %o , %o or with pro-inflammatory conditions when analysed for size and morphology. all evs displayed the tetraspanin markers (cd / / ) and internal proteins (alix, hsp ). evs from primed cells showed a > twofold increase of cc chemokines and a > sixfold increase in cxcl and csf- . protein cargo did not differ in evs from %o and %o . summary/conclusion: this study showed that proinflammatory culture conditions alter ev protein cargo, evidenced by the increased production of chemotactic and angiogenesis associated proteins. upcoming rnaseq analysis will show if ucmsc culture conditions also affect mirna expression in evs. ongoing functional studies will determine how changes in ev cargo correlates with changes in t-cell proliferation and polarisation. funding: this work is fund by the orthopaedic institute ltd, keele university and the rjah orthopaedic hospital charity. alzheimer's disease biomarkers in plasma extracellular vesicles of neuronal origin correlate with brain pathology in mice introduction: multiple studies have shown that neuronal-derived extracellular vesicles (ndes) in blood contain alzheimer's disease (ad) biomarkers, especially tau. however, the convergent validity of tau in blood ndes in relation to brain pathology is yet to be determined. to address this, we measured total and phosphorylated tau levels in matched nde and brain tissue samples from ad mouse models. methods: we collected the cortex, hippocampus and plasma of xtg-ad, xtg-ad, and wild type (total of mice; female, male; age: mean = . , sd = . , - months) . plasma samples were collected retro-orbitally for weeks and at euthanasia via heart puncture. ndes from the pooled serial blood collections (nde ) and the single endpoint (nde ) were immunocaptured by targeting the neuronal marker l cam. we measured human total tau and pthr -tau (p-tau) in ndes and cortex and hippocampus homogenates using a luminex multiarray. results: overall, there were strong positive correlations for both total tau and p-tau between ndes and brain tissues across mice types. total tau in ndes showed positive correlations with levels in the cortex and hippocampus (r = . and . , p < . , cortex vs nde and nde ; r = . , p = . , hippocampus vs nde ; r = . , p = . , hippocampus vs nde ). levels of p-tau in nde showed positive correlations with levels in the cortex (r = . , p = . ) and hippocampus (r = . , p = . ); however correlations were not observed for nde (r = . , p = . vs cortex; r = . , p = . vs hippocampus). summary/conclusion: tau levels in circulating ndes reflect levels in cortex and hippocampus across ad model mice, supporting their convergent validity as "liquid biopsy" biomarkers for ad. funding: this research was supported in part by the intramural research program of the national institute on ageing, national institutes of health. exosomal ceramide mediates neurotoxicity of amyloid beta (aβ) in alzheimer's disease. ahmed elsherbini a , simone crivelli b , alexander kirov c , michael dinkins d , zhihui zhu a , haiyan qin a , sanjib karki a , priyanka tripathi a and erhard bieberich a a university of kentucky, lexington, usa; b university of kentucky, lexington, usa; c augusta university, augusta, usa; d augusta university, augusta, usa introduction: amyloid beta is a pathologic hallmark of alzheimer's disease (ad), however, the mechanism of aβ neurotoxicity is not fully understood. it has been reported that exosomes associate with aβ, but it is not clear how this association affects aβ neurotoxicity. methods: here we utilized several techniques to isolate exosomes from the sera of wild type (wt) and ad transgenic mouse model. ( xfad) as well as alzheimer's patients and healthy controls. we used exoquick, exoeasy, sequential ultracentrifugation, and size exclusion chromatography. particles' size and number were characterized by nanoparticle tracking analysis (zetaview). results: we report that the sphingolipid ceramide mediates neurotoxicity of aβ. we show that sera from ad transgenic mouse model ( xfad) and ad patients, but not the wt or healthy controls, contain a subpopulation of astrocyte-derived exosomes that are enriched with ceramide and are prone to aggregation (termed astrosomes) as confirmed by nanoparticle tracking and cluster analyses. when taken up by introduction: multiple sclerosis is the most common chronic inflammatory demyelinating disease of the central nervous system, affecting more than million people worldwide. ms is a multifactorial, immunemediated disease caused by complex genetic and environmental interactions. in recent years, extracellular vesicles (evs) have been described as powerful mediators of the modulation of biological processes (e.g. inflammatory and immune response) following environmental exposures such as particulate matter (pm), and have been described altered in ms. we characterized evs in patients with ms and healthy subjects matched for age and gender and evaluated the effects of pm exposure on ev release patients with ms compared with controls. methods: evs isolated from blood samples were characterized by nanotracking analysis and by flow cytometry after labelling with the following markers: cd + (monocyte), cd + (platelet), cd + (neutrophil), cd + (t-reg), and cd + (endothelium). pm and pm . concentrations at the residency of each subject were obtained from the regional air quality monitoring network. results: we observed decreased concentrations of cd + (p < . ), cd + (p < . ), cd + (p < . ), cd + (p < . ), and cd + (p < . ) in patients compared with controls. in cases, pm was inversely associated with cd + evs (pm . , β = − . ; p < . ), cd + evs (pm . β = − . ; p < . ), and cd + evs (pm β = − . ; p < . ; pm . , β = − . ; p < . ). on the contrary, in controls pm was positively associated with cd + evs (pm β = . ; p < . ; pm . , β = . ; p < . ). summary/conclusion: our findings showed a different composition of blood-derived ev subpopulations in patients compared with controls. moreover, we observed that patients and controls react differently to pm exposure in terms of blood-derived ev release, suggesting the involvement of this mechanism in the modulation of both inflammatory and immune responses, and thus in ms pathogenesis. plasma neuronal and astrocyte-derived exosomes serve as biomarkers of neurodegeneration and systemic bioenergetic effects in male cynomolgus monkeys self-administrating oxycodone ashish kumar a , yixin su a , david soto-pantoja a , jingyun lee a , ravi singh a , cristina furdui a , michael nader b and gagan deep a a wake forest baptist medical center, winston-salem, usa; b wake forest baptist medical center, winston-salem, usa introduction: opioid use disorder (oud) is currently a health emergency in the usa affecting millions of people. oud is a complex issue requiring a multipronged strategy. at the biological level, there is an urgent need to understand the dynamic molecular changes and adverse effects associated with opioid addiction. here, we aimed at identifying the biosignature of brain cells-derived exosomes associated with opioid addiction in a non-human primate (nhp) model of oud in which cynomolgus monkeys perform cognitive tasks and self-administer (sa) intravenous oxycodone daily. we also characterized the systemic adverse effects of the brain cells-derived exosomes from drug-naïve and oxycodone sa monkeys. methods: we isolated total exosomes (te) by ultracentrifugation and exoquick methods from the plasma of male monkeys self-administrating oxycodone for years and naive monkeys. subsequently, from the te population, we isolated neuron-derived exosomes (nde) and astrocytes-derived exosomes (ade) using surface biomarkers l cam (l cell adhesion molecule) and glast (glutamate aspartate transporter), respectively. this novel method involved streptavidin coated magnetic beads and photo-cleavable (pc) biotin, providing us biologically intact exosomes useful for co-culture studies. we characterized the exosomes by nanoparticle tracking analyses (nta), western blotting, flow cytometry, immunogold labelling, transmission electron microscopy (tem), elisas and mass spectrometry. respirometric profiling in cardiac myoblasts and monocytes following exosomes treatment was performed by seahorse xf. results: the quality of isolated exosomes (te, nde, and ade) was confirmed by nta (size distribution and concentration), western blotting (e.g. cd ) and tem (size and shape). nta did not show any significant difference in exosomes size and concentration (number per ml) between control and oxycodone sa groups. flow cytometry (e.g. l cam and glast) and immunogold labelling (cd , cd and l cam) confirmed the purity of nde and ade isolated from te. proteomics analyses of te, nde and ade identified several unique proteins present in exosomes from the oxycodone sa group. interestingly, we observed significantly higher expression of neurodegeneration markers neurofilament light protein (nfl) and alpha-synuclein in nde and ade of oxycodone sa group compared to controls. furthermore, te treatment of h c cardiac myoblasts and raw . monocytes significantly compromised their mitochondrial metabolism (basal and maximum respiratory capacity). summary/conclusion: these results suggest the utility of plasma exosomes as biomarkers for better understanding of the neurodegenerative and systemic effects of oxycodone addiction. funding: da , da . vesicles released during mycobacterium tuberculosis infection: immunomodulatory (glyco)lipids and role in host-pathogen interactions emilie layre, pierre boyer and jerome nigou cnrs-université paul sabatier, toulouse, france introduction: the tuberculosis disease remains one of the top causes of death worldwide. mycobacterium tuberculosis (mtb) has evolved strategies to evade immune responses and to persist within the hostile intracellular environment of alveolar macrophages. the current lack of efficient anti-tuberculosis strategies is largely due to our incomplete understanding of the host-pathogen interactions of mtb infection. vesicles released by the bacillus itself (bacterial membrane vesicles, bmv) and by infected cells (host extracellular vesicles, hev) have immunomodulatory properties in vitro and when administered to animals. if vesicles likely play key role in host-pathogen interactions of the tuberculosis infection, their content in bacterial factors, their uptake, trafficking and interaction with host cells receptors remain incompletely deciphered. methods: bmv and hev have been purified by combining differential centrifugation, density gradient and exclusion chromatography. after characterization by microscopy, nanosight and western blot, their content in bacterial (glyco)lipids has been characterized by the use of high sensitivity mass spectrometry-based lipidomic approach. bmv have been tested for their capacity to activate reporter cell lines of pattern recognition receptors. in addition, fluorescent-labelled bmv have been used to study their uptake by host cells thank to super-resolution microscopy. results: we have undertaken to characterize the content, the trafficking and interaction with pattern recognition receptors of bmv and hev released during infection by mycobacteria of variable virulence. we have importantly optimized the purification of bmv showing that lipoproteins aggregates are co-purified with vesicles on density gradient. sfc-ms lipidomic analyses allowed the characterization of the repertoire of immunomodulatory bacterial lipids released by bmv and hev, which excluded a continuum between these two release pathways. preliminary, assays have shown that these vesicles are capable to interact with different pattern recognition receptors including tlr and lectins. finally, we have been able to visualize fluorescent-labelled vesicles uptake by macrophages using superresolution microscopy. summary/conclusion: during m. tuberculosis infection, the bacillus as well as infected cells release vesicles that harbour different content in immunomodulatory bacterial (glyco)lipids, including strain-specific lipids. these vesicles likely play important role in host-pathogen interactions by modulating immune response beyond the infected cells, in part through their interaction with different pattern recognition receptors. funding: fondation pour la recherche medicale, fondation fonroga. introduction: conventional diagnoses of mycobacterium tuberculosis (mtb) rely on quantifying bacteria in sputum samples, which make it incapable of measuring the body's total bacterial load and diagnosing patients that have difficulty producing sputumsuch as children and those that are hiv-positive. nanoscale ( - nm) outer membrane vesicles (omvs), which are shed from their bacterial cells of origin and circulate in the bloodstream, have been found to contain rich molecular information from their mother cells. despite the diagnostic potential, their nanoscale size in the presence of high background has complicated the use of these promising biomarkers for clinical diagnosis of tuberculosis. chair: amy buck -the university of edinburgh chair: cherie blenkiron -the university of auckland methods: here we report two complementary approaches to systematically discover and clinically detect mtb-derived omvs using protein and rna biomarkers. first, we employ a digital droplet elisa on whole, unprocessed samples to detect and quantify the presence of these omvs using surface protein markers. second, we have developed a platform to specifically enrich for mtb-derived omvs using our previously developed magnetic nanopore platform, wherein millions of nanofluidic devices are operated in parallel, increasing throughput relative to a single nanofluidic device by a million-fold. using this approach, we identify rnas that are specifically enriched in mtb-derived omvs and can be used to identify tb strain, infectious activity, and total body burden. results: using these platforms, we enriched for mtbderived omvs from plasma and profiled their cargo, both proteins and rna. we first determined a panel of protein biomarkers for multiplexed detection of omvs through a digital droplet sandwich elisa. we then tested our protein markers on spiked plasma samples as models for clinical tb samples. simultaneously, we performed rna sequencing and discovered a panel of rna biomarkers that are preferentially enriched in omvs. we picked ten of the most highly-expressed rna biomarkers and also tested for them on spiked plasma samples using our magnetic nanopore platform. summary/conclusion: these results demonstrate the capability of omv biomarkers in the development of novel liquid biopsy based mtb diagnostics. building on this work, we are working with clinical collaborators to test our assays on clinical samples from philadelphia and west africa. funding: nih ot . introduction: a dearth of knowledge exists regarding the molecular mechanisms by which host exosomes regulate immune response to infections caused by gram-negative pathogens. to address this gap in knowledge, our laboratory has been using two wellestablished model organisms; yersinia pestis (yp), and burkholderia pseudomallei (bp). yp and bp cause the emerging human diseases plague and melioidosis respectively. currently, no licenced vaccines or highly effective therapeutics are available for either disease. methods: ex were purified from naïve u monocytes (exu) and infected u (exi) by serial centrifugation followed by sucrose density gradient purification, and characterized by tem, zetaview nanoparticle tracking, and exosome markers (cd , tsg , . immune responses of naïve u cells and response mechanisms were analysed following treatment with equivalent amounts of exi or exu (as control). these included macrophage differentiation assays, multiplex measurements of inflammatory cytokines, bacterial clearance assays, quantitative protein microarray analysis of host signalling proteins, sirna knockdown of exi-induced cytokines in recipient cells, and mass spectrometry (ms) analysis of exi contents. for all assays, at least four biological replicates were performed. results: exi induce monocyte differentiation to macrophages and dramatic release of il- , il- and il- cytokines, effects that are also seen when monocytes are infected with the bacteria. the exi also induce a substantial increase in the capacity of the recipient monocytes to clear bacteria in an il- -dependent manner. specific host signalling molecules are strongly modulated by the exi, including p , jak and alk for exi-yp, influencing the observed phenotypes. ms analysis showed lack of lps in exi-yp and demonstrated the presence of specific bacterial proteins that have antigenic properties. summary/conclusion: we have identified some of the molecular mechanisms by which exi assist the host in clearing infection. exi prime distant naïve monocytes through modulation of distinct pathways such as p to mount immune responses similar to when they become infected. these include differentiation to macrophages and migration to infection site for increased il- -dependent bacterial clearance. introduction: recruitment of monocytes to sites of infection is important in restricting growth and invasion of various microorganisms such as pathogenic fungi c. albicans. beside complement supported phagocytosis and extracellular trap formation, human monocytes secrete extracellular vesicles which are crucial in cellular communication in physiology and pathophysiology as they transfer proteins, lipid, and nucleic acids. the current study attempts to shed light on immune evasion mechanisms by c. albicans via extracellular vesicles. methods: human monocytes were isolated by magnetic beads technique and extracellular vesicles were isolated using polymer precipitation or ultracentrifugation or size exclusion chromatography. vesicles were characterized by elisa, lc/ms-based proteomics, confocal laser scanning microscopy (clsm) as well as electron-and dynamic light scattering microscopy, etc. crispr-cas based genome editing was performed to knockout cd b in human monocytic thp- cells. effect of isolated vesicles were determined by using proximity ligation assay (pla), elisa, western blot, next generation rna sequencing, qpcr, immunohistochemistry, etc. results: here we show for the first time that human blood derived monocytes alone and in a whole blood model strongly induced and released extracellular vesicles in response to the pathogenic fungus c. albicans. one induced population carried the anti-inflammatory cytokine tgfβ- . release of these vesicles is triggered by binding of soluble β-glucan from c. albicans to the cr receptor on monocytes as demonstrated by crispr-cas based cr genome editing in thp- cells, and by using cr knock out mice. isolated tgf-β -transporting vesicles reduced the inflammatory response in human m macrophages and in a whole blood model. the anti-inflammatory effect by tgf-β transporting vesicles is investigated in detail and results in inhibition of il- β gene transcription. summary/conclusion: showing that human apoptotic bodies similarly induced tgf-β -transporting vesicles from human monocytes we hypothesize that c. albicans hijacks this new cr -dependent anti-inflammatory vesicle pathway for immune escape. funding: this work was supported by the "deutsche forschungsgemeinschaft" transregio funginet projects c , c , c and z . introduction: to date, most research involving extracellular rnas has focused in rnas encapsulated inside extracellular vesicles (evs) or in total unfractionated biofluids. it is known that exrnas also exist outside vesicles or in lipoprotein particles. however, nonvesicular exrnas remain widely uncharacterized despite being a feasible source of contaminants in ev preparations. our interest in nonvesicular exrnas arises from the observation that some small rnas, such as specific trna-derived fragments, have much higher relative representation in this extracellular fraction. at least in part, this enrichment seems to be a consequence of their differential extracellular stability. methods: to get a representative picture of the whole set of rnas released to the extracellular nonvesicular space by cultured human cells, we inhibited extracellular degradation by adding recombinant ribonuclease inhibitor to the cell-conditioned media and studied the kinetics of rna release and degradation. high-resolution iodixanol gradients were used to separate evs from extracellular rnps or vesicle-free rna. the conversion rate between parental ncrnas and their fragments was studied by high-throughput sequencing and northern blot. results: the inhibition of extracellular rnase activity revealed the presence of full-length trnas and ribosomes in the extracellular space of a variety of malignant and non-malignant cell lines. extracellular ribosomes co-isolate with evs purified by ultracentrifugation or size-exclusion chromatography, but not with evs purified by density gradients.these ncrnas are substrates of extracellular rnases, demonstrating an extracellular biogenesis route for the formation of ncrna-derived fragments, some of which achieve remarkable stability and can be detected in biofluids. we also highlight the immunoregulatory potential of purified rna-containing extracellular complexes. summary/conclusion: in conclusion, ribonuclease inhibition dramatically shapes extracellular rna profiles and uncovers a population of extracellular ribosomes, trnas and other coding and noncoding rnas which exists outside evs. although these rnas are prone to degradation, some of their fragments can accumulate in cell culture media and in biofluids. this dynamic view of exrnas impacts our understanding of rna secretion mechanisms and may offer a window to new molecules with biomarker potential. in contrast, evs confer an rnase-protected environment and contain more full-length ncrnas (trnas, yrnas, sl rnas, rrnas depending on vesicle size) than their fragments. introduction: cd is a ubiquitously expressed membrane protein that functions as a receptor for thrombospondin- and the counter receptor for signal regulatory protein-α in phagocytes. high expression of cd is associated with a poor prognosis for some cancers. conversely, cd blocking agents are in clinical trials for enhancing innate and adaptive antitumor immunity in cancer patients. these studies suggest utility of cd as a diagnostic and prognostic biomarker and as a therapeutic target. cd is also expressed on extracellular vesicles (evs), and we reported that cd expression identifies a distinct population of evs from those that express the traditional ev markers cd or mhc . cd -, cd -and mhc -enriched vesicles contain distinct small rna populations (pmid: ), and these differ in rna content from evs that lack any of these markers. the mechanisms by which cd directly or indirectly regulates which rnas are packaged into ev remain unknown. methods: to elucidate the mechanism by which cd regulates ev rna composition and function, we performed global mirna microarray analysis between evs produced by wild type and cd -deficient t cells. results were further validated using real-time pcr and rna-immunoprecipitation. interactions between cd and exportin- /ran complex was identified by mass spectrometry and confirmed by using co-immunoprecipitation, subcellular localization, flow cytometry, and confocal and electron microscopy. results: ev released from cd -deficient human t cells and in cd -/-mouse plasma were enriched in ʹ- -methylguanosine-capped micrornas and mrnas that depend on the exportin- /rangtp pathway. knockdown of cd in wild type cells or thrombospondin- treatment correspondingly enhanced levels of capped-rnas released in ev and re-expressing cd in null cells decreased their levels. mass spectrometry and co-immunoprecipitation identified specific interactions of cd with components of the exportin- / ran nuclear export complex and its known cargos and between the cd cytoplasmic adapter ubiquilin- and the exportin- /ran complex. interaction of cd with exportin- was inhibited by leptomycin b, which inactivates exportin- and increased levels of cap-dependent rnas in ev released from wild type but not cd -deficient cells. summary/conclusion: these findings indicate that cd -dependent thrombospondin- signalling regulates cytoplasmic levels of cap-dependent rnas in t cells at least in part through ubiquilin- -and gtpdependent physical interactions with the exportin- / ran transport complex, which regulate levels of specific pre-mirnas and mrnas available for sorting into evs. funding: this work was supported by the intramural research program of the nih/national cancer institute (zia sc ). role of membrane protein palmitoylation in extracellular vesicle biogenesis in squamous cell carcinoma introduction: desmoglein (dsg ), is a palmitoylated cadherin that is involved in cell-cell adhesion. interestingly, dsg promotes mitogenic cell signalling and is upregulated in many cancers, including scc, contributing to poor prognosis and survivability. we recently demonstrated that dsg promotes ev release, but the mechanism by which dsg enhances ev biogenesis and role of palmitoylation is poorly understood. methods: pharmacological drug inhibitors -bromopalmitate, gw , and bafilomycin a were used. stable scc cell lines were established by retrovirus infection expressing gfp, wild type dsg /gfp, or palmitoylation deficient dsg cacs/gfp. evs were isolated by sequential ultracentrifugation and iodixanol gradient separation and analysed by nta. proteins associated with the endocytic pathway were analysed by immunofluorescence and imaged by confocal microscopy or immunoblotting and signals were quantitated using imagestudio. results: here we demonstrate that the effect of dsg on ev release was reduced by the palmitoylation inhibitor -bromopalmitate. furthermore, mutations that prevented palmitoylation (dsg cacs) dramatically abrogated ev release by targeting of un-palmitoylated dsg to the lysosomes for degradation. dsg increased expression and subcellular localization of flot , a membrane lipid raft protein critical for membrane invagination. dsg also altered membrane localization of several early (eps and eea ), but not late (rab , rab , and hrs), endocytic pathway proteins. loss of palmitoylation in the dsg cacs mutants abrogated these effects. finally, dsg -induced ev release was abrogated by the sphingomyelinase inhibitor gw or augmented by the v-atpase inhibitor bafilomycin a . summary/conclusion: the combined results of the drug treatments and functional mutations of dsg suggest that dsg plays a critical role in ev biogenesis by modulating proteins involved in early endosome sorting and is dependent on post-translational palmitoylation. introduction: the translation initiation factor eif e ( e) is an oncogenic protein that is upregulated in % of cancers including a subgroup of acute myeloid leukaemia (aml) patients. eif e regulates post-transcriptional rna processing including the nuclear export and/or translation of mrna transcripts. in particular, it selectively increases the expression of genes that have a prominent role in cancer progression such as myc, cyclin d , and mcl . furthermore, our lab pioneered studies demonstrating that a subset of ehigh aml patients is clinically responsive to treatment with a e inhibitor (ribavirin) indicating the importance of e in aml progression and its relevance as a therapeutic target. we investigated an as yet unexplored perspective of e-whether the oncogenic role of e is in part mediated by its function as a master regulator of vesiculation. methods: to assess mrna export and identify ebound mrna targets that correspond to vesiculationrelated genes and associated cargo, we used cellular fractionation and rna immunoprecipitation techniques. to determine whether e regulates the number of extracellular vesicles (evs) released as well as their protein and rna cargo we used nanoparticle tracking analysis (nta) as well as mass spectrometry, antibody microarrays, and rna sequencing technologies. results: eif e upregulates cellular protein levels of the vesiculation marker cd by increasing its nuclear export. in addition to increased cellular expression, cd , cd , cd , and flotillin- proteins are elevated in evs released from e-high cells. this is also associated with an increased release of vesicles that are - nm in size. currently, we have validated the upregulation of several receptors and cytosolic proteins in evs isolated from e-overexpressing cells that function in cell growth, migration, invasion, and stemness. the most abundant rnas in our ev preparations are micrornas (mirs) and we have confirmed downregulation of several of these. summary/conclusion: our work shows that e reprograms the vesiculation of cancer cells changing the release and cargo of evs. this may impact cellular communication and tumour biology, which we are currently addressing in functional studies. we hope that these studies will highlight novel therapeutic strategies for aml patients. intranasal administration of neural stem cells-derived extracellular vesicles promotes neurogenesis and reduces neuroinflammation and amyloid plaques in a mouse model of alzheimer's disease introduction: cognitive and memory impairments worsen with time in alzheimer's disease (ad), likely due to a progressive loss of hippocampal neurogenesis, and escalation of neuroinflammation. these changes are also accompanied by increased deposition of amyloid plaques in the brain. methods: in this study, using the xfad mouse model, we examined the efficacy of extracellular vesicles (evs) shed from the rat subventricular zone neural stem cells (svz-nscs) for disease modification. we first purified evs from the rat svz-nsc cultures through ion-exchange chromatography and then administered intranasally to -months old xfad mice (~ billion/week for two weeks). two months later, the functional effects of ev treatment were quantified through a series of behavioural tests, and animals were euthanized for quantification of hippocampal neurogenesis, oxidative stress, neuroinflammation, and amyloid plaque deposition. results: in comparison to ad mice receiving vehicle, ad mice receiving nsc-evs displayed improved cognitive function to discern minor changes in the environment in an object location test, better spatial recognition memory in an object-in-place test, and improved pattern separation ability in a pattern separation test. besides, ev-treated ad mice displayed no anhedonia in a sucrose preference test. analyses of neurogenesis using the birth-dating marker ʹ-bromodeoxyuridine and the newly born neuron marker doublecortin revealed maintenance of a higher level of hippocampal neurogenesis in ad mice receiving evs, in comparison to vehicle-treated ad mice. moreover, analyses of brain tissues from ev-treated ad mice revealed decreased concentrations of oxidative stress markers malondialdehyde and protein carbonyls and elevated levels of antioxidants catalase and superoxide dismutase. also, the concentration of proinflammatory cytokines tumour necrosis factor-alpha and interleukin- beta and the extent of amyloid plaques were significantly reduced in ev treated ad mice. immunohistochemical analysis showed reduced hypertrophy of astrocytes. summary/conclusion: intranasal administration of nsc-derived evs restrains the deterioration of cognitive and mood dysfunction of ad by maintaining higher levels of neurogenesis and curtailing the progression of neuroinflammation. funding: supported by a grant from the national institute of neurological disorders and stroke ( r ns - to a.k.s.) ot . the case of mesenchymal stromal cells, opening new perspectives in the use of ipsc in regenerative medicine. the aim of this study is to evaluate the potential of ipsc-ev in the treatment of kidney disease. methods: the ipsc were generated from skin fibroblast after informed consent of healthy donors (cytotune®-ips . sendai reprogramming kit -protocol: clementino fraga filho uh . . . / . ). the ev were isolated from ipsc supernatants (cultured h in mtesr- medium) by ultracentrifugation ( , g for h at °c). characterization of ipsc-ev was performed using zetaview, tem, exoview™ tetraspanin kit and macsplex exosome kit. for in vitro injury, renal epithelial cells were cultured under hypoxia ( % o ). for in vivo injury, male wistar rats were submitted to bilateral renal arterial clamping ( min) followed by reperfusion without or with injection of ipsc-ev (protocol approval: federal university of rio de janeiro - / ). kidney damage was assessed by histological and immunohistochemistry analyses (pcna, tunel and ed- ). modulation of rna levels was assessed by rt profiler pcr array. results: the results show that ipsc-ev reduce renal cell death, tissue damage, macrophage infiltration, promote mitochondria protection and ameliorate renal function. the ipsc-ev mechanism of action is related to the regulation of key genes known to prevent damage caused by oxidative stress like gstk , sod , sod , txn and txnrd . characterization of ipsc-ev showed that ipsc-ev can carry important molecules that can support renal recovery as epcam and prominin- . summary/conclusion: ipsc-ev presents renoprotective properties, acting on different aspects of aki. this presents a new relevant application of ipsc as a source of ev for therapeutic purpose in kidney diseases. the hospital for sick children, toronto, canada introduction: incomplete lung development, also known as pulmonary hypoplasia (ph), is a recognized cause of neonatal death. we have previously shown that experimental ph can be rescued by the administration of extracellular vesicles derived from amniotic fluid stem cells (afsc-evs) through an rna-mediated mechanism. this effect was not observed with evs derived from mesenchymal stromal cells (msc-evs) . the aim of this study was to ) evaluate which rna species were responsible for ph rescue, and ) to define the mechanism behind this effect. methods: evs were isolated and characterized from conditioned medium of rat afscs and rat mscs (control group) using ultracentrifugation. evs were assessed for size (nanoparticle tracking analysis), morphology (tem), and expression of cd , hsp , flo- , and tsg (western). to identify the mediators of afsc-evs, we used deseq (fdr< . ) to differentially analyse rna from: a) asfc-ev and msc-ev cargo, isolated with seramir and sequenced with nextseq. b) lung epithelial cells from rat ph lungs treated with vehicle or afsc-evs. epithelial cell rna was isolated with mirvana and sequenced with nextseq. we correlated afsc-ev cargo mirna with validated mrna targets that were downregulated after ev conditioning in lung epithelial cells. results: of the rna species contained in asfc-ev and msc-ev cargo, mirnas were the most proportionally different between the two ev populations. afsc-evs were enriched for mirnas that are critical for lung development, such as mir ~ and their paralogues that control lung branching morphogenesis. afsc-ev administration to ph lung cells significantly downregulated genes, which formed mirna-mrna reported interactions. summary/conclusion: afsc-evs contain many rna species in their cargo, but mirnas are the main effectors of their ability to rescue underdeveloped foetal lungs. we have identified for the first time that afsc-ev biological effect on underdeveloped foetal lungs is in part due to the release of mir ~ cluster. funding: cihr-sickkids foundation grant. bottom-up assembly of fully-synthetic extracellular vesicles oskar staufer a , franziska dietrich a , jochen hernandez a , martin schröter a , sebastian fabritz b , heike böhm a , ilia platzman a and joachim spatz a a max planck institute for medical research, department for cellular biophysics, jahnstraße , heidelberg, germany, heidelberg, germany; b department for chemical biology, max planck institute for medical research, jahnstraße , heidelberg, germany, germany, germany introduction: extracellular vesicles (evs) are considered as key elements for future therapeutic and diagnostic procedures. however, despite enormous research efforts to understand their physiological relevance and several greatly successful clinical trials, evs are currently not authorized for clinical routines by american or european regulation and approval agencies. this is especially because therapeutic evs are produced or isolated from cell cultures or biofluids, both of which are subjected to batch-to-batch variations and ill-defined contaminations. therefore, complementary technologies that produce evs as reproducible and defined as state of the art nanotherapeutics, would revolutionize the application of evs in clinical settings and provide the scientific community with a holistic understanding of ev-mediated signalling processes. in our study, we achieve de novo bottom-up assembly of fully synthetic evs (fsevs) that comprise identical physiological and therapeutic functionalities to natural evs. methods: we applied droplet-based microfluidic synthesis to sequentially amalgamate synthetic lipids, proteins and nucleic acids into defined vesicles that display analogous therapeutic capabilities to natural evs. fsevs were characterized by electron and confocal microscopy, dynamic light scattering and mass spectrometry and tested on organotypic models or in vivo. results: using previously described evs as "naturegiven" blueprints, we assembled several fsevs in their exact molecular composition. in particular, we produced wound-healing promoting evs composed of several exosomal proteins, lipids and micrornas and showed that their therapeutic performance on human skin wounds is equivalent to that of natural evs. besides their high molecular complexity, being composed of dozens of different molecular building-blocks, the presented fsevs are completely defined on a quantitative level. based on this, we achieved a stoichiometric understanding of cell-vesicle interactions. summary/conclusion: by applying bottom-up synthesis of fsevs for quantitative studies on ev signalling, we not only provide innovative and safe compounds for ev-therapeutics but also a vastly new perspective on the application spectrum of extracellular vesicles in fundamental research. introduction: small extracellular vesicles (sevs) contain functional molecules from their cell of origin and can enter recipient cells for intercellular communication. ifnβ has been shown to induce some lncrnas to regulate host immune response and play a major role in the positive regulation of the activity of natural killer (nk) cells. here, we aim to clarify whether ifnβ induced sevs can regulate the cytotoxicity of nk cells by transferring specific lncrnas into nk cells. methods: evs were purified from a with/without ifnβ treatment by serial centrifugation followed by sucrose density gradient purification. elisa assay were performed to demonstrate the cytotoxicity of nk cells. qpcr and western blot were used to verify the expression of nkp . results: surprisingly, ifnβ induced sevs can strengthen the cytotoxicity of nk cells. through human transcriptome array (hta) we found the expression levels of lncrnas were significantly changed within sevs isolated from a cells following ifnβ treatment. additionally we found a specific sev cargo, linc-epha - , acted as a competing endogenous rna (cerna) for hsa-mir- which subsequently up-regulate the natural cytotoxicity receptor (nkp ) expression. furthermore, we verified over-expression of linc-epha - significantly enhance the cytotoxicity of nk cells against zika virus-infected a cells. summary/conclusion: our results demonstrated that ifnβ-induced linc-epha - wrapped in sevs can regulate the cytotoxicity of nk cells. our study provides a novel link between type i ifn and nk cells, which are two major players for the host innate immunity against pathogen infections. introduction: hiv-infected t cells release simultaneously viral particles and small extracellular vesicles (sevs) including mvb-derived exosomes and plasma membrane-derived evs. sevs and hiv share many physical and chemical characteristics, which makes their separation difficult. although several approaches have been used to obtain sevs free of virus they leave a majority of sevs within hiv preparations. for this reason, the function of sevs during hiv infection remains unclear. methods: we have developed a novel un-biased proteomic profiling approach to identify specific markers of the virus or sev subtypes released by a human t lymphoma cell line. our approach was to combine differential centrifugation of medium/small evs contained in the ccm with quantitative mass spectrometry to generate protein abundance profiles across the different sub-fractions. we generated an interactive database to define groups of proteins with similar profiles, suggesting their release in the same evs. results: we thus identified different categories of evs, which bear different surface proteins, e.g. different combinations of t cell surface markers, integrins or tetraspanins. in evs released by infected cells, we identified cellular proteins behaving like hiv proteins, and several that changed behaviour after infection, either moving towards or away from the hiv cluster. we identified two cell-derived proteins that are included in the viral particles and one that is specific of non-viral sevs that are modified by infection, and analysed their respective roles in controlling ev composition or virus infectivity. summary/conclusion: our approach presents a powerful tool for identification of common cargoes of given ev subtypes, and could be now used to identify modifications of ev composition in any given physiological or pathological situation. the encephalomyocarditis virus leader modulates autophagic pathways to promote the release of virions inside extracellular vesicles introduction: recent data indicate that naked viruses belonging to the picornaviridae family can be released from host cells via enclosure in extracellular vesicles (ev). ev cloak virus particles in a host-derived "envelope" and can thereby affect antiviral immune responses and disease severity. a better understanding of the formation and function of ev-enclosed viruses is therefore required. previously, we showed the presence of the autophagosome marker lc in ev isolates from encephalomyocarditis virus (emcv) infected cells, suggesting the involvement of a secretory autophagy pathway in ev-mediated virus release. however, little is known about the viral and host factors that regulate this process. here, we have assessed the role of the emcv leader, a viral protein that is dispensable for replication but is required for symptomatic disease. methods: cells were infected with wildtype virus or a mutant carrying an inactive leader. ev produced during the infection were isolated using differential ultracentrifugation and density gradient purification. ev were characterized by high resolution flow cytometry and their infectivity determined using end-point dilution assay. in addition, the fate of autophagosomes in infected cells was monitored using a reporter assay for autophagosome-lysosome fusion and analysis of the secretion of autophagosomal proteins. results: inactivation of the emcv leader strongly reduced the release of ev-enclosed virus. whereas autophagosomes are typically degraded, we show this is blocked by the leader. instead, autophagosomes fuse with the plasma membrane, as indicated by the secretion of autophagy marker lc during infection with wildtype but not the mutant virus. pharmacological reactivation of degradative autophagy in infected cells resulted in a strong reduction in the release of ev and ev-enclosed virus. similarly, the reduction in evenclosed virus release in the absence of the leader could be partially reversed by drugs that promote the secretion of autophagosomes. summary/conclusion: our data supports a role for secretory autophagy in the release of viruses in ev, a pathway that is regulated by the emcv leader. these findings highlight an unconventional route for ev formation that intersects with autophagosomal compartments and contributes to viral pathogenesis. introduction: zika virus (zikv) causes a public health emergency of international concern because of its correlation with microcephaly. during viral infection, the innate immune response quickly to produce some endogenous functional molecules which can prevent viral invasion or replication. extracellular vesicles (evs) contain molecules from their cell of origin under virus infection and can enter recipient cells for intercellular communication. here, we aim to clarify whether zikv induced evs can regulate viral pathogenicity by transferring specific rna. methods: evs were purified from a with/without zikv infection by serial centrifugation followed by sucrose density gradient purification. human transcriptome array (hta) was used to found rna expression within evs. flow cytometry was used to determine cell cycles. zikv replication was assayed by qpcr and western blot. flow cytometry was used to determine cell cycles. results: through hta we found the defensin alpha b (defa b) expression was significantly increased within evs isolated from zikv infected a cells. additionally, we found that the extracellular defa b but not the intracellular defa b exerts anti-zikv effect mainly before entry step. surprisingly, up-regulate defa b can retard cell cycles of host cell. we verified defa b could bind with the origin recognition complex (orc ) which is required to start dna replication during the cell cycle. furthermore, up-regulate defa b decreased the orc level in nuclear. interestingly, evs with defa b can internalize into recipient cells and inhibit their cell cycles. summary/conclusion: together, our results demonstrated that zikv infection can induce defa b wrapped in evs, and defa b not only exerts anti-zikv effect but also regulate cell cycles which may affect neurodevelopment. our study provides a novel viewpoint that defa b act as first-line anti-viral molecules during zikv infection also correlate with neurodevelopment by retarding cell cycles. extracellular vesicles mediate bacterial-immune cell interactions during respiratory viral-bacterial co-infections sidney w. lane a , matthew hendricks b and jennifer bomberger a a university of pittsburgh, pittsburgh, usa; b university of washington, seattle, usa introduction: respiratory infections are a major cause of morbidity and mortality worldwide and host-derived extracellular vesicles (evs) play important roles in mediating these infections. during respiratory infection, evs are shown to have a modulatory effect: promoting or suppressing infection dependent on the pathogen and cell type. in the age of next-generation sequencing, we now appreciate that many respiratory infections are polymicrobial in nature, with viral-bacterial co-infections correlating with worse disease outcomes. epidemiological studies correlate acute viral infections with the increased likelihood and severity of both acute and chronic secondary bacterial infections; however, the exact mechanisms of these interactions remain poorly understood. evs have been understudied in the context of respiratory viral-bacterial coinfections; thus, their role in mediating these infections is relatively unknown. unpublished data from the lab shows that in airway epithelial cells (aecs), viral infection induces the release of evs that associate with pseudomonas aeruginosa (pa) and promote biofilm growth. here, we aim to expand upon these findings and determine how aec evs mediate pa-immune cell interactions during respiratory viral-bacterial co-infection. methods: to determine how exposure to evs impacts pa-immune cell interactions, evs were isolated from the apical secretions of aecs and co-cultured with pa. ev-treated pa was then co-cultured with macrophages to evaluate ev impact on pa uptake and survival. results: in preliminary experiments using control evs, we observed that evs associate with pa. interestingly, during co-culture with macrophages, ev-treated pa are more susceptible to phagocytosis in comparison to non-treated pa. however, after hours of co-culture with macrophages, ev-treated pa are able to survive and replicate, while nontreated pa are effectively controlled by the macrophages. summary/conclusion: these findings suggest that while pa-ev association promotes pa uptake, it may ultimately enhance pa immune evasion and survival. ongoing experiments in the lab are evaluating the mechanism of pa-ev association and how evs from virus-infected aecs affect the phenotypes observed with control evs. notably, this is one of few reports of a mammalian ev influencing the pathogenesis of a bacterium; thus, results from these experiments will define the function of aec evs in regulating bacterial-immune cell interactions during respiratory co-infections. using machine learning with neuronal ev target proteins and clinical data to predict cognitive impairment in hiv infection lynn pulliam a , michael liston b , bing sun c and jared narvid d a university of california, san francisco, san francisco, usa; b veteran affairs, san francisco, usa; c ncire, san francisco, usa; d ucsf, san francisco, usa introduction: objective biomarkers are needed to assess and predict neuronal function and cognitive impairment. in people ageing with chronic infections, such as hiv, determining the mechanism of impairment will be important when therapies are available. methods: sixty plasma samples from hiv-infected people were obtained from nih-sponsored aids banks. clinical and epidemiological data were collected. all underwent neuropsychological testing and were considered impaired. neuronal extracellular vesicles (nevs) were isolated from plasma and assayed for high-mobility group box (hmgb ), neurofilament (nf-l) and phosphorylated tau- (p-tau) proteins. results: using different algorithms, support vector machines (svm) performed the best with an area under the curve (auc) value of . ± . . using different combinations of clinical data and the nev protein targets, selected clinical data and hmgb best predicted cognitive impairment (auc = . ). the most important features included cd count, hmgb , nf-l and education. summary/conclusion: specific clinical features plus nev hmgb , an inflammatory marker, were the best predictors of cognitive impairment. previous published data showed nev p-tau- elevated in alzheimer's disease and in this study p-tau had no importance in assessing hiv-associated cognitive impairment. nev target discovery can be improved to better identify neuronal damage, possibly to differentiate other neurodegenerative diseases and hopefully recovery after therapies are identified. in recent years, we have been able to separate and characterize extracellular vesicles (evs) from several different viruses including hiv- , htlv- , rift valley fever virus and ebolavirus. however, to date it is not clear whether there is a timing difference between ev and virus release from infected cells. methods: ev isolation by nanoparticle capture and differential centrifugation, ev quantification by nanoparticle tracking analysis, western blot, rt-qpcr, virus rescue assay. results: we have attempted to address the kinetics of ev and virus release from multiple-infected cells using serum starvation experiments from infected ( %) cells. these infected cells were initially put in g quiescent stage using serum starvation. both supernatants and cell pellets were collected postinduction release ( % fbs + pma/pha) at , , , , and hours and examined for the presence of ev, autophagy and viral proteins as well as viral rna expression. results from supernatants of uninfected cells showed a peak of tetraspanin proteins (cd , cd , and cd ) at hours and a gradual decrease of all ev associated proteins by hours. however, the ev from hiv- infected cells showed all three tetraspanins present at hours and expression gradually increased up to hours. when compared to htlv- infected cells, the three tetraspanin proteins peaked at hours and expression continued to decrease up to hours. htlv- infected cells also showed a unique pattern of cd expression. autophagy associated proteins (lc a, lc b and p ) from uninfected cells and htlv- infected cells plateaued at hours, whereas in hiv- infected cells their expression continued to increase and peaked at hours. hiv- viral proteins (p , gp , nef) expression was present at hours and continued to increase and peaked at hours. htlv- proteins (p and gp / ) peaked at hours and gradually decreased overtime. hiv- and htlv- rna gene expression analysis was performed, and data correlated with viral protein expression. additionally, evs release was quantified and showed significant increase of ev concentration overtime in both uninfected and infected samples. finally, experiments of infectivity from -and hour supernatants were performed on three naive cells. hiv- supernatant -hour sample was found not to be infectious. however, hiv- was successfully rescued from -hour sample. introduction: urinary extracellular vesicles (uevs) are important intercellular communicators. by systems biology integration, uevs prove to be relevant in genitourinary disease detection. however, it has recently been shown that labelled evs administered to the circulation can be detected in the urinary system, as well. thus, this pilot study aimed at phenotyping haematopoietic surface markers on uevs to create enough plausibility for future non-invasive biomarker studies of circulation and immune disorders that may translate into urine but are not yet timely recognized. methods: urine was obtained from healthy men signing a written informed consent (n = ). sampling was approved by the local ethics committee and in compliance with the declaration of helsinki. cell-free urine was obtained by serial centrifugation and ml, each, were utilized for the macsplex exosome kit, human (miltenyi biotec). the manufacturer's recommendations were followed to examine distinct uev surface markers of cd +/cd +/cd + vesicles in a multiplexed bead-based manner including respective controls. the accuri c (bd) was utilized for data acquisition. for further misev -compliant characterization, cd +/cd +/cd + uevs were isolated by immunoaffinity and analysed by fluorescence nanoparticle tracking (f-nta), transmission electron microscopy (tem) and western blotting (wb). urinary creatinine (ucrea) was determined to control for variances in urinary dilutions and used for data normalization. results: except cd , all other surface markers could be identified. the most abundant markers were cd and cd , which were detected in % of samples, followed by cd / ( %), cd ( %), cd and cd ( %, each). cd ( %), cd , cd ( %), cd e ( %) and cd showed similar relative median fluorescent intensities (rmfi), while cd yielded significantly higher (p = . ) and all other markers significantly lower rmfi (p < . ). tem and f-nta revealed cup-shaped vesicles ( ± nm) with . ± . e + particles/g ucrea. wb indicated uev isolates that were positive for alix, syntenin, tsg , cd , cd and cd without any uromodulin or calnexin contamination. summary/conclusion: our results imply that considerable quantities of circulatory evs are, indeed, filtered into urine and could serve as valuable non-invasive biomarkers for systemic dysfunctions. cardiovascular risk markers are strongly related to numbers of circulating extracellular vesicles ruihan zhou a ,esra bozbas a , plinio ferreira b and parveen yaqoob a a university of reading, reading, uk; b imperial college london, london, uk introduction: extracellular vesicles (evs) are small plasma membrane-derived vesicles released from various cells, which potentially affect many physiological and pathophysiological processes, and are emerging as a potential novel biomarker in cardiovascular diseases (cvds). however, there is little information about the association of circulating ev levels with traditional cardiovascular risk markers and cvd risk score. methods: • subjects (n = ) aged - yrs with moderate risk of cvds were recruited and assessed for body mass index (bmi), blood pressure (bp) and plasma lipid profile (triacylglycerol, total cholesterol and high-density lipoprotein). • evs were isolated from platelet-free plasma by size exclusion chromatography and analysed by both nanoparticle tracking analysis (nta) and flow cytometry (fcm). nta was used to measure the concentration and size distribution of evs population, and evs were phenotyped by fcm via a -colour panel, which included annexin v (for the majority of circulating evs), cd (for platelet-derived evs) and cd (for endothelial-derived evs). • the association between risk markers and ev numbers was examined by pearson's correlation coefficient and stepwise multivariate regression model. analysis of covariance (ancova) was performed after adjustment for various variables to determine the correlation between the quartile range of ev numbers and -yr cvd risk detected by qrisk . results: ev numbers, as determined by nta, were strongly associated with bmi (r = . , p < . ), blood pressure (systolic bp: r = . , p = . ; diastolic bp: r = . , p < . ) and plasma triacylglycerol levels (r = . , p < . ). plasma total cholesterol level was positively associated with platelet-derived evs, determined by fcm (r = . , p = . ). a multivariate regression model demonstrated that plasma triacylglycerol and diastolic bp independently predicted total ev numbers, with plasma triacylglycerol concentrations explaining . % of the variance for total ev numbers. an additional . % of the variance in total ev numbers was predicted by diastolic bp. ancova of the -yr cvd risk score in the quartile range of total ev numbers were positively and independently associated. summary/conclusion: bmi, blood pressure, plasma triacylglycerol and total cholesterol levels are strongly associated with ev numbers. plasma triacylglycerol and diastolic bp independently predict circulating ev numbers. elevated numbers of evs are independently associated with -yr cvd risk. introduction: extracellular vesicles from cardiospherederived cells (cdc-evs) are known to be anti-inflammatory in various disease models. to further dissect the mechanism, we examined the effects of cdc-evs on t lymphocytes. methods: naïve cd + t cells were isolated from secondary lymphoid organs of foxp -rfp reporter mice, using magnetic-activated and fluorescence-activated cell sorting. cells were subsequently polarized into effector subtypes (th , th , and th ), as well as regulatory t cells (tregs), and the effects of exposure to human-derived cdc-evs on proliferation and cytokine production were assessed. cdc-evs were isolated from serum-free, -day conditioned medium, using ultrafiltration by centrifugation. results: after polarization and culture for days, cdc-evs resulted in dose-dependent and cell-specific proliferative responses. effector t cells (th , th , th ) showed either no change in proliferation (th ) or decrease in proliferation (th , th ), compared to the vehicle control. in contrast, tregs proliferated much more than control (p < . ). next, we sought to characterize the changes in cytokine production by each effector t cell and tregs. compared to the vehicle control, exposure of polarized effector t cells to cdc-evs had little effect on the expression of characteristic cytokine genes, including ifnγ and tnfα (th ), il and il (th ), or il a and il f (th ). in contrast, exposure of tregs to cdc-evs resulted in~ -fold increase in expression of il , a key paracrine agent utilized by tregs in suppression of inflammation. this response was specific to cdc-evs insofar as it was not recapitulated with dermal fibroblast exosomes. concentrations of il- in the culture media of cdc-ev-conditioned tregs mirrored the increases in gene expression. summary/conclusion: cdc-evs potentiate tregs by increasing their proliferation and enhancing production of il- . this offers an attractive therapeutic approach to inflammatory diseases that relies on harnessing an endogenous mechanism of immunosuppression. funding: nih t hl . prostanoids impair platelet reactivity, thrombus formation and platelet extracellular vesicle release in patients with pulmonary arterial hypertension aleksandra gąsecka a , marta banaszkiewicz b , rienk nieuwland c , edwin van der pol d , najat hajji e , hubert mutwil f , sylwester rogula a , wiktoria rutkowska a , szymon darocha g , grzegorz opolski a , krzysztof j. filipiak f , adam torbicki g and marcin kurzyna g introduction: prostanoids (epoprostenol, treprostinil and iloprost) induce vasodilation in advanced pulmonary arterial hypertension (pah) but also inhibit platelet activation, thereby increasing the risk of bleeding. therefore, the platelet function and extracellular vesicle (ev) concentrations were measured in pah patients treated with prostanoids and compared to patients with pah not receiving prostanoids. methods: venous blood was collected from patients treated with prostanoids (study group; n = , ± years, % female) and patients not treated with prostanoids (control group; n = , ± years, % female). platelet reactivity was analysed in whole blood by impedance aggregometry using arachidonic acid (aa; . mm), adenosine diphosphate (adp; . µm) and thrombin receptor-activating peptide (trap; µm) as agonists. in a subset of patients, concentrations of evs from platelets (cd + and cd p+; pevs), leukocytes (cd +, levs) and endothelial cells (cd +, eevs) were measured in plateletdepleted plasma by flow cytometry (a -micro). platelet-rich thrombus formation was measured using a whole blood perfusion system. results: compared to the control group, patients treated with prostanoids had lower platelet reactivity in response to aa and adp (p = . ) and lower concentrations of pevs and levs (p ≤ . ). furthermore, thrombus formation was delayed (p ≤ . ) and thrombus size was decreased (p = . ) on prostanoids. epoprostenol did not affect platelet reactivity in vitro, but decreased the concentrations of cd + pevs (p = . ). in contrast, treprostinil and iloprost decreased both platelet reactivity in response to aa and adp (p ≤ . ) and the concentrations of pevs (p ≤ . ). all prostanoids delayed thrombus formation and decreased thrombus size (p ≤ . ). introduction: progressive lung disease is the leading cause of mortality in cystic fibrosis (cf), a chronic condition characterized by recruitment of polymorphonuclear neutrophils (pmns) into the airways. newly arrived pmns are exposed to extracellular vesicles (evs) from the airway epithelium and pmns recruited before them. in controlled experiments, these evs were necessary and sufficient to induce pathological changes including reduced bacterial killing and immunosuppressive activities towards macrophages and t-cells. however, children with cf do not always show a high pmn presence in their airways, which suggests that the balance between pmn recruitment and the activity of other cells is still in flux in early stage disease. methods: we utilized spectral nanoflow cytometry to profile the single ev content of the bronchoalveolar lavage fluid (balf) from cf children (< years of age). for nanoflow cytometry, evs were stained with di- -anepps, and with epcam, cd b and cd (to ascertain epithelial, pmn, and macrophage origins, respectively). violet side scatter and/or fluorescence threshold triggering were used for ev detection. results: the ratio of neutrophil-to epithelial-derived evs in cf balf correlated positively with the percentage of pmns that are present in the airways (p = . , spearman's rho = . ). this ratio also correlated with the pragma disease score, which quantifies airway damage by chest computed tomography (p = . , rho = . ). summary/conclusion: using a method to quantify evs from specific cell types in vivo, we demonstrated that the ratio of pmn-and epithelial cell-derived evs tracks with airway damage and neutrophil influx, suggesting a critical interplay between these cells in early cf disease. this ev-focused method can be applied to other diseases in which sampling cells is difficult. future experiments will use cf balf biobanks to strengthen data presented here. funding: cf foundation (tirouv a ), emory pediatrics flow core. the potential of crude extracellular vesicle micrornas for the diagnosis of community-acquired pneumonia and for the detection of pneumoniarelated sepsis as a severe secondary complication introduction: circulating cell-free micrornas (mirnas), often associated to extracellular vesicles (evs), are essential for cell-cell communication in the pathogenesis of infectious pulmonary disorders. as early pneumonia diagnosis is often clinically challenging, advances in disease detection could improve outcomes. we characterized crude ev mirnas as potential biomarkers for community-acquired pneumonia and sepsis as a severe secondary complication. methods: individuals were enrolled into our study, subdivided into a training (volunteer n = , pneumonia n = , sepsis n = ) and testing cohort (volunteer n = , pneumonia n = , sepsis n = ). after precipitating crude evs from sera (mircury exosome isolation kit-serum and plasma) and extracting total rna, small rna sequencing was performed. mirnas were selected as biomarker candidates by differential gene expression analysis (deseq ) and sparse partial-least-squares discriminant analysis (mixomics). technical and biological validation was performed by reverse transcription quantitative realtime pcr. group classification was predicted by partial-least-squares discriminant analysis. gene targets and causal networks were identified by ingenuity pathway analysis. results: differential gene expression analysis revealed significantly regulated mirnas in pneumonia compared to volunteers, and mirnas in pneumonia related to sepsis. based on sparse-partial least discriminant analysis, group separation was achieved by mirnas as discriminators with high sensitivity and specificity (area under the curve of the receiver operated curve: volunteer: . , pneumonia: . , sepsis: . ). mir- a- p (log fc = . , padj = . e- ) and mir- - p (log fc = . , padj = . e- ) differentiated between pneumonia and volunteers and mir- (log fc = − . , padj = . e- ) between pneumonia and sepsis. expression levels of mir- a- p and mir- were related to disease severity. mir- - p was higher expressed in pneumonia compared to volunteers and had equal expression in patient groups. prediction of group classification in the testing cohort was . %. signalling networks were constructed for "cellular and humoral immune response", "antimicrobial response" and "pathogen influenced signaling" involving the significantly regulated mirnas. summary/conclusion: crude ev mirnas are potentially novel biomarkers for community-acquired pneumonia and may help to identify patients at risk for progress to sepsis allowing early intervention and treatment. introduction: it remains unclear the specific mechanisms that lead to airways inflammation in asthma and the subsequent remodelling of the airways. exosomes, small extracellular vesicles, has become in an important mechanism of cell-to-cell communication and participate in diverse biological processes including inflammation. in this study, we hypothesize that exosomes and their mirna cargo play an important role in the proinflammatory status of the upper airway of asthma patients, especially in those patients with severe asthma. methods: in a pilot study, healthy subjects had induced sputum using standard methods. after several centrifugation steps, we were able to isolate exosomes from sputum supernatant by both precipitation and size exclusion cromatography (sec). exosome size was observed with transmission electron microscopy (tem) and the protein markers cd and cd were analysed by western blot (wb). then, total rnas were isolated from sputum exosomes and mirnas (mir- a-p, mir- - p, mir- a, mir- b- p, mir- - p, mir- - p, mir- - p, mir- - p, let- g- p) , were evaluated by rt-qpcr. after the optimization of the methodology, healthy adults subjects and patients with persistent moderatesevere asthma, matched by age and sex were selected and induced sputum was collected. results: exosomes isolated with both methodologies (precipitation and sec) were observe under the tem with a correct range of size. furthermore, wb assay displayed a coherent protein profile for the exosome markers cd and cd . however, sec displayed low signal and the variability of between subjects was to higher. using the optimized method of precipitation, we observed that after normalization, mirna- a showed a significant increased (p = . ) in asthma patients compared to control. this mirna has been linked with an active proinflammatory status. summary/conclusion: our results confirm the presence of exosomes in induced sputum with promising applications in the field of asthma. the upregulation of exosomal mir- a, which is related with inflammation, suggest that exosomes could play a crucial role in the chronic inflammation of airway described in asthma patients. human nrf -active multipotent stromal cell exosomes reverse pathologic delay in the healing of cutaneous diabetic wounds joseph kuhn a , absara hassan b , sonali sharma b , jennifer kwong b , montaha rahman b , salma adam b , jasmine lee b , alvaro villarreal ponce b and piul rabbani b a nyu langone health, new york, usa; b nyu langone health, new york, usa introduction: multipotent stromal cells (mscs) have attracted much attention for their capacity to accelerate wound healing. exosomes, nanosized extracellular vesicles, may be key to translating msc therapy. we previously found that nuclear factor erythroid -related factor (nrf ) regulates msc promotion of diabetic tissue repair. here, we explore a novel role of nrf in exosome biogenesis and investigate whether exosome treatment recapitulates the effects mscs have on healing. methods: exosomes were harvested by differential ultracentrifugation of conditioned bone marrow derived msc media. for nrf -active exosomes, mscs were incubated with potent nrf activator, cddo-im. exosomes and mscs were vigorously characterized. full-thickness humanized-stented wounds were created on adult leprdb/db diabetic mice (db/db). exosomes were injected intradermally and circumferentially to the wound margin. results: mscs adopt an adherent fibroblast morphology, demonstrate robust osteogenic, chondrogenic, and adipogenic differentiation, express > % positive msc markers (cd , cd , cd , and cd ) and < % express negative markers (cd , cd , cd , cd , or hla-dr). immunoblotting of msc exosomes shows enrichment for positive exosomal markers cd , cd and tsg . nanoparticle tracking analysis (nta) shows a nanoparticle population with mean diameter of . ± . nm. transmission electron microscopy of exosomes reveals flattened cup-like spheres. nta demonstrates that nrf -active human mscs increase exosome secretion by %, compared to nrf -baseline mscs (p < . ). both nrf -baseline and nrf -active exosome treatment significantly reduced closure time to . and days respectively, compared to . days for vehicle-treated wounds (p < . ). this reduction eliminated the delay in closure time compared to wounds of c /b mice. nrf -active exosome treatment of db/db wounds reduced closure time by a further . days compared to untreated c /b wounds. at day , exosometreated db/db wounds have significant decreases in epithelial gap, expanded granulation tissue, and greater density of cd + vessels compared to vehicle-treated wounds. introduction: obesity increases prostate cancer aggressiveness and adipose tissue (at) is a rich source of extracellular vesicles (ev) that have been shown to contribute to pro-oncogenic effects in various malignancies. twist is a key mediator of tumour cell metastasis.. the goal of this study was to determine molecular and phenotypic changes of prostate cancer cells in response to evs from obese human at and the role of different levels of endogenous twist . methods: ev were harvested from human at (atev) obtained from bariatric subjects or from at endothelial cells treated with proinflammatory cytokines (pic-ev) to mimic the obese at environment. evs were isolated by ultracentrifugation and characterized by electron microscopy, nta and protein markers. we determined the effect of atev and pic-ev on pc -ml prostate cancer cells proliferation and invasion. ev mirna cargo and transcriptome of pc -ml cells treated with atev or pic-ev were assessed using nanostring. to establish the contribution of twist to the ev-related phenotypic and molecular changes in recipient cells, we used pc -ml lines stably overexpressing or deficient in twist . results: atev from obese subjects and ev-pic from at endothelial cells both reduced invasion and increased proliferation in wild-type pc -ml cells. a molecular signature showing decreased expression of genes mediating invasion, adhesion and metabolism supported these functional effects. also atev and ev-pic shared a subset of mirna that target multiple mmps, inhibit glycolytic genes and target cell cycle inhibitory genes. pc -ml overexpressing twist showed an increase in both proliferation and invasiveness and this phenotype was supported by the transcriptomic analysis following ev treatment. summary/conclusion: ev produced by obese at or by at endothelial cells share a subset of mirna that in conjunction with increased twist expression contribute to tumorigenesis and metastasis of prostate cancer cells in vitro. funding: american heart association of symposium introduction: as researchers continue to explore the therapeutic potentials of extracellular vesicles (evs) for the treatment of many diseases, there is a growing unmet need for real-time in vivo monitoring of these therapeutic evs after they are injected into a subject to understand their safety, targeting, and effectiveness. while current optical imaging solutions like bioluminescence and fluorescence are useful for ev tracking studies in animal models, there is limited utility in clinical applications. here we present a novel ev tracking solution utilizing clinically applicable mri technology. methods: to generate trackable evs, cells were labelled with a clinically applicable novel magnetic agent. evs secreted by the labelled neural stem cells and amniotic fluid stem cells (afscs) were isolated by differential ultracentrifugation. the viability and morphology of labelled-cells were evaluated, and the in vitro mr properties of their derived evs were analysed by magnetometer. a proof of concept in vivo biodistribution study was conducted by injecting labelled evs into wt and alport mice (a model of chronic kidney disease) via retro-orbital and intra-cardiac routes and tracking them via mri at min and hr postinjection. results: the magnetic label did not affect the physiological characteristics of the cells. the mr detectability of labelled-evs was confirmed by in vitro/ex vivo mri phantoms. mri studies showed that homing of afsc evs to the kidney injected intra-cardiacally into alport mice were more efficient versus the retro-orbital route, and prussian blue staining of kidney sections confirmed the mr findings. introduction: a central question in ev biology is the fate of circulating ev. this can be evaluated by developing non-invasive ev bioimaging techniques in mice in order to benefit from transgenic and knock-out models. recent reports described ev biodistribution in vivo using optical (fluorescence) and nuclear imaging. but the physicochemical properties of the probes impact ev integrity, labelling efficiency, background signals and observation timecourse. methods: we developed the radiolabeling of red blood cells (rbc) and ev with [ f]fluorodeoxyglucose ( f-fdg). we used rbc-derived ev in their native, intact form, without pre-experimental processing (no centrifugation or filtration). we tracked f-fdg in vivo by pet-scan, within seconds of ev, rbc or free f-fdg injection, and during their dissemination in blood and recruitment by organs over one hour. ev and rbc biodistribution were confronted to the kinetics of free f-fdg. results: we collected images of the biodistribution of rbc, and rbc-derived ev. nuclear imaging was well suited for accurate studies of ev organotropism, with high sensitivity, excellent signal-to-noise ratio, very low signal absorption by tissues and an inherent quantitative tomographic nature. ev-specific signals were mostly accumulated within minutes of injection (tail vein), in the spleen and liver, with a small part in the bone marrow (femurs). signals in other compartments were largely transient and linked to tissue perfusion and blood volume. we selected the most drastic control conditions to secure a correct interpretation of the data. this made kidneys, hearts and brains unavailable for analysis. hence the new approach came with limitations, but we describe how "free" f-fdg signals can be used to draw sound conclusions for ev. summary/conclusion: we propose that three types of compartments coexist in control mice at rest: active ev-capturing organs with high capacity and specificity including the spleen, and to a lesser degree the bone marrow; passive ev-retaining organs with high capacity, including the liver; and ev-neutral organs where transient signals only mirror tissue perfusion. we also report how ev biodistribution patterns are altered in ageing animals, as an example. we hope that this novel, non-invasive, quantitative, dynamic wholebody imaging approach will help characterize native cell-derived ev and help set standards for the reproducibility of ev bioimaging in mice. funding: frm grant "biface", inserm copoc, cnrs. introduction: extracellular vesicles (ev) are important mediators of intercellular communication; however, basic principles of ev biogenesis and loading remain largely unknown. a limited repertoire of tools has thus far made these processes challenging to research. the development of an ev-transfer reporter in a genetically tractable organism such as drosophila has allowed us to study mechanisms of cargo loading in vivo and has provided us with a platform to explore fundamental aspects of ev biology. methods: we have developed a bioinformatic pipeline to analyse the properties embedded in the ʹutr of mrnas enriched in evs released by drosophila cells. in parallel, we have adapted a cre-loxp system for use in fruit flies that appears to be proficient to reveal the exchange of bioactive molecules between secretory and recipient cells. results: taking advantage of computational methods, we uncovered sequence motifs that preferentially appear in combinations along the ʹutr. these sequence motifs occur within characteristic secondary structures, in a way that is more variable and motif dependent than previously reported. identified motifs also show similarities to known binding sites for rna binding proteins; a feature potentially important for ev-loading. in parallel, we developed a drosophila in vivo system to detect cell communication in complex tissues and between different cell types. using this system, we studied the biological significance of specific sequence motifs and identified their ability to modulate mrna ev-transfer in a context dependent and evolutionarily conserved manner. summary/conclusion: in summary, we have developed a novel tool to study cell communication in complex tissues, and shown its effectiveness to study principles of ev biogenesis and loading. beyond improving our understanding of ev biology and providing a novel tool to the scientific community, we hope this knowledge will pave the way to harnessing evs as a means of remotely manipulating cell communication in many biological contexts. introduction: the idea of cross-kingdom, species and inter-individual transfer of bioactive compounds via extracellular vesicles (evs) is a recent avenue. however, the bioactivity and bioavailability of these dietary compounds upon consumption is highly debated. it has been proposed that evs from diet can absorbed by consuming organisms, be bioavailable in various organs and exert phenotypic changes. milk is the most vastly consumed beverage and is an abundant source of evs that may act as signalosomes. whether these milk-derived evs can serve as cross-species messengers and have a biological effect on host organism has been poorly understood. methods: bovine milk-derived evs were isolated by ultracentrifugation and optiprep density gradient centrifugation. the evs were characterised by tem, nta, quantitative proteomics and rna-seq. evs were orally administered to various mice models of colorectal, breast and pancreatic cancer. primary tumour burden was monitored, and the rate of metastases was measured by imaging and qpcr. immune cells were analysed by facs. mechanistic insights were obtained using quantitative proteomics, confocal microscopy and biochemical experiments. results: we demonstrated that upon oral administration, bovine milk-derived evs were able to survive the harsh degrading conditions of the gut and be bioavailable in peripheral tissues. interestingly, oral administration of milk-derived evs reduced the primary tumour burden in various cancer models and attenuated cancer cachexia. intriguingly, despite the reduction in primary tumour growth, milk-derived evs accelerated metastasis in breast and pancreatic cancer mice models. timing of ev administration was critical as oral administration after resection of the primary tumour reversed the pro-metastatic effects of milkderived evs in breast cancer. biochemical and quantitative proteomics analysis highlighted the induction of epithelial-to-mesenchymal transition and senescence upon treatment with milk-derived evs. summary/conclusion: taken together, we were able to demonstrate the capacity of bovine milk-derived evs in mediating cross-species communication and regulating cancer progression in a context-dependent manner. bacterial membrane vesicles (mvs)a bacterial innate defence system against viral infection xiaomei yan, qian niu and ye tian xiamen university, xiamen, china (people's republic) introduction: in order to survive the constant onslaught of phage, bacteria have evolved diverse defence mechanisms that act at every stage of the phage life cycle. it has been suggested that bacterial membrane vesicles (mvs) may play a key role in innate bacterial defence against phage infection by acting as a decoy to prevent phage adsorption. nearly a decade has passed since mvs were first proposed as a decoy, but details of how bacteria utilize mvs to defend against phages remain poorly understood. here we use the laboratory-built nano-flow cytometer (nfcm) to reveal details of the interaction between mvs and phages at the single-particle level, and to provide new insights into innate defence mechanisms of mvs. methods: s. typhimurium was used as the model system. differential ultracentrifugation and density gradient centrifugation were used to isolate and purify mvs and bacteriophage p . cryo-tem was used to determine the morphologies of mvs and phage p . the purity of mv isolates was validated by measuring the particle concentration before and after triton x- treatment. monodisperse silica nanoparticles were used as the size reference standards to measure the size distribution of mvs via single-particle light scattering detection. the purity of phage p was verified by concurrent detection of side scatter and fluorescence signals of single phages upon nucleic acid staining by syto . results: by incubating mvs and af -labelled p , the number of phages adsorbed on single mvs were accurately quantified. we found that s. typhimurium and mvs it secretes express different affinity for phage p attachment. the binding ability of p to mvs is greater than that of bacteria. we confirmed that p can inject their nucleic acids into mvs, and these nucleic acids can be degraded by non-specific nucleases inside mvs for the first time. besides, by labelling the nucleic acids of mvs with syto , we were able to distinguish three different subpopulations of mvs. summary/conclusion: taking advantage of the superior sensitivity of nfcm in single-particle analysis, we developed a novel approach to the characterization of the interaction between mvs and phages. our study revealed that bacteria produce mvs as bait to attract viral adsorption and nucleic acids injection. funding: this research was supported by the national natural science foundation of china (grants , and ). introduction: the development of evs for therapeutic applications requires an in-depth understanding of their in vivo biodistribution and pharmacokinetic profile. in this study, we have made a comprehensive comparison of nuclear, fluorescent, and bioluminescent imaging technologies to identify the most suitable in vivo ev tracking method. methods: evs were purified from expi f cell supernatant by differential centrifugation followed by iodixanol density gradient separation and further characterized following misev guidelines. engineered expi f cells were used to generate evs carrying mcherry or nanoluc (nluc) proteins. the membrane of naïve ev was labelled with indium (in )-dtpa or xenolight dir post-ev isolation. ct tumour-bearing balb/c mice were intravenously dosed with × evs followed by imaging at h, h and h using spec/ct and ivis systems. tissue distribution and blood circulation profile of evs were analysed from ex vivo samples up to h post-injection. results: xenolight dir and (in )-dtpa were the most suitable ev labels for live whole-body animal imaging, ex vivo organ imaging, and tissue lysate quantification. nluc was appropriate for ex vivo imaging and tissue lysates quantification, but suboptimal for live imaging with limited sensitivity. mcherry evs were found not suitable for in vivo tracking studies due to high background signal fluorescence. ex vivo organ quantification of in -dtpa and dir showed that naïve evs mainly accumulate in liver, followed by spleen, kidneys, and lungs at h post-dose, with less than % ev exposure to the tumours. interestingly, nluc-evs accumulated mainly in the lungs, regardless of the small size of the particles injected and the absence of aggregation. blood circulation profile of in -dtpa and nluc evs showed rapid clearance of vesicles from circulation, with % of injected dose detected in blood after min and less than % after h. summary/conclusion: radionuclide imaging is an excellent technology to detect evs in vivo and ex vivo with high resolution and sensitivity but requires advanced infrastructure for radiolabeling. the optical methods have limited tissue penetration and sensitivity but can be improved with the right selection of the dye. these results contribute to the understanding of the biodistribution and pharmacokinetics of evs and are highly relevant to exploiting their potential for targeted delivery to diseased tissues in vivo. symposium introduction: new methods for quantifying extracellular vesicles (evs) in complex biofluids are critically needed. we report the development of a new technology combining size exclusion chromatography (sec), a commonly used ev purification technique, with fluorescence detection of specifically labelled evs (flu-sec). methods: flu-sec was validated using red blood cell derived evs (revs). size and concentration measurements were performed by microfluidic resistive pulse sensing (mrps) using the ncs instrument (spectradyne llc, usa). pe-cd a (anti-glycophorin a) and alexa -wga (wheat germ agglutinin) were used to label revs. flu-sec experiments were performed on a liquid chromatography system using a tricorn / glass column filled with sepharose cl- b gel (ge healthcare). results: a log-normal size distribution was obtained for revs with a mean diameter of . ± . nm and standard deviation of . ± . nm. the concentration of revs measured by mrps was . * e particles/ ml. the fluorescence chromatograms of the rev samples labelled with pe-cd a and with alexa -wga show the typical features of the separation of evs from soluble proteins with sec and enables the determination of the labelling efficiency of the markers. the linear range for quantification of evs in our experiments spans over two orders of magnitude ranging from e particles/ml to e particles/ml. the lod depends on the type of the label. in our experiments the lowest lod was e particles/ml for alexa -wga. summary/conclusion: the results indicate that flu-sec is a quantitative technique with very good linearity over a wide range of concentrations, though the limit of detection depends largely on the employed label (sci. rep. , , ) . moreover, the ratio of ev-bound and free-antibody molecules can be also determined by flu-sec, which can be used to calculate the labelling efficiency of the used marker. funding: this work was supported by the national research, development and innovation office (hungary) under grant numbers pd and nvkp_ - - - . zv was supported by the janos bolyai research fellowship. the conan assay: purity grade and concentration of ev microlitre formulations by colloidal nanoplasmonics. (evs). control over such properties is constantly experienced by researchers to be critical for ev proper manipulation, engineering and translation. however, the need for characterization methods that strike the balance between robustness, working volume, cost and accessibility remains unmet. methods: the colorimetric nanoplasmonic (conan) assay we developed consists of a solution of gold nanoparticles (aunps) into which - μl of the ev formulation is added. the solution turns blue if the formulation is pure, while stays red if soluble exogenous single and aggregated proteins (saps) are present. the colour shift is visible by the naked eye and can be quantified by conventional uv-vis spectroscopy, providing a quantitative index of purity and an estimation the ev molar concentration (particle number). results: the assay specifically targets saps, and not the ev-related proteins, with a detection limit < ng/μl (an order of magnitude higher resolution than the bradford protein assay). for pure solutions, the assay also allows for determining the ev number, as the colour shift is linearly dependent to the aunp/ev molar ratio. instead, it automatically reports if the solution bears sap contaminants, thus avoiding counting artefacts. experiments, conducted on ev separated from milk and ascaris suum culture medium, are repeatable, with an error below %. summary/conclusion: conan proves to be robust and reliable, while displaying appealing performances in terms of cost (inexpensive reagents, run by standard microplate reader), working volumes ( - μl) and time (the procedure takes less than one hour). the ability to assign a quantitative purity grade is, up to date, a unique peculiarity of this assay. finally, the assay is potentially extendable to all classes of natural and artificial lipid micro-and nanoparticles. funding: evfoundry project, horizon -future and emerging technologies (h -fetopen), id: . marina cretich a , roberto frigerio b , alessandro strada b , greta bergamaschi b , marcella chiari c and alessandro gori c a consiglio nazionale delle ricerche (cnr), istituto di scienze e tecnologie chimiche (scitec), milano, italy; b consiglio nazionale delle ricerche (cnr); istituto di scienze e tecnologie chimiche (scitec), milano, italy; c consiglio nazionale delle ricerche (cnr); istituto di scienze e tecnologie chimiche (scitec), milano, italy introduction: small extracellular vesicles (sev) present fairly distinctive lipid membrane features in the extracellular environment. these include high curvature, lipid packing defects and a relative abundance in lipids such as phosphatidylserine and ceramide. sev membrane could be then considered as a "universal" marker, alternative or complementary to traditional characteristic surface-associated proteins. here we introduce the use of membrane sensing peptides as new, highly efficient ligands to directly integrate sev capturing and analysis on a microarray platform. methods: we designed and synthesized membranesensing peptide ligands as molecular baits for small ev and we demonstrate their use in a microarray platform as valuable alternative/complement to antibodies. evs from blood serum and plasma were isolated by ultracentrifugation, characterized by tem, nta, wb. samples were analysed by label-free, single particle counting and sizing on peptide microarrays coupled to fluorescence co-localization immune staining with fluorescent anti-cd /anti-cd /anti-cd antibodies. results: peptide microarrays were realized using a click-chemistry strategy for optimal peptide surface orientation and used to analyse evs from human blood. membrane sensing peptides showed a capturing capacity higher than anti-tetraspanin antibodies. in addition to purified vesicles, peptide ligands were tested with pure serum showing capacity to capture evs even from complex samples. in order to get insights into the ev-peptide binding mechanism and verify whether it is directly mediated by the lipid membrane, trypsin-treated evs were captured on peptide microarrays demonstrating that binding is not directly mediated by surface associated proteins. summary/conclusion: we introduced the use of membrane sensing peptides as a novel class of molecular ligands for integrated sev isolation and analysis, reporting for the first time on peptide microarrays for extracellular vesicles. given their affinity to the membrane of small ev, these molecules can serve as general baits, enabling vesicles capturing unbiased by differential surface protein expression. these new class of molecular probes may be integrated with the use of protein markers towards improved small ev isolation and characterization. compared to proteins and antibodies, peptides are characterized by low cost of preparation, remarkable stability and ease of chemical manipulation, offering virtually unlimited possibilities for experimental design. we anticipate that this new class of ligands, may greatly enrich the molecular toolbox for ev analysis. funding: hydrogex (regione lombardia&fondazione cariplo, grant n. - ) and index (european union's horizon research and innovation programme under grant agreement n° ) projects are acknowledged for partial financial support. high-resolution size-based profiling and morphological analysis of extracellular vesicles by scanning electron microscopy sara cavallaro a , federico pevere b , petra hååg c , kristina viktorsson c , rolf lewensohn c , jan linnros a and apurba dev b a kth royal institute of technology, stockholm, sweden; b uppsala university, uppsala, sweden; c karolinska institute, stockholm, sweden introduction: extracellular vesicles (evs) have been found to mediate intercellular communication in physiological and pathological processes. nevertheless, the understanding of evs bio-functionality remains elusive, mainly because of their high heterogeneity in molecular content, but also in size ( - nm) . therefore, accurate size measurements of evs are highly desired, particularly for exploiting their full diagnostic/therapeutic potential. currently available techniques, such as nanoparticle tracking analysis (nta), cannot accurately measure evs smaller than nm and are not capable to distinguish them from protein aggregates. on the contrary, electron microscopy (em) techniques allow high-resolution size-profiling and morphological analysis of evs over their whole size range. however, their low throughput combined with several long preparatory steps have prevented em from being routinely used for ev size profiling. methods: we shall present a method improvement in throughput and reproducibility of ev size-analysis by scanning em (sem). the technique is based on covalent ev capture onto a silicon wafer, using the protocol reported by cavallaro et al. up to the glutaraldehyde step. after immobilization, critical point drying (cpd) is performed to dehydrate evs before sem, while preserving their shapes. results: sem images, showing the comparison in densities of evs prepared by covalent and non-covalent coupling to substrate, indicated a good capture efficiency of our covalent protocol. the size distribution analysis showed good agreement between nta and sem for evs > nm. for smaller evs, sem is more sensitive than nta, thus more suitable to check the purity of ev-isolation techniques. last, atomic-force microscopy (afm) measurements was also used to validate our measurements. introduction: extracellular vesicles (evs) are membrane vesicles secreted into extracellular space, by almost all cellular populations, playing a major role in cell-to-cell communication. it has been already demonstrated that changes in luminal or surface protein cargos of these vesicles, may reflect the status of producing cells. for this reason, evs are considered as potential biomarkers in several types of diseases ranging from cancer diagnosis to heart rejection. periostin (postn) is a matricellular protein associated with evs, and its level is considered a possible biomarker, which indicate malignancy and poor clinical outcome in different types of cancer. here we extensively characterize the presence of postn associated on evs, showing how different isolation methods can drastically affect the amount of postn content in extracellular vesicles fraction. methods: serial ultracentrifugation steps or size exclusion chromatography were used to isolate evs from primary culture of cardiac progenitor cells. evs were characterize, according to misev guidelines, by western blot, nta, facs and cryotem analysis methods. postn amount, associated with evs, was analyses by western blot and elisa. furthermore, functional tests were performed on h c cardiomyoblast cell line, treated with the same amount of evs from different isolation methods; cells response were analysed by western blot. results: evs, from both the isolation methods, showed tsg , syntenin , cd positivity while grp was absent. nta showed no differences, in terms of amount and size of particles. by facs analysis evs resulted enriched with cd , cd and cd . cryotem showed a similar morphology in the two preparations with presence of protein contaminant in the ultracentrifuge pellet. in vitro, h c treated with evs showed activation of pfak after ʹ of treatment, this induction was . times higher in cells treated with evs isolated with ultracentrifuge compared to evs isolated with sec, confirming a drastic effect of postn protein contamination. furthermore, by phospholipase-c treatment, we found that postn is bound to evs surface through a gpi anchor. summary/conclusion: these results suggest that selection of a proper isolation method is critically relevant in evs studies, in particular when protein analysis is considered. different isolation methods dramatically influence protein amount in extracellular vesicles and consequentially their function. furthermore, in this study we show for the first time, that postn is actually bound to evs surface and not carried in their lumen as previously believed. members of the y-rna family have been detected in ev from various cell types and are among the most abundant non-coding rna types in plasma. we previously showed that shuttling of full-length y-rna into ev is modulated by tlr-activation of ev-producing immune cells. this suggested that y-rnas may have potential as biomarker for immune-related diseases. methods: we separated rna-containing structures in plasma based on differences in size, density, and resistance to protease/rnase treatment. using rt-qpcr, we quantified full-length y-rna subtypes (y , y , y ) in ev from various blood-related cell types cultured with or without lps-stimulation. inflammationinduced changes in y-rna were assessed in plasma samples from a human endotoxemia study. results: full-length y-rna in plasma was mainly found in ev (early sec-fractions, density . - . g/ml). in contrast, specific mirnas were either enriched in lpp (e.g. mir- ), in both ev and lpp (e.g. mir- and mir- ), or in ev (e.g. mir- ). evenclosed full-length y-rna was resistant to enzymatic degradation, while lpp-bound mirnas were degradation sensitive. we discovered that ev released by different blood cell types varied in y-rna subtype ratios. these ratios remained stable upon lps-stimulation of the ev-producing cells. in endotoxemia plasma samples, the neutrophil-specific y /y ratios and pbmcspecific y /y ratios changed significantly during systemic inflammation. importantly, the plasma y-rna ratios strongly correlated with the number and type of circulating immune cells during the inflammation process. summary/conclusion: cell type specific "y-rna signatures" in plasma ev can be determined without prior ev-enrichment, and may be further explored as biomarkers to diagnose inflammatory responses or other immune-related diseases. mining public ev small rna-seq data with mirev -insights into potential reference transcripts and abundant mirnas recently, extracellular membrane vesicle (mv) production has been proposed as a general secretion mechanism that could facilitate the delivery of functional bacterial nucleic acids into host cells. s. aureus produce membrane-bound, spherical, nano-sized, mvs packaged with a select array of bioactive macromolecules and they have been shown to play important roles in bacterial virulence and in immune modulation through the transmission of biologic signals to host cells. the present study sought to examine the nature of the association between nucleic acids and mvs produced by s. aureus. we also sought to analyse the immunostimulatory potential of mvassociated rna and dna, and to evaluate receptormediated recognition of mv-associated rna and dna molecules by innate immune cells. methods: by following a stringent purification protocol, we characterized the rna and dna content of mvs produced by actively growing s. aureus. nuclease protection assays were performed to determine whether mv-associated nucleic acids are protected from degradation. we assessed the immunomodulatory potential of mv-associated rna and dna by treating cultured mouse macrophages with mvs and measuring the induction of interferon-β mrna using qpcr. introduction: urinary extracellular vesicle (uev) transcriptome could potentially reflect the kidney gene expression profile and serve as virtual/liquid biopsy. in order to explore this possibility, we performed mrna sequencing of uevs from individuals with type diabetes to assess whether it can capture a "kidney enriched genes" expression signature that could lead to novel biomarker discovery for diabetic kidney disease. methods: the study included type diabetic individuals ( normoalbuminuric, microalbuminuric and macroalbuminuric). urine samples were collected either overnight (n = ) or during -hours (n = ) and uevs were isolated from - ml of urine by differential centrifugation. the evs quality was ensured by electron microscopy (em), western blotting and ev-rnasprofiling with the bioanalyzer. isolated rnas were subjected to rna sequencing after cdna library preparation (ultra-low amount protocols) using hiseq (illumina) pair-end protocol. the association between kidney specific gene expression levels (> fold higher compared to other tissues, n = ) and degree of albuminuria or glomerular filtration rate was explored. results: isolated ev quality appeared good by em and western blotting. rna quantity and quality were sufficient for sequencing of all samples with > million pair end reads. we detected on average expression of , genes. principal component analysis (pc) of the expression of all genes did not reveal any systematic batch differences between the overnight and -hour urine collections. comprehensive look-up of kidney-enriched genes revealed expression of > % (total ) of these genes in urine evs with high expression of five kidney-specific genes (slc a , slc a , nphs , aqp and slc a ). pc analysis combining the impact of kidney-enriched genes revealed that most macroalbuminuric patients clustered together along the pc axis, and the axis also correlated with the albumin-to-creatinine ratio (p = . ) explaining % of the variance (p = . ) in the whole data set. the pc axis also showed correlation with hba c (p = . ), but not with diabetes duration, bmi, age and egfr. introduction: due to their safety profile, tissue tropism and long-term transgene expression, adeno-associated viruses (aavs) have become the vector of choice for human gene therapy. however, pre-existing neutralizing antibodies (nabs) to many aav serotypes pose a critical challenge for the translation of gene therapies to clinic. here, we describe the use of exosomal aavs (eaav) as a robust cardiac gene delivery system that enhance transduction efficiency while shielding from pre-existing humoral immunity to the viral capsid. methods: we developed an ultracentrifugation-based purification strategy to obtain eaav specimens from aav-producing hek- t cells, and used electron microscopy-based visualization, confocal microscopybased colocalization studies, qpcr, immunoblotting, dynamic lights scattering, exoview technology and protease assays to characterize eaav morphology, contents and mechanism of action. we then evaluated efficiency of heart targeting for eaav or eaav and standard aav or aav encoding for egfp, mcherry or firefly luciferase in different human cell lines in vitro, in black mouse and in passive immunity nude mouse model in vivo using flow cytometry, confocal microscopy, langendorff perfusion system and methods: hlhs patients (n = ) after glenn procedure and swine (n = ) after pab were given rv injections of allogeneic/xenogeneic mscs. donor-specific, hla-i+, exosomes were isolated from plasma. in swine, exosomes were collected and rv fractional area change (fac) was measured post-msc-injection. in the elpis patients, exosomes were collected and outcome measurements (fac, stroke volume (sv), rv mass) were recorded and -months post-injection. exosomal mrna, microrna (mirna), and proteins were quantified and partial least squares regression (plsr) reduced the dimensionality of the datasets to build a swine model, upon which elpis outcome predictions were made. results: multiomics analysis of swine exosome cargo revealed mirna to be the largest contributor to overall variance. in swine and elpis patients, mirnas were similarly expressed ( %, fold-change< ). plsr reduced the dimensionality of the swine mirna dataset to mirnas with the highest weighted coefficients for changes in fac. pathway analysis of mirna targets revealed links to smooth muscle cell proliferation and cardiac chamber development. importantly, the swine mirna plsr model predicted elpis patient improvements in fac, sv, and rv mass with strong correlation (r > . ). summary/conclusion: these findings support the use of: ( ) swine pab model for rv failure in hlhs, ( ) circulating donor-specific msc-exosomal mirna as a novel, non-invasive biomarker of patient outcomes, and ( ) introduction: evs have been shown promising potential as a drug delivery vehicle, especially nucleic acid therapeutics. however, the overall short of specificity to target cancer cells has led to low therapeutic efficacy and potential toxicity. rna nanotechnology is the bottom-up self-assembly of nanometre-scale rna architectures. we previously discovered a stable phi prna three-way junction ( wj) motif and used it to construct multivalent rna nanoparticles with high chemical and thermodynamic stability. the resulting arrow-shape rna nanoparticles are homogenous, uniform in size and shape, and can harbour different functionalities while retaining their tertiary folding and independent functionalities both in vitro and in vivo. this flexible platform using rna nanotechnology to achieve tumour-specific targeting has been demonstrated over the last decade. here we introduce a strategy to take advantage of both evs and rna nanotechnology to develop a versatile platform for efficient target-specific delivery of sirnas for cancer treatment. methods: we design membrane-anchoring arrowtail wj rna nanoparticles to display tumour targeting ligand (psma rna aptamer or egfr rna aptamer or folate) on birc sirnas loaded evs (fig. ). nanoparticles were characterized by nanoparticles tracking analysis (nta), transmission electron microscopy (tem), dynamic light scattering (dls) and atomic force microscopy (afm). evs were produced by hollowfiber bioreactor and purify by tangential flow filtration (tff) follow by ultracentrifugation. cell binding were evaluated by flowcytometry and confocal microscopy and gene knockdown effect were assay by quantity reverse transcription-pcr (qrt-pcr). formulated evs were introduced to tumour (prostate, triple negative breast cancer, colon pdx) xenograft mice by tail-vein injection and evaluate in vivo tumour inhibition. results: ) we found the orientation of arrow-shaped rna can be used to control ligand display on evs membranes for specific cell targeting. ) by placing membrane-anchoring cholesterol at the tail of the arrow results in display of rna aptamer or folate on the outer surface of the evs and enhance cancer cell binding and uptake. ) taking advantage of the rna ligand for specific targeting and evs for efficient cytosolic delivery, the resulting ligand-displaying evs or plant derived evs-like nanovesicles were capable of specific delivery of sirna to cells, and efficiently blocked tumour growth in three cancer models. summary/conclusion: we developed an rna-evs based nanoparticles platform and shown the flexibility for different cancer type treatment. related publications: pi f et.al. nature nanotechnology. , : . li z et.al. sci rep. introduction: extracellular vesicles (evs) contain plasma membrane surface markers that provide insights into their cell source. until now, our understanding of the circulating ev-biome has been limited by the lack of celland size-specific ev quantitation methods. we have developed and validated a multiplex nanoscale flow cytometry approach to image cell-and size-specific ev populations using a novel human "ev-lyoplate" with differently coloured monoclonal antibodies per well in a well plate format (n = separate antibodies with isotype, stained pbs, unstained plasma, and quant-beads controls per plate). we hypothesized that platelet poor plasma samples from patients diagnosed with pancreatic cancer would have significantly different ev-biome profiles than screen negative study subjects. methods: study subjects were enrolled and sampled before clinically scheduled endoscopic ultrasoundguided biopsy (eus-fna) procedures to screen patients with symptoms of pancreatic duct obstruction who later had at least two years of clinical follow up, including surgical resection in cases of pancreatic neoplasia (n = ) or at least one follow up clinic visit to confirm resolution of symptoms. blood samples were uniformly collected, processed, and banked per isev recommended guidelines. uniform machine (facsymphony) settings to standardize light scatter and fluorescence detection were based on commercially available beads (eg. megamix). samples were coded and randomized for testing and results were reported as the mean cell-and size-specific ev events/ul of plasma. results: clinical outcomes confirmed cases of cancer and screen negative controls. principle component analysis suggested that a number of different celland size-specific evs were significantly more common in the cancer cases (adjusted p-value < . , with aucs > . ), including epcam+/cd + events likely from cancer cells and cd +/cd p+/cd + microvesicles from platelets, among others. summary/conclusion: in this proof of principle study employing an ev-lyoplate design and nanoscale flow cytometry, we could reliably discriminate the ev-biomes in patients with cancer from negative controls. ongoing studies will determine whether these discriminators will be validated in larger cohorts and provide at least noninferior predictive value compared with the current gold standard clinical testing assay (eus-fna introduction: small cell lung cancer (sclc) is an aggressive tumour type, usually metastatic at diagnostic leading to poor overall survival. interestingly, sclc tumours are composed by distinct subpopulations of cells that cooperate as an ecosystem to drive tumour survival. since the subtype of sclc may have prognostic significance, the aim of this study was to identify surface marker proteins as biomarkers of sclc. methods: a linear discriminant analysis (lda) model, implemented in python via sci-kit learn, was used to choose the best markers for distinguishing subtypes. this analysis was based on rna-seq data from a previous study. in order to identify ev-based biomarkers that would identify sclc evs and not normal evs, we excluded from this analysis proteins without a verified transmembrane domain and proteins associated with evs expected to be present in white and red blood cells, and endothelial cells (according to exocarta and vesiclepedia databases). we also prioritized proteins that could be pan markers for sclc and that might have prognostic significance. to validate our findings, we performed western blotting and flow cytometry in sclc cell lines from different subtypes. results: our rna analysis indicated that the best surface markers to distinguish sclc subtypes were ceacam , fam a, lrfn , epha . immunoblot analysis validated ceacam and epha but not fam a or lrfn . we also found that ncam , a commonly used sclc marker, only marks some of the subtypes. for further analysis, we chose proteins with antibodies validated for flow cytometry as our chosen biomarker platform. flow cytometry analysis of cd is suitable as a pan-sclc marker. however, the expression of non-ne cell lines was decreased compared to rna-seq data. summary/conclusion: protein analysis of ceacam and epha corresponded to rna-seq data. ncam was not detected as a pan marker for all sclc subtypes. however, we could see cd expression in all sclc subtypes, indicating it may be a useful pan marker for sclc. future studies will be performed to validate the expression of other surface markers in cells, purified evs, and plasma of sclc patients. funding: nih u ca and nih u ca . leukobiopsyexploiting extracellular vesicle-mediated leukocyte sequestration of cancer-specific signatures introduction: in cancer, extracellular vesicles (evs) act as a unique exit mechanism for mutant and oncogenic macromolecules (proteins, rna and dna) en route from malignant cells to blood . while this process has inspired major liquid biopsy efforts, the biology of circulating evs that carry oncogenic mutations (oncosomes) is still poorly characterized. it is also unclear what part (if any) of the tumour-related cell free dna (ctdna) , , a major liquid biopsy analyte, is linked to circulating evs and what is their fate, receptacles and biological activity. methods: we employed as series of cancer cell lines carrying mutations in major oncogenes (hras, her , egfrviii). ev-dna was analysed by digital droplet pcr (ddpcr), along with nuclear anomalies in donor cells (dapi, electron microscopy) and transfer of dna to recipient cells of endothelial (huvec, mmbec), astrocytic (nha) or myeloid (hl ) origin. blood underwent fractionation into red blood cells (rbc), white blood cells (wbc), platelets (plt), evs ( , g ultracentrifugation) and soluble plasma (sup) . results: hras-mediated cellular transformation (in ras- cells) triggers profound changes in the structure of nuclear chromatin, which is driven into the cytoplasm and released as cargo of evs. oncogenic dna is detectable in blood fractions of tumour bearing mice. while evs, ctdna and plts contain intermediate levels of mutant dna, rbcs contain only traces of this material. the highest hras copy number per ml of blood is found in wbcs (monocytes and neutrophils), which contain more cancer dna/cell than liver, spleen and bone marrow. depletion of neutrophils using anti-ly g antibody results in an increase in ev-and ctdna-associated mutant dna in blood, suggesting the role of these cells in regulating the circulating levels of cancer cell-derived particles. uptake of dna-containing evs impacts the phenotype of myeloid cells, which adopt thrombo-inflammatory properties. these cells also retain cancer-specific transcripts and other cargo. finally, normal astrocytes treated with oncogenic evs also exhibit phenotypic changes and signs of genomic instability including formation of micronuclei. summary/conclusion: we propose that the process of leukocyte sequestration of circulating particles containing tumour-related nucleic acids renders these cells potentially usable as a novel liquid biopsy platform (leukobiopsy) in cancer. introduction: early diagnosis of colorectal cancer (crc) and precancerous adenoma patients is of vital importance. previously we profiled small extracellular vesicles (sevs) derived mirnas isolated from plasma, proposed a new promising biomarker category of crc patients. here we further gave a full landscape of circulating sevs derived rnas to explore and evaluate sevs based rna biomarkers for early detection of both crc and adenoma patients. methods: plasma sevs were isolated from participants, including early-stage crc patients, adenoma patients, and normal controls (nc), and characterized according to misv guideline. the total sevs derived rna expression profile of all participants was investigated by next-generation sequencing (ngs). weighted gene coexpression network analysis (wgcna) was performed to categorize differentially expressed rnas, and t-distributed stochastic neighbour embedding (tsne) was adopted to distinguish crc, adenoma, from nc samples with the top-ranked genes in wgcna modules. rt-qpcr validation was performed in a cohort of additional participants. results: a total of rna species (including mirnas, mrnas, and lncrnas) were found differentially expressed between plasma sevs in crc and nc participants. additionally, rna species were differentially expressed between plasma sevs in adenoma and nc participants. rna species were differentially expressed between plasma sevs in crc and adenoma participants. wgcna categorized all rnas into modules, which exhibited different expression trends during the carcinogenesis of crc. a -gene combined tsne model consists of the top genes in each module could perfectly classify crc, adenoma, and nc samples. a -gene combined tsne model consists of the top gene in each module could roughly distinguish crc and adenoma from nc, with only sample misclassified. rt-qpcr assays also confirmed the potential classification ability of those genes in another validation cohort of participants. introduction: although the concept of systematic "liquid biopsy" using bodily fluids is simple and elegant, the path of clinical reality has been challenging. recently, numerous tissue-specific biomarkers have been discovered in evs derived from blood, urine, cerebrospinal fluid, cell culture media, and a variety of other fluids. however, tracing the lineage of evs to their tissue of origin remains challenging due to their minute amount of cargo and unavailability of matching biopsied tissue and bodily fluids from the same patient. we recently demonstrated in three separate publications (dogra et. al; smith et. al; murillo et. al) , a new device (nanodld) for ev isolations, it's comparison with current technologies, bioengineered vesicles, and a detailed study of rna types present in small/ large vesicles, lipoproteins, and ago protein in different biofluids. in the present study, we aim to investigate the lineage of prostate derived evs in biofluids. methods: using our chip technology, we have isolated exosomes from prostate cancer cell lines and patient tissue, blood and urine samples. after exosome isolation, small rna libraries were prepared, and sequencing is carried out at icahn school of medicine and new york genome center using illumine sequencer hiseq . our nanofluidic pillar array is manufactured in an sio mask using optical contact lithography and deep ultraviolet lithography. results: our study revealed i) rna markers, which are exclusive to their prostate tissue of origin and are secreted in evs; ii) approximately - % of prostate tissue-specific rna were discovered in evs; iii) over % ( of rna) of literature curated prostatespecific rna signatures were detectable in serum and urine evs from pca patients; iv) evs contained over - % of noncoding rna ( - % was mirna), while tissue predominantly yielded rrna (> %); v) finally, gene set analyses generated that over % of evs rna were enriched for signalling pathways, yielding mirna-associated, non-canonical wnt signalling, and androgen receptor pathways. this study enables us to noninvasively monitor prostate tissue-specific biomarkers, identify tumour-specific rna, and potentially may benefit in liquid biopsy by avoiding unnecessary surgical procedures. summary/conclusion: in summary, we have investigated patient matched tissue, serum, and urine derived evs in prostate cancer. we present a set of prostatic rna in evs, which are enriched in noncanonical wnt signalling, and androgen receptor pathways. this study enables us to noninvasively track prostatic biomarkers, identify tumour specific rna, and potentially may benefit in liquid biopsy by avoiding unnecessary surgical procedures. a multi-model, liquid biopsy approach for diagnosing and staging pancreatic adenocarcinoma introduction: pancreatic ductal adenocarcinoma (pdac) is the third largest contributor to cancerrelated death in the usa. since there is not yet a feasible technology to diagnose pdac early in the disease, % of patients are diagnosed at an advanced stage. moreover, for patients with confirmed pdac, standard imaging method has low sensitivity to detect early metastatic disease, which complicates the selection of therapy. to address these challenges, there has been great interest in developing minimally-invasive, extracellular vesicle (ev) based blood tests for pdac. to this end, we have integrated measurements of tumour derivedev rna cargo with circulating proteins and cell free dna (cfdna), and use machine learning algorithms to distill this multiplexed diagnostic to . diagnose pdac patients from healthy and disease controls and . distinguish pdac patients with distance sites of metastasis to guide their treatment. we make use of our lab's magnetic nanopore isolation technique to specifically enrich for tumour derived evs directly from patient plasma. methods: we have developed a high throughput nanofluidic sorting platform, which immunomagnetically isolates individual evs from plasma using magnetic nanostructures. however, our architectures is uniquely designed for massive parallelization allowing high throughput, robust processing of ml of plasma in minutes. we performed sequencing on a discovery set of patients and controls (n = ). subsequently, we trained our panel of biomarkers using a training set of n = . finally, we validated the performance of our platform using an independent blinded test set of n = . results: the results of a blinded test set achieved an accuracy = % and an auc = . on binary classification of pdac patients versus those that were healthy or disease controls. in addition, we achieved an auc = . and accuracy = % with sensitivity of % and specificity of % on detecting occult metastasist. summary/conclusion: we developed a highly sensitive pancreatic cancer diagnostics by combining our nanomagnetic isolation platform for tumourderived ev isolation, rna sequencing, and machine learning. we isolated tumour-derived evs and profiled their rna cargo, combined with cfdna and ca - for pancreatic cancer diagnosis. the predictive panels successfully distinguished non-cancer patients from pdac patients, and nodistant metastasis patients(m ) from distant metastasis patients(m ) for appropriate treatment. the resulting auc and accuracy from the independent blinded test set outperformed any individual biomarker, showing both the benefits and the robustness of combining multiple orthogonal biomarkers for pdac diagnosis. introduction: both hypertension and diabetes exhibit significant molecular changes to the vasculature that are associated with increased cardiovascular risk. here we examined the protein composition of large evs (l-evs) isolated from the plasma of hypertensive, diabetic and healthy mice to identify common and diseasespecific molecular changes. methods: we examined circulating l-evs isolated from transgenic mice expressing active human renin in the liver (ttrhren, a model of hypertension), ove type diabetic mice, and their wild-type (wt) littermates. at weeks of age mice were sacrificed and blood samples were obtained by cardiac puncture. l-evs were isolated from platelet-free plasma via differential centrifugation and protein content was assessed via mass spectrometry (ms). results: ttrhren mice exhibited increased blood pressure compared with ove mice or their wt littermates. ( . ± . vs . ± . [ove ] vs. . ± . mmhg [wt], p < . ). ms identified independent proteins with at least peptides per protein. of these, proteins were found in all groups studied, were exclusive to wt mice, were exclusive to ove mice and were exclusive to ttrhren mice. in addition, proteins were observed with > . fold change (fc) compared to wild-type mice, and proteins were reduced by > %. amongst the top ten differentially expressed proteins, fibrinogen was upregulated in both ove and ttrhren mice compared with wild-type controls. similarly trem-like transcript , sarcoplasmic/endoplasmic reticulum calcium atpase and junction plakoglobin were all downregulated in both ove mice and ttrhren mice suggesting molecular changes common to both conditions. conversely, arginase was up-regulated in diabetic, but not hypertensive mice while carboxypeptidase was upregulated in hypertensive but not diabetic mice. summary/conclusion: taken together, these results show that the protein composition of circulating l-evs is altered in diabetes and hypertension and that both common and disease-specific changes may be detected. further analysis of these changes may lead to the identification of novel pathways associated with the pathogenesis of vascular injury in hypertension and diabetes. funding: this study was supported by grants (to db) from the canadian institutes of health research, an ontario early researcher award, and the canada foundation for innovation. understanding the role of endothelial cell-derived apoptotic bodies in inflammatory signalling and cell clearance in an atherosclerosis model of inflammation. introduction: apoptotic bodies (apobds) are a class of large (~ - um) evs formed during apoptotic cell disassembly, that are becoming increasingly recognized as potential mediators of intercellular communication, e.g. via the transfer of proteins and other cargoes to target cells. during the inflammatory vascular disease atherosclerosis, endothelial cell (ec) apoptosis contributes to loss of barrier function and promotes the formation of plaques in regions of ec damage. although, experimentally, ecs generate an abundance of apobds, a specific role for ec-derived apobds (ec-apobds) in the progression of atherosclerosis remains poorly defined. methods: in the present study, a detailed in vitro characterization of ec disassembly was performed via flow cytometry, confocal live cell imaging and cytokine profiling, followed by function analyses of ec-apobds using a murine in vivo model of dead cell clearance. results: characterization of ec disassembly revealed that apobd formation in ecs is regulated by rho-associated, coiled-coil-containing protein kinase (rock ), a process that can be pharmacologically inhibited using a rock- inhibitor, thereby providing tools for functional in vivo studies. the specific cargo and role in clearance of ec-apobds were then investigated. profiling of ec-apobds was performed via cytokine antibody array to reveal that ec-apobds generated under inflammatory conditions contain high levels of pro-inflammatory cytokines including mcp- and il- , suggesting a potential role for ec-apobds in the propagation of inflammation during vascular disease. furthermore, the ability of ec-apobds to be cleared from the vasculature via phagocytosis was investigated, revealing that ec-apobds can travel to distal organs to undergo clearance. summary/conclusion: these findings provide important insights into the potential functions of ec-apobds generated under both non-inflammatory and inflammatory conditions and may contribute to future studies involving the therapeutic targeting of ec disassembly for the treatment of atherosclerosis. funding: this work was supported by grants from the national health & medical research council of australia (gnt , gnt ) adipose mesenchymal stromal cell derived evs foster cardio-renal protection in the doca-salt hypertensive rat model introduction: cardio-renal syndromes (crs) are disorders of the heart and kidneys whereby "acute or chronic dysfunction in one organ may induce dysfunction of the other". stem cell-derived extracellular vesicles (evs) mediates the protection of the kidney from development of chronic kidney disease (ckd). we here investigated the potential of adipose-mesenchymal stromal cells derived evs (asc-evs) as therapeutic tools for the treatment of crs. methods: adult wistar rats were uninephrectomized and treated with a high-na+ diet and deoxycorticosterone-acetate (doca-salt) for -weeks ( / ; a / - - ). evs were isolated by ultracentrifugation method. ev dimension, concentration and surface markers were characterized by nta, cytofluorimetric analysis and transmission electron microscopy. to characterize the role of evs in crs, doca-salt rats were injected weekly with asc-evs. systolic blood pressure was measured by the tail-cuff method. plasma creatinine and urinary protein excretion were determined by colorimetric assays and microalbuminuria by immune turbidimetric assay. qrt-pcr and western blot were conducted to evaluate fibrosis and inflammatory-related genes and proteins in the kidney and heart of doca-salt rats. immunohistochemistry was used to confirm matrix accumulation (a-sma) and immune infiltrate (cd + cells). results: multiple administration of asc-evs in doca-salt rats induced a protective effect on the kidney, by reducing tubular and vascular damage. kidney function was also conserved by ev treatment as detected by the normal glomerular filtration rate and the absence of proteinuria with respect to doca-salt untreated rats. ev administration significantly decreases the pro-inflammatory molecules mcp- and pai and reduce the recruitment of macrophages in the kidney. the mitigation of the inflammatory response by asc-ev infusion consequentially affected the development of fibrosis, as detected by the decrease in collagens (col a , col a ) and fibronectin (fn) expression in respect to doca-salt animals. asc-evs were able to act in multiple organs, preventing fibrosis and inflammation also in the heart, therefore alleviating blood pressure rise during the -weeks of treatment in doca-salt rats. summary/conclusion: our results indicate that asc-ev administrations in hypertensive-induced ckd rats promote protection from renal damage, reduction of the inflammatory response and prevention of interstitial fibrosis in the kidney. asc-evs are also able to protect the cardiac tissue and to control blood pressure increase, displaying complex and multiorgan beneficial effects. introduction: alveolar macrophages (ams) tonically secrete extracellular vesicles (evs) containing suppressor of cytokine signalling (socs ) protein. uptake of socs -containing evs by alveolar epithelial cells is critical for restraint of cytokine-induced janus kinasesignal transducer and activator of transcription (jak-stat ) signalling to promote homoeostasis in the distal lung. at steady state, ams exhibit suppressed glycolytic activity, a metabolic phenotype that promotes homoeostatic function. whether this glycolytic restraint is critical for am secretion of socs is unknown. in fact, to our knowledge, metabolic control over release of any ev cargo has never been explored in any cellular context. methods: immortalized mouse ams (mh-s) were treated with various doses of -deoxy-d-glucose ( -dg) and oligomycin, inhibitors of glycolysis and oxidative phosphorylation, respectively. primary rat ams collected by lung lavage were treated with an aqueous extract of cigarette smoke (cse) with or without -dg. metabolic activity was measured by seahorse assay, evs were quantified by nanoparticle tracking analysis, and vesicular (> -kda) socs secretion was determined by western blot of conditioned medium. additionally, ams collected from wild-type (wt) and lsl-krasg d mice bearing lung tumours weeks after intrapulmonary ad-cre were cultured ex vivo in the presence or absence of -dg. vesicular (> -kda) socs secretion was measured by elisa. results: in a dose-dependent manner, oligomycin inhibited, whereas -dg enhanced, socs and ev release by mh-s cells. treatment of rat ams with cse ( %) attenuated secretion of socs , an effect that coincided with increases in glycolytic activity, and co-treatment of ams with -dg abrogated the inhibitory effect of cse on socs release. finally, ams collected from lsl-krasg d mice exhibited a deficiency in socs secretion relative to wt ams, an effect that was reversible by overnight culture in the presence of -dg. summary/conclusion: in tandem, our data generated using in vitro and in vivo approaches demonstrate that am secretion of vesicular socs is down-regulated by glycolysis. we speculate that metabolic control over release of ev cargoes is a phenomenon of broad biologic relevance within and outside of the lung. introduction: bacterial extracellular vesicles (ev) are described to play roles in defence and resistance, pathogenesis and stress responses. cyanobacteria pioneered oxygenic photosynthesis, and are the ancestors of modern chloroplasts. we previously described that by deleting the gene encoding tolc (Δtolc) in the model cyanobacterium synechocystis sp pcc (s ), a key player in protein-mediated secretion systems, a hyper-vesiculating phenotype could be obtained. the goal of this work was to understand why Δtolc hyper-vesiculates. methods: isobaric tag for relative and absolute quantitation (itraq) was used for quantitative proteomic analyses of total cell extracts. ev were isolated as follows: cells were separated from the extracellular medium (em) by centrifugation ( g, min) and filtration ( . µm pore-size filters). cell-free em was concentrated using centrifugal filters (mwco of kda), and later ultracentrifuged for h at g. the final ev fraction was suspended in growth medium. ev characterization was performed using tem, dls, nanosight, and by the detection and quantification of lps (lipopolysaccharides). detection of specific proteins in ev was carried out by western blot. copper (cu) levels were quantified by atomic absorption spectrometry (aas). results: a large-scale quantitative proteomic analysis was performed, resulting in the identification of several metal-related proteins with differential regulation in s Δtolc. both wild-type (wt) and Δtolc cells were then challenged with different metals. compared to the wt, Δtolc showed impaired growth only when exposed to cu, a co-factor for several proteins with roles in primary metabolism. the intracellular cu levels were quantified and Δtolc accumulates threefold more cu than wt cells. we then asked whether the hyper-vesiculating phenotype observed could be linked to the stress induced by cu accumulation. in ev isolated from Δtolc we detected the metallochaperone copm, a periplasmic cu-binding protein involved in cu-resistance mechanisms in s . in addition, cu could also be detected in isolated Δtolc-ev. in addition, more ev were detected when s wt cells were challenged with cu, in a cu-concentration dependent manner. summary/conclusion: these results support the idea that bacterial ev represent an alternative cu-secretion mechanism to deal with cu-induced stress. funding: fct phd grant sfrh/bd/ / ; feder-compete -poci-fct project: poci- - -feder- . juan wang and maureen barr rutgers university, human genetics institute of nj, piscataway, usa introduction: extracellular vesicles (evs) function in intercellular communication. despite their physiological importance and biomedical relevance, knowledge of ev fundamental biology is not well understood, in part due to a lack of tractable animal systems. our analysis of environmentally-released c. elegans ciliary evs provides strong evidence that nematodes package cargo in evs that mediate inter-organismal communication, in analogy to intercellular signalling in mammals. we predict that conserved mechanisms underlie ev cargo sorting, biogenesis and signalling. cilia act as cell towers to both receive extracellular signals and to send information via ciliary evs. ciliary defects result in human ciliopathies including autosomal dominant polycystic kidney disease (adpkd). adpkd is a life-threatening disease that affects / and is caused by mutations in pkd and pkd , which encode polycystin- and − . in c. elegans and humans, the polycystins are architecturally similar, act in the same genetic pathway, function in a sensory capacity, localize to cilia, and are shed in evs, suggesting ancient conservation. moreover, ciliary ev biogenesis and shedding is an evolutionary conserved process from algae to worms to humans. by studying how cilia make and receive evs, we aim to uncover fundamental principles of how cells communicate using evs. methods: to study ciliary ev cargo sorting and biogenesis, we use genetically-encoded fluorescent-tagged ev cargo and superresolution zeiss airyscan confocal microscopy in living animals. results: we find that cargoes are sorted into distinct populations. in cilia, kinesin- motors and kinesin- klp- /kif transport different ev cargoes to the ciliary tip and generate an ev cargo enrichment zone. from here, evs are shed and released into environment in a spatially and temporally regulated manner. ciliary ev biogenesis and release is regulated by mechanical pressure and ph. our work revealsat the single cell levelthat different evs are made in response to environmental stimuli, which may be important for ev signalling properties. summary/conclusion: cells exploit the spatiallyrestricted cilium and its sophisticated transport system to generate distinct populations of ciliary evs. how these ciliary ev communicate cellular messages awaits decoding. introduction: we recently demonstrated that recycling endosomes marked by rab a generate exosome subtypes distinct in cargos and functions from late endosomes, which we collectively term rab -exosomes. these exosomes are preferentially released from cancer cells in response to metabolic stress and promote adaptive changes in a xenograft model. here we use comparative ev proteomics in hct colorectal and hela cervical cancer cell lines to identify rab -exosome signature proteins and screen for functional effects. methods: we analysed ev preparations by mass spectrometry using tandem mass tag® labelling to identify changes in ev protein cargo in response to glutamine depletion. candidate genes were subsequently knocked down in drosophila secondary cells, which permit visualisation of rab -exosome biogenesis using fluorescence microscopy, and in human cancer cell lines. results: we show that accessory escrt-iii proteins, chmp , chmp and ist , are enriched on glutamine-depletion-induced evs and play a selective and conserved role in generating rab -exosomes. they are, however, not required to traffic ubiquitinated cargos into late endosomes and lysosomes. escrt- components, thought to regulate trafficking of ubiquitinated cargos into intraluminal vesicles, are also required to make rab -exosomes. in flies the escrt- , hrs, localises to the limiting membrane of rab -endosomes. comparative proteomics reveals other proteins enriched in rab -exosomes, which also appear to be needed to mediate this novel exosome formation mechanism. summary/conclusion: we conclude that rab -exosome subtypes are formed via a distinct mechanism requiring accessory escrt-iii components, suggesting a route to selectively target these exosomes. introduction: the tumour microenvironment consists of a complex network of host cells embedded within extracellular matrix. communication between these cellular compartments is critical for tumour progression and exosomes have emerged as important regulators of intercellular communication. while a number of studies have implicated exosomes in cancer progression, mechanisms controlling exosome transfer are not well understood. we developed three-dimensional ( d) culture models to evaluate the role of cues provided by the extracellular matrix in exosome release and uptake. methods: exosomes were isolated from cells in two-and three-dimensional culture via ultracentrifugation and characterized by nanosight, qubit protein quantification, and flow cytometry analysis of exosome markers. exosomes were labelled with fluorescent lipophilic dyes and uptake in recipient cells quantified by flow cytometry. results: cells cultured in d display decreased exosome release and increased uptake compared to d cultured cells. exosome release in d culture was inhibited with the exosome release inhibitors brefeldin a and gw , but was not significantly altered by knockout of rab b. in addition, disruption of polarity signals provided by d culture did not impact exosome release or uptake in d, but induction of oncogenic hras increased both secretion and uptake of exosomes through activation of pi k signalling. summary/conclusion: release and uptake of exosomes is altered in d environments. these studies help provide insight into exosome production and uptake in vivo and have potential implications for therapeutically targeting exosome release and the development of exosome based therapeutic delivery vehicles. introduction: previous studies in our lab found that expression of r w-fibulin- induces rpe to undergo emt. the purpose of current study was to characterize the extracellular vesicles (evs) in rpe cells expressing wt-fibulin- versus rpe cells expressing r w-fibulin- and investigate the effects of these evs on rpe cell differentiation. methods: arpe- cells were infected with lentivirus with luciferase-tagged wild-type (wt)-fibulin- or luciferase-tagged r w-fibulin- . evs were isolated from the media of arpe- cells by conventional ultracentrifugation or density gradient ultracentrifugation. transmission electron microscopy (tem) and cryogenic electron microscopy (cryo-em) were performed to study the morphology of the evs. the amount and size distribution of evs were analysed by nanosight tracking analysis (nta). ev protein concentrations were quantified using the dctm protein assay (bio-rad). ev cargo were analysed by unbiased proteomics using lc-ms/ms with subsequent pathway analysis (advaita). migration ability was evaluated in arpe- cells with or without the exposure of evs by conducting scratch assays. results: morphologically, tem imaging showed concave-appearing vesicles and cryo-em imaging showed spherical vesicles with two subpopulations of evs: a small group with diameters around nm and a large group with diameters around nm. moreover, tem and cryo-em showed an increased amount of small evs (~ nm) in the mutant group compared to the wt group. this result was further confirmed by nta showing that, in the mutant group, the particle size distributions were smaller than the wt evs. no significant differences were shown in ev protein concentrations per particle between wt and mutant groups. our previous data suggest that the expression of r w-fibulin- causes rpe cells to undergo emt as evidenced by upregulated emt drivers and an increased migration ability. proteomic studies showed that evs derived from arpe- cells overexpressing wt-fibulin- contain critical members of sonic hedgehog signalling (shh) and ciliary tip components, whereas evs derived from rpe cells overexpressing r w-fibulin- contain emt mediators, indicating that ev cargo reflects the phenotypic status of their parental cells. ev transplant studies showed that exposing native rpe cells to mutant rpe cell-derived evs containing emt drivers, including tgf-β-induced protein (tgfbi), vim, and smad , leads to an enhanced migration ability of rpe cells in a dosedependent manner. introduction: despite of high expectations, mesenchymal stromal cell (msc)-based therapies still lack efficacy, partially due to loss of cell viability and function upon administration. msc-derived extracellular vesicles (msc-ev) emulate the regenerative potential of msc, shifting the field towards cell-free therapies. clinical applications require the establishment of a scalable and gmp-compliant processes for the production and isolation of msc-ev, combined with robust characterization platforms. methods: to develop a well-established process for the production of therapeutic msc-ev, we compared different msc sources (bone marrow, adipose tissue, umbilical cord matrix), culture media compositions (dmem supplemented with foetal bovine serum (thermo fisher scientific), dmem supplemented with human platelet lysate (aventacell biomedical) and stempro msc sfm xeno free medium (thermo fisher scientific)) and culture parameters (oxygen tension and shear stress) in two different culture platforms ( d static tissue culture flask vs d dynamic spinner vessels). subsequently, msc-ev were isolated by ultracentrifugation or a commercially available isolation kit and characterized according to isev guidelines. results: msc derived from different sources/donors were able to grow under normoxia and hypoxia in d t-flasks and d spinner vessel culture systems, while maintaining their immunophenotype and differentiation potential, according to the minimal criteria defined by the isct. the time point for pre-conditioning and collection of conditioned medium for msc-ev isolation was also optimized for both d and d culture systems. introduction: extracellular vesicles (evs) have great potential in prostate cancer (pca) diagnosis and progression monitoring to complement the inaccurate prostate specific antigen (psa) screening and invasiveness of tissue biopsy. however, current methods cannot isolate pure evs and therefor evs characteristics remain largely unknown. in order to develop an accurate approach for ev isolation, we aimed to compare three emerging methods with different characteristics of small evs (sevs) from human pca plasma samples and to choose the best one for diagnostic and functional studies methods: pca patients and age-matched healthy controls (hc) plasma (n = in each group) were used to isolate sevs with different isolation methods including commercial exoquick ultra kit, qev and qev size exclusion chromatography (sec). isolated sev were characterized by nanoparticle tracking analysis, immunoblotting, cyrogenic electron microscopy, flow cytometry (fc) and proteomics analysis. for fc characterizing surface marker expression, the sevs were further purified by cd and cd commercial immunoaffinity magnetic beads . lipoprotein was captured by streptavidin biotinylated apob magnetic beads to measuring the lipoprotein contamination results: the sev size, morphology, surface protein and protein cargo with proteomics were analysed between the three isolation methods. sevs isolated from sec methods had a lower particle size, protein amount, protein/sev marker ratio and apob+/sev marker ratio than those from exoquick ultra method. in addition, sevs isolated from qev demonstrated a significantly higher sev content, more up-regulated and down-regulated pca proteins from proteomics but lower sev marker/protein ratio and a higher protein contamination than those from qev . furthermore, sev marker signal also showed a good correlation with particle numbers instead of protein content in all the methods summary/conclusion: qev method demonstrated better performance in isolating relatively pure sevs from human plasma; qev has the better performance in isolating samples with higher sev content; exoquick ultra isolated samples with closely sev content to the qev but with the highest non-sev protein contaminations. introduction: extracellular vesicles (evs) are released to biological fluids from different tissues and organs and they contain molecules proposed as biomarkers for multiple pathological conditions. however, most ev biomarkers have not been validated due to the lack of sensitive techniques compatible with high-throughput analysis required for routine screenings. using immunocapture techniques, combining antibodies against tetraspanins and candidate tumour-specific markers we have recently optimized several assays that greatly facilitate ev characterization. methods: we have improved flow cytometry and elisa assays, increasing substantially the sensitivity for ev detection. using dls, em and analytical ultracentrifugation, we have characterised the biophysical basis of this enhancement. the final methodology can be performed in any laboratory with access to conventional flow cytometry or elisa reader. results: using combinations of antibodies specific for the tetraspanins cd , cd and cd , it is possible to detect evs in minimal volumes of urine and plasma samples without previous enrichment. additionally antibodies against other less abundant markers, like the epithelial marker epcam, have been used to capture and identify evs directly in minimal volumes of urine or plasma with sensitivity higher than western blot analysis of isolated evs. furthermore, we demonstrate that additives altering the biophysical properties of an ev suspension, increased detection of tumour antigens in these immune-assays. summary/conclusion: the development of sensitive, high-throughput methods, easily translatable to clinical settings, as elisa and flow cytometry described here, opens a new avenue for the systematic identification of any surface marker on evs, even scarce proteins, using very small volumes of minimally processed biological samples. these methods will allow the validation of ev biomarkers in routine liquid biopsy tests. introduction: when ev subpopulations are enriched on antibody microarrays and probed for their surface proteins, the detection signal is biased towards abundant subpopulations as it is dependent on both the protein expression level and the number of evs captured. to address this challenge, we developed a novel normalization approach allowing: ) the estimation of a target signal independent of ev subpopulation size through dye-based ev quantification, and ) the assessment of subpopulation target enrichment relative to the population average by leveraging tim as an unbiased, lipidbased ev capture. here, we investigated the expression of cancer-associated proteins, particularly metastasisassociated integrins (itgs), in breast cancer evs with varying metastatic potential and organotropism. methods: the relative protein enrichment profiles for various ev subpopulations were established from evs of skbr (her +), t d and mcf- (er+pr+), bt and mda-mb- (triple negative) breast cancer cell lines, as well as five mda-mb- -derived cell lines of four different organotropisms (brain, bone, lung, liver) using our custom antibody microarrays with our normalization approach. results: as expected, her was broadly detected in her + skbr evs. interestingly, her -t d and mcf- evs also expressed her where it was highly enriched in its epcam+ subpopulations. itg α , β and β were only found in triple negative and organotropic evs with itg β and β differentially enriched based on the organotropism. the population average of mda-mb- and lung-tropic evs had high expression of itg β , where subpopulations of cd + evs showed positive enrichment while cd + and cd + evs showed negative enrichment. itg α , β and β were absent in the bone-tropic cd + ev subpopulation, a profile atypical in other organotropisms. lastly, egfr was negatively enriched in tetraspanin+ subpopulations in mda-mb- evs, but positively enriched in these subpopulations in organotropic evs, especially for brain-tropism. summary/conclusion: following normalization, we were able to quantify specific protein associations, uncovering a multitude of co-enrichment profiles that characterize specific metastatic and organotropic cell lines. notably, we found enrichment signatures that distinguish between different organotropisms derived from the same parental cancer line. op . = pf . heparan sulphate proteoglycans are required for ev-mediated delivery of multiple growth factors sara veiga, alex shephard, alex cocks, aled clayton and jason webber cardiff university, cardiff, uk introduction: the tissue microenvironment surrounding tumours is complex and the cross-talk between cancer and non-cancer cells is essential for tumour growth and progression. we have previously shown that heparan sulphate proteoglycans (hspgs), on the surface of prostate cancer evs, are required for delivery of tgfβ and initiation of a disease-supporting fibroblast phenotype. however, hspgs are known to bind numerous growth factors, so here we have explored the repertoire of such proteins tethered to evs by hspgs. methods: evs were isolated from du prostate cancer cell conditioned media by ultra-centrifugation onto a sucrose cushion. vesicular hspgs were modified either by removal of heparan sulphate (hs) glycosaminoglycan (gag) chains using the enzyme heparinase iii (hepiii), or attenuation of hspg core protein expression using shrnas to knockdown specific hspgs within the parent cell. differences in proteins present in control vs modified evs were identified by a sensitive protein array, based on proximity-ligation technology, and selected targets validated by elisa. functional delivery of growth factors by ev-associated hspgs to recipient fibroblasts is being explored using a variety of in vitro techniques. results: proteome analysis identified targets that bind to hs-gag chains, and also different proteins that showed altered expression following the loss of one or more hspgs from evs. using elisa, we have been able to quantify selected candidates on wild type vesicles, some of these are lost following hsdigestion. we were also able to validate proteins on hspg-deficient vesicles. gene ontology analysis suggests that ev hspg-mediated delivery of growth factors is important for control of processes such as angiogenesis, tumour invasion and immune regulation. functional validation of proteins identified is ongoing. summary/conclusion: here we demonstrate that hspgs play a key role in loading of evs with a complex assortment of growth factors, and therefore subsequent ev-mediated growth factor delivery. we anticipate that loss or damage of ev- introduction: methamphetamine (ma) and related amphetamine compounds, which are potent psychostimulants, are among the most commonly used illicit drugs. neuroimaging studies have revealed that chronic ma abuse can indeed cause neurodegenerative changes in the brains of human ma abusers including prominent microglial activation throughout the brain. it is still unclear how chronic inflammation caused by ma abuse leads to long-term damage to the brain. with this in mind, we are particularly interested in studying the role of extracellular vesicles (evs) in eliciting chronic inflammation in ma exposed brains. in the present study, we focus on the role of a mirna, mir- a- p (mir- a) in chronic ma exposure. here, we present novel data that shows for the first time how chronic ma impacts not only the biogenesis but also the ev associated mirna cargo thereby affecting the overall health of the neurons and glial cells in the brain. methods: -density gradient centrifugation for isolation of brain-derived vesicles -characterization of bdes by western blotting, nanoparticle tracking analysis and transmission electron microscopy -quantitative rt-pcr -digital droplet pcr -confocal imaging of dendritic spines and synapses results: in the present study, we show from both in vivo and in vitro studies that chronic methamphetamine (ma) treatment alters ev biogenesis and microrna (mirna) cargo. brain-derived evs (bde) isolated from frontal grey tissue of rhesus macaques that were administered ma in a chronic regimen revealed a significant increase in both number and size. further analysis revealed increase in biogenesis genes and increased levels of mirna, mir- a- p (mir- a). in situ hybridization of the frontal brain area revealed that mir- a was exclusively expressed in microglia and neurons. further, in vitro studies revealed that ev associated mir- a elicited not only neuronal damage but also was able to activate microglia to release pro-inflammatory cytokines thereby inducing a chronic inflammatory cycle. finally, we show that an anti-inflammatory drug was able to rescue inflammation, mir- a levels and synaptodendritic injury. summary/conclusion: in summary, our results present for the first time show that chronic ma exposure in the brain affects ev biogenesis and mirna expression. we further confirm that mir- a can serve as potential marker to diagnose synaptic deficits for chronic ma addiction in humans. finally, we reveal that anti-inflammatory drug could rescue the ev biogenesis and reduces the secretion of mir- a, thereby rescues synaptodendritic injury. our data further supports the use of the anti-inflammatory drugs as therapeutic interventions for ma addiction. funding: nida funding # r da blood-borne and brain-derived ectosomes/microparticles in morphineinduced anti-nociceptive tolerance deepa ruhela, veena bhopale, ming yang, kevin yu, eric weintraub, aaron greenblatt and stephen r. thom university of maryland school of medicine, baltimore, usa introduction: opioid pain treatment is impeded because chronic administration decreases analgesia, a condition called tolerance that prompts dose escalation contributing to morbidity and mortality. inflammatory interleukin (il)- β is required for tolerance development, so we hypothesized that pro-inflammatory extracellular vesicles (evs) play a role. methods: evs with opioid administration were assayed in mice and humans. annexin v-positive, . - µm diameter microparticles (mps) were assessed by flow cytometry in murine and human blood and in murine deep cervical lymph nodes that drain brain glymphatics. blood-borne exosomes (< nm) were assayed by tunable-resistance pulse sensing (trps). anti-nociceptive tolerance following morphine administration to mice was assessed by speed of tail removal from warm water. results: repetitive morphine dosing of mice to induce anti-nociceptive tolerance increased blood-borne mps by eightfold, and by tenfold in cervical lymph nodes. mps expressed proteins specific to neutrophils, microglia, astrocytes, neurons and oligodendrocytes. il- β content of mps increased -fold. administration of an il- β antagonist to mice diminished blood and glymphatic mps elevations and abrogated tolerance induction. intravenous polyethylene glycol telomer b that lyses mps and intraperitoneal methylnaltrexone that binds peripheral opioid-mu receptors and myeloid differentiation factor- to inhibit toll-like receptors, inhibited mps elevations and tolerance. neutropenic mice did not develop anti-nociceptive tolerance, elevations of blood-borne mps or cervical node mps expressing microglial proteins. elevations of blood-borne exosomes were not identified based on trps analysis. patients entering treatment for opioid use disorder exhibited similar mps elevations as do tolerant mice. summary/conclusion: neutrophil-derived mps containing il- β are required for morphine-induced antinociceptive tolerance. funding: this project was supported by grant n - - - from the office of naval research and an unrestricted grant from the national foundation of emergency medicine. evs are a conveyor of toxic dipeptide repeat proteins in c orf als/ ftd models thomas jefferson university, philadelphia, usa introduction: amyotrophic lateral sclerosis (als) is a neurodegenerative disease characterized by loss of motor neurons. in als, motor symptoms initiate focally and then progress gradually, distal from the initial focus. abnormal forms of als-associated proteins are physically exchanged between neuronal cells. pathogenic als proteins like sod , fus and tdp are transmitted between cells by assisted mechanisms, mainly extracellular vesicles (evs), spreading toxicity and misfolding of native proteins within the recipient cells. an intronic g c aberrant nucleotide repeat expansion in c orf gene is the most common genetic cause of als. translation of this expanded region occurs by a process called repeat associated non-aug (ran) translation that produces five dipeptide repeats proteins (dprs), polyga, polygp, polygr, polypa and polyga. polyga, polygr and polypr are associated with toxicity in neurons. in this work we study the recruitment of these aberrant proteins into extracellular vesicles (evs) and the potential role of these evs in spreading toxicity between cells of the central nervous system. methods: to isolate the evs from cell culture media we isolated by ultracentrifugation the larger vesicles at , xg and the smaller evs at , xg. number, size and fluorescence of the vesicles were analysed by fluorescent nanotrack analysis (f-nta) and by cytoflex. the protein content of the vesicles was analysed by western blot (wb). to evaluate the potential toxicity of the evs, a transwell system (tw) was employed. neuron viability was assessed using live imaging techniques. results: nsc were transfected with reporter constructs expressing dprs tagged with gfp protein. by f-nta, cytoflex and wb analysis we assessed that all the five dprs were loaded in both the large and the small vesicles isolated from cell culture medium. by tw, nsc transfected with the dprs were put in contact with primary cortical neurons (cns) transfected with synapsin driven td-tomato for live imaging purposes. we observed that polygr+ nsc were able to cause a significant decrease in cns viability. we also observed that polygr+ evs associated toxicity was directly dependent on polygr length. this effect was reverted reducing the number of polygr+ evs treating nsc with gw . to understand the downstream effect of polygr+ evs in recipient cells we studied tdp mislocalization, ran-translation and activation of the integrated stress response, finding a dysregulation of all these potentially toxic pathways in neurons treated with polygr+ vesicles. summary/conclusion: concluding, dprs are actively secreted in evs and polygr+ vesicles cause the activation of toxic mechanisms in the recipient cells, possibly contributing to the spreading of als introduction: pregnancy is the a condition that profoundly mitigates symptoms of multiple sclerosis (ms) a complex disease characterized by immune dysfunction and neurodegeneration affecting . million people worldwide. serum exosomes, released by specific cells during pregnancy, modulate the immune and central nervous system function and contribute to pregnancy-associated suppression of experimental autoimmune encephalomyelitis (eae), an induced preclinical model of ms. extracellular vesicles (evs) are the new means for communication among cells. the aim of our study was to characterize the ability of human amniotic fluid stem cells-derived evs (hasc-evs) to antigen presenting cell function thus correcting immune dysfunction in eae. methods: amniotic fluids were obtained from human - -week pregnant women. hasc-evs were collected by ultra-centrifugation. evs were characterized for their specific proteins, lipids and nucleic acids expression. the ability of evs to modulate immune responses was performed in vitro, testing the ability of evs to induce a tolerogenic phenotype in mouse bone marrow derived dendritic cells, and in vivo for their potential to suppress eae, induced by immunization c /b female mice with mog - peptide. results: we found that hasc-evs expressed high levels of galectin- and promoted a significant increase of the immunoregulatory enzyme indoleamine , dioxygenase- enzyme in dcs. moreover in in vivo experiments administration of hasc-evs significantly reduced disease severity in eae. such effect was associated with reduced neurological deficits and suppression of pathogenic t helper (th ) cells, and increased percentage of regulatory t cells (treg-foxp +) cells. summary/conclusion: our findings unravel immunoregulatory effects of evs secreted by hascs. evs may represent a novel cell-free immune regulatory and regenerative therapeutic approach that can potentially mitigate immune dysfunction and promote remyelination. association of neuronal-derived extracellular vesicles cargo with cognitive decline in late middle life introduction: alzheimer's disease (ad) is characterized by a long preclinical stage during which phosphorylated tau pathology spreads in the brain leading to clinical symptoms. pathogenic tau spreads, in part, via extracellular vesicles (evs). we and others have demonstrated that tau cargoes of neuronal-derived evs (nevs) from blood can serve as biomarkers for ad. we aimed to examine whether nev tau cargo can predict cognitive decline in late middle age by leveraging samples from participants in the wisconsin registry for alzheimer's prevention (wrap) study. methods: we blindly immunoprecipitated nevs using antibody against neuronal l cell adhesion molecule (l cam) from serum samples of wrap participants who were cognitively unimpaired at baseline (mean age . ± . years old; . % females; . % apoe carriers), of whom half subsequently developed cognitive decline. we measured phosphorylated (p and p ) and total tau in nevs using electrochemiluminescence assays. we used linear regression models to identify differences between cognitive status groups including age, sex apoe status and the cognitive status*age interaction in the model. results: at baseline, we found trends for higher p -(p = . ) and p -tau (p = . ) levels in future decliners compared to stable participants. further, there were significant cognitive status*age interactions for ptau (p < . ), total tau (p < . ) and ptau (p < . ) with higher levels with increasing age in future decliners summary/conclusion: nev tau cargo differs between late middle-aged individuals at risk for ad with and without future cognitively decline even before decline occurs, presumably due to subclinical spread of tau pathology. further nev biomarker development may allow preclinical ad diagnosis. introduction: in the brain, circulating extracellular vesicles (evs) in the cerebrospinal fluid (csf) contain a variety of signalling factors, including proteins, enzymes, and rna transcripts. while evs have been implicated in many cell-to-cell signalling contexts, the vast majority of these studies are based on findings derived from cell culture conditions. thus, the ability to identify cell typespecific ev release from cellular subpopulations within the brain represents a critical barrier in the field. methods: to address this knowledge gap, we utilized a novel transgenic mouse model to determine the release of cell-type specific evs. here we report the exomap- mouse, which is designed to express an exosomal green fluorescent protein in response to expression of cre recombinase. specifically, the exomap- transgene was inserted at the mouse h locus and consists of (i) a broadly expressed cag promoter/enhancer, (ii) a floxed orf encoding mts-tdtomato, (iii) an orf encoding the exosomal protein acyltya fused to mneongreen (mng), and (iv) a ʹ utr containing the wpre element and polyadenylation signal from the bovine growth hormone gene. results: intracranial ventricular injections of the viral vector aav-ttr-cre, which drives cre recombinase expression from the choroid plexus-specific promotor of the transthyretin gene, leads to acyltya-mng expression in the choroid plexus. moreover, we observed that these mice released mneongreen-positive evs into the cerebrospinal fluid and also visualized the vesicles in the blood. furthermore, these mice displayed an accumulation of acyltya-mng fluorescence in the medial habenula. summary/conclusion: the results indicate that choroid plexus-derived evs are trafficked to the csf and the medial habenula, and more generally, that the exomap- mouse can be used to follow the trafficking of tissuespecific evs into biofluids and between tissues in vivo. introduction: large-scale colorectal cancer (crc) sequencing studies have shown that % of all tumours had at least one mutation in proteins implicated in the wnt signalling pathway. mutations in β-catenin have often been associated with the constitutive activation of wnt signalling pathway and has been established as a major driver of crc. one of the proposed mechanisms of activating wnt signalling involves extracellular vesicles (evs) as cellular couriers to transfer wnt ligands from one cell to another. however, the association of oncogenic mutant β-catenin with evs has not been studied. subpopulations of cancer cells with different mutational loads and behavioural variations lead to intra-tumour heterogeneity methods: integrative proteogenomic analysis showed the secretion of mutant β-catenin via evs. evs were isolated by ultracentrifugation and optiprep density gradient centrifugation. silac-based quantitative proteomics analysis, immunofluorescence, biochemical analysis, qpcr and xenograft models were employed to unveiling the role of evs carrying mutant βcatenin. results: an integrative proteogenomic analysis identified the presence of mutated β-catenin in evs secreted by colorectal cancer (crc) cells. follow up experiments established that evs released from lim crc cells stimulated wnt signalling pathway in the recipient cells with wild type β-catenin. silac-based quantitative proteomics analysis confirmed the transfer of mutant β-catenin to the nucleus of the recipient (rko crc) cells. in vivo tracking of dir labelled evs in mouse implanted with rko crc cells revealed its bio distribution, confirmed the activation of wnt signalling pathway in tumour cells and increased the tumour burden. introduction: there has been a significant increase in incidence of human papillomavirus (hpv ) driven oropharyngeal cancer (opc) in developed countries. there is evidence that hpv alters the molecular cargo of exosomes released by opc. emerging evidence suggests that hpv integration within the human genome is associated with both genomic and transcriptomic alterations. consistent with previous studies, the genomic viral-cellular junctions were identified using dips-pcr method in ( %) saliva samples collected from hpv -driven opc. methods: morphology and molecular features of exosomes derived from three different saliva sampling methods: unstimulated saliva; acid-stimulated saliva; and salivary oral rinses were examined using transmission electron microscopy (tem), nanoparticle tracking (nta) and western blot analysis. hpv- dna detection in salivary exosome was determined by using qpcr method. proteome profile of salivary exosomes derived from both cancer-free controls and hpv -driven opc patients was characterized using liquid chromatography-electrospray ionization-tandem mass spectrometry (lc-ms/ms). results: we demonstrate that unstimulated saliva had greater abundance of exosomes when compared to the other sampling methods. three common exosome markers (cd , cd and cd ) were higher in unstimulated saliva. only salivary exosomes derived from hpv-driven opc patients had a detectable level of hpv- dna. the proteomic signature of salivary exosome was significantly (p < . ) different between cancer-free controls and hpv-driven opc. we found elevated protein abundance of five main glycolytic enzymes (i.e. phosphoglycerate kinase (pgk ), glyceraldehye- -phosphate dehydrogenase (gapdh), aldolase (aldoa) and lactate dehydrogenase a (ldha) in salivary exosomes derived from opc patients, suggesting a functional role of salivary exosome in the reciprocal interplay between hpv-driven opc and glucose metabolism. summary/conclusion: our data suggest that the development of a low-cost non-invasive saliva-based test using both salivary exosomal dna and protein may offer an opportunity to detect hpv-driven opc, that may be clinically useful in managing these patients. continuous in vivo release of mast cell derived extracellular vesicles from an implanted device spreads pro-inflammatory response in mice introduction: mast cells are important players of the immune system and they secrete a wide range of mediators during bacterial infections. mast cells are also able to release extracellular vesicles (evs). here, we report that mast cells communicate with each other in vivo by evs. methods: we isolated bone marrow-derived and peritoneal mast cells from gfp-transgenic and wild type mice. evs were separated from the conditioned media of these cells cultured in the presence or absence of lipopolysaccharide (lps). evs were characterised according to the misev guidelines by flow cytometry, electron and fluorescent microscopy, trps, the spv lipid and the bca protein assays. separated ev-s were cultured with naïve mast cells, and tumour necrosis factor (tnf)-α production was tested by elisa and intracellular flow cytometry. gfp+ mast cells were seeded in diffusion chambers which were implanted into the peritoneal cavities of mice enabling us to investigate the continuous in vivo release of evs. uptake of gfp+ evs and tnf-α expression of peritoneal mast cells were tested by flow cytometry and fluorescent microscopy. results: here, we showed that bacterial lps-sensing mast cells release evs that in turn, induce tnf-α expression in resting mcs in vitro. moreover, we confirmed that evs are transmitted to other peritoneal mast cells in vivo spreading the pro-inflammatory response by inducing tnf-α secretion in peritoneal mast cells. summary/conclusion: ev communication between members of the mast cell network, play an important role in spreading and escalating pro-inflammatory responses to immune stimuli. our data may provide an explanation how the relatively rare tissue resident mast cells can play key roles in diseases such as autoimmune arthritis. the ability of small extracellular vesicles (sevs) to reprogramme cancer cells is known. integrins, receptors for extracellular matrix proteins, are major players in mediating sev functions. previously, we have reported that the αvβ integrin is detected in sevs of prostate adenocarcinoma (prca) cells and transferred into recipient cells in a paracrine fashion; however, its role and expression have never been explored in the most aggressive forms of prca, such as neuroendocrine prca (neprca). neprca does not express androgen receptor (ar) but does express neuron-specific proteins, such as aurora kinase a, synaptophysin and neuron specific enolase, that activate pro-tumorigenic pathways independently from the ar. methods: we isolated sevs from prca c - b cells using iodixanol density gradients and characterized them by immunoblotting and exoview. the experiments were performed in vivo by injecting subcutaneously, in nude mice, du cells treated with sevs expressing or lacking the αvβ integrin, and in vitro, by testing anchorage-independent growth of different cell lines treated with the same sevs. discarded human tissues from prca metastasis were analysed by immunohistochemistry (ihc). results: we demonstrate that a single treatment of prca cells with sevs significantly stimulates tumour growth and anchorage-independent growth. moreover, we show that one treatment with sevs, shed from c - b cells that express αvβ , but not from the control cells that lack αvβ , induces differentiation of prca cells towards a neuroendocrine phenotype and downregulates ar. finally, our ihc analysis shows coexpression of αvβ integrin and synaptophysin in neprca metastatic lesions. summary/conclusion: in conclusion, our current study shows, for the first time, that αvβ integrin expression in donor cells generates sevs that reprogramme recipient cells towards an aggressive tumour phenotype. funding: this study was supported by nci-p - , r - to lrl. introduction: exosomes are small extracellular vesicles (sevs) that carry a variety of cargoes and have been shown to promote tumour cell motility and metastasis. cell motility is influenced by dynamic formation and stability of filopodia: actin-rich protrusions that extend from the leading edge and perform directional sensing. filopodia regulators such as fascin are upregulated in multiple epithelial cancers and can promote invasive phenotypes. however, how filopodia are induced and controlled by extracellular factors is poorly understood. here, we describe a role for sevs in regulating filopodia formation and tumour cell motility. we utilized b f melanoma cells and ht fibrosarcoma cells for fixed-and live-cell imaging to quantify filopodia numbers and dynamics in control and exosome-deplete conditions. itraq proteomics was used to identify sev protein cargoes that contribute to filopodia formation. in vivo experiments were performed using a chick embryo model for metastasis. results: inhibition of exosome secretion in cancer cell lines, via rab a or hrs knockdown, led to decreased filopodia numbers. specificity to sevs was demonstrated by rescue experiments in which purified sevs but not large evs rescued the filopodia phenotypes of exosome-inhibited cells. live imaging of hrs-kd cells revealed that exosome secretion regulates formation and stability of filopodia. proteomics data and molecular validation experiments identified the tgf-beta coreceptor endoglin as a key sev cargo regulating filopodia formation, cancer cell motility, and metastasis. summary/conclusion: in this study, we identified exosomal endoglin as a regulator of filopodia formation and in vivo metastasis. these data are relevant to cancer as endoglin expression is altered in many cancers. in addition, endoglin is the disease gene for hereditary haemorrhagic telangiectasia, and may influence angiogenesis. overall, our data implicate sev-carried endoglin as a key cargo regulating filopodia. astrocyte-derived ev-mediated blood-brain barrier disruption shilpa buch, ke liao, susmita sil, fang niu and guoku hu university of nebraska medical center, omaha, usa introduction: the breach of the blood-brain barrier (bbb), resulting in ensuing neuroinflammation, is a key feature of hiv-associated neurological disorders (hands). while combination antiretroviral therapy (cart) has successfully suppressed peripheral viraemia, cytotoxicity associated with the presence of viral tat protein in tissues such as the brain, remains a significant concern. our previous study has demonstrated that hiv- tat can induce disruption of bbb by downregulation of tight junction (tj) proteins in human brain microvascular endothelial cells (hbmecs) and that this is regulated by the autophagic pathways. methods: evs were isolated from hiv tat-stimulated mouse/human primary astrocytes using the standard differential ultracentrifugation method and characterized by transmission electron microscopy, nanosight & western blot analyses. among the various mirs dysregulated in hiv tat -stimulated astrocyte ev cargo, mir- was found to be upregulated by realtime pcr. confocal microscopy identified uptake of astrocytic evs by hbmecs. functional assessment of astrocytic ev uptake by hbmecs involved cell permeability using transepithelial electrical resistance as well as trans-well endothelial cell monolayer permeability assays. results: hiv- protein tat-mediated induction of micrornas (mirs) in astrocyte-derived extracellular vesicles (adevs) regulated the permeability of bbb by targeting the expression of tj proteins in the hbmecs. exposure of hbmecs to tat-adevs resulted in down-regulation of the tight junction protein claudin , resulting in increased endothelial cell monolayer paracellular permeability. microarray data of tat-adevs demonstrated upregulation of several mirs compared to that of controls, among which upregulated mir- was identified to target the tj proteins using ingenuity pathways analysis. increased expression of mir- was validated in tat exposed astrocytes and tat-adevs. adevs loaded with mir- oligos showed similar effects as that observed with tat-adevs in inducing permeability in hbmecs. increased expression of mir- with downregulation of claudin- was also recapitulated in microvessels isolated from the brains of doxycycline-inducible hiv- tat transgenic mice (itat) mice and in lysates isolated from the frontal cortices of siv+ macaques/hiv+ autopsied brains. summary/conclusion: our findings demonstrated that tat-adevs containing mir- as an important mediator underlying tat-mediated disruption of the bbb. introduction: endogenous exosomes and related extracellular vesicles (evs) are potent nanoparticles released by all cells tested to date. the exploitation of their unique scaffolding for engineering next-generation drug delivery systems represents a major area of academic and commercial interest. the lag in exploiting this potential is in part due to our inability to measured extent and efficiency of modification, e.g., composition and drug loading. here we report a robust pipeline of optical tweezing combined with raman spectroscopy to molecularly characterize engineered evs and quantitatively assess extent of drug loading at single particle resolution. methods: evs derived from cell culture and isolated by ultracentrifugation were fused with synthetic liposomes to create engineered evs (eevs). these eevs were formed via well-established vesicle fusion techniques, namely ( ) mechanical extrusion, ( ) freeze-thawing, or ( ) probe-tip sonication. prior to formation, calcein was encapsulated in the liposomes and used as a surrogate for soluble drug loading. laser trapping raman spectroscopy (ltrs) was used to optically trap single evs, before and after synthetic manipulation. raman spectral analysis was used to assess trapped eevs compared to pure standards to quantify ratiometric variation in chemical composition. results: raman laser trapping experiments confirmed that each formation method results in largely varying ( ) extent of fusion between evs and synthetic calceinloaded liposomes, ( ) efficiency of calcein loading, and ( ) particle size. we could also quantify the molar amounts of liposome vs. ev molecules for single particles, revealing a great amount of variation from particle to particle. functional membrane proteins we left intact to varying degree across fusion methods. summary/conclusion: given the rising importance of analytical tools able to characterize extent of molecular loading for engineered evs, we believe this technology will be very useful, thus warrants further investigation for eev characterization across a variety of clinical applications. funding: randy carney, phd was supported by a research scholar grant, rsg- - - -cdd, from the american cancer society. extracellular vesicles containing host restrictive factor ifitm inhibited zika virus infection of foetuses in pregnant mice through trans-placenta delivery allen z. wu nanjing university, nanjing, china (people's republic) introduction: zika virus (zikv) infection can lead to neurological complications and foetal defects, and has attracted global public health concerns. effective treatment for zikv infection remains elusive and a preventative vaccine is not available yet. therapeutics for foetus need to overcome blood brain barriers to reach placenta and require higher safety standard. methods: in the present study, we engineered mammalian extracellular vesicles (evs) to deliver a host restrictive factor, interferon-induced transmembrane protein (ifitm ), for the treatment of zikv infection. results: our results demonstrated that the engineered ifitm -containing evs (ifitm -exos) were overall safe to the animals and suppressed zikv viraemia by log s in the pregnant mice. moreover, the engineered evs effectively delivered ifitm protein across placental barrier and suppressed overall zikv viraemia in the foetuses to the basal level with significant reduction of viraemia in key foetal organs as measured by q-pcr. mechanistic study showed that ifitm was delivered to the endosomes/lysosomes where it inhibits viral entry to the host cells. summary/conclusion: our study demonstrates that exosomes can act as a cross placenta drug delivery vehicle to foetus and ifitm , an endogenous restriction factor that is highly expressed in placenta, is a potential treatment for zikv infection during pregnancy. introduction: extracellular vesicles (ev) are natural and abundant nanoparticles capable of transferring complex molecules between neighbouring and distant cell types. translational research efforts have focused on co-opting this communication mechanism to deliver exogenous payloads to treat a variety of diseases. important strategies to maximize the therapeutic potential of evs include payload loading, functionalization of the ev surface with pharmacologically active proteins, and delivery to target cells of interest. methods: through comparative proteomic analysis (lc/ms) of purified evs, we identified several highly enriched and ev-specific proteins, including a transmembrane glycoprotein (ptgfrn) belonging to the immunoglobulin superfamily. leveraging ptgfrn as a scaffold for surface display, we generated evs with functional targeting ligands, including single domain antibodies (sdabs), single chain variable fragments (scfvs), single chain fabs (scfabs), and receptor ligands, on the surface to direct ev uptake to cell types of interest. biological activity of these engineered evs was assessed in an array of in vitro and in vivo assays and compared to untargeted controls. results: we engineered evs displaying anti-clec a scfabs to target conventional type dendritic cells (cdc s), anti-cd scfabs to target t cells, and cd ligand to target b cells. in mice, systemic administration of anti-clec a evs resulted in a % increase in the percentage of cdc cells that take up evs over controls. anti-cd evs resulted in both an increase in the percentage of ev positive t cells ( . and -fold for cd + and cd +) and the number of evs per cell ( and -fold for cd + and cd +) in the blood. furthermore, in primary mouse dendritic cells, anti-clec a evs loaded with sting agonist achieved a fold greater pathway induction compared to untargeted controls. preliminary in vivo data suggest that anti-clec a evs reduce the required sting agonist dose -fold to achieve efficacy and induce anti-tumour responses, compared to control evs. summary/conclusion: these results demonstrate the potential of our ev engineering platform to generate novel ev therapeutics targeted to cell types of interest for pharmacologic payload delivery. a novel method for the delivery of cell-free therapy to foetuses with congenital anomalies: a proof of principle study lina antounians, louise montalva, gabriele raffler, maria sole gaffi and augusto zani the hospital for sick children, toronto, canada introduction: antenatal cell-based therapies are currently considered invasive for the foetus. a promising cell-free strategy that holds great regenerative potential for several organs is the administration of stem cell derived evs, whose cargo contains bioactive molecules that epigenetically regulate target cells. herein, we aimed to ) assess the ability of evs to reach foetal organs when administered to the mother intravenously or intra-amniotically; ) compare these administration routes on normal foetuses and foetuses with a congenital anomaly. methods: evs were isolated from rat amniotic fluid stem cell conditioned medium using ultracentrifugation. evs were assessed for size (nanoparticle tracking analysis), morphology (tem), and expression of cd , hsp , flo- , and tsg (western). we injected rat dams with evs stained by exoglow™-vivo or saline (control) via maternal tail vein (iv) or intra-amniotically (ia) at e . . ia and iv injections were performed on dams carrying normal foetuses or foetuses exposed to nitrofen to induce congenital diaphragmatic hernia. after h, dams and pups were sacrificed. d high-sensitivity optical reconstructions of whole foetuses or micro-dissected foetal organs were imaged using the ivis® spectrum imaging system. ev fluorescence signal was compared between normal (n = ) and nitrofen-exposed (n = ) foetuses. results: both iv and ia injection routes were successful in delivering evs to foetal organs. no fluorescent signal was detected in saline only control. ia injections yielded higher signal than iv, and evs reached more organs with ia than iv injections. ia injected evs were detected in the lungs, gastrointestinal, and urinary tract of normal and nitrofen-exposed foetuses. nitrofen exposed foetuses had higher signal than normal foetuses. summary/conclusion: this proof of concept study shows that antenatal administration of stem cell evs is feasible with different routes. although maternally administered evs cross the placenta, ia injection is more effective at reaching foetal organs. further studies are underway to reproduce these findings in experimental models of various congenital anomalies. funding: cihr-sickkids foundation grant os . introduction: safe, efficient and specific nano-delivery systems are essential to the current cosmetic, nutraceutical and therapeutic medicine sectors. the ability to optimise the bioavailability, stability, and targeted cellular uptake of bioactive molecules while mitigating toxicity, immunogenicity and off-target/side effects is of the utmost priority. ves us is a european project, which aims to develop an innovative platform for the efficient production of extracellular vesicles (evs) from microalgae, which constitute a promising renewable bioresource (www.ves us.eu). here we present characteristics of evs from several microalgal lineages, which offer the opportunity for a potentially developing a new and scalable tailor-made biogenic nanotechnology. methods: we cultivated a number of ev-producing microalgal species and developed protocols for ev isolation both at laboratory (differential ultracentrifugation) and pilot scales (tangential flow filtration). the physico-chemical characterization of microalgal evs was carried out according to the minimal information for studies of extracellular vesicles (misev- guidelines): biochemical methods to verify the presence of specific ev-biomarkers, tuned for microalgal evs; dynamic light scattering (dls) and nanoparticle tracking analysis (nta) to assess the particles number and size distribution; electronic scanning microscopy (sem), atomic force microscopy (afm), and cryo transmission electron microscopy (cryo-tem) for imaging analyses; bilayer-specific fluorescence staining (f-nta) to test the purity of ev preparation. results: we identified microalgae as a novel natural source of evs that could constitute a cost-effective and sustainable way of mass-producing them. we screened strains of microalgae and generated an "ev identity card" for each, which contained a variety of ev features relating to their biophysical, biochemical and biological characteristics in line with the misev- . our approach will next focus on the scalable production, surface functionalization and bio-engineering of selected microalgal evs. at the same time, their bioactivity will be explored using both in vitro and in vivo biological models. summary/conclusion: the ves us consortium is investigating the potential of microalgae as novel ev bioresources. this research will attempt to bioengineer novel naturally-derived nanocarriers, microalgal evs, suitable for the development of future cosmetics, nutraceutical or therapeutic formulations. funding: this project has received funding from the european union's horizon research and innovation programme under grant agreement no . sequence-specific rna trafficking to extracellular vesicles is conserved across cell types several sequences have been identified that act as a zipcode for preferential rna targeting into ev (evtropic) or for retention in parental cells (cell-tropic) . in this work, we aimed to compare the ev-tropic capacity of specific rna sequence motifs in promoting loading into ev, across different cell models representing the main cell types found in the body. methods: immune, epithelial and mesenchymal cell lines were transiently transfected with xenogeneic c. elegans micrornas (mirnas) containing ev-tropic or cell-tropic sequences and grown in culture. ev were isolated from the supernatant by differential (ultra)centrifugation. rna was extracted from both cell pellets and isolated ev fraction, and target mirnas were quantified by digital droplet pcr. distribution of cargo mirna across cells and ev was also analysed for chimeras of ev-and cell-tropic sequences. results: the mirnas containing an ev-tropic sequence were highly enriched on the ev fraction, with - , higher levels than in parental cells. contrarily, cell-tropic mirnas were only - times higher in ev. no significant differences were observed in the ev loading efficiency for the various ev-tropic motifs tested. mutations in the ev-sorting motif resulted in reduced ev loading. ev-tropic sequences consistently promoted mirna loading into ev across all the cell models evaluated, suggesting conserved biological mechanisms. summary/conclusion: we showed that rna loading into ev is dependent on the presence of defined evtropic rna motifs, and that sorting mechanisms are conserved across the major cell types tested. the highest loading efficiencies resulted in . mirna copies per particle on average, suggesting a limited scope for ev-tropic motifs for therapeutic rna loading into ev. funding: as, os and eli are fellows of the astrazeneca postdoc programme. introduction: coordinated activity between pancreatic islet cells is critical for the regulation of glucose homoeostasis. chronic exposure to diabetogenic factors such as pro-inflammatory cytokines, perturb islet cell crosstalk and β-cell function in diabetes. extracellular vesicles (evs) derived from cytokine-exposed β-cells modulate physiological and pathological responses to β-cell stress. however, the mechanisms governing this process remain largely unknown. we set out to test the hypothesis that β-cell failure in diabetes is mediated in part through β-cell autocrine release of pro-inflammatory evs which promote inflammation and inhibit βcell function. methods: pro-inflammatory cytokine-exposed evs (cytoevs) were generated using conditioned media from mouse min β-cell line treated with diabetogenic cytokines (tnfα, il- β, ifnγ, h). evs were also isolated from human type diabetic (t dm) and lean non-diabetics (lnd) plasma. gw (n-smase inhibitor) was used in the presence of cytokines to determine the effect of reduced ev concentrations on the restoration of β-cell function. proteomic and rna-seq analysis was conducted on min β-cell cytoev (vs. control ev) and cytoev treated mouse islets, respectively. results: assessment of ev concentrations from cytoev and human t dm plasma revealed a~twofold increase (p < . , vs. control (ctl) and lnd ev). immunofluorescence staining of cd and cd expression was significantly elevated in human t dm pancreas (p < . , vs. lnd). while acute inhibition of ev formation with gw ( µm) showed significant restoration in β-cell function (glucose stimulated insulin secretion assay, gsis) in cytokine-exposed mouse and human islets (~ and fold vs. cytokines alone, p < . ). moreover, functional assessment of mouse islets exposed to cytoev ( h) resulted in suppression of gsis (~ %, vs. untreated, p < . ). identification of cytoev content through proteomic analysis revealed a significant upregulation of the chemokine, cxcl (~ fold vs. ctlev) and rna-seq analysis of cytoev treated mouse islets depicted a marked upregulation of transcripts associated with cxcl -cxcr signalling (p < . ) and downstream pathways (e.g. nfκb; p = . and jak/stat; p = . ). furthermore, inhibition of cytoev (gw ) with cytokines markedly decreased cxcl (~ %) and cxcr receptor (~ %) expression in min β-cells. summary/conclusion: these data suggests that cytokines elevate cxcl expression in β-cell ev to enhance inflammation-induced diabetes. this is mediated through ev-autocrine release of cxcl consequently activating cxcr signalling and downstream pathways to impair β-cell function in diabetes. synergy between -lipoxygenase and secreted pla promotes inflammation by formation of tlr agonists from extracellular vesicles introduction: damage associated molecular patterns (damps) are endogenous ligands that induce innate immune response, thus promoting sterile inflammation. during oxidative stress, stress-derived evs (stressevs) were found to activate toll-like receptor (tlr ), but the activating ligands were not fully determined. additionally, several enzymes, among them -lipoxygenase ( -lo) and secreted phospholipase a (spla ) are induced during inflammation and were suggested to promote damp formation. methods: stressevs were produced from hek cells exposed to um a and isolated with ultracentrifugation. : lysopi was oxidized for min with -lo. additionally, synevs were prepared from phospholipids (pls), oxidized with -lo and hydrolysed with spla . activity was measured by qpcr and elisa on wt and tlr -ko macrophages. -lo oxidized : lysopi was analysed by mass spectrometry. spla activity was measured in synovial fluid from rheumatoid and gout patients using fluorometric assay. k/bxn serum transfer induced arthritis model on wt and tlr ko mice (c bl/ mice) with spla -iia injection was used (approval no. u - / / by mkgp of slovenia). results: stressevs released after oxidative stress were found to activate tlr with a gene profile different from bacterial lipopolysaccharide (lps). stressevs, -lo oxidized synevs, but only -lo oxidized lysopls activated cytokine expression through tlr /md- . hydroxy, hydroperoxy and keto products of : lysopi oxidation were determined by ms and they activated the same gene pattern as stressevs. furthermore, spla activity, which we detected in the synovial fluid from patients, promoted formation of tlr agonists after -lo oxidation. injection of spla -iia into mice promoted k/bxn serum induced arthritis in tlr -dependent manner. summary/conclusion: both -lo and spla are induced during inflammation, therefore these results imply the role of oxidized lysopls in stressevs in promoting sterile inflammation through tlr signalling. the formation of tlr agonists is enzyme driven so it provides an opportunity for therapy without compromising innate immunity against pathogens. funding: h -msca-itn project tollerant (grant no. ), slovenian research agency (project no. j - to mmk, research core no. p - to rj). monocytes traffic extracellular vesicles to damaged muscle and adopt a novel immunophenotype to support muscle regeneration russell g. rogers, akbarshakh akhmerov, weixin liu, lizbeth sanchez and eduardo marbán smidt heart institute, cedars-sinai medical center, los angeles, usa introduction: extracellular vesicles (evs) are secreted membrane vesicles that carry bioactive molecules such as mirnas, mrnas, proteins, and lipids to modify recipient cell behaviour. we recently demonstrated evs secreted by cardiosphere-derived cells (cdc-evs) augment endogenous muscle regeneration in mdx mice, a model of duchenne muscular dystrophy, when delivered intravenously. in parallel, macrophages preferentially accumulate surrounding small regenerating myofibers in cdc-ev treated mdx muscle. however, it is currently unclear how intravenous cdc-evs home to dystrophic muscle and exert their therapeutic bioactivity. methods: fluorescently-labelled and unlabelled cdc-evs were infused into the contralateral femoral vein of wild-type mice with unilateral muscle injury induced by bacl . injured and uninjured muscles were dissected h following infusion and subjected to optical imaging, immunohistochemistry, and confocal microscopy. this experiment was repeated using clodronate liposomes to deplete endogenous monocytes/macrophages. next, rna-seq was preformed on bone marrow-derived m , m , and cdc-ev (mcdc-ev) polarized macrophages from mdx mice. conditioned media (cm) from these macrophages were tested in an in vitro model of myogenesis. lastly, small rna-seq was performed on evs secreted by m , m , and mcdc-ev macrophages. results: when delivered intravenously, cdc-evs naturally home to injured, but not uninjured, skeletal muscle. cdc-evs were detected in the interstitium adjacent to non-muscle cells, macrophages, and within surviving myofibers. after depletion of monocytes/ macrophages by clodronate liposomes, the presence of cdc-evs in the injured muscle was attenuated. bioinformatic analyses indicate cdc-evs confer a novel immunophenotype to mdx macrophages with features of both m and m . indeed, mcdc-ev cm promotes myoblast proliferation and supports myogenic differentiation. interestingly, mcdc-ev evs have a unique mirna signature and contain several mirnas with known roles in myogenesis. summary/conclusion: these data indicate circulating monocytes traffic cdc-evs to damaged muscle where they adopt a novel immunophenotype to support muscle regeneration. we propose mcdc-ev macrophages mediate their pleiotropic effects via paracrine factors, possibly including evs. introduction: microglia, the immunocompetent cells of the cns, play an important role in maintaining cellular homoeostasis in the cns. these cells secrete immunomodulatory factors including nanovesicles and participate in the removal of cellular debris by phagocytosis or autophagy. the contribution of microglial-derived extracellular vesicles (m-evs) to the maintenance of cns homoeostasis is unclear. in addition, knowledge of canonical signalling pathways of inflammation and immunity gene expression patterns in human microglia exposed to m-evs is scarce. methods: here, we analysed the effects of m-evs produced in vitro by either tnfα-activated or non-stimulated microglia bv cells. we showed that m-evs are internalized by both mouse bv and human c microglia and that the uptake of m-evs in microglia induced autophagic vesicles at various stages of degradation including autophagosomes and autolysosomes. consistently, exposure of microglia to m-evs increased the protein expression of the autophagy marker, lc b-ii, and promoted autophagic flux in live cells. to elucidate the biological activities occurring at the transcriptional level in c microglia exposed to m-evs, the gene expression profiles, potential upstream regulators, and enrichment pathways were characterized using targeted rna sequencing. results: inflammation and immunity transcriptome gene panel sequencing of both activated and normal microglia exposed to m-evs showed involvement of several canonical pathways and reduced expression of key genes involved in neuroinflammation, inflammasome and apoptosis signalling pathways compared to control cells. summary/conclusion: we demonstrate that in vitro produced microglial evs are able to influence multiple biological pathways and promote activation of autophagy in order to maintain microglia survival and homoeostasis. funding: this work was financed by hasselt university and by efro through the interreg v grensregio vlaanderen nederland project trans tech diagnostics. evaluation of plasma extracellular vesicles as biomarkers for longevity xin zhang a and virginia kraus b a laboratory medicine center, nanfang hospital, southern medical university, guangzhou, guangdong, , p. r. china, guangzhou, china (people's republic); b division of rheumatology, duke molecular physiology institute, duke university school of medicine, durham, usa introduction: extracellular vesicles (evs) have emerged as key indicators and effectors of ageing. although plasma concentrations of evs decline with age, the ev biomarkers associated with ageing and longevity are not fully understood. recently, our group found an age-related decline of plasma evs associated with immune cells during normal human ageing. our study aims to evaluate the association of plasma evs with longevity. methods: plasma samples were selected from the established populations for epidemiologic studies of the elderly study subjects (n = ): half dying within years (short-lived group) and half surviving ≥ years (long-lived group) after the blood draw; all matched for age (median age . ± . years, range - ), gender ( % female), and race ( % white/ % black). the samples were acquired under donor consent and irb approval of duke university. evs were separated from the plasma samples, and profiled based on the surface markers of haematopoietic stem cells (hscs), mesenchymal stem cells, immune cells, skeletal muscles, cardiac muscles and adipocytes (cd , cd , cd , cd , cd , cd , cd , cd , cd , cd , cd , cd a, cd a, cd , cd , hla-abc, hla-g, hla-drdpdq, cd , cd , cd , m cadherin, ryr , ryr , fabp , dlk ). the percentages of evs expressing each tested molecule were determined using a high-resolution multicolour bd lsr fortessa x- flow cytometer as we recently reported. graphpad prism . software was used for statistical analysis. results: we found significantly increased percentages of cd +, hla-abc+, cd + and cd a+ large evs ( - nm) in the long-lived compared to the short-lived group. none of the tested surface marker expressing medium ( - nm) or small (< nm) evs showed differential percentages between the shortand long-lived groups. summary/conclusion: evs carry surface markers from their parent cells. cd is expressed by hscs and immune cells. cd regulates homing of human cord blood cd + hscs, and delivers a potent cd independent costimulatory signal to activate t cells. hla-abc, the key human immunogen, is expressed by nucleated cells and platelets. cd is expressed by hscs, immune cells and epithelial cells, and cd + plasma evs declined with age in healthy people. cd a is expressed by hscs, megakaryocytes and platelets, and is functionally relevant for hsc maintenance and haematopoietic homoeostasis. our preliminary data suggest that hscs and immune cell associated plasma evs (cd +, hla-abc+, cd +, cd a+ large evs) inform on health status related to longevity. introduction: it is anticipated that stem/progenitor cells-derived extracellular vesicles (spc-evs) will rapidly progress towards clinical studies, and the development of reproducible, efficient, scalable and costeffective process for their production is expected to boost the therapeutic applications of evs-based products. in addition, the use of defined serum-/xenogeneic(xeno)-free culture medium formulations could result in substantial improvements for spc-evs production in terms of reproducibility, stability and quality, while ensuring the approval of regulatory agencies. the main goal of this work is to develop a full-controlled manufacturing platform for the spc-evs production. methods: human mesenchymal stromal cells (msc) were expanded in a xeno-free microcarrier-based bioreactor culture system operating in fed-batch feeding mode and after days the conditioned medium was collected. different methods for spc-ev isolation/purification from the msc-derived conditioned medium, including chromatography were compared and the the quality of the final product obtained was characterized by different methods according to misev, including nanoparticle tracking analysis, lipidomics and western blot. moreover fourier-transform infrared (ftir) spectroscopy was evaluated in terms of its implementation as a standard technique for the identification and characterization of evs. results: after days of msc expansion under dynamic conditions, we collected . l of conditioned medium with approximately . million evs/msc. a combination of a pretreatment with a nuclease for the digestion of dna/chromatin with a purification using strong anion exchange chromatography led to the best results so far in terms of evs isolation. of notice, by ftir spectroscopy, it was possible to define ratios of spectral bands, that can be used as biomarkers, enabling the discrimination of evs chemical fingerprint in function of the culture conditions tested. summary/conclusion: the platform established herein could be applied to the production of wellcharacterized spc-evs targeting their biomedical use in different settings (e.g. as drug delivery systems), as well as evs from other parental cells lines (i.e. dendritic cells) in therapeutic settings as cancer. ultrasensitive protein detection for quantification of extracellular vesicles in human biofluids enables comparison of isolation techniques dmitry ter-ovanesyan, maia norman, wendy trieu, roey lazarovits, george church and david walt wyss institute, boston, usa introduction: extracellular vesicles (evs) are released by all cells into biofluids and hold great promise as reservoirs of disease biomarkers. one of the main challenges in studying evs and using them in diagnostics is a lack of suitable methods to quantify evs that are sensitive enough and can differentiate evs from similarly sized lipoproteins and protein aggregates. we propose using ultrasensitive single molecule array (simoa) assays to quantify evs by immunoisolating and detecting ev transmembrane proteins in microwell arrays. we developed single molecule array (simoa) assays using the quanterix hd-x analyser for the quantification of evs using the tetraspanins cd , cd , and cd . simoa allows for the detection of single proteins using arrays of femtoliter wells, turning elisa into a digital immunoassay. we then used these assays, together with an additional assay for albumin, to compare commonly used ev isolation methods from plasma and cerebrospinal fluid (csf): ultracentrifugation, precipitation (exoquick), and size exclusion chromatography (sec) using the izon qev columns. we further used these assays to rapidly optimize and improve sec by comparing different sec resins and column dimensions in both plasma and csf. results: in comparing our simoa assays to traditional elisa with the same antibodies, we found that the simoa assays were more than times more sensitive, detecting the tetraspanins in samples where the proteins were undetectable by elisa. given the high dynamic range and high-throughput capabilities of simoa, we were able to comprehensively compare relative ev yields and ev purity for different isolation methods of evs from plasma and csf. we provide average tetraspanin and albumin levels to directly compare the methods. we also tested different sec resins and provide data for custom sec columns that outperform izon qev and allow for fine tuning of different ratios of evs to albumin. summary/conclusion: our results highlight the utility of quantifying evs using ultrasensitive simoa assays for tetraspanins. we were able to rapidly simoa to rapidly evaluate different ev isolation methods in csf and plasma. in general, the experimental framework we present could be easily applied to evaluate new ev isolation methods, or applied to any other biological fluid. thus, we think simoa is a powerful new tool for relative ev quantitation. introduction: the protein profile of extracellular vesicle (ev) subpopulations has been shown to contain valuable disease information, notably in cancer. currently, techniques aiming to find ev proteins that associate together mainly focus on transmembrane proteins, while methods that also probe cytosolic proteins generally resort to a combination of affinity capture, elution, and lysis, which limits throughput. to allow the high-throughput analysis of both membrane and cytosolic ev proteins, we optimized a total extracellular vesicle antibody microarray (tevam) incorporating fixation and heat-induced epitope retrieval (hier), then leveraged it to perform combinatorial protein profiling of evs from colorectal cancer (crc) cell lines ht and sw . methods: arrays of iggs targeting surface protein markers were incubated overnight with evs purified from cancer cell line supernatants. hier optimization was carried out through variation of buffer contents, presence or absence of prior permeabilization, as well as incubation time and temperature, for a total of conditions. a evs, previously profiled with other methods, were used as a model during the optimization. cytosolic protein hsp and membrane marker egfr, both with high expression in a evs, were probed and the results used to compare hier conditions. following hier treatment, protein targets were detected through incubation with primary antibodies and fluorescent secondary antibodies or streptavidin. the resulting optimized tevam workflow was used to phenotype ht and sw evs through probing of trios of surface ( ) and internal ( ) protein targets. results: the selected tevam protocol successfully maximized hsp signal while minimally affecting egfr detection, enabling simultaneous analysis of surface and internal proteins. profiles of more than combinations, featuring integrins, claudins, cytokines, and other key actors of cancer-relevant pathways, were obtained for ht and sw evs, revealing coexpression patterns that highlight the biomolecular heterogeneity both within and between crc cell line evs. summary/conclusion: using tevam, intra-and extravesicular proteins can be detected simultaneously in evs immobilized based on surface protein content, yielding extensive combinatorial protein profiles with significance for health and biomarker research. characterization of evs using orthogonal techniques identifies discrete ev populations from a mouse dendritic cell line bryce killingsworth a , timothy traynor b , joshua a. welsh c , aleksandra dakic a , jason savage a , kevin camphausen d , kenneth aldape a and jennifer jones a a laboratory of pathology, national cancer institute, national institutes of health, bethesda, usa; b laboratory of pathology, national cancer institute, national institutes of health, gaithersburg, usa; c laboratory of pathology, national cancer institute, national institute of health, bethesda, usa; d radiation oncology branch, national cancer institute, national institutes of health, bethesda, usa introduction: extracellular vesicles (evs) have the potential to serve as valuable biomarkers for patient response to cancer therapy. however, development of robust ev-based clinical assays relies on knowledge of ev concentration and diameter distribution. many different methods exist to measure the size and concentration of evs, and each method exhibits strengths and limitations. it is important to use orthogonal methods for determination of these important properties of ev preparations. here, we use dendritic cellderived evs to demonstrate that some ev analysis methods can give a biased interpretation of both diameter and concentration. through comparison, we highlight why orthogonal assays are essential in providing measurement reliability. methods: dc . mouse dendritic cells were cultured in flasks containing a total of . l of ev-depleted media ( % fbs, centrifuged hr. x , g.) when cells reached % confluency, conditioned media was collected, depleted of debris with two min. x , g spins, and concentrated down to~ ml using a pall jumbosep kda mwco filter. the ev concentrate was purified from protein using an izon qev- column, with ml fractions collected. the protein content of the ev-containing fractions was analysed by a , pierce bca, and bioanalyzer. the diameter distribution of the evs was determined by nanoparticle tracking analysis (nta), resistive pulse sensing (rps), flow cytometry (fcm), and electron microscopy (em.) concentration was compared using nta, rps, and fcm. evs were further analysed by protein mass spectrometry and rna sequencing. results: we have identified two distinct populations of evs with our dc . preparation, one highly abundant population with a power-law distribution, whose peak diameter is below nm, and a second, less abundant population with a peak diameter at approximately nm. these two distinct populations and their relative concentration were not detectable with all analysis techniques. based on cross-platform measurements, these populations appear to have distinct compositions that warrant further investigation. summary/conclusion: the use of orthogonal methods allowed the detection of two discrete populations of evs which was not possible on some platforms and would have resulted in a biased perspective of the sample composition. this work has highlighted the need for orthogonal measurements to be conducted by pairing techniques that do not have the same biases. introduction: extracellular vesicles (evs) are nanosized vesicles shed by all cells that serve vital roles in cell-to-cell communication. tumour-associated ev subpopulations vary in molecular content (lipids, proteins, nucleic acids, small molecules), enabling minimally invasive spectroscopic analysis for a wide variety of cancers. here, we use surface-enhanced raman spectroscopy (sers) in combination with a novel plasmonic substrate for global chemical composition analysis of cancerous and non-cancerous populations of evs to determine distinguishing surface characteristics. methods: evs were isolated from ovarian cancer (ovca) patient serum samples by differential ultracentrifugation. a new hybrid nanoplasmonic scaffold comprised of a microscale biosilicate diatoms embedded with silver nanoparticles (agnps) was used for sers measurements. the substrate was incubated with cysteamine to positively-charge the agnps (responsible for the sers enhancement) so that evs could attach (evs are naturally anionic). in a typical experiment, μl of~ particles/ml evs per sample were incubated with the porous substrate surface, which was inverted on a glass cover slip for raman interrogation. principle component analysis (pca) was used to compare the spectra and determine distinguishing characteristics between populations from tumour and non-tumour sources. we also trypsinized evs before sers analysis to see the extent of influence the surface molecules play in localizing the evs to the agnp "hot spots." results: a total of clinical samples ( ovca and non-malignant control) were tested in combination with ovca skov- cell line evs. simple pca was able to separate clinical samples according to disease subtype and major peaks were identified to provide chemical content analysis. each sample exhibited inherent heterogeneity but clustered together in a distinguishable way from the others. summary/conclusion: despite innate heterogeneity within single samples (i.e., evs isolated from a single patient sample), evs isolated from clinical samples could be easily distinguished from each other using our hybrid sers substrate, with minimal sample processing, a label-free approach, and only a few microlitres of sample. our study using this novel plasmonic material demonstrates its potential for use as a component in next-generation diagnostic platforms. introduction: single-particle analysis is critical for understanding extracellular vesicle (ev) heterogeneity. yet such techniques remain technically challenging due to low detection sensitivity and presence of variable amounts of "contaminants," including lipoproteins. the high degree of structural similarity between evs and lipoproteins in size, density, and chemical composition, results in their co-isolation using any of the standard ev isolation techniques. here we introduce laser trapping raman spectroscopy (ltrs) as a wellsuited, label-free, and non-destructive tool to distinguish evs from various lipoprotein species at single particle resolution. methods: ev samples were isolated from skov- cell culture supernatant by differential ultracentrifugation and their raman spectra measured. as the most abundant lipoproteins in ev isolations from human biofluids are sub-micron low density lipoprotein (ldl), very low density lipoprotein (vldl), and high density lipoprotein (hdl) particles, these were purchased as pure components and also measured by ltrs. ldl and vldl were then spiked-in to isolated evs to mimic "contaminated" post-isolation ev samples. raman spectra were analysed by principal component analysis (pca) using a custom matlab script. results: ldl and vldl have been observed to adhere to ev surfaces in vitro after standard isolation techniques. we could readily distinguish pure vldl, ldl, and hdl standards according to their raman spectra. pca revealed distinction of skov- evs from both ldl and vldl. pca also differentiated skov- evs incubated with ldl from skov- evs incubated with vldl. extent of ldl and vldl adherence to evs could be observed and quantified. summary/conclusion: through raman and pca, classes of lipoprotein and evs can be identified and quantified when co-incubated. ltrs is a quantitative single-ev analysis technique that can be used to differentiate between lipoprotein classes and evs when incubated together. this technique allows for analysis of evs where standard isolation methods fall short. introduction: extracellular vesicles (evs) are endogenous membrane-derived vesicles that shuttle lipids, proteins or nucleic acids between glia and neurons, thereby promoting neuronal survival and plasticity in the cns and contributing to neurodegenerative conditions. although evs hold great potential as cns theranostic nanocarriers, the specific molecular factors that regulate neuronal ev uptake and release are currently unknown. methods: we used a combination of patch-clamp electrophysiology and ph-sensitive dye imaging to examine stimulus-evoked ev release in individual neurons in real time. results: whereas spontaneous electrical activity and the application of a high-frequency stimulus (hfs) induced a slow and prolonged fusion of multivesicular bodies (mvbs) with the plasma membrane (pm) in a subset of cells, the neurotrophic factor bfgf (basic fibroblast growth factor) greatly increased the rate of stimulusevoked mvb-pm fusion events and, consequently, the abundance of evs in the culture medium. proteomic analysis of neuronal evs demonstrated bfgf to increase the abundance of the v-snare vesicle-associated membrane protein (vamp , cellubrevin) on evs. conversely, knocking-down vamp in cultured neurons attenuated the effect of bfgf on ev release. introduction: heat shock proteins (hsps) function as chaperones under both normal and pathologic conditions. as chaperones they assist in protein folding, in holding protein complexes for current or future activation, and in degradation of senescent proteins for recycling of components and display for immune surveillance. during stressful situations, hsp quantities and/or activities increase as cells and tissues seek protection from insults. these insults can result in the cell surface display of hsps, which can then lead to the surface display of hsps on extracellular vesicles (evs). hsps present on the cell surface or in the extracellular space are regarded as "danger signals" in an ancient biologic paradigm. hsp-accessorized evs may act as "danger boli", carrying not only the hsps, but hundreds of components of the stressed parental cell, capable of prompting differential responses depending on the status of the recipient cell. methods: clarified/filtered plasma from patients suffering from neurologic maladies (cancer, brain injury, multiple sclerosis) was incubated with peptides designed to bind hsps. the evs congeal under these conditions and are pelleted (microfuge) and washed with increasing-stringency buffers. we lysed the evs and subjected them to metabolomic analyses (focused on lipids) or assayed them on phosphokinase arrays. results: we show that evs from the blood of patients suffering from brain tumours, or from tbi, or from ms, possess distinct metabolomes compared to blood evs from healthy donors. we found hundreds of differentially-expressed lipids amongst the patients vs the healthy donors. the levels of annotation and identification for these compounds ranges from level (low, no matches in databases) to level (high, annotation matches to known database components). in addition, we found differences in phosphorylated kinases as cargo in these evs between patients with matched primary vs recurrent gliomas, and among tbi/stroke patients compared to healthy donors. summary/conclusion: hsp-accessorized evs present different metabolomic and phosphokinase content which may serve as biomarkers in a "liquid biopsy" setting, but may also play roles in the pathobiology of neurologic diseases. introduction: methamphetamine (ma) has deleterious effects to both peripheral organs and the central nervous system. the rewarding properties and addictive potential of ma are correlated with increased synaptic dopamine availability following alterations in dopamine and vesicular monoamine transporter function. in rodents, ma alters brain mirna expression and the mirna content of serum extracellular vesicles (ev). here we examined plasma evs isolated from human subjects actively using ma (ma-act) for size, concentration, protein markers, and mirna content. methods: plasma samples from ma-act, and controls (ctl) were obtained from the methamphetamine abuse research center. plasma evs were evaluated by vesicle flow cytometry (vfc) for size, concentration, and surface protein markers. vfc antibodies included markers for a pool of tetraspanins (cd , cd , and cd ), platelet evs (cd ), pro-coagulant evs (annv), and red blood cell evs (cd ). next plasma ev isolated by size exclusion chromatography were analysed by qpcr on taqman® array human microrna a + b card set v . . fold change was calculated by ΔΔcq between ma-act and ctl for mirna expressed in ≥ % of samples in at least group. we identified the top % of ranked mirna by fstatistic; of these, the mirna of interest for ma-act were identified by at least a (i) . fold change in expression, (ii) area under the receiver operating characteristic curve of . , and (iii) glass's Δ of . for mirna of interest correlations to additional ma variables were conducted, along with ingenuity pathway analysis of predicted gene targets. tobacco use was controlled for. results: vfc data show that the size (~ nm) and concentration (~ . x particles/ml) of all plasma evs is comparable between ma-act and ctl groups. in addition, the plasma evs primarily consist of tetraspa-nin+, annv+, or cd + evs, and to a much lesser extent cd + evs. of the mirna expressed in ma-act and/or ctl plasma evs, there were mirna that have at least a . fold increase or decrease in ma-act. mirna were identified to be of interest in ma-act based on fold change, effect size and diagnostic potential, compared to ctl. further, of the mirna correlate with a ma associated variable, including frequency of use and age of first use. together the mirna best cluster subjects based on ma-act status, not tobacco use. finally, the predicted gene targets of the mirna are associated with canonical pathways linked to ma. summary/conclusion: ev mirna expression in ma-act subjects was unique to ctl participants, suggesting that ma may affect ev communication among cells. the differential mirna expression also implicates a role for evs in behavioural and physiological effects specific to ma, and suggests that there may be changes in expression of mirna that are relevant to specific drugs of addiction, as well as to a spectrum of drug-mediated addiction disorders. bone marrow-derived extracellular vesicles may alter the ageing phenotype of murine haematopoietic stem cells. sicheng wen a , jill kreiling b , mark dooner c , elaine papa c , michael del tatto c , yan cheng c , mandy pereira c , peter quesenberry c and laura r. goldberg d in natural ageing of haematopoietic stem cells (hscs) is unclear. we tested the hypothesis that bone marrowderived evs (bm-evs) can modulate the ageing hsc phenotype. methods: we flushed bone marrow from old ( - month old) and young ( - -week old) c /bl mice and collected bm-evs by differential centrifugation ( × g for min, supernatant collected and centrifuged , × g for hour, bm-ev pellet collected and quantified by nanoparticle tracking analysis). we injected old mice with x ^ young bm-evs via tail vein, daily x days. control mice were injected with age-matched bm-evs or vehicle alone. we euthanized the mice one month post-injection, harvested whole bone marrow (wbm) and highly purified hscs (lineage negative/c-kit+/sca- +/cd +; lsk-slam) and tested stem cell function in competitive bone marrow transplants ( - recipients/group). results: at months post-transplant, wbm from old mice exposed to young bm-evs exhibited a statistically significant decrease in engraftment when compared to wbm exposed to age-matched bm-evs (percent average donor chimerism ± sem: % ± % (young evs) vs. % ± % (old evs)). for lsk-slam from old mice, we observed a trend towards decreased engraftment when exposed to young bm-evs and a trend towards increased engraftment potential when exposed to old bm-evs (percent average donor chimerism ± sem: % ± % (young evs), % ± % (old evs), % ± % (vehicle)). these findings are consistent with our previous data showing that, in contrast to highly purified hscs which develop impaired stem cell function with ageing, total un-separated old wbm actually has increased engraftment capacity when compared to young wbm. of note, we found that the classic myeloid skewing by old lsk-slam was partially reversed by exposure to both young and old bm-evs. finally, consistent with the known increase in highly purified hscs with age, our preliminary data showed that old mice exposed to young bm-evs had an approximately -fold decrease in the number of lsk-slam cells in marrow, indicating that bm-evs may influence agerelated changes in hsc number. summary/conclusion: these preliminary data suggest bm-evs may play a role in modulating hsc ageing phenotypes, potentially altering engraftment capacity, lineage fate, and lsk-slam population size. future studies delineating the molecular mechanisms underlying these ev-mediated effects could provide key insights into normal haematopoietic ageing. funding: this work was supported by the nih grants p gm , dk - a and by the savit foundation. oral with poster introduction: brain extracellular vesicles (evs) are heterogenous and include previously described microvesicles and exosomes. herein we characterized a formerly unappreciated population of mitochondriaderived evs that we term "mitovesicles". mitochondrial dysfunction is a well-established hallmark of ageing and neurodegenerative disorders as down syndrome (ds). hence, we examined mitovesicle levels and cargo under these conditions to characterize in vivo mitovesicle biology and responsiveness to mitochondrial stressors. methods: employing a high-resolution density gradient, distinct and novel populations of evs were isolated from murine and human ds and diploid control postmortem brains or from cell media. morphometric ev features were analysed by nanoparticle tracking analysis and cryogenic electron microscopy, while ev constituents were characterized by western blotting, mass spectrometry, lipid profiling and mitochondrial rna qpcr. results: we identified a population of double-membrane, electron-dense brain evs containing multiple mitochondrial markers ("mitovesicles") that are highly distinct from microvesicles and exosomes. proteomic data show that mitovesicles contain a unique subset of mitochondrial proteins while lacking others, such as tom . mitovesicles have a lipid composition that is unlike that of previously described evs and is consistent with mitochondrial origin. functionally, the complex-iii inhibitor antimycin-a stimulated in vitro mitovesicle release into the cell media, suggesting an interrelationship between mitochondrial dysfunction and mitovesicle biology. in mouse brains, mitovesicle levels increased with age and were found to be higher in ds compared to diploid controls. mitochondrial rna and protein levels were also altered in ds compared to diploid controls. summary/conclusion: we describe a previously unidentified type of metabolically competent evs of mitochondrial origin that we designate mitovesicles. our data demonstrate that brain mitovesicle levels and cargo are tightly regulated in normal conditions and are modified during pathophysiological processes in which mitochondrial dysfunction occurs, suggesting that mitovesicles are a previously unrecognized player in mitochondria quality control and may have a role in the trans-cellular tissue response to oxidative stress. introduction: alzheimer's disease (ad) is a devastating neurodegenerative disease leading to progressive memory loss and ultimately death with limited therapeutic options. growing evidence supports the theory that toxic proteins, like tau and amyloid, may propagate from diseased cells by packaging toxic proteins into extracellular vesicles (evs) and releasing them to infect other cells. one enzyme involved in the biogenesis of evs is neutral sphingomyelinase (nsmase ), which catalyzes the hydrolysis of sphingomyelin to produce phosphorylcholine and ceramide. several groups have reported improved cognition and reduced tau propagation when nsmase is pharmacologically inhibited or genetically knocked down in ad mouse models. unfortunately, current nsmase inhibitors are not suitable for clinical development due to poor solubility and inadequate pharmacokinetic profiles. methods: our group carried out a high-throughput screening campaign followed by extensive medicinal chemistry efforts leading to the discovery of phenyl (r)-( -( -( , -dimethoxyphenyl)- , -dimethylimidazo [ , -b] pyridazin- -yl) pyrrolidin- -yl) carbamate (pddc), an orally active, nm potent inhibitor with excellent selectivity and brain penetration. we tested pddc's ability to inhibit exosome release in cultured primary glial cells as well as an in vivo model of acute ev release. we then treated xfad mice with mg/ kg of pddc daily for six months and monitored their behaviour in the fear conditioning assay. results: pddc dose dependently reduced ev release from cultured primary glial cells and significantly reduced plasma ev numbers in an in vivo model. following chronic treatment with pddc, xfad mice demonstrated significantly improved cognitive function in the fear conditioning assay. summary/conclusion: these promising findings are currently being expanded using mouse models of tau propagation. if successful, these data would support pddc as a novel compound for targeting the pathological spread of tau as a therapeutic for ad. profiling evs in the anterior cingulate cortex of individuals with major depressive disorder introduction: major depressive disorder (mdd) is one of the leading causes of disability worldwide, affecting % of the population. the environment has been thought to play a role in the disease development, resulting in biological changes mediated by epigenetic mechanisms. microrna's (mirna) are well known epigenetic regulators that are disrupted in the depressed brain, and they are packaged into extracellular vesicles (evs). evs have emerged as means of intercellular communication, a process that is also disrupted in mdd. they are thought to transfer mirna between cells, which can alter gene expression in recipient cells. therefore, we hypothesize that ev cargo is altered in mdd subjects compared to healthy controls (hc). the aim is to extract evs from human post-mortem anterior cingulate cortex, a region previously associated with depression, and profile the mirna cargo and compare it between mdd subjects and hc. methods: post-mortem human brain tissue from the anterior cingulate cortex of mdd subjects and hc was mildly dissociated in the presence of collagenase type iii. residual tissue, cells, and large vesicles were eliminated, and evs were isolated using size exclusion chromatography. the quality was assessed by western blots and transmission electron microscopy (tem). rna was extracted and a small-rna library was constructed and sequenced using the illumina platform. differential expression analysis was then performed. results: western blots showed little to no endoplasmic reticulum (calnexin), golgi (bip), or mitochondrial (vdac) contamination, along with enrichment of the exosomal marker cd . tem images showed the typical cup-shaped morphology with sizes mostly between and nm. preliminary sequencing results revealed that mir- a- p, which is predicted to target glutamate receptors, is downregulated in evs from mdd subjects. summary/conclusion: high quality ev extractions can be obtained from post-mortem brain tissue using our method. this will be the first study to profile brainderived ev mirna in the context of depression. future studies will be needed to determine the effect of the different levels of mir- a- p. this could provide novel mechanistic insights into the pathophysiology of mdd and will serve as a starting point to examine the potential role of evs in mdd pathology. op . = ps . combining nanomagnetic isolation and artificial intelligence to guide the treatment of traumatic brain injury zijian yang a , kryshawna beard b , david meaney b , danielle sandsmark b , ramon diaz-arrastia a and david issadore c a university of pennsylvania, philadelphia, usa; b university of pennsylvania, philadelphia, usa; c department of bioengineering, university of pennsylvania, philadelphia, usa introduction: traumatic brain injury (tbi) is characterized by diverse primary mechanisms of injury that lead to the development of secondary pathological cascades that drive neurological deficit post-tbi. inability to separate patients based on the presence of these different endophenotypes represents a major challenge for diagnosis and treatment of tbi. extracellular vesicles including exosomes isolated from patient plasma have emerged as promising potential biomarkers for tbi due to their ability to cross the bbb into systemic circulation with molecular cargo intact for analysis. we have developed a novel microfluidic platform for rapid isolation of brain-derived evs providing a tool with which the biochemical state of neurons and glia can be directly assessed post-tbi. we used the ultra-sensitive, single molecule array (simoa) to quantify concentrations of protein biomarkers from the plasma and brain derived evs from mild tbi patients and controls. by combining multiple protein biomarkers, we could discriminate mtbi patients from controls in both the training and the blinded test set. building on this work, we are also characterizing single ev heterogeneity of neuron derived evs by developing novel droplet based digital assay for single ev quantification at ultra-low concentration. droplet based assay for single ev analysis would potentially be very informative for early disease diagnosis and therapy decision. methods: our microfluidic platform for ev isolation consists of tracked-etched membranes with millions of nanopores ( nm), coated with a magnetic film (nife) to precisely capture immunomagnetically labelled brain-specific evs from plasma. single molecule array (simoa) was used to quantify concentrations of the protein biomarkers (tau, uchl- , nfl, gfap, il , il , and tnf) in the plasma and brainderived exosomes of mild tbi (mtbi) patients and controls. to identify single ev, we applied droplet based enzyme-linked immunosorbent assay and encoded the fluorescent signal for single ev quantification within parallelized microfluidic platform. results: we report that concentrations of plasma and exosome gfap, nfl, and uchl were elevated in mtbi patients compared to controls (p < . ), and that each of these biomarkers are uncorrelated with one another. discrimination of mtbi patients from controls was most accurate when machine learning algorithms on the panel of biomarkers. specifically, combining plasma nfl, gfap, il and tnf-with tau from glur + evs showed % accuracy with % sensitivity and % specificity. summary/conclusion: this data suggests that neuronderived exosomes contain information that characterizes the injured and recovering brain. it also suggests that analysis of a panel of biomarkers from a combination of both blood and exosomal compartments could lead to more accurate diagnosis of mtbis. l cam is not associated with extracellular vesicles in cerebrospinal fluid or plasma wyss institute, boston, usa introduction: neurons in living psychiatric and neurological patients are inaccessible for cell type specific analysis of rna and protein. our understanding of these diseases instead relies upon imperfect sources of biochemical information such as post-mortem brain tissue analysis and animal models. furthermore, there is a paucity of biochemical assays available to diagnose and manage brain diseases. extracellular vesicles (evs) present an opportunity to noninvasively sample the contents of neurons in cerebrospinal fluid (csf) and plasma. in order to isolate neuron-derived evs (ndevs), a cell type specific transmembrane protein is necessary for immunocapture. l cam, a protein abundant on the surface of neurons, has been used extensively in the literature for ndev isolation. however, l cam exists in humans in several isoforms without a transmembrane domain, and as such it can be secreted as a free protein. additionally, the ectodomain of l cam can be cleaved off of the cell surface in physiological processes. it remains to be demonstrated whether the l cam found in csf and plasma is ev associated, or if it is instead a spliced or cleaved isoform behaving as a free protein. methods: using single molecule arrays (simoa), a digital form of elisa, as well as western blotting, we quantify ev markers (cd , cd and cd ) as well as l cam and albumin. we use these assays to determine in which fractions of size exclusion chromatography (sec) and density gradient the l cam appears. we also immunocapture l cam from csf and plasma and perform western blots for the internal and external domains of l cam. results: simoa and western blot analysis of sec and density gradient fractions demonstrated that while the ev markers peaked all together, l cam eluted in the free protein fractions along with albumin in both csf and plasma. when immunoprecipitation was performed, western blotting revealed different isoforms of l cam in csf and plasma. summary/conclusion: our data utilize a multitude of distinct techniques that converge to demonstrate that l cam is not associated with evs in csf or plasma. furthermore, our data suggest that the isoforms present in csf and plasma are distinct, which indicates that the l cam in plasma is likely not coming from the brain. this data call into question the utility of l cam as a ndev marker and point to the need to find novel candidates for immunoprecipitation of ndevs. introduction: in parkinson's disease (pd), α-synuclein (α-syn) aggregates known as lewy bodies (lb) are present in both the central and peripheral nervous system. furthermore, data showing that α-syn can spread from pd patients to transplanted tissue has led to a new theory postulating that pathological forms of α-syn can drive disease by "infecting" healthy cells and corrupting normal proteins. the exact routes and mechanisms involved in such spreading are yet to be fully understood but it is known that α-syn can be secreted from cells and transported via extracellular vesicles (ev). ev derived from erythrocytes (eev) are of particular interest in this regard as they have been shown to contain α-syn. methods: we first optimized a protocol for the isolation of fluorescently labelled human eev. the capacity of these eev to cross the blood-brain barrier (bbb) was then evaluated in vitro using a boyden chamber composed of primary human brain endothelial cells. next, eev were added to a more complex and physiologically relevant d human bbb model including ipsc-derived brain microvascular endothelial cells. in both in vitro protocols, flow cytometry was performed on media collect from each compartment to determine the number of eev. immunofluorescence was performed to assess the localization of fluorophore tagged eev. we are also using an in vivo paradigm for the extraction and testing of eev spread and an in situ cerebral perfusion (isbp) model in wt mice to investigate if and how eev cross the bbb using confocal microscopy. results: in both in vitro models, flow cytometry analyses showed that fluorescently tagged eev added to the luminal side traversed the endothelial cell barrier. confocal analysis revealed that some eev could also be found within endothelial cells themselves. ongoing experiments are being conducted in our newly developed d bbb to further confirm these results. our preliminary in vivo experiments showed that fluorescently labelled beads, similar in size to eev, used in the isbp experiments are detectable in the brain parenchyma of injected wt mice using confocal microscopy. preliminary work also includes isbp injections of eev in -month-old wt mice, (n = /groups) derived from pd patients (at different stage of the disease) and a healthy individual as a control. summary/conclusion: our preliminary data suggests that eev can indeed move across the bbb in both in vitro and in vivo experimental setups. ongoing experiments will determine the dynamics and processes involved in this transport and whether eev can precipitate and/or exacerbate disease-related features. introduction: neuroblastoma accounts for % of childhood cancer mortality. amplification of the oncogene n-myc is a well-established poor prognostic marker for neuroblastoma. whilst n-myc amplification status strongly correlates with higher tumour aggression and resistance to treatment, the role of n-myc in the aggressiveness of the disease is poorly understood. exosomes are released by many cell types including cancer cells and are implicated as key mediators in cell-cell communication via the transfer of molecular cargo. hence, characterising the exosomal protein components from n-myc amplified and non-amplified neuroblastoma cells will improve our understanding on their role in the progression of neuroblastoma. methods: in this study, comparative proteomic analysis, nanoparticle tracking analysis, transmission electron microscopy, rnai-based knockdown, migration and cellular survivability assays were performed to understand the role of exosomes isolated from cells with varying n-myc amplification status. results: label-free quantitative proteomic profiling revealed proteins that are differentially abundant in exosomes released by the n-myc amplified and nonamplified neuroblastoma cells. gene ontology-based analysis highlighted the enrichment of proteins involved in cell communication and signal transduction in n-myc amplified exosomes. treatment of less aggressive sh-sy y cells with n-myc amplified sk-n-be cell-derived exosomes increased the migratory potential, colony forming abilities and conferred resistance to doxorubicin induced apoptosis. incubation of exosomes from n-myc knocked down sk-n-be cells abolished the transfer of resistance to doxorubicin induced apoptosis. summary/conclusion: these findings suggest that exosomes could play a pivotal role in n-myc-driven aggressive neuroblastoma and transfer of chemoresistance between cells. op . = ps . introduction: quantification and characterization of single extracellular vesicles (sevs) based on surface markers can aid in dissecting the heterogeneous landscape of ev subpopulations. we and others have demonstrated the potential of imaging flow cytometry (ifc) to perform sev characterization. we recently showed release of protoporphyrin (ppix) positive sevs by -aminolevulinic acid ( -ala) dosed glioma cells, in vitro and in vivo. rickfels et al. also used ifc to demonstrate the enrichment of cd +/cd + evs in the plasma of glioma patients. herein, we performed in vitro studies to characterize ev subfractions using -ala as well as ev and cns specific surface markers. methods: we use ifc to characterize evs released by glioma using -ala, fluorescently labelled ev (cfda-se, cd ) and glioma specific (tenascin c and epidermal growth factor receptor viii, egfrviii) markers. furthermore, we characterized evs released by egfrviii positive glioma cells treated with dexamethasone, a steroid commonly used in glioma patients, to determine the effect of steroids on ev release. evs were quantified by ifc and results were confirmed by qpcr for the levels of egfrviii mrna. results: firstly, we optimized protocols to label glioma sevs using fluorescently labelled ev markers (cfda-se, cd ) and tumour specific markers (tenascin c and egfrviii). of the total evs (cfda-se), we demonstrate that % are tenascin c positive, . % are egfrviii positive and . % are -ala positive. there was only a minor overlap (< %) between the sub-populations. finally, we show that dexamethasone treated glioma cells release lower total evs ( . -fold), tumour specific evs ( . -fold; egfrviii), egfrviii mrna compared to mock treated cells. summary/conclusion: we demonstrate the potential of ifc to monitor sevs released by glioma cells exposed to different stimuli. this allows the characterization of ev sub-populations providing a working model to understand the dynamics of tumour evs at a single vesicle level. introduction: f. graminearum (fgr) and f. oxysporum f. sp. vasinfectum (fov) are severe fungal pathogens of cereals and cotton, respectively. fgr and fov cause economic losses and threaten food and fibre supplies worldwide. understanding host-pathogen interactions is crucial for developing new strategies for disease control. we are determining whether extracellular vesicles (evs) have a role in the interaction between fungal pathogens and their host plant. methods: we isolated evs from fgr and fov by sizeexclusion chromatography and characterized them by nta and tem. evs from fgr and fov are between - nm and have morphology similar to evs reported for other fungi. we performed label-free quantitative proteomics to describe the protein cargo of evs from fgr and fov, including a comparative study of evs from fov grown on different media: czapek dox (cd) and saboraud's dextrose broth (sdb). results: a total of proteins were detected in fgr evs and, according to prediction software effectorp, . % of these were potential effectors. similarly, % of ev proteins do not contain signal peptide indicating that packaging into evs is a novel mechanism of secretion for these proteins. notable fgr ev proteins include lipases, proteases and synthases for toxins and chitin. fov produced evs in similar quantities in both growth media tested, but ev protein cargo differed between them. there was a % overlap in proteins identified in the cd and the sbd ev proteins. in general, ev proteins were involved in metabolism, cell wall architecture and oxidoreduction, with . % and . % of potential effectors, respectively. polyketide and toxin synthases, proteases and effectors were present in both types of fov evs. summary/conclusion: this new fungal ev isolation method was rapid, yielded high-quality evs, and did not submit particles to high centrifugal forces. our data revealed that both fgr and fov produce evs enriched with proteins that could alter host immune responses or facilitate fungal infection. furthermore, the protein composition of fov evs was dependant on culture conditions. this supports a potential role for fungal evs in disease progression in plants and provides the foundations to pursue the role of evs in plant-fungal interactions with the potential to identify new targets for disease control. introduction: extracellular vesicles (ev) released by infective forms of trypanosoma cruzi, the agent of chagas' disease, modulate inflammatory response of macrophages through the activation of toll receptor (tlr ) via mitogen-activated protein kinase pathway. this induces the production of nitric oxide (no) and expression of the cytokines tnf-α, il- and il- , which could explain the inflammation observed in experimental chagas' disease, and eventually in the progression of human disease. evs released by the parasite are heterogeneous and it is unknown which factor, or factors present in the different vesicle populations act during the interaction with host cells. objectives. the goal of the present work was to characterize and isolate the different populations of evs released by t. cruzi and test their effects on macrophages. methods: ev released by trypomastigotes forms of t. cruzi (y strain) were purified by asymmetric flow fieldflow fractionation (af ) and characterized by nanoparticles tracking analysis (nta). the different populations of evs were incubated with host human monocytes cells (thp- ) and cytokines production determined by elisa and qpcr. the different ev populations were also incubated with llcmk- epithelial cells and the infection by t. cruzi determined. results: we found two distinct populations of evs. a population with to nm (ev ) and another with to nm (ev ). ev induced more tnf-alpha, il- , ip- and ccl than ev . it was also more effective in promoting t. cruzi infection in epithelial cells. summary/conclusion: t. cruzi released two ev populations that affects differently host cells. identification of these evs composition might help to better understand the role of evs in the modulation of t. cruzi infection funding: fapesp, cnpq and capes op . = ps . commensal bacterial extracellular vesicles act as carriers for norovirus sutonuka bhar, melissa jones, annalise galbraith and mariola edelmann university of florida, gainesville, usa introduction: human norovirus (hunov) are one of the most common causes of gastroenteritis and, along with inducing morbidity and mortality by diarrhoea, have a massive economic impact resulting in approximately usd billion each year in healthcare costs and missed worker productivity. development of anti-viral therapies for hunov has been hampered by the lack of robust in vitro cultivation systems. several cell types support viral replication but only produce modest amounts of virus due to unknown reasons, making these systems insufficient for use in drug development and infectivity assays. noroviruses are known to attach to gram-negative enteric bacteria and this facilitates infection in vitro. however, the microbiome-norovirus-host communication link is missing. noroviruses infect immune cells present in lamina propria during acute infection, but bacteria themselves are large enough to cross the mucosal and the tight epithelial barrier which separates gut lumen from lamina propria. we hypothesized that binding of noroviruses to bacteria enhances extracellular vesicles (ev) production. because commensal bacterial evs by themselves do not have any detrimental effects on host cells, we believe using evs in in vitro culture will enhance norovirus infection, thus producing higher titre of viruses for vaccine and anti-viral drug development. methods: attachment assay: purified norovirus was incubated with enterobacter cloacae, lactobacillus acidophilus and bacteroides thethiotaomicron, and grown to produce evs. the attachment was confirmed via qpcr. isolation of evs: clarified media supernatants were subjected to ultracentrifugation at varying speeds and . um filtration. co-purification of norovirus with the evs was checked. ev quantification and characterization: ev total protein content was measured by microbca. the number of vesicles were quantified by nanoparticle tracking analysis. scanning and transmission electron microscopy was performed to check quality of ev preparation and determine if virus was attached to the vesicles. internal ev protein content was evaluated using ms-hplc. the evs were also check for infectivity via tcid assay. results: incubation of noroviruses with commensal bacteria resulted in significant increases in production of evs compared to uninfected controls. murine norovirus (mnv), used as a surrogate, was found to be associated with evs. em analysis determine association of viruses with the bacteria as well as the mvs, while also showing certain surface structural changes in virus attached bacteria compared to mock bacteria. the evs were found to cause infection in naive macrophages. summary/conclusion: changes in ev production and content by bacteria exposed to noroviruses will provide insight into its pathogenesis and possible solutions to the low viral output from hunov culture systems. detection of bacterial extracellular vesicles in blood from healthy volunteers kylie krohmaly a , claire hoptay b , andrea hahn a and robert freishtat a a children's national hospital, washington, usa; b childrens national hospital, washington, usa introduction: bacteria constitutively produce biologically active extracellular vesicles (evs), which contain rna, dna, and/or proteins. bacteria use these evs for communication with other bacteria and recent research suggests bacterial evs can also affect host cells. given these findings, it is necessary to examine the role of bacterial evs in human disease. current methods of bacterial ev isolation from human specimens cannot distinguish between bacterial species. however, there is utility in examining evs from specific species, as bacterial species and their evs may have unique contributions to human disease. our objective was to isolate circulating evs specifically from escherichia coli (eevs) and haemophilus influenzae (hevs), two known colonizers and pathogens in the gut and airway, respectively. methods: total evs were isolated from the blood of six healthy volunteers via precipitation and size exclusion chromatography. evs were then selected via a novel latex bead-based fluorescent antibody construct targeting species-specific outer membrane proteins. we used flow cytometry to evaluate the isolated evs. results: the constructs were saturated with eevs at an antibody concentration of . µg/ml of plasma, as geometric means ≥ . µg/ml were nearly equal. hevs were detected at µg/ml of plasma, but saturation is yet to be determined. eevs were imaged by a fei talos f x electron microscope and measured between - nm, and hevs were between - nm. both types of evs were spherical. summary/conclusion: using this novel technique, we were able to isolate, detect, and visualize eevs and hevs. this technique enables the study of specific bacterial evs. in the future, ev contents will be assayed. furthermore, this technique will be modified so that specific bacterial evs from body fluids can be used for downstream functional applications. this is the first time that bacterial evs from targeted bacterial species have been detected in blood from healthy humans. introduction: new zealand (nz) has a population of just . million people with a remote geographical location in the pacific ocean. its unique culture, food-based industries and ethnic population make nz an invaluable place for extracellular vesicle research into all areas. however, as for many places in the world, standardization of methodologies, training and access to appropriate equipment is challenging. methods: the hub for extracellular vesicle investigations (hevi) is a virtual research centre established in with three-year seed funding from a university of auckland strategic research initiatives fund. two staff members are employed to support training, education and optimization of methods. the hevi is guided by a governance group providing valuable input from australasian experts in evs. results: since the hevi has organized research symposia, hands-on training days, hosted international students as well as providing one-on-one training for individuals from universities and institutes across nz. training is provided on multiple isolation and characterization methods and tailored to individuals access to essential equipment without bias towards individual manufacturers or techniques. travel funding has supported people to attend conferences and workshops for the purposes of education, networking and research dissemination. the hevi also provides support for project design with grants awarded to hevi members and a number of manuscripts in submission for publication. the embo practical course "extracellular vesicles: from biology to biomedical applications" is organized each year by a group of researchers active in the ev field in collaboration with the embl advanced training center in heidelberg. the course focuses on training phd students and postdoctoral researchers who enter or are already active in the field of ev research. given the large number of methods and protocols that is being used by researchers in the ev field, the organizers aim to provide practical guidance to new researchers and teach them appropriate skills. methods: participants obtain theoretical knowledge and hands-on experience on different ev purification and characterization techniques, such as fluorescent labelling, density gradient centrifugation, size exclusion chromatography, electron microscopy, flow cytometry and nanoparticle tracking analysis and on databases like ev-track and funrich. in addition, the organizers and invited lecturers from several different research areas explain which strategies are used to understand the role of ev in biomedical applications and give an overview of the current state of the field of ev research. results: the course therefore covers a broad range of topics important in the ev field, including heterogeneity in ev subpopulations, mechanisms of ev cargo selection, ev biogenesis, pre-analytical variables, therapeutic and diagnostic use of ev, and in vivo functions of ev. group discussions are facilitated and stimulated via assignments to analyse data obtained during the practicals and to critically evaluate literature. participants also have the opportunity to present their own research during poster presentations and ask for feedback from organizers and invited lecturers. summary/conclusion: among the participants selected for the course, a large geographical distribution is reached, including researchers from the newer eu member states and from outside of europe, to ensure a broad geographical distribution of the knowledge gained during this course. introduction: on october , we organized the st lugano exoday, first initiative in the southern switzerland to bring together resident researchers and european experts in the field of extracellular vesicles (evs). the workshop, centred on "technical challenges of extracellular vesicle research" aimed to highlight technical requirements and advances in the evs area, focusing on isolation, characterization and tracking. methods: the workshop started with a lecture by dr. cecilia lässer, from the university of gothenburg. the rest of the workshop was divided in two working groups (wg), each introduced by a keynote lecture followed by presentations by young researchers and a round-table discussion. wg , introduced by dr. mercedes tkach, from the institute curie in paris, focused on recent advances on evs characterization and isolation. wg was centred on evs tracking and introduced by dr. frédérik verweij, from the institute of psychiatry and neuroscience of paris. results: dr. lässer opened the workshop with a comprehensive review and introduced recent developments in the evs field. the first wg discussed different isolation methods, focusing on ultracentrifugation, size exclusion chromatography and immunoprecipitation-based techniques. supported by the keynote speakers, the participants agreed that the best approach to optimize the isolation process consists in the combination of different techniques. wg shared insights about new strategies to visualize and tracking evs, focusing on how to improve the routinely approaches used, defining optimal criteria for evs labelling and imaging. all the participants had an in-depth overview on the requirements and the state-of-the-art techniques currently in use for the isolation, characterization and tracking of evs. summary/conclusion: the transferable knowledge acquired during the workshop ensures participants to remain up-to-date with the advances in the field of evs. as our ultimate goal is to create a competence centre in southern switzerland around the field of evs, the workshop was an invaluable opportunity to intensify collaborations between resident laboratories and broaden the scientific exchange with laboratories of the experts hosted during the event. given the success of this first workshop we are already working to prepare the second edition and make the event a recurring appointment. funding: supported by the swiss national science foundation the role of core facilities and emerging technologies in maximizing rigour and reproducibility of ev quantification and characterization and following misev guidelines rachel derita a and andrew hoffman b a thomas jefferson university, philadelphia, usa; b university of pennsylvania school of veterinary medicine, philadelphia, usa introduction: it remains very clear in the field of extracellular vesicle (ev) research that the rapid rate of increase in publications and expansion of interdisciplinary clinical ev interest has created the need for increased standardization and access to the appropriate technologies to uphold these standards. as the first core facility in the usa with the sole intention of creating a space where users can both isolate and characterize evs, we provide a central location for the facilitation of ev research via access to multiple technologies (both established and emerging) such as resistive pulse sensing, nanoparticle tracking analysis, ultracentrifugation, high-performance liquid chromatography, flow cytometric analysis of evs and additional immune or fluorescence-based ev characteri zation techniques. methods: we surveyed a group of leading scientific investigators and researchers in varying stages of their scientific careers in the mid-atlantic region of the us. the survey data demonstrate applications of greatest current and future interest to be employed in a shared lab resource. results: the current demand is highest for isolation services, ultracentrifugation and nta, with a gradually increasing demand for immunophenotying analyses such as the exoview chip array, fluorescent nta and flow cytometry. we additionally present strategies and data-based examples of how shared resource facilities can facilitate multifactorial and rigorous ev characterization in accordance with misev guidelines, and encourage collaboration among ev researchers. summary/conclusion: in order to answer the larger remaining questions in the ev field such as the isolation of specific ev subsets, ev tracking between cells and the use of evs for biomarker discovery and drug delivery, it is essential that shared resource facilities interact not only with investigators, but with each other to integrate the necessary resources to progress. programme to assess the rigour and reproducibility of extracellular vesicle-derived analytes for cancer detection national cancer institute, rockville, usa introduction: cancer cells release more evs than normal cells and evs secreted from tumour cells can promote tumour progression, survival, invasion and angiogenesis. the ev cargo may mirror the altered molecular state of the cell of origin. therefore, evs have potential for the development of non-invasive markers for early detection of cancers. evs and their cargo also have the potential to be multiplexed with other molecular markers or screening modalities (e.g., imaging) to develop integrated molecular-based computational tools for the early detection of cancer. one challenge with using evs as a biomarker is the lack of robust and reproducible methods for the isolation of a pure vesicular population. there is a lack of clear consensus for an optimal method of isolation of a pure ev population that is devoid of contamination with similar-sized vesicles of different origins. there is also a lack of standards to ensure rigour reproducibility. methods: the current funding opportunity announcement (foa), par - , is promoting research on the isolation and characterization of extracellular vesicles (evs) and their cargo for the discovery of biomarkers to predict cancer and cancer risk. results: the previous cycle of this foa, par - / , successfully funded r and r grants. these awards are focused on proteomics profiling of evs, effect of methodological and biological variability, asymmetric-flow field-flow technology, therapeutic monitoring, lss and sers lab on a chip optical spectroscopic, evs in obesity-driven hepatocellular carcinoma, nanoscale structure and bio-molecular heterogeneity, urinary ev dna, and ev markers in paediatric cancers. progress from these awardees have shown separation of two discernible exosome subpopulations and identified a distinct nanoparticle, the exomere (nature cell biology, ); and have shown that large-evs contain the entire genome of the cell of origin, including cancer-specific genomic alterations (journal of extracellular vesicles, ). protocols that critically evaluate and refine the existing methodologies to improve utilization of evs in clinical use have been shared (nature protocols, ). summary/conclusion: drs. sudhir srivastava and matthew young are the program directors for the par which began accepting applications on january . this and other ev funding opportunities will be discussed. funding: this is a funding opportunity announcement offered by the national cancer institute introduction: early detection of cancer as well as monitoring cancer treatment are important to improve cancer care. diagnostics for cancer are mainly based on tissue biopsies and re-biopsy during treatment is challenging. moreover, current diagnostics are expensive, time-consuming and have low-throughput. therefore, liquid biopsies are expected to bring the next breakthrough in cancer diagnostics. in liquid biopsies tumour-secreted material is isolated from body fluids and subsequent analyses thereof allow for non-invasive diagnostics. one type of tumour-secreted materials are extracellular vesicles (evs), which are schedded from tumour cells. evs are surrounded by a lipid bilayer, whichs composition resembles the plasma membrane of their parental cell. as many tumours are driven by over-expression or upregulation of transmembrane proteins e.g. growth factor receptors, detection of the later on evs holds promise for early tumour detection and treatment monitoring. methods: for the immuno-pcr evs were first affinitycaptured on magnetic beads, allowing immobilization of purified evs as well as evs secreted into cell culture medium or spiked into plasma. afterwards each sample was divided and affibody-dna-conjugates directed against different targets were added. affibodies are small affinity proteins, which often are developed as high affinity binders for tumour imaging, making them suitable probes in the presented assay. after washing, the bead-ev-affibody-dna-complexes were analysed for the immobilized dna-amount via qpcr. results: via the presented immuno-pcr evs secreted from the non-small cell lung cancer cell line h as well as the ovarian cancer cell line skov were analysed. the immuno-pcr method allowed the detection of the tumour-associated membrane receptors epidermal growth factor receptor (egfr), receptor tyrosineprotein kinase erbb /her and insulin-like growth factor receptor (igf r). different levels of membrane receptors depending on the ev source and concentration were detected. summary/conclusion: the presented immuno-pcr showed to be a comparably fast and robust method for detection of tumour-associated membrane receptors on evs derived from cancer cell lines with medium through-put and is currently further developed into a method for liquid biopsy for non-small cell lung cancer patients. introduction: introduction. evs produced by cells can originate from different cellular compartments and evs in complex biofluids may originate from many different cell types. traditional biochemical analysis, which reports on the total composition of all evs in a sample can't adequately resolve this heterogeneity. single vesicle analysis methods can, if they have the necessary specificity, sensitivity and speed. flow cytometry (fc) is capable of rapid and quantitative analysis of individual particles, but conventional fc-based assays lack the specificity and sensitivity to measure individual evs. assays that combine sensitive instruments with ev-selective sample staining can measure individual evs with accuracy and precision. to better understand the nature and origins of ev diversity, we used single vesicle fc (vfc) to quantitatively measure vesicle number, size, and surface cargo expression on individual evs. methods: methods. evs in culture supernatants ( t, a , u , thp- , sh-sy y) were used neat or enriched by standard methods including differential centrifugation or ultrafiltration. evs from platelets (plt) and red blood cells (rbc) were induced by ionophore treatment of washed cells, and measured in diluted supernatant. evs were stained with a membrane-selective dye and fluorescence-labelled antibodies using a commercial vfc assay kit (cellarcus biosciences), measured using a commercial flow cytometer (cytoflexs, beckman coulter), and data analysed using fcs express v (de novo software). vesicle size, fluorescence intensity, and antibody binding were calibrated using appropriate vesicle and beadbased standards and essential controls performed as recommended by the miflowcyt-ev reporting guidelines. results: results. to assess the compositional heterogeneity of evs, we first characterized the expression of tetraspanins (tss; cd , cd , cd , cd , cd , cd , cd ) on evs released from cultured cell line and primary cell cultures. we find quantitative differences in the expression of ts on evs from different cell types that generally reflected the expression on the cell of origin, with most ev types expressing detectable amounts at least one of the common ts molecules (cd , cd or cd ) but generally not all three. in evs from some cell types, ts expression was uniform across the ev population (cd on evs), but evs from other cell types differentially expressed tss, with some evs expressing no detectable ts (rbc evs). intracellular cargo labelled genetically using fluorescent proteins (egfp or mneongreen) or fluorogenic enzyme substrates (cfse) were measured in individual evs and revealed distinctive associations between ev surface and internal cargo. summary/conclusion: conclusions. high resolution measurement of cargo on/in individual evs can help interpret ev heterogeneity in terms of cell of origin, signals carried, and effects on target cells. integrated omics reveal conserved and divergent modulation of cardiovascular disease by tissue-entrapped extracellular vesicles introduction: fewer than % of patients develop both vascular and valvular calcification, implying differential pathogenesis. while circulating extracellular vesicles (evs) act as biomarkers of cardiovascular diseases, tissue-entrapped evs are implicated in early mineralization but their contents and function are unstudied. we developed an innovative method to isolate and study evs from fibrocalcific tissue and investigated entrapped ev cargoes in human cardiovascular diseases. methods: human carotid artery endarterectomies and stenotic aortic valves were obtained from donors under irb-approved informed consent. tissues underwent enzymatic digestion, ultracentrifugation, and a -fraction optiprep density gradient. global proteomics was performed on intact tissue, each optiprep fraction, and ev-enriched pooled fractions; the latter also underwent mirna-seq. fractionated samples were also studied by cd immunogold electron microscopy (tem) and nanoparticle tracking analysis (nta). high confidence mir targets were predicted by targetscan, pathway analyses utilized the biocarta/kegg/reactome databases, and protein-protein interaction networks were built in string. results: vesicle-associated pathways were increased . x (p < . ; / vesicle-related top go terms) in proteins common to intact arteries and valves (n = , ). proteomics found ev markers to be highly enriched in the four least-dense optiprep fractions of arteries and valves, while extracellular matrix and mitochondria were predominant in denser fractions, as confirmed by tem/nta. proteomics and mirna-seq of tissue evs quantified , proteins and mir cargoes linked to , target genes. pathway networks of proteins and mir targets common to artery and valve tissue evs revealed a shared regulation of rho gtpase and mapk intracellular signalling cascades. proteins and mirs were significantly altered between artery and valve evs (q < . ); multi-omics integration found that evs differentially modulated cellular contraction and p mediated transcriptional regulation in vascular and valvular tissue. summary/conclusion: our findings delineate a novel strategy for studying tissue-entrapped ev protein and mir cargoes and identify critical roles that tissue-resident evs play in mediating cardiovascular disease. funding: this study was supported by a research grant from kowa company (ma) and nih grants r hl , r hl and r hl (ea). mir- a regulates exogenous cd expression on proliferation, invasion, migration and angiogenesis of gastric cancer zhengzhou university, zhengzhou, china (people's republic) introduction: to investigate the possible mechanism of mir- a regulating the expression of exosome cd on proliferation, invasion, migration and angiogenesis of gastric cancer, and to study the application value of cd in the early diagnosis and prognosis of gastric cancer. methods: the gastric cancer cell line mgc- was used as the research object. the exosomes were extracted from the culture supernatant of mgc- by exosome extraction kit. the extracted exosomes were identified by transmission electron microscopy and western blotting. the expression of cd in exosomes was detected by elisa. the expression of cd in exosomes and cd in whole blood and serum were detected by western blot. they were randomly divided into blank group (mock) and mir- a lentivirus experimental group (mir- a group). the lentivirus control group (mir- a/con) was transfected into cells. qrt-pcr was used to verify the status of mir- a after transfection; western-blot was used to detect the expression of cd and downstream erk / , akt and m tor proteins; mtt assay, cell colony formation assay, transwell migration assay for cell proliferation, invasion, and migration. a nude mouse xenograft model was constructed to observe the growth of transplanted tumours,microvessel density (mvd) was detected by immunofluorescence, and distant metastasis was recorded. results: the expression of cd in exosomes was detected by elisa and western blot. the expressions of cd , akt, erk / and m tor in mir- a group were significantly lower than those in mir- a/con and mock groups. cd protein is positively correlated with downstream protein levels.the growth rate and cell invasion ability of mir- a group were significantly lower than those of mir- a/con group and mock group. the weight of the nude mice in the mock group and the mir- a/con group decreased, while the weight loss in the mir- a group was not significant. the tumours in the mir- a/con group and the mock group showed invasive growth accompanied by abundant microvessels, while the mir- a group had smaller tumour volume and uniform cell distribution. only a small amount of microangiogenesis was observed, and no obvious necrotic area was observed. summary/conclusion: mir- a affects the proliferation, invasion, migration and angiogenesis of gastric cancer mediated by akt/erk/m tor signalling pathway by regulating the expression of exosome cd . streamlined detection and quantification of plasma extracellular vesicles and their protein cargo by high-performance nanoscale flow cytometry and label-free mass spectrometry introduction: nanoscale flow cytometry (fc) and mass spectrometry (ms) are useful for profiling ev surface proteins and performing bulk ev proteomics, respectively. this study sought to develop pre-analytical and analytical pipelines for ev protein profiling that are applicable to clinical studies. methods: to optimize plasma ev detection and quantification by fc, modifications of instrument settings and serial dilutions of platelet-free plasma (pfp) and antibodies were tested for improved separation of signal from noise and reduction of event coincidence and swarming. the high-performance flow cytometry (hpfc) platform was used to assess the effect of time ( , , , , , , or hrs) between blood draw (into acd, nacit, edta or heparin) and blood processing, on ex-vivo release of evs from blood cells. label-free ms was used to examine the intensity and breadth of identified proteins in plasma evs purified using several density and size separation methods, either manually or automated, along with various buffer conditions. results: ev event aborts were minimized at a pfp dilution, prior to staining, of : and by using a narrow cytometer window extension. target ev signals were distinct from noise and were triton x- labile. the most significant changes in plasma evs were associated with platelet-derived fractions, use of heparin and > -hour delay before blood processing. yet, platelet ev numbers did not significantly change for up to hrs in citrated and edta plasma. higher overall coverage of known ev proteins and a fivefold increase in number of uniquely identified proteins were observed in ms profiling of evs prepared by a combination of ultracentrifugation (uc) and manual size-exclusion chromatography (sec) compared to preparation by fplc on capto core /superose resins. uc/sec was better than direct sec at reducing contamination by excipient plasma proteins. column buffers with trehalose increased ev protein recovery while adding protease inhibitors had minimal effect. summary/conclusion: with our optimized hpfc protocol, we established that blood ev numbers remain stable for up to hrs in acd or edta and that uc+sec with trehalose-containing buffer result in high canonical ev protein recovery. we are applying these workflows to investigate cancer-associated changes in plasma ev protein cargo. the value of exosomes as a potential biomarker for devil facial tumour disease. university of tasmania, hobart, australia introduction: the tasmanian devil (sarcophilus harrisii), the largest living carnivorous marsupial is endangered because of two transmissible cancers: devil facial tumour disease (dftd) one and two. current efforts to manage dftd are hindered by the lack of a preclinical diagnostic test for dftd. detecting dftd infection is only possible once tumours are noticed, too late to stop dftd progression. a preclinal test could tell us about unknown components of dftd pathogenesis, such as latent period and host-tumour dynamics. exosomes are extracellular vesicles released by most types of cells under both physiological and pathological conditions. exosomes have utility as diagnosis and prognosis biomarkers in a range of diseases, including cancers. the aim of this study is to investigate exosomes-based approaches towards a preclinical and progression biomarker for dftd and in tasmanian devils. methods: exosomes were isolated from three different dftd- , dftd- and devil fibroblast cell lines by sizeexclusion chromatography. likewise, exosomes were isolated from plasma of healthy and diseased devils. to determine the size and morphology of exosomes, samples were imaged with transmission electron microscopy. exosomes isolated from cell lines and devil plasma were analysed with mass spectrometry to characterise proteins and determine their differential expression between the cell origins, and healthy and diseased animals. results: this study identified the presence of myelin proteins in exosomes from dftd cells relative to fibroblasts, which are diagnostic of dftd. additionally, we found that exosomes derived from dftd- abundantly express the inhibitory checkpoint molecule cd relative to exosomes from dftd- cells and devil fibroblasts, indicating a potential candidate for a differential diagnosis between tumours. moreover, exosomes from dftd cells present a greater amount of proteins related with metastasis in comparison with fibroblast exosomes, such as integrins. finally, we report the protein expression profile of exosomes from healthy and diseased devils, showing clear differences between them and the presence of immunosuppressive and metastasis proteins in animals in late stages of the disease. summary/conclusion: dftd-exosomes may provide a non-invasive diagnosis tool to detect early stages of dftd in tasmanian devils to facilitate the prevention of the disease. furthermore, dftd-exosomes may have utility as a prognosis biomarker, determining late stages of the disease using a simple a blood test, which would facilitate monitoring of wild populations. this project will provide long-term benefits for the future of the devils and encourage exosome-based solutions for other future wildlife disease outbreaks. introduction: despite the increased understanding of evs, from involvement in disease pathophysiology to therapeutic delivery, improved molecular tools to track biodistribution are largely lacking. current approaches used for ev labelling lacks sensitivity and specificity. here, we have explored bioluminescent labelling of evs to achieve a highly sensitive system for absolute in vivo quantification and tracking of exogenous evs at low cost and in a high throughput manner. methods: ev-producing cells were genetically engineered to express various tetraspanin-luciferase fusion proteins. evs purified by uf-sec from these cells were characterized by nta, multiplex bead-based array, tem and wb, followed by luciferase assay to determine the labelling efficiency. for in vitro applications cell lysate from treated cells or the conditioned medium were subjected to luciferase assay. for in vivo applications two different methodologies were applied to determine biodistribution; either by non-invasive real time in vivo imaging using ivis or by luciferase assay on harvested tissues for absolute quantification of injected evs. results: we initially performed a systematic comparison of five different luciferases for endogenous labelling of evs and identified nanoluc and thermoluc as lead candidates. we applied this technology to monitor in vitro cellular uptake and observed cell type differences in cellular uptake of engineered evs. in addition, we also observed an effect of different culturing conditions on exocytosis kinetics. for in vivo application, we applied the nanoluc labelling strategy to determine the pharmacokinetics and effect of different routes of injection on ev distribution. our results indicated a rapid uptake profile of administered evs in different tissues with liver, spleen, and lungs being the primary recipients. we also observed similar results upon tracking in vivo biodistribution in real time immediately after administration. finally, we show how different subpopulations of evs differ in their in vivo biodistribution. summary/conclusion: overall, nanoluc and thermoluc labelling of evs holds great potential for various in vivo and in vitro applications. in addition, it can enable the simultaneous detection of different subpopulations of evs in vivo, which may aid in our understanding of different sub-populations and their behaviour in vivo. apart from monitoring therapeutic evs, with one simple modification this platform offers great potential for tracking tumour derived evs both in vivo and in vitro and thus could aid in the development of anti-tumour therapies. biofunctional peptide-modified extracellular vesicles for cell targeting, macropinocytosis induction, and effective intracellular delivery ikuhiko nakase department of biological science, graduate school of science, osaka prefecture university, sakai-shi, japan introduction: [introduction] in our research group, developing therapeutic techniques based on extracellular vesicles (exosomes, evs) by effective usage of peptide chemistry to deliver therapeutic/diagnostic molecules into targeted cells has been focused. in this presentation, modification techniques using biofunctional peptides such as arginine-rich cell-penetrating peptides [ ] , artificial coiled-coil peptides with receptor targeting [ ] , and cell-penetrating sc peptides [ ] derived from cationic antimicrobial protein, cap for cancer targeting with macropinocytosis induction, on the ev membranes will be introduced. i will also show effects of lyophilization of the peptidemodified evs on their biological activity [ ] . methods: [methods] cd (ev marker)-gfpfusion protein expressed evs were used for cellular evs uptake assessments. all biofunctional peptides were synthesized by fmoc solid-phase method. results: [results] macropinocytosis with actin reorganization has been shown to be crucial for cellular ev uptake [ ] . we developed the methods for modification of arginine-rich cpps or sc peptides on ev membranes using chemical linker techniques, and for example, arginine-rich cpps modification can induce proteoglycan-clustering (e.g. syndecan- ) and macropinocytosis signal transduction [ ] . the artificial leucine zipper peptide-modified evs recognize the peptide-tagged epidermal growth factor receptor (egfr) on targeted cells, leading to macropinocytotic cellular ev uptake [ ] . in addition, lyophilization is a useful technique for long term storage, however, we found that lyophilization negatively affected biological functions of encapsulated proteins in the evs after their cellular uptake [ ] . summary/conclusion: [conclusion] these techniques and findings will contribute to development for the ev-based intracellular delivery systems. reference: [ ] sci. rep. , ( ), [ ] chem. commun. , ( ), [ ] chrmmedchem. , ( ) [ ] anticancer res. , ( ), [ ] sci. rep. , ( ) os . multi-compartmented microvesicles: novel extracellular secretory organelles that release exosomes and extracellular vesicles introduction: extracellular vesicles (ev) bud from the plasma membrane (pm) as microvesicles (mv) or arise from the fusion of multivesicular bodies (mvb) with the pm to release intralumenal vesicles (ilv) as exosomes. the variety of bioactive molecules carried by ev imparts diverse functionality to ev in intercellular signalling. the biogenesis and extracellular release of these specialized messenger organelles is not well understood. to investigate, we studied endothelial cells that line the inside of blood vessels, known to release ev that support angiogenesis. methods: cultured human umbilical vein endothelial cells (huvec) were examined by thin-section electron microscopy (em), serial sectioning and immunogold labelling to study the structure and composition ev release sites. to obtain optimal views of cellular ultrastructure, cells were preserved by fast-freezing and a freeze-substitution. results: a potential release site was identified in em thin sections as a discrete domain, up to several microns long, on the otherwise smooth huvec pm, where numerous bulbous membrane protrusions with thin necks were clustered. the cytoplasm in these protrusions was enriched with mvb and other vesicles and appeared to be on the verge of pinching off to release multi-compartmented mv (mcmv). consistent with this notion, in the neighbouring extracellular space, a plethora of mcmv of - nm with ultrastructural features matching the bulbous protrusions were observed, supporting the concept that mcmv bud from the release site. serial sections confirmed that these extracellular mcmv were independent of cells and not linked by nanotubes or other processes. remarkably, fusion of mvb with the mcmv membrane was directly observed, presumably caught in the act of releasing ilv (exosomes) from the mcmv. immunogold labelling for ev markers is being used to identify proteins enriched at release sites and on released mcmv. summary/conclusion: in summary, ) mcmv bud from localized sites on the endothelial pm, ) mcmv contain mvb, and ) fusion of mvb to mcmv to release exosomes occurs extracellularly. mcmv can now be evaluated as a potential source of exosome and ev release that occurs after budding from the cell of origin, adding new layers of regulation to when, where and how ev are assembled and released. funding: this work was supported by the division of intramural research of the nih. one size does not fit all: overcoming barriers to successful discovery and scaled manufacturing of therapeutic extracellular vesicles jieun lee a , wei guo b , hal sternberg c , mike west d and dana larocca d a stem cell team, seoul, republic of korea; b university of pennsylvania, philadelphia, usa; c agex therapeutics inc, alameda, usa; d agex therapeutics inc., alameda, usa introduction: extracellular vesicles have tremendous intrinsic therapeutic potential. however, the limited availability of production cell lines presents a barrier to scaled ev production and novel ev discovery. indeed, ev sources have been largely confined to a handful of cell types with the vast majority consisting of msc evs. to overcome this limitation, we developed a diverse library of hundreds of clonally pure and scalable progenitor cell lines that provides an alternative resource for ev drug discovery and production. methods: we harnessed the capacity of human pluripotent stem cells (hpsc) to differentiate into virtually any cell type by subjecting hpsc to a wide variety of media and culture conditions to maximize the diversity of partially differentiated cells. the resulting heterogeneous "candidate cultures" were plated at clonal density and further selected for self renewing and scalable clones. transcriptomic analysis indicated > distinct progenitor lines. cell fate potentials were mapped by screening for cell type specific marker expression in various differentiation conditions. evs were produced using cgmp methods (tff and sec) and characterized by nta, trps, surface marker analysis, rna and protein content. bioactivity assays included proliferation, migration, vascular tube network formation, senolysis, and oxidative stress. results: the progenitor library contained > distinct lines with diverse lineage fates including various types of bone, cartilage, muscle,and fat cells, as well as all blood vessel cell types. the lines displayed much longer replicative lifespans ( - pd) than primary cell lines like msc. clonal purity minimized phenotypic drift resulting in maintenance of cell identity, genome integrity, differentiation capacity and bioactive ev production over extended culture. evs were highly diverse in their rna and protein cargo and bioactivity displaying various degrees of migratory, proliferative, angiogenic and senolytic activity. library screening identified evs with higher angiogenic potency than primary adult stem cell evs. summary/conclusion: we demonstrated scalable and stable production of bioactive evs from a large progenitor cell library. library screening resulted in discovery of novel angiogenic and senolytic evs having diverse rna and protein cargo. we are currently creating a corresponding library of progenitor cell evs to accelerate discovery of novel evs and their production cell lines. funding: the initial establishment of the cell library was funded in part by grants from the california institute of regenerative medicine and national institutes of health. introduction: besides extreme potential in biomedical applications, extracellular vesicles (evs) are also promising candidates to expand biophysical understanding of membrane active biomolecules. their complex bilayer composition allows the better understanding of adsorbed proteins and protein coronas as well, which sets of macromolecules will likely be key for advanced ev targeted delivery. considering cargo, membrane active peptides are interesting as these can be both drugs to be delivered, but can also facilitate cargo insertion through lipid bilayers. however, at present very little is understood regarding interactions between the peptides and the ev lipid bilayer, and between peptides and membrane associated proteins on evs. methods: we have recently demonstrated, that ev membrane adsorbed proteins and their interactions can be studied by techniques such as polarized light spectroscopy, microfluidic resistive pulse sensing measurements and freeze-fraction transmission electron microscopy [ ] . furthermore, initially we studied several peptides with known antimicrobial properties and found that these strongly interact with the ev surface proteins, resulting in efficient removal of some from the lipid bilayer [ ] . results: here we present investigation of further evpeptide interactions also focusing on anticancer peptides, which may be promising drug candidates for targeted delivery. these studies allowed to gain insight to novel functions of several peptides, such as melittin, magainin, buforin, lasioglossin, temporin, but also provide a more detailed understanding on how ev protein coronas, or ev bilayers are affected, to such extent that they cannot exert their potential function as delivery systems. summary/conclusion: the above interactions are expected to be interesting both for applicability, i.e. for selecting suitable compounds for ev processing, and also for curiosity-driven understanding of peptide functions, and ev-biomolecule interactions. based on these we promote that peptide -ev interactions will receive increased focus in ev-engineering. introduction: our late-breaking finding is the identification of a non-coding rna (ncrna) in extracellular vesicles (evs) from neuronal cells that is a natural antisense transcript for the dbh gene and associated with epigenetic changes and gene silencing. dna methylation in neurons is involved in memory and neurological disorders (science ( )). earlier work found that during chronic brain infection with toxoplasma gondii induced a decrease in norepinephrine levels and expression of the host dbh gene; and the decrease is correlated with behaviours linked to noradrenergic signalling (infect immun. ( ); infect immun. ( )). dbh catalyzes the production of norepinephrine from dopamine in noradrenergic neurons. we found that evs from infected cultures suppress transcription of the dbh gene and hypermethylation of the gene in noradrenergic cells in vitro. in this study, we identify a ncrna in the evs from infected neuronal cells. methods: neuronal cells were induced by infection with toxoplasma gondii and evs purified on sucrose gradients. evs were characterised by electron microscopy and used to treat rat and human neuronal cells and expression levels of dbh mrna and nascent dbh gene transcription were measured. induced evs were injected into the locus coeruleus of rats and dbh gene expression was monitored. rna purified from evs was screened for natural antisense transcripts (nats) by strand-specific rt-pcr. results: we found that evs purified from infected neuronal cultures induced transcriptional gene silencing (tgs) and dna methylation of dbh in recipient neuronal cells. the induced evs down-regulated dbh gene expression > -fold and induced dna hypermethylation of the dbh gene. this could be induced in the brains of recipient rats by intracerebral injection of evs. using a panel of strand-specific primers, antisense transcripts for the dbh gene were identified in infected cells. this permitted us to examine the rna in purified evs and identify a lncrna in evs selective for evs from infected cultures. summary/conclusion: this is the first study to find a specific neurotransmitter antisense lncrna in evs associated with transcriptional gene silencing and epigenetic changes in the gene. this represents a different type of neuron-to-neuron signalling than the classic chemical and electrical neurotransmission. the findings will enhance our understanding of neurological disorders (ie. schizophrenia, epilepsy, drug addiction) and how memory works. human cd + t regulatory-derived extracellular vesicles and associated micrornas: role in cell-to-cell communication and involvement in the loss of immune tolerance during multiple sclerosis introduction: an impairment of immune tolerance is a determining factor in multiple sclerosis (ms) and dysregulation of cd + t regulatory (treg) cell function is believed to be a major pathogenic factor. micrornas (mirnas) released by treg cells in association with extracellular vesicles (evs) have been shown to participate in the block of pathological immune responses by inhibiting the growth and cytokine production of cd + t conventional (tconv) cells, but the molecular mechanism is still poorly characterized. aim of the present work was to evaluate whether treg cell-derived ev-associated mirna signature is dysregulated in ms and whether this defect may play a role in the development of autoimmunity. methods: human treg cells isolated from blood of naïve to treatment relapsing-remitting ms patients and healthy controls were in vitro stimulated and released evs were isolated by size exclusion chromatography and characterized by nanoparticle tracking analysis, electron microscopy and flow cytometry. evassociated mirnas were quantified by traditional rt-qpcr and droplet digital pcr for absolute quantification. the actual ev-mediated passage of rna molecules from cell to cell was followed through rnaspecific fluorescent staining and treg-derived ev effect on tconv cell transcriptome was evaluated by rnaseq. results: in healthy conditions, the treatment of tconv cells with treg-derived evs was shown to cause the specific repression of genes involved in the proteasome-dependent proteolytic process, known to be crucial for t cell activation. in ms, treg-derived evs may have lost this capability as a direct consequence of a significantly decreased expression of mir- - p, able to target key factors of the proteasome system. summary/conclusion: our results unveil a novel molecular mechanism for treg-mediated maintenance of self-tolerance based on ev-associated mir- - p and its potential alteration in human autoimmunity. funding: fondazione italiana sclerosi multipla, fism, # /r/ and # /r/ revealing the proteome of brain derived extracellular vesicles isolated from human amyotrophic lateral sclerosis post-mortem tissues. introduction: amyotrophic lateral sclerosis (als) is a neurodegenerative disease characterised by the deposition of misfolded proteins in the motor cortex and motor neurons. although a multitude of als-associated proteins have been identified, few have been associated with extracellular vesicle (ev) trafficking, a form of inter-cellular communication. additionally, the role of evs in als is undetermined, specifically in relation to pathogenic stress granule formation, a response to cellular stress involving aggregation of non-coding rnas and their rna binding proteins. therefore, this study aimed to determine the proteome of brain derived small extracellular vesicles (bdevs) isolated from als subjects and identify novel alsassociated deregulated proteins and their potential contributions to pathogenic pathways in als. methods: bdevs were isolated from human post-mortem als (n = ) and control (n = ) motor cortex brain tissues through an ultracentrifugation protocol (vella et al., ) . following thorough characterisation, bdevs that successfully met the minimum criteria required by the international society for extracellular vesicles were classified as evs. the bdevs subsequently underwent mass spectrometry analysis on the thermo scientific q-exactive hf with ultimate rslcnano. proteins identified to be statistically significant differentially expressed then underwent validation by western blotting. results: a panel of statistically significant differentially packaged proteins were identified in the als bdevs. this included several up-regulated rna binding proteins and a down-regulated cell adhesion molecule; dhx , stau and vcam , respectively. pathway analysis revealed that the bdevs were enriched in proteins associated with stress granule dynamics, exosomal and lysosomal pathways. summary/conclusion: the identification of the rna binding proteins in the als bdevs suggests there may be a relationship between als-associated stress granules and als bdev packaging. the packaging of stress granule associated rna binding proteins into als bdevs may be an attempt by the cells to compensate for lysosomal dysfunction caused by stress granule accumulation, a feature of als. thus, these results highlight a potentially novel role for evs in the pathogenesis of als for long-term cultivation . the whole cultivation process of tissue preparation, cultivation, and cryopreservation has been established using strict serum-free conditions under a good manufacturing practice. long-term-cultivated hmnpcs retained stemness and hmnpcs have excellent differentiation efficiency into dopaminergic neurons. hmnpcs reversed impaired motor function in a rodent model of parkinson's disease (pd). based on the promising results in animal experiments, the clinical trial is under way (nct ). multiple-system atrophy (msa) is one of fatal neurodegenerative diseases with a combination of progressive autonomic nervous system disorders, parkinson's syndrome, and cerebellar pyramid syndrome. there are three types of msa such as msa-a, msa-c, and msa-p. in case of a msa-p type, it is difficult to diagnose due to the similarity of symptoms with parkinson's disease (pd). methods: in vitro and in vivo animal msa model were established and rotational behavioural was performed. npc cells were isolated and cultured based on moon et al. mirna sequencing (bgi) was performed and several bioinformatics analyses were done. results: based on the finding that hmnpcs exhibited therapeutic effects on pd, we hypothesize that hmnpcs will have a therapeutic effect on msa-p, where sympotoms are largely common with pd. as expected, transplanted hmnpcs survived, integrated, and differentiated in to dopamine neurons in the host brain, consequently leading to the functional recovery in the msa-p model. to further investigate the therapeutic key factors of hmnpcs in msa-p, mirna sequencing of the extracellular vesicles (evs) secreted from hmnpcs was performed. we found that mir- a highly expressed in the npc-derived evs is one of key regulators of inflammatory response via nfkb pathway. we further experimentally demonstrated that mir- a had anti-inflammatory effect on cells of msa-p condition such that the level of cx cl expression and its receptor, cx cr were both decreased in the msa-p modelled cells and in severe inflammatory environment in msa brain. summary/conclusion: our study first showed that mir- a in hmnpcs-evs is one of key therapeutic factors for the recovery of brain damage through immuno-modulation in msa-p. introduction: oxidative insults are known to be involved in the pathophysiology of alzheimer's disease (ad). we have previously demonstrated that some blood-based redox-signature were associated to the cognitive scores in mild cognitive impairment patients and in ad (perrotte et al., ) . the aim of this study was ( ) to evidence the presence of some oxidative markers in circulating extracellular vesicles (evs), and ( ) to compare to their plasma levels. methods: plasma samples from healthy, mci and ad patients were from the memory clinic of sherbrooke (québec, canada). ad patients were stratified in three groups (moderate, mild and severe) according to the mmse and moca scores. total plasma extracellular vesicles (pevs) were isolated from plasma with the total exosome isolation reagent (invitrogen™ by life technologies inc.). pevs were then characterized by electronic microscopy, nta, dls and western blot. antioxidants apolipoprotein j, d (apo j, apod), the glyoxalase- and protein carbonyls were determined by western blot. results: in pevs, we found that apo d levels were higher in mci patients but not in ad patients. protein carbonyls levels were higher later, in pevs from moderate and severe ad while apo j levels were not different in pevs from the five groups of patients. in plasma, the pattern of apo j and apo d was different. the levels of apo d was not different in the five groups of patients while apo j levels were elevated in mci and in all ad groups. protein carbonyls were higher earlier from mild ad group, earlier than in pevs. the levels of the detoxifying enzyme glyoxalase- were higher in pevs than in plasma and were significantly decreased in early ad as compared to control subjects and mci summary/conclusion: these results demonstrate a differential regulation of redox homoeostasis in plasma and in pevs from ad patients. funding: acknowledgements: this work was supported by the chaire louise & andré on alzheimer's disease, foundation armand-frappier (cr) and cihr grant (tf). carlos j. nogueras-ortiz a , pavan bhargava b , sol kim b , francheska delgado-peraza a , peter calabresi b and dimitrios kapogiannis a a laboratory of clinical investigation, national institutes of ageing, baltimore, usa; b department of neurology, johns hopkins university school of medicine, baltimore, usa introduction: multiple sclerosis (ms) is a neurological disorder characterized by white matter demyelination and extensive synaptic pathology. recent studies have shown synaptic loss in the grey matter of ms brains in the absence of demyelinating lesions which could account for disease progression independent of demyelinating episodes. opsonization of synapses with complement components is a mechanism by which phagocytic cells normally prune synapses, but, when occurring in excess, it may underlie pathologic synapse loss. we sought to identify blood-borne biomarkers of hypothesized complement-mediated synaptic loss in ms using circulating neuronal-enriched and astrocytic-enriched extracellular vesicles (nevs and aevs). methods: nevs and aevs were immunocaptured in parallel from the plasma of ms patients ( with relapsing remitting, with progressive ms) and healthy controls, targeting the neuronal-specific marker l cam and the astrocyte-specific marker glast, respectively. we measured the protein levels of preand post-synaptic proteins synaptopodin and synaptophysin in nevs using elisas and multiple complement cascade components (c q, c , c b/ic b, c , c , c a, c , factor b, factor h) in aevs using a luminex array. results: synaptopodin and synaptophysin protein levels in nevs of ms patients compared to controls were markedly reduced ( . -fold; p < . for both), whereas multiple complement components in ms aevs were markedly increased (c q: . -fold change; c : . -fold change; c b/ic b: twofold change; c : . -fold change; c a: . -fold change; factor: . -fold change; p < . ); differences were not observed in total circulating evs or neat plasma. strikingly, we found the nev-associated synaptopodin/synaptophysin and the aev-associated complement levels to be negatively correlated in people with ms (synaptopodin vs: c q, r = − . and p < . ; c , r = − . and p < . ; factor h, r = − . and p < . /synaptophysin vs: c q, r = − . and p < . ; c , r = − . and p < . ; factor h, r = − . and p < . ), but not in controls. summary/conclusion: circulating evs provide markers of synaptic loss and complement activation in ms and suggest a link between astrocytic complement production and synaptic decline. funding: this research was supported in part by the intramural research program of the national institute on ageing, national institutes of health. methylglyoxal and glyoxal affect the protein cargoes in neuronal-derived extracellular vesicles introduction: advanced glycation end-products (ages) and their receptor rages are known to be involved in the pathogenesis of alzheimer's disease (ad). methylglyoxal (mg) or glyoxal (go) are the precursors of ages and particularly n-( -carboxymethyl)-l-lysine (cml), the most abundant ages. mg induced tau hyperphosphorylation and causes hippocampal damage and memory impairment in mice. the aim of our study was to analyse the effects of mg and go on the neuroprotective, neurotrophic factors, inflammatory and neurodegenerative markers in the human cell line sk-n-sh and their release into the neuronal derived-evs. methods: briefly, sk-n-sh cells were incubated in fbs free media with mg and go ( . mm) for hours. neuronal derived-evs (nevs) from culture media were isolated as previously described (haddad et al. ). nevs were characterized by electronic microscopy, nta and by western blot. cellular and nevs concentrations of bdnf, prgn, nse, app, mmp , angptl- , lcn , ptx , s b, rage, dj- and alpha synuclein were determined by a luminex assay from r&d systems, inc. aβ - , aβ - , ptau t and total tau levels were measured also with luminex assay from emd millipore corp. results: we found that both ages precursors, at non toxic concentration, reduced the neuronal levels of nse with no effect on bdnf, ptrx- , lcn- , dj- , on neurodegenerative markers and on cml. go decreased the levels of prgn, app, angpl- while the expressions of mmp- and angpl- were, respectively lower and higher in the presence of mg. mg and go greatly reduced the release of lcn- by neuronal cells in nevs. bdnf and prgn in nevs were reduced in the presence of go. both mg and go did not modify the release of nse, app, mmp , agntl- , ptx- , dj- , aβ, ptau and cml in nevs. summary/conclusion: our data demonstrated that mg and go differently affect the content of some protein cargoes in nevs and suggest that targeting mg and go may be a promising therapeutic strategy to prevent neurodegeneration. introduction: peripherally circulating brain-derived extracellular vesicles (evs) and their encapsulated rnas may serve as biomarkers for hiv-associated neurocognitive disorders (hand). however, rates of cigarette smoking are significantly higher in hiv+ individuals than the general population, and smoking can modulate the expression of these markers. to better understand how cigarette smoke might modulate rna expression and ev release, we examined several cnsderived cell lines, representing astrocytes (u mg), microglia (sv ), and oligodendrocytes (hog). methods: cigarette smoke extract (cse) was prepared by bubbling through culture medium using a standardized and published method. all cell types were exposed to either % or % cse for hours. cell viability was assessed by musetm cell analyser, and evs were isolated from culture conditioned media (ccm) by size exclusion chromatography. the void (fractions - ), ev ( - ), and protein ( - ) enriched fractions were pooled and concentrated. evs were characterized by transmission electron microscopy (tem), microfluidic resistive pulse sensing, and western blotting. total rna was isolated from cells and circular rna (circrna) expression was assessed with a circrna microarray. results: in response to cse exposure, cell viability was only slightly reduced for all cell types. tem images validate the presence of vesicles in the ev fractions, and their absence in the void and protein fractions. spectradyne particle counts indicated cse exposure substantially increased the ccm particle count in the ev fraction when compared with control. the presence of expected ev markers (cd , cd , and tsg ) in the ev fractions, and their absence in the void and protein fractions was observed via western blot. intracellular circrna expression was significantly altered in all three cell lines. summary/conclusion: cns cells display physiologic responses to cse that include vesiculation pathways and significant alterations in circrna expression. we are now studying the effects of cse exposure on circrna expression in released evs. funding: this work is supported by da , da , and ai . a method for exosomal rna extraction from paired human brain and blood specimens emily n. moya a , lillian wilkins a , esther cheng a , lisa linares b , brian kopell b , navneet dogra c , bojan losic a and alexander charney a a icahn school of medicine at mount sinai, new york, usa; b mount sinai hospital, new york, usa; c department of genetics and genomic sciences, department of pathology, icahn school of medicine, mount sinai, new york, usa introduction: diagnosis and treatment of neuropsychiatric disorders has made little progress in the last half-century likely in large part due to the absence of a scalable technique to profile the complex biological activity of the brain in a living person. exosomes are nanovesicles - nm in size that mediate intercellular communication and contain proteins, lipids, and nucleic acids. it has been shown that brain derived exosomes can be found in peripheral blood, but determining whether peripheral exosomes truly reflect ongoing brain processes has to date not been possible due to the absence of paired living brain and blood specimens. here, we present a novel method for paired sampling of the dorsolateral prefrontal cortex (dlpfc) and peripheral blood from living human subjects for exosomal rna profiling. methods: informed consent, approved by the irb at the icahn school of medicine at mount sinai, was obtained for patients undergoing deep brain stimulation (dbs). paired brain and blood specimens were collected from patients at two deep brain stimulation (dbs) electrode implantation procedures: left hemisphere followed by right hemisphere (total of samples). we developed protocols to profile rna from exosomes of brain tissue extracellular matrix (ecm) and peripheral blood. exosomes were isolated via our in-house protocol using ultracentrifugation. rna was then extracted from the exosomes using the qiagen mirneasy mini kit protocol. quality control (qc) was performed to determine whether rna obtained was sufficient for next-generation sequencing. results: we demonstrate the safety of a novel procedure to sample the brain in living human subjects. bioanalyzer traces and qc data show a mean total rna of . ng (range . - . ng) and no samples fell below the threshold required for library preparation and sequencing ( pg) determined by inhouse optimization on the smart-seq v ultra low input kit. summary/conclusion: to our knowledge, we have performed the first study to sample pairs of dlpfc and blood from living human subjects for exosomal rna for subsequent next-generation sequencing. ongoing analyses by our group promise to establish peripheral exosomal rna transcripts reflective of brain activity. this non-invasive approach to probing neurobiology in the living human brain may facilitate the development of exosome-based diagnostics for neuropsychiatric disorders. introduction: the relationship between obesity and dementia is complex. while obesity in middle age triples the risk of dementia years later, many patients with alzheimer's disease (ad) are cachectic, and a decline in adiposity portends progression of dementia. this suggests adipose-derived factors are important to nervous system homoeostasis. we previously showed that adipocyte-derived small extracellular vesicles (ad-sevs) induce pathologies critical to developing obesity-related diseases and may provide a mechanistic link between adiposity and dementia. we hypothesized that altered expression of ad-sev micrornas involved in neurodegenerative pathways is associated with more severe cognitive impairment methods: we studied serum and cerebrospinal fluid (csf) from participants with ad and non-ad controls. ad-sevs were isolated from samples by precipitation and immunoselection. ad-sev microrna expression was profiled in both biofluids and compared. results: serum and csf microrna expression correlated strongly (r = . ). in serum, micrornas were differentially expressed by a fold change ≥| . | in the ad and control groups (p ≤ . ) and micrornas were differentially expressed in csf. using ingenuity pathways analysis, we identified mrnas expressed in nervous system tissue that are targeted by the differentially expressed micrornas. the mrnas map to diseases and functions; neuronal cell death, neurodegeneration, and neuronal growth and developmental pathways are highly represented. of the differentially expressed micrornas in serum, were moderately correlated with participants' score on the mini-mental state exam, a test of cognitive function (rs = | . - . |). as validation, rencell cx cortical derived neuronal stem cells had decreased doubling time when exposed to ad-sevs from obese adipose tissue in vitro. summary/conclusion: these findings support our hypothesis that altered expression of circulating ad-sev micrornas are involved in neurodegenerative pathways associated with cognitive impairment. these findings support using serum ad-sevs as a surrogate for csf ad-sevs. functional validation is underway to define the connection between ad-sevs and ad. understanding the link between obesity and ad is crucial as the population ages and the global obesity epidemic grows. funding: supported by uw adrc (nih:p ag ) expression of extracellular vesicles after acute traumatic brain injury: an exploratory flow-cytometry study introduction: coagulation derangements related to disseminated intravascular coagulation (dic) are common after tbi and contribute to secondary neural injury. extracellular vesicles (evs) are released from all cell types, including platelets, endothelium, and lymphocytes, which are responsible for dic. we hypothesized that specialized flow cytometry techniques could identify a unique ev signature of dic in acute tbi. methods: using a modified flow cytometry instrument for detection of small particles, fluorescence panels were created to assess for evs from endothelial cells (cd , cd ), platelets (cd , cd p, cd a, cd b), and erythrocytes (cd ) as well as brain biomarkers (s b, uchl- , gfap, tau and nse) and t-lymphocytes (cd , cd , cd , cd ). samples were prepared in trucount tubes to determine volume and treated with triton to confirm presence of evs. results: / study patients and / controls were male. % of study patients presented with a glasgow coma scale of . in the hypercoagulability panel, of the subsets with statistically significant differential expression, involved s b+ and were elevated in patients. platelet-derived cd a evs and uch-l evs were significantly elevated in controls in ev subsets identified in the brain-specific panel. finally, cd +/ + evs, derived from t-cells and identified in the endothelial/t cell panel, are significantly lower in patients suggesting cns recruitment. summary/conclusion: endothelial and platelet/erythrocyte evs may be elevated early after tbi. s bcarrying evs are significantly elevated in circulation of tbi patients; if reproducible, this signature profile may be informative for diagnosis and risk stratification. further study is warranted to evaluate whether this expression correlates with secondary microvascular brain injury. funding: intramural award from the university of pennsylvania enrichment of mir- a in cns extracellular vesicles following impairment of the blood brain barrier nasser nassiri koopaei a , ekram-ahmed chowdhury b , lais da silva a , jinmai jiang a , behnam noorani b , ulrich bickel b and thomas d. schmittgen a a university of florida, gainesville, usa; b texas tech university, amarilo, usa introduction: extracellular rnas (exrnas) are present in essentially all biofluids and include all types of rna including mirna. to enhance their stability outside of the cell, exrnas are bound within ribonucleoprotein complexes or packaged into extracellular vesicles (evs). the blood brain barrier (bbb) is a dynamic interface between the systemic circulation and the cns and is responsible for maintaining a stable extracellular environment for cns cells. the intent of this study was to determine if evs and their contents are transferred from the peripheral circulation to the cns under conditions of an impaired bbb. methods: the bbb of mice was disrupted by hyperosmolar mannitol injections. to validate that the bbb has been disrupted with mannitol, intravenously-dosed [ c]-sucrose was increased in the forebrain by fold with mannitol compared to sham treated mice. evs were isolated from the forebrain, hindbrain and spinal cord following gentle tissue lysis and differential ultracentrifugation. evs were validated by nta, tem and western blotting. mir- a, a mirna that is highly abundant in erythrocytes, was measured in the evs by qpcr. results: qpcr showed that mir- a in cns tissue evs increased with mannitol treatment in the forebrain, hindbrain and spinal cord by -, . -and twofold respectively. qpcr analysis of mrna from reported mir- a target genes showed reduced target gene expression with mannitol. summary/conclusion: we demonstrate that evs containing mir- a, a highly abundant mirna present within erythrocytes and erythrocyte evs, is enhanced in the cns upon bbb disruption. astrocyte-derived extracellular vesicles in morphine tolerance guoku hu, rong ma, naseer kutchy, yuetong zhao, susmita sil and shilpa buch university of nebraska medical center, omaha, usa introduction: opiates, such as morphine are used extensively in the clinical setting owing to their beneficial effects. paradoxically, however, the prolonged use of morphine often results in the development of tolerance, drug addiction, and ultimately leading to various comorbidities associated with drug abuse. although great efforts have been made, at present there is no treatment. the sonic hedgehog (shh) plays a key role in brain development, and brain cells fine-tuning processes such as their proliferation, patterning, and fate specification recent findings have demonstrated that inhibition of the shh signalling prevents morphine tolerance in rodent models. we thus hypothesize that extracellular vesicles (evs) derived from morphine exposed astrocytes and their cargo such as shh are critical for the development of morphine tolerance. methods: mice were received either saline or chronic morphine injection with escalating doses of morphine for days (subcutaneously; mg/kg, day , mg/kg days - , and mg/kg days [ ] [ ] . the development of tolerance was assessed by measuring the tail-flick latency using tail flick analgesia metre (le , harvard apparatus). evs were isolated using either differential ultracentrifugation from astrocyte conditioned media or gradient ultracentrifugation from brain tissues. western blotting and qpcr were performed to determine the expression/activation of shh signalling pathway components. results: our data showed that the levels of shh protein were upregulated in morphine exposed astrocytederived extracellular vesicles (morphine-adevs). furthermore, shh containing morphine-adevs activated shh signalling in astrocytes. our in vivo study further demonstrated the upregulation of shh, as well as the activation of shh signalling, in astrocytes of morphine-administered mice. summary/conclusion: these findings thus demonstrated an autocrine mechanism for shh pathway activation in astrocytes associated with morphine tolerance. these findings could pave the way for the development of shh signalling pathway targeted strategies in the prevention and treatment for substance use disorders. biophotonics-based platforms for the evaluation of circulating extracellular vesicles as biomarkers of neurodegeneration in alzheimer's disease silvia picciolini a , cristiano carlomagno a , alice gualerzi a , monia cabinio a , francesca baglio a and marzi bedoni b a irccs fondazione don carlo gnocchi, milan, italy; b irccs fondazione don carlo gnocchi, milano, italy introduction: in the search for novel and non-invasive biomarkers of alzheimer's disease (ad), both circulating brain-derived extracellular vesicles (evs) and whole serum represent a valuable integration of the currently used classification system. to face the technological challenge of evs and serum analysis, we propose the use of biophotonics techniques as reliable, sensitive, fast and label free methods, potentially useful in tailoring pharmacological and rehabilitation treatments. methods: circulating evs, isolated by sec, and serum samples were collected from healthy subjects (hc) and ad patients. all subjects were asked to complete montreal cognitive assessment scale and mri examination. surface plasmon resonance (spr) was performed in order to detect evs coming from neurons, astrocytes, oligodendrocytes and microglia and to characterize each of them for the amount of ganglioside m (gm ), aβ and tspo expressed on their surface. serum analysis was performed using a raman microscope through the surface enhanced raman spectroscopy (sers) effect by mixing serum with ag nanoparticles. the pearson's correlation index was used to assess the linear correlation between spri data and clinical, mri data and data obtained from multivariate analysis (mva) of sers spectra. results: the spr analysis of evs showed that the selected bioactive molecules are differently loaded on neural ev populations and that their amount is increased on total evs in ad patients compared to hc. we observed a significant correlation between mva data from sers and the presence of aβ on neuronal and microglial evs and of tspo on neural evs, measured with the spr array. summary/conclusion: thanks to our methodological innovation we have verified the potentiality of evs as ad biomarkers, correlating biophotonics blood-based analysis with clinical data. this platform could provide a powerful tool for the evaluation of ad neurodegeneration. funding: the study was supported by the italian ministry of health (ricerca corrente - to irccs fondazione don carlo gnocchi). raman profiling of extracellular vesicles as new blood-based biomarker for brain disorders: focus on parkinson's disease introduction: extracellular vesicles (evs) play a pivotal role in brain homoeostasis and intercellular communication in both physiological and pathological conditions. in parkinson's disease (pd), evs are key players in the transfer of α-synuclein, with blood evs reported to undergo proteomic modifications. nonetheless, the detection and characterization of the ev cargo is technologically challenging, limiting the use of evs as biomarkers so far. herein, we propose raman spectroscopy for the label-free, bulk characterization of blood evs in pd patients. methods: evs were isolated by sec and ultracentrifugation from the serum of healthy subjects (hc) and pd patients. in all patients, the severity of pd was evaluated with the unified parkinson's disease rating scale (updrs) part iii and with hoehn and yahr scores (hy). after proper ev characterization following misev guidelines, raman analysis was performed. the raman microspectroscope was used with a nm laser in the spectral ranges - cm- and - cm- . data from hc and pd patients were compared by multivariate statistical analysis (pca-lda). results: the raman analysis of evs highlighted differences in the biochemical profile of the two groups, with the main variations in the spectral regions related to proteins, lipids and saccharides. a preliminary estimate of the accuracy of raman profiling of blood evs for pd diagnosis was obtained, demonstrating an accuracy of %. even more interestingly, we demonstrated the correlation between the raman spectra and the clinical scales (updrs and hy) used to stratify pd patients. summary/conclusion: in conclusion, the biochemical signature of blood evs can be detected by raman spectroscopy in pd patients and the ev spectral modifications can be related to their clinical status. these data suggest the possibility to use the raman profile of circulating evs as a biomarker for brain disorders, complementary to other specific molecular markers. funding: the study was supported by the italian ministry of health (ricerca corrente to irccs fondazione don carlo gnocchi) impact of circulating extracellular vesicles on brain functions and behaviours introduction: peripheral immune alterations have been described in psychiatric disorders such as schizophrenia, depression, and autistic spectrum disorders. in addition, behavioural changes have been observed in various immunodeficient animal models. however, the mechanisms by which peripheral immune system influences brain development and function are not well understood. in this study, we explored the mechanisms by which circulating extracellular vesicles (evs) mediate immune-brain communication and influence mouse behaviours. methods: mice deficient for rag or rag gene (rag ko mice) were used as a model to study the effects of loss of adaptive immune cells (t and b cells) on brain cellular phenotypes and behaviours. circulating evs were collected from their sera and analysed by using electron microscopy, nanoparticle tracking assay, and western blotting. brain cellular phenotypes were assessed by immunofluorescent staining and gene expression analysis. behavioural phenotypes of rag ko and wt mice were examined in social interaction test. in vivo transfer of evs was performed to see its effects on behavioural alterations of rag ko mice. results: rag ko mice displayed social behavioural deficits, accompanying by enhance c-fos immunoreactivity and altered microglia morphology in the medial prefrontal cortex (mpfc). circulating evs were also affected in these mice and lacked the expression of markers for t cells. a set of micrornas (mirnas) in circulating evs were diminished in rag ko mice. in vivo transfer of circulating evs rescues the social behavioural deficits of rag ko mice and ameliorate the c-fos immunoreactivities in mpfc of rag ko mice. summary/conclusion: our data showed that circulating ev profiles were altered in mice lacking adaptive immune cells and, accordingly, showing social behavioural deficits. notably, our in vivo experiments suggest that circulating evs may contribute to social behaviours. further study will provide a novel biological insight into the mechanisms underlying peripheral-to-brain immune communication via evs. introduction: the involvement of neuroinflammation on ageing process is widely recognized. extracellular vesicles (evs), such as exosomes, are able to cross the blood-brain barrier and were related to neuroinflammation. in this context, evs have been considered a potential mechanism of spreading molecules, including micrornas (mirnas) that can promote mrna degradation or inhibit translation of their targets. our aim was to investigate the mirna profile of circulating total evs during ageing process and their impact on canonical pathways. methods: the local ethics committee (comissão de Ética no uso de animais -ufrgs; n ) approved all animal procedures and experimental conditions. plasma was obtained from wistar rats ( and months-old) and total evs were isolated. ev microrna isolation and microarray expression analysis was performed to determine the predicted regulation of targeted mrnas. results: the analysis of global microrna expression revealed differentially expressed micrornas (p < . ; fold change of ≥ | . |); mirnas were up-regulated and were down-regulated in circulating total evs from aged animals compared to youngadult ones. a conservative filter was applied on ingenuity pathway analysis (ipa) and only experimentally validated and highly conserved predicted mrna targets were used. ipa showed that neuroinflammation signalling is ranked among the top canonical pathway impacted by differentially expressed micrornas and is upregulated in aged animals (p < . ; z-score: . ). the differentially expressed mirnas impacted molecules in the neuroinflammation pathway. interestingly, the ion channel grin b is predicted to be up regulated and is a target of many evs mirnas; in accordance with our results grin b was previously related to neurodegenerative diseases. moreover, let- a- p is predicted to be downregulated and target all the molecules of the neuroinflammation signalling pathway. previous studies have correlated let- a- p and neurodegenerative diseases. summary/conclusion: our data suggest that circulating total evs cargo, specifically mirnas, are altered by ageing and impact neuroinflammation pathway, suggesting the involvement evs mirna on ageinginduced susceptibility of neurodegenerative diseases. introduction: bidirectional cell-cell communication via paracrine mechanisms is critical for wound healing. a new paradigm involving exosome-borne distinctive repertoire of cargo such as mirnas has emerged as a predominant mechanism of cellular communication at the site of injury. unlike other shedding vesicles of similar size, exosomes selectively package mirna by sumoylation of heterogeneous nuclear ribonucleoprotein (hnrnp). methods: keratinocyte-derived exosomes (exoκ) were genetically labelled with fluorescent reporter (gfp) using tissue nanotransfection. purified, gfp-labelled exoκ were isolated from dorsal murine skin and wound-edge tissue by differential ultracentrifugation followed by affinity selection using magnetic beads. distributions of intact exosome were analysed using a prototype jarrold-geometry charge-detection mass spectrometer to directly measure differences in particle mass and charge distributions. complementary ms and ion mobility spectrometry (ims)-ms experiments have been used to characterize surface glycans and glycopeptides. to selectively inhibit mirna packaging within the exoκ in vivo, ph-responsive targeted sirna functionalized lipid nanocarriers (tlnκ) were designed using materials that have prior history of fda approval for human use. results: an increase in mass/charge ratio with glycan binding sites on the surface of wound-edge exoκ were observed compared to dorsal skin exoκ. wound-edge exoκ were selectively taken up by the macrophages in the granulation tissue (n = ). keratinocyte targeting sirnahnrnp functionalized lipid nanocarriers (tlnκ) were designed with encapsulation efficiency of . %. application of tlnκ encapsulating sirna of hnrnp (tlnκ/si-hnrnp) to murine dorsal woundedge significantly inhibited the expression of hnrnp by % in epidermis compared to control (tlnκ/sicontrol)(n = ). moreover, mice treated with tlnκ/si-hnrnp showed impaired barrier function, with significant presence of macrophage in granulation tissue at day , suggesting impaired conversion of macrophage in the granulation tissue. summary/conclusion: this work provides a novel insight wherein exosomes of keratinocyte lineage are recognized as a major contributor that directs macrophage conversion in granulation tissue for wound healing. multifaced effects of milk-exosome (mi-exo) as modulator of scar-free wound healing gna ahn, hyo-won yoon, yang-hoon kim and ji-young ahn chungbuk national university, cheong-ju, republic of korea introduction: recently, milk exosome (mi-exo) has been focused particularly on the possibility of oral distribution for therapeutic agents. however, studies related to the cosmeceutical effects associated with mi-exo are fairly limited. the purpose of this study is to suggest the anti-oxidant and antiinflammatory effect of mi-exo and possibility that can be induced by scar free healing by micro rna in mi-exo. methods: the characteristics of the extracted mi-exo were verified by size measurement, morphological characteristics through cryo-em and western blot. for antioxidant experiments, an abts assay was performed. next, mrna expression through four major cytokines (tnfα, il- , cox- , inos) was used to evaluate anti-inflammatory effects. finally, cell migration assay was performed to confirm the effect of scar-free healing and the detection of mir- b in mi-exo and vegf mrna expression confirmed. results: mi-exo using % acetic acid extraction showed the highest yield. the average size of the exosomes is approximately nm, confirmed the typical double membrane vesicle. as a result of antioxidant experiments, it was confirmed that the treatment of exosomes of ^ particles showed about % antioxidant activity. when ^ particles were treated, rna expression of cytokines showed about times more inhibitory effect than control. elisa test results also confirmed that the concentration-dependent decrease. the activation of the raw cell less proceeded as the treated mi-exo increased. the cell scratch assay cells did not close the cells as the number of milk exosomes increased (wound closing % of ^ particle = . %). and mir- b in milk exosomes was detected at ct value = . summary/conclusion: the antioxidant and antiinflammatory effects of mi-exo showed the greatest efficacy when ^ particles were treated. in addition, it induced to scar free healing rather than wound healing. mi-exo has great potential as a superior natural material in the future cosmeceutical field. extracellular vesicles in human milk expose tissue factor and promote coagulation introduction: tissue factor (tf), a transmembrane protein, initiates coagulation by binding and activating coagulation factor vii (fvii). tf is associated with extracellular vesicles (evs) in saliva and urine, but it is unknown whether also human milk (hm) contains evs exposing coagulant tf. methods: hm was collected from six healthy nursing women with informed consent. evs were isolated by ultracentrifugation and size exclusion chromatography (sec). the presence of tf antigen exposing evs was studied by western blot, flow cytometry, cryo-electron microscopy (cryo-em), and surface plasmon resonance imaging (spri). the ability of tf exposing evs to trigger coagulation was investigated with a plasma fibrin generation test (fgt), performed in the absence or presence of antibodies against tf or fvii(a). results: addition of hm to plasma shortened the plasma clotting time, even when hm was highly diluted. after ultracentrifugation of hm, both tf antigen and tf activity were detected in the ev-containing pellet. after sec, tf antigen and tf activity were present in the ev-containing fractions and . the presence of tf-exposing evs in these sec fractions was confirmed by western blot (cd , cd and tf), flow cytometry, spri, and fgt. in addition, the presence of evs in hm was confirmed by cryo-em. scalable isolation of evs from different probiotic strains with potential as cosmetic ingredients laura soriano-romaní, joaquin espí and begoña ruiz ainia, paterna, spain introduction: extracellular vesicles (evs) are increasing their application in a number of fields. recently, it has been shown that skin health may be affected not only by commensal skin bacteria, but also by the evs that they secrete. however, because most of the efforts have been directed to the characterization and evaluation of evs, the scaling up of the production process remains a bottleneck at the industrial level. in this work, the goal was to evaluate the potential applications of evs produced by different probiotic strains commonly used in the cosmetic field, considering the economic and technical viability of the process. methods: to meet our goal, a standardized workflow was defined to isolate evs from probiotic strains such as lactobacillus and bifidobacterium species, that have demonstrated cutaneous immuno-regulatory effects. the different bacterial strains were produced under standard culture conditions. to isolate the secreted bacterial evs, different chromatographic techniques were performed starting from clarified growth medium. then, evs were evaluated in vitro for a number of biological effects related with skin health. results: the ev yields obtained after downstream processing were calculated for each strain and isolation technique by means of nanoparticle tracking analysis (nta) and total protein content. moreover, evs were visualized by electron microscopy. the in vitro evaluation of isolated evs was based on changes in the expression of five biomarkers related with anti-ageing, anti-inflammatory and whitening effects using distinct skin cell types to identify possible cosmetic claims that could be associated to each probiotic source. summary/conclusion: the potential of evs obtained from probiotic strains as cosmetic active cell-free ingredients was preliminarily assessed with this work, where the process yield and cosmetic function were evaluated. however, additional experiments will be needed in order to increase and optimize the productivity of each step of the ev manufacturing process. acerola derived exosome-like nanovesicles enhances the repair of ultraviolet b-induced dna damage in cultured skin fibroblasts tomohiro umezu, masakatsu takanashi, yoshiki murakami and masahiko kuroda tokyo medical university, shinjyuku, japan introduction: acerola (melpighia emarginata dc.) is a fruit is known to contain not only high amounts of ascorbic acid but also various nutritional components such as carotenoids and polyphenols. previous reports showed the acerola juices are able to confer protection against ultraviolet radiation b (uvb), to improve barrier function of skin. uvb is the main cause of dna damage in epidermal cells, generating several types of pro-mutagenic lesions, like cyclobutene prymidine dimers (cpds) and prymidine ( - ) prymidinone photoproducts ( - pps): if not repaired, this dna damage leads to skin cancer. in this study, we investigated the biological property of the acerola derived exosome-like nanovesicles (adens), aiming to clarify the involvement of adens in repair of uv-induced dna damage. methods: normal human dermal fibroblasts (nhdfs) were purchased from lonza inc. the exosome-like nanovesicles were isolated from acerola juices using exoeasy maxi kit (qiagen). the morphology and size distribution of adens were checked by transmission electron microscopy (tem) and nanoparticle tracking analysis (nta, nanosight lm , malvern). nhdfs were exposed to uvb ( mj/cm ) with pre-or post-adens. effect of uvb was assessed by examining cell viability, cell morphology, and dna damage levels through biochemical assays, microscopy and protein expression studies. results: purified adens were compatible with nta or tem for assessing the nanovesicle size range and concentration ( - nm). when nhdfs were added with adens and incubated at °c for h, there was no effect of adens on cell proliferation of nhdfs. we found that adens treatment to uvb exposed nhdfs significantly reduced cpds and - pps dna adduct formation. present results showed that aden treatment prevented uvb induced dna damage in nhdfs. summary/conclusion: we confirm that adens have the effect of repairing dna damage caused by uvb. these results provide that adens can be a new source to protect human skin from uv-induced skin cancer. introduction: introduction: despite the development of a variety of therapies, complex wounds resulting from disease, surgical intervention, or trauma remain a major source of morbidity. extracellular vesicles (evs) derived from mesenchymal stem/stromal cells (mscs) have been shown to improve wound healing, especially via enhanced wound angiogenesis. however, despite their clearly established potential, evs have limitations that may limit clinical relevancy, such as low potency. hypothesis: increased expression of pro-angiogenic lncrna hotair within msc evs enhances their proangiogenic effects and thus their wound healing properties. methods: methods: hotair was overexpressed in human dermal microvascular endothelial cells (hdmecs) to determine any molecular or functional pro-angiogenic effects. anti-angiogenic mirnas and angiogenic mrna levels were quantified by rt-qpcr. effects of hotair on proliferation of hdmecs was also determined. hotair was then loaded into msc evs by delivering a cmv-based hotair plasmid to mscs for endogenous loading via a concentration gradient. evs were collected by differential centrifugation. hotair content within evs was confirmed by gel electrophoresis and rt-qpcr. effects on migration of hdmecs by hotair-loaded msc evs were determined using a scratch assay. results: results: overexpression of hotair decreased mir- c and mir- , while increasing vegf and hif- a. hdmec proliferation was also increased in hdmecs overexpressing hotair (p < . ). hotair was visually confirmed in hotair-loaded msc evs by gel electrophoresis, but was undetectable in unmodified msc evs. rt-qpcr confirmed a -fold increase of hotair compared to control msc evs. hdmecs showed a more statistically significant rate of gap closure when treated with hotair-loaded evs (p < . ) than compared to control msc evs (p < . ). summary/conclusion: summary: loading lncrna hotair into msc evs is achievable by a concentration gradient-dependent method and offers potential to enhance the angiogenic properties of msc evs. nanomaterial labelling of exosomes for cell biology introduction: exosomes are vesicles secreted by many, if not all, cell types and have been known about for decades. among larger micro vesicles that are produced directly from the cell membrane, the small ( - nm), exosomes are similar in size to a virus surrounded by a lipid bilayer. we and others have demonstrated that exosomes contain proteins, lipids, rna, and dna, making them promising materials for diagnosing and treating diseases, including many cancers such as brain cancer. in addition, exosomes from neurons and glial cells represent a novel type of intercellular communication. however, their size makes them hard to track with traditional fluorescence microscopy. to address this, we developed photothermal microscopy (ptm), which uses gold nanomaterial labelling to track exosomes' interaction with and effect on cells/tissue. methods: exosomes secreted by tumour cells and general exosomes found in the blood were isolated using differential ultracentrifugation or a commercially available kit (invitrogen). next, the exosomes were characterized by (tem), (nta), and western blotting to determine shape, size, morphology and the protein profile in the exosomal membrane. after characterization, the exosomes were labelled with gold nanoparticles via sonication. next, the samples were washed, and the exosomes were labelled with fluorescence dye to stain the membrane. after staining and labelling, the exosomes were added to u cells in culture and incubated for h. they were then fixed by % paraformldehyde and imaged by ptm. results: ptm found that exosome-cell interactions are exosome-type dependent, as u cells took up exosomes from other u cells but not human serum exosomes. this suggests that exosome uptake is a selective process and depends on the source of the donor cells. summary/conclusion: exosomes can be labelled with gold nanoparticles via sonication then successfully tracked by ptm to study the effect of exosome source on exosome-cell interactions and communication. cells incubated with u exosomes took the vesicles up rapidly, while cells incubated with serum exosomes had little uptake. ptm will help us design selective exosome-based strategies to treat different conditions, including brain cancer and cns damage. funding: nsf epscor riii award . loading of goat´s whey extracellular vesicles with spiked microrna and curcumin as an strategy for developing new nanocarriers for acellular therapies introduction: extracellular vesicles (ev) are involved in cell signalling and are present in a variety of cell secretions such as milk, from which enormous amount of ev can be purified, thus milk is an attractive raw material for scaling up ev production for therapeutic, cosmetic or other uses. here we isolated evs from the whey fraction of goat´s milk and demonstrated that such evs can be loaded with molecules like polyphenols and mirna. methods: to achieve this, milk was collected from lactating goats and fractionated by acidification and centrifugation into whey and caseins. evs were purified from the former fraction by serial centrifugation and precipitation with commercial kit (total isolation/ thermo fisher) and characterized by electron transmission microscopy (tem), western blot to identify surface markers and measurement of size through nanotracking analysis. once isolated, evs were loaded with different concentration of a spiked synthetic mir or with the polyphenol curcumin. mirna or curcumin were co-incubated over night with evs at oc, precipitated and purified as described above, with an additional washing and precipitation for curcumin. concentration of mirna uploaded by evs was quantified using mir specific qpcr. curcumin was measured using a spectrophotometer at nm. results: evs isolated from whey had an average size of nm, were positive for hsp , cd and alix. in tem, evs were identified with their natural conformation and corresponding size to exosomes. qpcr showed a significant difference of expression of mir in relation to control (loaded with shame) and the negative control (p < . ). curcumin presence was also confirmed after washing and precipitacion. summary/conclusion: in conclusion, milk evs and exosomes can be loaded with mirna and a polyphenol and can be used as alternative nanocarrier for acellular therapies. introduction: extracellular vesicles (evs) are cellderived lipid membrane nanoparticles that serve as messengers of intercellular communication, transferring bioactive molecules to recipient cells. evs have a natural therapeutic potential with high flexibility and biosafety for employing natural and synthetic biomolecules as therapeutic delivery vehicles. considering the importance of evs, their isolation methods are still a bottleneck. to get insights into the tissue-specific cargo in vivo for complete exploitation of evs as therapeutic, biomarker and diagnostic tools, ev purification methods are critical. the aim of the study was brought about to develop an efficient ev purification method both in vitro and in vivo and to further investigate function of evs in cellular senescence. methods: to isolate tissue-specific evs in vivo we developed recombinant evs by genetically fusing snorkel-tag to the cd . the snorkel-tag enables on-column protease treatment for purifying evs which does not rely on traditional immunoaffinity purification protocols using low ph or high salts solutions. results: we systematically evaluated the purification of evs harbouring snorkel-tag by employing different methodologies. our findings suggest that evs harbouring snorkel-tag indeed can be purified at high purity without altering ev biophysical properties. furthermore, we expressed cd -snorkel-tag under p ink a promoter and were able to purify evs derived from senescent cells. summary/conclusion: finally, we are developing an in vivo model with cd -snorkel-tag under p ink a promoter. this will provide us detail insights into the ev cargo secreted from senescent derived cells, by purifying evs harbouring snorkel-tag under pathophysiological conditions, allowing us to develop biomarkers and therapeutic tools. summarized, we have here developed novel tool for studying content and function of evs in the context of ageing and disease. this tool will now pave the way for studying the molecular mechanisms underlying these ev functions in vivo. funding: this work was funded by the austrian science fund phd program biotopebiomolecular technolgy of proteins (w ). engineering exosomes with gata- jie xu, christian paul, yi-gang wang and meifeng xu university of cincinnati, cincinnati, usa introduction: exosomes, are small vesicles ( - nm) secreted from cells that can transport and deliver of their components such as lipids, proteins, dna, mrna, and mirna to target cells. gata- , a cardiac transcription factor, has been shown to regulate differentiation, proliferation, and survival of a wide range of cell types. delivering gata- protein into ischaemic tissues may be one of the most straightforward approaches to improve cardiac function following myocardial infarction. here, exosomes were engineered with gata- by infusing gata- with exosome targeting peptide. methods: the open reading frame of mouse gata- cdna was ligated to xpack lentivirus vector (xpack-gata- ) and plvx-ef -ires-pouro lentivirus vector (plvx-gata- ), respectively. hek cells were transduced by lentivirus, then exosomes were isolated from conditioned medium of hek cells using ultracentrifugation. exosomes were identified using transmission electronic microscope (tem), and the expression of gata- was semi-quantified using western blot. the internalization of exosomes was tracked via treating bend cells with exosomes pre-labelled with pkh . introduction: chinese hamster ovary (cho) cells have dominated as the mammalian cell host for the manufacture of humanized biologics, in part owing to their genomic plasticity and robust growth in suspension culture. there is great interest surrounding the use of extracellular vesicles (evs) as novel therapeutics owing to their capacity to deliver bioactive molecules. however, much remains unknown about the mechanisms involved in ev cargo loading, limiting their development as novel biologics. to this end, we have engineered cho cells to stably express constructs enabling loading of gfp into evs. methods: tetraspanins are established markers of ev identity. accordingly, cd was selected as a tethering point to generate evs with gfp cargo and constructs were generated via golden gate assembly. cho cells were stably transfected by electroporation and expression was verified with fluorescence microscopy and western blotting. growth in batch culture was monitored to establish maximum viable cell densities for ev harvest and recovered evs were characterized by nanoparticle tracking analysis (nta). finally, uptake of gfp-evs was studied using time-lapse fluorescence imaging in co-culture experiments. results: strong localization of cd -gfp was observed at the cell membrane and blotting confirmed intact tetraspanin fusion present at the expected molecular weight. additionally, cells were confirmed to retain high gfp expression post-cryopreservation. stable cell pools were able to reach viable densities greater than million cells/ml in batch culture and nta allowed for detection of gfp cargo even prior to ev isolation. evmediated transfer of functional gfp to recipient cells was found to occur over a period of hours. introduction: extracellular vesicles (evs) are considered promising for therapeutic applications. evs resemble the cell membrane, allowing high biocompatibility to target cells, while their small size makes them ideal candidates to cross biological barriers. despite the promising potential of evs for therapeutic applications, robust manufacturing processes that would increase the scalability and consistency of ev production are still lacking. methods: in this work, evs were produced by mesenchymal stromal cells (msc), isolated from different human tissue sources (bone marrow, umbilical cord matrix and adipose tissue). msc were selected as these cells allow for a scalable production of evs, while displaying low immunogenicity. a vertical-wheel™ bioreactor system was implemented for the production of msc-derived evs and compared with traditional static systems. the obtained ev products were characterized by nanoparticle tracking analysis, atomic force microscopy, zeta potential and western blot. results: the bioreactor system allowed to obtain evs at higher concentration and productivity, as well as more homogeneous size distribution profiles, when compared to traditional static culture systems. functional studies were performed using breast cancer and lung cancer cell lines. proliferation assays allowed to determine the dose-response profiles of these cell lines when exposed to msc-derived evs. a bell-shaped profile was observed for most cases, since raising the ev concentration lead to increased cell proliferation until a certain point ( - µg/ml), after which cell proliferation was attenuated with increasing ev concentrations. summary/conclusion: the bioreactor culture system allowed a substantial improvement in the production of msc-derived evs, while the obtained dose-response profiles will be valuable to determine the most appropriate ev concentrations for anticancer drug delivery. overall, we demonstrate that this culture system is able to robustly manufacture human msc-derived evs in a scalable manner towards the development of novel therapeutic products such as anticancer drug delivery systems. biodistribution and cellular location of inhaled exosomes and liposomes in the lung introduction: increasing evidence reveals the potential role of extracellular vesicles, such as exosomes and liposomes, in lung regenerative medicine for the treatment of lung diseases. encapsulation and delivery of potential rna and microrna targets into liposomes and exosomes are attractive drug delivery methods, but remain difficult to deliver to the pulmonary parenchyma to reach target lung cell types. here, we demonstrate effective delivery and cellular uptake of exosomes and liposomes to the pulmonary parenchyma via inhalation treatment in a murine model of idiopathic pulmonary fibrosis. methods: human lung stem cells (lscs) were generated and expanded from healthy whole lung donors. lsc-exosomes were purified via ultrafiltration and diilabelled using vybrant☐ labelling solution according to the manufacturer's instructions. dsred-labelled liposomes were generated using lipofectamine™ rnaimax transfection reagent and block-it™ alexa fluor™ red fluorescent control according to the manufacturer's instructions. lsc-exosomes and liposomes were delivered via nebulization to cd mice with bleomycin-induced pulmonary fibrosis. exosome and liposome delivery and biodistribution were visualized -and -hours post-treatment through histological analysis. the study was approved by the institutional animal care and use committee of north carolina state university and complied with all national and state ethical standards. results: exosome and liposome delivery to the pulmonary parenchyma was confirmed by the presence of dii and dsred fluorescence in lung histological sections that penetrated the mucus-lined respiratory epithelium. more exosomes and liposomes surpass mucus-lined surfaces -hours post-treatment compared to -hours post-treatment. fluorescent colocalization of exosomes and liposomes with alveolar type i cells, alveolar type ii cells, basal lung cells, and cd + macrophages was observed through immunohistochemistry analysis. more exosomes and liposomes colocalize with these cell types -hours post-treatment compared to -hours post-treatment. summary/conclusion: lsc-exosomes and liposomes penetrate the mucus-lined respiratory epithelium and reach the pulmonary parenchyma through inhalation treatment. lsc-exosomes and liposomes are uptaken by alveolar epithelial cells, basal cells, and interstitial macrophages with improved biodistribution -hours post-treatment. funding: this study was supported by the nc state chancellor's innovation fund. transfection reagent artefact accounts for some reports of extracellular vesicle function codiak biosciences, cambridge, usa introduction: extracellular vesicle (ev) functions are frequently investigated by transiently transfecting cells with plasmid dna to produce evs modified with protein(s) or nucleic acid(s) of interest. however, evs and the dna-complexes used to transduce cells are physically similar, raising the possibility that they may co-purify during differential ultracentrifugation, the most common ev isolation procedure. activities attributed to evs may therefore be due to contaminating dna -transfection reagent complex. methods: ev producing cells were transiently transfected with plasmid dna encoding gene-editing or split enzymes fused to ev-targeting protein sequences. differential and density gradient ultracentrifugation were used to purify evs from cell culture supernatant or dna lipoplexes from cell-free culture media. protein expression and localization to evs was confirmed by western blot. cell lines stably expressing fluorescent or luminescent reporters were used to assess functional enzyme delivery in recipient reporter cells. results: reporter cells treated with ultracentrifuge pellet material (ucp) from media of transiently transfected cells showed robust and reproducible signal, however fractionating the ucp with an iodixanol density gradient revealed that reporter activity was associated with high-density fractions that were depleted in evs. ucp isolated from identical transfection conditions, but lacking cells (and exosomes), showed identical biological activity levels and distribution in iodixanol gradients, suggesting that the activity was due to contaminating transfection reagent complexes and not evs. serial media changes on ev producing cells post-transfection did not significantly reduce ucp activity on reporter cells. treatment with nucleases did not digest complexed dna, did not significantly reduce dna levels in the ucp as measured by qpcr, and did not decrease activity in reporter cells treated with ucp from either transfected cells or no-cell controls. summary/conclusion: we find that dna-transfection reagent complexes are not separated from evs using differential ultracentrifugation and that common approaches to remove such complexes, including media exchanges and nuclease treatment, are ineffective. due to the pernicious nature of the dna-complex in these cellular assays, it is likely that some reports of ev function are likely artefacts produced by contaminating dna-complexes. we find that density gradient centrifugation can effectively separate evs and dnacomplexes, highlighting the importance of validating elimination of contaminating transfection reagent complexes when using transient transfection to interrogate ev function. chair: suresh mathivanan -la trobe university cancer stem cell-derived exosomes: potential biomarkers for early diagnosis and prognosis in pancreatic cancer introduction: pancreatic cancer (paca) is the most deadly manlignancy, due to late daignosis and early metastatic spread, which prohibits surgery. it is urgently for relaible, early detection. research shows that tumour-derived exosomes, which had been present in the blood in the early stage of tumour formation and before metastasis, is the vanguard forces of tumour formation and metastasis; cancer stem cell-derived exosomes (csc-exos) has stronger migration ability, so the detection of blood csc-exos for early diagnosis and monitoring of progress for paca has great research potential and the value of application. methods: protein markers were selected according to expression in exosomes of paca cell line culture supernatants, but not healthy donors' serum-exosomes. according to these preselections, serum-exosomes were tested by flow cytometry for the pancreatic cancer stem cell marker cd v and tspan . results: the majority ( %) of patients with paca and patients with nonpa-malignancies reacted with anti-cd v and anti-tspan . serum-exosomes of healthy donors' and patients with non-malignant diseases were not reactive. recovery was tumour grading and staging independent including early stages. introduction: chronic traumatic encephalopathy (cte) is a tauopathy that affects individuals with a history of mild repetitive brain injury frequently seen in contact sports. initial neuropathologic change of cte include perivascular deposition of phosphorylated tau (p-tau) in cortical neurons and, in later stages, the formation of neurofibrillary tangles in neurons throughout the brain. extracellular vesicles (ev) are known to carry neuropathogenic molecules in neurodegenerative disease and able to cross the blood brain barrier. we therefore examined the protein composition of ev separated from cerebrospinal fluid (csf) and plasma in former national football league (nfl) players with cognitive dysfunction, and an agematched control group with no history of contact sports. methods: evs were separated from csf and plasma from former nfl players (n = , ) and controls (n = , ) by affinity separation method or size exclusion chromatography, respectively. the ev protein profiling was characterized by simoa for tau and ptau and mass spectrometry. the protein data was analysed for ev enrichment, differentially expressed proteins, pathway analysis and correlation with cognitive function, head impact and tau/p-tau levels by biostatistics and bioinformatics. results: the level of total tau and p-tau in csf evs was not significantly changed, but significantly elevated in plasma evs from former nfl players. the proteins were commonly identified between the paired plasma-csf from the same patients, but there was no significant correlation with disease status. collagen alpha- (vi) chain (col a ), − (vi) chain (col a ) and reelin (reln) were differentially expressed in former nfl players' plasma evs. a combination of these proteins in plasma ev can distinguish former nfl players from controls with % accuracy by machine learning. summary/conclusion: the interacting plasma-csf ev proteomes provide an original resource to ev biomarker development for neurodegenerative disease, and col a , reln and col a in plasma evs can be potential biomarker for monitoring the cte development. density-based fractionation of urine to unravel the proteome landscape of extracellular vesicles in prostate cancer introduction: current diagnostic tests are unable to discriminate indolent from aggressive prostate cancer (pca), leading to overdiagnosis and overtreatment, and an intense interest in biomarkers to improve clinical decision making. urine is considered an ideal proximal fluid for biomarker identification in pca due to its direct contact with the urogenital system. the discovery and translation of extracellular vesicle (ev) content into pca biomarkers remains challenging due to the difficulty of obtaining urinary ev (uev) with high specificity. methods: we developed a step-by-step protocol to separate uev by orthogonal implementation of ultrafiltration and bottom-up density gradient centrifugation (bu-odg). we implemented complementary particle and protein measurements to identify uev (lower density) and protein rich fractions (higher density) and assess the performance of bu-odg (specificity, efficiency and reproducibility). using mass spectrometry-based proteomics we interrogated uev and protein rich fractions from matched urine and radical prostatectomy tissue samples from pca patients (n = ), and urine from men with pca prior to (n = ) and after local treatment (n = ), benign prostatic hyperplasia (n = ) and other urological cancers (n = ). results: bu-odg separated uev from soluble proteins and tamm-horsfall protein (thp) complexes with high specificity and reproducibility, outperforming differential ultracentrifugation, exoquick and size-exclusion chromatography. comparison of the uev proteome from men with benign or malignant prostate disease, allowed us to expand the known human uev proteome and identify a pca specific uev proteome not uncovered by the analysis of the protein rich fraction. proteomic analysis of ev separated from prostate tumour interstitial fluid and matched uev confirmed pca specificity of the uev proteome. analysis of the uev proteome from patients with bladder and renal cancer provided additional evidence of the selective enrichment of protein signatures in uev reflecting their respective cancer tissues of origin. summary/conclusion: we identified hundreds of previously undetected proteins in uev of pca patients and developed a powerful toolbox to map uev and protein rich fractions, ultimately supporting biomarker discovery for urological cancers. immunoglobulin a coating of faeces-derived bacterial vesicles as a marker of inflammatory bowel disease in humans nader kameli a , frank stassen b , heike becker c , john penders c , daisy jonkers d and paul savelkoul b introduction: iga is the most abundant antibody in mucosal secretions and plays a crucial role in maintaining the balance between the host and the gastrointestinal microbiome. recent studies suggested that pronounced iga coating is especially prominent among inflammatory commensals which drive intestinal disease. membrane vesicles (mvs, nano-sized particles released by bacteria) have also been found to interact with the host and modulate development and function of the immune system. however, their interaction with iga has not been studied yet. here we developed a method to isolate and characterize the mvs from faecal samples and checked for possible differences in iga coating patterns of mvs in health and disease. methods: mvs were isolated by using a combination of ultrafiltration and size exclusion chromatography from faecal samples of healthy controls (hc), patients with active crohn disease (cd) and cd patients in a remissive state. quantification and verification have been done with tunable resistive pulse sensing (trpsbased analysis) bead-based flow-cytometer (bbfc) and transmission electron microscope (tem). mvs were selected with specific antibodies for capturing (gram +: lta, gram-: ompa) followed by pe-conjugated anti-human iga antibodies as detection. results: we could successfully isolate * - * particles/ml from mg of faeces. bbfc in combination with trps provide a valuable method for (semi-)quantitative measurements of mixed populations. intriguingly, remarkable differences were found between iga coating mvs derived from healthy controls and active and remissive cd patients as mvs derived from healthy controls were significantly more coated compare to both cd patient groups. in details, for selected g-ve derived mvs: % of the total population of mvs derived from hc were coated, % from remissive cd patients, and < % of active cd patients; and for selected g+ ve derived mvs: % of the total population of mvs derived from hc were coated, % from remissive cd patients, and % of active cd patients. (data are represented as the mean). summary/conclusion: here we demonstrate for the first time that mv isolated from the faecal samples are also coated with iga, and surprisingly mvs from healthy volunteers were more densely coated than mvs from diseased patients. the possible consequence of this difference remains to be determined in future studies. monitoring altered tetraspanin and psma expression in prostate cancer derived extracellular vesicles via advanced image flow cytometry (isx) lukas w. prause a , christopher millan b , natalie hensky c , tullio sulser c and daniel eberli c a universityhospital zurich, zurich, switzerland; b university of zurich hospital, schlieren, switzerland; c university of zurich hospital, zurich, switzerland introduction: new diagnostic and therapeutic options for patients with prostate cancer are urgently needed. prostate-specific membrane antigen (psma)-based imaging and therapy are increasingly used for prostate cancer management. unfortunately, as a membrane protein, psma is not found as a soluble protein in the blood and therefore has limited utility as a diagnostic biomarker. however, psma has reportedly been observed as a cargo protein of prostate cancer-derived extracellular vesicles (evs). we demonstrate altered psma expression on evs derived from prostate cancer cell cultures (c - , lncap) in response to novel next-generation androgen receptor inhibitor (enzalutamide), a standard chemotherapy agent (docetaxel), a novel experimental nonsteroidal antiandrogen (epi- ) that binds covalently to the n-terminal domain of the androgen receptor and dihydrotestosterone (dht). additionally, evs were isolated from the plasma of prostate cancer patients who participated in the prococ biobank campaign at the usz. plasma was taken and stored from patients both pre-and post-prostatectomy. results: transmission electron microscopy, nanoparticle tracking analysis and simple western (wes) analysis show stable size distribution and amount of evs produced by treated and non-treated cells. using advanced image-based flow cytometry, altered tatraspanin and psma expression could be detected in evs isolated from cell culture supernatants of lncap and c - prostate cancer cells following their treatment. summary/conclusion: measuring psma expression on extracellular vesicles might pave the way to use image flow cytometry of evs to develop a blood based diagnostic test for prostate cancer patients with a wide range of possible applications including: ) monitoring response to therapy and, ) early indications of potential relapse. funding: vontobel fondation. proteomic profiling of human neural cells derived extracellular vesicles to identify human brain cell-type specific markers introduction: alzheimer's disease (ad) is a common neurodegenerative brain disease which affects appropriately million patients worldwide. one of the major challenges in ad is to develop reliable biomarkers for early diagnosis and disease-modifying therapies, especially before the clinical symptoms. extracellular vesicles (evs) carry cargos of proteins, lipids and nucleic acids. there was no comprehensive characterization of evs isolated from specific brain cell types, which may be useful for cell type-specific biomarkers. the purpose of this study is to isolate evs from human induced pluripotent stem cell (ipsc)derived brain cells for proteomic profiling and characterization of cell type-specific molecules. methods: human ipscs-derived neurons, microglia and primary cultured astrocytes were differentiated in ev-depleted media. the evs were isolated by differential centrifugation combined with size exclusion chromatography, followed by characterization using nanoparticle tracking analysis and mass spectrometry. the proteomic data were subjected to bioinformatics analysis results: we identified proteins from neuronderived ev (nde), proteins from microgliaderived ev (mde) and proteins from astrocytederived ev (ade) by proteomics. gene ontology analysis indicated that most of these proteins are associated with evs. furthermore, , and proteins are present individually in ndes, mdes and ades. among them, high levels of atp a and syt in ndes, itgam and cd a in mdes, and eaat and gfap in ades were found, all of which are typically and highly expressed in the original cells. summary/conclusion: our results provide us the potential candidates for cell-type specific ev markers, which will be helpful to develop non-invasive tools to enrich ev originating from specific brain cells and may lead to the development of new biomarkers for neurodegenerative disorders. ) are a tremendous resource for extracellular vesicle (ev) research, but they are heavily focussed on mammalian evs, i.e. evs from humans and laboratory animals, where protein cargoes are well characterised, and a wide selection of antibodies are commercially available. protein markers can be used to identify and define the types of mammalian ev and to determine the presence of any contaminants that might confound functional studies. similar resources are not as readily available for bacterial evs as these are not as well characterised, commercially available antibodies are much less abundant and immunological variation between different bacterial species (and there are trillion bacterial species on planet earth!) means that each species, strain, or group of related species may require different antibodies. methods: to identify quality markers for bacterial evs, we have characterised the proteome of cells, crude evs (ultracentrifuge pellet from cell free culture supernatant) and size exclusion chromatography or density gradient centrifugation purified evs from two different (pathogenic vs probiotic) strains of escherichia coli grown under two different environmental conditions, and one strain of mycobacterium marinum grown in one medium. results: our results identify a selection of proteins enriched in purified ev preparations, and proteins that are depleted after purification steps. summary/conclusion: our results allow the identification of potential markers for ev purity and non-ev contaminants, but also highlight the variability in bacterial ev preparations and suggest potential targets that can be used to investigate the heterogeneity of bacterial ev populations. introduction: recent findings indicate an increase in mid-life mortality rates in the usa and persistent, significant race-related health disparities exemplified by differential mortality rates. this suggests that exploring new molecular markers that may be linked to mortality could provide novel insights into factors that are driving mortality rates. accumulating data suggests that extracellular vesicles (evs) circulating in blood may be potential biomarkers of age-related disease. evs are nano-sized membranous vesicles that bear molecular cargo and mediate intercellular communication between different cells and tissues. little is known about whether ev characteristics differ by race or whether evs are associated with clinically relevant mortality risk factors. methods: in this cross-sectional study, plasma evs were isolated from middle-aged african american (aa) and white males and females. results: we report no significant differences in ev size or concentration with race or sex. there were significantly higher ev levels of phospho-p , total p , cleaved caspase , erk / and phospho-akt in whites compared to aas. higher ev levels of phospho-igf- r were found in females compared to males. we examined ev characteristics and protein cargo in the context of well-established clinical mortality risk factors. ev concentration was significantly, and positively, associated with several mortality markers including, high-sensitivity c-reactive protein (hscrp), homoeostatic model assessment of insulin resistance (homa-ir), alkaline phosphatase, pulse pressure, body mass index, and waist circumference. the relationship of ev concentration and cargo with mortality markers differs by race. summary/conclusion: our data show that ev-associated proteins can differ by race and sex and are associated with mortality risk factors. this study provides insight into the characterization of evs in middle-aged aas and whites, which may aid in the development of ev-based diagnostics. funding: this study was supported by the national institute on ageing intramural research program of the national institutes of health. repurposing specialised cell-free dna blood collection tubes for extracellular vesicle isolation introduction: liquid biopsies offer a minimally invasive approach to patient disease diagnosis and monitoring. however, many plasma processing protocols have been designed with a single biomarker in mind. here we investigate whether specialised dna blood stabiliser tubes could be repurposed for the analysis of extracellular vesicles (evs). methods: peripheral blood (n = ) was collected into k -edta, roche or streck cell-free dna (cfdna) blood collection tubes and processed using sequential centrifugation immediately or after storage for days. microev were collected from platelet poor plasma by , g centrifugation and nanoevs isolated using size exclusion chromatography. particle size and counts were assessed by nanoparticle tracking analysis, protein by bca assay and dot blotting for blood cell surface proteins. results: major variations in micro and nanoevs were seen with delayed time to processing. nanoev counts did not change with processing delay or tube collection type but the associated protein amount increased, indicative of cell lysis or activation. the protein was predominantly derived from from platelets (cd ) and red blood cells (cd a). the increase in associated protein was seen more in the k -edta and streck tubes indicating that the roche tubes may offer improved cell stability. conversely, microevs increased in both quantity and protein content with delay to processing indicative of both lysis and cell activation, irrespective of tube type. epithelial cell surface marker epcam abundance remained the same across conditions in both micro and nanoevs demonstrating that epcam+ evs were stable. summary/conclusion: specialised cfdna collection tubes can be repurposed for micro and nanoev analysis, however simple counting or using protein quantity as a surrogate of ev number may be confounded by pre-analytical processing. the evs would be suitable for disease selective ev subtype analysis if the molecular target of interest is not present in blood cells. introduction: nutrigenomics and nutrigenetics have been defined as the effect of nutrients on gene expression and genetic variation on dietary response, respectively. here, we propose the isolation and characterization of exosomes from donors carrying different alleles of hla-dqa and hla-dqb , to investigate their involvement in coeliac disease (cd) management. methods: a chilean population (n = ) was investigated for snps mutations in hla class ii alleles associated to cd predisposition (as well as other mutations related to other food intolerances), using the genochip food technology. exosomes have been isolated from donors' serum by ultracentrifugation and characterized by sds-page, western blotting (cd and cd ), and transmission electron microscopy. exosomes were also studied for their interleukins (il- and il- ra) content. results: among the studied population, % present at least one of the alleles leading to cd development and % carry alleles encoding for αand β-chains heterodimers associated with very high risk to develop cd. in parallel, isolated exosomes from donors with low to extremely high risk for cd showed high il- ra content ( . ± . to . ± . ), as the persons were not following any treatment. however, values of il- ra decrease in exosomes isolated form persons receiving treatment for cd. a relationship between exosomes' content and genetic susceptibility for cd has been observed, which may suggest their possible use as biomarkers for cd as the diagnostic of this disease is still a big issue. summary/conclusion: until this point of this underway project, we demonstrate the existence of a relationship between the exosomes' content in il- ra and genetic susceptibility for cd. furthermore, the genetic predisposition to cd could also modulate the gut colonization process, another important player in intestinal homoeostasis. in the next step, extracellular vesicles from gut microbiota will be isolated and analysed to determine their role in cd management. nasibeh karimi a , razieh dalir fardouei a , jan lötvall a and cecilia lässer b a krefting research centre, institute of medicine, sahlgrenska academy at university of gothenburg, göteborg, sweden; b krefting research centre, institute of medicine, sahlgrenska academy at university of gothenburg, gothenburg, sweden introduction: the ability to isolate extracellular vesicles (evs) from blood is vital in the development of evs as disease biomarkers. both serum and plasma can be used but few studies have compared them in terms of amount and type of evs. we have previously developed a method to isolate evs from plasma with minimal contamination of lipoprotein particles (karimi et al ) . the aim of this study was to compare the presence of different subpopulations of evs in plasma and serum. methods: blood was collected from healthy subjects, from which plasma and serum were isolated. evs were isolated using a combination of density cushion and size exclusion chromatography (sec) (protocol ) or a combination of density cushion and density gradient (protocol ) or immune-capturing (anti-cd , anti-cd and anti-cd beads) (protocol ). purity and yield of evs were determined by nanoparticle tracking analysis (nta), western blot, electron microscopy (em), exoview, flow cytometry and mass spectrometry (lc-ms/ms). results: as determined by nta and protein measurement more evs could be isolated from plasma with protocol and the majority of the vesicles were cd / cd a positive as determined with exoview and western blot. additionally, flow cytometry and western blot showed that more cd /cd a positive evs where also identified with protocol . furthermore, western blot showed increased amount of cd a in plasma samples in protocol . when labelled evs were spiked in freshly collected blood, no difference in recovery was seen for plasma and serum. summary/conclusion: this study shows that a larger amount of evs could be isolated from plasma compared to serum when three different isolation methods were used. firstly, this suggests that more evs are present in plasma. secondly, it suggests that these vesicles are probably released by platelets and that evs are not trapped in the clot during serum formation. future studies are needed to answer how this affects the use of blood-derived evs as biomarkers from serum and plasma. tumour-derived extracellular vesicles contain distinct integrin proteins stephanie n. hurwitz a and david g. meckes b a university of pennsylvania, philadelphia, usa; b florida state university, tallahassee, usa introduction: cargo profiling, including proteomic analyses, of tumour cell-derived extracellular vesicles (evs) may provide ripe opportunities for further understanding cancer growth, drug resistance, and metastatic behaviour. accumulating data suggest that cancer-derived evs contain membrane-bound integrin proteins which may aid in cell detachment, migration, and homing to future metastatic niches. we have previously published an extensive proteomic profile of secreted vesicles from the nci- panel of human cancer cells. methods: here, we further examine the distinct integrin components in these cancer-derived evs, and additionally profile evs released from benign epithelial cells by liquid chromatography and tandem mass spectrometry for comparison. results: we demonstrate the enrichment of integrin receptors in cancer evs compared to vesicles secreted from benign epithelial cells. total ev integrin levels, including the quantity of integrins α , αv, and β correlate with tumour stage across a variety of epithelial cancer cells. in particular, integrin α also largely reflects breast and ovarian progenitor cell expression, highlighting the utility of this integrin protein as a potential circulating biomarker of certain primary tumours. other integrins including α , αl, and β are enriched in vesicles derived from leukaemia cells, and may provide a means to distinguish haematopoietic cell-derived evs. summary/conclusion: this study provides preliminary evidence of the value of vesicle-associated integrin proteins in detecting the presence of cancer cells and prediction of tumour stage. differential expression and selective packaging of integrins into evs may contribute to further understanding the development and progression of tumour growth and metastasis across a variety of cancer types. effect of nicotine and menthol on cytochrome p and antioxidant enzymes in rat plasma-derived extracellular vesicles introduction: tobacco products such as e-cigarettes pose potential adverse health effects caused by direct exposure to aerosolized nicotine, flavorants such as menthol, and other particulates. here, we aimed to study the hypothesis that whether nicotine and menthol modulate nicotine-metabolizing cytochrome p a (cyp a ), antioxidant enzymes (aoes), sod and catalase in plasma extracellular vesicles (evs). modulation of these enzymes would eventually lead to nicotine-induced toxicity and hiv- pathogenesis via evs-based cell-cell interactions. methods: we isolated and characterized evs from rat plasma before and after nicotine self-administration (nic) with audiovisual cue (av) and menthol and characterized using ev markers according to the isev guidelines. protein associated with cyp a , sod , and catalase were quantified by western blot. results: we measured size, total protein, and acetylcholine esterase activity of evs and found no significance difference in these characteristics before and after nic. to investigate the effect av, menthol alone or in combination in the absence and presence of nic, first we evaluated the expression of ev markers cd and cd . the results showed menthol and av together increased the levels of cd (p ≤ . ), the marker of small vesicles, in the presence of nic. the nic with menthol and av showed a pattern of increased levels of small vesicle but could not reach to significance. next, we demonstrated that the nic with av increased the level of sod (p ≤ . ), which showed a pattern of increased levels of catalase and cypa , though statistically non-significant. the expression of nicotine receptor did not change under any conditions used. the results showed an increased level of cyp a (p ≤ . ), sod (p ≤ . ), and catalase (p ≤ . ) in plasma evs in the menthol-nic group compared to menthol group only. nic group with a combined av and menthol, showed further increase in the levels of cyp a (p ≤ . ), and catalase (p ≤ . ). further analysis of plasma evs on inflammatory cytokines/chemokines in these groups, and the effect of plasma evs on nicotine-induced toxicity and hiv pathogenesis are underway. summary/conclusion: nicotine administration increased, though not statistically significant, the levels of circulatory evs. moreover, the study provided evidence that nicotine in the presence of menthol, av, and/or menthol+av increased nicotine-metabolizing cyp a in all the groups and aoes in specific groups. funding: we thank the national institute on drug abuse (grant #da , da- ) for supporting our work. introduction: biomarker discovery in breast cancer (bc) is a clinical need for therapeutics and non-invasive diagnostics. tumour exosomes are involved in premetastatic niche formation and drug resistance and represent a source of non-invasive biomarkers. the identification of tumour exosomal biomarkers provides, not only, the possibility to discriminate patient groups also potential targets to control cancer progression that could be exploited to develop innovate bc therapeutic strategies. methods: we have performed a comparative differenti. al proteomic profile of four bc cell lines and their derived-exosomes, representative of the most relevant bc subtypes in clinic to search non-invasive biomarker candidates. then, we have carried on two bioinformatics approaches: ) protein association network analysis interaction (string) and ) pathway inference analysis (hipathia), to characterize the functional profiling for each bc subtype. results: we have found differentially-expressed proteins, in both cells and exosomes, that include indicators of invasion, metastasis, angiogenesis and drug resistance. exosome proteome profile reflects their different bc cell origin suggesting potential indicators of bc subtype. further, bioinformatics analysis reveals a differential role of exosomes in bc signalling pathways in recipient cells, according to their protein cargo and cell origin. summary/conclusion: our results show a set of cells and exosome proteins that highly discriminate bc subtypes and may significantly contribute to further studies for the design of bc biomarker predictor to stratify bc patients and the development of novel therapeutic strategies. funding: a set of potential biomarkers to discriminate breast cancer subtypes. circulatory evs as potential biomarkers of hiv-drug abuse interactions and neurological dysfunction in hiv-infected subjects and alcohol/ tobacco users sunitha kodidela a , kelli gerth a , namita sinha a , asit kumar b , prashant kumar a and santosh kumar a a uthsc, memphis, usa; b university of tennessee health science center, memphis, usa introduction: abuse of alcohol and tobacco can exacerbate hiv pathogenesis and its associated complications. further, the diagnosis of neurocognitive disorders associated with hiv infection and drug abuse using csf or neuroimaging are invasive or expensive methods, respectively. therefore, extracellular vesicles (evs) can serve as reliable non-invasive markers due to their bidirectional transport of cargo from the brain to the systemic circulation. hence, we aimed to study the specific evs proteins, which are altered in both hiv and drug abusers to identify a physiological marker to indicate the immune status and neuronal dysfunction of hiv-positive drug abusers. methods: evs were isolated from plasma of the following subjects: a) healthy b) hiv c) alcohol drinkers d) cigarette smokers e) hiv+alcohol drinkers f) hiv +cigarette smokers. quantitative proteomic profiling of evs was performed by mass spectrometry and potential ev proteins associated with neuronal dysfunction were quantified by westernblot. results: the evs were characterized according to the isev guidelines. a total of proteins were detected in evs of all the study groups. comparison of proteins among all the study groups revealed that hemopexin was significantly altered in hiv+drinkers compared to drinkers and hiv subjects. further, our study is the first to show properdin expression in plasma evs, which was decreased in hiv+smokers and hiv+drinkers compared to hiv patients. though we couldn't identify the few other cns-specific proteins, g-fap and l -cam, associated with neuronal dysfunction in plasma evs by mass spectrometry, we could detect those by westernblot. the protein expression of gfap (p < . ) was significantly enhanced in plasma evs obtained from hiv-positive subjects and drinkers compared to healthy subjects, suggesting enhanced activation of astrocytes in those subjects. the l cam expression was found to be significantly elevated in smokers (p < . ). both gfap and l cam levels were not further elevated in hiv+smokers compared to hiv+nonsubstance users. summary/conclusion: the present findings suggest that hemopexin, and properdin show potential as markers for hiv-drug abuse interactions. further, astrocytic and neuronal-specific markers (gfap and l cam) can be packaged in evs and circulate in plasma, which is further elevated in the presence of hiv infection, alcohol, and/or tobacco and thus may represent as potential biomarkers for neurological dysfunction in those subjects. funding: we thank the national institute on drug abuse (da ) for supporting our work. . electrochemical detection of mirna- - p introduction: micrornas (mirnas) are small, single-stranded, non-coding rna species that regulate gene expression post-transcriptionally, and are transported by extracellular vesicles (evs). they play an essential role in biological processes, such as development, cell proliferation, apoptosis, stress response and tumorigenesis. thus, mirnas are considered relevant biomarkers in health. more particularly, mirna- - p is expressed in neurons after traumatic brain injury, being expectably transported to peripheral fluids by brain evs that cross the blood-brain barrier. the main goal of this work is to develop an electrochemical biosensor for the detection of mirna- - p in serum. methods: overall, the experimental assembly of the biosensor was made in three stages. the first one consisted in the electrodeposition of aunps, the second one in the incubation of anti-mirna - p on the carbon screen-s printed electrodes and the final stage in the incubation of mercaptosuccinic acid for blocking unspecific bindings. the probe was hybridized with the target mirna - p by a consecutive incubation of several standard solutions. each modification was evaluated with cyclic voltammetry (cv), electrochemical impedance spectroscopy (eis) and square wave voltammetry (swv). the electrochemical behaviour of the biosensor was followed in all steps by monitoring the electron transfer features of a standard redox system. the redox probe selected for this purpose was [fe (cn) ] -/[fe(cn) ] -. results: the results indicated that the electrodeposition of gold was more effective for − . v for s and could lead to better signals upon anti-mirna- - p hybridization. summary/conclusion: in general, the experiments showed increasing charged transfer resistance upon the incubation of higher concentrations of mirna- - p. in these experiments, ev concentration is a critical variable that must be carefully controlled to ensure scientific rigour and reproducibility: without controlling for concentration (dose), experimental outcomes will exhibit excess variability that could mask important biological discoveries. in this study, three orthogonal methods are compared for accuracy in ev quantification: microfluidic resistive pulse sensing (mrps) and nanoparticle tracking analysis (nta) were compared to each other and relative to the gold standard method, transmission electron microscopy (tem). the ability of nta to accurately measure particle concentration is shown to depend on the polydispersity of the sample itself. results validate the accuracy of mrps and emphasize the importance of using orthogonal techniques to quantify evs. methods: reference urinary vesicles were prepared and analysed with the three methods and the relative concentration accuracy of nta and mrps were compared as a function of particle size. the hypothesis that nta concentration accuracy was impeded by sample polydispersity was tested using polystyrene bead mixtures having a range of polydispersity. a theoretical argument based on fundamental physics explains the experimental observations. results: tem and mrps measurements of the evs were in excellent agreement and showed a broad, polydisperse particle size distribution with no peak on the measured size range ( nm - nm diameter). nta differed significantly from tem and mrps by reporting a steep decrease in measured concentration below about nm that resulted in a peak in the reported particle size distribution. bead measurements confirmed the hypothesis to be tested: sample polydispersity significantly affects the ability of the nta method to accurately measure concentration, even for particles as large as nm diameter. summary/conclusion: these experiments validate mrps as an accurate method for quantifying evs and highlight the importance of using orthogonal measurement methods in accordance with misev guidelines. clinically relevant synthetic reference materials to standardize concentration measurements of extracellular vesicles: state-of-the-art and future prospects introduction: there is an unmet need to standardize concentration measurements of extracellular vesicles (evs). flow cytometry is the clinically most applicable method, but the currently available reference materials for calibration are insufficient. for example, the refractive index (ri) between standard particles and evs substantially differs, whereas concentration and fluorescence calibration particles are too bright. the goal of this study is to ascertain the most desired properties of reference materials to standardise ev measurements. methods: an online survey was prepared within the meves ii project to measure the desired size, concentration range, optical properties, choice of fluorochromes, and stability of synthetic ev reference materials for flow cytometry (fcm) measurements. besides the desired properties of ev reference particles, also the available instrumentation was assessed in the survey, which was sent to the members of the stakeholder committee of metves ii project and members of the ev flow cytometry working group. results: the most desired size, concentration, and ri range for ev reference particles is nm to nm, e to e /ml, and . - . , respectively. based on mie-theory evaluation of the sensitivity of the available instruments, none of the respondents would be able to detect nm particles with ri = . with their current instruments. regarding fluorescence intensity, the most desired range according to the responses is from molecules of equivalent soluble fluorochromes (mesf) to mesf. considering the sizes of evs and fluorescent labels, the maximal mesf that can be obtained for ev reference particles with nm diameter and high molecular mass fluorescent dyes is in the range of several hundreds. typical antigen densities on evs fall below copies per ev with nm diameter, i.e. mesf values above are probably not physiologically relevant in this size range. summary/conclusion: a part of the desired properties of ev reference materials precludes either their physical feasibility of production or their detection at most currently available fcms, meaning that the intended reference materials will be future-proofed. funding: this work was supported under hlt metves ii project by the european metrology programme for innovation and research (empir). the empir initiative is co-funded by the european union's horizon research and innovation programme and the empir participating states. comparison of production and activity of amniotic fluid stem cell extracellular vesicles from d hollow fibre bioreactor and d culture. culture conditions may affect ev composition and potency. here we compare production, potency, identity and therapeutic potential of evs collected from cells grown in culture dish ( d) versus hfbr ( d). methods: human clonal afsc were derived from patient-consented amniotic fluids. x e hafsc were seeded in d ( cm ), and . x e hafsc on a small kd mwco hfbr (fibercell-c d, cm ) with fibronectin coating; both cultured in chang medium with % of es-fbs, starved for hr and then evs collected. the effect of harvest frequency was tested ( hrs, hr, hrs, wk). d-evs and d-evs were compared by nanosight, potency assay (by wb), identity (by exoview analysis) and therapeutic effect (in vivo in an animal model of kidney disease, alport syndrome). results: d production was~ . x e ev/ml/ hrs while d was~ . x e ev/ml (first four hrs) and . x e ev/ml (two days of hourly harvests). very little difference in ev concentration and very similar size distribution (~ nm) were observed during harvest intervals; possibly indicating either significant ev re-uptake or inhibition of ev secretion dependent upon free ev in the supernatant. d-evs trapped vegf (an in vitro established potency assay) as efficiently as d-evs, and expressed cd , cd , cd , cd , cd and vegfr as d-evs. summary/conclusion: d-evs had comparable properties and bio-activity to d-evs, but the hfbr produced x more evs. hfbr cell culture conditions for hafsc still need optimization, however an available . m cartridge provides a x scale up potential. the hfbr, a cgmp closed system, can produce sufficient numbers of ev to support pre-clinical and clinical applications with at least similar properties to evs produced by conventional d methods. funding: -intramural funding -intramural ev core pilot funding demonstration of high gain mode in combination with imaging flow cytometry for improved ev analysis luminex corporation, seattle, usa introduction: extracellular vesicles (evs) are membrane-derived structures that include exosomes, microvesicles, and apoptotic bodies. in recent years, the importance of evs has become apparent, as they are key mediators of intercellular communication. however, quantifying and characterizing evs in a reproducible and reliable manner is challenging due to their small sizeexosomes range from to nm in diameter. it is well-known that flow cytometers were originally designed to measure and detect cells, and due to the quantitative power flow cytometry offers, there has been a push to quantify and characterize evs using flow cytometric methods. however, these systems have not been designed to measure objects smaller than a cell. methods: here, we describe the use of high gain mode on the amnis® imagestream® imaging flow cytometer to address the challenges of measuring small particles. in this new high gain mode, the charge-coupled device (ccd)-camera is manually adjusted to higher gain settings, increasing the signal obtained from the ev. object thresholds and masking have also been adjusted to better identify and detect small particles. results: preliminary results using murine leukaemia virus-sfgfp reference particles have shown up to a fivefold increase in the number of gfp-positive objects collected in high gain mode, when compared to standard gain on the imagestream system. summary/conclusion: in this study, we demonstrate improved small particle detection, including evs, using this new high gain mode on the imagestream imaging flow cytometer. distance-controlled accelerated catalysed hairpin dna circuit for multiple and sensitive detection of exosomes-associated mirnas introduction: sensitive and simultaneous monitoring of multiplexed exosome-associated rnas is of great value for early cancer diagnosis remains a challenge. methods: here, we report a simple, multiple and sensitive exosomes-associated multiplex mirnas detection method that uses distance-controlled accelerated catalysed hairpin dna circuit (chdc) system without any complex operation or enzymatic amplification. the distance-controlled accelerated chdc can directly enter the plasma exosomes to generate fluorescent signal quantitatively by specifically targeting mirnas without any transfection means. results: we show that distance-controlled accelerated chdc strategy with signal amplification capability could selectively and sensitively identify low level rnas in serum evs, distinguishing patients with early-and late-stage breast cancer from healthy donors and patients with benign breast disease. summary/conclusion: this simple, accurate, sensitive, and cost-effective liquid biopsy by the distance-controlled accelerated chdc method is potent to be developed as a non-invasive breast cancer diagnostic assay for clinical applications. impact of isolation methods on biophysical heterogeneity of single extracellular vesicles university of california los angeles, ca, los angeles, usa introduction: current biophysical analysis of extracellular vesicles (evs) typically encompasses particle density and size distribution determinations using various techniques. however, variabilities in ev isolation methods and the structural complexity of these biological-nanoparticles (sub- nm) necessitate more rigorous nanoscale biophysical characterization of single evs to facilitate more reliable and comparable evbased assays. methods: combining atomic force microscopy (afm), super-resolution optical and conventional particle sizing light scatter and microfluidic techniques, we compared the unique sub-nanometre scale biophysical properties of breast cancer cell-derived ev isolates obtained using different isolation methods. results: afm and dstorm particle size distributions showed coherent unimodal and bimodal particle size populations in centrifugation and immune-affinity isolates respectively. more importantly, afm imaging revealed striking differences in nanoscale morphology, surface undulations, and vesicle-to-non-vesicle ratios among ev isolates from different isolation methods. our findings demonstrate the effectiveness of orthogonal high-resolution biophysical characteristics of single evs, not discernable via particle size distributions and counts alone. summary/conclusion: the identified nanoscale biophysical characteristics of ev isolates represent a strategic and complementary framework to resolve differences in the heterogeneity and purity of evs from introduction: extracellular vesicle (ev) concentrations measured by flow cytometry are incomparable. to improve comparability, the metves ii consortium is developing traceable reference materials and procedures, which require validation by test samples. in previous interlaboratory comparison studies, however, a main source of variation was introduced by pre-analytical variables and measurement artefacts introduced by test samples. to minimize variation introduced by test samples, our aim is to develop off-the-shelf biological test samples containing pre-labelled evs. methods: human urine and plasma were collected from healthy donors. evs were labelled with lactadherin-fitc, isolated by size-exclusion chromatography to remove free dye and minimize swarm detection, and mixed with dimethyl sulphoxide (dmso), exocap or trehalose, frozen in liquid nitrogen and stored at − °c . after thawing, ev concentrations were measured by a calibrated flow cytometer (apogee a -micro). results: compared to the ev concentrations measured in fresh plasma and urine, the concentrations decreased % in plasma (p = . ; mean of the cryopreservation agents) and % in urine (p = . ) after one day of storage. after months of cryopreservation, the concentration of plasma evs decreased % (dmso and exocap) and . % (trehalose) compared to one day of storage, whereas the concentration of urine evs decreased % (exocap) and % (dmso and trehalose). summary/conclusion: we have developed ready-touse, pre-labelled human evs that are stable up to months and dedicated for use in interlaboratory comparison studies. to further increase stability, other cryopreservation agents will be tested. our biological test samples will be key to validate the new reference materials and procedures developed by metves ii in . funding: this project has received funding from the empir program co-financed by the participating states and from the european union's horizon research and innovation program. understanding intracellular fate of ev-delivered content introduction: despite much work performed on evaluating the potential effects of extracellular vesicles (evs), the functional uptake of their cargo is still controversial. this project aimed to demonstrate that ev content (protein and mrna) is protected and can be subsequently transferred with functional activity into recipient cells, while also developing a tool to assess and quantify functional ev uptake. methods: fusion proteins used were mitochondrial localized coxviii-cfp-nanoluc(cox) and nuclear localized h b-rfp-nanoluc(h b). results: hek t cell-derived evs protected cox proteins from proteinase k digestion while demonstrating significantly improved efficiency of uptake when compared to free protein, as measured by bioluminescence that was still detectable in recipient cells hrs post-ev-exposure. to confirm functional uptake, recipient cells exposed to evs containing h b for hrs were imaged and some recipient cells manifested fluorescent red nuclei. to demonstrate the presence of functional mrna within evs, producer cells were transfected for such a duration as not to have detectable levels of protein in the evs while still containing detectable levels of mrna (qpcr) even after rnasea treatment. transfer of these evs to hela cells showed an increase in expression of h b which was blocked by cyclohexamide, confirming translation of the mrna ( . kb). to determine if recycling of ev delivered proteins occurs, recipient hela cells were exposed to evs containing cox for hrs. all extracellular evs were removed and cells were trypsinized ( . % for min) to remove any non-internalized cox protein. hrs later, evs (cd + and cd +) released from cells contained cox suggesting recycling of protein or possibly recycling of entire evs. lastly, an assay was developed to measure functional ev uptake. nanoluc protein was split in two and fused to mturquoise (n ) or mscarlet-i( c). expression of each fragment alone exhibited non-detectable levels of luminescence while expressing both together had a significantly increased signal. delivery of either fragment within an ev to a cell expressing the corresponding fragment worked as confirmation and quantification of ev uptake (hek , u , hela cells). summary/conclusion: this study robustly demonstrates ev delivery of functional mrna and protein to cells, while also establishing a simple assay to quantify and validate functional ev uptake. theoretical model of ev losses due to adsorption on the tube walls. application for immunomagnetic detection of the vesicles introduction: short-term storage of unfrozen samples of vesicles, mainly at °c, overnight or during a couple of days is rather common laboratory practice. however, it was found to lead to significant losses of vesicle concentration supposedly due to adsorption on the walls of the tube. the present work develops a theoretical model intended to describe the vesicle adsorption process. the experimental validation of the model was made using method of immunomagnetic precipitation. methods: the theoretical model considers the "diffusion-limited" case of vesicles storage. the maximal adsorption capacity of the surface of contact between the tube and the solution is given as the number of vesicles in hexagonally packed monolayer. for experiment, the vesicles were purified from ht cell culture supernatant by differential centrifugation, aliquoted and kept at − c. further the aliquots were consequently unfrozen, and placed into the tubes with different surface treatment and kept at + c. the kinetics of vesicles loss was measured by anti cd immunomagnetic capturing followed by cd , epcam and cd staining and flow cytometry. results: the model allows the estimation of the adsorption-associated losses as dependent on initial vesicles concentration, volume of the solution, tube geometry, the storage temperature and duration case of quiet vesicles storage (without mixing) and also accounts an expected effect of active agitation of the solution (ev-beads complexes formation). theoretical calculations were illustrated by analysis of ev at different storage conditions and during reaction of immunomagnetic precipitation of the vesicles. summary/conclusion: it was demonstrated that application of tubes surface treatment allows increasing sensitivity of immunomagnetic precipitation method to x ^ for cd +, x ^ for epcam+ and x ^ for cd + vesicles. introduction: it is now largely accepted that the intestinal microbiota plays a key role in intestinal bowel diseases (ibd). an imbalance in the composition and diversity of the intestinal microbiota (i.e. dysbiosis) of patients has been repeatedly pointed out by several teams. there are also indications that extracellular vesicles produced by bacteria and exosomes produced by epithelial cells might be increased in this family of diseases. methods: in order to differentiate healthy and ibd faecal samples on the basis of their vesicle profiles, we want to develop a means to enumerate rapidly particles in faecal samples, based on interferometric microscopy. the videodrop technology, developed by myriade, relies on the creation of single beam interferences between two signals from the same light path by nanoparticles such as small vesicles. it will permit to compare on large scales the viral load of healthy subjects and ibd patients. results: this fast and easy-to-use device was compared to the nta on several types of eukaryotic and prokaryotic vesicles and our preliminary results are encouraging. introduction: small extracellular vesicles (sevs) produced by mesenchymal stromal cells (msc-sevs) may be useful in cell-free therapies for immunomodulation and tissue regeneration. methods: to characterize msc-sevs produced ex vivo, human bone marrow mscs were cultured in mesencult-acf plus (macfp), an ev-free and animal component-free culture medium for days and spent medium collected to isolate sevs by ultracentrifugation (uc). analyses of sevs were performed by nanoparticle-tracking analysis (nta), western blot (wb), and human umbilical vein endothelial cell (huvec) tube formation assay. results: analysis of fresh uncultured macfp by uc, nta and wb for cd , cd , and cd confirmed the absence of sevs. msc-sevs isolated from spent macfp by uc ranged from - nm in size and were positive for cd , cd , and cd proteins. these sevs could be stored at − °c for > months in solution or lyophilized with minimal loss based on nta and wb analysis. the msc-sevs contained the msc-associated micrornas let a, mir , and mir a as per qpcr analysis. the biological function of ex vivo isolated msc-sevs was assessed using a human umbilical vein endothelial cell (huvec) tube formation assay. huvecs treated with msc-sevs generated tubes as early as h after seeding, which were not observed in control huvec cultures until h. moreover, the number of branch points present in such tube structures was >fourfold higher in huvec cultures (n = ) supplemented with msc-sevs versus control, with the former lasting > h and the latter lasting < h in culture. direct comparison of the performance of macfp medium to media containing non-depleted or ev-depleted foetal bovine serum demonstrated that only mscs cultured in macfp (n = ) were able to expand robustly with a doubling time of . , . and . days in these media, respectively. lastly, methods for isolating sevs using newly developed easysep-ev™ magnetic separation kits and size exclusion columns will be presented. summary/conclusion: taken together, these data demonstrate that msc-sevs can be produced in high yield in macfp medium and that these possess similar physical, phenotypic and functional characteristics as sevs in vivo. funding: this work was privately funded by stemcell technologies inc. introduction: extracellular vesicles (evs) are heterogeneous group of small vesicular structures released by different types of cells, including stem cells (scs). as recent studies demonstrate that they may enclose bioactive content and transfer it into the target cells, growing interest is placed on the utilization of evs in the field of biomedical research. however, there is still lack of standardized methods of evs characterization. as an example, typical flow cytometry-based protocols, commonly used for cells phenotyping, may be inadequate for the characterization of evs as particles with size close to the detection limit of conventional cytometers. thus, the aim of this study was to optimize and compare the use of different flow cytometry platforms for the multiparameter analysis of evs isolated from different types of scs populations. methods: ev samples were obtained by ultracentrifugation of conditioned media collected from selected scs types, including human induced pluripotent scs (ips) and mesenchymal scs (mscs). next, several high resolution flow cytometry systems: cytoflex, apogee (a and a micro-plus) and image stream mk ii were employed to compare their sensitivity and resolution, as well as influence of "swarm" effect. furthermore, we examined evs phenotype, including expression of tetraspanins and other surface markers. results: our results have revealed that tested flow cytometry systems may be utilized for the phenotypic characterization of evs secreted by scs populations. however, the conventional staining and gating strategy protocols have to be thoroughly optimized. additionally, depending on a type of tested cytometer, we have demonstrated the difference in a "swarm" effect and its influence on obtained results regarding evs phenotype. finally, imaging flow cytometry platform was also employed to visualize evs on the single particle level. summary/conclusion: in conclusion, we have demonstrated that tested high-resolution flow cytometry platforms are convenient methods for the multiparameter characterization of evs produced by different types of scs populations. however, careful selection of particular measurement parameters should be performed depending on a type of employed system. funding: this study was funded by ncbr grant strategmed iii (strategmed / / / ncbr/ ) to ezs. evaluation of atcc's exosomes from cell culture supernatant as reference standards in research and development. introduction: exosomes are subcellular particles - nm in size released from cells through a fusion of multicellular bodies with the plasma membrane. exosomes are stable carriers of cell-free cargo in the form of dna, rna, and proteins, thereby making them an attractive candidate for diagnostic and therapeutic applications. however, isolating a consistent population of exosomes can be challenging and there is an unmet need for highly characterized exosomes for use as reference standards in extracellular vesicle research (ev). methods: exosomes were isolated from cell culture supernatants of different atcc cell lines including stem cells and cancer cell lines representing the most prevalent cancer types -prostate, colorectal, breast, lung, cervical and glioblastoma, using tangential flow filtration (tff). these exosomes underwent sterility and mycoplasma tests as a part of their quality control. the morphology and size distribution of these exosomes were evaluated through multiple strategies including nanoparticle tracking analysis (nta), asymmetrical flow field-flow fractionation (af ), cryo-electron microscopy (cryo-em) and spectra dynetm particle analyser. exosome surface markers were also analysed through multiple strategies such as electro chemiluminescent elisa, flow cytometry and western blotting. also, stem cell exosomes and cancer exosomes were further evaluated for functionality through in vitro functional assays including migration assay, angiogenesis and anchorage independent growth assay. results: our optimized tff method resulted in high yields of > × exosomes/ml and average protein equivalent of more than mg/ml. more than % of the exosomes population had an average size distribution of - nm and median size of nm confirmed through a number of different size distribution instruments. although cell line dependent, we were able to obtain similar expression levels of different cell surface markers including tetraspanins (cd , cd , cd ) when evaluated through different methods. our functional data demonstrated stem cell exosomes were functionally active in promoting cell migration and tubule formation. additionally, cancer cell exosomes were found to promote a malignant phenotype in an anchorage independent growth assay. summary/conclusion: collectively, we demonstrated our ability to reproducibly manufacture production-scale batches of exosomes from multiple different cell types. our purified exosomes are of high yield, meet well-established quality control specifications, and are robust in maintaining size distribution, surface marker expression, and functionality in vitro. therefore, they can serve as ideal reference materials that can support different ev-based research applications. exo-cise: extracellular vesicles enriched from plasma post-exercise promotes myogenesis and neurogenesis bianca paris a , yaomeng liu a , vicente pagalday-vergara a , julie davies b , priya samuel a , ayman abu seer a , johnny collett a , laura gathercole a , ken howells a , karl j. morten b , zhidao xia a , daniel anthony b , david r f. carter a , helen dawes a and ryan c. pink a a oxford brookes university, oxford, uk; b university of oxford, oxford, uk introduction: physical activity brings about a widespread physiological response and elicits the beneficial adaptation of several tissues and organs. furthermore, regular participation in physical activity reduces the risk of developing major non-communicable diseases such as cardiovascular disease, diabetes, cancer, osteoporosis, and dementia. two important processes known to occur following physical activity are myogenesis and neurogenesis; both of which involve the activation and proliferation of specialised tissue-resident stem cells. the molecular mechanisms regulating these processes following exercise are poorly understood to date. here, we investigated the contribution of extracellular vesicles, which are released into the circulation after exercise, to benefit adult myogenesis and neurogenesis. methods: small extracellular vesicles were enriched from the blood of healthy participants before and following maximum and moderate intensity exercise. differentiation and proliferation using a range of methods was measured following vesicle treatment onto primary myoblasts and neuronal primary exvivo stem cells. activation of key cellular pathways were measured. results: we show significant proliferation and differentiation changes of both stem cell types. this is independent of extraction method, extracellular vesicle depleted fractions and is interestingly conserved across mammalian species. remarkably, we see an age-related effect. summary/conclusion: this advocates that short single bouts of exercise may promote myogenesis and neurogenesis via systemic signalling of extracellular vesicles which opens an interesting field in endogenous ev therapies. show promise as a cell-based therapy for retinal degeneration. while clinical trials are ongoing, the potential of extracellular vesicles (evs) as biomarkers for monitoring eye health and disease is not well studied. this study characterized the ev surface profile and cargo of hipsc-rpe to offer a baseline assessment in normal and disease conditions. moreover, we evaluated the importance of pnpla , a gene involved in membrane integrity and when mutated causes retinal degeneration, in ev biogenesis and secretion. methods: evs were isolated from serum-free culture medium of hips-rpe and identified with nanoparticle tracking analysis, transmission electron microscopy, and immunoblot analysis of exosomal markers, including alix, tsg , and cd . surface marker detection and proteomic profiling were completed using an ev surface marker kit and mass spectrometry, respectively. small interfering rna targeting pnpla was used to knockdown the expression in hipsc-rpe and evs were characterized. results: nanoparticle tracking analysis confirmed the presence of both microvesicles (> nm) and exosomes (< nm) by size distribution and the concentration of evs ( x particles/ml) from rpe. tem displayed typical morphological characteristics of evs. the presence of known ev markers, alix, tsg , and cd was confirmed via immunoblot and flow cytometry. surveillance of ev surface markers revealed enrichment of epithelial markers (cd ) and stem cell markers (cd / ) that depict donor cell origin and functional proteins including integrin-binding (cd ) and tgf-beta receptors (cd ). in addition, proteomic analysis revealed regulators of inflammation and rpe function, including hemopexin, clusterin, complement factor i, and pigment epithelium-derived factor. furthermore, reduction in pnpla expression reduced vesicle secretion and vesicle size compared to non-targeting controls. introduction: vascular endothelial growth factor (vegf) is a potent angiogenic factor and was first described as an essential growth factor for vascular endothelial cells. vegf plays a role in normal physiological functions such as bone formation, haematopoiesis, wound healing, and development. mesenchymal stem cell (msc) was found to secretes potential growth factors such as vegf when cultured in vitro. however there are some beliefs that foetal bovine serum (fbs) which usually used as serum in cell culture content vegf. methods: msc seeded in in -well plate in with concentration of , cell/well. cells were incubated for hours and fasted for another hours using only dmem. cells were treated with complete medium consist of dmem and % fbs. culture medium were collected after , , and hours after treatment. cell were culture in ºc dan % co . vegf concentration was detected using elisa technique. results: vegf concentration was not found in fbs which do not contact with msc. an increasing of vegf concentration in time-dependent manner was shown when culture medium was used in msc cell culture in normoxic condition. the result of vegf concentration when culture , , and hours were . pg/ml, . pg/ml, and . pg/ml, respectively. the mechanism of msc release growth factor is still under investigated. however, the classic growth factors and cytokines serves paracrine control molecules which were important in regenerative medicine. vegf was found to be an important molecules in angiogenesis process and determine the fate of cells. summary/conclusion: msc secreted vegf and concentration increased in time-dependent manner. isolation and characterization of exosomes from canine stem cells introduction: unlike induced disease models using laboratory animals, naturally occurring disease models display pathophysiologic attributes that are more similar to human diseases. unfortunately these models are underutilized in translational regenerative medicine research. this is partly due to the slow development of species-specific experimental therapeutics to investigate comparative efficacy. thus, we set out to isolate and characterize exosomes from canine adipose-derived mesenchymal stem cells (cad-msc) to use as a comparative therapeutic in dogs. to accomplish this, we optimized an isolation and purification strategy and characterized their molecular properties. methods: exosomes were isolated by sequential centrifugation and subsequent ultrafiltration. the proteome was characterized by tandem mass tag (tmt) mass spectrometry and the mirna cargo was identified using a canine specific pcr array with subsequent target and enrichment analysis using targetscan and the panther platform, respectively. also, nanoparticle tracking analysis and transmission electron microscopy were used to determine exosome size and structure. to investigate bioactivity, we measured the ability of exosomes to inhibit collagen production in an in vitro model of fibrosis. results: exosomes were purified by ultrafiltration using a kda cut-off. proteomic analysis by tmt mass spectrometry identified unique proteins. % of the exocarta top were identified from this list. additionally, we identified the mirna cargo within exosomes and found highly expressed mirnas. enrichment analysis identified multiple pathways of probable regulation including angiogenesis (fold enrichment = . ; p < . ) and transforming growth factor-beta (tgfb) signalling (fold enrichment = . ; p < . ). exosome size was quantified to be . ± . nm with a modal average of nm. lastly, in the presence of exosomes, tgfb stimulated fibroblasts deposited . % less collagen than vehicle controls (p = . ). summary/conclusion: in summary, cad-mscs exosomes display structural and functional features comparable to stem cell derived exosomes from other species. use of these exosomes in naturally occurring disease canine models may provide superior predictive value for human clinical trials. funding: support provided by the ccah, school of veterinary medicine, uc davis. mesenchymal stem cells-derived exosomes promote in vitro the progression of triple negative breast cancer cells introduction: mesenchymal stem cells (mscs) are multipotent stromal cells and have been described as key regulators of different aspects of tumour physiology. in tumour pathogenesis, mscs can integrate the tumour microenvironment after recruitment and are able to interact with cancer cells to promote tumour modifications by affecting epithelial-tomesenchymal transition (emt). it was revealed that exosomes derived from mscs are critical players in the tumour niche. exosomes are a novel way of cellto-cell communication and play crucial roles in the majority of pathways that contribute and affect response to therapy, cell-adhesion molecules and the progression of tumour cells. because of the known importance of this communication we decided to investigate the implication of mscs with triple negative breast cancer (tnbc) cell lines as well as exosomal profiles between the experimental conditions. methods: the interactions of mscs with triple negative breast cancer cell lines (mda-mb- and hs t) was performed by coculturing mscs (or tnbc cell lines) with exosomes derived from tnbc cell lines (or mscs). physical characterization of isolated exosomes was performed followed by their molecular investigations. cell proliferation was detected by mtt assay and migration was analysed by wound healing assay using d cultures. moreover, we also used d culture to assess the exosomes uptake and to observe their capability of internalization into a d structure. the alterations in expression level of some transcripts (mrnas and mirnas) and protein profile were investigated by qrt-pcr, western blot and immunofluorescence staining. results: we found that mscs-derived exosomes are actively incorporated by triple negative breast cancer cell lines ( d culture). in coculture, in tnbc cells the expression level of mesenchymal markers and emt markers (e-cadherin, vimentin) at mrna and at protein levels, as well as mirna-derived exosomes targeting mesenchymal genes were significantly affected. using bioinformatics tools, we highlighted the important biological processes which were activated by promoting tumour modifications. in addition, using d culture we provided a comprehensive understanding regarding exosomes internalization in d structures, which closely mimics in vivo conditions, compared to d culture. summary/conclusion: in this work, we focus on the investigation of mscs-derived exosomes in order to highlight their implication in several biological processes, including tumour proliferation and progression of triple negative breast cancer cells. all these alterations affect the response to therapy and should be considered for developing efficient therapeutic strategies. natural killer cell-derived extracellular vesicles have a potent anti-leukaemic effect and selectively target the cancer stem cell subpopulation introduction: natural killer (nk) cells of the immune system recognize and kill tumour cells. extracellular vesicles (evs) secreted from nk cells are capable of killing tumour cells independent of the cell to cell contact required for nk cell activation. cancer is a leading cause of death, primarily due to metastasis and recurrence. cancer stem cells (csc) within tumours are resistant to chemotherapy and immune attack, and cause metastasis and relapse. identification of the cancer types killed by nk evs is limited, and the effect of nk evs on cscs has not been described. here we determine whether nk-derived evs kill a myeloid leukaemia cell line and its csc subpopulation. methods: nk evs were isolated from our nk cell line, nk . , derived from normal human lymphocytes. nk . evs were characterized by immunoblotting, proteomics, and next generation rna sequencing. human k leukaemia cells were treated with nk . evs in vitro and analysed for proliferation and markers of cell death. results: nk . evs contain ev-associated proteins alix, cd , hsp , and tsg , nk effector molecules perforin, granzymes a and b, granulysin and nklam/ rnf b, an e ubiquitin ligase required for maximal nk cytotoxicity, and tumour suppressor mir- . nk . ev treatment of k significantly decreased its expression of proliferation markers cd and ki , and increased the frequency of apoptotic and necrotic cells, paralleled by elevated levels of active caspases − and − . non-tumorigenic cells were unaffected by nk ev treatment. most notably, nk . ev treatment significantly reduced the frequency of k cells highly expressing aldh, a csc marker. summary/conclusion: nk . -derived evs have a robust anti-tumour effect on k myeloid leukaemia cells and selectively target the csc population, suggesting they may circumvent the evasion and resistance mechanisms used by cscs. nk . evs therefore have introduction: due to their potential as a key bioactive agent in regenerative medicine applications, mscderived extracellular vesicles (msc-evs) are increasingly being investigated as a clinical therapy. manufacturing that generates enough evs for product development and clinical doses is currently a limitation in the field and clearly a scalable manufacturing solution will be necessary for successful translation. moreover, a complementary approach that increases the ev productivity, i.e. the number of evs produced per cell, could further help to accelerate the development of msc-evs as a therapy. methods: we developed a process that leverages a series of new cell culture reagents to couple to our established cell-media system for scalable manufacturing of msc-evs. briefly, human bone marrow-or umbilical cord-derived mscs were rapidly expanded under xeno-free conditions (i.e. > x expansion within days). cultures were then switched to our proprietary ev collection medium and evs were harvested for up to three additional days. at the end of culture, the evs in the conditioned media were concentrated using a tangential flow filtration (tff) system. to increase the productivity of mscs, two medium supplements were developed that increased ev yield by either increasing the number of evs generated per cell in a shortened culture process or increasing the number of collected evs by lengthening the ev collection culture period. results: this scalable msc-ev manufacturing method was implemented in both d flask and d bioreactor culture and generated over , particles per cell in d and over , particles per cell in d. with the addition of a medium supplement to increase evs produced per cell, the ev productivity was increased > x after hrs. alternatively, ev productivity was also increased > x by addition of the medium supplement that extended ev collection culture period. summary/conclusion: msc-ev success in clinical translation will be reliant on a manufacturing method that can scalably and reliably generate large amounts of evs. these results present one such solution. furthermore, increasing ev productivity, for instance by medium supplements that increase evs per cell or lengthen culture times could further address the limitation of generating the evs required for development and translation of clinical therapies. simplifying scalable msc ev production in a microcarrier-based bioreactor system divya patel, josephine lembong, katrina adlerz, jon rowley and taby ahsan roosterbio inc, frederick, usa introduction: the growpt ing numbers of msc-ev clinical applications drives the need for a scalable msc-ev production platform. while most msc-evs are generated while cells are attached to tissue culture plastic, such d cultures cannot be scaled up to meet the yields necessary for commercialization of ev-based therapeutics. we have shown that d bioreactors can be used to generate msc-evs and that paradigm can be scaled directly in terms of yield from the to l scales. the technical expertise of seeding cells onto microcarriers for expansion in bioreactors, however, requires technical expertise not available to all those in the ev field. therefore, our goal here is to simplify and expedite the ev collection process in bioreactors by cryopreserving cells on microcarriers, such that end users can merely thaw and then collect msc-evs. methods: mscs were expanded in d and then seeded on three different microcarriers and cultured in a bioreactor for days. when confluent, cells on microcarriers were cryopreserved. to evaluate the microcarriers and the cryopreservation protocol, the cells-microcarriers were thawed, cultured in a bioreactor in growth media for hours, then in ev collection media for additional days. cell recovery and ev production upon thaw was evaluated and compared to ev collection from fresh, non-cryopreserved cells. results: total cell counts hrs post thaw were comparable to those before cryopreservation and to fresh samples prior to ev collection. following -day ev collection, concentration of particles collected from cryopreserved cells on microcarriers were similar to those collected from the fresh cells ( e particles/ml). this process was validated for two different microcarriers using two separate cryopreservation solutions. summary/conclusion: our results show that cryopreserved hmscs on microcarriers can support ev collection in a d bioreactor process with a particle yield that is comparable to those collected from fresh cells. this cryopreserved product can simplify ev production, reducing cost and time by removing process steps associated with the hmsc expansion, with in a paradigm suitable for scale-up. the whitening, anti-wrinkle, and wound-healing effects of extracellular vesicles from orbicularis oculi muscle-derived stem cells. introduction: skeletal muscle-derived stem cells possess potent therapeutic activities in the treatment of muscle-related disorders. in our study, we tried to isolate and characterize orbicularis oculi muscle (orm)-derived stem cells (orm-scs) from the discarded human tissues which were obtained from the ocular surgery-subjected patients. we also prepared the natural extracellular vesicles (evs) from the cultured orm-scs and assessed the their therapeutic actitities including the skin whitening, anti-wrinkle, and wound healing effects. methods: we isolated the orm-scs from the patients subjected to ocular surgery and characterized the orm-scs by analysing cell morphology, proliferation, expression levels of the cell surface and stemness-associated markers, and tri-lineage differentiation and colony-forming capacities, confirming the stemness properties of the orm-scs. then, we prepared the natural evs from the orm-scs via the centrifugation and filtration of the media supernatants and their therapeutic activity was investigated. results: the isolated orm-scs showed spindle-like morphology and positive expression of cd , cd , and cd , but they were negative in expression of cd and cd . the orm-scs showed the capacity of osteogenic, adipogenic, and chondrogenic differentiations. the evs from orm-scs (orm-sc-evs) possessed the apparent inhibitory effect on the melanin synthesis in b f cells by blocking the tyrosinase activity, although orm-sc-evs treatment did not dramatically change the expression level of melanogenesisrelated genes, such as microphthalmia-associated transcription factors (mitf), tyrosinase (tyr), tyrosinaserelated protein (tyrp- ), and tyrp- . in addition, we confirmed that orm-sc-evs could stimulate skin cell migration and increase the expression level of antiwrinkle related genes and wound-healing properties. summary/conclusion: this study revealed the stem cell property of orm-scs and the whitening, antiwrinkle, and wound healing effects of orm-sc-evs, suggesting that orm-scs and orm-sc-evs can be successfully used for stem cell-based ev therapy and cosmetics, by regulation the melanogenesis, wrinkle, and wound. funding: this work was supported by grants from the national research foundation (nrf) funded by the korean government ( m a h ). use of stem cell extracellular vesicles as a holistic approach towards cns repair introduction: neurological diseases and disorders are leading causes of death and disability worldwide. many of these pathologies are associated with high levels of neuroinflammation and irreparable tissue damage. we have previously shown that extracellular vesicles (evs) from infected cells contain viral by products (noncoding rnas and proteins) and that these evs can exert deleterious effects on recipient cells - . therefore, in the context of neurotrophic viruses evs may contribute to or perpetuate processes relating to neuroinflammation and neurodegeneration. due to their multipotent properties, stem cells have broad applications for tissue repair; additionally, stem cells have been shown to possess both immunomodulatory and neuroprotective properties. in recent years it has been well-established that stem cell evs play a critical role in the functionality associated with stem cells. the diverse biological cargo contained within these vesicles are proposed to mediate their effects and, to date, the reparative and regenerative effects of stem cell evs have been demonstrated in a wide range of cell types. while a high potential for their therapeutic use exists, there is a gap of knowledge surrounding their characterization, mechanisms of action, and how they may regulate cells of the central nervous system (cns). methods: we have isolated and recovered high yields of evs from large scale cultures of both induced pluripotent stem cells (ipscs) and mesenchymal stem cells (mscs) using tangential flow filtration. our ev characterization includes both phenotypic (size, tetraspanin expression) and biochemical assays. ev functionality has also been assessed in vitro utilizing several cellbased assays related to cellular viability, migration, angiogenesis, and immunomodulation in both healthy and damaged recipient cells with relevance to the cns. results: our data suggests that evs from different sources of stem cells display unique phenotypes, exhibit differential association with various cytokines, proteins, and long non-coding rnas, and have the ability to significantly enhance processes that are critical for cellular repair . lastly, utilizing an ipsc-derived neurosphere model, we have observed a robust uptake of stem cell evs and have found that these evs are able to effectively penetrate these d structures. summary/conclusion: collectively, these results highlight the "holistic" properties of stem cell evs by demonstrating their ability to partially reverse or reduce damage in various cell types. funding: this work was supported by national institutes of health (nih) grants ai , ai , ai - , ai , and ns to fk and r ca and r ar to lal. the effect of cell culture media on extracellular vesicle secretion from mesenchymal stem cells and human pluripotent stem cell-derived neurons introduction: cell culture media and its supplements are known to affect the secretion and isolation of extracellular vesicles (evs) from cell cultures. identification of these effects is crucial especially when planning to use evs as therapeutic agents. here, we investigated the effect of cell culture media on ev yield from human mesenchymal stem cells (mscs) and human pluripotent stem cell (hpsc)derived neurons. methods: evs were collected from cell-conditioned media (ccm) and no cell control (ncc) media using size-exclusion chromatography (sec). mscs were cultured in dmem/f :neurobasal medium or in opti-mem reduced serum medium, both supplemented with exosome-depleted foetal bovine serum (fbs). the ev yield from hpsc-derived neurons was compared at two maturation time points (day and ), in dmem/f :neurobasal or in opti-mem, with and without -hour kcl stimulation. sec fractions were analysed by nanoparticle tracking analysis (nta), protein concentration assay and blinded transmission electron microscopy (tem). results: ccm samples had a clear peak of evs in sec fractions - , which was not detected with ncc. interestingly, a second population of evs eluted in sec fractions - in both ccm and ncc, indicating presence of evs in exosome-depleted fbs. moreover, this second population differed largely between used media batches. culture medium had no significant effect on msc ev yield (dmem: . e+ particles/ ml, opti-mem: . e+ particles/ml). with neuronal cultures, no significant differences in ev yield were found between culture media or cell maturation time points. in contrast to earlier findings, -hour stimulation of neurons by kcl resulted in significantly smaller ev yield compared to non-stimulated controls (stimulated: . e+ particles/ml, non-stimulated: . e+ particles/ml, p < . ). summary/conclusion: our results indicate that exosome depleted-media are not entirely devoid of vesicles, which can cause bias in downstream analyses. however, sec is a good method to separate cellsecreted evs from the contaminating medium-derived evs. culture medium did not affect the number of evs secreted by mscs or neurons; instead, we observed larger differences between media batches. this data emphasizes the importance of analysing the ncc as negative control in all cell culture experiments. mouse mesoangioblast stem cell extracellular vesicles are able to influence macrophage cell activity maria magdalena barreca a and fabiana geraci b a dept stebicef university of palermo, palermo, italy, palermo, italy; b dept stebicef, university of palermo, italy, palermo, italy introduction: it is largely demonstrated that stem cells release extracellular vesicles (evs) that are able to modify target cell behaviour. interestingly, there is a bidirectional signalling exchange between stem cell evs and damaged cells. moreover, it is well known that macrophages, could also play a role in wound repair and tissue regeneration. it was also demonstrated that stem cell evs are involved in immune cell regulation. for this reason, today takes hold the idea that evs could replace stem cells in regenerative medicine. the aim of our work was to evaluate if evs released by mouse mesoangioblast stem cells (a ) could have a role in immune cell regulation. specifically, we have investigated the possible a ev effect on murine macrophages (raw . ) in terms of cell proliferation, migration and phagocytic ability, and cytokines/chemokine release. methods: a evs were collected from conditioned milieu by ultracentrifugation. raw . cell proliferation with or without a evs was evaluated via cfse assay. scratch test was performed to assay their migration ability. to study raw . cell phagocytosis they were treated with μm beads. finally, cytokine array was used to monitor their secretion after ev treatment. results: we have found that a evs inhibited macrophage proliferation as proved by a proliferation index significantly reduced after ev treatment. simultaneously, we have noticed that evs increases raw . migration ability. furthermore, a evs are able to increase macrophage phagocytic activity. as it is known that hsp is involved in for macrophagic activity increase and a evs express hsp on their surface, we performed phagocytosis assays assay by blocking the protein or its receptor tlr , tlr and cd . our data demonstrated that a evs increase phagocytosis through hsp and its receptors. we have also proved that a evs modify the expression pattern of cytokines/chemokines released in the extracellular milieu by raw . cells. in particular, we observed an increase in anti inflammatory cytokines, and a decrease in some inflammatory ones, suggesting that evs could polarize macrophages towards an anti inflammatory m phenotype. summary/conclusion: in conclusions, our data show that a evs influence macrophage activity and additional studies could provide a new insight into understanding the underlying potential of evs in tissue regeneration. and ) . in a healthy kidney, the polycystins localize to renal cilia. mutations that abrogate ciliary localization of pkd (yet preserve its channel function) also cause cysts. besides cilia, pkd is also found in other subcellular locations including extracellular vesicles (evs) of human urine. how dysfunction of pkd trafficking and localization leads to the kidney pathology remains unknown. pkd is evolutionarily conserved across all members of eumetazoa. in c. elegans, pkd- is exclusively expressed in ciliated male-specific neurons, where it is trafficked to cilia and evs. gfp-tagged pkd- -containing evs play a signalling role in inter-organismal communication between animals. conservation of polycystin- cellular localization between worm and human suggests that their network of molecular interactions may also be conserved. we propose that pkd- plays distinct roles in cilia versus ciliary evs. methods: to understand the role of evs in c. elegans inter-organismal signalling, we aim to identify the pkd- -associated ev proteome, transcriptome, and metabolome. we established a pipeline for fluorescent labelling and tracking specific ev cargoes in a living animal using super-resolution microscopy. we used fluorescence of the pkd- carrying evs to optimize biochemical procedures for their enrichment. results: our initial analysis revealed two populations of pkd- -carrying evs that differ in their densities: . - . versus . g/ml. we are currently characterizing these two distinct populations using transmission electron microscopy and refining our enrichment procedure for protein identification by mass spectrometry, sequencing of their rna cargoes and metabolome analysis. summary/conclusion: what function human pkd plays within the cilia and within the urinary evs is not well understood. identification of molecular mediators of c. elegans pkd- ev signalling will inform on the interactome of human pkd and its function in cilia versus evs. introduction: ectosomes play roles in many physiological and pathophysiological processes, and their precise is dependent on molecular cargo and parent cell type. a single cell can release distinct subpopulations of evs enriched with different molecular cargo, which adds complexity to elucidating cargo sorting and biogenesis mechanisms. in the nematode c. elegans, ectosomes bud from sensory neuron cilia and are released into the environment to modulate animal behaviour. methods: c. elegans is genetically tractable and optically transparent, allowing for live imaging of fluorescently tagged ev cargo. we express all tagged cargo at endogenous levels, adding physiological relevancy. results: we discovered that the calcium homoeostasis modulator ion channel clhm- localizes to cilia of ev-releasing neurons and observed gfp-tagged clhm- in ciliary evs. using super resolution microscopy, we imaged evs released from animals coexpressing tdtomato-tagged clhm- and gfp-tagged pkd- (another vesicle cargo) in the same neurons. while the two proteins colocalize in the cilia, clhm- ::tdtomato and pkd- ::gfp rarely colocalize in evs. this indicates that separate subpopulations of evs are being released from the same neurons. to determine how the clhm- subpopulation is formed, we are investigating candidate genes. anoh- , a homolog of the ca + scramblase tmem f, localizes to neuron cilia and induces phosphatidylserine exposure on the outer membrane leaflet. in anoh- mutants, the number of clhm- ::gfp evs released is significantly decreased but the number of pkd- ::gfp evs does not significantly change. in addition, i am using facs to isolate clhm- and pkd- containing evs and analysing the respective proteomes with lc-ms/ms. summary/conclusion: we are elucidating mechanisms that give rise to distinct subpopulations of ciliary evs in c. elegans and defining cargoes being enriched in these ev subpopulations to gain insight into ev cargo sorting and biogenesis mechanisms in ciliated neurons. ceramide accumulation induces exosome secretion through lysosomal protein laptm b kohei yuyama, hui sun and yasuyuki igarashi hokkaido university, sapporo, japan introduction: exosomes, a type of extracellular vesicles originated from multivesicular bodies (mvb), are important carriers of cellular molecules and have critical roles in intracellular communication in both health and disease. ceramides (cer) are implicated in biogenesis of exosome, however the molecular machinery that mediates exosome secretion remains obscure. lysosome-associated protein transmembrane- b (laptm b) is a lysosome/late endosome-resident transmembrane protein, which has been reported to bind cer. we demonstrate here that laptm b is involved in the exosome secretion, which are induced by exogenous cer treatment or lysosomal ceramidase inhibition in cultured neuronal cells. methods: neuroblastoma sh-sy y cells were treated with cer (porcine brain-derived cer or synthetic d : /c : ~c : cer) for h. exosomes were isolated from the culture supernatants by sequential centrifugation and their amounts were measured using ps capture exosome elisa kit. to analyse mvb transport, mvb and recycling endosomes are visualized with gfp-cd and rab immunostaining, respectively. results: we found that exogenous treatment of cer, especially those with c and c fatty acids, resulted in a marked increase in exosome secretion. in addition, lysosomal cer accumulation induced by acid ceramidase inhibition also accelerated exosome production. knockdown of laptm b significantly prevented the ceramide-dependent exosome release. in addition, we showed that these cer loading promoted colocalization of cd -positive mvb with rab -positive recycling endosomes, further demonstrated that laptm b knockdown cancelled the cer-dependent increase of the colocalization. summary/conclusion: these data suggest that lysosomal cer binds to laptm b and promote the transport of mvb to plasma membrane, resulting in an increase of exosome secretion in neuronal cells. chloroquine-mediated lysosomal inhibition alters composition and function of cancer-derived extracellular vesicles jing xu a , kevin yang a , shane colborne a , elham hosseini-beheshti b , gregg morin a , emma guns b and sharon m. gorski a a bc cancer, vancouver, canada; b the vancouver prostate centre, vancouver, canada introduction: small extracellular vesicles (sev) are signalling entities released by many types of eukaryotic cells. sev are of special interest in cancer due to their reported roles in modulating the cancer microenvironment and facilitating cancer cell invasion. macroautophagy (hereafter autophagy) is a catabolic process well-known for the recycling of cytosolic cargos through lysosome-mediated degradation. in this study, we profiled the changes in sev content and function under lysosome inhibition and investigated the involvement of autophagy machinery in sev content. methods: chloroquine (cq) was used to inhibit lysosomal degradation and autophagy turnover in triplenegative breast cancer (tnbc) cell lines. sev were collected via precipitation after pre-clearing and concentration of conditioned media. western blotting, nanosight and transmission electron microscopy were used to profile sev. quantitative mass spectrometry was used to characterize cq-induced changes in the sev proteome. antibody-conjugated magnetic beads were used in immunoprecipitation of sev. results: cq treatment did not substantially alter the physical properties of tnbc-derived sev. however, cq treatment altered the sev proteome and growth effects of sev on normal and endothelial recipient cells. cq treatment induced co-localization of mammalian atg proteins with endolysosomal markers in the cytoplasm, which coincided with an enrichment of atg s and their adaptor proteins in sev. cq-induced enrichment of atg s in sev required lipidation, and occured preferentially in one subset of sev. summary/conclusion: our study reveals changes in the content and function of cancer cell-derived sev in response to perturbation of intracellular trafficking pathways, demonstrates the flexibility and heterogeneity of sev composition, and has implications for cq efficacy in therapeutic settings. introduction: introduction: argonaute (ago ) is the essential component of the rna-induced silencing complex (risc) that binds mirnas and promotes mrna degradation. extracellular vesicle (ev)-carried mirnas have been shown to influence gene expression and functional phenotypes in recipient cells. many investigators have found ago in evs and it is postulated that ago is a major transporter of mirnas into small evs (sevs), such as exosomes. others have reported extracellular ago that is non-vesicular. we set out to evaluate the effect of growth factor signalling and serum contamination on the detection of ago in sevs. methods: methods: wildtype kras colorectal cancer cells, dks , were conditioned with different culture media (serum-free dmem, dmem supplemented with ev-depleted fbs, and opti-mem). evs were purified from conditioned media by cushion-density gradient ultracentrifugation. western blot analysis of dks total cell lysates, large evs and density gradient fractions was performed, probing for ago and ev marker proteins. the size and concentration of the evs were determined by particle metrix analysis. results: results: in all conditions, we found the highest abundance of sevs in fractions and , as assessed by western blot analysis. ago was detected in the same fractions as sevs in both the serum-free dmem and opti-mem conditions, although the levels of ago was higher in the serum-free dmem fractions compared to that of opti-mem. in contrast, ago was present in both vesicular and non-vesicular fractions in the dmem supplemented with ev-depleted fbs condition. no significant differences were observed in the size and number of evs collected in the three conditioning methods. summary/conclusion: summary/conclusion: the presence or absence of ago in evs has been controversial. multiple factors may affect the ability to detect vesicular ago , including serum and growth factors in the conditioned media that may provide sources of extravesicular ago and also regulate the trafficking of ago into vesicles. introduction: cancer-associated glycosphingolipids have been utilized as tumour markers and targets of cancer therapy. we have investigated roles of gangliosides in cancers, and clarified that cancerassociated gangliosides enhance malignant properties of cells by forming complexes with membrane molecules in lipid rafts. in this study, we analysed contents of gangliosides and membrane molecules on extracellular vesicles (ecvs) secreted from melanoma cell lines. methods: melanoma cell lines with various ganglioside patterns were used for isolation of ecvs. gangliosidemodified melanomas with genetic engineering were also used. genetic modification was done by cdnas of ganglioside synthase genes. ecvs were collected by ultra-centrifugation, or by tim -beads. contents in ecvs were analysed by immunoblotting or flow cytometry. roles of lipid rafts in the generation and secretion of ecvs were analysed by treating cells with mm methyl β-cyclodextrin. results: using melanoma cell lines, ecvs were isolated by ultra-centrifugation, and their sizes were analysed by nanosight. all samples showed uniform sizes between and nm. protein amounts in ecvs were measured, showing heterogeneous levels at ~ μg/ ml. then, gangliosides expressed on ecvs from these cell lines were analysed using tim beads and flow cytometry. gd and gd were detected on ecvs almost proportionally with expression levels of those gangliosides on the cell surface. then, immunoblotting was performed to analyse integrin levels in ecvs from transfectant cells expressing high levels of gd , showing increased levels of integrins in ecvs from gd + cells compared with those from gd -cell lines. integrin levels in cell lysates from these cells (gd + and gd cells) were almost equivalent. treatment of a gd -expressing melanoma cell line by mm methyl β-cyclodextrin resulted in marked reduction of secreted ecvs and amounts of tsg in them. summary/conclusion: ganglioside expression patterns on melanoma cells were well reflected in the expression of gangliosides on ecvs. these results as well as increased levels of integrins in ecvs from gd + cells suggest that gangliosides and lipid rafts are involved in the generation and secretion of ecvs. introduction: hypoxia, or low oxygen tension, is a common feature associated with tumour growth and is known to regulate tumour cell function, especially through rewiring of cell metabolism. however, how hypoxia influences tumour cell interactions with surrounding cells is not fully elucidated. we sought to evaluate how hypoxia alters metabolite and metabolism-associated mirna packaging in exosomes. methods: exosomes were isolated from t breast cancer cells cultured in normoxia ( % o ) and hypoxia ( % o ) via ultracentrifugation, optiprep gradients, and size exclusion chromatography. exosomes were further characterized by nanosight, qubit protein quantification, and flow cytometry analysis of exosome markers. metabolite and mirna profiling was performed on exosomes and exosome-producing cells in normoxia and hypoxia. results: secretion of exosomes was increased under hypoxic conditions. metabolite profiling revealed alterations in metabolites specific to exosomes derived from hypoxic cells. profiling of exosomal mirna showed packaging of metabolism-related mirna into exosomes derived from hypoxic cells. summary/conclusion: hypoxia alters the metabolite and mirna profiles of cancer cells, with selective packaging of these molecules into exosomes. we identified metabolites and mirna that are depleted and enriched in exosomes compared to cells. these studies identify hypoxia-associated shifts in exosome cargo, providing insight into exosome cargo packaging with potential implications for understanding how cancer cell-derived exosomes regulate recipient cell function. lysosomotropic agents prompts the release of extracellular vesicles carrying autophagy-associated markers: evidence of a general mechanism of secretion driven by lysosomal impairment introduction: drug-induced lysosomal storage disorders (lsds) are due to the transient intracellular accumulation, mostly of phospholipids, into multilamellar inclusion bodies within late endosomal/lysosomal compartment. they represent a major side-effect for many drugs of several pharmacological categories. most lsds inducers are cationic amphiphilic drug (cad), but the molecular mechanisms leading to accumulation of undigested substrates are unknown. extracellular vesicles (evs) have been implicated in cell waste disposal, but it is unclear whether they might be involved in extracellular release of undigested substrates. methods: to investigate this aspect, we developed hek cells stably expressing the fluorescent fusion proteins egfp-cd and mcherry-cd , separated evs by differential ultracentrifugation and quantified by evassociated fluorescence and nta particle count. results: evs released by these models upon treatment with drugs inducing the accumulation of phospholipids (amiodarone) or glycosaminoglycans (tilorone), showed the release of fluorescent medium/large evs ( k fraction) and small evs ( k fraction), whose size and distribution were similar to the same vesicles released by control cells, but enhanced the recovery of medium/large evs and to a lower extent of small evs, analysis of evs associated markers revealed a dosedependent increase of autophagy-associated markers in medium/large and small evs. similar results were obtained when autophagic flux was impaired by drugs raising lysosomal ph by different mechanisms, such as chloroquine and bafilomycin, but not when autophagic flux was stimulated by drugs such as curcumin or overexpression of the endosomal/lysosomal regulator tfeb. summary/conclusion: overall results show that impairment of autophagic flux, either by indigested substrates or higher lysosomal ph, is associated with an increased release evs enriched in autophagy markers, compatible with autophagomes and/or amphisomes, unravelling a connection with secretory autophagy. tomofumi yamamoto a , yusuke yamawaki b , yutaka hattori c and takahiro ochiya a a tokyo medical university, shinjuku-ku, japan; b national cancer center research institute, chuo-ku, japan; c keio university faculty of pharmacy, minato-ku, japan introduction: multiple myeloma (mm) is a haematological tumour. last decade, the prognosis of mm has improved by the development of therapeutic drugs; however, mm cells acquire drug resistance by longterm exposure of these therapeutic drugs. one of the possible explanations of drug resistance is that cells with drug resistance transmit information to other mm cells and their microenvironmental cells. although the elucidation of the mechanism of drug resistance in mm have been desired, it remains poorly understood. methods: in order to understand the mechanism of drug resistance in mm, lenalidomide resistant cell lines were established by long-term exposure of low concentration of lenalidomide. drug resistance was assessed by mts assay and caspase assay. the amount of ev was measured by exoscreen, which is ultra-sensitive detection method of evs by measuring surface protein of evs, such as, cd and cd (yoshioka et al., nat commun., ) . to identify the genes which involved in drug resistance, rna sequence among the drug-resistant cell lines and their parental cell lines was performed. results: firstly, characterization of these cells was confirmed. we found that all of the lenalidomide resistant cell lines secreted more evs than their parental cell lines. in addition to this, the size of ev derived from resistant cells are smaller than those of parental cells. next, we collected evs from resistant cells and parental cells by using ultracentrifugation, and added them to parental cells in the presence of lethal dose of lenalidomide. compared with ev derived from parental cell lines, the evs derived from lenalidomide resistant cell lines increased a number of living parental cells. these results suggested that the evs derived from lenalidomide resistant cells can affect the lenalidomide sensitive cells. as a result of rna sequence, several genes highly expressed in resistant cell line we found, which associated with lysosome pathway. among them, attenuating the sort and lamp genes could significantly reduce the ev secretion in mm cells, leading to enhance the lenalidomide sensitivity. summary/conclusion: our results showed that ev secretion via sort or lamp could induce the drug resistance in mm. study on biological stimulate mechanism of stem cell-derived exosome generation by nanoparticles introduction: mesenchymal stem cells (mscs) are pluripotent stromal cells known to release extracellular vesicles (evs) containing various growth factors and antioxidants that can positively affect surrounding cells. nanoscale msc-derived evs, such as exosomes, have been developed as bio-stable nano-type materials, but had low yield and were difficult to quantify. we hypothesized that the mechanism of nanoparticleenhanced exosome production would stimulate intracellular molecules. the aim of this study was to elucidate the molecular mechanisms of exosome generation by comparing the internalization of surface-modified positively charged nanoparticles and exosome generation from mscs. methods: mesenchymal stem cells (mscs) were cultured in mem-alpha with % fbs and × antibiotics. the positively charged nanoparticles were synthesized by poly-lactide-co-glicolide (plga) and polyethylenimine (pei) with cy . for tracking nanoparticles. all of the exosome image were identified using an electron microscope. additionally, it was confirmed the internalization of the nanoparticles by if. the primary antibodies used were anti-eea , anti-rab and anti-gm . in order to prove the development of exosomes, rt-pcr using autophagy-related mrna was performed. real-time rt-pcr was performed using the applied biosystems sequence detection system . lastly, mirna from msc-derived exosome analysed automatically in the affymetrix data extraction protocol using the provided affymetrix genechip® command console® software (agcc). all statistical testing and visualization of differentially expressed genes was conducted using r statistical language . . results: we determined that rab , located in the mvb and autolysosomal membrane, was increased upon exosome expression and was associated with autophagosome formation. these results suggested that nanoparticles migrated to lysosomes during treatment; however, intracellular exosome-forming factors were stimulated during endosomal maturation simultaneously. summary/conclusion: therefore, msc-derived exosome research using nanoparticles is useful for increasing exosome yield and the discovery of nanoparticleinduced genetic factors. theoretical description of formation of extracellular vesicles by budding of membrane introduction: understanding mechanisms of extracellular vesicles (evs) formation is of utmost importance for their effective use in science, medicine and technology. in particular, the discovery of universal mechanisms explaining the phenomena taking place in vesiculation appears to be crucial and highly warranted. mammalian erythrocytes and giant phospholipid vesicles have been largely used as model systems to study principles of membrane budding and vesiculation. the mechanisms conveniently studied in these simple systems are then generalized to other types of biological membranes. we present a theoretical description of membrane budding and compare the theoretically obtained shapes with the observed ones. methods: in accordance with the fluid crystal mosaic model, membrane is considered as composed of constituents (inclusions) subjected to the local curvature field created by surrounding constituents. constituents can attain different in-plane orientations in the membrane which correspond to different energies. the thermal motion oposes the complete orientational ordering. the single-constituent energy expresses a mismatch of the curvature of the membrane at the position of the constituent and the intrinsic principal curvatures of the constituent and inplane orientation of their principal axes. the free energy of the whole membrane is obtained by summing up (integration) the contributions of the constituents and using methods of statistical physics, and minimized by using numerical methods. results: to outline the principle of (outward and inward) budding, respective sequences of shapes corresponding to a formation of one (outward and inward) spherical bud were calculated by minimization of the free energy. also the corresponding shapes observed in evs (imaged by electron microscopy) and in erythrocytes and giant phospholipid vesicles (imaged by optical microscopy) are shown. it can be seen that theoretically calculated shapes and experimentally observed ones agree well over up to orders of magnitude (the order of the size of giant phospholipid vesicles is between and micro metres, in erythrocytes it is about micro metres and in evs it is about nanometres). summary/conclusion: budding of the membrane is an universal mechanism in formation of external and internal vesicles. introduction: the release of extracellular vesicles (evs) from cells is important for many cellular mechanisms both in normal physiology and in disease. arrdc (arrestin domain containing protein ) is an adaptor protein known to facilitate the ubiquitination of target substrates by nedd family ubiquitin ligases. it also traffics cargo to extracellular vesicles. previous studies show the involvement of arrdc in the trafficking of the divalent metal ion transporter dmt to evs in a ubiquitin-dependent manner, and we aimed to further understand this mechanism. methods: we performed mass spectrometry to identify ubiquitinated lysine residues in arrdc . we then generated arrdc wt and lysine mutant clones and expressed these in cells to determine the effect on ev biogenesis and protein trafficking. results: mass spectrometry data identified potential ubiquitinated lysine residues. out of these, lysine appeared to be the most important for arrdc function. arrdc k r mutation caused a decrease in the number of ev released by the cell compared to arrdc wt, and a reduction in trafficking of dmt to evs. furthermore, we also observed a decrease in dmt activity and an increase in its intracellular degradation in the presence of arrdc k r. k also appeared to be ubiquitinated with k polyubiquitin chains by the ubiquitin ligase smurf . summary/conclusion: our data suggests that k polyubiquitin chains are the signal for arrdc mediated ev biogenesis and protein trafficking, and loss of this signal causes cargo to be rerouted to intracellular degradation mechanisms. chair: tanina arab -department of molecular and comparative pathobiology, johns hopkins university school of medicine a d-printed model to represent the structure and nature of extracellular vesicles, for public engagement and education events. christian burton a , sara veiga a , jason webber a , kate milward a , muireann ni bhaoighill a , lauren evans a , andreia de almeida b , rachel j. errington a and aled clayton a a cardiff university, cardiff, uk; b cardiff university, research associate, uk introduction: explaining the field of extracellular vesicles to the lay public and young audiences can often be challenging. whilst diagrams and images of evs may be helpful, conveying clearly the shape and composition of an ev by these means is not always a success. whilst many members of the audience may be familiar with concepts of cells and related structures, others will find such discussions very abstract and challenging. in order to aid interactions with lay audiences we embarked on the design of a physical hand-held plastic model, representing a typical ev. incorporating flexibility in the design allowing the community to adapt it to showcase their own research. the second goal was to ensure manufacturability using widely available dprinting technologies. methods: the basic model design was conceived by dr c. burton, and iteratively developed using solidworks, , then exported for use in any cad environment (stl format). a model showing a halved ev hemisphere, with a visible lipid-bilayer was developed. attachable rings allow trans-membrane-molecules to be represented, current designs include mhc class-i, hspgs, integrins, tetraspanins and supported by handouts accompanying the models. intraluminal cargo is included via removeable "pegs", and examples representing rna or simple globular proteins, and a template has been created. results: the design is free and open source, and available to the community at: https://www.thingiverse. com/thing: . instructions for d printing are available from the uk extracellular vesicle society website; https://www.ukev.org.uk/public-engagementmaterials/. models have been produced using entrylevel d printers and trialled at engagement events with good early responses. summary/conclusion: the authors hope the community will use and develop this d-model design and that the approach provides an additional and helpful tool for educating audiences about the complexities and roles of evs in biology and disease. centrifugal filtration-sec is promising for extracellular vesicle isolation from d and d her + breast epithelial cell lines introduction: despite recent developments in breast cancer therapy, there is still need for a more targeted approach. extracellular vesicles (evs), endogenous nanovesicles released from human cells, are an attractive choice as nanodrug carriers due to their size, stability and their unique targeting specificity. the aim of this study was to determine if centrifugal filtration (cf) combined with size exclusion chromatography (cf-sec) would be useful for ev isolation from two epithelial breast cell lines d and d her +, representing the tissue of interest, and the amount of cell culture needed to get measurable ev concentrations. methods: cell culture media (without serum) from the immortalized breast epithelial cell lines d and d her + was concentrated with centrifugal filtration (cf) followed by isolation with size-exclusion chromatography (sec) using hiprep / sephacryl s- column run with Äkta start ( nm), min runs. each fraction ( - ml) was collected with fraction collector. dulbecco's particle free pbs was used as mobile phase. the resulting particles were analysed with nanoparticle tracking analysis (nta, nanosight ns , camera gain , static mode, capture time sec), western blotting (wb), microbca and transmission electron microscopy (tem, samples fixed with % formaldehyse and stained with % uranyl acetate, run at kv). results: although sec did not show any prevalent peaks from early eluting regions previously shown to contain extracellular vesicles, these fractions (f -f , - min) were collected from d her + cell culture medium. interestingly, both nta and tem suggest that f and f contained evs as the isolated particles measured and nm, respectively and tem revealed spherical particles - nm in diameter. wb was unable to detect the ev associated protein alix (but was present in the whole cell lysate). soluble proteins and protein aggregates eluted late in the sec chromatogram ( min), with protein analysis (microbca), tem and wb confirming their presence. summary/conclusion: cf-sec is a promising method for ev isolation for pharmaceutical applications, but further work is needed to optimize the isolation process using Äkta start for these cell lines. customer stories from the ev core of university of helsinki introduction: the ev core, world's first ev-dedicated technology platform established in , is a joint venture of two extracellular vesicle (ev) research laboratories at university of helsinki. as an academic research/service facility, the ev core provides infrastructure, state-of-the-art and emerging ev-technologies for research groups, hospitals, companies and authorities in the ev-field. the ev core provides ev isolation, purification and characterization services and offers contacts to downstream analyses in other core facilities based on optimized ev protocols. here, we present and discuss the customer experiences and prospects with the aim to further develop ev core services. methods: our most wanted services are nanoparticle tracking analysis, electron microscopy, ev isolation and rna isolation and consultation. currently, the key down-stream analysis methods are (mi)rna sequencing, metabolomics, flow cytometry and functional assays. results: we present the stories from our customers starting with their research questions and need for the ev expertise/consultation and equipment. next, we show how the projects advanced and what types of ev core -derived or other downstream services helped them to achieve their aims. in the end, we will acknowledge the customers experience and current status of their research. summary/conclusion: narratives of customer stories are an effective starting point for fruitful discussions about the current status and next developments in the young ev service field. recent isev workshops: open, reproducible and standardized ev research (ghent, ) and evs in immunology (buenos aires, ) introduction: since its founding in , isev has sought to further extracellular vesicle research in various ways including scientific meetings. these events encompass annual meetings as well as smaller, topically focused workshops, with the first isev workshop (on rna and evs) organized in new york city in october, . in december, , the workshop "open, reproducible, and standardized ev research" was held in ghent, belgium. in march, , the workshop, "evs in immunology" was held in buenos aires, argentina, with a preceding education day. methods: the international organizing committees of the and isev workshops prepared scientific programs around key themes of ev rigour and standardization (ghent, belgium, workshop) and evs in immunology (buenos aires, argentina, workshop). abstract and application submissions were invited. applications were reviewed and ranked by panels of ev experts for each event, and participants were invited. results: the and workshops assembled a total of more than individuals for talks and discussions around the themes of rigour and standardization and evs in immunology. the buenos aires workshop was preceded by an education day, coordinated by the isev executive committees for education and science and meetings. during these two workshops, poster presentations were permitted for the first time, affording additional presentation and interaction opportunities. the rigour and standardization workshop also featured real-time discussant polling to facilitate discussion. summary/conclusion: isev workshops such as those addressing rigour and standardization (ghent, ) and evs in immunology (buenos aires, ) continue to provide opportunities for focused discussion of small groups of experts on key topics in the field. often followed by published products, isev workshops help to lead and coordinate progress in ev science. for future isev workshops, educational activities may again expand the reach of each event, while poster sessions and app-driven real-time responses should be considered for enhanced interactions and participant canvassing. ev journal club: exchanging pizza for a worldwide audience during covid- kenneth w. witwer johns hopkins university school of medicine, baltimore, usa introduction: a monthly journal club focused on extracellular vesicle science was established at johns hopkins university in , featuring lunch and presentations by academic and industry participants. when covid- prevented in-person meetings beginning in march, , the journal club was converted to a virtual, weekly format on the popular online meeting app zoom. the journal club has persisted despite initial problems with online vandalism. most sessions are also made public on a youtube channel, https:// www.youtube.com/c/extracellularvesicleclub. methods: weekly ev club sessions are arranged by the host. most focus on a specific manuscript related to evs, but some weeks feature presentations of published or soon-to-be-published research by the presenting authors. sessions are advertised one week to several days in advance on social media platforms such as linkedin, twitter, and facebook, asking interested parties to sign up to join a mailing list via surveymonkey. the log-in information is then sent to the mailing list. upon clicking the link, participants are placed in a virtual waiting room for vetting by the host and volunteers. after admission, all parties but the host and presenter are muted to avoid distractions. questions and comments may be placed in a chat box. contributions are monitored and compiled by the host and volunteers to build a question-and-answer session at the end of the presentation. recorded sessions-with or without editing as needed-are placed on the youtube channel for additional access. results: despite initial problems with online vandalism known as "zoombombing," the journal club has continued weekly during the covid- shutdown in the host country (us). an audience of between and individuals is typical. participants typically ask more questions than can be answered in a one-hour time frame. the online format also allows for debate-style events and polling of the audience. summary/conclusion: this ev journal club is an example of how online tools can be used to facilitate international scientific interactions. further development of such formats could provide alternative approaches for isev activities in the science, education, and communication areas. the study aim is to assess whether the exposure to pm and pm , , chosen as paradigmatic environmental stressors, could modify the composition of nasal microbiota (nm) and extracellular vesicle (ev signalling network, showing a role in allergic ar exacerbation). methods: nm analysis were performed on v -v s rrna gene regions amplified from upper-airway tracts of ar cases and healthy individual controls to perform nm analyses. ev size, concentration and cellular origin for each subject were assessed by nanoparticle tracking analysis (nta) and flow-cytometry (fc). information on daily pm and pm , concentrations at the municipality of residence in the days preceding nasal sampling (i.e. day − to day − ) was assigned to each subject by arcgis software. multivariable and logistic analyses were applied on nm, nta and fc outcomes. results: when taxonomy composition was considered, in controls actinobacteria ( . %) was the most represented, followed by firmicutes ( . %) and proteobacteria ( . %) while in cases proteobacteria were . %, actinobacteria were . % and firmicutes were . %. cases showed a higher concentration of all the investigated ev types, derived from platelets (cd +), activated endothelium (cd e+), monocytes (cd +), eosinophils (cd +), neutrophils (cd +), mastocytes (cd c+), epithelial cells (epcam+), gram+ bacteria (lipoteichoic acid+), gram-bacteria (lps+). the effect was greatest in the case of mastocytes evs which were increased . fold in cases versus controls (p < . ). evs were modified by pm exposure at several time lags. in particular, a negative association between pm and eosinophil evs was observed (beta = − , ; pvalue = , ). as we clustered subjects according to their nm, we observed this variable was a strong effect modifier of the association between pm exposure and ev release. summary/conclusion: our findings start to provide an insight on the effect of air pollution on evs, taking into account the effect of nm, in patients with ar. further research is necessary to disentangle the mechanism exerted by inhaled pollutants in modulating evs and nm, and therefore ar exacerbation. funding: gsk investigator sponsered study aryl hydrocarbon receptor activation induces the expression of specific microrrnas in th cells that are release into extracellular vesicules and associated with arthritis introduction: in rheumatoid arthritis (ra), an autoimmune disorder characterized by a chronic sinovial inflammation, smoking is a major risk factor contributing to disease progression, and poor response to therapy. th cell is actively involved in worsening smooking-associates inflammation mediated by aryl hydrocarbon receptor (ahr), a cytoplasmic transcription factor involved in xenobiotic metabolism. both, ahr and th cells, has important implications during ra development. considering that cigarette smoke is a potent epigenetic modifier, we hypothesized that ahr activation, by cigarette components, would transcribe specific micrornas in th cells as a molecular mechanism to exacerbate inflammation in arthritis. methods: microrna expression was evaluated by largescale approach or real-time pcr. c /bl and ahr null mice were submitted to arthritis experimental models and exposed or not to cigarrete smoke (ethical committee approved / ). extracellular vesicles (evs) were isolated by ultracentrifugation, and characterized by western blot and nanosight. rankl-induced osteoclasts (ocs) differentiation in vitro was stained for trap. inhibition of mirnas were performed using anti-mirs transfection. results: we identified a specific group of mirnas induced in th cells after ahr activation. during arthritis progression, the micrornas are expressed and increases after exposure to cigarette smoke. in the absence of ahr their levels were drastically reduced. interestingly, we found that these micrornas are released by th cells into evs, and are able to promote osteoclastogenesis. ocs differentiation in vitro increases in the presence of th -derived evs, and this process is reduced in the absence of micrornas. summary/conclusion: microrna-mediated gene regulation plays crucial roles in the immune system functions, and their abnormal expression is highly correlated with the pathogenesis of ra. evs are known to function in cell-to-cell communication and are able to transmit their contents and cause changes in the target cell. our findings demonstrate a new molecular mechanism by which cigarette smoke could aggravate inflammation in arthritis; through the activation of ahr receptor in th cells, inducing the transcription of specific micrornas that are released into evs, and act as pro-inflammatory mediators. introduction: chagas disease (cd) is caused by the flagellated protozoan t. cruzi. trypomastigote forms are capable of releasing extracellular vesicles (evs) that contain the major surface molecules of the parasite. the parasite has a complex life cycle that leads to it a rapid adaptation in the environmental changes in the hosts. however, the effects of stress on on evs release are not completely understood. objetive: we evaluated the release of evs by trypomastigotes incubated under different stress conditions and the immunomodulatory role of these evs in pre-activated bone marrow-derived macrophages (bmdm). methods: nanoparticle tracking analysis (nta) and scanning electron microscopy (sem) showed an increase in evs releasing by trypomastigotes at °c under acidic conditions, evs released was affected and triggered amastigogenesis process. results: treatment with sodium azide (nan ) also caused changes in the release of evs regarding size and concentration. nitrosative stress caused by sodium nitrite (in culture medium mildly acidic, ph . ; in this condition nano releases nitric oxide) stimulated an increase in production of evs by t. cruzi. when the parasites were treated with nm s-nitrosoglutathione (snog), we observed a reduction in size and concentration of vesiculate material by trypomastigotes. at a higher snog concentration ( µm), the concentration of the vesiculate material increased. t. cruzi-derived evs exposed to stress conditions increased the expression of inos, arg , il- and il- genes in ifn-γ and lps pre-activated bmms. summary/conclusion: results suggest that the viability and/or integrity of the parasite are necessary for the evs releasing. in those in vitro conditions they triggered a proinflammatory response in host cells. this may be a strategy developed by the parasite to favour its establishment in the host. funding: fapesp, cnpq, capes and fapemig ppm-x / . immuno-toxicological evaluation of human mesenchymal stem cell- introduction: mesenchymal stem cells (mscs) have been widely used to the field of autoimmune diseases or tissue regeneration therapy. recently, many research groups have reported that mscs showed their ability via secreted paracrine mediators including extracellular vesicles (evs) rather than cell-to-cell contact. mscs mainly exist on bone marrow, peripheral blood, umbilical cord and adipose and can mostly secrete evs. it has emerged that evs alone are responsible for the therapeutic effect of mscs in plenty of animal diseases models. hence, msc-derived evs may be used as an alternative msc-based therapy in regenerative medicine. methods: as part of safety programme for human therapeutics, we performed immunotoxicological assessment of evs obtained from human mscs (hevs) in mice and human peripheral blood mononuclear cells (hpbmcs). firstly, mice were treated intravenously with a negative control, a positive control (lps; . mg/kg), or low-dose ( x e paticles/head) and high-dose ( x e paticles/ head) of hevs every other day for days and then analysed lymphocyte subsets from collected spleen by facs. next, we treated the evs on hpbmcs for days with low conc. ( x e particles/ml), high conc. ( x e particles/ml), pma/ionomycin as a cell activator or cpt ( μm) as an apoptotic inducer. annexin v/pi and csfe were analysed by facs. results: as a result, splenic nk cells and b cells were slightly increased about ~ % in hevs-treated mice, without biological significance, compared with a positive control (lps) as an immunogenicity inducer. and there were no effects on serum levels of inflammatory cytokines in mice. in addition, hevs had no cytotoxic effect on hpbmcs at both low and high conc. under the culture medium with evs-depleted fbs, the hevs appeared minimal anti-apoptotic effect on hpbmcs. for the cfse assay, the hevs showed slight proliferation on hpbmcs and pbmc activation induced by pma/ionomycin. summary/conclusion: in conclusion, the hevs have little immuno-toxicological effects in mice and hpbmcs. further detailed studies to elucidate immunological response of hevs for development of human therapeutics are needed. funding: this research was supported by a grant ( mfds ) from ministry of food and drug safety. investigation of immune response to mesenchymal stem cell-derived extracellular vesicles in the cancer setting introduction: mesenchymal stem cell-derived extracellular vesicles (msc-evs) are thought to be a fingerprint of the secreting cell and therefore may retain the cancer targeting and immune privilege of mscs. thus msc-evs hold immense potential as tumour-targeted therapeutics for breast cancer. the aim of this study was to determine whether msc-ev administration in tumour bearing immunocompetent animals would initiate an immune response. methods: evs were isolated from conditioned media of both human and murine bone marrow-derived mscs through sequential differential centrifugation, microfiltration and ultracentrifugation. evs were characterized by nanoparticle tracking analysis (nta), western blot and transmission electron microscopy (tem). x ( ) human or murine msc-evs were administered intravenously into t breast tumour bearing balb/c mice (n = ) and healthy controls (n = ). tumour tissue, draining lymph node and spleen were then harvested, dissociated and flow cytometry performed targeting markers associated with a range of immune cells including t-cells, macrophages and natural killer (nk) cells. results: evs were successfully isolated from murine and human mscs with the appropriate size of small evs (sevs: - nm) and morphology including a lipid bilayer observed by tem. evs expressed tetraspanins cd , cd , cd ; cytosolic protein tsg and were negative for calnexin. ev concentrations ranged from . x ( ) - . x ( )/ml. in order to study a range of immune cell populations two antibody panels were created using complimentary fluorescent dyes. the proportion of t-cells (cd +, cd +, cd +), neutrophils (gr- +, ly- c+), dendritic cells (cd c+), macrophages (cd b+, mhci+, mhcii+), nk cells (cd +) and b cells (cd +) remained stable in the tumour, draining lymph node and spleen of all tumour-bearing animals that received either human or murine msc-evs, with no significant change observed in any category. summary/conclusion: the data presented supports the hypothesis that msc-evs retain the immune privilege of the secretory cell, with human cell-derived evs illiciting no immune response in mice. this is encouraging and reinforces the potential for use of msc-evs in the therapeutic setting. introduction: mycobacterium avium (m. avium) is a slow growth rate non-tuberculous mycobacterium (ntm). m. avium infection is a severe global health problem. but the mechanisms of pathogenicity of m. avium are poorly understood. outer membrane vesicles (omvs) that traverse the cell wall and contain a varied bioactive components inculding dna, rna, protein and toxins. previous studies have suggested that these omvs are produced in vitro and during animal infection, but the role of omvs secretion during the interaction of m. avium with host cells remains unknown. methods: in this study, m. avium were grown in middlebrook h medium (m h ) supplemented with % (v/v) oadc enrichment and . % (v/v) glycero. m. avium omvs were isolated by ultracentrifugation method. characterization of omvs by transmission electron microscopy (tem) and nanoparticle tracking analysis (nta). the raw . murine macrophages were incubated with the m. avium omvs to analyse inflammatory response and production of nitric oxide (no) and reactive oxygen species (ros) of macrophage. results: in this study, we demonstrate by fluorescence microscopy that murine macrophages can phagocytosis omvs produced by m. avium. incubation of m. avium omvs with murine macrophages resulted in increased levels of extracellular tumour necrosis factor alpha (tnf-α), interleukin- β (il- β), terleukin- (il- ) and interleukin- (il- ). meanwhile omvs stimulated macrophages produce no and ros. introduction: hospital associated venous thromboembolism (ha-vte) in paediatric patients is the second most common contributor to harm in hospitalized children. platelet-endothelial interactions are integral to the formation of vte, especially in inflammatory conditions such as sepsis. small extracellular vesicles (sevs) have the ability to reprogramme target cell phenotypes via their microrna contents and are known to contribute to vte formation. we hypothesize that sepsis alters platelet-derived sev micrornas capable of net upregulation of vascular endothelial procoagulant and downregulation of anticoagulant pathways. methods: using a precipitation solution and size exclusion chromatography, we isolated sevs from platelet poor plasma of children admitted to the paediatric intensive care unit for sepsis and from healthy controls. we positively selected platelet-derived sevs using immunomagnetic isolation for cd b platelet antigen and confirmed selection using flow cytometry. microrna was profiled using affymetrix genechip mirna . array. results: microrna from sepsis patients (median age . years; iqr: . - and % female) with a median psofa score of (iqr: . - ) and from healthy controls (median age years; iqr: . - . and % female) was isolated and compared. in septic vs. healthy patients mirnas were differentially expressed (false discovery rate (fdr)< . ; fold change ≥| . |) affecting mrna pathways. in septic children, pathways affecting chemotaxis and cell movement of leukocytes were predicted to be activated with z-scores ≥ . summary/conclusion: we developed a method to successfully isolate platelet-derived sevs. sepsis alters the platelet-derived sev microrna profile in paediatric patients with sepsis. these micrornas are predicted to target chemotaxis and cell movement pathways, important contributors in the formation of ha-vte. further analysis into specifically targeted pathways should be conducted as a potential target for the prevention of ha-vte in sepsis. introduction: sjögren´s syndrome (ss) is a systemic autoimmune disease that mainly affects salivary and lacrimal glands. mechanisms of ss pathogenesis are poorly understood. it is thought that inflammation leads to destruction of exocrine glands, however the triggers of autoimmunity and the mechanisms by which inflammation drives immunopathology are not characterized. our work identifies t cell-exosomederived mir- - p as a pathogenic driver of immunopathology in ss. micrornas (mirnas) are endogenous small noncoding rna molecules that regulate the expression of target genes through translational repression of mrnas. through transcriptomic profiling studies our group had previously documented a significant upregulation of mir- - p in patient ss tissues and in serum exosomes. methods: structured search for target genes of mir- - p involved in salivary gland (sg) physiology was performed with mirdip . serca b, ryr and ac were selected for further validation and functional analysis. binding of the mirna was confirmed by luciferase reporter assays in hsg cell lines and human-derived primary epithelial cells. the mrna and protein levels of serca b, ryr and ac were determined by qpcr and western blot, respectively. to investigate the cell-specific distribution of mir- - p in relation to the expression levels of serca b, ryr , and ac , a double fluorescent in situ hybridization was performed. ca + signalling and camp levels were measured using fluorescent sensor. to isolate exosomes, the t cell medium and serum of ss-patients and healthy volunteers (hv) were collected. results: we show that mir- - p is over-expression in the sgs of ss-patients. next, we demonstrated that mir- - p is contained in exosomes in serum of sspatients significantly more than serum of hv. we also show that activated t cells secrete exosomes containing mir- - p which transfer into glandular cells and affecting intracellular ca + signalling, camp production and protein production by mir- - p targets (serca b, ryr and ac ). summary/conclusion: this study provides evidence for a functional role of the mir- - p in ss pathogenesis and promotes the concept that t cell-activation directly may impair epithelial cell function through secretion of mi-rna containing exosomes. treg-derived il -coated extracellular vesicles promote infectious tolerance (p ) subunits, yet the forms that il assumes and its role in peripheral tolerance, remain elusive. methods: we induce cba-specific, il -producing t regulatory (treg) cells in tregebi wt c bl/ reporter mice, and identify il producers by expression of ebi tdtom gene reporter, plus ebi and p proteins. results: curiously, both subunits of il were displayed on the surface of tolerogen-specific foxp + and foxp neg (itr ) t cells. furthermore, il producers, although rare, secrete ebi and p on extracellular vesicles (ev) targeting a -to -fold higher number of t and b lymphocytes, causing them to acquire surface il . this surface il is absent when ev/exosome production was inhibited, or if ebi is genetically deleted in treg cells. summary/conclusion: the unique ability of ev to coat bystander lymphocytes with il , promoting exhaustion in, and secondary suppression by, non-treg cells, identifies a novel mechanism of infectious tolerance. funding: nih grants r -ai - (to w.j.b.), r ca and p ca (to d.a.a.v.) and the university of wisconsin carbone cancer center support grant p ca . unique formulated dual targeting antigen specific and delivered mirna- gene regulating exosomes acting at the immune synapse to induce apc-derived secondary suppressive exosomes introduction: an exosome-apc circuit we uncovered may be applicable beyond skin immunity we study in mice. methods: high antigen dose tolerized cd + t cells make suppressive antigen-specific exosomes due to chosen surface antibody light chains that enable targeting antigen presenting cells (apc) antigen-specifically for delivery of also chosen inhibitory mirna- to mediate specific functional gene alterations. results: both antigen and gene specificity aspects are lent to naïve but activated exosomes by simple in vitro incubations alone. for mechanism, these primary exosomes bind antigen peptides in mhc on apc that in turn make secondary suppressive exosomes that act peptide/mhc-specifically on the effector t cells at the immune synapse. they transfer another mirna for strong prolonged inhibition of active delayed-type hypersensitivity (dth) for days even, when the primary mirna- -pos exosomes are administered orally at the height of the in vivo response, in a physiological dose. summary/conclusion: it is shown possible to induce therapeutic exosomes with ag targeting of choice due to placed ab on the surface and that also target specific gene functions of acceptor cells due to carriage of a selected mirna. this dual ag and gene-specific therapy has applications in treatment of cancer, autoimmunity and allergies. introduction: previously, our group characterized distinct populations of extracellular vesicle (ev) released from neutrophilic granulocytes: ev formed spontaneously (sev) and upon activation with opsonized particles (aev). the aev differs in protein cargo and its ability to inhibit bacterial growth. we described that mac- integrin (cr receptor) plays key role in the aev production and extracellular calcium supply is crucial in this signalization. in the present work, our aim was to investigate whether mac- activation or casignalling on their own are sufficient for the initiation of the aev biogeneis. methods: we isolated neutrophil derived evs from peripheral human blood and murine bone marrow by two-step centrifugation and filtration. we tested the effect of ca-ionophore and examined the ev production on c bi coated surface and in soluble form. we quantified the vesicles by flow cytometry and determined their protein content by bradford assay. we examined their antibacterial effect in parallel with optical density-based measurement and our flow cytometry based method. results: on c bi coated surface, we observed an increased ev production, and these evs possessed antibacterial capacity. however, in soluble condition, c bi did not induce further ev production, and these evs did not show any antibacterial property. we found that ca-ionophore initiated ev formation, but these ev did not show antibacterial effect. we observed ev production increase after ca-ionofore treatment both in the presence and in the absence of extracellular ca. the ca-ionophore slightly increased the opsonized particle induced ev production, but did not potentiate their antibacterial capacity. summary/conclusion: mac- activation is not just crucial, but sufficient in initiation of the aev biogenesis. clustering of this receptor is required. while the ca-signal is crucial, it is not sufficient in the generation of aevs. extracellular vesicles and their microrna cargo in retinal health and degeneration: mediators of homoeostasis, and immune modulation yvette s. m. wooff, adrian cioanca, riemke aggio-bruce, joshua chu-tan, ulrike schumann and riccardo natoli the australian national university, canberra, australia introduction: photoreceptor cell death and inflammation are known to occur progressively in retinal degenerative diseases such as age-related macular degeneration (amd). however, the molecular mechanisms regulating these biological processes are largely unknown. extracellular vesicles (ev) are essential mediators of cell-to-cell communication with emerging roles in the modulation of immune responses. evs, including exosomes, encapsulate and transfer microrna (mirna) to recipient cells and in this way can modulate the environment of recipient cells. dysregulation of evs however is correlated to a loss of cellular homoeostasis and increased inflammation. in this work we investigated the role of isolated retinal small-medium sized ev (s-mev) in the regulation of homoeostasis and immune modulation in both the healthy and degenerating retina. methods: isolated s-mev from healthy and degenerative (photo-oxidative damaged) mouse retinas were characterized using dynamic light scattering, transmission electron microscopy and western blot, and quantified using nanotracking analysis. small rna-seq was used to characterize the mirna cargo of retinal s-mev isolated from healthy and degenerating retinas. finally, the effect of exosome inhibition on s-mev-mediated immune modulation was investigated using systemic daily administration of exosome inhibitor gw and analysed by in situ hybridization of s-mev-abundant mirna. electroretinography and immunohistochemistry were performed to assess functional and morphological changes to the retina as a result of exosome depletion. results: our results demonstrated an inverse correlation between s-mev concentration and photoreceptor survival, with decreased s-mev numbers following retinal degeneration. small rna-seq revealed that s-mevs contained uniquely enriched mirnas, however no differential composition in s-mev mirna cargo following photo-oxidative damage was observed. exosome inhibition using gw exacerbated photoreceptor degeneration, with reduced retinal function and increased levels of inflammation and cell death seen following photo-oxidative damage. further, reduced translocation of the photoreceptor-derived s-mev was demonstrated following exosome-inhibition in photo-oxidative damaged mice. summary/conclusion: taken together, we propose that retinal s-mev and their mirna cargo play an essential role in maintaining retinal homoeostasis through immune-modulation, and have the potential to be targeted using gene therapy for retinal degenerative diseases. impacts of agricultural dust exposure on human lung-resident mesenchymal stromal/stem cells and their extracellular vesicles introduction: agricultural dust is considered a high-risk occupational hazard by the cdc, with impacts reaching throughout the communities surrounding these industries, leading to increased incidence of respiratory illness and disease among individuals within this occupation and these communities. lung-resident mesenchymal stromal/stem cells (lr-msc) have an important role in maintaining homoeostasis in the lung, and mediating pro-and anti-inflammatory effects, particularly during exposure to inhaled irritants, like agricultural dust. one way in which these lr-msc promote lung homoeostasis is through the release of extracellular vesicles (ev), with a variety of cargo that elicit changes among target cells. we hypothesize that exposure to agricultural dust modifies the quantity and cargo of ev released by lr-msc to promote lung tissue homoeostasis. methods: primary human lung-resident mesenchymal stromal cells were exposed to extracts of dusts collected from swine confinement facilities (de) for or hrs and the media from these exposures were collected and enriched for ev by opti-prep density gradient ultracentrifugation. the quantity of these ev were assessed by nanoparticle tracking analysis. additionally, cytokine and chemokine release by lr-msc were analysed by enzyme-linked immuno assays. results: as assessed at hr following treatment, deexposed lr-msc released pro-inflammatory cytokines, il- and il- , with il- release reaching statistical significance at . %, . %, and % de concentrations (p = . , < . , and < . respectively) and il- trending a similar dose response but only statistically significant at % de (p = < . ). de exposure of lr-msc also induced changes in the lr-msc-derived ev populations when compared to vehicle control, where lr-msc released significantly more ev in the and % iodixanol fractions (p = < . and . , respectively) at hr following de treatment. alternatively, there were significantly less ev in the and % density fractions in the media of deexposed lr-msc versus vehicle control. summary/conclusion: following exposure to agricultural dusts, lr-msc-derived ev populations more likely consist of exosomes and ectosomes, which play an important role in promoting lung tissue homoeostasis during exposure-related pulmonary inflammation. introduction: during analyses of single extracellular vesicles (evs) by flow cytometry (fcm), particles below the detection limit may exceed the trigger threshold, which is called swarm detection and generates false-positive counts. serial dilutions are recommended to find the minimal dilution for which swarm detection is absent. however, because particle concentrations in plasma vary, the optimal dilution differs > -fold between donors, but it is unfeasible to do serial dilutions for each clinical sample. therefore, our aims are to ( ) develop a faster method to avoid swarm detection, and ( ) increase the number of detected evs per second. methods: we measured serial dilutions of cd stained evs in platelet free plasma (pfp), with and without spiking of fitc beads, by fcm (apogee a -micro). we triggered either on side scatter or fluorescence. results: for scatter triggering with our fcm, swarm detection consistently occurred for plasma samples exceeding a (total particle) count rate of , - , events/s. the cd + evs concentration scaled linearly over . orders of magnitude of the dilution and most donors required > -fold dilution to avoid swarm detection, thereby reducing cd + ev counts. for fluorescence triggering, the cd + evs concentration scaled linearly over > orders of magnitude of the dilution. for all donors, swarm detection was absent after -fold dilution (relative to pure plasma). the count rates of cd + evs were - -fold higher compared to scatter triggering. the spiked fitc beads confirmed that the median signals remained constant. summary/conclusion: we have developed two clinically applicable ways to avoid swarm detection. for scatter triggering, the count rate provides direct feedback on the presence of swarm detection in plasma samples. for fluorescence triggering, swarm detection was absent for all plasma samples diluted ≥ -fold and compared to scatter triggering, count rates of cd + evs were - fold higher, thereby improving statistical significance. funding: edwin van der pol is supported by the netherlands organisation for scientific research -domain applied and engineering sciences (nwo-ttw), research programmes veni . benchmarking flow cytometric analysis of nanoparticles: a cross-platform study for single extracellular vesicle detection introduction: despite flow cytometry being widely used to analyse cells in suspension, most commercial instruments lack sensitivity when measuring nanoparticles (nps) and extracellular vesicles (evs). furthermore, the use of appropriate reference materials (rms) for calibration and quality control are essential to compare results acquired with different instruments. to work towards successful clinical applications for ev biomarker profiling, benchmarking studies including state-of-the-art flow cytometers are required. we here investigated the ability of three different flow cytometers to detect nps and evs. methods: the instrument sensitivity of light scattering detection was evaluated by using synthetic nps of different sizes and refractive indices. fluorescent calibration was investigated by using molecules of equivalent soluble fluorophores (mesf) beads. biological recombinant evs (revs) were used to validate the detection and quantification of fluorescent evs in a side-by-side cross-platform study using an n nanoflow analyser (nanofcm), an optimized bd influx and a cytoflex lx. results: we found that when light scatter based detection was used, the nanofcm detected the smallest non-fluorescent nps, the bd influx was able to provide reliable fsc information from the smallest detected nps and the cytoflex performance was greatly improved by the use of violet-ssc. biological revs showed that the nanofcm could clearly resolve fluorescent evs while the bd influx and cytoflex were unable to fully resolve revs from background, although fluorescence threshold improved detection. in addition, our findings revealed that different concentrations are required to ensure single ev detection in these platforms. summary/conclusion: we identified several strengths and limitations for each platform with respect to single ev analysis. furthermore, our results showed that proper calibration and rms are of utmost importance to ensure reliable interpretation of ev flow cytometric data. caution when using membrane dyes for sequential extracellular vesicle analysis diana pham, michael wong, desmond pink and john lewis nanostics, edmonton, canada introduction: confirmation that particles detected by microflow cytometry are actually extracellular vesicles (evs), or at least membranous in composition, can be achieved through a variety of methods. positively staining particles with a membrane dye strongly suggest that the particle contains a membrane; loss of stain (or detection) after detergent solubilization of the membrane-dyed particles provides even stronger evidence that the particles were evs. it is important to recognize that the labelling protocol provided by the membrane dye manufacturer may not be ideal for all types of evcontaining biological samples, such as blood, urine, semen etc.). removal of excess dye from stained evs is very difficult and can be impractical depending on the nature of the experiment. however, this means that the potential for excess dye to contaminate subsequent sampling is high. therefore, it is important to determine optimal working concentrations and labelling conditions when using membrane dyes for ev detection to understand properties that may impact your analyses. methods: to assess the utility of membrane dyes, titration curves were generated to determine the optimal working concentrations of membrane dyes for ev detection in conditioned media and human serum samples. once the optimal concentration was determined the potential of dye carry-over from sample to sample during microflow cytometry detection was evaluated by tracking dye positive (dye+) particles in phosphate buffered saline (pbs) blanks and matched, unlabelled, sample replicates. results: we found that optimal concentration of any membrane dye is dependent on sample type. even with the inclusion of system washes to prevent sample carryover, there was carryover of low amounts of dye+ particles into sequentially analysed pbs blanks. if unstained samples were analysed following a stained sample, excess dye (or at least dye+ events) appeared in the data. a sample concentration effect was also seen; samples of lower concentrations were more susceptible to dye carryover. summary/conclusion: when using membrane dyes to stain evs in biological samples, especially if an autosampler is employed to run a series of tests, it is critical to determine the optimal concentration of dye for each type of sample, as excess dye can carry over to the next sample in the queue. in addition, determining the necessary steps to clean any excess dye following each sample run will improve the accuracy of ev detection and analyses. funding: nanostics alberta innovates alberta cancer foundation correlation between size and protein expression of single exosomes by combined atomic force and fluorescence microscopy introduction: there are no universal markers of extracellular vesicles, but often they are identified by the presence of tetraspanins in their membrane. based on this, products have been developed to precipitate or quantify evs by acting upon cd , cd , and cd . however, evs also carry proteins from their parent cells, and capturing evs based their presence allows for a more complete understanding of vesicle heterogeneity from a single cell type, and for evs derived from specific tissues to be enriched from other biofluids in support of biomarker assessment. for example, evs derived from the brain could be captured from the general population of serum evs for better assessment of cargo associated with proteinopathy. the goal of this study was to identify specific antibodies to capture and label evs bearing the neural markers cd , snap , α-synuclein, tau, and ncam. methods: the targets were overexpressed in hek t cells through transient transfection of plasmids (origene). media was conditioned for - hours, and then centrifuged to remove cell debris. cell lysates and concentrated conditioned media (cm) were analysed by western blot. unpurified cm, or cm after performing size exclusion chromatography (sec, izon), were analysed in the exoview r system. diluted cm was incubated on custom antibody microarray chips overnight. then the chips were labelled with a cocktail of labelled antibodies, washed and imaged. vesicles were counted, sized, and phenotyped. next, commercially available pooled human csf was analysed in a similar fashion to determine their abundance in a relevant biofluid. results: multiple antibody clones were tested in different combinations for capture and labelling for the five different neuronal enriched proteins of interest, and optimal combinations were identified. some markers were identified on particles > nm in size that were negative for tetraspanins, while others colocalized with tetraspanins. through comparing permeabilized and intact evs with and without sec to remove non-vesicular proteins, we found that tau could be on the vesicle surface, within the vesicle, and free in solution. summary/conclusion: the exoview platform can be customized to enable the detection of proteins of interest and to determine whether they are on the ev surface, intravesicular, or non-ev associated. methods: forty non-smoking male and female subjects ( - y) at moderate risk for cvd were recruited for the study. evs from platelet-free plasma (pfp) were isolated using size exclusion chromatography (sec). the concentration and size distribution of evs were measured by nanoparticle tracking analysis (nta) and flow cytometry (fcm). three ev markers, including annexin v for the circulating phosphatidylserinepositive (ps+) evs, cd for platelet-derived evs and cd for endothelial-derived evs were used for phenotyping. in addition, coagulation and fibrinolysis were assessed using a thrombodynamics analyser (hemacore). platelet aggregation to determine platelet function was assessed by a high-throughput platelet function assay with a wide range of concentrations of agonists, including adenine diphosphate (adp), collagen-related peptides (crp-xl), epinephrine, thrombin receptor activating peptide (trap- ) and u . the association between thrombogenic risk markers for cvd and ev numbers was tested by pearson's correlation coefficient and linear regression model using the statistical program, spss. results: circulating ev concentration with threshold of nm, measured by nta, were positively associated with coagulation-related risk markers, including rate of clot growth (r = . ; p = . ) and clot size at min (r = . ; p = . ). ps+ evs derived from endothelial cells, determined by fcm, were negatively associated with lysis onset time (r = − . ; p = . ), whereas they were found positively correlated with lysis progression (r = . ; p = . ). both mean and mode size of cevs, detected by nta, were significantly correlated with u -induced platelet aggregation (r = − . ; p = . , r = − . ; p = . , respectively). summary/conclusion: in subjects at moderate risk for cvd, cev numbers were positively related to rate of clot growth and clot size and size of cevs was negatively related to platelet activity. higher numbers of endothelial cell-derived ps+ cevs were associated with lower rates of fibrinolysis. this suggests that cevs promote clot growth and reduce fibrinolysis, and may therefore be an indicator for greater risk of cvd. beyond stem cells: extracellular vesicles from human induced pluripotent stem cells (hipsc) and hipsc-cardiomyocytes as therapeutic approaches for heart failure introduction: heart failure is caused by a variety of underlying diseases, the most common being myocardial infarction. initially regarded as an alternative to pharmacological approaches, stem cell transplantation has failed to demonstrate clinically meaningful results. instead, it has become increasingly apparent that the therapeutic effects of transplanted cells are largely mediated by their secretome, while mounting evidence suggests extracellular vesicles (evs) play a major role in cardiac repair. within this framework, evs from human induced pluripotent stem cells (hipsc) and hipsc-derived cardiomyocytes (hipsc-cm), hold a tremendous potential to treat cardiovascular disease. we isolated evs from conditioned culture media at key stages of the hipsc-cm differentiation and maturation processes, i.e. from hipsc (hipsc-ev), cardiac progenitors (cpc-ev), immature (cmi-ev) and mature (cmm-ev) cardiomyocytes, with the aim of studying their potential role as therapeutics, and whether their effectiveness was influenced by the state of their parent cell. methods: hipsc were differentiated into cardiomyocytes in a d culture approach, using the protocols developed by our group. ev isolation was performed on an iodixanol density gradient, and the evs were characterized in terms of particle size and particle size distribution, presence of ev-specific markers, and imaging through transmission electron microscopy. functional studies were performed using human umbilical vein endothelial cells (huvecs) to evaluate evuptake, cell migration and angiogenesis. results: evs from all hipsc and cardiac derivatives presented a typical cup-shaped morphology and expressed cd and cd . ev yield varied along differentiation, with a minimum for cpc and a maximum for cmi. pkh -labelled evs were uptake by huvecs, and colocalized with calnexin, a protein from the endoplasmic reticulum. wound healing assays showed an increased cell migration in huvecs treated with cardiomyocyte-derived evs, in comparison with control evs isolated from foetal bovine serum. summary/conclusion: our findings suggest a different ev secretion profile along cm differentiation and maturation, with preliminary assays showing ev functionality. ongoing work aims at elucidating the possible differences in function and cargo amongst these types of evs. endothelial cells differentially load and secrete extracellular vesiclederived micrornas into apical and basolateral compartments this may play a role in microcalcification in calcific aortic valve disease (cavd), but this is poorly understood. annexin a is thought to be a marker of membrane-derived evs, but because it can be found on the cytoplasmic or extracellular side of the plasma membrane, its localization within or on the surface of evs is unclear. the goal of this study was to determine whether annexin a is found on the surface of evs in two cell lines relevant to cavd, and develop an assay that can be used to determine whether this changes under pathogenic conditions. methods: evs were isolated by differential ultracentrifugation from the conditioned medium (cm) of smooth muscle cells (smc) and valvular interstitial cells (vic). total protein in the cell lysates and ev pellets was analysed by western blot. evs from cells treated with control sirna or anxa -sirna were enumerated and phenotyped using the exoview r platform. evs with surface expression of cd , cd , cd , and annexin a were captured using a customized antibody microarray chip. then evs were labelled with fluorescent antibodies to assess ev number, size, and colocalization of ev proteins. the knockdown of annexin a allowed us to assess the specificity of the selected annexin a antibody. results: the ev fraction was positive for cd , and lacked markers of other vesicle types. western blot on the ev pellet and supernatant in ± edta indicated that there is annexin a both on the surface of and within the evs. using the antibody microarray chips, numerous annexin a + evs were captured on the annexin a spots from the control cm, and there was a marked decrease in capture and labelling from anxa -sirna treated cells. under both conditions, vesicles were also captured on tetraspanin probes, with the greatest number captured on cd , then cd and cd . there was a significant population of annexin a + evs that was negative for tetraspanins. summary/conclusion: annexin a is found on the surface of evs. the assay developed in collaboration with nanoview biosciences is well suited for assessing the number and phenotype of annexin a + evs derived from smc and vic cell lines, which could provide a useful method for understanding ev populations in cavd patient cell lines. funding: this work was supported by hl and hl . possibility of exosomal micrornas associated with chronic limb-threatening ischaemia, the end stage of atherosclerosis, as a promising biomarker introduction: chronic limb-threatening ischaemia (clti), the end stage of peripheral artery disease (pad), has poor prognosis and is attributed to lifestyle disease. with increasing of atherosclerotic disease all over the world, establishment of biomarker for should play a pivotal role for early detection and preventing aggravation of the disease. the aim of this study is to explore the possibility of liquid biopsy for atherosclerotic disease by analysis of clti-associated exosomal micrornas. methods: clti due to pad was diagnosed by anklebrachial blood pressure index, skin perfusion pressure (< mmhg) and angiography. ten preoperative clti patients and control patients without pad were analysed (all patients with diabetes and % of patients had end-stage renal failure [esrd] ). to identify biomarkers associated with clti, exosomes were extracted from patient's serum after ultracentrifugation and total rna including small rna was isolated from the exosomes. the expression profile of exosomal micrornas associated with clti were evaluated using a next generation sequencing. results: forty-three exosomal mirnas associated with clti were identified. intriguingly, these mirnas were clearly categorized with esrd, which was well known as end-stage of life-style disease: these were stratified into micrornas for esrd patients and micrornas for non-esrd patients. since esrd is the most important factor significantly related to patient's prognosis in clti, exosomal micrornas reflected patient's comorbidity onto the expression profile. summary/conclusion: a portion of the expression profile of exosomal micrornas associated with clti was identified. exosomal microrna could be a biomarker to stratify patient's condition along with their comorbidities and is very promising for individualized diagnosis in atherosclerotic diseases with risk diversity. postoperative plasma exosomal mir- and mir- a signature in patients with left ventricular reverse remodelling after surgical mitral valve repair underwent implantation of a prosthetic mitral ring. lv remodelling was assessed by cardiac magnetic resonance imaging and pexos were isolated by optimized ultracentrifugation before surgery (t ) and six months after surgery (t ). isolated pexos were quantified by nanoparticle tracking analysis and mir- , mir- , mir- a, and mir- a were measured by rt-qpcr. the same analysis was performed on healthy subjects with normal cardiac function (n = ). local ethical committee approved the study (emigrate study, approval n° ) and informed consent was obtained from all patients. results: pexos levels at t were lower (− %, p = . ) in patients with worst postoperative lv function, while they were higher at t (+ %, p = . ) in patients with reversed lv remodelling after surgery. at t , the increase in pexos levels was associated to decreased heart mass index (− %, p = . ) and higher levels of exosomal mir- (+ %, p = . ) and mir- a (+ %, p = . ) were detected in patients with improved lv function. summary/conclusion: higher postoperative levels of pexos delivering mir- and a depict lv reverse remodelling after surgical mitral valve repair. monitoring of exosomal micrornas cargo might predict postoperative outcome in patients with mr. expression of lipocalin- (lcn ) in circulating extracellular vesicles (evs) and femoral plaque-derived evs of peripheral arterial disease patients. introduction: clinically, the drug resistance situation of acinetobacter baumannii is becoming increasingly serious, and its drug resistance has become a difficult problem for nosocomial infection and clinical treatment. in view of the relatively slow development of antibacterial drugs, exploring the resistance mechanism of acinetobacter baumannii is of great significance to improve bacterial resistance and help clinical treatment. studies have shown that outer membrane vesicles (omvs) can transmit resistance genes to mediate the spread of drug resistance, and recent studies have confirmed that high expression of efflux pumps play an important role in the multidrug resistance of a. baumannii. in this study, we want to explore whether the outer membrane vesicles of acinetobacter baumannii can transfer the efflux pump related substances. methods: first, ultracentrifugation and density gradient centrifugation were used to extract the omvs of acinetobacter baumannii antimicrobial-sensitive strains (atcc ) and antimicrobial-resistant strains. then, nanoparticle tracking analysis (nta) technology was used to analyse the particle size and distribution range of omvs. transmission electron microscopy (tem) was used to identify their morphology and structure. bradford method was used to determine the protein concentration of omvs. next, the omvs of antimicrobial-resistant strains were incubated with the antimicrobial-sensitive strains and then the drug susceptibility test was done to determine whether omvs of antimicrobial-resistant strains could transmit antimicrobial-resistance information to the antimicrobial-sensitive strains. finally, pcr, qpcr and mass spectrometry were used to determine whether the efflux pump related genes were higher expression in omvs of antimicrobial-resistant strains than those in antimicrobial-sensitive strains. results: nanoparticle tracking analysis (nta) detected the concentration and size distribution of omvs of acinetobacter baumannii strains. it showed that the extracted omvs have a relatively uniform particle size and a size between - nm. tem showed that omvs had a typical vesicle structure. omvs coculture experiments showed that omvs of the antimicrobial-resistant strains can indeed pass resistance to the antimicrobial-sensitive strains. and the efflux pump related genes were higher expression in omvs of antimicrobial-resistant strains than those in antimicrobial-sensitive strains. summary/conclusion: omvs of the antimicrobialresistant strains can indeed pass resistance to the antimicrobial-sensitive strains. the cause of acquiring antimicrobial resistance in sensitive strains may be caused by resistant strains passing efflux pump-related genes or proteins to sensitive strains. characterization of melanocytic extracellular vesicles during ageing of the choroid kelly coutant a , léo piquet a , nathan schoonjans b , philippe gros-louis a , julie bérubé c , stéphanie proulx a , alain r. brisson d and solange landreville a a université laval, quebec city, canada; b université de lille, lille, france; c centre de recherche du chu de québec-université laval, quebec city, canada; d université de bordeaux, bordeaux, france introduction: the choroid is located at the backside of the light-sensitive retina and is highly vascularized. it contains pigmented melanocytes, and their melanin protects them against oxidative stress. since ageing reduces the number of melanosomes in melanocytes and generates a stiffer extracellular environment, our hypothesis is that surrounding choroidal cells and the retinal pigment epithelium (rpe) are subject to more oxidative stress-related damages. this study aimed to characterize evs released by human choroidal melanocytes in the context of intercellular cooperation during ocular ageing. methods: melanocytic evs were recovered from the conditioned culture medium of young/old melanocytes grown on hydrogels of varying stiffness ( . - kpa) by differential centrifugation. the concentration and size distribution of melanocytic evs were determined by high-sensitivity flow cytometry. cryo-transmission electron microscopy combined with receptor-specific gold labelling were used to reveal their morphology, size and phenotype. the relative abundance of surface markers was evaluated with the exo-check exosome antibody array. the uptake of fluorescent melanocytic evs by the rpe and choroidal endothelial cells was assessed by confocal microscopy. results: choroidal melanocytes released evs positive for annexin- and the tetraspanin cd . young melanocytes produced more annexin- positive evs and evs larger than nm compared to older donors. the stromal stiffness impacted the concentration and size of melanocytic evs. we confirmed the uptake of melanocytic evs by endothelial and rpe cells. summary/conclusion: evs from choroidal melanocytes are internalized by surrounding endothelial cells and rpe. age-related stressors modify the phenotype of melanocytic evs. the identification of melanocytic factors that can protect retina/choroid cells from oxidative stress-induced cell death could lead to more efficient therapy for patients suffering from dry agerelated macular degeneration. introduction: owing to their proposed biocompatibility and ability to cross biological barriers, evs represent an attractive therapeutic delivery platform. however, evs are eminently heterogeneous. a better understanding of ev heterogeneity and its origins will allow for improved design of ev-based therapeutics. ev heterogeneity is mainly studied by focusing on distinct ev subpopulations. other sources of heterogeneity, such as heterogeneity within ev secreting cells themselves, have been investigated in lesser detail. in this study, we assessed the phenotypic drift of cell derived evs to explore the origins of ev heterogeneity and its potential impact. methods: three independent samples of two mda-mb- breast cancer cell sub-clones were cultured for six weeks. evs were harvested weekly and analysed using the macsplex exosome flow cytometry kit. at two time points the proteome of evs was analysed by lc-ms/ms mass spectrometry with subsequent gene ontology and reactome pathway analysis. results: the expression of over proteins was deregulated in evs derived from the two different cell clones. many de-regulated proteins were associated with biological processes predicted to affect potential ev toxicity (platelet activation, neutrophil degranulation, blood coagulation) and ev biological activity (antigen presentation, inflammation, tgf-beta/ mtor/wnt signalling). more surprisingly, within only two weeks, over ev proteins, many associated with immune modulation, apoptosis, interleukins, cytokines and cell signalling pathways (including those affecting t-cell/b-cell receptors) were de-regulated between the two ev isolation time points. summary/conclusion: results suggest that temporal changes can be observed in the ev proteome (potentially by clonal drift, epigenetic changes or cellular genomic instability) over short time periods. these changes could cause significant differences in biological effects and delivery capabilities between evs harvested from the same cells at different time points and conditions. in vivo tracking and biodistribution analysis of mesenchymal stem cellderived extracellular vesicles in a radiation injury murine model introduction: recent studies indicated that extracellular vesicles (evs) play key roles in intercellular communication and have great potential for clinical application. understanding the biodistribution of evs is therefore essential. our previous works have shown the ability of mesenchymal stem cell (msc)derived evs to protect haematopoietic cells from radiation damage. in this study, we evaluated the biodistribution of msc-evs in a radiated mouse model. methods: human msc-evs were harvested by ultracentrifugation and labelled with did lipid dye. the reliability of the labelling evs was confirmed by sucrose gradient fractionation analysis. the distribution of evs in radiation-exposed mice after ev intravenous administration were evaluated by fluorescence molecular tomography and further confirmed by flow cytometry and confocal microscopy analysis. results: we observed that did labelled msc-evs appeared highest in liver and spleen, lower in bone marrow in tibias, femurs, and spine, and were undetectable in heart, kidney and lung. we found the significantly increased msc-ev accumulation in spleen and bone marrow post-radiation appeared with an increase of uptake of msc-ev by cd b+ and f / + cells, but not b + cells, compared to those organs from non-irradiated mice. however, there was a predominant ev accumulation in lung and less accumulation in spleen and liver; in mice infused with human lung fibroblast cell derived evs (lfc-evs) and there was no significant lfc-evs accumulation change in the spleen or liver after radiation. we further found that increasing levels of irradiation caused a selective increase in vesicle homing to marrow and spleen. this accumulation of msc-evs at the site of injured bone marrow could be detected as early as hour after msc-ev injection and was not significantly different between and hrs. post-msc-ev injection. summary/conclusion: this study indicated the specific accumulation of ms-evs at the site of injury of haematopoietic tissue in radiation injury mice. funding: this work was supported by the nih grants uh tr , uh tr - s , p gm , and t hl . linking fat to colorectal cancer: extracellular vesicle crosstalk sheffield hallam university, sheffield, uk introduction: colorectal cancer is the third most common cancer worldwide, and fourth leading cause of malignancy related mortality. understanding the mechanisms of its growth and metastasis is key to elucidating new therapeutic targets and developing treatments in the clinical setting. epidemiological evidence indicates an increased risk of cancer in obese patients, pointing to bidirectional communication between colon and adipose cells. extracellular vesicles (evs) are small membrane enclosed packages released by cells, capable of transporting bioactive cargo from donor to recipient cells and inducing phenotypic changes. adipocytes are a key component of the tumour microenvironment and interactions between adipose tissue and tumour cells may be important in the growth and metastasis of cancer. in this study, we investigate the effects of colorectal cancer evs on adipocytes in vitro, and potential induction of dedifferentiation to a more fibroblastic, pro-inflammatory phenotype. methods: evs were isolated from sw and ht human colorectal cancer cell lines by differential ultracentrifugation and mature adipocytes generated by differentiation of the sgbs human pre-adipocyte cell line. adipocytes were treated with evs and their lipid content measured by oil red o to determine loss of lipids. inflammatory cytokine profile was measured by elisa to assess any increase in pro-inflammatory behaviour, and expression of late adipogenesis markers were determined by western blot. results: ev treatment was shown to reduce lipid accumulation in adipocytes, with up to % reduction in lipids observed at the µg/ml dose. treatment was also shown to reduce the expression of late adipogenesis markers, and increase secreted levels of proinflammatory cytokines il- and il- by over fold and fold respectively. these results provide evidence for colorectal cancer derived ev involvement in the dedifferentiation observed in cancer associated adipocytes in vivo, displaying an altered phenotype, releasing lipid energy stores to fuel tumour growth and increasing pro-inflammatory signalling. summary/conclusion: studies have shown colorectal cancer evs may be involved in signalling which induces functional changes in cells within the tumour microenvironment. our work indicates that ev mediated dedifferentiation of resident adipocytes may potentially contribute to a microenvironment favouring cancer cell growth and metastasis. further work aims to elucidate the specific ev cargo which mediates these effects. introduction: ageing is a major risk factor for many human diseases. it is a complex process that progressively compromises most of the biological functions of the organisms, resulting in an increased susceptibility to disease and death. senescence is a cellular phenotype characterized by a stable cell cycle arrest. senescent cells are accumulated in the body during ageing. it contributes to develop age-related diseases and cancer. the alteration in intercellular communication with age has been demonstrated to be due to senescent cells developing a phenomenon denominated senescenceassociated secretory phenotype (sasp). exosomes are small extracellular vesicles (sev) ( - nm) of endocytic origin whereas microvesicles are formed by shedding of the plasma membrane. they contain nucleic acids, proteins and lipid that generally reflect the status of the parental cell and can influence the behaviour of neighbouring cells. methods: in this study, we demonstrated that the small extracellular vesicles (sev) contribute for transmitting paracrine senescence to proliferative cells firstly, we evaluated the presence of exosome-like particles in the sev from senescent cells by detection of exosome markers (alix, tsg and cd ), transmission electronic microscopy (tem) and nanoparticle tracking analysis (nta). to determine that sev from senescent cells are mediators of the paracrine senescence, we performed functional assays using cre-loxp reporter system and high-throughput results: besides, we confirmed at a single-cell level that the proliferative cells internalizing sev from senescent cells activate senescence process using the cre-reporter system. sev protein analysis from senescent cells by mass spectrometry (ms) and validation of top candidates using a functional sirna screen identify interferon induced transmembrane protein (ifitm ), a component of non-canonical interferon (ifn) pathway, as partially responsible for transmitting senescence to proliferative cells. summary/conclusion: in conclusion, we found that sev are regulators of paracrine senescence and ifitm contained in senescent sev has an important role in the intercellular communication mediated through sev during cellular senescence . bin wu a , lei guan a , ye xu a , likang chin a , ting li a , youhai chen a , gordon mills b , jinqi ren a , ravi radhakrishnan a , rebecca wells a and wei guo a a university of pennsylvania, philadelphia, usa; b oregon health & science university, portland, usa introduction: extracellular matrix (ecm) remodelling and stiffening are associated with solid tumour progression. stiff ecm promotes cell proliferation, epithelial-to-mesenchymal transition (emt), metastasis and chemoresistance. hepatocellular carcinoma (hcc) appears frequently in patients with liver cirrhosis or fibrosis while the mechanism remains unclear. exosomes have been determined to serve as messengers to mediate intercellular communication and influence the extracellular. tumour-derived exosomes have been shown to influence tumour progression, metastasis, drug resistance, angiogenesis and immune regulation. thus, determining whether exosomes provide a mechanism by which stiff matrix modulates tumour microenvironment for tumour progression opens a new way to understand cirrhosis and oncogenesis. here we identified the molecular mechanism of matrix stiffening induced exosome secretion and showed the different effect of exosomes induced by soft or stiff matrix on tumorigenesis. methods: huh cells were cultured on acrylamide gels with the stiffness was modulated to pa (soft) or k pa (stiff). the exosomes in conditioned media were collected and analysed by nanoparticle trafficking analysis (nta) and immunoblotting. protein expression level in cells was screened by reverse phase protein array (rppa). inhibitor or shrna were used to inhibit target proteins function. in vitro phosphorylation and gef assay were used to verify rabin phosphorylation and activation. exosomes from cells on soft or stiff matrix were injected into mice to study their effect on tumour growth. results: ( ) stiff matrix promoted exosomes secretion. ( ) akt was activated by stiff matrix and was required for exosome secretion. summary/conclusion: matrix stiffening promotes exosome secretion via akt-rabin -rab pathway, contributing to tumorigenesis. tridimensional fibroblast culture revealed a novel exososome-dependent extracellular matrix secretion mechanism vincent clément a , bastien paré b , cassandra goulet a , thiéry de serres-bérard a , stéphane bolduc a , françois berthod a and françois gros-louis a a université laval, québec, canada; b norgen biotek corp., thorold, canada introduction: the extracellular matrix (ecm) is constituted of a variety of proteins and polysaccharides that are secreted locally and assembled into a thick d meshwork to provide biophysical and biochemical support to the surrounding cells, and regulate numerous cellular functions such as adhesion, migration and proliferation. dysregulation of ecm components or aberrant ecm remodelling can lead to various pathologies, as well as to play important roles in wound healing. although ecm secretion pathways are still largely unknown, the current paradigm is that ecmassociated proteins are synthesized in the endoplasmic reticulum and transported via the endosomes to the golgi apparatus en route to the cell surface and released by exocytosis. methods: to study ecm secretion pathway, we used dimensional ( d) cultured fibroblasts. this culture method technique has been used widely to generate tissue-engineered self-assembled stromal tissues, free of exogenous materials, and rely on long-term supplementation of sodium ascorbate into the culture medium. non-cancerous fibroblasts, grown in conventional two-dimensional ( d) cellular cultures, are known to be a poor source of secreted exosomes when compared to cancerous fibroblasts. results: here, we provide evidence that non-cancerous dermal fibroblasts can secrete high amounts of exosomes, containing different ecm proteins, when cultivated in a d fashion. we also demonstrated that dermal fibroblast-derived exosomes had the capacity to travel from one cell to another, induce cellular migration and promote wound healing. summary/conclusion: altogether, these findings reveal a novel exosome-dependent ecm deposition mechanism and suggest that the use of d-fibroblast cellular culture may emerge as an innovative approach in precision medicine to better study the role of patient-derived exosomes and ecm proteins in the establishment of cellular microenvironment in health and disease. anthony yan-tang. wu a , charles lai, yun-chieh sung b , steven t. chou c , vanessa guo c , jasper c. chien c , john j. ko c , alan l. yang c , ju-chen chuang c , hsi-chien huang b , syuan wu c , meng-ru ho d , maria ericsson e , wan-wan lin f , koji ueda g , yunching chen h , chantal hoi yin cheung i and hsueh-fen juan j introduction: bionanoparticles including extracellular vesicles and exomeres (collectively termed evs), have been shown to play significant roles in diseases and therapeutic applications. however, their spatiotemporal dynamics in vivo have remained largely unresolved in detail due to the lack of a limited suitable method. methods: we developed a bioluminescence resonance energy transfer (bret)-based reporter, palmgret, to enable pan-bionanoparticle labelling ranging from exomeres (< nm) to small (< nm) and medium and large (> nm) evs and larger evs (> nm). results: palmgret emits robust, sustained signals and allows the visualization, tracking and quantification of bionanoparticles from whole-animal to nanoscopic resolutions under different imaging modalities, including bioluminescence, bret, and fluorescence. using palmgret, we show that evs released by lung metastatic hepatocellular carcinoma (hcc) exhibit lung tropism with varying distributions to other major organs in immunocompetent mice. ev proteomics identified hcc-ev lung tropic protein candidates associated with cancer progression, in which slco a and clic expression on non-tropic evs conferred lung-tropism, while cd gave spleen tropism. our results further demonstrate that redirected lung tropism decreases ev distribution to the liver, whereas the spleen tropism significantly reduces over time delivery to most major organs distribution including the liver and kidney. summary/conclusion: we established a multimodal and multi-resolution palmbret method to enable pan-bionanoparticle labelling and imaging and therefore quantification in live cells, whole animals, and preserved tissues. the method can resolve the intricate spatiotemporal dynamics of evs. palmgret revealed that evs derived from lung metastatic hcc are lung tropic, and the tropism can be conferred to non-lungtropic ev- t by decorating evs with identified hcc-ev membrane proteins. importantly, the enhanced ev delivery to tropic organs also significantly alters its distribution to other major organs. our findings suggest that the dynamics of ev biodistribution and targeted design should be investigated at the organ systems level in ev biology and therapeutic developments, respectively. tracking mesenchymal stem cell-derived extracellular vesicles (evs) in a in vivo cancer model introduction: small extracellular vesicles (sevs) are nanoparticles ( - mn) encircled by a phospholipid bilayer, derived from the endocytic pathway and released by all cells. sevs have an inherent role in cell communication and deliver cargo to target cells. mesenchymal stem cells (mscs) and have a natural ability to home to tumours and metastases while avoiding the host immune response. it is hypothesised that msc derived sevs (msc-sevs) also possess tumourhoming and immune-evading capacities therefore could provide a novel targeted delivery vehicle for treatment of cancer. it is imperative to elucidate msc-sevs migratory itinerary in vivo to support translation to the clinical setting. methods: this study aimed to image the interaction of labelled msc-sevs with cancer cells in real time in vivo. sevs were isolated from wildtype mscs and mscs with stably expressing red fluorescent protein (rfp) (via lentivirus) by the combined techniques of differential centrifugation, microfiltration and ultracentrifugation. isolated sevs were extensively characterised by transmission electron microscopy (tem), nanoparticle tracking analysis and western blot. nod scid gamma (nsg) mice with dorsal skinfold window chamber (dsfwc) were injected with either mda-mb- luciferase (luc) expressing cells or ht- -luc cells. bioluminescence imaging was performed to confirm tumour formation. a dose of x ^ msc-rfp-sevs was directly added to the window chamber and rfp expression detected using a microscope with rfp filter attachments. x ^ evs were incubated with the radionuclide, technetium- m tagged duramycin ( mtc-dur) for minutes at room temperature. excess radiolabel was removed using exosome spin column (invitrogen™). the mtc-dur-sevs were then added directly to the window chamber and charged particle imaging carried out. results: hours post-administration; the rfp signal was localised at the tumour site. radiolabelled sev signal could be detected minutes and hours after administration. msc-sevs were successfully detected at the tumour site following direct administration using two different tagging and imaging approaches. summary/conclusion: this promising preliminary data supports the potential of this approach for tracking msc-sev migration in vivo. future studies will investigate systemic tracking of msc-sev migration. vaughn garcia ; aejez sayeed ; rachel derita ; shiv ram krishn ; peter a. introduction: tumor-derived small extracellular vesicles (sevs) have emerged recently as mediators of tumorigenesis. however, the role of sevs in response to irradiation, a widely used therapy in prostate cancer, is not fully understood. methods: our study involved the tramp mouse model of prostate cancer. we used plasma sevs isolated using differential ultra-centrifugation and further isolated using iodixanol gradient fractionation. we also used nanoparticle tracking analysis (nta) to analyze sevs. mouse pelvises were irradiated using gy, for consecutive days. results: we first observed that upon pelvic irradiation of tramp mice, the levels of the signaling oncogene c-src are reduced in plasma-derived sevs, while the average size of sevs is increased from - nms to - nms. furthermore, we show that the sevs from irradiated cells lose the ability to stimulate anchorage independent growth and migration of recipient cancer cells. additionally, sevs from irradiated mice increase the amount of dna damage in recipient cancer cells. summary/conclusion: overall, our data show that irradiation of tramp mice (and prostate cancer cells) significantly reduces the pro-metastatic and pro-anchorage-independent growth potential of sevs when tested on human cells. changes to the composition and behavior of a cancer cell sev population via radiation therapy offers promise for future therapeutic approaches for prostate cancer. introduction: there are emerging physiological and pathological functions of extracellular vesicles (evs) in neurodegenerative diseases including alzheimer's disease (ad). brain derived-evs contain pathogenic proteins, such as tau, amyloid beta (aβ), which have been reported to contribute to cell-to-cell propagation in those diseases. investigation of the brain-derived ev cargo, therefore, is important to further understand the mechanisms of progression in neurodegenerative diseases. we developed the ev separation method from unfixed frozen mouse and human brain tissues and assessed the protein composition. methods: to establish the ev separation method, we separated evs from frozen mouse brain tissue using sucrose density gradient ultracentrifugation (sg-uc) or size exclusion chromatography to compare the results from the particle number, morphology and protein profiling by nta, tem and mass spectrometry. evs were then separated from cortical grey matter of ad (n = ) and control (n = ) by sg-uc. tau and aβ in the evs were measured by immunoassay. differentially expressed ev proteins were observed by quantitative proteomics employing machine learning. results: the separated evs were enriched in ev molecules and devoid of contaminant proteins by sg-uc, showing our method was successful. the levels of ps tau and aβ - were significantly increased in ad evs. annexin a (anxa ), neurosecretory protein vgf, neuronal membrane glycoprotein m -a (gpm a), and alpha-centractin (actz) were differentially expressed in ad evs. a combination of these proteins were confirmed to predict ad with the % accuracy by machine learning. summary/conclusion: these data suggest our method were suitable for the separation of brain-derived evs and ev anxa , vgf, gpm a and actz can be potential biomarkers for monitoring the progression of ad. edta stabilizes the concentrations of extracellular vesicles during blood collection introduction: to establish reliable biorepositories for research on extracellular vesicles (evs) as disease biomarkers, the release of evs during blood collection and handling must be avoided. currently, citrate is recommended as the anticoagulant for blood ev research, but citrate does not inhibit the release of evs from activated platelets. the release of platelet-derived evs excludes pneumatic tube transport and makes assays time dependent, thereby limiting clinical compatibility. therefore, we aim to stabilize the release of platelet ev concentrations. methods: blood samples were collected from healthy individuals and subjected to common circumstances known to induce platelet activation. blood was (i) incubated with or without thrombin receptor-activating peptide (trap; n = ), a potent platelet activator, (ii) send to the lab by a routine blood transport (pneumatic tube system; n = ), and (iii) stored at room temperature or at °c for hours (n = ). the concentrations of evs from platelets (cd +), activated platelets (p-selectin+), erythrocytes (cd a+), and leukocytes (cd +) were determined by flow cytometry (apogee a -micro). results: following activation by trap, concentrations of platelet-derived and activated platelet-derived evs increased . -fold and . -fold in citrate-anticoagulated blood, compared to . -fold and . -fold in edta-anticoagulated blood (edta vs citrate: p = . and p = . , respectively). preliminary data show that during pneumatic tube transport and routine sample handling, both platelet-and activated platelet-derived evs were more stable in edta compared to citrate. the concentrations of evs from erythrocytes and leukocytes were unaffected under all studied conditions. summary/conclusion: to conclude, edta stabilizes platelet ev concentrations during and after blood collection, which would facilitate pneumatic tube transport, enhance reliability and thereby improves the establishment of reliable biorepositories for ev research. introduction: cancer-cell secreted extracellular vesicles, called exosomes, are an emerging biomarker for cancer liquid biopsy. profiling of cancer-associated exosomes usually required lengthy, and multi-step procedures; therefore simple and easy-setup sensing methods are urgently needed for diagnosing cancer in a timely manner. chirality, the foundational property of all biomolecules, including exosomal proteins, can be utilized for exosome detection and differentiation using recent advances in chiral nanostructures. we found that microfluidic sensors can be successfully implemented for successful detection of cancer-associated exosomes taking advantage of unusually high circular dichroism (cd) of chiral gold nanoparticles (aunps). circular dichroism-based exosome (cdexo) detection utilizes chiroplasmonic enhancement of cd signatures of cancer-associated exosomes. we first synthesized donut-shaped aunps conjugated with l-cysteine and immobilized the aunps on a glass slide using a layer-by-layer assembly. the aunps on slide glass were surface functionalized by the standard biotin-avidin reaction after mua treatment. biotinylated annexin v marker, targeting phosphatidylserine (ps) expression on cancer-associated exosomes, was conjugated to the aunp surface. μl of exosome samples from cancer cells (a and h ) or normal cells (mrc ) were injected into the pdms microfluidic device and incubated for minutes. the cd signal before and after exosome exposure was monitored, compared, and systematically analysed as a rapid technique for the detection of exosomes with high sensitivity. results: we showed that the cdexo signals from cancer exosomes showed . folds absolute cd peak value change and . folds shift, respectively, compared to that of healthy exosomes. importantly, the cdexo sensing method takes less than mins in terms of total scanning time and requires minimal sample volumes. from the preclinical studies using blood samples from cancer patients and healthy donors, we found that cancer patients show stronger band shift and signal change comparing to that of healthy donors, implying our platform could be used for cancer diagnosis. summary/conclusion: this new versatile and sensitive method based on chiroplasmonic exosome detection paves the way to profiling disease-associated exosomes in a timely manner for minimal volumes of liquid biopsies. ev classification and fractionation strategy using surface charge labelling takanori ichiki a , hiroaki takehara a , hirofumi shiono b and hiromi kuramochi a a the university of tokyo, bunkyo, japan; b innovation center of nanomedicine, bunkyo, japan introduction: the development of new classification technology is required based on the evaluation of physicochemical properties of exosome surfaces and the diversity of constituent molecules. in this presentation, we present the electric charge activated exosome sorting platform comprising microfluidic device technology and electric charge labelling technique. methods: the single nanoparticle analysis platform, which has been developed by our research group, images rayleigh scattered light (elastically scattered light) obtained by irradiating nanoparticles with convergent laser light and provides information of individual particles by image processing. the method that utilizes electrokinetic phenomena, unlike the method using fluorescent labels, measures the properties of the particle surface without serious difficulty in principle even if the particle size is on the order of tens nanometres, and further enables to perform fractionation. since the number of particles usually handled in exosome research or its envisioned application is enormous, it is not realistic to take an approach such as a cell sorter in which particles are sequentially manipulated one by one following the measurement results of individual particles. results: particles receive attraction or repulsion by an external field according to the charge density on the surface, so there is no need to control the external force, and it is possible to design a device that can autonomously fractionate particles according to the difference in zeta potential. summary/conclusion: in conclusion, we have proposed and demonstrated the new concept of electric charge activated ev sorter. funding: this research was partially supported by the center of innovation program (coi stream) from the japan science and technology agency. high throughput exosome analysis by using reversible microfluidic electrochemical sensor system introduction: exosome is one of the important extracellular vesicles (evs) released from parental cells and it contains various types of molecular cargos from its original cell including proteins, messenger rna (mrna), and micro rna (mirnas) [ ] . the exosomes have recently emerged as biomarkers for early stage cancer detection because the number of exosomes originated from cancerous cells are significantly higher than those from normal cells [ ] . since many different types of exosomes exist in the whole blood, it is necessary to isolate and detect disease-specific exosomes. for this reason, the isolation and the detection of exosomes is an important research issue and has been studied by many groups. however, limitations such as low throughput and low recovery still make it difficult to use exosomes in diagnostics and therapeutics. methods: in this study, we developed an integrated microfluidic electrochemical biosensor to extract plasma from whole blood and subsequently detect cancer related exosomes in a continuous manner. this consists of two parts. the first part is a channel for extracting plasma containing exosomes from whole blood, and the second part is a channel combined with an electrochemical sensor for multiple detection of various exosomes in the extracted plasma. previously, a multi-orifice flow fractionation (moff) channel that consists of a series of expansion and contraction structures has been developed in our group. in this channel, the blood cells are moved to sides of channels by hydrodynamic forces and then are eliminated to outlets. at this time, the plasma is moved to the electrochemical sensor part, the exosomes in the plasma are captured to the electrodes immobilized with the specific antibodies and are quantified the amount of cancer-related exosomes. results: using this chip, blood cells were eliminated from the whole blood with over % of separation efficiency at µl/min flow rate and exosomes were collected continuously with high recovery (~ %). in order to quantify various types of exosomes, a labelfree electrochemical biosensor with electrochemical impedance spectroscopy (eis) was used for the continuous detection of exosomes. the limit of detection was x ^ exosomes/ml. summary/conclusion: the developed device is an integrated device capable of separating exosomes from whole blood with high purity and quantitating exosomes through the electrochemical sensor in a continuous manner. , , ) . the development of highthroughput techniques capable of simultaneously monitoring physical and biochemical properties of evs would significantly simplify and accelerate the characterization process. in this context, microfluidic technology is emerging as an attractive platform. here, we present a microfluidic device based on the combination of diffusion sizing and multi-wavelength fluorescence detection to simultaneously provide information on ev size, concentration and composition. methods: the diffusion of evs in the microfluidic channel provides information on their size distribution, and four different staining protocols with high signalto-noise ratios track different ev native molecules. evs are separated from unbound fluorophores directly during the microfluidic analysis, therefore avoiding the need for sample pretreatments and allowing to operate the device as a single-step immunoassay. results: the microfluidic device coupled with complementary staining techniques allows to individually detect and size particle populations with different ev components such as lipids, primary amines and the ev marker cd . we demonstrate that this approach can probe the abundance of ev-specific markers and impurities such as lipoproteins with high throughput and low sample consumption. summary/conclusion: we present a microfluidic technique capable of characterizing and quantifying evs at low costs, in a time-scale of minutes and requiring only up to µl of non-pretreated sample. this method is an important complementary tool to the current array of biophysical methods for ev characterization, in particular for high-throughput screening applications. funding: h -eu. . . -fet open programme via the grant agreement . immunomagnetic isolation of specific subpopulations of exosomes for liquid biopsy via nano-architected porous materials introduction: exosomes offer the potential to reveal significant biological information in many areas of clinical importance by virtue of their rna contents and protein surface markers. this abstract reports the fabrication of a device for high throughput targeted immunomagnetic capture of exosomes via the use of highly-ordered nano-architected porous metal lattice materials. methods: we have invented a fabrication technique to precisely make millions of nanoscale exosome sorting devices that can operate on unprocessed plasma. each nanoscale device can precisely sort targeted exosomes from background vesicles but is too slow for practical use individually. however, the operation of millions of these devices in parallel preserves the precision of nanoscale sorting while also enabling high throughput and robust use on raw plasma samples. the metal lattice within which these devices are contained is assembled via metal electroplating onto a selfassembled polystyrene bead lattice with face-centred cubic (fcc) symmetry with nanometre pores. the devices feature a conformally-coated layer of nickel-iron with gold passivation atop a base layer of nickel, resulting in a lattice of millions of nanoscale pores capable of magnetic sorting of exosomes tagged via surface-marker-based immunomagnetic labelling with magnetic nanoparticles. results: compared to our previous work on immunomagnetic exosome capture via commercial track-etched membranes (tempo), this device offers superior capture due to increased surface pore density (> x) and three-dimensional pore density (> x) alongside lower required sample volume due to decreased noncapturing volume in the device. finite-element analysis simulations show that strong magnetophoretic traps emerge at the pore boundaries in this structure between higher-permeability metals such as nickeliron permalloy and the lower-permeability sample fluid in the device. preliminary experimental data shows that this device can isolate iron nanoparticles in solution with > x enrichment from input and x capture efficacy versus tempo. summary/conclusion: current methods of exosome isolation such as ultracentrifugation and column chromatography all suffer from low throughput and limited yield. the application of inverse opal materials towards exosome capture offers the potential for isolation of specific exosome populations from very low clinical sample volumes or sparse biological signals. micropatterned growth surface topography affects extracellular vesicle production colin l. hisey a , james hearn b , yohanes nursalim a , vanessa chang a , cherie blenkiron a and lawrence w. chamley a a the university of auckland, auckland, new zealand; b university of auckland, grafton, new zealand introduction: extracellular vesicles are micro and nanoscale packages released by all cells and play an important role in cell-to-cell communication by shuttling biomolecules to nearby and distant cells. however, producing enough evs for many in vitro studies using conventional tissue culture techniques can be challenging, and despite the success of some bioreactors in increasing ev-production, it is still unknown how many independent culture conditions like growth surface topography can alter the production and content of evs. methods: standard mm petri dishes were patterned with µm tall polystyrene microtracks spaced by , and µm across a mm area using standard microfabrication techniques including photolithography, soft lithography and microtransfer printing. the micropatterns were characterized with sem and profilometry, then activated with oxygen plasma and uv sterilized. mdamb cells were seeded onto patterned and smooth (control) dishes and grown in serum-free media for the final hours of culture. evs were isolated using sequential ultracentrifugation of conditioned media and characterized using nta, tem and western blot. cell morphology was imaged using immunocytochemistry and single cell migration was characterized using time-lapse microscopy and manual single cell tracking in fiji. results: we demonstrate the simple and repeatable fabrication of microtracks across a large surface area in order to culture cells on topographically patterned growth surfaces. furthermore, we show that the µm spacing produced significantly more evs than other patterns as well as the highest cell aspect ratio and average single cell migration speed (p < . ). summary/conclusion: these findings have implications in both biomanufacturing of evs and potentially in enhancing the biomimicry of evs produced in vitro. however, further experimentation to assess the differences in cargo on patterned growth surface topographies compared to conventional methods is still required. funding: this project was funded by the maurice and phyllis paykel trust. using miscroscale thermophoresis and surface plasmon resonance to measure the interactions of extracellular vesicles mst is a quick method, easy to handle, has a low sample consumption, has no limitation on molecule size, and enables measurements in solution, either in various buffers or complex biological liquids. these properties make mst an interesting tool for research of extracellular vesicles (evs); therefore, our aim is to apply this method to evs. methods: evs were isolated from jurkat cell line by differential centrifugation. microscale thermophoresis (mst) and surface plasmon resonance (spr) were used to analyse the interaction between antibody and evs. results: we have demonstrated that interactions of evs with antibodies could be analysed by mst. however, the tiny glass capillaries for sample mounting represent a challenge due to adhesion of evs to their surface. we have tested commercial capillaries as well as prepared capillaries in house coated by liposomes or bovine serum albumin. the interactions between evs and antibodies were confirmed by surface plasmon resonance (spr), which is an established method for studying the interactions of evs. introduction: the isolation of extracellular vesicles (evs) from cell culture supernatants and complex body fluids, such as blood and urine, is of high importance for ev research as well as for future medical applications in diagnostics and therapy. nevertheless, it is still challenging to reach the desired recovery, purity and specificity due to many manual and time intensive sample preparation steps. conventional centrifugation for ev isolation or sample preparation prior to affinity-based separation methods can damage evs and cells, leading to misinterpretation of results or inactive evs. alternative field flow fractionation methods employing acoustic fields are highly promising, but so far limited to laboratory usage, based on a complex (moulding) fabrication and/or hardly reproducible. here, we present an innovative surface acoustic wave (saw)-based acoustofluidic device for gentle sorting of cells and particles. methods: our device consists of interdigital transducers patterned on a piezoelectric substrate generating saw propagating on the substrate surface. upon interaction of saw with our on-chip structured, fluid-loaden microchannels, an acoustic pressure field is developed across the fluid wherein particles are suspended. this pressure field can be employed to simply manipulate cells and particles based on their intrinsic properties, such as size, density and compressibility in continuous flow. the device is manufactured using precise and low-cost microtechnological methods and is suitable for reproducible mass fabrication. results: we demonstrated the separation of blood components, i.e. the sorting of erythrocytes and thrombocytes. furthermore, we could also show results on thrombocyte activation indicating a gentle separation without damaging these shear-sensitive cells, as well as first results on plasma separation from whole blood samples and nanoparticle sorting. summary/conclusion: our unique acoustofluidic sorting technology for complex suspensions has the potential to overcome the need for time-effective, cheap and gentle separation of evs. funding: this work was supported by efre infrapro project "champ: chip-based acoustofluidic medtech platform". nanophotonic platform for cancer-associated exosomal microrna detection introduction: exosomes have an important role in intercellular communication at physiological and pathological processes. their cargo includes micrornas (mirs), single-stranded non-coding rnas, involved in alterations on recipient cells, such as development of tumourous phenotype and metastasis. more particularly, mir- excels due to its association with several cancers. determining exosomal mirs as cancer indicators demands selective and accurate methods, which are not currently available or entail high costs. colorimetric photonic-based assays are a promising label-free alternative, which dismisses complex apparatus for signal reading since biorecognition is detected by colour change. moreover, the clinical and economic systems have also been demanding a decrease on the green footprint of biosensors, requirement fulfilled with naturally derived biomaterials. methods: herein, the biosensor is constructed on a biopolymer matrix to meet the requirements of an eco-friendly disposable device, and it is based on a photonic structure obtained by imprinting a nanopattern on the polymer surface. then, the surface is functionalized with the complementary oligonucleotide sequence of mir- as sensing probe. a labelfree detection is thus envisioned and the sensor performance is evaluated by changes in the optical properties when the target is present. results: the combination of biological materials conducted to a biosensor support with great flexibility and low water permeability, allowing easy surface functionalization. the self-reporting ability of the photonicbased sensor enables high intensity colours detected by naked eye. summary/conclusion: the alliance with the high selectivity of oligonucleotide hybridization is expected to offer great exosomal mir- recognition ability and an optimistic perspective for utilization in clinical setups. funding: the authors acknowledge the financial support from the european commission/h , through mindgap/fet-open/ga project. introduction: urinary extracellular vesicles (uevs) are important intercellular communicators. by systems biology integration, uevs prove to be relevant in genitourinary disease detection. however, it has recently been shown that labelled evs administered to the circulation can be detected in the urinary system, as well. thus, this pilot study aimed at phenotyping haematopoietic surface markers on uevs to create enough plausibility for future non-invasive biomarker studies of circulation and immune disorders that may translate into urine but are not yet timely recognized. methods: urine was obtained from healthy men signing a written informed consent (n = ). sampling was approved by the local ethics committee and in compliance with the declaration of helsinki. cell-free urine was obtained by serial centrifugation and ml, each, were utilized for the macsplex exosome kit, human (miltenyi biotec). the manufacturer's recommendations were followed to examine distinct uev surface markers of cd +/cd +/cd + vesicles in a multiplexed bead-based manner including respective controls. the accuri c (bd) was utilized for data acquisition. for further misev -compliant characterization, cd +/cd +/cd + uevs were isolated by immunoaffinity and analysed by fluorescence nanoparticle tracking (f-nta), transmission electron microscopy (tem) and western blotting (wb). urinary creatinine (ucrea) was determined to control for variances in urinary dilutions and used for data normalization. results: except cd , all other surface markers could be identified. the most abundant markers were cd and cd , which were detected in % of samples, followed by cd / ( %), cd ( %), cd and cd ( %, each). cd ( %), cd , cd ( %), cd e ( %) and cd showed similar relative median fluorescent intensities (rmfi), while cd yielded significantly higher (p = . ) and all other markers significantly lower rmfi (p < . ). tem and f-nta revealed cup-shaped vesicles ( ± nm) with . ± . e + particles/g ucrea. wb indicated uev isolates that were positive for alix, syntenin, tsg , cd , cd and cd without any uromodulin or calnexin contamination. summary/conclusion: our results imply that considerable quantities of circulatory evs are, indeed, filtered into urine and could serve as valuable non-invasive biomarkers for systemic dysfunctions. cardiovascular risk markers are strongly related to numbers of circulating extracellular vesicles ruihan zhou a , esra bozbas a , plinio ferreira b and parveen yaqoob a a university of reading, reading, uk; b imperial college london, london, uk introduction: extracellular vesicles (evs) are small plasma membrane-derived vesicles released from various cells, which potentially affect many physiological and pathophysiological processes, and are emerging as a potential novel biomarker in cardiovascular diseases (cvds). however, there is little information about the association of circulating ev levels with traditional cardiovascular risk markers and cvd risk score. methods: • subjects (n = ) aged - yrs with moderate risk of cvds were recruited and assessed for body mass index (bmi), blood pressure (bp) and plasma lipid profile (triacylglycerol, total cholesterol and high-density lipoprotein). • evs were isolated from platelet-free plasma by size exclusion chromatography and analysed by both nanoparticle tracking analysis (nta) and flow cytometry (fcm). nta was used to measure the concentration and size distribution of evs population, and evs were phenotyped by fcm via a colour panel, which included annexin v (for the majority of circulating evs), cd (for plateletderived evs) and cd (for endothelialderived evs). • the association between risk markers and ev numbers was examined by pearson's correlation coefficient and stepwise multivariate regression model. analysis of covariance (ancova) was performed after adjustment for various variables to determine the correlation between the quartile range of ev numbers and -yr cvd risk detected by qrisk . results: ev numbers, as determined by nta, were strongly associated with bmi (r = . , p < . ), blood pressure (systolic bp: r = . , p = . ; diastolic bp: r = . , p < . ) and plasma triacylglycerol levels (r = . , p < . ). plasma total cholesterol level was positively associated with platelet-derived evs, determined by fcm (r = . , p = . ). a multivariate regression model demonstrated that plasma triacylglycerol and diastolic bp independently predicted total ev numbers, with plasma triacylglycerol concentrations explaining . % of the variance for total ev numbers. an additional . % of the variance in total ev numbers was predicted by diastolic bp. ancova of the -yr cvd risk score in the quartile range of total ev numbers were positively and independently associated. summary/conclusion: bmi, blood pressure, plasma triacylglycerol and total cholesterol levels are strongly associated with ev numbers. plasma triacylglycerol and diastolic bp independently predict circulating ev numbers. elevated numbers of evs are independently associated with -yr cvd risk. introduction: extracellular vesicles from cardiospherederived cells (cdc-evs) are known to be anti-inflammatory in various disease models. to further dissect the mechanism, we examined the effects of cdc-evs on t lymphocytes. methods: naïve cd + t cells were isolated from secondary lymphoid organs of foxp -rfp reporter mice, using magnetic-activated and fluorescence-activated cell sorting. cells were subsequently polarized into effector subtypes (th , th , and th ), as well as regulatory t cells (tregs), and the effects of exposure to human-derived cdc-evs on proliferation and cytokine production were assessed. cdc-evs were isolated from serum-free, -day conditioned medium, using ultrafiltration by centrifugation. results: after polarization and culture for days, cdc-evs resulted in dose-dependent and cell-specific proliferative responses. effector t cells (th , th , th ) showed either no change in proliferation (th ) or decrease in proliferation (th , th ), compared to the vehicle control. in contrast, tregs proliferated much more than control (p < . ). next, we sought to characterize the changes in cytokine production by each effector t cell and tregs. compared to the vehicle control, exposure of polarized effector t cells to cdc-evs had little effect on the expression of characteristic cytokine genes, including ifnγ and tnfα (th ), il and il (th ), or il a and il f (th ). in contrast, exposure of tregs to cdc-evs resulted in~ -fold increase in expression of il , a key paracrine agent utilized by tregs in suppression of inflammation. this response was specific to cdc-evs insofar as it was not recapitulated with dermal fibroblast exosomes. concentrations of il- in the culture media of cdc-ev-conditioned tregs mirrored the increases in gene expression. summary/conclusion: cdc-evs potentiate tregs by increasing their proliferation and enhancing production of il- . this offers an attractive therapeutic approach to inflammatory diseases that relies on harnessing an endogenous mechanism of immunosuppression. funding: nih t hl prostanoids impair platelet reactivity, thrombus formation and platelet extracellular vesicle release in patients with pulmonary arterial hypertension aleksandra gąsecka a , marta banaszkiewicz b , rienk nieuwland c , edwin van der pol d , najat hajji e , hubert mutwil f , sylwester rogula a , wiktoria rutkowska a , szymon darocha g , grzegorz opolski a , krzysztof j. filipiak f , adam torbicki g and marcin kurzyna g introduction: prostanoids (epoprostenol, treprostinil and iloprost) induce vasodilation in advanced pulmonary arterial hypertension (pah) but also inhibit platelet activation, thereby increasing the risk of bleeding. therefore, the platelet function and extracellular vesicle (ev) concentrations were measured in pah patients treated with prostanoids and compared to patients with pah not receiving prostanoids. methods: venous blood was collected from patients treated with prostanoids (study group; n = , ± years, % female) and patients not treated with prostanoids (control group; n = , ± years, % female). platelet reactivity was analysed in whole blood by impedance aggregometry using arachidonic acid (aa; . mm), adenosine diphosphate (adp; . µm) and thrombin receptor-activating peptide (trap; µm) as agonists. in a subset of patients, concentrations of evs from platelets (cd + and cd p+; pevs), leukocytes (cd +, levs) and endothelial cells (cd +, eevs) were measured in plateletdepleted plasma by flow cytometry (a -micro). platelet-rich thrombus formation was measured using a whole blood perfusion system. results: compared to the control group, patients treated with prostanoids had lower platelet reactivity in response to aa and adp (p = . ) and lower concentrations of pevs and levs (p ≤ . ). furthermore, thrombus formation was delayed (p ≤ . ) and thrombus size was decreased (p = . ) on prostanoids. epoprostenol did not affect platelet reactivity in vitro, but decreased the concentrations of cd + pevs (p = . ). in contrast, treprostinil and iloprost decreased both platelet reactivity in response to aa and adp (p ≤ . ) and the concentrations of pevs (p ≤ . ). all prostanoids delayed thrombus formation and decreased thrombus size (p ≤ . ). summary/conclusion: patients with pah treated with prostanoids have increased risk of bleeding both due to impaired platelet aggregation, ev release and thrombus formation, compared to patients not treated with prostanoids. antiplatelet effect of prostanoids varies: whereas epoprostenol decreases the release of pevs, treprostinil and iloprost impair platelet aggregation. funding: ag is supported by the national science centre, research project preludium / /n/ nz / . evdp is supported by the netherlands organisation for scientific research -domain applied and engineering sciences (nwo-ttw), research programmes veni . nanoflow cytometry identifies an imbalance of epithelium-and neutrophil-derived extracellular vesicles in the airway environment of paediatric cystic fibrosis patients brian dobosh, vincent giacalone, milton brown, lucas silva, lokesh guglani and rabindra tirouvanziam emory university, atlanta, usa introduction: progressive lung disease is the leading cause of mortality in cystic fibrosis (cf), a chronic condition characterized by recruitment of polymorphonuclear neutrophils (pmns) into the airways. newly arrived pmns are exposed to extracellular vesicles (evs) from the airway epithelium and pmns recruited before them. in controlled experiments, these evs were necessary and sufficient to induce pathological changes including reduced bacterial killing and immunosuppressive activities towards macrophages and t-cells. however, children with cf do not always show a high pmn presence in their airways, which suggests that the balance between pmn recruitment and the activity of other cells is still in flux in early stage disease. methods: we utilized spectral nanoflow cytometry to profile the single ev content of the bronchoalveolar lavage fluid (balf) from cf children (< years of age). for nanoflow cytometry, evs were stained with di- -anepps, and with epcam, cd b and cd (to ascertain epithelial, pmn, and macrophage origins, respectively). violet side scatter and/or fluorescence threshold triggering were used for ev detection. the ratio of neutrophil-to epithelial-derived evs in cf balf correlated positively with the percentage of pmns that are present in the airways (p = . , spearman's rho = . ). this ratio also correlated with the pragma disease score, which quantifies airway damage by chest computed tomography (p = . , rho = . ). summary/conclusion: using a method to quantify evs from specific cell types in vivo, we demonstrated that the ratio of pmn-and epithelial cell-derived evs tracks with airway damage and neutrophil influx, suggesting a critical interplay between these cells in early cf disease. this ev-focused method can be applied to other diseases in which sampling cells is difficult. future experiments will use cf balf biobanks to strengthen data presented here. funding: cf foundation (tirouv a ), emory paediatrics flow core. the potential of crude extracellular vesicle micrornas for the diagnosis of community-acquired pneumonia and for the detection of pneumoniarelated sepsis as a severe secondary complication introduction: circulating cell-free micrornas (mirnas), often associated to extracellular vesicles (evs), are essential for cell-cell communication in the pathogenesis of infectious pulmonary disorders. as early pneumonia diagnosis is often clinically challenging, advances in disease detection could improve outcomes. we characterized crude ev mirnas as potential biomarkers for community-acquired pneumonia and sepsis as a severe secondary complication. methods: individuals were enrolled into our study, subdivided into a training (volunteer n = , pneumonia n = , sepsis n = ) and testing cohort (volunteer n = , pneumonia n = , sepsis n = ). after precipitating crude evs from sera (mircury exosome isolation kit-serum and plasma) and extracting total rna, small rna sequencing was performed. mirnas were selected as biomarker candidates by differential gene expression analysis (deseq ) and sparse partial-least-squares discriminant analysis (mixomics). technical and biological validation was performed by reverse transcription quantitative real-time pcr. group classification was predicted by partial-least-squares discriminant analysis. gene targets and causal networks were identified by ingenuity pathway analysis. results: differential gene expression analysis revealed significantly regulated mirnas in pneumonia compared to volunteers, and mirnas in pneumonia related to sepsis. based on sparse-partial least discriminant analysis, group separation was achieved by mirnas as discriminators with high sensitivity and specificity (area under the curve of the receiver operated curve: volunteer: . , pneumonia: . , sepsis: . ). mir- a- p (log fc = . , padj = . e- ) and mir- - p (log fc = . , padj = . e- ) differentiated between pneumonia and volunteers and mir- (log fc = − . , padj = . e- ) between pneumonia and sepsis. expression levels of mir- a- p and mir- were related to disease severity. mir- - p was higher expressed in pneumonia compared to volunteers and had equal expression in patient groups. prediction of group classification in the testing cohort was . %. signalling networks were constructed for "cellular and humoral immune response", "antimicrobial response" and "pathogen influenced signaling" involving the significantly regulated mirnas. summary/conclusion: crude ev mirnas are potentially novel biomarkers for community-acquired pneumonia and may help to identify patients at risk for progress to sepsis allowing early intervention and treatment. introduction: it remains unclear the specific mechanisms that lead to airways inflammation in asthma and the subsequent remodelling of the airways. exosomes, small extracellular vesicles, has become in an important mechanism of cell-to-cell communication and participate in diverse biological processes including inflammation. in this study, we hypothesize that exosomes and their mirna cargo play an important role in the proinflammatory status of the upper airway of asthma patients, especially in those patients with severe asthma. methods: in a pilot study, healthy subjects had induced sputum using standard methods. after several centrifugation steps, we were able to isolate exosomes from sputum supernatant by both precipitation and size exclusion cromatography (sec). exosome size was observed with transmission electron microscopy (tem) and the protein markers cd and cd were analysed by western blot (wb). then, total rnas were isolated from sputum exosomes and mirnas (mir- a-p, mir- - p, mir- a, mir- b- p, mir- - p, mir- - p, mir- - p, mir- - p, let- g- p), were evaluated by rt-qpcr. after the optimization of the methodology, healthy adults subjects and patients with persistent moderatesevere asthma, matched by age and sex were selected and induced sputum was collected. results: exosomes isolated with both methodologies (precipitation and sec) were observe under the tem with a correct range of size. furthermore, wb assay displayed a coherent protein profile for the exosome markers cd and cd . however, sec displayed low signal and the variability of between subjects was to higher. using the optimized method of precipitation, we observed that after normalization, mirna- a showed a significant increased (p = . ) in asthma patients compared to control. this mirna has been linked with an active proinflammatory status. summary/conclusion: our results confirm the presence of exosomes in induced sputum with promising applications in the field of asthma. the upregulation of exosomal mir- a, which is related with inflammation, suggest that exosomes could play a crucial role in the chronic inflammation of airway described in asthma patients. human nrf -active multipotent stromal cell exosomes reverse pathologic delay in the healing of cutaneous diabetic wounds introduction: multipotent stromal cells (mscs) have attracted much attention for their capacity to accelerate wound healing. exosomes, nanosized extracellular vesicles, may be key to translating msc therapy. we previously found that nuclear factor erythroid -related factor (nrf ) regulates msc promotion of diabetic tissue repair. here, we explore a novel role of nrf in exosome biogenesis and investigate whether exosome treatment recapitulates the effects mscs have on healing. methods: exosomes were harvested by differential ultracentrifugation of conditioned bone marrow derived msc media. for nrf -active exosomes, mscs were incubated with potent nrf activator, cddo-im. exosomes and mscs were vigorously characterized. full-thickness humanized-stented wounds were created on adult leprdb/db diabetic mice (db/db). exosomes were injected intradermally and circumferentially to the wound margin. results: mscs adopt an adherent fibroblast morphology, demonstrate robust osteogenic, chondrogenic, and adipogenic differentiation, express > % positive msc markers (cd , cd , cd , and cd ) and < % express negative markers (cd , cd , cd , cd , or hla-dr). immunoblotting of msc exosomes shows enrichment for positive exosomal markers cd , cd and tsg . nanoparticle tracking analysis (nta) shows a nanoparticle population with mean diameter of . ± . nm. transmission electron microscopy of exosomes reveals flattened cup-like spheres. nta demonstrates that nrf -active human mscs increase exosome secretion by %, compared to nrf -baseline mscs (p < . ). both nrf -baseline and nrf -active exosome treatment significantly reduced closure time to . and days respectively, compared to . days for vehicle-treated wounds (p < . ). this reduction eliminated the delay in closure time compared to wounds of c /b mice. nrf -active exosome treatment of db/db wounds reduced closure time by a further . days compared to untreated c /b wounds. at day , exosometreated db/db wounds have significant decreases in epithelial gap, expanded granulation tissue, and greater density of cd + vessels compared to vehicle-treated wounds. summary/conclusion: enhancing nrf function in mscs multiplies exosome yield. our results demonstrate exosome-based therapies hold tremendous promise and warrant further investigation for rapid translation. introduction: obesity increases prostate cancer aggressiveness and adipose tissue (at) is a rich source of extracellular vesicles (ev) that have been shown to contribute to pro-oncogenic effects in various malignancies. twist is a key mediator of tumour cell metastasis. the goal of this study was to determine molecular and phenotypic changes of prostate cancer cells in response to evs from obese human at and the role of different levels of endogenous twist . methods: ev were harvested from human at (atev) obtained from bariatric subjects or from at endothelial cells treated with proinflammatory cytokines (pic-ev) to mimic the obese at environment. evs were isolated by ultracentrifugation and characterized by electron microscopy, nta and protein markers. we determined the effect of atev and pic-ev on pc -ml prostate cancer cells proliferation and invasion. ev mirna cargo and transcriptome of pc -ml cells treated with atev or pic-ev were assessed using nanostring. to establish the contribution of twist to the ev-related phenotypic and molecular changes in recipient cells, we used pc -ml lines stably overexpressing or deficient in twist . results: atev from obese subjects and ev-pic from at endothelial cells both reduced invasion and increased proliferation in wild-type pc -ml cells. a molecular signature showing decreased expression of genes mediating invasion, adhesion and metabolism supported these functional effects. also atev and ev-pic shared a subset of mirna that target multiple mmps, inhibit glycolytic genes and target cell cycle inhibitory genes. pc -ml overexpressing twist showed an increase in both proliferation and invasiveness and this phenotype was supported by the transcriptomic analysis following ev treatment. summary/conclusion: ev produced by obese at or by at endothelial cells share a subset of mirna that in conjunction with increased twist expression contribute to tumorigenesis and metastasis of prostate cancer cells in vitro. introduction: exercise is associated with various health benefits, including the prevention and management of obesity and cardiometabolic risk factors. however, a strong heterogeneity in the adaptive response to exercise training exists. differential response to exercise training might be mediated by myokines (proteins, nucleic acids, metabolites) that can be released directly into the systemic circulation, or packaged within extracellular vesicles (evs). the objective of this study was to evaluate if changes in evs after acute aerobic exercise (ae) were associated with the responders phenotype following -week resistance exercise (re) training. methods: this is a secondary analysis of plasma samples from the exit trial (clinical trial # ). eleven sedentary obese youth ( . ± . years, bmi ≥ th percentile underwent an acute bout of ae ( % heart rate reserve, min). blood was collected before [time (at) − , min], during [at , , min], and after [ min (at ), min (at )] exercise. afterwards, youth participated in -week re programme, and were categorized into responders or non-responders (nr) based on changes in insulin sensitivity (above or below percentile). primary outcome: evs were isolated using size exclusion chromatography (izon®) at baseline (at ), immediately after ae (at ) and after recovery (at ). ev protein concentration, size, and zeta potential were analysed in a single-blind fashion. results: responders had larger evs (~ . nm) as opposed to nrs (~ . nm) at at (p < . ) and this pattern was maintained at at and at , though not significant (p = . ). nrs displayed differential ev size distribution (peaks at nm or nm), while ev distribution was highest at nm in responders. no difference in average zeta potential or total ev protein yield was observed between groups. an increase in ev yield with exercise time and recovery was observed in both groups. summary/conclusion: our preliminary data suggest that ev size is significantly increased after an acute bout of ae in obese youth responders. further research to delineate the role of evs as predictors of exercise adaptation is warranted. funding: funded by dream and research manitoba. using dual-fluorescent reporter mice to track tissue-specific extracellular vesicles andrea estrada, gabriella hehn, zackary valenti, christopher allen, nicole kruh-garcia and dan s. lark colorado state university, fort collins, usa introduction: extracellular vesicles (evs) from tissues like skeletal muscle (skm) and adipose tissue (at) have been implicated in human disease but are understudied. skm is likely a major player in ev biology as it accounts for~ % of total body mass. tools to define cellular ev origin are needed because tissues like skm are comprised of a variety of cell types. here, we describe our ongoing efforts using the dual fluorescent mg/mt mouse as a tool to analyse skm-myocyte derived evs. methods: wild-type (wt) and mg/mt mice were used for these studies. mg/mt mouse cells express membrane-tagged red (mt) or green (mg) fluorescent protein in the absence or presence of cre, respectively. we made skm myocyte mg expressing mice using a mouse expressing cre on the human skeletal actin promoter. blood was collected via cardiac puncture and platelet-free plasma was obtained via centrifugation. plasma evs were isolated using exoquick, exoquick-tc or size exclusion chromatography. skm and at were dissected into~ mm chunks, placed in serum-free dmem and incubated for hours. tissuederived evs were isolated using exoquick-tc. ev abundance was determined with a horiba viewsizer. individual evs were analysed with a cytek aurora spectral flow cytometer. settings were optimized using polystyrene beads and spectral unmixing was performed to allow detection of mg and mt. results: in wt mice, skm releases > times more evs than adipose tissue per unit of mass (p < . using paired student's t-test). since skm is also a major component of total body mass, these data further emphasize the importance of skm-derived evs. skmderived evs from wt mice were not fluorescent (< . % of events). evs from mg/mt mouse skm overwhelmingly expressed mg (> % of events) with negligible (< %) expression of mt. at-derived evs robustly expressed mt but lacked mg. summary/conclusion: these data provide "proof-ofprinciple" that mg and mt are readily incorporated into evs secreted ex vivo. surprisingly however, plasma evs from mg/mt mice expressed very little mg (~ %) or mt (~ %). this observation was confirmed with three separate isolation techniques. we are currently exploring possibilities to explain this finding, including: ) modification of evs post-secretion, ) clearance of fluorescent evs by the liver or ) that evs secreted from tissues remain predominantly in the interstitial space. funding: this work was supported by an innovative project award from the american heart association ( ipa ) to dsl. endothelial cd delivery of fa loaded extracellular vesicles is critical for thermogenesis. introduction: membrane cd facilitates tissue fatty acid (fa) uptake. we recently found that endothelial cell (ec) cd controls muscle and adipose tissue fa uptake, and influences the tissue's metabolic phenotype. the mechanism for cd -facilitated fa uptake is unknown. here we examined the role of ec cd in thermogenesis and in fa delivery to brown fat tissue. methods: adult male mice were housed individually, restricted from food during acute ( hr) cold exposure ( °c) with core temperature monitored every minutes. after hours, animals were sacrificed and samples collected for analysis. for cellular studies, human microvascular (lonza) or primary murine microvascular ec were used. for primary cells, crude cell pellets from lung homogenates were purified using mouse-cd magnetic beads (miltenyi). for microscopy studies, alkyne fa (cayman) was added to cells and to enable visualization of internalized fas, click chemistry (invitrogen) used to label alkyne-fa with alexa . for radioactive studies, primary lung ec were serum starved for hrs and incubated overnight with h-oleic acid bound to fa-free bsa ( : ratio). media was collected, clarified by centrifugation to remove microvesicles and debris. small extracellular vesicles (sevs) were isolated from clarified media using total exosome isolation reagent (invitrogen) and counted for radioactivity. results: basal core body temperatures are similar in mice lacking ec cd (eccd -/-) compared to controls (cd fl/fl). however, during cold exposure at °c , eccd -/-are unable to maintain body temperature (p < . ). plasma free fa are higher in cold exposed eccd -/-indicating fa clearance by brown fat is impaired. mitochondrial function and expression of thermogenic and mitochondrial genes in brown fat from eccd -/-and cd fl/fl mice were similar. these data suggested that endothelial delivery of fas is necessary for thermogenic maintenance of body temperature. to examine fa handling by ecs we used alkyne fas to visualize the process. we found that fas are transferred by microvascular ec through caveolae-mediated transcytosis involving src signalling and cav- phosphorylation. the internalized cav- and cd positive vesicles containing fas are released as sevs. to determine the dependence of cd on this process, we treated primary microvascular ec with radiolabeled fa and found that sevs secreted by cd -/-cells contain less labelled-fa (p = . ). summary/conclusion: endothelial delivery of fa is critical for thermogenesis. our working model for the mechanism of fa uptake by brown adipose tissue is the following: endothelial cells transfer the fa through caveolae-mediated transcytosis and secrete small extracellular vesicles (sevs) that help deliver fas to brown adipocytes. funding: this work is supported by nih grants dk and dk . introduction: diet-induced obesity modifies intestinal permeability leading to bacteria infiltration and to a decrease in the number of immune cells protecting mucosa. as orange consumption is beneficial for human health and used in preventive medicine, we determined whether orange juice-derived nanovesicles (onv) might be recommended as nutritional strategies for the treatment of intestinal complications associated with obesity. methods: onv isolated from fresh orange juices were characterized by lipidomic, metabolomic, microscopy, nta and for their stability during digestion. intestinal barrier (ib = caco- cells+ht- cells differentiated with oleic acid) were treated with onv and co-cultured with adipocytes to monitor ib fat absorption and release. obesity was induced in mice fed for weeks with a high-fat high-sucrose diet (hfhs mice vs standart chow diet mice). then half of the hfhs mice were gavaged with micrograms/day for weeks. results: onv did not modify high-fat high-sucrose diet-induced obesity and insulin resistance but reversed diet-induced gut modifications. six hours post-gavage, onv accumulated preferentially in jejunum involved in lipid absorption. in jejunum, and no other intestinal region, onv increased villi size, restored immune response and decreased barrier permeability in hfhsd mice. in addition, onv-treated mice had increased expressions of acat , angptl and dgat , but a decreased expression of fabp , fatp , mtp vs hfhsd animals, which indicated that fat absorption, tg synthesis and chylomicron release were strongly reduced. similarly to other plant-derived nanovesicles, these results were likely associated with onv lipid and metabolite compositions (strong enrichment in bioactive phospholipids: pe, pa, pc, pi and leucine) as onv did not resist to harsh digestive conditions in vitro and were poorly incorporated in enterocytes. as the effects of onv on the decrease in tg content and epithelial cell growth were also observed in vitro, gut microbiota unlikely participate to these effects. summary/conclusion: onv are important bioactive compounds of orange juice and for the first time we demonstrated that they can modulate lipid metabolism in the intestinal barrier associated with morphological changes. interestingly onv treatment targets mtp and angptl mrnas, therapeutic intestinal targets to reduce plasma lipids and for attenuating inflammation in gastrointestinal diseases. therefore, onv might be used to reduce the development of dyslipidemia-associated diseases and to restore intestinal functions in obese patients. funding: olga triballat institut; benjamin delessert institut, inrae institut. association, structure, and function of fibronectin in extracellular vesicles from hepatocytes xinlei li, ruju chen, sherri kemper and david brigstock nationwide children's hospital, columbus, usa introduction: we have shown that extracellular vesicles from normal hepatocytes have anti-fibrogenic activity and that they preferentially bind to hepatic stellate cells (hscs, the principal fibrosis-causing cell in the liver) and hepatocytes. in this study, our goal was to determine the molecular nature of the ev components involved in cell binding. fibronectin (fn ) is a key component of extracellular matrix, functioning in processes including cell adhesion, differentiation, and wound healing. two types of fn are present in vertebrates, of which the soluble plasma fn is derived principally from hepatocytes, while cell-associated fn is produced by numerous cell types. here we describe a novel function of plasma fn in facilitating binding of hepatocyte evs to target cells. methods: differential ultracentrifugation was used to collect evs released by parental mouse aml hepatocytes, fn ko aml cells in which fn was ablated using crispr-cas , primary human or mouse hepatocytes, or human hepg cells, or from human or mouse serum. evs were characterized by nanosight tracking analysis (nta), western blot, iodixanol gradient ultracentrifugation, and mass spectrometry. the binding efficiency of pkh -labelled evs from parental (ev-hep) or fn ko (ev-hepfn ko) aml cells was analysed in hepatocytes or hscs. swiss webster mice were injected with ccl for five weeks to induce liver fibrosis, with some mice also receiving i. p. administration of ev-hep or ev-hepfn ko over the last two weeks, followed by determination of hepatic fibrogenic genes by qrt-pcr. results: ev-hep or ev-hepfn ko were - nm in diameter and positive for common ev markers (cd , cd , flotillin- ). mass spectrometry showed that fn was the most abundant protein in ev-hep and comprised principally the plasma form. the abundant presence of ev fn was verified by western blot and co-immunoprecipitation with anti-cd or antiflotillin- . western blot showed that fn was also abundant in evs from primary human or mouse hepatocytes, hepg cells, and human or mouse serum. fn and ev-hep co-sedimented at a density of~ . g/ml. ev-hepfn ko yield and size-range were similar to those of ev-hep, suggesting that ev biogenesis is fn -independent. as compared to ev-hep, the binding of ev-hepfn ko to target cells was highly reduced whereas ev binding was independent of fn expression by the target cells themselves. both ev-hepfn ko and ev-hep were anti-fibrogenic in vivo but only ev-hep attenuated collagen ⍺ expression in mouse hscs in vitro. summary/conclusion: fn is abundantly associated with hepatocyte evs and facilitates ev binding to target hepatocytes or hscs. additional studies are needed to clarify the functional role of fn in mediating ev-hep anti-fibrogenic actions in vitro or in vivo. elevated glucose increases soluble and aggregated forms of human islet amyloid polypeptide in islet-derived extracellular vesicles -implications in type diabetes and islet transplantation introduction: type diabetes (t d) is characterized by reduced beta cell mass and function. islet amyloid, formed by aggregation of human islet amyloid polypeptide (hiapp), contributes to progressive beta cell loss in t d. amyloid also forms in human islets during pre-transplant culture and following transplantation in patients with type diabetes (t d) which is associated with graft failure. the cellular mechanisms underlying islet amyloid formation are still unclear. in this study, we examined the potential role of islet-derived extracellular vesicles (ev) in the clearance of soluble and aggregated (pro)iapp species from beta cells and amyloid formation. methods: human islets isolated from cadaveric pancreatic donors (n = donors) and wild-type or hiappexpressing (hiapp+) transgenic mouse islets (n = / group) were cultured in normal ( . mm) or elevated ( . mm) glucose to form amyloid. ev (exosomes) were isolated from culture medium using classical centrifugation and ultracentrifugation. purified ev were analysed by nanoparticle tracking analysis. western blot analysis and double immunogold transmission electron microscopy were performed to verify the presence of ev markers as well as (pro)hiapp species and oligomers (aggregates). results: human islets formed amyloid during culture with elevated glucose which was associated with progressive beta cell apoptosis. (pro)iapp species were detectable in ev released from human islets cultured in normal and elevated glucose. the latter markedly increased (pro)iapp content in islet-derived ev. interestingly, hiapp aggregates (oligomers) were present in the majority of ev released from human islets cultured in elevated glucose but were not detectable in islets cultured with normal glucose. similarly, ev released from hiapp+ mouse islets which formed amyloid during culture had higher (pro)iapp content compared to wild-type islet-derived ev. moreover, hiapp oligomers were present in ev derived from hiapp+ islets but not wt islets. summary/conclusion: in summary, our data show that (pro)iapp species are present in islet-derived ev and that elevated glucose increases (pro)hiapp and its aggregates in ev released from islets. islet-derived ev may play a key role in the process of amyloid formation in t d and human islet grafts. funding: university of manitoba research grants program (urgp). on. contraction, but not glycolysis, regulates the size of skeletal muscle evs secreted ex vivo. colorado state university, fort collins, usa introduction: skeletal muscle (skm) is a metabolically active tissue and accounts for~ % of total human body mass. acute exercise increases secretion of extracellular vesicles (evs), but the mechanisms responsible are unknown. muscle contraction increases the demand for atp which requires intercellular communication in order to adapt. we hypothesized that this "metabolic stress" during contraction increases skm ev secretion. methods: we tested our hypothesis using an ex vivo ev secretion assay. all studies were approved by the colorado state university institutional animal care and use committee. vastus medialis muscle (skm) from male c bl/ j mice (n = ) or female mt/mg mice (n = ) was cut into~ mg pieces and added to well plates (~ mg/well) filled with ml of serum-free dmem and placed in a cell culture incubator at c for hours. skm from male mice was treated with -deoxyglucose ( -dg) ( . nm - mm) to induce metabolic stress via inhibition of glycolysis. skm from female mice was treated with um of blebbistatin (bleb), a contraction inhibitor. after incubation, skm mass was measured and conditioned media was centrifuged ( , x g for min) to remove cell debris. evs were isolated using exoquick-tc. nta was performed on isolated evs using a horiba viewsizer . ev secretion was normalized to tissue mass and culture media volume then reported as ([particle]/ml/mg tissue). statistical comparisons for -dg experiments were made using a repeated measures -way anova. bleb experiments were analysed using a paired student's t-test. results: there was a trend towards greater ev abundance (p = . ) as a function of -dg treatment, but no effect on ev diameter (p = . ). bleb treatment did not alter ev abundance (p = . ), but significantly reduced ev mean diameter (p = . ; % decrease; dmso: . ± . vs. bleb: . ± . ). summary/conclusion: contrary to our hypothesis, inhibition of glycolysis with -dg did not stimulate skm ev secretion. however, bleb did appear to promote the release of small evs and/or inhibit secretion of larger evs. ongoing efforts are focused on testing other metabolic stressors and defining how blebbistatin promotes small ev secretion. funding: american heart association grant to dsl (ipa ). introduction: extracellular vesicles (evs, exosomes) are nanovesicles ( - nm) secreted from various types of cells. because of vesicular encapsulation of mirnas and enzymes, the evs play crucial roles in cell-to-cell communication by delivering these functional molecules to other cells [ ] . on the other hand, the evs are highly expected as next generation therapeutic tools due to pharmaceutical advantages such as controlled immunogenicity, effective usage of cell-tocell communication routes, artificial modification and encapsulation of functional molecules. however, cellular targeting and uptake efficacy of the evs are insufficient to be utilized as therapeutic tools [ , ] . in this study, we newly developed evs decorated with cellpenetrating sc or (sc ) peptides, which are derived from the c-terminal domain of the cationic antimicrobial protein, cap , because the peptides can be efficiently internalized by breast cancer cells. [ , ] . methods: all peptides were prepared by fmoc-solid phase synthesis. secreted evs from cd -gfp stably expressing hela cells were isolated by ultracentrifugation. cellular uptake of evs was analysed using a flow cytometer and a confocal laser microscope. encapsulation of saponin in the ev was conducted by electroporation. results: sc peptide is known as one of cell-penetrating peptides, and branched structure of sc peptides, (sc ) , further enhances the cellular uptake [ ] . in this research, we examined the effects of the peptide modification on cellular ev uptake, and modification of the sc or (sc ) peptides on ev membranes was conducted via stearyl moiety. as our results, increased macropinocytotic cellular uptake by modification of the peptides was successfully attained. especially, the modification of (sc ) peptides showed higher cellular uptake and macropinocytosis induction efficacy than that of sc peptides. in addition, anticancer protein, saporin toxin-encapsulated evs modified with the (sc ) peptides significantly enhanced their biological activity with dependency of glycosaminoglycan expression on targeted cells. summary/conclusion: the cell-penetrating (sc ) peptide-modified evs shows high abilities to be effectively internalized by cells and are applicable for intracellular delivery of therapeutic molecules. this study is expected to contribute to development of intracellular delivery techniques based on evs. [ introduction: rna therapeutics possess high potential which is yet to be realised, largely due to difficulties involved in delivery to the cytoplasm of target cells. extracellular vesicles (evs) possess numerous features that may help overcome this hurdle and have emerged as a promising rna delivery vehicle candidate. despite extensive research into the engineering of evs for rna delivery, little is known about how their intrinsic rna delivery efficiency compares to current synthetic rna delivery systems. using a novel crispr/cas based rna transfer fluorescent stoplight reporter system, we here compared the delivery efficiency of evs to state-of-the-art dlin-mc -dma lipid nanoparticles (lnps). methods: evs were isolated from mda-mb- cells expressing either a targeting or non-targeting control sgrna and applied to hek t stoplight+ reporter cells. lnps containing targeting sgrna were titrated onto hek t stoplight+ reporter cells to determine the minimum effective dose. lnp and ev particles were characterized using nanoparticle tracking analysis, dynamic light scattering and zeta potential analysis. sgrna copy number was determined using rt-qpcr. results: evs were ± nm in diameter as measured by dls and possessed a negative surface charge of − . ± . mv. rt-qpcr and nta analysis indicated that sgrna ev loading was low, with only in . e ± . e evs containing a single sgrna copy. nevertheless, evs containing targeting sgrna induced significant reporter activation while evs containing non-targeting sgrna did not. lnps were ± . nm in diameter and possessed a neutral charge. these particles also induced significant reporter activation when loaded with targeting sgrna. when delivered via evs, only between to sgrna copies per cell were required to induce statistically significant reporter activation. in contrast, the minimal effective sgrna dose when delivered by lnps was considerably higher at approximately e copies per cell. summary/conclusion: mda-mb- evs deliver rna in a highly efficient manner and are functional at sgrna concentrations several orders of magnitude lower than those required for lnp mediated delivery. this underlines the potential of evs as rna delivery vehicles and highlights the need to study the mechanisms by which evs achieve their efficiency in order for improved development of rna therapeutics. the role of circulating extracellular vesicles in patients with chronic chagas disease introduction: chagas disease is a neglected tropical disease (ntd) caused by the flagellated protozoan trypanosoma cruzi. it is a major public health problem in latin america, and it is now expanding over the globe through immigration of infected individuals. eukaryotic cells release extracellular vesicles (evs) that circulate in body fluids and have an important roles in intercellular communication, both in physiological and pathological conditions. objectives. our study proposes to characterize and to compare the circulating evs isolated from plasma of the chronic chagas disease (ccd) patients with healthy individuals (controls). methods: peripheral blood was collected from patients and controls in the presence of edta and evs enriched from plasma by differential ultracentrifugation. the obtained evs were characterized and quantified by nanoparticle tracking analysis (nta) and added to human thp- cells. after h, the cell supernatants were analysed by elisa for the presence of cytokines. results: lower amounts of evs were obtained from ccd patients in comparison with control individuals. however, the same amount of evs of ccd were more capable of inducing cytokines such as ifn-gamma and il- in relation to controls. summary/conclusion: although less evs are present in the blood of ccd, these evs induce high inflammatory reactions on macrophages suggesting a possible role of these evs in the establishment of chronic disease. funding: supported by fapesp, cnpq and capes. extracellular vesiclesa trojan horse for therapeutic agent delivery introduction: extracellular vesicles (evs) may prove to be one of the optimal payload carriers for therapeutic agents. while they travel through the extracellular space, the ev's lipid membrane layer shields their luminal cargo from deleterious external factors. when autologous evs are used to protect this therapeutic cargo, little immunogenic effects are expected compared to viral vectors and artificial structures, such as liposomes. their usage is potentially manifold, and they are ubiquitously present in all body tissues and fluids. the key is to develop a manageable ev loading agent for adoptive transfer therapies. methods: to exploit the unique properties of evs, highly positively charged proteins were used to load them with multiple biomolecules, such as a cas protein or dicer substrate dsrna as a functional payload and to improve their apparently inadequate natural ability to deposit cargo into the cytoplasm of recipient cells. results: highly positively charged proteins can associate with and/or diffuse through a phospholipid bilayer (thompson et al. ) . when these kinds of charged proteins are mixed with isolated evs in vitro, they are loaded into the evs. the positive charge of the protein has the advantage that it can associate with negatively charged agents, such as rna species, and aids the associated molecule to also incorporate into the ev. moreover, the positive charge of the protein helps with cargo delivery, and thus overcoming the bottleneck of the ev's cargo to escape the endosome post-uptake in a recipient cell. self-quenching fluorescent lipid dyes demonstrated that discharge of the highly positive ev cargo into the cytoplasm is concomitant with lipid mixing between the membrane of evs and the membrane of the recipient cell. when egfp-expressing microglia were exposed to evs loaded with a dicer substrate dsrna able to silence egfp via the positively charged protein, the uptake of dicer substrate dsrna was concomitant with a decrease in egfp expression in the microglia. a similar result was achieved when evs were loaded with cas protein conjugated to the highly positively charged protein. post-uptake of these cas -loaded evs, microglia expressing anti-egfp sgrna (single guide rna) lead to decreased egfp expression. summary/conclusion: our ev delivery technology has the capability of delivering multiple biomolecules, such as protein and rna cargo and demonstrates postuptake of the ev functionality of the ev delivered cargo in the recipient cell. hybrid extracellular vesiclesbiomimetic tool for drug delivery to repair endothelial cell dysfunction introduction: traditional drug delivery systems (dds) are usually based on liposomes, micelles or dendrimers. unfortunately, many dds cause side effects including organ toxicity and/or unexpected immune response. in living organisms, extracellular vesicles (evs) are responsible for delivering biologically active molecules to distant cells. in vitro loading of therapeutic compounds into evs is still not effective and needs developing new strategies. for these reasons we aimed to design hybrid extracellular vesicles (hevs) with high loading capacity for dds. methods: for hev synthesis, we used human endothelial derived evs. using freeze/thawing method we fused them with liposomes composed of cholesterol and one of the three lipids: dopc, sphingomyelin or phosphatidylserine. to confirm membrane fusion, we applied a spectroscopy ruler -fret (förster resonance energy transfer) and cryotem imaging technique. we characterized hevs using nta (for size distribution evaluation), dls (zeta potential) and western blot (for detection of evs markers). we evaluated loading efficiency using calcein as a model drug. additionally, we performed cytotoxicity tests. results: in the cryotem imaging, pure and homogenous hev population with a diameter of ± nm was detected. additionally, we observed changes in zeta potential and in size distribution after fusion. fret measurements showed increased fusion efficiency with the increasing number of freeze/thawing cycles and dependence on a lipid-to-protein ratio in evs. additionally, hev had higher loading efficiency than liposomes and sole evs and that their internalization by endothelial cells did not cause a cytotoxic effect. summary/conclusion: based on cryo-tem and fret, we confirmed that our fusion method of hybrid evs is effective and can be applied as a delivery platform for dds to endothelial cells. response to a range of stressors. the functional activity of these evs in recipient cells may, in part, be driven by changes to their biological cargoes. however, the molecular details of the underlying ev biogenesis and loading processes, and how this may vary in different conditions, is poorly understood. methods: we first studied the effect of oxidative stress on the functional activity of evs in recipient cells using cell viability and mitochondrial membrane potential assays in drosophila s r+ cells. we then carried out total rna sequencing of ev and cellular rna under three stress conditions and compared results to existing data in mouse cells. further to this we have used a bioinformatic pipeline to identify sequence motifs enriched in evs under stress. results: functional assays indicated changes to cell viability and mitochondrial membrane potential in recipient cells, which were donor cell-stress dependent. subsequent characterisation of rna showed an enrichment of ribosomal rna in evs relative to cells, but no significant changes to other biotypes. comparative analysis has also uncovered a set of genes enriched in evs under oxidative stress, and a further subset whose enrichment may be evolutionarily conserved in mouse. we also identified potential ev-loading motifs which may assist in rna loading specifically under stress. summary/conclusion: we have shown that evs derived from oxidatively stressed cells show dose-dependent differences in rna cargo and identified potential sequence motifs that may have a role in its loading. we are now validating the biological significance of these findings by combining different in vivo approaches in drosophila. this will enable us to gain insights into the basic mechanisms which govern ev loading in different contexts, and ultimately the molecular mechanisms underlying ev-mediated intercellular communication. ishai luz a , bibek bhatta a , kanaga sabapathy b and tomer cooks c a ben-gurion university of the negev, beer-sheva, israel; b national cancer centre, singapore, singapore, singapore; c ben-gurion university, beer sheva, israel introduction: mutations in tp are considered one of the most frequent genetic alterations in human cancer. besides the abrogation of the wild-type (wt) p -mediated tumour suppression, a distinct set of missense mutations was reported to endow mutant p proteins with novel activities termed gain-of-function (gof). even though mutations in tp are typically thought to arise in the tumour cells rather than in the stroma, the non-cell-autonomous effects of these mutants over the tumour microenvironment are poorly understood. in the presented studies, focusing on colon cancer as well as on lung cancer microbiome, we investigated intercellular interactions mediated by exosomes and outer membrane vesicles (omvs) in the context of cancers harbouring mutant p . methods: p results: in the colon, tumour cells harbouring mutp were found to exert a non-cell-autonomous effect over macrophages. when exposed to tumour cells harbouring mutp , monocytes became polarized towards a distinguished subset of macrophages characterized by tams-related markers. the mutant p affected tam were characterized as tnf-αlow/il- high, over expressing cd- and cd , with decreased phagocytic ability and increased invasion and matrix degradation potency. investigating the exosomal transfer from mutp tumour cells to macrophages, revealed a mutp -specific mirs signature led by mir- promoting the tam phenotype and creating an invasive front together with tumour cells. mir- was also found to be the top mutp -associated mir in a cohort of human colorectal resected tumours. separately, in two lung cancer cohorts, we identified a signature of microbiome members associated with p mutations. acidovorax temperans, a gram negative bacterium, was found to be abundant in tumours of patients with mutant p . we found a significant increase in tumour volume in animals inoculated with acidovorax temperans as compared to sham treated animals, and increased lung weight as a percent of total body weight. these preliminary data indicate that acidovorax temperans contributes to lung tumorigenesis in the presence of activated k-ras and mutant p . omvs shed by acidovorax temperans promoted inflammatory signalling in lung carcinoma cells and elevated cd expression on tumour cells and sirpα levels on macrophages. summary/conclusion: altogether, these findings are consistent with a microenvironmental role for specific "hot-spot" gof p mutants tightening the interaction between the tumour cell and the immune compartment in colon cancer. in both colon and lung cancer, mutant p facilitates cellular interactions within the tumour microenvironmets mediated by vesicles. funding: intramural funding from the national cancer institute, national institutes of health. lori zacharoff and mohamed el-naggar university of southern california, los angeles, usa introduction: the metal respiring bacterium s. oneidensis creates outer membrane extensions and outer membrane vesicles that are sculpted by the novel bar domain protein bdpa. these vesicles and extensions incorporate mutliheme cytochromes involved in extracellular electron transfer to metals and electrodes. however, the physiological relevance of incorporating these cytochromes into the higher order d architecture of a vesicle or extension is unknown. given that bar domains serve as a protein sorting mechanism in eukaryotes, we investigated the pathway crosstalk between bdpa and outer membrane multiheme cytochromes as means to understand the physiological significance of membrane architecture. methods: o this end, vesicle morphology and content was measured using dry weights, dynamic light scattering, fluorescence microscopy and comparative proteomics from wild type s. oneidensis and deletion strains. results: cells lacking bdpa make large amorphous vesicles that are dense with protein. in contrast, a strain lacking outer membrane cytochromes recruits less total protein into smaller vesicles. proteomics to show that both bdpa and multiheme cytochromes are involved in recruiting other proteins to outer membrane vesicles and have a reciprocal relationship. summary/conclusion: in the absence of bdpa, protein crowding has to become the main driving force of vesiculation and bdpa is essential for efficient incorporation of cytochromes. however, multiheme cytochromes are not only vesicular cargo, but are also important for shaping and loading vesicles. both of these situations make it clear that vesicles play a role in increasing the respiratory surface area of s. oneidensis cells. moving forward, we hope to be able to control bdpa and cytochrome levels for selective recruitment of technologically relevant payloads. introduction: fascioliasis caused by fasciola hepatica represents a major economic loss and clinical burden in cattle farming worldwide. extracellular vesicles (evs) contain pathogen-derived molecules that represent novel biomarkers of disease. in the present study, we have identified potential new biomarkers of f. hepatica infection in evs present in sera of infected cattle. methods: parasites and sera were obtained from local abattoirs (valencia, spain, and medellin, colombia, respectively). sera from infected and from healthy animals. parasites were cultured, and evs obtained by sizeexclusion chromatography (sec) and characterized by nta, tem and proteomic profiling. recombinant proteins from f. hepatica evs (enolase and fh . tegumentary protein) were produced, and coupled to magnetic beads. measurement of bovine igg antibodies was performed using luminex bead array technology. results: a total of proteins were identified associated with evs as shown by the presence of typical ev-markers (tsg , alix, cd ). two parasite proteins, enolase and the fh . tegumentary protein were produced as recombinant proteins and used for detection of cattle igg employing luminex bead array technology. interestingly, significant differences were found in the fluorescence values of both recombinant proteins allowing discrimination between sera from infected and non-infected cattle. the use of the fh . protein generated a highly significant difference between the two groups (p value = . ); as did enolase (p value was . ). summary/conclusion: this study demonstrates the usefulness of ev proteins as new biomarkers for early diagnosis of helminth infections using multiplex assays, a technology that may also be applied to other parasite ev molecules. life stage-specific glycosylation of schistosome-derived extracellular vesicles introduction: glycans play an essential role in pathogen-host interactions. larvae and adult worms from schistosoma mansoni release distinct subsets of glycoconjugates as excretory/secretory (es) products. extracellular vesicles (evs) are also among the es products. we recently found that schistosomuladerived evs are glycosylated and bind human dendritic cells via c-type lectin receptor (clr) dc-sign, leading to increased il- and il- release. here we investigated the glycosylation profile of evs released by s. mansoni adult worms, compared this to schistosomula evs, and addressed how this may affect parasite-host interactions via clrs. methods: evs from cultured s. mansoni parasites were obtained by ultracentrifugation and purified with iodixanol density gradients. isolated evs were analysed by nta and cryo em. n-glycan and lipid glycan content was determined by mass spectrometry. density gradient fractions with evs were loaded onto sds-page gels followed by western blot (wb) analysis using anti-glycan monoclonal antibodies (mabs). results: cryo em showed that adult worm evs lacked the long thin filaments that are characteristic for schistosomula evs. additionally, in contrast to schistosomula evs, glycolipids could not be detected in the adult worm evs. mass spectrometry analysis showed that the most abundant n-glycans in the adult worm evs contained galnacβ - glcnac (lacdinac, ldn) motifs, which correspond to previously published overall glycan profiles of this specific life stage. other differences in ev glycosylation between the two life stages were observed by wb using anti-glycan mabs: adult worm evs showed a paucimannosidic glycan motif whereas in the schistosomula evs galβ - (fucα - ) glcnac (lewis x) was detected in line with previous ms analysis. introduction: phloem plays a central role in plant function, as it is the responsible for the translocation of photoassimilates from source-to sink-organs, and a long-distance route for signals distribution. due to the sap high nutrient content, sieve elements are primary target for plant pathogens and pests. in this work we aimed to isolate and characterize extracellular vesicles (evs) from cucumis melo phloem sap, derived from plant either exposed or not to the melon aphid, aphis gossypii (hemiptera: aphididae). methods: phloem exudates from -week-old melon plants, either uninfested or infested with adults of a. gossypii (n = , replicates each), were collected by cutting the stem with a sterile razor blade between first and second expanded leaf from the top. evs were isolated by size exclusion chromatography, and analysed by nanoparticle tracking analysis (nta) and transmission electron microscopy. evs proteome was determined by quantitative mass spectrometry. results: evs from phloem sap were successfully isolated in every condition. no significant differences were detected among distinct samples, neither in particle concentration and size by nta, nor in protein concentration. most importantly, a total of different proteins were identified in phloem sap evs, including present in exosome databases (exocarta). on top of that, differentially expressed proteins were identified in evs derived from aphid infested or uninfested plants (p value < . ). summary/conclusion: understanding how plants trigger their defences against pests and pathogens is important to develop new control measures. the characterization of several proteins in evs from the phloem sap provide valuable information on long distance signalling in plants. moreover, as plants lack an immune system comparable to animals, the different protein content in phloem sap evs after exposure to aphids could indicate their important role in delivering inducible defences against invading pests and pathogens. extracellular vesicles from nematode species heligmosomoides bakeri and trichuris muris contain distinct small rnas that could enable niche specificity in the host introduction: gastrointestinal nematodes are extremely prevalent parasites that infect most animals and % of human population. their success as parasites is attributed to their ability to secrete diverse molecules that modulate the host immune system. extracellular vesicles (evs) are one of the immune modulatory compounds they release that directly modulate host cells. our goal is to understand how the small rna (srna) cargo underpins ev function, using a comparative analysis of ev cargo from diverse nematode species. methods: we first compared how different ev isolation methodologies (ultracentrifuge (uc), size fractionation, sucrose gradient floatation) effect the small rnas detected in h. bakeri evs using different library preparation kits (cleantag, truseq), with or without polyphosphatase treatment. we then compared this to small rna libraries from t. muris evs using comparable methods, uc ev purification, with or without polyphosphatase treatment and using the cleantag library preparation kit. results: evs from both species contained mirnas, however the mirna gene familes in h. bakeri and t. muris evs are distinct. the mirna content detected in ev samples collected by different purification protocols is robust. the largest difference in detected mirnas was found when comparing different library preparation kits. although both h. bakeri evs and t. muris evs were dominated by srnas derived from intergenic or repetitive elements in the parasite genomes, only in h. bakeri evs were these secondary sirnas. summary/conclusion: h. bakeri and t. muris evs contain distinct small rna cargos, which may underpin their ability to colonise different host niches, and/or modulate the host immune system differently. t. muris evs do not contain secondary sirnas, in contrast to h. bakeri, however they are dominated by srnas derived from intergenic or repetitive regions. comparative analysis of helminth evs could help pinpoint the srnas involved in cross-species communication. please provide any keywords if applicable.: nematode, cross-species communication, small rna introduction: extracellular vesicles (evs) are secreted from various cells including cancer cells and known to contain protein and small rnas including mirna isoforms (isomirs). therefore we also focused on isomirs including other small non-coding rnas for biomarker discovery. although liquid biopsies using small rnas are promising biomarkers for early detection of cancer, current approaches to detecting and analysing mirnas in the blood are still inadequate. artificial intelligence (ai) data analysis may provide better algorisms for diagnosing cancer. methods: small rnas were isolated from serum or purified evs using a mirneasy mini kit (qiagen) and quantified by using the ion s ™ next-generation sequencing system. (thermo fisher scientific). evs were purified using total exosome isolation reagent (invitrogen™). ai data analysis was performed using jmp® genomics and datarobot enterprise ai platform. results: three small rnas, isomir of mir- - p, mir- a- p, and trf-lys (ttt) were significantly upregulated in breast cancer patients compared with the healthy cancer-free individual. the combination algorithm using these three small rnas allows for a more accurate diagnosis of the area under the curve (auc) . . to test the possibilities that these small rnas are derived from cancer cells, we isolated evs from the serum and performed ngs analysis to profiled serum small rnas in evs. interestingly we found that two small rnas, mir- - p and mir- a- p, also high in breast cancer evs, indicating that these small rnas were expected to be derived from cancer cells. in oesophagus cancer, we also performed ngs analysis and identified twenty-four mir/isomirs candidates for diagnostic biomarkers. a multiple regression model selected mir- a- p and two isomirs (mir- - p and mir- - p) . the auc of the panel index was . . we also performed ai data analysis and discovered the novel algorisms that can diagnose breast and oesophagus cancer more accurately. summary/conclusion: we demonstrated combinations of circulating non-coding rnas containing evs potentially useful for the detection of early-stage breast and oesophagus cancers. in addition, the datarobot enterprise ai platform enables us to the more accurate diagnosis of cancers at the early stage. identification of novel ev-associated mirnas as toxic biomarkers in mouse introduction: recent findings reveal that extracellular vesicles (evs), secreted from cells, are circulating in the blood. evs are classified into exosomes ( - nm), microvesicles ( - , nm) and apoptotic bodies ( - , nm). evs contain mrnas, micrornas, and dnas and have the ability to transfer them from cell to cell. recently, especially in humans, the diagnostic accuracy of tumour cell type-specific evs as biomarkers is more than %. in addition, micrornas contained in the evs are being identified as specific biomarkers in blood for chemical-induced inflammation and organ damage. therefore, micrornas contained in the evs released into the blood from tissues and organs in response to adverse events such as chemical substances and medicine are expected to be useful as novel biomarkers for toxicity assessment. in this study, we aimed to identify target organs by comprehensive analysis of ev rnas in the blood of mice after chemical exposure to establish a highly sensitive "next generation type" toxicity test for chemical substances and medicine using ev rna in blood as a biomarker. methods: all animal studies were conducted in accordance with the helsinki declaration and the guidelines approved by the animal care committee of the national institute of health sciences. c bl/ j male mice ( weeks) were orally dosed with ccl (vehicle, , mg/kg). serum were separated from blood after , , and hours after ccl administration. the serum was centrifuged at , x g to remove cellular debris and subsequently ultracentrifuged , x g. the pellet is resuspended in pbs and ultracentrifuged , x g again. the comprehensive small rna-seq of collected evs were performed according to the manufacture's protocols. results: we succeeded in isolating more than novel small rnas, which could be used as novel highly sensitive biomarkers for hepatotoxicity due to carbon tetrachloride (ccl : mg/kg & mg/ kg). well known hepatotoxicity biomarkers, mirna- and mirna- were upregulated more than -fold in the administration of mg/kg ccl , but not responded in the administration of mg/kg ccl . summary/conclusion: these results suggest that mir- and mir- are mainly released from liver to blood directly only in the administration of mg/kg ccl , while novel more sensitive hepatotoxicity biomarkers which responded in the administration of both mg/kg and mg/kg ccl should be included in the ev. our novel biomarkers will accelerate a rapid evaluation of chemical substances and medicine in nonclinical safety evaluation. introduction: advancements in sequencing technologies have allowed analysis of the genomic landscape of cancer using circulating cell-free(cf) dna. however, cfdna does not originate only from tumour cells. we recently demonstrated that most of the dna circulating in plasma of cancer patients is associated with large evs (l-evs), and that l-ev-associated dna reflects genomic aberrations of the cells from which l-evs arise. since l-evs are specifically released by tumour cells, we explore their potential to report cancer-specific genomic alterations in patient plasma and compare it to cfdna. methods: differential ultracentrifugation, tunable resistive pulse sensing, qubit dsdna high sensitivity assay, capillary electrophoresis, whole exome sequencing ( - x), targeted sequencing (qiaseqtm), flow cytometry. results: we show here that l-evs in the size range of > micrometre are present exclusively in plasma obtained from cancer patients and absent in plasma from healthy donors. in agreement with this finding, double-stranded(ds) dna is detected only in l-ev fractions of patient plasma and not in those obtained from healthy donor plasma using the same protocol. we also demonstrate that the fragments of dsdna associated with circulating l-ev are larger in comparison with cfdna (> , bp versus~ bp). a large-scale analysis of l-ev dna obtained from plasma of patients with metastatic castration-resistant prostate cancer (mcrpc) as well as with non-small cell lung cancer (nsclc) demonstrates that dna associated with circulating l-evs reports cancer-specific genomic alterations in both types of cancer. we further investigate if l-evassociated dna is intra-or extravesicular and demonstrate that it is present in both forms. we finally compare the purity of the tumour signal in intravesicular l-ev dna, total l-ev dna, and cfdna obtained from patient plasma. summary/conclusion: our results demonstrate that circulating l-evs contain high quality, large molecular weight dna that contains cancer-specific genomic alterations, supporting the use of l-evs as a source of tumour-derived dna in plasma. introduction: epidermal growth factor receptor (egfr) mutation driven lung adenocarcinoma (ac) represents a unique subgroup that lends itself to treatment with oral egfr tyrosine kinase inhibitors. current methods that are used to detect these mutations (e.g. l r or the resistance mutation t m) involve invasive tumour biopsies or blood circulating tumour dna (ctdna) and cell free dna (cfdna). the sensitivity of blood ctdna and cfdna is limited by the frequency of genomic alterations in the egfr gene; additionally, ctdna does not reflect changes in the egfr protein, against which novel therapies are in development. there remains a need to develop bloodbased biomarkers that can circumvent these disadvantages and replace the more standard, invasive tumour biopsies. we propose the study of exosomes for treatment monitoring as well as to identify egfr resistance related genomic and proteomic changes. methods: we enrolled patients with metastatic lung ac: with egfr mutations and without (control). from the patients with egfr mutant lung ac, we processed blood samples through the patients' treatment course, using ultracentrifugation to isolate exosomes. we then used both droplet digital pcr (ddpcr) to test exosomal rna (exorna) for the mutation of interest and western blots to test protein resulting from exon deletion or l r mutations. results: from patients with egfr exon deletion mutations, we detected identical mutations in exorna from / samples. exorna based mutational load increased and mirrored clinical progression in patients. three patients whose cancer remained stable demonstrated a decrease in their exorna. one patient had blood drawn only at points and was therefore not plotted. exorna from patients with l r and t m mutations demonstrated the corresponding mutations; however, exorna did not mirror their disease course. we also demonstrated mutant egfr protein presence in exosomes from patients. finally, we tested cfdna for egfr mutations from four matched samples using ddpcr. we detected matched mutations in exosomes in all four, while cfdna mutations were only detected in / patients. summary/conclusion: in summary, we detected egfr mutations in / exosome samples isolated from metastatic lung ac. our results set the stage for optimization of exorna methods and inform future experiments relating to exosomal cargo in patients with egfr mutant lung ac. identification of plasma-derived, ev-based biomarkers for glioblastoma introduction: glioblastoma multiforme (gbm) is the most malignant and aggressive primary brain cancer in adults, with an incidence of . per , people. currently, diagnosis is only performed via histopathological investigation of a tissue sample from a gbm lesion, complemented with molecular diagnostics for identification of select biomarkers. mri is the standard of care for follow-up and monitoring of treatment response. therefore, development of a "liquid biopsy" to obtain disease-relevant information from patient's body fluids is highly desirable. methods: we present the results from a clinical study in which extracellular vesicle (ev)-derived mrnas and long non-coding rnas were profiled from the plasma of gbm patients and control individuals. we obtained plasma from patients at the time of initial diagnosis, and matched controls by sex and age. ev-associated rna was isolated from - ml plasma and rna-seq was performed using our proprietary pipeline. sequencing data was analysed for differential gene expression. results: we observed mrnas as differentially abundant between gbm and control samples, with mrnas enriched in gbm samples and mrnas enriched in control samples (p < . ). correlation based on differentially abundant mrnas separated gbm and control samples into two unique populations. eight differentially expressed mrnas were previously identified as part of the mesenchymal gbm subtype. these data, while preliminary, provide a potential basis for the further development of a noninvasive gbm gene panel test. summary/conclusion: we have identified a novel rna signature for gbm from plasma derived evs, which differs from previously identified biomarkers isolated from tissue. further work will refine this signature to enable detection, characterization, and patient monitoring for gbm with minimally invasive techniques. introduction: sjogren's syndrome (ss) is a systemic autoimmune disease in which inflammation progressively damages the moisture producing glands of the afflicted. million americans are estimated to be suffering from the disease, % of which are women with an average age of . overlapping symptoms with other health conditions and co-morbidities make ss particularly difficult to diagnose, with average time to diagnosis of years. saliva exosomal rna profiling has been primarily focused on small rnas and has been limited thus far due to the large contribution of sequencing reads from the oral microbiome. a noninvasive saliva exosomal rna (exorna) based test capable of diagnosis would be highly desirable. methods: we began by first developing a novel long rna-seq workflow to selectively enrich and profile human exosomal mrnas and long non-coding rnas (lncrnas) from saliva. we then profiled salivary exorna obtained from ss patients and healthy matched controls. finally, we performed differential gene expression analysis to obtain an exorna signature for ss. results: rna-seq data analysis demonstrated highly efficient enrichment of human transcriptome, with over % of reads mapping to the transcriptome. further rna biotype analysis showed over % of transcriptome reads mapped to protein coding genes and lncrnas. we detected over , mrnas and approx. lncrnas. differential expression analysis (dex) of ss vs. healthy control exorna identified upregulated genes, including mrnas and lncrnas (p < . ). genes were found to be downregulated in ss, including mrnas and lncrnas. gene ontology analysis of dex genes revealed enrichment of genes involved in various immune system related pathways. most importantly, principal component analysis (pca) resulted in clear separation of ss patients from healthy controls. summary/conclusion: our optimized rna-seq workflow enables saliva-based liquid biopsy for biomarker discovery. the gene signature identified in this ongoing study could potentially provide a non-invasive molecular means of diagnosing sjogren's syndrome. introduction: increasing embryo implantation rates has become one of the greatest challenges in assisted reproduction techniques. usually an endometrial biopsy is done to identify a receptive endometrium, which prevents embryo transfer in the same cycle, as it is detrimental for the implantation. the implantation is a complex process, which requires a synchrony between the development of the embryo and the endometrium, but also, an adequate embryo-endometrial cross talk. the presence of extracellular vesicles (evs) as mediators of this communication has been describe in the endometrial fluid. therefore, we hypothesize that the molecular analysis of the content of the evs and companion molecules from endometrial fluid could be a non-invasive method to recognize an implantative endometrium and consequently improve the implantation rates. methods: the objective is to define a simple, sensitive and reproducible non-invasive ev-based method that allow the quick identification of an implantative endometrium by means of mirna analysis. for the establishment of a robust methodology for analysing evs from endometrial fluid in clinical settings, where the sample is limited and no sophisticated equipment is available, five different methodologies were compared in triplicate. two of them consisted in the direct extraction of rna while in the other three, before the rna extraction an enrichment of evs was done. smallrnaseq was performed to determine the most efficient method. once the best method was selected, it was applied in a set of real samples with different implantation outcome. the content of mirnas (mainly associated with evs) of endometrial fluid samples from women in whom the implantation was successful (n = ) and unsuccessful (n = ) were analysed. results: our results show that the protocols with a previous enrichment step of evs obtained a higher mirna expression. the results obtained from the differential analysis of the set of samples with different implantation outcome are being analysed and it is expected that the results will be available by the time this communication is presented. summary/conclusion: this work demonstrates that it is possible to obtain and analyse evs and evs-associated mirnas from a small volume of endometrial fluid samples, which allows the use of ev-mirnas as a low-invasive biomarkers for the detection of an implantative endometrium. funding: jip is supported by a predoctoral grant from the basque government. small rna cargo of evs is affected by hormone treatment in prostate cancer introduction: small rnas are recently reported as a regulator for prostate cancer progression to castration-resistant disease. our previous work has shown that evs protein cargo is affected by male steroid hormone, dihydrotestosterone (dht). in this study, we assess the small rna cargo of evs in response to androgen manipulations. methods: androgen receptor-positive lncaps are grown in css medium to deplete the androgens. media were then replaced with vesicle-depleted css medium ± nm dihydrotestosterone (dht) ± µm enzalutamide (enz) for h. evs were isolated using sequential ultracentrifugation ( g for min, , g for min, , g for h), washed once in pbs. protein and rna were collected from both parent cells and conditioned medium to allow direct comparison between s-evs cargo and cells. small rna ngs libraries were prepared using the illumina's truseq small rna library prep kit and single-end sequenced at a read length of nucleotides (nt). fastq library files were processed using a custom-designed pipeline. adapters were removed using the cutadapt tool, trimmed reads were mapped with high stringency against ribosomal sequences using bowtie . snorna and trna fragments were identified using the flaimapper software. remaining reads were mapped against the human genome hg using bowtie . results: we found that the presence or absence of androgens does not significantly change the amount of total rna in small evs (s-evs). however, hormone stimulation altered the small rna content of s-evs, in parallel with our previous published data on ev protein cargo. dht increased the abundance of snorna in cells, while a reduction of snornas was observed in the s-evs fraction. interestingly, dht induced the formation of cell filopodia that are not inhibited by androgen inhibitor enzalutamide. pathway analysis indicates the p mediated regulation driven by mirnas found in s-evs upon exposure to dht. the expression profile of snorna and trna fragments in dht treated cells resembles results from clinical prostate cancer specimens. summary/conclusion: our findings show that androgen manipulation alters both s-ev derived protein and rna cargo. changes in the s-ev rna profile due to treatment with androgens are not identical to small rna profiles in parental cells, indicating a specific sorting mechanism of s-ev small rna upon androgen manipulation. further, dht induces the formation of cell filopodia irrespective of enzalutamide, suggesting cargo selection of s-evs. we conclude that small rna ev cargo can be utilised to as prostate cancer biomarkers in androgen targeted treatments. introduction: cancer immunotherapy, such as pd-l blockade, is a method to eliminate cancer cells. ectopic expression of pd-l , on the surface of tumour cells, has been associated with tumour persistence and as an important predictor of therapy response. a test that, specifically and accurately, detects pd-l is critically important in order to identify patients that would benefit from these treatments. emerging evidence has shown that extracellular vesicles (ev) can carry immune checkpoint molecules, such as pd-l , and whose expression have been correlated with tumour immunity response. with a multitude of commercially available antibodies identifying appropriate clones and associated assay is important in order to standardize the diagnostic modality used. methods: pd-l expressing cancer cell lines were used to generate evs. pd-l -myc vector was transfected to generate an overexpression system. exoview® sensors containing different anti-pd-l clones were generated. samples (cell derived and plasma) were incubated on chips to allow the antibody to bind the antigen on the ev. after incubation, chips were immunolabeled with fluorescently labelled antibodies against pdl- or ev associated markers. exoview r reader was used to enumerate the evs captured on the sensor surface and analyse the expression of pdl- on single vesicle through fluorescence imaging. immunoprecipitation and mass spectrometry (ip/ms) were employed as an orthogonal method to verify the specificity of the assay. results: to study the detection efficiency of the antibodies, engineered pd-l -myc evs were used. under these circumstances, all the tested antibodies were able to capture evs. when testing endogenous pd-l positive evs from different cancer cell lines, only . and clones consistently bound to evs. in addition, evs derived from plasma demonstrated to be positive for pd-l , however, only clone . was able to immobilize these evs. the results suggested that clone . could be a potential pd-l antibody to detect pd-l positive evs originating from various sources. to confirm these results, and assure the specificity of the antibody targeted ip/ms was employed. summary/conclusion: in combination with the exoview platform, anti-pd-l antibodies can be screened and potentially used to generate a non-invasive ev-specific assay that could detect this protein in patients. differences in extracellular vesicle protein cargo is dependent on head and neck squamous cell carcinoma cell of origin university of michigan, ann arbour, usa introduction: head and neck squamous cell carcinoma (hnscc) is the sixth most common, eighth most fatal cancer worldwide and includes cancers of the oropharynx, larynx, hypopharynx, and oral cavity. in , there were over , new cases and , deaths estimated in the usa alone. despite recent advances in treatment, including radiation, chemotherapy, surgery, concurrent chemoradiation, and immunotherapy, many tumours develop resistance and progress. patients develop metastases or tumours recur locally or regionally; the -year overall survival rate for hnscc is only - %. factors that contribute to poor survival for patients with hnscc include late stage diagnosis, lack of reliable markers for early stage detection, high level of biologic heterogeneity, and local recurrence and distant metastases after treatment. methods: this study used representative hpv-positive and hpv-negative hnscc cell lines, one hpvtransformed cell line. and two non-cancer oral keratinocyte cell lines. evs were isolated using differential ultracentrifugation and peg precipitation/ultracentrifugation. evs were characterized by tem, nta, and wes protein analysis for reported ev markers. ev and whole cell lysates were assessed by lc-ms/ms analysis using the tandem mass tag- plex kit. cluster analysis was performed on the fold-change peptide spectrum matches (psm) for the evs from the hnscc lines compared to the evs from the normal keratinocyte line (noksi). protein was measured using a capillarybased electrophoresis instrument. results: cd and annexinv were detected in all of the ev lysates tested, while calnexin was detected in all of the whole cell lysates and none of the ev samples tested. selected proteins stat , hla-a, tenascin, e-cadherin, β catenin, cytokeratin , epha , and cd , and hpv-related markers p , p , rb, cyclin d , and egfr were tested using the wes platform. evs from hpv-positive cell lines showed higher protein levels compared to evs from hpv-negative cell lines in stat , hla-a, and tenascin. only kert demonstrated lower protein levels in evs from hpvnegative cell lines. of the common hpv-associated hnscc markers: egfr, p , rb, cyclin d and p , only egfr was positive in any the evs tested. the remaining proteins queried, e-cadherin, β catenin, epha and cd showed varying protein levels in evs from both hpv positive and hpv-negative cell lines. summary/conclusion: our findings suggest that these proteins may be potential hnscc ev markers that may be ) selectively included in ev cargo for export from the cell as a strategy for metastasis, tumour cell survival, or modification of tumour microenvironment, or ) representative of originating cell composition, which may be developed for diagnostic or prognostic use in clinical liquid biopsy applications. validation of antibodies on western blot for extracellular vesicles from biological human samples and cancer cell conditioned media the brady urological institute, johns hopkins university school of medicine, baltimore, usa introduction: one of the major challenges in extracellular vesicles (evs) research is to prove the particles that are isolated are true evs, rather than other co-isolated contaminants, like lipoproteins. isev recommends using multiple assays to characterize evs. this study aims to validate the positive and negative protein markers for extracellular vesicles from plasma, urine and prostate cancer cell conditioned media (ccm). methods: membrane and cytosolic fractions of mcf cells served as positive and negative controls for all antibodies validated. evs were isolated from plasma of healthy volunteers, urine of healthy volunteers and ccm of pc- cells using differential ultracentrifugation. eight protein markers were assessed: positive markers cd , cd , cd , flotillin (flot ), alix and tumour susceptibility gene (tsg ), negative marker calnexin (canx), and contaminant markers apo-a for plasma and thp for urine. tetraspanins are small transmembrane proteins expressed in evs. flot is membrane protein that forms microdomains in the plasma. alix and tsg , an accessory protein of the endosomal sorting complex required for transport, are involved in the biogenesis of evs. they are positive markers for evs. canx is in the membrane of the endoplasmic reticulum. apolipoprotein-a (apo-a ) is the protein components of lipoproteins, therefore it is marker of contamination for plasma ev. tamm-horsfall protein (thp) is contamination marker for urine ev, because it is most abundant protein in human urine. results: all antibodies were validated in the correct positive and negative control, thus confirmed as usable and reliable antibodies for western blot. in plasma ev, cd , cd , cd and flot were positive and canx and apo-a were negative. in urine evs, cd , cd , flot- , alix and tsg were positive and canx and thp were negative. in ccm evs, cd , cd , flot , alix and tsg were positive and canx was negative. summary/conclusion: we confirmed a high degree of ev purity from sample types: urine, plasma, and ccm. of particular importance, we confirmed that evs isolated from biologic patient samples, plasma and urine, had low contamination. future work will use these methods to confirm purity of ev samples prior to addition analysis, such as examining ev cargo and biologic significance. proteomic study of mesenchymal stem cells derived exosomes modified using mir. introduction: the project we are working on is to modify the immunogenic profile of human cmms from the umbilical cord stroma through its stable transfection with anti-mir- - p, and therefore of the exosomes that these cells generate, for use in free-cell therapy to treat inflammatory process. methods: evs released from a primary culture of human umbilical cord mesenchymal stem cells and from primary culture of human umbilical cord mesenchymal stem cells mir -/-modified through stable lentiviral transfection were isolated by ultracentrifugation processes, characterized by transmission electron microscopy (tem) and measured by nanoparticles tracking analysis (nta). protein extraction from evs was made using ripa buffer and after checking protein integrity the total ev proteins. we performed a shotgun proteomic study using a tmt ( -plex) label of the total mir -/-exosomes protein comparing it with normal exosomes. after labelling the ltq-orbitrap platform of proteored was needed for fraction injections and data acquisition. proteome discoverer . (thermo) was used for protein processing and quantification. results: a total of . proteins were identified at least with a unique peptide and we have able to establish the proteomic profile of mir -/-exosomes against normal exosomes. we found out several protein modulated by mir and related to inflammation. summary/conclusion: we have able to establish the proteomic profile of mir -/-exosomes against normal exosomes focusing on proteins involving inflammation process. all those results seem indicate that exosomes could be modified, which could be used as an anti-inflammatory free-cell therapy. funding: proteored concept test project grant. a novel extracellular vesicle isolation method used to discover urine liver disease biomarkers introduction: hepatocellular carcinoma (hcc) is the th most common cancer worldwide and the rd most common cause of cancer death; additionally, its incidence is increasing. while outcomes for early hcc are superior to those for late stage disease, early detection of hcc remains a challenge. current guidelines have suboptimal sensitivity and specificity. in this pilot study, we hypothesize that urine extracellular vesicles (evs) may identify candidate biomarkers towards the development of an inexpensive, widely accessible screening assay for the early detection of hcc. methods: urine samples from healthy subjects, subjects with cirrhosis, and subjects with cirrhosis plus hcc were collected and processed using ymatrix columns to isolate ev-associated protein and mirna. protein was analysed using a tandem mass tag method on a thermo scientific orbitrap fusion mass spectrometer with comet/paws and edger processing. mirna was analysed using a targeted firefly microarray from abcam. differential expression and predictive modelling for the presence of hcc and cirrhosis was performed to identify candidate mirna and protein biomarkers. results: for mirna, samples were eligible for analysis after low expression filtering. we used pair-wise ratios of cancer-associated mirnas by gradient boosting of decision trees to develop a predictive model for hcc. our best model had a sensitivity and specificity of . and . respectively using mirnas to distinguish hcc from cirrhosis. all samples were eligible for protein analysis. based on differential expression and biologic relevance, we identified protein candidate biomarkers. interestingly, we found liver-selective proteins and known hcc/cirrhosis plasma/tissue markers, demonstrating proof-of-concept for the method. summary/conclusion: urine extracellular vesicles contain liver-selective proteins and known liver disease serum biomarkers as well as novel mirna and protein biomarkers that are significantly up-regulated in disease samples. the described candidate biomolecules may be easily accessible biomarkers with which to develop a sensitive and specific universal screening diagnostic for the early detection of cirrhosis and hcc. introduction: the peptidergic g-protein coupled receptors (gpcrs) are cell-signalling transmembrane proteins, which in their native form comprise of seven segments embedded in the cell membrane. this structural advancement is believed to be maintained in extracellular vesicles (evs). in autoimmune diseases, the presence of autoantibodies towards gpcrs is not uncommon, and to detect plasma autoantibodies, evs carrying gpcr will be used as template in a novel microarray screening tool. methods: purified evs from hek cells were printed on different types of surfaces; polymer coated glass slides and hydrophilic and hydrophobic plastic well plates. five different print buffers were tested in a multiplex assays. spots containing evs were stained with biotinylated antibodies (cd , cd , cd , adrβ , hsp , epcam and flotilin- ) followed by binding of cy -labelled streptavidin and visualized microarray scanner. results: the outcome of these experiments was promising, as some of the chosen printing buffers showed increased tendencies to bind evs. the ev presence was verified with a panel of markers known to be present on small evs. in addition, the ev content of the adrenergic beta- receptor (adrβ -receptor), which is a gpcr of interest in autoimmune diseases, was verified in some of the experimental setups. summary/conclusion: the approach of using evs as template in a screening tool possesses the potential to easily screen for autoimmune illness markers in diagnostic purposes. using the microarray technology allows the screening to be multivariate, specific and highly sensitive. circadian variation of extracellular vesicles secreted in urine: analysis of time point collection and normalization strategy. introduction: urinary extracellular vesicles (uevs) are an ideal source of biomarkers for kidney and urogenital diseases. despite the great deal of interest generated by uevs, little is known about its collection time and normalization approach. the majority of the studies on uevs focus on spot urine collection based on the assumption that it accurately reflects the renal function, although time point of collection is not standardized. therefore the practice to collect spot urine does not allow for calculating and standardizing accurately the uev excretion rate which may vary during the day. in addition, no research has been carried out yet to show the quantitative and qualitative difference of uevs between spot urine and h collections.the aim of this study is to compare uevs excreted in all single voids during a hour collection period and compare it with hour collection performed. methods: uevs were enriched by differential centrifugation and electron microscopy, western blot, nanoparticle tracking analysis, tuneable resistive pulse sensing and imaging flow cytometry were used to quantify uevs and associated markers variation during the hour. creatinine, urine osmolality and particle concentration were used to normalize the assessed analytes. results: electron microscopy showed a heterogeneous population of evs and western blot confirmed the presence of ev markers (tsg , alix and cd ). rna was extracted by a column-based method (mirna extraction kit qiagen) and cel- mirna was spiked in each sample. a multiparametric detection of nephron markers podocalyxin, aquaporin- and uevs pan tetraspanins (cd + dc + cd ) was performed utilizing imaging flow cytometry. whereas the uev composition did not change across the hours analysis, the quantity of uevs and related markers fluctuated during the day depending on the hydration and excretion rate.the results of a hour urine collection reflected the average results of all single voids over a hr period. creatinine and particle count normalization failed to normalize "outliers". summary/conclusion: this study represents the very first report which compares single void urine versus hour uev analysis. we concluded that the hour collection is the preferred choice for a robust and rigorous assessment of uevs and its associated markers. porcine body fluids differ in small extracellular vesicle counts: comparison of blood plasma, seminal plasma and cerebrospinal fluid as vesicle sources for proteomic analyses helena kupcova skalnikova a , jakub cervenka b , jaromir novak a , karolina turnovcova c , bozena levinska a , jana juhasova a , stefan juhas a and petr vodicka a a institute of animal physiology and genetics, czech academy of sciences, libechov, czech republic; b institute of animal physiology and genetics cas, v. v. i. libechov, libechov, czech republic; c analysis tools were used to identify in silico biological pathways and functions governed by detected mirnas. expression of putative targets of selected mirnas was tested using qpcr after in vitro delivery of uterine evs to ptr cells. results: careful characterisation confirmed that uterine lumen is enriched with a diverse population of evs caring mirnas. interestingly, out of detected mirnas showed difference in abundance between tested days of pregnancy and half of them was exclusively detected on d . identified mirnas were characterized as potent regulators of cellular development, growth, proliferation, and movement, in addition to their involvement in organismal and embryonic development. the expression of genes identified as a possible mirna targets was tested after evs delivery to ptr cells in vitro. both down-(e.g., ptger ) and up-regulated (e.g., lifr) genes were found (p < . ); involved in the same molecular and cellular functions enriched by detected mirnas. methods: evs were harvested from wild type and arrdc -/-epididymal cells using differential ultracentrifugation, then characterised using nanoparticle tracking analysis and transmission electron microscopy. sperm motility was measured using computer assisted sperm analysis and imagej. fertilisation capacity was measured using the following assays: capacitation-associated tyrosine phosphorylation, calcium ionophore induced acrosome reaction, zona pellucida binding assay and in vitro fertilization with time-lapse imaging of embryo development. immunohistochemistry was also used to visualise two pronuclei formation and blastocyst morphology. arrdc -/-sperm was supplemented with wild type evs in the above assays to assess whether they could restore function. results: sperm from arrdc -/-mice develop normally through the testis but fail to acquire adequate motility and fertilization capabilities through the epididymis, as evidenced by reduced motility, premature acrosome reaction, reduction in zona pellucida binding and production of two-cell embryos. we observed a significant reduction in ev production by arrdc -/-epididymal epithelial cells, and addition of wild type evs to arrdc -/-sperm dampens the acrosome reaction and restores zona pellucida binding. introduction: gestational diabetes (gdm) is among the most common pregnancy complications. despite treatment, up to % of pregnancies complicated by gdm result in infants being born large-for-gestational-age (lga). this not only causes problems at birth but predisposes offspring to developing cardio-metabolic disease in adulthood. there are no treatments for lga as the cause is unclear, although it is associated with altered placental vascular development. micrornas (mirnas) regulate placental development; they are produced within cells but can be released into the circulation inside evs, which in turn can be transported into target cells and tissues to influence cellular processes. we aimed to characterise circulating evs in pregnancies complicated by gdm-lga and determine if ev-derived mirnas have the potential to influence placental development. methods: maternal serum and plasma samples were collected from women with pregnancies complicated by gdm at - weeks gestation; placental tissue was collected at delivery and birth outcomes recorded. serum and plasma evs were isolated and characterised by electron microscopy (shape), nanoparticle tracking analysis (nta; size/concentration), and western blotting (ev-enriched proteins). mirna qpcr arrays were performed on evs. mirnas were quantified in placental tissue via qpcr. results: em and western blotting confirmed isolation of evs and nta revealed no significant difference in size/ concentration in gdm-lga pregnancies (n = ) compared to gdm-aga (n = ; p > . ). several ev mirnas were altered in maternal circulation in gdm-lga compared to gdm-aga (n = /group; >twofoldchange; p < . ), including four skeletal muscle-specific "myomirs": mir- - p, mir- a- p, mir- b, and mir- a- p (all increased). all four myomirs were present in placenta but only mir- - p was significantly altered in gdm-lga compared to gdm-aga (n = - /group; p < . ). summary/conclusion: ev-bound myomirs could have predictive value for aberrant foetal growth in cases of gdm. mir- - p regulates vascular development in other systems, so we propose that mir- - p contributes to lga by influencing placental vascular development, however further work is required to establish this. introduction: seminal plasma is particularly rich in extra cellular vesicles. myelinosomes are membranous organelles described throughout the seminiferous epithelium of the testis but never reported in semen. the aim of this study was to look for the presence of myelinosome vesicles in human seminal plasma. methods: because of the viscosity of seminal gel and its water-holding capacity, classical transmission electron microscopy does not seem to be an optimal technique to reveal the presence of myelinosomes in this fluid. cryo-electron microscopy is a technique that allows visualization of nanosized structures without prior fixation or addition of heavy metals for contrast. the sample is therefore visualized as close to its native state as possible. using standard myelinosome preparation from tm sertoli cells, we first analysed the appearance of "standard" native myelinosomes by cryo em and then compared it with the vesicles from human seminal plasma samples. results: we have specified by cry-em the morphological aspect of "standard" myelinosomes isolated from the culture media of tm sertoli cells. the vesicles with the same morphological appearance were revealed in human seminal plasma specimens. summary/conclusion: myelinosomes are membranous organelles found in the seminiferous epithelium of the testis and secreted by the somatic sertoli cells in the lumen of the seminiferous tubules.the preparations from human seminal plasma contains a population of large ev (average diameter nm) whose morphological appearance resemble those of myelinosomes. defining the specific biomarkers and functionalities of myelinosomes in human seminal plasma are the concerns to be addressed in our further research. introduction: more than one million patients worldwide suffer from tuberous sclerosis complex (tsc) and have mutations in either tsc or tsc genes. together, the tsc proteins regulate mtorc activity. all tsc patient post-mortem samples exhibit renal disease and % of patients with tsc experience a premature loss of renal function. mouse and human studies are incongruity with the second somatic hit mechanism of disease, because of the low percentage of cystic cells exhibiting loss of tsc expression. we posited that the loss of a tsc protein expression may alter extracellular vesicle (ev) biology and contribute to disease. methods: we used crispr/cas to disrupt the tsc gene in mouse inner medullary collecting duct (mimcd) cells, and isolated evs using gel filtration from the isogenic cell lines. we characterized the evs using tunable resistive pulse sensing (trps), dynamic light scattering (dls), transition electron microscopy (tem), and wester blot analysis. we further performed mass spectroscopy on the ev proteins. results: loss of the tsc gene in mimcd cells induced a greater than three-fold increase in ev production compared to the same cells having an intact tsc axis. electron microscopy confirmed the purity and spherical shape of evs. both trps and dls demonstrated that the isolated evs possessed a heterogenous size distribution. approximately % of the evs were in the - nm size range. western blot analysis using proteins isolated from the evs revealed the cellular proteins alix and tsg , the transmembrane proteins cd , cd and cd , and the primary cilia-related hedgehog signalling-related proteins arl b. proteomic analysis of evs identified a significant difference between the tsc -intact and tsc -deleted cells that correlated well with the increased production. summary/conclusion: evs may be involved in tissue homoeostasis and cause disease by overproduction and altered protein content. the evs released by renal cyst epithelia in tsc complex may serve as a tool to discover the mechanism of tsc cystogenesis and in developing potential therapeutic strategies. introduction: we have shown that evs derived from amniotic fluid stem cells (afsc) of mouse origin present therapeutic effect in an animal model of chronic kidney disease, alport syndrome (as). in light of clinical translation, we isolated afsc-evs of human origin, characterized their cargo and evaluated thier therapeutic effect in vivo. methods: human clonal afsc were derived from amniotic fluid collected after volunteer donors provided consent. evs were obtained from afsc and identity and purity were assessed by rna-seq and proteomics. potency of hafsc-evs was evaluated by performing in vivo studies. ev biodistribution was evaluated by mri and therapeutic effect by measuring renal function and mice life-span. bulk rna-seq was performed on glomeruli obtained from injected and non-injected mice to identify potential ev regulating targets. results: proteomic profiling identified intact proteins and rna-seq data identified , mirs in hafsc-evs. hafsc-ev "fingerprint" was assessed by performing go analysis on the most highly expressed proteins and mirs. the results identified pathways involved in tissue homoeostasis such as mtor pathway, tgfβ and vegf pathways. when injected in vivo into as mice, biodistribution studies showed that hafsc-evs localized in the kidney, corrected proteinuria. no side effects (including teratoma) were noted in the treated mice. rna-seq of glomeruli obtained from treated as mice showed similar gene expression patterns to wilt type mice, by cluster analysis. our data indicated that hevs highly modulated pathways involved in collagen and matrix deposition remodelling, in addition to downstream targets of vegf, fgf, tnf, angiotensin and preserved glomerular cells structure and function. summary/conclusion: our protocol for hevs derivation is reproducible and allows derivation of ev lots with the same identity (specific cargo of proteins and mirs) and potency (present therapeutic effect in as). hafsc-evs modulated signalling pathways that are central to maintaining glomerular homoeostasis and preserved glomeruli structure with improved kidney function. this suggests the possibility of using hafsc-evs as a new therapeutic option for treating renal failure in humans. introduction: recent studies have shown that stem cell-derived extracellular vesicles (msc-ev) therapy improves renal outcomes in models of acute ad chronic renal disease. however, to better investigate the molecular mechanisms of ev-induced regeneration, and to define new ev sources, devices that mimic d organ architecture and flow conditions are needed. the aim of our work is to evaluate the regenerative potential of naïve and engineered ev in a millifluidic in vitro d model of glomerular damage in continuous perfusion. methods: methods: we set a millifluidic in vitro d model of glomerular filtration, a three-layers structure composed by human podocytes and glomerular endothelial cells, and, in between, of a basement membrane of collagen type iv. the barrier thus formed is set up inside a bioreactor, in a closed milli-fluidic circuit in which fluid flows continuously at a certain flow rate. we reproduced different pathological conditions and tested the localization and effect of evs in a dynamic system. : results: we obtained a standardized protocol and an adequate configuration of the milli-fluidic circuit subject to continuous reperfusion. renal damage was induced by doxorubicin or by hypoxia-reperfusion injury. we evaluated uptake, cargo transfer and effect of naïve and mirna engineered msc-evs or of klotho engineered ineffective evs administered into the dynamic co-culture system. evs were able to pass through the system and to deliver to podocytes proregenerative factors, promoting survival and limiting permeability. introduction: worldwide, renal cell carcinoma (rcc) is th most common cancer in men and th most common in women. new biomarkers are needed to aid rcc-diagnosis, provide prognostic information, and to predict response to modern targeted therapies. extracellular vesicles (evs) are an emerging source of cancer biomarkers because all cells, including cancer cells, secrete evs into biofluids as blood and urine. however, benign cells contribute to ev populations isolated from blood and urine reducing the diseasespecificity. we have developed a protocol for ev isolation directly from human rcc tissue that can increase tumour-specificity of biomarkers. methods: we obtained technical and biological replicates from normal kidney tissue and clear cell rcc tissue. serum-free media was incubated with the specimens. a combination of differential centrifugation, filtration, and ultracentrifugation was used for ev isolation. evs were quantitated using two methods, allowing for comparison between nanosight ns and nanofcm. tem was used to determine presence of intact vesicles in the ev samples. presence of ev introduction: urothelial carcinoma (uc) is a malignant cancer that affects the urothelial cells, representing % of all bladder tumours. at diagnosis % of bladder cancers are non-muscle invasive tumours. importantly, upon transurethral resection of the bladder tumour, nearly - % of these patients will experience disease relapse and - % will progress to muscle invasive tumour, requiring thereby, a rigorous and expensive follow-up. currently, this is performed through the frequent use of highly invasive cystoscopy and the low sensitivity urine cytology. thus, innovative liquid biopsy-based biomarkers that circumvent these drawbacks are highly desirable for improved uc clinical management. here, we aim to implement a protocol for the isolation and characterization of extracellular vesicles (evs) from uc patients' urine samples. methods: a two-step protocol involving ultracentrifugation (uct) and by size-exclusion chromatography (sec) was optimized for urine samples. the isolated urine-derived evs from uc patients were then characterized according to their size, concentration (nta), morphology (tem), protein amount (lowry method), presence of ev-associated and disease-associated protein markers (western blot). results: isolated urinary evs from uc patients had a size ranging from nm to nm with characteristic ev morphology, express ev-associated markers as cd and hsp and were negative for cell debris markers. the recovery yield and purity of isolated evs following each isolation technique was characterized. upon uct, sec was required to deplete most of the ev-associated thp and albumin protein contaminants. some disease-associated protein markers were highly enriched in isolated urinary evs compared to crude urine. summary/conclusion: taken together, these results indicate that a two-step ev isolation protocol was properly implemented and validated in uc patients' urine samples. notably, several ev-associated disease biomarkers were detected in the urine of uc patients. this ev-based liquid biopsy might provide the means for real-time monitoring of residual disease and relapse in uc patients. introduction: glioblastoma multiforme (gbm) is a very aggressive type of brain tumour. different gbm molecular subtypes (proneural, mesenchymal and classical) often co-coexist within the same tumour, with the mesenchymal subtype driving the tumour progression. recently, our lab demonstrated that the cargo of extracellular vesicles (evs) could mirror the molecular background of the gbm cells from which they were derived. altogether, we believe that gbm cell-derived evs can be directly involved in the expansion of the mesenchymal signature in tumours, thus supporting gbm aggressiveness. methods: non-mesenchymal (t & u ) gbm cells were "primed" using evs derived from mesenchymallike (u & ln ) gbm cells. ev-primed gbm cells were then co-cultured with their non-primed counterparts to determine whether the mesenchymal signature can "spread" from cell to cell via evs. effect on cell proliferation, migration and invasion (in hyaluronic acid hydrogels) was assessed following ev treatment and co-culture. the expression of mesenchymal gbm markers was measured by western blotting. further mass spectrometry analysis of cell and ev content was undertaken to describe potential underlying mechanisms. results: co-culture with ev-primed gbm cells significantly increased proliferation and hydrogel invasiveness of non-mesenchymal cells. interestingly, the stimulating effect of co-culture was even stronger on the proliferation of ev-primed gbm cells. moreover, further proteomic analysis revealed that expression of mesenchymal gbm markers such as cd was increased in non-mesenchymal cells following coculture. summary/conclusion: our data suggest that evs from mesenchymal gbm cells can be uptaken by gbm cells from different subtypes, thus stimulating tumour progression. overall, we think the present study provides with new insights for the understanding of gbm recurrence and the development of potential therapeutic strategies. introduction: triple-negative breast cancer (tnbc) is the most aggressive form of breast cancer. previously we reported that the heterogenous population of evs released from tnbc cells promotes the growth and aggression of recipient cells. here we investigated if, by using compounds proposed to inhibit ev release i.e. calpeptin and y (to block those budding at cell membrane) and gw and manumycin a (to block evs from mvbs), we could reduce the associated transmission of aggressive phenotype. methods: evs were separated from medium conditioned by tnbc cell line hs ts(i) , using a discontinuous optiprep density gradient, after the cells were treatment for hrs with the compounds listed above. evs (pooled fractions - with a density range of . - . g/ml) were characterised by nta, bca, lipid assay, immunoblot, tem and flow cytometry. to investigate the functional effects of the evs released, proliferation and migration assays were performed on hs t and mda-mb- cells using the ev to cell ratios of × evs/ x cells, × evs/ x cells, × evs/ x cells to evaluate doseresponse. ev-track id ev (score of %). results: gw significantly (p = . ) decreased ev release from hs ts(i) cells. manumycin a and a combination of calpeptin and y (combo) decreased ev release, but significance was not reached. conversely, calpeptin and y actually increased ev release; but not significantly. of the reduced numbers of evs released following gw treatment, hla-dr+ evs were significantly (p = . ) enriched. none of the evs analysed significantly changed hs t or mda-mb- growth rates. however, evs from cells treated with calpeptin (p = . ), gw (p = . ), manumycin a (p = . ) and combo (p = . ) caused significant reduction in mda-mb- migration compared to the effects of evs from untreated cells. similarly, ev from cells treated with gw (p = . ), and combo (p = . ) caused significant reduction in hs t migration. summary/conclusion: while gw was the only compound that caused a significant decrease in quantities of ev released, the evs that continued to be released following treatment with gw or calpeptin and y significantly reduced migration of both recipient cell lines. funding: phd funding: tcd scholarship and carrick therapeutics ltd extracellular vesicles from highly metastatic lung cancer cells induce barrier impairment, permeability, and epithelial-to-mesenchymal plasticity in a -day mature bronchial epithelium purdue university, west lafayette, usa introduction: epithelial-to-mesenchymal (emt) transition plays an integral role in cancer metastasis, which is responsible for as much as % of cancer mortality. cancer exosomes induce emt in bronchial epithelial cells, however, the epithelial cells inhibit emt when allowed to form a mature epithelial barrier with apicalbasal polarity. it is not known if cancer-derived extracellular vesicles (evs) can induce emt and more importantly, barrier disruption in a mature epithelium. here, we show that evs from a highly metastatic lung cancer cell line (calu ) are) are not only sufficient to induce emt in non-tumorigenic bronchail epithelial cells (beas- b), but are also capable of disrupting a -day mature bronchial epithelial barrier by significantly reducing teer, inducing sixfold increase in permeability and complete loss of e-cadherin at cellcell tight junctions. methods: beas- b and calu evs were characterized using electron microscopy, nanosight and western blotting for exosome-specific features. for permeability studies, beas- b cells were cultured in transwell for days to establish an intact epitheliumconfirmed by measuring teer (trans-epithelial electrical resistance). intact beas- b monolayers were treated with calu evs at , and μg/ml for hrs, and barrier intactness and permeability were evaluated by measuring teer, apical-basolateral translocation of dextran beads and confocal imaging of tight junctions (e-cadherin). for emt experiments, beas- b cells treated with calu evs at and μg/ml were evaluated for ecadherin and vimentin levels by qrt-pcr and western blot after hrs. results: beas- b and calu evs were enriched in - nm size range, and cd and cd were enriched in the ev fraction in contrast to the cell lysate and vice versa for gp . calu evs significantly impaired day mature beas- b monolayer's barrier properties, which at the highest dose caused % reduction in teer from . ± . to . ± . Ω.cm (n = ). this was further confirmed by~sixfold increase in dextran beads' apical-basolateral translocation in min ( . ± ng/ml in control vs . ± ng/ml in treated) (n = ) and complete loss of e-cadherin expression at cell-cell tight junctions (n = ). at the transcript level, calu evs induced significant downregulation of e-cadherin by % and upregulation of vimentin (mesenchymal marker) twofold (n = ) in beas- b cells, indicating transition into mesenchymal phenotype. summary/conclusion: we demonstrated the involvement of evs derived from highly metastatic lung cancer cells in inducing emt in bronchial epithelial cells and epithelial barrier disruptionthe initial stage of the intravasation process. grp plays a crucial role in the extracellular vesicle-promoted radioresistance of irradiated head and neck cancer cells introduction: small evs released from irradiated head and neck squamous cell carcinoma (hnscc) cells increase resistance of recipient hnscc cells to radiation in vitro. we have identified the glucose-regulated protein (grp ), a chaperone protein of the hsp family which is involved in cellular stress responses and associated with worse survival in head and neck cancer patients, as an essential component of the ev-mediated radioresistance. methods: small evs were isolated from conditioned medium from irradiated and non-irradiated bhy hnscc cells by combined microfiltration ( . µm) and differential ultracentrifugation. grp surface expression was measured by proteomic analysis, immunoblotting and bead-facs. radiation resistance of bhy cells was determined by a clonogenic survival assay. results: increased grp was identified on the surface of evs from irradiated cells. the increase in ev grp correlated with increased grp expression at the donor cell surface. the grp content of recipient cells also increased upon transfer of evs from irradiated, but not non-irradiated cells, ultimately leading to enhanced cell survival. to check a potential role of elevated grp in radiation resistance we overexpressed grp . here the modest ( x) overexpression of grp was sufficient to confer an enhanced radioresistant phenotype to the bhy cells. a correlation between grp -dependent increase of radioresistance and activation of the akt pathway is yet to be determined. summary/conclusion: our results suggest a pivotal role for ev-transferred grp in modulating the radiation response of recipient hnscc cells. radiation directly increases the cellular and vesicular grp levels, and subsequent ev-mediated transfer leads to enhanced grp levels and radioresistance in recipient cells. this study provides new mechanistic insights into the effects of evs in radiation response and elucidates an interesting target protein and novel strategies for the improvement of radiotherapy. d modelling of ev release in progressing prostate cancer introduction: the modelling of cancer progression should be capable to translate acquired knowledge of cell behaviour to the real human body conditions. however, the extracellular vesicles (evs) isolated from d cell models are commonly exploited in research. taking into account the specificity of the prostate cancer (pc) environment, and a strong need of early diagnosis of castrate-resistance by prostate cancer (crpc) patients, we suggest in-depth profiling of different ev subtypes isolated from d culture as a new tool to model the progressing pc. methods: cells from hormone-resistant prostate carcinoma -rv line were cultured in d and d conditions, using d coseedistm. acd plasma controlled for haemolysis and remaining platelets was taken from patients with pc and crpc. the fractions of ev subtypes from cell culture and plasma were obtained by differential centrifugation (dc) followed by iodixanol density gradient purification. each of the fractions was measured by nanoparticle tracking analysis (nta), tunable resistive pulse sensing (trps) followed by elisa. for that, cd and cd were used as ev markers, apob and apoa for lipoprotein contaminants control, and cd , cd and psma as tissue-specific biomarkers for determination of fractions containing evs of different origin. ev-contained fractions were subjected to next generation sequencing (ngs). results: in d conditions, the -rv cells produce up to -times higher ev number than in d. size and density distribution of evs derived from d cultures but not of d resembled plasma evs. size distribution and biomarker expression among different ev subtypes allowed distinguishing between pc and cprcderived samples, indicating a potential to translate these results into clinics for early cprc detection. summary/conclusion: this work demonstrates a new approach to study the secretome of a progressing pc under d conditions. the profiles of ev subtypes produced by cancer cells growing in a d spatial architecture resemble the profiles of plasma evs and can serve a useful tool for the establishment of new biomarkers. introduction: renal cell carcinoma (rcc) is the most common primary renal neoplasm, with over , cases in the us alone each year. early detection of rcc leads to consistently better patient outcomes, and extracellular vesicles (evs) isolated from patient samples may prove to be a valuable clinical tool in the future. evs are abundant in blood and urine and show a large amount of heterogeneity but are difficult to analyse due to their small size and difficulty in isolation. here, we employ a multiparametric analysis of ev surface markers to identify a set of markers that may prove clinically relevant in future studies. methods: rcc cell lines vok , vok , and vok were cultured in flasks containing ml of ev-depleted media ( % fbs, centrifuged hr x , g). when cells reached~ % confluency, the conditioned media was collected and spun at , g for mins two times to deplete any remaining debris, leaving~ ml of media. this media was concentrated to a final volume of~ ml using a pall jumbosep kda mwco filter. this concentrate was purified from protein by using an izon qev- column, collecting ml fractions. protein content of each fraction was analysed using a absorbance while concentration and diameter distribution were determined through nanoparticle tracking analysis (nta). pooled samples made of the three most concentrated fractions were concentrated to a final volume of~ µl using the pall microsep kda filter and then used for analysis in the miltenyi macsplex exosome kit. flow cytometric data were generated by the cytoflex s and analysed using flowjo and mpapass software. these positive signals were verified through bead-only controls and titrations. results: the mpapass software allowed for heatmap generation, data reduction, clustering and visualization of expression patterns. of the detection antibodies used across capture beads, cd , cd , cd , beta- microglobulin, and cd were found to be prevalent in these rcc evs. these markers were found to be co-expressed particularly with cd , cd , and cd . summary/conclusion: the use of multiplex analysis allowed for detection of five distinctive surface markers found to be prevalent in evs collected from rcc cell lines. these results demonstrate the utility of multiplex analysis and mpapass software for identifying potential markers of interest and provide proteins that are worth exploring further. the next steps to this work will be developing custom multiplex arrays that tailor capture and detection of evs specifically for rcc pathology. low molecular weight protein tyrosine phosphatase (lmwptp) carried by colorectal cancer cells-derived extracellular vesicles as a player in tumour-educated human fibroblast university of campinas -unicamp, campinas, brazil introduction: extracellular vesicles (evs) are doublemembrane-bound nanovesicles released by cells playing a key role as mediators of intercellular communication. low molecular weight protein tyrosine phosphatase (lmwptp) is upregulated in several cancers type, including colorectal cancer (crc), and it has been correlated with aggressiveness, chemoresistance and poor prognostic. methods: the aim of this study was to determine whether crc cells release lmwptp-enriched-evs and influence tumour microenvironment-associated cells as a representative tumour education. crc cells, hct and ht , were cultured in serum-free medium for hours. conditioned medium was concentrated by ultrafiltration (mwco kda) and evs were isolated by total exosome isolation reagent (invitrogen). evs were characterized by nanoparticle tracking analysis (nta), transmission electron microscopy (tem) and western blotting (wb). lmwptp levels were analysed by wb and sandwich-elisa. to evaluate tumour education, hff- fibroblasts were used as recipient cells. the uptake of evs (pkh fluorescently labelled evs), proliferation (viability) and migration (wound healing assay) were analysed in a co-culture model of crc-derived evs and hff- . results: nta showed a higher concentration of evs released by ht . hct and ht evs displayed a mean diameter around nm and a cup-shaped morphology. isolated evs were positive for evs-markers cd and tsg and negative for gm a non-evs marker. ht lineage as well as derived-evs are lmwptp-enriched in comparison to hct cells and evs. upon incubation, fluorescently hct and ht derived evs were internalized into hff- cells in a perinuclear region. evs derived from both cells increased the viability and proliferation of hff- cells. intriguingly, evs derived from ht promoted cell migration. summary/conclusion: in conclusion, for the first time, we showed that lmwptp can be carried by evs derived from crc cells and lmwptp-enriched-evs can modulate biological aspects of hff- fibroblast. overall, our findings point lmwptp out as important player in tumour-educated fibroblast. exosomal mir- a inhibition by vincristine and prednisone in paediatric acute lymphoblastic leukaemia. introduction: vincristine and prednisone are standard agents in treatment of paediatric acute lymphocytic leukaemia (p-all). mechanistically, vincristine induces apoptosis by blocking microtubules formation, while prednisone binds to cytoplasmic receptors and inhibits dna synthesis, both of which lead to apoptosis. the effect of these agents on exosomal micro-rna expression and its functional regulation is not yet investigated. elevated levels of mir- a in circulating exosomes (nanoparticles) has been shown to lead to progression in several cancers, including all. we have previously shown that leukaemia-derived exosomes induce leukaemia cell proliferation via up-regulating of mir- a expression and silencing of exosomal mir- a reverses this exosomeinduced cell proliferating effect. the objective is to investigate the effect of vincristine and prednisone on exosomal mi-r a expression in all. methods: jm , sup-b , and nalm- leukaemic cell lines were treated in vitro with vincristine ( . to . µm) and prednisone ( . to . µm) in exo-free medium and apoptosis was measured by mts assay. total rna of exposed cell lines was isolated and cdna was prepared for mir- a analysis. expression of mir- a was analysed by q-pcr. exosomes from conditioned medium of exposed cell lines were isolated by ultracentrifugation method. purity and particle size of exosomes were confirmed by western blot and nanoparticle tracking analysis (nta) assay respectively. total exosomal rna was isolated from exosomes (exo-rna) by trizol method. synthesis of cdna was carried out with the miscript ii rt kit (qiagen). results: vincristine and prednisone promote apoptosis in leukaemia cell lines (jm and sup-b ) in a dosedependent manner. both cellular and exosomal mir- a expression was down-regulated by vincristine and prednisone exposure in all three leukaemia cell lines (jm , sup-b , and nalm- ). these observations demonstrate that cellular mir- a down regulation in the parental cells is stable and can be transferred to exosomes, confirming the concept that exosomes are the fingerprint of parent cells. summary/conclusion: our data suggest that the vincristine and prednisone anti-proliferative effect in p-all maybe induced by another yet unexplored pathway, that suppresses mir- a at a cellular and exosomal level in p-all, resulting in apoptosis. funding: this project is supported by the dimartino family foundation. secreted extracellular vesicles from renal cell carcinoma cells anatoliy samoylenko, artem zhyvolozhnyi, eslam abdelrady, naveed ahmad, genevieve bart and seppo vainio oulu university, oulu, finland introduction: clear cell renal cell carcinoma (ccrcc) represents the most common form of kidney cancer and is among the most lethal of all genitourinary cancers. despite surgery and medication therapy, most patients with metastatic ccrcc have a poor prognosis. intratumoural hypoxia is a key factor involved in renal cancer progression and it is known to promote secretion of evs by many types of tumour cells. methods: rcc-derived renca cells, embryonic kidney derived ub cells, and primary mouse hepatocytes were used in the study. evs were purified from cell culture media by gradient ultracentrifugation, sequential ultracentrifugation and exo-spin™ columns. before ev isolation cells were kept for h either under normoxia or hypoxia ( % oxygen). evs were analysed by transmission electron microscopy with negative staining and immunolabeling, by nanoparticle tracking analysis (nta) and western blotting. cells proliferation and viability were assayed by live cell imaging using incucyte zoom (essen bioscience), cell metabolic activity by seahorse xf analyser (agilent), rna expression by qpcr and ddpcr. proteins were identified by ultra-performance liquid chromatography-mass spectrometry (uplc-ms). rna libraries were made using nebnext small rna library prep kit, and sequenced on nextseq (illumina). results: we showed that hypoxia induced production of evs by rcc cells, and characterized differences in protein and rna content of evs generated by renca cells cultured under normoxic and hypoxic conditions. we also showed that rcc-produced vesicles modify key features of tumorigenesis (gene expression, metabolic activity, motility, and growth) of target cells. these data were obtained by using two target cell types: model mouse kidney cells and primary mouse hepatocytes, which represent typical site of rcc metastasis with an exceptionally poor prognosis. we proposed that a possible mechanism of ev action in rcc is related to changes in caveolin- function. we also tracked renca-derived evs in a chick embryo model and in a novel kidney organoid co-culture assay developed by our group (xu et al., ) . summary/conclusion: hypoxia may influence tumorigenic properties of rcc by changing rates of production and composition of evs. funding: the study was supported by finnish cancer foundation grants. exosomes synthesizing her mirna and engineered to adhere to her on tumour cells surface exhibit enhanced anti-tumour activity introduction: exosomes are small extracellular vesicles averaging - nm in diameter. they serve as a means of intercellular communication. typically they consist of structural proteins as well as selected proteins, mirnas, mrnas, and long noncoding rnas. thus in an earlier report this laboratory designed a mirna targeting a major herpes simplex virus regulatory protein. as predicted by the nucleotide packaging signal the mirnas were packed in exosomes and on exposure to infected cells significantly reduced virus yields. her (human epidermal growth factor receptor ) plays an important role in the neoplasia of some breast cancers. the protein is exhibited on the cell surface and is the target of therapeutic antibodies. methods: firstly, we report on the construction of a mirna targeting the synthesis of her both in cells constitutively expressing her and in cells transfected with a plasmid encoding her . secondly, we report that the mirna targeting the synthesis of her reduced the viability of her positive cancer cells both in cell culture and in implanted tumours. lastly, we enhanced the anti-tumour activity of the exosomes by binding to the exosome surface a ligand with affinity for the her on the surface of tumour cells. the -mir-her exosomes package with mirna designed to block her synthesis and deliver to cells. these exosomes kill cancer cells dependent on her for survival but have no effect on cells lacking her or which were engineered to have her but do not depend on it for survival. the -mir-xs-her exosomes carry in addition a peptide which enables the exosome to adhere her on the surface of the cancer cells. in consequence, these exosomes preferentially enter and kill cells exhibiting her on their surface. the exosomes with -mir-xs-her are significantly more effective in shrinking the size of her -positive tumours implanted in mice than the -mir-her exosomes. summary/conclusion: our studies indicate that exosomes carrying mirna against her have no effect on her negative cells it was nevertheless desirable to increase the uptake of exosomes carrying the her mirnas by her -positive tumour cells. to this end we modified the exosomes to exhibit on their surface a peptide that bound the exosomes to the her on the surface of cancer cells. in consequence, we significantly enhanced the uptake of exosomes carrying the mirnas directed against her by her positive cells. funding: these studies were supported by grants from shenzhen overseas high-calibre peacock foundation kqtd , shenzhen science and innovation commission project grants jcyj , jcyj to shenzhen international institute for biomedical research. systematic characterization of ovarian cancer-derived exosomes unveil mirnas interfering with cd + t cell activation introduction: cd + tumour-infiltrating lymphocytes (til) have been widely reported to correlate with cancer patient survival, including ovarian cancer. even with the presence of tils, immunotherapy has limited success in ovarian cancer. understanding the interaction between cd + til and tumour cells is thus important. our hypothesis is that tumour-derived exosomes are released and taken up by cd + til such that specific mirnas contained within modulate physiological processes that inhibit cd + t cell activation. we aim to identify mirnas carried in tumour-derived exosomes that inhibit cd + t cell activation in ovarian cancer. methods: we purified exosomes from nine ovarian cancer cell lines and stocked in high concentration. interferon-gamma (ifn-gamma expression screening was performed after days of co-incubation of tumour derived exosomes, cd + t cells, and activators in conditioned medium. cell counts and viability were tested by trypan blue staining at day and day . rna-seq for exosomes were generated to identify mirnas critical in differentiation effects on cd + t cell activations. microrna target matching uncovered target mrnas while enriched pathway analysis predicted potential signalling pathways involved. results: our ifn-gamma screening results indicated the exosomes exhibit different behaviours in interfering cd + t cell activation owing to different donors. exosomes derived from peo. and ovca cells have consistent polarized results in ifn-gamma expression. exosomes derived from peo. remained a low ifn-gamma expression and from ovca stayed at relatively high level. small rnas profiling analysis between the two cell lines identified mirnas (p < . ), and mirnas have been reported with validated targeting information, and out of have targets involved in immune signalling. mrna targets were uncovered by target matching. cmap search identified complex connections among mrnas with the top enriched pathways actively involved in cell cycle and immune related behaviours. summary/conclusion: our ifn-gamma screening identified crucial mirnas in ovarian cancer exosomes interfering cd + t cell activation. computational modelling on both experimental and public multiomics datasets predicted promising signalling pathways of tumour-immune crosstalk for functional validation. irradiation of breast cancer cells alters the quality of dna cargo in the exosomes that they produce sheila spada, paul zumbo, doron betel, tuo zhang, nils-petter rudqvist and sandra demaria weill cornell medicine, new york, usa introduction: irradiation of breast cancer cells with an immunogenic dose ( gyx ) leads to accumulation of cytosolic dna that is sensed by cgas leading to interferon type i (ifn-i) signalling via cgas/sting pathway [ ] [ ] [ ] . we previously showed that tumour-derived exosomes (tex) secreted by irradiated ( gyx ) (rt-tex) but not untreated (ut-tex) tsa carcinoma cells carry dna that stimulates the production of ifn-i in recipient dendritic cells (dc) via the cgas/ sting pathway [ ] . moreover, mice vaccination using rt-tex, but not ut-tex, elicited anti-tumour immune response inhibiting tumour growth [ ] . here, we hypothesized that the differential ability of rt-tex and ut-tex to activate ifn-i in recipient dcs is due to qualitative differences in dna cargo of rt-tex compare to ut-tex. methods: the length of dna purified from tex and from the cytosolic fraction of tsa cells was measured by agilent bioanalyzer. the dna cargo of tex was analysed by whole-genome sequencing (wgs) and whole-genome bisulphite sequencing. the percentage of methylation of total dna in tsa cells was quantified by -methyl cytosine dna elisa kit. results: dna fragments with size between and bp were enriched in rt-tex compared to ut-tex, as well as in the cytosolic fraction of irradiated compared to mock-treated tsa cells. wgs revealed that the entire genome was represented in tex dna cargo, regardless of rt. more than % of tex dna was of nuclear origin, but mitochondrial dna was increased in rt-tex. interestingly, we found that rt decreases the level of methylation in both exosomal and total dna in tsa cells compared to the controls. summary/conclusion: these data support the hypothesis that immunogenic rt alters some characteristics of the exosomal dna cargo, mirroring molecular changes occurring in parent irradiated breast cancer cells. the enrichment in dna fragments of - bp in rt-tex is intriguing considering that cgas is optimally activated by dna in this length range [ ] . we are currently investigating which features of the cargo dna that differ between ut-tex and rt-tex may explain the differential ability to induce ifn-i pathway activation in recipient dcs. the identification of a dna signature associated with the ability of tex to activate the cgas/sting pathway could provide a circulating biomarker of the rt-driven immunogenic tumour response. introduction: triple negative breast cancer (tnbc) is among the most difficult cancer subtypes to treat and continues to cause a high number of cancer-related deaths annually. extracellular vesicles (evs) transfer cell type-specific cargo and have important implications in disease initiation, therapy and outcome. upon treatment of cancer cells with low-dose chemotherapy, released evs are able to transfer phenotypic traits to other cancer cells. new treatment strategies for tnbc, like inhibitors of the er stress pathway (ire ) might impact on ev biogenesis, cargo delivery and response of cells in the cancer microenvironment. our aim is to identify immune modulatory alterations in breast cancer cells and cancer derived evs upon treatment with inhibitors of the er stress pathway. methods: human tnbc cell lines were treated with ire inhibitor mkc and cells were analysed for immune modulatory surface markers, like hla-i, b -h molecules and different integrins. mitochondrial and lysosomal activities were investigated by the use of a mito-and lysotracker and analysed by imagestream (isx) technology. extracellular vesicles were isolated from cell culture supernatants by sequential centrifugation, quantified by nanoparticle tracking (nta) and characterized by exosome bead array. single ev analysis of total cell free supernatants and of isolated evs was performed by isx and marker positive evs were quantified for absolute fluorescence signals and total amount by objectives/ml. ev uptake into t cells was investigated by the use of different ev labelling strategies. results: several immune relevant surface markers (hla-i and cd ) are downmodulated by ire inhibition across different cell lines. cell surface expressed cd and b -h show cell line specific downmodulation profiles upon ire inhibitor treatment. other immunomodulatory marker such as b -h and b -h , integrin cd , cell adhesion-promoting cd and stemness/metastasis marker (cd and ssea) are unaltered on ire treated breast cancer cells. cancer cell derived evs were tetraspanin positive (cd , cd , cd ), similar in number and showed differential expression of immune markers upon ire treatment. mitochondrial and lysosomal activities were unaltered under ire inhibition, whereas cell proliferation was diminished. no breast cancer-derived ev uptake of externally labelled evs into healthy t cells could be detected. summary/conclusion: ongoing analyses focus on the multicolour analysis of multiple markers on single evs by imaging flow cytometry and on the functional impact of cancer derived evs on t cells delivered by ev receptor binding. funding: dagmar quandt is supported by the sfi (cÚram research centre, /rc/ ), the european regional development fund and the dr. werner jackstädt-stiftung. chair: uta erdbrügger -university of virginia chair: larry harshyne -thomas jefferson university comparison of three isolation protocols to search extracellular vesicles signature in sickle cell disease patients introduction: sickle cell disease (scd) is an inherited disorder characterized by chronic haemolysis and continuous activation of different cell types. extracellular vesicles (evs) were described to be at increased levels in scd patient's plasma compared to healthy subjects and were associated with several clinical manifestations such as leg ulcers and stroke. scd patient's plasma has increased concentrations of haem, free-hb and other proteins and lipoproteins as chronic haemolysis consequence. here, we report the comparison of three mostly used isolation protocols to search ev signature in scd patient's plasma by flow cytometry. methods: blood samples were obtained from scd patients (n = ) following wisgrill et al., ( ) protocol. three different ev isolation protocols were used: differential centrifugation (dc), ultracentrifugation (uc) and size-exclusion chromatography (sec). lactadherin and calcein-am were used to detect phosphatidylserine (ps)+ vesicles and membrane integrity, respectively. platelet-derived evs (pevs), endothelialderived evs (eevs), leucocyte-derived evs (levs) and monocyte-derived evs (mevs) were quantified. silica beads were used to define evs gate and samples were acquired in the cytoflex cytometer platform. results: the quantification of pevs in uc, dc and sec samples was, respectively, x , , x and , x events/ml mean, eevs was , x , × and , x events/ml mean, levs was x , × and , x events/ml mean and mevs , x , , x and , x events/ml mean. uc samples demonstrated a higher concentration of evs, which could be more useful to functional studies than dc and sec, however, it took more time to separate than dc. dc was the fastest method to separate evs from plasma, being useful to study large patients cohorts, but showed the smallest overall number of evs. sec also demonstrated high capability to detect evs in plasma and the possibility of obtaining a purer sample, although it is the most expensive and time-consuming method among all tested. all evs populations were detected in the three protocols tested. summary/conclusion: in summary, all protocols tested were efficiently to detect evs in scd patient's plasma and the definition of the best protocol may vary based on the research aim and time and budget available. funding: fapesp / - . gabrielle lapping-carr, joanna gemel, yifan mao and eric beyer university of chicago, chicago, usa introduction: aberrant cell-cell interactions involving the endothelium are central to the pathophysiology of sickle cell disease (scd), including acute chest syndrome (acs), a deadly and unpredictable complication. we previously demonstrated that the plasma of scd patients contains increased circulating small extracellular vesicles (evs) compared to controls and that those vesicles can disrupt endothelial integrity in vitro by affecting adherens junctions and ve-cadherin. the current study was designed to examine the effects of those evs on other cellular junctions including tight (zonula occludens , zo- ) and gap junctions (con-nexin , cx ) and to test the hypothesis that the junctions would be more severely affected by evs isolated from patients during an episode of acs than by ones isolated from the same patient at baseline. methods: we identified subjects with scd in our biobank who had plasma isolated at baseline and at the beginning of an admission for acs. evs were isolated from platelet free plasma using established methodologies. to determine the effects on endothelium, cultures of human microvascular endothelial cells were treated with evs for h and studied by immunofluorescence, immunoblotting and rt-qpcr. gap junction-mediated intercellular communication was assessed following microinjection of lucifer yellow and neurobiotin. results: the distribution and abundance of zo- at the plasma membrane were minimally affected by scd evs. while baseline evs did not affect the distribution of cx , evs isolated during an episode of acs caused loss of cx from the plasma membrane. the integrated intensity of cx membrane staining was decreased bỹ % following treatment with acs evs. cx protein decreased on average by %, cx mrna levels by % and neurobiotin transfer by - % in cells treated with acs evs, compared to baseline evs. summary/conclusion: circulating evs in scd affect multiple components of endothelial junctions. gap junctions composed of cx are the most sensitive of the cellcell junctions, since their abundance and function are reduced by acs evs even when the endothelial monolayer appears intact. cx -mediated intercellular communication may be an early and sensitive event in the endothelial disturbance caused by evs in scd patients. funding: nih ul tr , comer hospital rbc race funds, ted mullin fund. the effects of platelet concentrate storage time on extracellular vesicle interactions associated with fibrin clot formation in-vitro jamie nash a , christine saunders b , amanda davies a and philip james a a cardiff metropolitan university, cardiff, uk; b welsh blood service, velindre university nhs trust, cardiff, uk introduction: platelet concentrates (pcs) have been utilised for decades to prevent bleeding in thrombocytopenic patients and to stop active bleeding. the storage of pcs however is a logistical challenge due to the limited day shelf life under standard conditions. during storage, platelets undergo a number of mechanical and biochemical changes contributing to the short shelf life of a pc. these changes are collectively known as the platelet storage lesion. platelet extracellular vesicles (pevs) are known to increase throughout pc storage, due to an increase in platelet activation. as pevs have previously been shown to be pro-coagulant and increase in annexin v binding over pc storage. the aim was to investigate the effect of pc storage time on extracellular vesicle interactions on fibrin clot formation. methods: pcs were sampled on alternate days up to days of storage and centrifuged to achieve acellular plasma. the plasma was subjected to ultracentrifugation ( , xg) to pellet evs. the size and concentration of evs was assessed using nanoparticle tracking analysis software, followed by a western blot to confirm evs were of platelet origin. the pevs were added at a fixed number to a control pooled plasma sample with added thrombin and tissue plasminogen activator. the time to clot and % lysis time were recorded by using the turbidometry of the plasma over time. results: evs isolated from the pc were confirmed to be of platelet origin by western blot using cd as a marker of platelet origin and cd as an ev marker. pevs caused a significant increase effect on the fibrin clot formation (p < . ) when compared to the control plasma. pevs also had a significant effect (p < . ) on the fibrinolysis time, extending the time taken to lyse the clot. characterization of mirna from serum derived exosomes in a mouse tibia fracture model of introduction: complex regional pain syndrome (crps) is a debilitating chronic disease that occurs after trauma to the periphery and is intimately associated with nerve injury. its presentation is often described as an injury that is disproportional to the inciting event and manifests neuropathic pain, systemic inflammation, and immune dysregulation. owing in part to our poor understanding of disease aetiology, current treatments for crps are insufficient and as a disease of exclusion there is a lack of quantitative diagnostic markers. exosomes are small extracellular vesicles (sevs) - nm in size which provide a means of cellular communication through their cargo molecules (protein, mirna, mrna, lipids) , and have demonstrated promise in uncovering mechanisms of disease manifestation and identifying potential diagnostic markers. we have shown previously that crps patients have differential expression of several mirnas in serum derived sevs as compared to healthy controls, but little is known on how this compares to the established mouse tibia fracture model of crps. methods: mice undergoing fracture were anesthetized and subjected to a unilateral tibia fracture followed by casting of the injured limb. after confirming the establishment of pain hypersensitivity, serum samples were collected from fracture model and control mice three weeks post-injury. sevs were isolated by differential centrifugation and characterized using nanoparticle tracking analysis, transmission electron microscopy and western blotting. rna-seq analysis is being performed to identify differentially expressed mirnas. results: nanoparticle tracking analysis showed no significant difference in the number or size of sevs present in the serum from the fracture model and control mice. rna-seq is ongoing and differential mirna expression in sevs from fracture model will be compared to control samples. comparative studies identifying mirnas that are common between crps patients and the rodent model will facilitate the development of correlational outcomes between preclinical and human studies. summary/conclusion: identification of similarities and differences between crps patients and animal models will aid in directing future studies at clinically relevant aspects of crps aetiology and identifying potential diagnostic markers for crps patients. extracellular vesicle-based liquid biopsy in acute myeloid leukaemia: a reliable source of residual disease biomarkers? introduction: acute myeloid leukaemia (aml) is an haematopoietic stem cell disorder with a poor -year survival rate. monitoring of measurable residual disease (mrd) in aml patients receiving chemotherapeutic treatment is useful to assess therapy response and predict relapse. indeed, many different leukaemia associated immunophenotypic protein markers (laips) are presently useful to detect mrd. nevertheless, their analysis currently requires invasive bone marrow aspirates, thus severely hindering real-time monitoring of the disease. therefore, alternative peripheral blood-based methods are highly desirable for an easy, real-time and costeffective monitoring of aml progression. this work aims was to assess the feasibility of a peripheral blood ev-based liquid biopsy method for aml disease monitoring, based on the detection of laips with a known negative impact on the prognosis of aml. methods: the profile of evs isolated from paired samples from aml patients' blood plasma collected at diagnosis, complete remission (and some at relapse) was compared and correlated with clinical data. for that, a size-exclusion chromatography (sec) method was optimized to isolate the circulating evs from the blood plasma. the evs of the paired aml patients' blood samples were then characterized according to their size (dls/nta), morphology (tem), proteinto-lipid ratio (lowry/sulpho phosphovanillin assay), surface charge (zeta-sizer) and protein cargo (western blot). results: sec allowed the isolation of size-resolved plasmaderived evs from the peripheral blood of aml patients. isolated evs had a size ranging from nm to nm with an intact morphology, expressing ev-associated markers such as hsp , cd , cd and cd . size-resolved evs also had a differential expression of mitofilin, actinin- , syntenin- and annexin-xi proteins. several laips were detected in the isolated evs and their relative abundance changed throughout the stage of the disease. summary/conclusion: our preliminary data shows that aml patients' circulating evs carry relevant immunophenotypic protein markers, which might predict aml clinical outcome. introduction: cell plasticity regulated by the balance between the epithelial-to-mesenchymal transition (emt) and met is critical in the metastatic cascade. extracellular vesicles (evs) may play an important role in this balance by shuttling molecular cargos into recipient cells. this study aims to evaluate the feasibility of profiling mrnas of parental prostate cancer (pca) cells with different phenotypes and their daughter evs using the nanostring low rna input ncounter assay. methods: pc -epi and pc -emt cell lines representing epithelial and mesenchymal phenotype, respectively, were generated from original pc cell line. the cell culture supernatant was first pre-cleared for any dead cells and debris by centrifugation at × g for min. without disturbing the pellet, the supernatant was then transferred to a fresh ultracentrifuge tube and centrifuged at , × g for min at °c. the remaining supernatant was then centrifuged to isolate the evs at , × g for min at °c. the evs pellet was further washed in × pbs followed by a second centrifugation at , × g for min at °c. the final evs pellet was resuspended in × pbs for subsequent characterization (transmission electron microscopy, nanoparticle tracking analysis and western blot) and ncounter assays. the total rna of cells and their daughter evs were assayed by the ncounter pancancer progression panel to determine expression of selected mrnas. the nanostring ncounter low rna input kit with the multiplex gene primer pool was used for the pre-amplification of mrna and overnight hybridization with the pancancer progression panel. each sample type was submitted to the assay in biological triplicate. results: when comparing all samples, eisen cluster analysis separated all the cells and all evs into two groups, regardless of their phenotypes. in subgroup analysis, the expression patterns between pc -epi and pc -emt cells were significantly different. clec b, kdr, crip , il ra , cc d b were significantly upregulated in pc -emt cells, while cxcl , epcam, esrp , tgfb , cdh , s a , ovol were significantly downregulated in pc -emt cells. the expression patterns between pc -epi and pc -emt evs were also significantly different. tbx , cav , col a , slc a , myc, itgb , timp , camk b, ptgds, p h , itgb , vim, stat were all significantly downregulated in pc -emt cell derived evs. summary/conclusion: the nanostring low rna input ncounter assay can provide reliable mrna expression profiling of evs. the mrna expression patterns are very different between cells and their daughter evs. both cells and evs with different phenotypes have different gene expressions. cancer cell-derived evs containing alphav beta integrin regulate cd , il- and il- levels in peripheral blood mononuclear cells introduction: extracellular vesicles (evs) mediate communication in the tumour microenvironment and play an important role in cancer progression. previously, we have shown the enrichment of alphav beta integrin in small extracellular vesicles (sevs) isolated by differential ultracentrifugation and iodixanol density gradient from pc prostate cancer cells. we have also shown in the past that alphav beta -positive sevs induce peripheral blood mononuclear cell (pbmc) polarization by increasing the expression of pro-tumorigenic m markers, such as cd and cd . finally, we have demonstrated that down-regulation of alphav beta integrin up-regulates the stat -interferon stimulated genes (isgs) pathway in cancer cells and in sevs released by them. methods: in order to investigate whether prostate cancer cell-derived vesicular stat has a causal effect in pbmc polarization, we down-regulated alphav beta and stat in prostate cancer cells derived sevs using sirna as well as crispr-cas strategies. the sevs isolated from these cells were used to analyse m polarization by measuring the levels of cd in pbmc. the results show that sevs lacking alphav beta inhibit cd levels in pbmc in a stat -independent manner. analysis of cytokines released by pbmc upon incubation with sevs lacking alphav beta , show that pbmc selectively up-regulate the levels of il- and il- , which are predominantly anti-tumorigenic cytokines. in contrast, sevs lacking alphav beta do not upregulate pro-angiogenic cytokines, such as vegf. summary/conclusion: these findings suggest that cancer cell-derived sevs containing alphav beta integrin promote a pro-tumorigenic pbmc phenotype in the tumour microenvironment by regulating cd , il- and il- levels. introduction: the recognition of donor-mhc molecules by recipient t cells triggers the immune response leading to rejection of allografts. our recent studies have documented the presence of high numbers of recipient apcs displaying donor-mhc molecules (cross-dressed) on their surface in the lymphoid organs of mice after skin, heart or pancreatic islet transplantation. in addition, we have reported that acquisition of allogeneic mhc molecules by host apcs (mhc crossdressing) is mediated by donor-derived extracellular vesicles (evs) trafficking through blood and lymphatic vessels (marino et al. science immunology, ) . in the present study, we investigated the ability of allogeneic evs and allo-mhc-cross-dressed cells to initiate a t cell alloresponse in vitro and in vivo. methods: evs were isolated (using differential centrifugation) from balb/c bone marrow derived dendritic cells (bmdcs). these evs were used to cross-dress b splenocytes in vitro. the transfer of donor mhc class i and ii on b cells was analysed by imaging flow cytometry. next, t cells from b mice were cultured in vitro with either allogeneic bmdc-derived balb/c evs or b spleen cells crossdressed with allogeneic balb/c mhc. alternatively, × balb/c or b bm derived evs or × balb/c bm cells were injected iv to b mice. in both cases, the t cell response was assessed by activation markers detection, infg production and cell proliferation. results: apcs cross-dressed with allogeneic mhc molecules can trigger a pro-inflammatory direct alloresponse by t cells in vitro and in vivo. on the other hand, allogeneic evs alone were only able to induce early t cell activation but not proliferation in vitro. furthermore, injection of mice with allogeneic evs alone could induce some but suboptimal alloresponse in vivo and only when administered with complete freund's adjuvant. summary/conclusion: blocking donor evs release and subsequent recipient apc cross-dressing may represent a promising target to selectively inhibit anti-donor t cell inflammatory responses thus achieving long-term allograft survival. funding: r dk . antifungal antibiotic activity of outer membrane vesicles from adherent lysobacter enzymogenes c against therapeutic and biocontrol targets. rutgers university, new brunswick, usa introduction: lysobacter enzymogenes is a predatory gram negative bacterial species being studied for biocontrol activity against fungi. planktonic l. enzymogenes c produces outer membrane vesicles (omv) harbouring small molecule antifungal antibiotics (meers et al. ) . we show here that the more biologically relevant surface-associated c exerts remote antifungal activity via omv as well. the results have important consequences regarding the natural mechanism of biocontrol of fungal pathogens by c as well as isolation and delivery of therapeutically relevant antifungal compounds. methods: omv were isolated from scraped adherent c culture on agar by similar methods to meers et al . omv were stained in some cases with fluorogenic syto dna stain for microscopic observation. fungal growth was monitored via turbidity readings in liquid culture or photomicrographs on agar. c was also grown on polycarbonate filter membranes with defined pore sizes to monitor growth of fungal cells on the opposite side. vesicles were also labelled with an amine-reactive probe alexa- and washed x by sedimentation. binding of labelled omv to fungal cells was observed by epifluorescence microscopy. results: syto -stained vesicles from surface-adherent c were similar to previously observed~ nm vesicles (meers et al., ) . the isolated vesicles inhibited growth of saccharomyces cerevisiae or candida albicans in liquid cultures at similar potency and were active against the filamentous species fusarium subglutinans grown on agar or maize leaves. c cultures grown on filters with nm pore size but not nm were able to inhibit the hyphal growth of f. subglutinans on the opposite side. similarly c on filters with a nm pore size were able to inhibit growth of c. albicans. observation of fluorescently-labelled c omv after interaction with c. albicans showed binding specifically to hyphae or pseudohyphae and for f. subglutinans to the growing hyphal tips. summary/conclusion: the omv of c specifically bind and inhibit the growth of fungal hyphae of various species without direct c cell contact. these data elucidate mechanisms of biocontrol and suggest strategies for production of therapeutic antifungal antibiotics. meers et al. elucidating the cellular uptake and tissue distribution mechanism of cell derived vesicles, a novel therapeutic carrier hui-chong lau a , jae young kim a , jin-hee park a , jun-sik yoon a , min jung kang a and seung wook oh b a mdimune inc, seoul, republic of korea; b mdimune inc, seattle, usa introduction: cell derived vesicles (cdvs) are emerging as a novel therapeutic carrier. one of the crucial factors in the development and therapeutic applications of cdvs is to understand the precise mechanism by which vesicles find and enter the target cells. in this study, we aim to investigate the uptake mechanism of cdvs produced from natural killer (nk) cells using a manufacturing process established at mdimune inc. both in vitro uptake assay and in vivo distribution analysis were performed to provide precise insights into how cdv exert its effect at the cellular level. methods: nk cells were mainly used to produce cdvs. breast cancer cells, bt , and human and rodent endothelial cells, with a varying degree of icam- expression, were used to determine the effect of lfa- expressed on the surface of nk-cdvs in cellular uptake using facs and confocal imaging analysis. next, various inhibitors for uptake pathways, such as phagocytosis, dynamin dependent endocytosis, and receptor mediated endocytosis, were used to understand the underlying mechanism of cellular uptake of cdvs. biodistribution profile of cdvs were characterized using both normal and tumour xenograft models by ivis imaging. results: using a recently established manufacturing process, we demonstrate that nk-cdvs can efficiently enter the target cells. this study also shows that the cellular uptake depends on the molecular interaction between icam- and lfa- . in vivo distribution profile of nk-cdvs are also assessed using various tumour models. furthermore, we present a cellular uptake mechanism involved in the entrance of cdvs into the target cells. summary/conclusion: this study demonstrates that the cdvs produced at the manufacturing scale can be easily taken up by cells via specific cellular pathways. this finding will facilitate the development of more efficient therapeutics for cancer and other debilitating diseases. myofibroblasts-derived microvesicles increase dermal fibroblasts collagen production through plgf- syrine arif, sebastien larochelle and véronique j. moulin chu de québec -université laval, loex, québec, canada introduction: a proper wound healing of the skin involves angiogenesis, extracellular matrix (ecm) remodelling and re-epithelialization. these three mechanisms require well-organized interactions between different cell populations. a key role in this context is played by myofibroblasts (wmyo), a cell population mainly differentiated from dermal fibroblasts. these cells contract wound edges and synthesize new ecm. we previously showed that myofibroblasts predominantly produces microvesicles (mvs) and can favour angiogenesis. however, proteomic analysis of mvs from our previous studies indicated some molecules that can potentially be implicated in ecm remodelling. in this study, we evaluated whether myofibroblasts-derived mvs could affect dermal fibroblasts who are highly responsible for ecm regulation. methods: mvs were isolated by differential centrifugation of medium collected from wmyo cells. number and size of mvs were characterized by transmission electron microscopy and nanosizer. multiplex assays of cytokines were evaluated in mvs samples, wmyo and mvs-depleted medium. to examine the interaction of mvs with fibroblasts, we evaluated the uptake of mvs isolated from wmyo transduced with a fluorescent protein. we then treated fibroblasts cultures with mvs or a selected cytokine for days and evaluated collagen production. lastly, we neutralized the selected cytokine in mvs samples before evaluating collagen production. results: plgf- was the cytokine detected in mvs samples in large amount ( . ± . pg/µg proteins in mvs). fibroblasts treated with mvs or plgf- significantly stimulated pro-collagen i level production with a fold change of . ± . and . ± . . moreover, the neutralization of plgf- present in mvs significantly inhibited the production of pro-collagen i by dermal fibroblasts. summary/conclusion: our results indicated that mvs influence fibroblasts pro-collagen production through plgf- signalling. funding: this work was supported by natural sciences and engineering research council of canada (nserc) (rgpin - ); les fonds de recherche du québec-santé (frqs) via the research centre funding grant; the quebec cell and tissue and gene therapy network-thécell (a thematic network supported by frqs). structural insights on fusion mechanisms of extracellular vesicles with model plasma membranes introduction: extracellular vesicles (evs) represent a potent intercellular communication system. while their functional biological properties are more and more investigated, the biophysical aspects of their interaction with recipient cells are often overlooked. small size ( to a few hundred nanometres in diameter) of evs and their heterogeneous origin still pose a great challenge for their isolation, quantification and biophysical/biochemical characterization. in particular the complex network of interactions between differently classified evs and recipient cells remains to be further revealed. here we deeply investigate the fusion mechanism between evs and a model plasma membrane system by an interplay of different structural/morphological techniques to get a molecular description of the interaction helping to clarify the role of different membrane compartments on the evs uptake mechanism. standardized protocols and good manufacturing practice conditions were employed to derive highly stable vesicles of defined size and reproducible molecular profiles from umbilical cord multipotent mesenchymal stem (stromal) cells. after a thorough biophysical and biochemical characterization of evs non-contact liquid imaging atomic force microscopy (afm) and, in parallel, neutron reflectometry (nr), as well as small angle neutron scattering (sans) experiments were performed on evs to determine their interaction with model plasma membranes in the form both of supported lipid bilayers and suspended unilamellar vesicles of variably complex composition. results: we observed that evs tend to fuse with the model membranes with a preferential interaction with the external layer of the fluid membrane. moreover we revealed a stronger interaction with the liquid ordered domains, strengthening the hypothesis of a critical role of lipid rafts in fusion mechanisms. summary/conclusion: our results on the analysis of the interaction of evs with artificial lipid membranes could provide insights on the internalization mechanisms of evs. the approach shown here can be further extended to convey incremental complexity, adding glycolipid and membrane proteins to the model lipid bilayers. this approach combined with data on the specific biological function of each ev subpopulation as retrieved by standard functional assays, will turn useful to select the crucial molecular aspects of evs internalization by cells. introduction: platelet-derived extracellular vesicles (pev) are the most abundant circulating extracellular vesicle (ev) and exhibit platelet-like properties, hence the original term "platelet dust". direct phenotyping of ev surface markers within biofluids is challenging often requiring time-intensive purification steps that can significantly alter resultant ev population characteristics. the exoview™ (nanoview biosciences) specifically captures ev sub-populations and was used to characterise the ev content of platelet free plasma (pfp) and a potential novel haemostatic agent designed for the treatment of severe trauma and haemorrhage, platelet enhanced plasma (pep). methods: freeze-thaw cycling of platelet rich plasma/ expired platelet concentrates was followed by centrifugation to remove platelet remnants and yeilded pep. pfp controls were prepared by double centrifugation ( g for minuntes followed by , g for minutes). rotational thromboelastometry (rotem) and calibrated automated thrombography (cat) were used to assess ev driven haemostasis and thrombin generation. a dilutional and hypothermic model of coagulopathy was designed to assess pep. ev capture arrays comprised of anti-cd , anti-cd , anti-cd and anti-cd were used (exoview™, nanoview biosciences). captured vesicles underewent interferometric imaging and were quantified, sized and further probed with fluorescent tetraspanin markers, annexin-v and intravesicular markers. results: pep is highly procoagulant, exhibits enhanced thrombin generation and can restore haemostasis in a dilutional model of coagulopatic whole blood. pep can be generated from expired platelet concentrates, potentially allowing for upscalable production. the predominant vesicle population were pev with a large cd /cd population that contained a smaller subpopulation of phosphatidyserine positive procoagulant vesicles. pfp as expected has a much lower number of pev and a cd positive ev population. summary/conclusion: pep is a unique resuscitation fluid containing high pev levels for the potential treatment of severe trauma and haemorrhage. exoview measurements can be performed in unpurified plasma and may be useful for measuring circulating ev in health and disease. funding: defence and security accelerator, dstl therapeutic effect of exosomes in mice model of autism daniel offen a , reut horev a , nisim perets a , ehud marom b , uri danon b and yona gefen b a tel aviv university, tel aviv, israel; b stem cell medicine ltd., jerusalem, israel introduction: during the recent decade, exosomes that derived from mesenchymal stem cells (msc-exo) have been spotlighted as a promising therapeutic target for various clinical indications, including neurological disorders. we have previously shown that intranasal administration of msc-exo, cross the bbb and significantly ameliorate autistic-like behavioural phenotype in btbr and shank animal models of autism, representing a potential therapeutic strategy to reduce symptoms of autism spectrum disorder (asd). our objective is to study the mechanism of action and the cellular pathways in which the msc-exo activate their target, we performed rna sequencing analysis of primary neurons isolated from shank mice treated with msc-exo. methods: primary neuronal cell cultures were prepared from newborn shank homozygotes mice model of autism. cultures were treated with msc-exo ( ^ particles/ul), isolated from human adipocytes, followed by rna sequencing. the alterations in gene expression between the treated and intact neurons were analysed for gene ontology and pathways and were also compared to proteomics analysis of the msc-exo in order to find regulatory proteins that may lead to these differences. results: bioinformatic analysis revealed several up-regulators proteins that might be responsible for the increase in anti-inflammatory and protective factors seen in the mice neurons treated with msc-exo. one of them is bdnf which is known as an essential growth factor responsible for neuroprotection and neurogenesis. importantly, no difference in the genetic expression of cancer-related genes was identified following msc-exo treatment indicating for their safety. summary/conclusion: our data suggest that adipocytederived msc-exo carry therapeutic potential in asd via alternation in gene-expression related mainly to immuno-modulation, reduce neuroinflammation and increase neuroprotection and neurogenesis. the beneficial effects of the exosomes treatment in mice models is being translated into a novel, easy to administer, a therapeutic strategy to reduce the symptoms of asd. introduction: autologous blood-derived products gain increasing focus in regenerative medicine, especially in orthopaedics and osteoarthritis therapy. this disease is characterised by cartilage degradation and inflammation among other symptoms, which are targeted by conventional therapies, but genuine cartilage regeneration is rarely achieved. citrate-anticoagulated platelet rich plasma (cprp) is often clinically applied to stimulate soft and hard tissue healing. recently, cell-free alternatives to cprp including hyperacute serum (hypact™ serum) have been developed. cprp and hypact™ serum contain specific profiles of growth factors, however, they also contain extracellular vesicles (evs) that harbour signal molecules including mirna. methods: evs were enriched by ultracentrifugation (uc) followed by size exclusion chromatography (sec) to obtain purified evs. particle size and concentration of each fraction was measured by nanoparticle tracking analysis (nta). fractions with the highest amount of particles were pooled and concentrated via uc, before mirna expression was assessed via screening with a panel of mirna-specific primer pairs by rt-qpcr. presence of evs was confirmed by cryoelectron microscopy. results: the ev concentration tended to be lower in hypact™ serum than in cprp as determined via nta. similarly, lower diversity of mirna species was found in hypact™ serum than cprp evs. around % of detected mirnas were found in both blood products, whereas only % of mirnas were shared between evs from cprp and hypact™ serum. while mirnas such as mir- were consistently depleted in evs compared to the corresponding blood product, others like mir- a were in enriched in hypact™ evs, but not cprp evs, indicating release of specific mirnas via evs in response to clotting. summary/conclusion: although the purification resulted in high loss of evs, we identified specific mirnas enriched in evs from cprp and hypact™ serum. their functional spectrum with respect to osteoarthritis therapy focuses on inhibition of inflammation, inhibition of tissue remodelling via matrix degrading enzymes as well as preventing senescence. this renders blood product derived evs as interesting candidates for in vitro and in vivo testing with respect to cartilage regeneration. funding: the work was jointly supported by the european fund for regional development (efre) and the fund for economy and tourism of lower austria, grant number wst -f- / - . protective role of shiitake mushroom-derived exosome-like nanoparticles in d-galactosamine and lipopolysaccharide-induced acute liver injury in mice baolong liu, xingyi chen and jiujiu yu university of nebraska lincoln, lincoln, usa introduction: fulminant hepatic failure (fhf) is a rare, life-threatening liver disease with poor prognosis. new therapeutic interventions are urgently needed to treat this disease. administration of d-galactosamine (galn) and a low dose of lipopolysaccharide (lps) triggers acute liver damage in mice, which simulates many clinical features of fhf in humans and therefore is widely used to investigate the molecular mechanisms and potential therapeutic interventions of fhf. recently, suppression of the nucleotide binding domain and leucine rich repeat related (nlr) family, pyrin domain containing (nlrp ) inflammasome was shown to alleviate the severity of lps/galn-induced liver injury in animal models. therefore, the goal of this study was to identify food-derived exosome-like nanoparticles (elns) with anti-nlrp inflammasome function to potentially control fhf. methods: seven commonly consumed mushrooms were used to extract elns, which were examined for anti-nlrp inflammasome activities in primary macrophages. results: it was found that these mushrooms contained elns composed of biomolecules including rnas, proteins, and lipids. among these mushroom-derived elns, only shiitake mushroom-derived elns (s-elns) strongly inhibited nlrp inflammasome activation by blocking the inflammasome assembly. this inhibitory effect was specific for the nlrp inflammasome because s-elns had no impact on activation of the absent in melanoma (aim ) inflammasome. s-elns also inhibited the secretion of interleukin (il)- and both protein and mrna levels of the il b gene in macrophages. remarkably, pre-treatment of s-elns protected mice from lps/galn-induced acute liver injury. summary/conclusion: therefore, s-elns, identified as potent inhibitors of the nlrp inflammasome, represent a new class of agents with the potential to combat fhf. approaches to assess clinically available exosomes' quality and safety introduction: recent adverse events resultant from an exosome product use in a nebraska clinic, highlight the importance of assuring product quality and safety standards. an often-overlooked safety risk is ancillary reagents remaining within a finished product. when processes to obtain exosomes utilize cow proteins such as fbs or bovine sera albumin, failure to adequately remove these can result in significant adverse allergic reactions. we evaluated different exosome products to test the hypothesis that purity of some products may not be consistent with actual product quality and safety profiles claimed. methods: three different exosome products (manufacturer a, b, and c) were prepared per their instructions for use. sample source identity was blinded from assaying scientists. an independent cro service was used to conduct the experiments to ensure unbiased assay execution and data collection. exosome suspensions were sampled undiluted for bovine protein content using commercially available bovine secretome protein arrays from ray biotech. a total of different proteins found in bovine serum were quantified. results: six of proteins were not detected in any sample. of array antibodies were found to cross react with human antigens. of the bovine proteins that were acceptable for analysis, manufacturers a, b, and c exosomes contained of proteins, of proteins, and of proteins, respectively. concentrations of individual bovine proteins ranged from . to , . ng/ml. summary/conclusion: these results indicate manufactures a and b are selling potentially dangerous products. the successful implementation of exosome products into the clinic requires equivalent demonstrations of safety and quality. this requires adopting strict quality standards and safety testing during their production. physicians must require safety data prior to clinical use. engineering pro-healing ev cargo using a closed-system bioreactor. introduction: chronic wounds, including diabetic ulcers and pressure ulcers, are difficult and expensive to treat. while tissue engineering approaches have largely failed as a viable treatment for chronic wounds, we hypothesize that stem cell-derived extracellular vesicles (evs) may provide several unique advantages. zenbio, inc has developed a methodology to generate commercial-scale stem cell-derived exosomes using a closedsystem hollow fibre bioreactor capable of continuous ev production. additionally, we have shown that by manipulating the cellular environment, we can improve the pro-healing capacity of the evs.this technology leverages the complex healing capabilities of stem cells without the obstacles of replicating cells. methods: we have demonstrated that a mild heat shock resulted in evs enriched for stress-response proteins and increased pro-healing activities in vitro. we extended this innovative approach to include stimulating adipose stem cells with combinations of heat shock and growth factors to generate differential extracellular vesicle packaging that enhances pro-healing activity. to monitor reproducibility across lots and batches, we rigorously characterized tuned evs for particle size and number as well as surface marker and cargo composition. results: our results using tuned evs showed efficacy using cellular models of inflammation, motility, vascularization, collagen production and metalloprotease activity. we utilized an established murine model of pressure ulcers to assess the in vivo efficacy of the tuned evs. these studies showed a single injection into the wound site activated a more rapid wound closure, increased collagen deposition and reduced dermal thickness compared to saline control. summary/conclusion: these data strongly support our hypothesis that evs may be selectively modified to improve their wound healing activity by modulating the culture or tissue microenvironment. future studies will use chronic wound models to determine optimal dosing and routes of administration. introduction: mesenchymal stem cell-derived extracellular vesicles (msc-evs) can reduce inflammation, promote healing and improve organ function thereby providing a potential "cell-free" therapy. prior to clinical translation, there is a critical need to synthesize existing preclinical evidence supporting their efficacy. this systematic review provides the most comprehensive evidence map of methods, safety and efficacy for msc-ev research to date. methods: medline and embase were systematically searched for in vivo interventional studies using msc-evs. two reviewers extracted data for: ) methodology, ) study design, ) intervention details and ) efficacy/ adverse events. results: after screening articles, studies met our eligibility criteria. msc-evs were used to treat a variety of diseases including renal ( %), neurological ( %) and cardiac ( %) conditions. benefits were described in % of studies across all organ systems and adverse effects were seen in only three studies; two showing tumour growth. however, several key methodological concerns were evident. based on size criteria for ev subtypes (exosomes/small evs~ - nm, microvesicles~ - nm) only % of studies used appropriate nomenclature. ultracentrifugation ( %) and isolation kits ( %) were the most common isolation methods despite marked differences in yield and purity. evs were inconsistently dosed by protein ( %), particle number ( %) or cell count ( %), hindering inter-study comparisons. two-thirds of studies used xenogeneic evs suggesting immunocompatibility. techniques to determine size, protein markers and morphology was highly heterogeneous, and only and studies met the characterization standards recommended in the misev and guidelines, respectively. finally, % of studies did not incorporate randomization which represents a high risk for bias and only a quarter performed biodistribution studies. summary/conclusion: this systematic review reveals extensive heterogeneity in methods and intervention details for animal studies of msc-evs. nonetheless, nearly all studies showed significant benefits in a wide range of distinct conditions. the knowledge gaps we identified highlight important opportunities for improving preclinical design and the need for more standardized approaches in this growing field of ev therapeutics. msc-exosomes as next generation therapeutics for atopic dermatitis exocobio inc, seoul, republic of korea introduction: atopic dermatitis (ad) is a systemic inflammatory disease with unknown cause. recent approval of a targeted therapy, dupilumab, opens new era of ad management. however, current therapeutic options for ad are only targeting inflammation, a component of ad vicious cycle including itching and barrier disruption. human mesenchymal stem cells (mscs) have been highlighted as a novel therapy for suppressing allergic progress of ad in clinical studies. unfortunately, phase iii clinical study of human umbilical cord blood mscs for ad was failed with unknown reason. previously, our group reported that exosomes derived from human adipose tissue-derived mscs (asc-exosomes) alleviated the pathological symptoms in a murine ad model with concomitant reduction of inflammation. methods: our group has further investigated the therapeutic effects of human asc-exosomes in an alternative murine ad model with skin barrier defects. large scale isolation of asc-exosomes was performed by tangential flow filtration and isolated asc-exosomes were characterized according to the recommendation by the isev. the protein and lipid cargo were also analysed. results: we found that asc-exosomes induced restoration of skin barrier by inducing de novo lipid synthesis and reduced the levels of multiple inflammatory cytokines. in addition, asc-exosomes suppressed the expression of itching-causing cytokines. transcriptomic analysis of ad skin lesions revealed that asc-exosomes reversed the abnormal expression of genes functioning in skin barrier function, lipid metabolism, and cell cycle. summary/conclusion: taken together, asc-exosomes could be a promising cell-free therapeutic option for the treatment of ad, which affecting inflammation, skin barrier function, and itching. cell derived vesicles: unravelling the science of novel vesicles with therapeutic promises introduction: cell derived vesicles (cdvs) are nanosized vesicles produced by serially extruding cells through small pores. a growing number of studies have implicated their therapeutic potentials, with superior yield compared to other extracellular vesicles (evs). however, two key objectives remain to be accomplished to demonstrate the utility of cdvs in clinical applications. first, a manufacturing process has to be developed to allow a large-scale production of cdvs. next, these novel vesicles need to be thoroughly characterized at multiple levels. methods: manufacturing-scale extruders were developed to allow extrusion of large volume of cell suspension in a single process. cdvs with approximately - nm in diameter were obtained by a serial extrusion. crude samples were then purified using the tangential flow filtration method to further remove cellular impurities. finally, physical and biochemical characteristics of purified cdvs were analysed using dls, nta, cryo-em, and facs analysis. additionally, cdvs were subject to multi-omics profiling to comprehend our understanding in molecular contents of cdvs. both mesenchymal stem cells (mscs) and natural killer (nk) cells were used for this study. results: in this study, we first demonstrate that the large-scale extruder efficiently produce cdvs with consistent quality at the scale that are compatible for clinical applications. surface marker and membrane composition analyses show that the cdvs are primarily formed using plasma membrane of source cells, with characteristic cellular markers enriched on the surface. comprehensive profiling of molecular components reveals the unique properties of cdvs as well as the underlying mechanism of formation of cdvs. summary/conclusion: recently, we have established a manufacturing process to enable clinical applications of cdvs. this study also highlights key molecular features of cdvs that can be harnessed to offer a powerful tool for regenerative and anticancer medicine. antifibrotic properties of extracellular vesicles derived from human induced pluripotent stem cells introduction: fibrosis is a pathological condition resulting from abnormal healing of various tissues. it is triggered by activation of fibroblasts and their subsequent transition to myofibroblast. in consequence, excessive deposition of extracellular matrix proteins leads to impaired organ function. to revert this process, we employed extracellular vesicles (evs) derived from human induced pluripotent stem cells (hipscs). as a model system, we used human cardiac fibroblasts (hcfs), since heart fibrosis constitutes a serious socioeconomic problem worldwide. methods: we isolated evs from conditioned media from three hipsc lines using ultrafiltration combined with size exclusion chromatography methods. next, we analysed the evs by nanosight, transmission electron microscopy, mass spectrometry and western blot methods. finally, we treated tgf-b-stimulated hcfs with hipsc-evs and evaluated expression of fibrosisrelated genes using real-time qpcr, western blot and fluorescence microscopy. results: we detected anti-fibrotic properties of hipsc-evs exerted on hcfs pre-stimulated with tgf-b. the evs significantly decreased the expression levels of acta , fn, tnc, snai , col a and reduced the number of myofibroblasts. the canonical profibrotic tgf-b-dependent smad / pathway was significantly attenuated in response to ev-treatment. summary/conclusion: in this study we demonstrated strong anti-fibrotic function of hipsc-evs. our findings can further be exploited for future medical applications to treat fibrotic diseases, such as heart fibrosis. funding: this work was supported by the project sonata : umo- / /d/nz / from the national science centre of poland to sbw. induced pluripotent stem cells-derived extracellular vesicles ameliorates d-galactosamine and lipopolysaccharide induced acute liver failure tianjin third central hospital affiliated to nankai university, tianjin, china (people's republic) introduction: liver failure is among the most causes of death in patients with liver disease. promoting liver regeneration will help patients with liver failure recover on their own. extracellular vesicles (evs) can released by induced pluripotent stem cells (ipscs) through paracrine effects and play a pivotal role in inter-cellular communication in the treatment of disease. in this study, we investigated whether the ipscs-evs have therapeutic effects on acute liver failure. methods: the ipscs-evs were isolated by ultracentrifugation and identified using nanoparticle tracking analysis, transmission electron microscopy and western blotting. the isolated ipscs-evs were administrated d-galactosamine-injured heprg cells in vitro and tail intravenously injected into d-galactosamine and lipopolysaccharide induced acute liver failure model mice in vivo, respectively. the anti-apoptosis role and potential mechanism were evaluated using flow cytometry and immunofluorescence staining. and alanine transaminase (alt) and aspartate transaminase (ast) in serum, h&e staining and tunel staining were explored the effect of ipscs-evs on liver injured and liver function. finally, high throughput sequencing of small rnas was performed to investigate mirna expression profiles in ipscs-evs and ipscs. results: the ipscs-evs that were all - nm, doublelayered and oval or round cellular vesicles and expressed the marker proteins cd , tsg and hsp . in vitro, the ipscs-evs treatment inhibited heprg apoptosis induced with d-galactosamine in a time-and dosedependent manner and promote the proliferation of hepatic stem cells. in vivo results showed that ipscs-evs significantly alleviated liver failure, improved liver function and prolonged the survival period. tunel assay showed that ipscs-evs suppress apoptosis of hepatocytes. moreover, mirna expression profiles analysis found that mir - a cluster and mir - cluster were enriched in ipscs-evs and ipscs. summary/conclusion: these findings indicated that ipscs-evs could ameliorate d-galactosamine and lipopolysaccharide induced acute liver failure to attenuate hepatocyte apoptosis, which will be benefit for therapy of liver disease in the future. msc-derived extracellular vesicles promote human cartilage regeneration by control of autophagy introduction: osteoarthritis (oa) is a rheumatic disease leading to chronic pain and disability with no effective treatment available. recently, allogeneic human mesenchymal stromal/stem cells (msc) entered clinical trials as a novel therapy for oa. increasing evidence suggests that therapeutic efficacy of msc depends on paracrine signalling. here we investigated the role of bone marrow msc-derived extracellular vesicles (bmmsc-evs), an important component of msc secretome, in cartilage repair. methods: to test the effect of bmmsc-evs on oa cartilage inflammation the tnf-alpha-stimulated human oa chondrocytes were treated with bmmsc-evs and inflammatory gene expression was measured by qrt-pcr after h. to access the impact of bmmsc-evs on cartilage regeneration the bmmsc-evs were added to the regeneration cultures of oa chondrocytes, which were analysed after weeks for glycosaminoglycan content by dmmb and qrt-pcr. paraffin sections of the regenerated tissue were stained for proteoglycans (safranin-o) and type ii collagen (immunostaining). results: we show that bmmsc-evs promote cartilage regeneration in vitro. treatment of oa chondrocytes with bmmsc-evs induces production of proteoglycans and type ii collagen and promotes proliferation of these cells. msc-evs also inhibit the adverse effects of inflammatory mediators on cartilage homoeostasis. our data show that bmmsc-evs downregulate tnfalpha induced expression of pro-inflammatory cox- , pro-inflammatory interleukins and collagenase activity in oa chondrocytes. the anti-inflammatory effect of bmmsc-evs involves the inhibition of nfκb signalling, activation of which is an important component of oa pathology. autophagy, a cellular homoeostatic mechanism for the removal of dysfunctional cellular organelles and macromolecules, is essential to maintaining chondrocytes survival and differentiation. the expression of autophagy regulators is reduced in osteoarthritic joints, which is also accompanied by increased chondrocyte apoptosis. our preliminary data indicate that bmmsc-evs carry mrna of natural autophagy inducers and promote autophagy in oa chondrocytes. therefore, we hypothesize that msc-evs exert their beneficial effects on cartilage regeneration by restoring the expression of autophagy regulators. summary/conclusion: in summary, our findings indicate that bmmsc-evs have ability to promote oa cartilage repair by reducing the inflammatory response and stimulation of oa chondrocytes to produce extracellular matrix, the essential processes for restoring and maintaining cartilage homoeostasis. thus, msc-evs hold great promise as a novel therapeutic for cartilage regeneration and osteoarthritis. large-scale preparations of small extracellular vesicles from conditioned media of mesenchymal stromal cells modulate therapeutic impacts on a newly established graft-versus-host-disease model in batch dependent manners introduction: extracellular vesicles (evs) harvested from supernatants of humane adult bone marrow-derived mesenchymal stem/stromal cells (mscs) can suppress acute inflammatory cues in a variety of different diseases, including graft-versus-host disease (gvhd) and ischaemic stroke. furthermore, they can promote regeneration of affected tissues. following a successful clinical treatment attempt of a steroid refractory gvhd patient, we intend to optimize msc-ev production strategies for further clinical applications. as we observed functional differences of independent msc-ev preparations in vitro, we aimed to adopt an in vivo gvhd model for the more advanced functional testing of different msc-ev preparations. methods: to this end we set up a bone marrow transplantation mouse model in which endogenous bone marrow was myeloablated by ionizing irradiation (iir). gvhd was induced by the transplantation of major histocompatibility mismatched allogeneic spleen-derived murine t cells. if not treated otherwise, myeloablated mice developed severe gvhd symptoms. results: the gvhd symptoms were effectively suppressed, when msc-ev preparations were applied at consecutive days, which exerted immune modulatory effects in a mixed-lymphocyte reaction assay. msc-ev preparations lacking in vitro immune modulating activities, however, hardly improved the symptoms of the gvhd mice. thus, our results demonstrate that not all msc-ev preparations harvested from adult bone marrow-derived mscs contain the same therapeutic potential. summary/conclusion: thus, successful transplantation of msc-evs into the clinics requires a platform allowing identification of msc-ev preparations with sufficient therapeutic, most probably immune modulating activities. funding: this research was funded by sevrit leitmarkt lifescience.nrw ls- - - g. introduction: malnutrition impacts approximately million children worldwide and is linked to % of global mortality in children below the age of five. severe acute malnutrition (sam) is associated with intestinal barrier breakdown and epithelial atrophy. extracellular vesicles including exosomes (evs; - nm) can travel to distant target cells through biofluids including milk. since milk-derived evs are known to induce intestinal stem cell proliferation, this study aimed to examine their potential efficacy in improving malnutrition-induced atrophy of intestinal mucosa and barrier dysfunction. methods: mice were fed either a control ( %) or a low protein ( %) diet for days to induce malnutrition. from day to , they received either bovine milk evs enriched using differential ultracentrifugation and sucrose gradient purification or control gavage and were sacrificed on day , hours after a fluorescein isothiocyanate (fitc) dose. tissue and blood were collected for histological and epithelial barrier function analyses. results: mice fed low protein diet developed intestinal villus atrophy and barrier dysfunction. despite continued low protein diet feeding, milk ev administration improved intestinal permeability, intestinal architecture and cellular proliferation. summary/conclusion: our results suggest that evs enriched from milk should be further explored as a valuable adjuvant therapy to standard clinical management of malnourished children with high risk of morbidity and mortality. funding: cb was generously awarded a catalyst grant from the centre for global child health at the hospital for sick children to support this work. the impact of spheroids culture on mesenchymal stem cells and ev production introduction: mesenchymal stem/stromal cells (mscs) are now widely believed as bio-factories releasing bioactive products responsible for their therapeutic effect, i.e. cytokines, chemokines, and extracellular vesicles (evs). mscs are highly sensitive to physical stimuli from their surrounding microenvironment and can change their characteristics in response to their environment. the application of d spheroids cell culture allows mscs to adapt to their cellular niche environment which, in turn, influences their paracrine signalling activity. we aim to determine how d and d culture microenvironments can modulate the ev production and investigate their anti-fibrotic activity. methods: for d culture, bone marrow-derived mscs were cultured on standard tissue culture plastic. for d culture, mscs were aggregated into spheroids using non-adherent -well plates and cultured with addition of . % methylcellulose. to collect conditioned media, both d and d mscs were cultured using serum free medium for days. evs were isolated by serial ultracentrifugation and were characterised on exoview platform which allows simultaneous detection of particle size and expression of cd /cd /cd . cell lysates were collected for mirna isolation and qrt-pcr was performed to analyse expression of candidate mirnas. to model the progress of lung fibrosis, human lung fibroblasts (hlfs) were cultured with tgf-β to induce fibroblast activation, subsequently exposed to d and d evs, and collagen production was measured. further, d and d msc-evs were added into human lung mscs isolated from healthy and ipf patients and cell proliferation was assessed using mts assay. results: d and d msc-evs have similar ev characteristics in terms of particle size and ev tetraspanin markers expression. exoview analysis showed expressions of cd /cd /cd and average particle diameters of < nm. on a cellular level, we identified a panel of anti/pro-fibrotic mirnas which are differentially expressed in d and d mscs. d and d msc-evs have similar anti-fibrotic activity shown by their ability to reduce collagen deposition in hlf cultures. both d and d msc-evs could promote cell proliferation on ipf lung mscs but no overall effect on healthy lung mscs. summary/conclusion: this concept of engineering the cellular microenvironment to promote ev production is as yet untouched and we foresee that in d cultures, we can culture mscs for longer timeframe and therefore maximising the overall ev production process. the outcome presents future potential for d culture of msc to increase the efficiency and feasibility of scalable ev production. outer membrane vesicles from photobacterium damselae subsp. piscicida: characterization and antigenic potential introduction: photobacterium damselae piscicida (phdp) is a gram-negative bacterium that causes a septicaemia in > fish species worldwide. it represents a major drawback for aquaculture, whose importance has been sharply growing as a food supplier. given the phdp massive mortality and widespread antibiotic resistance, an effective vaccine is highly needed. extracellular products (ecps) have an essential role in phdp virulence, containing important antigens. however, the ecps' identity remain undisclosed. in our efforts to dissect their composition, we found that they contain high amounts of outer membrane vesicles (omvs). these particles are potent weapons for bacteria and are being explored in the field of vaccinology, since omvs present antigens in native conformations and are strongly immunogenic, without requiring adjuvants. this potential associated to the urgent need for an anti-phdp vaccine prompted us to isolate and characterize the omvs shed by phdp. methods: in order to harvest high amounts of pure phdp omvs, a reproducible optimized protocol was developed: the bacteria-free supernatant from a phdp overnight culture is concentrated, dialysed and ultracentrifuged to collect the omvs. results: analysis of the obtained omvs preparations by transmission electronic microscopy and dynamic light scattering indicate that the main population of vesicles has sizes around - nm. proteomic analysis of the vesicles revealed the presence of the apoptogenic ab toxin aip that is known to play a major role in phdp virulence, a putative pore-forming toxin, a putative adhesin/invasin and several outer membrane proteins (omps), including a kda omp, predicted to be involved in iron acquisition, and other omps ( - kda), with an ompa-like structure that may act as adhesins. moreover, preliminary in vivo studies suggest that some of those proteins may have important roles for virulence, since injection of knock-out strains in sea bass induced a decreased mortality comparing to the wt strains. summary/conclusion: our findings suggest that omvs are a promising vaccine candidate and we are currently studying their biological activities and determining the antigenic potential of the identified proteins. introduction: whole body exposure to high doses of ionizing radiation (ir) can potentially be lethal if radiation injury is not diagnosed and treated expeditiously. when considering a non-invasive approach for the identification of biomarkers of ir exposure, we and others have studied molecules in plasma, serum, saliva, and urine. however, these matrices can potentially have significant background noise, obscuring potential biomarkers of biological importance. extracellular vesicles (evs) are fast becoming a platform for biomarker discovery in radiation research as well as in other pathologies. however, no groups have investigated the use of metabolomics to analyse evs derived from urine in the context of ir exposure. furthermore, the dominant protocols for ev isolation from urine require a large (up to ml) amount of starting volume, which may not be available for many studies. the aim of this study was to optimize ev isolation from rat urine and assess radiation-induced alterations in urine ev number and metabolic content. methods: as a proof of concept, we compared and optimized several ev isolation methods on small volumes of urine from male wag/rijcmcr rats exposed to gy or gy x-rays to the whole body except the hind leg. starting with either µl or µl of urine, we isolated evs using ultracentrifugation (uc) with filtration, size exclusion chromatography (sec), and a proprietary bead-based isolation method developed by a rd party provider. ev samples were characterized using nanoparticle tracking analysis. metabolomics profiles were measured using lc-qtof-ms. results: we found that sec resulted in the highest yield of evs from as little as µl of urine, while uc was the poorest performing. lc-qtof-ms analysis revealed that sec and uc had the most consistent identification of features, whereas the bead-based method contained artefacts likely as a result of the extraction method. we next used sec to isolate evs from a larger cohort of rats exposed to ir and analysed with ms. ev metabolic content will eb related to differences in survival and organ function between sham and irradiated groups. summary/conclusion: we conclude that sec is the preferred method for isolating evs from small volumes of urine for broad-based mass spectrometric analysis, and that the ev metabolome may be a sensitive and specific early indicator of radiation injury. introduction: there is growing evidence that contents (including rna and proteins) of exosomes may serve as biomarkers for early diagnosis and prognostic prediction of cancers. here we aim to identify potential protein markers for oesophageal cancer. methods: using our newly developed label-free exosome automated preparation system (leaps), exosomes were isolated from ml culture medium of various oesophageal cancer cells with different differentiation profiles and different sources of metastasis. exosomes from µl plasma of cancer patients at different clinical stages or with/without relapse and healthy controls were also prepared by leaps. matrix-assisted laser desorption ionization time-of-flight mass spectrometry (maldi-tof ms) was employed to directly analyse exosomes. protein identities of exosomal fingerprint peaks were tentatively assigned by correlation with top-down and bottom-up proteomics. results: start from ml culture medium or µl plasma, high-quality exosomes rapidly isolated by leaps are sufficient for maldi-tof mass spectrometry. it seemed that poorly differentiated cells showed more exosome release. maldi-tof ms fingerprints of exosomes in cells is cell line specific. ms profiles from poorly differentiated cells showed more peaks than that from highly differentiated cells. fingerprints also allowed classification of cancer cell lines through software mathematical analysis. we identified different numbers of significantly differentially expressed peaks in exosomes of various cancer cells. fingerprints of exosomes derived from the poorly differentiated cells showed more elevated peaks. top four peaks ( , m/z, , m/z, , m/z, , m/z) were commonly down-regulated in exosomes of most cancer cells. top four protein peaks ( , m/z, , m/z, , m/z, , m/z) that might be correlated to the differentiation profile of cancer cells were also identified. maldi-tof ms detection of exosomes in the plasma and clarifying identities of potential biomarker peaks will be done in the future. summary/conclusion: the combination of leaps and maldi-tof mass spectrometry provides a fast and high-throughput tool for exosomal marker discovery. potential biomarker identified in exosomes derived from oesophageal cancer cells or from plasma of cancer patients by this tool might be useful in cancer diagnosis and prognosis. fraction-based proteomic profiling of serum extracellular vesicles derived from cervical cancer patients introduction: current evidence indicates that extracellular vesicles (evs) can release from most of cell types and affect adjacent or distant cells by circulating in all bodily fluids. proteomic analysis of evs from clinical samples is complicated by the low abundance of ev proteins relative to highly abundant circulating proteins. size exclusion chromatography (sec) has been overcome as a method to deplete protein contaminants and enrich evs. methods: we collected serum of healthy women and cervical cancer patients with stage i-iii and then counted concentration and size distribution of the evs using nanoparticle tracking analysis (nta). differential ultracentrifugation combined with sec was used to isolate and purify evs from contaminant proteins. isolated evs were investigated their characteristic based on morphology using transmission electron microscope (tem) and on expression of cd , cd , cd protein markers using western blot analysis. fraction no. - of isolated evs in among sample groups were profiled by nano-liquid chromatography tandem mass spectrometry (nanolc-ms/ms) analysis. results: nta shows that the concentration of evs is increased in patients compared with healthy women. proteome profiles of evs isolated by sec were compared in each fraction. moreover, we detected molecular evidence for fraction-specific molecular pathways in connection with cancer progression and complied a set of protein signatures that closely reflect the associated clinical pathophysiology. summary/conclusion: these unique features in each fraction among sample groups would be the informative considering in order to select for further analysis as in vitro. introduction: recently, diagnostic biomarkers from exosomes by proteomic analysis have been reported, but it is required to optimize the isolation protocol to screen out more effective biomarkers. for serum-originated exosomes, it has been also reported to isolate them selectively, however, it is observed that a different method resulted in different protein profiles in -d gel electrophoresis. methods: we isolated exosomes by two discrete methods, using ultracentrifugation and magnetic separation. before ultracentrifugation and magnetic separation, precipitation using polymer materials was perforemd. the isolation of exosomes by these two methods followed by comparison of their size, total vesicle number, morphology, and protein markers. to identify protein biomarkers, proteomic analyis using -d gel electrophoresis was performed. results: both methods induced enrichment of exosome-specific proteins, but protein profiles in each exosome fraction was totally different. the protein profiles showd that the magnetic seperation following a polymer-based precipitation step was more efficient to screen out candidate biomarkers, which showed nearly protein profiles originated from exosomes. summary/conclusion: in our study, magnetic separation of exosomes from serum fraction was optimized for -d gel electrophoresis to observe identifiable biomarkers. an extracellular small rna-seq data processing pipeline optimized for high-performance computing chenghao zhu and angela zivkovic department of nutrition, uc davis, davis, usa introduction: a variety of rna species is found in extracellular biofluids such as blood, bile, and urine, carried by extracellular particles including extracellular vesicles (evs) and lipoproteins (e.g high density lipoproteins (hdls)). the extracellular rna (exrna) carried by evs and hdls is of great interest for two reasons: ) the exrna within different carriers could be diagnostic of the state of the tissues from which the particles originate, and ) exrna has been shown to affect gene expression in target cells. although the origin and functions of exrnas remain largely unknown, there is growing interest in exrna research for the development of diagnostics and new therapeutic targets. small rna sequencing is widely used to estimate the abundance of exrnas in biofluid samples. here we present a data processing pipeline for extracellular small rna sequencing. sequencing data are pre-processed through quality control, and then aligned to the endogenous genome to obtain the gene counts for various rna biotypes, including microrna, trna, rrna, piwi-interacting rna, long non-coding rna (lncrna) and protein coding rna. it also aligns sequencing reads to exogenous databases, including the ribosomal rna sequence database silva, and all sequenced bacteria genomes available on ensembl, to estimate the abundance of exogenous genes. results: we analysed a publicly available small rna-seq dataset of hdl from three systemic lupus erythematosus (sle) patients and three healthy controls using this pipeline. the mirna hsd-mir- , lncrna al . and ac . were elevated in sle patients compared to controls. exogenous rna reads mapped to bacteroidetes were also elevated in sle patients. summary/conclusion: our pipeline is able to process exrna sequencing data and estimate the abundance of major exrna species, as well as exogenous rna taxonomy. the pipeline is optimized for the job scheduler slurm, and can therefore utilize the full computational power of high-performance computers. the pipeline is publicly available on github (www.github. com/zhuchcn/excernapipeline). introduction: ibd is a chronic hyperinflammatory disorder that severely compromises the intestines. the aetiology of ibd is poorly understood. however, it has been associated with a dysregulation of the immune system and gut microbiota and with genetic and environmental factors. cumulative evidence indicates that evs play an essential role in modulating immune responses. recent research suggests that evs derived from dendritic cells, saliva and intestinal epithelial cells may be involved in the progression of ibd inflammation. however, little is known about the contribution of immune cells-derived evs with this pathology. the goal of this study is to shed light on the contribution of pbmc-derived evs on ibd pathogenesis. here we characterized and compared the composition of evs derived from pbmcs of ibd patients and healthy control. evs were isolated by differential centrifugations from the supernatant of pbmc activated with cd -cd beads for days in serum-free media. size and concentration were analysed using a nano sight instrument, while the presence of known evs markers (cd , cd , hsp ) was analysed by immunoblotting. whole evs proteome was performed by ms/ms and functional-enrichment analysis was done using funrich with uniprot database. results: proteomics analyses identified a total of proteins in the four groups. of those, ( . %) were present in both the ibd patients and control. this group of protein was composed of several ras-related proteins, eukaryotic initiation factors, granzyme, cd , tubulin, and serpins among others. patients' evs shared proteins in common such as proteasome subunit beta type- , t cell receptor beta, and the amine oxidase containing copper . interestingly, each patient sample had a unique group of proteins. among these are myeloperoxidase, neutrophil elastase, proteasome subunit alpha type- , and signalling lymphocytic activation molecule (slamf ). summary/conclusion: these preliminary studies show that the ev composition from pbmcs of ibd patients is specific and differs from a healthy control. this exclusive composition has the potential to be used as a biomarker for diagnostics and progression of the disease, and it could also provide new insights into our understanding of the cellular pathways involved in the pathogenesis of ibd. the studies were performed with corresponding irb approvals. proteomic analysis of exosomes isolated using precipitation and columnbased approaches introduction: exosomes are a subtype of small extracellular vesicles (evs) involved in various physiological and pathological processes with huge potential as biomarker resources or as therapeutic tools. although several exosome isolation approaches are available, complementary studies focusing on optimizing the methods for human bloodderived exosomes isolation and method-specific comparative exosomal proteomic profiles will be of clinical value. methods: blood-derived evs were isolated through precipitation-and column-based methods and characterized by transmission electron microscopy, nanoparticle tracking analysis and western blot analysis. serumderived exosomal proteomes were analysed by mass spectrometry (ms). the resulting proteomes were then overlapped with the proteomes obtained from exosome-related databases, to determine the % of similar content. in addition, bioinformatic analysis, including gene ontology (go) was carried out. results: both methodologies tested isolated particles with the expected morphology and size range, although the column-based method isolated a higher number of particles. about % of the exosomal proteins identified through ms overlapped with the proteomes extracted from the databases. go terms were similar for the proteomes isolated from the column-and precipitationbased methodologies. the top go terms identified for molecular function were ion binding, peptidase activity and enzyme regulator activity and for biological process were immune system process, transport and response to stress. further, partial least square analysis revealed a clear segregation of proteomes obtained by the distint methodologies and complementary statistical analysis revealed the proteins differently expressed. summary/conclusion: no major differences were found in the top biological processes and molecular function based on go analysis. nonetheless, the two approaches result in different evs yields and significant proteome differences were identified. characterization of distinct methods for blood-derived exosomes isolation can be useful in the context of evs potential in disease diagnostics/therapeutics. introduction: we and others are developing biomarkers for neurodegenerative diseases using neuronalenriched evs immunocaptured from a suspension of total plasma evs. here we assess how the isolation method for total evs affects the yield, purity and enrichment of neuronal evs. methods: for n = subjects, total evs were isolated by ev precipitation solution (exq), ev precipitation solution plus bipartite resin columns (exu) and size exclusion chromatography (qev) from . , . and . ml plasma, respectively. then, neuronal-enriched evs were immunoprecipitated using anti-l cam antibody. in total and l cam evs, we measured particle concentration by nanoparticle tracking analysis, protein concentration, and novel multiplex electrochemiluminescence immunoassays for tetraspanins cd , cd and cd on intact evs. results: for total evs, yield followed the order of exq > qev > exu, assessed by particle (p < . ) and protein concentrations (p < . ). l cam evs immunocaptured after exq showed -fold higher particle (p < . ) and fivefold higher protein (p < . ) concentrations compared to l cam evs after exu, and -fold higher particle (p < . ) and -fold higher protein (p < . ) concentration compared to l cam evs after qev. l cam evs after ev precipitation (exq) showed , and -fold higher cd , cd , and cd concentrations (p < . ) compared to l cam evs after exu, and , and -fold higher cd , cd , and cd concentrations (p < . ) compared to l cam evs after qev. l cam evs following different methods had equal purity assessed by ratios of particle/protein concentrations (p = ns), and tetraspanin/particle concentrations (p = ns). summary/conclusion: l cam ev immunocapture preceded by exq exceeded the yield of immunocapture preceded by exu or qev. recovered l cam evs showed equal purity by particle/protein and tetraspanin/particle metrics. neuronal enrichment results will be available by the time of isev. immunoprecipitation following exq, often considered impure, purifies final isolates as effectively as more onerous methods typically considered purer. balancing sensitivity, purity and scalability is essential for implementation of blood biomarkers in the clinical setting and may be achieved by combining techniques. funding: this research was supported in part by the intramural research program of the nih, national institute on aging. characterisation of breath exosomes: towards non-invasive diagnosis deanna ayupova a , renee goreham b and paul teesdale-spittle c a school of chemical and physcial sciences, victoria univeristy of wellington, wellington, new zealand; b university of newcastle, newcastle, australia; c school of biological sciences, victoria univeristy of wellington, wellington, new zealand introduction: breath-derived exosomes present new potential for non-invasive diagnosis of lung cancer. however, breath-derived exosomes have not been well characterized and methodology for their purification has not been optimised. in order to exploit their potential for diagnosis, it is first necessary to develop methods that reproducibly provide high quality pure exosomes from breath. in this study, we optimise methods for their isolation and characterise them in comparison to exosomes derived from cell culture models. methods: in order to characterize exosomes from exhaled breath condensate (ebc) it was first necessary to optimize methods for isolation of pure, intact, and high quality exosomes. to this end, isolation methods were optimised on cell-derived exosomes and then applied to ebc, yielding high quality exosomes from size exclusion chromatography (sec). ebc exosomes were compared with those from a and wi-cells using dls, tem, and cryo-sem. an immunoblotting-grid technique was used to validate the presence of exosome-specific markers cd and cd . protein content of exosomes were quantified and compared. results: sec-based isolation was more effective at isolation of pure and intact exosomes than ultracentrifugation, with the highest purity exosomes obtained in the middle fractions of the exosome-containing eluate. exosomes from ebc had a size range ( - nm), protein content ( - ug/ml) and molecular markers typical of cell-derived exosomes. summary/conclusion: breath-derived exosomes isolated through size exclusion chromatography are sufficiently pure for diagnostic purposes and are phenotypically similar to exosomes derived from other sources. we foresee their use in non-invasive diagnostics for lung cancer as an important future application. ligand-based exosome affinity purification (leap) is a rapid and reproducible method for the enrichment of functional evs introduction: platelet-derived extracellular vesicles (pevs) represent the next generation of therapeutic biologics as they enable a more refined and targeted approach when compared to crude blood derivatives currently used for treating diseases such as cancer, thrombocytopenia and chronic wounds. however, development of an ev-based therapeutic is hindered by the lack of a scalable, validated and reproducible purification process. in this study, pevs were isolated from activated platelet concentrates and purified using exopharm's ligand-based exosome affinity purification (leap) technology to produce a functionally active ev therapeutic. methods: platelet concentrates (n = ) were obtained from the australian red cross blood service and were activated by exopharm's proprietary process. activation was verified by measuring cd p using flow cytometry. the resulting platelet releasate ( ml) was subjected to leap purification to isolate pevs. for characterization, protein concentration was determined by a bicinchoninic acid assay, microfluidic resistive pulse sensing (mrps) was used to perform a particle count and transmission electron microscopy (tem) enabled visualization of ev morphology. key ev markers were detected using mass spectrometry (ms) and western blots. to confirm biological activity, human dermal fibroblasts were subjected to serum starvation for hours before treatment with pevs ( µg/ml). cell growth was recorded by the real-time xcelligence system and differences in proliferation were statistically analysed using a one-way anova. results: mrps and tem both revealed isolated pevs to be - nm in size. the final product was positive for platelet markers (cd , cd p) and key ev markers (tsg , alix, cd ). treatment with purified pevs significantly increased proliferation in serumstarved fibroblasts over hours. summary/conclusion: exopharm's leap technology is a rapid and reproducible purification process which produces pevs that adhere to misev guidelines and are functionally active. funding: all funding was through exopharm ltd (asx:ex ) a novel but simple method to obtain purified exosomes by one-step ultracentrifugation introduction: exosomes are extracellular vesicles (evs) that are derived from endosome membrane. they are usually - nm in diameter, actively secreted in most living cells. originally, exosomes were thought to act as cellular garbage disposals. recent studies showed that exosomes not only can serve as biomarkers for diagnosis, but also can be used as an ideal delivery vehicle for drugs in therapeutics. exosomes are natural carrier for mrna, mirna, sirna, protein, dna and peptide for long distance intercellular communication. isolation of exosomes is challenging due to their small size and heterogeneity. traditional differential ultracentrifugation method is still the gold standard for exosome purification. to further explore the potentials of exosomes being as the therapeutic delivery vehicle or diagnostic reagent, it is an essential step to purify them in high quality at high yield. methods: here, we report a novel method to obtain intact shape, high-quality and high purity exosomes with one-step ultracentrifugation by using "exojuice". results: data of nanoparticle tracking analysis (nta) and western blotting showed "exojuice" can yield exosomes with a simpler method to obtain higher purity exosomes in comparison to previous method of cushion ultracentrifugation using optiprep. summary/conclusion: our method can be used to purify exosomes from cell culture medium, serum, urine, saliva, and other biofluids. a straightforward device to extract apoplastic fluid from succulent fruits for higher purity of extracellular vesicles introduction: edible plants are emerging as a sustainable source for extracellular vesicle (ev)-based drug delivery vehicles. however, current isolation methods (e.g. grinding or squeezing) may cause destruction of plants' biostructures, and in turn leads to unwanted effects in downstream applications and complicates the study of nanovesiclescell. therefore, we designed a simple device that allows the extraction of apoplastic fluid (af) from succulent fruits, facilitating ev isolation as well as effective downstream applications. methods: an inner filter tube was designed to extract af with a determined membrane pore size. af was collected by low-speed centrifugation method and then filtered to eliminate the impurities from the cytoplasm and damaged cells. minced juice (mj) was homogenized by a blender and then centrifuged to remove large fragments. subsequently, the differential centrifugation method was employed to extract evs from af and mj. fourier-transform infrared spectroscopy (ftir), nanoparticle tracking analysis (nta), and transmission electron microscopy (tem) were performed to discriminate af, mj and their evs. results: the "spectroscopic" protein-to-lipid (p/l) ratio of af ( . ± . ) is significantly lower than that in mj ( . ± . ), showing the higher lipid contents in af, which may result from the loss of lipids in mj obtained from grinding or juicing methods. similarly, ftir showed the difference in p/l ratio between af and its evs ( . ± . and . ± . , respectively). nta showed the sharper peak and smaller vesicle size in the following order: mj ( . ± . nm), af ( ± . nm), af-derived evs collected at , × g and , × g ( . ± . nm and . ± . nm, respectively). furthermore, tem study indicated that the collected evs exhibited a typical lipid bilayer of extracellular nanovesicles. summary/conclusion: by using a reusable filter device, we successfully isolated af from succulent fruits, paving the way to collect plant evs without an interference of significant biodestruction or damaged cells, hence improving the purity of evs and facilitating downstream applications. moreover, this method is straightforward, reproductive, and can be potentially used in a large-scale production. method to simultaneously capture multiple classes of intact extracellular rna carriers including extracellular vesicles and lipoprotein particles introduction: extracellular particles including extracellular vesicles (evs), lipoproteins, and free proteins are carriers of extracellular rna (exrna), which has been shown to regulate cellular function. because these particles have different physiological origins, they have different rna signatures, so the first step to understanding the biology of exrna is to isolate individual particle fractions with high purity and efficiency. current methods for isolating evs are optimized for increased yields and purity of ev fractions but typically require multiple millilitres of starting plasma and do not capture the other exrna carrier particle types. methods that can capture evs from low starting plasma volumes and can also capture other exrna carriers simultaneously are needed for analysing samples from previously conducted large cohort studies, biorepositories, and in populations where sample volume is limiting. methods: we have developed a method adapted from lipoprotein isolation that requires only µl of starting plasma, and uses brief ultracentrifugation (uc) followed by fast protein liquid chromatography (fplc) to capture classes of purified exrna carriers including evs, ldl, hdl, lipidated albumin, proteins, and vldl/chylomicrons. we have validated successful capture of evs by microfluidic resistive pulse sensing (mrps, spectradyne), transmission electron microscopy (tem), and single particle interferometric reflectance imaging system (sp-iris; exoview) with optional fluorescence. results: we have observed . × particles per ml from a ml fplc fraction of evs measured from , events by mrps, confirming that evs are being captured by this method. there were also . × particles/ml and . × particles/ml in the two subsequent ml fractions that are known to contain lipoprotein particles, though these were measured from , events each. by tem we confirmed these observations that evs are eluting before lipoprotein particles with some evs eluting later in fractions containing lipoproteins. summary/conclusion: these results confirm the efficacy of the method in isolating multiple exrna carrier fractions simultaneously from a single ul plasma sample, making it amenable for the analysis of exrna in samples from large cohort studies, biorepositories, and vulnerable populations such as the elderly and young children. funding: nih/nia r ag ; nih ug ca - optimizing the isolation of placental mesenchymal stromal cell-derived extracellular vesicles in a d bioreactor system leora goldbloom-helzner a and aijun wang baaaaa a uc davis, davis, usa; b uc davis medical center, sacramento, usa introduction: extracellular vesicles (evs) derived from placental mesenchymal stromal cells (pmscs) have the potential to provide neuroprotection at sites of injury. however, a rate limiting step in ev research is the low yield, high technical time, and high cost of current isolation procedures. to address this inefficiency, we cultured pmscs in a unique bioreactor system to increase the absolute yield of evs per ml of media and per cell. future studies will determine if this system can improve pmsc ev yield without altering the demonstrated neuroprotective properties of pmsc-evs. methods: pmscs were cultured in the bioreactor for ten weeks. ev-conditioned media was collected weekly and evs were isolated through differential centrifugation. nanoparticle tracking analysis (nta) measured ev size and concentration. western blots tested for normal ev markers (cd , cd , and cd , calnexin(-)) and enzyme-linked immunosorbent assays (elisa) measured levels of characteristic growth factors in conditioned media including vascular endothelial growth factor (vegf), brain-derived neurotrophic factor (bdnf), and hepatocyte growth factor (hgf). results: evs remained consistent until week eight, after which a decrease in both ev size and concentration was seen. western blots revealed normal positive expressions of cd , cd , and cd and negative expressions of calnexin. levels of vegf, bndf, and hgf were comparable after weeks. cost analysis revealed an overall increase in ev yield for shorter labour time and lower material cost. summary/conclusion: this initial study uses a bioreactor system for a unique source of cells and has brought us closer to optimizing pmsc ev isolation protocols for increased yield, lower cost and time commitment, and maintained sample purity. preliminary data suggests the ev phenotype and cell secretome are consistent with those present in current culture settings. future experiments will assess the preserved neuroprotective properties of the pmsc evs. a novel method for isolating extracellular vesicles from cell culture media and plasma using polyethylenimine introduction: due to their ability to transport dna, rna, and protein cargoes between cells, extracellular vesicles (evs) are becoming popular for biomarker discovery as well as for therapeutic delivery. here we describe the development of a novel precipitation method for the isolation of evs from cell culture media and plasma that is based on polyethylenimine (pei), an inexpensive, water-soluble, and biocompatible cationic polymer. pei is a group of hydrophilic cationic polymers that are synthesized as either linear or branched forms of varying molecular masses ( , to , da) and are widely used in the biomedical field as a coating and transfection agent. methods: linear and branched pei of varying molecular weights (mw) were tested for their ability to precipitate evs from either conditioned culture media (ccm) or human plasma. isolated evs were characterized by western blotting and nanoparticle tracking analysis (nta). the small rna profile of evs isolated using pei from human plasma was analysed by ngs and ev-specific mirnas were confirmed by digital droplet pcr (ddpcr). mass spectrometry (ms) was used to analyse the proteome of pei-captured evs from plasma. hek cells producing gfp+ evs were used to optimize conditions for release of evs from both linear and branched pei by fluorescent spectrophotometry and flow cytometry measure-ments. results: linear and branched pei were both able to precipitate evs as determined by western blotting for ev protein markers; however, branched pei with mw > , da and linear pei with mw > , da were more efficient for ev precipitation than lower mw forms. despite its known ability to bind nucleic acids pei was unable to capture cell-free dna from plasma, although rna and in particular ev-associated mirnas such as mir- - p were recovered. ms revealed that pei enriches extracellular exosome proteins from plasma. evs captured from ccm by pei could be released from the complex using heparin or high salt conditions. summary/conclusion: pei has an unexpected preference for associating with evs compared to nucleic acids in complex biological samples and has a hitherto unrecognized application for ev precipitation. introduction: there is ongoing debate about which is the most appropriate method for isolation of evs, with most labs using some combination of differential ultracentrifugation (uc), size-exclusion chromatography (sec), and/or density gradient ultracentrifugation (dg). here we applied a surface-enhanced raman spectroscopy (sers) analysis platform to compare chemical composition of the isolate from each method against lipoprotein standards to assess the relative purity of the ev preps. methods: - ml of plasma was separated from whole blood collected from head and neck cancer patients. each sample was split into batches and evs were isolated by either uc, sec, or dg. following isolation, samples were incubated on commercial sers substrates and raman spectra were collected. lipoprotein standards were purchased and also measured for comparison. using principle component analysis (pca), spectra were analysed for chemical variability. results: sers analysis of sec, uc, and dg isolated evs were chemically distinguishable using simple pca. the chemical changes could in large part be attributed to fitting the differences in spectra to lipoprotein standards. we found that uc isolated populations clustered with the high-density lipoproteins (hdl), sec populations with the low-and very low-density lipoproteins (ldl, vldl), and dg populations were more variable, but mainly clustered together with the highdensity-lipoproteins (hdl). summary/conclusion: this set of experiments matches our expectation that various lipoprotein would contaminate ev preps according to their relative size and density distributions. no single isolation method could separate pure ev samples. this study also illustrates the utility of label-free sers analysis for rapid chemical characterization of evs. bioreactors: lessons to develop an extracellular vesicle factory vanessa chang, priscila dauros-singorenko, lawrence w. chamley, colin l. the university of auckland, auckland, new zealand introduction: high density mammalian cell culture systems (bioreactors) provide valuable advantage for large scale production of secreted products such as extracellular vesicles (ev). however, optimisation of design selection, handling and operational costs can be quite challenging. here we provide our experience with a celline bioreactor system. methods: cultures of adherent cell lines were established in celline ad bioreactors and propagated for up to weeks. media was changed twice weekly and cells shed into serum-free conditioned medium were counted and assessed for viability. nanoevs were isolated by sequential centrifugation ( g - , g - , g) and size exclusion chromatography (sec). nanoevs were characterised in their protein (bca) and particle (nanoparticle tracking analysis) amount, ev markers (western blotting) and morphology (transmission electron microscopy, tem). results: the viability of shed cells varied between cell lines and through time, suggesting a changing dynamic during reactor establishment and continuous growth phases, that was specific to each cell line. hdfa, bt and bt consistently shed mainly dead cells ( - %), as opposed to mcf and mda-mb- which predominantly shed live cells. sec fractionation of nanoevs identified a dominate ev-rich peak and significant quantities of smaller proteins, highlighting the need for further purification. nanoev yields from each - day culture averaged - × particles, representative of yields obtained from cells grown in to conventional t tissue culture flasks. ev markers and tem confirmed the protein profiles and morphology of evs obtained from bioreactors. summary/conclusion: high density bioreactor cultures offer a physiologically relevant, cost and space efficient approach to produce significant amounts of evs, providing sufficient material for numerous experimental uses. in our hands, with careful twice weekly management, they can be propagated for up to weeks without significant changes to the evs. introduction: extracellular vesicles (evs) have potential applications for clinical theranostics. ultracentri-fugation is most commonly adopted to the evs isolation, which is recommended as a gold standard method. however, ultracentrifugation is time-consuming and expensive equipment requirement, resulting in the coisolation of contaminants such as protein aggregates. therefore, our aim is to develop a rapid and efficient platform to isolate heterogonous evs based on the insertion of lipid molecules into the evs membrane to avoid co-isolation of non-membranous protein particles. methods: herein, a defected nanoscale functional metal organic framework (mof) was constructed as an efficient platform for evs isolation. typically, one single-stranded dna was designed and modified with a phosphate group at the ʹ-end and cholesterol at the ʹ-end to form a capture dna named phosphate−dna−cholesterol (pdc). the phosphate group forms a strong covalent bond with the designed defeated site of zr (iv) in mof uio- -nh and the cholesterol inserts into the phospholipid bilayer to capture evs without non-membranous particles contamination. the formed mof−phosphate−dna −cholesterol−evs (mof@pdc@evs) system was further treated with dnase i for dna hydrolysis to give high pure evs. results: a rapid and efficient isolation platform of evs based on a defected mof functionalized with phosphate-dna-cholesterol (mof@pdc) has been constructed successfully. compared with ultracentrifugation, mof@pdc platform promises to isolate size heterogeneous evs i) without non-membranous particles contamination, maintaining evs intact membrane structure, protein components, and biological functions; ii) with the ability to capture evs with % isolation efficiency; iii) makes evs isolation process simple and fast, which could be finished in minutes without requirement of the expensive equipment. summary/conclusion: in conclusion, this rapid and efficient platform is suitable for isolation evs from biological fluid for downstream protein analysis. this work opens a new perspective in mof-based separation researches and may shed light on further studies towards evs isolation. introduction: incorporation of pharmacologically active molecules on the surface or the lumen of extracellular vesicles (evs) is an important strategy for maximizing the therapeutic potential of evs. genetic engineering of producer cells by introducing dna through random or site-specific integration are promising strategies for creating engineered evs. longterm stability with consistent transgene expression in the ev producer cells and therapeutic potency of resulting engineered evs are crucial for biomanufacturing. we present a comprehensive study to investigate stability of transgene expression and potency of two potential therapeutic engineered evs derived from stably selected pools transfected by either random integration (ri) or site-specific integration (ssi). methods: producer cells were engineered to make evs displaying interleukin (il ) or interferon gamma (ifng) by ri or nuclease-mediated ssi into aavs locus. following puromycin (puro) selection, longterm cellular stability and transgene expression without selective pressure was investigated. evs were generated from stable cell pools at , , and months post-thaw and purified by density gradient ultracentrifugation. purified evs were biochemically characterized by nta, bca, western blot, and cholesterol quantitation. transgene expression and biological activity of evs displaying il and ifng were assessed by alphalisa and in vitro reporter assays. results: transfection by ssi resulted in faster recovery in puro selection compared to ri. all stable cell pools, regardless of integration method, resulted in comparable cell culture performance, ev yield, and lipid and protein content at all time points tested. the engineered evs also demonstrated long-term stability of il and ifng transgene expression and in vitro activity from both integration strategies. summary/conclusion: both methods for generating stable cell lines were comparable in terms of cell stability, transgene expression, ev titre and potency, with ssi having the advantage of speed, allowing for more rapid iteration cycle times. thus, both methods are suitable for the precision engineering of therapeutic evs. this work demonstrates feasibility to manufacture therapeutic engineered evs from stable cells from either integration strategy for clinical development. transport of outer membrane vesicles as a model therapeutic delivery system in pathogenic and commensal bacteria introduction: outer membrane vesicles (omvs) in gram-negative bacteria have been shown to be important carriers of biomolecules, including toxins and other virulence factors, peptidoglycan, and nucleic acids. it has been shown that omvs play an important role in the delivery of these biomolecules to host cells and bacterial cells. while many thorough studies have explored omv delivery to host cells, few studies have explored the mechanisms of delivery of omvs to bacterial cells. our goal was to study the delivery of omvs to other bacterial cells. specifically, we were studying the oral pathogen aggregatibacter actinomycetemcomitans (a.a.), a gram-negative organism associated with localized aggressive periodontitis, to study the process by which vesicles from this organism communicate with other bacterial cells. overall, we want to understand the roles specific surface components of omvs play in the transport of these omvs to other bacterial cells. methods: we studied omvs from two strains of a.a.: jp , a highly pathogenic strain, and , a natural commensal strain. af -labelled omvs were incubated with fresh bacterial cultures. association of the omvs with the bacterial cells was quantified using flow cytometry. to examine the role of surface-associated dna in this process, dna was digested with dnase, and the amount of surface-bound dna was quantified with the membrane impermeable dna stain, toto- . results: using flow cytometry, we observed jp omvs were delivered to , cells, and at a lesser amount to jp cells. alternatively, , omvs associated readily with jp cells, more than to , cells. this suggests that the delivery of omvs to bacterial cells may be a targeted delivery mechanism. furthermore, we hypothesized surface-associated dna may play a role in this interaction. we next digested the surface-associated dna on the omvs with dnase, and observed a decrease in association between the omvs and bacterial cells. this supports our hypothesis that dna on the surface of the omvs plays a role in association. current experiments are investigating this interaction in more detail. summary/conclusion: we have demonstrated that omvs are selectively delivered to bacterial cells, and surface-associated dna plays a role in this process. we propose to investigate this process to further understand omvs delivery to bacterial cells. funding: r de & r de . utilizing a gaucher's disease cell line for the evaluation of a novel exosome-based replacement therapy annie k. brown a , jiayi zhang b , brendan lawler b and biao lu b a santa clara university, san jose, usa; b santa clara university, santa clara, usa introduction: engineered nano-scale exosomes have great potential as new and targeted delivery vehicles for the treatment of gaucher's disease, the most common lysosomal storage disease. recently, we have reported the design, production, and isolation of exosomes loaded with lysosomal β-glucocerebrosidase (gba). people suffering from gaucher's disease do not have functional gba, which results in toxic build-up of undegraded substrates within the cell. methods: to evaluate the efficacy of this exosomebased therapy, a human gaucher's disease model is required. here, we have utilized near-haploid human cells (hap ) modified via crispr-cas to model gaucher's disease in vitro. these cells contain a bp insertion in the th exon of the gba gene, resulting in non-functional gba. pcr, enzyme activity assays, and flow cytometry have been employed to confirm the diseased genotype and phenotype. results: characterization of gba-knock out cells shows a total loss of gba enzyme activity. further characterization demonstrates a normal growth rate but an increased number of lysosomes, indicating a diseased phenotype. summary/conclusion: the utilization of a human gba-knock out cell line will enable the evaluation of the efficacy of our engineered exosomes. disease models will be an important resource for the evaluation of new biologic therapeutics, including exosomes. funding: we would like to acknowledge the santa clara university school of engineering for their support. thrxosomes: a novel exosomes based theranostic for lung cancer introduction: chemotherapy is the first-line of treatment for lung cancer. however, inefficient bio-distribution and reduced accumulation of drugs in the tumour results in treatment failure. therefore, improved drug delivery and diagnostic systems are warranted. herein, we propose a novel theranostic system "thrxosomes" where exosomes are loaded with super paramagnetic iron nanoparticles (spions) conjugated to an anticancer drug via a phresponsive linker for controlled release. we hypothesize that thrxosomes will exert profound anticancer tumour activity that can be concurrently be monitored by magnetic resonance imaging (mri). methods: thrxosomes were produced by combining normal human lung fibroblast (mrc ) cell-derived exosomes with spions conjugated to and anti-cancer drug (chemodrug or mirna) via a ph cleavable linker. the physical and biological properties of thrxosomes were determined using transmission electronic microscopy (tem), nanotracker-analysis (nta), inductively coupled plasma mass spectrometry (icpms), western blotting, cell viability, and mri. results: exosomes used in preparing thrxosomes were nm in size with a typical lipid bilayer structure, and were positive for cd , cd , flotillin and negative for annexin a confirming presence and purity of exosomes. charge analysis, tem, and icmps data showed successful loading of spion-drug conjugate. biological studies showed selective and enhanced drug release under acidic condition (ph . ) compared to drug release at ph . . cell uptake and viability studies demonstrated increased uptake and killing of thrxosome-treated human a lung cancer cells compared to mrc- cells. in vivo studies demonstrated accumulation and detection of spions by mri in in-situ tumours of a tumour-bearing mice. summary/conclusion: our study demonstrates thrxosomes will produce profound anticancer activity in lung cancer that is measurable by mri. exosome-modified nanoparticles as an alternative delivery system for small rnas in cancer therapy petro zhupanyn a , alexander ewe b , thomas büch c and achim aigner a a independent division for clinical pharmacology at rudolf-boehm-institute for pharmacology and toxicology, faculty of medicine, university of leipzig, germany, leipzig, germany; b dr., leipzig, germany; c rudolf-boehm-institute for pharmacology and toxicology, faculty of medicine, university of leipzig, germany, leipzig, germany introduction: gene knockdown by rna interference (rnai) is an alternative, non-invasive method for inhibiting proliferation or promoting apoptosis in tumour cells. this technique allows the specific targeting of key signalling proteins or mutated genes. most of the available transfection compounds suffer from rather profound cytotoxicity in vitro. the aim of our study was to establish a novel targeted small nucleic acid delivery system to the cells, with good cellular biocompatibility and applicability for in vivo studies. for this aim, we used native, cell own vesicles-exosomes. since exosomes are known to transport peptides and different rnas between cells and tissues, these unique, small extracellular vesicles (ev) may also be useful as transport vehicles for therapeutic sirna. methods: as detected by multiple cell surface protein expression analysis, exosomes carry specific surface expression markers, allowing the cellular uptake by the most of tissues. we established an ev purification protocol from tumour cell culture supernatants and a strategy for the efficient ev loading with our test sirnas or antimirs. here we used the combination of polyethylenimine (pei)-complexation of the rnas with ultrasound treatment for their loading into the evs. our ev-modified, ultrasound-treated nanoparticles were tested in vitro by measuring knockdown efficacies in luciferase reporter cell lines or by rt-qpcr gene expression analysis. results: more efficient cellular sirna uptake was observed upon ev-modification of our pei/rna nanoparticles, accompanied by efficient inhibition of gene expression. biological efficacies were retained also after storage for several days at room temperature. the monitoring of the ev-based particles by facs revealed a different time resolution of cellular uptake and nucleic acid release compared to the classically formulated peinanoparticles. in an in vivo therapy study in tumour xenograft-bearing mice, high biocompatibility, significant biological knock-down and tumour inhibition were observed after injection of anti-survivin sirnas formulated in our ecv-modified pei nanoparticles. summary/conclusion: our data demonstrate the usability of ecv-modified nanoparticles as efficient delivery system for small rnas in cancer therapy. microglial extracellular vesicles as therapeutic vector for neuroinflammation giulia marostica a , annamaria finardi b and roberto furlan a a san raffaele scientific institute, milan, italy; b san raffaele scientific institute, milan, italy introduction: microglia is considered an eligible target against the progressive multiple sclerosis (ms), but currently available therapies do not allow its efficient targeting. as many cell types, microglia communicate with the neighbouring cells through a complex system of extracellular vesicles (evs) exchange. recently my group described that microglia derived-evs, engineered to encapsulate il , are taken up by microglia itself, mediating a phenotype switch to a protective phenotype. in vivo studies suggest that these evs can ameliorate established neuroinflammation, thus making them a promising drug-delivery tool to target cns in ms. my project focuses on understanding the mechanism of action and the signalling pathway of evs delivery and to exploit this knowledge to specifically deliver different potential therapeutic molecules. for this purpose, we decided to characterize the evs through trps technology. methods: a murine microglia cell line (bv ) was engineered to stably overproduce endogenous il . this cell line was cultured in exosome-depleted rpmi and stimulated with pma( mg/ml) for min. evs isolation was carried out by collecting supernatant and subjecting it to consequential centrifugation of g, min, rt and g, min, °c. the resulting supernatant was filtered ( µm) and ultracentrifuged at , g for h at °c. the evs pellet was re-suspended in ice-cold pbs. the evs analysis with trps shows two populations of evs, one with a mean diameter of - nm and a broad zeta potential ranging from − mv to − mv, while the second population has a mean diameter of - nm and a zeta potential of − /- mv. this difference can be consistent with the different pathway formation of exosomes and microvesicles. we demonstrated in vivo the strong phenotypic change induced by our evs to resting microglia in a dose-and time-dependent effect. then, impairing the physiological procedure of the endosome acidification, the effect of our evs on recipient cells is higher. thus, suggesting an endocytic pathway for the internalization of the vesicles. we further demonstrate with gradient ultracentrifugation the capability of our formulation to vehicle endogenous il inside the vesicles. even if some protein is co-purified in the procedure, we know that the half-life of this cytokine is too short to elicit a strong in vivo response. consequently, we assume that the anti-inflammatory effect of our evs in vivo is a result of the il internalized in our formulation. summary/conclusion: these data help us understand more in detail the process of internalization and phenotype change mediated by these evs. our next goals are to discriminate between different internalization pathways and further validate the efficacy of our therapy on the eae mouse model. targeting il- rα on tumour-derived endothelial cells blunts metastatic spread of triple negative breast cancer via extracellular vesicle reprogramming introduction: the lack of an approved targeted therapy and the early onset of metastasis highlight the need for new treatments for triple-negative breast cancer (tnbc) patients. interleukin- acts as an autocrine factor for tumour-endothelial-cells (tec), and exerts pro-angiogenic paracrine action via extracellular vesicles (nevs). il- rα blockade on tec changes tec-ev (anti-il- r-evs) microrna cargo and promotes the regression of established tumour vessels. as tec are the doorway for "drug" entry into tumours, we have aimed to assess whether il- r blockade on tec impacts tumour progression via their unique ev cargo. methods: human tnbc samples, mda-mb- , mda-mb- and mcf cell lines were evaluated for the expression of il- rα. nevs and anti-il- r-evs were characterized by electron-microscopy, macsplex-exosome-kit and western blot. proliferation, migration, apoptosis and sphere formation were evaluated. scid mice were used for in vivo experiments. results: we noticed that, besides tec and inflammatory cells, tumour cells from . % of the human tnbc samples expressed il- rα. mda-mb- and mda-mb- , but not mda cells, expressed il- rα. in vitro, nevs provide survival and migratory signals, while anti-il- r-evs promoted apoptosis as well as reduced cell viability and migration of human tnbc cell lines. in vivo anti-il- r-ev treatment induced vessel regression in established tumours formed of mda-mb- cells and almost abolished the spread of liver and lung metastasis. moreover, decreased β-catenin and twist were found in tumours from animals treated with anti-il- r-evs. in addition, anti-il- r-evs reduced lung metastasis that was generated via the intravenous injection of mda-mb- cells. nevs that were depleted of mir- - p (antago-mir- - p-evs) were effective as anti-il- r-evs in down-regulating twist and reducing lung-vessel density and metastatic lesions in vivo. summary/conclusion: overall, these data provide the first evidence that il- rα is highly expressed in tnbc cells, tec and inflammatory cells, and that il- rα blockade on tec impacts tumour progression. introduction: high-grade serous ovarian cancer (hgsoc) is the deadliest gynaecologic cancer. its lethality is explained for late diagnosis at advanced stages and frequent recurrences despite achieving complete response with standard therapy. most of recurrences occurs at abdominal cavity with multiple metastasis. therefore, identifying key determinants of metastatic process remains as priority to find better therapies. current evidence assigns a central role of the exosomes in conditioning the metastatic niche in epithelial cancers. recently, we demonstrated that statins reduce metastasis in hgsoc in preclinical models. here, we decided to study the effects of statins on hgsoc-derived exosomes and its capability to condition the metastatic niche. methods: exosomes were isolated from heya ovarian cancer cell line and primary tissue cultures established from advanced-stage hgsocs (with signed informed consent and irb approval) by differential ultracentrifugation and quantified by nanoparticle tracking analysis (nta). enriched-cancer initiating cells (cic) spheroids were established from heya cells by using stem-selecting conditions. the paracrine effect of exosomes on cic migration/invasion was studied using either d migration or boyden chamber invasion assays. previous to exosome isolation, heya cells were treated with simvastatin ( um, h) or solvent and proteins involved in exosome biogenesis/uptake (alix, tsg ), its trafficking (rab a, rab a) and in conditioning the metastatic niche (emmprin) were measured by immunoblotting. results: exosomes isolated from heya cells or hgsocs enhance the metastatic potential of heya established spheroids in d migration or boyden chamber invasion assays. upon simvastatin treatment, we observed a significant reduction in migration/invasion induced by equivalent number of exosomes in heya -derived cics. under same treatment, we observed a significant decrease in protein levels of alix and tsg and an increase in the inactive forms of rab a and rab a in heya cells. we also observed a decrease in emmprin levels in heya -derived exosomes. summary/conclusion: here, we demonstrated a paracrine effect of hgsoc-derived exosomes that favour the metastasis process. in addition, we demonstrated that simvastatin reduces metastasis induced by cancerderived exosomes. such an effect is partially explained by changes in the expression of proteins involved in exosome biogenesis/uptake, its endocytic trafficking and in the content of proteins conditioning the metastatic niche. thus, simvastatin arises as potential therapeutic target to improve outcomes in this disease. funding: this research was supported by fondecyt granted to mauricio a. cuello label-free optical imaging and characterization of cancer-associated extracellular vesicles in tissues introduction: cancer-associated extracellular vesicles (evs) visualized in the tumour microenvironment have been identified as a potential biomarker for cancer-related tissue changes. analyses of evs have traditionally been performed in cells or isolated evs, with no temporal or spatial information that could be critically important for elucidating their roles in carcinogenesis. since the unperturbed distribution and organization of evs in the tumour microenvironment is associated with their cellular function and can potentially serve as a diagnostic and prognostic biomarker, there is a strong need for visualizing evs in freshly isolated tissue specimens. currently, only fluorescent labelling methods enable visualization and tracking of evs. we used a custom label-free multimodal multiphoton optical imaging system to detect and characterize evs and classify them using their optical signatures both in isolated tissues and in situ tumours. methods: label-free multimodal multiphoton imaging was used to provide simultaneous, co-registered structural and functional images of evs in untreated samples. heterogeneous populations of evs could be identified from their unique optical signatures. results: the intrinsic metabolic and structural properties of evs enabled reliable visualization and optical characterization of evs from cell cultures and in situ imaging of tumour-bearing rats. unique optical signatures were then used for identification of cancer-related evs in tissues from human breast cancer patients, and their density was found to be highly correlated with clinical diagnosis. in the current study, evs were isolated from urine of tumour-bearing dogs, and urine and serum from breast cancer patients. analysis of ev content showed higher concentration of nad(p)h in evs isolated from cancer subjects, than from healthy subjects, which reflects the reprogramming of cellular metabolism in carcinogenesis. summary/conclusion: these results suggest a potential label-free optical methodology to detect and characterize evs by their optical signatures, which can be utilized as possible diagnostic and prognostic biomarkers for cancer. funding: this research was conducted under protocols approved by the iacuc and irb at the university of illinois and carle foundation hospital, and supported by funding from nih. novel potential anticancer therapies based on interference with nuclear entry of cancer cell-derived extracellular vesicle components in recipient cells introduction: the intercellular communication mediated by extracellular vesicles (evs) in the tumour microenvironment plays an important role in tumour progression. experimental evidence indicates that evs derived from highly metastatic cells influence the behaviour of less aggressive cancer cells. we have previously described a novel intracellular pathway where a fraction of endocytosed ev-associated proteins and nucleic acids is transported into the nucleoplasm of the host cell via a subpopulation of late endosomes penetrating into nucleoplasmic reticulum (nr). here, we better characterize this pathway and report that it is required for the induction of an aggressive behaviour induced by evs released from highly metastatic sw colon cancer cells in isogenic primary cancer cells. methods: super resolution-structured illumination microscopy and magnetic-based co-immunoisolation studies were employed to identify the protein components of the nuclear pathway and to monitor the entry of ev-containing late endosomes into the nucleoplasmic reticulum. human sw carcinoma cells expressing er-gfp and rab -rfp were exposed to evs from sw cells and then live imaged. results: we have previously reported that the tripartite protein complex, containing vap-a, orp and rab orchestrates the localization of ev-carrying late endosomes into nr. we now report that silencing of orp or vap-a, but not its homologue vap-b, reverses the pro-metastatic changes induced by evs isolated from metastatic cells on their non-metastatic counterpart, including transition to an ameboid phenotype, cell rounding and blebbing. moreover, we found that certain nuclear pore complex proteins and importin-beta are co-immunoisolated with orp , vap-a and rab suggesting the formation of a large protein complex at the entry of nuclear pores. summary/conclusion: interfering with the mechanisms regulating this novel intracellular pathway may find therapeutic applications particularly in ev field and oncology. educated osteoblasts regulate breast cancer proliferation via small extracellular vesicles thomas jefferson university, philadelphia, usa introduction: breast cancer commonly traffics to bone, where breast cancer cells (bccs) can survive undetected for years before metastatic outgrowth. in bone, bccs interact with surrounding stromal cells, including osteoblasts (obs), to shape the metastatic niche. our lab discovered there are at least two subpopulations of obs in the bone-tumour niche, based on protein marker expression. one group, "educated osteoblasts" (eos) have engaged in crosstalk with bccs whereas another group, naïve obs, have not. we have novel evidence that eos regulate bcc proliferation. the purpose of this study was to determine if extracellular vesicles (evs) produced by eos play a role in regulating bcc proliferation. we hypothesized evs produced by eos would decrease bcc proliferation. methods: eo-derived small evs from culture media were isolated via ultracentrifugation and characterized evs for size, protein marker expression, and density floatation to validate the purity of ev samples. the functionality of eo-derived evs on bcc proliferation was examined using edu and checkpoint proteins p and p . bcc protection from chemotherapy induced cell death was also examined. results: we found that evs produced by eos, but not naïve obs, decreased both triple negative and erpositive bcc proliferation in a concentration dependent manner. furthermore, using an edu assay, we found that exposure to eo-derived evs induces bccs to undergo cell cycle arrest. interestingly, the cell cycle arrest was reversible and bcc proliferation was restored upon removal of eo-derived evs. in addition, exposure to eo-derived evs leads to increases in bcc expression of the g checkpoint proteins, p and p . we next wanted to investigate proliferative signalling pathways that may be deregulated in bccs following exposure to eo-derived evs. we found that eoderived evs reduce bcc levels of erk / . because our data indicate eo-derived evs induce sustained cell cycle arrest in bccs, we desired to know if eo-derived evs protected bccs from chemotherapy-induced cell death. we found that bccs exposed to eo-derived evs and the chemotherapy drug, doxorubicin, have decreased cell death compared to bccs exposed only to doxorubicin. summary/conclusion: altogether, our data suggest eos play a crucial role in bone-tumour microenvironment by regulating bcc proliferation. funding: supported by nih r ca and commonwealth of pennsylvania -department of health sap for kmb. phosphorylation of tyrosine in annexin a is essential for its association with exosomes and for imparting invasive and proliferative capacity to other cells priyanka prakash desai a , pankaj chaudhary b , xiangle sun b and jamboor vishwanatha a a unt health science center at fort worth, fort worth, usa; b unt health science center, fort worth, usa introduction: triple negative breast cancer (tnbc) accounts for %- % of all breast cancer cases. the lack of targeted-based therapies highlights the importance of studying tnbc. elevated levels of annexin a (anxa ), a ca+ -dependent phospholipid binding protein, has been correlated with worse overall survival in tnbc patients. our previous data implicate that exosomal anxa is involved in creating a pre-metastatic niche and facilitating metastasis in tnbc. moreover, n-terminal phosphorylation of tyrosine (tyr) in anxa has been implicated in regulating several anxa activities in cancer progression. here, we demonstrated that n-terminal phosphorylation of anxa at tyr is important for its association with exosomes which imparts invasive and proliferative phenotype to other cells. hence, dissecting the regulatory pathway will be critical for verifying the value of anxa as a therapeutic, diagnostic or prognostic marker in tnbc. methods: pn -egfp plasmids expressing the constitutive phosphomimetic (anxa -y e) and non-phosphomimetic mutant (anxa -y f) gene expressing mutation at tyr site were overexpressed in mda-mb- tnbc cells. mutant cells were experimentally validated for anxa specific functions like migration, invasion and proliferation. exosomes were isolated from the mutant phosphomimetic (exo-anxa -y e-gfp) and non-phosphomimetic (exo-anxa -y f-gfp) cells and were analysed for exosomal surface expression of anxa by immunoprecipitation and flowcytometry. cal- breast adenocarcinoma epithelial cells were treated with exo-anxa -y e-gfp and exo-anxa -y f-gfp to analyse the rate of invasion and proliferation by transwell invasion and proliferation assay, respectively. transfer of exosomal anxa in cal- was studied using immunofluorescence and its implications on signalling pathways were studied by western blot. results: mda-mb- phosphomimetic tnbc mutant cells showed increased migratory, invasive and proliferative capacity compared to non-phosphomimetic tnbc mutant cells. exo-anxa -y e-gfp had elevated surface anxa expression compared to exo-anxa -y f-gfp. cal- cells treated with exo-anxa -y e-gfp showed high migratory, invasive and proliferative characteristics, with a higher expression of p-anxa (tyr ), p-src(tyr ) and p-paxillin(tyr ) compared to exo-anxa -y f-gfp treated cells. summary/conclusion: n-terminal phosphorylation of tyr in anxa in mda-mb- tnbc cells (phosphomimetic mutant cells) is essential for its association with exosomes and for conferring increased invasive and proliferative capacity to other breast cancer cells. funding: the above study is funded by national institute of health ro ca and nimhd's u md to dr.j.k.vishwanatha. a novel method for epithelial-derived extracellular vesicle isolation and enrichment in patients with advanced prostate cancer arpit rao, helene barcelo and bharat thyagarajan university of minnesota, minneapolis, usa introduction: evaluation of changes in prostate cancer biology is difficult due to presence of lymph nodal or bony metastatic disease in a majority of patients. a number of liquid biopsy assays have shown clinical utility in prostate cancer, but are limited by low sensitivity (e.g. circulating tumour cells-based assays) or inability to perform transcriptome sequencing (cellfree dna-based assays). epithelial-derived extracellular vesicles (epi-ev)-based assays are uniquely positioned overcome both these limitations as evs are abundantly secreted into the blood and have rnacargo that mirrors the cell of origin. however, a reliable method to enrich for epi-evs is currently lacking. methods: plasma was isolated from the peripheral blood collected from patients with metastatic prostate cancer enrolled in an institutional biobanking study before initiation of systemic antineoplastic therapy. evs were isolated from µl of plasma using a commercially available qev size exclusion column (izon inc.). without subjecting the evs to any physical stressors such as centrifugation, cd magnetic beads were used to fractionate the evs into cd + (platelet derived) and cd -(non-platelet derived) fractions. multiparameter flow cytometry was used to evaluate evs that expressed cd and epcam and were negative for calnexin. nanotracking analysis (nta) was used to quantify both total ev and cd + and cd fractions in all patient samples. the average ± standard deviation of total evs obtained from the patients was . x ^ ± . x ^ evs/ml of plasma (coefficient of variation [cv] : %) while the average and standard deviation of cd -evs was . x ^ ± . x ^ (cv: %). the cd -ev fraction represented a variable amount of the total evs in prostate cancer patients ranging from . % to . %. multiparameter flow cytometry showed that over % of total evs were cd + and calnexin-, suggesting an endosomal origin for a vast majority of the evs in these plasma samples. however, the proportion of evs expressing epcam (marker of epi-evs) was higher among the cd -fraction ( % - %) as compared to the cd + fraction ( . % - %). summary/conclusion: our novel method was able to isolate and enrich the epi-ev from the plasma of advanced prostate cancer patients. correlation between clinical characteristics and ev quantity is being evaluated to identify the reason(s) for large variations in cd -ev fraction. future studies are planned to use our method in improving the sensitivity of ev-based assays and increase the rna yield to facilitate transcriptome sequencing. funding: this work was funded by grants from randy shaver community and research fund, minnetonka, mn. exosomes drive medulloblastoma metastasis in a mmp and emmprin dependent manner introduction: recurrent/metastatic medulloblastoma (mb) is a devastating disease with an abysmal prognosis of less than % -year survival. the secretion of extracellular vesicles (evs) has emerged as a pivotal mediator for communication in the tumour microenvironment during metastasis. the most investigated ev's are exosomes, nanovesicles secreted by all cell types and able to cross the blood-brain-barrier. matrix metalloproteinases (mmps) are enzymes secreted by tumour cells that can potentiate their dissemination by modification of the extracellular matrix. we hypothesise that exosomal mmp and its inducer emmprin could enhance metastasis of mb. methods: proliferation, invasion and migration assays were used to evaluate the phenotypic behaviour of primary cell lines pre-treated with metastatic tumour cell-derived exosomes. gelatin zymography and western blotting were performed to confirm mmp functional activity in cell lines and exosomes. nanoscale flow cytometry was used to measure surface exosomal emmprin levels. exosomal mmp and emmprin were modulated at the rna level. results: number of exosomes is directly related to the migratory behaviour of parental mb cell lines (p < . ). notably, functional exosomal mmp and emmprin levels also correlate with this. furthermore, exosomes from metastatic cell lines conferred enhanced migration and invasion on their matched isogenic primary (non-metastatic) cell line pair bỹ . -fold (p < . ). exosomes from metastatic cell lines also conferred increased migration on poorly migratory foetal neuronal stem cells. summary/conclusion: together this data suggests that exosomal mmp and emmprin may promote medulloblastoma metastasis and supports analysis of exosomal mmp and emmprin levels in patient cerebral spinal fluid samples. introduction: exosomes secreted from cancer cells harbour the potential to regulate intracellular signalling and promote metastasis. wherein, metastasis suppressor genes (msgs) play a pivotal role in regulating such signalling cascades. however, the regulation gets hampered due to low expression of msgs under metastatic conditions. nm -h , product of first identified metastasis suppressor gene nme , is significantly downregulated under metastatic conditions. nm -h serves as a regulator of small gtpases. several evidences have highlighted an involvement of small gtpases (such as rab , rab and rab ) in the biogenesis of exosomes. in addition, bacterial homolog of nm has been shown to interact with rab and rab . however, experimental evidence supporting a relationship between exosomes and nm -h is lacking. our current focus is to deduce the relationship between exosomes and msgs. methods: breast cancer cell lines were used to assess the effect of exosomes isolated from highly metastatic cells (mda-mb- cells) on lower/non metastatic cells (mcf- cells). nme was overexpressed in mda-mb- cells and subsequently used to isolate exosome fractions. equivalent amount of isolated exosome fractions from mda-mb- cells and mda-mb- /nme cells were utilized to access their effect on migration and difference in exosome markers. results: we observed an enrichment of nm -h in the exosomes isolated from mda-mb- cells upon overexpression of nme . proteinase k protection assay confirmed the packaging of nm -h inside the exosomes isolated from mda-mb- /nme cells and excluded the possibility of membrane association of nm -h . additionally, overexpression of nm -h led to a significant reduction in the ability of mda-mb- exosomes to stimulate movement of mcf- cells as confirmed by wound healing assays. our data also highlights a clear reduction in the protein levels of exosome markers such as cd , cd and alix in the exosome fraction isolated from mda-mb- /nme cells as compared to mda-mb- cells. interestingly, rab a, a protein involved in the endosome-lysosome fusion was also present in lower amount in the exosomes isolated from nm -h overexpressing cells. summary/conclusion: our data highlights an antimigratory effect of nm -h via exosomes. these findings support a regulatory role of nm -h in the packaging or release of exosomes in highly metastatic breast cancer cells, and further suggest that metastasis suppressor proteins may be involved in the regulation or packaging of exosomes. additional studies will be required to decipher the downstream signalling of nm -h which affects the biogenesis of exosomes as well as to assess the effect of nm -h overexpression on the content of exosomes. these insights could help us delineate the complex exosome biogenesis pathway and provide new potential drug targets for exosome regulation. introduction: exosomes (exs) are emerging as novel players in the beneficial effects induced by exercise on vascular diseases. our recent study has revealed that moderate exercise enhances the function of circulating endothelial progenitor cell-derived exosomes (cepc-exs) on protecting endothelial cells against hypoxia injury. in this study, we aimed to investigate whether exercise-regulated cepc-exs contribute to the beneficial effects of exercise on ischaemic stroke (is). methods: c bl/ mice performed moderate treadmill exercise ( m/min, -wks) before is induced by middle cerebral artery occlusion surgery. acute injury was evaluated at day by determining neurologic deficit, infarct volume, cell apoptosis in the penumbra and neurologic recovery was assessed by determining angiogenesis/neurogenesis, sensorimotor functions at day . the correlations of cepc-exs and their carried mir- with neurological parameters were analysed. the underlying mechanism of the effects of cepc-exs isolated from exercised mice was explored in a hypoxia neuron model. cellular mir- level, apoptosis, axon growth ability and gene expressions (cas- , bdnf and akt) were measured. results: ) exercised mice had a smaller infarct volume on day , which was associated with decreased cell apoptosis and cleaved cas- level, and a higher microvessel density than those in control; ) the elevated cepc-ex level positively correlated with tepc-exs in ischaemic brain of exercised mice on day . the upregulated mir- level positively correlated with the numbers of tepc-exs in ischaemic brain; ) the numbers of cepc-exs and their carried mir- level negatively correlated with the infarct volume, cell apoptosis and positively correlated with the microvessel density in the peri-infarct area on day ; ) exercised mice had decreased infarct volume, increased microvessel density, promoted angiogenesis/neurogenesis and improved sensorimotor functions on day , accompanying with upregulated levels of bdnf, p-trkb/trkb and p-akt/akt; ) cepc-exs of exercised mice protected neurons against hypoxia-induced apoptosis and compromised axon growth ability which were blocked by mir- and pi k inhibitors. summary/conclusion: our data suggest that the protective effects of moderate exercise intervention on the brain against mcao-induced ischaemic injury are ascribed to cepc-exs and their carried mir- . funding: this work was supported by american heart association ( post ) and nih ( r ns ). syndecan- regulates alveolar type epithelial cell senescence mediating through extracellular vesicles during lung fibroproliferation tanyalak parimon a , changfu yao a , adam aziz a , stephanie bora a , marilia zuttion zuttion a , dianhu jiang a , melanie koenigshoff b , cory hogaboam a , paul nobel a , barry stripp a and peter chen a a cedars-sinai medical center, los angeles, usa; b university of colorado, denver, usa introduction: alveolar type epithelial cell (at ) senescence is implicated in the pathogenesis of lung fibrosis, a progressive fatal condition. syndecan- , a heparan sulphate proteoglycan, is overexpressed by at cells of human idiopathic pulmonary fibrosis (ipf) and bleomycin-injured wt mice and the overexpression of syndecan- is profibrotic. moreover, syndecan- deficient (sdc -/-) mice had less lung fibrosis after bleomycin injury. we reported that extracellular vesicles (evs) in bronchoalveolar lavage (bal) of bleomycin-injured wt mice augmented lung fibrosis whereas the sdc -/--bal-evs attenuated the process. moreover, wt-bal-evs expressed lower level of anti-fibrotic mirnas (mir- b- p, − - p, − - p, and − - p) compared to the sdc -/-bal-evs. these mirnas targeted genes in the cellular senescence pathway indicating that syndecan- altered microrna profiles in the bal-evs to promote cellular senescence during lung fibrogenesis. we investigate how syndecan- regulates at senescence through evs. methods: bleomycin was intratracheally given into wt and sdc -/-mice. at day , lungs were processed for single-cell rna sequencing (scrnaseq) and western blot (wb). evs were isolated using ultrafiltration centrifugation method. human (a ) and mouse (mle- ) lung epithelial cell lines were used for in vitro experiments. results: scrnaseq analysis indicated while bleomycin stimulated an overexpression of cellular senescencespecific genes on at cells of wt mice, these genes were significantly downregulated on sdc -/-at cells. senescence proteins, p and p , were also less expressed in the lungs of sdc -/-than of the wt mice by wb. to determine the functional effects of evs in bal, a cells were treated with human ipf or control lung wash-evs and evaluated for beta-galactosidase activity. we found that ipf-evs markedly increased beta-galactosidase enzymatic activity. corroborating with these data, bleomycin-injured bal-wt-evs also significantly upregulated senescence marker, p , by wb on mle cells whereas sdc -/--bal-evs inhibited p expression. summary/conclusion: our data indicate that syndecan- regulates lung fibrosis through the senescence signalling pathway on at cells. furthermore, syndecan- controls at senescence mediating through extracellular vesicles in the bal. lastly, the most likely cargo molecules mediating this process are micrornas. immortalized cardiosphere-derived cell ev-associated pirna, imev-pi, protects against ischaemic injury in the heart alessandra ciullo, ahmed ibrahim, liang li, chang li, weixin liu and eduardo marbán smidt heart institute, cedars sinai medical center, los angeles, usa introduction: cardiosphere-derived cells (cdcs) are a population of heart-derived progenitors with demonstrated therapeutic efficacy in preclinical and clinical settings. cdcs function by secreting extracellular vesicles (evs), lipid-bilayer nanoparticles laden with bioactive molecules. recently our group developed a strategy for immortalizing cdcs (imcdc) that retains their therapeutic potential and enhances cdc function indirectly through their secreted evs. imcdc show a different rna content(mirna, mrna, rrna, trna and pirna) compared to primary cdc. in particular, we focus on piwi rnas (pirnas), small rnas bound by piwi proteins, important regulators of both the epigenome and transcriptome. we seek to explore the role of a pirna highly enriched in imcdc-evs (imev-pi). methods: evs are prepared by conditioning cells for hrs in serum-free basal media, in hypoxic culture. after hrs conditioned medium is cleared of cellular debris and evs isolated using ultrafiltration by centrifugation (ufc). fractions were analysed in terms of particle size, number, and concentration and pirna content. in vitro, bone marrow derived-macrophages (bmdm) were exposed to imcdc-ev, imev-pi and control and transcriptomic profile and potentially activated pathways were assessed. in vivo, - week-old wistar-kyoto female rats received ^ imcdc-ev, imev-pi, scramble or vehicle intracoronary minutes after ischaemiareperfusion(i/r). cardiac troponin i levels, scar size and monocytes were assessed at and hrs. results: by small-rna sequencing analysis we found that pirnas are enriched in both cdc-ev and imcdc-ev. imcdc show a different pirna composition compared to primary cdc. imev-pi was identified as one of the most highly-expressed non-coding rnas (the number of reads were x higher in imcdc-ev compared to cdc-ev). in vitro, imexo-pi-conditioned bmdm exhibit a different transcriptomic profile compared with control, with upregulation of pathways involved in the inflammatory response, cell death, and cell-to cell signalling. in vivo, imev-pi is cardioprotective, as shown by reduced scar size and lower cardiac troponin levels compared to vehicleand scramble-injected animals at hrs post i/r. imev-pi only minimally alters neutrophil counts profile in blood but it alters monocytes profile with a decreased number at hrs and an increase at hrs. summary/conclusion: we posit that imev-pi is a key determinant of imcdc-ev therapeutic efficacy. our results indicate that target cells may be macrophages/ monocytes, given that imev-pi exposure modifies their composition and mrna profile both in vitro and in vivo. introduction: extracellular vesicles (exosomes, evs) are cell membrane particles ( - nm) secreted by virtually cells. during intercellular communication in the body, secreted evs play crucial roles by carrying functional biomolecules (e.g., micrornas and enzymes) into other cells to affect cellular function, including disease progression and tissue regenerations. literature previously reported that the macropinocytosis pathway contributes greatly to the efficient cellular uptake of evs. the activation of growth factor receptors, such as epidermal growth factor receptor (egfr), induces macropinocytosis. in this study, we demonstrated the effects of evs on demal papilla and hair follicle regeneration. methods: identification of distinct nanoparticles and subsets of extracellular vesicles from umbilical cord blood stem cell by asymmetric flow field-flow fractionation. results: the effects of evs from umbilical cord blood stem cell on the propagation of demal papilla and hair follicle regeneration were observed. summary/conclusion: the enhancement of extracellular vesicles from umbilicalcord blood stem cell the propagation of demal papilla and hair follicle regeneration were observed and confirmed. mechanisms of host resistance to plasma membrane damage induced by pneumolysin attack introduction: bacterial pore-forming toxins (pfts) are major virulence factors produced by pathogens. pfts target host plasma membrane (pm) and create transmembrane pores, which allow uncontrolled flux of ions and small molecules across the pm disrupting cellular homoeostasis. to survive, cells display poorly understood repair mechanisms to recover the cell homoeostasis. several mechanisms were proposed to participate in cell recovery: exocytosis of cortical lysosomes; endocytosis of pfts pores; pm blebbing and shedding. methods: we used increasing concentrations of purified ply to intoxicate cells. pm permeability was assessed by flow cytometry using propidium iodide dye. cytoskeleton rearrangements were investigated by confocal immunofluorescence microscopy. extracellular vesicles released during pm repair were isolated by high-speed centrifugation and characterized by nanoparticle tracking analysis (nta), transmission electron microscopy (tem) and mass spectrometry/ liquid chromatography analysis. results: ply triggers a complete reorganization of the actomyosin cytoskeleton inducing the formation of cortical actomyosin bundles at sites of pm remodelling. these structures assemble upon loss of pm integrity and disassemble as pm recovers. we detected the release of microvesicles during the recovery of pm integrity. vesicle population is heterogeneous with sizes ranging from to nm, with the majority of them measuring - nm. vesicle proteomic analysis revealed that they contain ply, suggesting they participate in pore removal, proteins involved in vesicle trafficking, pm repair and exosome biogenesis. summary/conclusion: our data demonstrate that cells are able to recover from the damage induced by sublytic concentrations of ply. actomyosin cytoskeleton undergo massive changes with the assembly of cortical bundles possibly at sites of pm damage. we showed that cells produced extracellular vesicles during the process of repair. we are now focusing on understanding the biogenesis of those vesicles and its importance during the process of repair. introduction: despite of high expectations, mesenchymal stromal cell (msc)-based therapies still lack efficacy, partially due to loss of cell viability and function upon administration. msc-derived extracellular vesicles (msc-ev) emulate the regenerative potential of msc, shifting the field towards cell-free therapies. clinical applications require the establishment of a scalable and gmp-compliant processes for the production and isolation of msc-ev, combined with robust characterization platforms. methods: to develop a well-established process for the production of therapeutic msc-ev, we compared different msc sources (bone marrow, adipose tissue, umbilical cord matrix), culture media compositions (dmem supplemented with foetal bovine serum (thermo fisher scientific), dmem supplemented with human platelet lysate (aventacell biomedical) and stempro msc sfm xeno free medium (thermo fisher scientific)) and culture parameters (oxygen tension and shear stress) in two different culture platforms ( d static tissue culture flask vs d dynamic spinner vessels). subsequently, msc-ev were isolated by ultracentrifugation or a commercially available isolation kit and characterized according to isev guidelines. results: msc derived from different sources/donors were able to grow under normoxia and hypoxia in d t-flasks and d spinner vessel culture systems, while maintaining their immunophenotype and differentiation potential, according to the minimal criteria defined by the isct. the time point for pre-conditioning and collection of conditioned medium for msc-ev isolation was also optimized for both d and d culture systems. msc-ev were characterized according to misev guidelines, using techniques as nta, protein and lipid quantification, western blot, imaging and fourier-transform infrared spectroscopy (ftir). the results indicate that msc-ev derived from different sources/donors have similar size distribution, however, ev yields tend to be higher for the d culture system. of notice, several spectral regions were identified by ftir, enabling the detection of differences in the biomolecules present in msc-ev, msc-conditioned media and cells produced under different conditions. summary/conclusion: in summary, this study contributes to the establishment of a scalable process for msc-ev production. evaluation of three different isolation methods for small extracellular vesicles from human plasma in prostate cancer diagnosis introduction: extracellular vesicles (evs) have great potential in prostate cancer (pca) diagnosis and progression monitoring to complement the inaccurate prostate specific antigen (psa) screening and invasiveness of tissue biopsy. however, current methods cannot isolate pure evs and therefor evs characteristics remain largely unknown. in order to develop an accurate approach for ev isolation, we aimed to compare three emerging methods with different characteristics of small evs (sevs) from human pca plasma samples and to choose the best one for diagnostic and functional studies. methods: pca patients and age-matched healthy controls (hc) plasma (n = in each group) were used to isolate sevs with different isolation methods including commercial exoquick ultra kit, qev and qev size exclusion chromatography (sec). isolated sev were characterized by nanoparticle tracking analysis, immunoblotting, cyrogenic electron microscopy, flow cytometry (fc) and proteomics analysis. for fc characterizing surface marker expression, the sevs were further purified by cd and cd commercial immunoaffinity magnetic beads . lipoprotein was captured by streptavidin biotinylated apob magnetic beads to measuring the lipoprotein contamination. results: the sev size, morphology, surface protein and protein cargo with proteomics were analysed between the three isolation methods. sevs isolated from sec methods had a lower particle size, protein amount, protein/sev marker ratio and apob+/sev marker ratio than those from exoquick ultra method. in addition, sevs isolated from qev demonstrated a significantly higher sev content, more up-regulated and down-regulated pca proteins from proteomics but lower sev marker/protein ratio and a higher protein contamination than those from qev . furthermore, sev marker signal also showed a good correlation with particle numbers instead of protein content in all the methods. summary/conclusion: qev method demonstrated better performance in isolating relatively pure sevs from human plasma; qev has the better performance in isolating samples with higher sev content; exoquick ultra isolated samples with closely sev content to the qev but with the highest non-sev protein contaminations. people can choose higher sev content or higher sev purity according to the downstream analysis. moreover, sevs may also be used for treatment monitoring, as recent studies suggested that the expression levels of certain markers may change during therapy, reflecting tumour response. for cancer diagnostics and therapeutic purposes in clinical settings, it is important to have a device which allows multiplexed measurements, in order to scan a large number of markers simultaneously and compare the expression levels of different patients, or same patients at different treatment stages, in a time efficient manner. methods: herein, we propose a multiplexed platform for label-free detection and surface protein profiling of sevs. the technique is based on the electrokinetic phenomena of streaming current and zeta potential (\zeta*) and measures the\zeta* change upon sev binding on functionalized microcapillary surfaces. for the purpose, we used sevs derived from lung cancer cells. in its current form, the platform can measure up to channels simultaneously, however, it can be further expanded. results: having demonstrated that our electrokinetic sensor successfully detects sevs in a specific way, we tested its ability to measure the expression level of membrane proteins. the analysis showed that it could detect differences in the expressions of egfr on sevs, with a sensitivity of %. we then extended the platform for multiplexed analysis, by connecting and measuring four capillaries, functionalized with different capture probes, simultaneously. for the purpose, we targeted specific tumour markers, i.e. egfr, and exosomal tetraspanin family proteins, such as cd and cd . the results showed successful multiplexed ev detection. summary/conclusion: being the sensor suitable for multiplexed sev detection, we shall present our investigation on a set of pleural effusion samples collected from a cohort of lung-cancer patients with different genetic makeup. introduction: extracellular vesicles (evs) are released to biological fluids from different tissues and organs and they contain molecules proposed as biomarkers for multiple pathological conditions. however, most ev biomarkers have not been validated due to the lack of sensitive techniques compatible with high-throughput analysis required for routine screenings. using immunocapture techniques, combining antibodies against tetraspanins and candidate tumour-specific markers we have recently optimized several assays that greatly facilitate ev characterization. methods: we have improved flow cytometry and elisa assays, increasing substantially the sensitivity for ev detection. using dls, em and analytical ultracentrifugation, we have characterised the biophysical basis of this enhancement. the final methodology can be performed in any laboratory with access to conventional flow cytometry or elisa reader. results: using combinations of antibodies specific for the tetraspanins cd , cd and cd , it is possible to detect evs in minimal volumes of urine and plasma samples without previous enrichment. additionally antibodies against other less abundant markers, like the epithelial marker epcam, have been used to capture and identify evs directly in minimal volumes of urine or plasma with sensitivity higher than western blot analysis of isolated evs. furthermore, we demonstrate that additives altering the biophysical properties of an ev suspension, increased detection of tumour antigens in these immune-assays. summary/conclusion: the development of sensitive, high-throughput methods, easily translatable to clinical settings, as elisa and flow cytometry described here, opens a new avenue for the systematic identification of any surface marker on evs, even scarce proteins, using very small volumes of minimally processed biological samples. these methods will allow the validation of ev biomarkers in routine liquid biopsy tests. introduction: when ev subpopulations are enriched on antibody microarrays and probed for their surface proteins, the detection signal is biased towards abundant subpopulations as it is dependent on both the protein expression level and the number of evs captured. to address this challenge, we developed a novel normalization approach allowing: ) the estimation of a target signal independent of ev subpopulation size through dye-based ev quantification, and ) the assessment of subpopulation target enrichment relative to the population average by leveraging tim as an unbiased, lipid-based ev capture. here, we investigated the expression of cancer-associated proteins, particularly metastasis-associated integrins (itgs), in breast cancer evs with varying metastatic potential and organotropism. methods: the relative protein enrichment profiles for various ev subpopulations were established from evs of skbr (her +), t d and mcf- (er+pr+), bt and mda-mb- (triple negative) breast cancer cell lines, as well as five mda-mb- -derived cell lines of four different organotropisms (brain, bone, lung, liver) using our custom antibody microarrays with our normalization approach. results: as expected, her was broadly detected in her + skbr evs. interestingly, her -t d and mcf- evs also expressed her where it was highly enriched in its epcam+ subpopulations. itg α , β and β were only found in triple negative and organotropic evs with itg β and β differentially enriched based on the organotropism. the population average of mda-mb- and lung-tropic evs had high expression of itg β , where subpopulations of cd + evs showed positive enrichment while cd + and cd + evs showed negative enrichment. itg α , β and β were absent in the bone-tropic cd + ev subpopulation, a profile atypical in other organotropisms. lastly, egfr was negatively enriched in tetraspanin+ subpopulations in mda-mb- evs, but positively enriched in these subpopulations in organotropic evs, especially for brain-tropism. summary/conclusion: following normalization, we were able to quantify specific protein associations, uncovering a multitude of co-enrichment profiles that characterize specific metastatic and organotropic cell lines. notably, we found enrichment signatures that distinguish between different organotropisms derived from the same parental cancer line. introduction: the tissue microenvironment surrounding tumours is complex and the cross-talk between cancer and non-cancer cells is essential for tumour growth and progression. we have previously shown that heparan sulphate proteoglycans (hspgs), on the surface of prostate cancer evs, are required for delivery of tgfβ and initiation of a disease-supporting fibroblast phenotype. however, hspgs are known to bind numerous growth factors, so here we have explored the repertoire of such proteins tethered to evs by hspgs. methods: evs were isolated from du prostate cancer cell conditioned media by ultra-centrifugation onto a sucrose cushion. vesicular hspgs were modified either by removal of heparan sulphate (hs) glycosaminoglycan (gag) chains using the enzyme heparinase iii (hepiii), or attenuation of hspg core protein expression using shrnas to knockdown specific hspgs within the parent cell. differences in proteins present in control vs modified evs were identified by a sensitive protein array, based on proximity-ligation technology, and selected targets validated by elisa. functional delivery of growth factors by ev-associated hspgs to recipient fibroblasts is being explored using a variety of in vitro techniques. results: proteome analysis identified targets that bind to hs-gag chains, and also different proteins that showed altered expression following the loss of one or more hspgs from evs. using elisa, we have been able to quantify selected candidates on wild type vesicles, some of these are lost following hsdigestion. we were also able to validate proteins on hspg-deficient vesicles. gene ontology analysis suggests that ev hspg-mediated delivery of growth factors is important for control of processes such as angiogenesis, tumour invasion and immune regulation. functional validation of proteins identified is ongoing. summary/conclusion: here we demonstrate that hspgs play a key role in loading of evs with a complex assortment of growth factors, and therefore subsequent ev-mediated growth factor delivery. we anticipate that loss or damage of ev-associated hspgs will result in attenuation of ev induction of a tumour-supporting fibroblast phenotype. introduction: ovarian cancer (oc) is the fifth leading cause of cancer-related death in women, partly due to difficulty in early diagnosis. extracellular vesicles (evs) show promise for use in early diagnostics of oc. here, evs from cervical mucus (cm) of ovarian cancer patients were used for discovery of oc biomarkers for diagnostics. machine learning was used to mine ev mirna data to develop an oc biomarker panel (validation via the cancer genome atlas). examination of the mirna targets reveal that the panel is a sufficiently accurate predictor of oc. methods: evs from the cm of patients ( highgrade serous, low-grade, benign) were isolated for small rna-sequencing. the top differentially expressed mirnas were used in a random forest and "voom" (variance modelling at the observational level) model. unsupervised approaches were used and then vetted against patient symptomology data. a tcga ovarian cancer dataset (n = ) was used for validation. results: an oc biomarker panel of micrornas (voom: . % accuracy; random forest: % accuracy) was generated. the panel consists of members from the mir- family and the mir- family, among others. the mirna targets are associated with molecular functions and pathways specific in oc progression. summary/conclusion: our method has identified ev mirna biomarkers that may be crucial for early, noninvasive detection of oc. data science has been used to develop a feedback system integrating biochemical experiments, smaller datasets, and previously available data to identify and verify a biomarker panel for oc diagnostics. introduction: liver disease has become a significant cause of morbidity and mortality among hiv patients. alcohol exposure can further exacerbate liver damage by activating hepatic stellate cells (hscs), leading to hepatic fibrosis or cirrhosis, often seen at all levels of alcohol exposure among people with hiv. due to the potentiating effects of alcohol on hiv-induced hepatocytes (hep) damage, as well as the effect of ethanol in hsc-mediated extracellular remodelling, it is imperative to understand the interplay of hep and hscs. here, we focus on the exosomes released by hiv-and ethanol exposed hep and how these exosomes modulate the functional behaviour of hscs. methods: human hepatocyte huh . cyp e [hepatoma cells stably transfected with cyp e designated as rlw cells] were infected with hiv in the presence or absence of alcohol metabolite, acetaldehyde using the acetaldehyde-generating system (ags). the conditioned medium was collected from groups of cells: untreated, hiv-, ags-and hiv+ags. quantification of exosomes number and size were evaluated with zetaview or nanosight and further characterized for exosome markers following the guideline from minimal information for studies of evs (misev ). the human hepatic stellate lx- cell line was exposed to hepatocyte-derived exosomes and assessed for the activation using pro-inflammatory markers il- β, il- , tnfα, and fibrotic markers acta , and timp using quantitative pcr. we also analysed exosome mirna content in primary human hepatocytes (phh), which potentially regulates the function of recipient cells by "programming" their inflammation/fibrosis status. the network analysis for mrna and mirna were carried out using gene ontology consortium, and mirror . and david bioinformatics resources . . results: ags treatment further enhanced the release of hiv-induced exosome from hepatocytes. size distribution assessed by zeta view or nanosight revealed that approximately - % of particles distributed in the range of to nm, with a peak at~ nm. enriched expression of hiv protein p was observed in fractions f -f . western blotting of hepatocytederived exosome demonstrated positivity for exosome-enriched proteins alix, tsg and cd specifically in f -f fractions and negative for endoplasmic reticulum protein calnexin. the uptake of hepatocytederived exosomes by hscs was apparent as demonstrated by immunofluorescence. the internalization of hepatocyte-exosome induced activation of hscs as evidenced by increased expression of pro-inflammatory il- β, il- , tnfα markers in the latter cells. summary/conclusion: we conclude that ags treatment in hiv-infected hepatocytes potentiates the release of exosomes, which, following uptake by the hscs, leads to their activation. funding: this work is supported by nih- r aa - a . antimicrobial peptide ll- induces neutrophil-derived extracellular vesicles with antibacterial potential and protects murine sepsis yumi kumagai, taisuke murakami, kyoko kuwahara and isao nagaoka juntendo university, bunkyo-ku, japan introduction: extracellular vesicles (evs) released from immune cells or other host cells upon microbial infection modulate the immune response and thereby regulate the infection. sepsis is a life-threatening multiple organ dysfunction caused by systemic dysregulated inflammatory response to infection. nevertheless, numerous therapeutic trails concerning immune dysfunction have still been disappointing outcomes. we have previously shown that ll- , a human cathelicidin antimicrobial peptide, improves the survival of caecal ligation and puncture (clp) septic mice. here, we investigated the induction of ev release by ll- and functions of ll- -induced evs in murine sepsis. methods: evs were isolated from peritoneal exudates of clp mice and the supernatant of ll- -stimulated mouse bone marrow neutrophils by differential centrifugation or size exclusion chromatography. isolated evs were analysed by flow cytometry, western blotting, and nano particle analysis. neutrophil-derived evs were injected into clp mice to assess the protective function of evs against septic mice. the antibacterial activity of evs was evaluated by incubating with escherichia coli. results: in clp mice, ll- augmented the level of evs. evs from ll- -injected clp mice contained higher amounts of neutrophil-derived antibacterial proteins (lactoferrin and cramp, cathelicidin-related antimicrobial peptide) and exhibited higher antibacterial activity compared to evs from pbs-injected clp mice. furthermore, ll- stimulated mouse bone marrow neutrophils to release evs with antibacterial potential, and administration of the ll- -induced evs reduced the bacterial load and improved the survival of clp mice. summary/conclusion: ll- induces the release of antimicrobial evs from neutrophils in clp mice, thereby reducing the bacterial load and protecting mice from lethal septic condition. identification of mirna profiles of serum exosomes in active tuberculosis introduction: tuberculosis (tb) has exceeded hiv as the most lethal infectious disease globally for two consecutive years, mainly due to difficulties in achieving early and definitive diagnosis, and timely treatment. exosomes carrying rna, particularly mirna, have demonstrated their functional and diagnostic potential in diseases including tb. however, few published studies have explored whether exosomal mirnas could be used for diagnosis of tb. thus, more systematic and comprehensive study of exosomal mirnas with regard to their potential as non-invasive tb biomarkers is still urgently needed. methods: we searched the gene expression omnibus database for datasets published before december , and performed meta-analysis on available exosomal mirna profile data for healthy control (hc) and active tb clinical specimens . reprocessing next generation sequencing data under uniform parameters and utilizing state-of-the-art bioinformatics analysis. results: we identified many distinct up-regulated and down-regulated differentially expressed exosomal mirna across multiple studies, and further screened the top , which might provide a potential panel for differentiation of hc and tb. we classified all differentially expressed mirnas into six expression patterns and identified two persistently up-regulated mirna (hsa-mir- - p, and hsa-mir- - p) as potential markers during tb progression. moreover, the differential expressed exosomal genes that we screened from the datasets were consistent with the genes overlapped with predicted mrna targets of differentially expressed mirna. pathway and function analysis further demonstrated down-regulated signalling pathways/immune response and up-regulated metabolism and apoptosis/necrosis. introduction: trypanosoma cruzi is a protozoan parasite that causes chagas disease, a relevant source of morbidity in latin america, which has spread to many countries as result of immigration of the people from endemic areas. many studies have been showed that trypomastigote forms of t. cruzi release extracellular vesicles (ev) that increase parasite infection. objectives. here, we aim to test if previous immunization with evs in adjuvant can generate a protective immune response by decreasing the effects of evs in experimental chagas disease. methods: female balb/c mice were immunized by intra peritoneal (ip) administration with × or evs isolated from trypomastigotes forms, with aluminium hydroxide adjuvant (aloh). injections were administered intravenous in doses during days ( days interval). after immunization, mice were infected intra-peritoneally with trypomastigotes forms. parasitaemia was quantified by counting motile parasites in fresh blood sample drawn from lateral tail veins. mortality and weight were analysed during the infection. in control group, the mice were immunized with aioh. results: the immunization with evs with aloh decreased the blood parasitaemia and the animals survived, while all animals died in the group aloh alone. the animals immunized with evs had an increase of f / + cd b+ and cd /cd expression in cells isolated from the peritoneum. summary/conclusion: these results indicate that t. cruzi ev antigens can induce an immune response that controls the development and establishment of the experimental chagas disease. introduction: acinetobacter baumannii (ab) is a nosocomial pathogen, of major concern due to its multidrug resistance (mdr) and the recent appearance of hyper-virulent strains in the clinical setting. the world health organization included ab as a critical priority pathogen for the development of novel antibiotics. ab pathogenesis is associated with a multitude of potential virulence factors (vf) that remain poorly characterized. there is growing evidence that outer membrane vesicles (omv) are used as vehicles to transport bacterial proteins that contribute to set up the conditions for the infections. in the present work we studied the physiopathology of mdr ab. we focused on the contribution of non-characterized outer membrane proteins (omps) associated to omvs, with special focus on lipoproteins (lp). methods: we conducted a bioinformatic prediction using available datasets to construct a list of omv-associated omps putatively acting as vf in ab . seven genes were selected and the corresponding mutants were obtained from manoil lab collection. physiological analyses of the mutants were performed, and the involvement of the selected proteins in ab pathogenesis was evaluated by adherence, invasion, and cytotoxicity assays on human lung cells a . results: biochemical analysis indicated similar growth rates in rich media, as well as similar levels of omv production for all the mutants as compared to wt. also, no differences in susceptibility to chaotropic agents were observed, indicating no alteration of the om function as a general permeability barrier. all mutants similarly reduced a cell viability, but to a lesser extent than the wt. moreover, three of them exhibited less adhesion and invasion compared to the wt, and omv isolated from these mutants displayed variable levels of cytotoxicity. summary/conclusion: these results suggest roles for the mutant gene products in ab pathogenesis and contribute to the better understanding of ab virulence mechanisms, revealing novel possible targets for therapeutic development. funding: agencia nacional de promoción científica y tecnológica (anpcyt, pict - ) medicine, nanfang hospital, southern medical university, guangzhou, , china, guangzhou, china (people's republic); d zhujiang hospital, southern medical university, guangzhou, china, guangzhou, china (people's republic) introduction: talaromyces marneffei (t. marneffei) grows as a mycelial form in the environment but multiplies rapidly as a yeast form in the host and within macrophages. the yeast can cause disseminated and progressive infections or lethal talaromycosis. but the mechanisms of pathogenicity of t. marneffei are poorly understood. fungal extracellular vesicles (evs) have previously been shown to transmit a proinflammatory message to macrophages. however, the characteristics and effects of t. marneffei evs on the progress of infection have not yet been investigated. methods: in this study, evs of t. marneffei yeasts were isolated by ultracentrifugation method. evs were detected and confirmed by electron microscopy and nanoparticle tracking analysis (nta). the raw . murine macrophages were incubated with the t. marneffei vesicles to observe the changes of macrophage morphology and function, especially in inflammatory response. the proteins, dnas, rnas of t. marneffei vesicles were respectively removed with protease, dnase and rnase. all treated evs were used to incubate with murine macrophages observe the effect on macrophages in inflammatory response. results: we observed that evs secreted by t. marneffei have a typical spherical shape with a diameter of to nm. t. marneffei evs were internalized by raw . murine macrophages and promoted the production of no and proinflammatory cytokine by macrophages in a dose-dependent manner. t. marneffei evs stimulate macrophages to generate reactive oxygen species (ros). addition of t. marneffei evs to macrophages also promoted transcription of the m -polarization marker cd and diminish that of the m markers cd . incubation of t. marneffei vesicles with murine macrophages resulted in increased levels of extracellular interleukin- β(il- β), interleukin- (il- ) and interleukin- (il- ). the proinflammatory effect of vesicles was weakened when the proteins of the vesicles were destroyed. in contrast, no similar changes were observed in degraded dna and rna. summary/conclusion: our results indicate that the extracellular vesicles of t. marneffei can stimulate macrophage towards to m polarization phenotype and promote proinflammatory function. plasma-derived extracellular vesicles as potential biomarkers in chronic chagas disease patients introduction: chagas disease (cd), caused by the parasite trypanosoma cruzi (t. cruzi), is a neglected tropical disease affecting about million people worldwide. currently, one of the main clinical problems is the lack of effective biomarkers for therapeutic response and disease prognosis during chronic infections. in that context, extracellular vesicles (evs) are raising attention as novel, minimally invasive, and inexpensive method for diagnostic and screening of diseases, as well as a new source to identify new biomarkers. the main objective of this study is to use evs derived from biological fluids of chronic cd patients for identifying novel biomarkers, specifically in the context of therapeutic response and disease prognosis. methods: plasma, saliva and urine from a cohort of chronic cd patients are being collected before and at the end of benznidazole treatment. as negative controls, healthy donors have been also included. the purification and characterization of the evs was performed by size exclusion chromatography, followed by nanoparticle tracking analysis, bead-based flow cytometry assay and transmission electron microscopy. a proteomic analysis of the evs was also performed. results: proteins associated with evs secreted by infective t. cruzi have been previously identified in cell culture, but never in human samples. our results, based on the analysis of a single heart-transplanted patient with chronic cd, showed the presence of t. cruzi and human proteins specifically associated with plasma-derived evs. noticeably, several human and parasite proteins identified in evs obtained from plasma samples, were present or upregulated before chemotherapy and were absent or downregulated following treatment. currently, proteomics analyses are being performed with higher numbers of cd plasma samples. summary/conclusion: to the best of our knowledge, this is the first proteomic profiling of plasma-derived evs from a heart-transplanted patient with chronic cd. these results thus open the possibility of using evs from biological fluids as a tool for the identification of new biomarker candidates in chronic cd. these biomarkers are essential for assessing disease introduction: eukaryotic cells communicate with one another through multiple pathways. an established route of communication between eukaryotic cells is via the production of a range of different membrane bound signalling "packages", called extracellular vesicles (evs). evs are produced by all domains of life and carry proteins, nucleic acid (rna and dna), and other biological material, travelling between cells and around the body to deliver a range of chemical messages. bacteria can also produce evs that communicate with each other to coordinate population behaviour, as well as with eukaryotic cells to stimulate host defence or induce tolerance. here i investigate the poorly explored axis where evs are the vehicle for communication between eukaryotic cells and bacteria. methods: as a first step, i have isolated evs from tissue cultured eukaryotic cells grown in advanced rpmi media with minimal ev-depleted fbs. nanoevs were isolated from spent culture media using sequential centrifugation ( , × g, , × g) and concentration ( kda filter) before purifying using size exclusion chromatography columns. nanoev-rich fractions were pooled based on particle (nanoparticle tracking analysis) and protein quantity data. nanoevs were characterised by electron microscopy and expression of exosomal markers. eukaryotic nanoevs were then characterised in their effect upon the growth of escherichia coli as a model bacterium, also grown in tissue culture media to mimic relevant in vivo conditions. results: further experiments with increased dosages are required to determine the effect of human evs on bacteria. summary/conclusion: our work will investigate whether human evs communicate with the resident and pathogenic microbiota, while examining the mechanisms behind this communication. escherichia coli pathogenic bacteria commensal bacteria hydrogen sulphide (h s) derived extracellular vesicles: a potential protective role in response to respiratory syncytial virus (rsv) infection methods: evs were isolated from untreated (control evs) and gyy treated (gyy-evs) a cells, a human alveolar type ii-like epithelial cell line. evs were purified using a two-step enrichment procedure. evs were characterized using particle sizing (size and concentration) and western blot for the ev markers. electron microscopy and immunofluorescence staining were used to investigate presence of multivesicular bodies (mvbs), evs precursors, in both groups. recipient a cells were cultured for hours in the presence or absence of control-or gyy-evs, then infected with rsv for hours. viral titres by plaque assay were measured in recipient infected a cells. results: we confirmed the presence and purity of our evs. we found that gyy reduced the particles number of evs, but did not change ev size. a cells treated with gyy showed an accumulation of mvbs/lysosomes-like structures, as well as an increase in cd expression, a mvbs marker, compared to untreated cells. recipient a cells treated with gyy-evs showed lower viral replication than control ev-treated cells in response to rsv infection. we are currently investigating the potential mechanism for this observation and characterizing the rna cargo composition of gyy-evs. summary/conclusion: no vaccine or effective treatment is currently available for rsv. cellular pretreatment with gyy-evs reduced the rsv replication in airway epithelial recipient cells, suggesting that h s could exert its antiviral activity in the context of rsv infection potentially through modulation of ev composition. therefore, gyy-evs could represent a future novel pharmacological approach for ameliorating virus-induced lung disease. effects of extracellular vesicle-mediated transmission on reoviridae infection results: taken together, these data suggest that multiple particles of reovirus and rotavirus egress in large, virus-modulated evs, and that transmission in evs increases segment complementation compared to transmission as free particles. summary/conclusion: these discoveries may be broadly applicable to viruses that travel in evs and will contribute to general principles of virus transmission and diversification. continued studies will illuminate the specific cellular pathways reovirus and rotavirus utilize for successful egress. these pathways may prove to be critical targets for the improvement of vaccines and oncolytic therapy. multiparameter flow cytometry analysis of the human spleen and its interaction with plasma-derived evs from plasmodium vivax patients introduction: the spleen is a secondary lymph organ that filters blood and elicits immune responses against blood-borne pathogens, such as malaria parasites. extracellular vesicles (evs) are membrane-bound particles involved in intercellular communication. evs play several roles in malaria ranging from modulation of immune responses to induction of vascular alterations. here, we report the first integrated characterization of human spleen cells using multiparameter flow cytometry (mfc) describing subpopulations of splenic leukocytes and red blood cells (rbcs), and studied their interaction with plasma-derived evs from p. vivax patients (pvevs). methods: human spleens were obtained from organ transplantation donors. myeloid, lymphoid, erythroid and haematopoietic stem cells (hscs) were immunophenotyped by mfc. t cells, dendritic cells (dcs) and rbcs were enriched by density centrifugation and immunomagnetic isolation. pvevs and healthy donors evs (hevs) were purified by size-exclusion chromatography (sec) and characterized by bead-based flow cytometry. enriched evs were labelled with fluorescent lipophilic dyes and incubated with total splenocytes or enriched populations. evs-cells interaction was assessed by flow cytometry. results: human spleen immunophenotyping showed that cd + cells included b ( %), cd + t ( %), cd + t ( %), nk ( %) and nkt ( %) lymphocytes. myeloid cells comprised neutrophils ( %), monocytes ( %) and dcs ( . %). erythrocytes represented % whereas, unexpectedly, reticulocytes were . % of total cells. in addition, we also detected hscs, which accounted for . %. sec separated evs from the bulk of soluble plasma proteins as shown by the enrichment of cd , cd l and cd markers. interaction studies showed an increased proportion of t cells (cd + -fold and cd + -fold), monocytes ( . -fold) , b cells ( . -fold) and erythrocytes (threefold) interacting with pvevs as compared to hevs. summary/conclusion: the integrated cellular analysis of the human spleen and the methodology employed here allowed in vitro interaction studies of human spleen cells and evs. a larger proportion of monocytes, t and b lymphocytes as well as erythrocytes was found to interact with pvevs compared to hevs. future functional studies of these interactions can unveil pathophysiological processes involving the spleen in vivax malaria. neuroblastoma-secreted exosomes carrying mir- promote osteogenic differentiation of bone marrow mesenchymal stromal cells introduction: bone marrow (bm) is the major target organ for neuroblastoma (nb) metastasis and its involvement is associated with poor outcome. yet, the mechanism by which nb cells invade bm is largely unknown. tumour microenvironment represents a key element in tumour progression and mesenchymal stromal cells (mscs) have been recognized as a fundamental part of the associated tumour stroma. here, we explore the potential role of nb-derived exosomes in induction of a pro-osteogenic phenotype on bm-mscs. introduction: extracellular vesicles (evs) are nanosized particles delimited by a lipid bilayer which transfer functional molecular cargos from the cells of origin to target cells. this intercellular crosstalk controls both physiological and pathological conditions. given their presence in body fluids and their characteristics, these nanocarriers might be potentially used in diagnostics and/or therapy. breast cancer is the most frequently diagnosed malignancy and ranks as the leading cause of cancer mortality in women worldwide; the triple negative breast cancer, in particular, is the most aggressive subtype with a poor prognosis. since it is recognized that cell stiffness of cancer cells play a crucial role during the metastatic spreading, we set ourselves the goal of clarify the effects and the activity of small-evs (i.e. with a diameter below nm) in metastatic breast cancer, with a special attention on their correlation with the biomechanical properties of cells. methods: functional assays were performed on the non-invasive mcf breast cancer cell line, before and after the cellular uptake of small-evs originating from the invasive mda-mb- triple negative breast cancer cell line. the mechanical properties (cell stiffness, cytoskeleton organization and focal adhesions) of mcf cells were investigated before and after the vesicle uptake. results: the uptake of small-evs derived from mda-mb- significantly reduces the young's modulus values of mcf cell line making them more invasive. moreover actin and focal adhesion variations were observed in mcf cells before and after small-ev's uptake, suggesting a molecular rearrangement inside mcf cells upon uptake. summary/conclusion: our results evidence that small-evs play a key role in altering biomechanical properties of target cells and underline their relevance in cell-cell crosstalk. our approach is very promising to identify new molecular mechanisms through which evs perform their oncogenic function. stratification of angiogenic or non-angiogenic lesions in colorectal cancer liver metastases patients using extracellular vesicle mirna introduction: colorectal carcinoma (crc) is the second leading cause of cancer death in the western world. over % of the crc patients develop liver metastasis (lm) and % will die from metastatic disease. in the current clinical setting, liver resection provides the only possible cure, but only % of crclm patients are resectable. the combination of angiogenic inhibitors with chemotherapy is used to downsize crclm with the goal of converting unresectable patients to resectable ones. however, only - % of these patients can be successfully converted to a resectable state. we have no way of identifying those crclm patients that would respond/benefit to the addition of anti-angiogenic therapies (e.g. bevacizumab: bev)). proper stratification of patients into angiogenic inhibitor responders and non-responders will permit a proper assessment of the efficacy of angiogenic inhibitors. crclm forms distinct histopathological growth patterns (hgp): angiogenic (desmoplastic) and nonangiogenic (replacement) hgp. we demonstrated that crclm patients with predominant angiogenic lesions receiving bev plus chemotherapy have a more than double -year overall survival compared to patients with non-angiogenic lesions. therefore, nonangiogenic lesions do not respond to angiogenic inhibitors. our study focuses on stratifying angiogenic vs non-angiogenic lesions of crclm through extracellular vesicle mirnas. we are using two approaches in the selection of mirnas to target: . text mining of published ev mirna from crclm patients; and . differentially expressed mirnas present in tumour tissue from both lesion types, we have obtained by sequencing - patients. these two strategies will generate a list of mirnas that we will target using qpcr on plasma-derived ev mirna from the patients used in approach , where we have classified the lesions in the patients. preliminary data on patients will be presented. methods: ev isolation was performed using the gold standard centrifugation method. rnaseq and qpcr are used to generate the expression profile for angiogenic vs non-angiogenic type of crclm. results: the research is under progress. summary/conclusion: the research is under progress. the introduction: it is known that bone metastasis causes a reduction in the quality of life of cancer patients due to fractures and nerve compression. therefore, it is important to elucidate the mechanism of bone metastasis and develop new treatments. metastatic bone tumours occur at particularly high rates in cancers of the prostate, breast, and lung. in this study, we focused on extracellular vesicles (evs) in bone metastasis, and investigated that the role of evs derived from cancer cells in osteolysis. methods: the prostate, breast, and lung cancer cellderived evs were added to osteoclast precursors with rankls. the osteoclast differentiation was evaluated by tartrate-resistant acid phosphatase (trap) stain and by measuring the expression level of osteoclast markers using by qrt-pcr. a proteome analysis (lc-ms/ms) and sirna approaches were used to identify molecules which are responsible for promotion of osteoclast differentiation in the prostate cancer cellderived evs. to investigate whether the molecules are suitable for the detection of bone metastasis in serum evs, we isolated evs from serum of prostate cancer patients, and analysed the protein level of the molecules by western blot analysis. results: we found that the prostate cancer and lung cancer-derived evs significantly promoted the rankl-stimulated osteoclast differentiation. our analysis revealed that cub domain-containing protein (cdcp ), which is a membrane protein on the prostate cancer cell-derived evs, was responsible for promotion of osteoclast differentiation. moreover, cdcp was markedly detected in the evs-derived from serum of prostate cancer patients who had bone metastasis than that of normal subjects. we also found that cdcp exits on the breast and lung cancer cell-derived evs. summary/conclusion: we showed that the evsderived from bone metastatic tumours have a role in activation of osteoclastogenesis. moreover, we revealed that cdcp in the evs is responsible for promoting of osteoclast differentiation. these evs could be the novel diagnostic and therapeutic target for bone metastasis. increased expression of chemokine receptor cxcr in non-invasive colorectal cancer cells after incorporation of platelet-derived extracellular vesicles. introduction: blood platelets and platelet-derived extracellular vesicles (p-evs) play a crucial role in tumour growth and metastasis. p-evs, also referred to as platelet microparticles, are recognized as a carrier for proteins and nucleic acids that control cell-to-cell communication, mediate the formation of metastatic niches and affect tumour invasion and metastasis. among the other factors, p-evs contain the chemokine receptor cxcr , known as a co-receptor for hiv entry but also regarded as important in cancer development due to the importance of cxcr /cxcl signalling. overexpression of cxcr was reported in various, especially in invasive cancers, including colorectal cancer (crc). crc, the third most commonly diagnosed cancer, is usually diagnosed at the late stage and patient's death is mainly related to metastasis. increased levels of cxcr has been reported as a poor prognostic factor for survival of crc patients and its blocking has been suggested as therapeutic approach. the aim of this study was to analyse the effect of p-evs on the levels of cxcr in crc cells on various epithelial-to-mesenchymal transition stage. methods: we used crc cell lines ht and sw , which represent distant invasive potential and different phenotypes, epithelial and strongly mesenchymal, respectively. p-evs were isolated from outdated concentrates of human blood platelets after activation by thrombin in the presence of calcium ions, by subsequent centrifugation and ultracentrifugation. the p-evs were labelled using pkh fluorescent dye to visualize their uptake into cell lines by confocal microscopy. we also quantified the levels of cxcr in ht and sw by western blot analysis. the effect of p-evs uptake on the migration of crc cells was studied by "wound healing" method. results: we found that the levels of cxcr in crc lines used in the study were correlated with their emt stage. we show here that p-evs released by activated platelets were incorporated into both ht and sw cell lines. the expression of cxcr in ht was increased after the uptake of p-evs. additionally we observed that migration rate of ht cells with incorporated p-evs was elevated as compared to control cells. summary/conclusion: we posit that circulating p-evs can be incorporated into yet not invasive crc cells to significantly increase the level of cxcr receptors and that may lead to the their more invasive characteristics. introduction: for cancer therapy it is important to identify markers and key processes induced during cancer progression. one of them is epithelialmesenchymal transition (emt) which is associated with cell acquisition of invasiveness, stem cell characteristics and resistance to apoptosis and therapy. also the extracellular vesicles (evs) released from tumour cells, which can be taken up by cells constituting pre-metastatic niches, can alter cancer progression by promoting cells' reprogramming. our group has recently reported that snail transcription factor, a key factor of emt, when overexpressed in crc ht cells, drives their early emt and alters the expression of microrna (mirs). in the present study we analysed the mirs profile of evs released from those cells. methods: evs from three ht clones stably overexpressing snail and from control ht -pcdna were isolated by differential centrifugation and ultracentrifugation of conditioned media after h of culturing in serum-free medium. total rna was isolated and nextgeneration sequencing (ngs) analysis of the mirnas was performed followed by gene ontology ( introduction: prostate cancer (pca) is the most common malignant tumour in male urinary system and osteoblastic bone metastasis is the most observed metastasis in prostate cancer patients. it has been demonstrated that circulating micrornas contained in extracellular vesicles are potential early biomarkers and therapy targets for many diseases. however, the potential role of micrornas in prostate cancer bone metastasis, is not yet to be fully explored. methods: after isolation and purification evs using ultracentrifugation from conditioned media of bone metastatic co-opting prostate cancer cells and normal cells, total rna was extracted. subsequent to library preparation and small rna-seq, differential gene expression analysis was performed. data were filtered by mean mirna expression of ≥ reads, two fold up or down regulation between . − . and adjusted pvalue ≤ . . the uptake of pca-sevs was performed. three candidate mirnas (has-mir- c- p; has-mir- ; has-mir- - p) were internalized and osteoblast differentiation were detected by qpcr, histochemical staining and protein activity detection. results: total reads of mirnas in bone metastatic co-opting pca-evs exceeded significantly than that in normal evs (p < . ), indicating that mirnas delivered by pca cells play critical role in pca bone metastasis. pca-cm enhanced osteoblast differentiation and can be reversed by gw . the uptake of pca-evs by mc t -e was efficient. the high expression of the three candidate mirnas in pca-evs was verified by qpcr. all the three candidate mirnas promoted osteogenesis, verified by mrna expression of osteoblastic markers (alp, ocn, runx , osx), alp activity, alp staining and aliza red s staining. summary/conclusion: these findings suggest that mirna cargos in pca-evs play a pivotal role in the development of osteoblastic bone metastasis of pca, which can be potential early biomarkers and therapy targets for prostate cancer bone metastasis. funding: this work was supported by grants from the national natural science foundation of china ( ); xijing hospital science and technology foundation project (xjzt ptk ). introduction: retinoblastoma (rb) is the most common intraocular cancer of childhood. despite recent advances in conservative treatment have greatly improved the visual outcome, local tumour control remain difficult in presence of massive vitreous seeding. thus, the identification of new biomarkers is crucial to design more effective therapeutic approaches. traditional biopsy has long been considered unsafe in rb, due to the risk of extraocular spread. exosomes, nano-sized vesicles containing nucleic acids and proteins, represent an interesting alternative to detect tumour-associated biomarkers. the aim of this study was to determine the protein signature of exosomes derived from rb tumours (rbt) and vitreous seeding (rbvs) primary cell lines. methods: exosomes from rbt (hsjd-rbt , hsjd-rbt , hsjd-rbt , hsjd-rbt ) and rbvs (hsjd-rbvs , hsjd-rbvs , hsjd-rbvs ) cell lines were isolated by high speed ultracentrifugation. vesicles number and size were confirmed by nanosight and scanning electron microscopy. protein content was analysed by bicinchonic-acid assay and high resolution mass spectrometry. results: a total of proteins were identified. among these, and were expressed in exosomes rbt and one rbvs group respectively. gene enrichment analysis of exclusively and differentially expressed proteins and network analysis identified identified in rbvs exosomes upregulated proteins specifically related to invasion and metastasis such as proteins involved in extracellular matrix (ecm) remodelling and interaction, resistance to anoikis and metabolism/catabolism of glucose and aminoacids. summary/conclusion: in conclusion, in this study, we isolated exosomes from rb primary tumour and vitreous seeding cell lines and characterized their content with a proteomic approach. this is the first evidence describing a proteomic exosome signature specifically associated with vitreous seeding in rb. this characterization may represent a starting point for future analyses that allow defining exosomal markers as promising diagnostic and potential prognostic markers in rb as well as therapeutic targets. activation of hepatic stellate cells by extracellular vesicles released by uveal melanoma cells introduction: uveal melanoma (um) is the main intraocular tumour in adults, and is particularly resistant to treatments when disseminated to the liver. our hypothesis is that extracellular vesicles (evs) released by the primary tumour are priming the liver stroma for metastatic cell colonization by activating hepatic stellate cells (hstecs). this study aimed to characterize evs from um cells, and to determine their interactions with liver cells. methods: evs were isolated from cell lines derived from ocular tumours and liver metastases by differential centrifugation. their concentration/diameter range were determined by high-sensitivity flow cytometry. cryo-tem combined with receptor-specific gold labelling was used to reveal the morphology/size of melanomic evs. the presence of melanoma and ev markers was assessed by western blotting. the internalization of fluorescent melanomic evs in hstecs and their subsequent activation were assessed by confocal imaging using alpha-smooth muscle actin (alpha-sma) and phalloidin stainings. ev impact on invasion was measured with a tumour spheroid model embedded in extracellular matrix. melanomic evs were inoculated into the retro-orbital sinus of immunodeficient mice to study their selective organ distribution. results: melanomic evs were positive for annexin- , tetraspanins, as well as some melanoma markers. stellate cells with internalized melanomic evs expressed more alpha-sma, reflecting their activation. adding evs on tumour spheroids increased the invasion process. melanomic evs were localized into different murine organs, but mainly into the liver, as observed by in vivo fluorescent imaging. introduction: exosomes are being tested for their use as therapeutic agents in degenerative and chronic diseases. however, the optimal source of exosomes is currently under investigation. amniotic fluid (af) is a naturally-rich source of exosomes that is easily obtained for use in regenerative medicine. organicell flow™ is a minimally-manipulated, acellular product derived from human af and consist of over cytokines/chemokines as well as exosomes derived from the amniotic membrane and surrounding tissues. we characterized the exosome fraction of our product to elucidate the protein cargo of af exosomes and demonstrate the therapeutic potential as a novel regenerative therapy. methods: the exosome fraction of our product was analysed using nanosight nanoparticle imaging and macsplex exosome surface marker array analysis. exosomes were precipitated using size-exclusion filtration followed by ultracentrifugation from independent products (in triplicate) and subjected to protein lysis and preparation for mass spectrometry analysis using the easy nlc and q exactive instruments. tune (version . ) and xcalibur (version . ) was used to collect data while proteome discoverer (version . ) was used to analyse data. protein expression lists were created by merging the sample replicates together and commonly expressed proteins were determined using vinny . vin diagram analysis. webgestalt tool kit classification system was used to identify top protein function and pathway hits. results: organicell flow™ contain a mean concentration of . x ^ particles/ml (n = ) with a mean mode size of . nm (n = ). surface marker analysis confirms the presence of exosome associated proteins cd , cd , and cd in addition to a high expression of cd (n = ). the completed analysis revealed commonly detected proteins across products. the top molecular functions of identified proteins included protein-binding, ion-binding, and nucleic acid-binding with enzymes, transcription regulators, and transporter proteins representing the most abundant protein groups. pathway enrichment analysis revealed top hits for integrin, pdgf, and p pathways. a deeper dive into the enzyme category of the protein cargo further demonstrates the presence of proteins that promote dna repair such as dna polymerase (beta and lambda), telomerase reverse transcriptase, and brca . summary/conclusion: organicell flow™ characterization demonstrates the therapeutic potential of afderived exosomes. proteomic analysis revealed protein cargo that may regulate various growth factor and cellcycle associated pathways. furthermore, the presence of dna damage response proteins suggests a possible mechanism for induction of cellular repair. generation of car-t and γδt cell-derived exosomes for future cell free immunotherapies γδt cells are a subset of t cells with dual innate and adaptive qualities. this duality provides various advantages over their more studied and used counterpart, αβt cells. in the present study, we sought to compare the immunotherapeutic potential of car-t cell and γδt cell-derived exosomes as novel cell-free based alternatives. methods: cd -targeting car-t cells were obtained following the isolation, expansion and transduction of αβt cells using a lentiviral vector bearing the car construct. γδt cells were isolated and expanded from peripheral blood mononuclear cells (pbmcs) following innate or adaptive stimulation. exosomes from both cell sources were isolated after a -day culture in serum-free media using ultracentrifugation-based methods. exosomes were characterized by nanoparticle tracking analysis (determination of size) and western blot assays (detection of the appropriate surface markers). nalm- (b cell precursor leukaemia) cells were used as target cells for assessment of exosome cytotoxic/ killing function. car-t cell and γδt cell-derived exosomes were incubated at particles/target cell for -hours. total viable cell counts were assessed via imaging-based cytometry (nc- ) utilizing acridine orange and dapi staining. results: exosomes derived from γδt cells activated via innate mechanisms showed significant killing of nalm- as compared to exosomes from non-activated or adaptively activated γδt cells. in comparison, car-t cell-derived exosomes showed minor killing capabilities of the target cells. summary/conclusion: here, we report for the first time that exosomes derived from cd car-t cells and innately activated-γδt cells show/exert inhibitory action on nalm- cells. further studies are currently underway to identify the underlying mechanism(s) responsible. introduction: age-related cognitive dysfunction is associated with increased oxidative stress, low-level chronic neuroinflammation, and waned hippocampal neurogenesis in the brain. from this perspective, biologics capable of modulating oxidative stress and neuroinflammation, and stimulating neural stem cell activity in the brain might be useful as anti-ageing interventions. methods: we investigated the efficacy of intranasal administration of extracellular vesicles (evs) generated from cultures of rat subventricular zone neural stem cells (svz-nscs) in the middle-aged mice to alleviate cognitive and mood dysfunction, increased oxidative stress, neuroinflammation, and neurogenesis decline in old age. mice were treated intranasally with nsc-evs once weekly for three weeks ( billion per administration) starting from . months of age. a month later, the animals were examined for cognitive, memory, and mood function using multiple behavioural tests, and brain tissues were examined for oxidative stress, neuroinflammation, and neurogenesis. results: object-based tests revealed that aged animals receiving vehicle displayed cognitive impairments for discerning minor changes in the environment as well as for distinguishing similar but not identical experiences. these animals also exhibited spatial memory dysfunction and anhedonia. in contrast, aged animals receiving nsc-evs showed improved cognitive and mood function. biochemical analyses of brain tissues revealed that nsc-ev treatment normalized elevated concentrations of oxidative stress markers malondialdehyde and protein carbonyls and the proinflammatory cytokine interleukin- beta. moreover, nsc-ev treatment stimulated increased production of antiinflammatory protein interleukin- and the antioxidant superoxide dismutase. immunohistochemical analysis revealed modulation of neuroinflammation typified by reduced activity of reactive astrocytes and activated microglia and improved hippocampal neurogenesis. summary/conclusion: the results suggest that the intranasal administration of nsc-evs is a promising approach for maintaining better cognitive and mood function in ageing through modulation of oxidative stress, neuroinflammation, and neurogenesis. funding: supported by a grant from the national institute of neurological disorders and stroke ( r ns - to a.k.s.) chemically modified myocytes-derived evs for the treatment of cardiac fibrosis. marta prieto-vila a , asao muranaka a and takahiro ochiya b a tokyo medical university, tokyo, japan; b tokyo medical university, shinjuku-ku, japan introduction: myocardial fibrosis is a disorder that may occur after cardiac injure due to a malfunction of the cardiac remodelling. fibroblasts resident in myocardium are erroneously activated causing an excessive accumulation of extracellular matrix, which decreases cardiac function and eventually, leads to death. it is known that cardiomyocytes communicate with the surrounding cells such as fibroblast and endothelial cells by extracellular vesicles (evs). the loss of this communication is thought to play a central role in cardiac fibrosis. therefore, cardiomyocytes-derived evs may be a promising a cell-free system for the treatment of fibrosis inhibition. methods: a novel culture medium was stablished to improve the expansion of primary cardiac myocytes. this was tested using two commercially available primary myocytes cell lines. evs were collected by serial ultracentrifuges, and their effect on fibrosis was tested. for that, prior to any treatment, and to mimic fibrosis, primary cardiac fibroblast were activated overnight with tgfβ. results: by the use of a defined conjunct of chemicals, mature cardiomyocytes culture was highly improved to ensure a high collection of evs. terminal differentiation markers, as well as senesce apparition was delayed in comparison to predetermined culture medium. interestingly, those primary cells secreted a rather large amount of evs, which expressed common evs membrane marker. tgfβ-treated cardiac fibroblasts were co-cultured with myocytes showing a decrease of fibroblast activation markers both at mrna and protein levels. similar results were found when activated fibroblast were treated with evs. summary/conclusion: our findings indicate that the use of evs derived from chemically modified myocytes is a promising treatment for ischaemic myocardial fibrosis. however, further molecular experiments have to be done to identify the molecules within evs responsible for the inactivation of fibroblast. evaluation of osteoinductive and anti-inflammatory properties of spinederived exosomes renaud sicard a , tania del rivero b , jonathan messer c , shabnam namin c and timothy ganey c a vivex, biologics, inc., miami, usa; b vivex, biologics, inc., miami, usa; c vivex, biologics, inc., miami, usa introduction: over the last decades, mesenchymal stem cell-derived exosomes have been shown to play a crucial role in a myriad of cell function such as extracellular matrix synthesis, proliferation, differentiation or cell migration. biological sources of exosome (heterogeneous or homogeneous cell population, serum, urine etc.) have a direct influence on the content of their cargo and their therapeutic application and potential. in this study, we evaluated exosomes excreted from cadaveric spine-derived cells. we hypothesized that exosomes derived from a bone source such as the spine, will drive the osteogenic differentiation of progenitor cells. we also investigated their effects on inflammation in nucleus pulposus cells using an in-vitro assay. methods: after their isolation and characterization, exosomes derived from cadaveric human spines were assayed for osteoinductive properties. a c c myoblast cell line was treated with different concentrations of exosomes and expression of alkaline phosphatase was measured after days incubation. treatment with bmp- was used as positive control. anti-inflammatory properties were assessed by incubating tnf-treated nucleus pulposus cells with exosomes for days. qpcr analysis of mrna expression of inflammatory cytokines (il- , il -beta, il- ) metalloproteinases (mmp and adamts ), and apoptotic genes (bax, bcl ) was used to determine the effects of exosomes on inflammation. results: spine-derived exosomes positively expressed the exosome flow cytometry markers tested (cd , cd and cd ). the mean number of exosomes per microgram of protein was . ± . x indicating a relatively high purity. osteoinductive (oi) testing was performed using different concentrations of exosomes. the oi index of treatment of c c cells with bmp- , x , x , x , × or × exosomes alone was . , . , . , . , . and . respectively. anti-inflammatory properties of exosome are currently being assessed and will be presented at the time of the poster presentation. summary/conclusion: administering exosomes alone or in combination with an exogenous scaffold has the potential to repair injured tissue and to restore bone function. the clinical significance of this application is aimed to promote the patients' bone healing process and provide a cell-free therapeutic platform that is safe and effective. administration of human mesenchymal stem cell derived extracellular vesicles modulates the abnormal plasticity of newly born neurons and neuroinflammation in a rat model of status epilepticus maheedhar kodali a , daniel gitai b , dong ki kim a , mariam atobiloye a , bing shuai c , sahithi attaluri c , raghavendra upadhya c , leelavathi n madhu a , olagide w. castro a , darwin j. prockop a and ashok k. shetty c decline in the percentage of newly born neurons displaying basal dendrites. besides, ev treated animals displayed higher percentages of resting microglia (ramified microglia), reduced percentages of activated microglia (microglia expressing iba- and cd ), in comparison to animals receiving vehicle after se. interestingly, diminished abnormal plasticity of newly born neurons was accompanied by the preservation of interneurons positive for reelin; a protein believed to guide newly born neurons to their correct locations. summary/conclusion: the results suggest that even a low dose in administration of msc-derived evs after se can limit neurons loss, dampen the abnormal plasticity of newly born neurons, and modulate the activation of microglia. introduction: autism spectrum disorders (asd) are neurodevelopmental disorders characterized by three core symptoms that include social interaction deficits, cognitive inflexibility, and communication disorders. they have been steadily increasing in children over the past several years, with no effective treatment. two percent of all asd patients are suffering from a disorder caused by a mutation in the shank gene. shank is an important synaptic protein, disruption of this gene directly leads to cognitive and motor impairments. during the recent decade, exosomes that derived from mesenchymal stem cells (msc-exo) have been spotlighted as a promising therapeutic target for various clinical indications, including neurological disorders. here we test three different autistic mice models. btbr as a multifactorial mice model of autism and two different shank mutated mice. the first is a complete deletion of exon ( q . ) and the second is a specific insertion mutation of guanine to position in the gene (insg ) that leads to stop codon. methods: exosomes were isolated using differential centrifugation protocol and characterized using the misev guideline recommendations. each animal received an intranasal administration of ul containing exosomes/µl. for intravenous administration, the same number of exosomes, were used, injected in µl. results: all three animal models showed significant improvement in their autistic behavioural phenotypes following intranasal administration. the improvement seems to be dose-dependent and was better achieved via intranasal vs intravenous administration. biodistribution of msc-exo showed accumulation in the brain within hours, yet the reduction of the signal was observed in the kidneys, heart and lungs. summary/conclusion: our data suggest that exosomes derived from adipose msc, carry a therapeutic potential in asd, via non-invasive intranasal administration in three different mice models. these data further emphasize our potential therapeutic strategy to reduce symptoms of autism in clinical trials. funding: stem cell medicine ltd. israel. equine tendon injury treatment by evs: an in vitro study introduction: current treatment options for tendinopathies (chronic, painful tendon disorders), are not able to restore the functional properties of native tendons. hence, new treatment options are sought. the efficacy of mesenchymal stem cells (mscs) therapies, which combined with a rehabilitation programme including controlled exercise is the current gold standard in equine tendon treatment, has been shown to be largely due to the cells´paracrine activity. the aim of this study was therefore to evaluate the effect of bone marrow msc derived autologous and allogeneic conditioned medium (cm, full secretome) and their extracellular vesicles (evs) on "tendon healing" in vitro. methods: to compare the "therapeutic" effect of msc derived evs and cm, a standardized scratch assay (wound healing assay) was performed. cm from equine tenocytes, ev depleted medium and medium with or without fcs served as controls. tendons and bone marrow aspirates were obtained from three horses ( , and years) which were euthanized for reasons unrelated to this study. mscs were isolated by ficoll density gradient centrifugation and tenocytes were obtained by migration from tendon explants. for cm and ev production, cells were cultured in ev depleted medium. evs were harvested by a stepwise ultracentrifugation approach and characterized by nanoparticle tracking analysis (nta), western blot (cd , cd ) and transmission-electron microscopy (tem). results: western blot, nta and tem confirmed successful isolation of evs from equine mscs. the strongest positive effect on wound healing (fastest gap closure) was achieved by msc-cm (p < . ). the gap closure achieved with msc-evs was slower than with msc-cm (p < . ) but faster than with cm of tenocytes (p < . ). donor specific differences in wound healing capability were shown for both autologous and allogeneic application. summary/conclusion: treatment with msc-cm resulted in significantly faster wound healing of adult tenocytes in vitro than msc-evs or tenocyte-cm. mscs donor age shows a significant effect on gap closure following autologous but not allogeneic administration. ev-enriched secretome fraction from gmp-compatible, scalable, human ipsc-derived cardiac progenitors improve heart function in chronic heart failure mice introduction: we have shown that research-use-only grade (res) human ipsc-derived cardiac progenitors (cpcres) can produce a secretome whose small-evenriched fraction (svf) can treat chronic heart failure (chf) in mice. gmp-compatible, scalable processes for a cpc-derived svf suitable for human therapeutic use is needed. methods: ipsc-derived cpc were produced and cultured using gmp-compatible, scalable processes (cpctx). media without cells were "cultured" in parallel for "virgin media" controls (mv). cpcres were cultured as previously described. as a proof of concept, svfs were isolated from conditioned media by ultracentrifugation: cpctx-ev, cpcres-ev and mv. particle size distributions/concentrations (nanoparticle tracking analysis), protein levels (bsa), and the presence of cd- (elisa) were determined. in vitro activity was assessed by huvec scratch wound healing assay, and by rat and human cardiomyocyte (cm) survival assays. c bl/ mice in chf received echoguided myocardial injection of pbs vehicle control ( ul, n = ), cpctx-ev ( ul, n = ), or cpcres-ev ( ul, n = ). change in cardiac function was assessed by echocardiography. results: cpctx-ev particle sizes were polydisperse (mode~ nm) at a concentration of~ . e particles/ml (~ , particles/cell) and~ . mu cd /ug protein. cpctx-ev increased wound healing, human cm survival, and rat cm survival in vitro by . x, . x, and x, respectively over mv controls. in chf mice, significantly less cpctx-ev mice, and less cpcres-ev mice had severely progressive heart failure (left ventricular end systolic volume, lvesv, increased > %) than pbs control mice (pbs vs cpctx-ev, p < . ; pbs vs cpcres-ev, p < . ), and the average ejection fraction of the pbs group deteriorated . x more than the cpctx-ev group (− % vs − . %, respectively; ns). summary/conclusion: we have a process for cpc differentiation and production of conditioned media suitable for use in human clinical trials from which can be made an svf with the potential to treat chf, possibly through re-vascularization or preservation of cm viability. introduction: exosomes are nanoscale vesicles that mediate cell-to-cell communication via exchanging molecular cargo. mesenchymal stem cell (mscs) modification towards an osteogenic path can occur by uptake of exosomes from other cells. it is less clear whether vesicle placement in the absence of cells will facilitate site-specific delivery through acellular transfer of osteogenic activity. an electrospun fleece was combined with bone marrow-derived exosomes in the absence of cells to evaluate osteoinductive potential that might be thermo-stable and be used in a biologically neutral collagen carrier. comparisons were made of standard laboratory assay of osteoinductivity (oi), and in vivo expression in a mouse calvarial defect model. methods: electrospun type-i collagen was prepared with and without hydroxyapatite (ha) (spinplant gmbh, leipzig) as a foundation base for application of the bone marrow-derived exosomes. individual discs of the collagen enhanced scaffolds ( -mm) were prepared and placed in a mouse calvarial skull defect. animals were followed for and weeks. exosomes were isolated from qualified cadaveric human spines by differential ultracentrifugation. microscopic observation, quantitative assessment of oi with an alkaline phosphatase assay, and flow cytometry were used to evaluate the composition, the hybrid nature of the addition to the nano-collagen fibres. a fluorescent protein reporter transgenic mouse model expressing osteocalcin, type-i collagen, phex, and sp (osterix) was evaluated at and weeks to determine bone formation across the defect. results: alp activity on the scaffold with ha demonstrated an approximate tenfold increase to that of the collagen scaffold alone. while a dose-dependent effect, with higher doses of exosomes resulting in a greater amount of alkaline phosphatase expression, expression that exceeded that of the ng bmp- control. dose escalation from . , , and e resulted in similar increases in expression that was statistically greater with the combination of the fleece with the exosome component. bone formation in the mouse calvaium did not demonstrate gap closure at or at weeks, but did demonstrate enhanced osteoclastivity and robust bone remodelling at the margins of the defect. summary/conclusion: bone marrow-derived exosomes dried into an electrospun fibrillar collagen demonstrated in vitro osteoinductive potential that might provide site-specific placement that could enhance biologic potential. with the capacity for ambient temperature storage, the provision of site-specific placement becomes a technical consideration. placement of the human tissue derived exosomes in a transgenic mouse calvarial defect model did not demonstrate bridging bone across the defect. exosomes loaded with pten-interfering rna enables functional recovery in rats after complete spinal cord transection daniel offen a , nisim perets a , shaowei guo b , oshra betzer c , rachela popovtzer c and shulamit levenberg b a tel aviv university, tel aviv, israel; b technion, haifa, israel, haifa, israel; c bar ilan university, israel, ramt gan, israel introduction: complete spinal cord transection is a debilitating disease that usually leads to permanent functional impairments, with various complications and limited spontaneous recovery. the current investigation of molecular mechanisms controlling axon regeneration, (e.g., signalling networks and environmental cues), led to new strategies to enhance axonal regeneration. we have previously shown that intranasal administration of mesenchymal stem cells derived exosomes (msc-exo), cross the blood-brain barrier and significantly ameliorate motor and behavioural phenotype in several animal models of neurotrauma and neuropsychiatric disorders. methods: msc-exo were isolated from human bone marrow and were loaded with phosphatase and tensin homolog small interfering rna (pten-sirna). the exosomes were given intranasally to rats two hours after complete spinal cord transaction. eight weeks later we followed the motor function and histology and electrophysiology study was performed in order to reveal the connectivity and the biochemical changes in the treated rats. results: we demonstrate that intranasal (in) administrations of msc-derived exosomes could penetrate the blood-brain barrier, home selectively to spinal cord lesion via chemotaxis, and integrated in neurons within the lesion. furthermore, in rats with complete spinal cord transection, msc-exo loaded with pten-sirna silenced pten protein expression in the lesion and promoted robust axonal regeneration and angiogenesis, companied with decreased astrogliosis and microgliosis. moreover, the intranasal treatment partially restored electrophysiological and structural integrity, and most importantly, enabled the remarkable functional recovery and significant improvement in their movements. summary/conclusion: this rapid, non-invasive, approach, using cell-free nano-swimmers carrying molecules to target pathophysiological mechanisms suggest novel strategy for clinical translation to spinal cord injury and beyond. a novel umbilical cord derived wharton's jelly formulation for regenerative medicine applications introduction: musculoskeletal injuries have traditionally been treated with activity-modification, physical therapy, pharmacological agents and surgical procedures. these modalities have limitations, as well as potential side-effects. over the last decade, there has been an increased interest in the use of biologics for regenerative medicine applications (rma), including umbilical cord (uc) derived wharton's jelly (wj). despite this increase, there is insufficient literature assessing the amount of growth factors, cytokines, hyaluronic acid (ha) and extracellular vesicles (ev) including exosomes in these products. the purpose of this study was to develop a novel wj formulation and evaluate the presence of growth factors, cytokines, ha and ev including exosomes. methods: wj was isolated from human-uc obtained from consenting c-section donors and formulated into an injectable form. randomly selected samples from different batches were analysed for sterility testing and quantified for presence of growth factors, cytokines, ha and particles in ev size range. the results showed all samples passed the sterility test. growth factors including igfbp , , , and , tgfα, pdgf-aa were detected. expression of several immunomodulatory cytokines, rantes, il- r, il- , were also detected. expression of pro-inflammatory cytokines mcsfr, mip- a; anti-inflammatory cytokines tnf-ri, tnf-rii, il- ra; and homoeostatic cytokines timp- and timp- were observed. cytokines associated with wound-healing, icam- , g-csf, gdf- , and regenerative properties, gh were also expressed. high concentrations of ha were observed. particles in the ev size range ( - nm) were detected and were enclosed by the membrane, indicative of true ev. summary/conclusion: our results confirmed the presence of numerous growth factors, cytokines, ha and ev in the wj formulation. more studies are underway to confirm the presence of exosomes in detected ev using exosome-specific markers. we believe the presence of multiple factors within one wj formulation may play a role in reducing inflammation, pain and augment healing of musculoskeletal injuries. this offers a potential expanded use for rma. funding: this study was funded by biointegrate llc, new york, ny, usa. collagen sponge loaded with mesenchymal stem cell-derived small extracellular vesicles promote robust bone regeneration shang jiunn chuah a , chee weng yong a , jacob ren jie chew a , ruenn chai lai b , yi ann cheow a , raymond chung wen wong a , asher ah tong lim a , sai kiang lim c and wei seong toh d introduction: mesenchymal stem cell (msc) therapy has demonstrated effective bone regeneration in clinical studies. however, the therapeutic efficacy of mscs have been attributed to the secretion of extracellular vesicles (evs), particularly - nm small evs (sevs). here, we investigate the efficacy of msc-sevs loaded in collagen sponge in the regeneration of critical-sized calvarial defects in immunocompetent rats. methods: sevs were isolated from conditioned medium of human mscs and stored at − c. calvarial defects of -mm diameter were surgically created on thirty-two -week-old male sprague-dawley rats. these rats were then randomly assigned to groups (n = rats/group): defects treated with collagen sponge containing μg of sevs in μl saline (cs/sevs) and defects treated with control collagen sponge containing an equivalent volume of saline (cs/control). at and -week post-surgery, the calvarial bone samples was harvested for analyses by micro-computed tomography (micro-ct), histology, immunohistochemistry and histomorphometry. results: at -week post-surgery, micro-ct analysis showed little bone formation at the defect site in both cs/sevs and cs/control groups. no statistical differences were observed in micro-ct and histology scores in both groups. interestingly, cs/sevs group showed significantly higher osteocalcin (ocn)+ area of . ± . % than that of cs/control group ( . ± . %; p = . ). cd + microvessels at sizes ≤ µm and > µm in cs/sevs group ( . ± . and . ± . microvessels/hpf) were also significantly higher than that of cs/control ( . ± . and . ± . microvessels/hpf; p = . and p = . respectively). by weeks, cs/sevs group displayed enhanced new bone formation that completely bridged the calvaria defect. in contrast, rats in cs/control showed limited bone formation. consequently, cs/ sevs group displayed a micro-ct score of . ± . which was significantly better than that of cs/control group ( . ± . ; p = . ). cs/sevs group also exhibited >twofold increase in bone volume, and improved bone quality with higher trabecular thickness and number, and smaller separation (p < . ), compared to cs/control group. consistently, cs/sevs group displayed a significantly better histology score of . ± . than that of cs/control ( . ± . ; p = . ). moreover, cs/sevs group showed significantly higher ocn+ area of . ± . % than that of cs/control group ( . ± . %; p = . ). summary/conclusion: this study demonstrates that single-stage implantation of collagen sponge loaded with ready-to-use msc sevs can promote robust bone regeneration in a rat calvarial defect model. funding: national university of singapore, r , national medical research council singapore, r . immunomodulatory potential of extracellular vesicles derived from mesenchymal stromal cells introduction: extracellular vesicles (evs) derived from mesenchymal stem/stromal cells (mscs) are promising new agents in regenerative medicine and immunotherapy. considering that independent msc-ev preparations might differ in their therapeutic function, we have set up a functional assay allowing testing for the potential immunomodulatory properties of independent msc-ev preparations. methods: human peripheral blood-derived mononuclear cells (pbmcs) were pooled from up to different healthy donors warranting high allogeneic cross-reactivity, even following an optimized freezing and thawing procedure. after thawing, mixed pbmcs were cultured for days in the absence or presence of msc-evs. thereafter, cell morphologies were documented, supernatants were harvested for cytokines quantification and cells were phenotypically characterized by flow cytometry. by analysing the expression of a collection of different lineage and activation markers, we selected a panel of antigens apparently being regulated by msc-ev preparations considered to be therapeutically active. results: we observed that in the presence of active msc-ev preparations more cd + (monocytes) are recovered from the mlr assay than in corresponding control samples. focusing on t cells, we learned that active msc-ev preparations reduced the content of cd and cd t cells expressing activation markers like cd and cd . summary/conclusion: the mlr assay allows elaborated functional testing of immunomodulatory activities of given msc-ev preparations. currently, we are comparing the immune modulatory capabilities of evs derived from distinct sources and optimize the marker panel to distinguish discrete immune cell subtypes such as different cd cell types, i.e. th , th , th and tregs. extracellular vesicles in platelet-rich plasma: dependency on sample processing zala jan a , saba battelino b , darja božič c , matej hočevar d , ales iglič e , marko jeran c , manca pajnič a , ljubiša pađen a , domen vozel f and veronika kralj-iglič a introduction: platelet-rich plasma (prp) proved effective in regenerative medicine. numerous protocols for its preparation and application are available in the published literature. prp possesses important immune, haemostasis and regenerative factors, however, the mechanisms of their action are yet poorly understood. extracellular vesicles (evs) could be one of the important factors that would contribute to the beneficial effects of preparations. this study was performed as a part of a registered randomised controlled clinical trial (nr: nct ). prp was used to treat chronic middle ear inflammations. here we present the results of prp analyses from blood samples of volunteers with no record of disease. methods: plasma obtained from ml of blood was depleted of erythrocytes and enriched with other particles by repetitive centrifugation of samples. flow cytometry (fcm) was employed to monitor particle contents (cells and smaller particles) throughout the sample processing. the platelet gate was divided into two parts: intact platelets and smaller particles. identity and morphology of particles in the preparations were examined by scanning electron microscopy (sem). standard laboratory tests of blood were performed. results: sem images revealed the presence of heterogeneous population of particles in the preparation of prp, most of which were activated and partially fragmented platelets. the population of smaller particles measured with fcm, was identified as evs. the erythrocyte sedimentation rate was statistically significantly correlated to the volume of plasma obtained in the initial centrifugation step (r = , , p < , ) and to the concentration of evs (r = , ; p < , ). time from sample collection to the preparation of prp was negatively correlated with the concentration of platelets in prp and positively with the concentration of evs (r = , , p < , ). platelet concentration in preparation samples was found to depend on the concentration of platelets in the blood and parameters of sample processing connected with larger centrifugal and shear forces on the samples during centrifugation. these include: sample volume, the size and shape of the centrifuge tube and the distance of the sample from the rotor axis. summary/conclusion: evs are gradually forming upon activation and degradation of cells in the sample throughout the sample processing. optimal processing may importantly contribute to the healing properties of preparation. funding: authors acknowledge support from the european union's horizon research and innovation program under grant agreement no. (ves us project) and slovenian research agency (arrs, grants p - , p - , j - ). satellite cell-derived extracellular vesicles as a therapeutic for mitochondrial dysfunction in duchenne muscular dystrophy duchenne muscular dystrophy (dmd). sc-derived extracellular vesicles (sc-evs) may unlock the therapeutic potential of scs by overcoming these limitations. to investigate their therapeutic potential, we assessed the ability of sc-evs to reverse mitochondrial dysfunction, a key pathological feature of dmd, in oxidatively-damaged c c and primary dmd myotubes. methods: scs from c mice were isolated and cultured. evs were isolated from the supernatant of scs via polyethylene glycol precipitation and characterized using nanoparticle tracking analysis. the ability of sc-evs to deliver protein cargo to c c myotubes, and the localization of the cargo once delivered, were analysed using fluorescence microscopy. to examine sc-ev potential to restore the function of damaged mitochondria, c c myotubes were treated with µm h o for h followed by treatment with . x sc-evs for h. separately, cultured dystrophic myotubes were treated with . × evs every h for h. in both sets of experiments, maximal oxygen consumption rate (max ocr) was measured via seahorse xf cell mito stress test. where appropriate, a t-test was performed to test for statistical significance (p < . ). results: based on estimated cell number and ev quantification, each sc released approximately . × ± . x evs/day. evs delivered protein cargo into myotubes within h. fluorescent labelling of intracellular mitochondria showed co-localization of delivered protein and mitochondria. incubation of myotubes with h o resulted in a % decline in max ocr relative to untreated myotubes. subsequent treatment with sc-evs resulted in a % increase in max ocr. treatment of undamaged myotubes with sc-evs had no effect on max ocr. primary dmd myotubes treated with sc-evs showed a % increase in max ocr relative to untreated dmd myotubes. summary/conclusion: sc-evs rapidly deliver proteins into myotubes, much of which co-localizes with mitochondria, and reverses mitochondria dysfunction in oxidatively-damaged and dystrophic myotubes. introduction: flow cytometry has been used extensively for analysis of ev particles stained with fluorescent antibodies directed to the known cell surface markers. quantitation of the surface markers in terms of the number of molecules or the number of antibodies bound per specific marker has remained one of the largest challenges in the ev research field. changes in instrument setup as well as changes in fluorescent antibodies from different vendors, all impact the relative mfi values for the same ev sample. in this work we report a standardization method of quantitating extra-cellular vesicle surface markers with mesf liposomes. methods: liposomes labelled with fitc fluorescent dye were prepared with a bd proprietary technology. dynamic light scattering analysis was used for size determination of the liposomes. bd facsaria™ fusion system, modified with a small particle side scatter module (sp ssc), was used for analysis of the labelled liposomes by flow cytometry. results: we created a set of nm fitc-modified liposomes of various fluorescent intensities with a known number of fitc molecules incorporated in each liposome intensity. the mfi values of each liposome population (intensity) had a linear relationship to the amount of fitc used for labelling the liposome nanoparticles, suggesting that no self-quenching of fitc fluorescence had occurred. the number for the fitc fluorophores for each liposome intensity was expressed in the units of molecules of equivalents soluble fluorochrome (mesf). a plot of mesf vs. the fluorescent intensity of the liposomes (mfi values) obtained from flow cytometry analysis provided a calibration curve, from which the fluorescent intensity (mfi value) of a stained ev sample can be converted to the number of fluorophores bound (mesf value) to the surface of the ev particles. summary/conclusion: by this approach, the mfi values of stained ev particles are converted to standardized mesf values that are independent of instrument variation, resulting in further improvement of inter-laboratory standardization. furthermore, utilization of liposomes with similar size and refractive index to ev particles simplifies the data evaluation and improves the accuracy of ev surface marker quantitation by flow cytometry. currently, other fluorescent dyes are being explored to expand the utility of mesf liposomes with other fluorescent colours. measuring cholesterol as a high-throughput method for quantifying extracellular vesicles introduction: the extracellular vesicle (ev) field currently lacks a high-throughput method for accurately quantifying evs in solution. ev quantification has traditionally relied on nanoparticle tracking analysis (nta), which is time intensive and indiscriminately counts non-ev particles, such as membrane fragments and protein aggregates. we have rigorously assessed two commercially available methods for measuring cholesterol, a major lipid component of the ev lipid bilayer, and evaluated the utility of these assays to quantify evs in minimally processed samples. methods: the amplex® red cholesterol assay and cedex bio ht were used to quantify cholesterol in ev samples via enzymatic oxidation, with dynamic ranges of - , ng/ml and - µl/ml, respectively. samples throughout various stages of purification were analysed, from clarified cell culture medium to highly purified evs separated on an iodixanol gradient. we evaluated several pre-processing methods, to remove non-ev cholesterol content prior to analysis. results: the amplex® and cedex bio ht assays were found to perform comparably for quantifying cholesterol in purified evs (r = . ). importantly, cholesterol quantification on purified ev samples, ranging from e to e particles/ml, correlated well with nta measurements (r = . ). both µm filtration or an additional , rcf centrifugation step following clarification removed cholesterol associated with cellular debris or other non-ev sources, allowing for accurate quantification of conditioned medium samples or ultracentrifugation pellets (ucp) instead of needing to rigorously purify samples with an iodixanol density gradient. summary/conclusion: cholesterol quantitation can be used to accurately estimate ev concentration, allowing for rapid characterization of samples from clarified cell culture supernatant to highly purified evs. this highthroughput analytical capability may enable more comprehensive assessment of methods to boost ev yield through mass screening of cell culture conditions. optimization of nanoparticle tracking analysis of extracellular vesicles isolated from plasma and bronchopulmonary lavage fluid of patients with non-small cell lung cancer introduction: recent studies show that tumourderived extracellular vesicles (evs) greatly influence the tumour microenvironment and impact the therapy. in non-small cell lung cancer (nsclc), bronchopulmonary lavage fluid (balf) appears to be a good source of tumour-derived evs, providing more accurate information about the tumour microenvironment than evs from plasma. so far there is a lack of accurate and standardized methods for ev quantification. fluorescence nanoparticle tracking analysis (fl-nta) is an emerging method of ev-analysis, allowing discrimination of evs and exosomes from impurities. here we perform an optimization of the fl-nta method to compare evs from plasma and balf of nsclc patients and healthy controls (nc). methods: evs were isolated using homemade sizeexclusion chromatography (sec) columns (plasma) and ultrafiltration or differential ultracentrifugation (balf). nta was performed using zetaview pmx (particle metrix) after ev-staining with membrane dyes or fluorescence-labelled antibodies against typical ev-marker (cd , cd , cd ). results: nta scatter measurements showed a higher total particle concentration in plasma than in balf. however, membrane-specific staining showed a much greater purity of ev-preparations from balf, where nearly % of the particles detected in scatter mode showed positive membrane-staining. in contrast, only around - % of particles in the plasma ev-preparations were positive for the membrane dyes. fluorescence-staining for ev surface marker requires further optimization to obtain reproducible results. summary/conclusion: classical nta using only the scatter mode fails to discriminate between evs, lipoproteins and protein aggregates. for ev-analysis from complex biofluids like plasma, fla-nta and staining for specific ev marker is necessary to receive reliable data. balf seems to be a better source of tumourderived evs than plasma, since the obtained ev-preparations show a higher purity. improving conditions for fluorescence-staining and nta measurement of evs from plasma and balf of nsclc patients will provide an additional method for quantifying and phenotyping of evs. introduction: the exoviewer platform currently enables the user to capture extracellular vesicles (ev) by means of surface antigen-specific antibodies (e.g. targeting tetraspanins), making possible the enumeration of individual particles using single-particle interferometric reflectance imaging sensor (sp-iris, interferometric) imaging as well as fluorescence. currently, through interferometric imaging particles smaller than nm cannot be detected, while fluorescently stained ev smaller than nm can be well resolved. further, it is conceivable that small ev contain antigen numbers in the single digits, making antigen-specific immunostaining a challenge. to further characterize ev populations of different sizes and surface marker composition, it would be highly advantageous to target the vesicular nature of the detected particles linked to a fluorescence readout. methods: the goal of this project is to detect ev with a probe that is ubiquitously distributed across the surface (or lumen) of the vesicle. small ( - nm) ev present fairly distinctive lipid membrane features in the extracellular environment, turning the ev membrane into a "universal" marker, and as such may serve as an alternative marker that is complementary to canonical ev surface markers. results: here we present data on successfully staining ev with the membrane dye di- -anepps (di- ) and the luminal dye calcein-am. we demonstrate that ev from different sources can be efficiently stained with either dye, allowing the quantitative characterization of ev in an unbiased manner using exoviewer's fluorescence mode. while both dyes certainly have their own unique strengths, they exhibit the wanted linear correlation of ev staining versus concentration. further, both dyes are compatible with subsequent immunostaining applications, allowing the user to target specific surface or luminal markers (di- ). summary/conclusion: while a large-panel screening featuring other powerful dyes is continuously ongoing, the current data support the notion of providing the experimenter with a reference for total particle count and at the same time fully exploring the larger dynamic range of the fluorescence mode. moreover, the universal probe will enable the user to correlate intensity and particle size measurements, thereby significantly improving the exoviewer platform and its applications. membrane labelling is essential for the identification and quantification of extracellular vesicles via facs introduction: extracellular vesicle (ev) research is challenged by the lack of standard protocols to identify and distinguish between exosomes and ectosomes being released via exocytosis or plasma membrane shedding, respectively. analysis of small ev populations requires high-resolution technology and can be further improved using fluorescent labels such as carboxyfluorescein diacetate succinimidyl ester (cfse). at the inner leaflet of the plasma membrane, cfse is cleaved enzymatically resulting in covalent binding of the dye. in this study we optimized the conditions for membrane labelling of evs and their subsequent detection by flow cytometry to obtain a maximum yield of intact evs. methods: using sequential centrifugation, we separated ev subpopulations from supernatants of colo pancreas carcinoma cells based on size and mass. after , x g centrifugation, we reconstituted evs from the pellet. we used cfse for ev detection and analysed the expression of tetraspanins by facs to confirm the lipid bilayer structure. furthermore, we determined size distribution of evs by nanoparticle tracking analysis (nta) and electron microscopy. detecting evs as cfse+ events, we quantified our samples and investigated the impact of threshold adjustment on ev quantification. results: after high speed centrifugation of cell free supernatants, we identified cfse+ events as evs, which appeared as round structures under the microscope, and ranged from to nm in size. interestingly, tetraspanin markers cd and cd were detectable only on a subpopulation of purified evs, suggesting heterogeneity of our preparations. for sufficient labelling of evs, minimal temperature variations and short incubation times correlated with ev stability. of note, threshold adjustment significantly improved the sensitivity of the flow cytometer for the detection of labelled evs and hence, is central for data comparability. summary/conclusion: protocol standardization is of major importance for the use of evs as diagnostic markers in liquid biopsies. funding: this project has been supported in part by annelise-asmussen foundation, luebeck (grant ), leo pharma germany (grant ). surface plasmon field-enhanced fluorescence spectroscopy (spfs) system for quantitative and qualitative extracellular vesicles total evaluation without any sample pretreatment introduction: the function of extracellular vesicle (ev) is interested in the immunology and oncology fields as a key transmitter for cellular communication. however, the conventional ev evaluation methods are required complicated evs preconcentration from the sample, its leads ev analysis uncertainty. in this study, we applied the spfs highly sensitive automated system for quantitative and qualitative ev evaluation without any sample pre-concentration and preparation step. methods: spfs automated system and plastic disposable sensor had been developed by konica minolta corporation in house. anti-membrane protein (cd , cd , cd ) antibody was chemically bonded on hydrophilic polymer which was immobilized through the gold thin film on the spfs sensor. the concentration of standard ev materials was evaluated by the qnano system before using. ev detection without preconcentrating was achieved by sandwich immunoassay step in microchannel round-trip flow reaction (tat min) with the spfs system, and elisa was adapted as a conventional standard method. after spfs highly sensitive fluorescent measurements step, extracted and detected ev were effectively recovered by using the recovery buffer reaction. results: the ev sensitivity performance between spfs and elisa clearly showed a significant difference, and the lod of spfs ( . particles/μl) method was estimated times superior to the lod of conventional elisa ( , particles/μl). the spfs calibration curve showed a wide dynamic range at least over logs as an additional specificity. spfs method also showed fine results in the dilution linearity test with high reproducibility under the serum/plasma sample condition. the data for recovery test of ev expected us that highly accurate measurement can be guaranteed under the condition of dilution about times or less even in the whole blood sample. after the spfs measurement, extracted ev on the spfs sensor chip could be effectively recovered and could be analysed nucleic acid which contains micro rna. summary/conclusion: spfs system might have great potential for quantitative and qualitative ev evaluation. our strategy with spfs system for ev proteomic and genomic profiling will be possible for applying to ev quality control as well as a novel biomarker development. identification of a novel compound that inhibits small ev secretion and tumour progression by a sensitive elisa screening. yunfei ma a , takeshi yoshida a , duc tuan nguyen a , kazutaka matoba b , katsuhiko kida b , taito nishino b and rikinari hanayama c a kanazawa university, kanazawa, japan; b nissan chemical corporation, tokyo, japan; c wpi nano life science institute, kanazawa university, kanazawa, japan introduction: small evs from tumour cells are known to promote tumour progression, therefore, it is expected to develop drugs that regulate small ev secretion, which can be used in clinical applications. methods: to identify such regulators, we first developed a sensitive elisa system for the quantification of small ev secretion using a high-affinity ev binding protein tim . by using this elisa system, we screened for small compounds that promote or inhibit small ev secretion using a drug-repositioning compound library (about , compounds). results: as a result, we identified eight promoters and two inhibitors, including compound a, which significantly reduced small ev secretion from various cell types without affecting cell growth. we further investigated the effects of compound a on a mouse model of osteosarcoma and found that compound a suppressed tumour progression efficiently. summary/conclusion: these data suggest that compound a would be useful not only for the characterization of small ev function but also for the clinical therapy against tumour progression, by inhibiting small ev secretion. introduction: for many years it was believed that several proteins such as cd , cd and flotillin- were unique for exosomes, however recent studies have shown that several of these markers also can be present in other subpopulations of evs (kowal et al pnas ) . furthermore, few markers have been identified as uniquely present in microvesicles. the aim of this study was to in depth compare the proteome of microvesicles and exosomes. methods: mda-mb- -luc-d h , -d h ln and -bmd a were cultured in ev-depleted media. microvesicles ( , x g, min) and exosomes ( , x g . h) were isolated using a combination of differential ultracentrifugation and a density cushion (~ . g/ml). purity and yield of evs were determined by nanoparticle tracking analysis (nta), western blot, and electron microscopy (em). quantitative mass spectrometry (tmt-lc-ms/ms) was used to identify differently enriched proteins in microvesicles and exosomes (n = x cell lines). results: in total proteins were quantified, with being quantified in all samples. in total and proteins were significantly upregulated in exosomes and microvesicles, respectively. go terms associated with the proteins significantly upregulated in exosomes were "extracellular exosome" and "plasma membrane", while the microvesicle proteome was associated with "membrane" and mitochondrion". in exosomes tetraspanins, annexins, escrt and rab proteins were significantly upregulated. in contrast, proteins that were upregulated in microvesicles were involved in protein translocation into the mitochondrial membrane (timm and tomm proteins), in cytokinesis, and in micos complex. however, flotillin- was not differently expressed in the ev subtypes. summary/conclusion: this study identifies several proteins to be differently enriched in exosomes and microvesicles. several of the proteins suggest recently by kowal and colleagues, such as adam and mitofilin could be validated. additionally several novel proteins could be identified. identifying markers separating microvesicles and exosomes is of high importance for the ev field and future studies will have to validate them also in other cells to determine if they are generic. introduction: the cellular elements composing the lining of brain ventricles have drawn much attention from neuroscientists, especially the role of subependymal cells in neurogenesis, but the role of ependymal cells in brain function and disease is still neglected. our objective is to study the morphological aspects of rat brain ventricles and the ependymal cells as analysed by transmission and field emission scanning microscopy in normal or ischaemic rats. methods: for this purpose, male wistar rats were submitted to minutes of global brain ischaemia and divided into two groups: a) sham-operated animals and b) saline-treated ischaemic animals. all animals were allowed to survive for seven days. all procedures were approved by the ethics committee of the federal university of são paulo ( / ). transmission and scanning electron microscopic analysis of lateral brain ventricles were done in buffered , % glutaraldehyde/ %formaldehyde perfused brains. cerebrospinal fluid was collected for nta analysis. results: the morphological characterization of brain ventricle revealed a slight rarefaction of ciliary tufts of animals submitted to ischaemia when compared to normal animals. field emission electron microscopy revealed the secretion of vesicles by the ependymal cilia in the lateral ventricle. size and concentration of particles in the cerebrospinal fluid was confirmed by nta and transmission electron microscopy. summary/conclusion: our results are unprecedented and bring innovative potential regarding the role of extracellular vesicles in both the physiology and pathogenesis of the nervous system. these data may also contribute to the development of new technologies for diagnosis and therapy of chronic degenerative diseases. introduction: the function of mitochondria relies on precise and effective quality controls. neurons have high metabolic demands and employ multiple mechanisms to ensure functional mitochondria. we investigated mitochondrial vesiclesa less understood quality control mechanism for mitochondriaand assessed the effect of cellular stress. methods: we surveyed mitochondrial vesicles in rat and planaria brains with electron microscopy. we quantified these vesicles with serial-section electron microscopy (fib-sem). we also conducted confocal microscopy with airyscan analysis of cultured neurons expressing fluorescently tagged mitochondrial markers. results: electron microscopy showed the ultrastructure of various types of mitochondrial vesicles. serial-section electron microscopy revealed the d ultrastructure of mitochondrial vesicles and their prevalence in neurons. confocal microscopic analysis showed increased numbers of mitochondrial vesicles in neurons under mild stress. summary/conclusion: our findings provide direct structural evidence for mitochondrial vesicles in neurons and their abundance in response to neuronal stress. their detection in the extracellular compartment (evidence for which is expected to be presented by the time of isev) may allow for development of biomarkers for mitochondrial health, with relevance to numerous pathologic conditions. from endosomes, might be involved in the impairment of rna, specific feature of als disease. combining high-resolution flow cytometry and surface marker analysis using an automated platform to study extracellular vesicle in cerebrospinal fluid unity health toronto, toronto, canada introduction: there is growing enthusiasm that extracellular vesicles (evs) carry the potential for a variety of applications in medicine. as biomarkers, evs may aid clinicians in the evaluation of diagnoses, disease progression, or even response to therapy. however, proper characterization of the amount, size, and phenotype of evs in a given sample remains challenging due to their sub-micrometre size and heterogeneity. over the last years, technologies, including high-sensitivity flow cytometry and automated platforms that simultaneously assess ev amount, size, and phenotype, have matured, providing new opportunities to study evs for future clinical applications. using such technologies to analyse cerebrospinal fluid (csf), which is in direct contact with the brain and spinal cord, may yield valuable insights into neurological disease processes. while there is often uncertainty about the exact source of evs in a biological sample, cd has emerged as a surface marker that suggests a neuronal origin. methods: csf samples that had been stored at - degrees celsius for advanced biomarker studies were analysed using two distinct approaches. a becton, dickinson and company (bd) aria iii flow cytometer was converted into using violet side scatter (ssc) for improved detection of evs with instead of nm ssc. for the combined analysis of amount, size, and phenotype, samples were analysed with the nanoview bio r platform. phenotype analysis included probing for the classic tetraspanins associated with exosomes (cd , cd , cd ) and the neural cell adhesion molecule l (cd ). results: flow of csf samples showed similar vesicle counts in control vs. disease and an increase of counts in later disease stages when neurodegeneration is thought to be more prominent. all csf samples showed some binding to classic exosomal markers (cd , cd , cd ). the sample taken at the latest time point showed relatively high vesicle counts, overall larger vesicle size, and abundant cd binding. interestingly, the cd positive evs were not positive for any of the classic exosomal markers (cd , cd , and cd ). summary/conclusion: this data supports the notion that analysing the amount, size, and surface markers of evs in csf can reveal intriguing dynamics in such basic ev characteristics over time and suggests important differences between ev populations in different disease stages. while previous studies indicated that cd could identify an ev to be of neuronal origin, it remains to be determined whether such specific surface markers will emerge as clinically relevant tools to support the evaluation of people affected by neurological diseases. a distinct microrna signature in plasma derived small extracellular vesicles of different neurodegenerative diseases introduction: exploring identifying robust biomarkers is essential for early diagnosis of neurodegenerative diseases. blood stream transports large (levs) and small extracellular vesicles (sevs), which are extracellular vesicles of different sizes and biological functions that are transported in blood. aim of our study was to investigate mrna/mirna signatures in plasma derived levs and sevs of amyotrophic lateral sclerosis (als), alzheimer's disease (ad), parkinson's disease (pdpd), fronto-temporal dementia (ftd) and alzheimer's disease (ad) patients. methods: levs and sevs were isolated from plasma of patients and healthy volunteers (ctr) by ultracentrifugation and rna was extracted. whole transcriptome and mirna libraries were prepared with truseq stranded total rna kit and truseq small rna library kit (illumina). results: our data suggested that the rna cargo in levs and sevs varies among different diseases. mirna analysis in sevs provided the most informative disease specific signatures, while whole transcriptome analysis did not show any specific signature. als was characterized by a small but specific group of circulating mirnas. mirnas profiling revealed that pd and ftd can be subgrouped in two classes while ad appears to be a homogeneous disease population. furthermore, mirnas profiling show the presence of overlaps in the signatures between the analysed diseases. mirna profiling in levs is similar to that observed in sevs, although in levs the overall differences between diseases are less marked. summary/conclusion: in this study we have demonstrated that mirnas are the most interesting subpopulation of transcripts transported by plasma derived sevs since they discriminate a disease from the other and they can provide a signature for each neurodegenerative diseases. may be linked with apoe genotype, we investigated the possible effect of apoe genotype on brain-derived evs (bdevs) and their protein and rna molecular cargo. methods: cortical brain tissues of ad patients with different apoe genotypes [ε /ε (n = ), ε /ε ( ), ε /ε ( ), ε /ε ( )] and non-ad controls (n = ) were obtained. bdevs were separated by size exclusion chromatography plus ultracentrifugation (uc) and characterized per misev . proteins were analysed by mass spectrometry. after protein identification, data were normalized using the cyclicloess method and analysed by principal component analysis (pca). nested factorial design highlighted differentially expressed proteins. rna from bdevs was extracted by mirneasy mini kit. small rna libraries were constructed using the ion total rna-seq kit and sequenced on the ion torrent s ™ using ion™ chips. reads were aligned to human reference transcriptomes using bowtie. differential gene expression was quantified by edger and limma. results: among proteins dysregulated in ad bd-sevs, several have reported roles in ad, e.g., microtubule-associated protein tau and peroxiredoxin- . regarding apoe genotypes, proteins were differentially expressed between ε carriers (ε /ε and ε /ε ) with non ε carriers (ε /ε and ε /ε ). however, ev markers did not differ by apoe genotype. in contrast to protein cargo of bdevs, the overall small rna expression pattern was similar among ad patients with different apoe alleles and non-ad patients. only a few mirnas showed different abundance level between ε /ε and ε /ε groups, or between ad and non-ad groups. summary/conclusion: bdevs carry proteins and mirnas related to ad development and apoe genotypes. further verification of protein and rna expression in brain and plasma derived evs may reveal mechanisms of ev function in neuroinflammation and develop biomarkers for ad disease. funding: this project was funded by mh . efficient pathology spread by extracellular vesicles from human brain tissues in mouse brain and tissue cultured neurons: transmission and propagation to gabaergic neurons however, whether human brain-derived evs induce tau pathology has not yet been characterized in the mouse brain. here, we assess the mechanisms of disease spread after intrahippocampal injection of human brainderived evs into the aged mouse model. methods: ev-enriched fractions were isolated from unfixed frozen human brain samples from ad, prodromal ad (pad), control (ctrl) cases, and tau knockout (tko) mouse brains. isolated evs containing pg of human total tau were sterotaxically injected into the right outer molecular layer of the dentate gyrus of months-old c bl/ female mice. . months after the injection, hippocampal slices were prepared for whole-cell patch clamp recordings of ca pyramidal neurons were undertakent. hippocampi were analysed with immunohistochemistry using phosphorylated-tau (p-tau) epitopes including at . evs were examined for protein composition by protein mass-spectroscopy, the neuronal uptake in vitro, and structural analysis by the atomic force microscopy (afm). results: semiquantitative brain-wide immunohistochemistry of p-tau revealed that inoculation of ad or pad-evs induced tau propagation throughout the hippocampus, including the dentate gyrus, ca and ca subregions. at was localized primarily in gad + gabaergic neurons in pad and ad evs groups, accompanied with reduced amplitude of inhibitory postsynaptic currents and excitatory-inhibitory ratio in amplitube of postsynaptic currents in ca pyramidal neurons in pad evs. afm analysis showed higher density of tau oligomers in both ad and pad evs while only ad evs showed significantly higher neuronal uptake compared to ctrl evs. finally, proteomic analysis showed that ad evs are enriched in disease and glia-related molecules compared to ctrl evs, which may contribute to their enhanced neuronal uptake. summary/conclusion: intracranial injection of ad or pad evs induced p-tau accumulation primarily in gabaergic neurons throughout the hippocampus, resulted in higher uptake by neurons, and tau oligomer conformation, indicating of their pathogenic potency as seeding factors. gabaergic neuronal dysfunction in the hippocampal neuronal circuitry reported in early ad brains could be attributed to specific ev mediated tau propagation in this cell type, a phenomenon meriting further investigation and validation. funding: nih rf ag , nih r ag , nih r ag , cure alzheimer's fund, brightfocus foundation, curepsp, coins for alzheimer's research trust introduction: extracellular vesicles (evs) are released by cells of the central nervous system as a result of injury, including mild traumatic brain injury (mtbi). since mtbi may alter circulating levels of evs, this study aimed to investigate differences in circulating ev numbers between contact sport athletes with and without acute mtbi. methods: circulating evs containing cd (cd + ev), cd (cd + ev), and neural cell adhesion molecule (l cam+ev) were analysed in young, male athletes with or without mtbi ( - yo, n = per group). sodium citrate-treated blood samples were obtained from athletes with mtbi within -hours of injury and from control athletes free of mtbi for one year. athletes were best matched for age and history of prior mtbi. samples were double-centrifuged to obtain platelet-poor plasma and stored at − °c until analysed. quantification of evs was performed using a spectral flow cytometer. the study was approved by temple university's irb, and all athletes provided written informed consent. results: mann-whitney u tests showed that population percentages of small size ( - nm) cd + ev, cd + ev and l cam+evs were significantly higher in mtbi athletes (mean rank: . , . , . ) than controls (mean rank: . , . , . ) (u = . , p = . ; u = . , p > . ; u = . , p > . , respectively). population percentages of large size ( - nm) cd + ev, cd + ev and l cam+evs were also significantly higher in mtbi athletes (mean rank: . , . , . ) than controls (mean rank: . , . , . ) (u = . , p = . ; u = . , p > . ; u = . , p > . , respectively). there were no significant differences between percentages of evs associated with blood brain barrier function (cd + ev) or platelets (cd a+ev) among mtbi athletes or controls. introduction: parkinson's disease (pd) is characterized by clinical heterogeneity, different rates of progression and absence of definitive biomarkers. extracellular vesicles (evs) are easily isolated from plasma and play a central role in intercellular communication which is highly relevant for inflammatory processes implicated in protein misfolding-related neurodegenerative disorders. thus, we characterized distinctive plasmatic ev subpopulations of pd and atypical parkinsonisms (ap) patients, with the aim to identify candidate biomarkers among evs surface membraneproteins. methods: plasmatic evs were collected from pd, matched healthy controls (hc), ap with multiple system atrophy (msa) and ap with tauopathies (ap-tau). evs were quantified by nanoparticle tracking analysis. the expression of ev-surface markers, related to inflammatory and immune cells, were measured by macsplex and correlated to clinical scales. a diagnostic model based on ev markers expression was built via supervised machine learning algorithms and validated in an external cohort ( pd, hc, msa, ap-tau). the cantonal ethics committee approved the study protocol. all enrolled subjects gave written informed consent. results: pd showed the highest ev concentration compared to others groups. pd and msa displayed a greater pool of overexpressed immune markers compared to ap-tau. ev antigens correlate to cognitive impairment and disease gravity in pd and msa. the roc curve analysis of a compound ev marker showed optimal diagnostic performance for pd (auc . ; sensitivity . %, specificity . %) and msa (auc . ; sensi-tivity %,specificity . %)andgoodaccuracyforap-tau (auc . ; sensitivity . %, specificity . %). a diagnostic model based on ev markers expression, cor-rectlyclassified . %ofpatientswithreliablediagnostic performance after validation in an external cohort ( % of accuracy). summary/conclusion: this analysis of multiple immune surface markers of circulating evs in pd and ap well captured the clinical heterogeneity of pd and showed optimal diagnostic performance. furtherly it suggests a different immune dysregulation in pd and msa vs. ap-tau, to be confirmed by functional analysis in experimental models of disease. funding: supported by abreoc. separation and characterization of extracellular vesicles from human cerebrospinal fluid introduction: extracellular vesicles (ev) are released from cells to the surroundings and are found in human biofluids, where they constitute promising targets for novel biomarker identification. ev have been found in cerebrospinal fluid (csf) where they may provide with markers for neurological diseases. here, we aimed at purifying and characterizing ev from human csf. methods: csf was collected by lumbar puncture from patients with amyotrophic lateral sclerosis. patients gave written consent and studies were agreed by the local ethics committee. csf was fractionated by ultrafiltration (vivaspin, cut-off , ), and size-exclusion chromatography (sec; qevsingle izon science). eluted fractions were analysed by dynamic light scattering (dls) and electron microscopy. proteins were analysed by immunoblotting and nano-liquid chromatographytandem mass spectrometry. results: ev eluted in early fractions ( + ) after the sec void volume as evaluated by detection of cd and cd markers (immunoblotting) and annexin a (peptide mapping by nanolc-ms/ms). there, nanoparticles around nm were identified by dls. in agreement, electron microscopy showed ev with characteristic shape and sizes typically between and nm, with average diameter ± nm. cd was visualized by immunocytochemistry at the surface of ev around nm. on the other hand soluble proteins igg and albumin eluted in later fractions. curiously, galectin- binding protein (lgals bp or k) was also partially detected in early-eluting fractions as nanoparticles of irregular shapes and heterogeneous sizes typically between and nm; some of those nanoparticles had ring-like appearance. occasionally k also appeared on ev of variable dimensions. summary/conclusion: in conclusion, ev from the csf may be separated from soluble proteins and small molecules by a combination of ultrafiltration with sec fractionation. however, using this strategy a population of k-containing nanoparticles co-eluted with ev from the csf. further separation techniques need to be applied to separate ev from k nanoparticles to investigate their individual physiological relevance and biomarker potential. introduction: extracellular vesicles (ev) are released from cells to the surroundings and are found in human biofluids, where they constitute promising targets for novel biomarker identification. ev have been found in cerebrospinal fluid (csf) where they may provide with markers for neurological diseases. here, we aimed at purifying and characterizing ev from human csf. methods: csf was collected by lumbar puncture from patients with amyotrophic lateral sclerosis. patients gave written consent and studies were agreed by the local ethics committee. csf was fractionated by ultrafiltration (vivaspin, cut-off , ), and size-exclusion chromatography (sec; qevsingle izon science). eluted fractions were analysed by dynamic light scattering (dls) and electron microscopy. proteins were analysed by immunoblotting and nano-liquid chromatographytandem mass spectrometry. results: ev eluted in early fractions ( + ) after the sec void volume as evaluated by detection of cd and cd markers (immunoblotting) and annexin a (peptide mapping by nanolc-ms/ms). there, nanoparticles around nm were identified by dls. in agreement, electron microscopy showed ev with characteristic shape and sizes typically between and nm, with average diameter ± nm. cd was visualized by immunocytochemistry at the surface of ev around nm. on the other hand soluble proteins igg and albumin eluted in later fractions. curiously, galectin- binding protein (lgals bp or k) was also partially detected in early-eluting fractions as nanoparticles of irregular shapes and heterogeneous sizes typically between and nm; some of those nanoparticles had ring-like appearance. occasionally k also appeared on ev of variable dimensions. summary/conclusion: in conclusion, ev from the csf may be separated from soluble proteins and small molecules by a combination of ultrafiltration with sec fractionation. however, using this strategy a population of k-containing nanoparticles co-eluted with ev from the csf. further separation techniques need to be applied to separate ev from k nanoparticles to investigate their individual physiological relevance and biomarker potential. release of extracellular vesicles from platelets requires platelet-platelet interaction aleksandra gąsecka a , naomi c. buntsma b , sytske talsma c , krzysztof j. filipiak d , rienk nieuwland e and edwin van der pol f introduction: arterial thrombosis is a major and global cause of human death and disability, but a biomarker for early-diagnosis of thrombosis is absent. platelet activation and aggregation are the first steps of plateletrich thrombus formation, but their relative contribution to platelet extracellular vesicles (pevs) release is unknown. methods: to study the relation between pev release and platelet interaction (aggregation), citrate-anticoagulated whole blood (wb) from healthy donors was diluted , , , and -fold and activated by μm thrombin-receptor activating peptide (trap). in addition, undiluted wb and -fold diluted wb, which totally blocked pev release, were activated with various trap concentrations. concentrations of pevs (cd + and cd +, cd p + > nm) and activated platelets (cd +, cd p+ > nm) were measured by flow cytometry (apogee a -micro). platelet aggregation was assessed using impedance aggregometry. results: a -fold dilution of wb blocked both aggregation and the release of pevs. compared to baseline, activation of undiluted wb with trap increased the concentrations of cd + . -fold and cd +-cd p + pevs . -fold. the concentration of cd + (r = . ) and cd +-cd p+ (r = . ) pevs as well as platelet aggregation (r = . ) scaled inversely (reciprocal) with the dilution of wb. further, we found a linear correlation between the % of activated platelets and the concentration of cd + (r = . ) and cd +, cd p+ (r = . ) pevs in undiluted wb, which was absent in -fold diluted blood (r < . ). summary/conclusion: the absence of aggregation and pev release upon platelet activation in -fold diluted blood shows that aggregation directly depends on the distance between platelets, which is confirmed by the reciprocal relationship between pev release and blood dilution. because pevs are only released when platelet activation is followed by aggregation, pevs are a potential early biomarker of thrombosis. funding: ag is supported by the national science centre, research programme preludium / / n/nz / . evdp is supported by the netherlands organisation for scientific research -domain applied and engineering sciences (nwo-ttw), research programmes veni . age-dependent alteration in concentration and size distribution of extracellular vesicles in plasma of normotensive and hypertensive rats kosuke otani, muneyoshi okada and hideyuki yamawaki laboratory of veterinary pharmacology, school of veterinary medicine, kitasato university, towada, japan introduction: spontaneously hypertensive rats (shr) are the most widely used animal model of human essential hypertension. we previously reported that plasma small extracellular vesicles (sevs) in shr regulate systolic blood pressure, however, the mechanism has not been clarified. in the present study, we compared the concentration and size distribution of plasma evs (sevs and large evs) from young and aged normotensive wistar kyoto rats (wky) and shr. methods: heparin-anticoagulated plasma was collected from male wky and shr at ~ -(young) and -(aged) week-old. large evs were isolated from the plasma by centrifugation ( x g). sevs were isolated by ultracentrifugation ( , x g) following precipitation with polyethylene-glycol. the concentration and size distribution of sevs and large evs were measured by a tunable resistive pulse sensing analysis. results: there was no significant difference in the total concentration of plasma sevs between wky and shr or between young and aged rats. the mean diameter of plasma sevs from aged rats was larger than that from young rats in both wky and shr. also, the number of particles with a diameter of smaller than nm in plasma sevs from aged rats was lower than that from young rats. the concentration of plasma large evs from aged rats was higher than that from young rats in both wky and shr. there was no significant difference in the size distribution of plasma large evs between wky and shr or between young and aged rats. summary/conclusion: the present results for the first time demonstrate that the concentration of plasma large-sized evs is increased by ageing, while there is no difference in the concertation and size distribution of evs between wky and shr. further research is required to clarify the cause of age-dependent alternation in plasma ev size distribution and its physiological meaning. microrna profiling of circulating extracellular vesicles is involved with susceptibility to age-related diseases: relevance to cardiovascular signalling in ageing process ionara rodrigues siqueira a , laura cechinel b , rachael batabyal c and robert freishtat c a universidade federal do rio grande do sul (ufrgs), porto alegre, brazil; b universidade federal do rio grande do sul, porto alegre, brazil; c children's national hospital, washington, usa introduction: ageing represents a central risk factor for several diseases, such as cardiovascular diseases. our hypothesis is that extracellular vesicles (evs) can be potential mechanism of spreading molecules, such as micrornas, involved with susceptibility to chronic age-related diseases and geriatric syndromes. in this context, the role of micrornas in age-induced detrimental changes in the cardiovascular system has been suggested. although evs can protect micrornas from endogenous rnases and internalization of these vesicles into cells is involved with cell communication, delivering micrornas even to distant tissues, the relationships between evs micrornas profile and chronic age-related diseases has not been evaluated. our aim was to investigate the microrna profile of circulating evs during ageing process and their downstream signalling pathways. methods: the ethics committee (ceua -comissão de Ética no uso de animais -ufrgs; nr. , ) approved all animal procedures and experimental conditions. male wistar rats of -and -month-old were used, and plasma was obtained from the trunk blood. evs were isolated with exoquick following the manufacturer's instructions. microrna was isolated from evs and then amplified. microrna was labelled using the flashtag biotin hsr rna labelling kit and profiled on affymetrix genechip microrna . arrays. ingenuity pathway analysis (ipa) was used to identify pathways regulated by significantly altered micrornas. results: microarray analysis revealed micrornas. of these micrornas, were differentially expressed between aged and young-adult animals, micrornas were significantly upregulated and were downregulated in aged animals compared to young adult (p < . ; fold change of | . |). a conservative filter was applied on ipa and only experimentally validated and highly conserved predicted mrna targets for each microrna was used. ipa analysis showed that cardiac hypertrophic signalling is ranked as highly predicted targets for these differentially expressed micrornas (p < . ). moreover, ipa demonstrated that this canonical pathway is upregulated in aged animals when compared to young adult. in addition to cardiac hypertrophic signalling, other relevant cardiovascular canonical pathways, such as endothelin- signalling and intrinsic prothrombin activation pathway have predicted targets. summary/conclusion: our results showed for the first time that micrornas profile in circulating evs has a potential role to drive heart senescence and consequent cardiac diseases which represents the leading cause of death. introduction: introduction: the vascular endothelium and smooth muscle form adjacent cellular layers that comprise part of the vascular wall. here, we examined the extent to which extracellular vesicles (evs) vesicles participate in endothelial-vascular smooth muscle cell communication. methods: methods: evs were collected from rat aortic endothelial and smooth muscle cell serumfree media by ultracentrifugation. vesicle morphology, size and concentration were evaluated by transmission electron microscopy and nanoparticle tracking analysis. endothelial cell and vascular smooth muscle cell cultures were subjected to various concentrations of evs for various times. functional assays were performed. results: results: western blot as well as shot gun proteomic analyses revealed sets of proteins common to both endothelial-and smooth muscle-derived ev as well as proteins unique to each vascular cell type. functionally, endothelial-derived evs stimulated vascular cell adhesion molecule- (vcam- ) expression and enhanced leukocyte adhesion in vascular smooth muscle cells while smooth muscle evs did not elicit similar effects in endothelial cells. evs from endothelial cells also induced protein synthesis and senescenceassociated β galactosidase activity in vascular smooth muscle cells. proteomic analysis of vascular smooth muscle cells following exposure to endothelial cellderived evs revealed upregulation of several proteins including pro-inflammatory molecules, high-mobility group box (hmgb) and hmgb . pharmacological blockade of hmgb and hmgb and sirna depletion of hmgb in smooth muscle cells attenuated nfkb (p ) phosphorylation and nuclear translocation, vcam- expression and leukocyte adhesion induced by endothelial cell evs. summary/conclusion: conclusions: these data suggest that endothelial cell-derived evs can enhance signalling pathways that induce a pro inflammatory in vascular smooth muscle cells. introduction: graft patency is one of the major determinants of long-term outcome following coronary artery bypass graft surgery (cabg). biomarkers, if indicative of the underlying pathophysiological mechanisms, would suggest strategies to limit graft failure. many studies have generated compelling data on the sensitivity of mvs as biomarkers of cardiovascular disease progression and events. the mv usefulness in cabg has been tested only in a study that highlighted their importance in surgical haemostasis. no information is so far available on the association between the amount or pattern of circulating mvs and cabg outcome. we aimed to evaluate whether mv pre-operative signature could predict mid-term graft failure. methods: this was a nested case-control substudy of the coronary bypass grafting: factors related to late events and graft patency (cage) study that enrolled patients undergoing elective cabg. of these, underwent coronary computed tomography angiography months post-surgery showing % graft occlusion. flow cytometry mv analysis was performed in patients ( /group with occluded [cases] and patent [controls] grafts) on plasma samples collected the day before surgery and at follow-up. results: before surgery, cases had two-fold (p = . ) and four-fold (p = . ) more activated platelet-derived and tf+ mvs, respectively than controls. the mv thrombin generation capacity was also significantly greater (p < . ). this mv signature predicted graft occlusion (auc of . [ %ci: , - , ], p = . ). by using a mv-score ( - ), the or for re-occlusion for a score above was . ( % ci . - . , p < . ). summary/conclusion: the pre-operative signature of mvs is an independent predictor of mid-term graft occlusion in cabg patients and a cumulative mvscore stratifies patient's risk. since the mv signature mirrors platelet activation, patients with a high mvscore would benefit from a personalized antiplatelet therapy. exosomes from engineered immortalized human heart cells improve ventricular function and attenuate fibrosis in mice with arrhythmogenic cardiomyopathy yen-nien lin, lizbeth sanchez, rui zhang, thassio ricardo ribeiro mesquita, chang li, ahmed ibrahim, eduardo marbán and eugenio cingolani heart institude, cedars sinai medical center, los angeles, usa introduction: arrhythmogenic cardiomyopathy (ac) is characterized by progressive loss of cardiomyocytes and fibrofatty tissue replacement. currently, there is no effective treatment for this disease. exosomes (imexos) secreted by heart stromal cells, engineered to be immortal and overexpressing β-catenin, exert anti-inflammatory and anti-fibrotic effects and improve ventricular function in models of ischaemic injury (ibrahim et al., nature bme ). methods: to investigate the effectiveness of imexos in a murine model of ac, four-week old homozygous dsg knockout (dsgko) mice and wild type (wt, age-and strain-matched) mice were compared. dsgko mice were randomized to receive weekly imexos or vehicle via intravenous injection for weeks. neonatal rat ventricular myocyte (nrvm) proliferation and apoptotic assays were performed to explore potential effects of exosomes. results: biodistribution studies of dir-labelled imexos revealed some cardiac uptake, along with strong signals in spleen. at weeks, dsgko mice which had received intravenous imexos showed improved cardiac function (echocardiographic ejection fraction ± vs ± % in vehicle mice, p = . ), with an underlying attenuation in myocardial fibrosis by histology. electrophysiology test showed shorter qrs duration ( . ± . ms imexo vs . ± . ms vehicle, p = . ) and effective refractory period. programmed ventricular stimulation showed dsgko mice which had received imexos were remarkably less prone to ventricular tachycardia induction ( . ± % vs . ± % in vehicle, p = . ). in vitro study showed nrvm exposed to imexos for days exhibited higher brdu expression relative to vehicle group, and less annexin-v expression after oxidative stress induced by -minute illumination with nm uv. summary/conclusion: intravenous administration of imexos improved cardiac function, reduced cardiac fibrosis, and suppressed arrhythmogenesis in ac. our findings motivate clinical testing of imexos in ac, an orphan disease with great unmet medical need. funding: nih r hl (to em) cardiac-derived extracellular vesicles contribute to communication between heart and brain in chronic heart failure (chf) and target nrf / are signalling changhai tian a , lie gao b and irving zucker b a department of cellular and integrative physiology, university of nebraska medical center, omaha, usa; b department of cellular and integrative physiology, university of nebraska medical center, omaha, usa introduction: mirnas regulate the translation of proteins that are involved in redox homoeostasis in the heart and brain. intra-and/or inter-organ communication takes place by multiple mechanisms including extracellular vesicular (ev) transport. our previous studies suggested that cardiac derived mirna-enriched evs contribute to the dysregulation of nrf /antioxidant enzyme (are) signalling in the myocardium via intercellular cross-talk, and result in the decreased nrf /are signalling in the sympatho-regulatory areas of the brain in chf. however, it is unclear if cardiac derived evs circulate to the central nervous system evoking sympatho-excitation by disrupting central redox homoeostasis. methods: cardiac-specific membrane gfp+ mice were generated to track the brain distribution of cardiac evs in rats with chf (coronary ligation). the isolation and characterization of evs were carried out by differential ultracentrifugation, tem, nanosight, western blotting, and qrt-pcr. transfection, labelling, and microinjection of evs into the rostral ventrolateral medulla (rvlm) were performed. results: nrf protein was reduced in the rvlm of chf rats consistent with an upregulation of nrf -targeting mirnas. nrf -targeting mirnas were enriched in cardiac and circulating evs of chf rats. nrf -targeting and cardiac-specific mirnas were abundant in brain-derived evs. circulating evs were taken up by neurons in sympatho-regulatory areas of the brain. mirna-enriched evs from chf animals increased sympathetic tone which was prevented by a cocktail of nrf -targeting mirna inhibitors. summary/conclusion: myocardial infarction-induced mirna-enriched evs mediate the inter-organ crosstalk between heart and brain in the oxidative regulation of sympathetic outflow through targeting the nrf / are signalling pathway. these findings suggest that cardiac-derived ev mirnas targeting nrf /are signalling may act as an endocrine signalling mediator of chf that has potential as a novel therapeutic target. introduction: a fine-tuned communication between cardiac cells is vital to maintain myocardial integrity and contractility. not only an impairment of gap junction (gj)-mediated intercellular communication, but also defects in ev-mediated communication have been associated with ischaemic heart disease, a major causative factor of heart failure. we have previously shown that cx , the main ventricular gj protein, assembles into channels at the evs surface, mediating the release of vesicle content into target cells.the main objective of this work was to characterize the signals underlying protein sorting into extracellular vesicles (evs) in a human pathophysiological context, using connexin (cx ) as a model substrate. methods: animal models of ischaemia/reperfusion (i/ r) injury by ligation of the left anterior descending coronary artery, ex vivo and in vitro ischaemia models and human patients were used to investigate the secretion of ev-cx . results: release of cx was downregulated in circulating vesicles from i/r-injured mice and patients with st-segment elevation myocardial infarction, as well as in intracardiac and cardiomyocyte-derived evs. additionally, we show that ubiquitin signalled the release of cx in basal conditions but appeared to be dispensable during ischaemia. depletion of the autophagy adaptor p partially restored the secretion of cx , suggesting an interplay between ischaemiainduced cx degradation and secretion. summary/conclusion: overall, we demonstrated that ischaemia impairs the sorting of cx into evs, which may ultimately affect long-distance communication. through the identification of the underlying molecular mechanisms and players, these results pave the way towards the development of innovative diagnostic and therapeutic strategies for cardiovascular disorders. introduction: remote ischaemic conditioning is a cardioprotective intervention which protects the heart against ischaemia/reperfusion injury. transient activation of toll-like receptor (tlr ) and its downstream regulators (tnfα and il- ) have been implicated in cardioprotective interventions. extracellular vesicles (evs) play a role in cardioprotection through the activation of the tlrs. however, since isolation of evs in high amounts with suitable purity from blood is a challenge, our aim was to develop a cellular model system from which tlr-inducing, cardioprotective evs can be isolated in a reproducible manner. methods: ev release from hek cells was induced by calcium-ionophore a . evs were characterized, cytoprotection by evs against simulated ischaemia/ reperfusion injury and its mechanism were investigated in h c and ac cell lines. results: a induction of hek cell induced ev release and the isolates contained mostly large evs. evs decreased cytotoxicity and apoptosis due to h ischaemia followed by h reperfusion in h c and ac cells in a dose-dependent manner. evs activated tlr and its downstream signalling pathway in h c and ac cells as well as the expression of cytoprotective haem oxigenase (ho- ) in h c cells. summary/conclusion: a -induced evs exert cytoprotection in h c and ac cells by inducing tlr signalling and ho expression. therefore, evs released via calcium-ionophore treatment may serve as a basis of an efficient carpdioprotective therapy. introduction: biliary strictures may be benign or malignant. the major malignant causes of biliary stricture are a primary cholangiocarcinoma (cca) or pancreatic ductal adenocarcinoma (pdac). there is ongoing debate about adequate diagnostics in biliary strictures of unknown aetiology. micrornas (mirnas) are small non-coding rnas important in tumourigenesis. mirna have been found to be enriched in exosomes, small membrane-bound extracellular vesicles (ev) of endocytic origin, which is a novel pathway for intercellular signalling within the tumour microenvironment and have been implicated in loco-regional pre-metastatic niche formation. this project aims to investigate circulating-free and ev mirnas as biomarkers that can aid diagnosis in patients with a biliary stricture. we will ( ) isolate and characterise evs in plasma and bile from patients with benign and malignant biliary strictures (i.e. pancreaticobiliary cancers); and ( ) identify differentially expressed circulating-free and ev mirnas in plasma and bile suitable for detecting malignancy. methods: sample size (n = ) was calculated for a study power of % and α error of % for the ability of extracellular mirnas to discriminate benign from malignant biliary strictures. prospective matched plasma and bile samples will be collected from patients with benign (n = ) and malignant (n = ) biliary strictures undergoing endoscopic retrograde cholangiopancreatography (ercp). evs will be isolated from the biofluids by ultracentrifugation and/or size exclusion chromatography and then characterised (tem, nta and immunoblotting). circulating-free and ev-associated mirnas will be profiled using small rna sequencing. extracellular mirna "signatures" will then be validated by rt-qpcr, and diagnostic accuracy confirmed (sensitivity, specificity, auc). results: evs derived from patient samples have been characterised using nta, western blotting and tem. sec derived evs appear to be more well-defined than uc evs with marker positivity for cd , cd and cd . ongoing work will be focused on rna profiles of evs from both malignant and benign cohorts. summary/conclusion: there is currently no effective method to differentiate benign from malignant biliary strictures. novel plasma and bile circulating-free and ev-associated mirna biomarkers may improve the speed and accuracy of diagnosis, resulting in considerable patient benefits. furthermore, as little is known about the ev-associated function of these tumours, candidate ev-mirnas could be taken from "bedside to bench" and their function further investigated using in vivo, vitro and silico models. introduction: urine is a source of extracellular rna (exrna) biomarkers that can be obtained non-invasively throughout pregnancy. several studies have profiled extracellular mirnas in biofluids during pregnancy, but few have profiled extracellular mrnas (ex-mrnas) in urine. objective: to optimize methods for ex-mrna isolation and rna-seq library preparation from urine of healthy pregnant and non-pregnant females. methods: rna was isolated from pooled non-pregnant urine using kits based on ev precipitation (mircury exosome kit for csf/urine, seramir), ev affinity purification (exorneasy), and protein precipitation (mirneasy serum/plasma advanced). next, long (> nt) and short rnas were isolated from ev enriched urine of pregnant (n = ) and non-pregnant (n = ) individuals using the mircury kit followed by the mirneasy micro kit. rna-seq libraries were prepared using the smart-seq v ultra low input rna (oligo(dt) priming) and the smarter stranded total rna-seq kit v -pico input (random priming) methods (takara). preliminary data were obtained using the illumina miseq, and aligned using star v. . . .a. results: overall, rna isolation using mircury followed by the smart-seq v library preparation kit yielded the highest % of mapped reads: % in pooled non-pregnant, % in individual non-pregnant, and % in individual pregnant urine. for rna extracted using the mircury kit, the smart-seq v libraries had higher % of mapped mrna reads compared to pico libraries (p < . , t-test). in contrast for mirneasy advanced it was reversed ( % vs %). summary/conclusion: early results from low-depth sequencing show the highest mrna mapping rates for mircury followed by the smart-seq v kit. high-depth sequencing data are now being generated, which will enable us to perform detailed comparisons of different rna species from the rna profiles obtained using different library preparations and rna isolation methods from urine of pregnant and non-pregnant subjects. funding: this study was funded by nih k hd - , nih u hl , and a ucsd igm-illumina mini-grant. il- mutein-induced changes of exosomal mirna cargo in a humanized mouse model emily lurier, erik sampson, patrick halvey, mike cianci and katalin kis-toth pandion therapeutics, cambridge, usa introduction: regulatory t cells (tregs) are key contributors to immune homoeostasis. decreased number and/or function of these cells are frequent features of many autoimmune diseases linked to the development of tissue inflammation. while interleukin- (il- ) is essential for pan t cell proliferation and performance, low dose il- treatment has been shown to preferentially affect tregs and is being evaluated as an intervention in autoimmune diseases. pt is a novel il- mutein fc fusion molecule (il- m) designed to selectively engage with tregs. using a humanized nod-scid il rn-null (nsg) mouse model we have shown that pt expanded tregs without significant effects on other immune cells. we have also shown that tregs from pt -dosed humanized mice exhibit increased expression of foxp and cd , and demethylation of foxp and ctla- genes, suggesting enhanced function and stability. in the current study we investigated the mirna content of plasma exosomes isolated from pt -or vehicle-treated mice in order to identify treg specific mirnas from the il- m treated animals. methods: cd + haematopoietic stem cell humanized nsg mice were dosed once subcutaneously with pt or vehicle. plasma samples from mice were collected at day and exosome isolation was conducted using the exoquick method. small rna was extracted and quantified using the bioanalyzer small rna assay. an illumina nextseq instrument was used for library preparation and sequencing with bp single end reads at an approximate depth of - million reads per sample. raw sequences were mapped to human genome grch and analysed via a pipeline provided by the university of california santa cruz. results: rna within the exosomes from vehicle and il- m-treated groups was mostly comprised of mirna and trna. plasma was pooled from animals per treatment group and differential expression was determined using a twofold change cut-off. we found that pt treatment actively altered the mirna content of plasma exosomes, compared to exosomes from vehicle-treated mice. many of the differentially expressed mirnas are involved in immunoregulation. summary/conclusion: plasma exosomes from pt treated humanized mice encapsulated treatment-specific mirnas which can potentially be used as systemic biomarkers of treg expansion and function. identification of potential biomarkers in microglial specific exosomes isolated from prion-infected serum introduction: transmissible spongiform encephalopathies (tse) are neurodegenerative disorders caused by the misfolding of the cellular prion protein (prpc) to the beta-sheet rich abnormal prion protein (prpsc). prpsc aggregates in the brain and causes amyloid plaques, neuronal loss, spongiform degeneration and microglial activation. currently, definitive diagnosis of tse diseases is only confirmed post-mortem thus a diagnostic test in accessible body fluid is of interest. exosomes are a good resource for biomarker discovery since they cross the blood-brain barrier easily and contain protein, lipids and nucleic acids from the cells of origin. the goal of this study was to look at biomarkers from brain-originating exosomes (specifically microglia) isolated in the serum of prion-infected animals. methods: westerns and nanoparticle tracking analysis (nta) were used to look at the composition of microglial-specific exosomes. as proof of principle, exosomes were isolated from a microglial cell line (bv cells). a cd antibody was labelled with a fluorophore and binding to exosomes was visualized via nta. exosomes were isolated from serum of both prioninfected and mock-infected mice throughout disease course. a macrophage specific antibody (f / ) was bound to beads which were used to isolate exosomes which includes those of microglial origin. microrna was extracted from these exosomes and next-generation sequencing (ngs) was performed using the illumina platform. clc genomics workbench was used for bioinformatics analysis. results: microglial and macrophage proteins (tmem and iba ) were identified in exosomes isolated from bv cells and prion-infected mouse serum. macrophage exosomes were isolated via a novel antibody-bead based system. results of the ngs analysis of the microrna isolated from these exosomes indicated a series of mirna that could differentiate between control and infected samples as well as age-specific markers. summary/conclusion: to our knowledge, this is the first time microglial-specific exosomes have been isolated from prion-infected serum from early and end stage disease. the results of this analysis could facilitate the diagnosis of prion disease in easily-accessible biofluids pre-mortem. comparison of urinary extracellular vesicle isolation methods for transcriptomic biomarker research in diabetic kidney disease introduction: urinary extracellular vesicles (uevs) are emerging as a source for early biomarkers of kidney damage, holding the potential to replace the conventional invasive techniques including kidney biopsy. several methods are available for uev isolation. our aim was to compare different workflows and isolation by hydrostatic filtration dialysis (hfd), ultracentrifugation (uc) and a kit based isolation method for their subsequent use in mirna-seq and rna-seq for biomarker discovery in diabetic kidney disease. methods: type diabetic patients (t d) with macroalbuminuria and normoalbuminuric healthy controls were included in the study. sample collection and all experiments were performed in accordance with the declaration of helsinki. evs were isolated from - ml of h urine collections by uc, hfd, or a commercially available kit (purification based on spin column chromatography, urine exosome purification and rna isolation midi kit, norgen biotech, canada) each with different established urine clarification steps. quality control of the evs was performed with negative staining em, nta and western blotting. isolated rnas were profiled with bioanalyzer pico kit and subjected to mirna and mrna sequencing. for rna-seq, cdna library was prepared using smart-seq v ultra low input rna kit for sequencing (takara bio, japan). rna-seq was performed using hiseq (illumina). mirna-seq library was prepared using qiaseq mirna library kit (qiagen, germany). mirna-seq was performed on the illumina hiseq platform (illumina). results: our data showed that uev yield, morphology and size distribution were closely similar in hfd and uc preparations, while lower yields were obtained using the kit. by western blot, ev markers were detectable in samples isolated by hfd and uc but not readily in samples isolated with the kit. tamm-horsfall protein was detected in all the samples and albumin levels appeared higher in hfd and kit isolated samples relative to uc samples. the number of paired-end reads for rna-seq in hfd and uc samples (in both > m) were closely similar. instead, rna reads were lower than m for the kit samples. for mirna-seq, the number of reads as well as the molecular biotype distribution were similar for the three methods. by principal component analysis of the rna-seq data, we observed that hfd and uc grouped together showing similarities. however, for mirna-seq data such similarities were not obvious. this suggests that the three different workflows and isolation principles may enrich different mirna-rich uev preparation components. summary/conclusion: our transcriptomics data shows that hfd and uc are suitable methods to isolate uevs for mirna-seq and rna-seq. the kit based method appears better suited for mirna-seq. introduction: exosomes contain a variety of biomolecules including dna. knowledge of cfdna distribution and localization in bioliquid is important for understanding both biological function of cfdna and exosomes. some publications state that a large proportion of plasma cfdna is localized in exosomes. to quantify cfdna content in free vs. exosomal form in human plasma, urine, and saliva, we employed subx technology, which allows affinity capture dna via phosphates groups of the polynucleotide chain and exosomes via membrane surface phosphate moiety clusters. subx is a proprietary compound that can simultaneously bind to both cfdna and exosomes in bioliquids, thus allowing precipitation of the [subx-dna/ subx-exosomes] complexes without ultracentrifugation. methods: detection of subx-dna and exosomes binding was done by measurement of particle sizes using zetasizer nano zs and nanosight ns . the samples were processed with the subx exo-dna isolation kit following the standard protocols. dna, protein and lipid concentrations were measured by fluorescent assays using qubit fluorometer. results: subx efficiently and selectively captures and co-precipitates cfdna and exosomes directly from bioliquids. exosomes are easily extracted from the pellet in exosome reconstitution buffer (erb), followed by subsequent isolation of tightly bound cfdna from the subx pellet. erb does not extract dna form the [subx -dna] pellet and thus does not contaminate reconstituted exosomes with cfdna. thus, we separate two distinct types of extracellular materialintact exosomes and purified cfdna in a single protocol from the same sample. over % of dna in plasma and urine exist as a free circulating pool, while in saliva up to % is associated with exosomes. thus, cfdna distribution is probably bioliquid-specific and must be evaluated by methods that eliminate cfdna-outer exosomal membrane aggregation. summary/conclusion: subx technology is suitable for simultaneous isolation of both cfdna and exosomes from the same bioliquid sample. subx separates cfdna fragments non-specifically attached to the outer lipid layers of the exosome membrane from the true intra-exosomal cfdna. in contrast, salting-out peg technique is associated with aggregation of macromolecules and vesicles and thus leads to overestimation of exosome-associated polymers content, including cfdna. tracing extrachromosomal dna inheritance patterns in glioblastoma using crispr eunhee yi, amit gujar, hoon kim, albert cheng and roel verhaak jackson laboratory for genomic medicine, farmington, usa introduction: glioblastoma multiforme (gbm) is the most lethal brain tumour; it is characterized by poor response to standard post-resection radiation and cytotoxic therapy, resulting in a dismal prognosis with a five-year survival rate of %. recurrence after therapy for gbm is unavoidable. there are substantial differences among the cells of gbm tumours in the abundance and types of genetic material. this heterogeneity likely is the major cause of therapy failure, the development of treatment resistance, and ultimately recurrence. a recent study has suggested that the amount of a particular type of dnaextrachromosomal dna (ecdna)differs substantially among different gbm tumours, and differs within a given gbm tumour over time. despite the speculation that ecdna is a key factor of tumour heterogeneity, how ecdna is propagated and distributed amongand how it behaves withincancer cells is completely unknown. methods: to address this gap in knowledge, this study focused on developing a novel cytogenetic crisprbased tool that enables visualization and tracking ecdna behaviour in live gbm cells. results: we found breakpoint sequences resulting from genome rearrangements during ecdna formation by performing computational analysis from whole genome sequencing data. and each breakpoint was regarded as a unique target sequence for ecdnaspecific labelling. the uniqueness of each breakpoint was validated by breakpoint-pcr (bp-pcr). furthermore, the location and the amount of each breakpoint were observed by breakpoint-fish (bp-fish) analysis in gbm cells. summary/conclusion: this results will be strong evidence to make ecdna-specific crispr system in further research. tracing ecdna dynamics will provide new insight into the impact of ecdna on cancer evolution. introduction: small extracellular vesicles (sevs) are - nm vesicles that mediate intercellular communication by transferring rna and proteins to the recipient cells. these cargo molecules are selectively sorted into sevs and mirror the physiological state of the donor cells. given that sevs can cross the bloodbrain barrier and their composition can change in neurological disorders, there is an increasing interest in elucidating the molecular signatures of sevs in circulation as disease biomarkers. however, circulating sevs are derived from multiple cellular sources and determining their source is challenging. information on sev composition can be beneficial in predicting whether these sevs are released predominantly from central nervous system cells. we hypothesized that differentially expressed mirnas between neuronal sevs and astrocytic sevs could be used as cell-typespecific signatures. methods: small extracellular vesicles were isolated from cell culture media of postnatal mouse primary neurons and astrocytes using differential centrifugation and characterized using nanoparticle tracking analysis, transmission electron microscopy and western blotting. rna from neurons, astrocytes, and their respective sevs were used for transcriptome and small rna sequencing. results: we observed that only a subset of cellular mirnas was packaged into sevs; differential expression of specific mirnas between sevs and their corresponding cells suggest that cells employ special mechanisms to sort mirnas into sevs. these mechanisms could be celltype specific since neuronal sevs showed a different mirna profile compared to astrocytic sevs. exomotifs, the short sequence motifs that control the loading of rna into sevs, were present in differentially expressed mirnas. we also observed that five rnabinding proteins, which are associated with passive or active rna sorting into sevs, were differentially expressed between neuronal and astrocytic cells. summary/conclusion: mirna signatures of sevs from neurons and astrocytes could be beneficial in determining if these cell types contribute to the alterations of sev composition in circulation in neurological disorders. cell-type-specific selectivity in rna loading might be attributed to the differential expression of rna-binding proteins. introduction: analytes present in the extracellular fraction of bodily fluids (ex. blood, urine) have utility as a tool for uncovering the molecular landscape of tumours and hold great potential for discovery of individualized cancer medicine. urine, being noninvasive as a sample type, has an obvious advantage over blood when used for liquid biopsy purposes. however, potential for microbial proliferation and the labile nature of host cells and extracellular vesicles (evs) at the point of sample collection/transport to the lab drives the need for stabilization of urine samples. development of such sample stabilization opens up capability for the detection of various biomarkers present in the extracellular fraction to be used in liquid biopsy. this is of particular concern as studies around urinary analytes for cancer diagnosis, progression and therapeutic effect are rapidly expanding in cohort sizes. multi-site collections and at-clinic collections are increasingly prohibitive for large scale recruitment and also lead to variability in the time between collection and processing. methods: in this study, we have analysed two commercially available ev extraction kits and compared them with ultracentrifugation technique for size, concentration and specificity of the isolated evs from human urine samples with and without our proprietary preservation solution using nanoparticle tracking analysis and western blot analysis for exosomal membrane markers. ev rna contents in various urine fractions (first morning first void, random first void and midstream) were compared using rt-qpcr assay to provide better understanding of the collection techniques and fractionations that are ideal for ev research work. results: in our current work, we have bench-marked human urine collection and ev extraction in order to provide recommendations in standardization of sample acquisition and processing for urinary ev studies. we have utilized these standardization in order to develop a novel and efficient sample stabilization principle for preservation of evs and ev rna in urine samples during an ambient temperature hold. summary/conclusion: taken together, we have established a framework for evaluating technologies and techniques in the ev sample processing space, which can be utilized by other research groups. vn -isolated plasma extracellular vesicles improve tumour mutation detection by next-generation sequencing compared to cell-free dna and correlate with tissue biopsy of nsclc patients introduction: liquid biopsy is a minimally-invasive diagnostic method that detects circulating biomarkers and has the potential to improve access to molecular profiling for nsclc patients when tissue biopsy material is unavailable or insufficient. although isolation of cell-free dna (cfdna) from plasma is the standard liquid biopsy method for detecting dna mutations in cancer patients, the sensitivity can be highly variable. vn is a amino acid peptide with an affinity for heat shock proteins that are exposed on the surface of extracellular vesicles (evs); peptide-ev aggregates readily sediment using a benchtop centrifuge and therefore the vn peptide provides a rapid, clinically-amenable procedure for ev isolation. in this study, we determine whether isolation of evs from nsclc patient plasma improves the sensitivity of single nucleotide variants (snvs) detection compared to cfdna and correlate genetic changes observed by liquid biopsy with tumour ffpe tissue biopsy. methods: blood was collected from stage iii/iv nsclc patients with informed consent in either edta or cell-free dna bct® collection tubes and plasma was harvested within minutes. total nucleic acid (tna) was extracted from either vn -isolated evs from edta plasma or directly from plasma collected in edta or cell-free dna bct® tubes (cfdna). snvs were detected by next-generation sequencing (ngs) results: vn isolation of evs from plasma resulted in higher recovery of dna than cfdna isolation. the snvs detected in both ev-dna and cfdna correlated well with those reported in matched ffpe tumour tissue using ngs, including % specificity for egfr mutations. no improvement in snv detection was observed using cell-free dna bct® collection tubes compared to edta tubes. isolation of evs with the vn peptide prior to sequencing improved a number of ngs parameters including library yield, total reads, median read coverage and molecular coverage, resulting in improved sensitivity of snv detection. summary/conclusion: in summary, our research demonstrates that vn -based ev isolation is useful for molecular profiling of nsclc patients for whom tissue biopsy is not an option, thereby improving access to molecular profiling and targeted therapies. funding: atlantic canada opportunities agency novel markers for neuroendocrine prostate cancer divya bhagirath a , michael liston b , theresa akoto a and sharanjot saini a a augusta university, augusta, usa; b veteran affairs, san francisco, usa introduction: prostate cancer (pca) is fuelled by androgens and androgen receptor (ar) signalling. therefore, ablation of ar signalling by androgen deprivation therapy (adt) is the goal of first-line therapy that results in cancer regression initially. however, two to three years post-adt, the disease develops into castration-resistant prostate cancer (crpc). as a second-line of therapy, next generation of ar pathway inhibitors (api) such as enzalutamide (enz) are used that are effective initially followed by emergence of drug resistance. a subset of api-resistant tumours emerges to an ar independent state via undergoing a trans-differentiation to neuroendocrine lineage, a process referred to as neuroendocrine differentiation (ned). due to lack of ar signalling, these pca variants, referred to as neuroendocrine prostate cancer (nepc), are impervious to anti-androgen therapy and constitute an aggressive variant of advanced crpc with poor prognosis. currently, there is a lack of effective molecular biomarkers for predicting api therapy resistance and emergence of therapy-induced ned. methods: exosomes/evs were isolated from sera of a patient cohort with/without ned. the study was conducted in accordance with ethical guidelines of us common rule and was approved by the institutional committee on human research. written informed consent was obtained from all patients. following extensive characterization of evs by electron microscopy, nanosight tracking analyses and western blotting of exosomal markers, small rna sequencing was carried out on illumina hiseq platform to identify differentially expressed transcripts. machine learning algorithms were applied to clinical sequencing data to train a "mirna classifier". further, we probed the proteomic profile of exosomes isolated from nepc cellular model nci-h and enzalutamide resistant crpc cell lines by mass spectrometry. results: we identified that transition from crpc-adenocarcinomas to neuroendocrine states is associated with significant ev-mirna dysregulation, with a specific dysregulation in certain mirna families. with the application of machine learning algorithm, we identified an ev-based "molecular classifier" that can robustly stratify crpc-ne tumours from crpc-adenocarcinomas. proteomic analyses identified novel nepc-specific, glycosylated proteins that can be exploited for nepc diagnosis. summary/conclusion: our data suggest that ev mirna and protein profile can predict neuroendocrine differentiation in advanced castration-resistant prostate cancer patients. exosomal mrna in diagnosis strategy for hepatocellular carcinoma aleksandr abramov, alisa petkevich, vadim pospelov and pavel ogurtsov peoples' friendship university of russia (rudn university), moscow, russia introduction: exosomal cargo is informative source illustrating the genetic events happening in cells, what can be especially advantageous in case of cancer development for disease progression or treatment effectiveness monitoring. methods: plasma samples of hepatocellular carcinoma (hcc) patients, plasma samples of patients with liver cirrhosis - on the hepatitis c virus (hcv) background, healthy donors' plasma samples. exosomes were isolated with ultracentrifugation, western blot (cd , cd ) was performed. total mrna was isolated with exosomal rna isolation kit, norgen biotec corp. sequencing was carried out on a minion sequencer. housekeeping genes (gapdh, b m, actb, tuba a). detected mutations were confirmed by real-time pcr with specific highly sensitive lna probes. results: significant changes in expression levels were identified for genes in hcc and liver cirrhosis groups (increasing up to x compared to control samples and decreasing up to no detected expression). in out of patients with hcc mutant burden was significant increased compared to mutant burden in groups with cirrhotic samples. in out of patients with hcc increased expression for mrna line- was identified compared to cirrhotic patients. summary/conclusion: exosomal mrna expression levels may serve as a prognostic and diagnostic marker for patients with liver cirrhosis caused by hcv for hcc risk development. funding: research is supported with federal funds " - " circulating extracellular vesicle signatures in small cell lung cancer michela saviana a , giulia romano a , giovanni nigita b , robin toft a , patricia le a , kai wang c , mario acunzo a and patrick nana-sinkam a a virginia commonwealth university, richmond, usa; b the ohio state university, columbus, usa; c institute for systems biology, seattle, usa introduction: lung cancer is the leading cause of cancer deaths worldwide and classified primarily as either non-small cell lung cancer (nsclc) or small cell lung cancer (sclc). compared to nsclc, sclc has a faster growth rate, earlier widespread metastasis, and shorter overall survival. the early diagnosis of sclc and the development of novel therapeutics have proven challenging. thus, progression and recurrence rates remain high. non-invasive methods for cancer detection are increasingly being used to inform clinical decision making. extracellular vesicles (evs) have recently emerged as potential carriers of genetic contents such as micrornas (mirs) to induce reprogramming of components of the microenvironment in cancer initiation and progression. moreover, extracellular mirs expression profiles have been shown to have signatures related to tumour classification, diagnosis, and progression. methods: we selected a cohort of patients divided into groups: high-risk smokers, adenocarcinomas, squamous carcinomas, and sclc. we extracted total circulating ev and plasma rna from plasma ( patients in total) and rna from plasma in a separate group ( patients in total). utilizing both next-generation sequencing (ngs) and nanostring platforms, we analysed for global microrna (mirs) expression patterns. candidate mirs were then validated by qrt-pcr. results: we identified several deregulated mirs in both evs and plasma of sclc patients compared to the other groups. for evs, we validated mir- - p as a significant biomarker for the late stage of sclc compared to controls. in the case of plasma, we validated the upregulation of mir- in sclc compared to controls. summary/conclusion: our results indicate that a potential combination of plasma (mir- ) and ev-based (mir- - p) mirs be valuable biomarkers for sclc detection and serve as a basis for a non-invasive sclc classifier. funding: virginia commonwealth university, doim -nih/nci introduction: the isolation of evs from milk is technically challenging due to the complexity of milk. currently used separation procedures allow for the removal of milk fat globules and cells (by low speed centrifugation of fresh milk), removal (by acidification), or disruption (by addition of edta) of casein micelles. using these protocols the integrity, composition and targeting of bovine milk evs has been evaluated and has led to believe that milk evs might withstand these conditions. however, the effects on functionality of milk evs (i.e. immunomodulatory properties) after processing and isolation have not been studied. therefore, we have set up an in vitro culture system using a human t cell line that allows for the rapid screening of milk ev functionality. methods: fresh bovine milk was defatted and cells were removed after x , g centrifugation, followed by differential centrifugation at , g and , g. this milk was either subjected to acidification with hcl, or edta was added, or the milk supernatant remained untouched. top down optiprep density gradient separation followed by sec was used to further purify evs. these highly purified milk evs were added to human jurkat t cells, which were simultaneously stimulated using anti-cd and anti-cd antibodies. after h t cell activation was measured by il- cytokine production. results: precipitation or disruption of casein micelles allowed for the substantial removal of proteins during isolation compared to directly isolated evs, which aids in the purification of milk evs. in vitro analysis revealed that in the presence of directly isolated, or edta isolated milk evs, jurkat cells were suppressed in their activation as measured by il- production. remarkably, evs isolated from hcl-acidified milk were impaired in their suppressive capacity to inhibit il- production. summary/conclusion: although casein removal from bovine milk greatly improves purity of isolated milk evs, the detrimental effects on ev functionality should be considered. interestingly, evs exposed to acidic conditions lost their ability to modulate t cell activation, which is in contrast with the general believe that milk evs could withstand the gastro-intestinal tract. funding: this work is funded by the european union's horizon framework programme under the grant fetopen- evfoundry. optimising methods for separation and characterisation of extracellular vesicles from skim milk and infant milk formula introduction: infant milk formula (imf) is intended to impart nutrition to infants, similar to breast milk. however, although industrial imf production involves harsh treatment, potential consequences on extracellular vesicles (ev) in imf are not yet established. this study aimed to optimise methods for separating evs from imf and skim milk (sm) and to characterise the evs in accordance with misev . methods: sm and imf were either not treated (nt) or treated with acetic (aa) or hcl acid (isoelectric precipitation, ip), to remove caseins. samples were then subjected to differential ultracentrifugation (duc) or gradient ultracentrifugation using iodixanol solution (guc). for duc, ml samples were centrifuged at k g, k g, k g, k g and k g sequentially for min each and pellets re-suspended in ml pbs. preparation of agarose microspheres for high-efficient separation of extracellular vesicles cheng-tai chen, chien-an chen, carolyn yen and nien-tzu chou industrial technology research institute, chutung, taiwan (republic of china) introduction: size exclusion chromatography (sec) is becoming a widely used technique for separating of extracellular vesicles. various commercially available products were launched on the market, however, their separation efficiencies were not fully disclosed. herein, novel porous agarose microspheres with the tunable diameter and pore size were synthesized by emulsion reaction. the performance was evaluated and compared with commercial products. the modified sec column packing materials were shown to exhibit advantages for rapid, high-recovery and high-purity separation of extracellular vesicles from cell culture-conditioned medium and human plasma. methods: the homemade sec column was packed by gravity flow. μl of the sample was loaded and the pbs buffer was used as eluent. factions were collected and analysed by cd /cd sandwich elisa assay and by micro bca assay for determining respectively extracellular vesicles and total protein content. results: agarose microspheres were prepared by emulsification. the particle size can be controlled by the types and concentrations of surfactants. the product was collected by desired screen meshes and used as packing materials of the sec column. our results showed that the extracellular vesicles were clearly separated from proteins. more than . % of proteins were removed while the recovery of extracellular vesicles was close to %, which is much higher than % of the commercial product. the total separation time was less than min. summary/conclusion: we have established an approach for generating spherical agarose microspheres as packing materials of homemade sec columns, which are capable of separating extracellular vesicles from complex samples with high efficiency. further validations with additional samples are currently ongoing. immunomagnetic sequential ultrafiltration (isuf) platform for enrichment and purification of extracellular vesicles from large and small volumes of biofluid eduardo reategui, jingjing zhang, luong t. h. nguyen, richard hickey, nicole walters and andre f. palmer the ohio state university, columbus, usa introduction: evs derived from tumour cells have the potential to provide a much-needed source of non-invasive molecular biomarkers for liquid biopsies. however, compromises have to be made when using a particular technology/methodology for the isolation of evs. currently, there is a trade-off between sample volume and specificity in ev isolation technologies that limits quantitative molecular analysis of ev contents, ultimately impacting the utility of evs in cancer diagnostics. here, we present an approach called immunomagnetic sequential ultrafiltration (isuf). our platform combines ultrafiltration and immunoaffinity separation. using isuf, we demonstrate that small or large volumes of biofluid can be processed (~ µl or > ml) while concomitantly removing . % contaminating proteins. we also processed serum from breast cancer patients enabling the characterization of different tumour and immune biomarkers on the isolated evs. methods: human samples were collected under an approved irb. size distribution and concentration of evs were measured using a tunable resistive pulse sensing (trps) method. ev proteins and rnas were extracted and quantified using a bca protein assay and uv spectroscopy. isuf and other ev isolation methods were compared for ev concentration, protein, and rna quantity. results: ml of cell culture media (ccm), . ml serum, and ml urine samples were processed with the isuf platform and recovered in µl. for all cases, evs were enriched with recovery efficiency greater than %. the processing time for a ml sample was min with over % of purity. we compared ev concentration and purity isolate from . ml serum using isuf and other commercially available methods, isuf demonstrated superior performance on isolating evs at high concentrations and purities. analysis of total rna amounts in the isolated evs using different methods was corresponding to higher ev recovery efficiency of isuf. we also compared protein and rna levels of evs enriched with isuf present in urine and serum samples from the same donors (n = ), and we found that for the same number of evs, the ev rna concentration from both biofluids showed no significant difference. finally, we have processed serum samples from metastatic breast cancer patients and demonstrated that their isolated evs have expression levels of her , cd and mir biomarkers at significantly higher levels than healthy controls. summary/conclusion: the isuf platform can be scale-down or -up to work with small or large volumes of biofluids for the isolation of evs. using the isuf platform with clinical samples shows the potential of our platform to be used for cancer diagnosis or monitoring treatment response. funding: national institutes of health (nih) grants ug tr (e.r.); r hl , r hl , and r eb (afp). challenges in exosomes isolation from primary biological samples derived from multiple myeloma patients introduction: multiple myeloma (mm) remains incurable despite advances in its treatment and research progress on the crosstalk between mm and surrounding host cells. exosomes are important regulators of the cellular niche. their importance for diagnostic and therapeutic applications has been proven in many cancers. in this context we hypothesized that a better understanding of the molecular role and features of mm-derived exosomes would provide a basis for their use for both risk stratification and as predictive biomarkers of response to anti-mm drugs already in use in clinical settings, given the optimization and validity of their isolation/purification method. methods: exosomes were isolated from human mm cell lines (hmcls) supernatants and peripheral blood plasma (pbpl) isolated from healthy donors, mm and mgus (monoclonal gammopathy of undetermined significance) patients. both fresh and frozen samples were tested. we evaluated commercially-available kits, density-based separation and ultracentrifugation. results: higher purity and recovery, evaluated by western blotting, nanoparticle tracking analysis and electron microscopy, were observed for supernatant density-based purification and for pbpl resin-based isolation. exploring the function of mm-derived exosomes, we observed an increase in proliferation of the immortalized stromal cell (sc) line hs treated with exosomes when compared to untreated cells, and a higher increase in proliferation of scs treated with mm-exosomes when compared to exosomes derived from normal and mgus pbpl samples. summary/conclusion: the method of isolation represents a critical step in the study of exosomes as many factors can affect the purity, yield and downstream application. our data demonstrated that density and resin-based isolation methods provided functional mm-derived exosomes with proliferative effects on scs. altogether our findings may serve as a guide to choose exosome isolation methods for mm studies. further optimization steps, including albumin-depletion from plasma samples and use/type of serum in cell cultures, should be taken into consideration when planning proteomics and genomics as downstream applications. funding: australian government rtp and monash departmental scholarship. a rigorous method for exosome isolation from post-mortem eyes introduction: in order to determine and validate the tissue-specific content of extracellular vesicles (evs) in biofluids, robust ev isolation methods from tissues must be developed. however, to date very few rigorous methods to isolate or enrich for intact evs from tissues have been reported. we present a comprehensive exosome isolation method with a sufficient level of characterization to unequivocally demonstrate true ev identity from ex vivo eyes. methods: iodixanol (optiprep) buoyant density gradient ultracentrifugation (dguc), cushioned dguc (c-dguc), and our newly developed c-dguc immunocapture (c-dguc-ip) method were used to compare yield and enrichment of exosomes isolated from porcine eyes between to hours post-mortem. yield was assessed by nanoparticle tracking analysis (nta) and immunoblotting for exosomal markers along with total protein quantitation. enrichment was assessed by comparison of exosomal markers, ocular-specific markers and known contaminant markers, plus in-depth proteomic mass spectrometry analyses. results: high enrichment of posterior eyecup small evs (sev) were achieved by dguc and c-dguc, with c-dguc resulting in an eightfold increase in yield by nta and two to fivefold increases of exosomal protein markers such as syntenin- and cd by immuno-blotting compared to dguc. interestingly, in-depth proteomic analyses revealed that a majority of these sevs with densities of . - . g/ml isolated by dguc and c-dguc were likely of endoplasmic reticulum (er) and golgi origin, suggesting er-to-golgi transport vesicles resulting from post-mortem tissue cell rupture. in order to enrich further for sevs (including exosomes) we subjected sevs isolated by c-dguc to anti-cd immunocapture. the resulting sev proteome was enriched . -to -fold for bona fide sev and exosome markers compared to c-dguc. summary/conclusion: the c-dguc method provides an enhanced yield and purity of sevs and exosomes from ex vivo eye tissue. however, to avoid significant contamination with er and golgi-derived vesicles from postmortem eyes, a final ev-specific immunocapture step is required to achieve sufficient purity for subsequent analyses. our highly rigorous method paves the way for identification and validation of ocular-derived exosomes in blood and their potential use as eye disease biomarkers. characterization of the extraction of extracellular vesicles using a lab-ona-disc filtration system introduction: personalized treatment for cancer is a promising way to face the multiplicity of the disease, to increase the efficacy of drugs and to decrease their toxicity. as part of this strategy, liquid biopsy explores a new non-invasive approach to diagnose cancer, guide treatment and monitor its efficacy. extracellular vesicles (evs) are nanometric lipid bilayers micelles with high potential as biomarkers. they are involved in the transfer of information (proteins, rna and dna) between cells. evs include a broad spectrum of particle sizes, from the tens to thousands of nanometres. the isolation of evs from complex matrices is the first step of any protocol and is particularly important for the reproducibility and fidelity of the results presented, as it could bring bias in further analysis. in order to explore the heterogeneity of evs, a full characterization (physical and biological) of the extracted evs is needed. we evaluate and compare evs purification methods, including ultracentrifugation, sizeexclusion chromatography (sec) column and an emerging microfluidic technology: labspinner filtration labon-a-disc device isolating evs between two filters of and nm. methods: a cell supernatant was used as a model matrix. we compared three methods of extraction of evs: ultracentrifugation with two cycles of h at , g at degrees celsius (rotor type ti, beckman floor ultracentrifuge optima l k), qev size exclusion chromatography columns from izon (qevoriginal/ nm) and lab-on-a-disc filtration system (labspinner, exodisc c). evs characterization was conducted with nta (nanosightns ), trps (izon), nanodrop (spectrometernd ), tem (fei tecnai kv) and custom micro-immuno-assay. results: in this study, we characterize a filtration system made of two serial filters of nm and nm pores for isolation of evs. compared to ultracentrifugation and chromatography columns, yield of extraction is up to times higher and the size of the extracted particles is smaller. tem imaging was used for assessment of the quality of the extracted evs. however, albumin concentration measurement tends to show that the purity of the solution is decreased. the immuno-labelling analysis shows that the proteomic signature of the extracted evs differs according to the extraction methods. the new filtration technology seems to give us access to a broader range of evs compared to standard methods. summary/conclusion: in this study, we characterized purification methods including lab-on-a-disc filtration, and were able to demonstrate an increase of the concentration of evs by a factor of , a decrease of the size of the accessible extracted particles and access to new proteomic signatures. funding: we acknowledge the support of génome québec and action marie skłodowska-curie. effects of sample processing on isolation of extracellular vesicles from blood plasma by centrifugation darja božič a , matej hočevar b , veno kononenko c , marko jeran a , urška Štibler d , immacolata fiume e , manca pajnič f , ljubiša pađen f , ksenija kogej g , damjana drobne c , ales iglič h , gabriella pocsfalvi i , veronika kralj-iglič f and darja bozic j introduction: the isolation of extracellular vesicles (ev) from body fluids is still controversial and the poor understanding of vesicle stability and effects of sample processing is probably one of the core issues preventing the breakthrough of this field into applicative practices. methods: we performed an in-depth study of sample changes in blood, blood plasma and samples throughout the increasing speed of centrifugation, considering the number, size, contents and shape of particles in the isolates. flow cytometry, light scattering, mass spectrometry and scanning electron microscopy were employed to reveal the properties of material in the samples. results: the particles of size about - nm with characteristic topology of membrane vesicles without internal structure were observed by the scanning electron microscope only in ev isolates prepared from fresh blood sample. inspection of the tube surface in which the isolation took place suggests that those particles are likely formed from activated platelets tearing at the tube wall due to the centrifugal pull. the isolates prepared from frozen blood plasma prepared by centrifugation with different forces contained different amounts of particles with similar protein contents, predominated by highly abundant human plasma proteins, including albumins and immunoglobulins. some lipoprotein clearance and fibronectin precipitation were however observed through increased speed and time of centrifugation. summary/conclusion: the results of this study [ ] contribute to the understanding of stability and dynamics of membrane particles. the reported evidence provides the support for viewing ev isolates as a product, shaped by uniqueness of the starting samples and the thermal and mechanical stress applied upon processing. we believe this kind of insights strengthen our ability of reading the story of evs. introduction: apoptosis is a form of programmed cell death with diverse roles in the tumour microenvironment and emerging data show that, besides its role in tumour suppression, it can also promote oncogenic proliferation. highly aggressive tumours such as burkitt lymphoma (bl) show high levels of apoptosis, which has a diagnostic and prognostic value for classifying and staging the disease. we hypothesize that amongst other elements, extracellular vesicles (ev) are key mediators of apoptotic cell-derived tumour microenvironment signals. here, we report on ev released in vitro by apoptotic bl cells (apo-ev) in relation to their potential use as cancer biomarkers. methods: basic physical properties of apo-ev such as structure, size distribution, surface charge and membrane fluidity are discussed using cryo electron microscopy (em) and tomography, nanoparticle tracking analysis, dynamic light scattering and fluorescence anisotropy respectively. for phenotypic analysis we apply immunocapture and flow cytometry, immunogold labelling on transmission em, fluorescence microscopy and quantitative pcr. in addition, we study the interaction of apo-ev with blood components such as platelets, leucocytes and red cells, in order to understand their effects in the circulation and therefore their potential for analysis in blood samples. results: looking at the differences between apo-and non-apo-ev, apo-ev have larger diameter, while structurally are not different. however, we have identified distinct apo-ev markers such as active caspase and histones, or dna and small non-coding rna-y. there is also strong interaction of ev with platelets and leucocytes but not with red cells, indicating potential routes of transfer of ev cargo in the circulation. summary/conclusion: it is concluded that for the characterization of the heterogenous ev populations, combination of multiple techniques is often required, and also, understanding the strengths and limitations of each method is essential for choosing the appropriate set of analytical tools. finally, we consider that monitoring free circulating apo-ev or blood cells with which they have interacted is a promising approach to improve cancer diagnosis, prognosis and evaluation of therapeutic response. casting a small netrin: functional roles of a novel surface factor on stroma-derived extracellular vesicles in pancreatic cancer kristopher s. raghavan a , ralph francescone b , janusz franco-barraza b and edna cuckierman b a drexel university; fox chase cancer center, philadelphia, usa; b fox chase cancer center, philadelphia, usa introduction: pancreatic ductal adenocarcinoma (pdac) is a devastating disease driven and supported by changes in its microenvironment, or stroma. here we dissect the intercellular communication that exists between the primary stromal component, cancer-associated fibroblasts (cafs) and pdac. pdac communicates with its microenvironment, in part, through the exchange of specific types of extracellular vesicles (evs). specifically, we focus on the mechanism by which caf-secreted evs support pdac survival, with an additional goal to identify biomarkers suitable to generate a future "liquid biopsy" test for early pdac detection and prognosis. methods: evs are isolated from patient-derived pdac-associated fibroblasts via differential ultracentrifugation and validated by isev standards. human pdac cell lines used as recipient cells are treated with caf-evs to assess their role in supporting pdac survival. recombinant proteins, neutralizing peptides, and non-functional mutant proteins are used to block ev interaction with target cells. results: we observe sub-types of caf-evs containing unique surface receptors. one ev sub-population of interest contains a novel surface protein (nsp) expressed on the plasma membrane of pancreatic cafs, but not their healthy counterparts. further, pdac cells up-regulate nsp's lone binding partner, suggesting a role for these factors in pdac-selective ev uptake. functional assays designed to test pdac viability suggest these nsp(+)-evs protect pdac cells from programmed cell death as a result of physiological stress. this ev-mediated survival benefit can also be inhibited by blocking the interaction of nsp and its binding partner, suggesting the engagement of these two factors is necessary for cafs to support pdac via evs. pursuing our biomarker goal we confirm stromal nsp expression increases during early panin stages prior to tumour development, and we are currently seeking to validate nsp(+)-evs in blood of pdac patients. summary/conclusion: this research shines light on a novel mechanism of tumour-stroma communication that may be crucial for cancer progression during early disease stages and a potential target for disrupting the supportive role of the tumour microenvironment. additionally, we describe a sub-population of nsp (+)-evs that have the potential to serve as biomarkers for identifying pdac development. exosomes carry distinct mirnas that drive medulloblastoma progression introduction: extracellular vesicles (evs) represent an ideal source of functional biomarkers due to their role in intercellular communication and their ability to protect cargo, including rna, from degradation. the most investigated ev's are exosomes, nanovesicles secreted by all cell types and able to cross the bloodbrain-barrier. here we characterised the rna of exosomes isolated from medulloblastoma cell lines, with the aim of investigating exosomal rna cargo as potential functional biomarkers for medulloblastoma. methods: exosomes derived from a panel of matched (original tumour and metastasis) medulloblastoma cell lines were isolated and characterised by nanosight, electron microscopy, western blotting and nanoscale flow cytometry. exosomal mirna and mrna from our matched cell lines and foetal neuronal stem cells, which were used as a normal control, were analysed by rna-sequencing technology. results: based on hierarchical clustering, malignant derived exosomes were distinctly separated from normal control exosomes. mirna profiling revealed several established oncomirs identified in our malignant derived exosomes compared to control samples. using interaction pathway analysis, we identified that our malignant exosomes carry numerous mirnas implicated in migration, proliferation, cellular adhesion and tumour growth. several previously identified oncomirs were also identified to be present at higher levels in metastatic exosomes compared to primary and normal, including hsa-mir- - p and hsa-mir- a- p. summary/conclusion: this study shows that exosomes from mb cells carry a distinct mirna cargo which could enhance medulloblastoma progression. the use of circulating exosomes as markers of metastatic disease could be an innovative and powerful noninvasive tool. introduction: inflammatory changes in the bone marrow (bm) and suppression of haematopoietic stem and progenitor cell (hspc) function during acute myeloid leukaemia (aml) significantly contribute to patient morbidity and mortality. our laboratory has previously shown that aml-derived extracellular vesicle (ev-aml) trafficking confers a state of enforced quiescence and leads to lasting dna damage in hspcs. here we explore the underlying cause. specifically, we hypothesize that ev-aml incite inflammatory regulators as potential mediators of dna damage. methods: as a validated model of aml, we utilized the murine tib cell line as a source of ev-aml. ev-previous work has indicated that mirnas, notably mir- a and mir- , play a critical role in scc tumour development. evs are membrane-bound vesicles involved in cell-cell communication carrying actively sorted cargo, protected from degradation. the potential pathways these vesicular mirnas modulate and the implication they have on cancer biology is under active investigation. we have previously shown that the cadherin dsg , a stem cell marker, modulates ev release. dsg is upregulated in a number of cancers, including scc, and correlates with poor prognosis. here we aim to elucidate the impact of ev-associated mirnas in sccs by bioinformatic analysis. methods: scc cells stably expressing dsg were generated and evs isolated by sequential ultracentrifugation. total cellular and ev rna was isolated by mirneasy, analysed using rnaseq and identified by grch alignment. results were confirmed by qpcr. altered pathways based on targets were identified using mirnet and kegg pathway analysis. potential cancerassociated cytokine targets were confirmed by antibody array. results: rnaseq revealed cellular and ev mirnas that were differentially expressed in response to dsg with overlapping. the highest altered mirnas were validated by qpcr. kegg pathway analysis determined that these mirnas have the highest number of shared targets in cancer, cell cycle, and p signalling pathways. interestingly, mir- was upregulated while mir- a was dramatically downregulated in evs. targets of mir- a, icam- , il- , and il- , cytokines critical for cancer progression were upregulated. summary/conclusion: these results suggest that the mirna content of evs is tightly regulated. by altering the mirna profile, dsg contributes to the pathogenicity of these evs by increasing levels of cytokines important for cancer stem cell renewal and metastasis. in addition, these mirnas may serve as non-invasive diagnostic markers for sccs. funding: nih r cancer cells grown in d release distinct extracellular vesicles during tumour growth and invasion jens c. luoto, sara bengs, leila coelho rato, lea sistonen and eva henriksson Åbo akademi university, turku, finland introduction: cancer cells secrete extracellular vesicles (evs) that affect tumour progression. the characteristics of evs produced during tumour growth and invasion are however poorly understood. in this study, we identify the composition and characteristics of evs produced by noninvasive and invasive tumours and correlate these characteristics with the invasive status of the tumour. for that purpose, we established a protocol for isolating evs from extracellular matrix (ecm)-based three-dimensional ( d) cancer cell cultures. methods: human prostate cancer pc cells were grown in d cultures using ecm-based hydrogel, in standard d culture conditions and in bioreactor. evs were isolated from these cultures with differential and density gradient centrifugation. the isolated evs were characterized with nanoparticle tracking analyses, electron microscopy, immunoblotting and mass spectrometry (ms). results: our results demonstrate that d ecm-based hydrogel cell cultures secrete evs that can be isolated from both the conditioned media and the hydrogel. the invasive d cultivated pc organoids were found to secrete large amounts of evs compared to the non-invasive organoids. interestingly, our ms results revealed that non-invasive and invasive organoids secrete evs with partially distinct protein cargo. summary/conclusion: we have established a novel protocol for ev production in a d cell culture system utilizing ecm-based hydrogel, in which invasive tumour growth can be mimicked. our method allows the specific isolation and characterization of evs derived from different stages of d culture, such as non-invasive and invasive organoids. importantly, we found that tumour-derived evs change in composition during the tumour progression. taken together, our method can be used to define the distinct ev characteristics involved in cancer invasion. we previously showed extracellular vesicles (evs) to be causally involved in transmitting drug resistance. this study aimed to evaluate compounds proposed to reduce/block ev release. specifically, we selected calpeptin and y (proposed to inhibit evs budding from the cell membrane) and manumycin a and gw (proposed to inhibit evs deriving from mvbs). associated effects on -and consequences of-ev release were then investigated. methods: suitable compounds concentrations that were non-toxic to cells were first selected by performing cytotoxicity assay and flow cytometry (fc). conditioned medium (cm) was collected from docetaxel-resistant pc (pc rd) cells after h incubation in dfbs-medium with or without the compounds. evs were separated from tangential flow filtration concentrated cm using optiprep density gradient. . - . g/ml fractions were then pooled and washed. evs were characterised using nta, immunoblot, tem and lipid assay and fc. influences on growth and migration, of evs continuing to be released (at x evs/ x cells, x evs/ x cells), were evaluated on recipient du and rv cells. evtrack id ev , score % results: calpeptin and y , alone and in combination, did not significantly affect quantities of evs released. however, gw significantly (p < . ) increased quantities of released evs, of a larger size; very high protein to lipid ratio; and carrying grp compared to control evs (p < . ). this effect was reverted when gw was combined with manumycin a (p < . ). following all compounds treatments, x evs/ x cells inhibited rv proliferation (p < . ), while at x evs/ x cells only evs from manumycin a (p < . ) and y (p < . ) treated cells reduce rv proliferation. evs following gw treatment significantly (p < . ) inhibited du migration compared to bulk non-treated control and compared to the effect obtained using the entire pool of evs (p < . ). summary/conclusion: while none of the proposed inhibitors significantly reduced ev release, the resulting evs were less potent in transmitting aggressive behaviour, such as proliferation and migration, to receiving cell lines. patient-derived organoids represent a novel tool to study the effect of intra-tumoral heterogeneity on ev release in non-small cell lung cancer introduction: lung adenocarcinoma (luad) is the leading cause of cancer-related death with a low -year survival. although the importance of intra-tumoral cellular heterogeneity of solid tumors in the clinical outcome and treatment is emerging, proper models to study its effects on ev release and cargo in human tissues still lack. the d organoid technology maintains the cellular and genetic heterogeneity of in vivo tissues and has proved to be so far the best ex vivo model of human cancers. by using patient-derived and mouse organoids we set out i) to compare the ev release from normal and tumor tissues and ii) to follow changes in ev secretion when the relative ratio of tumor cell subpopulations is shifted. methods: we used mouse and luad patient-derived normal and tumor organoids. the medical research council of hungary approved our experiments with human samples and informed consent was obtained from patients. evs were detected by antibody-coated beads, nta and tem. intra-organoid heterogeneity was proved by immunostaining and rt-qpcr. results: we provide evidence that both mouse and human normal organoids contain all the bronchiolar cell types. interestingly, luad organoids selected for tp mutation contained not only ki + proliferating cells, but differentiated cell types as well. furthermore, all the lung organoid cultures produced evs and this was shifted to the smaller size range. interestingly however, when modifying the proportion of organoid cell types, we observed an increased ev release when more ki + proliferating cells were present both in normal and in luad samples. summary/conclusion: our data show that patientderived lung organoids represent a novel model to study the role of intra-tissue heterogeneity in ev functions in the humans, leading to improved diagnosis. funding: this work was funded by the national competitiveness and excellence program nvkp_ - (national research, development and innovation office, hungary) and by the national excellence program in higher education (ministry of human resources, hungary). exosome mediate heart-adipocyte communication after myocardial ischaemia/reperfusion and impairs adipocyte endocrine function yajing wang, lu gan, dina xie, wayne lau, theodore christopher, bernard lopez and xinliang ma thomas jefferson university, philadelphia, usa introduction: by incompletely understood mechanisms, mi patients sustain systemic metabolic disorder. adipocytes are an important cellular type regulating energy homoeostasis. the impact of mi upon adipocyte function remains unknown. exosomes (exo) are critical vehicles mediating organ-organ communication. however, whether and how exo may mediate post-mi cardiomyocyte/ adipocyte communication have not been previously investigated. methods: adult male mice were subjected to mi/r. serum exo were isolated hours after r and incubated with t l cells for hours. the effects of exo upon adipocyte function were determined. results: compared to control, mi/r exo significantly altered the expression of genes known to be important in adipocyte function. go analysis revealed that genes associated with endoplasmic reticulum (er) function and adipocyte endocrine function are the primary two pathways altered by mi/r exo. venn analysis identified mi-rnas as cardiac-enriched, adipocyte-poor, and er function-related mirnas. rt-qpcr confirmed the mir- a/ a/ - family members are the most markedly increased mi-rnas in mi/r exo. incubation of t l cells with mi-r a mimic significantly downregulated edem , dsba-l, and pparn, and upregulated perk and chop. conversely, mi-r a inhibitor significantly decreased the impact of mi/r exo upon er function genes. additional studies demonstrated edem and pparγ (two critical molecules maintaining er function and adipocyte endocrine function) to be direct targets of mi-r a. one of the most significant endocrine molecules of adipocyte origin, adiponectin is regulated by pparn at the transcriptional level and by dsba-l at the post-translational level. we next determined whether mi/r exo may affect adiponectin expression/ assembly. incubation of t l cells with mi/r exo significantly inhibited total and high molecular weight adiponectin expression, an effect blocked by mir a mimic. finally, in vivo administration of gw (exo biogenesis inhibitor) or mir a inhibitor attenuated adipocyte er dysfunction and restored plasma adiponectin level in mi/r animals. summary/conclusion: we demonstrate for the first time that mi/r causes significant adipocyte er and endocrine dysfunction by exo mediated cardiomyocyte/adipocyte communication via mir- a/ a/ - . funding: nih and american diabetes association pancreatic cancer cell extracellular vesicles drastically alter the behaviour of recipient normal pancreas cells charles p. hinzman a , yaoxiang li a , meth jayatilake a , jose trevino b , partha banerjee a and amrita cheema a a georgetown university medical center, washington, usa; b university of florida health science center, gainesville, usa introduction: pancreatic cancer (paca) is predicted to become the rd leading cause of cancer-related deaths by . patients diagnosed with pancreatic ductal adenocarcinoma (pdac) have a -year survival ratẽ %. detection of pre-neoplastic lesions can potentially improve survival. however, there is currently no screening test for early stage detection. importantly, paca tumours are % non-tumorigenic cells. a better understanding of early paca oncogenesis is needed. cancer cells shed extracellular vesicles (evs) that are internalized by neighbouring and distant cells to induce a myriad of cancer progression events. we hypothesize that in early paca oncogenesis, evs mediate a behavioural change in surrounding normal cells, leading to the formation of this unique stroma. the purpose of this study was to develop a model to examine the phenotypic changes undergone by normal human pancreas cells when they are exposed to paca cell evs. methods: evs were isolated using differential ultracentrifugation with filtration from established (panc- , sw- , capan- and miapaca- ) and patientderived xenograft (ppcl- and ppcl- ) paca cell lines. cells were grown using ev-depleted fbs. ev isolations were validated and quantified using transmission electron microscopy, quantitative elisa, immunoblot and nanoparticle tracking analysis. normal pancreas cells (htert-hpne and hpde-h c ) were co-cultured with cancer cell evs for - hours. metabolic activity was measured using a mito stress test on a seahorse xfe extracellular flux analyser. results: we discovered that normal cells undergo vast behavioural transformations, including significant morphological changes, increased proliferation and an uncharacteristic invasive capability, when co-cultured with paca cell evs. these responses were ev dose dependent. further, paca cell evs metabolically reprogrammed normal cells, causing a bioenergetic switch, from a quiescent, aerobic profile to a highly energetic and glycolytic profile. summary/conclusion: our results indicate that paca cell evs confer enormous transformational properties to normal human pancreas cells in vitro. we hypothesize that evs impart distinct transformational properties to normal cells in vivo and this influence could unveil novel mechanisms regulating cancer onset and progression. these signals may be detectable before progression of early-stage paca to pdac, leading to the development of assays for earlier diagnosis in patients. further studies are underway to identify the biochemical mediators of these changes. plasma extracellular vesicles-mirnas released by hypoxic cells are associated to pro-tumorigenic and immunosuppressive microenvironment in lung cancer introduction: extracellular vesicles (ev) containing specific subset of functional biomolecules, such as micrornas (mirnas) are released by all cell types. it has been widely demonstrated that ev-mirnas from cancer cells can manipulate the tumour microenvironment modulating the gene expression of recipient cells. we previously identified a three levels risk classifier (msc) based on plasma-mirnas associated with lung cancer development and prognosis. the aim of this study was to investigate the potential role of ev- mirnas as mediators of pro-tumorigenic features. methods: evs were isolated from plasma of heavysmoker individuals with high (mscpos) or low (mscneg) risk of lung cancer by differential centrifugation method. purity of evs isolated was confirmed by sizing using nta and tem analysis and expression of ev-enriched proteins. mirna levels were analysed by dpcr. in vitro and in vivo analyses were used to assess the biological effect of plasma evs on different recipient cells. results: levels of mirnas in evs correlated with determination of whole plasma ( % of correlation with msc classifier). mscpos-evs stimulated d and d proliferation of non-tumorigenic epithelial cells through c-myc transfer into recipient cells. furthermore, mscpos-evs increased the ability of huvec to form tubular structures compared to mscneg-evs. in vivo co-injection of mscpos-evstreated huvec with a lung cancer cells resulted in an increase of tumour growth compared to mscneg-evs-treated huvec. mir- modulation in evs with mirna mimics or in recipient cells using mirna inhibitors demonstrated that this mirna is implicated in the autocrine proangiogenic modulation of huvec phenotype. mscpos-evs induced m polarization of macrophages both in vitro and in vivo. we demonstrated using synthetic oligonucleotides that mir- is responsible for the immunosuppressive modulation of these cells. regarding the potential origin of evs in mscpos individuals, we observed that hypoxia stimulated the secretion of evs containing c-myc from fibroblasts, mir- -evs from endothelial cells and mir- -evs from granulocytes. summary/conclusion: these data show that plasma evs of high-risk individuals display pro-tumorigenic features, as documented by their ability to induce a pro-angiogenic and immunosuppressive microenvironment suggesting an involvement of evs in lung cancer development. exploration of ev-associated metastatic targets on melanoma cells introduction: cancer cells secrete evs that may harbour metastatic proteins. previous studies have demonstrated the decrease of cd tetraspanin expression in melanoma cells are correlated with enhancing its metastatic potential. however, other proteins, such as cd , cd , met and nrp which are overexpressed in melanoma cells, are also associated with the spread of cancer. in this study, we sought to investigate the expression of metastatic proteins in evs derived from murine melanoma b f lineage. methods: b f cells were cultured in dmem supplemented with % fbs and antibiotics. the cells supernatant were harvested each hours, filtered through . µm and ultracentrifuged at x g for min at ºc to pellet evs. next, size and concentration was determined using nanoparticle tracking analyses technique, and the morphology of evs were analysed by negative-staining transmission electron microscopy (tem). the ev's surface protein were characterized by flow cytometry and protein content was profiled by mass spectrometry. results: our flow cytometry results have shown the presence of tetraspanins markers cd , cd and cd on vesicle´s surface. in addition, our assay demonstrated a diminished expression of cd molecule in comparison to cd and cd . we have profiled melanoma-evs by mass spectrometry, identifying the presence of proteins that may be associated to metastasis, such as cd , cd , met and nrp . summary/conclusion: these preliminary results are consistent with the literature and suggest that melanoma-derived evs harbour proteins, which may contribute to promote tumour metastasis. in our next step, we plan to generate b f lineages harbouring shrna vectors, in order to knockdown gene expression of cd , cd , cd , met and nrp to investigate the metastatic potential in vitro, in comparison to parental cells. we also may use syngeneic mice models to explore metastatic potential of genetically modified b f -derived cells. introduction: overexpression of her occurs in % of breast cancers and confers aggressive behaviour and poorer prognosis. thankfully, a number of drugs such as neratinib have been developed to target her , potentially providing substantial benefit for many patients. nevertheless, it is estimated that up to % of patients with her -overexpressing tumours do not gain benefit, as a result of innate or acquired drug-resistance. this study aimed to investigate if nano-sized membrane-surrounded extracellular vesicles (evs) released from drug-sensitive and drug-resistant cells reflect the her status of their cells of origin and thus have potential as minimallyinvasive biomarkers. methods: evs were isolated from conditioned media (cm) of her -positive cell lines (hcc and skbr ) and their neratinib-resistant counterparts (hcc -nr and skbr -nr) that we developed in our laboratory. evs from cm of a triple-negative breast cancer (tnbc) cell line variant, hs ts(i) , were evaluated as negative control for her . in brief, cm was centrifuged at g, for min x to get rid of any cells. the supernatant was then centrifuged at , g for h at ºc to collect evs. the resulting evs were washed in pbs, centrifuged as before, and resuspended in μl pbs. ev quantities were estimated by nanoparticle tracking analysis (nts). ev lysates were characterised by immunoblots, for established positive and negative ev markers. particle concentration as well as size distribution of evs were measured using the zetaview (particle metrix, germany). surface proteins on single evs were analysed by highresolution flow cytometry (fc), using an amnis imagestreamx mark ii. all data was submitted to ev-track (ev-track id: ev ). results: neratinib-resistant cell line variants were found to have reduced her protein expression compared to their respective drug-sensitive counterparts. neratinib-resistant cell line variants released fewer evs, when normalised per number of secreting cells, compared to their-drug sensitive counterparts. furthermore, evs from drug-sensitive cells carried her , while those from drug-resistant cells lacked her (similar to the evs from the tnbc cells); reflecting the her status of their cells of origin. summary/conclusion: this study indicates that a reduction in her protein expression is a mechanism by which cancer cells manifest resistance to her -targeted drugs (i.e. by making fewer her receptors available on the cells surface to accommodate the drugs activity). furthermore, ev-carried her seems to reflect the her status of their cells of origin. this suggest that analysis of her on evs in the peripheral circulation may help predict response to her -targeted drugs. thus, analysis of evs in appropriate cohorts of patients' specimens is warranted. introduction: rab a, a small gtpase involved in exosome biogenesis by regulating mve docking at the plasma membrane, and its expression level highly correlated with ohsv replication ability in vitro. oncolytic viruses is a newly promising therapeutic agent for cancer treatment. however, more than % of tumours naturally showed highly resistant to oncolytic viruses for unknown reasons. uncovering the underlying mechanisms of resistance to ohsv can offer potential therapeutic targets to enhance ohsv activity to kill tumour cells. in addition, it will give new insights into the identification of therapeutic biomarkers, which can be used to predict patient response to ohsv in clinical. methods: deep-sequencing data showed that lower expression level of rab a is present in ohsv resistant tumour cells compared to that in sensitive tumour cells. then an ohsv resistant mc tumour cell line was established by repeated injections with ohsv in mc tumour-bear mouse model. lastly, it was verified that ohsv resistance is associated with a downregulation of rab a and overexpression of rab a can rescue ohsv replication. results: ) the lower expression level of rab a is shown in ohsv resistant tumour cells compared to that in sensitive tumour cells shows in deep-sequencing data. ) furthermore, we established an ohsv resistant mc tumour cell line by repeated injections with ohsv in mc tumour-bear mouse model. similarly, in ohsv resistant mc cell line, rab a expression was lower than parental mc cells. and the release of exosomes and virus was decreased in ohsv resistant mc cell line. these results were confirmed by rab a sirna knockdown. ) we verified that in ohsv naturally resistant human cancer cell lines, ohsv resistance is associated with a downregulation of rab a and overexpression of rab a can rescue ohsv replication. summary/conclusion: downregulation of rab a expression restricts the efficiency of ohsv replication and the spreading ability of the released progeny virus which also provide rab a as a potential target and biomarker for ohsv cancer therapy. funding: these studies were supported by grants from shenzhen overseas high-calibre peacock foundation kqtd , shenzhen science and innovation commission project grants jcyj , jcyj to shenzhen international institute for biomedical research. inactivation of emilin- by proteolysis and secretion in extracellular vesicles favours melanoma progression and metastasis introduction: studies have demonstrated that melanoma-derived extracellular vesicles (evs) home in distal organs and sentinel lymph nodes favouring metastasis. although lymph node metastases are themselves rarely life threatening, they could be considered as one of the first step of metastasis in many cancer types. therefore, defining the mechanisms involved in lymph node metastasis and pre-metastatic niche formation in lymph nodes could bring the clue to block the metastatic process from the beginning. methods: we have characterized secreted exosomes from a panel of mouse melanoma models representative of low metastatic potential (b -f ), high metastatic potential (b -f ) and lymph node metastasis (b -f r ). we analysed the rna expression in cells and protein content of exosomes derived from mouse melanoma lymph node metastatic models by rna sequencing and mass spectrometry respectively. we validated expression by western-blot, qpcr and immunofluorescence. to define the mechanism of emilin- secretion cells were treated with the ev secretion inhibitor (non-competitive inhibitor of sphingomyelinase), gw . cell viability and cell cycle assays with an overexpression model (b -f -emilin- ) were also performed. in vivo experiments based on subcutaneous and intrafootpad injection for studying the role of this protein during melanoma progression were performed to define the relevance of our findings in vivo. human paraffin samples were analysed for emilin- expression by immunohistochemistry. results: we found a signature of over-expressed genes and hyper-secreted proteins in exosomes related to lymph node metastasis in the b mouse melanoma model. among them, we found that emilin- , a protein with an important function in lymph node physiology, was hyper-secreted in exosomes. interestingly, we found that emilin- is degraded and secreted in exosomes as a mechanism favouring metastasis. further, we found that emilin- has a tumour suppressive-like role regulating negatively cell migration. importantly, our in vivo studies demonstrate that emilin- overexpression reduced primary tumour growth and metastasis in mouse melanoma models. analysis in human melanoma samples showed that cells expressing high levels of emilin- are reduced in metastatic lesions. summary/conclusion: overall, our analysis suggests that the inactivation of emilin- by proteolysis and secretion in exosomes reduce its intrinsic tumour suppressive activities in melanoma favouring tumour progression and metastasis. funding: this work was supported by grants from mineco (saf r), asociación española contra el cáncer, fundación de investigación oncológica fero and mineco-severo ochoa predoctoral program. introduction: the amplification of erbb gene and the consequent overexpression of the encoded protein her have an important role in breast cancer classification at diagnosis and subsequent treatment decision with the anti-her monoclonal antibody trastuzumab. fish and ihc have been used so far to detect erbb gene amplification and her protein overexpression respectively in tissue biopsies. in this context, a major goal for liquid biopsies is to take advantage of the information carried by circulating tumourderived materials (such as extracellular vesicles (evs) and cell free dna (cfdna)) to noninvasively detect erbb gene status in the blood. however, the isolation of diverse tumour-derived materials from a single aliquot of patients' plasma and the accurate detection of cancer biomarkers is still challenging. methods: by adopting a recently published nickelbased evs isolation (nbi) protocol that allows for recovery of cfdna after evs isolation, we generated a high-sensitivity molecular assay to accurately detect erbb amplification and consequent her overexpression on a limited volume of plasma ( . ml) collected from breast cancer patients (stage i, ii and iii) at diagnosis. results: ) we detected erbb amplifications by droplet digital pcr (ddpcr) on the cfdna isolated from the plasma of erbb positive patients; ) we confirmed her overexpression on a subset of patients by setting up an antibody-based affinity reaction designed to detect her protein on the surface of the isolated evs; ) we succeeded in the quantification of her transcripts enclosed within evs by performing ddpcr in samples of patients showing a range of circulating tumourderived material. the specificity and sensitivity of these novel methodological assays were tested on a cohort of healthy individuals (n = ) and on a cohort of her positive (n = ) or her negative breast cancer patients (tnbc; n = ). summary/conclusion: here we report a pilot study on a novel multimodal method for erbb detection from a minimal amount of plasma. this approach integrates information from cfdna with evs-derived rna and proteins analysis. this proof of concept may ultimately translate into relevant clinical applications for disease diagnosis as well as for therapy selection and monitoring of disease progression. introduction: the biological and medical importance of exosomes recognized over the last decade has given rise to a crucial need for the discrimination between true evs and co-purified nanoparticles, such as lipoproteins, protein aggregates or debris. additionally, it is imperative to develop methods to identify and characterize ev sub-populations. considering ev biology and the reliability of labelled biomolecules, we developed both exogenous and endogenous labelling protocols, taking advantage of different dye properties which can target a multitude of compartments. this approach reveals key aspects of ev structure and integrity. methods: nanosized evs/exosomes were purified by size exclusion chromatography (sec) from model cell lines and human plasma. diverse dyes were orthogonally evaluated through different single particle and bulk analysis technologies, such as high-resolution cytometry, nanoparticle tracking analysis and plate fluorimeter. concomitant profiling of specific ev subpopulations was optimized using antibodies (abs) against tetraspanins and cell type specific targets and assessed by single particle analysis and elisa. to assess specificity of labelling protocols we used specific controls such as recombinant rfp expressing vesicles and purified lipoproteins. results: our ev staining protocols allowed for high labelling efficiency and unprecedent ev discrimination, quantification and characterization by combining single particle analysis and bulk measurements in simple matrices (saline buffers) and in complex biofluids (i.e. plasma). different approaches have diverse and complementary advantages (costs, capacity, sensitivity, informative readout) for implementation in research and diagnostic development flows, directly feeding in-house r&d and qc pipeline. summary/conclusion: overall, fluorescent evs are versatile and valuable tracers that can be applied in the optimization of pre-analytical and purification protocols, selection of target biomarkers and diagnostic assay calibration and validation. funding: endevor (por-fse - ) region tuscany and exosomics r&d program. subpopulations of tissue-derived extracellular vesiclesmethodological evaluation for vesicle size measurement introduction: introduction: subpopulations of extracellular vesicle (evs) are commonly classified by their different size, however, the ev size cut off is still under discussion. the aim of the study was to evaluate size range of six ev subpopulations using three methods: electron microscopy (em), nanoparticle tracking analysis (nta) and exoview™. methods: methods: ev subpopulations were isolated from melanoma tissues by a centrifugation based protocol recently established in our lab. large and small evs (levs and sevs) were isolated with differential ultracentrifugation and these were further separated into low and high-density fractions (ld and hd). size of ev subpopulations was then evaluated by: em introduction: small-extracellular vesicles have an important role in cell metabolism and cell-to-cell communication. moreover, sevs when secreted from cells are capable to act as mediators of various neurological diseases. however, sevs show heterogeneity and this may impact their functions. therefore, to characterize sevs at a single-particle level is important to better detail the associated biological activity. in this scenario, we innovatively propose the structured illumination microscopy (sim) as a technique able to complement the non-optical methods (transmission electron microscopy, tem) to analyse single sevs and their markers. methods: human plasma sevs were separated from healthy cognitive control (ctrl), mild cognitive impairment (mci) and demented subjects. the sevscontaining pellet was resuspended in % paraformaldehyde or % glutaraldehyde solutions. for sim, sevsenriched preparations were washed with the blocking solution and incubated with the primary antibody (l cam). the secondary fluolabelled antibody was then added. between the steps with the blocking solution, the primary and secondary antibodies, two ultracentrifuge steps were performed. the image acquisition was done on a nikon sim system with a x oil immersion objective. sevs were imaged with a d-sim acquisition protocol. tem was performed on mesh formvar copper-coated grids. sevs-enriched preparations were incubated first with the blocking solution and then, immunogold-labelled for cd . samples were counterstained with uranyl acetate and observed under a jeol ex electron microscope. data were recorded with a morada digital camera system. participation to the present study was approved by relevant local ethics committee of mendrisio and lugano hospital and written informed consents were obtained from subjects. results: for sim methodology, only vesicles in the range from to nm were detected, as the final resolution achieved was nm. instead, for tem, sevs under nm were identified. none of these methods provided relevant information about possible difference in morphology of the ctrl-, mci or demented subjects-derived sevs. summary/conclusion: even if both methods identified cd or l cam-positive vesicles, sim resolution and the complexity of the protocol represent some disadvantages respect to tem, that may be the first choice screening technique for evs analysis to be then completed by sim for particular tasks. fabrication of nanopore structures via conformal metal-film deposition for ev sensing kwanjung kim a , seung-min han b , soo-hyun kim c and sung-wook nam d luminex corporation, seattle, usa introduction: extracellular vesicles (evs) are membrane-derived structures that include exosomes, microvesicles, and apoptotic bodies. these evsreleased under normal physiological conditions as well as in the pathogenesis of neurological, vascular, haematological, and autoimmune diseaseshave been shown to transfer biological molecules such as protein and rna between cells, potentially transmitting signals. to understand more about these signalling mechanisms, there is a need for detecting and quantifying evs with cargo protein and rna in a reproducible and reliable manner. however, this has been challenging due to the small size of evs (ranging from to nm in diameter), and the lack of specific staining reagents. methods: here, we utilize the amnis® cellstream® flow cytometer, which enables high-throughput flow cytometry with increased sensitivity for detecting small particles. we demonstrate that a charge-coupled device (ccd)-based, time-delay-integration image capturing system can be used to detect and quantify evs and their cargo labelled with exoglow™-protein or exoglow™-rna. results: in this study, we show flow cytometry data quantifying ev samples that have been labelled with cargo markers for proteins and rna. the ev cargo contents along with the appropriate control samples will be shown. summary/conclusion: the ccd based detection of the cellstream flow cytometer has the sensitivity to quantify evs and their cargo. single ev imaging reveals novel ev biomarkers and dna cargo siobhan m. king a , ricardo bastos a and andras miklosi b a oni, oxford, uk; b oni (oxford nanoimaging ltd), oxford, uk introduction: extracellular vesicles (evs) are cellderived membrane-bound particles that range in size from - nm and carry active molecules such as dna, rna and proteins. upon secretion, evs can execute many biological functions such as initiating intracellular communication or regulating immune responses. depending on their origin evs have different characteristics and cargo, making them attractive candidates for early diagnostic and therapeutic applications. however due to their small size and heterogeneity, direct visualization and characterization of the surface markers expressed remains a challenge since these vesicles are below the resolution limit of standard light microscopy. methods: here, we describe a method that provides size analysis of single-evs, which falls below the diffraction limit of light. this was done with purified evs, immunostained using fluorescently labelled primary antibodies raised against ev surface markers (cd , cd , cd ), specific cargo such as dna and probed for tissue specific cargo. characterization of the molecular content and structural properties of surfaceimmobilized evs was performed using single-molecule localisation microscopy (smlm) on the nanoimager platform. results: multicolour smlm was used to detect up to three ev biomarkers showing successful characterization of the molecular signature for different ev subpopulations. the distribution of novel components on urinary evs were visualized for the first time using this approach. in addition, smlm revealed the presence of dna on both the surface and also as a cargo inside evs isolated from tumour cell culture media, which was validated using complementary biochemical characterization. summary/conclusion: smlm is a powerful technique for single-ev analysis and characterization. visualization of single-urinary evs enabled accurate sizing and further insights into novel components expressed on the subpopulation's membrane surface. together, the data demonstrates that the quantitative abilities of smlm can significantly enhance our understanding of evs, as structure, phenotypes, and cargoes can now be successfully resolved. introduction: working skeletal muscle is a common site for injury due to unaccustomed exercise with or without underlying pathology. direct analysis of skm injury requires invasive tissue biopsies. circulating extracellular vesicles (evs) are abundant in blood and have been shown to be enriched in microrna; profiles of which may reflect the state of tissues. evs may therefore serve as a non-invasive indicator of muscle injury and regenerative processes in vivo. methods: two consecutive bouts of muscle-damaging exercise (plyometric jumping and downhill running) were performed by healthy male volunteers. serum creatine kinase (ck) and plasma evs were analysed at baseline, and hr post-exercise. perceived muscle pain (pmp) was assessed at and hr post-exercise. large evs were isolated using a g centrifugation step, and small evs were isolated using qev columns. ev-enriched isolates were visualized using tem, and size and numbers were quantified using nta. based on nta results the highest particle fractions ( - ) were pooled for rna analysis. qpcr was done on plasma, large evs and small evs. a group of muscle and immune cell-important mirs were analysed by means of normalization to an exogenous control. results: ck and pmp increased post-exercise, providing evidence for muscle damage. tem revealed an abundant and heterogeneous pool of evs. a concomitant abundance of evs was seen with nta (mean = . x particles/ml plasma). mean ev diameters were ± nm across all timepoints. no change in ev size nor number was seen over time, however, mir- decreased at hr when compared to hr in the small ev isolate only. plasma displayed an immediate increase in myomirs- and − at hr, which returned to baseline at hr. in contrast, myomirs- b and remained elevated over the hr period. myomir- b and , as well as immune-mirs, did not change in evs or plasma as a result of the intervention. summary/conclusion: the decrease in mir- in small evs at hr is consistent with previous data. no decrease in mir- in large evs suggests specific packaging and hence a specific response to the muscle damage in small evs. more changes occurred in plasma myomirs suggesting less specific passive leakage into circulation from damaged cell membranes. funding: south african national research foundation pulsed electromagnetic fields potentiate the paracrine function of mesenchymal stem cells for cartilage regeneration yingnan wu a , dinesh parate a , eng hin lee a , zheng yang a and alfredo franco-obregón b a national university of singpaore tissue engineering program, yll school of medicine., national university of singapore, singapore, singapore; b biolonic currents electromagnetic pulsing systems laboratory, biceps, national university of singapore, singapore, singapore introduction: the mesenchymal stem cell (msc) secretome, via the combined actions of its plethora of biologically active factors, is capable of orchestrating the regenerative responses of numerous tissues by both eliciting and amplifying biological responses within recipient cells. mscs are "environmentally-responsive" to local microenvironmental cues and biophysical perturbations, influencing their differentiation as well as secretion of bioactive factors. we have previously shown that exposures of mscs to pulsed electromagnetic fields (pemfs) enhanced msc chondrogenesis. here, we investigate the influence of pemf exposure over the paracrine activity of mscs and its significance to cartilage regeneration. also, the subsequent extracellular vesicles analysis and isolation are processed for the understanding of how the pemfs affect stem cell evs and consequent differentiation induction. methods: conditioned medium (cm) was generated from mscs subjected to either d or d culturing platforms, with or without pemf exposure. the paracrine effects of cm over chondrocytes and msc chondrogenesis, migration and proliferation, as well as the inflammatory status and induced apoptosis in chondrocytes and mscs was assessed. the cms which have significant effects during chondrogenesis will be analysed by protein and mirna studies. results: we show that the benefits of magnetic field stimulation over msc-derived chondrogenesis can be partly ascribed to its ability to modulate the msc secretome. mscs cultured on either d or d platforms displayed distinct magnetic sensitivities, whereby mscs grown in d or d platforms responded most favourably to pemf exposure at mt and mt amplitudes, respectively. ten minutes of pemf exposure was sufficient to substantially augment the chondrogenic potential of msc-derived cm generated from either platform. furthermore, pemf-induced cm was capable of enhancing the migration of chondrocytes and mscs as well as mitigating cellular inflammation and apoptosis. the cms protein results in the significant promotion chondrogenesis condition showed an increase in proliferation and anti-inflammatory cytokines. summary/conclusion: the findings reported here demonstrate that pemf-stimulation is capable of modulating the paracrine function of mscs for the enhancement and re-establishment of cartilage regeneration in states of cellular stress. the pemf-induced modulation of the msc-derived paracrine function for directed biological responses in recipient cells or tissues has broad clinical and practical ramifications with high translational value across numerous clinical application. effects of extracellular vesicles from blood derivatives on osteoarthritic chondrocytes within an inflammation model introduction: the degenerative disease osteoarthritis (oa) is one of the leading causes of disability especially of elderly people. besides various treatment options depending on the severity of the cartilage degradation, the application of blood derived products such as platelet rich plasma (prp) are getting more and more popular in clinical practice due to its high concentration of platelets and the perceived high growth factor levels. drawbacks of using prp include high donor variability, discrepancies among preparation protocols and the presence of cells (platelets, leukocytes) which can evoke cellular processes, especially inflammation, when injected into the diseased tissue. one possibility is to isolate only extracellular vesicles (evs) from blood derivatives to overcome these problems. in the current study the effects of evs isolated from blood derivatives on oa chondrocytes within an inflammation model was investigated. methods: cd positive primary monocytes were isolated from citrate anticoagulated whole blood by magnetic bead sorting. monocytes were differentiated into resting m macrophages and activated into m macrophages according to published protocols. elisa measurements verified successful differentiation and activation as il β and tnfα levels increased. as control, thp monocytes were used. patient-derived oa chondrocytes were grown in well plates and co-cultivated with activated m macrophages which were seeded into thincerts and added to the wells representing the inflammation model. furthermore, cells were treated for hours with media containing fcs, ev depleted fcs or evs isolated from prp or hypact serum. results: successful differentiation and activation of monocytes (thp and primary monocytes) into m macrophages was demonstrated by elevated levels of the inflammatory cytokines il β and tnfα. within the inflammation model (co-culture of oa chondrocytes with m macrophages), addition of evs isolated from prp or hypact serum resulted in decreased secretion levels of il β and tnfα compared to media supplemented with either fcs or ev depleted fcs. summary/conclusion: taken together, evs from blood derived products might be chondroprotective and anti-inflammatory mediators which protect cartilage from being degraded during oa. funding: the work was jointly supported by the european fund for regional development (efre) and the fund for economy and tourism of lower austria, grant number wst -f- / - . α, (oh) d regulates growth cartilage matrix vesicle micrornas niels asmussen, michael mcclure, zhao lin, zvi schwartz and barbara boyan virginia commonwealth university, richmond, usa introduction: matrix vesicles (mvs) are small ( - nm in diameter) lipid bound extracellular organelles isolated from calcifying tissues including the growth zone (gc) of growth plate cartilage. α, (oh) vitamin d ( α, ) is a regulator of gc chondrocytes and the mvs they produce. these mvs are key players in the mineralization process and are selectively enriched with enzymes and growth factors. we found that mvs are also selectively enriched with micrornas (mir), including mir- , mir- and mir- . the aim of this study was to determine the regulatory role of α, in the packaging of mirna in mvs by gc cells. methods: gc cells were isolated by enzymatic digestion from costochondral gc cartilage harvested from wk-old male sprague dawley rats (iacuc approved). confluent fourth passage gc cell cultures were treated with - m α, or vehicle for h. media were removed, cell monolayers digested with trypsin and cells and mvs isolated by differential ultracentrifugation. rna was precipitated from cells and mvs. small rnaseq data were trimmed, aligned and counted before undergoing differential expression analysis. experimental groups had an n = per variable. significant differences (p < . ) were determined using r v . . . results: α, treatment altered expression of mv mirs compared to control mvs, whereas cell mirnas were differentially expressed. . % of significantly up or down regulated mir found in mvs overlapped between α, and vehicle groups with the remaining being uniquely differentially expressed. α, increased mv mir- and decreased mir- - p two mirs known to regulate osteoblast proliferation ( increases, decreases). summary/conclusion: α, regulates gc chondrocyte and mv behaviour and this study demonstrates that it also impacts the mir packaging within mvs. mir discovered in mvs have been demonstrated to impact chondrocyte behaviour and the present study indicates that α, regulates the growth plate through mir delivered by mvs. introduction: increasing evidence has proposed extracellular vesicles (evs) as mediators of many of the therapeutic features of mesenchymal stromal cells (msc) that have been widely studied in clinical trials over the last years. these evs have been recognized as nanocarriers of important biological information, which play a central role in cell-to-cell communication. in this context, evs can be used as an alternative to a cell-based therapy, with reduced risks. the present work aimed to evaluate the impact of different culture conditions on the msc-derived evs molecular composition through fourier-transform infrared (ftir) spectroscopy. methods: evs derived from msc from different sources, expanded in two different culture media ((xenogeneic -free (xf) vs serum-containing medium (fbs)) were characterized by ftir spectroscopy, a highly sensitive, fast and high throughput technique. moreover, principal component analysis (pca) of preprocessed ftir spectra of purified evs was conducted, enabling the evaluation of the replica variance of the evs chemical fingerprint in a reduced dimensionality space. for that, different pre-processing methods were studied as baseline correction, standard normal variation and first and second derivative. results: evs secreted by mscs cultured with serumcontaining medium presented a more homogenous chemical fingerprint than evs obtained with xf medium. the regression vector of the pca enabled to identified relevant spectral bands that enabled the separation of samples in the score-plot of the previous analysis. ratios between these spectral bands were determined, since these attenuate artefacts due to cell quantity and baseline distortions underneath each band. statistically inference analysis of the ratios of spectral bands were conducted, by comparing the equality of the means of the populations using appropriate hypothesis tests and considering the significance level of %. it was possible to define ratios of spectral bands, that can be used as biomarkers, enabling the discrimination of evs chemical fingerprint in function of the culture medium used for msc expansion and the msc donor. summary/conclusion: this work is a step forward into understanding how different culture conditions affect msc-derived evs characteristics. funding: fundação para a ciência e tecnologia (ptdc/equ-equ/ / , uidb/ / ). performance qualification for microflow cytometers: understanding technical limitations to improve your research desmond pink a , michael wong a , diana pham a , renjith pillai a , leanne stifanyk b , sylvia koch b , rebecca hiebert a , oliver kenyon c and john lewis a a nanostics, edmonton, canada; b dynalife, edmonton, canada; c apogeeflow systems, hemel hempstead, uk introduction: as microflow cytometry and other techniques mature as validated modalities for analysing extracellular vesicles (ev), there has been a concerted effort to improve reproducibility . in order for this reproducibility to occur there has to be a critical understanding of advantages and limitations for each technology. for microflow cytometry, several instruments are available to analyse evs. each platform has different limitations as well as advantages over other platforms. to provide the optimal data for your specific research, it is critical to understand the limitations of your platform. to accurately define these limitations, a performance qualification (pq) of your instrument should be undertaken. methods: an apogee a platform was used in these experiments. experiments were designed with expected ranges and cut-offs for acceptance criteria.initial tests included autosampling of a well plate with either single or double aspiration, single sample reproducibility and linearity proportional to flow rate. other experiments designed to show machine performance included minimal time to achieve valid data, sample volume required for double aspiration, determination of coincidence; detection sensitivity using a spiked sample; flow rate stability for extended periods ( - minutes) . tests should also be performed to determine carryover at a range of sample concentrations. if present, the means to remove contaminating samples should be determined. any performance tests should be applicable to any instrument in the field. results: auto-sampling helped demonstrate consistent data; reproducibility of total events and biomarkers was - % c.v. detected bead concentrations were linear with flow rates between . and . ul/min. double well aspirations provided similar data with aspirations between - ul. valid data was achieved for a low abundant target (~ - events/ul) after only s, < %c.v. detection sensitivity was determined to be~ / , . carryover ranges were determined in the presence of nominal unstained serum. an optimal number of machine washes was determined. some membrane stains, such as cell mask and cfse require much more rigorous cleaning to remove stain carryover. summary/conclusion: to improve data reproducibility, performance qualification of any instrument is key. operational limitations help define optimal performance parameters of any technology. understanding the types of experiments to perform for your particular type of characterization technology depends on the requirements you set for your research. a good performance test should be applicable to any related instrument in the field. funding: funding provided by nanostics, the alberta cancer foundation, and alberta innovates. introduction: cancer cells release more evs than normal cells and evs secreted from tumour cells can promote tumour progression, survival, invasion and angiogenesis. the ev cargo may mirror the altered molecular state of the cell of origin. therefore, evs have potential for the development of non-invasive markers for early detection of cancers. evs and their cargo also have the potential to be multiplexed with other molecular markers or screening modalities (e.g., imaging) to develop integrated molecular-based computational tools for the early detection of cancer. one challenge with using evs as a biomarker is the lack of robust and reproducible methods for the isolation of a pure vesicular population. there is a lack of clear consensus for an optimal method of isolation of a pure ev population that is devoid of contamination with similar-sized vesicles of different origins. there is also a lack of standards to ensure rigour reproducibility. methods: the current funding opportunity announcement (foa), par - , is promoting research on the isolation and characterization of extracellular vesicles (evs) and their cargo for the discovery of biomarkers to predict cancer and cancer risk. results: the previous cycle of this foa, par - / , successfully funded r and r grants. these awards are focused on proteomics profiling of evs, effect of methodological and biological variability, asymmetric-flow field-flow technology, therapeutic monitoring, lss and sers lab on a chip optical spectroscopic, evs in obesity-driven hepatocellular carcinoma, nanoscale structure and bio-molecular heterogeneity, urinary ev dna, and ev markers in paediatric cancers. progress from these awardees have shown separation of two discernible exosome subpopulations and identified a distinct nanoparticle, the exomere (nature cell biology, ); and have shown that large-evs contain the entire genome of the cell of origin, including cancer-specific genomic alterations (journal of extracellular vesicles, ). protocols that critically evaluate and refine the existing methodologies to improve utilization of evs in clinical use have been shared (nature protocols, ). summary/conclusion: drs. sudhir srivastava and matthew young are the programme directors for the par which began accepting applications on january . this and other ev funding opportunities will be discussed. funding: this is a funding opportunity announcement offered by the national cancer institute. introduction: traumatic brain injury (tbi) is characterized by diverse primary mechanisms of injury that lead to the development of secondary pathological cascades that drive neurological deficit post-tbi. inability to separate patients based on the presence of these different endophenotypes represents a major challenge for diagnosis and treatment of tbi. extracellular vesicles including exosomes isolated from patient plasma have emerged as promising potential biomarkers for tbi due to their ability to cross the bbb into systemic circulation with molecular cargo intact for analysis. we have developed a novel microfluidic platform for rapid isolation of brain-derived evs providing a tool with which the biochemical state of neurons and glia can be directly assessed post-tbi. we used the ultra-sensitive, single molecule array (simoa) to quantify concentrations of protein biomarkers from the plasma and brain derived evs from mild tbi patients and controls. by combining multiple protein biomarkers, we could discriminate mtbi patients from controls in both the training and the blinded test set. building on this work, we are also characterizing single ev heterogeneity of neuron derived evs by developing novel droplet based digital assay for single ev quantification at ultra-low concentration. droplet based assay for single ev analysis would potentially be very informative for early disease diagnosis and therapy decision. methods: our microfluidic platform for ev isolation consists of tracked-etched membranes with millions of nanopores ( nm), coated with a magnetic film (nife) to precisely capture immunomagnetically labelled brain-specific evs from plasma. single molecule array (simoa) was used to quantify concentrations of the protein biomarkers (tau, uchl- , nfl, gfap, il , il , and tnf) in the plasma and brainderived exosomes of mild tbi (mtbi) patients and controls. to identify single ev, we applied droplet based enzyme-linked immunosorbent assay and encoded the fluorescent signal for single ev quantification within parallelized microfluidic platform. results: we report that concentrations of plasma and exosome gfap, nfl, and uchl were elevated in mtbi patients compared to controls (p < . ), and that each of these biomarkers are uncorrelated with one another. discrimination of mtbi patients from controls was most accurate when machine learning algorithms on the panel of biomarkers. specifically, combining plasma nfl, gfap, il and tnf-with tau from glur + evs showed % accuracy with % sensitivity and % specificity. summary/conclusion: this data suggests that neuronderived exosomes contain information that characterizes the injured and recovering brain. it also suggests that analysis of a panel of biomarkers from a combination of both blood and exosomal compartments could lead to more accurate diagnosis of mtbis. ps . = op . l cam is not associated with extracellular vesicles in cerebrospinal fluid or plasma maia norman, dmitry ter-ovanesyan, wendy trieu and david walt wyss institute, boston, usa introduction: neurons in living psychiatric and neurological patients are inaccessible for cell type specific analysis of rna and protein. our understanding of these diseases instead relies upon imperfect sources of biochemical information such as post-mortem brain tissue analysis and animal models. furthermore, there is a paucity of biochemical assays available to diagnose and manage brain diseases. extracellular vesicles (evs) present an opportunity to noninvasively sample the contents of neurons in cerebrospinal fluid (csf) and plasma. in order to isolate neuron-derived evs (ndevs), a cell type specific transmembrane protein is necessary for immunocapture. l cam, a protein abundant on the surface of neurons, has been used extensively in the literature for ndev isolation. however, l cam exists in humans in several isoforms without a transmembrane domain, and as such it can be secreted as a free protein. additionally, the ectodomain of l cam can be cleaved off of the cell surface in physiological processes. it remains to be demonstrated whether the l cam found in csf and plasma is ev associated, or if it is instead a spliced or cleaved isoform behaving as a free protein. methods: using single molecule arrays (simoa), a digital form of elisa, as well as western blotting, we quantify ev markers (cd , cd and cd ) as well as l cam and albumin. we use these assays to determine in which fractions of size exclusion chromatography (sec) and density gradient the l cam appears. we also immunocapture l cam from csf and plasma and perform western blots for the internal and external domains of l cam. results: simoa and western blot analysis of sec and density gradient fractions demonstrated that while the ev markers peaked all together, l cam eluted in the free protein fractions along with albumin in both csf and plasma. when immunoprecipitation was performed, western blotting revealed different isoforms of l cam in csf and plasma. summary/conclusion: our data utilize a multitude of distinct techniques that converge to demonstrate that l cam is not associated with evs in csf or plasma. furthermore, our data suggest that the isoforms present in csf and plasma are distinct, which indicates that the l cam in plasma is likely not coming from the brain. this data call into question the utility of l cam as a ndev marker and point to the need to find novel candidates for immunoprecipitation of ndevs. introduction: in parkinson's disease (pd), α-synuclein (α-syn) aggregates known as lewy bodies (lb) are present in both the central and peripheral nervous system. furthermore, data showing that α-syn can spread from pd patients to transplanted tissue has led to a new theory postulating that pathological forms of α-syn can drive disease by "infecting" healthy cells and corrupting normal proteins. the exact routes and mechanisms involved in such spreading are yet to be fully understood but it is known that α-syn can be secreted from cells and transported via extracellular vesicles (ev). ev derived from erythrocytes (eev) are of particular interest in this regard as they have been shown to contain α-syn. methods: we first optimized a protocol for the isolation of fluorescently labelled human eev. the capacity of these eev to cross the blood-brain barrier (bbb) was then evaluated in vitro using a boyden chamber composed of primary human brain endothelial cells. next, eev were added to a more complex and physiologically relevant d human bbb model including ipsc-derived brain microvascular endothelial cells. in both in vitro protocols, flow cytometry was performed on media collect from each compartment to determine the number of eev. immunofluorescence was performed to assess the localization of fluorophore tagged eev. we are also using an in vivo paradigm for the extraction and testing of eev spread and an in situ cerebral perfusion (isbp) model in wt mice to investigate if and how eev cross the bbb using confocal microscopy. results: in both in vitro models, flow cytometry analyses showed that fluorescently tagged eev added to the luminal side traversed the endothelial cell barrier. confocal analysis revealed that some eev could also be found within endothelial cells themselves. ongoing experiments are being conducted in our newly developed d bbb to further confirm these results. our preliminary in vivo experiments showed that fluorescently labelled beads, similar in size to eev, used in the isbp experiments are detectable in the brain parenchyma of injected wt mice using confocal microscopy. preliminary work also includes isbp injections of eev in -month-old wt mice, (n = /groups) derived from pd patients (at different stage of the disease) and a healthy individual as a control. summary/conclusion: our preliminary data suggests that eev can indeed move across the bbb in both in vitro and in vivo experimental setups. ongoing experiments will determine the dynamics and processes involved in this transport and whether eev can precipitate and/or exacerbate disease-related features. introduction: neuroblastoma accounts for % of childhood cancer mortality. amplification of the oncogene n-myc is a well-established poor prognostic marker for neuroblastoma. whilst n-myc amplification status strongly correlates with higher tumour aggression and resistance to treatment, the role of n-myc in the aggressiveness of the disease is poorly understood. exosomes are released by many cell types including cancer cells and are implicated as key mediators in cell-cell communication via the transfer of molecular cargo. hence, characterising the exosomal protein components from n-myc amplified and nonamplified neuroblastoma cells will improve our understanding on their role in the progression of neuroblastoma. methods: in this study, comparative proteomic analysis, nanoparticle tracking analysis, transmission electron microscopy, rnai-based knockdown, migration and cellular survivability assays were performed to understand the role of exosomes isolated from cells with varying n-myc amplification status. results: label-free quantitative proteomic profiling revealed proteins that are differentially abundant in exosomes released by the n-myc amplified and nonamplified neuroblastoma cells. gene ontology-based analysis highlighted the enrichment of proteins involved in cell communication and signal transduction in n-myc amplified exosomes. treatment of less aggressive sh-sy y cells with n-myc amplified sk-n-be cell-derived exosomes increased the migratory potential, colony forming abilities and conferred resistance to doxorubicin induced apoptosis. incubation of exosomes from n-myc knocked down sk-n-be cells abolished the transfer of resistance to doxorubicin induced apoptosis. summary/conclusion: these findings suggest that exosomes could play a pivotal role in n-myc-driven aggressive neuroblastoma and transfer of chemoresistance between cells. ps . = op . results: murine ctl evs were broadly divided into two populations that were eluted at low salt (l-s: . m- . m nacl) and high salt (h-s: . m- . m nacl) concentrations. l-s ctl evs were abundant in late endosome-related proteins, integrins, rabs, and effective mirnas, indicating exosome characteristics, and had biological activity for preventing tumour metastasis after depletion of tumoural mesenchymal cell populations by intratumoral administration (see seo et al., nat. commun. : , ) . contrary, h-s ctl evs were rich in dna, core histones, ribosomal proteins, cytoskeleton proteins, and housekeeping proteins, considering microvesicles and apoptotic bodies, and easily phagocytosed by a kupffer cell line (kup : kitani et al., results immunol. : - . ). in addition, there were noticeable differences between ls and h-s ctl evs in the negative zeta potential width and membrane glycan structure. summary/conclusion: thus, ion exchange can be an optimal mass fractionation method for discriminating bioactive exosomes from cargos for nucleic acids in evs. funding: cryotem was conducted in nara institute of science and technology (naist), supported by nanotechnology platform program (synthesis of molecules and materials: # ) of the ministry of education, culture, sports, science and technology (mext). this work was supported by grants from the japan agency for medical research and development (translational research network program (nagoya univ. seeds a )) and the japan science and technology agency (crest [jpmjcr h ]). clic is essential for breast cancer metastatic competence and predicts disease outcome introduction: metastatic breast cancer is a consequence of complex interactions between cancer cells and the host. clic , a member of a conserved gene family in the glutathione-s-transferase superfamily, mediates crosstalk between tumour and host in breast cancer. tcga and metabric data indicated that elevated clic expression was associated with breast cancers from young women, those with poor prognosis, and those with early stage metastatic disease. methods: since bulk tumour analysis does not distinguish between cancer and host stromal cells, we used genetic modifications of established syngeneic breast cancer mouse models to evaluate the contributions of clic in the host or tumour cells to develop metastases. results: experimentally, the essential clic host contributions for metastatic competence were related to circulating levels of pro-metastatic soluble factors, neoangiogenesis, tumour cell attachment to lung tissue, myofibroblast differentiation, and leukocyte migration. clic was detected as cargo in circulating extracellular vesicles (evs) from breast cancer patients. similarly, circulating evs from tumour-bearing mice have abundant clic in comparison to those from mice bearing tumours that lack clic . tumour cells released evs that induced myofibroblast conversion of wildtype but not clic ablated lung fibroblasts. summary/conclusion: these results illuminate clic expression as a prognostic marker for breast cancer patients, and experimentally, clic is a critical host factor for metastatic competence and potential target within host tissues for anti-metastatic therapy. funding: this work was supported by the intramural program of the national cancer institute under project zia bc . the application of flow cytometry in an ev-based liquid biopsy for the detection of cancer multidrug resistance in myeloma gabriele de rubis a , krishna sunkara a , sabna rajeev krishnan and mary bebawy b a laboratory of cancer cell biology and therapeutics, discipline of pharmacy, graduate school of health, the university of technology sydney, australia, sydney, australia; b the university of technology sydney, sydney, australia introduction: multiple myeloma (mm) is an incurable cancer of bone-marrow plasma cells. it is characterized by unpredictable and highly variable therapeutic response and poor survival, attributed to the development of multidrug resistance (mdr) to chemotherapy. presently, no clinical procedures allow for a continuous, minimally invasive monitoring of mdr. we identified unique extracellular vesicle (ev) populations in the blood of myeloma patients, which serve as biomarkers of disease evolution and mdr to combination chemotherapy. we describe approaches used to optimise the use of flow cytometry (fcm) for ev summary/conclusion: although further investigation is required, our results potentially promise an effective and inexpensive priming agent (i.e., ethanol) for the production of anti-inflammatory msc-evs. this, combined with the significant increase in yield via d dynamic culture, presents practical solutions to both ev manufacturing scalability and potency issues. donor source affects potency of mesenchymal stem cell-derived extracellular vesicles introduction: mesenchymal stem cell (msc) therapies have been heavily investigated for their utility in applications such as wound healing and regenerative medicine due to their angiogenic, immunomodulatory and anti-apoptotic effects. recently, msc-derived extracellular vesicles (evs) have been implicated as primary effectors in msc-based therapies via protein and nucleic acid cargo transfer to patient cells. msc evs represent a superior alternative to msc-based therapies, as they lack the ability to replicate and are much smaller in size, circumventing related safety concerns such as immunogenicity, teratoma formation and blood vessel occlusion. however, a key drawback with msc therapies in general is their variable therapeutic potency, which is dependent on donor source. as a cell derived therapeutic, this crucial limitation is hypothesized to exist in msc evs as well. here, we demonstrate the varying bioactivities of isolated msc evs from differing donors and tissue sources. methods: six separate msc lines were obtained from different donors, with three msc lines derived from donor adipose tissue, and the other three from the bone marrow of separate individuals. evs were isolated from each msc line at passage via differential centrifugation and ultrafiltration. these isolated msc evs were then characterized for size/concentration via nanoparticle tracking analysis, and ev markers (tsg , alix, cd ) via western blot. pro-vascularization capacities of msc evs were determined by a gap closure assay using human umbilical cord vein endothelial cells (huvecs). results: characterization of msc evs revealed similar sizes and ev marker expression across donor groups, frontiers in chemistry, submitted funding: this work was funded by the momentum programme (lp - ), by the national competitiveness and excellence program catalan institution for research and advanced studies (icrea) proteomic profiling of retinoblastoma-derived exosomes reveals potential biomarkers of vitreous seeding angel montero carcaboso g , andrea petretto b , franco locatelli a and angela di giannatale a a department of paediatric haematology/oncology and cell and gene therapy, irccs, ospedale pediatrico bambino gesù ps : separation and concentration a laboratory of clinical biophysics, faculty of health sciences ps : diverse ev biomarkers chair: pia siljander -faculty of biological and environmental sciences urinary evs were isolated using low vacuum filtration method followed by ultracentrifugation. raman spectra of urinary evs were recorded using a renishaw invia raman spectrometer. data analysis was performed using principal component analysis (pca) and hierarchical cluster analysis (hca). the size distribution and morphology of evs were analysed by transmission electron microscopy and nanoparticle tracking analysis methods. results: average raman spectra obtained for urinary evs from studied groups showed differences in intensities of specific bands in the region of - cm- . we found significant correlations between mean area under curve (auc) calculated for raman bands (phenylalanine, dna, proteins, lipids and amide i) and selected clinical parameters such as: egfr, serum creatinine, glucose, urine creatinine. chemometric methods showed spectral pattern responsible for separation between studied groups. nta measurements visualized evs with size of . ± . nm. summary/conclusion: our results showed that characteristic raman spectra of urinary evs are promising candidates for new, non-invasive biomarkers for dkd isolation of circulating extracellular vesicles and cfdna allows for erbb detection in a single aliquot of breast cancer patients plasma michela notarangelo a , mattia barbareschi b , antonella ferro c , orazio caffo c , vito d'agostino a and francesca demichelis d a department of cellular results: results: tissue-derived large and small evs showed difference in size (mean nm vs nm) when examined by em, whereas nta and exoview™ were unable to show a clear difference between the populations (nta: mean . nm vs nm nta can only detected vesicles above nm and exoview™ only measures vesicles between - nm. of the three different methods, em analysis of single vesicles visualized in a significant number of micrographs was the only one able to distinguish ev subpopulations by size. funding: funding: swedish research council knut och alice wallenberg foundation imaging of human plasma-derived small-extracellular vesicles using transmission electron microscopy and structured illumination microscopy mitovesicles: a new extracellular vesicle of mitochondrial origin altered in ageing and neurodegeneration alldred b , chris goulbourne b , hediye erdjument-bromage d , monika pawlik b , mitsuo saito e , mariko saito f , stephen d. ginsberg b an in vitro and in vivo perspective on the role of erythrocyte-derived extracellular vesicles in parkinson's disease pathology frédéric calon c , Éric boilard f and francesca cicchetti b a centre de recherche du chu de québec and faculté de médecine, département de psychiatrie & neurosciences département de microbiologie-infectiologie et d'immunologie evidences on microalgal extracellular vesicles: a morphological assessment antonella bongiovanni i , ales iglič j and veronika kralj-iglič j a laboratory of clinical biophysics, faculty of health sciences a faculty of dentistry, national university of singapore, singapore, singapore, singapore; b institute of medical biology, agency for science, technology and research, singapore, singapore, singapore; c exosome of cancer-associated fibroblast induce anti-cancer drug-resistance of nsclc so-young kim a and yeon-ju lee b a chonnam national university hwasun hospital biomedical research institute, gwangju, republic of korea; b chonnam national university hwasun hospital biomedical research institute, gwangju, republic of korea introduction: the understanding of interaction mechanisms between cancer cells and the tumour microenvironment (tme) is crucial for developing therapies that can arrest tumour progression and metastasis. cafs are the major constituent of the tme in many cancers. recent studies indicate that exosomes harbour the potential to regulate proliferation, survival and immune status in recipient cells. most of the current studies are focused on cancer cell secreted exosomes; and little is known about cafderived exosomes and their influence on cancer cells. methods: nsclc cell lines (pc gr) and mrc (normal fibroblast cells) were grown in culture with exosome-free fbs. cutured media was filtrated by tangential flow filtration systems. exosomes in supernatant were isolated with the exoquick-tc™ system. considering the important role of cell extrinsic factors on cell growth and survival, we assessed whether factors contained in the mpa exosome could affect proliferation and survival of recipient cancer cells. cells were then treated with μm osimertinib or pbs for days prior to cell quantification of live cells.to investigate mechanisms of resistance to osimertinib mediated by ma or mpa-exosome in nsclc cell lines, we test cell viability by crystal violet assay in trametinib or osimertinib treated after pretreated ma or mpa-exosome, pc gr during days. we will investigate how mpa-exsomes activate erk signalling pathway in pc gr cells to induce antitumor effects by western blot. results: mpa exosome increased proliferation of pc gr cells by more than % compared to control pbs. pc gr cells grown in mpa-exosome and subsequently treated with osimertinib showed a significant increase in cell survival compared to pc gr cells grown in ma-exosome. osimertinib is used to treat egfr-mutant non-small cell lung cancer (nsclc) with tyrosine kinase inhibitor resistance mediated by the egfr t m mutation.these data show that "mrc -pc gr-crosstalk factors" affect proliferation and adaptive drug resistance of cancer cells. mpa-exosome mediates erk signalling activation and attenuated after treatment of um osimertinib. summary/conclusion: cafs support cancer growth and invasion. co-cultured nsclc with mrc lung fibroblast increased cell viability and exosomal mir- through the tgf-ß pathway in treatment osimertinib. exosomal mir- up-regulation in cocultured nsclc with mrc- induced drug resistance to drug-induced apoptosis. thus, exosomal mir- expression in co-cultured nsclc with mrc may support drug tolerance persister cells. introduction: neural stem cell (nsc) therapy has shown promise for brain repair after injury or disease mostly through bystander effects. nevertheless, the translation of nscs derived from human induced pluripotent stem cells (hipscs) to the clinic remain constrained due to safety issues, which include immunogenic risks, tumorigenesis potential, and incomplete differentiation. a way to avoid these issues is by using extracellular vesicles (evs) generated from nscs, as nsc-evs likely have similar neuroprotective properties as nscs and are amenable for non-invasive administration as an autologous or allogeneic off-the-shelf product. however, this would require reliable purification and characterization processes, and testing of evs for composition and biological properties. methods: we generated evs from hipsc-derived nscs using a combination of ion-exchange chromatography (iex) and size-exclusion chromatography (sec) and investigated their composition through small rna sequencing and proteomics. we also performed in vitro and in vivo experiments to determine their biological and functional properties. results: iex and sec facilitated purification of hipsc-nsc evs nearly to homogeneity, which expressed ev markers such as cd , cd , cd , and alix with a mean size of nm. small rna sequencing revealed enrichment of mirnas related to different neuroprotective signalling pathways and diverse metabolic functions consistent with their role in cell-cell communication. the proteomic analysis identified > , proteins, including ev markers and many other proteins involved in central nervous system function and cellular processes. the evs also displayed antiinflammatory activity in an in vitro mouse macrophage assay. intranasal (in) administration of nsc-evs resulted in their rapid incorporation by neurons, microglia, and astrocytes in virtually all regions of the brain. functionally, in administration of nsc-evs reduced inflammatory activity in the brain in a model of status epilepticus, and increased hippocampal neurogenesis in the adult brain. summary/conclusion: biologically active evs with antiinflammatory and neurogenic properties could be purified and harvested from hipsc-nscs. such evs also contain many mirnas and proteins that are of interest for brain repair after injury or disease. funding: supported by a grant from the national institute of neurological disorders and stroke ( r ns - to a.k.s.) introduction: extracellular vesicles (evs) generated from human bone marrow-derived mesenchymal stem cells (hmscs) display anti-inflammatory and neuroprotective properties. our recent study has shown that intranasally (in) administered hmsc-evs incorporate into significant percentages of neurons and microglia in virtually all regions of the intact as well as the injured forebrain within hours (kodali et al., int j mol sci, ) . in this study, using a rat model, we investigated the efficacy of a low dose of hmsc-evs administered intranasally for alleviating the abnormal plasticity of newly born neurons and the activation of microglia after se. methods: approximately billion evs were dispensed bilaterally into both nostrils of young f rats that experienced two hours of kainate-induced se. animals were euthanized seven days after se, and brain tissue sections were processed for immunohistochemical staining of neun (a neuronal marker), dcx (a marker of newly born neurons), iba- (a microglial marker), and parvalbumin (pv) and reelin (markers of subclasses of interneurons). in addition, activated microglia were quantified using iba- and cd dual immunofluorescence. results: in administration of evs reduced the seinduced loss of pyramidal neurons in the hippocampal ca subfield. also, ev administration after se maintained higher levels of pv+ interneurons in the dentate gyrus. furthermore, ev treatment after se modulated abnormal neurogenesis, which was evidenced by a the role of small extracellular vesicles in chronic neuropathic pain zhucheng lin a , renee jean-toussaint b , yuzhen tian b , ahmet sacan a and seena ajit b a drexel university, philadelphia, usa; b drexel university college of medicine, philadelphia, usa introduction: chronic pain is the most prevalent, disabling, and expensive public health condition in the usa. exosomes are - nm extracellular vesicles that can transport rnas, proteins, and lipid mediators to recipient cells via circulation. exosomes can be beneficial or harmful depending on their source and contents. we hypothesized that the composition of small extracellular vesicles (sevs) can be altered following nerve injury and these alterations can provide insight into how the body responds to neuropathic pain. methods: to characterize changes following nerve injury, small extracellular vesicles (sevs) were purified by ultracentrifugation from mouse serum four weeks after spared nerve injury (sni) or sham surgery. mirna profiling and proteomics analysis using tandem mass spectrometry were performed to determine differential expression of mirnas and protein cargo respectively. for in vivo studies, sevs were administrated intrathecally into the mouse lumbar region. animals were evaluated for mechanical and thermal hypersensitivity over days after injection. results: our mirna profiling showed a distinct mirna signature in sni model compared to sham control. proteomics analysis detected gene products. of these, were unique to sni model. neuropathic pain can induce the activation of the complement cascade and we observed significant upregulation of complement component a (c a) in sevs from sni model. intercellular adhesion molecule (icam- ), required for the leukocyte recruitment, adhesion and homing of exosomes was also upregulated in sevs from sni model compared to sham control. administration of sevs from sni model increased paw withdraw threshold in naïve recipient mice and inflammatory pain model, indicating a protective role for sevs in attenuating chronic pain. summary/conclusion: our preliminary studies suggest a critical role for sevs cargo in regulating pain. additional studies are ongoing to determine the functional significance of alterations in sevs composition using mouse models of pain. introduction: amyotrophic lateral sclerosis (als) is a progressive adult-onset neurodegenerative disease caused by selective motor neurons (mns) death. the rapid disease progression strongly suggests that cell-tocell spreading of noxious factors could take place in als pathogenesis. extracellular vesicles could potentially spread the disease. in this study, we characterized large (levs) and small extracellular vesicles (sevs) isolated from plasma of sporadic als patients and healthy controls and determined their different composition in order to understand their neuroprotective or neurotoxic role in als pathogenesis. methods: levs and sevs were isolated from plasma of als patients and healthy volunteers by differential centrifugation and characterized by nanosight ns . cd , cd , cd , cd a and annexin v were used for flow cytometry. sod , tdp , fus protein level was investigated by western blot. for raman spectroscopy, evs were dried on top of a caf slide and raman spectra were acquired using a nm laser line. mirna libraries were prepared by truseq small rna library kit (illumina). results: the mean size both for levs and for sevs resulted increased in als patients compared to controls. levs derived from als patients were enriched in sod- , tdp- and fus proteins compared to ctrls. sevs showed a distinct spectral pattern from levs. in addition, levs of als patients were richer in lipids and had less intense bands relative to aromatic aminoacids compared to healthy controls. we also found a great presence of leukocyte derived levs (lmvs) in als patients compared to ad patients and healthy donors and significant correlation with the progression rate of the disease. on the other hand, mirna and rna whole transcriptome sequencing identified a specific signature of mirnas in plasma derived sevs of als patients compared to a group of healthy controls and three neurological groups of control. summary/conclusion: these data may suggest that levs derived from als patients, enriched in lipids and toxic proteins, might play a role in prion-like propagation and immunity of als disease, while sevs, deriving ps . introduction: dendritic spines are actin-rich structures at the postsynaptic sites of most excitatory synapses in the central nervous system. they are highly important structures for higher brain functions such as learning and memory. several live imaging studies have shown that long, thin, actinrich protrusions called dendritic filopodia are precursors of dendritic spines in hippocampal and cortical neurons. so far, many intracellular factors that regulate filopodia formation have been identified. however, extracellular mechanisms of filopodia formation are largely unknown. also, detailed molecular mechanisms by which astrocyte secreted factors regulate synaptogenesis are not well understood. small extracellular vesicles (sevs)/exosomes have potential to regulate filopodia, spine and synapse formation in autocrine or paracrine manner due to their unique cargo composition. here, we examine role of exosomes in filopodia, spine and synapse formation. methods: primary rat hippocampal and cortical neurons were transiently transfected with the multivesicular body (mvb) docking regulator gfp-rab b or with shrnas against the exosome secretion and biogenesis regulators rab b and hrs. transfected neurons were immunostained for synaptic proteins and analysed for filopodia at day in vitro (div) or spines at div . for rescue experiments, exosomes were isolated using differential ultracentrifugation method from conditioned media of div cortical neurons or primary astrocytes and characterized for their size, common protein markers and morphology. results: here, we find that mvb docking factor gfp-rab b localizes to both the tips and bases of actin-rich filopodia and spines in primary neurons. furthermore, genetic regulation of exosome secretion by overexpression or knockdown of rab b or hrs leads to respective increases or decreases in the number of filopodia, spines and synapses. the defects of exosome-inhibited neurons in filopodia density are rescued by add-back of neuronal exosomes. additionally, treatment of primary neurons with exosomes isolated from primary astrocyte cultures leads to enhanced spine and synapse formation. summary/conclusion: these results indicate that autocrine and paracrine communication via exosomes are a key part of the process of neuronal filopodia, spine and synapse formation. effects of apolipoprotein e genotype on protein and small rna profiles of brain tissue-derived extracellular vesicles of alzheimer's disease patients introduction: multiple sclerosis (ms) is the most frequent chronic inflammatory disease of the young adult central nervous system. nevertheless, the pathogenesis remains largely unknown. it is therefore relevant to better characterise in cerebrospinal fluid (csf), which irrigates the brain, novel bioactive compounds whose dysregulation could be involved in ms pathology. the concentration of extracellular vesicles (evs) has been already found affected in ms patient fluids but the content in bioactive molecules, particularly the micrornas (mirnas), remains barely investigated. the mirna are short oligonucleotides that are major posttranscriptional regulators and we previously showed the dysregulation of specific mirnas in csf of ms patients. evs can potentiate mirna effects by allowing remote action through the shuttling within biological fluids such as csf while providing a protection from circulating rnase. nevertheless, csf remains a challenging fluid to analyse due to limited access, low volume and presence of lipoproteins (other putative mirna carrier) that can be co-isolated with evs. methods: we performed a comparative analysis of ev isolation from csf by size-exclusion chromatography (sec), density-gradient ultracentrifugation, ultrafiltration or chemical precipitation (chemp) to determine the optimal technique(s) to enrich ev. results: sec applied on csf of control patients showed optimal ev purification with sufficient evs from . ml of csf for downstream ev characterization. furthermore, we were able to isolate mirnas from csf and determined their enrichment in evs by rnase-sensitivity treatments. finally, we have combined chemp and sec to enable a fast and largescale isolation of evs from > ml of csf, which successfully provided an increase in particles detected by nanoparticle tracking analysis. we are currently characterising the particles to confirm that they are purified evs, cleared from contaminants. summary/conclusion: this work opens perspective to analyse evs from ms patients and to determine whether mirnas participates in ms pathogenesis through their transit in evs. funding: fondation louvain, charcot foundation. differences in circulating number of extracellular vesicles between contact sport athletes with and without acute mtbi: a pilot study meghan rath a , jacqueline sayoc a , soo-young choi a , karlee burns b , aja corchado c , jane mcdevitt b , jingwie wu d , ryan tierney b , michael selzer e , xiaoxuan fan f and joon-young park a for bottom-up guc, increasing iodixanol gradients with . ml of samples were centrifuged at k g for h. fractions were then pooled based on densities ( . - . g/ml). bca and sds-page were used to analyse total protein; nanoparticle tracking (nta) and transmission electron microscopy (tem) for ev presence; and immunoblotting and imaging flow cytometry (ifcm) to evaluate ev specific markers. (ev-track id: ev ). results: immunoblotting showed absence of actinin from all samples, while cd and tsg were detected for all samples; apart from imf_ip. nt_samples were not analysed reliably by nta and ifcm, due to the high concentration of casein micelles present (~ ^ /ml in milk) that otherwise would be co-counted with evs. as expected, following ip, which most efficiently removed casein micelles, bca showed that samples had lowest total protein. this was confirmed by sds-page. thus, most effects were then focused on the ip casein-depleted samples. ifcm indicated that, post-guc, sm_ip evs had significantly (p < . ) more cd -positive particles/ml of milk vs all other guc and kduc samples. while there were no significant differences in sizes of ev separated from sm or imf, directly comparing the ip pre-treated samples, sm had significantly (p < . ) higher quantities of evs when compared to imf. additionally, tem indicated that evs separated from sm by guc were intact with limited background debris, whereas those separated from sm by duc and all imf evs were not. summary/conclusion: in conclusion, regardless of the method used, imf has fewer intact evs compared to sm. also, to obtain purest sm evs, ip followed by guc separation is optimal. introduction: extracellular vesicles (evs) exist as subpopulations with heterogeneous content. the surface heterogeneity of evs may reflect differences in functionality between ev subpopulations, as interactions with recipient cells may differ between ev subpopulations with different surface profiles. however, it is currently challenging to study functional differences between ev subpopulations due to the lack of suitable techniques to purify intact evs based on their surface signature. here, we showcase a novel capture-and-release platform to enrich intact ev subpopulations by their surface profile and compare their characteristics. methods: mda-mb- and skov- cell-derived evs were isolated using size exclusion chromatography. ev subpopulations were enriched based on surface markers cd , cd , cd or phosphatidylserine (ps) using a novel magnetic bead-based capture-and-release platform. obtained evs were characterized by transmission electron microscopy (tem), nanoparticle tracking analysis (nta) and western blotting. evs were fluorescently labelled using pkh and celltracker deep red (ctdr) and their uptake by recipient cells was examined using flow cytometry. results: western blot analysis showed that ev subpopulations enriched for the selected tetraspanins and ps were successfully isolated using a novel capture-andrelease platform. interestingly, evs isolated based on ps exposure (ps+) lacked most canonical ev markers. all ev subpopulations showed intact, cup-shaped morphology when analysed by tem, but contained less protein contaminants compared to the initial ev isolate. ps+ evs were slightly larger than other ev subpopulations when analysed by tem and nta. to test the capacity of ev subpopulations to interact with recipient cells, evs were labelled with pkh and ctdr prior to subpopulation fractionation. after fractionation, ps+ evs showed a significantly higher ctdr/pkh ratio than other ev subpopulations as determined by fluorescence spectroscopy, suggesting higher esterase activity of ps+ evs compared to other tested subpopulations. furthermore, mda-mb- derived evs isolated based on cd and cd expression were taken up more efficiently by hmec- and mda-mb- cells than evs isolated based on presence of cd or ps. summary/conclusion: using a novel technology to isolate ev subpopulations based on their surface profile, we here show that composition and cellular uptake efficiency differs between ev subpopulations. theoretically, this technology is applicable to any surface marker of interest, allowing its use to further establish ev surface-functionality relationships and enrich evs with desirable characteristics for therapeutic purposes. funding: this work was supported by a veni grant (no. ) of the dutch research council (nwo). aml were harvested from tib cells cultured in evfree medium using serial ultracentrifugation. hspc (ksl; lin-sca + ckit+) clonogenicity and inflammatory responses were assessed using colony-forming unit (cfu) assay and real-time polymerase-chain reaction, respectively. ifn-alpha receptor (ifnar ) expression and intracellular reactive oxygen species (ros) levels were assessed by flow cytometry. dna damage were assessed by quantifying nuclear γ-h ax using immunofluorescent microscopy. results: similar to evs derived from aml patients, tib ev-aml elicited double-stranded breaks in hspcs, and actively suppressed hspc clonogenicity. transcriptional profiling revealed that exposure to ev-aml induced the upregulation of several inflammatory mediators in hspcs, including isg , il- , ifnα, ch h. inflammatory signalling triggered by ev-aml did not depend on ifnα signalling as evident from suppression of clonogenicity in ifnar -null hspcs as well as the lack of evs-induced stat phosphorylation or ifnar downregulation. instead, we found increased levels of ros following ev-aml exposure. summary/conclusion: our findings support a model whereby ev-aml inflammatory signalling and oxidative stress lead to dna damage in hspcs. introduction: basic leucine zipper atf-like transcription factor (batf ) is implicated in inflammatory response and anti-tumour effects. although the tumour suppressive function of batf has been reported, its extracellular role in maintaining a non-supportive cancer microenvironment has not been explored. methods: in this study, we established gbm orthotopic and subcutaneous tumour models in nude and balb/c mice and flow cytometry analysis determined the batf inhibitory effects of mdscs recruiting. we used transwell assay to determine batf -positive evs (evs-batf ) inhibitory of the chemotaxis of myeloid-derived suppressor cells (mdscs) in vitro. in addition, exo-counter detection during the development of the gbm-batf model to demonstrate evs-batf crosstalk with distant tissues. amd blocking in tumour model confirms that evs-batf dominated by the sdf- a/cxcr signalling pathway. in addition, exo-counter detection of evs in pairs of gliomas in different stages proposes plasma-evsbatf (plevs-batf ) as a prognostic marker. results: we found that tumour-derived evs-batf regulate crosstalk between glioma cells and tumour microenvironment by inhibiting mdscs recruitment. evs-batf can be detected in plasma and bone marrow of glioma-bearing mice, this provides direct evidence that glioma-derived evs can communicate with distant site by crossing blood-brain barrier. besides, evs-batf injection significantly reduced sdf- α expression in the tumour tissues. after blocking sdf- α signalling by amd , the inhibitory effects of batf overexpression on mdscs recruitment were rescued. evs-batf inhibit mdscs recruiting and secreting mmp , mmp , and vegfa which promote gbm progression. strikingly, exo-counter detection of evs in pairs of gliomas in different stages reveals that the number of plevs-batf can distinguish stage iii-iv glioma from stage i-ii glioma and healthy donors. summary/conclusion: our results suggest that evs-batf may be an effective circulating biomarker associated with glioma progression. of note, we are the first to determine the regulatory role of evs-batf in regulating tumour microenvironment and propose plevs-batf as a prognostic marker predicting glioma progression and candidate target for gbm therapy. introduction: electrofluidics is an emerging technology of combining electronics and nanofluidics. one important device in electrofluidics is an ion transistor in which the ionic current through a nanopore is regulated by gate voltage bias. here, we suggest a fabrication method of nanopore by introducing focused ion beam (fib) and atomic layer deposition (ald) to sense extracellular vesicle (ev) via metal electrode structures. methods: we deposited nm-thick silicon-nitrite layers on both sides of silicon wafer by low-pressure chemical vapour deposition (lpcvd). we fabricated rectangular patterns by photolithography followed by reactive ion etching (rie) on the backside of the wafer. anisotropic silicon etching by koh was performed. the front side of the chip was patterned by photolithography followed by ti/au deposition for the fabrication of electrode structures. we drilled ~ nm pores in the si n membrane by fib. by the ald process, we deposited highly-conformal metal film, either platinum (pt) or ruthenium (ru) to shrink nanopores by a self limiting process. results: we expect that the ion current through the nanopore is efficiently controlled by the gate-surrounding structures. the nanopore ion transistor can be used to count the number of evs. summary/conclusion: we suggest a fabrication method of nanopore ion transistors by introducing focused ion beam (fib) and atomic layer deposition (ald). this device will be applicable for single ev sensing. introduction: extracellular vesicles (evs) are key players in cell-cell communication and increasing evidence has shown that evs function in cancer by promoting cancer cell motility and metastasis. analysing tumour-derived evs in biofluids is attractive because it would be a novel approach to a non-invasive liquid biopsy. unfortunately, evs are highly heterogeneous. they vary greatly in size, lipid composition, and cargo and are difficult to distinguish from other small particles in complex biofluids. we have developed a novel flow cytometry method to generate a distinct ev fingerprint to profile biological specimens. methods: evs from cell culture media (purified and unpurified) and biological fluids (plasma and urine) were detected by flow cytometry using features on individual evs produced by intrinsic (cd -phluorin) and extrinsic (lipophilic dye, di- -anepps, and antibodies) fluorescent labels. ev subpopulations were visualized with dimensional reduction (t-sne and umap) of - features that defined the vesicle size, shape, and fluorescent emission spectra associated with the fluorescent marker. unsupervised density based clustering (hdbscan) in conjunction with supervised machine learning (xgboost) was subsequently used to define subpopulations. we refer to this method as "ev fingerprinting". results: ev fingerprinting was successfully used to detect evs in complex biological specimens and trace their differential enrichment through conventional purification methods. evs were readily distinguished from protein complexes, lipoproteins and non-lipid particles. calibration with externally validated purified ev, as well as size, lipid, and fluorescence standards enabled ev fingerprinting as a rigorous and reproducible method for resolving heterogenous ev samples. ev fingerprinting applied to conditioned medium from tumour cells and biological fluids from cancer patients reveals unique ev profiles generated by cancer, further supporting the potential of ev fingerprinting as a liquid biopsy. summary/conclusion: our single-ev analysis approach characterizes whole ev populations in complex biological fluids without the need for purification, reducing time intensive purification protocols and subsequent sample loss, permitting efficient analysis of liquid biopsy samples. detection and quantification of extracellular vesicles with cargo protein and rna using the amnis® cellstream® flow cytometer introduction: the particle size distribution (psd) of extracellular vesicles (evs) is commonly measured by tunable resistive pulse sensing (trps) and nanoparticle tracking analysis (nta). both trps and nta have limitations that hamper the accurate measurement of the psd of evs, specifically in the size range from to nm. an alternative technique for measuring the psd of evs is micro-fluidic resistive pulse sensing (mrps). because a standard operating procedure (sop) for characterizing evs by mrps is absent, we aim to establish a reliable sop to ensure reproducible psd measurements of evs by mrps. methods: measurements (n = ) of red-blood cell, prostate cancer cell line supernatant, and human urine and plasma evs were acquired in × s acquisitions. two microfluidic cartridges were used to study a dynamic range of - nm. samples were diluted into phosphate buffered saline with different concentrations of tween or bsa. because the excess of particles affects the detection limit, serial dilutions were performed to find the optimal dilution for each sample. data were evaluated using data viewer software. results: the optimal dilution was determined for each sample by maximizing the particle rate and minimizing the measurement time while preserving a robust detection limit of or nm. moreover, we developed a procedure to optimize the peak filter settings of data viewer by fitting data to normal distributions and identifying threshold values for signal-to-noise ratio, symmetry, and transit time within % confidence. summary/conclusion: we recommend to use . % w/ v bsa in dpbs as sample diluent, because tween affects evs as confirmed by flow cytometry. by using orthogonal techniques and well-characterized biological test samples, we developed and validated a sop for ev detection by mrps, thereby making mrps a valuable tool for ev researchers. real-time measurements of extracellular vesicles binding kinetics achieved through interferometric imaging in a multiplexed microarray modality introduction: extracellular vesicles are very promising diagnostic biomarkers. as a matter of fact, the properties of these biological nanoparticles depend on the health conditions of each individual. however, experiments that involve evs phenotyping are time consuming, due to h-or overnight incubations. in order to get accurate results, maximizing binding efficiency is a necessity; that normally involves ensuring the saturation of the capture reaction, which can result in an unnecessarily long incubation time. with the ability of labelfree kinetic binding measurements using interferometric reflectance sensing in a microfluidic chamber, we perform an optimization of the incubation time in different flow conditions, while demonstrating a new way of multiplexing for real-time evs specific capture and detection.methods: all the real-time binding measurements were performed with the interferometric reflectance imaging sensor (iris). iris chips were first coated with an organic polymer (mcp- ), which provides an active surface for probe immobilization. then, antibodies against cd , cd , cd markers were spotted at different densities in a microarray modality. the chips were then encapsulated with a glass window to form a microfluidic chamber that allows for imaging the sensor surface. samples of hek-derived extracellular vesicles were flowed across the sensor surface in the iris system and real-time images were acquired. incubation was performed at different flow rates, and in static and stopflow modalities. results: in this work, we focus on the specific capture of evs under different flow conditions to achieve an optimization of the incubation time. indeed, through the acquisition of real time binding data, we are able to precisely monitor the equilibrium point of the capture reaction. in this configuration of iris, low magnification optics allow for simultaneous detection of binding on hundreds of capture ligand spots. therefore, surface probes (surface density and specificity) as well as assay conditions can be optimized. we report on the optimization of antibodies against cd , cd , and cd markers. since the sensor chips are identical to the single-particle detection assays developed by nanoview biosciences, the optimization of binding assays will directly impact the phenotyping of individual exosomes. summary/conclusion: our method proved to be very efficient in optimizing the most crucial aspects concerning evs captureflow conditions, incubation time, surface density and sample concentration. introduction: diabetes is a life treating diseases extending its impairing influence on more than billion of people around the world within upcoming years. the most harmful complication generating high treatment and social costs is diabetic nephropathy, which develops in about % of patients suffering diabetes. still we do not have an effective and direct prognostic biomarker to diagnose renal complications in the primary stage of renal disease. methods: extracellular vesicles were concentrated from diabetic patients' urine and washed to perform spectral analysis: fourier transform infrared spectroscopy (ftir), based on the molecular absorption of electromagnetic radiation in the infrared region of the spectrum in a range from cm- to cm- and raman spectroscopy (rs) as a technique based on inelastic scattering of monochromatic light. both techniques provide information on the chemical structure of compounds by identifying functional groups with high molecular specificity. results: average spectral signature obtained for evs from urine samples of patients in the different stage of kidney damage allowed distinguishing specific bands, representative for amide (i/ii), lipids, cholesterol and nucleic acids. spectral parameters correlated with a clinical stage and a commonly used indicator of renal function (creatinine) in diabetic patients. summary/conclusion: infrared and raman spectroscopy are promising tools to diagnose and monitor renal function in diabetes. introduction: several existing bioanalytical strategies for purifying and characterizing exosomes have allowed for fundamental progress to be made. mixtures of evs can be enriched for exosomes by techniques such as ultracentrifugation and size-exclusion chromatography. but, these processes require large amounts of material that are often difficult to obtain and many different types of particles have similar sizes and densities. it is likely that unique subfractions within enriched samples exist, particularly in complex biological matrices such as blood, urine or milk which remain difficult to characterize and isolate with existing analytical technologies. methods: bovine milk exosomes were isolated via differential ultracentrifugation and resolubilized in mm ammonium acetate. these data were recorded using charge detection mass spectrometry (cdms). in cdms, individual particles are reflected back and forth through an electrostatic ion trap where they pass through a sensitive charge detector. each time a trapped particle enters and exits the detector, its charge (z) and mass-to-charge (m/z) ratio is measured. mass distributions are generated by multiplying the m/z values by the charge measured for each ion and binning the resulting masses.results: the masses of particles in a bovine milk extracellular vesicle (ev) preparation enriched for exosomes were directly determined for the first time by cdms. particle masses and charges span a wide range from m~ to~ mda and z~ to~ e and are highly dependent upon the conditions used to extract and isolate the evs. in total, , particles were detected from eight cdms measurements. a simple two-dimensional gaussian model suggests that eight unique subpopulations of particles may be resolvable based on charge and mass. complementary em and proteomics analyses confirm that samples are enriched for exosomes. particles associated with the s , s , and s families that are centred at~ . ,~ . , and~ . mda, respectively, appear too small to be ascribed to exosomes. the remaining , ( %) particles detected by cdms are within the mass range expected for exosomes. while cdms measurements are at an early stage of development, this approach appears to provide a new physical basis for separating and characterizing ev particles. summary/conclusion: this work describes a novel biophysical approach for measuring and characterizing the masses and charges of the extremely heterogenous population of exosomes and other extracellular particles enriched in bovine milk. as new sample preparation methods, aimed at purifying specific types of exosomes from different cell lines, tissues, and other body fluids continue to evolve, rapid and sensitive cdms measurements of the physical properties of mass and charge may become an important means of assessing the efficacy of different protocols. funding: nih (r gm - ). bab is supported by indiana university quantitative chemical biology fellowship (t gm ). in situ detection of exosomal microrna- b by fusion with liposomeencapsulated nanomotor introduction: breast cancer is the most common cause of cancer-associated death in women and has raised global health concerns. early diagnosis and treatment are crucial to improve the prognosis and survival rate of breast cancer patients. liquid biopsy is expected to provide a strategy for early diagnosis of breast cancer. exosomes have been regarded as novel liquid biopsy biomarkers due to their stable cargo of rnas, lipids, and proteins from their origin cells. exosomal micro (mi)rnas have recently been recognized as promising indicators of cancer occurrence and progression. however, most of the reported exosomal mirna detection methods require the lysis or extraction process, which increases the possibility of sample loss. in situ detection strategies avoid interference from body fluid. in this study, we developed a gold nanomotor fluorescence platform based on liposome fusion for breast cancer exosomal mirna in situ detection. the exosomal mirna detection platform was constructed using a gold nanomotor (detector) and liposomes (carrier). the dnazyme amplification sequences which could be especially triggered by mirna- b were identified by sds-page before modified on gold nano-motor and the capacity of the nanomotor was assessed using synthetic target sequence, breast cancer cell mda-mb- , mirna- b-encapsulated anionic liposomes, and mirna- b-expressing exosomes. three kinds of liposomes were synthesized, characterized, and assessed for loading ability. membrane fusion effect was evaluated by confocal laser scanning microscopy (clsm) and nanoflow cytometry. the performance of this method to discriminate between breast cancer patients and healthy individuals was investigated. results: the chosen dnazyme amplification sequences transformed "locked" status to "cleavable" status on target addition, releasing a fluorescence signal. the modified gold nanomotor showed a ten times higher fluorescence signal in the presence of mirna- b than the background and no noticeable fluorescence changes from a single-base-mismatch sequence. moreover, among the three different liposomes, cationic liposomes exhibited great stability, high loading efficiency, and excellent membrane fusion effect. furthermore, the fluorescent experiments confirmed that cationic liposomes could load and transfer the nanomotors into exosomes for mirna- b detection. finally, we were able to distinguish breast cancer patients and healthy individuals by sensing exosomal mirna- b directly from plasma samples without exosome isolation. summary/conclusion: a separation-free and sensitive assay based on dnazyme amplification technique and membrane fusion effect was established for breast cancer-derived exosomal mirna- b detection, which could be a promising tool for the liquid biopsy of breast cancer. isolation of exosomes by membrane affinity column increases non-exosomal rna recovery in comparison to differential ultracentrifugation introduction: exosome-based liquid biopsy is a potential aid in the diagnosis and prognosis of cancer patients. however, in order to incorporate exosomes into clinical routine, there is a need to compare different isolation methods. here we analysed the impact, in exosomal rna yield, of two intermediate recovery/ intermediate specificity methods: differential ultracentrifugation (ucd) and a membrane-affinity column (mac) kit. although mac has a faster performance which is more suitable to the clinic, we found that ucd results in a higher recovery of exosomes and less contaminating non-exosomal rna.methods: exosomes were enriched by mac and ucd from identical volumes of human plasma ( , xg, min/ . ) m filtration/ , xg, h)(n = ) and lymphoma conditioned medium( xg, min/ xg, min/ xg, min/ , xg . h/ , xg, h) (n = ). all exosomes were characterized by nanoparticle tracking analysis (nta), immunoblotting of cd /cd /flotilin/alix and electron microscopy (tem). exosome pellets were pre-treated with proteinase k ( mg/ml/ °c/ min) and rnase a ( mg/ ml/ °c/ min) before phenol-chloroform/glycogen rna extraction. rna yield was measured by both fluorometer and bioanalyzer.results: isolation of exosomes by ucd, in both plasma and medium, resulted in a higher yield in comparison to mac. this was shown by an augmented intensity of marker bands in the ucd samples (p = . , n = ) as well as by an increased number of exosomes in tem.in contrast, mac final exosomal fraction (from both plasma and medium), resulted in a -fold and fold increase in rna, respectively, in comparison to ucd when measured by fluorometer. this was confirmed by bioanalyzer. introduction: there is a need for better techniques for characterizing ev populations. we developed a sensitive multiplexed electrochemiluminescence (ecl)based assay format to characterize evs in cell-conditioned medium (ccm) and human biofluids. here we use the format to analyse ev samples for the presence of ev surface proteins, and to identify changes in ev phenotype associated with different cell lines, purification methods and growth conditions. methods: multiplex plates were prepared on msd's u-plex® platform with antibodies for putative evsurface proteins. each well displayed an array of nine specific capture antibodies and a negative control antibody. evs from samples were captured on the arrays and then detected with a cocktail of anti-tetraspanin antibodies (cd , cd and cd ) conjugated to an ecl label. three distinct cell types were grown at two sites, msd and atcc. resulting ccm were each purified by four common methods: tangential flow filtration, peg-based precipitation, size-exclusion chromatography and centrifugal ultrafiltration. all samples were also assayed without purification.results: fifty-five of the surface markers were detected on intact evs from at least one evaluated cell type. datasets were analysed using correlation matrices, hierarchical clustering, and machine learning. for each cell type, when comparing unpurified ccm grown at different sites or evs prepared by different purification methods, we typically observed correlations above . , indicating that the purification methods did not introduce bias to ev phenotypes, and that the assay format can provide robust phenotypic information without any purification of evs. two unsupervised clustering analyseshierarchical clustering and t-distributed stochastic neighbour embeddingboth generated wellseparated clusters for each of the cell types, regardless of purification method or source. summary/conclusion: we developed multiplex ev surface marker assays and demonstrated their use for multimarker ev phenotyping. this flexible format enables rapid assay development for new ev subpopulations with or without sample purification. these results also demonstrate ev surface marker phenotyping via multiplex ecl assays may be used to distinguish ev populations from various cell types, and characterize bias introduced by purification. detection of misev recommended ev protein-markers using automated western blotting method for isolation of evs and a simple western blotting platform for automated protein separation and immunodetection of misev-recommended proteins.methods: total evs were isolated by affinity-membrane spin columns from pre-filtered . - ml plasma or - ml urine, respectively. intact vesicles were eluted and the ev-depleted biofluid fraction was collected from the flow-through. a small fraction ( μl) was analysed by a simple western blot workflow providing automated capillary electrophoresis-based protein separation and immunodetection, characterizing each fraction for presence or absence of misevrecommended proteins.results: a range of specific antibodies were identified and the ev fractions were shown to be enriched in evproteins, whereas contaminating non-ev proteins were significantly reduced. isolation of evs was necessary to allow detection of the low abundant ev protein markers, whereas non-ev proteins were readily detectable both in the neat biofluids and in the ev-depleted flowthrough. we characterized the effect of washing on the purity of ev isolates and defined the dynamic range of the workflow using titrations of input volume of both plasma and urine ev isolations. summary/conclusion: simple western blotting protocols were established for quality control of isolated evs in accordance with misev-guidelines. evs isolated using affinity-membrane spin columns were shown to be enriched in ev markers and depleted for non-ev proteins. al-pha beads: a library of extracellular vesicle-associated metalloproteinase biosensors (adams) and a disintegrin and metalloproteinase with thrombospondin motifs (adamtss) are highly promising cancer biomarker candidates that have complex roles in cancer pathogenesis and metastasis. importantly, within the context of lung cancer, the detection of adam proteolytic activity might be more informative than the level of adam protein.therefore, the development of low-cost metalloproteinase biosensors could serve as useful biomarker research tools. methods: to this end, we developed advanced proteolytic detector polyhydroxyalkanoates (al-pha) beadsa library of biodegradable, biopolymer-based protease biosensors. broadly, these biosensors utilise phac-reporter fusion proteins that are bound to microbially manufactured bioplastic beads. these phac-fusions also incorporate specific protease cleavage sites. in the presence of a specific protease, reporter proteins are cleaved off of the al-pha beadsresulting in a loss of bead fluorescence that can be measured using flow cytometry. these biosensors were assayed using either metalloproteinases, conditioned media or evs from in vitro cancer models.results: human metalloproteinase recognition motifs were identified in the literature and a total of different al-pha bead biosensors were designed. a control, tev-specific biosensor detected . introduction: brain extracellular vesicles (evs) are heterogenous and include previously described microvesicles and exosomes. herein we characterized a formerly unappreciated population of mitochondriaderived evs that we term "mitovesicles". mitochondrial dysfunction is a well-established hallmark of ageing and neurodegenerative disorders as down syndrome (ds). hence, we examined mitovesicle levels and cargo under these conditions to characterize in vivo mitovesicle biology and responsiveness to mitochondrial stressors. methods: employing a high-resolution density gradient, distinct and novel populations of evs were isolated from murine and human ds and diploid control postmortem brains or from cell media. morphometric ev features were analysed by nanoparticle tracking analysis and cryogenic electron microscopy, while ev constituents were characterized by western blotting, mass spectrometry, lipid profiling and mitochondrial rna qpcr.results: we identified a population of double-membrane, electron-dense brain evs containing multiple mitochondrial markers ("mitovesicles") that are highly distinct from microvesicles and exosomes. proteomic data show that mitovesicles contain a unique subset of mitochondrial proteins while lacking others, such as tom . mitovesicles have a lipid composition that is unlike that of previously described evs and is consistent with mitochondrial origin. functionally, the complex-iii inhibitor antimycin-a stimulated in vitro mitovesicle release into the cell media, suggesting an interrelationship between mitochondrial dysfunction and mitovesicle biology. in mouse brains, mitovesicle levels increased with age and were found to be higher in ds compared to diploid controls. mitochondrial rna and protein levels were also altered in ds compared to diploid controls. summary/conclusion: we describe a previously unidentified type of metabolically competent evs of mitochondrial origin that we designate mitovesicles. our data demonstrate that brain mitovesicle levels and cargo are tightly regulated in normal conditions and are modified during pathophysiological processes in which mitochondrial dysfunction occurs, suggesting that mitovesicles are a previously unrecognized player in mitochondria quality control and may have a role in the trans-cellular tissue response to oxidative stress. introduction: alzheimer's disease (ad) is a devastating neurodegenerative disease leading to progressive memory loss and ultimately death with limited therapeutic options. growing evidence supports the theory that toxic proteins, like tau and amyloid, may propagate from diseased cells by packaging toxic proteins into extracellular vesicles (evs) and releasing them to infect other cells. one enzyme involved in the isev abstract book biogenesis of evs is neutral sphingomyelinase (nsmase ), which catalyzes the hydrolysis of sphingomyelin to produce phosphorylcholine and ceramide. several groups have reported improved cognition and reduced tau propagation when nsmase is pharmacologically inhibited or genetically knocked down in ad mouse models. unfortunately, current nsmase inhibitors are not suitable for clinical development due to poor solubility and inadequate pharmacokinetic profiles.methods: our group carried out a high-throughput screening campaign followed by extensive medicinal chemistry efforts leading to the discovery of phenyl (r)-( -( -( , -dimethoxyphenyl)- , -dimethylimidazo [ , -b] pyridazin- -yl) pyrrolidin- -yl) carbamate (pddc), an orally active, nm potent inhibitor with excellent selectivity and brain penetration. we tested pddc's ability to inhibit exosome release in cultured primary glial cells as well as an in vivo model of acute ev release. we then treated xfad mice with mg/ kg of pddc daily for six months and monitored their behaviour in the fear conditioning assay.results: pddc dose dependently reduced ev release from cultured primary glial cells and significantly reduced plasma ev numbers in an in vivo model. following chronic treatment with pddc, xfad mice demonstrated significantly improved cognitive function in the fear conditioning assay. summary/conclusion: these promising findings are currently being expanded using mouse models of tau propagation. if successful, these data would support pddc as a novel compound for targeting the pathological spread of tau as a therapeutic for ad. profiling evs in the anterior cingulate cortex of individuals with major depressive disorder introduction: major depressive disorder (mdd) is one of the leading causes of disability worldwide, affecting % of the population. the environment has been thought to play a role in the disease development, resulting in biological changes mediated by epigenetic mechanisms. microrna's (mirna) are well known epigenetic regulators that are disrupted in the depressed brain, and they are packaged into extracellular vesicles (evs). evs have emerged as means of intercellular communication, a process that is also disrupted in mdd. they are thought to transfer mirna between cells, which can alter gene expression in recipient cells. therefore, we hypothesize that ev cargo is altered in mdd subjects compared to healthy controls (hc). the aim is to extract evs from human postmortem anterior cingulate cortex, a region previously associated with depression, and profile the mirna cargo and compare it between mdd subjects and hc. methods: post-mortem human brain tissue from the anterior cingulate cortex of mdd subjects and hc was mildly dissociated in the presence of collagenase type iii. residual tissue, cells, and large vesicles were eliminated, and evs were isolated using size exclusion chromatography. the quality was assessed by western blots and transmission electron microscopy (tem). rna was extracted and a small-rna library was constructed and sequenced using the illumina platform. differential expression analysis was then performed.results: western blots showed little to no endoplasmic reticulum (calnexin), golgi (bip), or mitochondrial (vdac) contamination, along with enrichment of the exosomal marker cd . tem images showed the typical cup-shaped morphology with sizes mostly between and nm. preliminary sequencing results revealed that mir- a- p, which is predicted to target glutamate receptors, is downregulated in evs from mdd subjects. summary/conclusion: high quality ev extractions can be obtained from post-mortem brain tissue using our method. this will be the first study to profile brainderived ev mirna in the context of depression. future studies will be needed to determine the effect of the different levels of mir- a- p. this could provide novel mechanistic insights into the pathophysiology of mdd and will serve as a starting point to examine the potential role of evs in mdd pathology. methods: we use ifc to characterize evs released by glioma using -ala, fluorescently labelled ev (cfda-se, cd ) and glioma specific (tenascin c and epidermal growth factor receptor viii, egfrviii) markers. furthermore, we characterized evs released by egfrviii positive glioma cells treated with dexamethasone, a steroid commonly used in glioma patients, to determine the effect of steroids on ev release. evs were quantified by ifc and results were confirmed by qpcr for the levels of egfrviii mrna. results: firstly, we optimized protocols to label glioma sevs using fluorescently labelled ev markers (cfda-se, cd ) and tumour specific markers (tenascin c and egfrviii). of the total evs (cfda-se), we demonstrate that % are tenascin c positive, . % are egfrviii positive and . % are -ala positive. there was only a minor overlap (< %) between the sub-populations. finally, we show that dexamethasone treated glioma cells release lower total evs ( . -fold), tumour specific evs ( . -fold; egfrviii), egfrviii mrna compared to mock treated cells. summary/conclusion: we demonstrate the potential of ifc to monitor sevs released by glioma cells exposed to different stimuli. this allows the characterization of ev sub-populations providing a working model to understand the dynamics of tumour evs at a single vesicle level. introduction: extracellular vesicles (ev) released by infective forms of trypanosoma cruzi, the agent of chagas' disease, modulate inflammatory response of macrophages through the activation of toll receptor (tlr ) via mitogen-activated protein kinase pathway. this induces the production of nitric oxide (no) and expression of the cytokines tnf-α, il- and il- , which could explain the inflammation observed in experimental chagas' disease, and eventually in the progression of human disease. evs released by the parasite are heterogeneous and it is unknown which factor, or factors present in the different vesicle populations act during the interaction with host cells.objectives. the goal of the present work was to characterize and isolate the different populations of evs released by t. cruzi and test their effects on macrophages. methods: ev released by trypomastigotes forms of t. cruzi (y strain) were purified by asymmetric flow field-flow fractionation (af ) and characterized by nanoparticles tracking analysis (nta). the different populations of evs were incubated with host human monocytes cells (thp- ) and cytokines production determined by elisa and qpcr. the different ev populations were also incubated with llcmk- epithelial cells and the infection by t. cruzi determined. results: we found two distinct populations of evs. a population with to nm (ev ) and another with to nm (ev ). ev induced more tnf-alpha, il- , ip- and ccl than ev . it was also more effective in promoting t. cruzi infection in epithelial cells. due to unknown reasons, making these systems insufficient for use in drug development and infectivity assays.noroviruses are known to attach to gram-negative enteric bacteria and this facilitates infection in vitro. however, the microbiome-norovirus-host communication link is missing. noroviruses infect immune cells present in lamina propria during acute infection, but bacteria themselves are large enough to cross the mucosal and the tight epithelial barrier which separates gut lumen from lamina propria. we hypothesized that binding of noroviruses to bacteria enhances extracellular vesicles (ev) production. because commensal bacterial evs by themselves do not have any detrimental effects on host cells, we believe using evs in in vitro culture will enhance norovirus infection, thus producing higher titre of viruses for vaccine and anti-viral drug development. methods: attachment assay: purified norovirus was incubated with enterobacter cloacae, lactobacillus acidophilus and bacteroides thethiotaomicron, and grown to produce evs. the attachment was confirmed via qpcr.isolation of evs: clarified media supernatants were subjected to ultracentrifugation at varying speeds and . um filtration. co-purification of norovirus with the evs was checked.ev quantification and characterization: ev total protein content was measured by microbca. the number of vesicles were quantified by nanoparticle tracking analysis. scanning and transmission electron microscopy was performed to check quality of ev preparation and determine if virus was attached to the vesicles. internal ev protein content was evaluated using ms-hplc. the evs were also check for infectivity via tcid assay. results: incubation of noroviruses with commensal bacteria resulted in significant increases in production of evs compared to uninfected controls. murine norovirus (mnv), used as a surrogate, was found to be associated with evs. em analysis determine association of viruses with the bacteria as well as the mvs, while also showing certain surface structural changes in virus attached bacteria compared to mock bacteria. the evs were found to cause infection in naive macrophages. summary/conclusion: changes in ev production and content by bacteria exposed to noroviruses will provide insight into its pathogenesis and possible solutions to the low viral output from hunov culture systems.ps . = op . kylie krohmaly a , claire hoptay b , andrea hahn a and robert freishtat a a children's national hospital, washington, usa; b childrens national hospital, washington, usa introduction: bacteria constitutively produce biologically active extracellular vesicles (evs), which contain rna, dna, and/or proteins. bacteria use these evs for communication with other bacteria and recent research suggests bacterial evs can also affect host cells. given these findings, it is necessary to examine the role of bacterial evs in human disease. current methods of bacterial ev isolation from human specimens cannot distinguish between bacterial species. however, there is utility in examining evs from specific species, as bacterial species and their evs may have unique contributions to human disease. our objective was to isolate circulating evs specifically from escherichia coli (eevs) and haemophilus influenzae (hevs), two known colonizers and pathogens in the gut and airway, respectively. methods: total evs were isolated from the blood of six healthy volunteers via precipitation and size exclusion chromatography. evs were then selected via a novel latex bead-based fluorescent antibody construct targeting species-specific outer membrane proteins. we used flow cytometry to evaluate the isolated evs. results: the constructs were saturated with eevs at an antibody concentration of . µg/ml of plasma, as geometric means ≥ . µg/ml were nearly equal. hevs were detected at µg/ml of plasma, but saturation is yet to be determined. eevs were imaged by a fei talos f x electron microscope and measured between - nm, and hevs were between - nm. both types of evs were spherical. summary/conclusion: using this novel technique, we were able to isolate, detect, and visualize eevs and hevs. this technique enables the study of specific bacterial evs. in the future, ev contents will be assayed. furthermore, this technique will be modified so that specific bacterial evs from body fluids can be used for downstream functional applications. this is the first time that bacterial evs from targeted bacterial species have been detected in blood from healthy humans. introduction: nasopharyngeal carcinoma (npc) is characterized by a large presence of regulatory t cells (tregs) and the production of tumour-derived exosomes with immunosuppressive properties. our team showed that npc-derived exosomes favour the suppressive activity and recruitment of human tregs via ccl chemokine, thus contributing to npc immune escape (mrizak et al., jnci, ) . more recently, our team has shown that npc-exosomes could induce tregs by altering the maturation of dendritic cells (dcs) and promoting tolerogenic dendritic cells (tdcs) (renaud et al., herpas congress ). our main objectives in this study are (i) to define and compare the metabolic status of mature dendritic cells (mdcs), control tdcs and tdcs generated in the presence of npc-exosomes (exocnptdc) and (ii) to evaluate the chemoattractive potential of npc-exosomes on exocnptdcs, and notably to investigate the involvement of ccl in this recruitment. methods: dcs are generated from human monocytes in the presence or absence of npc-exosomes. the maturation status of dcs was evaluated at a phenotypic level by studying the expression of maturation markers using flow cytometry and at a functional level by analysing cytokines secretion using elisa. this cytokine analyse has been performed in both conditions, on treated dcs and during co-culture assays of autologous cd t lymphocytes with treated dcs. in a second step, a mitochondrial metabolic and glycolytic study was performed using the seahorse technology (ocr and ecar measurement). finally, the chemoattractive potential of npc-derived exosomes on the different induced dcs was analysed (i) using boyden chamber chemoattraction assays or real-time videomicroscopy (chemotaxis µslide ibidi) and (ii) using rt-qpcr analysis of the receptor expression of ccl (ccr ).results: npc-exosomes alter dc maturation, which gives rise to tolerogenic dcs that favour the induction of tregs. in addition, the metabolic analysis of dcs seems to put foward a specific metabolic signature of the tdcs induced by npc-exosomes. and finally, chemoattraction assay suggests that npc-exosomes preferentially attract tdcs and exocnptdcs in a ccl dependant manner. summary/conclusion: taken together our results should allow us to characterize the major role of npc tumour exosomes on the maturation and the recruitment of dc and so identify them as anti-tumoural therapeutic targets. cytotoxic t lymphocyte ev that prevents tumour metastasis by collapse of tumoural mesenchymal stroma is classified into exosome, but not microvesicle or apoptotic body.naohiro seo a , junko nakamura a , tsuguhiro kaneda a , takanori ichiki b , asako shimoda c , kazunari akiyoshi c and hiroshi shiku a a mie university graduate school of medicine, mie, japan; b the university of tokyo, bunkyo, japan; c kyoto university, kyoto, japanintroduction: recently, instead of ultracentrifugation, development of new preparation protocol is demanded for research of reliable bioactivity and drug discovery of extracellular vesicles (evs). in this study, we propose a novel method for large scale preparation of highperformance extracellular vesicles focusing on membrane negative charge. methods: murine cytotoxic t lymphocyte (ctl) evs in supernatant were concentrated more than times at over % purity without leaking by kda mwco ultrafiltration, and subjected to ion exchange deae column chromatography after replacing with pbs. after ion exchange, evs were characterized by bca assay, nta assay, cryotem observation, proteome analysis, dna content measurement, mirna microarray analysis, zeta potential measurement, lectin array analysis, and target cell analysis.biomarker detection and analysis and detail strategies for cross-platform analytical validation. methods: we conducted a cross-platform analysis using two commercially available flow cytometers designed for ev detection. scatter resolution, enumeration accuracy and precision were determined across both platforms by analysing submicron silica beads (apogeemix, - nm) of known concentration.we detected large evs, as established by reference size beads, electron microscopy, expression of phosphatidylserine and the presence of integral membrane proteins of cell of origin. we analysed evs isolated from plasma by high-speed centrifugation ( , g) as well performing analysis by direct plasma labelling followed by validation by detergent lysis of vesicular constituents. a clinical operating range was defined which ensures linearity and avoids swarm detection. we observed comparable scatter resolution, enumeration accuracy (error ≤ %) and precision (cv ≤ %) across both platforms used. we defined two ev size gates: a "latex" gate ( to nm polystyrene latex beads), and a "silica" gate ( to nm silica beads) for evs at the lower end of our size range of interest. to improve detection sensitivity, we identified common contributors to signal noise and applied workflow strategies to minimize these. finally, we identified linear ranges which avoid swarm detection, and which ensures reproducible ev counts (cv < %) across both instruments. summary/conclusion: we present an optimised, standardised and cross-platform reproducible working protocol which supports the use of fcm in an ev-based liquid biopsy application. funding: the project is funded by spark oceania and uts innovation commercialisation seed fund scheme to mb. metabolomic profiling of serum and exosomes isolated from head and neck cancer patients after radiotherapy introduction: cancer radiotherapy (rt) induces the response of the whole body that could be detected at the blood level. searching for new molecular signatures which could correlate with treatment response in cancer patients is of particular importance. radiation-induced changes in proteome and transcriptome of serum have been widely described. however, metabolomic changes in serum, exosomes and other classes of small extracellular vesicles (ev) of cancer patients after rt have not been given as much attention. metabolomics of serum and ev of cancer patients could provide a valuable insight into the response of both tumour and whole organism to the treatment. the aim of the study was to compare serum and ev metabolomic profiles in head and neck cancer (hnc) patients before and after rt. methods: serum samples from hnc patients were taken before (a) and after (b) rt. healthy volunteers were used as a control group (c). ev were isolated from ml of serum using size-exclusion chromatography (sec). selected sec fractions were subjected to extraction of metabolites. a mixture of meoh/h o was used for extraction of metabolites from serum and ev samples. samples were analysed by gas chromatography-mass spectrometry (gc-ms).the study protocol adhered to the tenets of the declaration of helsinki and was approved by the bioethical committee of the maria skłodowska-curie national research institute of oncology, branch gliwice, poland (permit nr. do/dgp/ / / / / /g). results: an untargeted gc-ms-based approach allowed the detection of metabolites in serum samples and exosomal small molecules, of which joint. the identified compounds included amino acids, fatty acids, carboxylic acids, sugars, and others. there were metabolites which levels discriminated compared groups (a,b,c) of serum samples and compounds that discriminate the ev isolated from hnc serum before and after rt from hc. summary/conclusion: rt caused significant changes in levels of serum and ev metabolites witch are involved in amino acid metabolism, lipids metabolism, energy metabolism and oxidative stress response. capable of contributing to intercellular communication and metastasis. numerous studies have focused on elucidating their role in cancer progression. we recently showed that sevs isolated from pancreatic cancer cells can function as an initiator in malignant cell transformation. here, using a mass spectrometry (ms)-based proteomics approach, we analysed the differences in the protein cargo of sevs secreted from normal pancreatic and cancer cells to better understand their biological characteristics. methods: sevs were isolated from human pancreatic cancer cell lines (capan- , mia paca- , and panc- ) and normal pancreatic epithelial cells (hpde) using a combined ultrafiltration-ultracentrifugation method coupled with a sucrose density gradient purification. proteomic profiling of sevs was carried out using an lc-ms/ms method. protein identification from resulting ms/ms spectra was conducted using proteome database search software followed by gene ontology (go) enrichment and reactome pathway analysis.results: a total of , unique proteins were identified confidently across the combined samples. the proteins present in all four sev types ( , proteins) consist of general housekeeping proteins. proteins were uniquely found in all cancer sevs but not in the normal hpde sevs. this group contains an enrichment of proteins that function in the endosomal compartment of cells responsible for vesicle formation and secretion and suggest their important role in driving the increased production of sevs from cancer cells relative to normal cells. moreover, this group includes a set of proteins that have been implicated in malignant cell transformation, consistent with our previous work showing that each of the cancer sevs analysed here could initiate malignant transformation of nih/ t cells. conversely, there were proteins uniquely found in normal hpde sevs. this group includes a number of immune response proteins that are not found in any of the pancreatic cancer cell sevs. summary/conclusion: the differences in the proteomes of cancer and normal sevs may be indicative of their varying roles in cell transformation and helpful in delineating the types of evs that are being produced. in addition, these differences point towards their potential value as cancer biomarkers. proteomic profile of tumour-derived exosomes in plasma of melanoma patients introduction: in the past years, extracellular vesicles (evs) have attracted considerable interest due to their ability to provide valuable diagnostic information from liquid biopsies. the high abundance in all bodily fluids and their cargo stability confers evs the potential as a powerful tool to not only obtain novel biomarkers from inaccessible tissues, therapy response and monitoring, but also to reduce infection risks of conventional highly invasive biopsies. virtually all cells continuously release vesicles into the extracellular environment, diverse in size, content and features depending on the biogenesis, origin and function. this heterogeneity adds a layer of complexity when attempting to isolate and characterize tissue-specific vesicles. methods: hence, we aimed to use a immunomagnetic capture approach for prostate-derived evs from cell culture supernatants, with further investigation into human plasma and urine samples. analysis was performed by nanoparticle tracking analysis, western blotting and electron microscopy. additionally, an in-house spotted antibody microarray is in development. here, we intend to detect different ev sub-populations based on their surface markers. results: isolated immunocaptured ev populations based on the classical ev marker cd show an increased signal for the luminal protein tsg . ev populations targeting the tissue-specific marker prostate specific membrane antigen (psma), were found positive for tsg in a lower extent indicating a subpopulation of evs. the microarray uses less than µl of sample (concentrated cell culture supernatant, human plasma, urine) and leads to a faster characterization within h for ev surface marker as compared to western blot. summary/conclusion: immunomagnetic isolation might be a promising approach for liquid biopsy and thereby the microarray could be valuable to identify potential capture targets. the current design for different surface marker from samples simultaneously could be easily extended for sample size and surface profiling allowing for a more economical way to multiplex samples. paving the way for implementing a feasible and reliable technique for assessing urinary extracellular vesicles as biomarkers for bladder cancer in clinical practice introduction: extracellular vesicles (ev) in urine have been proposed as biomarkers for bladder cancer (bc). however, at present there are no standardized methods for ev isolation or urine sampling. our goal was to evaluate the ev isolation performance between different methods, the effect of the sampling time and the importance of urinary creatinine (ucr) normalization. methods: two urine samples of ml were collected from patients with non muscle-invasive bc: one from the first micturition and another from any time of the day. twenty ml were used for ucr measurement and ml were used for ev isolation by either precipitation with polyethylene glycol (peg), concentration by filtration (uf, centricon plus- , k, millipore), sepharose size exclusion column (sec), or combinations of these methods. additionally, the effect of protease inhibitors (pi) and dtt treatment after collection or during processing was analysed. size and number of particles were evaluated by nanosight and the presence of exosomal markers was evaluated by western blot. results: among the methods evaluated, uf + sec showed the best performance retrieving the highest number of particles in the range of - nm, and the highest protein expression of exosomal proteins. uf alone showed the highest concentration of ev, but with a tendency to isolate larger particles. particle concentration was positively correlated with ucr, reflecting the importance of ucr normalization before journal of extracellular vesicles comparing between patients. finally, no differences in the performance according to the time of collection, nor in the use of pi or dtt were observed. summary/conclusion: uf + sec gave the highest ev yield and was not affected by the time of urine collection. the use of pi and dtt can be avoided, and normalization to ucr should be considered when implementing this technique for assessing evs as biomarkers for bc in clinical practice. funding: pida . the introduction: human tumours, including pancreatic ductal adenocarcinoma (pdac), often harbour a subpopulation of cancer cells with extra centrosomes. we found, that these cells secrete an increased number of small extracellular vesicles (sevs), within the - nm size range. sevs play a role in cancer signalling and progression and are widely studied for their diagnostic potential. we aim to understand the role of sevs secreted by cells with extra centrosomes in shaping pdac-associated stroma, particularly fibrosis. methods: to study the sev mediated changes in the pdac microenvironment, we purified sevs through serial ultracentrifugation and size exclusion chromatography, characterised the content through silac-based proteomics, and assessed phenotypic changes in pancreatic stellate cells (pscs) and extracellular matrix (ecm) production through immunofluorescence staining. results: our data indicates, that the sevs secreted by cells with extra centrosomes are exosomes due to their endocytic origin, and we found, that they can activate pscs, key mediators of fibrosis in pdac. indeed, we observed an increased level of collagen i produced by pscs activated by sevs from cells with extra centrosomes as compared to cells without extra centrosomes. interestingly, we found, that psc activation through sevs is not mediated by tgf-β, assessed by the level of nuclear smad accumulation downstream of tgfβ activation, suggesting a novel mechanism of pscs activation. summary/conclusion: pdac cells with extra centrosomes contribute to a novel type of psc reprogramming, which could alter their ecm deposition and contribute to the extensive fibrosis observed in pdac. we are currently characterising the signalling pathways associated with sev mediated psc activation and how it impacts padc progression to better understand the role of centrosome amplification in the cancer-stromal crosstalk. exosomal carboxypeptidase e confers and cpe-shrna loaded exosomes inhibit growth and invasion of hepatocellular carcinoma cells. methods: exosomes were isolated from the culture media of high metastic hcc h cells and incubated with low metastatic hcc l cells. in other experiments, cpe-shna loaded exosomes from hek cells were incubated with hcc h cells. the recipient cells were analysed for proliferation using mtt assay, colony formation, and matrigel invasion. results: analysis of exosomes derived from hcc h cells revealed cpe-wt mrna and protein. exosomes released from hcc h cells were able to enhance proliferation and invasion of hcc l cells. when cpe expression was suppressed in the hcc h cells before exosome isolation, the exosomes had no effect on proliferation and invasion. these data demonstrate the ability of exosomes to confer growth and invasion in hcc cells and the role of exosomal cpe in driving the process.previously it was shown that down-regulation of cpe expression by shrna can reverse tumour growth and metastasis in an hcc mouse model. we therefore loaded cpe-shrna into exosomes by infecting hek (human embryonic kidney) cells with adenovirus carrying cpe-shrna-gfp. these modified isev abstract book exosomes were used to transfer cpe-shrna to hcc h cells, resulting in significant reduction in proliferation and colony-forming ability of these cells. cpe-shrna loaded exosomes were found to down-regulate the expression of cyclin d and c-myc, two genes with high relavance to tumour growth and metastasis. summary/conclusion: our results demonstrate the ability of exosomal cpe to enhance proliferation and invasion in low metastatic hcc cells and the potential to use shrna loaded exosomes to target cpe as a therapeutic strategy to treat liver cancer.funding: intramural program of the eunice kennedy shriver national institute of child health and human development, and national cancer institute, national institutes of health, bethesda, md. . stress hormones promote prostate cancer aggressiveness through modulation of mir- - p expression and exosome release north carolina central university, durham, usa introduction: despite proactive screening and steady declines in mortality, prostate cancer (pca) remains one of the most prevalent cancers among men. evidence suggests that chronic activation of stress signalling pathways can result in an altered mirnas transcriptome and affect exosomal content and release. here, we study the interaction between leptin and mir- - p expression, previously shown to be downregulated in pca patients. in addition, explored the effect of stress hormones cortisol and leptin on exosomal release and content from pca cells.methods: we utilized normal prostate cell line rwpe- , and pca cells pc , lncap and mda-pca- b. proliferation of cells treated with leptin in the presence or absence of mir- - p mimic or negative control was assessed by mtt, colony formation, wound healing, and expression of targets affected by mir- - p was assessed by western blotting. moreover, exosomes were isolated via differential centrifugation from pca cells treated with leptin or cortisol and exosome number was determined by nanotracking analysis. exosome content was determined by western blotting and proteomic analysis by mass spectrometry.results: we observed that leptin significantly decreased expression of mir- - p in rwpe- cells.co-treatment with mir- - p mimic and leptin abrogated these effects in a cell dependent manner. we also observed that co-treatment with leptin affected mir- - p target jag and other molecules involved in epithelial to mesenchymal transition. in parallel, we demonstrated that cortisol increases exosome secretion particularly in pc cell exosomes with a . -fold increase at nm cortisol compared to untreated. western blotting revealed the presence of gr in exosomes particularly at nm cortisol. summary/conclusion: understanding epigenetic regulation through mirnas and exosomes may be the key to understand stress hormone influence in pca progression. these findings suggest that stress hormones effectively affect mir- - p expression and exosomal release and signalling.introduction: extracellular vesicles (evs) are promising drug delivery vehicles for therapeutic microrna (mirna). for the loading of exogenous cargo, researchers broadly seek to either manipulate the evs directly or the cell that produce them. electroporation, sonication, and direct ev transfection are common methods that work by physical disruption or irreversible chemical addition, which may irreparably damage the molecules intended for therapy. on the other hand, transfection into the producer cells is a simple option that does not imperil ev integrity.methods: there are multiple factors that contribute to ev loading efficiency, including transfection reagent used, timing, and dosage. thus, we sought to establish a basic protocol and improve understanding of the underlying dynamics involved in a basic system consisting of hek t cells and mir- a- p mimic.results: in this work, we examined how different reagents lead to variable ev loading. then we looked at variable dosages, specifically the relationship between rna amount added to reagent, amount present in cell, and amount exported to evs. summary/conclusion: these results will help future studies produce evs with exogenously loaded small rna, and suggest future optimizations. funding: national institutes of health. r and t (host pathogen interactions at university of maryland). we report a single ev trapping method via aptamermediated assembly between au nanoparticle (aunp) and au superlattice template. we propose a chip-based ev trapping technique based on semiconductor processes. methods: we introduce aptamer coated au nanoparticle (aunp) and au superlattices as a template to capture evs. first, we fabricated poly(methyl methacrylate) (pmma) hole pattern on au-coated si substrates by using electron beam lithography (ebl). we designed nm-diameter hole patterns to capture one ev in each hole. to connect the aunp and the au superlattice template, we used an aptamer molecule as a linker strand. also, to capture individual evs, the aptamer molecule is designed to have a hairpin structure to specifically bind to cd , a protein marker of ev. we modified ʹ-terminal and ʹ-terminal of the cd aptamer with thiol group for the formation of self-assembly monolayer (sam) on both aunp and au superlattice surface. results: first, we coat the cd aptamer on the surface of aunp. afterwards, we load the aptamer-coated aunp into au superlattice template. ev solution is specifically bound to cd aptamer. after washing step, each ev is expected to locate within a single hole due to the size confinement of the hole. to separate the evs from the aptamer, we use restriction enzyme, bamhi, to recognize specific dna sequence and cleave them. summary/conclusion: in this report, we propose a aunp -linked au superlattice chip by aptamer molecules for trapping evs. we selected cd aptamer for specifically binding with cd in evs. in addition, we designed cd aptamer as a linker strand to connect introduction: a hallmark of platelet activation is the release of internal granules as extracellular vesicles/ microparticles. thrombolux is a dynamic-light-scattering-based (dls) instrument that was developed for use in clinical setting to check for platelet activation before transfusion. compared to traditional dls, the thrombolux requires no cleaning (single-use capillary) and requires very little sample ( µl). hence the thrombolux may be a useful instrument beyond platelet pack test in blood transfusion laboratory. we have evaluated its use as an in-process monitoring tool for industrial ev manufacturing, for both quantifying cells (input) and evs (output). methods: the thrombolux was used to test the activation status of expired platelet packs (donated by arcbs for research purpose). the readout was compared with platelet swirling test and flow cytometry data (surface marker). furthermore, the thrombolux was also tested for process development and ev manufacturing monitoring purposes at different stages of the process for its ability to rapidly obtain particle presence and size information on evs. time to result was also compared between different particle analysis methods. results: the thrombolux was a better predictor of platelet packs variability compared to the traditional platelet swirling method. however, we did not observe a strong correlation between the activation status and the flow cytometry-based activation marker data. the thrombolux was able to provide a useful estimation of particle presence and sizing of evs in-process.results are obtained rapidly, within minutes, with minimal sample prep. summary/conclusion: although we did not observe a significant direct correlation between flow cytometry activation data and the % microparticles (within a small sample size), the thrombolux has shown potential to become a useful tool for in-process monitoring for ev manufacturing and other ev research, in particular through its speed and ease of use. funding: all funding was through exopharm ltd (asx:ex ). secreted introduction: a major manufacturing challenge related to exosome bioprocessing is that of robust and scalable purification. as efforts to translate exosomes into clinics grows, the more important the design of quality systems which can reproducibly purify the product becomes. the current gold-standard, ultracentrifugation, was adopted from the viral vaccine industry, but remains imperfect in terms of scale up and manufacturing due to labour and time intensive process requirements. in order to follow the preferential adoption of more standard bioprocesses, as previously achieved by the viral vaccine industry, we show the development of two monolith chromatography steps which can be used to purify exosomes from a clinically relevant, allogeneic stem cell product (ctx e ). methods: t-flask expansion of ctx e cells was performed to yield batches of - l of conditioned medium. the medium was subsequently clarified by benchtop centrifugation, and concentrated into a crude concentrate by tangential flow filtration [tff], using a combination of . µm dead-end filtration prior to concentration in a kda hollow-fibre tff system. tff retentate was loaded onto ml hic or aex monoliths, for further purification. potency was assessed by a fibroblast wound healing assay in vitro. results: exosome presence was verified in the tff material by detection of cd and cd . exosomes recovered in this manner could achieve full wound closure in vitro over hours, when dosed at µg. further purification by monolith chromatography showed high levels of reduction of albumin, detected by western blot, as well as heightened ratios of particles to both total protein, and total dna. the results indicate that neither aex nor hic steps cause detrimental loss to product function, either alone or in combination with one another. introduction: custom-made platelet pellet lysate (ppl) and heat-treated ppl (hppl) exert strong neuroprotective effects of neurotoxin-exposed dopaminergic luhmes neuronal cell culture. this effect is significantly enhanced using hppl, which was also highly protective of th-expressing neurons in mice parkinson's disease (pd) model. introduction: there is a critical unmet medical need for new therapies to treat age-related diseases including cardiovascular diseases such as stroke. exosome derived from stem cells have shown intrinsic therapeutic potential in a variety of animal models of ischaemic diseases. we have identified scalable exosome production cell lines (purestem) as a source of angiogenic exosomes and are aiming to generate good manufacturing practice (gmp) grade therapeutic exosomes that can effectively mediate angiogenesis and tissue regeneration. we are developing exosome production and purification protocols that combine methods of tangential filtration flow (tff) and size exclusion chromatography (sec). the particle number and size were measured by both tunable resistive pulse sensing (trps) as well as nanoparticle tracking analysis (nta) for comparison. exosomes were characterized by detection of exosome surface markers and absence of cellular markers. purity was assessed by measuring particles per ug of total protein content. the angiogenic activity of purestem-exosomes was assessed using live-cell imaging to measure endothelial wound-healing and tube formation assays. we further investigated the molecular cargo of purestem-exosomes by screening mirnas targets, rna-seq analysis, and mass spectrometry analysis.results: the isolated purestem-exosomes using our developed protocols were highly purified, resulting purity in the range of e - e particles/ug. we selected angiogenic exosome-producing cell lines from our purestem library by screening for functional activity and characterizing their molecular cargo. we found that purestem progenitor-derived exosomes showed higher angiogenic potency than primary mesenchymal stem cell (msc)-derived exosomes. furthermore, angiogenic micrornas such as mir- were enriched in purestem-exosomes from certain producer cell lines. summary/conclusion: these data demonstrate the potential for using purestem lines as a highly scalable source of therapeutic exosomes. we were able to obtain highly pure exosomes that retain their angiogenic activity. we anticipate that purestem-exosomes will be a valuable resource for developing ev therapies for stroke and other ischaemic diseases. we have developed purification methodologies aimed at achieving a robust and scalable exosome production compatible with gmp for clinical grade purestem-exosomes. these developments have great potential as therapeutic agents for future preclinical in animal model of stroke and clinical trials. neuronal introduction: the hallmark of parkinson's disease (pd) is a-synuclein accumulation, predominantly in dopaminergic neurons, causing neurodegeneration. pd is also associated with insulin resistance, a condition characterized by phosphorylated insulin receptor substrate- (irs- ). besides motor symptoms, some pd patients develop mild cognitive impairment (pd-mci) or dementia (pd-d). given the importance for prognosis, there is an urgent need to develop biomarkers for distinguishing pd with normal cognition (pd-n) from pd-mci/d. neuronal-origin extracellular vesicles (nevs) contain cell signalling and pathogenic proteins (including a-synuclein), which may serve as biomarkers for alzheimer's disease, pd and other dementias.methods: from . ml of plasma from pd-n, pd-mci, and pd-d patients, we immunocaptured nevs using anti-l cam antibody. then, irs- pser and irs- ptyr and a-synuclein were measured in nevs using electrochemiluminescence immunoassays.results: a-synuclein was lower in pd-mci and pd-d compared to pd-n (p < . ) and significantly decreased with increasing motor symptom severity measured by mds-updrs iii score (p = . ). irs- pser was lower in pd-d than in pd-n. irs- ptyr significantly decreased with increasing mds-updrs iii score (p < . ). no biomarker was associated with disease duration. summary/conclusion: pd patients with cognitive impairment exhibited lower nev levels of a-synuclein than cognitively intact pd patients, whereas a-synuclein and irs- ptyr were inversely associated with pd motor symptom severity. additional biomarkers and measurements will be available by the time of isev. plasma nevs is a valuable tool for discovering biomarkers in pd and investigating aspects of disease progression. introduction: despite decades-long advancement in transplant medicine, there is a necessity for personalized approach regarding early kidney allograft injury recognition and immunosuppression therapy towards improved transplant outcomes. biopsy, a gold standard for assessment of kidney allograft injury, cannot be serially used for the diagnosis of subclinical injury due to it's invasiveness and possible sampling errors. instead, urine is easily obtainable and bearing extracellular vesicles (evs), potential carriers of pathological signals related to kidney injury. our aim was to set up a urinary ev (uev) isolation protocol that would allow consistent and reliable identification of their characteristics and cargo. methods: second morning urine sample ( ml) was collected from patients and processed within hours. oxalate precipitation, ph and dilution variability, uromodulin polymerization and high protein content were taken into account. isolated evs were defined by transmission electron microscopy (tem) and nanoparticle tracking analysis (nta). uev specific proteins and mirnas were analysed by western blot and qpcr, respectively. results: the optimal protocol relied on low speed urine centrifugation ( . x g, rt) for cell removal and storage at − °c prior to further analyses. after urine thawing at rt, added edta averted cryoprecipitate and uromodulin polymer formation, while concentrated pbs neutralized the ph. filtration through . µm pores was used for large particle removal, while centrifugal kda membrane units (amicon®, milipore) served for sample concentration followed by particle separation on sizeexclusion chromatography (sec; qevoriginal, izon q). protein vacant sec fractions (as rated at a ) were pooled and concentrated to a volume of µl. tem micrographs revealed high sample purity and cup-shaped morphology of uevs. as per nta results, the average mean size of evs was , nm with concentration range of × particles/ml of starting urine. uevs were positive for the tested marker proteins hsc , flotillin, tubulin, gadph and cd . qpcr verified mirna presence in uevs, with ct for mir let- i at . summary/conclusion: we successfully isolated pure uevs. the set up protocol will be used to assess uevs as non-invasive biomarkers of allograft injury in kidney transplant recipients. astrocyte-derived extracellular vesicles regulate dendritic spine formation and neuronal network connectivity introduction: recent advancements in the biology of extracellular vesicles have begun to implicate glial released microvesicles as mediators of glia to neuron communication, suggesting that alterations in the release and/or composition of astrocyte microvesicles could impact neuronal function. methods: astrocytes were allowed to constitutively release extracellular vesicles (adev-cr), or stimulated with atp (adev-atp). adevs were isolated by ultracentrifugation followed by proteomic analysis. we developed a normative whole transcriptome database using primary neurons exposed to adev-cr, and identified changes in neuronal gene expression produced by exposure of neurons to adev-atp. we identified a number of pathways associated with the biological response of synapse, spine and neurite outgrowth that were regulated by adev-atp. the molecular cargo of adev-atp responsible for regulating synaptic functions in neurons were characterized by biochemical, molecular, and functional assays. results: adev-atp enhanced the maturation of dendritic spines and produced functional enhancements in neuronal activity and network connectivity. the mechanism for this effect involved the delivery of integrin- and epha that were enriched in adev-atp. integrin- facilitated binding of adevs to the neuronal surface, and epha -receptor signalled through ephrin to the tyrosine kinase erbb / that regulated the phosphorylation and activation of trkb without increasing expression of the natural ligands bdnf or ntf . this direct activation of trkb increased the expression of the synaptic scaffolding proteins disc , arc, and cplx to promote the maturation of dendritic spines. this increase in mature dendritic spines was associated with increased neuronal activity and network connectivity demonstrating a functional strengthening of synapses. summary/conclusion: these data identify a molecular mechanism whereby modifications in adev protein cargo produced by the stimulation of astrocytes with atp regulates synaptic maturation through activation of trkb in a manner independent of growth factors. stephanie kronstadt and steven m. jay university of maryland, college park, college park, usa introduction: mesenchymal stem cell extracellular vesicles (msc-evs) have been shown to have an immunosuppressive effect in both autoimmune and inflammatory disorders. despite this, clinical translation of ev therapies is hindered by potentially low potency in vivo and the lack of a scalable biomanufacturing process. cell culture parameters are critical in modulating both yield and bioactivity of evs. thus, we hypothesized that the combination of chemical priming and d dynamic culture would enhance the yield and potency of immunosuppressive msc-evs. methods: bone marrow-derived mscs cultured in flasks were chemically primed using ethanol or curcumin. mscs were also cultured using a d-printed scaffold-perfusion bioreactor using a flow rate of ml/min. anti-inflammatory effects were assessed following application of msc-evs to lipopolysaccharide (lps)-stimulated murine macrophages. subsequent inhibition of the production of the pro-inflammatory cytokine il- , quantified using an elisa, was used to characterize evs as anti-inflammatory. in addition, both chemical priming and the bioreactor will be simultaneously utilized to potentially uncover any synergistic effects on ev immunomodulation abilities. nanoparticle tracking analysis (nta) was used to assess ev size and concentration while protein mass was measured via a bca assay. results: preliminary data suggests that priming mscs with µm ethanol for hours prior to ev collection results in a strong inhibition of il- production in stimulated murine macrophages. nta revealed that msc-ev yield increased by about two orders of magnitude in the bioreactor ( . e ± . e ) when compared with flasks ( . e ± . e ). protein measurements also indicated that ev production in the bioreactor (~ µg) was much greater compared with production in the flasks (~ µg). additionally, average protein content per ev was reduced in the bioreactor when compared with flask evs. regardless of tissue source. furthermore, comparison of adipose tissue-derived (ad) msc evs from three donors indicates varying pro-vascularization bioactivity between those donors evaluated in vitro via gap closure assay. similar results were observed for the bone marrow-derived (bm) msc ev donor groups. summary/conclusion: this work highlights the need for screening of donor derived-mscs before use for therapeutic ev production. additionally, standardized criteria for msc donor selection are needed before isolated msc evs can be used as a large-scale, repeatable therapeutic treatment. analysis of extracellular vesicle populations from malaria-infected erythrocytes by field-flow fractionation reveal distinct sub-sets alicia rojas a , paula abou-karam a , anna rivkin a , yael fridmann-sirkis b , yifat ofir-birin c and neta regev-rudzi c a department of biochemical sciences, weizmann institute of sciences, rehovot, israel, rehovot, israel; b wis, rehovot, israel; c weizmann institute of science, rehovot, israel introduction: malaria is one the most devastating infectious disease in the world and plasmodium falciparum (pf) represents the deadliest species. this parasite invades human red blood cells (rbcs) and releases extracellular vesicles (evs) carrying dna, rna and protein cargo components which are involved in the pathogenesis of the disease. recently, it has been shown in mammalian systems that evs are subdivided into different subpopulations, each with a distinct biological function. however, it is still unknown whether pfinfected rbcs (pf-evs) release different ev subpopulations with distinct cargo. methods: we isolated evs from pf-infected and uninfected rbcs, pf-evs or ui-evs, respectively, using differential centrifugation. the ev pellet was subjected to field flow fractionation (fff). the different subpopulations were collected, concentrated with size-exclusion filters and evaluated by nanoparticle tracking analysis. additionally, the presence of ev markers (sr and hsp ) were examined by western blot analysis. results: the fff analysis showed four particle subpopulations derived from the pf-evs and five in the ui-evs. the first three subpopulations were similar in their detection signals in both samples, but the fourth subpopulation was consistently higher in ui-evs than in pf-evs. moreover, hsp was detected in subpopulations and of both pf-evs and ui-evs, whereas sr only in subpopulation . isev abstract book summary/conclusion: pf-ev and ui-ev have similar separation profiles and proteins markers in their subpopulations, consistent with the fact that both samples are derived from host rbcs. additional data regarding the dna and rna cargo, as well as microscopic observations of the pf-ev and ui-ev subpopulations is necessary. this will clarify how malaria parasites sort their components into evs and which fractions are associated to immune evasion and pathogenesis. we have established a small size laboratory production of the microalgae culture in order to harvest the extracellular vesicles (evs) for pharmaceutical and medical uses. in this work we report on globular particles in the isolates from media of microalgae of two types, that we recognize as evs. we observed changes in their production at different temperatures and conditions. methods: samples were fixed by various combinations of aldehyde fixatives and/or osmium tetroxide. they were dehydrated in a graded series of ethanol, hexamethyldisilazane, and air dried. they were au/pd coated for inspection with scanning electron microscopes (sem) crossbeam fib-sem gemini ii (zeiss, germany) and jsm- f field emission scanning electron microscope (jeol ltd., tokyo, japan). results: microalgae were incubated overnight at °c and °c in growth medium and in growth medium supplemented with detergent. the samples obtained from the microalgae culture contained particles that we recognized as extracellular vesicles, however, these particles do not correspond to characteristic shapes of membrane enclosed entities without internal structure. increased temperature and/or presence of surfactant (triton x- and sodium dodecyl sulphate) stimulated formation of evs of different shapes and sizes. the isolates of these samples were rich with evs. in the presence of surfactant, the cell-walls detached from the cell and collapsed upon dehydration. this was documented by sem. summary/conclusion: focused ion beam technique revealed complex internal structure of the algae. it seems from the shapes of the observed structures that the particles deposited on the surface of the microalgae do not derive from budding of the membrane surface, but are instead shed by the cells from the cell interior upon the rupture of the cell wall. key: cord- -ezrn cva authors: nan title: physicians – poster session date: - - journal: bone marrow transplant doi: . /bmt. . sha: doc_id: cord_uid: ezrn cva nan hematopoietic stem cell transplant unit, hematology department, hospital universitario de donostia, donostia/san sebastián and informatics and automatics department, university of salamanca, spain the immature platelet fraction (% ipf) is a relatively new parameter that measures young (reticulated) platelets in peripheral blood (pb). ips rise as bone-marrow (bm) production of platelets increases. several clinical utilities of the %ipf have been already proved, as the treatment response monitoring in aplastic anemia or immune thrombocytopenic purpura. in this study, we aimed to found if ip measurement might be useful during the grafting phase of hsct. this study includes patients who underwent allo-hsct in our center during the last . years. were male ( %) and female ( %). median age was years (range: - ). baseline diseases were: acute leukemias ( ), lymphoproliferative disorders ( ), myelodysplastic syndromes ( ), chronic myeloproliferative diseases ( ), multiple myeloma ( ) and bone marrow failures ( ) . donor was unrelated in cases, and related in (including haplo-identical). conditioning regimen was: busulphan-based ( ), melphalan-based ( ), tbi-based ( ) and others ( ) . progenitors source was pb in , and bm in . platelet count, %ipf and absolute ip count (aipc) from day + to the day of stable graft were analyzed. . % patients reached plat ⩾ /mcl at day + , . % at day + and . % at day + . median first day of plat ⩾ /mcl was day + (range: . median %ipf was . % (range: - . ), . % (range: - . ) and . % (range: - . ) at days + , + and + , respectively. median aipc was /mcl (range: - ), (range: - ) and (range: - ) at days + , + and + , respectively. among the time points analyzed, aipc at day + showed the best positive correlation with platelets counts at day + (r = . ). interestingly, patients with lower aipc at day + showed a delayed platelet graft (see table ). contrarily, patients with higher aipc at day + had an earlier platelet graft. absolute immature platelet count before the graft seems to predict the precocity of the platelet graft for the majority of patients undergoing allo-hsct. this finding might help physicians for the patient management (anticipation of hospital discharge and so on). disclosure of conflict of interest: none. [p ] p analysis of genetic polymorphism for cardiovascular diseases (cvd) in placental and maternal blood in hypertension and hypercholesterolemia c khalil , a azar and a ibrahim , reviva stem cell research and application center, lebanese university, middle east institute of health hospital and faculty of medical sciences, lebanese university, lebanon cardiovascular diseases are the world's leading cause of death representing % of the total global mortality. the genetic polymorphism of the cvd genes, especially the ace: angiotensin converting enzyme gene risky alleles (ins/del) which are associated with a high and inappropriate level of ace can be considered as a genetic model in the development of hypertension and its complications in cvd. we evaluated the mutation impact of the cvd genes in the lebanese population, based on samples derived from placental blood (pb) and samples derived from peripheral blood of postpartum mothers. adult females (age ⩽ years) were divided (n = per group) into group (normotensive, normocholesterolemia: nn), and group (hypertension, hypercholesterolemia: hh). buffy coat were extracted from the pb. all tests on pb and maternal blood were done by using the test strip assay to identify the most relevant genetic variations to estimate the risk for cvd. the presence of a double mutation (ins+/del+) related to the ace gene in the hh group was %. the presence of a single mutation (ins − /del+) was only associated to the hh by %. (ins − /del − ) was absent in % of the pb and nn. despite the presence of double mutation ins/del for cvd in maternal blood, pb was free of this mutation. therefore, beyond genetic mutations, other factors can play a major role in the occurrence of cvd. disclosure of conflict of interest: none. s b e mt automated red blood cell depletion in abo incompatible grafts in the pediatric setting c del fante, l scudeller, s recupero, g viarengo, f compagno, m zecca and c perotti fondazione irccs policlinico san matteo red blood cell (rbc) depletion by apheresis is employed to reduce the rbc content from abo major or bidirectional mismatch bone marrow (bm) grafts mainly to avoid severe haemolysis . rbc depletion results in a significant volume reduction (due to both rbc and plasma depletion) and buffy coat concentration . . in pediatric setting, both rbc depletion and volume reduction before transplantation or cryopreservation can avoid fluid overload and renal impairment, especially in low/very low body weight recipients. the aim of this study was to evaluate the quality of the graft and immediate post infusion complications in rbc depleted bm in major and minor abo mismatch recipients using an automated device. patients and methods: bm aspirates for transplantation in pediatric setting were processed at our centre using the spectra optia (terumo bct) automated device. the initial collection preference was set at level and then was adjusted in order to maintain a haematocrit of % (colorgram) in the collection bag. flow speed was set at ml/min for cycles. mean recipients' body weight was kg (range: - ). pre and post procedure bm bag volume, hct%, mononuclear cells (mncs) count, (including b and t lymphocytes), cd + cell and cell viability were calculated. moreover, post procedure rbc volume and procedure time were registered. on the patient's side, post infusion complications (renal impairment, fluid overload, fever and haemolitic reactions) and time to engraftment were evaluated. results: a total of rbc depletion procedures were consecutively performed on bm grafts ( major and minor abo incompatibility, mud and related donors). data about pre and post procedure graft composition are reported in table . mean time to engraftment for pmn was . days (range: - ) and for plt was . (range: - ). pre and post-procedure cell viability were always %. mean procedure time was . minutes (range: - ). no bacterial or fungal contamination was detected. no infusion complications were recorded. one graft failure was observed. conclusions the spectra optia automated system is efficient in rbc depletion of abo mismatched grafts, permitting an effective volume reduction and an excellent mncs and cd + cell recovery in pediatric setting. automated rbc depletion may be proposed in low/very low body weight recipients both in abo major and minor incompatibility setting to minimize graft infusion side effects. building up a stem cell transplantation program in an emergent country, in the public setting, with limited economic resources, is not an easy work to do. international cooperation may be essential for the development of the program, in training, technological support and implementation of international guidelines. after years, we show an experience of international cooperation between a highly developed center in france (institut paoli calmettes, marseille) and the stem cell transplantation department of hospital maciel, a public assistance service in montevideo, uruguay. fourteen persons between doctors and nurses have been trained in france in stem cell collection and processing, patient's clinical handling, nursing, outpatients care and quality management. french missions of experts have been also received in hospital maciel every year since for in situ human resources training. in last years we developed a program for optimizing transplant results and reducing transplant related mortality (trm), based on several measures: improvement of patients selection, applying the sorror comorbidy index; adjustment of conditioning regimen doses, in order to reduce toxicity; development of a program to improve interaction with the intensive care unit; protocolization of the standard proceedings treatments; and initiating a program of quality and safety at the national institute of quality of uruguay inacal. adult patients have been treated with autologous (asct) ( ) or allogeneic (allosct) ( ) sct, with hematological malignancies. different modalities of allosct have been included progressively, becoming the only center accredited by the national regulation authorities (fnr) to perform unrelated donor sct and the haploidentical donor sct. this increased the proportion of allogeneic transplants from the historical % until % in last years. regarding patients health coverage, % comes from the private assistance system and % from the public health system. the major indications are lymphoid malignancies and acute leukemia, for asct and allosct, respectively, showing the same trend than cibmtr. three-year overall survival (os) for acute myeloid leukemia after allosct is %. considering asct for diffuse large b cell lymphoma, years os after autologous sct is % and % for chemo sensitive and resistant disease, respectively. threeyears os after asct for hodgkin disease is and % for sensitive and resistant disease, respectively. asct in multiple myeloma shows an os of and % at and years, respectively. in trm, results during the last years (after the described strategy) are shown in figure . the development of the-program of continuous improvement in quality-and the impact of results was locally recognized by two annual prices from inacal in (bronze) and (silver) in the category ‛commitment to public service.' a successful mirna- and the level of proangiogenic cytokines: angiopoietin- (angpt ), matrix metalloproteinase- (mmp- ) and vascular endothelial growth factor (vegf) in patients with lymphoproliferative malignancies prior to autologous hematopoietic stem cell transplantation (hsct) and in early posttransplant period. twenty-four patients were enrolled to the study ( f, m). the median (me) age was years. the investigated group consisted of multiple myeloma and lymphoma patients. the plasma samples were collected on time points: before chemotherapy-‛bc', on the day of hsct -‛ ', days after hsct-‛+ ' and days after hsct-‛+ . ' the cytokines were evaluated using elisa method, while mirna levels were estimated by qpcr method. the wilcoxon matched-pairs test was used to compare groups of dependent continuous variables: mirna's relative quantification (rq) levels or cytokines expression at two different time points. spearman rank correlation coefficient (r) was used to compare independent variables. we observed continuous decline of cytokines and mirnas level after conditioning treatment. the deepest decrease of expression was marked on ‛+ ' day ( table ) . we noticed a positive correlation between mirna- , mirna- cells in pbsc product. among the autologous transplanted patients between march and october , we have selected according to diagnosis, conditioning regimen and number of infused bags of cryopreserved pbsc. this group included females and males with median age of (range: . most of them, ( . %), had multiple myeloma (mm), ( . %) had non-hodgkin's lymphoma (nhl) and ( . %) had hodgkin's disease (hd). after harvesting, cd + cell and leukocyte number in pbsc product were enumerated on flow cytometer and blood cell counter, respectively. pbsc were cryopreserved with % dymethil sulfoxide (dmso) and cell viability was measured with trypan blue exclusion test before and after adding dmso, and as well after thawing in water bath on °c. as a conditioning regimen for the mm patients, melphalan was used and for the nhl and hd patients we used beam regimen. all received one bag of cryopreserved pbsc and pegfilgrastim mg on the first or the second post-transplant day. time to hematopoietic recovery was measured; for neutrophils . × /l, leukocytes × /l and platelets × /l with at least days without platelet transfusion. the median number of total leucocytes infused was . × /l (range: . - . × /l) of which cd + cells were - . × /kg of patient's body mass (median . × /kg). pre-freezing cell viability before and after adding dmso was with a median of % ( . - ) and , % ( . - ), respectively, and post-thaw viability . % ( . - ) . the average time to engraftment was . days ( - ) for neutrophils, days ( - ) for leucocytes and . days ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) for platelets. our results confirmed the known correlation between the number of infused cd + cells and engraftment of neutrophils (po . ), leukocytes (p o . ) and platelets (p = . ). we found inverse correlation between the infused leukocytes and cell viability with dmso (p = . ) and after thawing (p = . ). no correlation was found between prefreezing and post-thaw viability with hematopoietic recovery, and also between the cd + number and these viabilities. no differences were found considering patients' age, gender, diagnosis, conditioning regimen or day of applying pegfilgrastim. we can indirectly infer good survival of cd + cells and higher sensitivity of other nucleated cells to preparation of pbsc product. trypan blue exclusion assay, due to its inability to distinguish type of stained cells, is not relevant for cd + cells survival determination. disclosure of conflict of interest: none. chronic granulomatous disease (cgd) is a kind of primary immunodeficiency disorder of phagocytic cells which resulting in failure to kill a defined spectrum of bacteria and fungi and in concomitant chronic granulomatous inflammation. allogeneic hematopoietic stem cell transplantation is the only treatment proved to be potentially curative in cgd. unrelated umbilical cord blood (ucb) is increasingly used as an alternative to bone marrow. methods: unrelated ucbt was performed consecutive cgd children at our center between and . median age was . months (range: - months), median body weight was . kg (range: - kg). all patients received myeloablative conditioning regimen consisting of busulfan, fludarabine, cytarabine, cyclophosphamide and g-csf. all patients received tacrolimus as prophylaxis for graft-versus-host disease (gvhd). median nucleated cells were . × /kg (range: . - . × /kg), and median cd + cells were . × /kg (range: . - . × /kg). median follow-up time was . months (range: - months) results: of patients engrafted. median time to neutrophil engraftment was days, and median time to platelet engraftment was . days. / patients were alive, and / had full donor engraftment. overall survival rate was . %. disease-free survival was . %. of patients had grades iii-iv acute gvhd. no patients developed chronic gvhd. only one patient died from multi-organ failure related to adenovirus infection. conclusion: unrelated ucbt should be considered as potential curative methods in children with cgd. myeloablative conditioning regimen has improved the engraftments of the ucb. disclosure of conflict of interest: none. reduced muscular mass and excess visceral fat in patients undergoing hsct are associated with higher mortality, longer hospitalization, longer use of immunosuppressive drugs, graftversus-host disease (gvhd) and comorbidities leading to shorter survival time. a recent study of patients undergoing allogeneic hsct showed that occurrence of enlarged areas of visceral and peripheral fat is inversely associated with the disease-free interval after the transplant. reduced muscle mass has also been associated with higher prevalence of chronic gvhd and low rates of success following allogeneic hsct. objectives: to investigate whether amount muscle mass and muscle strength (ms) as well as the amount of visceral fat (vf) of patients undergoing hsct would influence the duration of the engraftment time (en). we evaluated hsct patients (⩾ years) at hospital israelita albert einstein, são paulo, brazil, on their first day of hospitalization, before hsct. the thickness of the right femoral quadriceps muscle (rfq), measured at cm from the top edge of the patella was measured using ultrasound (us) in b-mode. the dominant upper limb strength of the patients was evaluated by the hand grip test. the vf was measured in the abdominal region, by the thickness of the fat layer between the linea alba and the anterior wall of the aorta. most patients were women ( %) with a mean age of years (± years) and % of our patients were elderly (⩾ years). the haploidentical ( %) was the predominant hsct, autologous ( %) and allogeneic ( %). most patients were overweight, with body mass index (bmi) of kg/m (± kg/m ). the average time en was days (± days). rfq was . cm (± . cm), ms was kgf (± . kgf) and the vf was . cm (± . cm). patients with lower rfq had a longer engraftment time that was statistically significant as the negative correlation between rfq and en was rs = . , p o . ), independent of the age and the hsct type as analyzed by linear regression. no significant correlation between vf or ms with en was found. in this cohort of patients we found that longer engraftment times were significantly correlated to reduced muscle mass but no positive or negative correlation was found with superior limb muscular force or with the amount of visceral fat. disclosure of conflict of interest: none. hematopoietic stem cell transplant unit, hematology department and pharmacy department. university hospital of donostia. donostia/san sebastiań introduction: lymphocytes are the cells responsible for the cellular and humoral immunity and, consequently, critical for hematological patients. the aim of this study was to analyze the eventual conexion between lymphocyte recovery and survival (srv) after allogeneic hematopoietic stem cell transplantation (allo-hsct). patients and methods: we retrospectively analyzed data from consecutive patients who underwent allo-transplants in our unit. in total, patients were male ( . %) and female ( . %). median age was years old (range: . baseline disease was: acute leukemia ( . %), lymphoma ( . %), myelodysplastic syndrome ( . %), chronic myelogenous leukemia ( . %), multiple myeloma ( %), aplastic anemia ( . %), chronic lymphocytic leukemia ( . %) and others ( . %). . % of allo-hscts were from an unrelated donor, and . % from a family donor ( % of them haplo-identical). the sc source was pbsc in . %, and bm in , %. a variety of conditioning regimens were employed, including: busulphan-based ( . %), melphalan based ( . %), tbi-based ( . %) and others ( . %). evolution of absolute lymphocyte counts (alc) and subpopulations during the first year after allo-hsct were analyzed. results: as shown in table , alc decreased abruptly during conditioning therapy and recovered up to baseline at days + and + ; at day + median alc had clearly improved compared with admission values. median cd + cells were lower than /mcl in two thirds of pts at day + and in only one third at day + . as shown in table , we found a significant link between alc at day + and srv, as well as between cd + cells at day + and srv. in our series, immunity recovery was a late event for the majority of patients undergoing allo-hsct. in addition, in our experience, the precocity and quality of the alc and cd + recovery was clearly linked with long-term survival. disclosure of conflict of interest: none. although there is experimental evidence suggesting the presence of a common mesoderm cell as origin of both hematopoietic (hsc) and mesenchymal progenitor cells (msc) in an animal model, it is still controversial if durable engraftment of native donor-derived mscs without ex vivo treatment can occur in the recipient of allogeneic hsct. to assess the presence of donor-derived msc following hsct. between july and july , a total of recipients of hsct were analyzed for hsc and msc chimerism. eighteen patients received bm grafts ( %), patients had peripheral blood as stem cell rescue ( %) and finally patients had a cord blood transplantation ( %). patients received myeloablative ( %) or reduced intensity conditioning ( %) for malignant ( %) or nonmalignant disease ( %). bm aspirate cells were plated and expanded in α-mem with % human platelet lysate at cells/cm . after - days, nonadherent cells were removed, while the adherent cells were expanded until they reached confluence. after weeks we quantified msc precursors as colony forming unit fibroblast (cfu-f). finally the amplified sequences were resolved by capillary electrophoresis ( ruo genetic analyzer, applied biosystems) and analyzed by comparing genotypes of bmt recipell detachment, nuclear dna was extracted (dneasy blood and tissue kit-applied biosystems) and specific polymorphic tandemly repeated regions (strs) were amplified by means of the polymerase chain reaction(pcr) following the specific manufacturers' instructions. (ampfℓstr identifile kit, applied biosystems following hsct (hsc and msc) to those of donors. we cultured whole bm aspirates from patients following hsct with a median time of day (range: - ). cfu-f/ × growth was observed in a majority of bm the prevalence of human pegivirus in recipients of allogeneic hematopoietic stem cell olga koroleva , e parovichnikova , l kuzmina , m drokov , v vasilyeva , z konova , ekaterina mikhalcova , d dubnyak , n popova , tamara romanova , d tikhomirov , t tupoleva and v savchenko bone marrow transplant department, national research center for hematology and virology department, national research center for hematology human pegivirus (hpgv; previously named as gb virus c/hepatitis g virus) was discovered more than years ago. it is an rna virus referred within the genus pegivirus of the family flaviviridae. hpgv rna is found in liver, spleen, bone marrow and peripheral blood mononuclear cell, including t-and b-lymphocytes, nk-cells and monocytes. despite of the fact that it is a molecular structure, mechanism of replication and transmission routes are very well understood but the clinical significance of hpgv is still not determined. recipients of allogeneic hematopoietic stem cell have a high risk infection of hpgv. it is known, that hpgv is a nonpathogenic virus, however, it may play a role in immunocompromised individuals. to investigate the frequency of occurrence of hpgv and its clinical significance in recipients of allogeneic hematopoietic stem cell. blood samples were obtained from patients who underwent allogeneic hematopoietic stem cell transplantation (allo-hsct): all n = , aml n = , mpn n = , cll n = , mm n = , lpd n = , aa n = , mds n = . a median of age was years ( - years) . forty five patients were males and patients were females. conditioning regimen was ric in cases, mac in . bone marrow as a graft source was used in , pbsc- . all patients received multiple transfusions of blood components at the previous stages of treatment. hpgv rna had been assayed by polymerase chain reaction real time (rt-pcr) on plasma samples before started pre-transplantation conditioning. despite the diagnosis incidence of hpgv was high . % (rna-hpgv was positively in patients). patients with piercings and tattoos had incidence of hpgv in % that was not statistically significant (p- . ). hpgv is known as nonhepatotropic virus. in our study there was also no statistical reliability of specific changes in liver function test such as elevating the levels of alanine aminotransferase, aspartate aminotransferase, alkaline phosphatase and total bilirubin due to the rna-hpgv. liver enlargement was also not statistically significant according to ultrasound scan results in patients infected with hpgv. we also analyzed the co-infection with hepatitis b and c virus. results are presented in table . coinfection was not statistically significant. however, only one patient with hepatitis c was coinfected hpgv. leukocytes recovery median was days ( - ). thrombocytes recovery median was ( - ). the presence of rna-hpgv did not affect the recovery of peripheral blood cells in patients after allo-hsct. according to our study the frequency of hpgv infection in recipients of allogeneic bone marrow was quite high ( . %), and it did not depend on the presence of any other hepatotropic viruses. clinical significance of hpgv infection in recipients of allogeneic hematopoietic stem cell has not been revealed, it is possible due to the short follow-up. it needs further clinical research. disclosure of conflict of interest: none. quantification of cd + recent thymic emigrants and t cell receptor excision circles (trecs) in umbilical cord blood transplanted patients v devlia , , j gridlestone , m raymond , s tulpule , d tewari , , r hough , c navarrete , a madrigal , , b shaw , , r danby and a saudemont , anthony nolan research institute, london, uk; ucl cancer institute, london, uk; nhsbt colindale, london, uk and ucl, london, uk reconstitution of t lymphocytes is a limiting factor in the regeneration of an effective immune system in adult patients following hematopoietic stem cell transplantation. cd (pecam- ) is a transmembrane glycoprotein expressed on naive t-cells that have recently emigrated from the thymus into the periphery. in peripheral blood, cd + t lymphocytes also contain high numbers of t-cell receptor excision circles (trecs); excision loops of dna excised during t-cell receptor gene rearrangement during t cell maturation within the thymus ( ) ( ) ( ) . however, quantification and correlation of cd and trec has not been formally investigated in patients following umbilical cord blood (cb) transplantation. quantification of cd and trecs post cb transplant will provide an insight into the immune reconstitution of t cells from the thymus. we therefore sought to measure cd and trecs in patients after cb transplant and assess whether these markers provided evidence of thymic recovery. we followed adult patients (median age . years) who underwent cb transplant in the uk. patient samples were collected , , , , and days post transplant. using flow cytometry, we determined absolute counts of cd +cd +cd ra+ and cd +cd +cd ra+, and quantified the copy numbers of trec genes in peripheral blood mononuclear cells (pbmcs) via real time pcr. results: at the six time points, the number of samples collected were the following: , , , , and . in all of the samples, the overall median number of cd +cd +cd ra+ was cells/μl (range: - cells/μl). the median level of cd +cd +cd ra+ cells increases from to cells/μl from day to day . absolute counts of cd +cd +cd ra+ at all of the six time points is -fold lower compared to healthy controls (median: cells/μl, range: - cells/μl). the overall median number of cd +cd +cd ra+ cells is cells/μl (range: - cells/μl). there is an increase in the median number of cd +cd +cd ra+ cells between days and posttransplant from to cells/μl. however, the absolute median counts of cd +cd +cd ra+ cells in patients are twofold lower, years post transplant, compared to healthy controls (median: cells/μl, range: - cells/μl). in the majority of the patient samples throughout all time points the trec gene copy numbers were undetected (n = ). in a few patient samples (n = ) trec gene copy numbers were quantified but with this limited sample size no correlations can be made between the absolute counts and trec gene copy numbers. our data suggests that cord blood transplant patients within the uk have reduced levels of cd +cd introduction: common variable immunodeficiency (cvid) is a highly heterogeneous group of primary immunodeficiency characterized by defective antibody production, recurrent infections, lymphoproliferation and autoimmunity. autosomal recessive mutations in lrba, encoding lps-responsive beigelike anchor protein were first described as a cause of cvid-like disease in . although hsct is accepted as a standard treatment modality for long-term resolution of severe primary immunodeficiencies, its role is less established in patients with lrba deficiency. patients and methods: whole exome sequencing of patient's genomic dna obtained prior to the hsct revealed a homozygous deletion in lrba (c. delt:p. c fs). immunological analyses including serum immunoglobulin levels, flow cytometry analyses of lymphocyte subsets, cytotoxicity/proliferation assays, vaccine responses were studied at several time points throughout the disease course, prior to and after hsct. a -year-old boy, born to consanguineous healthy parents of turkish origin became symptomatic at the age of months. he hospitalized several times due to recurrent pulmonary infections. he developed pancytopenia, lymphadenopathy, hepatosplenomegaly and autoimmunity (autoimmune hemolytic anemia and thyroiditis) with low serum immunoglobulin levels at the age of . as a result, he received several courses of steroid and prophylactic immunoglobulin and wide-spectrum antibiotics. over time he manifested growth failure and diagnosed with ibd-like colitis. due to the cumulating severe cvid-related complications, a hsct was performed at the age of years with the bone marrow stem cells from his hla identical brother after a conditioning regimen including fludarabine, busulfan and atg. severe intractable colitis with hypoalbuminemia continued till the engraftment despite vigorous fluid-electrolyte replacement therapy and accompanied with severe episodes of acute gastrointestinal bleeding. after the achievement of full donor chimerism, diarrhea episodes resolved. he received three doses of abatasept because of persistent cytopenia thinking about unresolved immune dysregulation. he is in complete remission at -year post-hsct with no signs of graft versus host disease. allogeneic hsct should be considered in patients with lrba deficiency prior to the development of disease-related severe cumulative manifestations. disclosure of conflict of interest: none. inflammatory bowel disease (ibd) is a chronic disorder of the gastrointestinal tract. very early onset ibd (veo-ibd) represents those severe children with disease onset occurring before -years-old. interleukin- receptors (il- ra, il- rb) mutation are considered to be one of the very important genes for veo-ibd. currently variant treatment, such as steroid medication, immunosuppressive agents and biological agents could not get complete remission. allogeneic hematopoietic stem cell transplantation (allo-hsct) was reported to induce remission in those with veo-ibd. we performed unrelated umbilical cord blood transplantation (ucbt) in five consecutive children with veo-ibd due to il- receptor mutation between and . median age of five children was months (range: - months), and median body weight was kg (range: . - . kg). all patients received reduced intensity conditioning (ric) regimen consisting of busulfan, fludarabine and cytarabine. prophylaxis for graft-versus-host disease (gvhd) was tacrolimus. most patients ( %) received a or hla alleles-mismatched cord unit. median nucleated cells of the cord blood were . × /kg (range: . - . × /kg), and median cd + cells were . × /kg (range: . - . × /kg). median follow-up time was months (range: - months). all patients engrafted, median time of neutrophil engraftment was days, and median time of platelet engraftment was days. four of five patients were alive with continuous donor engraftment, and achieved complete clinical remissions. colonoscopy at months after transplantation in two children revealed the mucosa healing. two children had grade iii acute graft-versushost disease (gvhd). one child developed severe chronic gvhd of both lungs and died of ards at months after transplantation. it is the first clinical trial that unrelated ucbt was performed in veo-ibd children in china. our data should unrelated ucbt with ric should be considered as a potentially curative therapeutic option in children with veo-ibd. disclosure of conflict of interest: none. patients with refractory primary induction failure and resistant relapse are poor candidates for hematopoetic stem cell transplantation (hsct) . additional attempts at remission induction with various combinations of chemotherapy will unlikely improve the outcome and will contribute to excess toxicity. a major goal of sct has been to develop strategies to reduce the risk of gvhd while maintaining or enhancing gvl. tcrαβ+/cd +lymphocytes depletion is a technology of graft manipulation with a potential to increase gvl effect and improve gvhd control and immune reconstitution in this group of patients. a total of pts with refractory aml (primary induction failure (n = ), refractory relapse (n = )), female/ male, median age . years ( . [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] , underwent allogeneic sct between may and august , median fu . years ( . - . ). pts were transplanted from haploidentical donors and from mud. all pts had active disease (ad) at the moment of sct and received treosulfanbased high-intensity conditioning regimen. three regimens of gvhd prophylaxis were used. regimen (n = ): atgam mg/kg with (n = ) or without (n = ) post-transplant tacro/ mtx; regimen (n = ): thymoglobulin mg/kg, rituximab mg/m and post-transplant bortezomib on day+ ,+ (n = ); regimen (n = ): tocilizumab mg/kg on day- and post-transplant bortezomib (n = ), pts receive additional abatacept mg/kg on day+ , + , + , + . tcrαβ+/cd +-depletion of sct with clinimacs technology was implemented in all cases. the median dose of infused cd + cells was × /kg (range: . - ), tcra/b- × /kg (range: - ). all engrafted pts received additional post-transplant courses of low-dose chemotherapy, including hypomethylating agents and dli. primary engraftment was achieved in of pts(three pts had disease progression, one died at the moment of engraftment), the median time to neutrophil and platelet recovery was days ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) . early mortality within days was . % (one pt with aml had acute lung injury after engraftment on day + ), . -years ptrm- . % ( %ci: . - ) . there were no allergic or infusion-related adverse events associated with tocilizumab or abatacept. ci of gvhd grades ii-iv and iii-iv was . % ( % ci: - ), and . % ( %ci: . - ), respectively. ci of cgvhd was % ( % ci: - ). ci of acute gvhd was lower in a group with prophylaxis regimen without serotherapy: % ( % ci: - ) vs . % ( % ci: - ) in atg group. no correlation between graft composition, donor type with the incidence of agvhd and cgvhd was noted at . years ppfs (event = death or relapse or progression) was % ( % ci: - ), . -years pos - % ( % ci: - ). median time of fu for survivors is . years (range: . [ ] [ ] . we confirm that the depletion of tcrαβ +/cd +lymphocytes from the graft ensures high engraftment rate and low transplant-related mortality in pediatric pts with refractory aml. we suggest that tocilizumab and abatacept can be safety administered to children with acute leukemia in the context of treosulfan-based conditioning regimens. long-term follow-up will demonstrate if the gvhd prophylaxis without serotherapy and combined administration of tocilizumab, abatacept and bortezomib post-tcrαβ+/cd +depleted grafting will improve gvl effects without extensive gvhd-related morbidity and mortality in pts with refractory aml. disclosure of conflict of interest: none. the jacie experience at the university hospital of amiens l marie-noelle , w brigitte , f isabelle , g bérengère , h muriel , h anne , v elsa , m jean-pierre and c amandine lacassagne; oncopôle, chu amiens picardie; hématologie clinique et thérapie cellulaire-chu amiens picardie; oncopôle-chu amiens picardie and the jacie (joint accreditation committee of isct and ebmt) accreditation aims to improve the management of patients benefiting from autologous or allogenic hematopoietic stem cell (hsc) transplants. usually, candidates' centers for jacie accreditation have already existing clinical activity when they have willingness to comply with jacie standards. here, we present our new experience in the implementation of jacie quality process, at the same time as allograft clinical activity. implementing process autograft clinical activity existed at amiens hospital, since , but in lack of center cellular therapy laboratory and hsc collection that were outsourced. the collection activity for autologous transplant was set up in , the cellular therapy laboratory in november and then the allogenic transplant was started in july . as early as march , we set up a steering committee with hematological clinicians, managers of each sector, a transplant coordinator nurse, the head of the processing laboratory and a part-time quality engineer recruited part-time. each actor had to become familiar with standards to obtain information from accredited centers in order to evaluate objectives and their prioritization. steering committee decided on deadlines and established a roadmap including the following: the list of jacie required standard operating procedures and their writing; assignment of the tasks for each actors in order to evaluate, writing and approval each document; organization of documents diffusion; information to all staff on the approach; creation of feedback committee for adverse events management; establishment of morbidity and mortality review; formalization of initial and continuing training for medical and paramedical staff; and organization of cross audits with external teams. at the same time, we assessed requirements for starting activity: training for medical and paramedical staff; training for the transplant coordinator nurse; circuits for taking care of donors; the organization of the in-patient department; the organization of follow-up of post-transplant patients; and authorizations of the national regulation agencies for processing facility on manipulations and cellular qualifications and for regulatory collection and transplant. allogenic transplant clinical activity started in july . accreditations jacie visit occurred on and june and our center has been officially accredited since march . neither quality approach, nor clinical activities were easy to implement. medical and paramedical staff had to get acquainted with a new organization and restrictions. despite difficulties, implementing jacie quality process, concomitantly with allograft activity allowed to create a true team dynamics with a common reflection on the means to be implemented. moreover, quality approach has assured us best ensure to care graft patients. the result is true satisfaction, which be credited to all. disclosure of conflict of interest: none. previously published p three-dimensional co-culture of peripheral blood monocytes supports and expands functional hematopoietic stem/progenitor cell without immobilization y xu , x li , b wang , w shan , h chen , s liu , r tie , y long , s cai , h xu , x yu and h huang bone marrow transplantation center, the first affiliated hospital, school of medicine, zhejiang university very low numbers of circulating hematopoietic stem/progenitor cells (chspcs) are found in normal human peripheral blood (pb) without mobilization. here, we developed a three dimension co-culture system to seize and expansion chspcs from pb monocytes without mobilization. flow cytometry analysis was carried out to identify chspc phenotypes. multipotential properties of chspcs were determined using colonyforming unit assay in methylcellulose and reconstitution ability in the compromised animals. the critical regulation mechanism underlying chspcs was identified with transcriptome analysis based on next-generation sequencing technology at total or single cell levels. loose cobble stone colonies (lcs), round or vessel-like compact colonies (rccs or vccs) were presented in three dimension co-culture system after about weeks. the colonies lasted for at least six passages with no obvious apoptosis sign, and expanded more than~ fold during the period. we studied the niche-mediated regulation mechanism of chspc fate at molecular level compared to the conventional method of two dimension culture. furthermore, chspcs were capable of forming all types of hematopoietic colonies, including cfu-gemm, and especially held short term engraftment capacity for compromised nogs by radiotherapy. transcriptome analysis by deepsage identified genes significantly associated with regulating the function of chspcs. figure : the cellular morphology in three dimension culture system for peripheral blood monocytes without mobilization during the culture for - weeks. figure : short transplantable potential analysis of chspcs. figure : (a) static of differentially expressed genes between three-and two-dimensional culture systems for peripheral blood cells. (b) go functional analysis classifies those genes by biological process, cellular component and molecular function. (c) the significant differences between the molecular phenotypes of three-and two-dimension chspcs indicating that chspcs from three dimension culture hold stem properties. our system may provide a more ideal and balanced approach which not only seizes circulating chspcs, promotes selfrenewal and expansion of chspcs, but also holds phenotypic and functional attributes of chspcs. . to wash or not to wash? comparison of neutrophil and platelet engraftment after infusion of cryopreserved autologous stem cells before and after the implementation of bedside thawing am halldorsdottir , s atladottir , m thorsteinsdottir, na arnason , g runarsson , t jonsson , oe sigurjonsson , and s reykdal the blood bank, landspítali, the national university hospital of iceland; department of hematology, landspítali, the national university hospital of iceland and school of science and engineering, reykjavik university cryopreserved autologous peripheral blood stem cell (pbsc) grafts are widely used after high-dose chemotherapy in the treatment of patients with myeloma or lymphoma. prior to infusion, cryopreserved grafts can be thawed at the bedside, or thawed and washed at the cell therapy laboratory. at our institution the practice of routine washing of stem cell grafts in the laboratory was discontinued in april and bedside thawing implemented instead. this was done to minimize the time thawed cells are exposed to toxic dmso. this study was performed at a single center, at landspítali-the national university hospital of iceland, which is the only transplant center in iceland. autologous pbsc transplants have been performed in iceland since . the study compares outcome for two groups of patients, who received either; (a) thawed and washed autologous pbsc cell grafts from january to [p ] april , or (b) autologous pbsc grafts thawed at the bedside from april to november . the following outcomes were compared; days to neutrophil engraftment (absolute neutrophil count (anc) . per μl), and platelet engraftment ( and × e /l). data on mean cd + cell content/kg of the infused grafts, measured prior to cryopreservation, were also compared. all patients have received premedication with solucortef, clemastine and ondansetron prior to infusion of the graft. from january to april a total of patients received thawed and washed autologous pbsc grafts, and between april and november patients received autologous pbsc grafts thawed at the bedside. majority of the patients were diagnosed with either multiple myeloma or related disorders (n = ) or lymphoma (n = ) whereas the remaining patients (n = ) had miscellaneous diagnoses. days to engraftment and the dose of cd + cells infused are compared in table . there was no significant difference in the mean cd content of infused autologous stem cells in the two groups ( . vs . × e cd +cells/kg, p = . ). there was also no difference in the mean number of days to engraftment of neutrophils ( . vs . days, p = . ), platelets at days ( . vs . days, p = . ) or platelets at days ( . vs . days, p = . ) after transplant. one hundred day mortality was comparable in the two groups or . %. additional data on transfusion requirements, infections and use of granulocyte-colony stimulating factor will be presented. [p ] there was no difference in neutrophil or platelet engraftment after changing the autologous stem cell graft thawing procedure from post-thaw washing in the laboratory to bedside thawing. bedside thawing of stem cells is a safe procedure that results in acceptable cellular engraftment. disclosure of conflict of interest: none. the procedure of autologous hematopoietic stem cell (hsc) transplantation requires cryopreservation of hscs. addition of dmso (dimethyl sulfoxide) is necessary to secure the viability of such cells, but this cryoprotectant causes adverse reaction during infusion into patient. the concentrations of dmso in cryopreservation mixture vary strongly between different transplant centers. usually, the hscs are stored in mixtures containing % dmso, however, many centers successfully use lower concentrations. the main aim of the study was to evaluate the clinical impact of different dmso concentrations in cryopreservation mixture ( %, . %, %) on reconstitution of hematopoiesis after autologous hsc transplantation. the project was approved by the local bioethics committee. written informed consent obtained from all of patients. the study is registered to clinicaltrials.gov (identifier: nct ). between january and july , consecutive patients with hematological malignancies or solid tumors, referred for autologous hsc transplantation, were recruited in the study. the patients were randomly assigned to one of three study arms ( patients each). hscs obtained by leukapheresis were cryopreserved in three concentrations of dmso: %, . %, %, respectively. study groups did not differ significantly with regard to the diagnosis (mostly mm, nhl or hl), age or conditioning regimen (chemo-or radiotherapybased). all patients received granulocyte-colony stimulating factor (g-csf, filgrastim) starting from day + after transplantation to support neutrophil recovery. in case of patients, the transplantation was cancelled due to progression or other medical reasons. four patients died shortly after transplantation, due to refractory infections. data for patients were subjected to statistical analysis. the viability of nucleated cells on the day of transplantation was similar in all groups (median %, range: - % for % dmso group; %, range: - % for . % dmso; %, range: - % for % dmso; p = . ). the dose of transplanted cd + cells was comparable in all group: (median . × /kg of recipient body weight for % dmso, . × /kg for . % dmso and . × for % dmso, p = . ). the median time to leukocyte recovery, defined as the first day with wbc count exceeding . × /l was days in all groups (ranges: - for % dmso; - for . % dmso; and - for % dmso; p = . ). similar results were obtained in case of neutrophil recovery-the median day, when the anc exceeded . × /l, was in all arms (ranges: - ; - and - , respectively; p = . ). the day when the platelets level were greater than or equal to × /l (sustained without transfusion within days) was similar in all groups: medians were days in %, . % and % dmso (ranges: - ; - ; - ; p = . ). no serious adverse effects were observed during hscs infusion and during h after transplantation. reduction of dmso concentration from in cryoprotective mixture % to . % and % has no negative impact on cell viability during cryopreservation and engraftment after auto-hsc transplantation. disclosure of conflict of interest: none. a real-world cost-effectiveness analysis demonstrates that introducing plerixafor to improve mobilization in multiple myeloma patients who behave as poor mobilizers is cost-effective considering the whole mobilization and transplant procedure r touzani , , a-m stoppa plerixafor, a cxcr -antagonist, is efficient to improve cd + cell mobilization and collection in candidates for autologous transplantation who behave as poor-mobilizers. the cost of the drug is however of concern. published medico-economics studies were mostly conducted in the us, and few including detailed and comprehensive micro-costing of the collection and transplantation process; conclusions may thus not apply to european countries where cost structures are different. to compare costs and effectiveness of plerixafor-free and plerixafor-replete management strategies for multiple myeloma patients who behaved as poor-mobilizers after adequate administration of a standard rhg-csf mobilization regimen. sixty patients diagnosed with multiple myeloma were consecutively identified during years - , immediately before and after ema granted marketing authorization for plerixafor. poor-mobilizers were defined as having circulating cd + cell counts below /μl. plerixafor was introduced or not as a result of the attending physician's decision, reflecting progressive changes in medical practices over this transitional period. the historical and study groups were matched over four criteria: disease stage at diagnosis, age, gender and number of chemotherapy treatments received before mobilization. two cost-effectiveness analyses (cea) were conducted; the primary cea looked at the criterion ‛collecting at least × cd + cells'; a secondary cea looked at the criterion ‛successful autologous transplant administered'. detailed micro-costing evaluations ( figures) did not or did include transplantation costs for the first and second cea, respectively. the two groups were similar in terms of age, sex distribution, disease characteristics or previous treatments. / and / patients proceeded to high-dose melphalan and autologous transplantation in the study and historical groups, respectively. there was a trend to a higher number of collected cd + cells in the control group; however, the proportion of patients who met the minimal target number of × collected cd + cells/kg was identical ( / ). length of hospitalization, times to neutrophil and platelet recoveries, numbers of prbc and platelet transfusions were identical in the two groups. mobilization and collection costs per patients were more important in the plerixafor group that in the historical group ( . vs . €, p o . ), and proportionally higher in patients who received plerixafor as part of a remobilization treatment rather than pre-emptively ( . vs . €, respectively). the main cea concluded to a . € increase in costs for the same number of patients achieving a minimal target number of × collected cd + cells/kg. the second cea found a decrease in the cost of transplant, with . € in the study group vs . € in the historical group (ns). in total, the . € increase for the complete procedure cost ( . € per successfully autografted patient in the study group vs . € in the historical group) was not statistically different. cost-effectiveness arguments should not been used against the administration of plerixafor in multiple myeloma patients in the european context. future prospective researches looking at patients reported outcome criteria and labour organization in apheresis facilities are needed. disclosure of conflict of interest: this work was supported by a grant from sanofi s.a.; cc: research support, honorarium & hospitality from sanofi s.a. administration of plerixafor for peripheral blood cd + stem cell content of o × /l for autologous stem cell mobilization leads to decreased apheresis days and increased total yield m kamdar , s abebe , gr gonzalez fontal, l gates , a hammes , d abbott , j gutman , b haverkos , d sherbenou and c smith division of hematology and transplantation and department of biostatistics and informatics, university of colorado, denver, colorado, usa autologous stem cell transplantation (asct) is an effective treatment for lymphoma and plasma cell neoplasm (pcn) (multiple myeloma and amyloidosis). granulocyte-colony stimulating factor (g-csf) is the most commonly used upfront mobilizing agent with plerixafor-based higher cost approaches reserved for poor/unsuccessful mobilizers. several mobilization algorithms utilizing g-csf and plerixafor have been published however the most efficient and cost effective strategy is yet to be determined. most transplant centers administer plerixafor for peripheral blood (pb) cd + stem cell content of o × /l on day (d) of g-csf mobilization. at the university of colorado (uch) we changed our programmatic approach in and administered plerixafor for pb cd + count of o × /l on d of g-csf mobilization. in this study we evaluate the impact of this novel mobilization algorithm on apheresis days and total stem cell yield. patients (pts) with lymphoma and pcn who underwent asct at uch until / received plerixafor if pb cd + cells on d of g-csf mobilization was o × /l. based on our institutional review of poor/unsuccessful mobilizers and using logistic regression analysis this algorithm was revised in / . in the new algorithm all pts received plerixafor if pb cd + cells on d of gcsf mobilization was o × /l. demographics were compared between pts with lymphoma and pcn before (group : / - / ) and after (group : / - / ) the new algorithm was implemented. the primary goal of this analysis was to assess the total days of apheresis and total stem cell yield between the two groups. we also sought to analyze days to wbc engraftment and platelet engraftment. a total of pts were included in this analysis. group consisted of pts ( pts had lymphoma and pts had pcn). group consisted of pts ( pts had lymphoma and pts had pcn). we found that there was a significant increase in total yield (p = . ) in group as compared to group . on further disease subtype assessment we noted that pts with pcn in group had a significant increase in total yield (p = . ). in lymphoma pts on univariate analysis group showed a significant decrease in apheresis days ( . days, p = . , % ci: (− . , − . )). on multivariate analysis there was still a marginally significant decrease in group ( . days, p = . , % ci: (− . , − . )) compared to group . in pcn pts on univariate analysis group showed a significant decrease in apheresis days ( . days, p = . , % ci: (− . , − . )). on multivariate analysis group continued to show a significant decrease in apheresis days ( . days, p = . , % ci: (− . , − . ) compared to group . we found no significant difference between the two groups in days to neutrophil engraftment and platelet engraftment. our analysis showed that a mobilization algorithm of administering plerixafor for a pb cd + stem cell count of o × /l on d of g-csf mobilization led to a decrease of roughly . days in the lymphoma cohort and a significant decrease of . days in the plasma cell neoplasm cohort. we also noted a significantly increased total yield of stem cell collection in group . overall our programmatic approach led to decreased chair-time for apheresis and better resource utilization. pharmacoeconomic impact of this approach will be updated at the meeting. disclosure of conflict of interest: mk: speakers bureau, seattle genetics; remaining authors declare no conflict of interest. administration of stem cell boosts (scbs) from the original donor offers a therapeutic option. we report on pediatric patients with pgf who received a total of boosts with cd + selected peripheral blood stem cells (pbsc) after transplantation from matched unrelated (n = ) or mismatched related (n = ) donors. median time between hsct and infusion of the scbs was days ( - ). boosts contained a median number of . × cd + progenitor cells/kg body weight (range: . - . × ) with a median number of /kg (range: - ) residual cd + t cells. within weeks after application, a significant increase in median neutrophil counts ( vs /mm , po . ) and a decrease in erythrocytes and thrombocytes transfusion requirement (median frequencies and vs , p o . and o . ), were observed, and . % of the patients resolved one or two of their initial cytopenias whereas . % had a complete hematological response. additionally median lymphocyte counts for cd +, cd +cd +, cd + and cd + increased . fold, . fold, . fold and . fold, respectively. the rate of de novo acute gvhd grade i-iii was only % and resolved completely after treatment. no gvhd iv or chronic gvhd occurred. patients who showed a response to scb displayed a trend toward better overall survival (os) (p = . ). administration of cd + selected scbs from alternative donors is a safe and effective procedure. we hypothesize that the cd + progenitor boosts may have an enhancing effect on maturation of committed lymphoid precursors already present in the host or generate another wave of thymic seeding with accelerated t-cell differentiation process in the absence of any immune suppression. further studies are warranted to better define the impact on immune reconstitution and survival. disclosure of conflict of interest: none. plerixafor plus granulocyte-colony stimulating factor (g-csf) has been shown to mobilize more cd + cells than g-csf alone for autologous hematopoietic stem cell transplantation (hsct). however, there are few studies that analyze the impact of this strategy in engraftment. the aim of our study is to compare mobilization and engraftment between patients who received a combination of plerixafor plus g-csf and patients (pts) who mobilized with g-csf alone. a retrospective casecontrol analysis was performed in pts with myeloma who mobilized with plerixafor plus g-csf (group p/g-csf) and was compared with matched for sex and age controls who mobilized with g-csf alone (group g-csf). all pts underwent hsct between and . mobilization with g-csf at dose of μg/kg/day was used in all pts. the aphaeresis was scheduled on day + . plerixafor ( . mg/kg) was added if the number of cd +cells on day + was o /μl for × cd +/kg requested (or o /μl for × cd +/kg), or if the number of cd +cells collected in the first apheresis was o % of cd + requested. conditioning and supportive care were similar in both groups. in p/g-csf group, were male and female. median age was . years (range: - ). in group g-csf, were men and female. median age was . years (range: - years). there were no differences between both groups. disease status at time of mobilization was different between groups (p = . ). in p/g-csf group: ( . %) pts were in complete remission (cr), ( . %) very good partial responses (vgpr), ( . %) partial response (pr) and ( . %) had no response to treatment. in g-csf group: ( . %) pts had reached cr, ( . %) vgpr and the remainder in pr. sixteen ( . %) pts in p/g-csf group had received ⩾ lines of treatment vs ( . %) pts in g-csf group (p = . ). no difference was seen on mean day-dose of g-csf ( μg/kg/ h in p/g-csf group vs μg/kg/ h) (p = . ). there was no difference on cd +/kg requested ( / pts in p/g-csf were requested × /kg vs / in g-csf group) (p = . ). p/g-csf group needed more apheresis sessions, ( . %) pts required ⩾ sessions against ( . %) pts in group g-csf (p o . ). we obtained enough cd + cells to carry out hsct in all patients, although mean number of cd + cells obtained in p/g-csf group was lower than in g-csf group ( . × /kg vs . × /kg, respectively) (p o . ). also, mean number of cd + infused in p/g-csf group was lower ( . × /kg vs . × /kg) (po . ). however, engraftment results were similar in both groups, as represented in table . patients who required mobilization with plerixafor plus g-csf got an engraftment as good as patients who do not require the combination despite of worse baseline parameters. given that the number of cd + infused in the p/g-csf group has been lower than g-csf group, these results might suggest that the different composition of graft cell with plerixafor plus g-csf mobilization, described in some studies, could impact on engraftment outcomes. high-dose chemotherapy following autologous hematopoietic stem-cell transplantation (autohsct) is an effective method of treatment both recurrent and primary refractory lymphoma patients. however, some patients have mobilization failure (‛poor mobilizers') with inadequate collection of peripheral blood stem cell (pbsc). aim: to evaluate the efficacy and factors influencing pbsc mobilization and collection for the autohsct in patients with lymphomas. thirty patients were included in this study: -with hodgkin lymphoma, -with non-hodgkin lymphoma, -with multiple myeloma; women and men of them. the median age of patients was years ( - years). the mobilization of pbsc with only colony-stimulating factors (csf) was carried out for patients, chemotherapy (cyclophosphamide, etoposide) in combination with csf-for patients. only one patient had plerixafor mobilization. the concentration of cd + in peripheral blood (pb) was studied on the day of the intended cytapheresis. cytapheresis was commenced when cd + concentration had been greater than . × cells/ml. twenty-four patients ( %) from had collection of pbsc. the collection was not performed in six patients ( %) because the concentration of cd + in pb on the day of the intended cytapheresis was lower than . × cells/ml. there was no possibility to use plerixafor in these cases for economic reasons. the median concentration of cd + in pb on the first day of the intended cytapheresis in the group of patients that had cytapheresis was . × cells/ml whereas in the group of failed- . × cells/ml (p o . ). fifty-nine tests of cd + in pb were done. distribution and test results by days from the first day of the intended cytapheresis are presented in table . the total number of the cytapheresis was . the majority of patients had procedure of pbsc collection (n = ), patients had procedures and only had . the last patient had had two previous failed cytapheresis procedures and the adding of plerixafor helped him to collect necessary number of cells. the median of cd + cells on patient's kilo was . × cells/kg. sex, age, mobilizing regimen, previous radiation therapy, the count of lines of chemotherapy before autohsct were not significantly associated with poor pbsc mobilization and collection. only tumor response before autohsct (complete/ partial response or stabilization) was significantly associated with cd + cell count in the product of cytapheresis. patients with complete or partial response had significantly better cd + count. [p ] disclosure of conflict of interest: none. factors associated with failure in mobilization of peripheral blood hematopoietic progenitor cells in autologous transplantation je dulon-tarqui, bl acosta-maldonado, l rivera-fong, sa sánchez-guerrero, jf zazueta-pozos, ja padilla-ortega, wj ladines-castro and lm valero-saldaña high dose therapy followed by autologous stem cell transplantation (asct) obtained from peripheral blood is currently the standard model for treatment consolidation in various hematologic malignancies. a global incidence of - % of failure to mobilization is reported, and some factors associated with poor mobilizers in hodgkin's lymphoma (hl), non-hodgkin's lymphoma (nhl) and multiple myeloma (mm) when the yield in peripheral blood stem cells (pbsc) collection is unsatisfactory, the effects for the recipient can be serious. the donor's age, gender, body surface area (bsa), processed blood volume and the method of g-csf dose calculation may affect the cd + yield. as g-csf has a low distribution volume in the peripheral blood (pb), it might be appropriate to calculate the doses by using the bsa instead of per kg body weight. consecutive allogeneic pbsc donations performed in healthy donors at the karolinska university hospital in stockholm were included. a complete medical history, physical examination, electrocardiogram, chest x-ray and laboratory testing were done before pbsc donation. relevant data for analysis were collected from the institutional quality database for a retrospective review. the total blood volume was calculated using the formula by nadler et al. the bsa was calculated using the formula by du bois and du bois. the concentration of cd + cells in the pb and the processed volume of blood were significantly correlated to cd + cells yield (po . and po . , respectively, see table ). the g-csf dose per m was significantly correlated to the concentration of cd + cells in the pb (p = . ) and in the product (p = . , see table ). smaller bsa (p o . ) and less processed volume (p o . ) were found among female donors, who were given lesser g-csf dose per m (p o . ) and showed lower yield compared to men (po . ). however, multivariate analysis of the yield showed that only the concentration of cd + cells in the pb and the processed volume remained independent significant (see table ). [p ] in this study, we found the concentration of cd + cells in the pb and the processed volume of blood to be independent predictors of yield. we recommend to get a high concentration of cd + cells in the pb, and to process adjusted volumes of blood when needed. an evaluation if the calculation of g-csf dose per m is more appropriate than per kg body weight should be done in future studies. autologous stem cell transplantation (asct) has been widely used in the treatment of hematological malignancies over the last two decades. despite its broad use, some characteristics that might influence engraftment have not been exhaustively investigated, particularly graft purity with respect to contamination by platelets (plts) and white blood cells (wbc). here we report collection characteristics and engraftment kinetics of a single center consecutive series of asct. we retrospectively collected clinical records of patients who underwent leucapheresis procedures (la; followed or not by asct) and data on asct at our institution over years ( - ) ( table ). the impact on engraftment kinetics of conditioning chemotherapies, amount of infused cd + cells and wbc/plts graft contamination were analyzed. absolute neutrophil count (anc) engraftment was defined as the duration of neutropenia (from day to the first of consecutive days of anc /μl post asct). regarding cd + cell collection, no impact of mobilizing regimens and wbc count during la was observed. on the other hand, we observed a difference in the number of total cd + cells collected among different diagnoses: the median overall collection was . ( . - . ) × /kg cd + cells for nhl patients, . ( . - . ) × /kg for mm patients, . ( . - . ) × /kg for hl patients and . ( . - . ) × /kg for aml patients) (p = . ). considering cd + cells/kg harvested on the first day of la, . % of nhl and hl, . % of mm patients and % of aml patients harvested ⩾ × /kg cd + cells. of note, among aml patients, . % collected o . × /kg. the differences were statistically significant (p = . ). moreover, an inverse correlation between collected cd + cells and age was shown (p = . ). anc recovery after asct was not influenced by conditioning regimen whereas diagnosis impacted on the duration of neutropenia (aml patients displayed a longer aplasia, po . ). we observed that the median days with anco /μl were , and in patients who received . × /kg, . - . × /kg and o . × /kg cd + cells, respectively (po . ). furthermore, the same finding was observed considering the duration of thrombocytopenia (median number of days with plts o /μl: , and in patients who received . × /kg, . - . × /kg and o . × cd + cells, p o . ). looking at the apheresis product, we analyzed the impact of harvest contaminating wbc and plts on engraftment kinetics. notably, when the asct collection contained × /μl wbc, anc engraftment (days with anc o /μl) lasted longer (median days ) compared to patients who received a graft with lower wbc count (po . ). a faster anc engraftment was also observed in patients receiving harvests with plts levels × /μl compared to those who infused a collection bag with plts o × /μl (p = . ). herein, we confirmed that the disease and the amount of infused cd + cells significantly influence time of anc and plts engraftment; furthermore, we observed for the first time that quality and purity of the graft have a substantial impact on engraftment kinetics. a combination of chemotherapy with growth factor is a commonly used strategy for hematopoietic stem cell (hsc) mobilization. the collection of timely and adequate numbers of hscs is a prerequisite for proceeding to transplantation. a variety of mobilization strategies are currently used. the knowledge of efficacy, safety and predictability of different hsc mobilization strategies might help blood and marrow transplantation (bmt) programs to effectively schedule patients for mobilization. given the many variables associated with the mobilization of hsc, collecting an adequate stem cell dose in a timely and effective manner is an art and science. factors that might affect the process includes type of disease and mobilization protocol, financial clearance, availability of chemotherapy beds, scheduling various diagnostic procedures and transplant urgency. to evaluate the effectiveness and related coordination efforts of ‛just-in-time' strategy of hsc mobilization and collection, we performed a retrospective study comparing all patients in whom peripheral hsc mobilization was attempted at khcc from january through november . data collected included the disease type, mobilization protocol, days to and number of collections, cd + cell dose, calendar of the mobilization and collection. the records of a total of mobilizations were reviewed. were of healthy allogeneic donors, and the remaining were of patients undergoing autologous transplantation. table depicts the overall summary of number of days and collection procedures per each protocol. detailed mobilization kinetics per disease type and mobilization protocol were also captured and evaluated. [p ] s [p ] detailed analysis of mobilization kinetics comparing different mobilization strategies aids in prediction of number of days of mobilization and anticipated number of collections. this helps in proactively scheduling patients based on collection predictability. a seamless communication through a shared calendar between key parties, primarily bmt physicians and nurse coordinators, bmt and flow cytometry laboratories and chemotherapy unit can be achieved. autologous stem cell transplantation is still a standard of care in the treatment of multiple myeloma. lenalidomide-based regimens are commonly used in both transplant-ineligible as well as -eligible patients. prolonged lenalidomide-exposure is known to affect mobilization of cd + cells, although the basic mechanisms are poorly understood. limited prospectively collected data is available on the effect of lenalidomide in the capacity to mobilize cd + cells for transplantation as well as graft cellular composition and post-transplant hematological recovery compared to the lenalidomide-naive patients. this prospective study included newly diagnosed myeloma patients who received mobilization with low-dose cyclophosphamide + g-csf, were successfully apheresed and transplanted before the end of . twenty-six patients had received a median of three cycles of lenalidomide-based induction ( %), whereas patients were lenalidomide-naive and served as the control group. both baseline characteristics and collection targets were similar between the groups. cd + mobilization and apheresis yields were analyzed and compared between the groups. blood graft cellular composition was analyzed from the thawed cryopreserved samples with a flow cytometry. graft function was evaluated by collecting engraftment data as well as by total blood counts at day + and at , , and months after post-transplant. the patients in the lenalidomide group had both lower median peak b-cd + counts and about % lower cd + yields of the first apheresis but without statistical significance ( table ). the median number apheresis was significantly higher in the lenalidomide arm ( . vs . , p = . ). the number of cd +cd +cd -, cd +cd +, cd +cd + cells and nk cells in the cryopreserved grafts were comparable between the arms. time to neutrophil engraftment was days in the both groups. the median time to platelet engraftment was d in the lenalidomide group and d in the control group. hematological recovery was comparable between the groups within months post-transplant. lenalidomide-based induction therapy seems to have an impact in the number of apheresis needed, but not in the total yield of cd + cells in the graft. neither the graft cellular composition nor posttransplant recovery in myeloma patients was affected by the limited duration of lenalidomide used before mobilization and collection of blood grafts. between september and november . siemens hematek system was used for luc count. luc numbers and percentage was measured before leukapheresis. we used pearson test for the correlation and roc curve for cut off value. patients' characteristics were shown in table- . there was not a correlation between luc number and mobilized cd positive stem cell number. but luc percentage was positively correlated with mobilized stem cell number (p: . ). a count of × /kg collected stem cells are optimal for autologous stem cell transplantation. we found % luc percentage as a cut-off value for prediction of collecting optimal number of stem cells with % sensitivity and % specificity. as expected luc percentage was negatively correlated with white blood cell count. there was no correlation between mobilized cd positive stem cell number and age. both luc percentage and mobilized cd positive stem cell number did not differ with underlying disease. we found only one study in the literature that evaluated luc percentage as a tool for the prediction of successful stem cell collection. they found that baseline luc numbers negatively correlated with stem cell mobilization in healthy donors ( ) . but we measure luc on apheresis day and found a positive correlation between luc percentage and stem cell mobilization. and we found a cut-off value for optimal stem cell mobilization with acceptable sensitivity and specificity. in our study we demonstrate that luc percentage measurement on apheresis day may be a very simple and cheap tool for the prediction of optimal stem cell mobilization. the spectra optia (so) apheresis system performs a wide range of therapeutic procedures, including peripheral blood stem cell (pbsc) collection in mobilized donors and patients (pts). the device was studied to evaluate the cellular composition of pbscs harvested in pts with multiple myeloma (mm), non hodgkin's lymphoma (nhl) and hodgkin's lymphoma (hl) planed for autologous peripheral stem cell transplantation (apbsct), and to optimize the collection of pbscs using the cd + precount and collection efficiency (ce ) of apheresis device which is calculated as follows: ce = total cd + cells collected × /kg; cd + precount/ μl × blood processed (liters). the blood volume processed is calculated as follows: desired cd + × /kg × recipient weight (kg): ce × cd + precount/μl in our study enrolled pts undergoing pbsc mobilization and planed for apbsct. we evaluated so system's mononuclear cell (mnc) collection performance, with respect to cd + cells and mnc collection efficiency, platelet reduction pre to post apheresis, and product purity in view of using prediction algorithms to optimize the procedure and predict the cd + yield, blood volume processed and platelets loss. we also evaluated neutrophil and platelet recovery in pts who underwent apbsct. results: between / / and / / , pts underwent pbsc harvesting by so device. median age was years ( - ). there were females and males. diagnosis was mm in pts, hl in pts and nhl in pts. the number of ahereses procedures was . mobilization consisted in g-csf alone in pts, chemotherapy and g-csf in pts, and g-csf + cxcr inhibitor in one patient. median count of cd + cells pre-collection was /μl ( . - ) . median total blood volume processed was . l ( . - . ). median count of cd + cells collected was . × /kg ( - . ). median mnc collection efficacy was % .median cd + cell collection efficacy was . % ( - %). median platelet reduction pre to post apheresis was % ( - %). median product hematocrit and granulocytes product was % ( - ) and % ( - ), respectively. twenty-six of the pts underwent myeloablative high dose chemotherapy followed by apbsct which was performed for mm in pts, hl in pts, and nhl in pts. the median count of cd + cells infused was . × / kg ( . - . ). all the pts received g-csf post-apsct until neutrophil recovery. the median day for neutrophil recovery was ( ) ( ) ( ) ( ) ( ) ( ) ( ) . median duration of severe neutropenia (anc o . × /l) was days ( - ). the median day for platelet recovery was ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) . median duration of severe thrombocytopenia (platelets o × /l) was . days ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) . conclusion: the study results confirm that the so apheresis system's mnc collection protocol is safe and effective. the neutrophils and platelets recovery in pts auto-transplanted was not inferior compared to historical controls. in addition, this system help to use prediction algorithms for whole blood processing to achieve a desirable and optimal yield based on cd + precounts and ce of the apheresis device. disclosure of conflict of interest: none. peripheral blood stem cell apheresis in small children is difficult! aa hedayati-asl , m emam-jome, p dinarooni, v fallah, a mehrvar and r zangooei in low-weight children with cancer and healthy donor children, peripheral blood progenitor cells (pbpcs) have largely replaced bone marrow as source of autologous and allogeneic stem cells in part because of their relatively easy collection. however, there is a concern regarding medical, psychosocial and technical difficulties in small children. we retrospectively analyzed peripheral blood stem cell apheresis in collections. patients were with cancer ( patients = neuroblastoma, patients = retinoblastoma, patients = germ cell tumor, patient = hepatoblastoma, patient = wilm's tumor) and healthy children donors. the study was conducted between and . peripheral stem cell apheresis was performed in the mahak cancer children's hospital in a nice room for children where the patients stayed with their families. patients s were not routinely sedated. pbpc were collected by a cobe spectra cell separator (cobe, denver, co, usa). harvesting was performed after days mobilization. mean body weight was . kg (range: - kg) for a median age of years (range: months- years). mean duration of harvesting was min (range: - min). mean volume of stem cell collection was ml (range: - ml). the mean number of total nucleated cells collected was . × /kg (range: . - . × /kg recipients). no side effects occurred. children didn't require an additional haematopoietic progenitor mobilization or additional apheresis in other day. pbsc collection was without transfusion in healthy donor children. pbsc collection may be difficult in small children owing to the large volume apheresis compared to the child's weight. various problems, such as metabolic or haemodynamic disorders may be were seen. peripheral stem cell harvest can be performed in low-weight children under safe and effective conditions even when systematic priming by blood is avoided. processing with increase of blood volume may to increase in the yield by recruiting progenitor cells. disclosure of conflict of interest: none. peripheral blood stem cell collection in low body weight children: a single centre experience g del principe*, g leone, s lazzaro, a meschini, k feri, p marchitelli, d carasso, f locatelli and m montanari department of pediatric hematology/oncology and transfusion medicine, irccs bambino gesù children's hospital, rome, italy pbsc became preferred source for autologous transplantation because of easier collection and faster engraftment. however apheresis for low body weight children ( o . kg) is affected by some issues: venous access, extracorporeal volume, metabolic and hemodynamic complications, citrate toxicity, so is crucial to standardize harvesting procedure both maximizing stem cells collection and reducing adverse events. a dual lumen central venous catheter was used to obtain a minimal blood flow of - ml /min and pbsc collection was performed with spectra optia mnc v . apheresis system, starting with cd + cell ⩾ μl in peripheral blood. the priming of extracorporeal circuit was made with compatible, irradiated, leucodepleted packed red cell to avoid hypovolemic state. citrate dextrose solution a(acd-a), with a ratio of : to whole blood, and a bolus of heparin ui/kg were used as anticoagulants. all patients, treated without sedation, were monitored by ecg, pulse oximetry and non invasive blood pressure; electrolytes panel (na, k, ca) and act (activated coagulation time) were assessed at the beginning, minutes after and then every hour during apheresis. hypocalcemia was managed by mg calcium gluconate slow infusion. we report our experience of pbsc collection in low body weight children ( o . kg) treated in our apheresis department between january and november . a total of pbsc collections were performed in children ( m/ f, median age months, median weight . kg) affected by medulloblastoma (n = ), germ cell tumor (n = ), neuroblastoma (n = ), retinoblastoma (n = ), brain cancer (n = ). total blood volume processed ranged from . to . tbv (median . ) and median count of cd + collected was . × /kg(range: . - ). all procedures were performed with a median duration time of minutes (range: - min) and no serious adverse events occurred. in our experience pbsc collection is safe and feasible also in low body weight children using a tailored apheresis procedure. disclosure of conflict of interest: none. plerixafor on demand in the first or in the second attempt of cd mobilization j romejko-jarosinska, e paszkiewicz-kozik, l targonski, m szymanski, z pojda and j walewski high-dose chemotherapy and autologous hematopoietic cell transplantation (auto-hct) is a recommended strategy for patients with relapsed, refractory or high risk lymphoma. mobilization failure of cd + cells after granulocyte colonystimulating factor (g-scf) with or without chemotherapy is a factor limiting patient access to this potentially curative procedure. the use of plerixafor with g-csf may improve cd + cell harvest in poor mobilizing patients. we evaluated the clinical effectiveness of plerixafor and g-csf ± chemotherapy administered on demand in the first and second attempt of mobilization in lymphoma or myeloma patients who were eligible for auto-hct. we evaluated data on consecutive patients with hodgkin lymphoma ( ), dlbcl ( ), mantle cell lymphoma ( ) , myeloma ( ) and other lymphoma subtypes ( ) who were mobilized with plerixafor between january and october . median (range) age of patients was ( - ). patients received a median of ( - ) chemotherapy lines. radiotherapy was applied in patients. all patients received g-csf ( μg/kg/day) ± chemotherapy and plerixafor ( μg/kg/day) on demand in the absence of increase in the number of cd + cells in peripheral blood above /μl on the day of the scheduled apheresis (within days following the chemotherapy and after at least days of g-csf). plerixafor was given to patients in the first attempt of mobilization and to patients during the second mobilization. the mobilization was considered effective if the harvest cell dose was × /kg cd or more. after plerixafor administration circulating cd + cells increased to /μl in patients ( %) and in patients ( %) in the first and in the second mobilization, respectively (p = . ). the cd + cell collection was performed in / patients ( %): in / ( %) patients in the first and in / patients ( %) during the second mobilization cycle. the median number of apheresis was (range: - ), for both mobilizing cycles. the median (range) cd cell dose collected in the first and second cycle was . (range: . - . ) × /kg and . (range: - . ) × /kg, respectively (p = . ). the harvest was successful in / patients ( %) in the first and in / patients ( %) in the second cycle (p = . ). three patients ( %) who failed the collection with plerixafor in the first attempt, succeeded in the second cycle. additional second mobilization with plerixafor was successful in five patients ( %) who failed the first mobilization. in total, / ( %) and / ( %) of patients given plerixafor in the first or in the second mobilizing cycle harvested at least a minimum cd cell dose for auto-hct (p = . ). these results show that plerixafor administered on demand is an effective rescue strategy for poor mobilizing patients. each mobilization cycle with plerixafor resulted in the increase of circulating cd cell count. successful harvest is more frequent if plerixafor is administered in the first than in the second mobilization attempt. the evaluation of the prognostic factors for mobilizing failure with plerixafor is necessary to identify the poor mobilizers precisely. disclosure of conflict of interest: jr-j, ep-k, lt and jw: sanofi (travel grants); ms and zp: none; jw: lecture, honoraria cryopreserved stem cell grafts are still widely used both in the autologous or allogeneic settings. cryopreserved grafts can be thawed at the bedside or thawed and washed at the cell therapy laboratory. we recently reported that post-thaw washing did not impair hematopoietic engraftment, in a cohort of autologous transplanted patients receiving either unwashed or washed grafts (calmels b et al, bone marrow transplant. ). post-thaw washing can be implemented using various methods such as manual centrifugation, automated centrifuge-based (sepax , biosafe) or spinningmembrane devices such as lovo (fresenius kabi). we here report a step by step implementation of the lovo biomedical device (bmd) for washing thawed stem cell grafts. having defined a washing program, we aim to compare this protocol to our routine process, using the sepax bmd. we took advantage of apheresis products intended for destruction and cryopreserved in identical bags; after dry-thawing (plasmatherm, barkey), bags were connected to the sepax or to the lovo bmd, diluted volume to volume with + - °c % hydroxyethylstarch / . (voluven, fresenius kabi) and processed using the smartwash program (sepax ) or a cycles standard wash protocol on lovo (a cycle referring to one pass through the spinning membrane). the lovo settings were customized for this application: reduction retentate pump rate ml/min, desired inlet pcv %, and automated volume to volume dilution. after processing, cd and cd absolute counts and viability were evaluated by single platform flow cytometry (stem-kit, beckman coulter) and dmso was quantified by capillary zone electrophoresis (p/ace, beckman coulter). post-wash data show comparable cd + cell recovery, viability and effective dmso depletion. we conclude that lovo enables high efficiency dmso depletion while preserving optimal cd viability and recovery. comparison with sepax , a widely used automated centrifugebased device, reveals comparable efficiency. moreover, the length of the procedure when using the lovo does not significantly delay the process as compared to bedside thawing. we are currently evaluating lovo for the processing of multiple bags and higher cell contents, due to its ability to concentrate large volumes of cells suspension. post-thaw washing using automated cell processing systems have thus to be preferred over bedside thawing, since they provide multiple benefits including a short processing time, efficient dmso and cell debris removal, precise determination of infused cd + cell dose, and improved cellular stability. [p ] disclosure of conflict of interest: none. using bone marrow (bm) as the graft source results in lower graft-versus-host disease incidence, which is particularity important in haploidentical (haplo) stem cell transplantations (sct). nonetheless achieving adequate cd + cell count might be complicated in cases of donor-recipient weight differences. priming with g-csf may partly solve this problem. also there are reports of immunomodulatory effect of bm priming. in the retrospective study we have evaluate the effect of priming on stem cell yield and the outcomes of sct. patients and methods: patients with primed bm graft were matched in the ratio : to non-primed grafts. the criteria for matching were type of the donor, age of the recipient, underlying disease and disease status at the time of sct. priming was performed with three injections of filgrastim - mcg/kg daily for days prior to bm harvesting. median recipient age was years (range: - ). % of patients received the graft from haplo donor, % from matched related donor (mrd). % had acute lymphoblastic leukemia, % had acute myeloid leukemia, % had aplastic anemia, % had other malignancies. % were classified as salvage patients. % received myeloablative conditioning, % received reduced intensity. post-transplantation cyclophosphamide (ptcy) was used as graft-versus-host disease prophylaxis in % of patients. results: the yield of cd + × cells /kg of recipient weight was only non-significantly higher in the priming group: . ± . vs . ± . , p = . . the yield of cd + cells per kg of donor weight was also not different: . ± . vs . ± . , p = . . there was no difference in the incidence of primary graft failure ( % vs %, p = . ). median time to neutrophil ( vs days, p = . ) and platelet ( vs days, p = . ) engraftment was shorter in nonpriming group. there was no differences between priming and non-priming groups in the incidence of acute grade ii-iv gvhd ( % vs %, p = . ), moderate and severe chronic gvhd ( % vs %, p = . ), -year non-relapse mortality ( % vs %, p = . ), relapse incidence ( % vs %, p = . ), overall survival ( % vs %, p = . ), event-free survival ( % vs %, p = . ) and gvhd-relapsefree survival ( % vs %, p = . ). conclusions: priming of the bone marrow with reported schedule did not result in higher cd + cell yield and was not associated with any differences in the outcomes of sct. nonetheless, these results should be interpreted with caution, because our study included large proportion of pediatric patients, patients with active disease and ptcy as gvhd prophylaxis, and they may not translate to the other groups of patients. disclosure of conflict of interest: none. priming with granulocyte-colony stimulating factor preserves the contents and abundant ifn-γ production capacity of γδ t cells z bian , q fu , m huo , xj huang and j liu peking university people's hospital the increasing evidences indicate that removal of αβ t-cell and b-cell from grafts was efficient and reproducible in allogeneic hematopoietic stem cell transplantation (allohsct). γδ t cell is one of the functional subpopulations preserved by this graft manipulation and supposed to play a role in improving the transplant outcomes. thus, comprehensive understanding the subsets and functional capacities of γδ t cells in graft becomes important. although there is increased attention paid on this special t-lymphocyte subpopulation, the contents and cytokine production capacities of peripheral γδ t cells before and after granulocyte-colony stimulating factor (g-csf) mobilization for allohsct have not been reported. peripheral blood (pb) before g-csf treatment, g-csf-primed pb and bone marrow (bm) grafts were obtained from healthy donors. the proportions of total γδ t cells and various γδ t-cell subsets were detected by flow cytometry. furthermore, effects of g-csf on the contents and cytokines production by γδ t-cell subsets were also determined. the percentages of most γδ t-cell subsets including cd +, cd -, vδ +, vδ +cd +, vδ +cd -, vδ +, vδ +cd +, vδ +cd -, and non-vδ /δ were preserved in the g-csf-primed pb grafts compared with those before g-csf mobilization. interestingly, we found that peripheral γδ t cells and various subsets all predominantly expressed ifn-γ in response to stimulation. this abundant ifn-γ production capacity of peripheral γδ t cells were maintained after g-csf treatment. in contrast, production of il- by γδ t cell and its subsets were decreased in the same context. priming with g-csf preserved the contents and abundant ifn-γ production capacity of γδ t cells. our data suggests a reasonable role of γδ t cells in preventing from allohsct associated complications and may help establish an effective γδ t cell-based immunotherapeutic approach to improve the overall survival of allohsct. disclosure of conflict of interest: none. processing of hematopoietic stem cells grafts: towards automation of cryopreservation/thawing steps a-l chateau , j gaude , c malenfant , a autret , c lemarie , c chabannon and b calmels centre de thérapie cellulaire-institut paoli-calmettes and unité de biostatistiques-institut paoli-calmettes autologous hematopoietic stem cells (hsc) support is still widely used to allow for high-dose chemotherapy in the context of myeloma and lymphoma treatment. in the autologous setting, mobilized aphereses are systematically cryopreserved. currently, cryopreservation and subsequent thawing rely on manual and largely operator-dependent processes such as manual addition of dmso for cryopreservation or thawing in standard water baths. these operations are thus hampered by significant intra-and inter-facility variability and have to be replaced whenever possible with automated and harmonized processes. the aim of our study was to evaluate a recent, versatile device: smartmax (biosafe, eysins, switzerland), based on the peltier-seebeck effect, for its ability to automatically add the dmso-containing solution to the cell product and to thaw hsc bags. we thus compared three different cryopreservation/thawing protocols ( figure ). we first evaluated the use of the smartmax at the thawing step by comparing cryopreserved apheresis products thawed using our routine device: the plasmatherm (barkey), an automated dry-thawing device that contains water (protocol a), with products thawed with the smartmax (protocol b); after thawing, all products were washed using the smartwash program of the sepax (biosafe). we then evaluated the smartmax for its ability to automatically add the dmso solution: autologous grafts were processed with the smartmax, both for cryopreservation and thawing (protocol c); we compared these ‛fully automated processes' to apheresis processed with protocol b. absolute cd + and cd + cell counts and viability were measured before cryopreservation and after washing using single platform flow cytometry. for all three protocols, the quality of the collected product was comparable in terms of median cd + cell and neutrophil contents. when comparing protocols a and b, viable cd + cell recovery after thawing and washing was slightly lower in the smartmax group ( %) as compared to the plasmatherm group ( %, p = . ). when comparing protocols b and c, viable cd + recovery was comparable (p = . ) when the cryopreservation solution was automatically added by the smartmax ( %), as compared to the manual technique ( %). these preliminary data need to be validated on larger numbers of procedures, however suggest that smartmax use can safely be substituted both to the manual addition of the cryoprotectant and to the traditional thawing step in water baths; potential advantages include complete water removal from sensitive clean rooms and gmp environments. full automation of previously manual and operatordependent technical processes will ultimately allow for improved standardization and reproducibility across cell processing facilities. [p ] disclosure of conflict of interest: none. reduced efficacy of mobilisation using gdp compared to ive a hunter, w merrison, am martin, k hodgson, f miall, r moore and r lewin university hospitals of leicester, nhs trust the use of ive ± rituximab for relapsed/refractory disease in lymphoma is well established. stem cell mobilisation using g-csf post ive administration has been the standard of care in our unit for years. recent interest in cisplatnin-based treatments has seen a change in practice with the use of gdp ± rituximab increasingly common. we have assessed the success of stem cell mobilisation post gdp and compared it to ive using g-csf. patients were eligible for augmentation with plerixafor if their peripheral blood cd levels were between - × cells/l at the time of collection. from sept to oct patients with progressive or relapsed lymphoma underwent stem cell collection. patients characteristics: dlbcl, follicular and t-cell nhl. had a median age ( - years). received gdp, ive. overall % patients failed to mobilise a sufficient cd cell dose to proceed to hdt. all the patients who received ive mobilised successfully but / ( %) patients receiving gdp failed to mobilise. of the patients who did mobilise the average cd collection was higher in the patients who received ive . ( . - . ) and the number of apheresis procedures was lower, median ( - ) compared to . ( . - . ) and ( ), respectively, in the gdp group. patients in the gdp group who failed to mobilise were not eligible for plerixafor because cd levels were below × /l. taking age into account the median age in the ive group was higher ( - ) than the gdp group ( - ) and the lines of previous therapy were not different. patients who had successful stem cell collections went on to receive hdt with leam and all patients engrafted. in this small collection of patients we have experienced a higher failure of mobilisation post a cisplatnin-based protocol compared both to our historical controls pre plerixafor usage (data not shown) but also to current patients. further investigation is needed to ascertain the impact of cisplatnin on stem cell mobilisation and its impact of treatment strategies. disclosure of conflict of interest: none. single centre experience of zarziotm biosimilar granulocyte-colony stimulating factor (gcsf) for the mobilisation of healthy donors demonstrates good leukapheresis yields and safety profile at month median follow-up jg taylor , , t seddon , k alizadeh , c agrawal , l kempster , jg gribben , and sg agrawal , centre for haemato-oncology, barts cancer institute, dept. haemato-oncology, st bartholomew's hospital, london, uk and experimental pathology, blizard institute, queen mary university of london, uk biosimilars have led to significant improvements in the affordability of growth factors such as granulocyte-colony stimulating factor (gcsf). data has shown similar performance and efficiency to parent drugs but concern has been raised about their use in healthy donors due to lack of data examining adverse effects in this setting. we conducted a retrospective analysis investigating mobilisation and adverse effects in healthy sibling donors of adults undergoing an allogeneic haematopoietic stem cell transplant at st bartholomew's hospital from to . harvest data were gathered from hospital records. adverse effects data were gathered from hospital records and telephone follow up. % of donors were male with a median age at harvest of ( - ). all donors were mobilised using zarziotm biosimilar gcsf at a dose of μg/kg/day. median number of apheresis required was ( ) ( ) ( ) . median cd + cell count was . × /kg bodyweight ( . - . ) with × cd +/μl ( - ) in peripheral blood. the target cd + count ( × /kg) was achieved in % of donors and an adequate yield ( - × /kg) in %. in four donors ( %), the harvest was deemed to have been unsuccessful as the cd + count was o × /kg. the patients with donor harvest yields o × /kg proceeded to transplant; all four patients engrafted and one patient had mixed chimerism at day but was fully donor by day . median cd + cell count was . × /kg bodyweight ( . - . ) . median days to neutrophil engraftment ( . × /l) was . median days to platelet engraftment ( × /l) was ( - ) with one patient never engrafting. forty ( %) of donors were contacted at a median of months ( - ) post mobilisation to establish incidence of adverse effects. three donors were uncontactable as they had moved overseas. eight donors were not contacted to avoid distress as their sibling had died since transplant. among contacted donors . % reported side effects including bone and lower back pain controlled with analgesia, constipation and low mood. other side effects included chest pain which was considered to be musculoskeletal in origin on day of gcsf administration associated with taking an increased dose due to patient error (n = ) and abdominal contractions like labour while receiving gcsf (n = ). three ( . %) reported side effects lasting beyond one month post mobilisation: lower back pain lasting months (n = ), fatigue of months duration (n = ), and cough of months duration (n = ). our data demonstrates good mobilisation using μg/kg/day zarziotm biosimilar gcsf without significant adverse effects at years median follow up. this supports its ongoing use for the mobilisation of healthy donors. disclosure of conflict of interest: sga has received honoraria from sandoz and grant support from sandoz and amgen. stem cell mobilization in poor mobilizers with multiple myeloma (mm) or non-hodgkin lymphoma (nhl) before and after introduction of plerixafor: single center comparative analysis using a cost-efficient single fixed-dose schedule r wäsch , c greil , c kiote-schmidt , s hildenbeutel , k kühbach , r bosse , j duyster and m engelhardt department of hematology, oncology and stem cell transplantation, university medical center, freiburg, germany collection of hematopoietic stem cells (hsc) from the peripheral blood (pb) is routinely conducted prior to highdose chemotherapy and autologous transplantation. despite safety and efficiency of current apheresis procedures including mobilizing chemotherapy and granulocyte colony-stimulating factor (g-csf), there is a significant rate of mobilization failures due to different patient-dependent factors necessitating additional agents like plerixafor. while plerixafor is approved for patients with mm or nhl based on prospective studies using steady state mobilization with g-csf − /+ plerixafor, prospective studies using chemo-mobilization are lacking. here we compared the outcome of poor mobilizer from the pre-plerixafor era with poor mobilizers who received additional plerixafor in a real world analysis. we analyzed consecutive patients with mm or nhl who were mobilized at our academic center between and and received plerixafor, because they were expected to be poor mobilizers, due to . low counts of cd + cells in pb samples prior to apheresis, . after a first apheresis day with insufficient yield or . as a rescue strategy after insufficient harvest with previous mobilizing chemotherapy (greil c,…engelhardt m, wäsch r. leukemia & lymphoma , in press). we examined cd + cell counts in pb and in apheresis products to identify those patients who were able to collect a sufficient cd + cell count for transplantation after application of plerixafor. we compared these data with consecutive poor mobilizers from the pre-plerixafor era, who were mobilized between and without plerixafor. the median pb cd +/μl count at first apheresis was significantly higher after the first dose of plerixafor when compared to the pre-plerixafor group with . vs . (p o . ). accordingly, the median collected cd + cells/d (× /kg bw) and total cd + cells (× /kg bw) were significantly increased with . vs . (p o . ) and . vs . (p o . ), respectively. the rate of × cd + cells/kg bw in first apheresis (%) increased from % in the pre-plerixafor era group to % after the first dose of plerixafor in the plerixafor group. consistently, the successful transplantation rate increased from % in the preplerixafor group to % in the plerixafor group. successful stem cell mobilization could be achieved with only a single fixed-dose of plerixafor in % of poor mobilizers as previously reported by our group. the addition of plerixafor to chemomobilization in poor mobilizers with mm or nhl significantly increased pb cd +/μl counts, apheresis yields and transplantation rates when compared to poor mobilizers from the pre-plerixafor era. these favorable apheresis results can be obtained using our cost-efficient, single fixed-dose plerixafor schedule in the majority of the patients leading to a % transplantation rate in poor mobilizer. disclosure of conflict of interest: rw received research funding, advisory and speaker's honoraria from sanofi-aventis. high-dose chemotherapy followed by autologous peripheral blood stem cells transplantation (pbsct) is the standard of treatment for patients with hematological malignancies. recombinant granulocyte colony-stimulating factors (g-csfs) are widely used alone or in combination with chemotherapy, in order to mobilize patient's stem cells (cd +) for autologous and allogeneic peripheral blood stem cells transplantation. aim: the aim of our study was to compare effectiveness and safety of different biosimilar products of filgrastim used in autologous pbsc mobilization in patients with hematological malignancies. our retrospective analysis included patients ( women and men) with median age years ( range: ,who underwent the procedure of autologous pbsct in years - in the haematology, blood neoplasms, and bone marrow transplantation clinic of medical university in wrocław. there were three different biosimilar products of filgrastim used: tevagrastim (teva) in patients, nivestim (hospira) in patients and zarzio (sandoz) in patients. ( %) patients were diagnosed with plasma cell neoplasms, ( %) with hodgkin's and non-hodgkin's lymphomas, ( %) patients had acute myeloid leukemia and ( %) had other hematological malignancies. statistical analysis was conducted using statistica (statsoft polska) statistical software. for quantitative variables arithmetic means and standard deviations were calculated for the estimated parameters in the studied groups. distribution of variables was tested using w-shapiro-wilk test. p . ). there were also small variations in the mean number of leukapheresis necessary to obtain the minimum cd + cell count: . in zarzio group, . in nivestim group and . in tevagrastim group. however, there were no difference between biosimilar g-csfs. the highest rate of successful mobilizations (defined as × /kg cd + cells collected) was observed in . % patients received zarzio, in . % received nivestim and in . % patients received tevagrastim. the safety profile was comparable between the biosimilar g-csf and included bone pain in ( %) patients and headache in ( %) patients. the results are shown in table . all three used biosimilar g-csfs demonstrated similar efficacy and safety in stem cell mobilization in patients with hematological malignancies. therefore, it seems that all the analyzed products can be used interchangeably. presented observations should be verified with wider prospective research. [p ] disclosure of conflict of interest: none. use of g-csf stimulation of bmt donors might prove to be beneficial in many respects, improving tnc yield but also through immunomodulatory effect on donor t cell function and apcs . we analyzed outcomes of consecutive patients receiving bone marrow transplants from hla-haploidentical related donors that received g-csf stimulation prior to harvest. fourteen patients received hla-haploidentical bmt with pt-cy between / and / . five donors were siblings, children, mothers and father. donors received g-csf at the dose of mcg/kg bw sc. on days − , − and before bm collection. twelve patients received nonmyeloablative conditioning according to baltimore protocol , while two patients received myeloablative conditioning (bucy). along with post-transplantation cyclophosphamide, all patients received tacrolimus and mmf form day + , as described earlier . median age was years (range: - ), female and male patients. eight patients had aml, cml, mh and one all. ten of them were in remission, while mh patients were in pr, and aml patients had residual disease as evident by immunophenotyping. median number of infused tnc was . × /kg bw (range: . - . ); cd + cells . × /kg bw (range: - . ) and cd + cells . × /kg bw (range: . - . ). median follow up was days (range: - ). eleven patients engrafted ( %), one patient had primary rejection, one had overt disease relapse at day + and one patient died in aplasia due to sepsis. median day to neutrophil recovery (anc . × /l) was (range: - ), median days to platelet recovery (plt × /l) was (range: - ). in all patients mmf was discontinued at d + . two patients developed acute gvhd in our cohort ( %), one after receiving dli for falling chimerism at day + . one patient ( %) developed chronic gvhd, after having received dli due to disease progression. at the time of analysis patients are evaluable; patients had disease relapse/progression ( %), patients are alive and in remission. one patient died due to sepsis in aplasia (accounting for % non-relapse mortality). one patient that rejected the graft was transplanted again from the same donor, using myeloablative conditioning and peripheral stem cells as graft source and engrafted. overall survival median is . years, with significantly shorter survival if patient was not in complete remission at time of transplant (p o . ). even though the experience with g-csf mobilized bm graft in the hlahaploidentical setting with pt-cy is relatively small, in our series it has been beneficial in terms of tnc yield. also, the incidence of acute and chronic gvhd in our patients has been low, particularly agvhd with one case developing only after dli. whether the observation is the result of limited number of patients, or it reflects the immunomodulatory effect of g-csf on bm graft as previously suggested remains to be seen as further studies are warranted. autologous transplantation of haematopoietic stem cells (ahsct) is usually perceived as a fully standardized and safe procedure; however, a minority of patients experience a delayed engraftment and seldom even an engraftment failure, possibly related to a poor quality of the graft. therefore the current policy in many centers is aimed to increase the target dose of collected cd + cells up to an ‛optimal' level of × /kg. plerixafor was introduced in the clinical practice to maximize the mobilization of hsc, in order to collect an optimal number of cd + cells in a limited number of collections also in poor and slow mobilisers. we carried out a retrospective analysis of our case series aimed to individuate mobilization predictors optimize the ‛on demand' use of plerixafor. we analyzed patients who underwent mobilization with cyclophophamide ( g/sqm) and filgrastim mcg/kg from + in our unit from and . diagnosis were multiple myeloma (mm) ( . %), non-hodgkin lymphoma (nhl) ( . %), hodgkin lymphoma (hdg) ( . %) and ( . %) autoimmune disease (ms . %; ssc . %). median age (range) was years ; male/female ratio / . circulating cd + cell count was started at white blood cells (wbc) recovery, which was defined as the first day when their count exceeded × /l. the primary goal was to identify at wbc recovery one or more factors predicting a suboptimal mobilization, which was defined as the failure to exceed cd +/mcl circulating cells in the day after the wbc recovery. patients were excluded from this analysis if ) showed a cd + count /mcl at wbc recovery (very good mobilizers) and/or ) had received plerixafor and/or ) did not proceed to another cd + count the day after wbc recovery. binary logistic regression was used to obtain the factors that increased the odds for an optimal mobilization. overall out ( . %) patients were shown as very good mobilisers as their cd + count exceeded /mcl at wbc recovery. on the remaining , were excluded for the lack of a second assessment and for the lack of data. among the remaining patients, the threshold of cd +/mcl cells on the second day was reached by ( . %) of patients (group a) while the remaining ( . %) failed the goal (group b). median (range) wbc × /l and cd +/mcl counts in group a and b at wbc recovery were . ( - . ) and . ( . - . ) and . ( - ) and . ( - ), respectively, with a statistically significant differences among group (mann-whitney u test with p = . and p = . , respectively). wbc (or = . ; % ci: . - . ) and cd +/mcl (or = . ; % ci: . - . ) in first day count, but not gender, disease category and time from mobilization chemotherapy to first cd + count, were predictors of optimal mobilization. combining these two predictors we found that wbc/cd + ratio has a sensitivity of . % with an auc . in roc analysis. assessment of circulating wbc, cd + and their ratio at wbc recovery in a chemo-based mobilization strategy can predict sub-optimal mobilization of hsc and support the decision of adding plerixafor. these data will be prospectively validated in a broader set of patients. disclosure of conflict of interest: none. human platelet lysate (hpl) is rich in growth factors (gf) and nutritive elements and represents a powerful xeno-free alternative to fetal bovine serum (fbs) notably for mesenchymal stem cell (hmsc) proliferation. however, there is a large variability in hpl preparations (various sources, use of different and non-standardized production protocols, with variable and limited number of donors), resulting in discrepancies in product quality, low management of product safety and poor batch-to-batch standardization. we describe here the development and the characterization of a standardized hpl prepared from outdated transfusional grade screened normal human donor platelet concentrates (pcs), manufactured on an industrial scale (batch size of donors) and following a highly qualified process (clean room, trained operators, validated aseptic filtration). pcs were frozen at − °c and thawed at + °c to lyse platelets. cell debris were removed by centrifugation and the supernatant (hpl) was recovered. clinical grade l batches of aseptic filtered hpl were characterized. first, we showed that hpl prepared from a limited number of donors displayed a variability in terms of gf contents. on the contrary, we observed a robust standardization between industrial batches of hpl ( donors) in terms of gf contents (bfgf, egf, vegf, pdgf-ab, tgf-beta and igf- ), biochemical analyses (total proteins, albumin, fibrinogen, vitamins and iron) and efficacy on bone marrow (bm)-hmsc proliferation. secondly, we compared expansion and functional characteristics of bm-hmscs grown in clinical grade hpl vs msc-screened fbs batches. we showed a reproducible increase in cell growth kinetics using hpl, a maintenance of bm-hmsc clonogenic potential and membrane marker expression (with however a strong overexpression of cd ). we observed a similar adipogenic and osteogenic differentiation potential and finally that immunosuppressive properties of bm-hmscs (inhibition of t-cell proliferation) cultivated in parallel in both conditions also remained identical. finally, we demonstrated the stability over time of hpl stored at − °c and − °c. in conclusion, we demonstrated the feasibility to use a standardized, characterized, efficient and clinical grade hpl for research and cell therapy applications. disclosure of conflict of interest: sv, se, lc, pb, tb, al, fg and bd are employees of macopharma. previously published p alpha/beta t cell depleted donor lymphocyte infusion m karakukcu, e Ünal, l kaynar, s Özcan, g tezcan karasu and mb acar the main objective of this project is to improve a safe and efficient new donor lymphocyte infusion (dli) with depletion of αβ+ t cells which cause graft versus host disease (gvhd), and enrichment of anti-leukemic γδ+ t cells, nk cells and dendritic cells to build an effective and permanent anti-tumor effects for patients relapsed hematological cancers after allogeneic hematopoietic stem cell (hsc) transplantation who have blasts and mixed chimerism. this study is conducted with collaboration of erciyes university pediatric and adult hsct units, and bahcesehir university, medical park hospital pediatric hsct unit. the tcr αβ+ t cell depleted dli product that is used in the study was collected and separated at erciyes university apheresis unit. the cell contents obtained for tcr αβ+ t cell depleted dli used for patients were cd cells were reduced to . - . × cells/kg, γδ+ t cells were reduced to . - . × cells/kg, αβ+ t cells were reduced by . %, and were obtained at - cells/kg. a total of patients ( female, male) were included in the study, consisting of an adult and children. nine patients had hematological malignancies. five patients were referred for all, three for aml, one for mds and one for griscelli syndrome. efficiency: the clinical response to the αβ+ t cell depleted dli treatment was achieved in / patients ( %). in these patients, although the increase of chimerism was limited in patients, no recurrence was occurred. one of the two patients who previously responded to the treatment but experience of decreasing chimerism had relapsed after months, and months later. one of these two patients died after relapse. the other was managed by the second transplant. the most important objective of this study was to show that αβ + t cell depleted dli treatment is reliable. none of the patient showed severe gvhd except one patient with mild grade ii gvhd. despite the presence of severe gvhd after hsct in two patients, reactivation for gvhd was not observed after treatment with αβ+ t cell depleted dli. none of the patients had a bone marrow aplasia. as a result, αβ + t cell depleted dli treatment seems to be highly safe, and effective in selected patients. disclosure of conflict of interest: none. hematopoetic stem cell transplantation (hsct) is associated with several potentially lethal complications; for example, relapse of the malignant disease, graft rejection, infectious complications and graft versus host disease (gvhd). higher levels of cd + cells in the graft have clearly been associated with increased risk of gvhd, but also superior gvl effect and less infectious complications. to tackle post-transplant complications such as graft failure and relapse, donor lymphocyte infusion (dli) have successfully been used for decades but with an associated risk of gvhd. to decrease the risk of gvhd but still use facilitating cells in the cell product we performed αβ depletion of grafts for use as stem cell booster after allogeneic hsct to treat infections or poor immune reconstitution. in this study, patients were infused post-hsct with αβ t-cell depleted grafts. the indication for infusion of αβ t-cell depleted graft in all patients was poor immune reconstitution with associated infectious complications. for all patients, the original hsct donor was used for the αβ t-cell depleted boost. to characterize the αβ-depleted stem cell grafts, samples were stained for various cellular subsets and analyzed by flow cytometry. we could show a median log depletion of αβ cells of . and a median yield of γδ t-cells (%) of . . the median cd + cell dose (× /kg) was . . all patients were alive months after infusion. after year only one patient succumbed. despite that the majority of patients suffered from agvhd grade or before infusion of αβ t-cell depleted graft none showed increased symptoms afterwards. in more than % of the patients there was a increase in granulocytes, thrombocytes and white blood cells months after infusion. in conclusion, we describe the use of αβ t-cell depleted grafts as stem cell booster in patients suffering infectious complications due to graft failure after hsct with encouraging results. disclosure of conflict of interest: none. delayed engraftment or graft failure still remains a concern in bone marrow transplantation (bmt). graft composition may predict engraftment after infusion. this study aims to determine which quality control parameters used for the characterization of bone marrow grafts are the most predictive in order to minimize the risk of engraftment delay or graft failure. we conducted a multicenter retrospective study in pediatric patients who underwent first allogenic bmt at two centers in barcelona (catalonia, spain) between and . quantitative variables considered for the study were: total nucleated cells (tnc), mononucleated cells (mnc), cd + cells, cd + cells and granulocyte-monocyte (gm) colonies enumeration. qualitative variables considered for the study were viability assessed by flow cytometry and clonogenic efficiency of the cd + cells (clonegm) which is the ratio between gm colonies and cd + cells. patients were included (median age (range) were years old ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ). the median tnc(range) was . e /kg ( . - . e /kg) and . e /kg ( . - . e /kg) for mononuclear cells (mnc). on the other hand, the median (range) cd + cell dose was . e /kg ( . - . e /kg) and t-cell dose (cd +) was . e /kg ( . - . e /kg). the median (range) colonyforming unit granulocyte macrophage (gm/kg) dose was . e /kg ( . - . e /kg). the median (range) of cd + cell viability, was % ( - %) and the median(range) of the clonogenic potential of cd + cells (clonegm) was . % ( . - . %). the median (range) of engraftment was days for neutrophils and ( - ) days for platelets. patient was considered as primary graft failure. in the univariate analysis, cd + (p = . ) and mnc (p = . ) cell dose predicted a faster neutrophil engraftment and female donor a slower neutrophil and platelet engraftment (p = . and p = . ). cell viability also correlated to a better platelet engraftment (p = . ). in the multivariate analysis we observed a trend for a faster neutrophil recovery according cd + cells infused. again, female donor was associated with slower engraftment. in order to establish a safety threshold, we did a quartile analysis of cd dose and found . e /kg (quartile ) discriminates a faster neutrophil engraftment [median days vs days for those with higher cd + cells (p = . )]. in conclusion, we found an association between mnc and cd + cell dose and time to engraft, and established a safety threshold of . e cd +/kg. also, bm grafts from female donors were associated with slower engraftment. no other qualitative parameters were predictive of engraftment. disclosure of conflict of interest: none. plasma cell myeloma (pcm) is currently treated with chemotherapy and autologous stem cell transplantation (asct) but relapse rates remain high. adoptive transfer of mature haploidentical natural killer (nk) cells is a promising approach to provide pcm patients with highly immunocompetent effector cells with anti-myeloma function early post transplantation. here we report on the current clinical phase i/ii trial of multiple preemptive infusions of good manufacturing practice (gmp) expanded nk cells to pcm patients (clinicaltrials.gov nct ). ten pcm patients were recruited (seven males, three females, median age: y). all patients received four cycles of vtd chemotherapy (reaching cr: × , vgpr: × and pr: × ) before high dose therapy with melphalan mg/m and asct. after successful stem cell mobilization and cryopreservation of patients' stem cells after the third vtd cycle, nk cells from haploidentical family donors were purified from unstimulated leukapheresis by t cell depletion and nk cell selection using clinimacs. highly pure nk cells (mean: . × cells) were obtained with a minimal t cell contamination corresponding to a . log t cell depletion. nk cells were expanded ex vivo for days in gmp-medium containing autologous irradiated feeder and interleukin- and - . nk cell numbers increased -fold (range: - -fold). in three nk cell products t cell contents were × cells/kg body weight (bw: × above limit of clinical trial) and were successfully reduced by °cd -depletion to . × cells/kg bw. nk cell products were cryopreserved in escalating doses ( . × , . × and rest as multiple doses of maximal . × cells/ kg bw). the pcm patients received - × expanded nk cells (median: . × cells/kg bw, range: . - . × cells/ kg bw) as - infusions (median. dlis). the nk-dlis were administered between day and after asct and were well tolerated without any acute adverse events. no signs of acute or chronic graft-versus-host disease were observed in any of the patients after a total of nk-dlis. engraftment occurred between days - (median: days). infused donor nk cells were monitored by short-tandem repeats pcr. donor nk cells were detected in peripheral blood one and h post infusion (% donor nk of enriched blood nk cells: mean: %, range: - %, and mean: %, range: - %, respectively) indicating significant nk cell survival in recipients in the absence of il- support in vivo. clinical responses at last follow-up compared to a retrospective cohort of matched control patients will be presented. these results demonstrate the feasibility of large-scale gmp expansion and safety and immunotherapy with third-party leukemia-specific t cells (leuk-sts) represents an attractive approach for acute leukemia (al) patients lacking a fully matched donor or relapsing after allogeneic hematopoietic cell transplantation (hct). its application however, is limited by the demand for high numbers of antigen presenting cells (apcs), capable to produce clinically relevant numbers of leuk-sts. low volume, non-transplantable cord blood units (cbus) could theoretically serve as an easily accessible source to generate high numbers of dendritic cells (dcs) and subsequently leuk-sts, providing also the advantage of reduced alloreactivity, even in cases of partial matching. our goal was to generate clinically relevant doses of leuk-sts targeting al-related antigens, the wilms tumor protein (wt ) and the preferentially expressed antigen in melanoma (prame), through the exploitation of non-transplantable cbus. to generate dcs, immunomagnetically enriched cd + cells from cbus ⩽ ml were cultured in g-rex devices in the presence of scf, gm-csf and il- . dcs matured by toll-like receptor ligand and / were immunophenotypically characterized by flow cytometry (fcm). secreted cytokines were measured with elisa. matured dcs were activated with a peptide-mix of wt and prame and used as apcs to repeatedly stimulate naive t-cells (derived from the cd fraction of the same cbu). the phenotype and the specificity of generated leuk-sts were determined by fcm and ifn-γ/ elispot, respectively. starting from mean . × ± . × cd + cells, from non-usable cbus, we generated . × (range: . - . × ) myeloid dcs (cd +/cd c+: . ± . %) in days (fold change~ . ). the produced cells highly expressed maturation markers (cd +/cd +: ± %; cd c+/hla-dr+: ± %) and secreted high levels of th cytokines (Ιl- : ± pg/ml; il- : . ± . × pg/ml, tnf-α: ± pg/ml) and low levels of the th -cytokine, il- . the average number of cd -cell-derived leuk-sts after week-culture was . ± . × (~ logs above clinical doses). the produced cells were enriched in cd + polyclonal cells ( ± %), comprising of cd + ( ± %) and predominantly cd + cells ( ± %), expressing effector memory (cd ra − /cd l − : . ± %) and effector memory ra markers (temra: cd ra+/cd l − : ± %), while containing insignificant numbers of cd +/cd +cells ( ± . %). specificity was seen after the second stimulation at the earliest and was increasing after each stimulation [mean spot forming cells (sfc)/ × cells at second, third, fourth stimulation: ± ; ± ; ± ; respectively]. in particular, produced cells were highly specific for both targeted antigens (prame: ± , wt : ± ), while they expressed low the programmed cell death protein- (cd +/pd- +: ± %), implicating absence of cell exhaustion after repeated stimulations. we report a paradigm of ‛circular economy' in science, by the exploitation of non-usable cbus, towards scalable generation of cb-cd +-cell-derived dcs and cb-cd -cellderived leuk-sts from the same cbu and establishment of leuk-sts banks. whether similarly produced leuk-sts could significantly advance the treatment of al or leukemic relapse after hct, will be ultimately determined in vivo. disclosure of conflict of interest: none. comparison of two different methods to generate antifungal-specific t-cells under pre-clinical-scale conditions r geyeregger , s tischer , invasive infections with aspergillus fumigatus constitute a major cause of morbidity and mortality in immunocompromised patients after haematopoietic stem cell transplantation. although adoptive immunotherapies against viral pathogens are already in phase i/ii trials, clinical-grade methods for the generation of aspergillus-specific t-cells (asp-t-cells) from healthy transplant donors or even related or unrelated thirdparty donors are still under development. in this study, two different strategies interferon-gamma (ifn-g) cytokine capture system (ccs) vs short-term in vitro expansion (ste) were performed from the same healthy volunteers in order to evaluate the most suitable approaches for the in-time generation of clinical applicable asp-t-cells. pbmcs from leukapheresis of healthy donors (n = ) were first prepared in hannover for the ifn-g-ccs and then sent to vienna to prepare the ste. all donors belong to the allocell registry (www.allocell.com) of hannover medical school and the frequency of asp-t-cells was pretested by high-throughput ifn-g elispot assay. for the ifn-g-ccs, × cells were stimulated for h with gmp-conform aspergillus lysate followed by magnetic selection of ifn-g-producing t cells. cells were characterized for phenotype and function by multicolour flow cytometry. for the ste, × cells were cultured in g-rex devices and stimulated for days with either the aspergillus lysate alone or with pooled overlapping pepmixes (catb, crf , f , gel , pmp , shmt and sot) and il- . to further characterize the final cell products, multicolour flow cytometry, ifn-g elispot and ifn-g/granzyme b flurospot analyses were performed. ifn-g-ccs: frequency of ifn-g positive asp-t-cells pre-magnetic enrichment ranged between . and . %. recently we defined t-cell donors as eligible if ⩾ . % specific ifn-g+ t cells are detectable. the purity of asp-t-cells among cd + cells, obtained from three donors after magnetic selection was in mean % ± (range: - %). the absolute number of selected ifn-g+ cd + t-cells was ± . this could be approximately multiplied by a factor of , if × pbmcs are used for the generation of clinically applicable t cells using the ccs and the prodigy device. ste: after days, asp-t-cells (n = ) showed highly specific activity against the lysate (in mean ± spot forming colonies (sfc)/ cells) and pooled pepmixes (in mean ± sfc/ cells). in both methods (lysate vs pooled pepmixes), predominantly cd + t-cells were expanded ( % ± . vs % ± . of cd +) compared to cd + t-cells ( . % ± , vs . % ± . ). interestingly, whereas after ste, cd + t-cells include mainly central memory t-cells (mean %; cd l+cd ra − ), cd + t-cells include mainly effector memory t-cells ( %; cd l − cd ra − ). generated t cells were highly functional and cytotoxic as determined by the secretion of effector molecules granzyme b and ifn-g. based on the purity of up to % after the ifn-g-ccs and the high number of sfc received after ste with lysate and pepmixes, both methods seem to be suitable for clinicalscale productions. for patients who are in need for high asp-tcell numbers the application of first in-time ccs-purified asp-t-cells followed by the administration of ste cells might be a promising way to boost antigen-specific t-cell response. disclosure of conflict of interest: none. complete computerization of cell therapy product files (‛zero paper') in the qap software o christéle , r catherine , r aline , k mathias , m lavinia , d vincent , m jean-pierre and l marie-noelle , hematologie clinique et thérapie cellulaire-chu amiens picardie, simedia-ver, hématologie clinique et thérapie cellulaire-chu amiens picardie, simédia-ver, direction système informatique-chu amiens picardie, hématologie clinique et thérapie cellulaire, lacassagne and the computerized management of cell therapy products (ctp) is an obligation for processing laboratories to meet regulatory requirements. the software used is often independent of institutional systems in view of the specificity of cellular therapies and do not always allow the implementation of the ‛zero paper' policies that are being put in place. we report here our experience with the qap software (quality assurance partner) developed by the company simédia (www. qap .com) in open source (mit license) allowing the management of fully computerized ctp files. the qap software has been developed to ensure the traceability of ctp for both preparation and quality control by combining the product preparation environment (personnel, premises, reagents, consumables, equipment). initially, with the help of the company simédia, we parameterized the software in accordance with our procedures for the preparation and quality control of ctp. we built a file that we printed out for archiving on paper. it soon seemed necessary to reverse this mode of operation to add to the software the documents papers to obtain a file completely computerized and to avoid paper archiving. the close collaboration between the cell therapy laboratory staff, the software referent within the information system department of the amiens hospital and the company simédia enabled: set up a document backup server sufficiently proportionate in memory. have simedia carry out the necessary developments so that all documents can be integrated into the software, set up a coherent working circuit, organize the registration of documents, put in place a rigorous verification of the mandatory elements of the file. the reflection on the computer file made it possible to evolve the software to widen its use to all documents of management of the laboratory: maintenance of equipment, control of premises, housekeeping, staff training, quality control of automatons, reagents and consumables, process, reception, distribution. rigorous formalization was mandatory to ensure that the record was organized in a uniform manner. an intermediate paper record is still necessary for a period of about month: from the programming of the graft to the final validity of the injected product. this folder consists only of transient elements that cannot be integrated into the qap software immediately. the transition from the paper file to a computer file took place in several stages, calling into question our functioning. the difficulties of this implementation are of several natures: the heterogeneity of the documents components a cell therapy product file, the impossibility of benefiting from an interface between all computer software used on the hospital, the psychological barrier prompting us to keep a paper copy, work habits, the guarantee of computer backup quality as well as its verification. but the complete computerization of the ctp file has the following advantages: easy and secure accessibility of information, resolution problems archiving paper files, a single backup media folder. disclosure of conflict of interest: none. conditioned media from allogenic mesenchymal stem cell culture (msc-cm) enhances wound healing in an allogenic d skin model moyasasr al-shaibani, x wang , p lovat, a tulah and a dickinson newcastle university migration of the epidermal layer towards the wound centre is an important step in the healing process. full thickness in vitro skin models can be used to investigate epidermal migration towards an injury site. since wound healing therapies often require allogenic transplantation of primary keratinocytes, an allogenic d skin model was developed to investigate epidermal migration. the effect of mesenchymal stem cell conditioned media (msc-cm) was assessed for wound healing using this in vitro human d skin model. human mscs were derived from human hip joints, and characterised using standard protocols. at % confluence, msc secretions were collected in serum free medium and referred to as msc-cm which were then analysed for protein content using elisa. fully humanised allogenic d skin models were developed (n = ) and a mm punch was induced into each model followed by daily treatment with msc-cm to investigate the migration of the epidermal layer towards the punch centre over the dermal layer at different time points ( week, weeks, and weeks). intact and wounded models were characterised structurally by haematoxylin/eosin (h&e) staining and immunofluorescence (if) was used to validate the dermal and epidermal biomarkers such as collagen (col ), cytokeratin (k ), keratin (k ), loricrin and involucrin. mscs were characterised as stipulated by the international society for cell therapy, that is, fibroblast like cells with the ability to differentiate into tri-lineages (adipocyte, chondroblast and osteoblast). phenotypically, over % of the cells were able to express phenotypic markers for variant stem cells such as cd , cd and cd . over % of the cells were negative for the expression of cd , cd , cd , cd and hla-dr (p = . ). msc-cm contained different concentrations of a variety of growth factors such as keratinocyte growth factor (kgf), hepatocyte growth factor (hgf), platelet-derived growth factor (pdgf), stromal-derived factor- (sdf- ) and macrophage stimulating protein- (msp- ). h&e staining showed that the models had distinct dermal and epidermal layers similar to that of real skin. additionally, if showed that the models expressed dermal and epidermal biomarkers, for example, col , k , k , loricrin and involucrin. after treatment with msc-cm, the epidermal multilayers of the punched models started to migrate towards the punch centre and covered the whole punched area after weeks of treatment with recovered expression of the epidermal biomarkers, for example, k , k , loricrin and involucrin. a fully humanised allogenic d skin model is a useful tool to mimic the in vivo environment and evaluate the wound healing process. it could also be used as a screening method to test candidate wound healing drugs. allogenic keratinocytes could be used as a cellular sheet to cover the wound area with the ability to migrate towards the wound centre and promote wound healing. a possible explanation for promoting epidermal migration at the injury site is that msc-cm contains cytokines which accelerate cell migration such as kfg, sdf- and msp- , in addition to other cytokines which promote both migration and proliferation of epidermal cells, for example, hgf and pdgf. disclosure of conflict of interest: none. before each freezing and after each thawing, a quality control is performed including a minima: (i) cd + quantification; (ii) estimation of the percentage of hsc cd + viability, via aminoactinomycin-d ( -aad) staining and (iii) evaluation of hsc functional ability to form colony ‛cfu-gm' (colony forming unit-granulocyte macrophage). apoptosis, or programmed cell death, involves complex pathways in part the path fas-fas ligand (fasl), mitochondrial components and caspase enzymes. the involvement of apoptosis dependent on caspases activation pathway in hsc cd + after thawing remains unknown. here, we assess the extent of apoptosis caspase-dependent before and after cryoconservation of hsc cd +, using a fluorescent labeled inhibitor of caspases ‛flica. ' we tested the induction of apoptosis caspasedependent, before and after hsc cd + cryoconservation from patients with different hematological malignances: multiple myeloma (n = ), lymphoma (n = ). caspases pathway activation status was evaluated by flow cytometry, using a fluorescent labelled inhibitor of caspases ‛flica' staining test, in hsc cd +, lymphocytes cd +, monocytes cd + and natural killer cells cd +. in order to assess cell viability, cells were stained in parallel with -aad. we determined positive cells %, that is, showing caspase activation in viable cells (flica+ cells), before and after cryoconservation. caspase pathway activation level was then correlated with hsc functional ability to form colony ‛cfu-gm,' and day's number of clinical aplasia. in our cohort, we showed a significant caspases pathway activation, with . % cd + flica+ cells after thawing, compared with the . % described in fresh cd + cells (p o . ). moreover, caspases pathway was significantly activated in thawing cd +, cd + and cd + cells: flica+ cells % in thawing cells were, respectively, . %, . % and . % vs %, . % and o % in fresh cells. we also report a significant increase of apoptosis caspasedependent in lymphoma patients ( . % of cd + flica+) in comparison to myeloma patients studied ( . % of cd + flica+) (po . ). in contrast, no correlation has been established between observed caspases pathway activation and hsc cd + capacity to form cfu-gm, or still day's number of clinical aplasia. our results show substantial cell death, induced by the increase in caspases pathway activation, secondary to the thawing process, and across all study cell types. this advance of apoptosis caspase-dependent may affect the immune response quality during recipient aplasia, without detecting a clinical impact. moreover, caspases pathway activation through cd + and cd + subpopulations could modify the therapeutic result of donor lymphocytes infusion dli, though yet untested. thawing process in autologous graft induces apoptosis caspase-dependent in all apheresis product cells, particularly in hsc cd +, without clinical impact in graft fate. disclosure of conflict of interest: none. donor-derived nk cell infusion combined with hla halpoidentcial blood stem cell transplantation to decrease leukemia relapse for high risk acute myeloid leukemia patients b wu, y huang, j xu, y he, jxm zhang*, z wu* hematology department, zhujiang hospital of southern medical university, guangzhou, china *shenzheng hank biologoical engineering co.ltd. hla halpoidentcial blood stem cell transplantation have solved the donor deficiency for patient who need to treat by transplantation. the high relapse of leukemia especially for high risk patient post transplantation affect the outcome of haploidentical stem cell transplantation. natural killer (nk) cells are part of the innate immune system and play a scavenger role to detect targets marked by ‛missing self' induced by viral infection or malignant transformation. infusion nk cells into receipt prior to stem cell transplantation could decrease the gvhd in mouse bone marrow transplantation model. in an effort to decrease the leukemia relapse and gvhd after halpoidentical stem cell transplantation for high risk acute myeloid leukemia patients, we evaluated the addition of donor-derived nk killer cells before halpoidentical stem cell transplantation in high risk acute myeloid leukemia patient. here we report interim results for five patients enrolled last year. five high risk acute patients received halpoidentcial stem cell transplantation combined with donor-derived nk cells infusion. all patients received an fbca conditioning regimen, which consisted offludarabine ( mg/m /day, intravenous) on days − to − , busulfan ( . mg/kg/day, intravenous) on days − to − , cyclophosphamide( mg/kg/day, intravenous) on days − to − and rabbit antilymphocyte globulin (atg . mg/kg/day, intravenous) on days − to − . donor-derived nk cells were infused into patient prior to stem cell transplantation. gvhd prophylaxis was a combination of cyclosporine a (csa) and short term methotrexate. five high risk patients ( patients with aml m cr , patient with aml m nr, patient with aml m cr and patient with aml m cr ) enrolled from jan to nov. ; the donors are parents and sibling. hla were mismatched between donor and patients. median cd + dose infused was . /kg (range: . - /kg) and the nk cell dose infused was × /kg ( . - . × /kg). all five patients got hematology recovery and achieved hematology cr. only one patient occurred grade ii agvhd post transplantation and controlled by methylprednisolone. at a median time of months (range: - months) post peripheral blood stem cell transplantation, the incidence of acute gvhd grade ii is % ( / ) . no chronic gvhd observed. four patients are still cr and survival with event free survival with median year follow up. one patient with aml m who had not achieved remission before transplant relapsed after months and got cr with second nk infusion and still survival. nk infusion prior to transplantation was found to be safe and feasible. there was no increase acute gvhd or chronic gvhd risk. there was a trend towards increased -year survival for high risk leukemia patient. the potential benefit on overall survival remains to be further evaluated with additional patient enrollment and longer follow up. however, given the favorable safety profile of nk cells, future strategies to enhance efficacy such as repeat dosing or modification of nk cells are worth potential exploration. disclosure of conflict of interest: none. donor lymphocyte infusion (dli) is a therapeutic option in the treatment or prevention of relapse after allogeneic stem cell transplantation (allohsct).of note, the risk of graft-versus-host disease (gvhd) associated with the graft-versus-tumor (gvt) effect may be influenced by the level of hla disparity between donor and recipient. data on use of dli after unmanipulated haploidentical hsct (haplohsct) with post-transplant cyclophosphamide (pt-cy) are still currently limited. we report patients (pts) receiving dli between and for prevention or treatment of relapse after haplohsct. seven pts were given haplodli doses, as treatment for relapsed disease (n = ) or as preventive therapy of relapse for high risk disease (n = ). four pts had acute myeloid leukemia (aml), had acute lymphoblastic leukemia and lymphomas - hodgkin (hl) and non-hodgkin dlbcl. pt had intermediate risk disease features, adverse risk and pts had refractory disease at time of haplohsct. pts had a previous hsct ( allogeneic and autologous). of the pts received a ric regimen and the source of stem cells was peripheral blood s (n = ) and bone marrow (n = ). gvhd prophylaxis was cyclosporine and mycophenolate mofetil (mmf), atg and pt-cy. median follow-up after haplohsct was (range: months. median time to neutrophil and platelet ( g/l) recovery were and days, respectively. after haplohsct, pts developed acute gvhd (agvhd) of grade i (n = ) or ii (n = ), at a median of days after haplohsct. the median time from haplohsct to first dli was days (range: - ). all pts had full donor chimerism at time of dli. before dli pts relapsed at a median time of days (range: - ), of whom pts had aml and received salvage chemotherapy and pt with hl being treated by dli alone. of the relapsed pts, showed progressive disease after first dli dose and achieved a sustained cr (with duration of cr of and months at last follow-up). the remaining pts were given dli in cr, in case (of aml) associated with azacytidine. pts received dli dose and pts were given dli injections with escalating doses. the first dose of dli was × cd /kg in pts, × in pt and × in pts. the pts who received dli doses (lymphomas) were given: ( ) × - × - × ; ( ) × for doses followed by dose of × . four pts developed chronic gvhd (cgvhd, %) in a median time of days (range: - ) after dli ( of them had presented previously agvhd grade i-ii). cgvhd was limited in case, moderate in pt and severe in pts. of these pts presented features of an overlap syndrome (acute/chronic gvhd) with signs of agvhd de grade i,ii and iii in pt each. involved organs were skin/mucosal (n = ), liver (n = ), gastrointestinal tract (n = ), lung (n = ) and joints (n = ). all patients experiencing gvhd after dli were treated by systemic corticotherapy, extracorporeal photopheresis and cyclosporine or weekly low dose methotrexate. median follow-up after first dli was months (range: . none of the pts receiving prophylactic dli relapsed during the follow-up period. pts died, of relapse and of severe cgvhd. pts were in cr at last follow-up, with no signs of gvhd and with limited cgvhd. despite the limited cohort, dli after haplohsct appears to be a therapeutic option in high risk pts allowing enhancement of gvt in the setting of haplohsct with post-cy infusion. disclosure of conflict of interest: none. previously published p early and sequential ctla ig primed donor lymphocyte infusions (dli) following post-transplantation cyclophosphamide (ptcy)-based haploidentical pbsc transplantation for advanced hematological malignancies promote proliferation of mature natural killer (nk) cells with cytotoxic potential and markedly reduces relapse-risk without increase in gvhd sr jaiswal, s zaman, p bhakuni, s bansal, s deb, s bhargava and s chakrbarti we have earlier shown that cd enriched cell infusion following ptcy resulted in rapid proliferation of mature nk cells with attenuation of gvhd and early use of prophylactic g-csf mobilized dli resulted in improved disease-free survival. ctla ig has been shown to be effective in attenuating t cell activation and induce transplantation tolerance in preclinical models. it has recently been employed to induce transplantation tolerance and reduce early alloreactivity in patients with nonmalignant disorders undergoing ptcy-based haploidentical hsct. nk cells on the other hand are resistant to ctla ig and in fact might demonstrate better anti-tumour effect in presence of ctla ig as cd is a putative activation receptor. to explore this phenomenon, we employed sequential ctla ig primed dli following ptcy-based haploidentical hsct in patients with relapsed/refractory hematological malignancies. patients ( - years; aml- , all- , nhl- ) received abatacept (ctla ig) as a part of gvhd prophylaxis at mg/kg on day − followed by pbsc and sequentially on days + , + and + followed h later by dli of × cd cells/kg containing . - × /kg cd + cells. ptcy was administered on days + and + with cyclosporine from day + to day + and subsequent rapid tapering. the immune reconstitution of the study group (ctla ig-dli) was compared with the cohort of patients with both malignant and nonmalignant diseases who received abatacept but not dli (n = ; ctla ig group) and those receiving cd enriched donor cell infusion on day + (n = ; nki group). results: there were no acute infusion related toxicities. all patients engrafted at a median of days ( - days). the incidences of acute and chronic gvhd (all mild) were % and %, respectively. three patients reactivated cmv and there was only one non-relapse mortality ( . %). only patients relapsed ( . %) with a disease-free survival of . % at year. these cells had greater expression of cd a compared to normal healthy donors. the recovery of cd +, cd + + and cd + − cells were similar in the ctla ig-dli and nki groups at days , and post-transplant and this was significantly higher than the ctla ig group ( figure ). in contrast to ctla ig group, nk cells recovered at day + with predominantly cd dim cd + phenotype with significant population of cells expressing kir+nkg a phenotype in both ctla ig and nki groups with higher expression of cd a. interestingly, the patients who relapsed had attenuated recovery of cd + + cells at and days( /μl and cells/μl) without cd a expression, in contrast to the rapid and sustained recovery of this population of nk cells in those not experiencing relapse (cd + + cells /μl and /μl). however, the recovery of tregs was prompt and sustained in the comparator groups, which remained low in the ctla ig-dli group until day + . there were no differences in the recovery of other t cell subsets between the three groups. the study demonstrates the unique ability of ctla ig to augment nk cell proliferation, maturation and cytotoxicity and reduce relapse with attenuation of t cell activation and gvhd in the context of the early use of ctla ig primed dli following ptcy-based haploidentical hsct without ex vivo selection or expansion. we hope this novel strategy might offer less expensive and yet a viable alternative in the field of nk cell therapy. [p ] disclosure of conflict of interest: none. enhanced cytotoxicity of γδ-cytokine induced killer cells against hematologic malignancies n bloom, s eldror, s caspi, s teihuman,h vernitsky, e jacoby, b bielorai and a toren cik cells are ex vivo expanded by scheduled addition of anti-cd mabs and a cytokine cocktail that contains ifn-γ, il- or il- . cells represent an in vitro generated heterogeneous population consisting of different effector cells-cd poscd pos, cd negcd pos and cd poscd neg-t cells that mainly ( %) express α/β t-cell receptor (tcr) s and to a lesser extent (o %), tcr γδ phenotype. these nklike t cells product show a dual functional activity, retaining their original t cell specificity and nk cytotoxic capacity via marked up regulation of the nk cell receptor, nkg d. pre and clinical studies showed that the optimal cytotoxic effect of cik cells against different malignancies (target cells) is achieved at : e:t ratio, which means high numbers of αβ t-cells that might increase the risk of gvhd. here we produced ciks from αβ tcr depleted cellular products (defined as γδcik) and tested their phenotype expression and in vitro cytotoxic activity against hematological malignancies. fresh apheresis products were processed using the clinimacs depletion reagent, according to manufacturer instructions. target product was cultured with rpmi supplemented with % fcs and ex vivo expanded by scheduled addition of cytokine cocktail that contains ifn-γ ( iu/ml), anti-cd mabs ( ng/ml) and iu/ml il- . the cells were cultured for days. cytotoxic activity of the γδcik was evaluated against various target hematological malignant cell lines (k , reh, jurkat, and u ). after days, the αβ depleted cik cultures resulted in . % γδ t-cells ( folds expansion) compared to . % of γδ t-cells immediately after depletion, and compared to only . % in non-selected cik cells. the percentage of αβ t cells in γδcik cell cultures started from . % (immediately after depletion) to . % compared to . % αβ t cells were found in non-selected cik cells cultures. γδcik cells produced robust cytotoxic activity at a : e:t ratio against reh cells ( . ± . %), jurkat cells ( ± . %); u ( . ± . %) and k ( . ± . %), compared to nonmanipulated cik cell activity against the same targets ( ± . %; . ± . %; . ± . %; . ± . %, respectively). we found higher degranulation capacity of γδcik cells compared to non-selected cik cells against reh ( . ± . % vs . ± . %), jurkat ( . ± . % vs . ± . %), u ( . ± . % vs . ± . %) and k ( . ± . centre de thérapie cellulaire, institut paoli-calmettes; unité de transplantation et de thérapie cellulaire, institut paoli-calmettes; centre d'immunologie de marseille-luminy and laboratoire d'immunomonitoring, institut paoli-calmettes during the past years, the major improvements in the field of allogeneic hematopoietic stem cell transplantation (hsct) (reduced intensity conditioning regimen, high level hla typing, alternative donors, gvhd prophylaxis…) significantly extended the feasibility of this procedure. in contrast, disease recurrence after hsct remains a main issue. thus, many post-hsct prophylactic interventions are under investigation. unmanipulated donor lymphocyte infusion (dli) remains one of the most frequently used post-hsct treatment, but its potential benefit in increasing gvl effect may be counterbalanced by the induction of gvhd. in this setting, the use of adoptive transfer of ex vivo enriched and activated nk cell infusions from the same donor (dli-nk) may induce gvl effect without causing gvhd. we therefore report on a single-center phase clinical trial (nct ) evaluating the safety of ex vivo activated allogeneic nk cells infused between days and after hsct. the aim was to determine the maximum tolerated dose (mtd) of ex vivo highly purified and activated dli-nk after matched related donor hsct. the schedule plan a first phase of + dose escalation method using dose levels ( . e /kg, . e /kg and . e /kg). grade - secondary adverse events according to nctci classification and severe gvhd occurring within days after dli-nk were considered as dose-limiting toxicities (dlt). a second step allowed enrolling patients at the mtd. over a period of . years, patients with various hematological malignancies (aml, all, hl, nhl, mds) were infused with activated nk cells at a median time of days (range: - ) post-hsct. apheresis products were collected from the hsc donor, cd -depleted and cd -selected by immunomagnetic separation using clinimacs. selected nk cells were cultured for days in medium supplemented with % fetal calf serum in the presence of u/ml of il- in air-permeable cell culture bags. after immunomagnetic separation, cd enriched products had a median cd + cell purity of % (range: - ) and viability of % ( - ). after il- activation, the median cd + cell dose was . × e /kg ( . - . ) , with a viability of % ( - ) and a residual cd + cell content of . × /kg ( - . × /kg). all release criteria to be met were fulfilled for the preparations infused : viability %, negative microbiological testing, cd + cell count ⩾ × /kg, and cd + cell content o × /kg. standardized quality controls were employed at all steps of the manufacturing process, adding a level of consistency to the product testing before release. activated-nk cells were well tolerated in all patients, with no occurrence of dlt. thus, mtd was not reached. two patients presented with a moderate chronic gvhd, both of them during cyclosporine a dose reduction. relapse occurred in patients with aml. one patient died from idiopathic pneumoniae, without evidence of relapse, gvhd or infectious disease. with a median follow up of months ( - ), year os was % ( figure ). therefore, infusion of highly purified, activated-nk cells of donor origin as a substitute to standard dli does not induce gvhd nor other side effects after hsct: the demonstration that modulation of nk cell activity can achieve disease control after hsct deserves to be investigated in larger trials. [p ] disclosure of conflict of interest: none. feasibility, safety, rapid production and efficacy of institution-produced cd car staff were trained on site for collection, processing and cryopreservation by regional experts. a total of units were collected and processed as part of the initial validation of the project. ucb units were processed on either axp or sepax systems, and all cryopreserved in bioarchive (an automated, robotic cryopreservation system that can archive up to units). the characteristics of which as well as the post processing data are depicted in table . [p ] we shared a successful story of establishing the first public cord blood bank in jordan. the first units collected showed excellent sterility, viability, collection volume and total nucleated cells. a very good recovery of both nucleated and cd + cells were obtained using axp and sepax cell separation systems. the process of validation of equipments and methodology is complete. we anticipate moving to permeant facility of the cord blood bank in the new expansion in early . we look forward for steady progress in ucb recruitments, hla typing, cryopreservation and adherence to netcord-fact standards as well as participation in international registries. functionally active ifn-gamma secreting cmv pp specific t cell therapy as an alternative for clinically urgent cmv related diseases n kim, y-s nam , k-i im , j-y lim, y-w jeon , y song and s-g cho the catholic university of korea, seoul cytomegalovirus (cmv) related diseases are a serious cause of morbidity and mortality following hematopoietic stem cell transplantation (hsct). it has been reported over the last two decades that cmv-specific cytotoxic lymphocytes (cmv-ctls) can provide long-term cmv-specific immunity without major side effects as an alternative to antiviral drugs. however, its application has been limited by prolonged manufacturing process of cell therapy. in this study, we apply the ifn-γ cytokine capture system (ccs) using the fully automated clinimacs prodigy device to rapidly produce cmv-ctls that may be applicable in clinically urgent cmv-related diseases. five validation runs were performed using apheresis samples from randomly selected cmv-seropositive healthy blood donors. then, clinimacs prodigy automatically performed successive processes including antigen stimulation, anti-ifn-γ labelling, magnetic enrichment, and elution which took~ h. the original apheresis samples consisted of . % ifn-γ secreting cd + t cells in response to cmv pp antigen (cd +ifn-γ+ cells) which were mainly cd ra+cd l+ naive t cells. following ifn-γ enrichment, the target fraction contained . % cd +ifn-γ+ cells with reduction in naive t cells and the selection of cd ra − cd l − and cd ra +cd l − memory t cells. furthermore, extended culture of these isolated cells revealed functional activity including efficient proliferation, sustained antigen-specific ifn-γ secretion and cytotoxicity effect against pp pulsed target cells. therefore, we suggest ifn-γ ccs by clinimacs prodigy as a simple and robust approach to produce cmv-ctls, which may be highly feasible and applicable in clinically urgent cmvrelated diseases. disclosure of conflict of interest: none. in vitro generation of tumor antigen-specific t cells from patient and healthy donor stem cells s bonte , s snauwaert , g goetgeluk , b vandekerckhove and t kerre hematology, ghent university hospital, ghent, belgium and department of clinical chemistry, microbiology and immunology, ghent university, ghent, belgium acute myeloid leukemia remains a therapeutical challenge, as many patients relapse after chemotherapy. allogeneic stem cell transplantation is in most of these patients the only option for cure, but carries a high risk of morbidity and mortality and a suitable donor may be lacking. recently, advances are being made in the field of t cell immunotherapy. the classical protocol, in which peripheral blood lymphocytes (pbl) are transduced with a tumor antigen-specific t cell receptor (tcr), can generate t cells with low and possibly hazardous specificities (due to mispairing of the endogenous and introduced tcr α and β chains). therefore, we have developed a novel protocol in which we generate tumor antigen-specific t cells from cd + hematopoietic stem cells. we have already succeeded in generating large numbers of tumor-specific, naive and resting t cells that only carry the introduced tcr, starting from postnatal thymus and cord blood cd + cells. now we are optimizing this protocol for clinically more relevant samples, such as mobilized peripheral blood from healthy stem cell donors and from patients in remission after chemotherapy and/ or other treatments, and leukapheresis samples from patients at diagnosis. in our protocol, cd + cells were isolated from hla-a + fresh patient and healthy donor samples and cultured on op -dl in the presence of scf, flt l and il- , until t cell commitment. subsequently, the cells were transduced with a tumor antigen-specific tcr and again co-cultured until cd + cd + double positive cells were abundantly present. at that point, agonist peptide was added, which induces maturation. finally, cells were polyclonally expanded on feeder cells. for hla-a negative samples, cd + cd + double positive cells were co-cultured with a cell line (t pulsed with the agonist peptide or a cell line with endogenous expression of the agonist peptide) which can present the agonist peptide to the maturing t cells. using the above protocol, we were able to generate tumor antigen-specific t cells from out of healthy donor samples, / sample from a patient in remission and / samples from patients at diagnosis, who were all hla-a +. for most samples, multiple rounds of agonist peptide stimulation were necessary to obtain further maturation. in contrast, generation of mature t cells from cd + cd + double positive cells in postnatal thymus or cord blood co-cultures, requires only round of agonist peptide stimulation. for the hla-a negative samples, we were able to generate an adequate cd + cd + double positive population from / healthy donor sample, / samples from patients in remission and / sample from a patient at diagnosis. agonist selection using a cell line seems inefficient as cd is not upregulated and cells did not mature to cd + or cd + single positive mature t cells. we are currently co-culturing more samples using our protocol. furthermore, we are investigating the effect of freezing and thawing on the in vitro t cell generation process (cell numbers and efficiency). finally, we are also working on optimizing the protocol for generation of tumor antigen-specific t cells from hla-a negative patient and healthy donor samples. disclosure of conflict of interest: none. increase of polyspecific immune responses against leukemia-associated-antigens (laa) and reduction of regulatory cytotoxic t-cell (ctl) responses against malignant cells play a major role in maintaining remission and prolonging overall survival in patients with hematologic malignancies after allogeneic stem cell transplantation (allo-sct) and/or donor lymphocyte infusions (dli). graft versus leukemia (gvl) effects after allogeneic stem cell transplantation and/or dli are considered to be t cell-mediated. many groups described specific t-cell responses against several leukemia associated antigens (laa) in different hematological malignancies. however, t cell responses after allo-sct and dli are not well characterized. in this study, we analyzed laa-specific t cell responses after allo-sct and dli. to this end, we assessed the frequency and diversity of laa-specific cd + t cells using elispot analysis and tetramer assays in patients ( patients (pts) with acute myeloid leukemia, pts with chronic myeloid leukemia, pts with multiple myeloma and pts with chronic lymphatic leukemia) before and after dli. epitopes derived from prame, npm mut, rhamm, wt- and other laa were tested. moreover, the frequency of regulatory t (treg) cells was measured and the course of cytokine profiles before and after dli was analyzed. these immunological findings were correlated to the clinical course in the respective patients. in elispot and tetramer assays, an increase in frequency and diversity of laaspecific t cells was observed in all patients. importantly, there was a significant increase from a median of to laa-derived t cell epitopes (p = . ) in clinical responders (r) when compared to non-responders (nr). these positive results in r vs nr where s confirmed by tetramer-based flow cytometry assays, where an increase in frequency from . to . % in the r group of laaspecific t cell/all cd + t cells was observed. interestingly, the frequency of tregs in clinical responders decreased significantly from a median . % to . % (p = . ) while the frequency of tregs kept stable over time in non-responding patients. t cell subset analysis did not reveal significant differences before vs after dli administration. in cytokine assays using elisa for the detection of more than cytokines before and after dli we found a shift towards proinflammatory and t cell stimulating cytokines. taken immunologic surveillance of leukemia is employed for the prevention and treatment of relapse post allohsct. to augment this effect donor lymphocytes are infused (dli) in patients at risk. this procedure is associated with a high risk of agvhd and we believe that this route of administration may not make the direct contact between infused cells and blasts the optimal one. to address these issues, we started delivering donor lymphocytes directly to the bone marrow cavity (ib-dli) in patients post allohsct at relapse. three with aml and one with cll, all relapsed post allohsct: allosib: -year-old female aml patient (relapsed years post hsct), -year-old aml male q del (relapsed in years, traumatic brain injury), -year-old male aml flt itd+ received mud hsct (relapsed months) and -year-old cll male, tp del, ebv reactivation (progressed years). two patients ( % and % blasts in the marrow) received ib-dli up-front and two others due to higher proportions of leukemic cells received either flag (aml case) or anti-cd moab (cll case) followed by ib-dli. tcr clonotyping revealed in all patients the presence of the prevailing oligoclonal response on the polyclonal background (characteristic for each individual) which was identified in the marrow and in the blood. however, in two out of patients a distinct oligoclonal peak was seen at first in the marrow and then in the blood. microarray analysis of the transcriptome in the marrows of patients who received three ib-dli courses revealed in all patients preferential use of genes associated with lymphocyte or lymphocyte activation pathways. the patients who responded favorably (cr or pr) clustered with the transcriptomes of normal individuals, but those who failed to respond clustered separately. ib-dli was safe and not associated with gvhd. selective accumulation of cd +cd + as well as the presence of a distinct oligoclonal peak in the marrow suggest that tcrbeta clonotypes may be private to leukemia cells recognition. the response may result in cr or pr and the patients were in a good physical shape during the treatment, which makes it possible to deliver the salvage chemotherapy if required. broad spectrum antibiotics were started. after the orthopedic consultation, the fourth finger was amputated and amputation from the left ankle was recommended. a stem cell transplantation option was offered to patients and their relatives as one of the therapeutic approaches. upon acceptance by patient, μgr/kg of colony stimulating agent was started to patient. when the stem cell was /μl, the stem cells were collected. the obtained stem cell product was injected intra-lesionally (picture b). granulation tissue began to develop from the second week in the foot floor of the patient. after from th week, the necrotic tissue was disappeared and the granulation tissue was appeared. at weeks, % of the lesion healed. at th week, there was normal tissue instead of necrotic tissue on plantar surface at left leg (picture c). this case report suggests that diabetic foot/ulcer can be healed with intralesional application of stem cells in patients with diabetes mellitus. [p ] disclosure of conflict of interest: none. and third (n = ) cell infusions were cryopreserved. cells were infused following conventional chemotherapy (ia, mec, hdac) in cases ( %), chemotherapy plus hypomethylating agents in cases ( %) and hypomethylating agents alone in cases ( azacytidine, decytabine; %). the procedure was well tolerated, with mild and transient ‛haploimmunostorm syndrome' (fever %, rash %, diarrhea %). only the two patients with cmml received corticosteroid. one patient suffered early infusional reaction that was resolved with support treatment. none of the patients showed acute or chronic gvhd or persistent donor engraftment in chimerism tests. four patients had bacterial infections, but no other significant invasive fungal or viral infections were observed. all aml/raeb patients treated achieved complete remission with microhct treatment ( ; %). only one patient, with cmml, died during microhct induction ( %). four patients relapsed at , , and months after the infusion; two of them achieved a second sustained complete remission with another micro-hct from a different donor (one of them had developed anti-hla antibodies). as described in figure , median overall survival is months and overall survival at years is %. microhct is a well tolerated procedure in elderly aml/mds patients who are not candidates to allogeneic hct. infectious complications are insignificant and the remission rates are very encouraging in very high risk cases, with no evidence of gvhd. patients can undergo a second microhct from a different donor. in addition to the experience by ai et al, we have also shown that microhct can be safely administered following a hypomethylant agent course instead of conventional chemotherapy. a large, international, randomized clinical trial will address the safety and efficacy of microhct for elderly aml/ mds patients (nct ). [p ] disclosure of conflict of interest: none. wilms tumor protein (wt ) is expressed in a variety of solid tumors and is found in more than % of patients with acute myeloid leukemia which makes it an attractive target for immunotherapy. previously it was shown that t cells recognizing wt are suitable for adoptive t-cell therapy by increasing the graft versus leukemia effect. however, the efficiency of this therapeutic strategy is still limited due to the low precursor frequency and specificity of wt -specific t cells in the peripheral blood of healthy donors. the ubiquitous antioxidant inducible enzyme heme oxygenase- (ho- ) and its products have immunomodulatory effects, which render it as a potential target for the modification of t-cell responses. recently, we found that inhibition of ho- enzyme activity via tin-mesoporphyrin (snmp) results in activation and proliferation of antiviral t cells from healthy donors. in this study we aimed ( ) to identify the mechanism of ho- modification in the generation of wt -specific t cells and ( ) to develop strategies for the sufficient generation of wt specific t cells from healthy donors to augment effective t-cell immunity in leukemia patients and to broaden the applicability of adoptive t-cell therapy to the majority of patients. the frequency of wt -specific t cells in peripheral blood of healthy donors (n = ) was examined before and after snmp treatment via ifn-γ elispot using the wt -overlapping peptide pool (ppwt ). enrichment efficiency of wt -specific t cells after ho- inhibition was verified in response to ppwt and the hla-a* : -restricted wt peptides (vldfappga, wt ) and (rmfpnapyl, wt ) by ifn-g secretion assay and expression analysis of the t-cell activation marker cd . phenotypic and functional characterization of wt specific t cells were further assessed by multicolor flow cytometry, luminex assays and elisa with respect to t-cell subsets, cytotoxicity, proliferative capacity and secretion of effector molecules. in % of donors we found specific t cells against ppwt by ifn-γ elispot ( spots/ . pbmcs). the frequency of wt -specific t cells in these donors could be increased fivefold after inhibition of the enzymatic activity of ho- via snmp. to assess the possibility that ho- modulation might be clinically applicable in conformity with good manufacturing practice, enrichment of snmp-treated wt -s specific t cells was evaluated based on ifn-g secretion and cd expression. compared to snmp-untreated cells there was a . -fold higher response of ho- modified wt -specific t cells pre-enrichment and an up to -fold higher enrichment efficacy, while snmp treatment did not affected the t-cell functionality. in conclusion, modification of the enzymatic activity of ho- resulted in a more effective generation of functionally active wt -specific t cells suitable for adoptive t-cell therapy. this makes ho- a promising therapeutic target to boost antigen-specific t-cell responses for treatment we recently developed and characterized a standardized and clinical grade human platelet lysate (hpl) that constitutes an advantageous substitute for fetal bovine serum (fbs) for human mesenchymal stem cell (hmsc) expansion required in cell therapy procedures, avoiding xenogenic risks (virological and immunological) and ethical issue. because of the progressive use of pathogen reduced (pr) labile blood components, we evaluated the impact of the novel procedure theraflex uv-platelets for pathogen reduction on hpl quality (growth factors content) and efficacy (as a medium supplement for hmsc expansion). this technology is based on short-wave ultraviolet light (uv-c) and has the main advantage not to need the addition of any photosensitizing additive compounds (that might secondary interfere with hmscs). we applied theraflex uv-platelets procedure on fresh platelet concentrates (pcs) suspended in platelet additive solution and prepared hpl from these treated pcs. we compared the quality of pr-hpl with the corresponding non-pr ones, in terms of growth factor contents. then, we evaluated the efficacy of pr-hpl, in comparison with hpl and msc-screened fbs. we performed large scale culture of hmscs during passages and evaluated the proliferation of cells and the maintenance of their properties: profile of membrane marker expression, clonogenic potential, immunosuppressive properties (inhibition of t-cell proliferation) and potential to differentiate in adipocytes and osteoblasts. we showed no impact on the content in growth factors tested (egf, bfgf, pdgf-ab, vegf and igf) and a significant decrease in tgf-b (− %, n = , p o . ). a large scale culture of hmscs during passages showed that hpl or pr-hpl at % triggered comparable hmsc proliferation than fbs at % plus bfgf (n = ). moreover, after proliferation of hmscs in hpl or pr-hpl containing medium, their profile of membrane marker expression, their clonogenic potential and immunosuppressive properties (inhibition of t-cell proliferation) were maintained, in comparison with hmscs cultured in fbs conditions. the potential to differentiate in adipogenic lineage of hmscs cultured in parallel in the conditions, evaluated using oil red o and nile red stainings and the measurement of triglyceride accumulation, remained quantitatively identical. we also showed that the potential to differentiate in osteoblasts (quantified using alizarin red s and von kossa stainings and alp activity measurement) of hmscs grown in hpl or pr-hpl was not impaired, in comparison with fbs. in conclusion, we demonstrated the feasibility to use uv-c treatment to subsequently obtain pathogen reduced hpl, while preserving its optimal quality and efficacy for hmsc expansion for cell therapy applications. although it is still not used widely in clinical practice. in this paper, we demonstrated a case of ada-scid who received hsct as an adolescent from matched unrelated donor (mudd) after termination of her peg-ada treatment due to severe intractable thrombocytopenia induced by peg-ada. patient showed good engraftment and incremental clinical improvement. her post transplantation course was complicated with multiple complications including: grade i gut gvhd as well as hemorrhagic cystitis (btk related) and ebv infection, additionally, she developed several cns complaints like headache, vomiting and dizziness which were found to be due to increased intracranial pressure with multiple enhancing cerebral lesions found on brain imaging. further investigations for the brain lesions confirmed the diagnosis of malignant diffuse large b cell lymphoma (dlbcl) involving the brain. the lymphoma was highly suggested to be originated from donor cells giving the timing relationship between transplant and establishment of the diagnosis. this lymphoma was successfully treated with full recovery and good final immune reconstitution but with lack of b cell engraftment and need for monthly ivig. we conclude that, peg-ada can rarely induce thrombocytopenia in an autoimmune manner by forming antibodies against platelets and good recovery of thrombocytopenia can be achieved after discontinuation of peg-ada. hsct can be considered as modality of treatment even in older patients with scid due to ada deficiency keeping in mind high possibility of complications including, autoimmunity and malignancy. disclosure of conflict of interest: none. ( ) ( ) ( ) ( ) . based on the pre and post-apheresis cd + cell counts, the collection efficiency of the apheresis amicus device was median . % ( - ) and of the comtec median % ( - ). in mm the apheretic collections were started on median day ( - ), while in lymphoma patients, due to chemotherapy, the day of apheresis start was ( - ). after cryopreservation and thawing, viability ( -aad, bd) was median . % ( - ). with these cell products, up to now we engrafted patients following high-dose chemotherapy ( mm autografted after mel , hl and nhl autografted after beam). engraftment was prompt and stable in all with anc . and . × /l on median day ( - ) and . ( - ), respectively, and with platelet count and × /l on median day ( - ) and . ( - ), respectively. these results are similar to those obtained by most experienced centers in europe and us, and confirm the fact that autologous transplantation may be implemented also in developing countries when appropriate technology and application of standard procedures are employed. with this experience our center is also developing allogeneic transplantation, and the initial results in thalassemia will be reported in a separate abstract. disclosure of conflict of interest: none. extracorporeal photopheresis (ecp) is a safe and effective immunoregulatory therapy for steroid-refractory graft-versushost disease (gvhd) but its mechanism of action is poorly understood. ecp is a non-immunosupressive therapy whose modulating mechanism is thought to result in an increase in t-regs in the patient and in inversion of the cd /cd ratio at the end of treatment. in this study, we evaluated the effect of ecp on t cell response in a cohort of steroid-refractory gvhd patients. from november to november , patients ( con acute gvhd and with chronic gvhd) treated with ecp in our unit, were retrospectively evaluated. patient characteristics are shown in table . we performed an ‛off-line' system ecp using a cell separator (spectra optia, teruno bct) for the cmn apheresis; after -methoxypsoralen was added, the product was photoinactivated in the ultraviolet a irradiator (uvamatic-g , macopharma). ecp procedures were performed for two consecutive days, initially weekly (agvhd), or every two weeks (cgvhd) and afterwards monthly according to clinical response. anthracycline-induced cardiotoxicity (aic) is irreversible, which has limited the use of this anthracycline in cancer chemotherapy. to explore the therapeutic effect and its possible mechanism of bone marrow derived mesenchymal stem cells (bmscs) on cardiac damage induced by anthracyclines in a rat model. study selects sd rats aged - weeks to isolate and culture bmscs, and flow cytometry was used for phenotypic identification of bmscs. female sd rats were first randomly divided into groups: the sham control, bmscs control, . mg/kg daunorubicin (dnr), dnr with bmscs, dnr with dexrazoxane (dzr), dnr with bmscs and dzr. left ventricular (lv) function before, during and after chemotherapy were assessed by echocardiography. at the end of weeks, animals were euthanized and organs were collected in % buffered formalin for histopathology using hematoxylin and eosin staining and immunohistochemical analysis was used to identify the cellular subpopulations that infiltrate the cardiac tissues. after the construction of microrna- (mir- )modified bmscs with lentiviral vector, sd rats were randomly assigned into groups: the normal control, the empty vector control, dnr, dnr with bmscs, dnr with mir- -modified bmscs. the density of new blood vessels of rats in each group was detected by immunohistochemical method. mir- , bcl- , bax and vegf mrna expressions were detected by qrt-pcr. bcl- , bax and vegf, cx , troponin t and bnp protein expressions were detected by western blotting. all procedures performed in studies involving animals were in accordance with the ethical standards of the institutional. an animal model of drug-induced cardiomyopathy was built in the dnr treated rats.lv ejection fraction (lvef) and lv fractional shortening (lvfs) were significantly decreased compared to that of the sham control (p o . ), and the signs of the myocyte injury (myocytolysis, vacuolization and disruption) in paralleled with the inflammatory infiltrates, marked by cd and hla-dr, were observed in the dnr group, while bmscs alone or synergistic with dzr facilitate the anthracycline-induced lv dysfunction returning to the baseline values and the recovery of myocarditis (p o . ). in the mir- -modified bmscs transplant group, mir- expression, cell migration and proliferation ability were higher than that in the bmscs and empty vector groups (p o . ). the cardiac regenerative capacity of bmscs following significant myocardial injury were further enhanced by mir- compared to that of the dnr group and the control groups (all po . ), revealed by the significantly higher density of new blood vessels and upregulation of vegf expressions, during which the pro-apoptotic protein bax were down-regulated and the anti-apoptotic protein bcl- function were upregulated in the mir- overexpression group compared to that with the bmscs, dnr group and the control groups (all po . ). western blotting demonstrated that the expression of c × were significantly decreased, while expressions of troponin t and bnp were significantly increased in the mir- overexpression group in contrast to that with the dnr group (all p o . ). these results showed that bmscs could reverse cardiac damage induced by anthracycline, and the cardioprotective efficacy was further enhanced by mirna- -mediated regulation of apoptosis and angiogenesis. disclosure of conflict of interest: none. effective adoptive t cell therapy against cancer is dependent on long-lived tumor-specific stem cell-like t cells with the ability to self-renew and differentiate into potent effector cells. however, current protocols for ex vivo generation of tumorspecific cd + t cells result in terminally differentiated effector t cells. it was found that minor histocompatability antigen (miha)-specific cd + t cells with an early memory-like phenotype and long-lived memory transcription profile could be expanded from naive precursors using akt-inhibitor viii . importantly, these akt-inhibited tumor-specific cd + t cells showed a superior expansion capacity and anti-tumor effect multiple myeloma bearing mice. for the clinical exploitation of ex vivo generated akt-inhibited tumor-specific cd + t cells, we tested the effect of potential clinical grade akt-inhibitors azd , gdc , gsk , gsk , mk and triciribine in polyclonal stimulations, allogeneic mixed lymphocyte reactions (mlr), and antigen-specific t cell assays. polyclonal stimulation with anti-cd /cd beads on cd naive t cells was used for a first screening of the akt-inhibitors. for all inhibitors, a dose dependent effect on the naiveassociated receptors ccr , cd l and cxcr was observed. this had limited effect on viability, activation and proliferation except for triciribine, which was therefore excluded for further assays. moreover, in the mlr, treatment of naive cd + t cells with remaining akt-inhibitors resulted in a dose dependent effect associated with higher ccr , cd l, cxcr and cd expression. furthermore, the akt-inhibited cd + t cell products showed a - fold increased expansion capacity upon restimulation in vitro. when expanding miha-specific cd + t cells from the naive repertoire in the presence of one of the akt-inhibitors, the miha-specific cd + t cells showed a more early memory phenotype compared to controls. this was displayed in higher levels of the naive-associated receptor cd l ( figure ). in addition, these miha-specific cd + t cells were shown to be functional, as antigen-specific restimultation resulted in degranulation (cd a) and ifn-γ production. based on this ifn-γ production, the akt-inhibited antigenspecific cd + t cells can be selected using the cytokine capture assay (miltenyi, for enriched infusion in patients suffering from hematological malignancies. using aktinhibition in the generation of tumor-reactive t cells results in a more early memory tumor-specific cd + t cell product. this adoptive immunotherapy product retains superior proliferation capacity upon infusion, and its potential selfrenewal capacity could result in a long-term anti-tumor effect in patients suffering from a hematological malignancy. chimeric antigen receptors (cars) are composed of an extracellular domain-derived from a tumour-reactive monoclonal antibody, linked to one or more signalling endodomains. in early clinical trials, cd car-t cells have demonstrated impressive anti-tumour activity against different b-cell malignancies, including chronic lymphocytic leukaemia, acute lymphoblastic leukaemia (all) and non-hodgkin lymphoma. conventional alpha-beta car-t cells are however hla-restricted and could cause graft-versus-host disease (gvhd) when used across major mismatches, as expected in the highly anticipated setting of off-the-shelf car-t cells from third-party donors. besides being non-hla restricted, gammadelta t cells possess intrinsic anti-tumour reactivity, making them attractive effectors for next-generation car-t cell therapies. so far, however, attempts at exploiting gammadelta t cells in patients have been largely disappointing, possibly because of sub-optimal ex vivo culture conditions. the aim of our study was to optimise the generation of gammadelta car-t cells and to test their anti-tumour potency both in vitro and in vivo. starting from peripheral blood mononuclear cells of healthy donors, we stimulated gamma-delta t cells with zoledronate and il- /il- , and transduced them with retroviral vectors encoding for cd cars carrying either cd .z or - bb.z signalling endodomains. we assessed antitumour activity in vitro by measuring killing, secondary expansion and cytokine production after co-culturing gamma-delta car-t cells with different cd + all cell lines, and in vivo in nsg previously engrafted with a b-all semi-cell line. although allogeneic hematopoietic stem cell transplantation (allosct) is a curative option to treat hematologic malignancies, disease recurrence remains a concern in the setting of high risk diseases. thus, post allosct therapeutic strategies are needed to treat and/or prevent disease progression. in this setting, donor lymphocytes infusion (dli) is an option as post allosct immunotherapy aiming to enhance graft versus leukemia (gvl) effect. although dli may induce persistent remission, graft versus host disease (gvhd) is a potential complication following dli. because of the suspected higher incidence of gvhd in the presence of hla mismatches, few series focused on dli following haploidentical stem cell transplantation (haplosct) so far. we therefore report our experience of dli following haplosct using post-transplantation cyclophosphamide (pt-cy) platform. we included in this single center study all consecutive adult patients with hematological malignancies who received dli after haplosct with pt-cy as part of gvhd prophylaxis from to (n = ). conditioning regimens were non-myeloablative (low dose tbi-based) or with reduced toxicity (various dose of busulfan according to disease and patient characteristics). ciclosporine a and mycophenolate mofetil were given as additional gvhd prophylaxis in all cases. dli were given at escalating doses, expressed as cd +cells/kg, without gvhd prophylaxis, and ranged from × our study suggests that dli following haplosct with pt-cy is feasible. gvhd is frequent but with a relatively low incidence of severe forms. no response rate was achieved in the context of hematological relapse, underlining that preemptive or prophylactic strategy might be preferred. indeed, the overall good outcome in patients receiving prophylactic dli is promising taking into account the poor prognostic of the diseases indicated for alternative donor transplantation. further prospective studies are needed in specific disease settings to assess the benefit for using such post allohsct immune-intervention. [p ] disclosure of conflict of interest: none. dual specific cytokine-induced killer cell therapy as a treatment option for life-threatening ptld-a case report of the frankfurt experience l-m pfeffermann infection with epstein-barr virus (ebv) is a frequent complication after allogeneic hematopoietic stem cell transplantation (hsct) and besides relapse remains a significant cause of morbidity. prolonged immunosuppression or delayed t-cell recovery may favor ebv reactivation after transplantation, which under these circumstances can lead to life-threatening lymphoproliferative disease (ptld). consensus is lacking on the optimal treatment of ptld. adoptive immunotherapies with both anti-tumor capacity and restored virus-specific cellular immunity may represent optimal treatment options especially when considered in the context of ptld. in this case report we applied in vitro activated t-cells namely cytokineinduced killer (cik) cells with dual specific cytotoxic capacities transferring both anti-cancer potential and donor t-cell memory against ebv infection for the treatment of ebvassociated ptld which progressed to highly proliferative large b cell lymphoma during delayed t-cell recovery after allogeneic hsct. the reported patient had received an allogeneic hsct for secondary myelodysplastic syndrome following acute myeloid leukemia, and due to delayed t-cell recovery had developed ebv-related ptld two months after transplantation. treatment with rituximab, conventional ebvspecific t-cells and wildtype cik cells failed, therefore the patient was offered ebv-specific cik cells on a compassionate use basis. ebv-specific cik cells were generated from peripheral blood mononuclear donor cells. cells were activated and expanded in the presence of ifn-γ, il- , anti-cd antibody and il- . on day and of culture an ebv peptide pool was added for additional priming. follow-up analysis included in vitro and in vivo monitoring of ebv-specific cik cells. with above mentioned protocol we were able to generate cik cells containing × cd + ebv-specific t-cells/kg body weight of the patient. infusion of ebv-specific cik cells resulted in rapid clearance of plasma ebv dna level and sustained disappearance of large (vol. cm ) ptld-malignant lymphoma. during one-year follow-up analysis we were able to detect ebv-specific cik cells (cd + and cd + ) in vivo by flow cytometry using specific mhcdextramers. facs-monitoring of the patient´s blood revealed besides cd bright t-cells also an increasing cd dim t-cell population with a remarkable percentage of t emra cells within this compartment (up to %) indicating virus-specific t-cells. no cytokine release syndrome appeared after ebv-specific cik cell treatment, but cytokine secretion patterns, analyzing serum of the patient, reflected cytotoxic and anti-virus capacity provided by this treatment. cytotoxic potential, as well as t h cell differentiation and function offered by ebvspecific cik cell treatment were further confirmed by in vitro analysis. ebv-specific cik cells revealed an . s hematologic disease. the global survival ratio in the follow-up was . % (with . %, . % and . % survivals in , and months, respectively). the variables significantly associated with greater survival were: type of gvhd (cgvhd), number of affected organs (an organ had to be moderately or severely affected to be included in this category) and steroid dependence as the main reason to initiate ecp (see figure ). there was a trend towards significance for the degree of gvhd and cutaneous involvement to be factors associated to enhanced survival ratios. extracorporeal photopheresis is a safe treatment option for patients with gvhd, generating a response and decreasing immunosuppression in an important percentage of them. the presence of cgvhd rather than agvhd, a lower severity degree of the condition, having a lower number of affected organs, skin as main affected organ and steroid dependence as the reason to start the ecp treatment were all factors associated with greater survival in our sample. disclosure of conflict of interest: none. tx) . we report the case of a patient refractory to chemotherapy treated with ibrutinib as debulking therapy before allo-tx. in june , a years old woman was diagnosed with mcl. the staging performed by whole body ct scan, colonoscopy, egds and bone marrow (bm) biopsy was conclusive for stage iva with bulky lymph node over and below the diaphragm, bm, enteric and peripheral blood (pb) localization. the planned treatment included cycles of r-chop, cycle of high dose (hd) cy, cycles of hd arac and autologous sct. after completion of hd cy, the restaging showed progressive disease, with a thyroid involvement and histologic switch in a blastoid variant. disease continued to progress even after cycles hd arac, so we tried to control the disease with r-bendamustine ( mg/mq on days - of -d cycle), but after the first cycle the neck circumference increased. we shift to lenalidomide ( mg on days - of -d cycle) without any response after two cycles. we excluded patient from autologous sct programme because of chemo-refractoriness and we searched matched unrelated donor because no hla identical sibling was available. we started a therapy with ibrutinib ( mg/die on days - of d cycle). after the first cycle we observed a rapid response with decrease of neck size and the disappearance of superficial lymphnode; we performed cycles of ibrutinib, and we reached a good partial remission with lymphnode of max cm, and a bm and pb involvement of %. meantime an unrelated donor with / hla matching was identified, so in december we performed allo-tx with reduced-intensity conditioning (thiotepa mg/kg-fludarabine mg/m -melphalan mg/m ) and cyclosporine and short term methotrexate as gvhd prophylaxis. engraftment was at day + . in the first days after allo tx she experienced a clostridium enteritis, transient cmv reactivation and acute gastrointestinal gvhd on day + with rapid response to steroid therapy. main complication happened on day , when sudden fever and stupor, progressive to coma, occurred; subsequently pneumococcal encephalitis was diagnosed, with positive csf microbiological exam and two signal alteration in the right cerebral hemisphere at mri. the patient was treated with ampicillin and ceftriaxone with a favourable outcome. the mcl revaluations performed at , and months showed complete remission with disappearance of all pathological lymph node and pb involvement. currently the patient is at year post asct, she is enrolled in a rehabilitation program and mcl is in complete remission. our experience seems to indicate that ibrutinib is safe and can be used as bridge to allo-tx therapy in refractory mcl. we will investigate side effects of this platform of therapy, and, given the early occurrence of pneumococcal infection, we will consider to perform capsulated bacteria vaccination before allo-tx. disclosure of conflict of interest: none. [p ] pt was diagnosed with c-all in and received a mud-hsct (tbi gy, cyclosphosphamide) in / due to persistend mrd. following early rel / , cycles of blina led to mrd+ cr, for which a nd hsct from a haploidentical family donor (busulfan, thiotepa, fludarabine) was performed in / . molcr lasted months and rel was treated with cycles of weekly io followed by one dli ( x - cd + cells/kg), resulting in mrd+ cr and complete donor chimerism. five weeks after the last io cycle, the pt was admitted with ascites, hyperbilirubinemia and reduced general condition. vod was suspected, but diagnostic paracentesis revealed malignant ascites demonstrating fatal progressive disease. conclusion. our data suggest that the sequential use of io and dli is feasible even for heavily pretreated patients with r/r all after hsct and can induce molecular remissions. we observed an unusual case of late onset, severe vod responding to defibrotide and one all relapse manifesting itself with ascites in our patients. we therefore suggest close monitoring of liver function tests in the setting of this therapy and extensive diagnostic work-up for any developing liver abnormalities or ascites. disclosure of conflict of interest: ng: advisory board (pfizer, amgen); research support (amgen); gb: honoraria (amgen). [p ] p while allogeneic hematopoietic stem cell transplantation from matched related and unrelated donors has become a standard of care treatment for patients with hematological malignancies, transplantations from mismatched or haploidentical family donors remain challenging. currently t-replete and t-deplete transplantation strategies are applied aiming to improve the outcome after haploidentical transplantation. despite high rates of relapse many centers regard post-transplant cyclophosphamide, a t-replete strategy, as a standard of care approach. we have developed a t-depleted transplant approach where donor lymphocytes selectively depleted of alloreactive t-cells (atir ) using th , arhodamide-like dye, are infused after cd -selected haploidentical hsct, to overcome the challenges of infectious complications, gvhd and relapse. in phase i (cr-air- ) we have demonstrated safe infusion of these lymphocytes at doses up to × viable t-cells/kg. recently, we reported a promising -year grfs was % from a phase ii trial (ash ),that is awaiting final results soon. here, we introduce a randomized, multicenter phase study (cr-air- ), where patients with acute myeloid leukemia (aml), acute lymphoblastic leukemia (all) or myelodysplastic syndrome (mds) are planned to undergo a haploidentical hsct with either a t-cell depleted graft and adjunctive treatment with atir , or with a t-cell repleted graft and use of posttransplant cyclophosphamide. inclusion and exclusion criteria are listed in table . all patients will undergo myeloablative conditioning consisting of either tbi ( gy) or melphalan/ busulfan, in combination with thiotepa and fludarabine. patients in the atir study group will receive atg ( . mg/ kg once daily for days) during conditioning and atir infusion at a dose of × viable t-cells/kg is given between and days after the hsct. patients in the ptcy group will receive cyclophosphamide ( /mg/kg) on day and (or ) with subsequent use of immune suppression up to months post-hsct. the primary endpoint of the study is gvhd-free, relapse-free survival (grfs). grfs is defined as time from randomization until grade iii/iv acute graft-versus-host disease (gvhd), chronic gvhd requiring systemic immunosuppressive treatment, disease relapse, or death, whichever occurs first. this endpoint captures both safety and efficacy. additional secondary endpoints are overall survival (os), progression-free survival (pfs), relapse-related mortality (rrm) and transplantrelated mortality (trm). patients are planned to be randomized in study centers in europe and north america. a number of - sites are planned to participate in the study. enrolment is expected to continue until mid- with initial results being available first half . results of this study will determine which transplant regimen provides most clinical benefit in haploidentical donor transplantation, with the promise of an effective regimen without the use of post-transplant immune suppression. disclosure of conflict of interest: jr is an employee of kiadis pharma. multipotent mesenchymal stromal cells (mscs) are used for prevention and treatment of graft versus host disease after allogeneic hematopoietic stem cells transplantation due to their immunomodulatory properties. mscs fate in vivo after infusion is unknown. the aim of this study was to analyze the changes in mscs and allogeneic lymphocytes properties when co-cultured in vitro to simulate their interactions in vivo. the bone marrow from donors ( male and female aged - years, median years) was used. mscs were cocultured with allogeneic lymphocytes in a ratio of about : for days and their basic properties were analyzed over time. lymphocytes were activated by adding to the culture medium mg/ml of pha (pha-lymphocytes). some mscs were treated for h with u/ml ifnγ (γmscs). determination of gene expression levels was performed by reverse transcription polymerase chain reaction in real time (modification of the taq-man) and of antigen expression on mscs and lymphocytes by flow cytometry. significant reduction in the proportion of viable cells was observed in mscs co-cultured with pha-lymphocytes. in γmsc co-cultured with pha-lymphocytes no reduction in the proportion of living cells was revealed. this indicates the sensitization of mscs by ifnγ to factors secreted by pha-lymphocytes. in mscs co-cultured with pha-lymphocytes and lymphocytes mean fluorescent signal intensity level (mfi) of cd gradually decreased. ifnγ treatment and co-cultivation with lymphocytes led to significant increase of hla-dr mfi on mscs. co-cultivation with lymphocytes increase the hla-dr mfi on mscs much stronger than ifnγ treatment. relative expression level (rel) of ido gene increased dramatically in both mscs and γmscs when co-cultured with lymphocytes. at a day in γmscs rel of ido increased fold, and then gradually declined. in mscs cocultured with lymphocytes il- rel increased almost -fold and then decreased -fold at the fourth day. the csf rel in γmscs showed twofold increases, upon incubation mscs and γmscs with lymphocytes csf rel increased fourfold and sevenfold, respectively. co-culture of msc and γmscs with lymphocytes led to decrease in the proportion of cd , cd , cd , hla-dr, and pd- positive cells (both cd and cd ) after one day, compared with pha-lymphocytes without mscs. proportion of cd +, hla-dr+ and pd- + cells also decreased after days of co-culturing with msc or γmscs (compared with pha-lymphocytes without mscs), but anyway number of activated cells increased . - folds compared with first day. number of cd + lymphocytes after days of co-culturing with mscs or γmscs did not vary significantly from control and decreased in comparison with first day. main inhibition of activated lymphocytes by co-culturing with mscs occurs during the first day of their interaction, and then the inhibition became less effective, moreover in mscs decreased the rel of the main immunomodulating factors, and most of them were eliminated. mscs treatment with ifnγ resulted in improved survival and resistance of these cells to lymphocytes action. the results indicate that the effect of mscs injected intravenously to patients is limited to several days. disclosure of conflict of interest: none. autologous adipose-derived mesenchymal stem cells (admscs) embedded in platelet-rich fibrin (prf) promote healing in different types of wounds. by avoiding the needlerelated complications, prf-embedded autologous admscs graft provides a new effective stem cell-based therapeutic strategy for wound healing. adult male (age ⩽ yo) were equally divided (n = per group) into group (prf only), and group (prf+admscs). regular dressing (without any agent) was used for both groups with a frequency of changing every days. rpf with or without admscs was patched on the wound (maximum surface area cm ). all patients were followed up until complete healing. a complete healing was noted in both groups; however, the healing in group was very slow (after weeks), compared to a quicker one in group (after weeks). control of the moisture was very well noted in group , less in group . group showed a lot of exudates on the wound; less exudate in group were noted. infections were absent. both groups had a colonized wound. signs of inflammation were very well noted in group ; no signs of inflammation in group . admscs embedded in prf offered rapid wound healing responses then prf alone. keywords: mesenchymal stem cells, platelet-rich fibrin, engineered tissue wound healing disclosure of conflict of interest: none. stem cell source p additionally cryopreserved g-csf primed pbsc can substitute the second transplantation for the patients with acute leukemia who lately relapsed after hematopoietic stem cell transplantation y lee , j moon , ih lee and s sohn department of hematology, kyungpook national university hospital; department of hematology,kyungpook national university hospital and kyungpook national university hospital although allogeneic hematopoietic cell transplantation (allo-hct) is a potentially curative therapy for acute leukemia, survival outcomes of the patients relapsed after transplantation remains poor with high early mortality and a small percentage of second remission. this study evaluated the efficacy of dli using g-csf primed pbsc additionally cryopreserved for the patients who relapsed after allo-hct. we retrospectively reviewed the medical records of the patients who received allo-hct for acute myelogenous leukemia (aml), myelodysplastic syndrome (mds), and acute lymphoblastic leukemia (all) between and in kyungpook national university hospital. among the patients who had relapsed after allo-hct, the patients received dli using the additionally harvested cells. at the time of harvest for the first hct, collecting targeted pbscs (greater than × /kg cd + cells) allowed us to cryopreserve surplus pbscs, including cd + cells with dimethylsulfoxide in a nitrogen tank. then, we analyzed the efficacy of dli for the patients who were classified into early relapse or late relapse group by the median time of relapse after transplant. the median age at transplant was . years (range - years) and male was patients ( . %). primary diseases for allo-hct were aml/mds (n = , %) and all (n = , %). one hundred forty three patients ( . %) were in cr (complete remission), ( %) in further cr, and ( . %) in relapsed and refractory status. one hundred seventy one patients ( . %) received myeloablative conditioning regimen. the median dose of cd + cell was . × /kg (range: ~ . × /kg). almost % of patients achieved the neutrophil engraftment with a median time of days (range: - days). the -year overall survival (os), relapse free survival (rfs), non-relapse mortality (nrm) and graft-versus-host disease (gvhd)-free/relapse-free survival (grfs) since hct was . ± . %, . ± . %, . ± . %, and . ± . %, respectively. there was no significant difference according to s the infused cd + cell dose (lower o × /kg vs higher ⩾ × /kg). the incidence of chronic gvhd was more frequent in higher cd + group ( . % vs . %, p = . ). median time from hct to relapse was days (range: ~ days). after relapse, patients ( . %) were treated with salvage chemotherapy, patients ( . %) with second allo-hct, and patients ( . %) with dli. the median number of cd +t cell was . × /kg (range: . ~ . × /kg). fourteen patients ( . %) achieved dli induced cr, patients progressed, and patients were not evaluable for response. dli induced acute gvhd was observed in patients ( . %) and chronic gvhd developed in patients ( . %). in late relapse group, the -year os since post-transplant relapse was significantly higher in dli group than non-dli group (figure , . ± . % and . ± . %, p = . ) but, early relapse group had no difference. the patients treated with dli showed significantly survival benefit in late relapse group (median days vs days, p = . ). the incidence of dli-induced gvhd does not differ between two groups. dli for the patients who lately relapsed after allo-hct can be a feasible and an effective option in terms of response, donor convenience and it's cost. in the late relapse group, g-csf primed dli may replace second transplantation. disclosure of conflict of interest: none. cord blood transplantation- years of experience c alves , f amado , f bordalo , s ferreira , s lopes , c pinho , t rodrigues , l antunes and s roncon serviço de imuno-hemoterapia; serviço de terapia celular and registo oncológico do norte, instituto português de oncologia do porto francisco gentil, epe allogeneic haematopoietic stem cell transplantation (hsct) is a well-established treatment for patients with malignant and non-malignant haematological disorders. cord blood transplantation (cbt) has extended the availability of hsct to patients that would not otherwise be eligible for this curative approach because of the lack of human leucocyte antigen (hla) identical donor. the aim of this retrospective study was to analyse and characterize years of cbt activity in our institution ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) . we examined patient electronic files and created a database in excel to register cord blood unit (cbu) parameters and patient characteristics. after thawing, cbu was washed/diluted with validated procedures; the cellular content was evaluated by immunophenotyping and followed ishage recommendations; sterility was assessed by bacterial/fungal cultures, viability by trypan blue exclusion assay and functionality by clonogenic capacity. the transfusion of blood products after transplant was quantified and the hematological recovery (hr) established using cibmtr criteria. correlation between continuous variables was assessed with spearman coefficient. overall survival (os) was determined according to cellular content and hla-disparity by the kaplan-meier estimator. survival between groups was compared using the log-rank test. a total of cbu were administered to patients (table ) : / female, the main diagnosis was acute leukaemia ( / ). a sibling cbu was used for patients; the unrelated were imported from europe ( %), usa ( %) and oceania ( %). hla-matching was / , / , / and / for , , and patients, respectively; % were abo-identical. after thawing, % were washed and presented no microbial growth. the majority of patients were submitted to a bussulfan-based myeloablative conditioning regimen; graft versus host disease prophylaxis was performed with a calmodulin inhibitor+mycophenolate mofetil. complete and mixed chimerism was verified in % and % of patients; % had graft failure; the rest were unknown results ( %). at the moment, we reported patients alive ( in complete remission, with evidence of disease relapse) and dead at a median of ( . - ) months after cbt; the most frequent cause ( %) was recurrence of the initial clinical condition. the correlation between nucleated cells (nc) and cd + cells per kg/hr (p = . ; p = . ) and number of cd + cells per kg/os (p = . ) was not statistically significant. however, the engraftment and os was associated with hla-mismatch (p = . ; p = . ) and os was related to nc per kg (p = . ). our clinical results suggest that despite increased hla disparity, ucb offers promising results. ucbt is feasible in patients when the unit contains a high number of cells. there are several strategies for the future, related to cbu expansion and homing techniques, nurturing procedures, selection of optimal cbu unit and enhancement of immune recovery, in order to improve the application of cbu. s received sirolimus and mmf. median time to neutrophil and platelet engraftment was days ( - ) and days ( - ), respectively. one patient died from parainfluenza pneumonia (d ), one patient from ptld (d ), one patient from late pulmonary vod (d ), and one patients from relapse (d ). with a median follow up for survivors of months, one year survival is %. three patients had grade - gvhd and none of the survivors have chronic gvhd. though unrelated donor chimerism was dominant early after transplant and contributed to early count recovery, definitive engraftment was dominated by ucb chimerism in all but one patient. conclusion: among older adult patients with hematological malignancies,~ % lack haplo-identical relatives. for these patients, double or single ucb transplant is challenging because of delayed engraftment. cd selected partially matched grafts from unrelated donors hasten hematopoietic recovery and are over time outcompeted by ucb grafts which provide robust hematopoiesis with low risk of chronic gvhd. the combination of mismatched unrelated hematopoietic progenitors and ucb grafts provides an attractive alternative for older patients lacking hla-idental donors or haploidentical relatives. in planned trials, mismatched donors may be selected based on kir type to further enhance gvl effects. disclosure of conflict of interest: partially supported by miltenyi biotec. haploidentical stem cell transplantation (haplo-sct) is an attractive option for patients who do not have an hlamatched donor. historically it has been associated with high rates of graft rejection, relapse and low incidence of graft versus host disease (gvhd). to decrease these issues we have considered the use of primed bone marrow as stem cell source, early withdrawal of immunosuppressive therapy and the use of donor lymphocytes infusions (dli) in haplo-sct with high-dose post-transplantation cyclophosphamide (ptcy) as main gvhd prophylaxis. to analyse our incidence of acute and chronic gvhd and overall survival (os) in patients with haplo-sct with short course of inmunosupressive therapy. we retrospectively analyzed a cohort of patients who underwent haplo-sct with primed bone marrow as stem cell source, between years and in our centre. gvhd prophylaxis consisted in ptcy ( mgr/kg on days + and + ) and tacrolimus plus mycophenolate (mmf) from day + as recommended by baltimore group. mmf was stopped on day + . tacrolimus was tapered off from day + with withdrawal on day + in patients without gvhd or with active disease. dli were considered if mixed chimerism, relapse or disease progression appeared. the characteristics of the patients are shown in table . results: there was no primary graft failure. eight of patients ( . %) developed agvhd (grade ii-iv) and it was severe (grade iii-iv) in patients ( . %). cutaneous agvhd location was the most common presentation ( patients ( %)) and it was associated with intestinal gvhd in patients. twenty two patients were evaluable for cgvhd. thirteen patients ( %) developed chronic gvhd that was mild, moderate and severe in: ( . %), ( . %) and ( %) patients, respectively. the median time of onset cgvhd was months (range: - ) and it was related with previous withdrawal of the immunosuppression in ( . %) patients, tapered off immunosuppression in ( . %) patients and dli in ( %) patients. systemic treatment was required in / patients but only patients were treated with high doses of steroids ( mg/kg/day). the median days of is therapy in patients who developed gvhd was days (range: - ). dli were used in ( %) patients because of: relapse/disease progression in ( . %) and secondary graft failure in ( . %). two patients achieved complete remission and patients developed cghvd. the median number of dli per patients were ( - ) with a median cd +cell of × /kg (range: × - × /kg). with a median follow-up of months (range: - ), the estimated os at and years after haplo-sct were % and %, respectively. at the moment of this study patients ( . %) were alive, patients in complete remission, in partial response and in progression. eleven ( . %) patients died due to: disease ( ), infections ( ), pleuropericarditis ( ), hepatic veno-occlusive disease ( ) and refractory gvhd. five patients ( . %) are without is therapy and without gvhd symptoms. in our experience, early withdrawal of immunosuppression following haplo-sct with primed bone marrow and posttransplantation cyclophosphamide facilitates the development of chronic gvhd and can decrease the relapses in patients with high-risk hematological malignancies. it is necessary more follow up and more studies to confirm this preliminary results. [p ] disclosure of conflict of interest: none. s myeloid malignancy (n = except patient with saa) received fludarabine (flu)/busulfan ± tbi gy, while lymphoid malignancies (n = ) received flu/tbi gy or cy/ tbi gy. all patients received g-csf-mobilized t-cell replete pbsc from a haplo donor. gvhd prophylaxis was ptcy mg/ kg on day + /+ , tacrolimus (d+ to + ), and mycophenolate (d+ to ). the median duration of follow up of surviving patients is months. median age was ( - ) years, patients ( %) were male, ( %) were african american, and patients ( %) had comorbidity index (hct-ci) ⩾ . all patients had hematological malignancy (except patient with saa) including patients ( %) not in cr. disease risk index was high/very high in ( %) and intermediate in ( %). on the day of transplant, patients ( %) did not receive steroid premedication ( = no-steroid group), while patients ( %) received mg of methylprednisolone minutes prior to pbsc product infusion ( = yes-steroid group). all the following outcomes are described in the ‛no-steroid' vs ‛yessteroid' group, respectively. cumulative rate (ci) of anc engraftment (⩾ /μl) on day + was % ( % ci - %) and % ( % ci - ) (p = . ). ci of platelet engraftment (⩾ /μl) on day + was % ( % ci - %) and % ( % ci - %) (p = . ). primary engraftment failure was observed in patients; in yes-steroid and in no-steroid. no primary engraftment failure was observed with myeloablative tbi ( - gy) (n = ). ci of agvhd gii-iv (day+ ) was % ( % ci: - %) and % ( % ci: - %) (p = . ). ci cgvhd ( year) was % ( % ci - %) and % ( % ci - %) (p = . ). ci of relapse ( year) was % ( % ci - %) and % ( % ci - %) (p = . ). ci of non-relapse mortality (nrm) ( year) was % ( % ci - %) and % ( % ci - %) (p = . ). post-infusion noninfectious fever (d to + ) was observed in / ( %) and / patients ( %). median tmax was f and f (p = . ). only patient in the no-steroid group developed life-threatening cytokine release syndrome and survived. no difference of viral reactivation was noted between groups. cmv reactivation occurred in ( %) and patients ( %), bk reactivation in % (in both groups), hhv in % and %, ebv in % and %. the -month overall survival was % ( % ci - %) and % ( % ci - %) (p = . ). the -month disease-free survival was % ( % ci - %) and % ( - %) (p = . ). t-cell replete haplo pbsc transplant is effective therapy for patients with high-risk hematological malignancies. high-dose steroid premedication with pbsc infusion neither influences transplant outcome nor prevents post-infusion febrile reaction. disclosure of conflict of interest: as discloses grant support (american porphyria foundation), consultation (medpace inc), research support (astellas and fate therapeutics), honoraria (alxion, and spectrum), and royalty for licensing of intellectual property (incysus biomedical). intrabone transplant of unwashed cb in hematological malignancies: engraftment and safety f giglio , s marktel , r greco , m morelli , mt lupo-stanghellini , e xue , l lazzari , m marcatti , m zambelli , c parisi , r milani , s piemontese , a assanelli , c corti , m bernardi , f ciceri and j peccatori unit of hematology and bone marrow transplantation, irccs san raffaele scientific institute, milano, italy and immunohematology and transfusion medicine unit, irccs san raffaele scientific institute, milano, italy cord blood transplant (cbt) in adult patients (pts) is limited by the risk of graft failure or delayed engraftment due to low cell counts. to improve the capacity and speed to engraft, intrabone (ib) cbt technique has been investigated, showing high rate of engraftment and low acute gvhd, also when compared with double cb transplant. cb units washing procedure has been suggested to remove dmso toxic potential effect. we report our experience in adult pts with hematological malignancies receiving ib unwashed cb in an attempt to reduce the loss of progenitor cells and the risks associated with cell-washing procedure. between and we performed allogeneic hematopoietic stem cell transplant (hsct) using unwashed cbu as a source and infusing them ib. all pts were adult and suffering from hematological malignancies. this population was characterized by very high-risk and advanced phase disease. all pts received a cb hsct because of unavailability of sibling or matched unrelated donors. eleven pts received a treosulfanbased myeloablative conditioning regimen and a sirolimusbased ghvd prophylaxis; four pts received busulfan-based myeloablative conditioning and a cyclosporine-based ghvd prophylaxis. cb units were thawed and diluted with albumindextran solution immediately before the transplant. this ‛nowash' dilution was implemented to reduce product manipulation that may results in cell loss. furthermore, graft manipulation risks potential contamination, requires increased technologist time, and delays time to infusion. the ib infusion was performed under local anesthesia and with short conscious sedation, at bedsite in the bmt ward. the infusion was preformed monolaterally or bilaterally according to the volume to be infused. starting from a % dmso concentration in the cb units before the dilution, the graft products contained a median of . % dmso (range: . - . ) at ib-hsct. the median cd + cells infused were . × /kg b.w. (range: . - . ). the median mono-nucleated cells were . × /kg b.w. (range: . - . ). the median cd + t-cells were . × /kg b.w. (range: - . ). the median infused volume was ml (range: - ). no procedure-related adverse events were observed, nor related to the ib technique, neither to the sedation. of the transplanted pts, were evaluable for engraftment ( patient rejected the graft and patients died before day + because of severe infections). all achieved anc . × /l after a median of days (range: - ) and achieved plt × /l at a median of days (range: - ). three patients developed grade iii-iv acute gvhd grade. according to extreme heterogeneity of the population no correlations with relapse incidence and diseasefree survival could be evaluated. ib infusion of unwashed cb is feasible, safe, easy to perform. no adverse events related to the procedure were documented. no dmso toxicity was documented. engraftment was obtained in all evaluable pts. our data confirm that direct ib cbt overcomes the problem of graft failure even when low numbers of cb cells were transplanted, thus leading to the possibility of using of this technique in a large number of adult pts, for whom this approach represents the sole possibility of long-term survival. the ‛no wash' cb dilution can also help the implementation of ib transplant thanks to the easier graft manipulation. [p ] disclosure of conflict of interest: none. lower incidence of cgvhd after cord blood transplantation for hematological malignancies in comparison with hematopoietic stem cell transplantation from other donors: years' experience in a single institute m yoshino, m obiki, m osakie, s ikeno, t sato, m nasashima, y kagaya, n kawashima, t morishita, y ozawa and k miyamura department of hematology, japanese red cross nagoya first hospital the outcome of cord blood transplantation (cbt) for hematologic malignancy was investigated. however the incidence of gvhd is not accurately known. the goal of this study was to compare the outcome of cbt with allogenic hematopoietic stem cell transplantation (allo-hsct) from other sources, mainly unrelated bone marrow (urbm). patients' characteristics: patients who underwent allo-hsct, between and in our hospital were retrospectively analyzed. donor sources were cord blood cell (n = ), urbm (n = ), hla matched sibling bone marrow (sibling bm) (n = ), and hla matched sibling peripheral blood stem cell (sibling pbsc) (n = ). in cbt, the median age was . ( - ), and the diagnosis included aml ( ), all ( ), mds ( ), cml ( ) and other ( ). the disease risk was good in and poor in . disease risk was slightly higher in comparison with other sources. prophylaxis of acute gvhd was tacrolimus, short-term methotrexate ( ), cyclosporine, short-term methotrexate ( ) and others ( ) . the -year overall survival (os) rate after cbt ( cbt ( - . %, engraftment failure . %, acute gvhd . %), relapse . % and other . %. in cbt cases, engraftment failure after allo-sct was observed in cases ( . %) which is higher than that among urbmt ( . %), out of . cbt cases underwent the second allo-hsct and patients achieved engraftment and patients were alive at days after allo-hsct. of them survived at years after allo-hsct. our results suggest that the outcome of cbt has improved in recent years. moreover, cbt has an advantage in the least cumulative incidence of acute/chronic gvhd. cbt may well create the best outcome in the future. disclosure of conflict of interest: none. chronic active epstein-barr virus (ebv) infection is a major type of ebv-associated t/nk-cell lymphoproliferative disorders (lpd) in childhood. however, young adults rarely develop chronic active ebv infection (caebv), and shows more aggressive features than that of childhood. umbilical cord blood transplant (ucbt) is a possible treatment option for caebv patients who have no hla-matched donor, but there is little information available about the efficacy and safety of ucbt for adult patient with caebv. we analyzed six adult patients with caebv who underwent a single-unit ucbt between and at our institute (including a case reported in [ ] ). the diagnosis of caebv was made according to the criteria proposed in [ ] ; persistent infectious mononucleosis (im)-like symptom and detection of increased ebv genomes in peripheral blood mononuclear cells (pbmc). ebv-dna load was measured using real-time quantitative pcr. median patient age at diagnosis was ( - ) years. target cells of ebv-infection were cd +t cells (n = ) or nk cells (n = ). median ebv-dna load was . × copies per microgram of dna in pbmc (range: . × - . × ) at the diagnosis. all patients were given prednisolone and cyclosporine, and then etoposide (n = ) or combination chemotherapy (n = ) before transplant. ebv load has slightly decreased to a median of . × copies (range: . × - . × ), but disease status was active at ucbt in all. median time from the diagnosis to ucbt was days (range: - ). one patient received total body irradiation (tbi) gy + cyclophosphamide (cy) mg/kg + cytosine arabinoside g/m , and the other five patients received fludarabine (flu) + melphalan (lpam) - mg/m or cy mg/kg with tbi gy before ucbt. umbilical cord blood (ucb) was / hla-matched to the recipients. median number of infused ucb cd + cells was . × /kg (range: . - . × ). gvhd prophylaxis was consisted of tacrolimus + methotrexate or mycophenolate mofetil. neutrophil engraftment and complete donor chimerism were achieved in four patients, but two of them developed secondary graft failure (gf) early after engraftment. the other two patients developed primary gf. second ucbt was successfully performed in the patients with gf a median of . days (range: - ) after the first ucbt. ebv genomes in pbmc became undetectable immediately after ucbt. at a median follow-up of days (range: - ), ebv-dna was undetectable or very low, and im-like symptoms were resolved in all cases. however, at - months after ucbt, two patients developed ebv+ b-cell lpd derived from donor cells, that was successfully treated with rituximab therapy. this study suggested that ucbt could eradicate ebv-infected cd + t cell-or nk cell-clones. ucbt can be a treatment option for adults with caebv. rituximab monotherapy was effective for post-transplant lpd from donor b cells. however, a high incidence of gf was observed in patients receiving reduced-s intensity conditioning of flu/lpam or cy /low-dose tbi. further studies are needed to find more optimal regimens for stable engraftment of ucb in adult patients with caebv. there is an increased incidence of ab incompatibility- - %, in allogeneic hematopoietic stem cell transplantation (allohsct) in patients who are russian citizens as a result of the variability of genetic polymorphism in the multi-ethnic population and a significant number of unrelated donors from international bone marrow registries. ab incompatibility in different types of allohsct may be an additional aggravating factor for the development of immunological complications and decrease effectiveness of treatment, but the data is still controversial [ ] . from may to december in raisa gorbacheva memorial institute for children oncology, hematology and transplantation patients with leukemia, malignancies and hereditary diseases were included to the study, who were performed hsct: allogeneic unrelated - ( %); allogeneic related- ( . %); haploidentical - ( . %); umbilical cord blood in patients ( . %). age was - , median- years. patients were predominantly with acute myeloid leukemia- % (n = ), acute lymphoblastic leukemia- % (n = ) and chronic myeloid leukemia - % (n = ). results: in . % of cases (n = ) АВ incompatibility was determined: major- . % (n = ); minor- . % (n = ); combined- . % (n = ). АВ incompatibility in allohsct did not influence overall survival (p = . ) and frequency of acute graft versus host disease (gvhd) (p = . ). also there was no difference in overall survival depending on combination of condition regimen and ab incompatibility: reduced intensity (ric) or myeloablative (mac) (p = . ). an increased frequency of acute gvhd was observed in ric and АВ incompatibility ( . %) compared to mac ( . %, p = . ). ab incompatibility was not a major factor (log worth . ) which influenced the fact and speed of donor's transplant engraftment in comparison to level of hlacompatibility ( . ), hematopoietic stem cell source ( . ) and type of hsct. but the presence of major ab incompatibility increase the period of erythroid recovery (p = . ) as reflected in the higher amount of blood transfusions. complications caused by ab incompatibility were identified in . % of all cases (n = ) including acute and delayed hemolysis, partial red cell aplasia and immune thrombocytopenia. conclusion. the presence of АВ incompatibility is not a limiting factor to perform allohsct, however, it demands high quality prophylaxis and accurate transfusion therapy depending on ab incompatibility type to prevent immune complications. keywords: allogeneic hsct, ab -incompatibility. poor graft function or graft failure have become common indications for infusion of immune-selected cd + cells (‛boost') or second unprocessed allo-hsct, creating the need for remobilization of the same related or unrelated. we retrospectively compared the results of two consecutive cycles of rh-g-csf treatment and peripheral blood progenitor cell collections in related donors cared for at our institution between and . mobilization consisted of the administration of rh-g-csf at a dose of μg/kg per day injected in the evening, and apheresis was started in the morning of the fifth day after the fourth dose of rh-g-csf. collection was performed with a spectra or spectra optia cell separator (terumo bct). eleven out of were haplo-mismatched donors and were hla matched donors. four donors were re-collected because of recipient graft failure and because of poor graft function; in the latter situation, immunomagnetic selection of cd + cells was performed on the collected cell product prior to infusion into the recipient, using the clinimacs medical device, as previously published. median donor age was years (range: - ) at time of first donation, median weight kg ( - ) and bmi ( - ). median delay between mobilizations and was days ( - ). interestingly, the median delay between collections was days ( - ) in the haplomismatched setting and ( - ) in the matched setting. median number of circulating cd + cells/μl after the first injections of rhg-csf was vs at the first and second mobilization cycles (po . , table ). seven out of donors ( %) requested more than one apheresis session to obtain the target number of collected cd + cells during the first cycle, as compared to out of ( %) for the second cycle: this is largely due to the higher target of cd + cells for the second collection, expecting that the median cd recovery after immunomagnetic selection is % in our experience. our study shows that a second cycle of mobilized peripheral blood progenitor cell collection from related donors is associated with a significant reduction in response to hematopoietic growth factors and mobilization capacity. this information allows planning the number of aphereses at the second cycle-and subsequently the number of immunoselection procedures to be carried out-taking into account the higher cd + cell dose target needed for subsequent immunomagnetic selection. cmv reactivation remains one of the main complications after allogeneic stem cell transplantation (hsct), requiring antiviral therapy, causing myelosuppression, prolonged hospitalization, higher treatment costs and mortality. cmv seronegative donors are recommended for cmv seronegative recipients. however data about donor selection for cmv positive (cmv-pos) recipients is not conclusive. some studies showed that selecting cmv-pos donors for cmv-pos patients might be beneficial. cmv-seropositivity is very high in lithuania among healthy blood and bone marrow donors ( %) and even higher among hsct recipients (up to %), so donor selection for cmv-pos recipients is an object of interest. retrospective analysis of cmv reactivations in cmv-pos allogeneic hsct recipients (transplanted during - year in vilnius university hospital) who survived at least months post hsct was performed. data about cmv reactivation frequency, time post hsct, duration, maximal cmv dna copy number at each reactivation collected. cmv reactivation was considered when cmv dna copies detected /ml in patient's blood. statistical analysis conducted using sas . ; student's tests for statistical significance; kaplan-meier methods for overall survival. among allogeneic hsct recipients ( . %) were cmv-pos. cmv-pos allo-hsct recipients were further analysed. of them received graft from cmv-pos (pos/pos group) and -from cmv-seronegative donors (pos/neg group). more patients in pos/neg group experienced cmv reactivation in first months post hsct in comparison to pos/pos group ( . % vs . %, p o . ). pos/neg group patients had more cmv reactivations ( . vs times in months post transplant period, po . ), reactivations were diagnosed earlier post transplant ( . vs days post hsct, p o . ), had longer duration ( . vs . days, po . ) and larger maximal cmv dna copy number ( , vs . copy/ml, p o . ) in comparison to pos/pos group patients. pos/pos group patients showed tendency for better survival than pos/neg group patients, however did not reach statistical significance. in a univariate analysis only hla mis-match and donor cmv seronegativity were factors statistically significantly associated with cmv reactivation. donor cmv serostatus is significant factor selecting donor for allo-hsct recipients. according to our findings, selecting cmv-pos donor for cmv-pos recipient may reduce cmv reactivation frequency and duration. disclosure of conflict of interest: none. selection of the best hsc donor when a matched donor is not available is still a matter of debate, and reports in pediatric population are scarce. this is a retrospective study conducted by brazilian society of hsct (sbtmo), including centers, aimed to compare matched unrelated (matched-urd), mismatched unrelated (mm-urd) and unrelated cord blood (ucb) hsct. all or aml/mds patients o y/o who have received first unrelated hsct between - were included. hla -digit typing was available for urd; for ucb, hla class-i -digit typing. overall survival (os), and cumulative incidence (ci) of agvhd, cgvhd, nrm and relapse were analyzed. on an unplanned analysis, we fitted a lognormal bayesian survival model with random effects, imputing the probabilities of ucb matching at loci. a total of patients were included ( matched-urd, mm-urd and ucb). median age was . y/o. most patients had all ( %). proportion of early disease in matched-urd was higher ( %, against and %). matched-urd were / -digit hla matched (except one, for whom dq was not available), while most of mm-urd ( %) were / hla matched. ucb were loci ( %) or -loci typed ( %). based on previous report on extending -loci hla typing to -loci ucb, we estimated that ucb were - mismatched at loci, had mismatches and had or more mismatches. conditioning regimens were mainly myeloablative, tbi-( %) or bu-( %) based. grafts were in vivo t-cell depleted in % of the patients, not balanced between groups (p = . ). with median follow-up of . years, y os was %, % and % for matched-urd, mm-urd and ucb, respectively (p = . , log rank test). for matched-urd, mm-urd and ucb, ci of grades iii-iv agvhd at months were %, % and % (p = ns); moderate/severe y-cgvhd, %, % and % (po . ); y-relapse, %, % and % (p = ns); and y-nrm, %, % and % (p = . ). we found out that primary graft failure occurred in ( %) of ucb, compared to % in mm-urd and % in matched-urd. when ucb matching probabilities at loci were imputed and analyzed in a bayesian model (controlled for age, gender, disease status and diagnosis, and t-cell depletion), survival was inferior in ucb with + mismatches ( . -times lower median survival, ci . - . ), but not with up to mismatches ( . -time higher median survival, ci . - . ). of note, in vivo t-cell depletion marginally impaired survival ( . -times decrease, ci . - . ). discussion: in our population, overall survival achieved with mm-urd was not different to / matched-urd, despite higher incidence of moderate/ severe cgvhd. on the other hand, survival with ucb was significantly lower. recent report have shown excellent os with ucb compared to / hla-matched urd. ucb cohort inferior results may have been due to hla disparity degree, since survival in ucb with up to mismatches (out of ) was not worse. one limitation of our study is that tnc and cd from ucb units were not available, impairing primary graft failure analysis. we have also found that in vivo t-cell depletion might have a detrimental effect on survival and should be studied further in prospective trials. in conclusion, mm-urd, especially hla / , is a suitable option when a fully hla / matched-urd is not available. ucb matched at least / may also be a good option. disclosure of conflict of interest: none. allogeneic hematopoietic stem cell transplantation (hsct) is a proven treatment for patients with high risk or relapsed hematological malignancy. the probability of having a hla matched family donor is about %. in populations with high consanguinity rates, the probability of having non-sibling matched family donor(mfd) is much higher. to explore the impact of msd vs non-sibling mfd on outcome of hsct recipients, we undertook a single center retrospective analysis of pediatric patients transplanted with the diagnosis of hematological malignancy at our center in the last five years. a retrospective cohort from to current included pediatric patients with hematological malignancies transplanted from family donors, of which were from msd and from non-sibling mfd. hla matched family donors were identified by high resolution allelic typing and were matched of hla loci. diseases were all (n = ), aml (n = ), mds (n = ), jmml (n = ), kml (n = ), nhl (n = ) and hodgkin's disease (n = ). conditioning regimens were tbi or busulphan-based myeloablative in all patients. the median age of the patients was . years (range: month- . years). although peripheral stem cell seemed to be used more commonly in non-sibling mfd recipients ( % vs %), the difference was not statistically significant. the median follow up time for alive patients months ( - months) . two year overall survival and leukemia free survival did not differ between patients with transplantations from msd or nonsibling mfd ( % ± . vs ± . , p = . ) similarly, leukemia free survival was not different between msd and non-sibling mfd transplants ( . % ± . vs . % ± . , respectively). the incidences of grade ii-iv acute gvhd in msd and nonsibling mfd transplants were % and % , respectively. the incidences of relapses were % in msd transplants and % in mfd transplants and the difference was not significant (p = . ). these data show that the results of hsct from nonsibling mfd is comparable to hsct of msd in children with hematological malignancy. our data emphasize the need for extended high resolution family typing for patients in regions where there is high rate of consanguinity. disclosure of conflict of interest: none. [p ] p donor-recipient rh incompatibility is a risk factor for mortality after pediatric matched related allogeneic hematopoietic stem cell transplantation k ghanem , n hariss , z merabi, n kreidieh , n tarek, r saab , s muwakkit , h el-solh and m abboud american university of beirut, department of pediatrics and adolescent medicine and american university of beirut, optimal donor selection is critical to achieve the best outcome after allogeneic hematopoietic stem cell transplantation (allo-hsct). there is no consensus regarding the effect of donor-recipient rh incompatibility on survival after matchedrelated donor (mrd) allo-hsct in children and adolescents. this abstract aims to study this effect in a single-institution cohort over a period of years. this is a retrospective chart review for all patients aged o years who underwent allo-hsct at the american university of beirut medical center between august and june . a total of patients with a median age of years (range: . - years) underwent allo-hsct from mrd for the following diseases: leukemia (n = ), bone marrow failure (n = ), thalassemia (n = ), scid (n = ), metabolic diseases (n = ) and lymphoma (n = ). the stem cell source was bone marrow for patients ( %) and mobilized peripheral blood stem cells for patients ( %). the grafts contained a median of . × /kg total cd cells. tbi was used in patients ( %). all but patients achieved sustained neutrophil and platelet engraftment. after a median follow-up of months (range: - months), the -year overall survival rate was % ( % ci: - %). by multivariate analysis using cox proportional hazard regression model looking at the following factors for overall mortality: diagnosis, recipient's age, donor's age, the use of tbi, stem cell source, cd count, donor-recipient abo incompatibility, donorrecipient rh incompatibility, and donor-recipient sex-mismatch, the only statistically significant risk factor for mortality was donor-recipient rh incompatibility (hr: . , p: . ). this risk was not statistically significant when looking at transplantrelated mortality (hr: . , p: ) and relapse-related mortality for malignant diseases (hr: , p: . ). there was no association between the incidence of acute or chronic gvhd and rh incompatibility. donor-recipient rh incompatibility was associated with an increased risk of mortality in children and adolescents undergoing mrd allo-hsct. further studies with larger number of patients are needed to confirm this finding. disclosure of conflict of interest: none. effect of iron or vitamin b deficiencies on in vitro colony forming capacity of peripheral blood-derived hematopoietic stem cells in children ny Özbek, mm zabun, y köksal and m Özgüner iron deficiency (id), id anemia (ida)and vitamin b deficiency (vit-b d) are common disorders in developing countries. in urgent situations, children with these disorders could be donors before treatment. in this study, we investigated capacity of peripheral blood-derived hematopoietic stem cells to develop colony-forming units (cfu) in children with id and vit-b , in vitro. patients and methods: we included children (age months- years) in the study in groups: children with id (n = ); children with ida (n = ); children with vit-b d (n = ); children with both id and vit-b d (i/ vit-b d; n = ); and control children (n = ) who has normal peripheral blood findings, and normal ferritin and vit-b levels. from each child complete blood counts (cbc), and levels of ferritin, vit-b , and cero-reactive protein (crp) have been obtained. who criteria, adjusted for age and sex, have been used for definition of anemia, id, ida and vit-b d. four ml peripheral blood drawn into tubes with edta has been used for cfu analysis. mononuclear cell suspension ( . × cell/ml), obtained from peripheral blood by ficoll-hypaque density gradient separation method, has been cultured in dishes containing semi-solid agar culture medium (methocult, h classic, stem cell technologies, canada) in appropriate conditions. after weeks, number of cfu colonies [burst forming erythroid (bfu-e); colony forming unitgranulocyte macrophage (cfu-gm); colony forming unitgranulocyte-erythrocyte-monocyte-megakaryocyte (cfu-gemm)] have been investigated by an inverted microscope. results: statistical analysis showed no difference between groups for age, sex, crp levels, and cfu-e, cfu-gm and cfu-gemm numbers. however, expected differences between groups were present concerning mean values of hemoglobin, ferritin and vit-b levels, mean corpuscular volume (mcv), and red cell distribution width (rdw) ( table ) . discussion: this study shows in vitro proliferation capacity of peripheral stem cells has not been influenced by id, ida, vit-b d, or i/vit-b d. our results may indicate normal grafting ability of peripheral stem cells obtained from donors with iron, vit-b or i/vit-b deficiencies for hematopoietic stem cell transplantation. however, in vivo analysis should also be performed in order to reach a definite conclusion. [p ] disclosure of conflict of interest: none. efficiency of day compared to day stem cell mobilization in allogeneic donors h al-gaithi , s al-mamar , m al-huneini , d dennison , s al-kindi , k al-farsi and m al-khabori hematology residency training program, oman medical specialty board; hematology department, sultan qaboos university hospital granulocyte colony stimulating factor (g-csf) given for - days is commonly used for mobilization of allogeneic stem cell donors. the optimal days of g-csf administration is still debatable. the primary objective of this study is to compare the yield of stem cell mobilization, assessed using cd + cell count, between day and day . secondary objectives include the assessment of the impact of donor's age, weight, mean corpuscular volume and blood group on the difference in the cd + cell count. in this retrospective study we included all allogeneic stem cell donors mobilized with g-csf for days from january till october in the bone marrow transplantation unit at sultan qaboos university hospital. of donor records reviewed, were with available data and selected for the study. descriptive and analytical statistics were performed using stata . . we included donors with median age and weight of years and kg, respectively. the median day wbc and cd + cell count were . × /l and × /l respectively; while the median day wbc and cd + cell count were . × /l and × /l, respectively, (figure) with a statistically significant difference from day (p o . ). in the multivariable model, there were no significant impact of donor's age (p = . ), weight (p = . ), height (p = . ) and mean corpuscular volume (p = . ) on the difference in cd + cell yield. however, donor's blood group ab predicated a significantly higher difference (p = . ). six days of g-csf mobilization achieves higher cd + cell count than days in allogeneic stem cell donors especially in donors with blood group ab. however, cd + cell count on day is high enough to allow for successful mobilization. appropriately designed prospective trial is needed to confirm these results. disclosure of conflict of interest: none. there are known differences between individuals on an unrelated hsc donor register who decide to proceed with verification typing (vt) vs those who choose not to. in the anthony nolan registry, white british donors are more than twice as likely as other ethnic groups to continue with testing at vt (or . ; po . (unpublished data)). the purpose of this study was to explore differences in key characteristics between white british donors and british donors from other ethnic groups with a view to developing interventions to reduce vt stage attrition. study recruitment occurred april -may . all donors not proceeding at vt were invited to participate, and a stratified random sample of those proceeding at vt were recruited to meet pre-determined targets for each ethnic group. data were collected via structured interview (telephone or online). broad categories of participant characteristics were assessed: demographic, culturally related, psychosocial, and donation-related. measures were previously validated scales with established psychometric properties either created for, or used in other donation-related settings. for analyses donors were divided into two groups based on ethnicity: white british (wb), and non-white british (nwb). results: wb donors and nwb donors completed interviews donors proceeding at vt were more likely than their counterparts to participate in the study ( % vs %, p o . ). mean donor age was . with no difference between ethnic groups and % of donors in both groups were female. nwb were statistically more likely to have completed higher education, and have a stronger religious affiliation. in contrast they were less likely to be blood or organ donors. nwb also described greater mistrust of the medical system and of hsc allocation. nwb donors were more likely to have joined the register at a recruitment event (p= . ) or a place of worship (p= . ), while wb donors were more likely to have joined online (p= . ). wb donors reported significantly higher scores regarding feeling well informed about donation both at the point of joining, and at the point of vt and were more likely to remember joining the register and the two donation methods. this study highlights important differences in demographics, culturally related variables and donor interaction with the register between white british donors and donors from other ethnic backgrounds. given the higher rate of vt attrition in nwb donors, these findings could be used to tailor interactions/information given to donors on the register to ensure their priorities are addressed. disclosure of conflict of interest: none. data on mismatched family donor transplants for myelofibrosis are scarce due to the risk of poor engraftment, gvhd and exclusion from trials. outcomes from such transplants performed between and reported to the ebmt are presented. sixty-nine patients, median age ( - ) years; ( %) male, ( %) had primary, ( %) had secondary myelofibrosis ( from et, from prv and others) and unknown ( %). jak v f was mutated in / . karnofsky performance status was % in %; median time from diagnosis to allograft was . (range: . - ) months. the donors were predominantly male ( %), median age ( - ) years, hla mismatched at locus in ( %) and or more loci in ( %). donor-recipient serology was cmv − / − in ( %) ± in ( %), − /+ in ( %) and +/+ in ( %) missing ( %) . bone marrow was used in ( %) and peripheral blood in ( %). the median total nucleated cell count (tnc) was . × /kg (range: . - × /kg) (n = ). the median cd + cell dose was . × /kg [p ] s (range: . - . × /kg)(n = ). patients. conditioning was myeloablative in ( %) and ric in ( %). predominant conditioning regimes were fludarabine, busulphan, atg (fbatg) and thiotepa, busulphan, fludarabine (tbf n = ). tbi was administered in ( %) and t cell depletion in vivo in ( %) and ex vivo in ( %) patients. gvhd prophylaxis varied with post transplant cyclophosphamide administered in / ( %) and atg in / patients ( %).neutrophil engraftment occurred in ( %) patients at a median of days (range: - ). primary graft failure ensued in ( %) and secondary graft failure in ( %) patients at a median of (range: . - ) months. eleven patients had a second allograft at a median interval of ( - ) months. responses to the first allograft censoring for a second allograft, data available in patients, showed that complete remission was achieved in patients ( %), ( %) were never in cr and ( %) were not evaluable. relapse occurred in ( %) of patients at a median interval of ( . - . ) months. the cumulative incidence (ci) of grade ii-iv acute gvhd (agvhd)was % ( % ci - %) and for grade iii-iv agvhd at was % ( % ci - %). data for chronic gvhd (cgvhd) was valid in patients of whom % developed cgvhd. the ci of cgvhd at years was % ( % ci - %):ci of limited cgvhd was % ( % ci - %) whereas the ci of extensive cgvhd was % ( % ci - %). median follow-up was ( % ci - ) months. the and year os was % ( % ci - %) and was % ( % ci - %). the and year rfs was % ( % ci - %) and % ( % ci - %). the -year ci of relapse was % ( % ci - %). the year nrm was % ( % ci - %), which increased to % ( % ci - %) at years. thirty patients died due to infection ( , %), gvhd ( , %), organ damage or failure ( , %), relapse/disease progression ( , %) and secondary malignancy or ptld ( , %) unknown . there was no significant effect (univariate analysis) of recipient or donor gender, degree of hla mismatch, cmv matching, primary or secondary mf, chronic vs advanced disease at transplant, conditioning intensity or regimen, gvhd prophylaxis with atg or post transplant cyclophosphamide or stem cell source on overall survival. the data are encouraging for patients with myelofibrosis, with engraftment, pfs and os being attained with limited severe chronic gvhd from family mismatched donors. disclosure of conflict of interest: none for all other authors, fc consulting with molmed. feasibility of salvage second allogeneic stem cell transplantation for disease relapse or graft failure: a single centre experience g battipaglia , , d salvatore , , r dulery , f giannotti , f malard , e brissot , s sestili , f isnard , s lapusan , a-c mamez despite high rates of toxicity and mortality, a second salvage allogeneic stem cell transplantation (second allohsct) might be an option to consider in patients experiencing disease relapse or graft failure after first allohsct. we retrospectively analyzed outcomes after second allohsct in a cohort of patients ( males and females) transplanted either for disease relapse (group , n = ) or graft failure (group , n = ) between and in a single centre in france. median age at second allohsct was (range: - ) years. diagnoses were acute myeloid leukemia (group : n = ; group : n = ), acute lymphoblastic leukemia (group : n = ; group : n = ), myelodysplastic syndrome (group : n = ; group : n = ), myeloproliferative neoplasm (group : n = ; group : n = ), bone marrow failure (group : n = ; group : n = ). median time from first allohsct to second allohsct was (range: . - ) months in group and . (range: - ) months in group . graft source for the second allohsct were: haploidentical bone marrow (group : n = ; group : n = ), haploidentical pbscs (group : n = ; group : n = ), cord blood (group : n = ; group : n = ), matched unrelated pbsc (group : n = ; group : n = ). at time of second allohsct, patients were in cr and presented active disease in group . conditioning regimen was myeloablative in patients (group : n = ; group : n = ), reduced intensity (ric) in cases (group : n = ; group : n = ). a sequential schema consisting of a combination of thiotepa, etoposide and cyclophosphamide followed by a fludarabine and busulfanbased ric was used in out of patients with active disease in group . sixteen patients received atg as part of the conditioning regimen for second allohsct (group : n = ; group : n = ). all but one patient engrafted, at a median time of (range: - ) days. cumulative incidence of acute and chronic gvhd were ± % and ± %, respectively, - ) months, non-relapse-mortality (nrm) and relapse incidence (ri) were ± % and ± %, respectively, while disease-free (dfs) and overall survival (os) were ± % and ± %, respectively, for the entire cohort. in all, patients died of infections (n = ), hematological disease (n = ), gvhd (n = ), hemorrhage (n = ) and for unknown causes (n = ). main outcomes of patients in group were: ri ± %, nrm ± %, agvhd ± %, cgvhd ± %, dfs ± %, os ± %, respectively. main outcomes of patients in group were: ri ± %, nrm ± %, dfs ± %, os ± %, agvhd ± %, cgvhd ± %. historically, a second allohsct was hampered by significant morbidity and mortality. however, the advent of reduced-toxicity conditioning regimens and improved supportive care allowed to significantly improve the results of patients receiving a second allohsct as suggested from the above results. therefore, a second allohsct could be considered as an option to rescue a certain number of patients experiencing disease relapse or graft failure, for which prognosis is very poor. decision is to be discussed on a case-by-case basis. disclosure of conflict of interest: none. haploidentical hematopoietic stem cell transplantation with post-transplant cyclophosphamide for patients with high-risk hematologic malignancies am carella department of oncology and hematology, irccs casa sollievo della sofferenza, san giovanni rotondo allogenic hematopoietic stem cell transplantation (sct) has been increasingly used for treatment of adult with high risk hematologic malignancies. for patients lacking an hlamatched related or unrelated donor, unmanipulated haploidentical (haplo)-sct is a potential alternative. haploidentical transplantation performed with post-transplantation cyclophosphamide (ptcy)-based graft-versus-host disease (gvhd) prophylaxis has been associated with favorable outcomes for patients with acute leukemia and lymphomas we analyzed outcomes of patients with hematologic malignancies who received t-cell-replete haematopoietic stem cells and posttransplantation cyclophosphamide after myeloablative or nonmyeloablative hla-haploidentical donor transplantation. the median age was years ( - ); twelve patients were in first remission (cr ), in second remission (cr ) and had an active disease. ). the diagnosis was acute leukemia (n = ), myelodisplastic syndrome (n = ), hodgkin disease (n = ) non hodgkin lymphoma (n = ) and multiple myeloma (n = ). median follow-up was days. stem cell source was bone marrow (bm) for patients, and peripheral blood (pb) for . myeloablative conditioning (mac) was used in patients and reduced intensity regimen (ric) in patients. thirty one patients were first grafts, the others underwent previous autologous sct (n = ) or mud (n = ). gvhd prophylaxis s consisted in pt-cy on days + and + , cyclosporine (from day + ), and mycophenolate (from day + ). the median day for neutrophil engraftment was day + ( - ). no graft failure was observed. chimerism was evaluable in patient; on day + all patients had % donor chimerism on marrow cells median follow-up was days. the cumulative incidence of acute gvhd grade ii-iv was %, grade iii-iv % and chronic gvhd %. one-and -years os was . % and . %, respectively. with a median follow-up for the surviving patients of days ( - ), the cumulative incidence of transplant-related mortality (trm) is %, and the relapserelated death is %. thus, we demonstrate excellent rates of engraftment, gvhd, and trm in adult patients treated with haploidentical hematopoietic stem cell transplantation with post-transplant cyclophosphamide. this approach is a widely available, safe, and feasible option for adult patients with high risk hematologic malignancies, including those with a prior history of myeloablative bmt and/or those with co-morbidities or organ dysfunction, also for patients with active disease at the time of transplant. disclosure of conflict of interest: none. it has recently been shown that t-replete allogeneichematopoietic stem-cell transplantation (allo-hsct) from a haploidentical donor (haplo-id) could be a valid option when a matched donor is not available. unfortunately, the worldwide donor registries comprise mainly donors of caucasian origin and patients of non-caucasian origin have a much lower chance of finding a matched unrelated donor (mud). the lengthy period of international search when required and the financial burden of this process are considered as additional significant limitations. at the american university of beirut medical center (aubmc) in lebanon, we started the mud program in and haplo-id hsct program in . we report here our experience in this two groups of patients. patients and methods: we have transplanted patients from a haplo-id donor since and compare their outcome with the patients transplanted from a mud since . the patients and transplant characteristics are listed in the table . the groups were comparable except for conditioning. patients in haplo-id group received two days of posttransplant high-dose cyclophosphamide (pt-hdcy) followed by cyclosporine a (csa) and mycophenolate-mofetil while patients in the mud group received pre-transplant antithymocyte-globulins and csa starting on day- . all patients engrafted in the mud group, while one patient did not engraft in the haplo-id group, the patient had refractory all transplanted with progressive disease, and died on day + . the median of anc /mm was days ( - ) vs days ( - ) in the haplo-id and mud groups, respectively. fourteen patients from the haplo-id group developed grade acute graft-versus-host disease (agvhd) vs one after mud-hsct. two patients haplo-id group developed limited cgvhd and none after mud grafts. six patients relapsed in the haplo-id group vs three patients in the mud group. two and three patients died from non-relapse mortality in the haplo-id and mud group, respectively. at the last follow-up, patients are still alive in the haplo group vs patients in mud group and all of them are in cr. we conclude that t-replete haplo-id hsct followed by pt-hd cy is associated with promising results or at least comparable to patients transplanted from mud. haplo-id hsct seemed to be safe and feasible in patients with high risk hematological malignancies. finally, because of the obvious advantage in rapidly finding a donor ( haplo transplants in three years vs mud transplants in years), development of haplo-id hsct is warranted to satisfy the regional needs. [p ] disclosure of conflict of interest: none. haploidentical hematopoietic stem cell transplantation (haplo-hsct) using t-cell-replete (tcr) grafts and posttransplantation cyclophosphamide (ptcy) provides a curative approach for patients with high-risk mds/aml lacking a conventional hla-matched donor. in children and adults haplo-hsct using ptcy as gvhd prophylaxis seems to be safe with low treatment related morbidity and mortality (trm). however, few data are available for elderly patients with advanced disease. we retrospectively analyzed the outcome of patients with mds (n = )/aml (n = ) age - years (median age years; patients - years, patients ⩾ years; male), who underwent tcr haplo-hsct with high-dose ptcy at our institution between january and november . disease was active in patients while had achieved cr. patients failed previous allo-hsct. pretransplantation risk factors were scored using the hematopoietic cell transplantation-specific comorbidity index (hct-ci) which was ⩾ in patients (median hct-ci = , range: - ). a sequential therapeutic concept using either flamsa (n = ) or clofarabine (n = ) as cytoreduction was used prior to reduced intensity conditioning (ric) in all but patients. ric consisted of fludarabine/cyclophosphamide combined with either melphalan (n = ), busulfan (n = ) or gy tbi (n = ). post-grafting immunosuppression consisted of cyclophosphamide, tacrolimus and mmf in all patients. % received a bone marrow graft. one graft rejection occurred. neutrophil and platelet engraftment was achieved in % and % of evaluable patients, respectively at a median of ( - ) and ( - ) days. acute gvhd grade i-iii occurred in % of the patients whereas no grade iv agvhd was observed. chronic gvhd presented in %. it was most frequently assessed as mild to moderate ( pts). only patients developed severe cgvhd; no gvhd related death was observed. cmv reactivated in of patients at risk, one patient developed cmv disease (pneumonia). no ebv reactivation or ptld occurred. one-year trm was %. / ( %) patients relapsed, three within the first days after haplo-hsct. at a median follow up of months (range: - months) estimated one-and two-year overall survival (os) was / %, respectively. when stratified by age, estimated one-and two-year os was / % in patients o years and / % in patients ⩾ years (p = . /p = . ). one-and two-year progression-free survival (pfs) was / %, respectively. stratified by age estimated one-and two-year pfs was / % in patients o years and / % in the elderly (p = . /p = . ). unmanipulated haploidentical allografting using ptcy-based gvhd prophylaxis in high-risk mds and aml patients aged over years is safe and well tolerated resulting in acceptable trm. a remarkable survival outcome can be achieved in elderly high-risk aml/mds patients with significant comorbidities. disclosure of conflict of interest: none. key performance indicators to assess the quality of a collection facility: experience of a single center s roncon*, c pinho , f bordalo , s lopes , s ferreira and f amado allogeneic hematopoietic stem cell transplantation (allo-hsct) has evolved into an effective immunotherapy for the treatment of a variety of disorders. when patients do not have a familiar matching donor, transplant centers (tc) search for an unrelated and volunteer donor. this one must be previously evaluated by the collection center (cc) to donate peripheral blood stem cells (pbsc) or bone marrow (bm); lymphocytes can also be asked after allo-hsct. this work aims to evaluate our performance as cc, ensuring donor safety, quality of cell therapy products (ctp) and the accomplishment of tc requirements. we retrospectively analyzed all the requests of ctp collections sent by the portuguese registry from to . countries of destination, number and type of ctp were determined. we established eight key performance indicators (kpi) classified into four categories: response time; product quality; satisfaction of patients and donors; and on-site donor motivation. the intended target was defined by the mean result obtained in the first half of (excluding kpi- ). written comments from donor center (dc) and tc were received by email or written in the local notebook. the donor's answers were obtained through a survey given on the collection day. a total of requests were assessed: pbsc, bm, lymphocytes and cancellations; % were sent to europe ( / to portugal), % to america and % to oceania; / were withdrawn by tc ( patients died, presented progressive disease and had a better hlamatched donor) and / by dc ( donors not cleared and refused). the results obtained with kpi- , - , - and - exceeded the intended target (table ) . after the first kpi- results, we verified a positive evolution. we took an average of days of delay in sending donor clearance. however, there is no holdup in the ctp delivery, as demonstrated by kpi- . regarding kpi- it is important to notice that % of ctp with a cell number less than requested were bm and lymphocytes; when pbsc was considered separately, the result increased ( % vs %). analyzing kpi- , % (n = / ) of the contaminated ctp were bm. concerning kpi- , - acknowledgments and - commitment, we recognize that our initial targets were too ambitious ( %). the kpi- shows a low number of complaints (n = ): one due to a misreading of the request and three to communication failures; all were properly examined and rectified. a good general status was guaranteed in almost all the donors (kpi- ). the decrease of kpi- is due to the fact that one donor refused to proceed after three postponements of the collection date by tc. table -key performance indicators of the quality of our activity as a cc. the overall good level of our results reflects an extremely professional performance as a cc. we consider that these kpi should be continuously monitored with the purpose of earlier detect any deviation of the stated goals and assess the progress against settled strategies. we further suggest the establishment of universal indicators in order to standardize [p ] practices, share expertise and improve the quality of services and products provided to patients and donors. two year later, the patient had a genoidentical allogenic stem cell transplant (from the bone marrow stem cell of his sister, who was years old, hla compatible). he had a reduced intensity conditioning, associating busulfan, fludarbine and antilymphocyte serum. months from the asct, he was in complete remission with % donor chimerism. years after the asct, the patient presented a progressive thrombocytopenia without any other peripheral causes. the bone marrow aspiration initially showed a refactory cytopenia with multilineage dysplasia. the patient was followed up during months, and then a second bone marrow aspiration has shown a refractory anemia with excess blasts raeb . a cytogenetic study has every time demonstrated a female karyotype ( ,xx) on mitoses out is , and chimerism was % donor. the diagnosis of the myelodsplastic syndrome of the donor cells was approved. the patient was treated by azacitidine ( mg/m , from j to j , j = j ). after cycles, the patient was in complete hematologic response (normalization of the platelet count) and a partial bone marrow response (normalization of the blasts rate but persistence of the signs of dysplasia). he received more cycles, and presented hematologic relapse (reemerging of thrombopenia). a phenoidentical allogenic stem cell transplantation was suggested. conclusion the occurrence of mds on the donor cells is rare. these anomalies are secondary to intrinsic factors (of donor) or extrinsic factors )of the transplant recipient). the treatment is not definitely determined. disclosure of conflict of interest: none. nk-cell alloreactivity based on kir/ligand mismatch in the donor vs recipient direction provides better graft-versustumor effect in patients with active hematological malignancies undergoing allogeneic t-replete haploidentical transplantation followed by post-transplant cyclophosphamide a wanquet , , s bramanti , , s harbi , s fürst , f legrand , c faucher , a granata , p-j weiller , , c picard , b calmels , c lemarie , c chabannon , , , l castagna , , d blaise , , and r devillier , haplo-sct have been developed in the past years with very interesting results in high risk patients. gvhd prophylaxis using post-transplant cyclophosphamide (pt-cy) recently allowed extending the use of unmanipulated haplo-sct. it was shown that nk alloreactivity, triggered by donor-recipient inhibitory kir gene-gene mismatches, could lead to better outcomes and survival in the setting of in t-cell-depleted haplo-sct. however, few data is available on the impact of kir-ligand mismatch on the outcome after t-replete haplo-sct with pt-cy. we thus assessed the impact of nk alloreactivity on the outcome of patients who received haplo-sct followed by pt-cy. we retrospectively collected the data from patients from two centers who were treated for various high risk hematological diseases and underwent a haplo-sct with pt-cy from december to december . we assessed the kir-binding epitope in hla-c and hla-b molecules for all patients, and we predicted nk cell alloreactivity in the donor vs recipient direction via the immune polymorphism database kir ligand calculator, based on the kir-ligand mismatch between donors and patients. because disease status at the time of haplo-sct is one of the most important predictor of outcome, we separately analyzed two cohorts of patients: those transplanted in complete remission (cr group) and those transplanted with active disease (no cr group). using a multivariate cox model (adjusted by disease type, age and conditioning), we therefore evaluated the impact of nk alloreactivity on outcome in both cr and no cr groups. we analyzed patients with a median age of y ( - ). they were mostly transplanted for lymphoma (n = , %) or aml/ mds (n = , %). patients mostly received a tbi-based nonmyeloablative conditioning regimen (n = , %) and pbsc as graft source (n = , %). eighty one and patients were transplanted in cr and in no cr, respectively. nk alloreactivity was found in / cr patients ( %) and / no cr patients ( %). with a median follow up of months ( - ), cr patients had a significantly better outcome than those in the no cr group ( -year pfs % vs %, respectively, p o . ). in no cr patients, multivariate analysis showed that nk alloreactivity was significantly associated with reduced the risk of relapse (hr = . , p = . , figure a ) with no increase of both acute (hr = . , p = . ) and chronic gvhd (hr = . , p = . ), and nrm (hr = . , p = . ). this led to significantly better pfs (hr = . , p = . , figure b ) and a trend for better os (hr = . , p = . ). in contrast, in cr patients, we found no difference in outcome according to nk alloreactivity for all end points (acute gvhd: hr = . , p = . ; chronic gvhd: hr = . , p = . , nrm: hr = . , p = . , relapse: hr = . , p = . , figure c ; pfs: hr = . , p = . , figure d ; os: hr = . , p = . ). our results suggest that nk alloreactivity provides better disease control with no increase of gvhd, especially in patients transplanted with active disease. thus, donor selection should rely on the prediction of nk alloreactivity. this may contribute to improve outcome of these patients with high risk of relapse after transplantation, underlining the need of a specific strategy of donor search, and the promising perspective of early post-transplant nk-cell-based immunotherapy. haploidentical bone marrow transplantation (haplo-bmt) with post-transplant cyclophosphamide (pt-cy) is being increasingly used, in the last five years, for patients lacking a suitable hla-matched donor. genoa study (eudract number: - - ) provides for a modified gvhd prophylaxis platform compared to the original baltimora protocol. aim of the study: in this study we assessed outcomes in consecutive patients transplanted from a haploidentical donor for haematological malignancies. all patients received a uniform gvhd prophylaxis: cyclosporine (csa) starting on day , mycophenolate (mmf) starting on day + , and post transplant cyclophosphamide (pt-cy) mg/kg, on days + and + . all patients received a myeloablative conditioning consisting of thiotepa, fludarabine, busulfan (three doses n = or two doses n = ), or tbi, fludarabine (n = ). the median age was years ( - ); at transplant ( %) patients were in remission of disease (cr and cr ), and had an active disease ( %); all patients were first grafts. the diagnosis were acute myeloid leukemia (n = ), myelodisplastic syndrome (n = ), acute lymphoblastic leukemia (n = ), myelofibrosis and myeloproliferative diseases (n = ), non hodgkin lymphoma (n = ), chronic lymphocytic leukemia (n = ) and multiple myeloma (n = ). the median follow up was days (range: - days). the median infused mononucleated cells was . × e /kg (range: . - . ). seven patients died before engraftment, and ( %) had autologous recovery: ( %) after conditioning with doses of busulfan. full-donor chimerism on day + was reached in ( %) patients. the median day for neutrophil engraftment was day + (range: - days). the cumulative incidence of grade ii-iv and iii-iv acute gvhd (agvhd) was % (n = ) and % (n = ), respectively. two years cumulative incidence of moderate-severe chronic gvhd (cgvhd) was % (n = ).sixty one ( %) patients experienced haemorragic cystitis. at years the cumulative incidence of non relapse mortality (nrm), relapse and relapse related death was % (n = ), % (n = ) and % (n = ), respectively. causes of death were infections (n = ), hemorrhage (n = ), gvhd (n = ), secondary neoplasia (n = ) and relapse (n = ). at years of follow up overall survival and disease free survival was . % and %, respectively. at the same time overall survival rate was % for patients in remission and % for patients with active disease at transplant(p o . ). in conclusion, a modified pt-cy as gvhd prophylaxis and ma conditioning regimen followed by haploidentical bmt results in a low risk of agvhd and cgvhd and encouraging rates of trm and dfs. disclosure of conflict of interest: none. the italian bone marrow donor registry (ibmdr), in collaboration with admo (associazione donatori di midollo osseo) since has implemented, as part of the donor enrollment strategy, public enrollment events (pe). our donor center (dc) has taken part to those events since the first years. one or more clinician (or trained biologist) has been present to pe to inform the potential donors, evaluate the candidates and supervise the collection of biological fluids. all the local permission where obtained. aim: aim of this study was to compare the compliance of the donor enrolled in pe with donors enrolled at our dc institutional site. we prospectively evaluated all the donors recalled for further evaluation and/or for requalification in the years and at our dc itmi . we defined possible results for the call: ‛success' (the donor was eligible and accepted to be evaluated, or only temporarily ineligible) ‛not eligible' (the donor was definitively ineligible) and ‛consent denied'. results: a total of donors were called back in the years and ( not found). eightyfour recalled donors had been enrolled after . among them ( . %) had been enrolled at the dc and during pe ( . %). the two populations were not different for age at the call, age at enrollment and gender (table ) . [p ] when evaluating the probability of obtaining a "success", no significant difference was found between the two populations: . % vs. . % (chi square p= . ). no significant difference was also found for the "not eligible" and the "consent denied" categories. of note, when we turned to the whole donor population we had called back (median age , range - ), the probability of "success" and "consent denied" were not related to donor age, and time from enrollment to recall, whereas donor ineligibility was (spearman test p= . and . ). public events with the presence of an adequate trained medical team represent a valid option for the enrollment of new unrelated donors. disclosure of conflict of interest: none. the search for hematopoietic stem cell unrelated donors in patients with malignant hemopathies with not-sibling matched family donor: the experience of a center a pérez , r goterris , m gómez , s blanco , a segado , c arbona , jch boluda , m poch and c solano hematology department, hospital clínico universitario, valencia unfortunately, as few as - % of patients will have an hlaidentical matched sibling donor available for hematopoietic stem cell (hst) donation. the search for an unrelated donor (urd) (adult or cord blood) is often the best option for those patients lacking a suitable matched donor. below we describe the experience with the search for an unrelated donor in our center. between september and march the search for urd was activated for patients. the median age of the patients was years (range: . - ), % were under years and % were males. acute myeloid leukemia (n = ), acute lymphoblastic leukemia (n = ), non-hodgkin's lymphoma (n = ), chronic/prolymphocytic lymphocytic leukemia (n = ), hodgkin's lymphoma ((n = ), multiple myeloma (n = ), chronic myeloid leukemia (n = ), philadelphianegative myeloproliferative neoplasms (n = ), myelodysplastic syndrome (n = ), aplastic anemia/paroxysmal nocturnal hemoglobinuria (n = ), others (n = ). the disease status in hematological malignancies was: first cr (n = ), second cr (n = ), pr (n = ) and refractoriness (n = ). the donor type requested at the activation of the search was an adult (n = ), umbilical cord blood (n = ) and two options (n = ). results: a compatible donor was found in patients ( % of the series) after a median of days (range: - ) from the activation of the search. the degree of adult donor compatibility (not available in cases) was: complete hla identity ( / : n = , / : n = ); an hla difference ( / : n = , / : n = ); lower degree of compatibility (n = ). the degree of umbilical cord blood compatibility: identity ⩾ / (n = ). a total of patients ( %) were transplanted, from adult donor and from umbilical cord blood. the median time between the activation of the search and the hst transplantation was months (range: . - ), being . months for acute leukemia and . months for other pathologies, and between the location of the donor and the hst transplantation days (range: - ), being days for umbilical cord blood and days for an adult donor. there were cancellations of the urd search ( % of the total) for the following reasons: clinical status of the patient (n = ), performing a haploidentical transplant (n = ), transplant center does not consider (n = ), norms of the registry (n = ) and loss of indication of transplantation (n = ). the median time from the beginning of the search to its cancellation was . months (range: . - ). at the time of analysis, the median follow-up of the patients is months. the survival of the series in the years is % and % for patients transplanted from urd. % of the searches activated in our center allowed the localization of a urd with an adequate degree of hla compatibility. however, only % of the patients for whom the search was activated were finally transplanted. the most frequent cause of cancellation of the procedure was the clinical deterioration of the patient. disclosure of conflict of interest: none. the leukemic transformation of otherwise healthy donor stem cells provides a useful in vivo model to study the mechanisms involved in leukemogenesis. we report two cases of donor cell-derived haematological malignancy in which wholeexome sequencing (wes) was performed in bone marrow (bm) samples from recipient at different times after allogeneic hematopoietic stem cell transplantation (allo-hsct) in order to study the dynamics of emergence of mutations that precede the development of donor cell leukemia (dcl) and donor cell myelodysplastic syndrome (dc-mds). case : a -year-old female diagnosed with lymphoblastic leukemia-b t( ; ), who developed acute myeloid leukemia (aml) with normal karyotype, npm +of donor origin months after unrelated cord blood transplantation (ucbt). case : a -year-old male diagnosed with mantle cell lymphoma, who developed mds ,xx,- ,del( )(p ) of donor origin, months after allogeneic bm transplantation from his hla-identical brother. the donor also developed mds several months later. wes (sureselect-xt human-exon mb) was performed by next generation sequencing (hiseq) on donor stem cells (scs) infused as well as on bm samples from recipient after allo-hsct. the exome of donor scs and bm samples, from case , were aligned to the human reference genome (grch /hg ) and donor scs and bm samples were aligned to grch / hg in the second case. in both cases non-synonymous variants in the coding regions or synonymous variants in splice regions of genes related to leukemia were selected. in addition, bm samples were matched to their scs and to prior bm samples to identify the acquired variants. variants meeting such criteria were evaluated with functional predictor software's (sift, polyphen and mutation taster). wes analysis revealed progressive emergence of multiple somatic mutations probably related to the development of leukemia in bone marrow samples post allo-hsct ( figure ). both scs showed alterations that may be involved in leukemogenesis. (case : sh b and case : kmt c, kmt a, arhgap and monosomy ). somatic mutations, acquired over time, fall into genes that play well-established roles in signalling pathways (ras-mapk, pre-mrna splicing factor, apoptosis, dna doublestrand break repair, dna replication and so on). mutations in leukemic subclones that disappear after chemotherapy were indentified, as well as the acquisition of new mutations in resistant subclones. we propose a possible model of leukemogenesis in these cases ( figure ). the present study reveals a process of sequential clonal expansions, promoted by the acquisition of additional somatic mutations in donor hematopoietic cells. detection of heritable or acquired gene mutations in donor associated with predisposition to haematological malignancies could have clinical implications for the patients undergoing to allo-hsct. although the cause of donor cell-derived haematological malignancy onset seems to be multifactorial, the infusion of a scu with pre-leukemic potential in a context of residual toxicity in recipient as a result of pre-transplant chemotherapy, a post-transplant environment characterized by a decreased immune surveillance may well have played role in these cases. the study of a greater number of dcl cases by next generation sequencing could help to understand this process and to detect new mutations involved in the emergence of aml. disclosure of conflict of interest: none. the impact of donor and recipient sex in allogeneic stem cell transplantation-single center experience (cic ) y petrov , p ganeva , g arnaudov , s lozenov , y davidkova , v stoeva , i tonev , m guenova and g mihaylov national hospital for active treatment of hematological diseases allogeneic hematopoietic stem cell transplantation (hsct) has been one of the most effective therapeutic modalities for patients with hematological malignancies and bone marrow failure syndromes. optimal donor selection is one of the key factors to enhance the success rate of this procedure. we [p ] s retrospectively investigated whether and how donor-recipient sex affects transplantation outcomes of patients transplanted between and in our center. the median age of the patients was years (range: - ). thirty-nine of the patients ( %) received a pbsc from a hla-identical sibling, and patients ( . %) received pbsc from matched unrelated donor. forty-six percent were male recipients with male donors (m-m), . % were female recipients with male donors (m-f), . % male recipients with female donors (f-m), and . % female recipients with female donors (f-f). we performed a crosstab analysis and χ tests to observe whether the donor sex affects our study population. patients with male donor had superior overall survival and progression-free survival compared to those with female donor ( . % vs . % p = . for os, and . % vs . % p = . for pfs; cramer`s v = . ). we further investigated how the disparity of the donor in the four groups (m-m, m-f, f-m and f-f) affects the os, pfs and nrm. the f-m group had a worse overall and progression-free survival comparing the other groups ( % -year os and % pfs; p o . ).this group had % relative increase in the non-relapse mortality compared with m-m group (p = . ). for m-m group there was a % relative increase in the subdistribution hazard of nrm compared with m-f group (p = . ). the f-f group and m-f group had similar subdistribution hazard of nrm ( % vs % p = . ). the incidence of acute gvhd and chronic gvhd for the groups was: % and % (m-m), % and % (m-f), % and % (f-m), % and % for the (f-f) group. the appearance of either acute or chronic gvhd did not show statistical significance regarding the os and pfs in the groups (p = . ). we examined the effect of donor-recipient sex incompatibility on the outcome of hsct in out center. our results showed inferior os and pfs for f-m group and a higher incidence of nrm compared with other groups. these effects might be associated with allogeneic immune responses against h-y antigens. key words: stem cell transplantation, donor sex, recipient sex, overall and progression-free survival [p ] disclosure of conflict of interest: none. from to , % of the patients affected by hematological malignancy searching for an unrelated donor through the italian registry successfully identified a suitable donor. this proportion increases up to % when searching for a cord blood unit was considered, corresponding to total transplant efficiency of %. from april , the rome transplant network adopted a unique policy for the identification of a potential alternative donor, following a hierarchical selection that considered as first choice a volunteer unrelated donor, secondly a cord blood unit and last a haploidentical related donor. before starting the unrelated donor search, a preliminary query through the bone marrow donor worldwide database was performed for all the patients referred to the rome transplant network. based on the low resolution hla typing (a, b and drb ) it was possible to arbitrary assign a good or poor score that might predict the identification of a full matched ( / a, b, c and drb ) donor. therefore, aims of the present study were to assess the utility of the preliminary query and the impact of the use of high resolution hla typing since the starting of donor search on the timing for the unrelated donor identification. moreover, the final aim was of comparing donor identification and transplant efficiency between the national registry, that considers only the unrelated donor and the rome transplant network, whose policy includes also haploidentical donor as third choice in the donor search process. at rome transplant network % out of adult patients met criteria of a good preliminary query corresponding to a matched unrelated donor identification in % of cases vs only . % for patients with poor preliminary query. our policy led to % and %, respectively, of alternative donor identification and transplant efficiency, significantly higher than the corresponding data of % (p = . ) and % (p o . ) reported by the national registry. moreover, the median duration of search process for mud identification has been significantly reduced by the use of hr hla typing patient at the start of the formal search activation from (range: - ) to (range: - ) days at ibmdr (po . ) and from to days ( - ) at rtn (po . ). in conclusion, the preliminary query represents a useful tool to address the search towards the best donor choice and to perform transplant in adequate time. moreover, the timing of donor identification has been significantly reduced with the use of high resolution typing at the start of donor search. a search and selection donor policy should be basically established and should include the haploidentical donor to improve the transplant efficiency. disclosure of conflict of interest: none. the long term prognosis of elderly acute myeloid leukemia (aml) patients remains poor. advances in the uses of alternative donors and reduced intensity conditioning regimens have extended the use of allogeneic hematopoietic stem cell transplantation (hsct) to a wider number of patients. however, few studies have reported data on the efficacy of hsct from alternative donors in elderly aml patients. we retrospectively analyzed the transplantation outcome in consecutive elderly aml patients aged years who received hsct ( hsct ( - at the catholic blood and marrow transplantation center. donor types were autologous (n = ) or hla matched related (mrd, n = ), unrelated (mud, n = ), or haploidentical (n = ). for graft-versus-host disease (gvhd) prophylaxis, methotrexate and cyclosporine (mrd) or tacrolimus (mud/haploidentical donor) were used. mud and haploidentical donors were given antithymocyte globulin. the median age was years, with patients ( %) years. intermediate-or adverse cytogenetic risk was observed in % of patients. with a median follow-up of . months, overall survival (os) and disease-free survival (dfs) at years after transplantation were % and % for autologous, % and % for mrd, % and % for mud, and % and % for haploidentical hsct, respectively. the -year relapse was significantly higher for autologous hsct compared to allogeneic hsct ( % vs %, p = . ), while it was similar among allogeneic donors: mrd, %; mud, %; haploidentical, % (p = . ). the -year non-relapse mortality (nrm) for mud ( %) or haploidentical donor ( %) hsct was comparable to that of autologous hsct ( %), while it was relatively higher for mrd hsct ( %, p = . ). of the patients receiving allogeneic hsct, the -year cumulative incidence of moderate to severe chronic gvhd was significantly increased for mrd ( %) compared to alternative donor hsct ( %, p = . ). in multivariate analysis, patient age (hr . , % ci . - . , p = . ) and donor type (hr . % ci . - . , p = . for mud; hr . , % ci . - . , p = . for mrd compared to haploidentical donor) were significantly associated with the cumulative incidence of moderate to severe chronic gvhd, while female-to-male hsct showed a borderline significance (hr . , % ci . - . , p = . ). incidence of acute gvhd was similar according to donor type. in the multivariate analysis for nrm, patient age (hr . , % ci . - . , p = . ), mrd (hr . , % ci . - . , p = . ), and hematopoietic cell transplantation-comorbidity index high risk (hr . , % ci . - . , p = . ) were significantly associated. in conclusion, our results showed significantly higher relapse rate for elderly aml patients receiving autologous hsct compared to allogeneic hsct, responsible for the lower survival rate in autologous hsct. we observed that nrm rate for mud and haploidentical donors for elderly aml patients were lower than expected and similar to autologous hsct. relatively higher incidence of nrm for mrd hsct seemed responsible for the low long term dfs. these results suggest a need for strengthening of gvhd prophylaxis in mrd hsct for elderly aml patients. our results suggest a potential role of alternative donor hsct to improve long term survival rates in elderly patients with aml. disclosure of conflict of interest: none. for patients with saa, transplantation from an unrelated donor (ud) is usually considered after failure of at least one course of immunosuppression. this strategy is based on a relatively high risk of complications for ud transplant recipients, such as graft rejection, graft-versus-host disease (gvhd) and infections. however, the outcome of unrelated donor transplants has significantly improved in recent years, due to better donor selection, conditioning regimen optimization and better supportive care. the authors describe results from patients with saa who receive unrelated allogeneic transplants in a single reference institution from to . data was retrieved from the center databasis and there were females and males. median age was years old . median total number of cells infused was . × /kg. % of the patients have received more than transfusions previously. conditioning regimen were: cy + tbi ± atg in ( %) patients, bu mg/kg+ cy + atg in ( %), and fludarabine + cy+atg in ( %), fludarabine, cy+tbi in ( %) patients. stem cell source was marrow in %, cord blood in % and peripheral blood in % of patients. transplants were full matched in ( %) patients, had one mismatch (out of ) in ( %) and mismatches in ( %) patients. engraftment was complete as evaluated by donor chimerism at day and post transplant in patients ( %), partial in ( %) and graft failure was observed in ( %) patients. acute gvhd grade ii-iv was seen in patients ( %) and nih moderate to severe chronic gvhd was seen in ( %) patients. median overall survival was days ( - ) and estimated years overall survival was %. risk factors for survival identified were: hla mismatch and stem cell sources other than marrow. unrelated transplants are a feasible salvage therapy for patients with saa refractory to immunosuppression, being hla compatibility and marrow stem cell source factors with a positive impact on survival. disclosure of conflict of interest: none. use of haploidentical stem cell transplantation continues to increase, the european society for blood and marrow transplant activity survey report jr passweg , h baldomero , p bader c bonini , rf duarte , c dufour , , a gennery , n kröger , j kuball , f lanza , s montoto , a nagler , ja snowden , j styczynski and m mohty for the european society for blood and marrow transplantation (ebmt) hematopoietic stem cell transplantation (hsct) is an established procedure for many acquired and congenital disorders of the hematopoietic system, including disorders of the immune system, and as enzyme replacement in metabolic disorders. the annual activity survey of the ebmt describes the status of hsct in europe and affiliated countries and has become an instrument used to observe trends and to monitor changes in technology use. teams were invited to report their transplant activity for by indication, stem cell source and donor type using a single paged survey. a record number of ' hsct in ' patients ( ' allogeneic ( %), ' autologous ( %)) were reported by centers in countries in . trends include continued growth in transplant activity during the period and , with the highest percentage increase seen in middle income countries (allo %, auto %), and the lowest in very high income countries (allo %, auto %), for both allogeneic and autologous hsct. in contrast the absolute growth is highest in the very high income countries (growth allo rates transplants per × inhabitants, auto rates for very high income countries; allo rates , auto rates for middle income). main indications for hsct were myeloid malignancies ' ( %; % allogeneic); lymphoid malignancies ' ( %; % allogeneic); solid tumors; ' ( %; % allogeneic); and non-malignant disorders; ' ( %; % allogeneic). remarkable is a decreasing use of allogeneic hsct in cll from patients in to in and is most likely due to the development of potentially very effective cll drugs. use of haploidentical donors for allogeneic hsct continues to increase ' in ; a % increase since . the highest growth is seen in myeloid malignancies ' , with lymphoid malignancies , nonmalignant disorders and others. in aml, haploidentical hsct increases similarly for patients with both advanced disease and those in cr . both marrow and peripheral blood is used as stem cell source for haploidentical hsct with higher numbers reported for the latter. this year's activity survey shows continued increase in the use of haploidentical hsct across europe within the main indication groups and cell source. it reflects in a timely manner current trends in stem cell transplantation and is an essential tool for health care planning and health policy makers. human bone marrow mesenchymal stromal cells derived exosomes (hbmmdes) are small membrane vesicles secreted from mesenchymal stromal cells that may serve as a vehicle for protein, mrna and microrna (mirna) transfer to distant cells; affecting gene expression, proliferation, and differentiation of the recipient cells. therefore, mdes may possess some of the immunoregulatory properties of their parental cells. in the present study we aim to explore the immunomodulatory function of mdes and understand the molecular mechanisms enabling it. for this purpose, we co-cultured hbmmdes with activated human lymphocytes. using ultracentrifugation, hbmmdes were isolated from expanded human bone marrow derived mesenchymal stromal cells (hbmmscs). using em and zeta sizer, particles were shown to be in the range of - nm. pha activated human peripheral blood lymphocytes (pbls), r- /il activated b cells and anti cd /cd activated t cells were co cultured with purified mdes. cell proliferation was tested using thymidine incorporation assay. we found that exosomes derived from × to × mscs exhibited a dose-dependent inhibition of lymphocyte proliferation. exosomes derived from × mesenchymal stromal cells co cultured with pha activated pbls, activated b cells and activated t cells showed proliferation inhibition of %( p ⩽ . ), . % (p ⩽ . ) and . % (p ⩽ . ), respectively. in order to understand the molecular mechanism behind the immunomodulatory effect of mdes, we have profiled mde's mir content using illumina hiseq platform and we are currently profiling co cultured activated lymphocytes mrna content using next-generation sequencing system, illumina. preliminary results demonstrate some higher abundance of specific mscs derived mirs in the mdes. hbmmscs have been shown to serve as immune modulators in patients with acute and chronic graft versus host (gvhd). in the future, mdes may provide an alternative therapy for gvhd. compared with bmmscs, mdes are more stable, have no risk of aneuploidity or ectopic proliferation and have less probability of immune rejection. additional studies are needed to explore the applicability of mdes to serve as modulators of the immune response. disclosure of conflict of interest: none. graft-versus-host disease (gvhd) is the major complication after allogenic haematopoietic stem-cell transplantation s (hsct). extra virgin olive oil (evoo) is a source of phenolic compounds such as glycoside oleuropein, hydroxytyrosol and tyrosol. olive oil polyphenols have shown antioxidant, immunomodulatory, antiproliferative, anti-apoptotic and antiinflammatory properties that might be useful in the prophylaxis and treatment of gvhd. polyphenolic extract (pe) of evoo was obtained by the method described by vazquez roncero et al. with some modifications. briefly, fifty grams of evoo (oleoestepa, seville, spain) was extracted with methanol/water ( : , vol/vol, ml ). the mixture of evoo, methanol and water was decanted and the methanolic extract was concentrated and lyophilized. then, the effect of pe in cell viability and activation of t lymphocytes from healthy donor's buffy coats either resting or activated with anticd plus anticd was analyzed by flow cytometry after staining with aad, anexin-v and cd . proliferation assays were performed with pkh and the quantification of il- , il- , il- , il- , tnf-α and ifn-γ cytokines in cell culture supernants with bd cytometric bead array (cba). signaling pathways were analyzed by western blot. finally, in a mouse model of acute gvhd (c bl/ in balb/c), mice were randomized into two experimental diet groups: standard diet ( s harlan laboratories) and standard diet ( s harlan laboratories) supplemented with ppm of pe obtained of evoo. the severity of gvhd was assessed by a scoring system described by cooke et al. that incorporates five clinical parameters: weight loss, posture (hunching), activity, fur texture, and skin integrity. pe did not affect t cell viability. by contrast, pe decreased t-cell activation and proliferation of t-lymphocytes stimulated with anticd plus anticd . in addition, there was a decreased production of th (ifnγ, il- and tnf) and th cytokines (il- , il- and il- ) in the presence of pe. regarding the signaling pathways analyzed, pe inhibited phosphorylation of akt and nuclear translocation of nfkb in activated t cells. in the mouse model of acute gvhd, animals which received the pe supplemented diet had an increased survival as compared to mice receiving a standard diet. also, gvhd incidence was significantly lower among mice receiving the pe supplemented diet as assessed by both the presence of gvhd signs as well as pathological examination. polyphenols obtained from evoo are an important immunomodulatory agent capable to reduce the proliferation and activation of activated t cells and the production of proinflammatory cytokines. in a mouse model of acute gvhd, pe supplemented diet reduced the incidence and severity of the disease and increased the survival of mice. disclosure of conflict of interest: none. graft-versus-host disease (gvhd) is a leading cause of postallogeneic haematopoietic stem cell transplantation (hsct) morbidity and mortality ( ) . extracorporeal photopheresis (ecp) is an alternative therapeutic strategy that appears to act in an immunomodulatory fashion, potentially involving regulatory t lymphocytes and dendritic cells in patients who are refractory to steroids. dendritic cells (dcs) are the most important antigen-presenting cells, playing a pivotal role in t-cell function and in the link between innate and adaptive immunity. moreover, dcs are also critical mediators of immune tolerance and energy. they can be divided into two major subsets, plasmacytoid dcs (pdcs) and myeloid dcs (mdcs) which have distinct functions. pdcs play a pivotal role in peripheral tolerance through generation of regulatory t (treg). on the other side mdcs promote, as well as pdcs, th and th /tr responses ( - ). our study was performed to understand the mechanism of action involved in immunomodulatory effect of ecp. as the modulation of dcs and tregs number and function ( , ) may be a central mechanism of ecp in maintaining self-tolerance, down-regulating immune responses, and limiting inflammation ( ). eight patients affected by gvhd were included in this pilot study. in ecp apheresed mononuclear cells are exposed to methoxypsoralen and uva radiation. after this photoactivation, which induces dna damage and apoptosis, the cells exposed are re-infused into the patient inducing an immunomodulatory effect. all patients or their legal guardians gave their consent for this study. a sample of peripheral blood (pb) (basal condition), a sample of apheresis pre-uva photoactivation (pre-pa) and a sample of photoactivated apheresis (pa) were collected at the first day of ecp and every week for the first month of treatment. circulating dcs, mdcs (cd / -cd +cd +), pdcs (cd / -cd +cd +) and tregs (cd +cd +foxp +) were directly enumerated and phenotypically characterized. the assays were performed at day+ ,+ , + ,+ ,+ data are expressed as mean ± s.d. of absolute number of cells/μl. at day + there were no differences in the absolute number of both mdcs and pdcs between pre-pa and pa. consequently there were no differences between pb and pa. from day + till + we observed an increase of these two cellular populations at every date of treatment. comparing the basal pb of day + vs day + we observed an increment of % and %, respectively for mdcs and pdcs (mdc from cell/μl to cell/μl; pdc from cell/μl to cell/μl). comparing the basal pb of day + vs day + we observed an increment of % of tregs (from cell/μl to cell/μl) while we observed a median increment of % calculated between pre-pa and pa of each day of treatment from day to day + . no firm conclusions can be drawn from a clinical point of view, however a biological effect has certainly highlighted. in particular no substantial differences in basal pb mdc or pdc emerged during the first month of treatment while a significant increase of mdc and pdc can be observed since day + following uva photoactivation. regarding tregs we observed an increment of % of tregs between pb from day + to day+ and a median increment of % calculated between pre-pa and pa of each day of treatment. disclosure of conflict of interest: none. [p ] p impact of th cells on xenogeneic graft-versus-host disease l delens, s servais , g ehx , l vrancken , g fransolet , c gregoire , m hannon , s dubois , c daulne , f baron and y beguin giga i : hematology, university of liege acute graft-versus-host disease (gvhd) is a severe complication of allogeneic hematopoietic stem cell transplantation. its pathophysiology is complex and not yet fully understood. in particular, the impact of th cells on murine acute gvhd has yielded conflicting results, while demonstration of increased levels of th cells at the site of acute gvhd provided only indirect evidence of their involvement in humans. here, we assessed the potential implication of th cells in a humanized mouse model of xenogeneic gvhd (x-gvhd). methods: x-gvhd was induced by infusing human peripheral blood mononuclear cells (pbmcs) into nod-scid il- rγnull (nsg) mice given . gy total body irradiation day prior transplantation. th cells were generated by culturing naive cd + t cells with anti-cd /anti-cd coated beads under th -skewing cytokines (tgf-β , il -β, il- , il- , il- , neutralizing anti-il- and anti-ifnγ antibodies) in hypernatremic conditions (nacl mm). results: after days of culture, a median of . % of il- a+ cells was obtained. we confirmed the expression of il- a, rorc and il- r by these cells by rt-qpcr. we next assessed the co-injection of human pbmcs ( . ) with in vitro differentiated cells under th skewing conditions ( × ) (co-injection group, n = ), in comparison with the injection of pbmcs alone ( × cells, pbmcs group, n = ). we observed higher x-gvhd score (p %) of cells expressing both il- a+ and ifnγ+ cells (th / th -like phenotype) among cd + il- a+ cells while coinjected mice had higher blood concentration of il- a (p = . ) than pbmc mice. these results demonstrate that addition of th cells worsened x-gvhd confirming their role in acute gvhd pathogenesis. disclosure of conflict of interest: none. although survival from allogeneic stem cell transplantation (hsct) has significantly improved, acute graft-versus-host disease (gvhd) remains a major cause of death. intestinal dysbiosis has been associated with acute gastrointestinal gvhd and poor outcome after hsct. we reported a correlation between microbiota (gm) composition and short chain fatty acid (scfa) production and gvhd in transplanted children. to assess how the metabolic pathways of gm change during transplantation and identify modulators of immune response, we perform first longitudinal metagenomic analysis in children undergoing hsct. patients (pts) ( male; mean age: y) with hematologic malignancies ( all, aml), who received busulphan-based myeloablative conditioning and t-cell replete bone marrow graft were enrolled. pts were prospectively enrolled in a protocol with at least specimens fecal samples collected: one before and two after hsct, in order to build a proper trajectory. gvhd prophylaxis was cyclosporine for pts receiving a matched related donor and cyclosporine, short-term mtx and atg for pts receiving a matched unrelated donor. non-gvhd and gvhd patients had similar exposures to antibiotics during the stool collection. of these pts, % developed gvhd within the first days. we applied shotgun metagenome sequencing to total fecal dna from samples collected. functionalities were assigned by reads mapping at different levels of the kegg database. relative abundance was calculated and statistical analysis was performed. according to our findings, core functional profiles were overall conserved through the time-points in all patients ( figure a ), in contrast to the phylogenetic profiles behavior, this finding confirming the overall redundancy of gut microbiome core functionalities. analyzing the single metabolic pathways in subjects who developed gvhd, we found in the pre-hsct period a higher relative abundance of nucleobasis (purine and pyrimidine) metabolism (p o . ) and branched-chain amino acids biosynthesis (p o . ). functions related to the production of branched-chain amino acids are involved in the biosynthesis of the cell wall of gram-negative bacteria, microorganisms including subgroups with well know opportunistic pro-inflammatory. in addition, post-hsct samples of gvhd patients showed a lower abundance of genes involved in polysaccharides metabolism, as glycan biosynthesis and glycosaminoglycan degradation (p o . ) ( figure b ). glycosaminoglycan degradation activity gets bacteria able to survive during extreme situations, as fasting using mucus polysaccharides as energy source, contributing to maintain a mutualistic composition of gm and scfa production by the saccharolytic functions of the endogenous mucus polysaccharides. this study detects functional peculiarities in the gm of non-gvhd pts. the gut metagenome configuration of non-gvhd patients is structured to derive scfa after hsct. the production of these metabolites promotes peripheral regulatory t-cell generation , potentially explaining the protective role of gm from gvhd. although intestinal epithelial cells (iecs) are crucial regulators of barrier function and immune homeostasis, they also facilitate inflammation in exaggerate responses to proinflammatory mediators by pretransplant conditioning regimen, which plays a critical role in amplifying graft-versus-host disease (gvhd). thus inhibition of the converting to pathogenic iecs by conditioning may represent a novel approach to inhibit gvhd. aryl hydrocarbon receptor (ahr) is the ligandactivated transcription factor which has the ability to mediate the biochemical, metabolic, and toxic effects of environmental chemicals. recently, it has been demonstrated that ahr is an important regulator of cell development, differentiation, and function of both innate and adaptive immune cells. the ability of ahr is induced by respond to endogenous ligands generated from the host cell, diet, and microbiota. here, we investigated the regulatory role of ahr in iecs under inflammatory responses and its therapeutic activity for modulation of gvhd. ahr and cyp a expression in mouse iecs were determined by real-time pcr. mouse iecs were pretreated with endogenous ahr ligands l-kynurenine (l-kyn, mm) or pbs for h and then stimulated with lps or il- b for h. cytokine levels were measured using the mouse flex-set cytokine bead array or real-time pcr. b d f (h- b/d) recipients were administrated l-kyn daily by i.p. injection for days. then the recipients were lethally irradiated and transplanted with × tcd-bm plus × t cells from b (h- b) donor. mice were monitored every other day for survival and clinical score. colons were collected and stained with hematoxylin and eosin (h&e) for histopathological scoring. we found that ahr was constitutively expressed in the mouse iecs. cyp a (an ahr target gene) was significantly increased by treatment of l-kyn under un-stimulatory condition. we further observed that l-kyn completely abrogated il- β-mediated il- or lps-mediated tnf-a expression in iecs. administration of bdf recipient mice with l-kyn before transplantation significantly reduced the lethality and severity of gvhd. histopathology clearly revealed that treatment of l-kyn inhibited intestinal gvhd. our results demonstrate that ) ahr is constitutively expressed in iecs, ) treatment of endogenous ligand l-kyn induce ahr activation in the steady status, ) ahr activation blocks conversation of the epithelial cells into pathogenic cell type, and ) pre-administration of ahr ligand reduces gvhd. our study suggests that activation of ahr pathway in iecs before allogeneic hematopoietic stem cell transplantation (hsct) is a possible strategy to reduce intestinal gvhd. disclosure of conflict of interest: none. [p ] s relating with acute graft-versus-host disease (gvhd) after allogeneic hematopoietic stem cell transplantation (allo-hsct), protecting endothelial cells (ecs) from damage may be a potent prophylaxis and therapeutic strategy of acute gvhd (agvhd). conventional agvhd therapies may cause many adverse side effects because of their multiple targets. therefore, we explored the therapeutic efficacy of simvastatin, a lipid-lowering drug, which has been demonstrated endothelial protection. our previous clinical observation has found patients with agvhd had lower angiopoietin- (ang- ) level at day but higher ang- level at day than those without agvhd. in this study, we explored changes in ang- and ang- expression in an agvhd mouse model and determined whether simvastatin prevents gvhd through regulating ang- and ang- expression. we preincubated ea.hy ecs with simvastatin ( mmol/l) h before stimulated with tnf-a, then ang- and ang- concentration in the cell supernatant was measured by elisa. ang- and ang- mrna and protein level of treated and untreated cells were examined simultaneously. in vitro simvastatin increased ang- production and release but conversely inhibited ang- release from ea.hy ecs. donor mice spleen cells were injected along with bone marrow cells into recipient mice after lethal irradiation to induce agvhd. simvastatin was administered orally once daily to mice ( mg/kg) for days after allo-hsct and started − day after allo-hsct. then mice survival time was monitored and organ damage was evaluated. the plasma level of ang- and ang- was measured by elisa, expressions of ang- and ang- in aortic endothelium were assessed by immunohistochemistry. simvastatin improved the survival and attenuated the histopathological gvhd grades of agvhd mice. plasma levels of ang- were significantly decreased, while plasma levels of ang- obviously increased in agvhd mice after transplantation. simvastatin reduced plasma levels of ang- , elevated the plasma levels of ang- as well as the aortic endothelial levels of ang- and ang- . in summary, simvastatin represents a novel approach to combat gvhd by increasing ang- production while suppressing ang- release to stabilize endothelial cells. there is a growing evidence of safety and efficacy of posttransplantation cyclophosphamide (ptcy) in stem cell transplantations (sct) from different donors and graft sources. still the optimal combination of immunosuppressive agents with ptcy should be elucidated for different types of scts. we report the -year update of the prospective nct single-center trial that evaluated risk-adapted graft-versushost disease (gvhd) prophylaxis with ptcy in related, unrelated and haploidentical scts. adult patients (median age y.o., range: - ) with hematologic malignancies, including aml ( . %), all ( . %), cml ( . %), mds ( %), and lymphomas ( . %), were enrolled in the study. % of patients were classified as salvage. % received the graft from matched related (mrd), % from matched/mismatched unrelated (mud/mmud), and % from haploidedntical (haplo) donor. % received bone marrow graft (bm) and %peripheral blood stem cell (pbsc) graft. . % had myeloablative conditioning and . %-reduced-intensity conditioning. gvhd prophylaxis for matched bm grafts consisted of single-agent ptcy mg/kg days+ ,+ , for matched pbsc graft-ptcy+ tacrolimus+ mycophenolate mofetil (mmf) mg/kg days - , and for any mismatched graft-ptcy+ tacrolimus+ mmf mg/kg days - . median follow-up was months (range: - ). grade ii-iv ( % vs % vs %, p = . ) and grade iii-iv acute gvhd ( % vs % vs %, [p ] p = . ) were not different in mrd, mud/mmud and haplo groups, respectively. moderate and severe chronic gvhd was infrequent in all groups with slightly lower incidence after mud/mmud graft: and % vs % vs %, p = . . nonrelapse mortality (nrm) was not different after mrd, mud/ mmud and haplo sct ( % vs % vs %, respectively, p = . ), while relapse incidence was higher after mrd and haplo grafts: ( % vs % vs %, p = . ). -year overall survival (os), event-free-survival (efs), and gvhd-relapse free survival (gfrs) were % vs % vs % (p = . ); % vs % vs % (p = . ); % vs % vs % (p = . ) for mrd, mud/mmud and haplo groups, respectively. in the multivariate analysis only disease risk index (hr . %ci . - . , p = . ), severe sepsis (hr . %ci . - . , p = . ) and chronic gvhd (hr . %ci . - . , p = . ) were predictive for efs, while type of donor was not a significant factor (hr . %ci . - . , p = . ) (figure ). the incidences of complications were: hemorrhagic cystitis- %, sepsis- %, severe sepsis- %, invasive mycosis- %, cmv reactivation- %, veno-occlusive disease- . %, transplant-associated microangiopathy- . %, grade - liver toxicity- %, grade - kidney toxicity- %. more than one third of patients experienced poor graft function during days after sct, and in % of them cmv, hhv and bk virus reactivations were identified as the cause. the reported risk adapted strategy alleviates the risk of gvhd and nrm after mmud and haplo grafts. the observed differences in the relapse incidence, os and efs were predominantly due to unbalanced disease risks in the groups. the relapse of underlying malignancy with this prophylaxis still significantly influences the outcome. substantial number of patients experience poor graft function, which doesn't translate into nrm. disclosure of conflict of interest: none. a high migratory capacity of donor t-cells in response to the lymph node homing receptor ccr increases the incidence and severity of graft-versus-host disease vg garcía de soria , ip sainz , e jiménez , a arriero , c fernández-arandojo , c cuesta , b colom , a marcos , a rosendo and cecilia muñoz calleja department of hematology hospital universitario de la princesa and department of inmunology. hospital universitario de la princesa graft-versus-host disease (gvhd) pathogenesis involves migration of the donor t-cells into the secondary lymphoid organs (slo) in the recipient, which is steered by two homing molecules: cd l and ccr . therefore we investigated whether the migratory capacity of donor t-cells is associated with gvhd. this single center prospective study included donor-recipient pairs. in vitro chemotaxis assays of the lymphocytes of the apheresis product were performed in parallel with the analysis of cd l and ccr by flow cytometry. the potential of activation of ccr + t-cells was assessed through ex vivo activation assays with peripheral blood monuclear cells (pbmc) from healthy donors using anti-cd and anti-cd mabs. the migratory index to the ccr ligands, ccl and ccl , was higher in t-cells from donors whose recipients will develop gvhd. these data indicated that the migratory capacity of the donor t-cells is clearly related to the development of gvhd. this prompted us to study the relationship between gvhd and the expression of two of the most relevant molecules in the trafficking of lymphocytes towards slo, cd l and ccr ,as a subrogate index of the migratory potential of t-cells. consequently, we quantified the numbers of cd l+ and ccr + t-cells in the graft. the initial transversal analysis of our data revealed that the percentage of cd l+ lymphocytes in the apheresis product was very low compared to healthy lymphocytes. the analysis also confirmed that cd l undergoes plasma membrane shedding after g-csf mobilization thus making it a non-valid biomarker. the analysis of ccr molecule revealed that the acute gvhd group received higher percentage of cd +ccr + t-cells, whereas chronic gvhd patients were transplanted with higher percentage of cd +ccr + t-cells compared to the non gvhd group. these results were confirmed when patients were subdivided into degrees of severity. a multivariate analysis was performed to investigate the real value of ccr to predict the development and severity of gvhd, and confirmed that ccr expression is a risk factor for the development of gvhd. thus, the percentage of ccr +cd + t-cells increases the probability of developing acute gvhd (or = . , c.i ( %) = . - . , p = . ) and suffering a higher degree (or = . , c.i ( %) = . - . , p = . ). similarly, the or of the percentage of ccr +cd + t-cells was . (c.i ( %) = . - . , p = . ) and . (c.i ( %) = . - . , p = . ) for the development of chronic gvhd and its degrees, respectively. finally, to study the potential of activation of ccr + t-cells, we carried out ex vivo activation assays with pbmc from healthy donors using anti-cd and anti-cd mabs and the expression of cd l on cd + t-cells and of cd on cd + t-cells as markers of activation, demonstrating that ccr + t-cells exhibited higher potential of activation than ccr -t-cells. to our knowledge this is the first analysis of the influence of the migratory capacity of the donor t-cells on clinical outcome following allogeneic hsct. our data show that ccr could be considered a subrogate biomarker of the migratory capacity of the donor lymphocytes for predicting the risk of suffering gvhd. based on the previous findings, we propose that the selective depletion of ccr expressing cells could be an effective preventive therapy for gvhd. disclosure of conflict of interest: none. previously published p a single center research for outcome in patients receiving imatinib for steroid-refractory chronic gvhd after allogeneic stem cell transplantation l ni, y luo, y tan, y hu, y zhao, j shi and h huang despite of major progress in allogeneic stem cell transplantation over the last decades, steroid-refractory chronic graftversus-host disease (sr-cgvhd) remains a leading cause of late morbidity and mortality. pre-clinical evidence confirms cgvhd has antibodies activating the platelet-derived growth factor receptor (pdgf-r) pathway. since this pathway can be inhibited by imatinib, we performed a study including patients with sr-cgvhd given imatinib at a dose of mg per day. all patients with a median age of years (range: - ) underwent allogeneic hematopoietic stem cell transplantation in our single center between and , and chronic gvhd occurred at a median time of months (range: - ) after transplantation. patients had active cgvhd with measurable involvement of skin, lung or other districts and had previously failed in first-line immunosuppressive therapy. the major organs involved were lung (n = ), skin (n = ) and mouth (n = ), including cases involving both lung and skin, cases involving or more organs. according to the national institutes of health (nih) criteria and nih global severity, patients were evaluated as severe cgvhd, and the other three were moderate. meanwhile, the nih working group had updated its recommendations for overall responses, consisting of complete remission (cr), partial remission (pr), and lack of response (unchanged, mixed response, progression). cr was defined as resolution of all manifestations in each organ or site, and pr was defined as improvement in at least organ or site without progression in any other organ. after months treatment, patients receiving sufficient dose of imatinib revealed overall response rate (orr) at . %, and orr remained unchanged at months assessment, but with cr rate increased to . %. two patients couldn't meet the response of cr or pr were considered as a lack of response, including one evaluated as unchanged and one mixed response because of pr in lung accompanied by progression in eyes. with a median follow-up of months, patients were alive, with a year estimated overall survival was . %. patients eventually died of pneumonia. except patient discontinued imatinib because of grade toxicity as gastrointestinal discomfort at the first month, no one had imatinib-related grade to toxicity. this study suggests that imatinib is a promising and better tolerated treatment for patients with sr-cgvhd. disclosure of conflict of interest: none. acute graft-versus-host-disease (agvhd) is a major complication after allogenic hematopoietic transplantation (allo-sct). in recent years, a number of tissue-specific proteins have been described as biomarkers that could contribute to anticipate and/or diagnose this complication earlier and more accurately. reg α (regenerating-islet-derived- -alpha) has been directly related to gastrointestinal (gi) agvhd. our objective was to analyze plasma levels of reg α at days + and + in patients who underwent unmanipulated haploidentical transplantation with reduced conditioning regimen (haplo-ric), and to correlate the results with the development of agvhd. we retrospectively analyzed consecutive patients ( - ) who underwent haplo-ric with post-transplant cyclophosfamide (days + , + ), mmf and csa as gvhd prophylaxis. seven cases were excluded due to early death (before day + ) and cases due lack plasma sample. characteristics of the patients included in the analysis are described in table . reg α detection was performed by elisa (mbl international corp, woburn, ma) according to manufacturer's instructions on μl of plasma obtained at day + and + . the association of the incidence of agvhd with known clinical variables and plasma reg a levels were performed by cox regression and mann-whitney u-test, respectively. the determination of the best cut-off of reg α levels to stratify patients with gi agvhd was performed with roc curves. the stadistical program used was r v . . . the cumulative incidence of grade ii-iv and grade iii-iv agvhd was % and %, respectively. characteristics of agvhd are shown in table . no association was found between agvhd and usual clinical variables (stem cells source, age, sex, conditioning regimen, donor/recipient sex and number of infused cd + cells), and with plasma reg α levels at day + .plasma reg α levels at day + were higher in patients who devolved gi agvhd compared to patients who did not showed gi agvhd (median [p ] s and range: ( - ) vs ( - ) pg/ml, p = . , figure ).the best cut-off selected on day + was pg/ml (s %, e %). patients with levels higher than pg/ml at day + had a significantly higher incidence of gi agvhd grade ii-iv (hr . , p = . , figure ). plasma levels of reg α at day + after haplo-ric correlated with the occurrence of gi agvhd grade ii-iv. therefore, plasma levels of reg α could be use for the prediction and/or diagnosis of gi agvhd. disclosure of conflict of interest: none. anti-fibrotic treatment with pirfenidone in patients with gvhd-associated bronchiolitis obliterans syndrome ke hostettler, s gerull, g nair , j passweg , m tamm and j halter hematology, university hospital basel, switzerland and pneumology, university hospital basel, switzerland prognosis of lung gvhd remains poor due to progressive decrease of lung function and repeated infections. pirfenidone exhibits anti-fibrotic effects and has been shown to reduce disease progression in patients with idiopathic pulmonary fibrosis. five patients with established bos (nih criteria ) and stable or deteriorating lung function under standard immunosuppressive treatment without active infection were treated with pirfenidone ( mg/d) in addition to their current therapy. clinical assessments and pulmonary function tests were performed every three months. five patients ( m, f), median age y (range: - y) that were diagnosed with bos at a median time of . months post-transplant started pirfenidone at a median time of months ( - ) after diagnosis of bos. two patients are currently still under treatment after and days. two patients had to stop treatment due to financial reasons after and days of therapy. one patient never reached more than % of the planned dose due to gastro-intestinal symptoms and was excluded from further analysis. at the start of treatment median fev was . l ( . - . ); . % predicted (range: - %) and median fvc . l ( . - . ); - % predicted. median fev trajectory was − . % predicted/ month during median months before start of pirfenidone (median − ml/month) and + . % predicted/month (+ . ml/month) during treatment with pirfenidon. the treatment was well tolerated except in one patient with gastrointestinal complaints, no phototoxic reactions or serious drugrelated adverse events occurred. in our small number of patients pirfenidone was rather well tolerated and generally safe. the observed, albeit small trend in change of fev trajectory justifies further studies of anti-fibrotic therapy as a new therapeutic option in bos after allogeneic hsct. disclosure of conflict of interest: none. anti-thymocyte globulin has been widely used for the prevention of severe graft versus host disease in patients undergoing hsct from unrelated donor. however, the optimal dose remains to be defined. in samsung medical center (seoul, korea), institutional strategy for the atg use has been changed since april , and we hypothesized that the incidence of chronic gvhd may differ by atg strategy. before april , atg . mg/kg was routinely used in allogeneic hsct from unrelated donor, whereas, the dose of atg was escalated to . mg/kg since april . in this study, a total of patients who underwent allogeneic hsct from matched or unmatched unrelated donor between jan and dec were retrospectively analyzed. peripheral blood was used as the source of stem cells in all patients. after a median follow up of . months, the cumulative incidence of moderate to severe chronic gvhd was . % ( % confidential interval [ci], . to . ) in the low-atg group and . % ( % ci, . to . ) in the non-atg group (p = . ). the rate of year overall survival (os) was not significantly different between the groups ( . % in low-atg group vs . % in high-atg group, p = . ), as was the rate of disease free survival (dfs) ( . % in non-atg group vs . % in atg group, p = . ) and cumulative incidence of relapse (cir) ( . % in non-atg group vs . % in atg group, p = . ). in allogeneic hsct from unrelated donor, larger atg dose ( . mg/kg) did not reduce the incidence of chronic gvhd when compared to lower atg dose ( . mg/kg). disclosure of conflict of interest: none. allogeneic hsct provides a curative chance for patients with hematological fatal disease. however, substantial risks remain for morbidity and mortality caused by disease relapse and graft-versus-host disease. in samsung medical center (seoul, korea), institutional strategy for the atg use has been changed since april , and we hypothesized that the incidence of chronic gvhd may differ by atg strategy. before april , atg was not routinely used in matched sibling donor (msd) transplantation, whereas, atg mg/kg has incorporated into hsct process in transplantation from msd thereafter. in this study, a total of patients who underwent allogeneic hsct from msd between jan and dec were retrospectively analyzed. peripheral blood was used as the source of stem cells in all patients. after a median follow up of . months, the cumulative incidence of moderate to severe chronic gvhd was . % ( % confidential interval [ci], . to . ) in the atg group and . % ( % ci, . to . ) in the non-atg group (p = . ). the rate of -year overall survival (os) was not significantly different between the groups ( . % in non-atg group vs . % in atg group, p = . ), as was the rate of disease free survival (dfs) ( . % in non-atg group vs . % in atg group, p = . ) and cumulative incidence of relapse (cir) ( . % in non-atg group vs . % in atg group, p = . ). in allogeneic hsct from msd, atg use was significantly associated with less occurrence of chronic gvhd, but not linked to increasing risk of relapse, with showing similar os and dfs between atg and non-atg group. disclosure of conflict of interest: none. long-term follow-up from the prospective randomized phase iii multicenter trial comparing a standard gvhd prophylaxis with cyclosporine a and methotrexate with or without additional pretransplant atlg (grafalon, previously atg-fresenius s) (given mg/kg/day, days − to − ) in unrelated donor hematopoietic cell transplantation after myeloablative conditioning resulted in a significant reduction of acute and chronic gvhd without compromising relapse rate and survival [ , , ] . here we report on a subsequent prospective non interventional observational study evaluating the outcome of patients receiving atlg in unrelated donor transplantation in day to day clinical practice without the selective measures of a clinical trial (german clinical trials register drks ). thirteen transplant centers included patients with haematological malignancies (median age years, iqr - years, range: - years) in early (n = , %), intermediate (n = ; %) or advanced (n = ; %) disease status receiving marrow (n = ) or pbsc (n = ) from / matched ( ; %) or mismatched ( ; %) unrelated donors (n = related) after myeloablative (n = , %) or ric (n = , %) conditioning. gvhd prophylaxis consisted of calcineurin inhibitors, mainly csa (n = , %) with mtx or mmf and atlg. different dosing regimens were allowed according to current practise of centers. median total atlg dose was mg/kg (iqr - mg/kg, range: - mg/kg). median follow-up was months (range: - months). as compared to patients in our randomized phase iii multicenter trial [ , , ] , patients in this study were older; advanced disease status, / match, pbsc transplantation were more frequent, and given median atlg dose was lower. acute and chronic gvhd, nrm, relapse risk, dfs and os at one year were similar to the results obtained in our randomized trial: incidence of°ii-iv agvhd: %, iii-iv agvhd: %; moderate/severe cgvhd: %; nrm: %; risk of relapse: %; relapse mortality: %; os: %. the experience in day to day clinical practice confirms the results shown in our randomized trial, namely the gvhd protective effect of atlg without compromising nrm or relapse rates. baseline calprotectin as a predictor for acute gastrointestinal graft versus host disease (gvhd)-a prospective study n schmidlin , a holbro , , jp halter , d heim , l infanti , , a plattner , r plattner , c rothen , a buser , , c bucher and jr passweg division of hematology, university hospital basel, switzerland; blood transfusion center, swiss red cross, basel, switzerland and rothen medical laboratories, basel, switzerland graft versus host disease (gvhd) is a major complication after allogeneic stem cell transplantation. so far there is no good validated predictor for the incidence and severity of gvhd. fecal calprotectin (cpt) is a protein in leukocytes with antibacterial properties. it has been shown to be elevated in acute gastrointestinal gvhd. additionally, cpt may be predictive for treatment response. the aim of the current prospective study was to investigate the role of baseline cpt in predicting incidence and severity of intestinal gvhd. in this prospective study conducted at the university hospital basel, switzerland, we included all adult patients undergoing hsct. the institutional review board approved the study. data were collected prospectively. cpt was measured twice before conditioning and at transplantation. fecal samples for cpt were obtained before conditioning and on the day of transplantation and assessed twice by standard elisa. between march and april a total of patients ( % males, patients with both baseline and transplant cpt values) were included. patient, disease and transplant characteristics are described in table . median age at transplant was years (range: - years). most patients had myeloid neoplasia and % received myeloablative conditioning. gvhd prophylaxis consisted mainly of cyclosporine containing regimens ( %). cpt levels ranged from to μg/g both at baseline (median: μg/g) and at transplantation (median: μg/g), with a good consistency between the two measurements performed (internal quality control). on the other hand, cpt did not correlate with c-reactive protein. the two measurements were taken in median days apart, depending on the conditioning regimen. eighty-five patients had an increase of at least μg/g between baseline and transplantation. overall ( . %) patients developed acute intestinal gvhd (grade : ; grade : ; grade : , and grade : patients, respectively). cpt both at baseline and at transplantation was not predictive for the incidence of gvhd, acute intestinal gvhd, and for acute intestinal gvhd grade - ( figure) . additionally, we did not find a significant association between cpt levels and the above mentioned endpoints for patients showing an increase of cpt of at least μg/g between baseline and transplantation. in the current prospective study, we didn't find any correlation between baseline cpt values and the incidence and severity of gvhd and intestinal gvhd. further studies identifying early markers and predictors of gvhd are urgently needed. [p ] disclosure of conflict of interest: none. calcinuerin inhibitor (ci) free graft-versus-host disease (gvhd) prophylaxis: its effects on resource utilization, renal function, and the cost of care m muilenburg , k cole, m abidi , , s williams and as al-homsi , spectrum health blood and marrow transplantation and michigan state university, college of human medicine effective gvhd prevention following allogeneic hematopoietic stem cell transplantation (ahsct) is vital to reducing transplant morbidity and mortality and improving overall outcomes. several strategies are currently utilized for gvhd prophylaxis including mtx, mmf, cis, post-transplant cyclophosphamide (cy), and proteasome inhibitors. recently, we described the results of a phase i-ii trial of cyclophosphamide (cy) and bortezomib (bor) where patients (pts) received cy ( mg/kg) on days (d) + & + and bor on d & + . the incidences of grade ii-iv and grade iii-iv acute gvhd were % and %. the incidence of chronic gvhd was %. in addition to gvhd, there are other factors that affect patients' quality of life and cost of care and that should be considered. it is well documented that cis have an unfavorable toxicity profile. this includes nephrotoxicity and electrolyte disturbances. furthermore, the cis need serial level monitoring. thus, we endeavored to compare the effects of cybor combination against ci-based regimens by focusing on electrolyte requirements, specifically mg, and renal function. we also sought to better understand financial considerations surrounding the need for ci drug level monitoring. sixteen pts were randomly selected from the cybor group and patients from an internal control group of patients who received mmf and cyclosporine or tacrolimus following reduced-intensity ahsct. the groups were well matched in regards to age, sex, disease status, pam score, and baseline renal function. on each pt, mg results from d to + were compiled. based on institutional protocol, a mg replacement value was assigned as well as the corresponding drug and infusion charges. next, the number of immunosuppressant (is) trough levels from d to + was tallied and the internal lab charges calculated. to compare renal function, gfr was calculated at baseline, d , and d + . χ tests and wilcoxon rank square tests were used to analyze the data. for the cybor group, median mg value was . mg/dl (iqr . ) vs . ( . ) in the control group (po . ). cybor pts required a median of grams ( ) vs grams ( ) in the control group (p = . ). the cost of mg replacement and infusion was significant (p = . ) ( table ) . for is checks, drug levels were checked a mean of . times per patient in the cybor group compared to . times in the control group (po . ), which also translates to significant savings ( table ) . considering these costs, the cybor group saved~$ . for gfr, cybor pts and control pt had reduced gfr at baseline. on d + , cybor pts had better renal function in comparison to the control group (p = . ) ( figure ). in summary, cybor significantly reduced the use of resources post-transplant and thereby the associated cost related to mg replacement and need for drug level monitoring. furthermore, cybor preserved renal function at d + . these findings could also impact patient's quality of life. although our cost analysis was restricted to certain aspects of care and did not take into account other factors, it highlights specific important benefits of ci-free gvhd prophylaxis and supplicates further study. a formal prospective comparison of cost and qol is warranted. . related donor n = and unrelated donor n = (compatibility / n = ), with conditioning regimen: myeloablative n = and non-myeloablative n = . median interval between transplantation and diagnosis of cgvhd of months( . - . ); and between cgvhd diagnosis and sativex months ( . - . ), with a median of prior treatment lines ( - ). at the time of beginning, the cgvhd was extensive in all patients, severe cgvhd n = and moderate cghvd n = . all patients except one had cutaneous involvement (n = with sclerodermal features). in addition, other organs were affected: digestive n = , pulmonary n = , hepatic n = , ocular n = , oral n = , genital n = and muscular n = . drug was started because of pulmonary affectation in patients and due to sclerodermal/muscular involvement in patients. concomitant therapies during treatment were: topical cutaneous treatment n = , topical ocular treatment n = , pulmonary n = , sirolimus n = , tacrolimus n = , oral corticosteroids n = , extracorporeal photopheresis s n = , ruxolitinib n = , imatinib n = , mesenchymal stem cells n = . the mean dose were three puff/day ( ) ( ) ( ) ( ) , with good tolerance, only two discontinuations of treatment because of adverse effects. median time of treatment days ( to ). at the time of the analysis patients were still under treatment. responses mainly occurred within the first days, s with a median time of duration of days ( in ). responses after two months of treatment were: partial organ response, mixed responses, unchanged and organ progressions; at th day ( / ) only two patients maintained their responses (one pr and one mixed response). it must be pointed out that one patient who reached pr with sativex in monotherapy maintain response after months of treatment. in addition, cramps were resolved in patients. sativex appears to be an effective treatment option in patients with chronic gvhd, particularly in those having cramps, sclerodermal features and pulmonary affectation. as seen in multiple sclerosis context, the main issue with its use is the loss of response in the long-term follow up. the median dose is inferior to the one described in ms, leaving the question if higher doses can deepen the response. these results should be confirmed in prospective trials. disclosure of conflict of interest: none. several risk factors associated with acute and chronic graftversus-host-disease (gvhd) have been identified in multiple studies. most commonly associated factors are human leukocyte antigen (hla), mismatch between recipient and donor, as well as several other characteristics such as age, conditioning regimen and prior acute gvhd. objective: the aim of this study was to evaluate the characteristics of acute and chronic gvhd in patients who underwent an allogenic hematopoetic stem cell transplantation (hsct), identify differences in the profile risk factors for acute and chronic gvhd and their impact in post-transplant morbidity and mortality. this retrospective study included mexican adult patients who received an allogenic hsct between january and march , at instituto nacional de cancerologia. we analyzed patients with a median age of years ( - ), from which, % were male patients. among the participants with hematologic malignancies, were previously diagnosed with acute lymphoblastic leukemia, acute myeloid leukemia, chronic myeloid leukemia, lymphoblastic lymphoma and with myelodysplastic syndrome. because bone marrow transplants are not performed at this institution, all transplants were from peripheral blood stem cell harvest. acute gvhd prophylaxis consisted in a triple immunosuppressive drug regimen for all patients. . % of the patients had high risk disease prior to hsct. myeloablative conditioning represented % of the applied regimens, which consisted of iv busulfan in . % of the cases. . % of patients, were transplanted within months from diagnosis. the cumulative incidence of acute gvhd at days was . % ( patients). patients with acute gvhd had % grade a, % grade b and % grade c, according to the ibmtr grading system. patients had skin involvement, with grade - acute gvhd in % of the cases, patients developed liver involvement and patients had gastrointestinal tract disease. % of the patients developed chronic gvhd, from which, % were classified as severe, . % as moderate and . % as mild. % of the patients who developed chronic gvhd had a single organ involvement, while . % had or more organs/sites. prior acute gvhd was associated with de development of chronic gvhd. the multivariate analysis identified hla unrelated donor as the only risk factor associated with the development of acute gvhd (hr, . ; % ci, . - . , p = . ). the overall survival at years was of % poor patients who developed acute gvhd and of % for those who didn´t (p = . ). our analysis showed that the incidence of acute and chronic gvhd at our center is lower than the reported at other centers, but we were not able to identify risk factors usually associated with the development of gvhd, perhaps due to the small population that we evaluated. graft versus host disease (gvhd) remains one of the main obstacles to broader application of allogeneic stem cell transplantation (sct). despite the routine use of prophylactic therapies, chronic gvhd (cgvhd) occurs in to % of patients undergoing allogeneic sct. ciclosporin a (csa) remains the backbone for gvhd prophylaxis in both myeloablative (mac) and reduced intensity conditioning (ric) sct. however, in a significant proportion of patients, csa causes important side effects and needs to be discontinued. in this study we have evaluated the impact of substituting csa for mycophenolate mofetil (mmf) as immunosuppression (is), on the incidence of cgvhd. we have compared the outcome of consecutive patients that underwent allogeneic sct from march to november at the bmt unit of the hammersmith hospital and received csa as part of the gvhd prophylaxis. of them, patients ( %) remained on csa prophylaxis for the duration of the planned post sct immunosuppression period and patients ( %) required a switch to mmf before day + . the reason for changing the is was nephrotoxicity in the majority of cases (n = , %), neurological toxicity (n = , %), disease relapse (n = , %), intolerance (n = , %) or not determined (n = , %) . we excluded from the analysis those patients whose is was changed due to the presence of acute gvhd. both groups had similar patient and transplant characteristics (see table ). [p ] however, distribution according to diagnosis showed a predominance of aml ( %) in patients that remained on csa and mds ( %) for those that switched to mmf. the mean survival rate of the entire cohort was . days (± ) . the mean survival of each group was: csa . days (± . ) and mmf . (± . ). this difference in survival reached statistical significance (p: . ). we graded cgvhd using the nih scoring system as mild, moderate and severe. out the patients that continued with csa, . % (n = ) had no cgvhd; . % (n = ) had mild cgvhd; . % (n = ) had moderate and . % (n = ) had severe cgvhd. in patients that switched to mmf . % (n = ) did not develop any cgvhd; . % (n = ) developed mild; . % (n = ) moderate cgvhd and . % (n = ) developed severe cgvhd. (p: . ). the cumulative incidence of any cgvhd at years post sct was % for the csa/mmf group and % for the csa only group (p = . ). csa is one of the standards of care for gvhd prophylaxis in both ric and mac sct. in our cohort of patients, those who remained on csa had a better overall survival and a reduced incidence of chronic gvhd compared with those patients that stopped csa and replaced it by mmf. csa toxicity should be prevented to avoid gvhd-related complications. disclosure of conflict of interest: none. although the outcome of allogeneic stem cell transplantation (sct) from an unrelated donor (ud) has considerably improved over the recent years, graft versus host disease (gvhd) still represents a severe and potentially lethal complication. in vivo t-cell depletion with anti-thymocyte globulin (atg) has been shown to significantly decrease the risk of both acute and chronic gvhd without compromising survival, however the optimal dose has not been defined yet. aim of present retrospective study was to evaluate the impact of two different doses of rabbit atg (thymoglobulin) on gvhd incidence, infectious complications and outcome of patients undergoing sct from ud. between february and september , patients received thymoglobulin mg/kg (atg- group) and received thymoglobulin mg/kg (atg- group) in addition to cyclosporin and short course mtx as gvhd prophylaxis. the two groups were comparable regarding sex, age, diagnosis and disease phase at transplant, comorbidity index, stem cell source and antimicrobial prophylaxis. conditioning treatment was myeloablative in % of atg- group patients and in % of atg- group patients. donor and recipient pairs were / hla matched in % of the cases of the atg- group and in % of the cases of the atg- group (p . ). netrophil engraftment occurred in ( %) patients at a median of days post transplant (range: - days); six patients ( in the atg- group and in the atg- group) died before engraftment. overall, patients ( %) developed grade ii-iv acute gvhd, without significant differences between the two groups (atg- % and atg- %, p . ). similarly, chronic gvhd was not significantly different between the two groups: moderate to severe chronic gvhd occurred in % of the patients in the atg- group and in % of the patients in the atg- group (p . ). univariate logistic regression analysis didn't show any significant differences between the two groups respect the incidence of bacteremia, invasive fungal infections acute and chronic gvhd. with a median follow-up of . months, patients ( %) are alive, in complete remission and after disease relapse. transplant related mortality was superimposable in the two groups (atg- % vs atg- %). kaplan-meier estimates of overall survival and event free survival were % and %, respectively, without statistically significant differences between the two groups and between hla matched and hla mismatched sct. the results of our study suggest that different doses of atg tailored on hla compatibility might be effective for preventing gvhd with any detrimental effect on overall survival and incidence of infectious complications. a prospective randomized study is mandatory to confirm our preliminary results. disclosure of conflict of interest: none. c-reactive protein levels at acute gvhd diagnosis predict steroid-resistance, treatment related mortality and overall survival after allogeneic hematopoietic stem cell transplantation l minculescu , ls friis , bt kornblit , sl petersen , i schjødt , ns andersen and h sengeløv department of hematology, rigshospitalet, copenhagen, denmark acute graft versus host disease (agvhd) remains an excessive cause of morbidity and mortality after allogeneic hematopoietic stem cell transplantation (hsct). primary treatment consists of high-dose corticosteroids, but a small group of patients are steroid-resistant and their prognosis is especially poor. a predictor of patients at risk of steroid-failure would aid the decision of additional immunosuppressive treatment at an early stage. there is experimental evidence that co-existing inflammation aggravates agvhd. since c-reactive protein (crp) is a systemic inflammatory marker, we aimed to investigate whether crp levels at agvhd diagnosis could predict the risk of failing first-line therapy and developing steroid-resistance. we retrospectively studied patients transplanted between and , table . acute gvhd was diagnosed in patients, of whom had grade ii-iv. crp, total white blood cell-, lymphocyte-and neutrophil counts were available for all patients at the time of agvhd diagnosis. according to local protocol, patients with failed response to high-dose steroid ( mg/kg) were treated with the tumor necrosis factor (tnf) alpha inhibitor infliximab and categorized as steroid-resistant. of grade ii-iv agvhd patients ( %) developed steroid resistant disease. crp levels at diagnosis among these where between o and mg/l. crp levels where significantly higher in patients who developed steroid resistance compared to patients responding to high-dose corticosteroids, p = . , hr . ( % ci . , . ). this translated into significantly increased transplant-related mortality (trm) and decreased overall survival in patients with high crp levels, figure . total white blood cell-, lymphocyte-and neutrophil counts were not associated with steroid resistance in agvhd patients. cxcr is chemokine receptor expressed on activated t lymphocytes, in particular on th cells, nk cells, dendritic cells, and subsets of epithelial and endothelial cells. cxcr ligands attract th cells into inflamed tissues and concomitantly block the migration of th cells. furthermore, inhibitory functional autoantibodies against cxcr occur in humans which play an important role in cxcr -dependent immune regulation. in addition, cxcr regulates endothelial cell homeostasis. there are two variants of cxcr : cxcr -a and cxcr -b. overexpression of cxcr -a on endothelial cells is associated with an increase in cell survival, whereas overexpression of cxcr -b dramatically reduced dna synthesis and up-regulated apoptotic endothelial death. here we have studied if a dysfunctional cxcr axis might be involved in gvhd pathogenesis and could link endothelial and t cell pathology in acute gvhd. we assessed concentrations of the cxcr ligands cxcl , cxcl and cxcl as well as anti-cxcr autoantibodies in patients with high grade ( ) ( ) acute intestinal gvhd for whom serum was available at gvhd onset. furthermore, anti-cxcr autoantibodies and cxcl levels were measured in sera stored before conditioning therapy. all variables were tested for influence on post-gvhd survival using cause-specific cox regression analysis. at gvhd onset, we observed a strong inter-correlation of cxcr ligands, but no correlation with anti-cxcr auto-antibodies. compared with pre-conditioning probes, cxcl levels strongly increased (median to pg/ml, p o . ), whereas anti- these results suggest crp levels at diagnosis as a valid predictor of developing steroid resistant disease in agvhd grade ii-iv and survival in allogeneic hematopoietic transplant recipients. [p ] s cxcr decreased (median . to . u/ml, po . ). anti-cxcr levels before conditioning and at gvhd onset correlated (coeff. . , p o . ), whereas cxcl levels did not. in multivariable analyses, low anti-cxcr and high cxcl measured at disease onset were strongest predictors of survival after acute gvhd. notably, high levels of the proinflammatory chemokine cxcl were particularly prognostic of an adverse outcome of gvhd in the presence of a high endothelial risk as assessed by the previously published easix score, while high anti-cxcr levels were most protective in patients with low easix score (that is, low endothelial risk). a score based on cxcl , anti-cxcr , and easix allowed an effective prediction of acute gvhd outcome ranging from mortality % (high cxcl + high easix) to mortality o % (low cxcl , low easix, high anti-cxcr . our data suggest a strong role for the cxcr axis in the pathology of acute high grade gvhd. the opposing effects of cxcl and anti-cxcr indicate a functional, attenuating role for these auto-antibodies. the overall prognostic impact of the immunemodulating cxcr axis appears to depend on the underlying integrity of the patients' endothelial homeostasis. despite some progress in acute lymphoblastic leukemia (all) treatment including modern chemotherapy modalities, monoclonal antibodies and newer tyrosine kinase inhibitors (tki) for ph positive cases, the final success is still difficult to reach. allogeneic hematopoietic stem cells transplantation (allohsct) has remained an essential approach in attempts to cure all. tki routinely used for all ph(+) pre-and post-transplant treatment are also described as an alternative and adjunctive approach for chronic gvhd especially with fibrotic features due to their antifibrotic activity targeting the platelet-derived growth factor receptor (pdgfr) pathways. in this study we have tried to estimate the potential influence of pretransplant tki treatment on gvhd occurrence comparing all ph(+) and all ph(− ) cases treated with allohsct. a cohort of all patients consisted of all ph( − ) and all ph(+) cases treated with allohsct was retrospectively analyzed. all patients were transplanted from sibling or unrelated donor (no haploidentical procedures were included). all ph(+) patients achieved pretransplant treatment with imatinib and chemotherapy, and ph( − ) patients with chemotherapy alone. the median age in ph( − ) and ph(+) group was vs (p = . ), the percentage of hla mismatched transplantations - ,. vs . (p = . ), the percentage of acute gvhd cases- . vs . (p = . ) and extensive chronic gvhd cases- . vs . , respectively. there were no significant difference between groups in patients sex (f/m- / vs / respectively), ric/mac conditioning, unrelated/sibling donor, donors age, bm/pbpc transplantation, number of cd cells and chronic gvhd incidence. all patients received cyclosporine-and methotrexate-based gvhd prophylaxis. gvhd occurrence was analyzed in subgroups as previously described: all ph( − ) and all ph(+). as mentioned above the incidence of acute gvhd was higher in ph(+) group (higher number of hla mismatched transplantations in this group) but the incidence of extensive chronic gvhd was higher in ph (-) group. cox proportional hazard model analysis revealed death risk caused by gvhd higher in ph negative group (hazard ratio = . ; ci % = . - . ; p = . ). the analysis of competing events was performed to estimate the probability of death caused by gvhd vs other complications (transplant related mortality, infections and relapse). the impact of conditioning was not significant on gvhd related deaths vs other complications (p = . vs . , respectively- figure ). the same results were achieved with donor cmv status (p = . vs . - figure ). we have not found any significant difference either in gvhd or other complications related deaths taking into account patient s sex/age, donor sex/age, patients cmv status, number of cd cells transplanted. on the other hand, the influence of agvhd and chgvhd on deaths related to other complications was not significant (p = . vs . ). cumulative probability of overall survival was higher in ph(+) group but the difference was not significant. the impact of pretransplant treatment with imatinib on gvhd occurrence has not been estimated so far. we are aware of our results to be preliminary and variety of data is still to be evaluated. however our results, if confirmed, may suggest the influence of imatinib on decreasing the extensiveness of chronic gvhd. disclosure of conflict of interest: none. seta-tsukinowa, otsu, - , japan; department of pathology and laboratory medicine, emory university hospital, atlanta, ga, usa; department of biostatistics and bioinformatics, rollins school of public health, emory university, atlanta, ga, usa; pathology and pediatrics, emory university school of medicine, atlanta, ga, usa, aflac cancer center and blood disorders service, children's healthcare of atlanta, atlanta, ga, usa and bloodworks northwest research institute, seattle, wa, usa more than % of allogeneic hematopoietic stem cell transplant (allo-hsct) patients receive red blood cell (rbc) and platelet (plt) transfusions in the peritransplant period. preclinical models indicate that rbc and plt transfusions trigger inflammation, raising the question of whether such transfusions are associated with development of severe acute graft-versus-host disease (grade iii/iv agvhd) and mortality in allo-hsct recipients. we conducted a retrospective analysis of rbc and plt transfusions, agvhd incidence, and mortality among consecutive adult patients receiving non-t celldepleted allogeneic bone marrow ( %) or g-csf-mobilized blood stem cell grafts ( %). common underlying diseases were acute myeloblastic leukemia ( %), myelodysplastic syndrome ( %), and acute lymphoblastic leukemia ( %) . underlying disease risk was ranked as low ( %), intermediate ( %) or high ( %). allografts were obtained from / hlamatched sibling donors ( %), unrelated donors ( %), or from donors mismatched at - hla alleles ( %). graft sources were bone marrow ( %) or mobilized pbsc ( %). the cumulative incidences of grade iii-iv agvhd and mortality prior to day without developing grade iii/iv agvhd were estimated using the cumulative incidence function and a cox proportional hazards regression model. covariates included in multivariable analysis was limited to baseline covariates associated with grade iii/iv agvhd at the p median number of rbc or plt transfusions ( figure ). univariate analysis showed a lower hematocrit on admission (median of rbc units transfused (p = . ) were significantly associated with the risk of developing grade iii/iv agvhd, while a longer time to neutrophil engraftment was inversely associated with grade iii/iv agvhd ( ⩾ median of days, hr . , p = . ). multivariate cox regression analysis showed only larger numbers of rbc units transfused and hla mismatch independently associated with severe agvhd (p = . and p = . , respectively), while underlying disease risk and larger numbers of transfused rbc units were independently associated with overall survival in a multivariate analysis that excluded agvhd grade. overall mortality rate was lowest for the group with fewer rbc and plt transfusions ( %), and greatest for the group with more rbc and plt transfusions ( %). groups that received more rbc units had higher rates of mortality due to gvhd, while patients who received more plt transfusions and fewer rbc transfusions had greater mortality from relapse ( figure ). these data support the hypothesis that peritransplant rbc transfusions are associated with the risk of developing severe agvhd and worse overall survival following allo-hsct. prospective studies are warranted to whether rbc transfusions promote t-cell activation and inflammation in allo-hsct recipients, leading to increased severe agvhd. disclosure of conflict of interest: none. early high umbilical cord blood cd chimerism associated with acute gvhd at time of onset in haplo-cord transplantation h choe , j hsu , s mayer , u gergis , a phillips , t shore and k van besien department of hematology/oncology, weill cornell medicine, new york, ny , usa introduction: haplo-cord transplantation is a combined haploidentical and cord blood transplant that allows for more rapid engraftment by the haplo with eventual loss of the haplo graft upon engraftment of the cord. haplo-cord transplants are associated with an approximate - % incidence of agvhd. reported, using chimerism assessments at approximately day after transplant for aml and mds, that lower umbilical cord blood (ucb) chimerism in the cd or cd lineages were associated with increased rates of relapse. we did not find a statistically significant association between day chimerism and risk for acute gvhd.( ) here we report our analysis of chimerisms at the onset of agvhd. patients and methods we retrospectively reviewed all patients who underwent haplocord sct for all hematologic malignancies between july and march . ucb for haplo-cord transplants were selected based on hla-typing and cell count. grafts were matched for at least of hla loci by the standard criteria and contained a minimum cell count of × nucleated cells per kilogram (kg) of the recipient's body weight before freezing. the haploidentical donor was a relative in the large majority of cases. we identified patients evaluable for agvhd (onset before day ) without preceding relapse or early death. of the total patients, patients were diagnosed with agvhd of any stage and grade. fractionated chimerisms including cd and cd components were routinely sent to evaluate for engraftment of the recipient vs haplo vs ucb. chimerism data was collected for both agvhd and no agvhd patients. the two-sided student's t-test was used to compare the agvhd cohort to the no agvhd cohort. chimerisms collected on patients with agvhd were within median ± days of onset of agvhd. the median time to onset of agvhd was days (range: - days). the median post-transplant chimerism recorded for comparison with the no agvhd patients was days. the agvhd cohort had significantly lower cd recipient (p = . ) and higher cd ucb engraftment (p = . ). all other fractions, including the cd chimerisms, were not significantly different between the two cohorts. the agvhd vs no agvhd cohorts were further compared by degree of hla mismatch ( - out of vs - out of ). the frequency of agvhd was similar in the - out of ( / , %) and the - out of ( / , %) groups. within these subgroups, cd ucb chimerism was higher for those with agvhd (p = . and p = . , respectively). conclusion the onset of agvhd in haplo-cord transplantation is associated with a significantly higher cd ucb chimerism and lower cd recipient chimerism. higher ucb chimerism may indicate that full ucb chimerism poses a higher risk of agvhd development. vice-versa persistent recipient chimerism may protect from acute gvhd. il- is a pleiotropic cytokine with both pro-and antiinflammatory properties (scheller ). the proinflammatory properties are mediated through trans-signaling that depends on the soluble il- receptor. il- trans-signaling is involved in several autoimmune diseases and in regulation of tissue regeneration of the gi-tract. specific snps in the il- receptor have been associated with increased baseline crp levels, severity of autoimmune diseases and response to interleukin- inhibition in rheumatoid arthritis. so fare little is known about the role of trans-signaling in graft-versus-host-disease (gvhd). in this study we investigated how specific snps in the il- receptor influence pretransplant levels of crp, il- sil- r and the risk of grade ii-iv acute gvhd in allogeneic stem cell transplantation (asct) in patients with family donor. dna was available for patients ( male, female median age , range: - ) and donors, that underwent asct with a matched related donor ( sibling) at haukeland university hospital in the period - . the majority received conditioning with either bycy ( ) or flubu ( ) and only patients were transplanted with tbi-based conditioning. four different snps in the il- r gene (rs , rs , rs , rs ) were chosen on the basis of (i) documented or suspected roles in autoimmune disorders; and (ii) allele frequency between . - . and r o . between the different snps. genotyping was done using kaspar assays with viia instrument (life technologies). the overall genotype call rate was %. no departures (p-values o . ) from hardy-weinberg equilibrium were observed. pretransplant serum levels of il- , sil- r and were analyzed with bio-plex kits (bio-rad, hercules, usa). both serum and dna analyses were performed in duplicates. patients being homozygous for the rs minor allele had significantly higher pretransplant serum sil r levels but lower crp levels compared with patients homozygous for the major allele. the overall incidence of agvhd requiring high-dose steroid treatment (grade ii gastrointestinal, grade iii-iv liver and skin) in the cohort was %. when analyzing the conventional clinical and laboratory parameters only transplantation with a non-sibling donor was associated with increased risk of agvhd (p-value o . hr , confidence interval . - . ). the presence of the rs in donor or recipient was associate with a significant increase in the rate of aghvd (p-value . hr . confidence interval . - . ). the snp rs (p-value . hr . , confidence interval . - . ) was also significant in an adjusted model including both donor type and rs . none of the evaluated snps were associated with an increase in early or late trm and did not influence os either. this study suggests that snps in the il- r influence pretransplant biochemical characteristics and clinical outcomes after asct. future studies investigating the effect of il- inhibition as gvhd prophylaxis or treatment should include analyses of il- receptor snps to investigate their possible influence on treatment outcomes. graft-versus-host disease (gvhd) continues to be the major cause of morbidity and mortality in allogeneic hematopoietic stem cell transplant (allo-hsct). the prophylaxis scheme varies according to the center and the country. in ours institution we use triple-prophylaxis based on cyclosporin a (cya), metrotexate (mtx), and mycophenolate mofetil (mmf). this scheme has been used for more than one decade in asian centers where it has proven adequate effective and safe to prevent gvhd. we evaluated patients undergoing allogeneic hematopoietic stem cell transplantation treated at the national cancer institute from january to december . the triple-prophylaxis scheme consists in cya (adjusted serum levels, mtx ( mg/m days + , + , + , + ) and we evaluated different doses of mmf, one of them includes mg bid × days and the other has high doses ( mg/kg bid × days), as gvhd prophylaxis. the response characteristic was analyzed using the pearson test, fisher's exact test on categorical variables and student's t-test, mann-whitney u on continuous tests. kaplan-meier method was used to estimate the probabilities of os, sle with the differences compared by the log-rank test. we analyzed patients with median of age of years (range: - ), % male gender, all were transplant with peripheral blood progenitor cells as a source. . % were acute lymphoid leukemia and . % acute myeloid leukemia, . % chronic myeloid leukemia, . % myelodysplastic syndromes, . % aplastic anemias, . % non-hodgkin's lymphomas and . % hodgkin's lymphomas. myeloablative conditioning was used in % (bucy, cfm-gat) and % reduce intense conditioning (flubu, flucy, flucy-gat), . % related hla compatibility. mmf mg twice daily (bid) for days (group ) and of mmf mg/kg bid for days (group ), in the group the . % developed febrile neutropenia vs . % in group (p = . ). the frequency of gvhd was . % group vs . % group (p = . ), chronic gvhd was . % vs . % respectively (p = . ). at the moment of analysis . % vs . % were free of disease (p = . ). there no difference at -year overall survival was % (group ) vs % (group ) (p = . ), neither free-survival disease (p = . ). the mmf regimen shows noninferiority scheme for gvhd. the low doses and for shorter administration did not show differences in the incidence and severity of acute or chronic gvhd, os, dfs compared to the mmf regimen at days with high doses. the high doses shows higher incidence of febrile neutropenia, but there were no differences in documented infections. disclosure of conflict of interest: none. a protein-losing enteropathy can develop due to conditioning regimen related gut toxicity and can cause albumin decline during peritransplant period in allogeneic stem cell transplantation (allohct). damaged intestinal mucosal barrier results in alloactivation of donor t cells and this situation is considered a primary event in the pathogenesis of acute graft-versus-host disease (agvhd). peritransplant albumin decline, as a result of conditioning regimen related protein-losing enteropathy, may predict agvhd ( ) . in this retrospective study we tested this hypothesis. we evaluated patients who received allohct between and . albumin decline from the day of conditioning initiation until its nadir in the first weeks of post-transplant period was calculated as delta albumin. acute gvhd was proven by biopsy in all patients. chi square and mann-whitney test were used for statistical analysis. patients' characteristics were shown in table- . acute gvhd was developed in patients and severe agvhd was developed in patients. delta albumin was not different between agvhd patients and no agvhd patients. delta albumin was not related with severe agvhd. delta albumin was not different between patients who received myeloablative and reduced intensity conditioning regimens. when we used a cutoff value of . gr/dl for delta albumin, we could not find a relation between delta albumin and development of both agvhd and severe agvhd. we repeated the analysis for acute myeloid leukemia (aml) and myelodysplastic syndrome (mds) patients who receive myeloablative conditioning regimen and we found the same results, there was no difference between agvhd patients and no agvhd patients in terms of delta albumin. there was a number of studies that used albumin as a predictive and prognostic marker in the setting of agvhd. but albumin may decrease in patients due to many reasons like malnutrition, proteinuria, enteropathy, liver disease or being negative acute phase reactant. because of albumin value can show variability between patients, albumin decline may be a more objective criterion. rashidi et al. showed that . gr/dl decline in albumin may be a predictor of severe agvhd in patients who was diagnosed with aml and mds and received myeloablative conditioning regimen. we repeat this analysis in our mds and aml (n = ) patients but we couldn't find this relation. when we evaluated all our patients, again there was no relation between delta albumin and development of both agvhd and severe agvhd. in conclusion, our study did not support rashidi et al.'s findings. because serum albumin level shows variability due to many reasons, it is hard to use albumin as a predictor of agvhd. sclerodermatous chronic graft-versus-host disease (scl-cgvhd) in its severe manifestation affects the patient quality of life and, due to complex pathomechanism, does not respond to standard immunosuppressive therapy-calcineurin inhibitors (cni) with corticosteroid. methotrexate (mtx) and rituximab appeared to be effective in some patients but the novel strategies, including extracorporeal photopheresis (ecp), imatinib, m-tor inhibitors (for example, sirolimus) and ruxolitinib seem to become the real breakthrough. we retrospectively analysed data of patients with scl-cgvhd, who underwent allogeneic hematopoietic cell transplantation (hct) between - in transplant centres. the study group consisted of patients with haematological malignancies and one with aplastic anaemia, female and male, with the median age ( - ). donors' median age was , with predominance of matched sibling donors ( donors) and even distribution of the donors' gender. in patients ( %) acute gvhd (agvhd) was diagnosed with skin involvement observed in ones. acute gvhd directly progressed to cgvhd in cases. in patients ( %) cgvhd developed ‛de novo' and in cases cgvhd was induced by dli. median time from hct to cgvhd diagnosis was months and to scl-cgvhd diagnosis- months. seven patients ( %) were scored as moderate cgvhd and patients ( %) as severe cgvhd according to nih- cgvhd activity classification. in patients sclerotic features had superficial form and in ones deep sclerosis was observed. chronic gvhd manifestation in other organs includes: mouth ( %), joints and fascia ( %), liver ( %), eyes ( %), gi tract ( %) and lungs ( %). patients were treated with ecp and/or sirolimus and /or imatinib with % response rate (complete-cr, partial-pr or minimal-mr). median duration of ecp therapy, sirolimus and imatinib treatment was months ( - ), months ( - ), and months ( - ), respectively. sirolimus was added more likely ( patients) as the first in case of suboptimal response to ecp after median weeks and in patients was subsequently replaced by imatinib with no favourable outcome in cases. in patients imatinib was initially used in combination with ecp therapy, leading to pr or mr. mtx without novel therapies was effective in patients with limited skin involvement, patients responded to mtx plus imatinib and patient to mtx plus sirolimus. two patients, after failure of other therapies, have been receiving ruxolitinib with improvement. only patient ( %) were nonresponsive to ecp (progressive or stable disease), patients ( %) to sirolimus and patients ( %) to imatinib. toxicity incidence was equally observed in case of sirolimus and imatinib and lead to the therapy discontinuation in altogether patients. infectious complications were observed in patients ( %). ecp confirms to be the most effective therapeutic strategy in severe forms of scl-cgvhd with favourable safety profile. imatinib and sirolimus, targeting different fibrotic pathways, both play important role in nonresponsive patients, improving the outcome in ecp and non-ecp group. in case of limited access to ecp, mtx remains to be beneficial in combination therapy of moderate scl-cgvhd and an alternative to cni. disclosure of conflict of interest: none. post-transplant morbidity and mortality are majorly determined by gvl effect counter-balanced by gvhd. treatment with systemic steroids represents the first-line therapy for gvhd, but is associated with increased incidence of infection and relapse. ecp can reduce the extent of gvhd while preserving anti-virus/-tumor activity. to elucidate this clinical phenomenon on an immunological level, we correlated clinical data with immunological findings in patients under ecp treatment. nine patients with acute gvhd (agvhd) of the gut ii-iv suffering from severe diarrhea were treated by ecp in addition to triple-drug immunosuppressive therapy. furthermore, patients with chronic gvhd (cgvhd) of the skin or lung despite triple-drug received ecp treatment. patients were evaluated according to their individual response and clinical condition. phenotypical analysis of different cellular subsets of patients and healthy donors was performed by multicolor flow cytometry. functional properties of virus-specific cd + t and nk cells were evaluated by inf-γ-elispot and cr-release assay. about patients were treated by ecp in this study. however, two agvhd and two cgvhd patients had to be withdrawn from ecp treatment after a few ecp cycles due to pancytopenia or poor clinical condition. for patients with agvhd up to ecp cycles were needed for response. all patients achieving a complete response (cr) were still alive year after initiating ecp therapy. overall response, that is, cr or partial response (pr) according to nih criteria, was obtained in of patients with agvhd ( . %) including cr in of ( . %). out of cgvhd patients ( . %) reached pr, and ( . %) remained stable under ecp treatment. after year, overall survival (os) was % for agvhd patients responding to ecp, while only % for non-responders. os for cgvhd patients was %. during intensive ecp treatment for patients with agvhd of the gut, the average stool volume and frequency decreased and consistency changed from loose to formed stool. steroids could be tapered down to a mean of % of the initial dosage. cgvhd patients were stabilized under ecp treatment and steroid dosage could be reduced to a mean of %. clinically responding patients showed increased numbers of regulatory cells including mdscs, foxp +cd + and foxp +cd +cd + tregs, as well as cd − cd − cd + t, vδ + t cells and regulatory b lymphocytes. furthermore, loss of cd l expression on effector cells like cd + te, cd + te, nk and nkt cells was observed under ecp treatment. interestingly, ecp treatment did not dramatically influence the frequency of cd +cd +cd + t, γδ t cells and nkt cells, which possess anti-virus/-tumor function. elispot and cr-release assays revealed stable anti-viral activity of cd + t cells as well as functional cytotoxicity of nk cells. moreover, cd + t, cd + tem, cd l+cd + temra, cd +cd − nk and cd brightcd − nk cells could serve as reliable biomarkers for prediction of response to ecp. conclusion: ecp treatment might stabilize or even improve clinical situation of patients suffering from gvhd. in clinically responding patients an immunomodulation was observed in terms of increasing numbers of regulatory cells with loss of migratory capacity of effector cells while anti-virus/-leukemia t-cell function was preserved. disclosure of conflict of interest: none. extracorporeal photopheresis affects dendritic cells by reducing total numbers and blunting cytokine production in patients with graft versus host disease tj altmann , , m bickerton , am flinn , u cytlak , p milne , s pagan , m collin , , v bigley , and ar gennery , institute of cellular medicine, newcastle university, newcastle upon tyne, united kingdom and newcastle upon tyne hospitals nhs foundation trust, newcastle upon tyne, uk graft versus host disease (gvhd) and concomitant immunosuppression is a leading cause of morbidity and mortality post hematopoietic stem cell transplantation (hsct). the pathophysiology of gvhd is complex, involving presentation of histo-incompatible antigen by activated recipient dendritic cells (dcs), activation and proliferation of donor t cells and resultant tissue damage. extracorporeal photopheresis (ecp) is a second-line treatment for steroid refractory or dependent gvhd that facilitates the reduction of immunosuppression. ecp's mechanism of action is unclear and is likely to be multifaceted. apoptosis of lymphocytes, induction of a th favoured environment and increased numbers of regulatory lymphocytes have been implicated . although ecp has been shown to modify the function of in vitro monocyte-derived dcs , its effect on primary (non monocyte-derived) dcs has not been studied. our aim was to determine whether ecp had immediate or long-term affects on primary dc numbers or function. we enumerated monocyte and dc subsets (cdc , cdc myeloid dcs and plasmacytoid dcs) in whole blood before, during and after ecp cycles, and developed a novel dc function assay, suitable for use on clinical samples. four adults with immunosuppression withdrawal gvhd and four children with acute gvhd, received ecp during the study. all received ciclosporin gvhd prophylaxis and corticosteroid treatment at onset of gvhd. children received ⩾ dose of infliximab prior to starting ecp. adults received two ecp treatments (one cycle) every weeks and children received two ecp treatments (one cycle) weekly. whole blood was taken before and after each cycle of ecp. trucount flow cytometry analysis of whole blood was used to enumerate mononuclear leukocytes. to assess function, peripheral blood mononuclear cells were isolated by density centrifugation and stimulated with toll-like receptor agonists. cell-specific cytokine production was then analyzed by flow cytometry. samples were compared to healthy controls and pre-ecp samples. median time to first ecp treatment from gvhd diagnosis was . days. no gvhd flares were experienced during study period. ( ) adult had a cycle of treatment delayed due to intercurrent pneumonia. numbers of cdc , pdcs and classical monocytes were significantly reduced after each ecp treatment in the adult group. dc numbers followed the same trend after ecp in the paediatric group but were not significantly different before and after ecp. this is perhaps due to initial lower dc count compared to adults in the children before the first ecp cycle. functional analysis showed a reduction in cytokine production in dcs and monocytes in both groups over the course of ecp treatment. our data support a cell-intrinsic effect of ecp on monocytes and dcs, with numerical and functional consequences. this may contribute to the beneficial effect of ecp both through reduction of inflammatory effector function and through modulation of interactions with other immune cells. correlation with immunosuppression withdrawal and clinical events during treatment may provide further insight into the role of monocytes and dcs in gvhd and ecp, which may aid in the development of novel targeted therapies for gvhd. extracorporeal photopheresis as early second-line treatment for patients with steroid-dependent or refractory acute graft-versus-host disease: a single-centre experience i sakellari , i batsis , e gavriilaki, a-k panteliadou, a lazaridou, k leontopoulos, d mallouri, a bouinta, v constantinou, e yannaki, c smias and a anagnostopoulos department of hematology bmt unit, g. papanicolaou hospital, thessaloniki, greece acute graft-versus-host disease (agvhd) remains a severe complication of allogeneic haematopoietic cell transplantation (allohct). corticosteroids as the backbone of initial therapy for agvhd result in varied complete responses ( - %). traditional secondary treatments lead to profound immunosuppression without improved survival. on the basis of our experience in chronic gvhd, we aimed to prospectively assess the role of extracorporeal photopheresis (ecp) as early secondline treatment in steroid-dependent and refractory agvhd. we enrolled consecutive patients with steroid-dependent or refractory grade (gr) ii-iv agvhd post allohct from january to august . all patients with unrelated or haploidentical donors received thymoglobulin (atg) mg/kg as prophylaxis. post-transplant gvhd prophylaxis included cyclosporine-methotrexate in myeloablative and cyclosporine-mycophenolate mofetil in reduced toxicity or intensity regimens. ecp was commenced after assessment of response to days of steroid treatment according to our protocol: two sessions per week for month, one session per weeks for months, evaluation of response and one session per month for months. we studied patients, aged ( - ), post allohct with myeloablative ( ), reduced toxicity ( ) and intensity ( ) conditioning, from sibling ( ), matched ( ) or one locus mismatched ( ) volunteer unrelated and haploidentical ( ) donors. disease risk index was high ( ), intermediate ( ) and low ( ). acute gvhd was observed at day + ( - ) in patients, late onset at + ( - ) in patients and induced at + post donor lymphocyte infusion in a relapsed aml patient. skin, intestine and liver involvement was evident in patients, skin and intestine in and skin only in patients. nine patients ( with grii, with griii agvhd) were steroid-dependent and ( with griii, with griv) steroidrefractory. atg was administered simultaneously with ecp initiation in six refractory patients that further developed ebv reactivation (p = . ) treated pre-emptively with rituximab. ecp was commenced at day + for ( - ) sessions. the majority of patients ( / ) presented partial ( ), very good ( ) or complete ( ) response to ecp. with . ( . - ) months of follow-up, immunosuppression was reduced in / and ceased in patient. clinically significant bacterial infections were found in patients, fungal in , cmv and ebv reactivation in and , respectively, and other viral in patients. cumulative incidence (ci) of chronic gvhd was . at year. one-year ci of agvhd-related mortality was %. one-year overall survival (os) was % and significantly increased in steroid-dependent vs refractory patients ( % vs %, p = . ). reduction of immunosuppression (p = . ) and response to ecp (p = . ) were associated with improved os, irrespectively of other factors. our data indicate that ecp should be considered early in the course of steroiddependent or refractory agvhd, before irreversible end organ damage has been established. optimal timing of intervention, frequency, duration and tapering schedule of ecp remain important unanswered questions. [p ] disclosure of conflict of interest: none. extracorporeal photopheresis for treatment of chronic graft versus host disease m lanska, a zavrelova, j radocha and p zak faculty of medicine, th department of internal medicine-hematology, university hospital hradec kralove, czech republic allogeneic stem cell transplantation represents a curative approach to many hematologic disorders. graft versus host disease (gvhd) is a complication with significant morbidity, mortality and decreased quality of life. extracorporeal photopheresis (ecp) represents possible treatment approach. mononuclear cells (mnc) collected by apheresis are photosensibilized with -methoxypsoralenem ex vivo, irradiated with uva and transfused back to the patient. aim of the study: evaluation of patients treated with ecp for gvhd at our center. thirteen patients ( females and males, median age years) were treated with ecp. about patients (pts) had matched sibling donor and patient had unrelated donor. about pts had sclerodermic form of gvhd, had concomitant pulmonary gvhd, had pulmonary gvhd alone. one pts had mild, seven moderate and five severe gvhd according to nih. about patients were treated with steroids. mnc separation was prepared on cobe spectra and spectra optia (terumo bct, usa). -methoxypsoralen was added, irradiation was done on macogenic g (macopharma, mouvaux, france). about procedures in pts were performed (median procedures, - procedures). the schema was as follows: ecp on consecutive days every - weeks first months with subsequent increase in interval. median follow up was months. in sclerosing form two pts reached cr, six pts pr, one is stable and one patients progressed. in pulmonary gvhd one reached cr, two partial improvement, one is stable. seven pts are still alive, six died (two due to relapse, one secondary malignancy and three infections). it was possible to withdraw steroids in pts. adverse events were clinically negligible. ecp is an effective treatment for chronic gvhd. especially sclerodermic form responds to ecp very well. it is safe and well tolerated procedure with minimal toxicity. supported by prvouk p- . disclosure of conflict of interest: none. allogeneic haematopoietic stem cell transplantation (hsct) is a potentially curative treatment option for children with a variety of haematological, oncological and immunological diseases. graft versus host disease (gvhd) represents a major cause of post-transplantation mortality and morbidity affecting multiple organs including skin, gut, liver and lungs. gvhd is considered a succession of inflammation and donor t-cell activation initiated by translocation of gastro-intestinal microorganisms through impaired mucosal barriers after chemotherapeutic conditioning and/or infection. diagnosis of gvhd is based on clinical symptoms and histological findings, necessitating invasive and potentially harmful procedures including endoscopy and biopsy. as yet, no non-invasive markers are available for diagnosis or treatment monitoring in children with gvhd. faecal calprotectin (fc) reflects intestinal mucosal inflammation of any origin. in the setting of allogeneic hsct in adults, fc has shown to be a marker for acute (steroid-resistant) gvhd. we aimed to evaluate the feasibility of prospective fc measurement as a non-invasive marker for diagnosis and treatment in children with gvhd. a prospective, observational, single centre study was started in july . by december , paediatric allogeneic hsct patients (age - years) were included after informed consent. faecal samples were collected from weeks before to months after hsct. fc levels were measured by elia, according to manufacturer's instructions. clinical symptoms were prospectively evaluated and managed according to local guidelines. if gvhd was suspected on clinical grounds, histological confirmation was obtained. first-line therapy for gvhd consisted of corticosteroids. in case of steroid-resistant disease, more advanced immune modulation was applied. a total of five patients developed histologically confirmed gvhd: acute gvhd of skin and gut (n = , one patient with steroidresistant disease), acute gvhd of skin only (n = ); chronic gvhd of lung only (n = ) and acute gvhd of skin followed by chronic oromucosal gvhd (n = ). without exception and regardless of gut involvement, gvhd occurrence was accompanied by rises in fc levels to values μg/g (range: - μg/g). fc levels correlated with clinical and histological grading. moreover, adequate response to therapy was consistently reflected by return of fc levels to values o μg/g. sensitivity of fc levels to diagnose gvhd was poor due to increased fc levels in patients with posttransplant complications other than gvhd such as viral reactivation and pulmonary or gastro-intestinal infections. fc levels reflect gvhd occurrence and correlate with clinical and histological grading in paediatric allogeneic hsct patients. fc levels increase in case of gvhd regardless of gut involvement, supporting a central role for (subclinical) intestinal inflammation in gvhd initiation. although, in this interim analysis, fc lacks sensitivity to diagnose gvhd, fc may serve as a noninvasive marker for monitoring therapy response and, thereby, reduce the need for repeated invasive procedures including endoscopy and biopsy. acute graft-versus-host disease (agvhd) is a major complication of allogeneic hematopoietic cell transplantation (hct), and glucocorticoids are typically used as first-line treatment. the aim of our study was to evaluate the effect of first-line ecp +/ − steroid therapy in order to reduce the incidence of infections and toxicity. from december to january , of pts ( %), were diagnosed with agvhd grade ⩾ following allosct. pts were treated with ecp +/ − steroid as first-line therapy. about ( %) pts were treated with ecp only and ( %) with ecp + steroid - mg/kg/day. we compare this cohort with an historical group of patients, transplanted between and , who were treated with steroid only for grade - agvhd (n = out of ). the two cohorts were well balanced in terms of median age (p = . ), disease type (p = . ), disease status (p = . ), graft source (p = . ), conditioning regimen (p = . ) and hct-ci (p = . ). there were more female patients (p = . ) and more haploidentical transplant (haplo-sct) (p = . ) in the cohort treated with ecp+/ − steroid. ecp was performed using the offline technique, and was started as soon as possible with a treatment schedule consisting of four rounds of two procedures per week, three rounds of two procedures every other week and finally two procedures every month. steroid was tapered as soon as possible after starting ecp. the clinical response was evaluated at day + . median follow-up for alive patients was months for ecp group and months for control group. there was no difference in terms of median time of agvhd onset ( vs days) and number of pts with grade or - agvhd ( figure ). ecp was started after a median of ( - ) days from agvhd diagnosis. every patient underwent a median of ( - ) ecp procedures, during a median time of months. on day after starting agvhd treatment with ecp+/ − steroid, pts ( %) achieved cr or pr, pts did not respond and experienced agvhd relapse to front-line therapy. one year cumulative incidence (ci) of agvhd relapsed/refractory was . % for ecp+/ − steroid. these percentages were not different from the cohort receiving steroid alone. ci of moderate-severe cgvhd was lower in the ecp group, probably due to the higher frequency of haplo-sct with pt-cy in the ecp group. about days after agvhd onset, ci of infection ( % vs %), especially cmv reactivation ( % vs %), was lower in the ecp group, but was not statistical significant. ecp allowed a faster taper of steroid: ( - ) vs days ( - ) (p o . ). overall survival, progression-free survival, non-relapse mortality and ci of relapse rates did not differ in the two groups. in multivariate analysis, visceral involvement by agvhd was associated with an increased risk of failure to front-line therapy (hr: . ; range: . - ; p = . ). this observational study suggests that the overall response rate of ecp +/ − steroids is similar to steroid alone for front-line treatment of grade - agvhd, but is potentially associated with lower incidence of infection, and in particular of cmv reactivation. a prospective phase clinical trial is warranted to address whether augmentation with ecp may be beneficial for agvhd frontline treatment. [p ] a -year-old girl with neuroblastoma received autologous stem cell transplant (asct), followed by antibiotic prophylaxis and filgrastim. her transplant preparative regimen consisted of busulfan and melphalan. engraftment of neutrophil took place on day after asct. twentieth day after asct, she experienced nausea and diarrhea. there was neither skin rash nor elevation in liver enzymes. the diarrhea continued to worsen day by day and reached to a daily volume of ml/ m . infectious studies for stool and blood including testing for influenza a and b, parainfluenza, adenovirus, epstein-barr virus, amebiasis, cryptosporidium parvum, cytomegalovirus, clostridium difficile, salmonella, campylobacter, yersinia and shigella were all negative. colonoscopy and endoscopy were performed by an experienced pediatric gastroenterologist and findings were suspicious for severe graft versus host disease (gvhd). colonoscopy and rectoscopy revealed severe inflammatory changes, friability and patchy dark exudates on the mucosa of rectum. endoscopy revealed erosions, ulcers in the esophagus and a pale mucosal surface with reticulated submucosal vessels accompanied with erosion and erythema in the antrum of stomach. grade gvhd was confirmed by pathologic analysis that revealed diffuse crypt dropout and mucosal erosion on rectal mucosal biopsy. mucosal erosions, apoptosis of epithelial cells and small lymphocytic infiltration of the lamina propria were found on duodenal biopsy. after these results, we started methylprednisolone intravenously at a dosage of mg/kg/day. on the fourth day of treatment we increased the dosage to mg/kg/day and added cyclosporine to treatment. because of unresponsiveness to treatment we decided to administer thirdparty mesenchymal stem cells (msc) ( × cd +/cd + cells per kg). these were given intravenously at day + asct as single infusion. the second dose was given at day + . within days after first application of mscs, the frequency of diarrhea decreased to one-third. at day + after second dose of mscs, the patient's stool became nearly normal. we tapered the steroids first and stopped cyclosporine at + th days after asct. discussion and conclusion: to our knowledge, this is the second case report of spontaneous severe autologous gvhd in a child with a solid tumor malignancy. regarding the pathogenesis of autologous graft-versus-host disease, there may have multiple causes for the loss of tolerance to self because of disrupted immune system. alteration of t regulatory cells by previous chemotherapy may be key point. endogenous cells that survive conditioning and assist in post-transplant maintenance of self-tolerance may be affected. microchimerism due to maternal cells transmitted during fetal development and persisting throughout adult life has also been postulated as a cause. however it is not very clear for factors that may contribute to the pathogenesis of this rare disease. autologous gvhd has the potential to cause critical illness in the hematopoietic stem cell transplantation patient population. in patients with multiple myeloma some experts report pathologically verified gastrointestinal gvhd as high as %. responses to steroids are variable. however, a significant proportion improve dramatically after early therapeutic intervention. so clinicians and pathologists should be aware in suspecting and recognizing gvhd in patients with diarrhea to guide therapy as soon as possible. disclosure of conflict of interest: none. haplosct patients did not receive additional gvhd prophylaxis aside from the ex vivo t-cell depletion (tcd) with clinimacs system. of the bud bmts out of patients engrafted, % of which had gvhd. the % who did not have any gvhd, relapsed. eighty one percent had agvhd, of which majority ( %) were grade ( table ) three patients did not have agvhd ( / mud and two cord blood grafts), but developed cgvhd. one of the patients who had grade agvhd died and one still has intermittent cgvhd years post bmt. there were % who had cgvhd. currently, all of our haplosct receive a cd /cd ra tcd grafts (n = ). the depletion techniques for the others were either cd tcd, cd / cd ra /tcrab tcd+cd +/cd ra tcd; tcrab tcd. all patients who received haplosct engrafted and % had agvhd ( % grade ) ( table ). we noted that some of the patients presented with nonclassical agvhd signs (upper gut gvhd, oral gvhd, blood). there were no patients who presented with grade agvhd. cgvhd in the cohort was %. of note, % of patients did not have any gvhd and did not receive any form of immunosuppression post bmt. only eight patients received further immunosuppression for agvhd, median duration (range: - ) days. at the time of this report, there are four patients with agvhd still receiving immunosuppression all o days and all on tapering doses. haplosct using ex vivo tcd techniques has a lower risk of gvhd with comparable, if not superior outcome to bud. the degree and duration of immunosuppression is also much less. this may translate to earlier immune reconstitution and less viral reactivation. [p ] disclosure of conflict of interest: none. allogeneic hematopoietic stem cell transplantation (allo-hsct) offers a potential cure for several hematological diseases, but it is burdened by severe life-threatening complications, being gvhd the major cause of morbility and mortality. recently, more have been understood of the physio-pathologic relationships between endothelium and graft-versus-host disease (gvhd), showing that vascular endothelium is an early phase target of gvhd. in recent years, the direct count of circulating endothelial cells (cec) has emerged as a valuable biomarker of endothelial damage in a variety of disorders. however, due to their rareness and complex phenotype, different published techniques have showed variable degrees of uncertainty, reporting a wide range of cec values in healthy subjects. by means of the commercially available rare cell isolation platform cellsearch system, for cec identification and count, we correlated cec count changes to gvhd onset and response to treatment in allo-hsct patients. cec were analysed in allo-hsct patients ( aml, all, hd, nhl, cll, mds, cms, mm, saa) at the following time points: t (pre-conditioning), t (pre-transplant), t (engraftment), t (day+ or onset of gvhd), t ( week after steroid treatment). the median cec/ml at t was (range: - ), in comparison to a value of (range: - ) in healthy controls (p %: or . , % ci . - . ; p = . ). we confirm that cec count represent a valid biomarker to monitor endothelial damage in patients undergoing allo-hsct and can be a valuable tool in supporting the diagnostic definition of gvhd and in monitoring responsiveness to treatment. moreover, the use of the cellsearch system can be crucial in order to move routinely cec monitoring into clinical practice of allo-hsct. reference clinicaltrials.gov nct . disclosure of conflict of interest: this research was conducted with the support of the investigator-initiated study program of janssen diagnostics, llc to ca. kb is employee of janssen diagnostics. results of hla mismatched unrelated donor (mmud) hematopoietic cell transplants (hct) are worse than results of fully matched hct due to higher risk of gvhd, infection and graft failure. atg during conditioning reduces incidence of gvhd but can increase risk of infection and relapse. high doses posttransplant cyclophosphamide ( × mg/kg) prevent gvhd in haploidentical hct. we initiated this approach instead of atg in hct from one alelle or antigen mismatched unrelated ( / )mmud-hct in . here we present outcome of patients (cy-group) transplanted between and , comparing it with outcome of patients transplanted between and from / mmud with atg-f (fresenius) mg/kg given during conditioning. patients in cy-group ( males, females) were transplanted from mmud mismatched for hla ( a- , b- , c- , dr- ). about patients had aml, mds, cll, cml, all and mps. med. age of patients was years ( - ). about patients received myeloablative (flu- mg/m + iv bu . mg/kg) and nonmyeloablative(flu- mg/m+mel - mg/kg +-tt mg/kg) conditioning. about patients received pbpc and bm as a graft. graft versus host prophylaxis consisted of cyclophosphamide ( mg/kg aibw) on d+ and + , cyclosporine a from d and mmf from d+ . all patients received antibacterial, antifungal, hsv and pcp prophylaxis. historical control (atg) group consisted of patients ( males, females), med. age y ( - ) who had mmud-hct for aml- , mds- , nhl- , mf- , all- , cll- , cml- , h.d- , saa- , mps- and mm- . there were mismatches for a, for b, for c and for dr. myeloblative conditioning was used in and nonmyeloablative in patients. all patients received atg-f mg/kg × given d- and- . cyclosporin was initiated d- and mmf d- . all patients received anti-infectious prophylaxis as described previously. three of patients from cy group died so far. two of them due to relapse and one due to toxicity and infection during aplasia. five patients relapsed . two achieved cr after dli and one is alive in relapse expecting second hct. about patients are alive, of them in cr. eight patients experienced agvhd (gr.i- , gr.ii- ,gr.iii- , gr. iv- ) and eight developed clinically mild cgvhd). about patients from atg group are alive - m (med. m) posthct. about patients died - m postransplant (med. m) due to vod, gvhd, infections and relapse. -day mortality is % ( / ) in cy group and % ( / ) in atg group. one year mortality is % ( / ) in cy and % ( / ) in atg group. patients from cy group have % probability of os at months posthct vs % from atg group. cyclophosphamide × mg/kg instead of atg fresenius( mg/kg) for gvhd prophylaxis reduced -day and -year mortality, and improved probability of m os significantly in our cohort of patients. this approach seems to be safe and effective in / mmud-hct. disclosure of conflict of interest: none. high transplanted cd + cells are not associated with beneficial effect on graft-versus-host disease-free, relapse-free survival (grfs) after allogeneic hematopoietic cell transplantation y lee and ih lee department of hematology, kyungpook national university hospital and inhee lee the success of allogeneic hematopoietic cell transplantation (allo-hct) is comprehensively assessed by individual comorbidity, relapse, graft-versus-host disease (gvhd) and death. besides, inconsistent results have been reported regarding the dose of cd + cells. in the current study we have addressed the issue of the potential effect of stem cell dose on the of gvhd-free/relapse-free (grfs) associated with cd + cells doses. we retrospectively reviewed the medical records of the patients who received allo-hct for acute myelogenous leukemia (aml), myelodysplastic syndrome (mds) and acute lymphoblastic leukemia (all) between and in kyungpook national university hospital. the grfs included grade - acute gvhd, systemic therapy-requiring chronic gvhd, relapse or death. the patients were reclassified into two groups according to the targeted cd + cell doses ( × per kg) by knuh protocol. a lower cd + group (n = , . %), patients who underwent allo-hct with cd + cell dose o × per kg; and a higher cd + group (n = , . %) patients who underwent allo-hct with cd + cell ⩾ × per kg. the median age at transplant was . years (range: - years) and male was patients ( . %). primary diseases for allo-hct were aml/mds (n = , %) and all (n = , %). one hundred forty-three patients ( . %) were in cr (complete remission), ( %) in further cr and ( . %) in relapsed and refractory status. one hundred seventy-one patients ( . %) received myeloablative conditioning regimen. gvhd prophylaxis consisted of methotrexate and cyclosporine a or mtx and tacrolimus. the median dose of cd + cell was . × per kg (range: . - × per kg) in lower cd + group and . × per kg (range: . - . × per kg) in higher cd + group. there was no significant difference in neutrophil, platelet engraftment between two groups. the incidence of chronic gvhd was more frequent in higher cd + group ( . % vs . %, p = . ). the median follow-up duration was . months, with a range of . - . months. the -year overall survival (os), relapse free survival (rfs), nonrelapse mortality (nrm) and graft-versus-host disease (gvhd)free/relapse-free survival (grfs) since hct was . ± . %, . ± . %, . ± . % and . ± . %, respectively. there was no significant difference according to the infused cd + cell dose (figure ). the relapse rate was not proportionally affected by the cell dose ( . % vs . %, p = . ). and there was no significant correlation between the number of cd + and cd + cells infused (spearman correlation coefficient: p = . ). in a univariate analysis, patients transplanted with the higher cd + cell doses and higher cd + cell doses had no increased grfs (p = . and p = . ). an independent factor associated with worse grfs was risk status at transplant (hr = . , % ci: . - . , p = . ). these results suggest that careful assessing the cd + and cd + graft content and tailoring the cell dose infused may help in reducing cgvhd risk without negative impact on grfs. a large and prospective study in a homogenous population will be needed to confirm the effect of stem cell dose. disclosure of conflict of interest: none. imatinib associated with extracorporeal photopheresis can fully reverse severe sclerotic-type lesions in patients with chronic graft-versus-host disease: the lille university hospital experience l magro , j gauthier, b catteau , l mannone , a lionet , v coiteux and i yakoub-agha lille university hospital and nice university hospital severe sclerotic-type chronic graft-versus-host disease (cgvhd) is difficult to reverse and can dramatically alter the quality of life of patients after allogeneic hematopoietic cell transplantation (allo-hct). imatinib or extracorporeal photopheresis (ecp) used separately yield sustained responses in s only about % of patients with steroid-refractory cgvhd. given their respective modest efficacy we hypothesized that the combination of imatinib with ecp could lead to higher response rates. we are reporting here on seven patients with severe steroid-refractory sclerotic-type cgvhd treated at our institution using this combination. we retrospectively analysed all patients treated at our institution (n = ) with the combination of imatinib with ecp for severe steroidrefractory scgvhd. imatinib was started at mg/day and increased to mg/day if well tolerated. the cellex closed system was used for ecp. ecp was initiated twice weekly during weeks. after this « induction » period, ecp sessions were scheduled less frequently according to the response to treatment. additional immunosuppressants were tapered gradually in responding patients. initial grading and response evaluation was determined according to the nih criteria. steroid-refractoriness was defined as progression of gvhd on high-dose steroids (⩾ mg/kg) or progression during corticosteroid tapering. patient characteristics are displayed in table . patients received an allo-hct between may and april . median age at allo-hct was (range: - ). a variety of myeloablative (n = ) and non-myeloablative conditioning regimens were used (n = ). antithymocyte globulin was used before allo-hct in one patient. gvhd prophylaxis consisted of ciclosporine and methotrexate in six patients. one patient received tacrolimus and methotrexate. five patients had prior history of acute gvhd. nih global severity grade was severe in all patients (n = ) due to severe sclerotic features. the median number of previous therapies was (range: - ). all patients were steroid-refractory. after a median follow-up of months (range: - months) the overall response rate was %. the complete response rate was %. median time on ecp associated with imatinib was months (range: - months). median time to best response was months (range: - months). corticosteroids could be discontinued in all patients after a median time of months (range: - months). patients # , # and # received maintenance therapy with ecp upon discontinuation of imatinib. in four patients, both ecp and imatinib led to complete response and could be discontinued after , , and months for patients # , # , # and # , respectively. patient # and # passed away after due to a myocardial infarction and the development of a solid tumour, respectively. patient # was off therapy while patient # remained on maintenance with ecp. both remained in complete response. patient # remained in response during months before progression of cgvhd while on imatinib and ecp. none of our patients experienced adverse events related to either imatinib or ecp. despite the limited number of patients in this report, we observed that the combination of imatinib and ecp can lead to complete and sustained reversal of severe steroid-refractory sclerotic-type cgvhd. these encouraging results should be confirmed in a larger cohort. disclosure of conflict of interest: lm: therakos (honorarium). allogeneic hematopoietic cell transplantation (allo-hsct) is an established treatment modality that is potentially curative for many patients (pts) with acute myeloid leukemia (aml). aml itself is the most common indication for pts undergoing hsct nowadays. for pts with high-risk disease, allo-hsct is, perhaps, the most effective curative treatment and is considered the standard post-remission therapy in first complete remission (first cr). this is a retrospective study to analyze those variables which were associated with patients' overall survival (os) after allo-hsct. the study population consisted of pts who were diagnosed of aml from january to july at the hospital universitario central asturias, and submitted to allo-hsct in first cr. risk status based on validated cytogenetics and molecular abnormalities following recommendations of european leukemianet was performed. sixteen ( . %) were male. median age was years old (range: - ). clinical characteristics at transplantation are represented in table . median follow-up was months ( - ). considering the donor type, os at year was higher in pts receiving sd ( . %) compared to % in those who received urd (p = . ). regarding graft source, os at year was . % who received pbsc compared to % in pts receiving bmsc (p = . ). gender also showed significant association with os, which was higher among men, os at year was %, compared to . % for women) (p = . ). the presence of minimal residual disease (mrd) detected using multiparametric flow cytometry was performed prospectively after induction and consolidation, and before transplantation. thirteen pts had negative mrd before transplantation. median os was greater in pts with negative mrd before transplantation compared to the group with positive mrd ( vs months, respectively) (p = . ). this difference did not reach statistical significance probably because the low number of the sample. thirteen pts developed agvhd. only ( . %) pts receiving sd developed agvhd compared to ( %) pts among those who had an urd; however this association was not statistically significant (p = . ). also, we observed higher incidence of agvhd in bmsc group ( pts; %) whereas only ( . %) in pbsc group developed agvhd. this tender did not reach significant association (p = . ). one year os was . % in pts who developed agvhd and . % who did not (p = . ). all factors that had a significant influence on pts survival were included in a multivariate analysis (cox regression model): graft source, donor type, pts gender and agvhd development. developing agvhd kept an independent association with mortality (or . , % ci . - . , po . ) and male gender also persisted as an independent protective factor (or . , % ci . - . , p = . ). in our series, agvhd has shown a significant and independent association with os over other parameters such as graft source, type of donor or mrd before transplantation. identifying reliable predictors for agvhd development, controlling well known risk factors for this disease, as well as improving management of immunosupressors should still be the key to potentiate longer os in our patients. larger studies are needed to confirm our results. acute and chronic graft-versus-host diseases (gvhd) are associated with increased morbidity and mortality after allogeneic hematopoietic stem cell transplantation (allo-hsct). older patients undergoing allo-hsct may experience a high degree of transplant-related complications and this concern has historically limited the use of allo-hsct for some older patients. in many studies, age has been shown to be a negative prognostic factor for survival and associated with higher transplant-related mortality (trm). however, in others, age was not shown to be a significant factor if appropriate adjustments for other comorbidities are incorporated in the analyses. there are very few studies that evaluated the relationship between patient's age, the presence of gvhd and long-term transplantation outcome. the aim of this study is to evaluate the impact of age in patients who develop acute and/or chronic gvhd after allo-hsct for hematological malignancies on the trm incidence. we included in the study patients with hematological malignancies who received allo-hsct and were followed in our center between january and january . for the purpose of this study, only patients who developed grade ii-iv acute gvhd and/or limited or extensive chronic gvhd where considered for analysis (n = ). patients were split into three homogeneous groups according to age at transplantation taking into consideration the underlying disease, type of conditioning and disease response at transplantation. group (younger) included patients aged o years (n = ), group (intermediate) included patients aged between and years (n = ) and group (older) included patients older than years (n = ). gvhd evolution over time was followed as well as the cumulative incidence of trm was calculated in case of acute or chronic gvhd in each group. thirty seven percent of grade ii gvhd occurred in the younger group (n = ), % (n = ) in the intermediate group and % (n = ) in the older group; majority ( %) resolved in the younger group as well as in and % in the latter two groups, respectively, while trm rates at year were %, % and %, respectively, sdhr = . , p = . . among patients who had acute gvhd grade ii, , and % in the three respective groups developed chronic gvhd later. grade iii-iv gvhd occurred in % (n = ) in the younger group, % (n = ) in the intermediate group and % (n = ) in the older group; with a respective resolution in %, % and % of patients and were associated with comparable trm rates at year of %, % and %, respectively, p = . . among patients who had acute gvhd grade iii-iv, , and % in the three respective groups developed chronic gvhd later. de novo chronic gvhd was observed with a higher rate in the intermediate and in the older group (table) while patients with extensive chronic gvhd older than years had significantly higher trm at years ( %) compared to % in those younger than years, sdhr = . , p = . . patients who develop acute gvhd grade iii-iv could incur over % of trm at year independently of age. resolution of acute gvhd grade ii was significantly better in younger patients while older patients with grade ii acute gvhd or with extensive gvhd had higher mortality compared to younger ones. in addition to an adapted prophylaxis, a better preemptive gvhd strategy should be warranted in older patients. [p ] disclosure of conflict of interest: none. in vivo effects of nilotinib on lymphocyte subpopulation and function following allogeneic stem cell transplantation e marinelli busilacchi , a costantini , j olivieri , n viola , s coluzzi , e pirro , g mancini chronic graft versus host disease (cgvhd) is a major complication of allogeneic stem cell transplantation and is characterized by frequent multiorgan involvement resembling autoimmune diseases; its pathogenesis is still incompletely defined and a standard treatment is lacking. donor-derived cd + and cd + t lymphocytes have been considered the main effector cells mediating cgvhd pathogenesis; however, recent studies suggest that b cells might also play an important role. in vitro data indicate that tyrosine kinase inhibitors (tkis) such as imatinib and nilotinib affects both innate and adaptive immune response by interacting with different cell populations (t cells, b cells, dendritic cells, mast cells and macrophages). we sought to evaluate the impact of different doses of nilotinib on the distribution and function of lymphocyte subpopulations. we analyzed samples obtained from patient with steroiddependent/refractory cgvhd enrolled in a phase - study with nilotinib in steroid-refractory cgvhd (nct ): triplets of patient were treated with escalating doses starting from mg/die ( ), mg/die ( ), up to mg/die ( ) . blood and plasma were collected at baseline and at day and of therapy. trough plasma nilotinib concentrations had been previously determined by hplc (abstract c , haematologica: evaluation of nilotinib safety in patients with steroid-refractory chronic graft-versus-host disease: a phase i-ii gitmo study). peripheral blood mononuclear cells were isolated by density gradient centrifugation using ficoll biocoll. six color flow cytometry analysis (facs canto ii) was performed using conjugated antibodies (anti-cd , cd , cd , cd , cd , cd ). inflammatory cytokine analysis was performed on plasma samples according to the instruction of bioplex pro human cytokine plex assay (bio-rad). statistical analysis was performed by -tailed student's t-test; differences were considered statistically significant for po . . flow cytometry analysis showed that nilotinib did not exert any significant impact neither on the proportion of t lymphocytes subpopulation (cd +cd + t helper, cd +cd +cd + t regulatory, cd +cd − t cytotoxic), nor on b lymphocytes and nk cells. on the contrary, a statistically significant and dose-independent decrease of pro-inflammatory and th- cytokine production was observed ( figure ): reduction of il (po . ), il (po . ) and ifnγ (po . ) were already significant after days; decreases of il (po . ) and tnfα (po . ) become significant after days. interestingly, after days of therapy, among the patients enrolled (according to the itt criteria) ten patients showed cgvhd improvement and the other five remained stable. this study shows that therapeutic doses of nilotinib can reduce plasma levels of inflammatory cytokines without affecting the proportions of lymphocyte subpopulations. these findings correlate with clinical response and suggest that besides the previously demonstrated anti-fibrotic effects, nilotinib has also potent anti-inflammatory and immune regulatory properties, supporting its role in patients with cgvhd. disclosure of conflict of interest: none. [p ] previously published p infectious gastro-enteritis after allogeneic hematopoietic transplantation after reduced intensity conditioning (allo-ric): incidence and possible role in gastro-intestinal acute gvhd i garcía-cadenas , r martino , a esquirol , a bosch , n rabella , s saavedra , c muñoz , j briones , s brunet and j sierra hematology and microbiology departments and hospital de la santa creu i sant pau, autonomous university of barcelona, spain enterotoxigenic c. difficile-associated associated disease or infection (cdi) is a common cause of diarrhea after hematopoietic stem cell transplantation (sct). recent studies have suggested the relationship of cdi with gastro-intestinal (gi) graft-versus-host disease (gvhd). the possible role of other types of infectious gastro-enterocolitis (g-ec) in gvhd development has not been studied. as a prior investigation to a national prospective observational study on this issue, we conducted a single-center retrospective analysis including all adult patients who received an allo-ric sct between january and march . the aim was describing the cause(s) (if known), timing and outcomes of recipients with possible g-ec (defined as new onset acute diarrhea grade ⩾ ) in the first year after sct. of the patients studied (median age: years, % male, % aml or mds as underlying disease), ( %) had a total of episodes of acute diarrhea, with ( %) developing more than one event. these acute diarrheas occurred at a median of days (range: - ) after sct. overall, a g-ec causing pathogen was identified in of stool specimens ( %) and included: cdi ( ), c. jejuni ( ), rotavirus ( ), adenovirus ( ), norovirus ( ), b. hominis ( ), s. stercoralis, g. lamblia, a. caviae, salmonella enterica and cryptococcus (one in each case). most postransplant diarrheas ( / ; %) occurred during the weeks after infusion and were attributable to mucosal damage caused by the ric (negative microbial screening and no evidence of gvhd).the rate of infectious g-ec among the diarrheas occurring after day + was % ( / ). the overall incidences of enteric infection were . % ( % ci: . - . ) and . % ( % ci: . - . ) at + and + months after sct, respectively. all the infected patients had mild to moderate disease, and no deaths were attributable to this complication. there were no differences in year-os and nrm between the infected and uninfected patients ( % vs %, p = . and % vs %, p = . , respectively). in univariate analysis age o years, prior sct, donor type, atg administration and prior grade - agvhd were associated with development of infectious gastro-enteritis. in multivariate analysis, unrelated donor and grade - agvhd were the only factors significantly associated with gastrointestinal infection (hr . ; % ci: . - . , p = . and hr . , % ci . - . , p = . ; respectively). acute gvhd occurred in % of patients (n = ), with a median onset of days (range: - ). the cumulative incidences of - acute gvhd at days and months post-sct were % ( % ci: . - %) and . % ( % ci: . - %), respectively, and there was a trend toward a higher risk of - gvhd in the group of patients with an enteric pathogen ( . % vs % at year, p = . ). more importantly, an enteric infection occurred just before the onset or aggravation of gvhd in / infected patients in our study ( %) at a median interval of days after the infection (range: - ). in summary, our results confirm that enteric infections are a common complication after allo-ric, representing at least % of the episodes of acute diarrhea during the first year post-sct. a possible interplay between infectious g-ec and gvhd was observed in this study. disclosure of conflict of interest: none. long-term efficacy of extracorporeal photopheresis in chronic graft versus host disease m nygaard , t karlsmark , n smedegaard andersen , i schjødt , s lykke petersen , l smidstrup friis , b kornblit and h sengeløv department of dermatology, bispebjerg hospital, copenhagen, denmark and department of hematology, rigshospitalet, copenhagen, denmark chronic graft versus host disease (cgvhd) activity is known to fluctuate over time, so we evaluated cgvhd continuously throughout the extracorporeal photopheresis (ecp) treatment course and after stopping ecp. patients with at least year follow-up, who were treated with ecp at department of dermatology, bispebjerg hospital between and were evaluated. a single investigator retrospectively evaluated response to ecp monthly for months, every months until years and every months until years. prednisolone doses were recorded every months. responses were defined as complete remission (cr) if no symptoms of cgvhd were present, partial remission (pr) as improvement in cgvhd or stationary cgvhd with more than % reduction in prednisolone, no change (nc) as no difference in symptom burden and o % reduction in prednisolone. progressive disease (pd) was defined as worsening of symptoms with unchanged or intensified immunosuppressive medication. ecp was performed with therakos uvar xts or cellex. there were evaluable patients with moderate (n = ) or severe (n = ) steroid-refractory, dependent or -intolerant cgvhd. the median age was years (range: - ) and there were females and males. conditioning regimen was myeloablative (n = ) and non-myeloablative (n = ). seventeen had related donors and had unrelated. stem cell source was peripheral blood (n = ), bone marrow (n = ) or umbilical cord blood (n = ). number of organs affected by cgvhd was one (n = ), two (n = ), three (n = ), four (n = ) or five (n = ) and involved organs were skin (n = ), eyes (n = ), mouth (n = ), lungs (n = ), genitals (n = ), liver (n = ), musculoskeletal system (n = ) or gastrointestinal tract (n = ). time from diagnosis of cgvhd to first ecp was median days (range: - ) and time from referral to ecp and the first ecp procedure was median days (range: - ). at the time of the first ecp procedure patients were also treated with prednisolone (n = ), sirolimus (n = ), calcineurininhibitor (n = ), mycophenolate mofetil (n = ), imatinib (n = ), methotrexate (n = ) or rituximab (n = ). one patient received no immunosuppression. total number of ecp cycles was median (range: - ). responses over time are shown in figure . overall response to ecp was seen in ( %) of the patients. most responses were seen after more than months ecp treatment. in univariate analysis of possible baseline predictors of response, no significant associations were found. prednisolone dose was significantly reduced at every months after start of ecp (p o . ). additional cgvhd treatment was administered to ( %) patients during ecp treatment (sirolimus n = , calcineurininhibitor n = , uva n = , methotrexate n = , rituximab n = , mycophenolate mofetil n = ). about ( %) patients had more than additional treatment. prednisolone dose was increased at least once in ( %) patients during ecp treatment. overall survival at years was %. follow up was median days (range: - ). more than half the patients with cgvhd ( %) improve overall after treatment with ecp, but flares in cgvhd activity still occur. prednisolone dose is significantly reduced at all time points after starting ecp, but short term increased doses or additional immunosuppression was necessary in more than one-third of the patients. larger prospective studies with long-term end points are warranted. disclosure of conflict of interest: marietta nygaard has received a travel grant and speaker's fee from therakos/ malinckrodt. chronic graft versus host disease (cgvhd) remains a major cause of morbidity and mortality after hematopoietic stem cells transplantation despite the improvement of the immunosuppressive prophylaxis. skin, buccal, lacrymal and hepatic disorders are the most frequent. sclerotic gvhd remains a severe form and often refractory to standard treatment lines such as corticosteroids and calcineurin inhibitors. the antifibrotic activity of imatinib by the inhibition of pdgf-r and tgfb-β pathways has been used in the treatment of refractory gvhc with sclerotic features and systemic scleroderma. here, we report the results of imatinib treatment in patients (pts) with refractory cgvhd. over a period of years (january -december ), pts received allogeneic stem cells transplantation from related donors, of whom were treated with imatinib for refractory cgvhd: pts for malignant diseases ( cml, aml, nhl) and pts for aplastic anemia. the median age is years ( - ), the sex ratio m/f: . . conditioning regimen used with chemotherapy alone: myeloablative ( pts with gvhd prophylaxis combining ciclosporin and methotrexate), reduced intensity ( pts with prophylaxis combining ciclosporin-mycophenolate mofetil). all pts received peripheral blood stem cell transplant with an average of cd cell count: . × /kg ( . - . ). the median duration of the cgvhd is months ( - ). the firstline treatment consisted of the combination of steroidsciclosporin with or without mycophelonate mofetil. imatinib was administered to these pts after median treatment duration of months ( - ) for moderate ( pts) and severe ( pts) cgvhd according to the nih classification. treatment with imatinib, at doses ranging from to mg/d, was introduced in the second line for all pts. the evaluation is conducted in october after a median follow-up of months ( - ). tolerance was good except in a one pt with severe thrombocytopenia that led to a transient cessation of treatment. after months, analysis of pts who received imatinib according to couriel criteria and nih criteria: complete remission (cr): pt ( %), partial remission (pr): pts ( %), stable disease (sd): pts, failure: pts ( %). a long-term evaluation performed after a mean duration of treatment months ( - ) finds similar results with a cr: pts ( %), pr: pts ( %), sd: pts ( %) and failure: pts ( %). corticosteroids were tapered or discontinued in pts (cr or pr). at october , pts ( %) were alive and pts ( %) died of severe infections. treatment with imatinib seems to be a good therapeutic option in the treatment of cgvhd in its moderate or severe form refractory to a minimum of two immunosuppressive agents according to the nih criteria as shown by our results in terms of response and survival with good tolerance. disclosure of conflict of interest: none. atg significantly reduces the risk of cgvhd both in unrelated and in hla identical sibling. the finke's study randomised pts undergoing an allogeneic unrelated stem cell transplant (sct) after a myeloablative regimen to receive or not mg/kg atg-grafalon reporting a significant reduction of cgvhd without increase of relapse and no os and dfs effect. however a successive study didn't confirm those results (significant reduction of acute and chronic gvhd but poorer survival mainly due to higher relapse probability in the atg arm). the conflicting data reported on urd sct have several explanations, one is about the dose and the timing of atg. the timing of atg infusion has been demonstrated to be crucial for cb transplant : an earlier administration is still active in preventing gvhd while ensuring engraftment and low hampering of immune reconstitution. here we report a large ( sct) retrospective monocentric analysis on low atg doses (and - mg/kg for bm according to the degree of hla matching and mg/kg for all pbsc sct) given early (from day − to − ). pts in the study were aml (n = , %), all (n = , %), hr mds (n = , %), cr (n = , %) cr or (n = , %), active disease (n = , %) for al; median age was (range: - ). myeloablative conditioning were bu-cy (n = , %), bu-flu (n = , %), edx-tbi (n = , %), other (n = , %); pbsc was used in % (n = ); sct were performed between and at the bologna transplant center. sct were performed from hla / identical urd (n = , %), or from / (n = , %), / (n = , %) and o / (n = , %). median follow up was months. overall, grade - agvhd was %, grade - agvhd %; cumulative incidence (ci) of cgvhd of any severity was %, for moderatesevere cgvhd %. ci of relapse and nrm was % and %, respectively. the -year overall and disease-free survival were % ( % ci: - %) and % ( % ci: - %). the gvhd (agvhd grade - and moderate-severe cgvhd) and relapse free survival (grfs) of the entire population ( figure ) was % at years ( % ci: - %). restricting the analysis to patients in cr - , we found that cgvhd (any severity), gfrs and os at years were %, % and %, respectively. comparing transplants with / urd to mismatched ones ( / or less) we found a trend for increased mod/sev cgvhd in pts undergoing transplant with mismatched urd (shr . , % ci: . - . , p = . ); agvhd grade - and cgvhd overall were not significantly increased; relapse incidence according to hla mismatches resulted % and % in / and ⩽ / , respectively; grfs was % in / and % in ⩽ / . the data reported show that low and early administration of atg is able to effectively prevent acute and chronic gvhd without increasing relapse thus ensuring really convincing grfs, even for o / matched urd transplants. graft versus host disease (gvhd) is one of serious complications in patients after allogeneic hematopoietic stem cell transplantation. the application of mesenchymal stem cells (msc) represents a promising method for the treatment of severe steroid refractory gvhd. we present the data from an interim analysis of clinical trial, within which we applied msc in patients with acute or chronic gvhd after allogeneic transplantation. the diagnoses included aml ( pts), mds ( pts), all ( pts), cll/nhl ( pts), mpn ( pts). the patients underwent sibling hla-compatible ( ), haploidentical ( ), unrelated hla-compatible ( ) or hla-mismatched ( ) transplants. the median interval between the transplantation and msc was months ( - ). the indications for msc infusion were steroid resistant acute gvhd ( pts), steroid-dependent gvhd (agvhd pts, cgvhd pts) or chronic gvhd with the need for long-term immunosuppression and corticosteroid intolerance ( pts). msc were applied as a single infusion at a median dose of . ( . - . ) × /kg. response to treatment was assessed on day , , and . the severity of gvhd prior to msc was graded as clinical stage ( - ) in acute and stage ( - ) in chronic gvhd, respectively. the median dose of corticosteroids was . ( . - . ) mg/kg/day in agvhd and s . ( . - . ) mg/kg/day in cgvhd patients. on day + the partial response (pr) was achieved in % of patients with agvhd, the stabilisation of gvhd (sd) was found in % of patients with cgvhd. the dose of corticosteroids was reduced in most patients with agvhd (to % of the starting dose; - %), while the early reduction was possible only in % of cgvhd patients. on day+ only patients were evaluable. the agvhd patients ( pts) achieved a significant clinical response: pr, cr and dose of corticosteroids was reduced in all of them (to %; - %). the minor responses were achieved in cgvhd patient ( pts.) with pr and sd. however the dose of corticosteroid was reduced in % of these cases (to % of the initial dose; - %). a total of patients died because of infectious complications. most of them ( / ) were agvhd patients who expired early up to day + . there were observed no side effects of msc application neither during the infusion nor later during the follow-up of ( - ) months. the analysis of lymphocyte reconstitution revealed the changes of kinetics of some subsets as compared to the day + benchmark. the b-lymphocyte count tended to decrease in % of patients from chronic gvhd subgroup (vs % in agvhd). conversely nk cells declined in most agvhd patients ( % vs % in cgvhd). also the pro-inflammatory th cell was affected especially in agvhd (decrease in % pts vs % pts in cgvhd). the counts of myeloid/plasmocytoid dendritic cell increased in %/ % agvhd and %/ % ccvhd patients. the screening testing of cytokines (raybiotech, cytokines, pts, day + to + ) revealed changes of some analytes after msc infusion, including a decrease of proinflammatory cytokines such as ifn-γ, tnf-α, il- . our experience with the treatment of gvhd using msc confirmed the safety of this immunotherapy. the favourable clinical effect with reduction of severity of gvhd and steroids dose was observed, especially in patients with acute form of gvhd. methotrexate day + omission is not associated with higher incidence of acute graft-versus-host-disease mm rivera franco, e leon rodriguez and a campos castro allogeneic hematopoietic stem cell transplantation (allo-hsct) remains a high-risk procedure due to its related morbimortality, limiting the broader application of this important treatment modality. despite extensive research over the years, acute graft-versus-host-disease (agvhd) affects the majority of patients undergoing allo-hsct, and up to % will develop clinically significant grades (ii). over the years, several methods for gvhd prophylaxis have been implemented, including immunosuppresive agents. methotrexate (mtx) is one of the earliest drugs used for gvhd prophylaxis. frequently, a short course of intravenous methotrexate (given on days + , + , + and + after hsct) is combined with a -month tapered course of cyclosporine. there is no consensus on which drugs and schedules for prevention of gvhd are best and clinical practice varies by institution. further, it is not clear whether omission of the day + dose of mtx has a negative effect on outcome in terms of morbidity. to describe the frequency of acute and chronic gvhd, mucositis and engraftment in patients receiving methotrexate (plus csa) as prophylaxis, omitting day + . ninety-five consecutive patients who underwent allo-hsct from to september , and received mtx as immunosuppressive prophylaxis were included. all patients received three doses of mtx, always excluding day + . mtx was administered iv, either mg/m day + , + , + or mg/m day + , and mg/m during days + and + . we included patients ( % male). the most frequent underlying diseases were aplastic anemia ( %) and acute lymphocytic leukemia ( %). ninety-nine percent of patients had a matched related donor. forty patients ( %) had gender disparity with their donor, and % presented abo incompatibility (major in %). most of the patients received myeloablative conditioning regimens (n = , %). the median of cd + infused cells were × (range: . - . ). the median neutrophil and platelet engraftment was ( - ) and (range: - ) days, respectively. from all the cohort, only patients ( %) developed acute gvhd ( % grades i-ii) ( figure ). thirty patients ( %) developed chronic gvhd, which was limited in %. most of the patients, % (n = ), presented acute toxicity after the conditioning regimen, from which % (n = ) corresponded to superior mucositis ( % grade i-ii and % grade iii-iv. the -year overall survival was % and the -year relapse free survival was %. our results showed a low incidence of acute gvhd, mostly grades i-ii, and similar survivals compared to previously reported studies, proposing that the administration of day + mtx as gvhd prophylaxis is not mandatory, however, prospective studies might be necessary to test our results. [p ] disclosure of conflict of interest: none. outcome of refractory graft versus host disease (gvhd) treated with extracorporeal photopheresis (ecp) as second line: a single-center experience j cornago navascues, b aguado bueno, av arriero garcía, edc jimenez barral, i vicuña andres and a alegre amor hematology department, university hospital la princesa, madrid, spain gvhd is a common and, sometimes, life-threatening entity related to hematopoietic stem cell transplantation (hsct). steroids remain the first-line therapy but they are not always enough to control it, or their side effects are simply unacceptable. both acute and chronic gvhd are responsible of impairment occurred in different organs that can lead to increase morbidity and mortality in our patients. different options are available as second line, but it is a well known fact that ecp, due to its inmunomodulatory mechanism, yields satisfactory response rates and presents excellent safety profile. from may to october , patients with steroid-dependent or refractory gvhd have been treated in our centre with ecp. we have performed ecp procedures with the therakos cellex device, an integrated 'on line' system. the transplant was from a sibling donor in cases and from an unrelated donor. the median of cd + infused was . × cd /kg. eight patients ( . %) presented agvhd, ( . %) cgvhd and finally, ( . %) had an overlap gvhd syndrome. most of patients ( . %) with agvhd had a severe intestinal involvement as the main manifestation of the disease. however, all patients with cgvhd had a multiorgan involvement with a median of four organs affected, being skin, mouth, eyes and lungs the most common implicated. ten patients in our series have died, for gvhd complications or infections and due to relapse of aml. as firstline treatment they all received steroids and cyclosporine or mycophenolate mofetil. median ecp per patient has been ( - ). ecp procedures were performed for consecutive days, in initial phase weekly (in those with agvhd), or every weeks (cgvhd) and then monthly according to clinical response, evaluated by clinical assessment and reduction in immunosuppression. about % of patients with agvhd had a significant clinical response to ecp so that steroid doses could be tapered and even in . % of them withdrawal was possible. in the cgvhd group overall response rate (orr) to ecp was . %. in % of these patients steroids could be suspended after a median of . ecp procedures. all patients who responded to ecp in cgvhd are still alive. independently of gvhd type, . % of patients responded to ecp and % of them even could stop steroid therapy. those who had no response are dead. in cgvhd, . % of patients remain alive, in contrast with agvhd or overlap syndrome patients whom survival is around %. about adverse events, % of patients did not present any complication associated with ecp. complications were mostly related to central venous catheter, with cases of bacteremia and thrombosis, easily recovered. in our experience, ecp is effective as second line treatment in gvhd, obtaining the best results in the chronic gvhd group. in fact, cgvhd patients with a good clinical response to ecp, specially when steroid doses can be tapered, have the better outcomes and longer survival. the tolerance to the procedure is excellent without severe adverse events. more experience is required to determine the best scheme of ecp and its role as prophylactic treatment. mesenchymal stromal cells (mscs) possess immunomodulatory properties and may play important roles in graft-versushost disease (gvhd) and engraftment. this study examined co-transplantation of mscs and hscs (hematopoietic stem cells). we investigated co-administration of ex vivo expanded mscs along with hla-identical sibling-matched hscs in β thalassemia major patients. we recruited patients from january to january in our study. all participants received cyclophosphamide-based or fludarabine-based conditioning regimens and short-course methotrexate and cyclosporine as gvhd prophylaxis. mscs were administered intravenously ( . - . × /kg) into patients (n = ) h before infusion of hscs. the outcomes were then compared to those of patients transplanted with hscs alone. the median follow-up in the msc and non-msc group was . and . years, respectively. median time to wbc engraftment . × /l was . days (range: - days) in both groups (p-value = . ) and median time to platelet engraftment × /l was . days (range: - days) in the msc group, while it was . days (range: - days) in the non-msc group (p-value = . ). fifty-six percent of patients had acute gvhd in the msc group compared to the non-msc group where . % developed acute gvhd (p-value = . ). meanwhile, chronic gvhd was % in the msc group and % in the non-msc group (p-value = . ). although the incidence of acute and chronic gvhd was lower in co-transplantation of hscs and mscs, no statistically significant difference was noted between the two groups. three-year overall survival rate was % and % in the msc and non-msc group, respectively (p-value = . ). three-year thalassemia-free survival rate was % in the msc group and % in the non-mscs group, showing no statistically significant difference (p-value = . ). the -year rejection incidence in the msc and non-msc group was % and %, respectively (p-value = . ). there was no statistically significant difference between the two groups in terms of -year transplant-related mortality (pvalue = . ). this study indicates that co-transplantation of hla-identical sibling hscs with mscs does not inflict harm on bone marrow transplantation procedure and seems to be safe and secure. on the other hand, differences between the two groups in acute and chronic gvhd, engraftment, overall survival, thalassemia-free survival and rejection incidence did not reach statistical significance. therefore, despite the immunomodulatory activity of mscs and their role in gvhd amelioration and engraftment improvement resulted from in vitro studies, their efficacy in the clinical setting has not been conclusively proven which indicates further multicenter randomized clinical trials are required. keywords: β-thalassemia major, co-transplantation of mesenchymal and hematopoietic stem cells, engraftment, graft-versus-host disease. hematology-oncology and stem cell transplantation research center, tehran university of medical sciences, shariati hospital, tehran, iran. disclosure of conflict of interest: none. a number of studies were published with contradictory results comparing tacrolimus (tac) and cyclosporine a (csa) for graftversus-host disease (gvhd) prophylaxis, but there are only few that accounted for pharmacokinetic (pk) parameters. in this retrospective study we have identified pk parameters that affected gvhd incidence and incorporated them in the s multivariate comparison of tac-and csa-based prophylaxis. the retrospective study included consecutive patients with csa and consecutive patients with tac prophylaxis. % were grafted from matched related donor (mrd) and % from unrelated donor (ud). about % received busulfanbased myeloablative conditioning (mac) and % reducedintensity conditioning (ric). second agent for gvhd prophylaxis was short-course methotrexate (mtx) - mg/m on days + , , , in % of patients and mycophenolate mofetil mg/kg days − to + in %. unrelated graft recipients also received antithymocyte globulin (atgam, pfizer, ny, usa) mg/kg. the pk parameters analyzed were mean and median concentrations, pk variability parameters and number of concentrations below the targeted limit (nlow) within , and days after hsct. for tac the highest predictive value for acute gvhd was observed for median concentration during first days (auc = . ), and for absolute skewness (auc = . ) of concentration data. for csa parameters with highest predictive value were median concentration (auc = . ) and variability coefficient (auc = . ) during first days. nlow was also a significant parameter for tac current gvhd prevention regimens are partially effective, delay immune reconstitution, impair graft versus tumor effect and are cumbersome to use. therefore, there is a pressing need to develop innovative approaches for the prevention of gvhd. we completed a phase i-ii study employing a calcineurin and mtor inhibitor-free regimen based on a combination of post-transplant cyclophosphamide and bortezomib (cybor) in patients receiving fludarabine and busulfanbased reduced-intensity conditioning followed by peripheral blood, matched related or unrelated transplant. patients receiving grafts from unrelated donors also received ratg. we reported that the regimen was feasible and safe and yielded promising outcomes. ( , ) herein, we compare the results to those of a quasi-contemporaneous matched group of patients receiving a calcineurin-based gvhd prophylaxis. the experimental and control groups were well-matched in terms of age, sex, donor type, disease status, renal function and pam score. the cybor group (n = ) was treated during a timeframe spanning from to and the control group (n = ) from to . gvhd prophylaxis for the control group was mmf and csa (n = ) or tacrolimus (n = ). both groups received supportive care according to standard institutional protocols. median follow-up for the cybor group was . months as opposed to . for the control group. median times to neutrophil engraftment for the cybor and control groups were days ( - ) and ( - ), respectively (p = . ). two patients from the cybor arm died before achieving platelet engraftment. for the remaining s patients, median time to platelet engraftment was days ( - ). for the control group, five patients never dropped their platelet count below × /l. for the remaining patients, median time to platelet engraftment was days ( - ) (p = . ). there was no primary or secondary graft failure in either of the two groups. the incidences of acute grade ii-iv and grade iii-iv for the cybor group were . and . %. for the control group, the incidences were (p = . ) and % (p = . ). the incidence of chronic gvhd for the cybor and control groups were % and . %, respectively (p = . ). treatment-related mortality was . % and % for the cybor and control groups, respectively (p = . ). the incidences of cmv, ebv and bk reactivation for the cybor group were . %, . % and . %, respectively. for the control group, the incidences were . % (p = . ), . % (p = . ), % (p = . ). the -year progression free survival and overall survival were . % and . % for cybor group and . % and . % for the control group ( figure ). the year gvhd and disease-free survival (grfs) were . % and %, respectively. despite the limitations of our study that include its size and its design and the delayed neutrophil and platelet engraftment associated with the cybor regimen in comparison to calcineurin-based prophylaxis, our data confirm the promising outcomes previously reported with the cybor combination and reaffirm the need for a large randomized study comparing cybor to a standard calcineurin-based regimen. clostridium difficile infection (cdi) causing enterocolitis may represent a serious clinical problem in patients undergoing allogeneic hematopoietic cell transplantation (allo hct). the reported prevalence varies substantially among heterogeneous patient cohorts. although cdi has been proposed as a risk factor for the development of gastrointestinal (gi) acute graft-versus-host-disease (agvhd), limited knowledge on the prevalence of cdi, occurrence of gi agvhd in cdi patients, relapse incidence and mortality of cdi patients in large patient cohorts is available. the aim of this analysis was to study the implications of cdi in a homogenous cohort of patients with either aml or mds undergoing allo hct. at our center all patients undergo stool test once a week for clostridium difficile antigen while in aplasia until discharge, irrespective of clinical symptoms for enterocolitis. patients with positive stool antigen tests (that is, toxin test) in the absence of clinical symptoms were referred to as cd+, in contrast to patients without a positive test and without clinical symptoms which were referred to as cd − . we retrospectively analyzed the data of a total of n = patients with either aml or mds undergoing allo hct in our institution between and . overall survival (os) was measured from allo hct to the date of death or last follow-up. after hsct, relapse and nonrelapse mortality were considered as competing events. eventprobabilities were calculated according to kaplan-meier for os and using competing event statistics for the cumulative incidence of relapse (cir), non-relapse mortality (nrm) and agvhd. % confidence intervals (ci) were provided for major endpoints. statistical analyses were performed using the r environment for statistical computing version . . (r core team , vienna, austria, www.r-project.org). from a total of n = patients with either aml or mds who underwent allo hct, we identified n = ( %) who were cd − , n = ( %) who were cd+, and n = ( %) who had cdi. interestingly, n = ( %) of patients with cdi were diagnosed having gi agvhd as compared to n = ( %) of patients who were cd+ and compared to n = ( %) of patients who were cd − , p = . . the three groups harbored no differences when comparing incidences of liver and skin agvhd or chronic gvhd, respectively. when dissecting gi agvhd according to ctcae criteria, only n = ( %) of cdi patients vs n = ( %) of cd+ patients, and n = ( %) of cd − patients had grade - gi agvhd, p = . . with regard to os and trm, no statistical differences were observed between the three groups. the cir was % for patients with cdi, % for cd+ patients and % for cd − patients, p = . , respectively. this analysis represents the largest published analysis of clostridium difficile in patients with aml or mds who underwent allo hct. the prevalence of cdi in this patient cohort was %. patients with cdi developed significantly more often gi agvhd as compared to patients who were either cd+ or cd − , respectively. however, this did not translate into differences in os or trm. disclosure of conflict of interest: friedrich stölzel has received research funding from astellas. the majority of studies on cytokines in allogeneic hsct were performed with classical gvhd prophylaxis, consisting of nonspecific immunosuppressive agents. with this type of prophylaxis almost in all studies published, higher levels of proinflammatory cytokines are associated with development of acute gvhd, while lower levels indicate the success of immunosuppressive agents in abrogation of alloreactive response. currently, there is no data, whether the dynamics of cytokines after ptcy is similar to the situation of classical gvhd prophylaxis. out of adult patients transplanted at first state medical university with ptcy between and we have identified cases with acute gvhd and plasma samples available. these patients were matched in the ratio : to patients who did not develop acute gvhd. the study group was comprised of adult patients with hematological malignancies who underwent hsct. all patients received ptcy-based gvhd prophylaxis. five plasma biomarkers were studied by elisa: il- a, il- , il- , tnf-α and ifn-γ. blood samples were obtained from patients on days − , , + , + . the fifth time point varied between day + and + to represent the sample after engraftment, but before onset of acute gvhd. about ( %) out of gvhd patients had a grade i, ( %) grade ii, ( %) grade iii agvhd, ( %) patients developed multiorgan agvhd. about patients ( . %) had chronic gvhd. there was no difference between gvhd + and gvhd − groups in any of the clinical parameters. the median of engraftment for all patients was ( - : range). the median agvhd was days ( - : range). neither of the cytokine levels was significantly different in patients with agvhd grades i − iv and without gvhd. however, for patients with agvhd grade ii − iv we found that low levels of il- on day + ( . ± . vs . ± . pg/ml, p = . ) and ifn-γ on day + - ( . ± . vs . ± . pg/ml, p = . ) were associated with increased risk of gvhd. the roc analysis was performed to determine the cut off values for il- - . pg/ml (auc = . ) and ifn-γ - . pg/ml (auc = . ). the incidence of agvhd grade ii-iv was significantly higher in patients with levels of cytokines lower than cut off ( % vs . %, p = . and . %, p = . for il- and ifn-γ, respectively). the same pattern was observed for patients with agvhd grade iii-iv. low levels of il- ( . ± . vs . ± . pg/ml, p = . ) and ifn-γ ( . ± . vs . ± . pg/ml, p = . ) on day + were especially predictive. the cut off values for il- was . pg/ml (auc = . ) and for ifn-γ- . pg/ml (auc = . ). the incidence of agvhd grade ii-iv was also significantly higher in patients with levels of cytokines lower than cut off (p = . and p = . for il- and ifn-γ, respectively). for chronic gvhd only higher level of il- at day + ( . ± . vs . ± . pg/ml, p = . for patient with and without gvhd, respectively) was significantly predictive. in this pilot trial we have demonstrated that dynamics of cytokines after gvhd prophylaxis with ptcy may be different from conventional one, and well-known predictive biomarkers might not work after ptcy. further large prospective trials are warranted to elucidate reliable biomarkers for gvhd after this type of prophylaxis. disclosure of conflict of interest: none. graft versus host disease (gvhd) remains one of the main obstacles to broader application of allogeneic transplantation. gvhd prevention and treatment techniques are poorly standardized. the st-line treatment of newly diagnosed chronic (c) gvhd is corticosteroid. there is no standard ndline treatment for cgvhd. approximately - % of patients (pts) with cgvhd require secondary treatment within y after initial systemic treatment. recently the jak / inhibitor ruxolitinib emerged as an efficacious treatment for corticosteroid-refractory (sr) acute and c-gvhd with a % of sr-cgvhd patients reporting a long lasting immunosuppression-free complete response. the current study seeks to analyse the efficacy and safety of ruxolitinib in highly pre-treated sr-cgvhd pts in our centre. ruxolitinib treatment was given off label after provision of an informed signed consent and in the absence of alternative therapeutic options including clinical trials. we analysed data prospectively collected at our long-term follow-up clinic between and . a written consent was given by pts allowing the use of medical records for research in accordance with the declaration of helsinki. overall pts (median age y-range: - years; mean karnofsky score %) with sr-cgvhd were treated s with ruxolitinib. median time from transplant was months (range: - ). ruxolitinib was initiated at a starting dose of mg twice daily-median time on ruxolitinib months (range: - )- / pts increased the dose up to mg twice daily. four pts had a classic and an overlap sr-cgvhd. all of them had skin sclerodermatous involvement and / joint and fascia involvement with significant decrease of range of motion and limitation of adl. all pts were previously treated with several lines of immunosuppression ( - ) including high-dose prednisone in st line ( / ), rapamycin ( / ), tk-inhibitor imatinib ( / ), extracorporeal photopheresis ( / ). all pts were pre-screened for risk of infection and regularly checked on a fortnightly basis. all pts were under active prophylaxis according to recommendation for gvhd pts and ruxolitinib therapy. after a cumulative follow-up of days we reported only one serious adverse event represented by a cmv pneumonia requiring hospitalization with complete recovery. early time point evaluation ( / pts evaluable) at + month underlined how all pts were reporting subjective improvement at the patient global ratings according to nih . data were confirmed at the health care provider global ratings. month evaluation ( / ) confirmed meaningful responses (partial responses / ) according to nih , with both patient and health care provider global ratings improvement and concomitant enhancement in lee skin symptoms score and sf- health-related qol. at last followup no evidence of myelosuppression, infections, pml, nonmelanoma skin cancer was registered. considering the concomitant treatment (with reference to azoles and rapamycin or cyclosporine) no cases of toxicity due to drug-druginteraction was reported. ruxolitinib is well tolerated in highly pre-treated sr-cgvhd. its safety profile seems to be reassuring. the efficacy data observed also at this early time point is preliminary but promising in this subset of pts with a long history (⩾ lines) of treatment for cgvhd. confirmatory study in a larger number of patients is underway on a multicentre basis. disclosure of conflict of interest: none. severe acute enteral graft-versus-host-disease (gvhd) is a lifethreatening complication of allogeneic bone marrow transplantation. in case of resistance to corticosteroids as the firstline treatment severe enteral gvhd harbors a high morbidity and mortality. retrospective analyses indicate efficacy of the jak / -inhibitor ruxolitinib in the treatment of acute or chronic gvhd in adults, but experience in paediatric patients is limited. here, we report a small cohort of paediatric patients with stage steroid-refractory gvhd of the gut who received ruxolitinib as salvage therapy within a multimodal immunosuppressive regimen. we retrospectively analysed four patients aged - years with severe, steroid-resistant acute gvhd of the gut who were treated with ruxolitinib in our institution. all patients were transplanted for non-malignant haematologic disorders, graft source was × mmud, × mud, × msd. the conditioning regimen consisted of treosulfan, fludarabine and thiotepa. serotherapy with thymoglobuline was administered in all patients transplanted from unrelated donors. all patients received mtx and cyclosporine as gvhdprophylaxis. gvhd was staged according to the glucksberg-scale. ruxolitinib was added to the immunosuppressive regimen when acute stage gvhd was reached and became resistant to treatment with methylprednisolone ( mg/kg/day) as well as infliximab and mycophenolate (mmf) as second-line immunosuppressants. acute stage enteral gvhd developed at a median of days after transplant ( - days) and ruxolitinib was started at a median of days post-transplant ( - days). the starting dose varied between mg/day and mg/day, that is, . - . mg/kg/day, taking into account the expectedly low bioavailability of the oral drug during severe diarrhea. upon improvement of gvhd symptoms and/ or increasing side effects the dose was gradually tapered and ruxolitinib was discontinued after a median of treatmentdays ( - days) . after addition of ruxolitinib to the immunosuppressive regimen, the symptoms of acute gut gvhd gradually improved in all four patients with decreasing abdominal pain and stool volumes. immunosuppression with steroids and mmf could slowly be tapered. all patients are alive after a median follow-up of days ( - days) from diagnosis of acute stage gut gvhd. the most prominent side effect attributable to ruxolitinib was thrombocytopenia with a nadir in platelet counts after days of ruxolitinib treatment in / patients. platelets recovered within weeks after ruxolitinib was discontinued. neutropenia was observed in one patient with anc dropping o . /nl after days of ruxolitinib treatment. mild to moderate elevation of liver transaminases was observed in all four patients during ruxolitinib treatment. one patient developed imminent acute renal failure, another patient showed symptoms of hemolytic uraemic syndrome. however, due to the multimodal treatment of these critically ill patients, these complications could not clearly be attributed to ruxolitinib. ruxolitinib is potentially beneficial in severe acute enteral gvhd in children refractory to corticosteroids as well as second-line immunosuppressants. however, randomized trials are warranted to verify safety and efficacy of ruxolitinib in this patient cohort. disclosure of conflict of interest: none. steroid refractory acute gvhd is a major cause of mortality after allogeneic stem cell transplantation. until date, no agent or treatment strategy has demonstrated superior efficacy in this patient group. the dose and duration of steroid treatment is associated with several short and long-term side effects, therefore concepts facilitating rapid steroid taper may be beneficial. both ruxolitinib and ecp have been reported to be effective in treatment of steroid refractory (sr) agvhd. we analyzed data from consecutive adult patients who received ruxolitinib for sr agvhd between march and august in our institution overall, patients (male n = ; female n = ) with a median age of years (range: - ) were included. donors for allogeneic sct were msd (n = ), mud (n = ) and mmud (n = ). median time to gvhd onset after stem cell transplantation was days (range: - days). about patients had agvhd grade iii or iv (all with gi involvement), while patients had skin grade involvement. sr agvhd was diagnosed if agvhd manifestations were progressive after days or persistent and without improvement after days or no partial remission after days of treatment with mg/kg bw of systemic steroids. patients received additional ecp (n = ), if response to ruxolitinib was lacking or slow (n = ) or instead of ruxolitinib due to cytopenias (n = ). ruxolitinib was first-line treatment for sragvhd in patients ( %). median initial dose of ruxolitinib was mg (range: - mg) twice daily. steroids were tapered and stopped, even if agvhd was still active. primary end point was non-relapse mortality at months. secondary end point was response on day after initiation of ruxolitinib. response occurred relatively slowly, resulting in a day overall response rate of % (cr = , pr = ). however, a total nine patients ( %) attained a complete response (cr), five with ruxolitinib alone and four others in combination with ecp. about patients ( %) required dose reduction or interruption of ruxolitinib mainly due to cytopenias. after a median follow-up of days, patients are alive. causes of death were relapse of malignant disease (n = ), gvhd (n = ), infections (n = ) and other (n = ). median survival from diagnosis of sr agvhd was days for non-responders and days for responders ( figure , p = . ). in univariate analysis, non-response was associated with higher risk of nonrelapse mortality (rr; . , % ci: . - . , p = . ). ruxolitinib and ecp are two effective promising treatment options, which may be complementary in patients with sr agvhd. cytopenia is the most frequent side effect of ruxolitinib while infections remain the major cause of death. [p ] disclosure of conflict of interest: ayuk-therakos: honoraria; kröger: novartis: honoraria, research funding. steroid-refractory acute graft-versus-host disease (sr-agvhd) is associated with a dismal outcome. janus kinase (jak) / signaling has been shown to be instrumental in multiple steps leading to inflammation and tissue damage in gvhd. jak / inhibitor ruxolitinib was studied in the treatment of sr-gvhd by zeiser et al. (leukemia ) , and the overall response rate was reported to be . %. we have now studied ruxolitinib in the treatment of six adult patients with steroid-refractory, grade iii-iv, intestinal agvhd. all the patients were male. the median age of the patients was (range: - ) years. three of the patients were transplanted for aml, one for all, mds and mm each. all the patients had been given a myeloablative conditioning treatment (cytbi , treosulfan+fludarabine ). two patients had a sibling donor and four a matched unrelated donor. the graft was from peripheral blood in all the patients. gvhd prophylaxis consisted of cyclosporine and a short course of methotrexate, and in addition antithymocyte globulin in the unrelated donor setting and methylprednisolone in one sibling recipient. agvhd of the intestine manifested on days + , + , + , + , + and + with diarrhea. in two patients it was preceded by agvhd of the skin by and days, respectively. gi-biopsy showed acute gvhd of grade iii and of grade iv in three patients each. treatment of intestinal gvhd was started with methylprednisolone mg/ kg/day, tapering the dose to and mg/kg after doses each. gastroduodenoscopy and colonoscopy were performed at the onset of symptoms indicating intestinal gvhd. biopsy confirmed the diagnosis in all cases. because the diarrhea continued in spite of methylprednisolone treatment, ruxolitinib was started , , , , and days from the first day of diarrhea. the dose of ruxolitinib was mg × per day orally. four patients showed a clear response to ruxolinitib, normalization of bowel function, after , , and days from the start of ruxolitinib treatment. the healing of the intestinal lesions was verified by biopsy. two of these patients had received extracorporeal photopheresis simultaneously. two patients did not benefit from ruxolinitib treatment. one of them had continuous infectious complications and therefore ruxolitinib was only started after days from the start of diarrhea. the other patient died of fulminant diarrhea after weeks of ruxolitinib treatment. cmv reactivation was detected in three of the responders, and two of them had also polyoma virus cystitis. one patient developed a pulmonary aspergilloma, which is under control with drugs. corticosteroid-resistant gastrointestinal acute gvhd was treated in six patients, out of whom four showed a good response. disclosure of conflict of interest: none. although methotrexate (mtx) is commonly used in the prophylaxis of graft-versus-host disease (gvhd) after allogeneic hematopoietic stem cell transplantation (allo-hsct), some small studies have also reported its use in the treatment of chronic gvhd. the aim of this study was to evaluate the efficacy and safety profile of low-dose mtx for treatment of sclerodermatous chronic gvhd (sgvhd) after the failure of first and second line treatments. we retrospectively evaluated adult patients who received low-dose mtx as salvage treatment of sgvhd during the period elapsed between june and june in a tertiary referral university hospital in spain. there were ( %) males and ( %) females. the median age was years (range: - ). all had received an allo-hsct for hematologic malignancies. the median time from allo-hsct to sgvhd was days (range: - ). thirteen patients ( %) had presented previous acute skin gvdh. superficial skin lesions mimicking lichen planus (lichenoid gvhd) were diagnosed in ( %) patients, while lesions resembling lichen sclerosus, morphea or fasciitis (sgvhd) where seen in all ( %) patients. the total body surface area was affected by more than % in patients ( %). besides the skin, other organs/tissues involved were the eyes ( %), mouth ( %), nails ( %), lungs ( %), liver ( %) and gastrointestinal tract ( %). treatment lines prior to mtx administration were: prednisone (pdn) in patients ( %), phototherapy (pht) in ( %), cyclosporine (cya) in ( %), mycophenolate mofetil (mfm) in ( %), pht + pdn + cya in ( %), pdn + mfm + cya in ( %), extracorporeal photopheresis + pdn in ( %). the median time from sgvhd onset to mtx treatment was days (range: - days). mtx was administered subcutaneously in patients ( %) and orally in patients ( %). median dose of mtx was . mg/week (range: . - . mg/week) and median length of treatment was weeks (range: - weeks). in two patients ( %) early withdrawal of mtx occurred (one due to early death secondary to septic shock and other due to rapid disease progression). mtx-related toxicity occurred in three patients ( %): megaloblastic anemia, asymptomatic increase of liver enzymes and mucositis, respectively. response to mtx was evaluated in the patients ( %) s who did not suffer early mtx discontinuation. seventeen patients ( %) presented a partial response; of them, two are still under mtx treatment for and weeks, respectively. fourteen patients ( %) received pdn concomitantly to mtx (median dose mg/day, range: - ); year after mtx treatment, only four patients were receiving pdn (median dose mg/day, range - ). seven patients have finished mtx treatment without reappearance of symptoms, receiving only topical treatment with emollients, tacrolimus or corticoids for short periods. in four patients ( %) sgvhd progressed despite mtx administration. our data suggest that mtx is a safe, inexpensive and effective alternative for refractory sgvhd. its potential used in earlier phases of sgvhd deserves further investigation. disclosure of conflict of interest: none. severe chronic gvhd has a major influence on late morbidity and mortality after hematopoietic stem cells transplantation (hsct). ecp is a good approach to treat refractory-gvhd: leucocytes are obtained from peripheral blood by apheresis, incubated with -mop, irradiated and then infused to the patient where they undergo apoptosis and induce tolerance. it is a promising alternative that reduces doses of immunosuppressive therapy and their side effects in the treatment of gvhd. this study shows its efficacy in persistent refractory cgvhd. the procedure was applied to three patients (pts) aged , and years (two aml and one cml), sex ratio ( m/ f) who underwent allogeneic-hsct with myeloablative conditioning regimen based on chemotherapy alone from a peripheral blood stem cells with cd levels: . , . and . × /kg respectively. prophylaxis of gvhd combined ciclosporin and methotrexate in short cycle. severe extensive cgvhd (according to nih criteria) was observed in the three cases after an average delay of . months ( - ) with involvement of - organs (mouth, eyes, skin, liver, joints and lungs). all pts are refractory to three lines of immunosuppressive agents (ciclosporin-corticosteroids, mmf and imatinib), with an average of thrusts/pt ( - ) over an average period of months ( - ). ecp was performed under the open system or dissociated system (macopharma) for two sessions per week for weeks, one session per week for weeks, one session every weeks for weeks and one session per month for months. after a median period of months ( - ), an average of sessions/pt ( - ) was performed. in terms of tolerance, a red blood cell transfusion was required in one pt, spontaneously resolved lymphopenia was observed in another pt, and a poor venous approach led to the pause of a central catheterization in one pt. the month and -month evaluation according to the couriel response criteria shows a partial response observed as of the first month with net improvement especially on skin sclerotic features and joints retractions initially refractory to all therapeutic lines. this allowed gradual reduction doses of corticosteroids. pce is recommended in the curative treatment for refractory chronic gvhd from the second line. this encouraging study on a small series shows its efficacy in persistent and late refractory forms. it is nevertheless necessary to evaluate it on a larger number of pts. disclosure of conflict of interest: none. successful treatment of steroid-refractory acute gastrointestinal graft-versus-host-disease by fecal microbiota transplantation p neumeister steroid-refractory acute gastrointestinal (gi) graft-versus-host disease (agvhd) is a severe complication of allogeneic hematopoietic stem cell transplantation (allo-hsct) associated with a high mortality rate. loss of intestinal bacterial diversity is thought to be associated with severity of gi-agvhd and an impaired intestinal microbiota with reduced diversity is an independent predictive factor for mortality. fecal microbiota transplantation (fmt) is the application of a fecal suspension derived from a healthy donor into a patient's gi tract. it has been successfully applied in recurrent clostridium difficile associated diarrhea including patients who underwent allo-hsct. we report the complete resolution of lower gi-agvhd following colonoscopic fmts in three patients that had been refractory to - lines of immunosuppressive therapies. microbiota analysis by s rdna before fmts revealed a severely depleted microbiota in all patients. donors (different persons for each patient) were healthy adult subjects. repetitive ( - ) colonoscopic fmts were necessary to permanently establish the donor's microbiome. all patients responded clinically by reduction/normalisation of stoll volumes, stopping total parenteral nutrition and tapering of steroids. a possible causative relationship of fmt in the reversal of severe intestinal dysbiosis and subsequent resolution of gi-agvhd can therefore be hypothesized. the establishment of donors' microbiota and increase in bacterial richness was associated with disease control. no immediate procedure-related infections or other side effects were observed. besides restoration of an initially severely reduced microbial richness by fmts, response of gi-agvhd was sustained despite reduction and discontinuation of concomitant immunosuppressive treatments. restoration of dysbiosis by fmt might represent a promising novel therapeutic approach for refractory lower gi-agvhd. disclosure of conflict of interest: none. tear film proteomics reveals important differences between patients with chronic ocular gvhd and healthy controls k plattner , n gerber-hollbach , s moes , p jenoe , d goldblum and j halter ophthalmology, university hospital basel, ch- basel and proteomics core facility of the biozentrum, university of basel, ch- , basel chronic gvhd frequently involves the eyes, leads to important decrease of quality of life and may threaten visual capacity. sicca syndrome is one of the hallmark of ocular cgvhd. analysis of tear protein composition may help to identify biomarkers for early diagnosis and prognosis of ocular cgvhd. tear fluid of patients with ocular cgvhd were compared with healthy individuals in this single center study. results of the first patients are reported here. tryptic digests from schirmer strips were analyzed on an orbitrap mass analyzer. clinical examinations included slit lamp examination, fluorescein staining, schirmer test, break-up-time (but) and a quality of life questionnaire (osdi). outcome measures were differences and consistency of proteins in human tear fluid s between patients with ocular gvhd and healthy controls. statistical analysis was performed by one sample wilcoxon-tests, p-values o . was considered significant. ten patients (eight males, two females) with a median age of years (range: - ) were analyzed. all underwent pbsct, eight from an unrelated donor. cgvhd overall score was moderate in three and severe in seven. eye organ score was in six and in four patients. all patients had more than one organ manifestation of cgvhd. eight were under systemic immunosuppressive therapy at the time of analysis, two had topical treatments only. in total different proteins were detected in tears analyzed. compared to controls, were differentially expressed in ocular cgvhd. expression was highly significantly different in proteins. compared to controls, expression of proteins was at least -fold increased, representing different categories. among them, more than % of all proteins belong to one of three categories: cytoskeletal proteins, nucleic acid binding or structural proteins. albumin, cluster or keratin (keratin type i-iii) and cluster of pyruvate kinase were most highly overexpressed. expression of proteins was decreased to o to %, belonging to different protein classes. half of them belong to defense/immunity proteins, enzyme modulators, hydrolases, nucleic acid binding and carrier proteins. expression of lactotransferrin, proline-rich protein and prolactin-inducible protein was most profoundly decreased. compared to healthy controls, a high number of protein is found to be differentially expressed in tears in ocular cgvhd. among them high expressions are observed for proteins that may indicate disturbed integrity of ocular surface and leakage of conjunctival capillaries. most profoundly decreased proteins include proteins with important functions in host defense and immunomodulation. more detailed pathway analysis is necessary to identify biomarkers for ocular cgvhd. disclosure of conflict of interest: none. steroid-dependent chronic gvhd after allogeneic peripheral blood stem cell transplantation is a great problem. nonresponders to corticosteroid therapy are at high risk of mortality. we hypothesized that such patients could benefit from treatment strategy using in patients with primary severe autoimmune diseases like multiple sclerosis and crohn's disease. patient z., y.o. was diagnosed in july with myelomonocytic leukemia (jmml). initially he was treated with low-dose of cytarabine and epigenetic agents. in september , jmml progression was observed with leukocytosis, thrombocytopenia, splenomegaly. bone marrow aspirate showed . % monocytes and . % blast cells. splenectomy was performed in november due to refractoriness to blood components transfusions. in december unmanipulated haploidentical peripheral blood stem cell transplantation from mother with . × /kg cd + and . × /kg cd + was performed. the conditioning regimen was myeloablative including melphalan mg/m day − and treosulfan mg/m days − , − , − . no organ toxicity grade was observed. gvhd prophylaxis consisted of hatg mg/kg on days − , − , − , + , i.v. tacrolimus from d − and mmf mg/kg from d . engrafted was fast and prompt ( % donor) with wbc . × /l on d + , plts × /l on d + . acute gvhd of stage ii was observed in early posttransplant period and treated with steroids and tnf-α inhibitor (infliximab). patient also received five procedures of ecp. all attempts of immunosupression tapering failed and the patient was staying on high dose of tacrolimus, mmf and courses of steroids till october . in october , gvhd stage ii flare with blood eosinophilia occurred after another attempt of steroids withdrawal. clinical examination showed that the patient was in complete remission with full donor chimerism. mild response of gvhd to steroids was observed. in april and may patient received two doses of rituximab mg/m with no significant response. in order to restore naive immune system first course of chemotherapy with cyclophosphamide mg/m was performed in the end of may . no toxicity grade was observed. the patient recovered wbc . × /l on d + , plts × /l on d + . in the phase of hematological recovery he was mobilized with g-csf and two leukaphereses of pbscs were performed. in june our patient was transplanted with previously collected . × cd +/kg following nonmyeloablative regimen including cyclophosphamide mg/m on day − and fludarabine mg/kg, on days − , − , − . second dose of cyclophosphamide was not administered because of severe hyponatriemia with seizures due to the cpm administration. no other significant toxicity was observed. the patient did not require either blood product or i.v. antibiotics. doses of tacrolimus and mmf were picked on months late and no more steroids were given. the patient is well in cr with no signs of gvhd for months. we speculate that pbsc collection from patients under massive immunosupression underwent allogeneic transplant is difficult but feasible. the nonmyeloablative regimens in such group of patients could be well tolerated and ensure the restoration of naive recipient immune system. this option could be discussed as an attractive alternative for treating resistant gvhd in steroid resistant patients. disclosure of conflict of interest: none. severe acute gi-gvhd is a serious early complication of allotransplants, and still remains a clinical diagnosis.( ) endoscopic biopsies provide the best supportive evidence, but are invasive and morbid in patients who are already medically compromised. f-fdg pet/ct may be able to stratify patients who require endoscopy and biopsy. to evaluate the performance of fdg pet/ct in differentiating moderate to severe gi-gvhd from no or mild disease in pediatric patients with suspected gi-gvhd. retrospective chart review of all paediatric allo-transplant patients referred for f-fdg pet/ct with suspected gi-gvhd from to . clinical follow-up, endoscopy and biopsy findings were correlated with f-fdg pet/ct. regional suv parameters were extracted by placing rois around stomach, duodenum, distal ilium, caecum, ascending, transverse, descending colon, recto-sigmoid colon and rectum. regional, and average large and small bowel suv data were statistically compared between patients with no or mild git-gvhd vs moderate to severe disease. the clinical and biopsy-supported diagnosis of acute gi-gvhd was taken as the true positive diagnosis for acute gi-gvhd. roc curves were generated for whole bowel suvmax values. about scans in patients, median age of years ( mths to y), were performed at a median of days post bmt. there were stage , stage - and with no acute gi-gvhd. transverse colon suvmax was significantly higher in the stage - gi-gvhd compared to no or stage disease (mann-whitney-u-test p o . ). there was a non-significant trend for average large bowel suvmax to be higher in the stage - group than the no or stage disease group (mean suvmax . compared to . , p = . ). a cut off whole bowel suvmax . had a sensitivity of % and specificity of % for detecting moderate to severe gi-gvhd. f-fdg pet/ct is a feasible and potentially useful non-invasive tool in the diagnosis and monitoring of therapeutic efficacy in acute gi-gvhd ( ) . large bowel suvmax may be higher in patients with stage - gi-gvhd, and transverse colon suvmax could have the ability to differentiate children with no or stage gi-gvhd from those with stage - disease. a negative fdg-pet/ct could serve as a criteria to avoid invasive endoscopic procedures and observe for the persistence of gastrointestinal symptoms before subjecting these patients to an imageguided biopsy. in patients too unwell for endoscopy, suvmax (roc curve specificity %) and a high suvmax in the transverse colon could serve as supportive evidence for moderate to severe acute gvhd, in the absence of biopsy findings. a major advantage of a pre-endoscopic f-fdg pet/ ct is to guide the procedurals to sample areas with the best diagnostic yield. prospective controlled studies are needed. oral mucosal progenitor cells (omlp-pcs) possess immunomodulatory and antibacterial properties, suggestive of their in vivo function in healthy tissue and their potential contribution to scarless wound healing in the buccal mucosa ( , ). our aim was to establish whether the function of oral stromal progenitors is impaired in chronic graft versus host disease (cgvhd) and restored with response to treatment. a patient with grade oral cgvhd was treated with systemic thalidomide for weeks ( mg/day). punch biopsies of buccal mucosa were taken before and after treatment. oral progenitor cells were isolated and expanded in vitro. numbers of progenitors was assessed using colony forming unitfibroblast (cfu-f) assays. stem cell markers (cd , cd , cd , cd , cd , cd , hla i and ii) were evaluated by flow cytometry. wound healing and antibacterial potential were assessed using a collagen gel lattice assay and bacterial cocultures as previously described ( , ) . secreted levels of relevant cyto-and chemokines associated with wound healing were assessed by elisa. significant clinical improvement with reduced inflammation in the oral mucosa and healing of ulcers was seen after weeks of thalidomide treatment, with continued improvement after weeks. cell surface expression of cd and cd on omlp-pcs was elevated postthalidomide; markers correlated with stemness and angiogenesis in mesenchymal stromal cells. this correlated with a restoration of wound healing potential and antibacterial function after thalidomide treatment ( figure ). figure : antibacterial testing demonstrated a loss of antibacterial function against (a) gram positive and (b) gram negative micro-organisms in the cgvhd omlp-pcs that could be completely or partially restored to levels comparable with healthy controls after thalidomide treatment. *p ⩽ . , **p ⩽ . , ***p ⩽ . . we demonstrate, for the first time a correlation between clinical improvement of oral cgvhd with thalidomide treatment and restoration of endogenous progenitor cell function. this study highlights the importance of a dysfunctional oral mucosal stroma in the pathogenesis of cgvhd. further studies should focus on the role of the stroma in promoting cgvhd and the precise mechanisms by which thalidomide is able to restore its functions. chronic graft-versus-host disease (cgvhd) is a major cause of late morbidity and treatment-related mortality in patients undergoing allogeneic hematopoietic stem cell transplantation (hsct). cgvhd is driven by a th biased t-cell mediated alloreactive immune response that leads to chronic inflammation and fibrosis in various organs. thymic stromal lymphopoietin (tslp) is an epithelial cell-derived cytokine that mainly affects myeloid cells. upon stimulation with tslp, dendritic cells are polarized towards a dc phenotype driving th biased immune response. we hypothesized that tslp is involved in the pathogenesis of cgvhd and that elevated levels of tslp post-transplant may lead to an increased risk of cgvhd. in the present study, we measured plasma tslp levels during hsct to study associations with clinical outcomes including cgvhd. about adult patients undergoing myeloablative hsct at rigshospitalet, denmark, from to were included. diagnoses included aml (n = ), all (n = ), myelodysplastic syndrome (n = ), other malignancies (n = ) and anemias (n = ). donors were either hla matching siblings (n = ) or mud (n = ). grafts were either bmsc (n = ) or pbsc (n = ). conditioning included tbi (n = ) or high-dose chemotherapy alone (n = ). plasma tslp was measured by elisa (abcam) before transplantation, at the day of transplantation and at day + , + , + and + post-hsct. monocytes were counted daily, and t, b and nk cells were measured at day + and + using flow cytometry. about ( %) patients engrafted; acute gvhd grade - was seen in ( %) patients, and ( %) patients developed severe cgvhd. oas was . %, trm . % and relapse rate . %. median plasma tslp levels increased from before conditioning ( pg/ml) to reach a peak at day + ( pg/ ml, p = . ), followed by a gradual decline. the plasma levels of tslp at day + were positively correlated with same-day monocyte counts (ρ = . , p = . ). approximately half of the patients (n = ) experienced an overall rise in tslp from baseline ( pg/ml) to day + ( pg/ml). this increase in tslp was not significantly associated with any transplantrelated baseline characteristics. however, patients, who had an increase in tslp levels from baseline to day + , had a significantly higher risk of extensive cgvhd compared to those in whom tslp levels at day + were similar or below baseline levels (cumulative incidence of cgvhd: % (increased tslp at day + ) vs % (normal/low tslp at day + ), p = . ). development of cgvhd was also associated with the nucleated cell dose infused (p = . ) and transplant using pbsc (p = . ). tslp plasma levels were not associated with acute gvhd, oas, trm, relapse rate or numbers of t cell, b cell or nk cells posttransplant. we have found that increased levels of tslp from baseline to day + were associated with an increased risk of extensive cgvhd. this association may be due to the ability of tslp to polarize the immune system toward a th response. importantly, the increase in plasma tslp levels was not associated with any transplant-related characteristics suggesting that tslp may be an independent predictor of cgvhd. these findings indicate that anti-tslp treatment may be a new approach to fight severe cgvhd. disclosure of conflict of interest: none. thymopoiesis following hct: a retrospective review comparing interventions for agvhd in a paediatric cohort c roberts , am flinn , ma slatter , , r skinner , h robson , j lawrence , j guest and ar gennery , institute of cellular medicine, newcastle university and great north children's hospital, newcastle-upon-tyne, uk acute graft-versus-host disease (agvhd) is a life threatening complication of allogeneic haematopoietic cell transplantation (hct), treated with topical and/or systemic corticosteroids. in steroid-refractory agvhd extracorporeal photopheresis (ecp) can be effective. ecp exposes apheresed mononuclear cells to -methoxypsoralen and ultra-violet radiation. systemic corticosteroids and agvhd are damaging to thymic tissue. delayed immune reconstitution, especially of the t lymphocyte compartment, is associated with increased morbidity and mortality. therefore, management strategies must be effective in treating agvhd but endeavour to minimise resulting thymic damage. we compare the effect of topical steroid therapy, corticosteroids and ecp on thymic reconstitution following hct in paediatric patients. statistical analysis was performed using the kruskal-wallis test. about paediatric allogeneic hcts were performed between june and april , at the great north children's hospital, newcastle for malignant and non-malignant disease. we reviewed computerised records to categorise patients into four groups: no agvhd, mild agvhd treated with topical steroid, agvhd treated with systemic steroid, agvhd treated with ecp. laboratory data were reviewed to provide values of naive (cd + and cd − )cd ra+cd + t-lymphocytes at , , and months post-hct. values for thymic output for the ecp group were additionally recorded at , , and months during ecp. excluded were patients with no available data, those with o months follow-up, those with chronic gvhd, recipient of hct or received dli post-hct. about patients were included, ( . %) had no agvhd, ( . %) had agvhd treated topically or systemically, ( . %) had agvhd and received ecp. for analysis, the group treated with steroids were divided into those treated with topical therapy and those given systemic steroids. the median values of all groups at each time point ( , , and months) are shown ( figure ). there was a significant difference between the rate of thymopoiesis (measured by the addition of cd + and cd − cd +cd + cells) between all groups (no agvhd, agvhd treated with topical or systemic steroids, and agvhd treated with ecp) at , , and months post-transplant (p = . , p o . , p o . , p = . respectively). further analysis excluded those treated with ecp (so including the no gvhd (n = ), topical treatment (n = ) and systemic steroid treatment group (n = )). at each time point p = . , p = . , p = . and p = . , respectively, demonstrating a statistically significant difference in time to thymopoiesis between those that had developed agvhd and those that had not. acute graft-versus-host disease (agvhd) is a significant cause of morbidity and mortality following allogeneic hematopoietic stem cell transplantation. which despite first line treatment is well-established (esteroids), second line is not well defined. evaluate the results with different second line treatment used and the risk factors associated with of sr-agvhd. we review the clinical records of consecutive patients undergoing allogenic hsct from to in our hospital. about % presented agvhd. sr-agvhd was defined as progression after days, no clinical change in days or incomplete response after days of treatment. about patients ( %) met s criteria for sr-agvhd. there were no significant differences between both groups (sr-agvhd vs no sr-agvhd) respect to age (recipient/donor), unrelated donor, prophylaxis of gvhd, cd lymphocyte and cd cell. the median time between transplantation and agvhd diagnosis was days ( - ). patients who did not respond on fifth day of steroid treatment have an % rate mortality vs % on no sr-agvhd group (p = . ). sr-agvhd: patients presented sr-agvhd and this was related to: hla mismatch ( % sr-agvhd vs % no sr-agvhd, p = . ), male recipient/female donor ( % sr-agvhd vs % no sr-agvhd, p = . ) and advanced underlying disease ( % sr-agvhd vs % no sr-agvhd, p = . ). second line: basiliximab ( . %); extracorporeal photopheresis (ep) ( . %), timoglobulin ( . %) and others therapies ( . %). two patients ( %) obtained complete response (cr) and patients ( %) partial response (pr). global response (cr, pr) after second line (mainly basiliximab) showed better overall survival (p = . ). third line: basiliximab ( . %); ep ( . %), mesenchymal cells (msc) ( . %), ruxolitinib ( , %) and others ( . %). ruxolitinib improve gvhd cutaneous and hepatic but not intestinal. the best results were achieved with ep ( cr, pr) and basiliximab/msc ( pr, respectively). only patients who achieved cr survived. fourth line: msc ( %)/ ruxolitinib ( %) does not improve the prognosis. no serious adverse effects were observed with msc therapy, basiliximab and ep. about % of patients showed cmv reactivation with basiliximab. about patients died ( %), patients with early mortality ( o months) due to refractory agvhd ( %) or secondary infections ( %). overall survival at months and year was ± % and %, respectively. in multivariate analysis the main factor for trm was the steroid-refractory vs steroidsensitive (hr . , % ci . - . ; p = . ) and was unfavorable the association of hepatic and intestinal agvhd (hr . , % ci . - . ; p = . ) no sr-agvhd: patients. trm- was % (n = ), mainly due to infection ( %). trm- year was % (n = ), mainly by gvhd ( %) and infections ( %). median follow-up of months, os- months and year were ± %/ ± %, respectively. trm was associated with not obtained cr/pr after second line (p = . ), no cr after third line (p = . ) and relapse of gvhd despite achieving cr initially (p = . ). in our series only the patients that obtained cr/pr after second-line or cr after third-line improved os. the best results in sr-agvhd were obtained with basiliximab and extracorporeal photopheresis. trm was associated with relapse of gvhd and advanced disease to the transplant. randomized clinical trials are needed to assess different treatment modalities for sr-agvhd. [p ] disclosure of conflict of interest: none. extracorporeal photopheresis (ecp) is a therapy for steroidrefractory chronic graft versus host disease (cgvhd). therapeutic response to ecp has been linked with a progressive increase in circulating granulocytic myeloid-derived suppressor cells (g-mdsc) in acute gvhd, but not in cgvhd . low density neutrophils (ldn) phenotypically resembling g-mdsc (putative g-mdsc) show marked flux in cgvhd patients receiving ecp, and a reduction in their frequency is associated with a sustained therapeutic response to ecp . recent data has identified lectin-type oxidized ldl receptor- (lox- ) as a specific marker of ldn with t-cell (tc) suppressive activity . using this marker we have conducted a cross-sectional study to assess whether putative g-mdscs in this patient cohort have suppressive activity. about patients with steroid refractory or steroid-dependent cgvhd (mean treatment duration of months) receiving ecp and healthy controls were recruited. patients had gvhd affecting skin ( / ), liver ( / ) and gut ( / ). pbmc were isolated and immunophenotyped by flow cytometry for markers of g-mdscs (cd − ve , cd , cd b, hla-dr − ve , cd int ) and lox- expression. suppressive function was determined by measuring the inhibition of proliferation of anti-cd /cd -activated purified cd tc from healthy donors by -day co-culture with g-mdscs from patients. statistical analysis was conducted using graphpad . ecp patients had substantially greater frequencies of circulating putative g-mdsc than healthy controls (p o . ; median: % and iqr %- % vs . % and iqr . %- . %, respectively). while there were substantially greater frequencies of circulating lox- + cells in pbmc from ecp patients than healthy controls (p o . ; median: . % and iqr . %- % vs . % and iqr . %- . %, respectively), these were mainly the minority population within the putative g-mdsc fraction with no significant difference between ecp patients and healthy controls in the proportion of lox- + cells ( % ± % vs % ± %, respectively). ecp had no significant effect on circulating putative g-mdsc frequency measured before and the day after treatment (median: . % and iqr %- % vs % and iqr %- %; n = , respectively) nor on lox- frequency (median: % and iqr . %- % vs . % and iqr . %- . %; n = , respectively). at a tc:g-mdsc ratio of : , isolated g-mdscs from ecp patients suppressed cd tc proliferation (mean ± sd: % ± %; n = ). however, the potency of suppression was highly variable (min-max: - %). the pattern of lox- expression suggests that only a subset of putative g-mdscs in ecp patients are suppressive and may explain why suppressive function in this cell fraction is so highly variable. however, the relatively high frequency of lox- cells in this patient cohort might contribute to immunosuppression resulting in increased susceptibility to opportunistic infections. according to the revised eortc/msg criterion, patients were diagnosed with cns-ifd. among those patients without cns-ifd ( patients), cns-ifd were matched in a : ratio for analyzing the risk factors of cns-ifd. and among ( . %) patients who occurred pulmonary ifd without cns involvement, patients were selected as control group for analyzing the risk factors associated with involvement of cns in pulmonary ifd. we selected the control group using a : ratio matched-pair method with the variates of ( ) age; ( ) sex; ( ) underlying disease. we retrospectively reviewed patients complicated with cns-ifd after hsct in our single center during a years period. most patients received haploidentical stem cell transplantation. the median onset time of cns-ifd was ( - ) after hsct, and most ( . %) of them have prior pulmonary ifd. the most frequent pathogen was aspergillus, while no crypoccosis and candidas were found. the most common clinical presentation was space-occupying symptoms and signs. brain abscess were the most common imaging finding. prior pulmonary ifd (po . , hr . ( % ci, . - . )) was the only risk factors associated with occurrence of cns-ifd. while poor response at weeks (p = . , hr . ( % confidence interval: . - . )) was the only risk factor predicting the involvement of cns in pulmonary ifd. the response (complete and partial response) at weeks and last follow-up was . % and . %, respectively. the overall survival was . % at the last follow-up with a median ( - ) days after transplantation. in conclusion, patients with pulmonary ifd had higher risk of cns-ifd, especially in those with poor response after weeks of treatment. and the prognosis of cns-ifd was very poor after hsct. disclosure of conflict of interest: none. adv may cause severe infections in hsct recipients, especially from unrelated donors or cord blood particularly in pediatrics. disseminated infections usually occur after digestive reactivations. at mo.post-hsct, the incidence of adv digestive infection and viremia in pediatric hsct is about % and %, respectively. therapeutic strategies to control adv infections are limited to the use of infusion of cidofovir (cdv) or ex vivo anti-adv selected cytotoxic lymphocytes (ctl). however cdv is not labeled for adv treatment, presents a renal toxicity and has shown limited efficacy. specific-ctl remain difficult to produce. brincidofovir (cmx , bcv, chimerix, usa) is an orally-available lipid conjugate of the nucleotide analog cdv that has demonstrated broad clinical antiviral activity against double-stranded dna viruses (that is, herpes-, adeno-, orthopox-and polyomaviruses. the drug has an increased bioavailability compared to cdv and has shown encouraging results. we report here the results obtained with this compound in patients treated in six centers from january . there were pts ( m/ f), median age at hsct: mo. ( - ). hsct indication was all in nine, pid in six, aml in two, fa in two and ibmf in one. donor was / or / mud in four and six pts, respectively; haplo-identical familial donor in ; / or / unrelated cb in two and three pts, respectively; msd in one. stem cell source was bm for pts, cb in and pbsc in . two pts underwent a second hsct. cond' regimen were mac in pts. all pts received either ex vivo or in vivo t-cell depletion. three pts presented with adv-disseminated disease, seven pts with blood + other site (throat, urine or stools) adv infection, three with adv-related gut disease, three with blood infection and three with gut infection. the remaining patient received bcv for jc viremia with fever. median time for virus infection diagnosis was d post-hsct (range: d- to d+ ). about pts experienced other viral infection episodes after hsct (cmv: ; ebv: ; bk: ; hsv: ; hhv : ; influenzae: ( ). about pts received - injections of cdv prior to bcv treatment. one pt received specific-adv ctl before bcv without efficacy. the reason to switch from cdv to bcv was uncontrolled adv infection (n = ) or cdvinduced renal failure (n = ). two additional pts experienced renal impairment after cdv. about pts received - lines of immunosuppressive therapies (including ecp) in addition to calcineurin inhibitor at time of bcv therapy due to grade iii and iv acute gvhd in seven and seven pts, respectively. median adv load at time of bcv initiation was . log copies/ ml (range: - ) in blood and log copies/ml ( . - . ) in stools. median duration for bcv therapy was weeks (range: - ). about seven pts with blood adv infection or disseminated disease experienced adv disappearance as well as four pts with gut disease or infection. three of them experienced adv infection relapse and received thereafter cdv, bcv or advspecific ctl. five pts presented with grade - diarrhea during bcv treatment. about were alive at end point where seven died from septis (n = ), multi-organ failure (n = ), gvhd (n = ) and adv disseminated infection (n = ). adenovirus infections occur often in immunocompromized pts receiving concomitant nephrotoxic drugs that may avoid cdv use.bcv appears as efficient therapy against adenovirus infection in such pediatric pts since here out of pts where alive after adv infection and bcv treatment. in this study we retrospectively analyzed cmv reactivation determined by pcr and response to pre-emptive therapy in patients receiving an haplo (n = , %) comparing them with a control group of non haplohsct ( mrd and mud) (n = , %). median age was years (range: - ), for haplohsct and for control group. conditioning regimen was myeloabaltive (mac) in . % and reduced intensity (ric) in . %. haplohsct characteristics: haplo conditioning was fludarabine ( mg/m or mg/m × days in ric or ma regimen) and busulfan ( . mg/kg × in ric or days in ma) ( . % mac, and . % ric). cyclophosphamide-post was used for gvhd prophylaxis in %. median of days to reach more than × granulocytes and more than × platelets were ( - ) and ( - ), respectively. incidence of acute gvhd was % (grade i-ii . %, and iii-iv . %), with two steroid-refractory cases. cmv reactivation: . % of haplohsct patients presented cmv reactivation, vs . % in control group (p = . ). median number of cmv reactivation episodes was in both groups. median time to cmv pcr detection was days ( - ) and ( - ) in haplohsct and control group respectively (p = . ). average maximum cmv iu by pcr was . in haplo vs . in the control group (p = . ). first antiviral pre-emptive therapy (valganciclovir in . %) was effective in % in haplohsct vs % in control group (p = . ). main reason for antiviral treatment switch was failure in cmv iu reduction, and foscarnet was the most used therapy in refractory cases. twenty patients developed cmv disease ( in haplo and in control group) (gi disease % and pulmonary disease % in both groups). in a multivariate cox-regression model, receiving an haplohsct, serological cmv status (positive patient/negative donor), mac regimen and development of acute gvhd grade i/ii or grade ii/iv were variables associated with a higher risk of cmv reactivation. based on these results, haplohsct is associated with a higher cmv reactivation compared to non-haplohsct, despite a lower incidence of all other risk factors as agvhd or mac in the haplo group. although it is not statistically significant, response to pre-emptive therapy is higher in haplohcst and no differences in cmv disease were observed. disclosure of conflict of interest: none. although a number of patients with hiv infection and hematological disease have successfully undergone allogeneic hsct together with combination anti-retroviral therapy (cart), short and long-term outcomes remain not well known. we report the spanish experience treating hiv-infected adult patients with high-risk hematological malignancies with allogeneic hsct. we retrospectively reviewed hiv-positive patients who received allogeneic hsct in three institutions in spain within geth (grupo español de trasplante hematopoyético y terapia cellular). seventeen patients have been transplanted between and . median age was ( - ), % male. diagnosis and transplant characteristics are summarized in table . cumulative incidence of neutrophil and platelet engraftment were % at days (median days), and % at days (median days), respectively. with a median follow-up of months ( - ), os and efs were %. trm was % at months and % at months. grade ii-iv agvhd rate was %, and moderate/severe cgvhd rate was %. all patients received cart. two patients showed severe toxicity related to interaction of immunosuppressive s drugs and protease inhibitors. about % of patients showed infectious complications. viral infections were the most frequent cause: cmv ( ), bk ( ), adv ( ), hhv- ( ), hcv ( ), hhv- ( ), parainfluenza ( ). two patients had invasive aspergillosis and one patient presented disseminated tuberculosis. causes of death were: relapse ( ), infection ( ), gvhd ( ) and toxicity ( ). all surviving patients showed undetectable hiv load after hsct. allogeneic hsct is an effective therapy for high-risk haematological malignancies in patients with hiv infection, and long-term hiv suppression with cart is feasible. however, interactions between immunosuppressive agents and anti-retroviral drugs, high rates of significant gvhd, and frequent infectious complications account for a complex procedure in this population. selected hiv-infected patients with hematologic malignancies should be considered for allo-hsct when indicated, in experienced centers. disclosure of conflict of interest: none. clostridium difficile infection (cdi) is one of the most common causes of nosocomial infectious diarrhea in europe and usa, which results in high morbidity and mortality among hospitalized patients. allogenic hematopoietic stem cell transplant (hsct) recipients remain at high risk for cdi. incidence rate ranges from to %. numerous risk factors including acute graft-versus-host disease (agvhd), hla matching status, conditioning-intensity, use of total body irradiation (tbi) may play an important role in the course of cdi in these patients. the aim of this study was to evaluate the prevalence of cdi in children, and to assess the influence of such factors as gender, age, diagnosis, hla matching status, conditioning-intensity, use tbi-containing regimen, source of graft (bone marrow/bm/ vs peripheral blood/pb/)or agvhd on course, duration of treatment and outcome in children undergoing hsct. between and a total of hscts were performed in five polish pediatric transplant centers, including allogeneic and autologous. all patients were followed up to months post hsct. we analyzed retrospectively episodes of cdi infection in the group of children. twenty-one of children were diagnosed with hematological malignancies: acute lymphoblastic leukemia (all), acute myeloblastic leukemia (aml), myelodysplastic syndrome (mds), two were diagnosed with severe aplastic anemia (saa), one with chronic granulomatous disease (cgd) and of them-with solid tumors. the median age was . years (range: . - . years). majority of patients underwent myeloablative conditioning protocol ( / ). in allogeneic setting / patients underwent mud-hsct, / pts msd-hsct and one patient was given a haploidentical pbsct. in this series, in out of cases bm was a transplant source, and pb in out of . cdi was defined as having diarrhea that tested positive for c. difficile via pcr, cytotoxin assay, or dual enzyme immunoassays. kruskal-wallis test, wilcoxon test and χ -test were used to estimate the influence of risk factors on severity of disease, duration of treatment and outcome. we observed episodes of cdi ( . %) in hsct recipients: in allotransplant recipients ( . % of all transplants) and in autotransplant recipients ( . % of all auto-hsct). nine patients responded to therapy with metronidazole, seven patients responded to vancomycin alone, and in two patients rifaximine was administered. six children required adding second drug: vancomycin or metronidazole, five patients were not given any medications. there was no significant correlation between such factors as diagnosis, gender, age, conditioning regimen, hla matching, agvhd and severity of disease, and duration of treatment. recurrence rate was difficult to assess due to lack of data. we observed three deaths. one of them was connected with cdi. there was one -year-old boy with saa (mud-pbsct, hla / ) with no agvhd. the other two deaths were due to progression of s disease. cdi occurred in nearly % of pediatric patients undergoing hsct, surprisingly often in autologous hsct too ( . %). almost all patients experienced mild cdi with adequate response to antibiotic therapy. cdi is a rare cause of death among transplant recipients. disclosure of conflict of interest: none. antifungal prophylaxis in high-risk paediatric patients with haematological malignancies: a monocentric experience k perruccio, i capolsini , a carotti , n albi , l pitzurra , a velardi and m caniglia pediatric haematology oncology section; haematology and clinical immunology section; transfusion medicine and the choice of antifungal prophylaxis in high risk paediatric haematological patients (according to the latest ecil /seifem guidelines) remains an open question. a recent retrospective survey from associazione italiana ematologia oncologia pediatrica (aieop) showed that, in these patient categories, the only variable which significantly impacted on invasive fungal infection (ifi) occurrence was the presence or not of antifungal prophylaxis at the ifi onset (unpublished data). from january , in our pediatric hematology oncology unit, allogeneic hematopoietic stem cell transplantations (hsct) were performed (median age: years; range: months- years), mainly for acute leukaemia (median follow-up: months; range: - ). patients received liposomal amphotericine b (n = ), micafungin (n = ), or fluconazole (n = ) as primary antifungal prophylaxis until neutrophil recovery ( × /l). seven patients developed acute gvhd ( %) which evolved in gvhd in ( %). as outpatients, they continued with posaconazole (n = ), voriconazole (n = ), or micafungin (n = ) until cd +t-cell recovery ( /cmm) or gvhd immune suppressive prophylaxis/treatment withdrawn. during the last year, according to ecil /seifem guidelines , we administered primary antifungal prophylaxis also to / high risk (hr) acute leukaemia patients. two patients with aml were treated with posaconazole, four patients with hr-all received micafungin, four relapsed all patients received micafungin (n = ), or liposomal amphotericine b (n = ), or posaconazole (n = ). one aml patient was then transplanted; all relapsed patients are waiting for transplant. no differences were observed in terms of breakthrough proven/probable (pp)-ifi incidence, according to antifungal prophylaxis in the various patient groups. in particular, in the early phase, we observed a pp-ifi incidence of % in both treatment arms (micafungin vs liposomal amphotericine b, p = ns). in the late phase, we observed case of pp-ifi who were receiving posaconazole as prophylaxis. overall survival (os) was %, with % mortality rate. in hr leukaemia patient group, we observed pp-ifi in the only two patients who were not receiving any antifungal prophylaxis at the ifi onset. antifungal prophylaxis is strongly recommended in paediatric patients with haematological malignancies who are at high risk of ifi. the choice of antifungal drug depends on the treatment phase, drug interactions (particularly for azoles), patient compliance and clinical conditions which interfere with intestinal absorption. in our experience, as no differences were observed in term of efficacy, micafungin resulted the best choice in terms of tolerability, toxicity, compliance and cost saving. antifungal prophylaxis with micafungin and bridging to inhaled liposomal amphotericin b after engraftment in patients undergoing allogeneic hematopoietic stem cell transplantation d rivera* , , c de ramon , a avendaño , j carrillo , d caballero , l lopez , i ormazabal , a navarro , a martin micafungin is an effective antifungal for prophylaxis, active against candida spp. including those resistant to other antifungals (c. glabrata and c. krusei) and also active against aspergillus spp. guidelines focused on antifungal prophylaxis, recommend its use during preengraftment and early postengraftment period in allogeneic hematopoietic stem cell transplant (allo-hsct) recipients. moreover, its profile of low drug interactions and side effects, makes it a suitable alternative for patients who need concomitant treatments, present hepatic insufficiency and in those who do not tolerate oral drug administration. the addition of inhaled liposomal amphotericin b (lamb) after engraftment, provides an alternative way to effectively prevent mold infections, that are acquired mainly by inhalation. inhaled lamb has good tolerance with absence of drug interactions and low toxicity. the aim of this study was to describe the experience in the hsct unit of the university hospital of salamanca with micafungin and lamb as primary prophylaxis in patients undergoing allo-hsct with reduced intensity conditioning (ric) and graft-versus-host disease (gvhd) prophylaxis with tacrolimus and sirolimus. thus evaluating efficacy and tolerability in our population. retrospective observational study from january to august , including all adult patients undergoing allo-hsct with ric and gvhd prophylaxis with tacrolimus and sirolimus, in whom an azole derivative is not indicated, due to drug interactions. therefore received prophylaxis with micafungin during the preengraftment period and bridging to lamb after engraftment at discharge, and continuing it during the first days post-transplant. data from patients from our hsct unit. ten ( . %) patients who had invasive fungal infection before undergoing allo-hsct, and ( . %) patients who received prophylaxis with drugs other than micafungin-lamb were excluded. underlying disease was grouped by leukemia in ( . %) patients, lymphoma in ( . %), myelodysplastic syndromes in ( %), multiple myeloma in ( . %) and other diseases in ( . %) patients. eighty patients underwent peripheral blood allo-hsct, of whom were related donor in ( %) patients and unrelated donor in ( %). prophylaxis with micafungin in ( . %) patients, dose of mg per day, with a mean of days (± days) with postengraftment bridging with lamb mg weekly, continuing it during the days posttransplant. days of neutropenia during preengraftment, o days in ( . %) patients, - days in ( . %), more than days in ( . %). during follow-up there were three cases ( . %), two catheter related candida infection, and one esophageal candidiasis. there were no reported aspergillosis cases (possible, probable or proven), according to the european organization for research and treatment of cancer (eortc) criteria. finally, prophylaxis with micafungin and inhaled lamb, was considered an effective and safe strategy in ( . %) patients, with no side effects reported. according to our experience with micafungin and the addition of inhaled liposomal amphotericin b, the results indicate that, this is an appropriate alternative for antifungal prophylaxis, in patients undergoing allo-hsct, because of their efficacy, few side effects and drug interactions. disclosure of conflict of interest: none. recipients of allogeneic hematopoietic cell transplantation (allohct) are at high risk of developing invasive fungal infections (ifi). in the early phase (o days) after allohct, the use of antimold prophylaxis has been generalized, although there is no consensus on the best therapeutic strategy. the use of nebulized liposomal amphotericin b and fluconazole has been shown to be effective, safe and associated with low economic costs in lung transplantation . however, the use of this prophylactic strategy in the early phase of allohct setting has not been evaluated. we included all consecutive patients who received their first allohct in our center from january to august and who underwent antifungal prophylaxis according to the prospective ambineb protocol (nebulized liposomal amphotericin b mg administered three times per week as loading dose and once per week and fluconazole mg per day until day + ). patients with a previous ifi were excluded. patients with graft-versus-host disease (gvhd) receiving high dose corticosteroids were allowed to be changed to voriconazole or posaconazole at physician's discretion. the primary objective of the study was the incidence of ifi at day + . the secondary objectives were to assess adherence and toxicity of the ambineb protocol. only cases with proven or probable ifi according to eortc-msg criteria were considered. a multidisciplinary team of experts in hematology, infectious diseases, microbiology and radiology prospectively evaluated and categorized each case. we included patients with a median age of years (range: - ) and a median follow-up for survivor of months (range: - ). patients received allohct mainly for acute leukemia ( %), non-hodgkin lymphoma ( %) and myelodysplastic syndrome ( %) . patients received allohct from hla unrelated ( %) or related donors ( %) mostly using a reduced intensity conditioning ( %). graft-versushost disease (gvhd) prophylaxis was performed with calcineurin inhibitors mainly in combination with sirolimus ( %) or methotrexate ( %). after the comprehensive review, only one case of proven or probable ifi at day + was diagnosed. prophylaxis with ambineb was completed in patients ( %) while ( %) stopped the treatment. the most frequent causes of discontinuation were possible ifi ( %), gvhd ( %), admission in intensive care unit ( %) and toxicity ( %) (figure) . ninety-four patients ( %) did not have adverse advents associated with ambineb. eight patients presented organ toxicity which was at least partially attributed to ambineb, including gastrointestinal symptoms (n = ), liver function test abnormalities related to fluconazole (n = ) and cough (n = ). of the patients who discontinued ambineb, ( %) were switched to other antifungal drugs including (echinocandins ( , %) posaconazole ( , %), voriconazole ( , %) or others ( , %)). overall survival and non-relapse mortality of all patients at median follow-up were % ( % ci - ) and % ( % ci - ), respectively. the combination of nebulized ambisome and fluconazole is effective in preventing ifi in the early phase of allo-hct and is associated with high adherence and low toxicity. neutropenia-related infections is a common complication of apsct in patients (pts) with mm and dlbcl. our study aims are to: ( ) assess antibacterial susceptibility patterns of isolated organisms, to guide antibiotic prophylaxis ( ) identify the epidemiology of bacteremia with susceptibility patterns to direct empiric therapy of febrile neutropenia ( ) assess the interval between the occurrence of neutropenia and the isolation of resistant bacteremia to identify the best timing to start prophylaxis ( ) identify contributing factors for the development of bacteremia and mortality. our retrospective study included adult pts who underwent apsct for mm and dlbcl between and . we recorded the following: age, gender, basic illness, comorbidities, number of cd + cells infused, a central venous catheter, duration of neutropenia, diarrhea and mucositis, mechanical ventilation, positive bacterial cultures with susceptibility profiles and history of broad-spectrum antibiotic intake for more than h for the past months on hospital setting, and mortality. statistical analysis was carried out using spss (version ). about isolates were obtained: urine ( . %), blood ( . %), sputum ( %), wound ( %), venous catheter ( %) and stool ( %). gram-negative (gn) species were predominant ( . %) with e. coli ( . %), klebsiella (k) ( %) and pseudomonas (pseudo) ( %). isolates sensitive to third generation cephalosporins ( gc) represented % of the enterobacteriaceae (entero) including % in e. coli and % in k. all entero isolated were susceptible to carbapenems (carba), pipercillin/ tazobactam (pip/taz), amikacin and ciprofloxacin (cipro). all pseudo (n = ) and acinetobacter (n = ) isolates were susceptible to carba, pip/taz, amikacin, cipro, colistin and tigecycline. as for gram-positive (gp) bacteria ( . %), coagulase negative staphylococci (cns) were predominant ( %) . oxacillin susceptibility reached % and two isolates methicillin resistant s. aureus were identified. all gp were susceptible to glycopeptides. a total of bacterial isolates were identified ( episodes of bacteremia) from pts. gn were predominant ( . %) with e. coli being most common ( %). all gn were susceptible to gc, carba, pip/taz, amikacin and cipro. as for gp ( . %), cns predominated ( %) including % oxacillin-susceptible causing seven episodes of bacteremia with six central line-associated. no glycopeptide resistance was identified. none of the clinical features and pts' characteristics reached statistical significance as risk factor for bacteremia. however, the need for mechanical ventilation and mortality were higher in bacteremic vs non-bacteremic pts ( . % vs %, p = . , and . % vs %, p = . , respectively). all bacteremic episodes occurred after developing neutropenia (median = . days, range: - ) except for one case of clabsi caused by e. coli occurring day before neutropenia. pip/taz was prescribed in % of bacteremia episodes followed by quinolones ( %) and carbapenems ( %). no previous use of third and fourth generation cephalosporins was observed. we recommend quinolone prophylaxis in apsct pts. for empiric therapy, antibiotics recommended by international guidelines including, cefepime and pip/taz still fit. thus, we could spare the use of carba and other last-resort antibiotics to other conditions. we also recommend continuous surveillance of resistance. disclosure of conflict of interest: none. fever is an almost universal complication in patients undergoing autologous stem cell transplantation (asct), however, microbiological documentation is only achieved in - % of such febrile episodes (fe). this low diagnostic efficiency makes epidemiological assessment in transplant units difficult, and may lead to a suboptimal empirical treatment. we have studied the utility of strict blood culture (bc) extraction as a mechanism to improve microbiological documentation of fe in these patients. we conducted a retrospective study over consecutive asct performed in our centre between june and may ( year). about patients were male and female, with age between and years (mean . ). diagnosis was hodgkin lymphoma, non hodgkin lymphoma and multiple myeloma. ascts were performed in reverse isolation conditions, in rooms equipped with hepa and pall filters. prophylaxis against herpes virus and p. jirovecii with acyclovir and pentamidine was used. no prophylaxis against gram negative bacteria or filamentous fungi was performed. bc were extracted at the beginning of every fe and every - h if fever persisted (or more frequently, following clinical criteria). blood samples from intravascular devices and peripheral blood were collected in two bactec bottles each (for aerobic and anaerobic microorganisms). complementary diagnostic techniques and empiric antibiotic therapy were performed following our institution's guidelines. fe were classified in microbiologically documented infections (mdi), clinically documented infection (cdi) and fever of unknown origin (fuo) following his criteria. fe were studied (average . fe per patient, . days of fever duration per fe). about % of fe were classified as mdi, % as cdi and % as fuo. mdis were cause by gram positive bacteria ( %), gram negative bacteria ( %) and polymicrobial infections ( %). no viral or fungal infections were observed. an average of . bc per fe and . per patient were extracted. the proportion of fe classified as mdi was related to the number of blood cultures extracted during the episode. only % of fe with three or less bc extracted were classified as mdi, % if - bc were extracted, and % if - bc were extracted (p o . ). no significant difference in proportion of mdi classified fe between the extraction of or more bc and - . all patients were discarded in good clinical conditions. according to our experience, a strict - blood culture extraction is related to a high rate of microbiological documentation of febrile episodes. moreover, we have not observed the rise in gram negative bacteria reported by other studies and gram positive cocci persist as the main infection cause in our centre. candida which has been traditionally related to duration of neutropenia, emerges as a pathogen beyond the aplastic period in allogeneic haematopoietic cell transplantation (allohct). in the setting of alternative transplants and aggressive immunosuppressive therapy, these infections are a challenging problem. there is scarcity of data regarding the significance of breakthrough candidaemia in allohct. to that end, we aimed to determine the incidence, clinical and microbiological characteristics and outcome of candidaemia in allohct recipients. we studied consecutive allohct recipients from january to june . blood cultures were obtained from peripheral vein or central venous catheters (cvcs) routinely and on febrile patients. well-known risk factors for candidaemia were studied: neutropenia, type of transplant, moderate to severe graft-versus-host disease (gvhd) and coexisting infections. among allohct recipients, we identified seven patients with candidaemia: five post matched unrelated (four myeloablative and one reduced intensity conditioning) and two post haploidentical transplant. in median time of . ( . - ) months, episodes of candidaemia were noted, despite antifungal prophylaxis with echinocandins or azoles. infections with non-albicans candida spp. occurred more frequently ( / ) and c. parapsilosis was the predominant microorganism ( / ). other species were isolated: c. famata ( ), c. krusei ( ) and c. haemulonii ( ). all candida spp. isolates were phenotypically susceptible to antifungal agents already administered to patients. there was no resistance to echinocandins indicated by minimum inhibitory concentrations (mics). all patients had severe acute or late-onset gvhd with intestinal involvement and cvcs prior to candidaemia. although cvcs were removed in / and patients were treated with echinocandins, new cvcs were re-contaminated in / with the same or other species. all patients presented well known risk factors for candidaemia (use of broad spectrum antibiotics due to severe bacterial infections, total parenteral nutrition due malnutrition, long-term high-dose corticosteroids and other immunosuppression), but no neutropenia. one patient survived, whereas five patients succumbed to gvhd and multi-organ failure and one patient to sepsis due to bacteremia. candidaemia was observed in non-neutropenic patients with agvhd and cvcs on antifungal prophylaxis, despite difficulties in diagnosis due to poor sensitivity of blood cultures. the epidemiology of candidaemia has changed in the last decade and its risk is more diverse and complex. the irreversible intestinal gvhd lesions might be the main source of candida in patients receiving antifungal prophylaxis. our data show that candidemia remains an important issue in profoundly immunosuppressed patients contributing to excessive morbidity. our aim was to compare the rate of neutropenic sepsis, defined as fever of o c and a neutrophil count of o . × /l, before and after the introduction of ciprofloxacin prophylaxis. one hundred and eight adult patients, of which had acute myeloid leukaemia, had acute lymphoblastic leukaemia, had lymphoma and had other haematological malignancies, were identified through our admission database. of these patients, received oral ciprofloxacin during their neutropenic phase. the median duration of neutropenia was days in both the no-prophylaxis and ciprofloxacin groups. there was a significant reduction in the rate of neutropenic sepsis from . % ( / ) in the no-prophylaxis group to . % ( / ) in the ciprofloxacin group (p = . ). prolonged infection, suggested by the use of broad-spectrum antibiotic treatment for more than days, was more common in the group which did not receive prior ciprofloxacin prophylaxis ( . % vs . %, p = . ). rate of intensive care admission ( . % vs . %, p = . ) was also reduced by the use of ciprofloxacin. however, there was no significant difference in the length of stay (mean of vs days, p = . ), or in the -day infection-related readmission rate ( . % vs . %, p = . ) between the two groups. in terms of the cause of neutropenic sepsis, escherichia coli, klebsiella pneumoniae and pseudomonas aeruginosa were the most common bacteria isolated from cultures in the no-prophylaxis group. eighty percent of these organisms showed sensitivity to ciprofloxacin. in the ciprofloxacin group, staphylococcus epidermidis was the most frequently found bacteria. with regards to treatment related adverse effects, none of the patients who received ciprofloxacin prophylaxis developed clostridium difficile diarrhoea. in conclusion, ciprofloxacin is still an effective antibacterial prophylaxis during neutropenia following allogeneic stem cell transplantation. clinicians should have a high suspicion of a gram-positive infection in patients who develop neutropenic sepsis on ciprofloxacin prophylaxis. disclosure of conflict of interest: none. s hematopoietic stem cell transplantation (haplohsct) to cure leukemia, malignancy and some inherited diseases, different additional reasons interfere microbiota metabolism and integrity. among them are radiation and chemotherapy, mucositis, infection and graft versus host disease (gvhd). the curative mechanism of fmt is based on the ability of donor intestinal microbiota to substitute and to provide all necessary functions of altered patient's microbiota. three patients ( , and years old) after haplohsct, who observed pseudomembranous colitis (toxin b-positive) as gvhd of intestine outcome, were enrolled to the study and performed fmt. relatives (mother, father and brother) were used as microbiota donors. donor and patient examination have included routine clinical and biochemistry laboratory data, microbiota cultural methods, pcr of most common intestinal microorganisms. additional for patient-level of fecal calprotectin by elisa was tested, identification of drug resistant bacteria and histology of intestine were made. patient's preparation for fmt included-probiotic (inulin) administration h prior procedure, discontinuation of all antibiotics h prior procedure and antiemetics ( ht agonist), prokinetics and proton pump inhibitor. delivery of donor's microbiota was performed in two consecutive steps under total intravenous anesthesia: with esophagogastroduodenoscopy-to the duodenum; with colonoscopy-to the caecum. all patients observed complete clinical response in - days after fmt (table ). in days we have revealed significant quantitative and qualitative changes in microbiota composition, which was matched to donor's microbiota. in days after fmt we identify microbiota changes in oropharynx and urogenital tract similar to donor microbiota. this leads to substitution of multidrug resistant klebsiella pneumoniae strains by drug sensitive microorganisms and helps to treat severe infection complications after haplohsct. platelet aphereses were carried out in donors ( males and females with median age . years) using haemonetics instrument with simultaneous leucoreduction. quantitative detection of cmv, ebv and hhv- dna was performed by multiplex real-time quantitative pcr kit (interlabservice, russia) in donors' whole pb, plasma and platelet aphereses at the time of platelet collections. viral load in hsct recipients was monitored weekly after hsct and days before hsct by the same pcr kit. lower limit of detection (llod) of the applied kit for all viruses was copies/ml. in specimens of platelet donors we additionally performed ultra-sensitive pcr with llod copies/ml. only one patient ( . %) was cmv-positive by pcr prior to hsct. cmv reactivation after hsct ⩾ copies/ml was noted in whole pb of patients ( . %) with median time of days (range: - ). donor source in cmv-reactivated patients was as follows ( mud, msd, haplo). cmv viremia ⩾ copies per ml was detected in seven patients ( . %). cmv disease was found in five cases ( . %). none of patients were positive by pcr for ebv or hhv- prior to hsct. ebv reactivation ⩾ copies/ml was found in six cases ( . %), ⩾ copies/ml in ( . %) with median time of days (range: - ). no signs of ptld or other ebv-dependent clinical symptoms were observed. hhv- level after hsct ⩾ copies/ml was detected in patients ( . %), ⩾ copies/ml in ones ( . %) with median time of reactivation days (range: - ). hhv- disease was observed in one patient. none of platelet donors were cmv-positive in plasma, whole blood or platelet aphereses products. ebv ⩾ copies/ml was detected in whole pb specimens of five platelet donors ( . %). application of ultra-sensitive pcr revealed low level of ebv-viremia in additional pb cases with median ebv level copies/ml (range: - ). none of platelet donors have any clinical signs of ebv disease. there is no any ebv-positive case among platelet concentrate specimens. in two cases low levels of hhv- was found in a whole pb ( and copies/ ml). none of hhv- -positive case was observed among plasma and platelet concentrate specimens. despite high incidence of cmv, ebv and hhv- reactivation after hsct in pediatric patients we could not show that source of viral reactivation was contamination of platelet apheresis products by donorderived herpes viruses. disclosure of conflict of interest: none. conventional respiratory virus (crv) infections are known to be major causes of morbi-mortality after stem cell transplantation (sct) due to the increased risk of progression to lower respiratory tract infection (lrti) in this setting. risk of developing severe lrti is mostly related to factors specific to the patient and the underlying disease, although the intrinsic virulence of crvs may also determine their outcomes. we conducted a single-center retrospective study including all adult sct recipients who had crv disease (defined as patients with symptomatic respiratory disease and crv identification) during a -year period ( - ) with the main objective of evaluating epidemiological changes over time and their association with infection outcomes. during the study period episodes of crv disease were diagnosed in patients (median age: years, % male, % aml or mds as baseline disease). patients ( %) received an allogeneic-sct (allo-sct) ( % had a prior sct) and ( %) an autologous sct. crv disease was diagnosed at a median of days after sct (range: - ), with cases ( %) occurring before day + . during the infectious episode of allo-sct recipients ( %) had active gvhd and ( %) were under s prednisone (pdn) mg/kg. most of the patients ( %) had symptoms compatible with an upper respiratory tract infection (urti), with of them ( %) progressing to a lrti, while ( %) had a lrti only. hospital admission was required in episodes ( %) with a median duration of hospitalization of days (range: - ), % required supplemental oxygen, % were transferred to the intensive care unit and % required mechanical ventilation. the most commonly identified pathogens over time are shown in figure . twenty-four cases ( %) had concomitant bacterial or fungal infections. influenza a virus was the most frequent crv detected ( episodes, %) followed by human respiratory syncytial virus ( episodes ( %) and human parainfluenza virus type ( episodes, %). during the flu pandemic, only of the crv infections diagnosed in sct recipients ( %) were associated to influenza a virus h n . antiviral treatment was started in episodes ( %), antibiotics in % and combined therapy in % during a median of days (range: - ). the rv resolved in cases ( %) at a median of days ( - ) from onset, with crv being considered the leading cause of death in only patients ( % of all cases and / ( %) in those with a lrti). predictors of severe crv infection (including icu admission, need for supplemental oxygen, need for mechanical ventilation requirement or death) in multivariate analysis were lymphocyte count o cells/μl (hr: . , % ci: - , p = . ) and co-infection with other pathogens (hr: . , ci %: . - , p = . ). no specific crv nor period post-sct of the infection influenced the risk of severe infection. our results confirm that crv infections are a frequent cause of morbidity after sct with a high need for hospital-based care. temporal changes in the principal circulating crvs has been identified during the -year study period, with influenza a virus being the most common. profound lymphocytopenia and presence of co-pathogens are associated with infection severity. [p ] disclosure of conflict of interest: none. the consecutive hsct performed from to are being analysed retrospectively. out of them ( %) performed in hepa filter room and ( %) in non-hepa filter room, criterion was purely financial to make this decision. / ( %) were allogeneic and / ( %) were autologous hscts. blood cultures both bacterial and fungal were taken at onset of fever and with every change of antibiotics till patient became afebrile. chest x-ray and if required hrct chest was done for all patients who had respiratory complaints. we did not use antibacterial prophylaxis; however, antifungal prophylaxis was administered along with conditioning; and at the onset of fever systemic antibiotics were started. antifungal agents were added if fever persisted for days pre empatively. extremely well trained nurses were looking after both the groups. all treatment protocols, antibiotic/antifungal policies were same in both the groups. median time for neutrophil engraftment was days in hepa filter room and days in non-hepa filter room. total / ( %) patients did not engraft till days. out of them / ( %) were in hepa filter room and / ( . %) in non hepa filter room. blood cultures were positive in total / ( %) patients, were positive for bacterial and for fungal organisms. in hepa filter hsct / ( %) were positive and in non-hepa filter hsct were / ( %) positive. total / ( %) patients developed pneumonia, out of them / ( %) were in hepa filter and / ( %) hsct were in non-hepa filter room. statistically not significant. no central venous access cather issues or infections were documented in any groups gr - agvhd : hepa rooms / ( . %),non hepa rooms / ( . %) :was not statistically significant the -day mortality was / ( %), / ( %) patients were from hepa filter rooms and / ( %) were from non-hepa filter rooms. cost : average cost of allogeneic hsct in hepa room : usd . average cost of allogeneic hsct in non hepa room: usd . average cost of auto hsct in hepa room: usd . average cost of auto hsct in non hepa room : usd . incidence of blood culture positivity & incidence of pneumonia was not different. these are two very important issues in outcome of hsct. agvhd incidence did not depend on the room type. these are significant findings from this study. results were slightly better in hepa filter rooms compare to non-hepa filter rooms, which was statistically insignificant. our study had few confounding factors hence we could not be concluded that hepa-filtered rooms are not necessary. nevertheless, our experience suggests that availability of dedicated hepa units with special air-handling equipment should not be considered a critical and essential precondition for providing allogeneic hsct to patients even in developing world with financial constraints. these would otherwise succumb to potentially curable hematological illnesses with background of financial constraints and wait list of hepa filter rooms. early hsct in a clean patient in non hepa rooms is extremely cost effective with comparable outcomes. nursing care, experience of the team, experience in hsct program & well established protocols are more important in outcome of hscts. disclosure of conflict of interest: none. we present five cases of cytomegalovirus (cmv) pneumonitis occurring in patients after recent allogeneic stem cell transplantation (allohsct). these cases were complicated by an organising pneumonia (during the recovery period) with a predominantly central peribronchial pattern. all patients presented with evidence of active cmv pneumonitis which was treated successfully with anti-viral therapy but was followed by persistent severe dyspnoea, cough and hypoxia. high resolution computed tomography (hrct) imaging showed widespread central peribronchial consolidation with traction bronchiectasis. in most cases there was a marked clinical and physiological improvement after treatment with systemic corticosteroids. however, in all patients the lung function remained abnormal and in some cases imaging revealed a fibrosing lung disease. these cases represent a previously undescribed central peribronchial pattern of organising pneumonia complicating cmv pneumonitis that can result in chronic lung damage. disclosure of conflict of interest: none. cytomegalovirus reactivation in pediatric acute leukemia after stem cell transplantation has an effect on relapse and survival in aml but not in b-precursor all j-s kühl , l sparkuhl and s voigt department of pediatric oncology/hematology/sct, charité universitätsmedizin berlin, berlin, germany several studies have indicated better survival after stem cell transplantation (sct) for acute leukemias, especially acute myeloid leukemia (aml), in case of cytomegalovirus (cmv) reactivation. here, we investigated if cmv reactivation had an impact on survival after sct for aml or acute lymphoid leukemia (all) in children. pediatric allogeneic stem cell transplant recipients from our institution who received myeloablative conditioning were included. transplant indications included aml, t-all and b-precursor all. cmv reactivation was correlated with relapse, mortality as well as acute graft-versus-host disease (gvhd) and was analyzed by fisher's exact test or χ -test (if n ). from the patients included, were transplanted for aml ( %), for t-all ( %), and for b-precursor all ( %). mortality and relapse rates ( - % and - %, respectively), cmv reactivation rates ( - %) as well as numbers of negative cmv serology status ( - %) of donor and recipient were comparable between different acute leukemias. when patients were analyzed altogether, cmv reactivation had no effect on relapse rates or mortality. however, a tendency towards fewer relapses after cmv reactivation was observed in aml patients (no relapse ( %) with cmv reactivation vs relapse cases ( %) without cmv reactivation; p = . ). in those leukemia patients capable of reactivating cmv (that is, donor or recipient cmv seropositive prior to sct), cmv reactivation had a protective effect on relapse rates in aml (no relapse ( %) with cmv reactivation vs relapse cases ( %) without cmv reactivation; p = . ). a similar tendency could be seen in t-all whereas no effect in patients with b-precursor all was documented. numbers of acute gvhd cases grade i between aml and t-all with or without cmv reactivation were similar. different effects of cmv on relapse rates and mortality in aml vs b-precursor all were noticed in patients who were either not capable of cmv reactivation or who did reactivate cmv post sct. in aml patients, there were no relapses ( %) and deaths ( %) in contrast to relapse cases ( %) and deaths ( %) in children with b-precursor all (p = . and p = . , resp.). latently cmv infected aml patients without documented cmv reactivation after sct have a significant worse prognosis compared with all other aml patients. this is also likely to be the case in patients with t-all, however, patient numbers in our cohort were too few. the protective effect of cmv reactivation in aml and possibly t-all does not appear to be gvh-related since the rate of relevant acute gvhd cases was comparable. cmv reactivation after sct for b-precursor all lacks significance. disclosure of conflict of interest: none. infections are among the most frequent and relevant complications of hematopoietic stem cell transplantation (hsct). little is known about the role of dental foci for the prevalence of infections in hsct. dental status was prospectively evaluated in all patients at our center before undergoing hsct. a total of different patients before undergoing hsct ( allogeneic and autologous), with a median age of years (range: - years) were evaluated. for evaluation a panoramic x-ray evaluation was performed. dental findings included the status of third molars and root fillings as well as caries, periodontitis, destructed teeth and apical bone loss. as non-dental parameters we used age, sex, type and status of central venous line, mucositis, and type of transplantation. these were correlated with neutropenic fever, bacteremia and pneumonia in a bivariate manner before a multivariate analysis was performed. no correlation of initial dental status to neutropenic fever, bacteremia or pneumonia was found. however, bacteremia and suspected infection of central venous lines was a significant predictor of neutropenic fever. in conclusion, dental surgery should only be performed prior to hsct if urgently required and limited to those individuals with overt infection. disclosure of conflict of interest: none. [p ] early experience with clinimacs prodigy ccs method in generation of virus-specific t-cells for pediatric patients with severe viral infections after hematopoietic stem cell transplantation k kallay viral reactivation especially in children is a frequent complication of allogeneic hematopoietic stem cell transplantation. most of these episodes can effectively be controlled by an antiviral or antibody therapy; in refractory cases a novel virusspecific t-cell therapy could be a promising management option. in our pediatric cohort of allogeneic transplantation during year patients fulfilled criteria for virus-specific t-cell therapy ( boys, girls, median age of ( . - ) years). six patients were transplanted because of hematological malignancies and for inborn errors. donor distribution was the following: matched unrelated, sibling and haploidentical donor. in cases bone marrow, cases peripheral blood and case cord blood was used as a stem cell source. the underlying viral illness was cmv in , ebv in and adenovirus in case, while more than one virus was detected in cases (cmv+adenovirus cases, cmv+ebv cases). viral diseases necessitating a t-cell therapy were cmv pneumonitis and colitis, adenovirus enteritis and cystitis and ptld. patients initially received cidofovir for adenovirus, rituximab for ebv and a combination of gancyclovir and foscarnet for cmv infections. the indication for t-cell therapy was progressive viral disease in of the cases and uncontrollable viral load in case. the procedure was performed on a median of ( - ) day post transplant. donors were st degree relatives in cases, nd degree relatives in cases and an unrelated person in case, the best hla match was haploidentical. the median age of the donors was ( - ) years. cells were produced by the clinimacs prodigy cytokine capture system (ccs) method after mononuclear leukapheresis. the system produced a median of . ( . - ) times /kg cd + and a median of . ( - . ) times /kg cd + interferon producing cells while the non-interferon producing cells were far below gvhd limit with a median of . ( . - . ) times /kg cd + and a median of . ( . - . ) times /kg cd + cells. the t-cell products were administered uneventfully in all but one case. we observed a manageable cytokine storm in one patient. glucocorticoid treatment was ongoing due to acute gvhd in children; however we could manage to keep the steroid dose below mg/kg in all cases. eight patients became completely asymptomatic, while also cleared the virus. we experienced decreasing viral load in all cases, the first negative viral results were achieved on a median day of ( - ). six patients are alive without viral illness or sequale, and complete viral dna clearance in peripheral blood with a median follow up of ( - ) days. one patient with cmv pneumonitis improved during the first week but deteriorated on the second week and died of respiratory insufficiency despite of mechanical ventilation. in cases the viral illness improved or cleared, but the patients died of invasive aspergillosis. no cases of gvhd, rejection, organ toxicity or recurrent infection were noticed. virus-specific t-cell therapy produced by the clinimacs prodigy ccs is a feasible, fast, safe and effective way to control resistant viral diseases after pediatric hematopoietic stem cell transplantation. this treatment can be implemented within a week in most cases. in order to define the appropriate place of this approach for patients with viral reactivations more data should be collected. disclosure of conflict of interest: none. central venous catheter (cvc) is essential for the treatment of recipients of stem-cell transplant. it is usually placed for the administration of conditioning regimen, stem cell infusion, intravenous antibiotics, immunoglobulins, electrolyte and nutritional support and blood concentrates. this patient group is at high risk for catheter-related bloodstream infections that can result in substantial morbidity and mortality. the neutropenia secondary to the conditioning regimen determines the risk of catheter-related infections, which may serve as an entry into the blood circulation, leading to bacteremia, fungemia, and consequently to septic shock and death. the risks of infection and the spectrum of infectious syndromes differ according to the type of transplant, conditioning regimen, type of implant of stem cells and therapies used after the procedure. gram-positive bacteria, particularly coagulase-negative staphylococcus spp, remain the leading cause of catheter-related bloodstream infection, although an increase in gram-negative bacteria as the causative agent has been noted. aim of the study: to evaluate the impact of the early cvc removal on the frequency of febrile episodes and infections in our group of patients. during a years period we have treated patients with hematologic neoplasm with high-dose chemotherapy and stem cells transplantation. patients were treated in sterile room conditioned with hepa filtration. in every patient was introduced double-lumen cvc ( subclavia, jugular, and femoral). % were febrile ( % fuo), catheter-related infection was present in %, while positive culture from cvc was present in %. the most frequent isolated bacteria from cvc were gram positive-staphylococcus coagulasa negative. the catheter was removed on the day of discharge. trm is . %. from january to november we have transplanted additional patients. to aim to decrease infection related mortality we perform strategy to remove cvc on day + after stem cell transplantation. the febrile episodes decreased on % ( / ), there were no early post-transplant mortality due to infection. early removal of the cvc and adequate handling from the nursing staff is essential for outcome of this patient population in regard of infective complications efficient prevention, early diagnosis, and effective treatment of catheter related infection are essential to providing the best care to these patients and can minimized morbidity and mortality. disclosure of conflict of interest: none. fever in patients with agranulocytosis during autologous hematopoietic stem cell transplantation (autohsct) can be associated with non-infectious causes due to g-csf, vancomycin, engraftment syndrome. in this case biochemical markers, such as presepsin (psp), procalcitonin (pct) and c-reactive protein (c-rp), can help in differential diagnosis of fever of infectious and non-infectious genesis. psp, pct and c-rp were assessed on the day of admission to the hospital (da), on d+ , on d+ , on d+ and on the day of discharge (dd). if patients developed neutropenic fever (nf), the markers were assessed at the beginning of the fever, h after, then on the second, third, fourth days after. if patients developed nf immediate empirical antibiotic therapy (at) was implemented with meropenem. in cases of ineffective st line ab, nd line at was added or totally changed. there were patients included in the study: patients with hodgkin lymphoma, with non hodgkin's lymphoma, with multiple myeloma, out of patients there were women and men. the median age was years ( - years). the conditioning regimens were cbv, beeac or hd melphalan. patients developed infectious complications (ic): of them had sepsis and others-nf. the median of nf development was . days. depending on the efficiency of at therapy patients were divided into two groups: group : patients that have had effective at (they 've had fast clinical response and they haven't needed to change medicine (n = )); group : patients that have had ineffective st line at, they haven't had response to st line at and they've needed to change another at (n = )). there were significant differences in psp levels on the third day after ab had been admitted: . pg/ml in group and . pg/ml in group (p = . ). similar differences between the analyzed groups were observed on the fourth day: . and . pg/ml, respectively (p = . ). pct and c-rp didn't show any significant changes between group and on each day of the study (table ) . disclosure of conflict of interest: none. enterovirus related immune reconstitution inflammatory syndrome (iris) following haploidentical stem cell transplantation in an mhc class ii deficient child r shah , s waugh , k foong ng , z nademi , t flood , m abinun , s hambleton , a gennery , m slatter and a cant paediatric immunology and bmt, great north children's hospital, newcastle upon tyne, uk and department of virology, great north children's hospital, newcastle upon tyne, uk immune reconstitution inflammatory syndrome (iris) has been described after hsct in association with fungal, viral and bcg infections. we describe a case of post-hsct iris associated with enterovirus infection. case: a girl with mhc ii deficiency (rfxp c. mutation) underwent treosulfan/fludarabine/ thiotepa/atg conditioned tcrαβcd + depleted haploidentical hsct at . years of age. pre-transplant work up did not reveal any viral or fungal infections except norovirus in stool. cyclosporine (csa) was given as gvhd prophylaxis. neutrophil and platelet engraftment occurred on d+ and d+ , respectively. on d+ , her stool was tested positive for enterovirus (taqman pcr), however; she was asymptomatic. the child started having fevers and irritability from d+ which persisted despite the use of antimicrobials. no evidence of fungal or bacterial infection was found. enterovirus pcr in blood was found positive on d+ (cycle threshold value, ct . ) and further typing showed it to be echovirus . at this time, symptoms progressed with diarrhoea, developmental regression and signs of radiculopathy. mri (brain and spine) was normal and csf showed pleocytosis ( wbc/mcl- % lymphocytes, protein . g/l) with positive enterovirus pcr (ct ). subsequently, immunoglobulin prophylaxis was increased to . g/kg bi-weekly, and with supportive measures, the patient slowly recovered. blood enterovirus pcr remained positive. with no evidence of gvhd, csa was tapered off by day+ and child was discharged on d+ on a bi-weekly ivig replacement. she presented days later with signs of raised intracranial pressure. mri showed hydrocephalus, and vp shunt was placed and broad spectrum antibiotics administered. csf showed wbc o /mcl, protein . g/l and enterovirus positive. methylprednisolone mg/kg/day was started suspecting iris. in subsequent csf testing days later, enterovirus was negative. enterovirus pcr remained positive in blood during this period. patient's clinical deterioration correlated with a rise in cd /cd counts and c reactive protein with clearance of enterovirus from csf, blood and stool ( figure ). subsequently, the child showed gradual but marked improvement and discharged home. discussion: the clinical features of index case fits into criteria for iris . markedly raised crp suggests high il- levels without any bacterial or fungal pathogens being isolated. in addition, iris occurs at the site of prior active infection (brain in index case) and viral clearance and clinical recovery demonstrated with the continuation of steroids. the incidence of enterovirus infection in hsct recipients is around % . iris, in this case, had a temporal correlation with discontinuation of csa, and it has been shown that discontinuation of immunosuppression is associated with higher risk of iris. a high index of suspicion for iris is necessary during immune recovery post-hsct especially when immunosuppression is being tapered in a patient with pre-existing infection. aggressive antiviral treatment (when available) and judicious immunosuppression are the keys to managing iris complications. posttransplantation lymphoproliferative disease (ptld) is a significant cause of morbidity and mortality in allogeneic stem cell transplant patients. identifying high risk patients, routine pcr screening, early diagnosis and therapy are crucial for successful management. patients and methods primary objectives of this study were to describe epidemiology of ebv associated ptld and to assess risk factors in our paediatric cohort. additionally, role of immunoglobulin (ig) levels as a possible diagnostic/prognostic marker was analyzed. between january and june , allogeneic transplantations were performed in pediatric patients ( boys and girls) at our center. median age was . years ( . - ). underlying diseases were hematological malignancies ( %), nonmalignant hematological conditions ( %), immunodeficiencies ( %) and others ( %). stem cell source was bone marrow ( %), peripheral blood ( %) and cord blood ( %). donors were unrelated ( %), sibling ( %), haploidentical ( %) or other matched family donors ( %). routine ebv pcr screening and ig level detection were performed weekly. rituximab prophylaxis was given only in nine cases. results ebv dnaemia was found in / patients ( . %), while ptld was diagnosed in / patients ( . %). all ptld cases were related to ebv infection, median of highest viral load was copies/ml ( - ). diagnosis was confirmed by biopsy in / cases, further five fulfilled criteria of probable ptld (positive pcr with appropriate clinical symptoms). ptlds occurred at a median of day + ( - ) after transplantation. all patients received rituximab treatment along with a reduction of immunosuppressive therapy. four patients died of ptld (mortality %), all confirmed by autopsy. a higher incidence of male gender ( / ; . % vs . %), bone marrow graft ( / ; . % vs . %), hematological malignancy ( / ; . % vs . %) and second transplantation ( / ; . % vs %) could be detected among ptld patients when compared to the non-ptld group. elevated igg, iga or igm levels were observed in / patients. nine out of had positive ebv pcr testing, eight of them developed ptld. five of the ptld patients had monoclonal or biclonal immunoglobulin elevation, two of them died. in cases, elevated ig level preceded the positive ebv pcr results by at least week. conclusion: at our centre incidence and mortality of ptld was similar to published data. we observed a tendency that a higher representation of male gender, hematological malignancy, bone marrow graft and second transplantation could be confirmed in the ptld group however due to small number of patients, a correlation and statistical significance could not be calculated. elevation of immunoglobulin levels do not seem to be specific for ptld but in selected cases it could predict ebv disease earlier than pcr testing. disclosure of conflict of interest: none. autologous peripheral hematopoietic stem-cell transplantation is a procedure of a stem cell rescue with patients' own previously collected hematopoietic stem cells, after myelotoxic therapy. the purpose of stem cell reinfusion is to ensure adequate recovery of hematopoiesis, shorten the period of profound neutropenia and to reduce the risk of infections. the transplantation itself carries a moderate risk for infection but some patients have higher risk due to the nature of underlying disease, earlier treatment and in case of severe mucositis. for these reasons, all treated patients are in isolated clean rooms and receive ciprofloxacin, fluconazole and acyclovir prophylaxis. in the . -year period, autologous transplantations were performed. the patients were - years old, with median of . years. of all transplanted patients, or . % had multiple myeloma, or . % had lymphoma and or . % had acute myeloid leukemia. all of the patients received pegfilgrastim mg on the first or the second posttransplant day. febrile neutropenia (ne o . × /l) was reported if patient's temperature was above . °c in one measurement or above °c in two consecutive measurements. these patients were treated empirically with piperacillin/tazobactam . g four times a day with the addition of vancomycin in the case of severe mucositis or pulmonary infiltrates. in all cases blood and urine cultures were performed, as well as testing for seasonal flu. time to neutrophil recovery (ne . × /l) was - days, with a median of days, and average of . days. febrile neutropenia was reported in patients ( . %) and in ( . %) patient's samples pathogen was isolated. gramm negative bacteria caused sepsis in . % of patients. we had to change empirical therapy according to antibiogram in . % patients. in month follow-up period, there were two ( . %) infection related deaths. our data on incidence of infections is consistent with literature data but large number of papers show satisfactory results of safety of patients discharged from hospital immediately after the autologous stem cell transplantation and who were treated at home during the phase of profound neutropenia. there is still an ongoing debate whether it is possible to conduct this procedure in such manner in our health system. disclosure of conflict of interest: none. fluconazole was equal to mold-active drugs in preventing early invasive fungal disease after allogeneic stem cell transplantation regardless of transplantation type y sun, j hu, h huang, j chen, j-y li and x-j huang there are still controversies that whether mold-active drugs is better than fluconazole in preventing invasive fungal disease (ifd) after allogeneic stem cell transplantation (hsct). we hypothesis that the optimal prophylaxis might be different in patients with different risk profile, such as in different time period after hsct or received alternative donor transplantation. in the prospective china assessment of antifungal therapy in haematological disease (caesar) study database, out of patients received primary antifungal prophylaxis were analyzed. the ifd incidence of different time period after transplantation (early, late and very late) and survival were compared among different drug groups. in patients with fluconazole, itraconazole, voriconazole or micafungin prophylaxis, the overall incidence of ifd after transplantation were . %, . %, . % and . %, respectively (p = . ). however, there is no difference in early ifd (o days post hsct) among groups of patients. the risk factors associated with occurrence of ifd were neutropenia duration days (po . , or . ( . - . )), adult (p = . , or . ( . - . )) and alternative donor (unrelated donor or haploidentical donor) transplantation (p = . , or . ( . - . )). in the sub-group analysis with only alternative donor (unrelated donor and haploidentical donor), it also demonstrated that fluconazole is equal to other mold-active drugs in preventing early ifd. patients received fluconazole prophylaxis has even better overall survival. the overall survival in patients with fluconazole, itraconazole, voriconazole or micafungin prophylaxis were . %, . %, . % and . %, respectively (p = . ). our current [p ] study suggests that fluconazole is equal to mold-active drugs to prevent early ifd in hsct patients, even in high-risk patients received transplantation from alternative donors. however, further prospective randomized study was warranted to confirm this conclusion. disclosure of conflict of interest: none. ( . %) received autologous hsct and ( . %) allogeneic hsct. sixty-five out of patients ( . %) were affected by different haematological diseases: by lymphoma, by multiple myeloma, by chronic lymphocytic leukaemia and by others diseases including mastocytosis, amyloidosis and essential thrombocythemia. hbv reactivation prophylaxis prescribed was entecavir for hbsag+ inactive carrier patients and prolonged lamivudine (lmv) course for ( %) patients. in patients ( . %) lmv prophylaxis was withdrawn - months after the end of immunosuppressive therapy. eight out of patients ( . %) experienced hbv reactivation: of them during lmv treatment and then they were switched to entecavir or tenofovir therapy, patients reactivate hbv after lmv interruption ( . %). in these patients reactivation was observed after an average time of months (range: - ) after discontinuation of lmv prophylaxis. median duration of prophylaxis was months (range: - ) after the end of immuno-suppression. three out of patients ( %) underwent allogeneic hsct and patients ( %) received rituximab. one out of ( %) seroreverted in hbsag positive and hbsab negative status, with hbv-dna ui/ml (table ) . two patients out of ( %) experienced hbv-dna detection below ui/ml. disclosure of conflict of interest: none. [p ] [p ] the severity is measured on grades (grade : microscopic hematuria to grado : clots cause urinary tract obstruction). the treatment is based on support measures: hyperhydration, continuous bladder irrigation, instillation of topical agents and in severe cases must be performed a cystoscopy for clot evacuation. in the case of the presence of poliomavirus virus (bk virus) the use of cidofovir had been demonstrated in vitro studies to have activity against bk virus. we performed allogenic transplants of which are haploidentical from to october . we realized a retrospective case study to analyses the experienced in the management of hc. results: of a total of allogenic transplants realized, developed a hc: patient received an identical hla transplant and the patients remaining haploidentical allotrasplant. all cases were male, with an age range of - years. the status of the disease was: were in complete remission and had visible disease. of the patients received cyclophosphamide as immunosuppressive therapy and all patients received cyclosporine and mofetil micofenolate also. the onset of the symptomatology was between day and day post transplant and the range of duration was from to days. the four patients precised continuous bladder irrigation but because of the poor response they received instillation of hialuronic acid ( doses). two patients required the use of cidofovir ( doses). one of the four patients required urinary tract catheterization because of hydronephrosis and renal impairment. in our review we confirmed that this entity is more frequent in the haploidentical transplant and bkv is the most prevalent cause in the late hc. -the three patients received doses of cidofovir ( mg/kg) without probenecid and had a good response. -three patients present acute renal failure associated to hc. the four patients needed bladder instillations with saline but they had poor response and received at least doses of hyaluronic acid. disclosure of conflict of interest: none. hsv infection in allo-hsct setting is mostly reactivation of latent virus. hsv disease commonly presents as mucocutaneous lesions of the oral cavity. however some patients develop serious fatal visceral dissemination. prophylactic use of acyclovir has markedly reduced the incidence of hsv disease during the period of neutropenia after allo-hsct. in this study, our aim is to demonstrate the incidence, clinical outcome and risk factors for hsv disease in adult allo-hsct. between and , patients who underwent allo-hsct in our center were included to the study. all hsct candidates and donors were tested for hsv- / immunoglobulin g (igg) antibodies prior to transplantation. all patients received acyclovir prophylaxis (related transplants mg tid, unrelated transplants mg tid) during conditioning and after allo-hsct up to months. chlorhexidine oral solution as well as bioadherent oral protective gels was used for oral hygiene. all patients were followed for symptoms of reactivation. hsv / igg seropositivity was detected in recipients ( %) and donors ( %). the distribution of hsv status was as follows: recipient and donor seropositive in ( %), recipient and donor seronegative in ( %), recipient seropositive and donor seronegative in ( %), recipient seronegative and donor seropositive in ( %) transplants. the median age of the patients was (range: - ), patients were male ( %) and ( %) had malign disease. the stem cell source was peripheral blood in ( %) patients and ( %) received grafts from related donors. sixty four patients ( %) received myeloablative conditioning regimen. the most common graft-vs-host disease (gvhd) prophylaxis administered was cyclosporine (csa) and methotrexate (mtx) in patients ( %). acute graft vs host disease was detected in patients ( %).four patients from seropositive patients ( %) had hsv reactivation, the patient characteristics are given in the table. all patients had hsv reactivation within month of allo-hsct except one patient had symptoms at sixth month posttransplant when he suffered from oral gvhd. all patients s and donors were seropositive prior to allo-hsct and responded well to antiviral treatment. the incidence of hsv reactivation in allo-hsct was detected as % which is lower to previous studies. successful primary prophylaxis and oral hygiene might reduce the incidence. all patients were responded to antiviral treatment and no visceral dissemination was detected. disclosure of conflict of interest: none. patients who have received hematopoietic stem cell transplantation (hsct) may suffer, to some extent, losses in humoral and cell immunity against antigens to which they had been previously exposed naturally (infection caused by wild microorganisms) or artificially (through vaccination). the conditioning regimen for hsct replaces the patient's immune system and involves the loss of previous immunity. this study analyzed patients included in the vaccination program for hsct recipients in the salamanca health care complex during the period - . we assessed the serological status prior to hsct for the following immunopreventable diseases (hepatitis b, hepatitis a, varicella), and the study after hsct also included measles, rubella and parotitis, prior to their inclusion in the hsct vaccination program. the study included patients, . % of which (n = ) were men. . % of the patients (n = ) were allogeneic hsct recipients with an average age of ± years, and . % ( ) were autologous hsct recipients with an average age of ± years. prior to hsct, % of the patients showed immunity against hepatitis b (hbv antibodies ui/l), . % against hepatitis a (positive for hav igg) and % against varicella (positive for varicella igg). no statistically significant differences were observed regarding this variable hepatitis b anti-hbs ui/l, hepatitis a, igg positive, varicella igg positive, measles igg positive, rubella igg positive, parotitis igg positive. table compares the serological status before and after transplantation. in the pre-transplant serological study we observed that less than half of the patients are protected against hepatitis b, while over % of them are protected against hepatitis a and varicella. regarding the diseases in which we know the serological status before and after transplantation (hepatitis a, hepatitis b and varicella), we observed that most patients maintain immunity. in the case of rubella, measles and parotitis we only have access to the serological status after transplantation, and we observed that parotitis is the disease with the lowest seroprotection. therefore, vaccination would be indicated, just as in the case of hepatitis b. the clinical results support the need to adapt the vaccination schedule to the immunological status of the patients after hsct individually. disclosure of conflict of interest: none. impact of cumulative steroid dose on infectious diseases after allogeneic hematopoietic cell transplantation m watanabe , j kanda, t kitano, t kondou, k yamashita and a takaori-kondo after allogenic hematopoietic cell transplantation (hct), highdose steroids are used to treat transplantation-related complications such as graft-versus-host disease (gvhd). however, the use of high-dose steroids is associated with an elevated risk of infectious diseases. information on the association between cumulative steroid dose and infectious diseases after hct is scarce. a total of patients who underwent their first hct in kyoto university hospital from to and survived at least days after transplantation were included in this study. we analyzed the association between cumulative steroid dose used within days after transplantation and the occurrence of infectious diseases, including invasive fungal infection (possible/probable/proven cases), cytomegalovirus (cmv) antigenemia, and bacteremia through days after transplantation. sixty-three patients received transplantation from a related donor, received unrelated bone marrow grafts, and received unrelated cord blood units. their median age was (range: - ) years and median day of neutrophil engraftment after transplantation was . patients were categorized into groups according to their cumulative steroid dose within days: no steroid administration (n = ), low-dose cumulative steroid administration under mg of prednisolone in total (n = ), and high-dose cumulative steroid administration over mg of prednisolone in total (n = ). reasons for steroid administration were treatment for gvhd in patients, engraftment syndrome in , and other reasons including lung complications in . the rate of invasive fungal infection was % ( possible cases with pneumonia and proven case of candida blood stream infection) and we found no apparent association between fungal infection and steroid use regardless of dose. cmv antigenemia was diagnosed in %, % and % of patients in the groups respectively, and both low-dose and high-dose steroid groups were significantly associated with a high risk of cmv antigenemia (low-dose group, adjusted hazard ratio (ahr), . , p = . ; high-dose group, ahr . , p = . ). bacteremia was diagnosed in . %, % and % of patients in the groups, respectively. high-dose steroid use was a risk factor for bacteremia (ahr . , p = . ). seven patients died from infection (fungal, ; viral, ; bacterial, ). two of three bacterial infection-related deaths occurred in the highdose steroid group, although the number of events was too small to analyze. our data confirmed that steroid administration is itself a risk factor for cmv antigenemia and close observation to detect cmv antigenemia is mandatory for patients using steroids regardless of its cumulative dose. high-dose cumulative steroid use is a risk factor for bacteremia. contrary to our expectations, steroid administration showed no apparent association with invasive fungal infection in our study, perhaps because of its generally low incidence in our hospital. disclosure of conflict of interest: none. impact of different t-cell depletion techniques on the incidence of infectious complications after allogenic hematopoietic stem cell transplantation k aikaterini , s federico , d-l vu , e boely , c dantin , a pradier , y tirefort , a-c mamez , o tsopra , c stephan , y beauverd , e roosnek , s masouridi-levrat , c van delden , y chalandon department of oncology, hematology unit, university hospital of geneva and department of medicine specializations, infectious diseases, university hospital of geneva t-cell depletion (tcd), obtained by either in vivo antithymocyte globulin (atg) administration or ex vivo depletion, is a well-established strategy for graft-versus-host-disease (gvhd) prevention after allogeneic hematopoietic stem cell transplantation (hsct) - . however, the prolonged lymphopenia associated with tcd can result in increased incidence of disease relapse and infections. although many studies investigated the impact of tcd on disease relapse - , little is known about the impact of tcd strategies on the incidence of infectious complications after allogeneic hsct. we retrospectively evaluated the incidence of infectious complications in consecutive patients who underwent allogeneic hsct at our center from september to december . patients received tcd grafts obtained by in vivo atg administration as part of the conditioning regimen (atg group). patients received partially tcd grafts obtained through incubation with alemtuzumab in vitro washed before infusion followed on day + by an add-back of donor t cd + cells (ptcd group). patients received grafts tcd by both methods combined. patients did not receive any form of tcd (no-tcd group). cumulative incidence estimates of infectious complications were calculated and compared using the gray test. given the increased risk of infection associated with gvhd and its treatments, gvhd or death from other causes were defined as competitive events in the analysis. we didn't observe any significant difference in the -year cumulative incidence of bacterial infections in patients receiving tcd by atg ( % ( % ci - . %)) ptcd ( . % ( % ci . - . %)) or both ( % ( % ci . - . %)) compared with patients receiving no tcd ( . % ( % ci . - . %)). similarly, the -year cumulative incidence of viral infections or reactivations was comparable in patients receiving no-tcd grafts ( . % ( % ci . - . %)) compared with patients receiving tcd grafts (atg: . % ( % ci . - . %); ptcd: . % ( % ci . - . %); atg+ptcd: . % ( % ci . - . %)). finally, no significant impact of tcd was observed on -year cumulative incidence of fungal (no-tcd: . % ( % ci . - . %); atg: . % ( % ci . - . %); ptcd: . % ( % ci . - . %); atg+ptcd: . % ( % ci . - . %)) and parasitic (no-tcd: . % ( % ci . - %); atg: % ( % ci . - . %); ptcd: . % ( % ci . - . %); atg +ptcd: . % ( . - . %)) infections. image/graph: -year cumulative incidence estimates of infectious complications depending on the tcd strategy employed. the results of our retrospective analysis indicate that the cumulative incidence of bacterial, viral, fungal and parasitic infectious diseases are similar in patients receiving tcd grafts compared to those receiving no-tcd graft, suggesting a favorable toxicity profile of different tcd strategy in respect of infections. these results should be confirmed by similar analysis in large scale, prospective clinical trials assessing the potential benefits of tcd on transplantation outcomes. patients with aml were considered eligible for hsct, died before transplantation. patients ( %) underwent transplantation from hla-identical sibling, ( %) from haploidentical family donor and ( %) from matched unrelated donor, while patients ( %) received unrelated cord blood cells. twenty ( %) out of eligible patients have had an ifi episode before transplant: were proven, probable and possible; ( ( %) pneumonia, ( %) gastroenteritis, ( %) sinusitis, ( %) candida sepsis, ( %) meningitis and ( %) cutaneous abscess were registered). five ( %) out of patients with a previous ifi and ( %) out of without previous ifi did not receive hsct (or . % ci . - . , fisher test p: . ). the majority ( %) of patients with a previous ifi waited hsct more than months from the date of eligibility in comparison with those without a previous ifi ( % vs %; or . , % ci . - . , p-value . fisher test) overall a post transplant ifi episode was diagnosed in ( %) of transplanted patient; ( %) had a relapse of a past ifi vs ( %) of the patients without a previous ifi who had a new episode. (or . , % ci . - . , p-value . yates test).a higher number of patients with ifi ( out of , %) respect to those without a previous ifi ( out of , %) died in a median time of days(range: - ) after hsct. furthermore, those who had a previous ifi had a lower median survival ( days (range: - )) compared to patients without a previous ifi ( days period (range: - )) (student's t-test p: . )). a previous ifi episode in the pre transplant period slows and limits the accessibility to hsct, and is significantly associated with an increased mortality. disclosure of conflict of interest: none. s delayed immune reconstitution has been described for haploidentical hematopoietic stem cell transplantation (hsct) compared to conventional hsct, nevertheless the incidence of invasive aspergillosis infections (iai) in haploidentical sct and the efficacy of primary prophylaxis are not well defined. our objective is to describe the incidence, risk factors and mortality of iai in our patients, using as prophylaxis micafungin during the conditioning and neutropenia period, switched to posaconazole or voriconazole when oral intake is feasible. we retrospectively analyzed consecutive patients from to who received haploidentical grafts: unmanipulated for adults, tcrab depleted in children and cd ra depleted in children. the stem cell source was peripheral blood in all cases. adults ( - yo) were treated for aml/mds (n = ), all (n = ) and lymphoma (n = ). children ( mo- yo) were treated for aml (n = ), all (n = ), aplastic anemia (n = ) and immunodeficiencies (n = ). conditioning regimen was bu-flu-cy (n = , adults), thio-bu-flu (n = , adults), flu-mel-thio for all pediatric patients; atg was used in children and tli in children. median follow up was months ( - ) for adults and months ( - ) for children. we used eortc criteria for iai and analyzed probable or definite as cases. there were events of iai, with a bimodal presentation: events ( . %) during neutropenia period and ( . %) after months of hsct ( figure ). five of them were probable and one definite (aspergillus niger). site of infection was mainly pulmonar; cns was suspected in two adult patients and skin was proven in one adult patient. all patients at the late period had chronic gvhd at diagnosis. one patient had primary graft failure. severe cmv disease (hepatitis and colitis) was present in one adult. mortality related to iai was high ( / ), patients died at a median of days. figure . iai patients characteristics the global incidence of iai in haploidentical hsct is similar to conventional hcst. primary prophylaxis with micafungin switching to oral triazole is successful ( . %) during the early period. late cases ( . %) had clearly known risk factors (chronic gvhd, steroids and cmv), and primary prophylaxis had been modified due to toxicity or interactions. iai mortality in our patients is very high ( %) despite effort in prophylaxis, diagnosis and treatment. visceral intractable abdominal pain prior to skin lesions from herpes zoster can be misdiagnosed as gvhd post stem cell transplantation which may lead to initial increase in immunosuppression and hence high mortality if we don't suspect. case report and literature review through pubmed results: year-old male with relapsed all post mud pbsct ( / ) transplant in following cy tbi atg conditioning presented at day + with intractable diffuse abdominal pain with constipation. no history of nausea, vomiting or skin rash. on physical examination his abdomen was soft, diffuse tenderness but no rigidity, muscle guarding and rebound tenderness. laboratory tests including liver function test, amylase, lipase were normal. usg abdomen and mri abdomen showed no abnormalities, except for presence of fecolith. during the stay his pain worsened needing morphine infusion, pca and later ketamine. he had previous history of acute gut gvhd controlled on budesonide and cyclosporine which was later being weaned once his symptoms were controlled. in view of previous history of gvhd, gi consultation was sought and he underwent ugi endoscopy and biopsy which was non-significant. on day of his admission he developed a pustular skin lesion on thigh and scrapping from that showed vzv and his blood pcr was also positive, he was started on intravenous acyclovir. his lesions improved and crusted and his abdominal pain subsided after h of acyclovir and was discharged on oral acyclovir after days of intravenous therapy. review of literature illustrated in table . severe abdominal pain in patients who received an allogeneic stem cell transplant has a broad differential. here we describe a case of vzv presenting with intractable abdominal pain needing opioids. because of the poor prognosis and life-threatening nature of disseminated vzv disease, it should be considered and included in the patient's workup. intravesical cidofovir ( mg/kg, diluted in ml sterile water) was once weekly applied until symptom control for min. via a transurethral catheter, i.v. cidofovir was initiated if no symptom control was achieved after local applications. in patients with hc or a lavage catheter was added. bkv cystitis (dysuria (n = ) or dysuria combined with hematuria (n = )) developed in out of transplants ( %). median age was years, % were female and % received a mismatch transplantation after mac or ric conditioning regimens. in % of bkv cystitis cases also cmv reactivation within the first days could be detected. % had acute gvhd ii°-iv°at the onset of bkv cystitis and % received steroid medication. the median time to symptom occurrence was day + after hsct (iqr - : - ). patients ( with dysuria and one either hc °and °) didn´t require therapy due to self limiting symptoms. ( %) of treated patients showed only dysuria, ( %) hc °, ( %) hc °, ( %) hc °and ( %) hc °. the first patient was treated with i.v. cidofovir twice and symptoms relieved. all the following patients were exposed to intravesical cidofovir as st line therapy. patients ( %) achieved a complete remission with a median of intravesical procedures (range: - ). patient showed symptom improvement and all patients didn´t require further therapy. patients had to be switched to i.v. application due to bladder spasms during intravesical application (n = ) or to insufficient symptom control (n = ). out of these responded to i.v. treatment, whereas patient receiving nd transplant didn´t respond at all. in patients with spontaneous symptom relieve the median bkv concentration at the time of symptom onset was log lower compared to those requiring antiviral therapy. local therapy reduced bkv viruria by log. pain during cidofovir instillation in % of patients was the only significant side effect of local therapy compared to creatinine increases by % in . % of i.v. treated patients. intravesical treatment of symptomatic bkv cystitis with cidofovir ( mg/kg) is safe and effective with an % symptom improvement rate and no systemic side effects. in patients without sufficient symptom or bleeding control i.v. cidofovir is still an option, which however induces significant renal toxicity. we therefore recommend intravesical cidofovir as st line therapy in case of dysuria or hematuria induced by bkv after hsct. disclosure of conflict of interest: none. haemorrhagic cystitis is a recognised complication of stem cell transplant (sct), with a reported incidence of - % of cases ( ) . the majority of cases are associated with bk polyomavirus (bkv), and less often adenovirus and cytomegalovirus. there are a lack of high quality studies on the optimal prevention and management of haemorrhagic cystitis. treatment options are restricted by conditioning toxicity, immunosuppression and other co-morbidities such as renal impairment. cidofovir has an inhibitory effect on bkv replication and has been used extensively in the treatment of haemorrhagic cystitis. however, severe nephrotoxicity limits routine intravenous use in sct patients. alternative options include using low dose intravenous cidofovir or intravesical administration. we conducted a retrospective case review of post sct patients presenting with bk virus associated haemorrhagic cystitis in our institution between january and november . we identified patients in total ( male, female). the indications for stem cell transplant were as follows: severe aplastic anaemia high risk aml relapsed aml relapsed all onset of symptoms (haematuria and painful micturition) ranged from day − to day + , and the time to resolution of symptoms varied from days to days. four of the patients were treated with intravesical cidofovir only, with the number of doses required varying from to . one patient received combination treatment with both intravenous ( doses), and intravesical cidofovir ( doses). all patients had a good clinical response with complete resolution of symptoms and no major complications. however, the level of bk virus in the urine did not always correlate with clinical response. some of the patients did not tolerate urethral catheterisation and required a general anaesthetic for the placement of the urethral catheter; patient required a supra-pubic catheter. currently out of patients are alive and well; patients died from causes not related to bk virus associated haemorrhagic cystitis. our experience shows that intravesical administration of cidofovir is a safe and effective option for the treatment of bk virus associated haemorrhagic cystitis. an allogeneic stem cell graft from a cytomegalovirus (cmv) seronegative donor puts recipients at high risk of cmv reactivation which can lead to cmv disease and mortality. based on the immunogenicity of cmv phosphoprotein (cmvpp ) we initiated a clinical phase i trial with a novel vaccine designed by our group: a cmvpp -derived peptide in water-in-oil emulsion (montanide) plus administration of granulocyte-macrophage colony stimulating factor. ten patients received four vaccines s.c. at a biweekly interval after allogeneic stem cell transplantation. we monitored the patients for their clinical outcome and cmvpp antigenemia. multi-color flow cytometry test were performed to assess cmvspecific cd + and gamma-delta t cells. novel neutralizing anti-cmv antibody assays were established and correlated to clinical parameters. findings: in general, patients tolerated the peptide vaccination well, no drug-related adverse events others than rash or induration at the site of injection were detected. seven of nine patients with cmvpp antigenemia cleared the cmv after four vaccinations and were hitherto free from antigenemia. two patients with cmv reactivation showed persisting cmv antigenemia. one of these two refractory patients received additional four injections and remained hitherto free from cmv antigen. another patient obtained a prophylactic vaccination and did not develop antigenemia. an up to six-fold increase in frequency of both cmv-specific cd + t cells or vdelta -gamma-delta t cells was detected in five patients. moreover, titers of neutralizing antibodies increased in four patients up to -fold over the time of vaccination. humoral and cellular immune responses correlated with clearance of the cmv load. cmvpp peptide vaccination was safe and well tolerated in patients after allogeneic stem cell transplantation at high risk for cmv reactivation. the vaccine showed encouraging immunological and clinical results. a prophylaxis study using the vaccine in solid-organ transplant patients is ongoing. disclosure of conflict of interest: none. sporopachydermia cereana is a rare yeast found in necrotic cactus tissue, predominantly in the americas. infection in humans has only been reported in neutropenic patients with fatal course, either directly from the pathogen or other complications of immunosuppression. treatment is complicated by difficulties in pathogen-identification with conventional diagnostic techniques and by resistance to echinocandins. here we present a patient with acute myeloid leukemia (aml) and s. cereana infection. this is the first patient who was successfully treated with antifungal therapy and who survived s. cereana infection. case presentation we present the case of a -year-old female patient who was diagnosed with normal karyotype aml with dnmt a and idh mutations in december . she achieved complete remission after two cycles induction chemotherapy. during the nd induction cycle the patient developed persistent fever in neutropenia despite broad-spectrum antibiotics and the replacement of prophylactic fluconazole to caspofungin. blood cultures showed growth of s. cereana, shown to be sensitive to azoles (mic fluconazole o mg/l, mic voriconazole o . mg/l) as well as amphotericin b (mic o . mg/l), but resistant to caspofungin (mic mg/l). following the susceptibility profile the treatment was changed first to liposomal amphotericin b, and with the availability of mic results to voriconazole. metastatic fungal infection (that is, endocarditis, endophthalmitis, hepatosplenic candidiasis) was excluded. after regeneration of peripheral blood values the treatment was switched to oral voriconazole. a ct scan of the chest and abdomen prior to allo-hsct after weeks of treatment with voriconazole revealed new multiple necrotic mesenteric lymph nodes. an ultrasound-guided biopsy of a node revealed no growth on fungal cultures, a grocott stain revealed no hyphae or spores. a panfungal pcr of an its (internal transcribed spacer) fragment revealed fungal dna, which could be confirmed as s. cereana. at this time the level of voriconazole in serum was found to be sub-therapeutic ( . mg/l), and the dosage was increased accordingly. subsequent ct scans and weeks later revealed a regression of the affected abdominal lymph nodes. in the further course non-myeloablative conditioning with fludarabine and busulfan prior to allo-hsct using pbsc from her hla-matched brother was performed. under prophylaxis with cyclosporine, methotrexate and antithymocyte globulins (atg) graft-versus-host disease (gvhd) remained absent. the allo-hsct was performed under voriconazole treatment with no further complications and the patient engrafted at day . the treatment was changed to fluconazole mg daily before discharge. due to the complete radiological regression of the infection in follow-up scans and excellent general condition of the patient months after hsct, fluconazole was discontinued. the patient remains in morphological complete remission months after hsct and has a % donor chimerism. the first published case of survival of infection with s. cereana exemplifies the continual progress made in treating infections in the severely immunocompromised patient. diagnosis via its sequence-analysis seems reliable but a high index of suspicion is required for neutropenic patients who do not respond well to standard antimycotic therapy. the increased availability of the technology may lead to more frequent diagnoses in the future. disclosure of conflict of interest: none. neutropenic enterocolitis (ne) is a clinical syndrome characterized by fever and abdominal pain in patients who received chemotherapy for hematological malignancies and who treated with stem cell transplantation (sct). the aim of this study was to determine the incidence, risk factors and outcome of ne after autologous sct (auto-sct). we retrospectively evaluated patients with non-hodgkin lymphoma (nhl), hodgkin lymphoma (hl) and multiple myeloma (mm) who underwent auto-sct between january and december in our center. patients with lymphoma were conditioned with carmustine, etoposide, cytarabine, melphalan (beam) or thiotepa, etoposide, cytarabine, cyclophosphamide, melphalan (tecam). patients with multiple myeloma were treated with melphalan as conditioning. diagnosis of ne was established in case of neutropenic fever, abdominal pain or diarrhea, and bowel wall thickening mm on abdominal ultrasonography. febrile neutropenia was seen in ( %) patients of all. the median time from transplantation to neutropenia was . days (range: - days). ne occurred in ( . %) in all neutropenic patients. the median time to ne after auto-sct was days (range: - days). the median neutrophil engraftment time was . days (range: - days). abdominal pain was seen in all patients with ne. twenty one patients ( %) had diarrhea. ileus was seen in ( . %) patient and septic shock was developed in ( . %) patients. five ( . %) of patients had bloodstream infection. klebsiella pneumoniae in , pseudomonas aeruginosa in , escherichia coli in , staphylococcus aureus in and coagulasenegative staphylococcus in patient were documented in patient's blood stream. early diagnosis was made by abdominal ultrasonography in all patients at a day of median days (range: - ). twenty ( %) patients were resolved completely with good supportive care and proper antibiotherapy. two ( %) patients died of septic shock and ileus. ne is a rare but serious complication in patients underwent high dose chemotherapy followed by auto-sct. gramnegative bacteria are the main causative pathogens. abdominal ultrasonography is the simple, cheap, fast diagnostic and noninvasive procedure that allows the early diagnosis and effective treatment. disclosure of conflict of interest: none. [p ] neutrophil transfusions in the treatment of neutropenic patients submitted to allogeneic hsct: possible role on graft failure s giammarco, p chiusolo , l laurenti , f sorà , n piccirillo , l teofili and s sica hematology department, università cattolica del sacro cuore and hematology departement, università cattolica del sacro cuore granulocyte transfusions (gtx) from g-csf-stimulated donors have been shown to increase the absolute neutrophil count (anc) before expected haematopoietic recovery in neutropenic patients after chemotherapy or haemopoietic sct. thus gt offers a therapeutic option along with antimicrobial agents and growth factors to improve clinical outcome of neutropenic patients with severe infections. the primary limitations of gt include low component cell dose and leukocyte incompatibility. the transfusion of g-csf-mobilized, hla-matched granulocyte components resulted in sustained anc increments, but the efficacy of this procedure has not been established by convincing randomized control trials. aim: we focused our attention on gt in the setting of allogeneic hsct, in particular on the feasibility and safety of this procedure on the rate of engraftment. between and our centre performed allogeneic hsct. we analyze data from transplanted patients receiving gt at some point during their disease. indication for gt was severe sepsis mainly due to mdr gram-bacteria. patients received a median of gt ( - ), in different phase: patients during induction therapy, during hsct, at diagnosis and during hsct and after hsct. patients' characteristics are summarized in table . median cd + cells dose was . × /kg (range: . - ). donor source was in patients g-csf mobilized peripheral blood, bone marrow and cord blood. median neutrophil recovery ( /mmc) was days and platelet recovery ( / mmc) was days. sepsis were documented in pts and pts developed fuo. relapse was documented in pts ( %). twenty-two pts are still alive and in complete remission ( %), death occurred in pts: due to trm and the remaining for disease relapse. graft failure occurred in of the pts submitted to hsct. among the patients ( %) who experienced graft failure, six ( %) received gt before hsct, because of sepsis during the induction therapy, and the remaining after hsct, during aplasia period. in the remaining group ( pts) not receiving gt, only ( %) graft failure were observed. thus a statistically difference (p = . fisher's exact test) increase in the rate of graft failure was detected in patients receiving gt. the role of gt in the treatment of infections in neutropenic patients remain still unclear for several reasons including the lack of clinical trials convincingly and consistently demonstrating efficacy, by availability of gt donors and by center's experience. gt has been successfully used in our center in patients with severe sepsis from mdr gram-bacteria during severe neutropenia but an increase number of graft failure has been registered in patients subsequently receiving hsct. alloimmunization to hla antigens in patients receiving gt might lead to an excess of graft failure requiring hla antibodies detection and attempt to reduce titer prior to hcst and maximizing stem cell dose. disclosure of conflict of interest: none. viridans streptococci are microorganisms frequently isolated from blood cultures of patients undergoing myeloablative allogeneic hematopoietic cell transplantation (allohct). poor dentition status has been associated with an increased risk of streptococcal bacteremia in the immediate post-allohct neutropenic period. the objective of this study was to evaluate the impact of oral health status on bacteremia risk in a cohort of patients undergoing therapy for acute myeloid leukemia (aml). a retrospective study was conducted in patients with aml treated at dana-farber/brigham and women's cancer center (df/bwcc) from to . there was no formal dental assessment prior to aml induction therapy. all patients underwent protocol directed pre-allohct dental evaluation that included a standardized examination, comprehensive dental radiographs, and detailed treatment planning guidelines. poor oral health status was defined as presence of acute or chronic odontogenic infection, and it was assumed that oral health status at the time of induction therapy was the same as the pre-allohct evaluation findings. oral health status at the time of allohct was determined by the completion of required dental treatment. positive blood cultures were recorded from aml induction to day + post allohct. organisms that caused bacteremia were classified as 'of possible oral source' by a blinded microbiologist. two-sided fisher's exact test was used to compare the oral health status of the entire cohort to patients with blood cultures of potential oral source. from january to january , patients with aml underwent myeloablative allohct at df/bwcc and were s followed through today + , and of these, patients met the inclusion criteria and were included in the cohort. the median age was years (range: - ) and there was similar distribution of genders. the most common aml induction regimen was daunorubicin and cytarabine ( / ; %) and of those that received consolidation therapy ( / ; %), almost all patients were treated with cytarabine. nearly all patients ( / ; %) received cyclophosphamide and total body irradiation for allohct conditioning and the majority of patients ( / , %) received tacrolimus/methotrexate (n = ) or tacrolimus/sirolimus (n = ) for gvhd prophylaxis. over half of patients ( / , %) experienced mucositis during their course of therapy for aml. pre-allohct dental evaluations were completed in / ( %) of patients. of the / ( %) patients identified as having poor oral health status, / ( %) completed all required dental treatment prior to allohct. bacteremias occurred in / ( %) patients, and / ( %) had positive blood cultures of potential oral source. of the patients with positive blood cultures of potential oral source, / ( %) patient developed bacteremia during induction and / ( %) patients developed bacteremia during allohct. of the / ( %) patients identified as having poor oral health status, one patient ( / ; %) had a positive blood culture with a bacteria of potential oral source during induction/consolidation (p = . ). oral health status was not associated with risk of bacteremia of potential oral source at either aml induction/consolidation or allohct. risk of such bacteremia in the setting of myeloablative allohct may be related more to overall gastrointestinal translocation. disclosure of conflict of interest: none. is one of the main alternatives to trimethoprimsulfamethoxazole (tmp-smx) for prophylaxis of pneumocystis pneumonia (pcp)(maertens et al. jac ). ato is less effective than tmp-smx to prevent pcp but the reasons of this lower efficacy are not well understood. ato acts on pneumocystis, plasmodia and toxoplasma species by inhibiting mitochondrial pyrimidine biosynthesis. ato is highly lipophilic and its absorption in volunteers is improved by a fatty meal. there is a wide inter-individual variability in bioavailability and many drug interferences. the aim of this study was to assess the plasma concentrations of ato in patients under pcp prophylaxis with ato oral suspension and explore the factors which might impact its bioavailability. all adult patients receiving ato for pcp prophylaxis in the hematology and clinical immunology wards between may and september were included in the study. the prescribed dose was mg of oral suspension twice a day. blood samples were collected around h after the evening dose (cmin) and - h after the morning dose (cmax). plasma was immediately separated after each sample and frozen at − °c until proceeding to the assay. ato plasma levels were measured by uv-high-performance liquid chromatography. clinical and biological data, exact timing and modalities of intake (during a meal or not), and concomitant medications were collected. cmin and cmax results are presented as median (iqr - %) and compared by mann-whitney u-test or signed rank test when appropriate. patients: a total of measurements were performed in patients (allogeneic hsct patients: ; hematology non-transplanted patients: ; hiv-infected patients: ). the mean age (range) was years ( - ), the m/f ratio was / . only two patients were neutropenic. the median cmin was . μg/ml ( . - . ) and the median cmax was . μg/ml ( . - . ). thirteen of the ( %) patients had a cmin. disclosure of conflict of interest: none. presepsin as a marker of infectious complications during high-dose chemotherapy following autologous hematopoietic stem cell transplantation in lymphoma patients y dubinina, v sarzhevskiy and v melnichenko national pirogov medical surgical center lymphoma patients, who undergo high-dose chemotherapy following autologous hematopoietic stem cell transplantation (autohsct), are at high risk of developing infectious complica- tions (ic). mortality from ic during the transplantation, according to various data ranges from to %. thus the development of models of early prognosis of ic during autohsct has become more urgent. it's reasonable to include the dynamics of biochemical markers of inflammation in these models. presepsin (psp), procalcitonin (pct) and c-reactive protein (c-rp) were assessed on the day of admission to the hospital (da), on d+ , d+ , d+ and on the day of discharge (dd). if patients developed neutropenic fever (nf), the markers were assessed at the beginning of the fever, h after, then on the second, third, fourth days after. there were patients included in the study: patients with hodgkin lymphoma, with non-hodgkin's lymphoma, with multiple myeloma, out of patients there were women and . the median age was years ( - ). the conditioning regimens were cbv, beeac or hd melphalan. depending on the presence of ic, the patients were divided into groups: group patients without infectious complications (n = ), group patients with the development of infectious complications (n = ). the median of the nf development was . days. patients from group had no microorganism growth in blood stream, either in repeated studies. gram+ flora was detected in patients, patient had gram-, patients had mixed flora and patient had pneumocystis jirovecii infection with respiratory insufficiency grade . significant differences in psp level between groups and were determined on d+ , on d+ and the dd after autohsct. considering the median day of the nf appearance ( . days), it's supposed both the prognostic value (differences on d+ , that is, days before the clinical manifestation of infection) and the diagnostic value of psp (differences on d+ and on the dd) ( table , graph ). [p ] disclosure of conflict of interest: none. hc is often a serious complication and occurs in % of allo-hsct recipients. early bleeding is usually the result of chemotherapy toxicity however late occurring hc is multifactorial. bk virus infection has been shown to be related with hc. most studies demonstrate bk virus at the time of bleeding therefore not allowing the risk imposed by asymptomatic infection to be estimated. in this study, our aim is to show the effect of risk factors as well as pre-transplant bk viral load in asymptomatic recipients on development of hc in allo-hsct. between and , we prospectively evaluated allo-hsct. in order to detect the bk viral load, we performed quantitative bk virus pcr (altona diagnostics, germany) from blood samples at days , , and after allo-hsct. informed consents were obtained from all participants. bk virus pcr was considered positive if any number of copies were detected above the analytical sensitivity of the tests. the patients were monitored for signs and symptoms of hs. the risk factors for the development of hs were evaluated by univariate and multivariate analysis. p o . was considered statistically significant. the median age of the group was (range: - ), of the patients ( %) were aged . male to female ratio was . ( / ). fifty two patients ( %) had diagnosis of malign hematological disease. stem cell source was peripheral blood in ( %), bone marrow in ( %) allo-hsct. patients received stem cells from related donors ( %) vs ( %) unrelated or haplo donors. myeloablative conditioning was administered in patients ( %). forty-four of the conditioning regimens ( %) included cyclophosphamide. hc was diagnosed in patients ( %) at a mean of days (range: - ), early hc was detected in of patients ( %). the frequency of bk viremia and number of viral copies are given in detail in table. the frequency of bk viremia increases during transplantation in relation to clinical hc ( %, %, %, %; p = . ). acute graft vs host disease (agvhd) was diagnosed in patients ( %) at a median time of posttransplant day : grade i-ii gastrointestinal/skin/liver in ( %), grade iii-iv gastrointestinal/ skin/liver in patients ( %). the most common gvhd prophylaxis preferred was cyclosporine and methotrexate in patients ( %). in univariate and multivariate analysis (age , sex, diagnosis, stem cell source, donor type, conditioning regimen, agvhd, cy administration, bk virus pcr at days , , ) bk virus titer positivity at day , , (p = . , p o . , p o . ), myeloablative conditioning (p = . ), the presence of agvhd after day (p = . ) and conditioning regimen that includes cyclophosphamide (p = . ) are found to be related with increased risk of hs. patients with hc and clots were treated with continuous bladder irrigation as well as of patients with bk viremia received cidofovir and six of them responded to treatment ( %). our study showed that, bk titer positivity, myeloablative conditioning, presence of agvhd, cyclophosphamide containing conditioning are associated with hc. detection of bk viremia in later transplant period is more sensitive for clinically proven hc. prophylactic treatment might be considered in patients with asymptomatic bk viremia in pretransplant period. [p ] disclosure of conflict of interest: none. this project has been granted by ankara university scientific research committee numbered as b . high-dose chemotherapy with peripheral blood progenitor cell (pbpc) collection followed by a myeloablative conditioning and autologous stem cell transplantation (asct) is considered the standard of care of relapsed/refractory non hodgkin/hodgkin lymphoma (nhl/hl). a widely adopted conditioning regimen is the combination of carmustine etoposide cytarabine and melphalan (beam), whose feasibility and efficacy has been largely demonstrated. high dose fotemustine plus etoposide, cytarabine and melphalan (feam) has in some cases replaced beam conditioning. neutropenic enterocolitis (nec) is a life threatening complication of patients (pts) treated with chemotherapy (cht) with mortality rate up to %. it's a clinical syndrome in neutropenic patients (pts) characterized by abdominal pain (ap), fever (f) and diarrhoea (d). ultrasound (us) was used to evaluate bowel-wall thickening (bwt), and mm is considered diagnostic of nec. early diagnosis is crucial to start conservative medical management (cmm), which appears the optimal strategy for most cases. objective: . to evaluate if nec incidence and outcome differs in beam vs feam and . to evaluate prospectively if bed-side-us (bus) can detect early signs of nec and guide a prompt treatment (cmm or surgical) in order to reduce mortality. in the last years all pts with nhl/hl admitted in our bmt unit wards at university of pisa (italy), undergoing asct were prospectively enrolled. abdominal us was performed, baseline before treatment, and as only one symptom (or a combination) appeared within h from onset: f and/or d and/or ap in cht-related neutropenic pts. pts were conditioned with beam and pts with feam. nec was diagnosed in n = / feam and in n = / beam patients. incidence was % and % respectively, without a statistically significant difference (p = . ). two pts died/ in feam arm ( . %) and pts/ in beam arm ( . %), without a statistically significant difference (p = . ). at time of diagnosis (dx) symptoms were: f+ap+d %, f+d %, f+ap %, ap+d %,d %,ap %. f alone was never present at diagnosis of nec. at dx, f was absent in / nec episodes ( %). all pts were treated promptly as bus allowed diagnosis with cmm except one pts who underwent surgery, guided by us features, during neutropenia. the likelihood of nec dx in a discriminant st model (bayes theorem) for pts with bwt and ap = . %, ap+d = . %, ap+d+f = %, ap+f = . %, d+f = %. bus allowed to detect early signs of nec and to start prompt treatment in this life threatening complication, of nhl/hl pts undergoing asct. this is a prospective study thus the true incidence of nec in nhl/hl undergoing asct should not be underestimated. there is not a statistically significant difference in incidence and outcome of nec in pts conditioned with beam in respect to feam. with bus pts do not live the isolation room. fever is not a condition sine qua non for nec diagnosis. early diagnosis allows most of pts to be treated with cmm. images of bus and ct were superimposable with lower costs, and less radiation exposure. a low mortality rate in pts with a - % chance of developing this life threatening complication suggests that a prompt bus in neutropenic patients as just one symptom presents allows to make early diagnosis of this life threatening complication and guide prompt treatment (conservative or surgical), reducing mortality. disclosure of conflict of interest: none. quantiferon-cmv in the evaluation of cmv-specific immunity after autologous and allogeneic hsct j moreno , ltesta , l zanetti , l serra , b pereira , m souza , a carolina souza , mp souza , vr colturato and cm machado , hsct program, amaral carvalho foundation and virology laboratory, institute of tropical medicine, university of são paulo cytomegalovirus (cmv) is a major cause of morbidity and mortality after allogeneic hsct. the same is not observed in autologous hsct recipients who do not need to receive immunosuppression after transplantation. in the present study, we compared the reconstitution of cmv-specific immunity in autologous and allogeneic hsct recipients. patients were invited to participate in the study and signed the informed consent. cmv surveillance with the antigenemia (ag) test (cmv brite, biotest, germany) was done weekly in the first months of transplant in allogeneic hsct recipients. preemptive ganciclovir therapy was initiated whenever a positive antigenemia was detected. the presence or absence of cmvimmunity was determined by a commercial interferon (inf) gamma release assay (quantiferon cmv, qiagen) before hsct and monthly thereafter up to d+ . from january to october , hsct recipients ( auto and allo) were included in the study. ag was positive in ( %) of the allohsct recipients at a median of (range: - ) days. ag recurrences occurred at a median of . ( - ) days, in of the pts ( . %) who had at least one episode of positive ag. hsct recipients were included in the analysis of qtf-cmv. in the pre-hsct sample, qtf-cmv was reactive in of the allohsct ( . %) and in of the autohsct ( . %). significantly less allo hsct recipients recovered cmvimmunity at day + ( . %) and day+ ( . %) in comparison with autohsct ( % and %, respectively, p o . ). up to day + , all autohsct have recovered cmvimmunity, in comparison to % of the allohsct recipients (p = . , figure ). the qtf-cmv test performed at d+ , d + and d+ did not predict the risk of cmv reactivation in the following month. similarly, the test did not anticipate the risk of ag recurrences: % of the hsct recipients with undetermined or non-reactive qtf-cmv test at d+ had ag recurrence after this period, in comparison with % of the patients with a reactive result (p = . ). in the present study, the qtf-cmv test alone could not predict the risk of cmv reactivation or recurrences. [p ] disclosure of conflict of interest: qiagen. recovery of vδ + γδ t cells is critical to epstein-barr virus reactivation after haploidentical hematopoietic stem cell transplantation j liu, z bian, q fu, l xu, x zhang, y wang and x-j huang peking university people's hospital, peking university institute of hematology, beijing, china epstein-barr virus (ebv) reactivation and its related disease are life-threatening complications in patients undergone haploidentical hematopoietic stem cell transplantation (haplohsct). our previous studies found that impaired cd − cd − t-cell recovery correlated to the increased occurrence of ebv infection after haplohsct. γδt cells make up - % of cd − cd − t cells in the peripheral blood of healthy donors. expansion of vδ + γδt t cells after hsct has been reported and this subset could respond against autologous ebv-lcl in vitro. selective activation and expansion of vγ vδ -t cell could inhibit ebv-lpd development in humanized mice. however, the association of γδ t-cell recovery with ebv reactivation after allohsct remains unknown. this is a prospective cohort study including consecutive patients who were diagnosed as hematological malignancy and underwent haplohsct. recovery of t lymphocyte and a panel of subsets, including cd +, cd +, cd +, cd -cd -, tcrαβ+, tcrγδ+, vδ +, and vδ + t cells, were determined by flowcytometry at , , , days after haplohsct. all recipients and donors were tested negative for ebv dna in the peripheral blood before transplantation. recipients were monitored weekly for ebv dna load until day after transplantation. recipients with peripheral blood plasma ebv dna load copies/ml at least on two consecutive occasions were diagnosed as ebv reactivation (ebv +). ebv − cohort generally represents patients whose ebv dna loado copies/ml in peripheral blood. within days after haplohsct, of ( . %) recipients were diagnosed as ebv reactivation. compared to recipients with negative ebv dna load, the counts of cd +, cd +, and tcrαβ+ t cells were not statistically different in the ebv+ cohort from to days after haplohsct. in contrast, recoveries of cd + and cd -cd -t cells in ebv+ patients were significantly hampered at days after transplantation (p = . and p = . , respectively). although the tcrγδ+ t-cell counts were also decreased at and days in the ebv+ cohort, the comparisons did not reach the statistical significance (p = . and p = . , respectively). notably, recoveries of vδ + γδ t cells at , and days were continuously delayed in recipients with ebv reactivation (p = . , p = . and p = . , respectively). whereas the counts of vδ + γδ t cells were similar between the two groups from to days in this context. in this prospective and large cohort study, we showed that the occurrence of epstein-barr virus (ebv) reactivation was associated with the hampered recovery of vδ + rather than vδ + γδ t cells after haplohsct. our findings will help explore γδt subset-dependent therapeutic strategies to control the serious complications due to ebv infection post transplantation and improve the overall survival of haplohsct recipients. disclosure of conflict of interest: none. in particular, bloodstream infection (bsi)is a frequent complication in the pre-engraftment phase with an impact on the morbidity and mortality of these patients. objectives: to analyze the incidence of bsi in patients undergoing hsct in our center, and to identify predisposing factors for the development of bsi in pre-engraftment phase patients after hsct. fifty-one consecutive patients undergoing hsct were analyzed retrospectively in our center during the period of july and june . the characteristics of the sample are shown in table . we have reported all the bsi between day and day after stem cell infusion. . % ( patients) received antibacterial prophylaxis with ciprofloxacin, five patients with broad spectrum antibiotics and five did not received any drug. the average days of fever have been . days ( - days). a total of blood cultures has been collected ( . per patient). there have been bsi ( . % of the patients) with ( . %) of cases caused by gram-negative organism ( escherichia coli, klebsiella pneumoniae, acinetobacter baumanii, proteus vulgaris and delftia acidovorans) and ( . %) by gram-positives ( enterococcus faecium, enterococcus faecalis, staphylococcus epidermidis, streptococcus mitis and streptococcus viridians group). one patient presented different episodes of bsi, two patients independent episodes and the rest eight, only one microorganism isolated. we have identified two bsi by extended-spectrum betalactamases (esbl-producing organism) and one isolation of carbapenem-resistant gram-negative bacteria. the rate of quinolone-resistant is % in all the sample. in univariate analysis, several factors like presence of comorbidities, presence of severe mucosits, type of catheter and antibacterial prophylaxis modality don't increased the risk to develop bsi (p . ). the place where the procedure is performed does not influence the development of bsi. although the presence of previous infections is not a risk factor, hospitalization for infection in the days before hsct does influence the development of bsi with statistical significance (po . ). the crude mortality rate of the sample has been very low ( %), with only one death related to bloodstream infection. bsi are a common relative complication in the patient undergoing hsct but with an extremely low mortality in our sample. hospitalization for infection in the days before hsct does influence the development of bsi. it is important to note that outpatient model and conventional rooms don't increased the incidence of bsi. although the use of quinolones in prophylaxis does not result in an increase in infections caused by multiresistant micro-organisms (esbl and carbapenemias) with acceptable resistance rates ( %), it also does not reduce the incidence of bsi in our sample. according to our analysis, his routine employment still throws light and shadows. [p ] disclosure of conflict of interest: none. septic episodes with multiple bacterial strains during antithymocyte globulin (atg) therapy for conditioning for allogeneic stem cell transplantation under rifaximin gut decontamination d markel , c schultze-florey , t brockmeyer , v panagiota , c lück , a schwarzer , m beck , e dammann , a ganser , g beutel and m eder recent evidence demonstrates the importance of the enteric microbiome for the development of gastrointestinal graftversus-host disease (gvhd) and mortality after allogeneic stem cell transplantation (sct) ( , ) . accordingly, the usage of the non-absorbed rifamycin derivate rifaximin for gut decontamination has been reported to preserve the intestinal microbiota composition with a positive effect on overall survival in a single centre retrospective analysis ( ). we here report severe septicaemia requiring therapy at the intensive care unit (icu) during atg application for conditioning in three patients with rifaximin used as single agent for gut decontamination within months. after changing our gut decontamination from a chinolon-metronidazole regimen to rifaximin, three cases of severe septicaemia by gram-negative and gram-positive bacteria during atg treatment occurred within months. patient # was a -year-old woman with tmds/aml after breast cancer conditioned according to the flamsa-bu protocol. the second (# ) and third (# ) patient were and -year-old males with a complex karyotype secondary aml after omf and relapsed inv( ) aml with meningeosis leucaemica, respectively. patients # and # were treated with flamsa-bu and flamsa-tbi, respectively. all patients received rabbit atg (atg fresenius/grafalon) at a dose of × mg/kg body weight and rifaximin ( × mg) for gut decontamination. patient # developed severe escherichia coli and pseudomonas aeruginosa septicaemia on day − of the conditioning regimen and had to be transferred to the icu with septic cardiomyopathy for therapy with vasopressants and levosimendan. in patient # escherichia coli, klebsiella oxytoca, staphylococcus hemolyticus and staphylococcus epidermidis were simultaneously detected in blood cultures at day − . the patient was transferred to the icu and treated with vasopressants for septic shock. patient # developed septic shock due to klebsiella pneumoniae and enterobacter cloacae on day − under atg therapy. mechanical ventilation and vasopressor therapy were required. fortunately, all three patients survived and completely recovered without any sepsis related disabilities under escalated anti-infective and intensive care therapy. all were discharged from the hospital in the outpatient clinics. interestingly, all isolated gram-negative pathogens were found to be sensible for a chinolon based gut decontamination. the reasons for these septic complications under atg therapy are not exactly understood but raise a note of caution on the use of rifaximin as single agent gut decontaminant during atg application in conditioning for allogeneic sct. infections with mycobacterium genavense were described for the first time in . since then, several cases have been reported, but almost exclusively in patients with aids. most patients who underwent hsct have insufficient cellular immunity. here we report a mycobacterium genavense infection in a patient mimicking a lymphoma-relapse after hsct. a year-old female patient was diagnosed in july with stage ivb alk-negative anaplastic t-cell-lymphoma with cervical, retro-/supraclavicular, mediastinal, axillary and retroperitoneal lymphadenopathy as well as pulmonary manifestation. two chemotherapy treatment lines and autologous stem cell transplantation resulted in a partial remission. to improve remission prior to hsct the patient received courses of brentuximab-vedotin. after conditioning therapy with fludarabine, busulfan, cyclophosphamide and atg, hsct from a hla compatible unrelated donor was performed in april . a pet-ct-scan in november confirmed complete remission. after hsct the patient remained lymphocytopenic with cell count of cd + cellso /μl. after acute stage iii gastrointestinal graft-versus-host disease (gvhd) low dose immunosuppressive therapy was maintained due to mild chronic gvhd of the liver and the upper gastrointestinal tract. beginning in june the patient experienced increasing fatigue, general weakness, loss of appetite, nausea, night sweating and fever. abdominal ultrasound, urine and blood culture as well as ct scans revealed no focus of infection. different lines of empirical antibiotic therapy resulted only in short term improvement. several blood culture tests remained sterile. a fdg-pet-ct scan showed a paraaortal and parailiacal lymphadenopathy with a high fdg uptake (suv between . and . ), highly suggestive of lymphoma relapse. endoscopic evaluations revealed two polypoid lesions in the bulbus duodeni. histology of duodenal biopsies revealed a massive accumulation of weakly pas-positive bacilli. pcr analysis confirmed an infection with mycobacterium genavense. despite several attempts mycobacteria were not recoverable on solid media even by long term culture. treatment was started with rifampicin, ethambutol, ciprofloxacin and clarithromycin. lymph node manifestation responded to therapy with decreasing fdg-uptake (suv . ) in a control fdg-pet-ct scan months later. after months treatment was terminated due to therapy refractory nausea. lymphocytopenia was persisting with cd + cellso /μl. six weeks after stopping the antibiotic therapy, symptoms as fever and weakness reappeared. duodenal biopsy could not confirm persistent mycobacterial infection. fdg-positive intraabdominal lymph nodes (suv . ) and spleen (suv . ) were detected in a control fdg-pet-ct-scan. five lymphnodes were surgically removed. immunohistology detected histiocytic cell proliferation with no sign of lymphoma relapse. pcr confirmed the presence of mycobacteria-dna. consequently, antibiotic treatment was resumed. mycobacterium genavense can present with all the symptoms of a lymphoma relapse and should be considered in immune compromised patients. reliable diagnosis can only be obtained from lymph node biopsies and/or endoscopic evaluation. treatment has to be accompanied by restoring cellular immunity and should only be stopped after pcr-negative biopsies. disclosure of conflict of interest: none. stratification of patients with multiple myeloma and lymphoma undergoing autologous hematopoietic stem cell transplantation in term of antifungal prophylaxis r moghnieh, s khaldieh, l awad, d abdallah, n droubi, a youssef, a mougharbel, t jisr and a ibrahiim makassed university hospital, beirut, lebanon autologous hematopoietic stem cell transplantation (ahsct) is at intermediate risk for invasive fungal infections (ifi). the recommendations of international scientific societies are not homologous regarding prophylaxis against ifi in patients (pts) undergoing ahsct. the primary end point was to assess risk factors for the need of empiric/preemptive antifungal therapy in ahsct recipients, and to extrapolate to the subgroup of pts that requires antifungal prophylaxis in our population of ahsct pts. the secondary endpoint was to determine the fungal species distribution infecting or colonizing the pts. our study included adult pts ( yo) who underwent ahsct for lymphoma and multiple myeloma (mm) between and . all febrile neutropenic pts are being managed according to the infectious diseases society of america (idsa) guidelines regarding the use of antimicrobial agents in neutropenic pts with cancer. eligible pts were divided into two groups: those who received empirical antifungal therapy and those who did not need it. we recorded demographic and baseline clinical characteristics including: age, gender, comorbidities, stage, disease status at ahsct, high-dose therapy regimen, the presence of mucositis and its grade, the number of cd + cells transfused, the presence of central line or portacath, the need for mechanical ventilation, the presence of diarrhea, the duration of neutropenia, and the presence of bloodstream infections. pts who had lung infiltrates suggestive of ifi were analyzed separately. the causative fungal pathogens and colonizers were analyzed. univariate and multivariate analysis of potential risk factors to assess further significance was performed using spss. patients were included. pts ( %) had lymphoma and pts ( %) had mm. the need of empiric antifungal therapy was statistically more significant in lymphoma than mm pts (po . ).the presence of mucositis grade ⩾ showed a statistical significance for the need of antifungal therapy (p = . ). in the lymphoma group, remission status (pr vs cr) was not a significant factor for the need of empiric antifungal therapy (p = . ).the presence of mucositis grade ⩾ was at the limit of significance ( p = . ). in the mm group, remission status (pr vs cr) did not affect the need of empiric antifungal therapy (p = ). however, mucositis grade ⩾ was found to be a significant risk factor for the need of empiric antifungal therapy (p = . ). following factors: the number of cd + cells transfused, the presence of central line and portacath, the need for mechanical ventilation, the presence of diarrhea, the duration of neutropenia, and bloodstream infections did not show any significance for the need of antifungal prophylaxis in both groups. all recovered fungal isolates (n = ) were not from deep seated tissues biopsies or blood, and were identified as candida albicans in with lymphoma, and in with mm. they reflected the candida ecology in this pts series rather than deep seated fungal infections. we suggest to give antifungal prophylaxis to all lymphoma pts because of the higher need of empirical antifungal therapy, and give antifungal prophylaxis to mm pts having a predisposition for severe mucositis. fluconazole is the antifungal of choice for prophylaxis since all the fungal isolates were candida albicans. keywords: autologous hematopoietic stem cell transplantation, antifungal prophylaxis. disclosure of conflict of interest: none. a -year-old previously fit woman from a rural area of eastern europe was admitted to the hospital for severe aplastic anemia. steroids, csa, antinfective prophylaxis and supportive therapy were administered without response; therefore rabbit atg was then administered, with minor response; the year later, she underwent allogeneic-hsct (mud / , ric: tbi, cyclophosphamide and fludarabine; gvhd prophylaxis: atg, csa, mtx). several days after transplantation she developed left migraine with ipsilateral back-eye pain. brain mri and ct showed a diffuse opacification of paranasal sinuses, mainly in the sphenoid sinus. the symptoms gradually improved with a specific treatment. the patient achieved a quick and complete haematological recovery and she was discharged. at follow-up visits she complained a flare of the migraine, with a left-sided headache that did not improve with nsaids. the headache gradually intensified until vision in the left eye became blurred with conjunctival injection. after consultation with ophthalmologist, for suspected toxoplasma retinitis, administration of intravitreal steroids and clindamicine was begun with partial benefit. however days after (d + ) she was admitted in hospital because of worsening headache, irradiated in the occipital area, and weakness in the right hemibody. tests on csf were negative for neurotropic pathogens. an mri showed a complete occlusion of the intracranial tract of left internal carotid artery, with likely infectious material localized in the left lateral cerebral fissure. a chest tc showed a nodule with initial excavation in the right superior pulmonary lobe. for suspected tuberculosis she started antitubercular therapy. despite a second lumbar puncture confirmed pleocytosis compatible with acute purulent meningitis, microbiological research for bacteria, fungi and bk were negative. so antitubercular and antitoxoplasma therapy were stopped and the patient underwent surgical biopsy within the sphenoid sinus. pathological examination of the biopsy specimens showed acute and chronic inflammation of the respiratory mucosa, periodic acid. schiff and grocott staining ( figure ) highlighted several septate fungal hyphae. cultural analysis revealed colonies of scedosporium apiospermum so the patient started targeted voriconazole intravenous therapy. nevertheless, days later, she developed aphasia and right hemiparesis. a brain angio-mri confirmed the appearance of new lesions compatible with infectious localizations associated to an increased defect of left internal carotid artery vascularisation and complete left choroid detachment. after weeks of voriconazole a significant clinical improvement have been observed and she was discharged, continuing oral antifungal therapy with voriconazole. at the last follow-up she achieved a complete resolution of neurologic symptoms, with permanent left eye blindness. months later (d + ) she was asymptomatic, with normal haematological and neurological conditions and was able to stop the antifungal therapy. this case-report confirms that the risk of invasive fungal infection (ifi) is relevant in patients receiving hsct for aa, probably due to the prolonged neutropenia and association of other risk factors such as the immunosuppressive therapy and the iron overload. in this very poor prognosis infection, the early diagnosis of cns ifi remains challenging, but the administration of voriconazole was extremely effective. disclosure of conflict of interest: none. in this study, we aim to present the seroprevalence of ebv and incidence of posttranplant lymphoproliferative disease as well as to evaluate the relation with gvhd. between and , the ebv serology of patients that underwent allogeneic hematopoietic stem cell transplantation and their donors were evaluated in the study. ebv ig g (vca-igg, ebna ig g, ea-igg) and igm (vca-igm) antibodies were detected by chemolluminesance method (abbott, abd). all patients were followed for reactivation. ebv igg seropositivity was detected in patients ( %) and donors ( . %). there was no statistically difference in related vs unrelated transplants in seropositivity. the median age of the patients was (range: , patients were male ( %) and ( %) had malign disease. the stem cell source was peripheral blood in ( %) patients and ( %) received grafts from related donors. myeloablative conditioning regimen was received by of patients ( %) (table) . all patients received acyclovir prophylaxis (related transplants mg tid, unrelated transplants mg tid) during and after allo-hsct up to months. twenty six-yearold pretransplant ebv seropositive aplastic anemia patient had ebv ig m positivity after months of allo-hsct and developed lymphoproliferative disease. he was in complete remission after courses of rituximab and methylprednisolone. three patients were ebv igm seropositive in th, th and th months of allo-hsct and received symptomatic treatment. acute gvhd was detected in patients ( %) whereas patients ( %) had chronic gvhd. acute gvhd and chronic gvhd incidences were similar in comparison of donor ebv seropositive vs seronegative status ( % vs %, p = . ; % vs %, p = . ). ebv seropositivity was detected in . % of patients. the donor ebv serology was not related with acute or chronic gvhd. [p ] disclosure of conflict of interest: none. the umc utrecht pediatric experience with brincidofovir after allo hsct ca lindemans, m bierings and jj boelens pediatric blood and marrow program, dept. of pediatrics, university medical center utrecht, the netherlands viral reactivation with dna viruses form a considerable complication of allogeneic hematopoietic stem cell transplantation (hsct). there are little effective antiviral therapies and most have considerable toxicity. especially for adenovirus, there is no satisfactory therapeutic option. recently a new oral antiviral agent, the cidofovir prodrug brincidofovir became available to european patients only on the basis of urgent medical need and after a case by case approval by the health authorities. the aim was to describe our single center experience with brincidofovir in the pediatric allogeneic hsct setting. in the umc utrecht, pediatric patients receive t-replete bone marrow or unrelated cord blood (ucb) as the donor source after mostly myeloablative conditioning regimens (+ serotherapy in unrelated-hct). as gvhd prophylaxis patients receive cyclosporine a (csa) and mtx for bone marrow, csa and prednisone for ucb. patients are by standard weekly monitored for the presence of adenovirus, ebv, cmv en hhv viremia by rt pcrs in the plasma. extensive immune reconstitution measurements are performed every weeks. since , patients that developed viral reactivation with adenovirus, or a combination of other dna viruses (cmv, bk or hhv ) were offered brincidofovir if the viremia was progressive or in the context of poor immune reconstitution. brincidofovir was given in suspension ( mg/ml) at the dose of mg/kg biw, or mg biw for larger children. de drug was discontinued when the viral load was below detection level. in total, six pediatric patients (age range: - ) received brincidofovir ( patients tablets, the suspension). four received it for adenovirus reactivation, a th patient for cmv and bk and a th patient for cmv en hhv . the median day post-hsct of the first administration was days post hsct (range: − to ), the median day post detection of viral reactivation days . the median duration of administration was days ( - ) with two patients being discontinued because of death. in no patient the drug was discontinued due to toxicity issues. the patients that died had multi-organ failure due to a combination of severe agvhd and multiple infectious issues. the patients were discontinued when the viral load was low and when they had cd counts of at least /μl. none of the four alive patients reactivated after the drug was discontinued. urgent medical need administration of brincidofovir is feasible. in our limited series we found the drug was well tolerated. disclosure of conflict of interest: i am a medical consultant for brincidofovir (chimerix). reactivation of herpes simplex virus (hsv- ) or varicellazoster virus (vzv) occurs frequently after allogeneic stem cell transplantation (asct). here, we report three unusual cases, two with reactivation of hsv- and one with vzv. patients and methods: patient (pt) ( -year-old, male) was allografted for high risk acute lymphoblastic leukemia in first complete remission after conditioning with total body irradiation ( gy) and etoposide ( mg/kg). graft-versus-host disease (gvhd) prophylaxis was performed using cyclosporine a, short course methotrexate and anti t-lymphocyte globulin (atg). pts ( year-old, female) and ( -year-old, male) were allografted for acute myeloid leukemia in second and first complete remission, respectively. conditioning regimens used were flamsa-ric in pt and fludarabine/busulfan in pt . in both cases, gvhd prophylaxis consisted of cyclosporine a, mycophenolate mofetil, and atg. pts and had already experienced hsv- -positive oral mucositis following induction chemotherapy and had successfully been treated with acyclovir. both developed hsv- -positive oral mucositis again after asct. in both cases, initial therapy with acyclovir i.v. at a dose of up to mg/kg t.i.d. was ineffective. to explore the mechanism leading to clinical acyclovir resistance, the thymidine kinase genes of both viral strains were sequenced. pt presented with severe abdominal pain and nausea months after asct. in this case, acyclovir prophylaxis post asct had been stopped months before due to side effects. moreover, low dose prednisolone therapy was necessary for chronic gvhd. the hsv- -strain from pt showed a single base pair deletion in the region from nucleotide position to of the thymidine kinase gene (which consists of a guanosine repeat). in pt a single base pair insertion in the same region was found. both genetic alterations lead to a loss of enzyme activity and acyclovir resistance. in both pts treatment was changed to foscarnet which led to rapid improvement. in the case of pt , multiple mucosal erosions were found on endoscopy of the esophagus. in these vzv dna was detected by polymerase chain reaction (pcr). only days later, a vesicular skin eruption developed, which did not follow a dermatomal distribution. again, in the vesicular fluid vzv dna was detected by pcr. in this patient, acyclovir ( mg/kg i.v., t.i.d.) resulted in rapid improvement. reactivation of hsv- and vzv after asct is a frequent finding. usually, hsv- strains respond well to acyclovir. in some cases, resistance can develop, especially in patients that had been treated with acyclovir before. acyclovir resistance of hsv- caused by mutations in the thymidine kinase gene can be overcome by treatment with foscarnet which directly inhibits the viral dna polymerase. disseminated vzv reactivations after asct have been described. clinical presentation can be misleading, for example, beginning with severe abdominal pain that precedes the vesicular eruption by several days. disclosure of conflict of interest: none. toxoplasmosis is a rare but severe complication after hematopoietic stem cell transplantation (hsct) ( ) . it can involve the central nervous system alone or can manifest as a disseminated disease. in the paediatric population the mortality rate is high and sequelae are often severe. new diagnostic tools, such as the pcr assay, may allow for rapid diagnosis and preemptive therapy ( , ) . we retrospectively analysed all children who underwent allogeneic hsct in our centre between january and december . patients lost to follow up before day + were excluded. patients and donors were tested before transplant in order to assess their immunological status against t. gondii. a total of allo-hsct were analysed. before transplant, . % of recipients (r) were toxo-igg positive and . % were toxo-igg negative. among donors (d), serology was available only for / : % were toxo-igg positive, % were toxo-igg negative. we found a high number of not tested donors ( . %, / ) which included, in most cases, mud from foreign registries. the group at higher risk for toxoplasmosis, d − /r+, included . % pairs, whereas d − /r − were . %, d+/r-were . % and d +/r+ were . %. in our series the cumulative incidence of toxoplasmosis disease was . %, with cases out of transplants. two of them (case and ) had cerebral toxoplasmosis, one (case ) had disseminated toxoplasmosis and case had toxoplasmic chorioretinitis. mortality rate was %: two patients died because of multiorgan failure and disseminated toxoplasmosis respectively. in no case localized cerebral toxoplasmosis was the main cause of death. no complications were seen in surviving patients. all patients who developed toxoplasmosis were toxo-igg positive before hsct and three of them were transplanted from a toxoplasma igg negative donor (fourth donor not tested). in the two fatal cases the interferon-gamma releasing assay (igra) never became positive, confirming the absence of specific cellular immunity. toxoplasmosis disease can affect hsct outcome in paediatric recipients and pre-hsct seropositivity is the most important risk factor for toxoplasma disease in the post transplant period. in our cohort seroprevalence was higher than expected, probably due to the high number of patients coming from eastern europe. in order to reduce the burden of toxoplasmosis disease in our population we decided to implement a real-time pcr screening protocol for d − /r+ pairs, to provide rapid diagnosis and early therapy. all positive recipients with a seronegative donor will undergo real-time pcr screening starting on the day of stem cells infusion, and regularly until cd + t cell recovery. in the future we will analyse the impact of this strategy in this particular subset of immunocompromised patients. treatment with brincidofovir for adenovirus disease in pediatric hematopoietic transplants introduction adenovirus may cause serious morbidity and mortality after allogeneic hematopoietic transplants in children. severe lymphopenia is the main risk factor associated with progression to disseminated and often fatal disease. treatment with unlicensed cidofovir is based on monitoring of plasma viral load by pcr. however, cidofovir is only moderately effective at controlling adenovirus and it is associated with significant renal toxicity. brincidofovir is a lipid conjugate of cidofovir. it has a good oral bioavailability and achieves higher intracellular levels of active drug than cidofovir with a better safety profile. it is a potent inhibitor of viral dna synthesis so it could be indicated in immunocompromised patients with adenovirus disease. patients and methods we present three children of , and years old diagnosed of acute lymphoblastic leukemia (all) in nd complete remission (the first two patients) and severe aplastic anemia the last one. there were girls and boy. they underwent a peripheral blood hematopoietic stem cell transplantation using αβ/cd depletion with a haploidentical donor in the two patients with all and cd ra depletion with a matched unrelated donor in the other patient. patients that underwent haploidentical transplants developed early acute graft versus host disease grade iii with gut and skin involvement so immunosuppressive treatment with corticoids was started. they developed severe lymphopenia ( o / mm ). in the first month after transplant an adenovirus disease was diagnosed in the three patients from the weekly monitoring of plasma viral load by pcr. adenovirus was also tested in stools, urine and respiratory sample. in all patients adenovirus was also detected in urine sample. in one of them adenovirus was detected in nasal exudate too and in the other the virus was isolated in stools and in a skin biopsy. results: all of them were initially treated with cidofovir with poor results. foscarnet and gancyclovir was also used without improvement. finally they started a treatment by compassionate use with oral brincidofovir twice a week. with the first dose of brincidofovir plasma viral load started to go down until its complete disappearance. brincidofovir tolerance was good with only mild and limited diarrhea in two cases in the day they were taking brincidofovir. two of the three patients were alive without signs of adenovirus disease. in the other patient blood adenovirus load by pcr decreased below /ml, but remain high in urine. she died of respiratory failure due to pulmonary graft versus host disease. conclusion brincidofovir may be a promising therapeutic option for the treatment of severe adenovirus disease in immunocompromised patients with a good toxicity profile. disclosure of conflict of interest: none. table . all patients were transplanted with pbsc for haematological malignancy, and s received reduced intensity conditioning (ric) regimens with in vivo t-cell depletion. the proportion of patients with baseline and post-vaccination hi titres ⩾ : were . and % for a(h n )pdm , . % at both time points for a (h n ), and . and % for b/phuket. pre and postvaccination geometric mean titres gmt) were higher by mn than hi for a(h n )pdm and a(h n ), but lower for b/ phuket (p = . ). no post-vaccination seroconversions were detected by hi, while a single seroconversion to a(h n ) pdm was detected by mn in a patient vaccinated at - months. the mn assay did not detect any additional low-titre seroresponses (negative to detectable titre) below hi threshold. none of patient age, lymphocyte count, days from transplant to vaccination, donor type, and gvhd or ist at vaccination correlated with baseline or post-vaccination titres by either assay. response to iiv was virtually absent throughout the first year post-hsct, with a single seroconversion to a(h n )pdm detected by mn but not hi, although the sample size was small and half of patients were vaccinated at - months. there is a clear need for a novel, immunogenic seasonal iiv and/or novel vaccination regimens in this population. vaccination of recipients' relatives and close contacts, and hsct healthcare workers should be strongly encouraged. pre-and post-transplant iron overload (io) has been associated with considerable long-term morbidity and mortality in pts undergoing transplantation. classically, management of io in the post-allo-hsct setting has been based in the performance of therapeutic phlebotomies (tp), which are inconvenient for the patient and are often not feasible due to ongoing anemia. we recently published the first prospective study of deferasirox in adult allo-hsct pts with io (vallejo, et al. haematologica ). in this retrospective analysis, we analyzed the real-life management of io in the post-allotransplant setting. this study includes the last pts with a minimum follow-up of weeks, who underwent allo-hsct in our center (october -october ). pts were male ( . %) and female ( . %). median age was years (range: - ). baseline diseases were: aml ( . %), lymphoproliferative disorders ( . %), mds ( . %), all ( . %), chronic myeloproliferative diseases ( . %), mm ( . %), and bm failures ( . %). donor was unrelated in cases ( %; of them hla mismatched), and related in ( %; of them haplo-identical). conditioning regimen was: busulphan-based ( . %), melphalan-based ( . %), tbi-based ( . %), and others ( . %). progenitors source was pb in ( . %), and bm in ( . %). pre-hsct: pts had been transfused with a median of prbc (range: - ), and their median serum ferritin (sf) was ng/ml (range: - ). day + post-hsct: pts had died, and pts had not reached that day yet, so pts were evaluable. they had been transfused with a median of prbc (range: - ), and their median serum ferritin (sf) was ng/ml (range: - ). % pts had sf superior to ng/ml. liver mri (by sir method) to assess liver iron concentration (lic) was performed in pts at day + . seven pts ( . %) had no io (lic - mg/g), pts ( . %) had moderate io (lic . - . mg/g), and pts ( . %) had severe io (lic superior to . mg/g). median lic was . mg/g (range: . - . ). among the cases with history of more than prbc transfused and sf higher than ng/ml at day + , ( . %) were proved to have liver io by mri; the other pt had io in spleen. pts started some kind of therapy to treat the io: pts with severe io initiated a tp program and pts ( out of with moderate io, and out of with severe io) initiated chelation therapy with deferasirox. the drug was started at low dose ( . - mg/kg/ day), and was increased if tolerated up to a maximum of mg/kg/day. of note, the majority of pts were also taken a number of medications (immunosuppressants, statins, antimicrobials, etc). of those pts ( . %) did not tolerate the drug, and were changed to tp. for more details, see the table. ( ) the combination of the history of prbc transfusions and serum ferritin levels was, in the majority of cases, enough to assess the io in the post-allo-hsct setting. ( ) liver mri (by sir method) helped to assess io in doubtful cases. ( ) deferasirox, initiated at low doses and increased if tolerated, was safe and its use helped to avoid the need of therapeutic phlebotomies for the majority of patients. this study reproduces, in a real-life setting, our previous findings in a prospective clinical assay. [p ] disclosure of conflict of interest: none. a case-control study of risk factors of primary graft failure with a focus on associated early-onset severe infections v alcazer , a conrad , f-e nicolini , s ducastelle-lepretre , f barraco , x thomas graft failure (gf) is a rare but devastating event after allogeneic haematopoietic stem cell transplantation (ahsct), exposing the recipient to disease relapse, drawbacks of marrow aplasia, infections and death. the aim of this study was to analyse the risk factors associated with graft failure after ahsct, with a specific focus on early-onset severe infections (esi). we conducted a retrospective, observational, single-centre, matched case-control ( : ) study among adult s ahsct recipients transplanted at the haematology department of our institution between and , with a subsequent follow-up of months. engraftment was assessed at day+ post-ahsct. gf cases were classified as primary gf (pgf), defined as failure to achieve donor-derived absolute neutrophil count (anc) ⩾ . × /l or lasting more than consecutive days without evidence of disease relapse and early-secondary gf (esgf), referring to the loss by day post-ahsct of a previously functioning graft associated without evidence of disease relapse. each case was matched with two controls according to underlying haematological disease, hla matching, stem cell source, intensity of conditioning and temporal proximity of ahsct. demographics, haematological and graft characteristics as well as esi report were retrieved. esi were classified in invasive fungal infections, viral infections (cmv, ebv, hhv- , other viruses), toxoplasmosis and severe sepsis of bacterial origin. during the study period, ahsct were performed at our center. seventeen ( . %) gf cases were identified, of which pgf and esgf, and were matched with controls. in the descriptive analysis, gf and control populations did not significantly differ when considering demographics, haematological characteristics and hematopoietic stem cell source. regarding pretransplantation status and graft characteristics, only disease status (progressive disease) and cell dose (both cd + and cd + cells number/ kg) were associated with graft failure. the proportion of patients with ⩾ esi before day was significantly higher in cases than in controls ( / vs / , p = . ), with an overall number of esi events of and among cases and controls, respectively. five cases had ⩾ concurrent esi. the median time from ahsct to the first esi event for gf cases was days (interquartile range (iqr), - ) vs (iqr, - ) days for controls (p = . ). in the gf setting, the most prevalent infections were herpesviridae infections (n = including hhv- n = , ebv n = , cmv n = ), probable ifi (n = ), severe sepsis of documented bacterial origin (n = ), toxoplasmosis (n = ) among whom one patient developed haemophagocytic syndrome. when further analysing subsets of esi using logistic regression, only toxoplasmosis was a significant risk factor for gf (p = . ). death related to an infection was proven for gf patients vs control patients (p = . ). the overall survival probability at months was significantly lower in the gf setting than in control patients (hr = . ( % ci . − . ), p = . ). the survival rates at months were . % and . % for gf and control patients, respectively. at our center, graft failure is statistically associated with early-onset severe infections, and already known graft characteristics such as cell dose and disease status. however, our study would need more power to increase its significance. disclosure of conflict of interest: none. allogeneic stem cell transplantation (asct) is a curative option for hematological disorders, especially malignancies. in immunosuppressed women after asct, the progression from cervical dysplasia to invasive carcinoma is accelerated, and cervical cancer is likely a more aggressive disease. therefore, follow-up protocols after asct should include regular gynecologic evaluation with papanicolaou (pap) smears. we retrospectively evaluated pap smears in women who underwent asct and searched the risk factors for abnormal cervical cytology. the median age at transplantation was . years (range: - years). the most frequent indication for asct was leukemia ( %), and % of the patients received a transplant from a sibling hla-matched donor. stem cell source was peripheral blood in all patients. myeloablative conditioning regimen was used in % of patients. cyclophosphamide, busulfan and fludarabin were used in ( %), ( %) and ( %) patients, respectively. acute graft versus host disease (gvhd) occurred in patients ( %) and chronic gvhd in patients ( %). secondary cancer ( breast cancer) was reported in only one patient at months after asct. the follow-up time was months (range: - months). after asct, benign and abnormal pap smears were found in ( %) and ( %) women, respectively. the median time between asct and development of abnormal cytology was months (range: - months). four ( %) women had at least one smear with atypical squamous cells of unknown significance (asc-us), one ( %) had a low-grade squamous intraepithelial lesion (lsil), one ( %) had atypical squamous cells/high-grade lesion (asc-h) and one ( %) had asc-us and asc-h. one ( %) patient had malign smear. two patients with asc-h showed high-grade atypia mimicking cancer but had a negative follow-up. patient who had malign smear died because of aorta dissection. cervical biopsy showed cervical intraepithelial neoplasia (cin) i in ( %) women who had asc-us or asc-h. one patient was hpv-positive. we did not find any relationship between cervical cytological abnormality and clinical factors. after asct, patients are high risk for abnormal cervical cytology and secondary gynecological cancer. regular surveillance of patients is the most important factor for decreasing the risk of developing cervical and other secondary cancers. gynecologic examinations and cervical cytological testing after asct allows early diagnosis and effective management of cervical abnormalities. disclosure of conflict of interest: none. kidney dysfunction is a frequent complication of allogeneic stem cell transplantation (sct) and contributes to the morbidity and mortality of the procedure. incidence of severe acute kidney injury (aki) in patients undergoing nonmyeloablative allogeneic sct for malignant diseases ranges from to %. lymphoma patients are often heavily pretreated through both chemotherapy and autologous sct and may be at increased risk of developing kidney injury. we performed a retrospective analysis of consecutive patients with lymphoma undergoing nonmyeloablative allogeneic sct between and (table ) . acute kidney injury (aki) within days of allogeneic sct was diagnosed and staged according to rifle-criteria, and severe aki was defined as rifle stage i-e ( doubling of creatinine or % decrease of egfr). chronic kidney disease was defined as an estimated glomerular filtration rate (egfr) o ml/min/ . m year after allogeneic sct. we performed multivariate logistic regression to evaluate potential risk factors for severe aki. severe aki developed in patients ( . %). reduced overall survival was observed in these patients, although not statistically significant. no significant associations were seen with age at transplantation, baseline kidney function or prior autologous sct. severe aki was associated with acute graft versus host disease (gvhd) (or . , p = . ) and the use of an unrelated donor (or . , p = . ). chronic kidney disease was observed in ( . %) of patients alive after year. we report a substantially higher incidence of severe aki after nonmyeloablative allogeneic sct for lymphoma than has been reported for other malignancies. acute gvhd and unrelated donor stem cell s source were associated with severe aki, while prior autologous sct, age and baseline kidney function were not. [p ] disclosure of conflict of interest: none. patients with acute myeloid leukemia who are treated with conventional chemotherapy still have a substantial risk of relapse. we, therefore, retrospectively analyzed data to investigate the effects and some risk factors of allogeneic hematopoietic stem cell transplantation in relapsed and refractory acute myeloid leukemia patients, and to provide some suggestion for the clinical treatment. a total of refractory and relapsed acute myeloid leukemia patients receiving allogeneic hematopoietic stem cell transplantation in our center between february and december were retrospectively analyzed, including patients in no-remission (nr) and patients in second complete remission (cr ) at the time of transplant. the median age was years (range: - ). conditioning was myeloablative using cyclophosphamide, busulfan and total-body irradiation (bu/cy, n = ; tbi/cy, n = ), and others were underwent nonmyeloablative stem cell transplantation. patients had successful engraftment. acute-gvhd and chronic-gvhd appeared in and patients. the year overall survival (os), relapse rate and disease-free survival (dfs) of the cases was ± . %, . ± . % and . ± . %, respectively. the -year dfs were higher for patients in cr patients ( . ± . %) than in nr patients ( . ± . %), and the relapse rate in nr group and cr group were . ± . % and . ± . % respectively. there was no significant difference in treatment-related mortality compared cr group with nr group. sex, age, related-donor graft were not independent factors affecting os, dfs and relapse rate. it is concluded that allo-hsct is an effective salvage therapy for patients with refractory and relapsed aml. non-remission before transplant and severe agvhd are high risk factors of poor prognosis for allo-hsct. patients in cr group who accept reinduction chemotherapy before transplantation have better prognosis than those in nr. the overall outcome seems related to the disease status. hsct during refractory and relapsed can achieve long-term survival in selected patients with individual therapy. disclosure of conflict of interest: none. the incidence of most hematologic malignancies increases with age. aging is related with a greater prevalence of impaired functional status and comorbidities. although cure of malignant and non-malignant hematological diseases is potentially possible with allo-hsct, it could lead to significant transplant-related mortality. decision making about referral to allo-hsct in older adults is a challenging task. in this study we aim to present our geriatric allo-hscts. from to , [p ] patients (age ) underwent allo-hsct in our center included to this retrospective study. pre-transplant status as well as posttransplant toxicities, complications and outcomes were determined. the age distribution of the group: patients was aged and o , patients was aged and o , patient was years old. the median age of donors was (range: - ). the pre-transplant patients' characteristics are given in the table. remission was achieved in twenty-three ( %) patients. twenty-six patients ( %) had neutrophil engraftment ( . × /l) at a median day of (range: - ) and platelet engraftment ( × /l) at a median day of (range: - ). post-transplant complications are detailed in the table. acute graft vs host disease (gvhd) was occurred in patients ( %) and chronic gvhd in patients ( %). eight patients ( %) were diagnosed with a relapse and year relapse-free survival was %. the -year and -year os were detected as % and %. the most common reason for mortality was sepsis. the -year os was higher in patients who had reduced intensity conditioning regimen and remission status pre-transplant however they were not statistically significant ( % vs %, p = . ; % vs %, p = . ) (figure) . since increasing number of older patients being diagnosed with hematologic malignancies, this trend of increasing number of allo-hsct will continue. tolerability and effectiveness are lesser, toxicity is higher in older adults. although study population is relatively small, reduced-intensity conditioning and pre-transplant remission status may be related to better survival. comprehensive geriatric assessment may be considered prior to allo-hsct for global evaluation. disclosure of conflict of interest: none. allogeneic hematopoietic stem cell transplantation (asct) is a procedure with high morbidity and mortality ( - %) requiring a complex hospital infrastructure. improved support measures and development of homecare units has allowed that asct at-home programs may be possible. our center has launched a pioneering program in our country in patients with asct to perform at home the following of aplasia, control of immunosuppressive therapy (ist) and intravenous support from the d+ of asct until the engraftment and independent ambulatory patient. to evaluate the patient safety, we compared the group of patients at-home (asct-op) with a cohort of asct 'in patient' with similar characteristics (asct-ip). asct patients between january and october at the hospital clinic of barcelona. patients performed asct-op and had an asct-ip. all patients received conditioning (myeloablative-mac-or reduce intensity-ric-) in the hospital with fludarabine mg/m (d - ) and busulphan . mg/kg ( - doses), prophylaxis of gvhd was performed with tacrolimus/mycophenolate (mmf) in asct-op group and cyclosporine(csa) and methotrexate (mtx) or mmf in asct-ip group. in all patients, the infectious prophylaxis was conventional (levofloxacin, fluconazole and acyclovir). moreover, the asct-op group received prophylaxis with ceftriaxone g intravenous (iv) once daily and liposomal amphotericin b inhaled mg twice a week during neutropenia. the asct-op group from d+ received a nurse visit once daily and physician visits twice a week in the hospital. baseline characteristics were analyzed those related to toxicity and patient outcomes. the median age (range) was years ( - ), male/female / ; ( % male). the source of the progenitors was peripheral blood in all cases and analysis of the results detailed in the table: disclosure of conflict of interest: none. an increase in rdw-sd after allogeneic hematopoietic transplantation is associated with a poor prognosis s leotta, a cupri, a di marco, a spadaro, l scalise, g sapienza, mg camuglia, g avola, g moschetti and g milone istituto oncologico del mediterraneo red cell distribution width (rdw), is an erythrocyte index influenced by stress erythropoiesis, inflammation and antioxidants. rdw predict mortality in sepsis, chronic kidney diseases and in cardiovascular disease. no data are available on rdw after hematopoietic transplantation. in a retrospective study we collected data on changes of rdw-sd in a group of patients who received allogeneic hematopoietic transplantation. fortyeight patients were affected by acute leukemia, by lymphoma, by mm, and by other diagnosis. rdw was studied at baseline and monthly for the first months. a subset of patients were studied prospectively for clinical and laboratory signs of microangiopathy. at baseline before the transplant a rdw-sd higher than normal upper limit was observed in % of allogeneic candidates. a high co-morbidity score (htc-ci score - ) at the pre-transplant screening was a factor associated to high rdw-sd (χ p = . ). a value of rdw-sd higher than normal range, at baseline, was not associated to any other factors, such as age, diagnosis, phase of the disease, previous transplantation, c-reactive protein, bilirubin, creatinine and arterial hypertension. early after allogeneic transplant we noticed at day + a significant reduction of rdw-sd but subsequently (at day + ) the proportion of patients showing an abnormal rdw-sd increased to %. an abnormal rdw-sd at s day + was registered in % of allogeneic transplant patients who presented an acute gvhd while in only % of patients who did not presented during the first months an acute gvhd (χ p = . ). in allogeneic transplantation group, patient who, at day + , had a rdw-sd higher than normal value had a inferior outcome in respect to patients having a rdw-sd within normal ranges (os was % vs %; logrank: p = . ;), (ci of trm: % vs %).these two groups were not significantly different for pretransplant features in the subset of patients studied prospectively, abnormal rdw-sd was associated to presence of schystocytes in pb (chi test: . ) and patients having ⩾ % schystocytes had a median rdw-sd of (iqr ) vs a median rdw-sd of (iqr . ) in patients who did not show schystocytes in pb (mann-whitney u-test p = . ). rdw-sd was significantly correlated also to serum triglycerides (r = + . , p = . ) and to red blood cell mean corpuscular volume (r = + . , p = . ). abnormal rdw-sd is frequent after allogeneic transplantation. abnormal rdw-sd is associated to acute gvhd and its value obtained at day + marks a group of patients with poor prognosis because of high trm. this simple parameter warrant further studies to determine its clinical usefulness in monitoring of patients suffering acute-gvhd and in diagnosis and monitoring transplant associated microangiopathy. [p ] disclosure of conflict of interest: none. sickle cell disease (scd) poses a lot of psychological burden for the patient and the caregiver. it also poses a significant financial burden over the family. ohaeri et al. developed a point questionnaire to asses sickle cell disease burden called as sickle cell disease burden index (scdbi) and its impact on caregiver's quality of life (qol). we used this questionnaire to assess the impact of hematopoietic stem cell transplant (hsct) on caregiver's qol. point questionnaire was sent to set of parents whose child underwent hsct between january and june . scdbi contained questions in various domains ( :family finances, :family interactions, :routine family activity and :parental coping ability). answers were graded on a score of - ( :never occurred and :occurred regularly or had a severe impact on the family). the results were interpreted in two headings a. family finances and interactions ( : no impact; - : insignificant impact; - : moderate impact; - : severe impact) and b. routine family activity and parental coping ability ( : no impact; - : insignificant impact; - : moderate impact; - : severe impact). all these domains were assessed before and after hsct. ten parents replied with duly filled questionnaire. mean age at hsct was . years (range: - ), m/f: / . all were symptomatic for months before hsct with % having more than hospital admissions. majority of parents were from middle class with median family income of usd per annum (range - usd). median score for family finances and interactions (a) before hsct was (range: [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] which decreased to (range: - ) after hsct. median score for routine family activities and parental coping ability (b) before hsct was (range: which decreased to (range: - ) after hsct. our results suggest that before hsct there was a moderate impact on family finances and interactions which reduced to no impact after hsct. similarly there was severe impact on family activities and parental coping ability before hsct which changed to no impact after hsct. our study suggests that hsct not only improves the qol of the child but also of the caregivers. chronic graft versus host disease (cgvhd) is a late complication of allogenic hematopoietic stem cell transplantation (hsct) that affects many tissues and organs and manifests with polymorphic clinical features similar to autoimmune diseases. poorly understood pathophysiological mechanisms are implicated in inflammation and tissue fibrosis which is a hallmark of cgvhd. the affection of lachrymal glands is frequent and contributes to ocular manifestations presenting as dry eye syndrome. autologous serum eye drops (aesds) are used topically to facilitate tissue healing and ease the symptoms in a variety of ocular diagnosis. it is unclear if the serum of a patient with cgvhd is suitable for remedy preparation and if the transplanted patient himself can meet the criteria for autologous donation. aim is to show the safety, feasibility and efficacy of autologous serum preparations in ocular lesions after allogenic hsct. donors should meet criteria for autologous blood donation (infectious disease status, complete blood count hgb g/l, hct %, adequate venous access). aesds are prepared from ml of autologous blood left to clot, irradiated and centrifuged to separate serum which is diluted with saline in : ratio or : if requested. product is dispensed into . ml ampules, stored at − °c and a -month supply is released to the patient after receiving negative results of sterility testing. in period from to . in the aesds program patients ( female, male) with ocular symptoms were included. all met required predonation criteria. of collections performed, one failed due to venous access problem and one product had to be discarded due to hemolysis. cgvdh global nih score of the patients at start of the program was: severe, moderate, mild and not scored. all patients presented with moderate to severe dry eye symptoms. in ( %) patients aesds alleviated dry eye symptoms. in ( %) out of patients referred to aesd program, more than autologous blood collections were performed (range: - ) and aesds were used regularly through period of - months, which points to the beneficial effect of the long-term use of the serum. three patients dropped out because aesds showed no advantage compared to commercial lubricant eye drops preparations. one patient dropped out because of a venous access problem, patients had disease s progression and needed other therapies: cases of amniotic membrane application of which continued with aesds to facilitate the healing effect. one patient was recently included and the effect of aesd is still evaluated. autologous donations in cgvhd patients are feasible, safe and autologous serum preparations can help relieve symptoms of dry eyes. it needs to be further elucidated specifically in which patients and at what point of the disease course the effect of the aesds is the most beneficial to make optimal use of these preparations. disclosure of conflict of interest: none. idiopathic pneumonia syndrome (ips) is a non-infectious pulmonary complication with diffuse lung injury that develops in - % of patients who undergo hematopoietic cell transplantation (hct) and the mortality rate remains high at % . the major aim of this study was to identify prognostic biomarkers for ips and establish positive and negative predictive values (ppv and npv) of ips. in a case-control study, we compared patients with ips with available samples (transplanted between and at fhcrc) with hct control recipients who did not require bronchoscopic examination and who did not grow any bacterial or fungal blood cultures. for each subject, plasma samples at day post hct and onset of ips or matched time points for controls were analyzed. the 'onset sample' for controls was the sample closest to day (median day of onset for patients with ips). we measured six proteins by elisa: suppressor of tumorigenicity (st ), tumor necrosis factor receptor (tnfr ), interleukin- (il- ), lymphocyte vessel endothelial receptor (lyve)- , endothelial protein c receptor (epcr), and herpes virus entry mediator (hvem). multivariable logistic regression models were used to evaluate the association of each protein with ips vs controls. cytokine cutoff values that maximized discrimination between ips and controls were identified using receiver operating characteristic (roc) analysis. ppv and npv of ips were calculated using the identified cytokine cutoffs across a range of hypothetical ips prevalence values ( - %) day weighted kaplan-meier survival curves were estimated for high/low cytokine subgroups. similarly, a weighted log-rank test was used to evaluate p-values. a multivariable logistic regression model including six cytokines showed that st and il- were significantly important markers to identify ips at the onset (table ) . st value at day post hct was significantly associated with occurrence of ips and il- had a marginal association. predictive values for ips by a plausible percentage of the actual hct population (up to %) are shown in figure . of the six proteins, st showed the highest ppv both at onset and day post hct followed by tnfr , and il- . npv were high in all the markers. to analyze whether st and il- at day after hct can predict survival following ips, we dichotomized the patients into cytokine high and low groups (cutoff level: st , ng/ml; il- , pg/ml) and compared survival after downweighting the observations to represent a plausible percentage of the actual population (ips prevalence, %). day survival rate were significantly lower in st high value group than in st low value group ( % vs %, p = . ). similarly, il- high value was associated with high mortality (day survival rate, % vs %, p = . ). st , il- , and tnfr were good prognostic markers for occurrence ips. especially, st and il- at day after hct can be a predictor for both ips occurrence and survival following ips. these results require validation in an independent prospective hct population. body composition parameters are sensitive nutritional indicators that influence response to treatment and mortality in cancer patients. research is not conclusive on the changes in muscle attenuation and adipose tissue areas in the stem cell transplantation (sct) phases. objective is to assess the changes in adipose tissues, skeletal muscle index (smi) and waist circumference (wc) among stem cell recipients in the peri-transplantation phase. study design: institutional review board approved this retrospective study with adult patients (age years) having b and t lymphoma who underwent sct. each patient was imaged by pet/ct scan pre-sct and months post transplantation. a cross sectional image was analyzed at the level of the l to calculate total adipose tissue (tat), visceral adipose tissue (vat), intra-muscular fat (imf), smi and wc. data was analyzed by gender since body composition parameters differed significantly between the two categories in the literature. the study sample consisted of patients (mean age: . ± . years, ( %) males, ( . %) autologous sct, median overall survival in months: . in males and . in females). death was observed in ( . %) males and ( . %) female. patient characteristics were similar for males and females except for weights (kg) and body mass index (kg/m ): . and . vs . and . in males and females respectively. changes from pre-sct to months post sct revealed that tat, vat, smi and wc decreased with mean differences of ± . cm , . ± . , . ± . cm /m and . ± . cm, respectively in males (po . ). in females, tat and wc significantly decreased with mean differences of . ± cm and . ± . cm, respectively (po . ). in females, vat and smi decreased clinically but did not reach clinical significance. in multivariate analysis, no significant associations were shown with mortality and progression rates. this study fills a research gap by providing data on the evolution of body composition parameters in the peri-transplantation phase. tat, vat, smi and wc decrease months post transplantation. future studies should evaluate the associations of these parameters with major outcomes on larger sample sizes. [p ] disclosure of conflict of interest: none. . patients received tbicontaining preparative regimen. all these patients were exposed to calcineurin inhibitors for prevention and treatment of gvhd. patients suffered from cgvhd-grade moderate or severe. all patients required systemic corticosteroids, because of gvhd ( pts) or during basic treatment of lymphoma ( pts). all patients had deficient states of vitamin d initially and required replacement. all of them, except for patients, had balanced adrenal insufficiencies and patients had balanced hypothyroidism. all women had premature ovarian failure ( received hrt). according to measurements of bone mineral density (bmd), low bone mass was detected in patients; osteopenia ( pts), osteoporosis ( pts). bone loss of femoral neck ( -osteopenia, -osteoporosis) occurred more often than lumbar vertebral ( -osteopenia, -osteoporosis) or radius ( osteopenia, -osteoporosis). presence of avascular necrosis of bone (avn), confirmed by mri, was detected in patients and the most common site of involvement was the femoral head(all patients), knee( pts) and shoulder( pts). one of the first symptoms of avn was pain and functional limitation. all patients required intensive analgesic treatment, usually nsaids and patientsfentanyl. fractures occurred in patients. the femoral neck ( pts) and thoracic or lumbar vertebral ( pts) were two most common fracture sites. all patients were qualified for surgery; patients required hip replacement, patients still awaited to perform surgery or were disqualified because of severe, skin cgvhd. bone complications may occur in about % of allo-hct survivors (including % patients with gvhd, and up to % patients with severe or moderate cgvhd) within first years after allotransplantation. bone loss, particularly at the femoral head, is the most common complication. avascular necrosis usually requires surgical intervention because of fractures. exposure to higher doses of corticosteroids (during treatment of gvhd) increases risk of bone complications. early diagnosis by mri and dxa may help to detect bone complications. ( %) and (e) ( %). a total of ( %) pts failed to meet these criteria but remained alive on day and ( %) died before day . the overall survival (figure ) for the pts was % at years with an overall mortality in icu of % ( / ) compared to % ( / ) for those who did not meet our criteria. the overall survival for pts that met our criteria at fifth day and were discharged to the haematology ward (n = ), was % at years. in this study, % of patients survived their icu admission. patients could be stratified according to the reason for admission and given an individualized -day trial: those who met our criteria for successful icu trial ( %) had a low icu mortality ( %) and those who were subsequently discharged home had a overall survival of % at years. this study raises the possibility of offering a short-term icu stay to oncohematologic patients and perhaps allows for the ceiling of intensive care for those who fail these criteria. [p ] disclosure of conflict of interest: none. in contrast, only . % of patients without cgvhd showed a thromboembolic complication in the later time course, with one patient showing an additional thrombotic risk factor. in multivariate analysis cgvhd was an independent risk factor for thromboembolic complications after hsct. . % of patients with thrombosis before hsct showed one afterwards. thrombosis before hsct was not found as risk factor for thromboembolic complication after hsct. our retrospective analysis showed an increased risk for thromboembolic complications after allogeneic hsct, with substantial higher risk in patients with chronic gvhd ( . %). in ongoing studies we currently investigate a vascular screening procedure with additional biomarkers according to inflammation and endothelial damage in patients with cgvhd prospectively. we hope to identify patients at risk for thromboembolism and prevent future complications on an individualized basis. disclosure of conflict of interest: none. ( ). pts with cns involvement received intrathecal therapy with cytarabine and in one case additional cns irradiation was applied. / pts died after a median time of mts (range: - mts) due to resistant systemic relapse, infectious complications or extensive graft-versus-host disease following allohsct. patient remains alive and disease-free at + mts following secondary allohsct. conclusions: our data indicate that em disease following allohsct affects a significant proportion of pts with aml. sites of em relapses vary widely among the pts with skin and cns being frequently involved. an aggressive approach combined of local and systemic therapy including secondary allohsct may produce favorable response in a small proportion of pts, however, overall prognosis for pts with isolated em relapses still remains poor. due to the lack of effective treatment strategies, there is a need for novel approaches to manage isolated em relapses after allohsct. disclosure of conflict of interest: none. hematopoietic stem cell transplantation (hsct)-associated thrombotic microangiopathy (tma) is a multifactorial complication, and has variable incidence in study populations due to different diagnostic criteria. aim: our aim was to identify pediatric patients with hsct associated tma using different diagnostic tma criteria published in literature and to compare the various groups for tma parameters and outcomes. we enrolled pediatric patients who underwent allogeneic hsct using treosulfan based or reduced intensity conditioning therapy. different tma diagnostic criteria, the bmt ctn toxicity committee consensus definition ( ), the overall thrombotic microangiopathy grouping ( ), the diagnostic criteria created by city of hope ( ) and the criteria proposed by jodele et al. ( ) were used to startify the patients. we determined and registered the following tma activity markers: presence or development of increased ldh and decreased haptoglobin levels, new onset anemia, thrombocytopenia, fragmentocytes, coombs test, kidney function, proteinuria, hypertension and terminal complement complex (sc b- ). complement pathway activities, components and sc b- were measured during early hsct period. two/ ( ), / ( ), / ( ) and / ( ) subjects met the different tma diagnostic criteria according to the four different systems on day and ( ) and on median ( ), ( ), ( ) post-hsct days. all of the / patients who were defined with the first three criteria, met the forth definition. due to normal haptoglobin levels and kidney function, / patients fulfilled only the forth criteria. tma coexisted with acute graft-versus-host disease in / cases ( / vs / ; p o . ). patients who met any of the different tma diagnostic criteria had higher sc b- level on day ( vs ng/ml; p = . ) compared to those without. all of the / subjects defined with tma had elevated sc b- ( ng/ml) level during the early hsct period. two patient died before day after hsct, out of which one patient met all of the four tma diagnostic criteria. after a median . ( . - . ) year follow-up time, overall survival was / . / patients with tma survived, compared to / patients without tma. relapse related mortality was the most common cause of death (n = / , po . ), while tma was not a significant cause of mortality after reduced toxicity conditioning therapy. hsct-associated tma has a variable and complex pathophysiology. using the different diagnostic criteria may influence the incidence and the time of diagnosis of this transplant-related complication. monitoring all of the published tma activity parameters, including complement terminal pathway activation marker, may help to guide physicians to recognise tma after hsct. disclosure of conflict of interest: none. hematopoietic stem cell transplantation (hsct) is associated with a risk of non-relapse mortality (nrm). it's important to assess the risk of complications and mortality before the hsct. some indexes quantify the impact of patients' comorbidities on hsct outcome. the most frequency used is the hct comorbidity index (hct-ci) and the european group for blood and marrow transplantation score (ebmts). this study tried to determine which of the two indexes best predicts the outcome in a series of patients submitted to hsct in a single center. between and , hsct were performed in our center. a total of hsct have been analyzed (we excluded patients o years (yr), nd hsct, haploidentical donors and hsct for specific diseases with very low number ( o %) of hsct performed: aplastic anemia, cll, prolymphocytic leukemia, mycosis fungoides, sezary syndrome, dendritic cell neoplasia, plasma cell leukemia and poems syndrome). the hct-ci and ebmts were calculated retrospectively (yr - ) and prospectively (yr - ). overall survival (os), relapse incidence (ri) and nrm were analyzed in the overall series and separately according to the type of hsct: autologous hsct (auto-hsct) or allogeneic hsct (allo-hsct). male: ( %) patients. median age: yr (range: - ). diseases: aml ( %), all ( %), mm ( %), nhl ( %), hl ( %), mds ( %), cmpd ( %). disease status: st complete remission (cr) or st chronic phase ( %), ⩾ second cr ( %), first partial remission (pr) ( %), ⩾ second pr ( %), no response ( %) and without previous treatment ( %). auto-hsct in patients ( %) and allo-hsct in ( %) patients. related and unrelated donor were ( %) and ( %), respectively. the conditioning regimen was standard in ( %) cases and reduced intensity in ( %). hct-ci and ebmts grouped - , and ⩾ were ( %), ( %), ( %) and ( %), ( %), ( %) in auto-hcct and ( %), ( %), ( %) and ( %), ( %), ( %) in allo-hsct, respectively. median follow-up was . yr ( . ; . ) for the overall series, . yr ( . ; . ) for auto-hsct and . yr ( . ; . ) for allo-hsct. significant differences in os and nrm were found according to the ebmts in patients submitted to auto-hsct. one-yr-os and -yr-os were % ( % ci: %; %) and % ( % ci: %; %), respectively, in patients with ebmts - , vs % ( % ci: %; %) and % ( % ci: %; %), respectively, in patients with ebmts ⩾ (p = . ). one-yr-nrm and -yr-nrm were % ( % ci: %; %) in patients with ebmts - , vs % ( % ci: %; %) in patients with ebmts ⩾ (p = . ). no significant differences were observed for ri according to ebmts in patients submitted to auto-hsct. no significant differences in os, ri and nrm were observed according to ebmts in patients submitted to allo-hsct. no significant differences regarding os, ri or nrm were found when the hct-ci was assessed. in our series, only the ebmts was predictive of os and nrm in patients submitted to auto-hsct. failure to find statistically significant differences for the hct-ci and for ebmts in allo-hsct recipients could be due to an insufficient number of patients or to a partial retrospective collection of data. infertility is common after hct predominantly as a result of the chemoradiotherapy used in conditioning. nonetheless, some patients do retain or recover fertility. newer reduced intensity regimens may be less gonadatoxic. in addition, patients are increasingly encouraged to store gametes, or embryos before transplant. we sent questionnaires to ebmt centers requesting retrospective details of number of pregnancies and pregnancy outcome for all patients treated between - . centers responded from countries detailing patients who became pregnant/partners conceived. the most frequent underlying diagnoses were acquired bone marrow failure (n = , f) aml (n = , f), hd (n = , f), cml (n = , f), all (n = , f) and b nhl (n = , f). other diagnoses included mds, mps, solid tumours, autoimmune disease, cll, t-nhl, haemoglobinopathy. of females (f), ( %) involved assisted reproductive techniques (art). f had tbi ( seven o gy) of which ( %) had art. f had reduced intensity conditioning of whom ( %) had art. f were specified as having standard conditioning of whom ( %) had art. f had allogeneic ( art, %) and f had autologous transplants ( art, %). of men (m) whose partners conceived, ( %) had art. m received tbi of which ( %) had art. where specified, had reduced intensity hct ( art, %) and had standard conditioning ( art, %) . had had allogeneic hct ( art) and autologous ( art). men had reduced intensity transplants. men received tbi (two o gy) of whom ( %) had art compared to men without tbi, ( %)of whom had art. data on return of menstruation was available for . indicated yes and ( %) had art. indicated amenorrhoea of whom ( %) had art. specifying number of children had live births (lb) and ( %) patients had more than one child after hct. lb occurred in female patients ( art, %) and lb were in partners of male patients ( art, %). the median gestational age for female patients was weeks (range: - ) and the median birth weight was kg (range: . - . ). there were / congenital anomalies. the median follow up of the offspring was y (range: - ). developmental problems were indicated for / (fine motor skills) and learning difficulties in / (adhd). in partners of male patients the median gestational age for offspring was weeks (range: - ). the median birth weight for offspring was kg (range: . - . ). congenital malformations occurred in / . one infant died of pulmonary infection. in women, several methods of assisted conception were used including hormone stimulation, ivf, cryopreserved embryos, donor embryos and cryopreserved ovarian tissue. the most frequent method was use of donor embryos ( / ) in which a minimum of attempts led to lb. the median number of attempts was (range: [ ] [ ] [ ] [ ] [ ] . art were frequently used in this group of posttransplant patients particularly in male patients vs female, tbi vs non-tbi, amenorrhoeic vs menstruating women, standard conditioning vs ric. in patients who conceive after hct, successful pregnancy leading to healthy offspring is the likely outcome. disclosure of conflict of interest: none. reduction of trm after sct was observed over the transplant periods and supportive care with danaparoid was found to be significantly effective to reduce trm. therefore, prophylactic administration of danaparoid is considered to be a reasonable option to improve the transplant outcomes for children. [p ] disclosure of conflict of interest: none. the attainment of transfusion independence after transplant is sometimes hampered by a combination of factors, ranging from infections to the need of combined therapy for clinical complications, as well as control of gvhd. iron overload is frequently observed in hematological patients before and after hematopoietic stem cell transplantation (hsct). whereas several reports have focused on iron overload before transplant, up to now, this is the only report that show full recovery of hematopoiesis and correlate this to deferasirox chelation performed on this particular subset of patients. we report on patients, transplanted for hematological diseases ( acute leukemia, aplastic anemia, multiple myeloma) heavily transfused before transplant that, considering the iron overload, were treated with deferasirox after hsct. before starting deferasirox, the patients were fully engrafted and in complete remission, although transfusion dependent, and with incomplete hematological reconstitution after allogeneic hsct. patients were selected according to the following inclusion criteria: ( ) transfused pre-transplant with more than rbc units; ( ) incomplete hematological recovery; ( ) transfusion-dependence; ( ) serum ferritin ng/ml; ( ) normal creatinine value. the workup for other aetiologies resulted negative. all patients received an initial dose of deferasirox mg/kg/day, later adjusted according to side effects. all patients experienced an increase in hemoglobin levels, with a reduction in the frequency of rbc transfusions, followed by transfusion independence (median time: days from the first dose of deferasirox). in addition, it was promptly (median time: days) associated with hematological improvement, with sustained values and no further platelet support or growth factors administration. no relevant modifications with immunosuppressive or myelosuppressive drugs were made during deferasirox treatment. deferasirox was well tolerated. basing on our results, we think that deferasirox determined stimulatory, and/or depressive effects on hematopoiesis after allo-hsct. this clinical experience raises the possibility of a potential additive benefit on hematopoiesis after transplant following iron chelation therapy with oral deferasirox. further long term studies, in larger cohorts of patients are needed to confirm these data and to design an efficient strategy to reduce iron loading after transplant. disclosure of conflict of interest: none. supported in part by ail pesaro onlus. ( ) hypertension (n = ). all five patients had normal adamts levels and negative testing for shiga toxin. complement mutation genetic studies were obtained for four patients including genes (n = ) and genes (n = ) and were all negative. testing for complement pathway including c b- were obtained for patients and were normal. all five patients were treated with eculizumab with induction treatment at mg weekly × doses, followed by one dose of mg on the fifth week, and mg every weeks thereafter. patients had a recovery of hemoglobin and platelets and a rise in haptoglobin and a normalization of ldh within - weeks from the start of eculizumab. eculizumab was discontinued for of the patients without recurrence of their tma; they are now - months since the discontinuation of eculizumab. in summary, there is a subacute syndrome of thrombotic s microangiopathy that can occur late post transplant. this syndrome appears to be complement mediated as shown by its response to a terminal complement inhibitor. it also appears to be transient without recurrence following treatment discontinuation. disclosure of conflict of interest: none. transplant associated microangiopathy (tam) is a very severe complication occurring after allogeneic bone marrow transplantation (bmt), burdened by a high case-fatality rate. it is characterized by abnormal complement activation, triggered by various agents (calcineurin inhibitors, acute gvhd, infections) with subsequent endothelial damage. in the literature, cases of mutations in recipient complement genes are described, but none in donor dna. here we describe for the first time patients affected by tam, carrying mutations in donor complement genes. in our lab, we studied patients affected by tam; they were screened for cfh autoantibodies, adamts function and variants and macro-rearrangements in cfh (and related), cfi, cfb, cd , c , dgke, thbd genes and at-risk haplotype (cfh-h and mcpggaac) by means of next-generation sequencing (ngs) and multiplex ligationdependent probe amplification analysis (salsa mlpa p armd mix- ; mrc holland). ngs was used to sequence dna by haloplex kit (agilent) on a miseq (illumina) platform with -fold coverage of every target base. the bioinformatic analysis was performed using sophia genetics and the pathogenicity was assessed by means of in silico predictions (polyphen , sift, mutationtaster, aligngvgd). all of the predicted pathogenetic variants were confirmed using sanger sequencing. the same genetic screening was extended also to donor dna in all cases. the screening for known causes of tam revealed mutations in recipient complement genes in one case; no mutations were found neither in recipient nor in donor dna in two cases; instead, donor genetic alterations were found in patients whose characteristics are summarized in table s donor hematopoietic cells. in the three cases presented, tam was relatively delayed with respect to hsct, in particular in two cases ( months) and this timing is compatible with the concept of reticulo-endothelial 're-population' by donor cells of monocytic lineage, responsible for the production of regulatory proteins of the alternative pathway of the complement. we also underline the response to anti-c inhibition in the patients who were treated with eculizumab; this fact further supports the hypothesis that the disease was related to complement dysregulation. we therefore suggest that both the recipient and the donor should be screened for complement gene mutations, so that more cases could be identified and the pathogenesis of tam could be further clarified. among these we observed one autologous engraftment, one death due to septic shock before engraftment and two primary gf. we used a desensitization treatment based on plasma exchange procedures, intravenous immunoglobulin ( g/kg) and rituximab ( mg/sm) in patients. one of these patients (aml, haploidentical donor) had dsa against hla-b (mfi ). she experienced primary gf with increasing titles of dsa (maximum mfi ); so, on day , a second transplant from the same donor was performed after a desensitization treatment. a progressive decrease in dsa was documented (up to mfi ⩽ ). on day patient achieved neutrophil count over /μl and on day platelet count over /μl. the second patient (mds, haploidentical donor), instead, received a desensitization procedure before the first transplant. she had dsa against hla-a (mfi ), and after desensitization dsa levels decreased and reached . on day patient achieved neutrophil count over /μl and on day platelet count over /μl. dsa were detected in / of usct candidates ( %) and / of haplosct candidates ( %) and they were associated with failure to obtain allogeneic engrafment in cases. desensitization treatment achieved dsa clearance and engraftment in the patients in which it was performed, underlining the potential benefit of this procedure in the setting of hsct with dsa that has to be validated by prospective and controlled studies. disclosure of conflict of interest: none. early complications and late effects and quality of life at myeloma multiplex patients z trajkovska-anchevska, a pivkova, s genadieva-stavrich, l chadievski, z stojanoski, l chevreska and b georgievski university hematology clinic, skopje, macedonia the subject of this research is the quality of life at patients with myeloma multiplex at diagnosis and during therapy within - months. the research aims to analyze patients to be able to continue activities which will contribute for improving their quality of life as a priority task placed before the patient, his family, health institutions and social environment. this research was conducted at the university clinic for hematology skopje in the period from june to march . it covers patients infected with multiple myeloma, diagnosed and treated during this period. a total of patients analyzed, using the eortc qlq c ver. . standardized questionnaires for hr quality of life that analyzed the physical, cognitive, emotional, personal and social functions related to the patients. it also analyzed and general health and quality of life. analysis of physical functioning at diagnosis is . during treatment . , significantly improved. personal functioning at patients at the diagnosis is . , during therapy − . . analyzing emotional functioning in patients at diagnosis is . , during the therapy over . significantly improved. in examining the cognitive functioning is also a significant difference at diagnosis . , during treatment . . social functioning of the patients was . at the diagnosis; during the treatment grow to . . significant improvement was notices in these patients' symptoms like fatigue, nausea and vomiting, pain, dyspnea, insomnia, loss of appetite, constipation and diarrhea. the analysis of the financial difficulties of patients at diagnosis is . and . during treatment, meaning no significant difference in the time given. the analysis of the overall health and quality of life at patient has a value of . , and during therapy . . quality of life at patients with myeloma multiplex that makes the research group was significantly improved as a result of on time diagnosis and treatment with modern medicaments and the role of social worker with the application of certain social skills, continuous counseling, guidance and education for their reintegration in the community. installing the quality of life as a separate category and investigating the factors that affect its expression in the daily functioning of the patients within the changed framework of action, as like this example for malignant disease. the needs of clearly defined interactions patient illness and treatment, quality of life and specifying the segments where it can effectively act and improve in order to achieve positive progression towards improving the qualitative features of this category is a clear and primary objective that must be inserted into the current approaches to monitoring patients with malignant hematological diseases. acute graft-versus-host disease (agvhd) is a common and severe complication after allogeneic stem cell transplantation. since the current first-line treatment is based on treatment with systemic glucocorticoids (gc), steroid-induced hyperglycaemia develops frequently in patients with (agvhd) potentially impacting on their outcome. we performed a retrospective analysis on patients who received systemic gc for agvhd and thoroughly investigated the consequences of aberrant glucose metabolism. in particular, we focused on glucose parameters early after initiation of gc. with a median of (range: - ) blood glucose measurements during gc treatment, increasing mean, median and maximum glucose levels as well as the need for insulin treatment were associated with decreased overall survival (os) in simple and multiple survival analysis. early hyperglycaemia, as defined by mean blood glucose levels mg/dl during the first days of gc therapy, was also found to be highly associated with adverse outcome: in multivariate analysis, the hazard ratio (hr) for death was . ( % ci . - . , p = . ) in patients with early hyperglycaemia. while the risk of death due to relapse was not increased, the hr for death due to non-relapse mortality was . ( % ci . - . , p = . ) in a competing risk analysis. a score based on early hyperglycaemia and non-response to gc within days allowed the identification of three risk groups: patients with both risk factors had an inferior os at years of . % as compared to . % in patients with none. patients with one risk factor had a -year os rate of . % (p = . for trend). in this retrospective study, we identified early hyperglycaemia after gc initiation as a prominent factor predicting increased nonrelapse mortality in agvhd patients. in addition, a score based on early hyperglycaemia and lack of response to gc was highly predictive for overall survival in these patients. disclosure of conflict of interest: none. early toxicity because of infectious complications not relapse is the main cause of death after allogeneic transplantation in aplasia for patients with refractory or relapsed acute myeloid leukemia high-dose cytarabin was given in / pts with induction failure. the search for a stem cell donor was started immediately after results of high-risk cytogenetic, no achievement of bone marrow aplasia on day of induction therapy, or immediately after diagnosis of relapse. four patients had a related / donor, for patients a / matched unrelated donor was identified and patients received a transplant from a / unrelated donor. the interval between diagnosis of primary disease or relapse and tx was ( - ) months (mo) for both groups . in patients melphalan ( - mg/m ) was used to induce an aplasia before starting conditioning therapy. the interval between melphalan and conditioning therapy was ( - ) days. three pts started the conditioning therapy while in aplasia after previous chemotherapy. the conditioning therapy was of reduced intensity in all pts. and consisted of treosulfan ( g/m )/fludarabin(flu) in pts, tbi( gy)/flu in pts and busulfan( m/(kg)/flu in pts, respectively.atg was given to all pts with an unrelated donor. most pts ( / ) had a severe neutropenia with a median of . /nl ( . - . ) before starting melphalan because of refractory leukemia. after a median follow-up of ( - ) mo pts ( %) were alive without relapse. ( %) pts died because of a relapse after a median of ( - ) mo. the nonrelapse mortality was % ( / pts). most of these pts ( / , %) died because of infectious complications early after transplantation (med ; - mo). in pts graft versus host disease was the main cause of mortality. in this retrospective 'real-life' analysis, we showed that an early allogeneic transplantation is feasible for patients with primary refractory or relapsed aml. a reduced intensity conditioning after induction of aplasia with melphalan offers a chance of long-term relapse-free survival for about % of patients with an otherwise dismal prognosis. nrm is high, especially because of infectious complications early after transplantation, probably related to the long period of severe neutropenia. therefore, the focus has to be set on early recognition and intervention of infectious complications. disclosure of conflict of interest: none. recent evidence supports the effector role of complement activation in several types of thrombotic microangiopathythe atypical hemolytic uremic syndrome (ahus) as well as the transplantation-associated thrombotic microangiopathy (ta-tma). the blockade of the terminal complement complex formation by anti-c monoclonal antibody eculizumab provides an effective treatment option in severe and devastating cases of ta-tma. the experience with the use of eculizumab in this indication is slowly accumulating in the hsct community, however the published data originate from small case series or uncontrolled trials and sharing of emerging real-life observations may be valued. on case reports of two pediatric patients treated with eculizumab for ta-tma with very detailed followup of multiple complement parameters, including terminal complex sc b- and eculizumab drug levels we would like to demonstrate: ( ) achieving therapeutic levels of eculizumab ( μg/ml) may be unsuccessful even with initially intensified dosing interval. furthermore, we documented rapid eculizumab clearance from circulation which allowed only for short periods ( o h) of efficient drug levels during the weekly dosing. ( ) we did not observe tightly correlated sc b- and eculizumab levels within the dosing intervals; however the long-term sc b- formation suppression was achieved concomitantly with improved eculizumab levels and slowed drug clearance. ( ) classical complement pathway activity assay (ch ) may not reliably substitute for therapeutic efficiency monitoring in case of hypocomplementaemia due to protein losses (profound diarrhea, proteinuria, gi bleeding, catabolism). this holds true also for the alternative pathway activity which remained low during treatment in both patients. ( ) mycotic infections may represent serious therapy related risks in eculizumab treatment after hsct (both patients achieved control of complement activation after multiple doses of eculizumab, however suffered fatal infections subsequently). besides, we observed a significant increase in c a concentrations correlated with clinical onset of infection which invites for further investigation of this complement cascade product as early indicator of mycotic infection. in conclusion, we would like to highlight the great added value of timely available complement assay results, including sc b- and especially eculizumab drug level values-to be used together with detailed clinical parameters for directing effectively these highly personalized (and also costly) treatments. [p ] disclosure of conflict of interest: none. table . no difference in terms of drug-related adverse events was observed in the three patient cohorts with no reported serious adverse events. similar results were obtained performing two separate sub-analysis only for lymphoma or myeloma patients. despite the limitations due to the non-randomized nature of the study, from our data on a large cohort of patients s with a long-term follow-up biosimilar filgrastim (zarzio®) could be considered substantially equivalent in terms of efficacy and safety to lenograstim (myelostim®) and peg-filgrastim (neulasta®), when used for hematological recovery and febrile neutropenia prophylaxis after asct in adult patients with hematologic malignancies. disclosure of conflict of interest: none. we studied all adults who underwent allo-hsct during a -month period ( january to november ) in our center. a total of pts ( . %) received epag for pfg with thrombocytopenia. three pts were male, and three female. median age was years ( - ). the baseline diagnoses were: alm ( ), mds-raeb ( ), idiopathic myelofibrosis ( ), aa ( ), and cll ( ). three transplants were from family donor (all of them haplo-identical), and from unrelated donor (the three of them hla / ). sc source was pb in cases, and bm in . epag was started at mg/day and escalated each weeks to , and mg if platelet count was o × /l. we analysed the platelets, anc, and hgb at epag initiation and days after being with the maximum dose. median time between allo-hsct and eltrombopag initiation was days ( - ). median maximum dose used of epag was mg/day ( - ). median platelets, anc and hgb before starting treatment were × /l ( - ), × /l ( . - . ) and . g/dl ( . - . ), respectively. five patients ( %) were severely thrombocytopenic (platelet count ⩽ × /l), ( %) were anemic (hbg o g/dl), and ( %) were neutropenic (anc o . × /l). median platelets, anc and hgb at day + of maximum dose were: × /l ( - ), . × /l ( . - . ) and . g/dl ( . - . ), respectively. the thrombocytopenic pts ( %) responded to epag, with increases of , , , and × /l in the platelet count. three anemic pts ( %) responded and achieved increases of hgb of . , . and . g/dl. finally, the neutropenic pts ( . %) responded and achieved increases of anc of and × /l. at the moment of the analysis close, pts are at a median of + . months post-hsct ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) , and all but one (who died from a septic shock) are alive and outpatient. this survival is striking for subjects who develop a complication with such a high expected mortality as pfg. pgf is a life-threatening complication, relatively frequent after alternative donor hsct, whose treatment has been very disappointed. we report our experience in pts who developed pgf during the last years. epag induced responses in platelets in all pts of the studied group. bilineal and trilineal responses were also seen. in our opinion, prospective studies are warranted in order to confirm epag as a new efficient treatment of post-hsct poor graft function. disclosure of conflict of interest: none. s ing patients who developed es with an equal number of patients who did not between january and november . we analyzed variables such as cd + cells per kg infused, use of granulocyte colony-stimulating factor (csf-g) and engraftment day. analytical data, including baseline and maximum determination of serum glutamic oxaloacetic transaminase (got) and glutamic pyruvic transaminase (gpt), c-reactive protein (crp) and procalcitonin (pct), as well as clinical data fever, weight gain, digestive and respiratory symptoms, pulmonary infiltrates were analyzed. sixty-eight patients were women. median age was years old (range: - ). patients were conditioned with beam ( %), melphalan mg/m ( %) and bcnu-tt ( %). nineteen patients developed es in our series, which correspond to eight percent of all asct. case and control groups were matched according to age, sex, diagnosis and conditioning regimen. the most prevalent baseline disease in the group with es was myeloma ( . %), followed by mantle cell lymphoma ( . %). all patients who developed es had fever, % skin rash, % respiratory symptoms, % pulmonary infiltrate an % digestive symptoms. a summary of the comparison of data analyzed in subgroups is shown in table . we found significant difference in the percentage of weight gain (p = . ), increase of tgo (p = . ), increase of tgp (p = . ) and increase the number of cd + cells per kg infused (p = . ), we found an inverse correlation between the number of cd + cells per kg infused and incidence of es. however, in terms of post-transplant csf therapy (p = . ) and crp and pct valor (p = . and p = . , respectively) we did not find significantly difference to develop es. in our series, weight gain and tgo and tgp rise were risk factors for es development. therefore, we should be aware of es in patients who develop fever, elevated liver enzymes and weight gain during graft phase. we did not find a significant difference in crp and pct suggested in other studies. further studies are required to better characterize risk factors of es development. busulphan-based ( ), melphalan-based ( ), beam ( ), tbi-based ( ), and others ( ). weight at hospital discharge was significantly lower than at admission ( . % in allo-hsct, and . % in auto-hsct). weight at day + was also significantly decreased compared with the admission ( . % in allo-hsct, and . % in auto-hsct). weight at day + was lower than the ideal for their sex and height in the allo-hsct setting. contrarily, among the patients undergoing auto-hsct, the weight at day + remained higher than the ideal for their sex and height in a high proportion of cases. regarding serum albumin, it was significantly decreased at discharge ( % in allo-hsct, and . % in auto-hsct), but recovered values similar to admission at day + . in the auto-hsct setting, prealbumin levels were significantly reduced at discharge ( %), and in lower proportion at day + ( %), compared with admission values. in the allo-hsct patients, prealbumin levels were significantly reduced at discharge ( %), but had been recovered at day + , compared with admission values. disclosure of conflict of interest: none. recently, blood and marrow transplant clinical trials network has proposed a composite endpoint: gvhd-free, relapse-free survival (grfs) for hsct outcomes. this endpoint includes as event: iii-iv acute gvhd (agvhd), relapse, death or chronic gvhd (cgvhd) requiring systemic treatment. in the last embt annual meeting a redefinition of this endpoint was proposed changing cgvhd event from those patients with cgvhd requiring systemic treatment (the original one) to those with just severe cgvhd (the redefined one). we retrospectively analysed patients consecutively transplanted ( - ) excluding non-malignant diseases, second allo-sct and those o years old age. we had generated two composite endpoints: in both iii-iv agvhd, relapse or death were considerated events but we defined grfs as the one with cgvhd event including those who required systemic treatment (as the original one) and in grfs just those with severe cgvhd (the ebmt redefined one). the median age was years ( - ) and % ( ) were males. other characteristics of patients are resumed in table . with a median follow up for patients alive of months , the median estimated survival in months and the % at + year and + years was: months, % and % overall survival (os); months, % and % event free survival (efs); months, % and % grfs ; months, % and % grfs . ( %) and ( %) hadn't any event in grfs and in grfs , respectively. in grfs , event's incidence was: ( %) for iii-iv agvhd, ( %) for cgvhd, ( %) for relapse and ( %) for death; in grfs was ( %), ( %), ( %) and ( %), respectively. considering those patients with cgvhd as event in grfs , of them hadn't the event as cgvhd at the same time in grfs (since they had cgvhd requiring systemic treatment but not severe cgvhd). for these patients, the alternative event in grfs was: without any event, relapsed and died. in the multivariate, the factors associated with better outcomes were: in grfs early ebmt stage (p o . with early as reference; intermediate p factor with more impact in both, but it is interesting to point it out that haploidentical donor had an advantage in grfs . these results are being validated in a large series and the definitive results will be available at the moment of the meeting congress. [p ] disclosure of conflict of interest: none. steroid refractory acute graft-versus-host disease (gvhd) remains a major complication of allogeneic hematopoietic stem cell transplantation (allo-hsct). affected patients have a very poor prognosis. gvhd has been associated with transplant-associated thrombotic microangiopathy (ta-tam). endothelial damage mediated by radiation, viral reactivation, drug exposure or alloreactivity results in exposure of subendothelial collagen, activation of coagulation and small vessel occlusion to a degree that results in organ failure. complement is thought to be a major mediator of endothelial damage. although a consensus exists about the exceedingly high morbidity and mortality of ta-tam and diagnostic criteria have been converging to a consensus, no biomarkers to diagnose tam and predict outcome have been established. we hypothesize that a ta-tma, related to dysregulation of the alternative complement pathway correlates with organ damage. a retrospective analysis of consecutive patients with hematological malignancies receiving an allo-hsct at the university hospital basel in the period from to was performed. data on the occurrence, risk factors and outcome of patients with ta-tma and the correlation with acute gvhd was collected. available biopsies of organs suspected to be affected by tam and/or gvhd will be performed. routine bone marrow biopsies for histological, immunohistochemical signs of ta-tam and complement activation will be analyzed. serum samples will be used to characterize markers of complement activation using plasma levels of c b- and c b- deposition in tissues biopsies. patients (aml n = ; all n = ; mds/mpn n = ; lymphoid neoplasm n = ; plasma cell disorder n = ; bone marrow failure n = ) underwent myeloablative (n = ) and non-myeloablative (n = ) allo-hsct at a median age of years (range: - years). forty-eight ( . %) patients matched the established diagnostic criteria for tam (increased ldh, platelet count o g/l or o % of normal baseline, schistocytes per high power field, creatinine increase). the median time to onset of tam was days post-transplant (range: - days). subjects with ta-tam had significantly higher -year nonrelapse mortality compared to those without ( . % vs . %, p o . ). grades - agvhd and cytomegalovirus viremia were independent risk factors for ta-tam, and serum ldh level u/l as well as arterial hypertension were early signs of ta-tma occurrence. patients with clinically relevant agvhd (⩾ grade ) had more ta-tam than patients without agvhd ( % vs %; po . ). tam correlated with agvhd severity; the higher the agvhd grade, the more the patients who suffered from tam. allo-hsct recipients with grades - agvhd or cytomegalovirus viremia should be closely monitored for the presence of ta-tma. at the meeting first results of histological, immunohistochemical and complement activation analyses will be presented. disclosure of conflict of interest: none. hemorrhagic cystitis (hc) after stem cell transplantation (sct) can cause significant morbidity and prolonged hospitalization. early bleeding occurs almost exclusively when using cyclophosphamide (cy) ( - % of cases), while late onset hc are classically attributed to bkv infection, and occurs up to % of patients (pts) receiving myeloablative haplo-sct who had positive bk viruria ( , ). we retrospectively studied hc cases among pts submitted to haplo-hsct in our department. thirty-eight pts receiving an haplo-sct with post-transplant cy (pt-cy) were included (table ) . prophylaxis for cy included hyperhydratation ( l/m of . % saline) and mesna administration ( mg for each mg of cy/daily divided into three doses). hematuria was graded as follows: grade i, microscopic; grade ii, macroscopic; grade iii, with clots; and grade iv, leading to urinary retention or requiring surgical intervention ( ). pts with hc and clots were treated with continuous bladder irrigation. twenty-three pts ( . %) developed hc at a median of . days post-sct (range: - ). clinical severity was grade i in cases ( . %), grade ii in cases ( . %), grade iii in cases ( . %) and grade iv in cases ( . %). at the onset of hc diagnosis, bk viruria was investigated in / pts. five pts ( . %) had bkv negative (bkv − ) hc and pts ( . %) bkv positive (bkv+) hc. bkv-hc occurred after a median of days (range: - ) while bkv+ hc after . days (range: - ), respectively (p = . ). among bkv+ pts, received iv cidofovir mg/kg once a week for weeks and then once every weeks. median number of administrations was (range: - ). oral probenecid was given at the dose of g h before and g and h after cidofovir administration. two pts obtained a complete response (cr) after and days, respectively, one patient reached a partial response after days and one pt failed to obtain a response. no pts developed renal toxicity during treatment. one pt received ganciclovir for concurrent cmv viremia and bkv+ hc resolved in days. three patients did not receive any treatment for mild or asymptomatic cystitis. all of them achieved remission after a median of days from the onset (range: - .) among bkv-hc, pts obtained spontaneous resolution after a median of days (range: - ), while two pts died early after sct. finally, among pts for whom bk viruria was not available, a remission was reached in of them after a median of . days (range: - ), while pts died early after sct. in our cohort of pts, hc occurrence was of . % and bkv was responsible for the . % of cases. contrary to its high incidence, hc showed a relative benign course, with an overall remission rate of . %, regardless of treatment. finally, we found a trend for a longer interval between sct and hc onset in pts with bkv+ hc, as compared to cy-related hc (p = . ). sos is a rare and serious complication of hematopoietic stem cell transplantation (hsct). it is diagnosed using the modified baltimore criteria of hyperbilirubinemia, weight gain or ascites % over baseline, hepatomegaly or right upper quadrant pain of liver origin. only defibrotide has been approved for the treatment of veno-oclusive disease. hdmp has been described as effective sos therapy in a few case series ( , ). we describe our experience of treating adult sos using hdmp. objective is to retrospectively analyze the treatment efficacy and overall survival of patients diagnosed with sos after hsct and treated with hdmp. we used vilnius university hospital data base to identify patients diagnosed with sos under baltimore criteria and treated with hdmp over - period. patient demographics, transplant and clinical data, response, survival (kaplan-meier survival analysis) and hdmp infusion related complications were analyzed. we identified patients ( males) of whom had had allogeneic hsct ( reduced intensity conditioning) and one had received a double autologous hsct. sos was diagnosed on the median day + (+ to + days). the median bilirubin value was . μmol/l ( . - μmol/l). all patients had liver enlargement of median mm ( - mm) on ultrasound. two patients had normal bilirubin values but displayed the remaining signs and symptoms of sos at diagnosis. patients received intravenous methylprednisolone mg/m every h for days. none received defibrotide. seven ( %) patients responded on median day + (+ to + days) after the start of hdmp. four responded by decrease in serum bilirubin by % and resolution of symptoms without the need of further treatment. the remaining three responders received maintenance treatment after one course of hdmp with reduced doses of methylprednisolone until resolution of symptoms. four patients failed to respond and died of multiorgan failure on median day + (+ to + ). the median observation time was months ( - months). the median overall survival for the sos group was months (range: - ) and it was months among the responders. no adverse reactions related to hdmp infusion were observed. hdmp therapy in adult sos results in clinically relevant response rate. further prospective trials are required to assess hdmp efficacy in comparison to defibrotide or as add on therapy. prevalence of hypertension (ht) in general pediatric population is~ %, while in children treated with hematopoietic stem cell transplantation (hsct) it is up to %. we assessed factors contributing to the development of ht in children treated with hsct and usefulness of ambulatory blood pressure monitoring (abpm) in this population of patients. the study included children ( boys, girls; mean age . years) treated with hsct for neoplasms (n = ; %) or non-neoplastic disorders (n = ; %). control group included children ( boys, girls; mean age years). abpm measurements (spacelab device) were performed before hsct and after a mean of months after hsct (in of the children). blood samples were collected from children treated with hsct and all controls. total rna extraction was performed and microarray analysis was conducted using genechip human gene . st arrays (affymetrix). in patients after hsct no antihypertensive treatment was used. mean systolic blood pressures (sbp) before and after hsct did not differ significantly from the control group. mean diastolic blood pressures (dbp) before and after hsct were . ± . mm hg and . ± . mm hg, respectively, and mean dbp percentiles were . ± . and . ± . , respectively; the differences between the study group and the control group were significantly higher before hsct. mean -hour arterial pressure (map) percentiles were . ± . and . ± . , respectively; the differences between the study group and the control group were significantly higher before hsct. before hsct and after the procedure, the european society of hypertension criteria for high normal blood pressure (bp) and ht were fulfilled in %/ % patients and %/ % patients, respectively. nocturnal bp decrease o % was found in %/ % patients and % nocturnal bp decrease in %/ % patients, respectively. in the control group o % nocturnal bp decrease was found in % of children and % nocturnal bp decrease in % of children. when the groups of patients before and after hsct were compared, highly significant differences were found in gene expression levels for mthfr ( in children referred for hsct a trend towards higher bp values was seen. in children assessed months after hsct more abnormalities in nocturnal bp measurements were seen, which may be a predictor of ht. in children treated with hsct significant differences in the expression of ht-related genes were found. abpm was useful in bp monitoring in children treated with hsct. hypothyroidism may complicate of allogeneic hematopoietic stem cell transplantation (allo-hsct); risk factors are analysed. we studied patients with aml who underwent an allo-hsct between and with different conditioning regimens (myeloablative, reduced-intensity, chemotherapybased, total body irradiation-based). thyroid stimulating hormone (tsh) and free thyroxin levels (ft ) were available in patients before and after allo-hsct. median age at transplantation (n = ) was years (iqr - ), ( . %) were female and overall mortality was . % (n = ) ( table ) ursodeoxycholic acid (udca) has been shown to have a protective effect in the liver complications after allogeneic stem cell transplantation (allo-sct), but it also has other immunomodulatory effects; it has been described also a potential benefice as graft-versus-host disease (gvhd) protection. we retrospectively analysed patients consecutively transplanted between - excluding second allo-sct and those o years old. we analysed the differences between those with and without prophylactic udca using spps v . results: the median age was years ( - ) and % ( ) were males. other patient characteristics are resumed in table objective of study was to evaluate the impact of disease status on the outcomes of allogeneic hematopoietic stem cell transplantation (allo-hsct) in the treatment of patients with refractory and relapsed acute lymphoblastic leukemia(all). patients with refractory and relapsed all, including cases in advanced stage (nonremission, nr) and cases in more than or equal to second complete remission(⩾ cr ), received allo-hsct after myeloablative conditioning regimen in our department. results: patients engrafted successfully. the transplantation-related mortality (trm) rate of nr and ⩾ cr was . % vs . % (p = . ). the incidence of agvhd was . % vs . % (p = . ), including . % vs . % (p = . ) with mild (grade i-ii) and . % vs . % (p = . ) with severe (grade iii-iv) agvhd. the incidence of cgvhd was similar also( . % vs . %, p = . ). with a median follow-up of ( . - . ) months, the cumulative relapse rate of nr and ⩾ cr was % vs . %(p = . ), respectively. the estimated year overall survival (os) and year leukemia-free survival (lfs) rate were . % vs . % (p = . ) and . % vs . % (p = . ), respectively. multivariate analysis results showed that cgvhd was independent favorable risk factor for os and lfs of r/r all. for relapsed patients, os was significantly better with first cr duration months and time to transplant ⩽ months. alio-hsct is an effective salvage treatment option for patients with refractory and relapsed all. our retrospective analysis showed that r/r all with different status prior transplant had similar outcome post transplantation. disclosure of conflict of interest: none. the deleterious effect of intensive care unit (icu) admission during hematopoietic stem cell transplantation (sct) on patient survival is well established. however, it is unknown whether admission into the icu during the chemotherapy for the underlying disease has any impact on survival after sct. we reviewed patients who had received a first sct between the years and in our institution, and we compared the overall survival (os), relapse incidence (ri) and non-relapse mortality (nrm) between patients who required icu admission during the chemotherapy prior to the sct (icu group) with matched patients ( : ) who did not need it (no-icu group). sixty-six patients were included, of them in the icu group and in the no-icu group. as shown in table , the main clinic-biologic variables and the sct procedure were comparable between the patient groups. the causes of icu admission for the icu group patients were: ( %) respiratory failure, ( %) septic shock, ( %) neurological disturbance, ( %) post-surgery and ( %) tumor lysis syndrome. seventeen patients ( %) needed mechanical ventilation. the median time between icu admission and the sct procedure was days (range: - ), and the median days of icu stay were . . with a median follow-up after sct of . years ( . - . ) for the icu group and . ( . - . ) for the no-icu group, the year os (ic %) probabilities were % ( - %) and % ( - %) in the icu and no-icu patients (p = . ), the -yr probabilities of relapse were % ( - %) and % ( - %)(p = . ) and the -yr probabilities of nrm were % ( - %) and % ( - %)(p = . ), respectively. there were no differences in either os, ri or nrm between icu and no-icu in the allogeneic or autologous subgroups considered separately. at the moment of the study, s ( %) of icu and ( %) of no-icu group had died. the causes of death in the icu group were: relapse in ( %), infection in ( %), gvhd in ( %) and gvhd plus infection in ( %). the causes of death in the no-icu group were: relapse in ( . %), infection in ( . %), gvhd in ( . %), gvhd plus infection in ( . %) and veno-occlusive disease and secondary malignancy, one each ( . %). in this series, admission to the icu before sct did not have an impact on outcomes after sct. these results suggest that these patients benefit from this treatment as much as the other patients, without expecting worse outcomes as a result of a previous icu admission. supported in part by grants rd / / (rticc, fejer), pi / , instituto carlos iii, and sgr (gre), spain. disclosure of conflict of interest: none. autologous stem cell transplant (asct) is a well established treatment for several haematologic and non haematologic malignancies, either as front-line or rescue therapy. however it is associated with toxicity and complications which might lead to treatment-related mortality (trm). although decrease in trm has been reported, data about the precise reduction and detailed analysis of causes of mortality throughout years are scanty. the aim of this study was to evaluate early trm and its causes in patients who underwent an asct in a single center along the last three decades. data of all consecutive adults ( years old) asct recipients were prospectively collected at a single center from december to august and then retrospectively analysed. trm was defined as mortality happened into the days post asct or during conditioning treatment due to any cause except relapse or progression of main diagnosis. demographic characteristics, diagnosis, conditioning regimen and cause of death were analysed. data were compared for two periods: from december to december and from january to august . a total of patients were included, median age was years ( - ) and . % were male. diagnoses were: lymphoma (n = ), multiple myeloma (mm) (n = ), acute myeloid leukaemia (aml) (n = ), amyloidosis (al) (n = ), acute lymphoblastic leukaemia (all) (n = ), solid tumours (including breast cancer and germ-cell tumours) (n = ), chronic myeloid leukemia (cml) (n = ), thrombotic thrombocytopenic purpura (ttp) (n = ) and autoimmune disease (n = ). the most frequent indication for asct was lymphoma ( . %) and mm ( . %). twenty patients died within d from asct (trm). demographic characteristics and causes of death for this patients are shown in table . the cumulative incidence of trm at day + was . % ( % ci . - . ). comparing both periods, trm cumulative incidence was . % ( % ci . - . ) in first period ( - ) vs . % ( % ci . - . ) in last period ( - ) po . . (figure ). according to main diagnosis trm cumulative incidence was higher in patients diagnosed with solid tumour . % ( % ci . - . ) and al . % ( % ci . - . ) followed by acute leukaemia (aml/all) . % ( % ci . - . ), mm . % ( % ci . - . ) and lymphoma . % ( % ci . - . ) p o . (figure ). sepsis ( %) was the main cause of death in both periods of time, and the only one cause of death in the last period. the second cause was sinusoidal obstruction syndrome (sos/vod) ( %), which only appeared in the first period. this study shows a low incidence of trm in asct recipients, with a significant decrease in the last period ( - ), despite the higher risk in some groups of patients such as those with amyloidosis and solid tumours. in our experience, infection is the main cause of early death in asct recipients and sos/vod has disappeared in last years as a cause of early transplant related mortality. disclosure of conflict of interest: none. incidence and risk factors for hepatic sinusoidal obstruction syndrome after allogeneic hematopoietic stem cell transplantation: a retrospective multicenter study of turkish hematology research and education group (threg) hepatic sinusoidal obstruction syndrome (hsos) is a potentially life-threatening complication of allogeneic hematopoietic stem cell transplantation (allo-hsct). the mean incidence of hsos was found to be . % ( - %) in the literature. we examined the incidence and risk factors for hsos after allo-sct. eight centers from turkey were enrolled in the study. we retrospectively evaluated the medical records of patients who were treated with allo-sct between january and december . a baltimore criterion was used for assessment of hsos. two hundred eighty three ( %) of patients who were treated with prophylaxis with defibrotide alone or one or more of the n-acetylcysteine, diuretics and heparin used defibrotide ( - mg/kg/day). the study included patients ( males/ females) with median age of ( - ) years. the demographic and clinical characteristics of patients were summarized in table . the incidence of hsos was . % ( ). prophylaxis for hsos was used in ( . %) of patients, who developed hsos. defibrotide as prophylaxis was received by of ( %) of patients. hsos developed in a median of ( - ) days after stem cell infusion. seventy-five ( . %) of patients who developed hsos had infection at the time of diagnosis. forty-five of them had ascites, had hepatomegaly and, had weight gain. seventy-two ( . %) of patients with hsos were treated with defibrotide after diagnosis. the median time of starting defibrotide in these patients was ( - ) days. thirty-six ( . %) of patients with hsos recovered completely and forty-seven ( . %) of them died as a result of multi organ failure. the incidence of hsosrelated mortality in allo-hsct cohort was found to be . %. in univariate analysis, statistically significant associations were not found between hsos incidence and age/sex of recipient, type of conditioning regimen, stem cell source and type of gvhd prophylaxis. on the other hand primary diagnosis of myelofibrosis, donor type, engraftment status and prophylaxis for hsos were significantly associated with hsos development. hsos prophylaxis was significantly decreased hsosassociated mortality (p = . ). hsos still remains a serious life-threatening complication of allo-sct. although the incidence is low, hsos is associated with increased -day nonrelapse mortality. hsos prophylaxis especially with defibrotide, seems to reduce hsos associated mortality in high risk patients. [p ] disclosure of conflict of interest: none. hemorrhagic cystitis (hc) is a serious complication occurring after allogenic hematopoietic stem cell transplantation (hsct) more frequent on haploidentical (haplo) hsct, with an incidence of - % associated mainly with the effect of cytotoxic agents such as cyclophosphamyde (cy). the conditioning regimen, bkpyv infection and graft versus host disease have an implication in the incidence. other authors related the reactivation of cmv and a previous transplantation as risk factors to hc development . with this study we aim to describe the hc incidence and risk factors in all haplo-hsct performed in the canary islands. we analyzed all consecutive haplo-hsct from family donors performed at our hospital between and . the conditioning regimen used for the transplant was the hopkins haplo protocol with high dose cy ( mg/kg on days and ) posttransplantation (ptcy). we used as hc prophylaxis intense hydratation on the cy administration day and the following h (using bladder wash only in patient with cardiac dysfunction) and perfused mesna at % of cy dose beginning min before the cy administration on pts and at % of the last dose at , and h on all pts. we used spss v. to determine the cumulative incidence (ci) of hc. we performed haplo-hsct, of which were males ( was transplanted times) and were women. the mean age was (range: - ). the pts presented the following diagnosis: aml ( ), all ( ), eh ( ), nhl ( ), am ( ). % of pts received the haplo-hsct in remission, % with refractory disease and % of pts did not receive previous treatment. pts developed hc ( . % ci at day + ) (figure a ) with a median time from haplo-hsct to onset of days (range: - ), ( %) was grade i, ( %) grade ii and ( %) grade iv. the grade i case did not received the mesna infusion like most of the other pts. no pts died due to hc and all cases resolved without sequelae. pts received cy pre-and post-transplant and only pts received ptcy. the ci at day + for the pts with ptcy was . % and for cy preand post-transplant . % (figure b) . we found no statistically significant difference on the ci of hc between these two groups. the development of hc was related to cy in patient, who suffered from this complication on the second and third haplo-hsct. for the rest of the pts (after day + ) the hc was related to bkpyv infection, as a consequence of the immunosuppression state of the patient, we also observed all these pts had positive serum viral load for cmv. the incidence of hc associated to post-hsct high cy dose in our series is % lower than other ones. most of them on grade or and without mortality associated. the risk of hc is high, particularly in the setting of highly pre-treated patients (especially those undergoing a nd transplant). the development of hc after day + is evidently associated to bkpyv as a contributing factor for continuous inflammation and cmv reactivation (as an immunosuppression marker). in our study, hc did not have an impact on mortality of high-risk patients after haplo-hsct. the hc remains frequent with a high morbidity in particular when it is severe, often causing prolonged hospitalization and resource use. we need further studies to recognize the at-risk population early. [p ] ta-tam is not a rare post-transplant complication and it is potentially fatal. in survivors, it was often associated with longterm morbidity and chronic organ damage, mostly to the kidney with poor renal prognosis. our retrospective study showed ta-tam associated risk factors included t reg haplo hsct as the incidence was highest in this group, tbi-based conditioning or tbi based conditioning plus cyclophosphamide. although acute gvhd and infection were associated with ta-tam in retrospective studies, no association emerged between acute gvhd or infection preceding diagnosis in our series of patients. in order to prevent ta-tam we need to understand its underlying biological mechanism so we are investigating its pathogenesis by means of cytokine assays, histology and murine models. disclosure of conflict of interest: none. mortality in children requiring invasive mechanical ventilation (imv) after allogeneic hematopoietic stem cell transplantation (hsct) is known to be high. little is known about the longterm outcome of those who survive imv. we therefore reviewed the medical records of children who survived s imv after they had received a hsct between and in the two pediatric hsct centers in the netherlands. retrospective multi-center cohort study in two university hospitals that perform all pediatric hscts in the netherlands. long-term survival of hsct recipients who had received imv was assessed. health status was reviewed more in detail for those who were still alive years after discharge from the pediatric intensive care unit (picu). in the absence of standardized use of quality of life questionnaires, health status was expressed as the number of affected domains (cardio-respiratory, motor and miscellaneous, regardless of the degree of dysfunction) and level of education. health status was categorized as follows: no health problems when all four domains were normal; mild health problem when there was an abnormal score in one of the four domains; moderate health problems when there was an abnormal score in two domains; severe health problem when there were abnormal scores in three or all four domains. between january and december , patients underwent a hsct in the two study centers together. a total of hsct recipients received imv within year after hsct ( % of all hsct recipients). median time between hsct and picu admission was days (iqr - days). the most common indication to start imv was respiratory failure ( %). median duration of imv was days (iqr - days). patients ( %) died during their picu admission. of the patients who were discharged alive from picu, patients were still alive years after picu discharge ( % of those who survived picu admission). health status of these long-term survivors was assessed in december by hospital database review, using the most recent hospital contact. follow-up time varied from to years (median . years) after picu discharge. two patients ( %) had no health impairment, eight patients ( %) had mild health problems, five patients ( %) had moderate health problems, and nine patients ( %) had severe health related problems. very little is known about long-term mortality and morbidity of hsct recipients who survived imv. survival of picu treatment in pediatric hsct recipients is limited. however, long-term outcome of those who survive picu treatment seems promising: a considerable proportion of them still is alive years later without obvious sequelae. this is the first study which assessed long-term outcome of imv after hsct. further studies in this population are urgently required to counsel parents and to optimize quality of life outcomes in these children. disclosure of conflict of interest: none. long-term surveillance data support lack of increase in mortality or cancer risk in brincidofovir clinical trial participants m morrison, k fitzgerald , t brundage, a harrison and wg nichols chimerix brincidofovir (bcv) is an orally bioavailable lipid conjugate of cidofovir (cdv), with broad-spectrum activity against doublestranded dna viruses, including cytomegalovirus (cmv), adenoviruses (adv), polyomaviruses (bk and jc viruses), and orthopoxviruses. bcv is being evaluated for prevention of cmv and other dna viruses in high-risk hematopoietic cell transplant (hct) recipients, and for the treatment of serious adv infection. bcv is also being developed for the treatment of smallpox under the us fda's animal efficacy rule. because bcv, cdv, and other marketed nucleoside analogs are reported to be carcinogenic in rodents, a registry was established to evaluate the long-term safety of bcv in subjects who have participated in bcv clinical studies. to date, the registry includes prior participants from study (a placebo (pbo)-controlled study of bcv for cmv prevention) and study (a single-arm study of bcv for adv treatment). subjects are encouraged to consent for long-term follow-up in the registry following participation in bcv clinical studies. registry participants are followed at -month intervals for a minimum of years from the time of completion of the parent study. development of malignancies (new or relapsed), lifethreatening or fatal adverse events (aes) assessed as potentially related to bcv, and subjects' vital status are collected. a total of subjects were enrolled in the parent studies ( bcv and pbo from study , bcv from study ). of these, are enrolled in the registry as of october ( bcv and pbo from study , bcv from study ). bcv recipients in the registry are % male, % white, with a median age of ( o - ) years, similar to the bcv recipients in the parent studies. the median duration of follow-up is ( - ) months, with % of subjects continuing in follow-up at the time of analysis. all-cause mortality from the time of first dose in the parent study through current registry follow-up is % for bcv vs % for pbo (p = . ) in study , and % for bcv in study . all-cause mortality in the registry since completion of the parent study is % bcv vs % pbo for subjects from study (p = . ) and % bcv for subjects from study (figure ) . the incidence of a new malignancy in registry subjects from study is % bcv vs % pbo (p = . ), and the incidence of relapsed primary malignancy is % bcv vs % pbo (p = . ). in registry subjects from study , % developed a new malignancy and % had a relapse of the primary malignancy. no bcv-related life-threatening or fatal aes have been reported to date in the registry. registry data collected to date support no increase in late mortality or increased risk for carcinogenicity in patients treated with bcv. long-term surveillance for cancer risk and other drivers of mortality is important for novel compounds undergoing development in hct and other immunocompromised patient populations, with high background risk for these outcomes. [p ] disclosure of conflict of interest: all authors of this abstract are employees and stockholders of chimerix. hematopoietic stem cell transplantation (hsct) is a medical procedure that allows the cure of many paediatric diseases. it has been described an increased risk of new malignancies in this population and it represents an important cause of late mortality. we analyzed the late evolution of patients submitted at pediatric age to hematopoietic transplantation (hsct) (allogeneic or autologous) in santa creu i sant pau hospital between and . a total of hsct was analyzed. it has been calculated the cumulative incidence of secondary malignancies at years of follow-up. it has been done univariate and multivariate analysis of risk factors through χ -test and binary logistic regression method (odds s ratio, or). it has been studied the relative risk (rr) for new malignancies through comparison of observed cases in our cohort with the expected cases in the general population. we observed cases of secondary malignancies with a cumulative incidence of % at years, % at years and % at years of follow-up. the risk was higher of expected in general population for each tumor type and in the different range of age, being the rr for malignancies in our cohort of . at years of follow-up. solid tumors were the most prevalent malignancies ( out of cases). the median time of latency from hsct to diagnosis of malignancy was years ( - years) . the thyroid tumors were the later ones and hematologic malignancies the earliest to be developed. chronic graft versus host disease was a statistically significant risk factor in univariate (or = ; p = . ) and multivariate analysis (or = . ; p = . ). total body irradiation of conditioning was a significant risk factor only in multivariate analysis (or = . ; p = . ). previous radiotherapy was a significant risk factor only in univariate analysis (or = . ; p = . ). mortality was % ( out of ) between patients with a new malignancy and it was the cause of death for all the cases. we observed an incidence of secondary malignancies after hsct of . % that is significantly higher compared to the expected in the general population (p = . ). the factors that have been related to an increased risk were chronic gvhd, tbi and previous radiotherapy. microalbuminuria defined, as urinary albumin: creatinine ratio (acr) of - mg albumin/g creatinine is a marker of endothelial dysfunction and inflammation. in general populations albuminuria predicts development of chronic kidney disease (ckd) and cardiovascular disease ( ). in the general population microalbuminuria is associated with the metabolic syndrome ( ) . in patients with hypertension, diabetes and the critically ill, it is a marker of adverse events and poor outcomes. following hematopoietic cell transplant (hct), microalbuminuria at day was associated with a four-fold increased risk of chronic kidney disease (ckd) at year in a single centre study; macroalbuminuria at day was associated with a . -fold increased risk of non-relapse mortality ( ) . international guidelines for adult and children survivors of hct recommend that proteinuria is assessed at least annually ( , ) . there is a paucity of data on the prevalence and implications of micro and macroalbuminuria in long-term survivors ( years) of adult hct, however. this was a single-centre retrospective study conducted in patients attending a dedicated clinic for long-term (minimum years) survivors of hct. we investigated prevalence of albuminuria and its association with renal disease, cardiac disease and the metabolic syndrome. of patients, were treated for acute leukemia, for aplastic anaemia and for cml. the median follow up time was years (range: - years) and the median age at follow up was years (range: - years). for / urinalysis was normal (group a) and ( %) had microalbuminuria (group b). none had macroalbuminuria. group b were significantly more likely to have ckd grade - (egfr o ) compared to those in group a (p = . ). group b patients were significantly more likely to have diabetes or impaired glucose tolerance / ( %) vs / ( %) in group a (p = . ). group b patients were also significantly more likely to have dyslipidaemia (p = . ) with / ( %) affected vs / ( %) in group a. cardiac disease and hypertension were more frequent in group b, / ( %) and / ( %), respectively vs group a / ( %) and / ( %) but these data were not statistically significant. the more features of the metabolic syndrome present, however, (elevated hba c, /glucose, dyslipidaemia, hypertension) the more likely a patient was to have microalbuminuria (p = . ). our data demonstrates that microalbuminuria is a frequent finding in long term survivors of hct. patients with microalbuminuria are more likely to have ckd grade or below. they are also more likely to have diabetes and dyslipidaemia. as this was a retrospective study we are not in a position to comment on whether microalbuminuria is predictive of the development of renal disease, metabolic syndrome or cardiovascular disease in this group of patients. this warrants further study as intervention, for example with ace inhibitors, may have the potential to reduce morbidity. the purpose of the study is the improvement of transplantation techniques and supportive care lead to an increasing number of long-term survivors after allogeneic hematopoietic stem cell transplantation (ahsct). recipients of ahsct have a higher prevalence of cardiovascular risk factors. ambulatory blood pressure measurement (abpm) is the 'gold standard' to diagnose arterial hypertension (ht). the prevalence and treatment control of ht by abpm is unknown in ahsct patients (pts). this prospective single center study at university hospital basel included all pts ⩾ year after ahsct in complete hematological remission during annual follow-up consultation. office blood pressure (obp) was measured on both arms after minutes rest. abpm by noninvasive continuous bp monitoring (pulse transit time method) was performed on the same day. ht was defined as obp ⩾ / mm hg, mean systolic bp ⩾ mm hg on abpm s (bp ) and/or current use of antihypertensive drugs. pts ( % female) were included with median age of years (range: - ) and years (range: - ) after transplantation. ( %) pts received total body irradiation-based conditioning, ( %) pts had chronic graft-versus-host disease, and ( %) required immunosuppression. mean bmi (kg/m ) (± sd) was ± , with ( %) pts . twenty-seven ( . %) pts were current smokers. fourty-three ( %) pts had chronic kidney disease (egfr o ml/min/ . m ) and ( %) diabetes. ( %) pts were on antihypertensive drugs consisting of ace/at-ii-inhibitors in ( %), calciumchannel blockers in ( %), beta-blockers in ( %) and diuretics in ( %) pts. thirty-nine ( %) pts were on ⩾ drugs. among our cohort ( %) pts were normotensive without antihypertensive treatment (mean age ± years, % female and mean bp (systolic/ diastolic bp) ± / ± mm hg). ( %) pts were hypertensive and/or on antihypertensive treatment. untreated ht was diagnosed in ( %) pts (mean age ± years, % female and mean bp of ± / ± mm hg), including ( %) with white-coat hypertension and ( %) masked hypertension (normal obp, high abpm). in the group of pts with current antihypertensive medication / ( %) were controlled (mean age ± years, % female, and mean bp ± / ± mm hg) whereas / ( %) were hypertensive on abpm (mean age ± years, % female, mean bp ± / ± mm hg). thirty-four ( %) pts with uncontrolled ht were already hypertensive at obp. although long-term survivors after ahsct are known to be at elevated cardiovascular risk, diagnosis of arterial hypertension was missed in every fifth patient. the proportion of controlled hypertension is poor with only %. disclosure of conflict of interest: none. myasthenia gravis (mg) is a rare complication of allogeneic stem cell transplantation (sct) and is often associated with graft-versus-host disease (gvhd). we report a -year-old man who presented with oculobulbar and neck weakness months after an unrelated donor, allogeneic sct for chronic myeloid leukaemia (cml). he was diagnosed in with chronic phase cml. this responded poorly to tyrosine kinase inhibitors (tkis) and he was found to carry the t i mutation with additional monosomy . he underwent a fully hla matched unrelated donor sct with y -anti cd targeted radiotherapy, fludarabine, melphalan and alemtuzumab conditioning. he had grade cutaneous gvhd on ciclosporin withdrawal but no other significant gvhd. he has an immune mediated neutropenia since months post sct and has reduced immune reconstitution as demonstrated by a sub-normal absolute cd level. he remains on pneumocystis prophylaxis and has not experienced increased infection. chemotherapeutic agents have a cytotoxic effect on the oral mucosa and is a major problem following cancer treatment. cooling the oral mucosa in conjunction with chemotherapy infusion, using ice chips, is known to reduce the severity of oral mucositis ( , ) . although effective, ice chips are perceived as uncomfortable. the aim of the present study was to determine the optimal cooling temperature to prevent adverse effect of chemotherapeutic agents using tissue engineered oral mucosal models (teom). teom were incubated at °c, °c, °c or °c for min followed by exposure to μg/ml of -fu for h (control models were incubated at °c). teom were then washed and further incubated for h at °c co . cell viability and inflammatory cytokine production (il- and tnf-α) were measured using (prestoblue) and (elisa), respectively this study demonstrates an increased capacity to restore cell viability with decreasing temperature (figure a ). teom treated with -fu further showed an increased secretion of the pro-inflammatory cytokines tnf-α and il- at all temperatures compared to un-treated controls. for il- , secretion increased markedly when cells were incubated with μg/ml -fu at °c and °c compared to cells incubated with medium alone at °c (figure b) . for tnf-α, secretion was significantly higher (p o . ) in cells treated with μg/ml fu at °c compared to untreated mucosal models and mucosal models treated with μg/ml fu but incubated at °c (figure c ). teom models incubated at °c has an increased capacity to restore cell viability following exposure to -fu. incubation at °c further reduces the release of pro-inflammatory cytokine compared to those incubated at °c. ( ) and one received fludarabine and cyclophosphamide. all patients received campath- h as part of the conditioning regimen. stem cell source: peripheral blood stem cells patients and bm. comorbidity was assessed using the haematopoietic cell transplantation co-morbidity index (hct-ci), with patients ( %) having no co-morbidities, ( %) a co-morbidity index of - and ( %) had a score ⩾ . follow up of survivors ranged from to months (median: months). at the specified end point patients had relapsed ( %) with an actuarial -year relapse rate of %. there were deaths ( %). relapse ( ) was the main cause of death with transplant related mortality of % ( ) at day , % ( ) at months and % ( ) at year. the actuarial os at years was %, with a -year dfs of %. of the surviving relapsed patients all received chemotherapy and donor lymphocyte infusions resulting in effective recovery of remission, showing the utility of this approach. in terms of co-morbidity, actuarial survival rates were % in those with an hct-ci index of , % with an index of - and % with an index ⩾ . the results of this retrospective study indicate that allosct using reduced intensity conditioning regimens can be an effective treatment strategy for older patients with high risk myeloid malignancies including those with significant co-morbidities. relapse remains the main cause of treatment failure and strategies to reduce relapse risk are required. patients that relapse post allosct may respond to further treatment such as azacytidine or intensive chemotherapy and donor lymphocyte infusions. ( ) whether patient-related variables were associated with disagreement. this is a secondary analysis of a cross-sectional multicenter study where patients and clinicians completed an identical qol questionnaire (fact-bmt) at day . clinical and demographic variables as well as anxiety and depression (hads) were collected. agreement was analyzed with the intraclass coefficient correlation (icc). rates of under-and over-estimation were calculated. logistic regression models identified predictors of disagreement. we analyzed pairs of questionnaires, filled in by patients and clinicians. patients' median age was years, ( %) were men, and ( %) received an allogeneic hct. clinicians' median age was years, were men and had worked on the transplant field for a median of years (range: - ). agreement on qol was moderate (icc = ). exploratory analyses revealed that agreement for emotional (icc = ) and social (icc = ) wellbeing was poor, whereas it was moderate for physical (icc = ), functional (icc = ) and bmt concerns (icc = ). patients' wellbeing was overestimated in - % of the categories of wellbeing parameters, and underestimated in - %. patient-related variables explained - % of the variance on disagreement across scales. specifically, anxiety contributed to disagreement in all subscales, except in social wellbeing, where non-significant univariate associations were observed (p . ). type of transplant (allogeneic vs autologous), performance status, and graft-versus-host disease were not associated with disagreement (p . ). patients and clinicians agreement on qol is suboptimal, particularly on emotional and social wellbeing. patients' wellbeing cannot be estimated from other sources than themselves. these results highlight the unmet needs of hct recipients with respect to qol-related issues; an outcome that must be addressed by hct programs since their wellbeing is as important as survival endpoints. disclosure of conflict of interest: none. . we wanted to test the function and the safety of picc device as alternative to standard cvc in patients submitted to autologous stem cell transplantation (abmt). the primary end point of the study was to individuate the cause leading to the failure of picc (its removal or the needing of another cvc during the abmt procedure). secondary end points were the correct function of the device and its praticity. twenty patients submitted to abmt for multiple myeloma ( ) or lymphoma ( ) experienced a double lumen picc device ( ) or a single lumen ( ) if the patient already carried a permanent single lumen cvc such as hickman or port-a-cath. we excluded from this experience patients with high risk of life-threatening situation or high risk of intensive care already before abmt. picc devices were placed from a specialistic nurse team by ultrasound identification of a deep venous vessel in upper arms. melphalan or ceam were the standard conditioning regimens employed in myeloma and lymphoma abmt respectively. we considered a failure all the causes leading to the removal of picc or requiring another cvc before the end of the transplant procedure. at last we collected nurses and clinicians opinions about the picc functionality. no complication has been recorded in positioning phase. / patients maintained the picc device for all the time of transplant procedure. only one patient needed to remove the device for infection. the opinion of nurses and clinicians about the picc device was a significatively slower speed of infusion and resistance to the flow; in fact, / patients needed an infusional pump. the idraulic resistance of the catheter was particular evident against cellular fluids (stem cells suspension, transfusions of blood and platelets). for this reason picc seems to be less indicated in patients requiring many endovenous infusions (nurses' opinion). the rate of infection of picc devices seems to be lower compared to cvc, but the number of cases tested in this experience is too limited for definitive conclusions about it. for other aspects picc is similar to other cvcs. picc seems to be a valid alternative to standard cvc in patients who do not require intensive care, and in particular in patients with low intensity abmt who do not present a high number of endovenous infusions. maybe picc is less burdened of infections respect to normal cvc. this fact, summed to the lower risk during the positioning of the device, leads to consider the use of this device in abmt setting for standard risk patients. disclosure of conflict of interest: none. there are only few algorithms for the selection of hlamismatched unrelated donors, when no fully matched donor is available. indirect recognition of hla-mismatches can be predicted using the model of 'predicted indirectly recognizable hla epitopes' (pirche). the pirche model is a recently developed computer-based strategy, which classifies hladerived epitopes that are potentially presented by patientdonor shared hla-molecules. we performed a multicenter retrospective study evaluating the impact of pirche on outcome after allogeneic stem cell transplantation from hla / matched unrelated donors. the study cohort included adult patients who had undergone allogeneic stem cell transplantation for aml or mds. pirche scores were computed for recipients of hla / matched unrelated donor transplants ( / mud) using a web-based tool. primary endpoint was overall survival at years. patients with a / mud were divided into groups according to the sum of pirche i+ii values (pirche score). eighty-five ( ) patients had a pirche score of (no pirche detected), a pirche score . km estimate of year os was higher for / mud with pirche score = compared to pirche score : % ( % ci: - %) vs % ( % ci - %), p = . . os was similar for / mud with pirche score = and / mud ( % vs %). cox regression analysis revealed poorer os for pirche scores (rr . , % ci: . - . , p = . ). cumulative incidence of nrm at years was lower for / mud with pirche score = compared to pirche score ( % vs %, p = . ). multivariate cox regression analysis revealed poorer nrm for pirche score (rr . , % ci: . - . , p = . ). cumulative incidence of agvhd grade - at months was not significantly different for / mud with pirche score compared to pirche score ( % vs %, p = . ). cumulative incidence of cgvhd at years was lower for / mud with pirche score compared to pirche score ( % vs %, p = . . our findings require confirmation, ideally in a large prospective cohort study. if validated, the pirche model would allow selection of permissible hla-mismatches that may be associated with an improved transplant outcome in terms of reduced nrm and better os. [p ] disclosure of conflict of interest: none. this study was supported by a research grant from pirche-ag to the university medical center, hamburg-eppendorf. pretransplant liver dysfunction has been recognized as a risk factor for complications and mortality after allogeneic hematopoietic cell transplantation (allo-hct). however, there is no consensus on the optimal way to evaluate liver function in hct candidates. transient elastography (te) is a noninvasive method for diagnosing liver damage and cirrhosis. while elastography is widely used in the setting of viral hepatitis, its possible role in allo-hct recipients has not been deeply evaluated. patients receiving allo-hct in our center from may are scheduled to receive pretransplant evaluation by a hepatologist under a prospective protocol. the evaluation includes a hepatologist consultation, liver function and infectious serology tests and te. all patients receive ursodiol from hct admission to day + . this study constitutes the first evaluation of the ongoing protocol for patients receiving their first allo-hct from may to august . sixty patients received a first allo-hct during the study period. sixteen patients did not undergo hepatologist evaluation due to timing issues (n = ), unstable medical condition (n = ) or other reasons (n = ). finally, patients received pretransplant evaluation by a hepatologist under the current protocol and constitute the study population. median age at transplantation was years (range: - ). most patients received a transplant for acute leukemia (n = , %) or non-hodgkin's lymphoma (n = , %) mainly from hla matched unrelated donors (n = , %). thirty-two patients received reduced-toxicity regimens ( %). graft-versus-host disease (gvhd) prophylaxis consisted of tacrolimus in combination with another agent. median follow-up for survivors of months (range: - ). median elastography was . kpa (range: . - . ). considering the hct-ci categories on hepatic dysfunction, , and patients scored , and points, respectively. there were two cases of veno-oclusive disease (vod). overall survival and non-relapse mortality of all patients at median follow-up were % ( % ci - ) and % ( % ci - ), respectively. in the univariate analysis, median elastography was not associated with a higher risk of nrm (p-value = . ), os (p-value = . ) or hepatic chronic gvhd (p-value = . ). the two patients with vod had normal pre-hct transaminase levels and te. this first analysis of an ongoing protocol with universal pre-hct evaluation of hepatic function indicates that increased values of transient elastography are not associated with higher nrm or lower os after the procedure. further studies including a larger number of patients are needed in order to clarify the possible role of elastography in the hct setting. disclosure of conflict of interest: none. allogeneic hematopoetic stem cell transplantation (hsct) remains associated with a high morbidity and mortality in spite of advances in hsct management. specifically, pulmonary complications account for a substantial proportion of deaths within the first days after hsct. therefore, identification of lung dysfunction and additional comorbidities are crucial for preventive strategies in hsct. given the inconsistent association of pretransplant lung function s parameters on mortality after hsct and the significant changes in hsct care over the last decades, the aim of our study was to assess the effect of pulmonary function and comorbid conditions on mortality in patients undergoing hsct for hematological disorders. we retrieved relevant clinical data of all consecutive patients at the hematology division of the basel university hospital with a transplant for hematological disorders between and . we examined the lung function at baseline and , and months after hsct-including the s forced expiratory volume (fev % of predicted), fev /vcmax and diffusing capacity for carbon monoxide (dlco, adjusted for hemoglobin concentration). in addition, we assessed pretransplant conditions such as age, sex, karnofsky performance status (kps), donor type and various risk scores in hsct (hematopoietic cell transplantation comorbidity index (hct-ci), european society for blood and marrow transplantation (ebmt), revised pretransplant assessment of mortality score (pam)). using uni-and multivariate cox proportional-hazards regression analysis, we evaluated patient-and transplant-related risk factors for all-cause mortality by including the following categorical candidate variables: fev (⩾ % vs - % vs o % of predicted), kps ( o % vs ⩾ %), age ( o vs ⩾ years), conditioning intensity and donor type (matched-related vs mismatchedrelated vs matched-unrelated vs mismatched-unrelated). within the study period, patients with predominantly acute leukemia ( %) or lymphoproliferative disorders ( %) underwent myeloablative (n = ) and non-myeloablative hepatic veno-occlusive disease/sinusoidal obstruction syndrome (vod/sos) is a potentially life-threatening complication of conditioning for hematopoietic stem cell transplantations (hsct). recombinant thrombomodulin (rtm) is a new drug for treating disseminated intravascular coagulation (dic) and is an endothelial anticoagulant cofactor that promotes the thrombin-mediated formation of activated protein c (apc). rtm has been used to treat vod/sos, but its ability to prevent vod/sos has not been established. we evaluated the cases of pediatric hematology and oncology patients ( ( %) acute myeloid leukemia, ( %) acute lymphoblastic leukemia, and ( %) neuroblastoma patients, and ( %) patient each with myelodysplastic syndrome, rhabdomyosarcoma, hemophagocytic syndrome (hlh), and wiskott-aldrich syndrome) who underwent hsct at our institution between and and had ≧ risk factors for vod/sos. these risk factors included previous treatment with gemtuzumab ozogamicin (go), receiving hsct, undergoing conditioning with busulfan (bu), and being diagnosed with hlh. the patients who received hsct after (n = ; rtm group) were treated with rtm as a prophylaxis against vod/sos ( u/kg per day for days; from days to ) together with ursodeoxycholic acid (urso) and low-molecular-weight heparin (lmwh), and the others (n = ; control group) were only treated with urso and lmwh. the incidence of vod/sos was evaluated, and various coagulation parameters and markers of endothelial injury (plasminogen activator inhibitor type (pai- ) and apc) were measured in both groups. the patients' median age was (range: - ) years, and ( %) were male. clinical characteristics, including vod/sos risk factors, were wellmatched in both groups. the risk factors possessed by the patients included receiving hsct ( / , %), previous go treatment ( / , %), conditioning with bu ( / , %), and a diagnosis of hlh ( / , %). although vod/sos occurred by post-hsct day + in ( %) patients in the control group, vod/sos was not seen in the rtm group. two of the former patients ( : previous treatment with go, : a diagnosis of hlh) suffered severe vod/sos, and (a diagnosis of hlh) died of the condition. no grade / adverse events involving bleeding or severe organ damage were reported in the rtm group. interestingly, the mean peak value of pai- and apc (markers of endothelial injury) were significantly lower in the rtm group (table ) . [p ] disclosure of conflict of interest: none. protective effect of early human cytomegalovirus reactivation on relapse of myeloproliferative disorders after allogeneic hematopoietic stem cell transplantation z peric , , j wilson , n durakovic , , l desnica , a ostojic , vv rezo , v marekovic , , r serventi-seiwerth and r vrhovac , school of medicine, university of zagreb, zagreb, croatia and university hospital centre, zagreb, zagreb, croatia there have been conflicting results regarding the association between early cytomegalovirus (cmv) reactivation and decreased incidence of relapse after allogeneic hematopoietic stem cell transplantation (allo-hsct). this prompted us to retrospectively evaluate the potential impact of cmv reactivation on transplantation outcomes in a study population of consecutive adult patients who underwent allo-hsct in our institution and were treated and followed in a homogenous manner. patients were monitored for cmv reactivation once weekly for the first days after allo-hsct. monitoring was done with a real time qpcr with lower limit of detection of genome copies per ml of blood. when cmv viremia was detected, all patients were treated with intravenous ganciclovir or oral valganciclovir untill two consecutive negative qpcr assays. univariate and multivariable proportional hazards models using the fine and gray approach were considered to evaluate the variables for relapse, treating death as competing event. between and , male and female patients underwent allo-hsct at a median age of years (range: - ). among them, most patients were treated for myeloid malignancies ( aml, mds and mpn with cml, mf and cmml), while the rest had lymphoproliferative disorders ( all, nhl, mm, mh and cll) and one patient had aplastic anemia. the donors were unrelated in cases, related in patients and haploidentical in patients. most of the patients ( %) received peripheral blood stem cells after a reduced-intensity conditioning regimen ( %). with a median follow-up of months, early cmv reactivation occured in % patients at a median of days after transplantation and did not affect relapse incidence in patients with lymphoproliferative disorders. on the contrary, the cumulative incidence (ci) of hematologic relapse in patients with myeloproliferative disorders (aml and mpn) at months after allo-hsct was % ( % ci, - %) in patients without, opposed to % ( % ci, - %) in patients with cmv reactivation (p = . ). however, cmv reactivation did not significantly affect (p = . ) overall survival between patients with ( %; % ci - %) and without cmv reactivation ( %, % ci - %). a striking and previously unreported correlation between cmv reactivation and relapse was found in patients with mpn; the ci of relapse was % ( % ci, - %) in patients without, opposed to only % ( % ci, - %) in patients with cmv reactivation (p = . ). a substantial and independent reduction of the relapse risk in myeloproliferative disorders (aml+mpn) associated with early cmv reactivation was confirmed by multivariate analysis using time-dependent covariate functions for high-risk disease, use of atg, chronic graft-versus-host disease (hazard ratio . ; % ci, . - . , p = . ), and cmv reactivation (hazard ratio . ; % ci, . - . , p = . ). in summary, this report supports an independent role of cmv reactivation on relapse in patients with myeloproliferative disorders. to our knowledge, we are the first to show a significant reduction of relapse incidence in patients with mpn, even though our findings are based on a relatively small number of patients. however, this putative virus-versus-myeloproliferation effect definitely warrants further research. [p ] disclosure of conflict of interest: none. final result of fact-bmt is score ranged - point (the higher the score, the better qol). for qualitative assessment of donor-recipient relationship, the adult sibling relationship questionnaire (asqr) in polish version was used. the asrq-s consists of items which are spread over eight scales designed to investigate three factors: warmth, conflict and rivalry. the questionnaires were given to both subgroups, donors and recipients of msd-hsct and the results were compared to each other. the overall result of the fact-bmt questionnaire was . ± . points, which means that the examined group generally described their qol as 'quite good'. the best results were found in functional well-being ( . ± . ), while the worst in emotional well-being ( . ± . ) dimension. statistically, the qol score was not influenced by age at hsct (p = . ), current age (p = . ) or gender (p = . ) of the respondents. the recipients scored highest on warm factor ( . ± . ), while donor respondents scored slightly higher rivarly ( . ± . ) than warm ( . ± . ). the second dimension scored by recipients was rivarly ( . ± . ). conflict scores were lowest, although donor respondents scored higher on these than recipient respondents ( . ± . in donors vs . ± . in recipients). statistical analysis revealed that the being a donor or recipient of msd-hsct determines the level of rivarly in the sibling relationship (p = . ) with no impact on warm and conflict dimension. health-related qol in transplanted patients is quite good. sibling donor-recipient relationship is unbalanced with recipient respondents being more likely to assess a warm relationship, while rivalry was more likely to be present among donor. further multicenter studies based on larger cohort of patients are necessary to assess sibling relationship after transplantation life experience. disclosure of conflict of interest: the authors have nothing to disclose. this work was supported by grant from poznan university of medical sciences ( - - - ). rate of re-admission in patients undergoing allogeneic transplants from identical siblings, unrelated donors or haploidentical donors f sora , s sica, l laurenti, p chiusolo, s giammarco, i innocenti, e metafuni, a corbingi and a bacigalupo department of hematology, catholic university of rome hla identical siblings (sib), unrelated (ud) and family hla haploidentical donors (haplo) are currently being used for patients undergoing an allogeneic transplant (hsct) for hematologic disorders. gvhd prophylaxis is usually different, and is commonly based on a calcineurin inhibitor (cni) and methotrexate (mtx) with or without atg for sibs and uds, wheres post-transplant cyclophosphamide (pt-cy)+a cni and mycophenolate (mmf) is used for haplos. we will refer as sib, ud, haplo platform, the combination of a given donor and a given gvhd prophylaxis. the outcome of these three different platforms is usually measured in terms of gvhd, non relapse mortality (nrm) and survival. days of admission and readmissions are important in terms of morbidity, but also of costs, and are usually not reported. aim of the study: assess the duration of the first admission and the incidence of a new re-admissions, in the first days after the transplant. we retrospectively analyzed patients from to . sixtyone received peripheral blood stem cell graft from an ud, and gvhd prophylaxis with cya+mtx+atg; received a peripheral stem cell graft from a sib and gvhd prophylaxis with cya +mtx; patients received bone marrow hsct from haplorelated donor and pt-cy+cya+mtmf. patients characteristics are shown in table . relapses were excluded from the readmission analysis. the median time from the transplantation to discharge was days for ud, for haplo and days for sib: there was no significant difference between haplo vs ud (p = . ), whereas the admission of both haplo and ud was longer than sibs (po . ). first readmission. fiftyone patient out of required of a new admission for complications after tranplant ( out of after mud ( %), out of ( %) using a sibling donor and out of using an haploidentical donor ( %)). there were significantly more re-admissions in the ud vs sib group ( . ) and a trend for more ud readmissions vs haplo (p = . ); siblings had the lowest number of readmissions. time to neutrophil engraftment was comparable in haplo vs ud patients (p = . ) and in sib vs ud (p = . ); the time was longer in haplo vs sibs (p o . ). the reason to re-admitted the patients in the hospital after tranplantation was fever in out of ( %) new admissions in ud setting, out of ( %) in sib and out of ( %) in haplo; acute gvhd was the cause for re-admission in out of ( %) ud, out of ( %) sib and none in haplo. the other causes for re-admission in the hospital were hemorragic cistitis, thoracic or abdominal pain. second re-admission. of hospitalization is registered in out of patients in ud ( for aghvd and fever), out of ( %) in sib ( episodes of fever) and out of ( %) patients in haplo ( for fever and progressive disease). also for second episodes, ud grafts had significantly more admissions compared to haplo and sibs. third re-admission was recorded only in ud patients ( out of - %). this study shows a comparable duration of admission for transplant for haplo and ud patients, both significantly longer than sib grafts. the number of re-admissions is comparable in haplo vs sibs and there is a trend for lower number of re-admission as compared to uds. we interpret this outcomes with caution given the relatively small sample size and heterogeneous disease population included. future studies need to confirme our results. disclosure of conflict of interest: none. prolonged thrombocytopenia (pt) is frequent event after allogeneic haematopoietic stem cell transplantation (hsct), especically in haploidentical transplantation, which could be up to % according to our previous report. pt has significant negative impact on long-term outcomes, mainly due to increased non-relapse mortality. however, there are no efficious treatment. in this study, we report the preliminary results of recombinant human thrombopoietin (rhtpo) in treating this kind of patients. from . to . , patients were enrolled under the following inclusion criterion: ( ) diagnosed with dpe or sfpr after allogeneic stem cell transplantion; ( ) no sign of minimal residual disease or recurrence of hematological malignancy; ( ) not using other tpo receptor agonist or il- within month of enrollment. pt include delayled platelet engraftment (dpe) and secondary failure of platelet recovery (sfpr). the former was defined as failure to achieve platelet counts ⩾ /μl for consecutive without transfusion until days after transplantation, while the latter was defined as a decline in platelet counts below /μl for consecutive days, or requiring transfusion support after achieving sustained counts without transfusions for consecutive days after hsct. the prescription of rhtpo was iu once daily for days, or if patients achieve platelet ⩾ /μl for consecutive days with a duration o days. response was defined as success of achieve platelet counts ⩾ /μl for consecutive days. the response time was defined as the first day achieve response from the start of prescription. the primary end point was response rate, and the secondary end point was reponse time. a total of patients were enrolled, including males and females. the median age was ( - ) years. all patients received haploidentical transplantation. among these patients, patients were dpe and were sfpr. all patients received a -day prescription. the overall response rate was % ( out of ) in the overall population, while % ( out of ) in dpe and . % ( out of ) in sfpr, respectively. among the patients with response, the median response time was ( - ) days from the first dose of rhtpo. after weeks of the last dose of rhtpo, none of the responsed patient lose response. since the followup time is too short, the impact of relapse, gvhd were not reported. this single-arm preliminary result suggest that rhtpo could be a efficious method to manage pt after stem cell transplantation. however, these result need further confirmation. disclosure of conflict of interest: none. reproductive health in long-term female survivors after allogeneic hematopoietic stem cell transplantation z peric , , a samardzic , n durakovic , , d tina , , l desnica , r serventi-seiwerth and r vrhovac , school of medicine, university of zagreb, zagreb, croatia and university hospital centre zagreb, zagreb, croatia most female recipients of allogeneic hematopoietic stem cell transplantation (allo-hsct) suffer from premature menopause, infertility and endocrine imbalance owing to gonadal damage from myeloablative conditioning. in order to evaluate ovarian recovery and long-term endocrine complications in our institution, we performed a retrospective study of female patients who received a myeloablative allo-hsct during their reproductive age. we identified female patients who underwent myeloablative allo-hsct in our institution between and and were still alive with available follow-up at the time of this study. among them, patients accepted to participate and responded to a query designed for this s purpose. the median age of our patients at transplantation was years (range: - years). they were interviewed at a median of years (range: - years) post allo-hsct. the majority of patients were transplanted for a myeloid malignancy ( acute myeloid leukemia, chronic myeloid leukemia, myelodysplastic syndromes and chronic myelofibrosis), while patients had aplastic anemia and had acute lymphoblastic leukemia. all patients received bone marrow transplant from a hla-matched related donor after a myeloablative conditioning. conditioning regimen consisted of cyclophosphamide with or without total body irradiation (tbi) or in combination with busulfan. only patients ( %) resumed a normal menstrual cycle after allo-hsct, without the need for hormonal replacement therapy (hrt). all these patients were transplanted for aplastic anemia and none of them received tbi in the conditioning regimen. eight patients ( %) remained amenorrheic indefinitely and never started hrt, even though most of these women were transplanted under the age of years. % of these patients were diagnosed with osteoporosis later in life. the remaining patients ( %) started hrt at a median of months after allo-hsct (range: - months). however, only seven patients on hrt ( %) resumed regular menstrual cycle. a median duration of hrt therapy was years (range: - years). none of the women receiving long-term hrt had severe cardiovascular complications or breast cancer. finally, five women gave birth to eight healthy children in our study population. three unassisted pregnancies were observed in two female patients after spontaneous recovery of ovarian function (both patients with aplastic anemia). the remaining two patients restored ovarian function with the use of hrt and gave birth after an assisted pregnancy (one woman gave birth to triplets after an in vitro fertilization (ivf), while other became pregnant with a donated oocyte). in spite of the fact that almost all women who undergo allo-hsct develop an ovarian failure, spontaneous recovery is sometimes possible, particularly following conditioning regimen without tbi. in patients without spontaneous recovery, hrt should be initiated promptly to prevent the early and late unwanted effects related to estrogen deficiency. moreover, recovery of normal ovarian function and even a viable pregnancy is a realistic possibility in patients placed on hrt, particularly with the use of potential therapeutic interventions as ivf or oocyte cryopreservation. it is therefore crucial to provide adapted pre-transplant counselling and recommendations for regular post-transplant follow-up in female patients who undergo allo-hsct. disclosure of conflict of interest: none. transplant-associated thrombotic microangiopathy (ta-tma) is a multifactorial disorder caused by systemic vascular endothelial injury leading to end-organ damage often involving the kidney. ta-tma occurs in up to % of patients undergoing hsct, and may be associated with poor outcome. although pathogenesis has not been fully clarified, activation of the complement system has been suggested to play a central role, and eculizumab, a monoclonal antibody (mab) that mediates terminal complement blockade, has shown therapeutic benefit in cases unresponsive to immunosuppression modulation. we report the case of a pediatric allogeneic hsct recipient with severe ta-tma, who did not tolerate treatment with eculizumab, now successfully treated with oms , a novel human mab targeted to the mannan-binding lectin-associated serine protease- (masp- ), a molecule central to the activation of the lectin pathway of complement. a -year-old girl received an allogeneic hsct from a hla-compatible unrelated donor for the treatment of diamond-blackfan anemia. at month + of the posttransplant course, she developed progressive deterioration of renal function, microhematuria and serositis, that prompted the cyclosporine discontinuation. from month + , the patient experienced progressive trilinear cytopenia, elevated ldh, schistocytes, undetectable haptoglobin, hypertension, increased serum creatinine, nephrotic range proteinuria, and serositis, and a diagnosis of ta-tma was established. laboratory investigations documented no abnormalities in the patient but identified a stop-codon heterozygous variant in cfhr c. _ dupaa (p.glu lysfs* ) in the donor's dna. the patient was initially treated with eculizumab, but she developed acute pulmonary edema soon after eculizumab administration as the consequence of a possible reaction to the drug which had to be discontinued. the patient was subsequently treated with plasma exchange, with only limited benefit. upon ta-tma relapse at month + , eculizumab was re-administered at lower doses, but she developed a new episode of acute pulmonary edema, preventing further eculizumab continuation. renal function progressively deteriorated and she was started on hemodialysis, reaching a times weekly regimen. the patient received oms , kindly provided on a compassionate use basis by omeros corporation, seattle, usa, starting with an iv dosing schedule. she did not experience any adverse events, and was able to tolerate the treatment well. at months from oms initiation, she has shown improvement in ldh and haptoglobin levels, and, more importantly, her creatinine levels have normalized, allowing for complete discontinuation of hemodialysis and partial outpatient management. anti-masp- mab oms is a promising new option for the treatment of ta-tma occurring after hsct, and seems to have a safe profile also in the pediatric/adolescent setting. disclosure of conflict of interest: none. severe cytokine release syndrome after t-cell replete haploidentical transplantation with post-transplant cyclophosphamide is associated with increased death rate d taurino , j mariotti , b sarina , l morabito , s bramanti , c carlo-stella , a santoro and l castagna bone marrow unit, humanitas cancer center, istituto clinico humanitas, rozzano, italy and hematology department, humanitas cancer center, istituto clinico humanitas, rozzano, italy haploidentical stem cell transplant (haplo-sct) represents a potential curative strategy for several hematological malignancies. haplo-sct may represent an alternative option when a hla matched-identical sibling (hlaid) or a matched unrelated donor (mud) is not available. the syndrome of systemic inflammation, characterized by fevers, vascular leak, hypotension, and respiratory and renal insufficiency, in the context of elevated inflammatory markers and cytokine levels was previously described as cytokine-release syndrome (crs) . recent publications have elicited the occurrence of crs after haploidentical transplant, especially after peripheral blood stem cell graft, and its high-related mortality - . here we report the experience of our institution with crs after haplo-sct. between march and october , we treated patients with haplo-sct with a graft source represented by peripheral blood stem cells. we monitored the occurrence of crs symptoms and utilize a previously described grading system , starting from day , up to day after transplant. severe crs is defined as grade or higher because it requires aggressive interventions and is characterized by oxygen requirement ⩾ %, l nasal cannula, hypotension requiring high dose or multiple vasopressors, grade renal toxicity or grade transaminitis. other characteristics comprise newonset altered mental status without other explanation and new cardiomyopathy without wall motion abnormality. results: out of patients experienced fever between day and day post transplant with most episodes ( patients) occurring between day and day . on day after transplant, patients had grade , grade and grade crs, respectively. by day post haplo-sct, patients had crs grade , grade and grade . overall, the incidence of crs any grade was % ( % ci - %). year after transplant patients died because of non-relapse related side effects. with a median follow-up for alive subjects of months, -year overall survival (os) was % ( % ci: - %). -year os was % for patients with a crs on day (p = . ). conclusions: crs represent an important complication after haplo-sct. crs score on day after hst apparently correlates with long-term survival. better strategies need to be implemented for an early detection of severe crs in order to develop effective treatments, such as tocilzumab, for this important side effect. further studies are ongoing at our institution in order to correlate post-haplo crs with graft composition, laboratory parameters and immunereconstitution. hematopoietic cell transplantation (hct) is associated with significant morbidity that impairs survivor's sexual functioning. however, few studies have specifically addressed it. thus, we examined ( ) sexual functioning during the first year post hct, ( ) differences between allogeneic and autologous hct, and ( ) whether demographic, clinical and psychological variables were associated with sexual functioning. this is a prospective multicenter study assessing patients before hct, at day , and . sexual functioning was assessed with the changes in sexual functioning questionnaire, which yields a total score, along with scores for the dimensions of frequency, pleasure, orgasm, desire and arousal. anxiety and depression (hads) were also collected. we included consecutive hct recipients: ( %) were men, with a median age of years (range: - ), ( %) received an allogeneic hct and ( %) an autologous hct. sexual functioning was significantly affected: % of the sample reported impairment at pre-hct, % at day , % at day and % at day . mixed model analysis indicated that sexual functioning was not associated with time from hct (p = . ) or hct type (p = . ). however, there was an interaction between these two variables (p = . ), particularly at day , since sexual functioning had improved among autologous survivors and worsened among allogeneic survivors leading to nonsignificant differences between hct type (p = . ). frequency of sexual functioning improved during the study period (po . ), and no differences were observed between hct type (p = . ). again, there was a borderline interaction between post-hct time and hct type (p = . ), since autologous survivors reached higher frequencies than allogeneic survivors, with significant differences at day (p = . ). pleasure significantly improved during the study period (p = . ), without observing differences between hct groups (p = . ). again, however, autologous survivors reported significant improvements in pleasure at day (p o . ) and a trend at day (p = . ) when compared with allogeneic survivors. orgasm did not improve during the study period (p = . ), and no differences were obtained between hct groups (p = . ). allogeneic survivors had higher orgasm scores at pre-hct (p = . ), which worsened during the study period, particularly at day (p = . ). in contrast, autologous survivors reported improvements in orgasm by day . non significant results were obtained in the sphere of sexual desire and arousal (p . ). bivariate analyses indicated that women, older age and depression were associated with impaired sexual functioning at all assessed time-points (p o . ). chronic graft-versus-host disease (gvhd) was associated with worse sexual functioning at day (p = . ) and (p = . ). no differences were obtained when considering diagnosis, having received previous hct, intensity of the conditioning regimen and whether patients lived with a partner (p . ). stepwise multivariate regression analyses indicated that gender (p = . ) and extensive chronic gvhd (p = . ) predicted for worse sexual functioning at day . sexual functioning should be routinely assessed and considered for eventual targeted intervention in both hct populations, particularly during the first year post transplant. additional clinical efforts should focus on patients more vulnerable to impaired sexual functioning. disclosure of conflict of interest: none. significant improvement of qol by using atg as part of the conditioning regimen followed by hla-identical peripheral stem cell transplantation in acute leukemia patients. results from a prospective, randomized phase iii study (atg family study) b francesca , s carlos , w christine , s mariarosaria , p massimo , s carmine , m giuseppe , b wolfgang , cm angelo , p francesca , m nicola cgvhd is a major complication after allogeneic sct. we previously demonstrated that the addition of anti-tlymphocyte globulin (atlg neovii, formerly atg-fresenius) to a myeloablative preparing regimen followed by peripheralblood sct from an hla-identical sibling for pts with acute leukemia resulted in a significant reduction of cgvhd, without increasing the risk of relapse or infection. the study protocol included quality of life (qol) questionnaires (eortc qlq- and hdc ) before and after sct (day+ , , and mos). the qlq-c includes a global qol scale, five functional scales (physical, role, emotional, cognitive and social function) and nine symptom scales (fatigue, nausea-vomiting, pain, dyspnea, insomnia, appetite loss, constipation, diarrhea and financial problems). the qlq-hdc includes six multi-item scales and eight single items that describe impairment through highdose treatment. mixed models for repeated measures (mmrm) and linear mixed models (lmm) were used to analyze the time courses and the slopes of the outcomes depending on treatment arm (atg vs non atg), age, country, sex, and cgvhd. (clinicaltrials.gov: nct ). pts with a qol form returned decreased by visit ( % pre-sct, % at days and % at mos after sct). forty-nine percent in the atg and % in non atg arm provided any qol forms after sct. return of any post-sct qol forms by country was % for germany, % for italy and % for spain. pts with cgvhd were more likely to return qol questionnaires ( % vs % w/out cgvhd) while neither age nor sex were closely associated with qol form return. the majority of subscales of the qlq- indicated an average improvement of qol and reduction of symptoms over time, notably in the atg group. in an mmrm model controlling for country, age, sex and cgvhd, pts treated with atg showed significantly more pronounced improvement of global health status/qol over time compared to non-atlg (p = . ), with a treatment group difference of . ± . points (marginal mean ± sem) at day and increasing to . ± . points at month favoring atg. significant superiority of atg (po . ) was also observed for four of the five functional scales as well as for several symptom scales scores including appetite loss, insomnia, nausea-vomiting and dyspnea. for the qlq-hdc , significant treatment effects favoring atg were observed for gi side effects and impact on family. lmm analyses of qol by country indicate that patients from italy generally gave more favorable ratings for all functional scales and lower scores for most symptom scales than those from germany while the time courses and slopes were similar for most scales. these results underline the importance of the habits and cultural environment which are distinctive of each country. males and females showed similar qol ratings at pre-and post-sct. patients up to years tended to provide more favorable functional ratings and less severe symptom scores than older patients and also showed more pronounced improvements of qol. pts receiving atg in a randomized study have significantly less cgvhd and improved grfs, resulting in an improved qol regarding global health status and most functional scales. notably, we also observed a significant difference in qol assessment between pts from germany and italy. oral mucositis (om) is a well-known side effect of high-dose chemotherapy and radiotherapy in hematological patients, which influences the health-related quality of life (hrqol) of the affected patients. the purpose of this study is to demonstrate the impact of om on hrqol in stem cell transplanted patients in routine care. prospective, noninterventional single-center observational study was performed at a german university hospital. inpatient allogenic and autologous stem cell transplant patients ⩾ years with high-dose chemotherapy. om was assessed with the who oral toxicity scale, pain using the numeric rating scale (nrs) and the performance status with the ecog score. hrqol was captured with the eortc qlq-c and the qlq-oh questionnaires ( days before hematopoietic stem cell transplantation (hsct); days after hsct; days after hsct). statistical significance was assumed p o . . a total of patients ( autologous and allogenic) was included from august to december . a total of ( %) patients developed om. of these patients, suffered from grade , from grade , from grade and from grade om. three days before hsct, the mean qol of all patients was %, the mean qlq-c summary score . % and the mean oral health related quality of life . %. most of the patients suffered from om around day after hsct. after days, quality of life (qol) was higher in patients with no om ( . %) than in patients with om ( . %). the qlq-c summary score was significantly (p = . ) lower in patients affected by om ( . %) than in patients who did not develop an om ( . %). om affected patients had significantly more limitations in emotional (no om . %; om . %; p = . ) and cognitive functioning (no om . %; om . %; p = . ) and in fatigue (no om . %; om %; p = . ), pain (no om . %; om %; p = . ) and insomnia (no om . %; om %; p = . ), they had a significantly higher rate of problems. oral health-related quality of life was significantly (p = . ) lower in patients who were affected by om ( . %) compared to patients who did not develop an om ( . %) and patients with an om had significantly more problems with a sore mouth (no om . %; om . %; p = . ), sticky saliva (no om . %; om . %; p = . ) and sensitive mouth (no om . %; om . %; p = . ). after days, qol was higher in patients with no om ( . %) compared to patients with om ( . %). patients with no development of om had a higher but not significant physical functioning, cognitive functioning and social functioning. patients affected by om had higher levels of fatigue and pain and more often suffered from a sore mouth. oral health-related quality of life was higher in patients without om ( %) compared to patients with om ( . %). comparing all assessed days patients with om had higher scores on the nrs increasing with a higher grade of om (mean nrs score grade ; - . , grade ; - . ), the ecog index was higher in om affected patients during episodes with om (mean ecog score- . ) compared to episodes without om (mean ecog score- . ). om has a major impact on the hrqol, health-related symptoms and functionality. in the future, there has to be a higher awareness from clinicians and patients of the prevention, assessment und causes of om. more research has to be initiated to ease the symptomatology and to improve patients' quality of life. disclosure of conflict of interest: none. according to ebmt data, chronic gvhd (cgvhd) occurs in - % of all patients after allogenic hematopoietic stem cell transplantation (allo-hsct). pulmonary cgvhd is the most severe form. but it is very unpredictable to use due to the fact that many factors can affect it (breath-dependent; need experience not only from physician but from patients also and so on). here we show that routine software-based image analysis algorithm can provide data that highly correlated with pft results and have excellent sensitivity and specificity in pulmonary cgvhd diagnosis. we blindly analyzed ct scans (made without additional expiration) in allo-hsct patients at different time points. all scans were performed on ct scanner aquilion , toshiba, japan. according to hounsfield units (hu) definition, − hu ('air') have approximate density at g/ml; hu ('water') have approximate density at g/ml. the analysis of ct scans (heart, vessels and bronchi were excluded from analysis) was based on automated software conversion (image-analysis algorithm providing by multivox software, msu, moscow, russia) of each ct-image pixel from hu to density units (g/ml). pft were performed using standard procedures at same as ct scans time points (spirolab iii, italy). all patients with hematological malignancies (acute leukemia- , aplastic anemia- , chronic myeloid leukemia- , t-cell lymphoma- , chronic myeloproliferative disorder- , myelodysplastic syndrome- ) were transplanted in national research center for hematology between and . median of age was . years (range: - years). eight patients were males, -females. seventeen received reduced-intensity and -myeloablative conditioning regimen. graft from match unrelated donor (mud) were used in cases, 'mismatch' mud- , match related donor (mrd)- , 'mismatch' mrd- . median follow-up is . months. we analyzed lung tissue experimental density in patients before and after allo-hsct at different time points. median of lung tissue experimental density were . (interquartile range (iqr), . - . ), . (iqr, . - . ) and . (iqr, . - . ) for patients before allo-hsct, after allo-hsct with cgvhd (except pulmonary cgvhd) and with pulmonary cgvhd, respectively. mann-whitney u test was used to reveal significant differences between these groups (see figure ). also, we found strong correlation between pft and experimental density (spearman's correlation coefficient r = . ) (see figure ). forty-five ct scans of patients with pulmonary cgvhd and ct scans of patients without pulmonary cgvhd at the time of ct scan as control subjects were included in roc analysis to assess the clinical values of our model. we generated an roc curve and found that the area under the curve (auc) was . ( % ci, . - . ) (p o . ) (figure ). standard ct scan is presented as easy to perform, breath-independent, standardized and wide spread method for every patient after allo-hsct. it can be performed many times during all their post-hsct life. ct scan with a simple software analysis allows to select a group with high probability of pulmonary cgvhd and who can be suspected of cgvhd development by this method with sensitivity- % and specificity- . %. disclosure of conflict of interest: none. the choice of effectiveness criteria affects conclusions of economic evaluation of newer allogeneic bone marrow transplantation modalities :example based on a randomised multicenter trial comparing two reduced intensity conditioning regimen (flu-bu-atg) vs (flu-tbi) for matched related allo-sct s le corroller*, anne-gaelle , c siani , , r tabrizi a re-evaluation of the per-diem hospitalization cost was performed in and included the utilization of hospital technical facilities and a more precise estimation of overheads costs. we performed three separated cost-effectiveness analysis, using, respectively, pfs, os and qaly as end point. when using pfs as effectiveness, relapse costs were not included. weighting coefficients for the cost per qaly analysis came from the literature. at years, os and pfs were % and %, respectively, and did not statistically differ between groups. the mean total cost per patient was not statistically different between groups ( € for fba vs € for ftbi, ns). using pfs as end point, the icer of fba compared to ftbi is € per year of pfs gained. using os, the icer became non-statistically significant, signifying that when handling uncertainty, no difference in term of cost-effectiveness was observed between fba and ftbi with os as end point. using s qaly, the icer was statistically ns again, showing no advantage in terms of cost per qaly of one conditioning regimen over the other. this result was obtained both considering three weighted health states (dfs, progression and death) and four weighted health states (dfs without gvhd, dfs with gvhd, progression and death) for the qaly calculation. using os and qaly, the two conditioning regimens were not different in terms of cost-effectiveness, while fba may be considered as more cost-effective using pfs as effectiveness criterion. using intermediary end points allows economic evaluation to be available earlier in the life cycle of an innovation. however, it implies strong hypotheses about the predictive value of the pfs over the os. longer period evaluation and qaly may reverse preliminary results. this situation is likely to exist in the hematology setting where alternatives between chances of cure and toxicities of treatment are often observed. research about allogeneic sct modalities is archetypical of such situations and decisions makers should be aware of the necessity of further economic re-evaluation along the development and diffusion process of innovative treatments. disclosure of conflict of interest: none. the impact of corticosteroids prophylaxis for the engraftment syndrome incidence during autologous stem cell transplantation in multiple myeloma and amyloidosis the es is a complication of asct characterized by an inflammatory response during peripheral blood recovery. the standard treatment is based on corticosteroid therapy. the incidence of es after asct increases in chemotherapy lowtreated patients such as those with multiple myeloma (mm) and amyloidosis (al).moreover, the es is associated with the use of g-csf after infusion of stem cells. therefore, our bmt team does not use g-csf since in this population reducing the incidence and severity of es. therefore, it makes sense to use low-dose prednisone to prevent this complication. in this study, we compared two consecutive cohorts of patients with mm/al that performed an asct while evaluating the corticosteroids prophylaxis (cp) in the es incidence and its effect on other clinical variables. we included patients with mm (n = ; %) and al (n = ; %) that performed an asct between january and november in a single institution. the median age (range) was . ( . - . ) years. during the procedure, all patients received melphalan as conditioning chemotherapy and none received g-csf. fortyseven patients ( %) received intravenous methylprednisolone or oral prednisone . mg/kg/day from day + until reaching a neutrophil count ⩾ per mm for consecutive days (cs group), and ( %) patients did not receive corticosteroids (noncs group). the characteristics of patients in both groups (age, gender, status performance and previous treatment were similar (p . )). the cs group, received higher doses of cd + than the noncs group ( . × /kg vs . × /kg, respectively, p = . ). the median (range) days of neutropenia ( o per mm ) was ( - ) days. es was diagnosed in ( %) patients. fifty-seven ( %) patients had fever, showing infectious focus or microbiological isolation in ( %) cases, whereas the incidence of grade iii-iv oral mucositis and relevant gastrointestinal toxicity was % and . %, respectively. the complete analysis between groups (cs versus noncs) for the whole series and in the mm/al subgroups is detailed in table . the administration of corticosteroids as prophylaxis seems to reduce the incidence of es in the overall series or in the analysis for the subgroups (mm and al) without increasing infection. [p ] disclosure of conflict of interest: none. chronic gvhd is a condition that might occur after allo-hsct and has been proved to impair long-term survival and quality of life of patients. graft failure is also a major potential complication for patients undergoing transplant for an aplastic anemia/bone marrow failure (bmf). partial in vivo t-cell depletion, employing anti-thymocyte globulin (atg) during conditioning, has been proved to successfully prevent the mentioned potentially life-threatening complications in highrisk patients. however, the possibility of developing epstein-barr virus (ebv)-induced post-transplant lymphoproliferative disorders (ptlp) has been a limiting factor to use atg. this study includes the last pts with a minimum follow-up of days, who underwent allo-hsct in our center (november -august ). a total of pts were male and female. median age was years (range: - ). baseline diseases were: acute leukemias ( ), lymphoproliferative disorders ( ), myelodysplastic syndromes ( ), chronic myeloproliferative diseases ( ), multiple myeloma ( ) and bone marrow failures ( ) . donor was unrelated in cases, and related in (including haplo-identical). conditioning regimen was: busulphan-based ( ), melphalan-based ( ), tbi-based ( ) and others ( ). progenitors source was pb in and bm in . patient/donor ebv pre-transplant serology was: +/+ in cases, +/ − in and − /+ in . rabbit atg (thymoglobuline) was employed in cases: at . - mg/kg (urd transplants) (low dose), and cases at . mg/kg (all of them pts with bmf) (intermediate dose). family donor (including haplo-identical) transplants of those pts with diagnosis different from bmf ( cases) did not receive atg. systematic monitoring of ebv using quantitative pcr was employed. ebv reactivation was considered when dnaemia was superior to copies per ml. a total of pts presented ebv reactivation: / ( %) in cases without atg, / ( . %) in cases with low-dose atg and / ( %) in cases with intermediate-dose atg. median time of reactivation was the day + (range: + to + ). there was one single case of ebv-induced ptld which belonged to the intermediate-dose atg group. all cases (including the one with ptlp) were successfully treated with rituximab at mg/m /week. median number of doses employed were (range: [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] . mortality due to ebv was % in our series. limited donor availability in the form of either matchedrelated or unrelated donors drew attention to haplo-hct. donors of haplo-hct shares an exact haplotype with the recipient but is mismatched for hla genes on the unshared haplotype. most studies have shown promising results in terms of graft success and survival. in this study our aim is to present the early and late outcome of our haplo-hct patients. between and , we retrospectively evaluated haplo-hct in terms of post-transplant outcome, survival and complications who diagnosed and followed in our center. the median age of patients was (range: - ), ( %) of them were male recipients. the patient characteristics were given in table . thirteen patients ( %) had pre-transplant active disease. neutrophil and platelet engraftment was achieved in patients ( %) at a median day of (range: - ) and (range: - ). eight of patients ( %) died within month after transplant because of sepsis without achieving engraftment. haplo-hct is the second transplant in four of patients ( %): patient relapsed after full-matched related transplant, patient relapsed after / matched unrelated transplant, patient had engraftment failure after full-matched unrelated transplant, patient underwent haplo-hct in another center, followed in remission for years and relapsed. acute graft vs host disase (agvhd) was diagnosed in patients ( %), whereas chronic gvhd in patients ( %). four patients were relapsed ( %) during follow-up with median rfs of months. three patient had bk virus-positive hemorrhagic cystitis ( %). the distribution of infections is shown in figure, viral infections were detected later than fungal and bacterial infections. previous history of invasive pulmoner aspergillosis was detected in of the patients ( %) ( of them were re-transplanted) and received secondary prophylaxis. overall survival (os) of months and year were % and %, respectively. the choice between alternative graft sources depends on the urgency of the transplant on each institutional preference. higher complication and infection rates in addition to decreased survival compared with previous studies since our patient population consisted of refractory patients with comorbidities. preferable patient profiles undergoing haplo-hct may have better outcomes. disclosure of conflict of interest: none. the third month risk factor score: detection of disease at day + of allogeneic stem cell transplantation is the most important risk factor of worse prognosis m celis , c fernández , l yáñez , , a bermúdez , , a insunza , m colorado , m lópez-duarte , i romón , s garcía-Ávila , a cabero , a casado , m sánchez-escamilla , c richard and e conde , hematology department, hospital universitario marqués de valdecilla and university of cantabria before allogeneic stem cell transplant (sct), several index can provide prognostic information (ebmt risk score and hcti score). however, there is scarce data for the impact of the procedure during the first days of transplant, in which opportunistic infections and the acute graft versus host disease (gvhd) can induce harmful effects. our purpose is to create a risk factor score, measured at day + post sct, to give information about the prognosis of the patient. we retrospectively analyzed seven clinical (disease, fungal and cmv infection, acute gvhd, treatment with corticosteroids, karnosfsky status and length of hospitalization) and eight analytical (related to immune status, liver and lung function, nutritional status, iron overload and platelet count) risk factors in patients who underwent sct in our center between and and were alive at day + . data were collected as categorical variables and compared by χ -test. significant variables (p o . ) were evaluated in a multivariate logistic regression model. those who maintained statistical significance were then assigned a point value calculated with their β-coefficient. summation of the points resulted in a weighted risk score. median age was years (range: - ) and were males ( . %). the most frequent disease was aml, patients ( . %). the conditioning regimen was myeloablative in patients ( %) and bone marrow was the principal stem cell source ( %). donor was mrd in ( . %), mud in ( . %) and mmd in ( . %). the median followup was months (range: - ). the univariant model identified five prognostic variables: detection of disease by molecular, cytogenetic or flow cytometry asses in leukemias, myelodisplastic syndrome and multiple myeloma or image (ct scan ± pet) in lymphoma, dose of corticosteroids ⩾ . mg/kg/ day, ferritin ng/ml, albumin o . g/dl and platelet o per mm . table shows variables evaluated. in the multivariate model, the detection of disease (hr . , % ci . - . , p ng/ml (hr . , % ci . - . , p = . ), and platelet o per mm (hr . , % ci . - . , p = . ) were associated with higher risk of death and according with their-coefficient , and points were, respectively, assigned. the third month risk score (tmrs) was calculated in all patients and they were stratified into three groups: low risk of death (a, - points), intermediate risk (b, points) and high risk (c, ⩾ points). at years post sct, the estimated overall survival according with the tmrs was . % ± . in group a, . % ± . in group b and . % ± . in group c, po . . although the harmful effect of the first months of transplant can impact in the survival, the detection of disease at day + is the most determinant risk factor of death. this fact gives us the need of transplant in the best response and, in those who cannot, to plan promptly rescue strategies. the next objective is to confirm our risk score in a validation group. disclosure of conflict of interest: none. recombinant human soluble thrombomoduline alpha (rhtm) is a novel anticoagulant agent and approved for disseminated intravascular coagulation in japan. the aim of the study is to evaluate the therapeutic potential of rhtm for sinusoidal obstructive syndrome/hepatic veno-occlusive disease (sos/ vod). we retrospectively studied times of allogeneic hematopoietic cell transplantation in toranomon hospital from june to june . we extracted the patients who used rhtm for dic and satisfied the diagnostic criteria of sos/ vod around the same time, because the use of rhtm for sos/ vod alone is off-label. data on the patients who used rhtm for days within days after transplantation were analyzed. the patients who were already treated with rhtm before the emergence of the first symptom or sign of sos/vod, and who started rhtm over days after the emergence of the first symptom or sign of sos/vod, were excluded from the [p ] analysis. to diagnose classical sos/vod (⩽ days after transplantation), we used two classical criteria of the modified seattle and the baltimore. for late-onset sos/vod ( day of transplantation), we used the criteria of ebmt. we defined as severe sos/vod, if the patients had renal (cr ⩾ times of baseline), respiratory (spo ⩽ % or the need for positive pressure) or central nervous system failure until weeks after the diagnosis of sos/vod. complete response (cr) was defined as the resolution of all the symptoms and the signs in sos/vod diagnostic criteria. a total of patients were extracted. the median age was years (range: - ) and patients ( %) was male. donor cell sources were ucb (n = ) and ubm (n = ). most of the prophylaxis regimen was the combination of ursodeoxycholic acid and dalteparin in patients ( %). classical sos/vod was diagnosed in ( %) and patients ( %) by the criteria of the modified seattle and the baltimore at the median day of (range: - ) and (range: - ), respectively. twenty-eight patients ( %) were diagnosed as late-onset sos/vod at the median day of (range: - ). severe sos/vod developed in patients ( %) (renal, n = ; respiratory, n = ; central nervous system, n = ). the elevation of transaminase was observed in patients ( %). the median interval from the emergence of the first symptom or signs of sos/vod to rhtm administration was days (range: - ). the median duration of rhtm use was days (range: - ). rhtm was used alone in patients ( %), in combination with dalteparin in ( %), with atiii in ( %), with dalteparin and atiii in ( %), with atiii and pge in ( %), and with pge in ( %). corticosteroid was used concomitantly in patients ( %). finally, patients achieved cr of sos/vod. the cumulative incidence of cr of sos/vod was . % at year after the administration of rhtm ( % confidence interval, . - . %). the median interval from the administration of rhtm to cr of sos/vod was days (range: - ). at year after transplantation, overall survival was . % ( % confidence interval, . - . %). from the administration of rhtm to weeks after the cessation of rhtm, hemorrhagic adverse events were observed. seven out of events were at grade - , and out of events were fatal (intra-abdominal in , gastrointestinal in , lung in and brain in ). we concluded that rhtm had a therapeutic potential for sos/vod. disclosure of conflict of interest: none. thrombopoietin receptor agonists for delayed and prolonged clinically-relevant severe thrombocytopenia after allogeneic hematopoietic stem cell transplantation v bosch vilaseca , i garcía cadenas , e roldán , s novelli , r martino , p barba , a esquirol, l díaz polo , g orti , d valcárcel and j sierra hematology department, hospital de sant pau, barcelona, spain and hematology department, hospital de la vall d'hebron, barcelona, spain persistent thrombocytopenia is a common complication after allogeneic stem cell transplantation (allosct), which dramatically increases the patients' dependence on hospital-based healthcare. thrombopoietin receptor agonists (tpoa) increase platelet counts in other clinical settings; however, the experience regarding their use after allosct is limited. we retrospectively evaluated tpoa efficacy in consecutive adult allosct recipients who received tpoa as a compassionate use for severe thrombocytopenia post-engraftment. five patients ( %) had primary and prolonged failure of platelet recovery, while had secondary thrombocytopenia: in seven of these cases, gvhd and/or a viral infection were the 'attributed' cause, while three were classified as post-allosct itp. all patients were dependent on platelet transfusions (median: times per week, range - ), with severe bleeding episodes in nine cases ( %) before tpoa onset. tpoa was started at a median of days after allosct (range: - ). romiplostim was used in ( %) cases. the median starting dose was μg/kg once a week (range: - μg/kg), while the final dose identified as most beneficial was μg/kg (range - μg/kg). eltrombopag was used in cases ( %), with an initial dose of mg daily; while the final doses were and mg daily. overall, / patients responded to tpoa therapy (defined as a stable platelet recovery to ⩾ /μl without transfusion support). the -day cumulative incidence of successful platelet recovery to ⩾ /μl and ⩾ /μl was % ( % ci, - %) and % ( % ci, - %), respectively, which were reached at a median of and days from start of therapy. five of the patients ( %) with severe bleeding at onset responded to tpoa ( of them without further hemorrhages) at a median of days (range: - ). at a median follow-up of days from start of therapy, three patients who responded continue tpoa treatment, while four other responders were able to discontinue it without recurrence of thrombocytopenia. among these patients, s the median total duration of treatment was days (range: - ). one patient lost his response within months after tpoa onset when he developed thrombotic microangiopathy associated with progressive gvhd. the remaining responder experienced disease relapse on day + after allosct. among the non-responders, had leukemia relapse during tpoa treatment, switched from romiplostim to eltrombopag without success and the remaining cases had active severe infections at tpoa onset ( hemorrhagic cystitis and cmv colitis) or non-controllable intestinal bleeding due to progressive gvhd. tpoa were well tolerated, with only patients showing adverse events (grade liver toxicity and grade fatigue), which did not lead to any change in therapy. six patients ( %) underwent follow-up bone marrow biopsies that did not display any increase in marrow fibrosis, including the patient who had myelofibrosis prior to allosct. although six patients in the study had active gvhd when tpoa was started, no patients showed worsening of gvhd. our results support the safety and efficacy of tpoa for the treatment of persistent thrombocytopenia in allosct recipients. further studies should compare the efficacy of romiplostim and eltrombopag and identify surrogate clinical and laboratory variables that are predictive of response to one (or both) of these tpoa. disclosure of conflict of interest: none. . clinical response in both groups was defined as improvement of organ function (no neurological residues; normalization of kidney function) and independence of red blood cell and platelet transfusions. results: the median time of ta-tma onset was . months ( . - . ) after hsct. thirty-five of patients ( %) were under treatment with calcineurin-inhibitors or sirolimus at the time the ta-tma occurred. in all cases, the immunosuppressive drug was stopped promptly. in patients, classical treatment was the primary therapy with a response rate of % (including four patients who switched to ec), whereas the response rate to ec treatment was significantly higher with % (p = . ). all patients receiving ec showed sufficient blockade of the terminal complement pathway after the second ec application (ch o %). despite the increased response rate for ec therapy, there was no difference seen between these two groups according to overall survival in weeks: classical treatment ( % ci - . ) vs ec treatment . ( % ci . - . ) p = . . the main cause of death differed significantly between this two treatment approaches with a therapy-related mortality due to infection with % in the ec group during tma therapy and none seen in the classical treatment group (p = . ). progressive gvhd was identified as an adverse prognostic factor in both groups (p = . and p = . ). conclusion: in our analysis, we show that ec shows a significant higher response rate in severe ta-tma patients compared to the classical treatment approach. however, in both groups the outcome remained very poor. since most patients presented with advanced, severe ta-tma, especially in the ec group, we hypothesize that earlier diagnosis and treatment of ta-tma and more effective prevention and treatment of infections will improve the outcome of patients with this complication. however, randomized studies are essential for comparison of these two treatment strategies to identify patient groups that benefit from a treatment with ec. disclosure of conflict of interest: none. tocilizumab as an effective treatment in cytokine release syndrome as an early peri-transplant complications in patients subjected to allogeneic stem cell transplantationproinfammatory/autoimmune patient/donor hla haplotype life-threatening early allogeneic hsct complication risk factor hypothesis m-g patrycja , , p-j beata , , s marcin , k ksenia , s-k agnieszka and sb aleksander , bone marrow transplantation unit, department of haematology, krakow university hospital and jagiellonian university collegium medicum cytokine release syndrome (crs) is classical complication of car t cells therapy, but also can be connected with early peritransplant complications in patients subjected to allogeneic stem cell transplantation. it can be connected with atg infusion, but also with inflammatory response during periengraftmetnt period (pre-engraftment syndrome and engraftment syndrome) and septic infections. severity of these complication can differ depending on patient's performance status and therapeutic options from just observation and vigilance to mechanical ventilation need. we would like to present small patient series (n = ) subjected to msd (n = ) and mud (n = ) with early transplant-related complications treated with combination of steroids (dexamethason) and tocilizumab. in two of them, tocilizumab was used after second dose of atg. both patients present hypotonia with decreased urine output, prompt increase of creatinine level and presence of acute inflammatory parameters crp, beta microglonulin and procalcitonin level, fluid retention and decreased oxygen saturation. in another one patient, these symptoms were connected with pbsc infusion from unrelated donor. in later two patients, we observe almost the same clinical presentation in preengraftment phase. in every of patients infection was ruled out-blood cultures were negative. all these patients were treated with tocilizumab in a single dose of mg/kg. in all patients, we observed prompt response-normalization of clinical state, renal function, oxygen saturation and decrease of inflammatory factors-crp, procalcitonin and beta microglobuline. discussion: crs is a rare complication connected with early phase of allogeneic stem cell transplantation. there were no results of treatment with steroids, reduction of a dose of cyclosporine a according to decreased renal function, but all patient completely/fully recovered after single dose tocilizumab treatment. all our patients were subjected to reduced intensity protocols, what might be a risk factor to develop crs because non complete depletion of the patient origin monocytes/macrophages active population. we also analyzed other factor connected with crs in early peritransplant period finding possible connection with s proinflammatory hla phenotype. it was obvious in the patient one our patients with peri-engraftment phase crs-he was diagnosed previously with rheumatoid arthritis b pos, dr . in three of five, we have found sle predisposition in hla phenotype (drb * /dqb * or drb * / dqb * ), in later one-ra associated hla antigen drb .these patients were analyzed correlating with historical cohort of additional five patients with mortal and another three with very severe early peri-transplant complications and in all we have found the same 'sle or ra hla phenotype'. because small number of analyzed patients and documented high frequency of these haplotype in population, this is still an opened question is proinflammatory/autoimmune hla phenotype connected pathogenically with predisposition to develop severe transplant complications and are we able to treat all these patients with combination of steroids with tocilizumab. further analysis is needed. disclosure of conflict of interest: none. transplant-associated thrombotic microangiopathy (ta-tma) is a severe complication post haematopoietic cell transplantation (hct) leading to high mortality rates. however, outstanding questions regarding its diagnosis, pathophysiology and treatment remain in the literature. recent studies suggest evidence of complement activation, implicating that complement inhibition may be an effective alternative treatment strategy in refractory patients. therefore, we hypothesized that increased complement activation can be detected in ta-tma patients using a functional assay, the modified ham test. we enrolled consecutive patients with ta-tma according to the international working group criteria from january to june . as controls, we studied patients with graft-versushost-disease (gvhd). complement activation was detected using the modified ham test, a cell proliferation assay based on the susceptibility of a pnh-like cell line to complement activated serum. normal human serum was used as a negative control and lipopolysaccharides(lps)-incubated normal serum as a positive control. all samples were tested in triplicates and twice. we studied ta-tma patients transplanted from unrelated / matched ( ) or / mis-matched ( ) donors, identical ( ) and haploidentical ( ) siblings. all patients presented severe acute and/or chronic gvhd. ta-tma presented at median + ( - ) day post-transplant. in the control group, we studied two patients with steroidsensitive grii and two with steroid-refractory griv acute gvhd. we were able to detect significantly increased complement activation in the serum of ta-tma compared to gvhd patients (p = . ). based on previous studies and present controls, percentage of non-viable cells higher than % was considered a positive modified ham test, indicating increased complement activation in four ta-tma patients. regarding treatment outcomes, two patients with a negative modified ham test responded to cyclosporine cessation and steroid administration. plasma infusion with/without plasma exchange was initiated in seven patients. however, only three of them responded to second-line treatment. the modified ham test result was significantly increased in refractory patients (p = . ). the terminal complement inhibitor eculizumab was administered in one refractory patient with a positive modified ham test and renal failure at presentation. despite delayed initiation ( days post ta-tma diagnosis), response was observed after three doses of eculizumab including evidence of reduced hemolysis, schistocytosis and transfusion needs. however, the patient succumbed to complications of end-stage renal disease ( days post ta-tma diagnosis). among ta-tma patients, succumbed at a median + ( - ) day to transplant-associated complications, related to gvhd and infections from multi-resistant pathogens. ta-tma is associated with increased morbidity, mortality and severe complications, including gvhd. unlike gvhd, increased complement activation was observed in a significant portion of ta-tma patients. complement inhibition seems an encouraging therapeutic option in these patients. given the lack of robust functional assays for complement activation, the modified ham test may be useful for early recognition of patients that would benefit from complement inhibition. . this proposal includes, along with the 'classical sos' (cases diagnosed before day + ), the new type 'late onset sos' (cases diagnosed afterwards). new ebmt criteria for severy grading classify cases of sos into four grades (mild, moderate, severe, and very severe). the aim of this retrospective study is to analyze the cases of severe/very severe, both classical and late onset sos, occurred in our unit during the most recent period of time. we studied the last pts, with a minimum follow-up of days, who underwent allo-hsct in our center (november -august ). pts were male and female. median age was years (range: - ). baseline diseases were: acute leukemias ( ), lymphoproliferative disorders ( ), myelodysplastic syndromes ( ), chronic myeloproliferative diseases ( ), multiple myeloma ( ), and bone marrow failures ( ) . donor was unrelated in cases, and related in (including haplo-identical). conditioning regimen was: busulphan-based ( ), melphalan-based ( ), tbi-based ( ) , and others ( ). all patient received prophylactic [p ] s ursodeoxycholic acid. progenitors source was pb in , and bm in . five patients developed severe/very severe sos ( % incidence); were classical (at days + , + and + ), and were late onset (at days + and + ) (see table ). four cases had received conditioning with a busulphan (iv)-based regimen (doses from . to . mg/kg), and one case with tbi plus cyclofosfamide at high doses. all cases presented with right upper quadrant pain, jaundice, ascites, weight gain, hiperbilirrubinemia, and renal function impairment. all but one had increased transaminases. the five cases were treated with defibrotide, in spite of which all of them died. considering that overall day + mortality was %, severe/ very severe sos was the most important cause of death of the series. [p ] although milder forms of sos might resolves within weeks, the most severe forms are still associated with a very high mortality rate. prophylaxis with defibrotide (the drug currently licensed for treatment) for high-risk patients has not been sufficiently studied yet. therefore, a high index of suspicion, early detection and early therapy are the only ways to try to reduce mortality due to sos in the hsct setting. disclosure of conflict of interest: this research has been performed entirely with public financial support. the royal marsden hospital, sutton, uk; anthony nolan research institute, london, uk and university college london, london, uk secondary poor graft function (spgf) complicates up to % allogeneic hcts, and is associated with increased mortality and poor quality of life due to recurrent infections and the need for ongoing blood product support. potential interventions include a second allograft using further conditioning, however many patients with spgf have a reduced performance status and are at an increased risk of complications from this procedure. unconditioned haematopoeitic progenitor cell (hpc) top-ups are associated with a high risk of gvhd if unmanipulated cellular products are used. cd + selection offers an attractive alternative, but incurs a loss of up to % hpcs and is an expensive procedure, unavailable to many centers internationally. alemtuzumab, a monoclonal anti-cd antibody, is routinely used in allogeneic transplant conditioning in the uk to prevent gvhd. we report the results of a retrospective study examining the efficacy of alemtuzumab conditioned hpc top-ups for spgf. data pertaining to patients who had undergone a second infusion of hpcs from their original donor were identified from our hospital-specific promise database. those who met the criteria of spgf defined as ⩾ of hemoglobin × /l without support. patients ( pediatric, adult) who underwent initial allogeneic transplants for malignancy ( ) or bone marrow failure ( ) received an alemtuzumab conditioned hpc top-up for spgf at our center - . the diagnosis of spgf was made at a median . months post allograft (range - ) with trilineage cytopenias in patients and bilineage cytopenias in patients. all patients had received transplants from / ( patients) or / ( patients) matched unrelated donors. the median interval between initial transplant and top-up was days (range - ), and a median cd dose of . × /kg recipient weight (range . - . ) was infused. % patients achieved haematological improvement (hi) at a median days post-top-up (range - ), with the only failure to achieve hi seen in the patient who had received the lowest cd dose ( . × /kg). one patient developed grade i agvhd post top-up but no grade ii-iv agvhd was observed. year os was % and year os % following hpc top-up. deaths occurred due to infection at , and months post top-up, and one due to relapse of a prior non-haematological malignancy. patients had an aplastic or hypocellular bm trephine pre-top up, which was repeated at days post topup in patients, of whom had a normocellular bm trephine, while remained hypocellular. alemtuzumab conditioned hpc top-up appears an effective intervention for spgf with results comparable to those of cd selected top-ups, and therefore represents a feasible alternative. larger studies are needed to exclude complications including viral reactivation and to investigate immune reconstitution following this procedure. disclosure of conflict of interest: none. high dose chemotherapy (hdt) followed by autologous stem cell transplantation (asct) has shown to improve outcome in patients with relapsed/refractory diffuse large b cell lymphoma (dlbcl). in the rituximab era, the benefit of asct has been debatable as prior study (coral study) has shown that patients who received r-chop as induction chemotherapy & responded to salvage chemotherapy had a poorer outcome following asct compared with those who received chop alone. in addition, it remains unclear whether addition of rituximab to standard high dose beam regimen provides any additional benefit. we retrospectively analyzed dlbcl patients receiving high dose beam (n = ) or rituximab +beam (r-beam) (n = ) followed by asct for relapsed/ refractory dlbcl since . all patients who received chop (n = ) ± rituximab (n = ) as first line therapy and who received ⩽ lines of salvage chemotherapy before asct were analyzed. rituximab was given at the dose of mg/m on day + and + of asct. twenty-two ( %) patients in beam group and all the patients ( %) in r-beam group received rituximab-based salvage chemotherapy prior to asct. the year overall survival (os) was % and event-free survival (os) was % for the whole cohort. r-chop induced patients did not fare any worst after asct than chop induced patients ( year os vs %; p = . ). there was a trend towards better survival in patients with pre-transplant disease free interval (dfi) months compared to those with dfi /μl) time was days and days, respectively. median platelet recovery ( /μl) time was days and days, respectively (p = . ). ten year os ( % r-beam vs % s beam, p = . ) and efs ( % r-beam vs % beam, p = . ) were also comparable between both groups. hdt with beam and asct remains beneficial for patients with relapsed/ refractory dlbcl. it should be offered to all patients who respond to salvage chemotherapy with the expectation that they fare no worse than patients who do not receive rituximab in the induction chemotherapy. addition of rituximab following the standard beam for hdt and asct does not compromise haematopoietic recovery, but does not result in improved outcome in our study. prior use of rituximab during first-line or salvage therapy in most of the patients of r-beam group might have negated the beneficial effect of r-beam over beam. ( ) . in this study, we aimed to develop a cns targeted chemotherapy regimen, which has lower toxicity and higher complete remission rates, in combination therapy. eight patients with secondary cns lymphoma (scnsl) and two with primary cns lymphoma (pcnsl), followed between the years and , were included in the study, retrospectively. the patients were histologically diagnosed with biopsy and underwent autologous stem cell transplantation (apkht). all patients were treated with r-idaram/ rt (radiotherapy)/subsequently autologous stem cell transplantation (apsct) with r-beam protocol. the r-idaram regime consists of the following substances: rituximab mg/m , cc/h infusion, day ; cytosine arabinoside . gr/m i.v., h infusion, days and ; dexamethasone mg, h infusion, days , and ; idarubicin mg/m i.v., min infusion, days and ; methotrexate gr/m ( gr/m at years old-patients), h infusion, day ; and cytosine arabinoside mg plus methotrexate mg, intrathecally, days and . the patients included seven males and three females. the median age was years (range: - ). six scnsl patients were diagnosed in the application and two of them were diagnosed during r-chop chemotherapy (ct) protocol. five patients ( %) were stage ivb, and the others ( %) were stage iiib at diagnosis. after two or three chemotherapy cycles, patients were mobilized with growth factor support and median . cells per kg (range: - ) stem cells were collected. then, at a dose of - cgy cranial rt was administered for days. after the third cycle of r/idaram, the state of remission was evaluated by cranial mri and lumbar puncture (lp). all patients achieved complete remission. neutrophil engraftment occurred at a median of days (range: - ) and platelet engraftment occurred at a median days (range: - ). after apkht, three patients relapsed and died at the fourth, ninth, and thirteenth months. grade i-ii manageable neurological toxicity occurred in two patients. the median follow-up time was (range: - ) months. the five-year overallsurvival (os) was %. serious signs of infection were not observed in patients during transplantation. in pcnsl and scnsl, a standard treatment regimen has not yet been found. apsct with r-beam following modified r/idaram/rt is a curative and applicable therapeutic regimen with low toxicity, which can provide high rates of long-term survival and disease-free survival. despite the advent of novel therapies, autologous hematopoietic stem cell transplantation (ahsct) following melphalan (m)-based conditioning remains the standard of care for patients with multiple myeloma who are eligible. still, the majority of patients experience disease progression and ultimately succumb to their disease. we hypothesize that integrating novel agents in the conditioning is feasible and safe and may increase complete remission rates and overall survival. we completed a phase i, dose escalation study of carfilzomib (c) added to a backbone of bendamustine (b) and melphalan. all patients received a fixed dose ( mg/m ) of c on days (d) − , − , − , − , − and − . in addition, patients were conditioned as described in table . due to dose-limiting toxicity in cohort , the study was amended after the first patients. subsequently, the dose of m was reduced to mg/m and the d + dose of c was omitted, per oversight of a data safety monitoring board. fifteen patients were enrolled, males and females. median age was years ( - ). performance status was ⩾ % (kps) in all patients. per the international staging system (iss), patients had stage i disease, had stage ii, had stage iii, and had unknown staging. three patients had high-risk cytogenetics: with t( ; ) and with deletion p. four patients had undergone a prior ahsct. disease status at enrollment was stable disease (sd) (n = ), partial response (pr) (n = ), or very good partial response (vgpr) (n = ). median cd + cell dose infused was . × /kg ( . − . × ). median follow-up was . months ( . - . ). all fifteen patients are evaluable s for engraftment. median time to neutrophil engraftment was d ( - ). one patient died before achieving platelet engraftment. for the remaining patients, median time to platelet engraftment was d ( - ) . non-hematologic toxicities included grade acute mucositis (n = ), lower gi complications (n = ), electrolyte disturbances (n = ), transaminase elevation (n = ) renal insufficiency (n = ), atrial fibrillation (n = ), hypoxia (n = ), prolongation of the qtc interval (n = ), and grade acute sepsis (n = ), including death (cohort ) on d + . eight patients went on to receive maintenance therapy: with bortezomib, with lenalidomide, and with lenalidomide, dexamethasone, and c. posttransplant disease status was assessed per protocol by spep, spif, serum free light chains, and light chain ratio. twelve patients were evaluable on d + . two patients had sd, had vgpr, and had complete response (cr). eight patients were evaluable on d + . two patients had progressive disease, had pr, had vgpr, and had cr. the combination of cbm prior to ahsct appears feasible, with manageable toxicities, at the doses described in cohort b. a prolonged follow-up and a phase ii study are warranted to determine response rates and long-term outcomes. disclosure of conflict of interest: none. beam (carmustine, etoposide, cytarabine, melphalan) is the most frequently used high-dose chemotherapy regimen for patients with lymphoma referred for autologous hematopoietic cell transplantation (autohct). in recent years a novel conditioning protocol containing bendamustine instead of carmustine (beeam) has been proposed in order to potentially increase the efficacy. so far, however data on its safety are limited. the aim of this study was to retrospectively compare the safety profile of beam and beeam based on single center experience. consecutive patients with lymphoma treated with beam and patients treated with beeam between year and were included in the analysis. the median age was ( - ) years and ( - ) years, respectively (p = ns). clinical characteristics of both groups were comparable. patients with hodgkin's lymphoma constituted % in the beam group and % in the beeam. among those with non-hodgkin lymphoma the diagnosis of dlbcl predominated. beam treatment consisted of carmustine mg/m on day − , etoposide mg/m /d on days − to − , cytarabine mg/m /d on days − to − , and melphalan mg/m on day − . in the beeam regimen carmustine was substituted by bendamustine administered on days − , − at the total dose of mg/m i.v. peripheral blood was used as a source of stem cells. cd + cell dose was . ( . - . ) × /kg in the beam group and . ( − . ) × /kg in the beeam group (p = ns). time to engraftment and the rates of adverse events up to day + after autohct were the study endpoints. all patients engrafted in both study groups. median time to neutrophil . × recovery was ( - ) days after beam and ( - ) days after beeam (p = . ). median time to achieve platelet count × was ( - ) days and ( - ) days, respectively (p = . ). two patients died without progression before day + in the beam group, both due to bacterial infections. no early deaths were reported in the beeam group. the rates of grade or adverse events were comparable (see: table ). administration of bendamustine instead of carmustin as part of conditioning does not affect engraftment as well as toxicity profile of the regimen. therefore beeam may be safely used in patients with lymphoma undergoing autohct. its efficacy requires evaluation in prospective studies focused on homogenous patient populations. [p ] disclosure of conflict of interest: none. the baltimore group reported a low dose tbi-based nonmyeloablative conditioning regimen followed by t cell replete bone marrow, with post-transplantation cyclophosphamide (pt-cy) to control gvhd and graft rejection. based on the fact that in our facility conventional low dose tbi was not available, we wanted to explore whether tmi/tli could be a potential substitute the aims of our study was to explore if tmi/tli can be considered an effective substitute of tbi in terms of os, pfs and nrm. retrospective analysis was applied in cases of haploidentical hsct from april to october . all patients underwent baltimore conditioning associating fludarabine ( mg/m /day) day − to − , cy ( . mg/kg/day) on days − and − , and tbi gy in patients and tmi/tli gy in patient at day − . unmanipulated bone marrow graft was infused at day . postgrafting immunosuppression consisted of cy ( mg/kg/day) on day + and + , and mycophenolate mofetil for days, and tacrolimus or cyclosporine. no differences between the two groups was observed in term of age, gender diagnosis, disease status and donor type. % of patients engrafted in both arm ( / and / ). in tbi cohort vs tmi/tli cohort, the median time to anc /μl and platelet recovery /μl was not different ( and days vs and . days, p = . and . , respectively). in all tmi/tli evaluable patients, full chimerism was observed at days + . after a median followup of months in tmi/tli cohort and months in tbi arm, -year nrm was . % and . % (p = . ), respectively. the years os and pfs were not statistically different in the two groups % vs . %, p = . and . % vs . %, p . , respectively). the -year relapse incidence was % in tmi/tli group and . % in tbi group, p = . . no difference in incidence of both agvhd and cgvhd was observed between the two groups. this retrospective analysis suggests that tmi/ tli could be considered an effective substitute of low dose tbi, with a sufficient degree of immunesuppression of recipient, allowing engraftment and full chimerism. the gvhd both acute and chronic as well as the -y nrm were not different. disclosure of conflict of interest: none. comparison of the beeam conditioning regimen and the beam conditioning regimen in the autologous transplantation for hl and nhl s lozenov , p ganeva , y petrov , g arnaudov and g mihaylov the beam has established itself as a standard of care conditioning regimen in the autologous lymphoma hsct setting for most transplant centres in europe. yet however various other regimens are being compared with it in order to achieved better safety profile, better os and dfs, in order to improve results with chemoresistant and unfavourable patients. one such regimen is beeam (bendamustine, etoposide, cytarabine, melphalan).we aimed to compare the efficacy of the beam and beeam conditioning regimens and to compare their myelotoxicity profile. we evaluated retrospectively adult patients (mean age . with sd . ), receiving auto-hsct at the national specialized hospital for active treatment of hematological diseases in sofia, bulgaria for relapsed/refractory hl or nhl (of them mh - , dlbcl - , pmbcl - , fl - , lbl - , ptcl-nos - , aitl - , alcl - , mcl - , mzl - ) for the period from . . to . . with a follow-up of patients up to . . . ninety-two of the patients received the beam (as previously described -bcnu mg/m i.v. day − , etoposide mg/m i.v. days − to − , cytarabine mg/m i.v. days − to − , and melphalan mg/m i.v. day − ) regimen and received beeam regimen (bendamustine on days − and − ( mg/m ); cytarabine, mg/m intravenously daily, from day − to day − ; etoposide, mg/m intravenously daily, from day − to day − ; and melphalan, mg/m intravenously on day − ). the overall survival at the second and third years of follow-up (os- , os- ) and dfs at the third year, the cr rates and the average time periods to hematological recovery, were compared. the os at and years, respectively, was . % and . %, for beeam and . % and % for beam, the dfs at years was . % for beeam and . % for beam, provided that the differences did not have statistical significance (p . for os and p . for the dfs). the cr rate was . % in the beeam group versus % in the beam group. from the patients who received autologous hsct in stable disease or progression pre-transplant status (chemoresistnat patients), . % of the patients receiving beeam achieved cr at the first post-transplant evaluation versus . % respectively for the beam group. the mean time to hematological recovery for neutrophils was . ± . days (beeam) versus . ± . days (beam) and . ± . days (beeam) versus . ± . days (beam) for platelets. beeam appears to be a non-inferior alternative conditioning regimen to the standard beam, it shows a trend towards higher myelotoxicity, but also a trend towards better response rates in chemoresistant patients. [p ] disclosure of conflict of interest: none. autologous hematopoietic stem cell transplantation (asct) is widely used as a consolidation therapy in aggressive non-hodgkin's lymphoma (nhl) and recurrent or refractory classic hodgkin's lymphoma (hl). in mexico, the use of carmustine (bcnu) in the conditioning regimen of these patients is limited due to the lack of access to the drug and its high costs. this study aims to compare results in terms of toxicity, disease-free and overall survival between a group of patients treated with the standard regimen beam and another group treated with a scheme in which carmustine was replaced by cisplatin (peam regimen). a comparison of two groups with lymphoma was performed and the clinical aspects of cisplatin mg/m d . the characteristics were well balanced between the two groups. the mean time for neutrophil grafting ( per mm ) was significantly slower with beam than with peam ( vs days, p = . ), hospitalization time was longer with beam compared to peam ( vs , p = . ). on the other hand, proportion of patients who require red blood cell s transfusion was significantly higher in beam group ( %) versus peam group ( %) (po . ), but total amount of platelet transfusion did not differ between groups. about the toxicity, beam patients had significantly more frequent incidence and severity of nausea/vomiting ( % vs . %) and diarrhea ( . % vs %) compared to peam (p o . ). no significantly differences were observed in incidence of mucositis (p = . ). at the moment of the analyses, % of patient of the peam group were in complete response versus % of the patients treated with beam, but it did not represent a significant difference. disease-free survival and -year overall survival in the peam vs beam scheme were similar with % vs % (p = . ) and % vs % (p = . ) respectively but with less toxicity using the peam scheme. peam regiment is not inferior scheme compared with beam, because it shows similar outcomes in disease-free survival and overall survival. additionally, peam is a well-tolerated regime and beam scheme was associated with greater gastrointestinal toxicity such as nausea, vomiting and diarrhea, also greater hematology toxicity such as more requirement of red blood cell transfusion. [p ] disclosure of conflict of interest: none. cumulative busulfan exposure is associated with relapse following busulfan and cyclophosphamide myeloablative allogeneic stem cell transplantation for acute myeloid leukaemia e wong, d kliman , m chau , j szer , c nath , p shaw , d ritchie , d gottlieb and a bajel westmead hospital, new south wales, australia and royal melbourne hospital, victoria, australia the optimal busulfan exposure to reduce disease relapse in adult patients with acute myeloid leukaemia (aml) undergoing busulfan/cyclophosphamide myeloablative allogeneic stem cell transplant (allosct) is poorly defined. we retrospectively analysed busulphan pharmacokinetics (pk) and outcomes of patients who underwent busulfan/cyclophosphamide conditioned allosct for aml from to . busulfan was administered intravenously over days ( . mg/ kg/d for days followed by . mg/kg for days). peripheral blood was obtained for busulfan pk after the first dose. subsequent doses of busulfan were decreased if daily busulfan exposure (area under the curve; auc) was anticipated to exceed μm per min/day. cyclophosphamide was dosed at mg/kg. the primary outcome was the cumulative incidence of relapse (cir) accounting for non-relapse mortality (nrm) as a competing risk. independent variables analysed included age, sex, cytogenetic risk group, disease risk index (dri), donor type, stem cell source, t-cell depletion, and cumulative busulfan auc (cumauc) calculated as previously described. (figure ) . t-cell depletion was also associated with increased cir (hr . ; p = . ). patient age, sex, cytogenetic risk, dri and graft type were not significantly associated with cir. on multivariate analysis, cumauc μm per min remained significantly and independently associated with lower cir (hr . ; p = . ). cumauc was not associated with nrm, rfs, os, or the incidence of acute or chronic gvhd. figure . cumulative incidence of relapse in patients stratified by total busulfan exposure. [p ] cumulative busulfan exposure μm per min is independently associated with reduced relapse following busulfan/cyclophosphamide allosct for adults with aml. these findings support further evaluation of the optimal busulfan exposure to reduce aml relapse in a prospective clinical trial, whereby patients could be randomised to target cumauc μmol per min versus standard practice. hematopoietic stem cell transplant with busulfan and cyclophosphamide (bucy) based conditioning has a relatively high incidence of liver toxicity and sinusoidal obstruction syndrome (sos). busulfan and cyclophosphamide metabolites share the same glutathione conjugation in the liver metabolism. a small number of studies addressed different sequence of both drugs bucy vs cybu during conditioning. differences in liver toxicity, sos, transplant related mortality (trm), relapse incidence (ri) and overall survival (os) were reported favoring cybu conditioning. we decided to address the above issues at the umc ljubljana, slovenia. this was a retrospective study following patients with myeloid malignancies (aml, mds, mpn) with bucy (n = ) and cybu (n = ) conditioning through a three year period in a single institution. primary endpoint was detecting difference in liver toxicity by measuring levels of liver enzymes. secondary endpoints were incidence of sos, difference in trm, ri and os. patients characteristics between groups at the time of the transplant did not differ significantly. we observed significantly higher liver toxicity through elevated bilirubin and alt in the bucy . % than cybu . % patient group (picture ). the highest probability of liver toxicity was around d in the bucy group and in the second week after the transplant in the cybu group. the incidence of sos, trm and ri were comparable between the groups. there was no difference in os between the patient groups during the -month follow-up. bucy conditioning for hematopoietic stem cell transplant causes higher incidence of liver toxicity compared to cybu conditioning. there is no difference in sos frequency, trm, ri and os between bucy and cybu conditioning. prospective controlled comparison would be needed for further study of the subject. disclosure of conflict of interest: none. early monocyte recovery is associated with better overall survival after busulfan containing myeloablative conditioning allogeneic hematopoietic cell transplantation in patients with acute myeloid leukemia a lojko-dankowska the outcomes of allogeneic hematopoietic cell transplantation (allohct) in acute myeloid leukemia (aml) depend on different patient-, disease-and transplant related factors, including the dose and combination of agents used for conditioning. the aim of the study was to analyze the outcomes of allohct in patients with intermediate or high risk aml according to disease risk index (dri) who received myeloablative conditioning consisted of intravenous busulfan ( . - . mg/kg) combined with cyclophosphamide ( mg/ kg) or fludarabine ( mg/m ) between and in our institution. the published data indicate that the combination of busulfan (bu) and fludarabine (flu) seems to have more favorable toxicity profile than combination of bu and cyclophosphamide (cy), so bucy regimen has been substituted with buflu as the myeloablative conditioning for aml patients in our institution practice since . we evaluated the influence of type of regimen on transplant outcomes along with the impact of other potential prognostic factors, including age of patient, dri, donor type, hla and gender mismatches, stem cells source, and lymphocyte and monocyte recovery. the study group consisted of aml patients, median age years (range: - ), classified as intermediate (n = ) or high (n = ) risk according to the dri, who were conditioned with bucy (n = ) or buflu (n = ) followed by allohct from hla identical sibling (n = ) or - / matched unrelated donor (n = ). the stem cell were collected from peripheral blood (n = ) or bone marrow (n = ). gvhd prophylaxis consisted of calcineurin inhibitor combined with mtx plus atg in allohct from unrelated donors. engraftment was observed in all patients. the median time to neutrophil count ( . g/l) and platelet count ( g/l) recovery was shorter after buflu in comparison with bucy ( days vs days; p = . and days vs days; p o . ), however peripheral blood stem cells were used more often after buflu regimen than after bucy ( % vs %, p /mm on+ day after transplant ( -year os % vs %, p = . ) and intermediate vs high dri ( -year os % vs %, p = . ). in multivariate analysis higher amc after allohct remained the only independent favorable prognostic factor for os (rr . ( % ci . - . ), p = . ). our results suggest that early monocyte recovery after myeloablative bu containing conditioning allohct is significant favorable predictor of outcome. in our experience both bucy and buflu myeloablative regimens result in similar long-term survival after allohct in aml patients. [p ] disclosure of conflict of interest: none. the use of t-cell depletion as part of the conditioning protocol has the potential to improve the tolerability of allogeneic stem cell transplantation (hsct) through the reduction in graft versus host disease (gvhd). despite the wide spread adoption of this practice in many parts of the uk and europe, definitive recommendations regarding the most appropriate dose remain elusive. previous experience by our group with mg of alemtuzumab combined with fludarabine and busulfan based conditioning demonstrated good long-term outcomes with low rates of gvhd. however, due to concerns of high relapse risk especially in patients with high-risk myelodsypastic syndrome and acute myeloid leukaemia, we instituted a policy change in to reduce the dose of alemtuzumab in the conditioning protocol from a total of - mg. we conducted a retrospective analysis of all consecutive patients undergoing reduced intensity unrelated allogenic stem cell transplantation with fludarabine ( mg/m ), busulfan ( . mg/kg iv or . mg/kg iv) and alemtuzumab (fb c or fb c, respectively) conditioning for neoplastic myeloid disorders between and . patients were subsequently analysed in two cohorts; those receiving mg of alemtuzumab (n = ) and those receiving mg of alemtuzumab (n = ). apart from a decreased proportion of females in the mg alemtuzumab group, the cohort was balanced across the different dose levels ( table ). the longterm overall survival (os) of the entire cohort was good with a year os of %. no significant differences in overall outcomes across the two groups were observed with a year os of % in the mg group vs % in the mg group (p = . ). cumulative incidence of relapse (cir) and nonrelapse mortality (nrm) was % and % and % and % in the mg and mg groups, respectively. interestingly, age had a significant effect on nrm in the mg ( % age o , % age - and % age p = . ), but not in the mg group ( % age o , % age - and % age p = . ). the effect on relapse rate was not significant in either group (p = . and p = . , respectively). this retrospective analysis did not demonstrate an overall improvement in transplant outcomes with dose de-escalation of alemtuzumab from to mg. in particular, we did not see the anticipated improvement in relapse rate in this cohort. notably older patients seem to tolerate the mg dose better due to the lower nrm. prospective trials with accompanying translational work are required to determine the optimal dosing and schedule for this group of patients. disclosure of conflict of interest: none. bendamustine was given at mg/m /d for the first pts then mg/m /d for the subsequent pts and finally at mg/m /d for the remaining pts ( pts). among the beam group, % had non-hodgkin's lymphoma (nhl) and % hodgkin's lymphoma (hl) compared to % and %, respectively, in the beeam group (p = . ). hhv- detection was performed by pcr for symptomatic pts (fever, rash or prolonged cytopenia). patients were housed in single bedrooms with air filtration and received the same supportive care. median age was ( - ) and ( - ) in the beam and beeam groups respectively and median of previous chemotherapy regimens was (range: - ). fifty two out of patients were male ( / in the beam group and / in the beeam group). pts were in cr ( . % vs . %) or pr ( . % vs . %) at time of transplant. there was no difference in terms of hematologic recovery (median = days (range: - )), blood and platelets transfusion, mucositis toxicity. there was no statistical difference in the incidence of acute renal failure when comparing the two groups. however, there was a very striking difference when considering the highest dose of bendamustine when compared as well to the two others doses of bendamustine (po . ) as to the beam group (p = . ). additionally, we also observed a high incidence of symptomatic hhv- infections ( . % vs . %, p o . ), digestive toxicity ( . % vs %, p = . ) and a longer hospitalization duration ( days (range: - ) vs days (range: - ), p = . ) for patients in the beeam group overall. with a median follow up of . and . months for beam and beeam respectively, overall survival ( % vs %), transplant related mortality ( % vs %) and event free survival ( % vs %) were comparable. overall, beeam regimen was associated with longer duration of hospitalization, higher rate of digestive toxicity and increased risk of symptomatic hhv- infection as compared to the beam regimen. in addition, higher doses of bendamustine ( mg/m /d for two consecutive days) were associated with unacceptable high rate of acute renal toxicity. with a still short follow-up, the absence of benefit on disease control together with higher short term toxicity does not allow to recommend the use of beam instead of classical beam. should it be used, we suggest that pts should be carefully monitored for renal toxicity and for hhv- infection in case of symptoms. disclosure of conflict of interest: none. high-dose treosulfan and melphalan for consolidation therapy in high-risk ewing sarcoma me abate, a paioli, a longhi, m cesari, e palmerini and s ferrari musculoskeletal department, rizzoli orthopaedic institutes, bologna, italy common toxicities observed after high dose chemotherapy with busulfan and melphalan for high risk ewing sarcoma (es) are generally well managed by current supportive care but some patients can develop severe complications. treosulfan is an alkylating agent that has recently been used as a substitute of busulfan to prevent potential serious complications related to busulfan. medical records of es patients undergoing autologous peripheral blood stem cell (apbsc) transplantation after intravenous treosulfan (treo) and melphalan (mel) from / / to / / were analyzed with regard to toxicity and outcome. patients were included into the study if they were eligible for the protocols activated in our institution for es and presented reasons that did predict potential complications related to busulfan, such as previous radiotherapy on axial skeleton/pelvis or coexistence of high risk of epilepsy. as consolidation treatment patients received intravenous treo g/m over days and mel mg/sqm with support of apbsc transplant and use of granulocyte colony stimulating factor. in those patients with lung metastases total lung irradiation was performed at least months after treomel. frequency of toxicity for treomel was recorded with at least months of follow-up and was evaluated according to nci ctg common toxicity criteria. the median age at diagnosis of patients receiving treomel was years (range - years), males and females. patients had localized disease at diagnosis with poor radiological or histological response to standard chemotherapy; one patient had lung metastases at diagnosis and one patient had relapsed disease with lung metastases. before receiving treomel the primitive tumour underwent radiation therapy in cases ( pelvis, cervical vertebra, sacrum), surgical resection in one case(tibia) and surgical resection plus radiation therapy in one case (fibula). patients showed eeg abnormalities at high risk of developing epilepsy. the median number of infused cd + cells was . × /kg (range . - . ). febrile neutropenia occurred in / patients and lasted one day in patients and days in patients. median time to granulocyte engraftment was days (range - days); median time to platelet engraftment was days (range - days). only one patient needed red blood cells transfusions; patients needed platelet transfusion and patients needed platelet transfusions. none developed grade - stomatitis or grade - [p ] hematuria or grade - liver toxicity. surprisingly, a patient became pregnant after year and months from transplantation. with a median follow-up of months (range - months) patients are alive in complete remission, one patient is alive with relapsed disease and one patient died for disease progression. these results, related to a limited cohort of patients, confirm the lower toxicity observed for treosulfan with respect to busulfan. although more data are needed to clarify the role of treosulfan in es, the impact of potential severe complications observed with busulfan, including infertility, should suggest its replacement with treosulfan in selected cases. disclosure of conflict of interest: none. immunoadsorption procedures prior to haploidentical allogeneic pbsct could prevent graft failure in patients with hematological malignancies displaying anti-donorspecific hla antibodies donor-specific anti-hla antibodies (dsa) have been shown to be associated with a high risk of rejection in solid organ transplantation and with graft failure (gf) in allogeneic hematopoietic stem cell transplantation. a combination of anti-cd (rituximab), plasma exchange (pe), and ivig in patients with additional buffy coat infusion in among them prevented graft loss in all patients that became c q negative before sct. we addressed the question whether immunoadsorption in combination with rituximab can also be applied in patients with dsa to prevent graft failure in haploidentical pbsct. four patients with acute myelocytic leukemia or myeloma in second complete remission were enrolled. the presence of dsa was determined by luminex at pre bmt checking. immunoadsorption was performed with polyclonal sheep anti-human igg adsorbers (miltenyi biotec gmbh, germany) on life apheresis system. in addition all patients received rituximab mg/kg bw in a single dose. patients were conditioned with a reduced intensity regimen comprising tbi gy, cyclophosphamide mg/kg, and fludarabin mg/m . all patients received cyclophosphamide post bmt (ptcy) mg/kg. non-t-cell depleted pbsct were transfused in a sequential manner in doses each. the data of the patients and treatments is summarized in table . two patients had a normal hematopoietic reconstitution and are alive at + and + months post-transplantation, one with hepatic gvhd; chimerism was % in peripheral blood on last follow up. one patient died following a graft failure. by a combination of rituximab and repeated immunoadsorption prior to allogeneic pbsct the titer of dsa could be lowered sufficiently to enable engraftment. ia turned out to be a safe procedure without relevant clinical side effects. hematopoietic reconstitution was in the normal range in of evaluable patients. disclosure of conflict of interest: none. allogeneic hematopoietic stem cell transplantation (hsct) is the only curative option for patients with beta thalassemia major. however, the availability of hla-matched related donor remains the main obstacle for allogenic hsct. although, a few studies have been reported, experience with hla matched unrelated donors is limited. we present the result of children with beta thalassemia major who received allogeneic hsct from hlamatched unrelated donors with using a novel conditioning regimen. we retrospectively assessed unrelated hsct in children with beta thalassemia major. all patients received busulphan (bu) based myeloablative conditioning regimen. busulphan was used according to weight adjusted doses. in addition, all patients received fludarabine mg/m in days, cylophosphamide mg/kg in days, thiotepa mg/kg in one day and atg mg/kg in days. cyclosporin-a and mtx were used for graft versus host disease (gvhd) prophylaxis. donor chimerism was evaluated in the peripheral blood on days + , + and + . the median age of the patients was . years (range month- year). two of the patients were grouped in class i and rest of them were class ii. the median serum ferritin level was . ng/ml (range, - ). all of s the donors were matched / with high-resolution hla typing in gvhd direction but three of them / with graft failure direction. twenty-three of them received bm (median tnc: . x /kg) and pbsc (median mnc: . x /kg) with median cd + cell number . x /kg. the median neutrophil and platelet engraftment days were and days in pbsc and and days in bm group, respectively. grade i-iv acute gvhd was observed in patients ( %) and only one experienced limited chronic gvhd with only skin involvement. mild to moderate vod was seen in patients ( %) and treated with defibrotide successfully. all patients except one are alive with full donor chimerism (between - %) with a median months (range - months) follow-up. one patient died because of cmv pneumonia. these data show that the results of hsct from unrelated donors in selected low risk thalassemia patients may be comparable to hsct of matched sibling donors. however, it needs further studies with long term follow up and larger study population. disclosure of conflict of interest: none. table . data about cytogenetic risk of group patients were available only in individuals. differences between groups were analyzed by t-student and chi square tests. survival was analyzed by kaplan-meier method and differences in survival between groups were evaluated by log rank test. no differences were found between groups regarding gender, sc source, disease status at sct, type of donor and number of cd + cells infused. patients in group were significantly older (median age for groups and : vs , p = . ). gvhd prophylaxis protocols included atg in a higher frequency in group . no differences between groups and were observed in neutrophils recovery (median days to anc /μl: vs respectively, p = . ) and platelets recovery (median days to platelets /μl: vs respectively, p = . ). patients in group required more red cell transfusions (median packed rbc: vs , p = . ). no differences were observed regarding platelets transfusion requirements or length of hospitalization. post-sct os was significantly better in group ( years-os group : %; group : %; p = . ) (figure ). there were no significantly differences between groups regarding frequency of mucositis, diffuse alveolar haemorrage, sepsis, acute and chronic gvhd. vod was more frequent in group ( / vs / , p = . ). trm mortality was higher in group ( / vs / ), being this difference no statistically significant (p = . ). as it was reported by others, the use of fludarabine-based conditioning regimen was associated with a significantly better post-sct os and a reduced frequency of vod in aml patients. reduction in trm and differences in the frequency of described complications are not statistically significant probably due to the small size of this sample. since march , given the limited availability of melphalan, we administer the beac regimen (carmustine, etoposide, cytarabine, and cyclophosphamide), instead of the gold standard conditioning regimen beam, followed by autologous haematopoietic cell transplantation (ahct) in relapsed or refractory hodgkin (hl) and non-hodgkin (nhl) lymphoma patients. the primary goal of this analysis was to assess the immediate related toxicity of this alternative regimen. we used beac (carmustine mg/m , etoposide mg/m , cytarabine mg/m , and cyclophosphamide mg/kg) in consecutive lymphoma patients ( hl, nhl) who underwent ahct for relapsed or refractory disease. the median age of the patients was . years ( - ). they all received peripheral stem cell grafts with a median cd + cell dose of . × /kg cells ( . - . ). disease status post salvage treatment (at ahct) was complete remission (cr) in , partial remission (pr) in and progressive disease in . the disease was chemosensitive to salvage therapy in / patients. median follow up was days ( - ). toxicity was assessed according to the who toxicity scale grading. all patients engrafted successfully. median time for engraftment was day + (d+ ) for neutrophils ( /mm ) and d+ for platelets ( /mm , without transfusion within the previous days). patients were hospitalized for a median of days ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) . no treatment-related mortality occurred. two patients died due to disease progression (both nhl patients, on d+ and d+ ). toxicity assessment until d+ is presented in table : moreover, no hemorrhagic cystitis or macroscopic hematuria, and no cardiac events were encountered. febrile neutropenia was recorded in and bacteremia in patients ( gram+, gram-, / related to central venous catheter), with fever ≤ grade in all cases. during d+ - two patients presented fever of unknown origin, and patients had upper or lower respiratory infections, with no other adverse events being recorded. in terms of disease best response within months post ahct ( / patients evaluated), patients achieved or sustained cr, pr ( of these patients eventually died due to disease progression), relapsed and succumbed due to disease progression (no response). according to our preliminary results, the early toxicity profile of beac is very low, the regimen is easily tolerable for the patients, and without any treatment-related mortality. its use as an alternative conditioning regimen in ahct for lymphoma patients seems feasible. further investigation including more patients and comparative analysis to other conditioning regimens are warranted for reliable conclusions on the toxicity and efficacy of beac. disclosure of conflict of interest: none. veino-occlusive disease (vod) is a potentially fatal adverse event caused by intravenous (iv) busulfan used in bone marrow transplantation (bmt) conditioning. the objective of this study was to identify determinants of vod in children treated by iv busulfan. this was a retrospective analysis of data collected in children from two bmt centers over years. vod was diagnosed according to modified seattle criteria. individual pharmacokinetic data, including busulfan area under the concentration-time curve (auc) and maximal concentration (cmax) were estimated in all children by using a validated bayesian approach. we examined the relationships between the occurrence of vod and available data in a learning (n = patients) and validation set (n = patients) obtained by random splitting. logistic regression was used as a continuous statistical model. in addition, we used classification and regression tree (cart) analysis, a machine learning and binary partitioning technique, to identify determinants of vod and their optimal cut-off values. the predictive performance of variables within both models was assessed by these results are compared with historical data from our service using beam as conditioning followed by auto-sct in lymphoma patients. nine patients were enrolled to receive neam: mitoxantrone mg/m day - to - , etoposide mg/m every hours and cytarabine mg/m every hours day - to - , and melphalan mg/m day - , followed by auto-sct. the median age was years ( - ); five non-hodgkin lymphomas (nhl) and four hodgkin lymphomas (lh). six patients were in partial remission (pr), two in complete remission (cr), and one with progressive disease at time of auto-sct. neam patients were compared with a historical control group of patients receiving beam regimen (n = ). differences between groups were analyzed by t-student and χ -tests. median cd + cells infused in neam and beam groups was . × ⁶/kg ( . - . ) and . × ⁶/ kg ( . - . ), respectively (p = . ). the median time to neutrophil recovery ( /μl) was days ( - ) and days ( - ) (p = . ) and median time for platelets recovery ( /μl) was and days ( - ) and days ( - ) (p = . ) respectively, for neam and beam patients. median duration of hospitalization was days ( - ) with neam and days ( - ) with beam (p = . ). among neam patients, % had one or more febrile episodes during neutropenia. no case of grade iii or iv mucositis was described. there was no transplant-related mortality (trm: %) associated with the use of neam regimen. at the present, all neam patients are alive, two of them in relapse ( %). due the difficulties in obtaining carmustine in our region, neam can be considered as a feasible alternative to beam. however, despite the sample was small enough to draw conclusions, we find that neam presents prolonged aplasia of significant value, we are currently exploring conditioning regimens followed by auto-sct in hodgkin's and non-hodgkin's lymphomas based on bendamustine, etoposide, cytarabine and melphalan. disclosure of conflict of interest: none. at present, decision-making about conditioning regimens for allogeneic hsct is based on patient's and donor's features, and disease characteristics. during the last years, terms as 'myeloablative/non-myeloablative/reduced-intensity' have been frequently employed in a confusing and unequal way among the different centers. knowing the expected intensity and myeloablative effect from each regimen is very useful and constitutes the aim of this analysis. we have analysed the severe neutropenia (anc o per mcl), and thrombocytopenia durations (platelets o per mcl), the need for platelet concentrates transfusion and the duration of the inpatient period of the allo-hsct carried out during the last four years in our centre. these data are reported according to the conditioning regimen used and to the type of transplant performed. then, they are compared among them in order to stablish intensity ranks. results: population characteristics are described in table : conventional intensive regimens (bu-cy , cy-bu , tbi-cy) reported more days of severe neutropenia and greater need of platelet concentrates transfusion. the regimens with less days of severe neutropenia, less need of platelet concentrates transfusion and fewer days of admission were flu-bu and flu-bu . allotransplants carried out with stem cells from bm presented more days of severe neutropenia and longer hospital stay. similar platelet transfusion need was reported. haplo-identical allotransplants reported more days of severe thrombocytopenia, but were not asociated to longer neutropenia or longer hospital stay than the others. these data are described in detail in table . flu-bu: the number ( , or ) expresses the doses of busulphan administered at . mg/kg/day. pc: platelet concretates data is expressed in medians. within the analysed conditioning regimens, an intensity rank is stablished regarding the myelosupression induced (in descending order): conventional intensive regimens (cy-bu , bu-cy , tbi-cy), flu-mel, flu-bu , flu-bu and flu-bu . all of them induced severe neutropenia and thrombocytopenia, and for that reason they must be considered myeloablative regimens. disclosure of conflict of interest: none. myeloablative allogeneic stem cell transplant for aml and mds: the impact of advanced age in the outcome m sánchez-escamilla* , , s garcía-Ávila , l yáñez san segundo , , ma bermudez rodriguez , , mm colorado araujo , , a casado diez , m celis alvarez , a cabero martinez , c fernandez martinez , a insunza garminde , , c richard espiga , and e conde garcía , allogeneic hematopoietic stem cell transplantation (allo-hsct) is the only curative option in high risk myeloid hematological malignancies. myeloablative conditioning (mac) regimen has been proven to be effective in the control of high risk diseases in advanced age patients. objective: the aim of this study was to analyze the efficacy of myeloablative allo-hsct in two cohorts of patients considering their age at transplant. we also analyzed the incidence of acute and chronic graft versus host disease (gvhd) and procedure related outcomes who underwent to myeloablative allo-hsct were retrospectively analyzed. the median age was years (iqr - ). both groups were divided regarding their age at allo-hsct [group , age ⩾ years (n = ) and group , age o years (n = )]. patient´s characteristics are shown in picture . data were collected as either continuous data and compared by two-tailed unpaired t-test or mann-whitney test, or as categorical variables and compared by chi-square. the procedure related outcomes were analyzed with the kaplan-meier test. the incidence of acute gvhd grade ii-iv was similar in both groups ( . % in group and . % in group , p = . ). the mean day to acute gvhd (grade ii-iv) development was days in group and days in group . the most involved organs in both groups were skin (group : . % and group : . % [p = . ]) and gut (group : . % and group : . % [p = . ]). at day + post-transplant patients were alive and evaluable for chronic gvhd. the incidence of cgvhd development was similar between group and ( . % versus . %, respectively, p = . ). however, severe grade s of cgvhd was high in group patients ( . % versus . %). with a median follow up of months (iqr, - ) the probability of os was significantly low (p = . ) in group ( . % +- . ) compared with group ( . % ± . ). pfs was also significantly low (p = . ) in group ( . % +- . ) compared with group ( . % ± . ). trm at months was higher in group compared with group ( . % versus . %). mortality due to relapses was also higher in group ( . % versus . %). most of the patients died during the first month. comparing both groups at this time ( months post-transplant), trm was higher in group compared with group ( . % versus . %). deaths due to relapse were also higher in this group ( . % versus . %). in our series, myeloablative conditioning regimen provides good survival rates and disease control in high risk hematopoietic diseases, however in patients aged ⩾ years confers high toxicity. it may be necessary to evaluate other strategies in this group of patients. disclosure of conflict of interest: none. allogenic hematopoietic cell transplantation (hct) is reserved for a group of high risk multiple myeloma (mm) patients having relapsed after high-dose melphalan and autologous transplantation. in general, reduced-intensity conditioning (ric) regimen are applied in this patient group with the aim of reducing transplant related mortality (trm). however, relapse of disease remains a major challenge after allogeneic hct. to address this issue, we added radioimmunotherapy (rit) to a conventional ric regimen. we have used a rhenium anti cd antibody in combination with a ric conditioning regimen. this ß-emitter leads to a so called 'cross-fire' effect allowing for bone marrow doses of gy and in parallel may target cd on myeloma cells. we hypothesized that this strategy may decrease the incidence of relapse. so far, we have treated nine patients with high risk relapsed multiple myeloma. all patients had received one (n = ), two (n = ) or three (n = ) high-dose regimens and autologous hct. conditioning therapy was flu/mel (n = ), flu/ bu (n = ) or flu/treo (n = ). flu/cy and gy tbi were applied before haploidentical transplantation. patients received g-csf mobilized pbsc from unrelated (n = ) or haploidentical (n = ) s donors. either tacrolimus/ methotrexate/ bortezomib or cyclosporine a/ methotrexate were used for gvhd prophylaxis. early extramedullary toxicity was limited. neutrophil and platelet engraftment was timely and complete in time in seven of nine cases. all patients achieved full donor chimerism around day fifteen after hct. severe acute graft-versus-host-disease (gvhd grade iii-iv) occurred in two patients and was lethal in both cases. two patients have experienced extramedullary relapse, one of them in the central nervous system and the other in the soft tissue. in two patients, a transplantation-associated thrombotic microangiopathy (ta-tma) was diagnosed. four patients are alive and in complete remission. we conclude that the combination of a ric regimen with a rhenium anti-cd radioimmunotherapy is save and feasible. the incidence of gvhd, ta-tma and extramedullary relapse will be monitored closely and will be presented in a larger patient cohort. disclosure of conflict of interest: none. the sirolimus/tacrolimus (sir/tac) combination has been associated with a better outcome after allogeneic hematopoietic stem cell transplantation (allo-hsct) when compared with conventional prophylaxis for graft vs host disease (gvhd) as cyclosporine/methotrexate in the true reduced-intensity conditioning (ric) setting but not in the myeloablative setting. in moderate-intensity regimens as thiotepa/busulphan/fludarabine (tbf), the sir/tac combination has not been evaluated. from january to december , all consecutive ric-allo-hsct recipients who received sir/tac combination to prevent gvhd in three spanish institutions were included in the study. the reduced-toxicity regimens used in this study where: (a) intravenous busulphan ( . mg/kg) and fludarabine mg/ m (bf), or (b) thiotepa days - and - and mg/kg if yrs old or mg/kg if o yrs old) on days - and - added to the bf regimen (tbf). the gvhd prophylaxis with sir/tac was given as detailed elsewhere (cutler c blood ) and was consistent within the center. the outcomes of the procedure according to the conditioning regimen were analyzed. overall, patients were included: tbf and patients in the bf group, with a median follow-up of months (range - ) and no difference in the median age ( vs years old). there were more males ( % vs %, p = . ) and more female donors to male recipients ( % vs %, p = . ) and more patients with lymphoid diseases and previous asct in the tbf group ( % vs %, p = . ), whereas there were more unrelated donors in the bf group ( % vs %, p = . ). other baseline characteristics were balanced between the groups (table ) . sir/tac prophylaxis had to be discontinued in % and % patients in the tbf and bf groups, respectively. toxicity was the main reason for discontinuation in the tbf group. the most frequent toxicities were renal injury (tbf % and bf %) and neurologic impairments (tbf %, bf %). in the bf group, the main reason of discontinuation was relapse or a mixed chimera. patients who received tbf presented higher incidence of extensive chronic gvhd ( % vs %, p = . ), higher nrm at days ( % vs %) and at years ( % vs %, p = . ). there were no differences in os ( years) between both groups ( ± . % vs ± . %, p = . ) (figure) . there were no differences regarding to acute gvhd - ( % vs %, p = . ), acute gvhd - ( % vs %, p = . ), or relapse (up to years, % vs %, p = . ) between the groups, either. the combination of sir/tac as gvhd prophylaxis was associated with higher incidence of chronic gvhd and nrm in patients receiving conditioning regimen with tbf compared to those receiving bf. there were no differences in os between both groups. [p ] cr complete remission, pr partial remission, nr non remission, hct-ci hematopoyetic cell transplant comorbility index. disclosure of conflict of interest: none. reduced-intensity conditioning regimen with fractionated total body irradiation of gy and cyclophosphamide mg/kg for allogeneic hematopoietic stem cell transplant is well tolerated and offers a potential disease control as treatment of acute leukemia and lymphoproliferative disorders m adler, t girinsky , s koscielny, g ferini, s wittnebel, s mayeur, c chahine, m vanghele, s pilorge, c castilla-llorente and j-h bourhis the use of reduced-intensity conditioning regimens (ric) before allo-hsct is widely extended since it preserves the graft-versus-leukemia effect but reduces treatment related mortality. however, there exist different ric regimens with diverse outcomes and the choice of the ric regimen relies on the type of disease treated, experience of the center and previous therapies. this is a retrospective study of patients treated in our institution within / and / . the ric regimen consisted of fractionated total body irradiation (ftbi) of gy administered in consecutive days ( gy/day) and cyclophosphamide mg/kg given in days ( mg/kg/day). post-transplant immunosuppression consisted of csa started the day before allo-hsct and short mtx on days , and after transplantation. for patients receiving transplant from unrelated donors, anti-thymocyte globulin at a dose of mg/ kg ( . mg/kg/day for days at day - and - ) was used as part of the immunosuppressant therapy. patients (median age: years: range: - years) were included. the median hct-ci was . (range: - ). primary disease was multiple myeloma (mm) in ( %), al/mds in ( %), cll in ( %), nhl in cases ( %) . patients ( %) received transplant from matched related donors, ( %) from matched unrelated donors and ( %) from mismatched unrelated donors. female to male mismatch incidence was % (n = ). most of the patients (n = ) received a peripheral blood graft. patient received a second allogeneic transplant. mm patients were transplanted in a "tandem" autologous-allogeneic hsct program in cases. the median number of chemotherapy lines prior to transplant was . in cll, . in mm and . in nhl. patients ( %) engrafted by day post transplant. neutrophil engraftment occured at a median of days (range: - days) and platelet engraftment at a median of . days (range: - days). full donor chimerism was observed in out of patients ( %) having survived by day . primary graft rejection was observed in patients. treatment related toxicities consisted of grade / mucositis in patients ( %), grade (range: - ) cardiac toxicity in patients ( %), grade (range: - ) hemorrhagic complications in patients ( %) including cases of hemorrhagic cystitis and secondary malignancies in patients, this within a median follow-up of . years. infectious complications during aplasia included fever of unknown origin (n = ), bacteremia (n = ) with cases of bacteremia with severe sepsis and cases of infections defined by bacterial foci. incidence of agvhd was % with cases of grade / refractory agvhd. cgvhd occurred in pts ( %). the non-relapse mortality (nrm) at days was % including cases of septic shock, case of acute cardiac toxicity and case of agvhd. the nrm at year was %. -year survival rates were % in al, % in cll and % in nhl with extended survival benefit. in al patients, the relapse incidence was % comprising patients who progressed during conditioning. the -year survival rate in mm patients was %. in mm patients who were in complete response prior to transplant, median overall survival was . years. the used ric regimen resulted in durable donor engraftment with an acceptable toxicity profile permitting efficient disease control in the described cohort. disclosure of conflict of interest: none. graft manipulation using selective depletion of αβ-t cells provides a source for haploidentical hematopoietic stem cell transplantation (haplo-hsct) enriched in effector cells. initial reports demonstrated safety and rapid immune reconstitution using this method, in malignant and non-malignant disorders using several proposed conditioning regimens. no specific considerations were given to hematologic malignancies. we reviewed a total of twenty seven pediatric patients who underwent haplo-hsct using αβ-t cell depletion between - in a single tertiary referral center. we report the results of procedures performed in eighteen patients transplanted for malignancies. twenty two haplo-hsct were performed in eighteen patients. the indication for hsct was acute leukemia in sixteen (all = , aml = ) and neuroblastoma in two. median age at hsct was . years. six patients had failed a prior hsct, and the remainder had no matched donor. the initial reduced-toxicity conditioning regimen consisted of melphalan, fludarabine, thiotepa and atg, and resulted in a high rate of graft rejections ( of ). thus, a totalbody irradiation (tbi)-based regimen was implemented, with prompt engraftment in all the patients. we observed rapid neutrophil and platelet engraftment kinetics (median time to engraft, days and . days, respectively). significant treatment related complications were all due to graft failure in patients receiving reduced-toxicity conditioning, with two infection-related mortalities in the presence of prolonged neutropenia. none of the patients developed hepatic sinusoidal-obstruction syndrome, and no grade - acute graft-versus-host disease (gvhd) or chronic gvhd were observed with either regimen. importantly, the majority of patients with acute leukemia were free of immunosuppression in the first days post hsct. the -year actuarial event-free and overall survival of the entire cohort were % and % respectively, with results for tbi-based conditioned patients being % and %. overall, we demonstrated that a tbibased conditioning for haplo-hsct using αβ-t cell depletion for malignant diseases resulted in acceptable outcomes in these high-risk patients without increased toxicity. disclosure of conflict of interest: none. high-dose chemotherapy conditioning regimens followed by autologous stem cell transplantation (auto-sct) generally provide good results in relapsed and refractory lymphomas. we evaluated the efficacy and safety of tecam regimen as conditioning with autologous stem cell support in patients with relapsed/refractory lymphomas. thirty-two ( patients were refractory, patients were relapse and one frontline treated) patients ( m, f) with lymphoma at various stages (stage ii, %; stage iii, %; stage iv, %) who underwent asct were included in this retrospective study. the median age at transplantation was . years (range, - years). the diagnosis were as follows: diffuse large b-cell non-hodgkin lyphoma (nhl), hodgkin lymphoma (hl), mantle cell lymphoma, follicular lymphoma, marginal zone lymphoma and t-cell nhl. all patients received tecam as conditioning regimen that consist of thiotepa ( mg/m × days), etoposide ( mg/m × days), cyclophosphamide ( mg/ kg × day), ara-c ( mg/m × days) and melphalan ( mg/m × days). median cd (+) cells were . × /kg (range; . - . × /kg) which were infused at day , followed by recombinant human granulocyte colonystimulating factor (rhug-csf) at a dose of μg/kg/day. the median time between mobilization and auto-sct was months (range; - months). the median time to recovery of absolute neutrophil and platelet counts independent of transfusion were (range; - ) and (range; - ) days, respectively. the median stay in hospital was days (range, - days). bacterial, sitomegalovirus and invasive fungal infection were detected in ( %), ( %) and ( %) patients, respectively in first days of auto-sct. three s patients ( . %) died from transplant-related complications. the overall response rate was % ( cr, . %; pr, . %) after auto-sct. relapse developed in patients during median follow-up period of . months (range; - months) after auto-sct. the -year estimated dfs ( figure ) and os were % and %, respectively. no statistical significance was observed for os and pfs in terms of gender, patient age ( o and ⩾ years) and nhl and hl lymphoma group (p ⩾ . ). the tecam regimen for auto-sct in lymphoma seems to provide encouraging results in terms of response and its good tolerance with acceptable toxicity. [p ] disclosure of conflict of interest: none. allogeneic hematopoietic cell transplantation (allosct) is the only curative treatment for myelofibrosis. however, its widespread use is limited by early non-relapse mortality (nrm). the optimal modalities of the conditioning regimen are a major unmet clinical need. in an attempt to reduce early nrm, we used a tbf conditioning regimen (thiotepa, busulfan (bu), fludarabine (flu) and antithymocyte globulin (atg)). our aim was to reduce nrm and improve engraftment by using such tbf conditioning. thirty consecutive patients with a median age of years (range, - ) who underwent allosct for primary (n = ) or secondary (n = ) myelofibrosis were included. according to the refined dynamic international prognostic scoring system (dipss-plus), patients were stratified as intermediate- (n = ), intermediate- (n = ), and high (n = ) risk. five patients had blast transformation. ruxolitinib was given to patients ( %) prior to allosct. graft source was pbscs in patients ( %) and bm in patients ( %). donors were matched related (mrd, n = ), unrelated (n = ) and haploidentical (n = ). conditioning regimen was tbf in patients ( %). in our historical cohort patients ( %) received fb (flu, bu, atg). in addition, patients received a 'tec-ric' sequential conditioning (thiotepa, etoposide, cyclophosphamide, and after days rest, flu, bu and atg) for blast transformation (n = ) or refractory proliferative myelofibrosis (n = ). gvhd prophylaxis consisted of cyclosporine (csa) and mycophenolate mofetil in patients ( %), csa and short course methotrexate in patients ( %) with abo mismatch and csa alone in patient ( %) with mrd. high dose posttransplant cy (pt-cy) was added in haplo cases. no significant difference was observed between tbf, fb and tec ric patients in terms of age, gender, karnofsky score, comorbidity index, number of previous treatment line, history of ruxolitinib administration and source of stem cells. median follow-up was months (range, - ). two tbf patients died of septic shock before engraftment at day + and + after allosct, respectively. one fb patient died of graft failure at day + post allosct. median time to neutrophils and platelets ( g/ l) recovery was days (range, - ) and days (range, - ) with tbf, days (range, - ) and days (range, - ) with fb, and days (range, - ) and days (range, - ) with tec ric. grade ii-iv acute gvhd occurred in . % of tbf patients, . % of fb patients, and % of tec ric patients (p = . ). moderate chronic gvhd developed in / evaluable tbf, / fb and / tec ric patients. no severe forms of chronic gvhd were observed. at last follow-up, patient relapsed, died and are still alive. main causes of death were disease progression (n = ), infection (n = ) and gvhd (n = ). nrm at years was . % in tbf patients, % in fb patients, and % in tec ric patients. the -year os were . % in tbf patients, . % in fb patients, % in tec ric patients, respectively. cd +-selected stem cell boost without further conditioning allowed to patients for poor graft function, with significant hematological improvement in patients. tbf conditioning regimen seems to be efficient in allosct for patients with myelofibrosis and compares favorably with previously published fb regimens. these preliminary results give a rationale to support a prospective evaluation of this platform. disclosure of conflict of interest: none. we proposed here to compare the outcome of patients receiving either thymoglobuline (atg), a rabbit anti-human thymocyte immunoglobulin or campath, a recombinant dna-derived humanized monoclonal antibody directed against cd . campath and atg are both commonly used as in vivo tcd before hsct, respectively in united kingdom and france but very few comparing data are available. all consecutive patients with acute myeloblastic leukemia (aml), myelodysplastic syndrome (mds) or myeloproliferative neoplasia (mpn) who received a reduced intensity hsct from an unrelated donor transplanted between and were included in this study. a propensity score was used to identify and control potential confounding to relate the treatment group to the outcomes. in the matched sample, cox regression model was used to describe the association between treatment and outcomes. patients have been included. all patients received fludarabine and busulfan with either atg (n = ) or campath (n = ). patients treated by atg received cyclosporine plus mycophenolate mofetil or methotrexate and patients treated by campath received cyclosporine alone as gvhd prophylaxis. comparing patient and transplant characteristics, atg patients were older ( vs years), had less often aml ( vs %), had higher disease risk (adverse dri: vs %; poor cytogenetics: vs %; high cibmtr score: vs %), were less often in complete remission at time of transplant ( vs %) and were transplanted less often from a mismatched hla donor ( vs %). cumulative incidence of sustained engraftment was in % and % campath and atg patients. time to neutrophil engraftment was longer in atg patients ( vs days). acute gvhd ii to iv rate were higher after atg ( % vs %) as well as chronic extensive gvhd ( % vs %). relapse rate was higher after campath ( % vs %). disease-free survival (dfs) was higher after atg ( vs %) and the gvhd-free relapse free survival (grfs) was similar ( % vs %). according to the prognostic factors for outcome, a propensity score was developed selecting patients from the original cohort. the estimation of tcd effect was than studied. relapse risk was higher in patients treated by campath while there is a non-significant advantage for atg in dfs (table ) . [p ] tcd with atg or campath gives similar os, dfs and grfs. severe acute or chronic gvhd is lowered by campath but the higher relapse risk counterbalances the potential benefit of campath finally given similar os. nevertheless, lower risk disease patient might benefit from campath while higher risk patients might benefit from atg. disclosure of conflict of interest: none. high-dose chemotherapy (hdt) with autologous stem cell transplantation is the standard of care for relapsed/refractory (rr) or high grade non-hodgkin-lymphoma (nhl) and hodgkin-lymphoma (hl) . the standard hdt in autologous stem cell transplantation (asct) for lymphoma is carmustinebased hdt using a combination of carmustine, etoposide, cytarabine and melphalan (beam); this standard conditioning programme is used by most groups worldwide . we have designed novels hdt regimens in which carmustine was substituited by an equal dose of fotemustine (feam) or thiotepa (team) and we compared these two hdt regimens in terms of engraftment times, toxicity, tolerability and frequency of relapse after asct. from february to september we consider a total of relapsed/refractory patients affected by hl and nhl respectively hl and nhl with different grade of initial disease (grade i-iv) and different response to prior treatments. the all other drugs were administered according to a standard beam regimen . after a day of rest, autologous peripheral blood progenitor cells were infused on day , followed by s.c. g-csf ( mg/kg) from day of asct until consecutive days when the ancs were × /l . the primary objectives of the study were to assess the feasibility and safety of the feam and team regimens in terms of acute toxicity, grade of mucositis, hemopoietic engraftment and relapse after asct. acute toxicity include chemotherapy-induced nausea and vomiting, diarrhea, hepatotoxicity, nephrotoxicity and infection complication. in all patients cd + cells were collected from peripheral blood and the median number of infused cells per patient was . × e /kg. the median time of engraftment was days for neutrophil recovery (n × /l) and days for plt recovery ( × /l). acute toxicity occurred in total patients ( . %), mucositis grade - occurred in patients ( % of cases). frequency of relapse in all cases was . %. feam conditioning regimen was used in cases showing a median time of neytrophil recovery of days and a median time of plt recovery of days. acute toxicity occurred in of these cases ( . %), mucositis grade - occurred in patients ( . % of cases). frequency of relapse in feam group of patients was . %. team conditioning regimen was used in cases showing a median time of neytrophil recovery of days and a median time of plt recovery of days. acute toxicity occurred in of these cases ( . %), mucositis grade - occurred in patients ( . % of cases). frequency of relapse in team group of patients was %. relapse/progression of lymphoma and conditioning regimen toxicities remain limitations to treatment success. the two novels hdt regimens feam and team are safe and feasible and show similar engraftment times, tolerability and frequency of relapse. maybe the team regimen shows toxicity slightly higher than feam regimen but longer follow-up is needed to evaluate fully its efficacy and long-term safety. disclosure of conflict of interest: none. treosulfan is a prodrug of a bifunctional alkylating cytotoxic agent. there are few reports regarding toxicological side effects of treosulfan-based conditioning prior to hsct. here we report on incidence of early potential treosulfan-related toxicity in patients treated with treosulfan-based conditioning before hsct. treosulfan was given at a dose of g/m /d for days in combination with fludarabin mg/m /d for days prior to hsct. most patients (n = ) had a haematological malignancy, while patients had a non-malignant disorder as hsct indication. an hla-a, -b and -dr matched unrelated donor (mud) was used in cases, patients had a hla-identical sibling donor and received an hla-a, -b or -dr allele mismatched unrelated donor. as graft versus host-disease (gvhd) prophylaxis, most patients (n = ) received cyclosporine and methotrexate. patients medical records were scrutinized retrospectively to collect laboratory tests (aspartate aminotransferase (ast), alanine aminotransferase (alt), creatinine) before hsct and then weekly until weeks after hsct. levels of ast and alt were significantly increased week after hsct compared to before hsct. however, only a few patients had transaminase levels over or times the upper normal level (unl) levels decreased sharply after the first week. most of the cases with high levels of ast/alt at one week had normal or close to normal levels before hsct. creatinine levels increased after week but no patient had levels ⩾ × unl. clinical features of all oral mucositis (om) were recorded using the world health organization (who) scoring system. most patients ( %) had no or very limited (grade i) om, % had grade ii and % had grade iii or iv of om. according to our toxicological results this is low-toxic protocol. however, all patients became neutropenic, % already at the time of graft infusion, indicating that the protocol has a myelo-toxic effect comparable to conventional mac protocols. all patients engrafted, except three patients who died very early. median time to neutrophil and platelet engraftment was (range - ) and days ( - ), retrospectively, which is significantly later when compared to engraftment data for other ric protocols used at our centre (data not shown). median duration of neutropenia (o . × /l) was days , comparable to what is expected after conventional mac conditioning. secondary graft failure (gf) occurred in ( . %) patients, all having a nonmalignant disorder and / having a urd. non-relapse mortality (nrm) was . % ( % ci . - . %) at days and . % ( . - . %) at one year after hsct. causes of death within one year after hsct were: relapse , epstein-barr virus associated posttransplant lymphoproliferative disease (ptld) , other infections , organ failure , gvhd , hemophagocytic lymphohistiocytosis (hlh) . other infections occurring within days after hsct were cytomegalovirus (cmv) reactivation ( %), invasive fungal infection ( . %) and blood stream infection ( %). veno-occlusive disease of the liver or sinusoidal obstruction syndrome (vod/sos) occurred in one patient and haemorrhagic cystitis in two patients. this study shows that early regimen-related toxicity after hsct was low despite similar marrow toxicities compared to mac regimens. disclosure of conflict of interest: none. allogeneic stem cell transplantation from haploidentical donors (haplosct) is an increasingly adopted option for patients (pts) with high-risk hematological malignancies. in our institution, we previously described a platform for unmanipulated peripheral blood stem cell (pbsc) haplosct using a calcineurin-free gvhd prophylaxis with sirolimus, micophenolate and anti-human t-lymphocyte immunoglobulin (atg) after conditioning with treosulfan and fludarabine (trramm; peccatori et al., leukemia ) . as an attempt to decrease relapse rate, especially in advanced-phase diseases, we designed a new phase ii prospective clinical trial intensifying conditioning regimen with the addition of gy total-body irradiation (tbi) (trramm gy; eudract# - - ). we report results on pts. pts affected by aml (n = ), other myeloid (n = ) and lymphoid (n = ) malignancies were prospectively enrolled from may to june . median pts age was y (range - ). revised disease risk index (r-dri) was low or intermediate in pts, high in pts and very-high in pts. twenty-five pts had previously received an allogeneic stem-cell transplantation with a median time from st to nd sct of months . median hct-comorbidity index by sorror et al. was ( ) ( ) ( ) ( ) ( ) ( ) . pts received a myeloablative conditioning regimen consisting of treosulfan ( g/m /d from - to - ), fludarabine ( mg/m /d from − to − ) and tbi gy (fractionated in doses, from − to ). source of stem cells were unmanipulated g-csf-mobilized pbsc from haploidentical donors. gvhd prophylaxis consisted of atg-fresenius (grafalon, neovii) mg/kg/d from − to − , rituximab mg/m on − , mycophenolate mofetil mg/ kg from − to + and sirolimus (target concentration - ng/ ml) from − . median infused cd + and cd + cell doses were . × ⁶/kg and . × ⁸/kg, respectively. median follow-up for survivors was months ( - ). neutrophil engraftment occurred in % of pts with a median of d ( - ), platelet engraftment was reached in % of pts with a median of d ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) . the -d cumulative incidence (ci) of grade ⩾ acute gvhd (agvhd) was ± % and of grade ⩾ agvhd ± %; the years ci of chronic gvhd was ± %. the ci of transplant-related mortality (trm) at y and y were ± % and ± %, respectively. the ci of relapse at y and y were ± % and ± %, respectively, with a median time to relapse of d . interestingly, we did not observe any extramedullary relapse; loss of mismatched hla-haplotype occurred in % of relapses. among pts who were in active disease at time of haplosct and who were evaluable, % achieved complete remission (cr) and full donor chimerism at day+ . the y and y probabilities of disease-free survival (dfs) were ± % and ± %, respectively. at y, % of pts are alive, disease-free and immunosuppression-free minimal residual disease, tolerance, chimerism and immune reconstitution the number of human leukocyte antigen (hla)-mismatched hematopoietic cell transplantation (hct), including cord blood transplantation, has been increasing. hla-flow method can discriminate mismatched hla antigens between donor and recipient by using flow cytometry, and can evaluate minimal residual disease (mrd) or chimerism after hla-mismatched hct. by developing more simple methodology, hla-flow might be more widely applicable. we have developed modified -colorbased hla-flow method. the aim of this study is to evaluate the utility of the -color-based hla-flow for monitoring of mrd and chimerism after hla-mismatched hct in children. from june to november , serial monitoring of mrd or chimerism by the -color-based hla-flow was performed in twelve patients undergoing hla-mismatched hct ( tests). nucleated cells obtained from bone marrow were stained by immunofluorescent antibodies against hla antigens mismatched between donors and recipients. these cells were also stained by immunofluorescent antibodies against surface antigens such as cd , cd , cd , cd and cd /cd for determining lineage of the cells. these surface antigens were also used as a marker of leukemic blasts in the mrd study. we used -color-based flow cytometry (facs-navious) and the data were analyzed with flow jo. erythroblasts and dead cells were excluded from the analysis. in each study, at least cells were analyzed. for mrd analysis, we concurrently tested real-time quantitative polymerase chain reaction (pcr) of peripheral wt mrna or leukemia-specific fusion genes. pcr of polymorphic short tandem repeats or fluorescent in situ hybridization of x/y chromosomes was concurrently tested for chimerism study. age of patients ranged from to years. donor sources included bone marrow (n = ) and cord blood (n = ). for mrd monitoring of acute leukemia (n = ), the -color-based hla-flow could detect mrd in three patients. five patients have not experienced relapse. no discordance with other mrd markers was observed in these patients. hla-flow could not separate donor-derived cells from recipient-derived ones in one patient receiving bone marrow transplantation. as for chimerism testing (n = ), the -colorbased hla-flow could successfully evaluate quantitative lineagespecific chimerism in all patients. there is no discrepancy between hla-flow and other methods. we could complete evaluation of the -color-based hla-flow within two days in all tests. the -color-based hla-flow is a simple, quick and useful method for the quantitative evaluation of mrd and lineagespecific chimerism after hla-mismatched hct in children, irrespective of donor sources. it is thought that our method is applicable in all institutions owing -color-based flow cytometry. acknowledgement: we thank drs. nobukazu watanabe, eri watanabe and natsuko sato (university of tokyo) for their technical advices. we also thank drs. tomoko okunushi (chiba university), hidefumi hiramatsu and katsutsugu umeda (kyoto university) for their care of patients involved in this study. disclosure of conflict of interest: none. survival (pfs), cumulative incidence of relapse (cir) and acute/ chronic gvhd incidence in aml patients (pts) submitted to allo-hsct at our institution between january and december . this retrospective study evaluated aml pts submitted to allo-hsct from matched sibling donors (msd) and matched unrelated donors (mud) who provided bone marrow (bm) or peripheral blood as stem cell grafts.ir was evaluated at , and days post-transplant in all pts. cmv-dna copies were determined in peripheral blood by quantitative pcr twice weekly in the first days post-transplant and subsequently once weekly. cmvi/r was analyzed as a timedependent covariate.effect of cmvi/r on os and pfs was estimated by cox proportional hazard model. cir and gvhd incidence were analized with a competing risk approach, considering death from any cause as a competing event. effect of cmvi/r on cir and gvhd were evaluated by fine & gray model. median age at allo-hsct was . years ( . - . ). in our population % of donors were seropositive for a previous cmv infection and pts transplanted from these donors showed a significantly lower cumulative incidence of cmvi/r than pts transplanted with seronegative ones (shr = . , %,ci: . our study demonstrates that cmvi/r influences the success of allo-hsct by determining a better ir characterized by a higher cd + cell number that might exert an immune protective control on disease outcome by improving os,pfs and cir with no effect on gvhd. another factor of utmost importance to achieve this same goal might be constituted by the significantly increased nk cell number six months after allo-hsct. to assess the dynamics of molecular response to treatment in aml adult patients with concomitant flt and npm mutations. this retrospective single center studystudy was approved by the institutional review board of american university of beirut medical center. twelve consecutive newly diagnosed (n = ) or relapsed (n = ) aml patients received idarubicin/cytarabine induction and one or two consolidation (s) ( table ) . seven patients received allogeneic stem cell transplant (allo-sct) and had haploidentical-sct (hap-lo_sct); all followed by post-transplant sorafenib maintenance. median follow-up was . ( - ) months. all transplanted patients remain alive and disease free.flt mutation was tested on dna using a qualitative method with a sensitivity of . %. npm- mutation was tested on cdna using a qualitative or a quantitative rt-pcr with a sensitivity of . % and . ncn respectively. patients were tested at diagnosis, after induction, after each consolidation, before and s at days , and after allo-sct for kinetics of npm and flt molecular response. after induction, flt became negative in all tested patients (n = ). after first consolidation, flt- was not tested in patients who had a negative result after induction, was negative in patients including the patients who were not tested after induction, whereas a molecular relapse was noted in one patient who developed a hematological relapse and rapidly died. another patient died after the third consolidation, in complete remission, due to septic shock. no molecular positivity for flt- was noted later on, whether after second consolidation or post-transplant. conversely, npm- mutation became negative in out of tested patients after induction, in additional patient after first consolidation and in additional patients after sct, mostly after starting sorafenib. npm- mrd value remained elevated in out of patients with quantitative assessment at diagnosis and post induction (figure ). flt become negative early after induction while npm negativity lags behind. persistent npm- mrd does not seem to predict post-transplant outcome and may indeed become negative after sorafenib. these results need confirmation in larger studies. disclosure of conflict of interest: none. in allogeneic stem cell transplantation (allo-sct), an early detection of the transplant outcomes such as overall survival (os), event-free survival (efs), cumulative incidence of relapse (cir) and non-relapse mortality (nrm) is fundamental regarding the use in time of additional therapy after sct. therefore, we investigated the association between early immune reconstitution (ir) on day + after allo-sct and outcomes in children suffering from acute leukemia or myelodysplastic syndrome (mds). this study collected data from allo-sct from january until december in our institution. the median survival follow-up was months. indications of allo-sct were all (n = , %), aml (n = , %) and mds (n = , %). the median age was years (range, . - ). patients were transplanted in cr (n = , %) and pr/nr (n = , %). patients included in the study received st sct (n = , %), nd sct (n = , %) or sct (n = , %). grafts were from sibling (msd; n = , %), matched unrelated (mud; n = , %), haploidentical (n = , %) or mismatched unrelated (mmud; n = , %). conditioning regimens were tbi-based (n = , %) or chemo-based (n = , %). stem cells were from bone marrow (n = , %) or peripheral blood (n = , %). we analyzed the absolute count of lymphocytes (alc), monocytes, cd + t cells, cd +cd + t-helper cells, cd +cd + cytotoxic t-cells, cd -cd + natural killer (nk) cells and cd + b cells assessed on day ± after sct. we used the percentiles of the lymphocyte subsets of the same cohort to categorize the samples throughout the study. patients with alc over the th percentile of alc (alc o cells/μl) had a . -fold increased hazard ratio (hr) to develop relapse (p = . ). nk cell counts on day after sct were strong associated with os, efs, cir and nrm. patients with nk cell count over the th percentile (nk . cells/μl) had increased hr for os (hr = . , p = . ) and efs (hr = . , p = . ) compared to patients with nk count under the th percentile. patients with nk cells over the th percentile (nk o . cells/μl) had a hr = . (p = . ) for relapse and hr = . (p = . ) for nrm compared to patients with nk cell count under the th percentile. monocyte cell count on day correlated with os, efs and cir. patients with cd + cells count under the th percentile of cd + (cd + o cells/μl) has an increased hazard ratio for os, efs and relapse compared to patients with cd + cell counts over the th percentile. no association between absolute cell count of cd +, cd +, cd + and cd + on day + after allo-sct and any outcomes either os, efs, cir or nrm was found. the study confirms the strong association between early ir and outcomes after allo-sct in children. our study suggests that especially nk cell and monocyte cell count on day + may have significant prognostic implications. our findings suggest that the cells count of alc, nk cells and monocytes on day + after allo-sct could be useful to predict outcomes after allo-sct and should be taken into account in considering alternative treatment. disclosure of conflict of interest: none. early immune reconstitution (eir) has proven to be a significant determinant for the outcome of allogeneic hematopoietic stem cell transplantation. in the setting of unmanipulated haploidentical transplantation (haplo-hsct), some groups have identified the absolute leukocyte count on day + (alc ) as an independent prognostic factor in terms of overall survival (os), disease free survival (dfs) and infectious mortality (im). the aim of this study was to evaluate the impact of eir on os, dfs and im among patients who underwent haplo-hsct with postransplant cyclophosphamide (ptcy) at our institution. from july to april , haplo-sct were performed at our institution. threedied before day after haplo-sct, and patients had missing data. conditioning regimen consisted of fludarabine, cyclophosphamide and busulfan. twenty-nine patients received a reduced intensity conditioning regimen ( - days of busulfan) while a myeloablative regimen ( - days of busulfan). gvhd prophylaxis comprised ptcy, cyclosporine and mycophenolate mofetil. patients were assessed for eir by means of alc , cd + t lymphocyte count on + (cd ), nk lymphocyte count on + (nk) and nk cd bright percentage on + (cd br ). we analyzed pts, with a median follow-up of months ( - ). the median age of the pts was (range - ), %men. diagnosis were: aml( %), hl ( %)non-hl ( %), all ( %),mds( %), cml( %), others( %). % were in complete remission at the time of transplant, % in partial remission and % had overt disease. in terms of infectious complications, cmv reactivation was documented in % of the pts, % developed a proven invasive fungal infection and % suffered from bk+hemorrhagic cystitis. median os and dfs were ( - ) and months ( - ), respectively. im rate was % at the end of follow up. median follow-up was months ( - ). roc curves were used to determine the optimal cut-off values for each of the studied parameters: cells/μl for alc , cells/μl for cd , cells/μl for nk and % for cd br were chosen. pts with alc ⩾ /μl had better os (p = . ) and dfs (p = . ), than those with alc o /μl. median os and dfs were months vs not reached (nr) and months vs nr, respectively. pts with cd br ⩾ % had better os (p = . ) than those with lower values. median os was months vs nr; however no difference was seen in terms of dfs. we didn´t observe statistically significant differences in os or dfs, among pts with different levels of cd and nk on + . cumulative incidence of im was significantly lower in pts with an alc ⩾ (p = . ), pts with cd ⩾ /μl (p = . ) and pts with nk ⩾ (p = . ); patients with cd br ⩾ % showed tendency to have lower cumulative incidence of infectious mortality (p = . , non-significant). cumulative incidence of relapse was not affected by alc , cd , nk or cd br . our study supports the independent prognostic value of early immune reconstitution after unmanipulated haploidentical transplantation with ptcy, especially in terms of lower infectious mortality. os and dfs were better among patients with alc ⩾ cells/μl. pts with cd br ⩾ % also showed better os. no correlation was found between cd or nk on + with os or dfs. cumulative incidence of infectious mortality was affected by alc , cd and nk on + ; while cd br seems to have less impact. [p ] disclosure of conflict of interest: none. an early absolute lymphocyte count (alc) recovery after autologous stem cell transplantation (asct) for hematologic malignancies has been related with an improved transplant outcome due to a faster autologous immune restoration. in this retrospective study we analyze post-transplant survival of non hodgkin lymphoma (nhl) patients and its relation with alc at day + post-asct. we analyzed consecutive adult nhl patients who underwent asct at the hematology and sct department of hospital maciel (montevideo, uruguay). only individuals with at least months post-transplant follow up were included. all patients received beam (bcnu, etoposide, cytarabine and melphalan) conditioning regimen followed by peripheral blood stem cells previously collected by apheresis. median cd + cell dose was . × e /kg ( . - . ). median alc at day + was /μl. patients were divided into two groups: alc at day + inferior than /ul (group ) and alc at day + superior or equal than /ul (group ). differences between groups were analyzed using t-student and chi-square tests, with statistical significance determined at p o . . disease free survival (dfs) and overall survival (os) were analyzed by kaplan meier method. differences in survival between groups were determined by log-rank test. no differences were observed between groups regarding gender, histology, disease status at transplant and cell dose. patients in group were older and more heavily pretreated. neutrophils and platelets engraftment were significantly faster in group (table ) . after a median follow up of months, disease-free survival (dfs) and overall survival (os) were superior in group . median dfs was months and not reached (p = . ) and os was months and not reached (p = . ) in groups and respectively (figure ). an early alc recovery after asct was associated with a superior dfs and os in nhl patients. individuals with alc major or equal than /ul had also a shorter time to neutrophils and platelets recovery and a shorter hospital stay. in this study, cd + cell dose does not seems to be a determinant factor for lymphocyte recovery. the load of previous treatment may influence lymphocyte recovery after asct. these results support the association between early post-asct lymphocyte recovery and improved survival in nlh patients. [p ] disclosure of conflict of interest: none. t cell depletion (tcd) reduces the risk of graft versus host disease (gvhd) but also the graft versus leukaemia (gvl) effect, thus increasing the risk of relapse. donor lymphocyte infusions (dli) can be given to boost donor chimerism, with the intention of enhancing the gvl effect. it is not currently known whether giving dli based on bone marrow chimerism (bmc) influences survival, or whether certain groups of patients benefit more from dli than other groups. in addition, it is not known whether the overall aim of achieving % bmc associates with improved survival. we investigated whether day (d ) bmc was predictive of survival, and whether giving dli based on this result was associated with improved overall survival. data were retrospectively collected from case notes and laboratory reports for patients who underwent allogeneic stem cell transplant (allosct) for aml or mds at the northern centre for bone marrow transplantation between and . patients who died prior to d were excluded from the analysis. of the patients analysed (aml , mds ), % were male and % female. the median age was years (range - ). conditioning was with flu/bu/ alemtuzumab ( ), flu/mel/alemtuzumab ( ), cy/tbi alemtuzumab ( ), flamsa tbi/bu atg/alemtuzumab ( ), other ( ) . ( %) received a graft from an unrelated donor, ( %) a matched sibling donor and ( %) another source. ( %) received mobilised pbscs, ( %) bone marrow and ( %) cord blood. statistics were performed using graphpad prism. p values were calculated using the chi square test and taking po . to determine significance. bmc was divided into groups %, - % and o %. % bmc at d was associated with a significant increase in year overall survival (os) ( . % vs . % and . % for - % and o %, respectively, p o . ). patients with a d bmc o % had a year os of o % (with relapse the cause of death in %). in patients whose d bmc was o %, there was a significant improvement in year os seen in those who received dli ( . % survival at years vs % with no dli, po . ) (figure : os by d bm chimerism (with and without dli). attainment of % bmc at a subsequent time point also significantly improved survival in those with a d bmc of - % ( . % year survival vs . % who never attained %, p o . ) and o % ( % year survival vs . %, p o . ). we found d bmc to be predictive of os in this population. in addition, dli was associated with an improvement in os, especially in patients whose bmc at d was o %. there was also a statistically significant improvement in os seen in patients who subsequently attained a % bmc, where it was o % at d . the objectives of this analysis were to examine the optimal alc recovery cutoff utilizing receiver operator characteristics (roc) analysis and to examine infused allograft characteristics associated with early alc recovery. after due irb approval, patients (pts) with aml and all who underwent hct at our institution between - were identified. pts with t-cell depletion or maintenance post hct were excluded. data were collected retrospectively from the patient's records. cellular contents of infused products (cd , cd , tnc, mnc, alc and amc) in addition to alc post hct were analyzed and optimal cutoff, if present, was established using roc analysis for the end point of relapse. time to end point analysis was computed using the kaplan-meier with log ranks. for competing events, cumulative incidence was computed using grey's model. univariable and multivariable analyses were performed using cox proportional hazard regression. a total of pts met the inclusion criteria and were analyzed. optimal alc cutoff by roc analysis was established to be on day + (d ) with alc . × /l and was subsequently defined as early lymphocyte recovery (erl). pts with alc ⩽ . × /l were deemed to have delayed lymphocyte recovery (dlr). patients were subsequently stratified accordingly and patient, disease and transplant related factors were well balanced between the groups. median follow up of the entire cohort was ( - . ) months. graft characteristics: roc analysis established optimal cellular cutoff, if present to predict elr. pts in the elr group were more likely to receive cd × /kg o ( . ), cd × /kg ( . ) and alc . × /kg (p = . ). we did not find a significant threshold for other allograft variables i.e. (tnc, mnc or amc). post hct outcomes: at years, corresponding cumulative incidence of relapse (cir) and non-relapse mortality (nrm) was . % vs . % (p = . ) and . % vs . % (p = . ), for elr and dlr cohorts, respectively. there was a trend towards improved progression free survival (pfs) and overall survival (os) in favor of elr vs dlr at . % vs . % (p = . ) and . % vs . % (p = . ), respectively. median time to neutrophil and platelet engraftment was and days, respectively for both groups. incidence of acute graft vs host disease (agvhd) was similar (p = . ); however, chronic gvhd (cgvhd) was more prevalent in the elr group at % vs %, respectively (p = . ). on s multivariable analysis for relapse, elr retained its prognostic significance with hr . ( . - . ; p = . ). cgvhd and first complete remission (cr ) at the time of hct were also protective factors from relapse in multivariable analysis. we observed that elr is an independent predictor for relapse in patients receiving allogeneic hct for acute leukemia with a trend towards improved os. this is possibly related to higher incidence of cgvhd. elr was influenced by infused allograft characteristics particularly cd count. given the sample size and retrospective nature of the analysis, these important observations should be examined prospectively. disclosure of conflict of interest: none. allosct is the only curative option for the treatment of hematological disorders with depression of hematopoiesis and primary immunodeficiencies.non-myeloablative conditioning (mac) regimens lead to long persistence of mixed chimaerism (mc) in the majority of patients. purpose: to estimate the relationship between type of hematopoietic chimaerism and appearance of gvhd in patients with non-malignant diseases after allosct eleven patients ( boys and girls) with median age of years (range - ) were included in the current study. among them there were patients with severe aplastic anemia (saa), with fancony anemia (fa), with thalassemia, with nijmengen syndrome, treated in our center from to . donors' sources were as follows: siblings in cases, mud ( / ) in ones. in cases bone marrow aspirate were used, in mobilized peripheral blood hematopoietic stem cells. conditioning regimens included fludarabin, cyclophosphamide and horse atg for saa patients, in fa and nijmengen syndrome patients this scheme was augmented by low-dose busulfan. in thalassemia patient we used mac with busulfan, fludarabin and horse atg. in majority of case gvhd prophylaxis consisted of tacrolimus and methotrexate combination. when allosct was performed form mud patients were additionally administered with mycophenolate mofetil. median of follow-up period was mo (range - ). quantitative evaluation of chimerism was done by multiplex amplification of str loci with subsequent fragment analysis using «cordis plus» kit («gordiz llc», russia). we analyzed whole bone marrow and peripheral blood together with cd + and cd + lymphocyte subpopulations. presence of ⩾ % donors' hematopoietic cells was considered as complete donor chimerism (cc), less than % was considered as mc. all patients engrafted in time and all of them are alive at the time of current analysis. there were no severe life-threatening complications, infections or graft rejections. only patients achieved cc at day + . at day + only these patients stayed in cc. at this time point mc was mainly revealed in cd + lymphocytes. in year after hsct proportion of cc patients enlarged to % ( patients did not achieved this time point). there is no any correlation between time of engraftment and chimerism value at day + , either between the dose of transplanted cd + cells and chimerism level ((p . in both cases). severe gvhd was noted only in female patients with cc at day + . in the first case it was acute gvhd grade iii after hsct from mud, in the second case extensive form of chronic gvhd in year after hsct from sibling was observed. there are no other cases of grade iii-iv acute gvhd in the observed cohort of patients. localized form of chronic gvhd [p ] was revealed in ( %) patients. in other patients there were no signs of chronic gvhd. despite limited number of observations we assumed that fast achievement of cc corresponds to severe gvhd. and vice versa, long persistence of mc prevented emergence of gvhd. however our findings need to be confirmed in a larger group of patients and preferably in a multicenter setting. disclosure of conflict of interest: none. interest of quantitative assessment of hematopoietic chimerism by real-time quantitative polymerase chain reaction after hematopoietic cell transplantation for hematological malignancies: a retrospective analysis on adult patients from rennes university hospital g laure , c mathilde , b marc , n stanilsas , d charles , l christine , a mehdi , lb laetitia, s gilbert , l thierry and r virginie department of hematology, chu pontchaillou, rennes; immunogenetics and histocompatibility laboratory, etablissement français du sang, rennes and etablissement français du sang-bretagne chimerism (percentage of recipient versus donor-derived blood cells) is used to document engraftment after hematopoietic stem cells transplantation (hsct). detection of persistant host cells, - as well as an increase in recipient cells chimerism has been associated with impaired dfs and os. quantitative real time pcr (qrt-pcr) is a highly sensitive, reproducible method, which can detect very low levels of recipient cells. the aim of this study was to evaluate the prognostic impact of early chimerism days (d ) and days (d )) after hsct and the meaning of detection of an increase of chimerism, even at low levels, during follow-up. adult patients who underwent hsct in rennes between and were included in this retrospective study. all chimerism analyses were done with qrt-pcr using whole blood sample. complete chimerism (cc) was defined by less than . % recipient cells detected. with a median follow-up was days, patients relapsed with a median time of . days after hsct. both d and d mixed chimerism (mc) ( . % recipient cells detected) were associated with an increased relapse risk (p = . and p o . respectively) compared to patients with cc in univariate analysis. however, when looking at subgroups analysis, d and d mc vs cc was significantly associated with increased relapse risk in this cohort for myeloid diseases (p = . and p o . ) but not for lymphoid diseases (p = . and p = . ). no difference in os was observed (p = . and p = . ). more important, detection of an increased of mc (imc) was associated with an increased relapse risk in univariate and multivariate analysis (or = . [ . ; . ], or = . [ . ; . ]), (po . ), as well as impaired os (p = . ) and dfs (p o . ). among the patients with aml and at least chimerism analysis available, only relapsed without imc detected but the patients' last available chimerism analysis was , and days before relapse respectively. median levels of recipient cells detected was . %. altogether, these results indicate that serial analyses of chimerism with qrt-pcr are a useful tool for post-transplant monitoring and might help identify patients at highest risk to relapse after transplant, especially in myeloid disease. monitoring frequency is critical in order to obtain the highest clinical impact, and the timing of monitoring as well as the safety and type of pre-emptive interventions still need to be explored. considering the kinetics of the disease, frequent analysis in myeloid pathology might improve the detection of impending relapse. although an approximately -log higher sensitivity of quantitative pcr (qpcr) compared to short tandem repeat (str) has been documented in different studies, the latter remains the standard procedure for hc assessment to date. we hypothesized that qpcr could be superior to str for monitoring the molecular kinetics of donor cell engraftment, response to donor lymphocyte infusions (dli) and development of relapse post-hct. we analyzed patients (pts) who underwent mainly / hla-matched unrelated hct mostly for acute myeloid or lymphatic leukemia at the university hospital essen between and . transplant conditioning was mostly myeloablative and gvhd prophylaxis was by cyclosporin a and methotrexate without anti-thymocyte globulin (atg). median follow-up of pts was days (d) ( - ). cytomegalovirus (cmv) reactivation in the first d posttransplant was measured by pp (n = ) or pcr (n = ). a total of retrospective genomic dna samples from peripheral blood (pb; n = ) or bone marrow (n = ) collected between d and d post-transplant were available for hc analysis in parallel by str (mentype chimera, biotype) and qpcr (alleleseq, abbott). threshold for hc positivity in qpcr was set at . % following published protocols. concordance in hc analysis between qpcr and str was found in / ( . %) samples, with all discordant cases positive in qpcr but negative in str. engraftment could be assessed in samples drawn at d -d from pts without relapse in the first months post-hct. these samples showed concordant negative or positive qpcr and str results reflective of full donor engraftment or persistent mixed chimerism (pmc) in and pts, respectively. in pts, qpcr but not str documented delayed conversion to full donor chimerism until d . in the remaining pts, positive results in qpcr but not str during early engraftment were observed exclusively in bm, in particular those drawn before d post-hct. qpcr but not str was also able to document the kinetics of conversion to full donor chimerism which took d and d in pts receiving dli for treatment of pmc and relapse, respectively. informative relapses could be assessed in samples drawn at least d before onset. / bm and / pb were positive in qpcr, compared to / bm and / pb in str, with a sensitivity of / ( %) and / ( . %) relapses, respectively. consistent with previous reports on a protective effect of early cmv reactivation on relapse in gvhd prophylaxis regimens without atg, relapse occurred in / ( %) pts who experienced cmv reactivation in the first d post-transplant, compared to / ( %) pts who did not. no apparent associations were observed between early cmv reactivation and engraftment kinetics post-hct. hc assessment by qpcr is highly concordant with str, but markedly superior for molecular monitoring of engraftment kinetics and relapse. positive qpcr results in bm should be interpreted with caution during early engraftment, while both bm and pb were highly informative for relapse in our series. these results advocate the feasibility and clinical utility of qpcr for post-hct hc monitoring in routine use. disclosure of conflict of interest: the commercial assays for qpcr chimerism analysis were provided by abbott molecular free of charge. tyrosine kinase inhibitor (tki) has become the standard of care in patients (pts) with chronic myeloid leukemia (cml) and an unavoidable tool in the combined therapy for pts with philadelphia chromosome positive (ph+) acute lymphoblastic leukemia (all). nevertheless, allogeneic stem cell transplantation (hsct) remains the standard therapy of all ph+ and of cml pts failing st line therapy with tki, with failure or insufficient response or intolerance or mutations resistant to nd generation tki, or in the advanced phase at diagnosis. in the past decade the feasibility and safety of post-hsct imatinib administration as prophylactic or therapeutic strategy was confirmed. second and rd generation tki administration after hsct is under investigation. here we are reporting our experience in post-hsct treatment with the rd generation tki ponatinib in pts treated between and at our institution. pts data and information were collected from institutional database and chapters revision. a written consent was given by pts allowing the use of medical records for research in accordance with the declaration of helsinki. pts and diseases features are reported in table . pre-transplant treatment for the all ph+ patient consisted of chemotherapy combined with dasatinib, followed by a st mud hsct and dasatinib in maintenance. the patient relapsed year after hsct with documentation of mutation v l. ponatinib was introduced as salvage treatment to bridge nd haplo hsct. pre-transplant treatment for the cml patients consisted of tki therapy with combination of chemotherapy in case of uncontrolled progression of disease. two pts received a st mud hsct but relapsed respectively months and years later. ponatinib was introduced as salvage treatment to bridge nd haplo hsct. four pts received ponatinib mg daily before the last hsct: one patient achieved sustained major molecular response, pts obtained transient response. all pts were presenting nd generation tki resistant mutations (table ) . ponatinib was started at a median of days after hsct (range, - ) as salvage treatment in overt relapse ( cases), prophylaxis ( case) or preemptive therapy ( case). acute gvhd was diagnosed in pts before ponatinib administration, of them also experienced chronic gvhd. no new cases of gvhd were observed after initiation of ponatinib. immunosuppressive treatment and azoles treatment were discontinued before ponatinib in all but one patient who was under combined treatment for chronic gvhd: therapeutic drug monitoring was closely performed without evidence of drug-drug interaction. pts were regularly evaluated for toxicities and monitored for cardiovascular events. no serious adverse events were reported in our experience: we administered ponatinib at a median maximum dosage of mg daily (range, - mg), for a median of weeks (range, - weeks). at last evaluation one patient maintained the status of molecularly undetectable leukemia (follow-up post hsct: months) and one major molecular response (follow-up post hsct months). three patients who received therapeutic ponatinib in overt relapse did not respond and died for progressive disease. ponatinib is safe and well tolerated as bridge to hsct and to maintain disease control after transplant. prophylaxis targeted therapy and preemptive therapy with ponatinib may lead to the reduction of disease relapse for high-risk ph+ leukemia. disclosure of conflict of interest: none. at nuh singapore we have adopted the cd ra depletion to ameliorate graft versus host disease. materials and methods: we have transplanted leukemia patients with cd depleted hsct followed by cd ra depleted donor lymphocyte infusion. no additional gvhd prophylaxis or gcsf was used. results: % patients achieved primary engraftment. median time for neutrophil engraftment ( /μl without gcsf) was days (range - days), platelet engraftment ( ) was days (range to days). immune reconstitution was rapid with median cd and cd cell counts /μl at day . by day median cd count was /μl (range - /μl). no patient developed grade iv acute gvhd. there was a significantly reduced incidence of invasive viral infections as compared to conventional transplants. importantly, all patients achieved complete remission (cr) on day + and remained in cr for longer time as compared to conventional transplants. conclusion: our preliminary data suggests that rapid immune-reconstitution of nk cells and t cells with this strategy correlates with reduced infection related mortality without loss of graft versus leukemia effects. disclosure of conflict of interest: none. the use of post-transplantation high-dose cyclophosphamide (pt-cy) has overcome the need for extensive depletion of t lymphocytes from haploidentical donor grafts, which traditionally resulted in severe and prolonged immunosuppression. it is therefore relevant to investigate the degree and the tempo of immune reconstitution after t cell replete haploidentical stem cell transplantation (haplo-sct) by use of pt-cy. we prospectively monitored cellular immunity in consecutive adult patients (male/female: / ), who underwent haplo-sct with pt-cy for myeloid (n = ) or lymphoid (n = ) malignancies. the median age at transplant was (range, - ) years. the conditioning regimen was myeloablative in , reduced-intensity in , and non-myeloablative in case. the source of the graft was peripheral blood (n = ) or bone marrow (n = ). in addition to pt-cy, graft-versus-host disease (gvhd) prophylaxis included tacrolimus and mycophenolate mofetil. absolute counts of cd +cd +, cd +cd +, cd + and cd +cd + cells were measured by flow cytometry at , , , , , and months post transplant. the median doses of infused cd + and cd + cells were . (range, . - . ) × /kg and . (range, . - ) × /kg, respectively. neutrophil engraftment ( /ul) was achieved at a median of (range, - ) days, whereas platelet engraftment ( /ul) was observed at a median of (range, - ) days. seven patients developed acute gvhd (grade i/ii: , grade iii: ). chronic gvhd occurred in patients, and was extensive in the of them. cytomegalovirus infection was detected in / cases at a median interval of (range, - ) days post transplant. two patients were administered rituximab for epstein-barr virus reactivation at months, whereas one patient developed bk virus-associated hemorrhagic cystitis at . months following haplo-sct. there was death due to gvhd and infection at months post transplant. at a median follow-up of (range, - ) months, / patients remain alive and disease-free. the absolute counts of t and b cells were extremely low early post transplant, while nk cells recovered from the first month (mean count, /μl). the number of cd + t cells started to increase beyond the first month, and exceeded lower normal limit at months (mean count, /μl). cd + t cells remained in general low ( o /μl) for the first months, increased moderately by months (mean count, /μl), and approached lower normal values at months (mean count, /μl) [ figure ]. of note, cd +cd ra+ naïve t cells remained significantly impaired (absolute count range, - /μl) in all patients in which they were assessed beyond the st year from transplant. cd + b cells were suppressed for the entire first trimester (mean count at months, /μl), but increased rapidly between and months (mean counts, /μl and /μl, respectively). in haplo-sct with pt-cy, reconstitution of cellular immunity can be achieved at adequate levels by - months following transplant. the observed deficit in the recovery of naïve t-helper cells may be related to a possible effect of pt-cy on thymopoiesis and warrants further investigation. [p ] disclosure of conflict of interest: none. hematopoietic stem cell transplantation (hsct) is a curative therapy for patients with sickle cell disease (scd). hemoglobin a in scd ameliorates the manifestations of the disease and this could be achieved with stable mixed chimerism after a reduced intensity hsct. this study aims to estimate the proportion of patients who develop mixed chimerism after hsct for scd and to characterize its progression in patients who develop it. this is a retrospective cohort study conducted at sultan qaboos university hospital (squh) bone marrow transplant unit in oman. we included all patients with scd who received hsct over the course of years between may to may . patients who received second hsct were excluded. short tandem repeat polymerase chain reaction was used for chimerism assessment. mixed chimerism was defined as − % chimerism at months from hsct. the data was analyzed by r program . . . χ or student t-test were used to assess the impact of acute graft versus host disease (agvhd) prophylaxis, age at transplantation, gender, red blood cell antigen alloimmunization, preparative regimen, and ferritin on the development of mixed chimerism. we included eligible patients. the median follow-up time after hsct was months (interquartile range: . − . months). the mean age at transplant was . years (standard deviation: . ). fifty-nine percent of patients were male. most patients had s/s genotype ( %), followed by s/beta-thalassemia mutation ( %). the indications for bmt were: stroke in %, acute chest syndrome (acs) in %, recurrent vaso-occlusive crisis (voc) in %, stroke and acs in %, acs and voc in %, orbital compression syndrome in %, stroke and moyamoya disease in %, and moyamoya disease in %. the two most frequently used preparative regimens were busulfan/fludarabine/atg in % and thiotepa/treosulfan/fludarabine in %. twenty-five percent of patients developed mixed chimerism at six months after hsct. on follow up of patients with mixed chimerism, % rejected the graft, % developed complete chimerism, and % continued to be in mixed chimerism. preparative regimen and the development of agvhd were statistically significant predictors of mixed chimerism at months (p values: . and . respectively). age at transplant, gender, red blood cell antigen alloimmunization, and ferritin were not statistically significant predictors of the mixed chimerism (p . ). the study confirmed that mixed chimerism can commonly be achieved in patients with scd after hsct and in majority, it remains stable on long-term follow-up. reduced intensity preparative regimen and lack of agvhd predicts the development of mixed chimerism. larger prospective studies are needed to confirm these results. disclosure of conflict of interest: none. there was a majority of male patients ( %). the median hbf level was % ( - ), median monocyte count was . /l (range: - . ), median platelet count was . /l ( - ), median marrow blasts was % ( - ) above patients who were explored by marrow karyotype, % of them had a monosomy . mutations in ptpn were detected in patients. fifty patients ( %) were treated with the bu/cy/mel regimen, whereas patients ( %) received the bu/flu/mel regimen. at years, the overall survival (os) was % ( % ci: - ). nineteen and patients developed vod after bu/cy/ mel and bu/flu/mel conditioning regimen, respectively. the cumulative incidence of agvhd - was % ( % ci: - ). the -year cumulative incidence of relapse and non-relapse mortality was % ( % ci: - ) and % ( % ci: - ), respectively. the median delay of relapse was days (range - ). among relapsing patients, were transplanted twice and one underwent hsct. in multivariate analysis, female donor to male recipient sex-mismatch, cmv status, total body irradiation and ras-double mutation/other additional mutation predicted poorer outcomes. the bu/flu/mel conditioning regimen was associated with a decreased risk of relapse. however, there was no statistical difference for os between the two main preparative regimens, bu/cy/mel vs bu/ flu/mel. our results show that allogeneic hsct may cure approximately % of patients with jmml and are similar to the best results published by other groups. relapse represents the main cause of treatment failure and a second hsct should be proposed. despite a decreased risk of relapse with the bu/ flu/mel regimen, there was no statistical difference in terms of os between the two main conditioning regimens, bu/flu/mel vs bu/cy/mel. disclosure of conflict of interest: none. allogeneic hematopoietic stem cell transplantation (ahsct) is the only curative treatment modality for the majority of pediatric patients with myelodysplastic syndrome (mds) and myeloproliferative neoplasms (mpn). the purpose of this study is to evaluate overall (os) and failure (relapse or death from any cause) free survivals (ffs), non-relapse mortality (nrm) and relapse incidence in children who underwent ahsct for mds or mpn in a single center from turkey. we retrospectively analyzed ahsct carried out in patients (median age: . years; range: . - ; males). thirty four had primary mds and had secondary mds. according to the modified who mds and mpn classification, had refractory cytopenia (rc), , refractory anemia with excess blast (raeb), , refractory anemia with excess blast in transformation (raeb-t) and , juvenile myelomonocytic leukemia (jmml). amongst patients with secondary mds, had been treated for acute myeloid leukemia, had been treated for non-hodgkin's lymphoma and had been treated for acute lymphoblastic leukemia, retinoblastoma and osteosarcoma, each, previously. donors were related in transplantation ( haploidentical transplantation) and the stem cell resources were bone marrow (n = ), peripheral blood (n = ), cord blood (n = ) and bone marrow +peripheral blood (n = ). three-year ffs and os for patients with mds were % and . %, respectively; and for patients with jmml, % and %, respectively. crude incidence of nrm and relapse for entire group were % and %, respectively. ahsct offers durable ffs and os for a significant group of pediatric patients with mds and mpn. less toxic conditioning regimens could result in better results in some patients. disclosure of conflict of interest: none. allogeneic stem cell transplantation in children with autism z antonella, g alessandra, ma beatriz and r vanderson bone marrow transplantation unit, hospital sirio-libanes, são paulo, brazil autism spectrum disorders (asd) are severe heterogeneous neurodevelopmental abnormalities characterized by dysfunctions in social interactions and communication skills, restricted interests, repetitive, and stereotypic verbal and non-verbal behaviors. the etiology of asd remains unknown, but recent studies suggest a possible association with altered immune responses and asd. inflammation in the brain and central nervous system has been reported with microglia activation and increased cytokine production in postmortem brain specimens of individuals with asd. other studies have established a correlation between asd and family history of autoimmune diseases, associations with mhc complex haplotypes, and abnormal levels of various inflammatory cytokines and immunological markers in the blood. the paracrine, secretome, and immunomodulatory effects of stem cells would appear to be the likely mechanisms of application for asd therapeutics. we describe two cases of patients with asd who underwent hsct for acute lymphoblastic leukemia (all) and whose symptoms were markedly decreased like an improvement of social interaction, communication, and behaviors. the first patient is an -year-old girl with asd who was diagnosis with ph-positive all in october (at the end of treatment, bcr-abl remained positive). she underwent a matched sibling hsct in march . the conditioning regimen was total body irradiation (tbi) and cyclophosphamide. during the -month follow-up period, we observed improvement in social interaction, communication, and behaviors (according to the childhood autism rating scale-cars). the second case is a -year-old boy with asd, asperger syndrome, who was diagnosis with all in september . he presented with bone marrow and testicular relapse in may and underwent a matched unrelated hsct in november . the conditioning regimen used was etoposide, atg and tbi. during the -month follow-up period, we observed improvement in social interaction, communication, and behaviors (according to cars). there is no treatment for asd thus every effort to minimize the symptoms are valuable. in both cases, social interaction was significantly increased, and the aggressive behaviors decreased. clinical cases have reported responses in autistic children receiving cord blood cd + cells. several incurable neurological disorders have shown benefits with cellular therapy. thus, autism should be explored as an indication. clinical studies are an immediate need to fully explore its potential in autism. disclosure of conflict of interest: none. conditioning is the initial phase of hematopoietic stem cell transplantation, based on high dose chemotherapy, combined or not with total body irradiation, aiming to eradicate the disease and prepare the environment of the bone marrow for the new cells. conditioning regimens can be characterized as myeloablative or non-myeloablative. during the period of conditioning and immunological reconstitution, signs and symptoms of the gastrointestinal tract are frequent, negatively influencing oral food intake, and may require the use of complementary nutritional therapies, aiming at an adequate caloric intake with the objective of avoiding decreasing in the nutritional status. the study aims to describe the association between the regiment intensity and the nutritional aspects during hospitalization of children and adolescents undergoing allogeneic hematopoietic stem cell transplantation (hsct) at a tertiary hospital. a retrospective study with medical records of patients undergoing allogeneic hsct, aged between and years of age (incomplete) between january and december . data were collected (regimen intensity, clinical signs of mucositis and nutritional therapies used) during the hospitalization and analyzed by the relative risk (rr). sixty-three patients were evaluated, being % male, with a median age of years. nineteen types of conditioning protocols were used. of these, % were high intensive regimen and % were low intensive regimen. the four most applied ( % of cases) were bucy (busulfan + cyclophosphamide) with and without atg (thymoglobulin), as well as cytb (cyclophosphamide+total body irradiation), also with and without atg. mucositis were observed in % of patients, being % grade and grade . the association was positive when analyzed the regimen intensity (myeloablative) with mucositis (rr = . ( . - . )) as well with the use of parenteral nutrition (rr = . ( . - . )). patients showed high prevalence of mucositis during hospitalization decreasing food intake, being necessary to use the parenteral nutrition. myeloablative regimen needed more nutritional therapy intervention when compared to non-mieloablative regiment. results demonstrate that an appropriate nutritional screening tool considering these aspects could help to intervene earlier maintaining an adequate nutritional status. autoimmune cytopenia (aic) is a potentially serious complication of hematopoietic stem cell transplantation (sct). autoimmune hemolytic anemia (aiha) is the most common aic, followed by immune thrombocytopenic purpura and autoimmune neutropenia. aic after sct is considered difficult to treat and associated with high morbidity and mortality. the aim of this cohort study is to evaluate incidence, outcome, potential risk factors and current treatment strategies and to explore the immune dysregulation predisposing to aic. the ebmt-promise database was accessed to identify all pediatric scts between and complicated by aic at our center. potential risk factors (i.e., age, gender, diagnosis, donor type, stem cell source, conditioning regimen) for aic after sct were assessed using univariate and multivariate cox regression analysis. in addition, we summarized treatment decisions of all aic patients. a nested matched case-control study was performed to search for possible biomarkers for aic. of consecutive scts, were complicated by the development of aic (cumulative incidence . %) at a median of months post-sct (figure) . aiha was the most common aic ( %), followed by combinations of two or more aics (evans syndrome, %). non-malignant disease, young age, alemtuzumab serotherapy pre-sct, non-tbi based conditioning regimen and cmv reactivation were associated with aic in univariate analyses. using multivariate cox regression analysis, non-malignant disease (hr . , p = . ), alemtuzumab use (hr . , p = . ) and cmv reactivation (hr . , p = . ) were independently associated with aic (figure) . for patients with cmv reactivation, diagnosis of aic was made at a median of months (iqr [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] ) after detection of maximum viral load. in our nested case-control analysis, serum levels of individual anti-and proinflammatory, and regulatory cytokines did not differ significantly between patients and controls. however, the cytokine profile of aic patients appeared to skew towards a more pronounced th response, compared to controls. firstline treatment, usually with prednisone and/or ivig, or a waitand-see approach led to resolution of aic in ( %) cases. second and subsequent-line therapies, often in combination with continuation of other treatments, consisted of rituximab (n = ), bortezomib (n = ) or sirolimus (n = ) and eventually led to resolution of aic in %, % and % of cases, respectively. overall survival of aic patients was %. in this retrospective cohort study, we identified cmv reactivation post-sct, alemtuzumab use and non-malignant disease as independently associated clinical risk factors for the development of aic. treatment with first-line therapy was mostly insufficient. for patients with severe aic, rituximab, bortezomib or sirolimus can be regarded as promising step-up therapies. figure (bkv) may cause polyomavirus-associated nephropathy or polyoma virus-associated hemorrhagic cystitis in bone marrow-transplant patients.we present patients with bk polyoma virus (bkv) ascociated hemorrhagic cystitis and patients with bk polyoma virus associated hemorrhagic cystitis and nephritis. between and , patients received an allogeneic bmt at acıbadem adana hospital pediatric bone marrow transplantation unit. patients occurred bkv associated hemorrhagic cystitis and nephritis. bkv was detected in the urine analysis and blood by pcr (polymerase chain reaction) in all patients. we presented patients with bkv infection, age ranging from to with a average of . years. they underwent allogeneic bmt due to thalassemia major ( patients), aplastic anemia ( patients) and acute lymphoblastic leukemia ( patients). the patients were treated with hydration, continuous bladder irrigation, ciprofloxacin, and weekly intravesical hyaluronic acid instillation for four weeks, and cidofovir. fourteen patients showed complete resolution of hematuria. one patient with refractory above these therapy also received hyperbaric oxygen and recombinant factor viia (rfviia, novoseven; novo nordisk,bagsvaerd, denmark). hemodialysis was performed in two patients who developed renal failure due to nephritis. bkv is ubiquitously present in the general population. reactivation of infection occurs under conditions of immunosuppression, particularly hsct or renal transplantation, and causes late-onset hc. bkv the management of bkv cystitis and nephritis sometimes may be very difficult and refractory all treatments, we presented our experience of bkv infection and management in transplanted patients in our center. patients with high-risk hematologic malignancies (hrhm) are among those in the highest risk group for developing invasive fungal disease (ifd), especially mold infections. allogenic hematopoietic stem cell transplantation (alsct), acute myeloid leukemia (aml), refractory and relapsed acute lymphoblastic leukemia (all), myelodysplastic syndromes and chronic extensive graft-versus-host disease are considered hrhm. ifd are a major cause of morbidity and mortality in these patients, however, the optimal strategy for antifungal p s prophylaxis in this population is not well defined yet. we performed a retrospective, observational study to investigate documented ifd during antifungal prophylaxis in children with hrhm who were admitted in our unit between and . demographic and clinical data were collected from patient's electronic medical records. all patients were treated with prophylactic voriconazole (vcz) according to our local practice. oral administration was preferred when available. vcz therapeutic drug monitoring (tdm) was not available in our center until june . breakthrough ifd was defined as occurrence of a proven or probable ifd according to eortc/ msg criteria while on vcz prophylaxis (⩾ days of treatment) or within days after discontinuation of prophylaxis. during the study period, hrhm patients were treated with prophylactic vcz in our unit. patients out of developed a breakthrough ifd. patient's demographic characteristics, main diagnosis and treatment are collected in table . initial and maintenance vcz doses are adjusted by weight in all patients except in patient- (adjusted according to vcz plasma level). adherence and tolerance to treatment was excellent in all patients. disclosure of conflict of interest: none. ( ) stable mixed chimerism (smc) when fluctuations of ac were o %; and ( ) mixed progressive chimerism (pmc) when ac were ⩾ %. - hscts performed in patients (pts) were included: children with a median age of . yrs (iqr . - . yrs) at diagnosis and . yrs (iqr - yrs) at hsct received one hsct ( . %), pts two hsct ( . %), and pts three hscts. primary diagnosis were bone marrow failures in pts ( . %), primary immunodeficiencies in ( . %), inborn errors of metabolism in ( . %) and haemoglobinopathies in ( . %). the donor was match related in ( %) procedures, match unrelated in ( %), and haploidentical in ( %); stem cell source was bone marrow in ( %), peripheral blood in ( %) and cord blood in ( %). conditioning regimen (cr) included busulfan in hscts ( . %), treosulfan in ( . %), while hscts ( . %) were conditioned with reduced intensity crs (including low dose of tbi in ); pt did not received cr. gvhd prophylaxis was based on csa/mtx (or mmf) in association with atg ( ) or alentuzumab ( ) ; recipients of tcrαβ/cd depleted haploidentical graft did not received post transplant immunosuppression. engraftment was observed in hscts ( after st , after nd and after rd hsct) after a median of day (iqr - days). acute gvhd occurred in hscts at risk ( %), and it was severe (gr. iii-iv) in ( %), chronic gvhd in ( %). at last follow-up (median . yrs), ( %) pts were alive, while pts are dead for infections (n = ), vod (n = ), c-gvhd (n = ) and vascular event (n = ). figure reported the evolution of chimerism over time. in our experience in children with non malignant disease, cc increased from % to % at subsequent analyses. % of pts with mc at st evaluation became cc, % remained smc, % evolved in pmc, and % rejected. only pts with cc at first time point rejected the graft. this study highlight the extreme variability of chimerism in the early post transplant course of children with non malignant disease and confirmed the relevance of performing serial analysis to monitor and, if necessary, improve graft function. naive t-cells identified by cd ra expression are believed to cause graft-versus-host-disease (gvhd), while cd ra-t-cells are memory cells that provide anti-infection and anti-tumoral effects. depleting cd ra+ naïve cells and retaining memory t-cells in the graft is a novel approach to haploidentical hsct for children. children with high risk leukemia ( aml, all) received cd ra-depleted haploidentical hsct following non-myeloablative conditioning. cell-selection performed on g-csf-mobilized peripheral blood. two cellular products obtained using clinimacs device, infused to each patient: a cd selection and a cd ra depletion from the cd negative fraction. product infused contained a median of . (range . - . ) x /kg cd + cells and a median of . (range . - ) × /kg of cd + cells in the cd -selected s graft. the second product was the cd ra depletion, cd ra +/kg was a median of . × /kg (range - × /kg) and a median . (range . - . ) depletion log of cd ra + cells. median dose of cd ro+ cells (memory t-cells) infused was (range . - ) × /kg. seventeen patients achieved neutrophil engraftment at median of days (range - ) post-transplant. one patient could not achieve engraftment, died at day + due to sepsis. two patients presented secondary graft failure (day + and + ), both received a second hsct. three patients developed agvhd grade ii with gastrointestinal tract involvement, all steroids responsive. three patients presented clinical features of cgvhd. patients have an extensive skin involvement, with hepatic findings in one and pulmonary affection in other, at day + , + and + post. ten of patients ( . %) remain alive in remission with median follow-up (range - ) days post-transplant. eight patients died, due progression at day + , + , + ( presented positive minimal residual disease at hsct), due to infectious complications (days + , + , + , + ) and due to cardiogenic shock at day + . four patients relapsed, of them died afterward with progressive disease. t-cells led immune recovery, achieved values higher than , , and cells/mcl at day , , and respectively. most of t cells were cd +cd ra-(median of , and × /mm respectively on day + , + and + ) and cd +cd ra-t cells (median of , and × /mm respectively on day + , + and + ), while cd + ra+ and cd + ra+ cells remained low recapitulating the cd ra depleted graft composition. six patients presented cytomegalovirus reactivation, one progressed to cmv disease. five patients with hhv- encephalitis. probable aspergillosis in patient (aml-m with secondary graft failure) at day + after second hcst. two cases of toxoplasmosis ( cns, pulmonary). cd ra-depleted haplo-hct showed acceptable tolerability with rapid and sustained engraftment as well as a full donor chimerism, minimal risk of acute gvhd and accelerated inmunologic reconstitution. to note the high incidence of hhv- encephalitis seen. disclosure of conflict of interest: none. collection of peripheral blood stem cells in teenager sibling donors: a single center experience c carvalho , f amado, f bordalo, s ferreira, s lopes, c pinho and s roncon serviço de terapia celular, instituto português de oncologia do porto francisco gentil, epe human leukocyte antigen (hla) compatibility is important in allogeneic haematopoietic stem cell transplantation in order to reduce post-transplant complications; however, siblings only present a % chance of hla-match with the patient. the well-known advantages and the low risk of complications associated to peripheral blood stem cells (pbsc) collected by apheresis made this procedure the first option in teenagers. the aim of this retrospective study was to analyse and characterize the paediatric sibling pbsc donor population assuring safety during the collection procedure, providing a high-quality product and accomplishing patient needs. we consulted the clinical files of donors under years old since - ; a database in excel ® was created to register population characteristics, collection parameters and graft requirements. the informed consent was obtained from parents before procedure. the leukapheresis were performed with a cobe spectra system; since , we use a spectra optia apheresis system. the donor/patient weight ratio (proposed by styczynski et al.) was determined for each pair. the collection was programed based on clinical and analytical donor's features as well as transplant requirements. the analytical assays were done by a certified laboratory. we performed pbsc apheresis in healthy donors, females and males ( table ) . all of them started on the th day after mobilization with granulocyte colony-stimulating factor (g-csf) administered subcutaneously, bidaily. the weight ratio was o in eight situations. most of donations were performed by peripheral vein; a central venous catheter (cvc) was placed into a femoral vein in six adolescents. a median of ( ) ( ) ( ) blood volemias were processed during ( - ) minutes; the anticoagulation used was citrate+heparin (ratio : ). in general, one-collection day was enough to obtain the number of cd + cells required; six donors had to perform a nd collection. in cases, we cryopreserved the exceeding cells after graft infusion. the procedure was well tolerated, with only adverse reactions registered (one hematoma in the puncture local; one paraesthesia due to hypocalcaemia induced by citrate). no blood products were used after the procedure or needed for the priming of the extracorporeal circuit. so far, no serious long-term adverse events were observed. table . median (range) of donors and leukapheresis products data. our long lasting experience shows that pbsc collection in the teenage population is safe and feasible, allowing us to obtain a high-quality product for the patients. there were no adverse events associated with the g-csf mobilization or cvc placement which is different from the experience of other groups. we recognize that leukapheresis by peripheral vein is a lengthy procedure but no complaint was reported to the collection team. [p ] disclosure of conflict of interest: none. correspondence between clinical and hystological grading of gastro-intestinal grading acute graft versus host disease in children m faraci , a rizzo , p gandullia , s arrigo , , a barabino , e lanino , s giardino and c coccia hematopoetic stem cell transplant unit, institute g gaslini, genoa, italy; pediatric department, institute g gaslini, genoa, italy; gastroenterology and digestive endoscopy unit, institute g gaslini, genoa, italy; gastroenterology and digestive endoscopy unit, institute g gaslini, genova and department of pathology, institute g gaslini, genoa, italy diagnosis of gastro-intestinal acute graft versus host disease (gi-agvhd) is frequently confirmed by apoptosis findings on mucosal biopsies. aims of this single center retrospective study is to evaluate the correlation between clinical and histological grading of gi-agvhd in children undergoing allogeneic haematopoietic stem cell transplantation (allo-hsct), and to describe histological findings obtained by gi endoscopies in order to evaluate usefulness in the diagnosis of gi-agvhd. allo-hscts were performed in our department between january and december . gi biopsies were performed in pts ( . %) because of suspected gi-agvhd. pts were transplanted for malignant ( . %) and for not malignant diseases. the median age at hsct was . years ( . - . ). pts ( %) received myeloablative and ( %) reduced intensity conditioning regimen. pts ( . %) received an unrelated donor (ud), pts a related donor (rd) ( . %), and an haploidentical donor ( . %). at onset of diarrhea, microbiological examinations of stool were performed and pcr research for cmv, adenovirus, hhv , ebv were evaluated in blood and in mucosal biopsies. mucosal biopsies were obtained with esophago-gastro-duodenoscopy in pts ( . %),esophago-gastro-duodeno-colonscopy in ( . %), pancolonscopy in ( . %), flexible sigmoidoscopy in ( . %), and rectal suction biopsy in pts ( . %). all mucosal biopsies, except in case of rectal suction, were obtained under sedation. the interval between mucosal biopsies and onset of gi acute symptoms was days (from − to days). biopsies were taken from different sites in the gi tract, were stained using hematoxylin-eosin and evaluated using histological grading of agvhd. in these pts the maximum grade of agvhd was: grade in one ( %), grade in ( %), and grade in pts ( %). at time of histological evaluation, diarrhea was the most common gi symptom ( . %); children had also cutaneous agvhd and hepatic agvhd. pcr-cmv was positive in mucosal biopsies obtained with pancolonscopy, pcr-adenovirus in other obtained with upper and pancolonscopy, pcr-hhv in rectal biopsies, and pcr-ebv in one with upper and pancoloscopy. the comparison between clinical and histology grading of gi-agvhd is shown in table . mucosal biopsies were positive in / pts evaluated with esophago-gastroduodenoscopy ( %) (grade agvhd), in / pts undergone esophago-gastro-duodeno-colonscopy (grade in and grade in ), in / ( %) who received a pancolonscopy (grade in , grade in , grade in ), and / ( %) of rectal biopsy obtained by sigmoidoscopy or rectal suction biopsy (grade in , grade in , grade in , and grade in ). one patient developed duodenal intraparietal hematoma after upper endoscopy. in our experience, we did not demonstrated a overall correlation between clinical and histological grading of agvhd showing that hystological examinations underestimated the grade mild or moderate of agvhd. we confirmed , that rectal biopsies represent to be more effective and safe diagnostic method for the confirm of diagnosis of gi-agi. during the past few decades, hematopoietic cell transplantation (hct) as a treatment modality for primary immunodeficiencies (pid) has undergone remarkable advancement mainly due to better availability of alternate donors resulting in increase in not just matched unrelated donor (mud) but also increased haploidentical (haplo) and cord blood transplants (cbt). additionally, refinement of the conditioning regimens and better graft versus host prophylaxis have presumably led to better survival outcomes. however, a literature gap is identified in evaluation of these outcomes in general with respect to donor and conditioning regimens. we conducted a systematic review by performing a comprehensive search of the pubmed and ovid library from its inception to august . mesh terms included 'primary immunodeficiency (immunodeficiencies)', 'stem cell transplant', 'bone marrow transplant' and 'hematopoietic cell transplant'. all pid studies which used hct as a treatment modality were included. experimental cellular therapies were excluded. both cellular immunodeficiencies (e.g. scid, was, a-t), and innate immunity disorders (e.g. ifngr, cgd) were included in the search. reviews, case reports, meta-analysis and non-english language articles were excluded from our electronic search. publication bias was excluded by performing a methodological search of unpublished conference abstracts from the annual meetings of cis, aspho, asbmt, ebmt, and siop from to . the data were analyzed considering the outcomes -overall survival and gvhd. studies fulfilled the strict selection criteria for the electronic search comprising of pid patients. in majority of the hcts, a myeloablative conditioning regimen (mac) was utilized ( % of the evaluable) but a shift towards more reduced intensity conditioning (ric) was observed in the later years. cbts were identified. % of patients developed some degree of acute gvhd, whereas chronic gvhd was identified in % of the patients. total number of haplos was . overall survival was found to be % post-hct. a meta-analysis could not be performed due to the heterogeneity of both the predictor variable data (combined stem cell sources were also used for hct) and due to the extremely small number of the patients when categorized in subgroups (e.g. for omenn syndrome, rag deficiencies). this is the largest study of hcts in pid, and we observe that alternate donor hcts have increased significantly over the past decade for the treatment of pid. while the incidence of chronic gvhd was low, acute gvhd still remains a problem in about a third of the pid patients transplanted. disclosure of conflict of interest: none. hepatic veno-occlusive disease (vod) is a common and serious complication of hemotopoietic stem cell transplantation (hsct) in children. we aimed to assess prospectively the use of prophylactic defibrotide in pediatric patients undergoing hsct. in this study, patients who underwent hsct were given defibrotide prophylaxis as mg/kg per day in four divided intravenous infusions over h, starting on the same day as the pretransplantation conditioning regimen. the mean duration of use of defibrotide is days as a prophylaxis. in this study, patients were recruited, male patients and female patients, with the average of . years, range - ; % infants, % children and % adolescent. there were patients with thalassemia major, patients with leukemia, patients with aplastic anemia, one patient with diamond blackfan anemia, two patients with congenitale dyserythropoetic anemia, one patient with osteopetrosis, four patients with famial hemophagocytic lymphohistiocytosis, two patienrs with severe immune deficiency and one patient with kostman syndrome. all transplants were allogeneic. no serious side effects were seen. in eight patients developed clinical vod (seattle criteria). in these patients, defibrotide dose was increased to a treatment dose of - mg/kg per day. one infant patient with kostman syndrome died due to hepatic and pulmonary veno-occlusive disease. after months of follow up, patients who developed vod are being well and no patient have transplant related complications. hepatic veno-occlusive disease, which is caused by hepatocyte and sinusoidal vessel endothelium damage, can ocur early after hsct, and in its severe form, may lead to liver faillure, hepatorenal syndrome, portal hypertension, and eventually death from multiorgan faillure. in this prospective study, we demonstrated that the use of defibrotide is safe and effective in preventing and treating vod in pediatric patients at high risk. immune reconstitution (ir) is critical for clinical outcome after allogeneic hematopoietic stem cell transplantation (hsct). host and proceeding-related factors affect the ir dynamics and survival. isolated ir parameters are commonly correlated and proposed to predict clinical outcomes after hsct, but these approaches only confer prognostic value at single time points or for single markers. we aim to demonstrate an appropriate methodology to assess the capability of combined serial measurements of lymphocyte subsets to reflect the impact of infections on ir after paediatric hsct. retrospective data of patients receiving a first hsct for any indication with any cell source in the paediatric hsct program from to were included. to characterize the kinetics of immune reconstitution, cd +, cd +, cd + t-cells, b-cells, nk-cells and their naive and memory subsets were measured and analysed at various time points at years post-hsct to stablish a joint model for the evolution of cell subpopulations. slope per month (cellular increase or decrease) of each lymphocyte subsets were calculated and compared with clinical outcomes and cumulative risk of infections. a total of children (range from - y.o. median y.o.) were included, with cb (n = ) pb (n = ) and bm (n = ) as cell sources. the cumulative incidences after early period were % for viral infections (ebv %, cmv %, bk %, adv %) and % for bacterial infections. data on ir were available for %, of the diseasefree survivors. in a exploratory multivariate analysis we detected mainly differences in cd +, cd +cd ro+ memory and nk cells at year after hsct, with dependent tendency according on the cell source and hla compatibility. analysis of the slope tendency patterns were stablished for the analysis of the impact of infections in the ir. delay in cd + and cd +ra+ appearance (mean slope/m = − . % and − . % respectively) remarks the ir profile for bacterial infections, and delayed in nk, cd and cd ro+ (− . %, − . %, − % respectively) for overall viral infections. additional correlations allow differences in ebv (cd +ra+ high mean slope/m = . %), cmv (delayed in cd ro+ slope/m = %), and bk infection (cd +ra + plus cd ro+ and nk high mean slope/m = . %, % %). understanding the dynamics of reconstitution by integrating information from the monitoring of lymphocyte subpopulations allows the establishment of kinetic profiles that may help to evaluate the risk of infections and adjust infection prophylaxis in the follow-up of transplanted patients. mortality rate in hsct patients admitted to intensive care unit (icu) is still as high as % to %. this rate increases when respiratory complications progress to acute respiratory failure (arf) requiring mechanical ventilation (mv). the aim of this study was to determine the feasibility and effectiveness of early continuous positive airway pressure (cpap) delivered in a pediatric hematology-oncology ward to prevent occurrence of arf requiring mv. we retrospectively analysed children treated with cpap in our pediatric hematology-oncology ward between october and october . thirty-two patients received cpap delivered with helmet during the study period. data were available for patients, males and females, median age years [range - ]. eighteen patients underwent allogenic hsct: from sibling donor, from matched unrelated donor, from haploidentical family donor, from cord blood unit. seven patients had a malignant disease: all, aml, ewing sarcoma. infectious pneumonia was the main cause of arf in / patients ( . %): viral pneumonitis ( rhinovirus, parainfluenzae virus, respiratory syncitial virus and cmv). five patients had proven/ probable invasive fungal infection according to eortc criteria ( aspergillosis and mucormycoses). other causative agents were pneumocystis jiroveci ( ), bacillus of calmette and guerein ( ), toxoplasma gondii ( ) and st. mitis ( ) . non infective causes of arf were acute transfusion related lung injury ( ), hemorragic alveolitis ( ), cryptogenic organizing pneumonia ( ), tumor lysis syndrome ( ), and alveolar oedema due to renal failure ( ). according to chest imaging, / patients ( %) presented with pulmonary consolidations, while % had both interstitial infiltrates and pulmonary consolidations. at baseline median neutrophil count was . × /μl (range - . × /μl), mean heart rate bpm, pulsiossimetry saturation in room air %. h-cpap was applied in / patient with a curative aim, in / patients as palliative support to reduce respiratory distress. median positive pressure delivered was cmh o ( - cmh o), median fio was % ( - %). h-cpap was applied for a median of days . no patient failed h-cpap because of agitation or adverse events (skin breakdown, conjunctivitis, gastric distension or epistaxis). ten patients were transferred to icu ( . %), / because of hsct complications. median icu stay was . days ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) . only patients required mechanical ventilation, in cases associated to ecmo. nether psao in room air (p . ci %) nor cpap level (p . ci %) correlated with the need of icu admission. patients requiring higher fio during cpap demonstrated a not statistically significant trend to higher icu admission rate (p = . ).there was a higher rate of mv in patients with higher cpap fio level (p = . ). mv prolonged icu stay (p . ). cumulative mortality was . % ( / ); only patient died in icu ( %), because of post hsct parainfluenza virus pneumonitis requiring ecmo. helmet cpap delivery in pediatric hsct ward is feasible and safe, both for curative and palliative aim. if applied early, cpap could reduce icu admission rate for mv and icu mortality. veno occlusive disease (vod) and graft versus host disease (gvhd) are both dreadful complications of hematopoietic stem cell transplantation (hsct). although they have different clinical signs, it is suggested that they share similar pathophysiological pathway. defibrotide is used in the treatment of vod for a long time but it is very less known about its effect on gvhd. in this study, we analyzed a 'high risk' patient population in pediatric hsct to show the effect of defibrotide on acute gvhd. between june -august totally 'high risk' pediatric allogenic hsct procedures were enrolled in this study. 'high risk' definition involves busulphan/ melphalan usage in conditioning regimen, second myeloablative hsct, pre-existing liver disease, allogenic hsct for leukemia with second relapse and diagnosis of hemophagocytic lymphohistiocytosis (hlh) or osteopetrosis. defibrotide prophylaxis group (n = ) received mg/kg/day per day and continued for at least days after transplantation. the control group (n = ) received only continuous infusion of low-dose heparin until days after transplantation. for the comparison between groups, the fisher's exact test was used. all analyses were performed using spss and p-value of . was considered statistically significant. we analyzed totally hsct procedures with different diagnosis; beta thalassemia major, leukemia, hlh, primary immunodeficiencies, osteopetrosis, fanconi aplastic anemia (faa), myelodysplastic syndrome, neuroblastoma, congenital amegakaryocytic thrombocytopenia, krabbe disease, aplastic anemia, hypereosinophilic syndrome and sickle cell disease. all the procedures meet the 'high risk' definition; most of them (n = ) have busulphan for conditioning, also there are hlh and osteopetrosis patients, neuroblastoma patients had the second myeloablative regimen, faa and aplastic anemia patients had pre-existing liver disease, and of the leukemias had beyond second relapse. the mean age was . years old ( . - . ), hsct performed from match sibling donor (msd) ( %), hsct from match family donor (mfd) ( %), hsct from match unrelated donor (mud) ( %) and hsct from haploidentical mother ( %). we especially focused on gvhd and vod. totally vod cases ( %) in these hsct procedures were detected. only two of them detected in the prophlaxis group ( %) and cases in the control group ( %). there were cmv reactivation cases detected in hsct procedures ( %). in the prophlaxis group there were cases ( %) and in the control group cases ( %). we detected acute grade i-iv gvhd cases in hsct procedures ( %). only of them were in the prophlaxis group ( %) and cases were in control group ( %). the prophlaxis group's agvhd ratio was significantly lower than the control group (p = . ). defibrotide for vod prophylaxis is confirmed by several studies, but its benefits for agvhd is not clear. in this study, we show the significant effect of defibrotide on agvhd. we speculate that the protective effect of defibrotide on both vod and agvhd could be explained by the similar pathophysiology of these complications. we need larger studies on the pathophysiological pathways, then we could invent more effective interventions. disclosure of conflict of interest: none. conventional extracorporeal photopheresis (ecp) has proven efficient for the treatment of graft-versus-host-disease (gvhd) but is limited to patients with sufficient body weight. a mini buffy coat ecp (mini-ecp) 'off line' technique that allows treatment of small children has been developed, using various methods for mononuclear cell (mnc) separation from whole blood. we present treatment of low body weight child with mini-ecp 'off line' technique using sepax system for mononuclear cell (mnc) selection and macogenic irradiator. a toddler with juvenile myelomonocytic leukemia (jmml) developed acute gvhd, after a matched unrelated stem cell transplantation (sct) performed at the age of months. acute gvhd of the skin occurred three months after sct and responded to high dose steroids, but recurred six months after sct (biopsy of the skin confirmed acute gvhd) together with gvhd of the liver. because of the resistance to steroids and cyclosporine, mini-ecp was introduced as therapy. the patient weighed kilograms. blood was collected from tunneled central venous catheter, and collected volume was replaced with saline infusion. the cord blood collection bag (macopharma, france), which contains ml citrate phosphate dextrose (cpd) anticoagulant solution was used for whole blood collection. whole blood was processed using sepax system separator (biosafe, switzerland), and final volume of buffy coat was set on ml. extracted buffy coat was transferred into the uv-a illumination eva bag (macopharma, france) and diluted with saline solution up to ml. methoxypsoralen (gerot, austria) was injected directly into the uv-a illumination bag, and cells were irradiated by the uv-a illumination device macogenic (macopharma, france). irradiated cells and autologous residual blood were reinfused back to the patient. during the whole procedure patient's vital signs were monitored. ecp procedures were performed times per week for weeks, followed by times per week at weeks intervals for months. in month period mini-ecp procedures were performed. median of collected whole blood was ml (range - ). median of total nucleated cell (tnc), and mononuclear cell recovery after sepax separation were . % (range . - ), and . % (range - ), respectively. median of hematocrit in final irradiated product was % (range . - %). patient was reinfused with median of . (range . - . ) tnc × /kg bw, and median of . (range . - . ) lymphocyte × /kg bw. after one month of ecp together with steroids and cyclosporine, gvhd of the skin improved, and the steroids were gradually weaned, with no recurrence. gvhd of the liver showed no improvement, and other therapies had to be introduced, but without steroids. for the first time in croatia, mini-ecp was performed in a child with gvhd, in whom conventional ecp could not be used because of insufficient body weight. mnc separation using automated closed system sepax separator has proven efficient and safe. mini-ecp treatment was continued for three months, without technical difficulties. positive effect was experienced concerning the skin gvhd, but not the liver gvhd. after the first experience in our country, in future we plan to use this technique for low-weight patients or patients with contraindications for apheresis, which are in need of second-or third-line therapy for gvhd. disclosure of conflict of interest: none. gonadal failure represents one of the late effects of haematopoietic cell transplantation (hsct) with a negative impact on quality of life in young patients (pts) undergoing hsct , . the aim of this retrospective multicentre ebmt study was to assess gonadal function in untreated pts undergoing allogeneic hsct between to years (yrs) of age, after a preparative regimen with busulphan (bu) or treosulfan (treo). eighty-seven pts ( females, males) were reported from out of contacted ebmt centers: / ( %) received allogeneic hsct during pre-pubertal and / ( %) in pubertal phase. of the pts, ( . %) received bu in myeloablative dose [ pre-pubertal, (median age of . yrs) and pubertal, (median age of . yrs)] and pts ( . %) received treo ( in pre-pubertal and in pubertal period). underlying diseases were primary immunodeficiency ( . %), chronic myeloid leukemia ( . %), myelodisplastic syndrome ( . %), familial haemophagocytic lymphohistiocytosis ( . %) and shwachman-diamond syndrome ( . %). / of prepubertal pts ( %) developed spontaneous puberty ( . % in the bu group and % in treo group). / ( %) females undergoing hsct during puberty completed their pubertal development ( . % in bu group and % in treo group). none of females ( / ) with bu during pre-pubertal phase developed spontaneous menarche (sm), while . %( / ) of females who received bu in pubertal period had sm. all females (n = ) treated with treo during pubertal phase had sm ( %). for both conditioning regimens, the . % ( / ) s of females treated during the puberty experienced sm. among the remaining females (for pts the information is missing) who did not developed sm, received hrt . yrs after hsct and of them had ovarian recovery after a median of . yrs from hsct ( . - . ). the median age at last follow up was . and . yrs in bu and treo pre-pubertal group, and . and . yrs in bu and treo pubertal group respectively. in the pubertal group, females ( . %) are still receiving hormonal replacement therapy (hrt) ( in the bu group and in treo group). pts ( . %) had spontaneous pregnancy. no problems in newborns are reported. sperm analysis was performed in . % of pubertal pts ( / ) of males, and % (n = / treated with bu) were azoospermic (data regarding pts were missing). the sperm analysis was repeated in half of the males. until now no paternity was reported. in this experience, the pubertal development in pts who received treo (n = ) was normal, and in the bu group the majority of females ( %) had normal puberty. the rate of sm is higher ( %) in females after treo than bu ( %). the hrt is ongoing at last follow-up in % of females treated with bu and in % of those who received treo. our data suggests that treo may have a better outcome than bu in young girls receiving allogeneic hsct and larger studies are warranted. male patients require longer follow-up. prevention in patients transplanted from partially matched donors. we report the single centre experience in haploidentical sct. in years - in the department of pediatric bmt, oncology and hematology at wroclaw medical university, children underwent sct from partially matched, haploidentical parental donors. graft manipulation in patients consisted of cd sel, in patients the cd immunomagnetic depletion (tcd-cd ) was performed, and in -tcr alpha-beta depletion (tcd-ab). we analysed the impact of type of manipulation procedure, conditioning regimen, demographic factors, and kir genotype on survival and probability of neutrophil recovery. the probability of engraftment and neutrophil recovery was % vs % in cd sel group (p = ns). probability of year overall survival in the tcd group was similar to the cd sel group ( % vs %, p = ns). in the tcd patients, neither use of busulfan vs treosulfan, nor kir genotype, nor donor sex had noticeable impact on sct result and survival. patients transplanted after tcd due to non-malignant disease had higher survival probability, than those with malignancies ( % vs %, p = . ). the trm in tcd patients was reduced in comparison to cd sel ( vs %, p = . ). the trm after tcd resulted mostly from severe viral infections in tcd-cd patients. in / tcd patients spontaneous acute, skin (stage ) gvhd was diagnosed and successfully treated. two patients received unmanipulated donor lymphocyte infusions (dli) and developed severe acute steroid-resistant (grade ) gvhd, in one of them with fatal outcome. tcd methods are superior to cd sel due to significant reduction in treatment related mortality. haploidentical sct after tcd can result in durable engraftment, but warrants intensive post-transplant monitoring for infectious complications and cautious approach to dli therapy. disclosure of conflict of interest: none. median of days for neutrophils in both groups, for platelets ( in ptcy, in αβ+cd +/cd +depleted, p . ). donor chimerism was complete in patients ( . %). in αβ +cd +/cd +depleted group, patient rejected ( . %: primary and secondary reject, , , and days after haplo, respectively) and were rescued with a second transplant. seven patients ( %) developed acute (a-) gvhd in ptcy group (grade - in ; grade - in ), compared to one ( . %: grade ) in αβ+cd +depleted group (p . ). among patients at risk, out of in ptcy group developed chronic (c-) gvhd ( . %: score- , overlap, score- ), compared to / patients in αβ+cd +/cd +depleted group (p . ). the cumulative risk of cmv-reactivation was % and % in ptcy and αβ+cd +/cd +depleted groups, respectively (p . ). t-cell reconstitution was significantly different in the two groups,with a median absolute number of cd + +cd -and γδ+cd + higher in αβ+cd +/cd +depleted group on day + (p . ) and a median number of cd +cd + higher in ptcy group on day+ (p . ). length of hospitalization was shorter in the αβ+cd +/cd +depleted group, with a median time from haplo to discharge of days compared to days in the ptcy group (p . ). some children have not donor and an urgent need to proceed to transplantation because of disease status. we reviewed the role of haploidentical transplantation in children and report our single center experience. ten children were transplanted from haploidentical family members donors (median age: . years). we performed alfa beta t cell depleted transplantation in three patients and unmanipulated bone marrow transplantation with posttransplant cyclophosphamide in seven patients. the diagnosis were eight high risk leukemias (three all and five aml) and two severe aplastic anemia. patients were myeloablative conditioned with cyclophosphamide, fludarabine and total body irradiation in aplastic anemia received alfa beta t cell depleted grafts with a median cd cell dose of . × /kg (range: . - . ) and busulphan, cyclophosphamide in high risk leukemias received unmanipulated bone marrow grafts with posttransplant cyclophosphamide in rd and th day of posttransplant with a median cd cell dose of . × /kg (range: . - . ). median follow up of our patients months. six of patients are alive and in disease free follow up. four patients were relapsed and dead median . months of transplantation. the rate of relapse was % for leukemia patients in remission and % for patients with active disease. myeloablative conditioning regimen followed by haploidentical bone marrow transplantation with posttransplant cyclophosphamide may be an option in high risk leukemia patients need urgent transplantation because of desease status who have not donor. table . all patients received hd-cy mg/kg on d+ and d + . cyclosporine a mg/kg/d i.v., then mg/kg/d p.o. adjusting for blood levels - ng/ml and mycophenolate mofetil mg/kg times daily po were started on d+ . mmf was discontinuated on d+ , csa-after d+ . all pts received anti-microbial prophylaxis for bacteria, fungal, herpes infection and pneumocystis according to institutional practices. analysis for donor chimerism and mrd were performed at d+ , + , + , + . pts, donors and stem-cell harvest characteristics are described in table . pts had high risk hematological malignancies, and relapsed after auto-sct neuroblastoma (hr-nb). pt was transplanted in st cr (aml m ) and others in nd cr. pts had full engraftment (neutrophil engraftment at , and days). pt (hr-nb) was concerned as a primary failure for achieving neutrophil and platelet engraftment by d+ , despite of complete donor chimerism in bone marrow. he was transplanted additionally with the same donor at d+ after st transplant. pt died before engraftment at d+ (fulminant ps. aeruginosa-sepsis). pts remain alive in cr ( ndcr-aml and st cr-m aml) with follow-up of and days ( / / ) without cgvhd with complete donor chimerism. pts relapsed after d+ ( were transplanted in nd cr-flt aml and nd cr-nb) and died. pt died because of infectious complication at d+ (transplanted in d cr-all). / pts had grade acute gvhd. hla-haploidentical hsct with post-transplant t-cell in vivo repletion grafts by using hd-cy is feasible and effective in children with hr-haematological malignancies. [p ] who were match unrelated donor. thalassemic reconstitution occurred in three patients. acute graft-versus-host disease (gvhd) of grade ii-iv occurred in % and chronic gvhd in %. acute and chronic gvhd were seen more frequently in patients with class - compared to class . mortality rate was also higher in these groups. seven patients died. one patient died on post-transplant day due to intracranial bleeding. the other patients with chronic gvhd died between and days, on average days post-transplant. these data suggest that allogeneic bmt remains an important treatment option for children with beta-thalassaemia major, particularly when compliance with iron chelation is poor. the society to support children suffering from cancer, also known as mahak, was set up in as a non-governmental and non-profit organization. in the past two decades, the organization has attracted a vast public support and fulfilled a great part of its mission which is to support children with cancer, reduce the child mortality rate and create an appropriate environment that empowers families who have children with cancer. pediatric stem cell transplant also is used to treat many types of conditions affecting children and adolescent, including cancer and certain hematologic, immune reconstitution inflammatory syndrome (iris) is a clinical condition emerging after immune recovery of an immunocompromised status, mostly after the initiation antiretroviral therapy (art) in human immunodeficiency virus (hiv) infected patients, but also in several other settings, such as the recovery from the severe combined immunodeficiency (scid) status after hematopoietic stem cell transplantation (hsct). herein, we report a patient transplanted for scid who developed iris for two times, namely shortly after transplantation and after donor lymphocyte infusion (dli) ( table ) in our patient, t cells passing from the donor probably contributed to the immediate post-transplant increase in the size of granulomas. this inflammatory response waned after the institution of immunosuppressive and methylprednisolone therapy. however, immunosuppressives were stopped due to lowered chimerism at follow-up, and the inflammatory response re-appeared after administering stem cell support containing a large amount of t cell from the donor for dli purpose. although the mechanism by which dli results in clinical responses is unclear, it is presumed to be a t cellmediated process. several studies have been performed to strengthen our understanding of the immunopathogenesis of iris. while some of those studies put forth t cell-associated causes, others implicated cytokines and non-t cells. the reaction that developed in our patient is suggestive of t cellassociated causes. immune reconstitution inflammatory syndrome remains a poorly understood entity. the dli procedure in our case provides a unique clue supporting a t cell mediated process. pediatric transplant teams need to be s aware of the previous iris phenomenon of bcg-adenitis while making the decision of dlis. [p ] disclosure of conflict of interest: none. pediatric patients treated with a hematopoietic stem cell transplantation (hsct) often suffer from late side effects caused by the treatment. the aim of this study is to investigate the late effects of a hsct on dental development. in addition, patients and parents awareness on this topic was investigated. young adults treated and followed at the ghent university hospital who were under the age of y at the time of hsct were examined clinically and radiographically (planmeca promax d). transplants ( autologous/ allogeneic) were done for malignant disease in pts. eight patients received a hsct for a non-malignant disease. twelve patients underwent a conditioning regimen with total body irradiation (tbi), patients with busulfan and patients with other chemotherapeutic agents. patients were o y, patients were - years and patients were years at hsct. every patient was evaluated on dental agenesis, microdontia and rootcrown ratio. patients and their parents were asked about their knowledge and interest for dental screening at the follow-up clinic using a questionnaire. overall, the prevalence of agenesis and microdontia of one or more dental elements is respectively . % and . % in our study population. . % of patients have a strongly aberrant root-crown ratio of at least one element. patients treated o years of age show significantly more microdontia ( . %; po . ) as well as agenesis ( . %; p o . ) compared to patients treated at an older age. the first premolar of the mandible is the most vulnerable element for agenesis as well as for microdontia. more microdontia is found in patients treated with a busulfan conditioning regimen compared to the other conditioning regimens ( . % versus %). patients older than years, treated with busulfan have statististically more microdontia compared to patients y treated with tbi conditioning regimen (p = . ). there was no difference of the conditioning regimens on agenesis nor on root-crown ratio. almost all patients/parents find it important to receive information about the dental late effects of a hsct and are interested in dental screening at the follow-up clinic. treatment with hsct has an explicit negative impact on dental development. the degree of this effect depends on age at hsct and used conditioningregimen. dental follow-up of these patients is essential and should be incorporated in the follow-up program. disclosure of conflict of interest: none. importance of body composition in the outcome of hematopoietic stem cell transplantation in elderly patients l koch , n hamerschlak , r garcia , c prado , c silva and a pereira hospital israelita albert einstein the loss of muscular mass is a well recognized cause of the decline in muscle strength and functionality that accompany the aging process. in , irwin rosenberg proposed the term 'sarcopenia' to describe the decline in muscle mass associated with aging. changes in body composition after hematopoietic stem cell transplantation (hsct) have been the subject of previous studies. immunosuppressive therapy and corticosteroids are known to alter skeletal muscle metabolism. infections and graft-versus-host disease (gvhd) that can occur after hsct may also affect body weight and composition. therefore, both the treatment and complications after hsct exert large negative effects on lean muscle mass, especially in elderly patients. patients with hematologic malignancies are usually well nourished before undergoing hsct. objective: the aim of this study is to determine in an elderly population whether parameters of body composition could be correlated to outcomes after hsct. we performed a retrospective longitudinal study through review of medical records of patients ⩾ years old undergoing hsct at hospital israelita albert s einstein, from to , that were subject to tomography scans (cts) in a period ranging from days before and days after hsct. table . there were no differences between groups with respect to age, gender, diagnosis, stage of disease, and source of stem cells. in ly patients, the quantity of peripheral cd + cell dose (× /kg) infused was different between groups (group ly-ct the incidence of nf was significantly higher in group mm-g ( ( . %) vs ( . %); p = . ). no differences were observed in the incidence and severity of mucositis, first day and duration of fever, documented bacterial infections or readmission rate between mm patients groups. this study suggests that in at home asct, the use of piperacillintazobactam prophylaxis significantly reduces the incidence of neutropenic fever and hospital readmission in patients with ly, and also that no administration of g-csf in mm patients reduces significantly the incidence of neutropenic fever. disclosure of conflict of interest: none. [p ] allogeneic stem cell transplantation (sct) has been recognized as a curative treatment for patients with wiskott-aldrich syndrome (was). in sct for was, myeloaberative conditioning (mac) has been indicated to avoid a mixed chimera. however, risk factors for a mixed chimera in patients with was who have received sct have not been evaluated. here, we analyzed the outcomes of sct and risk factors for a mixed chimera in patients with was who underwent sct in japan since . we reviewed medical records of consecutive was patients who received sct since january who were registered with the japan society for hematopoietic cell transplantation. the age of the patients at transplantation ranged from months to years, and the mean age was . years. the origin of the stem cells was related bone marrow (bm) or peripheral blood stem cells (pbsc), unrelated bm or pbsc, and unrelated cord blood (cb) for , and patients, respectively. a preparative conditioning regimen consisting of mac was provided to patients, and reduced-intensity conditioning was provided to patients. fifty-one patients received prophylaxis against graft-versus-host disease (gvhd) with cyclosporine in combination with methotrexate (mtx) or a steroid, and patients received tacrolimus (tac) with mtx or a steroid. chimerism analysis had been performed in patients. neutrophil engraftment was achieved in patients ( . %). the engraftment rate was significantly higher in patients who received tac for gvhd prophylaxis, (p = . ) overall survival rate was significantly higher in patients with complete chimerism than in patients with mixed chimerism ( . ± . % and . ± . %, respectively, p = . ), though there was no significant difference in stem cell sources. using multivariate analysis, the rate of complete chimerism in patients who received mac including cyclophosphamide (cy) at more than mg/kg was significantly higher (p = . ) than the other conditioning. not only patients with mixed chimerism but also patients with complete chimerism were complicated with auto-immune diseases. in this study, achievement of complete chimerism after sct was important for survival in patients with was. we found that patients who underwent mac including cy at more than mg/kg had a higher rate of complete chimerism. we also found a higher neutrophil engraftment rate in patients who received tac for gvhd prophylaxis. thus, mac including cy at more than mg/kg and tac for gvhd prophylaxis are optimal conditions of sct for patients with was. disclosure of conflict of interest: none. adenosine deaminase (ada) deficiency is an inherited autosomal recessive immunodeficiency which represents about - % of scid. since we diagnosed patients affected by ada-scid: underwent hematopoietic stem cell transplantation (hsct), were treated with replacement therapy with peg-ada and received gene therapy; patients died before or after treatment. maternal t lymphocyte engraftment is frequently detected in scid patients, but this is never been found in ada deficient patients. a -months-old italian girl, from non-consanguineous parents, presented to our hospital with a history of frequent bronchiolitis associated with dermatitis, mycosis, hypogammaglobulinaemia, marked lymphopenia (t cells cd , /mmc; cd /cd , /mmc; cd / cd , /mmc, b cells . /mmc, and nk cells, /mmc) and in vitro absence of proliferative response to pha. level of immunoglobulins was almost normal (igg mg/dl, iga mg/dl, igm mg/dl). high levels of toxic metabolites were found: axp, . micromol/ml rbc; daxp, . micromol/ml rbc; %daxp, . . ada activity in rbc lysates was abnormally high for scid-ada ( . u/g hb). molecular analysis confirmed diagnosis: the sequencing of exon revealed two mutations: a missense mutation previously reported called p.ser leu (c. c t) and a new missense mutation defined p. leu pro (c. t c). t-cells str analysis of patient showed . % maternal t lymphocytes engraftment never reported before in ada-scid patients. the girl was transferred to the isolated bmt unit and the respiratory symptoms progressively improvement. replacement therapy with peg-ada was started immediately at a dose of u/kg/twice per week. ig therapy was started at a dose of mg/kg every two weeks. after three months of treatment patient showed an increase in t cells count (cd , /mmc), and a decrease of toxic metabolites: axp, . micromol/ml rbc; daxp, . micromol/ml rbc; %daxp, . maternal t-cell engraftment persists, despite a good response to the peg-ada therapy. the last examination before hsct reveals maternal t-cell engraftment of . %. patient underwent hsct from mud hlaidentical donor after a myelo-ablative reduced intensity conditioning regimen protocol d ebmt/esid guidelines. the number of infused cd + cells was . × /kg and . × cd /kg. she is actually at day+ post hsct, is doing well and shows % engraftment of donor cells. disclosure of conflict of interest: none. graft versus host disease (gvhd) is a frequent complication in patients undergoing haematopoietic stem cell transplantation. while the exact pathophysiology of gvhd is not known, the gut microbiome has been implicated in its development since it was shown that total gut decontamination (tgd) decreases the incidence of gvhd. with this study we aim to get insight into the diversity of the gut microbiota before, during and after total gut decontamination in comparison with selective gut decontamination (sgd). secondly, we want to identify changes in microbiota composition that relate to the occurrence of graft-versus-host disease. for this prospective cohort study we recruited children (o y) that were eligible for a stem cell transplantation at the leiden university medical center between january and december . of these, % (n = ) received tgd (consisting of piperacillin/ tazobactam and oral amphotericin b), whereas the other % (n = ) received selective gut decontamination with polymyxin /neomycin and oral amphotericin b. in total, fecal samples were collected, weekly during admission for the stem cell transplantation and monthly thereafter up to months after transplantation. also samples were collected from family stem cell donors as healthy controls. samples were processed within hours and stored in the - freezer, after which s v amplicon sequencing (illumina hiseq, rapid mode, bp read length) was applied. data analysis (taxonomy and shannon diversity) was primarily done using qiime (ref). compared to microbiota diversity in stem cell donors (mean shannon index (si) . ), we observed an overall lower mean si during tgd ( . ) and slightly higher mean si during sgd ( . ). microbiota diversity months after sgd ( . ) was similar to diversity during sgd ( . ), while diversity months after tgd ( . ) was higher than during tgd ( . ). further analysis of repopulation dynamics demonstrated no differences in repopulation duration after both decontamination strategies. however, we did observe differences in the type of bacteria that repopulated, with bacteroidales being more prominent in sgd and lactobacillales more prominent in tgd patients. actinomycetales (genus rothia) was exclusively present in tgd patients during decontamination. also, the clostridiales (blautia, lachnospiraceae and peptostreptococcaceae) were bacteria that appeared after the decontamination period. four patients ( %) in this cohort developed gvhd grade or more. in these patients we did observe individual compositional changes of the gut microbiota at the time of ghvd diagnosis, e.g very low diversity or dominance of enterobacteriales. considerable microbiota diversity is observed in patients that received tgd. different repopulation dynamics were observed between tgd and sgd. no common pattern was found in the gvhd cases. disclosure of conflict of interest: none. minimal residual disease (mrd) pre-and post-hct for children with aml is highly predictive of event-free survival: a pediatric blood and marrow transplant consortium study d jacobsohn johns hopkins all children's hospital, children's hospital los angeles, usc keck school of medicine multicenter data regarding the significance of mrd in children with aml pre-and early post-hct are lacking. we hypothesized that pre-and post-hct mrd assessments using wt pcr combined with multi-dimensional flow cytometry (mdf) would be predictive of disease relapse and event-free survival (efs) at -yrs post-hct. subjects were o yrs with aml in morphologic cr undergoing ma allogeneic hct. stem cell sources included bm, pbsc, or cb. bm and pb samples were collected at time points: baseline ( o weeks prior to preparative regimen); day+ (± days); and day+ (± days). bm samples were analyzed for both wt expression and mdf mrd (single reference lab using a 'difference from normal' approach without access to diagnostic phenotype); pb samples were analyzed for wt only. mdf detection limit was . %; however, we required that independent analysts certify that the abnormal population was aml. in addition, sorted mrd+ cells were tested for chimerism. wt positivity was defined as ⩾ copies for bm and ⩾ copies for pb. results were not available to the treating clinician. subjects were enrolled at centers in us and canada. enrolled subjects did not undergo hct and were excluded for progression prior to hct or other ineligibility. in eligible subjects, -yr efs and os were % and %, respectively. the -yr ci of relapse and trm were % and %, respectively. mdf identified subjects pre-hct having . - % residual disease. the -yr relapse rate in subjects with +mrd by mdf pre-hct was % vs % ( - %) in those who were negative. -yr dfs and os were % and % ( - %) for positive mdf pre-hct, and % ( - %) and % ( - %) for negative mdf. pre-hct mdf sensitivity for -yr dfs was %; specificity was %. mdf mrd at days and were similarly predictive of outcome. sorted mrd+ cells from post-hct samples were all noted to be of recipient origin. pb wt had no correlation with dfs or relapse; bm wt at day+ correlated with -yr os ( % ( - %) low/neg vs % ( - %) high). other wt cutoffs studied demonstrated no correlation with outcomes. figure : relapse probability by flow cytometry mrd at time points. mdf mrd pre-hct and at days + and + was significantly associated with lower efs and os in children with aml undergoing hct. mdf is specific but not sensitive, as many negative mdf patients relapsed. our goal was to define a reproducible assay that did not depend on having the initial aml profile. this would facilitate multi-institutional studies aimed at decreasing relapse. given that constraint, we were able to detect clear mdf mrd in a small percentage of patients that was highly predictive and can be used in trials. wt level was not predictive in this multi-institutional trial. the sensitivity of flow was significantly affected by not having the initial flow available. future attempts to improve sensitivity should include initial flow and/or test higher channel flow or molecular pcr techniques. in addition, we confirmed that mrd + cells obtained by cell sorting post-hct were of recipient origin. future testing of 'suspicious' sorted cells by fish, molecular, or comparative genomic hybridization could possibly increase mfd sensitivity. novel cellular or targeted therapies should be tested in clinical trials to improve outcomes in patients with mfd mrd noted either pre-or post-hct. [p ] disclosure of conflict of interest: none. novel mutations were identified with ngs and low intensity of conditional regimen succeeded in children with fanconi anemia who received allo-hsct s hu , h hou, j lu, p xiao, x bian, h liu, y hu, j ling, l li, l kong, z zhai and y yao children's hospital of soochow university to explore the possiblity of applying next-generation sequencing (ngs) to diagnose the disease of fanconi anemia (fa) and evaluate the efficiency and safety of low intensity conditional regimen on children with fa receiving allogenic hematopoietic stem cells transplantation (allo-hsct). five patients initially suspected as severe aplastic anemia were diagnosed as fa by the method of next-generation sequencing (ngs)-based genetic diagnosis panel. one patient received hla-identical sibling donor hematopoietic stem cell transplantation (mrd), three patients underwent unrelated donor matched (ud) hsct, and one patient received unrelated cord blood transplantation (ucb). the conditional regimen consisted of either cgy tbi or . - . mg/kg of busulfan with - mg/kg of cyclophosphamide. meanwhile, atg at mg/kg and fludarabin at - mg/m were included as well. cyclosporin or tacrolimus as well as mycophenolate mofetil (mmf) were used for the prophylaxis of graft versus host disease (gvhd). engraftment of neutrophil and platelet and complications followed transplantation such as infection, gvhd, and hemorrhagic cystitis (hc) were observed. of cases diagnosed as fa by ngs, only case showed the abnormality of chromosome fragility test which has been regarded as golden criteria in the diagnosis of fa. meanwhile, we found novel mutations in cases of fa which enriched chinese national database with data of rare diseases by ngs. the counts of mononuclear cells (mnc) were ( . - . ) × /kg for non-ucb and . × /kg for ucb. the counts of cd + were ( . - . ) × /kg for non-ucb and . × /kg for ucb. all cases succeeded in allo-hsct with the low intensity of conditional regimen. the median time for neutrophils engraftment was days (range ~ days), median time to platelets (plt) engraftment was days (range ~ days). one case occurred with grade i of agvhd, cases with hemorrhagic cystitis. after transplantation, all patients were monitored the copies of ebv-dna and cmv-dna of whole blood, and five case with ebv positive and cases with cmv positive. no patient suffered of ebv or cmv disease. the hepatic veno-occlusive disease (vod) and hc were observed in fa patients after transplantation. ngs showed much more specific and facilitated for the diagnosis of fa. low intensity of conditional regimen is efficient and safe which should be recommended for the treatment of fa patients. disclosure of conflict of interest: none. outcome of alternate donor stem cell transplantation in children: an indian experience sp yadav , , n rastogi , , d thakkar , , s kohli , , s nivargi , , r misra and s katewa in india due to lack of donor registries and cord blood banks very few alternate donor stem cell transplants (sct) are performed. haploidentical sct has become feasible with availability of post-transplant cyclophosphamide (ptcy) technique. here we present our experience of setting up alternate donor program for sct for children in india and report the outcomes of the same. we collected data retrospectively of all children who underwent alternate donor sct during jan to dec in two centres. a total of sct were performed for children; median age years ( - years) and were boys and girls. of these, underwent haploidentical ( ptcy and tcr alpha-beta/cd depleted), matched unrelated donors (mud) and unrelated cord blood (ucb) sct. the diagnosis was: primary immunodeficiency- , thalassemia major- , sickle cell disease- , inherited bone marrow failure- , acquired aplastic anemia- , acute lymphoblastic leukemia- , acute myeloid leukemia- , neuroblastoma- , ewings sarcoma- & leukodystrophy- . the conditioning was with fludarabine, cyclophosphamide and total body irradiation backbone in children (thiotepa added in ), fludarabine and treosulfan in , fludarabine and busulfan in , busulfan and cyclophosphamide in . serotherapy was part of conditioning, rabbit anti-thymoglobulin . mg/kg in and campath mg/kg in . graft vs host disease (gvhd) prophylaxis was ptcy along with tacrolimus and mycophenolate mofetil in patients ( haploidentical, mud & ucb) and ex-vivo tcr alpha-beta depletion in and cyclosporine and methotrexate in . all were transplanted after a signed informed consent. a median of million of cd cells/kg was infused (range - million/kg).graft source was peripheral blood in and bone marrow in and ucb in . five children died before engraftment. the remaining had neutrophil engraftment by median of days (range - ) and platelet engraftment by median of days (range - ). chimerism at day+ was available in cases; of them had full donor hematopoiesis. one had mixed chimerism and fully recipient. four children underwent a second haploidentical sct after rejection of which are alive and disease free. the median follow-up of remaining patients is months (range - ); the cumulative incidence of graft versus host disease (gvhd) acute and chronic extensive was % and % respectively. grade-iii-iv acute gvhd was seen in patients. a total of patients have died (sepsis- , stroke- , gvhd- , vod- , encephalitis- and progressive disease- ). among encephalitis deaths, one child had undergone ucb with ptcy and another tcr alpha-beta depleted second sct.; both had bk virus in the csf.there were / deaths in haploidentical (ptcy- / & tcr alpha-beta- / ), / in mud and / in ucb sct. overall survival is % and disease free survival is % at last follow up. alternate donor sct is an acceptable curative option for children lacking a matched sibling donor. haploidentical donor sct is more feasible in the setting of lack of donor registries having indian ethnicity donor. disclosure of conflict of interest: none. hematopoietic stem cell transplantation (hsct) from an unrelated donor (ud) is largely used for pediatric patients with all in second complete remission (cr) lacking an hlaidentical sibling. in this study, we retrospectively analyzed outcome of patients (pts) given ud-hsct in centers affiliated to the associazione italiana di ematologia ed oncologia pediatrica (aieop) network between and . three hundred fifty-six pts with all in second cr experiencing either bone marrow (bm), isolated extramedullary or combined relapse were included in the study; were males and females, median age at hsct being . years (range . - ). bm, peripheral blood (pb) and cord blood (cb) were the stem cell source in %, % and % pts, respectively. all children received a myeloablative conditioning regimen, either tbi-( pts) or chemotherapy-based ( pts). as gvhd prophylaxis, the combination of cyclosporine a, short-term mtx and atg was employed in most pts. according to the berlin-frankfurt-munster (bfm) classification of first leukemia recurrence, % and % of pts were assigned to the s +s and s +s groups, respectively. level of pre-hsct minimal residual disease (mrd), measured within days before hsct through flow cytometry (fcm) in the laboratory of padova, is available in children; more data will be presented during the s meeting. with a medium follow-up of . years (range . - ), the overall survival (os) was %, while the event-free survival (efs) was %. the cumulative incidence of transplant-related mortality (trm) and leukemia recurrence were % and %, respectively. the efs probability for children transplanted in the time period [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] and - was %, % and %, respectively (p = ns). patients who received a tbi-based conditioning regimen had a significantly better outcome in comparison to children who received chemotherapy-based treatment, efs being % and %, respectively (p = . ). efs of pts belonging to s +s and s +s groups was % and % respectively (p = . ). the difference in efs is largely explained by an increased incidence of leukemia recurrence in s +s ( %) compared to s +s pts ( %) (p = . ). efs of pts who experienced grade ii acute gvhd was %, while that of pts with either absent/grade i acute gvhd or grade iii-iv acute gvhd was % and %, respectively (p = . ). pts with limited chronic gvhd had a better efs as compared to those with either extensive or absent chronic gvhd ( %, % and %, respectively; p = . ). the choice of stem cell source (bm, pbsc, cb) did not influence the probability of efs, which was %, %, % respectively (p = ns). importantly, among pts with evaluable mrd before hsct (n ), the group with detectable levels . % (n ), respectively % and % (p = . ). conclusions. outcome of children with nd cr all who underwent transplant from an ud is significantly influenced by the presence of tbi in the conditioning regimen, limited severity of acute and chronic gvhd and bfm classification at time of st relapse. notably, mrd level before transplant, namely with a cut-off of . %, influences efs. disclosure of conflict of interest: none. the median mononuclear cell dose was . × /kg. the median time to reach absolute neutrophil count . × /l was days, and the median time to platelet count × was days. grade and grade mucositis was seen in % of our patients. transplant-related mortality at days not occurred. only three patients relapsed , and months after transplant (mean . m.). with a median follow-up of months ( - months) after transplant the event free survival were %. only one patient had death, two years after transplantation. no significant different between cbv group vs ceam group in engraftment day. high-dose therapy with stem cell rescue can lead to durable remissions in children and adolescents with advanced hd. future investigations should focus on strategies designed to decrease relapse after auto-transplantation, particularly in patients at high risk for relapse. our analysis suggests that these regimens (ceam, cbv) are feasible in pediatric patients with acceptable engraftment and toxicity. pbsc collection may be difficult in small children owing to the large volume apheresis compared to the child's weight. various problems, such as metabolic or haemodynamic disorders may be were seen. peripheral stem cell harvest can be performed in lowweight children under safe and effective conditions even when systematic priming by blood is avoided. processing with increase of blood volume may to increase in the yield by recruiting progenitor cells. disclosure of conflict of interest: none. outcomes of children with hemophagocytic lymphohistiocytosis given allogeneic hematopoietic stem cell transplantation in italy allogeneic hematopoietic stem cell transplantation (hsct) is the only curative treatment for patients with familial hemophagocytic lymphohistiocytiosis (hlh) or relapsed/ refractory hlh. we analyzed outcomes of a cohort of patients ( m, f) with hlh given hsct between and . median age at hsct was years (range . - ). genetic testing was performed for / patients ( %). mutation of prf was found in patients ( %), of unc d in ( %), of stxbp in ( %), of rab a in ( %), of sh d a in ( %), of birc in ( %) and of lyst in patient ( %). no known gene abnormality was found in patients who had recurrent/ refractory hlh. central nervous system (cns) involvement at diagnosis was recorded for patients ( %) and was present in of them ( %). the primary endpoint was event-free survival (efs), defined as the probability of being alive and in continuous complete remission (cr) at last follow-up. in order to determine efs, death from any cause, relapse or graft failure were considered events. ninety-five patients received one transplant, while received more than one hsct, because of rejection in patients or disease relapse in (preceded by rejection in case): hsct were performed in cases, while and hsct were performed in case each. donor for first transplant was an hla-matched sibling for patients ( %), an unrelated donor for patients ( %) and a partially matched family donor for patients ( %). conditioning regimen was busulfan-based for patients ( %), treosulfan-based for patients ( %) and fludarabine-based for patients ( %). the -year probability of overall and efs were % and % respectively (fig. ) . twenty-six ( %) patients died due to transplant-related causes, while ( %) and ( %) patients experienced graft rejection and/or relapse, respectively (see also fig. ). twelve out of children ( %) given a nd hsct after graft failure/relapse are alive and disease-free. active disease at hsct was not statistically associated with adverse outcomes, while patients had a trend for a worse outcome if the interval between diagnosis and hsct was months. patients transplanted from partiallymatched family donors (pmfd) had a significantly worse efs ( %) than recipients of a matched family donor transplant ( %) or a matched unrelated donor allograft ( %, po . ). the main reason for the dismal efs of pmfd recipients was graft rejection, which, however, was largely rescued by a nd hsct. patients given peripheral blood stem cell transplantation had a lower efs probability ( %) as compared to bone marrow ( %) or cord blood recipients ( %, p = . ). children given hsct o o/u months from diagnosis had a better efs as compared to those transplanted months from diagnosis ( % vs %, p = . ). in multivariate analysis, only the use of a pmfd predicted a worse efs probability (relative risk: . , p = . ). these data suggest that in patients with hlh allogeneic hsct is able to cure / of patients. haploidentical hsct in patients with hlh is currently associated with unsatisfactory rate of engraftment, new approaches being needed to ameliorate this outcome. active disease does not preclude the chance of benefiting from transplantation, which should be ideally performed within months from diagnosis. [p ] defibrotide shows efficacy in the prevention of sinusoidal obstruction syndrome (sos) after allogeneic hematopoietic stem cell transplantation: a retrospective study on patients. disclosure of conflict of interest: none. standard gvhd prophylaxis regimens impair the graft-versustumor (gvt) effect, delay immune reconstitution and are associated with high rate of infections. high-dose posttransplantation cyclophosphamide (ptcy) targets alloreactive donor t cells proliferating early after bmt, promotes regulatory t cell and permits rapid immune reconstitution. in this pilot trial we evaluate the safety and effects of ptcy in unmanipulated haploidentical and matched unrelated transplantation (mud) in pediatric patients with all. fifteen pediatric patients with high risk all underwent unmanipulated allogeneic bone marrow (bm) (n = ) or peripheral blood stem cell (pbsc) (n = ) transplantation followed by ptcy between april and march with a median follow-up of months ( - ). eight patients were transplanted from haploidentical donors and from mud. the median age was . years (range . - ) and were in complete remission (cr) at the moment of bmt. in patients this was a second bmt. all pts. received myeloablative conditioning regimen (treosulfan-based n = , tbi based n = ) and ptcy on day + , + , posttransplant prophylaxis consisted of tacrolimus from day + (n = ), tacrolimus/mmf (n = ), atg (rabbit, thymoglobuline) at mg/kg without other posttransplant prophylaxis(n = , both from mud). primary engraftment was achieved in % of pts., the median time to neutrophil recovery was days and to platelet recovery was ( - ) days. all pts. had full donor chimerism on day + . causes of death included viral infections (n = ); gvhd and viral infection (n = ). cumulative incidence (ci) of acute gvhd grade ⩾ ii was % ( % ci: - ), grade iii-iv- . % ( % ci: . - ) and chronic gvhd- . % ( % ci: . - . ). two-year event-free survival (efs) and overall survival (os) were . % ( % ci: . - ) and were equal. median time of follow-up for survivors is years (range . - . ). we demonstrate that unmanipulated hsct and posttransplantation cyclophosphamide allows for high rate of engraftment with acceptable transplant-related mortality in pediatric patients with all. all major outcomes were equivalent between transplantation from unrelated and haploidentical donor. gvhd prophylaxis including ptcy was effective. event-free survival was high despite chemotherapybased conditioning in most patients. disclosure of conflict of interest: none. serotherapy with atg is frequently used in allogeneic hsct to prevent gvhd and rejection. however, the choice of the two most frequently used rabbit atg brands depends on country, disease protocol, national recommendations and/or physician's preference. atg-genzyme (atg-g, thymoglobulin) is prepared by immunizing rabbits with human thymocytes, whereas rabbits are immunized with a jurkat cell line for production of atg-fresenius (atg-f, recently named as antihuman t-lymphocyte immunoglobulin atlg, grafalon, noveii biotech). the recommended dose of both brands differs a factor - . we have previously reported the pharmacokinetics/ pharmacodynamics (pkpd) of atg-g in a large cohort of pediatric hsct recipients and concluded that the clearance of the active component of atg, which is the portion of atg binding to lymphocytes, had a major impact on immune recovery post-hsct, while total atg did not. both atg brands have frequently been compared according to disease outcome, without detailed analysis of composition and clearance of the active components. in the present study, we compared clearance of the active component and immune recovery after atg-g and atg-f, respectively. the serum concentrations of total and active atg were measured longitudinally after hsct in children ( atg-g, atg-f), transplanted with bm or pbsc of unrelated donors for all or aml between january and june in leiden (n = ) or copenhagen (n = ). atg-g treated patients received a total dose of - mg/kg and atg-f was given at a total dose of - mg/kg in both cohorts administration was divided over - days. serum samples (pre-conditioning, day of hsct, + ; + ; + ; + and + weeks and + and + months after hsct) were analyzed by elisa for total atg and by quantitative flow cytometry on hut cells for active (lymphocyte binding) atg. lymphocyte (sub-)populations were analyzed at + , + and + months post-hsct by flow cytometry. as reference group for immune recovery, children transplanted for all or aml with an hla-identical donor and not receiving serotherapy were included. the median serum concentration of total atg at the day of hsct was times higher for atg-f (atg-g μg/ml, atg-f μg/ ml; figure a) as the result of the higher dose of atg-f given. the active atg concentration was twice as high for atg-f (atg-g . au/ml, atg-f . au/ml figure b ). three weeks later at the expected time of engraftment, the total atg concentration was decreased with the same factor for both atg brands (atg-g from to μg/ml, factor . ; atg-f from to μg/ml, factor . ). however, the active atg concentration showed a much faster decline for atg-f (atg-g from . to . iu/ml, factor . ; atg-f from . to . iu/ml, factor ). correspondingly, the number of cd t-cells at month post-hsct was higher after atg-f than after atg-g (atg-g, atg-f and no-serotherapy , and cells/μl, respectively. figure c) . this is the first study to compare the pkpd of total and active atg-genzyme and atg-fresenius. active atg-f showed a much faster clearance than atg-g, which was associated with a significantly faster cd t-cell recovery at month post hsct. thus, atg-f is not only quantitatively but also qualitatively very different from atg-g, which will clearly impact hsct outcomes. reduced toxicity myeloablative conditioning regimen in pediatric hematologic malignancies not associated with improved outcomes s chaudhury , , i helenowski , r duerst , , wt tse , , m kletzel , , j schneiderman , and d jacobsohn ann and robert h. lurie children's hospital of chicago; northwestern university feinberg school of medicine, chicago and children's national health system, washington dc allogeneic (allo) hematopoietic cell transplantation (hct) is the only curative potential therapy in refractory and relapsed pediatric leukemias. poor outcomes in allo hct are associated with treatment-related mortality (trm), mostly due to regimen-related toxicities (rrt) and graft-versus-host disease (gvhd) after myeloablative conditionings (mac), but high relapse rate with reduced-intensity or nonmyeloablative regimens. to improve trm, without compromising conditioning intensity, we prospectively explored the feasibility and efficacy of a mac but reduced-toxicity conditioning (rtc) regimen, consisting of fludarabine mg/m /d (given first) × d, daily busulfan dosed to target an auc of microm*min/d × , ratg . mg/kg/d × and cgy of total body irradiation in patients (table ) with hematologic malignancies. gvhd prophylaxis was cyclosporine and mmf. all patients tolerated the rtc well, with no graft failures. rrt included moderate mucositis ( %), infections (bacterial %, viral reactivation %, fungal %) and cases of venoocclusive disease (vod). cumulative incidence d ⩾ gr acute gvhd was % ( % confidence interval [ci], - ), extensive chronic gvhd was . % ( % ci, . - ). mortality at days was . % ( % ci - ), due to infections with agvhd and vod. with a median follow-up of . y (range, . - ), the cumulative incidences of relapse at years was % ( % ci, - ). mortality due to severe agvhd was %. overall survival (os) and progression-free survivals (pfs) for year was % ( % ci, - ), and % ( % ci, - ) respectively. on univariate analysis there was no association of outcomes with donor type, graft source, disease or busulfan exposure except significantly higher cgvhd in unrelated donors, agvhd severity with peripheral blood. in summary, the use of the myeloablative rtc resulted in comparable trm, with high relapse rate was high, including in those developing chronic gvhd. this suggested a less robust graft-versusleukemia effect resulting in poor pfs and os. nonetheless, this regimen may be used as a lower-trm platform to combine with other strategies, intensive disease monitoring pre and post hct, addition of post hct maintenance therapy in combination with marrow as the stem cell source to decrease relapse or gvhd. specific immune response to vaccinations decline after hematopoetic stem cell transplantion (hsct). re-vaccination of all hsct recipients is recommended in all guidelines but bcg vaccination is not recommended due to safety concerns after hsct. mycobacterium tuberculosis can cause severe disease in children including meningitis and milliary tuberculosis (tb). the bacille-carmette-guerin (bcg) is a liveattenuated vaccine with documented efficacy against milliary disease and meningitis. routine vaccination of all infants residing in countries with high tb incidence is recommended by world health organization. however, there is no data in literature regarding its safety in post hsct setting. here, we report children who underwent matched related allogeneic hsct at ankara university pediatric bone marrow transplant (bmt) unit and received bcg -months post-transplant. all patients were free of graft versus host disease (gvhd) and immunosuppressive therapy (ist) and had negative ppd skin test prior to vaccination. none of the recipients developed local or disseminated tuberculosis as a complication of bcg with a median follow up of years. we conclude that the bcg vaccine is safe in the post hsct period when administered at least months out of transplant to a selected group of patients who are free of gvhd and ist. disclosure of conflict of interest: none. single centre experience of harvesting bone marrow from donors o years of age r raj, r uppuluri , d subburaj , d jayaraman , k mullanfiroze , v swaminathan and l vaidhyanathan department of paediatric blood and marrow transplantation, apollo speciality hospital harvesting bone marrow for allogeneic marrow transplantation from donors o kg presents special challenges. we present data on sibling donors from our institution between and . the mean age was months with a range between months to months. children less than one year accounted for % of our donors with the youngest being months of age and the smallest donor weighed . kg. all aspirations were performed from iliac crests and all donors were given general anaesthesia by a paediatric anaesthetist. irradiated blood was transfused in % of the donors during the procedure. the volume of marrow obtained ranged from to a maximum of ml/kg donor weight. the product contained an average cd count of . × /kg recipient weight with a range from . to × /kg. only on one occasion was a second harvest needed, where the donor weighed kg and recipient kg with major blood group incompatibility requiring red cell reduction. the yield of cd cells per ml of bone marrow was on average % higher than children above years of age. all recipients showed brisk engraftment in weeks. none of these donors experienced major difficulties following the aspiration procedure. thus, very young children may safely donate marrow for allogeneic transplantation and the yield of stem cells obtained is substantial. this data is particularly relevant in transplantation for haemoglobinopathies like thalassaemia major and sickle cell anaemia, where families are being counselled about a target of kg for the donor in order to plan transplantation. disclosure of conflict of interest: none. sinusoidal obstruction syndrome-veno-occlusive disease in pediatric patients given either autologous or allogeneic hematopoietic stem cell transplantation (hsct). a retrospective study of the aieop-sct (italian haematology-oncology association-stem cell transplantation) group m faraci , r luksch, e calore , f saglio , a prete , mc menconi , v trevisan , g de simone , v tintori , s cesaro , s santarone , mg orofino , e lanino , m zecca and a bertaina sinusoidal obstruction syndrome (sos), known as venoocclusive disease (vod), is a potentially life threatening complication that can develop after hsct. although sos progressively resolves within few weeks in most patients, the severe forms result associated with multi-organ dysfunction and high mortality rate ( %). aim of this survey is to evaluate incidence and management of sos in a large cohort of children receiving either allogeneic or autologous hsct. we retrospectively reviewed pediatric hscts performed in ( %) out of aieop affiliated centers, between january and april . new ebmt criteria have been used for the diagnosis of sos (serum total bilirubin ⩾ mg/dl and of the following criteria: painful hepatomegaly, weight gain %, and ascites) and for the classification of severity grading. , among a total number of hsct procedures ( autologous and allogeneic), we identified ( . %) patients with sos. this complication occurred in and cases after autologous and allogeneic hsct, respectively. fiftytwo pts ( %) received iv busulphan (bu) at myeloablative dose, ( %) oral bu, while ( %) were treated with different conditioning regimen. the median time of sos occurrence was days after hsct. details about prophylaxis and therapy are reported in figure . out of the children, ( %) fulfilled all sos-ebmt criteria. bilirubin ⩾ mg/dl, gain of weight %, ascites, and painful hepatomegaly did not occurred in , , and patients, respectively. thrombocytopenia was present in pts ( %), thickening of gallbladder in ( %) and abnormalities of coagulation parameters in ( %). according to sos ebmt severity grading, levels of transaminases were mild in pts ( %), moderate in ( %), severe in ( . %), and very severe in ( . %). notably, creatinine was mild in pts ( %), while ( . %), ( . %), and ( %) children showed moderate, severe and very severe grade of renal failure. thirty-three pts ( %) had respiratory failure, and ( %) of them experienced right pleural effusion. six out of the patients who developed acute kidney injury, required dialysis. severe encephalopathy occurred in pts ( . %) and ( %) out of the pts evaluated, were admitted in intensive care unit. as therapy of sos, pts received defibrotideâ (df); the dosage was mg/ kg/day in % of them. the median duration of df treatment was . days (range - ). thirty-three ( %) pts received methylprednisolone (median dose of mg/kg). fifteen pts ( . %) died due to mof ( in moderate, in severe, and in very severe group) at a median time of days from sos diagnosis (range - gg). our multicenter survey showed that, at least in our experience, there is a significant variability in the management approaches to sos/vod in children, while, diagnostic evaluations are more homogeneous. interestingly, in our cohort, the increase of bilirubin may be an absent criteria, while thrombocytopenia and abnormalities of coagulation parameters are more frequent. as expected, mof occurred mostly in patients experiencing severe sos. df represents first strategy to treat sos in the majority of patients, even if steroids and ursodeoxycholic acid are still used. the hyper-ige syndromes are characterized by marked elevations in plasma ige levels and eosinophilia with impairment in t cells which clinically results in combined immune deficiency. dock deficiency, the autosomal recessive form, brings about allergic/atopic manifestations and unusual susceptibility to infections with herpesvirus family members (herpes simplex virus, human papilloma virus) and molluscum contagiosum. symptoms in patients with dock deficiency typically emerge during childhood, and the majority results in death because of infections and malignancy by the third decade. hematopoietic stem cell transplantation (hsct) is now considered a standard of care for dock deficiency when an appropriate donor is available. in this study, we present the unrelated hsct results of children with dock mutation. the demographic and clinical data of the patients with transplantations studied are shown in table . hsct was administered between august and august at bahçeşehir university medical park antalya hospital and the clinical data of the hscts are presented in table . all patients were transplanted from unrelated donors with bone marrow, except one with cord blood. the cord blood transplantation´s regimen was non-myeloablative which resulted with rejection. despite existence of serious morbid problems before transplantation, all the patients engrafted successfully. majority of the complications mentioned in the table were improved and they are in the follow-up in an outpatient basis. discussion dock deficiency has high mortality, and hsct should be considered as early as possible before development of significant organ damage. despite myeloablative conditioning and high morbidity before the transplantation, survival was very good in our patients. myeloablative and nonmyeloablative transplants have been performed from related and unrelated donors and have reported successful results even without the preparative regimen. in our center, all transplants performed from unrelated donors by myeloablative regimen have been successful but have resulted in transplant rejection with cord blood transplantation after nonmyeloablative regimen. in all of our patients, stable full chimerism has been detected, however mixed chimerism have also been shown to be useful in several reports. whether hsct also cures the autoimmune complications and reduces the risk of cancers is as yet undetermined. however, a myeloablative conditioning regimen followed by allogeneic hematopoietic stem cell transplantation from unrelated donors in dock deficiency results in improvement of the clinical phenotype with a low incidence of regimen-related toxicity. disclosure of conflict of interest: none. successful bone marrow transplantation after myeloablative conditioning in a child with ipex syndrome b kuşkonmaz , d ayvaz , mh abur , fv okur , g karagüzel , f orhan , İ tezcan and du Çetinkaya immune dysregulation, polyendocrinopathy, enteropathy, x-linked (ipex) syndrome is a rare disorder. although most patients present in infancy with a clinical triad of intractable diarrhea, insulin-dependent diabetes, and eczematous dermatitis, some patients present with severe food allergies and other autoimmune manifestations. the disease is caused by mutations in the forkhead box p (foxp ) gene, a transcription factor that is essential for the development and function of regulatory t (treg) cells. this cells plays an essential role in controlling immune responses and preventing autoimmunity. patients usually die in the first years of life without treatment. the only effective cure is hematopoietic stem cell transplantation (hsct). here we report a patient with ipex syndrome who underwent hsct after myeloablative conditioning. months of age boy with the history of diarrhea, insulin-dependent diabetes, eczematous dermatitis, pneumonia, coombs positive hemolytic anemia, referred to our hospital for investigation of immunodeficiency. on admission physical examination showed eczematous skin rash, submandibular lymphadenopathy, hepatosplenomegaly. before hsct the patients treated with immunosuppressive agents including methylprednisolone, mycophenolate mofetil and monthly intravenous immunoglobulin. complete blood count revealed anemia (hb: . g/dl), and eosinophilia ( /mm ). serum immunoglobulins were: ig g: mg/dl ( - ), igm: mg/dl ( - ), iga: . mg/dl ( - ), ige : iu/ml. lymphocyte subset analysis showed cd %, cd %, cd %, cd + %, cd %. foxp gene analysis showed c. _ delaag mutation. at the age of year, patient underwent hsct from his hla matched sibling. myeloablative conditioning regimen including busulfan ( . mg/kg) and fludarabine ( mg/m ) was given to the patient. cyclosporine a and methotrexate (day + , day + , day + ) were used as graft versus host disease prophylaxis. bone marrow was used as the stem cell source and the number of cd + cells was . × /kg. neutrophil and platelet engraftment were achieved on day + and + [p ] s respectively. acute and chronic gvhd were not observed, but patient developed veno-occlusive disease treated with defibrotide, sepsis treated with broad spectrum antibiotics. chimerism analysis showed % donor profile at the third month of hsct. after hsct, autoimmune hemolytic anemia, eczematous dermatitis, food allergies, diarrhea and type diabetes resolved completely within two months after hsct. now the patient is in good clinical condition without any symptoms months after hsct. early hsct provides better outcome in patients with ipex, before the organ damage due to autoimmunity and/or adverse effects of immunosuppressive therapy. myeloablative conditioning is associated with substantial transplantation-related mortality whereas nonmyeloablative conditioning carries an increased risk of rejection because of dysregulated effector t-cell function. in this patients, myeloablative conditioning was preferred because of the risk of rejection. although the required levels of donor chimerism and conditioning intensity are unknown, engraftment of donor treg cells seems to be sufficient to control the disease. the patient is well without any symptoms of ipex after hsct with full donor chimerism. disclosure of conflict of interest: none. interferon gamma receptor deficiency (ifnr ) is a rare autosomal recessive immune deficiency disorder associated with very poor outcome secondary to severe and disseminated mycobacterial infections. hematopoietic stem cell transplantation (hsct) has been proposed as a curative option. however, hsct for these patients is particularly difficult owing to a high rate of graft rejection. the use of a non t-cell depleted transplant from an hla-identical sibling and fully myeloablative conditioning regimen has been shown to have improved outcomes. we report the first successful hsct with a t-depleted haplo-identical donor, performed in a girl with severe ifnr deficiency. we reviewed the medical chart of a -year-old hispanic girl with ifnr deficiency who was diagnosed at birth, since her brother had previously been diagnosed with the same complete ifnr deficiency. they were found to have a novel mutation variant detected at c. - g t. as expected with this disorder, she developed disseminated infection with mycobacterium abscessus infection at months of age and was subsequently found to have mycobacterium abscessus osteomyelitis. she was treated with multiple antibiotics including: amikacin, linezolid, meropenem and clarithromycin while tigecycline was added a few weeks prior to admission for hsct. she was continued on this therapy until day + following which antimicrobials were gradually weaned off. she was enrolled on the bp- trial, a multicenter, prospective phase i-ii trial (enrolling both malignant and non-malignant diseases) evaluating αβtcr +/cd + depleted haplo-transplantation followed by administration of bpx- t cells containing the ic suicide gene, (clinicaltrials.gov nct ). her conditioning regimen included busulfan ( mg/kg/day for days) and cyclophosphamide ( mg/kg/day for days). fludaragbne, tli ( cgy) . gvh prophylaxis with atg/rituximab. the patient received a graft with: tnc- . × cells/kg, cd + cells- × cells/kg, and αβtcr+ t cell content of . × cells/kg. as per protocol, since the αβ tcr+ t cells in the product was below threshold of × cells/kg, she did not receive any post-transplant immune suppression. bone marrow recovery occurred at day + with anc /mm and platelet recovery at day + . full engraftment with % donor chimerism based on cytogenetic analysis was observed at day + after transplantation and has remained stable. she is currently months post-transplant, and has done well without major complications and or signs of mycobacterial infection. there is limited data in patients receiving hsct for ifnr deficiency with very poor outcomes either relating to graft failure, transplant complications and progressive mycobacterial infection. to our knowledge, this is the first patient with ifnr deficiency transplanted successfully with a haploidentical donor and alive without any active mycobacterial infection. this report suggests that using a highly immunopotent graft depleted of only αβtcr+ t cells while retaining other immune effectors might offer a potential strategy to engraft these high risk patients using haplo-identical donors thereby allowing access to virtually all patients in need. disclosure of conflict of interest: none. tandem autologous stem cell transplantations for high risk pediatric embryonal central nervous system tumors: a single center experience k rosenfeld , r dvir , s constantini, j roth , s edelman , a tal , d levin , m manisterski , s achituv and r elhasid , department of pediatric hematology-oncology, tel aviv medical center; department of pediatric neurosurgery, tel aviv medical center and sackler faculty of medicine, tel aviv university pediatric embryonal central nervous system tumors are highly malignant tumors, which tend to disseminate through the cerebrospinal fluid to the brain and spinal cord and include: medulloblastoma, pinealoblastoma and primitive neuroectodermal tumors (pnets). the recommended treatment for these tumors is a complete surgical excision, craniospinal radiation and chemotherapy. the use of high dose chemotherapy with tandem autologous hematopoietic stem cell transplantation (hsct) has been advocated for high risk patients, and infants who could not be irradiated. between july and november , pediatric patients ( males, females) suffering from high risk medulloblastoma, pnet or pinealoblastoma underwent tandem autologous hsct. they were treated according to two protocols: group a consisted of ten patients with median age of . years (range . - . years) received the st jude sjmb protocol, while group b consisted of six patients with median age of . years (range . - . years) who received the children's oncology group -acns protocol. all patients engrafted with median time for neutrophil engraftment of days (range - days) and for platelets engraftment ( ) of days (range - days). median follow-up was . years (range week- years). neurological toxicity: two group a patients had convulsions episodes, one occurred during infusion of cryopreserved stem cells, and the other was a result of progressive disease during the last course of hsct. gastrointestinal toxicity: seven patients required total parenteral nutrition due to mucositis. diarrhea occurred in seven patients, two of them were diagnosed with rota virus and two with clostridium difficile. infectious complications: all patients suffered from at least one episode of neutropenic fever which was treated with broad spectrum antibiotics. there were documented bacteremia in patients. ( klebsiella pneumonia, proteus mirabilis, staphylococcus aureus, streptococcus viridans and staphylococcus epidermidis). metabolic complications: four patients in group a developed reversible syndrome of inappropriate anti-diuretic hormone secretion (siadh) during chemotherapy, and all group a patients developed hypomagnezemia. four patients died, one due to progressive disease, one due to early relapse months post treatment, one due to late relapse years post treatment and one due to sepsis months post treatment. another patient relapsed . years s post treatment, underwent surgery and radiotherapy and is now years post therapy. late effects: four group a patients developed endocrinological sequelae at a median of months (range - months) and require hormone replacement therapy. tandem autologous hsct is a feasible treatment for pediatric high risk embryonal tumors, with good engraftment and acceptable toxicities using sjmb and acns protocols, with overall survival of %. long follow-up is needed in order to diagnose and treat late effects. disclosure of conflict of interest: none. the diagnostic role of liver stiffness measurement in predicting hepatic veno-occlusive disease (vod) in pediatric hematopoietic stem cell transplantation (hsct) k kleinschmidt , f ravaioli , r rondelli , g marasco , r masetti , a prete , a colecchia , d festi and a pession pediatric oncology and hematology unit, department of pediatrics, university of bologna, sant 'orsola-malpighi hospital and department of medical and surgical sciences, university of bologna vod is a potentially life-threatening complication associated with hsct in which immediate therapeutic action is crucial for patients' outcome. liver stiffness measurement (lsm) using fibroscan represents a non-invasive method to detect the grade of liver fibrosis and portal hypertension as in case of vod. to evaluate the predictive potential of lsm in pediatric patients (pts) at risk for developing vod, a prospective, ongoing, single-center study has been performed at the university hospital of bologna. lsm was performed by using the fibroscan device, which consists of a . mhz ultrasound transducer probe that transmits low-frequency vibrations ( hz) to the liver tissue. the propagation velocity is proportional to the stiffness (elasticity) of tissue. lsm will obtain pathological high values ( . kpa) when the tissue is altered like in liver fibrosis, or post-sinusoidal portal hypertension. from november -september , pediatric pts ( male, female), aged - years (mean . ), affected by hemato-oncologic disease, eligible to allogeneic ( ) or autologous ( ) sct conditioned with busulfan-based chemotherapy, were enrolled. pts were scheduled for study examinations with lsm: at t (baseline) before chemotherapy, t (day - after sct), t (day - ) and t (day - ). the diagnosis of vod was defined according to modified seattle/baltimore criteria. twenty-five pts were enrolled in the protocol, of which were evaluable for the study (pts characteristics table ). out of pts ( %) developed vod. the cumulative incidence (se) of vod in our setting was % ( . ). baseline lsm values on t of all pts were normal ( . kpa at t (p = . ) and t (p = . ). from our observations, an anticipating pattern of pathological lsm in presence of clinical and laboratory parameters within normal ranges in patients who develop vod can be derived. preliminary data indicate a high predictive potential of lsm in the diagnosis of vod, however the number of cases is not sufficiently representative to draw definitive conclusions. to optimize the predictive potential of the method, more frequent (daily) measurement in the critical time frame are currently investigated. [p ] all= acute lymphoblastic leukemia, aml= acute myeloid leukemia, bu= busulfan, treo=treosulfan, fluda= fludarabine. disclosure of conflict of interest: none. [p ] the exact role of extra-corporeal photopheresis in children with gvhd: an unanswered question ss anak, h bilgen , , , , , , , y yaman, et saribeyoglu, k ozdilli, v hazar, m elli, am kokrek, h hizli and k payalan ecp continues to be a controversial treatment, probably due to the mechanism of action not being identified, the varying photopheresis procedures and treatment schedules, and the difficulty of conducting trials on relatively rare diseases with involvement of clinically heterogeneous organs. ecp was performed in our pediatric transplant center to patients mean age of years ( - ) diagnosed to have all ( pts), thalassemia ( pts), aplastic anemia ( ), blacfan diamond ( ), refractory hodgkin disease ( ) following our internal protocol for ecp sessions. five of the patients had mud, had hla id sibling transplants. chronic gvhd was diagnosed in of the patients had acute gvhd. skin was involved in all the patients, liver in of the patients, lung in , gut in and mucous membranes in patients. the ecp treatment consisted essentially of three steps: ( ) collection of mncs from the patient, ( ) processing of mnc buffy coat, and ( ) return of mncs to the patient. collection was performed using a cell separator (haemonetics mcs plus), processing two blood volumes. our protocol provides for a maximum final mnc volume to be collected at ml, with a hematocrit (hct) value below %. the maximum procedure time was set at min. the mncs collected were adjusted to a constant volume of ml by the addition of saline and ml of -mop in aqueous solution, to always obtain a final concentration of the drug of ng/ml. the diluted buffy coat was transferred into a special uv-a-permeable bag (pit-kit medtech solutions), and uv-a radiation at j/cm was performed (uva-pit irradiator). the photoactivated mncs were returned to the patient within minutes using a blood transfusion set. during ecp procedure, patients' vital signs were monitored. anticoagulation consisted in acidcitrate-dextrose formula a set at a variable ratio ( : - : ) according to the patient's characteristics (clinical conditions, body weight, coagulation values) and platelet (plt) count. prophylaxis of hypocalcemia consisted of the administration of calcium gluconate ( ml diluted in - ml saline) every to minutes. all procedure related side effects were recorded. during the reinfusion and postreinfusion phases, the patients were monitored for fever, chills, headache, rash, erythema, urticaria, itching and edema. no serious complication was detected. all the patients had also steroids, had concurrent mesenchimal stem cells. ecp was applied on consecutive days every - weeks which is continued for approximately months followed by a maintenance schedule tapered to an every -to -weeks. the mean session cycle was ( - ) between february to november . the most commonly involved organ was the skin which demonstrated a response rate of %, followed by liver ( %), lung ( %), gut ( %) and mucous membranes ( %) the concurrent immunosuppression could be reduced during ecp therapy, and no increase in opportunistic infections was detected. / patient died after a relapse, / are alive with chronic mild gvhd. however, despite our good response rates, our understanding of ecp remains limited. patients who suffer from acute and chronic gvhd have limited treatment options. ecp remains an important therapeutic option. future basic, translational, and clinical research studies will provide a better understanding of its mechanism of action and optimize its therapeutic potential. disclosure of conflict of interest: none. tolerability and responses to ex vivo il activated nk cells from haploidentical parental donors in paediatric patients with refractory leukaemia/lymphoma pl tan prognosis for patients with refractory leukaemia/lymphoma ineligible for transplants and those who relapse posttransplant is poor. in adult settings, adoptive transfers of ex vivo il activated natural killer ('ank') cells from nk alloreactive donors, especially for nk sensitive cancers, has been successful in bridging patients to curative transplants.( ) this approach has not been reported in paediatric patients. we report our experience in consecutive patients, of median age (range, - ) years, with refractory leukaemia/ lymphoma (aml, ; all, ; mixed phenotype acute leukaemia, ; lymphoma, ) who received treatments with 'ank' from haploidentical parental donors on institutional protocol, between aug and . parents/legal guardians/patients provided informed consents as per institutional guidelines for donors and patients procedures. donor lymphocytes harvested at steady state were cd depleted followed by overnight culture in il before being infused into patients lymphodepleted with fludarabine and cyclophosphamide. additional rituximab were given to patients and another received tbi gy. subcutaneous il injections at doses - mu/m /dose started on d- and were planned for doses, as tolerated. nk alloreactive donors (kir-ligand mismatch) and kir b/x genotype were available to all except patients. two patients were treated for post-transplant relapse; of whom also received 'ank' pre-transplant; other patients had failed best conventional therapy including cd /cd bispecific t cell engager (blinatumomab) in . lymphodepletion was well tolerated. a median tnc and cd + dose of . (range, . to ) × /kg and . (range, . - ) × /kg, respectively were administered. cytokine release syndrome (crs) was observed in of treatments ( grade , grade , grade ). the patient with dock deficiency, disseminated ebv+ cerebral lymphoma had grade crs and robust tumour lysis syndrome but succumbed to neurotoxicity. of the treatments, there were responses, including the given posttransplant. excluding the treatments given post-transplant and non-responders, median peak donor chimerism was % (range, - %) occurring at a median of (range, - ) days. five patients ( responders, non-responder) proceeded to transplants at a median of (range, - ) days after 'ank.' responders had longer survival time compared to nonresponders (median vs days). two responders ( %) achieved sustained minimal residual disease (mrd) remission after transplants and are alive and days from 'ank.' five eventually died of their primary leukaemia/lymphoma; from crs. our preliminary experience in a small cohort of paediatric patients with refractory leukaemia/ lymphoma showed that adoptive transfers of ex vivo il activated nk cells from haploidentical parental donors were tolerable; with responses seen in % of patients; and % achieving prolonged mrd remissions after transplants. patients with cerebral diseases might be at increased risks of neurotoxicity with this approach; and care must be taken in patient selection and the design of the lymphodepletion therapy. alternative donor choices are limited in multi-racial, multiethnic societies with small families such as singapore. unrelated cord blood transplant provides a feasible alternative to patients lacking adult stem cell donors in children with primary immunodeficiency diseases. method: we describe our experience using unrelated cord blood transplant (ucbt) for children with pid from august to november . during this period we performed hsct for children with pid: with unrelated cord blood ( %); with msd and i mud. out of cases of ucbt there were severe combined immunodeficency (scid), chronic granulomatous disease (gcd), hyperigm syndrome and wiskott aldrich syndrome (was). the median age of transplant was . months (range . to . months). all presented with multiple infections ranging from disseminated bcg infection to parainfluenza /rsv /rotavirus infection to pseudomonas sepsis, staphylococcal endocarditis to pulmonary aspergillosis for scid. hyper igm presented with pnemocystitis carini pneumonia while cgd conditions presented with perianal abscess and fungal pneumonia. the child with was had life threatening git bleeding and a hemorrrhage trachaebronchial cast removed after a failed initial extubation for gastroscopy. conditioning regimes consisted of reduced intensive (fludarabine based) conditioning regime for scid and myeloablative regime for the rest. the median tnc dose was . × ( )/kg (range . to . ) and median cd + cells dose was . × ( )/kg (range to . ). results: all engrafted well except for one graft failure in cgd. he refused nd transplant and died . years post transplant from fungal pneumonia. median engraftment time for neutrophil was days (range to ) and platelet was days (range to days). grade skin aghvd occurred in one patient while another patient died of agvhd of liver and lungs. chronic gvhd was found skin and liver in one patient. trm was % (due to agvhd). median follow up was days (ranged to ). overall years survival was %. post-transplant complication with life threatening puemonitis was not uncommon. one patient developed biopsy -proven idiopathic interstitial pneumonitis and required ecmo for one month. he received immunosupressive drugs including methylprednisolone, infliximab, oral imatinib (tk inhibitor), azithromycin and nebulised becotide. he was weaned off oxygen after - months. conclusion: our limited experience showed unrelated cord blood is good source of stem cell for transplant in pid in a multiracial population. one case of graft failure was likely due too low cell dose cd +cells dose × ( )/kg. the expertise in icu has enabled us to support several patients who presented with infective pneumonia pre-transplant and post -transplant. with better technology like alpha/beta depletion haploidentical transplant may be a better option to achieve engraftment earlier so as to avoid stormy post-transplant infections seen in unrelated cord blood setting. disclosure of conflict of interest: none. in spite of these recommendations, literature from developing countries suggest that pbscs are used more and more frequently without compromising the transplant results, as they seem to be preferred graft source for donors in many countries incl. poland. therefore we analyzed the efficacy of mud-hsct in children with saa transplanted in our centre. clinical data of saa and pnh children and adolescents ( boys and girls), who underwent mud-hsct between october and july were retrospectively analyzed. the median age was . years (range . - years) according to the graft source, the patients were divided into pbsct group ( patients) and bm group ( patients). four patients required second mud transplant due to graft rejection. overall survival for all patients was %. estimated -year overall survival (os) was not statistically different between pbsct group and bm group [( % vs % ) p = . ]. there was no significant difference in os between group who had ist before transplant and the group, who had an upfront transplant as a first line of therapy [ % vs %, p = . ].the time to neutrophil and platelet engraftment was statistically longer in bm group than in pbsc group [(anc vs days, plt vs days, respectively) p = . ]. the incidence of grade iii-iv acute graft-versus-host disease (gvhd) in pbsct group was similar to that in bm group [ % ( / ) vs % ( / )]. the incidence of chronic gvhd in pbsct group was similar to that in bmt group [ % ( / ) vs % ( / )]. other transplantrelated complications like heart failure, central nervous bleeding, incidence of infections were comparable within the two regimens. there were deaths in the whole group. the main reason of death were infectious complications or multiorgan failure (mof) in severely pretransfused patients in this historical cohort of patients. unrelated donor pbsct in children and adolescents with saa seems to be not inferior to unrelated donor bmt. the incidence of chronic gvhd was surprisingly low in saa recipients of mud pbsc. increased morbidity and mortality due to infections was due to individual poor clinical situation of patients before transplant (i.e. fungal infections, contamination with resistant bacteria, prolonged neutropenia). disclosure of conflict of interest: none. dyskeratosis congenita (dc) is characterized by the clinical triad of reticular skin pigmentation, nail dystrophy, and oral leukoplakia. the majority of patients with dc develop bone marrow failure (bmf), which is the main cause of death in dc patients. allogeneic hematopoietic stem cell transplantation (hsct) is the only curative treatment for bmf associated with dc. transplant-related morbidity/mortality is common, especially after myeloablative conditioning regimens. hsct has been introduced into the management of dc, which has had remarkable clinical results. we report our experience in children with dc who underwent allogeneic transplantation at a single medical center. patients received a fludarabine-based reduced intensity conditioning (ric), and the graft source was unrelated peripheral blood stem cells. median age at the time of hsct was . years (range, - years). the numbers of infused mononuclear cells and cd + cells were . ± . × /kg and . ± . × /kg, respectively. the median time of neutrophil and platelet recovery were . days (range, - days) and . days (range, - days). two patients experienced grade ii-iii acute graftversus-host disease (gvhd), and chronic gvhd was only observed in one patient. all four patients remained alive and transfusion independent at the median follow-up of . months (range, - months). correction of previously existing physical defects was observed in two patients. unrelated peripheral blood hsct can be a curative option for dc. ric based on the type of disease is important to s achieve successful hsct. a larger sample size and extended follow-up of this rare patient population are needed to determine whether the changes in therapy will improve longterm survival. disclosure of conflict of interest: none. autosomal recessive hyper-ige syndrome due to dock mutation is a combined primary immunodeficiency, characterized by severe eczema, recurrent infections, and susceptibility to autoimmunity, malignancy, and multiple allergies, in addition to unusual high serum ige level. dock patients tend to have a progressive severe clinical course with mostly fatal outcome during second to third decade of life without hematopoietic stem cell transplantation (hsct). in our center we have a large number of dock patients. during a period of years ( - ), we transplanted patients with documented dock- mutation confirmed by molecular genetics. one patient did not receive any conditioning because of poor clinical condition and he died from severe cutaneous and gut gvhd and another patient received cbt with bu/flu with zero engraftment. the rest of the patients received hsct from hla full matched donor with chemoablation with bu/cy for all with % lymphoid and myeloid engraftment (str). among those patients who received chemoablation, gvhd developed in patients mostly grade i and ii. in addition patients died: one died of severe gvhd and the other two died of sepsis. for dock patients we highly recommend early hsct if fully matched donor is available to prevent the high mortality associated with the disease. alloreactivity triggered by interactions between killer cell immunoglobulin-like receptors (kir) and natural killer (nk) cells plays a role in graft-versus-tumor (gvt) effects after hematopoietic stem cell transplantations (sct). in particular, kir-ligand mismatching between the donor and recipient might promote nk cell alloreactivity after unrelated cord blood transplantations (ucbt) in adult patients with acute myeloid leukemia (aml). recently, it has been suggested that allogeneic nk cells could be the effector cells that mediate gvt effects after mismatched allogeneic transplants for refractory childhood solid tumors. however, there are few reports about the efficacy of kir-ligand mismatched sct in pediatric cases. here, we report the excellent outcomes of kirligand mismatched cbt (kir-cbt) in pediatric patients with refractory malignant disease. we evaluated the cases of pediatric hematology and oncology patients [ ( %) aml, ( %) myelodysplastic syndrome [mds] , and ( %) neuroblastoma [nbl] patients] who underwent kir-cbt between and at our institution. among the aml cases, one involved refractory disease (induction failure), and the other three involved relapsed aml (one patient relapsed after the st sct because of q-). all nbl patients underwent kir-cbt followed by auto-peripheral blood stem cell transplantation (pbsct) because of stage disease. the mds patient underwent kir-cbt because of refractory anemia with excess blasts. kir mismatching was defined as incompatibility between the donor kir and recipient kir ligand, and only inhibitory kir that interacted with human leukocyte antigen (hla)-bw , -c , or -c group ligands were considered. the median age of the patients was (range - ) years. all of the aml patients were in complete remission (cr) at the time of the hsct (cr = one case, cr = cases). the mds patient was in a non-cr state, and all of the nbl patients were in their st cr at the time of the hsct. the aml patients received total body irradiation (tbi)-based conditioning ( gy tbi and mg/kg cyclophosphamide [cy]), and the mds patient received busulfan (bu)-based conditioning ( . mg/kg bu and mg/kg cy). the nbl patients received reducedintensity conditioning regimens ( mg/m fludarabine, mg/m l-pam, and gy tbi). the cb exhibited hla - locus mismatches (dna typing), including at least one inhibitory kir gene mismatch. the prophylaxis for graftversus-host disease (gvhd) consisted of tacrolimus and shortterm methotrexate. anti-thymocyte globulin (atg) was not used as a gvhd prophylaxis in any case. after the median follow-up period of months (range: - months), all patients were alive, and none of them had relapsed after the kir-cbt. although grade ii-iv gvhd was observed in patients ( %), it was controlled with prednisolone. chronic gvhd was not seen in any case. the present findings suggested that nk cell alloreactivity plays a role in preventing childhood myeloid leukemia and nbl relapse after kir-cbt. although our results are limited, this report provides novel data to support further investigations into the use of kir-cbt for the treatment of pediatric refractory malignant disease. disclosure of conflict of interest: none. impact of fcm-based minimal residual disease on transplant outcomes in patients with aml in hematological complete remission t oka, j kanda , k ohmori , m hishizawa , t kitano , t kondo , k yamashita it is reported that the presence of minimal residual disease (mrd) before hematopoietic stem cell transplantation (hsct) is associated with poor overall survival in patients with acute myelogenous leukemia (aml) in hematological complete remission (cr). we retrospectively analyzed the association between flowcytometry (fcm)-based detection of mrd and transplant outcomes. we included adult patients with aml in hematological cr, who underwent their first allogeneic hsct between april and may at kyoto university hospital. mrd of bone marrow before hsct was measured using fcm. to search for target antigens to detect mrd, threecolor fcm analyses were performed using a differential panel for every disease and patient, which allowed us to detect ⩾ . % of mrd. of the patients (median age: . , range: - ), patients were included in the mrd-negative group (mrd o . %), whereas were included in the mrd-positive group (mrd ⩾ . %). in the latter group, patients were included in the mrd-low group (mrd o . %), and were included in the mrd-high group (mrd ⩾ . %). there was no significant difference in the patient background between the mrd-negative and mrd-positive groups. the -year overall survival rates for the mrd-negative, mrd-low, and mrd-high groups were %, %, and %, respectively (p = . , figure ). in a multiple regression analysis, the mrd-high group was significantly associated with higher overall mortality than the mrd-negative group (mrd-low vs mrd-negative, hazard ration [hr] . , p = . ; mrd-high vs mrd-negative, hr . , po . ). the -year relapse rates for the mrdnegative, mrd-low, and mrd-high groups were %, , and %, respectively (p o . ). there were no significant differences in non-relapse mortality among the three groups. the analysis of fcm-based detection of mrd revealed that an mrd positivity of ⩾ . % was significantly associated with high risk of relapse and death even in patients with aml with hematological cr. the stronger consolidation or conditioning therapy before hsct based on mrd could improve transplant outcomes in these patients. [p ] disclosure of conflict of interest: none. post-transplant cyclophosphamide (ptcy) and megadose t cell depleted (tcd) haplohsct for tolerance induction f aversa , e bachar-lustig , n or-geva , y zlotnikov klionsky , l prezioso , s bonomini , a monti , i manfra , c schifano , s pratissoli , f lohr , r lamanna , v sgobba , n giuliani and y reisner hematology and bmt unit, university hospital of parma, italy; department of immunology, weizmann institute of science, rehovot, israel; neurology department, stanford school of medicine, stanford, california; radiotherapy unit, university hospital of modena, italy; radiotherapy unit, university hospital of modena, italy; genetic unit, university hospital of parma, italy and hematology and bmt unit, university hospital of parma, italy the use of ptcy is associated with reduced risk for gvhd in t cell replete nma haplo-hsct; however, this intervention is still not sufficiently safe to justify treatment of non-malignant diseases or as a platform for organ transplantation. experimental data: in a total of mice, we showed that combining the power of megadose tcd hsct with high dose ptcy (fig. a) , enables marked and durable chimerism following nma conditioning, while each modality alone was ineffective (figure a) . chimerism included all myeloid and lymphoid lineages, and lda analysis of alloreactive t cells revealed specific immune tolerance towards donor stimulators (fig. b) , also associated with acceptance of donor but not rd party skin. clinical trial: a similar protocol was developed for clinical use. the first patient, a yr old male with high-risk multiple myeloma in cr after autohsct, received megadose ( . x cd + cells/kg) cd /cd depleted ( . x cd +t cells/kg) haploidentical pbpcs after atg, fludarabine and gy single frcation tbi. ptcy was given to control both hvg and gvh reactions (fig. c) . hematopoietic engraftment was achieved at day + with over % donor type chimerism during the first months in the myeloid and b cell lineages. t cells during this period were predominantly of host type ( - % donor type), gradually increasing to - % at - months post transplant (fig. d) . the patient overcame cmv and subsequently ebv reactivation without any treatment (fig. e- g) . dextramer facs analysis revealed that cmv and ebv specific cd t cells were exclusively of host origin (fig. f- h) . at + months, cr and normal free light chain ratio were confirmed. the second patient, a year-old male with high risk heavily pretreated multiple myeloma (tandem auto-hsct, yr maintenance with lenalidomide, salvage therapy with vd) received a similar hsct ( . x cd + cells/kg, . x cd +t cells/kg). despite transient engraftment ( % donor cell on day + ), graft failure with autologous recovery ( . % donortype chimerism) was documented on day + . this may be due to the extended treatment ( yrs) with lenalidomide, but rejection cannot be excluded. after months, this patient tolerated a second haplo-hsct (different donor) after myeloablative conditioning (atg, treosulfan, thiotepa and fludarabine) and alfa/beta tcr/cd -depleted pbpcs. at month follow up, he shows no sign of gvhd, good immunological reconstitution, excellent quality of life, and remains in complete remission. collectively, our murine proof of concept data supported by clinical experience in the first high risk mm patient. the marked level of host t cells persisting over the first year after hsct can provide anti-viral immune protection until thymus-derived donor t cells are generated. avoiding additional post transplant immune suppression ensures a robust anti-viral immunity and a graft vs tumor effect. the rejection experienced by the nd patient, although corrected by a nd myeloablative tcd hsct, indicates that the conditioning must be fine-tuned to optimize engraftment in every patient. we are therefore testing, increasing tbi from gy to gy. further studies will determine the efficacy of this approach in elderly mm patients, in non-malignant hematopoietic diseases, or as a prelude for organ transplantation and cell therapy. over the last decade the addition of alemtuzumab to fludarabine-based reduced intensity conditioning regimen is common practice in the unrelated donor allograft setting. in recent years, however, its use has extended to reduced intensity hla-identical sibling donor allografts with the aim of providing an additional prophylaxis against gvhd. it is difficult to assess though whether this practice has any negative influence in the relapse rate or whether it has any net benefit or disadvantage in terms of overall survival. in this retrospective study we have analysed a historical cohort of patients [ males, females, mean age . ( - )] who s received a ric fully matched unrelated donor ( patients) or sibling donor ( ) hsct as consolidation treatment for hr aml in transplant centres in uk and greece. the conditioning regimen included fludarabine in all cases, together with melphalan and alemtuzumab( patients), busulphan and campath ( patients), busulphan and thiotepa ( patient), melphalan ( patients), busulphan with and without atg ( patients) total body irradiation ( cgy, patients). in total, patients received alemtuzumab ( mud mg alemtuzumab and sibling donor hsct recipients mg alemtuzumab) and patients ( mud and sibling donor hsct recipient) received atg with patients receiving t replete allografts. gvhd prophylaxis was ciclosporin for patients receiving alemtuzumab based or atg based regimen and ciclosporin with low dose methotrexate for t-replete allografts. the median follow up was . months (range - months).all but four patients were transplanted in cr overall, patients receiving conditioning without alemtuzumab suffered more frequent (po . ) and more severe (po . ) acute gvhd. this group, however, had a significantly (po . ) lower relapse rate. the overall survival remained unaffected. the subgroup of patients receiving allografts from mud had a clear benefit in terms of a lower incidence (p o . ) and severity (p o . ) of acute gvhd: none of the patientsreceiving alemtuzumab experienced grade iv agvhd, but up to / patients not receiving alemtuzumab suffered severe grade iv gvhd. however, the use of campath was associated with a significantly higher rate of relapse or progression of the aml (po . ), so that none of the mud recipients not having campath relapsed, while / patients having alemtuzumab relapsed. although none of these factors had a net impact on survival, there was a nonsignificant (p = . ) trend towards a higher survival in patients who received alemtuzumab. in the sibling donor allograft setting, alemtuzumab had no significant impact on the incidence of acute gvhd, relapse or survival. finally, in diseases where cytogenetic or molecular markers of high risk were available, our results showed a better overall survival (po . ) in ric alemtuzumab conditioning undergoing fully matched unrelated donor hsct, probably as a result of the protection against graft versus host disease while maintaining graft versus leukaemia effect. overall, alemtuzumab is a highly protective agent against agvhd in mud hsct recipients while it maintains the graft versus leukaemia effect.however it did not show any clear benefit of its use in the identical sibling donor setting. larger prospective studies are required in order to determine the need for this agent in this particular setting. disclosure of conflict of interest: none. blastic plasmacytoid dendritic cell neoplasm (bpdcn) is a rare disease which constitutes o % of all hematologic neoplasms annually. majority of bpdcn present with diverse skin involvement prior to leukemic dissemination, whereas a minority (~ %) have systemic involvement at diagnosis. there are no established therapies for bpdcn and most pts receive acute leukemia, myeloid or lymphoblastic, induction regimens; but responses are short-lived and prognosis is poor upon relapse. allogeneic hematopoietic cell transplantation (allo-hct) is offered to bpdcn cases based on small retrospective or registry case series. we retrospectively analyzed outcomes of bpdcn pts who received an allo-hct at transplant centers in the usa. a total of pts were eligible for analysis ( table ). the primary endpoint was overall survival (os). twenty patients (m = , %), median age of ( - ) yrs, received an allo-hct from a matched related (n = , %), matched unrelated (n = , %), mismatched-unrelated (n = , %), umbilical cord (n = , %) or haploidentical (n = , %) donor using myeloablative (mac) (n = , %) or reducedintensity (ric) (n = , %) conditioning. fifteen pts received hyper-cvad as pre-allograft therapy (front-line = , salvage = ). the majority (n = , %) were allografted in cr . median f/u for survivors was . ( . - . ) months. median time-to-neutrophil and platelet engraftments were ( - ) days and ( - ) days, respectively. five pts never dropped s platelet counts below /μl. three pts (mac = , ric = ) relapsed at , , and months, respectively. all relapsed with marrow involvement ( had also skin involved). mean os was . ( . - . ) months. one-year and -year os were % ( % ci = - %) and % ( % ci = - %), respectively. there was no difference in -year os when comparing mac versus. ric (hr = . ( % ci = . , . ), p = . ). median time to onset of acute gvhd was ( - ) days; grade ii-iv acute gvhd occured in cases. chronic gvhd was seen in cases (mild = , mod/severe = ). allo-hct is an effective therapy for bpdcn resulting in durable remissions. encouraging outcomes observed in this analysis may be explained by offering allo-hct early in the disease course and in the setting of complete remission. larger studies are needed to better understand risk factors for relapse to develop post-transplant strategies to improve outcomes. disclosure of conflict of interest: none. a risk-factor analysis for overall survival in patients with acute leukemia that relapse following t-replete haploidentical transplantation: on behalf of the acute leukemia working party of the european society for blood and marrow transplantation s piemontese , , m labopin , , f ciceri , , c schmid , , a ruggeri , , w arcese , z gulbas , y koc , j tischer , b bruno , w depei , d blaise , d beelen , g ehninger , a boumendil , , m houhou , , m mohty , and a nagler , relapse of acute leukemia is the leading cause of transplantation failure with devastating results. relapse post t-replete haploidentical transplantations (haplo-sct) is not well characterized. the objective of this study was to identify riskfactors for overall survival in patients with al that relapsed after a haplo-sct. from to , haplo-sct were performed in ebmt centers as first allogeneic transplantations for adults with acute leukemia. out of patients for whom we were able to receive updated data, relapsed and were included in this analysis. median follow-up among survivors was months after haplo-sct ( - ) and . months ( - ) after relapse. median time from haplo-sct to relapse was months ( d- m). diagnosis was acute myeloid leukemia (aml) in % and acute lymphoblastic leukemia (all) in % of the patients, respectively .fifty-two ( %) patients were transplanted in first complete remission (cr ), ( %) in cr or cr , while ( %) were transplanted in active disease. ric regimen was used in ( %) patients and ( %) received bone marrow as stem cell source. post-transplant cyclophosphamide (pt-cy) was used for graft-versus-host disease (gvhd) prophylaxis in patients ( %). fifty-two ( %) of the patients who relapsed post haplo-sct experienced previously acute gvhd and ( %) chronic gvhd post transplantation. treatment of relapse varied and included: none in ( %), ist withdrawal only in ( %), chemotherapy (ct) only in ( %), tyrosine-kinase inhibitor (tki) only in'( %), tki and ct in ( %), dli only in ( %), subsequent transplant in ( %), ct and dli in ( %), ct and subsequent transplant in ( %), tki ct and subsequent transplant in ( . %), dli and subsequent transplant in ( . %) patients. donors for second allogeneic transplant were unrelated (n = ), haploidentical (n = ) and cord blood (n = ). second transplant was performed in cr for patients and in relapse for patients. only patients who received a second haplo were alive at and months post second transplant. the majority of patients who received dli were in relapse at time of dli ( %), and % achieved cr after dli. os y after dli was %, patients being alive at a median time of mo ( - ) post dli. overall, the one-year overall survival (os) following relapse was % ( % ci: . - . ). in univariate analysis disease status at haplo-sct (cr vs active disease), cytogenetics (good/intermediate vs poor) and median time from haplo-sct to relapse ( or o . months) were associated to a higher os at one year after relapse: % (p = . ), % (p = . ) and % (p o - ), respectively. in multivariate analysis complete remission at haplo-sct (p = . ; hr . ; ci: . - . ) and time from haplo-sct to relapse higher than . months (p = . ; hr . ; ci: . - . ) were risk factors for a higher os after relapse. in the patients transplanted in cr and relapsing more than month after haplo, and y os were respectively % and %. these findings suggest that similar to other transplantation setting os for acute leukemia that relapse post haplo-sct is dismal. disease status at transplant and time from transplant to relapse are the two important prognostic factors that can predict somewhat better survival. indication for second transplant should be carefully evaluated. integrations with novel therapies are in unmet need to prevent and treat relapse post haplo-sct. disclosure of conflict of interest: none. patients (pts) with aml who relapse after autologous stem cell transplantation (asct) have a dismal outcome but some can be rescued with an allogeneic transplantation (allohsct). yet, available evidence presently stems from analyses of limited patient numbers. we decided to analyze the ebmt registry to evaluate the outcome and determine the prognostic factors in a large series of such pts. the ebmt registry was screened for adult pts with de novo aml (non-apl) who received an allograft in cr or first relapse ( - ) after being autografted in cr . pts receiving ex vivo t cell depletion (tcd) were included only if they received a haploidentical allohsct. inclusion criteria were met by pts ( % female, median age [range - ] years). median time from asct to relapse was (range . - , iqr . - . ) months. at allohsct, pts were in st relapse ( %) or cr ( %). donors were matched sibling ( %), unrelated ( %), haploidentical ( %), or cord blood ( %), respectively. conditioning was myeloablative in % and reduced intensity in % of the pts, respectively. the median follow up was months (range o - months). at years post allograft (figure), leukemia free survival ( - ] of the pts. all factors significantly associated with ⩾ endpoint in univariate analysis were entered in a multivariate cox regression model (table ) . ri was lower in pts transplanted in cr rather than in relapse ( . % vs . %; hr . , p = . ) and in pts who relapsed later ( months, median value) as opposed to those who relapsed early post asct ( . % vs . %; hr (per month) . , p o - ). ri was lower in pts transplanted with an unrelated donor (ud) in comparison to those transplanted from a matched sibling donor ( . % vs . %; msd, hr: . , p o - ). patient age, poor cytogenetics, transplantation in relapse, previous tbi for asct, myeloablative conditioning (mac) vs reduced intensity (ric) and ud, haplo or cbt vs msd all significantly increased nrm. lfs was significantly better in pts with good risk ( . %) than in pts with intermediate risk or poor risk cytogenetics ( %; hr . , p = . ) or in pts who relapsed late (per month: hr . , p = . ) post asct. lfs was worse in pts who previously had received tbi ( % vs %; hr = . ; po - ). the same prognostic factors were significant for os. haploidentical (hr . , p = - ) and cord blood (hr . , p = . ) transplants resulted in lower os than those from msd. finally, date of transplant significantly influenced os which was higher in pts transplanted after january vs those allografted before; . % vs . %, hr (per year) . , p = . ). about one third of adult patients with aml who relapse post asct can be rescued with an allogeneic transplantation, especially if the duration of persisting cr post asct is long and no tbi was received in the past. transplantation from an msd while in cr rather than at relapse offers the best outcome. disclosure of conflict of interest: none. high incidences of graft-versus-host disease (gvhd) and relapse have seriously impeded the widespread application of haploidentical hematopoietic stem cell transplantation (haplo-hsct) for high-risk acute leukemia lacking conventional hla-matched donors. one hundred and ten high-risk acute leukemia patients underwent haplo-hsct with idarubicin (ida) intensified conditioning regimen (ida intensified bucy for acute myelocytic leukemia (aml) and ida intensified tbi-cy for acute lymphoblastic leukemia (all)). for donor-recipient hla / or / transplant, we separately administered a total of mg/kg or mg/kg antithymocyte globulin (atg) and basiliximab for gvhd prophylaxis. all enrolled patients were observed longitudinally until death or lost to follow-up. the -day cumulative incidences of Ⅱ-Ⅳ and Ⅲ-Ⅳ agvhd for all patients were . %, . %, respectively. the -year cumulative incidence of extensive cgvhd was . %. the relapse rate was . %. the -year probability of overall survival (os) reached . %. the patients in non-complete remission (nr) showed significantly higher relapse and worse survival than complete remission (cr) minimal residual disease (mrd) (-) and cr mrd (+) patients. however, the relapse, y-os and disease-free survival (dfs) of cr mrd (-) did not differ from cr mrd (+) patients, indicating our intensified transplant technique could overcome the poor prognosis of mrd. for whatever aml or all patients, the relapse rates, agvhd, cgvhd and the estimated -year os and dfs between two atg group were equivalent, except that all patients in atg mg/kg experienced higher relapse ( . % vs . %, p = . ). although the incidence of cytomegalovirus (cmv) reactivation in atg mg/kg and mg/kg was . %, . %, the average episodes of cmv reactivation were remarkably [p ] higher in mg/kg. our ida intensified haplo-hsct technique could improve the outcome of high-risk acute leukemia and could be recommended as a good alternate for patients lacking hla-matched sibling donors. diagnosed secondary aml were randomized : to cpx- or standard + therapy. cpx- induction was units/m on days , , (first induction) and days , (reinduction); + first induction was cytarabine mg/m /day × days and daunorubicin mg/m on days , , , and reinduction was cytarabine mg/m /day × days and daunorubicin mg/ m on days , . a dynamic allocation procedure stratified patients by age group ( - or - years) for each study arm. patients with complete response (cr) or cr with incomplete platelet or neutrophil recovery were considered for allogeneic hct, based on institutional criteria. overall survival (os) landmarked at the time of hct was assessed. a total of patients were enrolled on the induction trial. . patient and aml characteristics in the hct age subgroups were generally similar between arms. in both age subgroups of patients receiving hct, median os was longer in the cpx- arm than in the + arm (table ). in the - group, serious adverse events (saes) prior to hct in the cpx- and + arms occurred in % and % of patients, respectively; in the - group, in % and %, respectively. the most common sae was febrile neutropenia (cpx- , . %; + , . %), occurring in all age groups. relapse after allogeneic haematopoietic stem cell transplant (allo-hsct) for acute myeloid leukaemia (aml) and myelodysplastic syndrome (mds) remains the main cause of treatment failure. it is associated with dismal prognosis and short survival. proposed salvage strategies are tapering of immunosuppressive therapy, re-induction with chemotherapy and consolidation with donor lymphocyte infusion (dli) or second allo-hsct, although, results remain disappointing. azacitidine (aza) and dli has proved to be an effective and well-tolerated outpatient approach in this setting, and results in at least temporary disease control in the majority of patients, thus, representing a valuable alternative to current treatments. between january and november , patients with relapsed aml or mds after allo-hsct were treated with subcutaneous aza mg/m days - every days and escalating doses of dli if feasible at manchester royal infirmary, uk. aza was continued until cr or disease progression. patients characteristics: median age (range - ) years, % males, diagnoses were aml (n = ) and mds (n = ). five ( %) patients had either monosomal or complex karyotype. fifty percent of patients were in cr before transplant, . % in cr , . % had a partial response and % did not receive any chemotherapy before the transplant. fifteen out of received fludarabine-base reduced intensity conditioning regimen and all but one had a t-cell depleted graft. at relapse % had mixed donor chimerism. median time to relapse was . (range - ) months after allo-hsct. with a median follow up of . (range - ) months a median of (range - ) courses of aza were administered and median of (range - ) dli were infused. doses of dli were administered starting at . x /kg and escalating by log . aza and dli infusions were well tolerated; only two patients withdrew due to intolerance. seven patients were admitted at least once due to infections ( %) or progressive disease. only two patients developed mild gvhd grade . complete remission was achieved in . % patients and stable disease in %. patients in cr had full donor chimerism. median overall survival for patients in cr was months compared to months for those who did not respond (p = . ). patients with more than % blasts on bone marrow at time of relapse after allo-hsct had a worse outcome than those with less than % blasts ( months and months respectively, p = . ). no differences were seen when compared time to relapse ( o months vs ⩾ months) s and outcome, or disease and overall response, although numbers in this series are small. image/graph: overall survival following azacitidine and dli, patients in complete remission, stable disease and disease progression. azacitidine and dli can provide long term remissions in patients with relapsed aml/mds post allo-hsct with low toxicity. lower disease burden at relapse carries better outcomes. low rates of gvhd are seen following azacitidine and dli most likely showing the immunomodulatory effect of azacitidine described by other groups. acute myeloid leukemia (aml) is a frequent complication in patients affected by telomere maintenance disorders ('telomeropathies') such as dyskeratosis congenita (dkc). treatment of aml in dkc patients by chemotherapy and hematopoietic stem cell transplantation is characterized by frequent remission failure, high organ toxicity and poor outcome. a -yearold patient with aml was admitted to our hospital in december . he had been treated with cycles beacopp for hodgkin´s lymphoma (hl) in . on admission, the patient presented clinical signs of premature aging with hair greying and lack of fully recovered hair growth after chemotherapy (cx) for hl. flow-fish analysis revealed tl below the % percentile within leucocytes in line with the suspected diagnosis of telomeropathy. retrospective tl analysis by confocal q-fish from bm at hl diagnosis confirmed short tl before the start of any chemotherapy. he received standard aml induction cx ( + ), but follow-up revealed persistence of aml. salvage cx with flag-ida was applied resulting in partial remission with only weak regeneration of normal hematopoiesis. the patient received an allogeneic stem cell transplantation (asct) after conditioning with mg/m melphalan and fludarabin from his hlamatched brother whose tl was found to be normal. after asct, he developed sinusoidal obstructive syndrome and progressive liver failure treated with defibrotide and he was admitted to icu for sepsis. leucocyte count showed sufficient engraftment on day ; however, liver function recovered only partially. during critical care treatment, the patient showed cardiomyopathy, renal failure and extensive wound healing problems without epithelial proliferation indicative of severe replicative exhaustion. finally, he died due to sepsis with acute liver failure on day after asct. aml arising from dkc is a rare event with substantial impact on patients´prognosis. therapy remains challenging due to poor bm function and high risk of organ toxicity, especially liver failure and lung fibrosis. dose reduction of alkylating agents and avoidance of total body irradiation are necessary in conditioning prior to asct in patients with dkc and aml, however no clear data or recommendations exist for the management of these patients. tl screening can help to identify patients with suspected dkc related bm failure or aml and to identify family donors without telomeropathy. physicians should be aware of possible dkc related aml, especially in familial cases of aml or bone marrow failure, impaired or prolonged recovery following cytoreductive treatment or coincidence of solid (e.g. oral cavity carcinomas) and hematological malignancies. disclosure of conflict of interest: none. chronic graft-versus-host disease and donor lymphocyte infusions in patients with non-de novo acute myeloid leukemia or advanced myelodysplastic syndromes after allogeneic stem cell transplantation pg hemmati , k pfeifer , lg vuong , cf jehn , p le coutre , b dörken and r arnold medizinische klinik mit schwerpunkt hämatologie, onkologie und tumorimmunologie, charité-universitätsmedizin berlin, campus virchow-klinikum, berlin, deutschland aml with myelodysplasia-related changes and therapy-related aml (taml), collectively termed secondary aml (saml) in daily clinical routine, represent distinct subgroups in the revised who classification of myeloid neoplasm and leukemias. as compared to de novo-aml, saml is associated with a poor survival when using conventional chemotherapy approaches. this is mainly due to unfavorable cytogenetics, older age and/or the presence of comorbidities as well as poor response to induction therapy. furthermore, cumulative organ toxicity resulting from treatment of the antecedent solid malignancy in patients with therapy-related disease has to be taken into account. allogeneic stem cell transplantation (allosct) represents the only option to achieve long-term disease control and definitive cure. we retrospectively analyzed patients with saml or advanced mds (eb- according to who) transplanted at our center between and . at the time of allosct, patients ( %) were in complete hematologic remission (chr), whereas patients ( %) had active disease. cytogenetic risk was categorized according to the swog/ecog classification and was favorable (n = ; %), intermediate (n = ; %), unfavorable (n = ; %), or unknown/undetermined (n = ; %). standard myeloablative conditioning (mac) using gy total body irradiation (tbi) and cyclophosphamide was used in patients ( %), whereas fludarabin/busulfan/atg-based reduced intensity conditioning (ric) was applied in patients ( %). grafts were from related (n = ; %) or unrelated (matched: n = ; % or mismatched: n = ; %) donors. the median follow-up of the surviving patients was ( - ) months. a graft failure occurred in / patients ( %). at last day of follow-up / patients ( %) were alive and in chr. relapse occurred in / patients ( %) after a median interval of . (range: . - ) months. cause of death were either relapse or nrm (gvhd and/or infections) in / patients ( %) or / patients ( %). at , , , and years after allosct overall survival (os) or disease-free survival (dfs) of the entire cohort was %, %, %, and % or %, %, %, and %, respectively. at the same time points, the cumulative incidence of relapse (ci-r) or non-relapse mortality (ci-nrm) was %, %, %, and % or %, %, %, and %, respectively. extensive uni-und multivariate analyses revealed a number of factors associated with inferior outcome, e.g. poor-risk cytogenetics, the presence of taml, advanced age, reduced physical performance, and comorbidities, whereas donor type (unrelated versus unrelated), and remission status had no significant impact on overall outcome. furthermore, the development of gvhd, especially the presence of cgvhd, and the use of donor-lymphocyte infusions (dli), either in a prophylactic or pre-emptive setting, were identified as independent predictors for a reduced relapse incidence, which in turn, led to an improved os and dfs. our results indicate that allosct represents an important treatment option for patients with saml. however, a relapse rate of % at months prompts the development of novel approaches to prevent early disease recurrence. strategies to augment the graft-versus-leukemia (gvl) effect of allosct may help to improve the results. disclosure of conflict of interest: none. myeloid sarcoma (ms) is a rare hematologic myeloid neoplasm that can involve any site of the body. it can occur as an exclusively extramedullary form or it can be associated with an acute myeloid leukemia (aml), a chronic myeloproliferative neoplasm (mpn) or a myelodysplastic syndrome (mds) at onset or at relapse. the rarity of ms does not enable prospective clinical trials and therefore a specific multicenter register can be useful for the clinical and biological studies of this rare disease. we report the clinical characteristics and outcome of histologically confirmed ms, diagnosed and treated in italian hematological centers in the last years. the patient's median age was years. there were / de novo extramedullary ms, / de novo aml-related ms and / were secondary aml-related ms. the most common extramedullary anatomic sites of disease were: skin, lymph nodes and soft tissues. forty-three patients ( %) underwent a program of intensive chemotherapy including flai, hdac-ida, hypercvad and mec schemes, with a cr rate of % ( / ). twenty-two ( %) patients underwent allogeneic sct, from a mud, from an hla-identical sibling donor and from an haploidentical donor. the median os of the whole population ( pts) was . months. the os probability at , and years was %, % and %, respectively. the os was better in patients that underwent an intensive therapeutic program (median os: months vs months). among the intensively treated patients, in univariate analysis, the os was better in young patients (p = . ), in patients that underwent allo-sct (p = . ) and in patients that achieved a cr during treatment (p = . ), and was worse in pts with secondary aml-related ms (p = . ). age, response to intensive chemotherapy and allo-sct were the only three variables that significantly influenced dfs (p = . , p = . and p = . , respectively). in multivariable analysis, allo-sct and response to intensive chemotherapy remained significant in predicting a better os (p = . and p = . , respectively), and response to intensive chemotherapy was the only significant variable in predicting dfs (p = . ). after allo-sct we observe a survival advantage in patients who achieved a pre-transplant cr (p = . ) and in those who developed a chronic gvhd (p = . ). patients with ms, both with de novo and secondary forms, still have a very unfavorable outcome and require an intensive therapeutic program, that includes allo-sct, whenever possible. the outcome after allo-sct is positively influenced by the development of chronic gvhd suggesting a graft versus ms effect. disclosure of conflict of interest: none. relapse of acute lymphoblastic leukemia (all) after allogeneic stem cell transplantation (sct) is associated with poor prognosis. blinatumomab may enhance the efficacy of donor lymphocyte infusions (dli) in this specific situation but data on the concurrent use of dli and blinatumomab are sparse. the patient presented here was diagnosed with standard risk pre-b-all (presence of t( ; ); bcr-abl and cd negative) at the age of . during treatment according to the german multicenter all-study group (gmall) protocol he presented with molecular relapse and months after initial diagnosis he received a tbi-based myeloablative sct from an unrelated hla-identical ( / ) donor. post sct he was negative for minimal residual disease (mrd) with % donor engraftment. given the high relapse risk he received prophylactic dli without occurrence of graft-versus-host disease (gvhd). one year after st sct he presented with an extramedullary (testes) and molecular relapse. after remission induction resulting in negative mrd he received a nd sct from an alternative, hlaidentical ( / ) donor after reduced intensity conditioning. this again resulted in negative mrd with % donor chimerism without any gvhd. six months after nd sct he presented with bone marrow relapse. we decided on the concurrent use of blinatumomab and dli. the first cycle of blinatumomab was initiated at standard dose including dose escalation without relevant toxicities. on day of the nd cycle, i. e. in the infusion-free interval before the rd cycle the patient received the first dli at x cd /kg. no toxicities or gvhd occurred. the rd cycle of blinatumomab was initiated and a second dli at . x cd /kg was applied on day of the rd cycle. on day of the rd cycle, i. e. day after nd dli the patient presented with signs of overlap gvhd (mouth, skin) and topical steroids were started. upon progression of clinical gvhd systemic steroids were initiated with immediate response. steroids were rapidly tapered and a th cycle of blinatumomab was started. gvhd did not recur. current staging after the th cycle blinatumomab, i.e. on day + after nd sct and months after initiation of blinatumomab treatment revealed complete remission with negative mrd, % donor chimerism and no signs of extramedullary relapse. counts of cd -cells at that time point were /μl. no relevant infections or relevant blinatumomab-associated toxicities were present during the entire course after the nd sct. in this case concurrent treatment of blinatumomab and dli resulted in the longest disease-free interval for our patient compared to preceding chemotherapy or dli alone. together with the small number of reported cases (ueda et al.) this supports the concept of concurrent blinatumomab and dli as an effective post sct treatment. the objective of the study is to evaluate the clinical efficacy and safety of decitabine (dac) in combination with haag regimen [homoharringtonine (hht), cytarabine (ara-c), doxorubicin (acla) and recombinant human granulocyte colony stimulating factor (g-csf)] for advanced patients with acute myeloid leukemia (aml). thirty-six patients with advanced aml receiving dac combined with haag chemotherapy in our center from december to august were enrolled in this study. eighteen of them were refractory or relapsed aml, and another patients were those who didn't achieve complete remission (cr) after a course of induction chemotherapy. the therapeutic responses, side effects and longtime survival were retrospectively analyzed. after a course of treatment, the rate of cr and partial response (pr) was . % ( / ) and . % ( / ) respectively, while the overall response rate (orr) was . % ( / ) in the cohort. for the patients with refractory or relapse aml, cr was . % ( / ), pr was . % ( / ), and orr was . % ( / ). while for the other not getting cr after a course of induction chemotherapy, cr was . % ( / ), pr was . % ( / ), and orr was . % ( / ). grade hematological toxicities were observed in all patients, and . % cases experienced infection. and all non hematological side effects were mild and well-tolerated. with a median follow-up of . ( . ~ . ) months, the -year overall survival (os) rate was . %, . % for the refractory or relapsed aml patients, and . % for those not achieving cr after a course of induction chemotherapy. the difference was significantly (p = . ). conclusion dac combined with haag regimen is safe and effective salvage treatment for advanced stage aml patients. disclosure of conflict of interest: none. aml patients harboring flt -itd mutation are associated with decreased survival compared to patients without flt -itd mutation. nevertheless, whether flt-itd mutation also has negative impact on the post-transplant survival is less clear. for flt -itd mutated aml, a decreased leukemia-free survival (lfs) after allogeneic hsct was observed in ebmt analysis but not cibmtr. in this study, unlike studies of ebmt or cibmtr which only pre-specified populations of patients were analyzed (cr in ebmt, cr +cr in cibmtr), we examined the prognostic impact of flt -itd mutation on post-transplant outcome of "all" the adult aml patients reported to taiwan bone marrow transplant registry (tbmtr). tbmtr is a research collaboration affiliated to the taiwan society of blood and bone marrow transplantation. it comprises all the transplantation centers in taiwan that contribute detailed data on hsct. adults aged ⩾ years with a diagnosis of aml and with known flt-itd mutation status in the registry were included. patient characteristics and transplant outcome following allogeneic hsct for flt -itd mutated and nonmutated aml were compared. kaplan-meier estimates were used to calculate the probability of lfs and overall survival (os). multivariable analyses for lfs and os were performed using cox proportional hazards model. patients who met the eligibility criteria were enrolled for analysis. the median follow-up of survivors was months. of the patients, ( . %) were positive and ( . %) were negative for flt -itd mutation. flt -mutated patients had significantly more transplantation at cr ( . %), shorter time interval between diagnosis and hsct ( . months), and higher wbc count at diagnosis ( . × /l) comparing to patients without flt mutation ( . % at cr , . months from diagnosis to hsct, and . × /l wbc count at diagnosis). significant more flt mutated patients had intermediate-risk ( . %) and normal ( . %) karyotype at diagnosis. the age, donor type, stem cell source, conditioning regimen, and atg use were not significant different between flt -mutated and non-mutated patients. of the whole population, flt mutation status did not negatively impact the transplant outcome ( years os for flt mutated and non-mutated patients: . % vs %, log rank p = . ; years lfs for flt mutated and non-mutated patients: . % vs . %, log rank p = . ). when different pre-transplant conditions (cr , subsequent cr, and no cr) were analyzed separately, flt -itd mutation status is still not a significant prognostic factor of os and lfs for patients in cr (equally good) and no cr (equally bad). however, for patients in subsequent cr, flt -itd mutation is the only significant factor predicting poor os and lfs in multi-variable analysis (median os and lfs for flt mutated and nonmutated patients: vs days, log rank p = . ; vs days, log rank po . respectively). the incidence of non-relapse mortality, grade / acute gvhd and extensive chronic gvhd is comparable between flt -mutated and nonmutated patients. flt -itd mutation is a significant and strong predictor of poor survival for aml patients in subsequent cr at hsct. for flt -itd non-mutated aml, a sizable portion of patients can have disease free survival after allogeneic hsct at subsequent cr. however, allogeneic hsct at cr should be strongly recommended for flt -itd mutated aml. [p ] disclosure of conflict of interest: none. allogeneic stem cell transplantation (asct) is a curative strategy in acute myeloblastic leukemia (aml) and myelodysplastic syndrome (mds). however, relapse keeps being the main cause of treatment failure. extramedullary relapse (er) is a rare event and its management is not well standardized. we retrospectively analyzed patients who received asct from to and developed er in our centre. we performed a descriptive study to analyze characteristic of these patients, post-relapse treatment and survival. statistic analysis was performed using spss v. . we found a total of patients with er, one of them with er after consecutive asct, so we analyzed cases of er. patient and transplant characteristics are summarized in table . at day + , % of patients were in complete response (cr). er occurred after a median of ( - ) months post-asct. eleven patients ( %) presented with a bone marrow relapse concomitant with the er. er affected central nervous system (cns) in patients ( . %), bone in patients ( %), skin or soft tissue in patients ( . %), mama in patients ( . %), ocular globe in patients ( . %) and teste in patients ( . %). two of them presented with multiple sites affected. between the patients who developed cns relapse, of them had received intrathecal prophylaxis. regarding post-er management, immune modulation was conducted in patients (immunosupression tapering in , donor lymphocyte infusions in and both strategies in ). all patients except one received systemic treatment (salvage chemotherapy in , azacitidine in , low dose arac in and atra in patient with a promyelocytic leukemia). together with systemic treatment, received radiotherapy and intrathecal therapy was used in all patients with cns involvement. response: out patients treated, ( . %) achieved cr and ( . %) progressed. two responding patients received a nd asct. after a median follow-up of months ( - ), patients are alive and disease free, with an estimated overall survival of % at years. patients receiving salvage chemotherapy followed or not by a nd asct experienced a significantly better os than those receiving other therapies (median os vs months; p = . ). patients with bone marrow involvement at relapse show a worse prognosis (median os vs months; p = . ) although not statistically significant due to small number of patients (image ). ten patients died due to disease progression. er must be considered in patients receiving an asct in case of organ symptoms. patients can be rescued with salvage chemotherapy followed or not by a nd asct achieving good results in terms of long term os. it seems that involvement of bone marrow at relapse confers a worse prognosis, what should be confirmed in a larger series of patients. [p ] disclosure of conflict of interest: none. flag-ida regimen as bridge therapy to allotransplant in refractory/relapsed aml patients: a single-center experience c pasciolla, m delia, d pastore, p carluccio, a ricco, a russo rossi, a mestice, f albano and g specchia university of bari, italy although treatment outcome in acute myeloid leukemia (aml) adult patient has improved over the past decade, relapse still occurs in up to - % of cases. furthermore, - % of patients fail to achieve complete remission (cr) because of treatment-resistance. the management of primary refractory and/or relapsed disease remainschallenging for clinicians. in our study, we reviewed the outcome of refractory and/or relapsed aml patients who underwent salvage therapy with the flag-ida regimen between and at our institution. the study aim was to determine the efficacy of the flag-ida regimen in order to clarify which variables (who ps, ldh, bone marrow, peripheral blood blasts and platelets counts, white blood cells (wbc), pmn, molecular-cytogentic risk, duration of response and relapsed or refractory disease), present before starting flag-ida treatment, might have an impact both on cr and on os. we analyzed consecutive adult patients ( males, females; median age years, range - ) with newly diagnosed acute myeloid leukemia refractory to standard induction regimens or relapsed after cr, who received the flag-ida protocol as salvage therapy between january and december . sixty-eight of the patients ( %) were in first relapse, forty-seven patients ( %) were refractory to conventional chemotherapy. median wbc count before salvage therapy was . x /l (range . - ). median bone marrow and peripheral blasts counts were and %, respectively; median platelets count was x e /μl. according to the fab classification, patients had m , m , m , m , m , m , had biphenotype acute leukemia. according to molecular-cytogenetic risk stratification ( %), ( %) and ( %) patients belonged to poor, intermediate and good risk group, respectively. sixty-nine of patients ( %) achieved complete remission (cr); forty-seven %) patients were refractory to the salvage therapy. in multivariable analysis, variables with positive impact on response rate were lower wbc counts (o e /μl, p = . ), higher platlets counts ( x e /μl, p = . ), molecular-cytogenetic risk (p = . ), duration of response in relapsed aml (p = . ) and relapsed rather than primary refractory disease (p = . ), respectively. median os was months (m). cox regression analysis confirmed that both higher platlets counts, p = . ( ( x e /μl) vs m (o x e ul), log rank, p = . ) and relapsed disease, p = . ( (relapsed) vs m (refractory), gehan-breslow, p = . ) correlated with better survival. of note, molecular-cytogenetic risk evaluated before starting treatment was associated with cr, while no correlation was found with os. our data seem to confirm the value of flag-ida in relapsed amland may suggest its best usage as bridge-therapy in patients awaiting allotransplantation. disclosure of conflict of interest: none. s leukemia relapse is the major cause of death in patients received allogeneic hematopoietic stem cell transplantation (allo-hsct). the precise etiological mechanisms of leukemia relapse remain unclear. both leukemia cells themselves and hematogenesis micro-environment are involved in the relapse event. in our previous study, we reported a case of donor derived relapse of acute myeloid leukemia (aml) after allo-hsct. the patient and his donor-sister both harbored a germline mutation(c. - dup) in cebpa gene. donor hematopoietic cells transformed to aml by developing two somatic cebpa mutations ( dupc and - dup) in the patient's microenvironment. hence we suspect that - dup mutation of cebpa gene may altered hematopoiesis microenvironment and increased the survival of aml cells. to conform our hypothesis, we transfected mesenchyme stem cells (mscs) with cebpa - dup or wide type and took vector as control. aml cell line hl cells were co-cultured with transfected mscs and then treated with ng/ml doxorubicin. apoptosis and cell cycle were detected at day . mscs protected hl cells from toxicity of doxorubicin. this protection was enhanced by overexpression of cebpa - dup . apoptosis rates of hl cells in group of msc-vector and msc-cebpa - dup were . ± . % vs . ± . % (p＜ . ). a larger part of hl cells remains quiescent with s higher rate of g /g phase in msc-cebpa - dup group, which may reduce the sensibility of hl cells to doxorubicin. to explore mechanisms involved in the alteration of microenvironment, we performed rna sequence with each group of mscs. we found that col a , col a and col a were upregulated in msc-cebpa - dup group compared with msc-cebpa wt group (col a :cebpa wt vs cebpa - dup was . vs . , p = . e- ; col a :cebpa wt vs cebpa - dup was . vs . , p = . e- ; col a : cebpa wt vs cebpa - dup was . vs . , p = . e- ). furthermore, we found that ddit and herpud genes, which were important factors in cellular unfolded protein response(upr) and to topologically incorrect protein, failed to augment in cebpa - dup group (ddit : vector vs cebpa wt the cure rate of childhood acute lymphoblastic leukemia (all) has improved considerably and approaches % today. however, the outcomes of patients who suffer from leukemic relapse remain unsatisfactory. despite the high cure rate of children and adolescents with all a subgroup of patients benefit from allogeneic hsct. allo hsct remains the standard treatment for intermediate/high risk aml patients. patients, all = and aml = age to years with median age years, m/f = / (m/f all = / , aml = / ) underwent sct in our hospital (from to ). fifty-eight patients transplanted allo hsct and pt aml auto hsct. conditioning regimens consisted of busulfan (iv) +cyclophosphamide for allo and cyclophosphamide + vp +cytarabine for auto hsct. peripheral blood (pb) was the source of progenitor cells in patients, bone marrow (bm) in patients and cord blood in one patient. in allo hsct, patient transplanted / matched and patients / matched. gvhd prophylaxis regimen was cyclosporine + mtx. all patients engrafted. in allogeneic pbsct all patients' median time to absolute neutrophil count (anc) . × /l was days, and the median time to platelet count × was days vs and days in allo bm all patients. in allogeneic pbsct aml patients median time to anc . × /l was days, and the median time to platelet count × was days. (all patients with aml transplanted with pb). at present pts are alive ( all, aml) and pts died due to ards, vod, hemorrhagic stroke, sepsis and relapse. trm was % at days. median time of death after transplantation was days in all and in aml. in allo pbsct all patients hospitalization period were days vs in allo bm all patients. acute gvhd appeared in % pts. chronic gvhd appeared in % pts. with a median follow-up of months ( - months) after transplant the event-free survival were % and four years overall survival % in all patients. a median follow-up of . months ( - months) after transplant the event-free survival were % and three years overall survival % in aml patients. hematopoietic stem cell transplantation can lead to durable remissions in children and adolescents with leukemia and increase in survival of children. pbsct in childhood all was consistent with significant faster anc and platelet recovery in allogeneic pbsct, hospitalization was shorter. longer follow-up is required to evaluate fully efficacy and long-term results. disclosure of conflict of interest: none. group hla-c /c subtypes were defined as previously practiced. median age of patients was , % of them were male. allo-hscts were performed from % unrelated donors vs % related donors. remission status was detected in % of patients whereas % had active disease pre-transplant. stem cell sources were as follows: % peripheral blood, % bone marrow, % cord blood, % bone marrow plus peripheral blood. the most frequent fab subtype was aml-m . patients were grouped by hla-c status: % c /c homozygote, % c /c heterozygote and % c /c homozygote. the frequency of hla c donor/recipient mismatch allo-hscts was %. relapse was detected in % of patients. the relapse risk was significantly lower in c /c homozygote patients compared to c /c homozygotes ( % vs %, p = . ). lfs was similar between c /c homozygote group and c /c homozygote group (p = . ) (figure ). in multivariate analysis (age, sex, remission status, related/ unrelated transplant, aml subtype), lfs was increased by pre-transplant remission status (p year). there was no difference detected between -years os in c /c homozygote group and other groups ( % vs %, p = . ) (figure ) . when similar analysis were repeated with donor hla type results were not significant. our results confirm two earlier published reports on aml and all. even in the absence of kir genotyping, hla group c has a protective effect. if hla matched donor is not possible a donor-recipient hla-c mismatch favoring c to c may be preferable. disclosure of conflict of interest: none. immunomodulatory kits do not induce aml-blasts' proliferation ex vivo: ipo- is an appropriate and reliable marker to detect and quantify proliferating blasts c plett, dc amberger, a rabe, d deen, z stankova, a hirn, y vokac, j-o werner, j schmohl, d krämer, a rank, c schmid and h schmetzer aml-blasts can be converted to dcleu by immunomodulatory 'kits' (ex vivo). t-cells' energy can be overcome after stimulation with dc/dcleu and results in antileukemic reactivity. a potential induction of blast-proliferation (e.g. by immunomodulatory kit-application in vivo) in aml-pts has to be excluded. kits containing combinations of gm-csf with - additional factors (pge- / , picibanil, ifnα, tnfα, calciumionophore) were studied with respect to the generation of dc/dcleu from blasts, mediation of antileukemic reactivity (after dc/dcleu-stimulation) and with respect to their potential to induce blast-proliferation in a whole blood (wb) culture-system. we studied different markers (ipo- , ki- , cd ) and quantified blast proliferation before/after culture. we correlated findings with (ex vivo) antileukemic functionality, with disease-entities and the course of the disease. dc-generation: we could generate dc/dcleu regularly from wb culture from aml-pts. detection of blast proliferation: Ø . % (range - %) of uncultured blasts expressed ipo- , . % ( - %) cd , . % ( - %) ki- . induction of blast proliferation: pooling all results we found lowest amounts of proliferating blasts after culture with kit i (gm-csf+picibanil, % ± . ), kit k (gm-csf+pge , . % ± . ), kit m (gm-csf+pge , . % ± . ). amounts of proliferating blasts were lower compared to uncultured cells. highest expression of proliferating blasts was found with ipo- followed by cd and ki- .we found few individual aml-samples with increased blast-proliferation after ex vivo kit-culture. antileukemic activity: t-cells stimulated with dcleu (generated with kits) improved antileukemic activity. correlations between blast-proliferation and antileukemic activity will be presented. clinical correlations: pts with bad (vs good) cytogenetic risk were characterized by higher proportions of proliferating blasts in uncultured blasts; in some pts with iron-deficiency anemia (ida) proportions of cd +unculured blasts were lower than of ipo- /ki- + blasts. ipo- is a stable marker to be used to quantify proliferating blasts in aml-pts. cd is also a good marker, although not suitable for some pts with ida, ki- is no reliable marker for every given pt. subtypes of pts correlated with proportions of proliferating blasts. in general kit treatment of blasts did only exceptionally induce blast proliferation ex vivo. in general lowest risk for blast proliferation was seen after culture with kit i, k and m. t-cellstimulation with dc/dcleu generated after kit-treatment resulted regularly in antileukemic reactivity. we conclude that an in vivo treatment of aml-pts with kits i, k or m might be safe (no induction of blast proliferation). disclosure of conflict of interest: none. the occurrence of additional cytogenetic abnormalities (acas) is common in philadelphia chromosome-positive acute lymphoblastic leukemia (ph+ all), but is of unknown significance in the tyrosine kinase inhibitor era. recent study [aldoss et al., ] has revealed the acas appear to have a significant deleterious effect on outcomes post-hsct in adult ph+ all patients only. we retrospectively analyzed data from adult and pediatric patients with ph+ all who had undergone allogeneic hematopoietic stem cell transplantation (allo-hsct) at our university between and . among patients with ph+ all, patients had available data on conventional cytogenetics before allo-hsct. all patients and transplant characteristics are listed in table i . thirty-three of patients ( %) had isolated t( ; ). acas were revealed in / ( %) pts, including / ( %) pts with ⩾ cytogenetic abnormalities (with complex karyotype, ck). the median follow-up was ( - ) days. overall survival (os) and event free survival (efs) were % ( % ci - ) and % ( % ci - ) at years, respectively. in univariate analysis, prognostic factors associated with increased os and efs were donor type (match related/match unrelated vs haploidentical; p = . for both), the disease status at transplant (cr vs beyond cr ; p = . , only for efs), acas (aca-vs aca+; p = . , only for os) and, especially, the complex karyotype (ck-vs ck+; p = . , only for os) (figure ). multivariate analysis showed that the independent prognostic factors for os and efs remained the complex karyotype (hr- . , % ci, . - . ; p = . ) and disease status at transplant (hr- . , % ci, . - . ; p = . ), respectively. the study demonstrates the acas and disease status at allo-hsct to be independent prognostic factors not only for adult, but for pediatric ph+ all patients too. up to % of newly diagnosed acute myeloid leukemia (aml) patients (pts) present initially with hyperleukocytosis consequently placing them at increased risk for morbidity and mortality during induction. , whereas early publications have indicated that hyperleukocytosis is an adverse prognostic factor associated with poor long term outcome, it is currently unknown whether hyperleukocytosis still retains prognostic value for aml patients undergoing allogeneic stem cell transplantation. furthermore, it is unknown whether hyperleukocytosis retains prognostic value when modern molecular markers such as flt and npm are accounted for. we hypothesized that hyperleukocytosis at initial diagnosis is still an independent adverse prognostic factor influencing long term outcome in aml pts undergoing allogeneic stem cell transplantation. we performed a retrospective analysis using the multicenter registry of the acute leukemia working party (alwp) of the european society for blood and marrow transplantation (ebmt). pts included in the analysis were over years of age, with de-novo non-m aml, a presenting white blood cell count of over k, with an hla matched related or unrelated donor, transplanted between and . clinical outcome indices of hyperleukocyotosis pts namely, non-relapse mortality (nrm), graft versus host disease (gvhd), relapse incidence (ri), leukemia free survival (lfs), overall survival (os) and gvhd-free/relapse-free survival (grfs) were compared to a cohort of pts without presenting leukocytosis. multivariate analyses were used to assess whether hyperleukocytosis was independently associated with ri, nrm, os, lfs, and grfs. age, gender, number of chemotherapy inductions, cytogenetics, donor type, fms-like tyrosine kinase- (flt ) status, nucleophosmin (npm ) status, and conditioning intensity were covariates for regression modeling. a cohort of pts with hyperleukocytosis ( patients with wbc over k and less than k, and patients with wbc over k) was compared to pts without hyperleukocytosis. pts with hyperleukocytosis were younger, had an increased rate of favorable risk cytogenetics, were more likely to be flt and npm mutated, and had an increased rate of myeloablative conditioning. on univariate analysis pts with hyperleukocytosis had an increased rate of ri ( % vs . %, p = . ), and decreased incidence of grfs ( . % vs . %, p = . ). in multivariate regression analysis, hyperleukocytosis was significantly associated with increased ri (hazard ratio [hr] of . , % confidence interval [ci], . - . ; p = . ), s poorer lfs (hr of . , % ci, . - . ; p = . ), decreased grfs (hr of . , % ci, . - . ; p = . ), and poorer os (figure ) (hr of . , % ci, . - . ; p = . ). image/graph hyperleukocytosis at initial presentation retains a significant prognostic role for aml patients undergoing allogeneic stem cell transplantation even in the current era of advanced molecular prognostication. outcome after hematopoietic stem cell transplantation for philadelphia-positive aml: relatively favorable outcome in patients allografted in first complete response; a survey from the acute leukemia working party of the european society for blood and marrow transplantation (ebmt) v lazarevic , m labopin , w deipei , i yakoub-agha , a huynh , p ljungman , n schaap , d blaise , jj cornelissen , n maillard , p pioltelli , t gedde-dahl , s lenhoff , m houhou , j esteve , m mohty and a nagler , aml with t( ; ) and bcr-abl rearrangement (ph-pos aml) is a very rare aml subtype, recognized as a new provisional entity in the recent who classification. the role of stem cell transplantation (sct) in the era of abl tyrosine-kinase inhibitors (tkis) is mostly unknown. we analyzed long-term outcome in patients ⩾ years after allogeneic or autologous sct performed between - in ebmt centers responding to a designated survey. patients with blast crisis cml and philadelphia-positive all were excluded. primary end-point was os. secondary end-points were nrm, acute gvhd, chronic gvhd, lfs, ri, and the effect of tki on outcome. patients (median age, years, range: - ; males and females) with ph-pos aml undergoing sct (allogeneic, ; autologous, ) were identified. median wbc count at diagnosis was x /l ( . - ) and % had splenomegaly (data missing on patients). translocation t( ; ) was the sole abnormality in patients ( . %). the majority of the patients received one or two courses of chemotherapy before transplant and % attained cr after one course. the majority ( %) received a tki (mostly imatinib, / ) before transplant, with a median period of exposition of days (iqr - ), while ( %) received tki after the transplant either as maintenance (n = ) or treatment for relapse (n = ). at time of transplant, patients were in complete response (cr - , including all autosct; cr - ) and the remaining patients were allografted in advanced phase. among patients with available information, achieved a mrd negative status at transplant (ratio bcr-abl/abl o ).regarding allosct, conditioning regimen was myeloablative (mac) in / ( %) patients o years, while in patients years received a reduced intensity regimen (ric) and mac. cell source was peripheral blood stem cells in and bone marrow in allogeneic transplants. the donor was a hla matched sibling (msd) in cases and unrelated (ud) in , amongst whom were / and were / hla matched, respectively. in the patients undergoing autologous sct the majority received busulfanbased conditioning (n = ) and peripheral stem cells (n = ). median follow-up was . . %, . %, . %, and . %, respectively. by the univariate analysis, age ( o vs ⩾ ) was associated with ri ( -yr: . vs %), lfs ( -yr: . vs . %, and grfs ( -yr: . vs . %), whereas mrd-negative status before allosct was associated with an improved grfs ( . vs . %). in the patients autografted, ri, nrm, lfs and os at -year was % ( . - . ), %, % ( . - . ), and . % ( - ), respectively. outcome of patients with ph-pos aml who received allosct in cr in recent years was relatively favorable, especially among younger patients, probably reflecting the beneficial effect of tki. disclosure of conflict of interest: none. outcome of allografting for aml-cr is equivalent across the bsbmt and ebmt and is associated with encouraging os and dfs across all age groups j byrne, j perry , k kirkland , r pearce and g jackson bsbmt and newcastle university and freeman hospital, newcastle relapsed aml has a very poor prognosis with a high mortality, even if a second cr is achieved. the only curative treatment is with an allogeneic hsct but allografts for aml in cr are considered to have a worse outcome compared to those performed in cr , especially in older patients for whom this therapy may not be considered. the bsbmt undertook a bench-marking study analysing the outcomes for all patients allografted for aml-cr from to . the uk outcomes from paediatric and adult patients were compared to non-uk patients transplanted for the same indication reported to the ebmt during the same period. allogeneic transplants for aml-cr represent an important part of any allograft program and numbers referred for allograft were stable between - in both programs. the median age of patients was . yrs and . years in the bsbmt and ebmt cohorts respectively, with % and % of patients aged o years and % and % aged years in the groups. the length of first remission was missing in many of the ebmt registrations so time from diagnosis to transplant was used as a surrogate for this and was similar in both cohorts ( m and m respectively). similarly the presence of comorbidities was poorly reported in both databases but was similar. the bsbmt cohort included fewer patients undergoing ric conditioning protocols ( % vs %), fewer sibling transplants ( % vs %) and more pbsc allografts ( % vs %). transplant related morbidity and mortality were similar across the two cohorts (bsbmt v ebmt) with rates of severe acute gvhd (grade iii and iv) % v %, limited chronic gvhd % v %, relapse at year % v % and death in cr at year % v %. chronic gvhd (both limited and extensive) appeared more common in the bsbmt cohort ( % v %) although reporting of cgvhd was more comprehensive and complete within the bsbmt registry and may be under-reported in the ebmt registry. outcomes were excellent in both cohorts with outstanding rates of leukaemia free survival in this high risk cohort at day (bsbmt v ebmt) % v %, at year % v %, at years % v % and at years % v %. although os and lfs was significantly shorter in patients aged years, at % and % the results in this high risk age group are acceptable and warrant its continued use. multivariate analysis of the combined cohorts showed that age at transplant ( o yrs/ - / yrs), time from diagnosis to transplant and the presence of agvhd were important factors affecting dfs. risk factors for relapse included the type of conditioning used, presence of agvhd and time from diagnosis to transplant, whereas those for trm included age, agvhd, source of stem cells and time to transplant. there was no significant difference in outcomes between the bsbmt and ebmt for this indication. outcomes for patients allografted for aml-cr are excellent and appear superior to those reported for patients not undergoing an allograft in both the bsbmt and ebmt cohorts. the os and dfs observed are comparable to those reported for allografts in aml-cr and, although this study has not considered outcomes for patients who did not achieve a nd cr, it nevertheless supports the practice of risk stratification of aml patients such that only high risk patients are offered an allograft in cr with the remainder being offered an allograft as salvage after relapse. disclosure of conflict of interest: none. to evaluate the efficacy and safety of intensive chemotherapy followed by allogeneic hematopoietic stem cell transplantation (allo-hsct) for atl. a total of evaluable atl patients treated at our center from to are retrospectively analyzed. the htlv-i proviral dna load in peripheral blood mononuclear cells using pcr assay was developed, in comparison with htlv- tax protein expression measured by western-blot, to confirm the diagnoses and monitor the disease control. patients were male and patients were female. median age was . (range - ) years. all obtained patient samples were subjected to flow cytometric examination and karyotype analysis. patients received chemotherapy as the induction therapy while quit at the time of diagnosis, with da-edoch regimen while with other regimens such as chop, vcap and amp. da-edoch regimen is a variation of dose-adjusted epoch regimen with the replacement of prednisone ( mg/m per day) by dexamethasone ( mg/m per day). before the conventional regimens bucy followed by prophylaxis donor lymphocyte infusion, both received a course of salvage chemotherapy including fludarabine and cytarabine for days registered on http://clinicaltrials.gov (nct ). the gvhd prophylaxis consisted of atg, csa and mtx. the patient characteristics, therapeutic effect and survival data were collected. all patients came from the coastal area in the south-east of china. subtype classification of these atll were acute, lymphoma and chronic type. the main manifestations were characterized with cutaneous and respiratory involvement, hepatosplenomegaly, lymphadenopathy and the laboratory abnormalities as leukocytosis with atl cells, hypercalcemia and elevated serum ldh. typical morphological characterisitic of "flower cells" were observed in . % cases and most of the atll cells are cd +cd -. chromosomal abnormalities were detected in cases. all patients who didn't receive da-edoch regimen died of disease progress, while among patients with da-edoch regimen, achieved cr, pr and died. with a median follow-up of . ( - . ) months, patients respond to da-edoch are still alive. patients in cr achieved successful engraftment with complete donor chimerism in one month post haplo-identical transplant. both were received prophylaxis donor lymphocyte infusion and the immunosuppressive agents were abruptly discontinued for induction of a graft-verus-atl (gvl) effect. they keep remain alive and disease free longer than years so far without severe graftversus-host (gvhd), and the htlv- proviral dna became undetectable after allo-hsct. conclusion: it shows great promise of da-edoch regimen followed by allo-hsct to the long-term cure of atl with apparent clearance of the virus. haplo-identical transplantation can be an alternative option for the atl patients without increasing non-relapse mortality. [p ] disclosure of conflict of interest: none. in the absence of an hla-matched related donor or a good matched unrelated donor in time, haploidentical stem cell transplantation (haplo-sct) is an option for patients requiring an allogeneic hematopoietic stem cell transplant. substantial progress has been made in the last two decades with a dramatic improvement in patient outcomes, with some groups reporting preliminary beneficial effects similar to the ones in hla matched unrelated donor and cord blood transplant. several strategies have been adopted through the years for the procedure. the two strategies used in haplo-sct are ex vivo t-cell depletion and t-cell replete transplantation. the latter can be done with a combination of immunosupressive drugs ( beijing approach) or with post-transplantation highdose cyclophosphamide (post-cy). due to of its lower cost, feasibility and practicality, post-cy has become the most often used platform for haplo-sct in the majority of allogeneic transplantation units worldwide. we analyzed our experience in haplo-sct, since the first one in march to the last one that has just been done in october . we collected all complications reported, also mortality related to treatment and to the disease. we analyzed the overall survival (os). transplants were treated, with different sct indications, the most common being acute myeloblastic leukemia (n = , %), the rest of indications are exposed in figure . all our patients, independent of the conditioning receive post-cy as t cell depletion measure and stem cells were collected from peripheral blood. age at the time of transplant was . years, % were males, % females, the rest of patient characteristics are listed in table . in our series the treatment related mortality (trm) was low with only patient ( %) that died before the day + . as complications, we reported % of hemorrhagic cystitis, % of sinusoidal obstruction syndrome, % reports systemic inflammatory response syndrome, % of citomegalovirus (cmv) reactivation. neutrophils graft is . days (r = - ) and the platelets graft is . days (r = - ). in our series we haven't reported any case of graft failure, one of the transplantes the patient had antihla antibodies, this was treated with a plasmapheresis previous the stem cell infusion, and was infused with a high number of cd + cells ( × /kg), no graft problems, and has had no complications since then with the graft. the os for the whole group is months, with a median not reach at months, with patient's dead at time of analysis. two patients had a relapse after the haplo-sct ( %), both of them received lymphocyte donor infusion, sadly, neither of them responded. the haplo-sct procedure is been adopted by many centers for high risk hematology malignancies, mainly because the fast availability of donors, and because of the preliminary results that have been reported place it as good as the unrelated donor or cord unit transplant. our center is getting experience in these types of transplants, and our results reflect similar outcomes as larger studies. with longer follow ups we will be able to keep the trend of good results both in procedure safety and disease efficacy. os, toxicity and trm are expected for these high risk malignancies. in overall, the haplo-sct seems a reasonable technique that is reflecting in our short series, the results being reported in studies worldwide at bigger scale. disclosure of conflict of interest: none. complex karyotype, the presence of flt- itd and losses of genetic material in chromosome , are all considered high risk markers in aml. patients bearing these abnormalities could undergo a myeloablative allogeneic transplantation in cr whenever this is possible, although this could significatly reduce the chances for cure in older patients due to increased transplant related mortality. ric allografts could be performed in older patients in order to overcome the deleterious effects of these individual abnormalities but its effect still remains controversial in the high risk group. between and , a total patients [ males, females, mean age . ( - )] received a ric allograft ( from fully matched unrelated donors, and from an identical sibling) for high risk aml in transplant centres. high risk disease was classified according to their response to treatment, the presence of complex karyotype, the presence of individual cytogenetic/molecular abnormalities or a combination of these. in particular, patients presented one single karyotype abnormality and presented three or more. ten patients presented alterations of chromosome and patients presented flt- itr. the conditioning regimes included fludarabine in all cases, together with melphalan and campath ( patients), busulphan and campath ( patients), busulphan and thiotepa ( patient), melphalan ( patients), busulphan with and without atg ( patients) total body irradiation ( cgy, patients). graft versus host disease prophylaxis was ciclosporin for patients receiving alemtuzumab or atg but for patients who had a t-replete allograft ciclosporin and low dose methotrexate was the preferred prophylaxis. all but four patients were transplanted in cr patients were followed-up for a mean . months (range - ). thirty-one ( %) patients remain alive. the causes of death ( cases) include relapse or progression of the original disease ( ), transplant-related causes ( ) and unknown in cases. the influence of the genetic abnormalities on survival was analysed, showing there were no significant differences between patients with normal karyotype, single chromosomal abnormalities and two or more abnormalities. likewise, the kaplan-meier survival analysis of patients bearing flt- itd was not significantly different to the rest of the cohort (p = . ; / died, with only one case being related to relapse). patients bearing chromosome abnormalities (with or without other chromosomal aberrations) had a comparable, not significantly (p = . ) different survival to the rest of the cohort: / patients bearing this abnormality died although, most interestingly, none of these deaths were related to relapse. our results indicate ric allografts can provide an adequate consolidating effect in hr aml with complex karyotype, alteration of chromosome or flt- itr, yielding clinical results that are comparable to those obtained in patients with aml allografted for other indications. this is particularly important as these alterations are more frequent in patients whose age prevents them from having myeloablative grafts. disclosure of conflict of interest: none. early detection of inapparent replicative human cytomegalovirus (hcmv) infection together with its preemptive antiviral treatment has led to a marked reduction of life-threatening hcmv disease after allogeneic hematopoietic stem cell transplantation (allosct). we first reported in a retrospectively performed study that hcmv reactivation is associated with a reduced risk for relapse in patients with aml after transplant. now, we evaluate the impact of early hcmv replication on the risk for leukemic relapse in patients with aml after t cell depleted transplantation in a prospectively performed observational study (registration trial drks ). between january and march we enrolled patients with aml in this trial who were consecutively transplanted at the university hospital of essen. patients received a myeloablative regimen (tbi based conditioning n = , chemotherapy based conditioning n = ) and patients a ric (n = chemotherapy based regimen). patients were transplanted in .cr (n = ), .cr (n = ) or more progressive disease stages (n = ) from hla-identical sibling donors (n = ) or hlaidentical unrelated donors (urd) (n = ) or mismatched unrelated donors (n = ). patients who received a second transplant were excluded from the study. the median age of patients was years (range - ) and that of the donors years (range - ). gvhd prophylaxis was performed with mtx and csa, or csa and mmf with (n = ) or without atg (n = ) ( - mg total dose). the incidence of acute gvhd grade - was statistically not different in both groups ( % versus %). hcmv-reactivation (hcmv-r) detected by pcr occurred between and days (median days) after allo sct. only patients surviving day after transplant were included in the study for estimation of relapse incidence (cir) or overall survival (os). hcmv status of recipients (r) or donors (d) were in % r-/d-, % r-/ d+; % d+/r-and % r+/d+. patients with a documented hcmv-r had a cir at -year after transplant of only % as compared to % in patients without a hcmv-r (p = . ). estimates for -year os were in favor for patients with hcmv-r ( % for patients with hcmv-r versus only % for patients without hcmv-r), but this did not achieve statistical significance. non-relapse mortality was greater in patients with hcmv reactivation ( % versus %, ns) a substantial and independent reduction of relapse risk associated with early hcmv replication was confirmed by multivariate analysis including competitive factors as unfavorable cytogenetics according to eln, advanced disease stages of aml, hla-identical donor versus mismatched donor, sibling versus unrelated donor, presence of acute gvhd grades ii-iv, chronic gvhd, and hcmv-r [(hazard ratio: . , % ci: . - . , p o . ) for occurrence of hcmv-r]. the final result of this first prospective performed study confirms an independent advantageous effect of early hcmv replication on the leukemic relapse risk in patients with aml after transplant. disclosure of conflict of interest: none. . primary engraftment of wbc and platelets was achieved in pts, one patient (pt) died at day + after hsct, and one had a secondary graft failure. a median time to wbc engraftment was days ( - ), to platelets - days ( - ). cumulative incidence (ci) of acute gvhd (agvhd) grade ⩾ ii was % ( % ci: - ): from haplo- %( - ), from mud % ( - ),p = . . ci of agvhd iii %( % ci: - ): haplo vs mud - % ( % ci: - ) vs %( % ci: - ), respectively,p = . . ci of chronic gvhd (cgvhd)- % ( % ci: - ). ci of agvhd was significantly lower in a group with regimen ( - ) of gvhd prophylaxis: % ( % ci: - ) vs %( % ci: - ), po . . regimen was also effective in prevention of cgvhd: ci at year after hsct was % vs %, p = . . Сi of trm was %( % ci: - ): haplo- %( - ), mud - % ( - ). early mortality (before + -day) was relatively low (n = ): pt died from bacterial sepsis; two pts died due to adv infection. thirteen pts died after days: pts relapsed and died due to complications of second hsct; bacterial sepsis in pt and viral infection (adv and cmv) in pts ( with extensive chronic gvhd). ci of relapse was % ( % ci: - ) at year: from haplo- % ( % ci: - ), from mud- % ( % ci: - ),p = . . event-free survival (efs) at years was % ( % ci: - ): haplo - %( % ci: - ), mud - % ( % ci: - ), p = . . os was % ( % ci: - ) at years: haplo- % ( % ci: - ), mud - % ( % ci: - ), p = . . relapse-gvhd-free survival at years was different among recipients of haplo and mud hsct: %( % ci: - ) vs % ( % ci: , p = . . we confirm that the depletion of tcrαβ+/cd + depletion from the graft ensures high engraftment rate and acceptable transplant-related mortality in pediatric aml pts. there is a trend towards better efs for haploidentical transplantation. gvhd prophylaxis including ratg/rituximab/bortezomib improves gvhd control s in recipients of tcrαβ+/cd +depleted grafts in comparison to hatg/tacro/mtx apparently without loss of anti-leukemic activity. disclosure of conflict of interest: none. results of t-cell depleted haploidentical stem cell transplantation in adults with acute leukemia improve with time: a study from the acute leukemia working party of the european society of blood and marrow transplantation (ebmt) s simona , , m labopin , a ruggeri , , , a velardi , f ciceri , j maertens , l kanz , f aversa , d bron , d bunjes , m mohty , and a nagler , t cell-depleted (tcd) transplants from haploidentical donors are increasingly used in the absence of a hla full-matched donor for patients (pts) with high risk acute leukemia (al). progress has been made in optimization of conditioning regimens and post-transplant cellular therapy to potentiate the graft-versus-leukemia effect with no graft-versus-host disease (gvhd). however, relapse incidence (ri) and non relapse mortality (nrm) remain the main obstacles for pts outcomes. we report adults with de novo al, given a tcd haplo from to . to analyze the effect of transplant period on tcd haplo outcomes, pts were analyzed in two separate groups: - (n = ) and - (n = ). tcd haplo were performed in ebmt centers. median follow-up was months with no difference according to transplant periods. median age was different between groups, being and years respectively (p = . ). the majority of pts had acute myeloid leukemia (aml) ( % vs %, p = . ) and disease status at haplo was first complete remission (cr ) in % and % of pts respectively (p = . ). pts transplanted before had more frequently a karnofsky performance status o % ( % vs %, p = . ). conditioning was myeloablative in % and % of tcd haplo before and after (p = . ), mainly based on fludarabine(flu)-tbi, flu-melphalan-thiotepa or cyclophosphamide-tbi. as for ric it was flu-melphalan-thiotepa, flu-tbi or cyclophosphamide-tbi. the cumulative incidence (ci) of neutrophil engraftment, grade ii-iv acute gvhd and chronic gvhd were not different according to transplant period, being % and %, p = . ; % and %, p = . ; % and %, p = . , respectively. in the whole population -year ri and nrm were % and %, with no difference before and after ( % vs %, p = . ; % vs %, p = . , respectively). ri was % before versus % after . the main cause of nrm was infection, with no difference over time ( % vs %, p = . ). in multivariate analysis, disease status was the only factor associated with ri (hr . , % ci . - . , p = . ). tcd haplo after (hr . , % ci . - . , p = . ), younger age (hr . , % ci . - . , p = . ) and ric (hr . , % ci . - . , p = . ) were independently associated with lower nrm. -year os was % with a marked improvement for tcd haplo performed after ( % vs %, p = . ), while lfs and grfs were % and %, respectively. according to disease status, -year lfs, os and grfs were higher for pts transplanted in cr ( % vs %, p o . ; % vs %, p = . ; % vs %, respectively p = . ). in multivariate analysis, tcd to further establish the role of haplosct in high-risk aml, we performed a retrospective matched-pair comparison of hlamatched sct vs haplosct/ptcy in two german centers. highrisk aml was defined by any of the following criteria: refractory or relapsed aml, secondary aml, or genetic aberrations classified as intermediate-high or adverse accordingly to the eln classification. all consecutive adults, who fulfilled ⩾ of these criteria before either hla-matched or haplosct/ptcy were included (n = ). recipients of haplosct were pair-matched with patients receiving a matched donor sct. matching variables were ( ) stage at sct, ( ) genetic subgroups accordingly to eln, and ( ) age (± y). patients (pts) undergoing haplosct/ptcy could be successfully pairmatched (p = . for stage and genetic subgroup, . for age) with recipients of matched sct ( family, unrelated sct). within the entire cohort, median patient age was y ( - ). at start of conditioning, % of patients were in cr, % had refractory, % had relapsed, and % had untreated disease. genetics were favorable ( %), intermediate i ( %), intermediate ii ( %) and unfavorable ( %). hla-matched sct was uniformly performed following flamsa-ric. recipients of haplosct/ptcy ( %) received cytoreductive chemotherapy with flamsa (n = ) or clofarabine (n = ) before ric was started. median follow-up among survivors was months. overall cr rate at d+ was %, patients suffered from refractory disease or early death, (n = each). overall-survival (os) for the entire cohort was %/ . % at y/ y from sct. the corresponding y/ y leukemia-free survival (lfs) was . %/ . %. median time to engraftment was . and . days after matched and haplosct, respectively (p = . ). with respect to outcome, no difference was observed between the two groups: os at y/ y was . %/ . % after matched sct, and . %/ . % after haplosct/ptcy (p = , , figure ). lfs at y/ y was . %/ . % within the hla-matched group and . %/ . % within the haploidentical group (p = , ). recipients of haplosct showed a higher incidence of agvhd ⩾ ii°( % vs %, p = . ), as well as a trend towards increased y-nrm ( % vs %, p = . ), whereas y-relapse rates were comparable ( % after haplosct/ptcy vs % after matched sct, p = . ). relapse was the most frequent cause of death in both cohorts, main causes of nrm were gvhd and infections (no difference between the two groups). allogeneic sct following sequential conditioning can achieve excellent results in high-risk aml. in our study, results after haploidentical transplantation were comparable to those obtained after hla-matched sct. hence, haplosct/ptcy following sequential conditioning can be considered as a reasonable option for patients with high-riaml. [p ] disclosure of conflict of interest: none. a significant proportion of patients with acute myeloid leukemia ( aml) will either be refractory to initial chemotherapy or will suffer refractory relapse. the role of allogeneic transplantation (hct) in active disease is contentious. there is a growing body of literature that sequential chemotherapy, pioneered by the german flamsa regimen, followed by ric hct is a safe and efficacious modality in these patients, and there have been numerous modifications of this regimen, especially as amsacrine is not widely available. fludarabine, cytarabine and etoposide (vp ) (flav) have been reported as an an effective salvage regimen. here we report on single center outcomes of a variation of the flamsa regimen, substituting amsacrine for etoposide with mainly myeloablative conditioning. patients were consented for a clinical protocol if they had aml that was refractory to cycles of chemotherapy, or cycle and considered to be at risk of complications of a second cycle, and if they had a matched related donor. patients with myelodysplasia received flav if they had high or very high risk cytogenetics. cytoreductive chemotherapy consisted of fludarabine mg/m / day × days, cytarabine g/m /day × days, etoposide mg/m /day × days, commenced simultaneously. after days of rest, conditioning chemotherapy consisted of fludarabine mg/m × days and and iv busulfan . mg/ m q hours; the number of busulfan doses varied between - , depending on patient comorbidity. ten patients ( %) received myeloablative doses of busulfan. patient received doses of atg at . mg/m /day on day - and - . patients received gcsf mobilized peripheral blood hematopoietic cells. post-transplant gvhd prophylaxis was csa and mmf. csa was tapered from day+ and stopped at day + in the absence of gvhd. mmf was discontinued between day + and day + . donor lymphocyte infusions were collected for planned prophylactic dli. thirteen patients received a flav-sct between march and october . the median age was ( - ); male:female ratio was( : ). patients ( %) had aml and ( %) pts had mds. all patients had detectable disease prior to flav. the median time for plt engraftment was days ( - ). the median time for anc engraftment was days ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) . cytogenetic cr rate on a day - bone marrow was %, and morphological cr was %. patients ( %) developed veno-occlusive disease. acute gvhd grade ii-iv occured in pts ( %). ( %) patients developed chronic gvhd. death was due to disease relapse in ( %) and nrm in ( . %) patients, resulting from h n pneumonia. patients ( %) received dli for post transplant relapse, and one of these is in molecular remission. at a median follow up of . months post transplant ( . - m), year dfs was %. the year and year os was % (± %) (figure ). our experience, consistent with published data, demonstrates that for patients with active aml refractory to chemotherapy, transplantation is an effective modality of disease control and may be the only curative therapy in a significant proportion. etoposide appears to be a suitable alternative to amsacrine. our patients tolerated busulfan at myeloablative doses, and this may be required for adequate disease control. our report is limited by small numbers and relatively short follow-up, but is encouraging enough for us to continue offering flav hct for these high-risk patients. [p ] disclosure of conflict of interest: none. sequential high-dose chemotherapy reinduction followed by haploidentical transplantation in acute leukemias l brunello , , cm dellacasa , l giaccone , , e audisio , d ferrero , , s d'ardia , b allione , s aydin outcomes after t-cell replete haploidentical stem cell transplantation (haplo hsct) have been encouraging and haplo hsct has become an alternative option for patients without a hla-identical related or unrelated donor. , as previously published, the sequential use of intensive chemotherapy and allogeneic transplantation represents a possible approach to the treatment of high-risk acute myeloid leukemia (aml). , between and , acute leukemia (al) patients received sequential therapy (s.t.) consisting of high-dose chemotherapy reinduction and haplo hsct during the chemotherapy-induced neutropenia. median age at transplant was years (range: - ); median number of previous therapy lines was (range: - ) and / ( %) patients had received a previous allogeneic hsct. twelve and out of patients had de-novo aml and secondary aml, respectively; furthermore two patients presented blastic crises of chronic myeloid leukemia. all patients had active disease at the time of s.t. and median marrow blast count before reinduction was % (range: - %). hematopoietic cell transplantation comorbidity index was ≥ in / patients ( %). all patients received high-dose cytarabine (≳ g/sqm) containing regimens as reinduction therapy. the conditioning regimen was started after a median of days from the end of reinduction (range: - ). sixty-eight percent of patients ( / ) were conditioned with a myeloablative regimen (thiotepa tot. mg/kg, busulfan tot. . mg/kg, fludarabine tot. mg/ sqm), while / ( %) patients received a non-myeloablative conditioning. bone marrow was used as stem cell source in / ( %) patients. graft-versus-host disease (gvhd) prophylaxis consisted of post-transplant cyclophosphamide with calcineurine inhibitors and mycophenolic acid. all patients engrafted but one, who was rescued with a second haplo-hsct with peripheral blood stem cells from the same donor. median day of neutrophil recovery was day + (range: - ). median follow-up of survivors was . years (range: . - . ); -year overall and event-free survivals were % and %, while -year relapse incidence and non relapse mortality were % and %, respectively. overall cumulative incidences of acute and chronic gvhd were % and % at day + and + . among patients who developed gvhd, grade iii-iv acute gvhd and moderate-severe chronic gvhd were observed. at . years post haplo-hsct, % of patients are alive and disease free. in our cohort of heavily pre-treated and high-risk patients, s.t. with a myeloablative conditioning was safely used to reduce leukemic burden pre-transplant and enhance graft-versus-leukemia effects. only the prompt availability of a haploidentical donor allowed to implement this treatment modality. though small the patient cohort, our findings suggest that transplant-related toxicity was acceptable and early relapse was the major treatment-failure. however, long-term survival and disease-free rates of % in these very poor prognosis patients are highly encouraging. in elderly patients with acute lymphoblastic leukemia (all) and kinase activating lesions allogeneic stem cell transplantation (allo-sct) is considered to be the only curative option. however, high risk of relapse and non-relapse mortality (nrm) often withholds elderly patients with existing comorbidities from definitive therapy. even though, age alone as the most important eligibility criterion for allo-sct has become less important. a -year old patient with a medical history of adipositas, hypothyreosis, arterial hypertension, hypercholesterolemia and coronary artery disease with stent implantation was diagnosed with common-b-cell-all based on immunophenotype. cytogenetic analysis showed two coexisting clones with an abnormal karyotype of ,xx; t( ; )(q ;q ), + [ ] and ,xx; t( ; )(q ;q ),der( )( ; )(p ;q ) [ ] and no evidence of bcr-abl positive disease using fluorescence in situ hybridization (fish) technique. rt-pcr and sequencing of the fusion transcript was performed to validate the rcsd -abl t( ; )(q ;q ) fusion between exon of rcsd and exon of abl . western analysis of phosphorylated abl and its downstream target crkl was performed to investigate the in vivo activity of dasatinib. clinical monitoring of minimal residual disease (mrd) levels has been performed via rt-pcr of the rcsd -abl fusion transcript followed by nested pcr of the amplicon to detect early relapse or mrd. as positive control the plasmid pcr . -topo_rcsd -abl ( bp) was synthesized encoding rcsd -abl amplicon with the fusion site. our patient was enrolled on gmall elderly ( / ) and treated accordingly. thereby, no sustained remission could be achieved. flag-ida re-induction and study treatment with an oral pi k/mtor inhibitor remained futile. cytogenetics and verification of the rcsd -abl fusion gene prompted salvage treatment with the tyrosine kinase inhibitor (tki) dasatinib as single agent. the in vivo activity of dasatinib was highlighted by a decrease of rcsd -abl amplicon and inhibition of phosphorylated abl and its downstream target crkl was shown. clofarabine and cyclophosphamide complemented treatment and mrd negative remission was achieved due to administration of the bi-specific t-cell engager blinatumomab. consolidation with allo-sct was performed. ongoing remission has been achieved for more than months now. we demonstrate that monotherapy with tki like dasatinib is effective in refractory rcsd -abl positive all. to the best of our knowledge, this is the first elderly patient with rcsd -abl positive all with a sustained and ongoing complete remission. thereby, we suggest allo-sct after successful tki even for elderly patients with existing comorbidities and uncommon cytogenetics. relapse is the most important cause of failure of allogeneic hematopoietic stem cell transplantation (hsct) for flt -itdpositive acute myeloid leukemia (aml). in those cases, treatment with flt tyrosine kinase inhibitors (tki) constitutes a promising clinical approach to induce remission without conventional chemotherapy. forty-eight-year-old woman was diagnosed of aml secondary to myelodisplastic syndrome with npm mutation and internal tandem duplications of the flt gene (flt -itd). after achieving complete remission (cr), she received a sibling allogenic-hsct. four months later, aml relapsed at the medullary level, without central nervous system (cns) involvement, treated with conventional chemotherapy and donor lymphocytes infusions (dli). she achieved second cr and developed chronic graft-versus-host disease (cgvhd). nine months later, she suffered the first extramedullary relapse, at the mammary, cutaneous and probably pericardial levels. there was not medullary involvement. disappearance of the lesions at all levels was achieved with conventional chemotherapy and radiotherapy, and full donor chimerism. eight months later, she referred atypical precordial pain irradiated to the back. cardiac mri was performed in which several masses were visualized in a pericardial sac up to cm in diameter (dec- ). bm was maintained in cr. in study of pericardial fluid, infiltration by leukemic flt positive cells was observed. the patient was not considered candidate for further systemic chemotherapy nor radiotherapy treatment. then, treatment with the flt sorafenib inhibitor was started, by compassionate use, at dosage of mg/ h, which maintains after one year. after first month with sorafenib, pericardial lesions decreased considerably, ranging from cm in diameter to . cm (jan- ). in the subsequent ct controls, progressive decrease of the lesions has been observed and no new lesions have appeared in other locations. in the last ct (oct- ) pericardial thickening is almost non-existent, without new lesions. after one year of treatment, she maintains cr at medullary and extramedullary levels images. in our patient, treatment with sorafenib has achieved sustained control of extramedullary disease, which had escaped the mechanisms of action of conventional chemotherapy, allotransplant and dli. further studies are needed to corroborate the efficacy of flt inhibitors in the control of aml extramedullary disease and in the treatment of relapses after allo-hsct. all pts with tbi-based regimen received rabbit atg. tcrαβ+/cd + depletion of hsct with clinimacs technology was implemented in all cases according to manufacturer's recommendations. the median dose of cd + cells in transplant was × /kg (range: . - . ), tcrα/β- × /kg (range: . - ). primary engraftment was achieved in of pts. ( pts died before engraftment, one received second hsct), the median time to neutrophil and platelet recovery was and days, respectively. early ( day) mortality was %( % ci: - ), -year ptrm- % ( % ci: - ). the three early deaths were due to bacterial sepsis (n = ) and viral infections(n = ), seven late: viral infection in pts (adv = , adv+cmv = , cmv = ), bacterial sepsis in pts and rhinocerebral mucormycosis in pt, all late deaths were associated with cgvhd and prolonged corticosteroid therapy. ci of acute gvhd grades ii-iv and iii-iv was . % ( % ci: . - ), and % ( % ci: . - ), respectively. ci of cgvhd was . % ( % ci: . - ). regimen was more effective in prevention of agvhd ii-iv: ci at year after hsct was % vs , % in regimen , p = . and in cgvhd % vs . %, p = . . ci of relapse at years was % ( %ci: . - . ). two-year pefs(event = death or relapse) was . % ( % ci: - ), -year pos- % ( % ci: - ). in patients, who received tbi-based conditioning pefs was % ( % ci: - ), as compared with treosulfan-based % ( % ci: - ), p = . . median time of follow-up for survivors was . years (range: . - . ). we confirm that the depletion of tcr-alpha/ beta and cd lymphocytes from the graft ensures high engraftment rate and acceptable transplant-related mortality in pediatric all patients. viral infections and leukemia relapse await further improvement of control. all major outcomes were equivalent between transplantation from unrelated and haplo donor. disclosure of conflict of interest: none. the prognosis of patients (pts) with relapsed/refractory acute lymphoblastic leukemia (all), especially after allogeneic hematopoietic stem cell transplantation (allo-hsct), is very poor. therapeutic options for these pts are limited. blinatumomab is a bispecific t-cell engager (bite) antibody construct with dual specificity for cd and cd . bite therapy may help to overcome the resistance to chemotherapy (ct) with minimal toxicity, and may be a bridge to allo-hsct. we analyzed data of pts from hematologic centers in russian federation with relapsed cd positive all, who received bite. the median age was (range: - ) years, ( %) pts o yrs, ( %) pts ≥ yrs. the diagnosis was all b-i (egil) subtype in pts, b-ii-in , b-iii-in pts, and patient had mixed phenotype leukemia (m (fab) and b-all). three ( %) pts had philadelphia positive (ph+) all. in pts it was the first relapse of all, in -second, in -third. thirty pts had isolated bone marrow relapse, pts-combined relapse (bone marrow and extramedullary sites). the bone marrow blast infiltration was o % in pts, % in pts. disease relapse was revealed after ct in pts ( ( %) pts received allo-hsct after the therapy of relapse), after allo-hsct ( -from related, -from unrelated, -from haploidentical donors)-in pts ( pts received second allo-hsct after the therapy). in pts with posttransplant relapse donor lymphocyte infusion (dli) was used in combination with bite. every patient received from to cycles (median ) of bite. complete remission (cr) was achieved in ( %) pts (in ( %) pts it was minimal residual negative remission): in ( %) pts with all relapse after ct, in ( %) pts-after allo-hsct. pts with less than % blasts in bone-marrow at baseline experienced substantially higher response rates compared with patients with % blasts or higher ( % ( / ) vs % ( / )). response rates were similar although the number of relapse- % ( / ) in first relapse, % ( / ) in second relapse and % ( / ) in third relapse. pts with posttransplant all relapse who received bite in combination with dli had higher response rate than pts, who received bite as monotherapy: . % ( / ) vs % ( / ), respectively. one-year os was % ( % ci - %). one-year dfs was % ( % ci - %). grade ≥ hematological toxicity (neutropenia, thrombocytopenia) was observed in ( %) pts, grade ≥ liver toxicityin ( %) pts. five patients ( %) developed toxic neuropathy during the therapy. cytokine release syndrome occurred in ( %) pts. one patient after allo-hsct (but not after dli) developed grade i agvhd. there were no fatal treatment related toxicity. tree ( %) pts who responded to bite had relapse. eighteen ( %) pts died: pts-of disease relapse/ progression. the treatment of relapsed/refractory all with bite is effective and has acceptable toxicity. we demonstrated high efficacy in therapy of posttransplant all relapses, especially when bite was combined with dli, perhaps, due to additional immunological effect of the transplant. disclosure of conflict of interest: none. the mechanism of sorafenib anti-leukaemic effect seen in aml patients relapsing post allohsct involves the augmentation of alloreactivity which includes infiltration of the affected marrow by cd + cells having pd- receptor which presence characterize lymphocytes with antitumour potential multikinase inhibitor (mki) sorafenib is clinically active in acute myeloid leukaemia (aml) patients, especially in those with flt itd who receive allohsct as a part of their primary treatment. to throw some light on the mechanism of this antileukemic post-transplant sorafenib effect we studied the fate of two patients (flt itd-positive, npm -positive) who relapsed ( -year-old male) and ( -year-old female) days post-transplant and their salvage treatment included sorafenib. the multikinase inhibitor ( mg twice daily) was given either together with flag or da + . the response was prompt. the patients became, after completion of the chemotherapy, leukaemia free. both patients continued the sorafenib ( mg twice daily) treatment together with the aml standard maintenance chemotherapy (female case) or without any chemotherapy (male patient, substantial comorbidity and liver toxicity). ( ) . the patients responded well to the therapy and were free of leukaemia for and + months after initiation of the mki treatment (flt itd negative, % chimerism documented in the blood and in the marrow). ( ) . in both patients, and months on sorafenib, skin lesions appeared either in the context of cgvhd, which progressed to a life-threatening level in a male patient or as a photodermatitis-like cheek eruption. histopathology revealed the presence of severe cd + cells infiltration in affected tissues in both patients. ( ) . cd positive lymphocytes colonized the marrow of both patients. these marrows infiltrating cells co-expressed cd (pd- receptor) in proportions which were higher than those seen in the blood ( . % ± . % vs. . % ± . %, p = . ). a similar observation was made for cd +cd + cells ( . % ± . % vs. . ± . %, po . ). . transcriptome analysis of the marrow cells, which addressed the genes involved in lymphocyte activation, revealed the presence of proinflammatory profile which included a higher expression of tlr and il- . ( ) sorafenib given with or without moderate chemotherapy was effective in two patients in maintaining the anti-leukaemic effect of salvage chemotherapy. ( ) this was associated with the presence of alloreactivity (affected tissues infiltration with cd + cells) clinically seen as a severe fatal cgvhd aggravated by sorafenib treatment associated unwanted effects in one cases and with rather mild skin lesions appearing later during the treatment. the outcome of elder patients with acute myeloid leukemia or high risk myelodysplastic syndrome treated with allogeneic hematopoietic stem cell transplantation ch tsai , , division of hematology, department of internal medicine, national taiwan university hospital; tai-cheng stem cell therapy center, national taiwan university and genome and systems biology degree program, national taiwan university allogeneic hematopoietic stem cell transplantation (hsct) is a curative-intent treatment for patients with high-risk hematologic diseases, including acute myeloid leukemia (aml) and myelodysplastic syndrome (mds). both the incidences of aml and mds increase with age and patients elder than years of age were traditionally excluded from hsct because of high possibilities of therapy related morbidity/mortality. recently, with the introduction of reduced intensity conditioning regimens and the improvement of hsct care, more and more elder patients could undergo hsct for consolidation or salvage purposes. however, literature regarding the treatment outcome of elder patients receiving hsct is scarce. patients diagnosed as aml or high risk mds aged equal or more than years were recruited consecutively at national taiwan university hospital. the high risk mds was defined to include myelodysplastic syndrome with excess of blasts- and according to the world health organization (who) criteria. the cytogenetic risk stratification was based on original medical research council classification. from to , a total of patients were enrolled consecutively. the median age was . (range: - ) years and the gender distribution was even. among them, ( . %) patients had high risk mds, ( %) had de novo aml, ( . %) had secondary aml, and three ( . %) had therapy related aml. at diagnosis, four ( . %) patients had extramedullary disease. nine ( . %) had unfavorable-risk cytogenetics, ( . %) had either unfavorable-risk cytogenetics or intermediate-risk cytogenetics but with flt -itd mutations. regarding the pre-hsct disease status, nine patients had the first complete remission (cr), had the second cr, and patients were treatment naive or had refractory disease. the graft-versushost-disease(gvhd)-free/relapse-free survival (grfs) in which events include grade - acute gvhd, chronic gvhd with moderate severity according to cibmtr criteria, relapse, or death of any cause. with median follow-up of months (range: . - . ), the median overall survival (os) for all patients was . months, the relapse-free survival (rfs) was . months, and the grfs was . months. in univariate analysis for os and rfs, high-risk mds was a favorable prognostic factor but secondary or therapy related disease (p = . for os and . for rfs, respectively), unfavorablerisk cytogenetics or intermediate-risk cytogenetics but with s flt -itd mutations (p = . and . , respectively), pre-hsct refractory disease (p = . and . , respectively), and grade - acute gvhd (p = . and . , respectively) were unfavorable prognostic factors. however, for grfs, only unfavorable-risk cytogenetics or intermediate-risk cytogenetics but with flt -itd (p = . ) and pre-hsct refractory disease (p = . ) were unfavorable prognostic factors. in multivariate cox proportional hazards regression analysis for os and rfs, grade - acute gvhd was a significant unfavorable risk factor; for gfrs, pre-hsct refractory disease status was a significant unfavorable risk factor. our results showed that the choice of hsct should not solely based on the age factor and pre-hsct disease status. incorporating cytogenetics and genetic mutation status could risk-stratify elder patients with hsct. further prospective trials are warranted to validate these findings. disclosure of conflict of interest: none. children affected with acute lymphoblastic t cell leukaemia (t-all) and relapse after allogeneic stem cell transplantation (sct) have limited treatment options and a poor prognosis. immune checkpoint inhibitors targeting the programmed death (pd- ) receptor pathway may enhance the graftversus-leukaemia (gvl) effect by blockade of inhibitory signals to t cells mediated by its ligand pd-l . we report a -year old girl with refractory t-all after allogeneic sct, who was treated off-label with the pd- inhibitor pembrolizumab. the girl was diagnosed with t-all ( . g/l wbc, % bone marrow infiltration, cns negative, t ( ; )) and underwent hlahaploidentical bone marrow transplantation from her mother with post-transplant cyclophosphamide since she failed to achieve molecular remission despite an intensified chemotherapeutic regimen. on day post sct, she had a % donor chimerism and decreasing minimal residual disease (mrd) marker (minimal × − ). days post sct she had a molecular relapse with an mrd of × − and a subsequent morphological relapse as well as mixed donor chimerism. further treatment regimens included chemotherapy, intrathecal therapy and four donor lymphocyte infusions (dlis). initially, she displayed a good morphological response to dlis but the leukaemic burden eventually remained stable with an mrd of × − . considering . % pd- expression on cd + t cells in the patient's bone marrow and the encouraging data in other hematologic malignancies an off-label therapy with the pd- inhibitor pembrolizumab - was initiated. the patient and her parents gave informed consent and she received a single dose of pembrolizumab at . mg/kg days after sct. one week after administration of pembrolizumab, the patient developed acute gvhd grade iv of the skin, mucosa, liver, lung, central nervous system and eyes. she had a severe generalized inflammatory reaction with high inflammatory markers, increased hepatic transaminases and lymphocytic infiltration of the liver, cerebrospinal fluid and bronchoalveolar lavage fluid. magnetic resonance imaging (mri) of the brain revealed periventricular white matter lesions and hyperintensities of basal ganglia and bilateral temporal lobe consistent with autoimmune encephalitis. treatment with high-dose corticosteroids, cyclosporine and the anti-interleukin receptor antibody tocilizumab slightly improved her clinical condition. her mrd value significantly decreased to × − two weeks after administration and she achieved a % donor chimerism in bone marrow. despite this promising response her medical condition deteriorated and the severe inflammatory reaction caused fatal multi-organ failure. this is to our knowledge the first report on a remarkable and fast response to pd- inhibition in a patient with pediatric t-all refractory to multiple lines of therapy including allogeneic sct. this case illustrates the potential risk of checkpoint inhibitors to trigger severe gvhd that is not responsive to steroids. induction of inflammatory gvl responses without causing severe gvhd by therapeutic checkpoint inhibition needs to be addressed in future clinical trials. in recent years, there is a remarkable trend in the use of haploidentical-related hematopoietic cell transplantations (haplo-hct) in patients who do not have a hla matched related or unrelated donor. here, we report our single-center experience, in patients who underwent haplo-hct for acute leukemia. between and seventeen consecutive adult patients, seven males and ten females, median age years (range: - years) with high-risk acute leukemia underwent unmanipulated, bm or pbsc transplantation from an haploidentical family donor. eleven patients transplanted for acute myeloid leukemia ( in cr , in cr , in minimal active disease after cr , second trasplant in cr , transformed mds in cr , aml secondary to myelofibrosis in cr ), for acute lymphoblastic leukemia ( in cr , in active disease) and mastcell leukemia (secondary to aml) in active disease. sixteen patients received myeloablative conditioning, and reduced intensity, respectively. in five patients stem cells source was bm, in were g-csf mobilized pbsc. the median infused cd + cell dose was . × (range: . × - . × ). conditioning regimens were: bu-flu-mac (n = ), tbf-mac (n = ), tbf-ric (n = ) the regimens for gvhd prophylaxis were: ptcy as sole gvhd prophylaxis (n = ), mtx-csa-atg (n = ), methylpred-atg-tacrolimus (n = ), atg-csa-mtx-mmf (n = ). sustained trilineage engraftment occurred in patients ( %), two patients died of transplantation-related complications before day after transplantation without myeloid recovery. for patients receiving bm or pbsc grafts, the median time to neutrophils recovery was days (range: - ), and , platelets recovery was days (range: - ). / patients ( . %) and / ( . %) had ii-iv and iii-iv grade of acute gvhd, respectively.the incidence of grade ii-iv cgvhd was %. after a median follow-up of months, / patients ( . %), out patients transplanted in cr , are alive and disease free at , , , months (inclusing the patient transplanted for aml after imf). the -year probability of overall and progression-free survival was % ( % ci, . - . %) and . % ( % ci, . - . %), respectively. causes of death were: sepsis (n = ), fatal agvhd (n = ), pneumonia (n = ), toxicity (n = ), progression (n = ) and relapse (n = ). in our experience unmanipulated bm or pbsc transplantation from haploidentical family donor is feasible approach with high engraftment rates and acceptable trm ( %) and rate of grade iii-iv agvhd, associated with durable remission in a proportion of patients with high-risk acute leukemia, specially in patients with aml transplanted in first remission. it is generally recognized that allogeneic hematopoietic stem cell transplantation (allo-hsct) should not be administrated to patients with severe aplastic anemia (saa) or very severe aplastic anemia (vsaa), when they got active infection. however, without neutrophil, severe infection is usually difficult to control and even fatal. under these circumstances, rapid recovery of neutrophil by allo-hsct might be an alternative to control infection. from january to december , there were young patients received allo-hsct for saa or vsaa at shanghai children's medical center in china. among them, patients ( males and females) with a median age of . years (range: . - . years) received allo-hsct with refractory active infections. refractory active infection was defined as persistent neutropenic fever with nonresponse to standard doses of broad-spectrum antibacterial agents and antifungal agents for more than three weeks, with or without definite focus of infection. prior to allo-hsct, four patients had persistent fever of unknown origin, patients with singlesite infection, and patients with multiple-site infections. sites of infection included lung, sinus, cellular tissue, peritoneum, liver, spleen and skin. the conditioning regimen consisted of fludarabine, cyclophosphamide and rabbit-antithymocyte globulin with or without total body irradiation (tbi) ( - gy). twelve patients were transplanted from mismatched unrelated donors, from matched sibling donors, and from haploidentical donors. sixteen patients received g-csf mobilized peripheral blood stem cells, three patients g-csf mobilized peripheral blood stem cells plus g-csf primed bone marrow stem cells, two patients bone marrow stem cells, and patient umbilical cord blood stem cells. a median of . × /kg mononuclear cells with . × /kg cd + cells were transfused, except the patient who underwent ucbt with a total of . × /kg mononuclear cells and . × /kg cd + cells transfused. eighteen patients achieved recovery of neutrophil and finally control of infections, including one patient who suffered primary graft failure and had autologous marrow recovery. three patients died of infection and one patients died of acute renal failure before recovery of neutrophil. one patient died of pneumonia months after allo-hsct. one patient become thrombocytopenia after allo-hsct. the other patients are all disease-free. there were five patients developing grade i-ii acute gvhd, and patient grade iii-iv acute gvhd. all were cured at last. three patients had localized chronic gvhd and one patient had extensive chronic gvhd. with a median of years follow-up, the overall survival rate and disease-free survival rate are . % ± . % and . % ± %, respectively. allo-hsct could be a feasible way to control infection for children with saa or vsaa in the present of refractory active infections. disclosure of conflict of interest: none. paroxysmal nocturnal hemoglobinuria (pnh) may present hemolysis isolated (classical pnh) or associated with aplastic anemia (aa; aa/pnh syndrome). while classical pnh patients require anti-complement treatment (eculizumab), the treatment of aa/pnh patients should target their underlying aa by immunosuppression (ist), or even bone marrow transplantation (bmt). however, in a few patients clinically meaningful aa and hemolysis may be concomitant, eventually justifying both ist and eculizumab. to date there is no standard treatment for s this rare condition. amongst a large cohort of pnh patients (between and ) at our reference centers, st. louis hospital (paris) and federico ii university (naples), we retrospectively assessed characteristics and outcomes of patients diagnosed with aa/pnh who received intensive ist during or immediately before ( - months) eculizumab treatment. nine patients were identified. eight patients fulfilled the criteria of severe aa, and one had an immunemediated isolated agranulocytosis. since no patient had a hla-matched related donor for bmt, all patients received intensive ist according to institutional guidelines. six out of patients were already on eculizumab treatment at the moment of starting intensive ist (concomitant treatment) whereas patients received ist in the - months (median time of months) before the introduction of anti-complement therapy (sequential treatment). for all patients already on treatment, eculizumab was not discontinued to minimize the risk of rebound intravascular hemolysis and thrombotic complications. eculizumab was administered at the standard dose of mg fortnightly in all but one patient, who needed an increased dose ( mg) because of pharmacokinetic breakthrough. six patients ( aa and agranulocytosis), including the three undergoing a sequential treatment, received standard ist with horse-antithymocyte globulin (h-atg, mg/kg for four consecutive days) combined with cyclosporine a (csa). the remaining three aa patients received alemtuzumab ( - - - mg subcutaneously in four consecutive days) and csa within the prospective trial nct ; one of these patients a few months later also received a second ist course with rabbit-atg ( . mg/kg for five consecutive days) and csa. all the patients completed the scheduled treatment without any side effect, including infusion-related reactions. lymphocyte depletion (o /μl) was observed in all patients irrespective of sustained therapeutic complement blockade. all the patients were available for response assessment at months. among the six patients receiving a concomitant treatment we observed one partial response (pr) and two complete responses (cr), whereas the three remain patients were non-responders (nr). of them one was rescued with an unrelated bmt, while two remained on eculizumab treatment. one of the cr relapsed at years showing clonal evolution and finally died. all the other patients are alive, keeping their hematological response. patients receiving a sequential therapy were one in pr and two in cr months after introduction of eculizumab. in conclusion, for patients diagnosed with severe aa/pnh syndrome intensive ist and eculizumab treatment, can be safely delivered either concurrently or sequentially, with an overall response rate of nearly %. this is the first systematic description of the management of severe aa in hemolytic pnh patients receiving eculizumab treatment. disclosure of conflict of interest: none. ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) in aa, ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) in rcc, and ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) years in rcmd. sixty-five patients underwent bmt from hla-matched (related , unrelated ) and from hla-mismatched (related , unrelated ) donors. conditioning regimens were used as follows; cyclophosphamide (cy)+antithymocyte globulin (atg) ± total body irradiation (tbi) (n = ), fludarabine (flu)+cy ± atg ± tbi (n = ), and flu +melphalan (mel) ± atg ± tbi (n = ). all patients got engraftment after bmt. however, late graft failure was found in patients with rcc, and with rcmd, but none with aa. out of patients who developed late graft failure, patients used flu+cy ± atg ± tbi, used cy ± atg ± tbi, and used flu +mel ± atg ± tbi for conditioning regimens. five-year cumulative incidence (ci) of graft failure was higher in rcmd ( ± . %) than in aa ( %) and rcc ( ± . %), significantly (po . ). five-year ci of graft failure tended to be higher in flu regimen ( ± . %) than in cy+atg ± tbi regimen ( ± . %), but not significant (p = . ). five-year ci of graft failure did not differ between with ( ± . %) or without tbi ( ± . %) (p = . ). multivariate analysis revealed that the morphological classification was a significant risk factor for graft failure (po . ). five-year failure free survival rate ( ± %) in rcmd was significantly lower than in aa ( ± . %) and rcc ( ± . %) (p = . ). graft failure, second malignancy, and death were considered as failure events. one patient with aa died of infection, four with rcc died of infection (n = ), bleeding (n = ) and myocarditis (n = ), and one with rcmd died of infection. five-year overall survival rates were not different among groups (aa, ± . %; rcc, ± . %; rcmd, ± . %) (p = . ). high incidence of graft failure in rcmd may be due to higher bm cellularity than in aa and rcc. the optimal conditioning regimen of bmt should be established for children with abmf based on the bm cellularity and morphological classification. disclosure of conflict of interest: none. recent studies have suggested inferior outcome of patients treated with rabbit atg (thymoglobulin, sanofi) as compared to horse atg (atgam,pfizer or lymphoglobulin, genzyme), and a higher early mortality with rabbit atg has been suggested to explain this difference. aim: to assess early mortality, response rates at , and months and long term outcome, in a large cohort of aa pts, treated in europe or asia with rabbit atg and cyclosporin, as first line treatment. eligible for this study were pts with aa, treated with thymoglobulin between and in europe (n = ) or asia (n = ). median year of treatment, was : characteristics were comparable : median age and years, interval diagnosis treatment ( and days) and severity of the disease ( % and % with vsaa). early mortality was analyzed for all patients.long term outcome was also analyzed for pts for who response data (no, pr, cr) were available. mortality o days was . % and . %, respectively, in the time period - and - (p = . ). in these time periods, early mortality for patients aged - , was reduced from . % to . % and for patients over , from % to %. overall response was recorded in patients. at months the cumulative incidence of response was comparable in the time periods: % vs %, and at year, % vs % (p = . ). response rates at months were age dependent: %, %, %, % respectively in patients aged - , - , - , (p = . ). when non responders at months were reevaluated at year, % had responded, % were non responders, % had died, and % had received other treatment. responses at months, were %, %, %, in pts with very severe, severe and non severe aa (p = . ). the actuarial year survival for the entire population was %, and %, when pts were censored as surviving at transplant. actuarial year survival in univariate analysis was as follows: % vs % for day responders vs non responders (p o . ), % vs % for males versus females (p = . ); %, %, %, % in pts aged - , - , - , years (p o . ); %, %, % in pts with neutrophils o . × /l, - × /l and . × /l (p o . ); %, %, % for pts with an interval diagnosis-treatment of o days, - days or days (p = . ). finally pts treated had a year survival superior to pts treated before ( % vs %, p = . ). survival at years, in the recent period ( - ), was % for pts aged - and % for pts over years. in multivariate cox analysis the following variables remained independent predictors of survival: patient age, year of treatment, severity of the disease, interval diagnosis treatment, and gender. thymoglobulin +csa is effective and safe in patients with aa. the outcome is mainly age dependent. the inrerval between diagnosis and treatment remains a strong predictor: the earlier the better. for pts o years old current early mortality. . for pts years of age, current early mortality is higher ( %), response rate ( %) and year survival ( %) are lower. . the actuarial year survival for the enire population was %. survival at years has improved from % (o o/u ) to % ( - ), especially for pts over years ( % vs %, p = . ). [p ] s disclosure of conflict of interest: we thank centers for providing up date follow up of their patients . this study was supported by a grant of sanofi-genzyme. both of the patients have extremely low anc o . × /l, reto . %, plt o × /l. both was given antibiotic treatment with carbapenem, vancomycin/linezolid, voriconazole or amphotericin b liposome and got no response. no pathogenic bacteria or fungus was found from either of the patients. both of them had no full sibling or matched unrelated donor and had their father as their haploidentical donor. bone marrow combined with peripheral blood stem cell (pbsc) was adopted. conditioning: fludarabine days − through − , ( mg/m × ), intravenous busulfan ( . mg/kg q h) on days − to − . gvhd prophylaxis: high-dose cyclophosphamide mg/kg on days + and + , mmf and tacrolimus since days + . rabbit anti-thymocyte globulin (thymoglobulin) . mg/kg on days − to − . stable neutrophil engraftment (anc . × /l) occurred on day + and day+ respectively. platelet achieved × /l on day + and day + , respectively. both transplant course was complicated by febrile neutropenia without detected etiology, while both children have no fever since the first day anc . × /l.the facial swelling was resolved in both patients except for palatal fistula and fistula of maxillary sinus as the sequela of severe nasosinusitis. no acute or chronic gvhd. case had hemorrhagic cystitis on day + which last for about days, and suspected thrombotic microangiopathy (tma) with hypertension, thrombocytopenia, elevated ldh and creatine on day + which was resolved soon after discontinue of tacrolimus. case had delayed engraftment of platelets and herpes simplex virus encephalitis on days + which was cured by ganciclovir and high dose intravenous immunoglobulin. now they are and months post-hsct respectively and are doing well with % chimerism and no gvhd. alternative donor hsct may be considered as the first line salvage therapy for patients of vsaa with extremely low anc and active infection. fast reconstruction of neutrophil helped to control the infection. hallo-identical hsct make sure nearly every patients can find a donor. ptcy is proved to be efficient and safety in gvhd prophylaxis and facilitating engraftment in these two challenging cases. disclosure of conflict of interest: none. long-term outcome of patients with severe aplastic anemia receiving allogeneic hematopoietic cell transplantation using nonmyeloablative conditioning with fludarabine and low dose total-body irradiation l cheryl xiu qi , l yeh ching , p michelle li mei , t lip kun , h william ying khee , g yeow tee , t patrick huat chye and k liang piu department of haematology-oncology, national university cancer institute, singapore and department of haematology, singapore general hospital, singapore allogenic haematopoeitic stem cell transplant (ahct) offers the best prospect of cure in patients with severe aplastic anaemia (saa). the use of myeloablative hct is however limited by the toxicity of preparative regimens, the lack of matched sibling donors, transplant related mortality and graft rejection. the introduction of non-myeloablative (nm) conditioning offers the possibility of extending this potentially curative treatment to patients in whom ahct was previously contradindicated. in , we reported the outcome of patients with saa who have received ahct using nonmyeloablative conditioning comprising of days of fludarabine at mg/m and total body irradiation at gy (flu + tbi gy). here, we report a longer follow-up, with additional patients who had recevied ahct with this regimen. fourteen patients with a median age of years old (range: - years old) received filgastrim-mobilised peripheral blood stem cell transplant from either hla identical sibilings (n = ) or matched unrelated donor (n = ) after receiving nm conditioning consisting of flu + tbi gy. the first two patients received cyclosporine (cya) and mycophenolate mofetil (mmf) for the post-transplant immunosuppessive therapy. the remainining patients received cya, mmf and a short course of methotrexate (mtx) for additional graft-versus-host dsease (gvhd) prophylaxis. results all patients achieved prompt engraftment. the median time for engraftment of neutrophils ( . × /l) and platelets ( × ) were days (range: - days) and days (range: - days), respectively. chimerism analysis on day and subsequently showed % donor cells in all patients except , who developed secondary graft failure at months and required salvage hct. none of the patients experienced grade and above regimenrelated toxicity. five patients developed grade ii-iv acute gvhd and patients developed limited chronic gvhd. with a median follow-up of . years (range: . - . years), the estimated overall survival and event free survival were % and % respectively. the two patients who did not receive mtx developed acute gvhd of the liver and succumbed to infective complications. the remaining patients who had received triple immunosuppressive therapy were well, with limited chronic gvhd seen only in . our results suggest that the nm conditioning regimen comprising of flu + tbi gy provides sufficient immunosuppression to allow prompt and stable engraftment with minimal regimen-related toxicity. it is an attractive option for patients with saa who require ahct but are at increased risk of regimen-related complications from more intensive cyclophosphamide-based regimens. disclosure of conflict of interest: none. paroxysmal nocturnal hemoglobinuria (pnh) is a rare acquired clonal disorder of hematopoietic cells characterized by the triad of hemolytic anemia, cytopenias and high risk of venous thrombosis. due to the rarity of the disease, most reported data derive from multicenter studies. we describe the natural history of the disease in a -year (yrs) long single center series of pnh patients (pts). we performed a retrospective analysis of consecutive pts followed at our center from to . since , the diagnosis was made by ham test; starting from , flow cytometry (fc) analysis was used to diagnose new pts and to confirm pnh in pts previously diagnosed by the ham test. at diagnosis, pts had classic pnh, aplastic pnh and intermediate form. the cumulative incidences of thrombosis, cytopenia and clonal neoplasm were %, % and %, respectively. except for pt with aplasia, no severe infections were diagnosed, nor renal failures or pulmonary hypertention. the yrs overall survival (os) was %. a nonsignificant better os was associated to the absence of thrombotic events ( % vs %) and to a diagnosis made during the last decade ( % vs % vs %).up to the treatment options were supportive care or allogenic bone marrow transplantation. since , eculizumab was used in transfusion-dependent patients and/or in case of a thrombotic history. overall, pts were transfusion-independent for the entire period of the illness, were transfusion-dependent and/or had thrombotic events( pts). six of the latter pts never received eculizumab but only transfusion support ( pts) or allogeneic bone marrow transplant ( pts), while pts received eculizumab (the first pts were included in the phase iii triumph and shepherd trials).considering the increased risk of meningococcus infection for pts on eculizumab, vaccination with conjugated anti-meningococcus serotypes acwy was employed and, since , conjugated antimeningococcus serotype b was added. overall, pts treated with eculizumab became transfusion-independent and four remained transfusion-dependent. no thrombotic event was observed after eculizumab, even if pts had recurrent thromboembolisms prior to receiving the drug. no severe infection was documented. one patient developed extravascular hemolysis and receive a successfully selective splenic artery embolization. the yrs os in the eculizumab group was %.no pnh-associated death occurred. our study confirms that thrombosis is a major complication in pnh pts not receiving eculizumab, influencing os. the better os in the last decade is probably due to the use of eculizumab and to lack of thrombotic events. in particular, for pts on eculizumab the year os was %, even though half of the pts had thromboembolism and diagnosis made prior to the last decade. although kidney failure and lung hypertension have been reported, we did not observe these complications in our long follow-up case series. we can assume that the availability of a dedicated emergency room at our center allows to perform, promptly, hyper-hydration or transfusion support in case of hemoglobinuria crisis, reducing the risk or organ damage. no infections have been observed after eculizumab, probably due to the vaccination program schedule recommended in the literature, plus the addition of conjugated anti-meningococcus serotype b. however, shared guidelines are needed. disclosure of conflict of interest: none. mortality following hsct in saa pts over the age of is reported to be in the order of %, without taking in to account long term sequelae such as chronic gvhd, known to be more frequent in older patients. this has prompted international guidelines to recommend first line immunosuppressive therapy above years of age. the question is whether this is still true in . the aim of the study is to assess whether trm in saa patients grafted - is reduced,as compared to the era - . we used the wpsaa ebmt registry, and identified pts aged years or more, with acquired saa, grafted between and . we divided pts in transplant eras: - (n = ) and - (n = ). in the more recent period ( - ) pts were older ( vs year, p o . ), were more often grafted from alternative donors (alt) ( % vs %, po . ), with a greater use of bm ( % vs %, p o . ), and with a longer interval dx-tx ( vs days, p . ), and more often received a fludarabine containing regimen ( % vs %, p o . ). the os year of pts grafted in - was %, compared with % for pts grafted - (p = . ). in multivariate analysis, including the interval diagnosis transplant, patient's age, donor type, stem cell source and conditioning regimen, the lack of improved survival in - was confirmed (p = . ). a very strong age effect was shown both in univariate and multivariate analysis: survival of pts aged - years, - years and years, was respectively %, %, % (p o . ) and this was confirmed in multivariate analysis. the conditioning regimen, also proved to be a significant predictor, with improved survival for alt transplants receiving flu containing regimens ( % vs %, po . ). in general pts receiving either cy or a flu containing regimen, did significantly better than pts receiving other preparative regimens ( % vs %, p = . ). the use of a sibling donor (sib) did not prove to predict survival in multivariate analysis. pts receiving campath in the conditioning,did significantly better than pts not receiving campath ( % vs % po . ); similarly survival of patients with atg was superior % vs % compared to patients not receiving atg (p o . ). when pts receiving either campath or atg (n = ) were compared to patients not receiving either (n = ), the difference in survival was % vs % (p o . ), and this was significant also in multivariate analysis. combined primary and secondary graft failure was reduced from % to % in the two time periods (p = . ), acute gvhd grade ii-iv was reduced from % to % (p = . ) and chronic gvhd was also reduced from % to % (p = . ) infections remain the leading cause of death in both transplant eras ( % and % respectively), followed by gvhd ( % and %) and graft failure ( % and %), whereas ptld have been reduced from % to . %. hsct in pts with acquired saa aged and over, continues to carry a significant risk of trm also in - , ranging from % in younger pts ( - ) to % in older pts ( years). survival is predicted in multivariate analysis, by two crucial predictors: patients' age and the use of either campath or atg,the latter giving a % survival advantage over no campath/atg. alt and sib donors produce similar survival. this study gives further support to current guidelines, suggesting first line therapy with atg+csa, in pts over the age of . [p ] disclosure of conflict of interest: none. autoimmune diseases p allogeneic haematopoetic stem cell transplantation as curative therapy for early-onset, refractory crohn's disease e groene , p bufler , k krohn , s immler , g marckmann , t feuchtinger , s koletzko and m albert dr. von hauner university children's hospital and institute of ethics, history and theory of medicine, lmu results of a recent randomized trial suggest that autologous hsct is an option in adult patients with severe, therapyrefractory crohn's disease (cd) with an associated mortality risk of %. however, relapse of the disease is frequent ( ). in contrast allogeneic hsct has resulted in long-term cure of cd in affected patients transplanted because of haematological malignancy ( ). we report a year old girl who was diagnosed with severe cd at age seven (paris classification l , l a, b ). neither next generation sequencing nor immunological work up identified a monogenetic cause of cd. progressive chronic inflammation manifesting ubiquitously in the gastrointestinal tract resulted in severe complications, such as perianal fistulas with rectal stenosis, intestinal abscesses, dysphagia, severe weight loss, failure to thrive, delayed puberty and the need for ileostomy and long-term exclusive enteral nutrition via tube feeding. despite multiple lines of therapy, including repeated nutritional therapy, steroids, immunosuppressants (methotrexate, azathioprine) and biologicals (infliximab, adalimumab, certolizumab) a lasting remission could not be achieved resulting in poor quality of life. after careful risk/benefit assessment including ethical counselling allogeneic hsct was offered. she underwent allogeneic hsct from a matched ( / ) unrelated bone marrow donor ( . × /kg total nuclear cells). conditioning was performed according to a protocol successfully applied in adolescents with chronic granulomatous disease ( ) with alemtuzumab ( × . mg/kg/d), targeted busulfan (tauc ng × h/ml) and fludarabine ( × mg/m ). cyclosporine a and mycophenolate mofetil were used as gvhd prophylaxis. neutrophil and platelet engraftment were observed on days + and + , respectively. the post hsct course was complicated by grade i acute skin gvhd treated with topical steroids and toxic megacolon secondary to scarring stenosis on both ends of the unused colon on day + requiring surgery and a colostomy. at months post hsct the patient is well, off immunosuppressive medication, without gvhd and exhibiting % donor chimerism. the cd is in complete clinical and histological remission as proven by endoscopy and biopsies. stoma reversal with restitution of intestinal continuity is planned for the next months. refractory cd can lead to life-threatening complications and severely reduced quality of life. although long-term outcome in our patient will need to be carefully assessed, allogeneic hsct may offer a curative therapy in children and young adults with severe cd, even in the absence of an identified monogenetic cause. current ebmt recommendations include consideration of ahsct in exceptional circumstances for patients with severe refractory cd. the only randomised trial of ahsct in cd (astic) confirmed substantial short-term benefits but failed to meet its primary year endpoint. to further clarify the longterm safety and efficacy of ahsct in cd we performed a retrospective analysis of patients undergoing ahsct for cd outside the astic trial using the ebmt registry. patients were identified from the ebmt registry. all adult patients undergoing ahsct for a primary diagnosis of cd from to were eligible for inclusion. patients who were enrolled in the astic trial were excluded. from a total of patients (across centres) on the ebmt registry, data were obtained from patients transplanted in centres in countries. median patient age was yrs (range: - ) and % were female. median age at first diagnosis of cd was yrs (range: - ). patients were heavily pre-treated, having failed or been intolerant to a median of previous lines of therapy (range: - ). % had received experimental therapy prior to auto-hsct. % of patients had undergone at least operation. the median time from first diagnosis of cd to auto-hsct was . years (range: . - . ). all patients received peripheral blood stem cells following conditioning with cyclophosphamide mg/kg and % received anti-thymocyte globulin (atg). the median cd + dose infused was . (range: . - . ) × /kg. twelve percent of patients underwent cd + selection. neutrophil and platelet engraftment occurred at a median of day (range: - ) and day (range: - ), respectively. sixety-one percent received post transplant g-csf. median length of follow-up following auto-hsct was months (range: - ). at days post auto-hsct, % of patients were in clinical remission (cr), defined as no abdominal pain and normal stool frequency. a further % experienced significant improvement, defined as improvement in abdominal pain and stool frequency. for % there was no appreciable change in disease and in % the disease worsened compared to baseline. at year post auto-hsct, % were in cr, % were improved, % were unchanged and % had worsened. at last follow-up, % were in cr, % were improved, % were unchanged and % had worsened. overall % restarted medical therapy post auto-hsct and % required further surgery. overall % developed an infection requiring treatment post auto-hsct ( % bacterial, % viral). ebv and cmv reactivation occurred in % and % respectively and herpes zoster occurred in %. a secondary autoimmune disease developed in %, most commonly thyroid disease ( %). malignancy developed in %, of which skin cancer accounted for % of cases. one patient died at days post auto-hsct due to cmv infection, sepsis and multiorgan failure. this large retrospective series further supports the safety and efficacy of ahsct in a population with severe and treatment-refractory crohn's disease, % of patients experienced complete remission or significant improvement in cd symptoms with long-term follow-up. trm observed was similar to ahsct for other indications. in summary, ahsct appears to be an extremely promising therapy for severe refractory cd. further follow up of astic patients and future randomised trials are warranted. disclosure of conflict of interest: none. memory stem t cells (tscm) are long living self-renewing memory t cells with long-term persistence capacity, which play a relevant role in immunological memory and protection against infectious diseases and cancer , , , , , . the aim of this work is to investigate the potential role of tscm as a reservoir of arthritogenic t cells in rheumatoid arthritis (ra). we analysed the dynamics of circulating tscm (here identified as cd ra+ cd l+ cd + t cells) and other memory t-cell subpopulations by multiparametric -color flow cytometry in patients with active ra and in of them also during treatment with anti-tnfα biological agents (etanercept). to analyse cytokine productions, functional assays were performed stimulating peripheral blood mononuclear cells (pbmcs) with pma/ionomycin and brefeldin a. after the stimulation, cells were stained for surface markers, fixed and stained for intracellular cytokines. we traced circulating antigen specific cd + t cells for the vimentine-derived citrullinated peptide (vimcit) savracitssvpgvr , in hla-drb × : ra patients before and during the anti-tnfα treatment using custom mhc class ii tetramers. viral antigen specific cd + t cells were traced using mhc class i dextramers. age-matched healthy donors (hds) were used as control for all the experiments. we found a significant expansion of cd + tscm in patients with active ra both in terms of frequency and absolute counts. notably, cd + tscm significantly contracted upon anti-tnfα treatment, suggesting a role of tnfα in tscm accumulation. in contrast to cd +t cells, cd compartment did not show significative alterations compared to (hds). furthermore, cd +tscm in ra patients displayed an enrichment in the th phenotype, largely implied in autoimmune disorders, while the other t cell subpopulation were not enriched in the th phenotype. at the antigen specific level, we were able to trace in hla-drb × : patients antigen specific cd + t cells, comprising tscm, specific for the vimentin-derived citrullinated peptide. of notice, citrullinated vimentin specific cd + t cells, including tscm, contracted during anti-tnfα administration, while viral-specific cd + t cells (ebvbhrf- ) and antiviral cd specificities (cmvpp , flump, ebvbmlf- ) were not affected by etanercept administration, thus suggesting a possible role of cd + tscm as reservoir of arthritogenic autoreactive t cells. overall, our results suggest that tscm, by representing a long-term reservoir of undesired specificities, might play a non redundant role in sustaining ra and possibly other t cell mediated disorders, thus representing novel biomarkers as well as therapeutic targets. ongoing experiments will characterize the tcr repertoire on sorted tscm and cd + memory subsets in order to identify a possible oligoclonality in tscm repertoire. in conclusion, the analysis of tscm dynamics in autoimmune disorders could have relevant clinical implications as new biomarkers and for devising innovative therapeutic strategies. ebv and cmv reactivation following autologous haematopoietic stem cell transplantation (hsct) for autoimmune neurological diseases resolves spontaneously and rarely requires treatment c mapplebeck , b sharrack , h kaur , y ezaydi , h jessop , l pickersgill , l scott , m raza and ja snowden departments of haematology, neurology and virology, sheffield teaching hospitals nhs foundation trust, sheffield, uk autologous haematopoietic stem cell transplantation (hsct) for severe autoimmune diseases involves immunosuppressive conditioning regimens and current guidelines recommend monitoring for viral reactivation of cytomegalovirus (cmv) and epstein barr virus (ebv) (snowden et al ) . however, the incidence, degree and management of viral reactivation are not established. we performed a retrospective observational service evaluation study of all patients receiving cyclophosphamide mg/kg + rabbit anti-thymocyte globulin mg/kg (atg, thymoglobulin) followed by autologous hsct for various autoimmune neurological diseases between and at our centre. data collected included the baseline serological status of the patient prior to transplant and serial blood pcr quantitation (copies/ml). if ebv and cmv reactivation occurred details of further management was collected and descriptive statistics were used to summarise outcomes. twenty-three patients received autologous hsct between january and october ; patients with multiple sclerosis (ms), with chronic inflammatory demyelinating polyneuropathy (cidp) and with stiff person syndrome. twenty-two patients had positive ebv igg serology prior to transplant and patient had an equivocal result. seventeen patients had evidence of ebv reactivation and a further patient had ebv dna detected post-transplant but with less than copies/ml. the average time to peak ebv pcr was . (range: - ) days post-transplant and a range: in ebv pcr peak level from to copies/ml. the patients who had ebv pcr results of over copies/ml had ct scans of chest, abdomen and pelvis performed which did not demonstrate significant lymphadenopathy or hepatosplenomegaly. in all patients monitored for a detectable ebv reactivation, the ebv pcr spontaneously began to fall within months (average days, range: - days) post-transplant and no specific treatment was required. one patient had late ebv reactivation of copies/ml at months post-hsct associated with chronic tonsillitis and tonsillectomy specimens showed follicular hyperplasia without evidence of post-transplant lymphoproliferative disorder (ptld) and ebv pcr levels normalised without other treatment. ( %) patients had positive cmv igg serology prior to transplant and one patient had an equivocal result. only of patients had a significant reactivation of cmv with copies/ml at days post-transplant, successfully treated with intravenous immunoglobulins and valganciclovir. two other patients had low level cmv reactivation with and copies/ml, respectively which resolved spontaneously without treatment. ebv reactivation in patients with neurological autoimmune disease undergoing autologous hsct is common and usually resolves spontaneously without treatment. asymptomatic cmv reactivation occurs in approximately % of patients in this setting and may require treatment. autologous hematopoietic stem cell transplantation (hsct) has been utilised for the treatment of severe multiple sclerosis (ms). it results in significant improvement of neurological function, although patients can experience exacerbations of ms-related symptoms during the procedure. we reviewed patients with ms who underwent stem cell mobilisation and collection from march to november . the median age was years ( - ). nine patients ( %) were male. the interval from diagnosis to hsct was . months (range: . - . ). patients ( %) had relapsing-remitting (rrms), six patients ( %) secondaryprogressive (spms) and two patients ( %) primary-progressive (ppms) multiple sclerosis. only patients ( %) had not received any prior treatment, whereas patients ( %) received two prior treatments, three patients ( %) received three treatments and two patients ( %) received four treatments. the median expanded disability status scale (edss) score was (range: - ). peripheral blood stem cells were mobilised with cyclophosphamide (cy) g/m on day + and daily gcsf ( μg/kg subcutaneously) from day + until the completion of the harvest. hsct was performed at a median of days after mobilisation (range: - ). the conditioning regimen consisted of cy ( mg/kg/day from day − to − ) and atg ( mg/kg/day from day − to − ). exacerbation of ms symptoms was defined as the appearance of new or worsening of old symptoms for at least h duration in a previously stable ( weeks) patient. of the total cohort, patients ( %) underwent mobilisation with cy+gcsf uneventfully. only two patients ( %) had an exacerbation of ms requiring hospital admission after collection (one with fatigue and increase of spasticity, other with worsening weakness). no patient required hospital admission during the mobilisation procedure. the median cd + cell dose was . × /kg (range: . - ). the median number of apheresis was ( - ). a total of patients have undergone hsct at the time of this analysis. during transplant a total of patients ( %) experienced an exacerbation of ms. of these, % (n = ) before day and % (n = ) between day + and + . symptoms of exacerbation were: muscle spasms in patients ( %), weakness and reduced power of limbs in patients ( %), increase instability and tremor in two patients ( %) and one patient ( %) with worsening of neuropathic pain. only three patients ( %) received treatment with methylprednisolone for ms exacerbation and symptoms had fully resolved by discharge in all patients. other transplant complications included neutropenic fever in all, invasive fungal infection in , fluid overload in ( %) and atg related complications in ( %) such as fever (n = ) and pericarditis/serositis (n = ). the median time to neutrophil engraftment was days ( - ) and the median duration of hospital admission was days ( - ). exacerbation of ms symptoms is common during hsct and can also occur during mobilisation. in our hands, after cy and gcsf mobilisation only two patients ( %) developed an exacerbation of ms symptoms compared with patients ( %) after ct and atg conditioned hsct. it is possible that the addition of atg to cy triggers an immunological response involved in this transient deterioration of the ms symptoms. further studies are required to confirm this hypothesis. disclosure of conflict of interest: none. inflammatory immune response syndrome (iris) is a noninfectious worsening of neurological condition during immune recovery and has been documented to occur in hiv and in multiple sclerosis following alemtuzumab. the manifestation of iris includes headache, nausea, weakness, neurologic deficits, and mri enhancing lesion. we report three cases of iris after autologous non-myeloablative hematopoietic stem cell transplantation (hsct) in patients for which the transplant indication was an inflammatory neurologic disease: neuromyelitis optica (nmo), chronic relapsing inflammatory optic neuritis (crion), and multiple sclerosis (ms). mobilization was with cyclophosphamide gr/m and gcsf. conditioned regimen was mg/kg cyclophosphamide ( mg/kg/d) and . mg/kg ratg (thymoglobulin). the conditioning regimen for nmo and crion also included mg rituximab. case . a years old african-american female with systemic lupus erythematosus (sle) and nmo was discharged day and readmitted on day for fever, headache, progressive altered mental status with dysarthria and legs. brain mri had numerous t /flair hyperintense and enhancing lesions in the subcortical and periventricular white matter. a lumbar puncture was negative for infection including jcv. complete recovery occurred after treatment included high dose of steroids and plasmapheresis. case . a years old female with crion experienced blindness, weakness and slurring of speech three months post hsct. mri showed a large enhancing brain stem lesion. lumbar puncture was jcv negative. complete recovery occurred after solumedrol and rituximab. mri months later demonstrated complete resolution of the enhancement with return of vision to baseline. case . a ten year-old boy diagnosed with paediatric ms developed hemichorea seven days after hsc reinfusion. brain mri revealed a gadolinium-enhancing lesion in the contralateral basal ganglia. lumbar puncture was negative for infection including jc virus. symptoms resolved spontaneously after seventeen days. the appearance of new neurologic symptoms and mri enhancing lesions early after autologous hsct is unexpected and may be related to lymphocytes in the graft, immune recovery post engraftment, and or persistent auto-antibodies. it is mandatory to perform a lumbar puncture to exclude the possibility of infections including progressive multifocal leukoencelopathy (pml) due to jcv. the timing of presentation, the negativity of jc viral load, and the complete recovery with or without immune suppression suggest the hypothesis of iris, as an epiphenomenon of the immune reconstitution following autologous hsct for neurologic diseases disclosure of conflict of interest: none. hematopoietic stem cell transplantation is the effective method of therapy for cns autoimmune disorders in children. long-term outcomes and late effects estimation required. the aim of the study is to estimate long-term outcomes and late effects at children underwent auto-hsct for multiple sclerosis (ms) and allo-hsct for neuromyelitis optica (nmo). twelve pts. with ms and pts. with nmo were included to the analysis. ms pts. gender: female - % (n = ), male - % (n = allogeneic haematopoietic stem cell transplantation (hsct) remains the sole curative option for patients with myelofibrosis (mf). although a spectrum of conditioning regimens has been used, the optimal preparative treatment before hsct remains to be defined. we did a phase ii randomized study at transplant centers in italy with the aim of comparing the reduced-intensity conditioning (ric) fludarabine-busulfan (fb) (conventional arm), that had been already tested in the prospective ebmt study ( ) with the ric fludarabine-thiotepa (ft) (experimental arm), that has been widely used in italy in the last two decades ( ) . eligible to this study were patients with primary mf or a mf subsequent to a previous essential thrombocytemia or polycyhemia vera, an age ≥ ≤ years, a karnofsky performance status , a comorbidity index o o/u and with at least one of the following unfavorable prognostic factors: anemia (hb o g/dl), leukocitosis ( × /l), circulating blasts % or constitutional symptoms. patients were randomized to receive intravenous busulfan . mg/kg for doses or thiotepa mg/kg for two doses associated to fludarabine mg/m for impact of pre-transplant ruxolitinib in myelofibrosis patients on outcome after allogeneic stem cell transplantation syed abd kadir, sharifah shahnaz, author , , zabelina, tatjana, co-author , christopeit, maximilian, co-author , wulf, gerald, co-author , wagner, eva, co-author , bornhaeuser, martin, co-author , schroeder, thomas, co-author , crysandt, martina, co-author , mayer, karin tina, co-author , stelljes, matthias, co-author , badbaran, anita, co-author , wolschke, christine, co-author , ayuk ayuketang, francis, co-author , triviai, ioanna, co-author , wolf, dominik, co-author ruxolitinib (rux) is the first approved drug for treatment of myelofibrosis. because spleen size and constitutional symptoms may influence outcome after allogeneic stem cell transplantation (asct), rux is recommended before stem cell transplantation in order to reduce therapy-related morbidity and mortality and improve outcome (ebmt/eln recommendation, leukemia ) the aim of this retrospective study was to evaluate the impact of pretreatment with rux in comparison to transplantation of rux-naïve mf patients with regard to outcome after asct. we included myelofibrosis patients (pts) with a median age of years (range: - ) who received asct between and from related (n = ), matched (n = ) or mismatched (n = ) unrelated donor. all patients received busulfan-based reduced intensity conditioning. while pts ( %) did not receive rux, pts ( %) received rux at any time point prior to asct. the median daily dose of rux was mg (range: - mg) and the median duration of treatment was days (range: - days). in pts rux was stopped before stem cell transplantation because of no response or loss of response, while in pts rux was given until start of conditioning. gvhd prophylaxis consisted of cni plus short course mtx or mmf and anti-lymphocyte globulin. according to dynamic ipss (dipss) (n = ) the patients were either low (n = ), intermediate- (n = ), intermediat- (n = ), or high risk (n = ). as the median follow up was shorter for patients treated with rux ( vs months, po . ). primary graft failure was seen in pts in the rux and three in the non-rux group. the median leukocyte engraftment was days (range: - ) in the ruxolitinib and days (range: - ) in the non rux group (p = . ). the incidence of acute gvhd grade i to iv was significantly lower in the rux group ( % vs %, p = . ), while agvhd grade ii-iv ( % vs %, p = . ) and grade iii/iv ( % vs %, p = . ), did not differ significantly. the ci of nrm at year was % ( % ci: - %) for the rux group and % ( % ci: - %) for the non-rux group (p = . ), and the ci of relapse at years was % ( % ci: - %) vs % ( %ci: - %, p = . ). the years rfs and os was % ( %ci: - %) and % ( %ci: - ) for the rux group and % ( % ci: - %) and % ( % ci: - %) for the non-rux group (p = . and p = . , respectively). within the rux group (n = ), pts responded to rux (more than % spleen size reduction), while pts did not respond or lost response prior to stem cell transplantation. here, no significant difference could be seen between the responding and non-responding group for nrm ( % vs %, p = . ), relapse ( % vs %, p = . ), rfs ( % vs %, p = . ) and os ( % vs %, p = . ). in a multivariate analysis including rux treatment as variable there was a non-significant trend in favor for in the tyrosine kinase inhibitor (tki) era, allogeneic haemopoietic stem cell transplantation (allo-hsct) has become the later-line therapy but still remains the only known curative treatment for chronic myeloid leukemia (cml). since the introduction of tki in our centre in , the trend of allo-hsct among our cml cohort has changed over time. the purpose of this study is to examine hsct outcomes of our cml cohort who was either tki naïve or has received tki therapy prior hsct. between may and december , cml patients in our center received allo-hsct with % were tki naïve. the time of diagnosis to transplant was significant shorter among the tki naive group as compared to those received tki prior hsct ( . ± . months versus . ± . months, respectively). there were no gender different ( % males) but the median age at hsct was younger among tki naïve group, . years (range: - years) versus . years (range: - years) respectively. malays remained majority ethnic group but the percentage was reduced among patients received tki prior hsct ( . % versus . % respectively). the disease phase at hsct was significant different whereby majority of tki naïve group was in first chronic phase (cp ) ( . %) as compare to patients with prior tki exposure ( . %). all the patients in the tki naïve group received hla-matched related siblings donor (mrd) with . % marrow stem cell source whereas only . % of patients who have prior tki exposure received mrd with . % were from peripheral blood stem cell (pbsc). all patients in the tki naïve group but only . % among patients who have prior tki exposure received full myeloablative conditioning regimen. there was slower neutrophil and platelet engraftment ( . ± . days versus . ± . days and . ± . days versus . ± . days respectively) among tki naive group. at june , the -year overall survival (os) of cml at all disease status was % in tki naive group versus % for patients who have prior tki exposure and transplanted in more advance disease stage. in general, patients in cp have the best os. there was higher incident of grade to acute graft-versus-host-disease (gvhd) among the tki naïve group ( . % versus . %, respectively) likely due to intensity of conditioning regimen with no significant different in chronic gvhd incident. similarly, there was higher relapse rate among tki naive patients ( . % versus . %, respectively) as upfront post transplant tki was not routinely given to this group of patients in the past. further multivariate analysis to ascertain predictors of transplant outcome among this cohort of patients included disease status, donor-recipient gender combination, ethnic difference will be presented. in conclusion, despite emergent of effective and potent next generation tki, hsct still has it role as curative modality for patients who failed tki. as showed in our data, the transplant outcome is excellent for patients who remain in cp at the time of hsct and it is important to identify patients earlier, before disease progression, especially young patients, in order to optimize transplantation outcomes. disclosure of conflict of interest: none. the purpose of this analysis was to provide -year follow-up of the gcllsg cll x trial which aimed at evaluating reducedintensity conditioning (ric) hsct in patients with poor-risk cll. the cll x trial included patients (median age ( - ) years), of whom patients were allografted with blood stem cells from related ( %) or unrelated donors ( %) using fludarabine-alkylator-based ric regimens. % had refractory cll at hsct, and % had a tp deletion and/or mutation. the -year follow-up of the trial including the observation that genetic risk factors such as tp lesions and sf b and notch mutations had no prognostic impact has been previously reported. survival and relapse information was requested for all patients who underwent hsct within the cll x trial in german centres (the canadian centre was unavailable for follow-up) and were alive at the -year followup. results: follow-up information was received for / patients ( %) alive at the -year follow-up. of these, patients had died ( cll, chronic gvhd, secondary cancer), and had experienced disease recurrence. with a median follow-up of survivors of . ( . - . ) years, -year nrm, relapse incidence (rel), event-free survival (efs), and overall survival (os) of all patients allografted was %, %, % and %, respectively, without significant effects of tp lesions on outcome. absence of minimal residual disease (mrd) at the -month landmark post hsct was highly predictive for a reduced relapse risk, in particular if mrd eradication occurred only after immunosuppression withdrawal, suggesting of effective graft-versus-leukemia activity (gvl; -year rel %). in the patients who were alive and event-free years post allohct, nrm, rel, efs, and os years after this landmark (or years after transplant) was . %, %, %, and % with a median follow-up of . years ( . - . ) after the -year landmark. notably, no relapse event occurred beyond years post hsct. of those who remained event-free beyond years, all patients who were available for mrd assessment at their most recent follow-up were mrdnegative. altogether of the allografted patients had cll recurrence after transplant; between and , and from onwards. whilst the median survival of those patients who relapsed during the earlier period was months, all patients with late relapse are currently alive - (median ) months after the event. conclusions: long-term observation of patients allografted in the cll x trial confirms that ric hsct can provide gvl-mediated sustained disease control in a sizable proportion of patients with poor-risk cll independent of the tp status. patients who are in mrdnegative remission one year after hsct have an % probability of remaining disease-free at least for years. however, late relapses do occur but may benefit from strategies involving innovative pathway inhibitors. hallek: consultancy and speakers bureau for pharmacyclics, llc and an abbvie company; speakers bureau for janssen; m. kneba: consultancy, honoraria, travel grants and research funding from gilead and roche; consultancy, honoraria and travel grants from abbvie and janssen-cilag; research funding from amgen; travel grants from glaxo-smithkline; p.dreger: consultancy for roche and janssen; consultancy and speakers bureau for novartis and gilead. no evidence for an increased gvhd risk associated with post-transplant idelalisib given for relapse of chronic lymphocytic leukemia or lymphoma: first results of a survey by the ebmt chronic malignancy and lymphoma working parties p dreger , , a boumendil, l koster , c scheid idelalisib is a kinase inhibitor (ki) approved for the treatment of cll and follicular lymphoma (fl). idelalisib has a specific adverse effect profile including immune-mediated inflammatory conditions such as colitis and pneumonitis, raising concern about the safety of this ki if administered for treatment of malignancy recurrence after allogeneic hematopoietic cell transplantation (allohct). the purpose of this ongoing study is to provide information on the safety and efficacy of idelalisib in this setting. we included in this study adult patients who had been registered with the ebmt for an allohct for cll or lymphoma and who received idelalisib for treating disease relapse or persistence at any time after transplant as indicated by participating investigators upon request by the ebmt study office in leiden. baseline patient, disease, and transplant data were collected from med-a forms. centers were requested to provide additional treatment and follow-up information. as of november , , a total of patients have been registered, of whom a full dataset as required for this analysis was available for patients (cll , fl , diffuse large b-cell lymphoma (dlbcl) , peripheral t-cell lymphoma , unspecified ) who had undergone allohct between july and april . all patients except one were male. median age at transplantation was ( - ) years and the median interval from diagnosis to allohct was . ( . - . ) years. prior to allohct, patients ( cll and lymphoma) had received an autohct and two other patients had been exposed to ki (idelalsib , ibrutinib ). disease status at allohct was sensitive in % of the patients. conditioning was reduced-intensity in % of the transplants and included in vivo t cell depletion in the majority of cases ( %). donors were identical siblings in % with pbsc being the stem cell source in all cases. the interval between hct and idelalisib commencement was ( - ) months in the cll group but only ( - ) months in the lymphoma group. prior to idelalisib, grade ii-iv acute gvhd and chronic gvhd had been observed in % and % of the patients, but was still active at the time of idelalisib commencement in only two cases ( %) . four patients with cll had already failed ibrutinib given for post-hct relapse prior to idelalisib. the median time on idelalisib until documented withdrawal or event (progression, retreatment, death) was ( - ) days. after start of idelalisib, one patient developed grade acute gvhd and subsequently chronic gvhd, however, in this patient idelalisib was started as early as days after transplant. efficacy of idelalisib in this high-risk patient sample was limited with only one pr in the cll group (stable disease , progressive disease , not available ; lymphoma not available), translating into a median event-free survival after start of idelalisib of days. five patients with cll underwent a subsequent treatment with an alternate ki (ibrutinib , venetoclax ). altogether, there were five deaths, all due to diease progression (cll , lymphoma ). median overall survival was days for the whole sample and not reached for cll. this preliminary data does not support concerns about the safety of idelalisib in the post allohct setting. updated results of this ongoing study will be presented at the meeting. tested patients ( %) achieved a ccyr and at least a mmolr, respectively. the response to transplant by day assessment correlated significantly with the disease status before transplant. a higher percentage of patients who experienced cytogenetic response before transplant experienced molecular response post-transplant ( %) compared with those who did not ( %; p = . ). for the entire group, the -year cumulative incidence (ci) of acute gvhd grade ii-iv and grade iii-iv were % and %, respectively; -year ci of extensive chronic gvhd was %. there was no significant difference in the ci of severe acute or chronic gvhd between donor types. the ci of nrm at days and year was % and %, respectively. the ci of cytogenetic and molecular relapse at years was % and %, respectively. overall the -year os, pfs and gvhd-free, relapse-free survival (grfs) were %, %, and %, respectively. in multivariable analysis for grfs, transplant in cp and the use of haploidentical donor significantly associated with better grfs. the -year grfs of patients in cp , ap and bp before transplant was %, % and %, respectively (p = . ). ( figure a ) patients receiving a haploidentical donor had a better -year grfs when compared with hla matched transplants ( % vs %, p = . ). ( figure b ) for pfs, transplantation in cp , using a haploidentical donor and mac regimen associated with better pfs while age, cytogenetic and molecular response before transplant did not predict survival. ahsct is curative for a proportion of patients with advanced cml. pfs and grfs are favorably influenced by percentage of bm blasts and donor type, with haploidentical donor having at least as good outcomes as hla matched donors, while molecular and cytogenetic response before transplant do not appear to correlate with survival posttransplant disclosure of conflict of interest: none. allogeneic stem cell transplantation (sct) has been considered as the treatment of choice for younger patients (pts) with high-risk chronic lymphocytic leukemia (cll). role of allogeneic sct in era of novel drugs is widely discussed. here we present our results after sequential use of chemotherapy and reduced-intensity conditioning (ric) in cohort of high-risk cll pts. high-risk cll was defined by one of the following: disease refractory to purine analogs, short response or early relapse (within months) after purine analog combination treatment, and/or progressive disease with unfavorable genetic abnormalities (del [ p]/tp mutation). we analyzed pts with high-risk cll undergoing chemotherapy and ric sct in our centre from august to june . the median of pretransplant lines were (range: - ), novel drugs (idelalisib, ibrutinib) were used in % of pts ( / ). fludarabine ( mg/m ) and cytarabine ( g/m ) for days (fc) were used for cytoreduction in all pts. after days of rest, ric consisting of gy tbi, anti-thymocyte globulin - mg/ kg/day for days, and cyclophosphamide - mg/kg/day for days followed. median age of pts was years (range: five-year overall (os) and relapse-free survival (rfs) was % and %. ci for cgvhd in pts surviving more than months post-hct was % after years and % after years. in a multivariate cox-regression model the occurrence of cgvhd independently improved os (p = . , hr . ; % ci . - . %) as well as rfs (po . , hr . ; % ci . - . ). high risk dipss plus score demonstrated significant inferior survival compared to intermediate- (os p = . ; rfs p = . ), int- (os p = . ; rfs p = . ) and low risk (os p = . ; rfs p = . ) which could be confirmed in multivariate analysis for os (p = . , hr . ; % ci . - . ) and rfs (p o . , hr . ; % ci . - . ). rfs additionally was improved by splenomegaly (n = ) vs. normal spleen size (n = ) at time of hct (p = . , hr . ; % ci . - . ). ruxolitinib (n = ) or none (n = ) pre-treatment compared to other drug therapy (n = ) resulted in improved os (p = . ) and rfs (p = . ) and was an independent factor for rfs in multivariate analysis (p = . , hr . ; % ci . - . ). non-relapse mortality (nmr) was significantly determined by high-risk dipss plus score (p = . ) or dipss high and int- (p = . ). relapse incidence was significantly lower in pts with splenomegaly compared to asplenic or normalspleensized pts prior to hct (p = . ). our data point out that pre-therapy and dipss or dipss plus score are relevant pre-transplant outcome factors while chronic gvhd is the most important independent hct-related factor. furthermore, splenomegaly at hct reduces risk of relapse and therefore improves rfs. [p ] disclosure of conflict of interest: none. thalassaemia major affects new babies in india each year and haematopoietic stem cell transplantation offers the only chance of cure. we present data on children with thalassaemia major aged between months and years using a uniform conditioning regimen consisting of thiotepa mg/kg, treosulphan gm/m and fludarabine mg/m . equine antithymocyte globulin at a dose of mg/kg was added to children who were undergoing transplantation from an unrelated donor source. there were eight deaths before engraftment due to sepsis or bleeding and two related to graft versus host disease. all patients showed complete chimerism on day . however, in children there was an acute drop in donor chimerism between day and post transplantation. immunosuppression was abruptly stopped when donor chimerism dropped below % in all children. seven children responded well and re-established complete chimerism with this measure. seven children progressed to develop complete graft loss. donor lymphocyte infusion (dli) in the form of small aliquots of peripheral whole blood from the donor was administered in seven children. dli was used in a graded fashion every weeks starting from × /kg of cd , followed by × /kg and × /kg. all of them continued to maintain their graft with these interventions. drop in chimerism was seen particularly in children less than years at the time of transplantation comprising out of children. older children with lucarelli class iii were also prone to rejection in our earlier series and this complication has now been eliminated with pre-transplant immunosuppression and hypertransfusion. children above the age of years were more prone to graft versus host disease and required on average months of immunosuppression. treosulphan based protocol has been equally well tolerated by all age groups, all lucarelli classes of children with thalassaemia major and different donor sources. the transplant related mortality and graft rejection rates have been low at . % and . %, respectively. however, children less than years need to be monitored carefully during the first months of transplantation as early withdrawal of immunosuppression can prevent graft rejection resulting in excellent outcomes. disclosure of conflict of interest: none. institute of cellular medicine, newcastle university, newcastle-upon-tyne, uk hemophagocytic lymphohistiocytosis (hlh; hemophagocytic syndrome) is a rare syndrome of potentially fatal, uncontrolled hyperinflammation. allogeneic stem cell transplantation (allosct) is indicated in familial, recurrent or progressive hlh. additional recommendations include central nervous system involvement and unknown triggering factor. while data for allosct outcome are available for the pediatric setting, information for adults is very limited. the aim of this study was to retrospectively analyze the information from the ebmt databases about adult hlh patients who underwent allogeneic stem cell transplantation. we obtained data of adult (≥ years of age) patients transplanted due to hlh. additionally, an hlh-oriented questionnaire was sent to the clinical centers, with responses received so far. median age at transplantation was (range: - ). there was a slight male predominance / ( %). the majority of patients were reported with secondary hlh / ( %), the familial disease was reported in / ( %) patients. in two patients triggering factor was attributed to malignancy. the majority of patients received stem cells obtained from the peripheral blood ( / ; %) while for the remaining ones it was bone marrow. reduced intensity conditioning was used since in / ( %) of patients. thirteen ( %) patients received tbi. donor choice was: matched unrelated ( %), mismatched unrelated ( %), identical sibling ( %). engraftment was observed in / ( %). the cumulative incidence of acute graft versus host disease (gvhd) at days was % ( % ci - %). the cumulative incidence of chronic gvhd at year after allosct was % ( % ci - %) and increased to % at years ( % ci - %). the -year probability of overall survival is shown in fig. . the median survival time was months. the -year os was % ( % ci - %). for patients who survived until months, this proportion was more favorable with an os of % ( % ci - %) at years after transplantation. among patients with observation times longer than months, only one patient died (in the th month after allosct due to relapse which occurred in the th month. after months no more relapses of hlh were recorded-the cumulative incidence reached %. the non-relapse mortality reached % after months. the familial disease was associated with a better prognosis than secondary hlh (p = . ). unlike the pediatric population, where reduced intensity conditioning (ric) was associated with higher survival, in adult patients there was no difference between the conditioning types. data form the questionnaires confirm clinical picture typical for hlh at the diagnosis: fever in / ( %), splenomegaly in / ( %), hemophagocytosis in / ( %) and hyperferritinemia with median concentration of ng/ml (range: - ). image fig. overall survival after allogeneic stem cell transplantation for adult hlh patients until months ( % confidence intervals are shown in grey). the number of patients at risk is indicated below the time axis at the corresponding time points. to our knowledge, this is the largest group of adult patients with hlh who underwent allogeneic stem cell transplantation. relatively low relapse incidence shows that allosct can effectively cure hlh. patients who survive the first period after this procedure can expect a long disease-free survival. disclosure of conflict of interest: none. allogeneic hematopoietic stem cell transplantation (hsct) is the only curative option for children suffering from various life-threatening inherited non-malignant diseases with best results using hla-identical family donor. hsct from unrelated or mismatched family donors is associated with increased risk of agvhd and graft rejection.use of post-transplantation cyclophosphamide (ptcy) with or without additional immunosuppression has been shown to be effective prophylaxis against gvhd in patients with hematological malignancies. there are limited reports of hsct using pt cy for patients with non-malignant disorders. we retrospectively analyzed results of hsct in patients with life-threatening non-malignant diseases using ptcy-based gvhd prophylaxis. patients characteristics are presented in table . thirteen patients ( . %) were transplanted upfront, patients ( , %) were rescued after primary or secondary graft failure after first hsct. donors were hla-matched (n = ) or mismatched ( - / ) (n = ) unrelated, haploidentical (n = ) or hla-identical family (n = ). bone marrow was used as graft source in ( . %) patients and peripheral blood stem cell in ( . %). median cd +/kg recipient weight- . × ( . - . ), cd +/kg- . × ( . - . ). the conditioning regimen was myeloablative in patients (conventional- , reduced toxicity- ), reduced intensity- . the gvhd prophylaxis consisted of a combination of ptcy at dose of mg/kg on days + and + with calcineurin inhibitors (tacrolimus- pts, cyclosporine a- pts) or sirolimus ( pt) and mmf ( pts) starting on day + . all but one patients received also serotherapy with rabbit ( pts) or horse atg ( pst) and rituximab ( pts). with a median follow-up of months (range: - ), the kaplan-meier estimates of os − . %. one patient with thalassemia died before engraftment on day+ from severe vod. / pts ( %) achieved engraftment. the median time for neutrophil and platelet engraftment was ( - ) and days ( - ), respectively. primary graft failure was observed in patients ( was successfully retransplanted from another haploidenticle donor, was not eligible for a second transplantation, but alive). at last follow up, ( %) patients had full donor chimerism, ( %) had stable mixed chimerism without signs of disease progression. one patien with wiscott-aldrich syndrome had secondary graft failure with progressive loss of donor chimerism and were successfully rescued with second haploidentical transplant from the same donor. of engrafted patients, agvhd ii-iv was seen in ( . %) patients. one patient developed grade ii (gut stage ii) agvhd, which resolved with systemic steroids. severe (griii-iv) agvhd was observed in pts after second hsct, both had calcineurin and mtor-inhibitors induced toxicity leading to discontinuation of this drugs, but responded on combined (steroids and ruxolitinib) therapy. one patient with was developed grade iii gvhd (gut stage ) after severe cmv-colitis and died on day from multiple organ failure (suspected tma). one patient developed extensive chronic gvhd of kidney (minimal change [p ] disease) after tapering of immunosupression. one patient with hurler syndrome had seizures, died on day+ from multiple organ dysfunction syndrome. conclusion: ptcy is a promising option for agvhd prophylaxis in patient with non-malignant disease, lacking an hla-matched family donor. disclosure of conflict of interest: none. an exploratory, open-label study to evaluate the safety and feasibility of atir , a t-lymphocyte enriched leukocyte preparation depleted ex vivo of host alloreactive t-cells (using photodynamic treatment), as adjuvant treatment to a t-cell depleted haploidentical hematopoietic stem cell transplantation in patients with beta-thalassemia major c selim , w rob , l sarah , f josu de la previous studies demonstrated that donor lymphocytes, selectively depleted of alloreactive t-cells (atir), could be given safely in adult patients receiving a haploidentical hsct. in patients a single dose of atir, at doses up to × viable t-cells/kg, was given and no grade iii/iv acute gvhd has been reported. this confirms the efficacy of the elimination method of allo-reactive t-cells and attributes to its beneficial safety profile. in an ongoing phase study, cr-air- (nct ), infusion of atir at days post-hsct results in a reduction of transplant-related mortality (trm) and improvement of overall survival and event-free survival. adjunctive treatment with donor lymphocytes in patients receiving a t-cell depleted, haploidentical hsct for nonmalignant diseases such as beta thalassemia major, could provide early immunological support and better immune reconstitution in the absence of gvhd. in an open-label, multicenter phase study (cr-bd- ; eudract - - ), patients age ≥ years and ≤ years with beta thalassemia major will undergo a haploidentical hsct with adjunctive administration of atir . patients will receive a t-cell depleted graft (cd -selected, or cd /cd depleted, or tcr-αβ depleted, depending on the experience of the study center) from a related, haploidentical donor, patient conditioning will be myeloablative following standard practices at the study center. atir infusion at a dose of × viable t-cells/kg is given between and days after the hsct. to assess safety, patients will be evaluated for the occurrence of dose limiting toxicity (dlt), defined as acute gvhd grade iii/iv within days post hsct. efficacy will be primarily evaluated by transfusion-free survival (tfs), occurrence of severe infections, and time to t-cell reconstitution, taking into account hematologic and sustained engraftment. all patients will be closely monitored for cmv, ebv and adenovirus titers, with initiation of pre-emptive treatment upon rising blood titers. regulatory authorities in the united kingdom and germany have approved this clinical study protocol. enrolment of the study is expected to continue during , with first report of safety of atir to be expected first half . disclosure of conflict of interest: j. rovers is employee of kiadis pharma, sponsor of the study. sickle cell disease (scd) can be cured with haematopoietic cell transplantation (hct), yet progress in the practice and research of hct for scd has not come without risks and uncertainty. the information and decisions that families and physicians encounter in this field are complex and hanging. in this hermeneutic study, we analyze the case of one family who advocated for hcts for two of their four children knowing the potential risks. these experiences have had a profound impact on both the family and the medical team. this study was conducted through the research method of hermeneutic phenomenology. hermeneutic inquiry is described as the practice and theory of interpretation and understanding in human contexts and aims to make sense of the particulars of these contexts and arrive at deeper understandings. data collection: in-depth interviews were conducted with the mother of the family, the hct nurse coordinator, and the hct physician. the interviews were audiotaped and transcribed verbatim. the transcribed interviews were later reviewed by the physician, who then wrote an additional reflection. this work culminated in approximately pages of single spaced data in textual form. in hermeneutics, interpretation takes place through a careful reading and re-reading of the data, looking for statements and instances that resonate with the researcher. initial individual interpretations of researchers are then raised to another level of interpretive analysis in the research team's communal attention to the data. particular criteria guide the analysis: agreement, coherence, comprehensiveness, potential, and penetration. the following excerpts and interpretations are provided as examples of the analysis, with names changed for confidentiality. "being heard" arose repeatedly in this family's experience, including at the time of their request for a transplant without meeting the traditional criteria for hct. they persisted in their belief that their children would benefit from hct. "they gave us options to see if there was a chance for a transplant...how life would look…. and then we figured…a transplant for him was better at the time…worth the risks…. and you wouldn't even know. he plays basketball now, he plays sports, he's active and he can exercise and run. i never had any regrets because i felt it was better and the most important thing is his organs were really intact; none of the organs were destroyed…so i think it's the right decision we made" (mother). "this family has changed my career, and my life as a result. they challenged my practice and way of thinking. they did so in a considerate way, out of a duty to advocate for their children. we worked through the tension of different viewpoints with respect and all of us grew in the process. at least i can say our team did. i certainly did... i am humbled by their trust and respect…i am grateful to them" (physician). patients and providers are deeply impacted by their interactions. dr. robert buckman stated that it was the individual case that changed his practice always. he claimed he could not walk into a new patient's room without his practice being forever changed. in presenting this hermaneutic analysis, we aim to remind ourselves of the opportunity for growth that can result from reflection on this sacred patientprovider relationship. disclosure of conflict of interest: none. defibrotide (df) prophylaxis and adjustment of busulfan schedule to prevent veno-occlusive disease and thrombotic microangiopathy in an infant with a membrane cofactor protein (mcp) gene mutation and metachromatic leukodystrophy undergoing hematopoietic stem cell gene therapy (hsc-gt) v calbi , , f fumagalli , , , r penati , g consiglieri , m migliavacca , , d redaelli , s acquati , v attanasio , r chiesa , f ferrua , , f barzaghi , , m cicalese , , a assanelli , , p silvani , s tedeschi, r arora , a soman , f ciotti , m sarzana , g antonioli , , c baldoli , s martino , gl ardissino , mg natali sora , l naldini , , f ciceri , , a aiuti , , and me bernardo hepatic veno-occlusive disease (vod) and thrombotic microangiopathy (tma) are life-threatening complications that can occur after hsc transplantation. expert consensus guidelines support use of df for treatment and prophylaxis of vod due to its ability to restore thrombo-fibrinolytic balance and protect endothelial cells. presymptomatic monozygous twins affected by late infantile metachromatic leukodystrophy (mld) underwent investigational hsc-gt after conditioning with busulfan. no comorbidities were evident at baseline. the dose of transduced cd + cells was similar in both patients ( . × cd +/kg for patient and . × cd +/kg for patient ). patient (p ): at months of age, received conditioning with iv busulfan mg/m /dose for doses (target auc mg × h/l). on day (d) + after gt, he developed severe vod and was treated with diuretics, fresh frozen plasma, paracentesis and df. on d+ schistocytes in peripheral blood, marked proteinuria, complement factor consumption, and increases ldh and bilirubin were observed. the patient's condition worsened, with reduced urine output and generalized oedema with pleural effusion. stool, urine and blood cultures were negative and adamts activity was %; anti-complement factor h (cfh) antibodies (ab) were positive ( ui/ml). these findings led to the diagnosis of atypical hemolytic uremic syndrome (ahus; a form of tma) and eculizumab ( mg/weekly dose) was started on d + . patient subsequently developed pulmonary oedema and needed non-invasive ventilation. molecular analysis revealed a heterozygous deletion of cfhr -r and ala val mutation in the mcp gene, a defect previously shown to be associated with ahus. due to the presence of ab anti-cfh and antiplatelet, weekly doses of rituximab ( mg/m ) were administered. after treatment, p progressively improved although he showed prolonged severe anaemia and thrombocytopenia and bone marrow (bm) hypoplasia, secondary bleeding which required reinfusion of unmanipulated autologous bm cells on d + . nine months after hsct-gt p has shown good hematopoietic recovery, stable engraftment of the transduced hscs, no signs of renal damage or complement activation, albeit with neurodevelopmental delay. patient (p ): given his twin history and genetics, this month old infant was considered at increased risk of vod, so prophylaxis with df ( mg/kg/d) was administered from d- to d+ and the busulfan conditioning was modified by adjusting the auc to a lower target ( mg/kg/dose for doses; target auc . mg × h/l). the child had a good clinical recovery and didn't develop signs of vod or tma after hsc-gt. on d+ and + , respectively, anti-cfh and anti-platelet ab were positive. considering the history of the twin, weekly doses of rituximab were administered. p is currently months after gt with persistent engraftment of transduced hscs and no signs of tma. data from this case-control report of monozygous twins diagnosed with mld, and subsequently shown to also harbor mutations in complement regulator gene, suggest that df prophylaxis and busulfan adjustment may have helped prevent systemic microangiopathic damage in the second twin. patients with rare disease may have mutations in genes in addition to those that cause their disease. patients at risk of post-transplant tma following hsc-gt for genetic diseases may require tailored df prophylaxis and treatment. disclosure of conflict of interest: a. aiuti is the principal investigator of the tiget-mld clinical trial of gene therapy. the mld gene therapy was licensed to glaxosmithkline (gsk) in and gsk became the financial sponsor of the trial. all authors declare no other competing interests. hematopoietic stem cell transplantation (hsct) using an optimized conditioning regimen is essential for the longterm survival of patients with inherited bone marrow failure syndromes (ibmfs). we report hsct in children with fanconi anemia (fa, n = ), diamond-blackfan anemia (dba, n = ), dyskeratosis congenita (dc, n = ) and shwachman-diamond syndrome (sds, n = ) from a single hsct center. the graft source was peripheral blood stem cells (n = ) or cord blood stem cells (n = ). fa, dc and sds patients received reduced-intensity conditioning, while dba patients had myeloablative conditioning. the median numbers of infused mononuclear cells and cd + cells were . × /kg and . × /kg, respectively. the median time for neutrophil and platelet recovery was and days, respectively. there was one primary graft failure. after median follow up years the overall survival was %. the incidence of grade ii-iii acute and chronic graft versus host disease (gvhd) was % and % respectively. in a multivariate analysis, the type of conditioning regimen was the only factor identified as significantly associated with grade ii-iii acute gvhd (p = . ). we conclude that hsct can be a curative option for patients with ibmfs. disease specific conditioning regimen was important to disease the transplant-related mortality. [p ] disclosure of conflict of interest: none. homozygous sickle cell disease (scd) patients suffering from end-stage renal disease (esrd) show a variable outcome after kidney transplantation as underlying disease can cause poor allograft survival and disease-specific problems. we present a case of a -year old patient with severe scd and esrd who underwent haploidentical bone marrow transplantation (bmt) with consecutive living kidney transplantion (lkt). the patient suffered from multiple complications of scd including stroke with secondary hemorrhage, symptomatic epilepsy, esrd and uncontrolled hypertension. the patient had been on hydroxyurea without success and required regular blood transfusion due to severe renal anemia. the rationale for bmt was uncontrollable iron overload. a reduced intensity conditioning regimen was used with (fludarabine, cyclophosphamide and gy of tbi, dose-adjusted to esrd). graft-versus-host disease (gvhd) prophylaxis consisted of post-transplant high-dose cyclophosphamide, cyclosporine a (cya) and mycophenolate mofetil (mmf). the donor was her -year old mother with hbs trait, the stem cell source was bone marrow, the cell dose . × nucleated cells/kg. during conditioning daily hemodialysis was performed to keep drug levels stable. neutrophil engraftment occurred on day + , chimerism at day + was %. hbs increased from . % pre-hsct to . % months after hsct. hemoglobin values increased from g/l pre-hsct to g/l post-hsct and reticulocytes from g/l to g/l. erythropoietin levels increased from . iu/l pre-hsct to iu/l months after hsct. during the follow-up, the patient did not show any sign of acute gvhd or vaso-occlusive crisis, hemolysis or sickling. relevant complications were disease-related (therapy resistant hypertension and epileptic seizure due to former brain damage). on day + a lkt from the same donor was performed. the initial immunosuppressive treatment with mmf was continued, cya was switched to tacrolimus and steroids were added for months. the post-transplant period was uneventful. currently, months after haploidentical bmt and months after lkt there are no signs of gvhd, the blood chimerism is %, the kidney allograft function is very good (gfr ml/min/ . m ) and immunosuppression is withdrawn. iron overload is being corrected by regular phlebotomies. the patient no longer requires antihypertensive medication and there is evidence of vascular remodeling. this is the first report of a successful haploidentical bmt followed by kidney transplantation from the same donor in a patient with scd. disclosure of conflict of interest: none. allogeneic hematopoietic stem cell transplantation (hsct) can cure non-malignant diseases, such as primary immune deficiency (pid), severe aplastic anemia (saa) and osteopetrosis (op). in the absence of a well-matched donor, transplantation from a haplo-identical donor maybe considered. post-transplant cyclophosphamide (ptcy) is a new strategy derived from the treatment of malignant diseases in adults that has been little studied in high-risk pediatric nonmalignant diseases. fifteen children ( . years, range: . - . ) underwent hsct in the pediatric immunology and hematology unit of necker hospital, paris, between december and september . these children were suffering from op (n = ), saa (n = ), hemophagocytic lymphohistiocytosis (hlh) (n = ), immunodysregulation polyendocrinopathy enteropathy x-linked (ipex) syndrome (n = ), combined immune deficiency (n = ) and leukocyte adhesion molecule deficiency (n = ). three patients received a low-intensity conditioning regimen (cr) (based on fludarabine, cyclophosphamide, and total body irradiation) whereas the other received myeloablative cr (based on busulfan auc targeted and fludarabine). fourteen patients received serotherapy before hsct. post-transplant cyclophosphamide ( mg/kg/ day) was given on d and d and graft versus host disease (gvhd) prophylaxis with cyclosporine and mycophenolate mofetil was initiated on d . the transplanted stem cells were obtained from bone marrow in all cases. engraftment with full donor chimerism was observed in patients. the median cd + cell dose was . × cells/kg body weight (range: . - . × ). neutrophils recovered after a median of days (range: - ), and overall survival (os) was % after a median follow-up of year (range: . - . ). three patients died due to graft failure (n = ) or infectious complications related to gvhd (n = ). grade ii acute gvhd occurred in of the patients displaying engraftment ( %), and chronic gvhd and/or autoimmune complications were observed in four patients ( %). viral complications were frequent, occurring in patients ( %) with cmv infection (n = ) /disease (n = ), adenovirus disease (n = ) and bk virus cystitis (n = ). haploidentical transplant with ptcy is feasible in high-risk patients with non-malignant diseases. chronic gvhd and autoimmunity were more frequent than for more conventional approaches in such patients. infection rates were high. disclosure of conflict of interest: none. sickle cell disease (scd) remains associated with high risks of morbidity and early death. even best of supportive care fails to improve quality of life. hematopoietic stem cell transplant (hsct) can be considered for selected group of patients. in long run it is not just economical but also substantially improves quality of life (qol). we report our experience with hsct for scd from india. seventy three consecutive patients suffering from scd who underwent hsct between january and november were included in the study. fifty two underwent matched sibling donor (msd), matched family donor (mfd), matched unrelated ( / or / ), cord blood transplant cbt ( matched sibling cord blood and matched unrelated) and patient underwent haploidentical transplant. different conditioning regimens were used and so was the graft versus host disease prophylaxis depending on institutional protocols as depicted in table . a total of patients underwent sct. the median age was years ( months- years). m/f ratio was / . majority of patients were either from african union or oman. all patients suffered from one or other severe symptoms making them eligible for sct. graft source was bone marrow (bm) in with median cd count of . x /kg ( . - . ), peripheral blood (pb) in with median cd count of . x /kg ( . - . ), cord blood in with median cd count of . x /kg ( . - . ) and combined bm & pb in with median cd count of . x /kg ( . - . ). of the patients, are alive and disease free with lansky/karnofsky scores of . there were deaths ( msd/mfd/mud; haploidentical and matched unrelated cbt). four patients rejected the graft ( haploidentical and msd/mfd/mud). at the last follow up, the probabilities of survival, scd-free survival, and transplantrelated mortality were %, . %, and %, respectively. outcome of hsct in scd has improved significantly. with better conditioning regimens, improved supportive care, the outcome of alternative donor transplant and adult scd has improved and matches sibling donor transplant. hsct should be strongly considered as a curative modality for selected patients suffering from scd. disclosure of conflict of interest: none. s staff jointly defined more than local standard operating procedures. patients with low-risk characteristics (age ≤ years, liver size ≤ cm below costal margin) and a hla matched sibling donor were considered eligible in this initial phase of activity. a downstaging protocol with hydroxyurea and deferoxamine or deferasirox was adopted. conditioning regimen included iv busulfan and cyclophosphamide. gvhd and rejection prophylaxis included atg from day − to − and csa, mtx and methylprednisolone. gcs-f primed bone marrow was chosen as stem cell source. the first allogeneic hsct of the whole iraq was performed in a child with thalassemia at hiwa hospital in october . up to now, patients ( females, male) underwent hsct; median age at transplantation was years; median infused tnc . × /kg, cd + . × /kg. all of them engrafted. no major complication were observed. one of them developed grade ii agvhd (skin only) which resolved after increasing the dose of steroids. a huge number of patients with low-risk thalassemia are now in the waiting list and some of them have already started downstaging having planned hsct in a short time. a matched sibling transplant program in children with thalassemia is feasible and safe in kurdistan. such a program can provide many advantages: far less psychosocial and financial burden for the families and significant saving for the government. the estimated costs of performing locally hsct are much less than in the countries where patients were previously referred. the continuation of cooperation is of paramount relevance for further implementing the activity and extending the transplant accessibility to patients with other hemato-oncological disorders of childhood. disclosure of conflict of interest: none. long term follow-up after reduced-intensity conditioning and stem cell transplantation for thalassemia major r rihani, a natsheh, sm abu, e khattab, r najjar, f sheab, s sharma, n hussein, a tbakhi and m sarhan bone marrow and stem transplantation program-king hussein cancer center, amman, jordan hematopoietic stem cell transplantation (hsct) is the only curative treatment for thalassemia major (tm). reducedintensity conditioning (ric) before hsct for high risk tm patients results in fewer complications, when compared with myeloablative regimens. one hundred and three tm patients received hscts from an hla-identical related donor at king hussein cancer center, between january and november . of those, were high risk tm ( %) who received ric hscts. in this report, we describe follow-up beyond years (median, ; . - months) post ric hscts. forty-four class ii-iii patients ( %) were identified ( % with hepatitis c); with a median age of ( . - ) years. females accounted for % (n = ). conditioning regimen consisted of oral busulfan mg/kg, fludarabine mg/m ,tli cgy and atg followed by pbsct. gvhd prophylaxis consisted of mmf and csa. median infused stem cell dose was . × /kg. all patients attained neutrophil and platelet engraftment (median, . and . days, respectively). persistent mixed donor or full donor chimerism were observed in . % (n = ) and . % (n = ), respectively. immune-suppressive therapy for gvhd treatment was required in ( . %) patients (agvhd, n = ; cgvhd, n = ). moreover, veno-occlusive disease occurred in patients ( %) that resolved completely. secondary graft failure was noted in ( %) patients. the -year overall survival was %, while the -year probability of thalassemia-free survival was . %. other factors evaluated include: growth parameters, endocrine and other organ functions, in addition to functional status. this report confirms the safety and efficacy of ric regimens in hscts for high risk tm patients. those regimens are associated with excellent engraftment and sustained mixed donor chimerism; and lead to excellent thalassemia-free and overall survival rates. [p ] disclosure of conflict of interest: none. in-time hsct for pts. with hurler syndrome (hs) can significantly improve the results. long-term follow-up and late effects estimation required to prepare a special observation and rehabilitation programs. aim. to analyze our experience with hsct for hs in the field of special observation and rehabilitation programs. forty hsct during the - were performed for pts. with hs. median age at the diagnosis was months ( - months), at hsct- months ( - months). bm used in . % (n = ), pbsc- . % (n = ), cb- . % (n = ). mac conditioning was used for hsct, ric-for . ric regimen: flu+mel+atg, mac: bu/treo +flu+thio/mel (bu was used in early ) and atg +rituximab (in case of mud hsct). all pts. with ric received mud hsct, pts. with mac mud- pts., mrd- pts. pts. received csa/tacro-based gvhd prophylaxis. mmf/mtx was additionally added in all cases. in ric hsct immunomagnetic Сd /Сd + depletion of pbsc (by clinimacs) was used. a special observation protocol including somatic and neurocognitive estimation was developed. all pts. engrafted with full donor chimerism on d+ . median of engraftment day- ( - days). thirty three pts. survived. reasons of death-mac: infections- pts., ric: trali- pt., agvhd- pt. trm improved, over the years, with improving of supportive care and donor selection as well as pre-transplant screening. no early severe toxicity revealed. pulmonary infection episodes was registered in % of pts. in our study. gvhd: grade iideveloped pts., grade iii-iv- pts. (after ric), local cgvhd- pts. (ric). no extensive cgvhd. pts. rejected (mac and ric rejection rate was same). at median follow up of months ( - months), the estimated years pos was %. best response correlated with early hsct (and better status before hsct) and higher level of aidu after. late effects estimation showed that . % (n = ) of patients experienced late effects: cardio-vascular- pts., skeletal- pts., endocrine- pts. all pts. with cardio-vascular effects received mac. skeletal effects affected patients of older age, pts. transplanted in younger age do not have such effects. median period of late effects arising after hsct was month ( - months). only pts. experienced serious pulmonary late effects (infections), all episodes was before . no pts. in our study have progressive retinal degeneration. % of pts. improved in the neurosensory component and all pts. improved in neurocognitive status and development after hsct. best response correlated with neurocognitive rehabilitation based on unique computer model used by our group in russian national rehabilitation center "russkoe pole." in-time hsct is an effective and safe way to stop neurodegenerative process for pts. with hs. both mac and ric regimens can be used with the same effectiveness. mac regimens associated with bigger number of cardiovascular late effects. long-time follow-up showed that these patients require the special observational protocol including estimation of cardio-vascular, skeletal, endocrine and neurocognitive risks. better neurocognitive response correlated with intensive rehabilitation using computer model. russian joint study showed effective cooperation for treatment pts. with hs in the national setting. disclosure of conflict of interest: none. little is known about pathogenesis of solid tumors after hsct but, intensive cytotoxic conditioning therapy with defective dna repair of persisting stem cells/stromal cells, viral infection, and immunosuppression may play a role. / patients with solid tumors had a melphalan-based conditioning. melphalan was linked to sarcoma and lung cancer in animal model. there are few data linking parotid mec to infection by cmv and hhv which can remain dormant in the salivary glands. both affected patients had hhv during the transplant period. p and p had a family history of solid tumor pointing to a possible genetic factor. whilst secondary malignancy post-hsct for patients with malignant disorders is well recognised, non-ptld malignancy post-hsct for pid has not previously been reported. a larger study is needed to evaluate incidence and risks. allogeneic hsct is a treatment of choice for the bone marrow failure in patients with sds. hsct from unrelated or mismatched family donors is associated with higher morbidity and mortality compared with matched sibling. combined pgd and hla antigen testing is a possible option to preselect a compatible donor for an affected sibling requiring hsct. we describe a case report demonstrating first successful hsct for years girl with sds by using preimplantation genetic diagnosis and hla matching. diagnosis of sds was suspected at m.o., based on clinical features, family history, laboratory studies. at m.o., bone marrow (bm) aspiration revealed hypocellular marrow with signs of dysplasia and expansion of blasts ( . % blasts). the sanger sequencing of sbds gene showed c. - ta ct and c. + t c mutations. the patient had recurrent infections, including bilateral pneumonia caused by phaeohyphomyces, bloodstream infection, cmvdisease. due to the lack of matched related or unrelated donors, hsct with ric (flu, mel, atg) from haploidentical father was performed at months of age. after the st allo-hsct, engraftment was achieved on d+ , initial str study showed full donor chimerism. post-transplant period was complicated with severe cmv-infection and signs of secondary hlh. at d+ , graft rejection was registered. the girl became dependent on regular rbc and platelet transfusions, bm examination revealed hypocellularity with moderate signs of myelodysplasia without elevated blast count. due to lack of available hla-compatible donors, an option of in vitro fertilization (ivf) with preimplantation selection of a normal hla-matched embryo was considered. after controlled ovarian hyperstimulation embryos were hla-compatible and healthy (first, wild-type; second, heterozygous for sbds gene mutation c. - ta ct). hence, the only unaffected hla-identical embryo was transferred resulting into full-term pregnancy. at the age of . years after st hsct, the nd s transplant was performed with a combination of cb and bm as a source of hematopoietic stem cells. the donors' age was years a reduced toxicity conditioning regimen (rtc) based on flu mg/m , treo g/m , thiotepa mg/kg with serotherapy (thymoglobuline . mg/kg) was used. because of neurotoxicity, arterial hypertension, since d+ csa was changed to sirolimus +mmf for gvhd prophylaxis. the total number of infused nc was . × /kg; cd +, . × /kg; cd +, . × /kg. engraftment was achieved on d+ . any signs of gvhd, severe infectious or toxic complications were not observed. eight months later, the patient is alive, has full donor chimerism in bm and is not transfusion-dependent. in the absence of hla-identical donor, ivf with preliminary pgd and hla-typing could be a chance for matched donor to cure patients with non-malignant genetic diseases. in case of low cord blood cellularity, a combination of cb and bm from the same sibling could be used. our experience showed a successful engraftment of sds patient and stable donor chimerism after second hsct of cb and bm from pgd-selected sibling with rtc. disclosure of conflict of interest: none. the safety and efficacy of familial haploidentical (fhi) stem cell transplantation utilizing cd enrichment and cd addback in patients with high risk sickle cell disease (scd) ( figure a ). probability of yr efs is . % (ci : - %) ( figure b ). immune cell reconstitution has been robust and similar to rtc and msd allosct in scd (table ). there have been deaths, vod, steroid refractory agvhd and cgvhd. mac followed by fhi utilizing cd enrichment and t-cell addback in patients with high-risk scd is safe, tolerable and results in long-term donor chimerism and absence of scd symptoms or complications. a larger cohort and follow-up will be required to confirm these preliminary findings. disclosure of conflict of interest: none. supported by r fd - a . [p ] s lymphoma p a clinical prognostic index for assessing patients aged being considered for high-dose therapy and autologous stem-cell transplant in relapsed or refractory high-grade non-hodgkin lymphoma d edwards , k kirkland , r pearce, s robinson and g cook bsbmt patients with relapsed high-grade nhl or disease refractory to first-line therapy can still be cured with high-dose therapy and autologous stem cell transplant if they respond to salvage chemotherapy. this aggressive algorithm is accepted in younger patients but is less well established in the elderly. age has a negative predictive score in the international prognostic index (ipi) and there are concerns that the outcomes of hdt in these patients are significantly worse. deciding which older patients will benefit from hdt is challenging and there are no established predictive tools to guide physicians. we present a clinical prognostic index derived from information readily available at the time a patient is being assessed for asct the bsbmt audited the outcomes for uk patients aged transplanted between - (n = ) and benchmarked against the european bone marrow transplant (ebmt) database for the same period (n = ). the primary outcome was progression-free survival (pfs) but data was also analysed for overall survival (os), relapse rate (rr) and non-relapse mortality (nrm). we included all patients with a diagnosis of high grade nhl and the following demographic features were also analysed: age at diagnosis; age at transplant; m/f; year of transplant; cr/not cr at transplant; no. of prior therapies; no. of cells infused; clinical staging; karnofsky status at transplant; histology; ipi at diagnosis; mobilising regime and conditioning regime. candidate prognostic indices were factors achieving significance in univariate and multivariate analyses of the main outcomes by regression analysis. the best prognostic index was selected based on the bsbmt dataset and then applied to the rest of the ebmt dataset (the validation dataset). there were no significant differences in patient characteristics between the uk and non-uk groups nor in outcomes of pfs, os, rr or nrm. (figure ). in both univariate and multivariate analysis the following features were associated with a significantly worse outcome for pfs, os, rr and nrm : age , karnofsky score. disclosure of conflict of interest: none. underwent an allo-sct at our center after a treosulfan-based conditioning regimen. eleven pts received a mrd, pts a mud, and pts a haplo unmanipulated pbsc allo-sct. at allo-sct pts were in cr, pts were in pr, and pts had sd/pd. hct-ci was evaluable for pts, had a score ≥ . the backbone conditioning regimen consisted of treosulfan g/m from day − to − , and fludarabine mg/m from day − to − ; twenty-five pts were treated with this reduced toxicity conditioning (rtc) regimen. intensification with other alkylating agent (melphalan, thiotepa, or cyclophosphamide) or radiotherapy ( gy total dose) was applied on the remaining pts (myeloablative conditioning, mac). gvhd prophylaxis was based on cyclosporine a and methotrexate ( pts) or rapamycin and mycophenolate mofetil ( pts), plus anti-thymocyte globulin or post-transplant cyclophosphamide accordingly to donor type. median numbers of infused cd +/kg and cd +/kg were . × (range: . - . ) and . × (range: . - . ), respectively. median follow-up was months (range: - ). thirty-nine pts were evaluable for engraftment; median time to neutrophil ≥ . × /l was days (range: - ), and days (range: - ) to platelet ≥ × /l. treosulfan conditioning provided a cr in and pts respectively in pr and sd/pd at transplant. no graft failure was observed. one and years overall survival (os) was . % and . %, respectively. progression free survival (pfs) and gvhd-free/relapse-free survival (grfs) were respectively . % and . % at year, . % and % at years. one and years relapse/progression incidence (ri) was . % and . %, respectively. transplant related mortality (trm) was . % at days, . % at year and for the entire follow-up. the -day cumulative incidence (ci) of agvhd grade ≥ was . %; ci of moderate to severe cgvhd was . % at years. the outcome of pts in cr at years was significantly better compared to that of pts with active disease in terms of both os ( . % vs . %, p o . ), pfs ( . % vs %, p o . ), grfs ( . % vs . %, p o . ), and ri ( . % vs %, p o . ). no statistical differences in os, pfs, and ri were found when pts were stratified according to donor type and [p ] the use of rtc or mac regimen. at last follow-up, patients are alive and disease free; of them obtained a durable cr using chemotherapy and/or dli for disease progression after allo-sct. treosulfan-based conditioning regimen is effective and well-tolerated in patients with advanced b-nhl undetgoing allo-sct. disclosure of conflict of interest: none. systemic anaplastic large cell lymphoma (salcl) is a very infrequent well-defined histological entity that comprises around % of all t-cell non-hodgkin lymphoma. in the absence of prospective clinical trials, autologous stem cell transplantation (autosct) is considered the standard of care as consolidation therapy after first line therapy for those patients not expressing the alk protein (alk neg salcl) and for patients with relapsed disease. the objective of this retrospective analysis was to analyse the long-term outcome of patients diagnosed with salcl and being treated with autosct during the course of the disease, making special emphasis on the potential impact of the administration of brentuximab vedotin (bv). eligible for this study were patients years or above with salcl who underwent autosct between to and were reported to the ebmt. baseline patient, disease, and transplant data were collected from ebmt med-a standard forms. centers with potentially eligible patients were contacted to provide additional treatment and follow-up information including a written histopathology report for central review. seventy-nine patients ( males) with a median age at diagnosis of years (range: - ) and at transplantation of years were included in the final analysis. thirty-nine patients were alk negative, alk positive and in patients expression of alk protein was unknown. at diagnosis, patients ( %) presented with advanced stage and ( %), with b symptoms. sixty-three patients ( %) received - lines of therapy before autosct. ten patients were treated with bv at some point before autosct; two patients as second line therapy, three as third line, one as fourth line and four as fifth line therapy. the median number of bv doses was (range: [ ] [ ] [ ] [ ] [ ] [ ] . the median time between diagnosis and transplantation was months (range: . most patients had chemosensitive disease at autosct [ patients ( %)] and in all but patients peripheral blood was used as the source of stem cells. conditioning regimen consisted on beam / beam-like protocols in patients ( %). all patients engrafted. with a median follow up for surviving patients of months (range: - ), patients are alive ( %), patients died ( %) and patients ( %) are lost for follow up. disease relapse after transplantation was the most frequent cause of death after the procedure. cumulative incidence of non-relapse mortality for the whole series was % ( % ci, . - ) at days, year and years. cumulative incidence of relapse was % ( % ci - ) and % ( %ci - ) at and years, respectively. and years progression free survival (pfs) was % ( % ci - ) and % ( % ci - ), respectively and and -years overall survival (os) was % ( % ci - ) and % ( % ci - ), respectively. there were no significant differences in any of the outcomes between bv treated and non-treated patients. autosct results in a promising pfs and os in patients with salcl. the potential impact of the administration of bv as salvage strategy before the procedure needs to be further elucidated. disclosure of conflict of interest: none. coeliac disease (cd) is a t-cell immune-mediated enteropathy to dietary gluten, characterized by small bowel villous atrophy resulting in malabsortion. the enteropathy is reversible with a gluten-free diet (gfd), however symptoms and signs which persist year are defined as refractory coeliac disease (rcd). rcd is divided into type i and ii, depending on absence/ presence respectively of clonal intra-epithelial t-lymphocytes (iels) with an aberrant phenotype (cytcd pos, membranous cd , cd and cd neg). rcdii patients have a year survival of . , plts ) was successful at a median of . (range: - ) days and no transplant-related mortality occurred. all patients achieved a clinical complete remission, with normalization of nutritional indices at days, but persistently abnormal iels and clonal t-cells on duodenal biopsy. with a median follow-up of . (range: - ) months, patients remain in clinical remission, patient relapsed with rcd and no patient progressed to eatl. chemotherapy and asct is a safe and effective strategy for the treatment of rcd offering the possibility of sustained clinical responses. clonal tcr in duodenal biopsy/blood and iel flow cytometry form part of the patient evaluation prior to the chemotherapy/asct program. most patients with hodgkin lymphoma (hl) are cured with conventional chemotherapy. however, approximately % of patients relapse after primary treatment. for those, high-dose chemotherapy (hdc) followed by autologous stem cell transplantation (asct) is the standard of care. fifty seven adult patients with relapsed or refractory hl submitted to asct between and were reviewed. variables examined were sex, age, ann arbor stage (i-ii vs iii-iv), b symptoms, bulky disease, extranodal involvement, nodal areas involved (≥ vs vs ≤ months) and response to the treatment prior to asct. log-rank test was used to compare differences in survival for each factor. patients median age was ( - ) years at diagnosis. ann arbor stage iii-iv in ( %) patients, b symptoms in ( %), extranodal involvement in ( %) and bulky disease in ( %). all patients were treated according to the abvd protocol in first line. indications for asct were relapsed disease (n = , . %) and lack of complete response (cr) or progressive disease with st line treatment (n = , . %). there were a median of ( - ) treatment-lines before asct (protocols eshap, ice, beacoop, gvd and others). the disease was chemosensitive in % cases: cr in and partial response (pr) in patients prior to asct. refractory disease (rd) in % (n = ). in . % patients, the hematopoietic cells mobilization was performed under stimulation with granulocyte-colony stimulating factor in hematologic recovery after the cycle of nd line chemotherapy, and most of which required ( - ) apheresis. conditioning regimens were beam ( %) and gmb ( %). the median time to hematologic recovery was days ( - ) for neutrophils /ul and days ( - ) for platelets , / ul. three months after asct, thirty-nine ( . %) patients had cr, one ( . %) patient maintained pr and ( . %) patients had disease progression. status unknown in patients and four ( %) patients died. relapse rate % (n = / ). with a median follow-up time after asct of ( - ) months, median disease-free survival (dfs) was ( - ) months and overall survival (os) was ( - ) months. there were deaths ( . %), four ( %) related to early infectious complications of asct, two ( . %) due to late infectious complications, eleven ( . %) due to disease progression and ( . %) in context of secondary acute myeloid leukemia. response to the treatment prior to asct was the only factor with survival influence. the dfs and os differed significantly in chemosensitive disease compared with rd (dfs mean: vs months,p = . , os mean: vs months, p = . ). the response to salvage treatment prior to asct is the main prognostic factor for survival after asct. prognosis remains poor in patients with rd or early and disseminated relapses. for these patients, the therapeutic approach should include intensive treatment with tandem hdc and stem cell transplantation, allogeneic transplant or early consolidation with brentuximab-vedotin after asct. hodgkin's lymphoma (hl), although considered a curable neoplasm in adults, could be associated with a very poor prognosis when refractory to primary induction therapy or when it relapses within months from an autologous stem cells transplant (auto-sct). the optimal treatment of patients with heavily pretreated/refractory hl is controversial. brentuximab vedotin (bv) is an active single agent in this context; unfortunately, there are no well established therapies when patients fail to respond or progress after bv. encouraging results were recently described with checkpoint inhibitors. similarly, data pertaining to efficacy of bendamustine (benda) shows encouraging activity in various refractory lymphomas. we included in this study adult patients with hl who relapsed post auto-sct and were refractory to or progressed after salvage bv and were treated with benda as salvage therapy with an intention to proceed with an allo-sct. this study was [p ] conducted in two major centers in lebanon, the american university of beirut medical center (aubmc) and makassed university hospital. we identified eligible cases. the primary study endpoint was objective response rate (orr). the secondary endpoint evaluated successful rate of bridging into an allo-sct. the median follow-up times from auto-sct and from benda salvage were ( - ) and ( - ) months, respectively. the median age of patients was years ( - ). all patients had bv as salvage therapy post auto-sct, and all of them progressed after a median of ( - ) cycles. clinical characteristics are outlined in table . patients received a median of cycles ( - ) of benda. the treatment was well tolerated, with rather infrequent adverse events and transient and manageable toxicities. the orr was %, in of patients, with % obtaining a complete response. eventually, of proceeded to allo-sct using a matched related donor, and the remaining patients are planned for allo-sct. only one patient died from disease progression after months post allo-sct. two of patients who progressed following benda received salvage therapy with nivolumab and are being planned for haplo-identical transplant while the third one is being planned for therapy with nivolumab. from the initiation of benda, the median duration of response for the patients was months ( - ); all these patients had maintained a continuous response at the last follow-up examination. conclusion: notwithstanding the limitations associated with our analysis, namely a small sample size and its retrospective nature, these results suggest a role for bendamustine in post bv failures. these findings also provide the basis to evaluate the concept of benda as a bridge to allo-sct in a large prospective study. [p ] disclosure of conflict of interest: none. brentuximab vedotin for relapsed or refractory hodgkin lymphoma, single center experience king faisal specialist hospital and research center, riyadh, kingdom of saudi arabia ms rauf , i maghfoor , a badran , mn zahir and s akhtar hodgkin lymphoma (hl) patients with relapsed or progressive disease after high dose chemotherapy (hdc) and auto-sct have limited curative options. fda granted approval of brentuximab vedotin (bv) for the treatment of hl and anaplastic large cell lymphoma (alcl) patients who fail auto-sct or have had at least prior multiagent chemotherapy regimens and are not candidates for auto-sct. we are reporting single center experience of bv usage in this "approved" setting. medical records were reviewed to collect required data. kaplan-meier (km) method was used to calculate overall survival (os) and progression free survival (pfs) from date of first dose of bv. from - , patients received bv. / had hl ( classic hl-nodular sclerosis, hl-mixed cellularity) and alcl. / ( %) pts were primary refractory or had early relapse after initial treatment. / ( %) pts received bv were refractory to the last treatment. all the baseline characteristics of patients are mentioned in table . median bv cycles administered were ( - ). overall response rate (orr) was % ( patients): cr in ( %), pr in ( %) ( / were primary refractory or early relapsed). median pfs for whole group was months ( % ci, . - . ). km estimated -year os was % and year was %, median os has not been reached yet. for patients who responded, pfs at months was % ( % ci, %- %), median pfs not reached. for / patients with progressive disease (pd) or non responders after bv, median pfs was only months ( % ci, . - . ). there was no difference in os between patients with responders and non responders. median os has not yet been reached in either group as mentioned in survival curves. at the median follow up of months (range: - months) patients are alive, patients are alive without disease, patients received consolidation bone morrow transplant ( auto-sct and allo-sct). patients completed courses and achieved cr. rest of patients who are alive without disease; they had pd on bv but achieved cr with other treatments. patients are alive with disease; patient is on bv and are on another treatment. patients have died, because of pneumonia while being on bv and due to pd. / patients who received bv, achieved cr after failing all previous treatments and are in cr. peripheral sensory neuropathy developed in patients; one required dose reduction. patient stopped treatment due to pulmonary toxicity. we are reporting largest single center data from middle east which confirms that bv as a single agent is effective and safe. overall response rate is lower as compare to pivotal trial but cr rate is comparable to other reported case series. this analysis also concludes that bv can be used as bridge to transplant in patients who don't respond salvage chemotherapy. disclosure of conflict of interest: none. was used to diagnose hiv infections. cox proportional hazards models were used to evaluate risk factors of overall mortality. fifty-six patients with nhl ( . %) and patients with mm ( . %) were positive for hiv antibody. in patients with nhl, overall survival was significantly lower in the hiv-infected patients than in the hiv-negative patients [ - year overall survival: hiv-infected patients, % ( % confidence interval, %- %) vs. hiv-negative patients, % ( % confidence interval, %- %), p o . )]. in a multivariate analysis, hiv infection was significantly associated with an increased risk of mortality (hazard ratio . , p o . ), and this effect was consistent regardless of transplant year. on the other hand, overall survival in patients with mm was similar between the groups [ % ( % confidence interval, %- %) vs. % ( % confidence interval, %- %), p = . ]. previous studies in europe and the united states showed comparable survival rates between hiv-infected and hiv-negative patients with nhl. however, our study showed that hiv infection was associated with a higher risk of mortality in patients with nhl in japan. suppression of t cell-mediated immunity or hiv related diseases might affect transplant outcomes in japanese patients. [p ] disclosure of conflict of interest: none. while beam and beac regimens (bcnu, etoposide, cytosar in both regimens and melphalan or cytoxan, respectively) are commonly used as conditioning high-dose therapy (hdt) in patients with non-hodgkin lymphoma (nhl), there have been few reports comparing these regimens. a retrospective analysis found the superiority of beam over beac in terms of overall survival (os) and event-free survival (efs). toxicities were similar, except that beam was associated with more frequent lower gastrointestinal (gi) mucositis. other studies reported that these regimens had similar efficacy and outcome. recently, a concern regarding cardiotoxicity of beac has risen. the current study aimed to compare efficacy and toxicity of beac and beam as consolidation hdt in young patients with mantle cell lymphoma (mcl) undergoing autologous stem cell transplantation (asct). this is a retrospective analysis of outcomes in mcl patients who received hdt with beam or beac followed by asct at bone marrow transplant centers in israel. os, disease-(dfs) and progressionfree survival (pfs) and regimen toxicity were compared. seventy seven mcl patients who were diagnosed between - / and received consolidation with beac or beam were included in the analysis. forty nine patients were treated with beam and patients-with beac. no significant differences between the groups were revealed in terms of age, sex, the mantle cell lymphoma international prognostic index (mipi) risk score, induction protocol and% of patients transplanted in first complete response (cr ) (mean age yrs in beam vs yrs in beac group; % of patients in beam group had mipi risk score - vs % in beac group; % of patients in beam group were transplanted in cr vs % in beac group). the amount of infused cd cells was significantly higher in the beam group (median cd cells/ kg: . in beam vs . in beac groups; p = . ); the number of days to platelet engraftment was significantly greater in the beac group (median days in beam vs days in beac group; p = . ). there were no differences in the number of blood transfusions or hospitalization days between the groups. the rate of grade - upper mucositis was significantly higher in the beam group ( % in beam vs % in beac group; p = . ); no other differences in toxicity (grade - lower mucositis, pulmonary congestion, infections) were observed between the regimens. non-relapse mortality by day posttransplant was % in both groups. a median follow-up was (range: - ) months. the -yr dfs in beam and beac groups was % and %, respectively (p = . ). there was no difference in the -yr os between the groups ( % in beam and % in beac group; p = . ). there was a trend to improved dfs and os in patients transplanted in cr receiving beam (p = . , figure) . in multivariate analysis, low-to-intermediate mipi and transplant in cr were found to significantly increase pfs (p = . and. , respectively), while the hdt regimen did not affect pfs. beac and beam hdt regimens followed by asct had similar efficacy in mcl patients. there was a trend to improved dfs and os in patients transplanted in cr and treated with beam vs beac. the toxicity profile was similar in both groups, except a significantly higher rate of grade - upper gi mucositis. [p ] disclosure of conflict of interest: none. early or refractory relapsed ( o year) diffuse large b-cell lymphoma has a very poor prognosis especially for those not responding to salvage chemotherapy. allogeneic stem cell transplantation is potentially curative. even though this is less likely in those not responding or having frank progression pretransplantation. methods: at our institution we identified all patients with aggressive b-cell lymphoma (diffuse-large b-cell lymphoma and blastoid mantle cell lymphoma) who were refractory or progressive to salvage chemotherapy with r-dhap and who had peripheral blood stem cells ( × cd +/kg body weight) collected after the st or nd cycle. after high-dose melphalane and autologous stem cell transplantation patients had a partial and a complete remission. patient died due to neutropenic infection, patients died due to progressive disease leading to a transplant related mortality of . %. median progression-free survival after autologous transplantation was . months. proceeded to allogeneic stem cell transplantation. patients had a matched related sibling, had a matched unrelated donor and had a mismatched unrelated donor. transplant related mortality was % in this heavily pretreated population. -year overall survival of all patients intended for treatment is %. one of these patients with relapsed mediastinal lymphoma after allogeneic transplantation was cured by salvage radiotherapy and is in long-term remission ( years). conclusions: salvage high-dose melphalane and autologous peripheral blood stem cell transplantation for diffuse large b-cell lymphoma as a bridge to allogeneic transplantation is potentially curative for a minor fraction of these patients. however, the remission rate of % ( % pr, % cr) and progression-free survival of . months after high-dose melphalane and autologous stem cell transplantation provides a window of opportunity to use new drugs and cellular therapies in these poor prognosis patients. high dose chemotherapy and autologous stem cell transplantation is the treatment of choice for patients with relapsed refractory hodgkin lymphoma. several factors including number of chemotherapy lines received before conditioning, time of relapse and remission status before transplantation can predict survival and pfs in patients undergoing autologous stem cell transplantation. in , we reported on a patients who underwent high dose chemotherapy followed by autologous stem cell transplantation from to . all patients with relapsed or refractory hodgkin lymphoma in the period of - , who underwent high dose chemotherapy followed by autologous transplantation were retrospectively analyzed. the main outcomes of the study were complete remission (cr) at day , overall survival (os) and relapse-free survival (rfs). the impact of the following variables on os and rfs: (a) disease status at the time of transplant, (b) number of chemotherapy lines prior to conditioning and (c) time of relapse months and (d) age. a total of patients were identified. the median age was year. there were . % females and . % males. complete remission (cr) was achieved in . % of patients and . % with chemotherapy sensitive disease at the time of transplantation. prior to conditioning regimen, . % received two chemotherapy lines, and . % received more than two lines. % relapsed in less than months and % relapsed more than months after completion of therapy cr at day was . %. the median os for the whole group was . months; the median rfs was , months. the number of chemotherapy lines significantly impacted os and efs. cr status before conditioning, favorably influenced os and efs with a trend toward better os in favor of those who underwent abmt while in complete remission. the time of relapse and the age did not affect survival outcomes. [p ] the outcome of patients with relapsed or refractory hodgkin lymphoma is favorable and the number of chemotherapy lines received before conditioning is the only factor that had a statistically significant impact on os and efs. since the identification of human immunodeficiency virus (hiv), a clear association between hiv and specific malignancies has been recognized. high-grade b cell lymphomas are the most common malignancy complicating hiv infection and one of three aids defining malignancies. diffuse large b cell lymphoma (dlbcl) accounts for % of cases. before , lymphomas were the cause of % of all deaths attributable to aids. after the introduction of highly active antiretroviral therapy (haart) overall incidence of adm declined, however longer survival and exposure to environmental risk factors have increased the incidence of non adm (adm) such as hodgkin's lymphoma (hl). since haart has improved overall survival substantially, the aim of chemotherapy should be complete remission rather than palliation with careful consideration of drug interactions and side of haart. between and a total of patients were detected hiv positive. twenty-one of these patients were diagnosed with a malignancy and patients referred to our department with a hematologic malignancy were evaluated retrospectively. diagnosis, stage, treatment, survival data were recorded. haart during chemotherapy, nadir cd count and cd count at diagnosis of malignancy was evaluated. four patients were diagnosed with high grade b cell lymphoma, patients with primary central nervous system lymphoma (pcnsl), patient with hl and patient with multiple myeloma (mm). all patients were male and median age at diagnosis was . ( - ). hiv seropositivity was identified during evaluation of malignancy in both pcnsl patients. median duration of hiv seropositivity before diagnosis of malignancy was months for the remaining patients. patient characteristics, treatment modification and cd counts are summarized in tables and . lymphoma was fatal in and the cause of death was identified as lymphoma progression in all patients including one patient diagnosed with hodgkin's lymphoma. a patient presented with multiple plazmositomas was diagnosed with multiple myeloma is currently receiving induction treatment together with haart. hiv related lymphoma patients frequently present with extra nodal disease, incidence of central nervous system involvement is also higher and prognostic score tends to be in the intermediate or high-risk groups. prognosis is also worse than hiv negative population. degree of immunosuppression is implicated and the duration of immunosuppression is directly correlated with the risk of developing lymphoma rather than hiv itself. haart allowed the use of aggressive chemotherapy since it improved immune system and decreased infectious complications. multiple myeloma is a rare neoplasm observed in hiv infection and the treatment is based on data obtained from hiv negative patients. treatment of such patients as well as lymphomas should take into consideration the toxic effects of haart combined with chemotherapy. since hiv positive [p ] patients are excluded from most studies, there are no guidelines to direct treatment and avoid toxicities. drug interactions should be monitored closely and modifications should be made accordingly. interruption of haart may not be mandatory since studies have shown safety of continuation of haart during chemotherapy. for newly diagnosed hiv and malignancy, careful clinical and laboratory evaluation should be made before postponing haart until after chemotherapy. disclosure of conflict of interest: none. the outcome of hdct and asct in refractory hodgkin lymphoma (r-hl) is not as encouraging as in relapsed hl. ten years ago we analyzed and reported outcomes of asct in our r-hl patients, however the follow-up was short. now we a reporting long term outcomes in r-hl after asct in one of the largest numbers reported to date. between and , patients with hl who underwent hdc and asct for r-hl in adult medical oncology (age years) were identified. r-hl is defined as partial response (pr), no response (nr), stable disease (sd), progressive disease (pd), relapsing within months (relapse o m) of finishing the planned (chemotherapy + radiation therapy (xrt)) treatment or refractory to salvage chemotherapy. kaplan-meier (km) method was used to estimate progression free survival (pfs) and overall survival (os) from the day of asct while progression is defined as progression of disease, relapse and death from any cause. all percentages are rounded to nearest. patients underwent hdc and asct during - and of them met the criteria of r-hl. male ( %), female ( %), median age at diagnosis was . years ( - years) and at asct was years ( - years). initial therapy was abvd in ( . %), mopp/copp alternating with abv or abvd in ( %) and others in ( %). ( %) had xrt after initial chemotherapy. response to initial chemo + xrt was pr in ( %), pd in ( %), cr in ( %) ( / relapsed within months and others have refractory relapse) and no response in ( %) and others in ( %). prior to salvage chemotherapy, ( %) had stage iii-iv, ( %) extra-nodal involvement, ( %) bulky disease and ( %) had b symptoms, spleen involvement in ( %), performance status , in ( %). eshap was used as first line salvage in ( %) or rd line ( %). post salvage / prior to hdc and asct disease status was pr in ( %), cr in ( %) and nr/sd in ( %). ( %) patients had a fdg-pet scan prior to asct, ( %) were in cr. beam was used as conditioning regimen. median follow-up for all alive patients is months ( - ) from asct. response rate post asct: cr in ( . %), pr in ( %), nr/sd in ( . %) and pd in ( . %) patients, others /unknown in ( . %). ( %) patients had xrt post auto-sct. type of first post hdc auto-sct event was no event in ( %), persistent disease in ( %), pd in ( %), relapsed disease in ( %), treatment related mortality in ( %) and died of other cause ( %). at last follow-up in november , patients ( %) are alive with no disease, ( %) alive with disease, ( %) died of disease and ( %) died of treatment related mortality or other causes. for entire group, km estimated median os is months, , , , and year survival is %: %: %: %: % respectively. median pfs is . month, , , , and year pfs is . %: . %: %: %: % respectively. we are reporting a very high risk group of patients with a very long follow-up. in patients with r-hl, eshap + beam combination resulted in high response rate ( . %). these remissions are durable. a year os survival of greater than % in our population is higher than most reports with similar numbers. although our cohort has a year os survival of %, % patients have either relapsed or died underscoring need for improvements in the management refractory hl. [p ] disclosure of conflict of interest: none. here we update the previously reported results of our reduced-intensity conditioning (ric) allo-hsct experimental program, initiated in . as of november , in our centre patients underwent ric allo-hsct. donors were hla-identical sibling in , fullymatched unrelated in , or -mismatch-unrelated in and haploidentical relative in . median age was years (range: - ). all patients ( m and f) had stage iib/iv refractory mf (n = ) or refractory ss (n = ). median number of previous treatment lines was (range: - ). source of stem cells was peripheral blood in patients and bone marrow in . median time from diagnosis to hsct was months (range: - ). conditioning included flu/ctx/tbi , pentostatin +tbi and flu/mel in case of hla-identical or unrelated donor, whereas the tt/flu/ctx/tbi regimen was used in the haplo setting. gvhd prophylaxis included csa/mmf in all patients, with the addition of atg in cases with unrelated donor and post-transplant ctx ( mg/kg giorni + e + ) in cases with haploidentical donor. full donor chimerism was obtained in / of the evaluable patients, in a median time of months (range: - ). grade ii-iv acute gvhd occurred in patients ( %), while grade iii-iv was observed in patients ( %). chronic gvhd occurred in patients ( %), being extensive in ( %), all transplanted from hla-identical sibling (no atg). following transplantation, a complete remission (cr) was achieved in out of the evaluable patients ( %), of whom experienced relapse at + and + months, respectively. transplant-related death occurred in patients ( %), of whom were in cr. out of the patients who did not achieve cr, died from progressive disease (median follow-up of months, range: - ), from a secondary malignancy and is still alive with disease months after transplant. of note, all pts who died in progression had chemoresistant disease at time of transplant. at the last follow-up, patients were alive and ( %) maintained cr after a median time of months (range: - ). in the whole population, the -year overall survival was % ( % ci - ) and the -year disease-free survival (dfs) was % ( % ci - ). however, when mf and ss were analysed separately, -yrs dfs were % ( % ci - ) and % ( % ci - ), respectively (figure) . apart from diagnosis, outcome appeared to be primarily associated with the disease status at transplantation, with a -yr dfs of % in the group of patients (n = ) who were in cr before starting the conditioning. after a median follow-up longer than years, we confirm the efficacy of ric allo-hsct as a powerful therapeutic strategy in inducing and maintaining remission in selected patients with chemosensitive advanced-stage ctcl, with results particularly encouraging in ss. [p ] disclosure of conflict of interest: none. outcomes of allogeneic hematopoietic stem cell transplantation for hodgkin lymphomas: a retrospective multicenter experience of the rete ematologica pugliese (rep) f gaudio , p mazza , am carella , d pastore , g pisapia , a mele , p galieni , n cascavilla , g specchia and v pavone hematology, university of bari, bari, italy; hematology, ospedale "san giuseppe moscati", taranto, italy; hematology, ospedale "casa sollievo della sofferenza", san giovanni rotondo, fg, italy and hematology, ospedale "cardinale panico", tricase, le, italy; hematology, ospedale "c. g. mazzoni", ascoli piceno, italy hodgkin's lymphoma (hl) is a potentially curable disease, and modern therapy is expected to successfully cure more than % of the patients. second-line salvage high-dose chemotherapy and autologous stem cell transplantation (sct) have an established role in the management of refractory and relapsed hl, leading to long-lasting responses in approximately % of relapsed patients and a minority of refractory patients. patients progressing after intensive treatments, such as autologous sct, have a very poor outcome. allogeneic sct represents the only strategy with a curative potential for these patients; this study reports a retrospective multicenter experience of the rete ematologica pugliese (rep) over the past years aiming to define the impact of patient, disease, and transplant-related characteristics on outcomes. patients with histologically confirmed diagnosis of hl who received allogeneic sct from to were retrospectively studied. the median age was years (range: - years) and ( %) were male. the majority of patients ( %) had had a prior autologous sct. at the time of allogeneic sct, ( %) patients had a chemosensitive disease and ( %) were chemorefractory. most ( %) patients received reduced-intensity conditioning, % received matched sibling donor and % matched-unrelated donor grafts. the disease status at day post-transplant was reported in out of evaluable patients. of the patients with chemosensitive disease, ( %) achieved a cr, ( %) had a pr or stable disease and ( %) had progressive disease. of the patients with chemorefractory disease achieved a cr ( %), had a pr or stable disease ( %) and ( %) had progressive disease. although the overall survival has improved significantly in mantle cell lymphoma (mcl) according to advanced treatment options, relapsed or refractory disease remains a challenge. recently, lots of targeted agents actively have been tried clinical studies and adapted to clinical practice in indolent lymphoma. however, the role of frontline autologous hematopoietic stem cell transplantation (auto-hsct) has not been fully understood in patients with mcl, compared with a few impressive published data about auto-hsct as salvage treatment option for patients with relapsed mcl. so, we retrospectively evaluated consecutive patients diagnosed mcl, and compared the clinical outcomes of high-dose chemotherapy followed by auto-hsct and conventional chemotherapy alone. between january and december , consecutive patients with newly diagnosed with mcl at catholic blood and marrow transplantation center in south korea were included in this study. all of the patients received high-dose cytarabine-containing regimen or chop with/without rituximab regimen for induction therapy regardless of transplant eligibility. the treatment approach in our institution for patients was based on the physician discretion for transplant eligibility or ineligibility that depend on patient age, comorbidities, and disease status. seventy patients were included in the analysis. initial chemotherapy regimens were consisted of chop (n = , %), r-chop (n = , %), r-hypercvad (n = , %), and hypercavd (n = , %). demographics and disease characteristics of both groups are shown in table . patients received auto-hsct were superior s overall survival (os; p = . ) and progression-free survival (pfs; po . ). the subgroup analysis according to high-risk of mcl international prognostic index (mipi) or bone marrow involvement was performed. between the two treatment arms among the high-risk mcl group, the clinical parameters were not different. the high-risk mcl patients with frontline auto-hsct showed superior os (p = . ) and pfs (po . ) compared with conventional chemotherapy alone. although mcl is classified within indolent lymphoma, frontline auto-hsct can be considered for patients diagnosed with mcl in the group of high-risk mipi or bm involvement with the favorable survival outcomes. disclosure of conflict of interest: none. nasal type extranodal nk/t-cell lymphoma (enktl) is a very rare and agressive malignancy characterized by a poor outcome. current standard therapy is not yet established. the role of high dose therapy followed by haematopoietic stem cell transplantation (hsct) is still controversial. we evaluated the outcomes of all the enktl patients undergoing hsct in a multicenter analysis on patients registered by the société francophone de greffe de moelle et de thérapie cellulaire (sfgm-tc) and compared them with a population of french patients who received chemotherapy alone. sixty four enktl ( males and females) received hsct, including allogeneic (allosct) and autologous transplantations (autosct). median age at the time of hsct was years (range: to years). overall, % of the patients presented with disseminated disease ( % and % in the allosct and autosct, respectively), % were in complete response (cr) at the time of hsct ( % and % in allosct and autosct groups, respectively) and % had received l-asparaginase regimen prior to hsct ( % and % in allosct and autosct groups, respectively). five ( %) and ( %) patients of the allosct and autosct groups underwent upfront hsct therapy, respectively. four patients received tandem autologous/ allogeneic transplants. in allosct, stem cell source was a matched related donor in patients, an unrelated donor in patients and an umbilical cord blood in patients. reduced intensity conditioning regimens (based on fludarabine-busulfan combination) and beam regimen were used in % and % of patients from the allosct and autosct groups, respectively. median overall survival for the whole cohort was . months (range: to months). the -year non-relapse mortality was . % and . % in the allosct and autosct groups, respectively (p = . ). the -year overall survival (os) and progression free survival (pfs) were . % and . % in the autosct and . % and . % in the allosct group, s respectively ( figure a) . the absence of cr prior to hsct was associated with a poor prognosis (p = . ). as compared to allosct, autosct resulted in a better outcome in patients who didn't achieve cr before transplant (p = . ) and tended to have better outcome in high pink risk score (figure b-c) . finally, at years pfs and os of patients who have been treated by chemotherapy alone (ct) (n = ) or followed by allosct (n = ) or autosct (n = ) in cr were %, % and %, % and %, %, respectively ( figure d ). in this french cohort, more patients received autologous hsct in upfront therapy than allogeneic hsct. in cr , there is no evidence suggesting that transplantation is associated to a better outcome than chemotherapy alone. however, a precise matching based on the pink score will be evaluated to ensure that patients who were intensified were not of worst prognosis. in refractory patients there is also no clear advantage to perform allosct when compared to autosct. however, in relapsing disease after ct or autosct allosct, allowed to obtained durable control of the disease. disclosure of conflict of interest: none. high relapse rate is one of concerns for allo-sct in pts with relapsed/refractory aggressive lymphoma. an optimal conditioning regimen designed for aggressive lymphoma may reduce relapse, especially during early post-transplantation period. however, it is not established yet. results of a german phase study of allo-sct with conditioning regimen of fludarabine, busulfan ( mg/kg po or . mg/kg iv), and cyclophosphamide with or without post-transplantation rituximab for relapsed/refractory aggressive lymphoma suggested the role of myeloablative busulfan-containing regimen in reducing relapse rate in pts with aggressive lymphoma. based on these results, we conducted a single institution prospective study to explore feasibility of the bmf regimen consisted of full-dose busulfan, melphalan, and fludarabine in pts with relapsed/refractory aggressive lymphoma (umin ). patients with aggressive lymphoma who achieved at least sd with salvage chemotherapy after experiencing either pd during first-line therapy, early relapse ( o mo) after firstline therapy, late relapse (≥ mo) but refractory to salvage therapy, relapse after auto-sct,; age - ; ecog ps of - ; and without severe organ dysfunction were eligible. donor source could be / matched related or unrelated donor pb/bm or cb with ≤ antigen mismatch; the bfm regimen was consisted of busulfan . mg/kg iv, fludarabine mg/m , and melphalan mg/m (yamamoto h. bbmt ). gvhd prophylaxis was csa + mtx (related pb), tac + mtx (unrelated bm), and tac + mmf (cb). primary end point of the study was survival with engraftment at day , and secondary end points were engraftment rate at day ; nrm and relapse rate at day and y; progression free survival (pfs), overall survival (os), and gvhd at y. protocol was approved by irb and written ic was obtained from all pts. twelve pts (male , female ) with a median age of y ( - ) were enrolled. ps was - in pts. diagnosis were dlbcl (n = ), transformed fl (n = ), enktcl (n = ), ptcl (n = ), and aitl (n = ). median number of previous line of therapy was . ( - ) and pts had failed previous auto-sct. diseases status at transplantation was cr (n = ), pr (n = ), and sd (n = ). donor source was cb (n = ), unrelated bm (n = ), and related pb (n = ). survival with engraftment at day , primary endpoint of the study, was achieved in %. neutrophil engraftment was achieved at a median of day ( - ). full donor chimerism at day was achieved in all of the pts evaluated. two pts developed vod which was manageable. with a median follow-up of mo, pts had progression of lymphoma at , , mo. five pts died and cause of death were progression of lymphoma in , interstitial pneumonitis in (at mo), systemic adenovirus infection in (at mo), and agvhd in (at mo). os and pfs at y were % and %, respectively. relapse and nrm rates were % and % (day ), and % and % ( y), respectively. agvhd of grade ii-iv was observed in / pts and pts developed limited cgvhd. this prospective study shows that allo-sct using myeloablative conditioning regimen with full-dose busulfan, melphalan, and fludarabine for relapsed/refractory aggressive lymphoma is feasible and deserves further evaluation. disclosure of conflict of interest: none. for patients with advanced ctcl, the allogeneic hsct seems to be curative with graft versus lymphoma effect playing a major therapeutical role. in this retrospective study, patients with a median age of years (range: - ) affected by ctcl underwent allogeneic hsct after a median of (range: - ) lines of chemotherapy, including autologous transplant for of them. the median time from diagnosis to hsct was months (range: - ). the diagnoses were: sezary syndrome (ss, n = ). mycosis fungoides (mf, n = ), primary cutaneous cd + lymphoma (n = ), panniculitis-like t-cell lymphoma (n = ), nk t cell lymphoma (n = ). at time of hsct, patients ( . %) were in complete remission (cr), ( . %) in partial remission (pr) and ( . %) had active disease. the patients were transplanted from an hla-identical (n = ), mismatched (n = ) or haploidentical (n = ) sibling, from matched unrelated donor (n = ) or from a single cord blood unit (n = ). different pre-transplant regimens were used as myeloablative (mac) in (th-bu-flu, n = ; bu-cy, n = ) or as reduced intensity (ric) in (th-flu-cy, n = ; th-bu-flu, n = ). al patients engrafted for neutrophils at a median of days (range: - ) and patients engrafted for platelets at a median of days (range: - ). acute gvhd was of grade -i in patients and ii-iv in ( . %). skin was the most common organ involved. five of evaluable patients experienced chronic gvhd which was mild in and severe in . at a median of months (range: - ), patients died ( mac and ric) because of gvhd (n = ), vod (n = ), pneumonia (n = ) or multiorgan failure (n = ). all patients surviving at months from transplant were in cr. only patients prepared with a ric (n = ) relapsed respectively at , , and months from hsct. these patients received dli associated or not to chemotherapy. three achieved cr, which remained stable in , while one patient died in cr from post dli acute gvhd. one patient (nk-t cell) not achieving cr is still alive with active disease. for all patients the median survival was months (range - ). with a median follow up of months (range: - ), patients ( mac, ric) are alive, in cr and with active disease. at years, the os was ± %; at years dfs was ± %. according with the median time ( months) from diagnosis to transplant, the -year os was ± % for patients transplanted early and ± % for the others (p o . ), while dfs was respectively s ± % and ± % (p o . ). despite the small number of patients, our results confirm the high susceptibility of ctcl to the graft versus lymphoma effect and point out the time to transplant as a crucial prognostic factors for the outcome. finally, the long-term follow up of our series strongly supports hsct for the cure of ctcl. disclosure of conflict of interest: none. recently, a new prognostic score, the nccn-international prognostic index (ipi) has been developed to stratify patients affected by diffuse large b cell lymphoma (dlbcl), and in high-intermediate and high risk groups the survival was equal or less than %. the aim of this analysis was to evaluate the outcome of a cohort of dlbcl patients undergoing high dose chemotherapy (hdc) as consolidation following first line chemo-immunotherapy, after their re-classification according to the nccn-ipi. we performed a retrospective study on patients diagnosed with dlbcl, with a high/intermediate or high-risk disease according to the ipi ( - ), who received upfront hdc with asct, in institutions. the patients were then re-stratified according to the nccn-ipi and arbitrarily classified in groups: low risk (nccn-ipi ≤ ) and high risk (nccn-ipi ≥ ). the pre-transplantation disease status was assessed by positron emission tomography (pet) or computed tomography (ct). the primary endpoints were non-relapse mortality, progression-free survival (pfs), overall survival (os) and relapse risk. the estimated -year pfs for all patients was . % ( % confidence interval [ci] . - . ) and the -year os was . % ( % ci . - . ). of these patients, had a low risk ipi score (ipi = ) and were considered high risk (ipi ≥ ). subsequently, the whole cohort was re-stratified according to the nccn-ipi: patients were allocated to the high-risk (nccn-ipi ≥ ) group, and to the low-risk group (nccn-ipi ≤ ). the analyses were then carried out for both groups. the -year pfs was . % ( % ci . - . ) in the low-risk group and . % ( % ci, . - . ) in the highrisk group (po . ), whereas the -year os was . % ( % ci . - ) in the low-risk group and . % ( % ci . - . ) in the high-risk group (p = . ). the significant difference in os and pfs between the two groups was mainly due to the cumulative incidence of relapse at years (graph ): . % ( % ci . - . ) in the low-risk group and . % ( % ci . - . ) in the high-risk group (po . ). non-relapse mortality was comparable in both cohorts: % ( % ci . - . ) for all patients. figure : cumulative incidence of relapse following hdc and according to nccn-ipi. patients affected by high-risk dlbcl still have an unsatisfactory prognosis after treatment with conventional therapy regimens, even in the rituximab era. the -year os and pfs in patients with nccn-ipi score ≥ range: from % to % and from % to % respectively . although this is a retrospective analysis subject to all related biases, our results suggest that upfront intensive therapy with autologous stem cell transplantation may significantly improve the outcome of these patients compared to conventional chemotherapy. the role of hdc in the treatment of dlbcl is controversial. however, new entities or new risk stratifications, as the one reported here, could allow to identify high risk subpopulations that could benefit from this approach. enteropathy-associated t-cell lymphoma (eatl) is an exceedingly rare and often rapidly fatal subtype of peripheral t-cell lymphoma, arising from intraepithelial lymphocytes. eatl type i is associated with celiac disease; type ii occurs in patients without inflammatory pre-conditions (according to who classification now called monomorphic epitheliotropic intestinal t-cell lymphoma (meitl)). surgical debulking and anthracyclinebased chemotherapy (ctx) followed by high-dose chemotherapy (hdctx) and autologous cell rescue (asct) are pursued when possible in this often malnourished and frail patient cohort. yet, even with intensive consolidation relapse occurs in - % of patients. the value of allogeneic hematopoietic cell transplantation (hct) is not clarified as of today due to limited reports. here, we report on a patient with meitl who was rescued with an allo-hct for his nd relapse following prior asct. moreover, we summarize the available literature on the use and outcomes of allo-hct for eatl and meitl. a y old man with spontaneous intestinal perforation was diagnosed with meitl following emergency partial resection of the small intestine. histology revealed infiltration by monotonous medium-sized lymphocytes with abnormal immunophenotype (cd +, cd +, cd +, cd -, cd -, tia- +) consistent with type ii eatl. post-surgical f-fdg pet-ct scan showed abnormal uptake in gastric antrum and pyloric region but no other manifestations. ctx with cho(e)p ( × ) followed by beam hdctx and asct was performed and achieved a complete remission (cr ). however, m post asct disease relapsed and was treated with × dhap, and × dhaox. cr was achieved after the rd cycle of salvage therapy. due to anthracyclineinduced cardiopathy allo-hct could not be performed at that time. m after completion of salvage therapy, disease relapsed again, and was progressive under pralatrexat treatment ( cycle, infusions). by then cardiac function had recovered and therapy was switched to dose-reduced mini-beam ( × ). in cr reduced intensity conditioning (ric; fludarabine, busulfan, atg) and allogeneic hct from a matched sibling donor was performed. ciclosporin a (csa) and mycophenolate mofetil (mmf) were given as gvhd prophylaxis. prompt engraftment in blood (day+ ) and full donor chimerism in the marrow (d+ ) were achieved. immunosuppression was tapered and discontinued on d+ (mmf) and d+ (csa), respectively. f-fdg pet-ct scan at m post-hct showed cr, but at m relapse was suspected (under work-up). only few cases of patients with eatl/meitl treated with allo-hct are reported in the literature (n = , table ), and the value of this highly aggressive therapy is not clear at this point. of note, the patients listed in table were given allo-hct instead of asct. long-term complete remission (cr) could be achieved in / patients, while patients suffered from early relapse and died of the disease (n = before d+ post allo-hct). asct following surgery and ctx appears to cure - % of patients in available series. no treatment concept is available for relapse following asct, and no published data are available for allo-hct for relapse post asct. the disease is exceedingly rare and is afflicted with very poor outcomes. therefore, patients given this aggressive treatment should be reported, even when treatment outcomes are not positive. disclosure of conflict of interest: none. strong graft versus lymphoma effects with low toxicicty of haploidentical hematopoietic stem cell transplantation comparing with hla-identical in t cell lymphomas: a retrospective multicenter study s bramanti, r devillier, s fuerst, b reda, a granata, s harbi, c faucher, i legrand, a santoro, d blaise and l castagna istituto clinico humanitas rozzano consolidation treatment of relapsed/ refractory t-nhl with allogeneic stem cell transplant (sct) is considered a curative options but few patients manage to undergo this procedure, due to the highly refractory nature of the disease. the primary aim of this work is to evaluate the gvl effects among t-nhl with both hla identical and haploidentical donors. we have retrospectively analized the long term outcome of consecutive patients affected by t-nhl, received hlaidentical or t-cell replete haplo-sct with pt-cy, in european centers, between february and october . the patients received nonmyeloablative (nmac) or reduced intensity (ric) conditioning regimen. gvhd prophylaxis consisted of mg/kg of pt-cy (day + and + ) in haplo setting and atg plus cyclosporine a in the hla identical setting. patients characteristics were reported in the table . no differences were founded in the two groups . most of the patients were transplanted in complete remissions but only as consolidation of first line. no graft failure occurred. the cumulative incidence of acute gvhd grade - was % in the haplo setting vs % in the hla id .extensive chronic gvhd was seen in % of haplo, and in % in the hla id . patients had cmv reactivation, hemorrhagic cystitis, and ebv reactivation. after a median follow-up of years os was % and % and pfs was % and % in the haplo vs hla id group see figure . nrm was % in haplo setting and % in hla identical one. the years cir is % and % in haplo and hla id setting respectively. this study confirm a strong anti-lymphoma effect of allo hsct without prohibitive toxicities. haplo-hsct with pt-cy shows low rate of cgvhd in a contest of poor prognosis t-nhl patients. [p ] disclosure of conflict of interest: none. ibrutinib is the first-in class bruton tyrosine kinase inhibitor that has been approved for the treatment of relapsed mantle cell lymphoma. however, despite the high response rate of % including % of complete response, the median duration of response is relatively short with an overall survival of % at months (wang ml, et al, n engl j. med ). we report a single experience of patients with relapsed mcl who underwent allogeneic stem cell transplant (allosct) after ibrutinib monotherapy salvage. all patients had previous autologous stem-cell transplantation (asct) before and were given ibrutinib at a dose of mg daily after the second or subsequent relapse. all patients had to be at least in pr according to cheson criteria before allosct. patients had an unrelated / ( ) or / ( ) allo-sct from peripheral hematopoietic stem cells after a reduced conditioning regimen with busilvex, fludarabine and antithymocyte globulin in association with zevalin according to our recent published phase study protocol (bouabdallah k, et al. ann oncol ). graft versus host disease (gvhd) prophylaxis consisted on ciclosporine and methotrexate. patients ( m/ f) were aged from to years and received between and previous chemotherapy regimens including asct in their last treatment strategy before introduction of ibrutinib. all patients had extensive disease with gastric involvement in patients and pulmonary localization in patient. median time between diagnosis and ibrutinib introduction was years ( - ) and the median time between asct and allosct was years ( ) ( ) . median duration of ibrutinib treatment was months ( - ) and it was stopped one week before proceeding to allo-sct. it was not planned to restart btk inhibitor after transplant. patients were assessed for response after at least months of treatment with ibrutinib. at time of evaluation, all patients were in complete ( ) or very good partial response ( ) before allosct. the patient in partial response had % tumor reduction with persistent gastric ulcer where histology examination shows cd + but negative cycline d lymphoid cells. all patients engrafted (median duration of pnn o g/l = days ( - ) and median duration of platelets o g/l = days ( - )) with fulldonor chimerism at month. one patient had a grade ii cutaneous chronic gvhd (cgvhd) with favorable outcome and developed months later a bronchopulmonary obstruction syndrome related to cgvhd. with a median follow-up of months ( - ) after allo-sct, all patients are alive in cr. one patient, in complete metabolic response before transplant had a gastric relapse months later but achieved again a cr months after reintroduction of ibrutinib. after the first case reported by furtado m et al (leuk lymphoma ), we report here additional cases with longer follow-up after allogeneic transplantation. the excellent tumor control after treatment with ibrutinib together with a very good outcome after allosct should drive to consider this approach in young patients with mcl relapse after asct. disclosure of conflict of interest: none. autologous hematopoietic stem cell transplantation (autosct) is considered the standard approach for high risk or relapsed/ refractory non-hodgkin and hodgkin lymphoma. although a large variety of conditioning regimens are available, including the widely used beam (carmustine, etoposide, cytarabine, melphalan), there is no consensus regarding a standard approach. in the context of carmustine shortage, we have chosen to replace it by thiotepa. however, clinical data about thiotepa-based autosct conditioning are still sparse, except some retrospective data for primary central nervous system lymphoma. thus, we designed a multicenter prospective study (nct ) to assess the efficacy and toxicity of a team (thiotepa, etoposide, cytarabine, melphalan) conditioning regimen. team regimen consisted in total dose thiotepa of mg/kg on day- ; etoposide mg/m / h and cytarabine mg/m / h (day- to - ); melphalan mg/m on day- . patients underwent autosct with team conditioning, and were included in this analysis if they have fullfilled the following criteria: age older than years, biopsy-proven hodgkin or non-hodgkin lymphoma, hiv seronegative, and first autosct. thirty-three male and nine female with a median age of years (range: - ) were analyzed thus far. karnofsky score was g/l was days (range: - ). of note, patients received thrombopoietic agents after engraftment because of persisting thrombocytopenia. the most significant regimen-related toxicities were mucositis in % of patients (median grade = , range: - ) and diarrhea in % of patients (median grade = , range: - ). other non-hematologic grade adverse events occurred in patients ( %) and no grade adverse events were observed. central line-associated bloodstream infection occurred in patients ( %). surprisingly, / evaluable patients ( %) developed human herpesvirus reactivation. only patients required intensive care unit transfer. the median duration of hospital stay was days (range: - ). after a median follow-up of months (range: - ), the non-relapse mortality (nrm) was %. only one patient relapsed of refractory aitl months after autosct and died month after. the estimated -year overall survival and progression-free survival were % and %, respectively. a team conditioning regimen seems to be a safe and valid platform in autosct for patients with high-risk or relapsed/ refractory lymphoma. although mucositis and diarrhea were frequent, there were no grade adverse events and no deaths related to the treatment. updated results with updated followup will be presented. disclosure of conflict of interest: none. the cell of origin has no prognostic impact on high-dose chemotherapy with r-beam and autologous stem cell transplant for diffuse large b cell lymphoma s lozano cerrada, r saliba, s srour, s ahmed, c hosing, r champlin and y nieto university of texas, md anderson cancer center, department of stem cell transplantation and cellular therapy diffuse large b-cell lymphoma (dlbcl) is a biologically heterogeneous disease that can be classified according to its cell-of-origin (coo). the germinal center b-cell (gcb) subtype has better outcome with frontline r-chop than the activated b cell (abc) subtype. however, the prognosis of these two types of dlbcl after high-dose chemotherapy and autologous stem cell transplant (asct) is less clear. the purpose of our study was to evaluate progression-free survival (pfs), event-free survival (efs) and overall survival (os) in a cohort of dlbcl patients treated with r-beam (rituximab, carmustine, etoposide, cytarabine, melphalan) and asct according to coo. we have the dicep regimen effectively reverses the poor outcome for lymphoma patients with suboptimal response or failure post st salvage treatment p kaloyannidis the outcome of patients (pts) with refractory hodgkin's (hl) and non-hodgkin lymphomas (nhl) post st salvage treatment (salv ) is considered poor. the published data, have shown extremely low survival rates ( - %) even after nd salvage treatment (salv ) followed by autologous stem cell transplantation (asct), due to the low response rates post salv and the high relapse rates post asct, confirming that the management of these pts remains a major challenge. we herein evaluated the dicep regimen [dose intesified cyclophoshamide ( gr/m ), etoposide ( mg/m ) and cisplatin ( mg/m ), days - ] as a salv treatment, in terms of safety and efficacy regarding disease response and stem cell mobilization/collection. moreover, we evaluated pts' long term outcome post asct. we retrospectively analyzed the data of ( hl, nhl) pts, with a median age of ( - ) yrs. twenty-one had suboptimal response ( % reduction): and minor response (≤ % reduction): ). three pts had stable disease while experienced progression. overall / pts underwent asct after a median of days (range: - ) post dicep. no pt was considered ineligible for the asct due to unacceptable toxicity post dicep; did not undergo asct because of progressive (n = ) or stable (n = ) disease. the -yr overall survival (os) was % for the whole cohort of pts ( % for hl and % for nhl, p = ns) while the -yr progression free survival (pfs) from dicep administration (± asct) was % ( % for hl and % for nhl p = ns). in particular, for the autografted pts, the -yr os was % ( % for hl, % nhl p = ns) and the -yr pfs was similar, % ( % for hl, % for nhl, p = ns) our data demonstrate that dicep is an effective salvage regimen with acceptable toxicity and no negative impact on the cd + collection. the promising response rates post dicep in combination with the very encouraging pfs rates achieved post asct, in this unfavorable and heavily pretreated group of patients, strongly support the rationale for using dicep as st line salvage regimen in selected pts in order to proceed to a successful asct. for the treatment of aggressive lymphoma, high dose chemotherapy followed by autologous stem cell transplant (asct) is an important component. however, the role of upfront asct in patients with diffuse large b cell lymphoma (dlbcl) is still controversial. furthermore, there is currently no consensus on a single best conditioning regimen for asct in patients with dlbcl. we retrospectively analyzed the records of patients with dlbcl who underwent upfront asct in state of complete remission (cr) or partial remission (pr) from institutions in korea. we evaluated the outcomes and prognostic factors of upfront asct in patients with dlbcl. we compared the outcomes of most widely used two conditioning regimens for asct; carmustine based regimens and busulfan containing regimens. total patients ( . %) achieved cr after asct and overall response rate (orr) was . %. with median follow up of months, patients ( . %) had progression or relapse. the -year overall survival (os) rates and progression free survival (pfs) rates were % and %, respectively. infection events were found in patients ( . %) and treatment related mortality was . %. these outcomes were comparable with the results of previous other studies. cox multivariate analysis for os showed that eastern cooperative oncology group performance status (ecog ps) ≥ (p = . ) and rituximab based induction therapy (p = . ) were significant prognostic factors. in addition, the following factors were significantly associated with pfs in multivariate analysis; female (p = . ), ps ≥ (p = . ) elevated β -microglobulin (p = . ), failure to achieve cr with induction chemotherapy (p = . ), carmustine based conditioning regimen (p = . ) and melphalan based conditioning regimen (p = . ). there were no significant differences in os and pfs according to stage, b symptom, bulky disease, high lactate dehydrogenase, bone marrow involvement, high or high-intermediate international prognostic index (ipi), absolute lymphocyte count and absolute monocyte count. therefore, it is considered that upfront asct can overcome the poor prognosis in patients with advanced stage or high risk ipi. in the analysis with conditioning regimen, neutrophil and platelet engraftment were slower in the carmustine group compared to the busulfan group. there were no significant differences in os between busulfan group and carmustine groups with -year os rates of . % and . %, respectively (p = . ). pfs at years was . % in busulfan group versus . % in carmustine group (p = . ). however, carmustine based conditioning regimen was poor prognostic factors for pfs in multivariate [p ] s analysis (p = . ). in subgroup analysis, busulfan group had significantly higher pfs compared to the carmustine group especially in female patients ( . months vs. months, p = . ), with b symptom ( . months vs. . months, p = . ) and abnormal serum ldh level ( . months vs. . months, p = . ). the outcomes of upfront asct in patients with dlbcl after induction therapy were acceptable. it is considerable in selected high risk patients who achieve cr with induction treatment, and have good performance status at diagnosis. in cases of conditioning regimen, busulfan based regimen resulted in improved outcomes compared with carmustine based regimen especially in patients with disseminated disease or female patients. disclosure of conflict of interest: none. no heavy chain was present in %). the predominant light chain was kappa ( %). patients had bence-jones positive myeloma. received bortezomib as induction therapy before transplant. we analyzed overall survival (os) and progression-free survival (pfs) in groups of patients. we separated the groups according to improvement in grade of response from preasct to postasct. the post-asct grade of response was measured months after asct. the os and pfs were estimated by the kaplan-meier method. pfs was measured from diagnosis to disease relapse and os was measured from diagnosis to death by any cause. results by subgroups of patients are detailed in table . median os and pfs of the whole group was years and months, respectively. if we analyze groups only by their grade of response before asct we find the following results: rc ( years os rate . %, median pfs months); pr/vgpr (median os . years and pfs months); sd/progression (median os . years and pfs months). according grade of response after asct, instead: rc ( years os rate %, median pfs: months); pr/vgpr (median os , years, and pfs months); sd/progression (median os . years and pfs months). in our experience, the grade of response before asct is a capital predicting factor for patients os and pfs. patient in cr before asct that preserve it after transplant, have a median pfs of months, the years os rate being . %. patients in situation of progression after asct have a very dismal prognosis (median os . years, pfs: months), however, patients who change from sd/progression to pr after asct have a median pfs of months and a os of . years. comparing these results we observe that this second group is particularly benefited by transplant. autologous peripheral blood hematopoietic stem cell transplantation in elderly patients with multiple myeloma as a standard therapeutic procedure. is it feasible? a single-center experience l cadievski, s genadieva stavric, z stojanoski, a pivkova veljanovska, d miloska, b kocoski, l cevreska and b georgievski university clinic of hematology, department for hematopoietic stem cell transplantation, university ss. cyril and methodius, skoje, republic of macedonia autologous peripheral blood stem cell transplantation (pbsct) represents a standard therapeutic approach in the treatment protocol of myeloma patients. it is known that multiple myeloma is a hematological disease that is a characteristic for the older population. autologous pbsct ideally should be performed in every myeloma patient, but with the elderly myeloma patients the procedure might be risky if know the possible comorbidities, or the possibility of the body to fully compensate the side effects of the conditioning regimen, the procedure or its possible complications. we present our experience in using high dose conditioning with melphalan mg/m followed by autologous pbsct for elderly myeloma patients, using the age limit od years. our retrospective analysis of our data during years of experience, shows that we have performed autologous pbsct on patients with myeloma at the age of or older. males ( . %), and female ( . %). patients ( %), were diagnosed with igg type myeloma, patients ( %) with iga myeloma, and patient ( %) with light chain myeloma. median age of the patients was . years ( - ). all patients were initially treated with cy-thal-dex regimen. in ( %) patients complete response (cr) was achieved, in ( %) very good partial response (vgpr), and in ( %), partial response (pr). in all patients the mobilisation of hematopoietic stem cells was performed with g-csf, and a median of apheresis procedures were performed, and the average number of collected cells was . × /kg tt mononuclear cells (range: . - . ). days to confirmed engraftment in our group of patients was . (range: - ). the number of blood transfusions was on average . (range: - ), and the number of transfusion of thrombocytes . units (range: - ). in the majority of patients, mainly after the year (that represents patients of the whole group), we used noncryopreserved hematopoietic stem cells, kept under the temperature of c, for median of days, thus avoiding the toxicity of dmso. additionally, we used central venous catheter inserted in the femoral vein for apheresis and application of the stem cells afterwards. the day after, the catheter was removed, thus avoiding catheter associated infections. all patients received standard infectious prophylaxis with fluconasole mg/daily, ciprofloxacin mg/ two times daily, acyclovir mg/ three times daily, cefixime mg/once daily, and ursodeoxycholic acid for vod prevention. no serious infectious complications were reported. our transplant related mortality was %. in the group with noncryopreserved stem cells no graft failure was reported. in two patients we even performed tandem autologous pbsct with no major complications. of the group of patients, the majority, patients ( %), had hta as comorbidity, ( %) with cardiomyopathy, and ( %) with inserted prosthetic aortic valves. three patients ( %) have died because of relapse of the disease. our oldest patents were and years old, and are still alive year posttransplant, in cr. we can conclude the performing autologous pbsct in elderly myeloma patients can be safe and effective therapeutic option, but with careful selection of the patients, balancing the risk profile of the patient and the benefit, or the risk of the procedure. affective supportive care, monitoring and reducing the risk of complications is an imperative to a good result. disclosure of conflict of interest: none. autologous stem cell transplantation program for patients with multiple myeloma in an outpatient setting k lisenko , s sauer , g egerer , j schmier , m witzens-harig , a schmitt , ad ho , h goldschmidt , , j hillengass and p wuchter , department of medicine v, heidelberg university hospital, heidelberg, germany; national center for tumor diseases heidelberg (nct), heidelberg university, heidelberg, germany and institute of transfusion medicine and immunology, mannheim, german red cross blood service baden-württemberg-hessen, medical faculty mannheim, heidelberg university, germany the first and second authors contributed equally. high-dose chemotherapy with melphalan and autologous blood stem cell transplantation (absct) for treatment of symptomatic multiple myeloma (mm) is performed in the usa and canada mostly on an outpatient basis, whereas in germany and western europe an inpatient setting is the standard. we report on a german single-centre program to offer the procedure on an outpatient basis to selected patients. major inclusion and exclusion criteria for eligibility were defined as follows: patients had to be able to reach the hospital within minutes, had reliable support from their family at home, had an ecog performance score of - and were willing and able to comply with the demands of the program. patients with severe co-morbidities were not included. all patients were treated on our outpatients' clinic and examined on daily visits by a team of physicians. feedback from patients was obtained by means of a questionnaire. from september to september , patients with mm stage iiia were enrolled. all engrafted within the expected time range: (median time to leukocyte , /μl and neutrophil recovery μ/l: days; median time to platelet recovery /nl: days, /nl: days). twenty patients ( %) had an episode of neutropenic fever but only in patients ( %) blood cultures were found to be positive. there occurred no cases of infection with multiresistent bacteria. although rather liberal criteria for hospital admission were applied, of patients ( %) could be treated entirely on an outpatient basis. eight patients ( %) were temporarily admitted for inpatient treatment with a median duration of . days (range: - days), mainly because of neutropenic fever. no severe adverse events occurred. feedback from patients revealed a high level of satisfaction with the outpatient setting. high-dose chemotherapy and absct on an outpatient basis is safe and feasible if conducted in a comprehensive surveillance program. the feedback from patients was very positive, thus encouraging further continuation and expansion of the program. disclosure of conflict of interest: none. high dose of melphalan (bor-mel). we retrospectively analyzed patients with mm who underwent asct between january and march . in these patients, conditioning regimen consisted of a high dose of melphalan ( - mg/ m ) intravenously on day - and two doses of intravenous bortezomib at . mg/m administered on days − and + . this cohort was compared with patients underwent asct between and , conditioned with high dose of melphalan alone. response rate was evaluated according to imwg criteria. all patients were evaluated after induction therapy and months after asct. all patients were followed until death or reference date (november, ). results: patients' demographics and baseline disease-related characteristics are shown in table . [p ] no difference was found in terms of neutrophil and platelet engrafment, hospitalization days (p= . ) and use of mechanical invasive ventilation (p= . ). bor-mel regimen did not enhance severity of preexisting peripheral neuropathy (pn) in any patients, and only one presented de novo grade pn. non relapsed mortality was . % and % in the bor-mel and mel cohorts, respectively (p= . ). complete response rate after transplant was significantly better in the bor-mel cohort than in the mel cohort ( . % vs. . %; p= . ) ( figure b) . when the analysis was restricted to patients who received bortezomib-based therapy, this difference was also statistically significant ( . % vs. . %; p= . ) ( figure d ). median of follow-up was months in the bor-mel vs. months in the mel cohort. no difference was found in terms of overall survival (os) and progression free survival (pfs) between both groups. for all patients, a post-transplant deeper response was associated with better os and pfs (p= . and p o . , respectively). our results are in line with previous studies demonstrating that bortezomib combined with melphalan is a well tolerated conditioning regimen and may enhance the response rate after transplant, even in patients receiving bortezomib in the induction therapy. these results should be confirmed in a randomized trial. for newly diagnosed patients (pts) with multiple myeloma (mm), the triple-agent induction treatment based on bortezomib plus dexamethasone in combination with cyclophosphamide (vcd) or lenalidomide (vrd) represent extremely reliable regimens, which in combination with early autologous stem cell transplantation (asct) result in high response rates and prolonged long-term outcomes. however, though both regimens are widely used, there are extremely limited studies that compare the vrd vs. vcd in terms of safety and efficacy. in the present study we compared the outcomes of newly diagnosed mm pts who received induction treatment vrd (n = ) or vcd (n = ) and proceeded early to asct. the vrd and vcd pts groups were similar regarding age at diagnosis ( vs. ys, p = ns), interval between diagnosis-asct ( , vs. , months, p = ns) and maintenance treatment post asct ( vs. pts, p = ns). per revised international scoring system (riss), the vrd-group had slightly more advanced disease (stage i: , stage ii: and stage iii: ), compared to vcd-group (stage i: , stage ii: and stage iii: ), however this difference was not statistical significant. the conditioning regimen consisted of single agent melphalan: mg/m . the t-test, kaplan-meir and cox regression were utilized for the statistical analysis. following a median of cycles of treatment (range: - for vrd vs. - for vcd, p = ns), in the vrd-group pts achieved complete remission (cr), pts very good partial remission (vgpr ≥ % reduction of m-band) and pt partial remission (pr: - % reduction of m-band) while in the vcd-group cr: , vgpr: and pr: pts (p = ns). the toxicities in terms of peripheral neuropathy, myelosuppression, liver and renal function were well tolerated and no patient discontinued treatment due to severe side effects. the -yr overall survival (os) was % for the vrdgroup vs. % for the vcd-group; nevertheless, the difference was not significant due to the size sample of the pt groups. the stage at diagnosis, the disease status pre-asct and the maintenance post-asct did not influence the os. interestingly, the -yr progression free survival (pfs) was significantly superior for patients who had been induced with the vrd regimen ( % vs. % p = . ) and for patients who achieved cr or vgpr before asct (pfs: %) while no pts with pr pre-asct was progression-free yrs post asct (p = . ). in multivariate analysis, only the cr or vgpr status before asct favorably affected the long term pfs. our results are in line with the limited published data from other studies with larger series of patients. in our study, very low disease burden before asct proven to be an independent factor for prolonged pfs. taking into consideration that vrd resulted in more cr or vgpr status, it is reasonable to conclude that vrd is a highly effective regimen and could be first treatment choice for newly diagnosed mm patients who are fit for early asct post induction. lenalidomide cohort and in the maintenance bortezomib cohort. baseline characteristics and outcome data were obtained via chart review. the primary outcome was pfs. the secondary outcomes were overall survival (os) and treatment-related toxicities. the median follow-up time was months. median time to death ( . years vs . , p = . ) and median time to progression ( . years vs . , p = . ) were not significantly different in the maintenance lenalidomide cohort compared to the maintenance bortezomib cohort. in the multivariate analysis, pfs was worse in patients at international staging system (iss) stage at diagnosis compared to those at iss stages and (hr, . ; % ci, . to . ; p = . ) and worse in patients with less than very good partial response (vgpr) to last prior therapy compared to those with a response to prior therapy of at least vgpr (hr, . ; % ci, . to . ; p = . ) [see figure ]. pfs was improved in patients with more than two years of maintenance therapy compared to those with less than two s years of maintenance therapy (hr, . ; % ci, . - . ; po . ), but this result does not account for patients who ended maintenance therapy due to disease progression. os was worse in patients at iss stage at diagnosis compared to those at iss stages and (hr, . ; % ci, . to . ; p = . ). peripheral neuropathy was more common in the bortezomib cohort ( % vs %, p o . ), while cytopenias were more common in the lenalidomide cohort ( % vs %, po . ). figure kaplan-meier curve for pfs for the maintenance lenalidomide group versus the maintenance bortezomib group by log-rank test (p = . ). lenalidomide and bortezomib maintenance after transplantation have equal efficacy in prolonging progression-free and overall survival in patients with multiple myeloma. iss stage significantly affects time to progression and overall survival, and response to last prior therapy affects time to progression. length of maintenance therapy may be a significant predictor and warrants further analysis. these findings suggest that both lenalidomide and bortezomib are acceptable maintenance therapy options for post-transplantation multiple myeloma patients. autologous stem cells transplantation (auto-hct) is an accepted method in multiple myeloma (mm) patients, but usually it is not curative. the issue of allogeneic hematopoietic stem cells transplantation (allo-hct) is challenging yet for myeloma. we investigated allo-hct in mm and compared with auto-hct. in this retrospective study, we recruited patients from january to january ( ( . %) patients in autologous group and ( . %) in allogeneic group). we performed allogeneic hct with peripheral blood stem cells source in our center for patients who are relatively young (less than years old) with good performance, have match sibling donor and accepted allogeneic hct. the conditioning regimens in autologous group was melphalan mg/m only and in allogeneic groups was fludarabine mg/m plus melphalan mg/m in consequent days. gvhd prophylaxis consisted of methotrexate and cyclosporine. the outcomes then compared between two groups using log-rank and gray tests and cox proportional hazard regression. the median follow-up in the autologous and allogeneic group was . months. three years disease-free survival of auto-hct was . % (ci: . %, . %) and . % (ci: . %, . %) for allo-hct patients (p value = . ). three years overall survival of auto-hct was . % (ci: . %, . %) and . % (ci: . %, . %) for allo-hct patients (p value = . ) showing no significant statistical difference between two groups. mortality rate was . % for auto-hct and for allo-hct was . %. the most common cause of death between two groups was relapse of primary disease. three year relapse incidence was . % (ci: . %, . %) for allo-hct and . % (ci: . %, . %) for auto-hct (gray's test p value = . ). the three year trm incidence was . % (ci: . %, . %) and . % (ci: . %, . %) in allogeneic and autologous patients respectively (gray's test p value = . ). despite there was no statistically significant difference between two groups in terms of os but dfs and relapse incidence was meaningfully better in allogeneic group. so, perhaps the reason of non-significant os improvement in allogeneic group is higher early death due to higher trm. we suggest that this study needs longer follow up to see whether allo-hct resulted in os improvement. disclosure of conflict of interest: none. myeloablative allogeneic hematopoietic stem cell transplantation from unrelated donors for patients with relapsed or refractory multiple myeloma n tsukada , s shingaki, m ikeda, t ishida and k suzuki division of hematology, japanese red cross medical center allogeneic hematopoietic stem cell transplantation (allo-sct) for patients with multiple myeloma (mm) is increasing in number despite in the era of novel agents, especially as a second line treatment and beyond. it has been reported that allo-sct for patients with mm resulted in high incidence of treatment related mortality (trm). high incidence of disease relapse is also a major problem especially after reducedintensity stem cell transplantation (rist). it is an important issue to reduce the incidence of trm while preventing disease relapse. the use of stem cells from unrelated donors is required for those without hla-matched sibling donors. the purpose of this study is to evaluate the feasibility of an intensified conditioning regimen incorporating both mg/ m of melphalan and gy of total body irradiation (tbi), followed by allo-sct from unrelated donors for patients with relapsed or refractory mm. we retrospectively analyzed eight consecutive patients who received allo-sct from unrelated donors with the conditioning regimen including gy of tbi, fludarabine mg/m for five days, and melphalan mg/m between april and july at the japanese red cross medical center. six patients received unrelated bone marrow transplantation (bmt) and two patients received cord blood transplantation (cbt). graft-versus-host disease (gvhd) prophylaxis was consisted of tacrolimus and short term methotrexate. the median age at allo-sct, the time from diagnosis of myeloma to allo-sct, and the numbers of prior treatment lines were . years (range: - years), . months (range: - months), and . lines (range: - lines), respectively. five patients are female. no episode of either grade ≥ iii toxicity or non-relapsed mortality was documented during the median follow-up period of over two years. cumulative incidence of grade ≥ ii acute and severe chronic graftversus-host disease were . % ( % confidence interval [ci] . %- . %) at days and . % ( % ci . %- . %) at days, respectively. probabilities of progression-free survival and overall survival were . % ( % ci . %- . %) and . % ( % ci . %- . %), at years, respectively. the results suggest that allo-sct conditioned with this intensified regimen may be tolerable for patients with relapsed or refractory mm. disclosure of conflict of interest: none. the role of allogeneic stem cell transplantation (allosct) in the era of novel myeloma drugs remains controversial. it is the only curative treatment option but non-relapse mortality makes the decision making difficult as opposed to achievements with autologous sct and new mm drugs by which the median survival is nowadays nearing years. aim of this study was retrospectively evaluate the outcome of allosct for mm performed at our institute, including evaluation of factors affecting survival. all consecutive patients allotransplanted for mm between and were included. the data were collected from our transplant registry. frequencies and medians were produced as appropriate. kaplan-meier method was used to calculate os and pfs and log rank test for comparisons. univariate analysis for factors affecting survival was performed with cox proportional hazard model. median age of all patients was ( - ) years. half of the patients had igg myeloma, % had iss score (score available for patients), and % had high-risk cytogenetics (data available for patients). response to treatment at sct was at least vgpr in % of patients, and transplant timing was early (within months from dg) in % of patients. sibling donors were used in % and muds in % of transplants, and conditioning was ma for % and ric for another % of patients. acgvhd grade - occurred in % and grade - in % of patients; % of patients had extensive chrgvhd. posttransplant cr rate was %. % of pattients have relapsed after allosct, and % are alive with the median follow-up of , years. non-relapse mortality has been % ( % until , % since then). the median survival of patients up to age of years is . years vs . years for patients years (n = ) with survival plateau after years at % level. transplant period, cytogenetics, donor type, conditioning intensity or occurrence of chrgvhd had no statistical impact on survival. significant differences in os were observed between disease status at scr vgpr vs o months from dg vs later), grade of acgvhd - vs - , and best resonse post-transplant cr vs not less than cr. the respective differences for pfs were in sct timing, grade of chrgvhd, and best post-transplant response. in univariate cox regression analysis the only significant factors for os were severity of acgvhd and cr vs other responses after sct, and for pfs allosct timing, severity of chrgvhd, and best response to sct. with allosct ca. % of mm patients can be cured but at the cost of high non-relapse mortality. the occurrence of grade - acgvhd and less than cr response to sct predict poor survival. considering the increasing survival expectations with modern standard therapy for mm, allosct may be recommended for younger patients with high-risk features, and allosct should be done early in the disease course. disclosure of conflict of interest: none. angiogenesis plays an important role in the pathophysiology of hematological malignancies including plasma cell myeloma (pcm). microrna- (mir- ) is overexpressed and displays oncogenic activity in cancers. however, little is known about the role of mir- in pcm. the aim of the present study is to examine the expression level of peripheral mir- in pcm patients and to determine its role in angiogenesis. vegf serum levels and mir- in pbmcs was measured in patients with pcm directly before melphalan mg/m followed by autologous hematopoietic stem cell transplantation (auto-hsct) and months after hsct; and healthy controls. the study population was divided into two groups after therapy: responders (stringent complete response, complete response, very good partial response, partial response) and nonresponders (stable disease, progressive disease). gene expression of mir- was quantified by sybr green real-time fluorescent quantitative pcr. further tube formation of huvecs and vegf secretion was measured in mir- mimic or inhibitor transfected human plasma cell myeloma cell lines h and rpmi- . the expression level of mir- was significantly increased ( . ± . versus . ± . ; p o . ) in pbmcs of pcm patients compared with healthy controls. further, serum vegf levels were increased in pcm patients ( ± pg/ml versus ± pg/ml in normal controls; p o . ). after auto-hsct, the expression level of mir- was significantly different in responders compared to nonresponders. responders had a lower expression of mir- compared to non-responders. further, serum vegf levels decreased in responders to auto-hsct compared to nonresponders. vegf expression was increased in the supernatant from mir- mimic transfected human pcm cell lines h and rpmi- compared with the negative control, while s vegf was decreased in the mir- inhibitor transfected cell lines. the angiogenic ability of huvecs was increased under pretreatment with the supernatant from h and rpmi- cells transfected with mir- mimic compared with negative controls and decreased when pretreated with mir- inhibitor transfected cells (fig. ) . this study demonstrated that mir- was upregulated in pcm patients. responders to auto-hsct had a decrease of mir- expression and vegf levels. further, mir- regulated angiogenesis. therefore inactivation of mir- or activation of its target gene may be a potential therapeutic approach in pcm. fig. : in vitro matrigel tube formation assay. (i), normal control (ii, iii), mir- mimic transfected h cells (iv), mir- mimic transfected rpmi- cells (v), mir- inhibitor transfected h cells. original magnification × . disclosure of conflict of interest: none. [p ] pilot study of busulfan/thiotepa as conditioning regimen followed by allografting and post transplantation cyclophosphamide in advanced relapsed myeloma patients c wolschke, e klyuchnikov, d janson, m heinzelmann, m christopeit, f ayuk and n kröger university medical center hamburg-eppendorf despite the significant improvement in outcomes has been observed for myeloma patients, the disease still remains incurable. due to limitations, such as trm and gvhd, the role of allogeneic stem cell transplantation as salvage therapy in this setting remains unclear. in present pilot study we provide data on the use of post cyclophosphamide (ptcy) as gvhd prophylaxis after a busulfan/thiotepa based conditioning regimen in patients who relapsed after autologous stem cell transplantation. between / and / myeloma patients (male n = , female n = ) with a median age of years (range: - ) (pts), who relapsed after autologous stem cell transplantation received allogeneic stem transplantation with with ptcy as gvhd prophylaxis after busulfan ( . mg/kg for age y and . mg/kg for age years) and thiotepa ( mg/ m )and for haploidentical and mmud additional fludarabin ( mg/m ). all pts. were relapsed after one or two autologous stem cell transplantations. donors were haploidentical (n = ), mmud (n = ), mud (n = ) and hla-identical sibling (n = ). stem cell source was pbsc (n = ) or bm (n = ). all patients received cyclophosphamide mg/kg of body weight on day + and + , which was in pts (n = mrd, n = mud) the only gvhd prophylaxis, while patients with mmud and haploidentical donor received also cyclosporine a from day + and mmf (until day ) and patients (mrd and mud) received additional cyclosporine. we observed no primary or secondary graft failure. the median time for neutrophil and platelet engraftment was (range: - ) and days (range: - ), respectively. major toxicities grade and were: renal (n = ) and mucositis (n = ). major infectious complications were: cmv: n = cmv-reactivations (n = ), sepsis (n = ), pneumonia (n = ) rsv-(n = ) and hsv (n = ). acute gvhd grade ii to iv and ii/iv was noted in % and %, respectively and mainly seen in patients with cyclophosphamide as single gvhd prophylaxis. remission rate were n = complete remission, n = vgpr, n = partial remission, n = n.a. after a median follow up of months pts progressed and patients (n = relapse, n = trm) died. the year pfs was % (n = ). busulfan/thiotepa is an active conditioning regimen for advanced relapsed myeloma patients. post cyclophosphamide might increase anti-myeloma activity, but as single gvhd prophylaxis it causes significant agvhd in mrd and mud and additional immunosuppressive agents such as cyclosporine should be added. disclosure of conflict of interest: none. magnetic resonance imaging (mri) for multiple myeloma (mm) is a sensitive, non-invasive and non-toxic method for detecting myeloma lesions. the goal of the study was to assess whether quantitative mri metrics can detect treatment response and replacement of neoplastic cells by fat marrow. the study was hipaa-compliant and irb-approved. we retrospectively identified all patients who achieved a complete response (cr) after induction therapy between and . inclusion criteria for the study was total spine mri imaging at diagnosis and after achieving cr. cr was determined using the imwg criteria. spinal vertebrae t through l were outlined with imagej software. fractures and lesions were excluded. images were analyzed using histogram-based (entropy, skewness, kurtosis) and texture-based statistics. a two-sided t-test was used to compare quantitative mri metrics from before therapy and after achieving cr. cox regression was used to explore the association between progression free survival (pfs) and change in each quantitative mri metric based on a median split. pfs was defined as the time from the second mri to death or progression of disease. nineteen patients met the above criteria. median age was . years (range: . - . ). majority of patients ( %) were male. majority of patients had iss stage disease ( . %) and standard-risk cytogenetics ( . %). an induction regimen containing an imid and/or a proteasome inhibitor was commonly used ( . %). all patients received an autologous stem cell transplant (asct) consisting of high dose melphalan followed by autologous stem cell rescue. three patients received a planned second asct. seven patients ( . %) were in cr before asct. nine patients ( . %) were treated with imid maintenance after planned initial therapy. median time to repeat mri imaging after cr was months (range: . - . ). mean change in measurements of kurtosis, skewness, entropy and texture analyses are shown in table . no significant change was detected between preand post-cr mri. furthermore, no significant association was seen between the change in any quantitative metric and pfs. [p ] despite promising results by other groups, we could not find a significant association between quantitative t image analysis and cr or pfs. there was heterogeneity in the time of repeat mri imaging which may have limited our ability to study interval change. although no definitive conclusions can be made from this small sample, correlation between pfs and kurtosis or texture d may be promising and should be investigated in a larger group prospectively. multiple myeloma (mm) treatment (tx) has evolved in recent years. solid data on the impact of new tx on patient (pt) outcomes outside clinical trials, however, is lacking. this study aimed at investigating tx practices, pt journeys, and outcomes in the real-world in countries with different access to new tx. the study was conducted between / and / in bulgaria, croatia, czech republic, poland, romania, and slovakia. it consisted of a cross-sectional (x) and a retrospective (r) phase. for the x-phase, investigators included all symptomatic mm pts seen during a -week counting phase to provide a snapshot of where in the pathway pts were at a given moment. for r-phase, investigators collected data on current and past tx, including symptoms, dosages, administration schedule, tx durations, tx interruption, reasons for change/discontinuation, and tx response. pts were selected in reverse chronological order with a quota of a maximum of pts who completed first-line ( l) tx within the past months (mo), pts in second-line ( l) and pts in third or higher lines ( +l). pts included in the x-phase could also be included in the r-phase, if they met the respective inclusion criteria. in total, physicians included pts in the x-and physicians included pts in the r-phase. in the x-phase, % of pts were o , % were - , and % were years; the median time since diagnosis was mo. % of pts were currently undergoing tx, % were previously treated and % had never been treated. of currently-treated pts, % received l, % l, % l and % +l. in the r-phase, % of pts were o years. of pts receiving l, % continued to l, % to l, % to l and % to l. of the % of pts eligible for stem cell transplantation (sct), % ( = % of all pts) received sct at l; these proportions were similar across countries. the most frequently-used regimens in l and l were bortezomib-based ( % and %, respectively), in l and +l lenalidomide-based ( % and %, respectively). median duration of l was mo, followed by a median disease-free interval (dfi) of mo. median dfi was longer in pts with sct than in those without ( . mo vs . mo). time to progression (ttp) decreased with later tx lines, from median mo at l to mo at l. depth of response, as assessed by the treating physician, decreased with each additional line of tx: % of pts achieved at least very good partial response (≥ vgpr) in l, while only % achieved ≥ vgpr at +l. ttp was longer in pts with better response levels: in l, median ttp for pts with ≥ vgpr was mo versus mo for pts with mo for pts with ovgpr. the most common ( ≥ %, all grades) adverse events (aes) and co-morbidities in l were anemia ( %), thrombocytopenia ( %), neutropenia ( %), neuropathy ( %), and fatigue ( %). these aes disrupted treatment in % in l, % in l and % in +l. the study found that of sct eligible pts, only slightly more than half were transplanted. poorer outcomes and increasing ae incidence with each tx line highlight the challenges of mm tx. information on real-world pt management may be valuable for physicians to plan their tx strategies and can provide input for health economic evaluations of existing and new tx. disclosure of conflict of interest: daniel coriu declares to have received consulting fees or other remuneration (payment) from novartis, amgen, pfizer, takeda, janssen. ivan spicka declares to have received research grants from celgene, consulting fees or other remuneration (payment) from bms, takeda, celgene, janssen-cilag, and amgen, and to be a member of the speakers bureau of celgene, janssen-cilag, amgen, and bms. zdenka stefanikova declares to have received consulting fees or other remuneration (payment) from amgen, celgene, and takeda and be a member of the speakers bureau of amgen, celgene, and takeda. daniela niepel, krisztian szabolcs toka, and paul schoen are amgen employees and hold amgen stock. dominik dytfeld, and georgi mihaylov have nothing to declare. safety and efficacy of autologous stem cell transplantation in elderly patients with multiple myeloma t maia , c marini , p medeiros , e aguiar , j cancela pires , r bergantim , f trigo and je guimarães , hematology department, centro hospitalar de são joão and faculty of medicine, university of porto autologous stem cell transplantation (asct) is considered standard treatment for multiple myeloma (mm) patients under the age of years, but its safety and efficacy still uncertain for patients over this age. retrospective analysis from one single centre concerning mm patients under, equal or over years who underwent asct between january/ and july/ . it was also compared to - years old mm patients diagnosed in this period of time who were not transplanted. we analysed a total of patients, of which underwent asct. onehundred-and-six of the transplanted patients were aged years or less (median , iqr years), patients were aged more than years (median , iqr years) and patients were non transplanted (median , iqr ). the conditioning regimen for younger patients who underwent asct consisted mainly of melphalan mg/m (mel ) while half of the elder patients received melphalan mg/m (mel ). regarding transplant-related myelotoxicity there were no statistical differences between patients aged years or less and over years old, however the first group needed less days of g-csf (p = . ). non-hematopoietic toxicity measured by infections and mucositis was not influenced by age. patients years conditioned with mel had more days of aplasia (p = . ), greater need of g-csf (p = . ) and transfusional support (p = . ) than patients ≤ years. there were no differences on non-hematopoietic toxicity. in the elderly group, patients conditioned with mel presented more aplasia days (po . ), higher grade of mucositis (p = . ) and more days of iv antibiotics (p = . ) than those transplanted with reduced dose of melphalan. comorbidities had no effect on transplant-related toxicity, either by age or by dose of melphalan. days of hospitalization and post-transplant complications did not differ according to age group. transplant related mortality was % at day posttransplant. survival after transplant in patients years old or under vs older patients (median follow-up time, months), was not influenced by age (os, mo vs mo, p = . ; pfs, mo vs mo, p = . ). regarding the non-transplanted elderly group, these are patients with more renal disease (p = . ) and poorer performance status (p = . ) than the transplanted cohort. there is also higher cytogenetic risk (p = . ). induction regimens were similar in transplanted group and non-transplanted group years old, and response to first line therapy (before asct of transplanted group) revealed no differences. infections were the most common complication in both groups. transplanted patients needed less days of hospitalization (p = . ). comparing the long term outcome of these two groups, survival curves of the elderly patients transplanted were clearly superior to the nontransplanted (os, mo vs mo, p o . ; pfs, mo vs mo, p o . ) although one has to consider that the non-transplant group has worse features than the elderly transplant group. transplantation in the elderly still debatable but this study shows that it might bring benefit. globally, transplant related toxicity is not influenced by age. regarding dose of melphalan, higher dose in elderly patients has higher toxicity, without apparent benefit in survival. therefore, age should not restrict the access to asct, but instead selection must be based on individual clinical and functional status. disclosure of conflict of interest: none. second autologous stem cell transplantation for relapse after allografting in multiple myeloma using cd + selected donor cells without immunosuppression p novak number of patients receiving a second allogeneic stem cell transplantation (sct) in europe is increasing despite high treatment related mortality (trm). in multiple myeloma only very few reports of second allogeneic sct exist with limited number of patients and substantial mortality. while in most hematological malignancies, the donor cell chimerism is dropping down if patients are relapsing, in myeloma donor cell chimerism remains complete despite relapse. to reduce trm we thought that full donor cell chimerism may allow us to perform a second high dose busulfan based chemotherapy followed by "autologous transplantation" after stem cell mobilization and collection. however, because two consecutive patients failed to collect sufficient cd + cells for an autologous transplantation even with plerixafor, we used donor t cell depleted cd + selected cells and transplanted those patients in an "autologous" fashion without any immunosuppression. to enhance graft-versus-myeloma effect, we added donor lymphocyte infusion (dli) at day . we report here on myeloma patients with a median age of years (range: - ) who relapsed after allogeneic sct and underwent a second "autologous" sct with cd + selected donor cells. all patients had received one (n = ) or two (n = ) autologous sct before . allografting. patients received an upfront auto-allo protocol and patients received . allogeneic sct as a salvage therapy. % of patients received a reduced intensity melphalan based conditioning regimen for . allogeneic sct and the median pfs was months (range: - ). before . allograft patients had received overall a median of (range: - ) treatment lines. at the time of . allogeneic sct all patients had a full or nearly full donor cell chimerism and remission status was very good partial remission (n = ), partial remission (n = ), stable disease (n = ), progressive disease (n = ). % of patients received a myeloablative busulfan based conditioning regimen and all received cd + selected stem cells with a median number of . × /kg cd + cells (range: . - . ) and × /kg cd + cells (range: . - ). engraftment was noted in % at a median of days (range: - ). no further graft-versus-host disease (gvhd) prophylaxis was performed and no acute gvhd (agvhd) was observed. according to treatment plan, patients received escalating dli around day + , starting with a median dose of × /kg (range: . - ) in combination with lenalidomide maintenance in patients. patients experienced agvhd ii-iv after dli. two patients had a severe gvhd (grade iii) which resolved completely after steroid therapy. no nonrelapse mortality after sct and dli was observed. after a median follow up of months (range: - ) the median pfs was months (range: - ) which translates into a pfs for all patients of % at year and % at years. median os was months (range: - ) and an os of % at years and % at years was observed. for patients with advanced multiple myeloma relapsing after allografting, a second "autologous" sct with cd + selected donor cells without immunosuppression followed by dli is an encouraging treatment option with low toxicity. disclosure of conflict of interest: none. second autologous stem cell transplantation as treatment option for relapsed patients with multiple myeloma: a single center experience (cic ) p ganeva, y petrov , m mincheff , i tonev , m guenova, l gartcheva , a michova , g arnaudov and g mihaylov ya. petrov; m. mincheff; i. tonev; l. garcheva; a. michova; g. arnaudov and g. mihaylov the use of modern therapies such as bortezomib, lenalidomide, thalidomide coupled with upfront high-dose therapy and autologous stem cell transplantation (asct) has resulted in improved survival in patients with newly diagnosed multiple myeloma (mm). the role of second asct as salvage therapy for relapse is unclear because of the availability of new agents to treat progression in multiple myeloma (mm). as the treatment options for management of patients with relapsed mm has become increasingly complex, physicians must consider both disease-and patient-related factors when choosing the appropriate therapeutic approach, with the goal of improving efficacy while minimizing toxicity. we retrospectively reviewed all mm patients who received a second asct as salvage therapy at our center from to december . for this period we performed transplants for mm patients. twenty five ( . %) patients received second asct ( patients were relapsed) and for patients asct was performed as tandem transplant. we analyzed only second asct for relapse. the median time to relapse after first transplant was . months (range: - months). all patients received reinduction therapy before the second asct. conditioning was performed with melphalan with two different doses ( mg/m and mg/m ). the median age at second transplant was . years (range: - years), and female/man ratio was / . median interval between first and second asct was . months (range: - months). we have no observed early deaths. until now ( %) patients are dead because of progression disease. response rate was assessed three months after asct, nine ( %) patients achieved vgpr, three ( . %) patients achieved at least a partial response, three ( . %) had sd and three ( . %) progressed despite salvage asct.median overall survival (os) was . months ( relapse ≥ months = . ; ≤ months = ). second asct is a feasible and safe option for salvage therapy in mm, especially in bulgaria where the possibility of using novel agents such as carfilzomib, lenalidomide, daratumomab for relapsed patients is limited to clinical trials, because of no reimbursement. the best results were observed in patients whose time to progression was more than months after first asct. advances in treatment of multiple myeloma (mm) has improved overall survival in these patients (pts). a steady increase in the risk of secondary malignancies has been reported over the last decades in mm survivors. estimated incidence of secondary acute myelogenous leukemia or myelodysplastic syndrome (t-mds/aml) after treatment with alkylating agents is %- . % per year - years after primary chemotherapy. no specific risk factor has been recognized, but genetic instability, natural history of the disease as well as induction therapy and autologous stem cell transplantation (hct) have been implicated. recently, novel anti-myeloma treatments have been linked with an increase in secondary malignancies, but no solid relationship has been established yet. in a retrospective study, we analyzed the incidence of secondary malignancies (t-aml/mds and solid tumors) in patients suffering from mm who had undergone autologous hct using high-dose melphalan conditioning regimen in our bmt unit. study population consisted of consecutive pts with median age of years ( - ), . % of them being male, who were transplanted during a period of years . type of myeloma was igg/a/d in %, . % and . % respectively, while . % was light chain and % nonsecretory. the majority of pts presented with k light chain myeloma ( . %). there was almost equal distribution between iss stages i and ii ( %/ . %) and only . % were diagnosed with advanced stage myeloma. most pts received two lines of chemotherapy ( %) and all of them more than one. treatment regimens before autologous hct included vad ( pts), bortezomib-based ( pts), dcep ( pts) and rd ( pts) and pts received radiotherapy. chemotherapy administration for mobilization was used in pts ( . %). conditioning regimen before autologous hct consisted of high-dose melphalan ( mg/m ) and in case of renal insufficiency mg/m . incidence of a secondary malignancy was . % after a median follow up period of months. t-aml /mds was diagnosed in ( . %) pts and ( . %) were diagnosed with breast and lung cancer respectively. pts diagnosed with secondary malignancy were previously exposed in induction therapy to melphalan ( ), vad ( ), bortezomib ( ), high-dose cyclophosphamide as mobilization treatment ( ) and radiotherapy ( ) . cytogenetic analysis was available in patients diagnosed with t-mds/aml and the majority ( / ) presented complex karyotype. abnormalities mainly observed were deletions and insertions in chromosomes , , . pts with secondary malignancies had an overall survival of months ( - ), however, after malignancy diagnosis, survival was very poor, four months only . secondary malignancies in pts with multiple myeloma after autologous hct occur with a substantial frequency and have a dismal prognosis. the role of novel treatment agents has to be elucidated. further studies are needed to identify new risk factors and develop better surveillance strategies. [p ] disclosure of conflict of interest: none. survival analysis after allogeneic hematopoietic stem cell transplantation in patients diagnosed with multiple myeloma: a single center experience p patricia hernandez* , r maria calbacho , a laura posada , g fabio augusto ruiz , r anabelle chinea hospital universitario nuestra señora de candelaria, santa cruz de tenerife (spain); hospital universitario ramón y cajal, madrid (spain) and hospital universitario cruces, barakaldo (spain) allogeneic hematopoietic stem cell transplantation (allohsct) may provide long term remission cures for patients diagnosed with high-risk multiple myeloma. however, its use is limited since it has a high rate of treatment-related mortality (trm), and because its efficacy compared to autologous hsct is not fully established. we studied patients that underwent allohsct between - . population characteristics are in table . all patients were treated at least with one prior therapy lines ( ) ( ) ( ) ( ) ( ) , all including autohsct ( . % underwent prior autohsct). % had or prior therapy lines. of them received bortezomib as part of treatment regimens. donor characteristics: non-related; hla-identical. gvhd prophylaxis: methotrexate plus a calcineurine-inhibitor: cyclosporine and tacrolimus. median follow-up . months ( . - . ), average was . months. seven patients died ( . %); because of progression ( . %), and ( . %) due to trm, including infections and immediate complications of transplantation, such as toxicities, icu admission and agvhd: infections: cmv reactivations, invasive fungal and bacterial infections. disease status: patients were in cr prior to allohsct. of them maintained it after. remaining patients died before disease was evaluated. seven patients were in pr prior to transplant, and reached cr after allohsct. one had progressive disease and reached cr after the procedure. two had stable disease and progressed after allo; one of them is in cr after additional therapy lines, and the other one died months after due to it. donor characteristics: hla-identical sibling donors: . % ( hla-mismatch, passed away . months after allo due to trm). one of the nonrelated donors, had an hla-mismatch, and died months after allohsct due to trm, the other one is alive after months. gvhd: ( . %) developed agvhd and of them maintained it chronically. two suffer from cgvhd, plus that initiated it as agvhd. were refractory to steroids. longterm survivors: patients had overcome three years after allohsct. they were among and years old at the time transplant was performed. none of them received bortezomib as part of therapy protocols for the disease. all had therapy lines prior allograft. were submitted to prior autologous hsct. relapse: patients relapsed after allohsct ( %, median time to relapse . months), being alive % at the end of the study. allogeneic hsct is associated with a high incidence of nrm and a low incidence of relapse. rates of acute and chronic gvhd are high. in our cohort, besides that more than % are alive until now, they suffer from extensive chronic gvhd and are in need of treatment. long-term survival may be related with patient factors such as young age, but also low tumor burden, or less prior therapy lines; in our group there are no differences in this aspect. studies including high-risk abnormal cytogenetics should help to define which patients are best candidates to allohsct. high-dose melphalan followed by autologous haematopoietic cell transplantation (ahct) remains the standard of care for eligible multiple myeloma (mm) patients. the majority of patients in clinical practice and trials receive a melphalan dose of mg/m (mel ), but a reduced dose of mg/m (mel ) is often used in patients perceived to be unable to tolerate mel . it remains to be determined whether this considerable dose difference results in different clinical outcomes. we therefore analysed patients with mm who underwent a first single mel or mel -conditioned ahct between january and december . all patients were included in the calm study, an analysis of a prospectively defined cohort of patients with data reported retrospectively to the ebmt, covering ahcts for mm and lymphoma. patients in the mel group were older than patients in the mel group at the time of ahct (median years [range: - ] vs years ; p o. ). compared to the mel group (n = , . %), fewer patients in the mel group (n = , . %) were overweight or obese ( . % vs . %; p = . ). compared to the mel group, more patients in the mel group had received proteasome inhibitor-containing induction therapy ( . % vs . %; p = . ), had a karnofsky score of ≤ ( . % vs . %; p = . ), and were transplanted in less than pr ( . % vs . %; p = . ). overall survival (os) from the time of ahct was not significantly different between the mel and mel group ( - significantly different between mel and mel ( days in both groups for neutrophil recovery; vs days for platelet recovery). however, late neutrophil recovery was noted in a small proportion of patients in the mel group. neutrophil recovery days post ahct was not observed in any engrafting patient in the mel group, but occurred in ( . %) engrafting patients in the mel group (p = . ). a cox proportional hazards model that included melphalan dose, age, and remission status at ahct showed that melphalan dose had no effect on os, pfs and relapse risk. the findings suggest that mel is not inferior to mel in younger and older mm patients and may reduce the risk of delayed haematological recovery in some patients. further analyses in relevant subgroups such as patients with high-risk features or renal impairment are required. disclosure of conflict of interest: none. high-dose therapy (hdt) followed by autologous stem cell transplantation (asct) remains the standard of care for patients younger than years of age with multiple myeloma (mm). different agents are being used to control the disease before asct, including the older thalidomide based combination or the newer bortezomib and lenalidomide based combination. the relation between the initial induction regimen and outcomes after asct is not completely clear. to evaluate the effect of different induction regimens on asct outcome, we retropsectively evaluated the outcomes of a low cost older regimen of thalidomide based combination in doublets or triplets with newer novel agents like bortizomib or lenalidomide based combination in a low resources country in transplant-elegible patients with multiple myeloma who underwent autologous stem cell transplantation at king hussein cancer center bmt program we retrospectively reviewed the files of patients diagnosed with mm from january till december , who received induction treatment followed by hdt and asct and followed up in a single institution. we compared the effects of different induction regimens, disease stage, and remission status before transplantation on over-all survival (os), event free survival (efs) and progression free survival (pfs) using kaplan meier curves. a total of patients were included, ( . %) of them received thalidomide based induction (group ) and ( . %) received bortezomib and lenalidomide based induction (group ). patients also offered no consolidation nor maintenance therapy. ( . %) patients were stage i, ( . %) stage ii and ( %) were stage iii. stage was not documented for ( , %) of cases. ( . %) were in complete remission (cr) and ( . %) were in partial remission (pr). the estimated -year os for the whole group was . %. there was no statistically significant difference between both groups in regards to initial iss stage of disease (p = . ) or cr status before asct. patients ( . %) in group achieved complete remission ( cr ) or very good partial response (vgpr), while ( . %) patient in group achieved cr or vgpr. there was no statistically significant difference between group and group in -years os ( -year os was % vs %, p = . ), efs ( . % vs . %, p = . ) or pfs ( . % vs . , p = . ). the use of an old, low-cost, thalidomide-based regimen in a low-resources country achieved a favorable transplantation outcomes in patients with multiple myeloma who received hdt and asct. double autologous stem cell transplantation (asct) is a useful treatment for multiple myeloma (mm) patients. we can make the second asct ( asct) without reinduction treatment (tandem regimen) or after a reinduction treatment after first asct ( asct) relapse (salvage regimen). we have conducted a retrospective study over mm patients undergoing a double asct performed in our centre from to . we have compared the different conditioning regimens used, and if there are any difference between tandem or salvage asct. we do not use maintenance treatment systematically. characteristics of patients and conditioning regimens in table . the overall survivals (os) of our patients are months (m) from treatment start till last control. the most important prognostic factors are the duration of the progression free survival (pfs) after asct (hr: . ( . - . ); p = . ), and the use of bumel like conditioning regimen at the asct or at the asct vs another conditioning regimens (hr: . ( . - . ); p = . ). today there are patients alive ( %), but only ( %) are free of mm now. the patients who were treated with tandem have a little better os than salvage patients ( m vs m; p = . . not significative). patients at tandem group who received different conditioning regimen at the asct and at the asct live more time than patients treated only with melphalan (mel ) at both asct. at salvage group the duration of pfs after asct is better than the pfs after asct ( m vs m). the use of the same conditioning regimen at the both asct has worse results than if we use different treatment. patients who were treated with s bumel at the asct or asct have better os than patients treated with cbv or mel . patients who not responded to reinduction treatment before asct have worst pfs after asct (rc: m, response; m and not response; only m). attention is drawn to the fact that patients who received bumel at asct have large os, but they are very few ( ) patients. only one patient has died during the asct, and was a patient of salvage group treated with bumel. double autologous transplantation continues to be a useful treatment despite the new mm treatments, and allows to prolonged the os. tandem asct probably is a useful treatment in high risk mm patients. salvage treatment is most useful in patients with a large pfs after asct, and good response to reinduction treatment. although mel continue to be the standard conditioning regimen for asct in mm patients, we have observed that patients treated with different conditioning regimen at asct and asct have better prognostic, and bumel has the best results in our serie. disclosure of conflict of interest: none. allogeneic haematopoietic stem cell transplantation (allo-hsct) is an effective treatment for myelodysplastic syndrome (mds) and acute myeloid leukemia (aml). the prognosis of elderly patients with mds and aml after chemotherapy is poor. allo-hsct is feasible in these patients; however the management of elderly patients with mds and aml for allo-hsct is difficult. we performed a retrospective survey of allo-hsct for elderly patients with mds and aml in our institution. we retrospectively analyzed the records of elderly patients ≥ years with mds and aml who underwent allo-hsct in our hospital between january and december . in this study, we assessed the ipss-r (revised international prognosis scoring system) cytogenetic score and the ipss-r score against the outcome of elderly mds and aml patients who treated with allo-hsct. fifty-one elderly patients with mds and aml were treated with allo-hsct in our institution, patients with mds ( with mds overt aml) and with de novo aml. ages ranged from to years (median ), patients were female and were male. there was a history of malignant disease in patients. according to the ipss-r cytogenetic scores of mds patients, patients fell in the good risk group, were in the intermediate risk group, were in the poor risk group, and were in the very poor risk group. regarding the ipss-r score, patient fell in the low risk group, in the intermediate risk group, in the high risk group, and in the very high risk group. sixteen patients were in st complete remission (cr), patient was in nd cr, patients were in partial remission, and patients were not in remission (nr) upon administration of allo-hsct. all patients received a reduced intensity conditioning regimen. patients [p ] were treated with fludarabine (flu), melphalan and low dose tbi-containing regimens; patients were treated with flu, intravenous busulfan and low dose tbi; and one patient was treated with flu, cyclophosphamide and low dose tli. graftversus-host disease (gvhd) prophylaxis consisted of tacrolimus plus methotrexate in patients, and tacrolimus, methotrexate and mycophenolate mofetil in patients. thirty-four patients received anti-thymocyte globulin (atg). the donor source was sibling bone marrow (bm) in patient, sibling peripheral blood stem cell in , unrelated bm in and unrelated cord blood in . relapse-free survival (rfs) and overall survival (os) were . % ( % confidence interval (ci): . - . %) and . % ( % ci: . - . %) at year, . % ( % ci: . - . %) and . % ( % ci: . - . %) at years, respectively (figure .) . in this study, patients died before engraftment. non-relapse mortality (nrm) was . % at day . twenty-five patients developed chronic gvhd ( patients limited and extensive). the causes of death were disease progression ( patients), treatment-related mortality ( patients), infection ( patients) and other causes ( patients). we suggest that many elderly allo-hsct patients with mds and aml were in the very poor risk group when the ipss-r cytogenetics score was assigned, in the very high risk group when the ipss-r score was assigned and nr upon administration of allo-hsct. rfs and os were . % and . % at years, respectively. there is a need for novel treatment strategies to manage elderly mds and aml patients for allo-hsct. [p ] disclosure of conflict of interest: none. counting bone marrow blasts as a percentage of nonerythroid cells provides superior risk stratification for mds patients with erythroid predominance a sun , y yu , t zhang , q wang , d liu and s chen the first affiliated hospital of soochow university, jiangsu institute of hematology, suzhou, china patients with erythroid predominance (≥ % erythroblasts, mds-erythroid) compose a significant proportion of patients with mds. the erythroid/myeloid subtype was divided from the aml category into mds-erythroid by the who classification of myeloid neoplasms. at that time, there was no consensus on a more appropriate way of enumerating bone marrow (bm) blasts from tncs or necs in mds-erythroid patients. to clarify these questions, mds patients were retrospectively analyzed in our center. mds-erythoid was observed in . % of patients ( / ), and these patients had similar clinico-pathological features and overall survival, with cases of mds with o % encs. by calculating the percentage of bm blasts from necs, of patients ( . %) with mds-erythroid who were diagnosed within who subtypes without excess blasts (eb) were moved into higherrisk categories and showed shorter os than those who remained in the initial categories (p = . ). recalculating the international prognostic scoring system-revised (ipss-r) by enumerating blasts from necs, of ( . %) mdserythroid patients with relatively lower risk were re-classified as higher-risk and had significantly poorer survival than those who remained in the lower-risk category (p = . ). this was especially true for the intermediate risk group that was stratified by ipss-r (unchanged patients vs. shifted patients, p = . ). however, the impact of enumerating bm blasts from necs on classification and prognostication was not evident in all mds patients. in conclusion, our results suggested that enumerating the percentage of bm blasts from necs significantly improved the prognostic assessment of mds-erythroid, especially for patients within the intermediate risk group stratified by ipss-r. disclosure of conflict of interest: none. myelodisplastic syndrome (mds) is a group of clonal and heterogeneous diseases, characterized by ineffective hematopoesis. the incidence of mds is about % of all blood disorders in children, approximately % of them develops acute leukemia. allogeneic hematopoietic stem cell transplantation (allo-hsct) is effective curative treatment of mds in children, but depends on disease status, type of clonal chromosomal abnormalities presented at the time of allo-hsct and graft quality. the aim of this study: to analyze the influence of graft quality on the outcome of childhood mds after allo-hsct. allo-hsct were performed in patients (pts) p hypomethylating agents vs. allogeneic sct in elderly patients with advanced myelodysplastic syndromes: a single center study j cermak, a vitek, m markova-Šťastná, j soukupova-maaloufova and p cetkovsky institute of hematology and blood transfusion, prague, czech republic a group of patients older than years of age with myelodysplastic syndrome (mds) raeb ii or with acute myeloid leukemia with multilineage dysplasia with less than % of bone marrow blasts (mds raeb-t according to the fab classification) was treated with hypomethylating agents (hma) and the results were compared to those obtained in an age and diagnosis matched group of patients who underwent allogeneic stem cell transplantation (sct). in the hma group, patients received azacytidine (vidaza) in the dose of mg/ m × every days and patients were treated with decitabine (dacogen) in the dose of mg/m × every days. median number of cycles administered was . (range: - ). in the transplanted group, patients were transplanted upfront and patients were pretretated with combination chemotherapy, patients received myeloablative conditioning and patients were transplanted after reduced conditioning regimen. a hematologic response to hma (cr,pr, hematologic improvement) was observed in patients ( . %), cr was achieved in patients ( . %). in sct group, engraftment was achieved in out of patients, patients died after sct ( on complications related to sct, patients relapsed). no difference in year survival was observed between both the groups ( . % for hma vs. . % for sct), however, median overall survival (os) was . months in hma treated group compared to . months in sct group (p = . ). in a recent analysis performed at months after starting the treatment, patients treated with hma ( . %) and transplanted patients ( . %) were alive, patients in hma group and patients in sct group relapsed. estimated years survival was . % in sct group and only . % in hma group (p = . ). no significant differences in results and adverse effects of treatment were observed between patients aged - years and those older than years in both hma and sct groups. our results confirm previous observations showing that despite a promising effect of hma resulting in hematologic response in more than % of elderly patients with advanced mds, allogeneic sct still represents the only potentially curative treatment connected with long-term survival in a significant number of patients even in this mds patients subgroup. disclosure of conflict of interest: none. immunophenotypic assessment of erythroid dysplasia by a simplified cocktail in myelodysplastic syndromes in taiwan c-c li , p-f weng , c-t lin , j-l tang hypomethylating agent (hma) is commonly used as a bridge therapy to prevent leukemic transformation prior to selection of a donor for allogeneic stem cell transplantation (sct) in patients with myelodysplastic syndrome (mds), and showed low toxicity. although its roles are known, the underlying genetics and clonal dynamics upon hma treatment has not been systematically examined using serial samples, especially in allogeneic stem cell transplantation (sct) setting. in this study, we performed targeted serial sequencing on bone marrow samples from mds patients treated with hma for bridging of allogeneic sct. to perform targeted deep sequencing, bm mononuclear cells before hma treatment and, and fractionated t-cell samples (cd + fraction) as controls were taken before hma treatment. analysis of genetic mutations were performed using targeted resequencing by illumina hiseq (sureselect custom probe set targeting [p ] entire exon regions of a myeloid panel consisting of genes). all patients received hma (decitabine: , azacitidine: ), and the median number of cycles was four (range: - ). the overall response rate for hma pre-treatment was %: there were four cases of complete remission (cr) ( %), six cases of marrow cr ( %), and two cases of hematologic improvement ( %). targeted sequencing revealed mutations in patients ( / , %) with median of mutations per patient (range: - ). mutated genes were then grouped into biological pathways, defined in the cancer genome atlas (tcga) aml study. the most frequent biological pathway at diagnosis was dna methylation ( %), followed by activated signaling ( %), chromatin modifiers ( %), tumor suppressors ( %), spliceosome ( %), cohesin complex ( %), npm ( %), and myeloid transcription factors (tfs) ( %). when assessing the difference in pattern of variant allele frequency (vaf), we found the significant reduction of vafs in four ( %) patients after hma. with a median follow-up of . months, -year overall survival (os) were . % ( % ci, . - . ). there was no significant difference in os according to the presence of mutations in each biological mutational pathway (all, p . ). to identify prognostic value of mutational dynamics, we subclassified the change of variant allele frequencies (vafs) after median fourth cycles of hma [no mutated or reduction of vafs ( patients) vs. stable or increased ( patients)]. however, there was no significant association between the dynamic of mutation and os (p = . ). these data show that hma using as bridge therapy for allogeneic sct in mds patients is insufficient to achieve the sufficient molecular responses and, mutational pathway and dynamics may not prognostic in this clinical setting. to clearly demonstrate the role of hma pre-treatment in mds, systematic assessment on a larger cohort is necessary. disclosure of conflict of interest: none. any role of high-dose chemotherapy in mediastinal nonseminoma germ cell tumors? p pedrazzoli, s secondino, a necchi , f lanza and g rosti istituto nazionale tumori, milano and ospedale santa maria delle croci, ravenna among germ cell tumors, primary mediastinal nonseminoma germ-cell tumors (pmnsgcts) have the poorest outcome with -year overall survival ranging from to %. indeed, the presence of mediastinal location defines per se a "poor prognosis" category according to the igcccg classification. this clinically and biologically distinct disease entity is associated with lower complete response rates to chemotherapy (ct), high rates of relapse and disappointing results from salvage ct. current standard first line treatment for patients with mediastinal primary location is still four cycles of peb, as for all igcccg poor-prognosis patients. we have reviewed available data present in the literature, including recommendations and expert opinions, on the use of high-dose chemotherapy (hdc) with autologous stem cell support in pmnsgcts. the use of hdc as both early intensification (that is, first-line setting) and at disease recurrence (salvage setting) have been reported in small cohorts of patients. according to the largest retrospective comparison, it has been suggested that hdc, given up-front, may produce a % to % absolute improvement in survival compared with standard dose ct. studies of the ebmt suggest that responsive disease after induction therapy may have a better outcome. mediastinal primary had salvage rates by hdct of less than % based on an international multicenter analysis and an ebmt study. the use of hdct in pmnsgcts warrants further investigation, preferably with the use of modern hdct strategies (that is, multiple carboplatin and etoposide courses). while hdc cannot be routinely recommended in pmnsgcts, selected patients with chemosensitive disease may benefit from early intensification. a retrospective analysis evaluating the large ebmt database is ongoing; results will be presented at the meeting. disclosure of conflict of interest: none. high dose therapy and autologous stem cell transplantation in gynaecological malignancies: a monocentric retrospective study m nderlita , i vergote and d dierickx university hospitals leuven, department of gynaecology; university hospitals leuven and department of hematology high-dose chemotherapy (hdt) followed by autologous stem cell transplantation (asct) has been established as a treatment option in many relapsed hematologic malignancies. however, in spite of many small trials, there still is no proven role for this treatment in solid tumors including most gynaecological epithelial carcinomas. however, in some recurrent non-epithelial ovarian cancers, such as sex cord stromal tumors, germ cell tumors, neuroendocrine gynaecological tumors and gestational trophoblastic disease, some studies suggest a possible role for hdt followed by asct. we performed a monocentric retrospective descriptive analysis of all patients diagnosed with gynaecological malignancies and treated with hdct followed by asct in our center. clinical, laboratory, pathological and imaging data were collected and analysed, together with information on treatment and outcome. eleven patients were included in this analysis, with a median age of years (range: - ) at time of diagnosis. eight patients suffered from ovarian neoplasia. at time of diagnoses patients showed metastatic disease. first line therapy consisted of surgery (n = ), chemotherapy (n = ) or a combination of both (n = ). median time to progression after first line therapy was . months (range: - ) with a median time between primary diagnosis and start hdt of . months (range: - ). three patients underwent single ast, whereas the other patients had a tandem ast, with a median time of months between first and second hdt (range: - ). treatment related toxicity was manageable, although there was treatment-related death. at last follow up patients ( %) were still alive with a median follow up of . years (range: . - . ) after last asct for all patients. of the deceased patients died with progressive disease. although the number of patients is very small, this retrospective study shows that hdt and asct is feasible in heavily pretreated patients with relapsed/refractory gynecological malignancies, although further studies are mandatory for optimal selection of patients, histological subtype and timing of hdt during the disease course. disclosure of conflict of interest: none. the human endogenous retroviruses (hervs) are remnants of ancient exogenous retroviral infections of the humans: they represent about % of the human genome . the basic genes of hervs are group-specific antigen (gag), polymerase (pol) and envelope (env); there are also two regulatory regions, long terminal repeat (ltr), located at ' and ' ends. several reports have shown that hervs may play a role in the development of autoimmune diseases, such as multiple sclerosis . additionally the existence of a strong relationship between hervs expression and cancer, based on the mrna expression profile of hervs in normal and cancer tissues has been suggested . the increased level of expression level of herv-h in colorectal cancer (crc), a major cause of cancer death worldwide has been already shown. the aim of the study was to analyse the expressions of env genes of herv-r, herv-h, herv-k and herv-p in the peripheral blood mononuclear cells (pbmcs), in the tumor and in the adjacent normal tissues of colorectal cancer patients. a group of control composed by pbmcs from healthy subjects was also included. rna was isolated from the biological samples and a reverse transcription assay was conducted. quantitative real time pcr was performed to evaluate the expression of the hervs env gene. all the env genes were related to the expression of an housekeeping gene, gapdh. the quantification was carried out using comparative ct method and the difference between the levels of env gene expression in pbmcs, cancer and adjacent normal tissue was given by fold difference. fold difference values were relative to a calibrator: first the pbmcs of patients and then pbmcs of control healthy group. Δct values were analysed using the paired sample t-test, followed by a bonferroni correction. higher levels of expression of herv-h, herv-k and in particular herv-p were found in tumor tissues, as compared to pbmcs and to adjacent normal tissues of patients, with an increase of -, -and folds, respectively. the Δct distribution of herv-h, herv-k and herv-p in cancer tissues were statistically significant (po . ) ( table ). the expression of herv-h, herv-k and herv-p env gene resulted increased in the colorectal tumor tissues also when compared with the pbmcs of the healthy controls ( -, -and -folds, respectively). the Δct distribution of herv-h, herv-k and herv-p in tumor tissues were statistically significant ( ρ < . ). no difference of expression was observed between pbmcs of healthy controls, pbmcs and normal adjacent tissues of patients (figure ). hervs env gene expression cannot be used as a diagnostic biomarker, but it is conceivable that hervs are directly involved in the pathogenic process of cell transformation and, if the protein expression will be demonstrated, the protein of hervs env gene could be the target for new immunotherapy strategies against colorectal cancer. [p ] disclosure of conflict of interest: none. a biosimiliar g-csf filgrastim is as effective as a reference drug however itis not as cost effectiveas it supposed to be and by the way no impact on the health care system m kurt yüksel, g pekcan, u sahin, s bozdag, s toprak, p topcuoglu, o arslan, g gurman and m beksac ankara university school of medicine biosimiliars are up to times the size of small molecule generic drugs and far more structurally complex. additionally biosimiliars are manufactured in living cell lines using processes that cannot be exactly replicated from one manufacturer to the next. a biosimiliar cannot be identical to its reference biologic drug. with billion dollars in global sales of biologic medicines anticipated to go off patent by .this lead to fast production of biosimiliar drugs. besides, it is expected that biosimiliar drugs will be more cost effective than the reference drugs and will have a meaningful impact on health care systems around the world. aim: to compare biosimiliarfilgrastim (leucostim) with two reference g-csf filgrastim (neupogen) and lenograstim(granocyte) in the context of safety, efficacy and cost effectivity. records of patients with multiplmyeloma(mm) whom underwent autologous stem cell transplantation(asct) and received g-csf sc mikrogram/kg/day from +day until engraftment were [p ] retrospectively evaluated mm patients were treated with high dose melphalan and asct at the ankara university school of medicine bone marrow transplantation unit between and . the median age was ( - years) with % male. patients were divided into three groups (n = ) whom received reference filgrastim (neupogen), lenograstim (granocyte) and biosimiliarfilgrastim (leucostim): groups a, b and c respectively. the total cost of each g-csf in dollars was calculated by one package of g-csf multiplied by total used days . chi-square, mann-whitney u and kruskal-wallis tests were used for analyses of variance. the percentage of patients who received melphalan mg/m were% , , in groups a, b, c respectively (p = . ).there was no statistically significant difference between the engraftment day of neutrophil and ; platelet and in the groups. (p = . , p = . , p = . , p = . respectively) themedian numbers of g-csf administered days were ( - ), ( - ), ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) in groups a, b, c, respectively .eventhough there was no statistical difference between the numbers of days( p = . ), the total cost in dollars was statistically difference between a vs b and c vs b (both p o . ) and there was no statistical difference between a vs c (p = . ), total cost in dollars as follows: $ ( - $), $( - $) and $( - $) for the group a, b and c respectively. our results demonstrate that biosimiliar gcsf leucostim is highly similar to existing licenced biologic products in turkey with no clinically meaningful difference interms of safety and efficacy. on the other hand it as a biosimiliardoes not have a meaningful impact on the cost savings to the health care system as expected when compared with reference filgrastim. disclosure of conflict of interest: none. in this study, we investigated the roles of prx ii, one of critical peroxidases besides catalase and gpxs, in cml primary cells at diagnosis and remission while patients were treated with sti (signal transduction inhibitor) and tested the same roles in imatinib(im) sensitive ph+ cell lines and resistant cell lines as well. newly diagnosed cml cells, im resistant k cells and parental k cells were treated stis and analyzed western blot assay to detect bcr-abl, phosphorylated bcr-abl and prx protein expression level. we added n-acetylcysteine ( - mm, hr) to k cells to show antioxidant effect of imatinib and analyzed dcf-da detection for intracellular ros level and western blot for prx protein level. mtt assay was performed to detect cell death by nac time-dependent treatment of mm nac( , , , hr). imatinib resistant k cells were established by treatment of gradual increment of imatinib. we also repeatedly investigated the effects of im therapy using prxii overexpressed k cells by transfection. at diagnosis of cml, ros level was elevated and prx ii was either absent or significantly suppressed. as ph chromosomes were decreased with stis, suppressed or absent prxs levels were restored to the level of normal individuals. these findings were also inversely correlated with the level of ph chromosomes in the cases of disease progression and re-remission with further treatment. when sti were treated in ph positive cell line, we found deceased cell survival and ros level by mtt assay and dcf-da methods respectively, but elevated prx ii by western blot. by the treatment of nac into ph+ cell lines, the level of dcf-da was decreased and mtt level was down, but prx ii level was elevated. interestingly, the level of bcr-abl oncogene were decreased in prx ii tranfected cells. meanwhile, we observed that prx ii restoration was mild or weak in imatinib resistant k- , which we established in our lab. the importance of the roles of ros and its prx ii, antioxydant enzymes in cml is further established by our work. our finding may contribute to find a new pathway on which tkis are working besides the mechanisms of atp binding competitively, blocking the binding of abl-bcr kinase to the substrate resulting apoptosis of ph+ cells. furthermore, our finding may be useful to overcome the stis resistant cml in the clinics in the future. disclosure of conflict of interest: none. allogeneic hematopoietic stem cell transplantation for the treatment of mucopolysaccharidosis f shunqiao, s xiaodong and l junhui department of hematology, capital institute of pediatrics, china mucopolysaccharidosis (mps) is a lysosomal storage disorder caused by deficient activity of the iduronate-sulfatase.this leads to accumulation of glycosaminoglycans(gags) in the lysosomes of various cells,which causes progressive multisystem involvement with ensuing death.the aim of this study was to exploit the effect of treatment with allogenic hematopoietic stem cell transplantation and administration of high doses of cyclophosphamide early after haplobmt in these cases. we retrospectively reviewed data from mps patients ( cases mps ii, and case mps i). the two mps ii patients were -month-old and -month-old boy and the mps i patient is a -month-old girl at the time of transplantation. the reduced-intensity of bu+flu conditioning regimen in allo-hsct for these patients was as follows: busulfan mg/kg at - days before transplantion,fludarabine mg/m at - days before transplantion.graft-versus-host disease(gvhd) prophylaxis:rabbit antithymocyte globulin . mg/kg daily at - days before transplantation,shortcourse methotrexate,posttransplantation high-dose cyclophosphamide on days + and + was followed by mycophenolate mofetil and cyclosporine.the donors all were their hlahaploidentical father. these three patients' neutrophil engraftment occurred on + d, + d and + d after transplantation respectively, platelet engraftment occurred on day + d, + d and + d after transplantation respectively.complete donor type engraftment was confirmed by short tandem repeat-polymerase chain reaction(str-pcr) on day after transplantation. no regimen-related toxicity occurred,gvhd and graft failure were not observed. month after transplantation, the activity of the iduronate-sulfatase was increased to normal. the motion of metacarpophalangeal joints ameliorated, regression of hepatosplenomegaly, the neurocognitive function improved. allogeneic hematopoietic stem cell transplantation is an effective measure to treat patient with mps at least mps ii and mps i. the reduced-intensity conditioning regimen was helpful to decrease the regimen-related toxicity. posttransplant cyclophosphamide approach successfully used and reduced the incidence of gvhd. this study aimed to evaluate the feasibility of alternative donor hematopoietic stem cell transplantation (hsct) using busulfan, fludarabine, and thymoglobulin conditioning for patients with chronic granulomatous disease (cgd) who lack an hla-matched familial donor. medical records of consecutive patients who received alternative donor hsct between may and may were reviewed, and the transplant-related outcome measures were analyzed retrospectively. the donor source was unrelated peripheral blood (pb) in , unrelated cord blood (cb) in , and haploidentical father in patients. only transplants ( / allele-matched unrelated pb) were hla-matched according to current standards relevant to the donor type. the conditioning regimen was uniform; fludarabine mg/m on days - to - , busulfan . mg/kg/d (or mg/m /d) on days - to - , and thymoglobulin . mg/kg/d on days - to - (or on days - to - in cb recipients). all but one patient were male and their median age at transplantation was . y (range: . - . ). one patient who received a cord blood graft suffered from primary engraftment failure, while the other patients were successfully engrafted with their chimerism levels ranging from % to % (median %) at month post-transplant. the median days to neutrophil and platelet engraftment were . (range: - ) and (range: - ), respectively. among the patients engrafted, one patient experienced secondary graft failure which was rescued by a second transplantation. the remaining one patient who failed to engraft was also rescued with a haploidentical graft from his mother. eight patients ( %) developed cmv antigenemia, and one of those patients developed cmv hepatitis. three patients developed grade acute gvhd which were manageable. one patient who developed grade hepatic gvhd eventually died. two patients developed extensive chronic gvhd, but became free of immunosuppressants after a complete resolution in one and with remaining stable mild joint contractures in the other. including patients who were rescued by additional transplantation, patients are alive with their latest chimerism levels ranging from . % to % (median %). the estimated -y overall survival rate was . % with a median follow-up of months (range: - ). even though the majority of our cohort underwent a mismatched transplantation, the survival rate was excellent. while conditioning with busulfan, fludarabine, and thymoglobulin seems feasible for alternative donor hsct in patients with cgd, special attention needs to be payed on cmv infection and severe gvhd which might offset the high survival rate. disclosure of conflict of interest: none. diarrhea is a common infectious complication in patients who had hematopoetic stem cell transplantation so, we aimed to detect entamoeba histolytica ratio before engraftment, amoung patients who had diarrhea after periferic hematopoetic stem cell transplantation (phsct) in our clinic. allogenic phsct patients had a median age of (range: - ) and autolog phsct patients had a median age of (range: - ). diarrhea is described as an abnormal increase in the frequency ( or more times per day), volume or liquidity of stools. we based upon this description in this study. we made stool examination in the first day of diarrhea. as stool examination, we used direct microscopic evaluation and adhesin antigen test specific for e.hystolytica with enzyme linked immunosorbent assay (elisa), e. histolytica ii, techlab, blacksburg, usa). we accepted e.hystolytica positivity as detecting cyst or/and trophosoit in stool and antigen test positivity at the same time. in our study, of patients had diarrhea in the first days of phsct. diarrhea was found in of in autologous phsct patients (% ), of patients in allogenic phsct with non-myeloablative conditioning regiment (% ) and of patients in allo phsct with myeloablative conditioning regiment (% ). diarrhea occured at + th day of transplantation and the median duration of diarrhea was days. e. histolytica positivity was found of patients ( %) who underwent phsct within first days of transplantation. infection is an important mortality and morbidity factor for patients who had hematopoetic stem cell transplantation, when especially before engrafment (between - days). autologous phsct patients were elderly, with poor self-care and low socioeconomic status individuals. e. hystolytica is a frequent pathogen in posttransplant diarrhea at endemic regions. prophylactic metronidasole treatment should be used routinely for autologous phsct as in allogenic phsct. patients and companions sholud be tested for e.hystolytica before autologus/allogenic phsct in endemic regions. prophylactic treatment for amebiasis and scanning patient/companions could be a part of solution for post phsct diarrhea. despite the emergence of disease modifying therapies (dmts) for multiple sclerosis (ms) a cohort of patients with aggressive disease have ongoing progression/relapse, associated with progressive disability. autologous haematopoietic stem cell transplantation (ahsct) has been used worldwide for aggressive ms with inflammatory changes on mri. we update on a uk single centre experience of ahsct in ms. a retrospective audit of ahsct performed for ms from to at uk centre (king's college hospital) was undertaken. patients were selected for transplantation based on persistent clinical relapses (relapsing-remitting ms) or secondary progressive neurological disability with mri lesion activity despite use of at least dmt. primary progressive patients were also eligible if new/active mri lesions were demonstrable. followup included clinical evaluation, edss assessment and mri scanning. we report our preliminary findings. as of november , patients ( female, male, rrms, spms, ppms) had received ahsct. mean age at transplant was . years (range: - ). the mean baseline edss was . (range: . - . ). patients underwent cyclophosphamide/atg conditioning, while received beam/atg. whilst conditioning and stem cell infusion were well tolerated there was a high rate of infections, with / patients developing a culture confirmed infection. reactivation of ebv and cmv were observed in a number of patients ( and , respectively) while a number of delayed herpes zoster infections were also seen ( cases of shingles and of disseminated varicella infection in patients who had previously experienced it in childhood). median follow-up was for days ( - ). of patients with a formal month assessment (n = ), had a stable edss, had an improved score (median improvement . , range: . - . ) and had a deterioration in their score (median . , range: . - . ). at months (n = ), had a stable edss, had an improved score (median . , range: . - ) and had a deterioration in score (median . , range: . - . ). at months, two patients assessed both had improvements in edss scores (median , range: . - . ). for patients who underwent mri at month follow-up (n = ), had a stable lesion load, demonstrated improvement in lesions and had a new lesion (the remaining mri was difficult to read due to a high baseline lesion load in this patient). patients had mri's at months; were stable and demonstrated a reduction in lesion load. to date, no patients have developed secondary malignancies or autoimmune diseases. of patients with followup data, / rrms patients experienced suspected clinical relapses following hsct-only one had a new lesion on mri (with no gadolinium enhancement). of the received steroids to treat these relapses (it is unclear if the remaining patient received treatment). patient tried a new disease modifying therapy ( dose of rituximab) following hsct. ahsct in this cohort was feasible with universal mobilisation and harvest. whilst conditioning and stem cell infusion were well tolerated, there was a high rate of infectious complications in the neutropenic phase. however, the transplant related mortality was % despite significant levels of disability amongst this patient cohort. ahsct remains a treatment option to be further investigated in this difficult cohort of patients. disclosure of conflict of interest: none. peripheral blood (pb) stem cells (scs) mobilized with g-csf are the first-choice source for allogeneic transplantation. we carried out a prospective study on healthy donors (hds), to identify donor characteristics that could influence the effectiveness of mobilization with special focus on the value of the basal cd + cell count. sibling hds were analyzed in a prospective study. we tested somatic variables (sex, age, weight, height, volemia) and, basal blood counts (white blood cell, peripheral blood mononuclear cell, platelets, hematocrit, hemoglobin, cd + cell). hds received g-csf subcutaneously at a dose of μg/kg day. two different determinations of cd + cells were done in each donor: baseline (before g-csf administration) and in pb on the morning of the fifth day (after g-csf administration). consecutive hds ( males) with a median age of years were enrolled. the mean value of cd +on day was . cells/μl, while the median value was . cells/μl. we performed two multivariate analyses either by using median regression (to predict the median value of cd +on day ) according to the values of cd + at baseline, the first adjusted by gender, age and blood volume and the second by gender, age and bmi. results of both models indicate that from basal cd + values o = to values ranging between and cells/μl, predicted median values of cd + on day significantly increase, from . to . cells/μl for model adjusted by blood volume, and from . to cells/μl for model adjusted by bmi. baseline, pb cd + cell count correlated with the effectiveness of allogeneic pbscs mobilization and could be useful to plan the collection. disclosure of conflict of interest: none. comparison of efficacy between chemotherapy plus granulocyte colony stimulating factor (g-csf) and chemotherapy plus g-csf and granulocyte-macrophage colony stimulating factor (gm-csf) for mobilization of peripheral blood stem cells (pbsc) and hematological recovery post-transplantation in patients with multiple myeloma (mm). a retrospective study of autologous peripheral blood stem cell (apbsc) mobilization data of mm patients who treated with chemotherapy plus g-csf or chemotherapy plus g-csf and gm-csf from may to july . the mobilization efficacy and hematopoietic recovery were analyzed. a total of stem cell mobilizations were performed in mm patients. in the univariate analysis, successful collection rate of single harvest in female and in patients at iss stage iii, r-iss ii/iii and chemotherapy plus g-csf was lower(po . ). however, age(≦ yrs vs yrs), subtype, d-s staging (i+ii vs iii), cycles of chemotherapy before mobilization (≦ cycles vs cycles), disease phase before mobilization (pr vs cr) and interval diagnosismobilization (≦ months vs months) were not correlated with the cd + cell collection and successful mobilization rate(p＞ . ). in the multivariate model, rate of successful mobilization in patients who received chemotherapy plus g-csf+gm-csf mobilization regimen was high (or = . , %ci . - . ). the effect of mobilization regimen remained significantly (p = . ). all patients successfully underwent hematopoietic reconstruction without transplantation-related mortality. chemotherapy plus g-csf +gm-csf mobilization regimen can significantly increase the effect of apbsc mobilization and ensure the reconstruction of hematopoietic function after transplantation. this mobilization regimen is a safe and effective method of mobilizing apbsc. disclosure of conflict of interest: none. clinical efficacy of bk virus specific t-cells in treatment of severe refractory hemorrhagic cystitis after hla haploidentical transplantation om pello , a bradshaw , a innes , s-a finn , s uddin, e bray , e olavarria, jf apperley and j pavlu centre for haematology, imperial college at hammersmith hospital, london, uk and department of clinical haematology, hammersmith hospital, imperial college healthcare nhs trust, london, united kingdom hemorrhagic cystitis caused by bk virus (bkv) is a significant complication of allogeneic hematopoietic cell transplantation (hct). it is particularly common in the setting of hla haploidentical transplantation and can be challenging to manage. here we present a post haploidentical hct patient who developed severe bkv haemorrhagic cystitis resistant to standard therapy and who responded to adoptive transfer of donor t cells enriched with anti-bkv specific cells. a year old man underwent hct for acute myeloid leukaemia with inversion of chromosome and monosomy of chromosome while in first complete remission. as he had no related or unrelated hla identical donor, cells from his hla haploidentical sister were used. on day + of this procedure he developed haemorrhagic cystitis. supportive treatment was initiated and cystoscopy showed diffuse bleeding from his urinary bladder with blood clots. urine pcr for bkv showed . billion copies/ml. despite bladder irrigation, local therapy to s bladder mucosa and intravenous hydration, he failed to improve, so treatment with weekly intravenous cidofovir was initiated on day + . his symptoms improved, but on day + he again deteriorated on weekly infusions of cidofovir. his immunosuppression was slowly tapered off without any graft versus host disease (gvhd) but without any significant effect on his hemorrhagic cystitis. he underwent bladder diathermy, was treated with intravesicular hyaluronate and with intravenous cidofovir, but continued to have frank haematuria with blood clots and significant lower abdominal pain. although there was no evidence of obstruction his renal function deteriorated on cidofovir therapy. hence we elected to trial adoptive anti bkv therapy. a leukoapheretic lymphocyte collection was used to prepare an anti-bkv t cell enriched product using the clinimacs prodigy and the cytokine capture system from miltenyi biotec. the eluted product contained % and % of cd + and cd + lymphocytes expressing ifng+ respectively and the cd +/cd + dose adoptively transferred on day + of transplantation was . × /kg. in vivo expansion of anti-bkv t cells in the patient was analysed weekly for the first month using the research grade peptivators bkv lt and bkv vp and the rapid cytokine inspector (cd /cd t cell) kit. bk viral load was monitored by pcr in urine samples twice weekly. ifng+ anti-bkv reactive t cells were undetectable in the patient for the first two weeks after adoptively transfer of donor t cells. twenty days after the adoptive transfer an increase in the cd + ifng+ population was observed, in response to the bkv vp peptivator. this observation correlated in time with a substantial decrease of the urine bkv viremia from . million copies/ml to copies/ml and a complete resolution of patient's symptoms. no gvhd, recurrence of urinary symptoms or any other problems have been observed to date (day + of transplantation, + days after the adoptive transfer). we are not aware of any other reports of successful adoptive anti bkv cellular therapy. our experience suggests safety and efficiency of the use of anti-bkv t cell enriched products using the clinimacs prodigy and the ifng capture system in hla haploidentical hct where bkv cystitis constitutes a significant complication. this opens the possibility of further clinical trials. disclosure of conflict of interest: none. haploidentical donor (hd) has been used as an alternative stem cell source when patients do not have a hla-matched related or unrelated donor. to overcome the hla barrier, haploidentical stem cell transplantation (haplosct) using post-transplantation cyclophosphamide (ptcy) has been conducted. here, we compared the clinical outcomes of haplosct using ptcy with those of unrelated donor transplantation. eighty-two patients ( from hd and from unrelated donor [ud]) who underwent allogeneic hematopoietic stem cell transplantation (hsct) in seoul national university children's hospital from january to june , were analyzed. there were no significant differences between hd and ud patients with respect to median age of patients, sex distribution, and diagnosis [ . %], p = . ). the conditioning regimen of haplosct included targeted busulfan, fludarabine and cyclophosphamide using ptcy, tacrolimus and mycophenolate mofetil for graftversus-host disease (gvhd) prophylaxis. all patients showed engraftment except for a patient who underwent unrelated hsct. neutrophil engraftment of ud was faster than hd (median days versus . days, respectively, po . ). however, there was no significant difference of platelet engraftment. incidences of complications, such as hepatic venoocclusive disease, cmv infection, and hepatic dysfunction, between both groups, were comparable, except hemorrhagic cystitis (hd: . %, ud: . %, p = . ). moreover, cumulative incidence of acute gvhd (hd: . %, ud: . %, p = . ), severe chronic gvhd (hd: . %, ud: . %, p = . ), relapse (hd: . %, ud: . %, p = . ) and non-relapse mortality (hd: %, ud: . %, p = . ) were not significantly different. the overall and event-free survival of hd and ud were . % vs . % (p = . ) and . % vs . % (p = . ), respectively. the clinical outcomes of haplosct using ptcy were comparable with those of ud, and a trend of lower cumulative incidence of severe chronic gvhd and non-relapse mortality was encouraging. it could be a promising alternative therapeutic option in pediatric hsct. disclosure of conflict of interest: none. , median number of apheresis procedures was , ( - ), median amount od dmso infused ml ( - ). time to engraftment was median days ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) . statistical comparison between cryopreserved pbsc grafts and bm showed benefit for pbsc in the terms (po . ) of faster engraftment, less infective complications, less transfusion support and less hospital stay. in patients ( %) dmso related events were not registered during graft administration. in patients ( %) mild to moderate dmso related events were registered, as nausea in patients ( . %), vomitus in patients ( %), tachycardia in ( . %), hematuria in patients ( %) and patients ( . %) with bradycardia, hypotension, fever and high temperature during graft infusion. cryopreservation of stem cells is a feasible procedure at our institution. there are some issues that have to be improved. the process is standardized with achieved engraftment in all transplanted patients. disclosure of conflict of interest: none. effectivity of a fludarabine based conditioning regimen in allogenic hematopoietic stem cell tranplantation for patients with severe aplastic anemia and over twenty years old p mustafa , , k melya pelin , s handan haydaroglu and g ilknur gaziantep university, faculty of medicine, department of internal medicine; hematology and bone marrow tranplantation unit, gaziantep, turkey severe aplastic anemia (saa) is an anemia with bone marrow hypocellularity and caused by hematopoietic stem cell failure ( ) . allogenic periferic hematopoietic stem cell transplantation (aphsct) is a curative treatment choice ( ) . although cyclophosphamide (cyc) and anti thymocyte globulin(atg) is accepted as standart conditioning regimen, especially for patients with high rejection risk, using fludarabin (fu) based regimens show increased successful engraftment ratio with minimal toxic side effects ( ) . to the study, saa patiens who were transplanted from hla matched sibling donors between the years - were included. the patients comprised of male (% ) and female (% ). median age was (range: - ). the median time from diagnosis to transplantation was (range: - ) months. conditioning regimen consisted of cyc ( mg/m ), fu ( mg/m ), atg (fresenius rabbit, mg/kg). the median dose of stem cells was × stem cell/kg (range: - ). methotrexate ( mg/ m given four days) and cyclosporine (cyca) ( - mg/kg given months) were applied for graft versus host disease (gvhd) prophylaxis. all patients ecog performance status were good ( - ). prior to transplantation only one of the patients received atg-csa, the others received only supportive treatment. after aphsct, neutrophil engraftment was occured at a median of days (range: - ) and thrombocyte engraftment was occured at a median of days (range: - ). posttransplant graft failure was observed in only one patient at tenth month and this patient had aphsct again from the same donor with the same conditioning regimen. acute gvhd didnot occur in any patient. the (% ) of patients had common chronic skin/oral mucosa gvhd. these patients received methylprednisolone (mp) and/or mycophenolate mofetil (mmf) in addition to the cyclosporine treatment. extracorporal photopheresis was applied to the two patients with chronic gvhd. all chronic gvhd patients had complete response to the immunsupresive treatment with a median follow up time months (range: - ). one patient died from sepsis. at year overall-survival rate was %. fu based conditioning regimen in aphsct with young saa patients has favorable results. fu based regimen might be a gold-standard treatment in the future. cgvhd; tgfb-induced factor homeobox , interleukin receptor gamma, tetra trico peptide repeat domain , carbonic anhydrase i, serpin peptidase inhibitor clade a and myod family inhibitor. we established a -gene model (myod family inhibitor, tgfb-induced factor homeobox , tetra trico peptide repeat domain ) to diagnose cgvhd. our -gene model showed . % sensitivity, . % specificity, . % precision, . % accuracy and . % roc area in diagnosing cgvhd. tgfb-induced factor homeobox increased in expression after rituximab treatment in responders. myod family inhibitor was found to be able to predict rituximab responses in steroid-refractory cgvhd patients. we could demonstrate that gene expression studies were useful in the diagnosis of cgvhd after allo-hsct. we developed a -gene model to diagnose cgvhd. hematopoietic stem cell transplantation (sct) is physically and psychosocially demanding. however, exercise interventions may have positive impact on sentiment and psychological well-being in patients undergoing sct. we report on a prospective, randomized study comparing the influence of a multimedia sensor-based practice with classical physiotherapeutic treatment (pt) on psychological aspects and quality of life (qol). patients undergoing sct were randomized into the control group (n = ) receiving pt or the experimental group exercising on the nintendo-wii (n = ). patients of both groups performed the exercises under the supervision of a physiotherapist and completed the functional assessment of cancer therapy -bone marrow transplantation (fact-bmt), hospital anxiety and depression scale (hads-d) and distress thermometer at the date of hospital admission (t ) and on day (t ), (t ) and (t ) after sct. questionnaires were completed by the participants independently and without supervision. groups were compared using the mann-whitney u-test. a p value o . was considered statistically significant. the median age of patients was years in the control group and years in the experimental group. results of fact-bmt generally showed a decline of the qol domains measured on t and t and a raise at t in both groups. physical well-being (pwb) showed the strongest fluctuation of all domains. it declined significantly between t -t in both groups (pt p = . , wii p = . ), followed by a significant increase between t -t (both groups p = . ). however, only in wii-group results of pwb at t ranked significantly above t (p = . ). highest scores were proved for social and emotional well-being (swb/ewb) in both groups. in wii-group ewb increased significantly between t -t (p = . ) and ranked above pt-group at all times. functional well-being (fwb) scored lowest in both groups at all times. the score of bone marrow transplant scale (bmts), the second lowest score in both groups, was always higher in wii-group. the level of distress was comparable between both groups. however, at t distress increased above the cut-off level of in both groups (wii-group p = . , pt-group p = . ). this was accompanied by an increase of anxiety (p = . ) and depression (p = . ) in the pt-group, while both parameters decreased in the wii-group (p = . and p = . ), respectively. anxiety in intervention group , / , / , /, at t /t /t /t stayed below standard group , / , / , / , at all times. depression averaged out at , / , / , / , in physiotherapy group and , / , / , / , in wii-group. to the best of our knowledge, this is the first study to show that exergaming using the nintendo-wii is feasible in the immediate phase after hsct. exergaming may be regarded as beneficial since our data indicate less psychological distress and higher qol in sct recipients exercising with nintendo-wii. therefore, it may be used in addition to pt. disclosure of conflict of interest: none. acute graft versus host disease (agvhd) is the most frequent and serious complication following haematopoietic stem cell transplantation (hsct), with a high mortality rate. a clearer understanding of the molecular pathogenesis may allow for improved therapeutic options or guide personalised prophylactic protocols. circulating micrornas are expressed in body fluids and have recently been associated with the etiology of agvhd, but global expression profiling in a hsct setting is lacking. this study profiled expression of n = mature micrornas in patient serum, using the nanostring platform, to identify micrornas that were dysregulated at agvhd diagnosis. selected micrornas (n = ) were replicated in independent cohorts of serum samples taken at agvhd diagnosis (n = ) and prior to disease onset (day post-hsct, n = ) to assess their prognostic potential. sera from patients without agvhd were used as controls. dysregulated micrornas were investigated in silico for predicted networks and mrna targets. profiling identified micrornas that were differentially expressed at agvhd diagnosis. mir- a (p = . ), mir- b- p (p = . ), mir- - p (p = . ), mir- a (p = . ), mir- a (p = . ) and mir- a (p = . ) were significantly verified in an independent cohort (n = ). mir- a (p = . ), mir- a (p = . ), mir- (p = . ), mir- a (p = ), mir- b ( . ) and mir- (p = . ) were differentially expressed days post-hsct in patients who later developed agvhd (n = ). high mir- b expression was associated with improved overall survival (os) (p = . ), while high mir- a and mir- b- p were associated with lower rates of non-relapse mortality (p = . and p = . ) and improved os (p = . and p = . ). pathway analysis associated the candidate micrornas with haematological and inflammatory disease. circulating biofluid micrornas are dysregulated at agvhd onset and have the capacity to act as prognostic and diagnostic biomarkers. their differential expression in serum suggests a role for circulatory micrornas in agvhd pathology, which warrants further investigation. disclosure of conflict of interest: none. factors associated with medication adherence amongst allogeneic hematopoietic stem cell transplantation recipients j lehrer , e brissot , , a ruggeri , , r dulery , a vekhoff , g battipaglia , f giannotti , c fernandez , , , m mohty , and m antignac ap-hp, hôpital saint-antoine, service de pharmacie, paris, f- ; service d'hématologie et de thérapie cellulaire, hôpital saint antoine, assistance publique-hôpitaux de paris; sorbonne patients with median ages of years (range: - years) between december and march , which including case of primary hlh (homozygous missense mutation in unc d: n = ; homozygous missense mutation in prf : n = ; heterozygous missense mutation in prf in the combination with hemizygous missense mutation in sh d a: n = ; mutation in rab a: n = ; mutation in itk: n = ). cases of unknown causes hlh, cases of lympgoma -hlh (nk/t-cell lymphoma: n = , primary γδt cell lymphoma in skin: n = ; subcutaneous panniculitis-like t cell lymphoma: n = ; primary t cell lymphoma in skin: n = ) and cases of ebv associated hlh. patients achieved cr+pr before hsct, and patients nr. patients were transplanted from hla-haploidentical family donors, from hla-identical sibling donors, and from a matched unrelated donor. conditioning regimen include tbi and non-tbi. the median overall survival rate was . % with a median survival time of months (range: - months). os of primary hlh is . %, os of unknown causes hlh is %, os of lymphoma-associated hlh is %, os of ebv-hlh is . %. os of cr+pr is . %, os of nr is . % patients without engraftment died because of graft failure and toxicity of conditioning regimen. patients with engraftment died. of those, patient died of hsct-associated tma, patient died of grade iv agvdh, patients died of relapsed hlh or organ failure as results from unsuccessful treatment of the progressively elevated ebv-dna load. patient died of tumor relapse, and patient died of infection. acute gvdh occurred in patients with grade i-ii agvdh in patients and grade iii-iv agvdh in patients; chronic gvdh occurred in patients. patients achieved completed chimerism, patients appeared with mixed chimerism,and patient presented with graft failure. of ebv-hlh with engraftment, reactivated ebv infection was found in ( %) with the whole blood ebv-dna load at - copy numbers per ml. ptld occurred in patients confirmed by pathology. after reduced immunosuppressors, negative result of ebv infection was obtained while patients developed gvdh. for ebv-hlh, patients who carry with ebv loading ebvdna ≤ copies/ml before transplantation, overall survival rate was significantly higher than that of ebvdna copies/ml (po . ); who achieved cr+pr os was significantly higher than that of nr (po . ); who range: from diagnosis to transplantion ≤ months os was significantly higher than that of months (po . ). allogeneic hematopoietic stem cell is an effective method for primary hlh and lymphoma-hlh, ebv-hlh,even haploid transplantation. the remission status before transplantation is decisive for the prognosis. disclosure of conflict of interest: none. hepatic veno-occlusive disease after allogeneic hematopoietic stem cell transplantation in a single centre: revised diagnosis and incidence according to new ebmt classification s santarone, a natale, p olioso, g papalinetti, t bonfini, p accorsi, s angelini, g iannetti, m di ianni and p di bartolomeo dipartimento di ematologia, medicina trasfusionale e biotecnologie, bmt center, ospedale civile, pescara, italy sinusoidal obstruction syndrome, also known as venoocclusive disease (sos/vod), is a potentially life threatening complication that can develop early after hematopoietic cell transplantation (hct). in this study we retrospectively investigated the incidence, risk factors and outcomes of sos/vod in transplants, performed in patients between march and may , on the basis of the new diagnostic criteria and classification of the ebmt. the patient's median age was years ( to ). of them, were males and females. patients received one transplant and two transplants. a diagnosis of hematological malignant and nonmalignant disease was present in and cases, respectively. the disease risk at hct was standard in cases, intermediate in and high in . an hla identical sibling donor was used in in cases, an unrelated donor in and a haploidentical family donor in . conditioning was myeloablative in transplants and at reduced intensity in . source of hematopoietic stem cells was bone marrow in transplants and peripheral blood in . we did not limit the diagnosis of sos/vod to the classical days after hct, but all suspicious cases appearing in the first days were evaluated. sos/vod was diagnosed in cases, of which in the first days after transplant and between day and (median day ). their main clinical characteristics are shown in table . the severity of sos/vod was mild in patients ( %), moderate in ( %), severe in ( %) and very severe in ( %). the cumulative incidence (ci) of sos/vod was . + . %. among the most relevant variables studied in univariate analysis (recipient age and gender, ferritin level at hct, type of hematological disease, disease risk at hct, type of donor, number of transplants, time of transplant, drugs used in the conditioning regimen, intensity of the conditioning regimen, source of stem cells), there was no factor with an adverse impact on sos/vod incidence. of patients with diagnosis of sos/vod, ( %) died. sos/vod was the main cause of death in patients and a relevant contributing cause of death in . of relevance, of patients ( %) with severe sos/vod and of patients ( %) with very severe sos/vod died, whereas only one patient with moderate sos/vod died and no patient with mild sos/vod died. among patients with sos/vod, received defibrotide therapy and the best supportive available therapy. defibrotide was given for a median of consecutive days (range: to ), starting at day post-hct (range: to ) with a median total bilirubin level of , mg/dl (range: . - . ). the -year overall survival (os) of patients treated with defibrotide was better as compared to that of patients who received the supportive therapy ( % versus %) although the difference doesn't reach the significance (p = . ). the occurrence of sos/vod does influence significantly the -yr os considering that it was + . % for patients without sos/vod and + % for patients with sos/vod (p = . ). in conclusion, the new ebmt diagnostic and severity criteria for sos/vod has been very useful in identifying patients with severe and very severe forms of this complication. if validated in prospective studies, these criteria will allow an earlier selection of patients requiring immediate therapeutic intervention. [p ] disclosure of conflict of interest: none. the prognosis of patients with newly diagnosed ewing's sarcoma family of tumors (esft) has improved significantly over the last few decades. nonetheless, the long-term survival is still below % patients with high risk features.the role of s high dose chemotherapy and autologous stem cell transplantation (hdct and asct) for high risk and relapsed esft was analyzed. a retrospective medical chart review was done on patients with efst who underwent hdct and asct between september and january at seoul national university children's hospital. indications for hdct and asct included metastasis at diagnosis, bulky primary tumor ( ml), axial/ central primary site, and relapsed disease. single hdct and asct was performed in the earlier period, and the regimen was changed from mec (melphalan, etoposide, carboplatin), to topothiocarbo (topotecan, thiotepa, carboplatin), and to bumel (busulfan, melphalan). tandem hdct and asct was performed in the recent period, st hdct with bumel and nd hdct with modified mec (melphalan, etoposide, carboplatin). twenty-one patients who were diagnosed with esft at a median age of . years old underwent conventional chemotherapy, radiation therapy and/or surgery and received hdct and asct in complete response (n = ) or partial response (n = ). the overall survival of the patients was . % at median . years and the event free survival (efs) of the patients was . % at median . years from the last asct. the efs of the patients who underwent single hdct and asct with mec (n = ), topothiocarbo (n = ), and bumel (n = ) was . %, . % and % respectively. the efs of the patients who underwent tandem hdct and asct (n = ) was . %. seven patients relapsed at median . months from the last asct. despite further treatment, patients died of disease and patients are currently alive without disease. one patient developed acute myeloid leukemia at . months from the last asct and is currently alive without disease after additional chemotherapy, hla-haploidentical stem cell transplantation and donor lymphocyte infusions. one patient died of transplantation-related mortality due to septic shock and lung infection. hdct and asct may be a promising treatment option for patients with high risk or relapsed esft. further refinements may be needed to identify the optimal regimen and number of hdct and asct. disclosure of conflict of interest: none. post transplant cyclophosphamide (pt-cy) has expanded the use of unmanipulated haploidentical grafts which have a high hla disparity between host and donor. one of the consequences of hla disparity is the development of engraftment syndrome (es). this is an immunological reaction characterized by non-infectious fever and skin erythema that develops after neutrophil engraftment. es resembles an infectious process but treatment involves the use of high dose steroids. our hypothesis is that pts undergoing haploidentical transplants (haplo) with pt-cy should have a high rate of es given the high hla disparity between donor and recipient. objectives: to determine the incidence, symptoms, morbidity and mortality of es in patients undergoing haplo with pt-cy at our institution. retrospective analysis of patients with highrisk hematological diseases undergoing haplo with pt-cy at clinica santa maria between november and august . es was diagnosed using the spitzer criteria ( ). es was diagnosed if pts met major criteria or major plus minor criterion. symptoms could occur prior to or after neutrophil engraftment (neutrophils over cells / ul). all patients signed informed consent and the study was reviewed by our institutional review board. patients received haploidentical grafts ( table ) . all patients had neutrophil engraftment at a median of days. / patients ( %) had symptoms that met criteria for es ( table ). / were transferred to icu due to hypoxemia and patient died after diagnosis of es. / pts were treated steroids. all patients received broad spectrum antibiotics during the febrile period and neutropenia. blood cultures, ebv and cmv pcr were negative in all es pts. there were no significant differences in hospital stay or one-year overall survival (os) between patients who developed and pts who did not develop es (median vs. days respectively, p = . ; one-year os % vs. %, p = . , respectively). es is a frequent complication in patients undergoing hsct haplo with pt-cy. the incidence of es in our study was higher when compared to historical full match related donors series and lower when compared to cord blood transplant studies ( ) there was no increased morbidity and mortality associated with es diagnosis. prompt institution of steroids is recommended in es patients after ruling out an underlying infectious process to avoid further complications. haploidentical allogenic hematopoietic stem cell transplants (haplo-hsct) is an alternative transplant procedure for patients with hematologic malignancies that are in need of transplant and do not have a compatible donor. due to the broad hla disparity, the haplo-hsct can be performed with t-cell depletion and megadose of cd +. alternatively haplo-hsct can be performed with non t-cell depleted transplants (t-replete) either in combination with anti-thymoglobuline serum (atg) or post-transplant cyclophosphamide (pt-cy) as gvhd prophylaxis strategy. center effect is a known risk factor for outcomes of haplo-hsct in both t-cell depleted (tcd) and t-replete settings. however, many centers tend to specialize in one gvhd prophylaxis strategy making it difficult to differentiate the treatment effect from the center effect. the objective was to investigate the role of center effects in gvhd prevention strategy, on leukemia-free survival (lfs) and overall survival (os) in a population of adult patients with acute leukemia receiving haplo-hsct. a retrospective multicenter study was conducted on patients reported to the ebmt registry. inclusion criteria were: age years, lymphoblastic or myeloid acute leukemia (all or aml) in first or second complete remission (cr or cr ), receiving a haplo-hsct between and . in this population (n = ), in order to assess the interaction between center and gvhd prevention treatment, we then included in the study selected patients from the centers that had performed more than % of both tcd and t-replete haplo-hsct during the study period. center effects on the outcomes consisted of ) center effect on the baseline risk of event and ) interaction between center and strategy of gvhd prevention. these center effects were estimated using cox mixed-effects models and tested using permutation tests. all models were adjusted on age, cmv statuses, disease (all or aml), secondary leukemia, previous autologous transplant, disease status (cr or cr ), peripheral blood vs. bone marrow transplant, conditioning regimen. after selection, patients were available across centers in europe. one hundred and one ( %) patients received tcd, t-replete haplo-hsct ( ( %) using atg and ( %) using pt-cy). overall, ( %) patients had aml. there were ( %) peripheral blood transplants in the tcd group and ( %) in t-replete. median follow-up was . years. in adjusted analyses, without accounting for center effect, t-replete tended to be associated with better lfs (hazard ratio (hr): . ( %ci . - . ), p = . ) and os (hr = . ( %ci . - . ), p = . ). when center effects were included, there was significant heterogeneity across centers on the baseline risk of both outcomes (lfs: p = . and os: p = . ). when accounting for interaction between center by strategy for gvhd prevention, the effect of t-replete vs. tcd on the outcomes did vary across centers (p = . and p = . for interactions in lfs and os, respectively) ( figure ). we found an interaction between center and strategy for gvhd prevention on outcomes of patients who received a haplo-hsct. the difference between the strategies (tcd or t-replete) varied across centers, in size and direction. this could be in part related to the increase in expertise with each technique in some centers and with the different management of complications, such as infections-related and relapse. disclosure of conflict of interest: none. adherence included recognition of spuriously high levels (typically from contaminated lines) and delayed dose adjustment due to late reporting of levels by the laboratory. the most common cause of unjustifiable non-adherence was failure to increase the dose in response to a low level. inadequate or excessive dose adjustments may be due to lack of experience or unfamiliarity with the sop. two interventions were launched with the aim of improving adherence to the sop for therapeutic tacrolimus dosing. firstly, to provide a rapid and user-friendly calculation method, we developed a mobile phone application (tacrocalc, a dose calculator based upon the sop algorithm) for android and ios devices using python and swift, respectively. secondly, to reduce the number of spuriously high levels, all nurses responsible for specimen collection participated in an educational module delivered by medical and senior nursing staff. key messages included the need to: use only the dedicated colour-coded tacrolimus lumen to infuse iv tacrolimus; avoid sampling from this lumen; sample peripherally when other lumens are known to be contaminated (reasons for this are being explored); suspend infusion of iv tacrolimus minutes before taking a level; send only immediately pre-dose levels for oral tacrolimus. initial re-audit of episodes post intervention (data collection is ongoing) demonstrated a % increase in sop adherence (p = . ; fisher's exact test), with no cases of unjustifiable non-adherence and a significant reduction in spuriously high levels. in conclusion, the use of tacrocalc by doctors and the implementation of targeted teaching for nurses dramatically improved adherence to the tacrolimus sop. this should ultimately improve therapeutic dosing whilst avoiding toxicity, which may result in better transplant outcomes. tacrocalc is now being adapted to include an option for paediatric dosing, with the potential to dose related medications such as cyclosporine. disclosure of conflict of interest: none. king's college hospital, imperial healthcare, charing cross hospital and imperial healthcare, hammersmith hospital managed with calcium and vitamin d alone in / cases ( %) and together with bisphosphonates in / ( %). osteoporosis was managed with bisphosphonates ± calcium/ vit d in / and with calcium/vit d alone in / . / indicated that they would give bisphosphonates in the absence of osteoporosis, if a patient with osteopenia was receiving long term steroids. dissemination and implementation of existing guidance on investigation and managing low bmd post hct appeared to be poor amongst respondents to our survey. routine dxa scanning was underused; the trigger for dxa in the context of steroids is inappropriately high at many centres at mg/kg daily for months; in established osteoporosis, bisphosphonates were used less frequently than would be anticipated. these findings may reflect the limited data on which current recommendations have been made, or the large number of non-transplant guidelines for investigating and managing low bmd which confound management of this post-hct patient group. hematopoietic stem cell transplantation (hsct) still remains as the most efficient therapy for adult patients with acute myeloid leukemia. for older patients and those lacking a hlacompatible donor, autologous hematopoietic stem cell transplantation (auto-hsct) is a valid therapeutic option. authors aimed for determining the effect of auto-hsct for acute myeloid leukemia patients and analyze group of patients who underwent auto-hsct. the study has been set as a retrospective single center study. clinical information included age, gender, aml type and cytogenetic risk. pretransplantation treatment, mobilization and conditioning were analyzed and thus subsequently authors used kaplan and meier method to calculate the actuarial overall survival rate. table describes patients' characteristics. majority of patients received similar induction therapy based on combination of cytarabine and anthracycline. timespan from the diagnosis to auto-hsct varied from days to days, median was days. seventy ( , %) patients received a preparative regimen consisting of busulfan at mg/kg orally, four times daily for days for a total dose of mg/kg administered on day - through day - and melphalan - mg/m intravenously for over hours on day - . patients achieved an absolute neutrophile count (anc) of ≥ . × /l in between to days; median was days. patients achieved not transfused platelet count ≥ × /l in between to days; median was days. median of patients' discharge from hospital was days (range: from to days) since auto-hsct. hundred day mortality after autologous transplant was at . % ( / ). on the date of our evaluation (april , ), patients were alive and in continued cr. the relapse rate was . % ( patients) and patients ( . %) were lost from follow-up. the -year overall survival (os) was . %, so the target median of overall survival has not been reached. [p ] the development of dyslipidaemia is commonly observed after haematopoietic stem cell transplantation (hsct). few data are available concerning lipid profiles over a long followup period or with regard to the different transplantation types (autologous vs. allogeneic) or the effect of multiple transplantations on the development of dyslipidaemia. a retrospective, single center cohort study including adult patients ( years) who underwent hsct at the university hospital basel s - and who survived ≥ days was performed. patients with at least a baseline lipid measurement were included (n = ) and grouped according to the type of their first hsct (autologous or allogeneic). for the examination of the effect of subsequent hscts, patients with consecutive transplantations of the same type were included and other patients were censored when a different transplantation type was performed. serial lipid profiles (total-, ldl-and hdl-cholesterol and triglycerides) before and after transplantation were examined. of the patients, underwent a first, and of these at least one subsequent autologous hsct. underwent a first, and of these at least one subsequent allogeneic hsct. median age of patients at autologous hsct was y (iqr - ) and y ( - ) at allogeneic hsct. % and % were males, median bmi pre-transplant was ( - ) and ( - ). the majority of patients underwent s intensive conditioning before hsct. median follow-up time was . years in the autologous and . years in the allogeneic group, with a maximum follow up time of . and . years, respectively. table shows the number and percentage of patients with dyslipidaemia ( st autologous and allogeneic transplants). the distribution of exact total cholesterol values along with comparisons with baseline measurements according to group are presented in the figure . *% based on number of measurements available total, ldl-and hdl-cholesterol and tg increased within months of transplantation, regardless whether autologous or allogenic transplantation or a first or a subsequent transplantation was performed. the percentage of patients with dyslipidaemia accordingly rose significantly within months of transplantation and persisted throughout follow-up. although patients undergoing an autologous hsct presented with higher baseline values of total cholesterol, a significantly greater increase post-transplant was observed after allogeneic hsct. first and subsequent transplantations seem to behave similarly with respect to changes in lipid profiles. disclosure of conflict of interest: none. nuremberg, erlangen, germany; department of cancer immunology, institute for cancer research, oslo university hospital, radiumhospital, oslo, norway; kg jebsen center for cancer immunotherapy, institute of clinical medicine, university of oslo, oslo, norway; department of haematology and oncology, university hospital of the goethe university, frankfurt, germany and childrens hospital, goethe university, frankfurt, germany natural killer (nk) cells are lymphocytes of the innate immunity with a potent anti-tumor capacity. in tumor patients, such as multiple myeloma (mm) patients, an elevated number of nk cells correlates with a higher overall survival (os) rate. our study adressed nk cells characteristics and anti-tumor ability in mm patients. especially cytotoxicity of patientderived, cytokine-stimulated nk cells against mm cells has been analyzed at various time points (at diagnosis, before/ after chemotherapy and/or auto-sct). nk cells from patients were analyzed by facs after pbmcs isolation via ficoll separation at different time points: tp , before the start of high dose chemotherapy (hdc)/auto-sct; tp , after early leukocyte recovery (leukocytes /μl) and tp : at least weeks after tp . for testing nk cell cytotoxicity against mm cells, nk cells were purified via negative selection and expanded in vitro for - weeks in low doses il- and il- . nk cells were divided into the cd ++ cd − or cd + and cd + cd ++ subsets. while the major nk cell subset at tp was the cd + cd ++ nk cell subpopulation ( . %), after leukocyte recovery at tp cd ++ cd − /+ nk cells were the main subsets (cd − : . %; cd + : . %). we further evaluated the nk cell function upon tumor interaction at the defined time points. cd ++ cd − nk cells were the main subset to produce ifn-γ upon interaction with k cells at all different time points. the percentage of ifn-γ-positive cd + + cd − nk cells was slightly decreased at tp compared to tp but significantly increased from tp to tp (p-value: . ). similarly, mip- β-and cd a-positive cd ++ cd − cells remained constant between tp and tp , whereas their percentages increased from tp to tp [p-values: . (mip β) and . (cd a)]. moreover, in a small group of mm patients, we isolated nk cells and expanded them for - weeks prior to the functional assays. as expected, the expansion rate was reduced after chemotherapy compared to nk cells from healthy controls, but the patients nk cells increased their ability to kill mm cells due to the ex vivo cytokine expansion. conclusion: our data demonstrate that nk cells have an altered phenotype and function after hdc/auto-sct. remarkably, these nk cells were able to secrete cytokines and still displayed cytotoxic capacity against different types of tumor cells. however, as the proliferative capacity of nk cells seemed to be reduced following chemotherapy, innovative nk cell therapeutic approaches further improve the patients nk cell activity by an ex vivo cytokine stimulation procedure. finally, we suggest that an additive cell therapy with cytokinestimulated autologeous nk cells might improve the outcome of mm patients. lymphoid and myeloid acute leukemia are the most frequent type of cancer and the most frequent cause of cancer related death in children. relapse and refractory disease are the main clinical problems that current therapies are still unable to solve. one of the main nk cell activating receptors is nk cell group d (nkg d). nkg d receptor recognizes human mica/ulbp - ligands. these nkg d ligands are expressed in leukemia cells and constitute suitable targets for immunotherapy. the expression of nkg d ligands was analyzed in peripheral blood mononuclear cells from pediatric patients suffering from acute leukemia ( acute myeloid leukemia, b cell acute lymphoid leukemia and t cell acute lymphoid leukemia), as well as in leukemia cell lines (k , rs - , jurkat, nalm- , molt- , reh and cem), by flow cytometry using specific monoclonal antibodies directed against mica, micab, ulbp- , ulbp- , ulbp- and ulbp- , and by quantitative pcr using taqman probes. peripheral blood mononuclear cells from healthy donors were labeled with cd ra microbeads and depleted using automacs device. the hl i r-mndanticd bbz lentiviral vector was derived from the clinical vector cl i r-ef a-hgcopt but contained the extracellular domain of nkg d, the hinge region of cd a and the signaling domains of - bb and cd -z. the cassette was driven by mnd promoter. viral supernatant was produced by transient transfection of hek t cells with the vector genome plasmid and lentiviral packaging helper plasmids pcagg-hivgpco, pcagg-vsvg and pcag -rtr . cytogenetic studies and array comparative genomic hybridization were performed to analyze the genetic stability of lentiviral-transduced memory t cells. the in vitro cytotoxicity of cd ra − t cells against leukemia cells, healthy pbmc and mesenchymal stem cells (msc) was evaluated by performing conventional -hour europium-tda release assays or by flow cytometry using cfse and aad labeling of target cells. nkg dl were heterogeneously expressed in leukemia primary cells and cell lines. for b cell all primary samples, we found expression of mica/b, mica and ulbp decreased in refractory disease compared to remission (p = . , p = . and p = . , respectively). lentiviral transduction of nkg d- - bb-cd z markedly increased nkg d surface expression in cd ra − memory t cells, which became consistently more cytotoxic than untransduced cells against leukemia cells. additionally, no chromosomal aberrations nor cytotoxic activity against healthy pbmc or mesenchymal stem cells was observed in nkg d car expressing t cells. our results demonstrate nkg d-car redirected cd ramemory t cells target nkg dl expressing leukemia cells in vitro and could be a promising and safe immunotherapeutic approach for acute leukemia patients. peripheral blood stem cell mobilization and collection from elderly patients (≥ years) with multiple myeloma: a single center experience g cengiz seval , sk toprak , s civriz bozdag , m kurt yuksel , p topcuoglu , o arslan , m ozcan , t demirer , g gurman , h akan , m beksac and o ilhan clinic of hematology, yildirim beyazit university yenimahalle education and research hospital and department of hematology, ankara university school of medicine high-dose melphalan followed by autologous hematopoietic cell transplantation (auto hsct) has become the standard procedure for patients with symptomatic multiple myeloma (mm). the ability to mobilize stem cells from healthy donors shows little deterioration with age, the influence of patients' age on auto hsct is uncertain and studies in patients' ≥ years are scarce. severe studies specific to mm have failed to show an independent effect of patient age on cd + mobilization. we retrospectively compared myeloma patients below the age of with patients above years of age, analyzing cd mobilization into peripheral blood and the number of leukapheresis needed to collect at least one single stem cell graft. material and methods: from february through april , data from myeloma patients below the age of were compared to myeloma patients above years of age. all these data were obtained from the ankara university faculty of medicine center for therapeutic apheresis and written informed consent was signed according to our institution regulations. most of the patients received only gcsf at a dose of μg/kg bw twice-daily s.c. until stem cell procurement. patients underwent further pbsc collections until we obtained the target dose cd + cells/μl blood. a maximum of collections were performed in the first mobilization; if the cell dose was not achieved, we submitted patients to a second mobilization. fifty two of patients were above years of age (median age , range: - ) and patients were below the age of (median age , range: - ). baseline characteristics of the older and younger patient cohorts are summarized in table . mobilization regimens for the younger patient population were cyclophosphamide based (n: ), g-csf only (n: ) and +plerixafor (n: ). mobilization in the older population was with cyclophosphamide based (n: ), g-csf only (n: ) and +plerixafor (n = ). the chemotherapy regimens were not statistically different between both age groups. there were no significant statistical differences in time from diagnosis to mobilization, number of prior therapies or disease status between both patient groups. the number of cd + circulating cells before scheduled leukapheresis was mean . cells/μl (median cells/ μl, range: - ; sem ± . ) in all patients (including patients who failed mobilization). our data support the observation that after a standard mobilization regimen with anti-myeloma chemotherapy and once-daily growth factor support, patients above years of age show an impaired cd mobilization into peripheral blood compared to a younger population. this can be overcome by an increased number of leukaphereses. still the number of progenitor cells in the actual graft is inferior compared to the younger population. [p ] disclosure of conflict of interest: none. donor and/or recipient citomegalovirus (cmv) seropositivity has been associated with a poor overall survival (os) in patients who have received an allogeneic hematopoietic stem cell transplantation (allohsct). in comparison with seronegative donors, hsct from seropositive donors has been associated with decreased disease-free survival (dfs) and increased non-relapse-related mortality (nrm). we analyzed the prognostic impact of cmv serology status (donor/ recipient) in patients diagnosed with acute leukemia (al) [p ] s who had received an allohsct in our institution. retrospective unicentric study of patients diagnosed with al between and who received allohsct.the following outcomes were studies: os, dfs, and cumulated incidences of relapse (ri), nrm, acute graft-versus host disease (agvhd) and chronic gvhd (cgvhd). the series included patients ( males, females), median age of years . al type: ( %) all, ( %) aml. type of transplant: ( %) related donor, ( %) unrelated donor and ( %) unrelated umbilical cord blood. the majority, ( %), received myeloablative conditioning. stem cells source: peripheral blood ( %), cord blood ( %) and bone marrow ( %). cmv serology status: positive receptor ( %), negative receptor cases ( %); positive donor ( %), negative donor ( %). serology status combinations (d/r): +/+ ( %), +/ − ( %), − / − ( %), − /+ ( %). patients developed agvhd and ( %) cgvhd. the impact of donor/recipient cmv serology status on os, dfs, ri, nrm and incidence of agvhd and cgvhd for the overall series is reported in table . no statistically significant differences were detected in any of the analyzed variables. in this study, donor/recipient cmv serology showed no influence on the analyzed variables os, dfs, al relapse, nrm, acute and chronic gvhd. however, the sample size limits the validity of the results. disclosure of conflict of interest: none. supported in part with the grants pi / from fondo de investigaciones sanitarias and rd / / from rticc, instituto carlos iii and sgr (gre), generalitat de catalunya, spain. petersburg, russia during the last two decades ahsct has been used as a treatment option for ms with promising outcomes. qol is an important outcome of ms treatment. its assessment gives the patient's perspective on the overall effect of treatment. we aimed to study qol in ms patients before and after ahsct and search the value of the data obtained for decision-making. a total of patients with different types of ms were enrolled in the study: mean age- (range- - ) years old; male/ female- / ; mean edss- . (range: . - . ). all patients were treated by ahsct. reduced-intensity beam-like conditioning was used (bcnu mg/m , etoposide mg/m , ara-c mg/m and melphalan mg/m ). mean follow-up was months (range: - months). qol was assessed using generic questionnaire sf- . for comparisons t-test for independent samples or mann-whitney test was used. qol parameters in ms patients at months after ahsct improved in comparison to base-line: physical functioning- . vs . , role-physical functioning- . . further qol improvement was registered at long-term follow-up: integral qol index exhibited . at long-term follow-up as compared to . at base-line. qol improvement was more dramatic in relapsing-remitting ms than in progressive ms. we found a significant increase of all eight sf- scales in a year posttransplant as compared with base-line in relapsing-remitting ms patients (po . ). in progressive ms patients statistically significant improvement was registered for six out of eight sf- scales (except bodily pain and role-emotional functioning) (p o . ). improved qol parameters were preserved over the entire study period in all the patients who did not have disease progression or relapse. in conclusion, qol monitoring in ms patients after ahsct provides clinicians with the unique information regarding the changes in physical, psychological and social well-being of patients who have been treated with this new treatment modality. it allows to evaluate risks/ benefits of ms patients undergoing ahsct and might influence decision-making. further studies are needed to examine the trajectory of qol changes in this patient population to better define treatment outcomes after ahsct. disclosure of conflict of interest: none. pediatric patients with leukocyte adhesion deficiency type-i (lad-i), a rare autosomal recessive primary immunodeficiency disorder, experience severe and recurrent lifethreatening bacterial infections. allogeneic haematopoietic stem cell transplantation (hsct) offers the possibility of curative therapy although the conditioning regimen used for hsct in lad-i is still a controversial issue. this study provides evaluation of outcome of the lad-i pediatric patients who underwent reduced-intensity conditioning (ric) hsct. twenty four patients ( female) with severe lad-i who received hscts between februay and september at our center were enrolled. the median age at hsct was months (range: months- years). patients received bone marrow (n = ), peripheral blood progenitor cells (n = ) or umbilical cord blood grafts (n = ) from hla-matched related donors (n = ), mismatched related or unrelated donors (n = ), unrelated fully matched donors (n = ) and haploidentical relative donors (n = ). ric regimen was provided with fludarabine, melphalan and anti-thymocyte immunoglobulin. cyclosporine a and prednisolon were used as graft-versus-host disease (gvhd) prophylaxis. engraftment occurred in / , of which one patient experienced graft rejection.the median times to neutrophil and platelet engraftments were days (range: - days) and days (range: - days), respectively. with a median follow-up of months (range: - months), overall survival (os) was . %.the main causes of death were gvhd and infection. acute gvhd occurred in ten patients ( grade i-ii, grade iii-iv) and patients also developed chronic gvhd. there were no significant differences in acute gvhd occurence and also os regarding to the stem cell sources. at this time, patients with full chimerism and patients with mixed chimerism are alive and disease free. conclusion: hsct offers long term benefit in lad- and should be considered as an early therapeutic option if a suitable hla-matched stem cell donation is available. as pretransplant infections in primary immunodeficient patients especially those affected by lad- lead to rise in mortality rate, ric regimen is found to be safe and mixed donor chimersim appears sufficient to prevent significant symptoms. disclosure of conflict of interest: none. tregs based immunotherapy may be beneficial in several immune mediated diseases including graft versus host disease (gvhd). the possibility of cryopreserving tregs might lead to the administration of multiple doses, thus potentially increasing their efficacy in chronic diseases. however, there are few and controversial data on the functionality of tregs after cryopreservation. here, we evaluated the phenotype and the inhibitory capacity of thawed tregs. tregs were purified from leukapheresis of normal donors (n = ) by double immunomagnetic depletion (cd and cd ) followed by cd enrichment using the clinimacs system (miltenyi biotec) under gmp condition. the cells were cryopreserved in saline solution containing % human serum albumin (hsa) and % dmso with a controlled-rate freezing. cell viability was assessed by -aad staining. number/phenotype and function were evaluated on fresh and thawed tregs. cryopreserved autologous t effector (teff) cells were used in mlr assays. before cryopreservation the tregs enriched product mean viability was ± % and the mean percentage of cd +cd +cd +cd low and cd +cd +cd +cd lowfoxp + cells was ± % and ± %, respectively. we then analysed the tregs enriched product after thawing. mean viability of thawed tregs, by -aad staining, was ± %. the viable tregs were almost totally cd +cd + ( ± %). the mean percentage of cd +cd +cd low and cd +cd +cd lowfoxp + thawed cells was ± % and ± % respectively. the contaminant cells present in the treg enriched product were mostly cd +cd +cd + (around %). we further characterized the phenotype of the cd +cd +cd low population. this population was almost totally foxp + ( ± %) and expressed selected markers at various degree (cd l ( ± %), cd s ( ± %), cd ra+ ( ± %), hla-dr+ ( ± %), ccr + ( ± %), cd d ( ± %), cd + ( ± . %), cd +cd + ( ± %). notably, viable thawed tregs were able to induce inhibition of autologous teff cells in a : tregs:teff ratio as freshly isolated tregs: ± % (thawed) vs ± % (fresh) of inhibiton (p . ). in conclusion, here we demonstrated that thawed tregs from healthy donors mantain a stable phenotype. in addition, in our hands tregs show good suppressive ability after thawing despite lower expression of cd l and cd s relapsed and refractory malignant b cell diseases: evidence for therapeutic efficacy via subcutaneous administration of anti-cd × anti-cd antibody lymphomun r buhmann, p ruf, j hess, h lindhofer, u jacob and m dreyling the trifunctional antibody anti-cd × anti-cd lymphomun represents a chimeric immunoglobulin scaffold (mouse igg a/ rat igg b) with promising treatment outcome in patients suffering from malignant b cell diseases. by changing the lymphomun administration route from intravenous (i.v.) to subcutaneous (s.c.) the proinflammatory cytokine-mediated side effects were considerably slighter and generally welltolerated. most importantly, s.c. lymphomun showed outstanding responses in b cell depletion even in the absence of elevated cytokine levels (e.g. il- ) that are required for cytotoxic t cell activation. in summary, the clinical tolerability of s.c. lymphomun may result in a considerable improvement of the subjective well-being and in enhanced mobility due to decreased pain symptomatology. intestinal microbiota disruption is associated with acute gastrointestinal (gi) gvhd and inferior outcome in patients after allogeneic stem cell transplantation (asct). the wide use of systemic broad spectrum antibiotics adds a further risk factor contributing to major microbiota shifts. here, in a retrospective analysis of patients undergoing asct at the regensburg university medical center we assessed the relative expression of paneth cell antimicrobial peptides (amps) in human intestinal biopsies in relation to acute gi gvhd and systemic antibiotic treatment. the relative expression of paneth cell amps was significantly higher in biopsies of the upper gi tract than in the lower gi tract for reg α (p ≤ . ), human defensin (hd) (p ≤ . ) and hd (p ≤ . ). regarding the distribution of paneth cell amps in the gi tract we observed significantly higher expressions of all three paneth cell amps in the duodenum, jejunum and ileum compared to the stomach, colon and rectum (po . , figure ). in the presence of acute gi gvhd, paneth cell amps reacted contrarily in the upper and lower gi tract: we observed a decrease of hd , hd and reg α in the upper gi tract (p ≤ . ), similarly paneth cell count dropped in case of severe gi gvhd stage - (po . ). however in the lower gi tract severe acute gi gvhd was associated with an increase of paneth cell amps (p ≤ . ). initiation of additional systemic antibiotic treatment prior to day after asct correlated with a significantly higher expression of hd (p = . ) and reg α (p = . ) in intestinal biopsies compared to patients without or with initiation of systemic antibiosis after day . however, no significant differences were found in terms of hd expression in intestinal biopsies and start of systemic antibiotic therapy. the expressions of hd , hd and reg α in intestinal biopsies seem to respond to major microbiota disruptions caused by acute gi gvhd or systemic antibiotic treatment. while observations in the upper gi tract seem to reflect paneth cell damage, the relative increase in the lower gi tract may indicate inflammatory induction of amps in colonic epithelial cells in the course of gvhd. [p ] disclosure of conflict of interest: none. patients ( %) were in complete remission at the time of pcy haplo-sct. hematopoietic cell transplantation-comorbidity index was ≥ in patients ( %). thirteen patients ( %) received non-myeloablative conditioning regimen (as baltimore schema, luznik et al. bbmt ) prior to haplosct while remaining patients received busulfan-based regimen. all patients were given pcy and both csa and mmf as gvhd prophylaxis. day+ cumulative incidence of grade to and to acute gvhd was % and %. -year cumulative incidence of chronic gvhd was %. the cumulative incidence of non-relapse mortality and relapse at years were % and %, respectively. with a median follow up of months (range: - ), -year progression-free and overall survivals were % and %, respectively. disease status at the time of haplosct was a major determinant for outcome. indeed, year nrm and os were % and % in patients transplanted with active disease, respectively, while corresponding values in patients transplanted in cr were % (p = . ) and % (p = . ), respectively ( figure a and b) . we can conclude that in selected patients who could be candidate for second transplantation, haplosct is feasible and may represent a curative option. the overall incidence of relapse of % is promising in this situation for which no alternative for cure is available, with relatively good survival in patients transplanted in cr. however, the very high nrm ( %) in refractory patients should make us consider second transplant with caution in this setting. for these patients, specific developments are needed to avoid procedure-related toxicity. [p ] disclosure of conflict of interest: none. secondary solid tumors following hematopoietic cell transplantation for thalassemia major a natale, s santarone, a meloni, a pepe, m di ianni, s angelini, p di bartolomeo dipartimento di ematologia, medicina trasfusionale e biotecnologie-ospedale civile, pescara, italy secondary solid tumors (sst) have been described after hct, in particular for patients affected by hematologic malignancies. there is limited information about the incidence of sst following hct for thalassemia major (tm). the aim of this study was to determine the incidence of sst in patients with tm who received hct in our center between and . patients survived more than years after hct and were enrolled in the study. of them, were males and females. their median age at time of hct was years ( - ). as conditioning regimen, they received busulfan ( mg/kg) and cyclophosphamide ( mg/kg). the gvhd prophylaxis included cyclosporine and methotrexate. all patients received bone marrow cells from an hla identical donor. at time of this report, patients were cured, whereas patients rejected their graft and are now under regular transfusion treatment. overall, the median follow-up after hct was years ( - ). seven patients developed a malignancy . to years (median . years) after hct including carcinomas of the tongue, oral squamous cell carcinoma, colorectal cancer, thyroid carcinoma, carcinoma of the uterine cervix, and parotid carcinoma. the -yr cumulative incidence (ci) of developing sst was + . %. all patients underwent surgical resection of the tumor and in addition of them received chemotherapy and/or radiotherapy. of relevance, the patients with cancer of the oral cavity were affected by severe chronic gvhd with buccal cavity involvement. patients ( with parotid and with tongue carcinoma) died of tumor progression and are living. we compared these results with case control populations. first of all, we investigated the occurrence of solid tumors in the individuals ( males, median age years at time of marrow donation), who served as stem cell donors for hct. one donor developed breast cancer years after marrow donation at age of . the -yr ci of developing solid tumor for donors was . + . % with a statistically significant difference (p = . ) as compared to that of transplanted patients. the second case control population consisted of patients affected by tm treated with transfusions and iron chelation. the matching technique applied was based on the variables age and sex. one control per case (transplanted patient) was randomly selected from the miot (myocardial iron overload in thalassemia) registry and matched by sex and age with the transplanted patient population. patients developed an hepatocellular carcinoma (hcc) at age of and years, respectively. one patient died and one is living. using the event rate measure, we observed an event rate of . at years for the transplant group and . for the nontransplant group (p = . ). this study shows that the magnitude of increased risk of sst is twofold to threefold for patients treated with hct as compared with an age-and sex matched nontransplant tm patients or with stem cell donors. notably, among the transplanted patients we didn't observe any case of hcc, which is one of the most frequent solid tumor in nontransplant tm patients, whereas we observed cases of head/neck cancers. in our series, cgvhd seems to be a strong risk factor in the development of new solid tumors. patients with cgvhd, especially those with involvement of the oral cavity, must receive a very long careful monitoring and surveillance in order to prevent the development of secondary cancers. disclosure of conflict of interest: none. sequential treatment with bortezomib plus thalidomide plus dexamethasone followed by autologous hematopoietic stem cell transplantation (hsct); consolidation and maintenance therapy in patients with multiple myeloma a bachiri , ma bekadja , s talhi , s abderrahmani , h ouldjeriouat and r bouhass department of hematology, hmru oran, oran, algeria; department of hematology and cell therapy, ehu st november, oran, algeria and department of hematology and cell therapy, oran, algeria the management of multiple myeloma (mm) has been significantly improved in recent years in young patients, where ahsct and advent of new molecules was introduced as first line treatment. the sequential treatment (induction followed by autologous hematopoietic stem cell transplantation; consolidation and maintenance therapy) has increased rates response (cr and vgpr) as well as the overall survival. our purpose was to assess the efficacy and adverse effects of sequential treatment with vtd chemotherapy and autologous hsct followed by consolidation and maintenance therapy. in a prospective multicenter study, we evaluated this mm management strategy at oran, in two hematology centers. patients aged under years with de novo mm, were treated with induction included: bortezomib ( . mg/m , d -d -d -d ), thalidomide ( mg/ m d -d ) and dexamethasone ( mg, d -d ; d -d ). a total of to cycles where delivered every days. autologous stem cell was mobilized using g-csf alone ( μg/kg/day for days). leukapheresis to harvest stem cells were performed on day - and - . the conditioning regimen consisted of melphalan mg/m . a consolidation phase was initiated two months later with the same protocol (vtd), followed by a maintenance treatment with thalidomide mg/day given orally for months. this study was done over a -years period (january -december ). fifty patients were included. they include women and men (sex ratio = . ). the median age at diagnosis was years ( - ). according to durie salmon staging, % of patients were in stage iii, while % were in stages iii according to iss staging. the monoclonal component was igg in % of patients. postinduction overall response rate in the eligible patients was %, including % vgpr and % cr/ and % pr rates. the median of cd + rate was . x /kg ( . to ). all patients had engraftment on the median of day (range; to ) and platelet transfusion independence on the median of day (range; to ). there was no graft failure. one patient died following the procedure (trm). posttransplantation on day , cr and at least vgpr remained significantly higher ( %). in the evaluable patients, the estimated os at months was %, the estimated dfs at months was % and the pfs at months was . %. at the / / , ( %) patients are alive and ( %) without disease activity after a median follow-up of months (range; - ). the main hematological toxicities post transpland (grade / ) were thrombocytopenia ( %), neutropenia ( %), and anemia ( %). the most frequently observed nonhematological toxicities (all grades) included peripheral neuropathy ( %). our experience suggests that the sequential protocol used in first line produce a better outcome with fewer adverse events and is an interesting therapeutic option in term of efficacy and tolerance. disclosure of conflict of interest: none. micrornas are small, non-coding single-stranded rnas and regulate approximately % of all genes by repressing translation. they are present in bodily fluids, where they are protected from rnase-mediated degradation by encapsulation into extracellular vesicles (evs) and demonstrate a novel capacity to regulate the cellular differentiation of blood cells and immune function. candidate micrornas mir- , mir-- , mir- * and mir- have previously been associated with acute graft versus host disease (agvhd) in posthematopoietic stem cell transplant (hsct) patient plasma. however, validation in independent cohorts is necessary, and their presence within extracellular vesicles (evs) has not been explored. microrna expression was evaluated in a prognostic cohort (n = ) of day (d ) post-hsct patient serum samples by taqman qrt-pcr. further assessment in an independent cohort of serum samples taken at the time of agvhd diagnosis was also performed. expression was also assessed in serum evs at sequential time points (pre-hsct, d , d and d ) and an independent verification cohort of d serum samples by ev isolation, rna extraction and taqman qrt-pcr analysis. this study replicated elevated serum expression of mir- (po . ), mir- (p = . ), mir- * (p o . ) and mir- (p = . ) in agvhd, in a prognostic cohort of d post-hsct patient samples (n = ). expression was also associated with disease severity. further analysis at agvhd diagnosis in an independent cohort (n = ) confirmed high expression of mir- (p = . ), mir- (p = . ) and mir- * (p = . ) at disease onset. investigation of microrna expression patterns during early hsct at sequential time points (pre-hsct to d ) identified elevated micrornas at d post-hsct in all transplant patients. in a novel investigation of microrna expression in serum evs (n = ), mir- (p = . ), mir- (p = . ) and mir- * (p = . ) levels were lower at d in patients who later developed agvhd, and this was replicated for mir- (p = . ) and mir- (p = . ) (n = ). comparing serum to circulating evs, at d patients remaining agvhd-free had significantly higher expression of mir- (p = . ), mir- (p o . ) and mir- * (p = . ) in the ev fraction. results validate the capacity for circulating serum mir- , mir- and mir- * to act as diagnostic and prognostic biomarkers for agvhd. novel findings of differential expression between whole serum and the ev compartment prior to disease onset suggest a role for ev micrornas in the biology of agvhd, which warrants further investigation. disclosure of conflict of interest: none. prior data indicate similar outcomes after transplants from hla-haplotype-matched relatives, hla-idntical siblings and hla-matched unrelated donors. we used our dataset to answer a clinically important question: who is the best donor for a person with acute leukemia. we analyzed data from persons with acute leukaemia in st complete remission treated in a prospective, multi-centre study. patients were randomly divided into training (n = ) and validation (n = ) sets. consecutive subjects received a transplant from an hla-haplotype-matched relative (n = ) or an hlaidentical sibling (n = ). -year leukaemia-free survivals (lfss) were % ( % confidence interval [ci], , %) and % ( , %; p = . ). the multivariate model identified major risk factors for transplant-related-mortality (trm): older donor/recipient age (donor years/recipient years; hazard ratio [hr] = . ; [ . , . ]; p = . ), female-to-male transplants (hr = . ; [ . , . ; p = . ) and donor-recipient abo major-mismatch transplants (hr = . [ . , . ; p = . ). a risk score was developed based on these three features. trms were % ( , %), % ( , %) and % ( , %) for subjects with scores of - , and (p o . ). year lfs were % ( , %), % ( , %), and % ( , %; p = . ). the risk score was validated in an independent cohort. in recipients years, lfss were % and % (p = . ) after transplants from identical-sibling or children. our data confirm donor source or degree of hla-disparity is not significantly correlated with transplant outcomes. selection of the best donor needs to consider donor-recipient age, sex-matching and abo-incompatibility amongst persons with acute leukemia receiving transplants from family members. [p ] disclosure of conflict of interest: none. synergistic effect of kir ligands missing and cytomegalovirus reactivation in improving outcomes of haematopoietic stem cell transplantation for treatment of myeloid malignancies d cardozo, a marangon, r da silva, fj aranha, j visentainer, s bonon, s costa, e miranda, c souza and f guimarães. the lack of one or more hla class i alleles, whose protein products are the ligands for kir receptors, has been exploited as a prognostic factor for the outcome of patients with haematological malignancies treated by haematopoietic stem cell transplantation (hsct). although it has been accepted that kir-hla interactions may influence the outcome of the hlamismatched hsct, there is no consensus regarding the settings of hla-matched transplantation. there are studies that have reported either benefits, or no effects, under the influence of inhibitory kir-hla interactions. additionally, certain activating kirs and/or reactivation of cytomegalovirus (cmv) infection have been reported to affect the outcome of hla-matched transplantation. the goal of this study was to evaluate the influence of kir-hla genotypes on the outcome of patients undergoing treatment for haematological malignancies by non-t-depleted lymphocyte haematopoietic stem cell transplantation (hsct) from hla-matched sibling donors. the prospective study was conducted at the center of hematology, university of campinas, and patients and their donors were followed up from to . kir and hla class i genes were genotyped and patients grouped based on the presence of kir ligands combined with kir genotype of their respective donors. patients with all kir ligands present (n = ) had a significantly higher (p = . ) incidence of acute graft-versus-host-disease (gvhd) than patients with one or more kir ligands missing (n = ). the overall survival following transplantation of patients with myeloid malignancies (n = ) was significantly higher (p = . ) in the group with one or more kir ligands missing (n = ) than in the group with all ligands present (n = ). presence of kir ds was associated with a worsening of hsct outcome while reactivation of cytomegalovirus (cmv) infection improved the outcome of patients with one or more kir ligands missing. our results indicate that kir-hla interactions affect the outcome of the hla-matched transplantation, particularly in patients with myeloid malignancies. disclosure of conflict of interest: none. p = . ), lower disease-free survival (p = . and p = . ) and lower overall survival (p = . and p = . ). one-year cir of the above two groups were . ± . % vs. . ± . % in mrd negative and positive patients, respectively (p = . ). in addition, those who had consistent positive mrd prior to hlamatched sibling hsct showed even worse outcomes compared to patients without pre-mrd. unmanipulated haploidentical hsct might have the stronger graft-versus-leukemia effect compared to hla-matched sibling hsct. it suggested that those who received unmanipulated haploidentical hsct with pre-mrd might not need more intensive relapse intervention after transplantation. disclosure of conflict of interest: none. the retrospective study of allogeneic hematopoietic cell transplantation for patients with mixed-phenotype acute leukemia in toranomon hospital, japan in the real clinical setting, however, there are substantial number of patients who can not achieve cr after chemotherapy. we conducted a retrospective study including such patients to elucidate the outcome of allogeneic hct in toranomon hospital, japan. we studied the patients with mpal diagnosed from july to september . mpal was diagnosed according to who classification in . from june , we examined cytoplasmic myelo-peroxydase (cmpo) routinely for flowcytometric analysis in all the patients, to distinguish mpal from acute lymphoblastic leukemia (all). we included the patients who were diagnosed as mpal in toranomon hospital, regardless of their diagnosis or clinical course in the previous hospitals. a total of mpal patients underwent their first allogeneic hct with related bone marrow or peripheral blood stem cells (r-bm/pb) (n = ), unrelated bone marrow (u-bm) (n = ), and unrelated umbilical cord blood (u-cb)(n = ). the median patient age was years (range: - ). the immunophenotype of leukemia cells included cases of b and myeloid (b/my) ( %) and cases of t and myeloid (t/my) cell lineage( %).eleven patients( %) harbored philadelphia chromosome. the remission induction chemotherapy was performed with all-type regimens in patients, and acute myeloid leukemia (aml)type regimens in of patients, patients( %) were not in cr at the time of transplantation. myeloablative conditioning (mac) regimens were used in pantients( %). the -year overall survival (os) rate was . % ( % confidence interval (ci), . - . %). to identify the factors that influenced os, we performed univariate analysis and compared the following pre-transplantation factors: age at the time of transplantation ( o vs. = years), committed immunophenotype (b/my vs.t/my), karyotype (philadelphia chromosome (ph vs.non-ph), disease status at the time of transplantation (cr vs.non-cr), donor cell source (r-bm/pb vs.u-bm vs.u-cb, cb vs.non-cb), and conditioning regimen (mac vs.reduced intensity conditioning). cr at the time of transplantation was extracted as a significant predictive factor for the better os( -year os; cr vs. non-cr, . % ( % ci, . - . %) vs. . % ( % ci, . - . %), p = . ). the cumulative incidence of relapse rate (rr) at years after transplantation was . % ( % ci, . - . %). to identify the factors that influenced relapse rate, we performed univariate analysis and compared pretransplantation factors same as above. harboring philadelphia chromosome was extracted as a significant predictive factor for lower relapse rate ( -year rr; ph vs.non-ph, . %( % ci, . - . %) vs. . %( % ci, . - . %), p = . ). the older patients(p = . ) and the patients in cr (p = . ) also showed a trend towards lower relapse rate. allogeneic hct provided . % of -year os for mpal patients in cr at the time of transplantation. on the other hand, for patients not in cr, year os was approximately %. the use of tyrosine kinase inhibitors along with chemotherapy before transplantation might prevent relapse after transplantation in mpal patients with ph chromosome. disclosure of conflict of interest: none. allogeneic hematopoietic stem cell transplantation (allo-hsct) is a standard of treatment for many patients with hematological malignancies. however, the disease relapse and graft failure after first allo-hsct ( st allo-hsct) lead to poor outcomes almost in all cases. second allo-hsct ( nd allo-hsct) is one of primary options that can decrease the mortality in this group of patients. here we report our experience of patients who underwent nd allo-hsct. the aim of the study was to estimate a clinical efficiency and practicability of nd allo-hsct. we included patients ( males/ females) with acute myeloid leukemia (aml, n = ), acute lymphoblastic leukemia (all, n = ) and myeloproliferative disease (mpd, n = ) who underwent nd allo-hsct for relapse ( , %) or graft failure ( , %) from the same (n = ) or another donor (n = ) between november and october . median age was years (range: - years). three ( %) patients had a matched related donor (mrd), nine ( %) patients had a matched unrelated donor (mud) and three ( %) patients had a mismatched unrelated (mmud) at the second transplant. to evaluate time gap affecting outcomes all patients were divided into two groups: who underwent nd allo-hsct in more/less than months after st allo-hsct. in "less than months" group three patients were re-transplanted for relapse and one-for graft failure, in other group there were seven patients who received nd allo-hsct for disease relapse and four-for graft failure. fisher's exact test were performed to exclude probability of imbalance between groups (p . ). median of overall survival (os) and disease-free survival (dfs) after nd allo-hsct was . months and . months respectively. (see figure a , c) two patients ( . %) developed graft failure and three relapsed ( %). acute graft-versus host disease (agvhd) incidence was extremely low as . % (n = ) even despite use of mud/mmud in % of cases. mortality rate were . % in a group of nd allo-hsct. it should be noted that only ( %) patients died because of disease progression. five patients ( . %) died in complete remission due to severe infections or previous toxicity (e.g. heart failure). the effect of donor change on dfs was not significant (p = , ). our statistical analysis reveal significantly differences in os in patient with long-term interval ( months) between st and nd allo-hsct. median of os in patients who underwent nd allo-hsct in more/less than months after st allo-hsct was , vs , months respectively. (see figure b , d) for hazard ratio (hr) estimation mantel-haneszel approach were used hr for group who were transplanted in less than months from st allo-hsct was . , ( % ci, . s to . , p = . ). as for dfs difference was not significant (p = . ). according to our analysis, performing nd allo-hsct in a period less than months after st allo-hsct seemed not very reasonable due to extremely high mortality even in young patients (hr- . , p = . ). as for "more than months" group it can be considered even despite hla-disparity between donor-recipient pair due to extremely low agvhd rate ( . %). donor change was not associated with better outcome (p = . ) disclosure of conflict of interest: none. hemopoietic stem cell transplantation (hsct) is an effective treatment for many hematologic disorders, and globally over procedures/year are performed in more than countries. however, not all the countries have enough resources and expertise to establish an hsct program, and patients are often forced to emigrate for transplantation, with heavy social and economic consequences. in the year the iuc (an italian ngo) identified the hiwa cancer hospital (hch) in sulaymaniya (iraqi kurdistan) as a possible site for the establishment of a new hsct transplant center. a hsct expert from italy (mi) following a visit to the hch, reported a positive conclusion on the feasibility of an hsct project. this was mainly due to the fact that many of the required technologies were already available at hch, including a -bed positivepressure, hepa-filtered-air clinical unit, last-generation cell separators and a well equipped hla laboratory. following this preliminary survey, a capacity building project was rapidly made and submitted to the italian agency for development cooperation, that approved and funded it in march with the specific aim to cure thalassemia patients either of kurdistan and of the refugees population from syria and other parts of iraq. in april , the joint italian and kurdish team started the project. a first autologous transplant was done in june followed by more autografts (overall, myeloma and lymphoma patients). in october, following appropriate downstaging, a first low-risk thalassemia patient was allografted from her hla-id sibling, followed by more patients. all the patients engrafted promptly, with one death occurring on day + with acute cardiac failure and a major toxicity recorded in a single patient (nhl, severe enterocolitis with perforation) that was successfully treated. the full process for the start-up included the following activities developed during -month time: ( ) s of transplants, the hch group also submitted to ebmt an application for full membership, that was promptly approved. in all this project, the italian counterpart provided over highly-experienced volunteer specialists (physicians, nurses, technicians and one physicist), each with a specific mission plan. despite the many difficulties and obstacles encountered, the clinical results obtained so far appear encouraging, though there is still need to furtherly support the hch in order to make it totally independent. following this intervention, the hch is the only one center performing both auto and allo hsct not only in the iraqi kurdistan region, but also in all the iraqi nation. we conclude that international cooperation may be fruitful also in the field of high-technology medicine, and may contribute to improve the capabilities of centers even in critical geographic areas, representing a valuable instrument also to implement nation-to-nation scientific exchanges. disclosure of conflict of interest: none. the use of plerixafor with g-csf in conditioning regimen for hematopoietic stem cell transplantations with tcr alpha/beta and cd depletion of graft in wiscott-aldrich syndrome patients: a single-center experience b dmitry , l alexandra , s larisa , g elena , s irina , t pavel , k rimma , n galina , m michael and m alexei grade acute gvhd (agvhd) was % ( pts). no pt experienced a grade agvhd. three patients presented a limited form of chronic gvhd ( %). incidence of oral mucositis and gastrointestinal/liver toxicity has been extremely low in this population of patients, even in those with active disease and heavily treated at the time of transplant. eight out of fifteen pts ( %) are alive with a median follow-up of months (range: - m). seven ( %) are in cytogenetic/molecular remission. six out the eight patients who were transplanted in cr or cr are alive ( %), while two out the seven patients who were transplanted in advanced phase are alive ( %). in this preliminary clinical experience, we find that unmanipulated haploidentical transplants with post-transplant cyclophosphamide are a valid alternative and have outcome comparable to unrelated and match sibling transplants, in pts with hematologic malignancies. advanced disease is the only adverse factor for diseasefree survival. we therefore consider this therapeutic option when a match sibling or a / ag mud donor is not immediately available. disclosure of conflict of interest: none. autism spectrum disorder (asd) is a group of neurodevelopmental disorders characterized by impaired social communication and interactions with restricted and repetitive behaviors. although asd is suspected to have either heritable or sporadic genetic basis, its fundamental etiology and pathogenesis are poorly understood. recently researchers have suggested that stem cells have therapeutic potential for asd. wharton's jelly-derived msc (wj-msc) from third-party donors (tpd) have high proliferation and differentiation potential. this cell population has also non-immunogenic and immunomodulatory properties, thus seem to be a promising treatment stem cell source. the polish stem cell bank (pbkm) has provided wj-msc for clinical application in a medical therapeutic experiment for children with asd. twenty-three patients (pts) with asd aged from to . / (median age: years and months), after bioethical committee approval, received intravenous injections of wj-msc, obtained from tpd. the cells were previously collected from healthy newborns, then processed, screened for bacterial contamination as well as endotoxin content, and frozen in liquid nitrogen vapour. wj-msc immunophenotype was confirmed using flow cytometry assay. the pts received from to injections in intervals from to weeks. the average cell dose per infusion was . × ^ /kg of body weight (bw). each pt was examined by the same neurologist at the day of infusion. comorbidities present in some patients: unspecified speech disturbances, flaccid paralysis, flaccid tetraplegia, unspecified encephalopathy, epilepsy, sensorineural hearing loss. one patient was diagnosed with comorbidities: conductive hearing loss and intellectual disability. almost % of pts, after their treatment with wj-msc, revealed positive changes in neurological examination. an improvement in speech was observed in pts and improvement of cognitive functions ensued in pts. what is more, % of children showed progress in self-reliance, social interactions and improved their ability to concentrate. there was a reduction of aggressive behavior in pts and pts have experienced better quality of sleep. there was only one adverse event after wj-msc infusions -psychomotor agitation occurred in hours after the administration. five follow-ups have not yet been completed. the administration of thirdparty donor wj-msc seems to be safe and efficient procedure with promising preliminary results in patients with asd. hematopoietic stem cell transplantation between red cell incompatible donor-recipient pairs red blood cell depletion from bone marrow and peripheral blood buffy coat: a comparison of two new and three established technologies human bone marrow processing using a new continuous-flow cell separation device disclosure of conflict of interest: none. references . zama d, et al. gut microbiota and hematopoietic stem cell transplantation: where do we stand? bmt the kyoto encyclopedia of genes and genomes-kegg metabolites produced by commensal bacteria promote peripheral regulatory t-cell generation disclosure of conflict of interest: none antifungal prophylaxis in hematopoietic stem cell transplant recipients: the unfinished tale of imperfect success guidelines for preventing infectious complications among hematopoietic cell transplantation recipients: a global perspective differences in aspergillus-specific immune recovery between t-cell-replete and t-cell-depleted hematopoietic transplants toxoplasmosis following allogeneic hematopoietic stem cell transplantation diagnosis of toxoplasmosis after allogeneic stem cell transplantation: results of dna detection and serological techniques implementation of molecular surveillance after a cluster of fatal toxoplasmosis at neighboring transplant centers management of high blood pressure genes for blood pressure a prospective studyon the predictive value of plasma bk virus-dna load for hemorrhagic cystitis in pediatric patients after stem cell translantation cidofovir for bk virusassociated hemorrhagic cystitis:a retrospective study hemorrhagic cystitis after bone marrow transplantation bcsh/bsbmt guideline: diagnosis and management of veno-occlusive disease (sinusoidal obstruction syndrome) following haematopoietic stem cell transplantation drug safety evaluation of defibrotide defibrotide for prophylaxis of hepatic veno-occlusive disease in paediatric haemopoietic stem-cell transplantation: an open-label, phase , randomised controlled trial safety and effects of prophylactic defibrotide for sinusoidal obstruction syndrome in hematopoietic stem cell transplantation disclosure of conflict of interest: none university children's hospital basel, division of paediatric oncology/haematology late complications subcommittee of translated related complications and quality of life wp; clinic of paediatric haemato-oncology, department of women's and children's health, university of padova, italy; department of surgery, division of transplantation division of blood and marrow transplantation, the children's hospital at westmead ovarian function after bone marrow transplantation during childhood pregnancies following high-dose cyclophosphamide with or without high-dose busulfan or total-body irradiation and bone marrow transplantation unmanipulated haploidentical bone marrow transplantation and posttransplantation cyclophosphamide for hematologic malignancies after myeloablative conditioning haploidentical hematopoietic transplantation:current status and future perspectives t-cell replete haploidentical donor transplantation using post-transplant cy: an emerging standard-of-care option for patients who lack an hla-identical sibling donor hematopoietic stem cell transplantation in thalassemia major and sickle cell disease: indications and management recommendations from an international expert panel allogeneic stem cell transplantation for thalassemia major killer-cell immunoglobulin-like receptors reactivity and outcome of stem cell transplant kir b haplotype donors confer a reduced risk for relapse after haploidentical transplantation in children with all kir/hla interactions negatively affect rituximab-but not ga (obinutuzumab)-induced antibody-dependent cellular cytotoxicity reduction of minimal residual disease in pediatric b-lineage acute lymphoblastic leukemia by an fcoptimized cd antibody diagnoses: hodgkin's lymphoma(hl)- pts (refractory ; relapsed ); non-hodgkin's lymphoma(nhl disease status before asct: st (after refractority prior radiotherapy to the mediastinum - / ( . %); heavily pretreated patients with advanced disease (x lines previous treatment) / ( . %). grafts: pbsc - / pts with median of cd +cells- ccnu dose: / pts mg/m ; pt mg/m . engraftment: anc> cells/mkl: median=d+ ( ÷ ), / pts. plt> cells/ mkl: median=d+ ( ÷ ), / pts. full engrafted / pts / pt required a short-term mechanical ventilation ( of them died because of lung infection ad d+ and d+ ) aeruginosa associated sepsis on a background of graft failure); pts ( . %)-d+ and d+ (pulmonary toxicities +infection; both had prior mediastinal radiotherapy). relapse/ progression after asct- / pts ( . %), of them died. pt achieved secondary mds (diagnosed . mo after asct). for this group of pts with relapsed/refractory lymphomas (n= ) -year os= for nhl(n= ) efs= . (se ± . ) lomustine-containing conditioning regimen cem (lomustine, etoposide, melphalan) is effective and feasible in autologous stem cell transplant efficacy and toxicity of a ccnu-containing high-dose chemotherapy regimen followed by autologous hematopoietic cell transplantation in relapsed or refractory hodgkin's disease champlin re reduced-toxicity conditioning therapy with allogeneic stem cell transplantation for acute leukemia idarubicin-intensified bucy conditioning regimen improved survival in high-risk acute myeloid, but not lymphocytic leukemia patients undergoing allogeneic hematopoietic stem cell transplantation: a retrospective comparative study comparison of outcomes of idarubicin intensified tbi-cy and traditional tbi-cy conditioning regimen for high-risk acute lymphoblastic leukemia undergoing allogeneic hematopoietic stem cell transplantation: a single center experience inhibition of cd (il- r alpha) expression and t-cell proliferation by polyclonal anti-thymocyte globulins csf-primed bone marrow transplantation for patients with high-risk hematologic malignancies in an exploratory analysis, os after hct appeared to be longer in the cpx- arm in both age groups. these results suggest that cpx- may provide an effective bridge to successful transplant for a high-risk subgroup of aml patients. support: celator pharmaceuticals, inc., a subsidiary of jazz pharmaceuticals plc consulting ambit biosciences, amphivena therapeutics, ariad, astellas pharma sunesis, tolero; institutional research funding abbvie chiarella and louie: employment celator/jazz; stock jazz pharmaceuticals plc. hoering disclosure of conflict of interest: none. inkt-/nk-/cik-cell (subsets) are important for immunesurveillance. antibody b targets the vα -jα -invariant-t-cell-receptor (tcr) in the cdr -region, which is semiinvariantly rearranged in inkt-cells. we characterized: i.) inkt-/nk-/cik-subsets in pb-samples from healthy donors (n = subsets under stimulation with dendritic-cells of leukemic origin (dc leu ), generated from aml-blasts in mononuclear cells(mnc) and whole-blood (wb, containing soluble/cellular components of pts' pb) with 'cocktails' (dc-generating-methods/kits). . ) compared to healthy mnc (significantly) lower proportions of inkt-cells comparable correlations were seen in adultall-and cll-pts. . ) we quantified inkt-/nk-/cik-subsets before/after mixed-lymphocytecultures (mlc) of t-cell-enriched immune-reactive cells stimulated with mnc/wb (with or without pretreatment 'cocktails' inducing blasts' conversion to dc leu ) from aml-pts. our findings show, that )inkt-/nk-/cik-cells increase after mlc independent of the stimulator-cells-suspension (under the influence of il- ); ) pretreatment of mnc/wb-blasts with 'cocktails' increases inkt-counts and induces a shift in the composition of inkt-/nk-/cik-subsets after mlc, that might correlate with an improved antileukemic potential; ) individual samples showed varying, however higher inkt-, cik-cell-counts after pretreatment with different (especially prostaglandin-containing) 'cocktails'; ) dc-/inkt-/nk-/cikcells-values after mlc were comparable in physiological hypoxia vs normoxia; ) in cases with antileukemic blast-lytic activity after mlc t-/inkt-/nk-/cik-cells were significantly increased-pointing to an involvement of these cells in antileukemic reactions. in summary: ( ) healthy mnc present with significantly higher inkt-/nk-/cik-cells compared to aml/all/cll-leukemic mnc. ( ) subtypes of inkt-cells differ in healthy vs leukemic samples, resembling a shift in the composition of inkt-cells. ( ) amounts of inkt-/ nk-/cik-cells in aml/all/cll-mnc-samples correlate with prognosis. ( ) 'cocktail'-treated aml-blasts (resulting in dc leu ) lead to a shift in t-,inkt-/nk-/cik-cell-counts/compositions, what correlates with improved antileukemic activity against aml-blastspointing to a cross-talk of these cells. proportions of inkt-/ nk-/cik-cells management of philadelphia chromosome-positive acute lymphoblastic leukemia (ph+ all) outcome of allogeneic stem cell transplantation for aml and myelodysplastic syndrome in elderly patients (⩾ years) comorbidity-age index: a clinical measure of biologic age before allogeneic hematopoietic cell transplantation high rate of hematological responses to sorafenib in flt -itd acute myeloid leukemia relapsed after allogeneic hematopoietic stem cell transplantation phase i trial of maintenance sorafenib after allogeneic hematopoietic stem cell transplantation for fms-like tyrosine kinase internal tandem duplication acute myeloid leukemia haematopoietic cell transplantation with and without sorafenib maintenance for patients with flt -itd acute myeloid leukaemia in first complete remission quantitative monitoring of minimal residual disease (mrd) after sct was performed by four-colour flow cytometry and/or real time pcr. the median time of neutrophil engraftment (above . × e /l) was days, % of pts ( / ) engrafted, one patient died in aplazia. non-relapse mortality (nrm) after year and years was % ( / ) and % ( / ). causes of death were refractory gvhd (n = ), infection (n = ) and multiorgan failure (n = ). incidence of acute gvhd was evaluated in pts: % ( / ) of pts had gvhd (grade i+ii in pts, grade iii in pts). incidence of chronic gvhd was evaluated in pts, % ( / ) of pts had gvhd with median follow-up from sct months (range: - ), % of all pts ( / ) were alive ( in remission of cll with mrd negativity, with relapse), pts died ( from nrm, from cll relapse/progression), relapses ( %; / ) occurred. sequential use of chemotherapy and ric regimen with allogeneic sct is safety and effective treatment of high-risk cll with reponse rate % and low nrm. progression-free survival and overall survival at years from sct were % and % department of hematology, hemostasis, oncology and stem cell transplantation hannover deutsche klinik für diagnostik helios klinik wiesbaden, germany; imperial college london at hammersmith hospital du cane road centre for haematology london disclosure of conflict of interest: none. references . sibon d, brice p. optimal treatment for relapsing patients with at our institution, pr-hl is defined as partial response (pr), no response (nr), stable disease (sd), progressive disease (pd), relapsing within months of finishing the planned treatment. progression free (pfs) and overall survival (os) from the day of auto-sct was estimated by kaplan-meier (km) method. from to , patients with aethera trial criteria were identified. male ( %), female ( %), median age at diagnosis: yrs ( - ), at auto-sct: . yrs ( . - )( % o yrs). initial chemo: abvd in ( %). ( %) had radiation therapy (xrt) after initial chemo. response to initial chemo + xrt was refractory disease: ( %), relapse between - months: ( %) and relapse after months: ( %) aethera had % stable disease before sct vs we have only %. aethera months pfs ( % control arm, % brentuximab arm, investigator assessment) and our . % is not much different. despite having similar selection criteria, our median pfs is higher than both aethera trial placebo and experimental arm. clinically, rate of progression in both studies are very high and comparable at months. given the very high cost of this drug and while waiting for survival fifty-nine ( %) and ( %) patients had relapsed and primary refractory chemosensitive dlbcl, respectively. secondary ipi was - in ( %) patients, in ( %) patients and - in ( %) patients. fifty-one ( %) and ( % patients had gcb and abc tumors, respectively. abc patients received more prior lines of chemotherapy than gcb patients ( % vs % received lines of chemotherapy, p = . ). the rest of characteristics were equally distributed between both groups (table ) disclosure of conflict of interest: none disclosure of conflict of interest: none. p upfront autologous stem cell transplantation in patients with diffuse large b cell lymphoma: focused on risk factors for survival and conditioning regimens ds kim association between complete response and outcomes in transplant-eligible myeloma patients in the era of novel agents e jantunen and v varmavuo department of medicine disclosure of conflict of interest: none. and hematology department lenalidomide after stem-cell transplantation for multiple myeloma bortezomib induction and maintenance treatment in patients with newly diagnosed multiple myeloma: results of the randomized phase iii hovon- /gmmg-hd trial disclosure of conflict of interest: none we performed a retrospective study to investigate survival outcomes and toxicities of l maintenance therapy compared with b maintenance in mm patients post-ahct. this study included patients who received ahct for mm between and after induction with l-or b-based therapy. all patients received ahct within months of mm diagnosis and received melphalan mg/m conditioning. patients who received tandem transplantations (autologous or allogeneic) were excluded. only patients initiating maintenance therapy within months post-ahct were included. maintenance therapy was defined as monotherapy with either l or b. the primary outcome was pfs. secondary outcomes were overall survival (os) and treatment-related toxicities. patients received l maintenance and b maintenance post-ahct. at baseline there were no differences in iss stage, ds stage or cytogenetic risk between maintenance cohorts. at time of analysis, % (n = ) receiving l maintenance and % (n = ) on b maintenance experienced disease progression. median time to progression ( . vs . yrs, p = . ) was not significantly different between cohorts. by multivariable analysis, choice of maintenance (l vs b) was not significant for pfs or os. variables significant for improved pfs were iss stage i disease response improved while on maintenance in % (n = ) with l and % (n = ) with b. median os was not statistically different between maintenance cohorts ( . vs . yrs, p = . ). iss stage i/ii vs iii while cytopenias were more common in the l cohort ( % vs %, p o . ). the median follow-up time for survivors was months. these findings suggest that both lenalidomide and bortezomib are equivocal maintenance therapy options for post-transplantation mm patients. choice of maintenance therapy post-ahct for mm did not demonstrate a difference in survival outcomes. based on these data, maintenance choice should be guided by patient specific anticipated tolerance rather than drug type alone. iss stage and post-ahct disease response continue to be significant predictors for outcomes. toxicities recorded on maintenance were as anticipated. length of maintenance therapy may be a significant predictor and warrants further analysis. the analysis was underpowered to disclosure of conflict of interest: none. p real-world multiple myeloma management practice patterns and outcomes in six central and eastern european countries d coriu , d dytfeld , d niepel , i spicka second autologous stem cell transplantation as salvage therapy for multiple myeloma: impact on progression free survival and overall survival second autologous stem cell transplantation: an effective therapy for relapsed multiple myeloma second auto asct for treatment of relapsed multiple myeloma the role of second autografts in the management of myeloma at first relapse moving beyond autologous transplantation in multiple myeloma ebmt data office bortezomib-based versus nonbortezomib-based induction treatment before autologous stem-cell transplantation in patients with previously untreated multiple myeloma: a meta-analysis of phase iii randomized, controlled trials first-line therapy with thalidomide and dexamethasone in preparation for autologous stem cell transplantation for multiple myeloma mechanism of action of bortezomib and the new proteasome inhibitors on myeloma cells and the bone microenvironment: impact on myeloma-induced alterations of bone remodeling boys; girls) with following mds types: refractory cytopenia of childhood- ( %), refractory anemia with excess blasts - pts ( %), refractory anemia with excess blasts in transformation- pts ( %), juvenile myelomonocytic leukemia in pts ( %). the median of age was years ( - years) mac consisted busulfan (bu) mg/kg + cyclophosphamide mg/kg. ric included fludarabine (flu) mg/m + melphalan (mel) mg/m , flu - mg/m + bu mg/ kg. the bone marrow (bm) was used in pts ( %), peripheral blood stem cells (pbsc) in pts ( %), combination of bm and pbsc in pts ( %). -years overall survival (os) was % os was in pbsc group - %; bm group- %, combination of bm and pbsc- % there were two cases of mds, eb- , although erythroid aberrancy can not be found, fc did disclose significant aberrancy on myelomonocytic lineages. on the other hand, all the normal control bm samples revealed no any erythoid phenotypic abnormality. our study suggests this simplified cocktail of -tube, -color, fc is very sensitive and useful in the assessment of erythroid phenotypic abnormalities in mds we analyzed consecutive patients ( % were female, median age of (range: - ) allografted for mds (median ebmt risk score of , median disease risk index of intermediate risk) over a -year period ( - ) with mac conditioning for % and ric for % pfs ± %, grfs ± %, ri ± % and trm similarly, there was not difference between tdep and non tdep patients for -years pfs ( ± % and ± %, p value . ), -years gfrs ( ± vs ± , p value . ) (graph), -years ri ( ± % and ± %, p value . ) and -years trm ( ± % and ± %, p value . ). finally, tdep had no significant impact on -years grade - agvhd when compared to the non tdep ( ± % and ± %, p value . ). it had not either on -years cgvhd ( ± % and ± %, p value . ). our study shows that tdep is feasible on patients undergoing hsct for mds disclosure of conflict of interest: none. p mutational pathway and dynamics may not be prognostic in patients with myelodysplastic syndrome receiving hypomethylating agent pre-treatment for allogeneic stem cell transplantation republic of korea; department of computer science; the donnelly center for cellular and biomolecular research amebiazis after bone marrow transplantation use of a five-agent gvhd prevention regimen in recipients of unrelated donor marrow impact of age on outcomes after bone marrow transplantation for acquired aplastic anemia using hla-matched sibling donors treatment of acquired severe aplastic anemia: bone marrow transplantation compared with immunosuppressive therapy-the european group for blood and marrow transplantation experience disclosure of conflict of interest: none. leukemia, myelodisplastic syndrome, juvenile myelomonocytic leukemia and chronic myelomonocytic leukemia s bondarenko hla-mismatched unrelated (n = , %), and haploidentical (n = , %) donors. response was achieved in % (n = ) of pts after - (median ) courses of hma therapy: complete remission (cr) in ( %), partial remission (pr) in ( %) of pts. stabilization (s) was documented in ( %) pts, in ( %) pts there was disease progression (p) after beginning of hma therapy mismanaging the gift of life: noncompliance in the context of adult stem cell transplantation l'adhésion thérapeutique et at. des lieux en allogreffe de cellules souches hématopoïétiques (csh) dans des services de pédiatrie et d'adulte. rapport de la sfgm-tc predictive validity of a medication adherence measure in an outpatient setting data is limited to small case series, transplant registries and a single prospective multicenter observational study. here we report our institutional experience with auto-hct in patients with hrl. twenty patients with hrl [non-hodgkin = ( %), hodgkin = ( %)] and treatable hiv infection underwent hdt consisting of carmustine, etoposide, cytarabine and melphalan (beam) followed by peripheral blood auto-hct from / to / . in cases rituximab was administered as part of the preparative regimen. patient-, disease-, and transplant-related characteristics are summarized in table . median age was years (range: - ). the median follow-up for surviving patients was months (range: - ) abbreviation: n: number of patients; m: male gender; auto-hct: autologous hematopoietic cell transplant; nos: not otherwise specified; dlbc: diffuse large b-cell lymphoma ara-c), melphalan; cr : first complete remission; cr :second complete remission disclosure of conflict of interest: none. p incidence of secondary primary malignancies (spm) in patients with multiple myeloma m curly , g laurent and k nicolaus city of hope igm ( . %), lines of induction regimens prior to hsct one in pts ( %), two in pts ( . %), in pts ( . %), and missing in pts ( %). induction regimens included imids and proteasome inhibitor (pi)s with alkylating agents in pts ( . %), imids and pis with no alkylating agents in ( . %), and alkylating agents with no imids or pis in ( . %) and missing data in ( %). radiotherapy was used pre hsct in pts ( . %), no radiation in pts ( %) and missing data in ( . %). plerixafor (p) was administered mostly for poor hsc mobilization as defined by the centers number of hsc collected o × in pts ( . %), - in pts ( . %), × in pts ( %), and data missing in ( %). the number of cd + hsc infused o × in pts ( %), - × in pts ( % , × in pts ( %), and missing in ( %). a total of pts developed spm with cumulative incidence of . % ( %ci . , . ) at mo. data are missing in pts ( %) use of radiotherapy, type of induction, hsc cell dose did not influence the cumulative conflict of interest: f. sahebi, none declared, s. iacobelli, none declared, l. koster none declared l. gardaret none declared, n. kroger received research fund from sanofi, curly morris, none declared p interaction between center effect and strategy for gvhd prophylaxis on outcome of t-cell depleted and t-cell replete haploidentical transplant inserm u ecstra team expanding transplant options to patients over years-improved outcome after reduced intensity conditioning mismatched-unrelated donor transplantation for patients with acute myeloid leukemia: a report from the acute leukemia working party of the ebmt nkg d ligands in tumor immunity comprehensive analysis of nkg d ligand expression and release in leukemia: implications for nkg d-mediated nk cell responses nkg d cars as therapy for cancer russian federation high incidence of mixed chimerism with impaired graft function remains a significant issue in patients with wiskott-aldrich syndrome (was) after hsct. simultaneous use of plerixafor with g-scf is efficient in inducing stem cell release and opening of bone marrow niches. the use of plerixafor/g-csf in conditioning demonstrates better levels of donor chimerism in patients with acute myeloid leukemia. we report our experience of plerixafor/g-csf usage in patients with was as an addition to myeloablateive conditioning to improve stem cell engraftment p = , . events were considered: death in patients, graft rejection in patients, mixed myeloid chimerism (less than % donor) in patients. median time of event was , months after hsct ( . - . ) all patients are alive, median fu is months, range: . - . . patients had acute gvhd: -grade (gut), -grade (skin), in both cases resolved after a short course of steroids. all patients had more than % donor chimerism monthly till the time of last fu. the comparison of peripheral blood chimerism (% of donor cells) in was patients transplanted with and without plerixafor/g-csf in conditioning is shown (figure ). the additional use of plerixafor with g-csf references . moratto et al disclosure of conflict of interest: none. is undesirable. fifteen pts ( males, females, median age , range: - years) with high risk hematologic malignancies ( acute myeloid leukemia n. , %; acute lymphoblastic leukemia n , % pretransplant conditioning regimen consisted of thiotepa mg/kg in two days, busulfan . mg/kg in three days, and fludarabine. source of stem cells was g-csf stimulated bone marrow in all. dose of marrow nucleated cells and cd + were . (range: . - . ) × /kg and . (range: . - . ) × /kg respectively. post-transplant cyclophosphamide at mg/kg/ day was given on days and after transplantation, together with cyclosporine (starting at day − until day posttransplant) and mycofenolate (from day + to day + ) modeling autism spectrum disorders with human neurons autism spectrum disorders neurobiology and genetics of autism: a developmental perspective. the development of autism: perspectives from theory and research wharton's jelly-derived mesenchymal stem cells treatment in children with cerebral palsy: our second preliminary results of the clinical application in poland a mucha , k kosterna , m chroscinska-krawczyk , m kotarska , k mitosek-szewczyk , m murzyn the polish stem cell bank cases application potential of bone marrow mesenchymal stem cell (bmscs) based tissueengineering for spinal cord defect repair in rat fetuses with spina bifida aperta sensory neuron differentiation potential of in utero mesenchymal stem cell transplantation in rat fetuses with spina bifida aperta: sensory neuron differentiation of in utero mscs analysis of post allo-hct relapse in acute leukaemia patients, a comparative on second allo-hct and donor lymphocyte infusions g orti , j sanz , i garcia-cadenas , i sanchez-ortega , mj jimenez , p barba , c ferra , r parody , j sierra , ma sanz , s querol and d valcarcel hospital universitari vall d´hebron; hospital universitario la fe; hospital de sant pau i la santa creu; hospital duran i reynals ico, hospital germans trias i pujol ico; hospital germans trias i pujol and banc de sang i teixits acute leukaemia relapse after allogeneic hematopoietic cell transplantation (allo-hct) associates poor prognosis. in this scenario, lowering the tumour burden prior to a second allo-hct ( nd allo-hct) or donor lymphocyte infusions (dli) is essential to improve survival. thus, patients that respond to chemotherapy and subsequently receive a dli or nd allo-hct appear to associate better outcomes compared to patients receiving only chemotherapy, but data regarding this particular group of patients is lacking. we retrospectively analysed a cohort of post allo-hct relapsed acute leukaemia patients, who, after tumour reduction, were treated with either a nd allo-hct or dli. data was collected from centers, patients were consecutively included from to . patients were treated to reduce the tumour burden and received the nd allo-hct or dli on morphological remission or postchemotherapy aplasia. patients ( %) were diagnosed with aml and ( %) with all. patients ( %) underwent nd allo-hct and ( %) received dli. median patient age was ( - ) years. the median follow-up was ( - ) days. since data regarding time from first allo-hct to relapse was unavailable, we calculated the time from allo-hct to nd allo-hct or dli (time to nd allo-hct or dli). median time to nd allo-hct/dli was ( - ) days, and was days and days for nd allo-hct and dli respectively (p = . ). regarding the dli group, the median dli dose was . x / cd + ( . - x ) cells and the mean number of infused dli was . /patient. one-year os was % (se ± %). in os univariate analysis, longer time to nd allo-hct/dli associated better survival rates (p = . ). the -year dfs was % (se ± %). a longer time to nd allo-hct/dli (p = . ) and nd allo-hct compared to dli (p = . ) (figure ) associated better dfs. the -year nrm was % (se ± %). univariate analysis identified pb as stem cell source as linked to better nrm (p = . ). the -year relapse incidence (ri) was % (se ± %). ri univariate analysis related longer time to nd allo-hct/dli (p = . ) to lower ri. on os multivariate analysis, longer time to nd allo-hct/dli was associated to better survival (p = . ). this association was also observed on dfs multivariate analysis (p = . ). table summarizes nd allo-hct and dli univariate analysis. grade ii-iv acute gvhd was diagnosed in ( %) and ( %) patients post nd allo-hct and dli, respectively. chronic gvhd was diagnosed in ( extensive) and patients after a nd allo-hct and dli, respectively. in this study, longer time to nd allo-hct/dli associated better dfs. nd allo-hct (compared to dli) associated better dfs on univariate analysis, but this association was not observed on multivariate analysis. of note, the nd allo-hct group included more patients with longer time to nd allo-hct/dli. this might be explained by nd allo-hct patients relapsing later or by the fact that the preparation of a nd allo-hct might require longer time than dli. results of this analysis warrant further study with larger number of patients.advancing age is associated with worse prognosis in acute myeloid leukemia (aml). intensive induction chemotherapy in patients aged ⩾ years results in lower aml remission rates with increased induction mortality vs younger patients. cpx- is a liposomal formulation of cytarabine and daunorubicin encapsulated at a : molar ratio. a phase iii, randomized, open-label study of cpx- vs + (cytarabine and daunorubicin) in newly diagnosed older patients with high-risk secondary aml showed superior survival in the cpx- arm (hazard ratio . ; p = . ). in that trial, eligible patients went on to allogeneic hematopoietic cell transplantation (hct). an exploratory analysis of those patients by age strata is reported here. patients aged to years with newly p number, composition and/or antileukemic activity of (dc-stimulated) invariant nkt-, nk-and cik-cells is predictive for outcome of patients with aml, all and cll cl boeck # , dc amberger # , f doraneh-gard , w sutanto , t guenther , j schmohl , f schuster , h salih , f babor , a borkhardt myelofibrosis (mf) is a hematolgic malignancy which is characterised by extramedullary hematopoiesis due to bone marrow fibrosis resulting in spleno-and/or hepatomegaly. allogeneic stem cell transplantation (allo-hsct) is the only curative treatment for mf but is associated with therapy related morbidity and mortality. retrospective studies suggested an increase of liver toxcicity in mf patients in comparison to other diseases following allo-hsct. the aim of this prospective study was to evaluate the impact of liver stiffness measured by transient elastography (fibroscan) on liver toxicity after allo-hsct. between and we included patients (male %, female %) who underwent allo-hsct due to primary mf( %), postpv/et-mf ( %) or mf in transformation ( %). the median age of the patients was y@@@ears (range: - ). conditioning regimen was mainly busulfan based reduced intensity. all patients received atg. gvhd prophylaxis was csa/mmf in all patients. stem cell source was peripheral blood in % and bone marrow in % of the patients. donor sources were as follows: mrd ( %), mud ( %) and haploidentical relative ( %). fibroscan was performed prior to conditioning. elevated liver enzymes, bilirubin above the normal value or the onset of veno-occlusive disesae (vod) from the time of conditioning start and within the first post-transplant days were considered as indicators for liver toxicity. the median stiffness of the liver measured by fibroscan on the day before conditioning treatment start was . kpa (range: . - . ). six patients ( %) had prior liver diseases such as cirrhosis (n = ), viral hepatitis (n = ), steatosis (n = ), or vod (n = ). the median onset of liver toxicity was day (range: − until + ). the median bilirubin level of all patients was mg/dl (range: - ). the median ap level was u/l (range: - ), the median ggt level was u/l (range: - ), the median alt level was u/l (range: and the median ast level was u/l (range: - ). the pearson-test revealed a positive correlation between liver stiffness and the elevation of the ap (r = . , p = . ) and ggt levels (r = . , p = . ). the comparison of the median maximum enzyme and bilirubin levels is shown in table . in two patients who developed severe vod requiering defibrotide, the liver stiffness level was . kpa and . kpa, respectively. the patient with the highest stiffness level ( . kpa) developed acute gvhd of the liver, which completely resolved after steroid treatment. only one of those five patients who had stiffness levels kpa died due to liver toxcity and concurrent septic shock, he suffered from viral hepatitis prior to transplantation. liver stiffness measured by transient elastography (fibroscan) positively correlates with the elevation of the cholestatic enzymes ap and ggt in myelofibrosis patients after allo-hsct and may predict liver toxicity. disclosure of conflict of interest: none.[p ]in the era of tyrosine kinase inhibitors (tki) as superior first line treatment in the therapy of cml, the concept of allogeneic hsct has been pushed to the role of salvage therapy. to date, data on allogeneic hsct after tki-therapy are scarcely available. in this study, we report single center data on the outcome of cml patients, for the most part pretreated with tki, who underwent allogeneic hsct between and with a follow-up of months to years. upon obtaining written informed consent patients diagnosed with bcr-abl-positive cml and patients with bcr-abl-negative atypical cml were included in this analysis. the majority of patients underwent myeloablative conditioning regimen. the median age at time of hsct was years with a range: from to years. twenty-one patients were transplanted from a matched related donor, and received stem cell grafts from an unrelated hla-compatible donor. / patients received tki-therapy before transplantation, patients received more than tki prior to hsct. / patients were treated with interferon prior to hsct. twenty-two patients were transplanted due to acceleration or blast crisis. twenty-six patients received an allogeneic hsct in chronic phase (cp, n = ) or complete hematologic (chr, n = ) or cytogenetic remission (ccyr, n = ). kinase domain mutations could be identified in seven patients including t i-mutation in four patients. seven patients showed "major route" cytogenetic aberrations. next to advanced disease status, tki intolerance (n = ) and tki resistance (n = ) were the main indications for hsct after . after a median follow up of years and months, those patients transplanted in cp, chr or ccyr showed an overall survival (os) of %. / patients died in remission and two patients died after cml relapse. after none of the patients transplanted in cp, chr or ccyr died or relapsed so far, with a median follow-up of days. all of these patients received tki therapy prior to transplant. twenty-two patients transplanted in advanced stage cml (bc and ap) had after a median follow up of years an os of %. the difference between survival curves is significant (log rank test p = . ; hr . , % ci of ratio . - . ). prior to transplantation of these patients received a tki-therapy. in this group, four patients died due to cml relapse, one died after development of donor cell leukemia and five patients died in remission. one patient with atypical cml was transplanted in bc and died of progressive disease shortly after transplantation. the other three patients with atypical cml were transplanted in cp-phase. with a median follow-up of days these patients are in ongoing remission. even in times of tkitherapy allogeneic hsct remains a successful and safe therapy option for cml patients with tki intolerance or resistance. patients transplanted in cp or complete remission had an excellent long-term outcome. allogeneic hsct should be considered in tki resistance or intolerance before the development of blast crisis. despite tki therapy, overall survival deteriorates in patients with advanced disease. however, this treatment modality can improve survival rates substantially compared to other available therapies. tkimaintenance therapy could be a possible strategy to prevent cml relapse, although randomized data on tki-maintenance therapy after allogeneic hsct are still lacking.[p ]disclosure of conflict of interest: none. use of first or second generation tki for cml after allogeneic hematopoietic stem cell transplantation: a study by the cmwp of the ebmt y chalandon, s iacobelli , j hoek , l koster , l volin , j finke , jj cornelissen , i yakoub-agha patients (pts) relapsing with cml after allogeneic hematopoietic stem cell transplantation (allohsct) may be treated with tki and/or dli. as nowadays the majority of cml pts would have received at least imatinib prior to transplantation, we were interested in analizing (a) the type of tki used after allohsct, (b) the indication for tki treatment, (c) the outcome of this treatment and d) the temporal relationship with dli if given. pts received tki after first allogeneic hsct for cml. transplants had been performed in cp , n = , ap, n = or for more advanced disease (bc/ cp , n = ) from hla identical siblings (n = ) or ud (n = ) between and . tki given prior to transplant was imatinib (n = ), dasatinib (n = ), nilotinib (n = ), bosutinib (n = ) and ponatinib (n = ). median age at transplant was ( . - ) years, pts ( %) were male. tki post allohsct were given between and . first tki given was either imatinib (n = ), dasatinib (n = ), nilotinib (n = ), bosutinib (n = ) or ponatinib ( ). the indications for tki therapy were the same as for transplantation (n = ), for relapse/progression/ persistent disease (n = ), for prophylaxis/pre-emptive (n = ), planned (n = ), others (n = ) and missing (n = ). median follow-up from start of tki was ( - ) months. the median time interval from transplant to tki was ( . - ) months. it was longer for tki given for relapse/progression with ( - ) months and shorter for tki given for prophylaxis/pre-emptive with . ( . haematopoietic cell transplant (hct) is the only curative approach for scd. due to concerns regarding the toxicities associated with myeloablative conditioning regimens in adults, a non-myeloablative protocol was developed by hsieh et al. (national institutes of health, nih protocol). the use of this novel regimen was able to achieve a curative degree of mixed donor chimerisms with minimal transplant-related complications. the alberta children's hospital (ach) has adopted this conditioning regimen in children due to the efficacy and low rates of toxicities published by the nih group. with generally lower rates of gvhd in younger recipients, our group had no reason to believe rates of toxicities would be greater in a younger population with fewer comorbidities secondary to scd than those described in the nih cohort. to our knowledge, there is no published literature describing the utilization of the nih protocol in a paediatric population. we describe our experience in children with scd who underwent matched sibling donor (msd) peripheral blood hct using nih protocol. this retrospective cohort describes outcomes of msd hct in children with scd who underwent hct with the nih conditioning regimen between - . a total of potential subjects were identified. eight subjects have consented to the analysis to date. msds with either normal haemoglobin or sickle cell trait were considered appropriate for donation. the transplant procedure: the conditioning regimen consisted of alemtuzumab . mg/kg/dose administered subcutaneously daily for five days (days − to day − ). patients received a tbi dose of cgy on day - , with testicular shielding for male recipients. gvhd prophylaxis consisted of a sirolimus load of mg/ m /dose (po) on day − , followed by mg/m /dose once a day starting on day . unmanipulated peripheral blood stem cells were infused on day . sirolimus was used for gvhd prophylaxis post-hct and continued until at least one year. weaning of sirolimus was initiated no earlier than year post-hct and if donor t-cell chimerisms were greater than %. institutional supportive care protocols for scd hct were followed. patients were eligible for early discharge post-hct even prior to neutrophil engraftment. eight patients ( f, m) have been registered on this retrospective study. follow-up ranges from to months post-hct. there were no failed stem cell mobilizations. all patients had donor neutrophil engraftment at a median of days. all patients are currently alive. there have been no cases of graft failure to date and no sickling crises post-hct. one patient has dropping myeloid chimerisms but still % donor. no cases of veno-occlusive disease, idiopathic pneumonia syndrome, cerebral hemorrhage, pres, or posttransplant lymphoproliferative disease were observed. three cases of cytomegalovirus (cmv) reactivation required pre-emptive therapy. only one patient did not initiate sirolimus weaning at year post-hct due to donor t-cell chimerisms of %; this patient is months post-hct and is likely to start weaning sirolimus soon. there have been no cases of acute or chronic graft-versus-host disease. nonmyeloablative conditioning regimen is safe and effective as curative therapy for scd. long-term follow-up of these children to assess organ function post-hct is underway. disclosure of conflict of interest: none.the number of new hiv/aids cases has been declining in developed countries, whereas it is still increasing in japan, with the cumulative number reaching , as of june , . hiv infection is associated with an increased risk of hematological malignancies such as non-hodgkin lymphoma (nhl). autologous hematopoietic cell transplantation (auto-hct) is a treatment option for hiv-infected patients with nhl and multiple myeloma (mm). however, the prognosis after auto-hct in hiv-infected japanese patients remains unclear. the aim of this study is to evaluate the effect of hiv infection on transplant outcomes after auto-hct in japan. using the national database of the japan society for hematopoietic cell transplantation, we retrospectively evaluated patients with nhl (n = ) and mm (n = ) who underwent their first auto-hct between and . presence of hiv antibodyperipheral t-cell lymphomas (ptcl) comprise a heterogeneous group of diseases among which ptcl-not otherwise specified (ptcl-nos) represents the most common histology. patients with ptcl are typically offered high-dose chemotherapy followed by autologous hematopoietic cell transplantation (auto-hct) as front-line consolidation. allogeneic hct (allo-hct) is generally offered in the relapsed setting; however, in selected cases it is also offered as front-line consolidation. no randomized controlled trial (rct) have been performed to date comparing offering an allo-hct versus other treatment modalities either in the front-line or in the relapsed setting. thus, we performed this systematic review/meta-analysis to assess the totality of evidence pertaining to the role of allo-hct in ptcl. search of the literature was undertaken via pubmed and web of science from inception until september , . no search limits were applied but studies presented only in abstract form were excluded. data were collected on treatment benefits (complete remission (cr), progression-free survival (pfs), overall survival (os)) and harms (non-relapse mortality (nrm), grade ii-iv acute graft-versus-host disease (gvhd), and chronic gvhd). the search identified references; however, only studies ( in front-line (n = pts), in relapsed/refractory setting (n = pts)) were eligible based on our inclusion criteria and had extractable data. three studies included both frontline and relapsed/ refractory cases but data for certain outcomes were reported separately. the median follow-up time for studies evaluating allo-hct in the front-line or relapsed/refractory setting ranged from - months and - months, respectively. in the front-line setting, allo-hct resulted in cr rates of % (( %ci = - %), studies, n = pts), pfs rate of % (( % ci = - %), studies, n = pts), and os rate of % (( % ci = - %), studies, n = pts). nrm rate was % (( % ci = - %), studies, n = pts). acute (grade ii-iv) and chronic gvhd rates were % ( % ci = - %), studies, n = pts) and % ( % ci = - %), studies, n = pts), respectively. in the relapsed/refractory setting, allo-hct resulted in cr rates of % (( % ci = - %), studies, n = pts), pfs rate of % (( % ci = - %), studies, n = pts), and os rate of % (( % ci = - %), studies, n = pts). nrm rate was % (( % ci = - %), studies, n = pts). acute (grade ii-iv) and chronic gvhd rates were % ( % ci = - %), studies, n = pts) and % ( % ci = - %), studies, n = pts), respectively. notwithstanding the need to perform a rct to compare the efficacy of allo-hct versus auto-hct as front-line consolidation in ptcl, the results of this systematic review/meta-analysis show very encouraging os rates of % following allo-hct. moreover, allo-hct also offers an encouraging os rate of % in patients with ptcl in the relapsed/refractory setting. the higher nrm rate in the relapsed/refractory setting probably reflects the adverse effect of a higher number of prior prescribed therapies. one of the limitations of our analysis is the inability to analyze outcomes for individual histologic subtypes due to the aggregate nature of the published data. disclosure of conflict of interest: none. high-risk patients with relapse or refractory hodgkin lymphoma do significantly better after hdc auto-sct compared to control arm of aethera trial. mature results from a cohort of patients s akhtar, s rauf, tam elhassan and i maghfoor king faisal specialist hospital and research center, riyadh, kingdom of saudi arabia brentuximab vedotin use in hodgkin lymphoma (hl) patients who had hdc auto-sct has been reported to improve progression free survival (pfs) but not the overall survival (os) in a phase trial (lancet ; : - ). in this trial, after hdc auto-sct, high risk hl patients were randomized to receive placebo (control gp) vs brentuximab (experimental gp) as consolidation therapy. we are reporting our experience of patients with similar selection criteria as control gp. hl patients z yrs who received hdc auto-sct with similar selection criteria as defined in aethera trial were identified that is, patients had at least one of the following risk advanced lymphomas still represent a therapeutic challenge and allo-hsct is among treatment options. between march and august , seventy-three patients (pts) affected by r/r lymphomas ( nhl and hl) underwent an allo-hsct after a treosulfan-based conditioning regimen and sirolimus as calcineurin-inhibitor-free prophylaxis of gvhd. six pts received a mrd, pts a mud, and pts a haplo unmanipulated pbsc. at allo-sct pts were in cr, pts were in pr, and pts had sd/pd; sixty patients underwent autologous sct before allo-hsct. hct-ci was evaluable for pts, had a score ≥ . thirty-three pts received treosulfan and fludarabine reduced toxicity conditioning regimen (rtc) and intensification with other alkylating agent or with gy total body irradiation was added on the remaining pts (myeloablative conditioning, mac). all pts received a backbone gvhd prophylaxis with sirolimus and mycophenolate mofetil; atg or pt-cy or both were added in , , and pts respectively. median numbers of infused cd +/kg and cd +/kg were . × (range: . - . ) and . × (range: . - . ), respectively. median follow-up was months (range: - ); median time to neutrophil ≥ . × /l was days, and days to platelet ≥ × /l. sixteen out of patients with pre-transplant active disease obtained a cr after treosulfan conditioning; nine of them ( hl and b-nhl) achieved durable cr without post transplant treatment. oneand -years os was % and %, pfs was % and % at and years respectively; cumulative incidence of relapse/ progression was % and % at and years. grfs was % and % at and years, respectively. transplant related mortality (trm) was % at days, % at year and for the entire follow-up. the -day cumulative incidence (ci) of agvhd grade ≥ was % and ci of agvhd grade ≥ was %; ci of moderate to severe cgvhd was % at years and for the entire follow-up. no differences in ci of agvhd or cgvhd were found if pts were stratified according to donor type, but ci of moderate-severe cgvhd was significantly higher in pts after mac regimens (p o . ). as expected, the outcome of pts in cr was significantly better compared with active disease, in terms of os (p = . ), pfs (p = . ), ri (p = . ). in multivariate analysis, intensity of conditioning regimen (rtc vs mac), gvhd prophylaxis (use of atg, pt-cy or none), donor sex and age at allo-sct did not impact the transplant outcomes; both os and pfs were reduced by active disease at allo-hsct (hr = . , ci % . - . , p = . and hr = . , ci % . - . , p = . , respectively) and by nhl histology (hr = . , ci % . - . , p = . and hr = . , ci % . - . , p = . , respectively); grfs and ri were impacted only by active disease (hr = . , ci % . - . and hr = . , ci % . - . , p = . , respectively). allo-hsct after treosulfan conditioning and sirolimus gvhd prophylaxis is feasible even in heavily pretreated pts affected by lymphomas. complete remission status before transplant remains crucial for better outcomes and in the era of new targeted treatments should be pursued. disclosure of conflict of interest: none.university of eastern finland, kuopio, finland and department of medicine, kymenlaakso central hospital, kotka, finland autologous stem cell transplantation continues to have an important role in the treatment of patients with multiple myeloma (mm). in mm patients the most commonly used mobilization method is granulocyte-colony stimulating factor (g-csf) ± cyclophosphamide (cy). generally, up to - % patients mobilize poorly with these methods and plerixafor may be used to enhance mobilization. the most important parameter of graft quality has usually been the number of cd + cells, but there are also significant numbers of other cell subsets in the grafts and they may also be of special interest in regard to post-transplant recovery and outcome. for example, a higher number of lymphocytes and nk cells in the grafts has been associated with improved lymphocyte as well as nk cell recovery, respectively. the mobilization methods used seem to affect the graft composition. however, there is currently no prospective data on the effects of plerixafor on the graft composition, post-transplant hematological and immune recovery or outcome in patients with mm. altogether eighty-seven patients with mm were included into this prospective study. seventy-seven patients were mobilized with g-csf ± cy (control group) and ten patients received also plerixafor due to poor mobilization (plerixafor group). in the control group / ( %) and in the plerixafor group / ( %) of patients were mobilized with g-csf+cy (p = . ). there were no statistically significant differences between the groups according to age, gender, paraprotein type, initial iss, induction therapy used or disease status at the time of mobilization. by imwg risk stratification, there were more high risk patients in the plerixafor group ( / vs. / , p = . ). cryopreserved graft samples were analyzed with flow cytometry for t and b cells (cd /cd /cd /cd ) as well as for nk cells (cd /cd +cd ). also, cd + cell subclasses were analyzed (cd /cd /cd ). complete blood counts were evaluated at + days, , , and months posttransplant. to evaluate immune reconstitution, flow cytometry of lymphocyte subsets (t, b, nk) was performed in a subset of patients at , and months after the graft infusion using the same antibody panel as for graft analysis. there were no significant differences between the groups in the number of cd + cells in the grafts. also, the median number of aphereses was two in the both groups (p = . ). the proportion of the more primitive cd + cells (cd + cd + cd -) was significantly higher in the plerixafor group (p = . ). in addition, the number of various lymphocyte subsets analysed was significantly higher in plerixafor group table ). there were no statistically significant differences in the course of hematological recovery. the recovery of blood cd +cd + t cells was significantly faster in the plerixafor group at one at three moths post-transplant. there was no significant difference in the progression-free survival (pfs) (log rank, p = . ) with the median follow-up time of days in the plerixafor group and days in the control group ( . ). in the present study plerixafor added to g-csf ± cy seemed to significantly alter the cellular composition of autologous blood grafts in poorly mobilizing mm patients. hematological recovery was comparable but the cd +cd + t lymphocyte recovery was faster in the plerixafor group. the pfs was comparable between the groups. disclosure of conflict of interest: dr. valtola has received honoraria from sanofi and jansen-cilag. dr. silvennoinen has received a research grant from celgene and janssen, honoraria from genzyme and sanofi and participated in advisory board organized by amgen, janssen and takeda. dr. siitonen has received honoraria from amgen and celgene. dr. jantunen has received honoraria from genzyme, amgen and sanofi and has participated in eu leadership meeting organized by genzyme as well as medical advisory board meeting organized by genzyme and amgen. dr. varmavuo has received consultancy fees from abbvie, roche, celgene, amgen and sanofi. the other authors declare no conflicts of interest. bortezomib after high-dose melphalan as conditioning regimen before autologous stem cell transplantation in patients with multiple myeloma: a comparison with the historical conditioning regimen with melphalan alone ga ferini; ja arbelbide; al basquiera; e nucifora; n schutz; v otero; d fantl hospital italiano d buenos aires, buenos aires, argentinahigh dose of melphalan followed by autologous stem cell transplantation (asct) is the standard of care for younger patients with multiple myeloma (mm). to enhance the efficacy of the conditioning regimen, the intergroupe francophone du myelome added bortezomib to melphalan showing improved response rates, without significant toxicity. bortezomib has shown synergistic effects with melphalan, mainly if the bortezomib is administered hours after the melphalan. since , we have changed our conditioning regimen for patients with mm undergoing asct by adding bortezomib toallogeneic stem cell transplantation (allosct) is a potencially curative option for patients with multiple myeloma (mm). despite the improvement of reduced-intensity-conditioning (ric), transplant-related mortality (trm) remains high. there is no consensus on which graft versus host disease (gvhd) prophylaxis regimen is superior. some studies have suggested that tacrolimus-based prophylaxis is more effective than cyclosporine (csa) in terms of lower incidence of severe acute gvhd (agvhd), with no impact on overall survival (os). herein, efficacy and toxicity between two gvhd prophylaxis regimens is analyzed. we retrospectively analyzed patients (pts) with relapsed mm who received allosct ric in the period from to in a single centre (table ) . population: age, years ( - ); median follow-up: months ( - ). conditioning regimen: allo-ric (fludarabine + busulfan or melphalan regimens) and % was bortezomib-based in the tacrolimus group. donor: matched related ( pts), unrelated ( ), mismatch unrelated ( ) and haploidentical ( ) donor. gvhd prophylaxis: all patients received a short course of methotrexate + csa ( pts, %) or tacrolimus ( pts, %). complete response at transplant was % at csa group and % at tacrolimus group. all pts underwent toxicity related to chemotherapy (mainly mucositis and neutropenic fever) with organ impairment (renal or liver) in % tacrolimus arm as well as pts in csa group. the incidence of agvhd was % and . % in tacrolimus and cyclosporine groups, respectively (p = . ). grade iii-iv agvhd were reported in pts ( %,tacrolimus) and pts ( %, cyclosporine), with severe gastrointestinal and liver involvement. glucocorticoid resistance was observed in % in both groups. patients with refractory agvhd received other immunosuppressive therapies: more than second-lines agents ( ) ( ) ( ) ( ) were necessary in fifty percent of pts in both groups to control gvhd. two patients had to interrupt tacrolimus due to neurological toxicity and suspected thrombotic thrombocytopenic purpura. no patients had to discontinue treatment in the csa arm because of toxicity. the -months os was . % ( % in tacrolimus vs . % in csa (p = . )) and the -months was . %. a total of pts died because of gvhd. during follow-up, only patients relapsed ( and months after allosct, respectively) in csa group. no relapse were seen in tacro group. in our experience, no significant differences were observed between both calcineurin inhibitor in terms of os, toxicity and gvhd incidence. an explanation could be our small number of patients. allosct is an effective therapy for selected patients but it is associated with high rates of gvhd and trm. a long-termsafety and effective prophylactic regimen is necessary as main objective.[p ]disclosure of conflict of interest: none. several parameters, including early lymphocyte, neutrophil, platelet recovery, and infused dose of cd + cells, have been associated with clinical outcome of patients with haematological malignancies. however, their prognostic significance remains uncertain. the aim of current study was to evaluate prognostic significance of clinical and laboratory parameters that might influence survival after autologous stem cell transplantation (asct) in hodgkin lymphoma (hl) and multiple myeloma (mm). this retrospective study included a total of with hl and mm patients (median age years, years, respectively) who underwent asct between november and june . hl patients were conditioned with beam ( . %) and cbv ( . %) regimen, while mm patients received conditioning with high dose of melphalan. high ips (international prognostic score) at diagnosis had . % hl patients and high iss (international scoring system) had . % of mm patients, of which . % had renal impairment. the average of transplanted cd + cells in hl patients was . × /kg (range: - . × /kg), and . × /kg (range: - . × / kg) in mm patients. after asct, favourable treatment response (partial/complete remission) achieved . % hl patients, of whom . % had infused o × /kg cd + cells. median time to recovery of absolute lymphocyte count × /l or greater (alc ) was days (range: - days), recovery of absolute neutrophil count ≥ × /l (anc ) was (range: - days), and platelet recovery ≥ × /l (plt ) was days (range: - days). after asct, . % mm patients achieved favourable treatment response, of whom . % had infused cd + cell dose . × /kg. median time to alc was days (range: - days), anc was (range: - days), and plt was days (range: - days). median follow up of patients with hl was months, while after asct, median event free survival (efs) was months, and overall survival (os) was months. treatment response after asct strongly influenced both efs and os after asct (po . ). in patients who achieved favourable treatment response, os and efs after asct were influenced by infused cd + cell dose (o × /kg vs. ≥ × /kg), prolonged recovery of alc by day+ , plt by day + , and achieving of anc by day + (po . ). multivariate analysis among significant variables showed that infused cd + cell dose was the most important parameter that influenced os and efs (po . ). median follow up of mm patients was months, while after asct, median efs was months and os was months. regarding patients who achieved favourable treatment response, os and efs after asct were influenced by the presence of renal impairment, infused cd + cell dose (≤ . × /kg vs. . × /kg) and plt recovery by day + (po . ). among these significant parameters, multivariate analysis pointed out infused cd + cell dose as the most important parameter that influenced both os and efs (po . ). these data suggest that number of infused cd + cells is an independent factor that may contribute to outcome of patients with hl and mm. disclosure of conflict of interest: none. high-dose therapy with autologous stem cell transplantation (asct) has become the treatment of choice for symptomatic eligible patients with multiple myeloma (mm). we studied an induction regimen of cyclophosphamide, bortezomib and dexamethasone (cybord) and showed rapid and deep responses after cycles in patients with newly diagnosed mm and we subsequently done asct with melphalan (mel) conditioning. cost is the major limiting factor in developing world.all the drugs used are generic brands manufactured in india. a total of mm patients (median age: . years, % male and % female) were transplanted between and . in all, patients had igg kappa- ( %), igg lambda- ( %), iga lambda- ( %), iga kappa- ( %), kappa light chain ( %), lambda light chain ( %) patients. prior to autograft, all cases had received cybord with generic medicines. median time diagnosis to asct was . months ( to months). stem cell mobilization was done with g-csf alone in ( %), g-csf plus plerixafor in ( %) and chemo mobilization in ( %) patients. all patients received asct support after conditioning with mg/m generic melphalan alone (dose adjustment was done according to renal status). all patients received thalidomide maintenance from march . bortezomib used was manufactured by dr. reddy's lab, hyderabad and melphalan used was manufactured by emcure pharmaceuticals, pune, india. patients from to received cyclophosphamide, vincristine, adriamycin and dexamethasone (cvad) protocol of originator medicines followed by originator melphalan conditioning and asct (cvad-mel-asct). at the time of autograft, ( %) of patients were in complete remission, ( %) in partial remission, ( %) very good pr. median day of engraftment was for neutrophils and for platelet. transplant related mortality was % ( / ) out of which died of infection and deaths of cardiac events. the pfs and os rates were % and % at median follow up of . months. patients who were treated with cvad-mel-asct had efs of % at yrs and % at yrs. cost of bortezomib showed significant difference, generic was usd where as for originator drug was usd for cycles of chemotherapy. cost of melphalan also showed difference with usd for generic and usd for originator drug. generic cybord showed excellent response rate and allows excellent stem cell collection and transplantation which can further consolidate response and improve outcome. cybord induction and melphalan conditioning with generic medicines can be considered a standard regimen for transplant-eligible patients with newly diagnosed mm in resource constraint situation. generic cybord-mel-asct is more cost effective than originator cvad-mel-asct. generic medicines produced in india are of good quality and cost effective. this study needs long term follow up to assess survival parameters at a median. disclosure of conflict of interest: none. and an extra copy of one or more odd-numbered chromosomes and as intermediate risk(ir) if they had t( ; ) or del( ) (q).overall survival (os) and relapse-free survival (rfs) were calculated from the time of allo hsct and auto hsct on day , from diagnosis to death or disease progression. the median age at presentation was . (range: - ) years, and ( . %) were men. at a median follow-up time of months, % were alive. of the patients with available fish samples underwent auto hsct. patients ( . %) achieved cr and patients ( . %) relapsed. of the patients who had received allo hsct, five patients ( . %) achieved cr and five patients ( . %) remained alive. in patients who received auto hsct, the risk of relapse was % less than those never transplanted (p = . ), but the difference was not significant in patients who received allo hsct. the relapse-free survival in hr patients was months (po . ), in ir was months (p o . ) and in sr was . months (p o . ). in transplant patients, rfs in hr patients was . times more than sr group (po . ) and in ir group was . times more than sr (p o . ). the survival time in transplant patients was significantly better than non-transplanted patients (p o . ). the median overall survival (os) in hr patients was . months, in standard risk group months and in sr patients was months. cytogenetic abnormalities detected by fish are of significant value in classification, risk stratification and management of patients with mm. we can use cytogenetic data to provide prognostic information and also to guide management and clinical practice. these data indicate that autologous stem cell transplantation could potentially be of benefit to myeloma patients. disclosure of conflict of interest: none. chronic graft-vs-host disease (cgvhd) is the most troublesome complication developing after allogeneic hematopoietic stem cell transplantation (allo-hsct). diagnosis of cgvhd has largely been based on clinical features only. we previously reported gene expression profiles in patients with cgvhd after allo-hsct. we extended our study to develop a molecular diagnostic method of cgvhd. we selected six most commonly expressed genes from the former dna expression study. and, a home-made -gene pcr array were used to evaluate gene expression profiles in the peripheral blood mononuclear cells of patients given allo-hsct ( cgvhd patients, non-cgvhd patients) and normal controls. the gene expressions of the allo-transplanted patients were compared to those of the stem cell donors. sybr green qpcr and multiplexqpcr were performed to confirm the usefulness of the selected genes in the diagnosis of cgvhd. infogainattributeeal and ranker were used to develop a gene model to diagnose cgvhd. k-nearest neighbor model and weka classifiers lazy ibk module were applied to evaluate the performance of the gene model. in another steroid-refractory cgvhd patients ( responders, non-responders), the gene expression changes were analysed using our -gene pcr array before and days after rituximab treatment. we identified six genes most accurately delineating cgvhd patients from those without treatments after allogeneic hematopoietic stem cell transplantation (hsct) are long and constraining for patients. medical adherence in hsct patients is of major concern in daily practice but it has been not yet described. , the aims of our study were to evaluate treatment adherence and to identify factors associated with adherence behaviors. an observational single-center study was based on self-reported questionnaires completed by patients in a hematology day hospital between november and july . the patientreported adherence was evaluated using the eight-item morisky medication adherence scale (mmas- ). , individual item scores were summed: patients with a score of / were considered as good adherents to medication whereas a poor adherence referred to a score under . among the latter, medium adherence ranged to a score of - , while a score of o was considered low adherence. socio-demographic and medical characteristics were collected by health records. a univariate model was used to evaluate if some of patients' characteristics were associated with adherence. statistical analysis was performed using r software (version . . - - rstudio, inc). fifty-six patients were included in the current study. median age at transplantation was years (range: - years). diagnosis were aml (n = ), all (n = ), myelofibrosis (n = ) and other hematological diseases (n = ). patients received a hsct from a related donor ( haploidentical). myeloablative conditioning was used in patients and reduced intensity regimen in patients. a total of . % ( / ) of the patients were poor adherent according to mmas- . among these patients, / were low adherent and / were medium adherent. the results of univariate analysis showed that a poor adherence was associated with a longer time since hsct and discharge at home. however elderly patients, patients treated with cyclosporine and patients with daily hydration at home were associated with a better adherence (po . ). our study presents the first data on adherence among patients undergoing hsct. risk factors associated with a poor adherence have been identified in order to determine patients' profiles that will benefit more from interventions to improve adherence. particular attention has to be paid to younger patients. efforts to establish a regular follow-up of these patients are needed in order to sustain patients in the treatment adherence to prevent the occurrence of severe complications. we studied the effect of basic fibroblast growth factor (fgfb) and dexamethason on expansion and immune modulation of mscs in patients with lymphomas. mscs were generated from bone marrow aspirates obtained from the patients with hodgkin's lymphoma (hl; n = ) and non-hodgkin's lymphoma (nhl; n = ). the adherent fraction of marrow aspirate was cultivated with/without the basic fibroblast growth factor (fgf-b, ng/ml) or dexamethason ( − М or − М) to reach - % confluence. then mscs were passaged with accutase and used for experiments after - passages. the number of msc precursors (cfu-f) in bone marrow of lymphoma patients was found to be significantly decreased both in patients with nhl ( ± , p o . ) or hl ( ± , p o . ). the time until - % confluence was significantly increased and took on average ± days (vs . ± . in donors). finally, the immunosuppressive ability of patient msc was significantly lowered and was only registered at the high concentrations of mscs ( : and : ). the expansion of patient mscs was significantly promoted with fgfb resulting in a significant decrease of primary cell cultivation (from . ± . to . ± . days; p = . ) and a statistically significant twofold increase in the number of cells received at the first passaging. in addition, in cultures with fgfb there was a decrease in the relative amount of resting mscs and a threefold increase of cycled cells in cd + mscs. dexamethasone has also provided a moderate stimulating effect on the msc growth. in fact, the use of − М of dexamethasone resulted in the increase of the cell yield by . times and of − М-by . times. however, fgfb and dexamethasone differed in their effect on the msc ability to inhibit the proliferative response of t lymphocytes upon stimulation with mitogens or alloantigens. indeed, fgfb failed to correct the impaired immunosuppressive activity of patient mscs, and median percentage of suppression still remained lowered- % vs % without fgfb. in contrast to fgfb, dexamethason could increase the immunosuppressive activity of patient mscs by . times (in dose of − М) and by . times (for − М). our data indicate that fgfb and dexamethasone used during the generation of mscs exert a stimulating effect on the msc expansion. in contrast to fgfb, dexamethasone, in the broad range: of doses, was able to enhance the suppressive properties of mscs that are initially reduced in patients with lymphoma. these findings suggest the existence of at least two mechanisms of impairments in immunoregulatory function of mscs in lymphomas-dependent and independent of the msc proliferation. disclosure of conflict of interest: none. free nonabsorbable antibacterial digestive decontamination is associated with a low incidence of gastrointestinal acute gvhd and better gvhd-free/ relapse-free survival (grfs) in the atg-based conditioning regimens nabil yafour commonly antibacterial prophylaxis based of oral no absorbable antibiotic such as (neomycin colistin, gentamicin, vancomycin) used before and after engraftment, other fluoroquinolone such as levofloxacine were recently used to prevent invasive infection. however the exact interaction with gastrointestinal acute graft versus host disease (gi-agvhd) remains unclear. the objective of this study was to evaluate a novel composite endpoint of gvhd-free/relapse-free survival (grfs), in which events include grades - gi agvhd, chronic gvhd requiring systemic therapy, relapse, or death in atg based-conditioning regimens, with free no absorbable antibacterial digestive decontamination prophylaxis. a total of evaluable consecutive patients with hematological disease were included in period of february to mai . patients with malignancies disease (n = ) received myeloablative conditioning regimens plus atg ( mg/kg); including once daily busulfan ( mg/m ,- d to - d, iv ) + fludarabine ( mg/m /d, - d to - d, iv) (aml = , all = , cml = ) or melphalan ( mg/m , d- , iv) (all = ). six patients received cy/atg for saa. gvh prophylaxis consisted to; ciclosporine a (csa) + mtx. csa was maintain levels between - ng/ml and tapered at the discretion of the treating physician. all patients were received peripheral blood stem cells (pbsc) graft from a matched related donor. since december levofloxacine and voriconazole was administered as antibacterial and antifungal prophylaxis. diagnostic, clinical grading and treatment of gi-agvhd and gi-cgvhd were performed according to established criteria and nih recommendations. probability of grfs was estimate by kaplan-meier method. median age was years (range: - ). median dose of cd + and cd + cell doses were . × (range: . - ) and . × (range: , - , ). the median time to neutrophil and platelet recovery were days (range: - ) and days (range: - ) respectively. at time of transplant / ( %) had an intestinal colonization with extended-spectrum betalactamase (esbl) producing bacteria. only / ( %) developed infectious diarrhea during the period of transplant. incidence of grade iii/iv gi-agvhd and gi-cgvhd requiring systemic therapy were % and % respectively. for patients with malignancies diseases (n = ), ( %) were alive at a median follow up of months (range: - ). incidence of relapse, disease free survival rates were %, % respectively. the grfs rate as defined previously was % at months. these results confirm that free no absorbable antibacterial digestive decontamination and atg-based conditioning regimens were associated with very low incidence of gi-gvhd and better grfs in patients with malignancies diseases. diverse bacterial populations of the gastrointestinal tract remain important factors to promote immune tolerance after allogeneic sct. disclosure of conflict of interest: none. g-csf primed hla haploidentical transplantation from maternal or collateral donor using atg plus reduced dose of posttransplantation cyclophosphamide: results of a phase ii prospective trial y wang , x-j huang peking university people's hospital, peking university institute of hematologythe transplantation milieu using granulocyte colony-stimulating factor (g-csf), and anti-thymocyte globulin (atg) for hlahaplotype-mismatched transplants from related donors has resulted in favourable outcomes with low transplant-related mortality (trm), without increased relapse rate. however, in this transplant modality, the poorer outcome owing to high incidence of graft-versus host disease (gvhd) related to maternal donor or collateral donor remains a concern. meanwhile the use of post-transplant cyclophosphamide (pt/cy) in recent years appears to be protective against severe acute and chronic gvhd. we performed a prospective pilot study of hla haploidentical stem cell transplantation (sct) from maternal or collateral donors with intensified conditioning including g-csf and atg, followed by two lower doses of pt/cy ( . mg/kg × doses). outcomes were compared with those of controls from matched-pair analysis who undergone haploidentical sct from other donors than mother or collateral relatives at the same time period. a total of patients with myelodysplastic syndrome (mds) or leukaemia undergoing haploidenticla sct from maternal or collateral donors were enrolled in the study. incidence of grade ii-iv and grade iii-iv acute gvhd at day were comparable between the study group and the control group ( . % vs. . %, p = . ; . % vs. . %, p = . ). incidence of cmv and ebv reactivation at day were also comparable between the study group and the control group ( . % vs. . %, p = . ; . % vs. . %, p = . ). after a median follow-up of days and days, the incidence of trm and relapse at year were comparable between the study group and the control group ( . % vs. . %, p = . ; . % vs. . %, p = . ); the probability of overall survival and lfs at year were comparable between the study group and the control group ( . % vs. . %; p = . ; . % vs. . %, p = . ). in conclusion, conditioning with atg and low-dose pt/ cy might be a feasible option for patients undergoing hla haploidentical, t-cell replete sct from maternal or collateral donors. trial registration: the study is registered at www. clinicaltrial.gov as nct . disclosure of conflict of interest: none. hematopoetic stem cell transplantation (hct) is a lifesaving treatment option for eligible patients with hematological malignancies. hct is inherently associated with a risk of nonrelapse mortality that varies greatly depending on transplant and patient characteristics. the assessment of the risk of complications and mortality before the procedure is extremely important. the hct comorbidity index (hct-ci) introduced by sorror m. is one of the tools proved to predict hct outcomes and was shown to be significant in various disease and hct settings. the objective is to evaluate hct-ci index of hct recipients, determine impact of different variables on ci score, particularly those, showing pulmonary and cardiac function. data of hct-ci of autologous (auto) and allogeneic (allo) hct recipients, transplanted during period january -october were analyzed. impact of pulmonary and cardiac function values on ci score was evaluated: dlco (diffusing capacity of the lung for carbon monoxide), fev (forced expiratory volume) and ef (cardiac ejection fraction) are parameters, reflecting pulmonary and cardiac function, which values are included into hct-ci score. the statistical data analysis was conducted using spss program. the differences were considered statistically significant at p ≤ . . records of allo and auto hct recipients, transplanted during . - . in vilnius university hospital were revised. median age of allo hct and auto hsc recipients was ( - ) and ( - ) years respectively. main indication for allo hct was acute myeloid leukemia ( %) patients and for auto hct -multiple myeloma ( . %) patients. hct-ci was completely calculated (no values missing) in allo and auto hct recipients. only patients with available complete hct-ci data were further analyzed. hct-ci in hct recipients was as shown in table . hct-ci score o was calculated in ( . %) and ≥ in ( . %) allo hct recipients. hct-ci score o was calculated in ( . %) and ≥ in ( . %) auto hct recipients. hct-ci score did not differ statistically significant between male and female recipients in both hct categories as well as in different age groups of patients (below and above years in allo and below and above years in auto hct). dlco was found to be below normal values (o %) in ( . %) allo hct and in ( . %) auto hct recipients. fev was less affected and found to be lower % in ( . %) allo hct and in ( . %) auto hct recipients. ef below % detected in ( , %) allo hct and in ( . %) auto hct recipients. low dlco was found to cause the greatest impact on hct-ci score and was statistically significantly associated with higher hct-ci (po . ). the most common hct-ci in both hct groups was score . dlco was found to be below normal ranges in relatively large patient group and had the greatest impact on hct-ci score. further studies on reasons of pulmonary function impairment and it's impact on hct outcomes are warranted.[p ]disclosure of conflict of interest: none. haemophagocytic lymphohistiocytosis (hlh), a life-threatening hyper-inflammation syndrome, is classified into primary and secondary forms. primary hlh is caused by gene mutations resulting in impaired cytotoxicity of natural killer (nk) cells and cytotoxic t lymphocytes (ctls). secondary hlh arises in the setting of autoimmunity, infection, malignancy, or less commonly, may be idiopathic. treatment of hlh has two major goals: halting the triggering event and controlling the overactive immune system. however, patients with primary or recurrent secondary hlh should subsequently undergo allogeneic hct for long lasting disease remission. we retrospectively evaluated hematopoietic stem cell transplantation (hsct) might be a valid treatment option for adults suffering from aggressive t-cell malignancies providing long term disease control. since a suitable hla-matched donor cannot be identified for all patients (pts) in need for transplantation, alternative donors graft sources such as related hla-haploidentical donors are considered. through introduction of t-cell-replete (tcr) hlahaploidentical transplantation (haplo-hsct) using post transplantation cyclophosphamide (ptcy) successful treatment with low non-relapse mortality rate (nrm) has been observed in lymphoma patients (luznik et al., bmt, ) . however, less data are available on the outcome of this haplo-approach in the treatment of t-cell malignancies, in particular when disease is refractory. we retrospectively evaluated the outcome of haplo-hsct using tcr grafts and ptcy in pts with peripheral t-cell lymphoma treated between and at our institution (t-nhl = , t-all = ; male n = ; median age: years). disease was refractory/active at time of transplantation in pts, while one had achieved second cr. all patients received at least prior treatment lines and one patient failed previous allogeneic transplantation. while fludarabine and cyclophosphamide served as backbone for conditioning, pts received a tbi-based and a drug-based conditioning regimen which was myeloablative in %. if disease was active at time of haplo-hsct, a sequential therapeutic concept was performed involving intensive chemotherapy (clofarabine n = ) shortly preceding conditioning (zoellner ak et al., bmt, ) . post-grafting immunosuppression consisted of cyclophosphamide, tacrolimus and mycophenolate mofetil in all patients. graft source was bone marrow in pts. no primary graft rejection occurred; / pts engrafted, one died early in aplasia. neutrophil/platelet engraftment was achieved at a median of (range: - ) and (range: - ) days, respectively. acute gvhd grade ii-iii was observed in pts, whereas no patient developed grade iv agvhd. mild chronic gvhd occurred in one patient. % of the pts developed grade ii-iii treatment-related toxicities most commonly diarrhea ( %) and mucositis ( %); grade iv toxicity (mucositis) was observed in one patient only. no vod occurred. cmv reactivated in / pts at risk, whereas no ptld was seen. proven invasive aspergillosis was diagnosed in one patient. at day + seven pts achieved cr. pts relapsed and died (relapse n = , infection n = ). -year nrm was %. at a median follow up of months (range: - ) the estimated -year and -year overall survival (os) and progression-free survival (pfs) were %/ % and %/ %, respectively. three pts received haploidentical dlt pre-emptively (n = ) and therapeutic (n = ), leading to sustained cr in two, while no severe gvhd occurred. sequential therapy in the setting of tcr haplo-hsct using ptcy as gvhd prophylaxis is feasible, well tolerated and shows low rates of gvhd and acceptable nrm in patients with relapsed/refractory t-cell lymphoma/ leukemia providing an effective anti-lymphoma/leukemic activity. thus, we suggest that intensified tcr hapo-hsct using ptcy should be considered as an alternative for patients suffering from aggressive t-cell malignancies, lacking hlamatched donors. disclosure of conflict of interest: none. tacrolimus is a calcineurin inhibitor increasingly used as immunosuppression following allogeneic stem cell transplantation; maintenance of therapeutic serum levels is essential to reduce the risk of graft rejection and graft versus host disease. however, tacrolimus can be associated with serious side effects and potential drug interactions. regular monitoring of serum levels and appropriate dose adjustment is essential to ensure therapeutic levels and to avoid toxicity. in our adult bmt unit, an established standard operating procedure (sop) provides a prescriptive dosing algorithm for: (i) initiation of tacrolimus therapy; (ii) conversion between iv and oral routes; (iii) dose adjustment based upon tacrolimus serum level and interacting medications. we performed an audit assessing adherence to the sop dosing algorithm. inpatient tacrolimus dosing episodes from five consecutive haploidentical transplants were retrospectively analysed. episodes were excluded due to insufficient records. for the remaining episodes, tacrolimus serum levels and corresponding doses were identified. the response of the medical team to each serum level was compared with the sop dosing recommendation. to account for sensible rounding of doses, a margin of error of ± % was permitted. adherence to sop dosing was %. non-adherence to the sop ( %) was subcategorised as justifiable ( %) or unjustifiable ( %). justifiable non-autologous hsct is currently being explored for its efficacy and safety in the treatment of multiple sclerosis (ms). as more experience is gained in treating this cohort, treatment related mortality has steadily improved although the procedure still carries a degree of risk. ebv reactivation is well described in allogenic stem cell transplants although less so in autologous transplantation. we investigated the frequency of ebv reactivation in patients with ms undergoing autologous hsct at a single uk site. patients underwent autologous hsct for treatment of ms at king's college hospital between feb and aug . all were mobilised with cyclophosphamide g/ m and g-csf. were conditioned with cyclophosphamide and atg, and one with beam/atg. previous exposure to ebv (ebv igg) was assessed prior to transplant and local posttransplant ebv monitoring was performed on whole blood samples by means of quantitative pcr in patients. data was collected retrospectively. all ( %) patients were positive for ebv igg pre-transplant. overall, samples were tested for monitoring post-transplant. ( . %) patients demonstrated positive pcr post-transplant on local testing with one further patient being negative on local tests but later becoming positive on testing in their parent hospital (full results unavailable). of these , the median time to positive testing post-transplant was days ( - ). maximal ebv dna titre was reached at a median time of days post-transplant ( - ) with a mean maximum titre of . log ( . - . ). patients experienced symptomatic reactivation with an associated large paraproteinemia. one of these developed hyper-viscosity requiring plasma exchange and developed neurological symptoms mimicking an ms relapse (max ebv titre of . log). this patient received rituximab and ebv level is declining, the other was observed carefully but developed right leg weakness which is slowly improving. the patient with raised ebv at their parent hospital also received rituximab (unclear if this reactivation was symptomatic). we have developed a protocol to pre-emptively treat ebv reactivation with rituximab once a log titre is reached and one patient has so far been treated according to this. of the patients with locally confirmed reactivation who did not receive rituximab, ( %) self-resolved at a median time of days ( - ), ( . %) have ongoing re-activation ( with improving, with stable titres) and ( . %) have not had any local bloods performed ≥ months. patient with selflimiting reactivation later had a further positive titre ( days post-transplant and days post initial resolution). ebv reactivation appears to be common in patients with ms in the first months post autologous hsct. unlike in other patient groups such as aplastic anaemia patients receiving allogeneic transplants it can cause significant neurological symptoms which may be confused with ms relapse. the mechanism of this reactivation is probably related to atg administration but may be exacerbated by prior immune suppression in this heavily pre-treated group, the majority of whom have received highly active disease modifying therapies in the past. these results demonstrate the importance of monitoring for ebv reactivation following autologous hsct and the consideration of pre-emptive therapy. disclosure of conflict of interest: none. reduced bone mineral density (bmd) is a well recognised complication of hct. guidelines recommend scanning by dual energy x-ray absorptiometry (dxa) one year after transplant in all hct patients or else specific groups of high risk patients. , it is recognised that both dose and duration of steroids are risk factors for low bmd and it is recommended that prednisolone doses greater than or equal to mg/day for more than months should prompt a dxa scan. for patients with osteopenia it is recommended that calcium/vitamin d supplements are given together with lifestyle advice including diet, smoking cessation and weight bearing exercise. in this survey we have investigated the current practise in investigating and managing bone health in the context of hct. a survey was sent to all centres including countries registered with ebmt as of november . centres replied from countries. response numbers to each question were variable and are indicated by the denominators. / used a national guideline to guide their practise, and / used an international guideline. no single guideline was quoted more than once. low testosterone has been demonstrated to be an independent determinant of endothelial (dys)function in men. graftversus-host disease (gvhd) is a major contributor to nonrelapse mortality (nrm) after allogeneic stem cell transplantation (allosct). vulnerability of the recipients' endothelial cell system is a novel concept to explain why a proportion of patients with acute gvhd fail to respond to escalating immunosuppressive therapy and ultimately succumb to gvhd and related complications. this retrospective study investigated the prognostic impact of pre-transplant testosterone levels on nrm after allosct in male patients. between and , a total of male patients undergoing allosct at heidelberg university (median age years) provided informed consent to participate in this observational study (training cohort). a total of patients ( %) received transplants from related donors (rd). diagnoses were aml ( %), mds ( %), lymphoid malignancies ( %) and multiple myeloma ( %) . a total of patients ( %) received statin treatment post allosct as per institutional standard policy. for validation, an independent patient cohort of men allografted for aml and mds (median age years, % rd, no statin treatment) at essen university was analysed. pretransplant serum samples were collected between and months before allosct and cryopreserved at − °c. testosterone and suppressor of tumorigenicity- (st ) levels were measured by radioimmunoassay and elisa, respectively. median pre-transplant testosterone level in the training and validation cohort was . nmol/l (range: . - . nmol/l) and . nmol/l ( . - . nmol/l), respectively. in the training cohort, lower pre-transplant testosterone as continuous variable was associated with shorter os (p = . ). lower testosterone levels showed a trend towards higher nrm (p = . ) and a significant association with nrm after onset of acute gvhd (p = . ). multivariate analysis confirmed lower pre-transplant testosterone levels as a significant predictor of an increased nrm risk after gvhd onset (p = . ). in the subgroup of patients not receiving statins post-transplant, lower testosterone levels were associated with increased incidence of transplant-associated microangiopathy (p = . ), and, in addition, with higher pre-transplant st levels indicating endothelial vulnerability. in the validation cohort, similar results with regard to overall survival (os, p = . ), nrm (p = . ), nrm after acute gvhd onset (p = . ) in univariate analysis, and to nrm after gvhd onset (p = . ) in multivariable analysis could be observed. the association of pre-transplant testosterone levels (in quartiles) and incidence of nrm after gvhd onset in the training and validation cohort is depicted in figure a and b, respectively. our study suggests that low pre-transplant testosterone is associated with serological and clinical evidence for endothelial damage and is an independent risk factor for a fatal outcome of gvhd. prospective studies in the allosct setting investigating testosterone and testosterone supplementation in deficient patients are highly warranted. disclosure of conflict of interest: none. nk cells anti-tumor ability in multiple myeloma patients s tognarelli , , b jacobs , , , i von metzler , h serve , p bader, t klingebiel , a mackensen and e ullrich , department of pediatric stem cell transplantation and immunology, childrens hospital, goethe university, frankfurt, germany; cellular immunology, loewe centre for cell and gene therapy, goethe university, frankfurt, germany; department of hematology and oncology, university hospital erlangen-busulfan is one of essential drugs for hematopoietic stem cell transplantation (hsct). because of its narrow therapeutic range: targeted busulfan using therapeutic drug monitoring (tdm) has been used. generally, the initial dose of busulfan is determined by patients' body surface area as mg/m except for infants ( mg/m ). however, pharmacokinetic evidence of these initial doses is scarce. therefore, we investigated the full pharmacokinetics of busulfan in infant and child, and attempted to validate that these initial doses are acceptable. one hundred ninety-five pediatric patients undergoing hsct using four-day targeted busulfan were enrolled. of them, patients received hsct when their age was ≤ year old (infant group [ig]), and patients received when - years old (toddler group [tg]). the remaining patients were defined as a child group (cg). busulfan was administered intravenously once daily for consecutive days. tg and cg received mg/m as the first dose, and ig received mg/m . using daily tdm, we adjusted the next dose of busulfan. target daily and total area under the curve (auc) were μg*h/l/day and - μg × h/l, respectively. median first-day busulfan auc of ig, tg, and cg were , and μg × h/l, respectively, which was significantly different (p = . ). however, there was no significant difference in median total busulfan auc (ig; , tg; , and cg; μg × h/l, respectively, p = . ). the coefficient of variance (cv) of four-day busulfan aucs in ig and cg was similar (median cv: . % and . %, respectively), whereas cv of tg was . %. in sub-analysis of tg and cg who received equally mg/m as the first dose, there was an inverse correlation between age and first-day busulfan auc (r = − . , p = . ), as well as between age and cv of four-day busulfan aucs (r = − . , p = . ). initial busulfan dose as mg/m for infant could be acceptable in aspect of first-day auc and cv of four-day busulfan aucs. however, higher first-day auc and cv were shown in tg. although target total busulfan auc could be achieved safely by tdm, we suggest that reduction of initial dose less than mg/m is also necessary to patients with - years old to lower the relatively higher first-day auc. taken together, tdm is highly recommended to reduce busulfan toxicity, especially in younger children. disclosure of conflict of interest: none. post-induction treatment strategy of acute myeloid leukemia (aml) is currently driven by european leukemia net (eln) risk assessment at diagnosis. if it is well established that patients belonging to favourable-risk group can be treated with chemotherapy and/or autologous stem cell transplantation (sct) and that those belonging to the unfavourable-risk group should be addressed to allogeneic (allo) sct, for patients included in the intermediate-risk groups the best post-induction treatment has not been established yet. we report here a years ( - ) allo-sct single center experience in aml patients. median age was years (range: - ), %, %, % and % were grouped in the eln favourable, intermediate-i, intermediate-ii and unfavourable risk category, respectively and % of the patients were allotransplanted in advanced disease-phase ( nd complete remission). half of the patients received a sibling hla compatible donor, % of the cases received peripheral blood stem cells and half of the patients received a myeloablative conditioning regimen. graft versus host disease prophylaxis was conventionally based on cyclosporine and shor-course methotrexate, with the addition of antilymphocyte immunoglobulin in case of matched unrelated donor. the clinical and transplant characteristics of the patients according to the eln-risk group were well balanced. with a median follow up of months (range: - months), the projected years overall survival (os) and disease free survival (dfs) is % ( % ci: - %) and % ( % ci: - %). the median os and dfs in favourable/intermediate-i vs intermediate-ii/unfavourable is . and . months ( figure a ; p = , ) vs and , months ( figure b ; p = . ). the relapse rate (rr) and the non relapse mortality (nrm) at two years are % ( % ci: - %), and % ( % ci: - %), respectively. non differences were observed comparing the years rr and the years nrm of patients in the favourable/intermediate-i vs intermediate-ii/unfavourable eln risk group ( % vs %; p = . and % vs %; p = . ). interestingly, the percentage of patients allotransplanted in advanced phase of the disease was higher in those included in low/intermediate-i with respect to intermediate-ii/unfavourable eln-risk group ( % vs %; p = . ). our data suggest that allo-sct can cure approximately - % of aml patients, with no difference within the eln risk groups. disease recurrence remains the major problem and this is highly correlated to the percentage of patients in advanced phase of the disease at transplant, particularly in eln favourable/intermediate-i patients. we are currently collecting the data on minimal residual disease (mrd) status of these patients during chemotherapy and before transplant using moelcular biology on target genes and/or multiparametric flow cytometry on leukemia associated immunophenotype, in order to assess if the prognosis of these patients may be refine by the prospective application of mrd data.[p ]disclosure of conflict of interest: none. outcome of allogeneic stem cell transplantation for patients with high-risk acute leukemia according to donor type and graft-versus-host disease prophylaxis s lindner, t berg , j riemann , s ajib , z jedlickova , s gueller , f lang , a sackmann , , n goekbuget , , h martin , a bacigalupo , h serve , and g bug department of medicine ii, hematology and oncology, university hospital frankfurt, goethe university, germany; s german cancer consortium (dktk), german cancer research center, heidelberg, germany and università cattolica del sacro cuore, fondazione policlinico universitario gemelli, roma, italyin high-risk acute leukemia (hr-al), allogeneic hematopoietic stem cell transplantation (hsct) is the only potentially curative treatment. increasingly, hsct is being performed utilizing alternative donors. we retrospectively analyzed the outcome of consecutive patients (pts) with hr-al (aml/all, n = / ) undergoing first allogeneic hsct in our transplant unit between / and / according to donor type and graft-versus-host disease (gvhd) prophylaxis: in the matched related donor group (mrd, n = ), hsct was performed with standard immunosuppression (is), that is, calcineurin inhibitor (cni) plus methotrexate or mycophenolate mofetil (mmf). for / hla-allele matched unrelated donors ( / mud, n = ) or / hla-allele mud ( / mud, n = ) we used is and anti-thymocyte globulin (atg fresenius/neovii). hsct with a haploidentical family donor or an / hla-allele mismatched unrelated donor was performed using is with cni plus mmf and post-transplant cyclophosphamide (pt-cy, n = ). a myeloablative (n = ) or reduced-intensity (n = ) conditioning regimen was applied in complete remission (cr, n = ) or active disease (n = ). pts had a median age of years (range: - ) and hematopoietic cell transplantation comorbidity index of (range: - ). patient and treatment characteristics were well balanced between the groups except for a higher percentage of pts transplanted in cr in the pt-cy group ( % vs. - %, p = . ). peripheral blood stem cells were preferred for mrd, / mud and / mud ( %, % and %, respectively) and bone marrow for % of pt-cy based hsct. all pts engrafted. with a median follow-up of months (range: - ), probability of overall survival (os) at years was ± % for the mrd, ± % for the / mud, ± % for the / mud and ± % for the pt-cy group, without significant differences (p = . ). however, the probability of achieving the combined endpoint gvhd-and relapse-free survival (grfs) at years varied significantly between the groups (mrd ± %, / mud ± %, / mud ± % and pt-cy ± %, po . ), reflecting the high cumulative incidence (ci) of chronic moderate and severe gvhd at year in the mrd ( ± %) as opposed to the other groups ( / mud ± %, / mud ± % and pt-cy ± %, p o . ). of note, donor type had no impact on ci of transplant-related mortality (trm) at years ( ± %), acute gvhd g - at day + ( ± %) or leukemic relapse at years ( ± %). overall, aml pts years of age had a significantly inferior relapse-free survival compared to younger pts ( ± % vs. ± %, respectively, p o . ) without a higher ci of trm (p = . ). median time to aml relapse was months. our results suggest that pt-cy-based alternative donor hsct is safe in hr-al pts and provides a solid basis for a randomized clinical trial comparing hsct from haploidentical family donors and / mud, currently in preparation. while os did not vary between groups, grfs was dismal after mrd transplants without atg, due to high rates of severe chronic gvhd, consistent with published data. as leukemic relapse remains the major cause for treatment failure especially in elderly pts, maintenance strategies using novel drugs or cellular therapies are warranted. disclosure of conflict of interest: none. relapse following hematopoietic stem cell transplant (hsct) is the leading indication for a second transplant in patients with malignant disease. hsct has been shown to be superior to chemotherapy alone or palliative measures in these patients. for non-malignant disease a second transplant may be considered for graft failure after first transplant. data regarding the outcome of a second hsct for non-malignant disease is scarce. we retrospectively analyzed patients who underwent a second hsct, for survival and toxicity data. twentynine patients (age - years) who received a second hsct at our institution during - were included in the analysis. thirteen patients had an underlying malignancy and patients were transplanted for non-malignant indications, including inborn errors of metabolism, non-malignant hematologic diseases and immune deficiency. median follow up was months (range: - ). there were deaths ( %) in the malignant group, ( %) were due to disease relapse and ( %) were transplant related. fifty percent of deaths occurred within the first year following the second hsct. in the non-malignant group there were deaths ( %), of which ( %) were attributed to the underlying disease and ( %) were transplant related. all deaths but one occurred within the first year post hsct. treatment related mortality following second hsct is higher compared to first transplant. the higher survival rate in the non-malignant group suggests that transplant following graft failure should be considered ins patients with otherwise incurable underlying disease. though the outcome for patients with relapse of malignant disease following hsct is poor, a second transplant may benefit a subset of these patients. attempts to achieve complete remission prior to transplant should be made to improve outcome. due to the small number of patients in our cohort, further multi-center trials are needed. disclosure of conflict of interest: none. disseminated bcg infection (bcg-osis) is a rare but most serious complication in vaccinized especially immunocompromised children. severe combined immunodeficiency disorder (scid) is probably the commonest primary immunodeficiency associated with bcg-osis, though there is no such definitive data as most of the cases described in literature are in the form of reports. hematopoietic stem cell transplantation (hsct) is a life-saving treatment for patients with scid, especially if therapy is instituted early, prior to onset of infections.as bcg vaccine is routinely given to all iranian children at birth, the likelihood of having an active infection at the time of transplant would be significantly high. the main objective of this study was to evaluate the outcomes of hsct in scid patients with disseminated bcg infection . sixteen scid patients underwent hsct in our center since to , of which nine patients ( male, female) were enrolled in this analysis. all the patients had received bcg vaccination according to the national vaccination protocol, and had undergone anti-tuberculosis (tb) treatment prior to transplant due to disseminated bcg infection. the mean age at hsct was . months (range: - months). patients received bone marrow (n = ), peripheral blood progenitor cells (n = ) or umbilical cord blood grafts (n = ) from hla-matched related donors (n = ) and mismatched unrelated donors (n = ). three patients received unconditioned matched sibling donor transplants and ric regimen was provided with fludarabine, melphalan and rabbit anti-thymocyte immunoglobulin (thymoglobulin) in others . cyclosporine a and prednisolon were used as graft-versus-host disease (gvhd) prophylaxis. they also continued to receive anti-tb treatment. all patients but one engrafted. the median times to neutrophil and platelet engraftments were days (range: - ), and days (range: - ), respectively. engraftment with full chimerism ( %) occurred in patients and the other patients had mixed chimerism. with a median follow-up of months (range: - months), overall survival was . %. the main cause of death was disseminated bcg infection.three out of patients who achieved engraftment, developed acute gvhd (grade i-ii), while one patient developed extensive chronic gvhd. although anti-tb treatment continued, tuberculous dactylitis occurred in patients post-hsct that were successfully treated. on last post-hsct follow-up, patients with full chimerism and with mixed chimerism are alive and disease free. scid is called as a pediatric emergency as it invariably leads to fatality in infancy without early aggressive therapy and hsct. in hsct recipients, the impaired cellular immunity renders these patients more susceptible to infection. as previous reports suggest, our study demonstrates that with appropriate anti-tb cover, immunological reconstitution with complete recovery from bcg infection can be achieved by early hsct. disclosure of conflict of interest: none. paraproteinemia occurrence after allogeneic hematopoietic stem cell transplant as a possible marker for chronic gvhd onset f monaco , s tamiazzo , f dallavalle , l calcagno , m pini and m ladetto hematology and transfusion medicine, azienda ospedaliera ss. antonio e biagio e cesare arrigo, alessandria, italy transient monoclonal gammopathy is commonly reported after solid organ or stem cells transplant (sct) for hematologic malignancies. however the clinical significance of a paraproteinemia appearance is not fully understood, because the attempts to correlate its effect on survival rates, graft versus host disease (gvhd) occurrence and viral reactivations have led to controversial results. starting from these reports we decided to evaluate among our allogeneic transplanted patients the incidence of m-component and its possible relationship with chronic gvhd. one-hundred and one patients undergoing allosct at the hematology unit of alessandria (italy) between and were evaluated. % of patients were male and % were females. pretransplantation diagnosis included: acute myeloid leukaemia/ high-risk myelodisplastic sindromes ( %), acute lymphoblastic leukaemia ( %), lymphoproliferative disorders ( %) and other less common malignancies ( %) . patients with multiple myeloma were excluded from the study. all patients had, at least, two pre-transplantation serum electrophoresis with no evidence of pre-existing monoclonal component. serum electrophoresis was scheduled to be performed at , and days and years after transplantation. forty-nine patients were submitted to allo-sct from a sibling donor and from a matched related donor (mud); in vivo t-cell depletion with anti-thymocyte globulin was used in patients. thirty-four patients relapsed after allosct, ( %) developed chronic gvhd and patients ( %) are currently alive at the last follow-up. posttransplantation follow up ranged from to days with a median of days. paraproteins were detected in out of patients ( %), being monoclonal in patients, and bi or tri-clonal in the remaining cases; the immunoglobulin subclass most commonly observed was igg. ten-year overall survival of the whole population was %; splitting the population in two cohorts (with or without paraproteinemia) we did not detect any statistical differences in overall survival, gvhd development and relapse incidence at + and + days posttransplant; viceversa, after days, a statistically significant difference was observed in chronic gvhd occurrence in patients with or without paraproteinemia ( % vs %, respectively, po . ). ten-year overall survival curves were significantly better in patients with paraproteinemia as compared with the paraprotein-free group ( % vs %, p = . ), and an even more evident significance was seen in ten-year relapse free survival curves ( % for patients with paraprotein vs % for patients without paraprotein, p = . ). monoclonal gammopathy, also in our experience, is frequent following allo-sct. we observed a strong correlation between the occurrence of paraproteinemia, chronic gvhd and a significantly better overall and relapse-free survival. recently many evidences showed that b cells are involved in the pathogenesis of chronic gvhd (cgvhd) and anti-b-cell therapy has been suggested for the treatment of cgvhd. we speculate that the presence of a monoclonal gammopathy after allogeneic transplant is expression of the activation of the b-cell compartment. a prospective study with a larger population should be considered, in order to confirm our results and assay post-transplantation monoclonal gammopathy as an early marker for gvhd development. disclosure of conflict of interest: none.inherited bone marrow failure (ibmf) syndromes are rare pediatric disorders that characteristically associate physical abnormalities, progressive bone marrow failure and predisposition to cancer. the most common of these disorders is fanconi anemia (fa). stem cell transplantation (sct) using related or unrelated donors are the only curative therapeutically approach when severe marrow failure is established. the aim of the study was to analyze the results of sct for patients with ibmfs in a single center. we performed a retrospective study in pediatric patients with ibmf admitted in pediatric hematology and bone marrow transplant department, fundeni clinical institute between january and september . diagnosis and severityof ibmfs were established based on hematological results, bone marrow biopsy and clinical findings. genetic testing for ibmfs is not currently available in our country. indication for sct was established when patients developed moderate/severe aplastic anemia and became transfusion dependent.in case of dba, sct indication was established for steroid resistant disease. the donors were selected from family members or unrelated donors, / matched.the conditioning regimens used were reduced intensity (fludarabine - mg/m , cy mg/kg, f-atg mg/kg) for af, dc and myeloablative (busulfan i.v., fludarabine mg/m , thiotepa mg/kg, f-atg mg/kg) for dba. gvhd prophylaxis consisted of standard methotrexate and csa/tacrolimus. all parents signed informed consent forms. in our center, between and , patients with ibmf were diagnosed: ( %) patients with fa, ( %) patients with diamond blackfan anemia (dba), ( %) patients with diskeratosis congenita (dc), and ( %) patients with not classifiable ibmfs. the patient data is available in table . seven out of patients ( %) performed sct procedures: sibling patients ( patients with af, patient with dc), mud patients ( patients with af, patient with dba). all patients ( %) engrafted for pmn (median = , range: - days) and platelet (median , range: - days). / ( %) presented reactivation of cmv and received valganciclovir, / developed cmv disease (encephalitis and pneumonia), / ( %) developed bkv cystitis and required extensive hydration and levofloxacin. / ( %) developed grade i-ii skin acute gvhd day + , which responded to topical treatment and low dose of corticosteroids. / ( %) developed grade iii intestinal acute gvhd, which responded to high-dose corticosteroids. / ( %) developed grade iv intestinal chronic gvhd (day + ), without response to high-dose corticosteroids, mmf and later died on day + , due to infectious complications (severe pulmonary and cerebral aspergillosis). / patients ( %) are alive, with % donor chimerism / ( %) or stable mixed chimerism / ( %). median follow-up for sct patients was days ( days- y mo). conclusions in our study we observed a low incidence of severe complications associated with low mortality rate ( %) . sct is a procedure that associates multiple risk situations, but it remains the only curative cytomegalovirus (cmv) infections remain a significant cause of morbidity and mortality in patients whose immune systems are compromised, including hematopoietic stem cell transplant (hsct) recipients. although the adoptive transfer of third party cmv-specific t cells has proven both safe and clinically beneficial in treating even drug-refractory infections/disease, broader implementation and commercialization of this strategy has been hampered by (i) the postulated need for extensive cell banks generated from donors representing diverse hla profiles, and (ii) lack of large scale t cell manufacturing processes. to address these limitations we have developed a proprietary decision tool (cytomatch™) to identify a small panel of healthy donors who should provide almost universal hla coverage; and optimized a simple, scalable manufacturing process to generate large numbers of cmv-specific t cells. to assess the robustness of our strategy we generated a bank of cmv-specific t cells (viralym-c™) from carefully selected healthy donors. the lines were polyclonal, comprising both cd + ( . ± . %) and cd + ( . ± . %) t cells, expressed central cd ro+/cd l+ ( . ± . %) and effector memory markers cd ro+/cd l-( . ± . %), and were specific for the immunodominant cmv antigens ie and pp (ie : ± ; pp ± sfc/ × , n = ). a fixed-dose ( × cells/m ) phase i clinical trial was subsequently initiated to test the safety and efficacy of these "ready to administer" t cells in pediatric and adult hsct recipients with drug-refractory cmv infections. using our bank of just lines, we have identified a suitable line for of patients screened. of these, patients have been treated with viralym-c cells; received a single infusion and patient required infusions for sustained benefit. there were no immediate infusion-related toxicities; and despite the hla disparity between the viralym-c™ lines and the patients infused, there were no cases of de novo or recurrent graft versus host disease (gvhd). based on viral load (measured by quantitative pcr) and/or symptom resolution, viralym-c cells controlled infections in all patients with complete (cr) and partial responses (pr) achieved within weeks of infusion. one patient with cmv retinitis had complete resolution of symptoms following viralym-c™ infusion. our results demonstrate the feasibility, preliminary safety and efficacy of "ready to administer" viralym-c™ cells that have been generated from a small panel of healthy, eligible cmv seropositive donors identified by our decision support tool. these data suggest that cost-effective, broadly applicable t cell anti-viral therapy may be feasible for patients following hsct and potentially other conditions. disclosure of conflict of interest: drs. juan vera, ann leen and brett giroir hold equity and drs. ifigeneia tzannou, sunitha kakarla are employed by viracyte. haploidentical stem cell transplantation (hsct) protocols utilizing ex vivo t-cell depleted grafts have been proven efficient in preventing graft versus host disease (gvhd), but cause a delay in early t-cell recovery that increases the risk of graft rejection, leukemia relapse and viral infections. conventional donor lymphocyte infusion (dli) after hsct transplantation is conditioned because of the high prevalence of gvhd even with low dose of t cells. here we present preliminary data of escalating cd ro+ memory t cells as dli in three patients that received a selective graft depleted of naïve (cd ra+) t-cells. three children that were transplanted following nonmyeloablative conditioning regimen with a graft consisting of cd + and cd ra-cells, with mixed chimerism, lymphopenic and viral/opportunistic infections and minimal residual disease positive before hsct received dose scalating cryopreserved haploidentical cd ra-memory t cell starting with a initial dose of × /kg, until a maximal dose of × /kg with a days interval. we infused products with a naïve ( ra+) t-cell dose less than × /kg with . % purity of cd + cd ro+ memory t-cells in all cases. all infusions were well tolerated without any side effect during infusions neither gvhd. following the dli, a progressive increase in t cell counts was observed. our preliminary data suggest that dose escalating of haploidentical memory t cells ( ro+) as dli provides a safety platform, even with high dose of t cells ( × /kg), for adoptive immunotherapy in haploidentical ra+ depleted grafts with no gvhd complications, and allows an increase in t cell reconstitution. however, efficacy of this strategy requires longer studies. relapse after allogeneic hematopoietic stem cell transplantation (allohsct) remains a major therapeutic challenge: outcome is very poor, without curative option in most cases. second allohsct may be considered in few selected patients because of anticipated limitations: ( ) donor availability; ( ) high toxicity due to previous treatments; ( ) low efficacy considering the very advanced disease situation. we hypothesized that the use of post transplantation cyclophosphamide (pcy) haplo-sct after relapse following allohsct may deal in part with these limitations. in particular, the presence of full haplotype hla mismatch could provide a decisive antileukemic effect relative to alloreactivity. in absence of large series in this setting, we report here the outcome after haplosct for patients who relapse after a first allohsct. we retrospectively studied adult patients, who received a second pcy haplo-sct for hematological malignancies. patients were treated between and . the objective was to assess both the feasibility and the efficacy of haplosct in this setting. twenty seven patients were included: median time between first allohsct and relapse was months (range: - ). median age at second transplantation was years old (range: - ). most of patients had acute myeloid leukemia (n = , %) or hodgkin lymphoma (n = patients, %). fifteen the impact of minimal residual disease and its kinetics prior to different types of allogeneic hematopoietic stem cell transplantation on clinical outcomes in patients with acute myeloid leukemia z xiao-su , , l yan-rong , yan-hong , p xu-ying , qian-jiang , hao-jiang , lan-ping, xu , xiao-hui zhang , , yu-wang , , h xiao-jun , and c ying-jun this study investigated the impact of minimal residual disease (mrd) and its kinetics prior to different types of allogeneic hematopoietic stem cell transplantation (hsct) on clinical outcomes in patients with acute myeloid leukemia (aml) in complete remission (n = ). patients who received unmanipulated haploidentical hsct and patients who received hla-matched sibling hsct were enrolled. mrd measured using -color flow cytometry (fcm) at fixed time points before transplantation was retrospectively analyzed. the patients were divided into four groups based on mrd kinetics before transplantation: consistent negative, positive to negative, negative to positive and consistent positive. during the follow-up, total twenty ( . %) patients underwent relapse. through unique variate analysis, none of mrd status at various time points before unmaipulated haploidentical transplantation was associated with clinical outcomes, as well as the dynamic change of mrd before hsct (p . ), although the patients with consistent positive mrd before hsct seemed to have a relatively higher incidence of relapse (p = . ). one-year cumulative incidence of relapse (cir) were . ± . % vs. . ± . % in mrd consistent negative and consistent positive groups, respectively (p = . ). however, patients with positive mrd after the second chemotherapy or pre-mrd before hla-matched sibling hsct showed a significant poor outcomes including higher cir (p = . and both neuroblastoma (nrb) and rhabdomyosarcoma (rms) in childhood are the aggressive malignant disease with higher mortality. this paper aims to study the efficacy of autologous peripheral blood stem cell transplantation (apbsct) in the treatment of high risk advanced nrb and rms. patients with high-risk stage iv nrb and patients with advanced childhood rms were treated by apbsct in our hospital from october of to may of . in the subgroup of nrb patients, patients got complete remission (cr) and patient got cru while patients had tumor residual disease after intensive induction therapy before asct. the median age was . ( - ) years old. primary sites of the tumors included submaxilla (n = ), cervical (n = ), adrenal gland (n = )and retroperitoneal (n = ). the conditioning regimen consisted of busulfan and melphalan (busulfan mg/kg × d, melphalan mg/m x d) or cem regimen (carboplatin mg/m × d, etoposide mg/m × d, cyclophosphamide mg/ m × d); the pathology of stage iii childhood rms patients was embryonal rhabdomyosarcoma. there were cases in cr and case in partial remission (pr). the median age was . ( - ) years old. primary sites of the tumors included bladder (n = ), left forearm (n = ), retroperitoneal (n = ), pelvic (n = ) s and talus (n = ). the conditioning regimen consisted of melphalan, cyclophosphamide and dactinomycin (melphalan mg/m × d, cyclophosphamide mg/m × d, dactinomycin . mg/kg × d).there were double apbsct cases (nrb n = , rms n = ). all the relapse patients were treated with chemotherapy and radiation therapy. all the patients successfully underwent mobilization, collection and reinfusion. the time of hematopoietic reconstitution was ( . ± ) days, no severe toxicity was observed, no transplant-related death was found. with a median follow-up of . ( - ) months, one of the patients was lost to follow-up. in the subgroup of nrb patients (n = ): the -year event-free survival and total survival rate of all patients were . % and . %, respectively. the survival time of no recurrence was significantly different between the double transplantation group and single transplantation group (p o . ). in the subgroup of rms patients (n = ), patient died, patients live without pd( patients had double apbsct), patients suffered recurrence but still alive. apbsct achieved good outcome in patients with high risk advanced nrb and rms. transplantation-related toxicities were tolerable. double apbsct significantly improved the depth of remission. disclosure of conflict of interest: none. transplantation outcomes of a once-daily intravenous busulfan and fludarabine conditioning for allogeneic hematopoietic stem cell transplantation in pediatric aml and high risk mds: single center experience in korea y-t lim, e-j yang and k-m park department of pediatrics, pusan national university children's hospital, yangsan, koreathere have recently been some reports suggesting that oncedaily intravenous busulfan as a conditioning regimen for hematopoietic stem cell transplantation (hsct) possibly reduces the toxicities without influencing the clinical outcome as compared with the traditional times daily dosage schedule. but until recently there has been little research and limited data available on the safety and efficacy of oncedaily intravenous busulfan and fludarabine in pediatric allogeneic hsct. we report the outcomes for allogeneic hsc recipients, evaluating engraftment status, regimen related toxicities (rrt), and event free survivals (efs) after use of oncedaily intravenous busulfan and fludarabine conditioning for allogeneic hsct in children with aml and high risk mds in a single pediatric center of korea. from january to december , aml and high risk mds children who received once daily iv busulfan/fludarabine based conditioning regimen for allogeneic hsct were reviewed, bu/flu ± atg consist of intravenous fludarabine ( mg/m ) and busulfan ( ~ mg/m , once daily iv) on days - to - , and antithymocyte globulin (atg) ( mg/kg) on days - to - . all patients received tacrolimus and mini-dose methotrexate ( mg/m( )) for graft versus host disease (gvhd) prophylaxis. boys and girls were enrolled with median age of . years (range: . - . years). the median period from diagnosis to transplantation was months (range: - months). more than half of the patients had a matched sibling donor (n = , %), % patients (n = ) had a matched unrelated donor, % patients (n = ) had a mismatched unrelated donor, and the remaining patient had a mismatched family donor. as a stem cell source, peripheral blood stem cells (pbsc) were cases ( %), bone marrow and cord blood were cases in each. the median follow-up for patients was months. the median number of infused total nucleated cells and cd + cells except cord blood transplantation were . × ( )/kg and . × ( )/kg. all patients including who received cord blood were successfully engrafted. the median time to absolute neutrophil count (anc) recovery (anc × ( )/l) and platelet recovery (platelet , × ( )/l) were days, days in each. the incidence of acute gvhd was . %, while severe grade iii/iv gvhd was observed in only patient ( . %). there were only two cases ( . %) of extensive chronic gvhd in this study. transplant-related toxicities were acceptable, there was no case with cns toxicity, eleven patients ( . %) developed grade ii,iii mucositis and grade i-iii hepatic toxicity in twenty four ( . %), but transient. there was clinically diagnosed veno-occlusive disease (vod), but most recovered by fluid restriction and diuretics. nine patients ( %) showed positive cytomegalovirus (cmv) antigen/pcr but only one patient developed cmv colitis. eight patients died: due to relapse/disease progression, due to extensive chronic gvhd. the -year efs and overall survival were . % and . % respectively. at year, the cumulative incidence of relapse was . %. overall, once-daily intravenous busulfan and fludarabine was less toxic and effective as conditioning regimen in aml and high risk mds patients undergoing allogeneic transplantation in children. disclosure of conflict of interest: none. haploidentical stem cell transplantation from unmanipulated graft has becoming a practiced option for high risk hematological malignancies who lack a matched related or unrelated donor. lack of a matched sibling or unrelated donor (mud) can be a significant barrier to allogeneic transplantation in patients who stand to benefit from this procedure. hlahaploidentical donors are readily available for nearly all such patients. haplo transplantation has inherent advantages over mud transplantation including the lower cost of graft acquisition, greater availability of donors for ethnic minorities, and immediate access to the donor in patients in whom delay cerebral palsy (cp) is a heterogeneous group of conditions that result in permanent motor disability. it may occur due to perinatal hypoxic insults, developmental brain abnormalities, genetic diseases, traumatic or infectious causes. in general the condition is non-progressive, but improvement over time is rarely seen. various treatment methods have been used for the management of this disorder. however, there has been no absolute cure for cp. the ultimate goal of stem cells therapy is to use the regenerative capacity of the stem cells causing a formation of new tissues to replace the damaged tissue. the polish stem cell bank (pbkm) has provided wharton's jellyderived msc (wj-msc) for medical therapeutic experiment application in children with cp. wj-msc from third party donors were administered to patients (pts) with cp aged from . / to . / (median age: years and month). twenty two pts have received infusions intravenously (i.v.), pt intrathecally (i.t.), and pts via both routes (first i.v., next i.t.). the cells were previously collected from healthy newborns, processed, screened for bacterial contamination as well as endotoxin content, and frozen in liquid nitrogen vapour. msc immunophenotype was confirmed using flow cytometry assay. the pts have received from to infusions in intervals from weeks to months. median i.t. dose was × cells per infusion, while median i.v. dose was × cells/kg of body weight per infusion. each patient has been examined by the same neurologist at the day of each infusion and the result of examination has been described in a follow-up. twelve patients were diagnosed with epilepsy as comorbidity. eighteen pts ( %) showed positive changes in neurological examination after their treatment with wj-msc. almost half of the children experienced improvement of cognitive functions ( out of pts). muscle tension was reduced in pts. improvement in the ability to concentrate, better contact with others and improved social interactions were observed in % of pts. correction of motility was noticed in pts, pts have experienced better quality of sleep. in cases there has been a reduction in the number of epileptic seizures ( pt even discontinued some of his medicines). there were no s noticeable changes in neurological examination of patients. seven follow-up forms have been not received yet. the experiment data provide evidence that third-party donor wj-msc are suitable and efficient stem cells for treatment in patients with cp. however further and more extensive examination, with a greater number of patients is needed, which will be beneficial for far-reaching results. spina bifida (sb) is a congenital malformation resulting from failure of fusion in the spinal neural tube during embryogenesis. despite surgical repair of the defect, most patients who survive with spina bifida have multiple system damage due to neuron deficiency in the spinal cord. it has been confirmed that the mesenchymal stem cells (mscs) have the ability to survive, migrate and differentiate into cells of a neural lineage. wharton's jelly-derived mscs (wj-mscs) from third-party donors have high proliferation and differentiation potential along with non-immunogenic features, thus seem to be a promising stem cell source. the polish stem cell bank (pbkm) has provided wj-msc for clinical application in a medical therapeutic experiment for children with sb. eleven patients (pts) were qualified for administration of wj-mscs. three pts have been waiting so far for their therapy after bioethical committee approval. seven pts were in the middle of stem cell therapy (after or injections), pt had finished one cycle of stem cell therapy ( injections -ijs) and resumed therapy by administering a first dose of wj-mscs. the cells were previously collected from healthy newborns, processed, screened for bacterial contamination as well as endotoxin content, and frozen in liquid nitrogen vapors. six pts have received infusions intravenously (median dose: . × /kg body weight per infusion), and pt was given injection of × cells intrathecally. each patient has been examined by the same neurologist at the day of each infusion and the result of examination has been written in a follow-up. there were pts, who received at least doses of wj-msc, and all of them showed positive changes in neurological examination. the important improvement, declared by pts, was in areas: pronunciation and/or self-reliance ( pts), movement of arms and/or legs ( pts), quality of life ( pts), core stabilization ( pt). only one adverse event occurred after third injection of wj-msc: pt had nausea and a fever. in case of other pts it was too early to provide reliable feedback. the transplantation of wj-mscs could stimulate the mscs to differentiate towards sensory neurons. this could be one of the reasons of observed improvement of many vital functions in patients, after mscs treatment. this approach might have value in the experimental treatment of sensory neuron deficiency in spina bifida. key: cord- - qib authors: nan title: the th annual meeting of the european society for blood and marrow transplantation: physicians – poster session date: - - journal: bone marrow transplant doi: . /s - - - sha: doc_id: cord_uid: qib nan background: allogeneic hematopoietic stem cell transplantation is routinely offered to patients with high-risk or advanced all in the hopes of improving outcomes. use of truly non-myeloablative (nma) conditioning reduces toxicity in other contexts but outcome data for all patients after nma transplants is lacking. we report the outcomes of patients with all transplanted using a nma conditioning without t cell depletion. methods: first transplant patients between october and june were reviewed. these were consecutive patients until then only those considered unfit for fmc conditioning as per the ukall protocol. all patients were conditioned with fludarabine mg/m /day for days and cyclophosphamide g/m /day for days. short course mtx and ciclosporin were used for gvhd prophylaxis. standard supportive care was employed. thirty-one patients with a median age of ( - ) met the criteria for this case review. had b-all and were philadelphia chromosome positive. patients ( %) had high risk disease by standard diagnostic criteria. ( %) were in first complete remission (cr ). matched sibling donors were used in instances with the remaining being fully matched unrelated donors. % of patients had a hct-ci score of , % a score of or with patients having a score of or higher. median cd dose was . x /kg ( . - . ) with a median cd dose of . x / kg ( . - . ) results: trm was low at % at year and % at and years respectively. no factors included in a univariate analysis (which included age, diagnosis, disease status, hct-ci, donor type, cmv risk and cell dose) significantly impacted trm. the incidence of classical acute (a) gvhd grade - and - was % and % by day and % and % by day if late onset agvhd is included. out of eligible patients developed chronic gvhd of any stage. relapse incidence was low ( % at years in all patients, % in cr patients) and was not impacted by any pre-transplant factors including positive mrd post phase induction (present in patients). notably, in univariate analysis relapse was significantly lower in patients who developed chronic gvhd. background: allogeneic stem cell transplantation (allosct) is the treatment of choice for many patients (pts) suffering from acute myeloid leukemia (aml). the graft vs. leukemia effect (gvl), applied by immunocompetent cells of donor origin, is the most important effector mechanism for the eradication of leukemia, the presentation of leukemic or allospecific antigens by malignant blasts is regarded as a crucial trigger for an effective allogeneic immune response. conversely, insufficient stimulatory capacity by the leukemic blasts is thought to be a relevant escape mechanism from cellular immunotherapy (allosct or donor-lymphocyte infusion (dli)). the purpose was to test, whether the ability of malignant blasts to differentiate in vitro towards dendritic cells of leukemic origin (dc leu ) is associated with response to allosct or outcome after immunotherapy (second allosct or dli) for post-transplant relapse in aml. methods: leukemic blasts were isolated from peripheral blood (pb) or bone marrow (bm) samples of aml patients before allosct (n= ) or at relapse after allosct (n= ). a panel of different assays was used to generate dc leu in vitro ( of them containing gm-csf). finally, in vitro results were correlated with clinical characteristics and outcome of patients treated with donor lymphocyte infusion and/or allosct. results: dc leu could be generated in vitro from all samples. when correlating proportions of dc-subtypes generated ex vivo with clinical data, significantly higher mean proportions of dc leu in the dc-fraction were found in responders vs. non-responders to immunotherapy ( . % vs . %,p= . , range: %- %). vice versa, the chance for response to immunotherapy was significantly higher, if a dc leu /dc ratio of >= % could be reached in vivo (p= . ). those patientswere characterized by a longer time to relapse (p= . ) and by a higher probability for leukemia-free survival (p= . ). similarly, generation of higher amounts (> %, p= . ) of dc leu in the mnc-fraction, and generation of more mature dc (> % cd +, p= . using the best gm-csf containing assay) were associated with a longer time to relapse in the respective patients. moreover, overall survival was improved, if > % dc leu /dc could be generated with the best gm-csf containing assay (p= . ). conclusions: in vitro generation of dc/dc leu from leukemic blasts obtained in active stages of aml before allosct or at relapse post transplant were associated with clinical outcome. this observation supports a role of antigen presentation by leukemic cells for an allogeneic immune response in aml. disclosure: nothing to declare background: the role of autologous hematopoetic cell transplantation (hct) in the treatment of aml is not clear. trials in the past have shown that autologous hct consolidation lowers the risk of relapse, however the magnitude of this effect is limited . autologous hct is advocated in patients with aml with lower genetic risk in cr .many of these patients will eventually relapse and will undergo reinduction followed by allogeneic hct in cr . methods: the aims of this study is to analyze outcome of allogeneic hct performed in cr comparing patients with prior consolidation by autologous hct vs. patients with chemotherapy consolidation. primary outcome is non relapse mortality (nrm) of allogeneic hct in cr in patients with, or without prior autologous hct in cr . secondary outcomes include leukemia free survival (lfs), relapse rate (ri), graft versus host disease free relapse free survival (grfs), overall survival (os), and treatment related toxicities. results: adult patients reigstered with the alwp of the ebmt with de novo aml were included, receiving a first allogeneic hct in cr , in cr , in - or without (n= ) prior autologous hct. patient and transplant characteristics are shown in the table. patient groups were not entirely comparable, patients with prior autologous hct were younger, had less often a favorable cytogenetic profile, had more commonly donors other than matched siblings and more often received reduced intensity conditioning (ric) as compared to mac conditioning. univariate outcomes are shown in the table with slightly higher nrm risks in patients with prior autologous hct consolidation. in multivariate analysis nrm risks in patients with prior autologous hct were . ( . - . ), p= . after adjustment for patient age, cytogenetic risk category, year of transplant, donor type, conditioning intensity, sex matching, time from diagnosis to relapse and time from relapse to allogeneic hct as compared to patients with chemotherapy consolidation. similarly, risks of events in lfs and grfs were higher with prior autologous hct, . ( . - . ), p= . and . ( . - . ) p= . , respectively, risk of death was also higher . ( . - . ) p= . but this was not statistically significant. conclusions: we may conclude that some of the advantages of potentially higher anti-leukemic activity of high dose chemotherapy and autologous hct when given to patients with aml in cr (as was shown in a randomized trial by vellenga e et al with lower relapse and higher lfs by approximately % but no significant differences in overall survival) may be lost by higher toxicity of allogeneic hct in cr in case of subsequent relapse. background: although relapse is a major cause of mortality in patients receiving allogeneic hematopoietic cell transplantation (hct) for acute leukemia, limited and conflicting data exist on extramedullary relapse (emr). we aimed to describe the incidence, risk factors, outcomes and prognosis in relapsed hct recipients. methods: we retrospectively reviewed charts of consecutive allogeneic hct recipients transplanted in our center with the indication of acute leukemia ( / - / ). we recorded: age, gender, disease, previous extramedullary involvement, phase at transplant, type of transplant, donor, conditioning, graft-versus-host-disease (gvhd), infections, treatment-related mortality and relapse mortality. in patients with extramedullary relapses, additional data on clinical manifestations, imaging, cerebrospinal fluid testing, histopathology and management were additionally documented. incidence of isolated emr (iemr) and bone marrow relapse (bmr) was calculated using cumulative incidence (ci) analysis, with each and treatment-related mortality considered a competing risk. results: among allohct recipients followed for . ( . - . ) years, ( %) patients presented with emr. the majority of emrs involved the central nervous system (cns, %). isolated emr was observed in patients at . ( . - . ) months. -year cumulative incidence (ci) of . % for iemr was associated only with pre-transplant advanced disease phase (p< . ). bmr was observed in patients at ( . - months), with a -year ci of . %. in the multivariate analysis, bmr ci was independently associated with fungal infections (p< . ), pre-transplant disease phase (p< . ) and lines of treatment (p= . ). -year trm of our whole cohort was . %. the majority of iemr and bmr ( % and %, respectively) patients received systemic treatment combined with local radiation for iemr ( %) and donor lymphocyte infusions (dlis, % and % respectively) when feasible. extensive chronic gvhd was recorded in % of iemr and % of bmr patients. outcomes were poor in iemr, with -year overall survival (os) of . %. favorable os in iemr was associated only with sibling donors (p= . ) and not with other factors, such as treatment with dlis or presence of chronic gvhd. similarly poor outcomes ( year os of . %) were observed in bmr. favorable os was independently associated only with the diagnosis of aml (p= . ) and absence of bacterial infections (p= . ). in the whole cohort, both iemr and bmr were independent unfavorable predictors of os (p< . ) along with extensive chronic gvhd (p= . ). conclusions: in a large population with long-term follow-up, incidence of iemr was relatively high, developed at the late post-transplant period and associated only with disease phase at transplant. furthermore, iemr and bmr conferred similarly poor outcomes despite systemic treatment or extensive chronic gvhd. these independent predictors of survival highlight the unmet clinical need of novel approaches either as maintenance or treatment to reduce extramedullary or systemic relapse post allohct for acute leukemia. disclosure: no competing financial interest. impact of t-cell depletion on outcome in patients undergoing allogeneic hematopoietic stem cell transplantation for acute myeloid leukemia background: after a diagnosis of acute myeloid leukemia (aml) the majority of patients (pts) who achieve complete remission (cr) eventually relapse, with only approximately % of pts maintaining cr for years or longer. late relapses (after years in cr) occur rarely ( - %) in pts receiving hsct in cr and late effects are followed up by routine surveillance as well as preventative measures. the purpose of this study was to investigate long-term outcomes in pts with diagnosis of aml undergoing hsct at our institution in cr . methods: a standardized follow-up of hsct-survivors is applied at our center. we analyzed adult pts with aml in cr consecutively transplanted between january and december at our institution. a written consent was given for the use of medical records for research. a landmark analysis was adopted for patients in cr at -y after hsct (ltcr -long-term cr). results: ltcr was achieved after hsct in / patients (male , female ) transplanted in cr . the median follow-up was years and the median age at transplant years (r - ). the selected donor was a family haploidentical relative in cases, an hla identical relative in , a match unrelated donor in and a cordblood in . in this cohort of ltcr, the -year overall survival was % ( % ci - ). cumulative incidence of relapseevaluated in competing risk with transplant related mortality (trm) -and trm -evaluated in competing risk with relapse -were respectively % ( % ci - ) and % for the cr cohort. the event-free-survival (efs) was % ( % ci - ). the causes of death were relapse ( / pts), second cancer ( / pts) and sepsis ( / pts). the -year incidence of dyslipidemia -defined as cholesterol >/= mg/dl, and/or ldl >/= mg/dl, and/or triglycerides >/= mg/dl or need for specific treatment -was %. the -year incidence of osteopenia / osteoporosisdefined as t-score lower than - and greater than - . and t-score lower than . respectively -was %. the -year incidence of second cancer was %: nonmelanoma skin cancer, lung carcinoma, cervical intraepithelial neoplasm, thyroid cancer, gastric cancer and colon cancer. the -year incidence of chronic moderate-severe gvhd was % ( % ci - ), with the latest diagnosis performed on day . of note, / pts are still on active treatment at last follow-up. conclusions: relapse incidence is low for patient that reached ltcr: patients in cr at transplant can obtain excellent os and efs once reached the target of ltcr. a proactive long-term follow-up and strategy of counseling are essential to keep at best quality the survival advantage offered by hsct in patients with aml in cr . disclosure: chiara bonini has research contract with intellia therapeutics. the other authors declare that they have no conflicts of interest. background: relapse, graft-versus-host disease (gvhd) and gvhd-associated mortality are major obstacles to success of transplantation from unrelated (mud) donors in children with acute leukemia (al). negative depletion of αβ t cells and cd + b lymphocytes, conserves the mature donor-derived natural killer cells and γδ t cells in the graft, may improve gvhd control, immune reconstitution and prevent the relapse. we present a retrospect analyses of a cohort of pts with al in cr transplanted from mud with depletion. methods: a total of children with acute leukemia ( aml, all, female, male, median age , y) underwent allo hsct from matched unrelated donor between june and july . all pts were in complete remission (cr = , cr = , cr> = ). all pts, except one, received treosulfan-based conditioning. either melphalan (n= ) or thiophosphamide (n= ) or etoposide (n= ) were added as a second agent. fludarabine was used in all pts. two types of gvhd prophylaxis were used: type (n= ): hatg mg/kg and post-hsct tacro/mtx (n= ) or without prophylaxis (n= ); type (n= ): thymoglobulin(ratg) mg/kg, rituximab mg/ m with either bortezomib on days + , + (n= ) or tacro/ mtx (n= ) . aβ t cell depletion with clinimacs was used in all cases. the median dose of cd + cells was x / kg, aβ t cells - x /kg. median time of follow-up for survivors was , years (range, , - , ) . results: primary engraftment was achieved in % pts., the median time to neutrophil and platelet recovery was and days, respectively. all evaluable pts achieved sustained complete donor chimerism by day + . early ( day) mortality was , % ( pt -bacterial sepsis, pt -adv fulminant hepatitis), -years overall ptrm at years was , % ( %ci: - ). six late trm events were due to: viral infection in pts (cmv= , adv+cmv= ), bacterial sepsis in pts and pts had bacterial and viral infection, all late deaths were associated with cgvhd and prolonged corticosteroid therapy. ci of acute gvhd grades ii-iv was % ( % ci: - ), acute gvhd grades iii-iv , % ( % ci : , - , ) . ci of cgvhd was %( %ci: - ). regimen was more effective in prevention of agvhd ii-iv in comparison with regimen : % ( % ci: , - ) vs , %, respectively, p= , . all events with acute gvhd grades iii-iv had pts with regimen . ratg was also effective in prevention of cgvhd: ci at years after hsct was , % vs. %, respectively, p= , . cumulative incidence of relapse was % ( %ci: - ) without difference between ratg and hatg. event-free survival (efs) (event=death or relapse) at years was % ( %ci: - ), overall survival %( % ci: - ), there were no difference between age and diagnosis. conclusions: we confirm that the depletion of tcrαβ +/cd + t lymphocytes from the graft ensures high engraftment rate. transplant-related mortality is caused by infections, mostly associated with cases of chronic gvhd. gvhd prophylaxis including ratg/rituximab/ bortezomib improves gvhd control in recipients of tcrαβ+/cd +depleted grafts in comparison to hatg/ tacro/mtx apparently without loss of anti-leukemic activity. disclosure results: at baseline, r/r all with emd and lbl were diagnosed in and ino patients and and sc patients. median (range) age of the ino and sc patients was . ( - ) and . ( - ) years, with / ( . %) and / ( . %) males, respectively. the rate of cr/cri was significantly higher in the ino group ( / [ . %] , % confidence interval [ci] : . - . ) compared with sc ( / [ . %], % ci: . - . ; p= . ) (table) . allogeneic hematopoietic stem cell transplantation was carried out in / ( . %) ino and / ( . %) sc patients prior to any post-study induction therapy. the pfs hazard ratio [hr] was . ( . % ci: . - . ; p= . ), with median pfs of . ( % ci: . - . ) months among ino and . ( % ci: . - . ) months in sc patients. the os hr was . ( . % ci: . - . ; p= . ), with median os of . ( % ci: . - . ) months in ino versus . ( % ci: . - . ) months in sc patients (figure) . all patients had adverse events (aes). serious aes occurred in / ( . %) ino and / ( . %) sc patients; ( . %) ino and sc patients had grade ae. one ( / , . %) patient in the ino group died from veno occlusive disease. conclusions: among r/r all patients with emd and lbl, improvement in remission rates, transplant rates, and progression free survival was shown in the ino group versus the sc group. although patient numbers were small and limited the ability for a robust comparison, these results support the use of ino in patients in this difficult to treat population with r/r all and emd or lbl. background: bcr-abl-targeted tyrosine kinase inhibitors (tki) revolutionized the outcome of patients inflicted with ph+ b-all. moreover, addition of tki may be relevant strategy for ph-like all patients. methods: we hypothesized that overcoming the bm microenvironment-mediated protection of all cells from tki-mediated apoptosis may further enhance the responsiveness to tki therapy. results: in vitro treatment of bcr-abl-positive all cell lines nalm and nalm ) with dasatinib resulted in significant dose-dependent cell growth inhibition, with ic of - nm (p< . ). furthermore, dasatinib exhibited significant growth suppression of bcr-abl -negative all cells (nalm and reh), with ic of nm and nm, respectively. however, when cocultured with bone marrow stromal cells (bmscs), dasatinib-mediated effect was abrogated in both ph-and ph+ all cells. furthermore, dasatinib treatment promoted significant upregulation of chemokine receptor cxcr , on both mrna and cell surface levels. elevated cxcr expression was accompanied by increased responsiveness of all cells to cxcl stimulation, resulting in strong and sustained phosphorylation of erk / and akt and increased adhesion capacity to bmscs. therefore, dasatinib-induced upregulation of cxcr promotes stroma-mediated survival advantage of all cells upon tki therapy. next, in order to overcome the cxcr -mediated stromal protection, we choose to combine dasatinib with the histone deacetylase inhibitor panobinostat, for its known ability to deplete cxcr in aml cells. single-agent treatment with panobinostat demonstrated significant inhibition of ph-and ph+ all cell growth at low nanomolar concentrations (p< . ). importantly, combination of panobinostat with dasatinib synergized (ci< . ), effectively overcoming the protection provided by bmscs and inducing the apoptosis of ph-and ph+ all cells, as demonstrated by phosphatidylserine externalization, mitochondrial depolarization and dna fragmentation. furthermore, combining panobinostat with dasatinib significantly reduced cxcr surface levels in ph-and ph+ all cells. accordingly, cxcl mediated responses, including erk / and akt activation and adhesion to bmscs were significantly reduced upon combined panobinostat/dasatinib treatment. these data indicate that panobinostat effectively suppresses both basal and dasatinib-induced cxcr expression and function in all cells overcoming stroma-mediated resistance to dasatinib. to determine the molecular mechanism, we performed gene and protein expression analysis. panobinostat, alone or in combination with dasatinib, significantly down-regulated the protein levels of calcineurin, a serine-threonine protein phosphatase previously implicated in t-all and b-all pathogenesis, as well as of nfatc , a critical effector of the calcineurin signaling cascade, and nfatc -regulated target genes. it was previously found that calcineurin signaling positively regulates cxcr expression in t lymphocytes. additionally, cyclosporin a (csa) decreased both basal and dasatinib-induced cxcr surface levels in all cells, overcoming the protection of the bmscs which result in potentiation of the cytotoxic effect of dasatininb and panobinostat. combining csa with panobinostat resulted in deeper suppression of nfatc -regulated target genes. we thus link the effect of panobinostat with calcineurin-dependent downregulation of cxcr , blocking the ability of the leukemic cells to respond to cxcl mediated stromal support. conclusions: taken together, our results identify calcineurin signaling pathway as a novel target of panobinostat in all cells and indicate that hdac inhibition with panobinostat may be effective strategy for facilitating the anti-leukemic activity of tki therapy. disclosure: nothing to disclose background: the treatment of relapsed/refractory acute lymphoblastic leukemia (rr-all) remains a clinical challenge with a generally dismal prognosis. allo-sct using a sequential conditioning ("flamsa"-like regimen) has shown promising results in relapsed/refractory aml, but little is known about the efficacy of this procedure in rr-all. methods: we identified adult patients ( % females; median age: y; range, - ) with all in primary refractory phase ( %) or in relapse ( %), allografted between and from a matched sibling ( %), matched unrelated ( %) or haploidentical donor ( %) at ebmt participating centers. almost half ( %) of the patients had t-all and % had a positive philadelphia chromosome. six patients ( %) underwent a previous autotransplant. karnofsky score was above in % of patients. conditioning was myeloablative (mac) with high dose tbi in % of patients, reduced intensity (ric) including low dose tbi in %, or with chemotherapy alone in %. in vivo t cell depletion was performed in cases ( %). most patients ( %) and about half of the donors ( %) were cmv positive. % of patients were males who received a graft from a female donor. the median follow-up was (range, - ) months. results: overall, patients ( %) failed to engraft, ( %) died within days after allo-sct without relapse, and ( %) could achieve complete remission. at day , the cumulative incidences of grade ii-iv and grade iii-iv acute gvhd were % and %, respectively. the year cumulative incidences of chronic and extensive chronic gvhd were % and %, respectively. the -year relapse incidence (ri) and non-relapse mortality (nrm) were % and %, respectively. the -year leukemia free survival (lfs), overall survival (os) and gvhd relapsefree survival (grfs) were %, % and %, respectively. in a multivariable cox analysis, karnofosky score below negatively affected ri, lfs, os and grfs. also, conditioning with chemotherapy alone, compared to tbibased conditioning, negatively affected relapse rates (hr= . ; p= . ), lfs (hr= . ; p= . ) and os (hr= . ; p= . ). conclusions: allo-sct using a sequential conditioning regimen is proposed by different teams in rr-all, and could be an option, especially when considering a tbibased regimen. however, the overall -year lfs of % suggests that these patients still face extremely dismal outcomes, highlighting that other therapies (e.g. bite antibodies, inotuzumab, car t cells) need to be combined prior and/or after allo-sct in order to further improve outcome. disclosure: no conflict of interest, no funding received chemotherapy courses, only pts were not treated: pts for the worsening of the general status and the other for invasive fungal infection. results: forty-three pts ( %) were in complete remission (cr) and negative minimal residual disease (mrd) at the time of hsct; pts were in active disease ( %), and ( %) showed a morphological cr with positive mrd. pts ( %) developed chronic graft versus-host disease (cgvhd) as followed: pts ( %) mild, pts ( %) moderate, and only sever grade respectfully. only patient developed cgvhd after dli. the overall leukemia free survival (lfs) time was months, the absence of cgvhd (hazard ratio -hr: , ; p = , ) and the pre-hsct disease status (hr , ; p = , ) were the most important factors on lfs. all pts treated with chemo-based regimens died due to progression or infective complications. patient of aza/dli group is still alive with a extramedullary relapse; pts treated with bl/dli are in cr. os was better for the dli group compared to the chemotherapy group ( vs months respectfully; p < , ). conclusions: dli after allo-hsct has exhibited definite anti-leukemic effects in post-transplant patients. bl and aza were reported to increase dli's graft vs-leukemia (gvl) effect. although cgvhd could be the most important protective factor against the relapse but it remains the main cause of morbidity. maximising the gvl effect without putting the patient at risk of gvhd still represents an unmet need. our data show that the combination of either bl or aza with dli infusion is safe and might represent an improvement in disease control in the early phase of relapse. disclosure: nothing to declare p increased detection of (leukemiaspecific) adaptive and innate immune-reactive cells under treatment of amldiseased rats and one therapy-refractory aml-patient with blastmodulating, clinically approved response modifiers (pg-e ,kit-k) or +pge (kit-m),patent ) convert myeloid blasts into dendritic cells of leukemic origin (dc leu ). after stimulation with dc leu , antileukemic tcells can be generated ex vivo. the compounds are approved for clinical use and are therefore attractive tools for immunotherapy in myeloid leukemia. methods: dc/dcleu-culture from rats'/patients' wholeblood (wb) with kits, mixed lymphocyte culture (mlc) of tcells with kit-treated blood, functional blast-cytotoxicity and leukemia-specificity assays (csa/elispot/degranulation/intracellular cytokine-assays). in addition flowcytometric evaluations of cellular and (leukemia-specific) lymphocyte compositions were performed from rats'/pts' blood in the course of the disease. results: ) aml-diseased rats: each rats were treated with "i", "k" or "m" or were untreated (controls). a significant increase of dcleu could be detected in spleen/pb in kit-(esp. m) treated compared to untreated animals without induction of blasts' proliferation (ki positivity): a significant reduction of blasts was seen with "m" (p= . / . in spleen/pb) and "i", but not "k". successful treatment correlated with an increase of cd l+tcells, most likely representing tmem-cells, (p= . ) and a reduction of cd +treg (p= . ). ) therapy-refractory aml-patients (during the course of decitabine/ld-aractreatment): kit-m was shown to ex vivo generate dcleu, activate immunereactive cells and mediate leukemia-specific/antileukemic response. activated or leukemia-specific lymphocytes were monitored in low proportions in active stages of the disease as well as of two patients during the further course of persisting disease. one of these patients ( yo male), was offered an individual systemic salvage-treatment (kit-m, applied as continuous infusions) for refractory leukemia. after approval from the local ethical commitee,extensive information of the patient about the experimental nature of the treatment and obtaining his written informed consent. clinically the treatment was well tolerated and the patient improved clinically. neutrophils in wbc increased from % to %, thrombocytes reached g/l after days. after weeks of treatment, the patient was discharged in good clinical conditions. days later, progression of aml was seen with high blast counts in pb and bm. the patient developed severe sepsis and died few days later. immune monitoring showed (other than before treatment and in the patients without kit-m-treatment) a continuous increase of proliferating and non-naïve tcells, nk, cikand nkt-, th cells, bmem-cells and dc in pb. the production of ifnƔ producing t-, cik and nkt-cells was demonstrated, suggesting an in vivo production/activation of (potentially leukemia-specific) cells. immune stimulatory effects decreased after discontinuation of therapy. conclusions: treatment of wb as well as leukemically diseased organisms with blast-modulating kits (especially gm-csf and pge ) was well tolerated and induced clinical and immunological improvement (adaptive and innate immune system), whereas low counts of (leukemiaspecific) activated immune-reactive cells were found in non-kit-treated organisms. disclosure: nothing to declare p long-term outcomes after allogeneic hematopoietic stem cell transplantation for acute myeloid leukemia with non-myeloablative and myeloablative conditioning: a single-center cohort study of consecutive patients lars klingen gjaerde, niels smedegaard andersen , lone smidstrup friis , brian thomas kornblit , søren lykke petersen , ida schjødt , henrik sengeløv rigshospitalet, copenhagen, denmark background: since , we have at our institution used a non-myeloablative (nma) conditioning regimen for older (> years) or significantly comorbid younger patients undergoing allogeneic hematopoietic stem cell transplantation (allo-hsct) for acute myeloid leukemia (aml). we aimed to compare the long-term outcomes of nma conditioned patients with myeloablative (ma) conditioned patients. methods: we studied nma and ma conditioned adult (> years) consecutive patients receiving their first allo-hsct for aml from to at rigshospitalet. nma conditioning consisted mainly of gy total body irradiation (tbi) and fludarabine mg/m ( % of cases). ma conditioning consisted mainly of cyclophosphamide mg/ kg and either gy tbi ( % of cases) or busulfan . mg/ kg ( % of cases), or fludarabine mg/m and treosulfan mg/m ( % of cases). five percent and % of nma and ma conditioned patients, respectively, received anti-thymocyte globulin. patients were followed until death or end-of-followup on october st , . cumulative incidences with % confidence intervals (ci) of acute graft-versus-host disease (agvhd) grade ii-iv, chronic graft-versus-host disease (cgvhd), relapse and non-relapse mortality (nrm) were calculated and compared between nma and ma conditioned patients using gray's test with death as a competing risk (or relapse when comparing nrm). overall survival (os) was estimated by the kaplan-meier method. results: nma and ma conditioned patients were comparable when regarding sex ( % and % female, respectively) and donor (matched related donor in % and %, respectively), but differed, as expected by indication, with regards to age (median of versus years, respectively) and karnofsky score (< in % and %, respectively). nma conditioned patients had generally a lower aml stage at transplant ( st complete remission in % versus % of ma conditioned patients) and a lower aml cytogenetic risk (adverse risk in % versus % of ma conditioned patients). patients were followed for a total of person-years (median follow-up in surviving patients was . years). agvhd grade ii-iv occurred less frequently in nma conditioned patients ( % [ci: %- %] versus % [ci: %- %] in ma conditioned patients, p < . ), while cgvhd occurred in similar rates ( % [ci: %- %] in nma conditioned patients and % [ci: %- %] in ma conditioned patients, p = . ). there was a trend towards a higher relapse rate in nma conditioned patients ( % [ci: %- %] versus % [ci: %- %] in ma conditioned patients, p = . ), and nma conditioned patients had, however not with statistical significance, lower nrm ( % [ci: %- %] versus % in ma conditioned patients, p = . ). os ( figure) was comparable, with -year os rates of % (ci: %- %) in nma conditioned patients and % (ci: %- %) in ma conditioned patients. conclusions: patients with aml undergoing allo-hsct with nma conditioning at our institution were older and frailer than ma conditioned patients, but their overall survival after transplantation was comparable. this might be explained by a generally lower aml stage and cytogenetic risk at transplant in nma conditioned patients. jedlickova , saskia güller , rosa toenges , juliane steinmann , hans martin , hubert serve , gesine bug background: allogeneic hsct is urgently indicated in patients with aml in first complete hematologic remission (chr) after intensive chemotherapy with increasing or recurrent minimal residual disease (mrd). these patients are at high risk of hematologic relapse (hr) during preparation of their transplant and hsct with active aml was found associated with poor outcome. azacitidine has recently been shown to substantially delay or even prevent hr in > % of patients (relaza trial, platzbecker et al., lancet oncology ) . we here present the outcome of a small cohort of consecutive patients with mrd-positive aml who received low dose cytarabine (ldarac) as bridging therapy prior to hsct. methods: mrd was assessed by quantitative polymerase chain reaction (qpcr) using mutated npm (n= ), runx -runx t (n= ), cbfb-myh (n= ) or kmt a-ptd (n= ). mrd negativity was defined as ratio of oncogene to control gene (abl ) ≤ , % while increased or recurrent mrd required a ratio > % (shayegi et al., blood ) . primary endpoint of our retrospective analysis was progression to hr (≥ % bone marrow blasts or extramedullary disease); secondary endpoints were achievement of molecular remission prior to hsct, neutropenia g according to ctcae, thrombocytopenia g , anemia ≥g , admission to hospital, os and rfs. os and rfs were calculated from the first dose of ldarac. ldarac was self-administered subcutaneously by the patients at home at a flat dose of mg bid over days and repeated after weeks if necessary. results: between / and / , nine patients (median age , range, - years) with low (n= ), intermediate (n= ) or high-risk cytogenetics (n= ) according to eln criteria were treated in continuous chr for increasing (n= ) or recurrent mrd (n= ) starting at a median of (range, - ) days after the last consolidation therapy, i.e., duration of chr was > months in all pts. patients received one (n= ), two (n= ) or three cycles (n= ) of ldarac prior to hsct. in three patients, neutropenia g occurred and one patient needed platelet transfusion. all patients were managed in the outpatient setting. in eight out of nine patients ( %), hr was successfully prevented and patients ( %) even became mrd negative prior to hsct. one patient (runx -runx t positive aml) progressed to hr after one cycle of ldarac and received salvage therapy with high-dose arac and mitoxantrone (ham) prior to hsct. all patients proceeded to hsct from a matched related (n= ), unrelated (n= ) or haploidentical donor (n= ) and are still alive (median follow-up of days). conditioning regimens included fludarabine (flu)/melphalan (mel)/tbi (n= ), flu/mel (n= ), flu/tbi (n= ), flu/busulfan (bu) (n= ) and thiotepa/bu /flu (n= ). after hsct, only the ldarac-refractory patient relapsed, resulting in a probability of rfs of % at years. conclusions: our data suggest that a bridging therapy with up to three cycles of ldarac prior to hsct is feasible and was associated with favorable outcomes in patients with npm -mutated or core binding factor aml and molecular relapse > months after achieving a first chr. the treatment has low costs, can be administered on an outpatient basis and is very well tolerated. clinical background: allogenic hematopoietic stem cell transplant (hsct) is the only curative treatment for all the patients with aml. high risk disease qualifies for upfront hsct irrespective of the presence of matched sibling donor (msd). in the absence of msd, haploidentical stem cell transplant is easier option with success rates as high as msd in a high volume transplant centre. we present our experience from a single centre. methods: we analyzed retrospective data of aml patients who have undergone hsct at our centre between january- and august- . for msd transplant we used fludarabine + busulfan or fludarabine + melphalan conditioning regimen, in matched unrelated donor transplant (mud) regime used was fludarabine + busulfan + atg. we followed john hopkins's protocol for haploidentical hsct. cyclosporine + methotrexate was used as gvhd prophylaxis in msd and unrelated donor group and cyclophosphamide + tacrolimus + mycophenolate was used for haploidentical post-transplant. day survival, overall survival (os), incidence of gvhd and cmv reactivation was computed. results: a total of aml patients underwent hsct during the study period, the basic and clinical characteristics of the study patients are presented in table . conditioning regime did not have significant impact on os. survival at day was %. the os function and relapse free survival (rfs) function did not significantly differ between msd and haploidentical transplantation ( . % vs . %; p= . ) and ( . % vs . %; p= . ) (graph ). disease status at latest follow up showed that % were in remission and % had relapsed. overall one year survival and five year survival in the entire cohort was % and % respectively. the average cost of msd transplant at our centre is inr , , (€ - ), haploidentical transplant is inr , , (€ - ) and mud transplant is inr , , (€ , + for stem cell procurement). conclusions: our study showed comparable outcomes in msd and haploidentical transplant with respect to day survival, os, and rate of gvhd. in a developing country like india where patients are not covered under state health insurance, the additional cost of procurement of stem cells in a mud transplant would add to the financial burden to the patients. haploidentical transplant is a feasible option in case of non-availability of msd, due to ease of donor availability and strong motivation from the family donor to donate the stem cells. background: allogeneic stem cell transplantation (allo-hsct) is not indicated as consolidation of first complete remission (cr ) in favorable-risk acute myeloid leukemia (aml) bearing mutations in nucleophosmin (npm ) in the absence of flt internal tandem duplication (flt -itd). nevertheless, a substantial proportion of patients eventually proceed to allo-hsct beyond cr or for chemoresistant minimal residual disease (mrd) while in cr , which might compromise transplantation outcomes. the study aimed at examining the characteristics and results of allo-hsct in aml cases with mutated npm and wild-type flt (npm mut/flt wt), with special focus on molecular monitoring of mrd following transplantation. methods: from / until / , patients (women/men, / ) underwent allo-hsct for npm mut/ flt wt aml. at transplant, median age of patients was . years (range, - ) , and disease phase was cr (n= ), cr (n= ), or primary refractory (n= ). among the patients who were transplanted in cr and had available molecular mrd assessments, had detectable mutant npm transcripts by real-time quantitative pcr (rq-rcr). also, patients fulfilled criteria of molecular relapse (increasing levels of npm -mutated transcripts in two successive bone marrow samples), with mutant npm load of - , transcripts/ , abl transcripts). the conditioning regimen was myeloablative in the majority of cases (n= ) or reduced-intensity (n= ). the type of donor varied, namely hla-identical sibling (n= ), matched unrelated (n= ), haploidentical relative (n= ), or double umbilical cord blood (n= ). results: engraftment was achieved in all cases, with a median time to absolute neutrophil count > /ul of days (range, - ) . among the patients with posttransplant monitoring of mrd by rq-pcr, exhibited a stable molecular remission whereas a rising level of npm mutated transcripts was observed in cases due to either hematologic (n= ) or molecular (n= ) relapse of disease. the cumulative incidences (cin) of hematologic relapse and non-relapse mortality (nrm) were . % and % at months, respectively. no events of relapse or nrm were encountered beyond months from allo-hsct. out of patients with hematologic relapse post transplant, died of disease whereas one achieved a stable complete remission after withdrawal of immunosuppression. at a median follow-up time of months (range, - ), / patients continue to be alive in cr. the estimated disease-free background: cpx- (vyxeos®) is an advanced liposomal encapsulation of cytarabine/daunorubicin at a synergistic : molar ratio. cpx- is approved by the us fda and ema for the treatment of adults with newly diagnosed, therapy-related aml or aml with myelodysplasia-related changes. methods: safety data were pooled from studies of cpx- in adults aged - years with newly diagnosed or relapsed/refractory aml. cpx- induction consisted of units/m (cytarabine mg/m + daunorubicin mg/m ) on days , , and (second induction: days and ) . cpx- consolidation consisted of or units/m (varying by study) on days and . cpx- was evaluated against standard-of-care controls. results: baseline characteristics were generally balanced between cpx- (n= ) and controls (n= ); the majority of patients were aged ≥ years ( %; %) and had secondary aml ( %; %). controls included + (n= ) and salvage therapy with mitoxantrone/etoposide/ cytarabine (n= ), idarubicin/cytarabine (n= ), other cytarabine-based chemotherapy (n= ), and mitoxantrone/ etoposide (n= ). the treatment-emergent adverse event (teae) profile of cpx- units/m was comparable to induction controls, but associated with a greater proportion of patients with teaes, grade ≥ teaes, and serious teaes during consolidation (table) . therefore, the cpx- consolidation dose was reduced to units/m in latter studies; this dose demonstrated an improved teae profile similar to consolidation controls. the most frequent system organ class was gastrointestinal disorders for both cpx- and controls; a lower incidence was reported for cpx- ( %) versus controls ( %), with this difference driven by the lower incidence of diarrhea for cpx- ( %) versus controls ( %). the most frequently reported grade ≥ teaes were febrile neutropenia (cpx- : %; controls: %), pneumonia ( %; %), hypoxia ( %; %), and bacteremia ( %; %). early mortality rates, both overall and by treatment period, appeared lower with cpx- versus controls at day and day ( table) ; the majority of early deaths were attributable to teaes. conclusions: across the studies comprising the cpx- clinical development program, cpx- demonstrated a safety profile comparable to conventional chemotherapy in adults with newly diagnosed or relapsed/refractory aml. background: haploidentical hematopoietic stem cell transplantation (hsct) with post-transplantation cyclophosphamide (pgcy) marked improved clinical outcome. recent studies comparing allogeneic hsct using unrelated donors versus haplo donors in patients with acute leukemia have suggested equivalent outcomes. the depletion of tcells with pgcy was subsequently applied for unrelated hsct setting for patients with unrelated donor. methods: we performed a retrospective study on patients with acute leukemia in order to compare the outcome after hla haploidentical (n= ) and unrelated hsct (n= ) with pgcy. the main characteristics of patients were similar in both groups. baseline disease were: aml ( %) and all ( %) for haplo group and aml ( %) and all ( %) for unrelated group. disease state at time of haplo and unrelated-hsct were following: and patients in cr ( % and %) and and non cr ( % and %). for aml recipients mainly received thiotepa, busulfan and fludarabine and for all recipients received tbi and etoposide conditioning. all patients who received pbsc graft were treated with rabbit antithymocyte globulin (atg) on days - and - . results: at the time of analysis, the os and dfs did not differ between the haplo and unrelated groups ( % vs %, and % vs %). incidence of severe (grade [ ] [ ] acute gvhd was the same in two groups ( % versus %). recipients of haplo-hsct transplant were statistical significance less likely to experience disease relapse ( % vs %) and chronic gvhd ( % vs , %). however, gvhd free relapse free survival (grfs) rate was slightly higher after haplo-hsct ( % vs %). addition, cumulative incidence of trm rate was higher after haplo-hsct ( % vs %).for haplo and unrelated groups who underwent hsct in cr , the os were % and % versus % and % for those in non cr . for aml, the os was same in two groups (haplo % versus unrelated %). however patients with all, the os was higher in haplo group compared with unrelated group ( % versus %). the impact of pretransplant disease state have a more powerfull effect on survival in the haplo-hsct setting (for aml cr % versus non cr % and for all cr % versus non cr %). viral reactivations were significant concern in both groups. conclusions: our retrospective analysis suggests largerly similar os and dfs with haplo versus unrelated transplants with pgcy for acute leukemia. our data indicate that haplo-hsct results in a lower incidence relapse and of chronic gvhd and higher grfs compared with unrelated hsct. in addtion, the pretransplant disease state have the important effect on the outcomes in both groups. allo-pbsc with atg can be used safely and effective as graft source in haplo-hsct with acceptable post-transplant outcomes and replaced bm in this settings. more statistical data for transplant related characteristics will be provided at the presentation.we emphasize that use the same pgcy gvhd prophylaxis for all types of allogeneic transplant. based on our results, we recommend haplo-hsct with pgcy against unrelated transplant for patients with acute leukemia. disclosure: disclosure of conflict of interest: none. excellent efficacy and tolerability of inotuzumab ozogamicin in b-cell all relapsed after allo-hsct background: donor lymphocyte infusion (dli) could be used prophylactically to reduce relapse after allogeneic hematopoietic stem cell transplantation for very high-risk leukemia/lymphoma without effective targeted therapy. to compare the safety and efficacy of prophylactic dli for prevention of relapse after allogeneic peripheral blood stem cell transplantation from haploidentical donors (hid-sct) and matched-sibling donors (msd-sct) in patients with very high-risk acute myeloid leukemia (aml), we performed a retrospective, observational cohort study enrolled in hid-sct and msd-sct recipients. methods: the very high-risk features were defined as: (i) in the non-remission (nr) state prior to transplantation, including primary induction failure, relapse untreated or refractory to reinduction chemotherapy, or untreated aml evolution from mds; (ii) achieving complete remission with ≥ cycles of induction of chemotherapy; (iii) carrying tp , dnmt a, tet or flt -itd gene mutation. the scheduled time of the prophylactic dli was + - days after transplantation for msd-sct recipients and + - days for hid-sct recipients. the g-csfmobilized peripheral blood stem cells were infused to the recipient at a dose of × cd + cells/kg. csa was given at mg/kg b.i.d from day - to day + (hid-sct) or to day + (msd-sct), and then tapered at % per month to be discontinued on day + - (hid-sct) or on day + - (msd-sct) unless graft-versus-host disease (gvhd) developed. if the patients received dli before day + (hid-sct) or day + (msd-sct), csa was given weeks after dli in hid group and weeks in msd group at a though concentration of - ng/ml for dliassociated gvhd prophylaxis, and then tapered and discontinued within weeks unless gvhd developed. if gvhd occurred before the scheduled time of prophylactic dli, it would be delayed for weeks when gvhd was well controlled. results: prophylactic dli was administered at a median of ( - ) days for hid-sct recipients and ( - ) days for msd-sct recipients (p= . ), and both groups displayed similar baseline characteristics except for donor's gender distribution (table ) . grade - acute graft-versushost disease (gvhd) at -day post-dli was higher in hid-sct group than that in msd-sct group ( . % vs. . %, p= . ). grade - acute gvhd ( . % vs. . %), -year chronic gvhd ( . % vs. . %) and severe chronic gvhd ( . % vs. . %) were similar between two groups (p> . ). one-year non-relapse mortality was higher in hid-sct group than that in msd-sct group with marginal significance ( . % vs. . %, p= . ). one-year relapse rate was similar between hid-sct group and msd-sct group ( . % vs. . %, p> . ). estimated -year overall survival (os, . % vs. . %) and relapsefree survival (rfs, . % vs. . %) rates were both similar between hid-sct group and msd-sct group (p> . ). in multivariate analyses, non-remission status prior to transplant, poor-risk gene mutations and donor's age ≥ years predicted a higher risk of relapse after dli. nonremission status prior to transplant predicted inferior os and rfs. patient's age ≥ years also predicted an inferior os. conclusions: prophylactic dli after hid-sct demonstrated similar tolerance and efficacy for reducing relapse compared to that after msd-sct for very high-risk aml. disclosure: the authors declare no conflict of interest. prognostic impact of pre-transplant tim levels on transplant outcome in acute leukemia patients background: t cell immunoglobulin and mucin domaincontaining protein- (tim ), a negative regulator of t cells, is expressed on a variety of tumors including hematological malignancies like acute myeloid leukemia (aml) and some lymphoma types in which it was shown to be associated with an adverse prognosis. the aim of this study is to identify the prognostic impact of pre-transplant tim levels on early and late transplant related complications as well as post-transplant relapse and survival methods: a total of hematopoietic stem cell transplantation (hsct) recipients with an initial diagnosis of acute leukemia [median age: ( - ) years; male/ female: / ] were included in the study. aml was the initial diagnosis in patients ( . %), acute lymphoblastic leukemia (all) in patients ( . %), mixed phenotype acute leukemia in patients ( . %) and blastic plasmacytoid dendritic cell neoplasm in patient ( . %). soluble tim- levels in pre-transplant serum samples were measured with enzyme linked immunosorbent assay (elisa). results: median pre-transplant tim level was . ( . - . ) pg/ml in the whole cohort. pre-transplant tim levels were significantly higher in aml patients when compared to all [ . ( . - . ) vs . ( . - . ); p= . ]. tim levels were significantly lower in patients with abnormal cytogenetics when compared to normal karyotype (p= . ). cytogenetic abnormalities, including mainly a complex karyotype or chromosome abnormalities, were more frequent in patients with low tim levels (p= . ). pre-transplant tim levels were significantly higher in patients who developed post-transplant viral hemorrhagic cystitis (p= . ). a positive correlation was demonstrated between tim levels and acute graft versus host disease (gvhd) grade (p= . ; r= . ). at a median follow-up of . ( . - . ) months, overall survival (os) was found to be better in low-tim group when compared to high-tim group, without statistical significance (% . vs % . ; p> . ) ( figure ). probability of os was relatively better in both aml ( . % vs . %; p> . ) and all patients ( . % vs %; p> . ) representing low pretransplant tim levels in the subgroup analysis conclusions: in this study, elevated levels of pretransplant tim levels in aml patients were compatible with the previous reports which had underlined an increased tim expression on aml stem cells. the possible association of tim expression with cytogenetic features should be confirmed with further studies as there is no adequate data except its relationship with flt -itd mutational status. tim- is also expressed on exhausted t cells in patients with viral infections, including human immunodeficiency virus, hepatitis b and hepatitis c virus. it plays an essential role in the regulation of antiviral and antitumor immune responses which may be an explanation for the increased frequency of hemorrhagic cystitis in patients with higher tim- levels. the adverse prognostic impact of tim on gvhd and os was confirmed without statistical significance which may be related to small sample size. as tim has a wide spectrum of action in the tumor microenvironment including stimulatory and inhibitory activities, further clues are required to define the exact role of this molecule in the clinical course of allogeneic hsct in order to develop targeted therapeutic strategies clinical trial registry: n/a disclosure: nothing to declare p homozygous hla-c is associated with increased risk of relapse after hla-matched transplantation in recipients with acute lymphoid leukemia: a japanese national registry study background: after hematopoietic stem cell transplantation (hsct), the role of natural killer (nk) cells which express killer-cells immunoglobulin-like receptors (kirs) and recognize hla-class ligands is important. kir dl recognizes not hla-c asp (c ), but hla-c lys (c ) and has polymorphism based on the th amino acid of the transmembrane domain. low frequency of c and high frequency of strong kir dl are characteristics observed in japanese. by using large transplant database, we reported that homozygous hla-c (c /c ) recipients displayed lower relapse rates than did c /c recipients after hla-matched hsct for acute myeloid leukemia (aml; hr = . , p = . ) or chronic myeloid leukemia (cml; hr = . , p = . ). this effect seemed to be independent of acute graft-versus-host disease (agvhd) or cytomegalovirus reactivation occurrence (arima n et al bbmt ) . methods: relapse rates of japanese recipients who first underwent hla-matched hsct between and for the treatment of acute lymphoid leukemia (all) were compared between c /c pairs and c /c pairs, using data from japanese data center for hematopoietic cell transplantation and adjusting for transplant characteristics. cord blood transplantation was excluded. multivariable competing risk regression analyses were performed to evaluate relapses and relapse-free survival (rfs) was estimated using kaplan-meier method. results: after recipients who did not achieve remission or experienced graft failure and recipients not-expressing c were excluded, resting recipients aged - years (median, . years) were analyzed. the median follow-up period for survivors was . years. there were recipients expressing c /c and recipients expressing c /c , respectively. after hla-matched hsct, c /c recipients had higher relapse rates than c / c recipients (hr = . , p = . ), resulting in worse rfs among c /c recipients (hr = . , p = . ). the frequent relapse in c /c recipients than in c /c was noticeable among recipients with agvhd (hr = . , p = . ), those without cytomegalovirus reactivation (hr = . , p = . ), and those with ph-negative all (hr = . , p = . ). conclusions: kir dl -positive nk cells may promote graft-versus-leukemia (gvl) in c /c recipients with aml or cml but suppress gvl in c /c recipients with all. one interpretation is that transplant-activated nk cells impair antigen-presenting cells or deprive cytotoxic tlymphocytes of their gvl effects on all cells. this hypothesis may be explained by the fact that agvhd was necessary for the recessive relapse in c /c recipients with all. furthermore, ph-positive all cells sometimes mimic aml cells in terms of their frequent myeloid antigen expression and might be directly targeted by nk cells. it would be necessary to further clarify in vitro the character of nk cell-affecting in the transplant immunity against residual leukemia cells. disclosure: authors have nothing to declare. hematopoietic stem cell transplantation with sequential conditioning for children with relapsed/refractory acute leukemia nao yoshida , kazuki matsumoto , daiki yamashita , yiqing zhu , daichi sajiki , ryo maemura , hirotoshi sakaguchi , asahito hama children's medical center, japanese red cross nagoya first hospital, nagoya, japan background: patients with acute leukemia who fail to achieve complete remission show a dismal prognosis even with allogeneic hematopoietic stem cell transplantation (hsct). this study evaluated whether sequential conditioning approach that is cytoreductive chemotherapy applied shortly prior to the main conditioning followed by hsct can improve prognosis in such high-risk patients. methods: we retrospectively analyzed the outcomes of children (median , range - years old) with primary refractory (n = ) or refractory relapsed (n = ) acute leukemia (aml n = , all n = ) who received hsct in our department between and . the stem cell source was related peripheral blood (pb) in patients, related bone marrow in , unrelated bone marrow in , or unrelated cord blood in . the grafts were hla serologically matched (n = ) or mismatched (n = ) with the recipient. in total, patients received the sequential conditioning approach. as cytoreductive chemotherapy, fludarabine/cytarabine/idarubicin/g-csf (flag-ida) was used in patients, mitoxantrone or daunorubicin/cytarabin in , or other regimens in , and of them were combined with gemtuzumab ozogamicin. without waiting for hematological recovery, the patients promptly underwent hsct; therefore, the median interval between cytoreductive chemotherapy and main conditioning was days. the main conditioning regimens were total body irradiation-based myeloablative (n = ), busulfan-based myeloablative (n = ), or reduced intensity (n = ). results: in children with relapsed/refractory acute leukemia, the -year overall survival (os), leukemia-free survival (lfs), cumulative incidence of relapse (ri), and transplantation-related mortality (trm) were %, %, %, and %, respectively. in multivariate analysis, the use of sequential conditioning was identified as the most favorable factor for lfs (hazard ratio [hr] . ; p = . ), although there were no differences in the outcomes according to the types of cytoreductive chemotherapy or the main conditioning regimen. hla-matched donor (hr . ; p = . ) and pb blasts-negative at the beginning of conditioning (hr . ; p = . ) were also independently associated with better lfs. with sequential conditioning, leukemia burden prior to the hsct was significantly reduced; pb blasts became undetectable at the beginning of conditioning in % patients given the approach, while in % patients without the approach (p = . ). notably, the outcomes in the patients without pb blasts at the beginning of conditioning who received sequential conditioning were promising; the -year os and lfs reached % and % and the -year ri and trm were % and %, respectively. conclusions: our study reveals that hsct with sequential conditioning can be an effective and tolerable treatment option for children with relapsed/refractory acute leukemia. the treatment strategies that focus on the reduction of leukemia burden immediately prior to hsct may contribute to the induction of long-term remissions in patients with high-risk acute leukemia. disclosure: this research was funded by japanese red cross, nagoya st. hospital research grant nfrch - . use of blinatumomab to achieve remission and consolidation with haploidentical transplant with cyclophosphamide post for the treatment of children with refractory acute lymphoblastic leukemia (all) background: most of patients with all in relapse or refractory to conventional treatment have only % possibilities to achieve long term remission. this report refers to the therapeutic efficacy and adverse events from the blinatumomab to achieve molecular remission in patients with pre-b cd + which lead to haploidentical with cyclophosphamide post transplant as a consolidation. methods: a pilot study was conducted in children with refractory all preb-cd +. as a strategy to achieved remission blinatumomab was used at a dose μg/m for continous infusion of hours, increasing the dose to μg/m during days, patients with a mrd of < . log, after cycles received an haploidentical bone marrow transplant as a consolidation, the conditioning regimen was with total body irradiation scheme at cgy/day/ days, cyclophosphamide and etoposide. receiving prophylaxis for gvhd with cyclophosphamide. results: a total of patients were included, seven of them achieved complete remission after cycles of blinatumomab, one with partial remission (table ) , these seven patients, six received an haploidentic transplant achieving graft in of the transplanted patients. one patient had a bone marrow relapse in the first months of the follow-up and patients are free of disease with a follow-up to months (figure ). as a acute complication the patients presented cytokine release syndrome, during the infusion of blinatumumab patients presented tachycardia (table ) and the patients presented agvhd after hsct ( grade i-ii and i grade iv). conclusions: allogeneic bone marrow transplant constitutes a treatment option on those patients that relapse or become refractory to treatment, one of the major problem is basically to identify a hla-identical donor, the alternative is an haploidentical donor. the most important factor to get these results is the disease status before transplant. the use of blinatumomab has proven to be effective in achieving remission in relapse acute linfoblastic leukemia pre-b cd + or refractory to treatment. characteristics nº % male % median age at diagnosis, (range), years . ( - ) status of disease o + relapse % refractory to primary or salvage therapy % complete remission after blinatumomab % partial remission after blinatumomab % active disease % [[p table] . table n° . demographic characteristics of patients undergoing blinatumomab (n= )] disclosure: a. olaya-vargas, r. rivera-luna, y. melchor-vidal, h. salazar-rosales, g. lopez-hernandez, n. ramirez-uribe. we wish to confirm that there are no known conflicts of interest associated with this abstact, the only financial support was provided by mexican associations that helping children wiht cancer in a few patients. sequential high-dose chemotherapy reinduction followed by myeloablative allogeneic transplant for active acute myeloid leukemias methods: at our center, relapsed/refractory aml patients were transplanted during chemo-induced neutropenia after high-dose salvage chemotherapy. median age at transplant was years (range - ). patients suffered from de novo (n= / , %) or secondary aml (n= / , %). genetic risk stratification was reported using stardardized groups proposed by the european leukemia net (eln) in . favorable, intermediate i and ii and adverse risk category at diagnosis was observed in / ( %), / ( %), / patients ( %) respectively. all patients had active disease at the time of sequential therapy and median marrow blast count was % (range - %). patients received a high-dose cytarabine based (mec in / , %) regimen as salvage therapy. donors were haploidentical relatives for / ( %) patients, identical siblings and matched-unrelated for / patients ( %) and / ( %), respectively. a myeloablative conditioning was used to further implement anti-leukemic effects. conditioning, thiotepa-busulfan-fludarabine in % patients, was started at a median of days (range [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] after the last day of chemotherapy. bone marrow and peripheral blood stem cells were used as graft source in / ( %) and / ( %) patients. graft-versus-host disease (gvhd) prophylaxis and supportive care were administered accordingly to each hsct platform. results: all patients engrafted. median day of neutrophil recovery was day + (range - ). median follow-up of survivors was months (range - ). non relapse mortality and relapse incidences (nrm, ri) were % and % at year and % and % at years, respectively. overall cumulative incidences of acute and chronic gvhd were % and % at day + and + . one and year overall survival (os) were % and %, while and year event-free survival (efs) were % and %. significant better os and efs were observed in patients with favorable-intermediate i-ii versus adverse risk score ( - years os % and % vs % and % p= . ; - years efs % and % vs % and % p= . ). adverse risk had a significant impact on os (hr . , p= . ) and efs (hr . , p= . ) by univariate analysis and on ri (sdhr . , p= . ) by fine and gray test. conclusions: though small the patient cohort, our findings suggest that sequential therapy with a myeloablative hsct is feasible in treating relapsed/refractory aml. transplant-related toxicity was low ( %) and relapse was the major treatment-failure. however, even with this approach, patients with adverse cytogenetic features have a very dismal prognosis. for these patients, the use of new drugs before hsct and/or maintenance therapy after transplant is highly encouraged to improve outcomes. disclosure: alessandro busca: honoraria from gilead sciences, merck, pfizer pharmaceuticals and jazz background: in spite of satisfactory results of overall survival (os) after allohsct in st and nd cr aml, relapse free survival (rfs) and graft-versus-host-disease free/relapse free survival (grfs) require further improvement. the detection of mrd is one of the factors which influence on the outcome of allohsct in aml is unclear but identification is important to improve risk-adapted relapse prophylactic treatment after allohsct. aim. to evaluate outcomes of allohcst in st and nd cr pediatric aml depending on the level of mrd status before myeloablative (mac) or reduced intensity conditioning regimens (ric). methods: the data of children with aml in st and nd cr underwent allohsct between and were analyzed. median age at the moment of allohcst was years old ( - ). mrd negative status had ( %) patients, ( %) were mrd positive by flow cytometry. mac based on busulfan ( mg/b.w.) received ( %) patients, on treosulfan - ( %) patients. ric based on melphalan received ( %) patients, based on busulfan ( mg/b.w.) - ( %) patients. patients received prophylaxis of agvhd by atg ( %) or ptcy - ( %) patients plus csa - ( %) or tacrolimus ± sirolimus - ( %) patients that depended on source of transplant (related, unrelated or haplo donor) . results: at the median follow up years in the cohort of mrd positive patients os is % vs % in mrd negative (p> , ). rfs is % vs % accordingly (p= , ). graft-versus-host-disease free/relapse free survival (grfs) in mrd positive patients is % vs % in mrd negative (p> , ). os, rfs, grfs in mrd positive patients after mac is %, %, % vs %, %, % after ric accordingly (p> , ). os, rfs, grfs in mrd negative patients after mac is %, %, % vs %, %, % after ric accordingly (p> , ). os, rfs in mrd negative patients with/without ptcy is %, % vs %, % (p> , ); grfs is % vs % accordingly (p= , ). os, rfs, grfs in mrd positive patients with/without ptcy is %, %, % vs %, %, % (p> , ). conclusions: mrd status does not statistically significant affect on os that can be related to different approaches to the treatment of relapse after allohsct. mrd positive status statistically significant decreases rfs that underline the necessity of posttransplant therapy improvement. ric vs mac in all patients in first and second remission do not show statistically significant impact on os, rfs, grfs. ptcy significantly improves grfs in mrd negative patients. disclosure: none of the authors has anything to disclose. background: with increasing overall-survival (os) of lymphoma patients, higher incidences of therapy-related clonal bone marrow diseases, such as acute myeloid leukemia (aml) and myelodysplastic syndrome (mds) are occuring. generally, the outcome is considered poor. allogeneic hematopoietic stem cell transplantation (allo hsct) often remains the only potentially curative treatment option. nonetheless, there is only little data available concerning this patient group. methods: we retrospectively collected data from patients with therapy-related aml (taml) and mds (tmds) after treatment for hodgkin's lymphoma (hl; n= and n= ) or non-hodgkin's lymphoma (nhl; n= and n= ), who received an allo hsct between and . median follow-up of surviving patients was . years (range . months- . years). background: the prognosis of relapsed/refractory acute leukemia (r/r al) is poor and the treatment is challenging. in this setting, allogeneic stem cell transplantation (allo-sct) constitutes the only curative option although the high relapse rate and non-relapse mortality (nrm). the sequential conditioning regimen followed by allo-sct has been used for persistent disease and aims to improve disease control by intensified chemotherapy, thus conceding more time for the presumed graft-versus-leukemia effect to occur. methods: the clinical outcome of r/r al with the sequential conditioning regimen combining a chemotherapy rescue followed by ric allo-sct in our center is described. patients who underwent a sequential allo-sct from to are included. the primary endpoint was progression free survival (pfs) and overall survival (os) that were estimated by the kaplan-meier method. secondary endpoints were non-relapse mortality (nrm). background: recommendations of the european leukemia net (eln) for favorable-risk genetics (frg) acute myeloid leukemia (aml) favor consolidation over transplantation, although reviews suggest advantage of autologous stem cell transplant (asct) in event free survival. our objective was to compare the progression free survival (pfs) and overall survival (os) of normal karyotype npm mutated without flt itd or allelic ratio < . (npm +) aml patients treated with consolidation chemotherapy alone (cc), asct or allogeneic stem cell transplant (allosct). methods: retrospective review of npm + frg-aml patients, treated in one institution ( to ) with the following induction regimens: cytarabine (ara-c) and vp- with daunorubicin (ade) or mitoxantrone (mice). consolidation regimens were ara-c with daunorubicin (ac-d), idarubicin, vp- and ara-c (mini-ice) or highdose ara-c (hidac). in asct, conditioning regimens were bucy or bvac and in allosct were bucy or flubu. pfs and os were calculated from the start of the last consolidation or stem cell infusion. results: a total of patients were evaluated, with a median age of years (y) ( - y), % female, % with ecog performance status (ps) - and % with ageadjusted charlson comorbidity index (aacii) ≥ at diagnosis. patients were treated with cc in % (n= ), asct in % (n= ) and allosct in % (n= ) of cases. there were no differences between groups for age, aacii, ps, leucocytes at diagnosis or extra-medullary disease. flt -itd was more frequent in allosct group ( %) than cc ( %) or asct ( %; p= . ). at induction, ade was used in % and mice in % of patients, with a complete remission (cr) rate of %. there were no differences between groups for induction regimen or cr. in cc group, consolidation regimens were cycle ( %) and cycles ac-d ( %) and cycles mini-ice ( %). asct patients received consolidation with - cycles ac-d ( %) and cycle mini-ice ( %), while allosct patients received - cycles ac-d ( %), cycles hidac ( %) and no consolidation in %. [[p image] . figure . pfs at y in cc, asct and allosct groups.] median follow-up was months, pfs at y was % and os at y was %. pfs at y for asct group was superior then cc and allosct groups ( %, % and %, respectively; figure ), although not statistically significant. os at y was statistically similar between groups, although inferior in allosct comparing to cc and asct ( %, % and %, respectively). conclusions: in this historical cohort review, although there was no advantage in os for asct in npm + frg aml, our data suggests that there might be a pfs improvement in asct over cc, which needs to be further addressed in prospective studies. disclosure: nothing to declare background: acute myeloid leukemia is a hematological malignant disease that motivates the persistent struggle in the scientific world to provide effective cure that can establish acceptable survival rates in this group of patients. autologous stem cell transplantation with myeloablative conditioning is still a powerful weapon that can be used against this entity methods: we have evaluated retrospectively patients with aml where autologous stem cell transplantation was performed in the period from till . our group consisted of patients; male patients ( . %), female patients ( . %). median age at diagnosis was years ( - ). the average period from time of diagnosis to autologous sct was . months. results: in the majority of our group, we used myeloablative conditioning regimen with busulphan-cyclophosphamide, patients ( . %), in patients ( . %) we have added melphalan to bu-cy conditioning, in ( . %) patients we used beam conditioning and in the rest, patients ( . %) we used bam conditioning regimen. as auto graft we used peripheral blood stem cells (pbsc) in patients ( . %), and in patients ( . %) we used bone marrow. the main mobilising regimen for pbsc was g-csf + etoposide and it was performed in patients ( . %), and in the remaining patients ( . %) mobilising of pbsc was performed only with g-csf. the mean number od apheresis procedures done in our group was . , and the mean number of collected mononuclear cells was . x /kg tt. the mean time to engraftment was . days ( - ). the transplant related mortality (trm) was . %. the year overall survival of our patients was . patients. the main reason for death was relapse of the primary disease( %). patients ( %)were treated with salvage chemotherapy regimen (flag-ida) because with the standard induction regimen + there was absence of adequate therapeutic response, or predominantly no complete remission was achieved. all patients were transplanted in complete remission conclusions: autologous stem cell transplantation could be an acceptable therapeutic solution for patients with aml as a consolidation therapy, where neither suitable compatible donor is available nor allogeneic stem cell transplantation could not be performed from various reasons depending on the bone marrow transplant unit disclosure: nothing to declare p prophylaxis dli alone may not prevent relapse of flt -itd positive aml after allogeneic hct background: one of the most potent prognostic factors affecting outcomes in aml is the presence of cytogenetic and molecular markers which can guide the selection of post-remission therapies. recently, favorable outcomes of npm wt /flt -itd neg /non-cebpa dm group after allogeneic hematopoietic cell transplantation (allo-hct) have been reported, that is similar to those of favorable risk by the eln risk classification. however, the role of allo-hct compared to consolidation chemotherapy has not yet been elucidated. methods: the data of patients who were diagnosed with aml and received intensive induction therapy from march to july were included in the current study. to address the time dependence of the allo-hct, the simon and makuch method was used in the graphical representation and the mantel-byar test and andersen and gill methods for identifying risk factors for long-term survival. results: median age of the patients were years (range - ), and patients ( %) were male. npm mutation was detected in patients ( %), and flt -itd were none, low, and high ratio in patients ( %), ( %), and ( %), respectively. the eln risk classification divided the patients into favorable, intermediate, and adverse risk group in patients ( %), ( %), and ( %), respectively. npn and flt -itd both negative group included patients ( %). allo-hct was performed in patients ( %). overall, complete response (cr) after induction therapy achieved in patients ( %), and patients ( %) were primary refractory disease. cr rates did not differ between npm wt /flt -itd negative group (n= / , . %) and other intermediate risk group (n= / , . %; p= . ) . with median follow-up duration of . months (range . - . months), one-year os rate were %, . ± . %, . ± . % in favorable, intermediate, and adverse risk group (p < . ). among intermediate risk group, os rate of npm wt /flt -itd negative group was similar to other intermediate risk (p= . ). allo-hct was performed in patients of npm wt /flt -itd negative group. one-year os rates did not differ between npm wt /flt -itd negative and other for allogenic hematopoietic stem cell transplant (allo-hsct), as a strategy to prolong survival. methods: data from aml pts over years, who underwent ric allo-hsct in our institution between september and september , was retrospectively collected from clinical files to evaluate the overall survival (os) up to november . we calculated the os using kaplan-meyer curves. results: we identified pts, median age y.o. ( - ) and median htc-i score . the median follow-up was months. one patient (pt) had cml blast crisis and was on first major molecular remission. of the remaining aml pts, were in st complete remission (cr), in nd cr and with progressive disease (pd); the other pts could be classified as mds according to who diagnostic criteria and were in cr . donors (d) were: matched unrelated (mud), mismatched unrelated (mmud - / ), matched siblings and haploidentical. thirteen pts were infused with peripheral blood hsc and with bone marrow. conditionings were: flubcnumel in unrelated donor (ud) pts and siblings, flumel in ud pt and sibling, flubu in ud pts and flutbi gy in sibling and in the haploidentical. graft versus host disease (gvhd) prophylaxis was tacrolimus (tac) + mmf in ud pts and sibling, tac + mtx in ud pts and cyclosporine (cya) + mmf in ud pt and siblings. all mmud pts had atg. ptcy was done in the haploidentical setting with tac + mmf. the median time to neutrophil and platelet engraftment for the whole cohort was and days, respectively. one pt with secondary engraftment failure required re-infusion of selected cd + cells. ten pts presented with mild acute skin gvhd. eleven pts had chronic gvhd, classified as severe; required systemic therapy, of those beyond year. the median time on immunosuppressants was days. at years the os was . %. there were deaths: disease-related ( relapses at and months and pd at d+ ), infection complications ( septic shock) and to secondary neoplasia. other relevant complications were hypoxemic pneumonia in pts, urinary sepsis, cmv and ebv reactivation respectively in and pts; pulmonary and renal toxicity either in pts. at end of follow-up, pts were in remission, without negative measurable residual disease (mrd), the other mrd negative pt died of septic shock and severe intestinal gvhd. conclusions: in this small cohort, chronic gvhd and infectious complications were major causes of morbidity but there were no treatment related deaths before d+ . pts maintaining or achieving mrd negativity after transplant had better survival. although with only pts, these results suggest that allo-hsct is feasible as consolidation strategy in selected aml pts over years. [[p image] . overall survival] disclosure: nothing to declare. background: hematopoietic stem cell transplantation (hsct) is the only curative option for fanconi anaemia (fa); an inherited disorder characterized by congenital anomalies, progressive bone marrow failure (bmf) and a predisposition to develop malignancies. methods: we retrospectively analysed the data of consecutive patients that underwent hsct at this centre from till june . the data was analysed for variables affecting the outcome in terms of overall survival (os). results: median age at diagnosis was years ( - years). median age at transplant was . years ( - yrs). all patients at transplant were in aplastic phase. male to female ratio was . : . twenty-four ( . %) patients had congenital anomalies along with bmf while were phenotypically normal. twenty-three ( . %) patients were / hla matched with siblings, with parents and with cousins. eleven ( . %) patients had gender mismatch transplant. three patients had major and had minor abo mismatch. background: paroxysmal nocturnal hemoglobinuria (pnh) is a rare clonal non-neoplastic hematopoietic stem cell disease whose incidence is . - . cases/million of individuals worldwide. disease characteristics and natural history have been mostly analyzed by multicenter, retrospective studies, with the limit of heterogeneous approaches. herein we report the incidence of severe complications and outcome in a real life setting scenario of pnh patients consecutively diagnosed and managed at our pnh referral center between january and june . methods: patients received a homogeneous diagnostic and treatment approach according to the period of observation (availability of diagnostic tests and eculizumab). all patients treated with eculizumab received vaccination with conjugated anti-meningococcus acwyserotypes and, since , conjugated anti-meningococcus b-serotype. in the event of any complication, patients could refer to dedicated hematology emergency rooms (er) hours daily. the occurrence of renal failure and pulmonary hypertension was specifically evaluated. the renal function was studied according to the cockcroft-gault formula and the lung function was prospectively monitored by daytime-on exertion, nocturnal pulsoximetric profiles and complete spirometric tests, with dlco measurement. results: overall, pnh patients, median age years (range - ), were analyzed. at diagnosis, patients had classic pnh, aplastic pnh and an intermediate form. the cumulative incidences (ci) of thrombosis, and clonal hematologic neoplasm were %, and %, respectively. ci of pancytopenia in the patients with classic pnh was %. one patient showed a spontaneous disappearance of the pnh clone. since , eculizumab was administered in patients. after eculizumab treatment % and % of patients reached hemoglobin level > g/dl and > < g/ dl without transfusion, respectively, while % were nonresponsive. during eculizumab treatment no thrombotic event was observed while two severe infectious episodes (respiratory tract and urinary tract infection) were observed in only one of the patients. extravascular hemolysis was demonstrated in % of patients. no patient showed a significant reduction of the renal function.out of patients prospectively monitored for lung function no pathological alteration in any diurnal or nocturnal pulseoximetric test was observed. no patient showed obstructive or restrictive ventilatory deficiency, nor reduced dlco values. -years overall survival (os) was % ( patients who died for non-pnh related reasons were censored at the last follow-up).a better os, even if not statistically significant,was associated to the absence of thrombotic events ( %vs %), and the period of diagnosis ( % in - , % in - , % in - ) . conclusions: our study reports a better os and lower rate of severe complications in pnh compared to previous experiences. although renal failure and lung hypertension have been reported by other groups, we did not observe these complications along a prolonged follow-up. we can assume that the availability of a dedicated er service enabled an early diagnosis and prompt treatment in case of hemoglobinuria crises (reducing the risk or organ damage) or other complications. the use of eculizumab, together with improved supportive approaches, presumably accounts for the trend towards a better survival witnessed over the last decade in the management of pnh patients. disclosure: nothing to declare haploidentical and unrelated allogeneic stem cell transplantation in aplastic anemia:single center experience zafer gulbas , elif birtas atesoglu , meral sengezer , İmran dora , cigdem eren , suat celik , demet cekdemir background: aplastic anemia is a syndrome of bone marrow failure characterized by peripheral pancytopenia and marrow hypoplasia. allogeneic stem cell transplantation from hlamatched sibling is performed in the firstline setting in young aplastic anemia patients and in elderly patients who are refractory to immunosuppressive treatment. but if the patient does not have a hla-matched sibling, allogeneic stem cell transplantation is performed from unrelated and haploidentical donors. in this study, we analyzaed and compared the results of aplastic anemia patients who had undergone allogeneic stem cell transplantation either from matched unrelated or haploidentical donors. methods: we collected and analyzed data of aplastic anemia patients who had undergone allogeneic stem cell transplantation from matched unrelated or haploidentical donor between and . results: there were patients who had received allogeneic stem cell transplantation from unrelated donors and there were patients who had undergone haploidentical transplantation. but in patients who had undergone haploidentical transplantation, engraftment failure had occurred and they were transplanted from different haploidentical donors fort he second time. so a total of unrelated and haploidentical transplants were performed. the median age of patients who had undergone unrelated transplantation was ( - ) and the median age of patients who had undergone unrelated transplantation was ( - ) . the results of the haploidentical and unrelated transplantations are shown in table . the neutrophil and platelet engraftment times were significantly longer in haploidentical transplantations (p= , and p= , , respectively). however, vod, gvhd and day mortality rates were not different in the groups. similarly overall survival (os) of the patients who had undergone haploidentical or unrelated transplantation were not significantly different (p= , ) ( figure ) . conclusions: although the number of patients are low in this study, we can conclude that urelated and haploidentical transplantation in aplastic anemia have comparable toxicity and efficacy. background: autologous stem cells transplantation (ahsct) is an effective treatment for very aggressive autoimmune diseases such as multiple sclerosis (ms). however, toxicity remains the major concern to a wide application of this approach. post transplant viral reactivations may induce severe complications and a rigorous monitoring of peripheral blood viral load for a prompt and effective therapy is required. a higher rate of ebv and cmv reactivation has been observed in patients affected by ms and conditioned with beam plus atg compared with a controlled group of lymphoma patients without atg in the conditioning regimen [ ] . we report here the policy of our center about both monitoring and treatment of such side effect. methods: a series of consecutive patients with ms, transplanted between and is included in this analysis. all patients were mobilized with cyclophosphamide g/sqm + g-csf and conditioned with beam plus rabbit atg (thymoglobulin©, . mg/kg). monitoring of cmv/ebv dna on whole blood by quantitative pcr was performed after the engraftment, weekly for at least one month, then at longer intervals. pre-emptive treatment with valgancyclovir was started in case of cmv viral load ³ x ^ copies/ml. in case of ebv assay between x ^ and x ^ copies/ml further determinations were performed and rituximab-based treatment was started if the viral copies exceeded x copies. patients received treatment in case of symptomatic disease for any value of the pcr of both viruses or ebv titer ³ x ^ copies/ml. results: detectable dna for cmv was observed in / ( , %) patients at a median time from transplant of days (range - ) and / ( %) required pre-emptive treatment. all patients promptly responded to treatment within weeks. ebv viral load was detectable in / patients ( , %) at a median time of days (range - ). one patient out of started the treatment on first determination for high viral load (> x ^ /ml); nine presented an ebv viral load over x ^ copies/ml, three of them were treated thereafter for the persistent increase of the viral load (> /ml). six patients spontaneously recovered the ebv reactivation. previous treatments were not predictive of any higher risk of viral reactivation. no impact on engraftment related to the reactivation was observed. conclusions: this policy shows that, despite a high rate of cmv and ebv reactivation, no grade iii-iv adverse events were observed, suggesting the key role of viral monitoring in these patients and the efficacy of the preemptive treatment. ebv reactivation at low titers should be monitored to identify those cases that could achieve a spontaneous resolution and avoid the induction of further immunosuppression by rituximab. these data confirm that patients diagnosed with ad undergoing autologous hsct need a more intense pattern of care than hematological patients. background: autoimmune diseases are chronic serious conditions that are often refractory to standard therapies. since , autologous haematopoietic stem cell transplantation (hsct) has been a very promising alternative that has shown satisfactory long-term results. the aim of this study is to evaluate immune reconstitution and mortality following hsct in patients with autoimmune disease. methods: a retrospective study was conducted on patients with diagnosis of autoimmune diseases that underwent autologous hsct between july and january at a tertiary referral center in colombia, south america. descriptive statistics were used to analyze patient's demographic and clinic characteristics. results: seven patients were included, with a mean age of years (range - ). five patients were female ( %). the indications for hsct were systemic sclerosis (n= ) , multiple sclerosis (n= ), and myasthenia gravis (n= ). the conditioning regimen administered in patients with systemic sclerosis was cyclophosphamide + human anti-t lymphocyte immunoglobulin, beam (carmustine, etoposide, cytarabine (ara-c), and melphalan) + human anti-t lymphocyte immunoglobulin in patients with multiple sclerosis, and cyclophosphamide + human anti-t lymphocyte immunoglobulin in myasthenia gravis. median time to myeloid engraftment (neutrophils> . × /l) was days post-transplantation, and platelet engraftment, defined as > , platelets/mm untransfused, was days post-hsct. median time of hospitalization was days (range - ); longer in-patient management was due to infectious complications. infectious complications included bacteremia caused by e. coli and pneumocystis pneumonia that resulted in septic shock and acute respiratory failure, respectively. evaluating t-cell immune reconstitution, none of the patients had reached normal cd + cell value after one year of hsct follow-up. four patients ( . %) reached normal cd + cells value at months post-hsct. regarding bcell immune reconstitution, . % of the patients ( / ) had reached both igg and iga normal levels at one-month post-hsct, and four patients ( . %) had achieved normal igm background: multiple sclerosis(ms) is an inflammatory disease caused by autoimmune reactivity of t cells against myelin. there is accumulating evidence of the efficacy of highdose chemotherapy followed by autologous haematopoietic stem cell transplantation(ahsct) in ms patients who failed response to standard immunotherapy, despite a variability in eligibility criteria, conditioning regimens and outcome. methods: we retrospectively reviewed ms patients submitted to ahsct in our centre ( ) ( ) ( ) ( ) ( ) . patient eligibility criteria were active relapsing remitting(rrms) or secondary-progressive ms (spms), with prior failure to treatment with disease-modifying therapies and evidence of disease activity (clinical relapse or new active lesions in magnetic resonance [mr] ). mobilization of cd +cells to peripheral blood was performed with granulocyte colony-stimulating factor(g-csf μg/kg/day) and conditioning regimen according to beam protocol. the severity of ms disability was classified according to the expanded disability-status scale (edss) and ahsct toxicity was evaluated by ctcaev . . results: seven ms patients had undergone ahsct ( female/ male), with a median age at ahsct of . years ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) . median age at ms diagnosis was . years( - ) and median time from diagnosis until ahsct was . years ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) . four patients ( . %) had spms and ( . %) had rrms, with ( . %) having mr active lesions. pre-ahsct relapse rate per year was ( ) ( ) ( ) ( ) . median baseline edss was . ( . - . ). median number of previous dmts was ( ) ( ) ( ) ( ) ( ) ( ) ( ) . all patients had been treated with corticosteroids and copaxone, ( . %) received rituximab, ( . %) natalizumab, ( . %) alemtuzumab, ( . %) fampridine and ( . %) fingolimod. all patients collected enough cd + cells in a single apheresis session. median number of cd + cells infused was . ± . x ^ /kg, for a mean dmso volume of . ± . ml. median inpatient stay during ahsct was days( - ). all patients developed febrile neutropenia, one was admitted to intensive care unit due to sepsis. one patient developed an anaphylactic reaction to transfusion and another a self-limited encephalopathy. two ( . %) patients had grade≥ oral mucositis, and all had gastrointestinal toxicity (grade - ). median time until neutrophils> /μl was ( - ) days, to platelets> , /μl was days( - ), and to engraftment was days ( ) ( ) ( ) . patients were transfused with a median of ( - ) unit for erythrocytes and ( - ) for platelets. there was no treatment-related mortality and no long term side effects have been observed so far. for a median post-ahsct follow-up of . months, no patient developed new lesions in mr, but ( . %) experienced symptoms consistent with a clinical relapse, at a median time of . ( - ) months, effectively rescued with corticosteroids. the absence of evidence of disease activity at -months was . %. although there was no variation concerning edss punctuation, ( . %) patients self-reported significant benefits, especially concerning limb strength and sphincter continence improving. conclusions: our real life results claim a stabilization effect of ms patients with highly active/progressive disease after ahsct, with no significant toxicity. the failure in reporting benefits in edss punctuation is probably due to a small sample size and short follow-up. more studies are needed to establish the best patient selection criteria and define the ideal time to include these patients in transplantation programs, as well as to evaluate its long term outcome. disclosure: nothing to declare. elena poponina , elena butina , anna yovdiy , galina zaytseva , natalia minaeva , igor paramonov background: reactivation of epstein-barr virus (ebv) represents a potentially life-threatening condition in approximately % of patients after allogeneic stem cell transplantation, with no specific treatment available. methods: we have previously developed a manufacturing protocol for the expansion of cytomegalovirus (cmv) and ebv-specific t cells by stimulation of g-csfmobilized stem cell grafts with defined peptide pools (gary, ) . this advanced therapeutic medicinal product is currently investigated in an ongoing phase i/iia trial (eudract number: - - ) . however, the expansion of virus-specific t cells relies on a pre-existing virusspecific memory compartment in the stem cell donor. in virus-seronegative donors, no expansion can be achieved. we therefore aim to identify ebv peptide-specific t cell receptors (tcrs) that can be translated into off-the-shelf cell products for the treatment and prophylaxis of ebv infection and ebv-associated malignancies. leftover cells from five allogeneic stem cell grafts were expanded in vitro in the presence of hla-b * -restricted peptides (hpvgeadyfey from ebna , eplpqgql-tay from bzlf ) associated with latent and lytic ebv infection. after expansion, single ebv-specific t cells were facs sorted using peptide-mhc (pmhc) tetramers, and individual tcr αand β-chain pairs were determined with single cell sequencing (han , penter . to confirm peptide specificity, dominant tcr pairs were transfected into a murine αβreporter t hybridoma cell line with nfat-driven gfp expression (siewert, ) . functional tcrαβ candidates were transduced into human peripheral background: lymphoid and myeloid acute leukemia are the most frequent cause of cancer related death in children. interactions between nkg d receptor, expressed in cytotoxic immune cells, and its ligands (nkg dl), that are upregulated in many types of tumor cells including leukemic blasts, are important for anti-tumor immune surveillance. nevertheless, tumor cells may develop immune scape strategies like ligand shedding, which reduces nkg dl expression and may cause nkg d receptor downregulation. engineering t lymphocytes with nkg d car may overcome immune evasion and become an effective therapeutic strategy. methods: cd ra -t cells were obtained by depletion of non-mobilized apheresis with cd ra magnetic beads using clinimacs. nkg d-car t cells were generated by lentiviral (nkg d- bb-cd z) transduction of cd ra -t cells with moi= . the expression of nkg d ligands was analyzed in peripheral blood or bone marrow samples from a total of leukemia patients (aml= , b-all= and t-all= ), at different status of the disease (diagnosis, remission, relapse/refractory), and in different leukemia cell lines by qpcr and flow cytometry. cytotoxicity of nkg d-car t cells against leukemia cells was evaluated by performing conventional- hours europium-tda assays. soluble nkg dl (snkg dl) concentration was measured in the sera of leukemia patients by elisa. to evaluate the effect of snkg dl on nkg d-car t cells, those were cultured in the presence or absence of different concentrations of snkg dl for days. one week later, cell proliferation and car downregulation were measured by flow cytometry using cell trace violet and nkg d labeling, respectively. the production of ifn-g and tnf-a was measured in the supernatants by elisa. the effect on cytotoxicity was evaluated in a hoursdegranulation assay by co-culturing snkg dl pretreated nkg d-car t cells against k cell line. results: nkg d ligands were expressed in leukemia cell lines and leukemic blasts. nkg dl expression changed with disease status with a trend to decrease at diagnosis and relapse/refractory compared to remission. nkg d-car t cells were cytotoxic against / leukemia cell lines with a percentage of specific lysis over %. myeloid and t-all cell lines were more susceptible to nkg d-car t cells (specific lysis ranging from - %) compared to b-all cell lines ( - %). physiological concentrations of snkg dl caused an increase in nkg d-car expression. however, supra-physiological levels of snkg dl decreased nkg d-car expression up to times and increased cell proliferation up to times. cd + subpopulation was more affected by downregulation, while proliferation had more impact on cd + subset. the effects of snkg dl were dose-dependent and attenuated by il- . conclusions: nkg d-car t cells are cytotoxic against leukemia cells, specially aml and t-all, and thus could be a novel therapeutic approach for non-b leukemia, or those b-all that relapse with undetectable cd after cd -car treatment. nkg d-car expression may be downregulated only by supra-physiological levels of snkg dl, although antitumor activity is not affected. il- softens the negative effects of snkg dl inducing nkg d expression, cell proliferation and cytokines production. the changes observed in nkg dl surface expression at the different stages of the disease could be related to ligands release and immune escape. disclosure: nothing to declare denis-claude roy , , ines adassi , , céline leboeuf , , vibhuti p. dave , hôpital maisonneuve-rosemont research center, montréal, canada, university of montréal, montréal, canada background: for patients with high-risk leukaemia, allogeneic haematopoietic stem cell transplantation is the only curative treatment. the presence of alloreactive t cells in the donor graft, however, leads to a high probability of developing graft-versus-host disease (gvhd) . t-cell depletion minimises the presence of gvhd-causing alloreactive cells, but often results in an increased incidence of infections and disease relapse. photodepletion treatment (pdt) can specifically deplete activated alloreactive t cells while conserving resting t cells. pdt-treated cells have been utilised after t-cell-depleted haploidentical transplant to help reduce infection and relapse. the efficacy and safety of such pdt-treated cells is currently under clinical investigation in a phase iii trial (hatcy, nct ; kiadis pharma). here the reactivity of pdt-treated donor t cells was assessed toward tumour-associated and viral antigenic peptides derived from wilm's tumour protein (wt p), preferentially expressed antigen in melanoma (pramep), and from cytomegalovirus and epstein-barr virus (cmv/ ebvp). methods: healthy donor (hla-a* ) peripheral blood mononuclear cells (pbmcs) were co-cultured with irradiated pbmcs from another mismatched donor ( : ) in a -day mixed lymphocyte reaction. th , a photoactive rhodamine derivative, was added and cells were exposed to visible light to deplete the th -containing activated alloreactive cells. elimination of alloreactive cells post-pdt was assessed using cd and hla-dr as activation markers. an ex vivo expansion protocol was exploited to evaluate the impact of pdt on reactivity to tumour and viral antigenic peptides. post-pdt t cells were co-cultured with irradiated autologous monocyte-derived dendritic cells ( : ) pulsed with wt p, pramep or cmv/ebvp. antigen-specific t cells were re-stimulated on days and with wt p-or pramep-pulsed autologous pbmcs or with cmv/ebvp added directly to the culture. mhctetramer staining was performed on days and ; ifn-γ elispot was conducted on day . [[p image] . functional wt -specific and viralspecific t cells can be expanded post-pdt] results: pdt resulted in a drastic decrease of cd and/ or hla-dr activation marker-expressing cd + and cd + t cells. pdt-treated cells showed a significant increase in background: acute lymphoblastic leukemia (all) is the most common childhood cancer and relapsed or refractory all is still difficult to treat. engineered t cells equipped with a synthetic chimeric antigen receptor (car) targeting cd have demonstrated remarkable efficacy to treat all. however natural killer (nk) cells are known for their target-independent cytotoxic potential without induction of cytokine release syndrome (crs) or graft-versus-hostdisease (gvhd), car-nk cells can overcome the persisting problem in the therapy with car t cells. as the use of viral vector generated car nk cells is limited by theire genotoxicity, cost and regulatory demands, we are developing an innovative protocol using non-viral sleeping beauty (sb) transposition of third party nk cells as a source to produce 'off the shelf' car-engineered cell products. methods: nk cells are isolated from peripheral blood mononuclear cells (pbmcs) using cd selection kits. they are successfully expanded ex vivo with il- cytokine stimulation under feeder-cell free conditions. after few days of expansion nk cells are electroporated using pmaxgfp. transfection efficiency and percent of living cells after electroporation is analyzed by flow cytometry. transposition based nucleofection using an sb x mrna and a minicircles (mc) dna vector is performed at different time points after nk cell isolation. the transient mc-venus longtime expansion and the viability after sb x based nucleofection is measured over two weeks. furthermore, α-retroviral (α-rv) cd -car transduction of nk cells with different viral amounts (moi) is conducted and the cytotoxicity of the engineered cd -car-nk cells against the cd positiv cell line supb is addressed. results: for an α-rv cd -car transduction of maximal x nk cells we could show transduction efficiency of , % for moi . the α-rv cd -car modified nk cells had a high killing activity against cd positiv supb cells (e:t ration : , %) compared to cd negativ k cell lines (e:t ration : , %) and the non-transduced nk cells (e:t ratio : , %). in first experiments with pmaxgfp vector based nucleofection, we could show an increasing efficiency of , % h post electroporation with a only slightly decreas of living cells ( , %) comparing to the non-electroporated nk cell viability. using sb x mrna with mc-venus dna we electrotransfected x nk cells after fews days of cultivation and we reached , % of transfected nk cells h post electroporation and a transient expression of mc-venus positive nk cells up to , % efficiency with an increasing rate of live cells over days after electroporation. conclusions: the sleeping beauty based nucleofection of nk cells is a very promising non-viral method to generate more easy, safer and higher amounts of genetically modified third party nk cells for therapy of all and has also a broad range of clinical applications. disclosure: winfried s. wels is an inventor on a patent describing chimeric antigen receptors with an optimized hinge region. axel schambach is an inventor on a patent describing alpharetroviral sin vectors. michael hudecek and zoltan ivics are inventors on patents related to sleeping beaut gene transfer technology. the remaining authors have nothing to disclose. background: mature immune cells from the stem cell graft are essential for the graft-versus-tumor (gvt) effect to eliminate residual malignant cells after hematopoietic stem cell transplantation (hsct), but donor cells are also involved in complications such as graft-versus-host disease (gvhd). methods: we performed a prospective study of the detailed graft composition in recipients of peripheral blood stem cells (pbsc) or bone marrow (bm) in order to identify correlations to clinical outcomes, table . grafts were characterized with concentrations of t cells and nk cells together with a multi-color flow cytometry panel with staining for tcrαβ, tcrγδ, vδ , vδ , cd , cd , cd , hla-dr, cd , cd ro, cd ra, cd , cd , cd and cd for detailed immune phenotyping. cell contents in stem cell grafts were analyzed both as fractions of cd positive lymphocytes and as absolute concentrations converted to transplanted cells/ kg. fractions were evaluated in patients receiving both bm and pbsc (n= ), while concentrations (cells/kg) were only analyzed in patients transplanted with pbsc (n= ). table] . table ] [[p image] . figure ] results: we found, that patients transplanted with graft nk cell doses above the median of x /kg and fractions of nk cells out of lymphocytes above the median of . % had significantly increased relapse-free-survival compared to patients transplanted with grafts containing nk cell doses below these values, figure ; results stayed significant in multivariate analyses. relapse incidence was significantly lower in uni-and multivariate analyses in patients receiving grafts with high nk cell fractions compared with low fractions, p= . , with -year relapse rates of % versus % in patients transplanted with high versus low fractions of nk cells, p= . . peripheral blood concentrations of nk cells obtained from samples from pbsc donors before g-csf mobilization were significantly correlated to graft concentrations-and fractions of nk cells.. analyses of graft contents of nkt cells showed that the incidence of grade ii-iv acute gvhd were significantly lower in patients background: extracorporeal photopheresis (ecp) is an immunomodulatory treatment that has shown efficacy in steroid refractory acute gvhd, but the mechanism of action is only partially understood. there is no clear relationship between the ecp-treated mononuclear cells (mnc) or lymphocyte numbers and response to ecp. the objective of the study was to analyse the relationship between the infused subpopulation cellularity and response. methods: patients from different centers with a total of ecp procedures were retrospectively analized. ecp procedures were performed from january- to june- . all ecp procedures were performed with the off-line system. the response was defined as responder (complete and partial response) and non-responder. infused cell numbers for lymphocytes, monocytes and mononuclear cells (lym+mon, mnc) were calculated. for analytic purposes, the median number of cells infused per procedure until response (or until the median number of procedures until response for non-responding patients) and the cumulative number of cells infused until response (or until the median in non-responders) were calculated for all the subgroups. same procedures were performed with the number cells infused until day of ecp. finally, the response and survival impact of infusing a number of cells above or below the median and in different tertiles was assessed until the median number of procedures needed to achieve a response. [[p image] . results: the median number of procedures until response was . we observed a trend towards a higher median number of monocytes per procedure and cumulative infused monocytes in responding patients (median number infused . vs . x /kg p= . , cumulative infused median number . vs x /kg p= . ) that was lost in the day of treatment. there was also a trend toward higher median infused mnc until response for responders ( vs x /kg p= . ). we observed no differences in the number of lymphocytes infused, but patients who received a number of lymphocytes per procedure over the first tertile ( x /kg) presented higher response rates ( % vs %, p= . ). none of the other analysed parameters showed a significant impact in overall survival. conclusions: patients with acute gvhd who responded to ecp received higher numbers of monocytes and mnc in the early phase of the treatment (a median of the first processes). also the patients who received higher numbers of lymphocytes in the first procedures achieved a higher response rate. these findings suggest the possibility that higher number of treated and infused cells could influence the response to ecp, but specifically designed prospective studies are need to asses this possibility. disclosure: nothing to declare background: the field of kidney transplantation has made enormous progress over the last decades towards being a standard treatment for patients with end-stage renal disease. however, administration of immunosuppressive drugs is still one of the major limitations of long-term allograft survival. therefore, strategies for induction of donorspecific tolerance are highly desirable. to this aim, a clinical phase i study with donor-derived modulated immune cells (mics) was conducted. methods: donor-derived mics were manufactured under gmp conditions. potency of mics was tested by different in vitro bio-assays. mics were administered to patients with an escalation from . x mics/kg on day - (n= , group a), to . x mics/kg on day - (n= , group b) or on day - (n= , group c) before kidney transplantation accompanied by standard immunosuppressive medication post-transplantation. frequency of adverse events (ae) was assessed from day until day post-transplant. dynamic changes of various lymphocyte subsets in patients after mic therapy were detected by multicolor flow cytometry. donor-specific immunosuppression was assessed by measuring anti-donor antibodies and mixed lymphocyte reaction (mlr) against donor and thirdparty cells. results: in all kidney transplant recipients, we observed a median serum creatinine of . mg/dl at day which remained stable until day (median creatinine of . mg/ dl) without significant proteinuria. none of patients experienced rejection episode. aes were observed while three aes being severe. most importantly, none of them was associated with mics transfusion. besides two infectious complications, no post-transplant positive cross match results against the donor or titers of de novo donorspecific antibodies were recorded. notably, immunosuppressive therapy could be reduced without signs of rejection in group c. after infusion, we observed a dramatic increase of cd + b cells up to a median of cells/μl until day , followed by a reduction to cells/μl on day in group c. notably, regulatory b cells significantly increased from a median of % on day to % on day . in parallel, the plasma il- /tnf-α ratio increased from a median of . before cell therapy to . on day . after mic cell therapy recipient lymphocytes showed no or only minimal reactivity against irradiated donor pbmcs in vitro, while reactivity against rd -party-donor pbmcs was not impaired. moreover, in vitro mics product demonstrated their immunomodulatory potency by inducing tolerogenic dendritic cells (tdcs) characterized by low expression of costimulatory (cd , cd ) and maturation (cd ) as well as high expression of inhibitory marker cd . functionally, tdcs could inhibit not only the release of ifn-γ but also the proliferation of cmv specific cd + t cells. moreover, mic-induced tdcs showed the capacity to inhibit donor-specific allo-reactive cd + and cd + t cell proliferation. conclusions: mic cell therapy modulates the immune system of kidney transplant recipients by increasing the ratio of regulatory b cells and facilitates the reduction of conventional immunosuppressive therapy without allograft injury or rejection episodes. therefore, mic cell therapy represents a promising strategy in transplantation medicine. we currently prepare a phase ii trial with mic cell therapy. disclosure: nothing to declare p genome editing of graft-derived t cells for post- background: immunotherapy using car t cells has shown promising results to fight cancer. however, car-t cell production requires specialized infrastructure and operators, which implies high cost and centralized production. automated production of car-t cells in clinimacs prodigy device allows clinical-grade manufacturing of car t cells. methods: million cd ramemory t cells from healthy donors were cultured in texmacs supplemented with iu/ml il- . at day cells were activated with t cell transact for h. at day one, activated cd ramemory t cells were transduced with nkg d-cd tm- bb-cd z lentiviral vector at moi = . then, nkg d-car t cells were expanded for - days. nkg d-car t cell products were next harvested, counted and analyzed for viability, nkg d-car expression and anti-tumor cytotoxicity. different quality tests including sterility, vector copy number, genetic stability, quantification of viral particles in the supernatant, myc/tert expression and endotoxin detection were performed. spare cells were cryopreserved either in autologous plasma and % dmso, m % albumin and %dmso or hypothermosol. after months, cryopreserved nkg d-car t cell products were analyzed for viability, nkg d-car expression and cytotoxicity. results: nkg d-car memory t cells expanded up to ± million with , ± % nkg d-car expression and ± % viability. harvested car t cells showed ± % of specific lysis against jurkat cells and ± % against mii osteosarcoma cell line. no microbiological contamination was observed in final car t cells products. vector copy number was ≤ in all validations except for one. cgh and karyotype showed no genetic alterations. free viral particles were undetectable in the supernatants. no overexpression of myc/tert was found except for one validation. endotoxins were ≤ . eu/ml. all cryopreserved nkg d-car t cell products kept nkg d-car expression one year after freezing. however, viability and cytotoxicity was best preserved using autologous plasma %dmso. conclusions: automated production of large-scale clinical-grade nkg d-car t cells using clinimacs prodigy is feasible and reproducible, allowing a decentralized protocol to generate car t cells for clinical use. background: immune reconstitution (ir) is essential to control severe infections after hematopoietic stem cell transplantation (hsct). reconstitution of adaptive immunity may take up to years to recover t-lymphocytes (lt). delay in early lt recovery increases the risk of relapse, viral infections and transplant related mortality. adoptive transfer of selected t cell subset with low alloreactivity potential is emerging as a strategy to improve ir. methods: depletion of cd ra+ naive t cells, preserving cd ro+ memory t cells could provide functional lymphocytes to protect against infection and leukemia relapse with low risk of graft versus host disease (gvhd). we present our experience with high-dose donor cd ro + memory t cell as donor lymphocyte infusions (dli) to assess safety and outcome. a total of dli of cd ro+ after hsct was performed in cases of cmv/ebv reactivation ( %), mixed chimerism ( %), persistent lymphopenia ( , %), graft rejection ( , %) , relapse ( %) or to boost ir ( %). dli product was obtained performing a cd ra depletion on donor leukapheresis product using the clinimacs® device. results: twenty-two pediatric patients, median age years (range - ), with malignant (n= ) and nonmalignant diseases ( ), received cd ra+ (n= ), tcr alpha/beta (n= ) depleted grafts from haploidentical and cd ra+ depleted grafts from match unrelated (n= ) and match related (n= ) donors. at a median of days (range - ) after transplantation, patients received a total of dli of cd ro+ cells, median (range - ), containing a median of . x /kg (range . x - x /kg), cd +cd ro+ . x /kg (range . x - . x /kg) and cd ra+ cells x /kg (range - . x /kg). all infusions were well-tolerated and did not develop or worsen gvhd. a total of / episodes of cmv/ebv viral reactivations decreased viral load, / cleared viral load and / showed a clinical improvement. a total of / patients with persistent lymphopenia there was a slightly increase in total lymphocyte count, but not to normal levels. prophylactic dli of cd ro+ to boost ir increased lymphocyte count in of cases. none of the dli administered in cases of mixed chimerism, graft failure or relapse were effective in reverting those situations. conclusions: our preliminary data suggest that infusions of high dose cd ro+ memory t cells are a safe adoptive immunotherapy strategy. efficacy has been observed in patients with lymphopenia and cmv/ebv reactivation, with no positive results in patients with mixed chimerism, graft failure and relapse. however, to determine the real efficacy of this strategy, prospective studies are required. disclosure: nothing to declare. background: increasing clinical trials have confirmed that chimeric antigen receptor t cells (car-t) targeting cd antigen (car-t- ) is a promising effective approach for the treatment of relapsed/refractory(r/r) b-cell lineage malignancies. considering cd is frequently expressed in large part of t( ; ) acute myeloid leukemia (aml) cells, we suppose that car-t- may be used as an approach to rescuing r/r t( ; ) aml patients. methods: both patients received lymphodepletion chemotherapy with decitabine mg/m × d, fludarabine mg/m × d and cyclophosphamide mg/m × d (dac +fc). two days after chemotherapy, autologous/allogeneic cart- cells provided by the unicar-therapy biomedicine technology co.(shanghai, china) at a total dose of - × cells per kilogram(kg) were infused dose escalation within to days. the research protocol was approved by the institutional review boards of the first affiliated hospital of soochow university and both patients gave written informed consent. results: both cases responded well with transient and reversible toxicities. case presented with grade cytokine release syndrome (crs), manifested by intermittent fever and chill from day after car-t- infusion for half months associated with neutropenia. car-t cells expansion were observed in blood without obvious increase of cytokines. after infusion, case achieved and maintained molecular complete remission (cr) for more than months. case presented with grade crs manifested by continuous high fever, hypotension and grade liver disfunction from day after car-t- cell infusion for week. obvious cytokines releasing (peak il- serum concentration . pg/ml, peak crp serum concentration pg/ml) were detected which were associated with car-t- cell expansion in blood and no severe off-tumor effect was observed. after infusion, case achieved hematological cr and cytogenetic cr and got months disease free survival. conclusions: our report implicates that car-t- is a safe and promising approach to managing r/r t( ; )aml with cd expression, and may provide a salvage treatment approach for all aml patients with cd expression and benefit a certain population with aml besides b-linage malignancies. clinical trial registry: na disclosure: nothing to declare. this work was supported by research grants from the national key r&d program of china ( yfc ), national natural science foundation of china ( , , , ) background: chimeric antigen receptor engineered t (car-t) cells have emerged as a powerful cellular therapy to treat malignant disease, which is currently revolutionizing field of cancer immunotherapy. a cryopreservation step postmanufacture is not only a logistical necessity for large scale cell manufacturing processes but also a mandatory request by regulatory authorities. in case relapse after st car-t cell transplantation, a second application, maybe at a higher dose constitutes a therapeutical option. however, data concerning clinical grade car-t cell stability and functionality after months of cryopreservation have not been released by companies so far. to investigate the effect of cryopreservation on car-t cells, we performed this study. methods: different batches of cd car-t cells were manufactured according to gmp requirements at our institution. final car-t products were frozen at concentrations of x cells/ml (high batch) and x cells/ml (low batch) by a controlled freezing process with the biofreeze bv device and stored in liquid nitrogen tanks below - °c until release. quality control tests for sterility, endotoxin and mycoplasma were performed for each batch according to european pharmacopoeia and united states pharmacopoeia guidelines. stability of cd car-t cells in terms of viability, recovery, transduction efficiency and functional capacity were determined by microscopy, multi-parametric flow cytometry as well as chromium- release tests following our sops. results: all the results of quality controls fully met the requirements of the regulatory authorities. stability results were highly robust and reproducible over time for all our gmp car-t batches. duration of cryopreservation (up to days) had no negative influence on cell viability, recovery of viable cd car-t cells and transduction efficiency. however, the cell concentration for cryopreservation has a significant impact on the post-thawing viability (low batches vs. high batches: . ± . vs. . ± . , p < . ) and recovery (low batches vs. high batches: . ± . vs. . ± . , p < . ) of cryopreserved cd car-t cells, but not the transduction efficiency. moreover, we observed four transient side-effects of cryopreservation on the amount of cytokines released by car-t cells, the cytokine release on a per-cell basis, the multifunctionality of car-t cells and the killing capacity. of note, functional capacity of cryopreserved car-t cells after overnight resting was comparable or even enhanced for inf-γ and tnf-α release by cd + and cd + cd car-t cells when compared to fresh car-t cells. the multi-functionality of car-t cells could be preserved. furthermore, the killing capacity of cryopreserved cd car-t cells after overnight resting could reach the level of non-cryopreserved/fresh car-t cells. conclusions: cryopreservation up to days has no harmful effect on transduction efficiency and functionalities of car-t cells. however, the cell number per milliliter freezing medium matters. dose over x cells/ml should be avoided. for the conduction of in vitro bio-assays to determine the function of car-t cells, an overnight resting process could mimic the situation after clinical application and eliminate the transient side-effects of cryopreservation to fully regain the functional potency of car-t cells. disclosure: nothing to declare background: dc and specific t-cells are important mediators of ctl-responses. we could already show that allogeneic donor-or autologous t-cells obtained from amlpatients can be stimulated by dc leu , resulting in a very efficient lysis of naive blasts. methods: chemokine-release (cxcl , - , - , ccl , - , and il- ) was analysed by cytometric bead array in serum of aml/mds-pts as well as in supernatants from different dc-generating-methods and correlated with pts' clinical course, dc-and t-cell-interactions as well as specific t-cell-reactions. the lytic activity of dcleu/blast -stimulated t-cells in mlc against naive blasts was quantified in a cytotoxicity assay. results: minimal differences in median chemokine-levels in pts' serum subdivided in subtypes were seen, but higher release of cxcl , - , - and lower release of ccl and - tendentially correlated with more favourable subtypes (< years of age, < % blasts in pb). in persisting disease, a higher serum-release of ccl and at relapse a significantly higher ccl -release were found compared to first diagnosis -pointing to a change of 'disease activity' on a chemokine level. whereas chemokine-levels in dc-culture supernatants compared to serum were variable, clear correlations with lateron (after stimulating t-cells with dcleu in mlc) improved antileukemic t-cell activity were seen: higher values of all chemokines in dc-culture supernatants always correlated with improved t-cells' antileukemic activity (compared to stimulation with blast-containing mnc as control) -whereas with respect to the corresponding serum values higher release of cxcl , - , and - but lower values of ccl and - correlated with higher probabilities to improve antileukemic activity of dcleu-stimulated (vs. blaststimulated) t-cells. predictive significant cut-off-values could be evaluated separating the groups compared. moreover, correlations with lateron achieved response to immunotherapy and occurrence of gvhd were seen: higher serum values of cxcl , - , - and ccl and lower values of ccl correlated with achieved response to immunotherapy. predictive cut-off-values could be evaluated separating the groups compared in 'responders' and 'non-responders'. higher levels of ccl and - but lower levels of cxcl , - , - correlated with occurrence of gvhd. conclusions: we conclude, that in aml-pts' serum higher values of cxcl , - , - and lower values of ccl and in part of ccl correlate with more favorable subtypes and improved antitumor'-reactive function. since in dcculture supernatants higher values of all chemokines correlated with improved antileukemic t-cell reactivity we conclude a change of functionality of ccl and - from an 'inflammatory' or 'tumor-promoting' to an 'antitumor'reactive function. this knowledge can contribute to develop immune-modifying strategies that promote antileukemic adaptive immune-responses. disclosure: nothing to declare background: allogeneic hematopoietic cell transplantation (allo-hct) is a curative treatment option for patients suffering from hematologic malignancies. infusion of donor lymphocytes (dlis) can induce sustained remission in case of minimal residual disease or relapse through potent graftversus-leukemia (gvl) effects, although graft-versus-host disease (gvhd) represents a common dose-limiting toxicity. as invariant natural killer t (inkt) cells are known to prevent gvhd while promoting beneficial anti-tumor effects, we investigated the role of inkt cells for successful dlis. methods: we analyzed dli samples by flow cytometry. inkt cells were identified by staining with pbs -loaded cd d tetramers. culture-expanded and purified dli-inkts were then tested against tumor cell lines and primary leukemia cells in an ex vivo tumor control model. tumor cell viability after coincubation with dli-inkts was measured by flow cytometry using -aad. results: inkt cells represent . % (range . - . %) of donor lymphocytes and can be expanded fold following a two-week protocol with a preferential expansion of cd + inkt cells. tumor cell lines such as jurkat were efficiently lysed after coincubation with dli-inkts. cd a as a marker of degranulation was significantly upregulated on dli-inkts after stimulation by jurkat. in addition, increased concentrations of tnfα, ifn-γ, sfasl and perforin were measured after coincubation of dli-inkts with jurkat. we observed that tumor cell lysis correlated with the expression of the mhc-i-like molecule cd d. consequently, adding a cd d antibody to the coculture abrogated the dli-inktmediated kill of tumor cells. dli-inkts also efficiently lysed primary leukemia cells such as aml blasts: expression of cd d on these aml blasts significantly correlated with dli-inkt-mediated tumor cell lysis (r = . , p= . ). conclusions: ex vivo expansion of dli-inkts and subsequent dli enrichment is an immunotherapeutic approach that could improve leukemia control and thus, prevent relapse after allo-hct without exacerbating gvhd. disclosure: nothing to declare. generation of antigen-specific cytotoxic t lymphocytes targeting wt using activated b cells sun ok yun , kyung won baek , hee young shin , hyoung jin kang seoul national university, seoul, korea, republic of background: the wilms tumor antigen (wt ) is highly expressed in many malignancies including leukemia and targeting wt as a tumor associated antigen (taa) in cancer immunotherapy is attractive. in this study, we generated wt -specific cytotoxic t lymphocytes to confirm if activated b cells can act as a cancer antigen presenting cell and induce ctls. methods: for the induction of ctls against wt , activated b cells were used as an antigen presenting cells. b cells were isolated from pbmcs of normal healthy donors and activated with α-galactosylceramide (α-galcer) and nucleofected with wt -coding plasmid dna. activated b cells were the cultured with pbmcs for days in vitro and harvested for assay. results: cells expanded about times after days of culture. we examined characteristic of wt -specific ctls by their surface markers. wt -specific ctls had more than % cd + marker, and ratio of cd to cd was . - . . we also examined nkt cell markers to see if nkt cells were activated by il- , a cytokine used in the induction of ctls, and the portion of nkt cells was about %. the ctls showed a decrease in naïve cell (cd l+cd ra +) and an increase in effector memory (cd l+cd ra-) and central memory (cd l-cd ra-) compared with non-stimulated pbmcs. subsequently, the ifn-γ elispot (enzyme-linked immunospot) assay was performed to confirm the response of the induced wt -specific ctls to the wt antigen. when wt -specific ctls encounters a target that does not have a wt antigen, it did not produce ifn-γ, but when it encounters a target cells loaded wt antigen, it responded to secrete ifn-γ. killing assays were also performed to determine the immunogenicity of induced ctls. the induced wt ctls was found to be killing more than % when the e:t ratio was : when the autologous pbmc met the target with wt pepmix. in addition, we found that wt ctls has killing activity when it encounters leukemia cell lines that express wt and matched hla-a* . conclusions: in this study, we can induce antigenspecific ctls that specifically react to wt using activated b cells as antigen-presenting cells. these observations confirmed that b cells activated by α-galcer can act as a taa presenting cell to induce taa specific ctls as viral antigen, such as pp and ie , and consequently wt specific ctls could be induced. moreover, ctls induced activated b cells had ability to recognize and kill the target cells expressing wt correctly. our results demonstrate that these in vitro expanded wt -specific ctls using activated b cells can be a promising candidate for adoptive immunotherapy against cancer. disclosure: nothing to declare judith böhringer , michael schumm , christiane braun , marina schmidt , patrick schlegel , christian seitz , murat aktas , georg rauser , sandra karitzky , peter lang , rupert handgretinger university children's hospital tübingen, tübingen, germany, miltenyi biotec gmbh, bergisch gladbach, germany background: t cells with chimeric antigen receptors (cars) on their surface facilitate to target specific surface expressed antigens. research and clinical trials with cd -car t cells show impressive remission induction rates and increased survival in heavily pretreated patients. therefore, car t cells are introduced as new potent cellular therapeutics in the clinical routine. in order to establish the manufacture of cd -car t cells, validation runs with the fully automated clinimacs prodigy t cell transduction process have been performed using the miltenyi anti-cd -car lentiviral vector. methods: unmobilized leukaphereses from donors ( x healthy, x all) were used for the clinimacs prodigy t cell transduction process. leukocytes undergo a cd + / cd + t cell enrichment via magnetic beads, followed by stimulation with macs ® gmp t cell transact™, transduction with an anti-cd -car lentiviral vector, expansion with il and il , and final formulation to the cellular product. during and after the manufacture, facs analyses were performed as well as cytotoxicity assays after cd -car t cell production. results: total volumes of leukaphereses were between and ml with . - . x total mononuclear cells. after enrichment x cd + / cd + t cells were transduced with anti-cd -car lentiviral vector and were further expanded. cells were harvested on day . the final cell counts of the cellular products were . , . and . x mononuclear cells from two healthy volunteers and the all-patient, respectively. the transduction efficiency of the cd -car t cells was . %, . % and . % among viable cd + cells. the final count of car t cells was therefore . , . and . x cells. the final products exerted excellent cytolytic activity against cd + bcp-all cell line nalm- . importantly, cd -car t cells generated from the all patient demonstrated complete eradication of autologous blasts at . to e:t ratio after hours incubation. conclusions: the clinimacs prodigy t cell transduction process has been shown to run a fully-automated manufacturing process over days without any deviations in a clean room environment on a single device. the user interaction was reduced to activities at only days to set up the system and provide fresh medium and reagents. the transduction process yielded a high number of t cells with a high frequency of cd -car t transduced cells. the results were comparable for both unmobilized leukaphereses from healthy donors and showed expected slightly lower results in the patient. finally, these results demonstrate that the clinimacs prodigy t cell transduction process is well suited to provide the clinical mb-cart . r/r cd + bcm study with appropriate investigational medical products. disclosure background: allogeneic hematopoietic stem cell transplantation (allohct) is an effective strategy in the long term control of several hematologic diseases, however, patients could experience complications, as graft versus host disease (gvhd) and disease relapse. recently, the introduction of post-transplant cyclophosphamide (ptcy) allowed to significantly reduce gvhd, but disease relapse remains an important issue. donor-lymphocyte infusion (dli) is an established adoptive cell therapy for disease relapse after allohsct, but, in order to be efficient and safe, patients have to be off immunosuppression treatments and gvdh-free. here we report our data about efficacy and safety of dli infusion as treatment for disease relapse in patients who received peripheral blood stem cell transplantation (allopbsct) from hla-matched unrelated/related plus ptcy as gvdh prophylaxis in our clinical trial (nct ). methods: we collected data from patients, treated with ptcy ( mg/kg/die, days + + ), mofetil mycophenolate (mmf) and tacrolimus (t) as gvhd prophylaxis after allo-pbsct, who received dli infusions. they were treated between january and october . we report data about overall response rate (orr), disease control rate (dcr), and dli-related mortality and morbidity. diagnosis were as follow: had multiple myeloma, had acute myeloid leukemia, had acute lymphoblastic leukemia and had lymphomas. all patients but one, who had chimerism loss, received dli because of disease relapse. results: median time between transplant and dli was (range - ) months. median number of dli infusions was (range - ). patients ( %) received cyclophosphamide mg/m preparative regimen the day before the cryopreserved dli infusions, while in the other cases dli were associated with lenalidomide, ponatinib and -azacitidine. the overall response rate (orr) was %, while disease control rate (dcr) was achieved in %. the patient who received dli because of loss of chimerism converted it in full donor after infusions. after dli treatment the incidence of acute gvhd grade i-iii was %, while was % for grade ii-iii and patients were started on short course of systemic immunosuppression treatments . none of these patients died because of dli adverse events. estimated -year overall survival was % with a limited follow-up length ( months). conclusions: the infusion of non-manipulated lymphocytes from allogeneic donors is a valuable and safe strategy of treatment for patients relapsing after allopbsct with ptcy. ptcy showed high efficacy in gvhd prevention, allowing early discontinuation of immunosuppression drugs. because of this, we can reach the goal to transform transplant in a platform where we could add early dli infusions as a new strategy for disease control. clinical trial registry: nct disclosure: nothing to declare p extracorporeal photopheresis in the treatment of refractory chronic gvhd: analysis of mononuclear cell infusion gillen oarbeascoa , maria luisa lozano , , , luisa maria guerra , cristina amunarriz , nuria revilla , , , pastora iniesta , , , cynthia acosta fleitas , jose luis arroyo , eva martinez revuelta , andrea galego , dolores hernandez-maraver , mi kwon , , aurora viejo , jose maria garcia gala , concepcion andon saavedra , jose luis diez-martin , , background: extracorporeal photopheresis (ecp) is an immunomodulatory treatment that has shown efficacy in steroid refractory chronic gvhd, but the mechanism of action is only partially understood. in some studies, a correlation has been suggested between treated mononuclear cells (mnc) or lymphocytes and response to ecp. the objective of the study was to analyze the relationship between the infused cellularity and response in chronic gvhd. methods: patients from different centers with a total of ecp procedures were retrospectively analyzed. ecp procedures were performed from january- to june- . all ecp procedures were performed with the off-line system. the response was defined as responder (complete and partial response) and non-responder. infused cell numbers for lymphocytes, monocytes and mononuclear cells (lym+mon, mnc) were calculated. for analytic purposes, the median number of cells infused per procedure until response (or until the median number of procedures until response for non-responding patients) and the cumulative number of cells infused until response (or until the median in non-responders) were calculated for all the subgroups. same procedures were performed with the number cells infused until day of ecp. finally, the response and survival impact of infusing a number of cells over or below the median and in different tertiles (t , t and t ) was assessed until the median number of procedures needed to achieve a response. results: the median number of procedures until response was . we observed no differences in the median number of lymphocytes, monocytes or mncs infused until response or until day between responding and non-responding patients. there were no differences in response if patients received lymphocytes or monocytes above or below the median number. nevertheless, patients that received a total absolute number of mncs above the median ( x cells) showed a trend towards a higher response rate ( % vs %, p= . ). the patients that received a cumulative number of lymphocytes in the first ecp procedures above the median showed improved overall survival (os) ( y os % vs %, p= . ). patients that received a number of monocytes above the median showed a trend towards better survival (p= . ), that was significant when the number of monocytes infused surpassed the first tertile ( y os % for t , % for t , % for t , p= . ). finally, the patients that received a cumulative number of mncs above the first tertile also showed improved survival ( y os % for t , % for t , % for t , p= . ). conclusions: there were no differences in the infused cellularity between responding and non-responding patients with chronic gvhd. at the same time, we found that except for a trend toward better response with higher mncs infused, there was no relationship between lymphocytes and monocytes with the response rate as other previous studies have suggested. however, even if there is no relationship with the response rate, the patients receiving the highest numbers of lymphocytes, monocytes and mncs in the cohort showed an improved survival, suggesting that larger quantities of cells could exhibit a protective effect. nevertheless, prospective studies that address this relationship are needed. disclosure: nothing to declare comparative analysis of the cytotoxic potential of cytokine-induced killer and natural killer cells for neuroblastoma therapy annekathrin heinze , beatrice grebe , eva mudry , jochen früh , bushra rais , claudia cappel , sabine hünecke , eva rettinger , thomas klingebiel , peter bader , evelyn ullrich , university hospital frankfurt, frankfurt, germany, german cancer consortium (dktk) partner site:, frankfurt, germany background: neuroblastoma (nb) is the most common solid extracranial tumor in childhood. despite therapeutic progress, prognosis for high-risk nb is poor and innovative therapies are of medical need. therefore, we investigated the cytotoxic potential of interleukin (il)-activated natural killer (nk) cells compared to activated cytokine-induced killer (cik) cells against different human nb cell lines in vitro. methods: nk cells were isolated from peripheral blood mononuclear cells (pbmcs) using cd enrichment or cd /cd depletion kits. they were successfully expanded ex vivo with different cytokine combinations such as il- , il- , il- and/or il- under feeder-cell free conditions. in contrast, cik cells were generated from pbmcs by ex vivo stimulation with interferon-γ, il- , okt- and il- . a comparative analysis of expansion rate, purity, phenotype and cytotoxic activity against different nb cell lines following different culturing protocols was performed. results: cd enriched nk cells showed a median expansion rate of . -fold after to days in culture with a final frequency up to . % nk cells and a median frequency of . % cd + cd -t cells. in contrast, the starting cell product after cd /cd depletion consisted of a median frequency of . % nk cells that expanded significantly faster with . -fold and also reached up to . % purity without any relevant t cell contamination. cik cells expanded with a median rate of . -fold and contained . % nk, . % t and . % nk-like t cells. interestingly, nk cells, particularly after cd /cd , showed a significantly higher median cytotoxic capacity against nb cells depletion ( . % for cd enrichment, . % for cd /cd depletion) compared to cik cells that induced . % killing of nb cells with e:t ratio : in a hours' co-incubation assay. interestingly, prolonging the ex vivo stimulation after cd /cd depletion to days enhanced the median expansion rate to . -fold with a slightly reduced cytotoxic potential ( . % for days' ex vivo expansion, . % for days' ex vivo expansion, comparison of the same donors). the addition of an il boost prior harvesting increased the expansion rate to median . -fold (compared to . -fold for the same donors) with an improved cytotoxicity of . % (compared to . %) . fortunately, all nk cell products showed a high viability and no relevant t or b cell contamination (median < . %). interestingly, further optimization of the culturing procedure with use of another cell culture medium led to an improved median . -fold (compared to . -fold) nk cell expansion rate in days, also resulting in comparable cytotoxicity of . %. conclusions: nk and cik cell products may offer an innovative immune therapeutic option for patients with high-risk nb after allogenic stem cell transplantation. our study revealed that nk cells have a significantly higher cytotoxic potential to combat nb. interestingly, the use of il- expanded and il- activated nk cells developed from a cd / depleted apheresis product is highly promising as additional immunotherapy in combination with haploidentical stem cell transplantation of children with nb. disclosure: nothing to declare. quantitative determination of donor allo-reactive t-cells in haploidentical donor-recipient pairs by enzymelinked immunospot (elispot) and mixed lymphocyte culture (mlc) assays background: t-cell alloreactivity is responsible not only for graft versus host disease and morbidity, associated with hematopoietic stem cell transplantation (hsct) but also for graft-versus-leukemia (gvl) activity. in this regard, monitoring and quantitation of alloreactive t-cells (allo-t) may potentially provide valuable information for individualized clinical management of transplant recipients. the aim of this study was the optimization of allo-Т detection and comparison of the elispot and mlc assays. methods: allo-t were determined in haploidentical donor-recipient pairs before hsct. donor mononuclear cells (mnc) served as effector cells (ec) . patient cd depleted mnc were used as stimulatory cells (sc).the ratio ec:sc were : and : . the frequency of allo-t in donor peripheral blood was tested in elispot assay and mlc. elispot provides the detection and quantitation of activated t-cells on the basis of cytokine secreted by each cell. the co-incubation time was h for ifn-gamma and h for il- detection. in mcl assay donor mnc were labeled with cfse and allo-t, proliferating in response to stimulation with alloantigens, were determined by flow cytometry on day . results: the median number of ifn-gamma producing allo-t per donor mnc was , ( - ; ec:sc ratio - : ) and , ( - ; ec:sc ratio - : ). the median frequency of allo-t was , % ( , - , ; ec:sc ratio - : ) and , % ( , - , ; ec:sc ratio - : ) among lymphocytes. il- -producing allo-t were less frequent in donor mnc in comparison with ifngamma-producing allo-t. the median number per mnc was , ( , - ; ec:sc ratio - : ) and , ( - ; ec:sc ratio - : ). the median frequency of il- -allo-t was , % ( , - , ; ec:sc ratio - : ) and , % ( - , ; ec:sc ratio - : ) among lymphocytes. the ec:sc ratio : is enough for stimulation of il- producing by mnc in elispot assay, but for optimal stimulation of ifn-gamma producing cells ec:sc ratio : is preferable. this suggests that allo-t are predominantly ifn-gamma producing cells. alloreactive proliferating t-clones were detected in mlc in of donor-recipient pairs on day of cocultivation. median percentage of proliferating t-clones were , % ( , - , ; ec:sc ratio - : ) and , % ( , - , ; ec:sc ratio - : ) among lymphocytes. however, mlc assay only permit a qualitative analysis that confirmed the presence of alloreactive t-clones, giving no information on their frequency within the culture. results of elispot and mcl assay directly correlated. conclusions: allo-t were detected in , % of assayed haploidentical donor-recipient pairs by elispot and only in , % by mlc. this difference in detection is due to the fact that elispot allows to detect single cytokine secreting cell whereas mlc can reveal proliferating аllo-t clones. the analysis of allo-t in haploidentical donor-recipient pairs may provide rationale to manipulate the allo-immune response and to exploit the powerful ability of allo-t to control hematologic malignancies. disclosure: nothing to declare allogeneic mesenchymal stromal cell as rescue therapy in an infant with life-threatening respiratory syndrome due to a filamin a mutation background: cell-based therapy has gained attention in the respiratory system diseases and encouraging results are reported following mesenchymal stromal cells (mscs) administration. due to their capacity to produce and secrete a variety of paracrine factors and bioactive macromolecules, mscs became a key player in lung tissue injuries and function, reducing fibrosis, promoting the normal development of alveoli and pulmonary vessels. for the first time we used the msc infusions as rescue therapy in a pediatric patient with flna gene mutation and life-threatening respiratory syndrome. methods: a child with a new pathogenic variant of the flna gene c. _ del; (p.val alafster ) at the age of months, due to the serious and irreversible chronic respiratory failure and dismal prognosis, was treated with intravenous infusions of allogeneic bone marrow (bm)-mscs at the dose of × mscs/kg body weight. bm-mscs were produced at "cell factory", fondazione irccs policlinico s. matteo, pavia,isolated and expanded ex vivo from healthy donor bm, following a previously reported protocol. premedication with antistaminic drug, min before every infusion to avoid any potential reaction was performed. the evolution of the respiratory condition was detected. peripheral blood were collected before each msc treatment for treg and th monitoring. treg, defined as cd + cd neg cd + cells expressing the forkhead box p (foxp ) transcription factor, and th , defined as cd + cells expressing intracellular il- , evaluation was performed by flow cytometry (facscanto; bd biosciences, san diego, ca) as previously reported, following standard procedures. results: no acute adverse events related to mscs infusion was recorded. during follow-up, patient maintained a good general condition and showed a regular growth. no systemic or respiratory infections occurred. after the second infusion, the child experienced a progressive improvement of his clinical respiratory condition, with a good adaptation to mechanical ventilation, in the absence of episodes of respiratory exacerbations. the baby maintains adequate volumes of exchange with substantial reduction of the inspiratory support. a reduction of trigger sensitivity was also obtained. thorax ct scan showed a recovery of the basal parenchyma bilaterally and the improvement of the anatomical-functional alignment and aerial penetration. after the first msc administration, an enrichment of treg and th percentage in peripheral blood, was observed. while, after the second msc infusion a significant increase in treg/th ratio was noted. conclusions: this report suggest that msc serial infusions are a promising therapy in aiding the respiratory failure, even in a pediatric patient with flna mutation. intravenous administrations of allogeneic mscs are feasible and safe without toxicity. our results suggest that to mitigate lung injury, mscs may act as regulators of treg and th balance. further investigations are upcoming to establish the useful of this therapeutic proposal in interstitial lung diseases in children. disclosure: nothig to declare p feasibility of il- stimulated donor nk cells manufacturing for early infusion in patients with high risk acute myeloid leukemia undergoing haploidentical transplantation background: nk cells provide a potent antitumor effect in the setting of manipulated haploidentical hematopoietic stem cell transplant (haplo-hsct). we propose a novel strategy to enhance the antitumor effect of allogeneic transplant through the infusion of nk cells stimulated with il- exvivo in adult high-risk acute myeloid leukemia (aml) patients undergoing unmanipulated haplo-hsct. the objective of this study was to provide efficiency and productivity data obtained in the manufactured cellular products infused. methods: selection criteria included patients with highrisk aml undergoing unmanipulated haplo-hsct. lymphoapheresis of the haploidentical donor was performed using spectra optia (terumo® bct) on days + and + after transplant. from the obtained product a double immunomagnetic cellular selection with clinimacs system (miltenyi biotec®) was performed in two steps: cd + depletion followed by positive cd + selection. the obtained an enriched cellular product of cd -cd + nk cells was incubated with il- ( ng/ml) between and hours at ºc and % co in gmp conditions. quality and microbiological controls were performed at the end of each manufacturing step. dxh cellular counters (beckman coulter®) and multiparametric flow cytometry were used for lymphocyte subpopulations and viability analysis (navios cytometer; beckman coulter®, conjugated monoclonal antibodies; miltenyi biotec®). the final product was infused intravenously to the patient on days + and + if manufacturing conditions were met (range of . - x nk/kg, purity ≥ %, viability≥ % and < x cd + cells/kg). if not, it was discarded. nk cell activation in the product was measured by the expression of cd and cd . results: between november and april , patients were included in this ongoing trial. two products were manufactured for of the patients, and only one for the first patient, due to transplant complications between first and second infusion. one product did not meet minimum viability criteria and was discarded. in the infused final products mean and sem of nk cell purity, recovery and viability were . %± . , . %± and . %± . , respectively. log cd + depletion ranged between - . and - . . median infused doses of nk cells and cd + cells per kg were . x ( . x - . x ) and ( - ). complete manufacturing data of all procedures are shown in table background: cytokine-induced killer (cik) cells are a promising immunotherapeutic approach to combat relapse following allogeneic hematopoietic stem cell transplantation (hsct) for acute leukemia or myelodysplastic syndrome. to show safety and efficacy, a multicenter clinical study with pediatric and adult patients including up to eight cik cell applications with escalating doses is ongoing. methods: we favor single large scale cik cell generation with the aim to apply fresh cik cells and cryopreserve ready-for-use doses according to the study protocol in contrast to recurrent manufacturing. therefore cryopreserved cik cells were tested against freshly generated cik cells to approve equivalence. furthermore, an alternative medium supplement for cik cell culturing was investigated to avoid supply bottlenecks in ab-serum. results: fresh frozen plasma (ffp), platelet lysate (pl) and ab-serum in cik cell culture showed median expansion rates of -fold, whereas cultivation without medium additive resulted in significantly lower proliferation (p< . ). cik cell composition including t cells, nk like t cells and a minor part of nk cells was not significantly influenced by changing the medium additive. moreover, neither cytotoxicity against thp- cells nor cd expression on nk like t cells were significantly influenced by the different medium additives. for cik cell generation either ficollized peripheral blood (pb) or unstimulated leukapheresis (lp) products were utilized. with regard to repeated manufacturing within the clinical study, also cryopreserved lp and pbsc as starting material came into the focus of interest. comparing cik cell expansion rates, no significant differences for the entire cik cells and the subgroup of t cells were detected between the four starting materials. cryopreservation of cik cells had no significant effect on cik cell composition, cytotoxicity and cd expression on nk like t cells. a small, albeit not significant effect of cryopreservation on viability was detected, which was . % before and . % after freezing and thawing. conclusions: the challenge was an efficient time-, personal-and cost saving production of cik cells within the clinical study. introducing ffp enabled cik cell manufacturing for an increased patient cohort by avoiding supply bottlenecks in ab-serum. furthermore, cryopreservation allows the storage of ready-for-use cik cell doses fulfilling the demands of the clinical study. clinical trial registry: eudract number - - disclosure: nothing to declare. automated generation of cd ra depleted donor lymphocyte infusion (dli) with the clinimacs prodigy® cd ra system , methods: the current clinimacs cd ra system was developed for graft engineering. up to x e magnetically labeled cd ra+ cells from leukapheresis products can be depleted from up to x e white blood cells (wbc). we developed a new clinimacs prodigy® process in order to ease the procedure for routine-use, to reduce the specifications according to reported cell numbers for dli applications, and to enable the use of peripheral blood products with high amounts of red blood cells (rbc). the new system was tested by performance runs. an new fluorescent flow analysis protocol was developed. results: the resulting clinimacs prodigy pb- ra system is an automated procedure with integrated labeling and washing steps. the new application software pb- ra depletion enables to deplete up to . x e cd ra + cells from up to x e total wbc from peripheral blood products. a major difference of this process is the rbc removal option based on an integrated camera for cell pellet detection. the final cell product is provided in physiologic saline. verification runs with peripheral blood products (n= in total, n= with whole blood, n= with leukapheresis products) resulted in a mean depletion of . log (range . - . ) for cd ra + t cells in the cd ra depleted product. viability of the target products was always above %, and mean wbc recovery was %. the mean process time was h min (range h to h min) without including the manual steps, i.e. tubing set installation and downstream analysis of blood products by flow cytometry. this data were in line with preceding evaluation runs (n= ), and results obtained in cooperation with an external beta test site. the performance results were furthermore in line with results obtained on clinimacs plus instrument runs. for quality control of cd ra depleted products we developed a flow cytometric analysis strategy for fast, accurate, and convenient analysis of even rare counts of remaining unwanted cells. it allows to determine naïve t cells at two different levels of subset staining. the minimum requirement for the flow cytometric analysis includes colors to define viable cd +cd ra+ cells. for further evaluation of the naïve t cell subsets additional colors are used to define viable cd +cd ro-cd -cd l+cd + cells. conclusions: the automated clinimacs prodigy pb- ra system process is capable to deplete cd ra+ cells efficiently from peripheral blood products within hours. the new process is a fast, convenient, and regulatory compliant method for the preparation of ready-to-use cd ra-depleted cell products for clinical applications. the submission to an european notified body for ce certification is an important next step. myeloablative conditioning regimen was preferred for patients out of . gvhd prophylaxis regimens are csa +mtx: (% ), csa+mmf: (% ), csa only: (% ). atg was given patients. despite been given gvhd prophylaxis (% , ) patients out of transplanted patients had gvhd features. of patients, had experienced steroid resistant gvhd after transplantation, including (% ) grade and (% ) grade . ecp treatment was started mean days after diagnosis of steroid resistant gvhd and (% ) patients had complete response while (% ) patients had partial and (% ) patients had no response to ecp treatment on day . sixteen out of patients had also received mesenchymal stem cell therapy as salvage therapy. only one patient had experienced hypocalcemic tetany, a complication of ecp procedure. thirteen patients had died and were directly related with steroid resistant gvhd. other conditions like relapse of primary disease or pres syndrome also played role in death. conclusions: extracorporeal photopheresis is a reliable and effective second line treatment modality in steroid resistant gvhd. starting ecp sessions as soon as gvhd symptoms occur increases its effectivity. mesenchymal stem cell administration with ecp for (% ) patients limits our study to reach o conclusion for efficacy of ecp itself. need for hemodialysis catheters, the prolonged sessions while adequate flow is not possible and catheter related infections are the lmitations for feasibility of ecp. disclosure: nothing to declare donor lymphocyte infusion administrations after allogeneic stem cell transplantations in pediatrics: a single center experience background: loss of chimerism is one of the major problems after allogeneic stem cell transplantation(sct). donor-lymphocyte infusions(dli) are used as a treatment after taper or stopping immunosuppression. in this study, dli experience in patients with loss of chimerism after sct due to various benign and malign hematological diseases was presented. methods: between july -august , twenty patients, detected chimerism loss and received dli after sct were evaluated retrospectively. patients received myeloablative or reduced intensity conditioning, atg, cyclosporine a and methotrexate for gvhd prophylaxis. chimerism analyses were performed with short tandem repeat(str) method from peripheral blood. results below % were considered as mixed chimeric and below % were nonchimeric. when patients considered as mixed chimeric, immunosuppression therapy was ceased immediately and treated with dli. donor lymphocyte infusions were performed at two-week intervals with chimerism follow-up. student t, mann whitney u, ki kare tests and kaplan-meier analysis were used. results: between - ages (median ), female, male patients were evaluated. the initial diagnoses were thalassemia major( ), aplastic anemia ( ),all( ), aml ( ) . dli initiation time was . +- . days after sct, total number of dli administrations were . +- . . dose of dli was x - . x /kg (mean . x /kg). nine patients' chimerism out of , fell below % at first month after transplant; patients were nonchimeric, of them were complet chimeric and were mixed chimeric. eleven patients´chimerism were below % between - months after sct, patients were nonchimeric and were mixed chimeric. early mixed chimerism was found relevant with graft rejections (p= . ). patients were followed up for - days. eight patients' chimerism increased after dli infusion and continued to decrease in patients. after dli, acute gvhd has been seen in both group.the group with decreased chimerism after dli, dose was mean x ± x /kg while the group with increased chimerism had dli dose mean x ± x /kg. although the difference was not statistically significant, numerical value revealed significantly different. eventually patients out of were mixed chimeric, patients were complete chimeric and were none. in thalassemic patients, patients with thalassemia-trait donor were mixed chimeric, in patients whose donors were normal, of them were complete chimeric and one of them was nonchimeric.the difference was significant (p= . ). the cd infusion doses revealed mean . ± . x /kg in mixed chimeric patients, . ± . x /kg in complete chimeric patients and . ± . x /kg in the patients with loss of chimerism. cd amount was seen high as numerical value in complete chimerics but no statistical significance was found. overall survival was %, disease-free survival was %. conclusions: we evaluated the efficacy of dl for patients with mixed chimerism in our patient group. we concluded that chimerism loss in patients with early decreased chimerism is similar to those in literature in spite if dli practices. dose and application frequency were greater in patients with increased chimerism. the small number and the heterogeneity of the patients limited our study. in this regard, studies with larger series and homogeneous groups are acquired. disclosure: nothing to declare phase i clinical trial of repeated administrations of bone-marrow derived mesenchymal stem cells in steroid-refractory chronic graft-versus-host disease patients nayoun kim , young-woo jeon , jae-deog jang , keon-il im , nak-gyun chung , young-sun nam , yunejin song , jun-seok lee , seok-goo cho (cghvd) is the most common long-term complication of allogenic hematopoietic stem cell transplantation which is associated with poor quality of life and increased risk of morbidity and mortality. currently, there is no standardized treatment available for patients who do not respond to steroids. as an alternative to immunosuppressive drugs, mesenchymal stem cells (mscs) have been used to treat and prevent steroidrefractory acute gvhd patients. these studies and reports have also provided a basis for using mscs in steroid refractory cgvhd patients. methods: to evaluate the safety and efficacy of repeatedinfusions of mscs, we enrolled ten severe steroid-refractory cgvhds patients. steroid refractory was defined as either no response to steroids lasting at least weeks or progression of disease during treatment or tapering lasting at least weeks. patients were intravenously administered with mscs produced from third-party bone marrow donors at a -week interval for a total of four doses. each dose contained x cells per kg body weight and all four doses consisted of mscs from the same donor and same passage. results: we enrolled ten patients ( female/ male, with a median age of . (range - ). median of cgvhd affected organs was (range - ) including the skin (n= ), eyes (n= ), oral cavity (n= ), lung (n= ), liver (n= ) and joints (n= ). all ten patients received their planned four doses of mscs, administering a total of infusions. median time from initial cgvhd diagnosis to first msc treatment was days (range - ). msc infusions were well tolerated with no immediate or delayed toxicities. after weeks of the first msc infusion, all ten patients showed partial response showing alleviation in clinical symptoms and increased quality of life. organ responses were seen in skin (n= ), eyes (n= ), oral cavity (n= ), liver (n= ), and joint(n= ). however, one patient died of progressive gvhd and one patient relapsed from primary disease. conclusions: repeated infusions of mscs was feasible and safe and may be an effective salvage therapy in patients with steroid-refractory cgvhd. further large-scale clinical studies with long-term follow up is needed in the future to determine the role of mscs in cgvhd. background: the majority of pregnant polish women ( %) have heard of cord blood banking. however, most doctors do not have sufficient knowledge about the possibility of using cord blood in order to respond to their potential concerns. only . % of healthcare professionals were aware that cord blood could be used to treat haematological diseases. in order to make doctors aware of this issue and provide patients with the information they expect, we would like to present data on the use of cord blood stored in our blood bank for haematological and nonhaematological therapies. methods: the table presented below has been created using data from the general database of the polish stem cell bank, warsaw, poland. no data regarding umbilical cord blood data have been excluded. all patients were planned to be assessed on day , day , on discharge, days after transplantation and , , , , and years after transplantation, but in some cases, patients were lost from follow-up due to a persistent lack of reports from transplantation centres. results: in cases, the therapeutic use of cb was transplantation (replacement of patients' own tissue); in cases it was administration (infusion without destruction of patients' own tissue). thirty-three were administered as standard therapy and as experimental therapy. conclusions: the survey study cited above, indicated low awareness of cord blood use among healthcare professionals. on the other hand, one study indicated that the expectations placed in cord blood banking may be unreasonable. as a private cord blood bank, we support recommendations which underline the importance of patient education. in poland, cord blood has been approved as standard therapy in approx. diseases; most of them are rare, but polish law allows the use of cord blood for the siblings, parents and grandparents of a donor. additionally, active and planned clinical trials throughout the world evaluate the therapeutic efficacy of cord blood in such areas as haematology, neurology, cardiology, diabetology, congenital paediatric disorders, ophthalmology, dermatology, gastroenterology, hiv infection, and the quality of life during aging. therefore, further indications may be expected in the future. background: cytokine release syndrome (crs) has been identified as a clinically significant, on target, off tumour side effect of the chimeric antigen receptor (car) t-cell therapies. it is clinically increased in interkeukin and elevations in other cytokines, lactate dehydrogenase (ldh), c-reactive protein (crp), and ferritin. these side effects can include fever, fatigue, headache, encephalopathy, hypertension, tachycardia, coagulopathy, nausea, capillary leak and multi organ dysfunction. crs symptoms can appear as early as one day after infusion and can resolve quickly or last for weeks. it´s severity to be related to the disease burden prior to car t-cell therapy. methods: the bristol oncology and haematology centre will be providing car t-cell therapy to patients in early . on collating from the leading consultant on the ward and fellow nursing team members it was apparent that an effective way at managing patients post car t-cell therapy side effects is to provide an educational and informative poster depicting a flow chart that will aid the practitioner to recognise and effectively treat/manage a patient with crs symptoms. results: none as of yet as this is a prospective tool ready for our first patient in early conclusions: through continuing reading and study days prior to the ward receiving its first car t-cell patient it is increasingly important that the entire multi disciplinary team recognise crs and understand the importance of early detection, careful monitoring and early intervention. background: allogeneic stem cell transplantation (allosct) is the only curative procedure for primary and secondary myelofibrosis (pmf, smf). elderly people are mainly affected, limiting the feasibility of intensive myeloablative chemotherapy regimens. the introduction of reduced-intensity conditioning (ric) made allosct feasible and effective for old patients. nevertheless, the incidence of pmf and smf is not negligible in young patients, theoretically able to tolerate also high-intensity therapy. very few data are available about the efficacy of ric-allosct in the particular setting of young-aged mf patients. methods: this study includes myelofibrosis young patients (age < y) who received allosct between and at the university hospital hamburg/germany. four patients were previously splenectomized. patients mostly fall into intermediate risk groups according to dipss. four patients belonged to the high-risk triple-negative category (jak /calr/mpl-). asxl -mut was tested in patients (pos: ). in % graft source was pbsc, patients received bmsc. only % of patients had a / hla-matched sibling, the others were transplanted from fully-matched ( %) or partially-matched ( %) unrelated donor. all transplants were conditioned according the ebmt protocol with busulfan ( mg/kg po or mg/kg iv), fludarabine ( mg/m ), atlg (grafalon® neovii, germany) administered in days at a dose of mg/kg die for mud, mg/kg die for mrd transplants, followed by cylosporina, and mycophenolate in the first days. results: engraftment rate was %, median neutrophil engraftment time days. platelet engraftment was reached by patients ( %, median days). four patients ( %) developed poor graft function, successfully treated with cd + selected pbsc-boost. after a median follow up of . years, estimated y-pfs and os were % and % respectively. dipss-risk and donor hla-matching resulted the only significant impacting factors on os. neither cytogenetic nor molecular abnormalities were significantly related to os. twenty-five patients ( %) experienced agvhd grade > . c-gvhd was observed in patients ( %), mostly ( %) beginning in the first days after transplantation. cumulative incidence of trm was % at year, with a plateau after the first year ( y trm = %). trm was observed only in patients with maximal grade ( ) of marrow fibrosis. furthermore, trm never occurred in previously splenectomized patients (p= . ), but no significant impact from splenectomy on os was observed (p= . ). after transplant, patients ( %) relapsed: died without any treatment because of infection, received dli ( durable cr), patients ( after dli) underwent a second allosct, with long-term survival in cases. conclusions: ric followed by allogeneic sct is a curative treatment approach for younger patients with myelofibrosis with a low nrm. the most important outcome-determining factor is donor hla-matching. interestingly, marrow high grade fibrosis showed to significantly impact trm. biological markers such as asxl mutation and cytogenetics, largely known as highly predictive for poor prognosis in the disease natural course, did not show any impact on survival, suggesting that patients harboring these abnormalities could get the greatest benefit from allosct. further data collection, and a prospective randomized trial are needed to confirm our conclusion. disclosure: nothing to declare p abstract already published. splenectomy as a risk factor for relapse after allogeneic hematopoietic stem cell transplantation in patients with myelofibrosis -retrospective cohort study background: splenectomy is a common procedure in patients (pts) with myelofibrosis (mf) performed to achieve improvement in blood cell counts and reduce b-symptoms. however, it has also been shown that splenectomy may adversely predispose to leukemic transformation in this setting. aim: to evaluate in a single-center retrospective analysis the long-term impact of pre-or post-transplant splenectomy on transplant outcome regarding overall survival and relapse risk. methods: this retrospective analysis comprises the data of pts ( male and female) with primary (n= ) or secondary (n= ) mf after allo-hsct from hla-matched sibling (n= ) or unrelated (n= ) donors in our center between and . the median age was years (range, to years). a myeloablative conditioning regimen was performed in pts, while pts where treated with a reduced intensity conditioning. peripheral blood stem cells (n= ) or bone marrow (n= ) with a median of . x cd + cells/kg bodyweight (bw) (range, . to ) were transplanted. splenectomy was performed in of pts: pts were splenectomized prior to and pts after allo-hsct. relapse was diagnosed in ( %) of pts. the median duration to relapse after transplantion was months (range, - months). results: the median duration of follow-up of this cohort was months (range, - months), the -year overall survival (os) was %. pts died, including pts who relapsed and pts who died of treatment related causes. the observed probability of relapse was significantly higher in splenectomized pts than in non-splenectomized pts: % versus % (relative risk (rr) . , % ci, . - . , p= . ). at years, the os was % in nonsplenectomised and % in splenectomised pts (p= . ) (fig. ) . the relapse rate in splenectomised pts was independent of pre-( of pts, %) or post-transplant ( of pts, %) treatment (rr . , % ci, p= . ) . conclusions: on the basis of our cohort, we could assert that pre-and post-allo-hsct splenectomy was equally and significantly associated with an increased relapse ratio in patients with mf, which also tends to negatively affect overall survival. [[p image] . figure : the overall survival after allo-hsct in patients with myelofibrosis.] background: b-cell prolymphocytic leukemia (b-pll) is a very rare lymphoproliferative disorder. although allogeneic stem cell transplantation (allosct) could be a curative option, patients often do not qualify for this consolidation treatment due to an aggressive course of disease. in this case study, we report on three patients who failed ibrutinib therapy but achieved complete remission and even mrd negativity after treatment with the chimeric cd -antibody rituximab, enabling them to undergo allosct. methods: clinical data and follow-up data were collected by chart review. results: all three patients (pt# : male, years; pt# : female, years; pt# : female, years) were referred with b-pll harboring highly complex aberrant karyotypes, including p abnormalities in pt# and pt# . a tp mutation could be detected in pt# and pt# . all three patients had symptomatic disease with rapidly increasing hyperleukocytosis and massive splenomegaly. two of them were treatment-naive and one relapsed after chemoimmunotherapy. all patients were put on ibrutinib mg. despite initial response to treatment, two patients developed progressive disease after (pt# ) and months (pt# ) on ibrutinib, whereas pt# remained in partial remission with persisting leukocytosis, precluding consolidating allosct as originally intended. in pt# , ibrutinib was replaced by venetoclax, but without response. in order to control rapid lymphoproliferation, rituximab was added to venetoclax. grade infusion reaction / tumor lysis syndrome (tls) (fever, tachycardia and hypotonia requiring intravenous vasopressors) followed each rituximab administration despite fractionating rituximab to small doses. however, continuation of rituximab ( mg/d over d) led to complete and durable clearance of hyperleukocytosis (from /nl to mrd negativity) despite venetoclax cessation. a similar pattern was observed in pt# , who received rituximab while showing rapidly increasing leukocytosis upon ibrutinib. again, complete clearance of b-pll cells in the peripheral blood (from /nl to mrd negativity) occurred after initial grade tls despite only modest cd expression on tumor cells in this patient. also, pt# achieved profound b-pll cell depletion (from /nl to a mrd rate of . %) upon addition of rituximab to ibrutinib (without tls in this case). subsequently, all three patients were able to undergo allosct after conditioning with fludarabine and total body irradiation with gy. pt# received stem cells from a hla-ident sibling donor, whereas pt# and pt# had unrelated donors (hla-ident and hla-matched respectively). with follow-up times of and months post-transplant, pt# and pt# are currently in ongoing mrd-negative remission. pt# developed an acute graft-versus-host disease (gvhd) of the liver (grade ), nevertheless the clinical course was well controlled by immunosuppression. in pt# a chronic gvhd of the skin occurred. pt# , who achieved mrd negativity after allosct, developed acute and chronic steroidrefractory gvhd of the skin and gastrointestinal tract. nine months post-transplant, gvhd deteriorated and after further complications the patient died of pneumonia months post-transplant. conclusions: supplementary treatment with rituximab can induce deep remissions in patients with ibrutinibresistant, genetically poor-risk b-pll, thereby enabling them to undergo successful consolidation with allosct. a high risk of life-threatening infusion reactions / tls associated with the addition of rituximab has to be taken into account. background: there is little experience on the use of the newer targeted therapies in cll patients relapsed after allogeneic stem cell transplantation (allo-sct). against this background, we evaluate the safety and efficacy of the bcr inhibitors (bcri), ibrutinib and idelalisib, administered after allo-sct for the purpose of treating the cll relapse. methods: data from cll pts who relapsed after sct, and were subsequently treated with ibrutinib (n= ), idelalisib (n= ) or both (n= ),were retrospectively collected in collaboration with the spanish group of cll (gellc) and the spanish group of stem cell transplantation (geth). results: transplant characteristics are summarized in table . eight patients received the bcri as the first salvage treatment after sct relapse, whereas patients had received ≥ prior lines of treatment. at the time of the onset of the bcri, patients had rai stage and patients had a lymph node size ≥ cm. del p was present in patients and del q and complex karyotype in patients, respectively. tp gene mutation was detected in patients (all with del p ). median time from sct to bcri therapy was . months, being shorter in patients treated with ibrutinib (n= , median months) than in those treated with idealisib (n= , median months). median time on ibrutinib and on idelalisib was . months ( . - . ) and months ( . - . ), respectively. the best overall response rate (orr) obtained with ibrutinib was % ( cr, pr, pr+l) whereas it was of % for patients receiving idelalisib ( cr, pr+l). among the patients treated with ibrutinib, ( . %) presented an adverse event (ae), being diarrhea (n= ), asthenia (n= ) and infections (n= ) the most frequent. hypertension was seen in patient and none patient developed atrial fibrillation. five patients stopped ibrutinib treatment, due to toxicity (n= ) or progression (n= ). after ibrutinib discontinuation, patients were newly treated with idelalisib (n= ) or venetoclax (n= ). all patients treated with idelalisib developed at least one ae, being diarrhea (n= ), pneumonitis (n= ) and neutropenia (n= ) the most common. four patients discontinued idelalisib because progression (n= ) or toxicity (n= ). venetoclax was given after idelalisib in patients. although acute and/or chronic gvhd before bcri was documented in ( . %) and ( . %) patients, respectively, only one patient (treated with idelalisib) reactivated a mild chronic gvhd. none patient received infusion lymphocyte from donor after bcri and one patient underwent a second sct. with a median follow-up of . months ( . - . ) after the onset of the bcri treatment, patients had died, all of them due to cll progression ( richter´s transformation), whereas patients remained in response ( cr, pr). the overall survival probability of the whole series at months was . % ± . %. conclusions: in our study, ibrutinib and idelalisib, administered in cll patients relapsed after sct did not increase the risk of gvhd reactivation but they show high incidence of adverse events. nevertheless, bcri offers a possibility of disease control in these patients with poor prognosis. further studies are needed to confirm these data. background: prior to the introduction of tyrosine kinase inhibitors (tki), median survival of chronic phase chronic myeloid leukemia (cp-cml) patients was approximately months and the standard treatment with interferon-alpha resulted in complete cytogenetic responses in about % of the patients. autologous stem cell transplantation (auto-sct) was first attempted for patients in transformation in order to restore a second cp and was introduced secondarily in cp to try to prolong the response. the main rational for autografting in cp resides on the reduction of the tumor burden and the number of leukemic cells at risk of developing blastic transformation. nevertheless, auto-sct alone was not able to maintain a long-term remission. nowadays, tkis represent the state-of-the-art therapy for cml and the concept of auto-sct has only little interest while long-term follow-up and outcome in this setting are currently unknown. the aim of our study is to evaluate at a first time the longterm outcome of cml patients who received auto-sct in chronic phase, and to evaluate at a second time in a subgroup analysis, the outcome of those who received tki after having been auto-transplanted, mainly for disease progression/loss of response and/or to enhance disease response. methods: we found a total of patients who received auto-sct for cp-cml in europe between years and , ( %) were males, median age at auto-sct was years (range: - ), the median time between diagnosis and auto-sct was months, stem cells source was peripheral blood in % of patients, most frequent conditioning regimen was busulfan mg/kg/day days + day of melphalan mg/m² one day prior to the cells reinfusion. information about receiving tki post auto-sct was available only for patients, first tki was imatinib for ( %) patients, dasatinib for ( %), nilotinib for ( %) and ponatinib for one ( %) patient. results: after a median follow-up of . years (range: - ) from time of auto-sct for the whole population, the probability of overall survival (os) at years was % ( % ci: - ); there was ( %) patients who relapsed after a median time of months after auto-sct. there was a total of patients transplanted before the tki era and survived until the availability of tkis. when we performed a landmark analysis evaluating the outcome of patients who received auto-sct, survived until the tki era and received tki (n= ), the years os probability of these patients from tki treatment was % ( % ci: - ). additional data requests will be sent to centers querying about prognosis, molecular responses, treatment and disease details. conclusions: we demonstrate here with these preliminary results that the introduction of tki has improved survival of cml patients. in addition, patients who received auto-sct, survived until the tki era and also received tki, had encouraging rates of long-term survival. an extensive analysis will be performed when additional data will be available and the study will be updated with more results. disclosure: nothing to declare a year single center transplant experience in chronic myeloid leukemia background: allogeneic hematopoietic stem cell transplantation (hsct) has been considered for decades the only curative approach for patients with chronic myeloid leukemia (cml). in the tyrosine kinase inhibitors (tkis) era, hsct for cml has been reserved only to patients not achieving a cytogenetic remission or showing progressive disease after multiple tki treatment lines. however, a progressive improvement in the long-term survival has been obtained in the overall hsct population. the present study aimed at evaluating whether in cml patients transplanted at our center over a long time period -from to -the outcome improved over time. methods: consecutive patients who underwent a transplant between and were compared to patients who received the transplant between and . overall survival (os), leukemia-free survival (lfs) and graft-leukemia-free survival (glfs) were estimated using the kaplan-meier method and the log-rank test was used to compare risk factors categories. results: of the patients [median age years (range - )], ( . %) were in st or nd chronic phase, ( . %) in accelerated phase and six ( . %) in blast crisis. matched related donors and alternative donors (matched unrelated donors, cord blood or mismatched related donors) were used in and cases, respectively. as stem cell source, bone marrow was used in patients, peripheral blood in and umbilical cord blood in . tbibased conditioning regimens were used in patients, while in the other cases irradiation-free conditioning regimens were used. both in univariate and multivariate analysis, irradiationfree conditioning regimens (hr . ; %ci . - . , p=. ) and transplants performed in st chronic phase (accelerate phase hr . ; %ci . - . , p=. - nd chronic phase hr . ; %ci . - . , p=. -blast crisis hr . ; %ci . - . , p< . ) were associated with a better os. patients transplanted before had a worse os (hr . ; %ci . - . , p < . ) and dfs (hr . ; %ci . - . , p=. ). a trend for a worse glfs was observed in univariate analysis (hr . ; %ci . - . , p= . ), in the first period of observation. conclusions: our single center experience confirms that higher os, dfs and glfs are observed in cml patients allografted in more recent years. improvement of conditioning regimens, use of tbi-free conditioning regimens and supportive therapy, have presumably contributed to these results, together with the more recent strategy of close monitoring of minimal residual disease, and prompt use of tki or donor lymphocyte infusion in case of relapse. hsct is nowadays a safer therapeutic procedure in cml patients that should be considered timely in tki-resistant patients to avoid progression into a more advanced disease phase. disclosure: the authors declare no conflict of interest. reduced-intensity transplantation (rit) in patients with high-risk or advanced chronic lymphocytic leukemia in last years: improvement of transplant outcomessingle centre experience . hct-ci ≥ was in % of pts. source of stem cells was peripheral blood in % and bone marrow in % of pts. the median of infused cd + cells was , x ^ / kg. the conditioning regimen consisted of fludarabine and melphalan (+atg in unrelated donor). gvhd prophylaxis were cyclosporine and methotrexate. results: all pts engrafted. none of pts in cr before rit progressed at day + after rit and among pts beyond cr before rit all of them achieved at least pr at day + after rit. pts ( %) developed acute gvhd ( pts grade iii-iv) and among evaluable pts ( %) of them developed chronic gvhd ( mild, moderate, severe). with median follow-up months (range - months) pts ( %) are alive in cr. pts ( %) relapsed or progressed , and months after rit and of them died. last relapsed patient achieved next cr after ibrutinib. pts ( %) died due to nrm. nrm till day + after rit was %. the estimated probabilities of -years cgrfs, pfs and os are %, % and %. conclusions: in spite of relatively small number of evaluated pts and retrospective type of analysis our data show that rit in pts with high-risk or advanced cll has achieved promising results ( -and -years pfs and os % and % resp. and %) in recent years and these results are better than outcomes of our historical patient cohort from period - ( -and -years pfs % and % resp. os % and %, p= . ) or ebmt published data of pts transplanted for cll in period - ( and -years pfs % and % resp. os % and %). current results of transplantation should be taken into account in our future decision-making process on indications for transplantation in pts with high-risk cll, of course also in the context of new or updated results of targeted cll treatment and its complications. disclosure methods: retrospective data and treatment outcomes were collected from the singapore childhood cancer registry (sccr). most children with cancer in singapore receive therapy at one of the two public paediatric cancer centers (kkh or nuh). a total of thirty two cases were diagnosed with cml and received treatment in either of these centers over a twenty year period ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) . results: the age at diagnosis of the thirty two children ranged from to years (median . years). six patients in the pre-tki era were treated with an upfront hsct. the remainder twenty six patients were initially started on a tki. of these / ( %) had a hsct at a median period of . months from diagnosis (range - months). the reason for hsct in ten out of the twelve children was due to high risk features i.e. accelerated/blastic phase/ no ccr/no cmr. the remaining two patients had a hsct due to parent and patient preference for attempt at upfront cure rather than the use of life-long and expensive tki therapy. non-compliance to tki therapy was a major finding in our teenage cohort. eleven of the eighteen transplants used a matched sibling donor. three patients had cord blood as their stem cell source. one patient had a single antigen mismatched related donor and three patients had a mismatched unrelated donor for their hsct. all patients except one had myeloablative conditioning with busulfan and cyclophosphamide. atg was added according to physician preference. one patient had cy/tbi conditioning because of pre-transplant lymphoid blast crisis. anti gvhd medications included cyclosporine/ methotrexate or tacrolimus and methylprednisolone in the cord transplant patients. six of the eighteen ( %) patients who had a hsct died. four died due to treatment related mortality ( infections, acute gvhd and pulmonary fibrosis). one patient died due to an early relapse and one had a late relapse related mortality. for the pre-tki era, hsct related and year os was % and % respectively. post-tki era and year os was %. for the entire cohort, the year os was %. conclusions: the post-tki era transplant outcomes from our two centers is comparable to leading centers in the world. outcomes for patients with mismatched unrelated donors was poor in our cohort. we recommend a haploidentical related donor transplant or an unrelated cord blood stem cell source for patients when a matched sibling or unrelated donor is not available. clinical trial registry: na disclosure: we have nothing to disclose. fludarabine, busulfan, and thiotepa may be a promising conditioning regimen for myelofibrosis patients undergoing allogeneic stem cell transplantation background: allogeneic stem-cell transplantation (sct) is a curative therapy for patients with myelofibrosis. however, recurrent disease and non-relapse mortality (nrm) are frequent causes of treatment failure. the optimal conditioning regimen for sct in this disease has not been defined. methods: we retrospectively analyzed transplantation outcomes of all adult patients given sct for myelofibrosis between and at a single large academic medical center. patients (n= ) were treated with several conditioning regimens that were grouped according to conditioning intensity. myeloablative conditioning (mac) included busulfan . mg/kg and cyclophosphamide mg/kg (bucy, n= ), fludarabine and busulfan . mg/kg (flu/ bu , n= ) and fludarabine and treosulfan - g/m (flu/ treo, n= ). reduced-intensity conditioning included fludarabine and busulfan . - . mg/kg (flu/bu , n= ). more recently we adopted the tbf regimen including fludarabine, busulfan . - . mg/kg and thiotepa - mg/ m (n= ). all patients were also given anti-thymocyte globulin during conditioning, irrespective of donor source. results: the median age was years (interquartile range [iqr] - ). the majority of patients had documented splenomegaly ( %) and were not previously exposed to ruxolitinib ( %). donor type was an hla-matched sibling ( %), / ( %) or / ( %) matched unrelated donor. the dipps+ score distribution was intermediate- ( %), intermediate- ( %), or high (n= %). the median followup was . years since the success of tyrosine kinase inhibitors (tkis), transplant-related mortality is considered too high to justify allogeneic hematopoietic stem cell transplantation (allohsct) as first-line treatment for chronic myeloid leukemia (cml) patients in chronic phase (cp). allohsct is currently considered for patients failing to at least tkis or with disease in advanced phase. nevertheless, the optimal timing for transplant referral is still not well defined. methods: we performed a retrospective analysis on consecutive patients with cml in cp receiving first transplants from an hla-identical sibling donor with partially t-cell depleted grafts from to at our center. partial t-cell depletion (ptd) consisted of in vitro alemtuzumab incubation of a part of the graft for infusion at day while the rest, containing x cd + cells/kg was given as a t-cell add-back at day . donor lymphocyte infusions (dlis) were provided, in the absence of gvhd, in case of disease relapse or mixed chimerism. molecular monitoring was performed by -month bcr-abl rt-qpcr testing in peripheral blood during at least a -year period after hsct. thereafter, -month testing schedule was maintained where possible, or followed by a -month one. kaplan-meier method was employed to determine the probability of overall survival (os) and leukemia free survival (lfs) since allohsct. results: median age at hsct was years (range, - ). all patients were in first cp but one who was in second cp. twelve patients were tki-naïve at hsct ( hsct ( - , patients had presented suboptimal response or/ and intolerance to imatinib ( - period) , while the last seven patients had presented suboptimal response or/ and intolerance to imatinib, dasatinib and nilotinib ( - period) . the time interval from diagnosis to transplant was < months in / ( %) patients. ( %) patients had an ebmt risk score of - , while ( %) patients of - . the conditioning regimen was myeloablative for all but one patients. the stem cell source was pbsc for patients and bone marrow for one. all patients engrafted. patients presented molecular relapse and one patient hematological relapse with a median interval from transplant to relapse of months (range, - ) . patients received dlis ( for relapse and for mixed chimerism), while patients in relapse also received tki. without prior administration of dli, ( %) patients presented grade ii agvhd and patients moderate cgvhd. after dli, agvhd occurred in and cgvhd in patients. one patient died of disease progression years after hsct and one of myocardial infarction years after hsct. with a median follow-up of . years (range . - . ), -year os and lfs were %. at the time of the analysis / patients were alive and in major molecular response. conclusions: these results of excellent long-term survival and no transplant-related mortality suggest that ptcd improves the outcome of cp-cml patients transplanted from an identical sibling donor and they can be useful for deciding risk-adapted strategies. we believe that ptcd could allow earlier transplant referral of patients failing tkis and having an identical sibling donor. disclosure: nothing to declare single tertiary centre experience in allogeneic haematopoietic stem cell transplantation (allo-hsct) for primary and secondary myelofibrosis (mf) the only curative option for fit patients is allo-hsct. novel therapy is emerging but current recommendation is that eligible patients with life expectancy less than years should be considered for allografting. methods: we retrospectively looked at the clinical features and outcomes of all allo-hsct for mf performed in our centre since . results: patients ( male, female) aged between - years old (median age ) with intermediate- or high-risk mf as per the international prognostic scoring system (ipss) or dynamic ipps (dipps) were transplanted in our centre since . of them ( %) were diagnosed with pmf and the remaining % with secondary mf; post-et and post-pv mf. / of our patient group received a sibling allograft and / a matched unrelated donor allograft ( % received a / human leukocyte antigen (hla)-matched transplant, % a / hlamatched and % a / hla-matched graft). all patients received a reduced intensity conditioning (ric); / patients with fludarabine/ melphalan/ campath (fmc), / fludarabine/ busulphan/ atg (fbatg) and fludarabine/ ara-c/ campath (flag/ campath); all received peripheral blood as source of hsc. engraftment occurred between day - , with a median of d+ . one late graft rejection occurred. all patients were alive at d+ . patients are currently alive; overall survival (os) is %. transplant related mortality (trm) was . % at year, % at years. patient died of graft versus host disease (gvhd) and patients of septicaemia leading to multiorgan failure. acute gvhd grade ii skin occurred in patients, grade iii and above in patients. patients have limited chronic gvhd. / patients received donor lymphocyte infusion (dli) for mixed chimerism (one of which had nd graft failure). out of these patients developed acute grade gvhd and died. response rate: / alive patients i.e . % exhibit no fibrosis in trephine biopsies, / alive patients had residual fibrosis but % donor chimerism, / alive patients had residual fibrosis with mixed donor chimerism, other patient non-assessable. conclusions: allo-hsct remains the only potentially curable option for myelofibrosis. in our centre which serves . million population, with new cases per year, patients were transplanted since . our data suggest that close collaboration between mpn-treating haematologists and transplant physicians is required so that all suitable patients have a transplant assessment early in their disease course. novel molecular prognostic systems are likely to identify those best placed to benefit in future but this series currently supports allo-hsct survival and cure. (range, - ) . dynamic international prognostic scoring system (dipss) score at the time of hct was intermediate- or high risk in patients ( %), intermedate- in patient. molecular evaluation was available in out of : jak v f mutation was detectable in patients, mpl-w k in patient, carl in patient. patients were "triple negative" for driver mutations. cytogenetics information was available for out of ; among which patients had complex karyotype, trisomy and trisomy . patients underwent splenectomy before hct. ruxolitinib was administered in patients before hct. ( %) patients received stem cells from an hla identical sibling, ( %) from a matched unrelated donor and ( %) from an haploidentical sibling. graft source was bone marrow in patients ( %) and peripheral blood in ( %). conditioning was myeloablative in patients ( %), reduced intensity in ( %). all patients engrafted. acute graft versus host disease was absent in patients ( %), grade i-ii in ( %), grade iii-iv in ( %). in evaluable patients chronic graft versus host disease was limited in ( %), extensive in ( %) and absent in ( %). transplantrelated mortality at days was %. main causes of death were: acute gvhd in patients, chronic gvhd in , pancreatitis in , pulmonary aspergillosis in . relapse occurred in patients and was the main cause of death in of them. notably, patients experienced late relapse after . and . years after hct. both of them are living while receiving ruxolitinib therapy. after a median follow up of days (range, - ), out of patients are alive. of them ( %) are disease-free and are living. the kaplan-meyer overall survival and disease-free survival at years was % and %, respectively. conclusions: our experience confirms that hct is a valid option to achieve cure in one third of mf patients. two patients experienced very late (> years) recurrence of mf. the rarity of this condition limits the amount of data and cases available for evaluation and study. life-long follow-up of all mf transplanted patients is warranted to better understand this rare event. disclosure: nothing to declare methods: А -years old female was diagnosed with jak v f-positive pmf, xx, ipss low risk, dipssplus intermediate - risk, subacute budd-chiari syndrome and portal vein thrombosis four years before allohsct. to reduce the splenomegaly and constitutional symptoms we performed pre-transplant ruxolitinib therapy mg daily. after three months of therapy the patient achieved clinical improvement (eln criteria). contrast-enhanced computer tomography and magnetic resonance imaging showed enlarged intrahepatic collateral vessels and signs of portal vein thrombosis with cavernous transformation and multiple dilated collateral veins. gastroscopy documented enlarged esophageal veins. allogeneic stem cell transplantation was performed from / -hla matched unrelated donor with peripheral stem cells ( . x Сd + cells/kg). conditioning regimen consisted of fludarabine ( mg/ m ), busulfan ( mg/kg p.o.). post-transplant cyclophosphamide was administered at mg/kg at day + , + , and ruxolitinib mg was used from d+ till d+ as graft versus host disease prophylaxis. results: starting d+ the patient experienced eight episodes of ebv some of them with severe blood loss. to treat the bleeding episodes blackmore tube was placed six times with temporary effect. to place blackmore tube the patient was two times intubated and required mechanical ventilation. at d+ leukocyte and neutrophil engraftment, full donor chimerism and molecular remission were achieved. platelet engraftment was documented only at d + and poor graft function was present due to cytomegalovirus reactivation (d+ ) and parvovirus b reactivation (d+ ). evb was stopped at d+ only after two esophageal veins ligations, and two procedures of gastric veins sclerotherapy. soon (d+ ) the patient achieved complete platelet recovery (more than x /l) and became red blood cells transfusion independent. at day + complete remission was confirmed by splenomegaly resolution, regression of bone marrow fibrosis, full donor chimerism, jak v f-negative molecular status. cbc showed hb g/l, platelets x /l, leucocytes , x /l. ultrasound examination after transplant documented portal vein thrombosis recanalization. at day + she developed mild (nih) chronic graft versus host disease with eyes and mouth involvement, which was managed with topical steroids. at d+ after transplant the patient is alive in complete remission and has no recurrent bleedings. conclusions: splanchnic vein thrombosis can significantly complicate the course of allohsct in pmf. easy access to surgical, intensive care unit and endoscopic teams is required to make allohsct more feasible in this group of patients. disclosure all patients received treosulfan-based mac regimens, treosulfan(total dose, - gms/m ) was given in combination with different conditioning drugs. the most commonly used regimen was treosulfan, fludarabine ( mgs/m ) and thiotepa( mgs/kg) referred to as ftt that was used in %(n= ). serotherapy was given in % of patients(n= ), as either alemtuzumab or antithymocyte globulin in %(n= ) and %(n= ), respectively. post-transplant graft-versus-host disease (gvhd) prophylaxis was given in all patients, based mostly on ciclosporin. patients( %) received the transplant from identicalrelated donors, patients( %) received the transplant from matched-unrelated donors, and two patients( %) had haploidentical transplants. % of the patients(n= ) were fully hla-matched. all stem cell sources were used as bone marrow in %(n= ), peripheral blood stem cells in %(n= ), and umbilical cord blood in %(n= ). this treosulfan-based conditioning was given as the st transplant in %(n= ), and as the nd transplant after the failure of a first procedure in %(n= ). two patients received treosulfan-based conditioned transplant twice. results: neutrophil engraftment and platelet engraftment occurred at a median of days and days respectively. chimerism was full donor in %(n= ), high donor in %(n= ), and mixed donor in %(n= ). gvhd developed in % of patients(n= ), with acute gvhd grade i/ii and grade iii/iv developed in %(n= ) and %(n= ), respectively. chronic gvhd grade i/ii and grade iii/iv developed in %(n= ) and %(n= ), respectively. all chronic gvhd were mild, limited, non-extensive, and resolved completely. none of our patients had persistent gvhd necessitating long-term systemic immunosuppression. mild vod occurred in %(n= ), and severe vod occurred in %(n= ). one of them died but was believed to be related to the underlying disease (wolman syndrome). viral reactivation occurred in % of patients(n= ), with cmv, ebv, and adenovirus reactivation was found in %, %, and %, respectively. five patients had invasive adenoviraemia that contributed to death in two of them. primary graft failure happened in two patients( %) due to adenoviraemia. seven patients( %) had secondary graft failure with autologous reconstitution. graft failure was significantly lower (p . ) in the ftt group than other conditioning groups. at a median follow-up of months (range, two- months), eleven patients( . %) died, with overall survival of . %, and event-free survival of . %. five patients died due to complications related to their original disease, while six patients died due to transplant-related causes (transplant-related mortality . %). immune reconstitution in alive patients was achieved at a median of eight months. this time was significantly longer (p . ) in ftt group. conclusions: this study demonstrates that treosulfan is a safe and effective conditioning drug that can achieve engraftment, with low rates of graft failure, transplantrelated mortality and morbidity, even if it is used twice in the same patient. disclosure: nothing to declare background: high-dose chemotherapy (hdc) followed by autologous stem cell transplantation (asct) is the treatment of choice for the patients with relapsed or high risk nhl. although the high-dose conditioning regimens commonly used in patients with non-hodgkin lymphoma (nhl) are beam (bcnu, etoposide, cytarabine, and melphalan), beac (bcnu, etoposide, cytarabine, and cyclophosphamide), survival of patients with nhl received above high-dose chemotherapy followed by asct was still unsatisfactory. methods: we prospectively evaluated the efficacy and toxicity of busulfan, etoposide, cytarabine and melphalan (bueam) including iv busulfan instead of bcnu of standard beam as a conditioning for asct in patients with nhl. the high-dose chemotherapy consisted of bu ( . mg/kg i.v. q.d. from day - to day - ), e ( mg/m i.v. b.i.d. on day - and day - ) a ( g/m i.v. q.d. on day - and day - ) and m ( mg/m i.v. q.d. on day - ) at centers in korea. results: two hundred five patients were enrolled onto the study. main subgroup was diffuse large b cell lymphoma (n= , . %), t cell lymphomas (n= , . %), and nk/t cell lymphoma (n= , . %). upfront asct was performed in patients ( . %), and salvage asct in patients ( . %). the disease status of the patients before hdt/asct consisted of patients ( . %) with complete response and patients ( . %) with partial response. treatment related toxicities included nausea in patients ( . %), diarrhea in patients ( . %), anorexia in patients ( . %) and stomatitis in patients ( . %), which were grade i or ii in the majority of cases. the common grade iii toxicities were stomatitis ( . %), diarrhea ( . %), and anorexia ( . %). there were no vod, and transplant-related mortality occurred in patients ( . %), due to infection. one hundred fifty three patients ( . %) achieved a complete response and patients ( . %) after asct, while patients ( . %) showed progressive disease. at a median follow-up duration of . months, the estimated -year overall survival and progression free survival for all patients was . % and . %, respectively. conclusions: the conditioning regimen of bueam for asct was well tolerated and seemed to be effective in patients with relapsed or high risk nhl. disclosure: none of declare background: allogeneic hematopoietic cell transplantation (hct) is potentially curative for high risk acute myeloid leukemia (aml) and myelodysplastic syndrome (mds), however both gvhd and disease relapse remain major challenges. we recently introduced a combination of posttransplant cyclophosphamide (ptcy) and atg ( . mg/kg) as graft-versus-host disease (gvhd) prophylaxis. the purpose of our study was to compare outcomes between ptcy/ atg and other gvhd prophylaxis regimens for high risk aml and mds. methods: we retrospectively investigated outcomes of patients that underwent allogeneic hct between january and july for high risk aml (n= , %) and mds (n= , %). gvhd prophylaxis regimens were compared for overall survival (os), cumulative incidence of relapse (cir) and non-relapse mortality (nrm) in univariate and multivariable analysis. high risk aml was defined as secondary aml, therapy related aml, high risk cytogenetics (eln criteria) in cr , good/ intermediate cytogenetic risk aml in cr and primary induction failure; high risk mds was defined as high/very high risk wpss score. results: median age of patients was years (range - years). donors were matched related in ( %) patients, matched unrelated in ( %) patients and haploidentical in ( %) patients. graft source was peripheral blood stem cells in patients ( %). myeloablative conditioning was used in patients ( %), reduced intensity regimens in ( %) patients. ptcy combined with atg was used in ( %) patients, other gvhd prophylaxis regimens were used in ( %) patients. both donor and recipient were cmv negative in ( %) patients. median follow-up of survivors was months (range - months). univariate analysis demonstrated os of the entire cohort at years was % ( %ci - %), cir at years was % ( %ci - %) and nrm at years was % ( %ci - %). concerning gvhd prophylaxis regimen, -year os for ptcy/atg versus others was % ( %ci - %) versus % ( %ci - %) (p= . , figure) , -year cir for ptcy/atg versus other was % ( %ci - %) versus % ( %ci - %) (p= . ) and -year nrm for ptcy versus other was % ( %ci - %) versus % ( %ci - %) (p= . ). grade ii-iv acute gvhd was seen in % of ptcy/atg patients versus % using other regimens (p< . ). chronic gvhd was observed in % of ptcy/atg patients versus % using other regimens (p= . ). multivariable analysis for os confirmed that the gvhd prophylaxis regimen has no influence (p= . ), while the predominant predictor of survival was age at hct (hr . , %ci . - . , p= . ). for cir, the ptcy/atg combination had no influence compared to other gvhd prophylaxis regimens (p= . ), while ric conditioning was the predominant predictor of relapse (hr . for ric, % p= . ) . for nrm, the atg with ptcy combination demonstrated no significant difference (p= . ), while age at hct was the predominant predictor (hr= . , %ci . - . , p= . ). conclusions: the ptcy/atg combination for gvhd prophylaxis has demonstrated on multivariable analysis similar os, cir and nrm with other previously used regimens at our center. a decrease in atg dose may potentially decrease the relapse rate while retaining the advantage of decreased gvhd. [ background: the combination of fludarabine with myeloablative doses of busulfan (fb ) represents a standard of care conditioning regimen before allogeneic transplantation in patients with myeloid malignancies (giralt, s.: the lancet oncology ). fb has potent antileukemic activity and is associated with low transplantrelated mortality and acute gvhd. however, early after transplantation (days - ), a proportion of patients may not convert to a full donor haemopoietic chimerism, particularly if anti-t lymphocyte globulin (atg) is used as gvhd prophylaxis (rambaldi a, et al.: the lancet oncology ) methods: we retrospective analyzed patients who underwent an allogeneic stem cell transplantation after fb conditioning regimen at our hospital, from november to august . the median age was years (range - ) and diagnoses were aml %, mds % cml % mfi %). the disease status at transplantation was: cr in %, cr in % and active disease in % of patients. the stem cell source was represented by pbsc in more than % of cases and anti-t lymphocyte globulin (atg) was part of the conditioning regimen in more than % of cases at a dose of mg/kg. the donor was a hla identical sibling ( %), a matched unrelated ( %) or mismatched (one allele or one antigen mismatched) unrelated, %. hematopoietic chimerism was molecularly evaluated by variable number of tandem repeats (vntr) on bone marrow (bm) mononuclear cells or peripheral blood (pb) t lymphocytes, purified by immunomagnetic positive selection (miltenyi, biotec). the analysis was performed at day , , , and after transplantation results: after , and days from transplantation, the proportion of patients with a full bm chimerism was %, % and %, respectively. at the same time points, the pb t cell chimerism was %, % and %. before day , patients required the infusion of dli to treat a pending or overt hematologic relapse and patients to convert the lymphoid chimerism from mixed (median %, range - ), to complete (successfully in cases). after day , additional patients required dli to treat disease relapse or progression and patients to improve the chimeric status or the immune reconstitution. at years, the overall survival is %, with a relapse and non-relapse mortality of % and %, respectively ( figure ). by uni and multivariable analysis, aml diagnosis and a mixed bm chimerism before day were associated with relapse and overall survival while age > was the only factor significantly associated with nrm. a mixed pb t-lymphoid chimerism before day does not adversely impact on non-relapse mortality, cumulative incidence of relapse, leukemia-free and overall survival. conclusions: after fb and atg, a progressive increase of pb lymphoid donor chimerism develops gradually after transplantation, in most of cases without the need of dli. early mixed lymphoid chimerism does not compromise the main long-term clinical outcomes and may at least partially explain the low non-relapse mortality. an incomplete bm chimerism within the first months strongly correlates with early disease progression or relapse. background: busulfan (bu) is widely used as a component of myeloablative conditioning regimen before hematopoietic stem cell transplantation (hsct) in children. bu has a narrow cumulative exposure window. the relation of bu exposure with toxicity is well established, but the link between the exposure and efs is not clear due to conflicting reports especially in pediatric patients. obtaining the ratio of bu to its metabolite i.e. metabolic ratio (mr) may serve as an indicator of bu gsh conjugating capacity of an individual, thus cumulative exposure of bu for a particular day that could be used along with auc as a marker to predict efs. the present investigation is aimed at evaluating the utility of bu mr to predict efs in children undergoing allogeneic hsct. methods: two different cohorts with children receiving bu in four times daily (qid, n= ) and once daily doses (qd, n= ) at st. justine's hospital, montreal were studied. bu and su levels were measured on day of the conditioning regimen at the end of infusion (dose in qid or dose in qd dosing). efs was defined from the time of transplant until death, relapse, or rejection, whichever occurred first. a receiver-operator characteristic curve (roc) for bu mrs measured was plotted to show the trade-off in sensitivity vs. -specificity rates for efs, as the cut-off of the test was shifted from low to high. cutoff values were defined based on the youden´s j statistic (i.e. sensitivity+specificty- ). results: twenty-two males and females aged from . to . years (mean±sd: . ± . ) from bu qid cohort had the mean mr of . (sd: . ). a cut off value of . in mr was chosen in roc analysis in this cohort, with better sensitivity ( %) and specificity ( %) for efs prediction (p= . , auc= . ( % ci= . - . ). in qd cohort nine females, and four males aged between . and . years ( . ± . ) had the mean mr of . (sd: . ). in roc analysis, a cut off value of . was chosen with better sensitivity ( %) and specificity ( %) for efs prediction (p= . ; auc= . ). conclusions background: treosulfan is an alkylating agent increasingly used prior to hematopoietic stem cell transplantation (hsct). the main objective of this study was to develop a population pharmacokinetic model of treosulfan in pediatric hsct recipients and to explore the effect of different covariates on treosulfan pharmacokinetics (pk). also, a limited sampling model (lsm) was developed. methods: in this multicentre study, patients, receiving a dose of , or g/m treosulfan a day, administered during consecutive days, were enrolled. a population pharmacokinetic model was developed using nonlinear mixed effect modelling (nonmem version . . , using psn toolkit . . and piraña version . . as modelling environment). demographic factors, as well as laboratory parameters, were included as covariates. results: treosulfan pk was best described by a twocompartment model. a bodyweight-based allometric model improved the model more than a model incorporating body surface area (bsa). clearance (cl) and intercompartmental clearance parameters were . l/h/ . kg ( %ci . - . ) and . l/h ( %ci . - . ). typical volumes of distribution of the central and peripheral compartments were . l/ . kg ( %ci . - . ) and . l ( %ci . - . ). a model-based dosing table based on bodyweight is created to achieve a target exposure of mg*hr/l (table ) , which was the median exposure of our population. estimated glomerular filtration rate (egfr) was shown to be the only parameter that significantly reduced interpatient variability in cl from . % to . %. a limited sampling model with samples (taken at . , and hours after start of infusion) accurately estimated pharmacokinetic parameters of treosulfan. conclusions: to the best of our knowledge, this is the largest cohort of pediatric patients treated with treosulfan used for a population pharmacokinetic study. we developed a two-compartment model with weight and egfr as covariates influencing treosulfan pk. recently we showed a relationship between treosulfan exposure and early toxicity. patients with an exposure > mg*hr/l have an increased risk of developing grade or higher mucositis and skin toxicity. another study in pediatric patients with thalassemia major reported an association between treosulfan clearance (< . l/h/m ) and poor overall survival. our model, together with the limited sampling strategy, can be used to adjust the dose, prior to or during treosulfan administration. ongoing studies conducted in different disease settings will determine if treosulfan exposure can influence patient outcome. subsequently, the optimal target exposure can then be established. background: autologous stem cell transplant (asct) is an effective treatment method for non-hodgkin lymphoma (nhl). until recently, carmustine, etoposide, cytarabine and melphalan (beam) was the most commonly used conditioning regimen. despite acceptable efficacy with beam, carmustine is associated with major pulmonary toxicity. for this reason, the aim of this study was to investigate the safety and efficacy of beb conditioning regimen for asct in nhl. methods: we conducted a prospective, multicenter, phase ii study for beb conditioning regimen for asct in nhl patients. a total of patients were enrolled from centers. they underwent asct with beb conditioning regimen (busulfan . mg/kg for days, etoposide mg/ m for days, bendamustine mg/m for days) between and . [[p image] . two year progression-free survival and overall survival.] results: the median age was years (range - ) and patients ( . %) were men. the most common type was diffuse large b cell lymphoma (n= , . %) and more than half of patients (n= , . %) were classified as ipi score or . eight patients ( . %) had a history of relapse and patients ( . %) received more than lines of chemotherapy before asct. most patients (n= , . %) were complete remission (cr) state at asct. a median number of . x /kg cd cells were infused (range . - . ). all patients engrafted after a median time of days (range - ). twelve patients ( . %) experienced neutropenic fever and patients ( . %) had grade toxicities during asct. however, no one had a documented infection, veno-occlusive disease, or treatment-related death. three months cr rate was . %. during a median follow-up period of . month, patients ( . %) exhibited relapse or progression, while patient ( . %) died of the disease. the estimated -year pfs and os rate were . % and . %, respectively ( figure ). conclusions: the beb conditioning regimens for asct is a feasible with tolerable toxicity in patients with nhl. disclosure: nothing to declare long-term report of total marrow or total lymphoid imrt in advanced leukemia, myeloma and lymphoma background: during the last three decades, total body irradiation (tbi) continues to play an important role in the conditioning regimens for patients undergoing stem-cell transplant (sct) for a wide variety of advanced hematological malignancies. however, tbi showed boundaries in dose limits for toxicity in allogenic and moreover in autologous stem cell transplantation. currently, the choice of conditioning regimen is based on the use of the least-toxic regimen to achieve the optimal therapeutic result. this report aims to assess the feasibility of a conditioning strategy based on high dose chemotherapy and whole-body radiotherapy focused on selective extensive tumor burden irradiation, both in allogeneic and autologous stem cell transplantation. methods: since december , sixty-two patients (pts) have been irradiated by helical tomotherapy (ht) to extensive target before allogeneic or autologous transplantation. selected total marrow irradiation (tmi) schedules were planned to treat patients with high risk acute leukemia (all or aml) or multiple myeloma (mm) as a part of conditioning regimen. total lymphoid irradiation (tli) was planned for patients with refractory or relapsed (r/r) hodgkin (hd) or non-hodgkin lymphomas (nhl). results: tmi and tli allowed delivering therapeutic dose over extensive selected targets with wide reduction of toxicity to all the organs at risk (oars). the higher radiation doses rate to the oars is reduced from % to %. allogenic conditioning regimen was tli ( gy x fx) than fludarabine + endoxan for patients with hd ( pts). tmi ( gy x fx) + fludarabine + melphalan for patients with mm ( pts). tmi ( gy x fx) + thiotepa + fludarabine + busulfan for advanced lam patients ( pts). tmi as the boost ( - gy) after conventional tbi was ( gy in bi-fractionated doses) by cyclophosphamide ( pts). autologous preparation to sct consisted of tli ( gyx fx) followed by high-dose bendamustine and melphalan for patients older than years and conventional feam (fotemustine, etoposide, cytarabine, and melphalan) for younger patients, in hd e nhl ( pts). while tmi ( gy x fx) plus melphalan was delivered for autologous sct in mm and lam ( pts). no unexpected acute toxicity was found. in the allogenic setting, all the patients' engraftment was achieved in all patients. no acute graft versus host disease increasing was detected. within the autologous setting, only % developed grade / mucositis. none experienced grade / extra-hematological toxicity. outcomes of the specific disease will be reported. conclusions: the current report describes the clinical feasibility of using ht to deliver tmi or tli in the setting of autologous transplantation or during allogenic stem cell conditioning regimen, to allow all patients (old, fragile or with high tumor burden) to achieve an ablative regimen before sct. to our knowledge, this single institution experience describes data from one of the largest cohort of patients treated in europe since the development of this irradiation techniques. disclosure induction therapy in both groups of patients was based in polychemotherapy without the use of new drugs. case matching was performed according to age, clinical stage at diagnosis, and response to induction therapy. conditioning regimen consisted of iv bu at a dose of . mg/ kg once a day on days - to - followed by mel at a dose of - mg/m on day - in the bumel group versus mel in the control group. maintenance therapy after transplant consisted of interferon and steroids in the majority of patients. results: the cut-off date for this update was june , . after a median follow-up of and months in the bumel and mel groups respectively, patients had relapsed in the bumel group and patients in the control group. median pfs was ( % ci, . - . ) months in the bumel and ( % ci, . - . ) months in the mel group (p = . ) ( figure ). in this update, patients in the bumel group are in maintained response and of them are in continuous cr (two with negative status for minimal residual disease) between and years after transplantation. ten-year os was not significantly different between both groups, being ( % ci - ) months in the bumel and ( % ci - ) months in the control group.transplant-related mortality was similar in both groups of patients ( % in the bumel and % in the mel group). regarding toxicity, bumel was associated with a higher incidence of mucositis and liver toxicity than the melphalan-only approach but no patient in our series developed sinusoidal occlusive syndrome and the hepatic toxicity observed was only grade i/ii. finally, no long-term side effects have been reported among bumel recipients. conclusions: this long-term follow-up analysis confirms that a therapeutic strategy including bumel as conditioning regimen beforeasct in patients with newly diagnosed mm is highly active and safe in these patients. [[p image] . figure . progression free survival in the bumel (____) and control group (…… frequency of acute gvhd grade iii-iv [cc: %; ct: %; tt: %, p= . ], and transplant-related mortality was higher in tt-carriers (cc: %; ct: %; tt: %, p= . cc&ct vs tt) . ta-tma, cmv infection/reactivation and cgvhd were also not different according to donor genotypes. fungal infections occurred more frequently as causes of death in carriers (cc: . % vs. ct: . % vs tt: . %, p= . ). conclusions: our results suggest that donor tgfb - c>t may exert an adverse influence on the outcome of myeloablative conditioning. our finding might be explained by the combination therapy of calcineurin and mtor inhibition in gvhd prophylaxis in myeloablative conditioning. disclosure: nothing to declare. treosulfan-based reduced intensity conditioning in hla-haploidentical transplantation using ptcy as gvhd prophylaxis in high-risk mds /aml of the elderly background: standard conditioning regimens prior to allogeneic hematopoietic stem cell transplantation (allo-hsct) are often associated with a considerable risk of severe adverse events, especially in elderly patients suffering from high-risk (hr) mds/aml. previous clinical studies have demonstrated feasibility of treosulfan-based reduced-intensity conditioning (ric) by stable engraftment, low non-relapse mortality (nrm), and favorable survival in elderly patients undergoing hla-matched related or unrelated allo-hsct (beelen et al, ash # ). however, data for treosulfan-based conditioning in the t-cell-replete hlahaploidentical (haplo-hsct) setting in high-risk aml/mds patients are rare. here we report on the outcome of eleven patients treated with a treosulfan-based conditioning undergoing haplo-hsct using exclusively post-transplantation cyclophosphamide (ptcy) as gvhd prophylaxis. methods: eleven patients with high-risk (hr) aml (n= )/mds (n= ) who underwent haplo-hsct using treosulfan for reduced intensity conditioning (ric) and ptcy as gvhd prophylaxis were retrospectively analyzed with respect to outcome and toxicity. all patients were > years old and transplanted between january and february at our institution. the majority of the patients ( / ) suffered from active disease at time of treatment initiation, only two patients presented in cr. all but one received sequential conditioning with cytoreductive chemotherapy using flamsa applied shortly prior to treosulfan-based ric ( g/m over days). a bone marrow graft was used in / patients. post-grafting immunosuppression consisted of cyclophosphamide, tacrolimus and mmf. national cancer institute common terminology criteria for adverse events version . were used for nonhematologic toxicity assessment starting from sequential therapy initiation or conditioning until day + . results: median age of the entire cohort was years (range: - ). the hct-ci was ≥ in eight pts (median hct-ci= , range: - ). no graft rejection occurred. neutrophil and platelet engraftment were achieved in % and % of the patients at a median of ( - ) and . ( - ) days, respectively. acute gvhd grade ii-iv occurred in % of the patients, exclusively involving the skin. no one developed severe (°iii-iv) acute gvhd. no patient died prior to haplo-hsct. severe nonhematologic regimen-related toxicities (°iii-iv) occurred in / patients, predominately affecting the gastrointestinal tract. no patient suffered from ≥two iii-iv°toxicities. all patients developed fever during treatment course, four with positive blood cultures. cmv reactivated in / patients at risk. no ebv reactivation or ptld occurred. six patients had clinical and radiological signs of pneumonia (probable invasive aspergillosis) without detection of aspergillus/antigen in the bronchoalveolar lavage. ci of nrm at day + was %. four patients relapsed within the first year after haplo-hsct, with two of them dying due to relapse. at last follow-up (dec ) / patients were alive. with a median follow-up of months ( - ) estimated -year os and dfs were % and %, respectively. conclusions: treosulfan-based unmanipulated hlahaploidentical allo-grafting using ptcy as gvhd prophylaxis in hr mds and aml patients aged over years is safe and well tolerated resulting in stable engraftment and a favorable toxicity profile. our preliminary data further show promising outcome with low nrm, no severe acute gvhd and favorable survival offering an attractive alternative in ric for haplo-hsct of the elderly. disclosure: nothing to declare comparison of outcomes of total body irradiation (tbi) vs non-tbi conditioning regimens in acute lymphoblastic leukemia for allogeneic transplantation background: in adult patients diagnosed acute lymphoblastic leukemia (all) long-term results are poor with intensive chemotherapy. allogeneic stem stem cell transplantation is the potential treatment that provides cure for these patients. myeloablative preparation regimens include total body irradiation (tbİ)+ cyclophosphamide(cy) and busulfan + cyclophosphamide.in adult all patients wbi/cy widely used, but the toxicity rate is higher. the aim of this study is to compare the result and effect of the tbi/cy and busulfan/cy regimens in allogenic bone marrow transplantation in all patients. methods: between - there were all patients who underwent transplantation using myeloablative preparation regimen with or without addition tbi in the adult bone marrow transplantation units of medipol medical faculty, istanbul university istanbul medical faculty, sisli florence nightingale hospital, atakent acıbadem hospital adult bone marrow units . we analyzed overall survival (os), progression free survival (pfs), veno occlusive disease, acute and chronic graft versus disease development rates in these patients. results: demographic characteristics of patients summarized in table - there was no significant difference between groups in donor age, gender, stem cell source. it was observed that the relapse rate was not statistically significant in both group.there was no statistically significant difference between the patients who underwent myeloablative regimen and myeloablative regimen with tbi in relaps,death, os, pfs. (figure- ) [[p image] . figure ] in terms of transplant complications there was also no respectable difference in development of vod and acute and chronic graft versus disease but vod was more common in the group that did not use tbi (p: . ) ( conclusions: although there are contradictory data in the literature, in our multicentre study, it was revealed that the addition of tbi in the myeloablative preparation regimen compared with myeloablative preparation regimen alone did not have a positive or negative effect on overall survival.we think that if we can prepare a good vod prophylaxis approches, we can give up tbi in future. disclosure (n= ) . for gvhd prophylaxis, cyclosporine a was given either alone (n= ), with mmf (n= ) or with methotrexate (n= ). the graft source was bone marrow (bm) in most cases (n= ), pbsc in seven cases, matched sibling cord +bm in two cases and one matched related cord. twentyfive of the donors were family donors and ten were unrelated. twenty-nine of the donors were / hla matched, six were / mismatched and one haploidentical. four patients had engraftment failure and required a second transplant, two of them were re-transplanted with cyclophosphamide and tbi, one with fludarabine, busulfan and campath, and one with no conditioning. thirty of the patients are alive ( %). four patients died of transplant complications and one died of metastatic squamous cell carcinoma. eight survivors are mixed chimeras ( %- % donor) and are all doing well, none of them developed any gvhd. nine patients developed acute gvhd, four of them with grade - . seven of these patients later developed chronic gvhd, two of them have extensive disease. conclusions: our results show a high survival rate of %, with a low rate of engraftment failure and reasonable rates of gvhd. only one of our patients died of late effects of hsct for fa. mixed chimerism does not seem to present a problem. we conclude that reduced intensity fludarabine based conditioning regimens are a good treatment option for patients with fanconi anemia undergoing hsct. disclosure: nothing to declare total marrow irradiation + bendamustine as reducedtoxicity myeloablative conditioning prior to allohsct for younger patients with multiple myeloma background: the prognosis of patients with multiple myeloma (mm) has improved markedly over the last two decades. despite that, allohsct remains the only treatment option with curative potential. however, its use is limited due to high incidence of non-relapse mortality (nrm) after myeloablative conditioning while insufficient efficacy of reduced-intensity regimens. we developed a new protocol characterized by reduced toxicity while preserved myeloablative potential, based on the use of total marrow irradiation (tmi) in combination with bendamustine. the aim of this study was to evaluate its safety and efficacy in a singlecenter experience. methods: between years - , mm patients below years old were offered tandem auto-allohsct as part of first-line therapy. the decision was based on individual patient preferences after detailed description of potential risks. autohsct was preceded by melphalan mg/m iv. the conditioning prior to allohsct consisted of tmi performed using helical tomotherapy at the dose of gy/d on days - , - , - (total gy) and bendamustine - mg/m /d iv. on days - , - (total - mg/m ). the immunosuppressive therapy consisted of cyclosporine + methotrexate +/-atg. peripheral blood was used as a source of stem cells. results: the analysis included patients (women - , men - ). the median follow-up was ( - ) months. the median age at allohsct was ( - ) years. the disease stage before allohsct was as follows: cr- , vgpr- , pr- . patients were treated with hsct from either hlamatched siblings (n= ) or unrelated donors (n= ). the interval between autohsct and allohsct was ( - ) months. all patients engrafted after allohsct with median time of neutrophil and platelet recovery of and days, respectively. one patient ( %) experienced grade acute gvhd, while there were no cases of grade - acute gvhd. the incidence of mild, moderate and severe chronic gvhd was %, % and %, respectively. the rate of grade non-hematological toxicities was %. one patient died of late bacterial infection. the incidence of trm was %. grade adverse events were not reported. disease status months after allohsct was: cr- , vgpr- , pr- . the probability of os and pfs after months was % (+/- %) and % (+/- %), respectively. the incidence of progression and trm was % and %, respectively. conclusions: allohsct using tmi gy + bendamustine conditioning protocol is characterized by good tolerance and low risk of gvhd. it may be used for younger patients with mm as part of tandem auto-allohsct strategy. encouraging results reported in this study should be confirmed in prospective clinical trials. disclosure: nothing to declare p comparison between two reduced intensity conditioning regimens in patients with a myeloid malignancy: a single center experience comparing fb with flumel background: hematopoietic stem cell transplantation (hsct) remains the only curative option for high-risk myeloid neoplasms. the optimal reduced-intensity conditioning (ric) is still debated. methods: a single-center retrospective analysis was conducted at our institution to compare two different ric regimens in adult patients transplanted for myeloid malignancy from to . a total of patients were analysed, of them treated with busulfan-based (fludarabine mg/m , busulfan . mg/kg, fb ) and with melphalan-based conditioning regimen (fludarabine mg/m ,melphalan mg/m , flumel). antithymocyte globulin (atg) was administered in all patients while no one received tbi. partial in vitro t-cell depletion was performed using alemtuzumab for low risk patients. results: the two groups were well balanced with a median age of and years in the fb and flumel group, respectively, and a median follow up of months. the most frequent indication for transplant in both groups was aml ( . and . % for fb group and flumel group, respectively) and the stem cell source was peripheral blood in . and . % of patients. more patients in the first group had near to significant worst karnosfky status (< ) at transplant compared to second ( . vs %, p= . ) and more patients received a t-partial depleted graft ( . vs . %, p= . ). the neutrophil engraftment was significantly shorter after flumel ( vs days, p < . ). the -year overall survival (os) and disease-free survival (dfs) were of . and . %, respectively, after fb and . and . % after flumel, respectively, and were not significantly different (p=. for os and . for dfs), with a karnofsky >= being the only factor significantly associated in univariate analysis with better os and dfs (p=. for both). the cumulative incidence (ci) of grade to acute graft-versus-host disease (agvhd) was . % after fb and . % after flumel (p< . ) and was associated in multivariate analysis with both t depletion and ric type (p< . and . , respectively). the ci of chronic gvhd at years was . % in fb and . % in flumel group (p=. ) . the ci of non-relapse mortality at years was . % after fb and . % after flumel (p=. ). the ci of relapse at years was . % for the first and . % for the second group (p< . ) and was associated with conditioning regimen in multivariate analysis (p=. ). no difference in -years gvhd-free/ relapse-free survival (grfs) was observed between the two group ( . % for fb and . % for flumel, p=. ). conclusions: when comparing two ric regimens for myeloid neoplasms, we observed a higher incidence of agvhd after flumel whereas no statistical difference was noted for the cgvhd occurrence. while the toxicity appears to be higher after flumel, this result is counterbalanced by a higher proportion of relapse after fb , accounting for no difference in os, dfs and grfs between the two groups. these findings could be partially explained by a larger proportion of patients receiving a partial t-depletion after fb ric, but a larger trial is needed to clarify this issue. disclosure: nothing to declare. once-daily vs -times daily intravenous busilvex in conditioning regimen before allogeneic stem cell transplantation for patients with myeloid malignancies: safety and efficacy background: busilvex (bu) is part of standard conditioning regimen before allogeneic stem cell transplantation (asct) for patients with myeloid malignancies and usually administered as an intravenous (iv) infusion -times daily. this study aimed to compare the saftey and efficacy of this schedule to a once-daily iv bu. we conducted a retrospective study in adult patients (≥ years) with myeloid malignancies who received asct from hla-identical sibling donors between january and june following iv bu-based preparative regimens. graft-versus host disease (gvhd) prophylaxis consited of cyclosporine and short course of methotrexate. intravenous bu was administered -times daily ( . mg/kg every hours x to doses) or oncedaily in a -hour infusion ( . mg/kg x to days) since june . results: ninty-nine patients were enrolled ( men and women). median age was years (range, - y). the median time from diagnosis to asct was months (range, days - years). diagnosis were acute myeloid leukemia (n= , %), chronic myeloid leukemia (n= , %), myelodysplasic syndrome (n= , %), primitive myelofibrosis (n= , %) and chronic myelomonocytic leukemia (n= , %). thirty-seven ( . %) patients had ebmt-score ≥ . sixty-five ( . %) patients were transplanted in cr , ( %) beyond cr and ( . %) had active disease. conditioning regimens consisted of bu/cyclophosphamide in patients ( . %), bu/fludarabine in patients ( . %). four-times daily bu was given to patients ( . %, groupe ) and once-daily bu to patients ( . %, groupe ). stem cell source were bm in patients ( . %) and pbsc in patients ( . %). globally, patients characteristics were well balanced between the two groups. the rates of severe complications were similar between the two groups with no statistically significant differences except oral mucositis (table ). non-relapse mortality (nrm) was comparable in the two groups ( % and % in groups and , respectively, p= . ). the relapse rate was % and %, respectively (p= . ). after a median follow-up of years (range, days - years), the os was not significantly different between groups and : % vs % (p= . ). however, the rfs was significantly better in the groupe : % vs % (p= . ). conclusions: once-daily iv bu regimen seems to be an efficient and safe alternative to the -times daily protocol. however, results should be interpreted with caution because the historical comparison and lack of bu pharmacokinetics studies. disclosure background: standard therapy of the most patients with juvenile myelomonocytic leukemia (jmml) is allogeneic hematopoietic stem cell transplantation (ahsct). the choice of optimal conditioning regimen for patients with jmml is crucial as well as long-term observation. we aimed to estimate the long-term follow-up and survival rates of patients with jmml after ahsct with the help of busulfan or treosulfan-based conditioning regimens. methods: thirty eight patients with jmml underwent ahsct in - . we compared equal groups of patients received busulfan (n= ) and treosulfan-based (n= ) conditioning regimen. m:f= : . median of age at hsct was . ( . - ). donor type: hla-related / - % (n= ), hla-related / - . % (n= ), hlaunrelated / - . % (n= ), hla-unrelated / - . % (n= ), and haploidentical - . % (n= ). stem cell source: bm - . % (n= ), pbsc - . % (n= ), ucb - , % (n= ), and ucb+bm - . % (n= ) . disease status on hsct: cr - . % (n= ), refractory - . % (n= ). results: median follow-up . months ( - months) . the estimated -year overall survival (os) probability in patients received busulfan-based conditioning was , ± , % in comparison with , ± , % in patients with treosulfan-based regimen (р= , ). event-free survival (efs) was , ± , % in group with busulfan-based regimen and , ± , % in patients with treosulfanbased conditioning (р= , ). background: post-transplant relapse remains the leading cause of treatment failure in high risk (hr) acute myeloid leukemia (aml), myelodysplastic syndrome (mds), myeloproliferative neoplasia (mpns) receiving allogeneic hematopoietic cell transplantation (allo-hct), especially for patients with relapsed or refractory aml. recently, a sequential transplant approach, as developed by the munich group, comprising of intensive cytoreductive chemotherapy flamsa (fludarabine/amsacrine/cytarabine) to decrease leukemia cell burden shortly prior to conditioning regimen, has been successfully used for high-risk (hr) aml/mds with promising results. methods: we studied patients (median age years, range - ) with hr aml (n= ), as defined by refractory, relapsed disease, secondary leukemia, or high/ very risk disease risk index risk, and hr mds (n= ) according to ipss-r, undergoing allo-hct using the sequential transplant approach in institutions between january and october . the sequential transplant approach combined a cytoreductive chemotherapy, which consisted of either flamsa (n= ), flag +/-ida (fludarabine/cytarabine/granulocyte colony stimulating factor /idarubicin) (n= ), or clo-arac (clofarabine/cytarabine) (n= ), followed by reduced (ric) (n= ) or myeloablative (mac) (n= ) conditioning regimen. all patients received peripheral blood stem cell from matched related donors (n= ) matched unrelated donors (n= ), or mismatched unrelated donors (n= ). post-grafting immunosuppression consisted of calcineurin inhibitor and mycophenolate mofetil in all patients. thymoglobulin was added for gvhd prophylaxis for unrelated donor transplant. results: the median time to neutrophil > /μl was days (range, - ) . with a median follow-up of . months (range, . to . months), the kaplan-meier estimate of leukemia-free (lfs) and overall survival (os) at years were % ( % ci, - ), % ( % ci, - ), respectively. patients receiving flag or clo-arac based sequential regimen showed a trend towards more favourable overall survival (os) as compared to patients given flamsa ( year os: % vs %; p= . ). at years, the cumulative incidences of relapse and non-relapse mortality (nrm) were % ( % ci, - %) and % ( % ci, - %), respectively. in multivariate analysis, the type of sequential conditioning regimen did not show any significant impact on lfs, os, nrm or relapse. conclusions: sequential transplant conditioning with flamsa, flag or clo-arac followed by allo-hct is an effective strategy in overcoming the dismal prognosis of hr aml and mds, and enabling long-term disease free survival. more studies on effective strategies such as posttransplant maintenance therapy of prophylactic donor lymphocyte infusion, are needed to further eliminate the risk of relapse, without increasing risk of treatment related toxicity. disclosure: nothing to declare optimization of the blood sampling procedure for busulfan therapeutic drug monitoring (tdm) to optimize our sampling scheme ( minutes, , , , , , and hours after the end of a -hour infusion), we reduced the number of blood samples collected, reducing nursing and laboratory staff time and increasing patient convenience. this study aims to show the performance of a simplified sampling protocol which includes the first samples from the original protocol. methods: individual pk parameters were retrospectively estimated using samples (simplified protocol) and were compared with those obtained after samples (original protocol). individual pk parameter values for a one compartment model were estimated using a maximum likelihood estimation modelling algorithm (adapt . ) and the statistical analysis of the results was performed (statgraphics centurion xv). results based on the approved dosage recommendations, mean (sd) initial dose was . ( . ) mg. after tdm, mean (sd) calculated dose at day for the remaining days (to achieve the defined target cumulative auc) was , ( , ) mg obtained from the original protocol. according to the simplified protocol the result would be , ( , ) mg. the median and the mean variation of the calculated dose were % and % ( - %) between protocols. a strong relationship between the cl of the day - obtained from the original protocol and the simplified protocol is observed (r = . ). this high correlation is also observed for patients with busulfan t / > h (r = . ), a population were the reduction of sampling could be more problematic. anova test for the log cl with the factors: patient, day of busulfan and type of sampling protocol was performed. sampling protocol was determined as non-statistically significant (p = . ). conclusions: results suggest that both protocols are equivalent concerning to the busulfan cl estimation and calculated auc. variation between protocols regarding the calculated dose at day for the remaining days to achieve the defined target cumulative auc is considered acceptable. we verified a strong relationship between busulfan cl obtained from both protocols and sampling protocol doesn't influence cl statistically. a reduced sampling collection of determinations until h after the end of the infusion is shown to be sufficient for the tdm of busulfan, so this was implemented in our centre in line with published data. disclosure: nothing to declare p impact of anti-thymocyte globulin doses in unrelated hematopoietic stem cell transplantation for patients with myeloid neoplasm background: anti-thymocyte globulin (atg) is widely used for the prophylaxis of graft-versus-host disease (gvhd) in hematopoietic stem cell transplantation (hsct). however, there is still controversy regarding the optimal dose of atg. therefore, we analyzed the impact of atg doses in unrelated hsct for patients with myeloid neoplasm. methods: this was a retrospective multi-center study that assessed the impact of atg doses on clinical outcomes in patients with acute myeloid leukemia (aml) or myelodysplastic syndrome (mds) undergoing an unrelated hsct. the patients who received peripheral blood stem cells (pbsc) transplantation after conditioning regimens containing i.v. busulfan (bu), fludarabine and rabbit atg between and were included in this study. results: a total of patents, median age years, with aml (n= ) or mds (n= ) were included in our analyses. patients ( %) received a myeloablative regimen (i.v. bu> . mg/kg). high-atg (atg mg/kg), intermediate-atg (atg . - mg/kg) and low-atg (atg mg/kg) were given in , and patients, respectively. after a median follow-up of months, the cumulative incidence of extensive chronic gvhd was . % in the high-atg group, . % in the intermediate-atg group and . % in the low-atg group (p= . ). conclusions: our study shows that the incidence of extensive chronic gvhd was similar regardless of the doses of atg after transplantation of pbsc from unrelated donor for patients with aml or mds. however, the rate of relapsefree survival and the rate of a composite end point chronic gvhd-free and relapse-free survival were significantly higher in the intermediate dose ( methods: we retrospectively retrieved data from the electronic medical records for consecutive patients aged and older, who underwent an asct for lymphoma over the last years at our institution. results: forty four patients ≥ years old underwent asct between aug and aug . twenty eight of them received a reduced-dose conditioning (median %, range %- % dose reduction). the dose was reduced for % of patients ≥ years old and for % of patients aged - . the outcomes of the following three groups of patients were compared: a) age ≥ ; without dose reduction, b) age - ; with dose reduction and c) age ≥ ; with dose reduction (table ). only one patient aged received full-dose conditioning. there was no significant difference between the groups in the number of previous chemotherapy cycles (median , range - ). however, significantly more patients at the age of - were in complete remission (cr) pre-transplant in both full and reduced-dose conditioning groups (a and b). no significant intergroup differences were observed in the occurrence of complications (mucositis and infections), day transplantrelated mortality (trm) or engraftment day. similarly, no significant differences were found either in the -year progression-free survival (pfs), which was %, % and %, or -year non-relapse mortality (nrm), which was %, % and %, respectively for groups a, b and c. the -year overall survival (os) tended to be higher in group b ( %), compared to groups a ( %) and c ( %). conclusions: beam/beac conditioning dose reduction was not found to adversely affect -year pfs and os rates. despite the fact that / of the patients in the age group ≥ underwent asct in partial remission and had dose reduction, theier achieved trm, pfs and os rates were similar to those of patients aged - . beam/beac conditioning at a %dose may be a suitable option for patients in their seventh decade requiring asct. this strategy should be further evaluated in prospective clinical trials. background: the transplant related mortality in autologous transplants for lymphoma and multiple myeloma, reported worldwide ranges from - %. from - , the trm at our center for these two diseases was approximately %. we introduced changes in mobilization schedule, conditioning regimens and drug dosages to determine whether these changes affected the transplant related mortality and overall survival. methods: from april -december , we used beam (bcnu: mg/m on day - ; etoposide mg/ m on days - to - , cytarabine mg/m on days - to - and melphalan mg/m on day - as conditioning chemotherapy for patients admitted in transplant unit for autologous transplants in hodgkin's and non-hodgkin's lymphoma. in patients with multiple myeloma high dose melphalan ( mg/m ) was used. the mobilization protocol consisted of cyclophosphamide . gm/m followed by gcsf μgm/kg twice daily till stem cell collection was completed. from january , we changed the beam protocol to bendaeam with dose modifications that included: bendamustine mg/m on days - and - , cytarabine mg/m on days - to - , etoposide mg/m on days - to - and melphalan mg/m on day - . for multiple myeloma melphalan was reduced to mg/m . we used only gcsf for mobilization of stem cells, which was continued till stem cell harvest was complete. response to treatment was evaluated by comparing trm and overall survival for two time periods: - and from till date. results: from april till december , n= autologous transplants were performed. the male:female ratio was . : . fifty seven patients underwent transplant for lymphomas, n= for multiple myeloma and n= for other diagnosis. median age was ± . ( - years). the mean mnc was . × ± . /kg. engraftment was achieved in % of patients. the transplant related mortality was . % and overall survival was % (follow up: months). since january till march we have performed n= autologous transplants of which n= were males. fifteen transplants were performed for lymphomas (nhl: , hd: ) and n= for multiple myeloma. median age was ± ( - years). the mean mononuclear cell count was . x /kg and the mean cd count was . x /kg. engraftment was achieved in all patients. the transplant related mortality was % and the overall survival was % (follow up months). conclusions: we were able to reduce the autologous transplant related mortality to % by decreasing dosages of conditioning chemotherapy and changing the mobilization protocol. long follow-up is needed to determine late mortality and late relapse in comparison to standard chemotherapy dosages disclosure: nothing to declare risk and benefit of thiotepa based conditioning followed by autologous stem cell transplantation in high risk lymphomas years ( - ) . stage (ann-arbor) at diagnosis of hd/dlbcl/pcnsl: stage ie n= / / ( / / %), stage ii n= / / ( / / %), stage iii n= / / ( / / %), stage iv n= / / ( / / %). median time from diagnosis to asct hd/dlbcl/pcnsl: / / month ( - ). induction treatment in hd patients was abvd, in most dlbcl patients r-chop and in pcnsl patients high dose methotrexate and cytosin arabinoside. tumor status at asct hd/dlbcl/pcnsl: complete metabolic remission (cmr) n= / / ( / / %) and from pcnsl patient's n= ( %) were in first complete remission (cr ). type of stem cell graft was periferial blood stem cell in all case. conditioning: thiotepa ( mg/ m on days - to - , busulphan , mg/kg on days - to - and cyclophosphamide mg/kg on days - to - plus rituximab mg/m on day - in dlbcl and pcnsl. median follow up from asct days . tumor stage at asct was defined with computer tomography with positron emission tomography (pet-ct). results: median time of engraftment was days ( - ). thiotepa caused toxicoderma appeared at ( %) patients. cytomegalovirus (cmv) reactivation was seen in ( %) cases with low dna content ( , , copies/ml) and responded completely to oral valgancyclovir therapy. transplantation related mortality hd/dlbcl/pcnsl n= / / ( / / %), in cases bacterial sepsis and one systemic mycoses and one pulmonary fibrosis. incidence of long-lasting grade iii-iv thrombocytopenia and anaemia: n= ( , %) and n= ( %), median time of duration from transplantation days ( - ) and days ( background: this study evaluated the efficacy and toxicity of intravenous busulfan and thiotepa as a conditioning regimen for autologous stem cell transplantation (asct) in patients with multiple myeloma (mm). methods: we retrospectively analyzed the data of patients with mm who received the intravenous busulfan and thiotepa conditioning for asct between november and april in korea. results: the median time to transplant was . months, and patients ( . %) underwent asct within months of the diagnosis. the overall response rate after asct was . %, including . % with complete response, . % with very good partial response, and . % with partial response. the most common severe non-hematologic toxicity (grade - ) was infection ( . %). three patients ( . %) developed venous-occlusive disease. one patient ( . %) died due to severe pneumonia after asct. after a median follow-up of . months, the median progression-free survival (pfs) and overall survival (os) were not reached. conclusions: in conclusion, a conditioning regimen of intravenous busulfan and thiotepa was effective and tolerable. clinical trial registry: not applicable disclosure: the authors have declared no conflicts of interest. myeloablative haploidentical bone marrow transplantation with post-transplant cyclophosphamide in paediatric patients with haematological malignancies santanu sen , sameer tulpule background: haploidentical transplants have been shown to be safe and effective in treating haematological malignancies in the paediatric population. we have previously reported on our experience of using reduced intensity conditioning with post transplant cyclophosphamide in haploidentical patients. we herein report our experience of using a tbi based myeloablative conditioning to treat our first patients with haematological malignancies. methods: patients were enrolled in the study, with relapsed acute lymphoblastic leukemia (all) and with relapsed/resistant acute myeloid leukemia (aml). all aml patients had genetic markers of high risk disease and all all patients had very early relapses (either on therapy or within months of stopping therapy). all patients were conditioned with an identical protocol using tbi-based myeloablative preparative regimen (fludarabine mg/m /d × d and tbi cgy bid on d − to − [total dose cgy]) followed by an infusion of unmanipulated peripheral blood stem cells from a haploidentical family donor. postgraft immunosuppression consisted of cyclophosphamide mg/kg/day on days and , mycophenolate mofetil through day , and tacrolimus through day . results: median time of neutrophil and platelet engraftment was and days, respectively. all patients achieved sustained complete donor chimerism by day + . acute gvhd, grades ii-iv and iii-iv, was seen in % and %, respectively. disease progression occurred in patients: & months after transplant and there was one death due to severe fungal infection. estimated twoyear survival and relapse were % and %, respectively. patients had severe bk viremia and cmv reactivation occurred in patients. all patients were successfully managed with appropriate supportive and antiviral therapy. conclusions: we report good outcome with a myeloablative conditioning in haploidentical transplants with excellent engraftment and hopefully a longer life expectancy. with small number of patients, it is difficult to state whether using a myeloablative conditioning would lead to better long term outcomes in this cohort of patients with very haematological malignancies, but we certainly showed that it is possible to achieve excellent early results. disclosure: nothing to declare fludarabine in combination with melphalan and atg can be the best conditioning for hematopoietic stem cell transplant of children with hemophagocytic lymphohistiocytosis methods: in this prospective study, we analyzed the outcome of two pediatric patients with hlh who had received hsct, using reduced-intensity conditioning (ric) regimen. they received the same ric regimen based on the use of fludarabine ( mg/m /day for days) in combination with melphalan ( mg/m /day for days) and horse antithymocyte globulin (atg mg/kg/d for days). cyclosporine and methotrexate were used as graft-vs.-host disease (gvhd) prophylaxis. results: a months boy with primary hlh (fhl ) was transplanted from his mother and a years girl with secondary hlh was transplanted from her brother. both of donors were hla match with their recipients. they were received x /kg and x /kg cd + cells from the harvested peripheral blood stem cells, respectively. they achieved full neutrophil and platelet recovery. the time to neutrophil recovery was and days, respectively. full chimerism was achieved for both of them. in addition, they was developed grade and of acute gvhd, respectively. gvhd was completely controlled with prednisolone. they are alive and in complete remission without any significant complications after and months, respectively. conclusions: it appears that fludarabine in combination with melphalan and atg may be the best conditioning regimen for hematopoietic stem cell transplant of children with hlh. due to a few number cases of this study, a study with sufficient sample size is required. disclosure background: hematopoietic cell transplant (hct) recipients often report depression and impaired quality of life (qol) before transplant. mixed evidence suggests depression may be a risk factor for greater mortality and worse qol. inconsistent findings may be due to the fact that previous studies have not evaluated antidepressant use. the aim of the study was to compare pre-transplant patientreported physical functioning and post-transplant overall survival (os) between four groups of hct recipients: ) non-depressed/taking antidepressant (treated depression), ) depressed/taking antidepressant (undertreated depression), ) depressed/not taking an antidepressant (untreated depression), and ) not depressed/not taking an antidepressant (control). it was hypothesized that physical functioning and os would be worse among patients with untreated and undertreated depression relative to those with treated depression and controls. methods: this retrospective case-control study included patients completing depression (phq- ) and quality of life (sf- ) questionnaires at pre-transplant. analyses were conducted separately for allogeneic and autologous recipients. results: participants (n= , ) were % men, mean age years ( - ), % allogeneic recipients. regarding depression and antidepressant use, ( %) allogeneic patients were characterized as having treated depression, ( %) as untreated depression, ( %) as undertreated depression, and ( %) as controls. hierarchical linear regression models indicated that after adjusting for significant univariate factors (performance status, disease status, and regimen intensity), allogeneic patients with treated depression (b=- . , % ci=- . , - . ) reported better physical functioning than patients with undertreated depression (b=- . , % ci=- . , - . ) and untreated depression ) but worse physical functioning than controls (p values < . ). cox regression models indicated depression/antidepressant usage was not associated with os among allogeneic patients (p values> . ).among autologous patients, ( . %) were characterized as having treated depression, ( . %) as untreated depression, ( . %) as undertreated depression, and ( . %) as controls. hierarchical linear regression models indicated that after controlling for significant univariate factors (gender, performance status, diagnosis, and disease status), autologous patients with treated depression (b=- . , % ci=- . , - . ) reported better physical functioning than patients with undertreated depression (b=- . , % ci=- . , - . ) and untreated depression (b=- . , % ci= . , - . ), but worse physical functioning than controls (p values < . ). cox regression models showed depression/antidepressant usage was associated with os (p values < . ), with patients with treated depression demonstrating significantly worse os than other groups (p= . ), but this association was no longer significant in multivariate analyses controlling for diagnosis and disease status (p= . ). conclusions: patients with untreated or undertreated depression pre-transplant may benefit from depression screening and treatment to improve physical functioning. disclosure: hslj: consultant for redhill biopharma and janssen scientific affairs p eltrombopag (epag) induces a high percentage of responses in patients with post allo-hsct poor graft function (pgf) and no active gvhd lourdes aguirre , aitziber lizardi , pilar bachiller , brigida esteban , carmen gonzález , nagore argoitia , maría araiz , aranzazu aguirre , anunciación urquía , carlos vallejo background: persistent cytopenia is a life-threating complication after hsct. several causes can lead to this situation (viruses, gvhd, drugs, etc) . a specific entity is the one called "poor graft function (pgf)", which is diagnosed in pts with ≥ cytopenias after day + , in the presence of donor chimerism and the absence of gvhd or relapse. pgf is more frequent after alternative allo-hscts, such a haplo-identical, mismatched, or ucb. several therapeutic approaches for pgf, with poor results, have been tested. recently, epag has been shown to improve platelet counts in the post-allo-hsct setting. in this study, we analysed the efficacy of epag in pts with post-transplant persistent cytopenias. methods: the population analyzed includes all pts who underwent allo-hsct from june through may in our unit. median age was years ( - ). were male ( . %) and female ( . %). baseline diseases were: aml, lpd, all, mds, mpd, mm, and bmf. donor was unrelated in ( . %) and was family in ( . %) (including haplo-identical). conditioning was ric in ( . %) and intensive in ( . %). sc source was pb in ( . %) and bm in ( . %). median followup was months ( - ). epag was initiated at some point during the first -month post-hsct period in pts ( . % of the series) due to thrombocytopenia (< /mcl) plus, at least, one other cytopenia. patients characteristics shown in table . epag was started at mg/day and escalated each weeks to , and mg/day if platelet count was < /mcl. global response was considered when, after epag, the patient needed no transfusions and reached the three of the following: platelets > /mcl, hgb > g/dl, and anc > /mcl. epag was tapered off in responders and discontinued if no response was reached after weeks. results: at epag initiation, all the pts had thrombocytopenia (< /mcl), had anemia (hgb < g/dl), and had neutropenia (anc < /mcl). counts pre and post and response to epag are shown in table . among the responders, all but one (who relapsed from thrombocytopenia and died from bleeding) were alive at analysis close ( . %). among the non-responders, three pts had gvhd-associated cytopenias, and finally died from infectious complications; the other patient relapsed from her aml, reached a new cr after treatment, and is alive and well months afterwards. epag was tapered off and discontinued in / pts who responded; / responders are still on epag. epag was discontinued in the / pts who did not respond. rest of treatment details shown in table . conclusions: ) epag worked striking well in subjects with pgf, an otherwise a life-threatening situation for patients. ) epag induced impressive responses in platelets, but strong bilinear and trilinear responses were also seen. ) epag did not improve gvhd-associated cytopenias. ) to confirm these innovative and transcendent results, we have just initiated a multicenter prospective study on the role of epag for treatment of post-hsct pgf. * five out of the six urdt were mismatched ** the donor was a woman in the six cases: three sisters and three daughters background: hepatic vod/sos with multi-organ dysfunction (mod; typically, renal or pulmonary) may be associated with > % mortality. defibrotide is approved for treating severe hepatic vod/sos post-hsct in patients aged > month in the eu, and for hepatic vod/sos with renal or pulmonary dysfunction post-hsct in the us. this analysis provides an overview of the safety results from studies of patients with vod/sos, with or without mod, who received defibrotide mg/kg/day. methods: safety data were pooled from patients with vod/sos post-hsct treated with defibrotide in a phase trial (n= ) and a phase , randomized dose-finding trial (n= receiving mg/kg/day). safety data for historical controls (hc) from the phase study (n= ) also are provided. reported separately, due to differences in patient population and data monitoring protocol, are aes from the expanded-access program (t-ind) in patients with vod/ sos with and without mod (n= post-hsct). vod/ sos was diagnosed by baltimore criteria/biopsy for the phase / studies; diagnosis by baltimore or modified seattle criteria was permitted in the t-ind. results: median patient age at hsct for the phase / studies was . years, . years for the hc, and . years for the t-ind. in the phase / studies defibrotide-treated group (n= ), ( . %) experienced aes; most common (> %) were hypotension ( . %), diarrhea ( . %), and multi-organ failure ( . %). treatment-related aes were at least possibly related to defibrotide (table) . any hemorrhage (an ae of special interest) occurred in patients ( . %); most commonly epistaxis ( . %), gastrointestinal and pulmonary alveolar hemorrhage and hematuria ( . % each), and conjunctival hemorrhage ( . %). all hc experienced an ae; most common (> %) were hypotension ( . %), tachycardia ( . %), diarrhea ( . %), nausea ( . %), and pyrexia, agitation, and petechiae ( . % each). any hemorrhage occurred in patients ( . %): most common (> %) were petechiae ( . %); hematuria, epistaxis, and pulmonary alveolar hemorrhage ( . % each); and lip hemorrhage ( . %). in the t-ind (n= ), / patients with mod ( . %) and / patients without mod ( . %) had an ae; other than vod/sos and mod, most commonly (> % in either subgroup) hypotension ( . % and . %, respectively). traes occurred in patients ( . %) ( table) . any treatment-emergent hemorrhage occurred in patients with mod ( . %) and patients without mod ( . %); most commonly (> % in either subgroup) pulmonary hemorrhage ( . % and . %, respectively) and gastrointestinal hemorrhage ( . % and . %, respectively). conclusions: the incidence and type of aes were as expected in these critically ill patients. of the pooled patients, % had aes; . % had a hemorrhage. all hcs had an ae, with . % having a hemorrhage. in the t-ind, patients with mod had higher rates of aes. support: jazz pharmaceuticals event, n(%) phase / studies (n= ) disclosure: paul g. richardson has served on advisory committees and as a consultant, and has received research funding from jazz pharmaceuticals. angela r. smith and leslie lehmann have nothing to disclose. nancy a. kernan received grants from gentium during the conduct of the study, and her research was supported by the national cancer institute of the national institutes of health under award number p ca ; the content is solely the responsibility of the author and does not necessarily represent the official views of the national institutes of health. she has a research grant from jazz pharmaceuticals. robert ryan and william tappe are employees of jazz pharmaceuticals and hold stock and/or stock options in jazz pharmaceuticals plc. stephan a. grupp has served on a steering committee and as a consultant to jazz pharmaceuticals. defibrotide for treatment of adults with hepatic vod/ sos with or without multiorgan failure after hematopoietic cell transplantation: results of a systematic review/meta-analysis background: although hematopoietic cell transplantation (hct), autologous or allogeneic, is potentially curable in various hematologic malignancies, the procedure is associated with serious and potentially life-threatening complications, among them veno-occlusive disease/sinusoidal obstructive syndrome (vod/sos) of the liver. several studies, prospective or retrospective, have reported outcomes of defibrotide, when used as prophylaxis or treatment, in a mixed population of adult and pediatric patients. in this systematic review/meta-analysis, we analyze outcomes of defibrotide when specifically used for treatment of adult patients with hepatic vod/sos with or without multiorgan failure. methods: a comprehensive search of large databases (medline/pubmed, cochrane and embase) on november , identified publications. analysis was restricted only to adult patients (defined as median age older than years) who received defibrotide for treatment of vod/sos and were reported in prospective or retrospective (which included ≥ patients) studies published in full manuscript form. there were no limitations based on language. data were extracted in relation to benefits [complete remission (cr) rate and overall survival (os)] and harms (hemorrhage, any site or organ-specific). a total of studies (prospective= ; retrospective= ) with patients met inclusion criteria. results: the median year of publication of prospective studies was ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) and for retrospective ones ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) . the prescribed starting dose of defibrotide varied among studies ranging from . mg/kg/day to mg/kg/day, mostly for a -day course. the pooled cr rate was % ( %ci= - %) for prospective and % ( %ci= - %) for retrospective studies. the pooled day + os rates were % ( %ci= - %) and % ( % ci= - %) for prospective and retrospective studies, respectively. the pooled rates of hemorrhage (any site) were % ( %ci= - %) for prospective and % ( % ci= - %) for retrospective studies. when analyzing organ-specific hemorrhage, prospective studies (n= patients) reported pooled rates of pulmonary alveolar (pa) hemorrhage of % ( %ci= - %) and of % ( %ci= - %) for gastrointestinal (gi) hemorrhage. only one retrospective study (n= patients) reported an incidence of pa hemorrhage of % ( %ci= - %) and a different study (n= patients) reported an incidence of gi hemorrhage of % ( %ci= - %). none of the studies reported cerebral hemorrhage as a complication of defibrotide therapy. conclusions: this systematic review/meta-analysis confirms the efficacy of defibrotide for treatment of vod/sos with or without multiorgan failure, yielding cr rates of - % and day + os rates of - %. the purportedly higher pooled cr and os rates observed with retrospective (vs. prospective) studies are likely due to assignment-bias inherent to observational studies. moreover, although the pooled hemorrhage (any site) rates of - % is considered proportionally significant, the pooled rates of pa and gi hemorrhage were ≤ %, in prospective studies. clinical trial registry: not applicable disclosure: m.a.k-d: consultancy for pharmacyclics m.m: received lectures honoraria and research support from jazz pharma efficacy and safety of defibrotide in the treatment of hepatic veno-occlusive disease/sinusoidal obstruction syndrome following hematopoietic stem cell transplantation: interim results from the defifrance study background: hepatic veno-occlusive disease/sinusoidal obstruction syndrome (vod/sos) is a potentially lifethreatening complication of conditioning for hematopoietic stem cell transplant (hsct) but may occur after nontransplant chemotherapy alone. vod/sos with multi-organ dysfunction (mod) may be associated with > % mortality with supportive care alone. diagnosis of vod/sos was traditionally based on baltimore or modified seattle criteria; however, the ebmt recently published separate diagnostic criteria for adults and children. defibrotide is approved for treating severe hepatic vod/sos post-hsct in patients aged > month in the eu, and for hepatic vod/sos with renal or pulmonary dysfunction post-hsct in the usa. the goal of the defifrance study, requested by the french health authorities, is to collect real-world data on safety and efficacy in a broader patient population in france, including all indications. this is the first interim analysis of the largest current evaluation of defibrotide for the treatment of vod/sos in europe. methods: defifrance is an observational, multicenter, post-marketing study that includes any patient treated with defibrotide from hsct centers in france. this interim analysis is based on all patients treated with defibrotide, including those with severe and very severe post-hsct vod/sos. vod/sos was diagnosed using traditional criteria. day+ survival, complete remission (cr; total serum bilirubin < mg/dl and resolution of mod), and safety profile are reported. results: a total of patients treated with defibrotide were included retrospectively and prospectively between july and october from table] disclosure: mohamad mohty: has received honoraria and research funding from jazz pharmaceuticals, delphine lebon: nothing to disclose, ann berceanu: none, charlotte jubert: has received funding from jazz pharmaceuticals, ibrahim yakoub-agha: has received honoraria from jazz pharmaceuticals, stéphane girault: none, marie detrait: has received research funding from jazz pharmaceuticals, cécile pochon: none, fanny rialland: none, virginie gandemer: none, jean-hugues dalle: has received honoraria from jazz pharmaceuticals, régis peffault de latour: has received research grant / honoraria / board from pfizer, novartis, alexion; research grant amgen; and honoraria from jazz pharmaceuticals, david michonneau: has received honoraria from jazz pharmaceuticals, myriam labopin: has received honoraria from jazz pharmaceuticals, floriane delaval: employee of jazz pharmaceuticals and holds stock and/or stock options in jazz pharmaceuticals plc, gerard michel: none, anne sirvent: none, laurence clement: none anne-lise menard: none, anne huynh: has received honoraria from jazz pharmaceuticals, virginie bouvatier: employee of jazz pharmaceuticals and holds stock and/or stock options in jazz pharmaceuticals plc, raj hanvesakul: employee of jazz pharmaceuticals and holds stock and/ or stock options in jazz pharmaceuticals plc, zakaria medeghri: employee of jazz pharmaceuticals and holds stock and/or stock options in jazz pharmaceuticals plc p incidence and predictors of severe cardiotoxicity in patients with severe aplastic anemia after haploidentical hematopoietic stem cell transplantation zheng-li xu , lan-ping xu , yuan-yuan zhang , yi-fei cheng , xiao-dong mo , feng-rong wang , yu-hong chen , wei han , chen-hua yan , yu-qian sun , ting-ting han , yu wang , xiao-hui zhang , xiao-jun huang peking university institute of hematology, peking university people's hospital, beijing, china background: severe cardiotoxicity after hematopoietic stem cell transplantation (hsct) is a rare but fatal complication. the aim of this study was to evaluate the frequency of severe cardiac complications and to assess the ability of various factors to predict these complications in patients with aplastic anemia after haploidentical transplantation., this is the first study evaluating the values of both clinical and imaging factors in the prediction of severe cardiotoxicity among saa patients after haploidentical transplantation. methods: a retrospective study was conducted in consecutive aplastic anemia patients who received haploidentical transplantation from to . all patients received a unified regimen including busulfan, cyclophosphamide (ctx) and antithymocyte globulin at our single center. results: a total of ( . %) patients developed grade iii or iv cardiac toxicity. patients with cardiotoxicity had significantly poorer overall survival (os) than those without cardiotoxicity ( . % vs. . %, p< . ). our multivariable model identified four independent adverse predictors of severe cardiotoxicity, including pre-transplant ecog score (≥ ), abnormal st-t wave on -lead electrocardiogram (ecg), hyperlipemia and recalculated ctx dose (≥ . g/m /d). a predictive risk model was refined as low risk ( - factor), intermediate risk ( factors) and high risk ( - factors) . the respective incidences of severe cardiotoxicity were . %, . %, and . % in the high-, intermediate-and low-risk groups (p< . ). the corresponding os rates were . %, . %, and . % in the three groups (p< . ) at the last follow-up. conclusions: patients with high risk scores had the poorest outcomes and should be monitored closely. a reduced intensity conditioning might be recommended for these patients. disclosure: there are no conflicts of interest to declare. background: allogeneic stem-cell transplantation (allo-sct) is associated with significant transplant-related mortality (trm). acute renal failure (arf) is a frequent complication and usually presents early after the procedure, compromising its feasibility. the aim of this study is to analyse the incidence of arf, its risk factors and its potential impact on trm after allo-sct. methods: patients were included ( males [ %]; median age years, range - ) treated with allo-sct consecutively between january and april in a single institution. patient characteristics are detailed in table . median follow-up was . years (range, . - . ). renal function was evaluated using creatinine and data was collected pre-transplant (baseline) and at the point when arf was developed after allo-sct. arf was evaluated using akin criteria, being akin- an increase . -to . -fold from baseline, akin- an increase . -to . -fold and akin- an increase ≥ -fold. chronic renal disease was evaluated one year after the date of arf using kdigo criteria. results: cumulative incidence of arf at year was % (akin- , %; akin- , %; akin- , %). in the multivariate analysis, arf (akin- / ) was associated with: non-use of antithymocyte globulin in conditioning chemotherapy, p= . (hr . , . to . ) and development of severe agvhd, p= . (hr= . , to . ). in patients with arf akin- , the most important variables in the multivariate analysis were: use of methotrexate (mtx) plus cyclosporine vs mycophenolate mofetil plus cyclosporine as gvhd prophylaxis, p= . (hr= . , . to . ); myeloablative conditioning vs reduced intensity, p= . (hr= . , to . ) and use of total irradiation therapy in conditioning, p= . (hr= . , . to . ). trm at year increased significantly according to akin: akin- , %; akin , %; akin , %; p= , ; hr= . . overall survival at years according to akin was: akin , %, akin , % and akin , %; p= , (figure ). the incidence of chronic renal disease at year after allo-sct according to arf was: no arf ( %), akin- ( %), akin- ( %) and akin- ( %); p= . . conclusions: arf is a frequent complication during the first year after allo-sct and is associated with several factors. arf akin- was associated with more intensive strategies received during conditioning, meanwhile akin- / were related to development of gvhd. there is an association of arf (akin- , or ) with development of chronic renal disease. background: the introduction of cellular therapies such as car-t and modalities of gvhd-prophylaxis with posttransplant/cyclophosphamide (ptcy) that increase the number of admission days have boosted the pressure of available beds in the bm-units. in this sense, our centre started an at-home allogeneic stem cell transplantation (allo-sct) program to follow aplasia from the d+ until independent ambulatory patient. to evaluate the feasibility and safety of allosct, we compared two groups: allohsct/athome (ah-group) vs. allohsct/in-patient (ip-group). methods: we included patients receiving allosct (january -november ) in a single centre: patients, ah-group and , ip-group. all patients received conditioning at the hospital. gvhd-prophylaxis consisted in tacrolimus (tk) plus mycophenolate (mpm) or methotrexate, or ptcy (d+ , d+ ) plus tk (d+ ). all patients received prophylaxis with levofloxacin, fluconazole and acyclovir. besides that, ah-group patients received prophylaxis with ceftriaxone g/ h iv or ertapenem g/ h iv, and aspergillus-prophylaxis with inhaled liposomal amphotericin-b or posaconazole during neutropenia. patients of ah-group since d+ or d+ (in ptcyprophylaxis) received a nurse visit at-home once daily. the visits by the physician were performed at the hospital and only during complication events. first-line therapy of neutropenic fever was meropenem g/ h in both groups, using a portable infusion pump in ah-group. in this group, the absence of focal infection or signs of severe sepsis allowed returning home after the initiation of antibiotics. the platelets support was performed at-home and the red blood support at hospital. results: the median (range) age (years) of the series was . the median follow-up of the series has been not achieved. the source of the sct was peripheral blood in all cases. we didn't find statistical differences between two groups (ah vs ip) in terms of age, diagnosis, type of donor, intensity of conditioning, gvhd-prophylaxis, toxicity (mucositis, acute renal injury, neutropenia and thrombocytopenia), agvhd, aspergilosis and trm. interestingly, a significant reduction of neutropenic fever was observed resulting the lower use of meropenem in the ah-group than ip-group. the admission median days were similar in the both groups and it represented - days the reduction in the total economic cost of the ah-group. the whole analysis of the results are detailed in table: in-patient group, conclusions: in our experience, at home allosct, including ptcy-gvhd prophylaxis, is a feasible and safe procedure reflected in similar trm and aspergillosis incidence. at-home allo-sct is associated with a significant lower risk of neutropenic fever than in-patient group, as well as a very low readmission rate. disclosure: gonzalo gutiérrez-garcía: honoraria from gilead. grant from jazz pharmaceutical and janssen. laura rosiñol: honoraria from takeda, janssen, amgen and celgene. the others author do not have any disclosures to declare. background: renal complications in sickle cell disease (scd) include episodes of acute kidney injury (aki), progressive chronic kidney disease (ckd) and hyperfiltration, defined by abnormally high glomerular filtration rates (gfrs). hematopoietic stem cell transplant (hsct) from an hla identical sibling donor is a well-established curative treatment for scd, but traditional myeloablative conditioning (mac) regimens pose risks of kidney injury due to intensive use of chemotherapeutic agents, infectious risks, and use of calcineurin inhibitors (cnis). aki and subsequent fluid overload (fo) are common in pediatric hsct with reported aki incidence of %- % (kyung-nam koh et. al., ). we report renal outcomes in pediatric patients with scd who received hsct following a non-myeloablative conditioning (nma) regimen without cni exposure. methods: retrospective chart review describing renal outcomes in pediatric patients ( years of age or younger) with scd (hbss) who underwent nma hsct in alberta, canada from july to february . the nma regimen is illustrated in figure . reported renal outcomes: ) measured gfr (dtpa) pre-hsct, ) aki (kdigo definition) post-hsct by reviewing all serum creatinine levels from pre-hsct to one month post-hsct, ) %fo calculated: (max post hsct weight -baseline weight)/ baseline weight x for the two first weeks post-hsct, and ) estimated gfr (egfr) using the pediatric schwartz formula at last follow-up post-hsct, ckd defined as egfr < ml/min/ . m , mildly reduced gfr: - ml/min/ . m , and hyperfiltration: gfr ≥ ml/ min/ . m . [[p image] . results: eighteen patients ( % male, - years old at transplant) were included. most common pre-morbid events: vaso-occlusive crisis (n= ), acute chest syndrome (n= ), splenic sequestration (n= ), and cholelithiasis (n= ). median follow-up time: months (range: - months). all patients engrafted successfully with no acute or chronic gvhd. baseline measured gfrs were all > ml/min/ . m (range: - ) with mildly reduced gfr and hyperfiltration seen in one ( . %) and ( . %) patients respectively. at baseline (pre-hsct), the only aki event was one transplant related aki secondary to delayed hemolytic reaction after exchange transfusion in preparation for transplant. post-hsct, there were no aki events. additionally, no substantial %fo post-hsct was observed. average %fo week one post-hsct: + . % (range: - . % -+ . %) and week two post-hsct: + . % (range: - . % -+ . %). post-hsct egfr remained > ml/min/ . m at last follow-up in all patients. hyperfiltration was present in ( . %) of the patients. conclusions: this is the first study describing stable kidney function in children with scd after the present nma hsct regimen with alemtuzumab/ cgy total body irradiation (tbi) with prolonged post-hsct sirolimus. no episodes of aki or significant fluid overload were observed during the first month post-hsct, and no patient developed ckd during follow-up. further prospective studies are needed to confirm our findings and to determine if stable renal function persists during longer-term followup. disclosure: nothing to declare. lung microbiota in patients with idiopathic pneumonia syndrome (ips) after hct background: idiopathic pneumonia syndrome (ips) is a non-infectious pulmonary complication after hematopoietic cell transplantation (hct) and the etiology remains unknown. recent studies have reported that various diseases are associated with changes of microbiota. the aim of this study was to evaluate the lung microbiota in hct recipients with ips and identify microorganisms potentially associated with ips. methods: frozen bronchoalveolar lavage (bal) samples from hct recipients with ips (n= ) and research bal samples from asymptomatic hct recipients as controls (n= ) were retrospectively analyzed. all samples were negative for common viruses by quantitative pcr. sequencing libraries were made with ng of input dna per sample (nextera xt, illumina). samples were pooled and sequenced by hiseq to obtain -bp paired end data. sequence data analysis and read classification were performed with sunbeam and the quality control and read classification were performed using komplexity and kraken, which classifies bacterial, archeal, and viral genomes. we used sequence data of bronchoscope prewashes from a separate cohort as controls for environmental sources (n= ). bray-curtiss dissimilarity among samples was calculated using the vegan r packages. permanova and a two-sided wilcoxon rank sum test were used to compare between the study groups. results: bal samples started at a median of x raw read pairs per sample and reduced to x reads assignable to microbial taxa following quality control. the bacterial phyla proteobacteria and firmicutes were most abundant followed by bacteroidetes and actinobacteria in both bal and bronchoscope prewash samples. separation of bal and prewash microbiota using bray-curtiss dissimilarity plots showed that bal samples were distinguished by sequences assigned to staphylococcus, acidovorax, and bradyrhizobium species, while prewash samples were distinguished mostly by pseudomonas and elizabethkingia species, consistent with environmental sources (figure) . within bal samples, staphylococcus species were the main drivers of separation between ips cases and the controls (p= . , permanova, figure) . consistent with this, a linear discriminant analysis to identify taxa best distinguishing cases and controls identified staphylococcus, especially s. epidermidis, in ips cases with lactobacillus and streptococcus species in controls. we then compared relative abundances of s. epidermidis between all study groups. ips case samples were significantly enriched in s. epidermidis compared to control (p< . , two-sided wilcoxon rank sum test) and prewash samples (p< . ). viruses were classified by category as human pathogens, non-human pathogens, and bacteriophages. torque teno viruses (ttv) was the most commonly detected virus among viruses that replicate on human cells, and there was a trend towards higher abundance in ips case samples than controls. conclusions: lung microbial sequences in hct recipients predominantly consisted of proteobacteria and firmicutes, and had considerable overlap with environmental background. patients with ips had significantly more staphylococcus sequences detected than asymptomatic hct patients. these results suggest that patients with acute lung injury post-hct show distinct patterns of lung microbiota, although heterogeneity of sample collection and processing cannot be excluded and no singular organism was uniquely associated with ips. a prospective study is required to confirm these findings and define the clinical significance of differences in abundance patterns. disclosure: nothing to declare p abstract withdrawn. romiplostim for the treatment of thrombocytopenia after allogeneic stem cell transplantation background: thrombocytopenia is a common complication after allogeneic stem cell transplantation (allo-hct). with variable possible causes, such as drug side effects, infections, poor graft function, graft vs host disease (gvhd) and immune mediated. the purpose of this study was to evaluate the efficacy of romiplostim, a thrombopoietin receptor agonist, in patients with prolonged thrombocytopenia with no obvious cause after allogeneic transplantation. methods: retrospective analysis of allo-hct patients who received romiplostim at a single bmt unit between november and november . romiplostim was given because of prolonged (> weeks) thrombocytopenia (< , μl) that couldn't be explained by obvious causes such as administration of drugs (antibiotics/antivirals), infection or gvhd. all patients were in complete remission and had complete chimerism. response to romiplostim treatment was considered transfusion independence or plt> . /μl. results: in total, patients (median years, - ) received romiplostim. patients ( male, females) had aml ( pts), all ( ), mds ( ) or hodgkin ( ), received a myeloblative (busiphex-based: , tbi-based: ) or ric ( ) conditioning and were transplanted from a sibling ( ), vud ( ) or haploidentical ( ) donor with pbsc ( ) or bm ( ) . all patients revealed primary neutrophil (median days, range - ) and > . /μl platelet ( days, - ) engraftment. romiplostim was started at median day + (range - ) with a median dose μg/kg ( ) ( ) ( ) ( ) ( ) . the median platelet count before commencement of treatment with romiplostim was . /μl (range . - . ) and them ( %) were transfusion-dependent. in total / ( %) patients responded to romiplostim treatment. eight out of the ( %) transfusion dependent patients responded to the administration of romiplostim. six out of the patients ( %) who were transfusion independent at romiplostin initiation (plt median . /μl, range . - . ) responded. the median duration of treatment was days ( - ) and the median follow up from the commencement of romiplostim was days ( - ). three out of ( %) patients experienced relapse of thrombocytopenia after discontinuation of romiplostim and re-initiation of romiplostim was commenced in all of them, of which responded and didn't. the administration of romiplostim was done on an external basis and was well tolerated by the patients. two patients experienced gvhd during romiplostim treatment (both patients transplanted from / unrelated donor, and days after initiation treatment with romiplostim). / patients interrupted romiplostim due to disease relapse. / patients receiving romiplostim are alive in complete remission and died ( due to relapse, and due to trm). conclusions: we present high response rates to romiplostim in patients with prolonged thrombocytopenia after allogeneic transplantation. in this retrospective study there were no side effects from the administration of romiplostim. however, the administration of romiplostim after allo-hct should be controlled in prospective trials. disclosure we report a single-center analysis of adult patients (median age years, range - , m/f / ), receiving tpo agonists for isolated severe thrombocytopenia (n= ) and spgf (n= ) after allo-hsct. primary diagnoses were aml ( ), all ( ), mds ( ), pmf ( ), mds/mpn ( ), saa ( ), cml ( ), nhl ( ) . severe pgf was defined as cytopenia in ≥ lineages (platelet < × /l, anc < . × /l, hemoglobin < g/l any time after sustained engraftment), full or stable mixed donor chimerism > % and no signs of relapse. median dose of romiplostim was (range, - ) mcg/kg weekly, eltrombopag - (range, - ) mg/day. overall response (or) included cr (platelet ≥ × /l, anc ≥ . × /l, and hemoglobin ≥ g/l) and pr (platelet > × /l, anc ≥ , × /l, hemoglobin > g/l). results: median time from pgf diagnosis to treatment with tpo agonists was days ( - ), median treatment duration was weeks ( - ). tpo agonists were well tolerated with no cases of grade iii-iv toxicity. tpo agonists were combined with rituximab (n= ), rituximab and dli (n= ) and hsc boost (n= ) in ( %) patients. a total of ( %) patients met criteria of response (cr: n= , %; pr: n= , %). combination therapy showed no difference in or compared to tpo agonists alone. or was not depended on the tpo agonist used nor the time to therapy initiation. median increase in anc in responders was . × /l ( . - . ), in platelet count - × /l ( - ). a total of patients died due to relapse (n= ), gvhd iii-iv grade (n= ) and infection (n= ). two-year os from the start of tpo agonist therapy was % ( % ci, - ) with a significant difference between responders and non-responders: % ( % ci, - ) vs. % ( % ci, - ) (p= , ). conclusions: this study showed promising results of tpo agonists for management of spgf. further studies are warranted to specify optimal timing and dosing regimen, predictors of response. [[p image] . two-year os in responders and nonresponders to tpo agonist therapy] disclosure: there are conflicts of interest to disclose p cytomegalovirus reactivation kinetics and peak titers as novel predictors of survival and relapse after allogeneic cell transplantation for hematologic malignancies saskia leserer , evren bayraktar , nikolaos tsachakis-mück , michael koldehoff , lara kasperidus , esteban arrieta-bolanos , mirko trilling , katharina fleischhauer , dietrich w. beelen , amin t. turki university hospital essen, essen, germany, background: after allogeneic hematopoietic cell transplantation (hct), human cytomegalovirus (cmv) reactivation associates with non-relapse mortality (nrm) but also with reduced relapse in patients with leukemia, as shown by numerous studies that evaluated cmv reactivation as a qualitative yes/no parameter in the first months posttransplant. we hypothesized that longitudinal quantitative assessment of cmv reactivation kinetics and virus loads might improve patient-specific clinical outcome associations. methods: this retrospective study included patients with hct for hematologic malignancies treated between / and / at university hospital essen, germany. cmv titers were monitored weekly by quantitative pcr (qpcr); cmv reactivation was defined by a cutoff of > genome copies per ml. patients were included for analysis, if at least measurements were available during the first days after hct. in total, , samples were analyzed. subgroup analyses were performed according to the time of cmv reactivation (before/after + d) or the cmv viremia titer (> , , , - , and - , copies/ml). results: cmv reactivation was detected in (median age years; range - years) out of patients. baseline characteristics (age, gender, underlying disease, transplant) of patients without cmv reactivation were comparable. cmv reactivation kinetics followed a gaussian normal distribution with a median first reactivation at + d and peak titers at + d. all except patient reactivated before d, % before + d. overall survival (os) of the cmv reactivation group as a whole did not significantly differ from the non-reactivation group ( vs. months). however, in subgroup analyses os was significantly reduced in patients with very early (< + d) compared to later reactivation ( vs. months, p= . ). moreover and importantly, os was significantly reduced in patients with cmv reactivation at high titers of > , copies/ml compared to those with lower titers (( vs. months) p< . ).cox regression analyses confirmed significantly reduced os for patients with cmv reactivation > , copies/ml and < day + as compared to the other cohorts (hr . , %ci . - . , p< . and hr . , % ci . - . , p= . ) respectively). the nrm was consistently higher (hr . ; %ci, . - . , p< . ) for patients with cmv copies > , /ml. the risk of hematologic relapse was exclusively reduced in patients with a peak cmv viremia between , and , copies/ml (hr . , % ci . - . ; p= . ) as compared to patients without cmv reactivation. for other levels of cmv reactivation this effect was not observed. conclusions: our data showed that cmv reactivations before + d or with high titers of > , copies/ml associated with significantly reduced os, while cmv reactivations at intermediate titers between , and , copies/ml had a positive impact on relapse incidence. these findings underline the complexity of cmv reactivations after hct outcome, and support longitudinal evaluation of cmv titers and individualized quantitative kinetics models for risk assessment after hct to distinguish the advantageous from the detrimental aspects of cmv reactivation. disclosure: att has received lecture fees from jazz pharmaceuticals and travel subsidies from neovii biotech outside the submitted work. the other authors declare no competing financial interests within the submitted work. association of serum ferritin levels before start of conditioning with mortality after allosct -a prospective, non-interventional study of the ebmt transplant complication working party background: elevated serum ferritin levels occur due to iron overload or during inflammation and macrophage activation. a correlation of high serum ferritin levels with increased mortality after allosct has been suggested by several retrospective analyses as well as by two smaller prospective studies. methods: this international multicentric study aimed to study the association of ferritin serum levels before start of conditioning with allosct outcome. patients with acute leukemia, lymphoma or mds receiving a matched sibling allosct for the first time were considered for inclusion, regardless of conditioning. data were prospectively collected between / and / . a comparison of outcomes between patients with high and low ferritin level was performed using univariate analysis and multivariate analysis using cause-specific cox model. variables included in the multivariate analyses were age, sex mismatch, diagnosis, disease status, karnofsky score, number of cd cells given, intensity of conditioning, type of gvhd prophylaxis, atg use, time from diagnosis to transplant, year of transplant and cmv status. results: twenty centers from european countries reported data on allosct recipients. patient characteristics are given in table . the ferritin cut off point was determined at μg/l (median of measured ferritin levels). overall survival of allosct recipients with ferritin levels above cut off measured before start of conditioning was significantly shorter ( figure a , univariate hr= . ci= . - . p= . ; multivariate hr= . , ci= . - . , p= . ). progression-free survival was also shorter ( figure b , univariate hr= . ci= . - . p= . ; multivariate hr= . , ci= . - . , p< . ). excess mortality in the high ferritin group was due to both higher relapse incidence (univariate hr= . ci= - . p= . ; multivariate hr= . , ci= . - . , p= . ) and increased non-relapse mortality (univariate hr= . ci= . - . p= . ; multivariate hr= . , ci= . - . , p= . ). non-relapse mortality was driven by significantly higher infection-related mortality in the high ferritin group (univariate hr= . ci= . - . p = . ; multivariate hr = . , ci = . - . p = . ). acute and chronic gvhd incidence or severity were not associated to serum ferritin levels. conclusions: ferritin levels before start of conditioning can serve as routine laboratory biomarker to predict mortality after allosct. disclosure: the authors declare no confict of interest related to this study p prediction of reduced lung function and acute gvhd by surfactant protein d in allogeneic stem cell transplantation transplantation (hsct) and may progress to bronchiolitis obliterans that has a high mortality rate. surfactant protein d (sp-d) is an innate defense molecule involved in immune regulation at the epithelial surfaces, particularly in the lungs, and elevated levels have been associated with exacerbation of chronic obstructive pulmonary disease (copd). the aim of this study was to investigate, whether sp-d plasma levels and variants in the gene encoding sp-d may predict the development of reduced lung function after allogenic hsct. methods: we performed a population-based, singlecenter study of children (aged - years) treated with allogeneic hsct. the study consisted of ) a prospective study of serial plasma sp-d levels and rs genotypes in patients during the first months after hsct, and ) a retrospective study of rs genotypes within the sp-d gene in patients transplanted between - . pulmonary function tests were performed regularly as part of the clinical monitoring. results: at the day of graft infusion (day ) sp-d levels were reduced compared to levels before start of treatment with conditioning chemotherapy, defined as baseline ( ng/ml (quartiles - ) at day vs ng/ml ( - ) at baseline, p< . ). from day + sp-d levels increased and remained increased during the whole study period ( ng/ml ( - ) at baseline vs ng/ml ( - ) at months, p< . ). acute gvhd (agvhd) occurred in patients, of those patients with grade - . high sp-d levels at day + were associated with the development of agvhd ( ng/ml ( - ) vs ng/ml ( - ), p< . ) ( fig. ). the c/c genotype was associated with generally low sp-d levels and low fev /fvc at all time intervals compared to the other genotypes, significantly - months post-hsct (p= . ). there was no overall correlation between sp-d levels and lung function, but stratifying for genotype, high baseline sp-d levels were predictive for reduced fev /fvc at - months in cc and tt homozygous individuals. conclusions: patients with a genotype causing low capacity for sp-d production are at increased risk of developing pulmonary impairment after hsct. in addition, our data lend support to other studies indicating that spd production may increase during inflammatory pulmonary disease, acting as a reactive, protective mechanism. further research is warranted to define the role of sp-d levels and genotypes as a prognostic tool for lung function and agvhd. [[p image] . background: allogeneic hematopoietic cell transplantation (allohct) means a long period of restricted mobility and a range of therapy related side effects on muscle function. in this context patients demonstrated a huge decline of physical capacity and muscle mass in particular, accompanied with a decrease of quality of life (qol). resistance training could maintain muscle mass but is limited by patientsb lood values (platelet-count) and well-being. whole body vibration (wbv) was shown to maintain muscle mass during bed rest and has less impact on blood pressure than conventional resistance exercises. furthermore it was also shown to be feasible in patients during high dose chemotherapy. therefore the aim of our study was to examine the effects of wbv during allohct on patients physical and functional performance as well as qol. methods: patients receiving allohct were randomly allocated to either a wbv exercise group (ig) or an active control group (cg) doing stretching and mobilization. both groups exercised during the whole time of hospitalization for times per week and underwent pre-, post-and followup-assessment. physical capacity was determined by maximum oxygen consumption (vo peak ) and maximum power (p max ) during cardiorespiratory exercise test and by maximum strength of the knee extensors and flexors (ex max , flex max ) during isokinetic strength test. functional performance was assessed by jumping height during counter movement jump (cmj) and time of chair rising test (crt) as well as power output during both tests. qol was assessed by questionnaires of the eortc. results: during allohsct vo peak and p max decreased in both groups but till follow-up an increase is seen in the ig (p= . ; p= . ). at day + /follow-up a vo peak group difference is seen (p= . ). ex max (p= . ) and flex max (p= . ) were only reduced in the cg during hospitalization. jumping height and power output decreased in the cg during hospitalization (p= . , p= . ) and a difference between groups were seen in changes of jumping height from pre-to follow-up-assessment (p= . ): increase in the ig and decrease in the cg. the ig showed a decrease in time from baseline to follow-up (p= . ) in the crt and an increase of power output (p= . ). qol decreased only in the cg during hospitalization (p= . ) while during follow-up qol increased in both groups (ig: p= . ; cg: p= . ). in the cg physical functioning decreased during intervention (p= . ) whereas an increase was seen in the ig from pre-to follow-upassessment (p= . ). body image was significant worse in the cg compared to the ig at hospital discharge (p= . ) as well as at follow-up measurement (p= . ) where it got worse over time (p= . ). conclusions: wbv was shown to maintain maximum strength, jumping performance and qol during allohct. although cardiorespiratory fitness could not be maintained by wbv during hospitalization, it seems in the follow up period till day + that recovery of the cardiorespiratory system is enhanced by wbv carried out during allohst. nevertheless reasons for this changes in recovery have to be analyzed in further studies as well as treatment effects of wbv compared to conventional resistance training. disclosure: supported by a grant of the faculty of medicine and comprehensive cancer center freiburg respiratory virus infection within year after of allo-sct is the significant risk factor of obstructive ventilatory disturbance kosei kageyama , michiho ebihara , mitsuhiro yuasa , daisuke kaji , aya nishida , shinsuke takagi , hisashi yamamoto , go yamamoto , yuki asano-mori , naoyuki uchida , atsushi wake , akiko yoneyama , shigeyoshi makino , shuichi taniguchi toranomon hospital, hematology, tokyo, japan, background: obstructive ventilatory disturbance (ovd) is one of the major life-threading complication at the chronic phase of allogeneic stem cell transplantation (allo-sct). bronchiolitis obliterans has been the most established etiology as a part of chronic graft-versus-host disease and major cause of late non-relapse mortality of allo-sct. but other etiologies impact on respiratory function after allo-sct and risk factor of ovd have not been well understood. methods: to address these issues, we retrospectively reviewed the medical record of consecutive patients who first allo-sct at toranomon hospital between and . to detect ovd, forced expiratory volume in second (fev . ) showed less than % of predicted in spirometry test was defined as positive. in the recipients who showed fev . less than % in pre-transplant test, more than % reduction of fev . was regarded as positive. nasopharyngeal swab of those who had upper respiratory tract symptoms were tested for the presence of respiratory viral antigens (adv, piv, and rsv). patients with ecog performance status of , had active infection at transplant were excluded from this analysis. the cases of early death or relapse before days post-transplant, and the cases of graft failure were also excluded. results: the median age was years (range, - ). underlying diseases were aml in , mds/mpd in , cml in , all in , atl in , hl in , nhl in , and others in . five hundred twenty-nine ( %) were not in remission at the time of transplant. five hundred eightythree patients ( %) were conditioned with myeloablative regimens, whereas patients received reduced-intensity regimens. donor sources consisted of related peripheral blood /bone marrow (bm) (n= ), unrelated bm ( ) forty-six developed ovd on median of ( - ) days post-transplant. cumulative incidence of ovd was . % in total population. in recipients those who could spirometry, overall survival at years was . % in patients who developed ovd and was comparable with those who did not develop it ( . %, p= . ). in univariate analysis, disease status (cr/aa or noncr), recipient age (age< or ≥ ), prior autologous stem cell transplantation (yes or no), intensity of conditioning regimen (mac or ric), tbi dose (< gy or ≥ gy), busulfan dose (< . mg/kg or ≥ . mg/kg), donor source (cord blood or non-cord) had no impact on the incidence of ovd. patients who developed respiratory virus infection showed significantly higher incidence of ovd compared to those who did not developed it ( . % vs . %, p< . ). in multivariate analysis, respiratory virus infection was the only significant risk factor for the development of ovd (hr= . , % ci . - . , p< . ). conclusions: respiratory virus infection within year after allo-sct is the significant risk factor of ovd. disclosure: nothing to declare. background: metabolic syndrome (mets) is related to increased risk of cardiovascular disease and type- diabetes (dm- ) and usually seen in overweight individuals in the general population. we investigated mets and clinical risk factors two decades after hsct. methods: all male survivors treated with myeloablative allo-hsct during childhood (< years) between - in denmark were invited to a follow-up study. mets was defined as the presence of at least three ncep atp iii criteria: fasting plasma triglyceride (tg) ≥ . mmol/l, high density lipoprotein (hdl) < . mmol/l or medical treatment of hyperlipidemia; fasting plasma glucose (fpg) ≥ . mmol/l; abdominal circumference (ac) > cm; bp ≥ mmhg (systolic) / ≥ mmhg (diastolic) or medical treatment for hypertension. patients with overt dm- were included into the mets group. furthermore, patients were examined for chronic graft-versus-host disease (cgvhd) by the nih-criteria at the time of follow-up and high sensitivity c-reactive protein (hscrp) was measured. the prevalence of mets was compared to a nordic reference group (hildrum et al. ) . results: we included out of eligible males (participation rate %) aged - years, median years. median (range) follow-up was ( - ) years. of these males, % had a malignant diagnosis and % were treated with tbi-based conditioning. donors were matched siblings (n= ), matched relatives (n= ) or matched unrelated donors (n= ). mets was more prevalent ( %) in the young adult survivors compared to the prevalence reported for - year-olds in the nordic reference ( %). instead the prevalence was comparable to that reported for the - year-olds ( %). of the components of mets, elevated tg ( %), hypertension ( %), and decreased hdl ( %) were frequent, while fpg was elevated in %. importantly, only % of those with mets had increased ac and mean bmi ( . kg/m ) of the hsct survivors was within normal range in contrast to features of mets observed in the background population. having mets was significantly associated with tbi (rr = . , %ci ( . - . ), p= . ) as was the following single components of mets (mean in tbi group vs. mean in non-tbi group): elevated tg ( . mmol/l vs. . mmol/ l, p= . ), lower hdl ( . mmol/l vs. . mmol/l, p= . ) and higher diastolic bp ( mmhg vs. mmhg, p= . ). mets was only demonstrated in one patient who received non-tbi based conditioning. sixteen of patients had cgvhd of which nine were moderate to severe cases, but cgvhd was not associated with mets. however, low-grade inflammation measured by hscrp was related to increased ac (rho= . , p= . ) and tg (rho= . , p= . ). conclusions: our results indicate that male long-term survivors of allo-hsct during childhood have a high risk of mets at an earlier age than the general population. the presence of mets despite normal bmi in several patients suggests unconventional etiologies like the effect of tbi and low-grade inflammation. disclosure: nothing to declare. results: this survey was completed by transplant directors ( %), transplant consultants ( %), nonconsultant grade physicians ( %), hsct clinical nurses specialists (cns) ( %) and other ( %) from centres in countries. % of the centres are adult-only, % paediatric-only and % treat adult and paediatric patients (mixed centres). % are located higher than degrees latitude (northern countries) and % lower than this latitude (southern countries). at the time of the survey % were members of the european union (eu). measurement of serum vd is routinely performed in % of the centres prior and in % after allogeneic hsct. the main clinical indications are known osteopaenia/osteoporosis ( %), previous fracture ( %), treatment with steroids ( %), premature menopause ( %) and established menopause ( %). monitoring occurs every months ( %), every months ( %), once a year ( %) or at other time-points ( %). in this regard, seasonality is not taken into account in the majority of the centres ( %). local and national/international guidelines (nice) are only followed by % and % of the centres, respectively. the most common cut-off value of serum vd for commencing on replacement is nmol/l ( %). northern countries tend to use values of ≥ nmol/l whereas southern countries ≤ nmol/l. % do not use cut-off values. following hsct, % of centres prescribe vd supplements to maintain calcium metabolism and bone health ( %), enhance immune reconstitution post-hsct ( %), gvhd prevention ( %), enhance immune-suppression to treat gvhd ( %), treat depression/fatigue ( %) and reduce relapse risk %. a "loading" dose is administrated in % ( % adult, % mixed and % paediatric), with a mean duration of weeks . the median daily loading dose is , iu ( - , ). the median "maintenance" daily dose is iu ( - , ). there are not remarkable differences between adult and paediatric centres or northern and southern countries. vd replacement is prescribed by transplant physicians ( %), family physicians ( %), endocrinologists ( %), cns ( %), others ( %) and in % of the centres, patients are advised to buy it over-the-counter. vd is prescribed combined with calcium carbonate in % and alone in % of the centres. it is eventually discontinued by % of the centres when therapeutical levels of vd are reached ( %), dexa scan returns to normal ( %) and symptomatic improvement ( %). conclusions: this survey has demonstrated discrepancies in monitoring and replacement of vd across ebmt allogeneic hsct programmes. although awareness has arisen over the last decade, there is still lack of evidence about the optimal levels of vd required for immunemodulation post-hsct. this survey emphasises the need for specific guidelines to harmonise the current management of vd deficiency in adult and paediatric hsct setting. disclosure background: the use of unmanipulated haploidentical sct (haplo-sct) with post-transplant cyclophosphamide (pt-cy) as gvhd prophylaxis has widely extended. primary and secondary graft failure are relatively uncommon complications. however, poor graft function (pgf) after haplo-sct with pt-cy has not been described thoroughly. the objective of this study is to describe characteristics, treatments and outcomes of patients with pgf after haplo-sct with pt-cy. methods: we retrospectively analyzed haplo-sct with pt-cy consecutively performed between and in our centre. pgf was defined as either occurring after initial engraftment: persistent neutropenia (anc < /ul) with the need of at least doses of g-csf and/or thrombocytopenia (platelets < . /ul) with platelet transfusion dependence, with complete donor chimerism and without concurrent severe gvhd or disease relapse. results: nineteen patients were excluded from the analysis due to early mortality (death before day + ), primary graft failure (absence of neutrophil engraftment by day + , with mixed chimerism) or secondary graft failure (development of severe cytopenias and mixed chimerism after initial achievement of neutrophil engraftment). thirty one patients ( , %) were diagnosed with pgf. main characteristics of these patients are summarized in table . twenty six patients ( %) presented with neutropenia and were treated with g-csf, while patients ( %) only developed severe thrombocytopenia without neutropenia, and were treated only with platelet transfusion. twenty four patients ( , %) had at least cmv reactivation, patients ( %) had or more cmv reactivations and patients ( %) received valganciclovir for cmv reactivation treatment. although most patients achieved adequate peripheral blood counts (pbc) with initial salvage therapy, patients ( %) had persistent cytopenias in spite of g-csf, platelet transfusion, cmv reactivation resolution and myelotoxic drugs withdrawal. four of them were treated with a boost of cd + selected peripheral blood donor cells at a median of days after . median cd + cells infused was , x /kg. these patients achieved adequate pbc after salvage therapy and two developed gvhd. the other patients were treated with increasing doses of thrombopoietin (tpo) receptor agonist (tra) eltrombopag. one patient started treatment days after hsct with mg daily and increased dose to mg daily, with complete recovery of pbc months after initiating tra. the second patient started treatment days after hsct with mg daily and increased dose to mg daily, with complete recovery of pbc months after initiating tra. twenty one patients ( %) with pgf diagnosis had long term survival. conclusions: poor graft function is a frequent complication after haplo-sct with cy-post. cmv reactivation and myelotoxic drugs could be the most relevant factors associated with development of this entity. although most patients recover pbc without specific therapies beyond g-csf and platelets transfusion, there is a small group of patients with persistent cytopenias. boost of cd + selected cells is effective in reverting this condition, with gvhd as main complication of this procedure. use of tra seems to be an interesting option for these patients, although more experience is needed to draw definitive conclusions. disclosure: nothing to declare. were also frequently observed. the high risk patients for anxiety (hads-a score ≥ ) and depression (hads-d score ≥ ) was found in . % and . %, respectively. . % of patients was in high distress status (nccn dt score ≥ ). we found that younger age (< years) was significantly associated with poor quality of life score (fact-bmt) (p= . ) and high risk of fatigue (p= . ), anxiety (hads-a) (p= . ), and depression (hads-d) (p= . ). female sex was significantly related to lower physical well-being score and higher distress score (p= . and p= . , respectively). acute lymphoblast leukemia (all) survivors after allo-hct showed significantly worse quality of life score (fact-bmt) (p= . ) and higher depression score (hads-d) (p= . ) compared to those with other disease. chronic graft versus host disease (gvhd) and continuous immunosuppressant usage also have significant adverse impact on lower fact-bmt score (p= . and p= . , respectively) and higher hads-d score (p= . and p= . , respectively). but there was no significant difference in fact bmt, hads-a, hads-d, nccn dt according to donor type, conditioning intensity, anti-thymocyte globulin use, acute gvhd. smoking and alcohol drinking was continued in . % and . % of allo-hct survivors. . % of survivors did not exercise regularly. regular health screening tests have been done only in patients ( . %). conclusions: allo-hct survivors over years following allo-hct still have many physical and psychological symptoms. younger patients (< years), female, all, chronic gvhd, and sustained use of immunosuppressant were significant risk factors for poor quality of life and anxiety. we need to build more active survivorship care plan after allo-hct especially for those patients. disclosure: all authors have nothing to declare. evaluation of the new ebmt criteria for the diagnosis of vod/sos in consecutive transplant patients using an electronic patient record analysis system asha aggarwal , nicola gray , oliver lomas , katalin balassa , nadjoua maouche , robert danby , , andy peniket , grant vallance background: veno-occlusive disease (vod), or sinusoidal obstruction syndrome (sos), is a recognised complication of haematopoietic stem cell transplantation. hepatic vasculature endothelial cells are damaged by conditioning chemotherapy, leading to venous occlusion and centrilobar necrosis. the ebmt criteria for diagnosis of vod are bilirubin >= with two of painful hepatomegaly, > % weight gain and ascites. vod is often under-diagnosed, and as a result, treatment may be delayed. integrated electronic patient record (epr) systems are now widely used, and provide an opportunity to retrospectively audit practice to identify patients in whom vod may have been un-diagnosed or in whom treatment was delayed. in addition these systems have potential for alerting clinicians to the potential diagnosis of vod. methods: we have developed software to analyse the data downloaded from epr to identify patients in whom vod was a possible diagnosis according to the new ebmt criteria. in order to identify patients who may have had vod we first screened for patients with a bilirubin of >= mmol/l (which is an absolute requirement for the clinical diagnosis of vod) within the first days of transplantation. epr data was then used to assess whether patients had > % weight gain. radiology reports were reviewed for patients who had bilirubin >= mmol/l to ascertain if they revealed ascites or painful hepatomegaly. results: patients underwent transplant procedures (january st to july st ). of all transplant patients ( . %) were found to have a bilirubin of >= mmol/l. of ( . %) autograft patients and of ( . %) allograft patients had an elevated bilirubin at this level. these patients were assessed for evidence of % weight gain. this was the case in patients overall- % of autograft patients, . % of allograft patients. seven patients ( autograft and allograft) had radiological evidence of ascites. two patients had a recording of painful hepatomegaly (both post allograft). overall our analysis identified patients ( . % overall) fulfilling the ebmt diagnostic criteria for classic early vod all of whom received defibrotide. all patients had received allogeneic transplants. we failed to identify any cases of late onset vod or any undiagnosed patients over this period. conclusions: this analysis enabled us to efficiently perform a complete audit of our practice to identify patients with vod. we would recommend using electronic patient records to retrospectively audit practice in this way. the tool that we have created for this analysis will be made freely available for public use and the details will be presented at the ebmt meeting. we now plan to extend the function of our epr system to provide alerts to clinicians when vod is a possible diagnosis and may lead to more rapid treatment of these patients. our data suggests that elevation of bilirubin and weight gain of > % will be the most frequently occurring criteria on which to base these alerts. disclosure: g.vallance has performed consultancy work for jazz pharmaceuticals. endothelial activation and stress index in predicting outcome of allogeneic stem cell transplantation-a retrospective cohort analysis zinaida peric , tomislav taborsak , nadira durakovic , lana desnica , alen ostojic , ranka serventi-seiwerth , radovan vrhovac university hospital centre zagreb, zagreb, croatia background: endothelial dysfunction is a common pathophysiology of major complications after allo-sct, such as graft-versus-host disease, veno-occlusive disease, thrombotic microangiopathy and sepsis. endothelial activation and stress index (easix) is a simple score comprised of standard laboratory parameters (creatinine, ldh and thrombocytes) developed as a potential tool to predict allo-sct mortality by luft and colleagues. a recent validation of easix included three retrospective cohorts and showed that easix taken before start of conditioning can be used as an independent predictor of survival after allo-sct. methods: the aim of our study was to retrospectively evaluate pre-transplant easix in our cohort of consecutive patients who underwent allo-sct in the university hospital centre zagreb between and . with the use of a cut-off used in the validation cohorts, we compared two groups of patients for overall survival (os) and transplantrelated mortality (trm). group comparisons were done using the log-rank test or gray test for competing risks outcomes. a multivariate analysis evaluated the association of os with relevant variables by using a cox's proportionalhazard regression model. results: our study group included patients and comprised males ( %) and females ( %, with a median age of years (range, to years) at the time of transplantation. the most frequent malignancies in our population were acute leukemia ( patients, %) and myelodysplastic/myeloproliferative neoplasm ( patients; %). the donor was an identical sibling for patients ( %), matched unrelated donor for patients ( %) and haploidentical for patients ( %). patients ( %) received a myeloablative conditioning regimen while patients ( %) received a reduced-intensity conditioning regimen. with a median follow-up of months (range, - ) for the whole study group, the os at months was %, ( %ci - ) in the group of patients with low easix score and % ( % ci - ) in the group of patients with high easix score (p= . ). this difference was mainly attributed to higher trm in the group with high easix score ( %, %ci - at months) compared to the group with low easix score ( %, %ci - at months) (p= . ). in the multivariate analysis which included easix, patients' age, intensity of conditioning, diagnosis (lymphoid vs myeloid), status of the disease at transplant and type of the donor, worse os was independently associated only with older age of patients (hr . ; % ci, . - . , p= . ) and high easix score (hr . ; % ci, . - . , p= . ). conclusions: our retrospective data support previous data and suggest that easix could potentially serve as a valid tool for prediction of allo-sct outcomes. as a simple biomarker panel, easix could easily be implemented in clinical decision making in the field of allo-sct. these retrospective data need validation in a prospective study which is currently being conducted. clinical background: veno-occlusive disease (vod) is a potentially devastating complication that can occur after hematopoietic stem cell transplant (hsct) and in severe cases can lead to multi-organ failure. (mohty ) defibrotide has been proven to be effective to prevent and treat vod, and it is critical that clinicians are aware of how to diagnose and treat this serious complication of hsct. this study was conducted to determine if an online, simulation-based continuing medical education (cme) intervention could improve performance of hematologists/oncologists (hem/ onc) and advanced practice providers (nurse practitioners and physician assistants, apps) in the diagnosis and treatment of patients with vod. ( methods: a cme certified virtual patient simulation (vps) was made available via a website dedicated to continuous professional development. the vps consisted of cases presented in a platform that allows clinicians to assess the patients and make diagnostic and therapeutic decisions supported by an extensive database of diagnostic and treatment possibilities, matching the scope and depth of actual practice. clinical decisions were analyzed using a sophisticated decision engine, and tailored clinical guidance (cg) employing up-to-date evidence-based and faculty recommendations was provided after each decision. one case was about vod and the other case was about acute myeloid leukemia (aml). decisions were collected post-cg and compared with each user's baseline (pre-cg) decisions using a -tailed paired t-test to determine p-values (p < . indicates significance). data were collected between / / and / / . results: at the time of assessment, hem/oncs and apps had fulfilled the participation criteria for completing the vod case simulation. conclusions: this study demonstrates that vps that immersed and engaged clinicians in an authentic and practical learning experience improved evidence-based clinical decisions related to the management of vod. this vps increased the percentage of clinicians who utilized standardized criteria to diagnose vod and who ordered defibrotide and iv fluids for vod management. however, further education is needed to increase the competence and performance of clinicians, particularly apps, in these areas in order to positively impact patients. disclosure: nothing to declare. a nationwide retrospective study of hematopoietic stem cell transplantation in solid organ transplant recipients: on behalf of jshct, transplant complications working group background: the outcome of hematopoietic stem cell transplantation (hsct) in solid organ transplant remain unclear. to address this issue, we conducted a retrospective survey of the japan society for hematopoietic stem cell transplantation centers. methods: to address this issue, we conducted a nationwide retrospective survey of the japan society for hematopoietic stem cell transplantation (jshct) centers. a first questionnaire was emailed to jshct centers requesting information on cases of hsct in sot recipient. patients' data about sot were collected by sending a second questionnaire to the centers with the patient. based on these reports, patients' data about hsct was identified in the japan transplant outcomes registry database by the transplant registry unified management program (trump), confirmed in . results: of the jshct centers, responded to the survey ( . %). of the responding centers reported a total of patients who had undergone sot from living donor, and subsequent hsct. they consist of three autologous hsct (auto-hsct) and allogeneic hsct (allo-hsct). in auto-hsct, all patients had received liver transplant for hapatoblastoma. they achieved neutrophil engraftment at days after hsct, and two of three patients were alive at one year after hsct. in allo-hsct (n= ), seven patients had received liver transplants, and nine patients had received kidney transplants. five patients received hsct from unrelated donor, and patients received hsct from related donor; two donors were identical in sot. their stem cell sources were seven peripheral blood stem cell, six bone marrow, and three cord blood. all but one patients achieved neutrophil engraftment at days after hsct. five-year overall survival ( yos) was . %. while yos in patients with bone marrow failure (n= ) was %, that in patients with malignant disease (n= ) was . %; all but one patients with malignant disease received allo-hsct in non-remission. seven of nine kidney-transplant recipients experienced dialysis, and three patients experienced renal rejection after hsct. on the contrary, no liver-transplant recipient experienced hepatic rejection. conclusions: in sot recipients, the outcome of allo-hsct for malignant disease was poor, partly due to disease status before allo-hsct. severe renal complications were common in kidney-transplant recipients, suggesting renal care with caution during and after allo-hsct. disclosure: this work was supported in part by the practical research project for allergic diseases and immunology (research technology of medical transplantation) from japan agency for medical research and development, amed. high incidence but low mortality of ebv related ptld after t-cell replete allo-peripheral blood hct with aggressive monitoring and without pre-emptive rituximab background: the aim of the study is to report the incidence and outcome of post-transplant lymphoproliferative disorder (ptld) in the setting of allogeneic peripheral blood hematopoietic stem cell transplantation (allo-hsct) combining post-transplant cyclophosphamide (ptcy) and anti-thymocyte globulin (atg) as graft versus host disease (gvhd) prophylaxis. methods: between october and may , adult patients diagnosed with hematological malignancies underwent a first t-cell replete allo-hsct in our center. all patients received a reduced intensity conditioning regimen with fludarabine, busulfan, and cgy of total body irradiation, combined with rabbit-atg, ptcy and cyclosporine (csa). ebv titres were monitored weekly by quantitative pcr in plasma samples. the cut-off value for test positivity was > copies of ebv dna/ml of plasma. last follow up was november . median follow up for patients known to be alive was months (range - ). results: patient information is summarized in table . ebv reactivation was documented in ( %) patients. median time to ebv reactivation and the diagnosis of presumed/proven (p/p)-ptld were ( - ) days and ( - ) days [ ( - ) months], respectively. median time between first ebv reactivation to p/p-ptld was ( - ) days. seventeen ( %) of the patients developed p/p-ptld. median age was years . two ( %) received mrd, ( %) / mud, ( %) / mud, and ( %) haploidentical donor grafts. twelve ( %) were on therapeutic cyclosporine at diagnosis. pre-emptive therapy was not given to any case and only probable or proven ptld were given rituximab. treatment was based on reduction of the immunosuppression in patients and with the addition of weekly rituximab mg/ m in cases. fifteen ( %) achieved complete clinical responses with pcr negativity. two ( %) patients died secondary to ptld. conclusions: atg based conditioning is associated with increased viral reactivations. frequent ebv monitoring and pre-emptive treatment may lead to rapid disease control. further research is required to optimize monitoring and management strategies in allo-hsct recipients. disclosure: nothing to declare p acoustically enriched extracellular vesicles as potential markers for allogeneic hematopoietic stem cell transplantation complications hooi-ching lim , robert palmason , stig lenhoff , thomas laurell , stefan scheding , background: extracellular vesicles (evs) contain a number of condition-specific proteins, dna and rna types and might therefore be used for the early detection of posttransplant complications. however, traditional ev isolation (ultracentrifugation) is time consuming and requires large sample volumes thus making it difficult to perform longitudinal studies on larger patient cohorts. we therefore investigated whether recently-developed acoustic trapping could be applied to isolate evs from patient plasma for biomarker development. methods: plasma samples were collected from consecutive patients before and up to months after allogeneic hematopoietic stem cell transplantation. patients (age: - years) with high-risk or refractory/relapsed diseases were transplanted with mobilized pbsc from related (n= ) and unrelated donors (n= ) after standard conditioning. gvhd prophylaxis was cyclosporine and methotrexate. plasma samples were frozen and thawed for ev enrichment using a novel acoustofluidic-based technology (acoustic trapping). acoustic trapping uses ultrasound as a local λ/ acoustic standing wave produced by a piezoelectric transducer over a capillary. first, μm polystyrene beads are captured which serve as seeding particles. after washing, target particles (evs) are then captured ("trapped") in the acoustic field. a semi-automatic trapping device (acoutrap) was used to isolate evs from diluted plasma ( : in pbs). the number of evs and size distribution were analyzed by nanoparticle tracking analysis. mirna analysis was performed by qpcr.evs were enriched in duplicate from μl and μl of diluted plasma for nanoparticle tracking analysis and qpcr analysis, respectively. results: evs were successfully isolated from all plasma samples. a total of plasma samples were processed. numbers of trapped evs ranged from . x - . x before conditioning to . x - . x per μl diluted plasma after transplantation. the maximum change in ev numbers in individual patients compared to pretransplantation values ranged from -fold to -fold. most patients showed slight increases in ev size after transplantation. eight of the patients showed signs of infection and received i.v. antibiotics. increased levels of evs (> -fold) were recorded in three patients during these episodes. furthermore, increased ev numbers were observed in a patient who required i.v. antiviral therapy for cmv reactivation. acute grade i gvhd was observed in five patients of which two had increased ev numbers (> -fold). one patient developed grade iv gvhd which was accompanied by a -fold increase in ev numbers. interestingly, progressively increasing ev numbers preceded the detection of early relapse in a pre-b all patient by three weeks. rna isolation from trapped evs yielded sufficient material for mirna profiling. here, first mirna profiling data demonstrated that mirnas were detected in ev samples (mir- a, - a, - c, - and - a) , and that acoustically enriched evs were not affected by hemolysis in contrast to the corresponding whole plasma samples (dcq of mir- a and mir- a). conclusions: acoustic trapping allows for efficient and rapid enrichment of evs from small volume plasma samples. trapped ev samples contain sufficient amounts of mirna for downstream analysis and are thus promising candidates for biomarker development in transplantation. disclosure: laurell and scheding are founders and board members of acousort ab, a lund-based biotech sme that develops particle and cell sorting methods based on ultrasound. the incidence, risk factors and outcomes of primary poor graft function after allogeneic hematopoietic stem cell transplantation fei gao , , jimin shi , , yi luo , , yamin tan , , xiaoyu lai , , jian yu , , he huang , , yanmin zhao , background: allogeneic hematopoietic stem cell transplantation (allo-hsct) is a curative therapy for both hematologic malignancy and many other blood disease. while, primary poor graft function (pgf) is still a severe complication following hsct which lead to poor prognosis. up to now, the incidence and risk factors of pgf have not been totally revealed. methods: from january to december , a total of patients who received allo-hsct in our center were analyzed retrospectively. there were males ( . %) and females ( . %) with a median age of . years ( - years) . pgf was defined as persistent neutropenia (≤ . × /l), thrombocytopenia (platelets≤ × /l), and/ or hemoglobin≤ g/l after engraftment with hypocellular bone marrow and full donor chimerism, without concurrent graft-versus-host disease or disease relapse. incidence was calculated from all patients. of the total patients, nineteen ( . %) developed primary pgf. a : ratio of nested case control study using the good graft function (ggf) subjects transplanted in the same year with the same sex and age of ± years was carried out. results: data was analyzed by univariate and multivariate logistic regression, and univariate analysis identified disease species, the time from diagnosis to transplantation, disease states, myelofibrosis, splenomegaly, serum ferritin (sf) level, cmv infection, mononuclear and cd + cells in graft as potential risk factors (p < . ) for pgf. multivariate analysis identified elements as the independent risk factors (p < . ), including cd + cells < × / background: transplant survivors affected by cgvhd usually take one or more immunosuppressants, as well as prophylactic antimicrobials; use of multiple medication classes concurrently poses a risk for drug-drug interactions or amplified side-effects. the use of medications other than cgvhd-direct immunosuppressive therapies has not been well-characterized. this study aims to evaluate patterns of opioid analgesic use in a cohort of patients severely affected by cgvhd. methods: patients (n= ) with cgvhd were consecutively enrolled in a cross-sectional natural history study (nct ) from / - / at the nih. participants underwent a comprehensive evaluation including a detailed history and physical examination (including current medications), multidisciplinary evaluations, and laboratory and diagnostic testing. for this analysis, respondents were classified as receiving or not receiving an opioid analgesic. following the initial screening by univariate methods (n= ), multivariable logistic regression analysis (mlr) was used to identify a set of factors which could jointly impact opioid use. for mlr data were divided into a training (n= patients) and a validation set (n= ). results: study participants´median age was . years ( - ), % were female, % had severe cgvhd per nih scoring criteria, and % were currently receiving high or moderate levels of systemic immunosuppression. approximately one third ( %) were taking opioid analgesics (oa). based on the univariate screening results (p< = . ), a set of parameters was evaluated by univariate logistic regression in the -patient training set, and the following parameters retained their significance and were included in the mlr model: nih average score per organ, total lss, patient impression of severity, nih cgvhd severity, presence of skin erythema, karnofsky performance score (kps,) clinician's therapeutic intent, nih joint score, and with the presence of several cgvhd symptoms including rashes, mouth sores, avoidance of food, vomiting, weight loss, joint and muscle aches, joint limitation, energy loss, need for naps, fevers, anxiety. multivariable logistic regression identified kps < % as predictive of oa use, or . , % ci . - . . in the training set . % of pts using opioids were correctly identified, . % of those not taking opioids were identified, an overall fraction of correctly identified pts was . % ( % ci . - . %), while in the testing set, . % of those using opioids were correctly identified, and . % of those not taking opioids were correctly identified, with overall . % ( % ci: . - . %) classification accuracy. conclusions: this study showed the burden of oa in this cgvhd cohort. lower kps was significantly associated with oa use, as well as self-reported symptoms and a more severe cgvhd disease, which could be of interest in the development of non-pharmaceutical interventions in this patient population. additional, prospective studies are needed to explore the indications for and effectiveness of oa in this population of survivors. disclosure: no conflict of interest to declare. rcts that tested an internet-based program and patientcentered survivorship care plans for hct survivors. patient and caregiver input is essential to inform the design and features for the mobile app platform so that it is usable and engaging for those it targets. methods: using a qualitative research design, we conducted telephone focus groups of adult patients and caregivers in the united states. adult (age > years at the time of study entry) hct recipients had to be at least oneyear post-hct to participate. participants had to be able to communicate in english, and could have received a hct for any diagnosis, and from any donor source or stem cell type. those who had multiple transplants were included. participants were asked to review printed and online visual presentations of the mobile app before the focus groups so they were prepared to discuss their responses to the materials during the call. focus groups were conducted to saturation, when no new qualitative content was offered. results: three focus groups were conducted with total participants ( patients, two caregivers/patient advocates). all patients received an allogeneic hct; average time since hct was years (range: - years).the majority of participants were female ( . %). participants had differing perspectives on the usefulness of the app to track follow-up appointments, lab values, and other health care plans. there was high interest in having the app tailored to meet specific needs of patients, including tracking information over time (e.g. test results, medications), and having health information available specific to their needs. to minimize duplication of information and data entry, participants recommended syncing the app with their calendars and online patient portals they already use. reasons provided for not using the app included perception that the materials repeated information already received, side effects such as graft-versus-host disease that restricted vision or motor skills, and lack of comfort with apps for some older participants. conclusions: many health technology and mobile apps are being created to improve patients' health and survivorship care. in this study, hct survivors and caregivers identified a variety of features that they would want in an app or website, in particular, features tailored to individual needs. health technologies provide an opportunity to improve survivorship care, but patients and caregivers should be engaged in the process of developing these tools to assure the technology fits their needs and will be used. given the effort required to maintain these technologies, they require testing for health benefits in rigorous clinical trials. clinical background: thanks to allogeneic stem cell transplantation (allo-hsct) patients suffering from hematologic malignancies have seen an increase in there life duration expectancy, but they are many side effets including decreasing in physical performance and in quality of life. the intensity of physical performance decrease is variable between patients, and today we did not know why. the aim of our study was first to characterize the physical performance of subjects less than year following allo-hsct by the use of a cardiopulmonary exercise testing (cpet), and then to determine the predictive factors of exercise performance. methods: we did a retrospective analysis from patients who had an allo-hsct at hematology department of toulouse-oncopole and cpet from / to / . the cpet was performed using a cycle ergometer with o and co analyzer breath by breath, (masterscreen cpx carefusion, san diego, usa), a continuous -lead electrocardiogram, and a blood pressure monitoring. the protocol included a -min rest period, a -min warm-up of w pedaling followed by a w/min incremental phase, up to exhaustion, then a -min active recovery of w pedaling, then a -min passive recovery. three exercise markers were analysed: the peak of oxygen uptake (peak vo ), the ve/vco slope and the first ventilatory threshold (vt ). data relative to conditioning regimen, short-term complications, impairment at cpet day, and physical activity since allo-hsct were gathered. results: after allo-hsct, nearly over patients reported fatigue, a half reported dyspnea, and over or more reported pain, muscular, neurological or psychological impairment. more than % of patients suffer from moderate or severe physical intolerance, particularly when myeloablative conditioning regimen was used. only % of patients followed rehabilitation sessions supervised by a physiotherapist, and non-supervised physical activity has been performed by % of patients. despite normal lung function tests and echocardiography findings in most patients, % had exercise intolerance (ei), % exercise deconditioning, and % had abnormal ventilatory efficiency. patients with moderate and severe impaired exercise capacity were significantly younger at diagnosis and at allo-hsct, such as patients with severe deconditioning conclusions: based on a retrospective study, we reported for the first time complete results from cpet and detailed clinical evaluation concerning deficiency and disability following first year after allo-hsct. these results confirm that exercise impairment is very frequent with more than a half of patients suffering from alterations of one or more of the three performance markers, despite being active. disclosure: nothing to declare demyelinating disorders: a paradigm of immunity disorders after hematopoietic stem cell transplantation background: neurologic complications are a major problem in patients who undergone hematopoietic stem cell transplantation (hsct). given the higher survival of transplanted patients, the burden of neurological complications is increasing in the last years. a significant reduction in overall survival was demonstrated in patients who developed neurological complication after hsct, irrespectively of the hematopoietic stem cell (hsc) source. neurologic disorders in transplanting setting comprise a wide variety of ethiologies including demyelinating disease, which are caused by immune and non-immune mechanisms. here, we analyzed the clinical presentation and the underlie ethiologies of patients developing hsct-related demyelinating disorders in order to give diagnostic and prognostic clues useful to manage these severe but treatable complications in the transplant setting. methods: a total of patients of our department which developed neurological complications after hsct were consecutively collected and ( %) of them, namely those having a diagnosis of a demyelinating disorder, were grouped and described according to the ethiologies of their neurological disorder. results: in / ( %) patients, an immune-mediated process was found, while / ( %) were diagnosed as having an infective etiology and / ( %) were supposed to have a demyelinating disorder caused by toxic exposition. a definitive etiologic diagnosis was not formulated in the remaining / ( %) patients. when patients who developed an immune-mediated demyelinating disorder ( / ) were compared to those in which a clear immune pathogenic mechanism was not detected ( / ), a higher incidence of acute graft-versus-host disease (agvhd) was detected in the former than in the latter ( % vs %). moreover, comparison of these two groups revealed that those with no evidence of immune-mediated process have a slight higher prevalence of t-cell depleted hsct thanthose with an immune-mediated demyelinating disorder ( % vs %). finally, a lymphoproliferative disorder pre-existing the hsct was detected in / ( %) patients with immune-mediated demyelinating disorder but only in / ( %) of those without evidence of immune-mediated processes. conclusions: demyelinating disorders may be responsible of near % of neurologic complications in the posttransplant setting and, among them, an immune-mediated process is likely to be involved in more than % of cases. our results suggest that the immune mechanism that underliesthe agvhd may also be involved in developing demyelinating disease in transplanted patients. it also may be possible that the lymphoproliferative disorder preexisting the hsct is a risk factor able to increase the risk to develop an immune-mediated demyelinating disorder in the post-transplanting setting. using a t-cell depleted hsct can increase the risk of immune-mediated disorders in at least a small fraction of transplanted patients. despite our results should be validated on a larger cohort of patients, we can speculate on the possible connections between the wide range of complex and still poorly defined immunity disorders which can influence the prognosis and course of transplanted patients. disclosure background: injury to the mucosal barrier and subsequent development of oral mucositis (om) is among the most common toxicities of allogeneic stem cell transplantation (sct). despite the high prevalence of om and its debilitating nature, prospective studies evaluating determinants of om are scarce. we therefore prospectively evaluated the occurrence of om following sct. risk factors for om and its implications short and long-term outcomes were assessed. methods: om was prospectively evaluated on a weekly basis in patients undergoing allogeneic hsct. the grade of om was determined based on the national cancer institute common toxicity criteria for adverse events (ctcae) scale (v. . ). severe om was defined as grade ii to iv. conditioning regimens were evaluated individually and according to intensity; myeloablative (mac), reduced intensity (ric) or reduced toxicity (rtc). the latter category included only patients receiving fludarabine and treosulfan at dose of - g/m (flu/treo). risk factors for the development of severe om were initially identified by a univariate analysis and then analyzed in a multivariate logistic regression model. association of om with peritransplant infectious complications, iv morphine consumption, hospitalization length, neutrophil engraftment, acute and chronic graft-versus-host disease (gvhd), non-relapse mortality (nrm) and overall survival were assessed in a univariate analysis. competing events were considered in analyzing engraftment, gvhd, and nrm. results: patients who underwent an allogeneic sct between and were included. median follow-up was days. leading indications for transplantation were acute myeloid leukemia ( %), lymphoma ( %), and myelodysplastic syndrome ( %). the majority of patients received an allograft from a matched sibling or unrelated donor ( %) and methotrexate gvhd prophylaxis ( %). the median time to om onset was (interquartile range [iqr] - ) days. prevalence of grade ii-iv om was %. the median duration om was [ - ] days, and iv morphine was administrated for a median of [ - ] days for patients with grades iii-iv om ( %). in a univariate analysis a younger age (p= . ), lower bmi (p= . ), recent smoking history (p= . ), recent antibiotics exposure (p= . ), mac (p< . ), and use of methotrexate (p= . ) were associated with an increased risk for grade ii-iv om. in a multivariable model the risk for grade ii-iv om was lower with rtc (i.e., flu/treo) vs. mac (odds ratio [or] . ; p< . ) and rtc vs ric (or . ; p= . ), mycophenolate mofetil vs. methotrexate (or . ; p= . ) and recent smoking (or . ; p= . ). compared to lower grades, grade ii-iv om was associated with a longer hospitalization duration (median days vs. days; p= . ), delayed neutrophil engraftment (median vs. days; p=- . ), and more gastrointestinal related infections ( % vs. %; p= . ). grade ii-iv om was not associated with increased risk of bloodstream infections, acute or chronic gvhd, non-relapse mortality, and increased mortality. conclusions: oral mucositis is prevalent among allogeneic-sct recipients. importantly, fludarabine-treosulfan, which is considered a myeloablative is associated with a markedly reduced risk for om. consequences of om include prolongation of hospitalization, delay in neutrophil engraftment, and a tendency for gastrointestinal infections, but does not increase the risk for gvhd and mortality. disclosure: nothing to declare background: the advent of recent diagnostic techniques for the assessment of iron overload (t *-mri) and their systematic use as screening tools in the setting of secondary hemochromatosis have led to an increased awareness that focal nodular hyperplasia (fnh) represents a possible incidental finding after hematopoietic stem cell transplantation (hsct). methods: clinical and radiological features of patients undergoing hsct in a single pediatric institution have been retrospectively reviewed for fnh. in order to provide an estimate of the prevalence of fnh after hsct, we analysed all the t *-mri scans performed during the last years in our centre and recorded the number of patients with fnh (group a). in addition, data about patients incidentally diagnosed with fnh at abdominal imaging performed for different clinical indications have been collected (group b). results: eight out of ( %) transplanted patients who underwent at least one t *-mri scan from september to september were incidentally diagnosed with fnh. group b included subjects with fnh incidentally found at ultrasound or non-t * mri scans performed before . overall, transplanted patients ( males, %), transplanted for al ( cases) or bone marrow failure ( cases) at a median age of . ± . years, were diagnosed with fnh between . and . years after hsct, namely . ± . years in group a and . ± . years in group b. a variable degree of iron overload was demonstrated in all patient (lic: - ± microg/g; baseline serum ferritin: - ng/ml). the potential risk factors for fnh are reported in table . in / patients, the radiological finding was pathognomonic; in / the diagnosis of fnh was confirmed histologically, while / subjects were labelled as "fnhlike", although a potential diagnosis of hepatic adenoma could not be ruled out. in / patients, fnh presented with an isolated lesion, while / had to more than hepatic nodules at diagnosis. the size of nodules at diagnosis ranged from to mm. in unenhanced mri scans, lesions were predominantly hyperintense on both t -and t weighted sequences. in dynamic studies with contrast medium, all lesions strongly enhanced during the arterial phase, with a variable degree of wash-out in the late venous scans. hepatic function tests were normal in all the enrolled patients at diagnosis of fnh. among the / patients for whom at least a follow-up scan was available, presented a complete regression, a reduction and an increase in the size and/or number of lesions, while in patients the nodules remained substantially unchanged after a mean radiological follow-up of . ± . years. no malignant transformations were observed. conclusions: fnh represents a relatively frequent incidental finding after hsct. although a malignant transformation is rare, given the demonstrated variable evolution of the hepatic nodules, a radiological follow-up is highly recommended. disclosure: nothing to disclose. incidence and risk factors for hepatic sinusoidal obstruction syndrome after allogeneic transplantation: retrospective multicenter study of turkish hematology research and education group (threg), updated data methods: ten centers from turkey were enrolled in the study. we retrospectively evaluated the medical records of patients who were treated with allo-sct between january and december . a baltimore criterion was used for assessment of hsos. four hundred twenty six ( . %) of patients who were treated with prophylaxis with defibrotide alone or one or more of the n-acetylcysteine, diuretics and heparin used defibrotide ( - mg/kg/day). results: the study included patients ( males/ females) with median age of ( - ) years. the demographic and clinical characteristics of patients were summarized in table seventy-three ( . %) of patients with hsos were treated with defibrotide after diagnosis. the median time of starting defibrotide in these patients was . ( - ) days. thirty-seven ( %) of patients with hsos recovered completely and forty-nine ( %) of them died as a result of multi organ failure. the incidence of hsos-related mortality in allo-hsct cohort was found to be . %. in univariate analysis, statistically significant associations were not found between hsos incidence and age/sex of recipient, type of conditioning regimen, stem cell source and type of gvhd prophylaxis. on the other hand donor type, engraftment status and prophylaxis for hsos were significantly associated with hsos development. hsos prophylaxis was significantly decreased hsos-associated mortality (p= . ). conclusions: hsos still remains a serious lifethreatening complication of allo-sct. although the incidence is low, hsos is associated with increased -day non-relapse mortality. hsos prophylaxis especially with defibrotide, seems to reduce hsos associated mortality in high risk patients. disclosure: nothing to declare prophylaxis with defibrotide in adults at very high risk of veno-occlusive disease: results in patients background: hepatic sinusoidal obstruction syndrome/ veno-occlusive disease (sos/vod) is a life threatening complication that can occur after hematopoietic stem cell transplantation (hsct). severe sos/vod rapidly evolves in multiple organ dysfunction syndrome (mods), associated with a mortality rate exceding %. precocity of defibrotide (df) treatment is the leading factor for efficacy. prophylactic use of df is recommended in children, but its value has not been validated in the adult population, although factors for individual risk assessment for vod are debated. we here present a real-world experience of df prophylaxis in adult patients at very high risk of sos/vod receiving allogeneic hsct. methods: from to we treated with prophylactic defibrotide and ursodeoxycholic acid (udca) patients, median age years (range - ). nine patients received allogeneic hsct for acute lymphoblastic leukemia ( b-all and t-all), one patient for severe aplastic anemia, one patient for primary myelofibrosis. they were all at high risk for sos/vod because of previous hepatotoxicity ( patients), previous hsct ( patients), double alkylating agent ( patients) or previous treatment with inotuzomab ozogamicin (io; patients). of the patients treated with io, received cycles of io, and received cycle, with the last io dose administered a median . days before hsct (range - d). defibrotide was administered in daily doses for a total dose of mg/ kg per day and udca at the dose of mg twice per day, starting from day - prior transplant. all patients received treosulfan-fludarabine based conditioning. in patients thiotepa was added to the conditioning and in patients a low dose gy tbi. gvhd prophylaxis included posttransplant cyclophosphamide, rapamycin and mycophenolate in all patients, except one patient with aplastic anemia receiving atg, rapamycin and mycophenolate. donor source was pbsc in all cases. seven patients received family haploidentical (mmrd) transplant, patient a mrd transplant and patients a mud transplant. results: the median duration of defibrotide therapy was days (range - days). documented non-severe gastrointestinal bleeding occurred in patients requiring defibrotide temporarily discontinuation, no other significant bleedings were experienced. four patients developed grade ii-iv acute gvhd and no transplant-associated thrombotic microangiopathy were diagnosed. overall, sos/vod occurred in / cases within days after hsct (days , and ) and no late-onset sos/vod were diagnosed. sos/vod was very severe, causing mods and death in all cases. all patients were characterized by a common pattern of very high risk factors for sos/vod by prior hsct and salvage treatment for b-all with cycles of io close to hsct. furthermore, they all received a fully myeloablative conditioning regimen with treosulfan and thiotepa and a mmrd transplant. conclusions: defibrotide prophylaxis was safe and well tolerated with no severe related complications. sos/vod occurred despite continuous df prophylaxis in / patients treated with inotuzomab ozogamicin close before undergoing nd transplant. to reduce the incidence of severe vod, pre allo-hsct treatment with inotuzomab ozogamicin should prompt avoidance of other cumulative risk factors for vod, such as use of double alkylating agents. disclosure background: busulfan is the backbone of many preparative regimens administered to children undergoing allogeneic and autologous hematopoietic stem cell transplantation (hsct). among its many long-term adverse effects, busulfan can cause various degrees of pulmonary injury. although well described in adults, there are few large series exploring pulmonary toxicity of busulfan in children. we describe long-term pulmonary follow-up in a large group of children treated at a single center who had received high-dose busulfan and examine the relationship of systemic drug exposure and lung function over time. methods: all surviving children who had received highdose busulfan between - in the context of hsct at the schneider children´s medical center, were referred for serial pulmonary function monitoring (including spirometry, plethysmography and diffusing capacity for carbon monoxide [dlco] . pre-transplant testing was available for children who were old enough to perform the procedure. spirometry results were adjusted according to the revised global lung initiative formulas for age, gender, and height. pulmonary injury was defined as a z score below - . for spirometry, or < % of predicted for the other parameters. busulfan levels were monitored following the second drug dose. all patients received busulfan in four daily doses. area under the curve (auc) calculations were performed by bayesian calculations. results: between - , patients aged - years were diagnosed with malignant or non-malignant diseases and treated with high-dose busulfan. of shortterm survivors, had at least one post-transplant pulmonary function evaluation. the mean age at treatment with busulfan was . years (range, . - years). of these children, children had undergone autologous transplantation and children had an allogeneic transplant. of these patients eventually relapsed and died. children had one or more pulmonary risk factors before hsct -chest or upper abdomen radiation ( ), chest wall tumors or lung metastasis ( ), chest surgery ( ), prior administration of pulmonary-toxic drug ( ) or asthma ( ). during follow-up (up to years, median . years), fev and fvc spirometry tests both decreased significantly (p= . and . , respectively), while the decrease in dlco was not statistically significant. % of patients had abnormal pulmonary function tests and seven children had symptomatic disease which in two may have been manifestations of gvhd. interestingly, no correlation was found between busulfan auc, busulfan peak levels, the number of busulfan doses administered, the type of transplantation (autologous vs. allogeneic) or primary disease to pulmonary injury. even after censoring of children with pre-transplant pulmonary risk factors we noted a decrease of fev and fvc. conclusions: as in adults, pulmonary injury is observed in children treated with high-dose busulfan prior to hsct. no correlation was observed between busulfan auc and pulmonary injury. follow-up of children who receive this drug should include regular pulmonary monitoring, referral to a pulmonologist when subclinical pulmonary compromise is found, and counseling regarding measures that might prevent or ameliorate pulmonary damage. continued follow-up of this cohort of patients should inform our pretransplant patient information sessions, and the future use of busulfan in children. disclosure: nothing to declare background: transplant-associated thrombotic microangiopathy (ta-tma) is a specific complication of allogeneic hematopoietic stem cell transplantation (hsct). post-hsct tma has been attributed to the vascular endothelial damage caused by high-dose chemotherapy, calcineurin inhibitors (cnis), graft-versus-host disease (gvhd), infections. there is a little evidence published regarding the efficacy and factors influencing the outcome of withdrawal of cnis. methods: the analysis comprised a total of patients, with diagnosed hematologic malignancy (aml ( ), all ( ), mds ( ), hodgkin lymphoma ( ), cml ( ) and neuroblastoma ( ) received allo-sct, from a matched related, unrelated or haploidentical donor between and . patients were diagnosed with ta-tma based on cho criteria. the median age of patients was ( adults, children). gvhd prophylaxis was performed with tacrolimus (tac) in , cyclosporine a(csa) in , combination tacrolimus+sirolimus (sir) in . patients received atg and ptcy. withdrawal of cnis was accompanied by administration of systemic steroids ( patients) or substitution with sir after reaching levels of csa< ng/ml or tac < ng/ml in . the target concentration of sir was - ng/ml. in pediatric patients who received combination tac+sir, the tac was discontinued in one step while sir continued. median time to development tma was , days after allo-sct (range - ). median follow-up of surviving patients was days. the primary outcome was overall survival (os) up to years after development of ta-tma. results: the following significant predictors of -year os were identified: tac replacement with sir (p< , ), ptcy in prophylaxis (p< , ), acute gvhd (agvhd) grade - (p= , ), previous sepsis (p= , ), level of ldh in debut (p= , ), combination sir+tac in prophylaxis (p= , ), major ab -mismatch (p= , ), severity of cns symptoms (p< , ). there was no significant difference in os according to patients' age, sex, "salvage" disease status at transplantation, previous vod, viral (hhv , , , cmv, ebv) reactivations, count of cd + cells transfused, ldh level, shizocytes and creatinine in the debut of ta-tma. in the multivariate analysis replacement of cnis with sir (hr . , %ci . - . , p= . ) and baseline ldh level (hr . , %hr . - . , p= . ) were associated with survival differences. the cut off for ldh was xunl. agvhd grade - (hr . , p= , ) and use of ptcy (hr . , p= . ) were not significant in the multivariate analysis (figure ). ta-tma cases after ptcy were significantly less frequently associated with clinically significant agvhd ( % vs %, p< , ). the survival was higher after ptcy ( % vs %), but not significant due to sample size and other ta-tma factors. leading causes of death were: gvhd progression ( %), bacterial infection ( %), tma ( %) and other ( %) . conclusions: replacing tac by sir is an effective therapeutic strategy in a group of patients with debut of ta-tma at least after ptcy, where it is less likely to be associated with agvhd. there is a significant overlap of populations with ptcy prophylaxis and substitution with sir, thus the study is not powered to provide guidance for patients on conventional prophylaxis with ta-tma. [[p image] . disclosure: none of the authors has anything to disclose. donor-recipient ab mismatch effect on the allogeneic hematopoietic stem cell transplantation outcome: a single-center retrospective study background: because transmission of major histocompatibility complex and blood group system genes is independent from each other, approximately - % of all allogeneic hematopoietic stem cell transplantations (allo-hsct) are realized crosswise the ab -blood group boundary. however, due to the widespread expression of ab antigens on a variety of human tissues other than erythrocytes, ab incompatibility may have an impact on the outcome of allogeneic hsct that goes beyond the wellknown immune-hematological complications such as immediate hemolysis due to the presence of isoagglutinins and delayed hemolysis due to passenger b lymphocytes. here we aimed to assess the donor-recipient ab mismatch effect on the allo-hsct outcome, comprising non-relapse mortality (nrm), overall and relapse-free survival, posttransplant prc transfusion requirement, as well as relapse rate, incidence of graft-failure and acute gvhd. methods: clinical and laboratory data from consecutive patients undergoing allogeneic hsct between / and / at the fondazione irccs ca' granda maggiore policlinico hospital in milan, italy, were retrospectively collected. kaplan meier estimates were used for the analysis of survival outcomes while nrm, relapse and acute gvhd cumulative incidences were investigated by competing risk analysis. results: the patient series included ab -match, major ab -mismatch, minor ab -mismatched and bidirectionally ab -mismatch transplants. indication for allo-hsct were mainly aml/mds ( pts), all ( pts) and t-nhl/ctcl ( pts). mean overall survival for groups of patients undergoing ab -identical, major ab mismatch and minor ab mismatch hsct were months ( % ci [ ; ]), months ( % ci [ ; ) and months ( % ci [ ; ]), respectively. nrm in the three groups were significantly different, with point estimates of %, % and % at years, respectively, whereas no significant differences were observed for relapse rate and graft failure incidence. although not statistically different, incidence of acute grade iii-iv gvhd was twice as high in patients transplanted from minor ab mismatched donors than in the ab identical group ( % vs %). following transplantation, prc transfusion requirement was significantly higher in the major ab mismatch then in the ab -match transplanted patients (median vs , p= . ), with a marginal positive correlation between the anti-donor a/b igg titers measured prior hsct and the total number of prc transfused during the first year following transplantation. we observed only one case of prca occurring in a year-old + woman who was transplanted from a -yearold male a+ hla-identical sibling using peripheral blood as the stem cell source following a myeloablative conditioning for aml in first complete remission. anti-a igg isoagglutinin titers prior to transplantation were : . during the first year post transplantation, the patient required a total of prc transfusions, with gradual resolution occurring only after introduction of danazole treatment. conclusions: in our patient cohort, both major and minor ab mismatch associated to a significantly higher nrm. major ab mismatch associated to a higher prc transfusion requirement. a more frequently occurring severe acute gvhd was also suggested in minor ab -mismatch transplants. altogether, our results suggest that allo-hsct outcome may be significantly affected by ab blood group mismatch. disclosure: nothing to declare background: at-tma is a severe endothelial injury complication and it may involve the intestinal vasculature. intestinal tma could be fatal and missdiagnosed. clinical and pathological criteria to differentiate from intestinal gvhd are needed. the aim of this study was to analyze the incidence and histological characteristics of intestinal tma in patients diagnosed of systemic tma. methods: we analyzed the incidence of tma in patients who underwent allo-hsct in our institution between january -august . tma diagnosis was based on ho criteria. we do a pathological review in biopsies from out of patients in whom an endoscopy have been performed days before and days after the diagnosis of tma for suspicious of gvhd. review was performed by a pathologist expert in gvhd, who examined the biopsies in search of hystopathological features of gvhd, tma or viral infection. diagnosis of gastrointestinal gvhd was stablished according to mcdonald and sales criteria, while intestinal tma diagnosis was stablished by warren et al criteria. results: out of patients ( , %) were diagnosed of tma. transplant characteristics and tma data of patients with systemic tma are shown in image. out of patients with tma ( %) had been diagnosed with prior/ simultaneous acute gvhd, of them grade iii-iv, and % with gastrointestinal gvhd. intestinal tma have been reported only in out of patients ( %) at diagnosis, whereas when review based on warren criteria was performed, in patients ( %) the pathologist found at least of the criteria of endothelial damage and % of the patients or more warren criteria were founded. the most frequent features were endothelial cell swelling (n= , %) and perivascular mucosal hemorrhage (n= , %). review hystological features of biopsies are shown in table of the image. regarding gvhd, it was found in patients ( %) at diagnosis and in ( %) at pathological review. with a median follow-up of months ( - ) patients of the with systemic tma ( %) are dead. of the deaths ( %) were related to tma ( tma, tma +gvhd, and tma+infection). patients with or more warren criteria in pathological review had poor outcome compared with patients less than criteria ( % alive vs % at months, p= . ). conclusions: intestinal tma is a life-threatening underdiagnosed entity. only patients of patients were diagnosed of intestinal tma. we found that most of our patients had endothelial damage in the gastrointestinal biopsy pathological reviews. gvhd histological criteria were present in most of the patients, mainly histological grade i-ii. prognosis of these patients is poor and pathologist effords in diagnosed the entity is guarranted. disclosure: nothing to disclosure p strategies to reduce neutropenic fever and hospital readmission in multiple myeloma patients managed at home after autologous stem cell transplantation background: neutropenic fever (nf) is the most frequent cause of readmission in the outpatient autologous stem cell transplantation (asct) programs. in our at home model for multiple myeloma patients, we added primary prophylaxis with ceftriaxone, decreasing the incidence of fever during aplasia phase from % to . %. the aim of this study was to analyze the addition of two strategies to reduce the non-infectious nf: withdrawal of g-csf and the addition of primary prophylaxis for engraftment syndrome with corticosteroids after asct. methods: between january and august myeloma patients were managed at-home since day + of asct. all were conditioned with mel . all patients received prophylaxis with quinolone, fluconazole, aerolized pentamidine, low-dose acyclovir (hvs+), and ceftriaxone (since day + ). the patients were classified into groups: group a (n= ; g-csf since day + without corticosteroid), group b (n= ; no g-csf and no corticosteroid), group c (n= ; no g-csf with prednisone . mg/kg/day since day + until granulocyte recovery). first-line therapy at home of nf was piperacillin-tazobactam . g/ h i.v. using a portable intermittent infusion pump. fever was an indication of immediate medical consultation and those patients presenting signs of focal infection or severe sepsis were admitted. other indications for readmission were: willingness of the patient or caregiver, uncontrolled nausea, vomiting or diarrhoea, and mucositis requiring total parenteral nutrition or i.v. morphics. results: the main characteristics of the patients and outcomes are shown in table . there were no differences between groups regarding age, gender, immunological subtype, response before asct, hct-ci, and cd cell dose infused. there were more patients with advanced disease (iss iii) in group c compared to group a ( . % vs. . %; p= . ). the duration of neutropenia was longer in those groups that did not receive g-csf (a: days, b: days, c: days; p< . ). comparing group a with group c, we observed that the incidence of nf and the readmissions rates were lower in group c (nf: . % vs. . %; p= . ; relative risk reduction: . , and number needed to treat . ; readmissions: . % vs. . %; p= . , respectively). the -day cumulative incidence of nf were . % in group a, . % in group b, and . % in group c; p= . . the non-administration of g-csf with the addition of prophylactic corticosteroid did not modify the incidence and grade of mucositis, the first day and duration of fever, nor the number of bacterial infections documented. in the multivariate analysis, this combination (no g-csf with corticosteroid) maintained its protective effect for the development of nf and hospital readmission (or . ; p= . and or . ; p= . , respectively). conclusions: the non-use of g-csf and the addition of prophylactic corticosteroid in mm patients managed at home after asct minimize the incidence of non-infectious fever and optimize hospital resources by reducing hospital readmissions. disclosure: nothing to declare. background: antibody titers to vaccine-preventable diseases decline during the - years after allogeneic hematopoietic stem cell transplantation (hsct) if the recipient is not revaccinated. it is therefore considered best practice to try to offer hsct recipients the same level of protection against all vaccine preventable diseases as the general population. few data in the literature are available concerning vaccine-related problems in hsct recipients. we performed a farmacovigilance evaluation in a cohort of allotransplanted patients followed in our clinic during a year period. methods: from october to november we administered a list of recommended vaccines to hsct recipients attending our routine out patient clinic who fulfilled the following criteria: cd t cells> /μl, cd b cells> /μl, anti-cd antibody infusion> months, ivig therapy> months, no active and severe graft-versus-host-disease (gvhd), no chemotherapy or biological therapeutic agents on going. vaccines suggested were influenza, pneumococcal conjugate (pcv ), polio (inactivated polio vaccine), diphteria, tetanus, acellular pertussis, hepatitis b, hepatitis a, haemophilus influenzae type b, meningococcal quadrivalent (mcv ), human papillomavirus, meningococcal b, measles-mumps-rubella (mmr), varicella. live vaccines (mmr and varicella) were not recommended before years after hsct and in patients with chronic gvhd. all the patients were asked to take the list to the local health facilities in order to have the vaccines injected and a vaccination table arranged with the doses already received and those to receive. we checked the vaccination tables at each visit and monitored potential side effects and gvhd status at , , and months after the first vaccine injection. results: twenty-nine out of patients were evaluable (table ), without gvhd and with chronic gvhd ( mild, moderate, severe). median time after hsct was months ( - ). median number of vaccines received was ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) . as regards patients without chronic gvhd, out of experienced fever after vaccine injections; out of developed transient mild reduction of platelet count; patient reported headache and otalgia after vaccine injection, while another one transient joint pain; out of patients presented signs of mouth chronic gvhd (score nih) and transaminase increase (grade according to world health organization toxicity scale) months after the first vaccine dose, so that cyclosporine dose had to be augmented. as regards patients with chronic gvhd, out of experienced fever after vaccine injections; patients with mild chronic gvhd of the mouth presented hepatic flare two and three months after the first vaccine dose, respectively. in both cases a new increase of cyclosporin and methylprednisolone doses determined progressive normalization of liver enzymes. conclusions: these data show that vaccines were globally well tolerated in hsct recipients, even when they suffered from chronic gvhd. however, close monitoring is warranted in order to better evaluate possible vaccine side effects in this setting of patients. background: allogeneic hsct improves survival for aml patients over the age of years of age when compared to chemotherapy alone. the haematopoietic stem cell transplantation comorbidity index (hct-ci) and ebmt score predict for non-relapse mortality and overall survival, yet little is known about whether qol is preserved in this patient group and whether hct-ci and other performance scores pre-bmt correlate with qol post allo-hsct. methods: we conducted a retrospective analysis of patients years and older who underwent ric allo-hsct at the university hospital of wales, cardiff between september and december (n= ). hct-ci, karnofsky performance score (kps) and ebmt scores were calculated prior to transplant and qol measured using the fact bmt (version ) questionnaire, which was completed at , and months post transplant. patients were grouped at the -, -and -month time points for each of the different performance indices, allowing group comparison against compound sub scores using the mann-whitney u test. results: patients were included in this study, with median age years (range - ). patient characteristics, including conditioning, donor type, pre-transplant hct-ci and kps scores are summarised in table . the year and year overall survival (os) for the patient cohort was . % and . % respectively. hct-ci of ≥ vs was significantly associated with poorer bmt-related qol domains at months (p= . ) and general qol domains at months (p= . ) post-transplant. while ebmt score showed no correlation with qol parameters, patients with kps of vs ≤ showed significant differences in both general (p= . ) and bmt-related qol (p= . ) at months and in all qol domains at months (symptomrelated qol p= . , general qol p= . , bmt-related qol p= . ). importantly neither the hct-ci nor the kps pre-transplant predicted for qol at months post transplant. conclusions: patient selection is key to ensuring maximum benefit from allo-hsct both in terms of overall survival but also with regards to qol and survivorship. we note that while patients with hct-ci ³ or kps ≤ had significantly poorer qol at months post allo-hsct, qol was recovered by months post transplant, with this significant difference no longer seen. our data shows that in selected aml patients over the age of years with good performance status and low comorbidity index, a favourable outcome can be achieved with good qol maintained throughout the post transplant period. background: advances in allosct technology, supportive care, and use of reduced intensity conditioning regimens for older patients have led to significant improvements in longterm survival after transplant. the survivors have an elevated probability of late morbidity and mortality, including abnormalities in phosphocalcic metabolism and bone disease. rapid and progressive bone loss occurs within the first - months after transplant, and this is followed by a slow process of recovery, with bone loss persisting for to months. bone fractures can worsen the quality of life of allosct survivors, but the real burden of the disease is unknown. the objective of the study is to ascertain the prevalence of bone pathology and vertebral fractures early after transplant in our center. methods: this is a retrospective and observational study. forty-nine patients ( male/ female, median age y, range - ) that underwent allosct were included in the study in the period of to months after transplant (may -december ). pre-and post-transplant risk factors associated with bone disease were recorded: age > years, female sex, menopause, hormone replacement therapy, previous treatment with steroids, previous fractures, weight < kg, bmi < - , low physical activity, low calcium intake, smoking, alcohol intake, and history of femoral fractures in parents. in all patients laboratory data (including serum calcium, -hydroxyvitamin d, and pth), lumbar and femoral bmd (dxa), and spinal x-ray were also evaluated. a vertebral fracture was defined as a reduction of > % in the anterior, middle or posterior high of the vertebral body. results: we identified vertebral fractures in ( %) patients. five patients had fractures prior to transplantation, and patients presented "de novo" vertebral fractures following transplantation; therefore, the prevalence of "de novo" postransplant fractures was / ( %). most ( %) of these fractures were asymptomatic at the time of diagnosis. most patients ( %) with vertebral fractures had > pre-sct risk factors (median risk factors pre-sct , range - ), the most frequent being low calcium intake, steroid exposure, presence of previous fractures, and menopause. those patients with fractures and less than risk factors pre-tph, added new risk factors after transplant, mainly steroid treatment. forty-four patients ( %) had vitamin d insufficiency (< ng/ml), ( %) had osteopenia and ( %) had osteoporosis. vitamin d insufficiency and bone disease were more frequent in women than in men ( % vs. % for vitamin d, % vs. % for osteopenia, % vs. % for osteoporosis, and % vs. % for vertebral fractures, respectively). conclusions: the prevalence of post-transplant bone disease and vertebral fractures in our series is high. most fractures appearing "de novo" after allosct were asymptomatic and were diagnosed by x-ray. patients who presented vertebral fractures frequently had more than risk factors identified pre-sct. patients undergoing allosct should have their bone health assessed early in their treatment and, if indicated, should start preventative therapy to avoid bone loss and fractures. other measures such as physical exercise, vitamin d and calcium supplementation, and dxa and spinal x-ray at baseline and following transplantation are also highly recommended. disclosure: maría suárez-lledó received a grant from dkms-spain foundation. other authors have nothing to declare the use of g-csf in selected patients after autologous stem cell transplantation is associated with low incidence of engraftment syndrome background: the use of g-csf after autologous stem cell transplantation (asct) accelerates neutrophil recovery, however it has been related to an increased risk of engraftment syndrome (es) development in some studies. for this reason, we do not routinely prescribe g-csf after asct and we only use it in patients with significant complications (enterocolitis, severe sepsis, atrial fibrillation) after stem cell infusion. the main objective of this study is to evaluate the incidence of es in patients who receive asct for monoclonal gammopathies (mg), non-hodgkin lymphoma (nhl) and hodgkin lymphoma (hl) and receive g-csf only if needed. as secondary objectives we evaluate differences in the engraftment day as well as the length of inpatient stay. methods: we retrospectively analyzed patients with mg or lymphoma, who underwent asct conditioned with high dose melphalan ( - mg/m ) or beam, respectively, between and in our center. specific clinical features for es according to spitzer and maiolino criteria were evaluated between days before and days after the engraftment. statistical analysis was performed with spss v. . . results: thirty-one patients with mg and patients with lymphoma were analyzed. median age at transplant was . years ( . - . ) and patients ( . %) were male. median prior lines of treatment in patients with gm or lymphoma were ( - ) and ( - ), respectively. table shows patients´characteristics. mobilization with g-csf ± plerixafor was performed in patients ( %) and chemotherapy + g-csf ± plerixafor in patients ( %). median cd x /kg cells infused was . ( . - . ). eleven patients ( . %) received g-csf, due to infection ( enterocolitis, listeriosis, acute hepatitis, septic shock) and because of atrial fibrillation or fibrilloflutter. median time from sct to first day of g-csf was days ( - ) and median time on g-csf treatment was days ( - ). patients who received g-csf showed a short time to neutrophil engraftment (≥ . x /l), days vs. days, p< . but longer duration of hospitalization, days vs. days, p = . . non-relapse mortality at day + , + and + was %. es was diagnosed in ( . %) patients, amyloidosis, multiple myeloma and plasmablastic lymphoma. there was not statistical difference in the incidence of es between patients who received g-csf ( . %) and patients who did not ( . %), p= . . analyzed by disease, es appeared in of patients who received g-csf in the lymphoma group ( . %) but none of the patients with mg that received g-csf developed it. we did not find statistical differences between patients who developed es and those who did not in age ( years vs. years, p= . ), length of hospitalization ( days vs. days, p= . ) and the number of cd x /kg cells infused ( . vs. . , p= . ) . conclusions: the use of g-csf in selected patients is associated with low incidence of es. our study confirms that the use of g-csf accelerates neutrophil recovery but it is unclear if it can increase the incidence of es, especially in patients with lymphoma. [[p image] . background: graft failure is one of the top- problems of allo-hsct (after gvhd and relapse). the problem of graft failure becomes more significant due to increasing number of allo-hsct with ric conditioning regimen from haploidentical and hla-mismatched unrelated donors. role of t cells in graft failure is well known. here we report an impact of t-memory cell subsets count before antithymocyte globulin (atg) administration on primary graft failure after allo-hsct. methods: sixteen patients with acute leukemia transplanted in national research center for hematology were included on this prospective study. all patients received horse atg at dose mg/kg/day from day - to - before allo-hsct as gvhd prophylaxis and were balanced by other factors that could affect engraftment. detailed patients characteristics are listed in table . peripheral blood samples were collected on day - before allo-hsct (before atg injection) in edta-tubes. flow cytometry analysis was performed on bd facs canto ii (becton dickinson, usa) to define t-memory subsets: t-naive and t-stem cell memory (tnv+scm) -cd r -ccr +cd +; t-central memory (tcm) -cd r +ccr +cd +; t-transitional memory (ttm) -cd r +ccr -cd +; t-effector memory (tem) -cd r +ccr -cd -; t-terminal effector (tte) -cd r -ccr -cd -, among cd + and cd + t-cells . sysmex xe- was used to calculate absolute count of different t-cell subsets. mann-whitney u test was used for nonparametric data analysis between two groups. fisher's exact test was used for x tables. p-value less than . was considered statistically significant. results: an influence of t-memory cell subsets count before atg administration on primary graft failure is shown in figure . according to our data high absolute number of cd +ttm and cd +tte is associated with primary graft failure. conclusions: based on these findings high absolute number of cd +ttm and cd +tte could be one of the prognostic factors of primary graft failure after allo-hsct. optimizing atg dose due to recipient absolute t-memory cell subsets count before atg administering may prevent graft failure and improve posttransplant results. background: upper gastrointestinal graft-versus-host disease (gi gvhd) has been an increasingly recognised entity following allogeneic stem cell transplantation (sct). budesonide, widely used in inflammatory bowel conditions, has also been found beneficial in gi gvhd. the major benefit of budesonide is attributable to its poor absorption and extensive first-pass metabolism via cytochrome p (cyp) a , which translates to less systemic steroid-related effects. however, transplant patients are often exposed to multiple drugs, among which some agents act as cyp a inhibitors and therefore can increase budesonide bioavailability and might lead to systemic toxicity. azole antifungal drugs are probably the most common concomitantly used cyp a inhibitors in transplant recipients. methods: we reviewed allogeneic sct records for patients treated with oral budesonide for gi gvhd at our transplant centre between and retrospectively. the aim of the work was to assess the development of adrenal suppression with or without clinical features of iatrogenic cushing`s syndrome. the standard dose of budesonide was mg three times a day. patients receiving prednisolone or other glucocorticosteroids and those with no available serum cortisol level measurements were excluded. results: our analyses identified four allogeneic sct patients in whom adrenal suppression was diagnosed with undetectable serum cortisol levels during oral budesonide treatment. of these patients two developed iatrogenic cushing`s syndrome and both patients were treated with cyp a inhibitors concomitantly: . clarithromycin and fluconazole; . clarithomycin and voriconazole. the development was rapid (within and weeks). symptoms included morphological features such as moon face, high blood pressure, weight gain, peripheral oedema and proximal myopathy. symptoms resolved gradually following cessation of azole antifungal agents and on gradual weaning of budesonide. conclusions: although single agent budesonide treatment given for gi gvhd is rarely associated with systemic side effects, patients on azole antifungal drugs and macrolide antibiotics are at higher risk of systemic toxicity due to drug interactions. patients who are allergic to penicillin and receive macrolide-based prophylaxis can be especially vulnerable. to our knowledge the number of cases reported in literature about systemic effects of oral budesonide in transplant recipients is less than . our observation supports previous reports on the potential of oral budesonide to induce systemic effects. we therefore advise careful monitoring of patients treated with budesonide in combination with cyp a inhibitors, including antimicrobial agents routinely used in sct. disclosure: none implemented strategies to overcome barriers in the establishment of a consolidated hematopoietic stem cell transplantation program in a developing country background: the national institute of medical sciences and nutrition "salvador zubiran" is a national health institute located in mexico city. although mexico is considered an upper-middle income country, more than % of the population lives in poverty without health care coverage and patients within this social stratum are referred to our institution. the first hematopoietic stem cell transplantation (hsct) in mexico was performed at our institution in . from this year until , hsct were sporadically performed (n= ), showing a poor overall survival (os) and high non-relapse mortality (nrm). these outcomes resulted from an unstructured hsct program, limitedresources, patient low socioeconomic status, and paucity of population-adapted procedures. in , according to these results, a decision to establish a hsct program was made. therefore, in order to set up a successful hsct program, implementation of financial and medical strategies were necessary. the objectives of this study were to describe the barriers and implemented strategies for the establishment of a hsct program in mexico along with the outcomes of patients undergoing this procedure throughout the reorganization of the program. methods: this study is a health services research. barriers were detected based on the results of the hsct program from - (not shown). table shows the financial, medical, and research strategies that were implemented for each barrier. results: from november to november , hsct have been performed in patients at our institution. most hsct were autologous (n= , %). forty one patients underwent hsct. from the patients, most were males (n= , %) and the median age was . years (range, - ). the most frequent underlying diseases for auto-hsct were lymphomas (n= , %), non-seminomatous germ cell tumors (n= , %), and multiple myeloma (n= , %). acute leukemias (n= , %), aplastic anemia (n= , %), and myelodysplastic syndromes (n= , %) were the most frequent diagnosis for patients undergoing allo-hsct; and acute leukemia was the most frequent diagnosis for patients undergoing haploidentical hsct (n= , %). acute and chronic gvhd were present in % (grades i-ii %) and % (limited %), respectively. for allo-hsct, , day, and -year nrm was . %, %, and %, respectively; and -day nrm in auto-hsct was . %; year os was % and % for auto and allo-hsct, respectively. conclusions: future perspectives of the hsct program include the acquisition of funds for unrelated donors; to improve outcomes of patients undergoing haploidentical hsct, and to increase the number of in-patient rooms. we conclude that despite paucity of resources and other limitations, the implementation of financial, medical, and research strategies have shown that barriers can be effectively overcome in a developing country in order to establish a consolidated and nationally renowned hsct program, providing good outcomes for patients. disclosure: none of the authors have any conflict of interest to disclose. the effect of protective buffering on daily stress and relationship quality in dyads following hematopoietic stem cell transplantation: results from daily process methodology malgorzata sobczyk-kruszelnicka , aleksandra kroemeke , zuzanna kwissa-gajewska , sebastian giebel background: cancer-related support communication (e.g., protective buffering) may impact the risk for psychological and relationship distress in patients following hematopoietic stem cell transplantation (hsct) and their caregivers. previous studies have revealed that protective buffering (i.e., hiding one's concerns and denying one's worries) has mixed effects: is beneficial (for "protected" person), costly (especially for the person using it), or unrelated to dyadic wellbeing. there has been, however, little evidence linking dyadic protective buffering with distress using daily process methodology. we assessed ( ) the relationship between daily protective buffering, and same-and next-day stress and relationship quality in patient-caregiver dyads following hsct and ( ) whether similarity or complementarity in protective buffering between dyads is adaptive. methods: two hundred patients (after first autologous or allogeneic hsct) and their caregivers (spouse or another relative) independently completed measures of daily protective buffering, daily relationship quality, and daily stress for consecutive evenings after patients´hospital discharge. actor-partner-interdependence model (i.e., both partners' and caregivers' reports regarding support communication and distress were studied) was used to test study hypotheses. results: for both patients and caregivers, multilevel structural equation modeling showed a significant positive relationship between daily protective buffering and sameday relationship quality. association of protective buffering with same-day stress level was negative. in next-day analyses, patient-reported protective buffering was related to patient's higher relationship quality, whereas caregiverreported protective buffering increased patient's daily stress. complementarity in protective buffering was related to higher immediate same-day relationship quality for both patients and caregivers, while benefits from similarity have delayed effects, although only in patients. conclusions: contrary to previous studies, protective buffering rather has a beneficial effect in dyads following hsct. protection of the partner and relationship against revealing negative emotions and powerlessness was not related to costs in both parties. the findings suggest that the effect of daily protective buffering in dyads following hsct depends on support timing (same-or next-day effect) and differs for both parties. patients seem to benefit the most from the similarity in protective buffering, while caregivers from complementarity. the "fit" between patient and caregiver in support communication ought to be taken into consideration in the practical approach. disclosure: nothing to declare. virus reactivation and low dose of cd + cell were associatied with secondary poor graft function within the first days after allogeneic stem cell transplantation yuqian sun , xiao-jun huang background: secondary poof graft function (spgf) was defined as the secondary cytopenia after initial engraftment of hsct. it was shown to be associated with poor prognosis, however there are very few reports on the incidence, risk factors and outcomes of spgf. methods: patients who received transplantation from peking university people's hospitial during january, to december, were retrospectively reviewed if they fulfilled the following conditions: ( ) diagnosed with acute leukemia or myelodysplastic syndrome; ( ) received allo-sct from either matched sibling donor (msd) or haploidentical related donor (hid). pgf was defined as persistent neutropenia (≤ . × /l), thrombocytopenia (platelets ≤ × /l), and/or hemoglobin ≤ g/l for at least consecutive days, transfusion-dependence, associated with hypoplastic-aplastic bone marrow (bm), and complete donor chimerism without concurrent graftversus-host disease (gvhd) or disease relapse. primary pgf was defined as the failure to achieve initial engraftment by days after transplantation, while secondary pgf was defined as the fulfillment of the criteria after initial engraftment hsct. results: during january, to december, , patients who received transplantation from peking university people's hospitial were retrospectively reviewed. among the patients who achieved initial engraftment, patients developed spgf. the cumulative incidence of spgf on day was . %. the median time of secondary pgf was . ( - ) days after transplantation. low (< median) cd + cell dose (p= . , hr . ( %ci, . - . )), ebv reactivation (p= . , hr . ( %ci, . - . )) and cmv reactivation (p= . , hr . ( %ci, . - . )) were identified as independent risk factors with spgf. there is no significant difference of pgf incidence in msd group and hid patients (p= . ). the overall survival of patients with spgf at year after transplantation was significantly poor than patients with ggf ( . % versus . %, p< . ). conclusions: in conclusion, spgf develop in . % patients after allo-sct, especially in patients with cmv, ebv reactivation or infused with low dose of cd + cell. the prognosis of spgf is still poor due to lack of standard treatment. disclosure: there is no conflict of interet thiotepa with treosulfan and busulfan based conditioning are significantly more gonadotoxic than treosulfan previous studies suggest that busulfan results in long-term gonadal toxicity. no previous studies have compared gonadal toxicity outcomes after treatment with busulfan with treosulfan, a newer agent with similar marrow toxicity to busulfan but with reduced non-marrow toxcitiy. our aim was to determine whether there are differences in pubertal and fertility outcomes in paediatric patients treated with treosulfan compared with busulfan. methods: inclusion criteria were patients who had received either busulfan or treosulfan or treosulfan with thiotepa, only one hct and were aged years and above in august . eligible patients were reviewed in clinic as part of their routine follow-up, thus research ethical approval was not required. follice stimulating hormone, luteinising hormone, oestradiol, and pubertal history were noted. ovarian reserve was estimated in female patients by measuring serum anti-mullerian hormone (amh). male patients had serum testosterone measured and were also offered semen analysis. results: thirty-five patients met the inclusion criteria, of which twenty-five wanted to be reviewed ( %); seventeen females and eight males. mean age at hct was years, mean age at review was years and mean years since hct was years. female patients treated with busulfan or treosulfan with thiotepa (n= ) had minimal amh and none of these patients were having regular periods. females treated with treosulfan (n= ) had normal amh and regular periods without needing hormone replacement. only four male patients opted for a semen analysis and all had significantly reduced sperm counts. conclusions: our results suggest that females treated with treosulfan have minimal (if any) reduction in ovarian reserve compared to other conditioning regimens which casue significant compromise. although this was a small study, and thus not suitable for statistical analysis, the clinical findings are marked. future studies should further investigate optimal doses of treosulfan that could be used to achieve bone marrow engraftment and limit long-term effects on fertility. disclosure background: autologous and allogenic hematopoietic stem cell transplantation (hsct) are potentially curative treatments for hematological malignancies. patients with related complications may need admission to the intensive care unit (icu) for specific therapy and organ support. mortality risk factors, supportive care and principal causes of admission in icu are described in our cohort of patients (pts). methods: we retrospectively studied pts, male, with a median age of , years (range: - ) who underwent allo-hsct in our center between july and october . two hundred and twenty-seven( , %) pts received autologous hsct (auto-hct) and ( , %) allogenic hsct (allo-hsct); from unrelated donor, from identical sibling, and the remainder, mismatched related donor . twenty-three ( , %) out of pts were admitted in the icu in the transplant procedure admission. results: fifteen ( , %) out of pts were male with a median age of years (range: - ). patients' baseline diseases were: multiple myeloma ( , %), non-hodgkin´s lymphoma ( , %), hodgkin´s lymphoma ( , %), acute lymphoblastic leukemia ( , %), myelodisplasic syndrome ( , %), solid tumor ( , ) and acute myeloblastic leukemia ( , %). fifteen ( , %) pts received auto-hsct, ( , %) allo-hsct from unrelated donor, ( , %) allo-hsct from identical sibling, and the remainder haploidentical hsct ( ) ( , %). so, , % of auto-hsct pts and % of allo-hsct were admitted in the icu. the median stay in the icu was days (range: - ) and reasons for admission were: respiratory insufficiency ( , %), septic shock ( , %), renal insufficiency ( , %) and multi-organic failure ( , %). twenty-one ( . %) pts required respiratory support with: nasal cannula or oxygen mask (c/m) ( %), non-invasive mechanical ventilation (nimv) ( , %) and invasive mechanical ventilation (imv) ( , %). fourteen ( %) pts needed inotropic agents for shock treatment. finally, ( , %) pts required substitutive renal therapy with hemodialysis or haemofiltration (hd/hf). eleven ( , %) out of pts died, ( , %) were male with a median age of years (range: - ). ten of them ( , %) needed imv and were treated with inotropic agents. all patients who required hd/hf (n= ) died. imv and treatment with inotropic agents were associated with icu mortality (or , ; p= , , or ; p= , ; respectively). conclusions: in our series of pts, , % needed admission in the icu, presenting a mortality rate of % approximately. there were no differences in the prevalence of icu admission regarding hsct donor. main reason for admission was respiratory failure with imv requirement in , % of pts. imv and treatment with inotropic agents were associated with icu mortality. an early identification of pts at risk of icu admission could have a beneficial impact on survival improvement disclosure: nothing to declare is there any association between thrombotic risk factors and veno-oclusive disease in childhood allogeneic hematopoietic stem cell transplantation? background: veno-oclusive disease (vod) is a major complication of hematopoietic stem cell transplantation (hsct). in some studies levels of fibrinolytic factors especially plasminogen activator inhibitor- (pai- ) level were found associated with vod. however, little is known about the relationship between thrombophilia risk factors and vod. in this study we aimed to investigate association of major thrombophilic gene mutations on vod in pediatric hsct patients. methods: we reviewed retrospectively patients with vod who underwent hsct between - in ankara pediatrics and pediatric hematology-oncology training and education hospital, bone marrow transplantation unit, turkey. fifty-one patients who did not develop vod and transplanted during the study period were accepted as control group. we evaluated plasma homocysteine and lipoprotein a level, protein s and c activity and antigen levels and factor v g a mutation, prothrombin g a mutation, methylenetetrahydrofolatereductase (mthfr) c t and a c mutations, plasminogen activator inhibitor- - g/ g polymorphism before hsct. we also evaluated the patients' hospital files and noted the demographic values and complications of hsct. statistical investigations were done with spss statistics . for windows and p< . has been accepted as significant. results: there was no difference between control and vod groups as regard to age, sex, diagnosis, donor type, conditioning regimen, hsc source, and hla typing . there was no difference between the groups according to homocysteine, lipoprotein a, protein s and c activity and antigen levels. we did not find any relation between the genetic variations of thrombophilia and vod (table ). in vod group there were patients ( . %) with acute graft versus host disease (agvhd) and in control group there were ( . %) patients with agvhd (p= . ). febrile episodes were more frequent in vod group compared to the controls (respectively; n= , . % vs. n= , . %, p= . ). -year overall survival was % . in vod group and % in control group (p= . ). disease free survival was also different between vod and control groups (respectively; . % vs. . %, p= . ). conclusions: in literature there are recent studies showing higher pai- levels in patients with vod. however, in our study we did not find any relationship between congenital thrombophilia factors and vod. new studies with larger sample groups is necessary to better evaluate the association of congenital thrombophilia factors and vod. disclosure: nothing to declare p different strategies of chemotherapy-induced nausea and vomiting (cinv) prevention in hematological patients receiving an autologous hematopoietic stem cell transplantation: a single center experience ilaria cutini , riccardo boncompagni , chiara nozzoli , antonella gozzini , stefano guidi , chiara innocenti , massimo di gioia , lorenzo tofani , riccardo saccardi background: despite the improvements of pharmacological control, cinv still represents a major problem in patient undergoing hematopoietic stem cell transplantation (hsct). we present here a comparison of two pharmacological strategies for preventing cinv in multiple myeloma (mm), hodgkin (hl), and non-hodgkin lymphoma (nhl) patients who received an autologous hsct in our institution. methods: from january to july , we retrospectively analyzed consecutive patients, median age years ( - yo) , diagnosed with mm, hl, and nhl, who underwent an autologous hsct following a melphalan mg/sqm and beam/feam condition regimens, respectively. the first patients received cinv prophylaxis with palonosetron i.v and dexamethasone mg die (regimen a), whilst the following were administered with fosaprepitant iv, ondansetron iv and dexamethasone mg die (regimen b) both cinv prophylaxis was administered the day of melphalan infusion (day - form transplant). emesis breakthroughs were treated with alizapride and metoclopramide. nausea and vomiting were assessed through the ctcae . score system. categorical variables were compared with pearson chi-square test. results: the overall incidence of nausea was %, ( % grade , % grade , and % grade , respectively). in regimen a was shown to be %, ( % grade , % grade , and % grade , respectively) while in regimen b was % ( % grade , % grade , and % grade , respectively). pearson chi-square test did not show any differences between the groups (p= . ). the overall observed vomit was % ( % grade , % grade , and % grade ). in regimen a it was ( % ( % grade % grade , and % grade ), and % in regimen b ( % grade and % grade ). conditioning regimens didn't' have any significant impact on either nausea or vomit. patientsyounger then median ( yrs), were reported to have higher incidence of both nausea, (p= . ) not related to cinv treatment, and vomit ( % vs %, p= . ). in multivariate analysis the overall incidence of nausea is related to age (younger patients have higher probability to develop nausea (or , ; p= , ) whilst the higher incidence of vomit is related to: regimen a (or . ; p< , ), previously reported nausea (or , ; p< , ), and no smoking habits (or , ; p= , ). conclusions: both regimens are equally effective for nausea control however regimen b evidenced a better vomiting control. this finding is particularly relevant when the center policies include an early discharge program, therefore improving both patient's quality of life and procedure cost-effectiveness. clinical background: patients who underwent an allogeneic hematopoietic cell transplantation (hct) are challenged by medical, psychological and social complications. support groups might help hct-survivors to cope with these challenges. however, the existing literature about post-hct support groups is scarce. moreover, data on professionallyfacilitated support groups do not exist. the aim of this project was ( ) to establish a professionally-facilitated support group and ( ) to assess the discussed topics. methods: from / until / all patients who received an allogeneic hct at the adult stem cell transplantation program of the university clinic mannheim were invited to participate in a professionally-facilitated support group. additionally, spouses and life partners were invited. a theologian who is also a physician served as facilitator. he had no further function within the transplant team. the format of the group was unstructured without any rules regarding regular attendance. the facilitator did not provide topics or a curriculum. during the first year the group met every days followed by a monthly schedule. from the fifth until the th meeting the attendance and the discussed topics were minuted by the facilitator. the content of the minutes was analysed by a combination of an inductive and a deductive approach. all participants provided their informed consent for the study. results: altogether patients (female: n= ; male: n= ) and spouses/life partners (female: n= ; male: n= ) participated. patients ( %) and spouses ( %) attended more than one meeting. among those who participated in ≥ meetings the median time of participation was months. the median count of participations was eight. % of the participants attended the meetings longer than one year, % longer than three years. there was no sex difference with respect to the frequency and the duration of participation. however, the frequency of participation decreased significantly the longer a participant was attending the meetings. during group meetings the facilitator recorded thematically different contributions to the discussions divided in distinct topics. these topics were grouped into main categories [(a) medical topics, (b) private life and environment, (c) human relationships, (d) physical and mental condition and (e) the support group itself] and eight further categories [( ) compliance, ( ) economic issues, ( ) religion, ( ) sexuality, ( ) death and dying, ( ) support and coping, ( ) objectives and needs and ( ) not otherwise specified issues] which could not be grouped in one of the main categories. the most frequent issues were medical topics ( %), human relationships ( %), physical and mental condition ( %), private life and environment ( %), financial issues ( %), the support group itself ( %), support and coping ( %) and objectives and needs ( %). noteworthy, death and dying ( . %) were rare topics and sexuality was never mentioned. conclusions: to our knowledge, this is the first prospective and systematic analysis of a professionallyfacilitated support group for hct-survivors. these data might help to establish support groups and to identify psychosocial needs of patients and targets for specific support. disclosure: nothing to declare background: endothelial damage is associated with inflammatory complications that appear early after hsct, such as sinusoidal obstruction syndrome or acute gvhd. engraftment syndrome (es) is an inflammatory condition diagnosed by maiolino clinical score. potentially, es can exhibit high morbidity and mortality, especially after autologous-hsct in multiple myeloma (mm) patients since the introduction of new drugs such proteasome inhibitors and immunomodulatory drugs (imids). the objective of the present study was to evaluate if es is associated with endothelial dysfunction in patients with mm who underwent auto-hsct. methods: we included six patients with mm who received induction treatment including new drugs and consolidated their response with an autologous-hsct. we analysed comparatively the effect of incubating endothelial cells in vitro with serum samples from patients with es vs. no es. serum samples were collected before (pre), and after , , and days from the transplant. an additional sample was collected at the es onset and at the discharge day (no es group). endothelial cells (hmec) in culture were exposed to media containing % of serum from each patient for h. cell growth was controlled morphologically. expression of the adhesion receptor icam- on the cell surface was analysed by immunofluorescence, and activation of the inflammation related p- mapk signalling pathway was evaluated by sds-page and western blot. results: exposure of hmec monolayers to sera from patients who developed es (onset day, n= ) resulted in an increased icam- expression on the cell surface, higher that the observed with sera from patients who did not develop es (discharge day, n= ) ( . % of labelled area vs. . %, respectively). in addition, in experiments with sera from patients not developing es, icam- expression on cells exposed to sera from day + was reduced with respect to the observed with sera from day + , probably due to the corticosteroid used as a prophylaxis in our centre. this reduction was not observed in es patients. regarding phosphorylation of p- , it was significantly higher in cells exposed to sera from es patients than in response to sera from patients who did not develop es. conclusions: the increase in the expression of the adhesion receptor icam- on the surface and the intracellular activation of p mapk in endothelial cells exposed to sera from patients developing es indicates the existence of endothelial activation in association with es. interestingly, the prophylaxis of es with corticosteroid seems to be less effective in patients who developed es than in patients who did not develop this complication. these results need to be validated in a higher number of patients and modifications in additional markers of endothelial dysfunction should be investigated. disclosure: gonzalo gutiérrez-garcía: honoraria from gilead. grant from jazz pharmaceutical the other authors do not have any disclosure to comment. p association between uric acid levels before and after allogeneic haematopoetic stem cell transplant and transplant outcomes: a single centre experience background: uric acid (ua) is a known endogenous danger signal which activates the nod-like receptor protein (nlrp) inflasome.ua is released from injured cells during conditioning in allogeneic stem cell transplantation (hsct). a pre-clinical study has demonstrated that nlrp inflasome-mediated il- production regulates graft-versushost disease (gvhd). the ua role in inflammation and gvhd is unclear. there are discordant reports in the literature about a potential protective role of ua on gvhd after a hsct. methods: we performed a retrospective study to assess the association between serum ua levels pre-and post- table] . table ] results: the characteristics of the patients are shown in table . median age was years (range - ), and patients ( %) were male. twenty-seven patients ( %) received low doses atg as part of gvhd prophylaxis. allopurinol was from the day before start of conditioning therapy until day . the median levels of ua were , mg/ dl before conditioning, , mg/dl at day , , mg/dl at day + and , mg/dl at day + . there was no impact between the ua levels and os at any time of the hsct. ua levels at day + were associated with a higher ci relapse at years ( % [ % ci, - %] for ua level > , mg/dl, and % [ % ci, %- %] for ua level ≤ , mg/dl [p= , ]). there was a trend for a higher ci of grade ii-iv agvhd for the subgroup of patients not treated with atg with ua < , mg/dl ( % vs %; p= , ) on day - and a higher nrm with ua < , on day ( % vs %; p= , ). conclusions: in our study the ua levels showed no impact on os, and only a tendency for ci of grades ii-iv agvhd grades ii-iv and nrm for the subgroup of patients not treated with atg. surprisingly, high levels of ua at day + of hsct were associated with a significant higher incidence of relapse. disclosure: dkms foundation, pi / (instituto carlos iii) and sgr (grc), generalitat de catalunya. background: veno-occlusive disease (vod) is an early, uncommon but serious complication of stem cell transplantation (sct) that is associated with high morbidity and mortality. defibrotide is the only licensed treatment for vod, and time to start of treatment (tst) affects outcomes. minor differences exist between the seattle, baltimore and classical ebmt ( ) criteria, which may trigger different start points for treatment. late onset vod (> days) is less recognised and we hypothesize, may have worse outcomes with longer time to diagnosis, and more limited treatment options across different healthcare systems. methods: electronic patient records from sept. -oct. at king´s bmt centre and pharmacy databases were reviewed, timepoint to clinical and bio-chemical manifestation of vod, diagnosis, tst, survival and longterm outcomes were analysed. results: of the patients( . %) who underwent an allogeneic sct, developed vod, including paediatric cases. none of the autologous sct patients developed vod. the paediatric and autologous sct patients were not analysed any further. adult patients (male= ; . %) developed vod at a median age of years(range - ), of whom developed < days and patients had late-onset vod as per ebmt criteria(range - days). cases classed as severe and as moderate vod. patients received defibrotide at diagnosis, patients within days, patients between - days, and patients received treatment after days. overall mortality for this cohort was %( / ). / ( . %) of patients with early onset vod and / ( . %) patients with late-onset vod died. of the deaths, died of liver failure and a further patients had vod as a likely contributing factor in their deaths. patient died with subarachnoid haemorrhage and with relapsed disease. patients that received defibrotide after days, / patients( . %) died, as compared to / ( %) for treatments between - days, / ( . %) for treatments within days. the lone surviving patient who received treatment after days has severe chronic liver disease and it's complications. of the patients who fit seattle criteria for early-onset vod, only fit the baltimore or ebmt criteria for classical vod. of these patients met the baltimore criteria later than the seattle criteria were met(range = - days). conclusions: vod carries high morbidity and mortality, and beyond the known risk factors and with the caveat of limited numbers in this study, we strongly suspect this is further increased when time to definitive treatment with defibrotide is delayed, particularly beyond days. nearly a quarter of cases with vod are late-onset as per classical ebmt criteria. however contrary to our hypothesis, their overall outcomes and mortality do not appear worse, with time to treatment again emerging as a strong predictive factor. conditioning treatment related factors, which play a stronger role in endothelial dysfunction in the hepato-portal circulation, may not be as much at play, perhaps for late-onset disease. uniformity in the use of diagnostic criteria, and high degree of vigilance, even beyond days, leading to early treatments may improve outcomes in vod. disclosure: nothing to declare background: hsct-associated thrombotic microangiopathy (ta-tma) affects - % of patients receiving an allogenic sct, with a high mortality up to - % in severe cases. endothelial injury mediated by complement activation has been atribuited a major role in the pathogenesis, and blockade of c with eculizumab offers promising results. methods: we present our experience with pediatric cases of ta-tma treated with eculizumab. the diagnosis of ta-tma was stablished attending to jodele et al criteria. clinical data were collected retrospectively from medical records. results: all cases were diagnosed between august and april , with a median age of years ( . - ) at time of diagnosis. primary disease was acute leukemia in cases ( all and aml), severe aplastic anemia in , and primary immunodeficiency in . they received their first sct in all cases, from mud and from mmrd (cd ra+ depleted haploidentical grafts), with mac regimen in cases, and ric in cases. of them received calcineurin inhibitors (cyclosporine) as gvhd prophylaxis. all patients developed agvhd (grade or higher in cases). and patients presented viral reactivation. hypertension was present in cases at tma diagnosis, requiring or more antihypertensive drugs in of them. all patients had renal injury consisting of less-than-normal glomerular filtration rate (median of ( - )) and proteinuria, with urine protein-to-creatinine ratio higher tan mg/mg in cases (data not available in patients). serum haptoglobin was decreased in just cases at diagnosis, and schistocytes were detected in patients. cutaneos signs were present in all cases, digestive symptoms in , neurological affection in , and notoriously all of them developed polyserositis. c and c were normal in all cases, with sc b higher than ng/ml in patients and lower in (data not available in cases). all patients received defibrotide as treatment, and cases received also rituximab, associated to therapeutical plasma exchange in . all of them received eculizumab, as first line in cases (median of days between diagnosis and eculizumab start). treatment was correctly monitorized with ch levels in cases (not available quick enough in other ). median number of doses needed in induction therapy was , and median interval between doses was days. patients required reduced interval and higher doses to maintain ch supressed. patients did not respond, and died because of tma. patients had hematological response, with chronic renal injury in of them and resolution of acute renal failure in case. nevertheless patient responding to eculizumab died because of tma related complications, and because of an invasive fungal infection. patients are alive, with a median follow up of months from treatment start. conclusions: our experience supports promising results of eculizumab based treatment for ta-tma, highlighting the importance of an early treatment and a careful therapy monitoring by ch supression. prospective studies are needed to achieve a better knowledge of this pathology and its treatment. disclosure: nothing to declare background: approximately - % of allogeneic hematopoietic stem cell transplant (allo-hsct) are made with some sort of abo blood group system incompatibility. an hsct abo donor-recipient incompatibility implies risks of complications during the process of infusion as acute hemolytic anemia (ah), delayed graft and other later complications due to the presence of isohemaglutinins (pure red cell aplasia or passenger lymphocyte syndrome). also, abo incompatibility could impact on graft versus host disease (gvhd) incidence, and could be associated with not relapse mortality (nrm) and overall survival (os). there are not concluded evidence about the abo incompatibility impact, so the aim of this study was to identify complications and response associated with abo incompatibility in patients undergoing allogeneic hematopoietic stem cell transplantation. methods: a retrospective study was performed on patients who receive an allo-hsct between january and august . two groups were performed according to the presences or not of abo incompatibility. demographic and clinical information was collected from physical and electronic medical records, and information was analyzed in spss v results: sixty-eight patients were enrolled in the study, % male, the median age was years ( - ) with the following diagnoses: acute lymphoblastic leukemia %, acute myeloblastic leukemia . %, granulocytic chronic leukemia . %, myelodysplastic syndrome . %, dendritic cell neoplasm . %, aplastic anemia . %. ninety-one percent of the patients received a transplant from an identical hla donor and . % received a haploidentical transplant. fifty-two patients ( %) were abocompatibility (g ) and patients ( %) had aboincompatibility (g ). none patient with aboincompatibility received a haploidentical transplant. the contrast between groups didn't show differences in fever, infections, bacterial isolation, presence and degree of acute or chronic gvhd and relapse of the disease. graft failure was %(g ) vs %(g ) (p= . ), intermediate risk cmv serostatus %(g ) vs (g ) (p= . ). the most relevant characteristics and complications are described in table . contrast analysis between g vs g showed that within the whole group there were deaths ( % vs % respectively) (p= . ), the overall survival -year was % vs % (p= . ) with a median of vs months respectively; mortality associated with relapse was % vs % respectively, and mortality related with transplantation was % vs % respectively. conclusions: abo incompatibility did not show association with complications related with the infusion, but there was a higher tendency of graft failure in the abo incompatibility group. it has no statistical significance, but it is important to expand its study. disclosure: none declared methods: retrospective data for nhl patients who underwent asct between and was analysed. patients were identified using the swbmt database and data on mets was collected using paper and electronic hospital records. forty-eight patients were excluded due to loss of follow-up, inaccessible/incomplete records, or death. cause of death was not determined. the ncep-atpiii definition of mets was used. this requires ≥ of criteria to be met. a bmi of ≥ kg/m and hba c of ≥ mmol/l were used to replace central obesity and impaired fasting glucose, respectively. other criteria include triglycerides (tgs) ≥ . mmol/l or treatment, high density lipoprotein cholesterol (hdl-c) < . mmol/l (male), < . mmol/l (female) or treatment and blood pressure > mmhg systolic or > mmhg diastolic, or treatment. results: the prevalence of mets in the cohort was % (n= ). eighty-two percent of patients (n= ) met one or more criterion for mets. twenty-seven percent (n= ) fulfilled only one criterion, % (n= ) fulfilled two criteria, % (n= ) three criteria, % (n= ) four criteria, and % (n= ) five criteria. the greatest prevalence of mets was in the + age group, accounting for out of ( %) patients with mets. overall prevalence decreased with declining age ( table ). the number of patients aged < years was too small to make any judgement on risk. raised triglycerides was the criterion most frequently met ( / patients), followed by hypertension ( ), raised bmi ( ), low hdl-c ( ) and an increased hba c ( ). conclusions: the prevalence of mets in our cohort ( %) was higher than the estimated worldwide prevalence of %, with the majority in the + age category. this is in keeping with other post-transplant studies, which show an increase in prevalence of mets after transplantation. moreover, the overall prevalence of mets was greater in the older population, which could be associated with the cumulative effect of ageing on the decline of normal metabolic homeostatic mechanisms. background: acute renal failure (arf) is a frequent complication in the early post-allogeneic hematopoietic stem cell transplant (allohsct) period with either myeloablative (ma) or non-myeloablative (nma) conditioning regimens. the aim of this study was to compare the incidence of arf in both types of hsct and to evaluate its impact on overall survival (os) and non-relapse mortality (nrm). methods: all allosct performed in one center between and were included in this study. allohsct from cord blood and from haploidentical donors were excluded. the renal function and the incidence of the main complications after allosct from day to day + were evaluated. arf was defined according to kdigo (kidney disease improving global outcomes) classification; the relative increase of serum creatinine levels was considered a marker of kidney damage. results: seventy-seven patients received a ma allohsct and a nma allohsct. recipients of nma allohsct had a higher median age ( years [range: - ] vs. years , p< . ), higher frequency of arterial hypertension ( % vs. %, p< . ) and showed most frequently active disease at allosct ( % vs. %, p= . ). in both groups the most frequent graft-versushost disease (gvhd) prophylaxis regimen was cyclosporine a and methotrexate. the median follow-up time was . years for the nma group and . years for the ma group. patients from the ma group had higher incidence of grade - mucositis ( % vs. %, p< . ) and acute gvhd of any grade ( % vs. %, p= . ) than patients from the nma allohsct. the incidence of arf was similar in both groups ( % in nma and % in ma). in the nma group arterial hypertension (hr . , p= . ), obesity (hr . , p< . ) and prior pneumonia (hr . , < . ) were predisposing factors for arf by multivariate analysis, whereas any factor was identified in the ma group. arf had no impact on -year os in both groups ( % vs. % p= . for the nma group and % vs. % p= . for the ma group). however, worse os were observed in patients with grade - arf in the nma group ( % vs. %, p= . ) and in patients with grade arf in the ma group ( % vs. %, p= . ). in turn, arf had no influence on nrm in the ma group but was associated with a trend for higher nrm in the nma group ( % vs. %, p= . ). conclusions: arf is a frequent complication in patients receiving allohsct irrespective of the intensity of the conditioning regimen. moderate and severe arf had negative impact on os. disclosure: supported by grants from: asociación española contra el cáncer, aecc (gc biga), instituto carlos iii (pi / fi), -sgr (grc), cerca program from generalitat de catalunya, and "la caixa" foundation. treatment and risk factors of hepatic veno-occlusive disease after pediatric hematopoietic stem cell transplantation: a single-center experience barbaros sahin karagün , ilgen sasmaz , ali bülent antmen background: defibrotide emerged as a promising treatment option for hepatic veno-occlusive disease, a significant cause of mortality in recipients of hsct. as vod diagnosis is quite difficult even with the recently introduced ebmt criteria, studies which report treatment outcomes and response to prophylaxis are required. our aim was to evaluate the efficacy of defibrotide prophylaxis in hsct recipients at our center. methods: a total of transplants in patients from january to july were included in this study. all patients had factors that increased the risk of vod and all received mg/kg/day prophylaxis. patients' coagulation, renal and liver function test were monitored daily and all clinical findings and complaints were recorded. diagnoses were made via the ebmt vod criteria and patients who developed vod received treatment with increased df dose ( mg/kg/day) and supportive interventions. after complete remission of vod findings, patients were returned to the prophylaxis dose. close follow-up of patients was performed until days. results: in total, patients developed vod ( . %), none of the cases were severe ( mild, moderate). median age was . years and the most common clinical findings were weight increase, hepatomegaly, right upper quadrant pain and ascites development. in those with vod, treatment with mg/kg/day df was initiated and average duration of treatment with this dosage was . ( - ) days. no adverse events were reported in any of the patients. conclusions: our findings are consistent with previous studies on this topic, and we believe that the use of df as a prophylactic agent for vod is beneficial for pediatric patients with risk factors. disclosure: the authors report no conflicts of interest in this work. background: several factors might influence outcome of allo-hsct. analysis of the impact of donor-receptor blood group-incompatibility have been performed in different series not always showing the same results. as a consequence, its clinical impact remains controversial. minormismatch is characterized by the ability of donor b lymphocytes to produce anti-recipient antibodies. in majormismatch cases, antibodies against donor antigens are present in the recipient. methods: pts underwent allo-hsct between may and august in our center. median age was years (range: - ). pts were male ( . %) and female ( . %). baseline diseases were: aml, lpd, mds, all, mpd, mm, and bmf. donor was unrelated in , and related in cases (including haplo-identical). donor-recipient abo compatibility was as follows: ( . %) majormismatched (including bidirectional), and ( . %) nonmajor-mismatched (including minormismatched and matched). donor-recipient rh compatibility was as follows: ( . %) major-mismatched, and ( . %) nonmajor-mismatched (including minor-mismatched and matched). the impact of donor-recipient abo and rh compatibility on transfusion needs (prbc and platelet concentrates) and survival by day + was analyzed. results: for the global series the median number transfusions by day + was: ( - ) prbc and ( - ) platelets concentrates. day + overall mortality was . %. rh-incompatible and nonmajor abo incompatible cases showed no different results. however, major abomismatched cases needed more prbc transfusions (median: ; range: - ) and more platelet transfusions (median: ; range: - ), and had higher day + mortality ( . %) (p < . ) (see table) . conclusions: our analysis showed: ) donor-recipient rh-incompatibility, as well as minor aboincompatibility had no impact on prbc and platelet concentrates transfusion needs nor on -day mortality; ) contrarily, donor-recipient major abo-incompatibility had a significant adverse impact on prbc and platelet concentrates transfusion needs and -day mortality. ) donor-recipient rh-incompatibility and minor aboincompatibility.might be considered of marginal importance at the time to choose a potential donor. ) donorrecipient major abo-incompatibility should probably be a factor to be considered, along with other features, to choose the best donor background: survivors of haematopoietic stem cell transplantation (hsct) are at significant risk of developing treatment-related complications, including cardiovascular risk factors such as arterial hypertension, that could eventually lead to cardiovascular disease. the aim of this study is to evaluate the incidence and risk factors of hypertension following hsct in a colombian population. methods: a retrospective cohort study was conducted to assess the incidence and risk factors of hypertension in consecutive adult hsct recipients who underwent transplantation between and at a tertiary referral center in colombia, south america. blood pressure data, from two different measures, were collected at time points: day of mobilization for autologous hsct and day before infusion for allogeneic transplantation, day , and months , , and post-transplantation. hypertension was defined as having a systolic blood pressure >= mmhg and/or a diastolic blood pressure >= mmhg. patients with history of arterial hypertension were excluded. results: one hundred and seventy-five patients were included, with a mean age of years (range - ). ninety-one patients ( %) were male. one hundred and sixteen patients ( . %) underwent autologous hsct and ( . %) allogeneic hsct. the most common indication for hsct was acute leukemia ( . %), followed by non-hodgkin lymphoma ( . %) and multiple myeloma ( . %). twelve patients ( . %) had medical history of type diabetes mellitus (dm), ( . %) dyslipidemia, ( . %) alcohol consumption, and ( . %) tobacco smoking. only two of the patients with history of tobacco smoking were active smokers at time of transplantation. twenty-four patients ( . %) had developed hypertension by the end of the first year post-hsct follow-up. two patients ( . %) had systolic and diastolic, ( %) had only systolic, and ( . %) had only diastolic hypertension. only one patient was hypertensive at more than two time points. the incidences of hypertension at each time point were . % on day post-hsct, . % at first month, . % at three months, . % at months, and . % at one-year post-transplantation. allogeneic hsct (p< . ), therapy with calcineurin inhibitors (p< . ), pre-hsct fasting glucose levels (p< . ), acute gvhd (p< . ), chronic gvhd (p< . ), and media of diastolic blood pressure (p< . ) were significantly associated with the development of arterial hypertension. however, age, history of type dm, history of tobacco consumption, volume of infusion, prophylactic treatment for gvhd with mycophenolate, chronic gvhd, serum creatinine level on day of hsct, and being overweight or obese at time of transplantation were not significantly associated with the development of hypertension. conclusions: arterial hypertension is a fairly common complication in hsct recipients. similar to findings reported in previous studies, association between allogeneic stem cell transplantation, therapy with calcineurin inhibitors, and acute and chronic gvhd, and post-hsct hypertension was found in the present cohort. further studies are needed to assess the link between hsct and developing long-term cardiovascular complications. disclosure: nothing to declare tramadol-based pain management of oral and esophageal mucositis in pediatric hsct recipients background: mucositis is one of the most common early hsct complications seen in about % transplant recipients with % of patients developing gr iii-iv mucositis. mucositis is characterized by painful gastrointestinal mucosa lesions impairing the solid and liquid foods intake and increased risk of infections, bleeding, and intestinal paresis. thus, it greatly decreases the quality of life of a transplant recipient. according to who recommendations, the moderate pain control in pediatric patient is based on the use of low-dose morphine. however, there are some factors such as genetic polymorphisms causing variable morphine pharmacokinetics in children, side effects, and social factors (caregivers' general unwillingness to use narcotic analgesics), which cause the need for alternative pain relief options in pediatric practice. tramadol, which has both opioid and non-opioid mechanisms of action, may be a feasible option in mild to moderate pain. it may be delivered via patient-controlled analgesia (pca), although there is no consensus on its optimal parameters in pediatric practice. methods: a total of pediatric patients with a median age of (range to ) years receiving an autologous or allogeneic hsct in our clinic as part of the treatment regimen for solid tumor (n= ), leukemia (n= ), acquired aplastic anemia (n= ) or inherited condition (n= ) were included. conditioning regimens were myeloablative (mac) in and reduced-intensity (ric) in patients. all patients had oral and/or esophageal mucositis accompanied by moderate pain. the pain severity was assessed using the scales corresponding to patient's age and varied from to points. the pain control was based on intravenous tramadol administration using patientcontrolled analgesia (pca) approach. the following pca parameters were used: loading dose of . mg / kg (not exceeding mg), basal infusion rate of . mg / kg (not exceeding . mg), a bolus of . mg / kg (not exceeding . mg), lockout interval of min. the maximal daily dose was mg/kg/day. the pain control was considered adequate if a patient was satisfied or the basic and breakthrough pain score values were not higher than and, accordingly. in case of inadequate pain control nsaids were added. non-responders were switched to morphine. all patients were divided into groups based on conditioning regimen intensity. results: as a whole, % of patients did not require pain control measures escalation. the tramadol pain control rate was slightly higher for ric (n= , %) compared to mac (n= , %) recipients. in most cases the inadequate pain control was due to progressive mucosal lesions. the pca regimen used was characterized by very few complications. drowsiness was observed in ( %) of patients, in all cases the patients also had anemia. there was only ( %) patients with severe nausea requiring switching to morphine. conclusions: tramadol is an effective pain control option in transplant recipients with mild to moderate pain due to oral and esophageal mucositis without progressive mucosal lesions. the pca allows achieving a very low complication rate. therefore, this option may be considered for both mac and ric recipients. disclosure: no immune reconstitution of lymphocyte subsets after allogenic stem cell transplant (sct) and vaccination background: infectious diseases are a major cause of morbidity and mortality after allogenic stem cell transplant (sct). vaccines constitute an effective strategy to prevent infections but the optimal timing to start vaccinating is not well stablished. in order to individualize the early vaccination schedule, we studied the lymphocyte subsets involved in generating enough response to produce protective serological levels. methods: we studied retrospectively patients that had undergone allogenic sct at our hospital. patient distribution -age range: - years-old; diagnosis: acute leukaemia/myelodysplastic syndrome/ chronic myeloid leukemia ( patients), lymphoma ( patients). analytic parameters: tcd +, tcd +, nk, total b and functional b lymphocyte subsets (naïve igd+cd -, memory igd+cd + and igd-cd +, and effectors cd ++cd ++). immunoglobulin levels (igg, iga, igm) and specific igg for pneumococcus, tetanus, hbv, chickenpox, measles, rubella and mumps. clinical parameters were collected from medical records. results: we distributed patients in two groups, based on the timing of lymphocyte analyses: -less than months since sct ( patients) no patient showed complete immune reconstitution, although had enough t and functional b lymphocytes to generate response to vaccination. in these patients, vaccination for pneumococcus was completed and they generated sufficient protection antibody levels, despite being under immunosuppressive treatment. -more than months since sct ( patients) before the beginning of vaccination, we collected specific antibodies of patients. we compared the serological status before and after sct and observed that protection against tetanus was the most frequently preserved ( patients) and hbv the least frequent ( patients). other than one patient treated with alemtuzumab, all patients in this group had minimum absolute count of tcd + (> cells/microl), tcd + (> cells/microl), nk (> cells/microl) and b cells (> cells/microl). we also observed presence of b effector and b memory cells, with predominance of igd-cd + memory cells. immunoglobulin levels were within the normal range. in this group, we registered vaccination in patients. all of them were vaccinated against flu, and against pneumococcus and hbv. the rest of vaccines administered were heterogeneous in type and timing. patients were under immunosuppressive treatment at the time of vaccination and were able to generate enough specific antibodies for pneumococcus. conclusions: immune reconstitution was not completed months after sct, although minimal immunological reconstitution was observed tcd + and no-switching memory b lymphocytes were the last ones to reach minimum normal values according to patient age. some patients maintain serological protection after allogenic sct. immunoglobulin levels were normal, suggesting no need for immunoglobulin administration to prevent infections. flu, pneumococcus and hbv vaccines were the most frequently administered. pneumococcus vaccination generated a much larger serological response than hbv. this seroconversion occurred in patients under immunosuppressive treatment. the analysis of lymphocyte t, nk, b total and b functional subsets could be useful when programming an early vaccination schedule after sct. completion of the vaccination schedule was heterogeneous despite giving specific indications. therefore a more rigorous supervision of the process may be required. background: the significant advances that have been achieved in the allogeneic transplantation (allohct) field, have resulted in better post-transplant outcome and therefore complications other than the graft vs. host disease (gvhd) or disease recurrence become increasingly important. the post transplant metabolic syndrome (pt-ms), which caused by several factors (i.e. immunosuppressive agents, chemo-radiotherapy, anti-viral, and biologic therapies) is a well known post transplant complication in pediatric allografted long-term survivors however, only few studies have evaluated the prevalence of the pt-ms in adults. in this retrospective study, we sought to evaluate the incidence, the risk factors and the impact of the pt-ms on the allosct outcome. methods: since , patients ( males and females) with adequate clinical and laboratory data and a minimum follow-up of months were included in the study. their median age was . ( - ) years and after a myeloablative (n= ) or a reduced intensity (n= ) regimen they received either mobilized peripheral blood stem cells (n= ) or marrow graft (n= ), originated from full-matched siblings (n= ) or haploidentical donors (n= ). calcineurin inhibitors plus either short-term methotrexate or mycophenolate mofetil were given as gvhd prophylaxis. the diagnosis of pt-ms was based on the ncep-atpiii criteria; for patients with unknown data for abdominal circumference the body mass index (bmi) ≥ kg/m was consider as a criterion for pt-ms diagnosis. the independent t-test, logistic regression analysis and logrank tests were used for the statistical analysis. results: twenty ( . %) patients ( males, females) assessed to have pt-ms within the first months following the allograft. seventeen diagnosed after the st trimester post allosct and additional patients after nd trimester. sixteen out of patients had elevated glucose and bmi> kg/m , / elevated triglycerides levels, / low hdl levels and / hypertension. four ( %) had already known history of ms before allosct (for patients no data were available for ms diagnosis before allosct). interestingly, for / ( %) patients who had diagnosed with pt-ms either in the st or in the nd trimester the syndrome was reversible and did not fulfill the criteria for pt-ms beyond months post allosct. patients' gender, age, bmi, the type of conditioning regimen and gvhd co-existence evaluated as potential predisposing factors for pt-ms diagnosis. in univariate and multivariate analysis only the: bmi> kg/m and age> years were detected as significant risk factors (p< . ). the pt-ms did not affected negatively the survival or the nrm incidence post allosct conclusions: in our study, in agreement with other publications, we demonstrated that the pt-ms is not an uncommon complication post in the early post transplant period however, for a significant number of patients the syndrome was a reversible. for patients with high risk features (bmi> kg/m , age> years, known history of diabetes-mellitus, dyslipidemia, hypertension) apart of close monitoring, specific diet and encouragement for adequate exercise might help to reduce the incidence and the severity of pt-ms. nevertheless, prospective and well design trials are warranted to determine the accurate incidence, severity and the impact of pt-ms on the allosct outcome. disclosure: no conflict ofinterest experience of a single center in the humanization of the hospitalization process: technology and team training impact on the qol of the patient and family maria claudia moreira , marcia rejane , marcia garnica , andrea ribeiro , paulo cesar dias , ilza fellows background: hematopoietic stem cell transplantation (hsct) is one of the most aggressive therapeutic modalities of internal medicine, making it a highly stressful experience for the patient and his family. the duration of hospitalization can be prolonged by several intercurrences, frequently generating anxiety in the patient and their caregiver, which may lead to confinement and reactive depression. interventions in the hospital environment, in addition to the continuous training of the multidisciplinary team, can have a positive impact in this process with improvement in the process of discharge and quality of life of the patient and his / her family. methods: the objective of this research was to evaluate the impact of a reformulation in the unit, completed in may , which modified the facilities with availability of hermetic balconies in each room, with a view of an internal garden. there was also the addition of a screen in the corridor of the floor with images -technology known as videoowall, interconnected to motion sensors (kinects) that allow interaction between patients and families, besides facilitating physiotherapy and physical exercise. there was re-training of the multidisciplinary team with emphasis on the practice of humanization. the methodology consisted in the application of questionnaires of satisfaction to patients and their families during the period of hospitalization in a bone marrow transplant unit in the third quarter of . the items evaluated ranged from the quality of the information provided by the medical team and nursing, to the cordiality and agility with which the patient and his patient were treated by the global team. the results were compared with a similar period of the same unit in the previous year and with the indices collected simultaneously in another unit of the same hospital (cardio-intensive). results: overall and segmental satisfaction scores in the various items surveyed were higher when compared to the previous period of the same unit and were also higher in those obtained in a high complexity unit of the same hospital, composed of patients submitted to mental and psychological stress similar to onco-hematologicos.a reports of "free speech" were also obtained anonymously, in order to guarantee the authenticity and free expression of the subjects analyzed. conclusions: the results obtained allowed the validation of the technical and professional team initiatives, bringing indicators that will allow better monitoring and support of these patients and their relatives in this difficult time of treatment. they served as an initial tool in the continuous process of humanization and stimulated the multidisciplinary team to continuously improve this process. disclosure background: pure red cell anemia (prca) is a rare complication of abo-incopatible hematopoetic stem cell transplantation characterized by anemia, reticulocytopenia and absence of erythroid precursors in patient's bone marrow. most patients with prca resolve spontaneously within months, however a small number of patients requires continued red blood cell (rbc) transfusions. the treatment of this complication is difficult and not standardized. different approaches has been used such as rituximab, donor lymphocytes, plasma exchange with different outcome. recently, a remarkable response to treatment with bortezomib has been described in a case of prca. methods: we reviewed patients who received an allogeneic hematopoetic stem cell transplant (hct) between januar and august at our institution. sixty eight patients received a major abo-mismached hct. prca was defined as a completely absence of erythroid precursors on day + bone marrow puncture, with absence of donor red cells and the recipient requiring rbc transfusion. results: only one patient developed prca ( . %). a years old male received a myeloablative hla-matched abomismatched sibling donor transplant (brother, years) for acute myeloid leukemia (aml), with t( ; ) cr ,mrd positive (runx -runx t ). the donor was blood type a rh positive and the patient rh positive. the patient had no complication after transplant. the day + bone marrow puncture has shown only few erythroid precursors and day + puncture and biopsy no erythroid precursors, he had transfusion dependent anemia requiring a rbc transfusion every two weeks and retukulocytopenia. parvo virus and cytomegalovirus were negative. due to very high ferritin level (> . u/l) and increased luiver enzymes without signs of gvhd, the treatment with deferasirox has been started. the patient has achived cr , mrd negative, and has evidence of complete chimerism. high titers of anti-a and anti-b issohemagglutinin was present.we started the treatment with rituximab mg/m weekly, weeks, however without response. the pathogenesis of the prca is thought to be due to the recipients plasma cells, bortezomib, a proteasome inhibitor inducing apoptosis of plasma cells has been given s. c. , mg/m two times weekly, for two weeks. the patient responded to the treatment two weeks later with increase in hb, which was , g/dl and increase in retikulocyte number. the patient has continued to be well at the last control. conclusions: prca aplasia is a rare but serious complication after abo-incompatible hct. bortezomib is an effective treatment for this complication if mediated by residual host isohemeagglutinins after hct and should be recommended as standard of care. clinical methods: this work is retrospective, observational, cross-sectional and analytical. it included all patients who received hsct at stem cell transplantation unit (utmo, by its spanish acronym) at solca-guayaquil, between the years - .we use the kaplan-meier method to analyze the survival rate between the autologous and allogeneic transplant. the information collected for this study was obtained from the database of the solcay institute and the review of the files of the patients included. results: at least, patients have been undergoing to hsct between - years. according to the type of hsct, . % received an autologous transplant and . % received an allogeneic transplant, from which . % were from a related donor. the main source of transplant was peripheral blood in . %, followed by % obtained from umbilical cord blood and . % by bone marrow aspiration. the most frequently reported pathologies were acute lymphoblastic leukemia (all) ( %), multiple myeloma (mm) ( %) and acute myeloid leukemia (aml) ( . %). the overall survival was % (ic: %). the . % of patients that were undergoing to autologous transplant have survive, meanwhile the patients that were undergoing allogeneic transplant only the . % have survived (p< . ). the highest death rate occurred during the first year after hsct, and decreased considerably after that period. the main cause of mortality related to transplant (mrt) was the graft-versus-host disease (gvhd) ( %); however, the main cause of mortality in the study population (n= ) was relapse in . % of the patients, presented more frequently in all. conclusions: the results showed that % of patients undergoing to hsct have survived. a high rate of deceased patients in this study, have died in the first year before the transplant ( . %%), due to relapse. the main cause of deceased in the study is not related to hsct, and was the relapse in % of patients, in compare the gvhd was the main cause of mrt ( %). we consider that hsct is a technique that is still under development in ecuador, but despite the short time it has been taking and the institutional and medical limitations present in the health field, has presented excellent results comparable to studies conducted in developed countries. [ background: pigmented epithelioid melanocytoma (pem, early known as 'Аnimal type' melanoma) is a rare tumor with unpredictable clinical behavior and metastatic potential. pem generally has favorable prognosis. involvement of regional lymph nodes is not rare. extranodal and distant nodal metastases are extremely rare. we report about patient with fanconi anemia (fa) and pem with developed distant metastases in the early term after allogeneic hematopoietic stem cell transplantation (hsct). methods: -years old boy with fa was hospitalized for hsct. the blue-black painless nodulus х mm was noted on the left cheek. this lesion was observed from early childhood and during life only slightly increased in size. there were no distant and regional metastases on computerized tomography (ct) and scintigraphy with m tc. the nodulus and regional lymph nodes were radically removed before hsct. the resection margin was within the normal tissue. microscopically the derma and subcutaneous fat were infiltrated with epithelioid and spindle cells with total expression of s , melana, mhb , cyclind . ki- expression level was - %. histological structure was specific for pem. hsct with tcrαβ+/cd + graft depletion from match unrelated donor was performed. the conditioning regimen included total lymphoid irradiation gy, fludarabin mg/ m , cyclophosphamide mg/kg, rabbit atg mg/kg and rituximab mg/m . results: at + day after hsct was detected the tumor on the left cheek and parotid region with a histological structure identical to the primary lesion. on ct in s segment of the left lung was detected focus x mm with a cavity. invasive aspergillosis was suspected and empirical antifungal treatment was started. but in days the lung lesion increased in size to x x mm and penetrated in the bronchus. after bronchoscopy with biopsy, pem metastasis was histologically confirmed. moreover, the tumor on the face continued to grow. therapy with cobimetinib and vemurafenib was not effective and patient died from progression of pem on + day after hsct. conclusions: pem was early described as indolent tumor with rare distant metastasis and favorable prognosis. we suspect that pem may acquire an aggressive course in the absence of immunological control, especially in high immunocompromised patients after hsct. disclosure: nothing to declare p abstract withdrawn lidia gartcheva , antoaneta mihova , penka ganeva , margarita guenova , branimir spassov background: the main objective of the study is to assess the dynamics of quantitative and qualitative changes in the parameters of the b cell population and the production of immunoglobulins in patients after autologous transplantation of hematopoietic stem cells in the course of recovery of the immune system. methods: patients with hematological neoplasms undergoing autologous transplantation were included in the study: women and men, with an average age of years. patients were diagnosed with lymphoma (n = ), multiple myeloma (n= ), leukemia (n = ) and solid tumors (n = ). at the time of transplantation, patients were in complete clinical remission or at least with very good partial response, patients were in partial remission and patients -with progression. all patients were evaluated in nine time points through examinations by clinical-laboratory, flow cytometric and immunochemical methods. results: the percentage of cd (+) b cells reached the minimum values one month after transplantation then began to increase in the second month reaching a plateau around the mean values in the period - months after transplantation. the absolute number remained low during the entire period of observation. the amounts of igg and igm serum immunoglobulins gradually increased within the reference range throughout the entire period, while the iga level varied around the lower reference range. conclusions: implementation of an adequate humoral immune response is hampered by the reduction of circulating b cells, suppressed proliferative potential and functional deficits. restoration of b-cell function occurs over a period of months to years after autologous transplantation. clinical trial registry: no clinical trials disclosure: nothing to declare justyna background: allogeneic hematopoietic stem cells transplantation (allo-hsct) is a life-saving and well established therapy for wide range of diseases. however, it is still uncommon treatment for infants less than months of age. the data about indications and outcome of allo-hsct in the youngest group of patients is sparse. the primary objective of this study was to assess the incidence, indications, post-hsct complications and general outcome of allo-hsct among infants not older than months. latter sequelae of hsct such as physical and cognitive development were secondary aim of this study. methods: we retrospectively analyzed data of patients who underwent allo-hsct before year of age in department of pediatric hematology, oncology and bone marrow transplantation in wrocław during years - . clinical and epidemiological features as well as indications for transplantation, early and late complications and general outcome were assessed. results: infants who underwent hsct in our department comprise . % of all patients undergoing hsct in analyzed period of time. thirty-one ( . %) patients received stem cells from matched unrelated donor (mud), ( . %) from mismatched (haploidentical) related donor (mmrd) and ( . %) from a sibling donor (msd). non-malignant disorders were indication for transplant in ( . %) patients and malignant diseases in ( . %) . acute graft versus host disease (agvhd) occurred in ( %) infants, chronic graft versus host disease (cgvhd) in ( %). majority of graft rejections were seen in infants transplanted from mmrd ( . %), whereas the rest ( . %) was associated with mud. median follow-up in study cohort was days, days for alive patients (range days- . yrs) and days for those deceased (range days- days). overall survival (os) in study cohort was . and transplant related mortality (trm) was . . in children with malignancy ( . %) patients died comparing to ( . %) patients in non-malignant group respectively. main cause of death in analyzed group of infants was infection ( %). conclusions: . allo-hsct is rarely performed in children less than months of age. . majority of those patients receive stem cells due to non-malignant disorder. . among youngest hsct recipients, haploidentical transplant are more common than in general pediatric transplant population. . graft rejection is a significant problem in infants transplanted from mmrd. disclosure: nothing to declare unusual non-infectious lung complication after allogeneic haematopoietic stem cell transplantation claudia lucia sossa melo , , manuel rosales , francisco fernando naranjo junoy , , sara inés jiménez , , luis antonio salazar , , angela maría peña , , maría angélica chacón manosalva , maria luna-gonzález , claudia marcela chalela , manuel ardila-báez jirovecii infections, viral infections or nocardia. we describe the case of a patient with acute lymphoblastic leukemia (all) diagnosis with pap associated to a hsct and pulmonary pneumocystis. methods: a -year-old colombian female patient diagnosed with b-precursor all of high-risk in january , positive philadelphia chromosome, positive bcr / abl in february , infiltration to the central nervous system (cns), . % of lymphoblasts, and karyotype without legible metaphases. refractory to induction according to the pethema protocol (vincristine, daunorubicin, prednisone, l-asparaginase) with presence of . % blasts at the end of the induction. re-induction was performed with the flag-ida protocol (idarubicin, fludarabine, cytarabine) achieving complete remission, obtaining minimum residual disease (mrd) < . . dasatinib was initiated by bcr / abl expression and cns involvement at the time of diagnosis. an allogeneic hsct was performed, from a male brother donor, with low intensity conditioning tt buflu and prophylaxis of graft-versus-host disease (gvhd) with tacrolimus and sirolimus. patient showed early posttransplant complications, given the reactivation of cytomegalovirus and hemorrhagic cystitis grade i due to adenovirus. late complications such us gvhd at the cutaneous level and subsequent hepatic and gastrointestinal involvement were seen too, for which immunosuppressive therapy was administered with high doses of systemic corticosteroid. results: patient was hospitalized on day + posttransplantation due to febrile neutropenia and respiratory symptoms, with normal chest ct, and ct of paranasal sinuses with acute pansinusitis, for which she received meropenem gr intravenously every hours plus vancomycin gr intravenously every hours during days with symptom resolution. she remained hospitalized for cytopenias with normal bone marrow and % chimerism. on day + posttransplant she presented fever and leukocytosis, with acute respiratory failure with chest ct that showed bilateral alveolar occupation, "crazy-paving" pattern and frosted glass (see image), so diagnostic fibro-bronchoscopy was performed, reporting postoperatively for pneumocystis jirovecii. she received days of trimethoprim-sulfamethoxazole, with a torpid evolution requiring mechanical ventilation and tracheostomy, persisting with hypoxemia. the report of cultures for fungi, mycobacteria, and respiratory panel of filmarray were negative. a pathology report was obtained with % neutrophils, as well as pas staining with acellular pink material and elevated serum ldh, with a diagnosis of secondary pap. the patient continued with poor general condition, refractory hypoxemia, high ventilatory parameters and hemodynamic instability, due to which she was not able to be a candidate for treatment with total pulmonary lavage; leading to multi-organ failure and later death. [[p image] . high resolution chest ct. sample opacification in frosted glass (a) and pattern ''crazypaving'' (b)] conclusions: the importance of considering the diagnosis of pap as a noninfectious pulmonary complication in patients with allogenic hsct despite its low incidence is recognized. disclosure: nothing to declare methods: once the project was approved by the clinical trials and ethics committee, pairs of blood samples were drawn ( from picc line and from venepuncture) from voluntary allo-hsct recipients who were receiving continuous infusion tacrolimus from february through august . the pts had inserted a double-lumen polyurethane picc. tacrolimus was always administered through the red line, and the blood draw always performed through the purple line. all of the patients signed the informed consent. were male and women. median age was years ( - ). of the venepunctures were carried out in the arm where the picc was set, and the other from the contralateral arm. a limited group of nurses performed the extractions of the samples. results: as shown in the table, tacrolimus trough levels determined in blood from venepuncture were similar to those in blood drawn through the picc (median: . vs . ng/ml). when comparing one by one in the individual patients, the differences were not significant, and changed the dosing prescription in no cases. conclusions: in our experience, there are not significant differences in tacrolimus levels draw from the picc line, compared with a peripheral site. so, in our opinion, if the line for tacrolimus infusion is properly identified and the one used for the sample draw is the alternative one, venepunctures to obtain sample from peripheral sites are not justified for tacrolimus levels measurements. background: patients undergoing a hsct may require icu admission due to transplant-related toxicities. the aim of this study was to analyse a single centre experience with hsct patients requiring icu admission and the factors affecting outcome. methods: we included all adult patients (age >= ) who had an allogeneic or autologous hsct during (d between - - to - - ) at st. george's hospital. data was retrospectively collected from patients' notes. icu outcome and -day survival were analysed. for those patients who were admitted to icu more than once, outcome was analysed from their last icu admission. results: allograft patients were included. were male, with a median age years (range - years). were female, with median age years (range - years). diagnosis n (%) includes all ( %), aml ( %), acml ( %), cmml ( %), hl ( %), mds ( %), mds/mpn ( %), fl ( %), scd ( %). sixteen ( %) patients received their first transplant, ( %) received second transplant. eight ( %) patients had sibling donor, patients ( %) had unrelated donor. sixteen ( %) patients had / matched donor, ( %) patients had / matched donor, ( %) patients had / matched donor. nineteen ( %) received reduced intensity conditioning (ric), one ( %) received myeloablative (ma) conditioning. majority of ric allo-hsct patients were conditioned with fludarabine, mephalan, campath (fmc). a small number were conditioned with busulfan, fludarabine and atg. the ma allo-hsct patient was conditioned with tbi, cyclophosphamide. gvhd prophylaxis was ciclosporin alone starting on day - with a target level of - ug/l for all ric and ciclosporin and methotrexate for the ma patients. two ( %) allograft patients were admitted to icu on three occasions. both patients were male, and years old. one had mmud allograft for mds/mpn. the other had nd mud allograft for relapsed aml. the reasons for icu admission include sepsis, cardiac arrest and respiratory failure. the median duration of icu admission was days (range - ). there were deaths within days of transplant. one patient died on day + during his second icu admission with multi organ failure (mof). one patient died after icu discharge on day + with relapsed disease, bronchopneumonia with disseminated fungal infection. icu mortality rate was %, and -day mortality rate was %. nineteen autologous patients were included (median age (range - years)), ( %) were myeloma patients who were conditioned with melphalan, ( %) were lymphoma patients who were conditioned with beam. the icu admission was %. the -day mortality rate was %. conclusions: our centre's icu admission rate, icu mortality rate, cause of icu admission in allo-hsct patients and autologous patients is comparable to literature reports. autologous transplant is safe with no deaths and icu admissions despite an older age. the mortality rate for allo-hsct patients requiring icu admission remain high. all patients were appropriately referred to icu and there was no one who was denied icu admission. this analysis is being extended to preceding years. disclosure: nothing to declare liposomal doxorubicin for the treatment of iatrogenic kaposi sarcoma following hematopoietic stem cell transplantation background: iatrogenic kaposi's sarcoma (iks) represent a rare complication after hematopoietic stem cell transplantation (hsct), related to hhv- infection in hivnegative immunocompromised patients (pts). methods: we describe a case of iks occurred after an allogeneic hsct and we provide a review of the literature using pub med. results: a -year-old man, hiv-negative, received full hla-matched related hsct after a reduced intensity conditioning regimen for relapsed aml. gvhd prophylaxis was based on atg (fresenius mg/kg), cyclosporine (cya) and methotrexate. no severe complication occurred in the first days after transplant. shortly after cya withdrawal, he developed grade i acute gvhd. gvhd resolved after restarting cya. at fifth month after transplant, the patient developed several red and purple angiomatous plaque and nodules involving the skin of both lower limbs, right arm and the nose (figure ). skin biopsy revealed multiple localizations of iks and positive hhv- viremia was detected in the peripheral blood. a visceral involvement was excluded. patient was treated with cya tapering and nine courses of liposomal doxorubicin mg/m every days, obtaining a negativity of hhv- viremia and partial response of the skin lesions. at last follow up, at months after transplant, the patient was in complete remission (cr) for aml, cya-free without signs of gvhd recurrence and with his single stable residual iks lesion on his left limb, currently waiting for local radiotherapy. we found additional iks published cases after hsct. most of post-hsct iks were secondary to an allogeneic-hsct ( out of , . %) and occurred in adult ( , %) and male ( , %) pts. median age at the time of iks diagnosis was . years (range - ). thirteen pts ( . %) had mediterranean origin. the most frequent underlying disease was aml ( . %). gvhd prophylaxis was primary based on calcineurin inhibitor. half of the pts developed gvhd and were treated with steroid and other immune suppressive drugs. median time between the hsct and the occurrence of iks was . months (range . cutaneous iks was the prevalent form of manifestation, however visceral involvement was reported in pts ( . %). in four cases ( . %) an hhv- associated bm failure was report. immune suppression drugs tapering ( . %) and chemotherapy ( . %) were the most frequent actions taken after the diagnosis of iks. in most cases, liposomal doxorubicin was used as chemotherapy. cr rate was high, . %, whereas progression disease occurred in out pts ( . %), all of which had visceral involvement. in pts ( . %), iks was the cause of death. conclusions: withdrawn of immune suppression drugs and anthracycline based chemotherapy can represent a feasible treatment option for pts with iks after hsct. clinical background: acquired haemophilia a (aha) is an autoimmune disease caused by the spontaneous production of neutralizing immunoglobulin g (igg) autoantibodies (inhibitors) targeting endogenous fviii. treatment of these inhibitors presents additional challenges in a hematopoietic stem cell transplantation (hsct) recipient, because preservation of the graft that restores a normal hematopoiesis is critical. here we describe the management of a case of aha in an acute myeloid leukemia patient following hsct. methods: the clinical, laboratory and molecular aspects of a -year-old italian male who developed aha after allogenic bone marrow transplantation were collected and presented in order to show how we diagnose and manage this severe but rare complication within the special setting of hsct. results: a -years-old man with a flt- itd, npm- , runx -runx t and cbfb-myh negative, not differentiated, chromosomally normal acute myeloid leukemia (aml) in third complete remission (cr) was submitted to a hematopoietic stem cell transplantation (hsct) from his haploidentical son. the conditioning regimen consisted of oncothiotepa, busulfan and fludarabine and was followed by the infusion of a t-cell depleted bone marrow graft. gvhd prophylaxis consisted of cyclosporine a (csa) and mycophenolate mophetyl (mmf). neutrophil engraftment occurred on day + . recipient's autoimmunity was negative. at months post-transplantation the patient received an antipneumococcal vaccination. fifteen days post-vaccination the patient was admitted to our in-patient ward due to general malaise, diffuse muscle and joint pains, cutaneous bleedings, oedemas, hyperchromic urines and constipation. physical examination revealed diffuse ecchymosis, swelling of deep muscles with a progressive functional disability due to hematomas and hemorrhagic suffusions of the tongue frenulum. and anti-factor viii inhibitors . bu/ml (high titers > bu/ml). thus, a diagnosis of acquired autoimmune haemophilia a was made and treatment with feiba combined with prednisone was started. patient's clinical conditions dramatically improved as he referred an improvement of movements and the resolution of joint and muscle pains despite the persistence of deep hematomas just after one day of treatment that had determined an increase of fviii:c value to . % and an improvement of aptt to . seconds. on the following medical checks physical examination showed the progressive disappearance of deep muscle hematomas, and normal values of fviii:c. conclusions: aha is a rare but severe complication following hsct and it could appear years afterengraftment. a prompt diagnosis and an early treatment with feiba and corticosteroid are necessary to avoid life-threatening sequelae. the inclusion of the coagulation panel in the laboratory exams performed during the follow-up is advisable in order to early detect this life-threatening complication. disclosure: nothing to declare background: splanchnic thrombosis is an uncommon complication of myelofibrosis and a controindication to proceed to hematopoietic stem cell transplantation (hsct) due to the risk of additional vascular and endothelial complications. we present a patient with myelofibrosis (mf) that proceeded to hsct from an unrelated donor, despite splanchnic thrombosis unresolved after heparin treatment and unable to proceed to surgical treatment due to severe thrombocytemia. methods: a -year woman with mf secondary to essential thrombocythemia, with intermediate- score according dynamic international prognostic staging system (dipss) and with extreme splenomegaly (maximum diameter cm), refractory to ruxolitinib, showed an extensive thrombosis of the portal and splenic veins, unresolved after -week heparin therapy, at the time of availability of an hla ( / ) and abo matched unrelated donor. she received a conditioning regimen including fludarabine and thiotepa and a gvhd prophylaxis with atg thymoglobuline, cyclosporine and methotrexate, followed by the reinfusion of . x /kg cd + pbsc. at the time of transplant we were aware of an high risk of developing sos, on the basis of the older age of the recipient, the unrelated donor, the advanced stage of myelofibrosis and the ferritin serum level of . ng/mg. results: on day + after hsct sos complicated the aplasia phase, characterized by jaundice, ascites, weight gain, progressive increase in creatinine and bilirubin serum levels. an ultrasound of abdomen confirmed an unchanged thrombosis extension and the development of ascites. on day + the patient was categorized as very severe sos stage, according to ebmt severity criteria, because of doubling of bilirubin serum level in hours and a % increase in comparison with her baseline weight. therefore, defibrotide was promptly started in association with diuretic therapy. the treatment was continued for weeks and allowed gradual restoration of the water balance and normalization of bilirubin serum level. at the last follow-up, months after hsct, the patient shows the persistence of a non-transfusion dependent anemia, platelets . x ^ /ul, palpable spleen cm below the rib, > % allogeneic chimerism in the granulocytic compartment and % in the t lymphocyte compartment. splanchnic thrombosis is partially recanalized and replaced by collateral circles with cavernous aspects. the patient is on treatment with fondaparinux and has shown neither significant infectious episodes or acute or chronic gvhd. conclusions: we conclude that defibrotide treatment allowed to perform a successfull allogeneic transplant in a patient with mf associated with an overt picture of splanchnic thrombosis. background: hematopoietic stem cell transplantation (hsct) is associated with an increased incidence of secondary malignancies including skin cancer. squamous cell carcinoma (scc) is the most common type in patients who are receiving immunosuppressive therapy and chronic graft-versus-host disease (cgvhd) appears to be an important risk factor for its development. recent studies describe voriconazole exposure as an independent factor that may contribute to this increased risk as well. in our best knowledge, no cases of scc have been reported in pediatric allogeneic hsct to date. methods: we present a case report of a year-old boy who developed a scc with high-risk features six years after undergoing hematopopoietic stem cell transplant. results: a year-old boy with acute lymphoblastic leukemia (all) underwent a matched unrelated bone marrow transplant years ago. he developed grade iv agvhd followed by extensive cgvhd with generalized scleroderma. he required intensive and continued immunosuppressive therapy and was on prolonged antifungal prophylaxis with voriconazole. in march , he developed scc involving left temporal region that was completely excised. two months later, more lesions in scalp and nose were noted and intralesion treatment with methotrexate was started. however, an unfavorable evolution was noted and he was put on systemic treatment including cisplatin and cetuximab receiving the whole scheme from january to march and continuing only with cetuximab, ten doses in total, until may, for unaceptable and severe tubulopathy that required admission at the hospital in several ocassions. he achieved a very good partial response but progression was noted shortly in follow up. at this point, non curative therapeutic options were found and he was put on intralesion methotrexate and photodynamic theraphy in a weekly basis with palliative intention. unfortunately, tumor growth was fast and patient passed away in august , fifteen months after squamous cell carcinoma diagnosis, due to tumoral progression. conclusions: ) scc is a rare, non-previously described, secondary malignancy in children undergoing hsct. ) high-risk features scc constitutes an aggresive disease with a median overall survival below year. ) cgvhd appears to be an important risk factor for its development. ) voriconazole induced-photosensitivity might have played a role. ) cisplatin based regimens +/-cetuximab are a therapeutic option in disseminated and/or high risk cases. as outcomes are unsatisfactory in these cases, alternative therapeutic options need to be explored. disclosure background: pregnancy is a rare event after allogeneic stem cell transplantation (sct) for acute leukemia. here we report, to the best of our knowledge, for the first time on a successful pregnancy after treosulfan-based conditioning. methods: a -year old woman was diagnosed with acute myeloid leukemia (aml) secondary to chronic myelomonocytic leukemia in july . ovarian preservation was performed by leuprolide acetate depot injection prior to cytostatic chemotherapy. of note, no cryopreservation of oocytes or ovarian tissue was conducted. she received two cycles of chemotherapy consisting of idarubicine ( mg/m² on day - ) and cytarabine ( mg/m² b.i.d. on days , , and ). due to secondary origin of aml sct was performed in first complete remission of aml after conditioning with treosulfan ( g/ m² days - ) and fludarabine ( mg/m² days - ). she received . × cd -positive cells per kilogram body weight from a hla-matched unrelated donor. results: follow-up bone marrow aspirates showed continuous complete remission of aml. seven months after sct she became pregnant, but decided for induced abortion. in january , months after hsct she became pregnant again and desired the child. medical examinations were performed monthly on an outpatient basis in stringent cooperation with the maternity clinic. the course of pregnancy was unremarkable, although she was hospitalized due to premature labor in the th week of pregnancy. however, gynecological examination showed no clinical significant findings, so that section was planned and she could be discharged again. in the th week of pregnancy she gave birth to a healthy girl ( cm, g) by cesarean section. peripartum she developed hypoethesia of the left body half. neurological examination showed no abnormalities and she recovered immediately. there were no other postpartum complications. breastfeeding was established but additional food was necessary for a sufficient nutrition of the child. conclusions: this case of successful pregnancy following sct demonstrates that fertility can recover after treosulfan-based conditioning. however, detailed studies of ovarian function and fertility are necessary to gain more insight into the risk of premature ovarian failure. disclosure: nothing to declare. experimental stem cell transplantation p cd -cart therapy before allo-hsct in children and adolescents patients who diagnosed r/r b-all with e a-pbx background: b-all with e a-pbx in children and adolescents is described with favourable prognosis. but there are more than % patients with e a-pbx diagnosed as relapsed or refractory. the results of allo-hsct in children and adolescents with this group leukemia in our center was analyzed in order to understand the therapeutic effect of cd -cart on the patients. methods: retrospective analysis, from june st, to july , , all children and adolescents diagnosed relapse or refractory b-all with e a-pbx who received allo-hsct, total cases. all patients was divided into two groups depending on whether or not accepted cd -cart before allo-hsct. according to fcm-mrd and e a-pbx level before allo-hsct, os lfs and cumulative recurrence rate were analyzed. r . . was used as statistical analysis software. results conclusions: . for r/r b-all with e a-pbx in children and adolescents, fcm-mrd pre-transplant hasn't obvious effect on the outcome of allo-hsct, while the level of e a-pbx has obvious effect. the out come of e a-pbx negative group was obviously better than positive group. . cd -cart can obviously improve the os and lfs, it is mainly because of cd -cart can makes more patients fusion to zero. . for r/r b-all with e a-pbx in children and adolescents, if chemotherapy can't make the fusion to zero. it is suggested to accept cd -cart therapy to make the fusion zero. it can improve the outcome of os and lfs. disclosure background: currently, hematopoietic stem cell transplantation (hsct) represents the only curative treatment for numerous hematopoietic malignancies like leukemias, immune deficiencies or metabolic diseases. cd serves a quality marker for stem cell grafts, which is not solely expressed on stem cells but also on a variety of progenitors. the role and the impact of these subpopulations remains unknown. we made use of our genetic barcode system to analyze the influence and contribution during reconstitution on a clonal level. methods: fluorescence activated cell sorting (facs) was used to sort hematopoietic stem and progenitor populations, namely hscs, mpps, cmps and clps, which were lentivirally transduced with our previously established bc barcoding system. after mixing the marked cells with bone marrow support, lethally irradiated recipient animals were and transplanted and monitored over weeks. we focused on bone marrow, blood, spleen and thymus, on chosen endpoints ( w, w, w, w) and samples were used to analyze the contribution of the subpopulations during the reconstitution process based on fluorescent protein (fp) expression. to investigate the clonal contribution in different organs, we performed next generation sequencing (ngs) and frequencies of unique barcodes in a sample were analyzed by bioinformatical approaches. results: a maximum of % of cells expressed the encoded fps, which were mostly derived from the hscs and mpps. cmp-derived cells were only detected week after transplantation in the myeloid compartment. cells derived from the clps were not detected at any time point. we analyzed the barcode content of the differently marked cells after next-generation-sequencing. in accordance with the facs data, the majority of the clones during the weeks of observation are derived from hscs and mpps. cmp-derived clones were only contributing during the first weeks and clp-derived clones are barely detectable. we did not observe any major differences with regard to age of donor or recipient, despite the total number of clones is higher in the group, which received the "aged" graft, independently from the transduced cell population. conclusions: here we show the suitability of our highly complex multi-color barcode system to study the clonal contribution of hscs and three progenitor populations after hsct. our results will contribute to a better understanding how these different populations interact to support the establishment of a new hematopoietic system. emphasized by the variability in data of graft and recipient age, this comprehensive analysis gives rise to an impression to the necessity of personalized graft composition, by which treatment success could be influenced. disclosure: nothing to declare survival and fate of adipose derived mesenchymal stem cells in a rat brain injury model background: mesenchymal stem cells have been identified as promising candidates in the treatment of central nervous system (cns) injury through neurotrophic support and immunomodulation. adipose tissue is an attractive source of mesenchymal stromal/stem cells (ascs) for regenerative therapeutic applications because they can be harvested from autologous donors with minimally invasive methods, can be rapidly expanded ex vivo, show low immunogenicity if allogeneic, and can be used in autologous or heterologous settings. the present study examines the fate and effects of intracerebroventricularly (icv) transplanted ascs in a traumatic brain injury (tbi) model. methods: ascs were isolated from inguinal fat pad of adult wistar rats under sterile conditions and cultured according to standard procedures. ascs at passage ( x cells) were seeded and transfected with sleeping beauty transposase and pt venus-neo r plasmids. selection with g antibiotic resulted in the generation of a homogeneous asc population which expressed fluorescent venus protein for several passages, phenotypic characterization showed that these cells were . % double positive for cd and cd stem cell markers, verifying their mesenchymal origin. tbi was induced by stereotactic surgery under deep anaesthesia and subsequently icv transplantation of venus+ ascs was performed on adult wistar rats. normal ascs-transplanted and tbi-saline transplanted rats were used as controls. the proliferation, migration, survival and fate of transplanted ascs and their effect on injury restoration were examined six weeks post transplantation (pt). results: six weeks pt ascs expressed the fluorescence venus protein and therefore were identified in brain parenchyma. their presence into brain was also confirmed by masson trichrome staining, which revealed their collagen depositions. ascs were found in lesser numbers compared to those transplanted and exhibited no proliferative activity. ascs were found scattered distributed in brain as individual cells, and there were no aggregates of ascs or mass formation into lateral ventricles. extensive migration of ascs was mainly performed through white matter tracks in the corpus callosum and fimbria of hippocampus. six weeks pt ascs retained the characteristics of mesenchymal cells and did not differentiate into cells of neural lineage. ascs exhibited limited long-term survival, which is restricted in perivascular areas probably contributing to vascular formation. homing of ascs into peri-injured area was detected in half of the animals and achieved through the corpus callosum, as revealed by the collagen depositions, in this white matter track. transplanted ascs reduced the area of tbi cavity and did not enhance the astroglial scarring in peri-injured area. in tbi +ascs transplanted animals, the cortical injury site, showed a significantly smaller volume and lower % tissue loss compared to that of tbi+vehicle animals ( . ± . mm and . ± . % respectively, versus . ± . mm and . ± . %, p= . and p= . respectively). conclusions: considering the effects of ascs on inflammation and regeneration, we suggest that their transplantation after brain injury may promote host brain repair mechanisms. ascs transplantation may be beneficial in tbi, however some of its effects need careful and indepth evaluation. disclosure: nothing to declare xie-na cao , yuan kong , zhong-shi lyu , , qi wen , min-min shi , , qian-yu sun , yu-hong chen , yu wang , lan-ping xu , xiao-hui zhang , xiao-jun huang , background: poor graft function (pgf) remains a serious complication after allogeneic hematopoietic stem cell transplantation (allo-hsct). our previous work reported that abnormal bone marrow (bm) endothelial cells (ecs) were involved in the pathogenesis of pgf patients after allo-hsct (bbmt ; bmt ; blood ), but the explicit mechanism requires further clarification. autophagy is a self-degradative process responsible for the elimination of cytosolic components including proteins and damaged organelles. recent findings demonstrated that stimulation of autophagy could reduce oxidative status and angiogenic potential in ecsafter high-glucose exposure, from diabetic patients.however, little is known regarding the autophagy of bm ecs in pgf patients. therefore, the current study was performed to evaluate whether autophagy in bm ecs play a role in the pathogenesis of pgf. moreover, to investigate the effects of autophagic regulation on ecs and thereby regulating hematopoietic stem cell (hscs). methods: in the prospective case-control study, the autophagy levels were compared in bm ecs from pgf patients, and their matched good graft function (ggf) patients.the expression levels of autophagy-related markers (lc , beclin , and p ), and intracellular autophagosomes were detected by immunohistochemical staining, flow cytometry, western blot and transmission electron microscopy. subsequently, rapamycin (the autophagy activators) or hydroxychloroquine (hcq, the autophagy inhibitor) were administrated tothe -day cultivated bm ecs and human umbilical vein endothelial cells (huvecs), respectively.the autophagic vacuoleswere detected by monodansylcadaverine (mdc) staining assay. the bm ecsand huvecs were evaluated by cell counting, dii-ac-ldl and fitc-lectin-uea- double staining, migration, cell proliferation, and levels of reactive oxygen species (ros). to explore whether autophagy would affect the ability of bm ecs to support hscs in vitro, bm cd + cells from healthy donors were co-cultured with cultivated bm ecs and huvecs. colony-forming unit (cfu) and the apoptosis of co-cultured hscs were analyzed. results: the defective autophagy in bm ecs, characterized by decreased intracellular autophagosomes and autophagic vacuoles, decreased expression of lc -ii and beclin , and high level of p , were observed in pgf patients compared with ggf patients. moreover, the coculture of bm cd + cells with bm ecs showed significant deficient cfu plating efficiency, and increased apoptosis of cd + cells in pgf patients. in vitro upregulation of autophagy by rapamycin quantitatively and functionally improved bm ecsand huvecs, which manifested as more dii-ac-ldl and fitc-lectin-uea- double stained cells, increased capacities of migration, lower levels of ros and apoptosis via regulating beclin pathway, whereas inhibition of autophagy by hcq aggravated the huvecs and bm ecs from pgf patients. furthermore, in vitro upregulation of autophagy by rapamycin significant improved cfu plating efficiency, and decreased apoptosis in bm hscs co-cultured with huvecs and bm ecs from pgf patients. conclusions: these findings suggest that defective autophagy in bm ecs may be involved in the pathogenesis of pgf. the effect of rapamycin in pgfpatients is potentially mediated by improving the dysfunctional bm ecsto support hscs. therefore, it would be of value to investigate whether upregulating of cytoprotective autophagy of bm ecs may ameliorate pgf, thereby providing a novel clinical intervention for pgf in the future. clinical background: heparanase (hpse) in an endoβ-glucuronidase that specifically cleaves the saccaride chains of heparan sulphate proteoglycans (hs), leading to a loss of integrity of the extracellular matrix and to release of hs-bound cytokines, chemokines, angiogenic and growth factors. hpse gene is polymorphic and includes approximately snps. the combination of two snps, rs and rs , are involved in the regulation of hpse expression with an inverse correlation between mrna expression and protein levels: gg-cc, gg-ct, gg-tt, ga-cc (low group) expressed high hpse concentration; ga-ct and ga-tt (median group) expressed intermediate hpse levels; aa-tt and aa-ct expressed low hpse concentration (high group). we studied hpse snps in the allogeneic stem cell transplantation (hsct) setting to evaluate a possible association with post-hsct outcomes. methods: we enrolled patients submitted to hsct in our department since to . for each couple recipient-donor, rs snp was genotyped using restriction fragment lenght polymorphism assay, whereas for rs snp an allele-specific polimerase chain reaction was applied. hpse genotype distribution was compared in different groups according to post-hsct outcome: graft-versus-host disease (gvhd), transplantrelated mortality (trm), overall survival (os), infectious complication and disease-free survival (dfs). statistical analysis was performed using ncss . results: distribution of rs snp was as follows: gg . %, ga . % and aa . % among recipients and . %, . % and . % among donors, respectively. hardy-weinberg equilibrium (hwe) was respected. distribution of rs snp was as follows: cc . %, ct . % and tt . % among recipients and . %, % and . % among donors, respectively. rs snp distribution did not respect the hwe. an association was found between recipient rs snp and the cumulative incidence of agvhd among patients submitted to a reduced intensity conditioning (ric): . % for tt genotype and % for ct or cc genotype (p= . ). on the other hand, an association was identified between donor rs /rs snps combination and the cumulative incidence of agvhd: . % for low group donor, % for median group donor and . % for high group donor (p= . ). conversely, aa genotype for donor rs resulted independent risk factor for cgvhd de novo development (p= . , od . ) together to donor-recipient sex mismatch (female donor to male recipient vs. others: p= . , od . ) . considering cmv reactivation rate after hsct, an association was observed according to recipient rs snp: % for cc genotype, . % for ct genotype and . % for tt genotype (p= . ). multivariate analysis confirmed recipient rs snp as independent risk factor for cmv reactivation after hsct (p= . , od . ) together with recipient cmv serostatus at transplant (positive vs. negative: p< . , od . ). conclusions: hpse role was widely studied in the setting of inflammation, autoimmune diseases, hematological disease and tumor. however, it still remains debated the inducing or protective activity of hpse in the setting of gvhd. obviously, our results need to be confirmed in a validation cohort. clinical trial registry: na disclosure: nothing to declare novel protocol for autologous hsct in patients with high risk of complications: ambulatory chemomobilization and transplantation of fresh hematopoietic stem cells with backup storage background: autologous hematopoietic stem cell transplantation (ahsct) is standard of treatment in many patients with high risk of complications: dialysed patients, patients with heart and kidney amyloidosis or patients with systemic sclerosis. we introduced recently a novel protocol for ahsct: combination of ambulatory mobilization with very low doses of ara-c and g-csf connected with direct ahsct with fresh cells. this protocol allowed us to reduce the transplant risk in various patient groups traditionally connected with high risk of complications. in this work we summarize the experience in such high risk patients. methods: the prospectively collected database of patients after ahsct was searched for patients who underwent ahsct after chemomobilization with ara-c and transplantation with fresh cells and who fulfilled at least one study inclusion criteria: a) dependence on dialysis b) amyloidosis c) systemic sclerosis d) disqualification from transplantation at other centre due to the high risk of complications. there were together patients selected for this analysis - with amyloidosis ( with ≥ organs involved), dialysed, with systemic sclerosis, unfit at other centre. the database included prospectively recorded serious adverse events during the mobilization and transplantation. results: there were transplantations performed in this group of patients. mortality was % at days. all patients underwent successful ambulatory mobilization. all patients received mephalan conditioning with single infusion with median dose of mg/m (min , max ). mean engraftment was . days for white blood cells and . days for plt over g/l. the rate of complications was low with cases of neutropenic fewer, single bacterial culture with staphylococcus epidermidis without clinical signs of infection, median mucositis grade of . and without patients on parenteral nutrition. the median time of hospitalization was days (min , max ). conclusions: we present here novel protocol of transplantation combining chemomobilization and ahsct with fresh cells with excellent safety profile among most severely ill patients allowing for safe and efficient transplants. with this protocol we were able to overcome multiple risk factors and perform full intensity transplantation in very fragile patients. disclosure: nothing to declare single umbilical cord blood transplantation provides durable disease remission of advanced hematological malignancies in elderly patients background: although allogeneic hematopoietic stem cell transplantation (allo hsct) is potentially curative therapy in a variety of hematological malignancies, little has been reported of the outcome for elderly patients who are not in remission at transplantation. but it has been pointed out that recipient age alone can not be regarded as contraindication for allo hsct in the literature recently, supported by suitable donor, conditioning regimens and appropriate management of complications. we conducted a retrospective study of elderly patients who had advanced hematologic malignancies to elucidate the outcome of single umbilical cord blood transplantation (sucbt) in toranomon hospital kajigaya, japan. methods: we retrospectively investigated the outcomes of patients aged over who underwent their first ucbt from june to december in our medical center. results: diseases included acute myelogenous leukemia (n= ), myelodysplastic syndrome (n= ), adult t-cell leukemia/lymphoma (n= ), myelofibrosis (n= ) and chronic lymphocytic leukemia (n= ). the median age at transplantation was years (range, - ) and follow-up for survivor post transplantation was day (range, - ). all patients were not in complete remission (cr) at the time of transplantation. reduced intensity conditioning (ric) regimens were used in patients. all patients received tacrolimus and mycophenolate mofetil as graftversus-host disease (gvhd) prophylaxis. all cases except early death achieved neutrophil recovery at median days (range, - ). at year, overall survival (os) rate and disease free survival (dfs) were , % ( % confidence interval (ci), . - . ). we performed univariate analysis to identify the factor that influenced os at year, but no statistical significance was demonstrated at the age of transplantation (aged to vs. ≧ , . % ( % ci, . - . ) vs. . % ( % ci, . - . ), p= . ). the cumulative incidence of non-relapse mortality (nrm) at days was . % ( % ci, . - . %) and relapse at year was . % ( % ci, . - . %). only two patients developed acute gvhd(ii-iv) and one developed severe gvhd at days after transplantation. the main causes of death was infection (n= ), including sepsis (n= ) and viral encephalitis (n= ), followed by idiopathic pneumonia syndrome (n= ) and thrombotic microangiopathy (n= ) during the early phase of transplantation. in contrast, no patients died of recurrence. conclusions: although our report consisted relapsed/ refractory disease of elderly patients at the time of sucbt, durable remission and lower incidence of gvhd could be noteworthy compared with previous reports. further strategies to reduce the rate of nrm and longer duration of follow up would be warranted. disclosure background: pearson syndrome and kearns-sayre syndrome are metabolic disorders caused by a de-novo deletion in the mitochondrial dna (mtdna). allogeneic stem cell transplantation has shown to improve metabolic function in distal organs in several metabolic disorders, but bears significant morbidity and mortality, especially for patients with mitochondrial disorders. novel gene therapies may correct diseases rising from genomic dna mutations, but targeting the mitochondrial dna is complex. mitochondria are able to transfer into cells and between cells, as seen in preclinical models of mitochondrial and other metabolic disorders. here, we introduce a novel concept of mitochondrial augmentation therapy (mat) of autologous cd + cells in children with mitochondrial deletion syndromes. methods: patients were treated under a compassionateuse program, approved by the sheba medical center irb and the israeli ministry of health. briefly, mobilization was performed using gcsf alone (n= ) or in addition to plerixafor (n= ) . cd + cells were isolated via miltenyi clinimacs system and co-cultured with maternal mitochondria, drawn from peripheral blood and confirmed nondeleted, for hours, and re-infused to the patient without any conditioning. patients were followed for clinical and metabolic parameters. results: all four patients presented with different deletions in mitochondrial dna, and different baseline characteristics, and were treated at the age of . , , and years. despite normal cbc, significant bone marrow hypocellularity was seen in evaluated patients ( %, % and % cellularity at age , and ), which correlated with low colony forming unit capacity of patients and low yield of cd + mobilization in the leukapheresis product. patients received on average x enriched cells/kg (range, . - . ), and the median enrichment of cd + cells was % (range, - %). no infusion reactions occurred, and the only severe adverse events of this cellular therapy were leukapheresis-related anemia, hypokalemia, hypocalcemia and alkalosis, all resolved promptly with proper supplementation. follow-up duration is variable, ranging - months. we were able to show improvement in mitochondrial heteroplasmy (proportion of deleted mtdna of total mitochondrial dna) and in normal mtdna content, starting - months from cell therapy, which correlated with improved atp production in peripheral blood derived mononuclear cells. clinically, patients showed improvement in aerobic function and endurance (measured by the half-bruce protocol, sit-to-stand test and -minute walk test), muscle strength (hand-held dynamometry), and in quality of life, measured by the international pediatric metabolic disability scale. no metabolic crises occurred following cell infusion. conclusions: patients with deletion in mtdna have metabolic dysfunction, including poor bone marrow cellularity and function. hematopoietic stem cells in patients with mtdna deletions can be enriched with normal mitochondria, via mat, as first shown in our patients. this novel process is safe and results in increase in the normal mtdna in peripheral blood of patients, and in improved metabolic and clinical function. clinical trial registry: clinicaltrials.gov nct disclosure: moria blumkin, noa sher and natalie yivgi ohana -minovia therapeutics, employment p high cytotoxic efficiency of alpharetrovirally engineered cd -specific chimeric antigen receptor natural killer cells for treatment of acute lymphoblastic leukemia stephan müller , tobias bexte , annekathrin heinze , franziska schenk , axel schambach , winfried s. wels , , ute modlich , evelyn ullrich , background: autologous chimeric antigen receptormodified (car) t cells with specificity for cd showed potent antitumor efficacy in clinical trials regarding relapsed and refractory acute lymphoblastic leukemia (all). natural killer (nk) cells are cytotoxic lymphocytes that are capable to kill their targets in a non-specific manner and additionally do not cause gvhd. therefore, using cd -car-nk cells exhibits several advantages, such as safety in clinical use, possible allogenic settings and the potential to also attack heterologous leukemia cells which lost cd . previous approaches used cd -car-nk cells pre-stimulated by feeder cells, bearing potential risks. thus, we focused on the optimization of generating cd -car-nk cells by viral transduction under feeder-cell free conditions. methods: human nk cells were isolated from healthy donor peripheral blood mononuclear cells via cd negative selection. after a feeder-cell free expansion phase with interleukin , transductions were performed with an egfp or a cd -car encoding vector at different multiplicities of infection (moi). to optimize gene modification different transduction enhancers (retronectin and vectofusin- ) and viral vector systems (lentiviral and alpharetroviral) were compared. finally, generated cd -car-nk cells were tested in their ability to kill cd positive and cd -negative cell lines. results: nk cells transduced with a lentiviral egfp encoding vector or a lentiviral cd -car vector using retronectin and vectofusin- showed similar transduction efficiencies for both transduction enhancers (egfp: retronectin moi : . %; vectofusin- moi : . %; cd -car: retronectin moi : . %, moi : . %; vectofusin- moi : . %, moi : . %). the generated cd -car-nk cells showed increased cytotoxic capacity against cd -positive cells compared to nontransduced (nt) nk cells ( . % vs. . %, effector to target (e:t) ratio : ). both nk cell populations were equally efficient in killing cd -negative cells ( . % vs. . %). alpharetroviral transduction of nk cells with an egfp encoding vector showed higher transduction rates with vectofusin- than with retronectin (retronectin moi : . %, moi : . %; vectofusin- moi : . %, moi : . %). further using vectofusin- , similar transduction efficiencies could be achieved with an alpharetroviral cd -car encoding vector (moi : . %, moi : . %, moi : . %), outperforming the efficiencies of lentivirally generated cd -car-nk cells in the same experiments (moi : . %, moi : . %, moi : . %). additionally, alpharetroviral cd -car-nk cells showed a higher cell killing activity against cd -positive cells than lentiviral cd -car-nk cells or nt-nk cells ( . % vs. . % vs. %, e:t ratio : ). interestingly, similar killing activities were achieved with an e:t ratio of . : ( . % vs. . % vs. . %) and alpharetroviral cd -car-nk cells remained a stable cytotoxicity level at lower cell concentrations down to an e:t ratio of . : . all three nk cell populations were equally efficient in killing cd negative cells ( . % vs. . % vs. . %, e:t ratio : ). conclusions: cd -car-nk cells can be successfully generated under feeder-cell free conditions using different transduction enhancers and viral vector systems. these data suggest the usage of vectofusin- in combination with alpharetroviral vectors to genetically modify nk cells to achieve sufficient amounts of transduced cells. these cd -car-nk cells mediate high cytotoxicity and therefore may offer a new therapeutic option in the treatment of all. disclosure: axel schambach is an inventor on a patent describing alpharetroviral sin vectors. winfried s. wels is an inventor on a patent describing chimeric antigen receptors with an optimized hinge region. the remaining authors have nothing to disclose. graft-versus-host diseaseclinical walter spindelböck , bianca huber-krassnitzer , barbara uhl , gregor gorkiewicz , hildegard greinix , christoph högenauer , peter neumeister background: steroid-refractory acute gastrointestinal (gi) graft-versus-host disease (agvhd) is a severe complication of allogeneic hematopoietic stem cell transplantation (allo-hsct) associated with a high mortality rate. loss of intestinal bacterial diversity is thought to be associated with severity of gi-agvhd and an impaired intestinal microbiota with reduced diversity is an independent predictor of mortality. methods: the fecal microbiota transplantation (fmt) procedures were performed according to a protocol approved by the local ethical committee ( - ex / ) after obtaining informed consent. donors were healthy adult subjects screened for potential infections by serologic and microbiologic tests according to local standards. donor stool was diluted with saline and homogenized to a volume of~ ml fecal solution for instillation into the terminal ileum and caecum via colonoscope. microbiota sequencing analysis of s rdna was performed before fmts and afterwards at predefined timepoints. results: we report the outcome of nine patients refractory to - lines of immunosuppressive therapies with lower gi-stage iii (n= ) or iv (n= ) agvhd following repetitive fmts from a single donor. all patients had received an allo-hsct for mds (n= ) , aml (n= ), pmf (n= ) and mm (n= ) following a reduced intensity (n= ) or mac (n= ) conditioning regimen using pbsc as stem cell source. after an onset of lower gi agvhd between - days after allo-hsct, nine patients refractory to several lines of immunosuppressive therapies received - fmts ( patients were treated with more than fmts, in patients fmt was only administered once or twice) mostly in weekly intervals. five patients achieved a clinical complete response with resolved diarrhea and no gastrointestinal complaints, and four of these could be discharged without gvhd symptoms. two patients (pr, nc) were discontinued after or fmts in pr or nc due to concomitant infections (metapneumoviral pneumonia, cmv gastroenteritis), the other non-responders succumbed to gvhdrelated infectious complications. the establishment of donors' microbiota with the emergence of new taxa, an increase in bacterial richness/diversity, and the disappearance of the "enterococcus signature" were associated with disease control and response to fmt. except the possible transmission of adenovirus by fmt in one patient, no other immediate procedure-related infections or other side effects were observed. conclusions: restoration of dysbiosis by fmt might represent a promising novel therapeutic approach for a subset of patients with refractory lower gi-agvhd. vigorous donor screening for infectious disease is mandatory. clinical background: migration of allo-activated donor effector tcells from lymphoid tissues to target organs is an important step in acute graft versus host disease (gvhd). the sphingosine- -phosphate- (s p ) receptor plays a crucial role in lymphocyte trafficking. data from animal models suggest that pharmacological modulation of the s p receptor reduces gvhd and improves mortality. we investigated this mode of action by using the secondgeneration s p modulator krp for the prophylaxis of gvhd in a pilot clinical trial in patients undergoing allogeneic hsct. methods: a multi-centric, phase b, prospective, open label, two-part study was conducted to evaluate the safety, tolerability and pharmacokinetics of krp in patients undergoing allogeneic hsct for hematological malignancies. primary endpoint was safety. initial efficacy was explored based on the incidence of gvhd, mortality and relapse. part was a single arm open label study to investigate the safety of mg/day krp added to standard of care gvhd prophylaxis (csa/mtx) in patients. part was a randomized two-arm open label study to compare the safety, efficacy and pk of mg/day of krp in combination with tacrolimus/mtx to mg/day of krp in combination with csa/mtx in patients. in both parts, treatment with krp was initiated days before hsct and continued for an additional days. patients were followed up for up to years. results: patients were included in the study. of patients completed the -day treatment with krp at the assigned doses. median duration of follow-up was days (range to days). krp was safe and well tolerated. serious adverse events (saes) suspected to be related to krp were observed. macular edema (n= ) and peripheral edema (n= ) as s p related adverse events occurred and resolved without sequelae. of note, the incidence of macular edema in hsct recipients is unknown. neutrophil engraftment was confirmed in all patients with a median of days (range to days). of patients presented with grade iii or iv acute gvhd (on days , , , and ) . no gvhd or infection related death occurred during the first days. -day survival was %, with no death occurring during krp treatment. death occurred on study day due to lymphoma relapse. a second death occurred on study day due to liver gvhd. four patients died in the follow-up period due to gastrointestinal gvhd (day ), aspiration pneumonia (day ) and relapse (day and day ). the kaplan-meier estimate of overall survival at year was . . when comparing the data from the two dose groups ( and mg krp ), no major differences in safety, engraftment, gvhd rate or mortality were observed. conclusions: this clinical trial was the first to test s p modulation in this population. our data suggest that krp had no negative impact on engraftment and overall, was safe, and well tolerated. based on exploratory data, when comparing to matched historical mortality data, krp may have favorable effects on overall survival ( figure ). background: uric acid is a danger signal contributing to inflammation. relevance to allosct has been demonstrated in preclinical models: the depletion of uric acid led to improved survival and reduced gvhd (j exp med. sep ; ( ): - ). results of a clinical pilot trial suggested that peri-transplant uric acid depletion reduce acute gvhd incidence (bbmt may; ( ): - ). methods: this international multicentric study aimed to study the association of uric acid serum levels before start of conditioning with allosct outcome. patients with acute leukemia, lymphoma or mds receiving a matched sibling allosct for the first time were considered for inclusion, regardless of conditionning. data were prospectively collected between / and / . a comparison of outcomes between patients with high and low uric acid level was performed using univariate analysis and multivariate analysis using cause-specific cox model. variables included in the multivariate analyses were age, sex mismatch, diagnosis, disease status, karnofsky score, number of cd cells given, intensity of conditioning, type of gvhd prophylaxis, atg use, time from diagnosis to transplant, year of transplant and cmv status. results: twenty centers from european countries reported data on allosct recipients. patient characteristics are given in table . the uric acid cut off point was determined at . mg/dl (median of measured uric acid levels). overall survival (os) and progression free survival (pfs) of allosct recipients with uric acid levels above cut off measured before start of conditioning were significantly shorter ( figure a , os univariate hr= . ci= . - . p< . ; multivariate hr= . , ci= . - . , p< . ) ( figure b , pfs univariate hr= ci= . - . p= . ; multivariate hr= . , ci= . - , p= . ). nonrelapse mortality was significantly increased in allosct recipients with high uric acid levels prior to start of conditioning (univariate hr= ci= . - . p= . ; multivariate hr= . , ci= . - . , p= . ). in addition, there was a non-significant trend towards higher acute gvhd incidence (gvhd grades ii-iv univariate hr= . ci= . - . p= . ; multivariate hr= . ci= - . , p= . ) in allosct recipients with uric acid levels above cut off before transplantation. finally, the incidence of relapse after allosct was moderately increased in the cohort with higher uric acid levels (univariate hr= . ci= - . p= . ; multivariate hr= . , ci= . - . , p= . ). conclusions: high uric acid levels before start of conditioning correlate with high mortality after allosct. our results can serve as rationale for clinical trials on depletion of uric acid during allosct. results: we found significant correlation between donors' ctla- + a>g polymorphism and hsct outcome. genotype aa was present in donors, ag in donors and donors was homozygous for g allele. recipients who received graft from g allele carrier donors showed significantly increased cumulative incidence of relapse (at months aa: . %, ag: . % and gg: . %; p= . ). on contrary, the frequency of the acute gvhd grades iii-iv and cytomegalovirus (cmv) reactivation/disease decreased according to the presence of the g allele in the donor ctla- genotype [agvhd: aa: %, ag: %, gg: %; p= . ; cmv: aa: %, ag: %, gg: %; p= . ]. cumulative incidence of agvhd was also markedly decreased among patients with g allele carrier donors (at days aa: . %, ag: . %, gg: . %; p= . ). donor genotype similarly influenced hsct outcome in mud donor and mac conditioning subgroups. overall survival (os) was not different in patient subgroups according to donor genotypes [os at months: aa: . ± . %, ag: . ± . %, gg: . ± . %; p= . ]. we did not find any correlation between recipients' ctla- + a>g polymorphism and hsct outcome. conclusions: several ctla- snps have previously been described to be associated with relapse rate, incidence of agvhd and os, but results are often contradictory in the publications. in our study, ctla- + a>g polymorphism of hsct donors influenced risk of relapse, agvhd, cmv and cause of death, but not overall survival. the genotyping of ctla- + a>g polymorphism in donors may help in the risk assessment process and the choice of personalised therapy. disclosure: nothing to declare. background: although steroids remain first-line therapy for the treatment of acute graft versus host disease (agvhd), response rates in patients with grade iii-iv disease are poor, with no apparent improvement in survival over the past years. we performed a prospective, multicenter trial to assess the efficacy and safety of the combination of ruxolitinib and etanercept as a novel approach to treat grades iii-iv sr-agvhd . methods: forty malignant hematologic disease patients with grades iii-iv sr-agvhd after allo-sct from three centers in east china were enrolled from january to june . ruxolitinib was initiated at a dose of - mg bid for months, and then tapered gradually for another one month. etanercept was administrated at mg biw for - weeks. results: the median age of patients was (range - ) years. at day after the combination treatment, the overall response rate (orr) was % including crs ( %) and prs ( %). the median time to the optimal response was (range - ) days. the incidences of cr per organ were . %, . %, and % for skin, liver, and gut, respectively. the agvhd relapse rate was analyzed for the patients who had achieved cr or pr and survived beyond days. relapses in agvhd occurred in . % ( / ) of responsive patients. the patients who received ruxolitinib within days after agvhd onset have a significant higher cr rate that those with delayed ruxolitinib therapy ( . % vs. . %, p= . ). and the patients without gut infections have a significant higher cr rate than infected cohort ( . % vs. . %, p= . ). by logistic regression analysis, the time from agvhd to ruxolitinib (rr= . , p= . ) and gut infection (rr= . , p= . ) were independent predictors for incomplete response. thirteen patients ( / , . %) suffered from at least infectious episode after the start of the combination therapy, and pulmonary infectious diseases was a frequent complication ( / , . %). iii-iv cytopenia and cmvreactivation were observed in % and . % of patients. the -year overall survival (os) after initiation of the combination therapy were . %. the -year nrm and relapse incidence was . % and . %, respectively. patients with complete response on day had significantly higher os probability than non-cr patients ( -year os: . % vs . %, p= . ). compared with the historical cohort of basiliximab and etanercept for sr-agvhd in our center (n= ), no significant difference was found on the baseline. although the orr in patients treated with ruxolitinib and etanercept is identical with the historical cohort, ruxolitinib group achieved rapider remissions in liver agvhd and gut agvhd than the historical cohort (gut agvhd: days vs. days, p= . ; liver agvhd: days vs. days, p= . ), thus, with regard to hospital stay after agvhd onset, the ruxolitinib cohort stayed shorter (median: days vs. days, p= . ) than basiliximab cohort. conclusions: combined treatment with ruxolitinib and etanercept resulted in a rapid cr to visceral agvhd and meanwhile reserve graft anti-leukemia (gvl) effect as the relapse rate of primary disease is relatively lower. the various infection complications associated with ruxolitinib merit more attention. disclosure: nothing to declare background: graft-versus-host disease (gvhd) remains one of the main life-threatening complications after allo-hsct, especially in patients with non-malignant diseases. the standard gvhd prophylaxis strategy is mostly based on the use of calcineurin inhibitors alone or in combination with other immunosuppressive (is) post-transplant cyclophosphamide (ptcy) is effective gvhd prophylaxis optiont for adult patients (pts), but has limited data in children. methods: the study aim was to evaluate ptcy as gvhd prophylaxis in pediatric pts with inherited disorders undergoing allo-hsct. pts, the most of them are pediatric age (median age - y.o., range month - y.o.) with different types inherited disorders (β-thalassemia - , bone marrow failure syndromes - , storage diseases - , primary immunodeficiencydisorders - ) were inrolled in retrospective study. donor type was: matched/mismatched unrelated (mud/mmud) - , matched related donor (mrd)- , haploidentical (haplo) - . conditioning regimen was: myeloablative (mac) - , reduce-intensity (ric) - . graft sourse was: bone marrow (bm) - , peripheral blood stem cells (pbsc) - , combintions bm +pbsc/bm+cord blood - . ptcy mg/kg days + , + based gvhd prophylaxis recived pts., standart gvhd prophylaxis based on calcineurin inhibitors - pts. results: cumulative incidence (ci) of agvhd was %. grade - , - agvhd were % and % respectively. ptcy based gvhd prophylaxis reduced ci of agvhd ( % vs %, p= , ). another reduce ci of agvhd factors were mac ( % vs % in ric pts group, p= , ), mrd ( % vs % in haplo group vs % in mud/mmud group, p= , ), bm as a transplant source ( % vs % in pbsc group, p= , ). in a multivariate analysis mac (hr , %ci , - ,, p= , ), time from diagnosis to allo-hsct less then month (hr , %ci , - , , p= , ) were predictive for reducing ci agvhd. for agvhd - st. significant factor increase ci was female donor both in univariate ( % vs %, p= , ) and multivariate analysis (hr , %ci , - , , p= , ). years overall survival (os) was %. improving os factors were: transplant age younger then y.o. ( % vs %, p= , ), time from diagnosis to allo-hsct less then month ( % vs %, p= , ), engraftment ( % vs %, p= , ). in a multivariate analysis only transplant age younger then y.o. (hr , %ci , ( ) ( ) ( ) ( ) ( ) p= , ) and engraftment (hr , %ci , - , , p= , ) were predictive for os. conclusions: ptcy-based gvhd prophylaxis can be effective options for reduce risk of acute gvhd. using unrelated donors, bone marrow as transplant source and mac can reduce ci of gvhd. performing allo-hscr earlier from diagnos and in earlier age can improve os patients with inherited disorders background: diarrhea is a frequent complication after allo-sct. at onset it is often difficult to differentiate gi gvhd from other causes of enterocolitis. recently, non-invasive tests, such as fecal calprotectin (fc), have been validated as markers of gut inflammation in patients with inflammatory bowel disease, but only a few studies have been published regarding its use as a diagnostic marker in gi gvhd. methods: our aim in this study was to explore the levels of fc in allo-sct recipients with new-onset diarrhea. so far we have included allo-sct recipients who developed acute diarrhea ≥ stage - at a median of days (range: - ) post allo-sct. stool samples were analyzed as soon as possible after the onset of diarrhea. fc levels were determined in addition to an extensive microbiological panel for infectious enterocolitis (including norovirus pcr and c. difficile associated diarrhea). endoscopies for histologic analysis were performed according to the treating physicians' discretion (n= ). results: patients characteristics are summarized in table . median follow-up for survivors was days (range: - ). twenty-eight patients ( %) were diagnosed of gi-gvhd. the additional causes of diarrhea were: drug-related enterotoxicity (n= ), viral enteritis (n= ), food intolerance (n= ), c.jejuni-enteritis (n= ), and non-specific causes (n= ). the concentration of fc was higher in patients with gi gvhd vs. other causes of diarrhea ( μg/g +/- vs. μg/g +/- , p= . ). patients who did not develop severe enterocolitis had normal to slightly raised calprotectin at the onset of diarrhea [< - in out of ( %) cases], including % ( / ) of patients with enterotoxic drug-related diarrhea. among the patients with gi-gvhd, ( . %) were later found to be steroid-resistant. as shown in figure , we found a significant association between high fc (≥ μg/g) and severe-refractory gvhd (hr . , p= . ). of note, high values of fc were also found in patients with severe infectious enteritis (norovirus, adenovirus and c.jejuni infections), with baseline fc> μg/g, respectively. overall survival was % (ic %: - ) at months. hypoalbuminemia and thrombocytopenia were the only variables linked to -yr os in univariate analysis, regardless of the cause of enterocolitis. conclusions: in the absence of standarized (and expensive) biomarker panels for analyzing and predicting gvhd onset and outcomes, the fc test may be an useful tool in the allo-sct setting. our initial results show that fc is helpful in predicting mild causes of diarrhea and to identify patients with a high probability of developing severe (and potentially steroid-refractory) gi gvhd, although high levels are also found in severe infectious enteritis. background: there is an urgent need for effective therapy for severe acute gvhd. results of gvhd therapies beyond months are rarely reported. we here report a median follow-up of years. we introduced mesenchymal stromal cells as therapy for severe acute gvhd, with a dramatic response in some, but not all patients. the placenta protects the fetus from the mothers haploidentical immune system during pregnancy. we found that maternal stromal cells from the fetal membrane, so called decidua stromal cells (dscs) were more immunosuppressive than other sources of stromal cells. methods: we treated patients, median years of age (range . - ) for severe acute gvhd. all had biopsy proven gastro-intestinal gvhd. all were steroid refractory, after > days or with progression and after > days. we used an improved protocol where dscs were thawed and infused in a buffer with % albumin. dscs were given at a median dose of . ( . - . ) x cells/kg and ( - ) doses, given one week apart. viability of frozen and thawed dscs was % ( - ) and cell passage was ( - ). results: complete resolution of gvhd was seen in patients and had a partial response. the cumulative incidence of chronic gvhd was %. six had mild, moderate and one severe nih overall gvhd severity scoring. nine patients died, from relapse, acute gvhd and septicemia, zygomycetes infection, liver insufficiency, cerebral hemorrhage, multiorgan failure and chronic gvhd with obstructive bronchiolitis. four years transplant related mortalliy was . % and overall survival was %. survival was not significantly worse (p= . ) than % for all patients undergoing allogeneic hematopoietic cell transplantation during the same period - . conclusions: to conclude, dscs seems to be a promising therapy for severe acute gvhd. randomized trials are under way. disclosure: nothing to declare p anti-apoptotic protein bcl- is upregulated in graftversus-host disease stem cell transplantation (allo-hsct) with - % developing either acute or chronic gvhd. recently, bcl- inhibitor venetoclax was approved for treatment of chronic lymphocytic leukemia. induction of apoptosis and depletion of lymphocyte subpopulations e.g. follicular b-cells or cd + and cd + t-cells led to further exploration in autoimmune disease. methods: to establish expression levels of genes in the bcl- pathway, low-input rna sequencing was performed on t cells isolated from non-inflamed skin and peripheral blood of hsct recipients at different time points before until year after transplantation. furthermore, we analyzed blood, lung, gut and skin samples of patients post allo-hsct with and without previously untreated acute or chronic gvhd by rt-pcr, flow cytometry and tissue immunofluorescence. [[p image] . bcl- is up-regulated in t and b lymphocytes of acute and chronic gvhd lesions.] results: rna-sequencing revealed that t cells upregulated bcl- upon conditioning treatment (day ) and cells of patients who later developed gvhd failed to downregulate bcl- after transplantation (day+ , day+ ). bcl- protein levels were elevated in overall leukocytes and pathogenic cell subsets including monocytes, cd + t lymphocytes and nkt cells showed significantly higher expression of bcl- in peripheral blood of gvhd patients as compared to healthy controls. these results could be recapitulated in tissue samples, where disease-promoting lymphocytes (t, b, nk, nkt) were numerically expanded and expressed bcl- in acute and chronic gvhd skin lesions. notably, non-pathogenic cell types such as keratinocytes did not exhibit increased bcl- expression compared to control samples from hsct recipients and healthy donors. while bcl- rna expression did not depend on type of conditioning (mac vs. ric) or gvhd grade, it correlated to disease severity and was significantly elevated in biopsies of patients with steroidrefractory gvhd. conclusions: we could show exclusive upregulation of bcl- in gvhd-mediating cell types in peripheral blood and tissue samples affected by gvhd, correlating to gvhd severity and response to first-line therapy. thus, bcl- inhibition may present a novel and urgently needed targeted therapy in treatment of steroid-refractory acute and chronic gvhd. disclosure: supported by a docmed fellowship od the austrain academy of sciences background: graft-versus-host disease (gvhd) represents a major contributor to morbidity and mortality in recipients of allogeneic hematopoietic cell transplants (hct). several therapeutic strategies exist for gvhd prophylaxis and include post-transplant cyclophosphamide (ptcy) and antithymocyte globulin (atg). while several groups have described the use of ptcy in younger patients, there is a paucity of data about the efficacy of ptcy in older individuals, particularly when combined with atg. we investigated the combined effect of ptcy with atg on transplant outcomes in older patients at princess margaret cancer centre, toronto, canada. methods: this retrospective study included all patients age ≥ who underwent allogeneic hct for any indication at our centre between december and july . overall survival (os) was calculated using kaplan-meier analysis and multivariable cox proportional hazards regression. cumulative incidence of relapse (cir) and non-relapse mortality (nrm) were calculated using competing risk regression (fine and gray method). incidences of acute (agvhd) and chronic (cgvhd) were compared using the fisher's exact test. results: of patients, ( %) were male. median age was (range - ) and median follow-up among survivors was months (range - ). acute myeloid leukaemia (aml) was the most common indication for hct ( patients, %), followed by myelodysplastic syndrome ( patients, %) and myelofibrosis ( patients, %). eightyfour ( %) patients had a matched unrelated donor, ( %) had a matched related donor and ( %) had a haploidentical donor. one hundred twenty-five ( %) patients received reduced intensity conditioning. sixty-two ( %) patients received ptcy combined with atg ( . mg/kg) while ( %) received other forms of gvhd prophylaxis. os at years was % ( % confidence interval (ci) - ) in the entire cohort. patients who received ptcy with atg had a superior -year os compared with other gvhd prophylaxis regimens ( figure a ): % ( % ci - ) vs. % ( % ci - ), respectively (hr= . , % ci . - . , p= . ). the -year nrm for the entire cohort was % ( % ci, - ). patients who received ptcy with atg had a lower -year nrm compared to those who did not ( figure b ): % ( % ci - ) vs. % ( % ci - ), respectively (hr= . , % ci . - . , p= . ). the -year cir in the whole group was % ( % ci - ). use of ptcy with atg was associated with a modest increase in cir at two years ( figure c ): % ( % ci - ) vs. % ( % - ), respectively (hr= . , % ci . - . , p= . ). there was a trend toward lower incidence of grade ii-iv agvhd among patients who received ptcy with atg compared to those who did not: % vs. % (p= . ). the incidence of grade ii-iv cgvhd was lower in individuals who received ptcy with atg compared to those who did not: % vs. % (p= . ). conclusions: in older hct recipients, use of ptcy combined with atg is associated with improved os, lower nrm, decreased risk of both agvhd and cgvhd and a modest increase in relapse risk. therefore the ptcy with atg combination represents an effective strategy for gvhd prophylaxis in older allogeneic hct recipients. disclosure: the authors have no conflict of interest to declare. outcome of severe graft versus host disease in pediatric patients with nonmalignant diseases after allogeneic bone marrow transplantation. a single center experience irina zaidman , sigal grisariu , batia avni , ehud even-or , bella shadur , adeeb nasereddin , polina stepensky background: hematopoietic stem cell transplantation (hsct) remained the only curative option for many nonmalignant diseases in pediatric patients. survival after hsct has improved the last few years due to significant advancement in human leukocyte antigens (hla) typing techniques, less toxic conditioning regimens and better supportive care and resulted to % survival and cure in some non malignant diseases. graft-versus-host disease (gvhd) remains a major complication of hsct and leading cause of morbidity and mortality. prognosis of patients with high grade gvhd is dismal and survival rate varies between % to % in pediatric patients. methods: the retrospective study included patients with non malignant diseases who underwent allogeneic hsct at hadassah medical center from to . the collected data included patient´s clinical data and transplant characteristics. the study was approved by the institutional helsinki committee. results: children with nonmalignant diseases underwent allogeneic bone marrow transplantations in hadassah university hospital during ten years period. fifty seven patients ( %) developed agvhd grade - , twenty five of them ( . %) grade - . median age was . (range . - . ), most patients were males ( males, females). patients underwent bmt from fully matched family members, children were transplanted from matched unrelated donors and from mismatched donors. twenty one of patients with severe gvhd ( %) survived. four patients ( %) died from severe gvhd and complications of immunosuppressive treatment. of deceased patients were transplanted from mismatched donor, in of cases the age of donor was advanced, of patients developed severe gvhd and died after second hsct. all patients were refractory to different treatment modalities. three of patients died in and one in , it was no death from severe gvhd in patients that were transplanted and developed high grade gvhd after . conclusions: the results of this study show a high survival rate of % in pediatric patients with non malignant diseases and severe gvhd. significant risk factors for mortality in our group included mismatched donor, advanced age of donor and second transplant. trend to better survival was observed after . additional multicentral studies analyzed the outcomes of agvhd in pediatric patients with nonmalignant diseases are urgently required. background: chronic graft-versus-host disease (cgvhd) is a serious late complication after allogeneic hematopoietic stem cell transplantation (allohsct) with heterogeneous presentation and still poorly understood pathophysiology including inflammation and endothelial dysfunction. factor viii (fviii) and von willebrand factor (vwf) are coagulation factors but also known indicators of endothelial dysfunction and inflammation in different settings, and therefore could serve as interesting candidate biomarkers of cgvhd. methods: since patients after allohsct were assessed by the multidisciplinary cgvhd team at the university hospital center zagreb, croatia, using established nih cgvhd-related measurements. an extensive history, physical and laboratory evaluations were performed, including fviii, vwf:ag and vwf:ac analysis. descriptive statistic and non-parametric analyses were performed. variables that showed significant univariate correlations were used in multivariate logistic regression (mlr) to identify the most predictive for fviii, vwf:ag and vwf:ac in cgvhd patients. results: cgvhd patients and controls (subjects after allohsct without cgvhd) were analysed. median age of cgvhd patients was ( - ) years, % females, . % underwent allohsct for hematologic malignancies, . % had myeloablative conditioning and . % matched related donor. median time from hsct to study was . days and from cgvhd diagnosis to study days. there were no demographic neither transplant related significant differences between cgvhd patients and controls beside stem cell source (peripheral blood . % vs . %, p= . ) and history of acute gvhd ( . % vs . %, p< . ). majority of patients had moderate ( . %) or severe ( . %) nih global cgvhd score, . % active cgvhd by clinician´s impression. median number of organs involved by cgvhd was ( - ), and the most frequently involved organs were mouth, skin and eyes ( . % each). cgvhd patients compared to controls had higher fviii levels (median ( - )% vs ( - )%, p= . , reference range - %) and higher vwf:ag (median . ( . - )% vs . ( . - )%, p= . , reference range - %), while vwf: ac showed a trend toward higher levels among patients (median . ( - )% vs ( . - )%, p= . , reference range - %). patients had higher ggt (p= . ), lower anticardiolipin igg (p= . ) and igm (p= . ), and lower albumin (p= . ) than controls, without differences between other laboratory parameters. univariate analysis showed that among cgvhd patients higher fviii was associated with worse karnofsky score (ks) (p= . ) and performance score (ps) (p= . ), higher leukocytes (p= . ), cholesterol (p= . ), triglycerides, ast, alt, ggt, ldh, and lower albumin. higher vwf:ag and vwf:ac in cgvhd patients were associated with worse ks and ps (p< . ), with more active cgvhd (p< . ), worse nih cgvhd liver (p= . ; p= . ) and nih cgvhd mouth (p= . ; p= . ), higher total nih score (p= . ; p= . ), higher number organs involved (p= . ; p= . ), higher esr, monocytes, ddimers, ast, alt, ggt, ldh, triglycerides, β- -microglobulin, ferritin, total proteins, iga and lower albumin. mlr analysis showed leukocytes (p= . ) and cholesterol (p= . ) as the strongest predictor of fviii (r = . %; p< . ), while strongest predictor of vwf: ac was number of organs involved by cgvhd (r = . %; p= . ). conclusions: results of this study detected high fviii and vwf levels in cgvhd patients with possible reflections to cgvhd manifestations, what needs to be further confirmed in larger longitudinal studies. disclosure: this work was supported, in part, by the unity through knowledge fund project entitled "clinical and biological factors determining severity and activity of chronic graft-versus-host disease after allogeneic hematopoietic stem cell transplantation", and also, in part, by the croatian science foundation project entitled "new biomarkers for chronic graft-versus-host disease". antonela samardzic -work financed by the croatian science fondations`"young researchers`career development project -training of doctoral students" background: thrombotic microangiopathy (tma) is a severe complication of allogeneic hematopoietic cell transplantation (hct) with multisystem involvement. a few recent reports have recognized evidence of tma in the intestinal vasculature (intestinal tma/itma) of patients with graft-versus-host disease (gvhd) with or without tma. we aimed to identify patients with itma and describe histological, clinical and prognostic features. methods: we prospectively evaluated available endoscopic samples (stomach and/or colon) from consecutive adult hct recipients for previously described histopathologic signs of itma (january -september ). systemic tma was diagnosed according to the international working group criteria. we compared findings among clinical groups: gvhd/systemic tma, gvhd/no systemic tma and no gvhd/no tma. results: we studied patients, classified as gvhd/ systemic tma, gvhd/no systemic tma and no gvhd/no tma. baseline transplant characteristics (age, donor, hla matching, conditioning) did not differ significantly among groups. histological features of itma, including loss of glands, total denudation of mucosa, apoptosis and detachment of endothelial cells, intraluminal fibrin, intraluminal microthrombi and mucosal hemorrhage were found in patients. previously described features of intraluminal schistocytes were not observed in our patients. interestingly, loss of glands, total denudation of mucosa, apoptosis and detachment of endothelial cells were also found in patients with gvhd and no itma, suggesting that these features are not pathognomonic of itma. among itma patients, two patients were classified in the clinical group of acute gvhd/systemic tma, while the other patients had clinical and histopathological features of itma and severe grade iii-iv steroid-refractory acute gvhd ( patients) or extensive chronic gvhd ( patient) but no evidence of systemic tma. in the majority of patients ( / ), itma occurred during the early posttransplant period at . ( . - ) months. clinical features (gastrointestinal bleeding, diarrhea, pain, nausea) presented no differences between patients with or without itma. prognosis was poor for patients with itma who suffered from a significantly higher mortality rate of % compared to the rest patient population (p= . ). with a median follow-up of . ( . - . ) months, year overall survival probability (os) was . for itma, % for gvhd and % for systemic tma. unfavorable predictive factors for os were itma (p= . ), hla mismatched donors (p= . ) and gastro-intestinal bleeding (p= . ). conclusions: intestinal tma has emerged as a novel distinct entity in patients with gvhd and/or systemic tma. distinct histological features may be useful in differential diagnosis of these severe hct complications. mortality rates higher than those of systemic tma highlight the need of proper recognition of itma that needs to be further studied in terms of diagnostic and therapeutic potential. disclosure: e.g. was supported by the european hematology association clinical research grant. the remaining authors declare no competing financial interest. the beneficial effects of thrombomodulin gene polymorphisms after hematopoietic stem cell transplantation background: chronic graft-versus-host disease (cgvhd) remains the major cause of late morbidity and mortality after allogeneic blood and marrow transplantation. treatment options for cgvhd, particularly its sclerotic forms remain limited. active hedgehog (hh) signaling was shown as a therapeutic target in both mouse and human cgvhd, with limited efficacy and significant toxicities described in a published clinical trial (defilipp, ). methods: adult patients with steroid refractory sclerodermatous cgvhd, defined as requiring > . mg/kg/day of prednisone dose equivalent (pde), or need for second-or third-line therapy beyond corticosteroids and calcineurin inhibitors or sirolimus were eligible for this open label study of vismodegib, a first generation hh pathway inhibitor. primary endpoint was failure free survival, defined as absence of non-relapse mortality, no recurrent malignancy, steroid dose at months =< . mg/kg/day of pde, and no addition of new systemic treatment. vismodegib was administered orally for - months, with dose reductions at development of toxicities. peripheral blood mononuclear cells were isolated from samples collected at treatment initiation and every three months thereafter. the immune profile of circulating b cells was analyzed by flow cytometry and t helper polarization by qrt-pcr of sortpurified cd + t cells. results: at the time of interim analysis, patients were evaluated. patients completed months of treatment and five patients completed months of treatment. therapy was discontinued in patients prior to months due to treatment-related (n= ) and unrelated (n= ) side effects. most patients experienced grade toxicities (muscle cramps and dysgeusia), with only a single grade toxicity (weight loss). patients who completed months of therapy demonstrated partial response, and overall, the primary endpoint was reached in % ( / ) of patients. in patients who discontinued vismodegib, cgvhd worsened acutely after discontinuation. correlative analysis of immune cellular subsets in peripheral blood in paired samples (pre-treatment and month of therapy) documented modulation of b cell subsets pathogenic in cgvhd (pregerminal center and plasmablast-like b cells) and diminished t helper polarization in cd t cells. conclusions: overall, use of vismodegib was associated with potential clinical efficacy in sclerodermatous cgvhd with possible mechanistic evidence arising in correlative studies. while side effects were common, further studies of hh inhibition in cgvhd are warranted. future studies should employ adjusted dosing regimens, along with supportive care interventions to offset side effects, and testing of novel hh inhibitors with enhanced safety profiles. clinical background: graft-versus-host disease (gvhd) results from recognition of host antigens by donor t cells following allogeneic hematopoietic stem cell transplantation (sct). we tested the hypothesis that somatic neomutations occurring after sct from donor and/or recipient dna may trigger gvhd. methods: we longitudinally analyzed both constitutive and somatic mutations by whole exome sequencing (wes) in patients who received sct from a sex-matched hlaidentical sibling for npm mutated acute myeloid leukemia (pt# ) and jak v f mutated primary myelofibrosis (pt# ). both patients were initially refractory to alloreactivity, i.e. not displaying any signs of gvhd, even after several donor lymphocyte infusions. acute gut gvhd finally occurred after a further dli preceded by a lymphodepleting chemotherapy. in pt# , gvhd correlated with a graft-versus-tumor effect. wes was performed on dna from recipient saliva and donor pbmcs (germline samples) and from sequential post-sct pbmcs samples on a hiseq illumina with x bp paired-end reads at a mean depth of coverage of - x. germline and somatic mutations were determined using in-house bioinformatic pipelines (named ewok from the curie institute and smaug from the henri mondor hospital), using briefly gatk as variant caller for germline samples, and a combination of variant callers for matched normaltumor pairs. we adjusted parameters to detect somatic mutations at a minimal variant allelic frequency (vaf) of % compared to recipient and donor germline for all variations (minimal coverage = x for germline and x for tumor sample). results: wes allowed detecting somatic driver mutations explaining aml and pmf for both patients in the initial timepoint and all these driver mutations disappeared at the following timepoints. as expected, the somatic variant rate was x higher in pt# with aml than in pt# with pmf at each timepoint, except for the final gvhd timepoint. indeed, at this final point, the somatic variant rate dramatically decreased by % as compared to previous timepoints. by subtracting variants detected pre-and post-sct from those identified at the ultimate time-point of gvhd occurrence, we created sets of and variants respectively for each patient (keeping only variants with at least reads of mutated dna). these variants can be classified in categories: (i) those with only with a slight increase at time of gvhd, i.e. ≤ -fold compared to highest previous vaf (lrrc , or u , or g , alpp, frg , frg b and lilrb genes), and (ii) those with a significant increase at that time, i.e. > -fold compared to highest previous vaf (phf , smpd , ercc and krtap - genes). none of the variants or genes involved was common between the patients. ontology classification of mutated genes showed the implication of some of them in cell death, regulation of map kinase activity, mrna splicing and immune system process, making them good candidates for further studies. identification of variants appearing pre-gvh and turning off at time of gvhd is ongoing to unveil putative neoantigens that could trigger the alloreactive response. conclusions: using a comprehensive, pre-and post-sct, wes of donor/recipient pairs, we identified several neomutations from donor and/or recipient dna correlating with gvh/gvt effect development. disclosure results: a total of patients experienced cgvhd, and mild, moderate, and severe cgvhd were observed in , , and patients, respectively. the -year cumulative incidence of total cgvhd was . % ( % ci, . - . %), and the -year cumulative incidence of moderate to severe and severe cgvhd was . % ( % ci, . - . %) and . % ( % ci, . - . %), respectively. the patients who had loci mismatched had a higher -year cumulative incidence of total cgvhd ( . % vs. . %, p= . ) and moderate to severe cgvhd ( . % vs. . %, p= . ) compared to those of the patients who had - loci mismatched. the patients who had maternal donors had a higher -year cumulative incidence of moderate to severe cgvhd ( . % vs. . %, p= . ) compared to that of the patients who had other donors. the patients who had grade iii to iv acute graft-versus-host (agvhd) had a higher -year cumulative incidence of total cgvhd ( . % vs. . %, p< . ) and moderate to severe cgvhd ( . % vs. . %, p< . ) compared to those of the patients without agvhd. in multivariate analysis, grade iii to iv agvhd was the only independent risk factor for total cgvhd (hr= . , %ci, . - . ; p< . ) and moderate to severe cgvhd (hr= . , %ci, . - . ; p< . ). in the model excluding agvhd, maternal donor was the risk factor for moderate to severe cgvhd (hr= . , %ci, . - . ; p= . ). conclusions: we observe that severe agvhd was the most important risk factors for cgvhd after haplo-hsct, and further interventions should be considered in these patients to prevent severe cgvhd. disclosure: none of the authors have any potential financial conflict of interest related to this manuscript. background: extracorporeal photopheresis (ecp) has been successfully used for the treatment of graft-versus-host disease (gvhd). ecp therapy might restore the balance between effector and regulatory cells which is severely impaired in gvhd. nk cells are the first lymphocyte subset to be reconstituted after allogeneic hematopoietic stem cell transplantation (allo-hsct). as an important innate immune cell population, nk cells can temporally bridge the transient period of t-cell deficiency post allo-hsct, by protection from opportunistic infections and prevention of leukemic relapse by graft-versus-leukemia (gvl) effect. nk cells not only preserve homeostasis through targeted killing of allo-reactive t cells and thereby control gvhd but also enhance inflammation by secretion of tnf-α and ifn-γ and thereby promote gvhd. therefore, we investigated here the role of nk cells in gvhd patients under ecp therapy. methods: thirty four patients with steroid-refractory/ resistant agvhd ≥ ii°and moderate to severe cgvhd received ecp therapy which performed according to the guidelines. glucksberg and nih criteria were used for clinical staging of agvhd and cgvhd under ecp therapy, respectively. the comprehensive phenotypical analysis of nk cells was evaluated by multicolor flow cytometry. nk activity in terms of killing function, cytokine release capacity and proliferation function was monitored by chromium- release assay, intracellular cytokine staining and cfse staining, respectively. results: five different nk cell subsets were defined based on cd and cd expression. cd bri nk cells displayed an immature and activation profile with high expression of cd l and nkg d. agvhd patients had a higher frequency of cd bri nk cells when compared with hds and cgvhd patients, who were characterized by significant increase of the cd dim cd + and cd -cd + nk cell subsets with high expression of differentiation markers cd b and cd . of note, cd bri cd -nk cells could serve as a novel predictive biomarker for the response of agvhd patients to ecp treatment. in responding agvhd patients, an increase of cd bri nk cells was observed already during the early ecp treatment phase, suggesting immune reconstitution. after priming of the progenitors, ecp could differentiate immature cd bri nk cells into mature cd dim nk cells with reduction of cd l on cd bri nk cells. moreover, cd dim nk cells could further be matured through upregulation of cd expression by ecp. notably, ecp therapy could shift the nk cells from a cytotoxic to a regulatory phenotype within the cd bri nk cells. in spite the immunomodulatory effect of ecp on nk cells, nk activity could be kept intact under ecp therapy. the killing activity of nk cells was stable as confirmed by a cr release assay. ecp therapy had no negative effect on the quantity and quality of cytokine release by nk cells upon k stimulation. especially, the polyfunctionality of nk cells was not altered significantly by the ecp therapy. conclusions: nk cells play an important role in gvhd and could serve as a predictive cell population for the clinical response to ecp therapy. in the current study, ecp influenced the differentiation, maturation and education of nk cells ameliorating gvhd without comprising the antiviral immune defense and gvl effect. disclosure: the authors declare no competing financial interests, except the following: therakos mallinckrodt gave a financial support to as and ms for the documentation of the clinical course and for the analysis of immune cells of the patients, pw has honoraria and membership on advisory boards for sanofi-aventis. abstract withdrawn. cyclosporine levels > µg/l on day post-transplant was associated with significantly reduced acute graftversus-host disease following allogeneic hematopoietic stem cell transplantation monica bianchi , dominik heim , claudia lengerke , martina kleber , dimitrios tsakiris , jakob passweg , alexandar tzankov , michael medinger background: acute graft-versus-host disease (agvhd) remains a major complication of allogeneic hematopoietic stem cell transplantation (allo-hsct). affected patients, especially with steroid-refractory agvhd, have a very poor prognosis. prophylaxis with cyclosporine a (csa) is the backbone of gvhd prevention in most conditioning regimens. methods: in a retrospective analysis of patients treated with allo-hsct, we correlated csa levels at the day of transplantation (day ) and day + with the incidence of acute and chronic gvhd. we postulate that higher target csa levels > μg/l will result in a lower incidence rate especially of agvhd after allo-hsct. results: we assessed patients with either aml n= , lymphoma/myeloma n= , mds/mpn n= , all n= , cll n= , cml n= , or bone marrow failure n= . in patients with clinically relevant agvhd grade ≥ , mean csa levels was lower on day and day + ( ± μg/l; and ± μg/l; respectively) compared to patients without agvhd ( ± μg/l; and ± μg/l; respectively; day : p= . ; day + : p= . x - ). in patients with csa level < μg/l, the incidence of agvhd was significantly more frequent compared to patients with csa levels > μg/l [( / ; %) versus / ( %); p= . x - ]. in patients with cgvhd, there was no significant difference between csa levels < μg/l ( / ) compared to csa levels > μg/l ( / ; p= . ). the optimal csa cut-off level for the prevention (i.e. roughly % incidence reduction) of agvhd was > μg/l at day and > μg/l at day + ( figure ) in a competing risk analysis, time to agvhd grade ≥ (using death of other causes as competing risk) was associated with csa levels > μg/l on day and on day , unrelated donors, myeloablative conditioning (mac), and for the diagnosis lymphoma/myeloma. conclusions: our data support close monitoring with active adjustments of csa dosing to maintain therapeutic csa levels above μg/l in the first days of allo-hcst to reduce agvhd. disclosure: noting to declare. liposomal cyclosporine a for inhalation (l-csa-i) to treat bronchiolitis obliterans syndrome: novel formulation with therapeutic potential for patients with bos following allo-hsct noreen roth henig , emilie hofstetter , dominik kappeler , gerhard boerner background: bronchiolitis obliterans syndrome (bos) is a rapidly progressive lung disease caused by t-cell mediated inflammation that leads to blockage of bronchioles, leading to respiratory failure and death shortly after diagnosis. approximately % to % of patients who undergo allogeneic hematopoietic stem cell transplant (allo-hsct) will develop bos, with - % developing bos as a respiratory form chronic graft-vs-host disease (cgvhd) in addition to other signs of cgvhd. mean time to bos diagnosis ranges from to days post-transplant. the histopathology of bos after allo-hsct and lung transplantation is identical. early studies of l-csa-i for the prevention of bos in lung transplant recipients demonstrated therapeutic benefit. l-csa-i is a novel, liposomal formulation of cyclosporine administered via a pari investigational eflow â nebulizer which delivers a potent immunosuppressant to the site of disease. pharmacokinetics and tolerability of l-csa-i is presented. methods: retrospective review of two clinical studies of l-csa-i (isotonic, mg/ml) for bos associated with lung transplantation. both studies had a control arm and results reported here are for patients who received l-csa-i. subjects received mg (single lung transplant) and mg (double lung transplant) bid via inhalation. blood samples for pharmacokinetic analysis of cyclosporine a concentrations were collected before inhalation, immediately after inhalation, and thereafter in intervals of , , min and , , and hours. local and general tolerability of l-csa-i was investigated. results: between the two studies, subjects received either or mg bid of l-csa-i. pharmacokinetic models predict a constant drug level in the lung. maximum serum cyclosporine a concentration after inhalation was . ± . ng/ml. trough levels for up to -years of daily administration was - ng/ml with no evidence of accumulation following repeated exposure. tolerability data was assessed from patient-month exposure to l-csa-i. reported symptoms were: pharyngeal soreness %; cough %; dyspnoea %; and wheezing %. no subject discontinued due to intolerability. inhalation time is on average - min. conclusions: l-csa-i provides high and constant concentrations to the airways of the lungs and the site of bos. l-csa-i is well tolerated in lung transplant patients. use of l-csa-i instead of augmentation of systemic csa reduces the total drug exposure. a multicentre phase safety and exploratory efficacy trial for the treatment of bos in allo-hsct recipients is underway. disclosure background: there are a number of biomarkers that predict non-relapse mortality (nrm), graft-versus-host disease (gvhd) and relapse incidence (ri) after conventional gvhd prophylaxis based on calcineurin inhibitors with or without antithymocyte globulin. currently there is limited data whether the conventional predictive biomarkers work with posttransplantation cyclophosphamide (ptcy) prophylaxis. methods: prospective single-center study in - enrolled adult patients with acute leukemia in cr ( % with all, % with aml). received matched related bone marrow (bm) graft with single-agent ptcy and received unrelated peripheral blood stem cell graft (pbsc) with ptcy, tacrolimus and mmf. the grafts were studied by flow cytometry (facs aria ii, antibodies by miltenyi biotec). the following populations were analyzed: cd , cd , cd , cd cd , nkt, inkt, treg, double-positive t-cells, double-negative t-cells, tcralpha/beta, tcr v memory cells. the crypreserved plasma from were analysed by elisa (commercial kits by ebioscience and critical diagnostics) for vegf a soluble tnf receptor (stnfr), il- , il- , soluble il- receptor, st , il- and stnfr. the above mentioned biomarkers were tested in logistic regression with roc analysis, assays with auc> . were selected for analysis in fyne-gray regression with competing risks. cut off levels were determined for significant parameters. results: median follow-up was months (range - ). in the whole group overall survival (os) was %, eventfree survival (efs) %, grade ii-iv acute gvhd %, moderate and severe (m&s) chronic gvhd %, nrm %, mortality in patients with gvhd %, ri %. there was no difference between bm/related and pbsc/unrelated grafts in the incidence of gvhd, nrm and ri (p> . ). the only significant predictor of acute gvhd were low levels of il- level on day+ (p= . , % vs % with the cut off pg/ml). m&s chronic gvhd was predicted only by the high percentage of inkt cells in the graft (p= . , % vs % with the cut off . %). there was a correlation between il- levels and number of nk cells in the graft (p= . ). nrm was related to infectious complications, nonetheless high levels of vegf a on day (p= . , % vs % with the cut off ng/ml), st on day+ (p= . , % vs % % with the cut off ng/ml) and low percentage of cd +cd -cells in the graft (p= . , % vs % with the cut off . %). the identified biomarkers of nrm had no association with the pre-transplant crp and ferritin levels (p> . ). the only significant parameter for ri was the level of cd cells in the graft (p= . ). none of the identified biomarkers significantly predicted overall survival (p> . ). conclusions: in the related and unrelated grafts with ptcy the study of biomarkers has low clinical utility due to very low gvhd-related mortality. however st and vegf a can predict infection-related mortality. also the study verified previous observations that high level of il- is associated with reduced gvhd incidence after ptcy and identified the importance of nk and inkt cells in the induction of tolerance with ptcy. references background: hematopoietic cell transplantation (hct) is the only curative approach for many hematological malignancies but life-threatening toxicities, such as graft-versushost disease (gvhd) and infections, still limit its fullpotential impact on the disease. strategies for keeping allohsct more effective and safe are needed in order to reduce morbidity while improving its immunological effect to control disease relapse. post-transplant cyclophosphamide (ptcy) has been demonstrated to improve acute gvhd (agvhd) and chronic gvhd (cgvhd) control in allogeneic bone-marrow hct from identical and haploidentical donor. the use of ptcy, after peripheral blood stem cell transplantation (allopbsct) from hla-matched unrelated/related donors, has been investigated by our group in a clinical trial (nct ) and preliminary results were published last year. here we report updated efficacy and safety data about the expanded cohort of patients treated with ptcy followed by tacrolimus and mycophenolate mofetil (t/mmf). methods: we analysed data about consecutive patients with high-risk hematologic malignancies received allopbsct from hla-matched unrelated/related donors between march and august . gvhd prophylaxis was ptcy mg/kg (days + + ), tacrolimus from day + and mmf from day + to day + . primary objectives were cumulative incidence of agvhd and cgvhd. secondary objectives were event-free survival (efs), cgvhd-efs, overall survival (os) and non-relapse mortality (nrm). results: patients median age at transplant was (range - ) years. ( %) patients were transplanted in first complete response (cr), ( %) patients in second/third cr, the others in disease control. a median dose of . (range - ) x ^ cd /kg was infused. primary graft failure was observed in one patient. all patients were off mmf on day + , the median day of tacrolimus discontinuation was (range - ). eight out of ( %) patients developed agvhd, ( %) of them were grade ii-iii; median day of onset was day (range . no grade iv was observed. no cases of late-onset agvhd were reported. cumulative incidence of cgvhd was % ( / ), median day of onset was (range - ). systemic treatments were required, but all patients were able to discontinue immunosuppression (is). with a median follow-up of (range - ) months, efs was %, cgvhd-efs was % and os was %. non-relapse mortality (nrm) was % ( / ): patients died because of multidrug resistant bacteria septicemia. nowadays patients are alive with no evidence of disease, being continuously off is and completely reintegrated in their normal daily life activities. conclusions: the updated reported results confirm, in a larger cohort of patients with a longer follow-up, that ptcy after pbsc-hct is highly active in agvhd and cgvhd prevention with extremely limited nrm. this strategy, not only allowed earlier discontinuation of immunosuppression, but also reduced the overall time of exposure to is for most of the patients. all these features might contribute, in the future, to transform hct into a safe immunologic platform that may be combined with advanced form of cellular therapies (car-tcells), aiming to increase safely the graftversus-tumor effect. clinical methods: pediatric patients ( - years) with nmd undergoing unrelated hct were eligible for this single center, phase i trial. following reduced intensity conditioning, abatacept ( mg/kg iv on days - , + , + , + ) was added to standard gvhd prophylaxis (cyclosporine, mycophenolate mofetil [mmf]). patients were followed for years for standard hct outcomes. [[p image] . figure results: since june , patients have been enrolled and transplanted (table , excluding # ). donor source was bone marrow in all. with median follow-up of . years, of patients survive without disease. initial engraftment was successful in , at a median of and days, for neutrophils and platelets respectively. one patient ( ) had secondary graft rejection in the setting of viral reactivation (cmv/ebv), with successful engraftment following a nd unrelated hct. in engrafted patients, myeloid (cd ) chimerism was % at all timepoints; t-lymphoid (cd ) chimerism was mixed but reached >/= % (figure ). one patient ( ) with saa had primary graft rejection in the setting of inadequate tnc dose ( . x /kg) and died from marrow aplasia/infection despite nd hct. a second death from wilms' tumor occurred months post successful hct, in a patient ( ) with dba and constitutional chromosome abnormality. except patient , all patients received doses of abatacept, which was well tolerated, with all severe adverse events expected for a hct population. cmv and ebv reactivation occurred in patients each, with resolution using standard anti-viral therapy. one patient ( ) was diagnosed with ebv-driven post-transplant lymphoproliferative disease, which responded to rituximab and immune suppression withdrawal. no patients developed severe acute (grade iii-iv) or chronic gvhd (table ) , and no patients required systemic immune suppression at > year. conclusions: these preliminary data suggest that abatacept can be safely added to cyclosporine and mmf gvhd prophylaxis in pediatric patients with bone marrow failure undergoing unrelated donor hct, with encouraging rates of gvhd despite half of patients having a mismatched ( / ) donor. given the higher risk of graft rejection in this non-malignant cohort, rejection (in addition to gvhd) will be a primary focus in our subsequent multi-center, phase trial. clinical trial registry: clinicaltrials. gvhd and may to have be separated from from toxicity to infectious complications in the early phase after allohsct. methods: from our files we identified patients which had upper gastrointestinal tract endoscopy after allohsct in with biopsies were taken from the esophagus, stomach and duodenum simultaneously. of these patients were excluded because of infection, reflux disease or drug toxity and the remaining patients were included in our study. we evaluated the routine stained esophageal biopsies, applied a grading scheme and compared the histological findings with those within the stomach and duodenum, the endoscopic findings and the clinical course. results: in of biopsy samples of the esophagus, we identified histological features of acute gvhd, ranging from vacuolar degeneration (grade ) and single-cell apoptosis (grade ) to the formation of clefts (grade ) and mucosa denudation in advanced cases (grade ), resembling epithelial lesions in acute gvhd of the skin. these findings correlated with gvhd involving the stomach and duodenum and the clinical manifestations of gvhd in other organs. endoscopically patients with gvhd revealed signs of inflammation, ranging from erythema to ulceration in the more advanced cases, sometimes reminiscent of reflux or infection. clinically these patients had abdominal discomfort ranging from inappetence to nausea, accompanied by emesis or diarrhea and weight loss. conclusions: we have shown that acute esophageal gvhd occurs after allohsct and is correlated with acute gvhd in stomach and duodenum. it could be diagnosed and graded histologically. the endoscopic findings are signs of inflammation. our results may help to establish the histological diagnosis of acute gvhd using endoscopic biopsies from the esophagus and to explain the alterations observed in the esophageal mucosa in patients after allohsct. [ background: intestinal acute graft versus host disease (agvhd) is a major thread after allogenic hematological stem cell transplantation (allohsct), with a high mortality in patients which were refractory to steroid treatment in particular. recent papers point to a correlation of histological grading of intestinal gvhd and prognosis in patient after allohsct. however a comparison with clinical scores has not been performed so far. methods: in this analysis, retrospective data from patients who underwent endoscopy due to clinical signs of agvhd (day + to + after allohsct) were evaluated. of each patient least biopsies from different sites of the colon which were taken simultaneously. of each biopsy series the maximum histological grad of agvhd according to the lerner scheme was obtained and compared with the glucksberg stage of the lower gastro intestinal tract (gslgi) and the overall glucksberg grade (ogg). these three grades were compared for non-relaps related mortality using the log-rank test and for sensitivity to steroid treatment applying the receiver operating characteristic for the patients who received steroid treatment. for these patients the non-relaps related mortality for responder and non-responder were calculated using also log-rank test. results: the histological grade strongly correlated with the survival (p= . ). a statistical significant correlation was also found for the gslgi (p= . ), whereas the ogg revealed no significant correlation (p= . ). non-relaps related mortality was mainly related to infection or sepsis (in / patients who died). -eighty-one of the patients received steroid therapy. the sensitivity to the steroid therapy correlated with each of the three scores (p< . ) but was the strongest for gslgi (area under the curve (auc) . ), compared to ogg (auc . ) and the histological score (auc . ). the survival of the patients, which were sensitive to steroid treatment was significantly better than those of steroid refractory patients (p= . ). conclusions: we found that histological and clinical grading in patients after allohsct with intestinal gvhd was correlated with survival and respond to steroid treatment. histological scoring may predict survival more precisely than ogg and gslgi but did not add substantial information to the prediction of treatment response. [ emerging evidences suggest that regulatory b cells (bregs) play essential roles in inflammation, autoimmune diseases and tumors. few data exist about the role of bregs in the contest of hematopoietic allogeneic stem cell transplantation (hsct). some authors have observed that bregs from patients with chronic graft-versus-host disease (cgvhd) were less frequent and less likely to produce il- than bregs as compared to healthy donors or patients without cgvhd. these findings suggest that bregs may be involved in cgvhd pathogenesis. the purpose of our study was to evaluate a possible role of b cell subsets on gvhd occurrence. methods: lymphocyte subset enumeration was performed by aquios cl flow cytometer (beckman coulter), a quantitative automated analyzer that performs two diagnostic panels: tetra- cd -fitc/cd -rd / cd -ecd/cd -pc and tetra- cd -fitc/cd +cd -rd /cd -ecd/cd -pc . b cell subsets (memory, mature and transitional b cells) on peripheral blood samples were analyzed by aquios designer software, a tool for the creation of user-defined applications. panel- cd -fitc/cd -pe/cd -ecd/cd -pc /cd -pc and panel- cd -fitc/cd -pe/cd -ecd/cd -pc / cd -pc were specifically designed by beckman coulter for our center. the flow cytometric analysis was performed as follows: in donors and patients at basal level; on graft products and in patients at days + , + , + , + after hsct. statistical significance was assessed with prism software (graphpad) by mann withney test. p < . was considered statistically significant. results: actually we enrolled patients submitted to hsct in our center from november . a preliminary statistical analysis was performed on patients. stem cells source was peripheral blood (pb) in cases and bone marrow (bm) in the others . the conditioning regimen was myeloablative in patients and ric in patients. agvhd was diagnosed in patients ( %). no associations were found between b cell subsets in donors and patients at baseline and the occurrence of agvhd. however we found a higher median percentage of transitional b cells in graft products in patients without agvhd ( . %, . - . ) compared to patients with agvhd ( . %, . - . ) (p= . , fig a) . in addition, patients without agvhd showed a lower median percentage of memory b cells ( . %, range . - ) in graft product as compared to patients with agvhd ( . %, range . - . ) (p = . , fig. b ). finally in the subgroup of patients receiving pb as stem cell source we observed a higher percentage of cd + lymphocytes in graft product in patients with agvhd ( %; range - ) compared to patients without agvhd ( %; range - ) (p= . ). in the monitoring of b cells reconstitution we observed that cd + events did not appear before day + after hsct and these were b transitional immature events predominantly. conclusions: our data suggest a possible protective link between transitional b cells and agvhd development. these results data need to be confirmed in a larger cohort of patients. moreover, it will be interesting to evaluate the relationship between transitional b cells at day + and the occurrence of cgvhd. clinical background: agvhd is a major complication of allogeneic hematopoietic stem cell transplant (hsct) and a risk factor for post-hsct mortality. the objective of this analysis is to describe patients with agvhd who had a suboptimal response to corticosteroids. methods: patients who developed ibmtr severity index ii-iv agvhd after first hsct between / / to / / were included in an ongoing chart review at centers in the united states. patients who had ever participated in a gvhd prophylaxis trial or used jak inhibitors were excluded from the study. suboptimal response to corticosteroids was defined as use of additional systemic anti-gvhd therapy, inability to taper high-dose steroids (≥ mg/ kg) by ≥ %, or tapered corticosteroids by ≥ % but not to < mg/day. results: the analysis included patients with suboptimal response to corticosteroids. mean age was years; % were male. median time from transplant to agvhd diagnosis was days. at the time of maximum agvhd grade, % of patients were grade ii and % were grade iii-iv; % had lower gi involvement, and % had ≥ organs involved. from time of diagnosis to maximum agvhd grade, % of patients had new organ involvement or an increase in agvhd grade. median time from diagnosis to maximum grade was . days, and was . days for patients with lower gi involvement. systemic corticosteroids were initiated on the day of diagnosis for % of patients. average starting daily dose was mg ( . mg/kg) for prednisone and mg ( . mg/kg) for methylprednisolone. steroid dose was increased for % of patients during follow-up; % were unable to taper below mg/day. among patients who received additional systemic anti-gvhd therapy (n= ), % increased their corticosteroid dose before initiation of additional anti-gvhd therapy. median time from initiation of corticosteroids to additional therapy was . days. frequently used therapies were mycophenyalate mofetil ( %), atg ( %), extracorporeal photophoresis ( %), tocilizumab ( %), etanercept ( %), and sirolimus ( %). agvhd recurred in % of patients and was managed by increasing corticosteroid dose in % of patients. % had any infection within first days post-hsct. forty patients ( %) required hospital readmission(s); % had ≥ readmissions within days post-hsct, with a mean inpatient lengthof-stay of days. relapse of underlying malignancy was reported for ( %) patients. two-thirds ( %, n= ) patients died at a median of (interquartile range (iqr): - ) days from agvhd diagnosis; a higher proportion ( %) of patients with maximum grade iii-iv agvhd died at a median of (iqr: . - . ) days; majority ( %) of patients with lower gi agvhd died at a median of . (iqr: - ) days. conclusions: a majority of patients with agvhd who had suboptimal response to systemic corticosteroids had severe and rapidly progressing disease and resulted in a high mortality rate ( %); progression was more rapid and mortality increased for patients with lower gi involvement. most patients required readmission to the hospital with extended length-of-stay. an urgent need exists for effective and tolerable therapies that quickly resolve life-threatening agvhd in early stages of disease. disclosure results: median time to onset of bo from allohct was . months (range . - . ). previous acute gvhd in . % (n = ) [grades iii-iv . % (n = )]. in . % (n = ) cgvhd had exclusive lung involvement, while the other patients ( %) had other organs affected. at diagnosis of bo, . % (n = ) were under immunosuppressive treatment. . % (n= ) of patients with bo received ecp as second-line treatment. median duration of treatment was months ( . - . ) and time to response . months ( . - . ). median of sessions was ( - ). evaluation of response was based on the evolution of fev measurement: . % (n = ) complete response; % (n = ) partial response and % (n = ) stable disease. one patient did not get any response and another was not evaluable. . % of patients (n = ) could reduce immunosuppression, and in one case it was completely discontinued. there is a trend for early separation between survival curves in favor of ecp ( figure ). one patient had sepsis secondary to central venous catheter infection as complication related to ecp. conclusions: ecp has emerged as a promising treatment for bo after allohct. in our experience, ecp was effective to stabilize or improve the disease in many patients and allowed to taper esteroids with minimal associated complications. however, prospective studies and longer follow-up are needed to support these findings. disclosure: nothing to declare background: the key role of il- signaling in acute graft vs. host disease (agvhd) and cytokine release syndrome (crs) has evoked growing use of tocilizumab, an anti-il receptor (il -r) antibody, in these settings. apart from regulation of t-and b-cell differentiation, immune cells migration to inflammatory sites and t-cell recruitment, il- complex with il -r through gp upregulates production of fibrinogen (fg) and other acute phase proteins, including c-reactive protein (crp). methods: we retrospectively analyzed data of patients treated with tocilizumab ( mg/kg) due to steroid-refractory (sr) agvhd and patients because of crs. median age was and years, respectively. seven patients were transplanted from unrelated donors (mud/mmud) and from sibling donors. eight patients received myeloablative and reduced intensity conditioning regimen. analyzed data included concentrations of fg, crp, an incidence of infections at tocilizumab administration and in weeks following the infusion. results: stage ii agvhd was diagnosed in patient, stage iii in , and stage iv in patients. involvement of the gastrointestinal tract (gi) was observed in % of cases. the median fg concentration before tocilizumab administration was . g/l (range, . - ) and crp mg/dl (range, - ) and % of patients had an active infection. after infusion of the antibody, we observed a decline of fg and crp levels. the median level of fg was . g/l (range, . - . ) - days after the tocilizumab infusion with no severe bleeding complications. a median crp value was . mg/dl (range, . - ) despite confirmed infectious complications. three weeks after infusion of tocilizumab fg raised to the normal range in % of patients (fig ) . five patients with sr agvhd achieved a complete response, and had a partial response after tocilizumab therapy. [[p image] . fibrinogen levels in gvhd patients following tocilizumab infusion.] a group treated with tocilizumab due to crs had higher initial levels of fg . g/l (range, . - . ) and crp mg/ dl (range, - ) before administration of the drug. reduced fg and crp levels from a baseline value were also observed in this group. however, concentrations were higher than in gvhd patients: fg . g/l (range . - . ) and crp . mg/dl (range . - ). in all patients, a differential diagnosis of disseminated intravascular coagulation was excluded. conclusions: . fibrinogen declines after tocilizumab therapy due to its cytokine-regulated production in the liver. coagulation monitoring should be performed during the first weeks after administration of the antibody to avoid serious bleeding complications. . crp concentrations remain low despite the presence of active infections following infusion of tocilizumab. crp fails as a marker of infection during weeks following the therapy. . tocilizumab is an effective therapy in patients with agvhd, especially with the gi involvement. disclosure: nothing to declare vanishing bile ducts after allogenic hsct: is it really gvhd? antonio grasso , lorenzo d'antiga , aurelio sonzogni , massimo gregori , alessandra maestro , roberto simeone , natalia maximova background: evaluation of liver gvhd was historically based by elevation of bilirubin levels and by reduction and degeneration of small bile ducts on histological samples of post-transplant liver biopsy. however, there is a lack of studies that compared histological finding of ductopenia between post-autologous hsct and post-allogenic hsct. studying severity of ductopenia following allogenic hsct, we aimed to demonstrate lack of correlation between ductopenia and clinical signs of liver gvhd. methods: we retrospectively collected a series of allogeneic hsct performed from to in the institute burlo garofolo. all patients undergo percutaneous liver biopsy in most cases at three months, one year and three or more years after hsct. indications for biopsy were alteration noted at weekly follow-up assessments of at least one clinical or laboratory marker of liver impairment or cholestasis. ductopenia was defined by number of portal tracts with no interlobular bile duct divided by the total number (severe if the ratio was less than . ). clinical gvhd was defined by nih consensus criteria results: our population involved % males and % females with oncological ( %) and non-oncological underlying disease ( %). clinical signs of liver gvhd were present in % of the patients (n= ), % with contextual intestinal involvement, % with cutaneous and intestinal involvement. patients underwent biopsy at a mean time of +/- days after hsct, patients underwent a biopsy at months after hsct and patients after three or more years from hsct. results of biopsies are showed in table . no difference in incidence of ductopenia were found between liver gvhd group and no gvhd. table] . table : incidence of ductopenia - months, months and or more years after hsct in total population, gvhd group and no-gvhd group] the group that not received chemotherapy prior the hsct had an overall incidence of ductopenia of % (severe ductopenia of %) statistically significative in comparison with the oncological underlying disease group ( % of ductopenia and % of severe ductopenia). furthermore, a little sub-group of patients extrapolated from our population received liver biopsy before hsct for diagnostic assessments: of the with an oncological underlying disease % already showed ductopenia, while no signs of ductopenia were found in the others with a nononcological disease. conclusions: there is no correlation between incidence of gvhd and histologically finding of ductopenia on liver biopsy. ductopenia may be caused in the first place by chemotherapy treatment received before hsct and myeloablative conditioning for hsct and it's not related with gvhd. this hypothesis is strengthened by the subgroup analysis of pre-hsct biopsy. background: second and third line therapies for steroid refractory acute graft versus host disease (agvhd) after allogeneic stem cell transplantation (asct) are still lacking. ruxolitinib, a selective januskinase / inhibitor could show high efficacy in agvhd, as well as extracorporeal photopheresis (ecp). here we report a single center experience of combining both therapeutic approaches in severe steroid refractory agvhd with additional analysis of immune status of these patients to elucidate direct effects of this treatment on immune response. methods: from june to february , patients ( . % male, . % female, median age: . years, r: - ) with steroid refractory agvhd of lower gi-tract after asct were treated with ruxolitinib and extracorporeal photopheresis as third, fourth or fifth line therapy. some patients showed additional agvhd of skin (n= ), liver (n= ) or upper gi-tract (n= ). all patients had an overall grade iii ( %) or iv agvhd ( %). steroid refractoriness was defined as no improvement in days or aggravation after days of steroid treatment.median start of ruxolitinib or ecp was day . after asct (r: - ). medianduration of ruxolitinib therapy was . days (r: - ) with a median start dosage of mg per day ( x mg; r: - mg). all patients started with ecp treatments per week with an individual reduction of treatment frequency. median number of ecp treatments was . (r: - ) with a median frequency of ecp therapy once a week (r: . - . ). cytomegalovirus (cmv) status of all patients and immune status of ten patients (lymphocyte count with cd + t helper lymphocyte and regulatory t cell count) were collected previously, after four weeks of starting combined treatment and four weeks after stopping the treatment. results: one-year estimated overall survival (os) of all patients was % with a median estimated os of days. patients died because of relapse of underlying disease, one of severe therapy refractory agvhd of lower gi tract and due to infection complications in agvhd refractory setting. overall response was . % (complete remission rate: . %, partial remission rate: . %). . % (n= ) of the patients had cytopenia ctc i-iii during the treatment, no grade iv cytopenia was reported. cmv reactivation during ruxolitinib occured in . % of cases (n= ). tapering of steroids could be performed rapidly with a medium reduction time of . days for reducing to half of the dosage.remarkably, regulatory t cells significantly increased during combined ruxolitinib/ecp treatment compared to regulatory t cell count before treatment (p= . ) and after stopping treatment, regulatory t cell count decreased again (p= . , see figure) . significant changes in whole lymphocyte count or in cd + t helper cell count were not observed. conclusions: treatment of severe steroid refractory agvhd with ruxolitinib plus ecp could show a high complete remission rate of . % with an one year os of %. detecting increased regulatory t cell count during the treatment underlines its direct effects on immune response and encourages to pursue this promising therapeutic approach. [ background: due to increased immunosuppression infections remain the main cause of death followed by higher risk of relapse in patients treated for acute graft versus host disease (agvhd) after allogeneic stem cell transplant (sct). here we report a single-centre experience with extracorporeal photopheresis (ecp) for acute gvhd that was introduced in order to reduce steroid treatment. comparison of overall survival (os) for patients on ecp and patients that received standard first line therapy for agvhd was performed. methods: we retrospectively analysed patients ( %) with acute gvhd grade ii-iv treated from january to october out of total allogeneic sct in that period. all patients received calcineurin inhibitors or sirolimus while receiving steroid treatment for agvhd. twenty-five patients ( %) received ecp with steroid lowering intent. we defined response as ( ) reduction of steroid dose for at least % from baseline while not adding another immunosuppresive agent and ( ) not repeating second steroid treatment if the ecp was started after lowering of steroids to prevent agvhd flare. we checked separately patient responsive and refractory/dependent to steroids. on average patients received ecp procedure once weekly. results: tapering of immunosuppressive therapy as defined was successful in ( %) out of patients in ecp group. in a group of patients without ecp ( %) patients had steroid refractory or steroid dependent agvhd compared to ( %) patients in ecp group. four ( %) patients with steroid refractory or dependent agvhd showed improvement in ecp group compared to only one ( , %) in non ecp group. twenty ( %) patients died due to infectious complication and ( %) due to relapse in non ecp cohort. in ecp cohort ( %) patients died due to infection and ( %) due to relapse. median os was months in non ecp group (r., - ) compared to months (r., - ) in ecp group and os of % at years in non ecp compared to % in ecp cohort was observed. patients with agvhd treated with ecp and faster steroid tapering had longer os compared to patients without ecp (p= , ). conclusions: ecp enables successful tapering or withdrawal of steroid therapy in many patients, even in those who are steroid refractory or steroid dependent. reduction of immunosuppression leads to reduced incidence of infection and relapse which translates into a better overall survival. background: the curative potential of allogenic stem cell transplantation is hampered by graft-versus-host disease (gvhd). pre-clinical study showed an efficacity of jak / inhibitor, ruxolitinib, in treatment of steroidrefractory gvhd. methods: we reported in this monocentric retrospective study, ruxolitinib response and follow up of cases of chronic gvhd (cgvhd) not improved with standard immunosuppressive therapy. complete organ response (cr) was defined as the resolution of clinical manifestations of cgvhd in a specific organ. very good response partial (vgpr) was defined as an improvement of clinical manifestations of cgvhd with more than % decrease of corticosteroid, while a partial response (pr) was associated with less than % decrease of corticosteroid. treatment failure was defined by the absence of improvement of cgvhd, deterioration of cgvhd in any organ by at least one stage, the development of cgvhd manifestations in a previously unaffected organ, and the use of any additional agents to control the disease. results: median age at transplant was years (range, - ). % of patients presented an acute myeloid leukemia. donor type was sibling (n= ), unrelated (n= ) or haploidentical (n= ).two patients benefited a cord blood transplant. patients received either myeloablative ( %) or reduced intensity ( %) conditioning regimens. stem cell source was peripheral blood for % of patients. patients presented mild (n= ), moderate (n= ) or severe (n= ) cgvhd according to nih score. median number of regimens prior to ruxolitinib was (range, - ), among those corticosteroids (n= ). median follow-up after ruxolitinib was months (range, - ). overall responses rate (orr) at month was % with % cr, % vgpr and % pr ( figure ). % of patients failed at month after introduction of ruxolitinib. the rate of cr increased with time : % at months (n= ), % at months (n= ) and % at months (n= ). but vgpr rate was rather stable at months ( %), at months ( %) and at months ( %) vs % at month. among the patients under steroids, ( %) patients discontinued steroids. the -months overall survival (os) and diseasefree survival (dfs) after ruxolitinib was % ( %- %, % ci) and % ( %- %, % ci), respectively. severe cytopenia (grade and ) was observed in patients. after introduction of ruxolitinib, patients presented bacterial infections, patients presented an invasive pulmonary aspergillosis and patient developped a pneumocystis. cytomegalovirus reactivation requiring preemptive treatment was observed in patients. no toxicities required withdrawal of ruxolitinib. [[p image] . figure and partial response to mesenchymal stem cells (msc), as second-line therapy, varies from % to % in acute gvhd patients. we report our experience using mscs to treat refractory agvhd. methods: the study was a retrospective single center study. all data were collected from patients' files. twenty patients were enrolled (age ranging from months to years) between april and april . results: five of these patients received reduced intensity conditioning and patients received myeloablative regimens before hsct. one haploidentical, autologous, cord blood, mud, msd transplantations were performed. the patients were eligible if they developed grades ii-iv agvhd. all patients were treated with standard first-line treatment with corticosteroids and at least one second-line therapy. the definition of steroid resistant agvhd considered as either no response to steroid treatment lasting at least days or progression during treatment of at least one grade within the first hours. prophylactic treatment with calcineurin inhibitors continued at therapeutic dose level. totally, doses of mscs were infused. the median dose of msc was . × cells per kg body weight. the median duration between the diagnosis of agvhd and initiation of mscs therapy was days (range: - ). the received msc doses ranged from one to seven. none of our patients had severe side-effects during infusions of mscs. overall, complete response (ocr) was obtained in patients, partial response in patients and no response (nr) was documented in patients. in our study group, the complete response rates in liver, gastrointestinal, skin agvhd were %, %, % respectively. four patients ( %) died in days after using mscs from complications of agvhd. eleven of patients ( %) were still alive with a median follow-up of days (range: - days) after first mscs infusion. one year estimated probability of overall survival for patients achieving ocr and partial remission/no remission in th day of mscs were . % and %, respectively. conclusions: in conclusion, mscs appears to be a safe and effective treatment option for pediatric patients with steroid refractory agvhd. disclosure: nothing to declare effect of extracorporeal photopheresis on production of serum elafin in chronic graft versus host disease arun alfred , charlotte burton , kathryn goddard , nichloas matthews background: extracorporeal photopheresis (ecp) is a second line therapy for steroid refractory, dependent or intolerant chronic gvhd (cgvhd). in order to guide ecp there is an unmet need for predictive and diagnostic biomarkers. elafin is a serine-protease inhibitor primarily produced by epithelial cells, particularly keratinocytes in inflammatory skin diseases and plasma and epidermal elafin have been identified as biomarkers of skin gvhd ( , ) . since skin cgvhd is noted for a particularly high response rate to ecp, we conducted a study to investigate whether ecp affects the production of elafin. methods: serum samples were collected from cgvhd patients ( male / female; age range: - ) and age-matched healthy controls ( male / female) before ecp and at month intervals up to year. patients had gvhd affecting skin ( / ), mucosal membranes ( / ), liver ( / ), joints ( / ), gut ( / ), eye ( / ), genital ( / ), and respiratory involvement ( / ). serum elafin was assessed by elisa (r&d systems). data were analysed using graphpad prism . statistical tests performed include -tailed mann-whitney, pearson's correlation test, and -way anova with repeat measures, as appropriate. results: chronic gvhd patients presenting for ecp had significantly elevated serum levels of elafin (p= . ; median of ng/ml, iqr - ng/ml) compared to healthy controls (median of ng/ml, iqr . - ng/ml).while % of patients had skin involvement, only % had elafin levels above the iqr of healthy controls. where disease scores were available (n= ) there were no significant correlations with modified rodnans (r= . ) or nih bsa scores (r= . ).sub-analysis was performed by grouping cgvhd patients according to quartiles of serum elafin at pre-ecp baseline. retrospective analysis of patients after months of ecp (n= ) revealed that those with serum elafin levels in the upper quartile (elafin hi ) pre-ecp (min-max: - ng/ml), showed a significant reduction after months of therapy (p< . ; mean +/-sd : ng/ml +/- ng/ml vs ng/ml +/- ng/ml, respectively), which was sustained up to months of ecp (p< . ; mean +/-sd: ng/ ml +/- ng/ml). in contrast, patients with elafin levels below the upper quartile (elafin lo ) showed no significant change (mean +/-sd: ng/ml +/- ng/ml vs ng/ml +/- ng/ml, respectively). of note, pre-ecp patients with elafin below the median received significantly more corticosteroid (cs) than those above, (p< . ; mean +-sd: +/- mg/d vs +/- mg/d, respectively), which was significantly reduced after months of ecp (p< . ; to +/- mg/d), while cs dose was not significantly changed in elafin hi patients until months (p< . ; mean +/-sd: mg/d +/- mg/d vs mg/d +/- . mg/d, respectively). conclusions: consistent with recent data, we found that serum elafin is significantly elevated in a subset of cgvhd patients compared to healthy controls, but did not correlate with skin disease scores. ecp administration was associated with a reduction in serum elafin in the elafin hi subset. further, elafin lo and elafin hi patients tolerated different rates of ecp-mediated tapering of cs immunosuppression suggests pre-ecp elafin measurements may have predictive value. references : background: allogenic hematopoietic stem cell transplantation (hsct) is a potential curative treatment for many malignant and no malignant hematologic diseases, primary immunodeficiencies and some metabolic and deposit diseases in children. graft versus host disease (gvhd) is a major cause of morbidity and a leading cause of non-relapse mortality. corticosteroids are the standard first-line systemic treatment for both acute and chronic gvhd, whereas no second line option for corticosteroid-refractory patients is standardised. ruxolitinib is a potent inhibitor of jak / showing significant responses in refractory gvhd patients in recent reports. methods: we present two centres experience with ruxolitinib for gvhd treatment in pediatric patients. the study was conducted in two spanish pediatric hsct centres, hospital vall d'hebron (barcelona) and hospital universitario la paz (madrid). all patients receiving ruxolitinib since the drug was available were included for retrospective analysis. results: between march and december pediatric patients with acute or chronic gvhd with refractoriness to corticosteroids were treated with ruxolitinib, in different episodes (one patient received it in different moments, and one patient received it in ). patient's sex at birth was female in and male in cases. median age at hsct was , years ( , - , ). primary disease was malignant in patients and non malignant in . median time of gvhd diagnosis was , days ( - ). all gvhd episodes were treated with corticosteroids as first line, with maximum doses between - mg/kg/day (the main dose used was mg/kg/day, / episodes). patients received a median number of , ( - ) previous lines of treatment including steroids before starting ruxolinib; they were extracorporeal photopheresis ( / episodes), sirolimus ( / ), mesenchymal cells ( / ) ruxolitinib initiation was indicated for acute gut refractory/steroid dependant gvhd in episodes and chronic multisystemic in episodes. other indications were chronic lung ( / episodes), chronic skin ( / ) and acute skin gvhd ( / ) . median post-hsct time of ruxolitinib start was days. doses ranged between , - mg/ h depending on age, weight, and tolerance (hematologic and liver toxicities). average duration of treatment was days ( - ). complete response (cr) rate was , %, global partial response (pr) , %, and no response (nr) % (progression in one patient and recent treatment start in other patient). mean time to maximum response was weeks. treatment stop cause was cr in cases, infection in , liver toxicity in . no severe side effects directly related to ruxolitinib treatment were described. conclusions: ruxolitinib has been recently introduced as second line strategy for rescuing corticosteroid-refractory gvhd in pediatric patients. while results of randomized trials are lacking, we present our experience (two centres). the main indications for starting treatment were acute gut and chronic multisystemic gvhd. most patients achieved some grade of response (partial or complete), allowing stopping or tapering corticosteroids. toxicity profile appears to be acceptable. disclosure: nothing to declare. stability of tacrolimus concentration early after allogeneic hematopoietic stem cell transplantation reduces the risk of acute gvhd background: tacrolimus is used as an immunosuppressive drug after allogeneic hematopoietic stem cell transplantation (allo-hsct). it is well known that early concentration level of tacrolimus is correlated with the risk of acute graft versus host disease (agvhd), however, whether range of standard derivation (sd) of early tacrolimus concentration after allo-hsct also affect to the risk of agvhd still remains unknown. here, we investigate the correlation between the range of sd of early tacrolimus concentration after donor hematopoietic cells engraftment and the development of agvhd. methods: we retrospectively assessed patients who underwent allo-hsct in our hospital from - . all patients received standard gvhd prophylaxis by continuous intravenous (iv) tac with starting dose of . mg/ kg/day from day before allo-hsct (day - ) and iv methotrexate on day , , at dose of mg/m , mg/m , mg/m , respectively. tac dosage was adjusted to target the serum concentration of - ng/ml until at least day and then tapered. to evaluate the sd of weekly tacrolimus concentration, the range of sd of tacrolimus concentration at day - (week- ), day - (week- ), day - (week- ) and day - (week- ) were calculated. the difference of the range of sd between the groups that develop or did not develop agvhd was compared by using mann-whitney u test. multivariate analysis was performed by using multiple logistic regression analysis. patients had given written consent allowing the use of medical records for research, in accordance with the declaration of helsinki, and the institutional review board approved the study. results: there were males and females and the median age was years (range, to years). the risks of disease were low-standard in and high in pts. the number of donors were in hla-identical sibling, in hla-mismatched related donor, in hla-matched unrelated donor and in hla-mismatched unrelated donor. thirty-seven patients developed agvhd (grade i-ii; , gradeiii-iv; patients). as a result, the wide range of sd at week- significantly increased the risk of agvhd (agvhd-group; . ± . ng/ml, agvhd+ group; . ± . ng/ml, p= . ). multivariate analysis demonstrated that narrow range of sd of tacrolimus concentration at week- reduce the risk of agvhd (or= . ; % ci: . - . ; p= . ). there were no correlation between gender, age, disease status, hla with the development of agvhd. conclusions: the range of sd at week- , an engraftment phase of donor hematopoietic cells, was significantly correlated with the development of agvhd. fine tuning of early tacrolimus concentration with narrow range of sd reduces the risk of agvhd, resulting in improvement of the overall survival after allo-hsct. disclosure: nothing to declair p ruxolitinib treatment for steroid-refractory graftversus-host disease han-seung park , je-hwan lee , jung-hee lee , eun-ji choi , miee seol , young-shin lee , young-ah kang , mijin jeon , kyoo-hyung lee background: steroid-refractory graft versus-host disease (gvhd) is one of the most lethal complications after allogeneic hematopoietic cell transplantation. recent studies have shown that ruxolitinib, a janus kinase / inhibitor, is effective in patients suffering from gvhd. here, we report a retrospective result of ruxolitinib treatment for steroid-refractory gvhd. methods: all patients had received cyclosporine and a short course of methotrexate as gvhd prophylaxis. antithymocyte globulin was added for unrelated or mismatched familial donor hct. ruxolitinib mg twice daily was added to immunosuppressive treatment in patients with steroid-refractory gvhd. results: a total of patients with gvhd (acute, , including patients with donor lymphocyte infusion [dli]related; and chronic, ) were included in the analysis. all patients had grade / acute gvhd or severe chronic gvhd at the time of ruxolitinib treatment. six ( . %) of patients with acute gvhd responded to ruxolitinib, including with complete response (cr). the median time to response was . days (range, - ) . nineteen patients received ruxolitinib for severe chronic gvhd, with the median of involved organs (range - ). fourteen patients ( . %) showed response to ruxolitinib, including crs. the median time to response was days (range, - ). five responders discontinued ruxolitinib and patients are still on the agent. after a median follow-up duration of . months, died ( from relapse of disease, from infection). the -year survival probability was . %. eleven of responders discontinued ruxolitinib. gvhd relapsed in of patients at , , and days after ruxolitinib discontinuation. thrombocytopenia ( / , grade / ; ) was the most common adverse event of ruxolitinib. during treatment, with grade / infectious adverse events occurred; pneumonias, brain abscess, and liver abscess. conclusions: ruxolitinib treatment seems to be effective for the treatment of steroid-refractory gvhd including long-standing chronic gvhd. the agent was well tolerated and relatively safe. disclosure: nothing to declare. il -receptor antibody tocilizumab as salvage therapy in the treatment of severe chronic gvhd after stem cell transplantation: a retrospective analysis background: severe chronic graft-versus-host disease (cgvhd) remains the most relevant factor affecting survival and long-term quality of life after allogeneic hematopoietic stem cell transplantation (hct). besides corticosteroids there is no established therapy for cgvhd and many of the used immunosuppressive agents may lead to significant toxicity incl. infectious complications. tocilizumab (an il -receptor antibody) has shown efficacy in acute gvhd and cgvhd. we retrospectively analyzed the efficacy and safety of patients having received tocilizumab for treatment of advanced cgvhd at our center between the years and . methods: patients with severe steroid refractory cgvhd and a median age of years (range: - yrs) having received at least two prior lines of therapy for cgvhd (range: - regimens) were treated with tocilizumab for at least one cycle (q w, dosage: mg/kg iv, maximum: mg) with a median number of cycles (range: . nih consensus criteria grading for cgvhd and the immunosuppressive regimen were noted at the time of the first tocilizumab administration and after , and months of therapy. all patients received additional concomitant immunosuppressive agents already given at least weeks without response before start of tocilizumab. no new immunosuppression (is) was added in parallel to tocilizumab and response assessment was stopped at start of any additional new is. all patients had received peripheral stem cell allografts. gvhd prophylaxis consisted of a calcineurin inhibitor in combination with methotrexate or mycophenolate and in case of unrelated donors atg was added. / patients had quiescent onset of cgvhd, one patient developed de novo cgvhd. the median number of days between hct and onset of cgvhd was ( - ). the median number of days between hct and initiation of tocilizumab therapy was ( - ) days. at cgvhd onset, / patients had mild cgvhd and / patients had moderate cgvhd. the thrombocyte count was < /nl in / patients. organs involved at initiation of tocilizumab therapy were skin ( %, all grade ), eyes ( %), mouth ( %), fascia ( %), lungs ( %) and genitals ( %). / patients are still receiving tocilizumab at the time of analysis. results: as tocilizumab was given fairly recently in most patients, -and -month follow-up was only reached in / patients ( %). at three-month follow-up after initiation of tocilizumab therapy, / patients ( %) showed partial remission, / patients stable disease ( %), and / patients progressive disease ( %) of cgvhd. maximal response was partial remission ( %), stable disease ( %) and progressive disease ( %). patients required subsequent new immunosuppressive treatment. one patient has not yet reached -month follow-up. during tocilizumab therapy none of the patients suffered recurrence of underlying malignancy. two patients developed significant respiratory infection and one patient developed soft tissue infection, all requiring antibiotic treatment and pausing of tocilizumab administration, hospital admission was not required. the os and rfs was % with median follow up of . months (range - months). conclusions: tocilizumab appears to be a promising treatment option in advanced cgvhd but further evaluation within a phase ii trial is required. disclosure: nothing to declare background: allogeneic hematopoietic stem cell transplantation (hsct) is for many patients suffering from aml the only curative treatment option. one major complication is graft versus host disease (gvhd), caused by donor immune cells attacking healthy tissue. regulatory t cells (treg) have been getting huge attention during the past years because of their important role in maintaining immune balance. here we collected peripheral blood samples from patients at different time points after hsct to investigate immune-reconstitution of treg as predictive marker for the development of gvhd. methods: we collected blood samples from patients in the course of allogeneic hsct prospectively once a week from d+ up to d+ . all patients received conditioning regimen with fludarabine and melphalan, combined with alemtuzumab for t cell depletion. patients developed acute gvhd in the later course. after isolation of pbmc`s we performed facs multicolor staining of t cell and nk cells. treg were identified as cd + cd + cd ++ foxp + , nk cells were characterized as cd neg cd + cd + and divided in nk cell subpopulation due to their expression of cd dim or cd high . results: . cd neg t cells: all patients developing acute gvhd in the later course showed significant elevated levels of cd + cd neg t cells, especially cd neg treg at d+ . . cd neg treg / cd + cd + t cells: one patient not developing acute gvhd showed lots of cd neg treg but missed cd + cd + effector t cells. we recently showed that cd + cd neg effector t cells are of impaired effector function. these data suggest that cd neg treg are only of relevance combined with functional cd + cd + effector t cells in the development of agvhd. . t cell marker: patients without agvhd showed elevated expression of garp on treg. garp was significantly higher expressed on cd + treg, indicating a better suppressive capacity of cd + treg. this was detected throughout from d+ until d+ . tigit and ilt showed a heterogeneous expression profile without significant differences between the two groups. . nk cells: we detected a higher ratio of cd ++ /cd dim nk-cell population in patients without. we could also show that tigit is mainly expressed on cd dim nk cells. conclusions: we and others showed reconstitution of cd neg t cell subsets after alemtuzumab mediated t cell depletion -our data on effector t cells showed an impaired effector function for cd neg cd and cd t cells. recently we presented data on impaired suppressive capacity of cd neg treg and the association with acute gvhd retrospectively (wölfinger ebmt , ash ). here we provide prospective data on patients after the use of alemtuzumab in the context of hsct: our preliminary data suggest that the total amount of cd neg-treg and the ratio of cd neg treg to cd + cd + treg on d+ after allogenic hsct could predict agvhd. this data may be a basis for immune monitoring of patients at d+ to evaluate their risk for agvhd and could lead to the use of prophylactic treg dli in the context of alemtuzumab mediated t cell depletion. disclosure: medac -travel support, novartis -consultancy fee, pfizer -consultancy fee, shireconsultancy fee background: multiple factors such as disease activity and severity, therapy and/or dietary habits can cause changes in nutritional status independently or by interacting with each other. presence of malnutrition or significant weight loss in chronic gvhd (cgvhd) patients was reported in literature up to %. the aim of this cross-sectional study was to identify factors that affect nutritional status in cgvhd patients. methods: nutritional status in patients with cgvhd treated at the university hospital center zagreb, croatia from to was assessed. anthropometric measurements (height, body weight (bw), body mass index (bmi)) and clinical validated tool patient-generated subjective global assessment (pg-sga) (where patients were categorized as well-nourished (pg-sga a), moderately malnourished (pg-sga b) or severely malnourished (pg-sga c)) were used. all patients were evaluated according to nih criteria for cgvhd diagnosis. descriptive and correlation analysis were preformed. results: in total, adult cgvhd patients were included in the study, women ( . %), median age ( - ) years, with mild cgvhd in ( . %), moderate in ( . %) and severe in ( . %) patients. according to the pg-sga rating ( . %) patients had pg-sga a, ( . %) pg-sga b and ( . %) had pg-sga c, giving a total malnutrition or risk of malnutrition prevalence of . %. the mean bmi was . ± . kg/m with correlation to pg-sga rating (r= . , p= . ). malnutrition according to the bmi (defined as bmi< kg/m ) was found in patients ( . %). bw changes ( % or more in months) were significant in patients ( . %). according to the pg-sga assessment tool, oral symptoms reported by patient ( . %) and decreased appetite reported by patients ( . %) were associated with oral cgvhd nih score (r= . , p= . ; r= . , p= . ) but not with bw or bmi. gastrointestinal (gi) symptoms assessed with sga, were generally mild with no correlation to gi cgvhd nih score. no significant association was found between nutritional status and other nih cgvhd scores. corticosteroid therapy present in ( . %) correlated with pg-sga rating (r= . , p= . ) but not with bw, bmi or appetite changes. in patients ( . %) with altered pg-sga rating, bmi, appetite and body weight changes, dietary counseling and oral nutritional supplementation were initiated. conclusions: oral symptoms, decreased appetite and corticosteroid therapy in our cgvhd patients were associated with altered nutritional status according to the pg-sga, but not with bmi. therefore, pg-sga might be a more sensitive tool in assessment of changes of the nutritional status and detection of patients at risk of malnutrition than bmi since it includes different factors like physical examination, presence of gi symptoms and corticosteroid therapy in its scoring system. nutritional counseling and support are important in cgvhd patients especially in presence of oral symptoms. disclosure: nothing to declare. background: sclerotic skin changes are common features in chronic graft versus host disease (cgvhd). one of the most challenging aspects in the diagnosis and management of sclerodermoid cgvhd (scgvhd) is the differentiation between reversible symptoms related to active cgvhd and nonreversible symptoms related to residual permanent damage such as long-standing fibrosis. although several candidate biomarkers of cgvhd inflammatory activity have been proposed, none of them are currently validated. therefore, there is a need for the development of more quantifiable and reproducible measurements tools to guide clinical decisions. we report our experience evaluating the usefulness of high-frequency ultrasonography (hfus) plus doppler ultrasound (doppler-us) and serum fibrosis biomarkers to determine the inflammatory activity of scgvhd. methods: we report patients with scgvhd. hfus plus doppler-us were performed at diagnosis of scgvhd and at different time-points after treatment initiation. serum hyaluronic acid and pro-colagen-iii were measured as fibrosis biomarkers simultaneously with hfus and doppler-us. nih cgvhd consensus conference diagnosis criteria, scoring system, and response criteria were used to assess global and organ-specific cgvhd, and to measure overall response to therapy. abnormal ultrasound findings were defined as the presence of ≥ of the following: hypoechogenic dermis, dermo-epidermal junction effacement, hypoechogenicity of septa and/or hyperechogenicity of lobules in hypodermis, hypoechogenic fascia, or myositis, for hfus; and, vessels thicker than mm in dermis and/or hypodermis, systolic pressure > cm/sec, and index of vascular resistance > . , for doppler-us. inflammatory activity was classified as mild, moderate and severe according to the severity of doppler-us findings. results: hfsu showed abnormal findings in all patients at diagnosis with no changes except in two patients along the treatment follow-up. inflammatory activity by doppler-us was observed in / patients at diagnosis ( mild, moderate, severe). four patients responded to treatment ( complete responses, cr, and partial responses, pr), one presented clinical improvement less than pr, and one, progressive disease. all patients with clinical response had also a p-rom improvement or normalization. all patients achieving a response showed normalization (n= ) or improvement (n= ) of doppler-us findings. the patient with clinical improvement less than pr and the patient with progressive disease showed persistence of inflammatory doppler-us findings. most patients had normal or light increase of pro-collagen levels at diagnosis and no significant changes were observed during follow-up. levels of hyaluronic acid tended to be very high in patients with progressive scgvhd (patients and ) and tended to decrease or normalize in those who responded to therapy (patients , , and ). conclusions: in this exploratory study, hfsu was a reliable method for evaluating sclerotic skin changes in scgvhd. doppler-us showed a good correlation with disease activity and response to treatment. serum hyaluronic acid levels might be a biomarker of disease activity that deserves further investigation. hfsu plus doppler-us is a useful, non-invasive, repeatable device in monitoring patients suffering from scgvhd. according to our results, doppler-us may be a more sensitive parameter than hfsu in assessment inflammatory activity of scgvhd. disclosure: maría suárez-lledó received a grant from dkms-spain foundation. other authors have nothing to declare post-transplant cyclophosphamide versus antithymocyte-globulin in hla-matched unrelated and haploidentical transplantation for hematologic malignancies background: post-transplant cyclophosphamide(ptcy) and antithymocyte-globulin(atg) are the most commonly used regimens for the prophylaxis of graft-versus host disease(gvhd). we compared these two regimens in hlamatched unrelated (mud) and haploidentical transplantation for hematologic malignancies. methods: we retrospectively analyzed the consecutive adult patients with hematologic malignancies who received mud and haploidentical transplantation at chungnam national university hospital between january and january . patients who received second transplantation and had refractory disease were excluded. results: this study included patients with median age of (range, - ) years: ( . %) patients received mud transplant ( and patients in ptcy and atg group, respectively), and ( . %) patients received haploidentical transplant ( and patients in ptcy and atg group). graft source was peripheral blood stem cell in all patients. median follow-up duration was . months (range, . - . ). in mud transplant, the estimated -months survival rate were . % in ptcy vs. . % in atg (p= . ), the -months relapse rate were . % in ptcy vs. . % in atg (p= . ), the cumulative incidence of grade to acute gvhd were . % in ptcy vs. . % in atg (p= . ), and the estimated -month extensive chronic gvhd rate were . % in ptcy vs. . % in atg (p= . ). in haploidentical transplant, the estimated -months survival rate were . % in ptcy vs. . % in atg (p= . ), the -months relapse rate were . % in ptcy vs. . % in atg (p= . ), the cumulative incidence of grade to acute gvhd rate were . % in ptcy vs. . % in atg (p= . ), and the estimated month extensive chronic gvhd rate were . % in ptcy vs. . % in atg (p= . ). patients receiving ptcy had significantly longer neutrophil engraftment time than those receiving atg in haploidentical transplant [median(range); . ( . - . ) days vs. . ( . - . ) days, p= . ]. conclusions: ptcy might be a good option for the prophylaxis of gvhd in hla-matched unrelated transplant as well as haplo-identical transplant. disclosure: nothing to declare early fam therapy for post allo-hsct bronchiolitis obliterans syndrome background: bronchiolitis obliterans syndrome (bos) is a potential major complication after allogeneic hematopoietic stem cell transplantation (hsct). attributed to an allo-immune reaction against the small airways, bo is considered a pulmonary manifestation of chronic gvhd. reported incidence of bos ranges from to %, and bos-attributed mortality as high as %- %. a few years ago, a new therapeutic approach with fluticasone, azithromycin, and montelukast (fam) was described (norman bc, et al. bmt ) . our aim was to analyze the outcomes of pts who developed bos and were precociously treated with the fam scheme. methods: all the allo-hsct performed in our center from january and july were included in the analysis. baseline diseases were: aml, lpd, mds, all, mpd, mm, and bmf. day + and day + overall mortality were , % and , %, respectively. rest of characteristics of the series are shown in table. fam therapy was systematically started when any patient was first diagnosed with bo. results: eleven patients ( , %) were diagnosed with bos. at diagnosis of bos, the pts exhibited a fev % of predicted (median fev : %; range; - %) and/or a decline > % from pre-hsct . at day + , pts had already the syndrome. two of them died before the end of the first year: one due to invasive zygomycosis (cns plus pulmonary) and the other to baseline disease progression. at day + , more pts had bos. two more pts with bos died at and months post-hsct due to baseline disease progression. at the close of the analysis, of the pts were alive. so, with a median follow-up of months (range: - ), mortality and bos-attributable mortality of the pts with the complication were , % and , %, respectively. conclusions: ) bos is an infrequent but very severe complication of allo-hsct; ) bos seems to be less frequent in pts with prophylactic pre-transplant ratg or post-transplant cyclophosphamide, as well as in pts undergoing transplantation with bm (compared to pbsc). ) early diagnosis and therapy are critical to minimize the bos-attributable mortality. disclosure background: donor lymphocyte infusion (dli) is an established treatment for patients with hematological malignancies relapsed after allogeneic hematopoietic stem cell transplantation (hsct). however, it is associated with an increased risk of graft-versus-host disease (gvhd) and modest anti-tumor activity. compared to the infusion of nonmobilized lymphocytes, granulocyte colony-stimulating factor (g-csf)-primed dli might induce a stronger anti-tumor effect and reduce the risk of infusion-induced gvhd. due to the limited experience of g-csf primed dli in patients relapsed after haploidentical hsct, we conducted a retrospective study of all patients at our hospital who received dli for the relapsed hematological diseases following related hla-matched or hla-haploidentical hsct. methods: the institutional research board approved the study. we identified patients with hematological malignancies receiving dli following related allo-hsct at national taiwan university hospital between and aug. the infusate was obtained from the cryopreserved specimen, which had been collected and stored at multiple aliquots at the same time as the initial haploidentical peripheral stem cell graft. patients received dli for either hematological relapse, preemptive or prophylactic treatment. univariate and multivariate analysis was performed using cox proportional hazard regression model. results: for the patients following related hlamatched and the patients following hla-haploidentical hsct received and doses of dli, respectively. in comparison, the median cd + cell dosage of haplo-dli is significantly lower (p = . ) than that of dli from sibling donors, with median cell dosage . × /kg (range, . - × /kg) and . × /kg (range, . - . × /kg), respectively. the median time to dli from initial sibling hsct and haplo-hsct was days (range, - days) and days (range, - days), respectively. overall, ( %) of the patients following sibling hsct developed grade - acute gvhd after dli, whereas ( %) of the patients receiving haplo-hsct developed grade - acute gvhd after dli (p= . ). importantly, for patients receiving dli with cd + cell dosage less than × /kg, there is no difference in the risk of developing grade - acute gvhd between patients receiving dli from sibling or haplo donors ( figure a) . interestingly, for patients receiving dli with cd + cell dosage more than or equal to × /kg, ( %) of the patients following haplo-hsct developed grade - acute gvhd after dli, significantly more than ( %) of the patients following sibling hsct developed grade - acute gvhd after dli ( figure b) . the cumulative incidence of grade - acute gvhd at day after haplo-hsct and sibling hsct were % ( % ci: . - . ) and . % ( % ci: . - . ), respectively ( figure b , p = . ). [[p image] . conclusions: our study shows that the administration of g-csf mobilized dli is feasible after haploidentical hsct for relapsed hematological malignancies. however, dli with cd + cell dosage more than or equal to × /kg in patients receiving haplo-hsct is associated with significantly higher risk of developing acute gvhd than dli from the sibling donors. disclosure: the authors declare no competing financial interests. background: the fresenius phelix is a uva irradiation device used to photoactivate mnc collected on the amicus. the system is closed, utilizing a special mnc kit and modified instrument software. the preliminary results of a phase i safety trial involving three patients ( treatments) with chronic graft vs. host disease are presented. methods: reasons for transplantation for the patients ages , and years were: acute myelogenous leukemia, myelodysplastic syndrome, and myelodysplastic syndrome with pnh. stem cell source was peripheral blood with a / match for all. each developed chronic skin gvhd. inclusion criteria included wbc and plt counts > and x /l, gfr > ml/min/bsa, and ast - unit/l. exclusion criteria included active gi bleeding, nyha cardiac disease greater than grade iii, and the presence of light-sensitive diseases. amicus software . and phelix software . were used. settings included: ml/min max draw rate, ml fixed cycle volume, . mg/kg/min citrate infusion rate, and : acd-a ratio. venous access was peripheral or subcutaneous port. target uva dose was . j/cm and -methoxypsoralen dose was . ml. results: the following mean + sd procedure results were obtained: , + ml whole blood with acd-a drawn, + ml acd-a used, + ml saline used, + minutes procedure time, and , + ml total blood volume. minor alarms (n= ) on the amicus and no alarms on the phelix were encountered. all -day aerobic and anaerobic cultures were negative and mean endotoxin levels were . + . eu/ml. mean pre/post cbc and plasma hemoglobin levels were: . / . wbc, . / . neutrophils, . / . basophils, . / . eosinophils, . / . lymphocytes, . / . monocytes, / platelets x /l, / % hct, . / . g/dl hgb, and . / . mg/dl plasma hemoglobin. plasma hemoglobin delta in the product was . + . grams and the subject was - . + . grams. collected product hct. mean . + . %. yields are in the table. adverse events included one each: acute respiratory failure, respiratory failure, muscular weakness, musculoskeletal discomfort, and peripheral swelling. three of four events occurred in one patient two weeks after the study procedure. none of the adverse events were considered related to the procedure or investigational product. the patient who experienced acute respiratory failure was removed from the study because of death due to pneumonia, felt to be unrelated to the procedure. conclusions: results indicate the new closed photopheresis system is capable of collecting sufficient mnc and irradiating the cells producing high lymphocyte apoptosis, with minimal alarms and adverse reactions. ( . %)). ( . %) of the patients also had acute gvhd of the skin or liver. patients ( . %) could be treated and controlled with methyl-prednislone monotherapy, patients had steroid refractory gvhd of whom patients ( . %) could be salvaged with additional drugs (infliximab: ; tacrolimus: ); patients ( . %) had refractory acute gut gvhd and could not be salvaged despite more than three lines of therapy. at the time of reporting, patients ( . %) of the are alive. patients died due to transplant related mortality, while patients developed relapsed disease. on binary logistic regression analysis, no baseline clinical or treatment related predictor (disease indication, disease status at transplant, transplant type, graft source, type of conditioning) could be identified for developing acute gvhd of the gastrointestinal system. conclusions: acute gvhd of the gastro-intestinal system is a significant cause for morbidity in allo-hct patients at our centre. further studies are warranted in our cohort, and a prospective analysis of gut microbiome analysis, faecal multi-drug resistance organism surveillance, conditioning related toxicity and antibiotic usage is ongoing. clinical trial registry: not applicable disclosure: the authors declare no potential conflicts of interest benefits and precautions of ruxolitinib in steroidrefractory acute gvhd background: corticosteroids are the standard first-line treatment option for patients with acute graft-versus host disease (gvhd), but approximately half of patients become refractory to steroids and require second-line treatment. ruxolitinib has the potential to treat gvhd in steroidrefractory (sr) patients based on retrospective clinical data. the ongoing prospective trials are currently enrolling patients to evaluate the therapeutic potential of ruxolitinib for gvhd. methods: we analyzed retrospectively clinical experience with ruxolitinib in patients (n= ) with grade ~ steroid-refractory acute gvhd patients compared with the control group not receiving ruxolitinib. in addition, immune status was evaluated about weeks~ weeks after the administration of ruxolitinib using flow-cytometry. ruxolitinib was used as a third option for sr gvhd, combined with previously used immunosuppressive drugs. and steroids were gradually decreased according to the symptoms and discontinued. patients received ruxolitinib mg twice daily (bid), with increase to mg bid if hematologic parameters are stable and no treatmentrelated toxicities. results: fifteen patients all were assessable for response. seven patients achieved a complete response, had a partial response, and had no response at weeks after the first ruxolitinib dose. overall response rate was %. three were treatment failures. most adverse effects were manageable, except infectious complications. infectious complications were occurred in about % patients (n = ), resulting in two deaths. common cause of infectious events included cytomegalovirus (n = ), herpes-zoster (n= ), epstein-barr virus (n= ), fungal infection (n = ), pneumocystis jiroveci (n = ), bacterial infections (n = ), and pneumonia of unknown origin (n = ). t cell counts tended to decreased in the group with ruxolitinib compared with the control group, especially cd cell counts. conclusions: ruxolitinib is effective in controlling sr gvhd and can lead to clinical benefits. however, we need to be aware of the infectious complications because ruxolitinib may lead to increased risk of opportunistic infections or reactivation of latent infections. in addition, common infectious complications are presumed to involve t cell dysfunction. clinical background: graft versus host disease (gvhd), being one of most common life-threatening complication post hsct, contributes significantly to morbidity and mortality. when affecting gastrointestinal tract (gi) it is the major cause of death in early period post hsct. due to widespread tissue involvement in most patients diagnosed with gi gvhd, surgical treatment is rarely considered. methods: among allo-hsct performed in department of pediatric hematology, oncology and bone marrow transplantation in wroclaw, poland during years - , ( , %) cases were diagnosed with gi gvhd. in this study we present cases ( %) which were referred to and benefit from surgical approach. results: . male, years old underwent hsct from matched unrelated donor (mud) due to chronic myelogenous leukemia (cml) and subsequent molecular relapse succesfully treated with donor lymphocyte infusion, followed by agvhd (skin and gut involvement, grade iv). extensive immunosuppression (steroids, mycofenolate mofetile, atg, okt ) resulted in significant resolution of agvhd symptoms. however aggravating severe abdominal pain and lack of gut movement suggesting bowel obstruction. due to presence of acute abdomen patient was immediately directed for laparotomy. resection of constricted bowel segment followed by subsequent laparotomies for secondary obstruction provided complete resolution of abdominal symptoms. after years of follow-up patient is alive and well. . eleven years old male was diagnosed with skin and gut grade iv agvhd on day + post mud-hsct performed due to acute myelogenous leukemia (aml). he received pronlonged immunosuppressive treatment including steroids, antibodies, msc and ecp which led to resolving of skin leasions and diarhoea. nevertheless patient was suffering from severe paroxysmal abdominal pain and incidentally vomiting. ct enterography showed partial small bowel constriction. after numerous surgical consultations, eventually on day+ patient underwent laparotomy with constricted bowel resection. histopatological examination of resected tissue revealed moderate gvhd. immunosuppersion was tapered to low dose of steroids with ecp. for now, years post hsct patient is alive, rarely experiencing mild abdominal cramps . fourteen years old female developed severe abdominal pain and high volume diarhoea on day + post mud-hsct performed for severe anaplastic anemia (saa). despite extensive immunosuppression (steroids, anti-tnf, anti-il antibodies) patient condition did not improved. through consistent stomach pain, suspected subileus confirmed by ct enterography, laparotomy was performed (day+ ). resection of inflamated and obstructed bowel was made. microscopic evaluation confirmed prior gvhd diagnosis therefore immunosuppression including csa and tapered doses of steroids was continued. complete resolution of abdominal symptoms was almost immediately achieved post-surgery, however months after recurrent abdominal cramps were observed and are now well controlled by pain killers. conclusions: commonly gi gvhd is diffused inflammatory process. however in some cases it may be localized and may lead to partial bowel constriction. in case of severe and prolonged stomach pain, despite of partial resolving of other gvhd symptoms, ileus should be considered. ct enterography may be useful for diagnosis confirmation. in those patients, surgical intervention may improve quality of life or even be a salvage approach. disclosure: nothing to disclose is there any impact of the uric acid levels during the preand early post-graft infusion period, on the gvhd occurence and allotransplant outcome? . ( . - . ) years, who underwent allogeneic stem cell transplantation (allosct) from full-matched sibling donors for acute leukemia (n= ), very severe aplastic anemia/pnh (n= ), lymphoma (n= ), myelodysplastic/ myeloproliferative syndrome (n= ) . thirty-two patients were in remission at the time of allosct (cr : , cr : , beyond cr : ). for a better and more accurate assessment of the ua levels on the agvhd incidence, unlike to the other published studies which evaluated the ua levels only at day , we evaluated the ua levels in different time points during the the peri-transplant period (at the conditioning regimen initiation, and at days , + and + ). because the majority of our patients developed agvhd within the - days post-transplant, we did not incorporated in the study the of ua levels beyond the + day. we also investigated the effect of the ua on survival and the non-relapse mortality (nrm). the vast majority of patients received allopurinol from the st day of conditioning regimen till day - . the independent t-test, kaplan-meir method and logrank test were used in the statistical analysis. results: the median ua levels were . , . , . and . mg/dl at days - , , + and + respectively. for the statistical analysis purposes, we grouped our patients as low-ua if they had values < mg/dl or high-ua if they had > mg/dl. this threshold was chosen based on the ua values from all the collected samples (n= ). finally / ( %) patients developed agvhd; ( %) were assessed as gr ≥ii, while ( %) as gr iii-iv. the incidence of the agvhd gr ≥ii was similar (ranged from - %) in both groups of patients (low-ua and high-ua) and for all the estimated time points (days - , , + , + ). we noticed a better -years overall survival for patients with low-ua ( % vs. %) however without any statistical significance. ten patients succumbed to nrm causes; / deaths attributed to gvhd complications. the nrm was assessed higher in the high-ua group ( % vs. %) but also this difference was not statistically significant. conclusions: though our study bears the limitations of the small number of patients and the retrospective origin, at least to our knowledge is the first which evaluates the impact of ua levels at different time points in the peritransplant period, on the agvhd incidence. in our study the ua levels did not influence the incidence or the severity of agvhd. the higher nrm rates for patients with ua> . mg/dl merits further evaluation. definitely, the role of ua on the allosct outcome will be clarified through well designed prospective trials. disclosure results: five male patients ( %) had genital cgvhd manifestations presented by urethral stricture in / patients and phimosis requiring surgical treatment in one patient. all five patients had simultaneously cutaneous, oral, and/or ocular cgvhd manifestations. the first patient underwent urethroplasty of bulbomembranous part of urethra with termino-terminal anastomosis and urethroplasty of penile part of urethra with buccal mucosa autograft -bmg (dorsal onlay) that resulted in significant improvement of symptoms and normal miction afterwards. biopsy of the urethra showed mononuclear infiltration in lamina propria consistent with cgvhd. biopsy of the buccal mucosa was done prior to surgery and was negative for cgvhd involvement. the second patient underwent urethrotomy due to circular strictures, but symptoms reappeared again and he is now candidate for bmg. in two patients urethral dilatation was done, and the fifth patient presented with phimosis requiring circumcision, resulted in significant improvement of symptoms. conclusions: male genital cgvhd is an underrecognized and under-reported manifestation. patients after allo-hsct need to be actively asked about their genital symptoms and sexual function, especially if they are diagnosed with other mucocutaneous or ocular cgvhd. multidisciplinary approach, early recognition and frequent follow-up is necessary for timely start of treatment. new methods, such as bmg for cgvhd patients with urethral stricture seem promising and should be further investigated. disclosure: nothing to declare. p abstract withdrawn. heracles: a phase ii single-arm prospective study to assess the efficacy of fecal microbiota transfer in the treatment of steroid refractory gastro-intestinal agvhd post allo-hsct background: steroid-refractory acute graft-versus-host disease (sr-agvhd) is associated with an % mortality rate and reduced quality of life (qol). so far, there is no approved standard of care for agvhd second-line treatment. there is an urgent need to identify effective therapy for sr-agvhd to improve patients' outcomes. fecal microbiota transfer (fmt) might be beneficial to substantially improve the prognosis. higher gut microbial diversity is strongly associated with increased survival in gvhd patients. recent studies reported promising results of sr-agvhd patients treated with fmt. further evaluation to confirm the efficacy and safety of fmt for agvhd is warranted. the ongoing phase study (heracles) investigates the efficacy of allogeneic fmt in the treatment of patients with sr-agvhd. heracles was launched after the odyssee study showed promising results in the reconstruction of gut microbiota diversity after induction chemotherapy with fmt in acute myeloid leukemia patients. we expect that fmt-based biotherapeutic drugs could be effective treatments to contain sr-agvhd, and thereby reduce the risk of life-threatening complications after allogeneic hsct. methods: heracles is a single-arm, multicenter prospective trial in european countries. patients aged ≥ years-old, who underwent allogeneic hematopoietic stem cell transplantation (allo-hsct) and developed a first episode of stage or agvhd with gut predominance resistant to a first-line steroid therapy are eligible for inclusion. main exclusion criteria comprise the use of other second-line gvhd therapy, patients with grade iv hyperacute gvhd, late onset agvhd, and overlap chronic gvhd and agvhd after donor lymphocyte infusion. patients receive a first maat enema within days after sr diagnosis (v ) and additional ones week apart (v / v ) from each other. maat is a highly-diverse, microbiome-rich enema formulation obtained from pooled, rigorously screened faeces from healthy donors, manufactured with a standardized process using the signature maat microbiome restoration biotherapeutic (mmrb) platform. at inclusion (v ), before each dosing (v , ), and days post inclusion (v ), patients' faeces and blood are collected. safety monitoring will be performed with corresponding blood analyses. exploratory measures on faeces include characterization of gut microbiota composition and evolution, impact of maat on metabolism, and gut inflammation. immune system phenotyping will be performed by flow cytometry on peripheral blood mononuclear cells, and by elisa assay on plasma. patients' qol will be assessed using a standard, eq- d- l questionnaire. the primary objective is to assess the efficacy of maat by evaluating complete response (cr, according to modified glucksberg criteria) and very good partial response (vgpr, defined by martin et al., bbmt, ) days post-inclusion (primary follow-up). secondary objectives include fmt safety assessment and evaluation of fmt impact on several endpoints, such as overall, relapsefree or gvhd-free survival and chronic gvhd evaluation, as well as multi-drug resistant bacteria carriage. patients will be followed-up until year after inclusion. overall, patients are planned to be enrolled and treated, to assess overall response rates and maat 's safety profile. results background: anti-programmed cell death protein (pd ) monoclonal antibodies can be used as "bridge to" a subsequent allogeneic hematopoietic stem cell transplantation (hsct) in patients with relapsed/refractory hodgkin´s lymphoma (hl). this strategy has been reported to be effective, but a frequent onset of steroid-refractory graft versus host disease (gvhd) was also reported. we report clinical cases of patients affected by hl undergoing allogeneic hsct after having been treated with nivolumab. methods: the patients of , and years respectively had advanced hl and had relapsed after a previous autologous ( ) or allogeneic ( ) hsct. they underwent a rescue therapy with , , nivolumab cycles respectively, depending on the time of partial response achievement and the availability of a donor. two patients received a thiotepa-fludarabinecyclophosphamide conditioning, atg-based prophylaxis and pb cells from unrelated donors. the third patient received bm cells from an haploidentical donor using the "baltimora" nonmieloablative platform. results: at a follow-up of , , months after hsct, respectively, all patients achieved and maintained a complete remission by pet-ct scans. all the patients developed acute gvhd on day + , + and + , respectively. patient progressed to grade iv acute gvhd with hepatic and intestinal involvement unresponsive to first line mg/kg steroid therapy and second line etanercept plus extracorporeal photopheresis (ecp). third line therapy with ruxolitinib partially controlled the gvhd. gvhd onset in patients and was preceeded by a prolonged fever without microbiological findings. patient developed hepatic grade ii gvhd with high transaminase levels, initially responsive to steroid therapy, then it progressed to gut requiring second line therapy with etanercept. patient progressed to severe chronic gvhd with skin involvement and resulted unresponsive to steroids and ecp and it was partially controlled by ruxolitinib. immune reconstitution was delayed in all patients: at months post transplantation cd levels were /μl, / μl and /μl and cd levels were /μl, /μl and / μl respectively. only patient , that underwent haploidentical transplant and received post-trasplant cyclophosphamide (pt-cy), is off of immunosuppressive treatment at months after hsct, without evidence of gvhd and no history of infections. out of the patients receiving pbsc from unrelated donors and atg prophylaxis, patient developed a disseminated fusariosis on day + and died of cns fusarium localization year after hsct, despite targeted antifungal therapy. patient had pulmonary aspergillosis, sepsis by multidrug resistant psuedomonas aeruginosa and otomastoiditis: at + months after hsct, he is on ruxolitinib treatment with skin clinical partial response. conclusions: this case series confirms that nivolumab as "bridge to transplant" is effective in appropriately selected patients. however, risk of acute gvhd and delayed immune reconstitution may require a careful consideration at the moment of planning the transplant. a possible advantage of pt-cy gvhd platform and haploidentical donors should be addressed in larger studies. background: acute graft-versus-host disease (agvhd) is the most important complication after an allogeneic hematopoietic stem cell transplant (hsct). no standard secondline treatment has been established for the corticosteroid refractory agvhd. the anti-tnfα agents are a good option of treatment for these patients, especially when lower gi tract is involved. methods: from april to july we reviewed the outcome of patients with steroid-refractory (sr) agvhd treated with etanercept as at least, second line treatment. etanercept dose was mg twice a week for the first weeks, followed by weekly doses. results: median age was years (range - years), and patients ( %) were male. fourteen patients ( %) had a non-advanced disease status at hstc. eleven patients ( %) received a myeloablative conditioning, and the stem cell source was peripheral blood in patients ( %). sixteen patients ( %) were / hla matched. the characteristics of the patients, their agvhd stage previous to rescue treatment with etanercept and their outcome are shown in table . seventeen patients ( %) had a classic agvhd while had a late-onset agvhd. etanercept was given as a nd , rd and th line in ( %), ( %) and ( %) patients respectively. the median doses of etanercept administered were (range - ), and just patients ( %) completed the doses planned treatment, of whom were alive at , and months from the onset of rescue treatment. complications during etanercept treatment were: infection (n= [ %]: gram negative bacilli [n= ]), grade - neutropenia (n= ) and grade - thrombocytopenia (n= ). etanercept was indicated as a rescue treatment due to: progression after days of agvhd treatment (n= ), no response after days of treatment (n= ), no complete remission after days of treatment (n= ) and relapse due to decrease corticosteroid doses (n= ). at the end of treatment patient achieved a complete response and patients a partial response, all of them are alive. these patients received etanercept as a nd (n= ) and th line (n= ) treatment, all of them had lower gi agvhd without any other organ significantly involved. causes of death were: agvhd with or without infection in patients ( %) and leukemia relapse in patient. conclusions: although if etanercept is an option for treatment of sr agvhd in some patients, their prognostic remains poor and more effective alternative strategies are needed. a prompt initiation of etanercept as a rescue treatment for sr agvhd is crucial to improve the prognosis. ( ) ( ) background: although both cyclosporine (csa) and tacrolimus are calcineurin inhibitors, csa is more widely used in pediatric hematopoetic stem cell transplantation (hsct) as a prophylactic drug for acute graft versus host disease (agvhd). there are some clinical experience but very few data about the clinical efficacy of conversion to tacrolimus. here, we present our single center data on this arguable topic. methods: this study involves the data of pediatric hsct patients in medical park göztepe hospital between - . all patients had prophylactic csa therapy and for various reasons csa was converted to tacrolimus therapy. most of the patients had this conversion due to agvhd. as steroid is the first line therapy for agvhd, conversion to tacrolimus is done concurrently at the start of steroid therapy (within hours after the start of steroid). and also, patients who had any other immunosupressive therapy for agvhd are excluded. response is defined as resolution of symptoms within days after conversion. results: mean age of the study population is months ( - months), male/female ratio is , ( / ), donor types are mud patients ( %), mfd patients ( %), haplo patients ( %) and mean conversion time is days ( - days) . the rationales for conversion are agvhd for patients, unproper csa plasma levels for patients, allergic reaction for patients, nephrotoxicity for patients, hepatotoxicity for patients, severe headache for patients, high arterial blood pressure for patients and one each for refractory vomiting, autoimmune thyroiditis and visual disturbance. the subgroup analysis of agvhd patients reveals that mean conversion time for agvhd is days ( - days) and there are only responders whose agvhd resolve completely (% ) after conversion. all of the patients had proper tacrolimus levels after conversion due to unproper csa levels and also patients in allegic reaction, severe headache, visiual disturbance and refractory vomiting group responded to conversion completely but only one of the patients in nephrotoxicity group responded and also of the patients in hepatoxicity group responded. the only one patient suffered from autoimmune thyroiditis did not respond to conversion. conclusions: in this study, it is obvious that there are response to conversion for some specific adverse effects of csa and tacrolimus is a good alternative for the patients who have unproper csa levels. conversely, the high percentage (% ) of non-responders shows that it is not feasible to make a conversion to tacrolimus for acute gvhd. disclosure: nothing to declare background: capillary leak syndrome is caused by the dysfunction of the vascular endothelial cells,and is characterized by weight gain,generalized edemas,unresponsive to diuretic treatment,and hypotension.it usually develops in the first days post hsct.and it is of great difficuty to distinguish from other complications which are occured post the allo-hsct. to diagnose this complication at the early stage,it is very difficulty. methods: a -year-old man was admitted to ningbo first hospital for its abnormal in the peripheral blood .he was diagnosed with aml-m by the classical morphology and immunophenotype.cytogenetic evaluation showed a normal , xy( ).the patient achieved cr with induction therapy including idarubicin, cytarabine and etoposide. after consolidation therapy,an allo-hsct from hla identical related dornor( -year-old male, donorrecipient matched by high resolution hla typing at hlaa, -b, -c, drb , and dqb , / matches) was performed.the recipient received conditioning with busulfan, mg/kg/day injection for days; cyclophosphamide, mg/kg/day injection for days; cytarabine, g/m /day injection for day; semustine, mg/m /day orally for day; donor peripheral blood stem cells (pbsc:mnc: . × /l, cd +: . × /l) were mobilized, pheresed and administered to the recipient. gvhd prophylaxis consisted of traditional cyclosporine, short-course methotrexate ( mg/ m at day + , mg/m at days + , + , and + ) and cyclosporin a injection mg/kg qer day was mot reduced untill the hematopoietic reconstitute sucessfully . on day + , complete donor chimerism was acheieved. the csa was gradually reduced and tapered.on day + ,the patients was manifested with increasing in the time and volume of the faeces, he was diagnosed with ii°gvhd (gut).the standarded does of immunosuppressive drug including methylprednisolone and cyclosporin a was administrated. the immunosuppressive drug was gradually reduced when the gvhd was controlled.on day+ ,the patient felt distress and the distress was not related to with the exercise,the temperature was normal,and he did not gain weight.there was no edema in the body.laboratory test including routine blood test,c-reactive protein,procalcitonin,blood gas analysis,cmvdna,ebvdna was normal. the ct scan shows that the pleural is filled up with water, and could not be enlarged promptly, there is pericardial effusion in the body.pulmonary function test shows that reduced function in ventilation and diffusion fuction.the laboratory test of the pleural effusion was normal,the blast cell was not detected in the pleural effusion,the cd + cell count was below the dectable level,the next generation sequencing for minimal residual disease shows that there was no gene mutation .thus, post capillary leak syndrome was considered .sirolimus was adopted and taken the place of cyclosporin a,immunoglobulin was adminstrated to reduce the edema. results: taking together comprehensively,the effusion in the pleural and cardiac was absorbed well. conclusions: occurance of capillary leak syndrome is rare,there is limited data about capillary leak syndrome. comprehensively,the mechanism of cls has not been totally identified.and there is no standard treatment to treat the complication.at present,the cls of this patient was absorbed well by administrating sirolimus,closely followup is needed. disclosure: nothing to declare graft-versus-host diseasepreclinical and animal models p short-term krp and posttransplant cyclophosphamide for graft-versus-host disease prophylaxis emi yokoyama , daigo hashimoto , takahide ara , eko hayase , takanori teshima hokkaido university faculty of medicine, hematology, sapporo, japan background: post-transplant high-dose cyclophosphamide (ptcy) in combination with other immunosuppressants such as calcineurin-inhibitors (cis) has been increasingly used as gvhd prophylaxis after hla-haploidentical or matched hematopoietic stem cell transplantation (hsct). however,cis could hamper reconstitution of regulatory t cells (tregs) and tolerance induction after hsct, facilitating us to develop novel ci-free/ptcy-based gvhd prophylaxis. in the current study, we developed a novel gvhd prophylaxis in which ptcy was combined with short-term administration of krp , a selective agonist of sphingosine- -phosphate receptor type (s pr ), using murine models of mhc haploidentical bone marrow transplantation (bmt). methods: b d f (h- b/d ) recipients were lethally irradiated and transplanted with bone marrow cells and splenocytes from allogeneic b (h- b ) donors. cy at a dose of mg/kg was intraperitoneally injected into the recipients on day + , and krp at a dose of . mg/kg was orally administrated daily from day to day + after bmt. donor t cells in the target organs and secondary lymphoid organs were evaluated by flow cytometric analysis. plasma levels of tnf-α were determined using cytometric beads array. to evaluate graft-versus-leukemia (gvl) effects, recipient mice were intravenously injected with luciferase-transduced p cells (p -luc) on day , and in vivo bioluminescence imagingwas conducted weekly after bmt. results: severe gvhd was developed in allogeneic recipients and all mice died by day after bmt.ptcy alone at a dose of mg/kg significantly ameliorated gvhd and % of ptcy-treated allogeneic recipients survived. oral administration of krp alone enhanced contraction of donor t cells in the lymph nodes and also ameliorated gvhd as has been previously shown with multi-s pr agonist, fingolimod. next, we tested if shortterm krp on days to + added to ptcy enhances anti-gvhd effects of ptcy. we found that survivals of ptcy+krp group were significantly prolonged compared to those of ptcy-alone group ( figure a) . plasma levels of tnf-a, clinical gvhd scores ( figure b) , and donor t-cell infiltration into the target organs such as the gut and skin were also significantly reduced in ptcy +krp group compared to ptcy-alone group (figure c and d) . unlike cis, addition of krp to ptcy promoted treg reconstitution after bmt. finally, bioluminescence imaging demonstrated that proliferation of p -luc injected on day was significantly delayed in ptcy +krp -treated allogeneic recipients compared to control mice transplanted only with t-cell depleted bone marrow cells, suggesting that significant gvl effects persisted in ptcy+krp -treated recipients. conclusions: a combination of short-term krp and ptcy is a promising novel calcineurin-free gvhd prophylaxis in mhc-haploidentical sct. we recently showed that donor inkt cells can be expanded ex vivo and that they are able to prevent activation and proliferation of alloreactive donor t cells while promoting efficient graft-versus-leukemia effects (schmid et al. ). however, the underlying mechanisms how human inkt cells induce immune tolerance after allogeneic hct are not fully understood. methods: monocyte-derived dendritic cells (dcs) were cultured in a mixed lymphocyte reaction with mhcmismatched t cells and culture-expanded inkt cells. tcell activation and proliferation was analyzed by multiparametric flow cytometry and released cytokines were measured via multiplex analysis. transwell assays and imaging flow cytometry were performed to elucidate cellcell interactions. bead-controlled flow cytometry-based cytotoxicity assays were used to evaluate dc apoptosis. apoptotic dcs were then purified by fluorescence-activated cell sorting to investigate their tolerogenic potential to prime regulatory t cells (tregs). results: the addition of inkt cells to mixed lymphocyte reactions resulted in a significantly reduced activation and proliferation of mhc-mismatched t cells. transwell assays and imaging flow cytometry revealed a cell contactdependent mechanism between inkt cells and dcs leading to apoptosis with increasing dna fragmentation of dcs over time. interestingly, various fluorescence-activated single cell sorted inkt-cell subsets were all able to induce apoptosis of host dcs. multiplex analysis revealed that dcs triggered inkt-cell release of cytotoxic factors like perforin, granzyme b and granulysin. blocking the inktcell receptor engagement with a cd d antibody prevented inkt-cell degranulation as well as the subsequent induction of host dc apoptosis. inhibition of cytotoxic factors also abrogated apoptosis of dcs. in turn, sorted apoptotic dcs induced tolerogenic dcs characterized by a high expression of pd-l in mixed lymphocyte reactions. such tolerogenic dcs promoted the expansion of cd + cd + foxp + tregs and prevented activation and proliferation of mhcmismatched t cells. conclusions: we propose a novel mechanism how culture-expanded human inkt cells prevent gvhd after allogeneic hct. host dc apoptosis through donor inkt cells induces a tolerogenic immunoenvironment characterized by pd-l high dcs and expanding donor tregs inhibiting activation and expansion of alloreactive donor t cells. our findings pave the avenue for clinical translation of adoptively transferred culture-expanded inkt cells in humans. disclosure: nothing to declare results: vip-ko mice transplanted with allogeneic tcd bm alone had increased graft rejection with lower levels of donor chimerism and % day survival compared with % survival of wt recipients. transplanting tcd bm plus × e donor t cells from b .br or balb/c donors in vip kio mice led to > % donor chimerism and significantly increased gvhd-mortality compared with wt recipients, with % vs % survival in the b .br-->b model (p< . ), and % vs % survival in the balb/c-->b model (p< . ). donor-derived t cells in vip-ko recipients had significantly higher th and th polarization, with higher rorγt in both cd + (p< . ) and cd + (p< . ) t cells, and higher frequencies of ifn-γ (p< . ), tnf-α (p< . ), and il (p< . ) in cd + and cd + t cells compared to wt recipients. b .br-->b second allogeneic transplantation of radiation chimeras caused lethal gvhd mortality in vip-ko-->vip-ko and wt-->vip-ko mice, but not in wt-->wt or vip-ko-->wt b mice, demonstrating the protective effect of vip was due to synthesis by non-hematopoietic recipient cells. immunofluorescent imaging of allo-bmt recipients showed marked up-regulation of vip in lungs post-transplant and high vip production within neurons innervating the lungs. finally, we demonstrated that short-term administration of vip ( mcg/day) from day to day prevented gvhdmortality in vip-ko recipients transplanted with b .br-->b mhc donor bm & t cells. conclusions: the absence of vip in recipient cells led to increased graft rejection in the absence of donor t cells and increased lethal gvhd when donor t cells were transplanted, indicating vip induced post-transplant regulates allo-reactivity of host graft-rejecting lymphocytes and donor gvhd-causing t cells. the protective effect of parenteral vip administration suggests vip-mimetics represent a novel approach to prevent and treat gvhd. these data also suggest a mechanism of action for the mitigation of gvhd by alpha- anti-trypsin (aat) whereby aat inhibits the proteolytic inactivation of endogenous vip. disclosure: dr. waller reports personal fees and other support from cambium medical technologies, grants from celldex, personal fees from kalytera, grants and personal fees from novartis, grants and non-financial support from pharmacyclics, and equity ownership in cerus corporation and chimerix outside the submitted work. in addition, dr. waller has intellectual property related to vip signaling that has been licensed to cambium oncology in which he holds equity. low-density neutrophils expansion is associated with acute graft versus host disease in allogeneic hematopoietic stem cell transplant patients background: low-density neutrophils (ldns) are distinguished from normal-density neutrophils (ndns) by their anomalous sedimentation within the mononuclear cell fraction after density gradient centrifugation of peripheral blood (pb). by analysing ldns and ndns from g-csfstimulated donors or lymphoma patients, we have previously demonstrated that, depending on physiopathological conditions, immature cd b + cd -ldns can promote t cell survival and ifn-γ production, while mature cd b + cd + ldns can exert immunosuppressive proprieties. aim of this study was to establish the frequency of cd b + cd and/or cd b + cd + ldns in pb of allogeneic hematopoietic stem cell transplant (hsct) patients throughout immune reconstitution, and verify their potential correlation with acute graft versus host disease (agvhd). methods: patients undergoing hsct in our institution between december and june were prospectively enrolled in the study upon informed consent and after institutional board approval. criteria of inclusion were age ≥ years and absence of rheumatologic or viral diseases. pb samples were collected at day + , + , + , + and + after hsct and any time within day + in case of gvhd, before first-line therapy. eight healthy donors (hds) were enrolled as control. mononuclear, polymorphonuclear, and whole blood cells were analysed by flow cytometry after cd vioblue, cd apc-cy , cd b pe-cy , cd pe, cd b fitc staining. cd b + ldns were expressed as percentage of cd + pb mononuclear cells (pbmcs) or cd + whole blood cells and were further characterized based on cd expression. cd b + ndns, expressed as percentage of cd + whole blood cells, were also analysed for cd staining. results: patients (m/f / , median age ) were enrolled in the study. patients received hsct from hlaidentical ( ) or haploidentical ( ) related and from hlaidentical unrelated ( ) donors. after a median time of ( - ) days, patients developed grade ii-iv agvhd. no patients were receiving g-csf at agvhd onset. the scheduled assessments were interrupted in agvhd patients at the beginning of first-line treatment and in patients relapsed of their primary malignancy. no patients developed de novo late-acute or chronic gvhd. starting from day + the frequency of ldns within cd + pbmcs was higher in all patients as compared to hds. the patients that did not develop agvhd showed a decreasing frequency of cd b + cd -ldns, with a progressive increase of cd b + cd + ldns, from day + to + . interestingly, patients with agvhd showed a significantly higher frequency of cd b + cd -ldns as compared to patients without agvhd throughout the same time lapse (i.e. from day + to + ) ( . vs . , p= . ). consistently, patients with agvhd had a significantly lower frequency of cd b + cd + ldns ( . vs . , p= . ). the frequency of mature cd b + cd + ndns was normal in all patients since day + . conclusions: ldns are more represented in hsct patients than in hds, with a significant expansion of the cd b + cd subpopulation (with a parallel decrease of the cd b + cd + subpopulation) in patients with agvhd as compared to those without agvhd. according to the previously demonstrated t cell activating function of cd b + cd -ldns, it is tempting to speculate that the expansion of this subpopulation may contribute to agvhd development. disclosure: nothing to declare background: acute graft-versus-host disease (agvhd) is a major complication after allogeneic hematopoietic stem cell transplantation (allo-hsct) which has negative impact on the morbidity and mortality of the patients. accumulating evidences suggest that abnormalities of foxp + regulatory t (treg) cells contributed to the pathogenesis of gvhd, but the underlying molecular mechanisms still remain largely unknown. methods: in this study, we enrolled all the patients treated with allogeneic hsct at the institute of hematology, chinese academy of medical sciences between and ,as well as age-matched healthy adults as control samples. the ratio of tregs in pb and bm of healthy controls (hcs) and patients with and without agvhd was determined by flow cytometry. the transcription profile between tregs from patients with or without acute gvhd was measured,the pathway enrichment analyses were performed by the kyoto encyclopedia of genes and genomes (kegg) pathway database and geneset enrichment analysis (gsea).the expression of lkb at transcript levels and protein levels was measured by realtime pcr and analyzed by the nanopro tm system. a series of functional assays in vitro were performed to assess the function and stability of tregs from patients with and without agvhd.meanwhile, to assume the affect of lkb on gvhd outcome, we established a murine transplant model,which recipient balb/c animals were transplanted with the same amount of mixture made by bm, cd +cd -tcon cells from c bl/ and cd + foxp yfp+ tregs from either foxp crelkb f/f or foxp cre mice. results: in this study, we demonstrated that bm had decreased frequencies of tregs, accompanied with a reversed lower ratio of tregs frequencies between bm and pb in agvhd patients. meanwhile, the number and function of tregs in bone marrow also affected hematopoietic reconstitution. futhermore,to elucidate these mechanisms which regulate tregs homeostasis, we examined the role of lkb on tregs in patients with agvhd and in agvhd murine model. studies demonstrated that lkb deficient tregs lost foxp expression and weaken suppressor function during agvhd. transcriptional profiling and pathway analysis revealed that nf-kb signaling activation and the impairment of a wide spectrum of immunosuppressive genes in agvhd tregs. further mice experiments suggested that cns methylation might lead to the instability of tregs in agvhd group. transplantation with marrow grafts from foxp crelkb f/fmice exacerbates gvhd lethality. conclusions: these studies indicate that lkb is a critical homeostatic regulator for tregs during agvhd. targeting of lkb therefore represents a novel therapeutic strategy that promote immune tolerance to mitigates the severity of agvhd. disclosure: national program on key basic research project ( program) role of aryl hydrocarbon receptor in intestine after allogeneic hsct in mice won-sik lee , soung-min lee , sj-kil seo inje university, busan paik hospital, hemato-oncology, busan, korea, republic of background: aryl hydrocarbon receptor (ahr) is a ligandactivated transcription factor that is activated by various small molecules from the diet, microorganisms, host metabolism, and xenobiotic toxic chemicals. the function of ahr has been demonstrated as a crucial regulator in intestinal homeostasis. here, we investigated the regulatory role of ahr in intestine of recipients after allogeneic hematopoietic cell transplantation in mice. methods: wild-type (wt) b (h- b ), ido -/-(h- b ) and ahr -/-(h- b ) mice were lethally irradiated and transplanted with x tcd-bm plus x t cells from balb/c donor mice. ahr activation in colon tissue of recipients was determined by the ahr target genes cyp a and cyp b expression using real-time pcr. the recipient mice were monitored every other day for survival and clinical score. histopathology and pathogenic effector cytokine levels in colon tissue were analyzed for evaluating ahr function. results: we observed that cyp a was constitutively expressed in the colon tissue of naïve recipient mice. although the expression levels were increased by tbi conditioning, the additive up-regulation of its levels with donor t cell alloreactivity was not observed. in contrast, cyp b expression was markedly induced in the colon tissue by donor t cell alloreactivity. we further observed that the cyp b expression was significantly decreased in the colon of ido-/-recipients with donor t cell alloreactivity, but cyp a was not changed. ido-/-and ahr-/recipient mice showed higher histopathological score for intestinal gvhd and increasing pathogenic cytokine levels in the colon compared with wt mice. conclusions: our results demonstrate that ahr-induced target gene profiles might be differently induced in intestine by ligand dependent manner after hsct, which affect intestinal gvhd. disclosure: nothing to declare. abstract already published. abstract withdrawn. in vitro platelet activation evaluation in allogeneic hematopoetic stem cell transplanted patients in response to haemostatic stimulation and cytomegalovirus stimulation (gvhd), complication of which one of the risk factor is cmv reactivation. the resultant inflammatory platelet response during the high-risk period of gvhd after allogeneic hsct remains unknown. our study aimed to characterize spontaneous platelet activation during the d and d months after allogeneic hsct, and in response to haemostatic stimulation and cmv stimulation. methods: we compared a group of healthy volunteers to a group of allogeneic hsct patients followed between the th and the th days after hsct. platelet activation was determined by the platelet surface expression of cd p and cd using flow cytometer after stimulation by an haemostatic agent, thrombin-receptor activating peptid (trap) and after stimulation by cmv glycoprotein b. the inflammatory response was determined by the detection of immune mediators, rantes, cd ps, pf , cd l and ccl , using the elisa technique in the stimulated platelet supernatants. results: no platelet activation or molecules release were observed after stimulation by cmv glycoprotein b in both groups. rantes and cd ps baseline levels are spontaneously higher in allogeneic hsc patients than in healthy volunteers. platelets from allogeneic hsct patients can be activated after haemostatic stimulation and release cd ps and rantes. in this situation, platelets release more cd ps, rantes and pf than platelets from healthy volunteers. conclusions: although no platelet activation was detected in response to cmv glycoprotein b stimulation, our study revealed a chronic platelet activation condition during the d and d months after allogeneic hsct with an haemostatic inducible hyper-responsiveness. this leads to the release of molecules with immune-modulating properties involved in the pathophysiology of gvhd. as we move further away from the hsct, that phenomenon seems to gradually weaken. clinical trial registry: nct , fipalloc https://clinicaltrials.gov/ct /show/nct disclosure: nothing to declare p efficient process and characteristics of umbilical cordderived mesenchymal stromal cells as a feasible source for anti-inflammatory therapy background: recently, umbilical cord (uc) has become attracted source of mesenchymal stromal cells (msc), because of abundant sources and ease of collection of fetal origin without invasive process for the donor and low immunogenicity with immunosuppressive ability and tissue repair potency. objectives of this study were to explorer the efficient and safe products and to evaluate the antiinflammatory potency of uc-mscs for the application of acute graft versus host disease (gvhd). methods: informed consent was obtained from mothers planning to have cesarean sections. uc tissue was cut and once cryopreserved. the safety assessment including infections and baby's health and development were done after months of birth, and performed small-scale quality test of the frozen uc. then we initiated to isolate master uc-mscs from frozen-thawed uc by an improved explant method, which was passed for quality test. the master uc-mscs were cryopreserved once and thawed and expanded until p . product cells were cryopreserved in original serum-free cryoprotectant dba-d solution. mixed lymphocyte reaction (mlr) assay co-cultured with uc-mscs was carried out using responder mononuclear cells (mnc) stained with cfse, and proliferation and cytokine secretion were analyzed by flowcytometry. results: uc-msc cultured showed significantly higher proliferation ability compared with those from bone marrow-derived mscs, and positive for cd , cd , cd , and negative for cd , hla-dr. cd , and cd were negative even in the high concentration of ifn-γ, while bm-mscs became positive for hla-dr. pd-l was constitutively expressed in uc-msc, while pd-l was induced by the addition of ifn-γ. in mlr, responder t cell proliferation triggered by allogeneic dendritic cells was inhibited efficiently by rd party derived uc-mscs, in which was induced ido, pge , hgf, and tgf-β analyzed by rt-pcr, and inhibited ifn-γ and tnf-α in the supernatant by cytokine beads array. uc-mscs migrated toward the tnf-α treated mnc and increased regulatory t cells incidence in peripheral mononuclear cells by the coculture. conclusions: these results demonstrated that cryopreserved uc are feasible and efficient source of mscs and frozen-thawed uc-mscs have high anti-inflammatory background: a new protocol is under development on the amicus separator that enables the device to perform ecp procedures. the amicus separator is used with a photoactivation device, disposable kit and -mop to provide ecp therapy in a closed system. the objective of this study was to evaluate the safety and performance of the investigational amicus ecp system in healthy human subjects. methods: an irb-approved written informed consent was obtained from subjects ( male, female). the amicus ecp system processed either , or ml whole blood (n ≥ per arm) using double-needle access and acd-a anticoagulation at a : wb:ac ratio. after mnc collection was completed, the subject was disconnected from the device. -mop ( . ml, μg/ml) was injected directly into the collected mnc product and saline (approximately ml total), which was photoactivated with - j/cm uva light. post photoactivation, the amicus separator reinfused the treated mncs into a transfer pack. subject laboratory and safety parameters were evaluated; in vitro evaluations were performed on subject whole blood, collected mncs, treated mncs, and reinfused cells. lymphocyte and monocyte analysis were performed on samples purified using density gradient separation and cultured for up to days post treatment. results: in procedures, median (range) wb processed was . ( - ) ml using . ( - ) ml of acd-a. procedure time was . ( - ) minutes, including photoactivation. no adverse events were reported. subjects' vital signs and hematology values were unremarkable and within expected values. the wbc count of the collected mncs was . ( . - . ) x /μl, comprised of . ( . - . ) % lymphocytes, . ( . - . ) % monocytes and . ( . - . ) % granulocytes and platelet count was . ( - ) background: transfusion of white blood cells (wbc) causes a number of transfusion reactions and complications, for example transfusion-associated graft versus host disease (tagvht), which still does not have effective treatment and is a fatal complication of transfusions. the only effective method of preventing tagvht is irradiation of blood components with ionizing radiation (x-ray or gamma radiation). but the use of ionizing radiation sources has a number of technical and material difficulties. the emergence of pathogen reduction technologies (prt) in blood components targeted by nucleic acids has opened the possibility of using these technologies as an alternative to irradiating of blood components. several prt demonstrated effective inactivation of wbc in platelet concentrates and blood plasma. so, determination of the influence of prt based on the combined effect of riboflavin (rf) and ultraviolet (uv) on the viability and proliferating potential of lymphocytes in whole blood is important. methods: samples of whole blood were obtained in healthy volunteers. each sample was divided into three unequal parts: untreated control, gamma irradiated, and treated by rf and uv prt (mirasol, terumo bct inc.). mononuclear cells (mnc) were cfse stained, viability and proliferating activity were tested at intervals of hours for consecutive days by flow cytometry. statistical analysis was performed with xlstat . . levels of significance were calculated by mann-whitney test, expressed as p-values (p< , ). results: the median viability of mnc after application of both methods of treatment was over , % on day and decreased to day -median percentage of viable mnc were , % (control group), , % (after gamma irradiation) and , % (rf/uv prt). the median of spontaneous proliferative activity on day of untreated and gamma irradiated mnc did not differ ( , % and , % respectively, p< , ). phytohemaglutenin (pha) induced proliferation on day in gamma-irradiated samples was significantly lower in comparison with control group ( , % and , % respectively, p< , ). in samples treated with rf/uv, spontaneous and stimulated proliferating cells was not detected. median percentage of proliferating mnc was less than , %. the use of this prt on whole blood, as well as gamma irradiation, significantly reduces the viability of lymphocytes during storage for days. conclusions: inactivation of wbc using rf/uv prt is a useful and very necessary bonus for a number of reasons. in one procedure two effects are achieved: infectious and immunological safety. the use of prt on whole blood gives the potential for obtaining pathogen-reduced and immunological safety components of blood, which reduces their material cost and staff loading. the use of rf/uv system does not have such complex security requirements and difficulties in servicing as the use of sources of ionizing radiation. the results demonstrate a promising potential for using this technology as an alternative to irradiation disclosure: nothing to declare p influence of patients´serum after allogeneic stem cell transplantation on t cell proliferation and treg function background: acute or chronic graft versus host disease (a/ cgvhd) is one of the major complications after allogeneic hematopoietic stem cell transplantation (ahsct). application of regulatory t cells (treg) as "immunosuppressive dli" to prevent or treat gvhd is investigated in clinical trials. here we ask the question, if there could be clinical conditions (e.g. cytokines or drug effects) limiting the efficacy of this approach. to face this problem we tested the influence of patients´serum on t cell proliferation and treg function. methods: lymphocytes from healthy donors were incubated with t cell medium ( % aim v + % serum + il /okt ) containing serum from healthy donors or serum derived from patients after ahsct with or without gvhd (n= ). next we evaluated the suppressive function of treg by performing treg suppression assays, also comparing serum from patients suffering from gvhd versus serum obtained from healthy donors (n= ). proliferation of cfse stained t cells was measured after days. to test the effect of immunosuppressive drugs on treg we performed treg suppression assays after incubation of treg with corticosteroids or tacrolimus or the combination of both drugs. results: serum of patients with acute or chronic gvhd had a negative effect on t cell proliferation. to avoid bias tests were performed with samples from patients without or only with low levels of immunosuppressive drugs. incubation with serum of patients without gvhd or with serum of healthy individuals showed no differences in t cell proliferation. treg from healthy donors showed a stronger antiproliferative capacity when incubated with serum derived from patients with gvhd. treg previously incubated with immunosuppressive drugs showed no decreased suppressive capacity. conclusions: components of serum from gvhd patients seem to have an antiproliferative effect on t lymphocytes itself. this fact might influence the clinical course of gvhd, but should not be a limiting factor for therapeutic application of treg dli. even the systemic treatment with immunosuppressive drugs e.g. corticosteroids or calcineurin-inhibitors should not diminish the treg application. in a next step we will analyze serum components responsible for this immunosuppressive effect with multi cytokine assays and proteomic analysis. the aim of our project is to develop new strategies to avoid gvhd and to optimize clinical settings for treg dli. disclosure background: hypercalcaemia can be very severe following stem cell transplant (sct) in some osteopetrosis patients. denosumab is a fully human monoclonal antibody that binds the cytokine rankl (receptor activator of nfκb ligand), an essential factor initiating bone turnover. rankl inhibition blocks osteoclast maturation, function and survival, thus reducing bone resorption. we describe the effective management of hypercalcaemia in a patient with rank mutation osteopetrosis who received a haploidentical sct. methods: our patient was diagnosed with osteopetrosis at year of age with a defect in the tnfrsf a gene which codes for rank and received a maternal haploidentical sct aged years. the patients calcium levels were monitored regularly post sct. denosumab was administered for hypercalcaemia as per laboratory reports or clinical symptoms. the drug was diluted with water for injection to make mg/ml solution to facilitate subcutaneous administration. results: significant hypercalcaemia emerged on day + with a level of mmol/l and treated with hyper-hydration and diuretics. this was ineffective in reducing the hypercalcaemia; therefore denosumab was initiated on day + post-transplant. initial dosing was determined using the only available paediatric case report at . mg/kg. a repeated larger dose of . mg/kg was given days later due to an inadequate response (calcium decreased from . mmol/l to . mmol/l). the calcium decreased to . mmol/l after this dose. four weeks later a third dose was required at . mg/kg as the calcium level had increased to . mmol/l. the dose was further increased to . mg/kg for another four doses and then further increased to . mg/kg for another doses and repeated every weeks. normalisation, but not excessive drop in calcium was achieved with these larger doses. over the month follow up post-transplant there were three admissions lasting less than hours for symptoms of hypercalcaemia. these were managed with denosumab administration and hyper-hydration. the remaining doses were given in an outpatient setting. conclusions: denosumab can be safely used as a first line agent in treating post stem cell transplant hypercalcemia in patients with osteopetrosis. a dose of . mg/kg is required as an initial starting dose in order to control hypercalcemia. this is a new higher dose than previously suggested by the original report. denosumab can be effective even after dilution and safely given in children weighing less than kg. disclosure: nothing to declare methods: the clinical, laboratory and molecular aspects of this italian male patient who developed such a complication were collected and presented in order to discuss the origin, clinical outcome and management of this very rare post-transplant event. results: a -years-old man affected by a high-risk chromosomally abnormal, ph -, mll-pro-b (egil b-i) all relapsed during maintenance treatment, nonresponsive to re-induction chemotherapy, in second complete remission (ii cr) after blinatumumab treatment received a female cb transplant. according to sorror's and ebmt scores he was considered a high-risk transplant. the patient and the cb unit were sex-mismatched, shared the same blood groups and were both cmv+/ebv+. he received a tbf conditioning regimen that was followed by the infusion of . x /kg cd + cb cells. gvhd prophylaxis consisted of rabbit atg, cyclosporine a (csa) and mycophenolate mophetyl (mmf). neutrophil engraftment occurred on day + , whereas platelets were never > . /μl. on day + a cm bulged area became apparent on the left parietal region of the skull. an echotomography showed that the lesion adhered to the bone without infiltrating it and lacked blood vessels and suggested that it may be either a site of disease relapse or an area of infection. at the same time a bone marrow (bm) aspiration showed morphological cr confirmed by immune-phenotypic studies and x-y fish a complete chimera. since the patient was still febrile no biopsy was performed, but on day + the axial diameter of the lesion that on a ct scan showed the same appearance revealed by the previous echo-tomography increased to cm. thus, the lesion was surgically removed and histological examination showed cd +, cd +/-, cd +/-, cd /lca+/-, cd -, cd -, cd -, cd -, and s -neoplastic cells whose phenotype suggested a granulocytic sarcoma rather than a histiocytic sarcoma. immuno-chemistry confirmed this suggestion by showing a nuclear npm positivity. fish studies demonstrated that these neoplastic cells were of recipient's origin. a novel bm aspiration showed cr confirmed by immune-phenotypic studies and fish revealed a complete chimera. since the patient was still pancytopenic due to anti-cmv treatment, radiotherapy with gy in nine fractions were given and the lesion completely resolved. conclusions: a granulocytic sarcoma of recipient's origin occurring three months after a cb transplant is a very rare and unusual event. in order to explain such a complication we suggest that granulocytic sarcoma cells were dormant but already present at the time of pro-b all diagnosis and survived not only the initial all treatment but also the cb transplant conditioning regimen. we can't exclude that immune-suppressive treatments given early post-transplant might have promoted the outgrowth of these neoplastic cell population. disclosure: nothing to declare haemoglobinopathy and inborn errors of metabolism p abstract already published. addition of fludarabine on to anti-thymocyte globulin, busulfan and cyclophosphamide conditioning improves outcomes in low-risk matched-related bone marrow transplantation in children with severe thalassaemia flu-atg-bucy. atg dose was mg/kg in all patients except patients with splenomegaly > cm from costal margin and/or sex-mismatched/maternal donor in whom atg was increased to mg/kg. all patients were younger than years and had no hepatomegaly (liver ≤ cm from costal margin) at bmt. results: actuarial overall survival (os) in the atg-bucy and flu-atg-bucy groups is % and %, thalassemia-free survival (tfs) % and %, gvhdfree and thalassaemia-free survival (gtfs) at a median follow up of . and . months was . % and . % months respectively, which is a significantly improved outcome by log-rank statistics (p= . ) in the flu-atg-bucy group. there was no significant difference between the groups in pre-transplant characteristics and posttransplant complications except for the following: median cell dose more in nd group with total nucleated cell dose of . vs . x cell/kg with p< . ; csa taper started later in the new protocol ( day vs. p= . ); median age at bmt ( . vs. . years, p= . ); number of pre-bmt transfusions (p= . ) and ferritin at bmt ( . vs. . ng/ml, p= . ) were higher in the second group; day and chimerisms were also significantly higher in new protocol (p= . and . respectively). there was a trend towards increased incidence of veno-occulsive disease (vod) and posterior reversible encephalopathy syndrome (pres) on the second group but this difference did not reach statistical significance. conclusions: adding fludarabine and targeted dose increase of atg in the standard bucy context seems to significantly improve outcomes of thalassaemia transplants without contributing to excessive gvhd or infectious complications. this protocol can be easily administered in low resource setting without major additional costs. clinical is an acquired clonal disorder of the hemopoietic stem cells for which the only curative treatment is allogeneic hematopoietic stem cell transplantation. however, there are still few reports on the outcomes of allogeneic hematopoietic stem cell transplantation (allo-hsct) in patients with pnh compared to paroxysmal nocturnal hemoglobinuria-aplastic anemia (pnh-aa) syndrome. our study aimed to compare the outcomes of allo-hsct for pnh with pnh-aa syndrome. methods: the clinical data of pnh patients received allo-hsct (pnh = , pnh-aa = ) in our center from july to june were analyzed retrospectively to compare the outcomes of pnh group with pnh-aa group. the clinical data including male patients and female patients, the median age was years (range - ). all patients had received various treatments before transplantation such as steroids, androgens, cyclosporine (csa), antithymocyte globulin, and growth factors. the median interval from pnh diagnosis to hsct was months (range - ). the conditioning regimen was modified bu/cybased regimen in haploidentical donors and unrelated donors, csa, mycophenolate mofetil (mmf) and shortterm methotrexate (mtx) were administered for graftversus host disease (gvhd) prophylaxis. patients with matched sibling donors were treated with the flu/cybased regimen and csa were administered for gvhd prophylaxis. results: there were no differences of baseline between the groups (p> . ) except gender and haploidentical donors. the median values of absolute nucleated cell counts were . ( . - . ) × /kg in the pnh group and . ( . - . ) × /kg in the pnh-aa group (p = . ). the median doses of cd + cells infused were . ( . - . )× /kg and . ( . - . )× /kg (p = . ), respectively. all patients attained complete engraftment, no patient occurred graft failure. the median time for myeloid engraftment were (range, - ) days in the pnh group and (range, - ) days in the pnh-aa group (p = . ). the median time for platelet engraftment were (range, - ) days and (range, - ) days (p = . ), respectively. with a median follow-up of ( - ) months in the pnh group and ( - ) months in the pnh-aa group (p = . ). in pnh and pnh-aa groups the incidences of grade i-iv acute graft-versus-host disease (agvhd) were . % and . % (p = . ), grade ii-iv agvhd were . % and . % (p = . ); chronic gvhd were . % and . % (p = . ), moderatesevere chronic gvhd were . % and . % (p = . ). in haplo-hsct and msd groups the incidences of infection were . % ( / ) and . % ( / ) (p = . ). no patient occurred early death and relapse. -year estimated overall survival (os) of pnh and pnh-aa groups were . % ± . % and . % ± . % (p = . ), gvhd-free and failure-free survival (gffs) were . % ± . %、 . % ± . % (p = . ). conclusions: the preliminary results indicated that allo-hsct is a feasible choice for pnh with favorable outcomes, time for myeloid and platelet engraftment in pnh group were faster than pnh-aa group. there were no differences in os and gffs between pnh group and pnh-aa group. disclosure: no disclosure pattern of calcineurin inhibitor-associated neurotoxicity in sickle cell disease patients receiving a stem cell transplantation background: allogeneic hsct with a msd represents currently the only curative option for sickle cell disease (scd), limited by a donor availability < %. neurotoxicity (nt) contributes significantly to hsct-associated morbidity and mortality. calcineurin-inhibitor (cni) associated nt ranges from . %- . % (severe nt %- %). the elevated incidence of nt in scd (around %) might be triggered by the systemic vasculopathy of scd, with the brain being the primary target. although both cyclosporine a (csa) and tacrolimus (fk ) have a proinflammatory effect, it is more pronounced in csa. infusion modalities also might impact ( . % after bolus injections versus . % after continuous infusion). methods: in a pilot study, we compared t-cell depleted haploidentical hsct (t-haplo hsct) with msd hsct in patients (pts) with advanced stage scd, using almost identical conditioning regimens. pts ( - years; yrs) with homozygous scd or hbs /+ ß-thal were treated between and . nine pts received a msd bone marrow graft, pts received t-haplo-hsct ( second t-haplo due to graft rejection). immunosuppression consisted of either csa ( msd, t-haplo) or fk ( msd, , in combination with mycophenolate mofetil (mmf). fk was administered as a -hours continuous infusion, csa as -hours bolus injections; both target level adjusted (csa: - ng/ml; fk : - ng/ ml). duration of immunosuppression was > months in thaplo-sct and < months in msd, depending on chimerism. results: cni-related nt was observed in . %, severe nt (pres, visual disturbance, aphasia) in . %. nt was more prevalent in msd (n= , . %) than in t-haplo (n= , . %). the incidence of nt was identical under csa ( / ; . %) and fk ( / ; . %), however the majority of severe nt (all pres) occurred with csa. complete recovery of nt was achieved in all pts either spontaneously or after switching to fk /everolimus or withdrawal of fk . moreover, . % of pts with nt were > yrs, and . % > yrs, suggesting an increased risk with age. only . % of pts with pre-existing cerebrovascular disease experienced post-hsct nt. of note, . % of pts with severe nt also developed mild acute gvhd. the overall (os) and disease-free survival (dfs) with a median follow-up of months in t-haplo-hsct and months in msd hsct was % vs. %, respectively. conclusions: our data confirm an elevated nt risk in scd pts following allo-hsct. importantly, the incidence of nt seems to be related to age ( % of pts with nt were > yrs), donor source (msd . % vs. t-haplo . %) and type of cni inhibitor where almost all severe nt ( . %, particularly all pres) was observed under csa. continuous infusion of fk vs. bolus injections of csa might have levelled concentration peaks. the nt observed with csa could be the consequence of predominantly csarelated vascular toxicity inflicting pre-damaged vessels in scd. the mechanism of action could be related to other systemic endotheliopathies such as vod, tam and agvhd, which was observed in . % of pts with severe nt, compared to an overall agvhd rate of %. disclosure: nothing to declare background: matched-related bone marrow transplantation (bmt) may cure over % of low-riskchildren with severe thalassemia (st) defined as a thalassemia syndrome with inability to keep a spontaneous hemoglobin > g/dl. it is well known that patient status at the time of transplant is critical in predicting transplant outcome. liver size > cm is an established adverse prognostic factor in terms of transplant-related mortality and, in our own experience,a spleen size > cm from costal marginis associated with increase rejection rates (blood vol. no. suppl ) . optimising liver and spleen size prior to transplant is likely to improve transplant outcomes. methods: we retrospectively reviewed the effectiveness of our strategy to reduce liver and spleen size pre-transplant using hydroxyurea, super-transfusion and intensive iron chelation. we considered liver size < cm and spleen size less than cm below costal margins as good risk features. liver biopsies were not performed thus pesaro risk classification could not be assigned. all transplant candidates were started on hydroxyurea for a minimum of months and pre-transfusion haemoglobin was maintained > gm/dl while on hydroxyurea. if the child had hepatospenomegaly at enrollmentand no improvement in liver and spleen size after an adequate trial of hydroxyurea (minimum of months of treatment achieving maximum dose of mg/kg day or tolerable haematological toxicity, i.e. neutrophil count between and /μl and/or platelet count between . and . /μl) patients were given a trial of supertransfusion maintaining haemoglobin above g/dl) for a minimum of months prior to declaring the patient as having failed downstaging. results: out of transplants across collaborating centers in india, patients had no hepatosplenomegaly at enrolment and hence were not actively downstaged. twelve patients were excluded due to inadequate information on their records. all of the remaining patients with enlarged liver and/or spleen were downstaged to low-risk features. all patients received adequate hydroxyurea trial among which seven ( %) patients required super transfusion in addition to maximal hydroxyurea. out of the patients ( %) were successfully down-staged with the above strategy and proceeded to transplant as low-risk patients. among the remaining ( %) patients had liver > cm and one had a spleen > cm only. there was significant improvement in liver and spleen size from the time of enrollment to transplant (p value . and . respectively by wilcoxon test for paired samples -two tailed) with median duration of downstaging of months (range - months). there was no significant difference in overall survival (os) and disease-free survival (dfs) by log rank test between the downstaged group and those who did not have hepatosplenomegaly at enrollment (p value . . respectively). conclusions: in the majority of children with thalassaemia and high transplant risk features liver and spleen size can be reduced pre-transplant using hydroxyurea and supertransfusions thereby decreasing transplant risk. disclosure: nothing to declare p abstract already published. abstract withdrawn. longitudinal analysis of the effect of hematopoietic cell transplantation on ocular disease in children with mucopolysaccharidosis i shows ongoing disease progression background: corneal clouding is seen in nearly all patients with mucopolysaccharidosis- (mps- ) causing visual impairment. hematopoietic cell transplantation (hct) is able to stabilize disease in many organs including the brain. however, residual disease in peripheral tissues is often described. therefore, the aim of this study was to determine the long-term effect of hct on ocular disease in mps- patients. methods: corneal clouding (grade - ) and visual acuity (decimal scale) were prospectively collected from all consecutive mps- patients treated with hct between and at the umc utrecht. the primary outcomes of interest, the effect of time on corneal clouding and visual acuity, were analyzed using a linear mixed model. the correlation between corneal clouding and visual acuity was analyzed with pearson's rho. other parameters studied were clinical phenotype, age at time of transplantation and hematological enzyme level after transplantation. other outcomes of interest analyzed included intra-ocular pressure, refraction, and macula and lens abnormalities. [[p image] . results: successfully engrafted mps- patients were included ( % with > % chimerism and normal enzyme levels after hct). corneal clouding stabilized during the first years after hct, but increased rapidly beyond three years (figure ). other predictors for increased corneal clouding were age at time of transplantation ( . , %ci . : . ; p= . ) and clinical phenotype (- . , %ci - . :- . ; p= . ). visual acuity also worsened significantly over time (- . , %ci - . :- . ; p= . ). corneal clouding was strongly negatively correlated with visual acuity (ρ - . , p = . e- ). conclusions: after initial stabilization, ongoing ocular disease is seen in mps- patients despite successful hct. this hallmarks the shortcomings of current standard therapies. new therapies that overcome the weak spots of current therapies are necessary to improve the late outcomes of these patients. clinical trial registry: n.a. disclosure: b.t.a.v.d.b. was supported by a research grant from the sylvia toth charity foundation, the hague, the netherlands, while working on this study. the sponsors of this study are public or nonprofit organizations that support science in general. they had no role in gathering, analyzing, or interpreting the data. all authors would like to thank all parents and patients for participating in this study. all authors state they have no competitive (financial) interests in this study. background: paroxysmal nocturnal hemoglobinuria (pnh) is an acquired clonal disorder of the hemopoietic stem cells for which the only curative treatment is allogeneic hematopoietic stem cell transplantation. haploidentical donor hematopoietic stem cell transplantation (haplo-hsct) is now increasingly applied as a curative therapy for patients with hematologic diseases. however, there are still few reports on the use of haplo-hsct for the treatment of pnh. our study aimed to compare the outcomes of haplo-hsct with matched-sibling donor transplantation (msd-hsct) for pnh. methods: the clinical data of pnh patients received hsct (haplo-hsct = , msd-hsct = ) in our center from july to may were analyzed retrospectively to compare the outcomes of haplo-hsct group with msd-hsct group. the clinical data including male patients and female patients, classical pnh and pnh-aa syndrome, the median age was years (range - ). all patients had received various treatments before transplantation such as steroids, androgens, cyclosporine (csa), antithymocyte globulin, and growth factors. the median interval from pnh diagnosis to sct was months (range - ). the conditioning regimen was modified bucybased regimen in haplo-hsct group, csa, mycophenolate mofetil (mmf) and short-term methotrexate (mtx) were administered for graft-versus host disease (gvhd) prophylaxis. patients with msd-hsct were treated with the flucy-based regimen and csa were administered for gvhd prophylaxis. results: there were no differences of gender, age, patients of pnh-aa and median time from diagnosis to transplantation between the groups (på . ). the median values of absolute nucleated cell counts were . ( . - . ) × /kg in the haplo-hsct group and . ( . - . ) × /kg in the msd-hsct group (p = . ). the median doses of cd + cells infused were . ( . - . ) × /kg and . ( . - . ) × /kg (p = . ), respectively. all patients attained complete engraftment, no patient occurred graft failure. the median time for myeloid engraftment were (range, - ) days in the haplo-hsct group and (range, - ) days in the msd-hsct group (p = . ). the median time for platelet engraftment were (range, - ) days and (range, - ) days (p = . ), respectively. with a median followup of ( - ) months in the haplo-hsct group and ( - ) months in the msd-hsct group (p = . ). in haplo-hsct and msd-hsct groups the incidences of grade i-iv acute graft-versus-host disease (agvhd) were . % and . % (p = . ), grade ii-iv agvhd were . %、 . % (p = . ). chronic gvhd were . % and . % (p = . ), moderate-severe chronic gvhd were . % and . % (p = . ). in haplo-hsct and msd groups the incidences of infection were . % ( / ) and . % ( / ) (p = . ). no patient occurred early death and relapse. -year estimated overall survival (os) of haplo-hsct and msd-hsct groups were . % ± . % and . % ± . % (p = . ), gvhd-free and failure-free survival (gffs) were . % ± . % and . % ± . % (p = . ). conclusions: the preliminary results indicated that haplo-hsct is a feasible choice for pnh with favorable outcomes, haplo-hsct and msd-hsct had similar therapeutic efficacy. disclosure: no disclosure p pres in bmt for thalassemia major in india: lower incidence and limited impact background: posterior reversible encephalopathy syndrome (pres) is a relatively common complication seen after blood or marrow transplantation (bmt) for hemoglobinopathies with a reported frequency of - %. pres has also been associated with poorer survival rates. severe hemoglobinopathies are one of the most frequent indications for bmt in the developing world, particularly in india. given the risk of rejection in multiply transfused patients and the need to minimize gvhd risk, immunosuppression post-bmt for these non-malignant conditions can be particularly intense and prolonged. we sought to measure the incidence and impact of pres in developing countries. methods: we analysed successive transplants for thalassemia using protocol (atg-bucy+csa/mmf or csa/mtx) maintaining cyclosporine a (csa) blood levels - ng/ml for patients and protocol (flu-atg-bucy+csa/mtx) maintaining higher csa levels post, i.e. - ng/ml for patients from fully matched donors with g-csf-primed bone marrow. for patients this was the second transplant from a different matched related donor. pres was confirmed with brain ct/mri for all patients. results: all recipients who had pres had sibling donors, males and females. age median . (iqr . - years). the frequency of pres was . %; disease free survival for patients who had pres was %. pres resolved completely in all. csa was switched to mmf in patients who had received mtx and were on csa only at the time of pres occurrence, while csa was stopped but mmf continued in patients taking csa/mmf combination and csa was continued for patient. three patients with pres had grade acute gvhd, had grade gvhd and none developed chronic gvhd. csa levels at the time of pres were a median of ng/ml (iqr: to ) with patient having ng/ml. three patients had pres while they were thrombocytopenic. hypertension stage was observed in four patients, stage in one patient, one patient was not hypertensive and in one patient blood pressure values were not available. two patients were on methylprednisolone and . mg/kg/day and one was on dexamethasone mg/m /day. one patient was started on csa again after the pres episode and within weeks had another one while on csa (level ng(ml), methylprednisolone . mg/kg/day and ruxolitinib for gvhd. protocol had statistically significant improvement in disease free survival from % to % (p< . ) with probability of occurrence of pres increasing from . % to . % (p = . , see figure ), yet had a benign course in all patients. conclusions: not stopping immunosuppression may have been the key factor which could explain why we have better outcomes with pres than what is reported. intensifying immunosuppression pre-bmt did lead to more pres, albeit not significantly, and yet it was quite manageable. even with addition of fludarabine our pres incidence is lower than previously reported. [[p image] . background: sickle-cell diseases (scd) are a group of genetic hemoglobin disorders marked by brain vasculopathy. allogeneic hematopoietic stem cell transplantation (hsct) is a curative option able to stop vascular disease progression. diffusion-tensor imaging (dti) is a magnetic resonance imaging (mri) technique sensitive to the brownian motion of water molecules and cellular environment. this microscopic quantitative technique is able to detect white matter (wm) alterations before a conventional mri. the aim of this study was to use dti to evaluate axonal damage and structural connectivity in the brain of patients with scd submitted to hla-identical sibling allogeneic hsct. methods: sixteen scd patients with no extensive vasculopathy detected by conventional mri ( male, age range: - years) and age-matched healthy controls ( male, age range: - years) participated in this prospective study. mri acquisitions were performed in a t scanner two times for patients (before and - years after hsct) and at a single moment for controls. from dti acquisitions, fractional anisotropy (fa), mean (md), radial (rd) and axial diffusibility (ad) were calculated in the wm of the whole brain. structural connectivity was also analyzed, based on graph theory, obtaining efficiency, length path and clustering coefficients of the brain network. an anova test was applied to analyze fa differences among controls and patients, before and after hsct. a paired two-tailed t-test was used to determine statistical significance of changes in the fa, diffusivity mean values and network parameters before and after hsct. results: mean fa was lower in patients before hsct than controls (p = , ) and increased after hsct being not statistically different when compared to controls (controls = , ; patients before hsct = , ; patients after hsct = , ; post hoc dunnett's test -error , ; anova test). when patients were compared before and after hsct, md and rd decrease after hsct (p = , and , , respectively). on the other hand, fa increased (p = , ). after hsct, efficiency was higher (p = , ) and path length index was lower (p= , ) than at study entry (table ) . conclusions: this study indicates that, before hsct, patients with scd present axonal damage not detectable by conventional mri, when compared to healthy controls. we also suggest that hsct is able to promote axonal recovery and reorganization. partial diffusivity recovery could be associate to a still unidentified mechanism of myelin regeneration. in the future, longer follow up and comparisons with other forms of treatment are required. background: bmt is a well-established treatment modality for haemoglobinopathies, limited by the availability of related donors. unrelated transplantation has historically shown variable outcomes driven by gvhd and toxicity, and usually restricted to / matches, but the impact of reduced toxicity conditioning regimens is yet to be known. methods: from to twenty-five consecutive unrelated bone marrow transplants were conditioned with fludarabine mg/m , treosulfan g/m , thiotepa mg/ kg and atg (thymoglobulin) . mg/kg if the source of stem cells was marrow (n = ) or ptcy if pbsc (n = ). endogenous haemopoiesis was suppressed pretransplantation for a minimum of weeks. gvhd prophylaxis was provided with ciclosporin/sirolimus and mmf. thirteen patients were transplanted for b thalassaemia major, one of a thalassaemia major and sickle cell disease. the median age was years ( - ). ten patients were / matched ( thalassemia and sickle) and patients had a / match ( thalassaemia and sickle). the median cell dose was . x tnc/kg (range . - . ) and . x cd +/kg (range . - . ). the median survival was . months ( . - . ). patients with thalassaemia were pesaro class i or ii (pesaro class iii patients were intensively chelated pretransplantation to return to class i or ii). patients with sickle cell disease were transplanted for stroke or recurrent vaso-oclusive crises and/or acute chest syndrome not responding to hydroxycarbamide. results: all patients engrafted and achieved evidence of donor haemopoiesis on day + and achieved transfusionindependence and donor haematological values, but subsequently one / patient with thalassaemia suffered secondary graft failure on day + after macrophage activation syndrome. median neutrophil engraftment was days (range to ) and days ( - ) for / and / patients respectively. patient with sickle cell disease had the platelet count maintained > x /l at all times. the median platelet engraftment > x /l was days (range to ) and days (range to ) / and / patients respectively. there were three deaths, all in the / matched group: two with thalassaemia (day + due to idiopathic pneumonia syndrome and day + due to mas) and one with scd (day + due to ips). there were different trends of complications seen by degree of matching that did not segregate otherwise by disease. conclusions: in conclusion, unrelated bmt for haemoglobinopathies with reduced toxicity regimens is feasible. whilst gvhd caused significant morbidity during the transplant period, other alloreactive/endothelial complications (vod, macrophage activation syndrome, idiopathic pneumonia syndrome) were only seen in the / transplants. disease-free survival, dependent on transplantrelated mortality, and lack of long-term toxicity, including chronic gvhd, are determined by the degree of matching. / matched transplants have excellent long-term outcomes with no chronic gvhd > months and can be considered for patients without a related donor; whereas / transplant have significant toxicity and mortality, warranting a haploidentical approach. disclosure: no conflict. long-term safety and efficacy of lentiglobin gene therapy in patients with transfusion-dependent β-thalassemia following completion of the phase / northstar study patients with transfusion-dependent β-thalassemia (tdt) may benefit from gene therapy involving β-globin gene addition to hematopoietic stem cells (hscs) enabling production of functional hemoglobin (hb). lentiglobin gene therapy contains autologous cd + hscs transduced ex vivo with the bb lentiviral vector encoding β-globin with a t q substitution under transcriptional control of the encoding β-globin locus control region. the safety and efficacy of lentiglobin was evaluated in adults and adolescents with tdt in the -year phase / northstar study (hgb- ; nct ). methods: patients with tdt (≥ ml/kg/year of red blood cells [rbcs] or ≥ rbc transfusions/year) received g-csf and plerixafor for hsc mobilization. to generate drug product (dp), cd + hscs were transduced with the bb lentiviral vector. patients underwent single-agent, myeloablative busulfan conditioning, were infused with the dp, and were followed for safety and efficacy. results: eighteen patients have been treated in the completed northstar study. as of september , patients had a median follow-up of . (min -max: . - . ) months. the median age at consent was (min -max: - ) years including patients ≥ years old. patients received a median cell dose of . (min -max: . - . ) cd + cells x /kg with a median dp vector copy number (vcn) of . (min -max: . - . ) vector copies/ diploid genome. the median liver iron content (lic) at baseline was . (min -max: . - . ) mg fe/g dw. outcomes by age and baseline iron status will be presented. the median time to neutrophil and platelet engraftment was . (min -max: - ) and . (min -max: - ) days, respectively. four patients had platelet engraftment ≥ day and four patients had platelet counts of ≤ x /l at month . none of these patients had ≥ grade bleeding events post-lentiglobin infusion. transfusion independence (ti, defined as weighted average hb ≥ g/dl without rbc transfusions for ≥ months) was achieved in / patients with non-β /β genotypes and / patients with β /β genotypes. in patients who achieved ti, total hb at last visit was . - . g/dl. lic increased from baseline in patients who achieved ti by a median of . % and . % at month and then decreased from baseline by a median of . % and . % at month and , respectively. non-hematologic grade ≥ adverse events post-infusion in ≥ patients included stomatitis, febrile neutropenia, pharyngeal inflammation, and irregular menstruation. there was no transplant-related mortality, vector-mediated replication competent lentivirus, or clonal dominance. two patients experienced grade serious veno-occlusive liver disease (table ) . events resolved following treatment with defibrotide and were attributed to myeloablative conditioning. conclusions: in the northstar study, % of patients with tdt and non-β /β genotypes and % of patients with β / β genotypes achieved transfusion independence. the safety profile of lentiglobin remains consistent with myeloablative busulfan conditioning. longer time to platelet engraftment was observed in some patients, but no graft failure was reported. clinical background: sickle cell disease (scd) is an inherited hemoglobin disorder associated with high morbidity and mortality. currently, allogeneic hematopoietic stem cell transplantation (hsct) is the only curative therapy for scd. transplant outcomes with thiotepa, treosulfan and fludarabine (ttf) preparative regimen are encouraging but this regimen has not been directly compared to other preparative regimens in scd. we therefore planned to compare the event free probability for death, rejection and high grade acute graft versus host disease (agvhd) between ttf and busulfan and fludarabine (bf) regimens. methods: in this retrospectively cohort study, we included all patients with scd who received allogeneic hsct at our center or who were transplanted in other centers and referred to ours for follow up before day . patients were transplanted between july and december . we used kaplan-meier curve to estimate the event free probability for death, rejection and high grade agvhd (grades - ). cox regression was used to assess the impact of the preparative regimen on these outcomes. results: a total of patients were included with a median age of years (interquartile range [iqr]: - ) and a median hemoglobin of g/dl (iqr: - ). sixtytwo percent were males. the proportion of patients who had splenectomy, stroke and acute chest syndrome was %, % and % respectively. all patients received peripherally collected hematopoietic stem cells from a matched sibling donor with a median stem cell dose of x /kg (iqr: . - . ). most patients, %, received cyclosporine or tacrolimus based agvhd prophylaxis. most patients received ttf ( %) or bf ( %) preparative regimens. all patients in the bf group received atg. the median follow-up time was months (range: - ). four patients died during the follow-up period with an os of % ( % confidence interval [ci]: %- %) at years. the os was not different (hr . , p = . ) between the ttf ( %) and the bf ( %) regimens. the probability of high grade agvhd free survival at day was % ( % ci: - ) for all patients. this probability was % in the ttf group and % in the bf group and the difference was not statistically significant (hr . , p = . ). the rejection free survival at months was % ( % ci: - ) for all patients. no patients in the ttf group rejected while the rejection free survival at months for the bf group was %. this was not statistically significant (p = . ). conclusions: in patients with scd undergoing allogeneic hsct from a matched sibling donor, the ttf preparative regimen is not associated with improved os, rejection free or high grade free agvhd survival when compared to the bf preparative regimen. larger studies are needed to confirm these findings. disclosure: nothing to declare. novel strategy for haploidentical hematopoietic stem cell transplant in sickle cell disease methods: consecutive patients suffering from scd who underwent hhsct between jan till date were enrolled in the study. all underwent autologous backup (target dose> x /kg) followed by pre-transplant immune suppression (ptis) cycles at weekly intervals using fludarabine @ mg/m /day(d -d ) + cyclophosphami-de@ mg/m /day(d ) + dexamethasone@ mg/m / day(d -d ) along with hypertransfusion (target hb - gm/dl), hydroxyurea ( mg/kg/day) and azathioprine ( mg/kg/day) from day - . the graft was mobilized using gcsf@ mcg/kg/day(d -d ) + plerixafor@ . mg/kg s/ c on d - hours before the pbsch. conditioning included thiotepa mg/kg in two divided doses (d- ), fludarabine mg/m (d- to d- ), cyclophosphamide . mg/kg (d- , d- ), tbi gy with thymic shielding (d- ), ratg (genzyme thymoglobulin . mg/kg (d- to d- ). gvhd prophylaxis included ptcy mg/kg/day on d and , sirolimus (target levels - ng/ml) (till - months post hsct) and mmf (till d ) starting from d . results: the median age of patient's was years (range - years). before transplantation all patients had repeated episodes of one or other complication warranting a transplant, non-responsive to hydroxyurea. six had maternal donors, paternal and sibling. median age of the donor was years (range - years). all were dsa negative with a cutoff mfi of > iu. all patients received x /kg cd cells irrespective of harvested dose which ranged from ( . - . x /kg). median cd dose was . x /kg (range . - . x /kg). all patients engrafted with median time to neutrophil engraftment days (range - days) and median time to platelet engraftment days (range - days). median duration of hospital stay was days (range - days). one patient had cytokine release syndrome needing tocilizumab. five had engraftment syndrome treated with short course of steroids. two had cmv reactivation needing treatment with ganciclovir/valganciclovir. acute gvhd grade ii was seen in one patient. till date of analysis none had features compatible with chronic gvhd. of the patients, are alive without sickle cell disease with lansky/ karnofsky scores of . at median follow up of days (range - ) the probabilities of survival, sca-free survival, and transplant-related mortality after transplant were . %, . %, and . %, respectively. one patient died due to mdr klebsiella sepsis after being discharged initially while he was receiving iv ganciclovir on day care basis. he had full donor chimerism. none of the patient had primary or secondary graft failure. conclusions: pre-transplant immune suppression and upfront use of plerixafor for graft mobilization decreases the risk of graft failure and graft versus host disease leading to overall better survival in hhsct for sickle cell disease. disclosure: none. combined haematopoietic stem cell transplant and enzyme replacement therapy in wolman disease: outcomes and challenges jane kinsella , denise bonney , helen campbell , robert wynn , simon jones background: infantile lysosomal acid lipase deficiencymore commonly known as wolman disease -is an autosomal recessive lysosomal storage disease, characterised by storage of cholesterol esters in the liver, spleen and gastrointestinal tract. these children present under the age of months and traditionally had a poor prognosis, with almost all being dead by the age of months. bone marrow transplant has been used to correct disease manifestations, but limited by high procedure-related mortality with the significant co-morbidities. the survival has changed over the past few years due to pharmacological enzyme replacement therapy but still presents challenges for these patients and their clinicians. in these children haematopoietic stem cell transplant we have offered bmt with enzyme replacement therapy, in certain specific circumstances. methods: four children with wolman disease being treated with enzyme replacement therapy, limited by alloantibody, or poor venous access, received treosulfan-based, myeloablative conditioning with serotherapy followed by a matched haematopoietic stem cell transplant: two family donors, one sibling donor and one unrelated donor. results: three of the four children survived transplant. they have continued to receive enzyme replacement therapy but at reduced dose and frequency with improved tolerability. they have continues to grow and develop. growth and gastrointestinal histology is improved for children having received transplant compared to those receiving enzyme replacement alone. monitoring of peripheral blood chimerism has shown a disease-associated engraftment defect, with mixed chimerism in the surviving patient. conclusions: haemopoietic stem cell transplant is a suitable treatment option in children with wolman disease in whom receiving enzyme replacement therapy is not possible because of venous access, sensitisation or cost reasons. it improves their tolerability of the enzyme treatment and allows for a reduction in enzyme dose and frequency. however, the results of engraftment are not as good as expected for a transplant with myeloablative conditioning and a matched donor. an engraftment defect has been observed in lysosomal acid lipase deficient animal models. a further understanding of this poor engraftment in children with wolman disease is required as to determine whether the risks of transplant is beneficial in these patients and for the consideration of future treatment options including gene therapy. background: thalassemia major is the most common transfusion dependent hemolytic anemia in the world. the absent or reduced production of the β-chain of hemoglobin causes severe ineffective erythropoiesis, massive erythroid hyperplasia in the bone marrow and extramedullary hematopoesis occurs. patients require regular transfusion therapy lifelong. currently, the only proven curative treatment of thalassemia is allogeneic stem cell transplantation (sct). methods: we evaluated the immune reconstitution results of patients at year after hematopoetic stem cell transplantation at our pediatric bone marrow transplantation center between january and december . all patients were not receiving any immunosuppressive treatment at least for months and they have normal lymphocyte counts, immunoglobulin levels and transfusion independent. lymphocyte subtypes and chimerism percentages and the relationship with the donor type were evaluated at year of transplantation. results: ages of transplantation was ranged between - years (median: years). seven ( %) of them was male. matched unrelated donor type was chosen in patients while others ( patients) were transplanted from family matched donor (matched sibling: patients, matched family: patients). all patients received myeloablative conditioning regimen containing busulfan/treosulfan, cyclophosphamide, thiotepa and fludarabine. follow up time was between - months (mean: ± months). in patients, whole bone marrow product was used while peripheral stem cell harvest in remaining patients. cd levels were found low in only patients, in normal patients mean was % ± %. cd levels were severely low in patients while cd in only patient. cd levels were increased in total patients in as compensatory. cd /cd ratios were very low in all patients (range: . - . ). b cells (cd +) were low in patients while immunoglobulin levels were normal. chimerism values between - % (mean: ± %). donor and product types did not differ in cd + lymphocyte reconstitution at year (p= . , p= respectively). all patients were alive and well at year after transplantation. conclusions: after year of transplantation, although patients are in well condition regarding to infection frequency and transfussion dependency, it was seen that their lymphocyte subtypes reconstitution could not be achieved enough as in normal children. we can conclude that low cd + cell levels were an expected finding in almost all patients. so, these patients may have a tendency to suffer serious bacterial and viral infections, and close follow up be required in terms of infections as long as cd levels continue to be low. immunoglobulin replacement therapy did not required even in patients with low b cell levels. disclosure: nothing to declare p phase international, multicentre trial to assess haploidentical aß t-cell depleted stem cell transplantation in patients with sickle cell disease with no available sibling donor background: sickle cell disease (scd) is an inherited disorder with an estimate of , affected newborns per year worldwide. allogeneic hematopoietic stem cell transplantation (hsct) with a matched sibling donor (msd) is currently the curative standard of care for scd patients (pts). however, msd availability is < %. a t-cell depleted haploidentical hsct (t-haplo-hsct) from a relative, mostly a parent, expands the donor availability while exhibiting low gvhd rates and thus could offer cure to the remaining % of scd patients. in a pilot study, comparing t-haplo-hsct with msd hsct in advanced stage scd, using almost identical transplant regimens for both. the overall (os) and disease-free survival (dfs) was % vs. %, respectively. methods: these results led to the design of a clinical trial to assess tcd-haplo-hsct prospectively which aims to demonstrate that a hsct from a haploidentical relative is not inferior to a msd hsct with regard to major outcome parameter. this phase , prospective, stratified, open-label study is targeting enrollment of patients aged - years with homozygous hbs disease or heterozygous hbsc or hbs /+ ß-thal suffering from severe or moderate scd related complications. inclusion criteria are clinically significant scd related complications such as stroke, silent crisis, pathological angio-mri, transcranial doppler (tcd) velocity > cm/s, or more episodes of acute chest syndrome (acs) in a lifetime, chronic transfusion dependency, transfusion-refractory allo-immunization and others. pts fulfilling inclusion criteria will be stratified according to donor availability. pts with a msd will receive a bone marrow graft, pts requiring an alternative donor will be transplanted with an aß/cd depleted graft from a haploidentical family donor. the conditioning regimen for both groups will be identical with the exception that antithymoglobulin (atg-neovii ® ) is given upfront in thaplo-hsct versus day - to - in msd. chemotherapy consists of thiotepa, fludarabine and treosulfan. posttransplant immunosuppression will consist of mofetil mycophenolate and tacrolimus for a duration > months in t-haplo-hsct and < months in msd, depending on chimerism. (eudract number: - - ) results: primary efficacy endpoint: event free survival (efs). event is defined as incidence of acute gvhd, grade iii -iv, chronic gvhd, rejection (graft failure) or death (for any reason). key secondary endpoint(s) are os, dfs, graft failure, hematological and immunological reconstitution, quality of life (qol) assessment and fertility. the primary null hypothesis is: efs of scd patients treated with t-haplo-hsct is non-relevantly inferior to efs in the msd arm. conclusions: results will help to determine if an a/ß depleted t-haplo-hsct can be considered equivalent to msd hsct with regard to dfs, adverse events and safety, in order to offer this form of cure to the majority of patients with scd. disclosure: nothing to declare hit three birds with one stone: successful stem cell transplantation from one family donor to three siblings methods: in august , three thalassemic siblings were admitted to hospital for stem cell transplantation from a full match donor, their years old sister. the patients' general health conditions and specific health issues due to thalassemia were checked extensively. it was decided to perform first transplant to older sister whom the disease and transplant complications are expected more intense due to prolonged transfusion and chelation therapy. the oldest daughter of family, healthy, was planned to accompany her sisters in transplantation unit so parents can take care the others and organize this period for whole family. results: the years old sibling was first admitted to bone marrow transplantation unit in july . the conditioning regimen was busulfan, fludarabine, cyclophosphamide and thiotepa with antithymocyteglobulin(atg) and defibrotide prophylaxis was given. the healthy donor was admitted to hospital and received g-csf for continuous days before harvesting. stem cells were collected peripherally on day and viable cd + cells were /ul. patient received , x e /kg stem cell and the other cell products were divided into parts according to other recipients´weight. no infusion problems were recorded in stem cell transfusion. gvhd prophylaxis was given with cyclosporin and methotrexate. severe sinusoidal obstruction syndrome was observed and successfully managed with supportive therapy. neutrophils were engrafted + . day, and platelets were on day . full blood chimerism results were % in day , % in day and % in day consecutively. after months from first transplant the years old sister was admitted to hospital on october . same conditioning with defibrotide prophylaxis and gvhd prophylaxis were given and , x e /kg peripherally derived and previously frost stem cell was infused without any complications. mild sinusoidal obstruction syndrome was observed and managed with supportive therapy successfully. neutrophils were engrafted + . day, and platelets were on day . full blood chimerism results were % in day , % in day and % in day consecutively. the third transplant was performed on january with the same conditioning and prophylaxis regimen. although defibrotide was used mild sos was observed and treated with supportive therapy with success. neutrophils were engrafted + . day, and platelets were on day . full blood chimerism results were % in day , % in day and % in day consecutively. conclusions: the patients are being followed for over a year after first transplat, neither adverse nor gvhd symptoms were observed. we presented this case for being a unique example for match family donor transplant and the first successful example from one donor to three recipients. disclosure: nothing to declare results: our female patient admitted for anemia at rd month of birth and was transfused every - months from th months to . years of age. since investigations directed towards hemoglobinopathies or membrane defects like hereditary spherocytosis were unremarkable, she was not transfused for years after the age of . -years because hemoglobin level was constant over g/dl. her bm examination showed erythroid hyperplasia and feature of dyserythropoiesis with a few binucleated erythroblasts. it was decided to follow-up the patient with a diagnosis of cda ii. after the age of -years, the need for transfusion started again for every to months which led the parents of our patient to request for bone marrow transplantation, however, the diagnosis was not definite, and because of the insufficient data for the transplantations for cda ii patients, it was decided to go on to follow-up. nevertheless, after years, the frequency of transfusion gradually increased to every - weeks, and bone marrow transplantation was brought into question again. at that time, genetic examination was started and sec b gene was analyzed by direct sequencing. hsct decision from her hla / matched brother, carrying sec b mutation in heterozygous state, was taken. in the preparation regimen, busulfan (bu) at a myeloablative weight adjusted dose ( days), mg/kg cyclophosphamide (cy) ( days), and mg/kg antithymocyte globulin (atg fresenius) were used. graftversus-host disease (gvhd) prophylaxis was with cyclosporin a started on day - and short-term methotrexate on day + ,+ and + . she was transplanted with bm with a dose of total nucleated cells= . x /kg and cd = x / kg. neutrophile and platelet engraftment were achieved at + and + , respectively. indeed, grade hemorrhagic cystitis due to bk virus and a moderate veno-occlusive disease prolonged platelet transfusion days which concealed the exact engraftment day of platelet. the patient was discharged on day with no more need for any transfusion and followed up as a complete chimeric with no type of gvhd since then. now, she is years old, under regular surveillance at our transplant centre without any symptoms. conclusions: hsct data in cda ii patients is still insufficient, however based on data from tm patients with similar treatment approaches in td cda ii patients, it is seen that the hsct is reliable and effective. disclosure: nothing to declare hematopoietic stem cells background: the prognosis after frontline therapy in b-all patients have improved due to monoclonal antibodies (cd , cd , cd ) and approximately % of patients achieve complete remission. in relapsed and refractory (r/ r) b-all and also in mrd + outcomes are relatively poor. disease-free survival (dfs) in this cohort is - %. in this cohort allo-hsct is indicated and complete remission before transplantation is crucial for prognosis. conventional chemotherapy is associated with high failure rate and significant toxicity. immunotherapy with monoclonal antibodies and car-t are more promising approaches. the aim was to evaluate the efficacy (frequency of responses, os, dfs) and toxicity, especially neurotoxicity and cytokinerelease syndrome, of a bispecific monoclonal antibody blinatumomab in patients both children and adults with persistence of minimal residual disease (mrd + ) or r/r b-all as a bridge to allo-hsct. methods: this study included patients with high risk b-all blinatumomab treated in - , among them pts ( %) with t( ; ), ( %) with t( ; ), with mll ( %), pts ( %) who were refractory to previous chemotherapy, ( %) after allo-hsct from deferent type of donors. median age was y.o. (range m- y.o), children - y.o. ( %) and adults > y.o. ( %). r/r all had pts ( %), mrd + - pts ( %), median days of follow up were ( - ). blinatumomab was applied as -day cycles followed by a -day off-period before the start of the following cycle. majority pts received one cycle (n= , %). in r/r all group dose was of mcg/d during the first days and afterwards mcg/d. patients with weight less than kg received mcg/m /d and mkg/m /d accordingly. in mrd group dose was mcg/m /d. results: the frequency of responses to blinatumomab was higher in mrd + pts in comparison r/r all pts ( % vs % p= . ). in mrd + pts cr mrdwas achieved in pts ( . %), pts ( . %) were mrd+ after blinatumomab. two-year os in this group was %. twenty pts ( %) received allo-hsct. in rr all pts cr mrdwas achieved in pts ( %), pts ( %) were mrd+ after blinatumomab, pts ( %) had no hematological response . two-year os in r/r all was %. fifteen pts ( %) received allo-hsct. os in cr mrdpatients who received allo-hsct was not significantly different in comparison with patients who received blinatumomab as a monotherapy ( % vs %, p= . ). no significant differences in dfs were observed at two years in cr mrdpts depending status of the disease before therapy-mrd vs r/r ( % vs %). of the reported adverse events, febrile fever was the most common pts ( %), neutropenia ( %), thrombocytopenia ( %), infection ( %), neurotoxicity ( %), cytokine-release syndrome ( %). all complications were reversible. conclusions: blinatumomab is effective option in patients with high risk b-all especially in the group with mrd persistence after previous chemotherapy and facilitates effective bridging to hsct. blinatumomab therapy is generally well tolerated. disclosure here we address the transcriptional regulation of differentiated cells from human embryonic stem cells (escs) using self-assembling peptide hydrogel without stromal cells, and compare with embryoid body (eb) culture system. methods: esc differentiation was induced in eb culture system or three-dimensional ( d) hydrogel culture system. the engraftment potential of differentiated cells was evaluated by flow cytometry. cd + cells from mobilized peripheral mononuclear blood cells (mpbmcs) or differentiated from escs at different times (day , day , day ) were purified by fluorescent-activated cell sorting. sorted cells were captured on medium-sized microfluidic chips using the fluidigm c single cell auto prep system. sequencing was performed by hiseq x ten. results: self-assembling peptide hydrogel formed a d scaffold for cell culture, the pore diameter of which ranged from to nm. compared to eb culture system, escs in d culture system differentiated more potently. the differentiated cells from d system were short-term engrafted in the nog mice, and myeloid cells, b cells and t cells could all be detected in peripheral blood after transplantation. however, the engraftment was not obtained in differentiated cells from eb culture system. we obtained and analyzed escs, cd + cells from eb culture system, cd + cells from d culture system, and cd + cells from mpbmcs. the cells were divided into cluters ( figure a ). in both differentiation systems, the cd + cells from day were more heterogeneous than cd + cells from day and day ( figure b) . however, cd + cells from mpbmcs were more homogeneous, probably because the differentiated cd + cells contained several cell lineages, including hematopoietic cells, endothelial cells and mesenchymal cells. there is transcriptional overlap between individual cd + cells from eb and d culture systems. however, we found that cluster , which is composed mainly of cd + cells from d at day and day , expressed similar level of several hematopoietic regulator as hsc, such as tal , lmo , erg ( figure c ). the cluster , which is almost the cd + cells from d at day , also expressed the highest gata among the clusters from differentiated cells ( figure c) . conclusions: our study demonstrates that d hydrogel culture system facilitates hematopoietic specification of escs. disclosure: nothing to declare higher cd + cell dose increases overall survival in the setting of dual t-lymphocyte suppression with atg and ptcy in matched related and unrelated donor allosct background: there is no consensus on the cd + donor cell numbers required for optimal outcomes in allogeneic stem cell transplant (allosct). there is controversy on the benefits or harm in higher cell dose for allosct. this study aims to evaluate the impact of cd + cell dose in allosct patients receiving reduced intensity conditioning (ric) combined with anti-thymoglobulin (atg) and posttransplant cyclophosphamide (ptcy) using related (mrd) and / and / matched unrelated donors (mud). methods: this is a single-centre retrospective analysis of adult patients who received allosct for hematologic malignancies between october and may . all received ric using fludarabine ( mg/m /day: day - to - ), busulfan ( . kg/m /day: day - and - ) and total body irradiation ( cgy: day - ). all patients also received rabbit-atg ( . mg/kg: day - to - ), ptcy ( mg/kg/day: day + ,+ ) and cyclosporine (from day + ). unmanipulated peripheral blood stem cells were infused on day . analyses were done using thresholds: ( ) an arbitrary cd + cell dose of x /kg (as this was our target dose) and ( ) cell dose according to quartiles (< . , . - . , . - . and ≥ . x /kg). results: median cd + cell dose was . x /kg. median follow up was months (range - ). median neutrophil engraftment was (range - ) days and platelet engraftment was (range - ) days. a cell dose greater than x /kg was associated with an increased overall survival (os) at year ( . %; % ci, . - . vs . %; % ci . - . ; p= . , figure ). the higher dose was also associated with shorter platelet engraftment time (p= . , figure ). there was no significant difference in neutrophil engraftment, nonrelapse mortality (nrm), relapse free survival (rfs), grade ii-iv acute graft versus host disease (agvhd) and moderate to severe chronic graft versus host disease (cgvhd), (table ) . analyses using quartile cell dose thresholds showed a trend towards decreased os with a cell dose of < . x ^ /kg, however this was not statistically significant ( figure ). higher cd + cell doses were associated with shorter platelet engraftment time (p= . , figure ). there was no significant difference in neutrophil engraftment, nrm, rfs, agvhd and cgvhd (table ) . conclusions: cd + cell dose greater than x /kg significantly increases overall survival in the setting of ric and dual t-lymphocyte suppression with atg and ptcy in mrd and mud allohsct. further studies in a larger number of patients and longer follow up are recommended to validate these findings. disclosure methods: fifty two adult patients were included. median cd + cells requested for infusion were x ^ /kg. all patients received the same ric regimen including fludarabine ( mg/m /day day - to - ), busulfan ( . kg/m /day day - and - ), and total body irradiation ( cgy) (day - ) combined with rabbit-atg ( . mg/kg: day - to - ), ptcy ( mg/kg/day: day + ,+ ), and cyclosporine. unmanipulated peripheral blood stem cells were infused. last followup was november . median follow-up was months (range - ). median cell dose count infused was . cd +/kg. we arbitrarily divided the cohort in two groups with cd + dose of > x ^ cd /kg as cut-off point. results: findings are summarized in figure . the infusion of more than x ^ cd /kg dose had a significant worse impact on overall survival (os) (p= . ), relapse-free survival (rfs) (p= . ) and cumulative incidence of acute gvhd (p= . ). chronic gvhd could not be compared between the two cohorts due to the different median follow-up. conclusions: the infusion of a cd + cell dose count higher than x ^ cells/kg had a significant adverse impact in overall survival and grade ii-iv acute gvhd in the setting of ric and dual t-lymphocyte suppression with atg and ptcy for haplohsct. disclosure: nothing to declare p long-term thymic activity and immune-reconstitution after haplo-identical allografting with post-transplant cyclophosphamide background: the use of post-transplant cyclophosphamide (ptcy) has expanded the application of t repleted haploidentical stem cell transplantation (haplo-hsct). in this setting, to investigate thymus role in longterm clinical outcomes, evaluation of immune reconstitution kinetics was performed. methods: twenty-nine patients (median age ) were enrolled. blood samples were collected before conditioning and at , , , , , months after haplo-hsct. analyses of cd and cd t-cell subsets by flow-cytometry were correlated by generalized linear models with real-time pcr (rt-pcr) quantification of signal joint t-cell receptor excision dna circles (sjtrecs), specific marker of naive t-cells thymopoiesis. a) naive; b) central; c) memory; and d) revertant cd and cd t-cells were defined as follows: a) cd ra+cd l+; b) cd ro +cd l+; c) cd ro+cd -; and d) cd ra+/ ro +, respectively. sjtrecs rt.pcr was performed on genomic dna ( ng) extracted from sorted cd and cd t-cells. results: a gradual increase in absolute numbers of all cd and cd t cell subsets and of sjtrecs copies from the first month up to years post-transplant was observed ( figure ) . however, at years, cd and cd t-cell levels and sjtrecs levels were lower than those observed in healthy donors. sjtrecs kinetics was associated with the increase in cd naive t-cells (overall, p < . ). this correlation suggests that most of cd naive t-cells derives from thymic re-education of donor precursor stem cells, whereas cd naive t-cells undergo peripheral expansion after thymic production. furthermore, an increase in cd revertant memory t-cells was also significantly correlated with sjtrecs kinetic (p , ). central and effector memory t-cells showed a faster thymic-independent expansion in both cd and cd tcells. interestingly, sjtrecs levels and thymic dependent immune-reconstitution were higher in a cohort of patients undergoing hsct from hla identical donors (manuscript in preparation). clinical outcomes and thymic function were correlated starting at months after hsct. lower thymic output was significantly associated by multivariate analysis with low pre-transplant trecs values (p , and p < , in cd and cd , respectively), moderate-severe chronic graft-versus-host disease (gvhd; p < , in cd ), and age (≥ years, p , in cd ). conclusions: the thymus, despite age-dependent involution, substantially contributes to t-cell reconstitution after haplo-hsct. chronic gvhd and older age were significantly correlated with reduced thymic function. overall, lower production of sjtrecs after haplo-hsct as compared after hla identical sibling hsct may partly be due to a higher degree of "mismatching" of mhc molecules during thymic re-education. [[p image] . figure ] background: the use of allogenic hematopoietic stem cell transplantation (hsct) in the treatment of adolescents and young adults (aya) with philadelphia negative all is decreasing with the adoption of pediatric inspired protocols to treat this age group and the incorporation of minimal residual disease assessment in the routine care of all patients. previously, its use was defined mainly by disease risk features at presentation. methods: a study on aya (age - years), who underwent allogenic hsct at our institute for philadelphia negative all, between february and december . all the studied patients received calgb based adult chemotherapy protocol for induction, and underwent a matched related donor (mrd) transplant with cy/tbi conditioning and mtx/csa as gvhd prophylaxis. the patients were eligible for allogeneic hsct, if they have a mrd plus one or more of the following risk factors: ( ) age ˃ years, ( ) high presenting wbc count (> for b-all, > for t-all), ( ) high risk immuno-phenotyping (pro-b, pro-t, early t, and mature t), ( ) bulky splenomegaly or bulky lymphadenopathy, ( ) high risk cytogenetics ( ; , ; , low hypodiploidy/near triploidy, complex), ( ) cns involvement, ( ) relapsed or refractory disease at d of induction. in this study, we investigated the impact of those different risk factors on the long term outcome of allogeneic hsct. results: the median os of our studied patients was not reached at . years, with a median dfs of . years (figure ). in a univariate analysis, relapsed or refractory disease prior to transplant was the only independent risk factor for os and dfs (p-value= . , and . respectively) (figure ). in addition, patients who had or more risk factors ( , . %) prior to transplant had a significantly lower long term outcome compared to patients, who had one ( , . %) or two risk factors ( , . %) with a median os of months, and a median dfs of only months (p-value= . , and . respectively) ( figure ) . conclusions: our results show that the long term outcomes of hsct in aya with philadelphia negative all treated on an adult type chemotherapy regimen, were significantly better in patients who showed a good response to initial therapy and a limited poor prognostic factors at presentation, with worsening of dfs as the number of poor prognostic features increase. we can conclude that, using this risk score can be helpful in predicting the outcome of allogenic hsct in aya with philadelphia negative all treated with adult type chemotherapy protocol. disclosure: no conflict of interest a prospective single center survey on donor-specific anti-hla antibodies and desensitization strategy in patients undergoing an allogeneic stem cell transplant background: in the setting of hematopoietic stem cell transplantation (hsct), considering the risk of poor engraftment or graft failure (gf), the detection of antibodies (ab) directed against donor specific hla loci (dsa) represents a contraindication to proceed with the same donor, suggesting the search of other donors. in many cases, there is not sufficient time to search for alternative donors and it is necessary to plan an immunosuppressive strategy to decrease the dsa level, thus reducing the risk of gf. to date, there is no consensus on desensitization standards to manage dsas in hsct. the aim of this study was to determine the incidence of anti-hla ab and dsas in hematologic patients candidate to an allogeneic hsct, and the efficacy of our desensitization protocol. here, we present an update of the results obtained with our strategy. methods: between august and september , we prospectively screened for dsa consecutive patients candidates to an allogeneic hsct. anti-hla ab research was carried out using the luminex bead assay (lifecode screen and lsa i/ii-immucor). the results were expressed as mean fluorescence intensity (mfi); mfi > was considered positive. in case of a mismatched related donor, a flow cytometric crossmatch test (fcxm) was performed. if the patient had dsas and only one available donor, a desensitization strategy was employed, scheduled with rituximab on day - , single-volume plasmapheresis procedures (pp), usually on day - and - , intravenous immunoglobulins on day - , infusion of hla selected platelets for dsa absorption in case of persistent antibodies directed against class i hla antigens. the aim of this schedule was to avoid interferences with chemotherapy and anti-t-cell globulins, infused during condition regimen results: since august , patients have been prospectively screened. thirty-three patients ( . %) showed anti-hla ab and of them ( . %) had dsas: were treated with the desensitization strategy, applied according to the mfi score and the fcxm result, and all of them obtained an engraftment; in cases, an alternative donor was selected and in case the research for an alternative donor is still underway. dsa detection was performed every days after hsct for the first month and , and days following hsct. neither a dsa rebound nor other complications were observed during the follow-up. conclusions: our prospective analysis underlines the high frequency of anti-hla antibodies detection in hematologic patients, confirming the necessity to routinely evaluate the presence of dsas before an allogeneic mismatched hsct. our desensitization schedules based on the combination of pp, rituximab, ivig and platelet absorption proved successful in reducing dsas. we confirm the necessity of a prospective multicenter collaboration to better define the role of dsas against each hla locus and the critical mfi cut-off level associated with a higher risk of gf. transplant and transfusion specialists should joint to define a consensus for a standard desensitization strategy. disclosure the most frequent technique used for counterbalance partial incompatible hsct is cd + selection that is associated with sustained engraftment and effective reduction of t cells that minimizes gvhd. on the other hand, this approach could delay immune reconstitution and increase risk of viral and fungal infection. in mud setting the use of pbsc is the procedure that most centers have recently adopted. this implies the infusion of a relevant higher number of t cells to times more as compared with bone marrow (bm). since in our centre most part of our patients are primary immunodeficiencies, we applied a procedure to minimize the risk of severe gvhd infusing a controlled number of cd positive cells. methods: we report data about paediatric patients who received mud hsct ( patient received hsct) between and in the bmt unit of the children's hospital of brescia. patients received conditioning, according to the european group for bone marrow transplantation (ebmt) and the european society for immunodeficiencies (esid) guidelines. cd + selection has been realized by a milteny column with an ideal addback of cd positive cells of x /kg. stem cell source was bm in cases and pbsc in cases. results: median patients age at transplant was years (range . months- years). the mean number of infused cells were: x /kg cd + and x /kg cd + in bm product, while x /kg cd + and x /kg cd + in pbsc. mean time for engraftment was day post-hsct. as concerns acute gvhd overall incidence . % ( / ) of the children presented this complication, but only % ( / ) presented gvhd grade iii and none gvhd grade iv, while chronic gvhd presented in . % ( limited, extensive/ ). while acute gvhd incidence and severity weren't significantly different between bm recipients and pbsc recipients, the cases of chronic gvhd were prevalently in the latters. no major infections presented in the post-transplant period and immunological reconstitution both cellular and humoral was completed by months. overall survival at years is % ( / ). the results obtained show how it is possible control severity of gvhd if an addback of a controlled number of cd + lymphocytes. acute gvhd wasn't severe and only few children presented with limited chronic gvhd. the method allows to graft primary immunodeficiencies patients even with pbsc without infusing too many t cells. in fact, especially in very young children, the number could be excessive and risky. nevertheless in case of an oncohaematological patient, gvl effect is preserved. disclosure background: dc is a rare genetic disorder that results from a defective telomere length maintenance and is characterized by mucocutaneous features, bone marrow failure (bmf) and a high predisposition to cancer and pulmonary fibrosis. bmf remains the major cause of mortality and the hsct is the only definitive treatment to restore hematopoiesis but is limited by a high incidence of treatmentrelated mortality. methods: a retrospective analysis of patients (pts) with dc who underwent hsct at the bone marrow transplantation unit in the clinical hospital of federal university of paraná, brazil, between july- and november- . results: boys and girls, with a median age of y ( - y) received a hsct from a mds (n= ), mud (n= ) or mmrd (haploidentical, n= ). pts received bone marrow (bm) and pt received a cord blood unit (cbu). the median of tnc infused was , x /kg (range , - , x /kg) and in the cbu was , x / kg. two pts received a myeloablative preparatory regimen with busulfan (bu) mg/kg + cyclophosphamide (cy) mg/kg or fludarabine (flu) mg/m + antithymocyte globulin (atg). the remaining pts received a ric regimen with cy mg/kg (n= ), flu mg/m + cy mg/kg + atg mg/kg (n= ), and flu mg/m + cy + tbi rads (n= , haplo). graft versus host disease (gvhd) prophylaxis consisted of cyclosporin (csa) and methotrexate or steroids (cbu) and post-transplant cy + csa + mycophenolate mofetil in the haploidentical transplants. of evaluable pts engrafted with a median time to neutrophil recovery of days (range: - days). one patient experienced primary graft failure (haplo) while second graft failure occurred in other pts. all these pts went a second hsct and survived. acute gvhd grade ii-iv occurred in of pts at risk. moderate to severe chronic gvhd occurred in pts with cases occurring in pts who had previously presented acute gvhd. overall survival (os) was , % at a median follow-up of y. the y os was slightly better in msd transplants compared to the others ( , % x , % p= , ). causes of early death include adenovirus sepsis (n= ), toxicity to preparatory regimen and sepsis (n= ), primary graft failure (n= ). pts remain alive between - y after hsct with a median fu of y. among them only pt has developed organ involvement by the underlying disease: hepatopulmonary syndrome (hps). pts died due to pulmonary fibrosis (n= ), liver fibrosis(n= ), gi bleeding(n= ), hps (n= ); cgvhd and sepsis(n= ), infection (n= ), and pts were lost to fu. conclusions: early mortality from bmf can be reduced by hsct, but late outcomes remain a consequence of the underlying disease. long term fu is essential in order to detect late complications related to the hsct procedure or the underlying disease. disclosure: nothing to declare single intra-bone cord-blood transplantation with a treosulfan-based regimen, atg-free and sirolimusbased gvhd prophylaxis: fast hematopoietic engraftment and immune-reconstitution in patients background: cord blood transplants (cbt) require less stringent hla-matching, compared to peripheral blood stem cell or bone marrow. however, cbt has been associated with delayed engraftment and immune reconstitution, especially if in vivo t-cell depletion, such as antithymoglobulin (atg), is used. methods: from to , patients with high-risk diseases received intra-bone infusion of unwashed single cb unit with an atg-free gvhd prophylaxis; were in active disease at cbt and had received prior allogeneic stem cell transplantation. median age was y [range (r) . conditioning regimen was myeloablative, with treosulfan and fludarabine in all, intensified with melphalan in or with gy tbi in . hla matches was / , / , / in , and cases, respectively. gvhd prophylaxis included sirolimus and mycophenolic acid (mmf). results: after thawing, median cd + cells was . x /kg [r . - . ], median cd + cells . x /kg [r . - . ], and median cd + cells . x /kg [r . - . ]. of the evaluable patients all engrafted with a sustained full donor chimerism at day . median time to neutrophils ( / , anc> /μl for consecutive days) and platelet engraftment ( / immune-reconstitution of cbt patients (tables a-b ) was compared with two cohorts of patients transplanted at our center from any adult donor with ( ) or without ( patients, including post-transplant cyclophosphamide cohort) atg in association with sirolimus and mmf. profiles of immune-reconstitution at day - - showed a better cd + recovery at any time-point in both cbt and no-atg versus atg cohort, with no statistic significant difference in the first cohorts. moreover, cd +/cd + ratio at any time point was better in the cbt cohort vs the no-atg cohort. b cell recovery was faster in the cbt cohort; immunoglobulin recovery was superimposable across different platforms. focusing on late events (> days from cbt), / pts experienced ebv reactivation, median time days [ - ] treated with rituximab, and experienced late hhv and cmv reactivation, both solved at last visit. sirolimus was withdrawn after a median of days [r - ]. only patient developed severe chronic gvhd, solved at last visit. overall, after a median follow-up of days [r - ], pts are alive and well. conclusions: our data confirm that intra-bone cbt without in-vivo t-cell depletion is associated with fast hematopoietic engraftment and immune-reconstitution, with very low rate of chronic gvhd and late infective events. background: a promising improvement of hematopoietic stem cell transplantation (hsct) may lie in the transplantation of high numbers of pluripotent stem cells to minimize the time span between transplantation and immunological reconstitution. hence, an ex vivo platform is needed that supports hsc proliferation before application and, at the same time, the maintenance of pluripotency by diminishing hsc differentiation into lineage-specific progenitor cells. methods: to artificially model the natural hsc niche in vitro, we used d bone marrow (bm)-like scaffolds made of polydimethylsiloxane (pdms). these structures are based on a human long bone cross section as a representative of the bm. human cryoconserved hscs were cultured in distinct cultivation systems for days under different conditions. cell counting and facs analyses at day were conducted. for characterization of the cultivated hscs, we used antibodies against cd alone or in combination with antibodies against cd , cd , cd ra and cd f. results: for optimization of culture conditions for human hscs, a commercially available medium was supplemented with a panel of cytokines and valproic acid. we found a significant increase in the number of cd + hscs by simultaneously increasing their vitality using the d system compared with conventional d culturing. a further improvement was achieved by introducing a silicon oxidecovering of the d pdms structures, suggesting that hydrophilic surface properties offer superior attachment for semi-adherent hscs. for a more precise characterization of the cultivated hscs, we introduced a panel of facs markers reflecting the immaturity of the amplified hsc. surprisingly, with increasing immaturity of the cultivated hsc, non-covered d pdms revealed to be best suited for amplification: cell number of vital immature hscs was increased after cultivation on non-covered d pdms compared with silicon oxide-covered d pdms and the d system. conclusions: by establishing a d scaffold according to the human bm, we found a platform mimicking the natural niche of human hsc which is suitable to amplify human hscs in vitro and support their vitality, pluripotency and ability for self-renewal. [[p image] . with the introduction of sion covering of d pdms structures the maintenance of cd + hscs could be further improved. despite the better conservation of cd + hscs by using silicon oxide-covered d pdms, we found that immature human hscs obviously prefer more hydrophobic conditions found on non-covered d pdms. disclosure: nothing to declare. abstract already published. improvements in neutrophil engraftment following changes in freezing method background: in the setting of autologous haematopoietic progenitor cell (hpc) transplants for haematological disorders, peripheral blood stem cells are routinely collected via apheresis and cryopreserved. leicester royal infirmary had been using a controlled rate freezer (crf) to cryopreserve cellular therapy products up until . in a literature review of cryopreservation techniques was undertaken, since the crf required replacement. this review found consistent evidence that cryopreservation using minus o c is comparable to crf, and engraftment times should not be negatively affected by changing to a more simplified method of freezing. there would also be cost saving benefits from switching from a crf to minus o c freezers. methods: as a result two minus o c freezers were purchased following the acceptance of a preparation process dosier (ppd) which was prepared for the human tissue authority. validation was carried out, and from january stem cell laboratory at the lri switched from the crf method to minus o c for cryopreservation of cellular therapy products. briefly, cells are frozen using % dmso (wak-chemie) in g/l human albumin solution (grifols) in cyrobags (origen biomedical, inc). the cells are transferred on cold packs to the minus o c freezer. cells are packaged in between stainless steel heattransfer plates. the plates are placed within a bubble wrap bag, and are placed in a rack within the minus o c freezer, which allows air to circulate freely around each bag. in one plate a el-usb-tc thermocouple data logger (thermosense) is inserted between the bag and stainless steel plate, to record the freezing profile. this data is downloaded after each run. after an overnight freeze at minus o c, the cells are subsequently removed from the bubble wrap and plates, and are transferred to minus o c freezers the following morning. results: a total of patients have had autologous stem cells frozen using this method so far this year. in addition to engraftment data for neutrophils, post-freeze trypan blue viabilities were also compared to the previous year. during a total of patients, who had all their cells cryopreserved, underwent collections. the post freeze median viability was % ( - %). a total of median neutrophil engraftment was . days, with a median cd dose infused of . x /kg. during so far, patients have undergone collections. median viability is . % ( - %). subsequent median neutrophil engraftment is days, with a median cd dose infused of . x /kg. conclusions: ongoing savings of approximately £ per annum have been made by changing our procedure. the benefit of changing to a simplified method of freezing has also resulted in a reduction in staff working overtime. more importantly, this simplified cryopreservation method has resulted in an improvement in neutrophil engraftment times since changing the cryopreservation technique from the previous method using crf to mechanical freezing using a minus o c freezer. disclosure: nothing to declare low doses of granulocytes in the apheresis product predict a better outcome of autologous hematopoietic stem cell transplantation in multiple myeloma patients background: high-dose chemotherapy with autologous stem-cell transplantation (asct) remains the standard consolidation therapy for multiple myeloma (mm). peripheral blood stem cell collection may be contaminated with large quantities of granulocytes and its consequences on the outcome of asct are still unclear. on the other hand, the effect of performing apheresis with high levels of monoclonal component (mc) on outcome is unknown. the objective of this study was to analyze the effect of total nucleated cells (tnc) and granulocytes count (considered as contaminating components of apheresis products) as well as the influence of mc in the apheresis product on outcome of asct in mm. methods: eighty-two patients diagnosed with mm were mobilized with filgrastim μg/kg/day (plus plerixafor if insufficient mobilization of cd + cells on day ). apheresis collection was performed with cmnc program by spectra optia cell separator. cd + count was carried out according to ishage protocol (target: ≥ x e cd +/kg). subsequent cryopreservation were performed according to the local protocol. results: the medians (range) of collected cd +, tnc and granulocytes were . x /kg ( . - . ), . x / kg ( . - . ) and . x /kg ( . - . ), respectively. the medians (range) of infused cd +, tnc and granulocytes were . x /kg ( . - . ), . x /kg ( . - . ) and . x /kg ( . - . ), respectively. a successful collection after first line therapy was performed in % of patients. treatment for mm was continued after carrying out apheresis in % of patients, as per protocol. a significant reduction of mc was observed prior to asct, indicating a further improvement in responses after apheresis (p= . ). an optimal response (cr or vgpr) at the time of apheresis was achieved in % of patients and a suboptimal response (partial or minimal response) was observed in the remaining %. undergoing apheresis in optimal response did not result in lower number of tnc or granulocytes in the harvest. the subtype of mc did not influence on the number of tnc and granulocytes in the product of apheresis. no differences in collected tnc and granulocytes were observed when plerixafor was used as mobilizing agent. the type of chemotherapy given prior the apheresis did not have influence on the characteristics of the apheresis product. a significant improvement in overall survival (os) probability ( %ci) was observed when low tnc (< . x figure a ). lower incidence of relapse (p= . ) ( figure b ) and non-relapse mortality (p= . ) was observed in patients who received low granulocyte count in the graft. no significant correlation was observed between the time of engraftment and the number of tnc or granulocytes infused. similarly, no increase in the frequency of the engraftment syndrome was observed when higher number of tnc or granulocytes were infused. conclusions: in our series, low doses of granulocytes in the product of apheresis predicted a better outcome of asct in mm patients. the amount of mc at the time of apheresis did not have influence in the characteristics of the harvest. efforts for avoiding contamination in grafts are important for its impact on outcome of asct in mm patients. disclosure: dkms foundation, pi / (instituto carlos iii) and sgr (grc), generalitat de catalunya. background: our initial experience (from to years) with hematopoietic stem cell transplantation (hsct) from mud with tcrαβ+/cd + graft depletion in patients with cgd showed high rate of secondary graft dysfunction, the incident of graft rejection was %. to improve the outcome, we hypothesized that the use of plerixafor and g-csf as additional agents in conditioning regimen would offers advantages and better outcomes. this trial was registered at www.clinicaltrials.gov (nct ) methods: between april and september , patients with cgd underwent allogeneic hscts from mached unrelated donors with tcrαβ + / cd + graft. the conditioning regimen included -treosulfan g / m , fludarabine mg / m , thiotepa mg / kg, g-csf mcg / kg and plerixafor mcg / kg. all patients received rabbit atg -timoglobulin® ("genzyme europe b.v.", the netherlands) mg/kg for serotherapy. in all cases, tcrab +/cd + graft depletion was used with the immunemagnetic method (miltenyi biotec, bergisch gladbach, germany) according to the manufacturer's instructions. stem cell source was g-csf mobilized peripheral blood stem cells in all cases. posttransplant gvhd prophylaxis was not performed. minimal follow up was days after hsct. results: neutrophil and platelet engraftment occurred at - days post-hsct in all patients. patients had full donor chimerism in whole blood and in cases we observed predominantly donor mixed chimerism at last follow up. all patients had full donor chimerism in cd + compartment and mixed chimerism in cd + lineage, but it stable without any sign of graft dysfunction. acute gvhd is verified in of cases and was limited to skin grade . all patients are alive for periods from to months post-hsct with good graft function without severe clinical problems. conclusions: we presented first experience of g-csf and plerixafor addition to conditioning regimen before hsct with tcrαβ+/cd + graft depletion in cgd patients . we suppose, the preliminary results are encouraging, as the frequency of primary and secondary graft dysfunction in patients from this group is not observed today, there are no significant toxic complications, as well as clinically severe manifestations of gvhd. currently, the recruitment of patients is continuing, and estimation of the rates of immune reconstitution and a more detail analyses will be evaluated later. background: introduction -it is believed that aboincompatibility is of minor importance in allogeneic stem cell transplantation (allo-sct) and that the clinical outcome is equivalent to abo-compatible sct. therefore, we performed a single center retrospective study to characterize the impact of abo-incompatibility on the outcome of haploidentical stem cell transplantation (haplo-sct). methods: this analysis included consecutive patients who underwent haplo-sct for various hematological malignancies at our center between october and may . we used our institutional database to evaluate details and characteristics of patients and transplant outcomes. results: demographic features of the patients and donors have been summarized in table . all of the patients had advanced hematological disease with a high risk of relapse ( patients with acute leukemia). out of patients, early transplant-related mortality was seen in this cohort of patients. the remaining patients were followed in and months. donor type abo group switch was observed in a median of days ( - days) after transplant. we were not able to show any statistical difference in terms of blood group switch between minor and major abo incompatible transplant. the median red blood cell (rbc) transfusions in the first days for the abo compatible and incompatible transplants were median units (range, - ) and median units (range, - ) (p= . ). no statistical difference was also encountered for the rbc transfusion need for stem cell source, peripheral blood vs bone marrow. a total of patients were followed up for reticulocyte engraftment. the median time for reticulocyte engraftment was days (range, - ) for all patients. reticulocyte engraftment was tended to be faster in minor abo-mismatched group (p= . ) than major or abo-compatible ones. nineteen patients achieved independence from rbc support after a median time of days (range, - days) in abocompatible patients, days (range, - days) in minor abo-incompatibilityand days (range, - days) in major abo-incompatibilitygroup, respectively (p> . ). the engraftman kinetics due to major and minor aboincompatibilitytransplants were presented in table- . pure red cell aplasia was not developed in our cohort. conclusions: the present single center study provides new evidence for the importance of the abo system for erythrocyte recovery in haploidentic stem cell transplantation. it's important to note that, randomize prospective and larger studies are warranted. disclosure: nothing to declare low dose of anti-t lymphocyte globulin protects against severe forms of graft versus host disease in patients undergoing allogenic stem cell transplantation background: graft versus host disease (gvhd) is the most important complication after allogeneic stem cell transplantation (allosct). optimal dose of different anti-tlymphocyte globulin (atg) formulations in this setting has not been established yet. the aim of this study was to analyze the impact of a low dose of atg-fresenius (atg-f) in allosct outcomes. methods: we analyzed adult patients who received an allosct for hematologic malignancies from october to march . the gvhd prophylaxis included a total dose of mg/kg ( mg/kg on day - , - and - ) of atg-f for patients who received a graft from peripheral blood, an unrelated donor; with a mismatch, and/or were older than years; associated to a calcineurin inhibitor and mycophenolate mofetil/short course-methotrexate. statistical analysis was performed using spss v. and r software. results: median age was years ( - ) and . % of patients were males. seventy-four percent of patients underwent myeloablative conditioning. the stem cell source was peripheral blood in patients ( . %), % were from unrelated donors ( % mismatched). seventeen ( . %) patients had high risk cmv status (d-/r+) (see image b). engraftment was observed in patients ( . %). primary graft failure occurred in patients ( myelofibrosis, aml). twenty ( . %) out of evaluable patients developed grade - acute gvhd. the cumulative incidence of severe agvhd and moderatesevere chronic gvhd were . % ( % ci, . - . %) and . % ( % ci . - . %), only ( . %) patients developed severe cgvhd. twenty-nine patients ( . %) discontinued immunosuppression before the first year of transplant. the median duration of immunosuppression for patients with moderate-severe cgvhd was days ( - ). at years non-relapse mortality (nrm) was . % ( % ci, . - . %). thirty-nine ( %) patients developed relevant infectious complications. two ( %) patients died within the first days due to gram negative blood stream infection. eleven ( . %) had at least two episodes of cytomegalovirus (cmv) reactivation between day and . three ( %) patients developed cmv gastrointestinal disease, ( %) had probable invasive fungal infection and ( . %), post-transplant lymphoproliferative disorder associated to epstein barr virus. with a median follow up of months for alive patients , the gvhd and relapse free survival (grfs) at one year, overall survival (os) and progression free survival (pfs) at two years were . % ( % ci, . - . %), % ( % ci, . - . %) and % ( % ci, . - . %), respectively. the relapse incidence at two years was . % ( % ci . - . %). complete remission at transplant was associated with better long term survival ( % at years, p < . ). hla disparity did not affect os (see image a). conclusions: the use of low doses of atg-f is protective against severe forms of acute and chronic gvhd in a cohort with high prevalence of unrelated donors and a high median age. this strategy showed good results in grfs, os and pfs in a population at high risk for developing gvhd or relapse. disclosure: nothing to declare performance parameters of a ngs-product for chimerism monitoring -applicable in patients after hematopoietic stem cell transplantation methods: for this purpose, samples from patients with mixed chimerism (mc) with increasing amounts of recipient dna were analyzed and compared using realtime pcr of insertions/deletions (indels), fragment analysis of short-tandem repeats (str) and ngs of indels. results: whereas real-time pcr displayed excellent sensitivity down to , % mc, but poor precision above %, fragment analysis exhibited good precision with limited sensitivity (> , %). in contrast, ngs chimerism demonstrated good sensitivity, with a limit of detection (lod) of , % mc, and precision throughout the whole spectrum of patient/donor mixed chimerism. the ngs chimerism product (devyser chimerism) exhibited at least three (average eight) and at least two (average ) informative genetic markers (indels), suitable for monitoring mixed chimerism of patients with their corresponding matched unrelated ( ) or related ( ) donor samples. in order to establish the performance of the separate techniques for determination of mixed chimerism on retrospective patient samples, a cohort of patient monitoring samples ( - weeks post-hsct) with low (< %), intermediate or high mixed chimerism (> %) were included and analyzed. dna from all monitoring samples was extracted from sorted cell fractions. the results show that although all evaluated techniques are suitable for monitoring patient/donor chimerism after allogeneic hematopoietic stem cell transplantation (hsct), only the ngs chimerism product exhibits high sensitivity (lod , %) and a broad dynamic range (detection range , - %) with good precision and accuracy throughout the whole spectrum of mixed chimerism (% patient/donor). in addition, the ngs chimerism product employ non-population dependent highly informative genetic markers providing stable resolution power and thus suitable for monitoring mixed chimerism. disclosure: dan hauzenberger is medical adviser at devyser ab and shareholder in devyser holding gender distribution: male - % (n= ), female - % (n= ). age median - , years old ( months - ). stem cell source: bone marrow - % (n= ), peripheral blood stem cells - % (n= ). patients ( %) received / matched unrelated donors hematopoetic stem cells transplantation and patients ( %) - / matched unrelated donors hematopoetic stem cells transplantation. differences in the antigen blood system: single group % (n= ), minor % (n= ), major % (n= ), mixed % (n= ). age of donor: - years old - % (n= ), - - % (n= ), - - % (n= ), and more % (n= ). gender differences in donor/recipient: male/female % (n= ), female/male % (n= ), one sex % (n= ). we also took into account the impact of gender difference and cytomegalovirus serostatus in the donor/recipient pair. results: in / group the estimated probability of overall -years survive was % and in the / group -years survive was %. the increase in donor age of years reduces the -years survive by - % (p= , ), however, the -years survive from donors over years old was %. we have found no difference between -years survive in transplants from donors that are compatible/ incompatible with the antigen blood system, cytomegalovirus serostatus, or the gender differences in donor/ recipient. in the study of donor-related factors, we found the negative impact of an human leucincompatibility ( / ) on the incidence of chronic gvhd - % (p = . ). the combination of cytomegalovirus positive serostatus of the donor and the negative status of the recipient increases the risk of primary graft rejection up to %, in comparison with others (p = . ). conclusions: our study showed the role of genetic matching on the hla system between the patient and the unrelated donor, and the donors age value. / transplants have better outcome and lower incidence of severe a. gvhd and ch. gvhd. younger donor increases -years survive, but there is a significant increase in -years survive if the donor is over years old. disclosure: nothing to declare allogeneic stem cell transplantation in chronic myelomonocytic leukemia. a single center experience nine patients ( %) relapsed with a median of , months ( - ) with different strategies at this point: in all cases we modulated immunosuppression, in cases as the unique strategy, in cases with donor lymphocyte infusion (dli), in cases we employed hypometilating agents (hma) and in cases with intensive chemotherapy, reaching cr only in two patients, one of them after dli and the other one after hma and consolidation with a second asct. eleven patients ( %) died being the relapse the main cause ( %). transplant related mortality (trm) at + were % and global trm were %. in the last follow-up, patients ( %) are still alive, ( %) in cr and ( %) in relapse situation. with a median follow-up of months ( - ), the event free survival (efs) were months ( - ) and the overall survival (os) were months ( - ). we observed advantage in terms of os in those patients that reach cr at + post asct and in those who develop chronic gvchd (p= . and p= . respectively) conclusions: asct is still the only curative option despite the high relapse rates. to reach cr at + post asct and the development of chronic gvhd seems that they confer advantage in terms of os. the importance of knowing the molecular profile of the entities that we consider for asct. disclosure this study documents a first experience of a cell processing lab seeking to integrate process automation technology to wash and volume reduce products which can account for the initial material source volume variability, product characteristics, and number of bags. methods: here we report the pre-clinical assessment of the lab's initial work with the lovo cell processing system for a product experience over days with machine. this study used products intended for destruction. the workflow used parallel and sequential processing schedule. after water-bath thawing, bags were sampled, weighed to determine volume, and subsequently connected to lovo or pooled into a transfer pack and then connected to lovo. the bags were then diluted : at ml/min with lovo at + - c using % hydroxyethylstarch / . (voluven, fresenius kabi) and processed using a cycle procedure. after processing the bags were weighed for volume, sampled, and stored in a - c refrigerator in their lovo final product bags. samples were assessed from t= to t= hours. results: cd + viability and absolute counts were determined using flow cytometry. processing duration and solution volume consumed was determined by the lovo's sensors and confirmed by the operator. data is presented as a percentage relative to the post-thaw values. note, the values presented are not total process yields. the results focus on the lovo processing step. conclusions: the operators easily integrated into the software to drive the machine. the machine demonstrated it's flexibility with a wide-range of volumes, cell-inputs, and number of bags. the lovo produced products which meet our specifications in a quick and reliable manner. further work on this platform will be performed to validate and qualify this system for production use. properties. the aim of this study was to evaluate prospectively the efficacy of a fbm protocol for the prevention of om in patients undergoing a hsct. methods: all patients consecutively who underwent a hsct at our center from x onwards received five weekly fbm sessions with a bidiodic laser (lumix ®, prodent, italy), which simultaneously emitted at nm and nm with a power of mw and energy of j per point. the procedure started the day before the beginning of the conditioning regimen up to the tenth day post-transplant. the laser was applied in a defocused mode on each of the mucosal surfaces ( areas). at each session, the morphine dose, the om level (according to the who scale) and pain through a numerical rating scale (nrs) were recorded. results: consecutive patients ( male/ female) submitted to a hsct were analyzed. the median age was years (range - ). eighteen patients had acute leukemia, myelodysplastic syndromes, lymphoproliferative diseases. the median number of treatment lines before hsct was (range - ). at transplant, patients had advanced disease. the myeloablative conditioning regimen mac (thyotepa, busulphan, fludarabine) was employed in patients; the same conditioning, with a reduced dose of busulphan (ric), was infused in patients. seven patients ( %) had no evidence of om. the incidence of grade ii-iv om was % in the group of patients receiving mac and the median duration days (range - ); grade om was observed, for day, in patient. in the ric group the incidence of om was %, the median duration days (range - ); no patient had evidence of grade iv om. in the whole population, the maximum nrs value was . morphine administration was required in patients, due to the occurrence of non-oral complications. conclusions: in our experience, prophylaxis with fbm to prevent or reduce om was safe and effective, compared to results of previous experiences reported in the literature, which used no prevention against this complication that negatively affects the quality of life of transplanted patients. further studies on a large series of are necessary to confirm our results. disclosure: nothing to declare background: cytogenetic abnormalities are an essential part of prognostic systems in myeloid malignancies before hematopoietic stem cell transplantation (hsct), however, their role in posttransplantation prognosis is unknown. the aim of this study was to assess the prognostic impact of genetic risk stratification of aml and mds patients on posttransplantation course, which could be an additional tool in making decisions regarding preemptive therapy. methods: a retrospective analysis covering patients treated with allo-hsct between and . cytogenetic studies included karyotyping (c-and gbanding) and fluorescence in situ hybridization (fish). the number of analyzed cells exceeded european cytogenetics association guidelines (for each fish at least interphase nuclei were analyzed). cytogenetic risk group in aml was assessed based on the eln criteria. patients with mds were stratified into three groups; favorable (good and very good prognostic score), intermediate, and adverse (poor and very poor) prognostic score according to ipss-r . interestingly, the poorest survival was in patients with monosomy of chromosome , which was present in patients of whom succumbed to refractory disease, while all patients who had deletion of long arm of chromosome (del q)-are alive at the time of writing of this report after a median follow-up of months ( - ). relapse was diagnosed in patients ( %), including; ( %) with adverse, ( %) with intermediate and ( %) with favorable cytogenetic risk. among patients with a complex karyotype and/or cytogenetic evolution prior hct: patients ( %)relapsed, including ( %)-who died. follow-up cytogenetic studies in relapse after transplantation were performed for patients; of them ( %) had clonal evolutions of the original karyotype with additional abnormalities-( % died) and ( %) had new aberrations in cells without primary changes (all died). in patients ( %) ( unfavorable, intermediate group) cytogenetic relapse was diagnosed by fish analysis and they were treated with azacitidine (+/-dli) achieving cr (n= , %), stabilization-(n-= , %), transient response (n= ), while deceased). conclusions: cytogenetic studies in patients after transplantation may facilitate assessment of mortality. karyotype may undergo cytogenetic evolution after allo-hsct. patients with monosomy of chromosome seem to have a particularly poor prognosis. transplanted patients are vulnerable to new cytogenetic alterations. disclosure: nothing to declare methods: the primary end-points were the rate of complete response (cr), defined as no emesis and no nausea without rescue medications, for both acute (cr- ) and delayed (cr - ) cinv and rate of post-transplant complications until discharge. we prospectively analyzed patients undergoing autologous ( %) and allogeneic ( %) stem cell transplantation and receiving cinv prophylaxis with nepa and dexamethasone (schedules shown in fig. ). in our series, patients ( %) were female. patients median age was years ( - ). the most frequent diagnosis were myeloma ( %) and lymphoma ( %), while % of patients were diagnosed with aml or mds. myeloma patients received one day hd-ct with melphalan ( % mel / % mel ). lymphoma patients were conditioned with feam ( , %) or tt_flu_edx ( , %) hd-ct. busulfan-based mds-ct regimen was offered to aml/mds patients. results: the incidence of cr- and cr - observed was % ( / ) and , % ( / ), respectively. more than grade nausea and vomiting (according to ctae- ), was reported in % ( / ) and % ( / ) of patients, respectively. female sex was associated with an increased risk of acute (hr , ; p , % ci . - . ) but not delayed (hr , ; p , % ci - . ) cinv. similar rate of cr and cr - was observed in one-day hd-ct ( % and %) compared to mds-ct ( % and , %) group (pns). median lenght of stay was days ( - ). no case of cardiotoxicity and no exitus was observed. the incidence of febrile neutropenia was % ( % fuo; % sepsis; % pneumonia). only one patient experienced an agvhd on day + . neutrophil (> /mcl) and platelet (> /mcl) recovery occurred in median on day ( - ) and on day ( - ) respectively. conclusions: nepa seems to be safe and effective in preventing acute and delayed cinv in patients receiving both one day hd-ct and mds-ct as conditioning regimen for hsct. more studies are needed to define the better -ht ra and nk- ra combination and the better schedule in transplant setting. disclosure: "nothing to declare background: vod and ta-tma represent two early endothelial complications occurring after allogeneic stem cell transplantation (sct) sharing many pre-transplant risk factors. the aim of our study is to evaluate the impact of donor graft composition, engraftment kinetics and infections on the development of these endothelial complications (ec). methods: we retrospectively reviewed consecutive sct recipients at our institu-tion between january and june . the median age was years (range - ). acute leukemia was diagnosed in patients ( %). complete remission was documented in % of patients at transplant. donor source was from hla mismatched donor in % and from unrelated donor in % of the patients. hbv positive patients were % of the sample. conditioning regimen was busulfan based in % of patients. ursodeoxycholic acid and unfractionated heparin were given to all patients as vod prophylaxis. cyclosporine was used as gvhd prophylaxis. lymphocytic subpopulation analysis (cd +, cd +, cd + and cd +/cd +) and cd + cells count on the donor graft were performed using bd facs cantoll. all patients had routine monitoring for ebv and cmv pcr, hemolysis tests, creatinine and electrolyte panels, proteinuria, complete blood count, blood pressure and schistocytes by direct examination until day + . the fisher's exact test was used to compare categorical variables, while continuous variables were analyzed with anova test. diagnosis of vod and ta-tma were carried out by using ebmt and cho criteria respectively. results: the incidence of very severe vod and tma was % ( / ) and , % ( / ) respectively. cmv reactivations with viral load over . cv/ ml was % and % in patients with and without ec, respectively (p , ; hr , p , %ci , - , ). the median day to neutrophils (ns) engraftment ( /ml and /ml) was vs and , vs in vod/tma group vs control group (p , and , , respectively). more rapid neutrophils engraftment (ns > /ml and ns> /ml within days) was related to a higher risk of ec with a hr of , (p , ; %ci , - , ) and , (p , ; % ci , - , ). patients with ec received a donor graft with a higher median numbers (x e /kg) of cd + and cd + (p> , ) and a lower numbers (x e /kg) of nk cells (p> , ). patients who received a cd + cells count > x e /kg and nk cells count < , x e /kg presented a relative risk of ec of , (p , ; % ci , - , ) and , (p , ; % ci , - , ), respectively. there were no differences with respect to the other analyzed variables between patients who developed vod/tma compared with those who did not. (pic_ ) conclusions: cmv viremia, early neutrophils engraftment and donor nk and cd + cells infused are associated with the risk of vod and tma. very few studies evaluated the link between these variables and the risk of developing such two complications. it could be interesting to investigate these relationships on larger series. clinical trial registry: not applicable disclosure: nothing to declare p when the last hope turns out to be just as good as best: haplosct following tbf conditioning, pt cyclophosphamide and tacrolimus as gvhd prophylaxis background: hematopoietic stem cell transplantation is an effective therapy for a variety of severe hematological diseases. in last decades, haploidentical sct (haplosct) followed by ptcy as gvhd prophylaxis has been reported as a valid alternative for patients who lack an hla matched donor. we therefore analysed outcomes with this. methods: patients without hla-matched donor received a haplosct between / and / . thiotepa mg/kg ( days), fludarabine mg/m ( days) and oral busulfan mg/kg/ h ( days,with pkd dose adjustments) was used as conditioning regiment; in patients > years busulfan administration was limited to two days. gvhd prophylaxis consisted of cyclophosphamide mg/kg on day + and + , and tacrolimus as a continuous iv infusion from day + . all patients received pbsc as the stem cell source. outcomes analysed were overall survival (os), progression free survival (pfs); cumulative incidences (ci) of gvhd, relapse and non-relapsed mortality (nrm). results: median ages was (range - ). % male and % female. diagnoses were: aml ( %), mds ( %), all ( %), hl and nhl ( %), and mm ( %). % (n ) were transplanted in early disease status, while most cases ( %) were in advanced status, including, second/third cr ( %, n ), one ( %) in aplasia without progressive disease and % (n ) had active/progression disease, % (n ) had stable disease; four cases were second alosct. thus, % of patients had a high or very high rdri and % had intermediate. ebmt score was ≤ was in % of patients (n ) . the donor was as son/ daughter in %, % a sibling and % a patient's mother. median time to neutrophil ( . x e /l) and platelet (> x e /l) recoveries were + and + days, respectively (g-csf was not used). only one patient had a primary graft failure attributed to anti-hla donor specific antibodies. median follow up in survivor is months (range - ). overall survival is ± . % (at months) and ± % (at months). pfs is ± % and ± % respectively. the cumulative incidence of relapse was % and %, respectively, while nrm is % at months. day + , grade - acute gvhd were %, while mild/ moderate and severe chronic gvhd were % and % respectively. ebmt ≤ and first alosct were the only variables to clearly impact -months os in univariate analysis. a combined covariate of ebmt ≤ -no prior alosct vs other patients showed a month os ± % vs ± % (p . ), pfs ± % vs ± % (p . ) and nrm % vs % (p . ) but without impact on relapse % vs % (p . ). conclusions: haplosct with an age-adapted tbf conditioning regimen, pbsc and ptcy followed by tacrolimus, led to very encouraging results, mainly in patients with a low ebmt score and as a first alosct. although formerly considered as a last alosct strategy, we now agree that the time has come to compare this strategy with hla mud (and even elderly sibling donors) in ongoing prospective randomized multinational trials. disclosure: nothing to disclosure background: autologous hematopoietic stem cell transplantation (autosct) for acute myeloid leukaemia (aml) is increasing becoming a viable option for an increasing number of patients due to limited availability of matched sibling or unrelated donor for allogeneic hematopoietic stem cell transplantation (allosct). we examined the relevant long-term outcomes in our local patient cohort. methods: we retrospectively reviewed the data for all autosct done for aml in our centre over a -years period between st january until st dec from our electronic record. patients with acute promyelocytic leukaemia (apml) were excluded from this analysis. patients were further stratified based on the number of high risk features present; not achieving complete remission (cr) following induction chemotherapy, high presenting total white cell count (wbc > x /ml, adverse cytogenetics (example: complex cytogenetics) and adverse molecular mutations (example: flt -itd & mll gene arrangement). outcome data including mortality (overall survival (os) and non-relapse mortality (nrm)) and morbidity (leukaemia free survival (lfs)) were recorded and analysed. results: a total of patients were identified. median age at diagnosis is -years old. the cohort comprised of males and females. the overall median os and median lfs is . years and . years respectively. the nrm is . % ( / ). there was no difference in the median os and median lfs for the patients achieving cr following induction chemotherapy and those not in cr following induction chemotherapy; . years versus . years (log-rank, p= . ) and . years versus . years (log-rank, p= . ) respectively. the median os were statistically significant for patients with zero versus one and two and more high risk features present; . years versus . years versus . years (log-rank, p= . ) respectively. however, the median lfs were not statistically significant for these three patient cohorts; . years versus . years versus . years (log-rank, p= . ) respectively. conclusions: in our patient cohort, autosct appeared to be a feasible option for patents with aml without matched sibling or unrelated donor available. disclosure: none to declare methods: between - , thalassemic patientsunderwent hisct. the median age of patients was ( - )years with male preponderance (n= , . %). across the gender and abo mismatch transplants were done in . % and % of patients. stem cell source was bone marrow in ( %) while peripheral blood in ( %) of patients. mean stem cell dose was . ± . x cells / kg and mean volume of product was ± . ml. preparative regimen included anti-thymocyte globulin, busulfan, fludarabine and cyclophosphamide.graft versus host disease (gvhd) prophylaxis comprised of posttransplant cyclophosphamide on day + & + followed by tacrolimus and mycophenolate mofetil. patients were observed for hematopoietic recovery (neutrophil and platelet engraftment) and transplant related mortality including acute and chronic gvhd for skin, gut, liverand lungs, primary and secondary graft failure and infectious complications. results: nine( . %) of sixteenpatients were engrafted with full donor chimerism. twelve ( %) patients belonged to pesaro class i and ( %) toclass ii patients. median time to neutrophil and plateletengraftment were ( - ) and ( - )days respectively. average number of packed red cell and platelet transfusions were . ± . and . ± . respectively.primary graft failure was observed in ( %) and secondary graft failure was observed in ( %) patients. two patients received a second dose of stem cells and they engrafted at and days of infusion respectively. of patients with primary graft failure died, one with sepsis (day + ) and the other because of intracranial bleeding (day + ).acute gvhdof gut and skin (grade ii-iii) was observed in patients each, within first days post-transplant. none of the patients had grade iv gvhd. cytomegalovirus reactivation occurred in % of patients, all of them received pre-emptive therapy with intravenous ganciclovir. none of them developed cmv disease. invasive fungal infection was not observed in any of the patient. culture proven bacterialinfection was documented in % of patients requiring intravenous antibiotics during first days post-transplant.overall survival and relapse free survival were . % and . % over a median follow-up of ( - ) days. conclusions: haploidentical transplant is a suitable modality for thalassemic patients lacking a full matched donor in pakistan. in view of our results, we suggest that thalassemia patients should be offered hisctas an option for cure. clinical background: gemtuzumab ozogamicin (go) is an anti-cd monoclonal antibody with significant activity in de novo and relapsed/refractory (r/r) acute myeloid leukemia (aml). a relevant side effect consists of hepatotoxicity and especially sinusoidal obstruction syndrome (sos). the objective of this study was to analyze tolerability of go during the induction and reinduction therapy in patients with aml, and its possible impact on subsequent hematopoietic stem cell transplantation (hsct). methods: from to , patients who had received go in three hospitals were collected and their medical records were retrospectively reviewed. results: fourteen patients diagnosed with de novo aml received go ( mg/m ) on day + in combination with standard chemotherapy (idarubicin and cytarabine, x schedule) as induction therapy. hyperbilirubinemia (bilirubin > . unl) was detected in patients and increase of aspartate aminotransferase (ast) (> . unl) in . twelve patients achieved complete remission (cr) and one was refractory ( not evaluated). in the r/r setting, patients diagnosed with aml (n= ), biphenotypic acute leukemia (n= ) and acute promyelocytic leukemia (n= ) received go as rd or subsequent rescue therapy either as monotherapy (n= ) or in combination with cytotoxic chemotherapy (n= ). prior hsct was performed in patients (autologous [n= ], allogeneic [n= ] ). rescue therapy was indicated for refractoriness (n= ), relapse (n= ), partial response (n= ) or absence of donor (n= ). four patients received doses of go ( mg/m ) and patients, one dose. hyperbilirubinemia (> . unl) was observed in patient and increase in ast (> . unl) in patients. seven patients achieved cr, was refractory, obtained partial response and died early during induction). thirteen patients received subsequent hsct (autologous [n= ], allogeneic [n= ]) after go therapy ( in the de novo aml and in the r/r group). the reasons for not performing hsct in the remaining patients were: low cytogenetic risk (n= ), active chronic graft versus host disease (gvhd) in previous hsct (n= ), early death during treatment (n= ), relapse (n= ), severe complications in rescue treatments (n= ), and unknown (n= ). the conditioning regimen was myeloablative (n= ), non-myeloablative (n= ) and sequential (n= ), and the donors were matched sibling (n= ) or unrelated (n= ) . cyclosporine, methotrexate and thymoglobulin were administered as gvhd prophylaxis in patients and cyclosporine, mycophenolate and thymoglobulin in . hyperbilirubinemia was observed in patients belonging to the de novo aml group. death after hsct occurred in patients due to infection (n= ), relapse (n= ), gvhd (n= ) and traffic accident (n= ). three patients are currently alive in remission. no sos was observed in any patient. conclusions: in both de novo and r/r aml the administration of low dose go is feasible and does not have impact on subsequent hsct outcome. although some degree of hepatotoxicity was observed, no cases of sos were observed, either before or after hsct. disclosure: supported by grants from: asociación española contra el cáncer, aecc (gc biga), instituto carlos iii (pi / fi), -sgr (grc), cerca program from generalitat de catalunya, and "la caixa" foundation. outcome of allogeneic hematopoietic stem cell transplantation in patients with benign hematological disorders saqib ansari , tahir shamsi , uzma zaidi , saima siddiqui , tasneem farzana background: allogeneic hematopoietic stem cell transplantation is a potentially curative treatment modality for hematological disorders. we evaluated the outcome of patients suffering from benign hematological disorders, including aplastic anemia, fanconi's anemia and thalassemia after matched related allogeneic transplantation. methods: all patients having hematological disorders including aplastic anemia (aa), beta thalassemia (btm), fanconi's anemia (fa) and severe combined immune deficiency disorder (scid) with hla identical related donors who underwent allogeneic transplantation were included. donors were given g-csf at a dose of μg/ kg/day daily for four days prior to harvest. the conditioning regimens for thalassemia included cyclophosphamide (cy) + busulfan (bu) in ( %), bu + cy + thiotepa in ( %) and bu + cy + antithymocyte globulin (atg) in ( %). conditioning regimens for aplastic anemia included, fludarabine (flu) + cy in ( %), flu + atg in ( %) and cy in ( %), cy+ atg in ( %) patients. for fanconi's anemia flu + atg in ( %), flu in ( %), cy + atg in ( %) and flu+ cy + atg in ( %). flu + atg in ( %) and cy given in ( %) and no conditioning regimen was offered to ( %) patients with scid. results: a total of allogeneic transplants were performed for benign hematological disorders including aa (n= ), btm (n= ), fa (n= ) and scid (n= ) from to july . median age was . years (range . - ). across the gender and abo blood group transplants were ( . %) and ( %). the median time to neutrophil and platelet recovery was days (range: - ) and (range: - ). primary and secondary graft failure was observed in ( . %) and ( . %). overall survival in aplastic anemia ( / , %), beta thalassemia ( / , %),fanconi's anemia ( / , %) and severe combined immune deficiency disorder ( / , %). eighty four patients expired ( . %) among them patients expired within days post transplant main cause of deaths included sepsis ( %), multi organ failure ( %) and gut gvhd ( %) conclusions: in developing world scenario where non malignant disorders are leading cause of morbidity and mortality. bone marrow transplantation has been successfully implemented with better long term diseases free survival and quality of life. clinical background: hematopoietic cell transplantation (hct) remains the only curative therapy for many diseases, yet transplant survivors carry an unusually high burden of morbidities, primarily because of exposure to intense chemotherapy, radiation and /or gvhd. this study aimed to evaluate the burden of chronic diseases at the end of life after allogeneic-hct and to identify the disability-adjusted life years (daly). methods: the pubmed, medline, and ovid databases were queried utilizing specific mesh terminology (post, allo stem cell, hematopoietic, bone marrow, transplantation).we collected data on the impact of the hct on the variables affecting survivor's health in all aspects .the rates of late complications were compared to the risks in the general population (united states). results: a total of studies fulfilled the selection criteria totaling to patients (table ) . median os at -year mark varied widely between studies from % to %. majority of the patients at -year mark were found to have new comorbidities thereby indicating a huge burden of late effects at the end of life, though exact dalys could not be calculated due to incomplete data. hct survivors were found to have higher risk of premature arterial disease (pad) at . % compared to the general population, however gvhd or the addition of tbi to the conditioning regimen were not found to be significantly associated with pad. regarding the risk of new cancers, the cumulative incidence of their development at and years was . , and . , respectively. increased risks compared with the general population were seen for some solid cancer including cancers of the lip: p= . , tonsils: p= . , oropharynx: p< . , bone: p< . , soft tissue: p< . , and vulva: p= . . with respect to mental health, depression was prevalent in ( . %) survivors, in whom ( . %) were still on antidepressants at the last follow-up. cognitive impairment and other psychiatric disorders were found in ( . %) and ( . %) survivors, respectively. the most common cause of nrm in the first years was gvhd. however, after years, the leading cause of death in those conditioned with mac regimen was secondary cancer, but in the ric group, new cancers and gvhd contributed equally. conclusions: hct survivors remain at risk of significant complications which lead to premature death and their burden of comorbidities at the end of life is significantly more than that of general population. background: late onset hemorrhagic cystitis (hc) is a common complication of hematopoietic stem cell transplantation (hsct) frequently associated with reactivation of bk virus (bk-hc).there is no consensus as to the best therapy for bk-hc, and many different treatments have been reported. hyper baric oxygen therapy (hbot) is used as primary or adjuvant therapy in diverse clinical situations involving hypoxic injury to tissues and has been explored as a useful tool in treating bk -hc. we report our experience with hbot in combination with non-invasive supportive care in children and adolescents suffering from bk-hc following allogeneic hsct. methods: the computerized database of schneider children´s medical center of israel was reviewed for all patients aged to years who underwent hsct between january and june and developed bk-hc. hbot therapy consisted of hours sessions at atmospheres, with patients breathing oxygen by mask. parents accompanied patients during the treatment. results: fourteen patients with a variety of underlying diseases received (hbot) for treatment of bk-hc following hsct. the initial treatment for children with bk-hc at our center prior to included continuous bladder irrigation and intravesicular instillation of various medications. beginning in , we adopted a non-invasive strategy that included the administration of oral anticholinergics (oxybutinin), systemic pain management, hyperhydration and the administration of weekly cidofovir with probenecid. hbot was administered to patients who failed the above regimen. with this protocol, the average time of starting hbot dropped from (prior to ) to . days. the median onset of hc was days post hsct. all patients were receiving immunosuppressive treatment at the onset of bk-hc. all patients suffered from macrohematuria with blood clots (grade iii cystitis), ( . %) experienced severe dysuria and ( . %) urgency. bk viruria was present in all patients, and concurrent bk viremia was detected in % of those who were tested. patients reported symptomatic improvement at a mean of . days following the initiation of hbot. no patient experienced serious adverse effects due to hbot, but two patients required insertion of tympanic ventilation tubes. eleven of our patients ( . %) experienced complete remission of bk-hc following hbot, with an overall response rate of . % ( / patients).eight of our patients ( . %) eventually succumbed due to either hsct complications or disease relapse. conclusions: hyperbaric oxygen therapy is a safe, effective, non-invasive and well tolerated treatment modality for bk -hc and should be considered for first line therapy for this complication of hsct. clinical background: every year, almost one thousand cases of hematological malignancies in pediatric population are reported in peru. allogeneic hematopoietic stem cell transplantation (allo-hsct) is an alternative strategy in many of these cases. only between - % of the pediatric population that requires a hsct has a compatible human leukocyte antigen (hla) donor. the remaining % have to access international donor registries, extending the awaiting time and conditioning the progress of the disease. allo-hsct has the potential to help children with several hematological disorders with non-compatible hla donor. hla genotypically identical sibling donors are the best option when pursuing an hsct. nevertheless, patients' alternative sources of stem cells could be obtained from an haploidentical donor like one of their parents. the haploidentical transplantation program with macs was implemented in peru in to reduce the risk of graft-versus-host disease (gvhd), support the immune system reconstitution and to expand pool of donors. it allows patients to access a treatment that is efficient and safe, as shown in the depletion of positive tcrα/β+ and b cells procedures for allo-hsct. methods: the mobilized leukapheresis products (n= ) of haploidentical healthy donor was washed to remove platelets and preparations were performed according to miltenyi's clinimacs® manual for tcrα/β+ and cd + cell depletion. analysis of the initial leukapheresis product and tcrα/β + and cd + depleted graft (target and non-target product) was performed using flow cytometer. cells were analyzed for cd +, cd +, -aad, cd +, tcrα/β+, tcrγ/δ +, cd + and cd + with fluorochrome-labeled antibodies from miltenyi biotec using a novocyte cytometer. the results obtained with the ex vivo t-cell depleted allo-hsct procedures show an overall survival (os) over % with an ic % at the end of the first year with low incidence of gvhd. macs of tcrα/β+ and cd + cells are effective (logp . and logp . , respectively) obtaining minimum levels of depleted lymphocytes within clinical established parameters for diverse pathologies. in addition, tcrα/β+ and cd + cell depletion does not significantly affect hematopoietic stem cell populations such as cd + and cd + cells or tcrγ/δ+ cell population. cd + and tcr γ/δ cells are highly recovered ( . % and . , respectively), which contributes with a better engraftment after allo-hsct. conclusions: peruvian results oscillated within european ranges with an os over %. our data suggests that the macs method is an efficient, effective and safe strategy for haploidentical hsct which has a remarkable cost-benefit ratio and makes it viable in countries of the latin american region with peruvian socio-economic characteristics. evaluation of genoresistance for viral reactivation treatment has been implemented as a strategy to improve os. better results are achieved in patients after allo-hsct with the validation of these tests in peru. preliminary pharmacoeconomic evaluations allow us to establish magnetic activated cell sorting (macs) as a promissory strategy compared to other alternatives for haploidentical hsct. it is necessary to increase the number of procedures in order to confirm efficacy and safety of macs in a larger population. disclosure: all authors have no conflicts of interest. abstract already published. micro-costing study of hematopoietic stem cell transplantation in two hospital institutions from southern of brazil background: hematopoietic stem cell transplantation (hsct) is a potentially curative treatment indicated for patients with onco-hematological, hereditary and immunological diseases. considering the increase of patients indicated to the hsct and the lack of knowledge about the costs resulting from this treatment, is important to identify and detail the resources consumed in each phase of hsct and provide knowledge to the public health brazilian system. we aimed measure the total cost of related hsct, based on micro-costing study of patients assisted in two hospitals in the south region of brazil. methods: hsct costs were estimated using the timedriven activity based costing method (tdabc), which measured cost of services / products based on actual consumption of resources. we collected data from medical records of patients submitted to allogeneic hsct in from public and private (philanthropic) hospitals. we interviewed professionals involved in the tcth activities, we performed chrono-analysis and, we consulted financial and administrative systems reports of hospitals. in order to compare costs according to clinical complications observed in patients, we grouped into two ranges of complexity: low/ medium and high. the study was divided into stages: hsct processes mapping; costs measurement; and analysis of results. finally, the costs were compared: by activity, by resource and by hospital. this study was financed by psid-uhs, by an agreement signed between ministry of health and moinhos de vento hospital, through adjustment term number: / , and approved by research ethics committees. results: from the hsct processes mapping, the following steps were defined: (i) hospitalization; (ii) conditioning; (iii) transplantation; (iv) period of aplasia; (v) engraftment; (vi) observation; (vii) pre-and medical discharge. seven patients were classified in low / medium complexity level, with hospitalization median time of days and an median cost of usd , . , whereas the other five patients, classified as high complexity, presented median time of days and median cost of usd , . . the hsct costs evaluation identified that steps ii and iv presented greatest cost in high complexity patients. lower complexity patients presented, in steps ii and iv, median costs of usd , . while in higher complexity usd , . . in addition, median costs of materials and drugs were usd , . and usd , . in lower and higher complexity patients. conclusions: tdabc method allowed the identification of the moment when patients consume the most resources. of all the hsct stages, periods of conditioning and aplasia presented higher costs, representing . % of the total hospitalization value. in these stages, higher complexity patients presented three times higher the median cost. the resources that had the greatest impact were medicines and medical materials, costing times more than lower complexity patients. conclusion: this study allowed a detailed identification of the hsct costs in patients with different complexity ranges in two hospitals from southern brazil. therefore, the identification of service demand regarding the clinical complexity, allows the generation of important information for the management of the best care in the health service. disclosure background: immunodeficiency due to lrba deficiency is characterized by hypogammaglobulinemia and autoimmunity. hemolytic anemia, lymphadenopathy, autoimmune hepatitis and, above all, autoimmune enteropathy are the fundamental characteristics of these patients together with the history of recurrent invasive infections.the therapy includes immunosuppressants, endovenous immunoglobulins. bone marrow transplantation is the final therapy of these patients, especially in the most serious cases and in recent years, also on the light of increasingly targeted conditioning regimes, it is associated with ever better prognosis. methods: we present the case of caterina, an -year-old patient with lrba deficiency, diagnosed at years of age for a history of hypogammaglobulinemia, recurring invasive, bacterial and fungal infections and a picture of autoimmunity represented by ahia, myelitis (c -c ), autoimmune hepatitis and enteropathy in treatment with abatacept and sirolimus. it also presents leptin deficiency lipodystrophy.she presented to our observation for ostemielitis in multiple outbreaks (tibia, femur and left knee) secondary to sepsis from mrsa. broad spectrum antibiotic therapy and curettage surgery were performed during hospitalization.after months of broad-spectrum antibiotic therapy (daptomycin, rifampicin, ceftaroline, cotrimoxazole, levofloxacin, dalbavancin) there was resolution of the infections.because of the seriousness of the disease it is therefore decided to subject catherine to hematopoietic stem cell transplantation. results: therefore were performed bone marrow transplant from mud after conditioning at reduced intensity, delayed in days, with treosulfan, fludarabine and thiotepa. prophylaxis for gvhd was performed with atg ( mg / kg / day), sirolimus (already practiced for enteropathy) and mmf. because of the hepatic picture, we also performed prophylaxis for vod with defibrotide for days. transplantation was performed by peripheral stem cells with x ^ cd / kg and x ^ cd / kg.the patient had always presented good general conditions with engrafment of the pmn to the d + and the plt to the d + . the chimerism at d + was % donor both on pmn and on pbl. prophylaxis for gvhd was changed on d + by replacing the sirolimus with tacrolimus for the appearance of grade i cutaneous gvhd . conclusions: we have successfully performed bone marrow transplantation in patients with lrba deficiency. the new antibiotic molecules, used to induce infectious remission, the new low-intensity regimens, the prevention of the most fearful complications (vod) have been the key to success in such complicated case. the high number of cd cells infused with a controlled number of cd were the key then of rapid engraftment with minimal gvhd readily controlled by the immunosuppressant. disclosure background: in the absence of hla-matched related donor, allogeneic stem cell transplantation from haploidentical donors are potential alternatives for patients with hematological malignencies with an indication to allogeneic stem cell transplantation. herein, we retrospectively assessed the outcome of haplo-sct for patients with refractory hematological malignancies. methods: this analysis included consecutive patients who underwent haplo-sct for various hematological malignancies at our center between october and may . we used our institutional database to evaluate details and characteristics of patients and transplant outcomes. results: demographic features of the patients and donors have been summarized in table . all of the patients had advanced disease with a high risk of relapse. the majority of patients underwent haplo-sct from their parents. out of patients, early transplant-related mortality was seen in this cohort of patients. four patients treated with second haplo-sct and recovered hematopoiesis after second transplant. the remaining patients were followed in a median of months. donor type abo group switch was observed in a median of days ( - days) after transplant. the median time for engraftment was days (range, - ) for all patients. after the first transplant, patients developed acute gvhd ( . %) with patients having grade ii-iii acute gvhd. five ( . %) had chronic gvhd, none of them with extensive manifestation. the prepative regimen was relatively well tolerated with limited regimen-related toxicity. cmv reactivation occurred in patients ( . %) during the follow-up of the study. eight patients ( . %) relapsed after a median of days post transplant (range, - days). cr was achieved in ( %) patients after haplo-sct. mean estimated -year os and pfs are . %± . % and . %± . %, respectively. conclusions: given the growing data on the similarity of outcomes after hla-matched and haploidentical sct, further studies are required to determine whether factors may be more important for donor selection than hlamatching. clinical trial registry: -disclosure: nothing to declare outcome of allogeneic stem cell transplantation for hodgkin and non-hodgkin lymphoma: single center experince from turkey ayşe uysal , hale bülbül , nur akad soyer , mahmut tobu , murat tombuloglu , guray saydam , filiz vural background: allogeneic sct (allosct) is generally optionally treatment choice for young and fit patients with relapsed/refractory lymphoma who were heavily pre-treated and after the failure of autologous stem cell transplantation (asct). relapse after asct is associated with a poor prognosis and allosct is a potentially curative therapy for lymphomas which have relapsed after asct. methods: in this study, we evaluated patients with hl and nhl who had treated with allo-hsct between november and december in ege university adult hematology transplantation unit. results: patients, disease and transplant characteristics were illustrated in table. histologic subtype of nhl was evaluated as t cell lymphoma (n= ; , %), mantle cell (n= ; , %), diffuse large b-cell lymphoma (n= ; , %) and b-cell lymphoma, unclassifiable, with features intermediate between dlbcl and classical hodgkin lymphoma (n= ; , %). all histologic subtype of hl was determined as nodular sclerosing. the median number of prior treatments before allo-hsct was (range, - ). twelve ( , %) patients had refractory disease, ( , %) patients were in complete remission and ( , %) patients were in partial remission before allo-hsct. the median time from diagnosis to allosct was (range, - ) months. peripheral stem cell was used for stem cell source in all of them. total body irritation plus fludarabine plus cyclophosphamide and busulfan plus cyclophosphamide were preferred most frequently for conditioning as non-myeloablative and myeloablative, respectively. neutrophil engraftment was occurred median of (range, - ) days. graft versus host disease (gvhd) prophylaxis was applied all of them and cyclosporine plus methotrexate was preferred most frequently (n= ; , %). gvhd was occurred in , % of them ( , % acute gvhd, , % chronic gvhd and , % both). veno-occlusive disease (vod) was occurred in ( , %) patients. transplant related death was observed in ( , %) patients. overall survival (os) and disease-free-survival (dfs) were evaluated as median (range, - ) and (range, - ) months, respectively. analyze of os and dfs was illustrated in figure. six patients are alive without disease. after a rapid tapering of immunosuopressive therapy was underwent a therapy with ponatinibat dose of mg/die results: afther a month of therapy we observed a rapid decrease in minimal residual disease on molecular assessment with an mmr of p -bcr-abl/abl non detectable confirmed by bone marrow revaluations at days + , + nd + after the salvage therapy. the patient has not had experienced of graft-versus-host disease, ponatinib treatment was well tolerated and considered safe with easily manageable side. conclusions: maybe in the era of tyrosine kinase inhibitors (tkis), philadelphia chromosome positive acute lymphoblastic leukemia (ph+ all) it could benefit from a combined treatment between transpalnt and tkis however more studies are needed to confirm these hypotheses. disclosure: nothing to declare immunodeficiency diseases and macrophage background: although a number of patients with hiv infection and hematological disease have successfully undergone allogeneic hsct together with combination anti-retroviral therapy (cart), short and long-term outcomes remain not well known. we report the largest spanish experience of hiv-infected adult patients with high-risk hematological malignancies with allogeneic hsct. methods: we retrospectively reviewed hiv-positive patients who received allogeneic hsct between and in spanish centers within geth (grupo español de trasplante hematopoyético y terapia celular). results: baseline and transplant characteristics of patients are shown in table . median age was years and % of the patients were men. the most frequent underlying malignancies were non-hodgkin lymphoma ( , %) and aml ( , %). in half of the patients an hlaidentical sibling was the donor; and in the other half, an alternative donor was used. peripheral blood was used as graft source in % of the transplants. at the time of hsct, all patients had been receiving suppressive cart for a median of years and only of them showed detectable plasma hiv rna, one of them because of poor adherence to cart together with the accumulation of multiple resistance mutations; and the other patient had detectable hiv rna at low levels (< copies/ml). all patients received cart throughout the transplant procedure, being temporally stopped in two patients due to significant mucositis. after a median follow-up of months ( - ), -year overall survival (os) and event-free survival (efs) were %. nrm was % at months and relapse was % at months. grade ii-iv agvhd rate was %, and moderate/severe cgvhd rate was % at months. a significant proportion of patients ( %) showed infectious complications with viral infections as the most frequent cause. two patients had invasive aspergillosis and one patient presented disseminated tuberculosis. causes of death included infections ( %), relapse ( %) and toxicity ( %). among the patients who died due to infections, had severe chronic gvhd and were under immunosuppressive therapy. two patients showed severe toxicity related to drug interaction with anti-retroviral therapy. all survivors except one showed undetectable hiv load at last follow-up after hsct. conclusions: allogeneic hsct is an effective therapy for high-risk hematological malignancies in patients with hiv infection, providing long-term disease free survival together to long-term hiv suppression with cart. however, drug interactions with anti-retroviral agents, occurrence of gvhd, and frequent infectious complications account for a complex procedure in this population. selected hiv-infected patients with hematological malignancies should be considered for allo-hsct when indicated, in experienced centers, with a multidisciplinary care. disclosure background: primary immunodeficiencies (pid) are rare diseases often associated with genetic defects in the immune system, predisposing individuals to recurrent infections and increased risk of allergy, autoimmunity and malignancy. allogeneic haematopoietic stem cell transplantation (hsct) has been successfully used as a curative therapy for most severe forms of pid. because pid is a genetic disease, < % of these children will have a healthy, human leukocyte antigen (hla) matched sibling donor available, and umbilical cord blood grafts from unrelated donors are a suitable alternative cell source. we report the results of umbilical cord blood transplantation (ucbt) performed in patients with pid between and at children's hospital of fudan university in china. methods: patients included chronic granulomatous disease (cgd, n= ), severe combined immunodeficiency (scid, n= ), interleukin- receptor-a deficiency (il- rad, n= ), wiskott-aldrich syndrome (was, n= ), leukocyte adhesion deficiency (lad, n= ), severe congenital neutropaenia (scn, n= ) and other immunodeficiencies (n= ). all patients were assessed by clinical immunologist to confirm clinical phenotype and genetic diagnosis. median age of patients was months (range, to months), and median body weight was . kg (range, . to kg). all patients received a ≤ / hla alleles-mismatched cord blood unit, were hla fully matched, were / matched, were / matched and were / matched. median nucleated cells of the cord blood were . x /kg (range, . to . x /kg), and median cd + cells were . x /kg (range, . to . x /kg). results: median follow-up time was months (range, to months), the overall survival rate at year for all patients was . %, and was . %, . % and % for cgd, scid and il- rad, respectively. patients died, most deaths ( / , . %) occurred in + days after transplantation, the main cause of death was infection ( / , . %). / ( . %) patients engrafted, median time of neutrophil engraftment was days (range, to d), and median time of platelet engraftment was days (range, to d). the cumulative incidence of grade - acute gvhd was . %, and that of chronic gvhd was . %. conclusions: unrelated ucbt should be considered for pid patients without an hla -matched sibling donor. effective control of infection before and after transplantation is important for improving survival. disclosure background: dedicator of cytokinesis (dock ) deficiency causes a combined immune deficiency characterised by recurrent bacterial infections, susceptibility to viral infection, eczema, food allergies, vasculitis and increased risk of malignancy. due to the high morbidity and mortality of the disease hsct has been increasingly offered to patients as a potentially curative therapy . methods: we retrospectively reviewed the outcomes of hsct for patients with dock deficiency at great north children's hospital newcastle upon tyne between and ( in published reference). results: ten patients with dock deficiency were treated with hsct (median age . y range . - . y). median duration of follow up was . years (range . - . y). there were a range of donor sources ( msd, mud and tcr ab/cd + depleted haploidentical), conditioning regimens ( treo-flu, treo-flu-thiotepa) and serotherapy ( alemtuzumab, atg+rituximab, none). one patient who received a cd + tcr alpha beta/cd + depleted haploidentical transplant received add back t-cells with caspacide molecular safety switch (bellicum pharmaceuticals). skin only agvhd occurred in / patients ( x stage , x stage ). no patients had cgvhd. overall survival was % ( / ). survival was comparable regardless of donor source. all deaths occurred within months of transplant. the patients who died had significant burden of disease pre-transplant: patient had chronic liver failure secondary to cryptosporidial sclerosing cholangitis, had a cirrhotic liver secondary to cryptosporidium, cerebral vasculitis, an axillary aneurysm and aortic vasculitis requiring grafting of an ascending aortic aneurysm, was pn dependent for failure to thrive with a history of cryptosporidium infection and had candida in a bal pre-transplant. causes of death in these patients were: respiratory failure (n= ), progressive encephalopathy (n= ), multi-organ failure with septic shock and encephalopathy (n= ) and multiorgan failure and septic shock after treatment for tma (n= ). two of these patients had reactivation of cryptosporidium prior to their death. pretransplant cryptosporidium was associated with mortality (graph ). one patient who survived had suffered from stroke pretransplant. one suffered from a basilar artery aneurysm years post-transplant at yo. at the time of latest follow up donor chimerism was % in / survivors and high level mixed in the other( % cd , % cd and % cd ). conclusions: this single centre study of hsct for patients is consistent with literature indicating that hsct is a potentially curative therapy for patients with dock deficiency. the increased morbidity associated with cryptosporidial infection is likely to be a consequence of overall disease burden rather than an infection specific effect. this does however highlight the improved outcomes of transplant prior to development of multiple comorbidities and suggests that hsct should be considered early. it is unclear whether the late occurrence of vascular complications after transplant were caused by a manifestation of disease which is not corrected by transplant or a result of vascular injury sustained pre-transplant. reference methods: referred infants underwent testing for: immune phenotype (ab, gd, naïve and memory t cell, b cell and nk cell numbers); functional activity of t and nk cells; maternal engraftment; adenosine deaminase (ada) and purine nucleoside phosphorylase (pnp) enzyme activity; and genetic testing. those with a confirmed diagnosis of scid underwent either allogeneic hematopoietic stem cell transplant (hct) or (if eligible) gene therapy (gt). infants identified as having ada deficiency as the etiology of their scid received enzyme replacement therapy prior to proceeding to definitive therapy. results: twenty-three ( %) infants were confirmed to have scid. three ( %) of these infants had a family history of scid but would not have been identified without nbs. in addition, one infant, born prematurely at weeks, was diagnosed as having pnp deficiency only after developing infections. this infant was identified by nbs but repeat testing at weeks gestation was normal likely due to support of transient t cell production from exogenous enzyme provided by red cell transfusion. twelve infants with confirmed low trecs had a non-scid diagnosis: with transient lymphopenia of infancy who normalized trec, immune phenotype and function, with prenatal exposure to -mecaptopurine ( -mp), genetically confirmed with digeorge syndrome, and with prolonged lymphopenia. of the three with prolonged lymphopenia, two had recurrent infections: one ultimately diagnosed with ataxia telangiectasia and one with absent trec but near normal number of t cells, normal pha but no specific antigen responses, and absent b cells who will be undergoing transplant in the near future. the third continues with absent trec, short telomeres, low numbers of a/b t cells, presence of g/d t cells, vaccine responses and freedom from infection with no identified genetic etiology. in summary, % of the patients referred to msk with confirmed abnormal nbs for scid have a non-scid diagnosis. there is no uniform collection of data for these infants and the threshold trigger for repeat testing varies from state to state, so the incidence of significant non-scid disorders identified will also likely vary from state to state. although our institution specific experience is biased, as most infants had confirmation of a low number of trec prior to referral, the significant number of disorders in the non-scid cohort emphasizes the importance of full evaluations and follow-up for these infants. disclosure: none of these relate to the work being presented. susan prockop -research funding mesoblast and atara biotherapeutics. nancy kernan -research funding jaz pharmaceuticals. richard o´reilly research funding and royalties atara biotherapeutics. kevin curran consulting juno pharmaceuticals, novartis. j.j. boelens avrobio, magenta, chimerix and bluebird bio background: post-transplant autoimmune cytopenia (aic) is challenging and associated with substantial morbidity and mortality. we aimed to study the cumulative incidence (ci) of post-hct autoimmune cytopenia (aic) and its predictors in a cohort of children with primary immunodeficiency (pid). methods: in this retrospective study, we included children with pid who underwent their first hsct with fludarabine(f)-treosulfan(t)±thiotepa(thio) at great north children's hospital from - . main outcomes of interest were the ci of aic and its predictors. fine-and-grey regression models were used to analyse predictors of aic, considering death as a competing event. variables included were age at transplant (< . years vs > . years), gender, diagnosis (scid vs immune-dysregulatory disorders vs other pids), pre-transplant aic, pre-and posttransplant respiratory virus, donor (mfd vs mud vs mmfd/mmud vs haploidentical donor), abo incompatibility, conditioning (ft vs ftthio), serotherapy (none vs alemtuzumab . - . mg/kg vs alemtuzumab . - . mg/kg vs atg), stem cell source (marrow vs pbsc vs cord vs exvivo t depleted pbsc), infused stem cell doses (tnc, cd and cd ), agvhd (none vs any agvhd), cgvhd (none vs any cgvhd), viral infections (cmv/adenovirus/ ebv/hhv viraemia), chimerism (full vs mixed chimerism (wb < %) within first year post-hct). impact of thymopoiesis using naïve t cell recovery was studied. results: median age at transplant was . years (range, . - . years). primary diagnoses were scid ( %), immune-dysregulatory disorders ( %) and other pids ( %). donors were mfd ( %), mud ( %), mmfd/ mmud ( %) and haploidentical parents ( %). stem cell sources were marrow ( %), unmanipulated pbsc ( %), ex-vivo t-depleted pbsc ( %) and cb ( %). % received additional thiotepa and % had csa/mmf as gvhd prophylaxis. median duration of follow-up of survivors was . years (range . to . years). -year os for the entire cohort was %. -month and -year ci of aic were % and %. of developed aic, ( %) had aiha, ( %) had aiha±itp and ( %) had aiha ±itp±ain. median onset of aic was . months post-hct (range . - . months). patients were treated with a median of treatment modalities (range, - ). one ( %) had steroid, ( %) had steroid+high-dose-ivig, ( %) had steroid+high-dose-ivig+rituximab, ( %) had steroid +high-dose-ivig+sirolimus and ( %) had steroid +high-dose-ivig+rituximab+sirolimus. the median time to resolution in ( %) who achieved remission after first aic was . months (range . - . months). had one relapse and had two relapses. died after development of aic ( aspergillus pneumonia; multi-organ failure). of ( %) surviving patients after aic, had on-going aiha at median of follow-up . years post-hct (range . - . years). on univariate completing-risk analysis, age at transplant > . years (p= . ) and pre-transplant aic (p= . ) were associated with higher incidence of aic (figure a -ic). on fine-and-grey models, only age at transplant (hr . , %ci . - . , p= . ) was independently associated with aic. of with complete immune reconstitution data, naïve t cells > cells/ml at months post-hct was associated lower incidence of aic (hr . , %ci . - . , p= . ) (figure d) conclusions: younger age and thymopoiesis were associated lower incidence of aic in children with pid after hct clinical background: human heme-oxygenase- (ho- ) deficiency has been reported to present with tetrad of anemia, nephritis, inflammation and asplenia and is fatal if not treated. its an auto-inflammatory disorder. macrophages/ monocytes express ho- and are engaged in recycling of red cells. human ho- deficiency results in intravascular hemolysis and severe damage to the endothelial system, kidneys, and other organs. transplantation of either healthy wild type macrophages or new macrophages produced by sct from healthy donor has been proven to be curative for ho- deficiency in mice. in , we had reported first successful allogeneic sct for human ho- deficiency. here we report second successful non-myeloablative msd hsct for a child with ho- deficiency. methods: a -yr-old-girl presented with complaints of fever, anemia and severe hypertension. in the past, at the age of years she was admitted for high fever for month and needed blood transfusion for the first time for severe anemia and had high platelets and high ferritin. she was treated as macrophage activation syndrome with prednisolone alone and later cyclosporine was added. she had short stature, abnormal facies but normal development. hemoglobin g/dl, urine for haemoglobin was positive, platelets , /ul, ferritin mcg/l and urine albumin + and urine rbc - /hpf. ultrasound and ct scan abdomen showed asplenia. a diagnosis of ho- deficiency was suspected. mutation analysis showed homozygous missense mutations in exon (r x) on chromosome q , which would result in the absence of the functional ho- protein. both parents were carriers of this mutation. we managed her over next -years with prednisolone, hydroxyurea and mycophenolate mofetil (mmf). however she remained steroid dependent. hla-typing confirmed her healthy unaffected -year-old brother to be a fully matched donor. at the age of years she was taken up for msd sct after taking informed consent. she weighed just kg. we conditioned her with alemtuzumab- . mg/kg, fludarabine- mg/m , cyclophospamide- mg/kg and total body irradiation gray. we infused million/kg peripheral blood stem cells from her brother. graft-vs.-host disease (gvhd) prophylaxis consisted of tacrolimus & mmf. results: she tolerated procedure very well. her entire hospital stay was uneventful and lowest platelet count recorded was , /ul. her neutrophils engrafted on day + and she was discharged on day+ . his urine albumin was nil by day+ . she had no gvhd. her chimerism on day+ showed % donor cells, on day+ was % and on day+ was % donor. now he is day+ post-sct and doing well. she has no evidence of hemolysis, proteinuria, hypertension, fever. she has normal ferritin and platelets. she has gained cm height and kg weight in last months. she had no viral reactivation and her immune recovery at months post sct is good. conclusions: non-myeoablative allogeneic msd sct is a curative treatment option for human ho- deficiency. disclosure: nil two decades of excellent transplant survival in children with chronic granulomatous disease: a report from a supraregional immunology transplant centre in europe background: haematopoietic stem cell transplantation (hsct) confers life-long curative therapy for chronic granulomatous disease (cgd). the ability of donor-derived neutrophils to replace recipient's defective neutrophils makes hsct a superior therapy compared to conventional standard of care using antimicrobial therapy. methods: we examined the outcome of children with cgd who received a first hsct at great north children's hospital from to . outcomes included overall survival (os), event-free survival (es), toxicity endpoints, autoimmune disease, long-term survival and graft function. cox proportional-hazard models were used to analyse predictors of os and es. variables included for predictor analysis were age at transplant, donor, stem cell source, stem cell doses and conditioning. results: = children were included in this analysis. median age of transplant was . years (range, . - . years). ( %) had x-linked and ( %) autosomal recessive cgd. twenty ( %) had matched family donor, ( %) had unrelated donor and ( %) had parental haploidentical donor. prior to , various conditioning regimens were used, with ( %) patients undergoing conditioning with pharmacokinetic guided intravenous (iv) busulfan (bu) and iv cyclophosphamide with or without serotherapy. from , the conditioning regimen was switched to flu-treosulfan-alemtuzumab with gvhd prophylaxis using ciclosporin (csa) and mycophenolate mofetil (mmf) for family and unrelated donors (n= , %). flu-treosulfan-thiotepa-atg-rituximab was used for cd tcr alpha-beta cd depleted haploidentical grafts (n= , %). ten ( %) patients had grade ii-iv acute gvhd while had ( %) had grade iii-iv acute gvhd. none had chronic gvhd. the -year os for the entire cohort was % ( % ci, - %) (figure ). analysis by age at transplant revealed a -year os of % for children transplanted at < = years of age and % ( %ci, - %) for the children > years of age (p< . ) (figure ) . the os was comparable between match family donor ( %, % ci, - %) and unrelated donor transplant ( %, - %) ( figure ). all four haploidentical transplants were successful. the -year es for the entire cohort was % ( % ci - %). none of the variables was associated with es. all seven patients with slipping chimerism received a successful second transplant. the five deaths were all due to transplantrelated complications ( multi-organ failures; pulmonary haemorrhage; graft iv acute gvhd; post-transplant lymphoproliferative disease). the median age at transplant of deceased patients was . years (range . to years). the -year and -year cumulative incidence of autoimmune diseases were % and % respectively. three ( %) had immune cytopenia while ( %) had autoimmune endocrinopathy ( thyroid dysfunction; type diabetes mellitus). the median age of long-term survivors was years (range, to years) with the median duration of follow-up of . years (range, . to . years). there was no late death in the entire cohort. the median donor myeloid chimerism was % (range to %) conclusions: despite the limitations of a single centre study, our findings confirm that hsct is a safe and longlasting curative therapy for children with cgd disclosure: none non -medical challenges in the diagnosis and transplantation of patients with primary immune deficiency: an experience from a tertiary care center in india sagar bhattad , stalin ramprakash , raghuram cp , chetan ginigeri , fulvio porta , background: primary immune deficiencies (pid) are increasingly being recognized in several parts of india. despite being diagnosed, many patients fail to receive optimal care due to financial and social constraints. methods: case records of patients diagnosed and treated (including hematopoietic stem cell transplants) for pid diseases during feb -nov at aster cmi hospital, bangalore, india were analysed. factors leading to deferred or suboptimal care were assessed in detail. results: patients with various pids were diagnosed during the study period (details in table). of them warranted a hematopoietic stem cell transplant (hsct) as definitive curative treatment. a total of children received hsct. of them died while of them are alive and well. children ( with severe combined immune deficiency) died before a hsct could be carried out. of them were critically ill at presentation, while were stable but deferred further treatment citing financial and social constraints. children needing transplant continue to remain on follow-up and have not been transplanted to date ( of them have significant financial constraints, families are not convinced about the need for transplant and of them are being prepared for transplant). table: (scid -severe combined immune deficiency, vodi-veno-occlusive disease with immunodeficiency, cgd -chronic granulomatous disease, hlh -hemophagolymphohistiocytosis, was -wiskott aldrich syndrome, xlt -x linked thrombocytopenia, lad-leukocyte adhesion deficiency, msmd -mendelian susceptibility to mycobacterial disease, xla -x linked agammaglobulinemia, cvid -common variable immune deficiency, apeced -autoimmune polyendocrinopathy candidiasis ectodermal dystrophy, cmcc -chronic mucocutaneous candidiasis, at -ataxia telangiectasia). conclusions: we present our experience from a developing country and discuss non-medical factors leading to suboptimal care in children with pid. only % children warranting hsct could be transplanted in our cohort. among those where hsct is potentially curative % of children died before hsct could be offered. transplants in developing countries pose unique challenges due to the absence of government funding and/or universal insurance coverage. in addition to delay in diagnosis and critical state of patients at admission, financial and social factors significantly contributed to poor outcome. disclosure: none the outcome of hematopoietic stem cell transplantation (hsct) in pediatric patients with hemophagocytic lymphohistiocytosis (hlh) in korea methods: the korea histiocytosis working party retrospectively collected nation-wide data from the patients diagnosed with hlh and underwent allogeneic hsct between and . the clinical characteristics and treatment outcomes of the patients were analyzed. results: a total of patients were enrolled. there were patients with fhl ( fhl , fhl , and fhl ), infection associated hlh, and secondary hlh of unknown cause. all the patients were treated with hlh- protocol, and patients achieved complete response (cr) after treatment for weeks, while did not. the main reasons for receiving transplantation were fhl in , reactivation in , and refractory disease in . the conditioning regimens were busulfan-based in patients, fludarabine-based in , treosulfan-based in , and busulfan/fludarabine-based in . stem cell sources used for hsct were from peripheral blood in patient, cord blood in , and bone marrow in . the donor types of hsct were unrelated donor in patients and related in ( matched sibling donor, haploidentical donor, partially matched donor). the causes of death of patients were disease reactivation/ progression in , acute gvhd with/without vod in , and graft failure in . five year overall survival rates were . %, respectively. the disease status at the time of hsct was cr in patients, and non-cr in . the -year survival rate of patients who received hsct in cr was % and % for patients transplanted while in non-cr status (p= . ). patients who received hsct using peripheral blood stem cells had a better -year survival rate of % compared to % of patients who received cord blood stem cells, significantly. the presence of neurologic symptoms, disease status after intial week therapy, conditioning regimen, and cd positive cell count did not have statistically significant impact on survival. conclusions: hsct improved the survival of patients who had familial, or relapsed, or refractory hlh in the korean nation-wide hlh registry. these results are similar to other reports in the literature. the disease status at the time of hsct and the stem cell source of the transplant were the important prognostic factors that affected the survivals of the hlh patients who underwent hsct. clinical trial registry: no registry number. disclosure: to the best of our knowledge, the named authors have no conflict of interest, financial or otherwise p hematopoietic cell transplantation with reduced intensity conditioning regimen using fludarabine/ busulfan and fludarabine/melphalan for primary immunodeficiency diseases background: primary immunodeficiency disease (pid) is congenital disorders of innate or acquired immune system. hematopoietic cell transplantation (hct) was a treatment option for pids with life-threatening infections or immune dysregulations. reduced intensity conditioning (ric) was increasingly used to prevent complications in hct, but optimized regimens have not been established. we performed hct for pids with ric using fludarabine and busulfan (flubu) or melphalan (flumel) according to the guidelines of european society for immunodeficiencies (esid) / european society for blood and marrow transplantation (ebmt), and assessed the efficacy and safety of these ric. methods: from april to december , pid patients underwent ric-hct using flubu or flumel in tmdu were analyzed retrospectively. the auc of bu was set to mg*hour/l for severe combined immunodeficiency disease (scid) and mg*hour/l for non-scid. overall survival (os) was analyzed. results: the median age at hct was . ( . - ) years old ( male and female patients). flubu was used for patients ( scid, combined immunodeficiency disease (cid), ectodermal dysplasia (eda), and severe congenital neutropenia (scn)) and flumel was used for patients ( scid, cid, xiap deficiency, and eda). anti-thymocyte globulin was used in patients of flubu group and patients of flumel group. cord blood in and bone marrow in was used for donor sources. matched donor was used for and patients in flubu and flumel groups ( . % and . %), respectively. median follow up period was . years. two years-os of all patients, flubu group patients and flumel group patients was . %, . % and . %, respectively. neutrophil engraftment was . %, . % and . % (all patients, flubu group and flumel group). in scid, all patients in flubu group achieved engraftment and survived. seven out of in flumel group achieved engraftment, but patient had secondary graft failure and patients died. in non-scid, out of in flubu group achieved engraftment, but patients had secondary graft failure. all non-scid patients in flumel group were engrafted and survived. two and patients in flubu group and flumel group suffered from severe acute graft-versus-host disease (grade iii-iv). ten patients had hemophagocytic lymphohistiocytosis (hlh). viral reactivation or infection was observed in patients, and resolved in all but one patient. conclusions: the ric-hct using flubu or flumel was advantagous for neutrophil engraftment, and flubu for scid and flumel for cid with immune dysregulation may be an effective opinion. flubu regimen needs to be improved for secondary graft failure in non-scid. prevention of hlh after transplantation using dexamethasone palmitate will be considered. disclosure: nothing to declare. survival after hematopoietic stem cell transplantation with tcrαβ/cd graft depletion in older children with primary immunodeficiencies background: tcrαβ/cd depletion is a graft engineering method that proved valuable in increasing the survival rate after hematopoietic stem cell transplantation (hsct) in patients with primary immunodeficiencies (pid). decreased survival rate in older patients with pid was previously reported after transplantations with nonmanipulated grafts. methods: patients with various pid (excluding classic scid) who received allogenic hsct with tcrαβ/ cd graft depletion from to september in our center were analyzed. the median age at hsct was , years (range , - , ). patients were divided into age groups: - years - patients, - years - , - years - . patients received hsct from matched unrelated, -haploidentical donors, -siblings. conditioning regimens with - alkylating agents and antithymocyte globulin were used in all patients. patient received short courses of various posttransplant immunosuppressants. median follow up after hsct is , years (range , - , years). results: overall survival (os) in patients was , ( % ci , - , ). we observed similar os in the younger age groups: , ( % ci , - , ) in - years and , ( % ci , - , ) in - years of age. seven of patients in older group ( - years of age) died, the os was only , ( % ci , - , ), p= , . all patients from older age group who died had combined pid: -wiscott-aldrich syndrome, -undefined pid, -il rg deficiency, -dclre c deficiency, -nijmegen breakage syndrome (nbs), -kabuki syndrome, -icf syndrome. the median time of death after hsct was , years (range , - , ). six of those had transplant-related mortality (trm). five patients had hsct-associated viral infections: -cmv pneumonias, -adv infections ( -fulminant hepatitis, -multiorgan). interestingly, of them had prolonged history of disseminated viral infections (adv), with the reduction of viral load in blood and other fluids and tissues upon treatment. one patient with kabuki syndrome after hsct developed hhv associated kaposi sarcoma, was successfully treated. eventually all patients with reduction of viral infection and sarcoma symptoms developed multiorgan failure with some clinical and laboratory evidences of endothelium cell damage syndrome. one patient with nbs died of high grade lymphoma progression. conclusions: hsct with tcrαβ/cd depletion demonstrates high survival rate in patients with various pid. in our group patients' age older than years predisposes to decreased posttransplant survival. patients with combined pid are at higher risks of posttransplant mortality. we conclude that at least in some of our patients with prolonged history of viral infections after hsct the cause of death could be multiorgan failure due to endothelium cell damage syndrome resulting from persistent inflammation and drug toxicity effects. disclosure background: chronic granulomatous disease (cgd) is a primary immunodeficiency (pid) caused by a mutation in of the subunits of the nicotinamide dinucleotide phosphate (nadph) oxidase, which leads to a reduction in the microbicidal activity of phagocytic cell. starting at an early age, cgd patients suffer from severe recurrent infections, as well as inflammatory events. allogeneic hematopoietic stem cell transplantation (hsct) is a curative option for cgd in patients with insufficient benefit from supportive care and prophylactic antibiotics. we reported a series of patients with cgd who underwent unrelated umbilical cord blood transplantation (ucbt) at our center. methods: in this retrospective study, we observed a series of consecutive ucbt performed at our center in children with cgd between and .median age at transplantation was months (range, to months), median body weight was kg (range, to kg). / patients received a myeloablative conditioning regimen consisting of busulfan, fludarabine, cytarabine, cyclophosphamide and g-csf, / patients received another myeloablative conditioning regimen consisting of busulfan, fludarabine, cyclophosphamide and atg. prophylaxis for graft-versus-host disease (gvhd) was tacrolimus. results: engraftment occurred in / ( . %) patients. / ( . %) patients occurred graft failure, all of them received a myeloablative conditioning regimen without atg. median time to neutrophil and platelet engraftment were (rang, - ) and . (rang, - ) days. / ( . %) patients developed acute gvhd, with / ( . %) episodes of grade iii-iv agvhd. chronic gvhd occurred in / patients ( . %). at a median follow-up of months (rang, to months), the overall survival rate was . %, and event-free survival rate was . %. conclusions: unrelated ucbt should be considered as potential curative methods in children with cgd. cgd patients who used myeloablative conditioning regimen with atg shows better graft and survival. disclosure: nothing to declare background: invasive fungal infections (ifi) remain a major cause of treatment-related morbidity and mortality in aml patients. although not uncommon, the presentation of unusually severe clinical features might be indicative for an underlying immunodeficiency. caspase-associated recruitment domain (card ) is recognized to have a crucial role in effective antifungal response, leading to th and th differentiation and to the initiation of the inflammatory cytokine cascade. particularly interferon-gamma (ifng) increases macrophage activity. patients with homozygous card mutations are known to have a significantly increased susceptibility to life-threatening systemic candidiasis. however, some sequence variants may lead to increased ifi-susceptibility even in heterozygosity, e.g. under immunosuppression. ifn-γ has been described as an additive treatment option because of its immune stimulating effect on the leukocyte immune response in a situation of immunological "blindness". methods: here, we report the case of an -year old male with aml m with a severe systemic candida tropicalis infection, unresponsive to triple-antimycotic regimen, leading to multi-organ failure. he was discovered to bear a heterozygous card mutation, and ifn-γ immunotherapy leaded to complete response of all disseminated infections. results: the patient developed septic fever immediately after the first chemotherapy cycle. unexpectedly, candida tropicalis was confirmed in the blood culture within hours. liposomal amphotericin b (ambisome ® ) was started immediately, however candida rapidly disseminated to lungs, liver, spleen, kidneys and cns despite extended antimycotic therapy with caspofungin, voriconazole and fluconazole. the patient was splenectomized due to massive infiltration ( figure ). genetic testing for mycosis predisposition revealed a heterozygous mutation in the card gene, inherited from the father (c. a>t(p.glu val)). ifn-γ treatment was started ( μg subcutaneously, times per week), leading to an almost complete response of disseminated infections. due to the severe infection, chemotherapy had to be interrupted after one course. however, bone marrow remained in complete remission for almost one year. the patient experienced altogether two relapses requiring an unrelated allogeneic and a haploidentical hsct. under combined ifn-γ and ambisome/ fluconazole prophylaxis no further mycosis was observed despite extensive and prolonged immunosuppression. conclusions: ifi in aml patients are common, however an unusual presentation in presumably immune competent individuals should raise the suspicion for immunodeficiencies. in our case, an unexpected early candida-sepsis was completely unresponsive to an adequate multi-agent treatment. while ifn-γ is used in adults as an immune stimulatory cytokine, little data are available for children. to our knowledge, this is the first case of successful ifn-γ treatment of a pediatric aml patient with disseminated candida sepsis, bearing a card mutation. given the elevated mortality risk for ifi, and the apparently safe and well-tolerated application of ifn-γ, an adjuvant immunotherapy might be considered. further studies are needed to define the indication and duration of this kind of adjunctive immunotherapy. moreover, considering the wide heterogeneity of genetic mutations involved in ifi-susceptibility, genome-wide expression profiling might be useful for pediatric cancer patients, as the identification of specific immune pathways might help to identify individual ifisusceptibility in order to improve the outcome of those high-risk patients. [[p image] . background: chronic granulomatous disease (cgd) is curable by allogeneic hematopoetic stem cell transplant (hsct). recent reports of haploidentical donor hsct with with post transplant cyclophosphamide (ptcy) from family donors in pediatric primary immune deficiencies have shown encouraging results. however, it has not been reported in cgd. here we describe successful haploidentical hsct in a child with cgd with myeloablative conditioning and ptcy. a year-old, male child diagnosed with cgd showed oxidative activity . % by dihydrorhodamine (dhr) test. he had no matched related or unrelated donor available so underwent haploidentical hsct after taking informed consent of the parents in may . donor was his / hla matched healthy elder sister (oxidative activity % by dhr). he has had multiple admissions for recurrent pneumonia prior to hsct. the conditioning was with rituximab mg/m iv on day - , thiotepa mg/ kg/dose intravenous (iv) for day (day- ), busulfan . mg/kg/dose daily iv for days (day - to - ) and fludarabine mg/m /dose daily iv for days (day - to - ) and rabbit anti-thymoglubulin (thymoglobulin) . mg/ kg/dose daily for days (day- to - ). peripheral blood stem cells ( million/kg cd + cells) were harvested from his sister and transfused to the patient on day . graft vs. host disease (gvhd) prophylaxis was with ptcy mg/ kg on day+ & , intravenous cyclosporine from day- (targeting levels - ng/ml) and mmf from day+ . results: his neutrophils engrafted on day+ and platelets on day+ . chimerism on day+ , and months was fully donor. he developed no acute or chronic gvhd. at months his lymphocyte counts showed cd - /ul, cd - /ul, cd - /ul and cd / - /ul. his had no viral reactivation. he is disease free and gvhd free on day+ post hsct and is on tapering doses of cyclosporine. his dhr test showed oxidative activity of % on day + . background: primary immune deficiencies (pid) are a functional disorder of inheritance immune system that increase predisposition to infectious disease in number and severity. the incidence is : , birth live; its immunological dysregulation may increase the predisposition of autoimmune diseases and malignancy, the latter being more frequent ( - %). at present, the only curative treatment is hematopoietic stem cells transplant (hsct). methods: we describe all patients transplanted with primary immune deficiencies at instituto nacional de pediatria. the conditioning regimen depended on the type donor and pathology: myeloablative ( . %), reduced intensity ( . %) and non myeloablative ( . %) without modification statistically significantly in overall survival. results: a total of patients were included from to january/ . severe combined immunodeficiency (scid) is the pathology most frequently transplanted ( figure ) . seventy three percent have molecular diagnosis, and . % have cases of family pid. the most used sources were umbilical cord blood (ucb) with . % and peripheral blood ( . %), however the trend of the source of obtaining it has been modified a few years ago (figure ) . the median graft was days for ucb, days for bone marrow (bm) and days for peripheral blood (pb) (figure ) the main complications are infectious (bacterial . % and viral . %) and non-infectious as pre-graft syndrome ( . %). conclusions: the overall survival was . % survival according to pathology was: % chediak higashi syndrome, % scid, . % griselli syndrome, . % hyper igm syndrome, % was, % cgd, % hemophagocytic lymphohistiocytosis. disclosure: ramírez-uribe rosa maria nideshda, salazar-rosales haydeé, olaya-vargas alberto, lópez-hernández gerardo, del campo-martínez maria de los Àngeles we wish to confirm that there are no known conflicts of interest associated with this abstact, the only financial support was provided by mexican associations that helping children wiht cancer in a few patients. long-term outcome following hematopoietic stem cell transplantation of wiskott-aldrich syndrome in a single institute mamoru honda , , yukayo terashita , minako sugiyama , yuko cho , akihiro iguchi background: wiskott-aldrich syndrome (was) is an xlinked disorder of hematopoietic cells, characterized by thrombocytopenia with small platelets, eczema, and immunodeficiency. hematopoietic stem cell transplantation (hsct) is the only curative treatment, and it is recommended to be performed as soon as was is diagnosed. myeloablative conditioning before hsct is recommended because there is a high risk of development of autoimmune disease in patients with mixed chimera after hsct. however, there are few reports about late complications such as pubertal development and eruption of teeth in patients with was receiving hsct. thus, we evaluated late complications in patients with was receiving hsct at hokkaido university hospital. methods: we reviewed medical records of male patients with was who received hsct between and . results: mean age at hsct was . (range, . - . ) years, and median follow-up time after hsct was . (range . - . ) years. conditioning regimen in all patients comprised busulfan at mg/kg for days and cyclophosphamide at mg/kg for days or mg/kg for days. additionally, anti-thymocyte globulin at . mg/kg/day for - days was administered in patients. engraftment, normal platelet count, and complete chimera were confirmed in all patients. no patients showed complications such as severe chronic graft-versus-host disease, autoimmune disease, short stature (≤ - . sd) and second malignancy. however, high ige level was observed in patients. pubertal development has been confirmed in patients. lack of complete eruption of permanent teeth has been observed in patients who received hsct at age of < years. conclusions: although this was a small-cohort study in a single institute, complete chimera has been achieved in all patients who received hsct with busulfan-based myeloablative conditioning. however, late complications such as male infertility and incomplete eruption of permanent teeth remain major problems. disclosure: nothing to declare methods: we performed unrelated umbilical cord blood transplantation (ucbt) in consecutive children with lad-i. median age of children was months (range, to months), and median body weight was kg (range, to . kg). all patients received myeloablative conditioning regimen consisting of busulfan, fludarabine and cytarabine. prophylaxis for graft-versus-host disease (gvhd) was tacrolimus. all patients received a ≤ hla alleles-mismatched cord unit, was hla fully matched, were / matched, was / matched. median nucleated cells of the cord blood were . x /kg (range, . to . x /kg), and median cd + cells were . x / kg (range, . to . x /kg). results: all patients engrafted, median time of neutrophil engraftment was days (range, to d), and median time of platelet engraftment was days (range, to d). median follow-up time was months (range, to months), all patients were alive with continuous completely donor engraftment, and achieved complete clinical remissions. / patients developed grade ii/iii acute graft-versus-host disease (gvhd), and / patients developed chronic gvhd with skin. conclusions: it is the first successful unrelated ubct for lad-i children in china. our data shows ucbt provided excellent outcome for patients with lad-i. disclosure: nothing to declare p excellent outcome using 'nktm' enriched hematopoietic stem cell transplants for patients with inborn errors of immunity results: majority of patients in the cohorts had significant infective co-morbidities at the time of hsct with patients in the later cohort entering hsct earlier. patients in the later cohort were sicker at hsct. final engraftment occurred in all except patient who received a hla mis-matched cord blood hsct. graft failures occurred in patients ( in earlier and in later cohort); of these patients received unmanipulated hscts from hla mis-matched unrelated donors ( cb, bm). second hsct were with same donors in and different donors in patients. no grade ii to iv acute gvhd or extensive gvhd occurred. one patient (cbt) died of infections/ non-engraftment. all patients in the later cohort compared to of patients in earlier cohort are alive, engrafted and cured. performance status were % in all alive patients. of the patients in the later cohort, ( hla mis-matched related and hla matched related donors) received 'nktm' enriched hsct. in the hla mis-matched 'nktm' enriched hscts, patients received high cd +, cd +cd ro+ and nk cell doses, with median of . (range, . - . ), . (range, . - . ) and . (range, - ) x /kg, respectively. no invasive infections occurred in these patients and immune reconstitution in t, b, nk compartments were complete at year after hsct with cd > by months and tcrαb > by year after hsct. background: viral infections contribute to significant morbidity and mortality after allogeneic hematopoietic cell transplantation (allo-hsct), increasing both the human and the financial cost. antiviral agents are often ineffective or/ and associated with toxicity. methods: in view of t-cell anti-viral immunotherapy in greece, we evaluated the actual cost of conventional pharmacotherapy for cmv, ebv and bkv reactivations after allo-hsct, by calculating the costs of (i) the antiviral agents, (ii) the treatment (excluding transfusions) of antiviral drug primary toxicity (e.g. graft failure, cytopenias, renal or hepatic dysfunction) and secondary toxicity (e.g. leukopenia-associated bacterial infections), iii) the treatment (excluding transfusions, ie for bk cystitis) of infectionrelated complications, iv) the transfusions due to treatmentrelated toxicity (ie cytopenias) or infection-related complications (ie, bk cystitis), v) the inpatient or outpatient daily care. notwithstanding that blood and its products, as a common natural good, are provided free in our country, the costs related to blood and platelet collection, processing, storage, laboratory testing and infusions were included in our model. results: the treatment cost of cmv, ebv and bkv reactivations/infections for the first six months post allo-hsct was evaluated in / patients who reactivated viruses and were transplanted between / - / from matched related ( / ), matched unrelated ( / ), mismatched unrelated ( / ), haploidentical ( / ) and mismatched related donors ( / ). we detected cmv, ebv and bkv infections/reactivations in , and patients respectively, with a mean of ± . infection per patient from all three viruses ( - /patient). of note, / patients experienced reactivations from more than one virus, requiring repeated treatments with antiviral agents and/or rituximab. the cost of antiviral agents for all cmv, ebv and bkv reactivations/infections was , €, , € and , € respectively ( , €, , € and , €/patient, respectively). the treatment cost of toxicity related to antiviral drugs and infection-related complications was , € ( , €/patient) excluding transfusions and , € ( , €/patient) including transfusions. in particular, the cost of transfusions for bkv hemorrhagic cystitis reached , €/patient. repeated ( - ) and/or prolonged (Δm d, range - d) hospitalizations were needed, up to a total of and days of inpatient hospitalization and short-term outpatient treatment, respectively. hospitalizations further increased the cost of inpatient and outpatient post-transplant care by , € and , € respectively ( , € and €/patient, respectively), onthe basis of a, rather underestimating the true cost, fixed, unified hospitalization fee ( €/day and €/day). conclusions: overall, in a six-month study period, the treatment of cmv, ebv and bkv infections substantially increased the cost of post-transplant care by , € ( , €/patient). the actual cost is undoubtedly higher as the hospitalization fee for transplant recipients is largely underestimated in greece. considering not only the hematopoietic but also the solid organ transplant recipients, the financial burden of antiviral treatment for national economies is enormous. given that antiviral pharmacotherapy is often associated with suboptimal efficacy, toxicity, development of drug resistance, reactivation recurrences and repeated hospitalizations, it is expected that a one-time treatment with multi-virus-specific t cells, able to expand in vivo and provide a long-lasting protection without significant toxicity, will serve as a powerful and costeffective treatment over conventional pharmacotherapy. disclosure: nothing to declare methods: this is a single-centre retrospective analysis of consecutive patients who underwent tcd allo-hscts for myeloid malignancies between january -june . ebv-dna was monitored frequently on whole blood samples with standardised quantitative real-time pcr. serum protein electrophoresis was routinely tested with immunoglobulin subclasses identified by immunofixation electrophoresis. histological confirmation of ptld was based on standard who diagnostic criteria ('proven'), while those without biopsy were classed as 'probable' based on clinical & radiological criteria as defined by ecil- guidelines. results: majority of patients had aml(n- / ) and mds(n- / ) with a median age of years(range . median follow up of survivors was months(range - ). majority of patients(n- / ; %) developed ebv-r with a median time of days[inter quartile range(iqr) - days] &higher cumulative incidence with atg(n- ) versus alemtuzumab(n- )(p< . ). figure- a shows schematic representation of ebv and ptld events (cumulative incidence of . %( %ci- . %- . %) at months). significantly higher peak ebv dna viral load(evl) were noted in patients with ptld(p-< . ). development of post-hsct mg was observed in %(n- / ). roc curve identified peak blood evl> , copies/ml significantly correlated with risk of developing ptld (sensitivity- . %,specificity- . %;auc- . ,p< . ). based on these estimates, subgroup of patients with no ebv-r(n- / ), peak evl < , (< k)copies/ ml(n- / ) & > , (> k)copies/ml(n- / ) were categorised in groups along patients with/without mg accordingly (groups - ;figure- b). patients with ebv-r had significantly better os [ -year os of % vs %(no ebv-r);log-rank p< . ],with this survival benefit mainly driven by subgroup of patients with lower evl(< k)(p< . ). ptld patients had trend towards inferior -year os( % vs %;p- . ). patients with mg had a significantly better os irrespective of degree of evl (group - ,p< . ).we report a 'sweet spot' of low evl & presence of mg in these patients, with a clear survival advantage compared to those with no ebv-r and/or no mprotein (group- -year os % vs % in group- ; hr- . ; %ci: . - . ;p< . ;figure b). overall cumulative incidence of relapse (cir) was %( %ci: - ) and non-relapse-mortality(nrm) of %( %ci: . - ) at years. multivariate analysis(mva) revealed absence of m-protein,high evl (> k copies/ml) or no ebv-r and absence of any gvhd as significant factors for high cir. similarly, high evl or no ebv-r, absence of m-protein and itu admission were significant predictors of high nrm. conclusions: this study adds to our understanding of role of ebv viraemia & associated mg in tcd-hscts while highlighting its significant impact on risk of ptld, os, nrm & cir. low ebv burden and development of mg is protective with significantly better survival outcomes and we recommend pre-emptive approach of using rituximab for ebv-r /ptld is best employed at higher ebv burden (e.g. > k copies/ml dna) in high risk patients and be prospectively evaluated in future studies. clinical trial registry: n/a disclosure: nothing to declare p impact of early candidemia on the long-term outcome of allogeneic hematopoietic stem cell transplant in non leukemic patients: an outcome analysis on behalf of idwp background: to assess the incidence of, and risk factors for, candida infection in the first days post-allogeneic hematopoietic stem cell transplantation (hsct) and the impact on long-term survival. methods: outcome analysis of , patients, % male, median age years (range - ), with diagnosis of hemoglobinopathies in ( . %), bone marrow failure in ( . %), lymphoma in ( . %) and myelodysplastic/myeloproliferative disesases in ( . %) patients who underwent hsct from to : with candidemia by day + , and , without candidemia. the incidence of -day candidemia was estimated by using the cumulative incidence method. the univariate and multivariate risk factor analysis for -day candidemia was performed with the cause-specific cox regression model. the occurrence of candidemia was analyzed as a timedependent covariate. the overall survival and non-relapse mortality after day + were assessed in a land-mark setting, this analysis was restricted to patients surviving to day + post transplant. [[p image] . figure a - b] results: the incidence of candidemia by day + was . % ( % c.i. . - . ) ( / , ) and occurred at a median of days post-hsct (range - - ). considering the candidemia within -day from hsct as a time dependent covariate, a higher -day non-relapsemortality (nrm) (hr . ( . - . ), p < . ), and a lower -day overall-survival (os) (hr . , % ci . - . ), p< . ) were obtained from the cox model for patients with candidemia. factors significantly associated with candidemia occurrence in the multivariate analysis were: gender female, increased age at hsct, bone marrow failure, lymphoma or myelodysplastic/myeloproliferative diagnosis, bone marrow or cord blood stem cell source, t-cell depletion, less recent year of hsct. among patients alive at day + , the -year nrm and os with and without candidemia were . % vs. . %, p < . , and . % vs. . %, p< . , respectively, after a median follow-up of . years ( % ci . - . ) (figure ). in multivariate analysis, the occurrence of a candidemia episode within day + was an independent risk factor for higher nrm, hr . ( . - . ), p= . , and lower os, hr . ( . - . ), p= . . conclusions: despite the general improvements in prophylaxis and treatment, the occurrence of early post-hsct candidemia had a negative impact on transplant outcome as showed previously in leukemic patients. abstract already published. carbapenem-resistant enterobacteriaceae colonizationimportance in the risk of cre bacteremia and mortality in stem cell transplant (hsct) and acute leukemia patients marcia garnica , , marco a f bellizze , priscila g a de jesus , rafaela r c gomes , filipe m akamine , alan j marçal , luzinete co rangel , andreia assis , marcia rejane valentim , angelo maiolino background: spread of infections due to carbapenemresistant enterobacteriaceae (cre) is a worldwide phenomenon and has been associated with high mortality and clinical complications. gut translocation is the most important portal of entry of bacteria during neutropenia, and cre gut colonization is a possible risk factor for bacteremia during neutropenia. goals: in the present study, we describe the frequencies of cre colonization and analyzed its relationship with development of cre bacteremia and mortality in two different scenarios: stem cell transplant patients (hsct) and leukemia patients. methods: prospective cohorts of hsct (from to ) and leukemia patients (from to ). hsct patients were analyzed from conditioning until discharge (pre-engraphment phase) and leukemia patients from first induction chemotherapy until last intensification. if an hsct was performed in leukemia patient the patient was censored in leukemia cohort and included in hsct cohort. all patients had rectal swabs performed weekly during hospitalization for the identification of cre colonization. patients with at least one positive swab (cre colonization group) were compared to patients with no documentation of colonization (controls). the outcomes analyzed were bacteremia due to cre, and overall mortality. results: there were hsct performed during the study ( [ %] autologous and [ %] allogeneic). multiple myeloma and non-hodgkin lymphoma were the most frequent baseline diseases (n= ; %, and n = ; %), respectively. cre colonization was documented in % (n= ), and it was more frequent among allogeneic hsct and leukemia patients (p< . for both). cre colonized patients had longer hospitalization ( vs. days, p< , ), higher frequency of cre bacteremia ( % vs. . %; p= . ), and mortality ( % vs. . %, p< , ) compared to non-colonized hsct. negative and positive predicted values for cre bacteremia were % and %, respectively. thirty-one patients were analyzed in leukemia cohort, accounting to hospitalizations (median hospitalizations per patient, ranging from to ). the median age was years, and % aml vs. % all. cre colonization was documented in eight ( %), with a median time from leukemia diagnosis and colonization of days ( - days). cre bacteremia was documented only in colonized patients ( % vs. zero; p= , ). all eight colonized patients were submitted to other cycles of chemotherapy after colonization, in one of them cre bacteremia relapsed. conclusions: a routine surveillance of cre colonization showed colonization frequencies from % to % in hsct and leukemia patients respectively and was effective to stratify cre bacteremia risk as the predictive negative value was over %. colonization had association with cre bacteremia and overall mortality. efforts to minimize risks for colonization and mortality are necessary. the information of surveillance can be a tool to improve adequacy in empirical febrile neutropenia therapy in hsct and leukemia patients. disclosure: nothing to declare background: the incidence of hepatitis b virus infection is high to . % in asian population, so there is more and more attention to the risk of hepatitis b virus(hbv) reactivation in the hepatitis b core antibody positive patients during chemotherapy, anti-cd monoclonal antibody, hsct, or other intense immunosuppressive drug therapy (isdt). hepatitis b core antibody is associated with a significant risk of hbv reactivation in patients undergoing hsct. however, there are remain uncertain that the effect of anti-hbsag antibodies in hepatitis b virus reactivation among the hepatitis b core antibody positive patients undergo hsct. we aim to investigate the role of the anti-hbs and the necessity of anti virus in hepatitis b surface antigen(hbsag) negative, hepatitis b core antibody positive patients during hsct. methods: we enrolled hematological malignant patients received hsct in our center from to . we classified hbsag negative and undetectable hbv dna patients into groups as anti-hbc(-)anti-hbs(-) (n= ), anti-hbc(-)anti-hbs(+) (n= ), anti-hbc(+) anti-hbs(-) (n= ), and anti-hbc(+)anti-hbs(+) (n= ). results: hbv reactivation was identified in patients ( . %) after hsct. there was a significant difference in hbv reactivation rate in anti-hbc(+)anti-hbs(-) ( . %) vs anti-hbc(+)anti-hbs(+) ( . %) (p= . ) and anti-hbc (+)anti-hbs(-) ( . %) vs anti-hbc(-)anti-hbs(-) ( . %) (p= . ), anti-hbc(+)anti-hbs(-) ( . %) vs anti-hbc(-) anti-hbs(+) ( . %) (p= . ), but not among anti-hbc(+) anti-hbs(+) ( . %) and anti-hbc(-)anti-hbs(-) ( %) and anti-hbc(-)anti-hbs(+) ( . %). whereas there were no difference according to the donor viral profile(p= . ). the median time of hbv reactivation in hbsag negative patients accepted hsct was ( - ) days after hsct. all of the patients with hbv reactivation have been controlled with nucleos(t)ide analogues drugs, and of them achieved reverse seroconversion which detect persistent anti-hbsag antibodies in their bodies. conclusions: the anti-hbsag antibodies negative and anti-hbc positive patients have the highest risk of hbv reactivation after hsct in resolved hbv patients. the anti-hbsag antibodies play a protective role in resolved hbv patients receiving hsct. we recommend not prophylactic anti hepatitis b virus in hbsag negativity and anti-hbsag antibodies positive patients following hematopoietic stem cell transplantation. disclosure: nothing to declare methods: allo-hscts performed between and for acquired bone marrow failure ( . %) or hemoglobinopathies ( . %), with bm±cb ( . %) or pb±cb+bm ( . %) as a stem cell source were included in this retrospective registry megafile idwp ebmt study. results: demographics: the median age of recipient was . years (range: . - ), and . % were children. . % recipients and . % donors were ebv-seropositive. . % had hsct from a matched family donor, . % from a mismatched family donor, and . % from an unrelated donor. t-cell depletion was performed in vivo in . %, and ex vivo in . % patients. conditioning regimen was myeloablative in . %, ric in . %. median follow-up was . years ( % c.i. . - . ). transplant out-comes: ebv-seropositive recipients in comparison to ebv-seronegative recipients had lower os ( . % vs . %, p= . ), and higher nrm ( . % vs . %, p= . ). no other significant differences were found for: ri, rfs, and acute or chronic gvhd with respect to ebv pretransplant serostatus donor and/or recipient. multi-variate analysis: ebv serostatus as a risk factor did not reach significance, while a trend towards higher risk of development of cgvhd (hr= . ; %ci . - . ; p= . ) and better survival (hr= . ; %ci . - . ; p= . ) in allo-hsct from ebv-seropositive donors. allo-hsct in ebv-seropositive recipients had a trend towards lower risk of development of cgvhd (hr= . ; %ci . - . ; p= . ). when subgroups (r-/d-, r-/ d+, r+/d-, r+/d+ ebv serology) were analyzed, the ebv serostatus had no significant impact on os, rfs, ri, trm and development of acute or chronic gvhd. conclusions: allo-hsct from ebv-seropositive vs ebv-seronegative donors are at % higher risk of chronic gvhd in patients with non-malignant hematological disorders undergoing allo-hsct, however this difference is non-significant in multivariate analysis. disclosure: nothing to declare. results: twenty-eight ( %) pts ( male, female) were tested positive (group ) for subtype a (n= , %), b (n= , %) or a (h n ) (n= , %) while ( %) pts ( male, female) were negative (group ). vaccination rate in group ( %) was significantly lower compared to group ( %, p= . ). the median time after transplantation ( vs days), t-cell counts ( vs / μl), bcell counts ( vs / μl), igg-level ( , vs , g/ l), proportion of immunosuppressed pts ( % vs %), male/ female ratio was not significantly different between groups and . within group influenza subtypes were similarly distributed in vaccinated and not vaccinated pts (a % vs %, b % vs %, a (h n ) % vs %). pts. with subtype b infection had higher levels of t-( vs / μl) and b-cells ( vs / μl) and a longer follow up from sct ( vs days) compared to subtype a / a (h n ) infection but differences were not significant. conclusions: influenza could be proven in one third of all tested pts. dominance of b and a (h n ) pdm e subtype occurrence corresponded to the flu epidemic dissemination in the german population. the most important protective factor for outpatient sct recipients was influenza vaccination. disclosure: nothing to declare background: cmv infection is one of the most frequent complications after haplo. some risk factors are well known but the best strategy (prophylactic or preemptive treatment) to mitigate this complication is not still well defined. the primary endpoint in our study is to describe incidence and risk factors to develop cmv infection or disease in haplo. as secondary objective we analyzed efficacytoxicity of treatment and cmv related mortality. methods: we analyzed patients who underwent haplo in our center between may and may . all of them received ptcy (d+ and d+ ), tacrolimus and mycophenolate as graft versus host disease prophylaxis. a preemptive therapy based on viral load was applied. treatment was started when > ui/ml of cmv were detected in one determination or > ui/ml in two consecutive determinations. cmv analyses were made in plasma using cobas pcr technique® and positive viral load cut-off point was ui/ml. the cmv viremia was determined weekly until d+ and then every two weeks until immune reconstitution. results: the cmv infection and disease incidence at d + was . % ( episodes) and . % ( episodes), respectively. cmv disease was digestive (n= ), pulmonar (n= ), neurologic (n= ) and disseminated (n= ). the median time to first cmv infection was . days ( - ). thirty-six patients had at least one episode of cmv infection: of them ( . %) had one episode, ( . %) had two episodes and ( . %) had or more episodes, respectively. only pre-transplantation cmv status was significantly associated with cmv infection (p< . ). risk factors are shown in image . the median viral load in first cmv infection and disease was ui/ml ( - ) and ui/ml ( - ), respectively (p= . ).the median counts of cd lymphocytes at d+ in cmv infection and disease were /mm and /mm , respectively (p= . ). preemptive therapy for the first episodes of cmv infections (n= ) was valganciclovir ( , %), ganciclovir ( . %) or foscarnet ( . %), reaching a complete viral load clearance in %, with a median time to response of . days ( - ) and a median treatment duration of days ( - ). grade iii-iv toxicity (mainly hematologic) was observed in . % (n= ), % (n= ) and . % (n= ), respectively. three patients had an ul mutation, one of them with clinical and microbiological resistance to the mentioned drugs. three patients ( %) had a graft failure secondary to cmv infections. five patients ( . %) died as consequence of cmv infection: before d+ secondary to cmv disease ( pulmonar, disseminated) and after d+ due to graft failure and infectious complications. with a median follow up of . months, overall survival at months for patients who had cmv infection was . % compared to . % for those who had no infection (p= . ). conclusions: a high incidence of cmv infection in haplo with ptcy was shown in our series and it contributed to mortality in . % of patients. only cmv status (d-/r+ and d+/r+) was significantly associated with higher risk of infection. identification of high risk patients and new prophylactic and treatment strategies may improve these results. disclosure: nothing to declare. methods: consecutive patients admitted at the sct unit between january- to november- were reviewed. only first admission was analysed. screening consisted of rectal and perineal swap on admission and weekly until discharge. in case of detection of mdro, patients were isolated and infection control strategies were applied. results: patients were analysed, median age years ( - ). % were male (n= ). median duration of hospitalization was days( - ). swabs were performed, with a median of swaps/patient ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) . patient characteristics are shown in table . patients ( %) spiked fever in a median of days after admission . % (n= ) had previous documented mdro colonization. median neutrophil engraftment was days ( %ci - ), in % patients (n= ) of patients had a positive screen: in ( %) patients at baseline and in ( %) patients were detected for the first time beyond baseline screen. cumulative incidence of colonization at days was . % ( %ci . - . ), at days % ( %ci . - . ), and at days . % ( . - . %) (figure ). mdro identified were: with extended-spectrum beta-lactamases producing e. coli (esbl-ec), multidrug-resistant pseudomonas aeruginosa (mr-ps), vancomycin resistant enterococci (vre) and patient with carbapenemaseproducing (cp) citrobacter freundii. / colonized patients developed mdro infection ( %): patients mr-ps, site of infection was urinary tract infection (uti), urethritis, genital ulcer. two patients were treated with ceftolozane/ tazobactam, with meropenem+amikacin; patients esbl-ec both uti treated with meropenem; patient cpcitrobacter freundii uti treated with ceftazidime/avibactam. in % patients ( / ) antibiotic treatment at febrile episode was guided by positive screening. no mdro related icu admission or mortality was observed. in % patients (n= ) background: hepatitis e virus (hev) can cause chronic infection and liver cirrhosis in immunocompromised individuals. there is limited data on hev infections in patients undergoing allogeneic hematopoietic stem cell transplantation (hsct). the aim of this study was to investigate the frequency and clinical importance of hev in a swedish cohort of hsct recipients. methods: we analyzed serum samples from hsct patients ( adults and children), collected months after hsct. hev igg and igm were detected by elisa (dia.pro®), hev rna by reverse transcriptase pcr, and quantification of hev rna was performed by digital pcr. in all patients, who were positive for hev-rna and/or serology at months, also samples collected at the time of hsct from both the patients and their donors were analyzed. in the hev rna positive patients, additional samples were analyzed to determine the duration of viremia. three hev rna negative controls were selected for each case of hev infection, matched for age, diagnosis, conditioning regimen and donor type. results: hev rna was detected in / ( . %) patients. in three of the patients hev rna was positive during a period of - months, and two of these patients were infected already at the time of hsct. in five patients hev-rna was positive, at a low level, only at months. / ( . %) patients had detectable hev igg and/or igm, whereof eight patients were hev rna negative. in / ( %) patients with hev infection (hev rna positive) alanine aminotransferase (alt) was > upper limit of normal (uln), in / ( . %) patients > . uln, and in / ( . %) patients alt was normal, at months after hsct. bilirubin was elevated > . uln in / ( . %) patients, and > uln in no patient at months after hsct. two patients died with ongoing signs of hepatitis and hev rna detected in blood. one of them developed acute liver failure, at the time interpreted as drug toxicity, and died of multi-organ failure. the other patient died of unrelated causes. the remaining six patients had cleared the infection at - (median . ) months after hsct. active gvhd was present at months after hsct in / ( . %) patients with hev infection, involving the liver in of these patients. corticosteroid treatment was ongoing in / ( %) patients; the mean dose during the preceding days was > . mg/kg in / ( . %) patient, . - . mg/ kg in / ( . %) patients, and < . mg/kg in / ( %) patients. hev infection correlated to elevated alt > . uln, or . p= . ) and > uln, p= . ) at months, but not at months, after hsct, compared to hev rna negative controls. conclusions: hev infection was detected in . % of patients tested at months after hsct and was correlated to abnormal alt. spontaneous clearance was common but one patient died in acute liver failure, where hev may have contributed. hev infection is a differential diagnosis in patients with elevated alt months after hsct. disclosure: nothing to declare monitoring of t-cell responses to viral-coded antigens in pediatric patients receiving tcrαβ-depleted haplo-hsct followed by bpx- cell administration background: αβ t-cell-depleted haplo-hsct is an effective option for children with hematological disorders in need of an allograft. however, recovery of adaptive immunity is impaired in these patients. thus, in order to accelerate immune reconstitution, we developed a novel approach based on post-transplant infusion of a titrated number of donor t cells, transduced with the suicide gene inducible-caspase- , ic (bpx- cells, sponsor bellicum pharmaceuticals®; nct ). we previously reported on immune recovery of children transplanted at our institution, showing that bpx- cells infused after αβ t-cell-depleted haplo-hsct expand in-vivo and persist over time, contributing to fasten adaptive immunity recovery (merli, ash ). here, we report the results of lymphoproliferation assay to viral-encoded antigens to assess tcell function in patients transplanted with this approach. methods: we evaluated children, male and female. median age at transplant was . years (range . [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] . patients had either malignant ( children) and nonmalignant ( ) disorders. no patient was given any posttransplant graft-versus-host disease prophylaxis. nine children were enrolled in the phase i portion of the trial consisting of cohorts receiving escalating doses of bpx- cells. the remaining patients (phase ii portion) received the recommended dose of x bpx- cells/kg identified in phase i. bpx- cells were infused at a median of days post-hsct (range - ). antigendriven activation of peripheral mononuclear cells was evaluated by lymphoproliferation assay with h-thymidine pulsing at d+ and harvesting at d+ . stimuli included pha or cmv, ebv and adv whole viral lysate. results are given as stimulation indexes (si, cpm stimulated sample/cpm unstimulated control). thresholds for positive response were arbitrarily set at si> for viral-encoded antigens and at si> for mitogenic stimulation with pha. fractions of responders are indicated in the figure. results: patients were analyzed from d+ to d+ post-hsct. pha responders (a) increased to %, while cmv (b), ebv (c) and adv (d) responders were %, % and % at years after haplo-hsct. responses to ebv and adv antigens were slightly delayed but improved over time. responses to pha and to cmv (e,f) were analyzed in the cmv-reactivating and cmv-non reactivating groups (cmv-yes/cmv-no). significant differences in pha response were observed at d+ and d+ . moreover, increased cmv responses were observed in cmvreactivators at d+ , d+ and d+ , with approximately % of responders at d+ , as opposed to cmvnon reactivators which comprised % responders. neither primary disease, age nor tbi during the conditioning regimen influenced proliferative capacity of the two subgroups (not shown). conclusions: we showed a rapid recovery over time of t-cell function after αβ t-cell-depleted haplo-hsct followed by bpx- cells administration. when patients were grouped according to cmv reactivation (previously demonstrated as a strong driver of immune reconstitution), a significant difference in the number of responders among the patients experiencing viral reactivation was observed using the cmv lysate only but not the immunodominant pp protein (not shown), suggesting that other viral antigens account for increased t-cell responses. results of t-cell function after bpx administration complements the phenotypic data we already reported. clinical trial registry: nct disclosure: nothing to declare. background: cytomegalovirus (cmv) is associated with significant morbidity and mortality in allogeneic hematopoietic cell transplantation (allo-hct) patients (pts). cumulative incidence of cmv infection in high-risk patients such as cd -selected or haploidentical hct have been reported as high as . - . % and - %, respectively. letermovir (ltv) was approved in / for prophylaxis (ppx) in cmv-seropositive recipients (r+) of allo-hct. since / , ltv ppx was implemented at our center for both primary and secondary ppx. we report our real-world experience. methods: adult cmv r+ allo-hct pts who initiated ltv as primary and/or secondary prophylaxis were identified between / / and / / . cord blood transplants were excluded. the primary outcome was the incidence of clinically significant cmv infection (cmv viremia requiring preemptive treatment or cmv disease). pts were followed through / . results: pts initiated ltv. . % pts were at high risk for cmv reactivation and disease (primarily ex vivo t-cell depleted hct [n = ; %] or haploidentical t-replete hct [n = ; . %]). the most common indication for hct was acute myeloid leukemia (n = ; . %) and the majority of patients received myeloablative conditioning (n = ; . %). pts ( . %) received ltv as primary ppx after hct, with a median day of ltv initiation of d+ (range d+ ─d+ ). at ltv initiation, pts had an undetectable cmv dna, and had cmv < iu/ml. clinically significant cmv infection requiring preemptive treatment occurred in of pts ( . %). one patient was treated with valganciclovir (vgv) for persistent cmv < iu/ml and received ltv as secondary ppx. a nd patient developed persistently detectable cmv (< iu/ ml) and breakthrough cmv viremia with a mutation in ul at site c yltv successfully treated with vgv. the median duration of primary ltv ppx was days ( - ), with primary ppx continuing beyond weeks post hct in pts. the median additional follow-up in patients who discontinued ltv was days ( - ), without clinically significant cmv infection to date. an additional pts ( pts overall; . %) received ltv as secondary ppx after cmv pre-emptive therapy. the median duration of secondary ltv ppx was days ( - ), with no reactivation. ltv was not discontinued due to toxicity or intolerance in any patient. cmv outcomes are summarized in figure . all-cause mortality for the pts over the observational period was . %. conclusions: primary ltv ppx significantly reduced cmv reactivation, and high-risk patients may benefit from extended prophylaxis. in patients who received preemptive therapy for cmv, use of secondary ppx showed no recurrent cmv reactivation. ltv is well tolerated. additional studies are needed to determine optimal ppx duration and to clarify role of secondary cmv ppx in high-risk allo-hct. the future standard of care will likely include extended primary ppx and secondary ppx and result in decreased morbidity and mortality associated with cmv. disclosure: andrew lin -nothing to declare, molly a. maloy -nothing to declare, valkal bhatt -nothing to declare, lauren derespiris -nothing to declare, meagan griffin -nothing to declare carmen lau -nothing to declare, anthony j. proli -nothing to declare, juliet barker -angiocrine bioscience , letermovir primary prophylaxis (pp) has been shown to reduce clinically significant cmv infection with a favorable safety profile. letermovir pp will improve the outcome of seropositive patients. however, patients who did not benefit from pp and experienced > cmv episode (infection or disease) after hct may be candidate to secondary prophylaxis (sp). indeed half of them will have > recurrent episode after pre-emptive treatment (pet). letermovir is available since november as part of the french early access program for pp and sp. we report the outcome of patients who benefited from letermovir sp in the context of this program. methods: letermovir is granted, in a restrictive manner, by the french drug agency (ansm) on a case-by-case basis for prophylaxis of cmv episode, in cmv-seropositive adult allogeneic hct recipients. sp patients should have a negative baseline cmv pcr, have already experienced > cmv episode, in the context of a potentially harmful pet according to physicians. planned letermovir daily dose was mg in case of concomitant cyclosporine and mg otherwise. all patients were routinely screened by blood or plasma cmv pcr. results: between november -july , patients received letermovir in the early access program, for pp, and for sp. among the sp patients, had previous cmv disease (gut: ; cns: ). mean age was ± years, m/f ratio was / . the sp cohort included one cord blood and haplo-identical hct. main diagnoses were acute leukemia ( %) and myelodysplastic syndrome ( %). the conditioning regimen was myeloablative in % and included atg in %. based on available data ( missing data, md), previous gvhd was present in ( %) patients, and active at letermovir initiation in ( %). thirty two ( %) patients were planned to receive immunosuppressants. donor's cmv serology was negative in / ( %) ( md). at baseline, cmv pcr was detectable in / patients. letermovir was initiated a median of days (iqr: - ) after transplant for a mean duration of ± days. only one ( %) patient developed cmv breakthrough. the median follow-up from letermovir initiation was days. among the patients exposed to letermovir prophylaxis, two patients permanently discontinued because of letermovir-related adverse events (acute gvhd and nephropathy for one, loss of appetite, pruritus, diarrhoea and weight loss for the other); two deaths occurred with no causal relationship to letermovir. data were consistent with the known safety profile of letermovir. conclusions: letermovir is or will be soonly available in most european countries for cmv prophylaxis in hct recipients. pending its routine use, letermovir used as sp was well tolerated and effective, with only / patients developing a breakthrough infection. in this high-risk population for cmv recurrence, letermovir may provide a safe bridge between pet and specific immune reconstitution, pending tapering or discontinuation of immunosuppressants. whether sp may improve survival deserves further studies. disclosure: thierry allavoine is a former employee of msd france, nathalie benard and amir guidoum are employees of msd france, marion masure is an employee of icta pm, sophie alain and catherine cordonnier have participated in advisory boards and have been members of the speaker bureau of msd. ibrahim yacoub-agha has received honoraria from msd, other authors: nothing to declare p real-world data on letermovir prophylaxis for cytomegalovirus reactivation after allogeneic hematopoietic cell transplantation: a single center experience patrick derigs , maria-luisa schubert , paul schnitzler , carsten müller-tidow , peter dreger , michael schmitt heidelberg university hospital, heidelberg, germany, background: reactivation of cytomegalovirus (cmv) still contributes substantially to morbidity and mortality after allogeneic hematopoietic cell transplantation (allohct). recently, letermovir became available as the first drug approved in europe for prophylaxis of cmv reactivation in seropositive patients who have undergone allohct. letermovir is neither myelo-nor nephrotoxic, and significantly reduced the incidence of cmv reactivation in a pivotal phase iii trial (nejm ; : ) . therefore we adopted letermovir prophylaxis according to the label as standard policy in our institution: in seropositive recipients letermovir was initiated after engraftment and continued until day + or cmv reactivation. the aim of the present study was to investigate if the favorable trial results could be reproduced under real-world conditions. methods: the study cohort consisted of the first seropositive patients who received letermovir prophylaxis at our institution (between march and august ). these were compared with a control cohort transplanted between august and march before the advent of letermovir. study and control cohorts were matched for cmv donor/recipient sero-status, underlying disease and donor type source of stem cells and application of atg. cmv viremia was monitored by a quantitative pcr twice a week during the inpatient period and weekly thereafter. patients reactivating cmv prior to engraftment were not considered as event in both groups. results: no major side effects of letermovir intake were observed. with altogether reactivation events, the cumulative rate of cmv reactivation on day + was % ( %ci - %) in the letermovir cohort and thus significantly lower than in the control group ( events, % ( %ci - %); hr . ( . - . ); p= . ). the median time to reactivation was days for the control group and not reached for the letermovir group. the cumulative number of days on valganciclovir before d + was d for the letermovir patients vs d for the control patients. there were no hospitalizations for foscavir administration in the letermovir group compared to hospitalizations in the control group. there were deaths before d + in the letermovir group (one pd, one nrm) and deaths in the control group (all pd). conclusions: this observational study confirms the safety and efficacy of letermovir for the prophylaxis of cmv reactivation in seropositive patients after allohct in a real-world setting. our results are in good concordance with the phase iii trial. although letermovir appeared to reduce the need for therapeutic valganciclovir and foscavir tremendously, larger samples with longer follow-up are needed to assess the impact of letermovir prophylaxis on non-relapse and overall mortality as well as on resource consumption. background: cmv viremia occurs in %- % of cmv r + hct recipients. pet use has reduced the risk of cmv end-organ disease (eod) and associated mortality; however, pet use may lead to substantial antiviral use and healthcare resource utilization. limited real-world data are available on the outcomes of pet. therefore, we aimed to examine cmv outcomes (eod, resistance), cmv-related mortality by day (d) and healthcare resource utilization between pet and no-pet groups among cmv r+ recipients undergoing first hct. methods: we conducted a retrospective cohort study of adults, cmv r+ recipients of first peripheral blood or marrow allograft at mskcc identified from march through december . data was extracted from electronic medical records and hct databases. cmv+ recipients were monitored weekly by quantitative pcr assay starting on d through d post hct. use of antiviral therapy for cmv viremia defined pet. high cmv risk (hr) comprised t-cell depleted (tcd) hct by cd +-selection regardless of donor hla match or conventional hct from mismatched or haploidentical donors; low risk (lr) included conventional hct from matched related donors. cmv eod was scored by standard criteria. cmv resistance mutations were confirmed by sequencing (viracor-eurofins). length of stay (los) for hct admissions and readmissions were identified through d . stratified analyses were performed to examine outcomes by pet use and cmv risk. background: in a phase iii randomized, double-blind, placebo-controlled study of cmv-seropositive post-hsct recipients, letermovir prophylaxis significantly reduced the incidence of clinically significant cmv infection through week . the objective of this research was to assess the impact of cmv prophylaxis on rates of rehospitalization in adult cmv seropositive allogeneic hsct recipients from the letermovir phase clinical trial. methods: rehospitalization was recorded as an exploratory endpoint in the clinical trial at end of treatment (week ), time of primary endpoint (week ) and through an extended follow-up period (week ). cmv-related rehospitalization was assessed in the trial. prespecified analyses describe the observed rates of rehospitalization for the letermovir and placebo groups at the specified times. fine-gray cumulative incidence function(cif) regression models were used to explore the rate of all-cause, and cmv-related rehospitalization accounting for the competing risk of mortality. a multiple linear regression model was used to describe the cumulative length of stay (los) for all-cause rehospitalizations that occurred through week (excluding time of initial transplant stay). results: observed rates of all-cause rehospitalization were lower for the letermovir group compared to placebo at end of treatment ( . %vs. conclusions: letermovir was shown to significantly reduce the rate of clinically significant cmv infection in a placebo-controlled randomized clinical trial. these analyses suggest that there is also a reduction in the rate and cumulative days of rehospitalization. this trial was not sufficiently powered to detect differences in this exploratory endpoint. nonetheless, these data provide valuable insights into the economic burden of cmv. real world data and findings from future clinical trials are needed to better understand the nature of the association between cmv and rehospitalizations. clinical methods: all consecutive patients with hematologic disorders who received hsct at our center between january and august were included. among the evaluable patients, received levofloxacin as antibacterial prophylaxis (group a) while did not receive any fq prophylaxis (group b). baseline characteristics were similar in the two groups, except for the number of patients with advanced disease ( % in group a and % in group b, p , ). median duration of neutropenia was days (range - ) in group a and days (range - ) in group b. a positive rectal swab for carbapenem-resistant enterobacteriaceae (cre) was detected in patients in group a and patients in group b. results: overall, bsi was detected in patients ( , %), ( , %) in group a and ( , %) in group b (p= , ). the median onset of bsi was days post transplant (range - ), without significant differences between the two groups. in univariate analysis, fq prophylaxis (or , ; % ic , - , ) and bone marrow stem cell source (or , ; % ic , - , ) were significant factors associated with the risk of bsi. gramnegative bacteria accounted for , % (n= ) of bsi in group a and , % (n= ) in group b, and gram-positive bacteria for , % (n= ) of bsi in group a versus , % (n= ) in group b, without statistically significant differences (p = , ). polymicrobic bsi were , % (n= ) in group a and , % (n= ) in group b. mdrgram negative bsi were detected in patients ( %) in group a and in patients ( , %) in group b (overall, cre, esbl producing enterobacteriaceae and mdr-pseudomonas). death attributable to bsi occurred in of patients ( , %); of these patients did not receive fq prophylaxis, but of them had both a pre transplant kpc colonization and active disease at transplant. neither antibacterial prophylaxis (p = , ) nor bsi (p = , ) had a significant impact on overall survival (os). conclusions: the preliminary data of our study show that fq prophylaxis is associated with a reduced incidence of bsi, in particular gram-negative infections, with no impact on os. the limitations of our study may be the different group sizes and the retrospective nauture of the study. whether antibacterial prophylaxis should be avoided in the pre-engraftment period in still a matter of debate and needs to be evaluated in larger prospective studies. disclosure: nothing to disclose. gillen oarbeascoa , nieves dorado , , laura solan , , rebeca bailen , , pascual balsalobre , , carolina martinez-laperche , , ismael buño , , javier anguita , , jose luis diez-martin , , , mi kwon , hospital general universitario gregorio marañón, hematology, madrid, spain, instituto de investigación sanitaria gregorio marañón, madrid, spain, universidad complutense de madrid, madrid, spain background: incidence and outcome of invasive fungal infection (ifi) are not well characterized in the setting of peripheral blood, non-manipulated haploidentical stem cell transplantation with postransplant cyclophosphamide (haplosct). the aim of the study was to analyze incidence and risk factors of ifi in patients who underwent haplosct at our institution. methods: consecutive patients who underwent peripheral blood haplosct with postransplant cyclophosphamide between and at our centre were reviewed. ifi was classified according to eortc definitions. proven and probable ifi were included. results: patients´characteristics are shown in table . primary antifungal prophylaxis was performed with micafungin from day - until oral intake, followed by posaconazole until day + . patients on steroid treatment for gvhd received prophylaxis with micafungin or posaconazole. % of patients obtained neutrophil engraftment. twenty-two episodes of ifi were observed in patients: proven and probable, with a cumulative incidence of ifi of % at days. most commonly isolated organism was aspergillus spp (n= ), followed by candida spp (n= : c. kruseii and c. parapsilosis), and fusarium spp (n= ). isolated cases of inonotus spp, mucor spp and trichosporon ashii were observed. pulmonary involvement was the most frequent clinical presentation (n= ), followed by fungemia (n= : candidemia, trichosporon ashii) and skin-pulmonary involvement (n= ). among patients with lung involvement, showed probable ifi: with elevated serum galactomannan and positive galactomannan in bronchoalveolar lavage (bal). there were patients without galactomannan, one with a positive bal culture for penicillum spp and the other with an aspergillus spp. median time to ifi diagnosis was days. thirteen cases were diagnosed in the pre-engraftment period, after engraftment and cases after day + . among patients with late ifi, median time to development was days. all of them were associated with gvhd ( grade iii-iv acute gvhd and moderate/severe chronic gvhd). ifi outcome was favorable in out of the ifi. treatment was liposomal amphotericin b in cases, voriconazole in and combined treatment (amphotericin b and azole) in . there were ifi related deaths, with a cumulative incidence of ifi related death of . %. prior transplant (or . , p< . ), particularly allohsct was associated to ifi development (or . , p< . ). patients with previous allohsct presented ifi mainly from molds: aspergillus, fusarium, inonotus, trichosporon and mucor. there were also candidemia episodes. no other factors were significantly associated to ifi occurrence. conclusions: in our experience, cumulative incidence of ifi in the setting of haplosct with posttransplant cyclophosphamide was similar than observed in previous studies in allosct. having received a previous sct, especially allosct, was the most significant factor related to ifi development. this high risk population should be closely monitored and could benefit from prophylaxis with azoles. disclosure: nothing to declare. methods: rsv infection was diagnosed in nasal wash (nw) or bronchoalveolar fluid (bal) by dfa (millipore, usa) or pcr (seeplex, seegene, kor). urti and lrti were defined according to ecil- guidelines. death from all causes was assessed within days after rsv infection and was attributed to rsv if the patient had persistent or progressive rsv infection with respiratory failure at the time of death. neutropenia and lymphocytopenia were defined as an absolute neutrophil count (anc) < /ul and absolute lymphocyte count (alc) < /ul, respectively. results: median number of confirmed rsv infections per year was , ranging from to . an outbreak of rsv was detected in , possibly due to a lack of compliance with contact precautions in the unit. median patients' age was years and time to rsv infection was day (- to ). twenty-three patients (pts) had received an autologous transplantation ( . %) and were allogeneic hsct recipients ( , %). median time to engraftment was days, ranging from to days. at rsv diagnosis, pts presented with urti ( . %) and with lrti ( . ) . surprisingly, around % of the auto hsct recipients had rsv pneumonia at diagnosis. variables significantly associated with lrti at diagnosis were mud hsct (no/ yes, or . ; ci . - . ); anc < /ul (or . ; ci . - . ); alc < /ul (or . ; ci . - . ); and recent or pre-engraftment hsct (no/yes, or . ci . - . ). among the pts with urti at diagnosis, progressed to lrti ( . %). forty-four of the pts died ( . %) and mortality rate was significantly higher in pts with lrti in comparison with pts with urti ( . % versus . %, p= . ). death was attributed to rsv in of the pts who died ( %). conclusions: autologous hsct recipients are also at risk of lrti caused by rsv. risk of rsv lrti is higher in mud hsct, infection acquired pre-engraftment or early after hsct, and low neutrophil and lymphocyte counts. continued education is necessary to sustain compliance to contact precautions in hsct units. disclosure background: measles is a life-threatening infection after allogeneic hct. due to the decreased coverage of vaccination in many countries, the disease reappears, increasing the risk of outbreaks worldwide. allogeneic hct recipients have been shown to be seropositive for measles in roughly - % of the cases years after transplant. however, these data were obtained before the 's from hct populations mainly conditioned with myeloablative (ma) regimens. our aim was to assess measles immunity before considering vaccination in a cohort of hct survivors including patients conditioned with reduced intensity (ric) or non-ma regimens. methods: allogeneic hct adult recipients who had not been vaccinated for measles since hct were routinely screened for measles immunity. measles igg titers were determined with a chemiluminescence immunoassay (liaison measles igg kit, liaison xl analyser, diasorin, italy). patients were considered to be seropositive if the igg titer was > . ua/ml. risk factors for seropositivity were analyzed. qualitative variables were described as numbers (%) and compared using the chi- test or fisher exact test as appropriate. quantitative variables were described as median or mean (range) and compared using the kruskall-wallis test. ors were estimated separately for factor yielding a p-value < . in the univariate analysis using logistic regression models. results: eighty-six patients, transplanted . to years (mean: , years) ago, were included. the mean age was years (range: - ), the sex ratio m/f: , . the underlying diseases were acute leukemia: ( %), myelodysplastic syndrome: ( %), lymphoproliferative diseases: ( . %), myeloproliferative neoplasms: ( %) and non-malignant diseases : ( . %). the hct was performed from an hla-identical donor in , an unrelated donor in , and a cord-blood in . conditioning regimen were ma in ( %), ric in ( %) and non-ma in ( %) patients no patient had experienced measles or had received measles vaccination since transplant. fifty-seven of the ( %) patients were seropositive for measles. measles seropositivity was not associated with conditioning regimen, patient age at transplant, patient age at time of assessment, donor age at transplant, lymphocyte count or gammaglobulin levels, or type of transplant (hlaid. vs others) measles vaccination before transplant or previous measles before transplant. the only parameters significantly associated to seropositivity were absence of previous gvhd (any type or severity, p= , or , [ , - , ]), and absence of previous extensive chronic gvhd (or , [ , ] p , ). conclusions: sixty-seven percent of allogeneic hct are seropositive for measles at a median of years after hct before vaccination. the only risk factor strongly associated with seronegativity is extensive chronic gvhd. in patients background: cytomegalovirus (cmv) reactivation is a frequent complication after hematopoietic stem cell transplantation (hsct). extracellular vesicles (evs) have emerged as a promising new category of biological biomarkers in different scenarios, including inflammation, tissue damage, cancer and viral infections. we recently reported on the potential use of serum evs as biomarkers of agvhd (lia g. et al. leukemia ( ) , ) . here, we investigated the potential correlation of cmv reactivation with plasma evs in post-transplant cyclophosphamide (ptcy) haploidentical-hsct (haplo-hsct). methods: plasma samples were collected after mononuclear cell separation at given time-points (pre-transplant, on day , , , , , , , , , and after haplo-hsct) and evs were extracted by a protamine-based precipitation method and their concentration and dimension were characterized by nano-tracking particle analysis (nanosight). after extraction, evs were analyzed by flowcytometry (guava easycyte flow cytometer) with a panel of antibodies (cd , cd , cd , krt , cd a, cd , cd , cd , cd , cd , cd , cd , cd , and cd a). results: thirty-two patients with hematological malignancies underwent haplo-hsct between and . cmv reactivation was observed in / ( , %) and occurred at a median of (range: - ) days after transplant. preliminary analysis ( / patients) showed that cd a fluorescence (platelet-derived growth factor receptor-α or pdgfr-α), cd fluorescence (ki- antigen) and cd fluorescence (ve-cadherin) were associated with an increased risk of cmv reactivation (or . p= . ; or . p= . ; or . p= . ), whereas cd (platelet endothelial cell adhesion molecule, pecam- ) concentration level was associated with a decreased risk of cmv reactivation (or . , p= . ). all these biomarkers showed a signal change before cmv reactivation (an increase with cd a, cd and cd , a reduction with cd ). (figure ). conclusions: we observed a potential association of evs membrane proteins with cmv reactivation: cd a, cd , cd and cd . these proteins are crucial for endothelium and immune cells interaction. cmv can infect different cell types including endothelial cells (bentz gl. pnas ( ) ). moreover, cd a (pdgfr-α) has been shown to function as an entry receptor for cmv expressing gh/gl/go complex (wu y. et al. plos pathog ( ) ). we plan to implement our analysis characterizing evs contents (mirnas) and will be applied to investigate other viral reactivations (e.g. epstein barr virus and human herpes virus ). [[p image] . methods: to explore the value of cmv dna extracted from gi tissue for the diagnosis of cmv gastroenteritis, we retrospectively evaluated patients, aged - (median . years) who received allo-hct from sibling( ), matched unrelated( ) or haploidentical donors( ), after receiving myeloablative ( ) or reduced intensity conditioning( ). they all underwent endoscopy for gastrointestinal symptoms between - . cmv dna from tissue samples and parallel blood samples were measured by q-pcr. positive cmv dna on the tissue was considered cmv gi infection.cmv gi disease was proven with the identification of cmv inclusion bodies or positive immunehistochemical staining using anti-cmv antibodies. results: overall, endoscopic tests were performed ( gastro-, colonoscopies) at a median of days (iqr: ) post transplantation. symptoms included nausea, vomiting, diarrhea, abdominal pain and weight loss. cmv dna was positive in / tissue samples: median copies/ml, range: - x ^ . only half patients ( / ) had concurrent cmv viremia (plasma viral load> c/ml). cmv gi infection was not correlated to the type of transplant, acute or chronic gvhd. gi cmv disease was documented by biopsy in patients. cmv dna of the tissue, but not the plasma viral load, was a predictor of biopsy positivity (or: . , %ci: . - . , p= . ). thirty-six out of cmv dna positive patients received specific treatment for at least days. symptoms resolved in / patients ( %) and the gi viral load was not a significant factor to predict cure. gi gvhd was diagnosed in / patients, among which %( / ) with cmv dna positivity. median os was days ( %ci: - ) for patients with cmv infection, similar to those without (median os: , %ci: - days, p=ns). we studied separately endoscopies of the upper ( / ) or lower gi tract ( / ) . there was no significant relationship between cmv gastritis proven by biopsy and cmv dna levels in gastric tissue. however, the viral load of the colon was a predictor of cmv enteritis (or: . , %ci: . - , p= . ). the auroc of the q-pcr was . ( %ci: . to ), the sensitivity was . % and the specificity was . % with a cutoff value of copies/ml dna. conclusions: pathognomonic findings in the biopsy remain the gold standard for the diagnosis, especially for the upper gi tract. however, when the lower gi tract is involved, quantification of cmv viral load in the tissue may be a valuable tool to support the diagnosis. positivity of cmv dna of the gi tissue, in linearity to the cmv viremia, may guide to preemptive treatment for prevention of cmv disease . disclosure: nothing to declare background: clostridium difficile infection (cdi) is caused by cd overgrowth in antibiotic-disturbed intestinal microbiota. antibiotics targeting unselectively beneficial for t-regulatory cell formation strains of clostridiales may increase pro-inflammatory processes in the guts promoting or augmenting the development of graft vs. host disease (gvhd). the efficacy of cdi treatment has impact on the persistence of inflammation which might influence the alloreactive reactions. methods: we retrospectively and, from , prospectively analyzed the data from transplant centers concerning cdi occurrence, treatment efficacy, and gvhd development. the study included patients with hematological malignancies who underwent allogeneic hematopoietic cell transplantation (allohct) between - . results: median time to cdi was days post-allohct with detection of both toxins a and b in % of cases. disturbance of intestinal microbiome was confirmed by a % rate of colonization with multidrug-resistant bacteria (mdrb). the cdi symptoms resolved with the negative toxins after the first line treatment in . % of patients. the median time to remission and therapy duration was and days, respectively. fifteen therapeutic failures were observed after treatment with metronidazole ( ), vancomycin ( ) and a combination therapy ( ) . eleven patients responded to second line treatment. thirty-seven ( %) patients died due to infections ( ), relapses ( ) and gvhd/infections ( ). we noted recurrent cdi in cases. eight patients died with active cdi. we observed occurrence or exacerbation of gvhd in ( %) patients following cdi, including cases with gut involvement (gi-gvhd). treatment with metronidazole and failure of the first line therapy increased the development or escalation of gi-gvhd (p= . and p< . , respectively). the duration of cdi exceeding days also had impact on the gi-gvhd incidence (p= . ). conclusions: . patients colonized with mdrb are at high risk of cdi. . high mortality due to infections and/or gvhd in patients with cdi. . due to lower efficacy and harmful immunomodulatory impact, metronidazole should not be the first line treatment in cdi post-hct. . emphasis must be put on fast cdi resolution to interrupt a vicious circle of the intestinal inflammatory processes. disclosure: nothing to declare establishing optimal preemptive cytomegalovirus therapy threshold post allogeneic hct in a patient population with high prevalence of seropositive status background: preemptive therapy (pet) for cytomegalovirus (cmv) reactivation post allogeneic hematopoietic stem cell transplantation (hct) was shown to decrease the incidence of cmv disease. however, the optimal pet threshold is unknown and there are significant toxicities associated with anti-cmv therapy. at our institution, we initiate pet at cmv dna titer above copies/ml ( iu/ml). our aim was to examine the efficacy of this approach including the incidence of spontaneous clearance in a population with high prevalence of cmv seropositive status. methods: after due irb approval, patients that underwent allogeneic hct were identified and records retrospectively extracted.cmv reactivation was defined as the first detectable viral titer post hct from plasma samples whereas clearance of viremia as the first date of two negative pcr values obtained at least week apart. cmv monitoring was initiated post hct performed at least weekly during the first days and every - weeks thereafter. a high sensitivity assay abbott realtime cmv was used with detection threshold of copies/ml ( . iu/ml). analysis was computed using jmp v. . . results: a. baseline characteristics: a total of patients were identified and included with a median follow up of . ( . - . ) months. median age was ( - ) years and % were male. indication for hct was for a malignant disorder in % of cases. the majority had a matched related donor ( %) and cmv igg was positive in both donor and recipient in % of cases. myeloablative conditioning was given to ( %) and ( %) received tbi. in vivo t-cell depletion was given to ( %); atg in ( %) and alemtuzumab in ( %). b. cmv reactivation and pet: a total of ( %) patients had a positive cmv pcr with median days to reactivation post hct of ; ( %) patients had peak cmv titer < copies/ml (low titer) whereas the remaining ( %) had a peak titer ≥ copies/ml (high titer). patients with high titer were more likely to be older (p = . ), have malignant disease (p = . ), haploidentical or unrelated donor (p < . ) and higher incidence of agvhd grade ii-iv (p = . ) as shown in the table. median peak titers for the low and high groups were vs. , respectively (p < . ). ( %) patients with low titers cleared spontaneously with median time to clearance of days ( - ), ( %) received anti cmv therapy and the remaining died with active viremia (range - copies /ml) with active disease. one patient in the high titer group developed cmv disease. -year os and ci-nrm was . % vs. . % (p = . ) and % vs. . % (p = . ) in the low and high titer groups, respectively. conclusions: cmv reactivation was high in this cohort however of low titer viremia in over %. a pet threshold of copies/ml ( iu/ml) appears desirable as it was associated with spontaneous clearance in almost all patients while minimizing treatment related toxicity. validation of these observations is warranted. background: the risk of pneumocystis pneumonia often warrants antifungal prophylaxis for recipients of blood and marrow or solid organ transplantation. however, complications such as myelosuppression, nephrotoxicity, and intolerance with the existing standard, trimethoprim/sulfamethoxazole (tmp/smx), may hinder or interrupt prophylaxis. rezafungin (rzf) is a novel echinocandin in development for prevention of invasive fungal disease caused by candida, aspergillus, and pneumocystis species in blood and marrow transplant patients. rzf has a favorable safety and tolerability profile and a low risk of drug-drug interactions. furthermore, the stability and pharmacokinetics of rzf allow for once-weekly dosing and broad distribution to the lung and other target organs. rzf was shown to prevent in vitro pneumocystis biofilm formation and to reduce the viability of mature biofilms. a previous prophylactic study was conducted using a broader range of rzf doses. in the current study, the efficacy of rzf was evaluated to better understand the minimum doses necessary to prevent pneumocystis growth in a mouse model. methods: c h/hen mice were immunosuppressed (dexamethasone mg/l in acidified drinking water) and then infected intranasally with p. murina ( x / μl). given the slow growth of p. murina, test agents were administered at the same time mice were inoculated to test for prophylactic efficacy. mice received intraperitoneal injections of either vehicle (control/steroid [c/s]), tmp/ smx / mg/kg/ x/week (wk), caspofungin mg/kg once daily, or rzf mg/kg or . mg/kg once daily, x, or x/wk. after a -week dosing period, mice were sacrificed and lung homogenates were processed for analysis to quantify the nuclei (trophic) and asci (cyst) forms of pneumocystis. prophylaxis efficacy was based on reduction of organism burden compared with c/s. nuclei and asci counts were log transformed and analyzed by anova; individual groups were compared by the student-newman-keuls t test. survival rates were compared using graphpad prism v . results: all mice in the rzf groups had significantly reduced nuclei and asci burdens compared with the c/s group, and all but the lowest doses of rzf ( . mg/kg x or x/wk) worked as well as tmp/smx at reducing nuclei levels. similarly, all rzf groups except for the . mg/kg x/wk group showed reductions in asci levels comparable to that of tmp/smx. the survival rates were not statistically different between treatment groups. conclusions: rzf demonstrated potent in vivo efficacy for prophylaxis against pneumocystis in an in vivo mouse infection model at dose regimens much lower than the human equivalent phase regimen. these data support the development of rzf for the prevention of invasive fungal infections including pneumocystis pneumonia. disclosure: melanie t. cushion: research funding (cidara therapeutics) taylor sandison: employee, stockholder (cidara therapeutics) alan ashbaugh: nothing to declare. yuhua ru , , ziling zhu , , yang xu , , suning chen , , xiaowen tang background: immunocompromising period following allogeneic hematopoietic stem cell transplantation (allo-hsct) may allow opportunistic pathogens to thrive and result in fatal complications. epstein-barr virus (ebv) infects more than % of chinese population, and its reactivation after hsct is one of the major concerns due to the increased risk of ebv diseases and post-transplant lymphoproliferative disease. with the development of infection prophylaxis and supportive care after hsct, demographic data on ebv reactivation post-hsct needs to be updated. methods: we retrospectively analyzed the data of patients who received allo-hsct between july and july in the first affiliated hospital of soochow university. quantitative pcr (q-pcr) was used to monitor ebv-dna load in peripheral blood dynamically. ganciclovir (pre-hsct) followed by acyclovir was given as viral prophylaxis. the treatment protocol for ebv reactivation consisted of tapering of immunosuppressive agents, antiviral agents (including ganciclovir and sodium phosphonatel), and rituximab for persistent positive patients. results: totally cases from most of the provinces in china were enrolled (characterized in table ), among whom ebv reactivation developed in recipients. most reactivation events ( . %) occurred in the first year post-hsct, with a peak of . incidence rates per personyears at the second month. besides, more episodes of lateonset reactivation occurred in patients receiving grafts from haploidientical donors ( figure a ) . multivariate analyses revealed that the major impactors of ebv reactivation included atg as gvhd prophylaxis (p< . ), hlamismatched donor (p= . ) and the appearance of chornic gvhd (p= . ). cumulative incidence of ebv reactivation was low ( . %) among patients with no major risk factors, but increased to . %, . % or . % with , , and major risk factors, respectively ( figure b) . there was no statistical difference of overall survival between people with or without ebv reactivation (p= . ). conclusions: we concluded that there are similar ebv reactivation impactors in chinese population compared to literatures, including atg use, hla-mismatched donor and the appearance of chronic gvhd. additionally, incidences of ebv reactivation increased significantly with the accumulation of risk factors. however, ebv reactivation had no impact on overall survival in current virus management protocol. disclosure: nothing to declare background: several studies have shown loss of diversity of the gut microbiome in association with significant gut injury following hematopoietic stem cell transplantation (hsct). prolonged broad spectrum antibiotic use further promotes loss of microbiome diversity and increases the risk of intestinal colonization by multi-drug-resistant (mdr) bacteria. aims of this study were to prospectively evaluate the overall changes in gut microbiome composition after hsct and differences in patients colonized by mdr bacteria and treated with carbapenems. methods: we performed a prospective observational study evaluating the gut microbiota of hematological patients undergoing hsct, from admission (t ) through day + (t ). fecal microbiota was assessed by s amplicon-based sequencing. clinical, and microbiological data as well as fecal samples were collected every th day from admission. results: one-hundred fecal samples were analyzed. overall, we found a progressive decrease of bacterial richness from t to t , with a significant reduction of blautia, ruminococcus and dorea species, which are strictly associated with the production of short chain fatty acids (sca) (fig. ) . moreover, in the % (no. ) of patients who were colonized by esbl bacteria, we observed a significant reduction of clostridium spp and bifidobacterium species. as for antibiotic therapies, carbapenems were used as second line treatment of febrile neutropenia in % (no ) of cases, usually associated with aminoglycosides. in patients treated with meropenem, a strong decline of blautia and ruminococcus species was observed. this finding suggests a correlation between carbapenem regimens and increase of pro-inflammatory bacterial strains in the gut. conclusions: our data support the hypothesis that loss of intestinal commensals that produce short-chain fatty acids may increase dysbiosis. moreover, for the first time we report significant and progressive alterations in the composition of blautia, ruminococcus and bifidobacterium species in patients treated with meropenem and colonized by esbl bacteria, respectively. our findings offer potential modifiable targets to reduce risk of colonization by mdr bacteria and to promote a carbapenem-sparing approach in the hsct setting. clinical background: cmv is associated with significant morbidity after allogeneic hematopoietic stem cell transplantation. strategies to prevent cmv-related complications include universal prophylaxis and preemptive therapy, more widely spread. antivirals used for cmv reactivation (cmv-r) produces major toxicities and costs. rate and characterization of cmv-r after haploidentical transplantation with post-transplant cyclophosphamide (haplo pt-cy) is scarce. our goal was to analyze cmv-r rate after haplo pt-cy, outcome, complications associated to therapy, and to identify risk factors. methods: one hundred haplo pt-cy transplants using peripheral blood as stem cell source performed between and in our center have been retrospectively reviewed. gvhd prophylaxis consisted of pt-cy mg/ kg/day on days + and + , mmf and csa from day + for all cases. cmv pcr was performed in a biweekly basis during admission for transplant and treatment, and weekly thereafter. cmv-r was considered with any cmv dna level by pcr assay above copies/ml. prior four consecutive negative weekly pcrs were needed to consider a new reactivation episode. preemptive strategy was applied in all cases. data collected in relation to cmv-r included: cmv serostatus of donor/recipient (d/r), number of cmv reactivations, length of each reactivation, antiviral treatment used, need for admission to receive treatment and adverse events related to cmv reactivation and/or antiviral treatment. results: patients characteristics are summarized in table . among patients, of them with positive cmv serology, episodes of cmv-r were detected. seventysix patients ( %) had at least one cmv-r in a median of days after transplant. none of them had cmv disease or die as a consequence of cmv-r. median duration was days ( - ). valganciclovir or ganciclovir was used in episodes ( %). foscarnet was used in episodes ( %). six of the episodes occurred after initial discharge, and required re-admission for treatment, with a median length of hospitalization of days ( - ). cytopenias requiring transfusion or g-csf support occurred in episodes ( %) treated with ganciclovir or valganciclovir. three of them needed further cd + cells booster for graft rescue. mild acute renal failure and genital ulcers were found in ( %) and ( , %) events treated with foscarnet, respectively. no cases of severe renal failure were observed. serological status different than negative/negative (n/n) (p . ) and older age ( vs years, p . ) were significantly associated with cmv-r. no relationship was observed with gender, disease, donor relationship, conditioning, gvhd or cells infused. more than reactivations were more frequent among patients with grade ii-iv acute gvhd (agvhd) and moderate-severe chronic gvhd (cgvhd). conclusions: in our experience, rate of cmv-r after unmanipulated haplo pt-cy, using pbsc as stem cell source, is considerably high. a significant proportion of patients presented complications associated with cmv-r and its treatment. cmv serological status other than n/n and older age are associated with high risk of cmv-r. patients with grade ii-iv agvhd are at higher risk of multiple reactivations. this population could be benefited from primary prophylaxis, in order to decrease treatment´s complications, re-admissions and costs. disclosure: nothing to declare. impact of infectious events occurring during the first hundred days after hsct for hematological malignancy: a monocentric retrospective study over a five-year period marie-pierre ledoux , célestine simand , , karin bilger , annegret laplace , bruno lioure background: patients undergoing hematopoietic stem cells transplantation (hsct) for hematological malignancy often present with infectious events in the early stages of the procedure, some of which having a documented impact on the outcome of the graft. for instance, cytomegalovirus (cmv) has been shown by some authors to have a protecting effect against relapse, whose features remain to be elucidated. we conducted a retrospective monocentric study regarding the outcome in terms of graft versus host (gvhd), relapse and survival of consecutive patients over a period of years, whether they presented or not with an infectious event by day among the following: cmv viremia, epstein-barr virus (ebv) viremia, human herpes virus (hhv ) viremia, bk virus (bkv) viruria, bacterial bloodstream infection (bsi) or invasive fungal infection. results: a high proportion of cmv seropositive recipients underwent a viral reactivation of cmv by day of the hsct: % if the donor is seronegative and % if the donor is seropositive. we observed that cmv wasn't associated with a lower relapse rate in our cohort, and data weren't sufficient to conclude firmly, but showed a trend towards a worse acute gvhd (hazard ratio hr . , pvalue . ). no significant correlation was found for ebv viremia. occurring in % of our patients and mostly with an early timing, hhv strongly correlated with worse acute gvhd (hr . , p-value < . ) but its impact on survival was not significant. bkv ( % of our patients) and bsi ( % of our patients) both correlated with poorer outcome in terms of overall survival (logrank < . and . respectively) although not significantly associated with relapse or acute gvhd. fungal infections were too rare events to draw any conclusion. conclusions: thus, contrary to many studies, we found no protection against relapse induced by cmv, although the trend for worse acute gvhd was obvious. the mechanisms behind this discordance could include early treatment, but remain to be studied. whether hhv is a cause rather than a consequence of acute gvhd or its treatment is debated, but the correlation is strong and the sequence of events suggests hhv might act as a trigger for gvhd. the association between bkv viruria and a higher mortality is in contrast with previous observations, and the lack for association with gvhd and relapse could suggest bkv is a surrogate for poor immune recovery and therefore other causes of non-relapse mortality. in addition to the direct lethal risk of bacteriemia, bsi also are a promoter of late non-relapse mortality through indirect toxicity. through the expansion of immune effectors they promote, one could assume that infectious events play a role in gvhd and gvl, and therefore have an interference with relapse. however, the association between each infectious event and outcome remains to be clarified to guide our prophylactic and therapeutic choices by a better understanding of the bright and dark sides of infectious events. disclosure background: rezafungin (rzf) is a novel echinocandin in phase development for treatment of candidaemia and invasive candidiasis and for antifungal prophylaxis against invasive fungal diseases caused by candida, aspergillus, and pneumocystis in blood and marrow transplant patients. rzf is differentiated by stable, prolonged pharmacokinetics (pk) that allow for once-weekly dosing and a pk-pharmacodynamic (pd) profile correlating with efficacy. clinical in vivo evaluations of drug interaction potential were performed proactively to assess the risk of drug-drug interactions (ddis) with respect to the phase dose of mg once weekly and known pk exposure in healthy individuals. methods: this open-label study of healthy inpatients assessed ddis between rzf (as perpetrator) and drugs known to have interactions with cyp enzymes and transporters (probe drugs): repaglinide (cyp c ), metformin (oct/mate), rosuvastatin (bcrp/oatp), pitavastatin (oatp), caffeine (cyp a ), efavirenz (cyp b ), midazolam (cyp a ), and digoxin (p-gp), as well as tacrolimus, a drug likely to be coadministered with rzf. an initial dose of rzf mg was administered on the first dosing day, to approximate a steady state plasma concentration of multiple once-weekly -mg doses, followed by once-weekly -mg doses on days and . probe drug cocktails containing ≥ drugs were administered, once before and once after rzf administration, on a schedule designed to allow for washout between doses and to limit interactions with other probe and test drugs. samples were analysed to determine respective drug concentrations in plasma (except for tacrolimus which was in whole blood) to characterize the pk profile of each analyte. area under curve (auc) and maximum concentration (c max ) were calculated from the plasma/blood concentration-time profiles by noncompartmental analysis. ln-transformed pk parameters were statistically analysed using an analysis of variance model. the ratio of geometric least squared means between each substrate drug when administered with and without rzf and corresponding % confidence intervals (cis) were calculated for lntransformed c max and auc. results: when rzf was given concomitantly with the probe drugs, six of nine substrates (metformin, pitavastatin, caffeine, efavirenz, midazolam, and digoxin) statistically demonstrated the absence of drug-drug interaction, as their % ci were all included within the default - % noeffect boundary. three substrates had the upper (repaglinide and rosuvastatin) or lower (tacrolimus) bounds of their ci falling just outside of this range (figure ), and these changes are considered unlikely to be clinically significant. conclusions: no meaningful pk interactions were observed between rzf and drugs known to have ddis and/or likely to be coadministered with rzf. these findings provide evidence that no dose adjustment is expected when rzf is co-administered with these commonly used drugs, which stand in contrast with the ddi complications widely associated with azole antifungals. disclosure: voon ong: employee, stockholder (cidara therapeutics), michael boily: employee (altasciences), hong wong: employee (altasciences), taylor sandison: employee, stockholder (cidara therapeutics), shawn flanagan: employee, stockholder (cidara therapeutics) abstract withdrawn. background: cytomegalovirus (cmv) continues to cause morbidity following allogeneic hematopoietic stem cell transplantation (hsct). letermovir is a newly approved drug for cmv prophylaxis in cmv-seropositive allogeneic hsct recipients. however, there is a paucity of data for its efficacy in patients receiving in-vivo t-cell depletion (tcd). at weill cornell medical center, we perform in-vivo tcd with alemtuzumab for related and hla-identical unrelated transplants, and anti-thymocyte globulin for umbilical cord blood transplant supported by third party accessory cells (haplo-cord transplant).although these drugs reduce the frequency of graft-versus-host-disease (gvhd), they significantly delay t-cell immune reconstitution post hsct, and may cause higher rates of cmv reactivation. our historical rate of cmv reactivation in cmv seropositive recipients receiving high dose valacyclovir prophylaxis is approximately %. therefore, we implemented letermovir for cmv prophylaxis in february . the primary aim of this study is to determine the incidence of cmv infection (defined as cmv viremia warranting treatment or development of end-organ disease) in tcd cmv seropositive allogeneic hsct patients who received letermovir prophylaxis. methods: this is a single center, retrospective cohort study to determine the incidence of cmv infection in adult, cmv-seropositive recipients receiving letermovir prophylaxis after in vivo tcd hsct with atg or alemtuzumab for gvhd prophylaxis. all included subjects were at least days post-transplant. results: allogeneic hsct transplant recipients met inclusion criteria. median age was years, iqr [ , ] and % were male. eight ( %) had a matched related donor, six ( %) had a matched unrelated donor, and ( %) were haplo-cord transplants. their underlying malignancy and conditioning regimens are summarized in table . ( %) received atg and ( %) received alemtuzumab for gvhd prophylaxis. median follow up time for survivors is days, iqr [ , ] . the incidence of cmv infection in the first days post-transplant was % as only one patient reactivated with detectable cmv viremia. this same patient developed cmv pneumonitis with documented ul resistance, and was successfully treated with ganciclovir. the incidence of cmv infection within the first days post-transplant was % ( / patients) . six patients ( %) developed acute gvhd in the first days, and one ( %) had relapse of their malignancy. five patients ( %) died within days post-transplant, but none of these deaths were cmv related. background: infectious complications caused by endogenous adenovirus (adv) are common and associated with morbidity and mortality rates in patients after hematopoietic stem cell transplantation (hsct). adv infections occur in about % to % of hsct recipients, with significantly higher rates in pediatric patients. a better understanding of adenoviral-specific t-cells (advt) response in donors can serve as a basis to develop more effective strategies for antiviral therapy. methods: frequencies of cytomegalovirus (cmv)-and adv-specific t cells were determined by enzyme-linked immunospot (elispot) assays with adv hexon and cmvpp respectively in health donors. we used x of mononuclear cells (mnc) per well in elispot assays. all donors were divided into groups according to the number of spots per well (spw) as follows: high responders (hr) (≥ spots; n= ), low responders (lr) (> and < spots; n= ), nonresponders (nr) (≤ spots; n= ). the average spot area of adv-and cmv-specific lymphocytes was calculated by immunospot® multiplate autocount™. cd ra+ and cd ro+ t-cells were generated by immunomagnetic negative selection. hla typing for class i and ii was performed by sequence specific oligonucleotides technology. statistical analysis was performed using graphpad prism v . software. levels of significance were calculated by mann-whitney rank-sum test, expressed as p-values (p< . ). results: the median frequency per well of advts were in hr group, in lr group, in nr group. the median spw of cmv-specific t cells in donors mnc were and didn´t differ between groups. antiviral activity may depend not only on the amount of advt but also on their ability to produce ifnγ. the average spot area for advt did not differ between hr, lr and nr groups and were , , , and , mm respectively. the median of the average spot area for anti-cmv t-lymphocytes was equal to , mm . thus, the frequency of advt was lower than cmv-specific t-cells, but advt have the ability to produce more ifnγ per cell (p< . ). in order to evaluate the distribution of the advt between naive and memory t cell compartments, we evaluated response to adv in preselected cd ra+ and cd ro+ fractions of t-cells in a group of donors. the median frequency of advt in unfractionated mnc was ; the median frequency of advt in cd ra and cd ro fractions were and , respectively. the amounts of cd ra and cd ro tcells were normalized to their amounts in mnc. we evaluated the impact of hla-alleles on the anti-adv response of t-cells in different groups and found association: hla-a* with hr group (p-value= , ; rr= , ; % ci: , to , ) and hla-a* with lr group ; rr= , ; % ci: , to , ) . conclusions: in this study the frequency of donors with advt is , % which corresponds to the reported frequency of adv-seropositive people ( %) in population of russia. advt are exclusively cd ro-positive cells. the analysis of advt in potential hsct donors will allow to determine more accurately the amounts and functional activity of specific antiviral t-lymphocytes administered to patient and optimize antiviral therapy. disclosure: nothing to declare p abstract withdrawn. chemotaxis and exhaustion of γδ t cells in the allografts are associated with cmv reactivation after hematopoietic transplantation background: cytomegalovirus (cmv) reactivation and its related diseases remain the most common and serious complications in patients who underwentallogeneic hematopoietic stem cell transplantation (allohsct). we previously reported that the incidences of total and refractory cmv reactivation reached approximately % and % after haploidentical hsct. while majority of studies in the literatures focused on the adaptive cd + αβ t cellsand nk cells in anti-cmv immunity, increasing evidences highlighted the important role of γδt cells in this context. a progressive and prolonged expansion of vδ + t cells in response to cmv reactivation was observed after allohsct. the effect of vδ + t cells associated with cmv clearance has been reported in vitro and in vivo. in contrast to the reconstituted γδt cells post transplantation, whether the phenotypes of γδt subsets in allografts correlate to cmv reactivation in hsct recipients have not been documented. methods: the proportions and phenotypes of γδ t cells were detected inallografts those were unmanipulated g-csf-mobilized bone marrow (bm) and peripheral blood (pb) harvests from donors for haplohsct. bm grafts were collected by aspiration on the fourth day of g-csf treatment (filgrastim, μg/kg/day), and pb grafts were obtained on the fifth day by leukapheresis. immunophenotyping for γδ t-cell subpopulations, including the expression of cd , cd , cd , tcrγδ, tcrvδ , tcrvδ , hla-dr, nkg d, cxcr , ccr , pd , ki , ifnγ, tnfα, and il- , was performed using flow cytometry. for detection of the intracellular cytokines, bm and pb grafts were pre-stimulated with x cell stimulation cocktail ( x, ebioscience). cmv dna in the peripheral blood of recipients was routinely monitored by quantitative pcr. the association of γδ t-cell contents in allografts with cmv reactivation in haplohsct recipients was analyzed using the mann-whitney u test and spearman test. all calculations were performed using spss . statistical software. results: we found that the proportions of total γδ t cells, and vδ and vδ subsets in both bm and pb grafts for cmv+ and cmv-recipients were comparable. neither the expression of hla-dr nor nkg d in the allografts were significantly different in correlation to cmv reactivation after hsct. the productions of intracellular cytokines of γδ t subsets did not varied in bm and pb grafts for cmv+ and cmv-recipients. interestingly, the proportions of cxcr +vδ and ccr +vδ cells in bm grafts for cmv + recipients were significantly higher than those for cmvrecipients (p = . and . , respectively). meanwhile, pma-stimulated ki +vδ cells in bm grafts for cmv+ recipients were less than those for cmv-recipients (p = . ). in parallel, the concentration of pd +vδ cells in pb grafts for cmv+ recipients were significantly higher than those for cmv-recipients (p = . ). conclusions: this study is the first to connect the chemotaxis and exhaustion of γδ t cells in grafts to the risk of cmv infection after allogeneic hsct. future studies should explore how the expressions of chemokines and exhaustion marker on the effector γδ t cells in allografts facilitate cmv replication and/or dissemination in the setting of hematopoietic transplantation. disclosure: all authors do not have conflicts of interest. this study is supported by the national natural science foundation of china (grants no. and no. ) results: incidence of ic was , %: allo-hsct - % (n= ), auto-hsct - , % (n= ). the etiology: c. parapsilosis %, c. albicans %, c. krusei %, candida tropicalis %, candida dubliniensis %. the most frequent underlying diseases was acute leukemia - % (n= ). the median age was y.o. [ month - years] . the median day of onset of ic after allo-hsct was , auto-hsct - [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] . febrile fever was the main clinical symptom; septic syndrome develops in % cases. antifungal therapy was with echinocandins - %, lipid ampho b - %, azoles (fluconazole, voriconazole) - %, without therapy (the early mortality) - %. overall survival (os) at days from diagnosis of invasive candidiasis was %. the central venous catheter (cvc) removal was the only factor significantly improved os ( % vs %, p= , ). conclusions: incidence of invasive candidiasis in children after hematopoietic stem cell transplantation was . %. the main etiology agent was c. parapsilosis. invasive candidiasis infections most often affect leukemia patients, developed later after allo-hsct than auto-hsct. overall survival at days from the diagnosis was %. removing of cvc improved overall survival in children with invasive candida infections after hsct. disclosure: nothing to declare background: graft versus host disease (gvhd) and virusassociated enteropathy in allogeneic hematopoietic stem cell transplantation (allo-hsct) may cause severe quantitative and qualitative composition changes of intestinal microbiota, leading to the development of small intestinal bacterial overgrowth (sibo) on the background of immunodeficiency, which can have a negative impact on treatment effectiveness. the gold standard for diagnosis and the main criterion for sibo is the detection in the jejunum aspirate > /ml bacteria and/or the appearance of colonlike microbiota in small intestine. it is also acceptable to use an alternative non-invasive technique -hydrogen breath test, which could be especially important in patients with severe mucositis, grade iii-iv and thrombocytopenia grade iv. sibo diagnosis in the setting of the gastrointestinal tract damage and dysfunction in patients treated with allo-hsct is insufficiently studied. methods: the study included patients with acute myeloid leukemia (n= ), acute lymphoblastic leukemia (n= ), myelodysplastic syndrome (n= ), non-hodgkin´s lymphoma (n= ), hurler syndrome (n= ), who underwent allo-hsct from an unrelated (n= ) and haploidentical donor (n= ) , and which were complicated by enteropathy development. in cases, the enteropathy reason was a combination of intestinal gvhd and viral colitis (hhv- ), in cases -viral colitis (hhv- ). all patients had esophagogastroduodenoscopy with species aspiration from descending part of the duodenum and feces collection, with further bacteria pcr identification. hydrogen breath test was performed also in which patients were treated with oral lactose g/kg with subsequent hydrogen assessment after and minutes. the study was performed in the period from to days after allo-hsct. results: according to feces analysis data, colon microbiota composition significantly differed from the reference values. at the same time total bacterial mass of the duodenum was less in comparison with colon microbiota: e+ ( e+ / e+ ) and e+ ( e+ / e+ ), respectively (p< . ). quantitative composition of the duodenal microbiota was comparable to that of colon: lactobacillus spp. e+ ( e+ / e+ ) > . e+ ( e+ / e+ ), (p= . ); bifidobacterium spp. e+ ( e+ / e+ ) < . e+ ( e+ / e+ ), (p= . ); escherichia coli e+ ( e+ / e+ ) > e+ ( e+ / e+ ), (p= . ); bacteroides fragilis group e+ ( e+ / e+ ) > e+ ( e+ / e + ), (p= . ); faecalibacterium prausnitzii e+ ( e+ / e+ ) < . e+ ( e+ / e+ ), (p= . ), indicating the presence of sibo. in this case, the hydrogen breath test was completely uninformative: basal values - . ( . / . ) ppm, hydrogen concentration in minutes - . ( . / . ) ppm, in minutes - . ( . / . ) ppm, which is less than in healthy volunteers. conclusions: quantitative composition of the duodenal and colon microbiota is similar in the case of intestinal gvhd and/or virus-associated enteropathy in allo-hsct patients, which may be of diagnostic value for sibo confirmation. the hydrogen breath test is an uninformative method for sibo identification in patients after allo-hsct. disclosure: nothing to declare johannes schulte , patrick hundsdörfer , sebastian voigt background: adenovirus (adv) infections or reactivations frequently occur in the pediatric hematopoietic stem cell transplant (sct) setting and these infections contribute to increased morbidity and mortality. the nucleotide analog cidofovir might be effective in reducing adv load, however, nephrotoxicity is a considerable side effect. the new antiviral compound brincidofovir (bcv, cmx- ), a lipid-conjugate nucleotide analog with broad-spectrum antiviral activity in vitro, has been reported to be effective in cases where cidofovir treatment was unsuccessful. methods: data of eight pediatric patients undergoing sct for malignant and nonmalignant indications were analyzed. all patients were weekly monitored for adv viremia by pcr. in case two consecutive positive adv pcr results indicating a viral copy number > /ml were documented, patients received a weekly dose of cidofovir. if no reduction of adv load was seen within two weeks after the commencement of treatment or side effects demanded cidofovir discontinuation, bcv was obtained through an emergency expanded access programme. results: eight pediatric patients developed adv viremia with maximum viral loads ranging between and copies/ml. six patients had c type adv and two patients had non c type adv infections. five patients had viral co-infections: two had an additional cmv infection, one had an epstein-barr virus (ebv) and herpes simplex virus co-infection, one patient had an ebv co-infection and one patient had a bk virus co-infection. all eight patients initially received cidofovir, however, a substantial decrease in adv load could not be observed in any patient after a two-week administration course. except in one patient who had extensive intestinal graft-versus host disease (gvhd), adv infection was cleared in all patients within three weeks after the beginning of bcv treatment. in addition, all coinfections were cleared. no nephrotoxicity or other side effects were observed. conclusions: bcv was effective in all but one patient. oral bcv might not be effective in advanced upper gut gvhd, especially when applied via a gastric tube, yet this was observed in only one patient. eventually, without nephrotoxic side effects, bcv could be an useful alternative to cidofovir. disclosure: nothing to declare. background: the use of post-transplant high-dose cyclophosphamide (ptcy) has overcome the need for extensive depletion of t lymphocytes from haploidentical donor grafts, which traditionally resulted in severe and prolonged immunosuppression. however, reconstitution of cellular immunity may be delayed even after t cell replete haploidentical stem cell transplantation (haplo-sct) with ptcy. the study of the incidence and severity of viral reactivation is therefore relevant to the outcomes of haplo-sct with ptcy. methods: our study enrolled patients (women/men, / ), who underwent t cell replete haplo-sct from / to / and achieved hematopoietic engraftment. median age at transplant was . years (range, - ) . the underlying disease was aml (n= ), all (n= ), mds (n= ), myelofibrosis (n= ), cml (n= ), or cll (n= ). the conditioning regimen was myeloablative (n= ), reduced-intensity (n= ) or non-myeloablative (n= ). peripheral blood was the graft source in the majority of cases (n= ) and bone marrow in the remaining (n= ). recipient/donor cytomegalovirus (cmv) serostatus was -/-(n= ), -/+ (n= ), +/-(n= ), or +/+ (n= ). the combination of tacrolimus and mycophenolate mofetil was administered in addition to ptcy for prevention of graftversus-host disease. cmv, epstein-barr virus (ebv), and human herpesvirus- (hhv- ) reactivation was monitored by real-time quantitative pcr (rq-pcr) in blood twice weekly post haplo-sct. bk virus (bkv) reactivation was assessed by rq-pcr in urine and/or blood specimens in cases with symptoms suggestive of bkv-associated hemorrhagic cystitis (hc). results: with a median follow-up time of months (range, , the cumulative incidences (cin) of relapse and non-relapse mortality (nrm) were . % ( % ci, . - . %) and . % ( % ci, . - . %) at years, respectively. median disease-free (dfs) and overall survival (os) were . % ( % ci, . - . %) and . % ( % ci, . - . %) at years, respectively. the cin of cmv reactivation/infection (> copies/ml) reached . % ( % ci, . - . %) at months. cmv infection developed in out of patients who were at risk, whereas recurrent cmv reactivation was observed in patients with a median number of episodes (range, - ) per patient. the median total duration of antiviral therapy for cmv infection was days (range, - ) . cmv disease (pneumonia) was documented in patients. the cin of ebv reactivation (> , copies/ml) was . % ( % ci, . - . %) at months. no case of ebv-related post-transplant lymphoproliferative disorder was observed, however preemptive therapy with rituximab was required in patients with rapidly increasing ebv viral load. hhv- reactivation (> , copies/ml) was observed in patients (cin, . % at months; % ci, . - . %), with none of them requiring specific therapy. bkv-related hc occurred at a cin of . % ( % ci, . - . %) at months. cystoscopy for bladder hemostasis was required in / and nephrostomy in / patients with hc. conclusions: despite preservation of non-alloreactive memory t cells, haplo-sct with ptcy is associated with substantial rates of viral reactivation (especially cmv and bkv) resulting in the need for prolonged antiviral therapy and considerable morbidity as well. therefore, strategies to prevent viral reactivation and disease are still warranted in haploidentical stem cell transplantation. disclosure: nothing to declare. background: adenovirus(adv) infections are a wellrecognised cause of morbidity and mortality in children and adults receiving an allogenic stem cell transplant(hsct).the reported incidence of adv infection is higher( %- %) in paediatric hsct than in adults( - %),but we currently lack accurate data of adv infection burden among adults.cidofovir has been extensively used as a pre-emptive anti-adenoviral therapy and is current standard of care.we present our single centre experience of adv incidence and outcomes with pre-emptive approach in adult patients receiving t-cell depleted(tcd) hscts for myeloid disorders. methods: this is a single-centre retrospective analysis of consecutive hsct patients for myeloid disorders including aml, mds, mpn & aplastic anaemia between january -june using atg or alemtuzumab based tcd.adv screening was performed in all patients with standardised real time quantitative pcr on weekly basis during standard risk period. figure a - b] results: baseline characteristics (table ) of patients were similar across both cohorts with or without adv infection. overall . %(n- / ) patients were positive for adv dna on atleast one of the sanctuary sites(upper respiratory airway,blood,faeces,urine) and %(n- / ) of these experienced disseminated infection(defined by adv in ≥ sanctuary sites or rising adv dna copies in blood), while developed typical adenoviral disease (pulmonary). among patients with disseminated infection,majority had adv in gastro-intestinal( %), . % in genitourinary and % as both sanctuary site of infection,in addition to blood viraemia( % of all cases).cumulative incidence of adv infection was . %( %ci: . - . %) at months with median time of days(iqr: - days) to detect adv-dna post hsct.overall survival(os) at years for whole cohort was %( %ci: - ;median os- months) with no statistical difference between patients with disseminated adv infection vs those with none(log rank; p- . ; fig- a ). overall cumulative incidence of non-relapse mortality (nrm) was %( %ci: - %) and relapse(cir) was . %( %ci: - %) at years,but no statistical difference noted between patients with disseminated adv infection & those with none(nrm:p- . ;cir: p- . ;gray test).pre-emptive therapy with cidofovir ( mg/kg weekly iv infusion for weeks and fortnightly thereafter until infection free) was required in %( / ) of symptomatic adv infection patients and %( / ) with disseminated infections.one patient required brincidofovir therapy for refractory disease,but one patient died due to severe sepsis, before adv specific therapy could be given.remaining patients were monitored and all self-recovered on cessation of immunosuppression.all patients treated with cidofovir developed renal impairment(defined by atleast > % increase in baseline creatinine),however majority( %) recovered their renal function near their baseline (fig- b) . conclusions: adv infection remains a significant cause of morbidity in adult hsct patients, however pre-emptive management with cidofovir has improved os and nrm despite use of tcd conditioning.renal toxicity remains common with cidofovir but with use of intermediate doses, majority do recover their renal functions. clinical trial registry: n/a disclosure: nothing to declare background: publications on invasive fungal disease (ifd) in lymphoma patients are limited especially after allo-hsct. there are no data on outcome of allo-hsct in lymphoma patients with prior ifd. this study focuses on epidemiology of ifd before and after allo-hsct in children and adults with hodgkin's lymphoma (hl). methods: single center prospective study included patients with classical r/r hl who received allo-hsct from to . the median age was ( - ) y.o., children (< yo) - %. allo-hsct from mud was performed in , % (n= ), mrd - , % (n= ), mmud - , % (n= ), haplo - , % (n= ), with ric ( %) and predominantly ptcy-based gvhd prophylaxis ( %). primary antifungal prophylaxis was fluconazole in %, secondary -voriconazole ( %). eortc/msg criteria for diagnosis and bronchoscopy before allo-hsct in pts with ct-scan lung lesions were used. "active ifd" means ifd diagnosed just before hsct. median follow-up time was months . results: incidence of ifd before allo-hsct was , % (n= ). ifd prior to hsct were invasive aspergillosis (ia) with lungs involvement. antifungal therapy before allo-hsct was used in , % pts with median duration - months. complete response to antifungal therapy was in , % pts, partial response or stabilization - , %, and , % pts had an "active ifd". after allo-hsct all pts received voriconazole as an antifungal therapy or secondary prophylaxis. cumulative incidence of relapse or progression of ia after allo-hsct was , % with the median day after hsct, which were successfully treated with voriconazole in post hsct period. incidence of ifd after allo-hsct for naïve patients was , % (n= / ). etiology of ifd after allo-hsct was ia - %, invasive candidiasis (ic) - %, mucormycosis - % and % combined ifd caused by aspergillus fumigatus + rhizopus stolonifer. the median day of onset of ifd after allo-hsct was day+ and was associated with post-hsct relapse of hl (p= , ). the main site of infection were lungs ( %), the main clinical symptom -febrile fever ( %). antifungal therapy was used in all patients: voriconazole - %, micafungin - %, posaconazole - %, lipid amphotericin b - % and combination lipid amphotericin b with caspofungin - %. overall survival (os) at weeks from the diagnosis of ifd after allo-hsct was %. the -year os in children and adult with hl after allo-hsct was , %. development of ifd after allo-hsct do not decrease the -year os rate ( , % vs %, p= , ). the impact of prior ifd on -year os in allo-hsct recipients was not statistically significant in all group ( , % vs , %, p= , ) , and separately in children and adults. conclusions: incidence of ifd in children and adults with hodgkin's lymphoma before allo-hsct was , %. incidence of ifd after allo-hsct in patients with hodgkin's lymphoma was , %. the major etiology agents as before as after allo-hsct were aspergillus spp. ifd was a late complication after allo-hsct and associated with post-hsct relapse. despite the high incidence ifd before or after allo-hsct didn't influence the outcome in children and adults with hodgkin lymphoma. disclosure: nothing to declare our community has high cmv positive serostatus, which is a known risk for cmv infection or reactivation. we conducted a study to explore the incidence and outcome of cmv infection among post-hsct children. methods: medical records of pediatric patients (age ≤ years) undergoing single allogeneic hsct from january to december , at king faisal specialist hospital and research centre, riyadh, saudi arabia, were reviewed. all patients with active cmv infection or disease before and during transplant were excluded. a total of patients were included in the study; were female. median age at hsct was years. recipient cmv serostatus was positive in patients before hsct, and donors were cmvpositive. the recipient-donor (r/d) serology was . % r +/d+, . % r+/d-, . % r-/d+, and . % r-/d-. indication for hsct was immune disorders . %, hemoglobinopathies . %, bone marrow failure . %, malignant disorders . %, histiocytic . %, and metabolic disorders . %. source of stem cells was bone marrow in , cord blood in and peripheral blood stem cell in cases. donor was matched related among , unrelated matched/mismatched in , haploidentical , and related with -antigen mismatch in . total body irradiation (tbi) based conditioning was used for patients, while atg was used in patients. results: out of a total of patients, patients developed cmv infection post-hsct ( . %). incidence in female recipients was high ( . % versus . %, p-value . ). both recipient and donor cmv serology positive ( . %) developed cmv infection (p-value < . ). however, no cmv infection in both recipient and donor negative group (r-/d-). the incidence of cmv infection post-hsct was high in patients received tbi based conditioning ( out of , . %, p-value . ), and in haploidentical transplant with . % (p-value . ). source of stem cells, myeloablative versus nonmyeloablative conditioning, atg use in conditioning and agvhd, did not exhibit significant association with cmv infection. in multivariable setting, when adjusted for primary indication for transplant, donor hla type, tbi based conditioning and recipient and donor cmv serology at transplant, haploidentical donor (odds ratio: . , p-value: . ) and donor-recipient cmv sero-positivity (odds ratio: . , p-value: . ) were found to be significant risk factors. cmv infection resolution rate was . % ( ). with a median follow-up time of . ± . months from infusion, five-year overall survival of cmv infected group was lower ( . ± . ) as compared to non-cmv infected ( . ± . , p-value: . ). conclusions: incidence of cmv infection post-hsct in our center is comparable to other centers. our data suggest that donor-recipient cmv positive serostatus, haploidentical donor, and use of tbi based conditioning necessitate close attention and surveillance. background: toxoplasmosis is a rare and underestimated complication following allogeneic stem cell transplantation (allo-sct) with an often fatal course. this is in part due to limited diagnostics relying mainly on imaging and detection of parasite dna by pcr. we present here eleven cases of toxoplasma disease following allo-sct. methods: we retrospectively analyzed consecutive adult patients who received an allo-sct in our bone marrow transplant unit between july and july . eleven ( %) of these patients were diagnosed of toxoplasma disease. the main characteristics of the patients are shown in table . all patients, except two cord blood, have received peripheral blood stem cells. fludarabine-based conditioning regimes were used in all patients. only the two cord blood patients received thymoglobulin in the conditioning. graft-versus-host disease (gvhd) prophylaxis consisted on tacrolimus plus mycophenolate mofetil in ( %) patients and post-transplant cyclophosphamide followed by tacrolimus in ( %). before the allo-sct was performed the igg/igm toxoplasma serology of the recipient and donor. we reviewed the absolute lymphocyte count (alc) and cd + lymphocyte count within four weeks prior to the diagnosis of toxoplasmosis, and if the patients took effective primary prophylaxis for this parasite. toxoplasma disease was defined as the presence of toxoplasma infection plus clinical, radiological or pathological evidence. toxoplasma disease was considered the main cause of death when no other major life-threatening infection or other potential fatal complication occurred immediately before death. results: median (range) age (years) of the eleven patients diagnosed with toxoplasma disease was ( - ). for pancytopenia, no patient received trimethoprimsufmethoxazole (tmp-smz) but pentamidine for pneumocystis jirovecii-pneumonia (pcp) prophylaxis in cases and atovaquone in one. toxoplasma serology pretransplant was positive (igg+/igm-) in ten of the eleven patients. all donors were seronegative (igg-/igm-) except two. toxoplasmosis was diagnosed a median (range) of days ( - ) post allo-stc. the clinical presentations were as cerebral-encephalitis (n= ), chorioretinitis (n= ), pneumonitis (n= ) and disseminated toxoplasmosis (n= ). one case, patient and donor seronegative pre-transplant, was presented as a primary infection in form of chorioretinitis. all three patients with chorioretinitis were diagnosed after day + of allo-sct. at the time of toxoplasma disease, of ( %) of patients had an alc < cells/μl and all of them with immunosuppressive therapy and corticosteroids for acute or chronic gvhd. we had cd + lymphocyte count only in four patients and in three of them was < cells/μl. eight of the eleven ( %) patients died, with a median (range) of days ( - ) since diagnosis of toxoplasmosis, and in of them the toxoplasma disease was the main cause of death. conclusions: in our series, the incidence of toxoplasma disease after allo-sct is low and is related to high mortality, in accordance with what has been reported by other groups. positive pre-transplant serology and gvhd and its treatment were factors strongly related with toxoplasmosis. we encourage the use of tmp-smz instead of pentamidine for pcp-pneumonia prophylaxis in patients seropositive for toxoplasma gondii pre-transplant. clinical trial registry: data about its epidemiology in children are scarce. we retrospectively analyzed the incidence, the severity and the risk factors that contribute to the manifestation of this complication in a pediatric population. methods: during a -year period (january -june ) we performed in our center allogeneic transplantations, for malignant hematological diseases and for non-malignant. the majority of our patients received myeloablative conditioning regimens. diagnostic criteria of hemorrhagic cystitis were the detection of the virus with pcr in urine samples and/or in blood samples, in combination with hematuria and lower urinary tract symptoms (dysuria, urinary frequency, urgency, suprapubic pain) that couldn't be attributed to any other reason. we defined the hemorrhagic cystitis as severe when one of the following factors was present: formation of clots and continuous bladder irrigation, obstructive uropathy with creatinine elevation or need for urological intervention. results: a total of patients with median age , years ( , - ) were studied. children ( %, % ci, , - , ) manifested bk virus associated hemorrhagic cystitis with median age , years ( , [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] ) . onset of cystitis occurred at a median time of days (day +-day + ) after transplantation. in children cystitis was severe. the median duration of symptoms was days ( - ). the median time of hospitalization for children with severe cystitis was days ( - ) whereas for those who didn't manifest cystitis was ( - ) . in of the patients we examined the presence of the virus not only in urine but also in blood samples. in of them the test was positive and almost half of them ( ) manifested severe cystitis. the risk factors that were examined were age, administration of antithymocyte globulin, type of disease, graft source, type of donor and the presence of acute graft versus host disease (agvhd). in multivariable analysis, independent risk factors for the manifestation of hemorrhagic cystitis were age > years old (hr: , , % ci, , - , p< , ), transplantation for malignant disease (hr: , , % ci, , - , , p= , ) and the presence of agvhd (hr: , , % ci, , - , , p< , ). the overall survival of children with hemorrhagic cystitis was , % vs , % of those who didn't manifest this complication, but in multivariable analysis for survival cystitis wasn't a statistically significant risk factor. conclusions: according to our results, stem cell transplantation in children > years old who suffer from a malignant disease and the presence of agvhd consist independent risk factors for the manifestation of bk virus associated hemorrhagic cystitis. the identification of the risk factors of this serious complication will contribute to better management of transplanted patients. further research through prospective trials can contribute to the better understanding of the pathophysiology of hemorrhagic cystitis and to the establishment of appropriate diagnostic and therapeutic guidelines. disclosure: nothing to declare p impact of natural killer cell reconstitution on outcomes in patients with early cytomegalovirus reactivation after allogeneic hematopoietic stem cell transplantation background: cytomegalovirus (cmv) reactivation influences survival after allogeneic hematopoietic stem cell transplantation (sct) and induces natural killer (nk) cell expansion. we evaluated nk cell reconstitution and clinical outcomes following early cmv reactivation after sct. methods: lymphocyte subsets were measured by flow cytometry on day in patients with hematologic malignancies undergoing sct between january and december at kanagawa cancer center, excluding patients with graft failure or death within days. cmv reactivation was defined as initiation of preemptive cmv therapy following pp antigenemia surveillance. results: the subjects were males and females with a median age of years (range: - years). the median follow-up period for survivors was . years (range: . - . years). there were patients with acute myeloid leukemia, with acute lymphoblastic leukemia, with myelodysplastic syndromes, and with other diseases. at transplantation, patients were standard risk and were high risk. myeloablative conditioning and reduced-intensity conditioning were employed in and patients, respectively. bone marrow transplantation, peripheral blood stem cell transplantation, and cord blood transplantation was performed in , , and patients, respectively. cmv reactivation occurred in patients ( %) at a median of days (range: - days) after sct. grade ii-iv acute gvhd and chronic gvhd affected patients ( %) and patients ( %), respectively. among all patients, -year overall survival (os), cumulative nonrelapse mortality (nrm), and cumulative relapse (cir) rates were %, %, and %, respectively. in patients without cmv reactivation (cmvr-) versus patients with cmv reactivation (cmvr+), -year os, nrm, and cir were % vs. % (p < . ), % vs. % (p = . ), and % vs. % (p = . ), respectively. among all patients, the median level of cd -cd + cells, cd +cd cells, and cd +cd + cells on day was /μl (range: - /μl), /μl ( - /μl), and /μl ( - /μl), respectively. nk cell subsets showed no significant differences between cmvr-and cmvr+ patients. when patients were divided into low and high groups at the median level of each nk cell subset, cmvr+ patients with high cd -cd +, cd +cd -, or cd +cd + cells showed significantly better -year os than those with low cells ( % vs. %, p < . ; % vs. %, p < . ; % vs. %, p = . , respectively). high cd -cd + cells were significantly associated with lower nrm ( % vs. %, p = . ), while high cd +cd -cells were significantly associated with lower cir ( % vs. %, p = . ). multivariate analysis confirmed these nk cell subsets as prognostic factors in cmvr+ patients. conclusions: nk cell reconstitution may contribute to improved transplantation outcomes in subgroups of cmvr + patients. disclosure: nothing to declare background: rezafungin (rzf) is a novel echinocandin in development for prevention of invasive fungal infections caused by candida, aspergillus, and pneumocystis spp. in patients at high risk of infection. rzf has demonstrated in vivo prophylaxis efficacy and low risk of drug-drug interactions. furthermore, the stability and pk profile of rzf allow for once-weekly dosing. rzf is also in development for treatment of candidemia and invasive candidiasis using a dosing regimen of rzf mg followed by mg once-weekly, which achieved > % target attainment against candida. while lower doses might be useful to prevent candida and pneumocystis, invasive aspergillosis is a different challenge. we evaluated rzf dosing for prophylaxis against aspergillus fumigatus in blood and marrow transplant (bmt) patients using pk/pd simulations of the treatment dosing regimen. methods: a previous population pk model was refined using data from phase and phase trials of iv rzf (nonmem vers . ). stepwise forward selection (α = . ) and backward elimination (α = . ) were used to assess for relationships between interindividual pk variability and covariates, such as age, sex, bsa, albumin, liver and renal function markers, and infection status. the final model was validated by comparing model-based predictions to observed data. the model and demographic data from bmt recipients at stanford medical center were used for monte carlo simulation (n= , ) of expected rzf concentrationtime profiles in bmt patients receiving iv rzf mg on week followed by mg weekly x . of the patients included in the demographic dataset, were female (mean values at baseline: age, years [ - years]; weight, . kg [ - kg] ). the median (range) bsa in the demographic dataset was . m ( . - . ), and albumin was . g/dl ( . - . g/dl) . free-drug concentration-time profiles were evaluated ( . % human protein-binding) relative to the a. fumigatus minimal effective concentration required to inhibit % of isolates tested (mec ; jmi - sentry international surveillance data). results: the population pk model was a linear, compartment model with zero order iv input. albumin, sex, infection status, and body surface area were statistically significant predictors of interindividual variability; clinical significance of these factors was not determined. the model provided precise, unbiased fits to the observed data (r = . observed vs individual-predicted concentrations). rzf plasma free-drug concentrations at weeks , , and were above the a. fumigatus mec ( . mg/l) for the entire dosing interval in . %, . %, and . % of simulated patients, respectively, and in ≥ . % for all weeks based on the mec ( . mg/l). conclusions: these data modelled from bmt patients support the rzf dosing regimen of mg iv followed by mg once-weekly for prophylaxis against a. fumigatus. current antifungal prophylaxis may be limited by toxicity, ddis, or patient factors such as mucositis. the pk of rzf and its spectrum, safety, tolerability, and lack of ddis may address current unmet needs in ifi prophylaxis for bmt and other immunocompromised patients. disclosure: janice brown: research funding, cidara therapeutics, elizabeth lakota: research funding, cidara therapeutics, shawn flanagan: employment, cidara therapeutics, taylor sandison: employment, cidara therapeutics, voon ong: employment, cidara therapeutics, christopher rubino: research funding, cidara therapeutics p is fungal prophylaxis necessary in non myeloablative peripheral blood stem cell allogeneic transplantation in the pre-engrafment period? julien vaidie , jean-baptiste woillard , stéphane girault , marie-laure dardé , arnaud jaccard , daniel ajzenberg , bernard bouteille , pascal turlure background: non myeloablative peripheral blood stem cell transplantation (pbsc), by limiting toxicity, can be proposed to elderly patients or patients with comorbidities. however, fungal infections remain a key issue that can negatively impact outcome, and increase duration and cost of hospitalization. systematic fungal prophylaxis have demonstrated benefits in outcome in the context of myeloablative conditioning but are not currently in reduced intensity conditioning allograft with pbsc. fluconazole prophylaxis is currently recommended in this situation (ecil). methods: primary objective of this retrospective study was to evaluate fungal infection incidence after allograft procedure in patients who received a non myeloablative allograft with pbsc in limoges university hospital between june and june . patients received fludarabine mg/m /day between d- and d- before allograft and busulfan . mg/kg/day at d- and d- . gvh prophylaxis consisted in rabbit anti-lymphocyte serum at the dose of . mg/kg at d- and d- , and ciclosporin at the beginning dose of mg/kg per os twice a day. mycophenolate mofetil was adding for patients with hla-matched or mismatched unrelated donors. patients did not systematically receive antifungal prophylaxis during the neutropenic pre-engraftment period. when patients had fever during more than hours, an empirical fungal treatment (caspofungine) was added to empirical antibiotics. as soon as neutropenic recovered and in the case of apyrexia without microbiologic documentation, antimicrobial treatments were stopped while in the case of microbiologic documentation, treatments were adjusted to germ in term of dosing and time of administration following recommendations. however, some patients received antifungal azole prophylaxis during the neutropenic pre-engraftment period in case of history of previous invasive aspergillosis (ia), or a nasal colonization by aspergillus. in post-engraftment period, posaconasole prophylaxis was administered for patients with systemic corticotherapy for acute graft-versus-host disease. results: patients were evaluated (median [min-max] age of [ - ] years). % of patients received an hlaidentical related donor, % an hla-matched related donor and % an hla-mismatched unrelated donor. the five years overall survival and survival without relapse or gvhd were % ic [ %- %] and % respectively ic [ %- %]. the median time for neutrophil recovery was days. patients did not receive prophylaxis and only patients received systematic fungal azole prophylaxis in the pre-engraftment period. two patients received an empirical treatment by caspofungine. only ifi was documented during the neutropenic period : candida krusei in blood culture. in the post engraftment period, patients with acute gvhd treated by corticotherapy received an antifungal prophylaxis by posaconazole and only patient had a probable ia at day despite prophylaxis by posaconasole. conclusions: except for patients with previous history of ifi, our results provide additional arguments against systematic fungal prophylaxis after reduced intensity conditioning with pbsc allogenic transplantation in the pre-engraftment period with a very low incidence of invasive fungal infections. in post-engrafment period, posaconazole prophylaxis is required for patient with gvhd treated by corticotherapy. disclosure methods: a simple, rapid and sensitive method using hplc with a diode-array detector (dad) was developed and validated for the quantification of letermovir in human serum using sorafenib as internal standard. after pretreating serum samples by liquid-liquid extraction with tert-butyl methyl ether, separation was achieved on a x-terra rp- column (dimension x . mm, μm) at c using gradient elution with a mobile phase of mm ammonium bicarbonate ph . (mobile phase solvent-a) and acetonitrile: mm ammonium bicarbonate ph . (mobile phase solvent-b). samples were eluted at a flow rate of . ml / min throughout the -minute run. uv wavelength mode was used, detection was at nm. results: the calibration curve was linear (r > . ) in a concentration range of - ng / ml for letermovir. the hplc assay established for letermovir determination showed a high rate of accuracy and precision with an intraday variability of - . to % (accuracy) and . to . % (precision) and an interday variability of - . to . % (accuracy) and . to . % (precision), respectively. letermovir serum concentrations of patients ( male / female, mean age . years) were determined in daily clinical practice. the mean concentration was ng / ml (median ng / ml, standard deviation ng / ml, range - ng / ml). conclusions: the newly developed hplc method is useful for the determination of letermovir concentrations. patient samples analyzed in a routine clinical setting demonstrated considerable interindividual variability. all measured concentrations were above the ec of letermovir. monitoring the concentration of letermovir could help to prevent over-or underexposure, especially in patients with polypharmacy which is frequent in allogeneic hematopoietic stem cell transplant recipients. disclosure background: the use of preemptive strategy (pet) has lowered the incidence of cmv disease in allo-sct to - %. nonetheless the use of this strategy implies that more than % of seropositive patients will replicate cmv. several studies have shown that cmv replication is detrimental for patient survival although the viral load related to this bad outcome variates among studies. objective: to analyse thel impact of cmv replication in overall survival (os) in allo-hct patients. methods: to analyse the impact of cmv replication in os we perform a unicentric, retrospective study on consecutive first allo-hsct patients transplanted between jan- and oct- with a median follow-up of days ( - ). all patients were monitored post-hct with real time pcr cobas-taqman® /cobas ® (rtpcr) in plasma. the cut-off for inception of pet was iu/ml. cmv mutations (ul /ul gene), were studied in plasma samples by sanger sequencing, median cmv viral load iu/ml ( - ). results: patients ( ): women/men ( %/ %), median age was years (range - ). identical allogeneic scts ( %), haploidentical scts ( %). donors were related in cases ( %) and ( %) unrelated. progenitors source was % peripheral blood and % bone marrow. cmv status was (d+/r+) in %(n= ), (d+/r-) in %(n= ), (d-/r+) in % (n= ) and (d-/r-) in % (n= ), unknown cases. positive pcrs were detected in patients: one episode in ( %); episodes in ( %) and to episodes in patients ( %). fifty-five patients ( %) received preemptive therapy. fourteen episodes ( %) were refractory/ probable refractory cmv infections (according to the criteria of chemaly r. cid ). a resistant mutation (ul gene) was detected in one patient with refractory infection patients that developed cmv infection had an inferior non-significant os at years ( , % vs , % log-rank p , ). those patients that received pet for cmv had a significant inferior os compared with those that replicate cmv but didn't receive preemptive therapy ( , % vs %, log rank p= . ). os of patients that received pet was inferior compared with those without pet (with or without infection) ( , % vs , %, logrank p , ). no difference in survival was found for those patients treated pre-emptively that were refractory vs no refractory ( % vs , %, log-rank p , ). conclusions: patients that received preemptive therapy had a significant inferior overall survival compared with those that didn´t replicate and those that replicate cmv but didn't receive preemptive therapy. this reinforce the relevance of prophylactic strategies for cmv with drugs with good safety profile like letermovir that in a randomised trial proved to decrease the need for preemptive therapy. disclosure: rafael, de la camara: has received grants from astellas, gilead, janssen, merck, novartis and pfizer clinical evaluation of stenotrophomonas maltophilia infection in allogeneic hematopoietic stem cell transplant recipients -retrospective single-center data analysis negative bacillus that causes severe infections associated with high morbidity and mortality in immunocompromised patients. the aim of our study was to determine incidence, characteristics and outcome of s. maltophilia infection in patients (pts) who underwent allogeneic hematopoietic stem cell transplantations (allo-hsct) in institute of hematology and transfusion medicine between october and november . methods: we retrospectively evaluated incidence, clinical features and outcome of s. maltophilia infections in consecutive patients with median age- years (range - ), who underwent allo-hsct from unrelated donors - ( . %), matched sibling donors - ( . %) and haploidentical donors - ( . %) in our center. s. maltophilia was detected by culture-based microbiological tests. invasive infection was defined by isolation s. maltophilia from cultures in the presence of both clinical symptoms and signs of infection -blood stream infection (bsi), pneumonia with or without pulmonary haemorrhage. the only colonization status was defined as s. maltophilia culture-positive samples in the absence of infection symptoms. in vitro susceptibility tests to antibiotics were performed. results: pts ( . %) with median age- years (range - ) with s.maltophilia culture positive samples were identified. ( . %) underwent allo-hsct from unrelated donors, -from matched sibling donor and -from haploidentical donor. among them bsi developed in pts ( . %), pneumonia in pts ( %) -with fulminant and fatal pulmonary hemorrhage in pts ( . %). all patients with pneumonia demonstrated bsi. positive sputum cultures were detected in pts, in pts hemoptysis was observed. the rest of isolated strains were identified as colonization (throat -in pts, stool -in pts). all patients with invasive s. maltophilia infection before pathogen identification demonstrated persistent fever despite of the use of broadspectrum antibiotics (carbapenems, glycopeptides, aminoglycosides, colistin), prophylactic antifungals and antivirals. all of them received fluoroquinolone (ciprofloxacin) as a standard antibacterial prophylaxis before neutropenic fever occurred. all patients ( %) with bsi, pneumonia and pulmonary hemorrhage died before engraftment (anc - . g/l) - of them during - hours from the onset of a positive blood culture for s. maltophilia. the c-reactive protein (crp) concentration before identification of s. maltophilia invasive infection was > x- x upper normal limits (unl). susceptibility to antibiotics of isolated strains from blood and sputum was respectively: % and % for ceftazidime, % and % for trimethoprim-sulfamethoxazole, % and % for levofloxacin; while % and % strains were resistant to ciprofloxacin. -year overal survival (os) and -y os for this group was . % and . % respectively compared with . % and . % for group without s. maltophilia infection. conclusions: s. maltophilia invasive infections are associated with high morbidity and mortality in allo-hsct recipients especially in the period from conditioning therapy to engraftment. an exposure to broad-spectrum antibiotics in the treatment of neutropenic fever or confirmed bacteremia of other etiology is one of risk factors of breakthrough s. maltophilia infections. empiric therapy against s. maltophilia in selected patients in risk of such infection before pathogen identification may be lifesaving procedure. disclosure: nothing to declare. role of cmv reactivation following allogeneic stem cell transplantation in preventing relapses in patients with acute myeloid leukemia background: cytomegalovirus(cmv) reactivation is common in patients undergoing allogeneic stem cell transplantation. it has been shown recently that cmv reactivation is associated with reduced risks of relapse in patients undergoing allogeneic stem cell transplantation for aml. however the analysis of cibmtr data did not show any effect of cmv reactivation on relapse. with this background we conducted an analysis of patients suffering from aml who are undergoing allo-sct for their long term disease free survival with respect to cmv reactivation. methods: after obtaining permission from hospital medical records committee, we retrospectively analysed data from electronic medical records of patients undergoing allo-sct for aml at our center between january to august . patients who underwent matched sibling, matched unrelated and partially matched allo sct were included. all patients underwent cmv monitoring with weekly pcr starting from the time of engraftment till d+ following allo sct. value of ≥ copies/mcl was considered as cut off for initiation of treatment in matched sibling donor transplant but in unrelated donor or partially matched donor transplants, ≥ copies/mcl was used as cut off for initiation of pre emptive therapy. results: total of patients were included in study. median age was . ± . years ( - yrs). ( . %), ( . %) and ( . %) patients underwent matched sibling, haplo (partially matched) and mud transplantation respectively. median follow up was months( - months). (table ) acute gvhd (grade - ) was observed in ( . %) of patients. cmv reactivation occurred in ( . %) of patients. overall survival at last follow up was . % ( / patients). ( . %) patients relapsed during follow up. relapse free survival at at last follow up was . %. ( . %) of patients who had cmv reactivation didń t relapse, whereas ( %) of patients who didn´t have cmv reactivation relapsed which was statistically strongly significant p < . . (figure ) similar results were seen in recently published paper from japanese society for hematopoietic cell transplantation (jshct) transplantation-related complication working group. conclusions: . cmv reactivation following allo sct had beneficial effect on preventing relapse in patients with aml. . probable immune activation resulting due to cmv reactivation may result in better graft versus leukemia effect preventing subsequent relapses. [ background: human herpesvirus (hhv- ) causes lifethreating central nervous system disorders such as encephalitis after allogeneic hematopoietic stem cell transplantation (hsct). recent studies showed that cd , a member of the tumor necrosis factor receptor superfamily, has been implicated as a specific receptor of hhv- b, and that its expression levels in cd -positive t cells after hsct could be related to the reactivation of hhv- . real-time quantitative polymerase chain reaction analysis (qpcr) is the most commonly used method for detecting and evaluating hhv- reactivation after hsct, but more sensitive detection method is required. we recently developed a new monitoring method for hhv- reactivation using digital pcr (dpcr) which provides high sensitivity of detecting hhv- dna in clinical samples. in this prospective study, we evaluated the relationship between hhv- reactivation monitored by dpcr and expression of cd on cd + t cells before and after allogeneic hsct. methods: thirty-four patients who underwent allogeneic hsct for hematological diseases at keio university hospital (tokyo, japan) between january and march were consecutively enrolled into this study. peripheral blood samples of the patients were obtained before the conditioning (pre), the day of transplant (day ), and weekly during the first month after transplantation (days , , , and ) . hhv- viral load in plasma was quantitatively measured by dpcr. the primers and a probe of dpcr for hhv- b were selected from immediate-early (ie- ) protein transactivator region (u ). we evaluated the relationship between hhv- reactivation and the serial expression rates of cd in cd + t cells (cd /cd ratio) measured by flow cytometry before and after hsct. results: median age of the patients was . years. onethird of patients received cord blood as a stem cell source. hhv- reactivation was detected in patients ( %) with dpcr. a comparison of cd /cd ratio between the patients with and without hhv- reactivation after hsct revealed that cd /cd ratio was significantly higher in patients with hhv- reactivation than those without before conditioning ( in contrast, there was no such significant difference after transplant (days to ) . in multivariate analysis, higher cd /cd ratio before conditioning (odds ratio (or) = . , % confidence interval (ci): . - . , p = . ) and stem cell source from human leukocyte antigen mismatched donor (including all cord blood transplantation cases) (or = . , %ci: . - . , p = . ) remained to be significantly associated with the incidence of hhv- reactivation. conclusions: higher cd expression rate in cd + t cells before hsct was associated with higher risk of hhv- reactivation, which could be a promising marker for predicting hhv- reactivation after allogeneic hsct. careful observation and monitoring may be needed in cd highly expressed patients. it is a subject of further research to clarify the role of cd + cd + t cell in hhv- reactivation. disclosure: nothing to declare. methods: criteria for the administration of ici (vistide) were grade iii-iv (clinically significant hematuria with clots) bk-related hemorrhagic cystitis after allo-hct which showed no improvement after symptomatic therapy with hyperhydration and bladder irrigation. cidofovir was diluted in ml of normal saline and installed via a foley catheter which was blocked for hour. not knowing the level of absorption of the drug we decided to give probenecid prophylaxis in all patients. ici was repeated weekly according to severity of symptoms. urine and plasma bkv viral loads were quantified by rq-pcr results: six patients (median years, - ) received ici after allo-hct. patients had haematological malignancies (aml , all , mds ), received busilfex-based myeloablative conditioning and a graft (pbsc , bm ) from a / hla-matched ( pts), / ( pt) or haploidentical ( pt) donor. median time for the onset of bkv-hc after allo-hct were . days (range - ). all patients were under standard cyclosporine prophylaxis and none of the patients had any signs of acute gvhd at the time of onset of hc. the median pcr-bkv viral load at the onset of bkv-hc in urine and plasma were . x (range . x - x ) and . (range - ), respectively. the median maximum pcr-bkv viral load in urine and plasma were . x (range . x - x ) and . (range - . ), respectively. five patients had impaired renal function (median egfr ml/min, range - ) at first ici which was probably multifactorial. the median dose of intravesical cidofovir was mg/kg (range . - mg/kg) and a median number of . instillations (range - ) were given. in / cases symptoms of cystitis improved dramatically and hematuria resolved. virological response (at least log reduction) was observed in all cases. two patients experienced relapse of hemorrhagic cystitis and were retreated with ici which resulted in resolution of the symptoms and the hematuria. no deterioration of renal function of other systemic adverse effects were observed. after a median follow up of . days after transplantation (range - ), / patients are alive without cystitis symptomatology and died ( due to relapse and due to trm). conclusions: in this retrospective study we propose that local therapy of bkv-hc with ici is safe and has high clinical and virological response rates. the administration of ici after allo-hct should be controlled in prospective randomized trials. disclosure: nothing to declare background: since cmv-preemptive therapy approach was implemented, cmv disease frequency is very low. however, cmv reactivation and the need of using nephrotoxic plus/less myelotoxic drugs is very frequent. in addition to the toxicity of the medications to avoid cmv disease, other potential adverse effects of cmv have been mentioned in medical literature. in this study, we wanted to estimate how recipient/donor serologic status influences the outcome of allo-hsct in our most recent series of patients. methods: the population analyzed for this report is the all patients who underwent allo-hsct during the -year period from october september in our unit. median age at transplant was years (range: - ). one hundred and thirty were male ( %) and were female ( %). baseline diseases were: aml, lpd, all, mds, mpd, mm, and bmf. donor was unrelated in transplants ( , %) and was family in ( , %) (including haplo-identical). conditioning regimen was ric in procedures ( %) and intensive in ( %). stem cell source was pb in ( , %) and bm in cases ( , %). median follow-up was months (range: - ). patient's and donor's cmv igg were positive in ( , %) and ( , %), respectively. recipient/donor serology was +/-(risk group ) in ( , %), +/+ (risk group ) in ( , %) , -/+ (risk group ) in ( , %) y -/-(risk group ) in ( , %). results: two pts underwent a second transplant before day + due to graft failure. overall mortalities (om) at days + and + of the rest of the series ( pts) are shown in table. the highest risk group (recipient cmv + / donor cmv -) exhibited more than double om at day + and more than four times om at day + , when compared with pts at lowest risk (recipient cmv -). those striking differences were mainly due to nrm. om for risk group ii (recipient cmv + / donor cmv +) was intermediate. conclusions: in our studied population, mainly adult patients, the combination of cmv-seropositive patient with a cmv-seronegative donor had a very clear adverse impact on hsct outcome. as a result, we considered that the election of a cmv-positive donor for a cmv-positive patient continues to be strongly advisable, whenever is possible. on the other hand, once letermovir has proved to be efficient and well-tolerated and has been licensed for prophylaxis of cmv in high risk recipients, this approach appears to be very attractive to try to avoid the adverse impact of recipient cmv-seropositivity, particularly when finally chosen donor is cmv negative. disclosure: nothing to declare an active surveillance and an early and individualized management is critical to avoid mortality from respiratory viral infections in allo-hsct recipients background: respiratory viral infections (rvis) are frequent among the general population. in transplant recipients, rvis are known to cause an important morbidity and potential mortality. for this reason and several others, as the need of preventing other pts from contagious or avoiding misdiagnosis with other infections processes, a high index of suspicion of vris is necessary. during the last few years, we have implemented an active and systematic surveillance policy orientedto early detection and management of rvis in the hsct recipients. methods: the population analyzed for this report is the patients who underwent allo-hsct from january through march in our unit. median age at transplant was years (range: - ). one hundred and four were male ( . %) and were female ( , %). baseline diseases were: aml, lpd, all, mds, mpd, mm, and bmf. donor was unrelated in transplants ( . %) and was family in ( . %) (including haplo-identical). conditioning regimen was reduced in procedures ( %) and intensive in ( %).stem cell source was pb in ( . %) and bm in pts ( . %).median follow-up was months (range: - ); at the close of the analysis, majority of the series ( . %) had a follow-up superior to one year from hsct. a throat swab(ts) was taken from every patient with any, even minor, respiratory symptoms. the respiratorysamples were tested whith a complete pcr panel of human respiratory viruses: rhinovirus (rv), influenza a and b virus (iv-a, iv-b), parainfluenza virus (pivs - ), respiratory syncytial virus (rsv), metapneumovirus (mpv), coronavirus (cov), adenovirus (adv), and bocavirus (bov). results: day + overall mortality of the series was , %. day + overall mortality was , % ( , % nonrelapse mortality -nrm-, and , % progression/relapse mortality). causes of nrm reflected in table . no patients died due to rvis. from st july through th june (a -month period), ts samples were obtained from pts ( , %).the median number of samples/patient was (range: - ).a total of ( - ) rvis episodes were diagnosed in pts ( , %).the median presentation of the first rvi was at the day + ( - ) post-hsct. the viral distribution was: rv ( . %), iv ( . %), piv ( . %), rsv ( . %), mpv ( . %), cov ( . %), adv ( . %), and bov ( . %).there were mixed (two or more viruses) rvi episodes. the temporary distribution of vri episodes is shown in figure . conclusions: ) symptomatic infections due to respiratory viruses are very frequent among the allo-hsct recipients. ) a high level of suspicion, as well as an early and systematic screening and management policy, are critical to avoid potential attributable mortality and the nosocomial spread of rvis among the transplant recipients. ) in our series, rhinovirus, parainfluenza and adenovirus might be detected at any moment of the year; the rest of the viruses showed a clear seasonal pattern (november to april). [[p image] . background: trimethoprim-sulfamethoxazole (tmp-smx) is the most suitable drug for prophylaxis against pneumocystis pneumonia and infections with toxoplasma after allogeneic haematopoietic stem cell transplantation (allo-hsct). allergic reactions or hypersensitivities, mainly exanthemas, occur in about - % of the patients, usually resulting in the use of alternative prophylactic drugs (e.g. pentamidine or atovaquone). it has been hypothesised that allergies might be cured with allo-hsct. methods: we conducted a retrospective chart review of patients with tmp-smx re-exposition after allo-hsct from december to september . follow-up is current as of december . results: six patients (f/m: / , median age: years, range: - years) with a history of tmp-smx hypersensitivity prior to allo-hsct were re-exposed to tmp-smx after engraftment of a matched related (mrd, n= ) or matched unrelated (mud, n= ) donor. median time to re-exposition was . (range: - ) days after allo-hsct with one oral dose of tmp-smx. in four patients, tmp-smx was tolerated without any signs of hypersensitivity reactions and has been continued for a median of days (range - ) until last followup. one patient (mud, re-exposition at d+ ) experienced pruritus and erythema some hours after tablet intake. another patient (mud, re-exposition at d+ ) developed an exanthema one day after re-exposition which was later diagnosed as a cutaneous gvhd. conclusions: re-exposition of tmp-smx in patients with prior hypersensitivity is feasible after allo-hsct. after successful re-exposition, patients can be treated with the best-studied drug for prophylaxis of infections with pneumocystis and toxoplasma. disclosure: nothing to declare brincidofovir for adenoviremia in paediatric hsct for primary immune deficiency background: reactivation of adenovirus is a severe complication of hsct associated with significant morbidity and mortality, particularly for children with primary immune deficiency (pid). the only drug currently licensed to treat adenovirus infection is cidofovir. brincidofovir is a lipidlinked derivative of cidofovir which has been shown to be a safe and effective alternative treatment to cidofovir. there is limited data describing the use of brincidofovir in patients undergoing hsct for primary immune deficiency. we reviewed all patients who received brincidofovir after undergoing hsct for primary immune deficiencies between and at the great north children's hospital, newcastle upon tyne, uk. results: of patients transplanted for pid, developed significant adenoviraemia ( %). all were treated with cidofovir initially but were switched to brincidofovir because of a failure to respond or because of renal toxicity. of these, resolved their adenoviraemia within days of commencing treatment (figure ). donor sources were tcr alpha/beta/cd depleted haplo-identical (n= ), tcr alpha/beta/cd depleted mmud (n= ) and / mud (n= ). patients were conditioned with treosulphan/fludarabine/thiotepa/atg/ rituximab (n= ), treosulphan/fludarabine/atg/rituximab (n= ) or treosulphan/fludarabine/alemtuzumab/ gcsf/plerixafor (n= ). occurrence of agvhd and treatment of agvhd are outlined in table . patient died + days post-transplant of multi-organ failure, severe thrombotic microangiopathy and sepsis. although patient initially responded to brincidofovir, reactivation occurred after cessation of treatment; severe diarrhoea precluded the reintroduction of brincidofovir and the adenoviraemia persisted with poor immune reconstitution. treatment with addback t cells was attempted however the patient died days post-transplant after a cerebral haemorrhage. patient had long-standing chronic diarrhoea which was thought not severe enough to warrant cessation of brincidofovir treatment. conclusions: the complete resolution of adenoviraemia in / patients who had previously failed to respond to prior therapy with cidofovir suggests that brincidofovir may be an effective treatment option for adenoviral reactivation post-hsct for pid. however, resolution of adenoviraemia is influenced by many other factors, including the adequacy of immune reconstitution, the degree of induced immune suppression and the presence of comorbidities such as gvhd. due to the small sample size it was difficult to assess the relative importance of these factors in this cohort. brincidofovir was well tolerated however its effectiveness may have been limited by poor gastrointestinal function in one patient (patient ) and could not be used after a viral reactivation in another for the same reason. further studies of the use of brincidofovir in this specific cohort are needed to clarify the role and effectiveness of this treatment. background: there is a high prevalence of cmv seropositivity in algerian population. because of high morbidity and mortality in pts who underwent allo sct with cmv reactivation, effective surveillance and timely treatment using anti-viral therapy s required. the risk of cmv reactivation depends on the type of stem cell source, immunosuppression (is) and serological status of the donor/ recipient pair. methods: over a months period (from / / to / / ), pts underwent allo-hsct for malignant or non-malignant hematology diseases of which pts are evaluated for this study. cmv reactivation was observed in pts ( . %) (aml: pts, all: pts, cml: pts, multiple myeloma: pt, nhl skin: pt, primary myelofibrosis: pt, aplastic anemia: pts, fanconi anemia: pts, β-thalassemia: pt), with a median age of years ( - ), sex ratio (m/f) of . . allo-hsct done with sibling donors: pts, haplo-identical donors: pts and pheno-identical donor: pt. all pts were treated by chemotherapy alone with myéloablative conditioning (mac) in pts and reduced intensity (ric) in pts. all pts received peripheral blood stem cells with an average rate of cd + cells: , . /kg ( . - . ). additional bone marrow graft was used in pts that received a haploidentical graft without pt-cy. gvh prophylaxis associated cyclosporine (csa) and methotrexate (sibling and phenoidentical); csa-mtx-mmf or csa-mtx-cyclophosphamid (haplo-identical). before transplantation, donor/recipient pair is at high risk reactivation in pts ( . %). detection of cmv reactivation done by antigenaemia pp or by quantitative pcr weekly for the first months and during an is treatment for acute or chronic gvhd. pre-emptive therapy is initiated by ganciclovir as soon as positivity of antigenaemia or increased viral load in pcr. results: a first reactivation occurred on average day ( - ) in pts ( . %) of which pts under corticotherapy for acute gvhd ( pts), thrombotic micro-angiopathy ( pt) and renal failure ( pts) or due to a reinforced is for haplo-identical transplantation ( pts). one pt with chronic gvhd presented a late reactivation months after transplant. twenty pts presented a nd reactivation on average day ( - ) and pts a rd reactivation on average day ( - ). pre-emptive treatment is introduced in the first episode by a viral dna polymerase inhibitor (ganciclovir: pts; valganciclovir: pts, foscarnet: pt). the negativity of antigenaemia is observed on average at days of treatment ( ) ( ) ( ) ( ) ( ) . second line treatment was required in pts ( %) due to resistance ( pts), severe cytopenia ( pt) or renal failure ( pt). the onset of severe cytopenia imposed a dose reduction ( pts) or a therapeutic stop ( pts) before days. two pts received additional maintenance treatment for negativation delay. three pts ( . %) died from cmv infections resistant to antiviral treatment (pneumonia: , colitis: ). conclusions: cmv infection is a serious complication after allo-hsct. in the absence of vaccination, the systematic monitoring for cmv reactivation is strongly recommended for the establishment of a rapid and effective preemptive treatment. disclosure: nothing to declare p abstract withdrawn. results: in transplanted group, episodes of bkv reactivation occurred in patients ( %). in cases only urine colonization (c) found before hsct. in this group in patients ( %) virus was transmitted from urine to the blood (b) . dysuria and/or hc were observed in / ( %) patients . all of them ( %) had urine and serum involvement. in cases bkv replication was found after hsct ( -cases detected in urine, cases-bothserum and urine). dysuric syndromes and/or hc were found in / of cases ( %)-all in patients with serum and urine involvement. urinary tract was always first location of the virus. there was no case of isolated serum reactivation. the incidence of bk infection was higher in patients older than > yrs (p< . ), transplanted from family donor (msd) (p< . ). mud recipients had more often both serum and urine reactivation (p< . ) than isolated urine involvement. sex, day of neutrophil recovery, conditioning regimen, or use of total body irradiation were not significant risk factors for bkv infection, or hc . six patients were treated with cidofovir (range - doses) with good response. there was no death due to evident bkv infection. conclusions: bkv reactivation remains one of the most frequent infectious complication in children undergoing allogeneic hsct. most of patients experienced mild infection and age < years was the positive prognostic factor influencing its incidence. bkv monitoring and prompt treatment of hc resulted in excellent outcome. we observed surprisingly high rate of new bkv replication after hsct. disclosure: nothing to declare background: high-dose chemotherapy (hd-ct) and auto pbsct have been the standard therapy for multiple myeloma (mm) for more than two decades, despite a wide range of new therapeutic options. recurrent/refractory malignant lymphomas and recurrent/metastatic germ cell tumors (gct) also benefit from this intensive therapy. in comparison to allogeneic transplantation, this treatment is known for lower complication rates, e.g. infections. however previous studies have schown that treatment related toxicity may not be underestimated and depending on the conditioning regimen used. methods: we retrospectively analyzed patients ( cases) who underwent hd-ct plus auto pbsct between and in a single-center study. to anlyze the incidence of infections depending on the conditioning regimen, we formed the following categories based on the agiho: no infections, neutropenic fever, sepsis and severe sepsis. results: the median age in this analysis was years; . % were male. the most frequent diagnosis was mm ( . %) receiving high dose melphalan (mel), followed by malignant lymphoma ( . %) receiving beam (bcnu, etoposide cytarabine, melphalan) and relapsed/metastatic germ cell tumours (gct) ( . %) receiving high dose carboplatin/etoposide (ce). % of all patients developed severe sepsis, patients had to be ventilated and patients died. sepsis was documented in . % of all cases ( cases). the majority of patients ( . %, cases) developed neutropenic fever and . % ( cases) didn´t have any infection complications. the beam conditioning regimen showed the highest tendency to result in a septic course ( . %), followed by ce ( . %) and mel ( . %). the most commonly documented pathogen in blood cultures was s. epidermidis ( . %), followed by e. coli ( . %) and s. mitis ( . %). only in one blood culture we detected a multi-resistant pathogen ( mrgn e. coli). p. aeruginosa was detected in blood cultures ( . %), l. monocytogenes in ( . %) and s. aureus in ( %). . % of all patients developed diarrhea, only in . % of these cases we could detect c. difficile. the conditioning regimen shows no significant effect on the incidence of c. difficile. the mean neutropenic period was . days in malignant lymphoma patients, followed by . in mm patients and . days in gct patients. the hospital discharge, calculated from the day of transplantation, was significantly different: for malignant lymphoma the mean was . days, for mm . days and for gct . days. conclusions: our data correspond to former published results by many groups. the beam regimen shows the highest infectious complication rate followed by ce and mel. the duration of neutropenia and hospital stay depends on the conditioning regimen. the type of infectious complication doesn't effect the progression free-and overall survival in our analysis. disclosure: nothing to declare. impact of donor and recipient cytomegalovirus serostatus on outcomes of unrelated allogeneic haematopoietic stem cell transplantation background: cytomegalovirus (cmv) is an important cause of morbidity and mortality in allogeneic haematopoietic stem cell transplant (hsct) patients. the aim of our study is to evaluate the outcomes of our cmv seropositive recipients who received grafts from seropositive unrelated donors (d+r+) compared with grafts from seronegative unrelated donors (d-r+). methods: this is a retrospective single center study on a series of cmv seropositive recipients who underwent hsct from unrelated donors between febuary to july . a total of patients were analyzed. their clinical course and laboratory results were reviewed for evidence of cmv reactivation and/or cmv disease. we defined cmv infection as detection of cmv reactivation or primary infection by antigenaemia or polymerase chain reaction (pcr) assays, but was not accompanied by signs and/or symptoms suggestive of a systemic disease. cmv disease occurred when cmv was isolated from any site in association with organspecific signs and/or symptoms. monitoring for cmv infection commenced upon engraftment (approximately day + ). peripheral blood samples were sent twice a week for cmv antigenaemia or cmv quantitative pcr. the duration of twice weekly monitoring was at least about days. longer monitoring was performed in patients who experienced cmv infection after hsct. results: all patients received graft-versus-host-disease (gvhd) prophylaxis using anti-thymocyte globulin (atg) at . mg/kg in addition to cyclosporin or tacrolimus. among the entire cohort of patients, ( %) had cmv infection, including ( . %) out of patients from the d-r+ group and ( %) out of patients from the d+r + group. patients ( . %) from the d-r+ group and patients ( . %) from the d+r+ group had ≧ cmv reactivation above the threshold for preemptive therapy respectively; p= . . patients developed cmv disease, ( . %) from the d-r+ group and ( . %) from the d +r+ group. cmv resistance to both foscarnet and ganciclovir was detected in patients ( . %) from the d-r+ group but none from the d+r+ group. patients died due to cmv disease, both were from d-r+ group. year overall survival (os) were % versus % for d-r+ group and d+r+ group respectively; p= . . median survival was not reached at years. year non-relapse mortality (nrm) were % for d-r+ group and % for d +r+ group respectively; p= . . conclusions: the incidence of recurrent cmv infection was higher in the d-r+ group compared to the d+r+ group. there were no statistically significant differences between the groups in terms of os and nrm. however, there was a trend towards higher nrm in the d-r+ group compared to d+r+ group. our findings suggest that for matched unrelated hsct, it may still be important to select a seropositive donor for a seropositive recipient. disclosure: none background: it´s known that some patients submitted to allogeneic stem cell transplantation (asct) could present a greater susceptibility to infection even when they are in long term complete remission or potentially cured. this fact is related to the dynamic of immunological recovery that is variable in every single patients and it is dependent from many factors: the haematological disease, the conditioning regimen, the age of patient and donor, the number of stem cell and lymphocytes infused, the anti-gvhd prophylaxis, the use of anti-thimoglobulins and others. in clinical practise we can observe patients who are potentially cured, who tapered and stopped the immunosuppressive treatment months or years ago and who are suddenly graved from opportunistic infections. the largest part of these infections is represented from varicella-zoster virus (vzv) cutaneous eruption. methods: in this report we retrospectively analysed a monocentric cohort of patients submitted to asct for haematological malignancies from a median time of months. all of them were free of disease. they stopped the immuno-suppressive treatment in a median time of days after asct (range: - ) and did not present later chronic gvhd needing treatment neither other moderate or severe chronic post transplant complications nor other diseases. prophylactic treatment with anti viral agents (acyclovir or valacyclovir) has been conducted simultaneously to immuno-suppressive treatment and for a period ranging between to months after its suspension. in this cohort of patients we considered the incidence of vzv eruption occurred after the suspension of the immunosuppressive treatment, and we analysed the immunological recovery in terms of lymphocytes sub-population after , , and months from asct. results: of these patients considered, developed at least one vzv manifestation. all the vzv presentation were cutaneous, we did not observe neurological, ophthalmic or visceral presentation. all the vzv manifestation occurred in patients who ended the anti-viral prophylaxis. median time of presentation was days after asct (range: - ) the remaining patients did not present vzv manifestation nor other kind of opportunistic infection despite the absence of anti-viral prophylaxis. the analysis of lymphocyte sub-population after - - and months did not show a significant difference in b, t, t , t and nk lymphocytes in the different post transplant period. conclusions: vzv reactivation seems not to be correlated with the number of the different lymphocyte subpopulations in the post transplant period. actually it is not possible to distinguish patients more suitable of vzv reactivation on the basis of lymphocyte sub-populations analysis, so anti-viral prophylaxis should be prolonged for a medium period after suspension of immuno-suppressive drugs. in absence of anti viral prophylaxis a careful clinical surveillance should be performed in order to treat early eventual vzv manifestations. disclosure background: infection and disease cytomegalovirus (cmv) are common problems in patients undergoing hematopoietic stem cell transplantation (hsct). cmv infection has a high overall seroprevalence, therefore, during the first days post-hsct, it is important to prevent reactivation of cmv. the international clinical recommendation is the use of ganciclovir as prophylaxis in hsct patients; however, the cost of this treatment is not accessible for our population. in this respect it has been used as an alternative valganciclovir because of its lower cost and oral administration. our study´s aim was to assess the response and safety of valganciclovir in comparison with ganciclovir to prevent viremia and cytomegalovirus disease in patients undergoing allogeneic hsct methods: a retrospective study was performed on patients who receive an hsct-allo between january and august . participants were enrolled in two groups according to prophylaxis treatment: (a) ganciclovir mg/k once daily and (b) valganciclovir mg twice daily for days pretransplant, at day + ; viremia was measured by pcr. demographic and clinical information was collected from medical records and furthermore analyzed in spss v . results: sixty-eight patients were enrolled in the study, % male, the median age was years ( - ) with the following diagnoses: acute lymphoblastic leukemia %, acute myeloblastic leukemia . %, granulocytic chronic leukemia . %, myelodysplastic syndrome . %, dendritic cell neoplasia . %, and aplastic anemia . %. ninety-one percet of the patients received a transplant from an identical hla donor and . % received a haploidentical transplant. thirty-four patients received ganciclovir (g ) and thirtyfour valganciclovir (g ). median age was vs years (p= . ), intermediate risk cmv % vs (p= . ), associated bacterial infections was %vs % (p= . ), and fungal infections % vs % respectively (p= . ). the reactivation by cmv was presented in % vs % respectively (p= . ). there were no significant differences in fever, bacterial isolation, dysfunction or graft failure, presence and degree of acute or chronic gvhd and relapse of the disease. the most relevant characteristics and complications are described in table . within the whole group there were deaths, % in the ganciclovir group and % in valganciclovir group (p= . ), overall survival -year was % vs % (p= . ) respectively; in both groups % was associated with relapse and % associated with transplantation. conclusions: ganciclovir and valganciclovir were effective in preventing the reactivation of cmv, the only statistically significant difference was that the presentation of the disease appeared earlier in the valganciclovir group. no difference in toxicity between the groups was identified. disclosure: none declared background: invasive pulmonary aspergillosis (ipa) is a severe and serious complication that occurs in the immediate post-transplant period due to severe neutropenia or late usually following prolonged corticosteroid therapy during treatment of graft-versus-host disease (gvhd). the objective of this study is to analyze the epidemiological, diagnostic and evolutionary characteristics of this major complication over a period of years. methods: from january to december , patients (pts) received an allogeneic hematopoietic stem cell transplantation (allo hsct) for malignant and nonmalignant haematological diseases. during the transplant procedure, anti-infectious prophylaxis consisted of pts isolation, digestive decontamination, fluconazole and aciclovir. secondary prophylaxis done for pts with prior history aspergillosis. during the follow-up, a standard chest x-ray is performed systematically at each control or in case of clinical signs a thoracic ct scan is requested from suspicion. the diagnosis of ipa is made according to the criteria of the eortc-msg based on the predisposing criteria of the host and clinico-radiological criteria (possible infection). galactomannan antigen and histopathology criteria are not common practice. results: a total of ipa episodes ( %) were identified in pts (aml: , aa: , all , cml , mm ) of median age ( - ) , sex ratio: . . all of them had transplantation from a family donor (geno-identical: , haplo-identical: ) with conditioning by chemotherapy alone and a graft of csp ( pts) and peripheral stem cells-bone marrow ( pts). all pts had at least one predisposing risk factor: antecedent of aspergillosis ( pts), prolonged neutropenia> d ( pts), acute gvhd ( pts), chronic gvhd ( pts), prolonged corticosteroid therapy ≥ , mg /kg/day exceeding days ( pts). the diagnosis of api was possible on average at j ( - ) after appearance of clinical signs (in all cases) and evocative radiological in cases (in cases, the standard chest x-ray was normal). at the time of thoracic ct scan, pts ( %) had characteristic signs: halo sign ( pts), crescent sign ( pt) and cavity ( pts). other minor radiological signs are found in the other pts. empirical first-line antifungal therapy was started as monotherapy in pts (voriconazole: , caspofungin: pts) or in combination in pts. a secondline treatment was required in pts for failure after an average duration of days . three pts presented a second episode after an average delay of months ( ) ( ) ( ) ( ) with a favorable evolution of resumption of thetreatment. fourteen pts ( %) are alive with complete resolution after a median treatment time of months ( - ). twelve pts ( %) died rapidly on average days after diagnosis (ipa , relapse of his disease: ) conclusions: ipa occurring after an allograft of allo-hsct is a severe complication with high mortality. it is essential, in each case, to identify the pts with risk factors, perform a thoracic ct-scan, send serum serology for apergillus galactomannan antigenand start specific treatment as soon as possible while waiting to be able to reinforce the diagnosis by direct examination or sputum or brochoalveolar lavage with aspiration. disclosure: nothing to declare background: cmv (cytomegalovirus) has a prevalence varying between - %. its pathogenicity is relatively low in the general population, usually resulting in a selflimiting viral illness. in an immunosuppressed host, infection can lead to life threatening illness. disseminated cmv infection can manifest in a number of organs and is diagnosed using internationally accepted criteria. in the post solid organ and stem cell transplant (sct) setting, it is postulated that it is viral reactivation, rather than primary reinfection that leads to cmv viraemia. prevention of reactivation requires the presence of a competent immune system, mediated by t-cells. this accounts for the increased incidence in intensive and t-cell depleting sct conditioning regimens. despite improved outcomes following the introduction of cmv monitoring by pcr and pre-emptive treatments (current uk guidance), cmv pneumonitis still carries a high mortality. the use of cmv specific immunoglobulins (cmvig) for the treatment of this complication is generally not recommended post chemotherapy or sct in haematological cancers due to lack of evidence. however, cmvigs are widely used in the setting of cmv reactivation post solid organ transplants. we report the use of cmvig in patients with suspected cmv pneumonitis at a single uk centre. the aims of this retrospective study were to establish safety and review efficacy in this highly immunocompromised group of patients. methods: data was collected retrospectively on the use of cmvig in patients with haematological cancers post sct or chemotherapy alone between and at manchester royal infirmary, uk. all patients included had cmv positive pcr in blood (and or from bronchoscopy), as well as high resolution ct imaging evidence of cmv infection. the data was sourced from pharmacy database and crossreferenced with a departmental list. for each patient identified, case notes and prescriptions were sourced. data collected included patient baseline characteristics, timing of treatment, number of doses of cmvig and outcome. results: eight patients received cmvig for suspected cmv pneumonitis. seven patients were post sct and one patient was severely immunosuppressed with chemotherapy alone. median age was years (range - ). the cmvig regimen used was ml/kg of cytotect ® on days , , and , followed by ml/kg every four days until resolution of symptoms. there were no infusion related reactions observed. patients received a median of doses of cmvig. four out of patients responded to the treatment and showed full recovery but only are alive and well to date. conclusions: this study shows that the use of cmvig is safe in the post-sct setting of acutely unwell patients with multi-organ failure. despite limitations of retrospective studies, there appears to be benefit for the use of cmvig in our patient population, with % of patients showing a full recovery from that episode. allogeneic sct plays a confounding role in the outcome of patients although the numbers in our study are small. there is clearly a need for better treatments of cmv pneumonitis. cmvig is a promising treatment but further studies are needed to identify the optimal dosing regimen and provide evidence of efficacy. disclosure: biotest-honaria p abstract withdrawn. background: the risk of fungal infection related to allogeneic transplantation is a well-known cause of morbidity and mortality. the main agents implicated are yeast during the neutropenic period and filamentous fungi after this period. methods: we decided to evaluate the effectiveness of a prophylactic regimen containing fluconazole since day - . after discharge fluconazole was kept until day or or switched to posaconazole in high-risk patients. patients with gvhd under steroids were kept under prophylaxis.the group of high risk patients was defined by one of the following variables: -non related donors -atg, campath or fludarabine in the conditioning -presence of gvhd with need of steroids above . mg/kg we have analyzed the patients submitted to allobmt during and . all patients were first admitted to an isolation room with hepa filters.patients under secondary prophylaxis were excluded. breakthrough fungal infections during the first year and toxicity leading to discontinuation was evaluated. results: sixty six patients were included with transplants. male/female ratio was / . the age range was . - yo with a median of . malignant ( ) and nonmalignant ( )diagnosis were included. donor type was related ( ) haploidentical ( ) and non-related ( ). the conditioning regimen includes atg in , campath in and fludarabine in . fourteen patients were treated after discharge with fluconazole and with posaconazole. three patients fluconazole were switched to micafungin for hepatic toxicity, two cases to amphotericin due to persistent fever and in one case to caspofungin for a proven fungal infection (candida parapsilosis in blood stream in day + ). after discharge and during the first year of follow-up a single case of possible fungal infection was diagnosed, in a patient with gvhd with a lung nodule. conclusions: during the neutropenic period after transplantation the main risk of fungal infection is associated with candidiasis. the greatest risk of aspergillosis occurs later and have a significant relation with gvhd. except for candida parapsilosis the main source of yeasts are the gi tract. the main source of aspergillus are aerosolized particles retained by hepa filters. in patients without a previous episode of fungal infection the main risk of filamentous fungi occurs only after discharge. we conclude that fluconazole alone or followed by posaconazole in high risk patients is a feasible and effective regimen for primary prophylaxis, in allogeneic transplantation. disclosure background: bk virus-associated hemorrhagic cystitis (bkv-hc) has emerged as a serious infection after hematopoietic stem cell transplantation (hsct). it is characterized by painful hematuria due to hemorrhagic inflammation of the urinary bladder mucosa, this causes significant morbidity, prolonged hospital care with extensive nursing requirements and increases in healthcare costs. the purpose of this study is to determine the incidence, risk factors, and duration of treatment in our center. methods: we performed a retrospective review of hsct patients at luis calvo mackenna children´s hospital in santiago, chile diagnosed with bkv-hc, from st january to th november . we investigated the incidence, risk factors and duration of treatment of bkv-hc in paediatric patients undergoing hsct over a months period. bkv-hc was defined as bk virus (bkv) detection in urine by pcr testing in association with clinical symptoms and hematuria grade or higher. sixty-seven patients were trasplanted during this period. results: eleven patients were diagnosed with bkv-hc at our institution, only one with bk viremia. the cumulative incidence of bkv-hc in our series was %. all of them were treated with cidofovir. the median age at diagnosis was years old (range: - y.o.). the median time from hsct to hemorrhagic cystitis (hc) was days (range: - days), the median length of treatment was weeks (range: - ). all patients received myeloablative conditioning regimens and used cyclophosphamide ( %); ten ( %) were unrelated cord blood transplant recipients and nine ( %) used antithymocyte globulin. a concomitant viral reactivation (cmv/vh ) was demonstrated in six ( %) patients. no patient died due to bkv-hc or its complications, but in the follow up three patients died, one in relapse and two of other post transplant´s complications. conclusions: bkv-hc is the result of a complex interaction between patient characteristics, donor type and conditioning regimen intensity. these patients experienced significant morbidity and prolonged treatment. in our cohort bkv-hc of all patients but one were transplanted with an unrelated umbilical cord blood unit, all of them received myeloablative conditioning regimen with cyclophosphamide and most of them received anti-thymocyte globulin. we also observed frequently co-existence of viral infections from herpes family as cmv and vh . the main limitations of this work are its retrospective nature and it´s from a single center. more studies are necessary to better understand the epidemiology and risk factor associated with bkv-hc and the morbidities associated with its treatment. disclosure: nothing to declare how we manage hhv- reactivation in the posttransplant setting oscar borsani , anna amelia colombo , daniela caldera , paolo bernasconi university of pavia, san matteo hospital, pavia, italy background: hhv- encephalitis is a life-threatening complication in the post-transplant setting and it develops in about % of patients receiving traditional hsct. several risk factors were described. a differential diagnosis between hhv- encephalitis and other neurological complications is extremely important but often not-easy to achieve because of the highly heterogeneous clinical and radiological features and complexity of interpretation, especially in transplanted patients. here we described vignettes that represent and highlight distinct problems in the diagnosis and management of transplanted patients with suspected hhv- reactivation. methods: we collected the clinical, laboratory and radiological (electroencephalogram, brain mri and brain ct) data of transplanted patients who developed a neurological syndrome suspected for hhv- reactivation. hhv- was detected on serum and csf using rt-qpcr. results: ) a -years-old patient developed a diffuse erythema and subsequent encephalitic syndrome following hsct. the brain mri revealed clear signs of limbic encephalitic and searching for hhv- on serum and csf revealed . copies/ml and . copies/ml respectively. an antiviral therapy was started but no clinical benefit was achieved. ) a -years-old patient developed a typical neurological syndrome without brain mri findings of encephalitis and with no evidence of skin involvement. the lumbar puncture and csf analysis showed a total of . hhv- dna copies/ml. antiviral therapy with ganciclovir and foscarnet was promptly started with clinical improvement and a drastically reduction of hhv- dna on both csf and serum. a new brain mri revealed an acute limbic encephalitis. ) a slight neurological syndrome consisting of confusion and amnesia developed in a -years-old-patient. brain mri findings were compatible with a wernicke syndrome, but no improvement of neurologic symptoms were obtained with thiamine supplementation. csf analysis did not revealed hhv- dna, which was detected at low copies number on serum analysis. a second brain mri was conclusive for limbic encephalitis, so an antiviral therapy with foscavir was started and radiological but not clinical improvement was noted. the patient died after few days. ) in the last case we present a -years-old patient who developed a clinical picture of encephalopathy (i.e. amnesia, ataxia, drowsiness, weakness, depression) with rapid progression to coma after seventy-eight days from hsct. a brain mri showed a slight contrast enhancement in parietal-occipital regions. during the recovery phase from conditioning-induced cytopenia, an increasing in serum hhv- dna was detected. searching for hhv- dna on donor's follicles showed a chromosomally integrated hhv- (cihhv- ). cyclosporin a (csa) was interrupted and neurological improvement was observed in the following hours: a diagnosis of pres was made. conclusions: hhv- encephalitis should be suspected in transplanted patients with a clinical syndrome of encephalopathy. pcr detection of hhv- dna in csf associated with either typical brain mri abnormalities or a clinical diagnosis of nonspecific encephalopathy must lead to the urgent initiation of systemic antiviral treatment. if an increase of both serum hhv- dna and wbc is detected, a cihhv- should be confirmed. pres is an important differential diagnosis in transplanted patients which developed an encephalitic syndrome. disclosure: nothing to declare background: cytomegalovirus (cmv) infection is a major cause of morbidity and mortality after hematopoietic stem cell transplantation (hsct). it causes end-organ disease, multi-organ dysfunction syndrome, graft failure, increased susceptibility to infections and gvhd. greatest risk of cmv infection in a seropositive host is the reactivation of latent virus. methods: a prospective descriptive study performed at armed forces bone marrow transplant centre, rawalpindi, pakistan from dec to sep . hundred consecutive patients who underwent hsct were followed with weekly cmv dna quantitative pcr from engraftment till day for cmv reactivation. patients in whom cmv pcr showed more than copies/ml were treated with antiviral therapy. factors associated with cmv reactivation, outcome of antiviral therapy and effect of cmv on transplant outcome is studied. results: out of cases, were hla matched siblings, were matched family donors and were haploidentical transplants there were males and females. mean age was . ± . years. fourty-two transplants were done in thalassemia, in aplasia, in leukemias and in other hematological disorders and immune deficiencies. ninety-eight recipients and all the donors were cmv seropositive before hsct. cmv reactivation was seen in patients and of them had cmv viral load more than copies/ml and patients had cmv viral load less than copies/ml. nineteen patients had no cmv reactivation. mean time to reactivation since transplant was ± days. valganciclovir was given in patients due to ease of administration and six patients were treated with ganciclovir during their hospital stay. only one patient had resistant disease. mean time to clear viremia was ± . days. the patients having viral load less than copies/ml, subsequently cleared cmv without any treatment. antiviral agents; ganciclovir and valganciclovir were equally effective for treating cmv infection with % efficacy, however, more adverse effects were seen with ganciclovir. myelosuppression i-iii was seen in % patients treated with valganciclovir and in % treated with valganciclovir. renal impairment i-ii was seen in % of valganciclovir and % of ganciclovir treated patients. steroid administration was strongly associated with cmv reactivation (p = . ). no statistically significant association was found with the use of atg, gvhd, underlying disease, abo or gender mismatch. os was . % and . % in with and without cmv reactivation (p= . ) and dfs was . % and . % in with and without cmv reactivation (p= . ) conclusions: cmv reactivation was seen in % of the transplant recipients, this is higher compared to the western world due to high cmv seropositivity is this region. steroids administration in post-transplant period significantly increase the risk of cmv reactivation. preemptive therapy with valganciclovir effectively treats cmv reactivation with acceptable side effects. viral threshold for treatment should be decided considering the regional endemicity. cmv adversely affects the transplant outcome in terms of dfs and os. disclosure: no conflict of interest. acute nephritis requiring nephrectomy caused by adenovirus (hadv) and human polyomavirus bk (bkpyv) following allogeneic hematopoietic stem-cell transplantation in a patient with ph+ all background: adenovirus infection represents an important cause of morbidity and mortality after allogeneic hematopoietic stem cell transplantation (allo-hsct), with no established therapy. although different organs may be affected by disseminated hadv infections, kidney involvement has been rarely reported. co-infection of hadv and bkpyv are common complication in patients undergoing allo-hsct, but recent studies demonstrate that bkpyv may facilitate the replication of hadv and lead to elevated viremia with increased virulence and serious clinical consequences. here we report a case of an adult patient who required a monolateral nephrectomy due to hadv pyelonephritis as an early complication of allo-hsct for philadelphia-positive acute lymphoblastic leukemia (ph+ all). methods: in september , an ethiopian gentleman was diagnosed with ph+ all at the age of years. he was treated with polychemotherapy in association with the tyrosin kinase inhibitor imatinib mesylate achieving a complete remission (cr). one year later, due to disease relapse with cns involvement, he was started on vincristine and dexamethasone plus imatinib treatment and in april he was referred to our bmt center from ethiopia. upon confirmation of the p ph+ b-all diagnosis, therapy with the scr/abl dual inhibitor dasatinib associated to intrathecal chemotherapy was started and a salvage treatment with inotuzumab ozogamicin followed by an allogeneic hsct from a hla-identical brother was planned. having achieved a documented molecular cr disease status, in june the patient underwent allo-hsct following the fludarabine-melphalan reduced-intensity conditioning regimen. graft-versus-host prophylaxis included anti-thymocyte globulin, cyclosporine and mycophenolate mofetil results: on day + post-transplantation the patient developed macro-hematuria due to hemorrhagic cystitis and a ct scan unveiled a left pyelonephritis with marked kidney enlargement. kidney microbial investigations were all negative. at the same time, hadv viremia with very high copy number (> cp/ml) was documented and also elevated bkpyv (> cp/ml) viruria and viremia ( cp/ml). the genotyping of hadv evidenced serotype b mainly involved in infections of the urinary tract. treatment with cidofovir was immediately started; nonetheless, due to rapid clinical worsening despite maximal antibiotic therapy, on day + a left nephrectomy was performed, which led to a subsequent progressive resolution of the clinical symptoms and negativization of hadv and bkpyv viremia and viruria. pcr real time performed on the kidney tissue unveiled very high concentration of hadv copy number. conclusions: acute pyelonephritis due to disseminated hadv infection may represent a possible cause of severe complication following allo-hsct. monitoring of hadv copy number is helpful to evaluate infection severity and response to treatment. co-infection of hadv and bkpyv in immunocompromised patients should be always considered likely to worsen clinical course and outcome. disclosure: nothing to declare background: infection is a major cause of morbidity and mortality in patients (pts) receiving an allo-hsct. its severity is related primarily to the depth and duration of neutropenia. febrile neutropenia (fn) is defined as a neutrophil count below cells/mm and a fever ≥ . °c at a single measurement or≥ °c times at one hour intervals. the objective of our study is to analyze the epidemiological, clinical, biological characteristics of febrile episodes (fe) occurred in pts who benefited an allo-csh over a period of years. methods: from january to december , allo-hsct were performed in pts including sibling-hla identical, haplo-identical and phenoidentical for essentially acute leukemia ( pts, %), acquired and congenital aplasia ( pts, %). the median age is years ( - ) and sex-ratio (m/f): . . prophylaxis consisted on isolation sterile room with laminar flow, digestive decontamination, fluconazole and aciclovir. nine pts ( . %) were infected at the time of hospitalization (cellulitis , pneumoniae , bacterial angina , veinitis , bronchial pneumonia , furuncle cutaneous ) requiring treatment with antibiotics. conditioning regimen is myeloablative in all pts. anti-thymocyte globulin is used in pts ( . %). peripheral blood stem cells (pbsc) are used in pts ( %) with an average level of cd + cells: , . /kg ( . - . ) , bone marrow (bm) in pts with a mean level of nucleated cells: . x /kg ( . - . ) and the association of pbsc-bm in pts (haplo-identical). at each fe, are practiced: chest x-ray, procalcitonin test, blood culture, microbiological study of urine and stool (if diarrhea). results: all pts showed aplasia with an average duration of days ( - ), neutrophil engraftment was observed at day ( - ). one hundred and twenty-nine pts ( . %) presented fe with an average of . per pt. eleven pts ( %) had fe or more. forty nine ( , %) fe are clinically documented (digestive: , skin: , pulmonary: , urinary: , oto-rhino-laryngology: ). the blood cultures are made at fe, fe are microbiologically documented ( %): gram-positive bacteremia in % (mainly coagulase negative staphylococci) and gramnegative bacilli in % of cases. procalcitonin test performed during fe: normal ( cases), probable infection ( cases), probable sepsis ( cases), severe sepsis ( cases) and septic shock (one case). empirical double antibiotic therapy is initiated in pts without waiting for the results of the microbiological study. this association was sufficient in pts ( %). the transition to a second line was needed in pts ( . %) and third line in pts ( %). antifungal is added in cases ( %). eight pts benefited from g-csf. the evolution is favorable in fe ( . %), apyrexia obtained after an average of . days . three pts died ( %) by severe sepsis on a durable aplasia, of which had a cellulitis before the conditioning. conclusions: fe increase morbidity and mortality in allo-hsct so prophylactic measures are essential. empirical antibiotics treatment has to be instituted very quickly in the absence of documentation. disclosure: nothing to declare p abstract withdrawn. atsushi satake , masaaki hotta , ryo saito , akiko konishi , hideaki yoshimura , takahisa nakanishi , shinya fujita , tomoki ito , kazuyoshi ishii , shosaku nomura kansai medical university, osaka, japan background: cytomegalovirus (cmv) infection remains a common complication after allogeneic hematopoietic stem cell transplantation (ahsct), which results in increased morbidity and mortality. letermovir is a novel anti cmv drug that inhibits the cmv-terminase complex. the purpose of this retrospective study is to elucidate the efficacy and safety of cmv prophylaxis with letermovir early after ahsct in clinical practice. methods: we retrospectively analyzed the incidence of cmv infection, cmv disease, preemptive therapy, adverse events through week after ahsct, the rates of engraftment and overall survival. all patients underwent ahsct in our institution for hematopoietic malignancies between may and nov . data collected in this study included patient's characteristics such as age, sex, disease status, donor source and cmv disease risk. cmv infection was evaluated by cmv antigenemia. this study was approved by the research ethics committee of the faculty of medicine, kansai medical university. results: thirteen patients (male , female ) underwent ahsct and received cmv prophylaxis with letermovir. the median age was years (range, - years). overall, of patients ( . %) were considered to be at high risk for cmv, including patients ( . %) with haploidentical donors, and ( . %)with mismatched, unrelated donors. all patients began letermovir from day after ahsct, and achieved engraftment (median , - days). no patient developed cmv disease and required preemptive therapy. one patient died of treatment-related mortality, and patients died of acute gvhd. although one patient discontinued letermovir before day after ahsct because letermovir was suspected to be a cause of persistent nausea, severe adverse events were not observed. conclusions: it is still unknown whether cmv prophylaxis with letermovir improves os and reduces trm; however, our data suggests that cmv infection is considerably inhibited by administration of letermovir early after ahsct. clinical trial registry: not applicable. disclosure: the authors declare noconflicts of interest for this study. background: cytomegalovirus (cmv) is cause of increased morbidity and mortality after transplantation of hematopoietic cells. the pathogenesis of cmv disease or infection is complex with multiple interactions with the immune system, mainly in acute and chronic graft-versus-host disease (gvhd). the aim of this study is to analyze the risk factors for the reactivation of cmv in patients undergoing allogeneic hematopoietic cell transplantation (hct). methods: prospective descriptive study of the risk factors for the reactivation of cmv in the described population. univariate and multivariate analysis of the predisposing factors were performed: donor graft, treatment with corticosteroids, use of antithymoglobin, serologic status, conditioning regimen and the presence of gvhd. results: during the period between august until january , patients were evaluated. . % (n: ) had reactivation of cmv. average reactivation was days post transplant. both (recipient and the donor) had positive cmv igg in . %. in the univariate analysis, the reactivation of cmv was associated with haploidentical transplantation (p: < . ), with the use of corticosteroids (p: < . ) and gvhd (p: < . ). in the multivariate analysis, the haploidentical transplant maintained its statistical significance in comparison with the related allogeneic transplant (p: . , or: . ; ic %: . - . ) as well as the use of corticosteroids (p: . , or: . ; ic %: . - . ). % of patients receiving corticosteroid treatment had grade ii / iii gvhd. the serologicac status, myeloablative conditioning regimen and the use of atg showed no statistically significant association. conclusions: in patients undergoing allogeneic transplantation, were found as risk factor to reactivation, those who received haploidentic transplantation and treatment with corticosteroids. another risk factor that showed greater reactivation was the presence of gvhd. disclosure: nothing to declare methods: a y/o male was referred for allogeneic transplant following cycles of induction therapy for aml with complex karyotype and axsl mutation having achieved complete remission following the first cycle of chemotherapy. his first induction cycle was complicated by a perianal myeloid sarcoma which became infected and required surgical drainage and formation of a defunctioning colostomy. results: following allogeneic transplants, the first complicated by secondary graft failure and the second by primary graft failure he presented with two skin lesions, with a third lesion adjacent to his stoma developing shortly after admission. all lesions were erythematous with central necrosis and progressed rapidly in size over hours. biopsy of the skin and para-stomal lesions revealed fungal mycelia, with culture subsequently identifying rhizopus oryzae. initial treatment was with liposomal amphotericin b mg/kg/day followed by dose escalation to mg/kg/day due to the development of new skin lesions. the patient had been taking posaconazole (tablet) prophylaxis since his first allogeneic transplant and peripheral blood drug levels checked at the time of admission were therapeutic confirming that this was a breakthrough fungal infection. consequently posaconazole was stopped and isavuconazole added to the treatment regimen. surgical assessment was undertaken but surgery was deferred on the basis of high risk due to the extent of the infection and the patient´s profound pancytopenia. the organism was tested for in vitro susceptibilitiy and found to be resistant to posaconazole (mic > mg/l), with borderline resistance to isavuconazole (mic mg/l) and sensitive to amphotericin b (mic . mg/l) (phe mycology reference laboratory, england). isavuconazole was therefore stopped and the patient was managed with liposomal amphotericin b along with daily granulocyte infusions. he underwent a third allogeneic transplant using a different unrelated donor and stable engraftment was achieved. post transplant there was initially an increase in the size of the para-stomal lesion, but no new skin lesions developed. following engraftment he underwent resection of the stomal lesion, with primary closure and re-siting of his stoma. amphotericin b was replaced by isavuconazole prophylaxis on discharge and he continues to make an excellent recovery. conclusions: whilst aspergillus species remain the most common cause of invasive fungal infections in allogeneic transplant patients, other species including the mucorales are seen, and generally associated with poorer outcomes. whilst there are standardised methodologies for susceptibility testing, fungi specific cut offs based on clinical outcomes are only available for a limited number of species/ antifungal agents. in this case, susceptibility testing demonstrated resistance to posaconazole which was consistent with the clinical presentation of invasive infection despite therapeutic levels of posaconazole. it is also worth noting that an estimated % of r. oryzae isolates in the uk are resistant to posaconazole. treatment with high dose amphotericin b resulted in improvement in small skin lesions with stabilisation of the larger stomal lesion until count recovery allowed surgical resection. background: total depletion of innate and adaptive immune cell populations occurs after intensive chemotherapy and hematopoietic stem cell transplantation (hsct). both t and b lymphocyte pools are restored slower that myelomonocytic populations. hsct patients are at high risk for bacterial and viral infections at early terms (< days) post-transplant. the reconstitution of the immune system depends on the time required for stem cell recruitment, differentiation, expansion, maturation and release into the bloodstream. restoration terms for myeloid cells after hsct are usually defined as the st day with neutrophil count of ≥ . x ^ /l with mean recovery terms of to days. high occurrence of cytomegalovirus (cmv) in hsct patients mostly result from reactivation of a latent virus acquired in early childhood. however, delayed immune reconstitution and subsequent infections such as cmv, adenovirus (adv) or herpes (hhv- ) diseases are not unusual and still constitute a major cause of death in peru. methods: peruvian pediatric patients (n= ) diagnosed with aplastic anemia, mds, aml or all underwent a haploidentical hsct performed with the clinimacs device. patients treated were separated in two groups. the group of patients who received viral prophylaxis (ganciclovir) was compared to the group that did not receive any prophylaxis treatment. viral reactivation was confirmed by pcr test twice a week and clinical signs within days after hsct. results: in the group that didn´t received prophylactic treatment, engraftment occurred close to day post haplo-hsct and none of the patients developed gvhd (graft versus host disease). nevertheless, incidences of cmv, hhv- and bkv infections before day post haplo-hsct were still high. an overall survival (os) over % with an ic % was reached at the end of the first year. on the other hand, the group of patients that received prophylaxis with ganciclovir did not developed gvhd and reached the engraftment close to day with a very low viremia incidence after the first month post haplo-hsct. all viral reactivations were caused by cmv and the os was over % with an ic % at the end of the first year. previous prophylaxis to both the donor and the receptor with ganciclovir ( mg/kg) every hours before and during the conditioning regimen has allowed a better control of viral reactivation. conclusions: the attempts to improve immune function and reduce nonrelapse mortality from infectious complications without increasing gvhd have focused on a partial t cell depleted graft, such as t cell depletion (tcr α/β). this graft retains a large numbers of effector cells, such as tcr γ/δ and natural killer cells. however, delayed immune reconstitution and subsequent infections are a big issue. a novel partial t cell depletion strategy such as depleted naïve t cells (cd ra+ t cells) could enhance the recovery of immune function after haplo-hsct because donor pathogen memory t cells from the donor are retained. it is necessary to increase the studies and the database to set the scheme of previous prophylaxis to the recipient to contain the viral reactivation and to help a rapid immune reconstitution. disclosure: no conflict of interest is declared information was recovered from the medical records. results: thirty-four patients were included, of them with the following diagnoses: acute leukemia ( ), granulocytic chronic leukemia ( ), dendritic cell neoplasia ( ), aplastic anemia ( ). % of the patients received a transplant from an identical hla donor and % received a haploidentical transplant. mean age's patients was years ( - ). prophylaxis with posaconazole was performed on % of the patients with identical hla and % on haploidentical group; the rest of the patients received fluconazole. the posaconazole group presented: fever %, mucositis gi-ii %, gastrointestinal toxicity gi-ii % (p= . ), hepatic toxicity %, kidney toxicity %, oral candidiasis %. during this period none of the patients presented invasive fungal infection in any group. there were deceases, one on each group and none related to a fungal infection. the overall survival was of the % versus % on the posaconazole group and the fluconazole group respectively. conclusions: the prophylaxis with posaconazole and fluconazole is effective on the prevention of invasive fungal infection on the first days. the toxicity was similar on both groups. posaconazole can be effective on the prevention of the haploidentical type. is necessary to continue following the patients with infection risk on a long-term period associated with the chronic gvhd. disclosure: none declared lymphoma these results open the question whether allo-sct should still be offered to these patients. methods: we aimed to define the role of allo-sct in refractory or relapsing after two lines de novo or transformed dlbcl patients, and its comparison with zuma- car-t cells trial (neelapu et al nejm ). we analyse long-term allo-sct results in de novo (n= ) or transformed dlbcl (n= ) out of the allo-sct performed in our institution between october to october . results: patients and transplant characteristics are summarized in table . complete response (cr) at days was , % and % of them remain in cr at months. with a median follow-up of months, -year progression-free survival (pfs) was % and -year overall survival (os) %, with a -year transplant-related mortality of %. refractoriness at the time of the transplant was associated with a poorer prognosis, with only out of refractory patients being long term survivors (figure ). similar results were reported for zuma- trial, with a best response of % cr retained in % of them at months. with a median follow-up of months, -months pfs was % and -months os %. patients characteristics did not differ in our series and zuma- , except that all the patients in zuma- were refractory prior to therapy (table ) . conclusions: although very few patients with de novo or transformed dlbcl are offering an allo-sct ( % of all allo-sct), this is a curative option in chemosensitive patients and with more mature data and longer follow-up than with car-t therapy; for these reasons, it should still be offer to these poor prognosis patients. moreover, almost all patients have now available donor, better graft-versus-host disease prophylaxis will decrease trm and morbidity, and new therapies will make more patients in sensitive disease before allo-sct. therefore, allo-sct and car-t cells are strategies to be discussed in every young patient with available donor. disclosure: honoraria as advisor or speaker from gilead ( methods: consecutive patients transplanted for hgbl (excluding burkitts lymphoma) between - in the ebmt database were included. data collected included age, sex, pathology subtype (hgbl (including subtypes), tfl, dhl), disease status at sct, conditioning (ma vs beam cam vs flu-mel-cam/atg), engraftment, day outcome, trm, os and pfs and eligibility for emea licensed indication of car-t therapy. results: fifty patients ( m, f) with a median age of at diagnosis and at sct were included. the subtypes included hgbl (n= ), tfl (n= ) and dhl (n= ). indications for sct were: primary refractory (n= ), relapse < months after primary treatment (n= ), previous autologous-sct (n= ) and dhl (n= ). the median lines of therapy was (range to ). conditioning used was cytbi n= , bu/cy n= , etop/tbi n= , flubucy n= , beamcam n= , fmc/t, n= . all patients engrafted with neutrophil > . /l at median days and platelets > /l at median days. the day mortality was % (progressive disease %, nrm %) with a year os of % and mortality due to progressive disease % and nrm %. disease subtype influenced outcome with an os for primary refractory hgbl, relapsed hgbl, tfl and dhl respectively of %, %, % and %. patients were eligible for a licensed car-t product. conclusions: the outcome of these high risk hgbl patients have an acceptable os of %, with relapsed disease being the commonest cause of mortality. patients with dhl have a particularly good outcome in this series; recent evidence indicates that some of these patients with a non-immunoglobulin gene associated myc translocation could be managed more conservatively (ash sehn). the outcomes achieved with allogeneic-sct in this series will provide a baseline for outcome assessment with a cart program. disclosure: nothing to declare background: immune checkpoint inhibitors (ici) allow to achieve a durable remission in patients with resistant or refractory (r/r) classical hodgkin lymphoma. however, the information about optimal duration of therapy and the prognosis of the patients after ici cessation is limited (manson, blood ). therefore, the optimal role of hematopoietic sct (hsct) in this patient group is not defined. our aim was to determine remission duration in patients who discontinued ici monotherapy after achieving complete remission (cr). methods: this analysis included patients ( male/ female) aged to (median years) with r/r classical hodgkin lymphoma who were treated with nivolumab ( mg/kg every days) and achieved cr. response was assessed by positron-emission tomography/computed tomography (pet/ct) using lyric criteria every month. after nivolumab therapy had been stopped the patients received no other treatment and disease was assessed every months by pet/ct. median follow-up after therapy discontinuation was ( - ) months. results: at the moment of therapy initiation ( %) patients had stage disease, ( %) patients had progressive disease (pd), ( %) patients had stable disease, ( %) patients had partial remission and ( %)complete remission; ( %) patients had b-symptoms and ecog score > . the median number of previous therapy lines was ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) . before nivolumab initiation high dose chemotherapy with autologous sct was performed in patients ( %) and ( %) received brentuximab vedotin. the median number of nivolumab cycles was ( - ). cr was achieved after median of ( - ) cycles. the median duration of therapy after achievement of cr was ( - ) months. at the time of analysis, all patients were alive, ( %) out of patients relapsed after therapy discontinuation. the median progression-free survival (pfs) for the total group was not achieved. among patients with relapse, the median time before pd was ( - ) months. after relapse all patients were retreated with nivolumab monotherapy or with chemotherapy combination. one patient achieved complete remission; -partial remission; -indeterminate response type . other patients are continuing the therapy and their response has not yet been evaluated. conclusions: while complete response was maintained in some patients at median follow up of months after nivolumab therapy cessation, the pfs plateau was not reached. we report that patients with relapse after nivolumab discontinuation sustained sensitivity to nivolumab and achieved a response during retreatment with nivolumab monotherapy or with chemotherapy combination. in patients with unsatisfactory response to nivolumab retreatment, hsct option should be considered. disclosure: nothing to declare high dose chemotherapy with autologous stem cell transplantation in primary central nervous system lymphoma: data from the japan society for hematopoietic cell transplantation (jshct) registry center hospital, tokyo, japan, national cancer institute, bethesda, md, united states, okayama university hospital, okayama, japan, kanazawa medical university, uchinada, japan, kyoto university, kyoto, japan, aomori prefectural central hospital, aomori, japan, yamagata unversity school of medicine, yamagata, japan, tenri hospital, tenri, japan, hiroshima university, hiroshima, japan, japanese data center for hematopoietic cell transplantation, nagoya, japan, nagoya university graduate school of medicine, nagoya, japan, shimane university hospital, izumo, japan background: high-dose chemotherapy (hdt) with autologous stem cell transplantation (asct) has been shown to improve prognosis of patients with central nervous system (cns) lymphoma. whereas the common regimen of hdt for pcnsl in the europe and the us is thiotepa-based regimen, e.g. bcnu-thiotepa, tbc (thiotepa-busulfan-cyclophosphamide), thiotepa-based regimen was only available before discontinuation of thiotepa in in japan. we report the results of asct for pcnsl from the japan society for hematopoietic cell transplantation (jshct) registry. methods: data from the jshct registry were retrospectively analyzed. patients with pcnsl who received first hdt/asct between and were evaluated. distribution differences of clinical characteristics between groups were analyzed with fisher´s exact or mann-whitney u tests. overall survival (os) and progression free survival (pfs) were calculated using kaplan-meier method. two-group analysis of the cumulative incidence of relapse was conducted using the grey test. factors were analyzed in univariable analysis, and all factors with p≤. were retained in the multivariable model. all p values were sided, and values were regarded statistically significant if p< . . results: median age was months (range - ) with patients over years of age; males and females. ecog-performance status (ps) at diagnosis was better (ps - ) in patients and poor (ps - ) in patients. serum lactate dehydrogenase (ldh) levels at diagnosis were elevated in patients. karnofsky ps and cerebrospinal fluid (csf) protein concentration at diagnosis were not collected in the registry. patients were in complete remission (cr), patients were in partial response (pr), and patients were stable disease (sd) or progressive disease (pd) at the time of hdt/asct. after hdt/asct, additional patients achieved cr. with median follow-up period of months, the -year os and pfs were . % and . %, respectively. the was no significant difference in os and pfs between upfront and salvage hdt/asct. since thiotepa, a key agent in hdt/asct for pcnsl, has been unavailable after the discontinuation in japan, the hdt regimens used were not uniform. thiotepa-containing hdt was received by out of patients before , but by out of patients after . thiotepa-containing hdt showed improved pfs (p=. ), lower relapse (p=. ) and a trend toward a survival benefit. in the multivariate analysis, non-complete remission at hdt/asct was an independent predictor for os (hr= . , %ci: . - . , p=. ) and thiotepacontaining hdt remained significant for pfs (hr= . , %ci: . - . , p=. ). [[p image] . os(a),pfs(b) in all patients (n= ) and cumulative incidence of relapse in cr patients (c; n= )] conclusions: our results confirm the activity of thiotepacontaining regimen for hdt/asct in pcnsl patients. currently a pharmaceutical company re-develops thiotepa for new approval of hdt/asct in pediatric solid cancer and adult lymphoma in japan (japiccti- ). further evaluation with the thiotepa by prospective clinical trials is warranted. disclosure background: t-cell non-hodgkin lymphomas (t-nhl) are rare diseases and they are associated with worse prognosis when compared to their b-cell counterparts. allogeneic stem cell transplantation (allo-sct) may have a curative potential for these patients due to the graft versus lymphoma effect. however, data is limited on the efficacy of allo-sct for these diseases. methods: we identified patients ( % females; median age: y; range, - ) with t-nhl that underwent allo-sct at university hospital eppendorf between and . twenty-one patients (underwent allo-sct from a matched sibling donor (msd) and ( %) from a matched unrelated donor (mud). sixteen patients had ptcl ( %), n= ( %) anaplastic large-cell lymphoma (alcl), n= ( %) angioimmunoblastic large cell lymphoma, n= ( %) adult t-cell leukemia/lymphoma, n= ( %) hepatosplenic gamma/delta t-cell lymphoma, n= ( %) enteropathy associated t-cell lymphoma, n= ( %) tcell-prolymphocytic leukemia, and n= ( %) each extranodal t/nk-cell lymphoma, cutaneous t-cell lymphoma as underlying diagnosis. the median ann arbour stage at diagnosis was (range, - ). ten patients ( %) had bone marrow involvement at diagnosis. all patients were heavily pretreated, ( %) patients relapsed post autologous stem cell transplant (apsct) and one patient post allo-sct. fifteen patients ( %) were transplanted in complete remission (cr) ( in st cr, in nd cr), n= ( %) in partial remission (pr), and n= ( %) with advanced disease. most of the patients received myeloablative conditioning ( %). thirty-eight ( %) patients received total body irradiation based regimens and ( %) received chemotherapy based regimens. twenty patients ( %) received anti-t-lymphocyte globulin (atlg neovii), and most patients ( %) received g-csf mobilized peripheral stem cells. results: overall, patients ( %) had neutrophil engraftment (median days: ; range, - ) . at day , the cumulative incidences of grade ii-iv and grade iii-iv acute gvhd were % and %, respectively. after a median follow up of months (range, - ) the cumulative incidences of chronic gvhd was % distributed evenly between limited and extensive. twenty nine patients ( %) achieved cr after allo-sct. median overall survival (os) and disease free (pfs) survival were months and months respectively. the year os and pfs were % and % respectively. fourteen ( %; % ci [ . - . ]) deaths were due to non relapse mortality (nrm) and patients ( %; % ci [ . - . ]) died due to disease progression. patients with a male donor had improved os compared to those with a female donor ( year os male %, female %; p= . ). patient gender, disease subtype, bone marrow involvement, type of allo-sct, donor, patient cmv status, abo incompatibility, disease stage at diagnosis, previous transplant, disease status at transplant, conditioning regimen, atg and stem cell source had no effect on os, pfs, nrm, and post transplant complications. conclusions: acknowledging the retrospective nature, our study shows that allo-sct induces high rates of complete remission, and may have a curative potential even in diseases relapsing post asct. however our findings need to be confirmed in larger prospective studies. disclosure: no funding, no conflict of interest p abstract already published. at-home autologous stem cell transplantation in lymphoma patients: clinical impact of non-g-csf administration post-transplant background: severe neutropenia remains the main cause of morbidity and mortality after autologous stem cell transplantation (asct). g-csf administration after asct is a common practice, performed to reduce the duration of neutropenia and its complications. in a previous work in patients with multiple myeloma managed at home after asct, we did not observe a deleterious clinical impact in those patients that did not receive g-csf post-transplant (martinez-cibrian n. et al, bmt ) . despite the fact that lymphoma patients receive a more intensive conditioning regimen that multiple myeloma patients, we hypothesized that the use of g-csf in lymphoma patients managed at home during the aplasia phase of asct does not provide a significant clinical benefit. methods: lymphoma patients were managed at-home since day + of asct. between february and july , patients received at-home g-csf μg/kg per day since day + until their anc reached x /l (g-csf group) and, since august , patients did not receive g-csf (non-g-csf group). all patients were conditioned with beam and received prophylaxis with a quinolone, fluconazole, aerosolized pentamidine and low-dose acyclovir (hvs+). in all cases we added primary prophylaxis with piperacillin-tazobactam . g/ h i.v., using a portable intermittent infusion pump (iip), from an absolute neutrophil count (anc) < . x /l until the first day of fever or until attaining an anc of x /l. first-line therapy at home of neutropenic fever (nf) was refrigerated meropenem g/ h i.v using a portable iip. fever was an indication of immediate visit to the hospital, and those patients presenting with focal infection or signs of severe sepsis were admitted. other indications for readmission were: willingness of the patient or caregiver; uncontrolled nausea, vomiting or diarrhea and mucositis requiring total parenteral nutrition or i.v. morphics. results: the main characteristics of the patients are shown in table . there were no differences between groups with respect to gender, diagnosis, stage of disease, comorbidity index (hct-ci), source of stem cells (peripheral blood) and cd + cell dose infused. the median (range) age (years) was ( - ) in g-csf group and in non-g-csf group (p= . ). duration of neutropenia less than . x /l was significantly longer in non-g-csf group, with a median of days (range - ), compared with (range - ) in g-csf group (p < . ). severe neutropenia, less than . x /l, was also longer in the non-g-csf group ( days ( - ) vs. ( - ); p= . ). no differences were observed in the time to platelet engraftment. g-csf post-transplant avoidance did not influence the incidence of neutropenic fever, the first day and duration of fever, the incidence and severity of oral mucositis, bacterial infections documented and number of readmissions. the median duration of the whole procedure at-home was day shorter in the g-csf group ( vs. days; p= . ). conclusions: the policy of not administering g-csf post-asct in our home-based program for lymphoma patients, that include intensive bacterial prophylaxis, did not have a deleterious impact on the main results reviewed, which suggests that elimination of its use can be achieved. disclosure the aim of this study was to analyze the spanish experience with patients diagnosed of nhl who received haplosct with pt-cy. methods: sixty patients who received haplosct with pt-cy in spanish centers from to were analyzed. patients were followed-up until . gvhd prophylaxis consisted in cyclophosphamide mg/kg/d on days + and + , and mmf and a calcineurin inhibitor from day + . results: patients' characteristics are summarized on table . median age of patients was , % male, and diagnosed from t cell lymphoma ( %). most of them didn´t achieve complete response prior to transplant ( %), but only % with active disease. up to % of patients had received previous transplant, from which % was an allogeneic transplantation. source of stem cells was mainly peripheral blood ( %), and reduced intensity conditioning was the preferred ( %) regimen. donors were % siblings ( ), % offspring ( ), and % parents ( ). median neutrophil and platelet engraftment was ( - ) and ( - ) days, respectively. acute gvhd grade ii-iv rate was %, with only patients developing grade iii-iv ( %). chronic gvhd rate was %, and only in ( %) was extensive. median follow-up was months. the -year overall survival and event free survival was % and %, respectively. the -year cumulative incidence of relapse was % and -year cumulative incidence of nrm was %. conclusions: relapsed/refractory nhl are aggressive entities with a fatal course in a short period of time. haplosct with pt-cy permit a new treatment option among these patients, with acceptable outcomes. more studies are needed with a larger cohort of patients and longer follow-up to confirm these results. disclosure: nothing to disclose. higher suv at pre-transplant and day posttransplant pet scan both independently predict inferior survival in patients with diffuse large b cell lymphoma background: autologous stem cell transplant (auto-hct) can cure some patients with relapsed diffuse large b-cell lymphoma (dlbcl) but relapse occurs in about % of patients. while our center and others utilize routine surveillance imaging post-transplant, the utility in this setting is unclear. imaging is costly and exposes patients to radiation. novel interventions are now available for patients relapsing after auto-hct making early disease recognition crucial to intervene prior to clinical progression. hence, we studied impact of post-auto-hct surveillance ( )f-fdg-pet ct at day on transplant outcomes. methods: we analyzed a cohort of consecutive auto-hct recipients with relapsed/refractory dlbcl who then underwent pre-transplant pet/ct and surveillance pet ct at day (interquartile range (iqr): - days) post-hct at the university of minnesota medical center. univariate analysis was performed to analyze pet parameters including deauville score (d), standardized uptake values (suv), total lesion glycolysis (tlg) and total metabolic tumor volume (tmtv) as predictors of relapse and survival after auto-hct. in addition, we assessed outcomes of patients with clinically versus radiographically detected relapsed dlbcl after auto-hct. other pre-hct factors analyzed included age, gender, conditioning regimen, performance status, consolidation radiation therapy, tmtv, suv, tlg. results: five-year cumulative incidence of relapse after auto-hct was % ( %ci to ) and overall survival (os) was % ( % ci to ). twelve ( %) relapsed prior to day . d-score for patients with d pet/ct were d ( %), d ( %), d ( %), d ( %), d ( %) with median survival in years for d , d , d and d of . , . , . , and . , respectively. mean suv varied from . (d ) to . (d ). suv was predictive of relapse and os. risk of relapse increased with doubling of suv; -fold higher suv increased hr by . ( %ci . - . ; p= . ). mortality increased with doubling of suv in both pre-hct ( -fold increase in suv associated with hr . [ % ci . to . ]; p= . ) as well as post-hct pet (hr . [ % ci . to . ]; p= ) irrespective of the bulk of tumor. in addition, risk of death was times higher in d patients relative to d (hr . [ % ci = . to . ]; p≤ . ). patients with d (n= ) had higher tmtv ( cm ) compared to d (n= , tmtv . cm ). the hazard ratio for death following relapse was -fold higher (hr . [ % ci . to . ]; p= . ) if relapse was detected clinically versus only radiographically over a median follow-up time period of . years. other pretransplant patient and disease characteristics did not significantly influenced the outcomes. conclusions: in patients with r/r dlbcl undergoing auto-hct, surveillance pet/ct at day identified patients with poor survival~ year. higher suv in both pre-transplant as well as post-hct pet was predictive of increased mortality. these patients may benefit from novel treatments. [ there are concerns about the risks of nivolumab treatment before and after allo-hsct, due to the risk of heavy gvhd, thus the place of immune checkpoints inhibitors is not yet defined. this report include analysis of our center experience of nivolumab treatment in patients with r/r hl before and after allohsct. methods: we retrospectively evaluated the results of allohsct in patients with r/r chl who had undergone transplant from to . the analysis included patients received the flube conditioning and ptcy gvhd prophylaxis. in group a patients (n= ) received bridge therapy with nivоlumab and in group b patients (n= ) received bridge therapy with brentuximab vedotin or chemotherapy-based bridges. time from the last nivolumab administration to allohsct was at least months. results: at the time of analysis, median follow-up was ( - ) months for group a, and ( - ) months for group b. there was no difference in two-year os (p= , ) with significantly better efs (p= , ) for group a versus group b: % and % vs , % and % respectively. relapse incidence was % for group a versus , % in group b (p= , ). cumulative incidence of non-relapse mortality at years was , % and , % in group a and group b, respectively (p= , ). there was no difference in grade ii-iv ( % vs %, p= . ) and grade iii-iv ( % vs %, p= . ) agvhd, as well as extensive chronic gvhd ( % vs %, p= , ) in groups a and b, respectively. ten patients with relapse after allohsct were treated with different doses ( , - mg/kg) of nivolumab in cic center. at the median follow up of mo ( , - ) all patients remain alive. the objective response to therapy was assessed in patients noted in all patients ( %), disregard the dose of the nivolumab: cr in %, and pr in %. the response was lost in four patients, which required nivolumab retreatment. none of the patients developed gvhd after nivolumab administration. in this analysis, there was also no correlation between dose of nivolumab and incidence and severity of adverse events. conclusions: allohsct in combination with immune checkpoints inhibitors is a good option for patients with r/r chl. consideration for immune-mediated toxicities and the potential for increased graft-versus-host disease remain important. early data suggest that nivolumab may be an efficient therapy in patients with r/r chl relapse after allo-hsct. further research needed. disclosure: the authors declare no conflicts of interest. background: transformation to diffuse large b-cell lymphoma (dlbcl) is considered to be one of the most unfavourable events of lymphoma natural history with poorer outcome as compared to de novo dlbcl (alonso-Álvarez et al, bjh ). in patients suitable for salvage therapy, hematopoietic stem-cell transplantation (sct) could be an option, although its role is not well stablished. we analyse indication and outcome after transplant in transformed dlbcl at a single reference transplant unit. methods: out of total of transplants performed at our unit between and - autologous and allogeneic- were dlbcl transformed from an indolent nhl. of them, received an autologous sct (asct) and an allogeneic sct (allo-sct). results: median age was years old (range - ) and (range - ) for patients receiving asct and allo-sct, respectively. all asct received beam as a conditioning regimen and most of the patients in the allo-sct group received a fludarabine/melphalan combination ( %). gvhd prophylaxis consisted on tacrolimus/sirolimus combination in % and calcioneurin plus methotrexate in %. regarding transplant disease status, ( %) of the asct patients were transplanted in complete response (cr). in the allo-sct group, ( %) patients had received three or more treatment lines before transplant and patients ( %) had received a previous asct, being ( %) in cr, in partial response (pr) and in progressive disease. transplant related mortality (trm) was . % in the asct and % in the allo-sct group. overall survival (os) and progression-free-survival (pfs) at months were % (os), % (pfs) for patients receiving asct and % (os) and % (pfs) for allo-sct. with a median follow up of months for patients receiving an asct, ( %) remain in cr. in the allo-sct group median follow up is months for the whole group and months for alive patients; patients are alive and disease free and have died, due to trm ( %). regarding progression, ( %) have progressed after autologous transplant and after allo-sct. conclusions: indication for hematopoietic sct in transformed dlbcl is stablished in few patients. only % of the patients in our transplant unit receive a transplant due to transformed lymphoma, corresponding to a . % of autologous activity and . % of allogeneic activity. according to our results transplant should be considered a curative option. most of our patients were transplanted in cr, so new agents trying to reach best response before transplant should be considered. [[p image] . eva konirova , antonin vitek , marta krejci , edgar faber , katerina steinerova , david belada , jan novak , juraj duras , petr sedlacek , veronika valkova , andrea janikova , ludek raida , pavel jindra , pavel zak , tomas kozak , marie trnkova , michal karas , marek trneny management. however, differences in patient's characteristics as well as frequency of hsct indication in different lymphoma subtypes have been observed in the last decade. the aim of this study was retrospective analysis of hsct for lymphomas in czech republic. methods: data of adult patients transplanted between years - were retrospectively analyzed using ebmt database. results: between and , autologous hsct (asct) were performed in patients ( men, %) with different lymphoma subtypes. the median age was years (range - ). out of these, ( %) were patients with non-hodgkin lymphoma (nhl), ( %) with hodgkin lymphoma (hl). the nhl group comprised of diffuse large b-cell lymphoma (dlbcl, %), follicular lymphoma (fl, %), mantle cell lymphoma (mcl, %) and t-nhl ( %). the frequency of asct in lymphomas increased from to and has been constant since ( - transplants per year). differences in frequency of asct were observed among lymphoma subtypes -decreasing numbers of dlbcl and fl and increasing numbers of t-nhl and mcl, with asct as part of the induction therapy. between and a total of allogeneic hsct (allosct) were performed in patients ( men, %). median age was years (range - ). out of these ( %) were patients with nhl, ( %) hl. the most common nhl subtypes were fl ( %), mcl ( %), t-nhl ( %) and dlbcl ( %). in the last years the number of allosct for lymphoma is fluctuating around per year. the median age at asct was significantly higher in the years - vs - [ . ( . - . ) vs. . ( . - . ), p < . , fig ] , while the increase at allosct [ . ( . - . ) vs . ( . - . )] did not reach statistical significance (p= . ). with median follow up for allosct, y probability os for patient transplanted in the later period - was in relapsed dlbcl . %, in fl . %, in hl . % and in mcl . %, y os for asct as part of first line therapy in the same period was in mcl . % and in t-nhl . %. os was significantly better in all patients who underwent asct in the years - vs - ( .% vs. . %, p < . ) and there was a trend towards better os in patients after allosct (with . % vs . %, p= . ) (fig ) . conclusions: hsct remains important treatment modality for lymphomas in the era of targeted antibody and molecular therapy and we can transplant older patients due to better supportive treatment. acknowledgment: progress q - uk from the czech ministry of education youth and sports disclosure: nothing to declare background: disease chemosensitivity to salvage treatment has been proven to be a major predictive factor for a favorable outcome after autologous stem cell transplantation (asct) for patients with refractory lymphomas. therefore the importance of effective and safe salvageregimens is indisputable. methods: we retrospectively compared the outcomes in terms of safety and efficacy, in (hl: , nhl: ) patients, with a median age of . ( - ) years, who received as st salvage either dicep [cyclophoshamide ( mg/m ), etoposide ( mg/m ), cisplatin ( mg/ m ), days - , (n= )] or the widely used regimen eshap (n= ). rituximab was additionally given to all cd- positive lymphoma patients. the statistical analysis based on the independent t-test, kaplan meir method and logrank test. results: the reason for salvage treatment was primary induction failure (pif, n= ), early relapse (< months post induction-remission therapy n= ) and late relapsed disease (n= ). more specifically, / patients ( %) in the dicep-group, and / patients ( %) in the eshapgroup were assessed with pif or early relapsed disease, however this difference was not statistically significant. both regimens were well tolerated and no major organ toxicities were noticed. eleven patients ( %) from the dicep-group, while only ( %) from the eshap-group developed febrile infections. all patients were successfully managed with the appropriate treatment and only one, from the eshap-group, required for short period admission to the intensive care unit. after cycle of dicep and cycles of eshap the disease response was re-assessed by pet/ct scan. the overall response rate (> % tumor reduction) was significantly superior for the dicep-regimen, reaching % ( / patients) vs. % ( / patients) for eshapregimen (p= , ). eleven patients ( %) from the dicep-group and ( %) from the eshap-group achieved complete metabolic remission according to pet/ ct criteria (p=ns). the median hospitalization period was ( - ) days for the dicep-group compared to ( - ) days for the eshap-group. however, for the eshapgroup, an additional median of ( - ) hospitalization days were required, since of the non-responders patients received a nd salvage before asct. the mobilization and stem cell collection was successful for both groups, though significant higher number of cd + cells were collected in the dicep-group ( . x /kg vs. . x /kg, p= , ). all but two patients (due to refractory disease) underwent asct. noticeably, the median period from st salvage treatment to asct was significantly shorter for the dicepgroup ( vs. days, p= , ), apparently because non-responders patients from eshap-group treated with a nd salvage. the -years overall and progression free survival were similar for dicep-and eshap-groups ( % vs. % and % vs % respectively). two heavily pretreated patients from the eshap-group developed secondary myelodysplastic syndrome post asct conclusions: in our series of patients both regimens proved to be safe. interestingly, despite the fact that more patients in dicep-group had poor risk disease the dicepregiment was significantly more effective, resulting thus in an earlier asct, less exposure to chemotherapeutic agents, that might led in less long-term toxicity. nevertheless, prospective trials with large series of patients are needed to define the role of dicep in the salvage treatment setting. disclosure: no conflict of interest background: although autologous hematopoietic stem cell transplantation (auto-hsct) is one of the best curative strategies for patients with chemosensitive t-cell lymphoma, major limitation remains a tumor contaminated graft-related relapse or residual disease after chemotherapy. several purging methods were introduced in auto-hsct for these limitations, however there are few studies of ex vivo purging of the autograft in lymphomas, especially t-cell lymphoma. therefore, we retrospectively analyzed consecutive t-cell lymphoma patients receiving auto-hsct with/without ex vivo purging. methods: among them, patients underwent autograft manipulation with ex vivo purging by cd + cells selection using a clinimacs device. results: with median follow-up duration of months (range, - months), -year overall survival (os; . % vs. . %, p= . ) and -year progression-free survival (pfs; . % vs. . %, p= . ) in a purged and unpurged group, respectively. transplant-related mortality was observed in both groups ( patients of a purged group and patient of an unpurged group). neutrophil ( vs. days, p= . ) and platelet ( vs. days, p= . ) recovery were similar in both group and there was no engraftment failure. on subgroup analysis according to upfront and salvage auto-hsct, while survival outcomes were improved by stem cell purging in the upfront auto-hsct (os with p= . and pfs with p= . ), there were no different survival outcomes in salvage auto-hsct. the unmanageable late-infectious complications were few in both groups except for predominantly cytomegalovirus reactivation in a purged group ( vs. patient). conclusions: although cohort was a small number, ex vivo graft-purging method was feasible and safe in t-cell lymphomas. and this purging strategy observed the more favorable survival outcomes in the upfront auto-hsct than salvage setting. therefore, further randomized studies are needed to determine the firm efficacy of cd + purification with the large number of patients in auto-hsct for t cell-lymphomas. disclosure: nothing to declare nivolumab-based regimens in relapsed or refractory non hodgkin lymphomas: the role of hematopoietic stem cells transplantation methods: we analyzed data of patients with r/r nhl, among them n with diffuse large b-cell lymphoma (dlbcl), n with primary mediastinal b-cell lymphoma (pmbcl), n with gray zone lymphoma (gzl) and n with gamma-delta peripheral t-cell lymphoma (ptcl), who received nivolumab-based regimens. the median age was years (range, - years). most of the patients n ( %) had a primary chemoresistant disease, the rest patients n ( %) had a relapse. the median of lines of prior therapy was lines (range, - lines). all sixteen patients with dlbcl and pmbcl received - cycles of nivolumab in combination with bendamustine, gemcitabine and rituximab (begern). the patient with gzl received cycles of nivolumab in combination with brentuximab vedotin and epoch. and the patient with ptcl received cycles of nivolumab monotherapy. results: at median follow up months ( - ) objective response (or) after nivolumab-based regimens was noted in n ( %) patients, complete response (cr) and partial response (pr) in n ( %) and n ( %) patients, respectively. cr observed in n patients with dlbcl, n with pmbcl, n with gzl, n with ptcl. and pr observed in patient with dlbcl. two responding patients with dlbcl underwent auto-hsct. and four responding patients (n dlbcl, n pmbcl, n gzl, n ptcl) received allogeneic hematopoietic stem cells transplantation (allo-hsct). the median duration of response for all n patients with or was (range: - +) months. among n patients who achieved or without hsct, only n remain in cr. two patients who received auto-hsct had a relapse. one patient with dlbcl improved the response after allo-hsct from pr to cr, and all four patients with allo-hsct remain in cr. the probabilities of -year os and pfs rates were % and %, respectively. conclusions: nivolumab-based regimens can lead to an objective response in % patients with r/r nhl. however, the durability of response to therapy is not long. nivolumab-based regimens can be used as bridge to allo-hsct disclosure: there are no conflicts of interest to disclose background: patients with aggressive non-hodgkin lymphoma (nhl) who relapse after autologous stem cell transplantation have a dismal outcome and could benefit from radiotherapy, allogeneic stem cell transplantation or experimental treatments. systemic inflammatory parameters at diagnosis have demonstrated to be useful to predict lymphoma evolution. methods: we conducted a retrospective review of patients with aggressive nhl who underwent autologous stem cell transplantation (astc) to evaluate the relationship between ldh, β -microglobulin, inflammatory parameters (lymphocyte (alc) and monocyte count (amc), ferritin or c-reactive protein) and imaging techniques before and on day + post-astc and progression free survival (pfs), as well as the role of residual disease directed radiotherapy (rt). results: one hundred and sixty patients with aggressive nhl received asct as consolidation treatment in our center between and . the most common diagnosis was diffuse large b-cell lymphoma (dlbcl). one hundred and nine patients received upfront asct for high risk dlbcl (defined as age-adjusted ipi - )(n= ) or for having received two or more lines to obtain first complete remission (n= ), for t-cell lymphoma (n= ) and for mantle cell lymphoma (n= ). the rest was performed in relapsed lymphomas. forty-seven patients ( %) relapsed and pfs was months. pretransplant response was evaluated with ct scan in patients ( of this with partial remission (ct-pr) and patients were evaluated with fdgpet/ct ( were pretransplant positive (pet ); of these, patients maintained positivity at day after astc (pet ). pfs in patients with ct-pr was months, in pet positive ones months and in pet positive ones months. univariate analysis showed pet positivity as the most accurate predictor of relapse (hr , , p= , ) followed by amc at day + (hr , , p= , ), albumin at day + (hr , , p= , ), ldh at day + (hr , , p< , ) and pretransplant alc/amc ratio (hr , p= , ). multivariate analysis only demonstrated an association with pet positivity (hr , ) p< and ldh in day + (hr , ) p= , with pfs. five and ten years overall survival were % and % in pet negative patients vs and % in pet positive ones (p< , ). eight out of patients with pet positivity did not relapse. salvage radiation therapy was used in patients with positive residual mass and of them did not relapse. two patients relapsed: one patient had residual mass and another had remote affectation from primary site and could be considered as progression before day + . conclusions: post asct fdgpet/ ct is superior to conventional ct in predicting outcome in aggressive lymphoma after astc. pre and post asct systemic inflammatory parameters didn't help to improve the relapse risk prediction. addition of consolidative rt after astc has demonstrated improvement in pfs in patients with pet positivity. it would be neccesary to develop randomized trials to assess the role of rt in residual disease in advanced aggressive nhl with insufficient response to systemic treatment with pet response evaluation. disclosure: nothing to declare long term outcome of patients with lymphoid malignancy who underwent high dose chemotherapy followed autologous hematopoietic cell transplantation at a single institution over years joanna romejko-jarosinska , ewa paszkiewicz-kozik , lukasz targonski , lidia popławska , jan walewski background: high dose chemotherapy (hdt) and autologous hematopoietic cell transplantation (auto-hct) is a standard of care for relapsed/refractory lymphoma patients (pts) or it is used as a consolidation for myeloma and high risk lymphoma patients in first line treatment. we retrospectively evaluated long-term outcome including late effects and risk factors in patients with lymphoid malignancy who underwent auto-hct. methods: we collected data from consecutive patients with hodgkin lymphoma (hl) (n= ), aggressive b lymphoma (dlbcl) (n= ), myeloma (n= ), indolent lymphoma (n= ), mantle cell lymphoma (n= ) and peripheral t cell lymphoma (n= ) who underwent auto-hct at our institution between and . at transplant median (range) age was ( - ) years, clinical stage iii/iv was found in of lymphoma pts, complete remission, partial remission and stable/ progressive disease occurred in ( %), ( %), ( %) pts, respectively. beam regimen was used in pts ( %), mel in pts ( %) and other myeloablative regimens in pts ( %). results %) ], respectively. partial remission or stable disease at transplant, clinical stage iii or iv, and age more than , were identified as risk factors associated with inferior os and pfs in univariate and multivariate analysis. histopathologic diagnosis was not a risk factor for os and pfs (p=ns). the outcome of patients who underwent auto-hct between - was inferior to the outcome of patients treated in - or - . - year os was %, %, %(p< . ) and year pfs was %, %, % (p< . ), respectively. we recorded ( %) cases of second primary cancer ( solid tumors and hematologic cancers). acute cardiotoxicity occurred in patients from to years after transplant, and required heart transplant in patients. patients ( %) died. the main causes of death were progressive disease in pts ( %), second primary malignancy in pts ( . %) treatment related mortality was . % ( pts), and mortality within days was ( , %). [[p image] . pfs and os in patients underwent hdt and auto-hct - , - , - conclusions: more than % of patients who underwent hdt and auto-hct had long term survival without progressive disease. older age, non-complete remission at transplant, advanced stage are associated with poor outcome. patients recently transplanted had a better outcome than patients transplanted before . disclosure: nothing to declare outcomes after haploidentical and matched related hsct in lymphoma do not differ significantly: a single center study nadira durakovic , , zinaida perić , , lana desnica , ranka serventi-seiwerth , sandra bašić kinda , ivo radman-livaja , alen ostojić , ante vulić , dražen pulanić , , pavle rončević , zorana grubić , igor aurer , , radovan vrhovac , university of zagreb, school of medicine, internal medicine, zagreb, croatia, uhc zagreb, zagreb, croatia background: allogeneic hsct still offers patients with relapsed/refractory lymphoma the best chance of long-term survival. in most such patients timing of hsct is crucial, therefore a related donor is preferred. we analyzed acute and chronic gvhd incidence, relapse and overall survival, but also time to immunosuppression (is) discontinuation and hematopoietic recovery comparing transplantation using haploidentical (haplo) and matched related donors (mrd) in single center in this indication. methods: in the time period between / and / at uhc zagreb, croatia, mrd and haplo transplantations in lymphoma were done, for hodgkin and for nhl. all patients transplanted from haploidentical donors received ptcy. data were computed using the r package. the probability of gvhd was calculated using the cumulative incidence method and subgroups were compared using the gray test. results: median age was ( - ) years; ( - ) in haplo and ( - ) in mrd group. four patients were in pr and in cr in haplo group, while in mrd group patients were in cr and in pr. in haplo group patients ( %) received bone marrow (bm) and only ( %) peripheral blood stem cells (pbsc). in mrd group all patients received pbsc. all patients in haplo group received nma ("baltimore") conditioning with ptcy while in mrd group patients ( %) received flu-bu atg, and only one received flutbi as conditioning protocol. in haplo group % patients were previously treated with autologus transplantation, % in mrd group. there was no significant difference in time to is discontinuation, and days in haplo and mrd group, respectively. patients after haplo recovered slower, recovering anc after . days ( % ci, . - . ) and . ( % ci, . - ) (p= . ) and recovering platelets after . days ( % ci, . - . ) and . ( % ci, . - . ) (p< . ) in haplo and mrd group. with a median follow up of days, overall survival was % ( % ci, - ) in haplo and % ( % ci, - ) in mrd group. trm was % in haplo and % in mrd group. cumulative incidence of agvhd ii-iv was % ( % ci, - ) and % ( % ci, in haplo and mrd group, respectively (p= . ). cumulative incidence of cgvhd requiring treatment was % ( % ci, and mrd % ( % ci, - ) in haplo and mrd group, respectively (p= . ). all cases of cgvhd developed after dli. cumulative incidence of relapse was % ( % ci, - ) and % ( % ci, - ) for haplo and mrd group, respectively (p= . ). conclusions: we found no significant difference in overall survival, relapse incidence, agvhd and cgvhd incidence between these two groups. hematopoietic recovery was slower after haploidentical transplantation, but it did not influence trm as it was higher after mrd. even though limited in number, this data contribute to the growing body of evidence that use of haploidentical donors, particularly in lymphoma setting, is as worthy as using matched related donors and should be at least second choice in donor selection, and in older patients (with older donors) probably the first one. disclosure: nothing to declare. adjuvant involved field radiotherapy post autologous stem cell transplantation for refractory/relapsed lymphomas results in favorable outcome with low toxicity: a single center experience background: involved field radiotherapy (ifrt) to previous bulky or localized residual disease, is a widely used treatment approach to minimize the risk of relapse post autologous stem cell transplantation (asct). however, the proper time for irradiation treatment remains controversial. adjuvant ifrt (adj-ifrt) in pre-asct period could cause undesirable toxicity which might delays or even cancel the asct resulting in increased risk of relapse, or could affect the marrow environmental and marrow niche resulting thus in impaired engraftment. on the other hand, the ajd-iftr in the early post-asct period, upon marrow recovery, offers a potential advantage by delivering irradiation after sufficient disease response, without affecting the engraftment. in this retrospective study we evaluated the safety and efficacy of the ifrt as adjuvant treatment in patients who had previously treated with asct for relapsed or refractory lymphomas. methods: twenty-three patients (hodgkin= , non-hodgkin= ), aged of ( - ) years, underwent asct, for primary refractory (n= ) or relapsed (n= ) disease. patients who had bulky disease at the time of relapse or those with residual mass post salvage treatment, were considered as candidates for adj-ifrt, early (within - months) after documentation of autologous stem cells engraftment. all patients proceeded to asct with chemosensitive disease after a median of lines of salvage therapy. at the time of asct patients ( %) had residual disease while ( %) evaluated to be in complete remission. the preparative regimens were: single-agent melphalan (n= ), busulfan-etoposide-melphalan (n= ), beam (n= ) and bendamustin-etoposide-cytarabine-melphalan (n= ). filgrastim was given till neutrophills recovery, while prophylaxis against bacteria, fungus, viruses and pcp were administered till the completion of adj-ifrt. results: all patients engrafted promptly and successfully. no patient experienced any severe toxicity or active infection before adj-iftr. though our plan was to proceed with adj-ifrt within months post asct, finally it was delivered after a median of . ( - ) months; the median radiation dose was ( - ) gy. ten patients received radiotherapy in the mediastinum, in the abdomen/pelvis/ inguinal area in the neck, and in the left leg. the adj-ifrt was well tolerated. no patient experienced toxicity grade > and none required hospitalization. currently, after a median follow-up of ( - ) years, / patients are alive and well; the -years overall and progression free survival rates are % and % respectively. four patients died; due to relapsed disease and heavily pretreated patients due to secondary myelodyspalstic syndrome conclusions: in our study, the adj-ifrt in the early post transplant period demonstrated a safe and well-tolerated profile. taking into consideration the poor risk status of our patients (residual disease post salvage regimen or bulky disease at the time of relapse), the promising overall and progression free survival rates suggested that adj-ifrt post asct is also an effective approach. well designed trials are needed to clarify the role and the appropriate time of radiotherapy in the asct setting disclosure: no conflict of interest adverse prognostic impact of pre-transplant neutrophil/ lymphocyte ratio in lymphoproliferative disorders background: brentuximabvedotin(bv) is a chimeric anti cd igg antibody, conjugated to synthetic antitubulinmomomethylauristatin. bv is approved for the treatment of classical hodgkin lymphoma (hl) in relapse either after autologous stem cell transplantation (asct) or after two lines of combination chemotherapy in transplant ineligible patients. the aethera trial revealed increased pfs when bv is used as maintenance therapy for cycles in high risk patients after asct. however, this schedule is associated with a high cost and significant toxicity particularly in term of peripheral neuropathy. our primary objective is to assess the efficacy of cycles brentuximab as consolidation therapy after asct for relapsed/refractory (r/r) hl. secondary objectives include side effects, progression free survival (pfs), and overall survival (os). methods: this is a retrospective single center analysis approved by the irb of the american university of beirut medical center. we included in this study consecutive patients with r/r hl who underwent asct between and , and received bv consolidation post-asct. results: we identified consecutive adult patients with r/r hl treated with bv . mg/kg iv every weeks as consolidation therapy after asct. the indications for bv consolidation was primary refractory disease in patients ( %), early relapse in patients ( %) (after a median time of months; range, - ) andextranodalinvolvement in one patient ( %). the median number of lines of therapy pre-asct was (range, - ). the median time to bv initiation post-asct was days (range, - ). patients received a median of cycles (range, - ) of bv post-asct. after a median follow up of months (range, - ), five ( %) patients relapsed after asct. the median time to relapse was months (range, - ). median pfs and os were not reached. we did not observe any significant toxicities during or after therapy. conclusions: cycles of bv consolidation after asct seem to be safe and effective in preventing relapse, however our findings need to be confirmed with larger prospective studies. chemotherapy or who progress after autohsct is poor. despite introduction of novel agents like brentuximab vedotin (bv) or nivolumab, allohsct appears the most effective treatment option with curative potential. the goal of this study was to evaluate efficacy of allohsct for hl, including patients pre-treated with novel agents. methods: between years - , patients (including males) with hl were treated with allohsct in msc institute of oncology in gliwice, poland. median age was ( - ) years. median lines of preceding chemotherapy was ( - ); ( %) patients had been pre-treated with autohsct, ( %) with radiotherapy, ( %) with bv, ( %) -with nivolumab. disease status at allohsct was as follows: cr- , pr- , nr- . patients were treated with hsct from either hla-matched sibling donor (msd, n= ), unrelated donor (urd, n= ) or haploidentical donor (n= ). conditioning was myeloablative in ( %) cases. peripheral blood was used as a source of stem cells. results: all but one patient engrafted with median time of neutrophil recovery of ( - ) days. the incidence of grade - and grade - acute gvhd was % and %, respectively, while the incidence of chronic gvhd was %. the probabilities of os and pfs at years were % (+/- %) and % (+/- %), respectively. the incidences of progression and transplant-related mortality were % and %, respectively. the y pfs rates were % for msd, % for urd and % for haploidentical donors. in a univariate analysis pfs was affected by recipient gender (female - %, male - %, p= . ) and disease status at allohsct (cr - %, pr - %, nr - %, p= . ). in a multivariate model the disease status other than cr was the only factor associated with increased risk of treatment failure (reverse pfs) -hr= . , %ci . - . , p= . . neither donor type nor conditioning affected long-term outcome. conclusions: results of allohsct for patients with relapsed/refractory hl are determined by disease status at transplantation. efforts should be done to reduce tumour burden before transplantation, optimally to achieve cr. disclosure: nothing to declare background: brentuximab vedotin (bv), nivolumab and pembrolizumab have been assigned to chemorefractory hodgkin lymphoma treatment. impact of these agents on disease-free-survival after autologous stem cell transplantation (asct) remains under investigation. aim of the study is to compare bv-and nivolumab-treated patients with a control group. methods: clinical characteristics and outcomes of chemo refractory hodgkin lymphoma patients who underwent asct during - . results: a total of patients ( men; women, median age years old, - ) were treated with bv: pre-transplant, post-transplant and pre-and posttransplant. pre-transplant bv patients had primary refractory disease or early relapse in the majority ( %). post-transplant treatment occurred in the context of relapsed/refractory disease in patients; ( %) had an allogeneic stem cell transplant. among them, had additional chemotherapy and nivolumab, gaining a complete metabolic response. in the rest of patients, change of treatment due to eventual bv failure occurred. bv was administered as a maintenance treatment in patients. in six of them bv had already been administered pre-transplant as well. out of maintenance treatment patients, relapsed and subsequently received nivolumab. two patients died due to prior chemotherapy complications, whereas are currently on nivolumab treatment. pet-based response was available in patients, having a complete metabolic response (cmr) and a partial metabolic response. stable disease was achieved by ctbased response in the rest patients. no major toxicities were observed. one patient presented with grade asymptomatic hypothyroidism and one with grade anemia attributed to non-inflammatory upper gastrointestinal blood loss. in total, patients received anti-pd treatment, all post bv failure. with a median follow-up of . ( . - . ) months, -year overall survival (os) was . % in patients treated only with bv compared to . % in patients treated with additional anti-pd treatment (p= . , figure) . median os for patients treated only with bv was . months, whereas median os has not been reached for patients that received anti-pd treatment. conclusions: bv pre or post-transplant and anti-pd treatment post-transplant after bv failure have outstanding results in chemo refractory lymphoma patients. treatment sequence in allogeneic transplantation eligible patients remains to be further studied. disclosure: nothing to declare background: allogeneic hematopoietic cell transplantation (allo-hct) with reduced-intensity conditioning (ric) has been used in heavily pretreated lymphoma patients with the promise of decreased treatment-related mortality. despite improvements in outcomes of patients with lymphoid neoplasms, several new agents emerge as potential therapies. therefore, we aimed to describe our long-term experience in patients with hodgkin (hl), non-hodgkin lymphomas (nhl) and chronic lymphocytic leukemia (cll) post allo-hct. methods: in this retrospective study, we enrolled consecutive patients who underwent allo-hct for lymphoid neoplasms in our institution ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) . results: in total, patients (male:female= : ) aged ( - ) years, underwent allo-hct for hl (n= ), nhl (n= ) and cll (n= ). the majority of patients were diagnosed at stage iv ( %); % had bone marrow involvement and % had undergone autologous hct. most patients were heavily pretreated (median lines= , range - ), of them had received more than treatment lines and at the time of transplantation only had complete response, while had partial response and were refractory. according to disease-risk index (dri), patients were stratified at low (n= , . %), intermediate (n= , . %), high (n= , . %) or very high (n= , . %) category. among patients with hodgkin lymphoma, brentuximab vedotin was administrated in and of them were effectively bridged to allo-hct. all patients received ric, mainly fludarabine ( mg/ m )-cyclophosphamide ( g/ m ) in cll and nhl and thiotepa ( mg/kg)-fludarabine ( mg/m )-cyclophosphamide ( mg/kg) in hl from matched sibling (n= ), matched (n= ) or mismatched unrelated (n= ) donors. graft-versus-host disease (gvhd) prophylaxis consisted of cyclosporine or tacrolimus and mycophenolate mofetil or short-term methotrexate and additional low-dose antithymocyte globulin ( mg/kg) in unrelated donors. peripheral blood was the main cell source ( / ) and median number of cd + cells infused was . x /kg ( . - . ) . two patients succumbed to advanced underlying disease before engraftment; while the other engrafted successfully. median time until neutrophil and platelet engraftment was and days respectively. eighteen patients ( . %) developed acute gvhd (grade iii-iv, n= ), steroid sensitive in ( . %) and relapsed. one-year cumulative incidence (ci) of extensive chronic gvhd was . %, and patients required more than one additional line of immunosuppression (range - ). ten patients presented cmv reactivation successfully treated with antiviral medication and patient died from hsv encephalitis. with a median follow of years ( - years), -year os was . %, -year non-relapse mortality ci . % and year dfs %. there was no difference in survival according to original disease ( -year os for nhl= . %, hl= . %, cll= %%, p= . ). multivariate analysis revealed high and very high dri as the single predicting factor of os (hr . , ci . - . , p= . ), when assessing impact of disease, dri, prior treatment lines, gender and bone marrow infiltration at diagnosis. conclusions: our data suggest that ric allo-hct provides encouraging survival rates, potentially offering the chance of cure, with acceptable long-term mortality in selected high-risk patients with lymphoid neoplasms. dri that is mainly associated with disease stage at transplant independently affects survival. therefore, continued efforts are necessary for clinical application of novel agents aiming to lower disease stage pre-transplant. disclosure: nothing to declare results: six pts were identified, with a median age of years at diagnosis: five with hl nodular sclerosis and with lymphocyte depletion. the median number of therapeutic lines prior to allo-hsct was [ - ]; four pts were previously treated with brentuximaband two pts had been submitted to high dose chemotherapy with autologous bone marrow support. at the time of allo-hsct, pts had progressive disease (dp), was in partial response and in complete response (cr). five allo-hsct were performed with a related donor, of wich were haploidentical ( parents, sibling and descendant) and with an unrelated donor ( / ). prophylaxis for gvhd was performed with tacrolimus and mycophenolate mofetil (with post-transplant cyclophosphamide in haploidentical allo-hsct). on day + evaluations, pts had a cr and patient (pt) had dp. the median time to relapse after allo-hsct was of months. at the time of initiation of nivolumab, pts were under steroid therapy for disease control, without other immunosuppressive therapy. the median time between allo-hsct and the beginning of nivolumab was months. the initial dose was mg / kg (associated with corticosteroid therapy), escalated up to mg/kg biweekly, according to patient's tolerance. after the start of nivolumab, patients, with previous gvhd manifestations, presented a worsening of the cutaneous gvhd, which required an escalation of immunosuppressive therapy. as toxicity, pt had a grade pneumonitis, pt had a grade encephalitis/hypophysitis, pt had a grade pancreatitis, pts had headache (grade and ), pts had a grade - cutaneous reation. with a median follow-up of months since nivolumab treatment, the overall response rate was %: pt obtained cr and pts partial remission. nevertheless, there were deaths after the onset of nivolumab: pt at monts with dp and another one due to acute myocardial infarction at months. at the time of analysis, pts maintained response under nivolumab treatment (median cycles ) and pt had therapy suspended because of toxicity. conclusions: these results demonstrate the high probability of achieving response with nivolumab treatment in patients with rr-hl relapsing after allo-hsct, but adverse events of grade were frequent in this small group, and the treatment toxicity was significant. disclosure: nothing to declare background: intravascular large b-cell lymphoma (ivlbcl) is a rare form of large b-cell lymphoma with pathological findings of intravascular proliferation and/or sinusoidal involvement of lymphoma cells. according to their geographic distribution, ivlbcl could be dichotomized into asian and western variants. compared with the western variant, where skin involvement was common, the asian variant was reported to involve more frequently the liver, spleen and bone marrow, and hemophagocytic lymphohistiocytosis is more common in asian variant. diagnosis of ivlbl is still difficult because of the lack of overt lymphadenopathy and peripheral blood involvement. thus, timely diagnosis and immediate treatment remain as a challenge to improve outcomes for patients with the asian variant. therefore, we analyzed the clinical features and treatment outcomes of patients with the asian variant of ivlbcl. methods: we analyzed patients who were diagnosed with ivlbcl between and . all patients were treated with rituximab, cyclophosphamide, doxorubicin, vincristine, and prednisone (r-chop). results: forty-six patients were diagnosed with ivlbcl, and their median age at diagnosis was years (range: - years). male patients predominated (n= , %), and b symptoms were present in patients ( %). hepatomegaly and/or splenomegaly were observed in patients ( %), whereas lymphadenopathy was less common (n = , %). bone marrow and liver were the most commonly involved extranodal organs ( %, and %, respectively) and were the most common sites of biopsy for diagnosis in this study. all patients received r-chop as a first-line treatment after diagnosis with a median number of six cycles (range one to eight). at the end of treatment, patients achieved a complete response (cr), whereas eight patients showed progression. six patients died after the first or second cycle of r-chop, and the causes of death were treatment-related adverse events including cytopenia, infectious complications, and pulmonary hemorrhage. upfront asct was done for two patients including one patient with cns involvement at diagnosis, and these patients were still alive at the time of analysis without evidence of relapse. on the other hand, the outcome of six patients undergoing salvage asct after relapse was poor; thus, only one patient was alive. likewise, patients with disease progression at the end of treatment with r-chop showed dismal prognoses even after salvage chemotherapy except for one. at a median follow-up of . months ( % confidence interval, ci . - . ), the median overall survival was . months ( % ci . - . ). the treatment outcome of patients with the asian variant of ivlbcl is still not satisfactory. although upfront autologous stem cell transplantation might be effective for selected patients at high-risk of relapse, its role is still not clear, either. thus, further study should be warranted to develop more effective strategies for diagnosis and treatment. clinical trial registry: not applicable disclosure: nothing to disclare background: peripheral t-cell lymphomas (ptcls) are about % of non-hodgkin´s lymphomas usually with an aggressive clinical course and unfavorable prognosis.given their heterogeneity, consensus on the best first-line treatment and the role of autologous/allogeneic (asct/allosct) stem cell transplantation as consolidation is controversial. methods: we evaluated the overall survival (os), progression-free survival (pfs) and toxicities of a cohort of patients with ptcls submitted to asct/allosct intensification at our institution between january and july . os was calculated from the date of diagnosis until death. pfs was measured from transplant until relapse, progressive disease or last follow-up. os and pfs rates were estimated using the kaplan-meier method and compared with the log-rank test. results: twenty-six patients were identified, female ( %), median age was years (range: to ). ninetytwo percent of patients presented with advanced-stage disease at diagnosis (ann arbor stage iii or iv), % with b symptoms. according to the who classification, histologic ptcl subtypes included angioimmunoblastic tcell lymphoma (n = ); ptcl not otherwise specified (n = ); anaplastic large cell lymphoma, alk-negative (n = ); anaplastic large cell lymphoma, alk-positive (n = ); nodal peripheral t-cell lymphoma with tfh phenotype (n = ). extranodal nk/t-cell lymphoma, nasal type and primary cutaneous subtypes were excluded. the ageadjusted ipi (aaipi) was low/intermediate low in patients ( %) and intermediate high/high in patients ( %). twenty-seven transplants were performed ( asct, allosct); were consolidation in st response ( asct and allosct) with in complete remission (cr) and in partial remission (pr). nine transplants were performed as consolidation of nd response ( asct and allosct) with in cr and pr. in patient allosct was performed after asct, due to early relapse (< months). beam regimen was used in asct as conditioning and flumel in allosct. all patients engrafted, the median time to leukocyte recovery > , /μl was days (range, to ). four of the pts ( %), submitted to allosct had chronic graft versus host disease which was the most relevant complication of this analysis. considering the whole cohort, the median follow-up was . months (range, to ). the estimated -year os and pfs were % and %, respectively. seven patients relapsed ( early) all after asct, there were no relapses after allosct, however, the results were not statistically significant between the allosct and asct groups; the -year os rates were % and % (p = , ) and the year pfs rates were % and % (p = , ) respectively. for the all patients treatment-related mortality (trm) was , %; patients died, with progressive disease (asct) and for hepatic toxicity (allosct) before d+ . conclusions: the results of this retrospective study, taking into account the adverse risk profile of the population, suggest that autologous/allogeneic stem cell transplantation as an effective and safe option for the consolidation of patients with ptcls. these results need to be validated in prospective studies, including a larger number of patients. disclosure background: autologous stem cell transplantation is used as consolidation therapy in relapsed lymphoma patients. however, outcome of lymphoma patients relapsing after autologous stem cell transplantation is poor and allogeneic stem cell transplantation which can be curative is used in the transplant eligible patients in this setting. besides, allogeneic stem cell transplantation can be an option before autologous stem cell transplantation in some high risk patients. in this study, we aimed to compare the survival rates of lymphoma patients older than years of age and patients aged - who had undergone allogeneic transplantation in our center. methods: we collected the data of lymphoma patients older than years of age who had undergone allogeneic transplantation in our center and analyzed the results by grouping them into , namely the ones between - years of age and the ones over years of age. [[p image] . figure results: there were patients over the age of who had undergone allogeneic stem cell trasplantation with the diagnosis of lmphoma between and . of these patients were over years of age. patients had non-hodgkin lymphoma and patients had hodgkin lymphoma. the characteristics of the patients are summarized in table . patients' comorbidity indexes were calculated with augmented hct-ci which includes patients' pretransplant ferritin, albümin and thrombocyte counts as a variable. no difference could be found between groups regarding neutrophil and platelet engraftment times and comorbidity indexes. however, acute graft versus rate and documented bacterial infection rate during the hospitalization period were higher in the - years age group (p= , ). day mortality rate and non-relapse mortality rate were not different between groups. more importantly, progression free survival(pfs) and overall survival(os) of patients in the - years age group and over years of age group were not different (p= , ) (figure ) conclusions: in the present study, although the number of patients is low, we showed that lymphoma patients over years of age have similar outcomes and transplant related toxicity as the patients between to years of age. pfs and os were very close in this study. we think that this may be due to low relapse rate in the patients and high mortality rate in relapsing patients. in conclusion, allogeneic stem cell transplantation which has a curative potential may be employed in transplant eligible elderly lymphoma patients disclosure: nothing to declare background: follicular lymphoma (fl) histologic transformation consist on the development of an aggressive lymphoma, usually a diffuse large b cell lymphoma (dlbcl). histological transformation has been considered to have poor prognosis. in pre-rituximab era median os ranged between and years, however, in recent series of patients treated with chemotherapy plus rituximab, the outcome of transformed fl has improved, especially in those that receive autologous stem cell transplantation (asct), who reach -year os up to % in some series. methods: we have retrospectively studied consecutive patients undergoing asct for transformed fl between and in a tertiary center in the basque country, spain. patients were considered to have a transformed fl if they were diagnosed of a dlbcl and they have previous history of fl or histological evidence of a fl in another location. these patients were compared to a retrospective cohort of dlbcl patients with high ipi or stage that received asct in first remission according to our institution strategy. pfs and os were calculated from the time of the asct. in the case of transformed fl, both relapses of the aggressive or indolent lymphoma were considered. survival analysis was performed with kaplan-meyer estimator results: a total of transformed fl and dlbcl patients were studied, with a median follow up of . and . months respectively. patient characteristics are described in table . -year pfs was % in transformed fl and % in dlbcl, and -year os was % and %, respectively (picture ). there were no significant differences in pfs or os between this two groups (p = . ). in both groups all relapses occurred in the first three years after asct. among the patients with transformed fl relapses were observed. five of them ( %) were aggressive relapses, while only one patient presented relapse as an indolent lymphoma (fl histological grade a with an aggressive clinical course). [[p image] . image : transformed fl and dlbcl pfs after asct] conclusions: in our experience, asct in transformed fl offers good results, similar to those in dlbcl. fl presents a natural course akin to that of dlbcl, with relapses occurring early and survival reaching a plateau. this data suggests that some patients with transformed fl can be cured after asct. disclosure: nothing to declare. safety and efficacy of intensive preconditioning regimen containing cladribine in autologous peripheral blood stem cell transplantation of refractory and relapsed young highly invasive lymphoma background: autologous peripheral blood stem cell transplantation (apbsct) is one of the main treatments for patients with non-hodgkin's lymphoma (nhl). effective and safe conditioning regimens can improve the cure rate of nhl. beam is the most common pretreatment scheme, but for refractory and relapsed young highly invasive lymphoma, especially for dual-expression dlbcl, pretreatment needs to be strengthened. studies have shown that the cladribine (clad)+gemcitabine (gem)+busulfan (bu) combination provides synergistic cytotoxicity in lymphoma cell lines.we evaluated the the safety and short-term efficacy of intensive preconditioning regimen containing cladribine (clad+gem+bu) for refractory and relapsed young highly invasive lymphoma undergoing apbsct. methods: ten patients with nhl received apbsct. ca)ctx+ ara-c) therapy followed by g-csf was used for pbsc mobilization. sevenr patients received conditioning regimens of beam(beam group): bcnu mg/ m ·d - × d (- d), vp mg/m · q h× d (- d-- d), ara-c mg/m ·q h× d (- d-- d), mel mg/m ·d - × d (- d). three patients received intensive preconditioning regimen containing cladribine (clgb group): clad mg/m ·d - × d (- d-- d), gem mg/m ·d - × d (- d, - d), bu . mg/kg q h× d (- d-- d). follow-up date expires on december , . results: the age of patients in clgb group was , and years, respectively. two patients were diagnosed as diffuse large b-cell lymphoma with double expression and one was diffuse large b-cell lymphoma with two recurrences. the patients of beam group were all high-risk, relapsed and refractory nhl.all patients were successfully engrafted after infusing apbsc. the average lowest leukocyte in clgb group and beam group were ( . ± . ) × /l vs ( . ± . )× /l, respectively. the average lowest leukocyte in clgb group was lower than that in beam group. the average time to anc < . × /l in clgb group and beam group were . d ± . d vs . d± . d. the average time to anc≥ . × /l in clgb group and beam group were . d± . d vs . d± . d; the average time to plt≥ × /l of clgb group was not different to that of beam group ( . d± . d vs . d± . d) the average time of neutropenia wasn't significantly different in two groups ( . d± . d vs . d± . d). the adverse reactions of gastrointestinal tract and oral mucosa were close in tow groups.vod, hemorrhagic cystitis, pretreatment-related interstitial pneumonia, liver and kidney dysfunction were not happened in tow groups. the rate of infectious fever was close in two groups ( / vs / ). the median followup period in beam group was ( ~ ) months. in the beam group, a patient died days after transplantation, because he was diagnosed with recurrent nkt cell lymphoma and intracranial infection caused by severe sinus infection. another case of beam group was diagnosed as double-expressed dlbcl, which relapsed months after transplantation. the remaining patients in ebmt group survived disease-free. the follow-up time of patients in clgb group were months, months and months respectively. all patients survived without disease.however, the follow-up time is short and needs long-term follow-up. conclusions: the treatment of intensive preconditioning regimen containing cladribine (clgb) for refractory and relapsed young highly invasive lymphoma undergoing apbsct is safe. the time of hematopoietic reconstruction is short, and the adverse effects is tolerable for patients with refractory and relapsed young highly invasive lymphoma. the current short-term outcome is good, but the long-term effect need a longer time to follow-up. disclosure: this work was supported by national nature sciences found of china ( ). there is no disclosure of conflict of interest.the all authors name: xiang-li chen, yu-zhu zang, wen-hui zhang, yin zhang, zhong-wen liu, ping-chong lei, jing yang, yu-qing chen, kai sun. background: small part of children with hodgkin disease (hd) demonstrate initial resistance to the standard and even "salvage" chemotherapy and need innovative drugs for the treatment. methods: a -year girl was diagnosed with classical hd (nodular sclerosis)corresponding to stage ii e b (fever > °c) in april .after two cycles of oepa (vincristine, etoposide, prednisone and doxorubicin) and next two cycles copp (cyclophosphamide, vincristine, prednisone and procarbazine) the patient again had progressive disease. as the patient achieved a partial response (pr) after "salvage"therapy with two cycles of igev (ifosfamide, gemcitabine, vinorelbine, and prednisone), she received auto-sct in february (patient status before auto-sct was pr). we used ccnu-containing conditioning regimen cem: lomustine (ccnu) mg/m + etoposide mg/m + melphalan mg/m . at day + after auto-sct, the patient again demonstrated progression of the disease: pet/ct-examination showed mediastinal tumor mass enlargement with increased left lung nodule simultaneously to short metabolic activity. patient was under observation. at day + the disease had relapsed and progressed -examination by pet/ct demonstrated multifocal progressive disease with multiple pulmonary lesions and increased metabolism in comparison with the previous pet/ct scan. in july-october , the patient had salvage chemotherapy with a combination of brentuximab vedotin (bv) (bv dhap (dexamethasone, cytarabine, cisplatin) + bv (without chemotherapy due to suspected invasive mycosis) + bv dhap), however, only partial pet-positive remission was achieved. because of many times relapsed and progressed disease pembrolizumab therapy was started in october : mg / kg every three weeks four cycles totally. toxic effects and serious complications during and after therapy by pembrolizumab were not observed. in february , after pembrolizumab # , the patient showed complete metabolic remission of the disease by control pet-ct. in april , the patient received haplo-sct with post-transplant hd-cyclophosphamide. we used conditioning modes with reduced toxicity (fludarabin mg/m + treosulfan g/m ), high doses of cy ( mg / kg) on days + and + . tacrolimus and mycophenolate mofetil started on day + . mmf was terminated on day , tacrolimus -on day . patient did not have acute and chronic gvhd. results: at the moment the patient is alive and still in pet-negative cr with duration more than mo. conclusions: pembrolizumab has demonstrated high activity against resistant hd even after post-auto-sct progression with good tolerability for the sick child. disclosure: nothing to declare p high dose chemotherapy followed by autolougous peripheral blood stem cell transplantation (asct) in diffuse large b cell lymphoma (dlbcl) median age is , years ( to ) and sex ratio (m/f) . ; ann arbor stage iii-iv: pts. before hdt induction chemotherapy (chop, c h opa) was instituted and associated with rituximab in pts ( , %), pts ( , %) received more than treatment lines and pts ( , %) received complementary radiotherapy. transplant disease status before hdt was complete remission (cr) in pts, partial remission in pts (rp) and disease progression in pts. the delay from diagnosis to hdt is , months ( - ). the hdt protocols used are: tutshka : pts, tutshka+vp : pts, bam (busulfan +cytarabine+melphalan) : pts et beam : pts. all pts received, after thawing, mobilized pbsc obtained by g-csf mobilization ( μg/kg/d, days) alone and froze in liquid nitrogen. the median rate cd + cells infused is , x /kg ( . - . ) . the median follow-up at / / is months . results: the median time to graft (pnc > . x /l) was days ( - ). ten early deaths were observed including infection (trm: , %) and in disease progression at months. after months of hdt pts are assessable including pts in cr ( , %) and pts in pr ( , %). relapse was observed in pts ( . %) and it was earlier relapse in a period of months in pts ( %). deaths were among / pts ( %). persistent cr was achieved in / pts ( , %) including / ( , %) mlcl and / ( , %) others dlbcl. the overall survival (os) and event free survival (efs) at years are respectively % and %. the os and efs are better in patients who received rituximab in initial therapy : os ( % vs %; p< , ) et efs ( % vs %; p< , ). herein, we present one patient with refractory mcl, who were insensitive to chemotherapy and then experienced a dramatic improvement with ibrutinib mono-therapy but later developed ibrutinib resistance,ultimately resulting in the deterioration of disease and death. methods: we give the patient several examinations including ultrasound, bone marrow biopsy, lymph node biopsy, exome sequencing, sanger sequencing, and so on. for the treatment of lymphoma, the patient received chemotherapy, including course of chop(cyclophosphamide . g day , doxorubicin mg day ,vinorelbine mg day , and dexamethasone mg from day to ) and course of r-dhap (rituximab mg day , cytarabine g q day , cisplatin mg day ,dexamethasone mg from day to )in succession.because of the failure to control disease progression, ibrutinib mg qd was used until the patient died. results: the -year-old man initially referred to our hospital for complaints of abdominal pain and distention over months. ultrasound showed splenomegaly and multiple enlarged retroperitoneal lymph nodes.excisional biopsy conducted on the right neck lymph node revealed the presence of malignant cells.immunohistochemically, the neoplastic cells were positive for bcl , bcl ,cd , cd , cd a, cd , ki- ( %), sox , cd (fdc) and cyclin d and negative for cd , cd and cd ; fluorescence in situ hybridization(fish) showed igh/ ccnd ,t( ; ) %.thus a diagnosis of mcl was confirmed. course of therapeutic chemotherapy were applied to the patient but he did not respond well.he suffered recurrent fever, thrombocytopenia, left abdominal pain, splenomegaly and multiple enlarged lymph nodes. then he received ibrutinib mono-therapy, and experienced a dramatic improvement as his body temperature was controlled, his hemogram became normal and his spleen and lymph node tapered.after about months response of ibrutinib, the disease deteriorated rapidly and he died very soon. exome sequencing from the patient peripheral blood at this time detected one missense mutation in exon of tp at nucleotide g>a, resulting in an argnine to histidine change at amino acid (p.arg his). but sanger sequencing of the patient bone marrow ffpe sample at the time of original diagnosis did not detect this mutation. conclusions: thus, our study reported a tp r h mutation mcl patient who developed ibrutinib resistance and progressed aggresively, which may open new insight for future effort for alternative therapeutic strategies in ibrutinib-refractory mcl. disclosure: nothing to declare. minimal residual disease, tolerance, chimerism and immune reconstitution peripheral blood samples were obtained for routine analysis at several time points after hsct. all available blood samples between . and years were used in the analysis. to assess changes in the cd +b and cd +cd +memory b cell counts over time while accounting for the correlation between the repeated measurements of each patient, we used linear mixed-effects models. wilcoxon rank test, kruskal-wallis test and linear regression were used for univariate analysis. results: at one year after hsct, univariate analysis showed that patients transplanted with a cb graft compared to bm and pbsc had a significantly higher absolute number of b cells (median bm= , median cb= , median pbsc= cells/μl, p= . e- ) and memory b cells (median bm= , median cb= , median pbsc= cells/μl, . recipients with age under years had significantly higher absolute numbers of b (median= , median= cells/μl, p= . e- ) and memory b cells (median= , median= cells/μl, p= . e- ) than above years. increase in donor age was associated with a decreasing effect on b cell (r = . , p= . e- ) and memory b cell (r = . , p= . e- ) reconstitution as determined in regression analysis. following univariate analysis, we analysed these factors in a mixed effects model to assess the relation with differences in b cell or memory b cell numbers . - years after hsct. in our analysis we found significant decreasing b cell and memory b cell numbers with increasing donor age corrected for recipient age and source (both p< . ). increasing recipient age also showed a significant decrease in b cell and memory b cell numbers (both p< . ) but there was no significant influence of donor source ( figure ) . conclusions: b cell and memory b cell numbers after hsct are influenced by donor and recipient age but not by donor source. older donors and recipients show a decrease in b cells and memory b cells numbers . - years after hsct. [[p image] . figure . b cell development and donor age. green shows cb, red bm, blue pbsc at mean donor age.] copenhagen university hospital rigshospitalet, copenhagen, denmark, leiden university medical center, leiden, netherlands background: the outcome of allogeneic hsct is challenged by a delayed and long-lasting imbalanced t-cell reconstitution increasing the risk of acute gvhd, infections and disease progression. although the role of differentially and functionally distinct t-cell subsets in the development of complications has been addressed, little is known about the factors controlling their recovery. in this study, we investigated the impact of immuneregulating and homeostatic cytokines on the reconstitution of functionally distinct t-cell subsets and associated clinical outcomes. methods: we included children undergoing allogeneic hsct for all (n= ) or aml (n= ) with a median age of . years (range: . - . ). donors were either mrd (n= ), mud (n= ) or mmud (n= ). bm (n= ) or pb (n= ) were used as stem cell source. conditioning regimens were based on tbi (n= ) or highdose chemotherapy alone (n= ) and included atg in patients. thirty age-matched healthy children were included as controls. cytokines (il- , il- , il- , scf, il- , il- and tnfα) and active atg in plasma were longitudinally measured from before conditioning until months after hsct (n= ) along with an extended phenotyping of t-cell maturation and differentiation by flow cytometry (n= ). results: the homeostatic cytokines il- and il- increased from pre-conditioning to peak - weeks post-hsct and gradually declined thereafter. il- levels were shortly elevated, while il- and scf remained relatively stable, and il- and tnf-α levels were below threshold of detection at all time points. the early rise of il- and il- was strongly associated with the degree of t-cell depletion by atg, while il- also correlated with markers of systemic inflammation. il- and il- levels were significantly higher in children treated with atg (p< . ) and correlated with both longer exposure to atg (p< . ) and increased levels of active atg (day + : il- : r= . , p< . ; il- : r= . , p< . ), indicating that high levels of these cytokines reflected more pronounced t-cell depletion during lymphopenia. higher circulating levels of il- and il- were associated with a slow recovery of cd +, cd + and cd + t-cell counts at day + and + post-hsct (p< . ), while the remaining cytokines did not correlate with immune reconstitution. looking into t-cell subpopulations, increased levels of il- and il- during the first month post-transplant were associated with lower numbers of naïve t cells and correlated with an increased proportion of cd + and cd + effector memory cells ( figure) . no differential effect of cytokines on polarization of cd + t cells into th , th , th cells or treg cells was found. in atg-treated patients, il- and il- levels at day + were significantly lower in patients developing acute gvhd grade ii-iv (p= . and p= . , respectively). in the total cohort, increased il- levels were associated with a reactivation of ebv (p= . ). conclusions: these findings suggest that quantification of il- and il- can be indicative for the degree of t-cell depletion during the first weeks after hsct and predictive of complications. overall, these results indicate that the lymphopenia-induced elevation of il- and il- is a major driver of the initial expansion of donor t-cells. background: mathematical kinetic models were adopted to study immune cell reconstitution after allogeneic hematopoietic stem cell transplantation (allo-hsct). the associations between acute graft-versus-host disease (agvhd), relapse and the immune cell reconstitution kinetic models were explored. methods: from june , to may , , sixty-five patients with hematological malignancies after allo-hsct were recruited. peripheral blood was collected on + day, + day, + day and in + month, + month, + month, + month, + month, + month, + month. lymphocyte subsets were determined by flow cytometry, including in total t lymphocytes (cd + ), helper t cells (cd + cd + ), cytotoxic t cells (cd + cd + ), cd /cd ratio, nature killer (nk) cells (cd -cd + ), nkt cells (cd + cd + ), b lymphocyte (cd + ), naive t cells (cd + hla-dr + ), static t cells (cd + hla-dr -), and regulatory t cells (cd + cd high foxp + ). mathematical kinetic models were calculated for immune cell reconstitution with spss. results: after allo-hsct, a logarithmic curve model was observed for cd + t cells. cubic curve models were observed for cd + cd + t cells, cd + cd high+ foxp + t cell, cd + hla-dr -t cells, cd + cd + nkt cells, cd + b cells. cd + cd + t cells, cd + hla-dr + t cells, and cd -cd + nk cells showed s type curve models. considering t cells were the major mediators for agvhd and graft-versusleukemia effect after allo-hsct. with established immune cell kinetic models, we found that different curve models were observed between patients with and without agvhd after allo-hsct. although the kinetic models were almost the same for leukemia-free and relapsed patients in the first months after allo-hsct, significantly different kinetic curves could be observed thereafter. conclusions: the immune cell reconstitution showed different mathematical curve models after allo-hsct. kinetic reconstitution model of certain immune cell was associated with agvhd and relapse. hence, mathematical kinetic models of immune cell reconstitution may be potential indictor for predicting agvhd and relapse after allo-hsct. disclosure: nothing to declare lineage specific chimerism analysis in pediatric patients following allogeneic hematopoietic cell transplantation (hct background: the outcome of allogeneic hct is dependent on several variables that include patient age, disease and stage, cytoreduction, graft, graft manipulation, and graft versus host disease (gvhd) prophylaxis. one aspect of hct that remains poorly defined and studied is the donor/ host (d/h) chimerism post hct. since , we followed patients with d/h lineage specific chimerism post hct. analyses were performed by short tandem repeat (str) polymorphism analysis at the american red cross blood services (philadelphia, pa). studies were performed on blood total leukocytes, myeloid/neutrophil cells, t-cells, bcells, and nk-cells. methods: in this retrospective study, the charts of consecutive patients who underwent allogeneic hct between january to june on the pediatric bone marrow transplant service at mskcc were retrospectively reviewed. lineage specific donor chimerism post hct was studied including d/h chimerism trend, and factors with potential impact on chimerism including: age, disease, graft source, and t-cell depletion (tcd). preliminary analyzes performed on this cohort included wilcoxon rank test and cox proportional hazard analyses. results: patients were selected based on the number of analyses. the median age was . years. patients had hematologic malignancies (n= ) or non-malignant hematologic diseases (n= ), or immune disorders (n= ). cytoreduction included tbi-(n= ), or chemotherapybased regiments (n= ). patients were recipients of t-cell depleted marrow or peripheral blood grafts (n= ), unmodified marrow or peripheral blood grafts (n= ) or cord blood grafts (n= ). full donor chimerism of myeloid cells, b-cells and nkcells, but not t-cells occurred early post-transplant. there was no difference in the percentage of total donor leukocytes at months vs. months post hsct (n= ), while the median of donor t-cell chimerism was % at months and % at months post hsct (p< . , n= ). figure shows the impact of different factors including: (a) the use of tbi-or chemotherapy-based regimens, (b) age (< or > years), and (c) type of graft (t-cell depleted vs unmodified vs cord blood). donor total leukocytes chimerism was significantly lower at months as compared to months for patients < years of age (p= . ). for most grafts, full donor chimerism of t-cells occurred early, while for t-cell depleted transplants, it took up to one year to complete. cord blood grafts were associated with high t-cell donor chimerism throughout the post-transplant period. there was a significant difference in the % donor t-cells at and months post hct when comparing t-cell depleted and unmodified grafts (p= . ). conclusions: this preliminary analysis of lineage specific chimerism post-transplant showed that donor tcells may take one year to fully recover post-transplant, mostly following t-cell depleted grafts, without intervention. cord blood grafts were associated with high donor chimerism throughout the post-transplant period. lastly, factors associated with loss of donor chimerism posttransplant were younger age and non-malignant disorders. more in-depth analyses are being performed including the relation of chimerism and hct outcome. disclosure: eileen nicoletti -employee rocket pharmaceuticals, susan prockop -investigator atara biotherapeutics -research funding, susan prockop -mesoblast -research funding, nancy kernan -gentium -support; jazz pharmaceuticals -support, richard o´reilly -atara biotherapeutics -royalty, consultancy and research, jaap jan boelens -bluebird bio -consultancy, avrobio -consultancy; jaap jan boelens -chimerix -consultancy; magenta -consultancy background: the success of hematopoietic stem cell transplantation (hsct) lies with the ability of the engrafted immune system to remove residual leukemia cells via a graft-versus-leukemia effect. despite this, relapse remains the major cause of mortality among patients receiving hsct. one of the immune evasion mechanisms of leukemic cells to escape from donor t cell recognition in haplo-hsct is the genomic loss of the patient specific hla. it has been described in - % of acute myeloid leukemia (aml) and myelodysplastic syndrome (mds) relapses after haplo-hsct. the aim of this study was to analyze hla loss in a large cohort of patients who relapsed after t-cell replete haploidentical transplantation with posttransplant cyclophosphamide. methods: from december to september , patients with hematological malignancies who received a haplo-hsct were recruited. among them, patients presented a relapse after haplo-hsct. hla typing was performed by real-time pcr using hla-kmr kit (gendx, netherlands).nine patients were excluded from the analysis because the kit employed did not include the recipientspecific hla. thus, a total of relapse cases were analyzed. the analysis of chimerism was carried out using short tandem repeat pcr amplification (ampflstr sgm plus, thermo fisher, usa) with a sensitivity of %. results: genomic loss of the patient hla occurred in / patients ( %) ( table ) . these patients presented different hematological neoplasms. interestingly, patients presented lymphoid neoplasm ( acute lymphoblastic leukemia (all-t), dentritic cell leukemia (dcl) and hodking´s lymphoma (hl)). hla loss relapses occurred later than classical relapses ( vs. days). regarding the treatment received (table ) , four patients were studied retrospectively. three of them were treated with donor lymphocyte infusions (dlis) + chemotherapy or other drugs at the time of the relapse. the other patient did not receive any treatment. in the end, all patients died from disease progression. prospectively, we detected hla loss at relapse in other two patients. at the moment of relapse, the first case received brentuximab + haplo-hsct from alternative donor and the other case received daratumumab + haplo-hsct (pending). both patients are alive, the first one in complete remission (cr) and the second one in partial remission (pr). conclusions: the frequency of hla loss at relapse is similar in our cohort to what is described in the literature. hla loss has been identified in patients with lymphoid neoplasms, while this mechanism has not been previously described in such diseases. the analysis of this immune evasion mechanism should be implemented in the routine screening of patients transplanted from haploidentical donors in order to design effective rescue strategies. these treatments should not be based on dlis or second transplantation with the same donor, instead, alternative donors should be used. background: an adequate immune reconstitution (ir) is crucial to reduce transplant toxicity, relapse rate and mortality after allogeneic stem cell transplantation (allohsct). the aim of this, single center retrospective study was to investigate the correlation between the recovery of different lymphocyte subpopulations with the main transplant outcomes, including overall survival (os), disease free survival (dfs) and non-relapse mortality (nrm). methods: we analyzed the ir of adult patients (aml n= , all n= , mds n= , nhl n= , hd n= , cll n= , cml n= , mm, n= , mpn n= ) who underwent (allohsct) between january and march . median age at transplant was years (range . - . ) with male/female ratio of %. donors were hlaidentical siblings (n= , %), family haploidentical (n= , %), matched unrelated ( , %), mismatched unrelated (n= , %) and cord blood units (n= , %). the stem cell source was the bone marrow (bm) in patients ( %), the cord blood in ( %) and g-csf mobilized peripheral blood in ( %). the conditioning regimen was myeloablative in ( %) transplant, reduced intensity in ( %) and immunosuppressive in ( %). gvhd prophylaxis was based on calcineurin inhibitors in combination with methotrexate or mofetil mycophenolate. antilymphocytes immunoglobulins was used in patients ( %) (anti thymocytes globulin, atg sanofi-genzyme in or anti t-lymphocyte globulin, atlg -neovii biotech, in ). the peripheral blood lymphocyte subsets (cd +, cd +cd +, cd +cd +, cd + (b cells) and cd +cd + (nk) were analyzed by flow cytometry at , , , , and months after hsct. post-transplant engraftment was molecularly determined by vntr analysis. results: as detailed in table the proportion of full donor chimerism analyzed in the peripheral blood t lymphocytes improved progressively after transplantation and the same pattern was observed when the chimeric status was measured in bone marrow mononuclear cells. to favor the achievement of a full donor chimerism, dli were performed in patients starting at the median of days after transplant (range: - ). with a median follow-up observation of months (range - ), the one year os and nrm was % and %, respectively. at months after allohsct, the achievement of values higher than , and /μl for cd +, cd + and nk cells, respectively was significantly associated to a better os (figure ), dfs (p = . ), and to a lower nrm (p< . for cd + and cd +, p= . for nk). a better lymphoid reconstitution was observed after the use of either a sibling or a haplo donor than a matched unrelated or cord blood donors. the use of atg was significantly associated with a delayed cd + recovery but with a faster nk cells reconstitution. conclusions: at six months after allohsct, recovery of cd + and nk cells predicts survival. monitoring of immune recovery may help to guide pre and post-transplant treatment strategies. days infections and disease control. several groups have demonstrated the independent prognostic value of different lymphocyte subpopulations in hsct outcomes. posttransplant cyclophosphamide (pt-cy) effectively prevents gvhd after hla-haploidentical hematopoietic stem cell transplantation (haplo). the use of pt-cy in hla matched related (mrd) or unrelated (mud) donors hsct, although less explored, has also been introduced. the aim of this study was to compare the early immune reconstitution after allogeneic hsct from haploidentical and hla-matched donors using pt-cy. methods: one hundred and sixty-four hsct performed in our center were analyzed: haplo performed between and and hsct from hla-identical donors ( consecutive mrd sct performed with pt-cy between and and mud sct with pt-cy between and ). pt-cy was administered at mg/kg/d in days + and + postransplant, followed by mmf mg/kg/ d and a calcineurin inhibitor (ciclosporina a or tacrolimus) from day + ahead. we retrospectively compared early immune reconstitution at day + and day + among these three populations. early ir was assessed through the analysis of lymphocyte subpopulations including total t lymphocytes cd +, cd + and cd +subpopulations, nk cd -cd + cells, cd + bright immature subpopulation and total b cd + lymphocytes.. lymphocytes subpopulations were determined by multiparametric flow cytometry (fc and navios, beckman coulter®). results: all patients, but mud and haplo, received pb as stem cell source. patients received prior transplant in haplo group. patient´s characteristics are shown in table . patients who received hsct from mrd showed the fastest ir, with statistically significant differences compared to haplo in almost all lymphocyte populations at day + (cd +, cd +, cd + and nk cells), and also in cd +, cd + and b lymphocytes at day + . comparison between haplo and mud hsct showed better ir among haplo, demonstrated by higher counts in cd +,cd +, cd + and nk cell counts at day + . no differences were seen at day + . (figure ). percentage of immature cd bright nk cells was higher in mud hsct at + , with no differences between haplo and mrd hsct. conclusions: in our cohort of patients with pt-cy based gvhd prophylaxis regimen, those who received hsct from mrd showed the earliest immune reconstitution compared to haplo and mud at day + and + . haplo showed better ir compared to mud at day + . nk maturation at day + was a little better among haplo and mrd hsct recipients than mud hsct patients. in our experience, using mostly pbsc as graft source, type of donor influenced early ir in pt-cy based hsct, background: cell-free dna (cfdna) isolated from plasma or serum has received increasing interest for diagnostic applications. however, the reported clinical usefulness of cfdna in patients undergoing allogeneic cell transplantation (hsct) is scarce. methods: the chimeric status both in peripheral blood and in cfdna obtained from plasma was investigated in patients undergoing hsct. dna and rna were isolated from plasma within four hours of blood draw. patients were evaluated for chimerism at day + , + and + post-transplant. a panel of seven microsatellites was amplified by pcr for chimerism detection and pcr products were analysed by capillary electrophoresis. for further cfdna characterization the micro rna (mirna) c was analysed using digital pcr. mutations frequently used for minimal residual disease assessment such as flt -itd, npm and jak were also investigated in cfdna. results: the mean cfdna concentration in transplanted patients was ng/ml, while in healthy donors used as control group (n= ) was ng/ml. the mean cfdna concentration difference between both groups reached statistical significance (p= . ). when analysing cfdna from transplanted patients and in the control group we could not detect dna fragments larger than bp and the size range of the analysed fragments was between and bp. in out of patients a mixture of donor and recipient cfdna (mc) was detected. with the exception of three patients relapsing after transplant in which mc was detected both in peripheral blood and plasma in the rest of the patients (n= ) mc was detected only in plasma. the mean percentage of recipient cfdna in the plasma samples was % (range: - %). all the patients with acute gvhd (agvhd) (grade: i-iv) (n= ) showed mc in plasma at least in one of the time-point tested. no significant difference was found in the mean recipient cfdna percentage in patients with agvhd grade i-ii when compared with grade iii-iv. meanwhile in the group of patients with chronic gvhd (n= ) mc in plasma was detected in patients. in those patients with clinical improvement of agvhd (n= ) a decrease in the percentage of recipient cfdna was observed during treatment. in patients without improvement or even agvhd worsening (n= ) stable or increasing recipient cfdna percentage was detected. since recipient cfdna can be detected in patients without transplant-related complications we analysed the mirna c expression in all patients with recipient cfdna. a significant difference was found in the mirna c expression in patients with agvhd (mean mirna c: . mirna c copies/ u copies) when compared with patients without gvhd (mean mirna c: . mirna copies/ u copies). in those patients with extramedullary aml relapse (n= ) frequent mutations (flt -itd, npm ) were only detected in the cfdna fraction. conclusions: longitudinal analysis of cfdna represents a useful complementary tool in particular for those patients with clinical complications after hsct. disclosure: nothing to declare comparison of the impact of atg/pt-cy-based and tcr αβ-depletion as gvhd prophylaxis regimens on the recovery of memory t-cell compartment background: over recent years haploidentical and hlamismatched donors have been increasingly adopted as a valid donor source. modern graft-versus-host disease (gvhd) prophylaxis regimens such as drug-based (antithymocyte globulin (atg), post-transplant cyclophosphamide (pt-cy)) or graft-manipulated (tcr αβ-depletion) demonstrate effective prevention of gvhd. here we report our data about an influence of different gvhd prophylaxis regimens after allo-hsct with pbsc as a graft source on cd + memory t-cells recovery. methods: our study comprised leukemia patients who underwent allo-hsct with pbsc as a graft source in national research center for hematology, moscow, russia. detailed patients characteristics are presented in table . peripheral blood samples were collected on day + , + and + after allo-hsct. flow cytometry analysis was performed on bd facs canto ii (becton dickinson, usa) to define t-memory subsets: t-naive and t-stem cell memory (tnv+tscm) -cd +cd r -ccr +cd +; t-central memory (tcm) -cd +cd r +ccr +cd +; t-transitional memory (ttm) -cd +cd r +ccr -cd +; t-effector memory (tem) -cd +cd r +ccr -cd -; t-terminal effector (tte) -cd +cd r -ccr -cd -. sysmex xe- was used to calculate absolute count of different t-memory cell subsets. mann-whitney u test was used for nonparametric data analysis. a p-value less than . was considered as significant. results: results of mann-whitney u test (calculated pvalues) to compare absolute number of t-memory cell subsets in terms of different gvhd prophylaxis regimens are presented in figure . during all follow-up period the number of tnv+scm and tcm remains significantly reduced after atg+pt-cy or tcr αβ-depletion compared to atg-based immunosuppressive regimen. on day + we observe no difference in the number of tnv+scm and tcm cells after atg+pt-cy or tcr αβ-depletion. terminally differentiated cd + cells (ttm, tem, tte) count is significantly lowered in tcr αβ-depletion patients group in comparison to atg+pt-cy. nevertheless recovery of tnv+scm and tcm after pt-cy is faster than after tcr αβ-depletion. conclusions: according to our data the mechanism of pt-cy is seems to be more selective compared to tcr αβ-depletion due to its transient impact just on tnv+scm and tcm without affecting on the effector pool. through this it may lead to delayed reconstitution of adaptive immunity after tcr αβ-depletion compared to using pt-cy. clinical relevance of the quantitative characteristics of immune recovery in the context of different approaches to gvhd prevention remains to be established. background: immune effector cells, belonging to either innate or acquired immunity, play a key role on preventing disease relapse after allogeneic haematopoietic stem cell transplantation (hsct). most of known immune effector are cd + cd + t-cells and cd -cd + natural killer lymphocytes, while cd + cd + cells act as modulatory and regulatory cells. the early post-hsct ratio between these cellular subsets may be an indicator of graft vs-tumor (gvt) effect. methods: we retrospectively revised the immune recovery of allogeneic hsct performed at our institution from to , analysed on peripheral blood by multiparametric flow cytometry lymphocyte subpopulations panel. diagnosis were acute leukemias ( %), chronic myeloproliferative neoplasms ( %), lymphomas ( %), myelodysplastic syndromes ( %), multiple myeloma ( %) and severe aplastic anemia ( %). we established early time-points of evaluation, and days from the graft infusion, to analyse the differences in disease free survival (dfs) and overall survival (os) between patients according to the cd + cd + x cd -cd + / cd + cd + ratio. results: median ratio at + days was of , . at this time point, patients who showed the ratio higher than the median had both a better dfs (median dfs time not reached vs months; p = , ) ( figure ) and os (median os time not reached vs months; p = , ). likewise, ratio at + showed an advantage on dfs (p = , ), and not on os (p = , ). other factors possibly affecting both dfs and os were analysed in univariate analysis, such as the use of antithymocyte globulin (atg), conditioning regimen intensity, graft source, hla-matching and disease status at hsct, the latter being the only variable with a significantly detrimental impact on both os and dfs. disease status was confirmed an independent valriable associated with both dfs and os as well as + ratio both on dfs (hazard ratio [hr] - , ; p = , ) and os (hr , ; p = , ). conclusions: our data show that cd + cd + x cd -cd + / cd + cd + ratio assessed at + is and independent predictor of transplant outcome, possibly representing a row indicator of anti-leukemic immune surveillance. the integration of this index with other known outcome predictors may help in improving the management of post-transplant phase. [[p image] . figure background: allogeneic stem cell transplantation (alo-hsct) is a curative treatment but it is associated with lifethreatening complications. most deaths are due to relapse, graft versus host disease (gvhd) and infection. the pattern and quality of the immune reconstitution (ir) after transplantation may affect these outcomes. however, there are limited data on the association of the quality of the ir and either the development of gvhd and survival. methods: eighty-five patients who received a non t-cell depleted alo-hsct in our center from to were prospectively studied. most patients received hla-identical grafts. total cd + and cd + t cells, ccr +cd + and ccr +cd + (which include both naïve and central memory t cells) and naïve ccr +cd l+ t lymphocytes were quantified by flow cytometry. data were collected at days + , + , + , + and + after alo-hsct. the association between ir and the gvhd was studied through an anova. for the multivariate analysis, a logistic regression was performed including those confusing clinical variables that were significant in the univariate analysis (p≤ . ). the study of overall survival (os) versus ir was performed with a cox regression model. results: total cd + t lymphocytes reached normal numbers within the first two months. median t cd + count was cells/ul after one month, which is within the normal range. conversely, it took nearly one year to get normal counts of cd + t cells ( cells/ul). the only two clinical parameters conditioning a worse recovery of the cd + t cells were the previous alosensitization of the donor and the sex, being female donor and male recipient the worst combination for the ir. no parameters influenced the quality of the reconstitution of cd + t cells. of note, the age or the hla status did not influence the quality of the ir. when the patients were divided into gvhd and no gvhd, we found no differences in the recovery of either the proportion or absolute count of every t cell subpopulation, including total t cells as well as naïve/central memory t cells, both cd + and cd +. finally, a multivariant analysis confirmed that the absolute counts of cd +ccr + t cells at day + as well as the absolute counts of both cd +ccr + t cells and naïve cd +ccr +cd l+ at day + were associated with better os. conclusions: in conclusion, neither the development of gvhd nor other relevant parameters seem to play a determinant role in the quality of the ir. to our knowledge this is the first study which demonstrate a clear association between the recovery of naïve cd + t cells measured by flow cytometry and the os. disclosure: nothing to declare p abstract already published. azacitidine (aza) for prophylaxis or pre-emptive therapy for myeloid neoplasms after allogeneic stem cell transplantation whom were treated prophylactically and preemptively. median age was years [range, - ] and all patients had a diagnosis of aml or high-risk mds. prophylactic treatment consisted of aza mg/m for days in cycles of days. in the pre-emptive setting, patients received mg/m for days per cycle and patients mg/m for days per cycle. a median of cycles [range, [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] was administered in the prophylactic group and of cycles [range, - ] in the pre-emptive group. ten patients also received at least one dli after the third aza cycle: patients in the prophylactic group and patients in the pre-emptive one. results: during follow-up, patients had significant delays in treatment plan due to transitory mild complications. however, % of patients (n= ) presented infectious complications requiring hospitalisation and % of patients (n= in the prophylaxis group and in the pre-emptive group) presented some form of gvhd. in patients who developed gvhd, had to discontinue treatment (all in the prophylaxis group); also patients discontinued treatment due to disease progression. the overall drop-out rate was . % (n= ). survival was analysed from initiation of treatment with aza and median follow-up was months. one-year efs was % in the prophylaxis group, with only one patient relapsing and no deaths. in the pre-emptive group, the -year efs was % and the median efs was months; -year os was % and median os was months. conclusions: we conclude that post-transplant aza treatment is a well-tolerated therapy, but the incidence of side effects remains discordant in the literature. results in the prophylaxis group are excellent, but patients with positive minimal residual disease treated pre-emptively had a lower outcome with only stabilisation of the disease. randomised prospective trials are needed to define patients who would benefit the most from this treatment and at what timing, dosage and duration of treatment. disclosure: nothing to declare. abstract already published. results: all subjects experienced hematopoietic engraftment at a median of days (range - ) and demonstrated full donor myeloid chimerism. m-mdsc and pmn-mdsc recovery peaked at a median of days posttransplant. the median peak absolute m-mdsc count was , cell/ml (range , - , /ml) representing a range of . % to . % of pbmcs. the pmn-mdsc peak was more robust, with a median absolute peak of , cells/ml (range , - , /ml) representing a median of . % of pbmcs (range . - . %) . of note, the one patient who developed severe, life-threatening gvhd had the lowest absolute and relative pmn-mdsc recovery ( , cells/ml and . % of total pbmcs). recovery of m-and pmn-mdscs occurred at a similar tempo and magnitude in two recipients of standard gvhd prophylaxis (tacrolimus/methotrexate). however, while mdscs isolated from ptcy recipients exhibited clear t-cell suppressive capacity, those from the comparison patients did not (see figure) . conclusions: mdscs recover rapidly and robustly after allohct using ptcy as gvhd prophylaxis, and may play a role in mitigating gvhd risk by mediating t-cell suppression. this may be a mechanism by which ptcy results in donor-recipient tolerance. background: high dose chemotherapy followed by autologous stem cell transplantation (asct) offers a cure in the upfront and relapsed setting in both hodgkin (hl) and non-hodgkin lymphoma (nhl). asct also remains standard of care in previously untreated multiple myeloma (mm) patients after induction therapy, if eligible. the availability of new cellular or other immune therapies that can be used after asct underscores the potential importance of monitoring immune reconstitution after asct. methods: immune reconstitution panels (irp) were evaluated retrospectively in all lymphoma and mm patients over a -year span ( - ) whom underwent asct at our institution. patients were included if they had a pre-asct measured within days of asct and two other irp at any of the following timepoints ( ) day - , ( ) day - , and ( ) at -year post-asct. patients in the lymphoma cohort had their irp excluded if they had additional treatment within the first year post-asct (other than maintenance rituximab). mononuclear cells from peripheral blood were analyzed by flow cytometry for assessment of lymphocyte phenotype and numbers. absolute values were compared using the mann-whitney u test. results: the data on patients were available for analysis ( mm, nhl, hl) . all lymphoma patients were conditioned with beam. all mm patients were conditioned with a standard high dose melphalan regimen. the median pre-asct absolute cd counts in the lymphoma cohort were significantly lower than the mm cohort at cells/μl vs cells/μl, respectively (p= . ). however, the mm cohort exhibited a greater percent reduction in cd cells on day at . % vs . %, respectively which continued through day at . % vs . %, respectively. this led to nonsignificant changes in absolute cd count by day at cells/μl vs cells/μl, respectively (p= . ) (figure ). the median absolute cd count pre-asct for mm and lymphoma cohorts were cells/μl and cells/μl, respectively (p= . ). similarly, a greater percent reduction in cd cells led to comparable absolute counts on day at cells/μl vs cells/μl, respectively (p= . ). the failure of post-asct cd reconstitution to pre-asct levels was driven by lack of cd + cell recovery, namely cd +cd ra+ cells with a median of cells/μl and cells/μl in the mm and lymphoma cohorts, respectively at day (figures and ). this led to markedly diminished cd :cd ratios through day (figure ). [[p image] . conclusions: impaired t-cell reconstitution in both lymphoma and mm continues through -year post-asct. as shown, a larger percent reduction in median cd and cd counts through day was appreciated in mm compared to lymphoma leading to the nonsignificant differences in the post-asct absolute counts despite significantly higher pre-asct counts in the mm cohort. impaired recovery of cd t-cells may increase the risk of opportunistic infections, decrease the response to vaccination and lead to ineffective anti-tumor response. further prospective and larger retrospective studies like this should continue in the modern-era as they may help predict responses to further interventions requiring a robust t-cell repertoire for maximal efficacy such as car-t cell and bite therapies. disclosure: nothing to declare p peri-transplant detection of measurable residual disease by multicolor flow cytometry is highly predictive for relapse in acute myeloid leukemia patients background: presence of measurable residual disease (mrd) prior to allo-sct has been shown to be predictive for survival in patients in hematological cr of aml. in this study we analyzed the impact of mrd in such patients measured by -color multiparameter flow cytometry (mfc) prior to and on day + post-transplant. methods: the bone marrow samples immediately prior to allo-sct and on day + post-transplant were retrospectively analyzed. mrd evaluation was carried out with antibodies against: ( ) results: a number of aml patients (male, n= ) with median age of years ( - ) in hematological cr prior to allo-sct were enrolled in the study. we observed lower survival in patients with mrd by mfc pre-transplant ( y os: % ( - %) vs. % ( - %), p= . ) due to increased relapse incidence ( % ( - %) vs. % ( - %), p= . ). in multivariate analysis, mrd pos prior to allo-sct has strong significant impact on os (hr . ( . - ) , p= . ). of patients, a sample both before and on day + after transplantation was available in patients. of those patients, ( %) were mrd negative prior to transplant and on day + (mrd neg/neg ); ( %) patients were mrd positive prior to transplant and negative at day + (mrd pos/neg ); and ( %) patients were mrd positive at both timepoints (mrd pos/pos ). dfs and os for these three groups were as follow: y dfs: mrd neg/neg : % ( - %), mrd pos/neg : % ( - %); mrd pos/pos : % ( - %, p= . ); y os: mrd neg/neg : %; mrd pos/neg : % ( - %); mrd pos/pos : % ( - %, p< . ). upon multivariate analysis, the mrd status prior to transplant and on day + showed strong significant impact on dfs (hr . ( . - . ), p= . ) and os ), p= . ). we did not observe any significant impact of other factors included in the multivariate analysis (patient's age, patient's sex, and recipient/ donor sex constellation). conclusions: mrd positivity prior to allotransplant and at day + by mfc is highly predictive for survival after allo-sct. disclosure: nothing to declaire background: immune reconstitution is a critical factor for risk assessment of life threatening infections and long-term survival in patients undergoing allogeneic cell transplantation (hsct). methods: immune cell subsets (cd , cd , cd , cd , cd +cd -) were quantified by flow cytometry. trec and krec were quantified simultaneously using droplet digital pcr (dpcr). a total of patients were evaluated. mean age at transplant was years (range: - years) samples were obtained before hsct and at day , and after hsct. results: absolute numbers of cd and cd cells remained below pre-transplant levels until day , increasing further and eventually reaching pre-transplant levels one year after hsct. absolute counts of cd and cd +cd -cells remained below pre-transplant levels beyond one year after hsct. cd cells were characterized by fast reconstitution kinetics, reaching pre-transplant levels already at day . b cells correlated with krec levels at all time-points tested, whereas t cells correlated with trec levels only one year after transplantation. when we compared cell subsets, trec, krec levels and the reconstitution kinetics thereof between patients with reduced intensity conditioning (n= ) or full conditioning (n= ) no significant differences were observed. patients with pre-transplant trec levels above the mean ( trec copies/ml blood) showed higher trec levels and a faster t-cell reconstitution after hsct suggesting that tcell reconstitution can be predicted by analysing thymic functionality before transplantation. indeed, in patients with a pre-transplant trec above trec copies/ml blood, the positive predictive value for an efficient t-cell reconstitution was . (p= . ). we analysed the recovery kinetics of the cell subsets, trec and krec levels in patients with and without transplant-related complications. patients with either acute graft-versus-host disease or severe infections showed a slower trec reconstitution when compared with patients without complications. conclusions: our data suggest that the analysis of immune cell subsets together with trec and krec quantification can be used to evaluate the immune reconstitution process after hsct. pre-transplant trec levels allow t-cell reconstitution efficiency prediction after hsct. disclosure: nothing to declare background: falling donor / mixed chimerism after allogeneic haematopoetic stem cell transplant (sct) is associated with an increased risk of relapse and the potential for graft rejection. donor lymphocyte infusions (dli) are often administered in patients with mixed chimerism to achieve full donor chimerism but there is little data on long term outcomes for dli given for persistent mixed chimerism. methods: a retrospective analysis of all patients administered dli for mixed chimerism between to january was performed. all patients were transplanted at the university hospital of wales within the south wales blood and marrow transplant (swbmt) programme. patients were identified by the swbmt database and additional outcome data gathered by review of patients' medical records. results: patients were treated with donor lymphocyte infusions between and january . thirty one patients treated for relapse (with or without mixed chimerism) were excluded as was a further patient with a mismatched donor. the rest were / match. twenty six patients received a total of donor lymphocyte infusions for mixed chimerism alone. the median age was years (range: - ) with % women. fourteen ( %) of the patients had sibling donor transplants and twelve ( %) from matched unrelated donors. indications for transplant were: for aml or saml (n= ), myelofibrosis (n= ), mds (n= ), hodgkin lymphoma (n= ), non-hodgkin lymphoma (n= ) and all (n= ). escalating doses of donor cd + t cells were administered commencing at × /kg to × /kg then increased at half log increments according to chimerism results until full donor chimerism was achieved. the median number of doses administered was (range - ). the median interval was days (range - ). the median dose was × / kg (range × - × ). seventeen patients ( %) achieved full donor chimerism and remained so until most recent follow up (median months, range - ). one patient continued to receive dli after the study period and later reverted to full donor. two patients had ongoing mixed chimerism with no evidence of relapse. two patients relapsed; one of whom later achieved remission. there were six cases of gvhd; acute gvhd (grade ii n= , grade iii n= ) and cases of chronic extensive gvhd. one patient had gvhd features consistent with overlap syndrome. a total of five patients died, four due to infection (one in a patient with gvhd) and one due to cardiac toxicity from previous treatment (confirmed on post-mortem). conclusions: the results of our single centre study help reinforce the evidence for dli in establishing full donor chimerism when mixed chimerism is detected in the absence of relapse. incremental dli dosing is an effective strategy and associated with a low relapse rate. caution should still be given to the risk of gvhd following dli, however the risk appears to be low in this study. larger prospective studies are ongoing to address the optimal dosing strategy for dli post-transplant. disclosure: nothing to declare hypomethylating agents for the treatment of relapsed acute myeloid leukemia after allogeneic blood stem cell transplantation: a single center experience mariarita sciume , giorgia saporiti , elena tagliaferri , nicola fracchiolla , federica grifoni , giorgia levati , luca baldini , francesco onida the post-transplant period with well-balanced profile of good efficacy and moderate toxicity. we retrospectively evaluated the safety and efficacy of hma +/-dli in a reallife cohort of aml patients relapsing after allo-sct. methods: data from all patients with aml who underwent allo-sct at our institution in the last years and subsequently received hma as a salvage treatment for disease recurrence or preemptively for loss of complete donor chimerism were collected. results: eleven patients with a median age of years (range - ) were identified; median time between allo-sct and time to hma therapy was months (range - ). according to eln genetic risk stratification, patients were classified in the favorable group, in the intermediate-i, in the intermediate-ii and in the adverse one. six patients were treated with aza, whereas the remaining patients with dac. the cycles were repeated every days. ten patients ( %) started hma for morphological aml relapse, while one patient received aza as a sequential treatment after dli administered for loss of complete donor chimerism. median number of cycles was (range - ). treatment strategy included combination with dli in patients ( in the dac cohort, in the aza cohort), while in one case of flt -itd + aml sorafenib was also associated to dac and dli. no grade / toxicities and no acute gvhd occurred. a clinically significant response was observed in four patients ( %), all receiving at least cycles of hma therapy; in particular, a complete remission (cr) was achieved in / patients treated for morphological relapse, including the one who received the dac/dli/sorafenib combination and one (favorable eln risk) who received aza alone (not eligible for dli due to a concomitant lateonset cutaneous grade gvhd). of interest, the latter patient also displayed a resolution of the cutaneous gvhd. full donor chimerism recovery with no gvhd was observed in the patient who received aza for the progressive donor chimerism loss not responding to dli alone. with a median follow-up of months (range - ), the median os from hma treatment in responding patients was months (range - ); at the time of data collection responses were maintained in all four patients. seven patients had died, six from aml progression and one for severe intestinal gvhd occurring after failure of dli+aza and a following salvage induction chemotherapy treatment. conclusions: although arising from a limited number of patients, our real-life experience of treatment with hmas +/-dli in aml patients relapsing after allo-sct showed a general very good safety profile and promising antileukemic activity, altogether suggesting a facilitation of the graftversus-leukemia effect (gvl) associated to a possible suppression of the gvh reaction. disclosure: nothing to declare conclusions: in this study, cd -positive cell count and igg value had recovered about months after sct. in our institute, we have achieved a low incidence of infection by education and medication for patients until recovery of cd -positive cell count and igg. however, we found a higher incidence of infection after recovery of cd -positive cell count and igg. at - months after sct, administration of prophylactic medications such as sulfamethoxazole-trimethoprim were terminated and social comeback such as return to school or work were achieved in most patients. it is possible that the high incidence of community-acquired infection was associated with their comeback. thus, we should consider additional prevention of infection for patients in this period and further evaluation of immunological markers is needed. disclosure: no potential conflicts of interest were disclosed. effect of minimal residual disease before transplantation on the outcome of haplo-identical hematopoietic stem cell transplantation for high-risk acute lymphoblastic leukemia yehui tan , sujun gao , xiaoliang liu , long su , wei han , yu liu , yangzhi zhao background: to analyze the effect of haploid hematopoietic stem cell transplantation (hid-hsct) on high-risk acute lymphoblastic leukemia (all), and to explore the effect of minimal residual disease (mrd) before transplant on the prognosis. methods: a retrospective analysis was made on high risk all patients accepted hid-hsct in our hospital from january to january . the clinical features, stem cell implantation, complications, survival and recurrence were compared between pre-transplant mrd + and mrdpatients. results: all the patients got successfully implanted. the overall survival (os) was . %, the disease free survival (dfs) was . %, the incidence of acute graft versus host disease (agvhd) was . %, including . % ii~iv degree agvhd and . % iii~iv degree agvhd. there was no significant difference in stem cell implantation, gvhd, cytomegalovirus and hemorrhagic cystitis between mrd + and mrdpatients. dfs and os in mrd + patients were significantly lower than those in mrd -patients, and the cumulative rr rate increased significantly, there was no significant difference in cumulative trm. conclusions: hid-hsct was an effective method to treat high risk all, but mrd + patients had high recurrence rate and poor prognosis. strategy adjustment should be considered to reduce tumor residual and the transplantation strategy should be optimized for these kind of high risk patients, so as to improve survival rate. disclosure: nothing to declare background: lymphocytes are responsible for the cellular and humoral immunity and, consequently, its recovery after allo-hsct might be linked with the survival after the procedure. the aim of this study was to analyze this hypothesis in our series of patients. methods: all the allo-hsct performed in our center from january through july were included in the analysis. median age was years (range: - ). pts were male ( , %) and were female ( , %). baseline diseases were: aml, lpd, mds, all, mpd, mm, and bmf. donor was unrelated in ( , %), and was family in cases ( , %) (including haplo-identical). stem cell source was pb in ( , %) and bm in pts ( , %). conditioning regimen was reduced in procedures ( , %) and intensive in ( , %) (including just one non-myeloablative). overall mortalities at days + and + (the latter in patients with follow-up superior to year) were , % and , %, respectively. median follow-up was months (range: - ). evolution of absolute lymphocyte counts (alc) and subpopulations at pre-hsct and during the first year after allo-hsct were analyzed. results: as shown in table , alc and cd + lymphocytes decreased after conditioning therapy, and recovered progressively during the post-hsct period. at day + , majority of patients had > alc/mcl, clearly improved compared to admission values. cd + lymphocytes at day + was still very low, but at day + around half of the series had - /mcl. we found a strong link between alc, cd + lymphocytes, and cd + lymphocytes at days + and day + with overall survival at day + of the series (table ) . conclusions: in our series, immunity recovery was a late event for majority of patients undergoing allo-hsct. in addition, in our experience, the precocity and quality of the alc, cd +, and cd + cells recovery was clearly linked with long-term survival. background: the reconstitution of t and natural killer (nk) cells after hematopoietic stem cell transplantation (hsct) strongly influences the outcome of hsct including viral infection and graft versus-host disease (gvhd). the purpose of this study was to investigate the clinical efficacy of immune reconstitution including t and nk cells after hsct in children. methods: we reviewed the records of patients who undergoing allogeneic hsct in department of pediatrics, pusan national university children's hospital, from january to july . the counts of t lymphocyte subsets and nk cells was monitored in peripheral blood by flow cytometric technique during , , , and months post-hsct. blood samples for cytomegalovirus (cmv) and epstein-barr virus (ebv) monitoring were tested by real-time pcr assay. results: for total of patients, the mean age was . years (range, months- years), of the patients were boys and was girl. out of a total patients without pre-hsct cmv viremia or cmv infection, ( . %) recipients experienced cmv infection. the number of cd + t cells in and months post-hsct was significantly higher in patients with cmv reactivation compared to patients without (median . /μl vs. . /μl, p= . , and . /μl vs. . /μl, p= . ) . in ( %) recipients presented acute gvhd, the number of cd + t cells in and months post-hsct was significantly lower in patients with acute gvhd compared to patients without (median . /μl vs. . /μl, p= . , and . /μl vs. . /μl, p= . ) . the number of nk cells in months post-hsct was significantly lower in patients with cmv reactivation and acute gvhd compared to patients without ( . /μl vs. . /μl, p= . , and . /μl vs. . /μl, p= . , respectively) . in multivariable analysis, acute gvhd was shown to be the decisive factor influencing total t cells (p= . ) and cmv reactivation was independently associated with cd + t cells (p= . ). the cd + t cells counts were associated with prior hsct history and acute gvhd (p= . and p= . ), and the cd + t cells counts were also significantly associated with donor type (p= . ). conclusions: overall, our study documents that immune reconstitution of cd + , cd + t cells and nk cells is strongly associated with cmv reactivation and acute gvhd. additionally, we show that acute gvhd is influenced by lack of sufficient numbers of nk cells as well as cd + t cells early after sct. cd + t cells, on the other hand, significantly increase after cmv-reactivation and most likely play an important role in reactivation. disclosure: nothing to declare background: curative effect of allogeneic hematopoietic stem cell transplantation (allo-hsct) depends on the alloreactive t-cell immune response toward residual malignant cells -graft-versus-leukemia reaction. however, alloreactive population has not been phenotypically defined. recent studies suggest that alloreactive t cells express both costimulatory and inhibitory receptors simultaneously. exhaustion caused by the inhibitory signaling dampens tcell functionality, which could lead to the disease relapse. here we aimed to investigate the expression of costimulatory and inhibitory receptors on antigen-experienced t cells after transplantation, to isolate subpopulation specific for allo-hsct patients and analyze their t-cell receptor (tcr) repertoire. methods: expression of coinhibitory and costimulatory molecules on pbmcs patients at various time points after allo-hsct was analyzed for expression of: cd , cd , cd , cd ra, ccr , cd , cd , cd , klrg , tigit, pd , cd and ox by flow cytometry and compared to healthy donors. cd +cd +cd -cd +cd +pd +tigit+ fraction and cd + cd + control fractions were separated on facs aria ii cell sorter. double barcoded cdna libraries of tcr beta-chains for both fractions were prepared and analyzed by sequencing on illumina platform. sequencing results were processed by migec, mixcr and vdjtools software. enriched clones were identified by fisher's exact test (p> - ). results: we did not find any significant differences between patients after allo-hsct and healthy donors in single marker's expression, but, when considering coexpression of co-stimulatory and inhibitory molecules on t cells we found that cd +cd +cd -cd +cd +pd +tigit+ subpopulation was significantly increased in allo-hsct patients. moreover it increased with the time since the transplantation (fig. ). this population was isolated by cell sorting and alongside with total cd + fraction subjected to tcr beta-chain repertoire sequencing. the population contained clones significantly enriched compared with cd + fraction representing potentially alloreactive cells. this hypothesis is further supported by the notion that the level of expression of cd and cd co-stimulatory molecules is lower in the group of patients who subsequently relapsed, compared with the patients with complete remission, while the expression of inhibitory receptors was high in both groups. conclusions: according to our data patients after allo-hsct have a phenotypically distinct t-cell population characterized by simultaneous expression of costimulatory and inhibitory markers. this population contains specifically enriched clones, which may be specific for alloantigens. further functional assays are needed to confirm the alloreactive potential of this subpopulation. besides low expression of costimulatory molecules combined with high expression of inhibitory receptors on antigen-experienced t-cells of patients after allo-hsct might be associated with a disease relapse. fondazione mbbm, monza, italy, ospedale san gerardo, laboratorio stefano verri, monza, italy background: poor graft function (pgf) is a severe complication after hsct, with a high risk of morbidity and mortality, mainly due to infections. donor cd + scb seems to offer high chances of haematological recovery, not jeopardized by gvhd. however, pediatric reports remain scarce. methods: out of patients undergoing transplantation in our pediatric unit from to have been retrospectively evaluated for at least line persistent cytopenia (hb< . g/dl, plt< /mmc, n< / mmc) and/or transfusion-dependency beyond months after hsct in the presence of full donor chimerism. bone marrow cellularity was evaluated through biopsy as further indicator of pgf. ( / ) to donate or medical decision ( / ). bone marrow cellularity was < % in % of the patients who underwent scb for which the histology was available ( cases), and % in those who have not been treated ( / ). at days after scb / ( %) patients had hematological response, which was complete in % and partial in % of the patients. only patient had no response. the infusion was always well tolerated with no adverse events, and no worsening of gvhd. haematological recovery occurred spontaneously at days after bone marrow biopsy in a significantly lower proportion of patients ( / , %, p< . ) within the non-scb group. in two cases platelets engraftment was significantly delayed, up to one year after bone marrow biopsy and in one case thrombocytopenia persists and the patient is still receiving thrombopoietin agonists and red blood cells transfusions at months after bone marrow biopsy. conclusions: a stem cell boost matched the goal to yield count recovery in our cohort. viral infections and gvhd may be possible risk factors for pgf.bilinear or trilinear cytopenia with transfusion dependency and bom cellularity < % and full donor chimerism are good indications for scb, that can provide a significantly earlier hematological reconstitution, without risks of gvhd. due to the proved early efficacy and safety of cd + stem cell infusion, we suggest that this procedure should be taken in consideration in children with severe bone marrow hypoplasia and persistent cytopenia after hsct. disclosure background: as allogeneic hematopoietic stem cell transplantation (hsct) is sometimes performed despite erythrocyte's antigens incompatibility and mismatch, it is essential to carefully track patients' genotypes after it. methods: for the study we used erythrocytes (n= ) and dna (n= ) from patients undergoing abo-or rhesus-mismatch hsct and their donors. we used posttransplant no transfused patients on the periods according transplant protocol by hemagglutination methods in plate and tube using monoclonal antibodies to abo and rhesus antigens (hematolog, russia). we extracted dna with dna kit (bag, germany) and conducted pcr-ssp with kits abo-type, rh-type (bag, germany). chimerism was assessed by the str-pcr analysis with cordis plus multiplex kit for amplification of polymorphic strmarkers and amelogenin loci. the fragment analysis was performed on a genetic analyzer. informative loci were chosen by comparison of pretransplant patient's and donor's dna. the percentage of donor chimerism was calculated using standard formula. precise rhce and abo genotypes were determined by direct sanger sequencing. we revealed patients with unexpected erythrocyte abo, rhesus phenotypes and genotypes after hsct on + ( patients) and + days (all patients). chimerism analyses on str showed in a.e.kh. and g.l.v. patients % of donor's dna and less than % of recipient's one. b.n.a. patient was relapsed and chimerism analysis revealed % of recipient's dna and % of donor's one. using serological methods and pcr-ssp we revealed genotypes abo * a b ; rhd+; rhce * ccee in patient a. e.kh. before hsct, abo * a o ; rhd+; rhce * ccee in her donor, and abo * a a ; rhd+; rhce * ccee on + d after hsct. genotype a a was no recipient's neither donor's origin. direct sequencing did not prove this genotype, but revealed donor's one.on + d serological methods and pcr-ssp also revealed donor's genotype in this patient. patient b.n.a. had genotypes abo * o /o ; rhd+; rhce * c w cee before hsct, her donor -abo * b o ; rhd +; rhce * ccee. on + d this patient relapsed, but rhesus genotype has been detected as rhd+; rhce * c wcee (lack e gene). direct sequencing revealed gene rhce*ee. abo genotype was recipient's origin -o o . in patient g.l.v. using serological and pcr-ssp methods we determined genotypes abo * o /o ; rhd +; rhce * ccee genotype before hsct, and abo * a / o ; rhd+; rhce * ccee genotype in her hsc donor. on + d patient had unexpected genotype abo * o /o (a lack of a antigen); rhce * ccee (a lack of e antigen). in order to explain unexpected patient's genotypes after hsct we sequenced her rhce and abo genes and found donor's genotype ccee; a o that was in agreement with results of str analysis. to resolve discrepancies between serological, pcr-ssp and sequencing analysis data we sequenced patient's rhce cdna and observed only ce allele. at present time the molecular basis of selective inactivation one of the two rhce alleles is not clear. on + d patient had donor's genotype. conclusions: what kind of mechanisms led to discrepancies between results obtained by different laboratory methods are still not clear. an interesting case of expression of only one rhce allele in patient g.l.v. allows us to suggest involvement of some epigenetic mechanisms like dna methylation or histone modification in this process. clinical background: in relapsed patients with acute b -lymphoblastic leukemia (all-b) who achieved complete remission (cr) after re-induction therapy, minimal residual disease (mrd; ≥ - all-b cells/ul) is often detected. according to available data, such condition varies from % to even % of cases, as assessed by pcr or flow cytometry (fc), while the presence of mrd is the most important risk factor for all recurrence. in this abstract, we describe our experience with bridging therapy using blinatumomab infusion after re-induction regimens and before the planned allogeneic stem cell transplantation (allo-sct). the procedure was performed in three young men suffering from relapsed ph (-) all-b at the age of , and years. in the first case ( yo), relapse with previous mrd accounting for . % occurred months after cr mrd neg . in the next patient ( yo) the second relapse with central nervous system (cns) involvement occurred months after allo-sct performed in cr ( months after cr , mrd neg ), while in the third patient ( yo), recurrence with cns and testis involvement occurred years after cr (mrd neg ). all patients underwent chemotherapy (flam, hypercvad and dnr/vcr/pegasp/dexa regimens respectively) followed by one cycle of blinatumomab (at a dose of mcg/d on days - , followed by mcg/d on days - in a continuous infusion) and allo-sct (using eto/cy/tbi/atg/ conditioning regimen for ist and iiird patient and bucy for iind patient; using matched unrelated donor (mud, ist and iiird patient) or matched related donor (iind patient)). mrd status was assessed after each cycle of blinatumomab by fc. results: all patients achieved cr mrd pos after reinduction therapy followed by clearance of mrd after blinatumomab course (tab. ). the second patient, due to positive mrd months after allo-sct received donor lymphocyte infusions additionally. during the administration of blinatumomab, no adverse events (aes) were observed in grade or . one patient developed cytokine release syndrome in grade . the progression free survival, time to positive mrd and follow up are presented in tab . conclusions: the use of blinatumomab as a bridging therapy between re-induction regimens and allo-sct in patients with all-b and mrd pos appears to be safe and leads to the clearance of mrd which may be crucial in os and pfs prolongation after following allo-sct. future studies on larger groups of patients are necessary to confirm this thesis. background: haploidentical hematopoietic stem cell transplantation (hsct) is considered an alternative treatment for hematologic malignancies in patients who do not have an hla-identical sibling donor [ ] . since infections and disease relapse resulting from delayed immune reconstitution (ir) are the most common causes of mortality among patients undergoing haploidentical-hsct [ ], timely ir is essential in the recovery and survival of these patients. the aim of this study is to describe the evolution of ir after haploidentical-hsct and to estimate survival rates in patients with delayed vs. adequate reconstitution in a single center in colombia, south america. methods: a retrospective cohort study was conducted on consecutive adult haploidentical-hsct recipients at a tertiary referral center. cd +cells, cd +cells, cd +cells, and immunoglobulins levels were monitored before hsct, at first month, and then every three months for the first two years post-transplantation. descriptive statistics were used to analyze patient's clinical characteristics. the kaplan-meier method was used to assess overall survival (os) and relapse-free survival (rfs) rates. results: twenty-six patients were included ( % were male), with a median age of . years (range - ). the most common indication for haploidentical hsct was acute lymphoblastic leukemia (n= , . %), followed by non-hodgkin lymphoma (n= , . %) and myelodysplastic syndrome (n= , . %). all patients received gvhd prophylaxis therapy with cyclophosphamide, tacrolimus, and mycophenolate mofetil. fifteen patients ( . %) presented cytomegalovirus reactivation ( / at risk), patients ( . %) epstein-barr virus reactivation, and patients ( . %) developed adenovirus infection. median time to neutrophil engraftment (neutrophils> . × /l) was days (range - ) for the patients recipients of peripheral blood progenitor cells (pbpcs) and days (range - ) for the three remaining bone marrow recipients. platelet engraftment, defined as > , platelets/ mm background: daratumumab is a human monoclonal antibody directed against the glycoprotein cd that is overexpressed on the surface of plasma cells in multiple myeloma patients. it is approved as second line therapy either as single agent therapy or in combination with lenalidomide or bortezomib for the treatment of patients with relapsed/refractory multiple myeloma. despite the curative potential of an allo-sct, the high relapse rate remains a clinical problem. data addressing the choice of an optimal salvage therapy regime for these heavily pre-treated patients is missing. methods: from april till november a total of patients (male, n= ) with the median age of years ( - ) received daratumumab as a salvage therapy for relapse of multiple myeloma after allo-sct at the university of hamburg. prior to allo-sct all but one patient had received an autograft, patients even ≥ autografts and patients also a . allograft. the median number of salvage lines post-transplant and prior to first daratumumab infusion was ( - ). these salvage regimens included cyclophosphamide, etoposide, bortezomib, lenalidomide, pomalidomide and carfilzomib. daratumumab was started at a median of months ( - ) after relapse/ progress and initiated as single agent therapy in all patients. concomitantly, patients received either an immunomodulatory drug (lenalidomid, n= ; pomalidomid, n= ) or a proteasome inhibitor (bortezomib, n= ) during a later course of daratumumab infusions. combination therapy was initiated when a slow rise of paraprotein and/or free light chains or no response to monotherapy was observed (median at the th infusion). results: the median number of infusions was . twenty adverse reactions were observed in of ( %) patients: dyspnea (n= ), bronchospasm (n= ) shivering (n= ) , cough (n= ), musculoskeletal pain (n= ), acute coronary syndrome (n= ), skin rush (n= ), facial edema (n= ), pressure on eyes (n= ). all adverse reactions appeared during the first infusion and were mostly mild or moderate (ctc - , n= ). tolerance of the following infusions improved and in none of the cases therapy had to be stopped due to adverse events. three patients developed late onset infections (pneumonia, n= ; urinary tract infection, n= ) followed by temporarily therapy interruption. with a median follow-up of months after the first administration of patients remain alive . %). one patient died due to progress of myeloma and another died due to severe infection/sepsis. of patients responded ( %; pr, n= ; vgpr, n= ; cr, n= ) to the therapy with daratumumab. the responses (decrease of paraprotein and/or free light chains ≥ %) occurred at a median of days ( - ) after the first administration and lasted for . months ( . - . ). conclusions: daratumumab shows an encouraging efficacy and acceptable toxicity profile in patients with relapsed/refractory myeloma after allo-sct. further studies are needed to investigate the role of the combination therapy with immunomodulatory drugs or proteasome inhibitors in this setting. disclosure: nothing to declare p clonal plasma cell detection by high sensitive flow cytometry in aphaeresis product is poor prognostic and not increased by use of plerixafor alone background: in an earlier from our center we have demonstrated that residual clonal plasma cells (cpc) decrease both overall survival (os) and disease free survival (dfs) (ash ).plerixafor is a selective antagonist of cxc chemokine receptor (cxcr ) and able to mobilize human peripheral blood stem cell (pbscs) by acting synergistically with g-csf.the purpose of this study was to evaluate the safety and efficacy of plerixafor in myeloma patients who were proven poor mobilizers and specifically to assess the flow cytometric measurement of residual clonal plasma cells in the apheresis products. methods: patients with a diagnosis of mm who underwent auto hsct at our center between january -november were retrospectively analyzed.out of patients, patients received plerixafor as mobilization regimen due to poor mobilization with g-csf.pbsc grafts were tested for the presence of clonal pcs (cpc) and the number of normal pcs (npc) by multi-parameter flow cytometry (fcm).the acquisition of the cells was performed using the navios flow cytometer beckmancoulter) .upon the daily checks of the instrument, x cells for each sample were acquired and the collected data was analyzed using the kaluza software (beckmancoulter,usa). results: patient demographics are shown in table .the majority of patients were male and median age was years in the plerixafor group.the median interval from time of diagnosis to mobilization and follow-up from mobilization were . months and . months in plerixafor group, respectively. cpc contamination in the pbsc grafts was detectable in and patients with counts ranging between - . x - and - . x - in g-csf alone and g-csf+plerixafor groups, respectively (p= . ).there were no significant differences in the proportion of the patients with graft contamination between subtypes of mm in both groups. one hundred (gcsf/plerixafor; / ) patients had pre-asct pet-ct imaging done with (gcsf/plerixafor; / ) have active lesion at the time of mobilization. statistically significant association could not be demonstrated between the disease < cr status at mobilization and the number of apc in the apheresis product in both groups (p> . ).twelve of patients from plerixafor treatment arm proceeded to transplantation within median . months.the best overall response to induction treatment is shown in table .thirtyfour patients from the g-csf alone arm and patients from the g-csf+plerixafor arm died during the follow-up (p= . ).disease progression was seen in patients from g-csf alone group and patients from g-csf+plerixafor group of the study(p= . ).estimated mean os was better among patients w/o apc contamination in plerixafor group, respectively ( . ± . mos vs . mos; p= . ). conclusions: our results on and few plerixafor used patients show that clonal plasma cells are detectable by multiparametric flow more frequently when patients are poor mobilizers and require plerixafor.the clonal pc contamination can be attributed to the myeloma biology as manifested by higher number of lines induction regimens and pet positivity among the plerixafor-required patients. the overall and disease survival was impaired by residual clonal pcs in the graft but not by plerixafor per se. neither was the content of clonal pcs differed from others.thus the cxcr shared by hsc and myeloma cells do not cause a myeloma mobilization. clinical trial registry: -disclosure: nothing to declare prognostic factors for overall survival after allogeneic hematopoietic cell transplantation in multiple myeloma patients all factors with significant influence on pts survival were included multivariate analysis (cox regression model) but only re-admission in the first days demonstrated impact on os (hr , ; p= , ) . conclusions: we analyzed risk factors for survival in mm pts who received allo-hct. our study identified disease-related risk factors like iss and transplantationrelated factors such as hct-ci and pam, hospital readmission, days of hospitalization and cmv reactivation that were associated with worse long-term survival. in our series, the most frequent death and re-admission cause was infection, so focusing the efforts in reduction of infection could have a beneficial impact on improvement of survival in mm undergoing allo-hct. [[p image] . figure ] disclosure: there is no disclosure. novel protocol for autologous hsct in multiple myeloma: ambulatory chemomobilization and transplantation of fresh hematopoietic stem cells with backup storage background: autologous hematopoietic stem cell transplantation (ahsct) after melphalan conditioning is still a part of standard treatment of multiple myeloma patients. traditional transplantation of frozen stem cells poses additional risk for the patients connected with dmso and central venous catheter. the transplantation of fresh cells is an option -however, most mobilization protocols are either low-efficient (g-csf), expensive (g-csf + plerixafor) or toxic (standard dose chemomobilization) to directly proceed to transplantation in this fragile group of patients. we describe here the novel combination of ambulatory mobilization with very low doses of ara-c and g-csf connected with direct ahsct with fresh cells. methods: the prospectively collected database of patients after ahsct was searched for patients who underwent ahsct after chemomobilization with ara-c and transplantation with fresh cells (fc) and compared with control group of consecutive patients transplanted with standard protocol (sp) (transplantation with frozen cells) between july and october . protocol of ambulatory mobilization was: mg/m² of arac on days + and + and g-csf at the dose μg/kg/day from day + and escalated to μg/kg/day split into two doses + to + , apheresis started on day + (or later) and finished when at least . x e cd + positive cells were collected. the collected cells were split in three equal parts: ) for use as fresh transplant ) frozen for possible nd transplant ) frozen as backup. results: there were transplantations with fresh cells and transplantations with frozen cells compared. both groups had same mobilization protocol -ambulatory low dose ara-c. the median age and number of transplanted cells was similar in both groups ( vs , p= . ; . vs . cd +/kg, p= . conclusions: we present novel approach that allows direct ahsct after chemo mobilization in all patients who are treated with melphalan. we show that it is not only feasible to do ahsct directly after chemomobilization but also the results may favour this approach when compared with current standard. disclosure: nothing to declare background: high dose chemotherapy followed by autologous hematopoietic cell transplantation (hsct) is considered, since the nineties, the standard of care for patients aged less than - years old with newly diagnosed multiple myeloma (mm). however, the optimal induction treatment prior to hsct to reduce the tumor burden has changed during the last few years. improved regimens have shown to be able to increase the quality of the pre-hsct response, which might subsequently impact on the post-hsct response, which has been proved to be associated with a longer pfs. we recently changed the induction therapy for pts candidates to hsct. in this analysis, we aimed to check if newer regimens impacted on pretransplant responses, and how auto-hsct changed the pre-hsct status. methods: all the auto-hsct for mm patients performed in our center from january through august were included in the analysis. median age was years (range: - ). pts were male and were female. durie-salmon stage was distributed as follows: i ( . %), ii ( . %) and iii ( %); % had creatinine > mg/dl. iss was: ( %), ( . %), and ( . %). type of monoclonal component was: igg ( . %), light chains ( . %), iga ( . %) , and non-secretory ( . %). . % had bence jones proteinuria. conditioning regimen was melphalan mg/m in ( . %), melphalan - mg/m in ( . %), and other in ( . %). results: pre-transplant therapy was: vcd in (mostly in - ), vtd/vrd/krd in (mostly in - ), and others in cases. status of the disease at transplant was: cr/vgpr in , pr in , and sd in . distribution of pretransplant response based on the type of induction is shown in table . peri-transplant mortality was %. day + mortality was . % ( pts), due to progressive disease. as shown in table , all patients ( / ) who obtained cr pre-hsct, maintained the response at day + post-hsct. among the patients in vgpr at hsct, ( . %) became into cr, and ( . %) maintained the response. the cr rate at post-hsct increased % compared to pre-hsct ( versus pts). altogether, after hsct pts ( . %) improved and ( . %) maintained the pre-hsct response. during the last period of time, pts started on post-hsct maintenance/consolidation, mainly with lenalidomide. conclusions: ) with the new chemotherapeutic schemes, . % of patients underwent hsct in cr or vgpr; ) majority of pts ( . %) consolidated or improved the pre-hsct response; ) cr increased substantially ( . times) after transplant; ) optimized induction regimens, along with auto-hsct followed by the recently licensed use of maintenance therapy with lenalidomide, might result in a better pfs of patients with mm. background: autologous stem cell transplantation (asct) is commonly used in treatment of patients over years with multiple myeloma (mm), however the safety and efficacy of this procedure is debatable. methods: we conducted a retrospective review of mm patients who underwent asct from to at our institution. the purpose of this retrospective study was to compare the -day mortality, time to engraftment, and incidence of grade - toxicities in elderly mm patients with younger patients taking into account comorbidity information. other secondary end points measured were overall survival (os) and progression-free survival (pfs). results: a total of patients were analysed and categorized by age as young patients ( to y; n= ) or elderly ( to y; n= ). the compared groups did not differ in terms of gender, ecog, hct-specific comorbidity index (hct-ci), and disease status at asct. melphalan in a dose of mg/m was used as preparative regimen in % of younger patients, and in % of the elderly (p= . ). the remaining patients received mg/m of melphalan or lower dose (range, - mg/m ) due to hct-ci > or age, on the physician discretion. in the whole study group there were no transplant related deaths within the first days of asct. stratifying by age, there was no statistically significant difference concerning febrile neutropenia (fn) incidence, which was observed in % of younger patients, and % of elderly. in contrast, fn was observed more frequently in patients with hct-ci > ( % vs %, p= . ). grade - infections were more frequent in older patients ( % vs %, p= . ), but no difference was found in grade - infections incidence rate such as pneumonia, uroinfections and neutropenic enterocolitis ( % vs %, p= . ), nor grade - and - noninfectious toxicities ( % vs %, p= . , and % vs %, p= . , respectively) . the median time to granulocyte engraftment was days (range, - days) in elderly and was comparable with younger patients. the time to platelet recovery was also similar. after the median follow-up of months for survivors, os at months was % in both groups. pfs at moths was % for younger patients, and % for elderly (p= . ).however, the association between pfs and the dose of melphalan used in conditioning was observed. pfs probability at months for patients conditioned with the dose of mg/m , mg/m and mg/m was %, % and %, respectively (p= . ). conclusions: our data show that asct in transplant eligible mm patients ≥ years of age is safe and provides similar outcomes as seen in younger patients. disclosure: nothing to declare is mobilization with chemotherapy effect response in the multiple myeloma? background: high dose melphalan therapy with autologous stem cell support is a standart approach in symptomatic multiple myeloma patients. response rates increased with the novel anti myeloma agents and the use of chemotherapy for stem cell mobilization should be questioned. the purpose of this study is to determine the effect of cyclophosphamide used during stem cell collection on disease response and transplantation results. methods: we retrospectively collect data from myeloma patients who underwent autologous stem cell transplantation (asct) in ankara university medicine faculty, blood and bone marrow transplantation unit between january and november . patients who received cyclophosphamide protocol for stem cell mobilization were included in the study. disease response were determined according to international myeloma study group (imwg) criteria before and after the cyclophosphamide. transplant responses and their effects on survival were also indicated. results: after the diagnosis of mm, patients (male/ female: / ; median age: years (between - years)) with median follow-up of . months (between , - , months) underwent asct at a mean of , ± , months.. forty-one patients were evaluated before and after cyclophosphamide (table ). in % of the patients, the disease response was not altered by cyclophosphamide therapy, and % of the patients improved their response status. post-transplant response rates of patients who underwent stem cell mobilization with cyclophosphamide are also shown in table- . the mean survival of the patients was , ± , months. when patients were grouped according to changes in response status before and after cyclophosphamide; there was no statistical difference between mean calculated survival (improved response, disease progression and stable disease; , ± , months, , ± , months and , ± , months respectively, p= . ) (figure- ) . the rates of -year and -year overall survival (os) of the patients with no response to cyclophosphamide treatment were as follows; , %± , % and , %± , % respectively. thirteen patients who were followed up median months after transplantation died at an average of , ± , months; of these deaths were caused by the infection after transplantation. conclusions: in our study, it was observed that the use of cyclophosphamide for cd + stem cell mobilization did not change the disease response rates by %. the posttransplant survival rates of mm patients who had progressive disease after cyclophosphamide use were lower. however, these results warranted confirmed by randomized controlled trials. clinical trial registry: -disclosure: nothing to declare results of a single center experience: an attempt to augment conditioning regimen in first autologous stem cell transplantation treatment of multiple myeloma (mm) continues to evolve in the era of novel agents. the addition of bortezomib to highdose melphalan (bor-hdm) has been reported by several groups, and it has been outcome and toxicity profile is comparable to high dose melphalan (hdm) alone. the aim of this retrospective study was to evaluate the outcome of the bor-hdm conditioning regimen on overall response for patients with mm undergoing first single asct at our institution. methods: this retrospective single center study reviewed consecutive myeloma patients who had received the first asct either with bor-hdm (n= , m/f= / ) or single agent hdm (n= , m/f= / ) conditioning regimen. in the single agent hdm conditioning regimen, melphalan was administered intravenously at a total dose of mg/m on day - and - and stem cells were infused on day . in the bor-hdm group, melphalan mg/m was administered on day - . bortezomib was administered intravenously at a dose of mg/m on day's - , - , + , and + as described in a phase study by intergroupe francophone du myeĺome (ifm). results: all consecutive patients who underwent single asct from january to march using bor-hdm as conditioning or hdm were evaluated. conditioning regimen was hdm in patients and bor-hdm in patients. median age was significantly lower in bor-hdm conditioned asct compared to hdm group ( years vs years, p= ). there was no significant difference for mm subtype, iss stage at diagnosis, prior treatment line among hdm vs bor-hdm cohorts (p> . ). after a median of cycles of induction chemotherapy, patients in the bor-hd exhibited ≥vgpr of . % (n= ) compared to . % (n= ) in the hdm group (p= p> . ). pre-asct immune response (if (-)) was reported in . % of patients treated with hdm, higher than that seen in the bor-hdm group ( . %) (p= . ). nine ( . %) patients achieved post-asct immune response (if (-)) ≥vgpr compared to ( . %) in the hdm group. at the time of this analysis, ten patients in the bor-hdm group and in the hdm group had already died, respectively (p> . ). a total of ( . %) patients in the bor-hdm group and ( . %) patients in hdm group had already progressed (p> . ). estimated mean os and pfs was shorter for group treated with bor-hdm; . ± . mos and . ± . mos vs. . ± . mos and . ± . mos, respectively (p> . ) (figure- ) . we could not demonstrate the impact of pre or post transplant ≥vgpr immune response on survival and disease free survival. there was no engraftment failure observed on either treatment group and no worsening peripheral neuropathy was developed in the bortezomib arm. conclusions: this single center experience on a small patient pool was able to repeat the prospective randomized study results of ifm. further studies are warranted to explore this regimen, especially when induction treatment with novel agents are employed, with special emphasis on the high-risk myeloma patients where response rates are good but sustainability remains an issue. disclosure: nothing to disclosure the efficacy and safety of bortezomib plus busulfan/ melphalan as conditioning regimen in multiple myeloma undergoing autologous stem cell transplantation: phase / study background: bortezomib have a powerful antimyeloma activity and was regarded as backbone of therapy in the past decade but its safety and efficacy as a part of busulfan/ melphalan conditioning regimen of autologous stem cell transplantation is yet to be shown. methods: a phase / trial to explore the safety and activity of a bortezomib on days - , - , and + added to a conditioning regimen with busulfan and melphalan (bumel, . mg/kg/day and busulfan during day - and - , mg/ m /day of melphalan on the day - ), in multiple myeloma (mm) patients who received autologous stem cell transplantation following bortezomib-based induction chemotherapy. in phase , escalating doses ( . , . , and . mg/m ) of bortezomib with bumel were administered in each group with three patients. with determined maximum tolerated dose of bortzomib at a . mg/m /day, cohort with patients were analyzed for phase . results: in phase , no dose limiting toxicity was observed at a . mg/m /day of bortezomib. in phase , overall responses at months was shown as % of very good partial response (vgpr) or better and % of complete response (cr), whereas post-transplant overall best response included % of vgpr or better, and % of cr, respectively. with median follow-up duration of . months, median progression-free survival (pfs) was . months. the probabilities of years-pfs and overall survival (os) were . % and not estimable, respectively. especially, high-risk cytogenetics were associated adverse survival outcome compared to standard-risk cytogenetics, respectively (pfs, . vs. . months, p= . ; os, . vs. . months, p= . ) . with median days and days for neutrophil and platelet engraftments, any graft failure or delayed engraft was not observed. the common grade or severe non-hematological adverse events included neutropenic fever ( . %) and stomatitis ( . %). except three cases with transplant-related mortality due to sepsis, other adverse events were manageable. conclusions: these results demonstrate that bortezomib is safe and can be a part of conditioning regimen in combination with bumel, for patients with transplanteligible multiple myeloma. clinical background: allogeneic stem-cell transplantation (allo-sct) is one of treatment option for patients with multiple myeloma (mm) refractory to novel agents. the reports on allo-sct for mm are limited and it is an important issue to argue appropriate conditioning regimens and stem-cell sources, and patient population who will benefit from allo-sct. methods: we retrospectively analyzed consecutive patients who received allo-sct for relapsed and refractory multiple myeloma (rrmm) between oct and july at japanese red cross medical center. characteristics of patients, progression-free survival (pfs), and overall survival (os) were analyzed. results: median age at allo-sct was (range - ). twelve patients were male and were female. myeloma type were igg: , iga: , igd: , and bence-jones: . stem-cell sources were peripheral blood from hlamatched related donor (rpbsct): , bone mallow from hla-matched unrelated donor (mud): , bone marrow from hla-mismatched donor (mmud): , and cord blood (cb): . twenty-three of patients received flu/mel-base, one patient received bu/mel-based, and one patient received etoposide/cyclophosphamide-based conditioning regimens. twenty-two patients who transplanted after received gy of total body irradiation (tbi). responses before allo-sct were cr: , vgpr: , pr: , sd: . five-year pfs was % ( %ci: - ) and -year os was % ( %ci: - ). ten patients died during observation period and causes of death were primary disease: and treatment-related mortality: . patients with vgpr or better before allo-sct showed significantly better pfs (p= . ) and os (p= . ) as compared with others. female recipients showed significantly better pfs (p= . ) and os (p= . ) as compared with male recipients. recipients of mmud showed significantly better pfs (p= . ). among patients surviving, patients received treatments including maintenance therapy. conclusions: the reason for better pfs and os in female recipients is unknown. it is interesting that recipients of mmud showed better pfs, suggesting graft-versusmyeloma effects. allo-sct can be an effective treatment option if patients and stem-cell sources are appropriately selected. disclosure: authors declare that there are no conflicts of interest. second autologous hematopoietic stem cell tranpslant versus chemoimmunotherapy in relapsed multiple myeloma after first transplantation: single center data background: combination therapy, mostly triple, followed by autologous hematopoietic stem cell transplantation (auto-hct) is widely accepted as the first-line standard therapy for multiple myeloma (mm). despite the availability of agents such as new immunomodulatory drugs (imids), proteasome inhibitors (pis), histone-deacetylase inhibitors and antibodies, it is still possible to achieve longer and deeper responses, however, multiple myeloma is still not cured and relapse is inevitable. the availability of these novel agents has increased questions for determining optimal treatment of patients with relapse after the first auto-hct. methods: we retrospectively analyzed patients who relapsed according to international myeloma working group (imwg) criteria after st auto-hct. first group [salvage chemotherapy(ct)] (n= ) was treated with only chemoimmunotherapy because of early relapse or refractory first auto-sct (within months), ineligible to second transplantation because of co-morbidity, unwillingness to transplant. second group (n= ) (salvage transplantion) was treated with second auto-hct as a salvage therapy. consolidation and long term maintenance treatments were used in both groups. results: there was no difference in sex and age between salvage ct and auto-sct groups [female/male: vs / vs ; ]. the best response after salvage auto-sct was complete remission (cr) in , %, partial remission (pr) in , % patients, while cr in %, pr in , % patients treated with salvage ct. progression free survival (pfs) were significantly better in second transplant group (pfs; % on the first year; , % on the second year after transplant vs % on the first year; % on the second year after the salvage therapy in chemotherapy patients)[p: , ]. overall survival (os) in salvage auto-sct group was longer than salvage ct ( , % . %), although it did not reach a statistical significance (p> . ). time to achieving the best response after salvage auto-sct and salvage ct was ( - ) month versus , ( - ) months [p: , ]. grade or nonhematological toxicities were similar (auto-sct %, salvage ct %) in both groups. conclusions: salvage auto-hct may provides longer progression free survival with similar toxicity profile according to chemoimmunotherapy especially in patients with sensitive to first auto-sct. it is suggested that earlier and better responses, long-term progression free survival can be achieved with salvage auto-sct. we believe that there will be statistical significance in os such as pfs by increasing the number of patients. the authors believe that large scale randomized clinical trials are needed for optimal treatment of relapsing multiple myeloma after first auto-sct. disclosure: nothing to declare background: one of the conditions for successful transplantation of autologous hematopoietic stem cells (auto-hsct) in patients with multiple myeloma (mm) is the timely recovery of hematopoiesis, which is associated with the quantitative and qualitative characteristics of the graft. one of the key indicators is the content of cd + cells in the autograft, which depends on many factors. some of them are due to previous treatment, others are directly related to the patient: age, stage of the disease, features of the hematopoietic stem cells (hsc) microenvironment. the aim of the study was to assess the influence of the immune response genes on the autograft cellularity in patients with mm. methods: А retrospective analysis of the genotyping results was performed. evaluation of loci in genes immune response and harvesting of autologous hsc in patients with mm has been made. hematopoietic stem cell mobilization regimen included cyclophosphamide g/m with granulocyte colony-stimulating factor. genotyping of the immune response genes polymorphic regions was carried out by the polymerase chain reaction with allelespecific primers. the number of cd + cells was counted on a -color facs canto ii flow cytometer. results: according to the results of the autologous transplant harvesting, two groups of patients were identified. first included patients with an autograft cellularity of more than × /kg body weight. the second group consisted of patients examined with the number of cd + cells in the autograft ≤ × /kg of the patient's body weight. comparing the identified haplotypes of the immune response genes with the cellularity of the transplantation material, it was found that the presence of the mutant allele in the homo-and heterozygous haplotypes of the il β gene (t- c) increased the chances of harvesting cellular material with a higher content of cd + cells in times (χ = . , p= . ), and the carriage of the wild type allele in the homo-and heterozygous state of the tlr (arg gln) gene is more than in times (χ = . , p= . ). currently, it has been shown that single nucleotide or amino acid substitutions in genes can lead to changes in the expression pattern of their final products: increased secretion of interleukin β (il- β) or changes in the spatial configuration and functionality of the receptors (tlr ). thus, in the presence of mutations in the il β gene, the enhanced synthesis of il- β influences on fibroblasts, immunocompetent, endothelial, epithelial and other cells, by activating hemopoiesis. in turn, the mutational status of the arg gln locus located within the tir domain of the tlr receptor in the cytosol, determines the spatial configuration of the tlr acting as a co-stimulatory receptor of cd + cells, which ensure the engraftment of the graft. conclusions: identified haplotypical features of the il β and tlr genes in patients with mm may act as predictors of the response effectiveness to mobilization of hscs in their carriers, which may contribute to the mobilization regimen optimization and will contribute to harvesting the optimal cellularity of an autologous graft. clinical trial registry: none. disclosure: authors declare no conflict of interest. differentiating diffuse from focal pattern on computed tomography: added values of a radiomics approach background: focal pattern in multiple myeloma (mm) seems to be related to poorer survival and differentiation from diffuse to focal pattern on computed tomography (ct) has inter-reader variability. therefore the purpose of this study is to assess if a radiomic approach could help radiologists in differentiating diffuse from focal patterns. methods: we retrospectively reviewed imaging data of patients with mm between january and september of whom ( men and women; mean age . ± . ) with ct, pet-ct or mri available before bone marrow transplant. two general radiologist evaluated in consensus only ct images to define a focal (at least one lytic lesion > mm in diameter) or a diffuse (lesions < mm, not osteoporosis) pattern. radiomic analysis on ct thinslice images was then applied with regions of interest (rois) done by one researcher not expert in medical imaging or mm blindly to the condition of the patients. the reference standard to differentiate diffuse from focal pattern was done by radiological evaluation of two expert musculosketal radiologists blinded to the clinical data reviewing ct, mri and pet-ct images. n= radiomics features were extracted and evaluated with an open source software. mann-whitney u test for unpaired data with bootstraps samples was used to compare radiomics features of diffuse and focal patterns and then feature reduction was done to avoid over-fitting. receiver operator characteristic (roc) analysis with area under the curve was done to compare radiologists and radiomics evaluation against reference standard. reading time to perform radiomic analysis was also estimated. results: the pathological group included: diffuse and focal patterns. after feature reduction, features were different (p< . ) in the diffuse and focal patterns (n= / features were shape-based: majoraxislength and sphericity; n= / were gray level run length matrix (glrlm)). mg/kg). a number of eleven patients did not receive any additional immunosuppression except of post-cy. results: after a median follow up of . months (range . - . ) patients were alive. the -year probabilities of pfs and os were % ( - %) and % ( - %).the cumulative incidences (cis) of relapse and nrm at years were % ( - %) and % ( - %), respectively. lower serum albumin level at transplantation (≤ g/dl) was associated with increased relapses (hr . ( . - . ), p= . ) and nrm (hr . ( - ), p= . ) and resulted in poorer pfs ), p= . ) and os ), p= . ). mmud and haploidentical donors were associated with poorer nrm (hr . ( . - . ), p= . ), and resulted in decreased pfs ), p= . ). the high-risk cytogenetic at diagnosis showed no impact on survival. the cis of acute (grade ii-iv) at day + and chronic gvhd at years were % ( - %) and % ( - %), respectively. absence of immunosuppressive medication beside post-cy was associated with poorer os ), p= . ). conclusions: the conditioning with bu, tt and post-cy leads to a favorable pfs and os due to low incidences of relapse and nrm for patients with multiple myeloma relapsing after autografting. disclosure: nothing to declare methods: between january and may , we included patients with mm who underwent asct and received bortezomib/lenalidomide/dexamethasone (vrd) consolidation and maintenance therapy, mainly lenalidomide(r) mg/day for days every days. results: the median age at transplant was years ( - ). forty-six ( %) of patients received r maintenance, patients received vrd maintenance for higher risk features. median duration of r maintenance was months . r dose was changed for toxicity (grade i-ii) in ( %) patients. twenty-nine ( %) patients relapsed: ( %) patients were shifted to different treatment protocols (treatment change). patients ( %) were kept on the same r maintenance (observation group) and ( %) patients had increased lenalidomide dose with dexamethasone (r/ d group). patients ( %) of the last groups required change of treatment later. the median follow up was months . median tnt was months ( - ). at years, the estimated pfs and os were % and . % respectively. the median os and pfs (from change of therapy) were and months for patients in the observation group, versus and months in the r/d group, and and months with treatment change, respectively. no statistically significant difference was noted. conclusions: our small monocentric study is limited by its retrospective design and small sample size. however, it suggests that increasing lenalidomide dose as well as adding dexamethasone in selected patients can postpone change to different lines of treatment without affecting survival. disclosure: nothing to declare can the drugs used before autologous hematopoietic stem cell transplantation have impact on cmv reactivation that results in decreased os in myeloma patients after asct? more intensive treatment regimens, such as proteasome inhibitors (pi) and/or immunomodulatory (imid) agents. we performed a retrospective, single center study to evaluate the incidence, risk factors, and outcomes of cmv infection in patients with mm who underwent asct with a high-dose melphalan-based regimen. methods: this study involved a retrospective review of all patients with who underwent asct between january and november at our stem cell transplantation center. a total of consecutive adult patients with a diagnosis of mm (median age at diagnosis: , range: - ) underwent asct following induction treatment with novel agents (pis and/or imids). all patients received antiviral prophylaxis with acyclovir mg/day (n= ) or valaganciclovir mg/day (n= ). results: baseline patient characteristics, according to induction treatment, are summarized in table- . one hundred-five of the patients ( . %) were cmv iggpositive before asct. overall, . % (n= ) of cmvseropositive patients developed at least one episode of cmv viremia (cmv dna > copies/ml) after a median months (range; - mos) follow-up. persistent cmv viremia (detectable cmv dna load in more than sequential plasma specimens) occurred in . % ( of ) of the seropositive asct recipients and all of them were preventive treated with ganciclovir (n= ) or valganciclovir (n= ). the time from stem cell infusion to the development of cmv viremia ranged from days to days. none of the patients with untreated viremia developed identifiable cmv sequelae. no case of primary infection in seronegative patients at transplant was observed. adding to that none of the patients developed cmv disease post asct. if we analyzed the subgroups of patients according to induction therapy (pi-based, imids, pi+imid), the incidence of post-asct cmv reactivation was higher but not statistically significant, in patients who received only pi vs pi+imid ( ( . %) vs ( . %); p= . ). in univariate analysis, we could not demonstrate the importance of induction therapy with novel agents the occurrence of a post-asct cmv reactivation requiring antiviral treatment. however, statistically significant association found between the disease < vgpr status at asct and cmv reactivation ( . % vs. . %; p= . ). after a median follow-up . months (range; - . months), there was no significant impact on pfs, however there was significant decrease in estimated mean os who had cmv reactivation when compared to those without cmv reactivation ( . ± . vs. . ± . ; p= . ) (figure- ) . conclusions: cmv establishes lifelong latency within host cells and in the setting of impaired cellular immunity; cmv may reactivate from latency, disseminate, and directly cause life-threatening disease. our data suggests that mm patients treated with pi-based induction regimens and immunological response < vgpr at time of asct seem to have higher risk of developing symptomatic cmv reactivation. however, further studies on a large number of patients are warranted to clarify these findings. clinical background: high-dose therapy followed by autologous stem cell transplantation (asct) has been shown to prolong survival in patients with multiple myeloma (mm) in randomized trials. however, these trials only include patients aged < years. data regarding safety and outcomes in this patient population is lacking. methods: the aim of this study was to compare safety profile and outcomes in mm patients younger and older than years-old who underwent asct in our unit from july to october . patient's demographics, clinical characteristics, transplant related variables and probability of admission to the intensive care unit (icu) were analyzed. patients aged < and ≥ years-old would be called m and m , respectively, from now on. sorror index was used to estimate risk of mortality in the two cohorts. results: a hundred and eleven patients with mm underwent asct in the study period. median age was . years-old (range - ) and . % were male. thirtythree ( , %) patients were ≥ years. the probability of having a high risk comorbidity index was similar in both groups (m , vsm , %). the median cells obtained in the apheresis procedure was . x ( , - . ) in m compared to . x ( . - - ) in m . there were no differences in median admission lenght between the cohorts (m : days vs m : days). median days for neutrophil recovery above was days in both groups with a wider range in m ( - ) compared to m ( - ) . no differences were found in platelet recovery above . (m days vs m days). median packed red blood cells and platelets transfusions were ( - ) and ( - ), respectively, in m . in m cohort, they were ( - ) and ( - ), respectively. the incidence of grade - mucositis in m and m was . % and . %, respectively. there were no statistically significant differences in terms of using morphine for pain control between the two cohorts (m , , % vsm , , %). none patient requiered total parenteral nutrition (tpn) in group m and only one in group m . the incidence of icu admission was . times higher in patients aged ≥ than in patients < years-old , % vs , %), but differences were not statistically significant (p = . ). there were no deaths during the transplant procedure in any of the cohorts conclusions: ) in our series, high-dose therapy followed by autologous hematopoietic cell transplantation in mm patients aged ≥ was feasible. ) transplant procedure in older patients was as safe as in patients < years-old. ) no differences were found in terms of graft, transfusion support, transplant related complications and length of admission. ) age should not be a limiting factor in considering the modality of asct in this patient population disclosure: nothing to declare the correlation between the kinetics of peripheral blood counts and the response to treatment after high-dose melphalan with stem cell support in multiple myeloma patients background: the long-term survival of mm patients has dramatically increased in the last years, particularly for younger patients. this is attributable in part to the introduction and development of high dose chemotherapy with melphalan with stem cell support (hdm-asct). currently, frontline asct is still considered the standard of care for all eligible patients. many prognostic factors pre and post transplantation have been identified, e.g.: age, comorbidities, cytogenitcs, response to treatment and disease status prior to and post transplantation. to our knowledge there is no data correlating between kinetics of counts response to melphalan and prognosis. our aim was to assess the prognostic significance of the neutrophil and platelets decaying counts after high dose melphalan. methods -we retrospectively analyzed our cohort of multiple myeloma patients who underwent hdm-asct at the hadassah medical center bone marrow transplant department, between the years - . the kinetics of neutrophil and platelet decay during the first two weeks after melphalan administration was fitted using linear and exponential mathematical models. methods: we retrospectively analyzed our cohort of multiple myeloma patients who underwent hdm-asct at the hadassah medical center bone marrow transplant department, between the years - . the kinetics of neutrophil and platelet decay during the first two weeks after melphalan administration was fitted using linear and exponential mathematical models. results: factors associated with prolonged os in univariate analysis were: iss stage (p= . ), ≤ lines of treatment prior to asct(p< . ), favorable cytogenetics(p= . ), response to treatment (pr or better, p= . ) and rapid linear neutrophil decay (p = . ). in multivariate analysis, only ≤ lines of treatment before hdm-asct and rapid linear neutrophils count decay remained statistically significant for os prolongation. no predictive threshold value of the neutrophil decay incline was found. improved pfs was associated with ≤ lines of treatment prior to asct, and the response status after hdm-asct (p= . , p= . ). additionally, toxicity evaluation showed prolonged neutropenia to be associated with inferior os (hr = . , p= . ) and rapid exponential decay of neutrophil counts to correlate with higher incidence of mucositis (p = . ). fast platelet decay was associated with delayed platelet engraftment (p< . ) conclusions: we have shown that rapid linear decay in neutrophil counts predicts better os without a significant benefit in pfs in mm patients undergoing hdm-asct. this discrepancy might reflect the problematic estimation in a retrospective analysis of pfs. rapid decrease in neutrophils and platelet counts was associated with more toxicity: higher mucositis rate and delayed engraftment, respectively. therefore a rapid decay of blood counts after hdm-asct appears to be an in-vivo phamacodynamic marker of higher efficacy and toxicity of melphalan. disclosure: nothing to declare p do we need to freeze hematopoietic cells for autotransplants in patients with myeloma conditioned with melphalan? daniel garcia belmonte , beatriz aguado bueno , miguel herrero coderch , rafael de la camara background: multiple myeloma (mm) is the most frequent indication of auto-hsct, representing % of all auto-hsct in (passweg jr. bmt ; : - ) . nearly all are performed with peripheral blood progenitor cells (pbpc), and melphalan mg/m is considered the gold standard conditioning regimen. the standard procedure consists in obtaining progenitors, cryopreserved with dimethyl sulfoxide (dmso) and stored and subsequently thawed and re-infused in the patient on day . the procedure of cryopreservation is expensive and has some inherent toxicities (dmso) and loss of cells during the procedure. several groups have used non-cryopreserved progenitors showing some benefits compared with cryopreserved transplants, mainly a faster engraftment and a shorter length of hospitalization. objective: to compare noncryopreserved vs cryopreserved auto-hsct in mm methods: we perform an unicentric, retrospective study on consecutive first auto-hsct mm patients transplanted with pbpc between nov- and oct- , and conditioned with high dose melphalan ( mg/m ). the median follow-up was days (range: - ). patients received non-cryopreserved and cryopreserved auto-hsct. patients characteristics, without differences between non-cryopreserved vs cryopreserved: women/men ( / ); median age was years (range - ); in the majority auto-hsct was done as consolidation after first line therapy ( %); year of transplant ≤ ( %), ≥ ( %). the number of infused cd cells were not different: median . x /kg (range . - . ) in noncryopreserved patients and . x /kg (range . - ) in cryopreserved patients. results: we didn´t observe significant differences in the day of engraftment between non-cryopreserved vs cryopreserved although always was a little bit faster in the noncryopreserved group with a tendency to faster platelet engraftment (> /mm ): > platelets/mm (median day: vs . , p . ), > platelets/mm (median: vs days, p . ); > neutrophils/mm (median: vs . days, p . ). the media of days of hospitalization was shorter in non-cryopreserved patients ( vs days) although not statistically significant (p . ). transplantrelated mortality at day + was % in both groups. overall survival at years was not different: . % in in non-cryopreserved vs . % in cryopreserved patients (kaplan-meier, log-rank p . ). the accumulative incidence of relapse at the median follow up ( days) was similar: . % in non-cryopreserved vs . % in cryopreserved patients. conclusions: in our short experience, auto-hsct with non-cryopreserved pbpc in myeloma patients conditioned with high dose melphalan obtain similar results to those performed with classical cryopreserved pbpc and might has a faster platelet engraftment and shorter length of hospitalization. if no advantages are associated with cryopreservation, the simplicity of using fresh product is appealing. disclosure: nothing to declare p abstract withdrawn. . results: a early death is observed in one pt (group ) and pts(group ). the median delay of aplasia is days ( - ) and days ( - ) respectively. in group , among the evaluable pts, / ( %) are in cr, pts in pr and refractory. in group , cr: / ( %), pr: and refractory. a relapse is observed in pts/ ( , %) in group and pts/ ( %) in group with a frequency of % and % respectively in the first months. at months: pts/ ( %) in group and pts/ ( %) in group are dead. at months: pts ( %) and pts ( %). at months: pts ( , %) and pts ( , %). the overall survival (os) of the group and group pts were % and % at months; . % and . % at months; % and , % at months respectively (without significant difference). the event free survival (efs) of group and pts were % and , % at months, % and % at months and % and % at months respectively (without significant difference). conclusions: these protocols with equivalent toxicity allow obtaining of long-term equivalent results on the response rate early transplant, on the rate of relapse and on the overall survival. these results are identical to those of fermand ( ) . disclosure: nothing to declare background: dimethylsulfoxide (dmso) is a major intracellular cryoprotectant, used for cryopreservation of stem cells. it is toxic to both cells and patients at temperatures above o c. reduction of this effect is achieved by either washing of cells after thawing or by reduction of dmso during freezing and storage. the latter requires addition of extracellular cryoprotectants to the freezing media. we assessed the effect of low dmso concentration and different hematocrits of the frozen cells on cell viability and hematologic recovery in patients, transplanted for multiple myeloma. methods: cells were non-programmed frozen and stored at - o c in a cryoprotectant solution achieving final concentrations of % dmso, . % of hydroxyethyl starch (hes, weight average molecular weight da) and % of human serum albumin. the cell concentration in the frozen product for the first patients ( transplantations) varied between x and x cells/ml. in an attempt to reduce the amount of dmso infused, for the rest of the patients (n= ; transplantations) we further decreased the volume of the freezing suspension by removal of the entire plasma. the average age of the transplanted patients was ( - ). the cells were bedside thawed at o c water bath. the average cell dose was , x /kg ( , - , x /kg). results: viability of the stem cells following thawing assessed by trypan blue exclusion was , % . the hematocrit of the frozen cells had no effect on cell viability ( , %(low) vs , %(high)). the major complaints, if any, during stem cell infusion were coughing and an increase in nausea and vomiting induced by the prior conditioning. the average time for hematological recovery was , days (between and ) for the neutrophils, and , (between and ) days for the platelets. there was no significant difference in viability and hematologic recovery ( , and , vs , and , ) between patients receiving cells frozen with low or high hematocrit. conclusions: dimethylsulfoxide, despite its cryoprotective properties, is toxic for stem cells at temperatures above zero c and induces many side effects (cardiac, neurologic, respiratory, etc.) in the patients. to reduce those side effects we use lower dmso concentration, high hematocrit resulting in lower volume of the frozen cell suspension, thus reducing the final quantity of dmso to be infused to the patients. this does not affect the cell viability or the hematologic recovery of patients after transplantation. our easily performed method for unprogrammed freezing of stem cells with final dmso concentration % at - o c is safe, well tolerated, and provides cryopreservation, which allows high viability and stable cell engraftment, while reducing the undesired side effects of dmso. disclosure: nothing to disclose the conditioning regimen consisted of melphalan for most of the patients. the average age at the time of transplantation was years ( - ). patients were transplanted with an average cell dose of , x /kg ( , - , x /kg) for the first transplantation and , x /kg ( , - , x /kg) for the second one (every patient received the same cell dose as for the first) with average cell viability , % ( - %), with little difference between first and second transplantation. results: the average time for hematological recovery was , (between and ) days for the neutrophils, and . (between and ) days for the platelets. we found no correlation between the cell dose and the hematological recovery. there was no difference in the hematopoietic recovery between the first and the second transplantation in the patients, who underwent tandem or two transplantations. conclusions: recovery time is considered by some to be a function of the effective stem cell number. we did not find such correlation, probably because in the analyzed group all the patients, except four of them, received a dose greater than x /kg cell, which is accepted as a safe dose for autologous stem cell transplantation. disclosure: nothing to disclose p plerixafor-mobilized patients have a high risk of noninfectious fever during engraftment after autologous peripheral blood stem cell transplantation background: plerixafor enables rapid and efficient mobilization of hematopoietic stem cells. however, its impact on adverse clinical events after autologous peripheral blood stem cell transplantation (pbsct) is not fully understood. fever is one of the major complications in the preengraftment phase of pbsct. in this research, we focused on non-infectious fever around the time of bone marrow recovery and investigated whether plerixafor as mobilization therapy plays a role in engraftment fever. methods: we reviewed autologous pbscts for treatment of multiple myeloma at the japanese red cross medical center between - . non-infectious fever was defined as temperature ≥ °c with onset between two days prior to and two days after engraftment without clinical or microbiological documentation of infection. results: patients were mobilized by cyclophosphamide and filgrastim in . % (n = ) and filgrastim and plerixafor in . % (n = ). the median number of transfused cd + cells were . × /kg and . × / kg, respectively (p= . ). patients transfused with plerixafor-mobilized grafts had a higher risk of noninfectious fever ( . % vs . %, p< . ). cd + cell number or cyclophosphamide pretreatment had no relationship to non-infectious fever. the recovery of lymphocytes was more rapid in plerixafor-mobilized patients (p= . ). however, the number of lymphocytes was not associated with non-infectious fever. conclusions: combination of filgrastim and plerixafor as mobilization therapy resulted in an increased risk of noninfectious fever during engraftment comparing to mobilization with cyclophosphamide and filgrastim. while the mechanism remains unclear and requires further studies, plerixafor-mobilized grafts may result in an unintended increase in engraftment fever. clinicians should be aware of this possibility if patients are transplanted with those grafts. disclosure: ks received honorarium outside the submitted work from janssen, novartis, celgene, ono pharmaceuticals, takeda, fujimoto pharmaceuticals and srl. ti received honorarium outside the submitted work from janssen, celgene, ono pharmaceuticals and takeda. we assessed the efficacy of a new conditioning regimen consisted of decitabine (dec), busulfan (bu), cyclophosphamide (cy), fludarabine (flud) and cytarabine (ara-c) for allo-hsct in patients with mds and mds/ mpn. fifty patients were enrolled, including with mds and with cmml. patients received dec mg/m /day on days - to - , combining bu/cy/ flu/ ara-c modified preparative regimen. results: at a median follow-up of ( - ) days, the overall survival (os) was %, disease-free survival (dfs) was %, and relapse incidence was %. the incidence of severe acute (grade iii/iv) graft-versus-host disease (gvhd) was %, and that of (predominantly mild) chronic gvhd was %. os at years was % for mds patients with high risk, % for mds patients with very high risk, respectively. the survival was delightful in patients with poor-risk mutations, such as tp and asxl , ( %) and with three or more gene mutations ( %). among the total patients with poor-risk mutations in our research, only one patient ( %) with tp relapsed and one ( %) with asxl and tet died. result of continuous observation after transplantation, the percentage of nk cells in the peripheral blood of all patients who had received dec/flu/bu/cy/ara-c conditioning increased at day , which may essentially contribute to disease control post-transplantation. conclusions: in summary, the addition of a -day schedule of decitabine to a flu/bu/cy/ara-c conditioning regimen has proven feasible, with a low level of toxicity and promising early disease control especially in patients with high risk mds. disclosure: there are no conflicts of interest. the sfgm-tc mds score at day is associated with post-transplant outcomes in patients with myelodysplastic syndrome who underwent cd + selected allogeneic stem cell transplant conclusions: in patients with mds undergoing tcd-hct, the sfgm-tc score at day is significantly associated with survival. the lower incidence of acute gvhd in recipients of cd -selected transplants and the use of myeloablative condition regimens, with lower relapse, may explain the difference with the original finding that the sfgm-tc was predictive at day in unmodified grafts. disclosure the most frequent grade , toxicities were thromobocytopenia and neutropenia. infections developed in patients ( . %), neutropenic fever in ( . %). five patients ( . %) either developed or experienced exacerbation of acute graft versus host disease (gvhd), nonechronic gvhd. conclusions: azacitidine use is associated with only modest activity in patients who relapse after allo-hsct. however, in patients who respond to treatment it may allow for a durable disease control. disclosure: the authors declare no competing conflicts of interest background: somatic mutations in mds patients are closely related with clinical phenotypes and prognosis in mds patients. but whether mutations are prognostic for outcomes after allogeneic hematopoietic stem-cell transplantation (allo-hsct) remains to be elaborated. methods: targeted mutational analysis were performed on samples obtained before transplantation from patients underwent hsct. we analyzed the relationship of mutations and clinical outcomes. results: all patients carried more than one somatic mutations, most frequently in kmt d( . %), arid b ( . %), ccdc ( . %), pclo( . %), asxl / ( . %), srcap( . %), u af ( . %), dnah ( . %), ush a( . %) and tet ( . %). tp mutations were associated with higher ipss-r risk, complex karyotype and monosomal karyotype. dnah were more frequent in pediatric patients. in univariable analyses, tp mutations were related with decreased disease-free survival (p= . ); dnah mutations were related with increased disease-free survival (dfs) (p= . ). in multivariable analysis including ipss-r stratification, gvhd, hct-ci and candidate genes, dnah mutations were independently associated with better dfs(p= . ). conclusions: dnah mutations is independently associated with better outcomes in mds patients treated with allo-hsct while tp may predict unfavorable outcomes. accounting for these somatic mutations may help better selection of candidates for allo-hsct among mds patients. disclosure background: there is a controversy among experts if and how patients with mds and saml should receive cytoreductive therapy before transplant. while aiming to reduce disease burden in order to lower the risk of relapse after transplant cytoreductive therapy is associated with several drawbacks. besides a considerable risk for toxicity and mortality preventing patients to proceed to transplant cytoreductive therapy may also favour the selection of resistant clones which may be difficult to treat at relapse. methods: to address this hypothesis we retrospectively analysed the response and survival following salvage therapy in patients with mds and saml who had relapsed in median . months ( to months) after allo-sct according to their pre-transplant strategy (upfront transplantation n= %; induction chemotherapy [ctx] n= %; hypomethylating agents [hma] n= %). results: the majority of these patients received salvage therapy with hma (n= , %; aza n= , dac n= ) mostly in combination with dli, while the remaining received other salvage treatments (intensive chemotherapy n= , dli alone n= , nd transplant n= , bsc n= , miscellaneous n= , missing information n= ). when focussing on those patients treated with hma and dli it became apparent that a significantly higher proportion of patients in the upfront group ( %) achieved cr after salvage therapy in comparison to pre-treated patients ( % cr, p= . ; ctx group % cr; hma group % cr). accordingly, overall survival (os) calculated from the time of relapse was significantly longer in patients in the upfront group than in the group of pre-treated patients ( -year os % vs. %, p= . ). conclusions: overall, these findings imply that pretransplant therapy may favour the iatrogenic selection of resistant clones, which poorly respond to salvage therapy with hma and dli in case of relapse after allo-sct. furthermore, the results support the concept that an upfront transplant strategy is a promising alternative for patients with mds and saml that can be augmented by salvage therapy with hma and dli. disclosure: ts and gk received travel support, lecture fees and research funding from celgene gmbh conclusions: in our country, this procedure has shown to be feasible and we hope to improve it, with better infection control and by acquiring more experience related to the management of these patients. background: extramedullar relapse of mds is a rare complication after allogeneic stem cell transplantation. we present the case of a -year-old woman who was admitted into hospital because of insecure walking. paresis of both legs, hypaesthesia of the inner thighs, increased effort at urinating, reduced sphincter tonus, central paresis of the right arm and discreet paresis of the right facial nerve were documented at neurological exam. mri showed a large tumour of the dorsal thorax that immured the adjacent ribs and spine, affected the processus transversus of t - and invaded the spinal canal. the patient had undergone ric allogeneic stem cell transplantation five years ago for mds-eb with complex aberrant karyotype. following an uneventful course and no signs of gvhd, she had been off immunosuppression since , years. at the time of the admission the patient had slightly lowered wbc ( , gpt/l) and plt ( gpt/l) and clearly increased ldh ( u/l). methods: histology of a ct-based biopsy of the paravertebral tumour showed an infiltration of the muscles by blastous cells that were cd -, cd -, pax -positive, tdt and cd a were questionably positive. provisonal diagnosis therefore was lymphoblastic lymphoma, pox tested negative. the bone marrow was hypocellular with increased numbers of mature lymphocytes, but no definite signs of malignancy. cerebrospinal fluid revealed cells/μl with % blasts. immunotype was cd , cd , cd , cd , hla-dr positive, pox and lymphatic markers were negative. because of this we finally suspected meningeosis leucaemica. we completed the diagnostic workup with genetical and chimaerism tests and compared the result to the patients' mds before allogeneic stem cell transplantation. [[p image] . mri scan of the large thoracic tumour] results: cerebrospinal fluid (csf) cells consisted of % recipient cells, whereas peripheral blood cells were % donor. high risk mds at transplant displayed a complex caryotype including trisomy and tetrasomy , now % of the cells in csf showed trisomy and % tetrasomy . chimerism and fish of the solid tumour could not be performed, coexpression of myeloid markers within the tumour is pending. conclusions: in conclusion the patient has meningeosis as a result of exclusively extramedullary relapse of myeloid blasts originating from the initial high risk mds with blast excess and complex aberrant caryotype. the evolution of a trisomy clone to tetrasomy clone in relapse is linked to extramedullar manifestations. whether the solid tumour represents myeloid sarcoma with coexpression of lymphoid markers, extramedullary relapse of mds with lymphoid differentiation or, less likely, a separate lymphobastic lymphoma, is not yet clear. disclosure background: adoptive t cell therapy with genetically engineered t cells is a potent innovative immunotherapeutic approach for cancer treatment. unfortunately, the use of t cells redirected against tumor antigens, is severely limited by ) the difficulty in identifying appropriate cell surface antigens, that could be targeted by car t cells and ) the paucity of tumor-specific t cell receptors (tcrs) against shared, oncogenic antigens. methods: focusing on wilms´tumor (wt ), a tumorassociated antigen overexpressed by acute myeloid leukemia and several solid tumors, we designed and implemented an innovative protocol for the rapid isolation of wt -specific t cells and for the generation and characterization of a library of wt -specific tcrs displaying different human leukocyte antigen (hla) restrictions, to be exploited by tcr gene transfer and tcr gene editing. to this aim, we repetitively stimulated t cells with autologous antigen-presenting cells, including immortalized b cells, pulsed with overlapping peptides spanning the entire wt protein. t cell recognition was assessed by flow cytometry in terms of cd a expression and ifnγ production. recognized peptides were mapped by a deconvoluting grid and t cell clonotypes were longitudinally tracked by tcrαβ sequencing. results: we successfully expanded tumor-specific t cells from consecutive healthy donors, in an average of rounds of in vitro stimulations. the ability of wt specific t cells to recognize naturally processed epitopes and their on-target specificity was demonstrated upon coculture with antigen-expressing targets including primary leukemic blasts. tracking of the tcrαβ repertoire during culture led to the identification of clonotypes that recognize several tumor-associated peptides and are restricted by more than hla alleles, including hla-a* : . tcrs were then expressed via genome editing. briefly, simultaneous editing of endogenous tcr α and β chain genes was achieved using crispr/cas technology (efficiency > %), followed by transduction of t cells with lentiviral vectors encoding wt -specific tcrs (efficiency > % of cd + t cells). phenotypic characterization of edited t lymphocytes showed a major enrichment of cells harboring t stem cell memory properties. functional validation of the edited t cells is currently ongoing. preliminary results of a hours coculture experiment show that tcr edited t cells kill fresh wt + leukemic blasts, harvested from hla-matched patients, with an efficiency up to % at an effector to target ratio of to , while no killing of controls is observed. conclusions: we set up a protocol enabling consistent and efficient hunting for tumor-specific tcrs with no need for labor intensive t cell cloning. tcr genes can be easily and rapidly used to redirect t cell specificity against cancer cells by tcr gene editing. disclosure: chiara bonini: research funding from intellia therapeutics p car t cell therapy targeting relapsed or refractory cd + lymphoid disease with third-generation vector rv-sfg.cd .cd . - bbzeta maria-luisa schubert , anita schmitt , leopold sellner , , brigitte neuber , angela hückelhoven-krauss , kunz alexander , lei wang , gern ulrike , birgit michels , susanne hofmann , carsten mueller-tidow , , dreger peter , , michael schmitt , background: t cells genetically engineered to express chimeric antigen receptors (carts) directed against cd have demonstrated significant efficacy in patients with iymphoid malignancies including relapsed or refractory (r/r) b-lineage acute lymphoblastic leukemia (all) or r/r b-cell non-hodgkin's lymphoma (nhl). access to cart treatment for patients in europe has been limited so far given that the vast majority of cart trials have been performed in the united states and the p. r. of china. here we present the preliminary results of the first investigator-initiated trial (iit) cart trial in germany. hd-car- (eudract no. - - ; nct ) is a phase i/ii trial with in-house cart manufacturing which was initiated in september at the university hospital heidelberg. methods: adult as well as pediatric patients with r/r all and patients with chronic lymphocytic leukemia (cll) or nhl including diffuse large b-cell lymphoma (dlbcl), follicular lymphoma (fl) or mantle cell lymphoma (mcl) are treated with autologous t lymphocytes transduced with a third-generation car retroviral vector (rv-sfg.cd .cd . - bbzeta) targeting cd . the main purpose of hd-car- is to evaluate safety and feasibility of escalating third-generation car t cell doses ( - × transduced cells/m ) after lymphodepletion with fludarabine and cyclophosphamide. patients are monitored for cytokine release syndrome (crs), car-t-cell related encephalopathy syndrome (cres) and/or other toxicities. in vivo function, survival and anti-tumor efficacy of carts are assessed. results: to date, three patients (cll, dlbcl and mcl, respectively) have been enrolled and subjected to leukapheresis. high numbers of transduced carts were harvested on day of culture ( - x carts). transduction efficiency ranged between and %. cart products were sterile and free from mycoplasms and endotoxins. no production failure occurred and all patients received the cart product. no signs of crs or cres > grade have been observed. assessments of clinical responses are pending and will be presented at the conference along with updated technical results. conclusions: for hd-car- , gmp-conform leukapheresis as well as cart manufacturing was effective. administration, patient monitoring and follow-up were performed in-house providing independency from transport or production sites outside the university hospital heidelberg, altogether suggesting that academic cart iits are feasible in germany. clinical background: the prognosis of adult patients (pts) with relapsed/refractory (r/r) precursor b-acute lymphoblastic leukemia (all) is dismal, including with allogeneic hematopoietic stem cell transplantation (allo-hsct). blinatumomab, a bispecific cd -directed cd t-cell engager and inotuzumab ozogamycin (io), a cd -directed antibody-drug conjugate revolutionized the field, improving their outcomes. anti-cd chimeric antigen receptor t (cart) cell therapy has led to further progress and improved outcome (jacoby e; am j hematol, ). nowadays, patients with r/r b-all can be offered both therapies, but there are limited data on the safety and efficacy of cart -cell therapy post antibody treatment. we detailed our single center experience in this regard. methods: this report is a part of a single center, phase b/ study on therapy of b-cell malignancies with locally produced cart-cells (nct ). the approach uses autologous t cells with car construct that is composed of an anti-cd single-chain fv, cd co-stimulatory and cd -zeta intracellular domains. cd expression on the blasts was documented prior treatment in all pts by flow cytometry. all pts received x /kg cart-cells after lymphodepletion with fludarabin and cyclophosphamide. results: six pts ( males and females) with r/r b-all were enrolled, including one with ph-positive b-all. the median age was years ( - ). median number of prior therapy was ( ) ( ) ( ) . five pts had prior allo-hsct. four pts were given antibodies as the last therapy prior to cart cells. two pts received blinatumomab resulting in pr in one of them. two additional pts received io ( after failing blinatumomab) achieving mrdpositive cr. cytokine release syndrome occurred in all pts and was severe in only one patient who required tocilizumab treatment. this patient was also the only patient who experienced neurotoxicity (grade ), and was treated with dexamethasone. this patient eventually died days post infusion of cart cells due to severe pseudomembranous colitis, toxic megacolon and sepsis. all pts had prolonged neutropenia for a median of days ( - ) after the infusion of cart cells. at day after infusion of cart-cells the cr for the entire cohort was %: three pts with mrd-negative and one with mrd-positive response. among the four pts who received antibodies prior the cart-cells, one patient had mrd-positive and two pts had mrd-negative response. the patient with ph positive b-all had progressive disease during the treatment. two pts were referred to second allo-hsct from other donors. one patient with mrd-negative response relapsed after the second transplant and was treated by salvage therapy. the second patient with mrdnegative response demonstrated prolonged remission ( months) even without second transplantation. with a median follow-up of months ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) the median progression-free and overall survival for the entire cohort were . and months, respectively. conclusions: autologous anti-cd car t-cell therapy after debulking treatment with antibodies, including blinatumomab and/or io, is feasible and results in high response rates in pts with r/r b-all. patients may respond to anti-cd car t-cell therapy even after failure to their last salvage therapy with blinatumomab, which demonstrates similar mechanism of action. clinical background: genetically engineered t cells expressing a chimeric antigen receptor (car-t) targeting specific antigens present on acute lymphoblastic leukemia (all) blasts have generated promising results in children and adults with relapsed and refractory disease. the below report provides an insight of lineage switch occuring as a result of intense immunological selection after car-t cell therapy, even with a tumor clone that has no potential for this switch. methods: an eight year old caucasian male with precursor b (pb) cell lymphocytic leukemia was treated with cd directed car-t cell therapy in third remission, and after relapse after previous bone marrow transplantation (bmt) . he was diagnosed with t( ; ) pb cell all at years of age and treated with bfm protocol. he relapsed months after completion of maintenance therapy, and had a / mmud bone marrow transplant after etoposide, tbi and alemtuzumab conditioning therapy. he had cutaneous acute and chronic gvhd but months post-transplant, he relapsed again with pb cell all, with the same cytogenetic and immunophenotypic disease characteristics.. he was treated with lymphodepleting chemotherapy with fludarabine, cyclophosphamide and alemtuzumab followed by infusion of cd directed car t cells. he developed cytokine release syndrome of grade severity manifested as persistent fever, associated with car t cell expansion in the blood. after car-t infusion,there was no detectable b cell all clone in the marrow by pcr and the cytogenetics were negative for t( : ) translocation. months after the car t cell therapy, he was found to have a mrd positive disease which was monitored closely. results: we document clonal evolution from cd negative, mrd positive disease to aml, with the same ig rearrangement (the same clonal disease) but with complete myeloid phenotype mpo, cd , cd positive disease. there was cytogenetic evolution of the underlying clone but the original t( ; ) was retained within the evolved karyotype. sadly, our patient developed fludarabineneurotoxcity during an attempt to induce aml remission, and further curative-intent chemotherapy was not possible. conclusions: there are two case reports of mll rearranged b-all acquiring a clonally related myeloid phenotype associated with cd -negative escape after cd directed car t cell therapy,so far. but, this is the first post car-t cell therapy transformation of all to aml with etv -runx mutation, which is not recognised to have such lineage-switch potential. unlike mll, etv -runx translocation in the pathogenesis of acute myeloid leukemia is not been reported in the literature. the theory behind such transformation is an intense immunological selection of the tumor, driving it to myeloid differentiation with additional clonal cytogenetic events. disclosure: nothing to declare p ivac-all- : interim analysis of a phase i/ii clinical study on personalized peptide vaccination based on patient-individual tumor-specific variants in relapsed pediatric acute lymphoblastic leukemia armin rabsteyn , , christopher mohr , olaf witt , , roland meisel , , cristiane chen-santel , tobias feuchtinger , , christopher schroeder , jakob matthes , background: acute lymphoblastic leukemia (all) is the most common pediatric malignancy. standard chemotherapy is a successful treatment in % of patients, only about % develop a relapse, however these patients have a dismal prognosis. prevention of relapse after firstline chemotherapy or stem cell transplantation (sct) is therefore an urgent clinical need. we established a platform for the design of patient-individual peptide vaccination cocktails by combination of whole exome sequencing of tumor and normal tissue with in silico epitope prediction algorithms for individual patient hla types. we started clinical translation of this approach by starting a phase i/ii clinical trial in (nct ). besides feasibility and toxicity assessments, we aim to assess the capability of the peptide vaccination to induce neoantigen-specific t cell responses in high-risk all patients to target residual tumor cells and prevent leukemic relapses. methods: key inclusion criteria are: pediatric patients with all who suffered from second relapse after standard therapy or first relapse after sct. hematological remission has to be reached prior to vaccination. nonsynonymous mutations are identified by whole exome and transcriptome sequencing of patient leukemic blasts and healthy reference tissue. hla binding peptides harboring the altered amino acids are subsequently predicted in silico by algorithms syfpeithi, netmhc and netmhcpan for the patients' individual hla type. vaccine cocktails consisting of - individual peptides are produced and formulated under gmp conditions. the vaccination schedule is vaccinations over weeks using gm-csf and imiquimod as adjuvants. response to the vaccination is monitored by detection of t cells recognizing the vaccinated peptides occurring over time in peripheral blood of patients by prestimulation and intracellular cytokine staining. results: until now, patients were recruited, for of those, whole exome sequencing was performed to identify all-specific snvs using a comparative bioinformatics pipeline. we found an average of . mutations per patient on dna level. based on these data, an average of hla binders derived from neoantigens could be predicted per patient. an average expression of . % of mutations was assessed by rna sequencing. in all cases validated mutations could be identified and cocktail design was feasible. until now, patients received vaccinations. the vaccine was generally well tolerated and no or only mild side effects were observed. immune monitoring was performed for patients until now. in the first patient, we observed a transient cd + response against one vaccinated mhc class ii ligand and a sustained cd + response against the included wildtype control peptide derived from the antigen survivin. in the second patient, immune monitoring was performed for the first vaccination timepoints, a t cell response was not measurable at this timepoint of vaccination. conclusions: whole exome sequencing of pediatric all patients is feasible and yields small amounts of expressed, tumor-specific mutations. these few mutations are sufficient to predict hla-binding peptides that can be used to formulate individualized peptide vaccine cocktails. we currently conduct a clinical phase i/ii trial in a small cohort of high-risk all patients to assess safety, toxicity and immunogenicity. clinical background: chimeric antigen receptor t cells (cart) are considered as gene therapy medicinal products (gtmp) and genetically modified organisms (gmo). hence, carts manufacturing for clinical application is strictly regulated. appropriate methods assessing car transgene copy number (cn) in a cart product and definition of the frequency of carts in treated cart patient are mandatory. although quantitative real-time pcr-based (qpcr) analysis has been used for this purpose, no standardized procedure to minimize systematic errors and enable comparability has been established yet. here, we report on a single copy genebased (scg) duplex (dp) pcr (scg-dp-pcr) for the determination of the vector copy number (vcn) in cart products as well as patient samples following cart administration. scg-dp-pcr was validated and compared to the broadly used absolute copy number qpcr (acn) approach within the framework of a clinical trial treating patients with good manufacturing practice (gmp)-grade carts (hd-car- ). methods: for conventional acn, primers and probe targeting the car vector rv-sfg.cd .cd . - bbzeta were designed. standard curves were established via serial dilutions of the sfg.car plasmid. amplification of the standard curve as well as target genomic dna for vcndetermination was performed as singleplex (sp) pcr (sp-car) (method a). on the same qpcr plate, duplex (dp) qpcr reactions were carried out. additionally to the components comprised within method a, the experimental setup contained haploid human genomes as well as primers and probe targeting ribonuclease (rnase) p as human scg. the amplifications for the sfg.car plasmid (dp-car) and the rnasep gene (dp-rnasep) were performed simultaneously (scg-dp-pcr; method b). scg-dp-pcr was performed for standard curves and target samples. target-sample dna was extracted from carts prepared from leukapheresis products of three healthy donors (hd). results: for method validation, efficiency and linearity (correlation coefficient) of the qpcr reactions of method a (sp-car) and method b (dp-car, dp-rnasep) were assessed by linear regression of the pcr signal to the reference standard curve. overall, standard curves were only considered valid if a correlation coefficient (r ) of above . and efficiencies of % ± % were achieved. vcns applying method a and b to the same target sample were compared. sp-car pcr reaction displayed efficiency of . ± . %; . % ± . % and . ± . % efficiencies were achieved for dp-car and dp-rnase pcr reaction, respectively (table ) . applying scg-dp-pcr using formula for relative cn assessment ( -Δct (dp-car -dp-rnasep) ) on hd samples resulted in an average of . ± . increased cn when compared to method a (table ) . conclusions: in terms of efficiency and linearity by linear regression qpcr reactions were comparable. validation of scg-dp-pcr was achieved and represents an exact and less error-prone method to fulfil regulatory safety release criteria of cart products. besides of accurately assessing vcn of transduced cells, scg-dp-pcr is also a highly robust method to follow-up carts in treated patients. applying this approach, no standard curve is needed, hence significantly economizing required material as well as time. disclosure: nothing to declare background: t-cells' antileukemic responses in aml-pts need to be improved. dc leu effectively activate t-cells against leukemic blasts, resulting in blast-lysis ex-vivo. factors influencing these activities are not known. methods: we generated dc/dc leu from aml-blastcontaining mononuclear cells (n= ) using standard methods (mcm-mimic/ca-ionophore/picibanil/ifn-α, "mnc-methods") and from blast-containing heparinized whole blood (n= ) using modulatory kits (various combinations of - clinically approved response-modifiers, "wb-kits", patent ) and correlated statistically (t-/u-test, pearsons correlation, # means significant) proportions of dc-/t-cell-subtypes/cytokine-profiles with t-cells' antileukemic cytotoxicity (ctx), achieved after mixed lymphocyte culture (mlc) with/without mncmethod-("t*dc mnc "/"t*bla mnc ") or wb-kit-treated cultures ("t*dc wb "/t*bla wb "). ctx was given as proportions of cases with achieved or "improved" blast-lysis compared to control and as frequencies of viable blasts (bla via ) after effector-cell-influence. pooled data and data obtained with single methods in different cohorts are given. results: . generation of dc/dc leu : with a) mncmethods: Ø ± % dc and Ø ± % dc leu and b) wb-kits: Ø ± % dc and Ø ± % dc leu without induction of blasts' proliferation. t-cell-proliferation increased (vs uncultured t-cells) after mlc with a) mnc-methods: Ø ± % vs Ø ± % and b) wb-kits: Ø ± % vs Ø ± %. . antileukemic reactivity (t-effector-cell-cytotoxicity after mlc): pooling all data: a) mnc-methods ("t*dc mnc " vs "t*bla mnc "): we found a ) blast-cytotoxicity in Ø %(vs %) of cases with Ø %(vs %) bla via , a ) blast-cytotoxicity was improved (vs control) in % with Ø decrease of bla via of %; b) wb-kits ("t*dc wb " vs "t*bla wb "): we found b ) blastcytotoxicity in Ø %(vs %) of cases with Ø %(vs %) bla via , b ) blast-cytotoxicity was improved (vs control) in % of cases with Ø decrease of bla via of %. in general, these results could be confirmed with single methods: best mnc-methods were picibanil and mcm-mimic, best wbkits were kits containing gm-csf+picibanil or prostaglandins. . correlations: pooling all data: cases with "improved" lysis (vs "not improved" lysis) were characterized by a) mnc-methods: increased proportions of mature dc/cells (Ø ± % vs Ø ± %), dc leu /cells (Ø ± % vs Ø ± %) and proliferating t-cells (Ø ± % vs Ø ± %), b) wb-methods: dc/cells (r= . # ), dc leu /cells (r= . # ), dc leu /bla (Ø ± % vs Ø ± %), dc leu /dc (Ø ± % vs Ø ± %), cd + t-cells (Ø ± % vs Ø ± %), ifn-γ (r= . #) , mcp- ( ± vs ± pg/ml). conclusions: blasts are regularly converted to dc leu in the presence of mnc-methods and wb-kits (simulating the in vivo microenvironment). t-cells' coculture with dc/ dc leu after mlc induces and improves antileukemic t-cell activation compared to controls. blast-cytotoxicity correlates with proportions of dc/dc leu -and t-cell subtypes and released cytokines. these data support a role of antigen presentation by leukemic cells (dc leu ) for the stimulation of an immune response in aml in vitro and possibly in vivo. disclosure: nothing to declare evaluation year after the launch of the motion comic immuno-t, explaining patients and their caregivers how immunotherapy strategies work background: one year ago, the first version of immuno-t, a motion comic explaining to patients and caregivers how immunotherapy strategies work, was released. people were informed on and inspired to use the application during (inter)national meetings and events for the general public. meanwhile, the motion comic was further refined and adapted into a second version, based on the evaluations we've collected on the first version. adaptations included a multi-language tool (currently languages), increased user friendliness, and a new supporting musical score. also, a website was launched from which the second version could be downloaded on tablet or smartphone (both android and apple) and a new online evaluation form could be filled in. in months, people have evaluated the motion comic online, and these results are presented here, as well as our future plans within the immuno-t program. methods: through an online questionnaire, participants from belgium (n= ) and the netherlands (n= ) have evaluated the dutch version, and belgian participants evaluated the french version of the motion comic. results: the total group (n= ) consisted of patients (n= ) and their families (n= ), general public (n= ), students (n= ), health care professionals (n= ), researchers (n= ) and kindergarten teachers (n= ). participants' age ranged from to years, with an even distribution amongst the different generations. the majority of the evaluators ( , %, n= ) thought the motion comic is a good way to explain immunotherapy to patients. individuals ( , %) felt mainly interested after watching immuno-t, and a total of participants ( , %) felt hopeful or motivated. focussing on the patient group (n= ), all of the responders think the immuno-t motion comic is a good tool to use in a patient-doctor consultation. patients ( , %) felt hopeful and/or motivated after watching the whole motion comic, while of them ( %) felt combative and ( , %) felt gripped and intrigued. as for the new musical score, participants ( , %) think the music is suitable for the app, while evaluators ( , %) think the new music is not or not at all fitted to support the motion comic. conclusions: the detailed evaluations allow us to further improve immuno-t, and aid us in the development of other motion comics we plan to release under the cancer-t in motion umbrella. with the current version of immuno-t, a single-center pilot study is being set up, to test the efficacy and usability of immuno-t, based on qualitative research during the experience of the tool, and using validated questionnaires. with this study we want to evaluate the impact of immuno-t on patient empowerment, and the decision making process. the study protocol will be presented at ebmt. disclosure: the development of immuno-t was partly financially supported by celyad, calgene, novartis, roche, amgen, bms, but these companies did not by any means influence the contents and development of the motion comic. a therapeutic strategy to trespass the blood brain barrier for adoptive nk cell therapy in glioblastoma multiforme induced rat: a preclinical study background: glioblastoma multiforme (gbm) is among the most common and aggressive primary brain tumors with very poor prognosis. according to the central brain tumor registry of the united states, central nervous system (cns) tumors in pediatric patients (ages between - years old) are the second most common malignancies after blood-born malignancies, and the first amongst solid tumors, and known the most common cause of cancer-related deaths. although hematopoietic stem cell transplantation has been exploited to treat many kinds of malignancies, currently its success rate in gbm is limited. therefore, the gbm treatment paradigm needs shifting towards more effective treatments such as immune cell therapy. natural killer (nk) cells have been recognized as potential anti-cancer effector cells, as they can recognize and target tumor cells. since a small percentage of blood cells are differentiated as nk cells, the number of this group of cells is hardly enough to fight tumors, and so their multiplication and activation would be a potential effective cancer treatment. methods: this preclinical study was focused on setting up an optimal protocol for expansion and activation of naïve nk cells and assessing their efficacy towards induced gbm in rat models. ex-vivo expanded and interlukin- (il- )and heat shock protein- (hsp- )-treated nk cells have been exploited. after in vitro study and confirming the efficacy of treated cells through cytotoxicity assays, we induced gbm in male wistar rats (weighted - gr) using c tumor cells injection in rat brain through stereotactic surgery. the tumor formation was proven by mri imaging. following tumor establishment, we analyzed the effect of single injection of il- -and hsp- -treated nk cells compared with single injection of non-treated nk cells in two groups of rats. results: systemic intravenous delivery of il- -and hsp- -treated nk cells through tail's vein resulted in tumor shrinkage in different time intervals and complete remission in the first group of gbm-induced rat models, whereas in the other group of gbm rats receiving untreated nk cells, the tumor progressed. therapeutic efficacy of the treated nk cells was ascertained compared with non-treated nk cells considering tumor shrinkage observed in mri and survival rates between the two model groups. conclusions: the amelioration of tumor which has been confirmed by mri, proved the migration of activated nk cells through blood brain barrier and homing to cns, and finally targeting gbm tumor cells. our data suggest that nk-cells treated with il /hsp may be a promising immune cell-based therapeutic approach towards treating the fatal gbm. disclosure: nothing to declare p abstract withdrawn. long term sorafenib response for extramedullary flt + aml relapse after allogeneic stem cell transplantation since june , sorafenib dose has been tapered to mg/day, due to mild skin and gi toxicity. after years of treatment, she maintains cr at medullary and extramedulary levels, with no evidence of a disease that had escaped the mechanisms of action of chemo, hsct and dli. conclusions: in our patient, treatment with sorafenib has provided long-term control of this refractory extramedullary disease, even at adjusted doses. further studies are needed to confirm the efficacy of flt inhibitors in the control of relapses after allo-hsct, extramedullary disease and its potential role as maintenance agent. disclosure: nothing to declare background: although chemotherapeutic(ct) agents that used in the treatment of acute lymphoblastic leukemia (all) increase survival, the results are still weak. longterm survival with ct's in relapse all cases is difficult and the prognosis is very weak. inotuzumab ozogamicin is an anti-cd monoclonal antibody and it has the potential to reduce the overall toxicity of intensive regimens for all, as well as to possibly increase the number of patients who may achieve a state of minimal residual disease. methods: -year-old male patient was diagnosed with b-cell all in december .after the hoelzer kt protocol was started, maintenance treatment was continued. in the fifth month of treatment,flag ct protocol was started cause of recurrence was seen on % blast detection in peripheral blood smear. in august , inotuzumab ozogamicin treatment was started and six cures were completed because the patient was not in remission. in september , he had gone haploidentical bone marrow transplantation from his sibling donor( / )with defibrotid prophylaxis for veno-occlusive disease(vod)s. he engrafted succesfully and chimerizm was . % in th days of transplantation. he is th day of transplantation and in a remission. results: bone marrow transplantation cannot be performed since the complete response cannot be achieved in patients with relapse and resistant b-all.in these patients, new therapies targeting malignant lymphoblasts are needed. inotuzumab ozogamicinis a monoclonal antibody drug conjugate that targets cd antigen on malignant lymphoblasts.in many studies, it has been shown that inotuzumab ozogamycin is effective and reliable anti-tumor activity in adults with recurrent and resistant cd positive all. however, monoclonal antibody drug conjugates have been shown to be associated with vod's.for this purpose, we used defibrotid to protect our patient from vod. conclusions: treatment with combination ct regimens in b-all is suboptimal and long-term survival is achieved in only - % of patients.targeted molecular therapy and new regimens are needed in relapse and resistant patients.at this point, inotuzumab ozogamycin is an anti-cd- monoclonal antibody, as in our case, it provides remission in recurrent and resistant b-all patients and allows patients to complete their treatment with an allogeneic transplant from a fully compatible donor. disclosure: nothing to declare background: mesenchymal stem cells (mscs) are an attractive consideration for therapeutic cures of many difficult diseases on the cellular-level. due to the trophic effects of the cytokines and chemokines that they produce, mscs have shown multiple beneficial properties in the field of oncology. in this study, we will be investigating the effect of mscs derived from human bone marrow (bm), adipose tissue (at), and umbilical cord derived mscs (uc-mscs) on ovarian cancer. to differentiate the mscs, we performed a comparative analysis between the various sources for proliferative capacity, surface antigen expression, differentiation ability, tumor marker and paracrine activity, and their influence on ovarian cancer cell proliferation. methods: measurements of ovarian tumor marker proteins were computed by elisa. proliferative effects, immunomodulatory effects, and apoptosis of the mscs were measured by the culture and counting of colony formations. flow cytometry (fcm) was used to measure the variation of the immunophenotyping and cytokine secretions in co-culture, as well as gene expression. results: cells noticeably proliferated without any modifications to their immunophenotype during the third subculture. the colony-forming unit fibroblast (cfu-f) test showed a proliferation of the mscs along with healthy cells and cancer cell lines with no changes in their phenotype. the supernatant of mscs showed an increase in cellular death of the ovcar in ovarian cancer cell lines. a reduction in the level of ca- ( - %; p= . ) with ovcar in co-culture, and a decline of ldh ( - %; p= . ) and beta-hcg ( - %; p= . ) were observed in co-culture in caov , skov and igrov cell lines. a decrease in cd of the cancer cell lines in co-culture with the msc supernatant showed a reduction of the cancer tumorigenicity and aggressiveness, while the rate of the cd and cd asserted their stem state. msc supernatant decreased cell proliferation and mmp- , mmp- , and ca- mrna expression, while increasing timp , , and . this suggests that mscs have a role in cell death and inhibition of ovarian cancer cell proliferation. an increase of anti-inflammatory il- and il- cytokines, and a decrease in growth factor gm-csf along with their proinflammatory inf-a, tnf-a, il- , and il- a cytokines were also noted. conclusions: the gene and cytokine activity indicate a potential therapeutic anti-inflammatory and antiproliferative role of mscs on ovarian cancer despite their sources. the reduction of ca- , ldh, and beta-hcg in co-culture, along with the decrease in cd and amplified cellular apoptosis demonstrate the beneficial effects of stem cells in ovarian cancer cell lines. disclosure background: hematopoietic stem cell transplant (hsct) is the only cure in sickle cell disease (scd) so far. because of the risk of toxicity, its indication in france is restricted to severe patients with match sibling donor. this study compares the incidence of severe acute toxicity after hsct, between children aged less than years and teenagers aged to years old, treated for scd. methods: all patients suffering from scd, aged less than years at transplant, who received hsct in chu robert debré and necker, between / / and / / , were included. severe acute toxicity, defined by onset of severe acute gvhd, organ toxicity or infection, was compared between the two groups of age. results: patients ( children and teenagers) were included. all patients received a myeloablative conditioning regimen. bone marrow from a sibling donor was the main stem cell source (n= ; %). neither death nor rejection was observed with a median follow-up of . months (range, . the incidence of grade iii-iv acute gvhd was . % and was similar between the two groups; no risk factor was identified in univariate analysis. teenagers had more frequently acute skin toxicity ( . % vs %, p= . ). in univariate analysis, patients presenting severe organ toxicity were significantly older than others ( . vs . years old, p= . ). teenagers were more frequently treated for bacterial ( . % vs . %, p= . ) or bk virus ( . % vs . %, p= . ) infections. in univariate analysis, patients who developed infection were also significantly older at time of transplant (respectively . vs . years old, p= . ). no severe sinusoidal obstruction syndrome was observed. regarding long-term toxicity, patients presented an extensive chronic gvhd, they were both aged less than years old. no cut-off of age could have been defined. conclusions: this study confirms the excellent results of hsct in scd, with a -year event-free survival and overall survival of %. an older age at transplant seems to be associated with more frequent severe acute toxicity. these results are consistent with previous studies and suggest that hsct should be performed as soon as possible, without any defined "best age". prospective studies are needed, in order to define the place of each therapeutic in scd, with the aim of reducing treatment-related toxicity and developing alternative strategies for patients without match sibling donor. disclosure: nothing to declare p new insights into risk factors for transplant-associated thrombotic microangiopathy (ta-tma) in paediatric hsct (n= ) was associated with ta-tma in % vs . % vs . % respectively (p= . ). the presence of comorbidities at d (n= ) was significantly associated with an increased risk of ta-tma . % vs . % in absence of co-morbidity (n= ); p= . . use of csa/tac-based gvhd prophylaxis was associated with less ta-tma with an incidence of % vs % if these agents were not included (p= . ). in univariate analysis ta-tma was significantly higher among patients with agvhd grade ii-iv ( / ; . %) vs grade -i ( / ; . %) (p= . ). pres was recorded among cases and % of them developed ta-tma. two out of the patients with ta-tma had pathological gene mutations in their complement pathway. on multivariate analysis the presence of active comorbidity was a risk factor for ta-tma (or: . ; % ci: . - . ; p= . ) while the use of csa/tac-based gvhd prophylaxis did not increase the risk for ta-tma (or: . ; ci: . - . ; p= . ). in the presence of comorbidities the use of defibrotide as prophylaxis or therapy for vod (n= ) was associated with a drop in the incidence of ta-tma from % ( / ) in absence of defibrotide to % ( / ). -year overall survival was significantly lower among ta-tma cases ( %) in comparison to . % in absence of ta-tma (p= . ) (figure ). conclusions: active co-morbidity is a significant risk factor for ta-tma. use of defibrotide prophylaxis in patients with co-morbidities at the time of hsct might offer protection against ta-tma. surprisingly the use of csa/tac based gvhd prophylaxis is not a risk factor for ta-tma probably through limiting the development of high grades agvhd. the association between pres and ta-tma suggests a common pathway of endothelial damage background: gonadal impairment is a severe late effect of myeloablative conditioning regimes with significant impact on quality of life of cancer survivors. the aim of this study was to analyze gonadal function after busulfan (bu) or treosulfan (treo) containing regimens with regard to pubertal stage. methods: this was a retrospective, multicenter study involving patients treated in pediatric ebmt centers between - . patients receiving myeloablative doses of bu or treo as part of hsct conditioning were eligible for inclusion. analysis was conducted in two groups according to pubertal status at time of hsct. results: patients (pts) were treated in pediatric ebmt with bu or treo before allogeneic hsct. the median age at transplant was . years (range - ); / ( %) were males (m), / ( %) were females (f). / ( %) pts were pre-pubertal at hsct (f= ;m= ) and / ( %) were post pubertal (f= ;m= ). / ( %) patients received bu (f= ;m= ), / ( %) were pre-pubertal. / ( %)(f= ;m= ) received treo, / ( %) were pre-pubertal ( figure ). females who received treo in pre-pubertal stage (n= / ) reached more often spontaneous puberty ( % vs %; p= . ) compared to pre-pubertal bu group (n= / ) and occurrence of menarche was higher in treo group (p< . ) hormonal replacement therapy was given in / ( %) females transplanted in pre-pubertal stage and in / ( %) of those transplanted in post-pubertal stage. / ( %) males were pubertal at last follow-up and of them ( %) performed sperm analysis ( oligo-azoospermic, unknown). three pregnancies were reported in the population group, all received bu. regarding the evaluation of hormonal levels in pubertal patients at time of hormonal dosage (median . yrs) ( bu and treo), males treated with treo had significant lower lh levels (p = . ) compared to bu group. females treated with treo had significant lower levels of lh and fsh (p= . and p= . respectively). conclusions: gonadal damage related to treo was significantly lower compared to bu. we observed that: females transplanted during pre-pubertal period had spontaneous puberty more frequently after treo compared to bu and that hypogonadism hypergonadotropic was more frequent after bu than treo. these results must be further confirmed on a larger population. background: viral infections significantly contribute to both morbidity and mortality in patients undergoing hematopoietic stem cell transplantation. traditional antiviral therapy is associated with lack of efficacy, potential toxicity, prolonged hospitalization and increased patient costs. viral specific t cells can be manufactured from donor blood to treat viral infections post-transplant, and are associated with increased clinical efficacy and low toxicity. we postulated that direct costs of vst therapy are lower compared to traditional anti-viral medications methods: vsts were manufactured according to local protocols and fda requirements. total drug cost (as per institutional charges per drug) was calculated for patients who required treatment for viral infections post-hsct. manufacturing costs of vsts are fixed per fda requirements. patients who were treated with investigational antiviral medications (including brincidofovir) were excluded from analysis. patients treated with vsts +/anti-viral medications over a year period were compared to patients treated only using traditional anti-viral medications, including cidofovir, ganciclovir, valganciclovir, foscarnet and rituximab. results: demographics are shown in table . there were no major differences between the two groups treated. the number of anti-viral medications used in the vst group was lower compared to the anti-viral treatment group. median cost of vst treatment was significantly lower compared to those threated with traditional anti-viral therapy ($ , vs $ , , p-value= . ) . conclusions: treatment with vsts post-hsct for viral infections was lower in cost compared to anti-viral medical therapy. it is likely that overall costs are further reduced with vsts due to reduced inpatient hospital time, less monitoring of labs associated with anti-viral medication side-effects and reduced ancillary costs including nursing and pharmacy. more studies are needed to examine these indirect costs further. background: dock deficiency is an autosomal recessive primary immunodeficiency (pid) disease caused by loss-offunction mutations in the dock gene ( ) . patients with dock deficiency present with multiple abnormalities of the immune system, including defective t cell function and impaired production of antigen-specific antibodies. these lead to persistent viral infections of the skin, mucocutaneous candidiasis, recurrent sinopulmonary infections, atopic dermatitis, and other allergic disease, malignancies, and sometimes autoimmunity ( ). hematopoietic stem cell transplantation (hsct) is currently the only curative treatment option available ( ) . methods: we retrospectively evaluated our patients who underwent allogeneic hematopoietic stem cell transplantation due to dock deficiency in ege university pediatric stem cell transplantation unit between and . results: we identified patients transplanted at a median age of . years (range: - . years). of patients; (% ) received hsct from matched sibling, (% ) from unrelated donors and patient from haploidentical donor. we used busulfan-based myeloablative conditioning regimen to patients (% ), reduced toxicity myeloablative regimen with treosulfan to patients (% . ) and nonmyeloablative regimen to patients (% . ). eight of the recipients received bone marrow, of the patients received peripheral blood stem cells, of the recipients received cord blood as stem cell source. fifteen of patients (% ) had achieved engraftment and median follow-up of patients was months ( - ). grade iii-iv acute graft versus host disease (gvhd) occurred in % of patients and chronic graft versus host disease was seen % of patients. one patient received cord blood from unrelated donor did not engraft and died from septic shock. four patients died from transplant related toxicity. our patient's survival was % ; / patients alive. conclusions: hsct is the only curative treatment option for dock deficiency. in particular, patients with high comorbidity scores have a high risk of toxicity and toxic death. therefore, reduced toxicity conditioning regimens should be used for these patients. references : background: eltrombopag, a low-molecular-weight synthetic nonpeptide thrombopoietin receptor agonist (tpo-r), is a second-generation tpo. it is an oral thrombopoietin mimetic licensed in chronic immune thrombocytopenic purpura that induces platelet maturation and release by binding to c-mpl receptors on megakaryocytes. in a recent study; for patients with refractory saa, eltrombopag induced a response (at weeks) in at least one hematologic lineage in % patients and % no longer required platelet transfusions. and also % patients became rbc transfusion independent and % had a neutrophil response. trilineage responses were seen in % of patients; although surprising, this might indicate stimulation of c-mpl receptors on remaining stem cells. delayed recovery from thrombocytopenia is common after stem cell transplantation. in a study including adult patients, eltrombopag was used to enhance platelet recovery for post-hsct thrombocytopenia. it is well tolerated and efficacious offering transfusion independence. methods: in our retrospective study, eltrombopag ( mg/day) was started in pediatric patients (age ranging from to years with a median age . years) for posthematopoietic stem cell transplantation (hsct) thrombocytopenia. all patients fulfilled the following criteria: ( ) undergone hematopoietic stem cell transplantation (hsct), ( ) had improved total leucocyte counts after leucocyte engraftment, ( ) had prolonged thrombocytopenia (< . ) needing platelet transfusion. results: four of the patients have received ric while patients ma conditioning regimens before hsct. two haploidentic, autologous, mud, msd transplantations were performed. et ( mg/day) was started in patients who had thrombocytopenia despite neutrophil engraftment on the + th day of hsct a reduction in platelet transfusions and a platelet count of more than , were the primary endpoints. before et treatment, bone marrow biopsy was checked in / patients, / patients had decreased number of megacocyocytes. none of the patients had active bleeding at the start of eltrombopag but they were all at high risk of bleeding. according to the platelet monitoring, patients had a dose increase starting from the second week. the number of patients in need of platelet transfusions was at the end of the first month; and only at the end of the nd month. all patients had a thrombocyte count of more than . in the third month. in patients, et was discontinued after - months. no dose limiting toxicities have been observed. conclusions: as a conclusion, et was found highly effective for posthsct thrombocytopenia, with no drug related adverse effects. there was a gradual increase in platelet count, and none of the patients had any complication due to thrombocytopenia. disclosure background: isavuconazole (isa) is a new triazole approved for ifi treatment in the adult population. advantages are activity against both moulds and yeasts spp, excellent bioavailability after oral administration without relevant food or gastric ph effect, a water-soluble prodrug developed to facilitate intravenous administration without nephrotoxic excipients such as β-cyclodextrin, potentially poor drug-drug interactions. isa does not currently appear to require tdm. isa safety and efficacy have not been yet established in children and, in particular, no data are available in the pediatric hsct setting. methods: italian association pediatric hematology oncology (aieop) multicentric analysis of a cohort of allogeneic hsct pediatric patients who received isa as ifi treatment or prophylaxis. due to the lack of recommended dosing in pediatric patients and a clear target isa plasma trough-level range, the therapeutic monitoring (tdm) of isa concentrations was applied by a validated liquid chromatography-tandem mass spectrometry (hlpc-ms/ ms) assay technique. isa trough plasma concentrations (c ) and hours after drug intake (c h) were measured. results: a total of allo-hsct recipients were included, (m/f / ); median age: , years, range - , median body-weight , kg (range - ). isa was used as ifi treatment in cases and as prophylaxis in patients. donors were haploidentical in patients, matched-sibling in , allogenic-unrelated in cases. according to eortc criteria, ifi was proven in patients ( penicilum, mucor, aspergillus fumigatus), probable in and possible in patients. lungs were the main localization ( cases), associated with cns involvement in cases and paranasal sinuses in ; patient had possible hepatic candidiasis. all patients, but one, received isa as rescue treatment for previous therapeutic failure ( ambisome, voriconazole, combination therapy, posaconazole). seven patients received only iv isa, received only oral isa whereas patients received both iv and oral isa. patients under kg body weight received half isa dose ( mg tid loading dose on days and , mg/die manteinance). the others received adult schedule; only patients received loading dose. isa was administered for a median of days (range: - ). in patients isa was administered in combination with caspofungin. tdm was applied to patients including patient with severe vod and with renal failure secondary to ta-tma. the median isa concentrations were . ( . - . ) mg/l and . ( . - . ) mg/l for c and c h, respectively. ifi complete remission was achieved in cases, partial remission in ; treatment failure was experienced by patients. in cases fungal lesions remained stable. ctae grade ii-iii toxicity was observed during treatment in patients, with increased transaminase and/or creatinine levels which resolved after temporary isa withdrawal. no drug-drug interactions were observed in patients receiving csa as gvhd prophylaxis and no modification of csa daily dose was needed. conclusions: isavuconazole use may be considered in the pediatric population, even in the hsct setting, for its safety, efficacy, tolerability, no drug-drug interaction. of course these data deserve further evaluation. disclosure: nothing to declare p new treosulfan-based conditioning regimens including epigenetic agents in patients with very high-risk neuroblastoma background: the pts aged mos. or older with disseminated nb involving bone and bone marrow constitute a group of pts with very poor prognosis. although the majority of them are responsive to intensive conventional chemotherapy, most eventually relapse with efs at years of < %. at the beginning of the year we came up with a protocol for this very unfavorable group including epigenetic therapy ( -azacitidine) in the phases of consolidation. methods: seven pts with a median age of ( , - ) years completed the protocol and received hdct with autologous sct as a consolidation. hdct included two different epigenetic agent containing regimes according to tumor response to the induction therapy assessed by i-mibg and mri (ct-scan). three pts revealing large active residual tumor assessed by i-mibg scan or multiple active bone metastases received a conditioning regimen (regimen a) including i-mibg therapy at a dose . - . mbe/kg on d- , treosulfan mg/m /d, on d- ,- and - (total mg/m ), melphalan mg/m /d, on d- ,- (total mg/m ), -azacitidine mg/m /d on d- to d- (total mg/m ). four pts with cr or vgpr received a «split» conditioning regimen (regimen b) including treosulfan mg/m /d, on d and - , and on d- and d- (total mg/m ), melphalan mg/m /d, on d- and - (total mg/m ), and -azacitidine mg/m /d, on d - to d - and on d- to -d- (total mg/m ). a median number of . ( . - ) cd +/kg was infused on d . results: the median recovery times to wbc> . x /l and to an unsupported plt> x /l were ( - ) and ( - ) days, respectively. all pts experienced grade hematological as well as infectious toxicity assessed by nci-ctc score. there were episodes of severe organ toxicity of grade occurring in pts. in cases we observed a severe mucositis, in cases gi toxicity and episode of the erythema multiforme occurred. one pt revealed a lifethreatening episode of hypotension of grade . no transplant-related death occurred. the median number of transfused rbc and plt doses was ( - ) and ( - ), respectively. all pts are alive and well without signs of disease progression in complete hematological recovery with a limited follow-up of . ( - ) mos. from day of hdct. conclusions: although it is rather early to evaluate the efficacy of the epigenetic agent's inclusion in the induction and/or consolidation phases of a very high-risk nb treatment, we can assume that, first, the hdct combining mibg i and/or high dose of treosulfan with epigenetic agent such a -azacitidine was feasible and had an acceptable toxicity. second, the "split" modality of the treosulfan use in conditioning regimen would permit to increase the total dose of the alkylating agent with no inacceptable toxicity. disclosure: nothing to declare pre-and-post magnetic resonance imaging of hips and knees for detecting osteonecrosis in children undergoing hematopoietic cell transplantation: in whom is it necessary? ali y suliman , sue c kaste , ying li , dinesh keerthi , guolian kang , brandon m triplett , ashok srinivasan between april and august at the university medical center hamburg-eppendorf, germany, were included. total and active ratg plasma levels were analyzed by elisa and flow cytometry, respectively. primary endpoint of the study was exposure to ratg. secondary endpoints included transplant-related mortality, incidence of acute and chronic gvhd, immune reconstitution, chimerism, rejection and viral infections. patients were monitored at least days post transplantation. statistical analyses were performed using ibm spss statistics software, or graphpad prism software. results: median total grafalon™ and thymoglobuline™ peak plasma levels were . μg/ml and . μg/ml, respectively; median active grafalon™ and thymoglobu-line™ peak plasma levels (appl) were . μg/ml and . μg/ml, respectively. active thymoglobuline™ plasma levels showed highly variable pharmacokinetics compared to grafalon™. neither grafalon™ nor thymoglobuline™ exposure correlated with lymphocyte count prior to transplantation, cell count in the graft (wbc, mnc, t cells), age, body weight or body surface area (bsa). this is indicative for a saturation effect in both groups. to correlate high or low ratg exposure with clinical outcome parameters, we built two groups within each patient cohort by median appl. the incidence of gvhd was not dependent on high or low ratg exposure. until day post hsct, viral infections or reactivations (ebv, cmv, adv, hhv , bkv) occurred in the patients. interestingly, adv infections affected only children with high ratg exposure. the median time to leukocyte engraftment was not significantly longer in the high ratg groups compared to the low ratg groups ( to days for grafalon™, and to days for thymoglobuline™). there was a decreased and/or delayed recovery of cd + , cd + and cd + t cell reconstitution, but not of b cells and nk cells in the high thymoglobuline™ exposure group compared to the low thymoglobuline™ exposure group. overall survival was not statistically significant with % in the grafalon™ and . % in the thymoglobuline™ group without influence of ratg exposure. conclusions: high and low exposure to grafalon™ or thymoglobuline™ did not result in significant differences in outcome parameters as incidence of survival, agvhd, cgvhd, rejection, or mixed chimerism in this limited cohort. delayed and decreased immune reconstitution in the high ratg exposure groups did not translate into different clinical outcome parameters. adv infections only occurred in the high ratg exposure group. grafalon™ and thymoglobuline™ showed distinct pharmacological and immunological differences in children and larger cohorts are needed to detect clinically significant differences and adjust dosing regimens individually. disclosure: nothing to declare background: the optimal conditioning regimen for allogeneic hematopoietic cell transplantation (allohct) in children with myeloid malignancies remains undefined, particularly when reduced-intensity conditioning (ric) regimens are utilized. methods: we performed a retrospective review of children undergoing allohct for acute myeloid leukemia (aml) and myelodysplasia-related aml (mdr-aml) over a year period ( - ) at our institution, comparing the outcomes of those who received either a busulfan (bu)-or melphalan/thiotepa (mel/thio)-based conditioning regimen. results: a total of patients were analyzed. twentyone received fludarabine/melphalan/thiotepa and received myeloablative busulfan-based conditioning, either in combination with cyclophosphamide (n= ) or fludarabine (n= ). atg was used in patients depending on donor. recipients of mel/thio were selected for ric regimens due to pre-transplant comorbidities (cardiac dysfunction, n= , requiring peri-transplant milrinone), transplant during chemotherapy-induced aplasia (n= ), underlying diagnosis of treatment-related aml (t-aml) and significant pre-allohct chemotherapy exposure (n= ). proportions of patients with de novo aml (mel/ thio, %; bu, %) and mdr-aml ( % and %) were similar between groups; however, recipients of mel/thio were more likely to have t-aml ( % vs %). cytogenetic and molecular risk factors were similar between groups. the majority of patients were transplanted in cr (mel/thio, % vs bu, %) or cr ( % vs %). more recipients of bu conditioning ( % vs %) were mrd-negative at the time of allohct; both groups had comparable proportions of patients with ≥m marrow (~ %). donor types and stem cell sources were similar between groups, except unrelated umbilical cord blood which was more common in bu recipients ( % vs %). there were no graft failures in mel/thio recipients, compared to % (n= ) in those receiving bu-based regimens. engraftment kinetics and immune reconstitution were similar. overall acute and chronic gvhd incidence was higher in recipients of mel/ thio compared to bu ( % vs %, and % vs %, respectively), but rates of grade iii-iv acute or extensive chronic gvhd were comparable. vod requiring treatment was diagnosed in ( %) recipients of bu conditioning and no mel/thio recipients. median duration of follow-up was months (range - ) in the mel/thio group, and months (range - ) in the bu group. transplantrelated mortality (trm) was similar in both groups ( patient), occurring before day . relapse incidence was comparable (mel/thio, % vs bu, %); however, relapse occurred at a later time in mel/thio recipients (median d + vs d+ ). overall survival at and years was superior in mel/thio recipients ( % vs %, and % vs %, respectively). conclusions: in our single institution experience, use of a melphalan/thiotepa-based ric regimen was associated with similar outcomes compared to full-intensity bu-based conditioning, despite higher risk patient and disease characteristics. the majority of recipients of mel/thio conditioning had significant pre-transplant comorbidities, which did not translate into higher trm. while mel/thio recipients had less optimal leukemia control at the time of transplant and high-risk leukemia features (e.g. t-aml), relapse was similar between groups, occurring later in mel/ thio recipients, which may have contributed to better overall survival. disclosure: nothing to disclose methods: pubertal development and biological gonadal parameters were assessed in a retrospective monocentric cohort of pre-pubertal patients who underwent hsct after myeloablative conditioning with total body irradiation (tbi) or busulfan between and . results: seventy-four patients ( girls and boys) were included. no spontaneous pubertal development was found in % of girls and % of boys (p < . ) and delayed puberty or no spontaneous pubertal development was found in % of girls and % of boys (p= . ). hormone replacement therapy was used in % of girls and % of boys (p < . ). in univariate analysis, tbi conditioning (p= . ), female sex (p < . ), acute gvhd (p= . ), extensive chronic gvhd (p= . ), steroid treatment > months (p= . ), and malignant diseases (p= . ) were associated with no spontaneous pubertal development, whereas tbi conditioning (p= . ) and extensive chronic gvhd (p= . ) were associated with delayed puberty. in multivariate analysis, factors independently associated with no spontaneous puberty onset were female sex (p= . ) and age > years (p= . ). factors independently associated with delayed puberty were extensive chronic gvhd (p= . ) and age > years (p= . ). tbi was not an independent risk factor for pubertal complications. conclusions: this study confirms the toxicity of myeloablative conditioning on pubertal development and the role of older age and female sex in increased pubertal issues, and suggests a possible role of gvhd in delayed puberty. disclosure: nothing to declare p abstract already published. neutrophil elastase activity may serve as a marker for neutrophil extracellular traps formation following stem cell transplantation ronit elhasid , sivan berger-achituv , hila rosenfeld-keidar , szilvia baron tel aviv sourasky medical center -tel aviv university, tel aviv, israel background: post-transplant infections rise dramatically in patients with quantitative or qualitative neutrophil defects and constitute a major source of morbidity and mortality following hematopoietic stem cell transplantation (hsct). neutrophils protect the host from microorganisms via multiple processes including phagocytosis and formation of neutrophil extracellular traps (nets). although reactive oxygen species (ros) production seems to be essential for nets formation, the key enzymes of the process are neutrophil elastase (ne) and myeloperoxidase (mpo). methods: ne and mpo activity as well as nets formation were investigated following hsct in patients at week to and after neutrophil engraftment. neutrophils were isolated using easysep direct human neutrophil isolation kit (stemcell technologies inc.) by immunomagnetic negative selection. enzymatic activity of ne and mpo were measured using colorimetric assays. nets formation of phorbol -myristate -acetate (pma)activated neutrophils was investigated by confocal fluorescence microscopy. all results were compared to those of healthy volunteers. statistical significance was calculated using one way-anova with bonferroni post hoc test. results: patients (median age of . years [range - years]) were investigated, following allogeneic hsct ( acute lymphoblastic leukemia, acute myeloblastic leukemia, epidermolysis bullosa, rhabdomyosarcoma) and following autologous hsct ( ewing sarcoma, desmoplastic small round cell tumor). all patients experienced fever and neutropenia. at engraftment, average ne activity was significantly decreased compared to the average value of healthy individuals. ne activity improved week by week in patients, reached the lower reference range at weeks following transplantation (fig. a) and continued to increase. the enzymatic activity of mpo was comparable to the average value of healthy individuals (fig. b) and showed no significant difference between the distinct time points. at neutrophil engraftment, nets formation was absent and comparable to those of non-activated neutrophils (fig. c) . although nets formation increased week by week, it did not reach the average of normal controls during the monitored time period. also linear correlation between ne activity and nets formation (r = . ) was demonstrated. conclusions: impaired ne activity following hsct corresponds to decreased nets formation and could serve as a marker for netosis. strategies to accelerate the recovery of ne function post transplantation might improve nets formation and thereby induce better infection control. a) the average of ne activity (n= ) during weeks following hsct. reference range was measured and calculated from measurements of healthy volunteers using. b) the average of mpo activity (n= ) during weeks following hsct. reference ranges were measured and calculated from measurements of healthy volunteers using the quartile method. c) the average of netosis activity after nm pma activation for h (n= background: to have a better understanding of incidence, treatment, outcome and risk factors of immune cytopenia in children after allogeneic hsct. methods: between january and september , pediatric allogeneic hsct have been performed in patients at the ghent university hospital (ghent, belgium). autoimmune hemolytic anemia was defined by a positive direct agglutinin test (dat). dat was performed at moment of engraftment and in case of hemolysis or unexplained anemia. platelets antibodies were evaluated in case of no otherwise explained thrombocytopenia. results: the cumulative incidence of post allo sct autoimmune cytopenia is . % ( / ). in cases there were positive antibodies against red blood cells, and one patient against had antibodies against platelets. of these cases, only ( . %) were clinically relevant and needed treatment. the median observation period post sct for the whole cohort was months ( - ) . the clinically significant immune cytopenia started at a median time of day+ and day + in the group without symptoms. the patient who presented the autoimmune thrombopenia developed antibodies against anti-gpiib/iiia, this was resolved after days, the treatment consisted intravenous immunoglobulins (ivig). two of the patients with autoimmune hemolytic anemia had igg mediated antibodies, and had complementmediated dat. these patients were treated with ivig, steroids, rapamune and rituximab. one patient has still dat positive after months, but clinical stable. the other two are also dat positive and have some hemolysis, but the follow up is much shorter ( months). treosulfan-contained conditioning regimens were more frequently used in patients with significant immune cytopenia. conclusions: immune cytopenia is an infrequent complication after allogeneic hsct. however, its treatment can be challenging, and the hemolysis can persist for years. the association of rapamune and rituximab was adequate to treat this problem in our patients. background: approaches to the management of refractory and relapsed classical hodgkin´s lymphoma (r-r chl) are changing and become more effective. the role of anti-cd targeted immunochemotherapy with brentuximab vedotin (bv) has been extensively investigated in adults with r-r chl and is only to be elucidated in children. the study included children and adolescents with r-r chl that were sucessfully treated with bv-based therapy prior to hematopoetic stem cell transplantation (hsct). median age of patients was years ( - ), main histological variant -nodular sclerosis ( %, n= ), advanced stage at diagnosis - % (n= ). most were heavily pre-treated (median number of previous therapies - ) and progression after autologous hsct was documented in ( %). refractory disease was diagnosed in ( %) and relapsed in ( %). among relapsed patients ( %) were with multiple episodes, ( . %) -early and ( . %) -late relapse. treatment regimens consisted of bv in monotherapy . mg/kg triweekly (n= ) or combination of bv . mg/kg on day with bendamustine - mg/ m on days and of -week cycles (n= ) or combination of bv . mg/kg on day with dhap (n= ). median number of bv infusions was . ( - ). all selected patients achieved complete (n= , %) or partial remission (n= , %) prior to hsct. consolidation with autologous hsct was performed in ( %) and with allogeneic hsct -in ( %). primary end points were overall (os) and progression free survival (pfs). response to bv was not assessed in the study as only responders to the bv-based treatment were included. results: with median follow-up of days ( - ) os and pfs for all patients are % and %, respectively. pfs after autologous hsct and allogeneic hsct are % and %, respectively (p= . ) at present moment ( %) patients are alive and are in remission. three patients died ( %): disease progression (n= ), postransplant idiopathic pneumonia syndrome (n= ) and posttransplant pneumonia (n= ). bv was generally well tolerated with only mild polyneuropathy in patients ( %) as the main reversable documented adverse event. conclusions: in prognostically unfavourable heavily pretreated children and adolescents with r-r chl achievement of response to bv-based therapy prior to hsct is assosiated with promising rates of os and pfs. disclosure provides a treatment by restoring thymidine phosphorylase function and improving disease manifestations. here we report the outcomes of affected siblings who underwent transplantation using an unaffected sibling donor to highlight important experiences in the transplant of such a rare condition. methods: four siblings of consanguineous pakistani descent aged , , and years underwent myeloablative hsct using fully hla-matched ( / ) peripheral blood stem cells harvested in a single apheresis from an unaffected year old sibling. the oldest sibling, a year old male, first presented in having emigrated from pakistan with a history of growth failure and abnormal movements. biochemical, nerve conduction and imaging studies confirmed a diagnosis of mngie. testing on three other siblings identified similar biochemical abnormalities, though the youngest children had minimal clinical manifestations of the disease. based on the progressive nature of the disease and the availability of a fully matched donor, a decision was made to pursue transplant for all affected siblings. results: due to the severity of their disease, the oldest siblings were transplanted first using a myeloablative conditioning regime of fludarabine, thiotepa and treosulfan with alemtuzumab. neutrophil engraftment occurred on day + for both, with % donor chimerism achieved. there were no significant transplant related complications. the post-transplant course of the year old sibling was complicated by a major stroke-like event characterised by dramatic imaging changes and requiring ventilation, though no cause was identified and the patient's neurologic deficits have since resolved. gastrointestinal symptoms have persisted and both remain tpn dependent, though symptomatically have shown gradual improvement. following the neurologic complications in their older sibling, the younger siblings were conditioned with auc-targeted busulfan and fludarabine plus alemtuzumab. neutrophil engraftment occurred on day + , with full donor chimerism achieved. progression to enteral feeding has been much more rapid, with nutrition now fully enteral for both. there were no significant transplant related complications. conclusions: stem cell transplantation represents the only curative option for mngie. due to its rarity and relative infancy as a condition, little is known of the expected course following transplant or the best approach to transplantation itself. despite previous challenges with graft failure in mngie recipients, we were able to gain rapid and sustained donor engraftment using different myeloablative conditioning regimes with minimal transplant-related morbidity and no mortality. in keeping with previous reports, resolution of established gastrointestinal symptoms has been slow, though the siblings transplanted earlier in their disease course have shown more rapid improvement supporting the role of early recognition and access to transplant. it is essential moving forward that specialised transplantation centres collaborate so as to guide clinicians in the management of such a challenging condition. disclosure: there are no conflicts of interest to disclose. g pc congenital neutropenia -biology of inflammatory colitis associated with gcsf use, and disease response to allogeneic transplant, a report of cases background: an autosomal recessive disease, glucose- phosphatase catalytic subunit (g pc ) deficiency is a relatively recently identified cause of chronic severe neutropenia. there can be a spectrum to the disease and patients may also present with non-haematological features including prominent chest veins, cardiac, endocrine or urogenital abnormalities. we describe in our patient cohort a response to gcsf but an inflammatory, incapacitating, biopsyproven colitis associated with that g-csf response. we have transplanted children with such colitis, and describe a similar colitis with intestinal failure in a fourth. methods: we investigate the biology of the neutropenic colitis, and demonstrate necrosis of the stimulated neutrophils. in vitro studies demonstrated that unstimulated neutrophils from patients with g pc d exhibited significantly increased production of il , reactive oxygen species (ros) and neutrophil extracellular traps (nets) alongside significantly higher expression of cd b, cd b and cd . in contrast, neutrophils from patients with g pc d produced significantly less ros, mmp- , neutrophil elastase and nets upon stimulation. neutrophils from patients with g pc d also exhibited significantly accelerated apoptosis and secondary necrosis which was exaggerated upon stimulation with live escherichia coli bacteria but could only be partially rescued with supplemental exogenous glucose. results: patients have undergone hsct for g pc neutropenic enterocolitis ( unrelated donor and msd) after fludarabine treosulfan and thiotepa conditioning therapy. alemtuzumab was given as as serotherapy. all patients are alive and well, immune suppression has been discotniuned and there is no gvhd with normal organ function, and resolution of colitis. we describe a th patient with no good donor who has continuing intestinal failure with g-csf use. conclusions: we describe the aetiology of intestinal inflammation and failure with an extensive study of neutrophil biology in this metabolic neutropenia. we describe a novel indication for hsct in this "g-csfresponsive neutropenia". disclosure: nothing to declare p does body mass index (bmi) pose a risk to outcome for pediatric non-infantile patients undergoing hematopoietic cell transplantation (hsct)? mona al-saleh , khawar siddiqui , amal al-seraihy , abdullah al-jefri , ali al-ahmari , hawazen al-saedi , awatif al-anazi , mouhab ayas , ibrahim al-ghemlas with no evidence of toxicity. as benefits of stoss therapy in hsct remain unknown, and safety has yet to have been studied extensively in the pediatric population, we hypothesize that stoss therapy is an effective and safe method to reach and attain sufficient levels of vd in pediatric patients undergoing hsct. methods: this is an ongoing prospective, randomized clinical control trial at phoenix children's hospital that commenced december st , . following consent, subjects are randomized to the intervention (stoss) or control arm prior to hsct. stoss therapy consists of a single oral dose of vd (ranging , iu - , iu), given based on baseline -hydroxyvitamin d [ (oh)d] level and age, followed by standard weekly supplementation. subjects enrolled on the control arm receive standard of care based on endocrine society guidelines of weekly vd supplementation. data collection includes demographics, (oh)d levels at baseline, day + , and day + , vd toxicity (hyperphosphatemia, hyperkalemia and renal calculi), as well comorbidities were collected. at each time point and for each trial arm, the mean (oh)d level and changes from baseline were computed with corresponding % confidence intervals (cis) to indicate variability. results: presently, subjects have completed baseline assessment, with day + and day + follow-up completed for and of these, respectively. at baseline, the mean ( % ci) (oh)d was . ng/dl ( . , . ) among stoss patients and . ng/dl ( . results: total hrqol scores of transplanted patients were significantly improved compared to those on supportive care and also compared to healthy siblings (p < . and . respectively), the same was true for physical (p < . and . respectively) and emotional functioning (p < . and . respectively). social and school functioning of transplanted children were not different from healthy siblings (p . and . respectively) while were very significantly improved compared to children with st on supportive care (p < . in both cases). conclusions: bmt in a lower-middle income setting may be even more impactful compared to high-income regions. our analysis clearly indicates normalization of hrqol in all major areas of children transplanted for st. a possible resilience effect was noted for physical and emotional scores which were improved compared to healthy sibling controls. we could not however quantify the effect of longer-term issues like fertility impairment after bmt which may eventually adversely impact hrqol, particularly in the indian culture. disclosure: none allogeneic hematopoietic stem cell transplantation in ataxia telangiectasia patients without malignancy background: ataxia telangiectasia (a-t) is a primary immunodeficiency with mutations in atm-gene. besides a slowly progressive neurodegenerative course, a-t leads to increased susceptibility to malignancies which affects % of patient (median: . years) with a high mortality mainly due tocomplications of conventional radio-chemotherapy. the incidence of cancer correlates with the extent of immunodeficiency. patients often develop severe progressive granulomatous skin disease with evidence of vaccine-strain rubella-virus in the lesions. prolonged survival, neurologic improvement and malignancy prevention was observed in atm-deficient mice after treatment by syngeneic hsct. nevertheless, pre-emptive hsct is not routinely performed in a-t patients due to concerns about neurodegeneration and toxicity. methods: we present three a-t patients with severe immunodeficiency phenotype, undergoing successful hsct as an individual treatment strategy intending to restore immunodeficiency for long-term malignancyprevention (patient- ) and to treat progressive skin/joint granulomas (patients- and - ). results: patient- underwent a reduced intensity conditioning (ric) regimen at years of age including fludarabine ( mg/m ), cyclophosphamide ( mg/kg), and atg-fresenius ( mg/kg/d) which was tolerated well. hematopoietic engraftment occurred by day + . there was an expansion of naïve and memory cd + t-cells and cd + cells. while initially a mixed donor chimerism in patient's pbmcs ( - % donor) was observed, patient's tcells (cd + ) reached over % of donor origin over time. at last follow-up ( years) he is well, without signs of gvhd and organ toxicity, off immunosuppression with normal levels of atm-protein; his granulomas resolved. patient- is a year-old male who was transplanted from his hla-identical sibling, conditioned with fludarabine ( mg/m²), cyclophosphamide ( mg/kg), and atg-fresenius ( mg/kg). hematopoietic engraftment was observed by day + . t-cell reconstitution started by day + with > μl cd + t-cells. his mixed chimerism rapidly turned to donor origin ( % donor cd +) over time. there was no acute toxicity, however, he developed lumbosacral pain episodes with evidence of urine bk-virus with spontaneous remission. an intermittent metapneumovirus associated pulmonary hypertension was observed with pericardial effusion. treatment included sildenafil and oxygen. at last follow-up ( months) patient is well without immunosuppression. patient- suffered from recurrent chest infections, failure to thrive and progressive and debilitating rubella positive progressive granulomas of the skin. she received allohsct from an hla-identical family donor at years of age. conditioning included busulfan ( . mg/kg), fludarabine ( mg/m²), cyclophosphamide ( mg/kg), and alemtuzumab ( x mg/m², x mg/m²). hsct was complicated by intermittent acute renal failure, cmv reactivation and tma. hematopoietic recovery was observed by day + . t-cell chimerism increased rapidly over time (> % donor). at last follow-up ( months) patient is well, off immunosuppression and ivig. her skin granuloma resolved with scarring residues. conclusions: pre-emptive allohsct is feasible in a-t when reduced intensity conditioning is used and can correct the immunodeficiency. it might be a treatment option for some a-t patients at high risk of hematological malignancy and severe granulomatous skin disease. to what extent the restored immune system and the increase of atm-protein in these patients could prevent the development of other malignancies needs to be evaluated further. disclosure: nothing to disclose p abstract withdrawn. hematopoietic stem cell transplantation in diamond blackfan anemia: brazilian experience background: diamond-blackfan anemia (dba) is a rare inherited red cell aplasia caused by an intrinsic defect of erythropoietic progenitors. the main therapeutic approach is based on repeated red blood cell transfusions and/or corticosteroid therapy. hematopoietic stem cell transplantation (hsct), a potentially curative treatment for dba, is indicated for patients that do not respond to first-line therapy. methods: the aim of our retrospective study is to report the outcomes of brazilian dba patients transplanted between and in bmt centers. the median age of the patients was ys (range - ) and % were male. seventeen patients ( %) were transplanted with matched related donors (mrd) and thirteen ( %) from matched and mismatched unrelated donors (mud/mmud). in the mrd group all patients received bone marrow as hsc source, while in the mud/mmud, eight patients received bone marrow and five received cord blood. all patients with incompatibilities (mismatched) were ucb ( / ). nineteen recipients were conditioned with busulfan plus cyclophosphamide, while the remaining received fludarabine and busulfan, which has been the preferred regimen in brazil in the recent years. after transplant, most (n= ) of the mrd and mud recipients received cyclosporine and short course methotrexate as graft versus host disease (gvhd) prophylaxis. results: twenty-two out of the patients were alive and disease-free at a median follow-up of months (range to months). the -year overall survival (os) was % (ci - %) (fig ) . similar results have been demonstrated in studies from europe and from the united states. when analyzed according to donor type, os was % (ci - %) and % (ci - %) in mrd and mud/mmud respectively (fig ) . three out of the patients who were transplanted with ucb died. these results are in agreement with those of previously published data showing worse results in unrelated ucb transplants. twenty-nine out of the patients engrafted successfully. in of the evaluated patients, the median time to neutrophil engraftment was days (range - ). one patient experienced an early death from hemorrhagic shock on day , before neutrophil recovery, and another two patients experienced primary graft failure. post-transplant chimerism was available for patients. sixteen had complete chimera (> % chimerism), while patients presented with mixed chimerism. acute gvhd was observed in patients ( %), of which classified as grade iv. five patients developed chronic gvhd, considered severe in three of them. eight patients died at a mean of days (range - days) after hsct and the main causes of death were infections and hemorrhagic disorders. conclusions: hsct is a potentially curative treatment option for dba. in the present study, we report the outcomes of patients with dba transplanted in brazil with a os of %, with better results in mrd compared to mud, as expected. despite the small numbers, we observed lower survival after mud/mmud ucb transplantation. since dba is a rare disease, international collaborative studies are essential to better understand the benefits of the hsct in the treatment of these patients. disclosure: nothing to declare p treatment of the obliterant bronchiolitis in pediatric allogeneic recipients: two periods compared results: in group , the therapies administered for bo included prolonged treatment with steroids in all patients, anti-tnf in , azatioprine in ; while in the group , all patients received ima, montelukast and azitromicin, and received i.v. mpd. the median duration of imatinib therapy was years ( . - . years). after a median follow-up of . years (range . - . yrs), / patients of group ( %) died with bo in progress for transplant-related causes. while in the group , / ( %) died in presence of worsening bo. the estimated os at year after hsct was % ( % ci; - ) in group and % ( % ci, - ) in group (p= . ) (figure ), while the os after year decreased at % ( % ci; - ) in the group while remained stable in the group . conclusions: this experience shows a relevant improving in prognosis of children with bo with the use of this protocol including ima, since the significant improving of survival obtained, confirming as reported in adult populations. disclosure results: we presented patients with pres, age ranging from months to years with a average of . years. there were ten patients with thalassemia major, two patient with acute lymphoblastic leukemia, three patients with sickle cell disease and one patient with myelodysplastic syndrome, one patient with immune deficiency, two patients with acute miyeloid leukemia, one patient with aplastic anemia. ten patients were males, ten were female. all patients were treated with csa or tacrolimus and metilprednisolone for the prophylasix of gvhd. pres occurred at a median of days (range - ). clinical findings at onset of leukoencephalopathy were hypertension, headache, seizures, visual disturbance, and altered mental function. eighteen patients alive with normal neurological status. mri showed abnormalities in all patients including patchy bilateral cortical and subcortical lesions, especially in parieto-occipital lobes. conclusions: bmt is associated with several neurological complications that may be underlying diseases, bmt procedure, and severe immunosupression. pres is an uncommon but serious complication after bmt. we report cases of pres who received allogeneic bmt for thalassemia major to emphasize the importance of early recognition and institution of appropriate management of pres during bmt. disclosure: nothing to declare p continuous complete molecular remission using three different monoclonal antibodies followed by allogeneic bone marrow transplantation in an infant with chemotherapy-refractory acute lymphoblastic leukemia bernd gruhn , susan wittig , thomas ernst , jana ernst university of jena, jena, germany, background: a -week-old infant was diagnosed with very immature acute lymphoblastic leukemia (all) with myeloid markers in a foreign university hospital. at the end of induction therapy according to the current lal/shop protocol % leukemic cells were detectable in the bone marrow. treatment was changed to fludarabine, cytarabine and granulocyte colony-stimulating factor (flag) in combination with liposomal doxorubicin. after this re-induction still % leukemic cells were detected in bone marrow, so the bispecific t-cell engager antibody blinatumomab was given. due to an increasing portion of leukemic cells during the continuous infusion, antibody therapy was stopped and a cycle of clofarabine, cyclophosphamide and etoposide was administered. unfortunately, still % leukemic cells were detectable afterwards. because of chemotherapy-refractory leukemia a palliative oral treatment with mercaptopurine was started. however, the parents did not accept the palliative situation and searched for alternative therapeutic options in other university hospitals in europe. after plenty of refusals the infant was admitted to our hospital five months after diagnosis. methods: for molecular characterization genomic dna was isolated from leukemic cells. a mll-mllt /af rearrangement as a consequence of the translocation t( ; ) (p ;q ) was detected and used as a marker for minimal residual disease. for further molecular characterization targeted deep next-generation sequencing was performed for a panel of leukemia-associated genes. interestingly, no mutation was found. to allow precise immunophenotyping of the leukemic cells treatment with mercaptopurine was stopped. results: as in the first immunophenotyping the cd antigen was found, we administered the anti-cd monoclonal antibody gemtuzumab ozogamicin twice within two weeks. because of the detection of cd + leukemic cells after infusion of gemtuzumab ozogamicin, the anti-cd antibody daratumumab was given alternating twice within two weeks. unfortunately afterwards, leukemic cells reappeared being negative for cd und cd , but positive for cd . therefore, we administered the third antibody, the anti-cd monoclonal antibody inotuzumab ozogamicin, whereupon our patient developed a tumor lysis syndrome and a severe bone marrow aplasia. shortly after, allogeneic bone marrow transplantation from an unrelated donor using a special conditioning regimen consisting of thymoglobulin, busulfan, fludarabine and clofarabine was conducted. clofarabine was added because an additional antileukemic effect especially in infant all with mll rearrangement was described. after transplantation the patient suffered from a severe hepatic sinusoidal obstruction syndrome with massive ascites, renal and pulmonary dysfunction, but finally the patient recovered completely. the first bone marrow examination days after transplantation revealed a donor chimerism of % and a complete molecular remission using the mll-mllt /af rearrangement as marker for minimal residual disease. in all follow-up bone marrow samples we observed a complete donor chimerism and a complete molecular remission. currently, eight months after transplantation the patient is in a very good physical condition with normal development according to the age. background: paediatric chronic graft versus host disease (cgvhd) is a debilitating condition associated with substantial morbidity and mortality. to date, there are no approved therapies for paediatric patients with cgvhd, and current treatments often lack sufficient efficacy or lead to severe/life-threatening toxicities that limit their effectiveness. ibrutinib, a first-in-class, once-daily inhibitor of bruton's tyrosine kinase (btk), is approved in the us for the treatment of adult patients with cgvhd after failure of ≥ lines of systemic therapy. this phase / study will evaluate the use of ibrutinib in paediatric patients with moderate or severe cgvhd. methods: this open-label, multicenter, international phase / study (pcyc- ) includes patients with moderate or severe cgvhd as defined by the nih consensus development project criteria. it is divided into two parts: part a will determine the recommended paediatric equivalent dose (rped) of ibrutinib in patients aged ≥ to < years, and part b aims to evaluate the safety and efficacy of ibrutinib in patients age ≥ to < years. for part a, patients with cgvhd aged ≥ to < years who have failed ≥ lines of systemic therapy will receive once daily oral ibrutinib at a starting dose of mg/m to be escalated up to mg/m after days, if no grade ≥ toxicities occur, until the rped is determined. for part b, patients aged ≥ to < years with cgvhd who have failed ≥ lines of systemic therapy or have newly diagnosed cgvhd will receive once daily ibrutinib ( mg) until one of the following criteria is met: treatment is no longer required; new systemic treatment for cgvhd is initiated; progression of cgvhd; recurrence of underlying disease; or unacceptable toxicity. patients with newly diagnosed cgvhd will receive ibrutinib in addition to daily corticosteroids ( . - mg/kg prednisone). patients < years of age may be enrolled in part b and treated at the rped after it is determined in part a. key exclusion criteria include uncontrolled active systemic infection or active infection requiring systemic treatment; progressive underlying malignant disease or any post-transplant lymphoproliferative disease; or active hepatitis c/hepatitis b virus. patients must have adequate renal, hepatic, and hematologic function to be enrolled. the primary endpoint of part a is the rped of ibrutinib, as based on pharmacokinetic (pk) data; secondary endpoints include safety and pharmacodynamics (btk occupancy). the primary endpoints of part b are pk and safety of ibrutinib in paediatric patients with cgvhd. secondary endpoints for part b include response rate at weeks as defined by the nih consensus development project criteria; duration of response; overall survival; and late effects on growth, development, and immune reconstitution. results: this global study is currently enrolling. conclusions: this phase / study will explore the use of ibrutinib in paediatric patients with cgvhd to potentially meet the high unmet need for proven effective therapies for this population. disclosure enzyme replacement therapy (ert) is the treatment of choice in non-neuropathic hunter syndrome, but as the recombinant enzyme does not cross the blood brain barrier and neuropathic hunter syndrome is left untreated. hematopoietic stem cell transplantation (hsct) is the standard of care in patients with severe mucopolysaccharidosis (mps) type i (mpsih, hurler syndrome) as early transplantation halts cognitive decline in these patients and significantly improves survival. only few case studies have been published on the potential benefit of hsct in mps ii and mostly used busulfan-based conditioning regimens. in one comparative non-randomised multicenter study, hsct might to be superior compared to ert. here, we present our experience in hsct in three children with hunter syndrome using a treosulfan-based conditioning regimen. methods: a retrospective chart review was carried out in patients, who underwent hsct for hunter syndrome. the conditioning chemotherapy regimen included fludarabine, treosulfan, thiotepa and atg. all patients received bone marrow of either related and or matched unrelated donors. gvhd prophylaxis was performed with csa and methotrexat. results: three patients with hunter syndrome were transplanted in our department in . the age was six months, two years and four years, respectively. bone marrow donors were related in one patient and matched unrelated in two patients. the conditioning therapy was generally well tolerated. major complications were fever of unknown origin with need for antibiotic therapy and a mucositis. one patient developed a cmv reactivation. all patients engrafted successfully and recovered well from the hsct. there was no case of acute or chronic gvhd. in all three patients are alive. donor chimerism is complete in one patient; two patients have a mixed donor chimerism. after application of donor lymphocyte infusions in one patient, donor chimerism is stable at a low level of %. the donor chimerism of the other patient still slowly declines to currently %. after stem cell transplantation, two patients did not show further progression of the disease and even achieved psycho-motor improvements. interestingly, one of these patients is the one with the low donor chimerism of %. one patient suffers from a further progression of the underlying disease with psycho-motoric agitation, aggressive behavior and loss of speech, that occurred within the first year following hsct, but neurocognition stabilized thereafter. conclusions: we found a beneficial effect of hsct on the neuropsychological outcome or at least stabilization of neurocognitive function in our patients with a follow-up of eight years. despite low toxicity of the conditioning regimen, increased donor chimerism may further improve the neurological outcome. disclosure: nothing to declare. tandem sct in pediatric solid tumors, other than brain tumors, has no advantage in terms of efs over single procedure-single center experience , germ-cell tumour (gct), ewing sarcoma (es), nefroblastoma. patients were divided into groups according to the number of procedures: st group-single sct procedure, nd group-multiple procedures. regimens used for stem cell mobilisation were: topo-cy for nbl and epi-tax for gct, followed by g-csf±plerixafor. conditioning regimens: bu-mel and thiotepa-cy for pts with nbl, thio-tax and ice for pts with gct. patients received antibiotic, antiviral and antifungal prophylaxis, parenteral nutrition and supportive treatment. patients received consolidation treatment, followed up monthly in the first year, then yearly. patients were evaluated for residual disease by imaging tests. parents signed informed consent forms. results: we performed sct procedures to patients: . % nbl, . % es, . % gct and . % nefroblastoma. for this study only patients with nbl and gct were considered. in st group were % of pts, % in nd group. patients were diagnosed, staged and treated according to international protocols. sex ratio was f/ m. age distribution was - y % ( pts), - y % ( pts), > y % ( pts). peripheral stem cell (pbsc) mobilisation was more difficult in patients with multiple courses of chemotherapy±radiotherapy. we found no difference in the period of engraftment following a nd or rd procedure. hospitalization and supportive measures increased in nd and rd procedures ( to days). patients with multiple courses of chemotherapy and multiple hospitalizations had increased infectious risk and during the nd or rd procedures developed various infectious complications.incidence of severe oral mucositis after the first hsct was %, after tandem hsct was %. nbl patients : st group- / patients alive and efs, / receives anti g treatment; nd group- / patients-alive, / patients-not reached timepoint for mibg scan; / patients-mibg negative at first, relapsed after mo; / patient deceased due to pulmonary toxicity. gct patients: st group- / patients alive and ef, / high values in afp levels and receives metronomic therapy, / patients deceased due to progressive disease, but only had sct. only / patient had one procedure and died due to progressive disease. conclusions: in our study, tandem hsct in children with solid tumours lead to an increase in survival rates, at least in the first months after sct. most patients ( %) had progressive or relapsed/refractory disease when referred to our department. multiple procedures require a higher number of cd cells, very hard to achieve in patients with multiple courses of chemo± radiotherapy. new approaches have to be considered in these diseases, especially in high risk group. disclosure: nothing to declare background: antimicrobial prophylaxis for prolonged neutropenia occurred during the pre-engraftment period is a common practice in allogeneic hsct recipients. data on its effectiveness are few and generally from cases series and not from randomized clinical trials, especially in children. methods: all clinical records of allo-hsct performed from january st to november th at hsct-unit of istituto g.gaslini, genoa-italy, were retrospectively reviewed. collected data were underlying diseases, type of donor, antibiotic prophylaxis administration and type, development of fever and pathogen isolated from blood culture, if any, during pre-engraftment neutropenia. antibiotic prophylaxis, usually starting together with the conditioning regimen, was categorized in "standard" (with amoxicillin/clavulanate or ampicillin/sulbactam) or "tailored" (when based on previous bacterial isolations or colonizations). results: allo-hscts were performed in pediatric patients ( % males) with a median age at hsct of years (iqr: - ; range: - ). hscts were performed from alternative donor (ad) in % patients, from relative donor (rd) in %, and from haploidentical donor in %. table shows the pre-engraftment febrile neutropenia episodes according to type of antibiotic prophylaxis. ( %) hscts received standard prophylaxis, while ( %) the tailored one; only in ( %) did not receive any prophylaxis. fever occurred in ( %) of episodes in patients receiving standard prophylaxis, in ( %) of those treated with tailored prophylaxis and ( %) in the group without prophylaxis; only % of patients who received prophylaxis did not develop fever.in % of patients, the febrile episodes were diagnosed as bloodstream infections: staphylococcus aureus in %; cons in %; enterococcus spp in %; enterobacteriaceae in %; pseudomonas aeruginosa in %; other non-fermenting gram negatives in % and fungi in %. conclusions: the occurrence of fever in patients who received antibiotic prophylaxis suggested that it could not be effective in prevention of fever related to neutropenia after allo-hsct. the personalization of prophylaxis could be a possible path to follow these patients. disclosure methods: a total of patients with leukemia or neuroblastoma were included in the study. patients' mothers signed an informed consent for participation in the study. six of study participants were boys and girls, all aged to years. the control group consisted of healthy preschool children ( groups of children aged to years), boys and girls. results: in most of games the role of a doctor was played by a child. only one child declined to impersonate both a patient and a doctor. younger children mostly agreed to have for a "patient" a toy (proposed by psychologist or one of child's own), child's mother or a medical psychologist. the game lasted for - minutes. most patients preferred using real medical consumables and instruments (syringes, adhesive tape, winged infusion sets or, more rarely, pills). most often a syringe or an adhesive tape was chosen. as known from their mothers, among medical manipulations most unpleasant for children are injections and changing implanted catheter dressing. also, most healthy preschool children preferred using real medical instruments over toy ones. group more often used a syringe, a winged infusion set, adhesive tape, gauze or pills. group most often chose syringe or gauze. among medical instruments both groups more often chose a phonendoscope or thermometer.one patient refused to cause pain to a "toy patient". other children sympathized with a "toy patient", stroke injection or dressing location sites or used soothing terms ("wait a little", "it's going to be all right"), wished prompt recovery and hugged their "patients". one child was angry over his "patient" wishing him to "get hurt too". first preschool group children were mostly scolding a toy "patient" for "being guilty of getting sick". second group children were mostly compassionate, encouraged a "toy patient" telling that "all the procedures are needed to get healthy". from children's schoolmasters we know that all first group children received vaccination about a week before a test. children from second group had no injections. overall attitude towards toy "patients" was more mild in the second group. conclusions: . during a play children mostly use the medical devices which cause them most discomfort and/or pain. . manipulating the items children illustrate their own impression of medical procedures, which are most unpleasant. . children may express their negative emotions directed towards medical manipulations via their play actions, these negative reactions may be suppressed in different ways by parents or medical staff. . the intensity of child's own traumatic experience and an attitude of nearby adults may influence the child's attitude towards other patients. . the mother's wish for a child to tolerate all medical procedures with ease exceeds real capabilities of a small child. disclosure: nothing to declare. allogeneic stem cell transplantation in patients with mucopolysaccharidosis type iiia (sanfilippo): a case series methods: allogeneic sct was performed at the ages of , and years, respectively. all three patients received intrathecal enzyme replacement therapy within a clinical trial setting prior to hsct. the conditioning regimen consisted of treosulfan, fludarabine, thiotepa and thymoglobuline. gvhd-prophylaxis was carried out with csa and mtx in two patients and csa and mmf in one patient. stem cell source was bone marrow in two patients and peripheral blood stem cells in one patient. results: the conditioning regimen was well tolerated and all three patients successfully engrafted. two of three patients had an uncomplicated course without occurrence of acute or chronic graft-versus-host-disease (gvhd). at last follow-up and months after hsct, both patients are in good condition and show constant progress of psychomotor development. the third patient experienced severe steroid refractory acute gvhd of intestines (stage ) and skin (stage ), which resolved under intensive immunosuppression with cyclosporine, mycophenolate and ruxolitinib. around day after hsct, this patient showed clinical and biochemical signs of transplant-associated microangiopathy (tma) with cerebral seizures and acute renal failure. the cerebral mri showed progressive cerebral atrophy and leukoencephalopathy, also consistent with a progress of the mps iiia. at last follow-up months after hsct, this patient had recovered from tma and was in a stable clinical condition. conclusions: in consideration of the small case number and the short follow-up period in our cohort, allogeneic hsct might be considered as a salvage therapy for patients with mps iiia if other therapeutic options are unavailable for children with this otherwise unfavourable prognosis. however, the early psychoneurological course after transplant seems promising compared to the literature and hsct could become a treatment option for this rare disease. disclosure: nothing to declare methods: for the identification of underlying molecular mechanisms leading to the increased sensibility of rms cells, the activation status of different nf-kb signaling pathways were analyzed using western blot analysis and quantitative real time pcr (qpcr). further, flow cytometry was used to analyze the surface expression of death receptors on either sm treated or untreated rms cells. the overall effect on cell death induction was measured by pi/hoechst staining using a fluorescent microscope. results: treatment with sm led to the suspected degradation of iaps. followed by the activation of both the canonical nf-κb signaling pathway, indicated by the phosphorylation of iκbα and p , and the non-canonical nf-κb signaling pathway, as indicated by the accumulation of nik and the degradation of p to p . determination of selected target gene transcription revealed an upregulation of the inhibitor iκbα, nik, p , il- and at later time points the death receptors trail-r and trail-r . analysis of gene transcription also led to the finding of neither up-nor downregulation of ciap and p . to evaluate the involvement of trail-r and trail-r in the sm induced sensitization towards nk cell-mediated killing, surface expression of both death receptors was analyzed. treatment with sm led not only to an induced transcription of trail-r and trail-r , but also to an increased surface presentation of trail-r . subsequent ligation of trail-r by either wt-trail or a specific agonistic antibody (etr- ) resulted in a significant increase in cell death induction. the aforementioned analysis of gene transcription hints towards a bimodal feedback mechanism regulating both, the canonical and non-canonical nf-κb signaling pathway. on the one side, the canonical pathway is negatively regulated by the induced transcription of the inhibitor iκbα. on the other side, the induced transcription of nik, p and relb points towards a positive feedback loop of the non-canonical pathway. one mechanism of the increased rms cell sensitivity might be the induced transcription and surface presentation of the death receptor trail-r . the involvement of trail receptors is further validated by the cytotoxicity data, illustrating a sm mediated sensitization towards a trail induced cell death induction. this mode of cell death fits to the previous research, were trail transcription could be induced in nk cells by sm treatment. the graphical abstract shows the transcriptional upregulation of target genes leading to a putative bimodal nf-kb regulation and increased surface presentation of trail-r by treatment with smac mimetics. aim: to investigate the outcome of ucb transplantation in pediatric patients with malignant and non malignant diseases methods: data from patients underwent first allogeneic bone marrow transplantation with ucb from / until / were retrospectively analyzed. eighteen had malignant disease (md), of whom in complete hematologic response, and non malignant disease (nmd) (scid , chronic granulomatous disease , severe aplastic anemia , s.kostmann , osteopetrosis , wiskott-aldrich , amegakaryocytic thrombocytopenia ). the majority of the patients were male, for md and nmd, as well (m: /f: , m: /f: , respectively), of median age . years (range . - . years) and . years (range . - . years), respectively results: all patients but one, received ucb unit. hla compatibility in antigen/allele level was at least / and only in patients with md was / . conditioning regimens were myeloablative and tbi gy was given in / . gvhd prophylaxis consisted of cyclosporine and atg was given in all patients pre-transplantation. median value of nucleated cells for md was . χ /kg (range - . χ / kg) and for nmd was . χ /kg (range - . χ /kg). neutrophil and platelet engraftment was achieved in / and / patients with md respectively, in a median time of days (range - ) και days (range - ). in patients with nmd, neutrophil and platelet engraftment was achieved in / and / with median day of engraftment days (range - ) και days (range respectively. acute gvhd grade ΙΙ-Ιv presented in / patients with md and / with nmd, although none had cgvhd. the incidence of viral infections was cases in patients with md and cases in patients with nmd. disease relapse occured in / patients with md. after a median time of years follow up, overall survival (os) and event free survival (efs) for children with md were % and . % respectively, while for nmd, os and efs were %.treatment related mortality at d+ was % for md and % for nmd. among patients with md, are still alive, while the rest died from relapse (n: ), viral infections (n: ), septicemia (n: ) and agvhd(n: ). among patients with nmd, are alive, while the rest died from viral infections (n: ), septicemia (n: ) and multiple organ failure (n= ). the median time of hospitalization for patients with md was days (range - ), whilst for nmd was days (range - ). conclusions: transplantation of unrelated ucb in our unit was combined with high trm in children with nmd and higher probability of relapse for md. disclosure: nothing to declare p serum levels of -s cysteinyldopa is associated with stem cell transplantation related complications yukayo terashita , mamoru honda , minako sugiyama , yuko cho , akihiro iguchi hokkaido university hospital, pediatrics, sapporo, japan background: diffuse hyperpigmentation is common in patients who received chemotherapy or stem cell transplantation (sct). however, there are few reports of the relationship between skin reaction such as pigmentation and the other complications. pigmentation of the skin is thought to be the result of melanin stagnating in the dermic layer due to increased synthesis of melanin and destruction of the basement membrane due to inflammation induced chemoradiotherapy. melanin pigments are classified into two types: brown to black eumelanin and yellow to reddishbrown pheomelanin. -s cysteinyldopa ( scd) is precursors of pheomelanin, and its serum level has been used specific biochemical marker for malignant melanoma. here, we examined serially scd during the course of sct to determine association with sct related complications, because visual evaluation of skin color is difficult, and there have been no reports about scd as sct related biomarker. methods: we prospectively analyzed ( males, females) patients who received sct between may and march in hokkaido university hospital. the median age at transplantation of the patients was . years (range, - ). indication for sct were acute myelogenous leukemia in patients, acute lymphoblastic leukemia in patients, and other disease in patients; juvenile myelomonocytic leukemia( ), malignant solid tumor( ), immunodeficiency( ), anaplastic anemia( ), and diamond blackfan anemia ( ) . patients received allogeneic sct and received autologous sct. myeloablative conditioning (mac) was used for patients and reduced intensity conditioning (ric) was used for patients. sera were obtained from patients before conditioning therapy, on day , + , + , + , + and + . all blood samples were centrifuged at , rpm for min, and stored at - ˚c until used. we also examined sct related complications such as graft-versus-host disease (gvhd), viral infection, and pre-engraft syndrome (pes). statistical analyses were completed using the mann-whitney u test for unpaired samples, and kruskal-wallis test for three samples. each test was performed with a % level of significance. results: the average value of scd reached two peaks, day ( . nmol/l) and day ( . nmol/l), regardless of stem cell source and intensity of conditioning. in all patients, we found that the level of scd on day was associated with viral reactivation (p= . ), scd on day was associated with pes (p= . ), and scd on day was associated with malignant disease (p= . ). similarly, in patient who received allogeneic sct (n= ), the level of scd on day was associated with viral reactivation (p= . ), scd on day was was associated with pes (p= . ), scd on day was associated with malignant disease (p= . ). in addition, the level of scd on day was associated with gvhd of skin (p= . ), the peak level of scd was associated with acute gvhd (p= . ). conclusions: we found that scd can be a biomarker for sct-related complications such as aute gvhd. it is presumed that the production of pheomelanin could be induced by inflammatory procedure in sct. disclosure: nothing to declare p hsct in children with bone marrow failure: outcomes from a single singapore centre prasad iyer , michaela seng , vijayakumari k , ah moy tan , mei yoke chan , rajat bhattacharyya kk women's and children's hospital, paediatric haematology-oncology, singapore, singapore background: children presenting with pancytopenia often present a challenge to the paediatric haematologist. the underlying diagnosis can be hard to establish as many of the inherited bone marrow failure syndromes (ibmfs) can present with protean manifestations. the large majority of patients with bone marrow failure are often diagnosed with idiopathic severe aplastic anaemia (saa) despite extensive testing. we report our experience of hsct in patients treated with primary and acquired bone marrow failure. methods: we reviewed case notes of all the children who underwent hsct for bone marrow failure in our centre. results: a total of fifteen patients underwent eighteen stem cell transplants in our centre between and . three patients were diagnosed with fanconi anaemia, one with hoyeraal-hreidarsson syndrome, one with paroxysmal nocturnal haemoglobinuria and the remaining ten children had idiopathic saa. eight children had matched sibling donor transplant, had a matched related donor, had a matched unrelated donor, had umbilical cord blood transplants and the remainder were haploidentical transplants. four of the haploidentical transplants were t-cell depleted and one was t-cell replete. one child with fanconi anaemia had primary graft rejection with cord blood transplant and was successfully rescued with a haploidentical transplant. one child with saa had primary graft rejection twice (t-cell depleted graft) and then was rescued with an alternate haploidentical donor with a t-cell replete graft. of the two patients who died, one had a fatal fungal infection ten months after transplant, and the other died due to a severe influenza pneumonitis three and a half years after bmt. conclusions: haematopoietic stem cell transplant outcomes from our centre are comparable to leading centres in the world. the understanding of underlying conditions that present with bone marrow failure has improved our approach and the way we treat bone marrow failure syndromes. clinical trial registry: not applicable. disclosure: nothing to declare. methods: a retrospective study was performed in children treated with hsct who received pos or flu during early neutropenic period until engraftment from january to december at siriraj hospital in thailand. the efficacy, safety and tolerability of pos were compared to flu. results: there were hsct recipients (allo-hsct . %, auto-hsct . %) with mean age of . + . years. most of the patients were thalassemia ( . %) followed by hematologic malignancy ( . %) and solid tumor ( . %). seventeen and cases received pos and flu, respectively. all of patients in pos group were allo-hsct whereas . % in flu group were allo-hsct. in pos group, cases were diagnosed with suspected ifi and cases were probable ifi with total cases ( . %). in flu group, cases were diagnosed with suspected ifi and cases were probable ifi with total cases ( . %) which compared groups were not statistically significant (p= . ). no possible and proven ifi in both groups. in flu group patients received empirical antifungal treatment more than pos group but no statistical significance ( . % vs. . %, p= . ). both groups had similar rate of elevated liver function test (p= . ). no early discontinuation of antifungal prophylaxis for intolerance was found in both groups. only . % of patients achieved pos target trough level of . mg/l after days of treatment with started dose mg/kg three times a day. conclusions: pos and flu are comparably effective, safety and tolerability in ifi prophylaxis in neutropenic children treated with hsct. defining dose recommendation of pos in this setting requires larger studies. disclosure background: severe congenital neutropenia (scn) is typically characterized by anc of < /μl, maturation arrest of bone marrow myeloid precursors at the promyelocyte-myelocyte stage, and susceptibility to lethal pyogenic bacterial and fungal infections. scn is a rare group of disorders resulting from intrinsic defects in myeloid cell proliferation and maturation caused by mutations in several genes; elane, hax , gfi , was, and g pc are among the most common ones. almost % of patients are refractory to g-csf, and the only definitive curative approach for such patients is allogeneic hsct. the current absolute indications for hsct is failure to respond to g-csf treatment, or the development of mds/leukemia in patients with scn. here, we present the result of children with scn who received allogeneic hsct . methods: we retrospectively assessed allogeneic hsct in children with severe congenital neutropenia. all patients received busulphan (bu) based myeloablative conditioning regimen. busulphan was used according to weight adjusted dose. in addition, all patients received fludarabine mg/m in five days or cyclophosphamide mg/kg in days and atg mg/kg in days. cyclosporin-a and mtx were used for graft versus host disease (gvhd) prophylaxis. donor chimerism was evaluated in either bone marrow or peripheral blood on days + , + and + . results: the median transplantation age of the patients was months (range - months). six of them are male. two of the donors were matched siblings and were unrelated two of which were ag ag mismatched. stem cell source was bone marrow in patients, peripheral blood in and cord blood in patients. all patients engrafted. the median time of neutrophil and platelet engratment to was ( - ) days and ( - ) days, respectively. graft rejection was experienced in patients, one of them had received unrelated cord blood. all patients are alive, eight of which are with full donor chimerism (between - %) without any complication (no infection, no gvhd) with a median months (range - months) follow up. probability of disease free and overall survival were found % and %, respectively. conclusions: we concluded that hsct is a useful treatment for scn patients, especially those who are unresponsive to gcsf treatment and at high risk for leukemic transformation. however, a larger number of scn patients and longer follow-up are necessary to identify appropriate conditioning regimens and long-term prognosis. disclosure: nothing to declare background: prolonged thrombocytopenia (pt) or secondary failure of platelet recovery (sfpr) are a lifethreatening complications that occurs in - % and - % respectively of the patients following allogeneic hematopoietic stem cell transplantation (allo-hsct). management strategies, including the use of growth factors, cd +-selected stem cell boost, mesenchymal stem cell (msc) transfusion, and second allo-hsct, are not effective or possible for all patients. eltrombopag, is an oral non-peptide thrombopoietin receptor agonist, that leads to signal transduction and results in promoting the proliferation and differentiation of megakaryocytes. some recently studies show that also can promote haematopoiesis along all three lineages. methods: we described our experience in paediatric patients with poor graft function or secondary failure of platelet recovery after allogeneic stem cell transplantation treated with eltrombopag. results: patients characteristics are detailed in table . all the patients received and allo-hst. the median dose of cd + cells infusion was . x e /kg ( . - . ). neutrophils engraftment occurred in + day ( - d) and platelets in + day ( - d). all the patients had an hypoplastic bone marrow with complete chimerism. the median duration from transplantation to spcf diagnosis was months ( . - m) . one of the patients received a stem cell boost prior to eltrombopag, without response. the time onset from spgf/sfpr diagnosis to initiating eltrombopag was days ( - d). eltrombopag was started at a dose of mg/kg/d, requiring an increase dose in all cases. the median dose was mg/d ( - mg). the overall response rate was % ( / ). two patients achieved complete response (cr), as defined by platelet ≥ × /l. both patients already got neutrophil ≥ . × /l without g-csf. the time from eltrombopag initiation to achieving cr was ( - d) days. the treatment was given for a median of days ( - ). it was discontinued after and days respectively in the two responder patients. both patients maintain stable blood counts after discontinuing the treatment. the non-responders patients had to stop the treatment because of other reasons not related to eltrombopag. patient had to be rescued with a cd + cells boost with a good response. two patients that were in treatment with voriconazole for a fungal infection developed hyperbilirubinemia. there were no grade - toxicities related to eltrombopag. conclusions: in our experience, according to recently published studies, eltrombopag is a safe and efficacy drug in the treatment of secondary failure of platelet recovery post-hsct. it may be used successfully in children. sometimes higher doses may be considered if no response is achieved. further prospective trials are needed to increase the level of evidence and to identify predictors of response. disclosure: nothing to declare very slow clearance of busulfan in a child with infant leukemia background: busulfan is a drug with a high interindividual variability between dose and exposition. therefore, it is recommended to perform therapeutic drug monitoring (tdm) in the context of myeloablative conditioning, especially in children. methods: we report on a -month old boy ( . kg, cm) of caucasian decent born to non-consanguine parents with mll-rearranged prob-lymphoblastic leukemia. diagnosis was established one month after birth from peripheral blood and csf tap showed cns involvement. primary chemotherapy was commenced according to the interfant- protocol. however, mrd remained positive two months under treatment, leading to an indication for allogeneic stem cell transplantation. in the interfant- protocol, we opted for a conditioning regimen comprised of fludarabine ( . mg/kg for days), busulfan and thiotepa ( x mg/kg). in our institution, busulfan is applied once daily with a target auc of - h*mg/l in this very high risk situation. according to body weight, busulfan was given with . mg/ kg as a three-hour infusion on the first day. busulfan concentrations in plasma were measured with gas chromatography-mass spectrometry (gc-ms) and auc was calculated using bayesian curve fitting. results: exact busulfan quantification was not possible after the first dose due to technical reasons. as the levels were estimated to be very high, we decided to reduce the second dose of busulfan by %. this resulted in a very high auc of h*mg/l for the second dose, so that busulfan was discontinued after two days, because it was calculated that the patient already received busulfan with a cumulative auc of h*mg/ml. trough levels after the first and second dose were and μg/l, respectively. the patient showed a very slow clearance of . l/h/sqm, while the volume of distribution was in the usual range ( . l/kg). bilirubin and liver transaminases were in the normal range at the time of conditioning, while albumin and quick were decreased on day + after transplantation the patient developed clinical und biochemical signs of venoocclusive disease (vod). vod symptoms completely resolved under therapy with defibrotide. leukocyte engraftment was established on day + . unfortunately, the patients suffered from an early relapse of the leukemia from day + . attempts to induce a second remission with blinatumomab failed. the patient is currently under palliative chemotherapy. conclusions: busulfan tdm is very important especially in infants receiving myeloablative doses of busulfan to prevent under-or over-exposure. there is evidence that high busulfan trough levels contribute to the development of vod, but anti-leukemic activity of busulfan and cns permeability make it a valuable drug for very high risk patients in childhood leukemia. larger patient cohorts are needed to assess the exposure dependent risks of toxicity versus relapse in infants and toddlers. disclosure: nothing to declare blood (ucb) obtained at delivery from three children who received a diagnosis of cerebral palsy. methods: immunophenotyping of the ucb leukocyte fraction was performed using multicolor flow cytometry. the procedure was performed according to the protocol by shatorje and colleagues ( ) . briefly, the ucb samples were labeled with specific antibodies and incubated in the dark for minutes. afterwards, the samples were treated with ml of bd facs lysing solution for minutes to preserve the leukocyte fraction only. the cells were washed using pbs (roche) and then centrifuged twice ( rpm, °c, minutes). the results were analyzed using the facsdiva software (becton dickinson). results: we found an increased white blood cell (wbc) count, lymphocyte count, and cd :cd ratio in all ucb samples. one patient had a low nk cell count and percentage, and another had a low b-cell count and percentage. one sample displayed high t (cd +) and th cell (cd +) counts, but with percentages within the limits of the reference values. conclusions: we detected elevated wbc and lymphocyte counts in all ucb samples, despite a lack of intrauterine infection symptoms. many authors have described the pathogenesis of hypoxic-ischemic encephalopathy. briefly, after an acute hypoxia-ischemia insult, activated resting microglia show macrophage-like activity. this leads to a break-down of the blood-brain barrier, infiltration by peripheral leukocytes, and brain exposure, which further exacerbates inflammation. the role of systemic inflammation is being evaluated in the animal model. it is known that systemic inflammation plays a role in traumatic brain injury (tbi) and is an independent risk factor for poor outcome in isolated tbi patients. on the other hand, m -phenotype microglia inhibit inflammation and protect neurons from secondary damage and death. however, anti-inflammatory mechanisms in neonates are immature and expose them to extremely intensive inflammation. therefore, anti-inflammatory agents, including stem cells, may be beneficial in these patients. disclosure: three of four authors are employees of the polish stem cell bank, warsaw, poland reference: background: under the hypothesis that early natural killer cell infusion (nki) following haploidentical stem cell transplantation (haplo-sct) will reduce relapse in the early post-transplant period, we conducted a pilot study to evaluate the safety and feasibility of nki following haplo-sct in children with recurrent neuroblastoma who failed previous tandem high-dose chemotherapy and autologous sct. methods: we used the high-dose i-metaiodo benzylguanidine and cyclophosphamide/fludarabine/antithymocyte globulin regimen for conditioning and infused × /kg of ex-vivo expanded nk cells derived from a haploidentical parent donor on days , , and posttransplant. results: seven children received a total of nkis, and nki-related acute toxicities were fever (n = ) followed by chills (n = ) and hypertension (n = ); all toxicities were tolerable. grade ≥ii acute gvhd and chronic gvhd developed in two and five patients, respectively. higher amount of nk cell population were detected in peripheral blood until days post-transplant compared with reference cohort. cytomegalovirus and bk virus reactivation occurred in all patients and epstein-barr virus in six patients. six patients died of relapse/progression (n = ) or treatment-related mortality (n = ), and one patient remained alive. conclusions: nki following haplo-sct was relatively safe and feasible in patients with recurrent neuroblastoma. further studies to enhance the graft-versus-tumor effect without increasing gvhd are needed. disclosure: nothing to declare regenerative medicine p repeated administration of g-csf using stem cell mobilization protocol could induce improvement of cognitive functions of children with cerebral palsy: phase ii randomized placebo-controlled study background: we performed phase ii randomized placebocontrolled clinical study to reveal the safety and feasibility of repeated granulocyte colony-stimulating factor (g-csf) administration for improvement of cognitive functions of children with cerebral palsy. methods: forty-four children with non-severe type of cerebral palsy were enrolled, and their age were - years old. g-csf ( μg/kg) was administered for days subcutaneously every months during months. we compared their cognitive functions with the magnetic resonance imaging (mri) findings and the following tools between before and months after treatment; zoo location and picture memory as working memory index (wmi) in wechsler preschool and primary scale of intelligence (wpssi), receptive and expressive vocabulary test (revt), and visual motor integration (vmi)/visual perception (vp) test. mobilized stem cell count and cytokine levels were measured before (d+ ) and after (d+ ) g-csf administration for days every months. results: no significant findings in demography was noticed between g-csf (g-) and placebo (p-) groups. no serious adverse events were observed during the whole study period. the non-severe adverse events such as urticaria (n= ), itching sense (n= ), bone pain (n= ), headache (n= ), fever (n= ), and stomatitis (n= ) were tolerable. the parents felt the clinical improvements of cognition in cases of g-group and cases of p-group (p= . ), of language in cases of g-group and cases of p-group (p= . ). in zoo location test, we can not find out the significant score (expressed as age equivalent) differences between g-and p-groups. however, in picture memory test, there were significant improvement of age equivalent of months ( . ± . → . ± . ) during months of study period in g-group compared to months in p-group (p= . ). in revt, there were significant improvement of months of age equivalent in expressive tests of g-group ( . ± . → . ± . , p= . ) compared to months in p-group. no significant findings were noted in receptive test. vmi test showed the increasing tendency of months of age equivalent in g-group ( . ± . → . ± . , p= . ) compared to p-group. the increment of cd + cell counts in peripheral blood were significant in ggroup compared to p-group. the changed levels of interleukin (il)- , il- , vascular endothelial growth factor (vegf) as well as g-csf were noted in g-group. we also observed the correlation of cognitive function tests and white matter connectoms of several networks using functionally-defined white matter atlases. conclusions: the repeated administration of g-csf using stem cell mobilization protocol is safe and feasible to improve the language and cognitive functions in children with cerebral palsy. further studies for cellular and paracrine effect of g-csf and/or mobilized peripheral blood stem cells would be needed. background: while high-dose chemotherapy (hdct) with autologous hematopoietic stem cell transplantation (auto-hsct) is an integral part of multimodal therapy for highrisk neuroblastoma (hr nb), there are still subgroups, in which the results are extremely poor. for these patients allografting (allo-hsct) may offer some hope. methods: we summarize the experience of consecutive hr nb patients receiving therapy in our pediatric transplant department in - . the median age was years ( months to years). a total of auto-hscts and allo-hscts were performed. all auto-hsct recipients were characterized by one or several high-risk features: age of more than months at disease (onset n= ), primary disseminated disease (n= ), unfavorable biologic variant (n= ), poor st -line therapy response (n= ) or systemic relapse (n= ). most patients (n= ) received bu-mel hdct (in younger patients oral busulfan was replaced by busilvex), in primary resistant cases a d/ d regimen was used. a total of patients with st (n= ) or nd (n= ) chemosensitive relapse, resistant relapse (n= ) or poor mobilizers with locally advanced resistant tumor (n= ) received allo-hsct from haploidentical donor with fludarabine-based ric. in cases the transplant was modified via immunomagnetic positive or negative selection, patients received post-transplant cyclophosphamide (post-cy)-based gvhd prophylaxis. gvhd prophylaxis also consisted of calceneurin inhibitors and sirolimus. thirteen of allo-hsct recipients received posttransplant immunoadoptive (n= ) or targeted (n= ) therapy. results: the -year os and efs in auto-hsct recipients was % and %, accordingly. all but one patient engrafted with a median time of ( - ) days. bu-mel regimen was characterized by acceptable toxicity with most common toxicities being oral mucositis and infectious complications. the vod/sos incidence was only %. four patients dies due to infection (n= ), cns hemorrhage (n= ), and secondary leukemia (n= ). according to multivariate analysis the most important prognostic factors were response to st line therapy and post auto-hsct mibg scan results. the prognosis in initially resistant patients with good response to nd or rd -line therapy was still very poor (all patients relapsed with the median efs of months). all patients receiving a second auto-hsct after relapse died due to disease progression. with a median follow up of ( - ) months allo-hsct recipients are alive, of them with no signs of disease progression. all long-term responders received post-transplant therapy. one patient died due to transplant complications, other deaths were caused by disease progression. there was no obvious difference between outcomes in post-cy based and transplant modification-based transplantations. agvhd more often developed in modified transplant recipients ( patients vs in post-cy group, of these cases gr iii-iv), patients in post-cy group had grade iii-iv hemorrhagic cystitis. the median time to engraftment was longer for ptcm group compared to transplant modification group (d + vs. d+ , accordingly). conclusions: while single hsct with auto-hsct is a golden standard in hr nb patients, the relapse rate is still high and the prognosis in relapsed/refractory patients is dismal. the allografting has some limited effectiveness in these cases and post-transplant therapy has a potential for further improvement. disclosure: nothing to declare p abstract already published. veno-occlusive liver disease (vod) is frequent but well treatable with early defibrotide administration in children with neuroblastoma receiving high-dose busulfan and melphalan background: using high-dose intravenous busulfan and melphalan (bumel) prior to autologous stem cell transplantation (sct) in children with high-risk neuroblastoma, seems to decrease toxicity of the myeloablative regimen, except for vod. in this multicenter retrospective study we aimed to assess the outcome of bumel-associated vod with early defibrotide treatment intervention. methods: we retrospectively analyzed children with high-risk neuroblastoma who underwent autologous sct with i.v. bumel regimen in slovakia and prague, czech republic in the period / - / . busulfan was administered in q hour schedule, with therapeutic drug level monitoring in % of patients. all vod patients except one were treated with defibrotide starting at a standard dose of mg/kg/day, given in doses per day. patient was treated with supportive therapy only. ursodeoxycholic acid was used as prophylaxis in all patients. vod was established using the modified seattle clinical criteria (corbacioglu, lancet ). results: the incidence of vod was % ( / ) in patients treated with intravenous busulfan and melphalan. there was no significant difference in busulfan total dose/kg between patients with ( . mg/kg (sd= , )) and without ( . mg/kg (sd= , )) vod manifestation. vod developed at a median of days after sct (range - days). anicteric forms of vod were documented, although % patients with vod ( / ) presented with increased bilirubin. % patients with vod ( / ) developed ascites but only patients ( %) required ascites drainage. no vod patient received renal replacement therapy and only one needed mechanical ventilation. importantly, we successfully treated vod in all patients. relapse or progression of neuroblastoma was the cause of death in vod patients ( %) who died. conclusions: despite targeting busulfan levels to decrease toxicity of the regimen, vod is common (we observed vod incidence exactly in the range of the siopen hr nbl- multicenter study (ladenstein, ) ). early recognition and early treatment with defibrotide seems to be effective in vod associated with bumel regimen -none of our patients died due to vod. disclosure: nothing to declare results of high-dose chemotherapy (hdct) with autologous hematopoietic stem cell transplantation (auto-hsct) in the treatment of ewing sarcoma family tumors (esft) background: while current dose-intense treatment protocols allow achieving - % survival in localized esft patients, the long-term survival in high-risk cases is still unsatisfactory. although there is a considerable body of data on high-dose consolidation the real effectiveness and optimal indications for this option are still not completely clarified. therefore, a large prospective cohort analysis may still yield useful data. methods: the whole cohort includes consecutive highrisk esft patients with median age of (range - ) years receiving hdct with auto-hsct in to after obtaining st (n= ) or nd (n= ) cr, pr (n= ), or stable disease (n= ). the high-risk features included lung (n= ), bone (n= ) or bone marrow (n= ) involvement, inadequate local control in primary axial tumors (n= ), large lesions volume or poor treatment response (n= ), and chemosensitive relapse (n= ). most patients had several risk factors. disseminated disease patients were also evaluated according to prognostic score by r.ladenstein et al. highdose busulfan-melphalan followed by autologous stem-cell transplantation (hdt/sct) was used. results: the median observation time was (range - ) months. the -year overall (os) and event-free (efs) survival were % and %, accordingly. most important outcome predictors were inadequate local control in chemoresistant cases, a primary tumor volume more than ml, more than one bone metastatic site, bone marrow involvement and additional lung metastases. according to prognostic risk score in disseminated disease esft patients identified three groups with -year os rates of % for score ≤ ( patients), % for score to ( patients), and % for score ≥ ( patients), (p< . ). conclusions: while bu-mel hdct with auto-hsct may still be a feasible option with acceptable toxicity for chemosensitive patients with inadequate local control and some primarily disseminated cases it is ineffective in primary resistant or very high-risk metastatic patients. disclosure: nothing to declare efficacy of tandem high-dose chemotherapy with autologous hematopoietic stem cell transplantation in the treatment of infant embryonal brain tumors day + (range, - ), after the second auto-hsct was day + (range, - ). two-year overall survival (os) was % and disease free survival (dfs) was %. dfs was significantly better among patients with mb ( %) and pnet ( %) in compared to children with etmr ( %), pb ( %) and atrt ( %), (p= , ). dfs in patients who received tandem auto-hsct was % in compare to infants who received only one auto-hsct ( %), (p= , ). complications grade (according to common toxicity criteria ) were observed in % of cases. conclusions: employment of tandem hdct with auto-hsct in primary infant embryonal brain tumors may be a feasible option for patients after induction treatment. both conditioning regimens had acceptable toxicity. all patients who had tandem hdct with auto-hsct had better os ( %) in compare with single hdct ( %). patients with mb and pnet had better prognosis with os % and %, respectively, in compare with other embryonal tumors. disclosure: nothing to declare background: metastatic extra ocular retinoblastoma is carrying a poor prognosis. therapeutic intensification with high-dose, marrow-ablative chemotherapy and autologous hsct has been explored, but its role is not yet clear. this study aimed to evaluate the survival outcome of patients with extraocular retinoblastoma post autologous stem cell transplant, treated at single center methods: this is a retrospective study included all patients with metastatic extraocular retinoblastoma (stages a and b) that underwent autologous hsct at the children cancer hospital egypt (cche) from november to july , the treatment protocol was adopted from cog protocol (aret ) as all patients received cycles induction chemotherapy followed by consolidation myloablative conditioning, cem (vp . mg/m x , melphalan: mg/m x , carboplatin: mg/m x ) and stem cell rescue. patients data including initial disease characteristics, transplant data, and survival outcomes were collected and analyzed results: a total of cases were included with median age of . years, and male to female ratio . . nine patients ( %) were initially presented by extra ocular disease, while patients were presented by intra ocular disease and progressed to metastatic disease. according to cog staging of extra ocular disease, patients had stage a, and were stage b ( of them had trilateral disease). after induction therapy, ( %) showed complete response and ( %) had ≥ partial response. with average cd count of x / kg, the median time to anc and platelet engraftment were days and days respectively, and there was no transplant related mortality. post-transplant radiotherapy was given only to patients. with median duration of follow up of months, the overall and event free survival rates of whole patients were . % and . % respectively conclusions: high dose chemotherapy and stem cell transplantation is a promising potential curative option for patients with metastatic extra ocular only two primary gf ( . %) occurred, both without dsa. patients developed a primary pgf ( %). -years os, years pfs and -year nrm were analyzed according to the presence of dsa in comparison with negative population. no statistically difference was found. no impact of the presence of dsa on the risk of developing gf and pgf was revealed. major outcomes of transplant was analyzed separately in patients with pgf and good graft function (ggf). -years os, -years pfs and year-nrm in ggf and primary pgf populations were % vs % (p< . ); % vs % (p< . ), % vs % (p= . ), respectively. conclusions: the presence of low level of dsa in the absence of desensitization doesn't correlate with the risk of developing gf and pgf. patients who experienced a pgf had worse outcomes in comparison with patients with ggf. disclosure: nothing to declare. the impact of hla-dpb mismatch in t-cell replete unrelated donor allogeneic stem cell transplantation background: high resolution matching of donor-recipient hla improves outcome in allogeneic stem cell transplants. matching for hla-a, -b, -c, -drb and -dq is mandatory in our transplant centre, to identify / or / matched unrelated donors. high resolution matching for dpb has been added over the last - years. however, the role of dpb matching is not yet clearly defined. methods: in this study, we retrospectively analyzed the impact of hla-dpb matching on the outcome of t-cell replete allogeneic hematopoietic stem cell transplants with cya/mtx-and without atg as gvhd prophylaxis in patients with hematological malignancies at oslo university hospital between and . patients with an unrelated donor fully matched ( / ) at hla-a, -b, -c, -drb and -dqb loci were included. further, patientrecipient pairs were also fully matched on dpb ( / ); had permissive and had non-permissive mimatches of one or two dpb alleles. the three groups were comparable with respect to diagnosis, gender, age, cytomegalovirus serostatus and conditioning regimen. results: cumulative incidence of relapse at years were significantly higher in the dpb matched pairs compared with the permissive and non-permissive mimatched ones, at % vs % and % (p< . ) respectively. relpase free survival and overall survival were superior in the nonpermissive and permissive dbp mismatched groups vs the fully matched, at % and % vs % (p= . ) and % and % vs % (p= . ) respectively. no difference in frequency of acute gvhd grade ii-iv between the three groups were found; dp match %, permissive mismatch % and non-permissive mismatched % (p= . ). neither was there a difference seen in gvhd grade iii-iv; % vs % vs %, respectively. finally, there were similar outcomes between the three groups regarding chronic gvhd and trm. in corrected multivariate analysis, only dp matching had significant influence on mortality and survival. conclusions: our results show a favorable relapse free and overall survival following a mud allotransplant with a dpb permissive or non-permissive mismatched donor compared to a fully dpb matched. this is likely due to an increased gvl-effect in dpb mismatched groups without the counterbalance of increased acute gvhd and trm. disclosure: nothing to declare p a haploidentical may be a better choice than a female genoidentical donor to transplant a patient with high risk acute myelogenous leukemia in first remission norbert gorin , myriam labopin , didier blaise , goda choi , gerard socie , jean henri bourhis , fabio ciceri , emmanuelle polge , arnon nagler , mohamad mohty china, the first affiliated hospital of soochow university, hematology, suzhou, china background: despite the incidence of leukemia increases with age, currently the geriatric population is poorly represented in the standards of care concerning that older adults undergoing hematopoietic cell transplant (hct) may experience higher transplant-related mortality (trm). previous studies have demonstrated that donor age is vital for older patients by affecting trm and survival. accordingly a relevant question is whether outcomes can be improved with a younger hla-haploidentical offspring donor rather than an older hla-matched sibling (msd). in our previous multi-center report under atg+g-csf based protocol for haplo hct, offspring donor is correlated with lower trm and higher leukemia free survival (lfs) as compared with older msd in subgroup analysis for recipients > years although it did not reach statistical significance. on the contrary, in a recent report from ebmt and cibmtr under ptcy modality for haplo hct, among patients aged to years, despite lower chronic graft-versus-host-disease (gvhd), graft failure, trm, and overall mortality were higher after transplant from offspring compared with an msd although there were differences in transplant platforms between the groups. methods: we extended our multi-center dataset and a matched pair analysis was performed. outcomes of acute leukemia patients (>= years) transplanted in cr / cr who received hct from offspring (n= ) or msd (n= ) between jan, and june, present in the multi-center database were analyzed. because the patient population was small, a : ratio matched pair analysis was implemented with the following matching factors: underlying disease (acute myeloid leukemia, acute lymphoblastic leukemia), disease status (cr /cr ), age and sex of patients, year of transplant, blood group incompatibilities, and sex of donor. results: we were able to match offspring with msd patients. the two matched groups were comparable in baseline characteristics except for donor age due to the family relationship. all patients achieved myeloid recovery with a median time of d and d for msd cohort and offspring group (p= . ). the d platelet recovery rate was % in both groups. the cumulative incidence of grade ii-iv acute gvhd in msd cohort was significantly lower than in offspring group ( % vs %, p= . ) while the incidence of chronic gvhd in msd cohort was significantly higher than in offspring group ( % vs %, p= . ). the -year trm ( % vs %, p= . ) were significantly lower in offspring-hct compared with in msd-hct and relapse incidence was comparable ( % vs %, p= . ). as a result, the -year overall survival ( % vs %, p= . ) and lfs ( % vs %, p= . ) ( figure ) were significantly higher in offspring-hct compared with in msd-hct. in a multivariate analysis, msd-hct remained a significant factor for decreased overall survival (hr . ( . - . ), p= . ) by increased trm ), p= . ) in comparison with offspring-hct. conclusions: these data favor a young offspring over an older msd in patients > years. the current analyses confirm non-hla donor characteristics, rather than hla disparity, predominantly influence survival in older acute leukemia patients. validation of these findings requires a prospective trial wherein the transplant platforms can be closely matched. [[p image] . figure . lfs in offspring-hct compared with in msd-hct ( % vs %, p= . )] disclosure: nothing to declare. impact of sibling donor-recipient sex combinations on rejection after hla-matched bone marrow transplantation for severe thalassemia cure children foundation, florence, italy, sankalp india foundation, bangalore, india, people tree hospitals, bangalore, india, south east asia institute for thalassemia, jaipur, india, pakistan institute of medical sciences, islamabad, pakistan, central asiri hospital, colombo, sri lanka, nawaloka hospital, colombo, sri lanka, kokilaben dhirubhani ambani hospital, mumbai, india background: severe thalassemia (st), i.e. a thalassemia syndrome with inability to keep spontaneous hemoglobin > g/dl, is a common indication for bone marrow transplantation (bmt) in children in the middle east and south east asia. sex mismatch has been associated with increased risk of solid organ rejection but is not generally considered an important transplant-associated risk factor in the context of fully matched sibling bmt for st. methods: a total of consecutive sibling bone marrow transplants carried out between january and april after conditioning with busulfan ( mg/kg oral, not adjusted to serum levels) and cyclophosphamide ( mg/kg) ( patients) in addition to either thiotepa ( mg/kg) ( patients), or anti-thymocyte globulin (genzyme mg/ kg or fresenius mg/kg on days - to - ) ( patients) and fludarabine mg/m ( patients) were analysed. all cases received cyclosporine and methotrexate or mycophenolate mofetil as gvhd/rejection prophylaxis. in the thiotepa group methylprednisolone at . mg/kg/day was also used during the first days after bmt (lucarelli protocol i). bone marrow was the source of hematopoietic stem cells in all cases, in the atg group it was g-csfprimed ( μg/kg/dose twice daily for to days prior to harvest). all patients were considered low risk based on liver size < cm from costal margin and age less than years (median . years, range . to . ), all sibling pairs where hla-compatible. results: [[p image] . sibling donor-recipient sex combinations.] the lowest rejection rate ( %) was observed in the sister to sister (s s) group of cases, followed by brother to brother (b b) group of cases with %. in the sister to brother (s b) group of cases, rejection rate was %, and % in the brother to sister (b s) group of cases. on univariate analysis the only significant difference at the p . level by log rank test was b s vs. s s groups (rejection proportions of % and % respectively). interestingly, all patients with rejection and persistent pancytopenia were female recipients of male grafts. conclusions: even though several preparative regimens were employed over an -year period, our data suggests that sex mismatch among compatible siblings should be considered as a relevant variable related to bmt decisionmaking. we also recommend to consider autologous back up hematopoietic stem cell collection and storage in sibling sex mismatched transplants, particularly in brother to sister bmts. same-sex fully matched related bmt for severe thalassemia might be the best scenario in which reducedintensity preparation strategies aiming at maximizing fertility preservation might be explored. disclosure: nothing to declare. outcomes of t-cell replete hematopoietic cell transplantation from mismatched related or unrelated donors using high dose post-transplant cyclophosphamide based gvhd prophylaxis background: high dose post-transplant cyclophosphamide (ptcy) based gvhd prophylaxis overcomes immunological barriers in hla mismatched donor transplantation. ptcy has been adopted in many centers as de facto standard for hct from haploidentical donors (haplo hct). it's use in mismatched unrelated donor transplant (mmud hct) is less well established. methods: we analyzed retrospectively outcomes of contemporary cohorts of patients who underwent haplo hct or mmud hct using ptcy + cyclosporine (csa) and mycophenolate mofetil (mmf) at our center. we compared these outcomes with outcomes of cohorts of patients who underwent hct from matched unrelated donors (mud) using atg based gvhd prophylaxis or matched sibling donor (msd) with csa and mmf. patients and donors were considered matched if they background: hla-alloantibodies are a major risk factor for engraftment failure in allogeneic hematopoietic stem cell transplantation (hsct). particularly, complement fixing, donor specific antibodies were shown to be associated with early engraftment failure. prospective antibody-screening, although not currently required for donor search, could permit early identification of high risk patients for positive crossmatch. aim of this study is to set the basis for future applicability of antibody-screening-based definition of acceptable mismatches in donor selection, by creating a large prospective antibody-screening database of patients due to receive an hla-mismatched allogeneic hsct. methods: patients (n= ) diagnosed with mds/mps, nhl, mm, cll, cml, anaemia (aplastic anemia, hemoglobinopathies, pnh) and hl were prospectively screened for hla-antibodies whenever initial donor search indicated that no completely matched donor would be available. screening was performed with an elisa class i +ii screening assay. all positive screening cases were tested for antigen-specific antibody identification with luminex sab, and acceptable mismatches were defined. the results were subsequently considered in donor search and selection. we now report the frequencies of alloimmunization observed in these patients. results: the highest rate of alloimmunization was observed in patients from the anaemia disease group (overall . %) followed by those from the mds/mpn group (overall . %). the lowest immunization rates were observed in cll (overall . %) and hl ( . %) patients. alloimmunization rates for hla-class i antigens (p< . ) were significantly higher compared to hla-class ii antigens. overall hla-class i immunization rates ranged from . % to . %. hla-class ii immunization rates ranged from . % to . % (table ) . conclusions: our findings suggest that patients with high transfusion burden like anaemia and mds/mpn patients have the highest risk of hla-alloimmunization with . % and . % anti-hla prevalence rates, respectively. analysis of follow-up data, will enable us to confirm whether prospective definition and consideration of acceptable mismatches in donor selection may lead to similar engraftment failure rates between immunized and non-immunized patients undergoing hlamismatched hsct. background: mothers displaying a persistent fetal microchimerism (fm) proved to be the most suitable donor in t cell-depleted haploidentical stem cell transplantation (hhsct) in children. we presumed that fetal cells leave an imprint in the mothers' immune system which positively affects recognition and elimination of malignant cells in the child by maternal effector cells. distinct killer cell immunoglobulin-like receptors (kir)/hla constellations are not only associated with reduced relapse rates after hsct in children, but also supposedly influence the establishment of an fm. methods: after approval by the local irb and obtaining informed consent, we initiated a protocol to elucidate the factors that influence the establishment, persistence and effect of fm. we established a digital droplet pcr (ddpcr) protocol to determine the fetal microchimerism. for differentiation between maternal and fetal cells, biallelic short insertion/deletion polymorphisms were used. kir and hla-c genotyping was performed by ssp-pcr. parental nk cell alloreactivity against the respective leukemic blasts and kir phenotyping were analyzed by flow cytometry. results: we analyzed parents, whose children were treated for hematological diseases at the university medical center hamburg-eppendorf. a fetal microchimerism was detected in % of the mothers. the amount of fetal cells varies between individuals ( x - - x - ). we observed a positive correlation between a persisting fm and hla-c homo-and heterozygous mothers along with a maternal cen a/b and cen b/b genotype. additionally, fm positive mothers showed a higher surface expression of the hla-c respective receptors kir dl /s . the percentage of alloreactive maternal nk cells against fetal cells was higher compared to paternal nk cells; while alloreactivity of fm positive maternal nk cells was similar to nk cells from fm negative mothers. conclusions: persistence of fm was more frequent in mothers carrying at least one hla-c allele and a centromeric b/x motif. phenotypically, fm positive mothers had higher expression of kir dl /s indicating a role of these receptors on the persistence of an fm. in vitro, maternal nk cells showed a higher alloreactivity compared to paternal nk cells. there was no difference in alloreactivity whether the mothers were fm positive or negative, suggesting other mechanisms are responsible for the superior outcome in transplantation from fm positive mothers. disclosure: nothing to declare background: although there have been significant improvements with conventional therapies in beta thalassemia major, hematopoietic stem cell transplantation is only curative therapy. related donors are preferred to diminish transplant risks. in lack of identical related donor, identical unrelated donors are second best choice. in this study, thalassemia major patients transplanted from unrelated donors (mud) were compared with thalassemic patients transplanted from relative donor (mrd) retrospectively. methods: patients who were transplanted between june and december in bahçelievler medical park hospital pediatric bone marrow transplantation unit were evaluated retrospectively. all patients were classified according to pesaro risk classification. thirty four of received busulfan, fludarabine, cyclophosphamide, thioteopa for conditioning, patients received myeloablative preparation regimen with treosulfan, fludarabine, thiotepa, cyclophosphamide. all patients were given atg, cyclosporine and methoteraxate for gvhd prophylaxis. the patients were compared in terms of acute complications in first days, engraftment, chimerism, acute and chronic gvhd after transplantation. results were evaluated with ibm spss statistics (ibm spss) program. results: a total of patients, ( . %) male and ( . %) female, aged between and years (median years) were evaluated. patients were evaluated in two groups as "mud" (n = ) and "mrd" (n = ) groups. there was no difference between groups about given stem cells (mud , ± , x /kg and mrd , ± , x / kg). neither significant difference between different pesaro risk groups in terms of developing acute and chronic gvhd and nor decreased chimerism were detected. neutrophil engraftment time ( , days) in mrd group was significantly longer than mud group ( , days) (p = . ) but no difference between platelet engraftments were observed. gvhd ratio was . % in mud donor group and . % in mrd group and no statistically significant difference was found(p> . ). the incidence of engraftment loss in mud group was . % and . % in the mrd group, and there was no statistically significant difference (p> . ). the rate of decreased chimerism was found to be significantly higher in the mrd group ( %) than in the mud group ( . %) (p: . ; p< . ). the survival rate was . % in the mud group and . % in the mrd group. the disease-free survival rate was . % in the mud group and % in the mrd group. the disease-free survival of mud group was significantly higher than mrd group (p: . ). conclusions: in our study, transplant related complications and success of transplantation with both mud and mrds were found to be similar. it is promising for mud transplantations to found lower decreased chimerism and similar os and dfss. based on these results, it was concluded that hsct from non-family donors, especially for patients incompatible with chelation therapy and had organ damage, transplantation from unrelated identical donors can be a good choice. although the results of our study seem promising, larger patient groups and prospective clinical trials are required. disclosure: nothing to declare background: use of g-csf stimulation of bone marrow (bm) donors is beneficial in many aspects; it can enhance tnc yield, but also have an immunomodulatory effect on donor t cell function, particularly invariant natural killer t (inkt) cells expansion as well as apcs. we analyzed outcomes of consecutive patients receiving bone marrow from hla-haploidentical donors that were stimulated with g-csf prior to harvest. methods: in the time period between / and / , patients received bone marrow from donors stimulated with ug/kg bw of g-csf on days - , - and day of bm collection. four patients ( %) received myeloablative (bucy) conditioning, one ( %) received tec ric conditioning while ( %) received nma ("baltimore") conditioning. all patients received posttransplantation cyclophosphamide (ptcy) on days + and + , tacrolimus and mmf were started on day + . for patients donors were fathers, mothers, siblings and children. results: median age was years ( - ), there were female and male patients. twelve patients had aml, hodgkin lymphoma, all, mds, nhl and cml. median number of infused tnc in graft was . x /kg bw ( . - . ) and cd + cells . x /kg bw ( - . ) . after median follow up of days (range - ), overall survival was %, with median survival of months. engraftment was established in ( %) patients, ( %) had primary rejection and patients ( %) died in sepsis prior to engraftment. of patients that engrafted, further ( %) patients had secondary rejection, two of them were transplanted again from a haploidentical donor, both using pbsc as a source of graft. median time to neutrophil recovery (anc> ) was days ( - ), while median time to platelet recovery (plt> x /l) was days ( - ) in evaluable patients. cumulative incidence of agvhd ii-iv was . % ( % ci, - );of note is that of patients that developed agvhd only one had grade iii, while remaining patients had grade ii. cumulative incidence of cgvhd requiring treatment was . % ( % ci, [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] . cumulative incidence of relapse was . % ( % ci, - ) and trm was . % ( % ci, - ). conclusions: the use of g-csf mobilized bm graft in the hla-haploidentical setting with ptcy has proven to be useful to us, not only in terms of tnc yield which was more than satisfactory and contributed to adequate hematological recovery, but also in the excellent control of both acute and chronic gvhd, with most patients developing agvhd of grade ii and only one grade iii (actually developed only after dli given for decreasing chimerism). comparative studies are of course warranted to prove benefit, but this data contributes to the growing body of evidence that indeed donor stem cell stimulation with g-csf has potentially powerful immunomodulatory effect. disclosure: nothing to disclose p other-relative donors as a reliable bank for allogeneic hsct in countries with culturally accepted cousin-cousin marriages: a two-year report from a pediatric center in iran background: although the optimal donors for patients undergoing allogeneic hematopoietic stem cell transplantation (allo-hsct) are fully-matched siblings, the cousincousin (consanguineous) marriages in some countries have extended the chance to find a matched donor for the hsctrecipient. in this study, an outcome analysis of transplanted patients receiving stem cells from their relatives other than siblings (other-relatives or non-sibling donors) is provided. methods: in this retrospective cross-sectional study, a two-year report of patients who received allo-hsct from their other-relative donors during september to september at the department of stem cell transplantation of children's medical center in tehran, iran is presented. the patients were followed up until st december . results: during this time period, patients underwent hsct (both autologous and allogeneic) at this center, of which cases received allo-hsct. out of allo-hsct recipients, the donors of stem cells for cases ( . %) were their other-relatives. the median (range) age at hsct was ( - ) years and the majority of patients were boys ( / , . %). according to disease class, the patients were most commonly involved with non-malignant hematologic diseases ( / patients, . %) (figure) . the source of hscs for most patients ( cases, . %) was peripheral blood and for only patients the source was bone marrow. the donors for patients were fully matched and only one patient received the hscs from a one-locus mismatched donor. hsct was successful in patients with most of them achieving full chimerism ( patients, . %) followed by those developing mixed chimerism ( patients, . %) and only one patient ( %) experienced graft failure. post-hsct complications included cmv infection in patients ( . %), other infections in ( . %), hemorrhagic cystitis in ( . %) and pres in ( . %). acute gvhd occurred in patients ( . %) and chronic gvhd in ( . %). death occurred in cases and of them were transplant-related, while was due to disease relapse and due to graft failure. the median of overall survival was ( - ) days. conclusions: the likelihood of receiving hscs from an hla-matched other-relative donor in one-thirds of children undergoing allo-hsct, with comparable outcomes to sibling and unrelated donors (as evidenced in this study compared with other studies), introduces family bank as a reliable source for pediatric allo-hsct in countries with culturally accepted cousin-cousin marriages. hence, for transplant physicians, parental consanguinity would be an indication of an extended search for a potential matched donor among the patient's family. [[p image] . distribution of patients according to disease and disease class] disclosure: nothing to declare. abstract already published. update on the hla frequency distribution of the portuguese bone marrow donor registry eduardo espada , dário ligeiro , hélder trindade , joão forjaz de lacerda frequency distribution varied throughout the country, allowing for analyses of molecular variance and generation of relatively geographically accurate graphical representations of genetic distances between regions and districts. conclusions: with the most recent hla analysis of the portuguese bone marrow donor registry we were able to extrapolate high-resolution haplotype frequencies from the most common low-resolution hla-a/-b/-drb haplotypes (corresponding to % of the estimated haplotypes at that level), which will lead to an optimization of its use, hopefully limiting the time between donor search and allogeneic hematopoietic stem cell transplant. disclosure: nothing to declare. abstract already published. abstract already published. unmanipulated haploidentical donor transplantation compared to identical sibling donor had better antileukemia effect for refractory/relapsed acute myeloid leukemia in not remission status background: patients diagnosed with saa with no sibling donors and who are refractory to immunosupression are candidates to hematopoietic stem cell transplant using alternative donors. haploidentical donor transplants has been reported using cyclophosphamide (cy) post stem cell infusion as immunephrophylaxis. the present study has the objective of evaluating overall survival and engraftment rates after haploidentical stem cell transplant for saa in a reference center. methods: saa adult patients (≥ yo) received hsct from haploidentical donors from de january/ to august/ . median age was y ( - ); donor was the father in six, mother in five and a brother in four cases. stem cell source was marrow in cases ( %). conditioning: patients ( %) received cy mg/kg, fludarabine mg/m² e tbi cgy. the remaining received the same drugs but radiotherapy dose varied from - cgy, all them received immunephrophylaxis with post transplant cy mg/kg, cyclosporine and mmf. median of infused cells (tcn) was , x /kg ( , - , ). results: eight patients engrafted ( %). among seven graft failures four received a second haploidentical transplant and one received an unrelated donor transplant as salvage regimen. two patients were successfully rescued after the second haplo and the others died from infectious complications. three years overall survival was %. death causes included: five infections and two lung hemorrhage. median survival was days ( - ). no patient had acute graft-versus-host-disease (gvhd) and one patient had mild c-gvhd. conclusions: haploidentical transplant was feasible as therapy for saa refractory to immunessupression with an overall survival of % in this cohort. graft failure however is still a problem to be addressed in this setting. disclosure: no disclosure stem cell mobilization, collection and engineering p abstract already published. key performance and quality indicators for a successful bone marrow collection marco sampaio , , ana salselas , fátima amado , filipa bordalo , sérgio lopes , catarina pinho , susana roncon and one from ecc (staphylococcus spp.)presented positive microbiological results. conclusions: bm collection is a challenging strategy because it is a one-time procedure and manually operatordependent technique; simultaneously it is more difficult to control the final cellular content of the bm, which is a risk for donor volume depletion. bm collection is feasible even with donor and recipient weight difference. poorer performance may be found when higher tnc are requested. we respond efficaciously when the request is between and * tnc but we fail to accomplish higher tnc values. we must emphasise that icc tnc demanded was generally lower than ecc. deciding the appropriate tnc for each patient remains a dare and an art. disclosure: nothing to declare. impact of adding plerixafor to mobilization protocol in the immune reconstitution of vδt cells after autologous hematopoietic stem cell transplantation efrat luttwak , , yael chava cohen , , odelia amit , , irit avivi , , svetlana trestman , , esti rom , , rinat eshel , , ram ron , tel aviv medical center, tel aviv, israel, sackler faculty of medicine, tel aviv university, tel aviv, israel background: multiple myeloma has remained an incurable disease even in the era of novel therapies. front line treatment typically comprises of induction chemotherapy with - cycles of a bortezomib-based regimen, stem cell mobilization, and harvesting of peripheral blood stem cells (pbsc) by apharesis, followed by high dose melphalan with hct. while brotezomib-based induction regimens have demonstrated no adverse impact on hematopoietic cell harvest number and quality, no study analyzed the impact of timing of the last brotezomib dose prior to collection. in this study we aimed to determine the effect of the timing of the last dose of brotezomib before hematopoietic cell collection and the collection yield. methods: this was a single center historical prospective study, including all sequential newly diagnosed patients with myeloma between and that were given a bortezomib-based induction therapy (≤ cycles) followed by pbsc collection. we excluded patients who either received st line vtd-pace or lenalidomide-containing regimens. peripheral blood cd + cells were measured on the day of collection. patients with cd + levels of > cells/ microliter started collection on the same day, while those with lower levels were given plerixafor. we performed regression analyses to analyze the impact of a variety of precollection factors, including days from last bortezomib therapy on the collection yield. results: we identified patients who fulfilled the inclusion criteria, table. median time from last dose of brotezomib to first leukapheresis was (range, - ) days. a statistically significant correlation was found between the days from last dose of brotezomib and both the first collection day-cd + cells/kg (r= . , p< . ), and the total collected cd + cells/kg(r= . , p= . ), figure. the optimal cutoff point as indicated by the roc curve was . days according to collection success with sensitivity of % and specificity of %, youden´s index . . in multivariate analysis included other factors affecting collection yield (age, gender, status of disease at collection, and prior radiation) -timing of last dose of brotizomib remained significantly associated with the total collected cd + cells/kg (p= . ). increasing age, female gender, and prior radiation were associated with lower collection yield (p= . , . , . , respectively). based on this, we developed a model to predict the total collected cd pos cells = . + . (timing in days of last dose of brotezomib) - . (age) - . (if female) - . (≥pr) - . (if prior radation). conclusions: timing of last dose of brotezomib is an important factor for predicting a successful collection. a washout period of days is associated with a better collection yield. these results should be further validated in a prospective study. age (median, range) ( - ) gender -male (%) ( ) prior radiation treatment (%) ( ) disease status at collection (%) ≥pr - ( disclosure: nothing to declare p mobilization with plerixafor in "poor mobilizer" related and unrelated donors of hpc-a in case of failed mobilization with g-csf background: in the allogeneic hpc transplantation, both from related and unrelated donors, the most commonly used source is peripheral blood after mobilization with g-csf. it is however known that about % of donors are "poor mobilizers"; the rescue strategies are: a third apheretic collection; bone marrow donation. methods: in - in italy a procedure to be adopted in case of failed mobilization of peripheral blood stem cells has been defined and shared between ibmdr, cnt (transplant national center) and cns (blood national center) and the scientific societies simti, sidem and gitmo, using plerixafor, a selective reversible antagonist of the cxcr receptor with its binder (the stromal derived factor sdf- ), in combination with standard g-csf dose. moreover since , in accordance with this protocol, the competent authority (aifa) has extended the registration of plerixafor (law no. / ), also for the mobilization in "poor mobilizer" healthy donors. finally, in the protocol was extended to "poor mobilizer" family donors, making management equivalent in related and unrelated donors. at the time of the donor´s informed consent for the donation of hpc, the hypothesis of the lack of mobilization or inadequate collection was illustrated and the possible actions proposed as "back-up donation" were anticipated. failed mobilization of cse has been defined as the presence of one of the two criteria: a number of cd + circulating on peripheral blood lower than /μl on the th day of stimulation ( d), or the collection of cd + < . x /kg weight of the recipient at first apheresis. in these cases, a single dose of plerixafor is administered subcutaneously by health professionals under medical supervision, . mg/kg of body weight, - hours before the start of apheresis. in case of use plerixafor due to failed mobilization of hpc-a or collection of an inappropriate number of cd +, the notification is made by the collection center to ibmdr (for both family and non-family donors), and to the recipient transplant center : both donor and recipient express their consent; finally once the collection is completed, the collection center informs ibmdr, which in turn notifies cnt/cns/simti/sidem/gitmo. any adverse reactions/events are notified in real time, based on the sop specifications and current regulations. results: since the introduction of the national protocol, donors ( unrelated donors and related donors) were treated, presenting at least one of the two inclusion criteria (cd < /μl at d or cd < x e /kg after first collection)after use of plerixafor in all donors, the required dose of cd was obtained to ensure successful transplantation, with a sufficient increase in the cd + cells. no side effects or adverse reactions related to the administration of plerixafor occurred. conclusions: in cases of failed mobilization in the related and unrelated donor, the use of plerixafor according to the methods described in the shared protocol between ibmdr, cns, cnt, simti, sidem, gitmo, proved to be safe and effective. this protocol emphasizes the great value of the sharing of procedures between the register, institutions and scientific societies, ensuring the supervision of the process and the protection of the donor and recipient. disclosure: nothing to declare background: autologous stem cells transplantation (asct) is an effective treatment option for young patients with multiple myeloma (mm). a minority of patients may still experience untoward toxicity due to delayed engraftment. thus, the current policy in many centers is aimed to increase the target dose of collected cd + cells up to an "optimal" level of x /kg per procedure. therefore, an ideal mobilization, aimed to collect to cd + cells/kg in one apheresis, should achieve a number of circulating cd + cells > /mcl (very good mobilizers). plerixafor may help to maximize the cd + collection but its use is limited by high cost. we carried out a retrospective analysis aimed to predict the quality of mobilization and develop an algorithm to optimize both timing of collection and use of plerixafor. methods: we retrospectively collected data from mobilization procedure performed in our center between and for mm. all received the same mobilization protocol with cyclophosphamide (range - gr/sqm) and g-csf mcg/kg from + . cd + cell count was started when white blood cells (wbc) count exceeded x /l. patients were excluded from this analysis if ) showed a cd + count > /mcl (target achieved at first day count) and/or ) cd + count on second day was missing and/or ) plerixafor was administered on first day according to previous policies. sixty-eight patients were evaluable for the study. univariate and multivariate logistic regression analysis to study ccd + kinetics and assess predictors impact on mobilization was carried out. ratio cd +/wbc in first day count, gender, disease category and time from mobilization chemotherapy were also included results: among the patients included in the analysis, the threshold of cd +/mcl cells on the second day was reached by ( , %) of patients (groupa) whilst the remaining ( , %) failed the target (groupb). median (range) wbc x /l and cd + /mcl counts in group a and b were , ( - , ) and , ( , - , ), , ( ) ( ) ( ) ( ) ( ) ( ) ) and , ( , - , ) respectively, with a statistically significant differences among group (mann-whitney p= , and p= , respectively). only cd + /wbc ratio and cd + /mcl on first day count had an impact on kinetics and optimal mobilization. logistic regression model highlight cd +/mcl (or= , ; % ci: , - , ) on first count as an independent predictor of second day optimal mobilizer, with auc of . % ( , ) in roc analysis. two cd + thresholds were then calculated: < , /mcl (ppv , ; npv , ) that identified poor mobilizer, and ≥ , /mcl (ppv , ; npv , ) that exclude probability to fail on second day. for those with a cd + count between , - , the cd +/wbc ratio (or= , , % ci: , - , ) was a predictor of optimal mobilization (auc , ; , - , ); cut-off value was , (sensibility , ; specificity , ) conclusions: assessment of circulating wbc, cd + and their ratio at wbc recovery in a chemo-based mobilization is a valid tool to manage the collection strategy and the on-demand use of plerixafor. we have developed an algorithm aimed to the use of plerixafor to both rescue poor mobilizers and boost cd + count in intermediate mobilizers. background: successful autologous stem cell transplantation (asct) requires the infusion of a sufficient number of hematopoietic stem cells (hscs). peripheral blood (pb) is the most commonly used source of hscs, therefore, it is important to optimize methods used to mobilize the hscs. the most clinically used chemotherapeutic agents for effective mobilization are cyclophosphamide and etoposide. recent published studies suggest that etoposide has a better mobilization effect than cyclophosphamide even at lower doses, but it is not clear why this difference occurs. in this study, we tried to determine whether there is a difference in the mechanism of mobilization between cyclophosphamide and etoposide. methods: first, in order to confirm the clinical data for efficacy and toxicity of mobilization, we retrospectively analyzed the data of patients who were diagnosed with lymphoma and performed mobilization using cyclophosphamide or etoposide from january to december . second, mesenchymal stem cells (msc) were primarily cultured from the healthy controls, then treated cyclophosphamide or etoposide at a concentration of % inhibition of cell growth, and cytokine analysis was performed to identify cytokines known to be associated with mobilization. third, mobilization mouse model using cyclophosphamide or etoposide was generated, total blood was collected at the time of hscs collection, and cytokine and network analysis (using ingenuity pathway analysis) was performed. results: the mobilization yields for cyclophosphamide or etoposide were analyzed. etoposide miblized a significantly higher median number of cd +cells than cyclophosphamide. the rate of successful or adequate mobilization was also significanctly higher for etoposide in univariate and multivariate analysis (table ). in the analysis of toxicity during mobilization, the incidence of neutropenic fever was higher in the cyclophosphamide group (p = . ). during mobilization, cyclophosphamide maintained lower wbc counts than etoposide and showed a large increase in wbc counts at the start of collection ( figure ). the cumulative dose of cyclophosphamide or etoposide in patients who underwent autologous stem cell transplantation did not affect leukocyte (anc> /microl or platelet (plt > k/microl) engraftment. in msc treated with etoposide at a concentration of % inhibition of cell growth, il- , which is a cytokine that promotes hematopoietic stem cell mobilization, were shown a statistically significant increase (figure ). in the mouse model of mobilization (figure ), the levels of kc, one of the il- homologues in mice, had significantly increased in the etoposide-treated group compared with the levels in the cyclophosphamide-treated group. the levels of other il- homologues, mip- and lix, also showed increases in the etoposide-treated group compared with those in the cyclophosphamide-treated group; these differences, however, were not statistically significant (figure ). network analysis based on in vivo cytokine results identified that etoposide could promote mobilization in association with matrix metalloproteinase as compared to cyclophosphamide ( figure ) . conclusions: etoposide has a higher mobilization efficacy when compared to cyclophosphamide, which could due to the different mechanisms of mobilization through the elevation of il and the activation of matrix metalloproteinase associated therewith. background: high-dose chemotherapy followed by autologous blood stem cell transplantation (asct) is a standard therapy for wide range of hematologic and solid malignancies. although various methods have been introduced to improve the peripheral blood stem cell (pbsc) mobilization, autologous stem cell collection (ascc) is not successful in every patient. furthermore, even if the ascc is complete, not all of them lead to asct. we evaluated the result of ascc and actual use of pbsc grafts in current practical setting. methods: we retrospectively reviewed the all consecutive ascc procedures performed at the department of oncology in asan medical center, seoul, korea, between january and october . the targeted number of background: fanconi anemia (fa) is a rare inherited genetic bone marrow (bm) failure syndrome. while abnormal bm cells production occurs very early in life, the usual age of diagnosis is - years old. gene therapy (gt) might be an alternative to hematopoietic stem cells (hsc) transplantation, but harvest a large number of autologous hsc remains a challenge. we started a mobilization assay, fancomob, to evaluate the safety and the efficacy of fa patients' mobilization with granulocyte-colony stimulating factor (g-csf) and plerixafor. this study is part of the fa's european gt project "eurofancolen". methods: four patients with fanca mutations following the inclusion criteria were selected before pancytopenia. to note, fa was diagnosed before clinical manifestation through family screening. they received subcutaneous injection of g-csf ( μg/kg twice a day) from d- and plerixafor ( . mg/kg/day) from d- . the collection protocol targeted x e /kg of cd + cells, based on a predicted future weight in years. cd + cells and white blood cells (wbc) blood count were monitored tightly along the mobilization. patients with more than cd +/μl or between and cd /μl with a clustered aspect detected by flow cytometry after plerixafor injection underwent apheresis. cd + cells were immunoselected from the collection with clinimacs purification system (miltenyi) and cryopreserved for further gt manipulation. results: the mobilization target was not achieved for the first two included patients (fa years old and fa years old). the minimum value of cd +/μl required wasn't obtained for fa and the flow cytometry cd + aspect was not clustered for fa . cd + cells were mobilized quickly but transitionally after plerixafor injection for the last two patients, fa and fa , and years old respectively. both patients underwent apheresis procedures. no cd + cell rebound was observed after the apheresis was stopped.collection target was not achieved after four days of collection for fa . it was obtained the first day for fa (figure ). back-up for hsc transplantation could not be cryopreserved because of the limited number of cd + cells collected in patients fa and fa . no short-term adverse events were observed. following cd + immunoselection, cd + cell purity and recovery were poor but in the normal range described in the literature for fanconi patients ( - %)( table i) . one month after the collection hemograms were unchanged. conclusions: our clinical study offer new data showing that mobilization of fa patients with g-csf and plerixafor is safe and more efficient for younger patients, especially before clinical manifestations of bm failure. further efforts are required to establish an effective technic to purify the cd + cells after harvesting. basal cd + cell and platelets count are a strong predictor for mobilized peripheral blood stem cells on the th day of g-csf treatment in donors cryopreserved pbscs. this was associated with considerable efforts for the patients and caused additional treatment costs. on the one hand, having the therapeutic option of an autologous transplantation in the future may represent a clinically relevant advantage. however, a huge number of stem cell products are kept in storage for many years without ever been used for transplantation. our study provides cause for a careful reevaluation of the current clinical practice, which may help to focus more precisely on patients who actually benefit from a cryostored autologous stem cell graft. [[p image] . fig. : absolute numbers and relative distribution of stem cell grafts] disclosure: the authors confirm that there are no potential conflicts of interest to disclose, except the following: katharina kriegsmann: research funding from bms, celgene, and sanofi. patrick wuchter: membership in advisory boards for sanofi-aventis. reduction of dimethylsulfoxide (dmso) concentration from % to % in criopreservation of stem cells. influence on the kinetics of engraftment and tolerance to infusion patricia lopez-pereira , beatriz aguado , elena sola , carmen cámara , isabel vicuña , lorena vega , adrian alegre hospital universitario de la princesa, madrid, spain background: dmso is the cryoprotectant most used in the cryopreservation of stem cells. it is associated with adverse effects during the infusion of the product, its toxicity being proportional to the volume infused. the most common concentration used has been %, although recent publications report that reducing it to % leads to lower rate of side effects without impact on the product or the graft. we retrospectively analyzed patients recipients of autologous peripheral blood stem cell transplantation (hct) in our hospital from january to september . they are divided into two groups according to the concentration of dmso used in the freezing ( % until september or % since october ). the baseline characteristics of the patients, the infused product and the graft are shown in table . the cd count was performed by flow cytometry. all freezings were performed with a biological freezer for programmed controlled rate cryopreservation and stored in ultrafreezers at - ºc. results: both population groups are homogeneous. the t-test was used for statistical analysis. regarding the cd + variable, no statistically significant differences were observed (p = . ). neither for the variables leukocyte recovery and platelet recovery (p = . , p = . respectively). the difference in the variable viability is . units (ci %: [ . - . ]) and is statistically significant (p = . ) in favor of dmso %. regarding adverse effects, % (n = ) of the serious adverse reactions occurred in the % dmso group (hypotension and seizures). the mild and moderate ones were similar in both groups, most were mild nausea, vomiting and flushing. overall, no statistically significant differences were observed due to the low rate of adverse effects found. patients starting with until october , total of patients attempted collection of autologous pbscs, and poor mobilizers recieved plerixafor during first mobilization cycle. in total, patients required repeated mobilization cycles ( , %) of which were from the plerixafor group. in total patients recieved pleriksafor; females and males, median age ( - ) with following diagnoses: nhl, mh, multiple myeloma, neuroblastoma, nephroblastoma, sarcoma ewing and seminoma. of repeated mobilizations with plerixafor, patients ( , %) still failed to collect adequate transplant. in this period we had altogether unsuccessful mobilizations ( , % in repeated cycles, , % in total). this group of patients consisted of male and female patients, median age ( - ), diagnosis of nhl and failure to collect after leukapheresis procedures each. median number of leukapheresis needed for adequate collection was with preemtive plerixafor use, and in repeated mobilizations. conclusions: our expirience shows that preemtive use of plerixafor in poor mobilizers is efficient and has enhached success of the pbsc collections. due to drug high cost each institution needs to develop its own algorythm in management of poor mobilizers. the factors contributing to plerixafor mobilization failure still need to be elucidated. disclosure: nothing to declare. platelets recovered from mobilized leukapheresis units obtained from hla-haploidentical donors fulfill the criteria of a conventional hemocomponent and can be used for transfusion background: central venous catheter (cvc) related complications may lead to high morbidity and mortality. unlike cvc, peripheral cannulation offers a quick and inexpensive method for safe and non-traumatic vascular access (va) thus its utilization is strongly recommended whenever possible. the ultrasound (us) guidance for acquiring peripheral va is a useful tool for reduction or elimination of the need of using cvc for stem cells collection. we have made an attempt to introduce us method in our apheresis unit having no previous experience with us devices. the aim of the study was to measure the decrease of cvc insertions after introducing us and evaluate the quality of va by comparing average flow rate and confirming that the desired blood volume could be processed. methods: the theoretical education involved a free elearning course in peripheral ultrasound-guided va (pugva, usabcd, aarhus, denmark). subsequently, the personnel have implemented knowledge in practical training on gelatine and silicone phantoms and healthy volunteers. the practical activities also included a fiveday course in an apheresis centre with us-guided cannulation experience. the details concerning va were recorded, including va site, cannula size, average inlet flow rate, number of inlet pressure alarms reported by the apheresis device. the procedure details where traditional approach was applied i.e. palpable cannulation and cvcs have been collected. similarly, the necessary data for procedures where veins were assessed with ultrasound prior to apheresis were recorded. results: before introducing ultrasonography, stem cell collections have been performed in patients. of all these procedures, were accomplished with cvc ( %) and with peripheral va ( %). median cubital vein was the vessel of choice. out of the peripheral va procedures, ( %) were problematic, with or more inlet pressure alarms during every procedure. after the training stage, collection procedures were performed in patients. after introducing us we have observed a significant reduction of the number of cvc insertion required for successful apheresis from % to % (p= . ; chi-square test with fisher's exact). thirty one procedures were completed with peripheral va ( %). ultrasound device enabled cannulation not only the superficial veins but also for the deeper veins. cannulation sites included upper arm cephalic vein ( %), median cubital vein ( %), upper arm basilic vein ( %), median antebrachial vein ( %). out of the collections, were considered problematic ( %). no difference in an average flow rate was observed between procedures performed peripherally with and without ultrasound usage (p= . ; u mann-whitney test). conclusions: despite no previous experience in us guidance, we have successfully managed to introduce the new method in our apheresis unit. within months, we have reduced cvc usage threefold and as the personnel is gaining more experience, we suppose that the cvc usage may be reduced to episodic cases. despite slightly higher number of pressure alarms, all procedures with ultrasound guidance were completed as planned. ultrasound guidance is the most important tool for significant increase in peripheral va usage and may become the only option for patients with difficult va. disclosure: nothing to declare. abstract already published. donor blood management in healthy bone marrow donors: a retrospective single institution analysis background: over the last two decades mobilized peripheral blood stem cells (pbsc) have been established as the main source of stem cells because of improved engraftment and no necessity for hospitalization for the donors. nevertheless, due to the introduction of promising new transplant regimens, especially in the haploidentical transplantation setting bone marrow (bm) donations are regaining importance. although for both donation methods severe side effects are rarely described, bm collection is associated with considerable blood loss and hence symptoms of acute blood loss are commonly observed. therefore autologous blood are collected routinely in some institutions before donation. since the collected bone marrow amount depends on the target dose, the wbc yield in the product influences the required bone marrow volume. therefore we sought to investigate the relationship between collection volume, rbc volume removal, drop in hb and indications for blood transfusion. furthermore, we assessed wbc and cd +yields in relationship to various donor parameters and to product volume, in order to find prediction tools for collection volumes. methods: allogeneic bone marrow harvests from adult donors were performed at our institution and retrospectively analyzed. complete blood counts, serum iron and ferritin were assessed at work-up and weeks after donation. the bone marrow product quality including wbc, hematocrit (hct) and cd + cells were assessed by automatic hemocytometry and single-platform flow cytometry with ishage gating. results: besides local pain most of the side effects were related to blood loss. none of the donors received blood transfusions. the mean reduction of hemoglobin levels was . g/dl with a minimum hemoglobin level of . g/dl and a persistent anemia according to who criteria after weeks in . % and pathologically low ferritin levels in %. no donor presented symptoms with indication for blood transfusion. the median wbc concentration of the bm product was . /nl ( - % percentile: . - . / nl) the cd +cell concentration . /μl ( - % percentile: . - . /μl). in the linear regression analysis leukocyte counts of the donor before donation correlated significantly with wbc concentration in the product. thus in order to collect with % certainty the mio wbc which are a typical per-kg dose for an allogeneic recipient, . ml of bone marrow must be collected. collection volume did not systematically affect wbc or cd + cell concentration. conclusions: achieving high wbc yields in the bone marrow product allowed for collection of relatively modest bm volumes, thus protecting donors from excessive blood loss. acute adverse events were acceptable. optimization of perioperative management in healthy bone marrow donors may be achieved by good collection technique and reevaluation of wbc yields of each institution to calculate required bone marrow amount. the collection of autologous blood is not indicated. furthermore stringent pre-and postoperative hemoglobin management is predicted to limit adverse effects. disclosure: nothing to declare. donor-recipient weight ratio predicts successful stem cell mobilization on day four of gcsf mobilization results: in group median age of donor was years (range to years). in group median age of donor was years (range to years). table] . table ] elaborates other parameters analyzed between the two groups. one patient in group developed grade ii acute gvhd whereas patients in group developed acute gvhd grade ii-iv. at the last follow up no ( / ) patient in group has any symptoms of chronic gvhd whereas ( / ) patients in group have features of chronic gvhd (one extensive, one limited). conclusions: our observation suggests that upfront use of plerixafor in combination with gcsf modifies the graft favorably decreasing the risk of graft failure and graft versus host disease both acute and chronic. it also helps the donor by decreasing the total volume processed, amount of acd exposure and the duration of harvest. disclosure: none. impact of vitamin d levels on peripheral stem cell mobilization in autologous hematopoietic stem cell transplant recipients ferda can , zeynep arzu yegin , zubeyde nur ozkurt , orhun akdogan , lale aydın kaynar and total product cd + cell count [ . ( . - ) vs . ( . - . ); p= . ] were significantly higher in patients receiving chemotherapy+g-csf than g-csf only. the study group was divided into two groups based on peripheral cd (cut-off level: x /kg) as well as product cd levels (cut-off level: x /kg). vitamin d levels were found to be similar among these groups (p> . ). total product cd + cell count was found to be relatively lower in patients with vitamin d levels below μg/l [ . ( . - ) vs . ( . - . ); p= , ]. (figure ) conclusions: based on its effect on stem cells in in vitro studies, it may be considered that vitamin d may have a favourable impact on stem cell mobilization. statistically insignificant but relatively lower total product cd + cell count in patients who had lower vitamin d levels, which may indicate a role for vitamin d in stem cell mobilization, needs to be confirmed with larger studies. considering the high prevalence of vitamin d deficiency in the general population, the possible role of vitamin d in hematopoietic stem cell mobilization deserves further consideration. disclosure: nothing to declare background: one of the factors, affecting efficiency of autologous hematopoietic stem cell transplantation (autohsct) in hodgkin lymphoma (hl) patients is early recovery of graft, depending on cd + cell count and conditions of cell product cryopreservation and storage. it is well known, that dimethylsulfoxide (dmso), used for cryopreservation, can be cardiotoxic and cause diverse gastrointestinal, pulmonary, kidney, liver side effects and acute hemolysis. lethal for animals dose - mg/kg leads to life threatening arrhythmias and respiratory arrest. in order to improve dmso toxicity different ways of alternative cryoconservation modes are studied -lower dmso concentration ( % vs %), temperature - ˚c instead of ultra-low and washing of cell product. aim of the study is to evaluate the influence of dmso washing on hematopoietic recovery after autohsct. methods: retrospective analysis of hematopoietic recovery of relapse/refractory hl patients after autohsct was performed. mobilization regimen included second line chemotherapy for hl (dhap, begev, igev, ice) with consecutive g-csf administration. cd + cells were assessed, using -colour flow cytometer facs canto ii while cell collection, thawing and washing. cells with % dmso were stored at - ˚and washed in cases of transplantation with human albumin-dextran (reopolyglukin) and centrifugation. statistical data processing was performed by the χ method -pearson criterion; p -the level of significance of differences. results: patient groups had no difference in age, disease stage, gender, time from treatment start to autohsct and cd + cell count (p> , ). time to wbc recovery > х /л was - (median , ) days vs - (median , ) days, time to platelet> х /л recovery was - (median , ) days vs - (median , ) days in groups without and with cell washing respectively (p= , ). no difference in blood component consumption was observed (p= , ). in out of ( %) patients during cell reinfusion without washing nausea, vomiting, arterial hypertension was observed, no reactions were detected after cell washing (p = , ). conclusions: washing autologous mononuclear cells from cryopreservant dmso does not lead to low hematopoietic recovery rate after autohsct and can avoid toxicity, thus making autohsct more safe. disclosure: authors declare no conflict of interests. quality assesment of hematopoietic stem cells autografts after cryostorage, harvested using plerixafor background: the introduction of high-dose chemotherapy followed by transplantation of autologous hemopoietic stem cells (hscs) into the treatment program for multiple myeloma (mm) has significantly increased the frequency of achieving complete remissions and overall survival in patients. to obtain a sufficient amount of hscs, hematopoiesis is stimulated with granulocyte-macrophage factors (gm-csf) both in mono mode and after the administration of cytostatics followed by cytapheresis sessions (alone or after the cytostatics followed by cytapheresis sessions) . cryopreservation protocols are used to preserve cells in a viable state, followed by long-term storage of transplants in liquid nitrogen. however, in some patients it is not possible to obtain the necessary amount of hscs. the inclusion of plerixafor in standard mobilization schemes allows you to prepare the sufficient quantity of hscs in most patients with mm. methods: the study included samples of autografts from patients with mm from to (median . ± . ). hscs mobilization was performed on the background of unstable blood formation after high doses of cyclophosphamide g/m with the subsequent administration of g-csf at a dose of - μg / kg ( samples from patients) and with the addition of plerixaphor at a dose of . μg / kg ( samples from patients). immunophenotype viability of hscs in autotransplants after cryopreservation were determined by flow cytometry using the ishage protocol on a flow cytometer (facs cantoii, becton dickinson) by expressing surface markers of antibodies against cd , cd , cd , cd and staining with aminoactinomycin ( -aad). the colony-forming activity of hscs (cfu-cfu-mix, cfu-gm, cfu-g, cfu-m) was evaluated in methylcellulose (methocult h , stemcell technologies, canada) for x transplanted cells for days. results: the viability of hscs in autografts (cd + / cd + / add-) after cryopreservation in both groups was ± . %. in the group of samples using plerixaphor, a higher content of primitive hemopoiesis precursors (primitive cells) (cd + cd + cd -) was detected compared with the control group ( . ± . % and . ± %, respectively). the cfu count (cfu-cfu-mix, cfu-gm, cfu-g, cfu-m) in the plerixafor group was ± . per x explanted cells, in the control group - ± . ( figure a-d) . conclusions: the use of plerixafor against the background of standard protocols for the mobilization of hscs allows to obtain high-quality graft with a higher content of primitive cells and proliferative activity. disclosure: no conflict of interest. nothing to declare. comparison of effectiveness of plerixafor plus g-csf in poor and very poor-movilizers: efficacy of the combination of plerixafor and g-csf in poor-movilizer background: healthy donors ocassionally show a poor response to mobilization agents. plerixafor+g-csf can be a salvage strategy in poor mobilizers. some series describe the use of plerixafor to collect greater doses of cd + cells in hematopoietic stem cell transplantation (hsct) with tcell depletion. plerixafor use in the mobilization protocol could help collecting higher cd + dose in indirect t-cell depletion (cd + selection) for ex-vivo manipulated haploidentical transplantation, with less number of apheresis and a rapid engraftment. methods: data of fourteen healthy peripheral-blood donors was retrospectively collected. they received days mcg/kg/day g-csf and , mg/kg/day plerixafor on º day as mobilization treatment. fourteen pediatric patients (median age years, range - ) diagnosed with malignant and no malignant hematological diseases received haploidentical hsct with cd + selection and cd ra+ depletion between february and july . results: one leukoapheresis procedure was performed in all cases. median processed volume was liters (range - ). median of cd + cells obtained was , x /kg (range , - , ) . after positive selection, > x /kg cd + cells were infused in all cases (figure ). neutrophil engraftment was achieved after a median of days (range - ). few donors presented only plerixafor mild secondary effects. conclusions: our experience showed that a mobilization protocol using g-csf and standard dose of plerixafor (compasive use) is a safe strategy that allows collecting great cd + dose in one apheresis procedure. this could be useful for haploidentical transplantation with ex-vivo t depletion, especially if there´s a weight disproportion between donor and patient. background: mesenchymal stem cells (mscs) are selfrenewing multipotent progenitor cells with wide differentiation potential. their ease of isolation and expansion in vitro as well as their unique regenerative therapeutic properties suggest the use of msc as an approach for treating several disorders. extra-embryonic tissues as placenta have been proposed as potential sources of mscs due to the absence of ethical problems neither risks for the patients. furthermore, only protocols using fresh placental tissue have been described so far. a protocol for isolating mscs from delayed-manipulated tissue was designed and tested in order to optimize the use of placental mscs (mscs-p) in an advanced therapies context. methods: full term placentas (n= ) were obtained from healthy mothers in hospital universitario central de asturias (spain). informed consent was obtained from each mother prior to delivery. after dissection of gr decidual tissue it was washing with saline (b. braun, germany) and cut into small pieces. these biopsies were conserved hours in dmem media with % antibiotic solution x (gibco, usausa) until processing. the day after, tissue was mechanically minced and then enzimatically digested with a combination of ui/ml dnase i (sigma aldrich, usa) and . % tripsin-edta solution (w/v) (biochrom, germany) at ºc for hour. then, the mixture was filtered with μm cell strainer (bd bioscience, usa) and centrifuge at xg for minutes. finally, cells were resuspended in ml of dmem media suplemented with % fbs and antibiotic, seeded in -cm flask and incubated in forma stericult co incubator (thermo fisher scientific, usa) at ºc, %co . culture-expanded mscs cells were phenotipically characterized by flow cytometry (facs aria iiu, bd) with antibodies against cd , cd , cd , cd , cd , cd , cd , cd , hla-dr cd and cd using mesenchymal cell kit (immunostep, spain). afterwards, these cells were differentiated to adipogenic, osteogenic and chondrogenic lineages using stemmacs adipodiff media, stemmacs osteodiff media and nh chondrodiff medium (miltenyi biotec, germany) respectively. after three weeks of differentiation cells were fixed in % paraformaldehide (merck, usa) and analyzed. adipogenic, osteogenic and chondrogenic differentiation was visualized after staining with oil red o, alkaline phosphatase and hematoxilin-eosin (sigma-aldrich, usa). results: mscs-p isolated cells were characterized according to the isct criteria for mesenchymal stem cells. they were positive for cd , cd , cd , cd and cd and negative for cd , cd , cd and hla-dr, indicative of a typical msc phenotype ( figure ). all the markers showed a high percentage of expression between . and . %, meaning that msc population obtained with the designed method was very homogenous. similarly, staining for the three studied lineages was positive ( figure ). conclusions: the described protocol allows us to obtain mscs from decidual placental tissue stored and processed hours after the biopsy extraction using a unique enzymatic digestion. this circumstance permits to take advantage of placentas that are discarded after delivery giving us the option to obtain mesenchymal cells that could be used in clinical trials. disclosure: nothing to declare outcomes of umbilical cord transplant in high risk relapsed or refractory acute myeloid leukaemia background: high-risk relapsed/refractory acute myeloid leukaemia (aml) is a fatal disease. allogeneic haematopoietic stem cell transplantation represents the only chance of cure. as the transplant relies on the graft-versusleukaemia (gvl) effect, and if different donors exert different gvl effects, then choosing the right donor assumes great importance. in manchester, a large bmt centre in the north of england, our practice in such aml has been to choose unrelated cord blood (ucbt), without serotherapy in the conditioning therapy, as our preferred donor cell source. methods: we report the results of unrelated ucbt in patients (five boys and ten girls) with high-risk aml, defined as relapsed or refractory disease. thirteen patients ( %) received this as a st transplant, two patients ( %) received this as a nd transplant for relapsed aml post matched unrelated donor transplant, and one ( %) received ucbt twice, once in cr and once in cr . nine patients ( %) had mismatched ucbt, and the rest were fully matched at class-i (hla-a, -b, and-c) and class-ii (hla-drb ). conditioning was given as treosulfan, fludarabine and thiotepa in half of the patients (n = ), other treosulfanbased regimens were used in two patients ( %), and busulfan-based regimens were used in six patients ( %). no serotherapy was given. results: the median age at transplant was years (range, months - years). neutrophil and platelet engraftment were achieved in and patients at a median of and days, respectively. patients ( %) had engraftment syndrome. all engrafted patients achieved % donor chimerism, except one patient who had mixed lymphoid chimerism initially, that was corrected spontaneously to % at three months after transplant. acute gvhd grade i-ii developed in six patients ( %), and grade iii-iv developed in three patients ( %). all cases resolved, except two patients where acute gvhd evolved into chronic gvhd (one with grade i skin gvhd which fully resolved, and one with grade iii gvhd gut colitis who was parenteral nutrition dependent till death). two more patients developed chronic grade i skin gvhd and resolved (chronic gvhd developed in % in total). three patients ( %) developed veno-occlusive disease (vod), that completely resolved with defibrotide treatment and necessitated ascitic drainage in one of them. viral reactivations occurred in five patients ( %) and were successfully treated. at a median follow-up of months (range, seven months -four years), eight patients ( %) died at a median of (range, to days), with a transplantrelated mortality of % and relapse-related mortality of %. five patients ( %) relapsed post-ucbt; four died and one had a successful second ucbt (event-free survival was %). immune reconstitution in alive patients was achieved at a median of eight months. conclusions: very high-risk patients treated with ucbt with good overall survival and event-free survival, similar to aml treatment rate with low-risk disease. disclosure: nothing to declare in haploidentical transplants is the incidence of acute and chronic gvhd strictly related to the stem cell source? results: the odds ratio was . with a % confidence interval of . - . (p= . ). conclusions: the risk of infection of the uc is not related to the microbiological status of the ucb. a possible explanation for this is the presence of antibiotics in the medium used for uc, but not ucb, transport. this means that cryopreservation of ucs from which contaminated cord blood has been obtained is justified. comparison of turkish stem cell coordination center (turkok) with istanbul university bone marrow bank (tris); a single center experience in match unrelated donors azize mergen , selime aydoğdu , başak aksoy , yunus emre savcı , gürcan dikme , funda Çipe , ceyhun bozkurt , tunç fışgın older patients are increasingly being transplanted, thanks to improvement in allogeneic hematopoietic stem cell transplantation (allo-hsct) techniques. increasing donor age is associated with greater risk for mortality and graftversus-host disease (gvhd). since sibling donors are of similar age to recipients, we hypothesized that, in older patients, a young matched unrelated donor (mud) would be comparable to an hla-matched sibling donor (msd). methods: we retrospectively compared outcomes of allo-hsct from msd (n= ) and / hla mud (n= ) in patients aged ≥ years with hematological malignancies transplanted between - . all patients received reduced-intensity conditioning and graft source was peripheral blood. the primary outcome was overall survival. msds served as the reference category and were compared to muds split into three age groups (≤ [n= ], - [n= ], > [n= ] years) using univariable analyses and multivariable cox regression models adjusted for patient, disease, and transplantation features. results: the median age of hsct recipients was years and was similar across groups. median donor age for msd was years and , , and for the mud age groups ≤ , - , and > years. acute leukemia was the leading transplant indication ( %) followed by myelodysplastic syndrome, myeloproliferative neoplasms and indolent non-hodgkin lymphoma. disease risk distribution was similar across donor groups (low [ %], intermediate [ %] , and high [ %] in the complete population; p= . ). time from diagnosis to hsct was longer with mud compared to msd and increased with an older age of mud. in a univariate analysis, overall survival was % (msd), % (mud≤ ), % (mud - ), and % (mud≥ , p= . ). corresponding non-relapse mortality (nrm) cumulative incidence was %, %., %, and . % (p< . ) (figure) . gvhd-relapse-free (grfs) was %, %, %, and % (p= . ). in a multivariable cox model, young mud (≤ ) had a similar risk for mortality compared to msd (hr . , p= . ), while a monotonic increase in risk was observed with an older donor age (mud - y: hr . ,p= . ; mud≥ y: hr . , p= . ) (table) . findings were confirmed in a propensity score analysis, matched for key covariates. nrm and grade - acute gvhd were consistently higher with mud, with the greatest risk associated with older muds. the hazard for grfs was higher with mud aged or higher compared to msd; risk was not higher with younger mud. conclusions: in older patients receiving reduced intensity conditioning, msd remain the optimal choice. however, when not available, young mud provide comparable results. disclosure: nothing to declare background: there is growing evidence that community acquired respiratory virus (carv) increase the risk of pulmonary invasive fungal disease (ifd) in recipients of allogeneic hematopoietic stem cell transplantation (allo-hsct). to date, there is a lack of knowledge regarding the rate of ifd, risk factors (rfs) as well as the most critical period for the development of a later ifd after carv infections in allo-hsct recipients. methods: in this prospective observational study, we retrospectively analyzed the effect of carv on the development of a later ifd in a consecutive cohort of allo-hsct adult recipients who developed carv infectious episodes from december to december . respiratory virus in upper and/or lower respiratory tract specimens were tested using multiplex pcr panel assays. results: overall, out of allo-hsct recipients ( %) developed ifd within months after a carv episode at median of days (range - days) from the day of carv detection. all the ifds involved the lungs and in cases ( %) the diagnostic was ia accomplishing criteria of probable (n= ) or proven (n= ). of note, out of ifd ( %) occurred within the first year after transplantation. the overall rate of ifd after carv episodes was % whereas this rate was higher in recipients developing carv during the first year of transplant ( %). ifd was diagnosed in out of with carv lower respiratory tract disease (lrtd) episodes ( %) compared to out of carv upper respiratory tract disease (urtd) ( %) (p= . ). twenty-three out of carv episodes involving the lrtd during the first year after transplant ( %) developed ifd. we did not found differences in ifd rates according to the type of carv identified. multivariate analysis identified rfs for ifd: the use of atg as a part of conditioning [odds ratio (or) . , % confidence interval (c.i.) . - . , p= . ], carv lrtd (or . , % c.i. . - , p= . ), carv infection during the first year of transplant (or . , p natural killer cell alloreactive haploidentical stem cell transplantation for multiple myeloma patients catharina elssen , lotte wieten , peter von dem borne , ellen meijer , gerard bos maastricht university medical center, maastricht, netherlands, leiden university medical center, leiden, netherlands, amsterdam university medical center, location vumc, cancer center, amsterdam, netherlands background: in the past years many new drugs for multiple myeloma (mm) have been developed and are responsible for a increase in survival. notwithstanding such progress, mm remains incurable. results from allogeneic stem cell transplantation (sct), including haploidentical transplantation, in mm has shown clinical results. however, these responses are only observed in a minority of patients. we hypothesize that this observation might be due to differences in natural killer (nk) cell alloreacitvity, since we have shown in in vivo and in vitro models that mismatched alloreactive nk cells hold the capacity to kill mm cells. the aim of this prospective phase study is to evaluate if kir-ligand mismatched haploindentical bone marrow transplantation (bmt) with post-transplant cyclophosphamide will improve progression free survival (pfs) in poor risk mm patients. methods: poor risk mm patients, aged < years were enrolled if they were responsive to their last line of therapy. poor risk was defined as, high-risk cytogenetics, or relapse within a year after autologous sct, or treated with three or more previous lines of therapy. a prerequisite of enrolment was the possibility of an nk cell mismatch and availability of a mismatched family donor. patients were excluded if donor-specific hla-antibodies were present. patients received a haploidentical bmt with a non-myeloablative conditioning regimen and post-transplant cyclophosphamide. primary endpoint is pfs at , years. secondary endpoints are engraftment, bone marrow reconstitution, nk cell reconstitution and repertoire, graft versus host disease (gvhd), infections and non-relapse mortality (nrm) at , years. results: in total poor risk patients were included in the study of which could be evaluated for the primary end point. graft failure and disease progression before transplant rendered the remaining two patients not evaluable. at this interim analysis patients have already reached the , years of follow up, relapsed within , years and died due to treatment related infections, without showing progression of disease ( % nrm). average time of progression is days ( - days). two of the remaining patients at follow up, still show responsive disease (days en ). the average time to neutrophil reconstitution is days ( - days). all evaluated patients ( / ) show nk cell reconstitution with a mature phenotype in the bone marrow and peripheral blood by day . three patients developed acute gvhd ( %) of which / grade i-ii agvhd and / patient showed a grade iv agvhd. treatment related mortality was / ( %), which was in all cases due to infectious disease. conclusions: our interim analysis of mismatched haploidentical bmt in mm showed that the treatment is feasible and forms a possible platform for immunotherapeutic strategies. the majority of patients showed an early disease progression. we predefined that with a pfs of % at , years we would qualify this treatment option successful. with only two patients still in remission this goal will not be achieved and we hypothesize that the late nk cell reconstitution (day ) is responsible for the lack of response. clinical background: mscs are known to have immune modulatory capacity and may be effective in the treatment of patients with acute gvhd. however clinical studies yielded inconclusive results which was in part due to the great heterogeneity of the msc used. the off-the-shelf msc preparation "msc-ffm", generated by a proprietary pooling process, selection by plastic-adherence, expansion for an aggregate four weeks followed by cryopreservation until use, is available in germany through a national marketing authorization. "msc-ffm" is indicated in steroidrefractory agvhd, dosed at - x /kg bw i.v. in four doses one week apart. methods: we report seven consecutive pediatric patients (median age . y), who received "msc-ffm" from unrelated hla disparate donors between december and november in our institution. we gave msc infusions to patients with steroid-refractory grades iii-iv agvhd and one patient who had therapy-refractory background: regulatory t cells (treg) are known for their immunosuppressive function and have proven successful as graft-versus-host disease (gvhd) prophylaxis after allogeneic bone marrow transplantation in a number of preclinical as well as first clinical studies without compromising graft-versus leukemia (gvl) effects. in murine models of acute gvhd lymph node homing capacity via cd l (l-selectin) proved to be essential for disease prevention. yet, treg recruitment from lymph nodes to peripheral sites of ongoing gvhd also seems necessary to achieve maximum protective as well as therapeutic effects. the chemokine receptor ccr directs activated t cells to sites of inflammation, thus high ccr expression should facilitate treg homing to affected gvhd target organs. with this project we lay the foundation for future in vivo studies of treg therapy for gvhd by upregulation of ccr expression. methods: we performed systematic ex vivo analysis of ccr expression on murine naive and memory conventional (tconv) and regulatory t cells isolated from spleen, blood, bone marrow, lymph nodes, liver and lung. cells were stained for characteristic surface and intracellular markers and characterized by multiparametric flowcytometric analysis. ccr expression kinetics following stimulation were analysed in tconv and treg isolated from murine splenocytes by facs and polyclonally activated by anti-cd /cd -coated beads in the presence of exogenous il- . expression was monitored by daily flow cytometric analysis. ccr overexpression was induced by transduction of expanded treg with ccr mrna via electroporation. expression kinetics were monitored by facs, receptor function was tested in transwell migration assays using ccr ligands ccl- and ccl- . results: systematic analyses showed higher ccr expression on memory treg than on their naive counterpart in all examined organs with bone marrow samples displaying the greatest disparity. memory treg showed higher ccr expression than memory tconv in all analysed organs, except for lymph nodes where both memory populations revealed equal expression levels. stimulation of in vitro expanded treg and tconv lead to a strong increase in ccr expression with maximum levels on d and d respectively, whereas restimulation (d ) resulted in no further relevant ccr expression on treg. we performed systematic optimization of stimulation and mrna-electroporation conditions to reliably achieve highlevel short-term ccr expression. transduction of treg on d of in vitro expansion resulted in a strong ccr expression, with maximum levels h after electroporation and strong ccr expression being detectable for at least h. transwell migration assays showed enhanced migrational properties of mrna-electroporated treg towards ccr ligands. analyses performed h and h after electroporation showed persistent migration even though measured ccr surface expression had already declined significantly. conclusions: we showed that high ccr expression can be detected on memory treg in all analysed organs. since in vitro stimulation of murine treg did not reliably induce ccr expression, we established a protocol for ccr mrna-electroporation. electroporated cells showed stable short-term ccr expression and enhanced migrational properties towards ccr ligands in vitro. future studies will show whether the induction of short-term ccr expression will facilitate in vivo homing of adoptively transferred treg to sites of ongoing gvhd and thus mediate long-term inflammation suppression. disclosure: the authors have no conflict to disclose. survival and immune reconstitution of syngeneic, haploidentical and allogeneic hematopoietic stem cell transplantation in atm-deficient mice ruth pia duecker , patrick c. baer , stefan zielen , ralf schubert from allo-hsct. it´s not necessary to do chemotherapy before transplantation for patients with bone marrow blast cells more than % at the time of diagnosis. [[p image] . figure the hci-ct of patients before transplantation and occurance of grade iii-iv agvhd on overall surviv] background: . allogeneic haematopoietic stem cell transplantation (sct) offers the chance of cure for patients with transfusion-dependent thalassemia (tdt). based on the non-neoplastic nature of this condition sct approaches urgently require to prove both efficacious and safe. methods: . we report on children, adolescents and young adults (median age: years; range - years) with tdt receiving sct from an hla-matched donor (mud n= , msd n= , mfd n= ) in our center from - . all patients received the same treosulfan-based conditioning regimen (treosulfan x g/m , fludarabine x mg/m , thiotepa x mg/kg). gvhd prophylaxis was based on atg-fresenius™ ( x mg/kg, if mud or mfd as donor), csa (with taper from day + ) as well as mtx (day , , , ) in / and mmf in / patients with mtx toxicity, respectively. stem cell source was bone marrow in , peripheral blood stem cells in and cord blood in patient. prior to transplantation children received cytoreductive treatment with azathioprine, hydroxycarbamide and intensified erythrocyte transfusion. iron elimination therapy was carried out in / children with deferasirox. among the patients with available ferris-can™ analysis patients showed substantial liver iron overload (liver iron > mg/g) despite intensive chelation prior to sct. results: . all patients achieved leukocyte engraftment at median day + (range - ), however two patients required a cd -selected pbsc boost on day + and day + based on delayed platelet and/or erythrocyte engraftment. nine patients exhibited full donor chimerism in the bm at day + , the other showed mixed chimerism with < % autologous cells. on day + peripheral blood chimerism was complete in / patients with the remaining patient exhibiting stable split chimerism with % donor-derived erythrocytes and - % autologous myeloid cells. acute gvhd was observed in three patients (grade : n= , grade : n= ). however, all patients responded to immunosuppressive therapy with steroids ± ecp and re-initiation of cni (n= ). one patient suffered background: patients with relapsed or refractory acute myeloid leukaemia (aml) have a poor prognosis. allogeneic hematopoietic stem cell transplantation is the only curative option. however, allogeneic transplantation with active leukemia failed to improve significantly the longterm outcome. to improve the outcome of allo-hsct in such high-risk and refractory patients, sequential schedule of cytoreduction therapy followed by nonmyeloablative conditioning has been developed. methods: to evaluate the outcome of sequential intensified conditioning regimen followed by allogeneic hematopoietic stem cell transplantation (allo-hsct) for refractory acute myeloid leukemia (aml). results: a total of patients with primary or secondary refractory aml transplanted between june to july were included. refractoriness was defined as primary induction failure, relapse within months from induction/ consolidation chemotherapy or second relapse. median age is years ( to ). the salvage chemotherapy administered was flag-ida. two patients did not receive intensive chemotherapy because of no recovery after induction chemotherapy. seven days after the end of flag-ida, a reduced intensity conditioning consisting of fludarabine, mg/m , thiotepa, mg/kg and busulfan , mg/kg i.v. (n= ) for haploidentical donors or fludarabine plus busulfan (n= ) for hla identical sibling or unrelated donors was administered. graft-versus-host disease (gvhd) prophylaxis consisted of tacrolimus and mycophenolate mofetil. the mycophenolate was withdrawn at day + post-transplantation and tacrolimus at day + . donor lymphocytes (dli) were infused in patients without agvhd at day + post-transplantation. seventeen patients achieved complete donor chimerism, patients progressed early and patient died before engraftment. one of the patients which recovery was with persistent leukemia reached donor chimerism after immunosupression discontinuation. ten patients are alive in complete remission. median follow-up of survivors is months (range: - ). five patients died of leukemic progression, as result of gvhd and suffered intracranial hemorrhage. five patients received prophylactic dli. the incidence of acute moderate-severe gvhd and moderate-severe chronic gvhd were % (n= ) and % (n= ), respectively. the non-relapse mortality was % (n= ), mainly due to acute gvhd (n= ) . the -year cumulative incidence of relapse posttransplantation was . %. the probability of relapse was %± %. the -year os and dfs were % ± % and % ± % conclusions: the strategy of sequential chemotherapy followed by allohsct ± prophylactic dli has an acceptable toxicity profile and improves both the relapse rate and the survival for refractory aml patients. disclosure: nothing to declare background: the concept of immunological intervention to prevent relapse after hematopoietic stem cell transplantation is associated with the assessment of the chimerism status. distinguishing patient and donor hematopoiesis is usually performed by str-pcr, a powerful method developed for forensic purposes. however, this method shows restrictions with respect to detection limit, preciseness, and the possibility of automated read out. digital pcr could circumvent some of these limitations. methods: recently, validated for the bio-rad droplet digital platforms, the biotype mentype digitalquant assay was released. the assay uses indel polymorphisms on chromosomal dna to distinguish patient and donor hematopoiesis on a fret hydrolysis assay basis ("taqman assays"). thus the assay in principle is applicable on the chamber based d digital pcr system (thermo fisher). due to different reaction chemistry and physical properties of thermal transfer between the digital pcr platforms protocols are reasonably not fully compatible which would lead to lower fluorescence intensities and poor signal resolution on the solid chip based thermo fisher d platform. an adjusted pcr protocol was established and optimized using representative markers, followed by determination of tolerable and optimal amount of input dna. specificity, sensitivity and reproducibility testing with artificial mixed samples preceded the extensive verification by comparative measurement of clinical samples (n= ) and ring-trial samples (n= ). source to allogenic bm or pbsc. ucb units are immediately available for transplantation as they are frozen and banked with defined hla typing and it has an advantage for patients who need urgent transplantation. in addition, a higher degree of hla mismatch appears to be acceptable with a comparatively lower risk of acute and chronic gvhd. meanwhile, a higher incidence of engraftment failure, delayed neutrophil and platelet recovery, and posttransplant immune disorders including pre-engraftment immune reactions (pir) are major problems in unrelated ucbt. methods: in our institute, gvhd prophylaxis in ucbt was changed after march . between january and march , thirty-two patients received tacrolimus plus methylprednisolone (tac/mpsl) and between april and january , thirty-one patients received tac plus methotrexate (tac/mtx) for gvhd prophylaxis. to investigate better gvhd prophylaxis after ucbt, we compared transplant outcomes after ucbt using gvhd prophylaxis with tac/mpsl (n= ) and tac/mtx (n= ) in single-pediatric transplantation center. results: the cumulative incidence of neutrophil engraftment at day in tac/mpsl group was . % and . % in tac/mtx group (p= . ). median time of neutrophil engraftment was days earlier in tac/mtx group ( days) than tac/mpsl group ( days). according to pir, and acute gvhd, tac/mtx group showed superior outcomes; the incidence of pir (p= . ) and the cumulative incidences of acute gvhd at day ( . vs . %, p = . for grade ii-iv, . vs . %, p= . for grade iii-iv) was significantly lower in tac/mtx group than in tac/mpsl. however, the incidences of relapse (p= . ) and cytomegalovirus viremia (p= . ), and estimated overall survival (p= . ) and event-free survival (p= . ) were comparable between two groups. conclusions: our results indicated that gvhd prophylaxis with tac/mtx had favorable effects; reduced incidence of rip and acute gvhd after ucbt without any negative influences. disclosure: nothing to declare transfer of donor regulatory t-cells after atg reconditioning cures severe refractory gvhd and leads to long term persistence of regulatory t-cells in the recipient cells on a clinimacs® plus device (miltenyi biotec). the cell product contained % foxp + t-cells. the patient received , x /kg t reg on day + . subsequently intestinal gvhd decreased and finally resolved. three months after the first t reg transfer the patient got a second t reg transfer ( , x /kg) on day + due to decreasing t reg levels. thereafter t reg persisted and there was no recurrence of gvhd. the patient is well with low dose sirolimus and prednisone as the only immunosuppressants and is particularly recovering intestinal function. conclusions: this case illustrates an unusually severe acute gvhd after matched sibling sct. transfer of donorderived t reg was able to cure severe and refractory gvhd after t-cell ablation by atg. transferred t reg persisted in the recipient for a long period and did not lead to any adverse events. disclosure: no disclosures to declaim allogeneic hsct for patients with transfusion dependent anemia from matched and mismatched donors julia fekadu , andrea jarisch , jan sörensen , emilia salzmann , eva rettinger , andré willasch , shahrzad bakhtiar , thomas klingebiel , peter bader after first asct. the mean harvest for patients receiving dhap was , x cd (+) cells/kg, , x cd (+) cells/kg for cy, . x cd (+) cells/kg for igev, , x cd (+) cells/kg for ice, , x cd (+) cells/kg for choep. the patient mobilized with vtd-pace achieved , x cd (+) cells/kg after apheresis. of the patients achieved the target number of > x /kg cd + cells after apheresis, after two, and after three apheresis. the median time to apheresis was days ( - ) without significant difference between the regimens. the mean wbc count at the time of apheresis was , x /l after dhap, , x /l after cy, , x /l after igev, , x /l after ice, , x /l after choep. there was correlation between wbc and cd harvested cells (p= . ). grate - thrombocytopenia was found in patient ( dhap, ice, igev, vtd-pace). grate - anemia was registered in patients ( dhap and vtd-pace). no correlation was found between the cd + harvest and the age, number of previous lines chemotherapy, the response before mobilization, the type of the lymphoma and the clinical stage. conclusions: our results demonstrate that the chemo-g-csf protocols have comparable effectiveness with accep- background: cytomegalovirus (cmv) may cause severe complications in recipients of allogeneic haematologic stem cell transplantation (allohsct). letermovir (ltv, / mg daily without/with co-administration of cyclosporine) was recently licenced only for cmv prophylaxis in adult allohsct-recipients. paediatric data as well as data on cmv therapy are missing so far. methods: we administered letermovir mg orally once daily (with no co-administration of cyclosporine a) to paediatric patients after allohsct. edta-plasma were occasionally obtained at different time points and frozen for determination of letermovir levels using liquid chromatography/mass spectrometry (lc-ms/ms). results: for details on patients, treatment and cmv load see table . in short periods of letermovir administration, cmv blood levels became negative in both patients. considering the lacking safety data in paediatric patients, we stopped letermovir treatment in both patients, when liver parameters increased. in patient hepatopathy turned out to represent histologically proven graft versus host disease (gvhd). in patient liver parameters further increased despite withdrawal for another weeks, however, hepatopathy was only mild and self-limiting. both patients additionally received other possibly hepatotoxic substances (mycophenolate mofetil and trimethoprim/ sulfamethoxazole). letermovir plasma levels were . ng/ml ( h), . ng/ml ( h), . - . ng/ml (median . ng/ ml, n= , h) and . ng/ml ( h after administration). conclusions: during short letermovir treatment, we observed fast resolution of cmv viraemia as well as rising liver parameters in both patients. while elevated liver parameters represented gvhd in patient, a causal relationship with letermovir might be considered in the other patient. letermovir peak levels after administration of mg were within ranges reported in adults after administration of mg while trough levels were higher indicating differences in pharmacokinetics in terms of delayed clearance. inguinal lymphadenomegaly. after failure to respond to seven conventional treatment lines: methotrexate, cop (cyclophosphamide, vincristin and prednisone); gemcitabine; puva; interferon; acitretin and extracorporeal photopheresis), allogeneic hsct from an identical hla male donor was indicated. the non-myeloablative conditioning consisted of fludarabine ( mg / m ), cyclophosphamide ( mg / kg) and total body irradiation(tbi) ( cgy). prophylaxis of graft versus host disease (gvhd) was performed with cyclosporine ( mg / kg) and mycophenolate mofetil ( mg/kg). after conditioning, there was improvement of pruritus and involution of the skin. bone marrow infusion occurred on / / (d ). on d + he presented recurrence of skin lesions of fmf. donor lymphocyte infusion (dli) was performed ( x cd + cells / kg / recipient). he presented oral lichen and diarrhea respectively as manifestations of gvhd on d + and d + . as infectious intercurrence, hemorrhagic cystitis occurred by bk virus months after the first dli and he received conservative treatment and remained without systemic immunosuppression. nine months after hsct, a second dli ( x cd + cells / kg / receptor) was performed and at this time the patient is without clinical manifestations of fmf or gvhd. conclusions: the clinical response of the presented case confirms what has been reported in the literature. ctcls appear to be particularly susceptible to gvl effect, which makes hsct a potential cure for advanced ctcls in eligible patients. the timing to perform hsct in the clinical course of the disease remains a matter to be settled clinical trial registry: not applicable disclosure: no conflict of interest p abstract already published. methods: we applied next generation sequencing (pgm, ion torrent/ fischer lifetechnologies) to an unselected cohort of patients ( female, male, median age ( - ) years) who had been referred for allogeneic stem cell transplantation due to the presence of a high-risk myeloid disorder dnmt a r h ( . %) . ), cebpa ( . %; ceb-pap s ( . %) vus) kras ( . %; krasr r ( . %) vus), and kit ( . %; kitm l ( . %) . ). patients displayed a median of sequence variants (aml, mpn and cmml patients each ; mds, saa and patients with other, non-malignant hematologic diseases each sequence variants found most frequently in aml were cebpap s ( . % of all sequence variants in patients with aml, p< - , chi² test) in patients suffering cmml, dnmt ar h was particularly frequent ( . % of all sequence variants in cmml, p=. ). asxl e d ( . % of all sequence variants in other, non-malignant hematologic diseases, p=. ), idh r q ( . %, p=. ) and krasr r ( . %, p=. ) were frequent in other, non-malignant hematologic diseases. in saa, nrasg d ( % of all sequence variants in saa patients; p=. ) was frequently found, as were dnmt ak fs* ( %, p=. ), tet l w ( %, p=. ) and tet i v ( %, p=. ). conclusions: taken together, these data show that vus occur with high abundancy in this high-risk cohort of patients, and that they differ in frequency between various myeloid disorders methods: retrospective analysis of patientswho experienced either hematological relapse or progressed to aml after allo-hsct and were treated with azacitidine for this indication at hematological centers in poland. the primary end-point was overall survival (os), the secondary -response rate. results: patients, males ( . %), median age (range, - ), were enrolled. the primary indication for allo-hsct was aml median time from allo-( %ci: . - ); with -year os of . % ( %ci: . - . ). for patients stratified according to ebmt aza relapse prognostic score -year os was: prophylaxis of agvhd: with atg -atgam mg/b.w.- pts( , %), posttransplant cyclophosphomide (ptcy) mg/b.w. on d+ ;d+ - pts ( , %) conclusions: unmanipulated haplo-hsct in st - nd cr in children and adolescents with high risk al allows achieving the long-term survival in , %. the use of g-csf stimulated unmanipulated haplo-bm is associated with a satisfactory rate of engraftment. the main cause of death in our study was relapse after allo-hsct. the frequency of acute and chronic gvhd was acceptable, -years grfs rate of , % in st - nd cr represents good quality of life following unmanipulated haplo-hsct and therefore may be recommended as option for use in children and adolescents with high-risk al. disclosure: nothing to declare background: b -h (cd ) is thought to act as an immune checkpoint and regulates t and nk cell responses. it is highly overexpressed on many solid tumors while on healthy tissue protein expression is limited. this makes b -h an interesting target for cancer immunotherapy. in highrisk neuroblastoma patients, targeting disialoganglioside gd with the recently approved monoclonal antibody (mab) ch . after autologous or allogeneic sct, significantly improves survival. however, gd expression is heterogeneous and ch . causes severe adverse effects. thus, we evaluated b -h as an alternative or additional target antigen. we investigated different anti-b -h mab constructs and mab-cytokine fusions (immunocytokines) for their ability to elicit antibody-dependent cellular cytotoxicity (adcc) using expanded γ/δ t cells of healthy donors, chex lf-il was confirmed to be the most effective mab construct. interestingly, chek -il showed comparable target cell lysis -however, lysis was only transient while chex lf-il mediated permanent target cell lysis. using patient pbmcs after receiving allogeneic sct, chex lf-il and ch . mediated comparable lysis. calculated specific lysis of lan- (after hrs.; in ascending order): targets + effectors w/o mab ( %) conclusions: b -h is a suitable target antigen in case gd expression is low or absent. immunocytokines and fcoptimized mabs targeting b -h might increase the efficacy of immunotherapy in gd -negative tumors and in combinatory approaches. until now, the low-fucose immunocytokine chex lf-il seems to be the most promising anti-b great ormond street hospital, nhs foundation trust petersburg/ raisa gorbacheva memorial institute of children's oncology, hematology and transplantation, rehabilitation medicine, saint petersburg, russian federation, first i. pavlov state medical university of st. petersburg/raisa gorbacheva memorial institute of children's oncology, hematology and transplantation, bone marrow transplantation for pediatric solid tumor petersburg/raisa gorbacheva memorial institute of children's oncology, hematology and transplantation, pediatric hsct outpatients, saint petersburg, russian federation university hospital carl gustav carus james`s hospital diagnosis was aml in %, all in % and mds in % of patients. % of patients received pbsc grafts, % received unmanipulated bone marrow grafts. os at years was % in msd/mud-atg, % in haplo-ptcy, % in mmud-ptcy and % in mmud-atg groups (p= . ). in a multivariate cox model non-relapse mortality was %, %, . % and % in msd/mud-atg, haplo-ptcy, mmud-ptcy and mmud-atg groups, respectively. cumulative incidence of acute gvhd grade or was %, . %, % and % after in msd/mud-atg, haplo-ptcy cumulative incidence of chronic gvhd was % in the msd/mud-atg group uwe platzbecker , verena wais republic of china background: there are two most noteworthy strategies of haploidentical stem cell transplantations (haplo-hsct), the baltimore post-transplantation cyclophosphamide (ptcy) with or without anti-thymoglobulin (atg), and the beijing g-csf primed bone marrow (bm) plus peripheral blood stem cells (pbsc) (giac). however, the comparison of these two modalities is scarce. in this study, we aim to compare these two approaches for hematological malignancies based on the taiwan blood and marrow transplantation registry (tbmtr) with the comparable cd infusion amounts, the neutrophil engraftment time were statistically distinct among these three groups [d+ (group ) vs. d+ (group ) vs. d+ (group ), respectively as to the graft-versus-host disease (gvhd), the patients in group had more grade ii-iv but similar grade iii-iv acute gvhd compared with others (grade ii-iv: . % vs. . % vs. . %, respectively conclusions: haplo-hsct with different strategies is a feasible treatment modality for hematologic malignancies in taiwan. regarding the retrospective nature and limited patient numbers of this study republic of china background: we previously presented a low-resolution hla analysis of cedace, the voluntary portuguese bone marrow donor registry (ebmt , poster p ) and, more recently, its epidemiological characterization (ebmt , poster b ). currently, cedace is one of the largest bone marrow donor registries in the world, including nearly % of the country's population, twice the number of donors present in . the current work is an update on the most common hla haplotypes found in cedace results: of the donors in the cedace registry, . % were typed in at least loci (hla-a/-b/-drb ), . % in (including hla-cw), and . % in (including hla-dqb )the , and most common haplotypes accounted for, respectively, . %, . % and % of the haplotypes found in the entire registry. the five most common haplotypes at the low-resolution at the loci, low-resolution level, out of donors, individual genotypes were identified, leading to an hla matching probability at this level of . % hid-hsct) have a stronger anti-leukemia effect compared to identical sibling donor hsct(isd-hsct) in high-risk features .but in refractory/relapsed(r/r) aml patients who not in remission status, it is unclear whether it also augments the gvl effect antithymocyte globulin was used in haploidentical hsct. unmanipulated bone marrow and peripheral blood stem cells for all patients. cyclosporine, short-term methotrexate were employed for gvhd prophylaxis. mycophenolate mofetil included in hid-hsct. performed multivariate analysis for all patients of pretransplantation variables and developed a predictive scoring system for survival according to adverse factors. results: the total survivor median period of follow up was ( - ) months. hid -cohort had higher -year actuarial of os multivariate analysis showed isd-hsct,standard conditioning regimen and less than % proportional reduction in blast percentage pre-≥ . conclusions: haploidentical donor compared to identical sibling donor had better anti-leukemia effect in allo-hsct for r/r aml in nr status conditioning protocol was melphalan mg/m for mm and beam for nhl. the quantification and characterization of γδt cells in peripheral blood samples were performed by flow cytometry based on the expression of cd /cd /vδ /vδ /vγ /cd at , and days after ahsct. percentage (%) of γδt cells represented the proportion of these cells among all t cells. results: median age at ahsct was ( - ) years, % male. median time from diagnosis to mobilization was ( - ) months, after a median number of therapeutic lines of ( - ); pts ( . %) received radiotherapy. seventeen pts ( . %) were re-mobilized with plerixafor±g-csf ( % mm vs % nhl there was no difference in febrile neutropenia incidence (p= . ), timeto-engraftment (p= . ), time-to-neutrophils> /μl (p= . ) or erythrocyte transfusions (p= . ). however, there was more time-to-platelets> , /μl ( vs days; subpopulations did not affected pfs. conclusions: our results showed that pts mobilized with plerixafor need more collection volume (less cd +cells, higher dmso). plerixafor negatively affected platelet recovery, with similar hematologic and immune recovery for the remaining variables. its use was associated with higher %vδ +, suggesting that it induces an antineoplastic phenotype. more studies with larger samples and follow-up period are needed to evaluate plerixafor results: total ascc procedures were carried out in patients over years, once in patients, twice in patients, three times in patients, and four times in patient. non-hodgkin lymphoma (nhl) comprised . % of all cases (n = ) ), total number of chemotherapy cycles before ascc (or . , % ci . - . , p = . ), failure to achieve at least partial response before ascc (or . , % ci . - . , p = . ), total number of days receiving g-csf for mobilization (or . , % ci . - . , p = . ), and salvage use of plerixafor (or . , % ci . - . , p < . ) were found to be independent factors associated with failure of ascc. at the end of the study period, . % of successful collections (n = / ) were used for asct, . % (n= / ) were in storage awaiting transplantation hôpital necker-enfants malades melf methods: a total ahsct candidates [median age: ( - ) years; male/female: / ] were included in this study. twenty-seven patients ( . %) were diagnosed as non hodgkin's lymphoma, patients ( . %) hodgkin's lymphoma, patients ( . %) multiple myeloma, patients ( . %) acute myeloid leukemia, patients ( . %) plasmocytoma and patient ( %) testis cancer. premobilization serum -hydroxy vitamin d ( -oh d) levels were measured with immunoassay method peripheral cd + cell count background: some published data suggest a positive effect of iron chelators on the risk of post-transplant relapse, with an improvement in overall survival after allogeneic hematopoietic stem cell transplantation (hsct) conditioning regimen consisted to intravenous bu mg/m and, flu mg/m d- to d- . gvhd prophylaxis included atg . mg/kg on d- and d- , ciclosporin and methotrexate. all patients received peripheral blood stem cell transplant from an identical hla-related donor. iron chelation consisted on deferasirox (exjade ® ) - mg/kg/day, started at day , if serum ferritin level ≥ - ug/l and stopped when the level decreased below ug/l or normalized at day after transplant, / patients were evaluable (g = pts), (g = pts). patients were abo compatible ( %), had major incompatibility ( %), and % had minor incompatibility. median serum ferritin level at day , were ug/l ( - ) and ug/l ( - ) in g and g respectively with a median follow-up of months, ( - ), disease relapse incidence was higher in patients who did not received iron chelation treatment (g : . %) versus those who received oral deferasirox (g : . %) (figure ), but the difference was not statistically significant conclusions: these results deserve more investigation choep (n= ), and patient received vtd-pace μg/kg depending on the protocol. the aim was to collect at least x cd (+) cells/kg body weight. results: forty patients all patients were stage iii and iv at diagnosis. of the patients were mobilized after one line of treatment, six after two lines of treatment and five after and more lines of treatment alexandra martínez-roca , gerardo rodriguez-lobato , gonzalo gutierrez-garcia , , maria suárez-lledó background: hematopoietic stem cell transplantation (hsct) is an established procedure in lymphoma background: the role of inflammatory cascade in tumor microenvironment has been demonstrated in several studies. as a part of this issue, elevated neutrophil/lymphocyte ratio (nlr) was shown to be associated with an adverse prognosis, particularly in solid tumors. the aim of this study is to determine the impact of pre-transplant nlr on early transplant complications, as well as post-transplant relapse and survival.methods: a total of lymphoma patients [median age: ( - ) years; male/female: / ] who underwent autologous hematopoietic stem cell transplantation (hsct) were included in this retrospective study.results: the initial diagnosis was hodgkin lymphoma (hl) in ( . %), b-cell non-hodgkin lymphoma (nhl) in ( . %) and t-cell nhl in ( . %) patients. of patients who were evaluated for pretransplant disease status, patients ( . %) were in complete remission, patients ( . %) were in partial remission and ( . %) patients had refractory disease. median pre-transplant nlr was found to be . ( - ) . when the study population was divided into two subgroups as "low-" and "high-nlr", based on median nlr value, number of febrile days were found to be relatively higher in the low-nlr group (p= . ). a positive correlation was demonstrated between nlr and lactate dehydrogenase levels (r= . ; p= . ); and nlr and ferritin levels (r= . ; p= . ). at a median follow-up of ( - ) months, overall survival (os) was found to be better in the low-nlr group without statistical significance [ vs ( - ) months; p= . ]. in univariate analysis, pre-transplant nlr represented a significant impact on os (p= . ). other prognostic factors were age (p= . ), platelet engraftment (p= . ), post-transplant relapse (p= . ) and pre-transplant ferritin level (p< . ). the permanent impact of ferritin on os was confirmed in multivariate analysis (p= . ).conclusions: in this study, an adverse impact of elevated pre-transplant nlr on os was demonstrated in autologous hsct recipients with lymphoma. as a predictor of prognosis, nlr may be considered as a safe and cost-effective parameter. further studies are required in order to use this predictor in routine clinical practice.background: high cure rates in childhood diseases have been achieved by stem cell transplantation (sct). however, there is little knowledge concerning recovery of the immune system and community-acquired infection after sct. here we studied the long-term reconstitution of the immune system and incidences of community-acquired infection after sct.methods: we reviewed medical records for patients (m/f: / , median age: years (range: - years) who were treated in the department of pediatrics, hokkaido university hospital. we analyzed cd -positive cell counts, serum immunoglobulin g (igg) levels, and incidences of community-acquired infection until years after sct. indications for sct were all in patients, aml in , aa in , nb in , rms in , jmml in , nhl in , cgd in , was in , xscid in , apds in , cd ld in , and other solid tumor in patients. stem cell sources were autologous pb/bm in , allogenic bm in ( related and unrelated) and allogenic cb in patients. in this study, we excluded patients who relapsed after sct.results: the duration of cd -positive cell counts < / ml after sct was . ± . months in all patients. the durations were . ± . months in patients with hematologic malignancies, . ± . months in patients with hematologic disorders such as aplastic anemia and pid, . ± . months in patients with solid tumor, . ± . months in patients who received autologous sct, . ± . months in patients who received related bmt/ pbsct, . ± . months in patients who received unrelated bmt, and . ± . months in patients who received cbt. the durations of igg < mg/dl after sct were . ± . months in all patients, . ± . months in patients with hematologic malignancies, . ± . months in patients with hematologic disorders, . ± . months in patients with solid tumor, . ± . months in patients who received autologous sct, . ± . months in patients who received related bmt/pbsct, . ± . months in patients who received unrelated bmt, and . ± . months in patients who received cbt. there was a significantly higher incidence of community-acquired infection from months after sct. there were significant differences in the incidence of community-acquired infections between patients with cd -positive cell counts of < /ml and background: allogeneic hematopoietic cell transplantation (allo-hct) has the potential to cure subgroups of patients with multiple myeloma (mm) but its role is controversial due to high transplant-related mortality. while autologous hct is well established as consolidation after induction therapy using novel agents, allo-hct is still considered experimental due to excessive early toxicity.methods: we retrospectively studied mm patients (pts) ; ( , %) were males with a median age of years (range: - ), who underwent allo-hsct in our center between and .median time between diagnosis and allo-hct was months (range: - ).results: the international staging system (iss) was iss i ( , %), iss ii ( , %), and iss iii ( , %). twentyeight ( , %) pts received cells from unrelated donor and pts from identical siblings. stem cell source were: peripheral blood (n= ) and bone marrow (n= ). grouprisk distribution using hct-ci and pam index can be seen in figure . response was evaluated at day + : cr ( , %), vgpr ( , %), pr ( , %) and sd ( , %); response was not evaluated in pts. regarding hospitalization, ( , %) pts and ( , %) pts needed readmission in the first days and days post-allohct, respectively, and median days of hospitalization was days (range: - ). reasons for first re-admission were: infection ( , %), gvhd ( , %) and renal insufficiency ( , %). twenty-seven ( %) died and death causes were: infection ( , %), progression ( , %), secondary tumor ( , %) and the remainder, gvhd and intracranial hemorrhage. in addition, pts ( , %) suffered cytomegalovirus (cmv) reactivation.median overall survival (os) was months (range: - ). univariate analysis showed that re-admission in first days (hr , ; p= , ) , re-admission in first days (hr , ; p= , ) , cmv reactivation (hr , ; p= , ), iss (iss-i vs iss-ii and iii; hr , ; p= , ), days of hospitalization in the first days and response at + day (cr plus vgpr vs the remainder; hr , ; p= , ) were predictor factors for os. other factors like karyotype, response before allo-hct, hla-mismatch or baseline cmv serostatus, among others, do not impact on os.median os in pts with low hct-ci were months (range: - ) vs months in intermediate-high hct-ci background: allo-sct is a potentially curative therapy for patients with multiple myeloma as it provides a graftversus-myeloma effect and a myeloma-free graft. due to increased nrm and unclear os benefit the recent guidelines suggested allo-sct to be used in context of clinical trials focusing on the high-risk patients and those who relapsed early after autograft. reduced-intensity conditioning regimens may improve rate of nrm; however, optimal conditioning regimen is still to be determined. here we studied conditioning regimen with alkylating agents consisting of thiotepa (tt), busulfan (bu) and gvhd prophylaxis with post-transplant cyclophosphamide (post-cy) in high risk myeloma patients relapsing after autograft.methods: a total of patients (m, n= ) with median age of years (range - ) underwent an allo-sct (mud, n= ; mrd, n= ; mmud, n= , haploidentical, n= ) during a period from to in university of hamburg. the majority of patients had advanced disease (stage iiiab, %) and high-risk cytogenetics ( %). the median response durartion after autograft was . years (range . - . ). the conditioning included tt (cum. dosage mg/kg), bu (median cum. dosage . mg/kg i.v. or . mg/kg in elderly patients) and post-cy (cum. dosage mg/kg, day + and + ). eight patients (all of them received allografts from mmud or haploidentical donors) received additionally fludarabine (flu, cum. dosage of background: the overall incidence of active cmv infection in patients with multiple myeloma (mm) receiving background: several scoring systems have been developed to estimate outcomes in mds patients who undergo allohct. however, none of them have been specifically validated in t cell depleted grafts. the aim of this study is to investigate the prognostic ability of a recently published scoring system (sfgm-tc) in a cohort of patients with mds who underwent tcd transplants.methods: patients underwent a first tcd allohct for mds from to . the sfgm-tc score (caulier et al. curr res transl med. ) is performed at day and ranges from - , discriminating low ( ), intermediate ( ) ( ) ( ) , and high risk ( - ). additional analyses were performed at day and day . a landmark analysis was done at each time point for the day , , and analyses, respectively. background: hematopoietic stem cell transplantation (hsct) is the only curative procedure for the treatment of myelodysplastic syndrome (mds), but among several limiting factors for its accomplishment, such as the patient´s performance status, is a very relevant issue, i.e., the availability of a compatible hla donor and, when available, very often the donor´s age and comorbidities also constitute factors that hinder this medical conduct. considering this scenario, the possibility of a haploidentical transplantation (ht) has emerged as an option. in latin america, ht has been included as a treatment option since . since then, these patients have been included in the latin american registry of transplantation in myelodysplastic syndrome, making it possible to analyze the viability and results of these transplants.methods: from october to october , seventeen ( ) patients were transplanted with a haploidentical donor and included in the latin american registry. none of these patients had an identical hla ( / match) related or unrelated donor. data were obtained from the latin american registry of hsct in mds. the statistical analyses were performed using the software spss, version . and graphpad prism version . , with significance being set at p < . .results: table shows the patients and their characteristics. all donors were haploidentical. there was a predominance of reduced intensity conditioning, which was performed in patients ( . %), whereas the others received the myeloablative conditioning. cell source was peripheral blood in ( . %) and bone marrow in ( . %) of the patients. graft-versus-host disease (gvhd) prophylaxis in post-hsct was carried out with cyclophosphamide mg / kg on d+ and d+ , cyclosporin from d and mycophenolate from d+ . complete hematologic recovery was achieved in ( . %) patients. the incidence of grade ii-iv acute gvhd was . %, whereas chronic gvhd was . %. one death occurred due to graft failure and none of the patients showed autologous recovery. three other patients died. one on d+ due to a fungal infection, the second on d+ due to sinusoidal obstruction syndrome and a third on d+ due to pneumonia caused by pseudomonas. regarding overall complications, there was a predominance of mucositis ( %), overall infections ( . %) and reactivated cmv in . % of cases. of the total number of living patients, ( %) achieved complete remission and ( . %) showed disease relapse. the mean follow-up was months (ranging from to months). the lowest probability of disease-free survival at years was % ( % ci: . - . ).background: relapse is the most important cause of failure after allogeneic hematopoietic stem cell transplantation (hsct) for flt -itd-positive acute myeloid leukemia (aml). treatment with flt tyrosine kinase inhibitors (tki) constitutes a promising clinical approach to induce remission without conventional chemotherapy.methods: a year-old woman diagnosed with aml secondary to myelodisplastic syndrome (mds) with npm mutation and internal tandem duplications of the flt gene (flt -itd) in october . after achieving complete remission (cr) with conventional chemotherapy, she received a hla sibling allogenic-hsct in february , with bucy. four months later, aml relapsed only at medullary level (flt ratio: , %), treated with chemotherapy and donor lymphocytes infusions (dli). she achieved nd cr and developed limited chronic graft-versushost disease (cgvhd). nine months later (april ), she suffered the first extramedullary relapse only, breast and skin. disappearance of the lesions at all levels was achieved with chemo and radiotherapy. she always had full hematologic donor chimerism.in december , she referred atypical precordial pain irradiated to the back. cardiac mri was performed and several masses were visualized in the pericardial sac, up to cm in diameter. bm remained in cr with full donor chimerism.pericardial fluid showed massive infiltration by leukemic-flt positive cells (ratio: , %). she was not considered background: ta-tma is a severe complication that can reduce survival after hsct. risk factors have been variably reported in adults although data on children remains scarce. we aimed to identify a risk profile for development of ta-tma in children undergoing hsct.methods: we retrospectively reviewed clinical charts of children who underwent hscts between - : at great ormond street hospital (gosh) and the great north children's hospital (gnch). ta-tma was defined according to revised criteria (jodele et al. ) . risk factors were categorized into patient derived [age, gender, active co-morbidity at d of hsct (uncontrolled viral/ bacterial or fungal infection, pulmonary, cardiovascular instability, steroid therapy > . mg/kg beyond d , bcgiosis or autoimmune disease), transplant related factors (conditioning intensity, stem cell source, hla-matching, use of ciclosporin a (csa) or tacrolimus (tac), cd + dose, ex-vivo t cell depletion, use of defibrotide) and post-hsct factors (agvhd, post-hsct viral reactivation, venoocclusive disease (vod) and occurrence of posterior reversible leukoencephalopathy (pres).results: at a median of months post-hsct, ta-tma occurred among / transplants ( . %). there was no reported centre variation ( . % vs . % in gosh vs gnch; p= . ). gender, underlying disease -primary immune deficiency (pid) (n= ) vs haematological disease (n= ), use of myeloablative (n= ) vs reducedor minimal intensity conditioning (n= ), use of serotherapy or mega doses of cd ≥ x * /kg did not influence the development of ta-tma. donor type: msd/ mfd(n= ) vs mud (n= ) vs mmud/haplo-hsct background: we evaluated the outcome of haploidentical hct (hhct) using ex vivo αβ t cell-depleted (tcd) grafts after reduced-intensity conditioning (ric) containing low-dose tbi (ld-tbi) in pediatric patients with acute leukemia (al) in complete remission (cr).methods: between may and october , patients with acute leukemia ( all and aml) in cr received haploidentical hematopoietic cell transplantation (hhct) using tcrαβ-depleted graft at asan medical center children's hospital. eighteen patients received hhct between and (earlier time period) and the remaining between and (recent study period). the conditioning regimens, the dose of αβ+ t cells and gvhd prophylaxis are summarized in table. results: all patients achieved a sustained neutrophil engraftment at a median of days (range, - ) . of patients, patients ( all & aml) relapsed at a median of months (range, - ) after transplant. of the patients, patients died of disease. one patient died of disseminated tuberculosis at months after transplant, leading to the trm of % at year. as of december , of the patients survive free of disease at a median follow-up of months (range, . at a median followup of months (range, - ), efs and os at years for all patients were % and %, respectively. outcome of hhct in the recent study cohort was significantly better than that in the earlier study period (efs of % vs efs of %, p= . ). among the patients with all, the efs of patients, who received hhct in early time period after conditioning with tbi of cgy, was significantly worse than that of patients, who received in recent study period after a higher dose of tbi at cgy ( % vs %, p< . ). the efss of aml were similar between the two study groups ( % for earlier cohort vs % for recent study, p> . ).conclusions: in pediatric patients with acute leukemia in cr, our current haploidentical hct using ex vivo αβ tcd graft after ric containing ld-tbi without gvhd prophylaxis is feasible approach with a low trm. the background: anti-hla antibodies (ahab) have been recently recognized as an important risk factor for graft failure (gf), especially in hla-haploidentical stem cell transplantation (haplo-hsct). although, recently, ebmt consensus guidelines have been published [ciurea, bone marrow transplant ] , experience in pediatric t-cell depleted (tcd, another well-known risk factor for gf) haplo-hsct is lacking. in the present study, we report our experience on the use of a desensitization approach based on ebmt guidelines.methods: between june and august , all patients affected by non-malignant diseases and scheduled for a transplant from an hla-haploidentical donor after negative depletion of αβ t and b cells as previously described [li pira, biol blood marrow transplant. ], were tested for ahab with luminex® solid-phase immunoassay (one lambda, thermo fisher scientific) month before the hsct. all patients with a mfi higher than , which is considered a cutoff predicting for gf, were treated with a desensitization protocol based on the use of anti-cd rituximab ( mg/m before and immediately after the end of plasma-exchange cycle) and plasma-exchange (pe) ± infusion of irradiated buffy coat (bc) (if after pe ahab mfi was still > mfi). this latter was obtained by the non-target fraction of the αβ t-cell/b-cell-depletion procedure and consisted of * irradiated nucleated cells/kg of the recipient; this was infused - hours before the infusion of the tcd graft. pe was performed with miltenyi life tm apheresis unit (miltenyi, bergish-gladback) .results: in the study period, patients received αβ tand b-cell depleted haplo-hsct. eighteen ( %) resulted positive for ahab (mfi> ); ( %) of them had an mfi > for either anti-class i or ii ahab. these patients (see table i background: osteonecrosis (on) is a debilitating complication in survivors of allogeneic hematopoietic cell transplantation (hct). limited data is available on its course post-transplantation in children. the purpose of our study was to identify recipients of hct in whom pre-and post-magnetic resonance imaging (mri) is indicated.methods: the retrospective cohort consisted of patients who underwent first allogeneic hct from - , and prospectively underwent a total of pre-and post-transplant mri studies of the hips and knees done annually for years regardless of symptoms. surviving patients were followed for a median time of . (range . - . ) years. cases of on were compared to controls matched for age, sex, transplant type, and follow up in a : ratio for the following variables: ethnicity, underlying disease, on pre-hct, conditioning regimen, graft source, bone mineral density z-scores, body mass index, presence or absence of graft-versus-host disease, steroid use and dosage, and survival status.results: thirty ( . %) patients had mri findings confirming on post-hct. all patients with on except one were more than years of age. twenty ( %) patients were male. on pre-hct (p < . ) was the only factor associated with presence of on post-hct. epiphyseal on was seen in ( %) patients pre-hct, and ( %) post-hct. eighteen ( %) patients had involvement of more than % of articular surface, and were more likely to undergo surgery (p = . ).conclusions: the incidence of on in this large pediatric cohort was %. the only risk factor for on post-hct was pre-existing on. mri evaluation for on pre-hct is indicated in all patients. mri evaluation for on post-hct is only indicated for patients with on pre-hct and symptomatic patients. this will entail cost savings of usd , per surviving allogeneic hct patients per year. patients with more than % involvement of the articular surface need close follow up.clinical trial registry: none disclosure: none impact of rabbit anti thymocyte globulin exposure on immune reconstitution and outcome after stem cell transplantation in children background: rabbit anti thymocyte globulin (ratg) has been frequently used for many years as gvhd prophylaxis in pediatric stem cell transplantation. precise dosing regimens are crucial but remain challenging due to several pharmacological characteristics in children.methods: ratg levels were measured in pediatric patients undergoing allogeneic stem cell transplantation after obtaining approval by the local irb and informed consent by legal guardians. pediatric patients who received either thymoglobuline™ (n= ) or grafalon™ (atg-f) (n= ) as part of their conditioning regimen background: obesity among children is a growing health problem. malnutrition or being over-weight can be of prognostic impact among children who need hsct.scientific literature shows a lot of controversy in terms of effect of bmi at the time of infusion on the outcome of hsct.methods: we reviewed data of patients who underwent hsct at king faisal specialist hospital & research centre between - to correlate bmi with the outcome and complications of hsct. transplant naïve recipients with age at infusion between - years who received hsct from matched related donor or autologous hsct, were included in the dataset for analysis. a total of patients' profiles were reviewed of whom . % were boys. median age at transplant was . years. primary indication of disease was malignancy in . % followed by hemoglobinopathies . %, bone marrow failures . % and immune disorders . %. solid tumors accounted for . % among malignant disorders. myeloablative conditioning was used in . % transplants with . % regimens containing total body irradiation. majority of the patients . % underwent allogeneic transplantation using bm as the source in . % and pbsc in the remaining . % cases. donor was hla-identical sibling in %, parents in . % and others in the remaining . % patients. median tnc dose was . x ^ and cd was . x ^ per kg of the body weight at the time of infusion. age and gender specific bmi percentiles were obtained and classified according to the definition of centers for disease control and prevention (cdc); . % ( ) recipients were categorized as under-weight, . % ( ) normal, . % ( ) over-weight and remaining % ( ) as obese.results: based on chimeric studies at day- , our engraftment rate was . % ( ) out of evaluable cases. median time to neutrophil recovery was -days from infusion and -days for platelets. no statistically significant difference was found for engraftment rate on d- as it was % ( ) among . % ( ) in normal, % ( ) in under-weight and . % ( ) in the obese (p-value: . ). median time to neutrophil recovery from the infusion date was -days in over-weight patients and in the remaining three groups (p-value: . ). acute graft vs. host disease (agvhd) of any grade at day- was recorded in . % ( ). any-grade agvhd was more common in over-weight % ( ), followed by obese with . % ( ), % ( ) in under-weight and . % ( ) in normal bmi-category (p-value: . ). chronic gvhd was more frequent in over-weight ( . %, ), compared to ) background: hematopoietic stem cell transplantation (hsct) is the standard treatment for children with severe aplastic anemia (saa) who have hla-identical related donor. there is no standard conditioning regimen for children with saa secondary to non-fanconi anemia (fa) constitutional bone marrow failure syndromes such as telomeropathies. we report the outcome of a consistent reduced intensity conditioning regimen in patients with idiopathic saa or inherited bone marrow failure syndromes other than fa who underwent hla matched related hsct methods: children with saa underwent hsct using the following conditioning regimen: fludarabine mg/m , cyclophosphamide mg/m , and atg (thymoglobulin) ( mg/kg) . gvhd prophylaxis included cyclosporin and mycophenolate mofetil. donors were all matched related and bone marrow was the stem cell source. all patients had normal chromosomal fragility testresults: a total of children with saa underwent hsct, females and males. average age was . (range . - . years). all nine patients who were tested for telomere length had short telomeres. pathogenic or likely pathogenic mutations were reported in patients ( ercc l , ankrd , tinf , lztfl ). all donors had normal physical examination, normal cbc, and negative genetic testing if patient mutation is known. all patients engrafted successfully, median time to neutrophil engraftment was (range, - days) and platelet engraftment (range, - days). median infused nucleated cell dose was . (range, . - . x /kg) and cd cell dose was . (range, . - . x /kg). none of our patients had acute gvhd and one patient had mild classic chronic gvhd of the skin that was controlled with topical therapy for a short period. three patients had secondary graft failure in the first-year post transplant. first patient had pancytopenia with loss of donor chimerism and underwent successful second transplant using fludarabine, atg, and melphalan. the second patient had a nonfunctioning graft despite full donor chimerism suggesting that the related donor might be affected and had silent phenotype. the third patient had homozygous ercc l mutation and developed progressive cytopenia with myelodysplastic features few months post-transplant and subsequently underwent myeloablative matched unrelated transplant using busulfan, fludarabine, thiotepa and atg. however, the patient progressed to have acute myeloid leukemia six months post hsct. fifteen patients ( %) have normal cbc and stable donor chimerism. median follow-up duration of days (range - days). one patient with lztfl mutation developed chronic renal impairment five years post hsct.conclusions: hsct using lower dose cyclophosphamide ( mg/kg) as part of fludarabine based regimen was safe and effective in saa patients with shorter telomeres and described genetic abnormalities. optimal conditioning regimen in ercc l -associated bone marrow failure needs to be defined. larger study is needed to confirm our results.clinical trial registry: not applicable disclosure: nothing to declare late effects in patients with hemophagocytic lymphohistiocytosis treated with hematopoietic stem cell transplantation: a review of the literature background:hemophagocytic lymphohistiocytosis (hlh) is an inherited or acquired disorder of immunedysregulation. early diagnosis and immunosuppressive treatment can prevent significant organ-failure. the inherited forms, and some acquired cases can only be cured by hematopoietic stem cell transplantation (hsct). with modern transplant practices, a significant number of patients survive. the purpose of this literature review was to collect data on the frequency and type of late effects in hlh patients surviving after hsct and to examine the association with pre-existing hlh conditions (eg. involvement of the central nervous system (cns) before transplant) and with the pre-transplant conditioning regimens.methods: the medline, embase, web of science and pubmed databases were searched, by two librarians at the karolinska institutet, between may and september according to the preferred reporting items for systematic review and meta-analysis (prisma) statement. the search terms included "hlh", "fhl", "mas", multiple terms for "hsct" and late-effect conditions. inclusion criteria were publications in english that included children between january and may . authors of this review screened all the abstracts of studies against the inclusion criteria.results: only nine papers published between and , with information on late effects in hlh patients who had undergone hsct, were identified. three reports include only small numbers of hlh patients. the remaining papers contain data on long-term survivors with a median follow-up of . years. five papers address neurological sequelae with a reported incidence from - %. the highest incidence was found after a thorough neurological assessment of hlh patients compared to matched sibling controls. however, the association with cns disease before transplant and age at transplant was not clear. patients with ebvassociated hlh seem to have fewer long-term neurological problems. non-neurological late effects are described in papers only, with endocrinological problems, namely short stature, being the most frequent. one paper specifically analyzed poor growth, thyroid dysfunction and vitamin d deficiency in a cohort of patients with non-malignant disorders including hlh who had undergone hsct after a reduced intensity conditioning regimen and found significant abnormalities in all groups.conclusions: data on late effects in hlh patients is scarce and is mostly based on the retrospective evaluation of small national cohorts. the available information indicates that a significant number of patients suffers from problems which affect their daily life, but lack of information does not allow to analyze the association between pre-transplant conditions and long-term sequelae. therefore, a retrospective comprehensive analysis of patients registered in the ebmt and cibmtr registries is currently performed. it will be crucial to better define the frequency and type of late effects in a large cohort. this knowledge will aid counselling prior to hsct, provide guidance for long-term monitoring of these patients, and potentially identify specific risk factors for late effects in this rare patient population.disclosure: nothing to declare. allogeneic stem cell transplantation in patients with mucopolysaccharidosis type ii (morbus hunter)bernd hartz , nicole muschol , matthias bleeke , johanna schrum , ingo müller background: the transplantation of hematopoietic stem cells (hsct) is one of the leading methods of treatment in patients with blood system diseases, primary immunodeficiency syndromes and genetic diseases. at the same time, the quality of life in patients in the long-term after hsct significantly differs from the quality of life of healthy people of the same age. deformations in psychosexual development including problems in the gender identity formation cause social isolation of adolescents, which makes their sexual selfrealization impossible and significantly reduces the quality of their life. the purpose of our study was an assessment of the level of gender identity formation of adolescents and psychosexual development correlation to the normal adolescents of the same age.methods: in a prospective single-center study in , on the base of the department of rehabilitation medicine raisa gorbacheva memorial institute of children´s oncology, hematology and transplantation, we conducted a study of families. the respondents were: ) parents / guardians of patients accompanying them in the process of examination; ) adolescents who underwent hsct treatment and undergo planned examinations at the clinic in the posttransplant period (after d + ), (n = , of which girls and boys, age - years, from the date of hsct - years).the following methods were used to assess gender identity: specially developed questionnaires for teenagers and parents; questionnaire by sandra l. bem (sandra l. bem, ) ; projective techniques "the human picture", "the non-existent animal"; max lüscher´s color choices test.results: the traditional type of gender identity, which characterizes high masculinity among male respondents and high female gender indicators in % of cases, was not revealed. both among girls and among boys, the androgynous type prevails with a tendency toward femininity.on average, adolescents see themselves as a bit more courageous than their mothers, with rare exceptions, regardless of gender. this confirms the thesis that we received in a previous study that parents tend to see and encourage complacency of adolescents of both sexes, passivity instead of leadership, dedication and independence. all % of adolescents who participated in the test demonstrate a shift in the theme of aggression, % have some signs of preventing sexual self-determination, abandoning their body, gender, and age. % of patients do not communicate with their peers. in % of them, negative emotions prevail over positive ones. one third of the test participants demonstrate strong support for rest and minimizing their efforts.conclusions: the characteristics of family upbringing of adolescents who have undergone hsct often contribute significantly to limiting their social experience and lead to specific deformities of individuality, including in the sphere of gender identity. we consider advisable to introduce thematic group counseling of parents within the framework of the "patient's school" in psychological treatment support in the clinic. early diagnosis of the personal aspects of the psychosexual development of adolescents after hsct allows for timely identification of individual problems in this area and identification of general trends in the long term after hsct.disclosure: all authors -nothing to disclose. abnormalities in the morphology of the umbilical cord blood obtained at delivery from children who received a diagnosis of cerebral palsy maciej boruczkowski , izabela zdolińska-malinowska , maciej rojek , dariusz boruczkowski background: embryonal brain tumors are the most common malignancies in infants less than months of age. histologically characterized as undifferentiated small round cell tumors, all are similarly aggressive, have a tendency to disseminate throughout central nervous system and very poor prognosis. we tried to assess the effectiveness of tandem highdose chemotherapy (hdct) with autologous hematopoietic stem-cell transplantation (auto-hsct) in this patient group. methods: from to , infants under months with different primary embryonal brain tumors such as medulloblastoma (n= ), different pnet nos (n= ), pineoblastoma (n= ), atypical teratoid rhabdoid tumor (n= ), etmr (n= ) after surgical resection and induction chemotherapy were planned to receive tandem hdct with auto-hsct. nine patients were conducted only single transplantation because of the development of lifethreatening complications after the first hdct (n= ) or the emergence of early disease progression (n= ). at the moment of hdct patients were in complete remission (cr), patients were in partial remission (pr) and patient had stable disease (sd). the conditioning regimen for tandem auto-hsct were: the first hdct was carboplatin and etoposide, the second was thiotepa and cyclophosphamide, both with intraventricular methotrexate.results: the median follow-up is months (range, - ). the median time to engraftment after the first auto-hsct was background: a series of findings suggest that optimizing natural killer (nk) cell reactivity could further improve outcome after allogeneic hematopoietic cell transplantation (allohct). this could be achieved by killer cell immunoglobulin-like receptor (kir) genotype informed donor selection. an enhanced receptor-ligand model which used kir ds and kir dl donor genotype information to augment nk cell activation and minimize inhibition demonstrated improved survival in one large aml study (boudreau et al, jco ) . likewise, a second model built on the classification of centromeric and telomeric kir haplotype motifs, also predicted mortality after allohct for aml (cooley et al, blood ) . this joint ebmt and cibmtr study aimed at validating the two approaches in an independent cohort of patients with mds or secondary aml.methods: donor samples were retrieved from the collaborative biobank (dresden, germany) and mapped to patient outcome data extracted from the ebmt and cibmtr. genotyping of all kir genes by sequencing exons , , , , , and was performed by high resolution amplicon-based next generation sequencing. the impact of the classifiers on time-to-event outcomes was tested in cause-specific cox regression models adjusted for patient age, a modified disease risk index, performance status, donor age, hla-match, sex match, cmv match, conditioning intensity, type of t-cell depletion and graft type.results: clinical data from patients and corresponding donor genotype information were analyzed. the median age at allohct was . years (range, . to . years). the indication for allohct was mds for % and saml for % of patients. disease risk was low/intermediate and high/very high in % and %, respectively. donors were / matched for % of patients. myeloablative, reducedintensity and non-myeloablative conditioning regimens were used in %, %, and % of patients, respectively. peripheral blood stem cells were the predominant graft source ( % of patients). atg was administered in % and alemtuzumab in % of patients. during follow-up after allohct patients died. in univariable and multivariable analyses of the whole cohort, overall survival and the cumulative incidence of relapse of patients with kiradvantageous versus disadvantageous donors were not statistically significantly different. we could not replicate the pattern of outcomes predicted by the kir dl / conclusions: relapse incidence and overall survival after unrelated donor allohct could not be predicted using the kir dl /kir ds -receptor-ligand model and centromeric/telomeric kir-motif model in this large cohort of patients with mds or secondary aml. this points at the possibility of interactions between nk-cell mediated alloreactivity and disease type or procedural variations of allohct. available information on kir-genes, which have been sequenced but not yet analysed, will be investigated in exploratory analyses.disclosure: the authors have nothing to disclose in the absence of an alternative donor, it is recommended that patients undergo desensitization therapy, especially with high dsa levels (> , mfi). the aim of this study is to analyze the impact of dsa on risk of gf and poor graft function (pgf), and on major outcomes in a consecutive cohort of patients who were systematically screened for dsa before haplo-sct. methods: consecutive patients were candidates for unmanipulated haplo-sct with post-transplant cyclophosphamide (pt-cy) at our center from january to january and were analyzed for the presence of hla antibodies.results: patients underwent haplo-sct. hla antibodies were detected in patients, of them were dsa, while were non-dsa (ndsa). patients out of with dsa were transplanted using the same donor; underwent a desensitization program before transplant.background: a recent study from ebmt comparing matched sibling (msd) versus haploidentical donors transplantations, showed better outcome with msd in adult patients with intermediate risk aml in first remission (cr ). however, a female donor to a male recipient transplant combination is a poor prognostic factor and this study did not address the question whether in this situation, a haploidentical donor transplant might do better. the present study compared the outcomes of allografted male patients according to whether they received stem cells from a female msd or a haploidentical donor, in the intermediate and high risk cytogenetics groups (mrc classification).methods: the study included male patients with cytogenetics transplanted between january and june and reported to ebmt. received stem cells from a msd female donor and from a haploidentical donor ( male and female). the follow up was months ( - ). we studied separately intermediate and high risk patients. multivariate analysis was adjusted on factors differing significantly between the groups.results: -intermediate risk group: male patients received a female msd and a haploidentical transplant. the distribution of group characteristics was even except that in the haploidentical transplant group, donors were younger ( y versus ; p< . ), marrow was more frequently used ( % versus %, p< . ) and the interval from diagnosis to transplant was longer ( . versus . months, p< . ). by univariate analysis at two years post transplant, cumulative incidence (ci) of nrm post haplo was higher ( % versus %, p= . ) and ci of extensive chronic gvh lower ( % versus %; p= . ). lfs post msd and post haplo were % and % (p= . ), os % and % (ns), grfs ( % and %). by multivariate analyses the only significant poor risk factors were the haplo-identical transplant for nrm (hr: . ( . - - )) and the patient age for os (hr: . ( . - . ; p= . ). haploidentical transplantation resulted in less chronic gvhd (hr: . ( . - . ); p < - ), but a lower lfs (hr: . ( . - . ); p= . ). -high risk group: male patients received a female msd and a haploidentical transplant. in the haploidentical group, donors were younger ( y versus ; p< . ), marrow was more frequently used ( % versus %, p< . ) and the interval from diagnosis to transplant was longer ( . versus . months, p= . ). by multivariate analysis, haploidentical transplants were associated with a lower relapse incidence (hr: , ( . - . ; p = . ),a better lfs (hr: , ( . - . ; p = . ),os (hr: , ( . - . ; p = . ), and grfs (hr: , ( . - . ; p = . )(see figure) . the only other significant prognostic factor was patient age.conclusions: this study shows that in a male patient with intermediate risk aml, a genoidentical sister donor remains associated with a better lfs. in contrast, in a male patient with high risk aml in cr , a haploidentical donor may be a better choice than an hla genoidentical sister.disclosure: nothing to declare p abstract already published. hematopoietic transplant for older acute leukemia patients: improved survival with offspring donor in comparison with older-aged matched siblingsyu wang , sheng-ye lu , qi-fa liu , de-pei wu , xiao-jun huang background: post-transplant relapse remains the major cause of death of treatment failure. therapeutic options for relapse after first allogeneic stem cell transplant ( st hsct) include chemotherapy followed by donor lymphocyte infusion or second allo-hsct ( nd hsct) from the original donor or change to another donor. however, there is unclear outcome for different treatment approach. in this retrospective cohort study, we aim to compare the clinical outcome after different treatment strategy for relapse after first allo-hsct.methods: between jan and oct, consecutive patients receiving st hsct registered to the bmt database in national taiwan university hospital were analyzed. among them, cases had relapsed after first allo-hsct. one hundred and three patients who received no treatment after relapse or with incomplete data were excluded. their transplant data was collected following the ebmt registry data collection forms and manuals. overall survival rate and progression free survival rate were performed by the kaplan-meier method. univariate and multivariate analysis were performed using cox proportional hazard regression model.results: of the patients who experienced relapse after st hsct, total patients ( %) received chemotherapy followed by dli or nd hsct from the same donor (no change group), patient ( %) received chemotherapy followed by nd hsct from different donors (change group), and ( %) had conventional chemotherapy alone. the patients in "change group" were younger (median age vs , p = . ), and had more patients achieving complete remission (cr) prior to nd hsct ( % vs %, p = < . ) than patients in "no change group". after the nd hsct, the cr was % for "no change group" and % for "change group". the progression-free survival at -year and -year were . % and . % (fig a, p = . %), respectively, for "no change group" and . % and . %, respectively, for "change group". while the overall survival (os) at -year and -year were . % and % (fig b, p = . %), respectively, for "no change group" and . % and %, respectively, for "change group". those who achieved cr prior to nd hsct had a trend of better os than those without cr ( . % vs . % at -year; . % vs . % at year, p =. )( fig c) . there were cases survived for more than years in "change donor group" and cases survived more than years in "no change group". only one had developed relapse after nd hsct but achieved subsequent remission again.conclusions: our study shows that change donor had similar poor outcome comparing to those using the same donor after the st hsct. patients who achieved cr before nd hsct had a trend of better os than those without remission and the long-term survivors were only those who achieved cr prior to nd hsct. novel therapy for cr induction would be warrant for this poor prognostic population.disclosure: nothing to declare functional relevance of fetal microchimerism in nk cell cytotoxicity against leukemic blasts in children: a role for hla-c and kir dl /s ?background: allogeneic stem cell transplantation (allo-sct) remains the most effective curative intent therapy for patients with unfavorable risk acute leukemia. various donor options are available for the patient who lacks an hla-matched sibling donor, such as unrelated donors (urd) and hla-mismatched family (haploidentical) donors. in order to discover the exact role of transplantation type, there are many retrospective analysis, which compared these donor sources, have been reported. recent studies showed some promising results of haploidentical donor transplantation (hidt) using post-transplant cyclophosphamide in comparison with unrelated donor. the goal of this study was to compare the outcome of allo-sct from haploidentical versus matched unrelated (mud / ) or mismatched unrelated donor at a single hla-locus (mmud / ) for patients with acute leukemia in remission.methods: ninety-six adult ( - years) patients with acute leukemia in first or second remission who underwent allogeneic transplantation with a minimum days follow-up at florence nightingale hospital hematopoietic stem cell transplantation center between and were included in this study. patient characteristics and medical records of all patients were reviewed retrospectively. thirty-eight patients who received haploidentical donor transplantation were compared with patients receiving a mud / and receiving a mmud / . patients who completed minimum days post-transplantation follow-up were identified as eligible for survival analysis.results: the characteristics of the patients and transplant donors in this study are summarized in table . median age of patients was . ± years. proportion of male patients was . %, . % and . % for mud / , mmud / and hidt groups, respectively, which is significantly different (p= . ). the other baseline factors were similar, including patient age, donor age, recipient cytomegalovirus (cmv) status, donor cmv status, graft versus host disease incidence, median neutrophil and platelet engraftment times and disease status at post-transplant th day. no significant difference was identified in survival analysis among the mud / , mmud / and hidt groups, even if they were classified according to primary disease (aml vs all) and pre-transplant disease status (cr vs cr ). also, donor cmv status (cmv igg positivity or negativity) was not an important factor on survival analysis when compared between these three groups (p= . ).conclusions: in our study population, clinical outcomes of hidt patients were inferior to mud / and mmud / groups. when choosing an alternative donor for patients without an available hla-matched sibling, urgency of transplantation and host/donor features should be considered. we believe that hidt might be a feasible alternative choice in this subset of patients.disclosure: nothing to declare p g-csf primed bone marrow in hla-haploidentical transplantation using post-transplantation cyclophosphamide (ptcy) could promote tolerance and further reduce risk of gvhd nadira durakovic , , zinaida perić , , lana desnica , ranka serventi-seiwerth , mirta mikulić , brian melamed , alen ostojić , dražen pulanić , , pavle rončević , zorana grubić , radovan vrhovac , implementation, development, and coordination of unique quality management systems with evaluation audits, intrahospital and international accreditation and certification processes. quality of health care is a major focus for providers, patients, and accreditors; so, in this study, we aim to compare the quality of bm harvested at ipo-collection centre (icc) with the quality of bm received from external collection centres (ecc) during these last years.methods: this retrospective evaluation included the number of total nucleated cells (tnc) requested by the transplant centre, the tnc collected, and the results of bm microbiological analysis performed; donor age, weight and infectious disease markers (idm); patient demographics and diagnosis. bm collection technique in use at icc was validated according to jacie standards.we consider successful a collection (sc) with tnc between and % of the requested value, unsuccessful (uc) if lower than % and outstanding (oc) if over %.results: a total of bm was collected, for allogenic ( unrelated) and seven for autologous transplant; unrelated bm were received from ecc (nine from germany, seven from usa and five from portugal). patient main diagnosis were severe aplastic anaemia (n= ), acute myeloblastic leukaemia (n= ), and acute lymphoblastic leukaemia (n= ). donors idm were all negative and nonreactive.mean age (±standard deviation, sd) was (± . ) and (± . ) years for icc and ecc donors, respectively. at icc, we were asked to collect an average (±sd) of . * (± . ) tnc while ecc were asked for . * (± . ). we collected . * (± . ) and received . * (± . ) tnc. correlation between requested and collected tnc was . at icc and . for ecc.we had . % sc and . % oc meaning an accomplishment of . %. we failed to collect required tnc in . %. although % of received bm fulfil tnc requirements, bm processing lowered this value to % due to erythrocyte removal (seven patients with major abo incompatibility) and plasma reduction (two patients with abo minor incompatibility). these steps reduce final tnc available for transplant. weight difference between donor and patient had no significant impact on final tnc collection performance.sixteen bm from icc (seven staphylococcus spp., five propionibacterium acnesand fourcorynebacterium spp.) background: success of peripheral blood stem cell (pbsc) collections depends on patient biological parameters and stable apheresis device performance. peripheral blood cd + cell enumeration is the most reliable predictive factor of apheresis yield however, there are some unexpectedly poor cd + cell harvests despite successful mobilization. the aim of the study was assess total collections cd + yields and factors influencing main apheresis procedure outcomes including collection efficiency (ce).methods: of consecutive donors covering the period - - to - - were analyzed for the following parameters: pre cd count, cd yield per procedure, total cd dose collected per patient, cd collection targets requested by clinical teams. the efficiency of pbsc procedures was determined by calculating the ce and the correlation coefficient between pre cd count and yield per procedure. ce was correlated to preprocedure wbc, platelet count, pre cd count and blood volume processed. all pbsc collections were performed by optia spectra across units in uk.results: of the donors, were autologous and allogeneic. the autologous donors underwent in total procedures. the median cd target dose for these donors was x /kg. ( %) achieved the target dose with procedure and ( %) with procedures. the median pre cd count was /μl. the median cd yield per procedure was . x /kg and the median total cd dose collected per donor was . x /kg. ( . %) of autologous donors collected a total cd dose < x /kg, of those ( . %) had a pre cd count < /μl and ( %) > /μl.the allogeneic donors underwent in total procedures. the median cd target dose for these donors was x / kg. allogeneic donors ( %) achieved the target dose with procedure and ( %) with procedures. the median pre cd count was /μl. the median cd yield per procedure was . x /kg and the median of total cd dose collected per donor was . x /kg. ( . ) % of allogeneic donors collected a total cd dose < x / kg, of those ( . %) had a pre cd count < /μl and ( . %) > /μl. the median ce for autologous donors was % (range - ) and for allogeneic donors was % (range - ). the ce was negatively correlated to wbc (r= - . and - . ) and platelet count (r=- . and - . ) for auto and allogeneic donors respectively, but did not correlate to the pre cd and blood volume processed. the correlation coefficient between pre cd count and cd yield per procedure was r = . for the autologous and r = . for the allogeneic collections.conclusions: the majority of autologous and allogeneic donors achieved the target cd dose with one procedure. . % of autologous and . % allogeneic donors collected a transplantable cd dose of > x /kg. % of autologous and . % of allogeneic donors did not collect a transplantable dose despite a precd count of > /μl indicating suboptimal procedure performance. the ce was variable and was negatively correlated to the preprocedure wbc and platelet count. the ce and correlation coefficient are lower in allogeneic donors compared to autologous donors.disclosure: nothing to declare the outcome of autologous blood stem cell collection and its actual use in real world: the st century experiencekyoungmin lee , jung yong hong , dok hyun yoon , jae-lyun lee , shin kim , kyoung min lee , jung sun park , cheolwon suh background: mobilized peripheral blood stem cells (pbscs) have largely replaced bone marrow as the graft source for allogeneic stem cell transplantation. pbscs mobilization with g-csf is highly effective even on the th day in order to collect enough number of stem cells. a longitudinal, prospective, observational, single-center, cohort study on healthy donors (hds) was designed to identify predictors of cd + cells on the th day. methods: as potential predictors of mobilization, age, sex, body weight, height, blood volume as well as white blood cell count, peripheral blood (pb) mononuclear cells, platelet count, hematocrit, and hemoglobin levels were considered. two different evaluations of cd + cell counts were determined for each donor: baseline (before granulocyte colony-stimulating factor [g-csf] administration) and in pb after g-csf administration on day . a total of consecutive hds with a median age of . years were enrolled.results: the median value of cd + on day was cells/μl (iq - ). basal wbc, plt and basal cd +, are significantly higher for group with cd + on the th day over the median than below. a multivariate quartile regression analysis, adjusted by gender, age, basal cd + and basal plt, shows a, progressively steeper, relationship between baseline cd +, basal plt and cd + on the th day. the basal cd + cut-off for prevision of cd + on the th day was < = cells/μl and >= cells/μl whereas basal platelets count was < = x /l and >= x /l.conclusions: g-csf can be highly effective in hds on the th day in order to collect enough number of stem cells and we have developed a model for predicting the probability to perform pbsc collection after a short course of g-csf.disclosure: nothing to declare p pre-apheresis peripheral blood cd + cell counts highly correlates to actual stem cells collected background: prediction of stem cell yield on the basis of pre-apheresis cd + cell count and the processed blood volume is essential for the planning and executing of the apheresis process.methods: data analyzed included donor weight, complete blood count and cd + count on day of collection, total processed blood volume, cd + cell dose collected in the apheresis product and the number of aphereses performed. using the method described by pierelli et al, predicted cd + yields were calculated: predicted cd + yield x /kg = (benchmark ce x volume of blood to be processed x peripheral cd + count per μl) / (patient's weight in kg x metric conversion factor).results: in we established the method described by pierelli to predict the cd + cell yield. allogenic aphereses were performed in with this approach. mean processed volume was . liters. the mean cd + peripheral count before apheresis was /ul, the mean collected cd +count per kg bodyweight recipient was . . pearson´s correlation coefficient (r) between predicted yield using pre-apheresis cd +count and actually collected cd + cells per kg bodyweight recipient was . . the mean difference between predicted and collected cell dose was + . %.with knowledge of the predicted stem cell count, we were able to adjust apheresis procedure. in case of marginal predicted yield compared to the requested cell dose, we increased the blood volume to be processed. this proceeding led to a significant reduction of second day donations in by % compared to . in only cases we saw more than - % lower cd + doses collected than initially predicted. all donors showed mild iron deficiency with rbc microcytosis, a factor known to affect apheresis procedure.conclusions: pierellis method of calculating the stem cell yield shows a good correlation between pre-apheresis cd + count and actual collected stem cells, making planning and adjusting of the apheresis procedure more feasible and reliable. this proceeding led to significant reduction of second day donations. attention should be paid to iron deficiency anemia, leading to lower than estimated cd + dose.[ background: for more than a decade many transplant centers routinely collect and cryopreserve two or more peripheral blood stem cell (pbsc) grafts for a tandem and/ or salvage autologous blood stem cell transplantation (absct) in patients with hemato-oncological diseases. however, subsequent high-dose chemotherapy (hd-cht) and absct is in many cases not performed for a variety of reasons, specifically in patients with aml, all, mpn and burkitt lymphoma. data about the actual utilization rate of the cryostored stem cell products are lacking.methods: we retrospectively analyzed the collection, storage and disposal practices of pbsc products from a large cohort of patients who were treated at the university hospital heidelberg or at the university medical center mannheim during a -year period. disease entities included acute myeloid leukemia (aml, n= ), acute lymphoblastic leukemia (all, n= ), mpn (n= ; primary myelofibrosis [pmf], n= ; chronic myeloid leukemia [cml], n= ; secondary fibrosis/essential thrombocythemia [et], n= ; not specified, n= ) and burkitt lymphoma (n= ). patients between and were included and followed until .results: an adequate stem cell graft was defined as ≥ . x exp cd + cells /kg body weight. % of the patients were able to collect at least one stem cell graft and the median number of grafts per patient was (range - ). we could demonstrate that only % of all patients who had collected sufficient pbscs for transplant subsequently underwent an absct. among the disease entities the actual use of the stored pbsc grafts varied considerably from % to % (figure ) .conclusions: we could identify striking discrepancies between the collection/storage and actual utilization of background: biosimilars (bio) of granulocyte colony stimulating factors (gcsf) were approved several years ago on the basis of some studies that indicated similar efficacy to the already patented gcsf (neupogen®, neu) both in terms of shortening the neutropenic period after chemotherapy as well as peripheral blood stem cell mobilization in patients with lymphoma and multiple myeloma (mm) treated with autologous stem cell transplantation (auto-hct). however, all these studies are retrospective and there are still concerns about the real efficacy of bio and even more about the real benefit on final costs.methods: we have retrospectively compared the characteristics of the mobilization procedure in both patients with mm and lymphomas, and healthy donors that received either neu or bio (with no chemotherapy) in university hospitals in catalunya from december / to november . bio replaced neu in june in all institutions. primary objectives were the mobilization rate (defined as the percentage of patients that achieved ≥ x /ml cd + cells in peripheral blood on day ) and the use of plerixafor (plex) in each group as pre-emptive strategy. a multivariable analysis of risk factors influencing the use of plex and mobilization failure (defined as collection of < x /kg cd + cells) was also performed.results: we treated patients ( lymphomas and mm) and healthy donors. both groups of patients ( neu and bio) and donors ( neu and bio) were comparable regarding pre-mobilization general characteristics. there was a trend for a lower median cd + peak on day for bio patients ( vs , p value = . ). a total of patients received plex, although of them ( . %) out of strict theoretical indication, cd + cells > x /ml (range . - . ) and were removed for further analysis (n = , in the neu group and in the bio group). median number of cd + cells on day was significantly lower in the group bio who needed plex ( . vs . for neu+plex, p= ), as well as cd + cells finally harvested ( . vs . x /kg, p= . ). mobilization failure rate was higher in bio group ( vs %, p= . ). regarding no plex patients, median number of cd +cells on day was also significantly lower for bio patients ( . vs . , p= . ). risk factors for plex use were age, basal disease (lymphoma) and number of prior mobilization therapies. the use of bio was the only risk factor for mobilization failure patients receiving plex [hr . ( %ci . - . ), p= . ]. with respect to healthy donor mobilization, none of them needed plex but cases from the bio group ( %) needed more than one apheresis procedures ( and , respectively).conclusions: we found a lower efficacy of bio in the setting of stem cell mobilization of patients with only gcsf both in terms of a lower cd + cells peak on day and a lower number of cd + cells in final apheresis product. bio gcsf also seems to be less effective in healthy donors.disclosure: no conflict of interest to be declaredbackground: auto-sct is a common treatment in patients with mm or nhl. the aim of the prospective multicenter goa (graft and outcome in autologous transplantation) study was to investigate the impact of mobilization method used on the cellular composition of collected blood grafts and eventually hematological and immune recovery as well as long-term outcome post-transplant. altogether patients with mm or nhl transplanted between / and / at four university hospitals were included. the long-term goal of the study is to evaluate characteristics of optimal blood grafts in regard to post-transplant recovery and outcome. methods: altogether patients with mm undergoing first auto-sct were compared with patients with nhl. all nhl patients were mobilized with chemotherapy + g-csf, whereas % of mm patients were mobilized with g-csf only (p < . ). mobilization data, graft cellular composition including cd + cell subsets and lymphocyte subsets of the blood grafts, post-transplant hematological recovery and outcome were evaluated. the median followup time was months in mm patients and months in nhl patients.results: mm patients mobilized cd + cells better (median peak blood cd + vs. x /l, p < . ). the median number of aphereses was in both groups (p = . ). altogether % of the nhl patients received plerixafor compared to % in mm patients (p = . ). the median number of cd + cells collected was higher in mm patients ( . vs. . x /kg, p < . ).the median amount of cd + cells (with -aminoactinomycin) in the infused graft was . x /kg in mm group and . x /kg in nhl group (p = . ). the grafts contained more nk cells (median . vs. . x /kg, p = . ) and cd + cells (median . vs. . x /kg, p < . ) in mm patients. neutropenic fever tended to be more common in nhl patients ( % vs. %, p = . ) but mm patients had significantly more bloodstream infections ( % vs. %, p = . ). the median duration of hospitalization was longer in the nhl patients ( d vs. d, p < . ) and the nhl patients had significantly more often icu admissions ( % vs. %, p = . ).post-transplant neutrophil engraftment was faster in the nhl group (median d vs. d, p < . ). the median time to platelet engraftment was days in both groups. platelet count was higher in the mm group from day until year after auto-sct. there were significantly more early deaths (< d from the graft infusion) ( % vs. %, p = . ) and non-relapse deaths ( % vs. %, p = . ) in the nhl group.conclusions: mm and nhl patients differ in terms of cd + cell mobilization, graft cellular composition and post-transplant recovery as well as risk of non-relapse death. thus, the optimal graft may be different in nhl and mm patients.disclosure: the study was supported by vtr fund from north savo hospital district and study grant from sanofi. abstract already published. single-center experience in use of plerixafor for autologous stem cell mobilization: change in practice over years background: plerixafor has been proven to mobilize human periferal blood stem cells (pbscs) alone or acting synergistically with granulocyte-colony stimulating factor (g-csf). it has mainly been used for rescue mobilization after failed regimen of chemotherapy plus g-csf, but lately preemptive use in poor mobilizers has been established as cost-effective. we aim to show ten years of experience and change in practice with plerixafor use in our center.methods: we retrospectively evaluated the outcome of mobilization procedures and leukapheresis collections in our center in the period from january to october . practice from the first years, when plerixafor was mainly used as rescue agent after failed attempt, was compared to period from till present when preemptive use in poor mobilizers (defined as cd + cell counts < x /l blood) was established.results: in the period from to , total of patients underwent collection of autologous pbscs, and patients required repeated mobilization cycles ( , %). g-csf alone was used in patients and patients ( %) recieved combination g-csf with plerixafor. this cohort consisted of males and females with non-hodgkin (nhl) and hodgkin lymphoma (mh); and respectively. we noted unsuccessful mobilizations ( , % in repeated mobilization, , % in total) of which one patient was from plerixafor group ( , %).background: transplantation of hla-haploidentical hematopoietic stem cells (haplo-hsct) is an established procedure for the treatment of several different hematological diseases. one possible strategy to reduce the risk of graft-versus-host disease is represented by the selective depletion of ab t-lymphocytes (coupled with the depletion of cd + b-cells in order to reduce the risk of ptld) using the clinimacs device (miltenyi, bergish-gladback). before depletion, leukapheresis units are washed by lowspeed centrifugation, resulting in a platelet (plt) rich supernatant (prs) as a by-product generally discarded. we studied the possibility of recovering plt from prs of the haplo donor for transfusion to the recipient during the aplasia period occurring after hsct.methods: hsc donors were mobilized with g-csf (plus plerixafor in out of donors) as previously described [locatelli et al, blood ] . leukapheresis units, obtained with a spectra optia device, were washed twice (producing prs bags) at low speed to remove plt before starting the ab t-cell/b-cell depletion. the two prs were leukodepleted by filtration, centrifuged, resuspended and pooled in a total volume ranging from to ml intersol (is-plt) for overnight incubation at °c with agitation ( cycles/min). the is-plt samples were analyzed for the criteria established by the italian transfusion law. is-plt bags were examined for the following parameters: volume > ml, plt after leukodepletion > x , residual leukocytes < x , ph > . at °c at the end of the -day storage period. the sterility was tested using bd bactec culture vials. the evaluation of the residual leukocytes was performed with the bd leucocount kit. the absolute plt counts were determined using hemocytometer sysmex xn .results: prs bags from donors were processed to produce is-plt units. median resuspension volume in intersol was ml (range - ). the absolute mean value of plt counts measured at the end of the storage period was . x (range . - . x ). this value was found below the threshold fixed by italian regulation in cases ( . %). mean value of residual leukocytes was . x (range - x ); the ph value was always > . . sterility was observed in all cases. according to the work of slichter et al. conclusions: we demonstrated that plts recovered from leukapheresis bags can be accepted as a conventional hemocomponent according to the parameters fixed by italian transfusion law and thus can be administered to the haplo-hsct recipients early after transplantation. this strategy carries several advantages. indeed, apart from the obvious advantages in terms of reduced costs, is-plt can be used to desensitize the recipient by absorption of anti-hla class i antibodies, if present in the recipient. moreover, this strategy can avoid the risk of sensitizing the transplanted patients with hla alleles that differ from the donor's ones. finally, the is-plt unit is readily available. a clinical study aimed at testing the use of is-plt units in transplant recipients will be performed to confirm the clinical efficacy of the approach.disclosure: nothing to declare p ultrasound guidance as a powerful tool in increasing background: peripheral blood stem cells are generally the preferred graft source for allogeneic stem cell transplantation for malignant disease. in most centers first apheresis is performed on day after to doses of granulocyte colony stimulating factor (gcsf) up to ug/kg twice daily. the dose of gcsf and the number of apheresis procedures required contribute to symptom, travel and time burden donors are put through during the process. we hypothesized that taking donor-recipient weight differences into consideration may help reduce this burden methods: a total of healthy donors who donated peripheral blood stem cells on day of gcsf mobilization in the period between january and august at the university medical center hamburg-eppendorf were included in this quality control evaluation. the donors were divided into two cohorts. the impact of donorrecipient weight ratio on stem cell harvest was tested in the training cohort ( - ) and validated in the second cohort ( ) ( ) . for the training cohort, donors were grouped according to donor-recipient weight ratio < . vs. . - . vs. > . . for the purpose of this analysis a stem cells dose of x cd +/kg recipient weight was set for successful apheresis.results: in the training cohort including donors, ( %), ( %) and ( %) had a donor-recipient weight ratio of < . , . - . and > . respectively. the target stem cells count of x cd +/kg recipient weight was achieved in of ( %), of ( %) and of ( %) donors with donor:recipient weight ratio < . , . - . and > . respectively. the cut-off for the validation cohort was therefore set at a weight ratio of . .in der validation cohort including donors, ( %) had a weight ratio > . while ( %) had a weight ratio ≤ . . overall in this cohort target cell count of x cd +/kg recipient weight was reached in ( %) cases. this target was reached in of ( %) of donors with weight ratio ≤ . and in of ( %) donors with weight ratio > . , p = . .conclusions: a donor-recipient weight ratio of > . is seen in about % of peripheral blood stem cell donations for allogeneic stem cell transplantation. in these cases apheresis on day after doses of gcsf is reasonable. donors with lower weight ratios should preferentially donate on day after to doses of gcsf.disclosure: all authors declare no conflicts of interest background: the effect of a second mobilization and collection of peripheral blood stem cells (pbsc) on the cell yield is low, as we previously demonstrated. however, donor safety has been poorly addressed with no changes in the clinical practices.methods: second donations of unrelated and related donors performed between and were evaluated (n= ), including pbsc+pbsc (n= ), bone marrow (bm)+pbsc (n= ) and pbsc+bm (n= ). analytical parameters including leukocyte, lymphocyte, hemoglobin and ldh quantification, obtained on the pre-harvest evaluation of first and second donation, were retrospectively analyzed and compared for all donors. the portuguese bone marrow donors registry (cedace) recommends a time between donations no lesser than months. it also states that in very urgent situations like graft failure, donor should be clinical and analytical cleared and its safety ensured.in order to evaluate the impact of time between donations, donor population was divided in groups: < months, - months, > months; to determine the influence of donor age, donors were divided in groups: < and ≥ years.results: among the total of donors, were volunteer donors of cedace and were familiar. fifteen second donations were performed because of recipient graft failure and due to disease progression or relapse. at the time of second collection, median donor age was years (range - ). the median delay between both collections was days ( - ). time between donations did not seem to substantially impact the analytical donor evaluation: leukocytes, lymphocytes, hemoglobin and ldh results are kept within the reference values. however, donors with less than months between donations showed a slight decrease on leukocyte counts ( %) and hemoglobin values ( %), from the first to the second pre-harvest evaluation. donor age showed no significant influence on the analytical evaluation. nevertheless, when considering only the pbsc+pbsc donations, donors with ≥ years showed a small decrease on lymphocyte counts ( %) .conclusions: this study demonstrated that the analytical parameters, chosen based on literature, had no significant changes between first and second donation. however, particular attention should be paid when time between donations is lesser than months or donor age is ≥ years.as we concluded that no significant changes were observed in the group of - months, it is our opinion that the minimum of - months established by the registries can be shortened to months ensuring donor safety. an accurate donor risk assessment with a larger population should be accomplished in order to strengthen this recommendation.disclosure: nothing to declare. gaurav kharya , atish bakane , pratibha dhiman , anil khetrapal , vikrant bhar background: t cell replete haploidentical stem cell transplant (hhsct) is complicated mainly by increased risk of graft failure (gf) and graft versus host disease (gvhd). conventionally gcsf has been used to mobilize hematopoietic stem cells (hsc). in tcr hhsct gcsf mobilized graft with megadose of cd + cells expose the patient to higher doses of alloreactive t cells increasing the risk of gvhd. plerixafor based mobilization gives an advantage of giving high cd cell dose limiting exposure to high alloreactive t cell dose. we share our experience of gcsf + plerixafor based mobilization for tcr hhsct. methods: consecutive patients suffering from scd who underwent hhsct between jan till date along with the respective donors were enrolled in the study (group ). all underwent pre-transplant immune suppression (ptis) cycles at weekly intervals using fludarabine+cyclophosphamide+dexamethasone.the graft was mobilized using gcsf@ mcg/kg/day(d -d )+plerixafor@ . mg/kg(d ) - hours before the pbsch. gvhd prophylaxis included ptcy mg/kg/ day on d and , sirolimus and mmf starting from d . group included historical controls where graft was mobilized using gcsf@ mcg/kg/day(d -d ). various parameters pertaining to mobilization, harvest, engraftment, gf and gvhd were assessed between the two groups.background: poor mobilizers (pm) defined as those with a peripheral blood cd + count ≤ cells/μl on day+ is a significant risk factor for mobilization failure. within these, patients with < cells/μl are considered as very poor mobilizers (vpm). use of plerixafor in vpm patient is controversial. the aim of our study is to compare mobilizing and engraftment between pm and vpm who received plerixafor plus g-csf (p+g-csf).methods: in our center, mobilization with g-csf at dose of μg/kg/day was used in all pts. apheresis were scheduled on day+ . plerixafor ( . mg/kg) was added if the number of cd + cells on day + was < /ul for x cd + /kg requested (or < /ul for x cd + /kg), or if the number of cd + cells collected in the first apheresis was < % of cd + cells requested.between january and september , out of pts ( , %) received plerixafor for mobilization. we retrospectively studied pts who mobilized with p+g-csfdue to the number of cd + cells on day + was < /ul.results: twelve out of pts were pm, were females, median age , years (range: - ). patients' baseline diseases were: non-hodgkin lymphoma (nhl) ( , %), multiple myeloma (mm) and hodgkin lymphoma. median cd + cell count on day + was / ul (range: - ).there was no mobilization failure. eighteen out of pts ( %) were vpm, were females, median age , years (range: - ). patients' baseline diseases were: nhl ( , %), mm and solid tumor. median cd + cell count on day+ was , /ul (range: - ). two out of pts ( , %) were considered mobilization failure, in of them did not realized apheresis due to cd + cell count on day + was /ul. no difference was seen between both groups regarding gender, age, patients baseline disease or median cd + cells count on day + .vpm needed more apheresis sessions, / pts required sessions against / pts in pm (p=not significant (ns). we obtained enough cells to carry asct in % pts, although mean number of cd + cells obtained in vpm was lower than in pm ( , x /kg vs , x /kg, respectively) (p=ns).twenty-six pts underwent asct and mean number of cd + cells infused were , x /kg in vpm vs have been the only route used for chpc administration. the appearance of other catheters types made us to reconsider the exclusive use of the cvc for the infusion of chpc. we analyzed the use of a peripheral iv cannula (pivc) as an alternative to cvc for the infusion of chpc in patients with cardiovascular diseases.methods: medical records of patients who received an asct for hematological malignant diseases at the hospital clínic of barcelona from january to february were reviewed. of those, eight were infused through a pivc due to cardiac impairment related to previous treatments, ischemic cardiomyopathy or amyloid deposition.hpc were obtained from peripheral blood by apheresis after mobilization with g-csf using acd-a as an anticoagulant. cryopreservation was performed with autologous plasma and dmso % by mechanical means and stored in liquid nitrogen. analytical controls were performed including hematocrit, total nucleated cells, total polymorphonuclear neutrophils and platelets using the advia analyzer. the cd + / cd + population was analyzed by flow cytometry following the ishage single-platform protocol. viability of total nucleated cellularity was carried out by vital staining with acridine orange and the specific viability of the cd + population through the technique of -aminoactinomycin d. thawing was performed bag to bag by immersion in a water bath at ºc and transferred to the bedside of the patient for gravity infusion using an infusion set without filter through pivc of gauche. vital sings monitoring performed before, during and the end of the every infusion bag including: blood pressure, heart rate, oxygen saturation, body temperature and central venous pressure. other aspects assessed during the infusion were pain, cold sensation and signs of extravasation in the area of pivc insertion. after the infusion, the recovery time of the granulocyte series and platelet were evaluated.results: median volume and bags administered was ( - ) ml and ( ) ( ) ( ) . the median of total nucleated cells, total nucleated cells / ml and total cd + cells/kg was . x ( . - . ), . x ( . - . ) and . ( . - . ) respectively. vital signs were within the normal range and allowed to perform the infusion in an average of - minutes/bag. no patient required stopping the infusion due to pain in the area of peripheral catheter insertion and no extravasations were detected. all patients referred some cold sensation in the insertion vein and its path. median hematopoietic recovery was ( - ) days for neutrophils and ( - ) days for platelets, similar to the recovery experienced from patients who received chpc through cvc.conclusions: based on our data, we conclude that the administration of chpc, through pivc and by gravity is safe for the product and for the patient, being the preferred choice for patients suffering from some type of cardiovascular disease.disclosure: nothing to declare background: by selective depletion of potentially alloreactive cd ra + cells, t memory cells might be retained in the graft and could mediate pathogen specific immunity. however, cd ra expression is not restricted to naïve t cells, but also available on b cells, nk cells and cd + stem cells to some extent. methods: within this project we aim to analyze cd ra expression on stem-and nk cells by flow cytometric analysis to estimate the eventual loss of these cells during cd ra-depletion. furthermore, clinimacs depletion following a one-step approach of direct cd radepletion and a two-step approach with primary cd selection followed by cd ra-depletion of the negative fraction was investigated.results: cd ra expression on cd + stem cells was in median . %. with a median of . % cd ra expression was measurable on nearly all b cells, which obviates depletion via cd . a comparably high cd ra expression of in median . % was detected on nk cells. unfortunately, the amount of nk cells in the cd ra-depleted product was . %. clinimacs depletion following one-step approach resulted in a stem cell recovery of . %. memory t cell recovery was . % following one-step and . % applying two-step approach. depletion quality measured by log-depletion was . and . for cd ra + t cells and . and . for cd + b cells for one-and two-step approaches, respectively.conclusions: with regard to stem cell recovery, a previous cd -selection before cd ra-depletion is recommendable.background: current clinical practice of routine use of filters for infusion of autologous hematopoietic cell transplantation (ahct) at bone marrow transplant centers across north america and europe is not known. the use of "y" administration tubing without a filter could possibly increase the risk of infusion of macro-aggregates and cellular debris, which may result in increased side effects.methods: we carried out a retrospective chart review of patients (pts) at spectrum health who underwent ahct. group a (gp a) pts received ahct using a "y" administration tubing with -micron filter from / - / . these patients were compared to a control group (gp b) that received ahct without filter administration tubing from / - / .this change in clinical practice occurred due to a change in policy at our transplant center as a result of inorganic particles noticed during cryopreservation. we compared the neutrophil and platelet engraftment duration between these groups. we also studied the length of hospital stay and the effect of filter use on any immediate side effects after infusion. due to the retrospective nature of the study it was not feasible to evaluate the difference in duration of infusion between these groups results: the two groups were similar in their age, gender, primary disease distribution and median number of cd stem cells infused (table) . there was no difference in median neutrophil ( vs days) or platelet engraftment ( vs days) duration for the filter group and the nonfilter group respectively. the median length of hospital stay was also comparable ( days). there was no statistically significant difference in the immediate side effects (fever, cough, dyspnea, fluid overload, flushing, nausea, vomiting, hypertension, hypotension and anaphylaxis) or confirmed post-transplant infections (viral, bacterial, fungal) experienced by these two groups.conclusions: our results show that the routine use of filter does not prolong hospital stay, and neutrophil/ platelet engraftment duration, thereby, suggesting that viable stem cells are not affected. on the other hand, filter use failed to demonstrate any appreciable decline in the immediate side effects experienced after ahct. gp background: allo-hsct from related haplo-identical donors (haplo-hsct) with post-transplant high-dose cyclophosphamide is increasingly employed in patients who lack a matched related or unrelated donor. the current standard is to use bone marrow grafts (bm) as peripheral blood stem cell grafts (pbsc) have been associated with an increased risk of acute and chronic gvhd. thus, the aim of our study was to compare the main transplant outcomes and especially the incidence of acute and chronic gvhd in recipients of bm and pbsc grafts. methods: thirty-five unselected patients with hematologic malignancy who underwent an haploidentical transplant at our unit between and and received bm (n = ) or pbsc (n = ) grafts after the same tbf conditioning regimen were analysed in order to assess differences in transplant outcomes.our gvhd prophylaxis consisted in cyclosporine a (csa) from day - to + , a methotrexate "short course" and mycophenolate mofetil (mmf) from day + to + .results: no statistically-significant differences were observed between patients who received bm grafts and those who received pbsc grafts. at transplant fourteen patients were in first complete remission (cr), twelve in advanced cr and had active disease. according to sorror's risk, nine patients were low-risk, nine intermediate-risk and seventeen high-risk. twenty-eight cmv+ patients received the graft from twenty-three cmv+ and five cmv-donors, seven cmv-patients received the graft from five cmv+ and two cmvdonors. mean age at transplant was years (range - ), mean donor's age years (range - ) and mean follow-up . months (range . - . ). median cd + cell dose was . x /kg (range . - . ), . x /kg (range . - . ) in bm recipients and . x /kg (range . - . ) in pbsc recipients. median time to neutrophil recovery (> /μl) was days (range - ) posttransplant, days (range - ) for bm recipients and (range - ) for pbsc recipients. platelet recovery (> . /μl) occurred in all patients except one at a median of days (range - ) post-transplant, at a median of days ( - ) post-transplant for bm recipients and at a median of days (range ( - ) for pbsc recipients. seven patients never reached platelets > . /μl. three patients developed a poor graft function. acute and chronic gvhd incidence was . % and . % and the risks of acute (hazard ratio [hr], . ; p = . ) and chronic (hr, . ; p = . ) graft-versus-host disease were similar in the two patient groups. in addition, there were no differences in relapse risks post-transplant (hr, . ; p = . ); relapse-free survival was better with pbsc grafts but this difference did not reach any statistical significance. finally, no significant differences were noted in overall mortality by graft type (hr, . ; p = . ).conclusions: despite in haplo-hsct the incidence of acute and chronic gvhd is reported to be higher with pbsc than with bm our small patient series does not confirm this assumption that should be clarified by additional studies. instead, our data suggest that pbsc background: since , cord blood (cbu) has become an alternative source of stem cells for transplantation, with approximately , procedures currently performed. with a -year gs of %.objective: analyze the outcomes from all the patients transplanted with cbu in our hospital unit.methods: retrospective, longitudinal study. all patients transplanted with cbu in our hospital between and were included. we analyzed patients with ages from months to years.results: two of them received doubled cord transplantation the ratio male: female was . : . the transplanted pathologies were: bone marrow failure %, immunodeficiency %, aml %, all . %, osteopetrosis . %. ric regimens were used in patients with bone marrow failure and immunodeficiency and myeloablative conditioning regimens were used in patients with malignant hematology diseases. antithymocyte rabbit globulin (atg) based serotherapy was used. one case received cbu from a related donor (sister), the rest received unrelated cbu obtained from centro nacional de la trasfusión sanguínea. the infusion of cd + was in a range of . to of . x /kg with and average of . x / kg. compatibility was / in %, / in % and / in %. post-thawing cellularity was not measured. the hla-c was not analyzed. forty two point five percent of the patients had a successful engraftment; the average time of engraftment was days. primary graft failure was detected in . % and secondary graft failure in %, for a total success of . %. gvhd was detected in % of patients, of which % was grade i-ii and % grade iii-iv. the overall mortality was . %. causes of death were: infection % relapse %, hemorrhage % and gvhd %. the cbsct continues to be an essential alternative in our patients who required transplantation knowing that this stem cell source allows the procedure to be done with less histocompatibility requirements and it is available immediately, which facilitates the process considering the great diversity that exists within our population. however, in our experience, the cbsct has shown a higher mortality risk, which can be improved by analyzing the hla c, choosing in this way the units with better compatibility, and improving cellular dosage since this is key in success.disclosure: nothing to declare the risk of infection of the umbilical cord is not related to the microbiological status of the umbilical cord blood methods: the statistical analysis was carried out on data obtained from samples taken in poland between -jan- and -dec- . the samples were collected in hospitals by external midwifes and sent to the pbkm in accordance with the requirements of the american association of blood banks. after arrival in the laboratory, the blood samples were cultured and the ucs were assessed immediately for visual signs of infection, such as odor, altered color, or visible bloom. the status of both kinds of samples was introduced into the pbkm general database. for the purpose of this analysis, the ucs were considered as microbiologically pure if stored, destroyed after storage, or handed over to the pbkm. samples marked as infected or disqualified for unknown reasons (other than termination of the contract with the customer, viral infection of the mother, and lack of cell growth) were considered as infected. at the time of the statistical analysis, the samples of unknown ucb microbiological culture status were removed from the generated report. the data was summarized as percentages and the odds ratio was calculated. statistical significance was considered at p˂ . .background: match family donors are the preferable options in allogenic stem cell transplant. however, in the absence of donor relatives match unrelated donors have been an option. in this study, the donor screening, transplant preparation phases of turkish stem cell coordination center (turkok) and the İstanbul university bone marrow bank (tris), were compared.methods: the unrelated donor scanning data between march and november in pediatric stem cell transplantation unit of altınbas university bahcelievler medical park hospital were evaluated. unrelated transplants were performed in total. % of these transplants (n= ) were included from the donors of turkok registration system and , % of these transplants (n= ) were included by means of tris from donors outside of turkey. patients ( tris, turkok) were excluded from the study in consequence of screening update and postpone of transplantation. the statistics were carried out on a total of patients, , % of whom were in turkok (n= ) and , % were transplanted via tris (n= ). the day of application to stem cell transplantation unit, reply dates and the transplantation dates were examined for the transplant patients.results: in the current study, the average response time of turkok was found as , ± , day (median: ), the average transplant time after receiving a reply was found as , ± , (median: ) day, the average number of days from date of application to date of transplantation of patients was found as , ± , (median: ). the average response time of tris was , ± , (median: ) day, the average transplant time after receiving a reply from tris was , ± , (median: ) day, average number of days from date of application of tris to date of transplantation of patients , ± , (median: ) day.the average response time of turkok, the average transplant time after receiving a reply from turkok and the average number of days from date of application of turkok to date of transplantation of patients was shorter than tris. the difference between them was found statistically significant (p< . ).conclusions: in this study, it was determined that the transplantation processes with turkok were progressing more rapidly. the rapid progress of the process was attributed to the fact that all donor hla tissue groups in the turkok database were studied in high resolution. in international scans carried out through tris, it was thought that the examination of the donor castings coming from bone marrow banks and the time differences between the countries prolong the process. it was thought that hla tests of the registered donors in the tris database and some international bone marrow banks were studied in low resolution but not studied the all hla loci, the centers wanted high-resolution hla, and therefore the involvement of social security institutions and payment procedures were among the factors extending this process.disclosure: nothing to declare background: allogeneic hematopoietic stem cell transplantation (ahsct) is being performed for a group of hematologic diseases with a curative intent. outcomes after ahsct are influenced by the type of donor used. haploidentical transplantation is an emerging option when a fullmatched donor is unavailable. methods: we retrospectively analyzed our transplants performed between january and november , investigating outcomes and complications among haploidentical stem cell recipients.results: one hundred and nineteen patients underwent ahsct, of them ( . %) were recipient of a haploidentical stem cell and included in this study. one patient diagnosed with acute lymphoblastic leukemia (all) were performed a haploidentical ahsct for two times due to relapse. among those transplants, of them were diagnosed with acute myeloid leukemia, with all, with chronic lymphocytic leukemia, with myelodysplastic syndrome and with hodgkin lymphoma. the mean age of group was . ± . years. three patients ( aml, all) were in remission at the time of transplantation. patients were given a conditioning regimen based mostly on busulfan, fludarabin and total body irradiation with a myeloablative intent. patients were also given a various combinations of post-transplant cyclophosphamide, calcineurin inhibitors, mycophenolate mofetil and antithymocyte globulin for graft versus host disease (gvhd) prophylaxis; post-transplant cyclophosphamide administered on ( %) of those transplantations. peripheral blood was the source of stem cells in all patients. patients were infused with mean . ± . x /kg of cd + cells. hematological recovery was achieved with neutrophil engraftment at a mean of . ± . days and platelet engraftment at a mean of . ± . days. after a median month ( . - . months) follow up, the cumulative rates of grade - gvhd, relapse and non-relapse mortality were %, % (n= ) and %, respectively. one patient died due to relapse, at the end of the follow up two were still alive with remission. only one patient has died due to chronic gvhd affecting serosa and resulting with a fatal tracheoesophageal fistula. the mean overall survival was . ± . months in our study.conclusions: haploidentical transplant is a feasible option in hematologic malignancies with novel gvhd prophylaxis approaches, especially post-transplantation cyclophosphamide. however, these results need to be supported with further investigations with a larger patient group.disclosure: nothing to declare results: a total of patients between and years (median age: years). transplant was done for following disease: acute leukemia (n= , %), aplastic anemia (n= , %), lymphoma (n= , %), myelofibrosis (n= , %), myelodisplastic syndrome (n= , %), chronic myeloid leukemia (n= , %). ten donors were from turkey and fifteen donors were from different countries of europe and america. two of donors were / and the other was / hla matched. the conditioning regimen was mostly non myeloablative (n= , %) while eight patients were treated with myeloablative regimen. other than two patients who took tacrolimus and mycophenolate mofetile all of them got cyclophosphamide and methotrexate for graft versus host disease (gvhd) prophylaxis. the median time of neutrophil and platelets engrafman were days (range - ) and , days (range - ) respectively. acute gvhd was seen nearly half of the patients ( , %).overall survival was % for all patients and of patiens ( %) died within first month to months (median months). the mortality rate was more higher for the recipients who had donor source from countries other than turkey ( % vs % p= , ). transplant related mortality was the most common reason of mortality (n= / , , %) and other reasons were gvhd ( , %), infections and cirrhosis respectively.conclusions: we found the mortality rate more higher in the patients whose donors were from out of our country. however, we need to further multicentric and prospective investigations to confirm our hypothesis, it would be related with impact of ethnicity.disclosure: nothing to disclose late-breaking abstracts p targeted twice daily busulfan-based ric-conditioning for allogeneic hematopoietic stem cell transplantation in pediatric patients with chronic granulomatous disease: a -year experience with the zurich protocol matthias felber , mathias hauri-hohl , ulrike zeilhofer , federica achini , jana pachlopnik-schmid , janine reichenbach , seraina prader , tayfun güngör background: chronic granulomatous disease (cgd) needs sufficient myeloablation to avoid graft failure. for this purpose the ebmt inborn errors working party currently recommends treosulfan or busulfan-based conditioning regimens for cgd-sct. we analyzed the last years of targeted busulfan-based ric-conditioning including engraftment, gvhd rates, chimerism and late term effects in our pediatric sct center in zurich. methods: between and , n= consecutive pediatric cgd patients (median age years, range - years, n= female, n= autosomal recessive inheritance) have been transplanted. all patients received therapeutic drug monitoring of twice daily administered iv busulfan ( or hour infusions; d- to -d ) to achieve a targeted cumulative auc of - mg/l*h. fludarabine ( mg/ sqm; d- to -d ) and serotherapy (thymoglobuline . mg/ kg total, d- to d- ) or alemtuzumab ( . - . mg/kg total; d- -to d- ) were used for immunoablation. donors were matched unrelated ( / hla; n= ), mismatched unrelated ( / hla; n= ), mismatched unrelated (hla / ; n= ), matched sibling (hla / ; n= ) and haploidentical parental (hla / ; n= ). for patients with haploidentical donor post-transplant cyclophosphamide (d- and d- with mg/kg iv each) and upfront atg-grafalon ( mg/kg - to-d- ) was used. stem cell sources were bm (n= ) and pbsc (n= ). gvhdprophylaxis included iv csa (d- ; continuous infusion) and iv. mmf (d- , in - doses).results: follow-up was to months. good overall engraftment was noted, with n= secondary graft failure followed by successful retransplantation. in one patient a stem cell boost/dli was necessary due to decreasing myeloid donor chimerism during ebv reactivation, resulting in rapid myeloid donor reconstitution after intervention. low rates of gvhd were documented with n= agvhd grade iv and n= mild/limited cgvhd (nih criteria). with exception of one patient, myeloid donor chimerism at last follow-up was over %, mostly over %. overall survival was / ( %). deaths were due to gi-gvhd (n= ), autoimmune hemolytic anemia/sepsis (n= ) and thrombotic microangiopathy (n= ).conclusions: precision dosing of iv busulfan in combination with fludarabine and serotherapy results in excellent outcome of hsct for pediatric cgd-patients with good engraftment, low overall cgvhd rates and stable, mostly excellent donor chimerism. graft failure rate was as low as %. low dose alemtuzumab prevented gvhd in the majority of patients. this analysis demonstrates that targeted busulfan-based conditioning is a valid option for pediatric cgd-patients. serum alemtuzumab or atg monitoring could further improve gf and gvhd rates in the future.disclosure: the authors declare no conflict of interest. abstract already published. young hla-matched unrelated donors are comparable to matched sibling donors in elderly patients receiving reduced-intensity conditioning: an analysis on behalf of the ebmt scientific council % c.i. . - . , p= . ) and voriconazol prophylaxis during carv (or . , % c.i. . - . , p= . ). conclusions: we provide evidence that ifd after carv infection. allo-hsct recipients developing a carv lrtd during the first year after transplant may benefit from an adequate antifungal prophylaxis and a close monitoring for the development of a later ifd.disclosure: jose luis piñana has received both, advisory for preclinical/clinical research and financial support to assist to the spanish society of hematology annual meeting from msd. favorable outcome and engraftment following reducedintensity conditioned allo-hsct in children with primary haemophagocytic lymphohistiocytosis (hlh) and high-risk langerhans cell histiocytosis (lch) laura m. moser, emilia salzmann-manrique, andrea jarisch, jan sörensen, shahrzad bakhtiar, peter bader background: primary haemophagocytic lymphohistiocytosis (hlh) and high-risk langerhans cell histiocytosis (lch) represent two major entities of childhood histiocytoses, which are -although only of rare occurrence -severe in their clinical manifestations. patients present with multisystemic uncontrolled inflammation and multi-organ involvement requiring diverse courses of immunosuppressive and chemotherapy regimens. allogeneic haematopoietic stem cell transplantation (allohsct) is the only available curative option; however, the cumulative treatment toxicity and the underlying inflammatory disease often result in high organ toxicity and inflammatory complications of transplantation, such as graft versus host disease (gvhd) and/or graft failure. especially patients with unrelated donors often deal with high transplant-related mortality (trm) in the setting of conventional intensity conditioning.herein, we present the clinical course of the disease and transplant outcome of children diagnosed with primary hlh (n= ) and high-risk lch (n= ) who underwent allohsct at our centre from / to / .methods: the hlh cohort consisted of cases of familial hlh (fhlh), cases of griscelli syndrome, one xiap-deficient patient and one hlh-patient with inconclusive genetic testing. all hlh patients had developed clinical symptoms prior to transplantation and had been treated according to hlh-protocols , hlh-protocols , or median age at transplantation was months ( to months). stem cells were derived from hla-matched siblings (msd, n= ), matched unrelated donors (mud, n = ) or haploidentical donors (n= ).the majority of patients ( / ) received a ric regimen containing fludarabine, melphalan and thiotepa (n= ) and fludarabine plus cyclophosphamide (n= ). myeloablative treatment ( / ) included a treosulfan-based regimen (n= ) and busulfan-containing treatment (n= ). the entire cohort received serotherapy using either muromonab (n= ), atg-fresenius® (n= ) or alemtuzumab (n= ).results: the overall survival of the entire cohort was . % ( / ) on a median follow-up of . years ( figure a+b) .all lch patients, being treated with fludarabine, melphalan and thiotepa, survived transplantation and showed complete remission ( / ) . within the hlh cohort the overall survival was . % ( / ). fatalities (n= ) included two patients from the myeloablative group and one ric-treated patient. the cause of death were progressive disease activity during the conditioning phase, leading to multi-organ failure on day + despite immunosuppressive treatment (n= ) and complicated cerebral seizures followed by lung haemorrhage, possibly due to aspiration pneumonia with evidence of enterococcus faecium, resulting in septic multi-organ failure on day + (n= ). a third hlh patient developed a sudden cerebral edema and ensuing respiratory insufficiency on day + . whether this was caused by acute neurotoxic damage by fludarabine or a consequence of relapsed hlh could not be conclusively specified. none of our patients suffered from transplant failure or nonengraftment. there was neither severe acute gvhd (iii-iv) nor chronic gvhd observed in this cohort.conclusions: primary hlh and high-risk lch are lifethreatening medical conditions needing rapid allohsct. ric regimens are well-tolerated and sufficient for proper engraftment and disease clearance. disclosure: the authors have no conflicts of interest to disclose. abstract withdrawn. thrombocytopenia following allo-sct concomitant to stem cell boosts. steroid refractoriness was defined as: progression after three days or no response after days of steroid treatment. the median time from sct to the onset of agvhd was . d (range - d), and d (range - d) from the onset of agvhd to the first msc infusion, respectively.the majority of our patients (n= ) suffered from a malignant disease and received a graft from a matched unrelated donor (n= ), while one patient had a haploidentical donor. gvhd prophylaxis was performed in all patients except the patient with the haplo-identical graft. all patients with agvhd were treated with steroids and the patient with thrombocytopenia required regularly transfusions and romiplostin therapy. the median msc dose was . x cells/kg bw (minimum . x ; maximum x ). three patients received msc doses, two patients , one patient and another doses.results: at the time point of agvhd manifestation and msc application, two patients had cmv reactivation, one patient adenovirus infection and one patient ebvreactivation. by day , / agvhd patients responded to msc administration: with complete response and with partial response. at the last follow-up (median: . months, range , - . months), of patients were alive without acute or chronic gvhd. one patient died soon after msc treatment with no obvious response in the course of a systemic hyperinflammation syndrome. the other patient although complete responder to msc-ffm developed fatal adenovirus sepsis. this based on the profound tcell depletion induced by concomitant application of steroids. the overall survival probability at six month was . %. no acute side effects occurred after msc infusions. the previously mentioned patient suffering from thrombocytopenia did not need any further transfusions after receiving doses mscs combined with stem cell boosts while continuing romiplostin application.conclusions: our data confirm excellent tolerability and high efficacy of the licensed off-the-shelf msc preparation "msc-ffm" in pediatric steroid-refractory agvhd. in our center, current treatment algorithms have escalated "msc-ffm" to the second line, i.e. immediately after steroid refractoriness has been established. besides immunoregulatory properties, this product might facilitate hematopoietic stem cell engraftment.disclosure: novartis (consultancy: included expert testimony, speaker bureau, honoraria), medac (research funding, patents and royalties), riemser (research funding), neovii (research funding), amgen (honoraria) expression of ccr modulates migrational properties of in vitro expanded murine regulatory t cells laura m. moser , , ulrike tischler , christin riegel , julia minderjahn , rüdiger eder , jaqueline dirmeier , isabel zimmermann , evelyn röseler , petra hoffmann , , matthias edinger , background: hematopoietic stem cell transplantation (hsct) as it is carried out successfully at other genetic instability syndromes seems to be an encouraging opportunity for a curative therapy to restore immunity and prevent the development of hematologic malignancies in ataxiatelangiectasia (a-t). however, experience in the conditioning regimen is limited and no transplantation strategy for a-t patients exists, especially in an allogeneic setting. conditioning regimen and donor selection are critical factors in the clinical setting of hsct and incur substantial risks, especially in a-t. the aim of this study was ( ) to evaluate whether different approaches of hsct including allogeneic hematopoietic hsct are feasible in regard to graft versus host response (gvhd) and sufficient concerning immune reconstitution ( ) and to de-escalate the toxic effects of the conditioning regimen by reducing the dose of cyclophosphamide (cp).methods: t cells from syngeneic, allogeneic and haploidentical donor mice were used to determine gvhd induced t cell proliferation in a mixed lymphocyte reaction (mlr). atm-deficient mice were treated with cp or reduced cp in combination with fludarabine (flu) and transplanted with x cd . depleted bone marrow donor cells from /svev gfp-transfected wildtype mice (syngeneic) or from mice of the f generation of /svev wildtype mice and c bl/ mice (haploidentical), or from c bl/ mice (allogeneic). tracking of gfp-positive donor derived cells was performed using flow cytometry and atm pcr. oxidative stress and damage were detected by a rt profiler pcr array and -hydroxy- ′deoxyguanosine.results: mlr resulted in an increased proliferation of allogeneic donor t cells compared to syngeneic and haploidentical donor cells. response was lower on dendritic cells isolated from atm-deficient mice compared to wildtype controls. in vivo results showed the restoration of t cells in atm-deficient mice accompanied by a prolonged life span and through reduction of thymic tumors. however, allogeneic stem cell transplantation was accompanied with a higher mortality rate, compared to the haploidentical and syngeneic setting. decreased antioxidative capacity and a higher dna-damage were seen in cp treated atmdeficient mice.conclusions: haploidentical hsct seems to be a feasible strategy for a-t. our data provided further evidence for the high sensitivity against ros-inducing agents in a-t and this fact needs to be taken into consideration in the choice of the host-conditioning strategy.disclosure: nothing to declare this research received funding from action for a-t charity ( gou ) background: prognosis of pediatric patients and young adults suffering from refractory or high-risk soft tissue sarcomas remains poor with limited improvement over the last decades despite multimodal treatment strategies. replacing the immune system by an allogeneic hematopoietic stem cell transplantation (hsct) in refractory solid malignancies has been proposed as a potentially curative therapy due to its presumable graft versus tumor effect. based on this concept we additionally performed consecutive donor-derived lymphocyte infusions in allogeneic hsct-patients with refractory or relapsed solid malignancy to further increase anti-tumor efficacy post-transplant.methods: pediatric patients with relapsed and/or refractory cancers or with delayed responses to the respective induction therapies were offered donorderived cellular therapies after immune system replacement by an allogeneic hsct. cellular immunotherapies comprised of donor lymphocyte infusions (dli), natural killer (nk) cell or cytokine-induced killer (cik) cell infusions generated from the original stem cell donors. allogeneic nk cells were generated from unstimulated leukapheresis by a two-step purification procedure using immunomagnetic cd t cell depletion, followed by nk cell enrichment (cd +) with or without in vitro il- stimulation and expansion for - days. for cik cell generation peripheral blood mononuclear cells were isolated and activated by in vitro cytokine stimulation (inf-γ, anti-cd , il- and il- ) an expanded over - days. expanded cik cells represented a heterogeneous population of polyclonal t cells with in part shared phenotypic and functional properties of nk cells.results: between october st and january st a total of patients (eight patients with rhabdomyosarcoma, one patient with synovial sarcoma, two patients with ewing sarcoma, five patients with neuroblastoma, one patient with hepatoblastoma, and one patient with nasopharynx carcinoma) were enrolled. seven of ( %) patients in this study had achieved complete remission (cr) before hsct while another of ( %) patients had obtained at least very good partial or partial response (vgpr or pr). dli was applied in patients, nk cell treatment was offered to another patients, while cik cell therapy was given to patients. . -year probabilities of overall survival (os) and progression-free survival (pfs) were . % and . % for all patients with a median follow up of . months (range, . - . months). patients in cr at the time of immune cell therapy (it) showed estimated . -year os and pfs of . % and . %, respectively. the majority of patients relapsed and ultimately succumbed to their diseases with two of ( %) patients still being alive . and . years after it. cumulative incidence of relapse was . % at . years. t cell engraftment and immune reconstitution (ir) was improved by it, and correlated with treatment response. however, two of heavily pretreated patients ( %) died due to cumulative treatment-related mortality (trm). furthermore, acute graft-versus-host-disease (agvhd) occurred in of patients ( %) with agvhd grade i-ii observed in ( %) and agvhd grade iii seen in three ( %) patients.conclusions: altogether, the results of this study indicate that allogeneic donor-derived cellular therapy at its current state offers curative benefit in selected refractory childhood cancers but warrants further improvement. background: allogeneic stem cell transplantation (allo-sct) is the only curative treatment option for a variety of nonmalignant diseases. the success of allo-sct is strongly associated with rapid and sustained immune reconstitution (ir). we analyzed the ir in patients who received an allo-sct for nonmalignant diseases.ir was assessed on days + , + , + , + and + after allo-sct analyzing leukocytes, lymphocytes, monocytes, cd + t cells, cd + cd + t helper cells, cd + cd + cytotoxic t cells, cd -cd + natural killer (nk) cells and cd + b cells.methods: we analyzed ir-data of consecutive patients receiving allo-scts between september and november . indications of allo-sct were hereditary anemias (thalassemia, sickle cell disease, diamond blackfan anemia; ha, n= , %), inherited bone marrow failure syndrome (fanconi anemia, severe aplastic anemia, others; bmfs, n= , %), hemophagocytic lymphohistiocytosis (hlh, n= , %), immunodeficiency (id, n= , %) and metabolic disorders (n= , %). the median age at allo-sct was years (range, . - ) and at diagnosis . years (range, - . - . ).patients received st allo-sct from msd/mfd (n= , %), mud (n= , %), haploidentical mismatch family donors (n= , %) and mmud (n= , %). conditioning regimens were busulphan-based (n= , %), treosulphan-based (n= , %), flu-mel-thio (n= , %) or others (n= , %). graft sources were bm (n= , %) and pbsc (n= , %).in order to consider the age-dependency of ir we normalized each absolute cell count with its corresponding age-specific expected mean values. (huenecke et al.; eur j haematol; ) results: the ir pattern was similar between the ha and bmfs groups. the cd + t cells were recovering slightly faster in ha patients compared to the recovery of bmfs patients.monocytes and nk cells proliferate very fast. at day + half of the patients already reached their respective monocytes reference value except for id patients, who reached % of the reference value at the end of the first year.cd + cd + cytotoxic t cells recovered significantly faster in patients with hematologic diseases compared to patients with hlh (p< . ) and id (p= . ). half of the patients reached the reference value of cytotoxic t cells in the hematologic diseases group at day + . by far inferior was the ir for the hlh patients. in this group only % of the patients reached the th percentile of the healthy age-matched reference. in the id group % of all patients reached the th percentile of the age-matched reference group at day + .b cells are profoundly decreased at day + in all groups. however, the longitudinal expansion of b cells was significantly lower both in the id group and hlh group compared to the hematologic diseases group. at day + fifty percent of patients with id, hlh and hematologic diseases reached the th percentile, th percentile and the th percentile, respectively (p< . ; p= . ).conclusions: allo-sct is the only curative option for patients with nonmalignant diseases. ir is dynamic and revealed a complex diversity pattern with regard to the original disease. to investigate factors influencing ir after allo-sct is crucial to improve outcome of these patients.disclosure: nothing to declare. allogeneic hematopoietic stem cell transplantation in patients with myelodysplastic syndrome of relatively high-risk groups: curative effect analysis and optimal timing selection results: among the patients, patients were transplanted successfully. the -year overall survival (os) rate and disease-free survival (dfs) rate was . % ± . % and . %± . % respectively. the -year cumulative relapse rate (rr) and the non-relapse mortality (nrm) rate was . %± . % and . %± . % respectively. the incidence of grade ii-iv acute graft versus host disease (agvhd) was . %± . %. for the patients who survived more than days after allo-hct, the years cumulative incidence of chronic graft versus host disease (cgvhd) was . %± . %. univariate analysis showed that the hematopoietic cell transplantation comorbidity index(hct-ci) and grade iii-iv agvhd are the high risk factors for os( . ± . % vs . ± %, p= . and . ± . % vs . ± . %,p < . ). multivariate analysis demonstrated that grade iii-iv agvhd and hct-ci are independent risk factors for os(hr= . , p < . , % ci: . ~ . and hr= . ,p= . , %ci: . ~ . ). chemotherapy before transplantation did not improve os or dfs for patients with bone marrow blast cells more than % at the time of diagnosis.conclusions: allo-hsct is an effective treatment for mds patients of relatively high-risk groups. the physical condition of the patients and occurrence of agvhd are independent risk factors. for intermediate and high risk ipss-r mds patients, transplantation before the disease progressed into very high risk can achieve better prognosis, high-risk group can still benefit from rebound gvhd after cni tapering which was promptly responsive to treatment steroids, fk and ecp. aa one year after sct / patients were without gvhd and off all immunosuppression while one single patient was still on taper of immunosuppressant after rebound acute gvhd. no chronic gvhd occurred. sctrelated toxicity was common with mucositis in all patients (who grade : n= ), elevated liver enzymes (≥grade : n= ) and impaired renal function (gfr - ml/min: n= ). five patients developed neurologic symptoms (seizures n= , pres n= , pseudotumor cerebri n= ) which all resolved without sequelae. overall survival and transfusion-free survival was % with a median observation time of ( - ) years.conclusions: . treosulfan-based conditioning followed by sct from hla-matched related or unrelated donors represents a highly efficacious treatment approach for children, adolescents and young adults with tdt and exhibits an acceptable but not negligible safety profile. an individual risk-benefit assessment incorporating hazards such as secondary graft failure, gvhd and long-term toxicity including infertility and nd malignancy has to be executed in the informed consent process for every patient and his/her guardians.disclosure: "nothing to declare" p abstract already published. early iron chelation with deferasirox might prevent relapse after busulfan plus fludarabine and atg as a myeloablative conditioning for hla-identical sibling allogeneic hct in aml results: we show an excellent concordance between chimerism assessment on bio-rad and d platforms over the complete range of mixture ratios (r > , ) and proof the lower detection limit ( , %) compared to str-pcr.conclusions: our results promote the transfer of the established mentype assay to a more diverse instrument portfolio. that will allow to implement the analysis of patient and donor hematopoiesis by digital pcr methods in our lab.disclosure background: with the immense progress in therapeutic regimens in pediatric oncology and stem cell transplantation the survivor rates increased up to %. at the same time the field of reproductive medicine has achieved substantial advances to offer potential options for fertility preservation. therefore it is of great importance to implement fertility counseling and fertility preserving (fp) procedures for patients facing gonadotoxic therapy. in the report on the expert meeting of the paediatric diseases working party (pdwp) of the ebmt in counseling related to fp opportunities should be offered to each patient receiving stem cell transplantation (sct), as part of the pre-sct workup by a dedicated and trained team. yet there many medical, ethical, structural and financial issues to consider and overcome. we describe the setting up process to enable fertility counseling for all children with newly diagnosed cancer or those facing stem cell transplantation for malignant and nonmalignant diseases in our department of pediatric oncology and immunology/stem cell transplantation.methods: at our tertiary care center we assembled a multidisciplinary team involved in fertility preservation (pediatric hematology/oncology, pediatric immunology/ stem cell transplantation, reproductive medicine, andrology, psychology and pediatric surgery). we developed an internal grading system for recommendations regarding fertility preservation based on the current recommendations for fertility preservation of leading societies in this field like ebmt, gpoh and dggg. it is important to find a consensus within the team for the counseling to ensure reliable counseling. the third step is to implement structures needed for fertility counseling and performance of invasive procedures including legal aspects (amg). a detailed description of this process is given.results: after setting up structures for the counseling process we counseled oncology and stem cell transplant patients ( - years) between january and may . we analysed data of the patients including age subgroups and disease entities and the results of the counseling process. for those patients undergoing stem cell transplantation the risk of gonadotoxicity is very high, therefore even the very young children underwent fertility preserving procedures in alignment with our recommendations if they suffered from a nonmalignant disease. currently we discourage tissue preserving in malignant systemic disease due to possible contamination with malignant cells. postpubertal female patients were more likely to undergo invasive procedures such as ovarian tissue cryopreservation in the case of oncological diseases, while sperm cryopreservation was recommended in all postpubertal male patients. overall a high percentage of the patients and their family followed our recommendations.conclusions: fertility preservation should be considered as a very important part of the treatment plan for newly diagnosed children and young adults with cancer and those facing stem cell transplantation. unfortunately there is still a great need for setting up structures in institutions taking care of these patients. in addition fertility preservation sadly lacks funding by health insurance in some countries. with the presentation of our experience and data we want to facilitate incorporation of fertility counseling in other pediatric care centers to provide counseling for pediatric patients in need for fertility preservation.disclosure: no conflict of interest regulatory t-cells (t reg ) have been shown to play a role not only in autoimmune diseases and solid organ transplantation but also in gvhd. several mouse models showed a decrease of gvhd incidence after t reg administration. the few clinical trials regarding the application of t reg for the treatment of gvhd are encouraging, however the data is limited. methods: patient: a -year-old boy underwent allogeneic sct for chronic myeloid leukemia refractory to imatinib, dasatinib and nilotinib treatment from his / hla identical brother. freshly derived unmanipulated bone marrow was transplanted after conditioning with of fludarabine ( mg/m²/d, day - to - ), thiotepa ( x mg/ kg, day - ) and melphalan ( mg/m², day - ). cyclosporin a (csa) and mycophenolatmofetil (mmf) were used for gvhd prophylaxis. leukocyte regeneration (> /μl) was seen on day + , granulocyte regeneration (> /μl) on day + and thrombocyte regeneration (> . /μl) on day + . on day + after sct he developed acute intestinal gvhd that exacerbated to grade iv°(bloody diarrhea, ileus) and did neither respond to steroids, nor to different immunosuppressive drugs such as cyclosporin, tacrolimus, sirolimus, mycophenolatemofetil and ruxolitinib. extracorporal photopheresis and the administration of immunmodulatory antibodies (adalimumab and tocilizumab) did not succeed either.results: by administration of low-dose interleukin- (il- ) in vivo induction of t reg was expected but did not succeed. finally antithymocyte globuline (atg, mg/kg/ d) was administered on day + to + to eliminate the gvhd-triggering cells. hence, the gvhd declined to grade iii. finally, a decision was made to manufacture t reg from his stem cell donor. from an unstimulated leukapheresis t reg were selected by magnetic depletion of cd + t-cells and cd + b-cells followed by positive selection of cd + background: treatment of patients with transfusion dependent anemia like thalassemia major (tm), sickle cell disease (scd) and diamond-blackfan anemia (dba) has improved over the last decades. for the vast majority of patients, allogeneic hematopoietic stem cell transplantation (hsct) is the only available curative therapy. for a long time, hsct has only been performed from hla-identical sibling donors (msd) or matched family donors (mfd). however, approximately only - % of affected patients do have a matched sibling donor, therefore hsct from / (mud) and even / (mmud) matched unrelated donors has gained importance in recent years.methods: patients (age range: - years) with scd (n= ), dba (n= ) or tm (n= ), receiving hsct from a msd, mfd, mud or mmud between and were included in our analysis. patients received transplants from msd/mfd, patients from mud/ mmud. patients were identical for hla-a, b, cw, drb and dqb , patients shared only / genes. we analyzed extended haplotypes including drb , drb , drb and dpb for all patients with thalassemia. pairs showed non-permissive dpb mismatch and pair mismatch for dpb and drb .results: median time for granulocyte recovery was days in patients transplanted from msd/mfd and days in patients transplanted from mud/mmud. platelet recovery was reached after days after hsct from msd/mfd and days after hsct from mud/ mmud. / ( %) patients showed complete donor chimerism in all controls. / ( %) patients showed low level mixed chimerism up to % during follow up. patient died shortly after hsct, patient showed slowly increasing mixed chimerism and finally developed autologous recovery and one patient rejected the graft.cumulative incidence of grade ii-iv acute graft-versushost disease (agvhd) of mud/mmud was , %, whereas only cases of agvhd grade i occurred in patients transplanted from msd/mfd. as patient rejected the graft from a hla-identical parent, patient transplanted from a hla-identical grandparent developed autologous recovery after year and patient transplanted from a mud lost the graft due to hemophagocytosis, the probability of event-free survival was , % after hsct from msd/mfd and , % from mud/mmud.altogether / patients ( , %) are alive and transfusion-independent with complete donor chimerism two years after hsct; resulting in an overall survival probability of , %. in contrast, overall survival probability was % in the group of patients transplanted from msd/mfd and , % in patients transplanted from mud/ mmud after years.there were patients with thalassemia ( , %) who died from transplantation-related causes. the first patient died days after hsct from a mmud due to candida sepsis with pulseless electrical activity resulting from cardiac iron overload. the second patient died months after hsct from a mud due to graft failure.conclusions: hsct from mud and mmud is a feasible therapy option for patients with transfusion dependent anemia. nevertheless, it should be noted that iron overload can cause severe complications; therefore, measurement of liver and heart iron concentration through mri prior to hsct as well as phlebotomy after transplantation are advisable.disclosure: novartis (consultancy: included expert testimony, speaker bureau, honoraria), medac (research funding, patents and royalties), riemser (research funding), neovii (research funding), amgen (honoraria) background: the therapeutic options for patients with hodgkin´s disease who relapse after first high-dose chemotherapy with autologous stem cell ( st asct) support are limited. allogeneic stem cell transplantation in this setting is associated with a high level of transplant-dependent mortality rates in excess of - %. new agent, such as brentuximab vedotin, have been approved for the treatment of these patients, however, their efficacy to provide longterm control or cure is still unknown. a second autologous stem cell ( nd asct) has historically been considered as an option only in a small group of patients so the published experience is scarce. we report our institution´s experience with second autologous transplants in this patient population.methods: we evaluated the outcome of adult patients ( ( %) female and ( %) male), who received an nd asct between / and / . planned tandem asct were excluded. the median age at nd asct was years (range - ), ( %) patients had a karnofsky performance score ≥ %. ( %) patients were in complete remission (cr) and ( %) patients were in partial remission (pr) at day after st asct. seven ( %) relapses within months after st asct. patients received a median of ( - ) treatment lines between st asct and nd asct. only ( %) patients received brentuximab vedotin and none of the patients in our series received checkpoint inhibitors as salvage after st asct. the median interval from st asct to relapse/progression was , months (range , - , ). the median interval from relapse/progression to nd asct was , months (range , - , ). all patients received beam as the conditioning regimen for st asct, and beeam as the conditioning regimen for nd asct.results: the median time to neutrophil recovery (> . x /l) after nd asct were days (range - ). best response at day following nd asct included cr in ( %) patients and pr in ( %); ( %) had stable disease. ( %) patients received brentuximab vedotin and none of the patients received checkpoint inhibitors after nd asct. ( , %) patients are currently alive, with a median follow-up , months (range , - , ). patient died after nd asct. causes of death were hl progression. the -year overall survival was %.conclusions: the second asct in patients with a longterm response after the first asct may be the optimal therapeutic option, the effectiveness of which can be enhanced by using new drugs, such as brentuximab vedotin, at all stages of treatment.disclosure: nothing to declare effectiveness of chemo-g-csf protocols for mobilization of peripheral stem cells in patients with non-hodgkin lymphomas and hodgkin disease-single center experienceilina micheva , stela dimitrova , vladimir gerov , trifon chervenkov , liana gercheva , igor reznik background: high-dose chemotherapy and autologous stem cell transplantation (asct) play an important role in achieving long-term remission in patients with non-hodgkin lymphoma (nhl) and hodgkin disease (hd). granulocyte colony stimulating factor (g-csf) combined with high-dose chemotherapy is a frequently used mobilization approach; however, the optimal mobilization strategy has not been determined.the objective of the study was to analyze the mobilizing potential of different regimens used for the collection of peripheral stem cells in patients with relapsed or refractory (r/r) nhl and hd. methods: we retrospectively analyzed patients with r/r nhl and hd undergoing stem cell collection after chemo-mobilization in the transplant unit at the university hospital, varna. patients were mobilized after dhap letermovir is promising, even as a therapeutic agent. more paediatric data are urgently needed.disclosure: nothing to declare p development of paroxysmal nocturnal hemoglobinuria in a patient after mudallohsct due to jak v fpositive myelofibrosis-a case of successful treatment with the second transplantation from another donor agnieszka tomaszewska , barbara nasiłowska-adamska , iwona solarska , kazimierz hałaburda background: paroxysmal nocturnal hemoglobinuria (pnh) is a very rare disease associated with pig-a gene mutations in hematopoietic stem cells. there are only single case reports on evolving myeloproliferative diseases to pnh in the literature. there is no data concerning development of pnh de novo after allogeneic hematopoietic stem cell transplantation. in our report we describe a patient with recurrence of jak v -positive myelofibrosis years after matched unrelated donor allogeneic hematopoietic stem cell transplantation (mudallohsct) with simultaneous development of clinically significant pnh. a year-old-man with a history of mudal-lohsct in may due to jak v -positive myelofibrosis secondary to essential thrombocythemia was admitted to our department years later with mild anemia (hb- . g/dl) and elevated lactate dehydrogenase ( u/l). during last years he remained in complete remission of myelofibrosis with jak v mutation negativisation and % donor chimerism. suspecting disease recurrence we performed trephine biopsy confirming myelofibrosis (mf /mf ) with heterozygous jak v mutation and in flow cytometry analysis of bone marrow we identified cell membrane defect in myeloid line (loss of cd c). we decided to perform detailed diagnostic tests on pnh -multiparametric flow cytometry of peripheral blood revealed % granulocytes and % red blood cells with loss of gpi-anchored proteins -pnh clone. these results corresponded with donor chimerism -it was only % of donor dna in bone marrow and % in blood tests. molecular analysis didn't revealed any mutations in genes: calr, asxl and mpl. finally the diagnosis of myelofibrosis recurrence after mudallohsct with presence of pnh clone was established. the therapy with eculizumab was unreachable. so the second allohsct from another matched unrelated donor after fludarabinemelphalan-thymoglobuline-tbi cgy conditioning was performed on . . . we didn't observe any complications of this procedure, engraftment was slightly delayed: anc> . g/l on the day and plt> g/l on the day.results: at present, more than years after the second mudallopbsct, the patient remains in a very good condition with % of the second donor chimerism and without any features of pnh (clone is undetectable) and myelofibrosis.conclusions: presented case is the first in the literature well documented myelofibrosis recurrence after mudal-lohsct with concurrently development of clinically significant paroxysmal nocturnal hemoglobinuria. the second mudallohsct from another donor was safe and successful treatment strategy in this situation.disclosure: nothing to declare. abstract already published. cutaneous refractory t-cell lymphoma treated with allogeneic hematopoietic stem cell transplantationmarcia silva , ercole orlando , maria claudia moreira , simone lermontov , simone maradei , yung gonzaga , leonardo arcuri , renato araujo , decio lerner instituto nacional de cancer, cemo, rio de janeiro, brazilbackground: folliculotropic mycosis fungoides (fmf) is an aggressive clinical course variant of cutaneous t-cell lymphoma (ctcl) -classic mycosis fungoides (mf) , with distinct clinical and pathological characteristics, and it is less responsive to skin-directed therapies. for diseases in advanced stages, chemotherapy, autologous hematopoietic stem cell transplantation (hsct) or immunomodulator drugs may provide remissions with limited duration and the treatment remains substantially palliative , . these dismal results have induced to explore the therapeutical approach with allogeneic hematopoietic stem cell transplantation (hsct) in such patients. early studies have shown encouraging results also in patients with advanced disease, suggesting a major therapeutical role played by the graft versus lymphoma (gvl) effect , , . methods: this is a case report of the use of allogeneic hsct as a potential cure for cutaneous refractory t-cell lymphoma type folliculotropic mycosis fungoides .results: case presentation : a -year-old male patient with refractory subtype b fmf t-cell lymphoma , diagnosed in , clinically characterized by exfoliative erythroderma, widespread plaques on the trunk and limbs, solitary tumor on the right shoulder, pruritus and bilateral